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Synthesis and properties of N-acylated and N-alkylated polyamine derivatives and of agrobactin A, a naturally occurring siderophore

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Title:
Synthesis and properties of N-acylated and N-alkylated polyamine derivatives and of agrobactin A, a naturally occurring siderophore
Alternate title:
N-acylated and N-alkylated polyamine derivatives
Alternate title:
Agrobactin A, a naturally occurring siderophore
Creator:
Stolowich, Neal J., 1957-
Publication Date:
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English
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vii, 171 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Amides ( jstor )
Amines ( jstor )
Antineoplastics ( jstor )
Chlorides ( jstor )
Hydrochlorides ( jstor )
Polyamines ( jstor )
Protons ( jstor )
Reagents ( jstor )
Signals ( jstor )
Solvents ( jstor )
Dissertations, Academic -- Medicinal Chemistry -- UF ( mesh )
Medicinal Chemistry thesis Ph.D ( mesh )
Polyamines ( mesh )
Spermidine -- chemical synthesis ( mesh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida.
Bibliography:
Includes bibliographical references (leaves 165-170).
Additional Physical Form:
Also available online.
General Note:
Photocopy of typescript.
General Note:
Vita.
Statement of Responsibility:
by Neal J. Stolowich.

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University of Florida
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University of Florida
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Copyright Neal J. Stolowich. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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SYNTHESIS AND PROPERTIES OF N-ACYLATED AND N-ALKYLATED
POLYAMINE DERIVATIVES AND OF AGROBACTIN A,
A NATURALLY OCCURRING SIDEROPHORE






BY

NEAL J. STOLOWICH















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA 1983











































To Barb and Dan,

for their unending love and patience














ACKNOWLEDGEMENTS
I would like to express my sincere appreciation to my research advisor, Dr. Raymond Bergeron, under whose guidance this work was accomplished. I would also like to express my gratitude to the rest of my supervisory committee, Dr. Richard Streiff, Dr. Margaret James, Dr. Federico Vilallonga, and Dr. Cemal Kemal.

The uptake determinations of the polyamine derivatives were performed by Dr. Carl Porter and his group at Roswell Park Memorial Institute, Buffalo, New York. I wish to extend my appreciation to them for these determinations, as the data were a significant part of this work.

Finally, I would like to thank all my family and friends that have helped make this work possible: first, to my parents, Frank and Helen Stolowich, for their guidance to further pursue my academic career; secondly, to all my friends, past and present, in medicinal chemistry for their friendship and assistance. Special thanks to Dr. Kathy McGovern for those technical discussions at Joe's and for going through this at the same time. And, last but certainly not least, to my wife Barbara for her continual support and motivation to carry me through graduate school.











iii
















TABLE OF CONTENTS
PAGE
DEDICATION .................................................... ii
ACKNOWLEDGEMENTS.... ........................ iii

ABSTRACT...................................................... vi

CHAPTER

ONE INTRODUCTION AND BACKGROUND .......................... 1

Polyamines and Growth.............................. 4
Structural Requirements for Polyamine Uptake ....... 13 Spermidine Derived Siderophore Systems............. 16

TWO REAGENTS FOR THE SELECTIVE FUNCTIONALIZATION OF
SPERMIDINE, NORSPERMIDINE, AND HOMOSPERMIDINE....... 24

Experimental............................. 31
Results and Discussion............................. 49

THREE BIOLOGICAL EVALUATION OF POLYAMINE DERIVATIVES........ 64

Materials and Methods.............................. 64
Results........................................... 67
Discussion........................................ 71

FOUR PRELIMINARY INVESTIGATIONS TOWARDS THE DEVELOPMENT
OF SPERMIDINE-ANTINEOPLASTIC CONJUGATES ............. 76

Experimental..................................... 80
Results and Discussion............................. 86

FIVE SYNTHESIS OF TRIS-PROTECTED SPERMIDINES............... 90

Synthesis......................................... 92
Experimental.......... ............................ 97
Results and Discussion ............................ 121

SIX SYNTHESIS AND SOLUTION DYNAMICS OF AGROBACTIN A...... 127

Experimental Section............................... 127
Results and Discussion ............................ 132




iv









CHAPTER PAGE SEVEN CONCLUSIONS ... .................................. 162

REFERENCES ............... ................................ 165

BIOGRAPHICAL SKETCH ...... ..................*................. 171













Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

SYNTHESIS AND PROPERTIES OF N-ACYLATED AND N-ALKYLATED
POLYAMINE DERIVATIVES AND OF AGROBACTIN A,
A NATURALLY OCCURRING SIDEROPHORE

BY

NEAL J. STOLOWICH

December 1983


Chairman: Raymond J. Bergeron
Major Department: Medicinal Chemistry

The synthesis of reagents for the selective functionalization of

the polyamines spermidine, norspermidine, and homospermidine is described. With these reagents various N-acylated and N-alkylated polyamine derivatives are generated and their uptake characteristics studied in Murine L1210 leukemia cells. In general those polyamine derivatives having their terminal amines modified were unable to compete with 3H-spermidine for

uptake into those cells. Polyamine derivatives in which the central nitrogen is modified are found to compete with 3H-spermidine for uptake. This suggests that theprimary amines are critical for inferring uptake specificity while the secondary amine is less important. The synthesis of two antineoplastic spermidine conjugates is also described as a preliminary investigation into the potential of spermidine as a drug delivery device.

In a related synthetic sequence, Agrobactin A and a number of analogues are prepared. High field proton nuclear magnetic resonance


vi









spectroscopy is employed to evaluate the origins of the conformers in agrobactin A. The coalescence temperatures and activation energies between conformers of these polyamides are determined using 1H NMR. The role of steric and hydrogen bonding factors in these compounds is also discussed.










































vii













CHAPTER ONE

INTRODUCTION AND BACKGROUND

Although the existence of polyamines in human semen has been known since 1677,1 the majority of research regarding the polyamines' biosynthesis and physiological functions has occurred only in the last twenty years. These naturally occurring polyamines are, in general, linear diand triamines: the five most commonly associated with eukaryotes are presented in Figure 1-1. Of these, the amines 1,4-diaminobutane (putrescine), spermidine, and spermine are the most important and most commonly occurring polyamines in mammalian tissues, although others have been shown to occur in trace quantities as well.

The biosynthesis of putrescine, spermidine, and spermine proceeds through a common pathway in mammals as well as microorganisms, the primary precursors being the amino acids L-ornithine and L-methionine. As shown in Figure 1-2, L-ornithine is decarboxylated to form putrescine, and the ATP activated form of methionine, S-adenosyl-L-methionine, serves

as the propylamine donor, the moiety added to putrescine to form the higher polyamines spermidine and spermine. Four enzymes are primarily responsible for the synthesis of the polyamines in mammalian tissues-two decarboxylases, and two synthetases. The decarboxylases, L-ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAM-DC), have been extensively purified and studied. Ornithine decarboxylase, which catalyzes the conversion of ornithine to putrescine, is a pyridoxalrequiring enzyme, like most other mammalian decarboxylases. However,


1












H2NCH2C H2CH2NHCH2CH2CH2CH2NH2 Il2NCH2CII2CH2CH2NH2
Spermidine
1,4-diaminobutane

(Putrescine)
H NCH CH CH NHCH CH CH NH 2 2 2 2 2 2 2 2 Norspermidine H2NCH2CH2CH2CH2CH2NH
H2NCH2CH2CH2CH2NHCHCH2CH2CH2NH2 "1,5-diaminopentane

(Cadaverine) Homospermidine



H2NCH 2CH 2CH2 NHCH 2CH 2CH2CH 2NHCH2 CH 2CH2NH2 Spermine




Figure 1-1. Commonly occurring polyamines associated with eukaryotic cells










COOH
I Ornithine
H2NCH2CH2 CH2CHN2 H2NCH2CH2CH2CH2NH2
decarboxylase

L-Ornithine Putrescine


Spermidine
synthase




C00H
+ C SAM +
CH3- S-CH2CH2CHNH2 ) CH3---CH2CH2CH2NH2 H2NCH2CH2 CH2 2NHCH2CH2CH2 CH2NH2
C decarboxylase
Adenosine Adenosine
Spermidine

S-Adenosyl-Methionine


Spermine synthase Pi + PPi




L-Methionine + ATP H2NCH2CH2CH2NHCH2CH2 CH2CH2NHCH2CH2CH2NH2


Sperm i ne


Figure 1-2. Biosynthesis of putrescine,spermidine, and spermine in mammals.






4


unlike most mammalian enzymes, ODC is strikingly inducible2 and has a very short half life--lO0-30 min.3 Its basal activity is the lowest of the four; thus, it is considered to be the rate-controlling enzyme in polyamine biosynthesis.4

The other decarboxylase, S-adenosyl-methionine decarboxylase, decarboxylates the methionine residue that is covalently bound to the adenosine. It too has a short half life of about one hours but, unlike ODC and most other decarboxylases, does not need pyridoxal phosphate for catalytic activity. Instead, SAM-DC contains pyruvate residues within its peptide chain that serve as the carbonyl cofactor.6

The spermidine and spermine synthases catalyze the transfer of propylamine groups from decarboxylated SAM to putrescine and spermidine, respectively. These synthases have been only partially purified at the present time, but have been shown to be relatively stable enzymes with long half lives.7 The differences between the decarboxylases' and synthases' half lives are of significant importance in considering the inhibition of polyamine biosynthesis, as will be discussed later.

As mentioned earlier, polyamines have gained considerable interest in the past few decades, and this has been mainly on two fronts. First, polyamines have been implicated as being directly involved in cellular function and, secondly, the polyamine spermidine serves as a backbone

to a number of naturally occurring siderophores or iron chelators. The latter will be briefly dealt with later, while some of the aspects of polyamine involvement in cellular function will be discussed now.

Polyamines and Growth

As illustrated in Figure 1-3, there have been many suggested in vivo and in vitro functions of polyamines. Of all of these, this thesis is









Table 1-1. Suggested in vitro and in vivo functions
of polyamines

1. POLYAMINES AS GROWTH FACTORS

2. STABILIZATION OF WHOLE CELLS AND MEMBRANES

3. STABILIZATION OF SUBCELLULAR PARTICLES

4. ASSOCIATION WITH NUCLEIC ACIDS
Stabilization of DNA against denaturation
Stabilization of folded form of DNA
Association with tRNA
Stabilization of newly synthesized RNA
Stimulation of DNA synthesis Stimulation of RNA synthesis
5. EFFECTS ON PROTEIN SYNTHESIS
Binding of tRNA molecules to ribosomes
Stimulation of tRNA methylation
Replacement of Mg++ in aminoacyl tRNA synthetase reaction
Association with ribosomes Initiation of translation
Stimulation of tRNA nucleotidyltransferase

6. EFFECTS ON VARIOUS METABOLIC REACTIONS
Stimulation of nucleotide kinases
Enhancement of ADP-ribosylation of nuclear proteins
Activation of phosphorylase b
Stimulation of lipolysis
Activation of choline kinase
Inhibition of ATPase
Modification of acetylcholine esterase activity

7. PHARMACOLOGIC EFFECTS IN WHOLE ANIMALS
Nephrotoxic effects
Hypothermic and sedative effects

(from reference 10)






6

primarily concerned with the polyamines' role in cellular growth in both the normal and diseased states. Several excellent reviews8'-0 are available for information concerning the other biological effects and will not be discussed here.

The initial observations linking polyamines to cell growth were established in several different rapidly growing tissue systems during the mid-sixties. In studies involving chick embryos, marked increases in polyamine levels along with increased ODC and SAM-DC activities were observed during development relative to those of the mature animal.11-13 In the human fetal liver, putrescine concentration displayed a distinct peak in the fifth and sixth month of gestation.14,15 Although the embryonic data are limited, the higher polyamine levels were all attributed to higher ornithine and adenosylmethionine decarboxylase activities associated with rapid growth.

The regenerating rat liver has been a more widely used system to study the effect of growth upon polyamine biosynthesis. Experimental evidence from several groups has shown that enhanced accumulation of putrescine reaches a maximum approximately four hours after partial hepatectomy,16 resulting in a moderate enhancement of spermidine levels in 2-3 days.17 This early increase in putrescine coincides with enhanced ODC activity, which can be as great as 100- to 500-fold.12,18 This increase is extraordinarily high as most enzymes in vivo only fluctuate 2-, 3-fold. The enhanced ODC activity, along with recent data revealing that this enzyme is closely regulated by transcriptional and translational mechanisms,19,20 suggests that this early dramatic increase is the primary event responsible for triggering polyamine biosynthesis. The increased levels of putrescine and spermidine in the regenerating






7


liver are also usually accompanied by a decrease in spermine17 as spermine synthetase has been shown to be inhibited by high concentrations of putrescine. 21

More recently, a third type of rapidly proliferating cell and its

corresponding high polyamine levels havedrawn much attention-- the cancer cell. This was first shown in Ehrlich ascite carcinoma cells grown in mice by two separate labs.22,23 Both groups found that in the early stages of tumor growth, a remarkably high putrescine content was found that rapidly decreased after the first week following inoculation, whereas the concentration of spermidine peaked near the second week after inoculation. These data are comparable to that found in the regenerating rat liver system mentioned earlier. Williams-Ashman et al. were further able to show, by using hepatomas of vastly different growth rates, that the accumulation of putrescine was proportional to the rate of tumor growth.24'25 Putrescine levels in the most rapidly growing hepatomas were ten times as high as levels in the normal rat liver. Furthermore, ornithine decarboxylase activity also coincided well with rate of tumor growth.

Similar results have also been found for other types of cancer.

For example, markedly elevated levels of spermidine as well as ornithine decarboxylase activity were found in d/3 sarcoma as compared to normal muscle tissue.26 Ornithine decarboxylase activity was found to be six times higher in mouse L1210 leukemia tumor cells, as well as containing

more than double their normal values of putrescine and spermidine.27 Additionally, some rapidly growing tumors produce enough polyamines that

they are actually excreted into the surrounding interstitial fluid causing elevated polyamine serum levels.28,29 Accordingly, clinical researchers






8

are currently developing diagnostic tools to follow tumor regression and perhaps detect early tumor growth."3

From the above data it can be suggested that all cells, whether normal or neoplastic, undergoing rapid proliferation contain elevated levels of polyamines, notably putrescine and spermidine.

If polyamines are so intimately involved and required for rapid cell growth as implied so far, then the selective inhibition of their biosynthesis should have a pronounced detrimental effect upon cell growth. This concept of inhibiting cell growth by inhibiting polyamine synthesis would not only be valuable in determining the actual physiological significance of polyamines but could also represent a possible method of arresting cancer growth. As mentioned earlier, the short half lives of both ornithine and SAM decarboxylases are significant in that they should be the easiest enzymes to inhibit. This is in fact the case as most of the research in inhibiting polyamine synthesis in vitro and in vivo has been centered on finding selective inhibitors of these two enzymes utilizing various tumor cell lines.

A great majority of the inhibitors that have been developed in the last twenty years are congeners of ornithine and methionine. Ethionine, the ethyl analog of methionine, was probably the first compound used to inhibit polyamine synthesis. Although injections of it led to an initial decrease in spermidine concentration, chronic treatment eventually led to an overall increase in spermidine concentration,31 and its use as an inhibitor subsequently was abandoned.

In 1973, a-hydrazinoornithine, a potent reversible inhibitor of ornithine decarboxylase (ODC), was synthesized.32 Synthesis of a-hydrazinoornithine found it to be a competitive inhibitor with a Ki value of






9


2 UM.33 In vivo, the presence of 0.5 mM a-hydrazinoornithine added to the medium of hepatoma cells could prevent the normal rapid accumulation of putrescine and, likewise, partially prevent the increased accumulation of putrescine due to partial hepatectomy.34 In both systems, however, hydrazinoornithine did not disturb the enhanced synthesis of RNA, a negative observation as far as indicating the need of polyamines for cell

proliferation or RNA stabilization.

A close analog to hydrazinoornithine, D,L-a-hydrazino-6-aminovaleric acid was an even more effective inhibitor of ornithine decarboxylase than the former, having a Ki of 0.5 pM.35 Administration of hydrazinoaminovaleric acid blocked the increase in DNA synthesis, the accumulation of putrescine, as well as the weight gain usually seen in mouse sarcomas.36 Similar results were also obtained from cultures derived from hamster tumors.36 In all these cases spermidine and spermine levels remained

unchanged, and the effects of hydrazinoamino valeric acid could be reversed by the addition of putrescine, but not spermidine35 or cadaverine.36

Perhaps the simplest of ornithine analogs, a-methylornithine, synthesized in 1974, is also a potent competitive inhibitor (Ki value 20 M) of ODC. Addition of a-methyl ornithine to cultures of rat hepatoma cells completely prevented accumulation of putrescine, and likewise prevented increases in cellular spermidine.37 Incorporation of thymidine in DNA greatly declined after the first doubling time in its presence. Furthermore, a-methylornithine's growth inhibition could be immediately reversed by concentrations of spermidine, spermine, and putrescine lower than that of ornithine, offering good evidence that the intracellular polyamine levels are casually related to proliferation.






10

Recently, a difluoro analog of a-methylornithine, difluoromethylornithine (DFMO), has been synthesized which has been found to be a potent irreversible inhibitor of ODC.38 Difluoromethylornithine, which is nontoxic in mammals,39 has been shown to block multiplication of the parasite Trypanosoma b. brucei, and can cure mice infected with the parasite simply by administration in their drinking water.40 Furthermore, the cure can be reversed by injection of small doses of polyamines. Difluoromethylornithine has also been shown to inhibit growth of L1210 leukemia cells, again,its antiproliferative effects prevented by spermidine and putrescine.41 In fact, L1210 cells depleted in polyamines by pretreatment with DFMO show up to a 3-fold increase in polyamine uptake as compared to untreated cells,42 evidently in an attempt to restore normal levels. This observation will have additional importance in following discussions.

Up to this point, all the inhibitors of polyamine synthesis discussed, with the exception of ethionine, are inhibitors of ODC. However, an important group of SAM-DC inhibitors has been recently discovered and warrants further discussion. The first of this group, methylglyoxal bis(guanylhydrazone) (MGBG), was first synthesized in 1898,43 but only as recently as 1972 shown to be a potent and specific inhibitor of SAMDC by Williams-Ashman and Schenone.44 The discovery of MGBG as an inhibitor of SAM-DC grew out of an investigation of its known antitumor effects. Methylglyoxal bis(guanylhydrazone) was first shown to be active against L1210 leukemia,45 but has since been tested against a wide range of experimental tumors.46,47 However, leukemias exhibit the greatest sensitivity to MGBG and, accordingly, MGBG recently has been used to treat several forms of human leukemia clinically.48








If one were to look at MGBG's structure (Figure 1-3), one might

assume that the mode of action of inhibiting SAM-DC is by mimicking SAM's structure, and that this inhibition of polyamine synthesis might be responsible for the antileukemic activity. However, there appears to be no simple correlation between the ability to inhibit SAM-DC and the antileukemic activity. In general, minor modifications of MGBG, such as the dimethyl and ethylglyoxal analogues,do not have any pronounced effect against its ability to inhibit SAM-DC, but lack antileukemic activity.49

Recently, an alternative mechanism for MGBG's antiproliferative activity has been suggested. Several recent studies have shown that cultured L1210 cells treated with micromolar concentrations of MGBG develop excessively swollen mitochondria several hours before detectable changes in polyamine levels or cell growth.50'51 This suggested MGBG may act as a mitochondrial poison. The damage in most instances is reversible and normal appearance can be restored by the addition of spermidine. However, the irreversible inhibitor l,l](methylethane diylidene dinitrilo]bis(3-aminoguanidine) (MGAG) which is equally effective as MGBG in inhibiting cell growth, is much slower to produce mitochondrial damage.52

Of additional interest, MGBG is concentrated in a variety of cells,53 apparently through a saturable energy-dependent transport mechanism.54 In fact, in Ehrlich ascite carcinoma cells, MGBG is concentrated so effectively that a concentration gradient as high as 1000-fold across the cell membrane has been observed, producing millimolar intracellular MGBG levels.55ss Furthermore, the accumulation of MGBG could be blocked competitively by polyamines and, to a lesser extent, some diamines.56,57 For example, only micromolar concentrations of spermidine or spermine,






12









H
HHH
a) NH c


H2N. H N .NH H NH2 Methyigiyoxol-Bis (Guanylhydrozone)




H H H
b) NHz
N
+ NH2 ~~------I


H
COOH





S-Adenosyl- L-methionine




H
c) H
Spermidine





Figure 1-3. Structures of a) Methylglyoxal-bis-(guanylhydrazone), b) S-adenosyl-L-methionine, and
c) Spermidine.






13


but not putrescine, effectively block MGBG's uptake. Accordingly, it

is postulated that MGBG competes with the carrier or receptor site responsible for spermidine uptake. Additional evidence which supports this is that, in DFMO pretreated L1210 cells, which respond by increasing polyamine uptake, uptake of MGBG is also increased.42

Structural Requirements for Polyamine Uptake

Whatever MGBG's mechanism of action may be, whether it acts by inhibiting SAM-DC and diminishing spermidine levels, or by replacing spermidine at some intracellular site, perhaps in the mitochondria, it is of significant importance that MGBG and spermidine both compete for energy-dependent uptake. Moreover, little is known as to what structural requirements are necessary for a molecule to be actively transported into the cell via this carrier. Indeed, up until the present time very little has been done to investigate the effects upon cell growth and uptake by derivatizing or modifying the polyamines. Several N-modified spermidine and spermine compounds have been synthesized,as in the case of the monoand bis-acridyl-spermidine and spermines; however, these compounds were initially synthesized in order to investigate their ability to intercalate with DNA.58 Likewise, several novel branched spermidine and spermine homologues have been recently synthesized and screened for their antineoplastic activity,9,s0but the uptake characteristics of these compounds have not yet been investigated. From the studies discussed thus far, it is apparent that certain structural parameters are necessary for polyamine uptake; consequently, it would be desirable to further define the structural requirements for uptake.

Recent evidence exists that of the polyamines, spermidine, spermine, and putrescine, the carrier is more specific for spermidine.






14


Therefore, this study will limit itself to the syntheses of spermidine

derivatives only.

In considering the synthesis of spermidine derivatives, three questions can be asked concerning spermidine's structure relating to uptake:

1) What is the role of the hydrocarbon chain length?

2) What are the roles of the primary amines?

3) What is the role of the secondary amine?

To answer these questions, compounds having the general structures as illustrated in Figure 1-4 were synthesized. These compounds included both N-acylated and N-alkylated spermidine derivatives as well as homologues that differ in hydrocarbon chain length i.e. nor- and homospermidine derivatives. The synthesis of these compounds necessitated the generation of a series of reagents capable of efficient, selective functionalization of the spermidine backbone, the details of which are discussed .in Chapter Two.

Once in hand, this gave a broad range of N-modified spermidine

derivatives with which to investigate the effect of such parameters as chain length, functionalization of the primary versus secondary amine, size of the substituent added, and amide versus amine linkages upon uptake.

Since this energy-dependent polyamine transport system appears to

be more specific for spermidine, uptake of these derivatives was assayed by measuring the ability of the derivative to compete with radiolabelled spermidine for uptake in an in vitro system-murine L1210 cell cultures. These cells represent a good model as they efficiently accumulate polyamines, notably putrescine and spermidine, from their environment during periods of rapid growth.61,62 Accordingly, the cells are incubated in






15






RICONH --(CH2 )a NH (CH2)b- NHCORl









RICH2NH --(CH2)a- NH (CH2)b --NHCH2RI






R
1
CO

H2N (CH2)a- N (CH2)b NH2












H2N (CH2)a- N (CH2)b NH2





Figure 1-4. Generalized structures of polyamine derivatives
synthesized,






16

the presence of both the derivative and 3H-spermidine. Spermidine derivatives which successfully compete with spermidine for the carrier will accordingly reduce the amount of radiolabel taken up by the cells.

The uptake characteristics of these derivatives suggested that only a minor structural significance is placed on the secondary amine of spermidine as compared to its primary amines. For example, bis-acylated compounds such as NI,N8-bis(2,3-dihydroxybenzoyl)spermidine and N1,N8-bis(t-butoxycarbonyl)spermidine are unable to compete with 3H-spermidine for uptake. However, N4-benzylspermidine is as good as MGBG in inhibiting spermidine uptake.42

These findings suggested the possibility of conjugating several

small antineoplastics to spermidine via its secondary nitrogen. Since tumor cells apparently concentrate spermidine, this may serve as a novel method to target the delivery of antineoplastics to the tumor. Therefore, a preliminary investigation into this area was initiated with the synthesis of two spermidine antineoplastic conjugates: N4-chlorambucil spermidine and N4-[4-(2,3-dihydro-lH-imidazo]l,2-b]pyrazolo)carboxamido)butyryl]spermidine (Figure 1-5). Preliminary in vitro findings indicate that even the attachment of N4-substituents of this magnitude does not prohibit the competitive uptake of the conjugate.

Spermidine Derived Siderophore Systems

The second part of this dissertation deals with the synthesis and

solution dynamics of agrobactin A (Ia, Figure 1-7), a naturally occurring spermidine-derived iron chelator. There has been considerable interest in this family of siderophores since the isolation and identification of two other siderophores: N',N8-bis(2,3-dihydroxybenzoyl) spermidine (II) and N4-[N-(2-hydroxybenzoyl)threonyl]-Nl,NB-bis(2,3-dihydroxybenzoyl)






17





CIN, N C 1



4) 0





CO
HN N NH2








C N"
N

b) CO




co
H2N NN
NH2





Figure 1-5. Structures of a) N4-Chlorambucilspermidine,and
b) N -[4-(2,3-dihydro-1H-imidazo]1l,2-b]pyrazolo)
carboxamido)butyryl]spermidine.





18


spermidine (parabactin A, Ib) by Tait63 in 1975, Figure 1-6. For the most part, this is due to the lack of a satisfactory therapeutic device for the treatment of various iron overload syndromes,64-66 and the potential that these compounds have shown for clearing iron.67,68

For example, both compound II and parabactin A have been shown to

be more effective at removing iron from transferrin than desferrioxime,69,70 the drug which is currently being used clinically for chelation therapy. Since the initial isolation of parabactin A, it has been revealed that the product isolated may have resulted from the hydrolysis of the siderophore N4-[N-(2-hydroxyphenyl )-4-carboxyl-5-methyl -2-oxazol ine]-N1,N8-bis(2,3-dihydroxybenzoyl)spermidine (parabactin, IIIb) actually produced by the microorganism. Based on Neilands' and Peterson's study,71 it is likely that, during the original Tait isolation of parabactin A, the acidic workup employed would have been sufficient to hydrolyze parabactin's oxazoline ring to produce the open form Ib isolated. Both the open threonyl form and the closed oxazoline form can be easily differentiated, among other means, by high field proton nuclear magnetic resonance (1H-NMR) spectroscopy due to characteristic chemical shifts and coupling patterns of the threonyl residue versus the oxazoline residue.72'73 Furthermore, it has been recently observed that parabactin exists in at least two distinct conformers as determined by 1H-NMR spectroscopy.73 The existence of these conformers is supported by the threonine oxazoline proton signals, which exist in duplicate.

A similar system to the parabactin/parabactin A system has been

recently isolated from Agrobacterium tumifaciens B6 cultures, given the trivial names of agrobactin A (Ia) and agrobactin (IIIa),74 Figure 1-6. Like parabactin, agrobactin A is a spermidine-derived siderophore;






19










CH
CO

CH3CHCH H HO HOCO OH CONH CH2C H2CH2 NCH2CH2CH2CH2NH CO Ia R=OH
Ib R= H

HO CH HO OH
H: 'OH
CONHCH2C H2CH2CH24CH2CH2H2CH2NHCO
H

II





OH



HO CH OH HO CH3 OO'0 H CONHCH2CH CHC H2CHC H2Hi CH2NHCO IIIa R=OH IIIb R= H

Figure 1-6. Structures of the siderophores Agrobactin A and
Agrobactin (la and IIIa), compound II, and Parabactin A and Parabactin (Ib and IIIb).






20

however, agrobactin contains a third 2,3-dihydroxybenzoyl moiety in place of parabactin's 2-hydroxybenzoyl moiety. Likewise, agrobactin (IIIa) also exists in a series of conformers, and on activation energy for interconversion between conformers has been determined.72 However, similar information regarding agrobactin A is not available. Furthermore, no information exists concerning the nature of the conformers observed in these siderophores in generali.e., the contribution of steric and hydrogen bonding factors towards the activation energy controlling conformer interconversion. Accordingly, synthesis and high field 1H-NMR studies of agrobactin A were undertaken.

In considering a synthesis of agrobactin A, the ability to generate symmetrical homologues and other derivatives must be kept in mind. Agrobactin A has been recently synthesized by Neilands by reacting spermidine with the bulky reagent 2,3-dibenzoyloxybenzoyl chloride, followed by attachment of the N-(2,3-dibenzyloxybenzoyl)threonyl "centerpiece" and removal of the benzoyloxy protection groups.72 However, the yields

reported for the condensation steps and the limitations on the derivatives that can be generated make this synthesis somewhat undesirable.

If, on the other hand, a protected spermidine reagent could be
75
employed, such as NI,N8-bis(t-butoxycarbonyl)spermidine (IV), an efficient synthesis of agrobactin A can. be envisioned as illustrated in Figure 1-7. The reagent (IV) was easily condensed with N-(t-butoxycarbonyl)threonine using the coupling reagents dicyclohexylcarbodiimide

and N-hydroxysuccinimide. The resulting product N4-[N-(t-butoxycarbonyl)threonyl]-N1,N8-bis(t-butoxycarbonyl)spermidine was then quantitatively deprotected by brief exposure to trifluoroacetic acid generating N4threonylspermidine (VI). Agrobactin A could be subsequently synthesized






21






cl
Iv tBOC-NH2 N NH-tSOC






HO NH-tBOC V CH3 0
t C-NH N N H- t 80 C



I

H NH3

CH3 / =0
VI H3N s N
NN NH3





OH


OH

Ia
HHO OH
HOHO NH O C3 / OH O' NH N








Figure 1-7. Synthesis of agrobactin A.






22

by attaching three 2,3-dihydroxybenzoyl groups to the triamine (VI). Recently, Van Brussel and Van Sumere have shown it to be possible to generate the succinimide esters of a number of mono- and dihydrobenzoic acids in the presence of the unprotected phenols.76 Thus, when N4-threonylspermidine was reacted with excess succinimide 2,3-dihydroxybenzoate in the presence of triethylamine in aqueous THF, agrobactin A was obtained in 75% yield after chromatography.
Unlike previous synthesis of catecholamides which usually attached the "centerpiece" in the final stages of the synthesis, this synthesis of agrobactin A reverses this order, building the molecule from the "inside out" and attaching the catechols in the final step. This is important as any number of groups can now be attached to the triamine
(VI), making it possible to easily synthesize additional agrobactin A derivatives. Examples of some derivatives that were synthesized in this manner included the 2,3-dimethoxybenzoyl derivative of agrobactin A as well as an unsubstituted benzoyl derivative. Likewise, symmetrical analogues of agrobactin A can be generated as both nor- and homospermidine homologues of N',N8-bis(t-BOC)spermidine are available.75

With these compounds at hand, their 1H NMR spectra were subsequently investigated. Not surprisingly, agrobactin A also exhibited conformers in its 'H NMR spectrum as determined by the duplicity in the signals originating from the threonine residue. If, indeed, these duplicate signals are a result of an equilibrium between two interconverting isomers and not two distinctly separate compounds, the activation energy for this interconversion can be measured using 1H NMR. The rate of interconversion or exchange between conformer population can be estimated by determining the coalescence temperature of the duplicate signals.






23


At low temperature the rate of interconversion is slow on the NMR time scale and two separate signals are observed. As the temperature of the sample is increased, the rate of interconversion becomes faster, the signals broaden and move towards each other. Finally, as the temperature is increased above the coalescence temperature, the temperature at which the two signals coalesce into one broad signal, the conformers are interconverting freely and only one "averaged" signal is observed. The corresponding activation energy, AG, can be estimated from the coalescence temperature by the following equation:77 AG = 22.96 + log e (T /6V)
RTc c

where Tc is the coalescence temperature, 6v is the width of the signal at half-height in hertz, and R equal the gas constant.

,easurement of the coalescence temperatures of agrobactin A and its asymmetrical derivatives would thereby give the AG's for these interconversions. A comparison of the calculated AG's and their corresponding structures should indicate the structural significance attached to conformer interconversion. The results of such a study performed here indicate that, in polar solvents such as DMSO, steric bulk is the dominant influence controlling interconversion between the conformers of agrobactin A and its derivatives.













CHAPTER TWO

REAGENTS FOR THE SELECTIVE FUNCTIONALIZATION
OF SPERMIDINE, NORSPERMIDINE, AND HOMOSPERMIDINE

In order to investigate the uptake properties of various N-acylated and N-alkylated polyamines, one must consider the ability to selectively and efficiently modify the polyamines' primary versus secondary amines to produce the derivatives desired. Commercially available spermidine itself fails in this respect as previous studies have shown there is little difference in reactivity and hence, selectivity between its primary and secondary amines.78 Reactions of spermidine with even the most bulky reagents often lead to complex mixtures and poor yield of the desired product. Additionally, the symmetrical polyamines, nor- and homospermidine, are not commercially available. Therefore, current methodology has focused on the preparation of protected spermidine reagents,

derived usually from compounds simpler than spermidine itself.

A review of the literature reveals there are currently several reagents available for the selective acylation of spermidine. The first of these, designed by Eugster et al., N4-tosyl-N8-phthaloylspermidine, is designed to fix three different substituents to the spermidine backbone.79 The eight steps required for the synthesis of this reagent and the mode of protective group removal make it somewhat impractical. A second reagent, N4,N8-di-t-butoxycarbonyl spermidine (accessible in 49% yield in three steps) is acceptable for what it is designed for --introduction of an acyl group at the NI-position.80 A more recent development employs

1 -(4-aminobutyl)hexahydropyrimidine and l-(3-aminopropyl )hexahydropyridine for the selective acylation of the terminal primary amino nitrogens

24






25

of spermidine and homospermidine.81 This procedure represents an excellent protocol for functionalization of these two polyamines although it cannot be extended to norspermidine and the conditions required for the

opening of the pyrimidine can be restrictive.

Recently, Bergeron and coworkers have described the preparation of the benzylated polyamines N4-benzylspermidine, N4-benzylnorspermidine, and Ns-benzylhomospermidine as reagents for the selective acylation of the primary amines.82 This synthesis offers the flexibility of generating the varying chain lengths desired, as well as employing inexpensive starting materials. The scheme employs benzylamine as the common starting material for all three reagents, Figure 2-1. As illustrated, benzylamine is reacted with one equivalent of acrylonitrile, followed by alkylation with 4-chlorobutyrylnitrile which, upon reduction, generates N4benzylspermidine (VIIa). Alternatively, benzylamine can either be reacted with two equivalents of acrylonitrile or two equivalents of 4-chlorobutyrylnitrile, producing upon reduction the symmetrical reagents N5benzyl homospermi dine (VIIb) or N4-benzylnorsperamidine (VIIc), respectively. These three reagents represent an excellent method of efficiently generating the terminally bis-acylated spermidine desired as outlined in Figure 2-2. The benzylated polyamines (VII) can be reacted with two equivalents of the desired acylating agent, followed by removal of the

benzyl protecting group via hydrogenolysis over palladium chloride catalyst producing the desired derivatives (IX), figure 2-2.. In order to determine if there are any size restrictions placed on uptake by the receptor, in this study the acyl groups acetyl, propionyl and benzoyl were affixed to the reagents (VII) via their acid chlorides.






26































VIIc N sC Nc VI Ia NNH2

VIIb Figure 2-1. Synthesis of secondary N-benzylated polyamines.





27









CH2 H2N (CH \) -(CHI)j-NH2 VII









CH2 RICONH -- (CH (CH---- NHCOR VIII







Ix
RICONH (CH2;a--NH -(CHZ b- HCCR 1 IX



RICHzNH -l'H2)--- NH -- (C'H2) -- IHCHeR






Figure 2-2. General scheme for the synthesis of terminally
N-modified polyamine derivatives.






28

In turning our attention towards a reagent for the selective secondary N-acylation of spermidine and its homologues, the lack of a suitable reagent in the literature necessitated that a new approach be developed. Starting with the already available benzylated reagents (VII), this should easily be accomplished by first blocking the terminal amines followed by debenzylation. The only requirement in such a synthesis would be an easily

attachable/removable protecting group which is stable to the hydrogenation conditions employed. The t-butoxycarbonyl protecting group meets this requirement.

Hence, (VII) is reacted with two equivalents of t-butoxycarbonyloxyimine -2-phenylacetonitrile (BOC-ON) to form the terminally bis-tbutoxycarbonyl compounds in high yield, Figure 2-3. The benzyl group is then removed via hydrogenolysis as before producing the reagents (XII)

capable of selectively secondary N-acylation. These t-BOC protected reagents are then reacted with a variety of acylating agents, again of varying size such as acetyl, benzoyl, and hexanoyl acid chlorides. The t-BOC protecting groups are then removed quantitatively with trifluoroacetic acid generating the secondary N-acylated spermidine derivatives (XIV), as illustrated in Figure 2-4.

In considering the synthesis of N-alkylated spermidine derivatives, although the reagents N4-benzylspermidine and the symmetrical homologues are available, direct alkylation of these compounds would presumably result in over alkylation and poor yields. Therefore, it would seem more practical to reduce the N-acylated derivatives already on hand via the above two procedures directly to the N-alkylated compounds. Accordingly, the terminally bis-acylated derivatives (IX) could be converted directly to the bis-alkylated derivatives (X) as shown in Figure 2-2






29










H2NN NH 2 VII 2













tBOC-NH. N NH-tBOC XI










CI

t BO C -N H NH2 NNH-tBOC XII






Figure 2-3. Synthesis of N1,N8-bis(t-Butoxycarbonyl)spermidine,






30 HCI tBOC-NH --(CH2)a- NH (CH2)b- NH-tBOC


XII







R2

CO tBOC-NH (CH2)a N (CH2)b- NH-tBOC XIII







R2 Co H2N (CH2) -- N- (CH2)b- NH2

XIV






1


1 2 H2N (CH2 a N (CH2 ) b- NH2 XV Figure 2-4. Synthesis of secondary N-modified polyamine
derivatives.






31


employing an appropriate reducing agent. Likewise, the secondary Nacylated compounds (XIV) could be converted to the alkyl derivatives

(XV), Figure 2-4, in the same manner.

Experimental

Materials

The reagents N4-benzylspermidine, N4-benzylnorspermidine, and N5benzylhomospermidine were prepared as previously described."8 All other reagents were purchased from Aldrich Chemical Company and, except where

indicated, used without further purification. Dry methylene chloride (CH2C12) was obtained by distillation followed by storage over 3 A molecular sieves. Dry dioxane was obtained by distillation from sodium metal immediately before use. Sodium sulfate was used as the drying agent. Sephadex LH-20 was obtained from Pharmacia Fine Chemicals. Preparative thin layer chromatography (TLC) was performed on Analtech 20 x 20 cm

silica gel GF plates.

For the physical measurements, melting points were taken on a FisherJohns apparatus and are uncorrected. Proton nuclear magnetic resonance (PH-NMR) spectra were recorded on a Varian T-60 and, unless otherwise noted, prepared in deuterated chloroform (DCCl3) with chemical shifts

(6) given in parts per million relative to an internal (CH3)4Si standard. The infrared (IR) spectra were recorded on a BeckmanAcculab 1 spectrophotometer. Elemental analyses were performed by Galbraith Laboratories, Knoxville, TN,or Atlantic Microlabs, Atlanta, GA. Synthesis of Terminally N-modified Spermidine Derivatives General methods

The bis-acylated polyamines were prepared from the appropriate
secondary benzylated polyamine as described for the following synthesis






32

of N1,N8-bis(acetyl)spermidine. The procedure is the same regardless of the benzylated polyamine or acylating agent employed.

A solution containing 2.2 equivalents of acetyl chloride was reacted with N4-benzylspermidine in the presence of triethylamine as base to cleanly afford N4-benzyl-N1,N8-bis(acetyl)spermidine in 98% crude yield. The crude products are usually of sufficient purity that they can be debenzylated without further purification. Accordingly, N4-benzyl-N1,N8bis(acetyl)spermidine was hydrogenated overnight over palladium chloride catalyst in methanol/HCl to afford pure NI,N8-bis(acetyl)spermidine as the hydrochloride salt upon recrystallization. The corresponding amine was prepared by reduction of this bis(amide) with a suitable reducing agent.

Of the many reducing agents available, sodium borohydride-trifluoroacetoxy complex83 was chosen due to its relative ease and mildness of reduction. Other reagents, such as lithium aluminum hydride, are often less selective and may cleave tertiary amides, an important consideration for the preparation of the N4-alkyl derivatives. Therefore, NI,N8-bis(ethyl)spermidine was prepared by refluxing N1,N8-bis(acetyl)spermidine and sodium trifluoroacetoxyborohydride in dry dioxane for eight hours to afford the desired product in 78% yield after distillation. The resulting amines are then usually converted to their hydrochloride salts to prevent oxidation and improve handling. N4-Benzyl-N1,NB-bis(acetyl)spermidine (1)

A solution of acetyl chloride (725 mg, 9.2 mmol) in 10 dry CH2C12

was added dropwise to a cooled solution (ice bath) of N4-benzylspermidine (1.0 g, 4.2 mmol) and triethylamine (1.3 ml, 9.2 mmol) in 20 ml dry CH2C12 under a N2 atmosphere. After the addition was completed, the






33

ice bath was removed and the mixture allowed to stir for a total of 18 hours. Additional CH2zC1 (50 ml) was then added and the reaction mixture washed with cold 3% HC1 (3 x 20 ml), H20 (2 x 20 ml), 5% NaHCO3 (3 x 20 ml), H20 (2 x 20 ml), dried, and evaporated to afford 1.3 g (98%) of the desired product as an oil: IH NMR a 1.50 (m, 6H), 1.84 (d, 6H), 2.40 (m, 4H), 3.17 (m, 4H), 3.42 (s, 2H), 6.26 (br, 2H), 7.20 (s, 5H); IR (CHC13) 3310 (s), 2975 (s), 1650 (s), 1550 (s), 760 (s) cm1.
An analytical sample was prepared by preparative TLC eluting with 10% methanol/CHzC12. Analysis calculated for C18H29N302*.H20: C, 65.82; H, 9.21; N, 12.79. Found: C, 65.67; H, 9.24; N, 12.73. N4-Benzyl-N',N8-bis(propionyl)spermidine (2)
A solution of propionyl chloride and N -benzylspermidine was reacted and purified in the same manner as for (1). Yield: 1.35 g (93%); 1H NMR 6 1.10 (dt, 6H), 1.56 (m, 6H), 2.05 (quar., 4H), 2.38 (m, 4H),
3.2 (m, 4H), 3.46 (s, 2H), 6.14 (br, 2H), 7.23 (s, 5H); IR (CHC13) 3320 (s), 2980 (s), 1660 (s), 1550 (s), 770 (s) cm-'.
Anal. cal. for C20H33N30.H20: C, 67.38; H, 9.61; N, 11.79. Found: C, 67.61; H, 9.60; N, 11.75.
N4-Benzyl-N1,NB-bis(benzoyl.)spermidine (3)
A solution of benzoyl chloride and N4-benzylspermidine was reacted and purified in the same manner as for (1). Yield: 3.4 g (91%); 'H NMR
6 1.65 (m, 6H), 2.50 (m, 4H), 3.45 (m, 4H), 6.57 (br, 2H), 6.96-7.85 (m, 15H); IR (CHC13) 3340 (s), 1640 (s), 1520 (s), 1310 (m), 690 (m) cm-1.
Anal. cal. for Cz2H33N302: C, 75.81; H, 7.50; N, 9.47. Found: C, 75.73; H, 7.46; N, 9.37.





34

N1,N8-Bis(acetyl)spermidine hydrochloride (1)
Palladium chloride (100 mg) was added to a solution of (1) (1.15 g, 3.6 mmol) in methanol (50 ml) containing concentrated HC1 (seven drops). The resulting suspension was stirred under a hydrogen atmosphere overnight. The catalysts were then filtered, washed with methanol, and the filtrates evaporated. The resulting crude solid was recrystallized from ethanol/ether, the solid collected by filtration, and dried under high vacuum over P20s to afford 550 mg of the desired product as a white solid; mp 1330C. Concentration of the mother liquor and recrystallization afforded another 330 mg. Total yield: 880 mg (92%); IH NMR (D20) 6 1.70 (m, 6H), 2.04 (s, 6H), 3.20 (m, 8H); IR (KBr) 3300 (br), 1625 (s), 1530
(m) cm-1.
Anal. cal. for C1zH24N302C1: C, 49.71; H, 9.10; N, 15.81. Found: C, 49.45; H, 9.15; N, 15.48.
N1,N8-Bis(propionyl)spermidine hydrochloride (5)
A solution of N4-benzyl-N',N8-bis(propionyl)spermidine (2) was hydrogenated and purified in the same manner as described for (4). Yield: 780 mg (86%); mp 147-1480C; 1H NMR (D20) 6 1.10 (t, 6H), 1.72 (m, 6H), 2.35 (quar., 4H), 3.16 (m, 8H); IR (KBr) 3350 (br), 1630 (s), 1540 (m)
cm"1.
Anal. cal. for C13H28N3z0C1: C, 53.14; H, 9.60; N, 14.30. Found: C, 52.81; H, 9.50; N, 13.86.
N1,N8-Bis(benzoyl )spermidine hydrochloride (~)
A solution of N4-benzyl-N',N8-bis(benzoyl)spermidine (3) was hydrogenated and purified in the same manner as described for (4). Yield:
2.9 g (93%); 'H (D20) 6 1.74 (m, 6H), 3.20 (m, 8H), 7.06-7.82 (m, 10H); IR (KBr) 3300 (s), 1635 (s), 1540 (s), 690 (m) cm'.






35

N1,N8-Bis(ethyl)spermidine trihydrochloride ( )

A suspension of N,N8-bis(acetyl)spermidine hydrochloride (330 mg,

1.24 mmol) and sodium borohydride (500 mg, 13 mmol) in 20 ml freshly distilled dioxane was cooled to 10-150C under a N2 atmosphere. A solution of trifluoroacetic acid (1.5 g, 13 mmol) in 10 ml dry dioxane was slowly added dropwise with stirring. After the addition was completed, the suspension was slowly brought to a gentle reflux and the reaction continued for ten hours.

The reaction was then cooled, the excess reducing agent destroyed by careful addition of water (2 ml), and the solvent reduced under high vacuum. The residue was treated with 2N KOH (5-10 ml), the product extracted into CH2C12 (4 x 20 ml), dried, and concentrated to afford 240 mg crude product as a semisolid. The product was further purified by distillation to afford 190 mg (78%): bp 133-135, 0.25 mm; 1H NMR 6 0.881.82 (overlapping multiplets, 15H), 2.60 (m, 12H); IR (CHC13) 2980 (s), 1465 (m), 1120 (m), 750 (s) cm'.

The distilled amine was converted to the hydrochloride salt by dissolving in a solution of ethanol and ether (1:1), cooling, and then bubbling HCl gas through.

Anal. cal. for CllH30N3C13: C, 42.52; H, 9.73; N, 13.52. Found: C, 42.37; H, 9.68; N, 13.19.

N1,N8-Bis(propyl)spermidine trihydrochloride (8)

A suspension of N',NB-bis(propionyl)spermidine hydrochloride was reduced and purified as described for (7). Yield: 210 mg (71%); bp 144-147, 0.10 mm; 'H NMR a 0.90 (t, 6H), 1.16-1.80 (m, 13H), 2.55 (m, 12H); IR (CHC13) 2975 (s), 1460 (m), 1120 (m), 760 (s) cm-1.









































7.0 4.0 IF. 4.0 3.0 2. 1.0

Figure 2-5. 60 MHz 1H NMR spectrrum of N4-benzyl-Nl,N8-bis(acetyl)spermidine (1).














I I




















.. ..1 i .e ..a. a I a m i i g i I i 10 4.0 5.0 .O0 3.0 2.0 1.0 o Figure 2-6. 60 MHz 1H NMR spectra of N1,N8-bis(acetyl)spermidine(4).









































l . ..l ... lI . ..... I I . l. . .
1.0 4.0 S.0 q.0 3.0 2.0 1.0 o Figure 2-7. 60 MHz 1H NMR spectrum of N1,N8-bis(ethyl)spermidine(7).

















V/AVLLLNt.IH NI M #RONS
3 35 45 5 5.5 6 65 7 7.5 a 9 10 I| 12 14 16 I00.
ton o
!ihli! Ii li!! jii
.... : ~~l.l.(
P ti
,, .4 ii l a nio



,o

....!llii :lililllif llil'.'lill ,-soilLlllllllllilll~ill!:!LiUlUU~l~U II 7
MAl'~ll a ,,
.... Il I I

S30





: ,' ,s I" lm ;
0
50 -s






,o !u~li




4In! 00 2000 100 1600 1400 8200 1000 600 00


fiR WAVQ*IMS CM' N~a I~i$I l S)II I I. 4IOIII i lO*i~. II $ -1*.l 4 s*

Figure 2-8. IR spectrum of N4-benzyl-N1,N8-bis(acetyl)spermidine (1).
INIn
30


i~iif

flllll l ll l ll lll MIM 11 1

4000o 3000 A"0 1800 100 1400 1200 1000 goo 600
*~ur~t u mr* c~r o, ~l MAIn WAVINUMSERf CIIAlw*r.*,KIUI)Y(IO' r ~ ~ IDVL




Figure 2-8. IR spectrum of N4-benzyl-NI,N8-bis(acetyl)spermidine (1).


















WAVLIfNC.,* tI MCROlS
5 3 5 6 65 7.5 9 10 1 12 14 16

I UO k oo
Hi ii 111111111l

;I'll "H S


10 101
III ''~i i
i11iil




itri




ftt



60
"oo
30 .A, ..! (i
ilii
fill 111160














Figure 2-9. IR spectrum of N1,N8-bis(acetyl)spermidine (4).















WAViU4GIIt I4 MICOtIS
1,5 315 A 4 .5 6 6,5 7 7.5 a 10 II 12 14





40 [i






I :9
to
,o a;o
10 0l








0







4000 3000 2000 i1800 1600 1400 i 200 1000 800 600
Figure 2-0. IR spectrum of NN8bis(ethy)spermidne (7).
so 11: I





60 2

iii!

io U






4000 3000 2000 1800 1,6ao 1400 1200 1000 Boo 600




Figure 2-10. IR spectrum of 090N-bis~ethyl)spermidine (7).






42

Anal. cal. for C13H34N3C13: C, 46.09; H, 10.12; N, 12.40. Found: C, 45.59; H, 9.65; N, 12.07.

N1,Ns-Bis(benzyl)spermidine trihydrochloride (i)

A suspension of N1,N8-bis(benzoyl)spermidine hydrochloride was reduced and purified as described for (7)-. Yield: 1.5 g (89%); bp 183-185,

0.10 mm; 1H NMR 6 1.52 (m, 9H), 2.56 (m, 8H), 3.44 (s, 4H), 7.22 (s, 10H); IR (CHC13) 2970 (s), 1450 (m), 1130 (m), 760 (s) cm-1.

Anal. cal. for C21H3sN3C13: C, 68.07; H, 9.25. Found: C,67.82; H, 9.18.

Synthesis of Secondary N-modified Spermidine Derivatives

General methods

The reagents N1,N8-bis(t-butoxycarbonyl)spermidine, N1,N7-bis(tbutoxycarbonyl )norspermidine and N1 ,N9-bis(t-butoxycarbonyl )homospermidine were prepared by reacting the appropriate benzylated spermidine with

2.1 equivalents of BOC-ON84 in tetrahydrofuran. The resulting benzylbis(t-BOC) polyamines were then hydrogenated over palladium chloride catalysts in MeOH/HCl to afford the bis(t-BOC) spermidine reagents as the hydrochloride salts in 80-90% yields from the benzylated polyamine.85

Again, the preparation of N4-hexanoyl and N4-hexylspermidine are illustrative as the same methodology is employed regardless of the bis(t-BOC protected) spermidine reagent or acylating agent used. A solution of NI,N8-bis(t-butoxycarbonyl)spermidine.HC1 and 1.1 equivalents of hexanoyl chloride were allowed to react in the presence of base in dry dichloromethane to afford N4-hexanoyl-N,N8-bis(t-butoxycarbonyl)spermidine in 95% yield. Once again, the products of these reactions are usually clean enough to be used without further purification. Therefore, N4-hexanoyl-N1,N8-bis(t-butoxycarbonyl)spermidine is reacted with trifluoroacetic acid to afford N4-hexanoyl spermidine, quantitatively.





43

The resulting trifluoroacetic salts are very hydroscopic and therefore, commonly converted to the hydrochloride salts. Finally, N4-hexanoylspermidine dihydrochloride can be converted to the corresponding amine, again utilizing sodium trifluoroacetoxyborohydride as the reducing agent. N4-Benzyl-N1,N8-bis(t-butoxycarbonyl )spermidine (I1)

A solution of BOC-ON (5.4 g, 0.022 mol) in 50 ml of distilled tetrahydrofuran (THF) was slowly added dropwise with stirring to a cooled solution (ice bath) of N4-benzylspermidine (2.35 g, 0.010 mol) in 75 ml THF under N2. After the addition was completed, the ice bath was removed, and the reaction mixture allowed to stir for eight hours at which time the solvent was evaporated. The residue was dissolved in 150 ml of ether and washed with 5% NaOH (4 x 25 ml), water (3 x 25 ml), dried, and evaporated to afford 4.3 g (99%) of the desired product, as a viscous light yellow oil. Thin layer chromatography and 1H NMR analysis indicated the crude oil purity was in excess of 95%, and subsequently used without further purification.
An analytical sample was prepared by preparative TLC, eluting with 10% MeOH/CH2C12: 1H NMR 6 1.45 (s, 18H), 1.40-1.88 (m, 6H), 2.18-2.70 (m, 4H), 2.82-3.35 (m, 4H), 3.48 (s, 2H), 4.79-5.63 (br, 2H), 7.17 (s, 5H); IR (CHC13) 3390 (m) 1705 (s), 1510 (s), 1190 (s), 760 (m) cm-'.
Anal. cal. for C24H41N304: C, 66.18; H, 9.49; N, 9.65. Found: C, 65.93; H, 9.79; N, 9.38.
N4-Benzyl-N',N7-bis(t-butoxycarbonyl )norspermidine (11)
A solution of N4-benzylnorspermidine and BOC-ON was reacted and purified as described previously for (10): yield: 2.0 g (98%); IH NMR
6 1.41-1.83 (m, 4H), 1.44 (s, 18H), 2.11-2.68 (m, 4H), 2.77-3.35 (m, 4H), 3.50 (s, 2H), 4.75-5.68 (br, 2H), 7.21 (s, 5H); IR (CHCl3) 3400 (m), 1700
(s), 1510 (s), 1175 (s), 755 (m) cm-1.






44

Anal. cal. for C23H3N,304: C, 65.53; H, 9.32; N, 9.97. Found: C, 65.49; H, 9.35; N, 9.9.
N5-Benzyl-Nl,N9-bis(t-butoxycarbonyl )homospermidine (12.)
A solution of Ns-benzylhomospermidine and BOC-ON was reacted and purified as described previously for (10): Yield: 1.35 g (93%); 'H NMR
6 1.38-1.87 (m, 8H), 1.47 (s, 18H), 2.13-2.71 (m, 4H), 2.78-3.33 (m, 4H),
3.51 (s, 2H), 4.80-5.65 (br, 2H), 7.19 (s, 5H); IR (CHC13) 3420 (m), 1690 (s), 1520 (s), 1180 (s), 750 (m) cm-1.
Anal. cal. for C25H43N304: C, 66.78; H, 9.64; N, 9.35. Found: C, 66.52; H, 9.69; N, 9.25.

N',N8-Bis(t-butoxycarbonyl) spermidine.hydrochloride (13)
Palladium chloride (350 mg) was added to a solution of (10) (3.9 g,
9.0 mmol) in 50 ml methanol containing concentrated hydrochloric acid (0.75 ml, 9.0 mmol). The resulting suspension was stirred under a hydrogen atmosphere overnight (18 h). The catalysts were then filtered, washed with MeOH, and the filtrates evaporated. The resulting crude solid was recrystallized from ethanol/ether to afford 3.4 g (94%) of the desired product: mp 149-1500C; 1H NMR (D20) 1.50 (s, 18H), 1.66 (m, 6H), 3.14 (m, 8H); IR (KBr) 3380 (s), 1690 (s), 1520 (s), 1190 (m) cm-1

Anal. cal. for C17H36N304Cl: C, 53.46; H, 9.50; N, 11.00. Found: C, 53.41; H, 9.54; N, 10.99.
NI,N7-Bis(t-butoxycarbonyl )norspermidine.hydrochloride (14)
A solution of (11) was hydrogenated and purified in a similar manner as described for (13). Yield: 1.2 g (89%); mp 173-1740C; 1H NMR (D20) 6 1.48 (s, 18H), 1.78 (m, 4H), 3.16 (m, 8H); IR (KBr) 3400 (s), 1700 (s), 1515 (s), 1170 (m) cm-1.





45


Anal. cal. for C16H34N304Cl: C, 52.23; H, 9.31; N, 11.42. Found: C, 52.29; H, 9.31; N, 11.37.
N',N9-Bis(t-butoxycarbonyl)homospermidine hydrochloride (15
A solution of (12) was hydrogenated and purified in a similar manner as described for (13). Yield: 770 mg (92%); mp 187-1880C; 1H NMR (D20) a 1.47 (m, 26H), 3.19 (m, 8H); IR (KBr) 3380 (s), 1690 (s), 1510 (s), 1175 (m) cm1.
Anal. cal. for C18H38N304C1: C, 54.60; H, 9.67; N, 10.61. Found: C, 54.58; H, 9.67; N, 10.59.
N4-Acetyl-N1,N8-bis(t-butoxycarbonyl)spermidine (16.)
A solution of acetyl chloride (180 mg, 2.2 mmol) in 10 ml dry CH2C12 was slowly added to a cooled solution of (13) (760 mg, 2.0 mmol) and triethylamine (600 ul, 4.4 mmol) in 30 ml CH2C12 under N2. The solution was allowed to warm to room temperature and stirred overnight (18 h) at which time additional CH2C12 (25 ml) was added. The organic layer was washed with 3% HC1 (3 x 15 ml), H20 (2 x 15 ml), 5% NaHCO3 (3 x 15 ml), H20 (2 x 15 ml), dried and concentrated to afford 720 mg (93%) of the product as a colorless oil: 1H NMR 6 1.42 (s, 18H), 1.64 (m, 6H),
2.06 (s, 3H), 3.18 (m, 8H), 5.02 (br, 2H); IR (CHC13) 3320 (m), 2960 (s), 1690 (s), 1620 (m), 1170 (s) cm1.
An analytical sample was prepared by chromatography of silica gel
(70-230 mesh) eluting with EtOAc/CHC13 (1:1). Anal. cal. for C19H37N30sH20: C, 57.55; H, 9.66; N, 10.60. Found: C, 57.46; H, 9.30; N, 10.22. N -Hexanoyl-N1,N8-bis(t-butoxycarbonyl)spermidine (1.)
A solution of hexanoyl chloride and (13) were reacted and purified in the same manner as for (16). Yield: 620 mg (95%); IH NMR 6 0.90






46

(t, 3H), 1.12-1.95 (m, 30H), 2.31 (quar., 2H), 3.18 (m, 8H), 5.00 (br, 2H); IR (CHC 3) 3330 (m), 2970 (s), 1680 (s), 1625 (m), 1175 (s) cm'1.
Anal. cal. for C23H4sN305: C, 62.27; H, 10.22; N, 9.47. Found: C, 62.48; H, 10.25; N, 9.23.
N4-Benzoyl-N',Ns-bis(t-butoxycarbonyl)spermidine (18)
A solution of benzoyl chloride and (13) was reacted and purified in the same manner as described for (16). Yield: 470 mg (95%); 'H NMR 6
1.42-1.92 (m, 24H), 2.83-3.58 (m, 8H), 5.23 (br, 2H), 7.19 (s, 5H); IR (CHC13) 1710 (s), 1630 (m), 1515 (m), 1225 (s), 770 (s) cm-1.
Anal. cal. for C24H39N,305sH20: C, 62.86; H, 8.79; N, 9.16. Found: C, 62.98; H, 8.99; N, 8.85.
N4-Acetylspermidine hydrochloride (19)
To a cooled flask containing (16) (600 mg, 1.55 mmol) was added 20 ml trifluoroacetic acid. The reaction was allowed to warm to room temperature and stirred for 20 minutes at which time the solvent was quickly evaporated. The residue was dissolved in methanol and evaporated (twice). The residue was then dissolved in ethanol/ether, cooled, and HC1 gas bubbled through. The solid was collected by filtration, and dried in vacuo over P205 to afford 360 mg (89%) of the desired product: 1H NMR (D20) a 1.30-1.88 (m, 24H), 2.06 (s, 3H), 3.18 (m, 8H), 5.00 (br, 2H); IR (KBr) 3300 (br), 1690 (s), 1520 (m) cm-.
Anal. cal. for C9H23N30C12: C, 41.54; H, 8.91; N, 16.15. Found: C, 41.18; H, 9.04; N, 15.95.
N4-Hexanoylspermidine dihydrochloride ($0)
A solution of (17) was deprotected and purified in a similar manner as described for (19). Yield: 500 mg (89%); IH NMR (TFA) 6 0.96 (m, 3H),
1.40 (m, 6H), 1.60-2.36 (m, 6H), 2.66 (m, 2H), 2.96-3.90 (m, 8H), 6.92 (br, 6H); IR (KBr) 3300 (br), 1680 (s), 1530 (m) cm-1.






47


Anal. cal. for C13H31N30C12: C, 49.36; H, 9.88; N, 13.28. Found: C, 49.23; H, 9.77; N, 12.75.

N4-Benzoylspermidine dihydrochloride (21)
A solution of (18) was deprotected and purified in a similar manner as described for (19). Yield: 370 g (92%); 1H NMR (TFA) 6 1.62-2.18 (m, 6H), 2.78-3.52 (m, 8H), 7.20 (s, 5H), 8.15 (br, 6H); IR (KBr) 1700
(s), 1540 (m), 750 (s) cm-1.
Anal. cal. for C18H25F6N305H20: C, 43.64; H, 5.49; N, 8.48. Found: C, 43.79; H, 5.41; N, 8.17.
N4-Ethyl spermidine trihydrochloride ( 22)
To a cooled suspension (10-150C) of N4-acetylspermidine dihydrochloride (19) (500 mg, 1.92 mmol) and sodium borohydride (380 mg, 10 mmol) in 15 ml dry dioxane was added a solution of trifluoracetic acid (1.14 g, 10 mmol) in 5 ml dry dioxane. After the addition was completed, the suspension was slowly brought to a gentle reflux and the reaction continued for eight hours under N2.
The reaction was then cooled, the excess reducing agent destroyed by careful addition of water (2 ml), and the solvent reduced under high vacuum. The residue was treated with 2N KOH (5 ml), the product extracted into CH2C12 (4 x 20 ml), dried, and concentrated to afford 350 mg crude product. The product was further purified by Kugelrohr distillation to afford 280 mg (78%) of the desired product: 1H NMR (6)
0.94 (t, 3H), 1.64 (m, 8H), 2.12 (quat., 2H), 2.67 (m, 8H); IR (CHC13) 3320 (m), 2960 (s), 1430 (m) cm1.
The amine was subsequently converted to its hydrochloride salt by HCl gas. Anal. cal. for C9H26N3C13: C, 38.24; H, 9.27; N, 14.86. Found: C, 38.01; H, 9.31; N, 14.49.






48

N4-Hexylspermidine trihydrochloride (23)

A solution of (20) was reduced and purified in a similar manner as described for (22). Yield: 180 mg (82%); IH NMR 6 0.85 (m, 3H), 1.041.82 (overlapping m, 18H), 2.12-2.85 (m, 10H); IR (CHC13) 3300 (m), 2970 (s), 1425 (m) cm-.
Anal. cal. for C13H34N3C13: C, 46.09; H, 10.12; N, 12.40. Found: C, 46.37; H, 10.04; N, 12.17.

N4-Methyl-N1,N8-bis(t-butoxycarbonyl)spermidine (24)

Three hundred fifty milligrams of N',N8-bis(t-butoxycarbonyl)spermidine hydrochloride (13) were added to a 15% aqueous solution of Na2CO3. The solution was extracted with ether (3 x 30 ml), the organic layer dried, and concentrated to afford 300 mg of the free amine as an oil.

To a solution of N',N8-bis(t-butoxycarbonyl)spermidine (300 mg, 0.9 mmol) in 3 ml acetonitrile was added a 37% aqueous formaldehyde solution (0.4 ml, 5 mmol) and sodium cyanoborohydride (100 mg, 1.6 mmol). A mildly exothermic reaction ensued which subsided after a minute or so. After 15 min, the pH of the solution was checked, adjusted to pH 7.0 with acetic acid, and the reaction allowed to stir for an additional 1.5 h.
The solvent was then reduced in vacuo, the residue dissolved in 2N KOH (5 ml), and the product extracted with ether (3 x 15 ml). The ether layer was dried and concentrated to afford 400 mg of the crude product.
Further purification was effected by chromatography on silica gel (Merck 7734) eluting with MeOH/CHC13 (5% + 15% MeOH) to afford 210 mg (65%) pure (24): 1H NMR 6 1.43 (m, 24H), 2.24 (s, 3H), 2.42 (m, 4H), 3.06 (m, 4H), 5.14 (br, 2H); IR (CHC13) 3390 (m), 1700 (s), 1510 (s), 1180 (s) cm-.





49

N4-Methylspermidine trihydrochloride (25)

Trifluoroacetic acid (10 ml) was added to a flask containing N4methyl-N',N-bis(t-butoxycarbonyl)spermidine (24) (110 mg, 0.3 mmol), and the resulting solution stirred for 20 min. The solvent was then evaporated, the residue dissolved in MeOH (25 ml) and concentrated (repeated twice) to afford 120 mg product as the trifluoroacetate salt. The product was subsequently exchanged to the hydrochloride salt by dissolving in cold EtOH (3 ml), bubbling HCl gas through the solution, and collecting the solid via filtration. The solid was dried in vacuo over P205 to afford 70 mg (87%): 1H NMR (020) 6 1.75 (m, 4H), 2.10 (m, 2H), 2.87 (s, 3H), 3.15 (m, 8H).

Anal. cal. for C8H25N3C13: C, 35.63; H, 9.34. Found: C, 35.17; H, 9.52.

Norspenmidine trihydrochloride (26)

A sample of N',N7-bis(t-butoxycarbonyl)norspermidine hydrochloride

(14) was exposed to TFA and purified in the same manner as described for (25). Yield: 260 mg (83%); 1H NMR (020) 6 2.20 (m, 4H), 3.22 (m,

8H).

Homospermidine trihydrochloride (27)

A sample of N1,N9-bis(t-butoxycarbonyl)spermidine hydrochloride

(15) was exposed to TFA and purified in the same manner as described for

(25). Yield: 620 mg (91%); 1H NMR (D 0) 6 1.84 (m, 8H), 3.18 (m, 8H).

Results and Discussion

In order to further characterize the polyamine's receptor, a wide array of polyamine derivatives would be required to adequately judge the effects of functionalization on uptake. This necessitated a synthetic scheme capable of both flexibility and efficiency to deliver the desired














I,









I 1









1 1 I I I (I
1.0 4.0 5.0 4.0 3.0 2.0 1.0 Figure 2-11. 60 MHz 1H NMR spectrum of N1,N8-bis(t-butoxycarbonyl)spermidine
hydrochloride(13).























/










, 1 ,. .. 1 ,_ 1 ,.. ,...... l. .... .. .. .
1.0 .0 5.0 .0o 3.0 2.0 1.0 Figure 2-12. 60 MHz 1H NMR spectrum of N4-hexanoyl-N,N8-bis(t-butoxycarbonyl)spermidine (17).


































..I 8 5 I I t I I a a I ....... a g a g... I .... A A s a a
1.0 6.0 5.0 0 3.0 2.0 ,o

Figure 2-13. 60 MHz IH NMR spectrum of N4-hexanoylspermidine tris(trifluoroacetate)(20).





























I





1.0 4.0 S.o 4.0 3.0 2.0 1.0
Figure 2-14. 60 MHz 1H NMR spectrum of N4-hexylspermidine(23).


















WAVEfLNGi IN MICROtS4
2.5 3 3 4 4.5 5.5 6 65 7 75 8 9 10 II 12 14 16




I'll! H il l 10(
1 90



o so ,00










3u 30







0 0 4000 3000 2000 1800 1600 14.100 1200 11000 800 600
W Utoe04 1m aK (s41 lm l pai n WAVENUMEi CM' Pcil-l 0 V. A.sis. c musto..c s.. .ew A, ewoeI.




Figure 2-15. IR spectrum of N4-hexanoylspermidine (20).


















WAVELENGIH IN MICRONS
2.5 3 3. 4 4.5 5 5.5 6 65 7 7.5 9 10 11 1 14 16





9,0 90


80 so 70


o 60



050





30 430






In H0 4000 3000 2000 1800 1600 1400 2)00 1000 800 600
wn nO e otaVt.e I 1ot. 14s 1281 WAVOUMIER CM"' sman ssntuII N( IIIw0 (i u o nw*, r.n seUSs. *I




Figure 2-16. IR spectrum of N4-hexylspermidine (23).


71



































-Nn











1.0 6.0 5.0 .0o 3.0 1,0 0 Cr Figure 2-17. 60 MHz IH NMR spectrum of N4-methyl-N1,N8-bis(t-butoxycarbonyl)spermidine(24).







































1.0 4.0 5.0 4.0 3.0 2.00 .o Figure 2-18. 60 MHz 1H nmr spectrum of N4-methylspermidine (25).


































I lI fI t i t f I 1 9 1 1 t I i qi
1.0 4.0 5.0 q .0 3.0 1.0 1.0 0 Figure 2-19. 60 MHz 1H NMR spectrum of norspermidine trihydrochloride(26).


































I! . .I l. lI I
1.0 4.0 5.0 4.0 30 .0 1.0 Figure 2-20. 60 MHz 1H NMR spectrum of homospermidine trihydrochloride(27).






60


derivatives in the fewest steps in good yields. Utilizing the key synthons N4-benzylspermidine and its homologues, this objective could be realized.

Based on earlier studies,s" N4-benzylspermidine could be reacted with a wide variety of acylating agents and debenzylated to generate N',NB-bis(acyl)spermidines, both steps proceeding in high yields as outlined in Figure 2-2. Indeed, this was the case as,in all instances, the benzylated polyamines were condensed with the appropriate acylating agents in yields in excess of 90%. The reactions proceeded so smoothly that, in fact, the crude products obtained after simple acid-base workup could usually be debenzylated without further purification.

The benzyl bis(acyl)spermidines (VIII) were debenzylated as in earlier studies; however, the solvent acetic acid was replaced with methanol containing one equivalent of concentrated hydrochloric acid. In addition to the obvious improvement in ease of removing the solvent on workup, the hydrogenations proceeded at a faster rate compared to the earlier methods. Reaction times were generally on the order of 12-18 hours; however, it appeared that the reactions may be finished in six hours or

so.

The resulting terminally bis-acylated spermidine derivatives are thus generated in two high yield steps and can be converted directly

to bis-alkyl derivatives. Sodium borohydride-trifluoroacetic acid complex was used to accomplish this reduction of the amide to the amine. Lithium aluminum hydride is the reagent commonly used for the reduction of such amides; however, its extreme reactivity would be a detriment if other reducible groups were present. As it is the long term goal to conjugate antineoplastics (which may contain susceptible functionalities) to spermidine, a milder reducing agent was sought.






61

Sodium borohydride itself does not reduce amides; however, the addition of one equivalent of organic acid produces a reducing agent now capable of this.83 In fact, the order of reduction is now reversed-compared to most other reducing agents, i.e., tertiary>secondary>primary amides. Additionally, no cleavage of tertiary amides to the aldehyde and amine is noted, an important consideration for the reduction of secondary N-acylated derivatives.

The synthesis of secondary N-acylated spermidine derivatives was also easily accomplished, again using N'-benzylspermidine as the key

starting material.

In this case the benzylated polyamines (VII) were first reacted

with a transitory protecting group followed by debenzylation to produce the reagents (XII), Figure 2-3. The t-butoxycarbonyl group served as an excellent choice of protecting group for it is both quantitatively put on and removed. Accordingly, N4-benzylspermidine was reacted with a slight excess of BOC-ON, followed by hydrogenation, again employing PdC12 and MeOH/HCl to afford the bis-terminally protected reagents (XII), in 90% yield from (VII) after recrystallization.7s

Once generated, the bis-BOC spermidines were also capable of selective acylation of spermidine as shown by the synthetic scheme in Figure 2-4. As with the benzylated reagents, acylations with bis-BOC-spermidine (XII) likewise proceed smoothly to afford the N4-acyl NI,N-bis(t-butoxycarbonyl)spermidine cleanly and in yields in excess of 85%. The desired N4-acylspermidines (XIV) were then generated by brief exposure to trifluoroacetic acid. These N4-acyl derivatives are also efficiently reduced to the N-alkyl compounds (XV) using NaBH4.CF3CO2H. Although this reduction is a reduction of a tertiary amide, it proceeds smoothly and with no apparent cleavage of the amide to a secondary amine and aldehyde.






62

Synthesis of N4-Methylspermidine

As with the synthesis of other N4-alkylspermidines, the synthesis of N4-methylspermidine was envisioned as three steps: reaction of NI,N8bis(t-BOC)spermidine with an appropriate acylating agent, removal of the t-BOC protecting groups, followed by reduction of the amide. In the corresponding retrograde synthesis, N4-methylspermidine could be prepared by the reduction of N4-formylspermidine which, in turn, could be generated by deprotection of N1,N8-bis(t-BOC)N4-formylspermidine. Indeed, this last compound was easily synthesized by reacting NI,N8-bis(t-BOC)spermidine with acetic formic acid anhydride. However, its subsequent deprotection with trifluoroacetic acid always led to inseparable mixtures of the desired N4-formylspermidine and spermidine itself. This observation was not totally unexpected as the formyl group is commonly employed as a N-protecting group which is removable by acid conditions. Even when the reaction time was limited to two minutes at DOC, substantial loss of the formyl group still resulted.

Alternatively, other methods were available for methylation of amines. Direct alkylation of N1,N8-bis(t-BOC)spermidine with methyl iodide resulted in mixtures of unreacted starting material, product, and overalkylation to the quaternary salt. However, reductive alkylation with formaldehyde and sodium cyanoborohydride87 led to the formation of N,N-bis(t-BOC)-N4-methylspermidine in 70% yield after chromatography. The desired N4-methylspermidine was subsequently obtained by the usual deprotection with trifluoroacetic acid.

In summary, the reagents N4-benzylspermidine, N,N8-bis(t-butoxycarbonyl)spermidine and their symmetrical homologues were synthesized and used to prepare seTectively N-modified spermidine derivatives. The






63

synthesis of a variety of N-acylated and N-alkylated spermidines was described and illustrates that the number of polyamine derivatives that

can be generated by these methods are virtually limitless. Additionally, these reagents can and have been used toward the synthesis of other systems, such as the siderophores, an example of which is described in Chapter Five.













CHAPTER THREE

BIOLOGICAL EVALUATION OF POLYAMINE DERIVATIVES Materials and Methods

Polyamine Derivatives

The following compounds were synthesized as described in Chapter Two and the following abbreviations will be used: N4-benzylspermidine (BSpd), N4-benzylnorspermidine (BnSpd), Ns-benzylhomospermidine (BhSpd), N4-benzoylspermidine (Benzoyl Spd), N4-methylspermidine (MeSpd), N4acetylspermidine (AcetylSpd), N4-Ethylspermidine (EtSpd), N4-hexanoylspermidine (HexanoylSpd), N4-hexylspermidine (HexylSpd), N-(2-Cyanoethyl)-N-(3-cyanopropyl)benzylamine (BSpdN), N ,N8-bis(t-butoxycarbonyl)spermidine (BisBOCSpd), Nl,N8-bis(acetyl)spermidine (BisAcetylSpd), N1,N8-bis(ethyl)spermidine (BisEtSpd), N1,N8-bis(propionyl)spermidine (BisPropionylSpd), N,NB-bis(propyl)spermidine (BisPropylSpd), norspermidine (nSpd), and homospermidine (hSpd). The following were purchased from Aldrich Chemical Co., Metuchen, NJ: spermidine (Spd), spermine (Spm), and putrescine (Put). Available in this lab already was NT,N8bis(2,3-dihydroxybenzoyl )spermidine (DHBSpd). Ascites Leukemia Cells

Murine L1210 leukemia cells were maintained by weekly i.p. transplantation in female DBA/2J mice. For in vitro studies 106 leukemic cells were inoculated i.p., four days prior to use. Cells were collected by peritoneal lavage with RPMI-1640. The cells were washed twice, counted electronically, and adjusted to a density of 10 cells/ml for uptake studies. The effects of DMFO on Spd uptakewere studied by 64






65

giving mice bearing L1210 cells 2% DFMO by drinking water for 40-48

hours prior to removal of cells.

Cell Culture

Murine L1210 leukemia cells were maintained in logarithmic growth as a suspension culture in Roswell Park Memorial Institute Medium 1640 (RPMI-1640) containing 2% HEPES-MOPS and 10% fetal calf serum. Cells were grown in glass tubes in a total volume of 2 ml under a humidified 5% CO2 atmosphere at 370. Cultures were treated while in logarithmic growth (0.5 to 1 x 10s cells/ml) with the polyamines, Spd derivatives, or MGBG at concentrations ranging from 10-6 to 10-2 M. After 24 or 48 hours, cells were removed from tubes for counting and viability determinations. Cell number was determined by electronic particle counting (Model ZF Coulter counter; Coulter Electronics, Hialeah, FL) and confirmed periodically with hemocytometer estimates. Cell viability was assessed by trypan blue dye exclusion (0.5% in unbuffered 0.9% NaCl solution). Percent control growth was determined as the final treated cell number minus the initial inoculum ('5 x 104 cells/ml) divided by the final untreated cell number minus the initial inoculum, times 100. Growth data were further analyzed with a Hewlett Packard HP-85 microcomputer programmed to determine percent growth inhibition and 50% growth inhibitory doses (IDso) at 24 and 48 hours.

Uptake Determinations

The Spd derivatives and MGBG were studied for their ability to compete with 3H-Spdfor uptake into ascites L1210 cells in vitro. These determinations were performed by Dr. Carl Porter's group at Roswell Park Memorial Institute using the following procedures. Prewarmed L1210 cell suspensions (5 x 106/cc) were incubated in 1 ml of RPMI-1640 containing






66


2% HEPES-MOPS and 0.2, 0.5, 1.0, 2.0, 5.0 or 10 pM 3H-Spd (New England Nuclear Corp., Boston, MA) alone or in the presence of 10 or 100 uM polyamine, Spd derivative or MGBG. The cells were incubated for 20 min at 370 except for one tube containing 10 pM 3H-Spd which was not prewarmed and was incubated at 00 to measure surface binding. At the end of the incubation the tubes were centrifuged at 900 g for five min at 0-40. A 200 ul aliquot of supernatant was removed for scintillation counting and the remainder of the supernatant discarded. The pellet was washed twice with 5-7 ml of cold RPMI-1640 containing 1 mM Spd to displace nonspecifically bound 3H-Spd. The pellet was then dried with a cotton swab and dissolved in 200 Pl of IN NaOH at 600 for 20-60 min. The material was neutralized with IN HCl, diluted to 1 ml with distilled water and transferred to a vial for scintillation counting. Uptake was linear with time from one to 40 min and with 3H-Spd concentration up to 30 PM. Results were expressed as pmol 3H-Spd taken up per min per mg protein as determined by the method of Lowry.88 Uptake data were analyzed for kinetic characteristics by using a Hewlett Packard HP-85 microcomputer programmed for nonlinear regression curve fitting.89 Acute LD50 Toxicity Studies

Acute toxicity of Spd derivatives was determined in male CD-1 mice

at 24 or 36 hours. The Spd derivatives were dissolved in isotonic saline, the pH adjusted with NaHCO3 or H3PO4 when necessary. The animals were given one i.p. injection of the derivatives, the volume of which was adjusted with additional saline, so as to deliver the same volume for each dose. Generally, two mice per dose at several widely ranging values were used to determine preliminary toxicity. Once the approximate LD50 is determined, 10 mice at five doses closely around this value are used.





67


Results

Uptake of Polyamine Derivatives

The inhibition of 3H-Spd uptake in ascites L1210 cells by Spd derivatives, polyamines, and MGBG are summarized in Tables 3-1, 3-2, and 3-3. For the control experiments, ascites L1210 cells exposed to 10 UM 3H-Spd alone for 20 min at 37*C take up approximately 56 pmol Spd/107 cells/min, Table 3-1. However, at 40C this uptake is reduced to 10% of that at 370C and may represent the fraction of nonspecific binding to the cell surface. Also shown in Table 3-1, with the exception of putrescine, all polyamines are quite effective in preventing 3H-Spd uptake. The polyamines Spm and hSpd are equally effective as Spd itself in competing for uptake, inhibiting 90% of 3H uptake.

The uptake characteristics of terminally bis-modified Spd derivattives are reported in Table 3-2. In general, modification of Spd primary amines produces derivatives that are either weak inhibitors or unable to inhibit 3H-Spd uptake at all. All the bis-acylated derivatives tested could be considered noncompetitive in preventing Spd uptake, allowing 90% of the control uptake of 3H-Spd. Only Bis(ethyl)Spd was capable of

substantially inhibiting Spd uptake, having a Ki of 62 uM.

On the other hand, modification of Spd's secondary amine appears less restrictive. All of the secondary N-alkylated derivatives, Table 3-3, are quite competitive in inhibiting 3H-Spd uptake. Additionally, a preference in backbone chain length of the benzyl derivatives is shown. For example, BhSpd is more effective than BSpd in preventing uptake, whereas BnSpd is less effective. Of the secondary N-acylated Spd derivatives, only N4-AcetylSpd is capable of weakly inhibiting Spd uptake.





68


Table 3-1. Effects of Polyamines and MGBG on 3H-Spd Uptake fnto Ascites L1210 Leukemia Cells


3H-Spd Uptake*
Competing Agent pmol/107 cells/ Ki(uM)** (100uM) min %control



None 65 100 40C 6 8


Put 58 86 90 nSpd 16 29 19 Spd 6 10 10 hSpd 6 10 10 Spm 6 11 11 MGBG 37 66 53



Cells were incubated for 20 min at 370 with lOuM 3H-Spd
and 100 uM polyamine or MGBG

**Cells were incubated for 20 min at 370 with 0.2, 0.5, 1.0,
2.0, 5.0, or lOuM 3H-Spd and 10 or lOOuM polyamine or MGBG.






69



Table 3-2. Effects of Terminally Modified Spd Derivatives
on H-Spd Uptake into Ascites L1210 Leukemia
Cells



3-Spd Uptake*
Competing Agent pmol/10cells/ UptakeK pmol/l Ki(uM)* (lOOuM) min %control



None 56 100


BSpdN 49 88 163 BisBOC-nSpd 53 95 1103 BisBOC-Spd 51 91 521 BisBOC-hSpd 50 89 504 DHBSpd 51 91 256 BisAcetyl-Spd 51 91 508 BisPropionyl-Spd 51 92 550 BisEthyl-Spd 39 69 62 BisPropyl-Spd 45 80 117


* Cells were incubated for 20 min at 370 with lOuM 3H-Spd and 1OOuM Spd Derivative

**Cells were incubated for 20 min at 370 with 0.2, 0.5,
1.0, 5.0, or lOuM H-Spd and 10 or 100 uM of derivative.






70



Table 3-3. Eff cts of Secondary Modified Spd Derivatives
on H-Spd Uptake into Ascites L1210 Leukemia
Cells


3H-Spd Uptake*
Competing Agent pmol/l07 cells/ Ki(uM)**
(100uM) min %control



None 65 100


N4-AcetylSpd 53 81 115 N4-HexanoylSpd 57 88 151 N4-BenzoylSpd 60 92 500 N4-MeSpd 11 17 3.4 N4-EthylSpd 9 14 3.1 N4-HexylSpd 45 69 34 N4-BnSpd 57 88 135 N4-BSpd 44 67 39 N5-BhSpd 24 37 14


* Cells were incubated for 20 min at 370 with lOuM 3H-Spd
and 100lOuM derivative

**Cells were incubated for 20 min at 370 with 0.2, 0.5,
1.0, 5.0, or lOuM 3H-Spd and 10 or 1OOuM derivative






71


Acute LD50 Toxicity Studies of Polyamine Derivatives

The acute toxicity of some of the polyamine derivatives synthesized was investigated in vivo in white mice. For purposes of comparison, the LD50 of Spd was also measured. The results of this study are presented in Table 3-4. The LD50 for Spd was determined to be about 400 mg/Kg. This value is somewhat lower than a previously reported value of 470 mg/Kg;90 however, this difference can be attributed to the difference in duration between the two studies. In the earlier study, deaths were recorded only up to five hours while, in this study, deaths were recorded up to 36 hours. In fact, excluding the very toxic doses, the greatest percentage of animals given Spd died between 18 and 24 hours. The most likely cause of death appeared to be respiratory failure.

The toxicity behavior of MeSpd was virtually identical to Spd, both in LD50 and time course of death. However, the toxicity of BSpd and BenzoylSpd was very much different. Death usually resulted in five minutes or less marked by convulsions. With all four compounds, in almost every instance, if the animals survived the first minutes no ill effects were seen afterwards. Interestingly enough, the LD50 of the three benzyl homologues also showed a dependency on chain length with BhSpd being the most toxic of all compounds tested. Finally, all of the terminally Nmodified compounds were shown to be relatively nontoxic, the three bis(acyl)Spd having LD50's exceeding 800 mg/Kg.

Discussion

Several lines of evidence support the existence of an energy dependent transport carrier for the uptake of polyamines across the cell membrane. The anticancer agent MGBG has been shown to actively compete with spermidine for uptake and is concentrated intracellularly via this carrier.56






72



Table 3-4. Acute Toxicities of Spermidine and Polyamine
Derivatives



Compound LD50 (mg/kg)



Spd 400 DHBSpd >800 Bis(Acetyl)Spd >1000 Bis(Ethyl)Spd 425 N4-MeSpd 375 N4-BnSpd 250 N4-BSpd 200 N5-BhSpd 125 N4-BenzoylSpd 300






73

Furthermore, in cells having their polyamine pools depleted by pretreatment with DFMO, both polyamine and MGBG uptake is enhanced several-fold.42 Accordingly, it is hypothesized that several structural parameters must be recognized by this receptor or carrier for uptake.

In an effort to further define these necessary parameters for uptake, a wide array of spermidine derivatives were synthesized and assayed for their ability to compete with 3H-Spd for uptake. These studies suggest that the primary amines of spermidine are critical for recognition by the carrier for uptake. Acylation of both primary amines produces derivatives that are very poor inhibitors of 3H-Spd uptake. Reduction of these amides to the corresponding amines offers some improvement in the derivatives' ability to compete for uptake; however, this uptake appears severely restricted by the size of the alkyl substituent. For example, only BisEtSpd is as effective as MGBG in preventing Spd uptake.

Modification of spermidine's secondary amine appears less critical in conferring uptake specificity, as both N-alkylated and N-acylated derivatives inhibited 3H-Spd uptake. However, there is an obvious preference for the alkylated derivatives. Moreover, there is less restriction upon the size of the alkyl substituent as all four alkyl derivatives ranging up to hexyl and benzyl all have Ki's comparable or better than MGBG.

With respect to chain length, quite unexpectedly, the hSpd backbone competed more effectively than the Spd backbone as demonstrated by the order of preference BhSpd>BSpd>BnSpd. This finding is interesting since

hSpd is not found in mammalian systems. Whether this effect will be seen in other derivatives is currently under investigation.






74

The toxicity of the polyamine derivatives was also investigated.
None of the polyamine derivatives tested were unusually toxic; however, an interesting relationship was noticed. A comparison of LD50 values and Ki values reveals that the derivative's toxicity is proportional to its uptake. For instance the bis(acyl) derivatives, bis(acetyl)Spd and DHBSpd have LD50 in excess of 1000 mg/Kg and are unable to compete for uptake. On the other hand derivatives which are good inhibitors of 3HSpd uptake generally have LD50's less than 500 mg/Kg. Moreover, this relationship even holds true for the BSpd derivatives; BhSpd, which is the best inhibitor of the three, is also the most toxic. These data seem to suggest the toxicity of these derivatives is related to their cellular uptake.

In summary, it is apparent that the terminal amines of spermidine

are of critical importance for uptake. Modification of the terminal amines by either acylation or addition of an alkyl substituent produces a derivative which is ineffective in competing for uptake. In terms of substrate recognition by the transport system, this suggests that the positive charges carried by spermidine's terminal amines at physiological pH are necessary for uptake. Furthermore, although the bis-alkyl derivatives also carry this positive charge, recognition appears to be limited by the size of the alkyl substituent. In this case, large alkyl groups may prevent interaction of the positively charged terminal amines with the carrier by interacting sterically or by changing the polyamines' conformation.

The role of the secondary amine is less certain. Modification here appears to be less restrictive, although preference was again seen for the alkylated derivatives. The argument for requirement of positive charge may again be true; however, recently it has been shown that diamines such






75


as 1,8-diaminooctane, in which the central nitrogen is replaced by a carbon, are still very good inhibitors of 3H-Spd uptake having a Ki of 22.1 M91. Nevertheless, the central nitrogen may be the appropriate site to conjugate antineoplastics to.













CHAPTER FOUR

PRELIMINARY INVESTIGATIONS TOWARDS THE DEVELOPMENT OF SPERMIDINE-ANTINEOPLASTIC CONJUGATES

It is apparent from the uptake data presented in Chapter Three that several structural parameters are required for uptake of a Spd-derivative via the spermidine receptor. First, bis-acylation of the terminal amines of spermidine is limiting on uptake. However, acylation or alkylation of spermidine's secondary nitrogen appears less restrictive, having little negative effect on uptake. Accordingly, the secondary amine may represent a potential site in which antineoplastics can be attached. Therefore, the purpose of this chapter is to begin a preliminary investigation into the synthesis and uptake of several small antineoplastics conjugated to spermidine's central nitrogen. The results of these initial findings will hopefully shed some light on the feasibility of such an approach.

Of the wide variety of antineoplastics with which to conjugate spermidine's central nitrogen, two structural criteria should be met: it should contain a functionality or site capable of attachment to the t-BOC-protected spermidine reagent, and be stable to the trifluoroacetic acid needed to deprotect the intermediate. Two such choices are the al kylating agent 4-[p-[bis-(2-chloroethyl)amino]phenylbutyric acid (Chlorambucil) and 2,3-dihydro-lH-imidazo[1,2b]pyrazole (IMPY), an inhibitor of ribonucleotide reductase.92

The syntheses of such conjugates are straightforward, indeed.

Chloroambucil contains a free carboxylic acid which should render itself



76






77

to usual peptide condensation techniques. The reaction scheme is shown in Figure 4-1. The succinimide active ester of chlorambucil is first generated using dicyclohexylcarbodiimide and N-hydroxysuccinimide and then reacted with N,N8-bis(t-butoxycarbonyl)spermidine producing N4chlorambucil-N1, 8-bis(t-butoxycarbonyl)spermidine (28). The desired N4-chlorambucil spermidine conjugate (29) is prepared by brief exposure of intermediate (28) to trifluoroacetic acid.

Although IMPY does not contain a carboxylic acid with which to directly condense with the protected spermidine reagent, a short connecting bridge, such as glutaric acid, could be used. Subsequently, IMPY can be reacted with glutaric anhydride, thereby producing an intermediate (30) with a free carboxylic acid.93 This is then condensed with N,N8-bis(t-butoxycarbonyl)spermidine in the same manner as described above. The entire synthesis of N4-[4-(2,3-dihydro-lH-imidazo[1,2-b]pyrazolo)carboxamido)butyryl]spernlidine (32) is shown in Figure 4-2.

Once these antineoplastic-Spd conjugates are synthesized, they will be tested in vitro for their ability to compete with spermidine for uptake as well as their antileukemic activity. The former assay is performed in the same manner as for the polyamine derivatives in the preceding chapter. The antineoplastic-Spd conjugate will be incubated in the presence of 3H-Spd in ascites L1210 cells, and in inhibition of 3H uptake determined as before. Additionally, the uptake of the native antineoplastics themselves will also be determined.

The antileukemic activity of the antineoplastic-Spd conjugates

and the native antineoplastics will be ascertained by determining the dose necessary to prevent 50% of cell growth (ID50) of L1210 cell cultures. It is anticipated that if conjugation of spermidine to the






78



0

OH



0


C N C 1) DCC N-OH Suc. 2) 13
Et3N 28




TFA





0

NH3




O NH 29


Figure 4-1. Synthesis of N4-Chlorambucilspermidine.





79




\ o


HN 0
N












N (Ho d(30)


S1) DCC 2) 13 N-OH Suc. Et3N 31


i T FA

0 0

NN H 32



Figure 4-2. Synthesis of N4-GIMPY-spermidine(32 )






so


antineoplastic does indeed improve the uptake of the drug, this will be demonstrated by a lowering of the drug's IDso.
Experimental

Materials

Chlorambucil was purchased from Sigma Chemical Company, St. Louis, MO. Samples of IMPY were generously donated by Dr. Leonard Kedda, National Cancer Institute, Bethesda, MD. All other reagents were purchased from Aldrich Chemical Company, Metuchen, NJ and, except as noted, used without further purification. Physical measurements were performed as

detailed in Chapter Two.

N4-Chlorambucil-Nl,N8-bis(t-butoxycarbonyl) spermidine (28)

A solution of DCC (145 mg, 0.70 mmol) in 10 ml THF was added to a cooled solution of chlorambucil (185 mg, 0.61 mmol) and N-hydroxysuccinimide (80 mg, 0.70 mmol) in 20 ml THF. The ice bath was removed and the solution allowed to stir for 18 hours during which time a thick

white precipitate ensued.

The reaction mixture was then concentrated, 20 ml CH2C1 2 added, the DCU precipitates filtered and washed with an additional 10 ml of CH2C12. The filtrates were combined, cooled, and a solution of NI,N-bis(tbutoxycarbonyl)spermidine HC1 (13) (265 mg, 0.65 mmol) and triethylamine (90 pl, 0.65 mmol) in 10 ml CH2C12 slowly added. The resulting reaction was allowed to stir at room temperature for 48 hours, at which time additional (20 ml) CHC12 was added and the organic layer washed with 3% HC1 (2 x 10 ml) and H20 (2 x 10 ml). The CH2C12 layer was dried and concentrated to afford 340 mg crude product. Further purification was effected by chromatogrpahy on alumina (neutral, activity I) eluting with CHC13 to afford 280 mg (73%) product: 1H NMR 6 1.34-1.96






81

(m, 26H), 2.41 (m, 4H), 3.28 (m, 8H), 3.68 (s, 8H), 5.12 (br, 2H),

6.58 (d, 2H), 7.03 (d, 2H).
Anal. cal. for C31H52N405C12: C, 58.94; H, 8.30; N, 8.87. Found: C, 59.49; H, 8.42; N, 8.47.
N4-Chlorambucilspermidine-bis-trifluoroacetate (29)
Trifluoroacetic acid (10 ml) was added to a flask containing (28) (220 mg, 0.35 mmol) and the resulting solution stirred for 20 minutes. The solvent was then quickly evaporated, dissolved up in methanol (25 ml) and concentrated (twice). The residue was then dissolved in 10 ml H 0, washed with CH2C12 (2 x 5 ml), and the aqueous layer lyophilized to afford 150 mg (70%) as a beige solid: 1H NMR (TFA) 6 1.54-2.01 (m, 8H), 2.47 (m, 4H), 3.31 (m, 8H), 3.70 (s, 8HO, 6.67 (d, 2H), 7.06 (d, 2H).
Anal. cal. for C2sH38 N OsC F: C, 45.53; H, 5.81; N, 8.49. Found: C, 45.81; H, 6.32; N, 8.40.
N-(4-Carboxybutyryl)2,3-dihydro-1H-imidazo[l,2-blpyrazole (_~1 _) ()
Glutaric anhydride (250 mg, 2.2 mmol) was added to a solution of
IMPY (220 mg, 2.0 mmol) in 30 ml of dry CH2C12 under N2. After 18 hours the product was filtered off, washed with CH2Cl2, and dried in vacuo to afford 400 mg (90%) as a white powder. The physical and spectral characteristics were identical to those previously reported:93 mp > 2400C; 1H NMR (TFA) 6 1.52-1.98 (m, 2H), 2.04-2.61 (m, 4H), 4.35 (s, 4H), 6.28 (s, lH), 7.57 (s, LH). N4-[4-(2,3-Di hydro-lH-imidazo(l ,2-b]pyrazolo)carboxami do)butyryl]NI,NO-bis(t-butoxycarbonyl )spermidine (3)
Dicyclohexylcarbodiimide (DCC) (165 mg, 0.80 mmol) was added to a solution of (30) (150 mg, 0.67 mmol) and N-hydroxysuccimide (95 mg,

































7 6 5 U 5 2 1 0

Figure 4-3. 300 MlHz 1H NMR spectrum of N4-Chlorambucil-N1,N8-bis(t-butoxycarbonyl)spermidine (28).




































a ..... --. A a 0..0 J
1.o0 o s5.O .0 3.0 2.o .o a
Figure 4-4. 60 MHz 1H NMR spectrum of N4-Chlorambucilspermidine trifluoroacetate (29).


































7 Lj 3 2 1
Figure 4-5. 300 MHz 1H NMR spectrum of N4-[4-(2,3-dihydro-IH-imidazo[1,2b]pyrazolo)carboximido)butyryl]-N1-N8-bis(t-butoxycarbonyl)spermidine (31).
































I 6 .. I I I I I i

10 S.0 .0 3.0 1.0O Figure 4-6. 60 MHz H NMR spectrum of N4_4-(2,3-dihydro-H-imidazo[1,2b]pyrazolo)carboxamido)butyryl]spermidine trifluoroacetate (32).






86

0.80 mmol) in 50 ml dry pyridine. The resulting suspension was allowed to stir for 36 hours at which time a solution of (13) (300 mg, 0.75 mmol) in 10 ml CH2C12 was added and the reaction allowed to proceed an additional 36 hours. The solvent was then evaporated and the residue dissolved up in 100 CH2CI2, filtered, washed with ice cold 3% HC1 (2 x 20 ml), H20 (2 x 20 ml), 5% NaHC03 (3 x 20 ml), H20 (3 x 20 ml), dried and concentrated to afford 310 mg (83%) as a white crystalline solid; mp 155-1570C (CHC13/cyclohexane); 1H NMR 6 1.20-1.82 (m, 6H), 1.39 (s, 18H),
2.12-2.67 (m, 2H), 2.75-3.44 (overlapping m, 12H), 4.32 (s, 4H), 5.616.28 (m, 3H), 7.16 (s, 1H); IR (CHC13).
Anal. cal. for C27H46N,06: C, 68.89; H, 8.42; N, 15.26. Found: C, 59.17; H, 8.40; N, 15.07.
N4-[4-(2,3-Dihydro-lH-imi dazo[1 ,2-b]pyrazolo)carboxami do)butyryl]spermidine trifluoroacetate (N4-GIMPY Spd) (3j)
A solution of (31) and trifluoroacetic acid was reacted and purified as described previously for 29: 400 mg (91%); 1H NMR (TFA) 6 1.571.96 (m, 6H), 2.10-2.62 (m, 2H), 2.74-3.57 (overlapping, 12H), 4.37 (s, 4H), 6.30 (s, IH), 7.53 (s, 1H), 7.85 (br s, 6H).
Anal. cal. for Cz3H32zFN60,: C, 39.89; H, 4.80; N, 12.19. Found:

C, 39.76; H, 5.11; N, 12.03.
Results and Discussion

The antineoplastic Spd conjugates N4-Chlorambucil Spd (29) and N4GIMPY Spd (32) were successfully synthesized in a two-step fashion employing the bis(t-BOC)-protected reagent (13). Chlorambucil or GIMPY
(30) were condensed with (13) using the condensing reagents DCC and N-hydroxysuccinimide in 70-80% yields. The yields were lower than the corresponding secondary N-acylations illustrated in Chapter Two. This was probably due to the less active acylating agent employed (succinimide





87


ester versus acid chloride) and the bulkier size of the antineoplastic being condensed. However, it illustrates that a wide variety of acylating agents of varying size can be coupled to the reagent (13) in good yields.

The antineoplastic-spermidine conjugates were then tested for their ability to compete with 3H-Spd uptake and their cytotoxic effects on L1210 cells in vitro. The results of these preliminary investigations are given in Table 4-1. Both antineoplastic-spd conjugates inhibited 3H-Spd uptake; however, the N4-chlorambucil conjugate was much more effective (Ki = 6 um). Furthermore, N4-chlorambucil-Spd was found to be cytotoxic to L1210 cells, with an ID50 of 15 um. Chlorambucil itself was equally cytotoxic, thereby showing no preference for the Spd conjugate. In view of the fact that chlorambucil alone does not compete for Spd uptake, this suggests that it may be entering the cell via an alternate mechanism, perhaps by an amino acid specific carrier.94

On the other hand, the N4-GIMPY-Spd conjugate was found to be relatively noncytotoxic, having an ID50 of 350 pM. When this value is, however, compared to the ID50 of its precursor (30), a positive effect of the conjugation of the spermidine backbone can be inferred. Assuming that the mode of action between N4GIMPY-Spd (32) and GIMPY (30) is the same, the increased cytotoxicity of (32) can be explained by the improved uptake of the conjugate over (30). It should be pointed out that the cytotoxicity of the GIMPY-Spd conjugates is far below what is considered sufficient for antineoplastic activity. The mode of action of IMPY is by chelation of the iron in the ribonucleotide reductase, rendering it inactive. It is a distinct possibility that the amine which is reacted with glutaric anhydride may be involved in chelation. Functionalization of it to an amide






88


Table 4-1. Uptake and Cytotoxicity of Antineoplastics and Antineoplastic-Spermidine Conjugates in
L1210 Leukemia Cells in vitro.




Ki(uM) ID50(uM)



Chlorambucil 350 15 N4-ChlorambucilSpd (29) 6 15 GIMPY (30) >1000* N4-GIMPY Spd (32) 150 350


*Insolubility at higher concentrations prevented determination





89


may produce a less effective chelator, thereby decreasing its activity. Accordingly, the synthesis of an analogue in which this amide is reduced to an amine is currently being investigated.

Finally, although the in vitro data at this point are limited, it

indicates that these conjugates are indeed taken up via the spermidine carrier and represent a potential drug delivery system. Accordingly, further testing and synthesis of other systems are currently being evaluated at this time.













CHAPTER FIVE

SYNTHESIS OF TRIS-PROTECTED SPERMIDINES

The reagents described in Chapter Two, N4-benzylspermidine and

N1,N8-bis(t-butoxycarbonyl)spermidine, have demonstrated themselves as important reagents for the selective functionalization of spermidine's primary and secondary nitrogens. Using these methods, derivatives can be obtained that 1) contain only one substituent on the secondary nitrogen, 2) contain the two same substituents on both primary nitrogens, or 3) a combination of both; two identical substituents on the primary nitrogens and a third different substituent on the secondary nitrogen. However, it is not possible to place three different groups on spermidine or, for that matter, only one substituent on only one primary nitrogen via these methods. The latter group of derivatives, those modified at only one terminal nitrogen, pose an additional interesting question concerning uptake --how effective would these derivatives be in competing for uptake?

The answer to this question, based upon the uptake data presented in Chapter Three, is yes. The Nl,N8-bis(alkyl)spermidine derivatives were able to compete with Spd for uptake, and it is expected that modification of only one terminal amine should accentuate this uptake. This may offer another potential avenue in conjugating antineoplastics to spermidine. Accordingly, the synthesis of such a reagent is desired.

The ideal reagent would be one in which each of spermidine's nitrogens is protected with three different protecting groups, each removable separately under different conditions as illustrated in Figure 5-1.

90










Z
I
A-NH-(CH2)a-N-(CH2)b-NH-B




Z Z
H2N-(CH2)a-N-(CH2)b-NH-B A-NH--(CH2 )a- -(CH2)b-NH2






ANH-(CH2)a-- NH-(CH2)~-- NH-B






Figure 5-1. Spermidine reagents containing three different protecting groups,
A, B, Z, which can be independently removed.








Additionally, the boundary conditions of earlier synthesis must be still met in that the reagent should be generated in as few steps as possible, and the protecting groups removed cleanly and efficiently. The benzyl and t-butoxycarbonyl protecting groups employed earlier have already proven themselves as easily removable in the presence of one another and, accordingly, their use will be maintained. The choice of the third protecting group should therefore be one which is removable under basic conditions as the t-BOC and benzyl groups are removed under acid and neutral conditions, respectively. A prime candidate for the base removable protecting group is the trifluoroacetoxy protecting group.9" It can be easily attached via either the anhydride or the acid chloride, and removed under relatively mild basic conditions employing sodium bicarbonate.

Synthesis

In the simplest terms, the synthesis of the target tris-protected reagent would be nothing more than the synthesis of N4-benzyl-Nl,N8bis(t-butoxycarbonyl)spermidine however, replacing one of the t-BOC groups with the trifluoroacetoxy group. However, in reality the removal of one t-BOC protecting group would likely be impossible. If, on the other hand, this intermediate (XVI) figure 5-2, could be generated by some other means, the target compound (XVII) could be realized.

Recalling the synthesis of the benzylated polyamines in Figure 2-1, a synthetic scheme for intermediate (XVI) can be envisioned by incorporating a t-BOC protecting group early on in the synthesis. Such a scheme is outlined in Figure 5-3.

The key to the whole scheme lies in the ability to selectively react the terminal amine of N-(3-aminopropyl)benzylamine with BOC-ON to produce N-[N-(t-butoxycarbonyl)3-aminopropyl]benzylamine (XIX). The desired






93















tBOC-NH~ N NHCOCF3 XVII











0

tBOC-NH., N NH2

XVI







Figure 5-2. Synthesis of the desired tris-protected reagent
(XVII) from intermediate (XVI).




Full Text
60
derivatives in the fewest steps in good yields. Utilizing the key
synthons N4-benzylspermidine and its homologues, this objective could
be realized.
Based on earlier studies,85 N4-benzylspermidine could be reacted
with a wide variety of acylating agents and debenzylated to generate
N1,N8-bis(acyl)spermidines, both steps proceeding in high yields as out
lined in Figure 2-2. Indeed, this was the case as,in all instances, the
benzylated polyamines were condensed with the appropriate acylating agents
in yields in excess of 90%. The reactions proceeded so smoothly that,
in fact, the crude products obtained after simple acid-base workup could
usually be debenzylated without further purification.
The benzyl bis(acyl)spermidines (VIII) were debenzylated as in ear
lier studies; however, the solvent acetic acid was replaced with methanol
containing one equivalent of concentrated hydrochloric acid. In addi
tion to the obvious improvement in ease of removing the solvent on work
up, the hydrogenations proceeded at a faster rate compared to the earlier
methods. Reaction times were generally on the order of 12-18 hours;
however, it appeared that the reactions may be finished in six hours or
so.
The resulting terminally bis-acylated spermidine derivatives are
thus generated in two high yield steps and can be converted directly
to bis-alkyl derivatives. Sodium borohydride-trifluoroacetic acid com
plex was used to accomplish this reduction of the amide to the amine.
Lithium aluminum hydride is the reagent commonly used for the reduction
of such amides; however, its extreme reactivity would be a detriment if
other reducible groups were present. As it is the long term goal to
conjugate antineoplastics (which may contain susceptible functionalities)
to spermidine, a milder reducing agent was sought.


n "i i ~j i i
,3 7
Figure 6-?
a \
l i ] T~> 1 f | i i --"r -j ~r i T-T I i~ i | iir ( || i r ~t i
' :.S *1 h 7 i
. 300 MHz NMR spectrum of N^-[N-(t-butoxycarbony1)threony1]-N^ ,N^-
bis(t-butoxycarbonyl)spermidine(5J3).


SYNTHESIS AND PROPERTIES OF N-ACYLATED AND N-ALKYLATED
POLYAMINE DERIVATIVES AND OF AGROBACTIN A,
A NATURALLY OCCURRING SIDEROPHORE
BY
NEAL J. STOLOWICH
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1983


CHAPTER FOUR
PRELIMINARY INVESTIGATIONS TOWARDS THE DEVELOP
MENT OF SPERMIDINE-ANTINEOPLASTIC CONJUGATES
It is apparent from the uptake data presented in Chapter Three that
several structural parameters are required for uptake of a Spd-derivative
via the spermidine receptor. First, bis-acylation of the terminal amines
of spermidine is limiting on uptake. However, acylation or alkylation of
spermidine's secondary nitrogen appears less restrictive, having little
negative effect on uptake. Accordingly, the secondary amine may repre
sent a potential site in which antineoplastics can be attached. There
fore, the purpose of this chapter is to begin a preliminary investiga
tion into the synthesis and uptake of several small antineoplastics con
jugated to spermidine's central nitrogen. The results of these initial
findings will hopefully shed some light on the feasibility of such an
approach.
Of the wide variety of antineoplastics with which to conjugate
spermidine's central nitrogen, two structural criteria should be met:
it should contain a functionality or site capable of attachment to the
t-BOC-protected spermidine reagent, and be stable to the trifluoroacetic
acid needed to deprotect the intermediate. Two such choices are the
alkylating agent 4-[p-[bis-(2-chloroethyl)aminojphenylbutyric acid
(Chlorambucil) and 2,3-dihydro-lH-imidazo[l,2b]pyrazole (IMPY), an in
hibitor of ribonucleotide reductase.92
The syntheses of such conjugates are straightforward, indeed.
Chloroambucil contains a free carboxylic acid which should render itself
76


100
Anal. cal. for C2jH32N3O3F3: C, 58.45; H, 7.47; N, 9.74. Found:
C, 58.31; H, 7.49; N, 9.71.
N-[N-(t-Butox.ycarbonyl) -3-aminopropyl]-N-(2-cyanoethyl )benzvlamine (37)
A sealed vessel containing (^3) (6.0 g, 0.023 mole) and acryloni
trile (2.6 ml, 0.039 mole) under an argon atmosphere was heated in an
oil bath at 100C for 24 hours. The reaction mixture was cooled and
purified via chromatography on silica gel eluting with CHC13 to afford
7.06 g (98%) product as an oil: XH NMR 6 1.42 (s, 9H), 1168 (m, 2H),
2.58 (m, 4H), 3.13 (quar., 2H), 3.55 (s, 2H), 4.73 (br, 1H), 7.23 (s,
5H); IR (neat) 3000 (m), 2250 (w), 1715 (s), 1515 (s), 1180 (s), 740
(m) cm"1.
Anal. cal. for C18H27N302: C, 68.11; H, 8.57; N, 13.24. Found:
C, 67.96; H, 8.55; N, 13.19.
N4-Benzyl-NI-(t-butoxycarbonyl)spermidine (38)
A solution of (37) was reduced and purified in a similar manner
as described for (35). Yield: 9.4 g (97%); NMR 6 1.40 (2, 11H), 1.67
(m, 4H), 2.53 (m, 6H), 3.10 (quar., 2H), 3.48 (s, 2H), 5.28 (br, 1H),
7.18 (s, 5H); IR (neat) 2975 (s), 1710 (s), 1515 (s), 1180 (s), 740
(m) cm"1.
Anal. cal. for C18H31N302: C, 67.25; H, 9.72; N, 13.07. Found:
C, 66.98; H, 9.78p N, 13.04.
N4-Benzyl-N1-(t-butoxycarbonyl)-N -trifluoracetylnorspermidine (19)
A solution of (38) and trifluoroacetic anhydride was reacted and
purified in a similar manner as described for (36). Yield: 1.8 g (92%);
XH NMR 5 1.43 (s, 9H), 1.65 (m, 4H), 2.44 (m, 4H), 3.14 (m, 4H), 3.52
(s, 2H), 5.18 (br, 1H), 7.21 (s, 5H), 7.67 (br, IN); IR (neat) 3320 (m),
3000 (m), 1705 (s), 1620 (s), 1180 (s), 745 (m) cm'1.


PERCENT TRANSMISSION
V/AVtUNt.lM III MICRONS
3000
WMN tlOilCMC JMiiM CH*f NO
2000 I BOO
wavenumber cm1
1600 I 400 I 200
NiiMW njm-MiMij Inc rumion. (aihom*. u a
Figure 2-8. IR spectrum of N^-benzyl-N^ ,N-bis(acetyl)spermidine (1).
CJ
to
PERCENT TRANSMISSION


42
Anal. cal. for Cx3H31+N3C13: C, 46.09; H, 10.12; N, 12.40. Found:
C, 45.59; H, 9.65; N, 12.07.
N1 ,N8-Bis(benzyl)spermidine trihydrochloride (9j
A suspension of N1,N8-bis(benzoyl)spermidine hydrochloride was re
duced and purified as described for (_7). Yield: 1.5 g (89%); bp 183-185,
0.10 mm; NMR 6 1.52 (m, 9H), 2.56 (m, 8H), 3.44 (s, 4H), 7.22 (s, 1 OH);
IR (CHC13) 2970 (s), 1450 (m), 1130 (m), 760 (s) cm-1.
Anal. cal. for C21H35N3C13: c, 68.07; H, 9.25. Found: 0,67.82 ;
H, 9.18.
Synthesis of Secondary N-modified Spermidine Derivatives
General methods
The reagents N1,N8-bis(t-butoxycarbonyl)spermidine, N1,N7-bis(t-
butoxycarbonyl)norspermidine and N1,N9-bis(t-butoxycarbonyl)homospermi-
dine were prepared by reacting the appropriate benzylated spermidine with
2.1 equivalents of B0C-0N84 in tetrahydrofuran. The resulting benzyl -
bis(t-BOC) polyamines were then hydrogenated over palladium chloride
catalysts in MeOH/HCl to afford the bis(t-BOC) spermidine reagents as
the hydrochloride salts in 80-90% yields from the benzylated polyamine.85
Again, the preparation of N4-hexanoyl and N4-hexylspermidine are
illustrative as the same methodology is employed regardless of the bis-
(t-BOC protected) spermidine reagent or acylating agent used. A solution
of N1,N8-bis(t-butoxycarbonyl)spermidine-HC1 and 1.1 equivalents of hexan-
oyl chloride were allowed to react in the presence of base in dry di-
chloromethane to afford f^-hexanoyl-N1,N8-bis(t-butoxycarbonyl)spermi-
dine in 95% yield. Once again, the products of these reactions are
usually clean enough to be used without further purification. There
fore, N4-hexanoyl-N1,N8-bis(t-butoxycarbonyl)spermidine is reacted with
trifluoroacetic acid to afford N4-hexanoyl spermidine, quantitatively.


142
rise to a complex multiplet. Finally, the y-methyl should be split
principally by the single 8-methine proton resulting in a doublet with
an expected coupling in the order of 5.5-6.5 Hz, as long range a-y cou
pling is likely to be small.
Inspection of Figure 6-4b and 6-5b reveals that these predicted
coupling patterns do exist; however, they do exist in duplicate. For
example, the y-methyl group exists as a pair of doublets instead of a
single doublet, Figure 6-4b. These doublets are observed in a ratio
of approximately 2:1 in CDC13/DMS0-d6 and are both equally coupled to
the B-methine (J = 6.3 Hz). Likewise, the B-methine also exhibits an
additional set of signals. This is more clearly seen when the y-methyl
is decoupled, Figure 6-5b. Again, in the absence of any conformational
effects, irradiation of the y-methyl should result in the B-methine
collapsing to a doublet arising from the coupling with the a-methine
(J = 2.6 Hz). However, in agrobactin A irradiation of the y-methyl re
sults in a pair of doublets centered at 5 4.2, Figure 6-5b, also in
approximately a 2:1 ratio. Surprisingly enough, the a-methine, however,
does not show an additional set of lines, giving rise to the expected
doublet of doublets as one might anticipate i.e., split once by the
amide (J = 3.4 Hz) and once by the B-methine (J = 2.6 Hz).
The XH NMR data are consistent with the threonine substituent exist
ing in at least two distinct magnetic environments. Because the B-methine
and y-methyl protons are located further out on the threonyl side chain
i.e., closer to the terminal 2,3-dihydroxybenzoyl groups than the a-
methine, on rotation about the central amide bond they see more signifi
cant changes in magnetic environment. The a-methine, having a more
'internal' location, moves through a relatively smaller area upon


CHAPTER TWO
REAGENTS FOR THE SELECTIVE FUNCTIONALIZATION
OF SPERMIDINE, NORSPERMIDINE, AMD HOMOSPERMIDINE
In order to investigate the uptake properties of various N-acylated
and N-alkylated polyamines, one must consider the ability to selectively
and efficiently modify the polyamines' primary versus secondary amines
to produce the derivatives desired. Commercially available spermidine
itself fails in this respect as previous studies have shown there is
little difference in reactivity and hence, selectivity between its pri
mary and secondary amines.78 Reactions of spermidine with even the most
bulky reagents often lead to complex mixtures and poor yield of the de
sired product. Additionally, the symmetrical polyamines, nor- and homo
spermidine, are not commercially available. Therefore, current method
ology has focused on the preparation of protected spermidine reagents,
derived usually from compounds simpler than spermidine itself.
A review of the literature reveals there are currently several re
agents available for the selective acylation of spermidine. The first of
these, designed by Eugster et al., N4-tosyl-N8-phthaloylspermidine, is
designed to fix three different substituents to the spermidine backbone.79
The eight steps required for the synthesis of this reagent and the mode
of protective group removal make it somewhat impractical. A second re
agent, N4,N8-di-t-butoxycarbonyl spermidine (accessible in 49% yield in
three steps) is acceptable for what it is designed for--introduction of
an acyl group at the N^position.80 A more recent development employs
1 -(4-aminobutyl)hexahydropyrimidine and 1-(3-aminopropyl)hexahydropyri-
dine for the selective acylation of the terminal primary amino nitrogens
24


74
The toxicity of the polyamine derivatives was also investigated.
None of the polyamine derivatives tested were unusually toxic; however,
an interesting relationship was noticed. A comparison of LD50 values and
Ki values reveals that the derivative's toxicity is proportional to its
uptake. For instance the bis(acyl) derivatives, bis(acetyl)Spd and
DHBSpd have LD50 in excess of 1000 mg/Kg and are unable to compete for
uptake. On the other hand derivatives which are good inhibitors of 3H-
Spd uptake generally have LD50's less than 500 mg/Kg. Moreover, this
relationship even holds true for the BSpd derivatives; BhSpd, which is
the best inhibitor of the three, is also the most toxic. These data
seem to suggest the toxicity of these derivatives is related to their
cellular uptake.
In summary, it is apparent that the terminal amines of spermidine
are of critical importance for uptake. Modification of the terminal amines
by either acylation or addition of an alkyl substituent produces a deriva
tive which is ineffective in competing for uptake. In terms of substrate
recognition by the transport system, this suggests that the positive charges
carried by spermidine's terminal amines at physiological pH are necessary
for uptake. Furthermore, although the bis-alkyl derivatives also carry
this positive charge, recognition appears to be limited by the size of
the alkyl substituent. In this case, large alkyl groups may prevent in
teraction of the positively charged terminal amines with the carrier by
interacting sterically or by changing the polyamines' conformation.
The role of the secondary amine is less certain. Modification here
appears to be less restrictive, although preference was again seen for
the alkylated derivatives. The argument for requirement of positive charge
may again be true; however, recently it has been shown that diamines such


93
Figure 5-
XVII
NHCOCF3
Synthesis of the desired tris-protected reagent
(XVII) from intermediate (XVI).


25
of spermidine and homospermidine.81 This procedure represents an excel
lent protocol for functionalization of these two polyamines although it
cannot be extended to norspermidine and the conditions required for the
opening of the pyrimidine can be restrictive.
Recently, Bergeron and coworkers have described the preparation of
the benzylated polyamines N4-benzylspermidine, N4-benzylnorspermidine,
and N5-benzylhomospermidine as reagents for the selective acylation of
the primary amines.82 This synthesis offers the flexibility of generat
ing the varying chain lengths desired, as well as employing inexpensive
starting materials. The scheme employs benzyl amine as the common start
ing material for all three reagents, Figure 2-1. As illustrated, benzyl
amine is reacted with one equivalent of acrylonitrile, followed by alky
lation with 4-chlorobutyrylnitrile which, upon reduction, generates N4-
benzylspermidine (Vila). Alternatively, benzyl amine can either be react
ed with two equivalents of acrylonitrile or two equivalents of 4-chloro
butyrylnitrile, producing upon reduction the symmetrical reagents N5-
benzyl homo spermidine (VI lb) or N4-benzylnorsperr,iidine (Vile), respective
ly. These three reagents represent an excellent method of efficiently
generating the terminally bis-acylated spermidine desired as outlined
in Figure 2-2. The benzylated polyamines (VII) can be reacted with two
equivalents of the desired acylating agent, followed by removal of the
benzyl protecting group via hydrogenolysis over palladium chloride cata
lyst producing the desired derivatives (IX), figure 2-2.. In order to
determine if there are any size restrictions placed on uptake by the re
ceptor, in this study the acyl groups acetyl, propionyl and benzoyl were
affixed to the reagents (VII) via their acid chlorides.


spectroscopy is employed to evaluate the origins of the conformers in
agrobactin A. The coalescence temperatures and activation energies be
tween conformers of these polyamides are determined using NMR. The
role of steric and hydrogen bonding factors in these compounds is also
discussed.


148
the differences in anisotropy generated by the t-butoxycarbonyl and 2,3-
dihydroxybenzoyl groups. Additionally, the conformation of the threo
nine residue may be different between agrobactin A and (52). This is
supported by the observation that no coupling is seen between the a-
and 8-methines of (53) compared to the J = 2.6 Hz in agrobactin A
(Table 6-1), indicative of a difference in dihedral angle between the
a- and e-methines in agrobactin A and (53).
It was mentioned earlier that the chemical shifts of agrobactin A
and its related siderophores are extremely sensitive to the XH NMR
solvents employed. Similarly, the conformer population of agrobactin A
was found to be effected by solvent. As previously illustrated by agro
bactin A's y-methyl and B-methine signals in Figures 6-4b and 6-5b,
the conformers exist in approximately a 2 to 1 ratio in CDC13/DMS0-dg
(10:1). Upon switching to a more polar hydrogen bond acceptor solvent,
such as DMSO or DMF, the conformer population appears to even out, ap
proaching a 1:1 ratio as illustrated in Figure 6-7 for DMF. This sol
vent dependency suggests that hydrogen bonding may play a role in de
termining the conformer population. It is suspected that DMSO or DMF
is interacting intermolecularly with agrobactin A, reducing the intra
molecular interactions responsible for the conformer preference seen in
the less polar CDC13.
Determination of activation energies using NMR temperature coales
cence experiment
Since agrobactin A, agrobactin and N4-[N-t-butoxycarbonyl)threonyl]-
N1,N8-bis(t-butoxycarbonyl)spermidine (53) all exhibit duplicity in
their XH NMR spectra, the energy of activation (Ea) for the intercon
version between the conformers can be estimated by measuring the coal
escence temperature (Tc) of these compounds. A qualitative comparison


61
Sodium borohydride itself does not reduce amides; however, the addi
tion of one equivalent of organic acid produces a reducing agent now
capable of this.83 In fact, the order of reduction is now reversed
compared to most other reducing agents, i.e., tertiary>secondary>primary
amides. Additionally, no cleavage of tertiary amides to the aldehyde
and amine is noted, an important consideration for the reduction of
secondary N-acylated derivatives.
The synthesis of secondary N-acylated spermidine derivatives was
also easily accomplished, again using N4-benzylspermidine as the key
starting material.
In this case the benzylated polyamines (VII) were first reacted
with a transitory protecting group followed by debenzylation to produce
the reagents (XII), Figure 2-3. The t-butoxycarbonyl group served as
an excellent choice of protecting group for it is both quantitatively
put on and removed. Accordingly, N4-benzylspermidine was reacted with
a slight excess of BOC-ON, followed by hydrogenation, again employing
PdCl2 and MeOH/HCl to afford the bis-terminally protected reagents (XII),
in 90% yield from (VII) after recrystallization.75
Once generated, the bis-BOC spermidines were also capable of selec
tive acylation of spermidine as shown by the synthetic scheme in Figure
2-4. As with the benzylated reagents, acylations with bis-BOC-spermidine
(XII) likewise proceed smoothly to afford the N4-acyl N1,N8-bis(t-butoxy-
carbonyl)spermidine cleanly and in yields in excess of 85%. The desired
N4-acylspermidines (XIV) were then generated by brief exposure to tri-
fluoroacetic acid. These N4-acyl derivatives are also efficiently reduced
to the N-alkyl compounds (XV) using NaBH4-CF3C02H. Although this reduc
tion is a reduction of a tertiary amide, it proceeds smoothly and with
no apparent cleavage of the amide to a secondary amine and aldehyde.


128
added DCC (1.89 g, 9.16 mmol) in dioxane (20 ml) under N2. After 16 hours
the mixture was filtered and the DCC washed with dioxane (15 ml). The
solvent was evaporated in vacuo and the residue crystallized from Me0H/H2
to yield 1.8 g (95%) of product as tan crystals, mp 55-56C; XH NMR (d6-
DMSO) 6 2.93 (4H), 6.52-7.36 (3H), 9.82 (2H).
Anal. cal. for CnHgNOg: C, 52.60; H, 3.61; N, 5.58. Found: C, 52.68;
H, 3.66; N, 5.52.
N4[N-(t-Butoxycarbony1 )threony!]N1,N8-bis(t-butoxycarbonyl)spermidine 53
A solution of DCC (680 mg, 3.3 mmol) in 20 ml THF was slowly added
to a solution of 53^ (660 mg, 3.0 mmol) and N-hydroxysuccimide (380 mg,
3.3 mmol) in 30 ml THF. After 18 hours, the DCU precipitates were fil
tered, washed with fresh THF, and the filtrate evaporated. The residue
was dissolved in 30 ml CH3CN and slowly added to a solution of (13)
(1.26 g, 3.3 mmol) and Et3N (470 yl, 3.5 mmol) in 5% aqueous CH3CN
(50 ml). After stirring for 36 hours at room temperature, the reaction
mixture was then concentrated, the residue taken up in 100 ml EtOAC,
washed with H20 (2 x 20 ml), 3% HC1 (3 x 20 ml), H20 (2 x 20 ml), dried,
and concentrated to afford 1.5 g (91%) of the desired product.
An analytical sample was obtained by chromatography on silica gel
eluting with EtOAc/CHCl3 (1:1): *H NMR (CDC13) 6 1.20 (d, 3H), 1.44 (s,
27H), 1.75 (m, 6H), 2.80-3.58 (overlapping multiplets, 8H), 4.04 (m, 1H),
4.38 (d, 1H), 4.8-5.5 (br, 4H); IR (CDC13) 3000 (m), 1715 (s), 1180 (s),
770 (s) cm-1.
Anal. cal. for C26H50^48: C, 57.12; H, 9.22; N, 10.45. Found:
C, 57.25; H, 9.24; N, 10.17.


UNIVERSITY OF FLORIDA
3 1262 08554 7882


87
ester versus acid chloride) and the bulkier size of the antineoplastic
being condensed. However, it illustrates that a wide variety of acylat-
ing agents of varying size can be coupled to the reagent (13) in good
yields.
The antineoplastic-spermidine conjugates were then tested for their
ability to compete with 3H-Spd uptake and their cytotoxic effects on
LI210 cells in vitro. The results of these preliminary investigations are
given in Table 4-1. Both antineoplastic-spd conjugates inhibited 3H-Spd
uptake; however, the N4-chlorambuci1 conjugate was much more effective
(Ki = 6 pm). Furthermore, N4-chlorambuci1-Spd was found to be cytotoxic
to LI210 cells, with an ID50 of 15 pm. Chlorambucil itself was equally
cytotoxic, thereby showing no preference for the Spd conjugate. In view
of the fact that chlorambucil alone does not compete for Spd uptake, this
suggests that it may be entering the cell via an alternate mechanism, per
haps by an amino acid specific carrier.94
On the other hand, the N4-GIMPY-Spd conjugate was found to be rela
tively noncytotoxic, having an ID50 of 350 pM. When this value is, how
ever, compared to the ID50 of its precursor (30), a positive effect of
the conjugation of the spermidine backbone can be inferred. Assuming that
the mode of action between N4GIMPY-Spd (32) and GIMPY (30) is the same,
the increased cytotoxicity of (32) can be explained by the improved uptake
of the conjugate over (30). It should be pointed out that the cytotoxicity
of the GIMPY-Spd conjugates is far below what is considered sufficient
for antineoplastic activity. The mode of action of IMPY is by chelation
of the iron in the ribonucleotide reductase, rendering it inactive. It is
a distinct possibility that the amine which is reacted with glutaric anhy
dride may be involved in chelation. Functionalization of it to an amide


34
N1,N8-Bis(acetyl)spermidine hydrochloride (4)
Palladium chloride (100 mg) was added to a solution of (1_) (1.15 g,
3.6 mmol) in methanol (50 ml) containing concentrated HC1 (seven drops).
The resulting suspension was stirred under a hydrogen atmosphere over
night. The catalysts were then filtered, washed with methanol, and the
filtrates evaporated. The resulting crude solid was recrystallized from
ethanol/ether, the solid collected by filtration, and dried under high
vacuum over P205 to afford 550 mg of the desired product as a white solid;
mp 133C. Concentration of the mother liquor and recrystallization af
forded another 330 mg. Total yield: 880 mg (92%); XH NMR (D20) 6 1.70
(m, 6H), 2.04 (s, 6H), 3.20 (m, 8H); IR (KBr) 3300 (br), 1625 (s), 1530
(m) cm"1.
Anal. cal. for 1H24N302C1: C, 49.71; H, 9.10; N, 15.81. Found:
C, 49.45; H, 9.15; N, 15.48.
N1,N8-Bis(propiony1 )spermidine hydrochloride (5.)
A solution of l^-benzyl-N1,N8-bis(propionyl)spermidine (2) was hy
drogenated and purified in the same manner as described for (4_). Yield:
780 mg (86%); mp 147-148C; XH NMR (D20) 6 1.10 (t, 6H), 1.72 (m, 6H),
2.35 (quar., 4H), 3.16 (m, 8H); IR (KBr) 3350 (br), 1630 (s), 1540 (m)
cm"1.
Anal. cal. for C13H28N302C1: C, 53.14; H, 9.60; N, 14.30. Found:
C, 52.81; H, 9.50; N, 13.86.
N1 ,N8-Bis(benzoyl )spermidine hydrochloride (jj)
A solution of N4-benzyl-N1,N8-bis(benzoyl)spermidine (3) was hydro
genated and purified in the same manner as described for (4). Yield:
2.9 g (93%); XH (D20) 6 1.74 (m, 6H), 3.20 (m, 8H), 7.06-7.82 (m, 10H);
IR (KBr) 3300 (s), 1635 (s), 1540 (s), 690 (m) cm"1.


122
atmosphere in the presence of Raney nickel and NaOH to produce amine (35)
in 95% yield. Again, the reaction proceeds very cleanly, and the amine
(35) in many instances can be used without further purification. Final
ly, the desired tris-protected spermidine reagent (36) is prepared by
the high yield acylation of (35) with trifluoroacetic anhydride.
Thus, N4-benzyl-N1-t-B0C-N8-trifluoroacetylspermidine was conven
iently prepared in four high yield steps from N-(3-ami nopropyl)benzyl -
amine in an overall yield of 75%. Additionally, recent experiments sug
gest that the overall conversion of (33) -* (36) can be performed without
purification of the intermediates instead of only purifying the final
product (36). Also, it should be mentioned that the conversion of amine
(35) to (36) with trifluoroacetic anhydride, is in itself not necessary
if one wishes to functionalize the terminal amine of (35) with some
other desired group.
Synthesis of Tris-protected Homologues (39) and (44)
Once the feasibility of the target reagent (36) had been demon
strated, the synthesis of its symmetrical nor- and homospermidine homo
logues was required. The synthesis of the norspermidine derivative
could be effected by the addition of a three-carbon chain to inter
mediate (33). The typical means of accomplishing this is by the Michael
addition of acrylonitrile to amine (33). When (33) was reacted with a
slight excess of acrylonitrile at room temperature, little or no addi
tion occurred. However, when the reactants were placed in a sealed
vessel and heated to 100C, the reaction proceeded cleanly to afford
nitrile (37) in 98% yield. The desired norspermidine reagent (39) was
subsequently generated by reduction of (37), followed by trifluoroacety-
lation as for the spermidine homologue (36).


I


^LJ
>....1 nntnnn .11.-i i ...- i...1.--I t.i l i i l i i i > i i~t 1
10
4 0
50
MO
3 0
i.O
1.0
Figure 5-15. 60 MHZ NMR spectrum of N8-acety1-N^-benzoylspermidine (49 ).


166
19. Tomlinson, R.V., Ringold, H.J., Qureshi, M.C. and Forchielli, A.
(1972) Biochem. Biophys. Res. Comm. 46_, 552.
20. Ku, E.C., Wasvary, J.M. and Cash, W.D. (1975) Biochem. Pharmac. 24_,
641 (1975).
21. Hannonen, P., Janne, J. and Raina, A. (1972) Biochim. Biophys.
Acta 289, 225.
22. Siimes, M. and Janne, J. Acta Chem. Scand. 21_, 815 (1967).
23. Bachrach, V., Bekiarkunst, A. and Abzug, S. Isr. (1967) J. Med.
Sci. 3, 474.
24. Williams-Ashman, H.G., Coppoc, G.L. and Weber, G. (1972) Cancer
Res. 32, 1924.
25. Williams-Ashman, H.G., Coppoc, G.L., Schenone, A. and Weber, G.(1973)
in Polyamines in Normal and Neoplastic Growth (Russell, D.H., ed.)
pp. 181-197, Raven Press Publishers, New York
26. Niesh, W.J.P. and Key, L. (1967) Int. J. Cancer 2_, 69.
27. Russell, D.H. and Levy, C.C. (1971) Cancer Res. 31_, 248.
28. Russell, D.H., Levy, C.C., Schimpff, S.C. and Hawk, I.A. (1971).
Cancer Res. 31_, 1555.
29. Bartos, D., Campbell, R.A., Bartos, F. and Grettie, D.P. (1975)
Cancer Res. 35_, 2056.
30. Russell, D.H. and Durie, B.E.M. (1978) Polyamines as Biochemical Markers
of Normal and Malignant Growth. Raven Press, New York
31. Raina, A., Jnne, J. and Siimes, M. (1963) Acta Chem. Scand. 1_8,
1084.
32. Johansson, J.G., Alto, P. and Skinner, W.A. (1973) U.S.A. Patent
3754207.
33. Harik, S.I. and Snyder, S.H. (1973) Biochim. Biophys. Acta 327,
501.
34. Harik, S.I., Hollenberg, M.D. and Snyder, S.H. (1974) Mature 249,
250.
35. Inove, H., Kato, Y., Takigawa, M., Adachi, K. and Takeda, Y. (1975)
J. Biochem. 77, 879.
36. Kato, Y., Inove, H., Godha, E., Tamada, F. and Takeda, Y. (1976)
Gann 67, 569.


Figure 5-12. 60 MHz 1H NMR spectrum of N4-benzoyl-N1-t-butoxycarbonyl-N8-trif1uoro-
acetylspermidine (46).


CHAPTER ONE
INTRODUCTION AND BACKGROUND
Although the existence of polyamines in human semen has been known
since 1677,1 the majority of research regarding the polyamines' biosyn
thesis and physiological functions has occurred only in the last twenty
years. These naturally occurring polyamines are, in general, linear di-
and triamines: the five most commonly associated with eukaryotes are pre
sented in Figure 1-1. Of these, the amines 1,4-diaminobutane (putres-
cine), spermidine, and spermine are the most important and most commonly
occurring polyamines in mammalian tissues, although others have been
shown to occur in trace quantities as well.
The biosynthesis of putrescine, spermidine, and spermine proceeds
through a common pathway in mammals as well as microorganisms, the pri
mary precursors being the amino acids L-ornithine and L-methionine. As
shown in Figure 1-2, L-ornithine is decarboxylated to form putrescine,
and the ATP activated form of methionine, S-adenosyl-L-methionine, serves
as the propylamine donor, the moiety added to putrescine to form the
higher polyamines spermidine and spermine. Four enzymes are primarily
responsible for the synthesis of the polyamines in mammalian tissues
two decarboxylases, and two synthetases. The decarboxylases, L-ornithine
decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAM-DC),
have been extensively purified and studied. Ornithine decarboxylase,
which catalyzes the conversion of ornithine to putrescine, is a pyridoxal-
requiring enzyme, like most other mammalian decarboxylases. However,
1


60
anti neoplastic does indeed improve the uptake of the drug, this will be
demonstrated by a lowering of the drug's IO50
Experimental
Materials
Chlorambucil was purchased from Sigma Chemical Company, St. Louis,
MO. Samples of IMPY were generously donated by Dr. Leonard Kedda, Na
tional Cancer Institute, Bethesda, MD. All other reagents were purchased
from Aldrich Chemical Company, Metuchen, NJ and, except as noted, used
without further purification. Physical measurements were performed as
detailed in Chapter Two.
N4-Chlorambucil-N1,N8-bis(t-butoxycarbonyl)spermidine (28)
A solution of DCC (145 mg, 0.70 mmol) in 10 ml THF was added to a
cooled solution of chlorambucil (185 mg, 0.61 mmol) and N-hydroxysuc-
cinimide (80 mg, 0.70 mmol) in 20 ml THF. The ice bath was removed and
the solution allowed to stir for 18 hours during which time a thick
white precipitate ensued.
The reaction mixture was then concentrated, 20 ml CH^Cl2 added, the
DCU precipitates filtered and washed with an additional 10 ml of CH2CI2.
The filtrates were combined, cooled, and a solution of NL,N8-bis(t-
butoxycarbonyl)spermidine HC1 (13J (265 mg, 0.65 mmol) and triethyl amine
(90 yl, 0.65 mmol) in 10 ml CH2C12 slowly added. The resulting reac
tion was allowed to stir at room temperature for 48 hours, at which
time additional (20 ml) CH2C12 was added and the organic layer washed
with 3% HC1 (2 x 10 ml) and H20 (2 x 10 ml). The CH2C12 layer was
dried and concentrated to afford 340 mg crude product. Further purifi
cation was effected by chromatogrpahy on alumina (neutral, activity I)
eluting with CHC13 to afford 280 mg (73%) product: NMR 1.34-1.96


131
(350 mg, 0.60 mmol) and Et3N (90 yl, 0.65 mmol) in 5% aqueous CH3CN
(30 ml). After 48 hours the solvent was evaporated, and the product
chromatographed on silica gel eluting with 10% MeOH/CHCl3 to yield 400
mg (91%) as a white hygroscopic solid: NMR (CDC13) 5 1.16 (d, 3H),
1.58 (m, 6H), 3.42 (m, 8H), 3.56 (s, 18H), 4.0-4.6 (m, 3H), 6.62-7.7
(m, 9H), 7.74-8.4 (m, 3H).
Anal. cal. for C38H50N4On: C, 61.78; H, 6.82; N, 7.58. Found:
C, 61.84; H, 6.71; N, 7.47.
N4-[N-(Benzoyl)threonylj-N1,N8-bis(benzoyl)spermidine (60)
A solution of the succinimide ester of benzoic acid, prepared by
reacting DCC, N-hydroxysuccinimide and benzoic acid, was reacted with
(13) in a similar manner as described for (59). Yield: 225 mg (93%);
XH NMR 5 1.18 (d, 3H), 1.61 (m, 6H), 3.39 (m, 8H), 4.08-4.66 (br, 3H),
7.23 (s, 15H), 7.98 (m, 3H).
Anal. cal. for C32H38N405: C, 68.80; H, 6.86; N, 10.02. Found:
C, 68.57; H, 6.91; N, 9.84.
Agrobactin A (via scheme II)
A mixture of (D,L)-M4-threonyl-N1,N8-bis(2,3-dihydroxybenzoyl)sper-
midine-HBr73 (100 mg, 0.17 mmol) and triethylamine (24 yl, 0.17 mmol)
in dry DMF (20 ml) was cooled to 0C under N2. A solution of N-hydroxy-
succinimido-2,3-dihydroxybenzoate [52) (43 mg, 0.07 mmol) in DMF (20 ml)
was added dropwise over 15 min. After 12 hours, the solvent was removed
in vacuo and the residue chromatographed on LH-20 (10-^30% EtOH/benzene)
to afford 82 mg (75%) of product as a white solid. The TLC and 300 MHz
!H NMR spectral characteristics of this product were identical to that
of agrobactin A in Scheme 1.


Figure 6-7, 300 MHz ^ H NMR spectra of agrobactin A in DMF-d7
a) Y-methyl decoupled 8-methine region; b) y- '
methyl region.


94

XVI la
Figure 5-3. Entire synthesis of tris-protected spermidine
reagent (XVI la) and norspermidine analogue(XVIIb)


This dissertation was submitted
of Pharmacy and to the Graduate
fulfillment of the requirements
December 1983
to the Graduate Faculty of the College
School, and was accepted as partial
for/the degree of Doctor of Philosophy.
/ CX.
Dean, College of Pharmacy
Dean for Graduate Studies and Research


20
however, agrobactin contains a third 2,3-dihydroxybenzoyl moiety in place
of parabactin's 2-hydroxybenzoyl moiety. Likewise, agrobactin (Ilia)
also exists in a series of conformers, and on activation energy for in
terconversion between conformers has been determined.72 However, simi
lar information regarding agrobactin A is not available. Furthermore, no
information exists concerning the nature of the conformers observed in
these siderophores in general, i.e., the contribution of steric and hydro
gen bonding factors towards the activation energy controlling conformer
interconversion. Accordingly, synthesis and high field ^-NMR studies
of agrobactin A were undertaken.
In considering a synthesis of agrobactin A, the ability to generate
symmetrical homologues and other derivatives must be kept in mind.
Agrobactin A has been recently synthesized by Neilands by reacting sper
midine with the bulky reagent 2,3-dibenzoyloxybenzoyl chloride, followed
by attachment of the N-(2,3-dibenzyloxybenzoyl)threonyl "centerpiece"
and removal of the benzoyloxy protection groups.72 However, the yields
reported for the condensation steps and the limitations on the deriva
tives that can be generated make this synthesis somewhat undesirable.
If, on the other hand, a protected spermidine reagent could be
75
employed, such as N1,N8-bis(t-butoxycarbonyl)spermidine (IV), an effi
cient synthesis of agrobactin A can be envisioned as illustrated in
Figure 1-7. The reagent (IV) was easily condensed with N-(t-butoxycar-
bonyl)threonine using the coupling reagents dicyclohexylcarbodiimide
and N-hydroxysuccinimide. The resulting product N4-[N-(t-butoxycarbonyl)-
threonylj-N1,N8-bis(t-butoxycarbonyl)spermidine was then quantitatively
deprotected by brief exposure to trifluoroacetic acid generating N4-
threonylspermidine (VI). Agrobactin A could be subsequently synthesized


70
Table 3-3. Effects of Secondary
on 3H-Spd Uptake into
Cells
Modified Spd Derivatives
Ascites L1210 Leukemia
Competing Agent
3H-Spd Uptake*
pmol/107 cells/
Ki (uM)**
(lOOuM)
min
%control
None
65
100
-
N4-AcetylSpd
53
81
115
N4-Hexanoy1Spd
57
88
151
4
N -BenzoylSpd
60
92
500
N4-MeSpd
11
1 7
3.4
N4-EthylSpd
9
14
3.1
N4-HexylSpd
45
69
34
N4-BnSpd
57
88
1 35
N4-BS pd
44
67
39
N5-BhSpd
24
37
14
* Cells were incubated for 20 min at 37 with lOuM ^H-Spd
and lOOuM derivative
**Cells were incubated for 20 min at 37 with 0.2, 0.5,
1.0, 5.0, or lOuM 3H-Spd and 10 or lOOuM derivative


72
Table 3-4. Acute Toxicities of Spermidine and Polyamine
Derivatives
Compound
LD50 (mg/kg)
Spd
400
DHBSpd
>800
Bis(Acetyl )Spd
>1000
Bis(Ethyl )Spd
425
N4-MeSpd
375
N4-BnSpd
250
N4-BSpd
200
N5'BhSpd
125
N4- Benzoyl Spd
300


130
Ns-[N-t-Butoxycarbony1)threonylj-N1,N9-bis(t-butoxycarbonyl)homosper-
midine (56)
The active ester of (51_) was reacted with a solution of (1_5) and
purified as described for (53): 89% yield; XH NMR (CDC13) 6 1.18 (d,
3H), 1.48 (s, 27H), 1.40-1.71 (m, 8h), 3.02-3.56 (m, 8H), 4.03 (m, 1H),
4.36 (d, 1H), 4.72-5.48 (br, 4H); IR (CHC13) 3010 (m), 1715 (s), 1175
(s), 770 (s) cm-1.
Anal. cal. for C27H52N408: C, 57.83; H, 9.35; N, 9.99. Found:
C, 57.56; H, 9.41; N, 9.92.
N5-Threonyl homospermidine (57)
A solution of (56) was deprotected and purified as described for
(54): 98% yield; lH NMR (DMS0-d6) 1.18 (d, 3H), 1.36-1.99 (m, 8H), 2.82-
3.37 (m, 8H), 4.54 (m, 2H), 7.97 (m, 9H).
Anal. cal. for C18H31FgN408: C, 35.89; H, 5.19; N, 9.30. Found:
C, 35.54; H, 5.27; N, 9.14.
N5-[N-(2,3-Dihydroxybenzoyl)threony!]-N|,N^-bis(2,3-dihydroxybenzoy1)-
homospermidine (Homoagrobactin A) (58)
A solution of (58) was prepared and purified as described for (55);
72%yield; NMR (See Table 1).
Anal. cal. for 0338^40!!: C, 59.27; H, 6.03; N, 9.57. Found:
C, 58.91; H, 6.19; N, 9.37.
N4-[N-(2,3-Dimethoxybenzoyl )threonyl1-N1,N8-bis(2,3-dimethoxybenzoyl)-
spermidine (Hexamethyl agrobactin A) (j)
A solution of DCC (450 mg, 2.2 mmol) in THF (15 ml) was slowly
added to a solution of 2,3-dimethoxybenzoic acid (400 mg, 2.0 mmol) and
N-hydroxysuccimide (255 mg, 2.2 mmol) in THF (30 ml). The reaction was
allowed to stir at room temperature for 18 hours at which time the DCU
precipitates were filtered off and washed with THF. The filtrate was
then concentrated to approximately 20 ml and added to a solution of (54)


HNCH_CHCHNHCHCHCHCHNH
2 222 22222
h2nch2ch2ch2ch2nh2
I ^-diami nobutane
(Putrescine)
h2nch2ch2ch2ch2ch2nh2
I,5~d¡aminopentane
(Cadaver¡ne)
Sperm¡dine
H NCH CH CH NHCH CH CH NH
2 222 2222
Norspermidine
h2nch2ch2ch2ch2nhch2ch2ch2ch2nh2
Homosperm idine
H NCH CHCH NHCH CHCHCH NHCH CH CH NH
2 222 2222 2222
Spermine
Figure 1-1. Commonly occurring polyamines associated with eukaryotic cells


TABLE OF CONTENTS
PAGE
DEDICATION 11
ACKNOWLEDGEMENTS iii
ABSTRACT vi
CHAPTER
ONE INTRODUCTION AND BACKGROUND 1
Polyamines and Growth 4
Structural Requirements for Polyamine Uptake 13
Spermidine Derived Siderophore Systems 16
TWO REAGENTS FOR THE SELECTIVE FUNCTIONALIZATION OF
SPERMIDINE, NORSPERMIDINE, AND HOMOSPERMIDINE 24
Experimental 31
Results and Discussion 49
THREE BIOLOGICAL EVALUATION OF POLYAMINE DERIVATIVES 64
Materials and Methods 64
Results 67
Discussion 71
FOUR PRELIMINARY INVESTIGATIONS TOWARDS THE DEVELOPMENT
OF SPERMIDINE-ANTINEOPLASTIC CONJUGATES 76
Experimental 80
Results and Discussion 86
FIVE SYNTHESIS OF TRIS-PROTECTED SPERMIDINES 90
Synthesis 92
Experimental 97
Results and Discussion 121
SIX SYNTHESIS AND SOLUTION DYNAMICS OF AGROBACTIN A 127
Experimental Section 127
Results and Discussion 132


Figure 4-6. 60 MHz ]H NMR spectrum of N4-[4-(2,3-dihydro-1H-i mi dazo[1,2b]pyra
zo I o )carboxamido)butyry1]spermidine trif1uoroacetate (32).
00
t_n


7 <: S 4 3 2 1
Figure 4-5. 300 MHz NMR spectrum of N^-[4-(2,3-dihydro-lH-imidazo[l,2b]-
pyrazolo)carboximido)butyryl]-Nl-N8-bis(t-butoxycarbonyl )-
spermidine (31 ) .


170
98. Curran, W.V. and Angier, R.B. (1966) J. Org. Chem. 31_, 3867.
99. Bergeron, R.B., McGovern, K.A., Channing, M.A. and Burton, P.S.
(1980) J. Org. Chem. 45, 1589.
100. Bergeron, R.B., Stolowich, N.J. and Garlich, J.R., manuscript
in preparation.
101. Elliot, D.F. (1948) Nature 162, 657.
102. Pfister, K., III, Robinson, C.A., Shabica, A.C. and Tishler, M.J.
(1949) J. Amer. Chem. Soc. 71_, 1101.
103. Bergeron, R.B., Burton, P.S., Kline, S.J. and McGovern, K.A. (1981)
J. Org. Chem. 46, 3712.
104. Eng-Wilmot, D.L. and van der Helm, D.J. (1980) J. Amer. Chem. Soc.
102, 7719.


144
Figure 6-5. 300 MHz NMR spectra of the a- and B-methine~
region of a) Homoargrobactin A; b) agrobactin A;
c) 5_3 ; d) 56 in the solvents indicated in figure
6-4. (y-metTvyls decoupled to simplify spectra)


65
giving mice bearing LI210 cells 2% DFMO by drinking water for 40-48
hours prior to removal of cells.
Cell Culture
Murine L1210 leukemia cells were maintained in logarithmic growth
as a suspension culture in Roswell Park Memorial Institute Medium 1640
(RPMI-1640) containing 2% HEPES-MOPS and 10% fetal calf serum. Cells
were grown in glass tubes in a total volume of 2 ml under a humidified
5% C02 atmosphere at 37. Cultures were treated while in logarithmic
growth (0.5 to 1 x 105 cells/ml) with the polyamines, Spd derivatives,
or MGBG at concentrations ranging from 106 to 10"2 M. After 24 or 48
hours, cells were removed from tubes for counting and viability deter
minations. Cell number was determined by electronic particle counting
(Model ZF Coulter counter; Coulter Electronics, Hialeah, FL) and con
firmed periodically with hemocytometer estimates. Cell viability was
assessed by trypan blue dye exclusion (0.5% in unbuffered 0.9% NaCl
solution). Percent control growth was determined as the final treated
cell number minus the initial inoculum (_5 x 104 cell s/ml) divided by
the final untreated cell number minus the initial inoculum, times 100.
Growth data were further analyzed with a Hewlett Packard HP-85 micro
computer programmed to determine percent growth inhibition and 50% growth
inhibitory doses (ID50) at 24 and 48 hours.
Uptake Determinations
The Spd derivatives and MGBG were studied for their ability to com
pete with 3H-Spd for uptake into ascites LI210 cells in vitro. These
determinations were performed by Dr. Carl Porter's group at Roswell Park
Memorial Institute using the following procedures. Prewarmed LI210 cell
suspensions (5 x 106/cc) were incubated in 1 ml of RPMI-1640 containing


163
Secondary N-modified polyamines, however, were found to be less
restrictive than their terminally N-modified counterparts in competing
for uptake. Although the N4-alkylated spermidines were the best inhibitors
of spermidine uptake, the N4-acyl derivatives were also found to be capable
to competing for uptake.
The structural requirements for polyamine uptake as defined by these
data, therefore, indicated that the free primary amines of spermidine are
critical in inferring uptake specificity. On the other hand those poly
amine derivatives modified at the secondary amine were less limiting upon
uptake, suggesting only a minor role of the secondary amine in recognition
by the receptor for uptake. Based on these findings, two anti neoplastic
spermidine conjugates, N4-chlorambucil spermidine and N4-[4-(2,3-dihydro-
lH-imidazo]l,2-b]pyrazolo)carboxamido)butyryl]spermidine were synthesized.
Preliminary in vitro findings indicate a preference for these conjugates
over the native antineoplastic for uptake, and tin's specificity may be
responsible for the increased cytotoxicity seen in the N4-IMPY spermidine
conjugate over the glutaryl IMPY precursor. Those studies, although in
the early stages, suggest the feasibility of utilizing spermidine as a
carrier to target the delivery of anti neoplasties to tumor cells.
Of the polaymine derivatives tested for acute toxicity, an interest
ing relationship between the compounds LD50 and cellular uptake was
noticed. The terminally N-modified derivatives, which were the least
effective in competing for uptake, were also the least toxic, having
LD50S in excess of 800 mg/Kg. Likewise, the best inhibitors of spermidine
uptake, the N4-substituted derivatives, were also the more toxic of the
group, suggesting a link between uptake and toxicity.


Figure 5-14.
60 MHz 1H NMR spectrum of N^-acetyl-N^-benzoyl-N^-t-butoxycarbonyl
spermidine (48).


16
the presence of both the derivative and 3H-spermidine. Spermidine deriva
tives which successfully compete with spermidine for the carrier will
accordingly reduce the amount of radiolabel taken up by the cells.
The uptake characteristics of these derivatives suggested that only
a minor structural significance is placed on the secondary amine of sper
midine as compared to its primary amines. For example, bis-acylated com
pounds such as N1,N8-bis(2,3-dihydroxybenzoyl)spermidine and N1,N8-bis-
(t-butoxycarbonyl)spermidine are unable to compete with 3H-spermidine
for uptake. However, N4-benzylspermidine is as good as MGBG in inhibit
ing spermidine uptake.42
These findings suggested the possibility of conjugating several
small anti neoplasties to spermidine via its secondary nitrogen. Since
tumor cells apparently concentrate spermidine, this may serve as a novel
method to target the delivery of anti neoplasties to the tumor. There
fore, a preliminary investigation into this area was initiated with the
synthesis of two spermidine antineoplastic conjugates: N4-chlorambucil
spermidine and N4-[4-(2,3-dihydro-lH-imidazo]l,2-b]pyrazolo)carboxamido)-
butyryljspermidine (Figure 1-5). Preliminary in vitro findings indicate
that even the attachment of N4-substituents of this magnitude does not
prohibit the competitive uptake of the conjugate.
Spermidine Derived Siderophore Systems
The second part of this dissertation deals with the synthesis and
solution dynamics of agrobactin A (la, Figure 1-7), a naturally occurring
spermidine-derived iron chelator. There has been considerable interest
in this family of siderophores since the isolation and identification of
two other siderophores: N1,N8-bis(2,3-dihydroxybenzoyl) spermidine (II)
and N4-[N-(2-hydroxybenzoyl)threonylj-N1,N8-bis(2,3-dihydroxybenzoyl)


Figure 6-8. Effects of Temperature upon y-methyl region of agrobactin A in DMSO-d
(e-methine decoupled).


I
xxv > XXVI
Y
\jD
cr>
Figure 5-4. Synthesis of tris-protected homospermidine (XXVI).


-in
10
40
5.0
M.O
3 0
.0
1.0
Figure 4-4. 60 MHz NMR spectrum of N^-Chlorambuci1spermidine trifluoro-
acetate (29 ).
oo
CO


Figure 2-9. IR spectrum of N1,N8-bis(acetyl)spermidine (4).
-e*
o
PKCENT TRANSMISSION


102
was then cooled to room temperature, then to 0C, and the excess BH3
carefully destroyed by adding 6 N NH^Cl (15 ml). The THF was evaporated,
and enough solid NaOH added to the remaining aqueous solution to make
basic. The product was extracted into ether (3 x 25 ml), dried and con
centrated to afford 1.5 g crude product. Further purification was ef
fected first by distillation followed by preparative TIC to afford 850
mg (47%) of the desired product: XH NMR 5 1.41 (m, 14H), 2.58 (m, 2H),
3.06 (m, 4H), 3.70 (s, 2H), 4.85 (br, 1H), 7.23 (s, 5H); IR (neat) 2975
(s), 1710 (s), 1520 (s), 1180 (s), 750 (m) err1.
Anal. cal. for C16H26N202: C, 69.03; H, 9.41; N, 10.06. Found:
C, 69.13; H, 9.42; N,10.01.
N-[N-(t-Butoxycarbonyl)-4-ami nobutyl]-N-3-cyanopropylbenzyl amine (42.)
A solution of (£]_) was reacted with 4-chlorobutyrylnitrile and puri
fied in a similar manner as described for (34). Yield: 3.9 g (96%);
XH NMR 6 1.43 (m, 15H), 2.47 (m, 6H), 3.10 (m, 2H), 3.47 (s, 2H), 4.63
(br, 1H), 7.17 (s, 5H); IR (neat) 2990 (s), 2240 (w), 1700 (s), 1520
(s), 1190 (s), 750 (m) cm"1.
Anal. cal. for C20H31N302: C, 69.53; H, 9.04; N, 12.16. Found:
N5-Benzyl-N1-t-butox,ycarbony1 homospermidine (41)
A solution of (42_) was reduced and purified as described for (35).
Yield: 9.8 g (91%); :H NMR 6 1.50 (m, 19H); 2.43 (m, 6H), 3.02 (m, 2H),
3.50 (s, 2H), 4.74 (br, 1H), 7.18 (s, 5H); IR (neat) 2970 (s), 1700 (s),
1515 (m), 1170 (s), 740 (w) cm"1.
Anal. cal. for C20H35N302-H20: C, 65.36; H, 10.14; N, 11.43.
Found: C, 65.44; H, 10.13; N, 11.43.


PERCENT TRANSMISSION
Figure 2-16. IR spectrum of N4-hexylspermidine (23).
percent transmission


62
Synthesis of N4-Methylspermidine
As with the synthesis of other N4-alkylspermidines, the synthesis
of N4-methylspermidine was envisioned as three steps: reaction of N^N8-
bis(t-BOC)spermidine with an appropriate acylating agent, removal of the
t-BOC protecting groups, followed by reduction of the amide. In the
corresponding retrograde synthesis, N4-methylspermidine could be pre
pared by the reduction of N4-formylspermidine which, in turn, could be
generated by deprotection of N1,N8-bis(t-BOC)N4-formylspermidine. Indeed,
this last compound was easily synthesized by reacting N1 ,N8-bis(t-B0C)-
spermidine with acetic formic acid anhydride. However, its subsequent
deprotection with trifluoroacetic acid always led to inseparable mixtures
of the desired N4-formylspermidine and spermidine itself. This observa
tion was not totally unexpected as the formyl group is commonly employed
as a N-protecting group which is removable by acid conditions. Even when
the reaction time was limited to two minutes at 0C, substantial loss of
the formyl group still resulted.
Alternatively, other methods were available for methylation of
amines. Direct alkylation of N1,N8-bis(t-B0C)spermidine with methyl
iodide resulted in mixtures of unreacted starting material, product,
and overalkylation to the quaternary salt. However, reductive alkyla
tion with formaldehyde and sodium cyanoborohydride87 led to the formation
of N1,N8-bis(t-BOC)-N4-methylspermidine in 70% yield after chromatography.
The desired N4-methylspermidine was subsequently obtained by the usual
deprotection with trifluoroacetic acid.
In summary, the reagents N4-benzylspermidine, N1,N8-bis(t-butoxy-
carbonyl)spermidine and their symmetrical homologues were synthesized
and used to prepare selectively N-modified spermidine derivatives. The


Figure 2-11. 60 MHz NMR spectrum of M-bis(t-butoxycarbonyl )spermidine
hydrochloride(13).


66
n HEPES-MOPS and 0.2, 0.5, 1.0, 2.0, 5.0 or 10 pM 3H-Spd (New England
Nuclear Corp., Boston, MA) alone or in the presence of 10 or 100 pM
polyamine, Spd derivative or MGBG. The cells were incubated for 20 min
at 37 except for one tube containing 10 pM 3H-Spd which was not pre
warmed and was incubated at 0 to measure surface binding. At the end
of the incubation the tubes were centrifuged at 900 g for five min at
0-4. A 200 pi aliquot of supernatant was removed for scintillation
counting and the remainder of the supernatant discarded. The pellet was
washed twice with 5-7 ml of cold RPMI-1640 containing 1 mM Spd to dis
place nonspecifically bound 3H-Spd. The pellet was then dried with a
cotton swab and dissolved in 200 pi of IN NaOH at 60 for 20-60 min.
The material was neutralized with IN HC1, diluted to 1 ml with distilled
water and transferred to a vial for scintillation counting. Uptake was
linear with time from one to 40 min and with 3H-Spd concentration up to
30 pM. Results were expressed as pmol 3H-Spd taken up per min per mg
protein as determined by the method of Lowry.88 Uptake data were analyzed
for kinetic characteristics by using a Hewlett Packard HP-85 microcomputer
programmed for nonlinear regression curve fitting.89
Acute LD50 Toxicity Studies
Acute toxicity of Spd derivatives was determined in male CD-I mice
at 24 or 36 hours. The Spd derivatives were dissolved in isotonic saline,
the pH adjusted with NaHC03 or H3P04 when necessary. The animals were
given one i.p. injection of the derivatives, the volume of which was ad
justed with additional saline, so as to deliver the same volume for each
dose. Generally, two mice per dose at several widely ranging values were
used to determine preliminary toxicity. Once the approximate LD50 is
determined, 10 mice at five doses closely around this value are used.


147
in an attempt to resolve the problem, synthesized a symmetrical analogue
to agrobactin A, containing a glycine residue instead of the threonine
residue. As we have noted, the a-methine of agrobactin A does not appear
to 'see' a difference in conformation; hence, a model lacking the threo
nine side chain may not be the appropriate model to verify the symmetry
(or asyrcmetry) of the polyamine backbone in the *H NMR spectrum.
Accordingly, in order to verify the glycine work, the symmetrical
analogue of agrobactin A incorporating homospermidine as the backbone
was synthesized. As expected, the symmetrical homoagrobactin A result
ed in a simplified spectrum. The y-methyl of homoagrobactin A now ex
hibits only a single doublet, Figure 6-4a, as does its uncoupled e-
methine located at 5 4.2, Figure 6-5a. The appearance of the a-methine,
<5 5.0, is similar in both homoagrobactin A and agrobactin A, Figure
6-5, a and b, respectively, although the doublet structure of the latter
appears to be broader indicating that, perhaps, minor conformational
effect is being felt by the a-methine in agrobactin A. Similar results
are observed in the 300 MHz XH NMR of the asymmetrical t-butoxycarbonyl
precursor (53) and its symmetrical homologue (56). The y-methyl protons
of (53) are observed as two overlapping doublets centered at 5 1.18,
Figure 6-4c. However, in the symmetrical precursor (56), only one doub
let is observed for the y-methyl, <5 1.17, Figure 6-4d. Additionally,
the threonyl NH (6 5.48) is simplified in (56) and is observed as a
doublet, Figure 6-5d, as compared to a doublet of doublets for (53),
Figure 6-5c. On the other hand, the a- and 6-methines (<5 4.41 and 4.04,
respectively) of (5J3) experience little change in their magnetic envi
ronment as a result of conformation, Figure 6-5c.
It is interesting that the g-methine of (53) lacks the duplicity
of its counterpart in agrobactin A; however, this might be explained by


123
The synthesis of the homospermidine reagent (44), however, required
the preparation of four-carbon analogue to {33). Attempted alkylation
of benzylamine and 4-chlorobutyrylnitrile, even with a ten-fold excess
of benzylamine, resulted predominantly in bis-alkylation. Accordingly,
a new synthetic scheme for the preparation of (41_) needed to be developed.
Based on the general success of acylation followed by reduction to pro
duce N-alkyl derivatives in Chapter Two, an approach such as this was
undertaken.
Therefore, benzylamine was acylated with the succinimide ester of
t-BOC-GABA to produce the amide (40) in good yields. This was based on
the premise that the amide could be reduced selectively in the presence
of the urethane protecting group generating the desired four-carbon
analogue (41_) at (33). Such a reduction has been accomplished in the
literature using the reducing agent diborane. In this case a trifluoro-
acetamide substituent was reduced efficiently in the presence of an
ethoxycarbonyl group.98
When a suspension of (40) in THF was treated with BH3 THF complex
the desired reduction of (40) -* (41) did occur; however, a major byproduct
determined to be N-(4-aminobutyl)benzylamine was noted. Evidently,
either the reduction or the acidic workup employed was sufficient to
cause cleavage of the t-BOC protecting group. Even when NH4C1 was em
ployed in place of HC1 for the workup, substantial cleavage still occurred.
Based on the behavior of the t-BOC protecting groups in earlier reactions,
it seemed unlikely that the acid workup should be sufficient to cause
cleavage. However, diborane is a good Lewis acid and, perhaps, under
the conditions of reduction, the t-BOC group can be cleaved, unlike the


CHAPTER PAGE
SEVEN CONCLUSIONS 162
REFERENCES 165
BIOGRAPHICAL SKETCH 1 71
v


Figure 2-14. 60 MHz ^ H NMR spectrum of N^-hexy1spermidine(23).


4 7
Anal. cal. for C13H31N30C12: C, 49.36; H, 9.88; N, 13.28. Found:
C, 49.23; H, 9.77; N, 12.75.
N4-Benzoyl spermidine di hydrochloride (21)
A solution of (1_8) was deprotected and purified in a similar manner
as described for (19). Yield: 370 g (92%); XH NMR (TFA) 6 1.62-2.18
(m, 6H), 2.78-3.52 (m, 8H), 7.20 (s, 5H), 8.15 (br, 6H); IR (KBr) 1700
(s), 1540 (m), 750 (s) cm-1.
Anal. cal. for C18H25F6N305H20: C, 43.64; H, 5.49; N, 8.48. Found:
C, 43.79; H, 5.41; N, 8.17.
N4-Ethylspermidine trihydrochloride (22)
To a cooled suspension (10-15C) of N4-acetylspermidine dihydro
chloride (1_9) (500 mg, 1.92 mmol) and sodium borohydride (380 mg, 10
mmol) in 15 ml dry dioxane was added a solution of trifluoracetic acid
(1.14 g, 10 mmol) in 5 ml dry dioxane. After the addition was completed,
the suspension was slowly brought to a gentle reflux and the reaction
continued for eight hours under N2.
The reaction was then cooled,the excess reducing agent destroyed
by careful addition of water (2 ml), and the solvent reduced under high
vacuum. The residue was treated with 2N K0H (5 ml), the product ex
tracted into CH2C12 (4 x 20 ml), dried, and concentrated to afford 350
mg crude product. The product was further purified by Kugelrohr dis
tillation to afford 280 mg (78%) of the desired product: *H NMR (s)
0.94 (t, 3H), 1.64 (m, 8H), 2.12 (quat., 2H), 2.67 (m, 8H); IR (CHC13)
3320 (m), 2960 (s), 1430 (m) cm-1.
The amine was subsequently converted to its hydrochloride salt by
HC1 gas. Anal. cal. for C9H26N3C13: C, 38.24; H, 9.27; N, 14.86. Found:
C, 38.01; H, 9.31; N, 14.49.


154
signal broadened, and eventually displayed two signals that coalesced
at 18C, corresponding to a Ea of 15.2 0.15 kcal/mole. This low Ea
observed for (53) as compared to agrobactin was anticipated, at least
in direction if not in magnitude. The reason for this may not lie in
a difference in overall size between the t-butyl versus the 2,3-dihy-
droxybenzoyl group, as molecular models suggest that planar 2,3-dihy-
droxybenzoyl group to be only slightly bulkier. However, it may indi
cate that a small role is played by catechol hydrogen bonding in the
conformer populations of agrobactin A versus the precursor (53). Alter
natively, the ether linkage present in the t-butoxycarbonyl group may
provide the t-butyl group an additional degree of freedom, making it
easier for it to avoid the centerpiece as it rotates.
The coalescence data suggest, then, that in solvents such as DMSO
which can compete for intramolecular hydrogen bonds, steric factors pri
marily influence interconversion between conformers in the systems
studied. It further implies that the molecular hydrogen bonding in
agrobactin A is not unusually strong. It is certain, however, that
hydrogen bonding must play a role in conformer population in nonpolar
solvents. This was illustrated by the effect of solvent upon duplicity
of XH NMR signals in agrobactin A and, most dramatically, in the pre
cursor (53) in which DMS0-d6 spectra lacked any sign of the conformers
present in CDC13 at 23C. This effect on the *H NMR of (53), on chang
ing between DMSO and CDC13, is of additional interest in view of the
fact that the t-butoxy group is not able to form as many hydrogen bonds
as the 2,3-dihydroxybenzoyl group. This suggests that an even greater
solvent effect may be experienced by agrobactin A and its related si-
derophores, producing much higher Tc and Ea values in nonpolar solvents


68
Table 3-1. Effects of Polyamines and MGBG on ^H-Spd
Uptake into Ascites L1210 Leukemia Cells
Competing Agent
(lOOuM)
3h
pmo1/10'
mi n
-Spd Uptake*
cells/
%control
K -j (u M)
None
65
100
o
O
6
8
-
Put
58
86
90
nSpd
1 6
29
19
Spd
6
10
10
hSpd
6
10
10
Spm
6
11
11
MGBG
37
66
53
* Cells were incubated for 20 min at 37 with lOuM ^H-Spd
and 100 uM polyamine or MGBG
**Cells were incubated for 20 min at 37 with 0.2, 0.5, 1.0,
2.0, 5.0, or lOuM ^H-Spd and 10 or lOOuM polyamine or MGBG.


Figure 5-8. 60 MHz ^ NMR spectrum of N^-benzyl-N^-(t-butoxycarbonyl)-N^-trifluoro-
acetylspermidine (36).


81
(m, 26H), 2.41 (m, 4H), 3.28 (m, 8H), 3.68 (s, 8H), 5.12 (br, 2H),
6.58 (d, 2H), 7.03 (d, 2H).
Anal. cal. for C31H52N405C12: C, 58.94; H, 8.30; N, 8.87. Found:
C, 59.49; H, 8.42; N, 8.47.
N4-Chlorambuci 1 spermidi ne-bis-tri fl uoroacetate (9.)
Trifluoroacetic acid (10 ml) was added to a flask containing (28)
(220 mg, 0.35 mmol) and the resulting solution stirred for 20 minutes.
The solvent was then quickly evaporated, dissolved up in methanol (25
ml) and concentrated (twice). The residue was then dissolved in 10 ml
H20, washed with CH2C12 (2 x 5 ml), and the aqueous layer lyophilized
to afford 150 mg (70%) as a beige solid: !H NMR (TFA) 6 1.54-2.01 (m,
8H), 2.47 (m, 4H), 3.31 (m, 8H), 3.70 (s, 8H0, 6.67 (d, 2H), 7.06 (d,
2H).
Anal. cal. for C H N 0 Cl F: C, 45.53; H, 5.81; N, 8.49. Found:
J O O i O 4
C, 45.81; H, 6.32; N, 8.40.
N-(4-Carboxybutyr,yl )2,3-dih,ydro-lH-imidazo[l ,2-b]pyrazole (GIMPY) (30)
Glutaric anhydride (250 mg, 2.2 mmol) was added to a solution of
IMPY (220 mg, 2.0 mmol) in 30 ml of dry CH2C12 under N2. After 18 hours
the product was filtered off, washed with CH2C12 and dried in vacuo
to afford 400 mg (90%) as a white powder. The physical and spectral
characteristics were identical to those previously reported:93
mp > 240C; *H NMR (TFA) 6 1.52-1.98 (m, 2H), 2.04-2.61 (m, 4H), 4.35
(s, 4H), 6.28 (s, 1H), 7.57 (s, 1H).
N4-f4-(2,3-Dihydro-1 H-imidazop ,2-b]p,yrazolo)carboxamido)butyryl ]-
N^NS-bisU-butox.ycarbonyl )spermidine (3ll
Dicyclohexylcarbodiimide (DCC) (165 mg, 0.80 mmol) was added to a
solution of (30) (150 mg, 0.67 mmol) and N-hydroxysuccimide (95 mg,


14
Therefore, this study will limit itself to the syntheses of spermidine
derivatives only.
In considering the synthesis of spermidine derivatives, three ques
tions can be asked concerning spermidine's structure relating to uptake:
1) What is the role of the hydrocarbon chain length?
2) What are the roles of the primary amines?
3) What is the role of the secondary amine?
To answer these questions, compounds having the general structures as
illustrated in Figure 1-4 were synthesized. These compounds included
both N-acylated and N-alkylated spermidine derivatives as well as homo
logues that differ in hydrocarbon chain length i.e. nor- and homosper
midine derivatives. The synthesis of these compounds necessitated the
generation of a series of reagents capable of efficient, selective func
tionalization of the spermidine backbone, the details of which are dis
cussed in Chapter Two.
Once in hand, this gave a broad range of N-modified spermidine
derivatives with which to investigate the effect of such parameters as
chain length, functionalization of the primary versus secondary amine,
size of the substituent added, and amide versus amine linkages upon up
take.
Since this energy-dependent polyamine transport system appears to
be more specific for spermidine, uptake of these derivatives was assayed
by measuring the ability of the derivative to compete with radiolabelled
spermidine for uptake in an in vitro system murine L1210 cell cultures.
These cells represent a good model as they efficiently accumulate poly
amines, notably putrescine and spermidine, from their environment during
periods of rapid growth.6162 Accordingly, the cells are incubated in


163
56. Dave, C. and Caballes, L. (1973) Fed. Proc. 32, 736.
57. Seppnen, P., Alhonen-Hongiston, L. and Janne, J. (1980) Eur. J.
Biochem. 110, 7.
58. Canellakis, E.S., Shaw, Y.H., Hanners, W.D. and Schwartz, R.A.
Biochim. Biophys. Acta 418, 277 (1976).
59. Weinstock, L.T., Rost, W.J. and Cheng, C.C. (1981) J. Pharm. Sci.
70, 956.
60. Israel, M., Rosenfield, J.S. and Modest, E.J. (1964) J. Med. Chem.
7, 710.
61. Alhonen-Hongisto, L., Seppnen, P. and Jnne, J. (1980) Biochem. J.
192, 941.
62. Pegg, A.E. and McCann, P.P. (1982) Amer. J. Physiol. 243, C212.
63. Tait, G.T. (1975) Biochem. J. 146, 191.
64.Smith, S.R. (1964) Ann. N.Y. Acad. Sci. 119, 766.
65.Model 1, C.B. and Beck, J. (1974) Ann. N.Y. Acad. Sci. 232, 201.
66.Hilder, R.C., Silver, J., Neilands, J.B., Morrison, I.E.G. and Rees,
L.V.C. (1979) FEBS Lett. 102, 325.
67.Weitl, F.L. and Raymond, K.N. (1979) J. Am. Chem. Soc. 101, 2728.
68. Hoy, T., Humphreys, J., Jacobs, A., Williams, A. and Pooka, P.J.
(1979) J. Ahematol. 43, 3.
69. Jacobs, A. (1979) Br. J. Hameatol. 43, 1.
70. Jacobs, A., White, G.P. and Tait, G.T. (1977) Biochem. Biophys. Res.
Comm. 74, 1626.
71. Neilands, J.B. and Peterson, T. (1979). Tetrahedron Lett., 4805.
72. Neilands, J.B., Peterson, T., Falk, K.E., Leong, S.A. and Klein,
M.E. (1980) J. Am. Chem. Soc. 102, 7715.
73.Bergeron, R.J. and Kline, S.J. (1982) J. Am. Chem. Soc. 104, 4489.
74.Neilands, J.B., Ong, S.A. and Peterson, T.J. (1979) J. Biol. Chem.
254, 1860.
75. Bergeron, R.J., Stolowich, N.J. and Porter, C.W. (1982) Synthesis,
689.
76. Van Brussel, W. and Van Sumere, C.F. (1978) Bull. Chim. Belg. 87,
791. ~


78
1 ) DCC
N-OH Sue.
2) H
Et3N
>v
28
TFA
C 1
N H 3
N H 3
29
Figure 4-1. Synthesis of N^-Chlorambuci1spermidine.


5
Table 1-1. Suggested i_n vi tro and i_n vi vo functions
of polyamines
1. POLYAMINES AS GROWTH FACTORS
2. STABILIZATION OF WHOLE CELLS AND MEMBRANES
3. STABILIZATION OF SUBCELLULAR PARTICLES
4. ASSOCIATION WITH NUCLEIC ACIDS
Stabilization of DNA against denaturation
Stabilization of folded form of DNA
Association with tRNA
Stabilization of newly synthesized RNA
Stimulation of DNA synthesis
Stimulation of RNA synthesis
5. EFFECTS ON PROTEIN SYNTHESIS
Binding of tRNA molecules to ribosomes
Stimulation of tRNA methylation
Replacement of Mg++ in aminoacyl tRNA synthetase reaction
Association with ribosomes
Initiation of translation
Stimulation of tRNA nucleotidyltransferase
6. EFFECTS ON VARIOUS METABOLIC REACTIONS
Stimulation of nucleotide kinases
Enhancement of ADP-ribosylation of nuclear proteins
Activation of phosphorylase b
Stimulation of 1ipolysis
Activation of choline kinase
Inhibition of ATPase
Modification of acetylcholine esterase activity
7. PHARMACOLOGIC EFFECTS IN WHOLE ANIMALS
Nephrotoxic effects
Hypothermic and sedative effects
(from reference 10)


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Chairman
Raymond J. Bergeron, Chfairman
Associate Professor of Medicinal Chemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Margaret J James
Assistant Professor of Medicinal Chemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
\vt ¡7 C
Federico A. yilalloriga
Professor of Pharmacy
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy,
(JL£J A ;/
Richard R. Streiff
Professor of Medicinal Cfiemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Cc-/* >*7
Cemal Kemal, Assistant Professor of
Chemistry


I 1 I
IZI3 i~~i i .i.. I. ZH-1 1 i t i I t 1 i i i 1 . 1
\
10
4 0
5.0
*.0
3 0
1.0
1.0
o
-^1
Figure 5-6. 60 MHz NMR spectrum of N-[N-(t-butoxycarbony1)-3-aminopropy1]-N-
(3-cyanopropy1)benzy1amine (34).


121
Results and Discussion
Synthesis of N4-Benzyl-N1-t-B0C-N8-Trif1uoroacety1sDermidine (36)
In developing a scheme for the synthesis of a tris-protected sper
midine reagent, such as (36), it was recognized that the desired pro
tecting groups should be incorporated throughout the initial steps of
the scheme. Combining the methodologies that were so successfully em
ployed in the synthesis of the previous two spermidine reagents, the
synthesis of (36) could easily be envisioned, Figure 5-3.
The sequence begins with N-(3-ami nopropyl)benzyl amine, a key inter
mediate in the synthesis of N4-benzylspermidine. The utility of this
starting material is that it has already incorporated what will become
the secondary N-benzyl protecting group. Hence, N-(3-aminopropyl)ben-
zylamine was reacted with BOC-ON generating the desired terminally t-BOC
protected compound (33) in high yields. The reaction proceeds selec
tively, with no apparent involvement of the secondary amine. Further
more, the product is easily purified by distillation.
Intermediate (33) was then reached with an excess of 4-chlorobuty-
rylnitrile as in earlier syntheses to afford the mono-nitrile derivative
(34), again easily purified via silica gel chromatography. Subsequently,
it has been shown that crude (34) can usually be reduced directly to
amine (35) without further purification.
The reduction of nitrile (34J to amine (35) employs a recent im
provement over earlier methods used to reduce these nitriles. Earlier,
lithium aluminum hydride was the reagent of choice in the preparation
of benzylspermidine; however, recently it has been shown that a basic
solution of Raney nickel can accomplish this reduction more efficiently.97
Accordingly, the bis-protected nitrile (34) was reduced under a H2


'iL.r;
o 6 6 4 2 0 PPM
Figure 6-3. 300 MHz ]H NMR Spectrum of Agrobactin A in CDCL3/DMS0-d6 (10:1) at 23 C
The downfield phenolic region at -45 is shown in the insert.
1 39


141
lies between 6 6.6 and 7.4 and integrates to nine protons. Their
assignment, although similar to the aromatics of N1,N8-bis(2,3-dihydroxy-
benzoyl )spermi dine reported earlier,lc:3is further complicated by the
third 2,3-dihydroxybenzoyl moiety. The upfield overlapping multiplets
centered at 6 6.70 integrate to three protons and can be assigned to
the meta protons. Downfield to this are four lines centered at 6 7.00,
which correspond to the three para protons. Finally, the ortho protons
appear the furthest downfield as two apparent triplets centered at <5 7.19
and 7.25, the upfield triplet integrating to two protons versus one for
the downfield triplet. The NH protons make up the next group of signals
downfield from the aromatics; those resulting from spermidine's terminal
amides are located at 6 8.03 and 8.24 while the threonyl N-H is located
between them at 6 8.09. The phenolic protons lie the furthest downfield
and, like the threonine hydroxyl, undergo rapid exchange at 23C- This
exchange results in broad, undefined signals whose location is, again,
dependent upon such factors as temperature and amount of water present.
At -45C, however, the rate of exchange is considerably slowed, result
ing in sharper signals at approximately 5 12.0 and 13.0 as shown in the
insert in Figure 6-3.
Of particular interest, however, is the duplicity of signals ori
ginating from the threonyl moiety. The threonine coupling pattern, in
the absence of any conformational effects, represents a simple spin sys
tem. The a-methine should be split once by the amide resulting in a
doublet with a J => 6.9 Hz. This doublet should then be further split
by the S producing a doublet of doublets. The B-methine is coupled to
the three y-methyl protons, resulting in a quartet which is split again
by the a-methine producing eight lines which would be expected to give


z
I
A-NH (C H 2)aN (C H 2)b NH-B
ArNH (CH2) NH (CH2) NH-B
Figure 5-1. Spermidine reagents containing three different protecting groups,
A, B, Z, which can be independently removed.


125
The salt (45) was acylated with a slight excess of benzoyl chloride
in the presence of triethylamine to afford N4-benzoyl-N1-t-B0C-N8-tri-
fluoroacetyl spermidine (40) in 92% yield. The acylation proceeded smooth
ly and the product (46) could be used without further purification.
Next, the trifluoroacetyl protecting group was removed by treatment
of (46) with potassium carbonate in refluxing aqueous methanol for two
hours. The resulting amine was purified by chromatography on silica
gel to afford pure N4-benzoyl-N1-t-B0C spermidine (47) in 81% yield.
Intermediate (47) was then reacted with excess acetyl chloride, again in
the presence of triethylamine, to qualitatively (98%) afford N8-acetyl-
N^benzoyl-N^t-BOC spermidine (48).
The final deprotection, removal of the t-butoxycarbonyl group, was
effected by brief treatment of (48) with trifluoroacetic acid. The prod
uct, N8-acetyl-N4-benzoyl spermidine (49) was easily purified by washing
the acidic solution with CH2C12, adjusting the pH to 11 with Na2C03,
and extracting the product into CH2C12 to afford (49) in 75% yield.
Higher yields (>90%) of slightly impure product could be obtained by
treating the crude (49) with MeOH/NaOMe, followed by removal of the sol
vent, and extracting the product away from the salts with ether. A solu
tion of (49) was reacted with 2,3-dimethoxybenzoyl chloride to afford
the final product N8-acetyl-N4-benzoyl-N1-2,3-dimethoxybenzoyl spermidine
(50) in 93% yield. The overall yield for the conversion of (36) (50)
was 42% in six steps.
It should be noted that in the case of the synthesis of a product
such as (50), in which three acyl groups are added in random order,
intermediate (35) could be used in place of (36) as the starting material.
Instead of acylation with trifluoroacetic anhydride, (35) could alternately


:zzlizii: zztz z zzzzzzzziziiiiii x> nzzju.
-jii-
11 i i ~ i
| 0
ill
so
M 0
3 0
20
1.0
Figure 6-10. 60 MHz NMR spectrum of N^-threonylspermidine trif1uoroacetate (54).


49
N4-Meth,ylspermidine trihydrochloride (5.)
Trifluoroacetic acid (10 ml) was added to a flask containing N4-
methyl-N1,N8-bis(t-butoxycarbonyl)spermidine (24) (110 mg, 0.3 mmol),
and the resulting solution stirred for 20 min. The solvent was then
evaporated, the residue dissolved in MeOH (25 ml) and concentrated (re
peated twice) to afford 120 mg product as the trifluoroacetate salt.
The product was subsequently exchanged to the hydrochloride salt by
dissolving in cold EtOH (3 ml), bubbling HC1 gas through the solution,
and collecting the solid via filtration. The solid was dried in vacuo
over P205 to afford 70 mg (87%): XH NMR (D20) 6 1.75 (m, 4H), 2.10
(m, 2H), 2.87 (s, 3H), 3.15 (m, 8H).
Anal. cal. for C8H25N3C13: C, 35.63; H, 9.34. Found: C, 35.17;
H, 9.52.
Norspermidine trihydrochloride (26)
A sample of N1,N7-bis(t-butoxycarbonyl)norspermidine hydrochloride
(14) was exposed to TFA and purified in the same manner as described
for (25). Yield: 260 mg (83%); *H NMR (D20) 5 2.20 (m, 4H), 3.22 (m,
8H).
Homospermidine trihydrochloride ()
A sample of N1,N9-bis(t-butoxycarbonyl)spermidine hydrochloride
(1_5) was exposed to TFA and purified in the same manner as described for
(25). Yield: 620 mg (91%); XH NMR (D20) 6 1.84 (m, 8H), 3.18 (m, 8H).
Results and Discussion
In order to further characterize the polyamine's receptor, a wide
array of polyamine derivatives would be required to adequately judge
the effects of functionalization on uptake. This necessitated a synthetic
scheme capable of both flexibility and efficiency to deliver the desired


23
At low temperature the rate of interconversion is slow on the NMR time
scale and two separate signals are observed. As the temperature of the
sample is increased, the rate of interconversion becomes faster, the sig
nals broaden and move towards each other. Finally, as the temperature
is increased above the coalescence temperature, the temperature at
which the two signals coalesce into one broad signal, the conformers are
interconverting freely and only one "averaged" signal is observed. The
corresponding activation energy, aG, can be estimated from the coales
cence temperature by the following equation:77
= 22.96 + log e (TVv)
Tc
where Tc is the coalescence temperature, 6v is the width of the signal
at half-height in hertz, and R equal the gas constant.
Measurement of the coalescence temperatures of agrobactin A and
its asymmetrical derivatives would thereby give the AG's for these
interconversions. A comparison of the calculated AG's and their corre
sponding structures should indicate the structural significance attached
to conformer interconversion. The results of such a study performed here
indicate that, in polar solvents such as DMSO, steric bulk is the domi
nant influence controlling interconversion between the conformers of
agrobactin A and its derivatives.


10
to
5.0
<1.0
Figure 2-19. 60 MHz "* H MMR spectrum of
I
.i. > (ii i i... 1.... > i.
>.,i. .. ... 1
cn
00
3.0 2.0 1.0
norspermidine trihydrochloride(2f>) .


140
the chemical shifts previously reported for agrobactin A,75 and the
overall appearance of the spectra is similar to the previously pub
lished NMR spectra of agrobactin75 and parabactin73 noting, however,
the differences in the a, 8, and y threonine resonances between the open
and closed forms.
For purposes of analysis, the spectra can be divided into two re
gions, the upfield portion above 6 6.00 containing the resonances from
the spermidine backbone and the threonine residue, and the downfield
portion containing the aromatic, phenolic, and amido protons. The high
field end of the spectrum is characterized by a pair of doublets cen
tered at 5 1.20 which integrates to three protons. These protons are
assigned to the y-methyl of threonine, each doublet equally coupled to
the 8-methine (J^ = 6.3 Hz) as confirmed by decoupling experiments.
The envelope between 6 1.5 and 2.1 integrates to six protons, and ori
ginates from the three internal methylene groups of the spermidine back
bone, while the four external methylene groups adjacent to the amides
are responsible for the eight proton envelope observed from 6 3.15 to
3.75. The final set of peaks in the upfield region of the spectra in
clude an extensively split multiplet at 6 4.18 and an apparent doublet
of doublets at <$ 5.02, both integrating to one proton each. These have
been assigned as the 8- and a-methines, respectively, by the appropriate
decoupling experiments. Additionally, a broad hump is observed at about
6 4.7 resulting from the threonine hydroxyl which disappears on exchange
with D20. Furthermore, as expected the hydroxyl's intensity and loca
tion will vary with concentration and temperature.
The downfield portion of the spectra consists of the aromatic pro
tons, amido protons and the phenolic hydroxyls. The aromatic region


5 0
H.O
1.0
o
Figure 5-13. 60 MHz 1h NMR spectra of N^-benzoyl-N^-t-butoxycarbonylspermidine (47).


69
Table 3-2. Effects of Terminally Modified Spd Derivatives
on 3H-Spd Uptake into Ascites L1210 Leukemia
Cells
Competing Agent
(lOOuM)
3tj
pmol/I O'
min
-Spd Uptake*
cells/
^control
Ki (uM)**
None
56
100
-
BSpdN
49
88
163
BisBOC-nSpd
53
95
11 03
Bi sBOC-Spd
51
91
521
BisBOC-hSpd
50
89
504
DHBSpd
51
91
256
BisAcetyl-Spd
51
91
508
BisPropionyl-Spd
51
92
550
BisEthyl-Spd
39
69
62
BisPropyl-Spd
45
80
117
* Cells were incubated for 20 min at 37 with lOuM ^H-Spd
and lOOuM Spd Derivative
**Cells were incubated for 20 min at 37 with 0.2, 0.5,
1.0, 5.0, or lOuM ^H-Spd and 10 or 100 uM of derivative.


152
Y-methyl resonance into two lines at room temperature. Upon heating,
the two lines at first gradually broaden, then move rapidly as the coal
escence temperature is approached, Figure 6-8. Finally, the lines are
observed to coalesce at approximately 74C and, with a further increase
in temperature, the line width of the resultant single line now decreases
as its intensity increases.
The observed Tc of 74C for agrobactinA corresponds to activation
energy of approximately 18.2 .15 kcal/mole. This is quite similar to
the value reported for agrobactin by Neilands and was unexpected in view
of the difference between the relatively rigid oxazoline ring versus the
more flexible open agrobactin A. A second unexpected result was the high
coalescence temperature of the methylated derivative (59_). It was ini
tially anticipated that methylation of the catechols would lower the co
alescence temperature of the various conformers by destroying the stabi
lizing hydrogen bonding network. On the contrary, (59) coalesced at a
much higher temperature than agrobactin A. In fact, the Tc of (59) ex
ceeded the operating limit of our probe, preventing the precise measure
ments of (59^)1 s Ea. At 130C, the highest temperature that could be
measured, the decoupled y-methyl signals of (59j were nearing coales
cence. The Ea of (59) was, therefore, estimated based on a Tc of at
least 130C and 5v of 9.4 Hz, to be greater than 21 kcal/mole. Since
the Ea varies directly with temperature, the further increase in tem
perature above 130C can only increase the Ea. Therefore, 21 kcal/mole
represents the minimal Ea for (59_). This large increase in Ea for (59)
over agrobactin A can be explained by an increase in steric interactions
that are preventing interconversion between conformers. It does not,
however, necessarily mean steric factors are more important than hydrogen


SYNTHESIS AND PROPERTIES OF N-ACYLATED AND N-ALKYLATED
POLYAMINE DERIVATIVES AND OF AGROBACTIN A,
A NATURALLY OCCURRING SIDEROPHORE
BY
NEAL J. STOLOWICH
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1983

To Barb and Dan,
for their unending love and patience

ACKNOWLEDGEMENTS
I would like to express my sincere appreciation to my research ad
visor, Dr. Raymond Bergeron, under whose guidance this work was accom
plished. I would also like to express my gratitude to the rest of my
supervisory committee, Dr. Richard Streiff, Dr. Margaret James, Dr.
Federico Vilallonga, and Dr. Cemal Kemal.
The uptake determinations of the polyamine derivatives were performed
by Dr. Carl Porter and his group at Roswell Park Memorial Institute,
Buffalo, New York. I wish to extend my appreciation to them for these
determinations, as the data were a significant part of this work.
Finally, I would like to thank all my family and friends that have
helped make this work possible: first, to my parents, Frank and Helen
Stolowich, for their guidance to further pursue my academic career;
secondly, to all my friends, past and present, in medicinal chemistry for
their friendship and assistance. Special thanks to Dr. Kathy McGovern
for those technical discussions at Joe's and for going through this at
the same time. And, last but certainly not least, to my wife Barbara
for her continual support and motivation to carry me through graduate
school.
ii i

TABLE OF CONTENTS
PAGE
DEDICATION 11
ACKNOWLEDGEMENTS iii
ABSTRACT vi
CHAPTER
ONE INTRODUCTION AND BACKGROUND 1
Polyamines and Growth 4
Structural Requirements for Polyamine Uptake 13
Spermidine Derived Siderophore Systems 16
TWO REAGENTS FOR THE SELECTIVE FUNCTIONALIZATION OF
SPERMIDINE, NORSPERMIDINE, AND HOMOSPERMIDINE 24
Experimental 31
Results and Discussion 49
THREE BIOLOGICAL EVALUATION OF POLYAMINE DERIVATIVES 64
Materials and Methods 64
Results 67
Discussion 71
FOUR PRELIMINARY INVESTIGATIONS TOWARDS THE DEVELOPMENT
OF SPERMIDINE-ANTINEOPLASTIC CONJUGATES 76
Experimental 80
Results and Discussion 86
FIVE SYNTHESIS OF TRIS-PROTECTED SPERMIDINES 90
Synthesis 92
Experimental 97
Results and Discussion 121
SIX SYNTHESIS AND SOLUTION DYNAMICS OF AGROBACTIN A 127
Experimental Section 127
Results and Discussion 132

CHAPTER PAGE
SEVEN CONCLUSIONS 162
REFERENCES 165
BIOGRAPHICAL SKETCH 1 71
v

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
SYNTHESIS AND PROPERTIES OF N-ACYLATED AND N-ALKYLATED
POLYAMINE DERIVATIVES AND OF AGROBACTIN A,
A NATURALLY OCCURRING SIDEROPHORE
BY
NEAL J. STOLOWICH
December 1983
Chairman: Raymond J. Bergeron
Major Department: Medicinal Chemistry
The synthesis of reagents for the selective functionalization of
the polyamines spermidine, norspennidine, and homospermidine is described.
With these reagents various N-acylated and N-alkylated polyamine deriva
tives are generated and their uptake characteristics studied in Murine
LI210 leukemia cells. In general those polyamine derivatives having their
terminal amines modified were unable to compete with 3H-spermidine for
uptake into those cells. Polyamine derivatives in which the central ni
trogen is modified are found to compete with 3H-spermidine for uptake.
This suggests that the primary amines are critical for inferring uptake
specificity while the secondary amine is less important. The synthesis
of two antineoplastic spermidine conjugates is also described as a pre
liminary investigation into the potential of spermidine as a drug delivery
device.
In a related synthetic sequence, Agrobactin A and a number of ana
logues are prepared. High field proton nuclear magnetic resonance
VI

spectroscopy is employed to evaluate the origins of the conformers in
agrobactin A. The coalescence temperatures and activation energies be
tween conformers of these polyamides are determined using NMR. The
role of steric and hydrogen bonding factors in these compounds is also
discussed.

CHAPTER ONE
INTRODUCTION AND BACKGROUND
Although the existence of polyamines in human semen has been known
since 1677,1 the majority of research regarding the polyamines' biosyn
thesis and physiological functions has occurred only in the last twenty
years. These naturally occurring polyamines are, in general, linear di-
and triamines: the five most commonly associated with eukaryotes are pre
sented in Figure 1-1. Of these, the amines 1,4-diaminobutane (putres-
cine), spermidine, and spermine are the most important and most commonly
occurring polyamines in mammalian tissues, although others have been
shown to occur in trace quantities as well.
The biosynthesis of putrescine, spermidine, and spermine proceeds
through a common pathway in mammals as well as microorganisms, the pri
mary precursors being the amino acids L-ornithine and L-methionine. As
shown in Figure 1-2, L-ornithine is decarboxylated to form putrescine,
and the ATP activated form of methionine, S-adenosyl-L-methionine, serves
as the propylamine donor, the moiety added to putrescine to form the
higher polyamines spermidine and spermine. Four enzymes are primarily
responsible for the synthesis of the polyamines in mammalian tissues
two decarboxylases, and two synthetases. The decarboxylases, L-ornithine
decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAM-DC),
have been extensively purified and studied. Ornithine decarboxylase,
which catalyzes the conversion of ornithine to putrescine, is a pyridoxal-
requiring enzyme, like most other mammalian decarboxylases. However,
1

HNCH_CHCHNHCHCHCHCHNH
2 222 22222
h2nch2ch2ch2ch2nh2
I ^-diami nobutane
(Putrescine)
h2nch2ch2ch2ch2ch2nh2
I,5~d¡aminopentane
(Cadaver¡ne)
Sperm¡dine
H NCH CH CH NHCH CH CH NH
2 222 2222
Norspermidine
h2nch2ch2ch2ch2nhch2ch2ch2ch2nh2
Homosperm idine
H NCH CHCH NHCH CHCHCH NHCH CH CH NH
2 222 2222 2222
Spermine
Figure 1-1. Commonly occurring polyamines associated with eukaryotic cells

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4
unlike most mammalian enzymes, ODC is strikingly inducible2 and has a
very short half life10-30 min.3 Its basal activity is the lowest of
the four; thus, it is considered to be the rate-controlling enzyme in
polyamine biosynthesis.4
The other decarboxylase, S-adenosyl-methionine decarboxylase, de-
carboxylates the methionine residue that is covalently bound to the aden
osine. It too has a short half life of about one hour5 but, unlike ODC
and most other decarboxylases, does not need pyridoxal phosphate for
catalytic activity. Instead, SAM-DC contains pyruvate residues within
its peptide chain that serve as the carbonyl cofactor.6
The spermidine and spermine synthases catalyze the transfer of pro
pylamine groups from decarboxylated SAM to putrescine and spermidine,
respectively. These synthases have been only partially purified at the
present time, but have been shown to be relatively stable enzymes with
long half lives.7 The differences between the decarboxylases' and syn
thases' half lives are of significant importance in considering the in
hibition of polyamine biosynthesis, as will be discussed later.
As mentioned earlier, polyamines have gained considerable interest
in the past few decades, and this has been mainly on two fronts. First,
polyamines have been implicated as being directly involved in cellular
function and, secondly, the polyamine spermidine serves as a backbone
to a number of naturally occurring siderophores or iron chelators.
The latter will be briefly dealt with later, while some of the aspects
of polyamine involvement in cellular function will be discussed now.
Polyamines and Growth
As illustrated in Figure 1-3, there have been many suggested in vivo
and in vitro functions of polyamines. Of all of these, this thesis is

5
Table 1-1. Suggested i_n vi tro and i_n vi vo functions
of polyamines
1. POLYAMINES AS GROWTH FACTORS
2. STABILIZATION OF WHOLE CELLS AND MEMBRANES
3. STABILIZATION OF SUBCELLULAR PARTICLES
4. ASSOCIATION WITH NUCLEIC ACIDS
Stabilization of DNA against denaturation
Stabilization of folded form of DNA
Association with tRNA
Stabilization of newly synthesized RNA
Stimulation of DNA synthesis
Stimulation of RNA synthesis
5. EFFECTS ON PROTEIN SYNTHESIS
Binding of tRNA molecules to ribosomes
Stimulation of tRNA methylation
Replacement of Mg++ in aminoacyl tRNA synthetase reaction
Association with ribosomes
Initiation of translation
Stimulation of tRNA nucleotidyltransferase
6. EFFECTS ON VARIOUS METABOLIC REACTIONS
Stimulation of nucleotide kinases
Enhancement of ADP-ribosylation of nuclear proteins
Activation of phosphorylase b
Stimulation of 1ipolysis
Activation of choline kinase
Inhibition of ATPase
Modification of acetylcholine esterase activity
7. PHARMACOLOGIC EFFECTS IN WHOLE ANIMALS
Nephrotoxic effects
Hypothermic and sedative effects
(from reference 10)

6
primarily concerned with the polyamines' role in cellular growth in both
the normal and diseased states. Several excellent reviews8-10 are avail
able for information concerning the other biological effects and will
not be discussed here.
The initial observations linking polyamines to cell growth were es
tablished in several different rapidly growing tissue systems during the
mid-sixties. In studies involving chick embryos, marked increases in
polyamine levels along with increased ODC and SAM-DC activities were ob
served during development relative to those of the mature animal.11-13
In the human fetal liver, putrescine concentration displayed a distinct
peak in the fifth and sixth month of gestation.1415 Although the em
bryonic data are limited, the higher polyamine levels were all attributed
to higher ornithine and adenosylmethionine decarboxylase activities as
sociated with rapid growth.
The regenerating rat liver has been a more widely used system to
study the effect of growth upon polyamine biosynthesis. Experimental
evidence from several groups has shown that enhanced accumulation of
putrescine reaches a maximum approximately four hours after partial
hepatectomy,16 resulting in a moderate enhancement of spermidine levels
in 2-3 days.17 This early increase in putrescine coincides with enhanced
ODC activity, which can be as great as 100- to 500-fold.1218 This in
crease is extraordinarily high as most enzymes in vivo only fluctuate
2-, 3-fold. The enhanced ODC activity, along with recent data reveal
ing that this enzyme is closely regulated by transcriptional and trans
lational mechanisms,1920 suggests that this early dramatic increase is
the primary event responsible for triggering polyamine biosynthesis.
The increased levels of putrescine and spermidine in the regenerating

7
liver are also usually accompanied by a decrease in spermine17 as sper
mine synthetase has been shown to be inhibited by high concentrations of
putrescine.21
More recently, a third type of rapidly proliferating cell and its
corresponding high polyamine levels have drawn much attention the cancer
cell. This was first shown in Ehrlich ascite carcinoma cells grown in
mice by two separate labs.2223 Both groups found that in the early
stages of tumor growth, a remarkably high putrescine content was found
that rapidly decreased after the first week following inoculation, where
as the concentration of spermidine peaked near the second week after
inoculation. These data are comparable to that found in the regenerat
ing rat liver system mentioned earlier. Wi11iams-Ashman et al. were further
able to show, by using hepatomas of vastly different growth rates, that
the accumulation of putrescine was proportional to the rate of tumor
growth.2425 Putrescine levels in the most rapidly growing hepatomas
were ten times as high as levels in the normal rat liver. Furthermore,
ornithine decarboxylase activity also coincided well with rate of tumor
growth.
Similar results have also been found for other types of cancer.
For example, markedly elevated levels of spermidine as well as ornithine
decarboxylase activity were found in d/3 sarcoma as compared to normal
muscle tissue.26 Ornithine decarboxylase activity was found to be six
times higher in mouse LI210 leukemia tumor cells, as well as containing
more than double their normal values of putrescine and spermidine.27
Additionally, some rapidly growing tumors produce enough polyamines that
they are actually excreted into the surrounding interstitial fluid caus
ing elevated polyamine serum levels.2829 Accordingly, clinical researchers

8
are currently developing diagnostic tools to follow tumor regression and
perhaps detect early tumor growth.30
From the above data it can be suggested that all cells, whether
normal or neoplastic, undergoing rapid proliferation contain elevated
levels of polyamines, notably putrescine and spermidine.
If polyamines are so intimately involved and required for rapid
cell growth as implied so far, then the selective inhibition of their
biosynthesis should have a pronounced detrimental effect upon cell growth.
This concept of inhibiting cell growth by inhibiting polyamine synthesis
would not only be valuable in determining the actual physiological sig
nificance of polyamines but could also represent a possible method of
arresting cancer growth. As mentioned earlier, the short half lives of
both ornithine and SAM decarboxylases are significant in that they should
be the easiest enzymes to inhibit. This is in fact the case as most of
the research in inhibiting polyamine synthesis in vitro and in vivo has
been centered on finding selective inhibitors of these two enzymes uti
lizing various tumor cell lines.
A great majority of the inhibitors that have been developed in the
last twenty years are congeners of ornithine and methionine. Ethionine,
the ethyl analog of methionine, was probably the first compound used to
inhibit polyamine synthesis. Although injections of it led to an initial
decrease in spermidine concentration, chronic treatment eventually led to
an overall increase in spermidine concentration,31 and its use as an
inhibitor subsequently was abandoned.
In 1973, a-hydrazinoomithine, a potent reversible inhibitor of or
nithine decarboxylase (ODC), was synthesized.32 Synthesis of a-hydra-
zinoornithine found it to be a competitive inhibitor with a Ki value of

9
2 yM.33 In vivo, the presence of 0.5 mM ct-hydrazinoornithine added to
the medium of hepatoma cells could prevent the normal rapid accumulation
of putrescine and, likewise, partially prevent the increased accumulation
of putrescine due to partial hepatectomy.34 In both systems, however,
hydrazinoomithine did not disturb the enhanced synthesis of RNA, a nega
tive observation as far as indicating the need of polyamines for cell
proliferation or RNA stabilization.
A close analog to hydrazinoomi thine, D,L-a-hydrazino-6-aminova1eric
acid was an even more effective inhibitor of ornithine decarboxylase
than the former, having a Ki of 0.5 yM.35 Administration of hydrazino-
aminovaleric acid blocked the increase in DNA synthesis, the accumulation
of putrescine, as well as the weight gain usually seen in mouse sarcomas.36
Similar results were also obtained from cultures derived from hamster
tumors.36 In all these cases spermidine and spermine levels remained
unchanged, and the effects of hydrazinoamino valeric acid could be re
versed by the addition of putrescine, but not spermidine35 or cadaverine.36
Perhaps the simplest of ornithine analogs, a-methylornithine, syn
thesized in 1974, is also a potent competitive inhibitor (Ki value 20
yM) of ODC. Addition of a-methyl ornithine to cultures of rat hepatoma
cells completely prevented accumulation of putrescine, and likewise pre
vented increases in cellular spermidine.37 Incorporation of thymidine
in DNA greatly declined after the first doubling time in its presence.
Furthermore, a-methylornithine's growth inhibition could be immediately
reversed by concentrations of spermidine, spermine, and putrescine lower
than that of ornithine, offering good evidence that the intracellular

polyamine levels are casually related to proliferation.

10
Recently, a difluoro analog of a-methylornithine, difluoromethyl-
ornithine (DFMO), has been synthesized which has been found to be a po
tent irreversible inhibitor of ODC.38 Difl uoromethylornithine, which
is nontoxic in mammals,39 has been shown to block multiplication of the
parasite Trypanosoma b. brucei, and can cure mice infected with the para
site simply by administration in their drinking water.40 Furthermore,
the cure can be reversed by injection of small doses of polyamines.
Difluoromethylomithine has also been shown to inhibit growth of LI210
leukemia cells, again, its antiproliferative effects prevented by spermi
dine and putrescine.41 In fact, LI210 cells depleted in polyamines by
pretreatment with DFMO show up to a 3-fold increase in polyamine uptake
as compared to untreated cells,42 evidently in an attempt to restore nor
mal levels. This observation will have additional importance in follow
ing discussions.
Up to this point, all the inhibitors of polyamine synthesis discussed,
with the exception of ethionine,are inhibitors of ODC. However, an im
portant group of SAM-DC inhibitors has been recently discovered and
warrants further discussion. The first of this group, methylglyoxal
bis(guanylhydrazone) (MGBG), was first synthesized in 1898,43 but only
as recently as 1972 shown to be a potent and specific inhibitor of SAM-
DC by Williams-Ashman and Schenone.44 The discovery of MGBG as an inhi
bitor of SAM-DC grew out of an investigation of its known antitumor ef
fects. Methylglyoxal bis(guanylhydrazone) was first shown to be active
against LI210 leukemia,45 but has since been tested against a wide range
of experimental tumors.4647 However, leukemias exhibit the greatest
sensitivity to MGBG and, accordingly, MGBG recently has been used to
treat several forms of human leukemia clinically.48

n
If one were to look at MGBG's structure (Figure 1-3), one might
assume that the mode of action of inhibiting SAM-DC is by mimicking SAM's
structure, and that this inhibition of polyamine synthesis might be re
sponsible for the antileukemic activity. However, there appears to be
no simple correlation between the ability to inhibit SAM-DC and the anti
leukemic activity. In general, minor modifications of MGBG, such as the
dimethyl and ethylglyoxal analogues, do not have any pronounced effect
against its ability to inhibit SAM-DC, but lack antileukemic activity.49
Recently, an alternative mechanism for MGBG's antiproliferative
activity has been suggested. Several recent studies have shown that
cultured LI210 cells treated with micromolar concentrations of MGBG de
velop excessively swollen mitochondria several hours before detectable
changes in polyamine levels or cell growth.5051 This suggested MGBG
may act as a mitochondrial poison. The damage in most instances is re
versible and normal appearance can be restored by the addition of sper
midine. However, the irreversible inhibitor 1,1](methylethane diylidene
dinitrilo]bis(3-aminoguanidine) (MGAG) which is equally effective as
MGBG in inhibiting cell growth, is much slower to produce mitochondrial
damage.52
Of additional interest, MGBG is concentrated in a variety of cells,53
apparently through a saturable energy-dependent transport mechanism.54
In fact, in Ehrlich ascite carcinoma cells, MGBG is concentrated so
effectively that a concentration gradient as high as 1000-fold across
the cell membrane has been observed, producing minimolar intracellular
MGBG levels.55 Furthermore, the accumulation of MGBG could be blocked
competitively by polyamines and, to a lesser extent, some diamines.5657
For example, only micromolar concentrations of spermidine or spermine,

12
Mefhyigiyoxal-Bis (Guanylhydrczone)
Spermidine
Figure 1-3. Structures of a) Methylglyoxal-bis-(guanyl-
hydrazone), b) S-adenosyl-L-methionine, and
c) Spermidine.

13
but not putrescine, effectively block MGBG's uptake. Accordingly, it
is postulated that MGBG competes with the carrier or receptor site re
sponsible for spermidine uptake. Additional evidence which supports this
is that, in DFMO pretreated LI210 cells, which respond by increasing
polyamine uptake, uptake of MGBG is also increased.42
Structural Requirements for Polyamine Uptake
Whatever MGBG's mechanism of action may be, whether it acts by in
hibiting SAM-DC and diminishing spermidine levels, or by replacing sper
midine at some intracellular site, perhaps in the mitochondria, it is
of significant importance that MGBG and spermidine both compete for
energy-dependent uptake. Moreover, little is known as to what structural
requirements are necessary for a molecule to be actively transported into
the cell via this carrier. Indeed, up until the present time very little
has been done to investigate the effects upon cell growth and uptake by
derivatizing or modifying the polyamines. Several N-modified spermidine
and spermine compounds have been synthesized,as in the case of the mono-
and bis-acridyl-spermidine and spermines; however, these compounds were
initially synthesized in order to investigate their ability to inter
calate with DNA.58 Likewise, several novel branched spermidine and sper
mine homologues have been recently synthesized and screened for their
antineoplastic activity^9,5but the uptake characteristics of these com
pounds have not yet been investigated. From the studies discussed thus
far, it is apparent that certain structural parameters are necessary for
polyamine uptake; consequently, it would be desirable to further define
the structural requirements for uptake.
Recent evidence exists that of the polyamines, spermidine, sper
mine, and putrescine, the carrier is more specific for spermidine.

14
Therefore, this study will limit itself to the syntheses of spermidine
derivatives only.
In considering the synthesis of spermidine derivatives, three ques
tions can be asked concerning spermidine's structure relating to uptake:
1) What is the role of the hydrocarbon chain length?
2) What are the roles of the primary amines?
3) What is the role of the secondary amine?
To answer these questions, compounds having the general structures as
illustrated in Figure 1-4 were synthesized. These compounds included
both N-acylated and N-alkylated spermidine derivatives as well as homo
logues that differ in hydrocarbon chain length i.e. nor- and homosper
midine derivatives. The synthesis of these compounds necessitated the
generation of a series of reagents capable of efficient, selective func
tionalization of the spermidine backbone, the details of which are dis
cussed in Chapter Two.
Once in hand, this gave a broad range of N-modified spermidine
derivatives with which to investigate the effect of such parameters as
chain length, functionalization of the primary versus secondary amine,
size of the substituent added, and amide versus amine linkages upon up
take.
Since this energy-dependent polyamine transport system appears to
be more specific for spermidine, uptake of these derivatives was assayed
by measuring the ability of the derivative to compete with radiolabelled
spermidine for uptake in an in vitro system murine L1210 cell cultures.
These cells represent a good model as they efficiently accumulate poly
amines, notably putrescine and spermidine, from their environment during
periods of rapid growth.6162 Accordingly, the cells are incubated in

15
R,CONH (CH2)a NH (CH2)b NHCOR,]
R]CH2NH (CH2)a NH (CH2)b NHCH2Rj
CO
H2N (CH2)a N (CH2)b NH2
CH-
I
H2N (CH2)a N (CH2)b NH2
Figure 1-4. Generalized structures of polyamine derivatives
synthesized,

16
the presence of both the derivative and 3H-spermidine. Spermidine deriva
tives which successfully compete with spermidine for the carrier will
accordingly reduce the amount of radiolabel taken up by the cells.
The uptake characteristics of these derivatives suggested that only
a minor structural significance is placed on the secondary amine of sper
midine as compared to its primary amines. For example, bis-acylated com
pounds such as N1,N8-bis(2,3-dihydroxybenzoyl)spermidine and N1,N8-bis-
(t-butoxycarbonyl)spermidine are unable to compete with 3H-spermidine
for uptake. However, N4-benzylspermidine is as good as MGBG in inhibit
ing spermidine uptake.42
These findings suggested the possibility of conjugating several
small anti neoplasties to spermidine via its secondary nitrogen. Since
tumor cells apparently concentrate spermidine, this may serve as a novel
method to target the delivery of anti neoplasties to the tumor. There
fore, a preliminary investigation into this area was initiated with the
synthesis of two spermidine antineoplastic conjugates: N4-chlorambucil
spermidine and N4-[4-(2,3-dihydro-lH-imidazo]l,2-b]pyrazolo)carboxamido)-
butyryljspermidine (Figure 1-5). Preliminary in vitro findings indicate
that even the attachment of N4-substituents of this magnitude does not
prohibit the competitive uptake of the conjugate.
Spermidine Derived Siderophore Systems
The second part of this dissertation deals with the synthesis and
solution dynamics of agrobactin A (la, Figure 1-7), a naturally occurring
spermidine-derived iron chelator. There has been considerable interest
in this family of siderophores since the isolation and identification of
two other siderophores: N1,N8-bis(2,3-dihydroxybenzoyl) spermidine (II)
and N4-[N-(2-hydroxybenzoyl)threonylj-N1,N8-bis(2,3-dihydroxybenzoyl)

1 7
Figure 1-5. Structures of a) N4-C h1orambuci1spermidine ,and
b) N -[4-(2,3-dihydro-1H-i midazo]l,2-b]pyrazolo)
carboxamido)butyryl]spermidine.

18
spermidine (parabactin A, lb) by Tait63 in 1975, Figure 1-6. For the
most part, this is due to the lack of a satisfactory therapeutic device
for the treatment of various iron overload syndromes,64-66 and the poten
tial that these compounds have shown for clearing iron.6768
For example, both compound II and parabactin A have been shown to
be more effective at removing iron from transferrin than desferrioxime,6970
the drug which is currently being used clinically for chelation therapy.
Since the initial isolation of parabactin A, it has been revealed that
the product isolated may have resulted from the hydrolysis of the sidero-
phore N4-[N-(2-hydroxyphenyl)-4-carboxyl-5-methyl-2-oxazolinej-N1 ,N8-bis-
(2,3-dihydroxybenzoyl)spermidine (parabactin, 111 b) actually produced by
the microorganism. Based on Neilands' and Peterson's study,71 it is
likely that, during the original Tait isolation of parabactin A, the acidic
workup employed would have been sufficient to hydrolyze parabactin's oxa-
zoline ring to produce the open form lb isolated. Both the open threonyl
form and the closed oxazoline form can be easily differentiated, among
other means, by high field proton nuclear magnetic resonance ^H-NMR) spec
troscopy due to characteristic chemical shifts and coupling patterns of
the threonyl residue versus the oxazoline residue.7273 Furthermore,
it has been recently observed that parabactin exists in at least two dis
tinct conformers as determined by 3H-NMR spectroscopy.73 The existence
of these conformers is supported by the threonine oxazoline proton signals,
which exist in duplicate.
A similar system to the parabactin/parabactin A system has been
recently isolated from Agrobacterium tumifaciens B6 cultures, given the
trivial names of agrobactin A (la) and agrobactin (Ilia),74 Figure 1-6.
Like parabactin, agrobactin A is a spermidine-derived siderophore;

19
1 I i
conhch2ch2ch2nch2ch2ch2ch2i\jhco
la R=OH
Ib R= H
HO
HO
conhch2ch2ch,nch2ch2ch
WH
II
Ilia R=0H
111b R= H
Figure 1-6. Structures of the siderophores Agrobactin A and
Agrobactin (la and Ilia), compound II, and Para-
bactin A and Parabactin (I b and 111 b).

20
however, agrobactin contains a third 2,3-dihydroxybenzoyl moiety in place
of parabactin's 2-hydroxybenzoyl moiety. Likewise, agrobactin (Ilia)
also exists in a series of conformers, and on activation energy for in
terconversion between conformers has been determined.72 However, simi
lar information regarding agrobactin A is not available. Furthermore, no
information exists concerning the nature of the conformers observed in
these siderophores in general, i.e., the contribution of steric and hydro
gen bonding factors towards the activation energy controlling conformer
interconversion. Accordingly, synthesis and high field ^-NMR studies
of agrobactin A were undertaken.
In considering a synthesis of agrobactin A, the ability to generate
symmetrical homologues and other derivatives must be kept in mind.
Agrobactin A has been recently synthesized by Neilands by reacting sper
midine with the bulky reagent 2,3-dibenzoyloxybenzoyl chloride, followed
by attachment of the N-(2,3-dibenzyloxybenzoyl)threonyl "centerpiece"
and removal of the benzoyloxy protection groups.72 However, the yields
reported for the condensation steps and the limitations on the deriva
tives that can be generated make this synthesis somewhat undesirable.
If, on the other hand, a protected spermidine reagent could be
75
employed, such as N1,N8-bis(t-butoxycarbonyl)spermidine (IV), an effi
cient synthesis of agrobactin A can be envisioned as illustrated in
Figure 1-7. The reagent (IV) was easily condensed with N-(t-butoxycar-
bonyl)threonine using the coupling reagents dicyclohexylcarbodiimide
and N-hydroxysuccinimide. The resulting product N4-[N-(t-butoxycarbonyl)-
threonylj-N1,N8-bis(t-butoxycarbonyl)spermidine was then quantitatively
deprotected by brief exposure to trifluoroacetic acid generating N4-
threonylspermidine (VI). Agrobactin A could be subsequently synthesized

21
IV
V
VI
la
HO NH-1 BO C
*
Figure 1-7. Synthesis of agrobactin A.

22
by attaching three 2,3-dihydroxybenzoyl groups to the triamine (VI).
Recently, Van Brussel and Van Sumere have shown it to be possible to
generate the succinimide esters of a number of mono- and dihydrobenzoic
acids in the presence of the unprotected phenols.76 Thus, when N^-threonyl-
spermidine was reacted with excess succinimide 2,3-dihydroxybenzoate in
the presence of triethylamine in aqueous THF, agrobactin A was obtained
in 75% yield after chromatography.
Unlike previous synthesis of catechol ami des which usually attached
the "centerpiece" in the final stages of the synthesis, this synthesis
of agrobactin A reverses this order, building the molecule from the
"inside out" and attaching the catechols in the final step. This is im
portant as any number of groups can now be attached to the triamine
(VI), making it possible to easily synthesize additional agrobactin A
derivatives. Examples of some derivatives that were synthesized in this
manner included the 2,3-dimethoxybenzoyl derivative of agrobactin A as
well as an unsubstituted benzoyl derivative. Likewise, symmetrical ana
logues of agrobactin A can be generated as both nor- and homospermidine
homologues of N1,N8-bis(t-B0C)spermidine are available.75
With these compounds at hand, their XH NMR spectra were subsequently
investigated. Not surprisingly, agrobactin A also exhibited conformers
in its NMR spectrum as determined by the duplicity in the signals
originating from the threonine residue. If, indeed, these duplicate
signals are a result of an equilibrium between two interconverting iso
mers and not two distinctly separate compounds, the activation energy
for this interconversion can be measured using XH NMR. The rate of
interconversion or exchange between conformer population can be estimated
by determining the coalescence temperature of the duplicate signals.

23
At low temperature the rate of interconversion is slow on the NMR time
scale and two separate signals are observed. As the temperature of the
sample is increased, the rate of interconversion becomes faster, the sig
nals broaden and move towards each other. Finally, as the temperature
is increased above the coalescence temperature, the temperature at
which the two signals coalesce into one broad signal, the conformers are
interconverting freely and only one "averaged" signal is observed. The
corresponding activation energy, aG, can be estimated from the coales
cence temperature by the following equation:77
= 22.96 + log e (TVv)
Tc
where Tc is the coalescence temperature, 6v is the width of the signal
at half-height in hertz, and R equal the gas constant.
Measurement of the coalescence temperatures of agrobactin A and
its asymmetrical derivatives would thereby give the AG's for these
interconversions. A comparison of the calculated AG's and their corre
sponding structures should indicate the structural significance attached
to conformer interconversion. The results of such a study performed here
indicate that, in polar solvents such as DMSO, steric bulk is the domi
nant influence controlling interconversion between the conformers of
agrobactin A and its derivatives.

CHAPTER TWO
REAGENTS FOR THE SELECTIVE FUNCTIONALIZATION
OF SPERMIDINE, NORSPERMIDINE, AMD HOMOSPERMIDINE
In order to investigate the uptake properties of various N-acylated
and N-alkylated polyamines, one must consider the ability to selectively
and efficiently modify the polyamines' primary versus secondary amines
to produce the derivatives desired. Commercially available spermidine
itself fails in this respect as previous studies have shown there is
little difference in reactivity and hence, selectivity between its pri
mary and secondary amines.78 Reactions of spermidine with even the most
bulky reagents often lead to complex mixtures and poor yield of the de
sired product. Additionally, the symmetrical polyamines, nor- and homo
spermidine, are not commercially available. Therefore, current method
ology has focused on the preparation of protected spermidine reagents,
derived usually from compounds simpler than spermidine itself.
A review of the literature reveals there are currently several re
agents available for the selective acylation of spermidine. The first of
these, designed by Eugster et al., N4-tosyl-N8-phthaloylspermidine, is
designed to fix three different substituents to the spermidine backbone.79
The eight steps required for the synthesis of this reagent and the mode
of protective group removal make it somewhat impractical. A second re
agent, N4,N8-di-t-butoxycarbonyl spermidine (accessible in 49% yield in
three steps) is acceptable for what it is designed for--introduction of
an acyl group at the N^position.80 A more recent development employs
1 -(4-aminobutyl)hexahydropyrimidine and 1-(3-aminopropyl)hexahydropyri-
dine for the selective acylation of the terminal primary amino nitrogens
24

25
of spermidine and homospermidine.81 This procedure represents an excel
lent protocol for functionalization of these two polyamines although it
cannot be extended to norspermidine and the conditions required for the
opening of the pyrimidine can be restrictive.
Recently, Bergeron and coworkers have described the preparation of
the benzylated polyamines N4-benzylspermidine, N4-benzylnorspermidine,
and N5-benzylhomospermidine as reagents for the selective acylation of
the primary amines.82 This synthesis offers the flexibility of generat
ing the varying chain lengths desired, as well as employing inexpensive
starting materials. The scheme employs benzyl amine as the common start
ing material for all three reagents, Figure 2-1. As illustrated, benzyl
amine is reacted with one equivalent of acrylonitrile, followed by alky
lation with 4-chlorobutyrylnitrile which, upon reduction, generates N4-
benzylspermidine (Vila). Alternatively, benzyl amine can either be react
ed with two equivalents of acrylonitrile or two equivalents of 4-chloro
butyrylnitrile, producing upon reduction the symmetrical reagents N5-
benzyl homo spermidine (VI lb) or N4-benzylnorsperr,iidine (Vile), respective
ly. These three reagents represent an excellent method of efficiently
generating the terminally bis-acylated spermidine desired as outlined
in Figure 2-2. The benzylated polyamines (VII) can be reacted with two
equivalents of the desired acylating agent, followed by removal of the
benzyl protecting group via hydrogenolysis over palladium chloride cata
lyst producing the desired derivatives (IX), figure 2-2.. In order to
determine if there are any size restrictions placed on uptake by the re
ceptor, in this study the acyl groups acetyl, propionyl and benzoyl were
affixed to the reagents (VII) via their acid chlorides.

26
VI Ib
Figure 2-1. Synthesis of secondary N-benzylated polyamines.

27
R] COiMH (CHZ) M (CHo ) b NHCGR,
VIII
I
R,CCNH (CH2)aNH (CH-,) NHCCR,
IX
'I
R]CH,NH CH2)a- NH (CH2)b NhC'rUR,
X
Figure 2-2. General scheme for the synthesis of terminally
N-modified polyamine derivatives.

28
In turning our attention towards a reagent for the selective secon
dary N-acylation of spermidine and its homologues, the lack of a suitable
reagent in the literature necessitated that a new approach be developed.
Starting with the already available benzylated reagents (VII), this should
easily be accomplished by first blocking the terminal amines followed by
debenzylation. The only requirement in such a synthesis would be an easily
attachable/removable protecting group which is stable to the hydrogenation
conditions employed. The t-butoxycarbonyl protecting group meets this
requirement.
Hence, (VII) is reacted with two equivalents of t-butoxycarbonyl-
oxyimine 2-phenylacetonitrile (BOC-ON) to form the terminally bis-t-
butoxycarbonyl compounds in high yield, Figure 2-3. The benzyl group is
then removed via hydrogenolysis as before producing the reagents (XII)
capable of selectively secondary N-acylation. These t-BOC protected re
agents are then reacted with a variety of acylating agents, again of vary
ing size such as acetyl, benzoyl, and hexanoyl acid chlorides. The t-BOC
protecting groups are then removed quantitatively with trifluoroacetic
acid generating the secondary N-acylated spermidine derivatives (XIV),
as illustrated in Figure 2-4.
In considering the synthesis of N-alkylatod spermidine derivatives,
although the reagents N4-benzylspermidine and the symmetrical homologues
are available, direct alkylation of these compounds would presumably
result in over alkylation and poor yields. Therefore, it would seem
more practical to reduce the N-acylated derivatives already on hand via
the above two procedures directly to the N-alkylated compounds. Accord
ingly, the terminally bis-acylated derivatives (IX) could be converted
directly to the bis-alkylated derivatives (X) as shown in Figure 2-2

29
Figure 2-3. Synthesis of n"* ,N-bis (t-Butoxycarbonyl)spermi-
dine.

30
Figure 2
HC1
tBOC-NH (CH2)a NH (CH2)b NH-tBOC
XII
Y
I2
co
tBOC-NH (CH2)a N (CH2)b NH-tBOC
XIII
I
I2
CO
H2N (CH2)a N (CH2)b NH2
XIV
*
H2N (CH2)a N (CH2}b NH
2
XV
4. Synthesis of secondary N-modified polyamine
deriva ti ves .

31
employing an appropriate reducing agent. Likewise, the secondary N-
acylated compounds (XIV) could be converted to the alkyl derivatives
(XV), Figure 2-4, in the same manner.
Experimental
Materials
The reagents N4-benzylspermidine, N4-benzylnorspermidine, and N5-
benzylhomospermidine were prepared as previously described.82 All other
reagents were purchased from Aldrich Chemical Company and, except where
indicated, used without further purification. Dry methylene chloride
O
(CH2C12) was obtained by distillation followed by storage over 3 A mole
cular sieves. Dry dioxane was obtained by distillation from sodium metal
immediately before use. Sodium sulfate was used as the drying agent.
Sephadex LH-20 was obtained from Pharmacia Fine Chemicals. Preparative
thin layer chromatography (TLC) was performed on Anal tech 20 x 20 cm
silica gel GF plates.
For the physical measurements, melting points were taken on a Fisher-
Johns apparatus and are uncorrected. Proton nuclear magnetic resonance
^H-NMR) spectra were recorded on a Varian T-60 and, unless otherwise
noted, prepared in deuterated chloroform (DCC13) with chemical shifts
() given in parts per million relative to an internal (CH3)4Si stan
dard. The infrared (IR) spectra were recorded on a BeckmanAcculab 1 spec
trophotometer. Elemental analyses were performed by Galbraith Labora
tories, Knoxville, TN, or Atlantic Microlabs, Atlanta, GA.
Synthesis of Terminally N-modified Spermidine Derivatives
General methods
The bis-acylated polyamines were prepared from the appropriate
secondary benzylated polyamine as described for the following synthesis

32
of N1,N8-bis(acetyl)spermidine. The procedure is the same regardless of
the benzylated polyamine or acylating agent employed.
A solution containing 2.2 equivalents of acetyl chloride was react
ed with N4-benzylspermidine in the presence of triethyl amine as base to
cleanly afford N4-benzyl-N1,N8-bis(acetyl)spermidine in 98% crude yield.
The crude products are usually of sufficient purity that they can be
debenzylated without further purification. Accordingly, N4-benzyl-N1,N8-
bis(acetyl)spermidine was hydrogenated overnight over palladium chloride
catalyst in methanol/HCl to afford pure N1,N8-bis(acetyl)spermidine as
the hydrochloride salt upon recrystallization. The corresponding amine
was prepared by reduction of this bis(amide) with a suitable reducing
agent.
Of the many reducing agents available, sodium borohydride-trifluoro-
acetoxy complex83 was chosen due to its relative ease and mildness of
reduction. Other reagents, such as lithium aluminum hydride, are often
less selective and may cleave tertiary amides, an important consideration
for the preparation of the N4-alkyl derivatives. Therefore, N2,N8-bis-
(ethyl)spermidine was prepared by refluxing N1,N8-bis(acetyl)spermidine
and sodium trifluoroacetoxyborohydride in dry dioxane for eight hours to
afford the desired product in 78% yield after distillation. The result
ing amines are then usually converted to their hydrochloride salts to
prevent oxidation and improve handling.
N4-Benzyl-N1,N8-bis(acetyl)spermidine (1)
A solution of acetyl chloride (725 mg, 9.2 mmol) in 10 dry CH2Cl2
was added dropwise to a cooled solution (ice bath) of N4-benzylspermidine
(1.0 g, 4.2 mmol) and triethylamine (1.3 ml, 9.2 mmol) in 20 ml dry
CH2C12 under a N2 atmosphere. After the addition was completed, the

33
ice bath was removed and the mixture allowed to stir for a total of 18
hours. Additional CH2C12 (50 ml) was then added and the reaction mix
ture washed with cold 3% HC1 (3 x 20 ml), H20 (2 x 20 ml), 5% NaHC03
(3 x 20 ml), H20 (2 x 20 ml), dried, and evaporated to afford 1.3 g
(98%) of the desired product as an oil: NMR <$ 1.50 (m, 6H), 1.84 (d,
6H), 2.40 (m, 4H), 3.17 (m, 4H), 3.42 (s, 2H), 6.26 (br, 2H), 7.20 (s,
5H); IR (CHClg) 3310 (s), 2975 (s), 1650 (s), 1550 (s), 760 (s) cm'1.
An analytical sample was prepared by preparative TLC eluting with
10% methanol/CH2Cl2. Analysis calculated for Ci8H29N302-H20: C, 65.82;
H, 9.21; N, 12.79. Found: C, 65.67; H, 9.24; N, 12.73.
N4-Benzyl-N1 ,N8-bis(propionyl )spermidine (2_)
A solution of propionyl chloride and N -benzylspermidine was react
ed and purified in the same manner as for (1_). Yield: 1.35 g (93%);
:H NMR 6 1.10 (dt, 6H), 1.56 (m, 6H), 2.05 (quar., 4H), 2.38 (m, 4H),
3.2 (m, 4H), 3.46 (s, 2H), 6.14 (br, 2H), 7.23 (s, 5H); IR (CHC13)
3320 (s), 2980 (s), 1660 (s), 1550 (s), 770 (s) cm'1.
Anal. cal. for C2oH33N30-tH20: C, 67.38; H, 9.61; N, 11.79. Found:
C, 67.61; H, 9.60; N, 11.75.
N4-Benzyl-N1,N8-bis(benzoyl)spermidine (2)
A solution of benzoyl chloride and N4-benzylspermidine was reacted
and purified in the same manner as for (1_). Yield: 3.4 g (91%); XH NMR
<5 1.65 (m, 6H), 2.50 (m, 4H), 3.45 (m, 4H), 6.57 (br, 2H), 6.96-7.85
(m, 15H); IR (CHC13) 3340 (s), 1640 (s), 1520 (s), 1310 (m), 690 (m) cm'1.
Anal. cal. for C28H33N302: C, 75.81; H, 7.50; N, 9.47. Found:
C, 75.73; H, 7.46; N, 9.37.

34
N1,N8-Bis(acetyl)spermidine hydrochloride (4)
Palladium chloride (100 mg) was added to a solution of (1_) (1.15 g,
3.6 mmol) in methanol (50 ml) containing concentrated HC1 (seven drops).
The resulting suspension was stirred under a hydrogen atmosphere over
night. The catalysts were then filtered, washed with methanol, and the
filtrates evaporated. The resulting crude solid was recrystallized from
ethanol/ether, the solid collected by filtration, and dried under high
vacuum over P205 to afford 550 mg of the desired product as a white solid;
mp 133C. Concentration of the mother liquor and recrystallization af
forded another 330 mg. Total yield: 880 mg (92%); XH NMR (D20) 6 1.70
(m, 6H), 2.04 (s, 6H), 3.20 (m, 8H); IR (KBr) 3300 (br), 1625 (s), 1530
(m) cm"1.
Anal. cal. for 1H24N302C1: C, 49.71; H, 9.10; N, 15.81. Found:
C, 49.45; H, 9.15; N, 15.48.
N1,N8-Bis(propiony1 )spermidine hydrochloride (5.)
A solution of l^-benzyl-N1,N8-bis(propionyl)spermidine (2) was hy
drogenated and purified in the same manner as described for (4_). Yield:
780 mg (86%); mp 147-148C; XH NMR (D20) 6 1.10 (t, 6H), 1.72 (m, 6H),
2.35 (quar., 4H), 3.16 (m, 8H); IR (KBr) 3350 (br), 1630 (s), 1540 (m)
cm"1.
Anal. cal. for C13H28N302C1: C, 53.14; H, 9.60; N, 14.30. Found:
C, 52.81; H, 9.50; N, 13.86.
N1 ,N8-Bis(benzoyl )spermidine hydrochloride (jj)
A solution of N4-benzyl-N1,N8-bis(benzoyl)spermidine (3) was hydro
genated and purified in the same manner as described for (4). Yield:
2.9 g (93%); XH (D20) 6 1.74 (m, 6H), 3.20 (m, 8H), 7.06-7.82 (m, 10H);
IR (KBr) 3300 (s), 1635 (s), 1540 (s), 690 (m) cm"1.

35
N1,N8-Bis(ethy1)spermidine trihydrochloride ()
A suspension of N1,N8-bis(acetyl)spermidine hydrochloride (330 mg,
1.24 mmol) and sodium borohydride (500 mg, 13 mmol) in 20 ml freshly dis
tilled dioxane was cooled to 10-15C under a N2 atmosphere. A solution
of trifluoroacetic acid (1.5 g, 13 mmol) in 10 ml dry dioxane was slowly
added dropwise with stirring. After the addition was completed, the
suspension was slowly brought to a gentle reflux and the reaction con
tinued for ten hours.
The reaction was then cooled, the excess reducing agent destroyed
by careful addition of water (2 ml), and the solvent reduced under high
vacuum. The residue was treated with 2N K0H (5-10 ml), the product ex
tracted into CH2C10 (4 x 20 ml), dried, and concentrated to afford 240
mg crude product as a semi sol id. The product was further purified by
distillation to afford 190 mg (78%): bp 133-135, 0.25 mm; NMR <5 0.88-
1.82 (overlapping multiplets, 15H), 2.60 (m, 12H); IR (CHC13) 2980 (s),
1465 (m), 1120 (m), 750 (s) cm-1.
The distilled amine was converted to the hydrochloride salt by dis
solving in a solution of ethanol and ether (1:1), cooling, and then bub
bling HC1 gas through.
Anal. cal. for C11H30N3C13: C, 42.52; H, 9.73; N, 13.52. Found:
C, 42.37; H, 9.68; N, 13.19.
N1 ,N8-Bis(propy1)spermidine trihydrochloride (&)
A suspension of N1,N8-bis(propionyl)spermidine hydrochloride was
reduced and purified as described for (7J. Yield: 210 mg (71%); bp
144-147, 0.10 mm; *H NMR 0.90 (t, 6H), 1.16-1.80 (m, 13H), 2.55 (m,
12H); IR (CHC13) 2975 (s), 1460 (m), 1120 (m), 760 (s) cm-1.

Figure 2-5. 60 MHz ^ H NMR spectrrum of N4-benzy1-N1,N8-bis(acety1)spermi di ne (1).
co
en

Figure 2 6. 60 MHz Vi NMR spectra of NVN-bi?(acety1)spermidine(4).

t
1.0
to
so
M.0
Figure 2-7. 60 MHz NMR spectrum of N1
bis(ethyl )spermidine(_7).

PERCENT TRANSMISSION
V/AVtUNt.lM III MICRONS
3000
WMN tlOilCMC JMiiM CH*f NO
2000 I BOO
wavenumber cm1
1600 I 400 I 200
NiiMW njm-MiMij Inc rumion. (aihom*. u a
Figure 2-8. IR spectrum of N^-benzyl-N^ ,N-bis(acetyl)spermidine (1).
CJ
to
PERCENT TRANSMISSION

Figure 2-9. IR spectrum of N1,N8-bis(acetyl)spermidine (4).
-e*
o
PKCENT TRANSMISSION

2-10. IR spectrum of N1,N8-bis(ethy1)spermidine (7).
PERCENT TRANSMISSION

42
Anal. cal. for Cx3H31+N3C13: C, 46.09; H, 10.12; N, 12.40. Found:
C, 45.59; H, 9.65; N, 12.07.
N1 ,N8-Bis(benzyl)spermidine trihydrochloride (9j
A suspension of N1,N8-bis(benzoyl)spermidine hydrochloride was re
duced and purified as described for (_7). Yield: 1.5 g (89%); bp 183-185,
0.10 mm; NMR 6 1.52 (m, 9H), 2.56 (m, 8H), 3.44 (s, 4H), 7.22 (s, 1 OH);
IR (CHC13) 2970 (s), 1450 (m), 1130 (m), 760 (s) cm-1.
Anal. cal. for C21H35N3C13: c, 68.07; H, 9.25. Found: 0,67.82 ;
H, 9.18.
Synthesis of Secondary N-modified Spermidine Derivatives
General methods
The reagents N1,N8-bis(t-butoxycarbonyl)spermidine, N1,N7-bis(t-
butoxycarbonyl)norspermidine and N1,N9-bis(t-butoxycarbonyl)homospermi-
dine were prepared by reacting the appropriate benzylated spermidine with
2.1 equivalents of B0C-0N84 in tetrahydrofuran. The resulting benzyl -
bis(t-BOC) polyamines were then hydrogenated over palladium chloride
catalysts in MeOH/HCl to afford the bis(t-BOC) spermidine reagents as
the hydrochloride salts in 80-90% yields from the benzylated polyamine.85
Again, the preparation of N4-hexanoyl and N4-hexylspermidine are
illustrative as the same methodology is employed regardless of the bis-
(t-BOC protected) spermidine reagent or acylating agent used. A solution
of N1,N8-bis(t-butoxycarbonyl)spermidine-HC1 and 1.1 equivalents of hexan-
oyl chloride were allowed to react in the presence of base in dry di-
chloromethane to afford f^-hexanoyl-N1,N8-bis(t-butoxycarbonyl)spermi-
dine in 95% yield. Once again, the products of these reactions are
usually clean enough to be used without further purification. There
fore, N4-hexanoyl-N1,N8-bis(t-butoxycarbonyl)spermidine is reacted with
trifluoroacetic acid to afford N4-hexanoyl spermidine, quantitatively.

43
The resulting trifluoroacetic salts are very hydroscopic and therefore,
conmonly converted to the hydrochloride salts. Finally, N4-hexanoyl-
spermidine dihydrochloride can be converted to the corresponding amine,
again utilizing sodium trifluoroacetoxyborohydride as the reducing agent.
N4-Benzyl-N1,N8-bis(t-butoxycarbony1)spermidine (10)
A solution of BOC-ON (5.4 g, 0.022 mol) in 50 ml of distilled tetra-
hydrofuran (THF) was slowly added dropwise with stirring to a cooled solu
tion (ice bath) of N4-benzylspermidine (2.35 g, 0.010 mol) in 75 ml THF
under N2. After the addition was completed, the ice bath was removed,
and the reaction mixture allowed to stir for eight hours at which time
the solvent was evaporated. The residue was dissolved in 150 ml of ether
and washed with 5% NaOH (4 x 25 ml), water (3 x 25 ml), dried, and evap
orated to afford 4.3 g (99%) of the desired product, as a viscous light
yellow oil. Thin layer chromatography and XH NMR analysis indicated
the crude oil purity was in excess of 95%, and subsequently used without
further purification.
An analytical sample was prepared by preparative TLC, eluting with
10% Me0H/CH2Cl2: XH NMR 5 1.45 (s, 18H), 1.40-1.88 (m, 6H), 2.18-2.70
(m, 4H), 2.82-3.35 (m, 4H), 3.48 (s, 2H), 4.79-5.63 (br, 2H), 7.17
(s, 5H); IR (CHC13) 3390 (m) 1705 (s), 1510 (s), 1190 (s), 760 (m) cm'1.
Anal. cal. for C24H41N304: C, 66.18; H, 9.49; N, 9.65. Found: C,
65.93; H, 9.79; N, 9.38.
N4-Benzy1-NI,N7-bis(t-butoxycarbonyl)norspermidine (11)
A solution of N4-benzylnorspermidine and B0C-0N was reacted and
purified as described previously for (JO): yield: 2.0 g (98%); XH NMR
6 1.41-1.83 (m, 4H), 1.44 (s, 18H), 2.11-2.68 (m, 4H), 2.77-3.35 (m, 4H),
3.50 (s, 2H), 4.75-5.68 (br, 2H), 7.21 (s, 5H); IR (CHC13) 3400 (m), 1700
(s), 1510 (s), 1175 (s), 755 (m) cm*1.

44
Anal. cal. for C23H3gN30l+: C, 65.53; H, 9.32; N, 9.97. Found: C,
65.49; H, 9.35; N, 9.9$.
N5-Benz,yl-N1>N9-bis(t-butox,ycarbony1) homos pe rmi dine (12)
A solution of N5-benzylhomospermidine and BOC-ON was reacted and
purified as described previously for (10): Yield: 1.35 g (93%); XH NMR
5 1.38-1.87 (m, 8H), 1.47 (s, 18H), 2.13-2.71 (m, 4H), 2.78-3.33 (m, 4H),
3.51 (s, 2H), 4.80-5.65 (br, 2H), 7.19 (s, 5H); IR (CHC13) 3420 (m),
1690 (s), 1520 (s), 1180 (s), 750 (m) cm"1.
Anal. cal. for C25H43N304: C, 66.78; H, 9.64; N, 9.35. Found:
C, 66.52; H, 9.69; N, 9.25.
N1,M8-Bis(t-butoxycarbonyl)spermidine-hydrochloride M31
Palladium chloride (350 mg) was added to a solution of (1_0) (3.9 g,
9.0 mmol) in 50 ml methanol containing concentrated hydrochloric acid
(0.75 ml, 9.0 mmol). The resulting suspension was stirred under a hydro
gen atmosphere overnight (18 h). The catalysts were then filtered, washed
with MeOH, and the filtrates evaporated. The resulting crude solid was
recrystallized from ethanol/ether to afford 3.4 g (94%) of the desired
product: mp 149-150C; lH NMR (D20) 1.50 (s, 18H), 1.66 (m, 6H), 3.14
(m, 8H); IR (KBr) 3380 (s), 1690 (s), 1520 (s), 1190 (m) cm1
Anal. cal. for C17H36N304C1: C, 53.46; H, 9.50; N, 11.00. Found:
C, 53.41; H, 9.54; N, 10.99.
N1,N7-Bis(t-butoxycarbonyl)norspermidine-hydrochloride (14)
A solution of (1J_) was hydrogenated and purified in a similar man
ner as described for (1_3). Yield: 1.2 g (89%); mp 173-174C; NMR
(D20) 6 1.48 (s, 18H), 1.78 (m, 4H), 3.16 (m, 8H); IR (KBr) 3400 (s),
1700 (s), 1515 (s), 1170 (m) cm-1.

45
Anal. cal. for C16H34N304C1: C, 52.23; H, 9.31; N, 11.42. Found:
C, 52.29; H, 9.31; N, 11.37.
N1>N9-Bis(t-butoxycarfaon.yl) hornos pe rmi dine hydrochloride (15)
A solution of (12) was hydrogenated and purified in a similar manner
as described for (13). Yield: 770 mg (92%); mp 187-188C; XH NMR (D20)
1.47 (m, 26H), 3.19 (m, 8H); IR (KBr) 3380 (s), 1690 (s), 1510 (s),
1175 (m) cm-1.
Anal. cal. for C18H38N3O4CI: C, 54.60; H, 9.67; N, 10.61. Found:
C, 54.58; H, 9.67; N, 10.59.
N4-Acetyl-NI>N8-bis(t-butoxycarbonyl )spermidine (16.)
A solution of acetyl chloride (180 mg, 2.2 mmol) in 10 ml dry CH2CI2
was slowly added to a cooled solution of (1_3) (760 mg, 2.0 mmol) and
triethylamine (600 yl, 4.4 mmol) in 30 ml CH2C12 under N2. The solution
was allowed to warm to room temperature and stirred overnight (18 h)
at which time additional CH2C12 (25 ml) was added. The organic layer
was washed with 3% HC1 (3 x 15 ml), H20 (2 x 15 ml), 5% NaHC03 (3 x 15
ml), H20 (2 x 15 ml), dried and concentrated to afford 720 mg (93%)
of the product as a colorless oil: NMR 6 1.42 (s, 18H), 1.64 (m, 6H),
2.06 (s, 3H), 3.18 (m, 8H), 5.02 (br, 2H); IR (CHC13) 3320 (m), 2960 (s),
1690 (s), 1620 (m), 1170 (s) cm-1.
An analytical sample was prepared by chromatography of silica gel
(70-230 mesh) eluting with EtOAc/CHCl3 (1:1). Anal. cal. for ClgH37N305-r
H20: C, 57.55; H, 9.66; N, 10.60. Found: C, 57.46; H, 9.30; N, 10.22.
N4-Hexanoyl-N1,N8-bis(t-butoxycarbony1)spermidine (17)
A solution of hexanoyl chloride and (1_3) were reacted and purified
in the same manner as for (1_6). Yield: 620 mg (95%); NMR 6 0.90

46
(t, 3H), 1.12-1.95 (m, 30H), 2.31 (quar., 2H), 3.18 (m, 8H), 5.00 (br,
2H); IR (CHC13) 3330 (m), 2970 (s), 1680 (s), 1625 (m), 1175 (s) cm1.
Anal. cal. for C23H45N305: C, 62.27; H, 10.22; N, 9.47. Found:
C, 62.48; H, 10.25; N, 9.23.
N^Benzoyl-N1 ,N8-bis(t-butoxycarbonyl)spermidine (18)
A solution of benzoyl chloride and (1_3) was reacted and purified in
the same manner as described for (16). Yield: 470 mg (95%); !H NMR 5
1.42-1.92 (m, 24H), 2.83-3.58 (m, 8H), 5.23 (br, 2H), 7.19 (s, 5H); IR
(CHC13) 1710 (s), 1630 (m), 1515 (m), 1225 (s), 770 (s) cm-1.
Anal. cal. for C24H39N305-jH20: C, 62.86; H, 8.79; N, 9.16. Found:
C, 62.98; H, 8.99; N, 8.85.
N4-Acetylspermidine hydrochloride (19)
To a cooled flask containing (16) (600 mg, 1.55 mmol) was added
20 ml trifluoroacetic acid. The reaction was allowed to warm to room
temperature and stirred for 20 minutes at which time the solvent was
quickly evaporated. The residue was dissolved in methanol and evaporated
(twice). The residue was then dissolved in ethanol/ether, cooled, and
HC1 gas bubbled through. The solid was collected by filtration, and
dried in vacuo over P205 to afford 360 mg (89%) of the desired product:
XH NMR (D20) 6 1.30-1.88 (m, 24H), 2.06 (s, 3H), 3.18 (m, 8H), 5.00 (br,
2H); IR (KBr) 3300 (br), 1690 (s), 1520 (m) cm"1.
Anal. cal. for C9H23N30C12: C, 41.54; H, 8.91; N, 16.15. Found:
C, 41.18; H, 9.04; N, 15.95.
N4-Hexanoy1spermidine dihydrochloride (20)
A solution of (17) was deprotected and purified in a similar manner
as described for (19). Yield: 500 mg (89%); XH NMR (TFA) 6 0.96 (m, 3H),
1.40 (m, 6H), 1.60-2.36 (m, 6H), 2.66 (m, 2H), 2.96-3.90 (m, 8H), 6.92
(br, 6H); IR (KBr) 3300 (br), 1680 (s), 1530 (m) cm"1.

4 7
Anal. cal. for C13H31N30C12: C, 49.36; H, 9.88; N, 13.28. Found:
C, 49.23; H, 9.77; N, 12.75.
N4-Benzoyl spermidine di hydrochloride (21)
A solution of (1_8) was deprotected and purified in a similar manner
as described for (19). Yield: 370 g (92%); XH NMR (TFA) 6 1.62-2.18
(m, 6H), 2.78-3.52 (m, 8H), 7.20 (s, 5H), 8.15 (br, 6H); IR (KBr) 1700
(s), 1540 (m), 750 (s) cm-1.
Anal. cal. for C18H25F6N305H20: C, 43.64; H, 5.49; N, 8.48. Found:
C, 43.79; H, 5.41; N, 8.17.
N4-Ethylspermidine trihydrochloride (22)
To a cooled suspension (10-15C) of N4-acetylspermidine dihydro
chloride (1_9) (500 mg, 1.92 mmol) and sodium borohydride (380 mg, 10
mmol) in 15 ml dry dioxane was added a solution of trifluoracetic acid
(1.14 g, 10 mmol) in 5 ml dry dioxane. After the addition was completed,
the suspension was slowly brought to a gentle reflux and the reaction
continued for eight hours under N2.
The reaction was then cooled,the excess reducing agent destroyed
by careful addition of water (2 ml), and the solvent reduced under high
vacuum. The residue was treated with 2N K0H (5 ml), the product ex
tracted into CH2C12 (4 x 20 ml), dried, and concentrated to afford 350
mg crude product. The product was further purified by Kugelrohr dis
tillation to afford 280 mg (78%) of the desired product: *H NMR (s)
0.94 (t, 3H), 1.64 (m, 8H), 2.12 (quat., 2H), 2.67 (m, 8H); IR (CHC13)
3320 (m), 2960 (s), 1430 (m) cm-1.
The amine was subsequently converted to its hydrochloride salt by
HC1 gas. Anal. cal. for C9H26N3C13: C, 38.24; H, 9.27; N, 14.86. Found:
C, 38.01; H, 9.31; N, 14.49.

48
N4-Hexylspermidine trihydrochloride (23)
A solution of (20) was reduced and purified in a similar manner as
described for (22). Yield: 180 mg (82%); *H NMR 5 0.85 (m, 3H), 1.04-
1.82 (overlapping m, 18H), 2.12-2.85 (m, 1 OH); IR (CHC1 3) 3300 (m),
2970 (s), 1425 (m) cm-1.
Anal. cal. for C:3H31+N3C13: C, 46.09; H, 10.12; N, 12.40. Found:
C, 46.37; H, 10.04; N, 12.17.
N4-Methy1-NI>N8-bis(t-butoxycarbonyl)spermidine (24)
Three hundred fifty milligrams of N1,N8-bis(t-butoxycarbonyl)sper-
midine hydrochloride (1_3) were added to a 15% aqueous solution of Na2C03.
The solution was extracted with ether (3 x 30 ml), the organic layer
dried, and concentrated to afford 300 mg of the free amine as an oil.
To a solution of N1,N8-bis(t-butoxycarbonyl)spermidine (300 mg, 0.9
mmol) in 3 ml acetonitrile was added a 37% aqueous formaldehyde solution
(0.4 ml, 5 mmol) and sodium cyanoborohydride (100 mg, 1.6 mmol). A mild
ly exothermic reaction ensued which subsided after a minute or so. After
15 min, the pH of the solution was checked, adjusted to pH 7.0 with acetic
acid, and the reaction allowed to stir for an additional 1.5 h.
The solvent was then reduced in vacuo, the residue dissolved in
2N K0H (5 ml), and the product extracted with ether (3 x 15 ml). The
ether layer was dried and concentrated to afford 400 mg of the crude
product.
Further purification was effected by chromatography on silica gel
(Merck 7734) eluting with Me0H/CHCl3 (5% -* 15% MeOH) to afford 210 mg
(65%) pure (24): lH NMR 6 1.43 (m, 24H), 2.24 (s, 3H), 2.42 (m, 4H),
3.06 (m, 4H), 5.14 (br, 2H); IR (CHC13) 3390 (m), 1700 (s), 1510 (s),
1180 (s) cm-1.

49
N4-Meth,ylspermidine trihydrochloride (5.)
Trifluoroacetic acid (10 ml) was added to a flask containing N4-
methyl-N1,N8-bis(t-butoxycarbonyl)spermidine (24) (110 mg, 0.3 mmol),
and the resulting solution stirred for 20 min. The solvent was then
evaporated, the residue dissolved in MeOH (25 ml) and concentrated (re
peated twice) to afford 120 mg product as the trifluoroacetate salt.
The product was subsequently exchanged to the hydrochloride salt by
dissolving in cold EtOH (3 ml), bubbling HC1 gas through the solution,
and collecting the solid via filtration. The solid was dried in vacuo
over P205 to afford 70 mg (87%): XH NMR (D20) 6 1.75 (m, 4H), 2.10
(m, 2H), 2.87 (s, 3H), 3.15 (m, 8H).
Anal. cal. for C8H25N3C13: C, 35.63; H, 9.34. Found: C, 35.17;
H, 9.52.
Norspermidine trihydrochloride (26)
A sample of N1,N7-bis(t-butoxycarbonyl)norspermidine hydrochloride
(14) was exposed to TFA and purified in the same manner as described
for (25). Yield: 260 mg (83%); *H NMR (D20) 5 2.20 (m, 4H), 3.22 (m,
8H).
Homospermidine trihydrochloride ()
A sample of N1,N9-bis(t-butoxycarbonyl)spermidine hydrochloride
(1_5) was exposed to TFA and purified in the same manner as described for
(25). Yield: 620 mg (91%); XH NMR (D20) 6 1.84 (m, 8H), 3.18 (m, 8H).
Results and Discussion
In order to further characterize the polyamine's receptor, a wide
array of polyamine derivatives would be required to adequately judge
the effects of functionalization on uptake. This necessitated a synthetic
scheme capable of both flexibility and efficiency to deliver the desired

Figure 2-11. 60 MHz NMR spectrum of M-bis(t-butoxycarbonyl )spermidine
hydrochloride(13).

Figure 2-12. 60 MHz Hi NMR spectrum of N^-hexanoy 1-NHfi^-bi s (t-butoxycarbony 1 )-
spermidine (17).

en
ro
Figure 2-13. 60 MHz ^ H NMR spectrum of N^-hexanoylspermidine tris(trifluoro-
a ce ta te)(20).

Figure 2-14. 60 MHz ^ H NMR spectrum of N^-hexy1spermidine(23).

PERCENT TRANSMISSION
WAVUNGIH IN MICRONS
3000
WMH IIOIMIMO VKtl (Hill HO. lomi
3000 ieoo
WAVENUMBER CM*
1600 1100 1300 1000
mccmam ti&iauMHiis **c. imita ion. CMifoaHM. u a a.
600
HHIIUHUIA.
Figure 2-15. IR spectrum of N^-hexanoy1spermidine (20).
cn
-P*
PERCENT TRANSMISSION

PERCENT TRANSMISSION
Figure 2-16. IR spectrum of N4-hexylspermidine (23).
percent transmission

1- 1 lit 1.1 I .1... 1 .11IIl1- 1 I t I I I ~ I I 1 I 1
TO iO 50 *4.0 3.0 i.O 1.0 O
Figure 2-17. 60 MHz Hi NMR spectrum of N^-methyl-n\N-bis(t-butoxycarbony1 )-
spermidine(24j ,

v .A
-i- z .i 1 nzi z: i -i 1.1i
zz z 1.... muzz fuzz i i 1
J.I u
-1 I L
10
4 0
5.0
M.O
3 0
2.0
1.0
Figure 2-18. 60 MHz ^ H nmr spectrum of N^-methylspermidine (25).

10
to
5.0
<1.0
Figure 2-19. 60 MHz "* H MMR spectrum of
I
.i. > (ii i i... 1.... > i.
>.,i. .. ... 1
cn
00
3.0 2.0 1.0
norspermidine trihydrochloride(2f>) .

\
~&/ ,.i. Hi1 i -1, i i 1.. iii .-I-- i i -1 i i 1 i i 1. i 1 i -1
1-0 0 5.0 M.O 3 0 J.O 1.0 O
0
Figure 2- 20. 60 MHz NMR spectrum of homospermidine trihydrochi oride (27 ).

60
derivatives in the fewest steps in good yields. Utilizing the key
synthons N4-benzylspermidine and its homologues, this objective could
be realized.
Based on earlier studies,85 N4-benzylspermidine could be reacted
with a wide variety of acylating agents and debenzylated to generate
N1,N8-bis(acyl)spermidines, both steps proceeding in high yields as out
lined in Figure 2-2. Indeed, this was the case as,in all instances, the
benzylated polyamines were condensed with the appropriate acylating agents
in yields in excess of 90%. The reactions proceeded so smoothly that,
in fact, the crude products obtained after simple acid-base workup could
usually be debenzylated without further purification.
The benzyl bis(acyl)spermidines (VIII) were debenzylated as in ear
lier studies; however, the solvent acetic acid was replaced with methanol
containing one equivalent of concentrated hydrochloric acid. In addi
tion to the obvious improvement in ease of removing the solvent on work
up, the hydrogenations proceeded at a faster rate compared to the earlier
methods. Reaction times were generally on the order of 12-18 hours;
however, it appeared that the reactions may be finished in six hours or
so.
The resulting terminally bis-acylated spermidine derivatives are
thus generated in two high yield steps and can be converted directly
to bis-alkyl derivatives. Sodium borohydride-trifluoroacetic acid com
plex was used to accomplish this reduction of the amide to the amine.
Lithium aluminum hydride is the reagent commonly used for the reduction
of such amides; however, its extreme reactivity would be a detriment if
other reducible groups were present. As it is the long term goal to
conjugate antineoplastics (which may contain susceptible functionalities)
to spermidine, a milder reducing agent was sought.

61
Sodium borohydride itself does not reduce amides; however, the addi
tion of one equivalent of organic acid produces a reducing agent now
capable of this.83 In fact, the order of reduction is now reversed
compared to most other reducing agents, i.e., tertiary>secondary>primary
amides. Additionally, no cleavage of tertiary amides to the aldehyde
and amine is noted, an important consideration for the reduction of
secondary N-acylated derivatives.
The synthesis of secondary N-acylated spermidine derivatives was
also easily accomplished, again using N4-benzylspermidine as the key
starting material.
In this case the benzylated polyamines (VII) were first reacted
with a transitory protecting group followed by debenzylation to produce
the reagents (XII), Figure 2-3. The t-butoxycarbonyl group served as
an excellent choice of protecting group for it is both quantitatively
put on and removed. Accordingly, N4-benzylspermidine was reacted with
a slight excess of BOC-ON, followed by hydrogenation, again employing
PdCl2 and MeOH/HCl to afford the bis-terminally protected reagents (XII),
in 90% yield from (VII) after recrystallization.75
Once generated, the bis-BOC spermidines were also capable of selec
tive acylation of spermidine as shown by the synthetic scheme in Figure
2-4. As with the benzylated reagents, acylations with bis-BOC-spermidine
(XII) likewise proceed smoothly to afford the N4-acyl N1,N8-bis(t-butoxy-
carbonyl)spermidine cleanly and in yields in excess of 85%. The desired
N4-acylspermidines (XIV) were then generated by brief exposure to tri-
fluoroacetic acid. These N4-acyl derivatives are also efficiently reduced
to the N-alkyl compounds (XV) using NaBH4-CF3C02H. Although this reduc
tion is a reduction of a tertiary amide, it proceeds smoothly and with
no apparent cleavage of the amide to a secondary amine and aldehyde.

62
Synthesis of N4-Methylspermidine
As with the synthesis of other N4-alkylspermidines, the synthesis
of N4-methylspermidine was envisioned as three steps: reaction of N^N8-
bis(t-BOC)spermidine with an appropriate acylating agent, removal of the
t-BOC protecting groups, followed by reduction of the amide. In the
corresponding retrograde synthesis, N4-methylspermidine could be pre
pared by the reduction of N4-formylspermidine which, in turn, could be
generated by deprotection of N1,N8-bis(t-BOC)N4-formylspermidine. Indeed,
this last compound was easily synthesized by reacting N1 ,N8-bis(t-B0C)-
spermidine with acetic formic acid anhydride. However, its subsequent
deprotection with trifluoroacetic acid always led to inseparable mixtures
of the desired N4-formylspermidine and spermidine itself. This observa
tion was not totally unexpected as the formyl group is commonly employed
as a N-protecting group which is removable by acid conditions. Even when
the reaction time was limited to two minutes at 0C, substantial loss of
the formyl group still resulted.
Alternatively, other methods were available for methylation of
amines. Direct alkylation of N1,N8-bis(t-B0C)spermidine with methyl
iodide resulted in mixtures of unreacted starting material, product,
and overalkylation to the quaternary salt. However, reductive alkyla
tion with formaldehyde and sodium cyanoborohydride87 led to the formation
of N1,N8-bis(t-BOC)-N4-methylspermidine in 70% yield after chromatography.
The desired N4-methylspermidine was subsequently obtained by the usual
deprotection with trifluoroacetic acid.
In summary, the reagents N4-benzylspermidine, N1,N8-bis(t-butoxy-
carbonyl)spermidine and their symmetrical homologues were synthesized
and used to prepare selectively N-modified spermidine derivatives. The

63
synthesis of a variety of N-acylated and N-alkylated spermidines was
described and illustrates that the number of polyamine derivatives that
can be generated by these methods are virtually limitless. Additionally,
these reagents can and have been used toward the synthesis of other sys
tems, such as the siderophores, an example of which is described in
Chapter Five.

CHAPTER THREE
BIOLOGICAL EVALUATION OF POLYAMINE DERIVATIVES
Materials and Methods
Polyamine Derivatives
The following compounds were synthesized as described in Chapter
Two and the following abbreviations will be used: N4-benzylspermidine
(BSpd), N4-benzylnorspermidine (BnSpd), N5-benzylhomospermidine (BhSpd),
N4-benzoylspermidine (Benzoyl Spd), N4-methylspermidine (MeSpd), N4-
acetylspermidine (AcetylSpd), N4-Ethylspermidine (EtSpd), N4-hexanoyl-
spermidine (HexanoylSpd), N4-hexylspermidine (HexylSpd), N-(2-Cyano-
ethyl)-N-(3-cyanopropyl)benzylamine (BSpdN), N1,N8-bis(t-butoxycarbonyl)-
spermidine (BisBOCSpd), N1,N8-bis(acetyl)spermidine (BisAcetylSpd),
N1,N8-bis(ethyl)spermidine (BisEtSpd), M1,N8-bis(propionyl)spermidine
(BisPropionylSpd), N1,N8-bis(propyl)spermidine (BisPropylSpd), norsper-
midine (nSpd), and homospermidine (hSpd). The following were purchased
from Aldrich Chemical Co., Metuchen, NJ: spermidine (Spd), spermine
(Spm), and putrescine (Put). Available in this lab already was N^N8-
bis(2,3-dihydroxybenzoyl)spermidine (DHBSpd).
Ascites Leukemia Cells
Murine LI210 leukemia cells were maintained by weekly i.p. trans
plantation in female DBA/2J mice. For in vitro studies 106 leukemic
cells were inoculated i.p., four days prior to use. Cells were col
lected by peritoneal lavage with RPMI-1640. The cells were washed
twice, counted electronically, and adjusted to a density of 107 cell s/ml
for uptake studies. The effects of DMFO on Spd uptake were studied by
64

65
giving mice bearing LI210 cells 2% DFMO by drinking water for 40-48
hours prior to removal of cells.
Cell Culture
Murine L1210 leukemia cells were maintained in logarithmic growth
as a suspension culture in Roswell Park Memorial Institute Medium 1640
(RPMI-1640) containing 2% HEPES-MOPS and 10% fetal calf serum. Cells
were grown in glass tubes in a total volume of 2 ml under a humidified
5% C02 atmosphere at 37. Cultures were treated while in logarithmic
growth (0.5 to 1 x 105 cells/ml) with the polyamines, Spd derivatives,
or MGBG at concentrations ranging from 106 to 10"2 M. After 24 or 48
hours, cells were removed from tubes for counting and viability deter
minations. Cell number was determined by electronic particle counting
(Model ZF Coulter counter; Coulter Electronics, Hialeah, FL) and con
firmed periodically with hemocytometer estimates. Cell viability was
assessed by trypan blue dye exclusion (0.5% in unbuffered 0.9% NaCl
solution). Percent control growth was determined as the final treated
cell number minus the initial inoculum (_5 x 104 cell s/ml) divided by
the final untreated cell number minus the initial inoculum, times 100.
Growth data were further analyzed with a Hewlett Packard HP-85 micro
computer programmed to determine percent growth inhibition and 50% growth
inhibitory doses (ID50) at 24 and 48 hours.
Uptake Determinations
The Spd derivatives and MGBG were studied for their ability to com
pete with 3H-Spd for uptake into ascites LI210 cells in vitro. These
determinations were performed by Dr. Carl Porter's group at Roswell Park
Memorial Institute using the following procedures. Prewarmed LI210 cell
suspensions (5 x 106/cc) were incubated in 1 ml of RPMI-1640 containing

66
n HEPES-MOPS and 0.2, 0.5, 1.0, 2.0, 5.0 or 10 pM 3H-Spd (New England
Nuclear Corp., Boston, MA) alone or in the presence of 10 or 100 pM
polyamine, Spd derivative or MGBG. The cells were incubated for 20 min
at 37 except for one tube containing 10 pM 3H-Spd which was not pre
warmed and was incubated at 0 to measure surface binding. At the end
of the incubation the tubes were centrifuged at 900 g for five min at
0-4. A 200 pi aliquot of supernatant was removed for scintillation
counting and the remainder of the supernatant discarded. The pellet was
washed twice with 5-7 ml of cold RPMI-1640 containing 1 mM Spd to dis
place nonspecifically bound 3H-Spd. The pellet was then dried with a
cotton swab and dissolved in 200 pi of IN NaOH at 60 for 20-60 min.
The material was neutralized with IN HC1, diluted to 1 ml with distilled
water and transferred to a vial for scintillation counting. Uptake was
linear with time from one to 40 min and with 3H-Spd concentration up to
30 pM. Results were expressed as pmol 3H-Spd taken up per min per mg
protein as determined by the method of Lowry.88 Uptake data were analyzed
for kinetic characteristics by using a Hewlett Packard HP-85 microcomputer
programmed for nonlinear regression curve fitting.89
Acute LD50 Toxicity Studies
Acute toxicity of Spd derivatives was determined in male CD-I mice
at 24 or 36 hours. The Spd derivatives were dissolved in isotonic saline,
the pH adjusted with NaHC03 or H3P04 when necessary. The animals were
given one i.p. injection of the derivatives, the volume of which was ad
justed with additional saline, so as to deliver the same volume for each
dose. Generally, two mice per dose at several widely ranging values were
used to determine preliminary toxicity. Once the approximate LD50 is
determined, 10 mice at five doses closely around this value are used.

67
Results
Uptake of Polyamine Derivatives
The inhibition of 3H-Spd uptake in ascites L1210 cells by Spd deriva
tives, polyamines, and MGBG are summarized in Tables 3-1, 3-2, and 3-3.
For the control experiments, ascites LI210 cells exposed to 10 uM 3H-Spd
alone for 20 min at 37C take up approximately 56 pmol Spd/107 cells/min,
Table 3-1. However, at 4C this uptake is reduced to 10% of that at
37C and may represent the fraction of nonspecific binding to the cell
surface. Also shown in Table 3-1, with the exception of putrescine,
all polyamines are quite effective in preventing 3H-Spd uptake. The
polyamines Spm and hSpd are equally effective as Spd itself in competing
for uptake, inhibiting 90% of 3H uptake.
The uptake characteristics of terminally bis-modified Spd derivat
ives are reported in Table 3-2. In general, modification of Spd primary
amines produces derivatives that are either weak inhibitors or unable
to inhibit 3H-Spd uptake at all. All the bis-acylated derivatives tested
could be considered noncompetitive in preventing Spd uptake, allowing
90% of the control uptake of 3H-Spd. Only Bis(ethyl)Spd was capable of
substantially inhibiting Spd uptake, having a Ki of 62 uM.
On the other hand, modification of Spd's secondary amine appears
less restrictive. All of the secondary N-alkylated derivatives, Table
3-3, are quite competitive in inhibiting 3H-Spd uptake. Additionally,
a preference in backbone chain length of the benzyl derivatives is shown.
For example, BhSpd is more effective than BSpd in preventing uptake,
whereas BnSpd is less effective. Of the secondary N-acylated Spd deriva
tives, only N4-AcetylSpd is capable of weakly inhibiting Spd uptake.

68
Table 3-1. Effects of Polyamines and MGBG on ^H-Spd
Uptake into Ascites L1210 Leukemia Cells
Competing Agent
(lOOuM)
3h
pmo1/10'
mi n
-Spd Uptake*
cells/
%control
K -j (u M)
None
65
100
o
O
6
8
-
Put
58
86
90
nSpd
1 6
29
19
Spd
6
10
10
hSpd
6
10
10
Spm
6
11
11
MGBG
37
66
53
* Cells were incubated for 20 min at 37 with lOuM ^H-Spd
and 100 uM polyamine or MGBG
**Cells were incubated for 20 min at 37 with 0.2, 0.5, 1.0,
2.0, 5.0, or lOuM ^H-Spd and 10 or lOOuM polyamine or MGBG.

69
Table 3-2. Effects of Terminally Modified Spd Derivatives
on 3H-Spd Uptake into Ascites L1210 Leukemia
Cells
Competing Agent
(lOOuM)
3tj
pmol/I O'
min
-Spd Uptake*
cells/
^control
Ki (uM)**
None
56
100
-
BSpdN
49
88
163
BisBOC-nSpd
53
95
11 03
Bi sBOC-Spd
51
91
521
BisBOC-hSpd
50
89
504
DHBSpd
51
91
256
BisAcetyl-Spd
51
91
508
BisPropionyl-Spd
51
92
550
BisEthyl-Spd
39
69
62
BisPropyl-Spd
45
80
117
* Cells were incubated for 20 min at 37 with lOuM ^H-Spd
and lOOuM Spd Derivative
**Cells were incubated for 20 min at 37 with 0.2, 0.5,
1.0, 5.0, or lOuM ^H-Spd and 10 or 100 uM of derivative.

70
Table 3-3. Effects of Secondary
on 3H-Spd Uptake into
Cells
Modified Spd Derivatives
Ascites L1210 Leukemia
Competing Agent
3H-Spd Uptake*
pmol/107 cells/
Ki (uM)**
(lOOuM)
min
%control
None
65
100
-
N4-AcetylSpd
53
81
115
N4-Hexanoy1Spd
57
88
151
4
N -BenzoylSpd
60
92
500
N4-MeSpd
11
1 7
3.4
N4-EthylSpd
9
14
3.1
N4-HexylSpd
45
69
34
N4-BnSpd
57
88
1 35
N4-BS pd
44
67
39
N5-BhSpd
24
37
14
* Cells were incubated for 20 min at 37 with lOuM ^H-Spd
and lOOuM derivative
**Cells were incubated for 20 min at 37 with 0.2, 0.5,
1.0, 5.0, or lOuM 3H-Spd and 10 or lOOuM derivative

71
Acute LD5Q Toxicity Studies of Polyamine Derivatives
The acute toxicity of some of the polyamine derivatives synthesized
was investigated in vivo in white mice. For purposes of comparison,
the LD50 of Spd was also measured. The results of this study are pre
sented in Table 3-4. The LD50 for Spd was determined to be about 400
mg/Kg. This value is somewhat lower than a previously reported value of
470 mg/Kg;90 however, this difference can be attributed to the difference
in duration between the two studies. In the earlier study, deaths were
recorded only up to five hours while, in this study, deaths were recorded
up to 36 hours. In fact, excluding the very toxic doses, the greatest
percentage of animals given Spd died between 18 and 24 hours. The most
likely cause of death appeared to be respiratory failure.
The toxicity behavior of MeSpd was virtually identical to Spd, both
in LD50 and time course of death. However, the toxicity of BSpd and
BenzoylSpd was very much different. Death usually resulted in five minutes
or less marked by convulsions. With all four compounds, in almost every
instance, if the animals survived the first minutes no ill effects were
seen afterwards. Interestingly enough, the LD50 of the three benzyl
homologues also showed a dependency on chain length with BhSpd being the
most toxic of all compounds tested. Finally, all of the terminally N-
modified compounds were shown to be relatively nontoxic, the three bis(acyl)-
Spd having LD50's exceeding 800 mg/Kg.
Piscussion
Several lines of evidence support the existence of an energy depen
dent transport carrier for the uptake of polyamines across the cell mem
brane. The anti cancer agent MGBG has been shown to actively compete with
spermidine for uptake and is concentrated intracellularly via this carrier.56

72
Table 3-4. Acute Toxicities of Spermidine and Polyamine
Derivatives
Compound
LD50 (mg/kg)
Spd
400
DHBSpd
>800
Bis(Acetyl )Spd
>1000
Bis(Ethyl )Spd
425
N4-MeSpd
375
N4-BnSpd
250
N4-BSpd
200
N5'BhSpd
125
N4- Benzoyl Spd
300

73
Furthermore, in cells having their polyamine pools depleted by pretreat
ment with DFMO, both polyamine and MGBG uptake is enhanced several-fold.42
Accordingly, it is hypothesized that several structural parameters must
be recognized by this receptor or carrier for uptake.
In an effort to further define these necessary parameters for up
take, a wide array of spermidine derivatives were synthesized and assayed
for their ability to compete with 3H-Spd for uptake. These studies sug
gest that the primary amines of spermidine are critical for recognition
by the carrier for uptake. Acylation of both primary amines produces
derivatives that are very poor inhibitors of 3H-Spd uptake. Reduction
of these amides to the corresponding amines offers some improvement in
the derivatives' ability to compete for uptake; however, this uptake
appears severely restricted by the size of the alkyl substituent. For
example, only BisEtSpd is as effective as MGBG in preventing Spd uptake.
Modification of spermidine's secondary amine appears less critical
in conferring uptake specificity, as both N-alkylated and N-acylated de
rivatives inhibited 3H-Spd uptake. However, there is an obvious prefer
ence for the alkylated derivatives. Moreover, there is less restriction
upon the size of the alkyl substituent as all four alkyl derivatives
ranging up to hexyl and benzyl all have Ki's comparable or better than
MGBG.
With respect to chain length, quite unexpectedly, the hSpd backbone
competed more effectively than the Spd backbone as demonstrated by the
order of preference BhSpd>BSpd>BnSpd. This finding is interesting since
hSpd is not found in mammalian systems. Whether this effect will be
seen in other derivatives is currently under investigation.

74
The toxicity of the polyamine derivatives was also investigated.
None of the polyamine derivatives tested were unusually toxic; however,
an interesting relationship was noticed. A comparison of LD50 values and
Ki values reveals that the derivative's toxicity is proportional to its
uptake. For instance the bis(acyl) derivatives, bis(acetyl)Spd and
DHBSpd have LD50 in excess of 1000 mg/Kg and are unable to compete for
uptake. On the other hand derivatives which are good inhibitors of 3H-
Spd uptake generally have LD50's less than 500 mg/Kg. Moreover, this
relationship even holds true for the BSpd derivatives; BhSpd, which is
the best inhibitor of the three, is also the most toxic. These data
seem to suggest the toxicity of these derivatives is related to their
cellular uptake.
In summary, it is apparent that the terminal amines of spermidine
are of critical importance for uptake. Modification of the terminal amines
by either acylation or addition of an alkyl substituent produces a deriva
tive which is ineffective in competing for uptake. In terms of substrate
recognition by the transport system, this suggests that the positive charges
carried by spermidine's terminal amines at physiological pH are necessary
for uptake. Furthermore, although the bis-alkyl derivatives also carry
this positive charge, recognition appears to be limited by the size of
the alkyl substituent. In this case, large alkyl groups may prevent in
teraction of the positively charged terminal amines with the carrier by
interacting sterically or by changing the polyamines' conformation.
The role of the secondary amine is less certain. Modification here
appears to be less restrictive, although preference was again seen for
the alkylated derivatives. The argument for requirement of positive charge
may again be true; however, recently it has been shown that diamines such

75
as 1,8-diaminooctane, in which the central nitrogen is replaced by a car
bon, are still very good inhibitors of 3H-Spd uptake having a Ki of 22.1 uM91.
Nevertheless, the central nitrogen may be the appropriate site to con
jugate antineoplastics to.

CHAPTER FOUR
PRELIMINARY INVESTIGATIONS TOWARDS THE DEVELOP
MENT OF SPERMIDINE-ANTINEOPLASTIC CONJUGATES
It is apparent from the uptake data presented in Chapter Three that
several structural parameters are required for uptake of a Spd-derivative
via the spermidine receptor. First, bis-acylation of the terminal amines
of spermidine is limiting on uptake. However, acylation or alkylation of
spermidine's secondary nitrogen appears less restrictive, having little
negative effect on uptake. Accordingly, the secondary amine may repre
sent a potential site in which antineoplastics can be attached. There
fore, the purpose of this chapter is to begin a preliminary investiga
tion into the synthesis and uptake of several small antineoplastics con
jugated to spermidine's central nitrogen. The results of these initial
findings will hopefully shed some light on the feasibility of such an
approach.
Of the wide variety of antineoplastics with which to conjugate
spermidine's central nitrogen, two structural criteria should be met:
it should contain a functionality or site capable of attachment to the
t-BOC-protected spermidine reagent, and be stable to the trifluoroacetic
acid needed to deprotect the intermediate. Two such choices are the
alkylating agent 4-[p-[bis-(2-chloroethyl)aminojphenylbutyric acid
(Chlorambucil) and 2,3-dihydro-lH-imidazo[l,2b]pyrazole (IMPY), an in
hibitor of ribonucleotide reductase.92
The syntheses of such conjugates are straightforward, indeed.
Chloroambucil contains a free carboxylic acid which should render itself
76

77
to usual peptide condensation techniques. The reaction scheme is shown
in Figure 4-1. The succinimide active ester of chlorambucil is first
generated using dicyclohexylcarbodiimide and N-hydroxysuccinimide and
then reacted with N1,N8-bis(t-butoxycarbonyl)spermidine producing N4-
chlorambucil-N1,H8-bis(t-butoxycarbonyl )spermidine (28). The desired
N4-chlorambucil spermidine conjugate (29j is prepared by brief exposure
of intermediate (28) to trifluoroacetic acid.
Although IMPY does not contain a carboxylic acid with which to
directly condense with the protected spermidine reagent, a short con
necting bridge, such as glutaric acid, could be used. Subsequently,
IMPY can be reacted with glutaric anhydride, thereby producing an inter-
mediate (30) with a free carboxylic acid. This is then condensed with
N1,N8-bis(t-butoxycarbonyl)spermidine in the same manner as described
above. The entire synthesis of N4-[4-(2,3-dihydro-lH-imidazo[l,2-b]-
pyrazolo)carboxamido)butyryl]spermidine (32_) is shown in Figure 4-2.
Once these antineoplastic-Spd conjugates are synthesized, they will
be tested in vitro for their ability to compete with spermidine for
uptake as well as their antileukemic activity. The former assay is per
formed in the same manner as for the polyamine derivatives in the pre
ceding chapter. The antineoplastic-Spd conjugate will be incubated
in the presence of 3H-Spd in ascites LI210 cells, and in inhibition of
3H uptake determined as before. Additionally, the uptake of the native
antineoplastics themselves will also be determined.
The antileukemic activity of the antineoplastic-Spd conjugates
and the native anti neoplasties will be ascertained by determining the
dose necessary to prevent 50% of cell growth (ID50) of LI210 cell cul
tures. It is anticipated that if conjugation of spermidine to the

78
1 ) DCC
N-OH Sue.
2) H
Et3N
>v
28
TFA
C 1
N H 3
N H 3
29
Figure 4-1. Synthesis of N^-Chlorambuci1spermidine.

79
Figure 4-2. Synthesis of N4-G IMPY-spermi di ne (_32 )

60
anti neoplastic does indeed improve the uptake of the drug, this will be
demonstrated by a lowering of the drug's IO50
Experimental
Materials
Chlorambucil was purchased from Sigma Chemical Company, St. Louis,
MO. Samples of IMPY were generously donated by Dr. Leonard Kedda, Na
tional Cancer Institute, Bethesda, MD. All other reagents were purchased
from Aldrich Chemical Company, Metuchen, NJ and, except as noted, used
without further purification. Physical measurements were performed as
detailed in Chapter Two.
N4-Chlorambucil-N1,N8-bis(t-butoxycarbonyl)spermidine (28)
A solution of DCC (145 mg, 0.70 mmol) in 10 ml THF was added to a
cooled solution of chlorambucil (185 mg, 0.61 mmol) and N-hydroxysuc-
cinimide (80 mg, 0.70 mmol) in 20 ml THF. The ice bath was removed and
the solution allowed to stir for 18 hours during which time a thick
white precipitate ensued.
The reaction mixture was then concentrated, 20 ml CH^Cl2 added, the
DCU precipitates filtered and washed with an additional 10 ml of CH2CI2.
The filtrates were combined, cooled, and a solution of NL,N8-bis(t-
butoxycarbonyl)spermidine HC1 (13J (265 mg, 0.65 mmol) and triethyl amine
(90 yl, 0.65 mmol) in 10 ml CH2C12 slowly added. The resulting reac
tion was allowed to stir at room temperature for 48 hours, at which
time additional (20 ml) CH2C12 was added and the organic layer washed
with 3% HC1 (2 x 10 ml) and H20 (2 x 10 ml). The CH2C12 layer was
dried and concentrated to afford 340 mg crude product. Further purifi
cation was effected by chromatogrpahy on alumina (neutral, activity I)
eluting with CHC13 to afford 280 mg (73%) product: NMR 1.34-1.96

81
(m, 26H), 2.41 (m, 4H), 3.28 (m, 8H), 3.68 (s, 8H), 5.12 (br, 2H),
6.58 (d, 2H), 7.03 (d, 2H).
Anal. cal. for C31H52N405C12: C, 58.94; H, 8.30; N, 8.87. Found:
C, 59.49; H, 8.42; N, 8.47.
N4-Chlorambuci 1 spermidi ne-bis-tri fl uoroacetate (9.)
Trifluoroacetic acid (10 ml) was added to a flask containing (28)
(220 mg, 0.35 mmol) and the resulting solution stirred for 20 minutes.
The solvent was then quickly evaporated, dissolved up in methanol (25
ml) and concentrated (twice). The residue was then dissolved in 10 ml
H20, washed with CH2C12 (2 x 5 ml), and the aqueous layer lyophilized
to afford 150 mg (70%) as a beige solid: !H NMR (TFA) 6 1.54-2.01 (m,
8H), 2.47 (m, 4H), 3.31 (m, 8H), 3.70 (s, 8H0, 6.67 (d, 2H), 7.06 (d,
2H).
Anal. cal. for C H N 0 Cl F: C, 45.53; H, 5.81; N, 8.49. Found:
J O O i O 4
C, 45.81; H, 6.32; N, 8.40.
N-(4-Carboxybutyr,yl )2,3-dih,ydro-lH-imidazo[l ,2-b]pyrazole (GIMPY) (30)
Glutaric anhydride (250 mg, 2.2 mmol) was added to a solution of
IMPY (220 mg, 2.0 mmol) in 30 ml of dry CH2C12 under N2. After 18 hours
the product was filtered off, washed with CH2C12 and dried in vacuo
to afford 400 mg (90%) as a white powder. The physical and spectral
characteristics were identical to those previously reported:93
mp > 240C; *H NMR (TFA) 6 1.52-1.98 (m, 2H), 2.04-2.61 (m, 4H), 4.35
(s, 4H), 6.28 (s, 1H), 7.57 (s, 1H).
N4-f4-(2,3-Dihydro-1 H-imidazop ,2-b]p,yrazolo)carboxamido)butyryl ]-
N^NS-bisU-butox.ycarbonyl )spermidine (3ll
Dicyclohexylcarbodiimide (DCC) (165 mg, 0.80 mmol) was added to a
solution of (30) (150 mg, 0.67 mmol) and N-hydroxysuccimide (95 mg,

I
Figure 4-3. 300 MHz NMR spectrum of N^-Chlorambuci1-N^,N^-bis(t-butoxy-
carbonyl)spermidine (28).

-in
10
40
5.0
M.O
3 0
.0
1.0
Figure 4-4. 60 MHz NMR spectrum of N^-Chlorambuci1spermidine trifluoro-
acetate (29 ).
oo
CO

7 <: S 4 3 2 1
Figure 4-5. 300 MHz NMR spectrum of N^-[4-(2,3-dihydro-lH-imidazo[l,2b]-
pyrazolo)carboximido)butyryl]-Nl-N8-bis(t-butoxycarbonyl )-
spermidine (31 ) .

Figure 4-6. 60 MHz ]H NMR spectrum of N4-[4-(2,3-dihydro-1H-i mi dazo[1,2b]pyra
zo I o )carboxamido)butyry1]spermidine trif1uoroacetate (32).
00
t_n

86
0.80 mmol) in 50 ml dry pyridine. The resulting suspension was allowed
to stir for 36 hours at which time a solution of (1_3) (300 mg, 0.75 mnol)
in 10 ml CH2C12 was added and the reaction allowed to proceed an addi
tional 36 hours. The solvent was then evaporated and the residue dis
solved up in 100 CH2Cl2 filtered, washed with ice cold 3% HC1 (2 x 20
ml), H20 (2 x 20 ml), 5% NaHC03 (3 x 20 ml), H20 (3 x 20 ml), dried and
concentrated to afford 310 mg (83%) as a white crystalline solid; mp
155-157C (CHC13/cydohexane); NMR 5 1.20-1.82 (m, 6H), 1.39 (s, 18H),
2.12-2.67 (m, 2H), 2.75-3.44 (overlapping m, 12H), 4.32 (s, 4H), 5.61-
6.28 (m, 3H), 7.16 (s, 1H); IR (CHC13).
Anal. cal. for C27H46Ng06; C, 68.89; H, 8.42; N, 15.26. Found:
C, 59.17; H, 8.40; N, 15.07.
N4-[4-(2,3-Dihydro-lH-imidazo[1,2-b]pyrazo1o)carboxamido)butyrylJsper-
midine trifluoroacetate (N4-GIMPY Spd) (32)
A solution of (31_) and trifluoroacetic acid was reacted and puri
fied as described previously for 29; 400 mg (91%); XH NMR (TFA) 6 1.57-
1.96 (m, 6H), 2.10-2.62 (m, 2H), 2.74-3.57 (overlapping, 12H), 4.37
(s, 4H), 6.30 (s, 1H), 7.53 (s, 1H), 7.85 (br s, 6H).
Anal. cal. for C23H32FgN608: C, 39.89; H, 4.80; N, 12.19. Found:
C, 39.76; H, 5.11; N, 12.03.
Results and Discussion
The antineoplastic Spd conjugates N4-Chlorambucil Spd (29j and N4-
GIMPY Spd (32) were successfully synthesized in a two-step fashion em
ploying the bis(t-BOC)-protected reagent (1_3). Chlorambucil or GIMPY
(30) were condensed with (1_3) using the condensing reagents DCC and
N-hydroxysuccinimide in 70-80% yields. The yields were lower than the
corresponding secondary N-acylations illustrated in Chapter Two. This
was probably due to the less active acylating agent employed (succinimide

87
ester versus acid chloride) and the bulkier size of the antineoplastic
being condensed. However, it illustrates that a wide variety of acylat-
ing agents of varying size can be coupled to the reagent (13) in good
yields.
The antineoplastic-spermidine conjugates were then tested for their
ability to compete with 3H-Spd uptake and their cytotoxic effects on
LI210 cells in vitro. The results of these preliminary investigations are
given in Table 4-1. Both antineoplastic-spd conjugates inhibited 3H-Spd
uptake; however, the N4-chlorambuci1 conjugate was much more effective
(Ki = 6 pm). Furthermore, N4-chlorambuci1-Spd was found to be cytotoxic
to LI210 cells, with an ID50 of 15 pm. Chlorambucil itself was equally
cytotoxic, thereby showing no preference for the Spd conjugate. In view
of the fact that chlorambucil alone does not compete for Spd uptake, this
suggests that it may be entering the cell via an alternate mechanism, per
haps by an amino acid specific carrier.94
On the other hand, the N4-GIMPY-Spd conjugate was found to be rela
tively noncytotoxic, having an ID50 of 350 pM. When this value is, how
ever, compared to the ID50 of its precursor (30), a positive effect of
the conjugation of the spermidine backbone can be inferred. Assuming that
the mode of action between N4GIMPY-Spd (32) and GIMPY (30) is the same,
the increased cytotoxicity of (32) can be explained by the improved uptake
of the conjugate over (30). It should be pointed out that the cytotoxicity
of the GIMPY-Spd conjugates is far below what is considered sufficient
for antineoplastic activity. The mode of action of IMPY is by chelation
of the iron in the ribonucleotide reductase, rendering it inactive. It is
a distinct possibility that the amine which is reacted with glutaric anhy
dride may be involved in chelation. Functionalization of it to an amide

88
Table 4-1. Uptake and Cytotoxicity of Antineoplastics
and Antineoplastic-Spermidine Conjugates in
L1210 Leukemia Cells in vitro.
Ki(uM)
ID50(uM)
Ch1orambuci1
350
15
N4-Chlorambuci1Spd (29)
6
15
GIMPY (30)
*
>1000*
N4-GIMPY Spd (32)
150
350
insolubility at higher concentrations prevented determina
tion

89
may produce a less effective chelator, thereby decreasing its activity.
Accordingly, the synthesis of an analogue in which this amide is reduced
to an amine is currently being investigated.
Finally, although the in vitro data at this point are limited, it
indicates that these conjugates are indeed taken up via the spermidine car
rier and represent a potential drug delivery system. Accordingly, further
testing and synthesis of other systems are currently being evaluated at
this time.

CHAPTER FIVE
SYNTHESIS OF TRIS-PROTECTED SPERMIDINES
The reagents described in Chapter Two, N4-benzylspermidine and
N1,N8-bis(t-butoxycarbonyl)spermidine, have demonstrated themselves as
important reagents for the selective functionalization of spermidine's
primary and secondary nitrogens. Using these methods, derivatives can
be obtained that 1) contain only one substituent on the secondary ni
trogen, 2) contain the two same substituents on both primary nitrogens,
or 3) a combination of both; two identical substituents on the primary
nitrogens and a third different substituent on the secondary nitrogen.
However, it is not possible to place three different groups on spermi
dine or, for that matter, only one substituent on only one primary ni
trogen via these methods. The latter group of derivatives, those modi
fied at only one terminal nitrogen, pose an additional interesting ques
tion concerning uptake --how effective would these derivatives be in com
peting for uptake?
The answer to this question, based upon the uptake data presented
in Chapter Three, is yes. The N1,N8-bis(alkyl)spermidine derivatives
were able to compete with Spd for uptake, and it is expected that modi
fication of only one terminal amine should accentuate this uptake.
This may offer another potential avenue in conjugating antineoplastics
to spermidine. Accordingly, the synthesis of such a reagent is desired.
The ideal reagent would be one in which each of spermidine's ni
trogens is protected with three different protecting groups, each remov
able separately under different conditions as illustrated in Figure 5-1.
90

z
I
A-NH (C H 2)aN (C H 2)b NH-B
ArNH (CH2) NH (CH2) NH-B
Figure 5-1. Spermidine reagents containing three different protecting groups,
A, B, Z, which can be independently removed.

o?
Additionally, the boundary conditions of earlier synthesis must be still
met in that the reagent should be generated in as few steps as possible,
and the protecting groups removed cleanly and efficiently. The benzyl
and t-butoxycarbonyl protecting groups employed earlier have already proven
themselves as easily removable in the presence of one another and, ac
cordingly, their use will be maintained. The choice of the third pro
tecting group should therefore be one which is removable under basic
conditions as the t-BOC and benzyl groups are removed under acid and neu
tral conditions, respectively. A prime candidate for the base removable
protecting group is the trifluoroacetoxy protecting group.9- It can be
easily attached via either the anhydride or the acid chloride, and re
moved under relatively mild basic conditions employing sodium bicarbonate.
Synthesis
In the simplest terms, the synthesis of the target tris-protected
reagent would be nothing more than the synthesis of N4-benzyl-N1,N8-
bis(t-butoxycarbonyl)spermidine however, replacing one of the t-BOC
groups with the trifluoroacetoxy group. However, in reality the removal
of one t-BOC protecting group would likely be impossible. If, on the
other hand, this intermediate (XVI) figure 5-2, could be generated by
some other means, the target compound (XVII) could be realized.
Recalling the synthesis of the benzylated polyamines in Figure 2-1,
a synthetic scheme for intermediate (XVI) can be envisioned by incor
porating a t-BOC protecting group early on in the synthesis. Such a
scheme is outlined in Figure 5-3.
The key to the whole scheme lies in the ability to selectively react
the terminal amine of N-(3-ami nopropyl)benzyl amine with BOC-ON to pro
duce fl-[N-(t-butoxycarbonyl)3-ami nopropyl]benzylamine (XIX). The desired

93
Figure 5-
XVII
NHCOCF3
Synthesis of the desired tris-protected reagent
(XVII) from intermediate (XVI).

94

XVI la
Figure 5-3. Entire synthesis of tris-protected spermidine
reagent (XVI la) and norspermidine analogue(XVIIb)

95
selectivity is expected based on the known preference in reactivity of
primary over secondary amines seen with t-BOC forming reagents.8096
Once (XIX) is obtained, the remainder of the synthetic sequence is essen
tially identical to the corresponding benzyl spermidine scheme. Deriva
tive (XIX) can either be reacted with 4-chlorobutyrylnitrile producing
the 3,4-precursor, or with acrylonitrile to produce the 3,3-precursor.
The nitriles (XX) are subsequently reduced with Raney nickel97 to generate
the intermediates (XVII a & b) which,when reacted with trifluoroacetic
anhydride, form the tris-protected reagents of spermidine and norsper-
midine.
The synthesis of the corresponding homospermidine reagent would
proceed along the same lines, requiring the synthon N-(4-aminobutyl)-
benzyl amine (XXI) in place of N-(3-ami nopropyl)benzyl amine (XVIII). The
preparation of (XXI), however, is not a trivial matter as compared to
the synthesis of (XVIII) from acrylonitrile and benzylamine. For in
stance, alkylation of excess benzylamine with 4-chlorobutyrylnitrile
affords very little of the nitrile precursor to (XXI), the major product
resulting from bis-alkylation instead.
Therefore, it seemed obvious that the synthesis of (XXI) should be
effected by a higher yield acylation of benzylamine, instead of alkyla
tion, followed by reduction. The requisite 3-cyanopropionic acid for
this acylation is not commercially available; however, N-(t-butoxycar-
bonyl)-4-aminobutyric acid (t-BOC-GABA) is available and offers an
attractive scheme as outlined in Figure 5-4.
Benzylamine can be reacted with the succinimide ester of t-BOC-GABA,
previously generated with DCC and N-hydroxysuccinimide, to afford the
N-benzyl butanamide derivative (XXII), which already contains the t-BOC

I
xxv > XXVI
Y
\jD
cr>
Figure 5-4. Synthesis of tris-protected homospermidine (XXVI).

97
protecting group. The remainder of the scheme lies on the key reduction
of (XXII) to N-[N-(t-butoxycarbony1)-4-aminobutyl]benzylamine (XXIII).
It has been recently reported in the literature on its ability to selec
tively reduce amides in the presence of urethane linkages using borane-
THF complexes.98 An approach such as this, if applicable to this system,
would generate (XXIII) from benzylamine in just two steps compared to
the three required for synthesis of (XIX).
The remainder of the scheme, Figure 5-4, is identical to that of
the 3,4-homologue. Intermediate (XXIII) is reacted with 4-chlorobutyryl-
nitrile, followed by Raney nickel reduction producing (XXV). Reaction
of amine (XXV) with trifluoroacetic anhydride would afford the desired
tris-protected homospermidine (XXVI) in five steps from benzylamine.
Finally, an example of the use of the tris-protected reagent is
given. The spermidine derivative N8-acetyl-N4-benzoyl-N1-(2,3-dimethoxy-
benzoyl)spermidine is synthesized via selective removal and acylation of
the spermidine reagent.
Experimental
Materials
The starting material N-(3-ami nopropyl)benzyl amine was prepared
as previously described.99 All other reagents were purchased from Aldrich
Chemical Company, Metuchen, NJ and, except where noted, used without
further purification. Dry THF was obtained by freshly distilling from
calcium hydride. The physical measurements were performed as described
in the materials section of Chapter Two.
N-[N-t-Butox,ycarbonyl )-3-aminopropyl jbenzylamine (33.)
A solution of BOC-ON (6.9 g, 0.028 mole) in 20 ml THF was slowly
added dropwise to a solution of N-(3-aminopropyl)benzylamine (5.0 g,

98
0.030 mole) in 30 ml at 0C. After the addition was completed, the
ice bath was removed, and the reaction stirred for 8 hours. The solvent
was then evaporated, the residue dissolved in 100 ml ether, washed with
5% NaOH (4 x 20 ml), dried, and concentrated to afford 3.2 g crude prod
uct. The product was further purified by distillation to afford 6.6 g
(89%) pure (33): bp 150-151C (0.13 nm); *H NMR 6 1.53 (s, 9H), 1.77
(m, 3H), 2.72 (t, 2H), 3.16 (quar., 2H), 3.70 (s, 2H), 5.26 (br, 1H),
7.23 (s, 5H); IR (CHC13) 2950 (m), 1690 (s), 1490 (m), 1160 (s), 750
(s) cm-1.
Anal. cal. for C15H24N202: C, 68.15; H, 9.15; N, 10.60. Found:
C, 68.14; H, 9.19; N, 10.55.
N-[N-(t-Butox,ycarbonyl)-3-aminopropyl ]-N-(3-cyanopropyl )benzylamine (34)
A solution of 4-chlorobutyrylnitrile (4.7 g, 0.095 mole) in 100
ml BuOH was slowly added to a suspension of (3J3) (10.0 g, 0.038 mole),
Na2C03 (4.4 g, 0.042 mole) and KI (1.4 g, 0.0095 mole) in 100 ml BuOH.
The reaction mixture was then gently refluxed for 48 hours, cooled,
and diluted with an equal volume of ether. The salts are filtered,
washed with ether, and the filtrates evaporated. The crude product was
chromatographed on silica gel, eluted with 5% Me0H/CHCl3 to afford 1.2 g
(95%) of the desired product as an oil: XH NMR 6 1.43 (s, 9H), 1.73
(m, 4H), 2.43 (m, 4H), 3.12 (quat., 2H), 3.47 (s, 2H), 4.83 (br, 1H),
7.23 (s, 5H); IR (neat) 2990 (s), 2250 (w), 1710 (s), 1520 (s), 1180
(s), 740 (m) cm-1.
Anal. cal. for C1?H2gH302: C, 68.85; H, 8.82; N, 12.68. Found:
C, 68.57; H, 8.85; N, 12.59.

99
N4-Benzy1-N1-(t-butoxycarbony1 )spermidine (35)
Raney nickel (3.0 g) was added to a solution of (34) (13.6 g, 0.0409
mole) and NaOH (4.0 g, 0.10 mole) in 100 ml 95% EtOH and the resulting
suspension was hydrogenated under 40 psi for 28 hours. The catalysts
were then filtered, washed well with 95% EtOH, and the filtrates concen
trated. The residue was taken up in H20 (250 ml), and the product ex
tracted into CH2C12 (4 x 50 ml), dried and concentrated to afford 13.4 g
(97%) of the desired product as a nondistillable oil. Thin layer chroma
tography and NMR analysis indicated the purity of the product was in
excess of 95% and could be used without further purification.
An analytical sample was prepared by chromatography on silica gel
eluting with 30% MeOH/CHCl3: NMR 6 1.50 (m, 17H), 2.47 (m, 6H), 3.10
(quar., 2H), 3.47 (s, 2H), 5.33 (br, 1H), 7.17 (s, 5H); IR (neat) 2940
(s), 1710 (s), 1510 (m), 1170 (s), 740 (m) cm-1.
Anal. cal. for C19H33N302: C, 68.02; H, 9.91; N, 12.53. Found:
C, 67.79; H, 9.96; N, 12.43.
N4-Benzyl-N1-(t-butoxycarbonyl)-N8-trifluoroacetylspermidine (36)
A solution of trifluoroacetic anhydride (2.3 g, 11 mmol) in 10 ml
dry CH2C12 was slowly added to a cooled solution of (35J, (3.5 g, 10
mmol) and triethylamine (1.4 ml, 10 mmol) in 20 ml dry CH^Cl9 under N2>
The reaction was allowed to warm to room temperature and stirred for
16 h, at which time additional CH2C12 was added (50 ml). The organic
layer was washed with cold 3% HC1 (2 x 15 ml), H20 (1 x 25 ml), 5%
NaHC03 (2 x 25 ml), dried and concentrated to afford 3.9 g (91%) of
the desired product: NMR 6 1.42 (s, 9H), 1.58 (m, 6H), 2.43 (m, 4H),
3.15 (m, 4H), 3.50 (s, 2H), 5.22 (br, 1H), 7.25 (s, 5H), 7.58 (br, 1H);
IR (CHC13) 3300 (m), 3020 (m), 1710 (s), 1620 (s), 1170 (s), 750 (s) cm'1.

100
Anal. cal. for C2jH32N3O3F3: C, 58.45; H, 7.47; N, 9.74. Found:
C, 58.31; H, 7.49; N, 9.71.
N-[N-(t-Butox.ycarbonyl) -3-aminopropyl]-N-(2-cyanoethyl )benzvlamine (37)
A sealed vessel containing (^3) (6.0 g, 0.023 mole) and acryloni
trile (2.6 ml, 0.039 mole) under an argon atmosphere was heated in an
oil bath at 100C for 24 hours. The reaction mixture was cooled and
purified via chromatography on silica gel eluting with CHC13 to afford
7.06 g (98%) product as an oil: XH NMR 6 1.42 (s, 9H), 1168 (m, 2H),
2.58 (m, 4H), 3.13 (quar., 2H), 3.55 (s, 2H), 4.73 (br, 1H), 7.23 (s,
5H); IR (neat) 3000 (m), 2250 (w), 1715 (s), 1515 (s), 1180 (s), 740
(m) cm"1.
Anal. cal. for C18H27N302: C, 68.11; H, 8.57; N, 13.24. Found:
C, 67.96; H, 8.55; N, 13.19.
N4-Benzyl-NI-(t-butoxycarbonyl)spermidine (38)
A solution of (37) was reduced and purified in a similar manner
as described for (35). Yield: 9.4 g (97%); NMR 6 1.40 (2, 11H), 1.67
(m, 4H), 2.53 (m, 6H), 3.10 (quar., 2H), 3.48 (s, 2H), 5.28 (br, 1H),
7.18 (s, 5H); IR (neat) 2975 (s), 1710 (s), 1515 (s), 1180 (s), 740
(m) cm"1.
Anal. cal. for C18H31N302: C, 67.25; H, 9.72; N, 13.07. Found:
C, 66.98; H, 9.78p N, 13.04.
N4-Benzyl-N1-(t-butoxycarbonyl)-N -trifluoracetylnorspermidine (19)
A solution of (38) and trifluoroacetic anhydride was reacted and
purified in a similar manner as described for (36). Yield: 1.8 g (92%);
XH NMR 5 1.43 (s, 9H), 1.65 (m, 4H), 2.44 (m, 4H), 3.14 (m, 4H), 3.52
(s, 2H), 5.18 (br, 1H), 7.21 (s, 5H), 7.67 (br, IN); IR (neat) 3320 (m),
3000 (m), 1705 (s), 1620 (s), 1180 (s), 745 (m) cm'1.

TCI
Anal. cal. for C20H30N303F3: C, 57.54; H, 7.24; N, 10.06. Found:
C, 57.62; H, 7.27; N, 9.95.
N-(N-t-Cutoxycarbonyl -4-aminobutyryl) benzyl amine (40)
A solution of DCC (2.47 g, 12 mmol) in 20 ml THF was slowly added
to a cooled solution of N-t-BOC-GABA (2.0 g, 10 mmol) and N-hydroxysuc-
cinimide (1.4 g, 12 mmol) in 20 ml THF. The ice bath was removed and
the reaction allowed to stir for 16 hours during which time a thick
white precipitate ensued. The DCU precipitates were filtered, washed
with THF, and the filtrates concentrated to a volume of 20 ml.
The above solution was then added to a solution of benzylamine (1.6
g, 15 imol) in 20 ml THF, and a white precipitate immediately resulted.
The suspension was stirred for 18 hours at room temperature at which
time the solvent was evaporated. The residue was dissolved up in 100
ml ether, and the insoluble matter filtered. The ether layer was washed
with 3% HC1 (2 x 20 ml), H20 (2 x 20 ml), 5% NaHC03 (2 x 20 ml), dried
and concentrated to afford the crude product. Recrystallization from
ether/cyclohexane afforded 2.5 g (87%) pure (40) as a white solid:
mp 110-111C; lH NMR 6 1.37 (s, 9H), 1.8 (quar., 2H), 2.18 (t, 2H),
3.06 (quar., 2H), 4.30 (d, 2H), 4.80 (br, 1H), 6.47 (br, 1H), 7.12 (s,
5H); IR (CHC13) 3010 (m), 1700 (s), 1660 (s), 1515 (s), 760 (s) cm'1.
Anal. cal. for C16H24N203: C, 65.73; H, 8.27; N, 9.58. Found:
C, 65.70; H, 8.30; N, 9.56.
N-(N-t-Butoxycarbony1-4-aminobutyl)benzylamine (H )
A solution of 1 M diborane-THF complex (13 ml) was slowly added to
a cooled suspenson of (40) (1.9 g, 6.5 mmol) in 20 ml dry THF under N2.
Once the addition was completed, the ice bath was removed, and the now
clear solution brought to reflux for 20 hours. The reaction mixture

102
was then cooled to room temperature, then to 0C, and the excess BH3
carefully destroyed by adding 6 N NH^Cl (15 ml). The THF was evaporated,
and enough solid NaOH added to the remaining aqueous solution to make
basic. The product was extracted into ether (3 x 25 ml), dried and con
centrated to afford 1.5 g crude product. Further purification was ef
fected first by distillation followed by preparative TIC to afford 850
mg (47%) of the desired product: XH NMR 5 1.41 (m, 14H), 2.58 (m, 2H),
3.06 (m, 4H), 3.70 (s, 2H), 4.85 (br, 1H), 7.23 (s, 5H); IR (neat) 2975
(s), 1710 (s), 1520 (s), 1180 (s), 750 (m) err1.
Anal. cal. for C16H26N202: C, 69.03; H, 9.41; N, 10.06. Found:
C, 69.13; H, 9.42; N,10.01.
N-[N-(t-Butoxycarbonyl)-4-ami nobutyl]-N-3-cyanopropylbenzyl amine (42.)
A solution of (£]_) was reacted with 4-chlorobutyrylnitrile and puri
fied in a similar manner as described for (34). Yield: 3.9 g (96%);
XH NMR 6 1.43 (m, 15H), 2.47 (m, 6H), 3.10 (m, 2H), 3.47 (s, 2H), 4.63
(br, 1H), 7.17 (s, 5H); IR (neat) 2990 (s), 2240 (w), 1700 (s), 1520
(s), 1190 (s), 750 (m) cm"1.
Anal. cal. for C20H31N302: C, 69.53; H, 9.04; N, 12.16. Found:
N5-Benzyl-N1-t-butox,ycarbony1 homospermidine (41)
A solution of (42_) was reduced and purified as described for (35).
Yield: 9.8 g (91%); :H NMR 6 1.50 (m, 19H); 2.43 (m, 6H), 3.02 (m, 2H),
3.50 (s, 2H), 4.74 (br, 1H), 7.18 (s, 5H); IR (neat) 2970 (s), 1700 (s),
1515 (m), 1170 (s), 740 (w) cm"1.
Anal. cal. for C20H35N302-H20: C, 65.36; H, 10.14; N, 11.43.
Found: C, 65.44; H, 10.13; N, 11.43.

103
N5-Benzyl-N1-t-butoxycarbony1 -N9-trifluoroacetylhomospermidine (44)
A solution of (43) and trifluoroacetic anhydride was reacted and
purified as previously described for (36). Yield: 1.8 g (76%); lH NMR
1.52 (m, 17H), 2.38 (m, 4H), 3.18 (m, 4H), 3.43 (s, 2H), 4.70 (br, 1H),
7.18 (s, 5H), 7.62 (br, 1H); IR (neat) 3320 (m), 2975 (s), 1710 (s),
1520 (s), 1170 (s), 740 (m) cm*1.
Anal. cal. for C22H34N3O3F3: C, 59.31; H, 7.69; N, 9.43. Found:
C, 59.12; H, 7.69; N, 9.41.
Nl-t-Butoxycarbonyl-N8-trif1uoroacetylspermidine (45)
Palladium chloride (90 mg) was added to a solution of (36) (1.0 g,
2.3 mmol) in 25 ml MeOH containing six drops concentrated HC1. The res-
sulting suspension was stirred under a H2 atmosphere for 12 hours at
which time the catalysts were filtered off, washed with MeOH, and the
filtrates evaporated. The crude product was recrystallized from EtOH/
ether to afford 710 mg (82%) of pure (45j: mp 140-141; NMR (D20) 6
1.54 (s, 9H), 1.80 (m, 6H), 3.30 (m, 8H); IR (KBr) 3380 (m), 2960 (m),
2800 (m), 1705 (s), 1530 (m), 1175 (s) cm"1.
Anal. cal. for C14H27N3C1 F303-H20; C, 42.50; H, 7.38; N, 10.62.
Found: C, 42.55; H, 7.32; N, 10.64.
N4-Benzoyl-N^t-butoxycarbonyl -N8-trifluoroacetyl spermidine (46j
A solution of benzoyl chloride (240 mg, 1.7 mmol) was slowly added
to a cooled solution of (45) 560 mg, 1.5 mmol) and trimethylamine (280
pi, 2.0 mmol) in 25 ml dry CH2C12 under N2. The reaction was allowed
to warm to room temperature and stirred for 18 hours. Additional CH2C12
was added (50 ml) and the organic layer washed with 3% HC1 (3 x 15 ml),
H20 (2 x 15 ml), 5% NaHC03 (3 x 15 ml), H20 (2 x 15 ml), dried, and
concentrated to afford 620 mg (92%) of the desired product as a puffy

104
solid. Thin layer chromatography and NMR analysis indicated product's
purity was in excess of 95%, and could be used without further purifica
tion.
An analytical sample was prepared by preparative TLC eluting with
10% MeOH/CHCl3: lH NMR 6 1.37 (s, 9H), 1.68 (m, 6H), 3.27 (m, 8H), 5.18
(br, 1H), 7.28 (s, 5H), 7.62 (br, 1H); IR (CHC13) 3310 (m), 3010 (m),
1710 (s), 1620 (s), 1170 (s), 750 (s) cm'1.
Anal. cal. for C21H30N304F3: C, 56.62; H, 6.79; N, 9.43. Found:
C, 56.50; H, 6.82; N, 9.20.
N^Benzoyl-N^t-butoxycarbonylspermidine (47)
Potassium carbonate (660 mg, 4.6 mmol) was added to a solution of
(46) (515 mg, 1.15 mmol) in 30 ml MeOH and 2 ml H20. The reaction mix
ture was refluxed for 2 hours, cooled, and the solvent evaporated. The
residue was dissolved in 50 ml CH2C12 washed with H20 (2 x 10 ml),
dried, and concentrated to afford the crude product. Further purifica
tion was effected by chromatography of silica gel eluting with 20% MeOH/
CHC13 to afford 325 mg (81%) product: XH NMR 5 1.20-1.94 (m, 17H), 256
(m, 2H), 3.30 (m, 7H), 5.26 (br, 1H), 7.33 (s, 5H); IR (neat) 3000 (m),
1710 (s), 1625 (s), 1510 (s), 1175 (s), 760 (s) cm1.
Anal. cal. for C19H31N303'H20: C, 62.10; H, 8.94; N, 11.43. Found:
C, 62.67; H, 8.69; N, 10.89.
N8-Acetyl-N4-benzoyl-N1-t-butoxycarbonylspermidine (4B.)
A solution of acetyl chloride (85 mg, 0.71 mmol) in 5 ml dry CH2C12
was added to a cooled solution of 47 (215 mg, 0.62 mmol) and triethyl-
amine (150 pi, 1 mmol) in 10 my dry CH2C12 under N2. The reaction was
allowed to stir for 18 hours, and then worked up and purified as described

105
for (46). Yield: 240 mg (98%); XH NMR 6 1.43 (s, 9H), 1.70 (m, 6H),
1.93 (s, 3H), 3.27 (m, 8h), 5.38 (br, 1H), 6.44 (br, 1H), 7.37 (s, 5H),
IR (2980) m, 1710 (s), 1630 (br, s), 1180 (m) cm'1.
Anal. cal. for C21H33N304: C, 64.43; H, 8.50; N, 10.73. Found:
C, 64.17; H, 8.5a N, 10.51.
N8-Acetyl-N4-benzoylspermidine (4)
Trifluoroacetic acid (10 ml) was added to a flask containing 48
(320 mg, 0.82 mmol) and the resulting solution allowed to stir for 20
min. The solvent was then quickly evaporated, the residue dissolved
in 25 ml MeOH and concentrated (twice). The crude product was then
treated with 15% Na2C03 (10 ml), extracted with CH2C12 (3 x 25 ml), dried
and concentrated to afford 180 mg (75%) product as a light yellow oil:
NMR 6 1.66 (m, 8H), 1.94 (s, 3H), 2.61 (m, 2H), 3.24 (m, 6H), 6.41
(br, lh), 7.35 (s, 5H); IR (CHC13) 2990 (m), 1650 (s), 1620 (s), 740 (s)
cm'1.
Anal. cal. for C1gH25N302: C, 65.95; H, 8.65; N, 14.42.
N8-Acety1-N4-benzoyl-N1-2,3-dimethoxybenzoylspermidine (50.)
A solution of 2,3-dimethoxybenzoyl acid chloride (95 mg, 0.47
mmol) in 10 ml dry CH^Cl2 was slowly added to a cooled solution of (49)
(125 mg, 0.43 mmol) and trimethyl amine (70 yl, 0.5 mmol) in 10 ml dry
CH2C12. The reaction was allowed to stir for 18 hours, and purified as
described for (46). Yield: 180 mg (93%); NMR 6 1.68 (m, 6H), 1.95
(s, 3H), 3.24 (m, 8H), 3.87 (s, 6H), 6.45 (br, 1H), 6.93-8.12 (m, 9H);
IR (CHC13) 3310 (m), 2980 (m), 1650 (br, s), 1530 (s), 740 (m) cm"1.
Anal. cal. for C9 H N 0 : C, 65.91; H, 7.30; N, 9.22. Found:
C, 65.88; H, 7.46; N, 9.05.

Figure 5-5. 60 MHz ^ H NMR spectrum of N-[N-t-butoxycarbonyl-3-aminopropyl]-
benzylamine (33).

I 1 I
IZI3 i~~i i .i.. I. ZH-1 1 i t i I t 1 i i i 1 . 1
\
10
4 0
5.0
*.0
3 0
1.0
1.0
o
-^1
Figure 5-6. 60 MHz NMR spectrum of N-[N-(t-butoxycarbony1)-3-aminopropy1]-N-
(3-cyanopropy1)benzy1amine (34).

Figure 5-7. 60 MHz ^NMR spectrum of N^-benzy1 -N1 -(t-butoxycarbony1)spermidine (35).
6

Figure 5-8. 60 MHz ^ NMR spectrum of N^-benzyl-N^-(t-butoxycarbonyl)-N^-trifluoro-
acetylspermidine (36).

I

5-10. 60 MHz NMR spectrum of N
amine (41).
(N-t-butoxycarbonyl-4-ami nobutyl)benzyl-
O

Figure 5-11. 60 MHz ^ H NMR spectrum of fP-t-butoxycarbonyl-N^-trif1uoroacetyl-
spermidine Hydrochloride (45 ).

Figure 5-12. 60 MHz 1H NMR spectrum of N4-benzoyl-N1-t-butoxycarbonyl-N8-trif1uoro-
acetylspermidine (46).

5 0
H.O
1.0
o
Figure 5-13. 60 MHz 1h NMR spectra of N^-benzoyl-N^-t-butoxycarbonylspermidine (47).

Figure 5-14.
60 MHz 1H NMR spectrum of N^-acetyl-N^-benzoyl-N^-t-butoxycarbonyl
spermidine (48).

^LJ
>....1 nntnnn .11.-i i ...- i...1.--I t.i l i i l i i i > i i~t 1
10
4 0
50
MO
3 0
i.O
1.0
Figure 5-15. 60 MHZ NMR spectrum of N8-acety1-N^-benzoylspermidine (49 ).

10 40 so *0 3 0 2.0 1.0 a
Figure 5-16. 60 MHz ^H NMR spectrum of N8-acetyl-N4-benzoyl-N1-2,3-dimethoxybenzoyl-spermidine (50).

TI
tQ
c
5
0>
en
i
tO l-H
O 70
fD
-5
3 "O
0)
Q. O
3 5
0> C
U> O
en -h
8LL
SNOH7IW NI MIOMIIIAVM

pkcenuransmission
Figure 5-18. IR spectrum of N-(N-t-butoxycarbonyl-4-aminobutyryl)benzylamine (40).
RRCENT TRANSMISSION

SPCNT TRANSMISSION
WAVtttNOIH III MICRO! (S
45 S<
3000
WMtM moimiwC incur chari mo iosa
2000 |000
WAVlNUMBCR CM*
60 *
1600 1400 1200
mcirran 45IRUWIMIS c. rimrttcHi. cmvoinm, u s a.
1000
M4I0 KUI
Figure 5-19. IR spectrum of N-(N-t-butoxycarbony 1 -4-ami nobutyl ) benzy 1 ami ne (4_]J
ro
O
PS*r-

121
Results and Discussion
Synthesis of N4-Benzyl-N1-t-B0C-N8-Trif1uoroacety1sDermidine (36)
In developing a scheme for the synthesis of a tris-protected sper
midine reagent, such as (36), it was recognized that the desired pro
tecting groups should be incorporated throughout the initial steps of
the scheme. Combining the methodologies that were so successfully em
ployed in the synthesis of the previous two spermidine reagents, the
synthesis of (36) could easily be envisioned, Figure 5-3.
The sequence begins with N-(3-ami nopropyl)benzyl amine, a key inter
mediate in the synthesis of N4-benzylspermidine. The utility of this
starting material is that it has already incorporated what will become
the secondary N-benzyl protecting group. Hence, N-(3-aminopropyl)ben-
zylamine was reacted with BOC-ON generating the desired terminally t-BOC
protected compound (33) in high yields. The reaction proceeds selec
tively, with no apparent involvement of the secondary amine. Further
more, the product is easily purified by distillation.
Intermediate (33) was then reached with an excess of 4-chlorobuty-
rylnitrile as in earlier syntheses to afford the mono-nitrile derivative
(34), again easily purified via silica gel chromatography. Subsequently,
it has been shown that crude (34) can usually be reduced directly to
amine (35) without further purification.
The reduction of nitrile (34J to amine (35) employs a recent im
provement over earlier methods used to reduce these nitriles. Earlier,
lithium aluminum hydride was the reagent of choice in the preparation
of benzylspermidine; however, recently it has been shown that a basic
solution of Raney nickel can accomplish this reduction more efficiently.97
Accordingly, the bis-protected nitrile (34) was reduced under a H2

122
atmosphere in the presence of Raney nickel and NaOH to produce amine (35)
in 95% yield. Again, the reaction proceeds very cleanly, and the amine
(35) in many instances can be used without further purification. Final
ly, the desired tris-protected spermidine reagent (36) is prepared by
the high yield acylation of (35) with trifluoroacetic anhydride.
Thus, N4-benzyl-N1-t-B0C-N8-trifluoroacetylspermidine was conven
iently prepared in four high yield steps from N-(3-ami nopropyl)benzyl -
amine in an overall yield of 75%. Additionally, recent experiments sug
gest that the overall conversion of (33) -* (36) can be performed without
purification of the intermediates instead of only purifying the final
product (36). Also, it should be mentioned that the conversion of amine
(35) to (36) with trifluoroacetic anhydride, is in itself not necessary
if one wishes to functionalize the terminal amine of (35) with some
other desired group.
Synthesis of Tris-protected Homologues (39) and (44)
Once the feasibility of the target reagent (36) had been demon
strated, the synthesis of its symmetrical nor- and homospermidine homo
logues was required. The synthesis of the norspermidine derivative
could be effected by the addition of a three-carbon chain to inter
mediate (33). The typical means of accomplishing this is by the Michael
addition of acrylonitrile to amine (33). When (33) was reacted with a
slight excess of acrylonitrile at room temperature, little or no addi
tion occurred. However, when the reactants were placed in a sealed
vessel and heated to 100C, the reaction proceeded cleanly to afford
nitrile (37) in 98% yield. The desired norspermidine reagent (39) was
subsequently generated by reduction of (37), followed by trifluoroacety-
lation as for the spermidine homologue (36).

123
The synthesis of the homospermidine reagent (44), however, required
the preparation of four-carbon analogue to {33). Attempted alkylation
of benzylamine and 4-chlorobutyrylnitrile, even with a ten-fold excess
of benzylamine, resulted predominantly in bis-alkylation. Accordingly,
a new synthetic scheme for the preparation of (41_) needed to be developed.
Based on the general success of acylation followed by reduction to pro
duce N-alkyl derivatives in Chapter Two, an approach such as this was
undertaken.
Therefore, benzylamine was acylated with the succinimide ester of
t-BOC-GABA to produce the amide (40) in good yields. This was based on
the premise that the amide could be reduced selectively in the presence
of the urethane protecting group generating the desired four-carbon
analogue (41_) at (33). Such a reduction has been accomplished in the
literature using the reducing agent diborane. In this case a trifluoro-
acetamide substituent was reduced efficiently in the presence of an
ethoxycarbonyl group.98
When a suspension of (40) in THF was treated with BH3 THF complex
the desired reduction of (40) -* (41) did occur; however, a major byproduct
determined to be N-(4-aminobutyl)benzylamine was noted. Evidently,
either the reduction or the acidic workup employed was sufficient to
cause cleavage of the t-BOC protecting group. Even when NH4C1 was em
ployed in place of HC1 for the workup, substantial cleavage still occurred.
Based on the behavior of the t-BOC protecting groups in earlier reactions,
it seemed unlikely that the acid workup should be sufficient to cause
cleavage. However, diborane is a good Lewis acid and, perhaps, under
the conditions of reduction, the t-BOC group can be cleaved, unlike the

124
ethoxycarbonyl group which is not susceptible to acid cleavage. For
tunately, an alternate method to prepare N-(4-ami nobutyl)benzylamine in
large quantities has recently been devised by our group.100
The remainder of the synthesis of the tris-protected homospermidine
reagent proceeds in an identical fashion to the spermidine reagent (36).
The t-BOC-protected intermediate (4]_) was reacted with excess 4-chloro-
butyrylnitrile, thusly producing the nitrile (42), once again in high
yields. Finally, N5-benzyl-N1-t-butoxycarbonyl-N8-trifluroracetoxyhomo-
spermidine (44) is produced by reduction of nitrile (42), followed by
acylation with trifluoroacetic anhydride.
Synthesis of N8-acetyl-Ntl-benzoyl-N1-2,3-dimethox,ybenzoyl spermidine (501
As an illustrative example of the use of the tris-protected spermi
dine reagents, the synthesis of (50) was demonstrated. The choice of
the acyl groups affixed: acetyl, benzoyl, 2,3-dimethoxybenzoyl, were
determined solely on their relative ease of identification in the NMR.
However, any other groups could have been used as well. Likewise, the
order and position in which these groups were attached was entirely random.
The synthesis of (50) was accomplished by three series of deprotec
tions and acylations as follows. The benzyl protecting group was first
removed by hydrogenolysis employing the same conditions described earlier.
Hence, a solution of the spermidine reagent, N4-benzyl-N8-t-butoxycar-
bonyl-N4-trifluoroacetyl spermidine (36) in methanol/HC1 was hydrogenated
overnight over PdCl2 catalysts to afford the debenzylated adduct (45) as
the hydrochloride salt in 85% yield after recrystallization. The reac
tion proceeded cleanly without any ill effects to the two remaining pro
tecting groups.

125
The salt (45) was acylated with a slight excess of benzoyl chloride
in the presence of triethylamine to afford N4-benzoyl-N1-t-B0C-N8-tri-
fluoroacetyl spermidine (40) in 92% yield. The acylation proceeded smooth
ly and the product (46) could be used without further purification.
Next, the trifluoroacetyl protecting group was removed by treatment
of (46) with potassium carbonate in refluxing aqueous methanol for two
hours. The resulting amine was purified by chromatography on silica
gel to afford pure N4-benzoyl-N1-t-B0C spermidine (47) in 81% yield.
Intermediate (47) was then reacted with excess acetyl chloride, again in
the presence of triethylamine, to qualitatively (98%) afford N8-acetyl-
N^benzoyl-N^t-BOC spermidine (48).
The final deprotection, removal of the t-butoxycarbonyl group, was
effected by brief treatment of (48) with trifluoroacetic acid. The prod
uct, N8-acetyl-N4-benzoyl spermidine (49) was easily purified by washing
the acidic solution with CH2C12, adjusting the pH to 11 with Na2C03,
and extracting the product into CH2C12 to afford (49) in 75% yield.
Higher yields (>90%) of slightly impure product could be obtained by
treating the crude (49) with MeOH/NaOMe, followed by removal of the sol
vent, and extracting the product away from the salts with ether. A solu
tion of (49) was reacted with 2,3-dimethoxybenzoyl chloride to afford
the final product N8-acetyl-N4-benzoyl-N1-2,3-dimethoxybenzoyl spermidine
(50) in 93% yield. The overall yield for the conversion of (36) (50)
was 42% in six steps.
It should be noted that in the case of the synthesis of a product
such as (50), in which three acyl groups are added in random order,
intermediate (35) could be used in place of (36) as the starting material.
Instead of acylation with trifluoroacetic anhydride, (35) could alternately

126
be acylated with the appropriate acylating agent, in this case acetyl
chloride. The synthesis of (50j would subsequently be accomplished by
debenzylation, acylation with benzoyl chloride, removal of the t-BOC
group, and acylation with 2,3-dimethoxybenzoyl chloride.
In addition to the preparation of triacylated spermidines as
described by the example here, a limitless number of mono- and bis-
acylated spermidines can be generated using these new reagents as well.
Moreover, the ability to remove the protecting groups in any order im
parts enormous flexibility into the types of substituents to be added.
This feature, for example, can 'custom tailor1 the synthesis so as to
add a particularly labile group last.

CHAPTER SIX
SYNTHESIS AND SOLUTION DYNAMICS OF AGROBACTIN A
Experimental Section
Materials
All reagents, with the exception of t-butoxycarbonyloxyimino-2-
phenylacetonitrile (BOC-ON, Sigma Chemical Co., St. Louis, MO) were pur
chased from Aldrich Chemical Co., Metuchen, NJ and, unless noted, were
used without further purification. The reagents N1,N8-bis(t-butoxycar-
bonyl)spermidine-HCl (13) and N1,N9-bis(t-butoxycarbonyl)homospermidine
(15) were prepared as previously described in Chapter Two. Sodium sul
fate (Na2S04) was used as the drying agent. Melting points were taken on
a Fischer-Johns apparatus and are uncorrected. Routine *H NMR spectra
were recorded on a Varian T-60 and prepared in DCC13 or DMS0-d6 with
chemical shifts given in parts per million () from an internal Me4Si
standard. The infrared spectra were recorded on a Beckman 4210 spectro
photometer. Elemental analyses were performed by Atlantic Microlabs,
Atlanta, GA.
N-(t-Butoxycarbonyl)threonine (511
This compound was prepared by reacting D,L-threonine with t-butoxy-
carbonyloxyimino-2-phenylacetonitrile:8t* mp 75-76C (Et20/Pet ether)
(lit. 74-77).
Succinimide-2,3-dihydroxybenzoate (52)
To a solution of 2,3-dihydroxybenzoic acid (1.16 g, 7.53 nmol)
and N-hydroxysuccinimide (1.04 g, 9.04 mmol) in dry dioxane (30 ml) was
127

128
added DCC (1.89 g, 9.16 mmol) in dioxane (20 ml) under N2. After 16 hours
the mixture was filtered and the DCC washed with dioxane (15 ml). The
solvent was evaporated in vacuo and the residue crystallized from Me0H/H2
to yield 1.8 g (95%) of product as tan crystals, mp 55-56C; XH NMR (d6-
DMSO) 6 2.93 (4H), 6.52-7.36 (3H), 9.82 (2H).
Anal. cal. for CnHgNOg: C, 52.60; H, 3.61; N, 5.58. Found: C, 52.68;
H, 3.66; N, 5.52.
N4[N-(t-Butoxycarbony1 )threony!]N1,N8-bis(t-butoxycarbonyl)spermidine 53
A solution of DCC (680 mg, 3.3 mmol) in 20 ml THF was slowly added
to a solution of 53^ (660 mg, 3.0 mmol) and N-hydroxysuccimide (380 mg,
3.3 mmol) in 30 ml THF. After 18 hours, the DCU precipitates were fil
tered, washed with fresh THF, and the filtrate evaporated. The residue
was dissolved in 30 ml CH3CN and slowly added to a solution of (13)
(1.26 g, 3.3 mmol) and Et3N (470 yl, 3.5 mmol) in 5% aqueous CH3CN
(50 ml). After stirring for 36 hours at room temperature, the reaction
mixture was then concentrated, the residue taken up in 100 ml EtOAC,
washed with H20 (2 x 20 ml), 3% HC1 (3 x 20 ml), H20 (2 x 20 ml), dried,
and concentrated to afford 1.5 g (91%) of the desired product.
An analytical sample was obtained by chromatography on silica gel
eluting with EtOAc/CHCl3 (1:1): *H NMR (CDC13) 6 1.20 (d, 3H), 1.44 (s,
27H), 1.75 (m, 6H), 2.80-3.58 (overlapping multiplets, 8H), 4.04 (m, 1H),
4.38 (d, 1H), 4.8-5.5 (br, 4H); IR (CDC13) 3000 (m), 1715 (s), 1180 (s),
770 (s) cm-1.
Anal. cal. for C26H50^48: C, 57.12; H, 9.22; N, 10.45. Found:
C, 57.25; H, 9.24; N, 10.17.

129
N4-Threon,y1 spermidine-trifluoroacetate (54)
Trifl uoroacetic acid (25 ml) was slowly added to a cooled flask con
taining (53J (650 mg, 1.2 mmol) and the resulting solution stirred for
25 min while warming to room temperature. The solvent was then quickly
evaporated, the residue dissolved in MeOH (25 nl), and evaporated (twice).
The product was then dissolved in 50 ml H20, washed with cold CHC13
(3 x 10 ml), and the aqueous layer lyophilized to afford 690 mg (97%)
as a light tan hygroscopic solid.
An analytical sample was prepared by chromatography on Sephadex LH-20
eluting with 20% MeOH/EtOAc: lH NMR (DMS0-d6) 6 1.15 (d, 3H), 1.36-2.08
(m, 6H), 2.80-3.32 (m, 8H), 4.52 (m, 3H), 7.94 (m, 9H); IR (KBr) 1685
(s), 1190 (s) cm-1.
Anal. cal. for C17H2gFgN40p: C, 34.70; H, 4.97; N, 9.52. Found:
C, 34.50; H, 5.03; N, 9.43.
N4-[N-(2,3-Dihydroxybenzoyl)threonylj-N1,N8-bis(2,3-dihydroxybenzoyl)-
spermidine (Agrobactin A) (5,I.)
A solution of succinimide 2,3-dihydroxybenzoate (52J (350 mg, 1.4
mmol) in 15 ml THF was slowly added dropwise to a solution of (54.) (275
mg, 0.46 mmol) and Et3N (210 ml, 1.5 mmol) in 5% aqueous THF (40 ml) un
der N2. After 36 hours the solvent was evaporated to dryness and the
residue preabsorbed on Sephadex LH-20, and eluted with an ethanol/ben
zene gradient (5-25% v/v) to yield 225 mg (75%) of the desired product.
The spectral characteristics were identical to those reported in the
literature.72
Anal. cal. for C32H38N4011*H20: C, 57.13; H, 5.99; N, 8.33. Found:
C, 57.10; H, 6.09; N, 8.26.

130
Ns-[N-t-Butoxycarbony1)threonylj-N1,N9-bis(t-butoxycarbonyl)homosper-
midine (56)
The active ester of (51_) was reacted with a solution of (1_5) and
purified as described for (53): 89% yield; XH NMR (CDC13) 6 1.18 (d,
3H), 1.48 (s, 27H), 1.40-1.71 (m, 8h), 3.02-3.56 (m, 8H), 4.03 (m, 1H),
4.36 (d, 1H), 4.72-5.48 (br, 4H); IR (CHC13) 3010 (m), 1715 (s), 1175
(s), 770 (s) cm-1.
Anal. cal. for C27H52N408: C, 57.83; H, 9.35; N, 9.99. Found:
C, 57.56; H, 9.41; N, 9.92.
N5-Threonyl homospermidine (57)
A solution of (56) was deprotected and purified as described for
(54): 98% yield; lH NMR (DMS0-d6) 1.18 (d, 3H), 1.36-1.99 (m, 8H), 2.82-
3.37 (m, 8H), 4.54 (m, 2H), 7.97 (m, 9H).
Anal. cal. for C18H31FgN408: C, 35.89; H, 5.19; N, 9.30. Found:
C, 35.54; H, 5.27; N, 9.14.
N5-[N-(2,3-Dihydroxybenzoyl)threony!]-N|,N^-bis(2,3-dihydroxybenzoy1)-
homospermidine (Homoagrobactin A) (58)
A solution of (58) was prepared and purified as described for (55);
72%yield; NMR (See Table 1).
Anal. cal. for 0338^40!!: C, 59.27; H, 6.03; N, 9.57. Found:
C, 58.91; H, 6.19; N, 9.37.
N4-[N-(2,3-Dimethoxybenzoyl )threonyl1-N1,N8-bis(2,3-dimethoxybenzoyl)-
spermidine (Hexamethyl agrobactin A) (j)
A solution of DCC (450 mg, 2.2 mmol) in THF (15 ml) was slowly
added to a solution of 2,3-dimethoxybenzoic acid (400 mg, 2.0 mmol) and
N-hydroxysuccimide (255 mg, 2.2 mmol) in THF (30 ml). The reaction was
allowed to stir at room temperature for 18 hours at which time the DCU
precipitates were filtered off and washed with THF. The filtrate was
then concentrated to approximately 20 ml and added to a solution of (54)

131
(350 mg, 0.60 mmol) and Et3N (90 yl, 0.65 mmol) in 5% aqueous CH3CN
(30 ml). After 48 hours the solvent was evaporated, and the product
chromatographed on silica gel eluting with 10% MeOH/CHCl3 to yield 400
mg (91%) as a white hygroscopic solid: NMR (CDC13) 5 1.16 (d, 3H),
1.58 (m, 6H), 3.42 (m, 8H), 3.56 (s, 18H), 4.0-4.6 (m, 3H), 6.62-7.7
(m, 9H), 7.74-8.4 (m, 3H).
Anal. cal. for C38H50N4On: C, 61.78; H, 6.82; N, 7.58. Found:
C, 61.84; H, 6.71; N, 7.47.
N4-[N-(Benzoyl)threonylj-N1,N8-bis(benzoyl)spermidine (60)
A solution of the succinimide ester of benzoic acid, prepared by
reacting DCC, N-hydroxysuccinimide and benzoic acid, was reacted with
(13) in a similar manner as described for (59). Yield: 225 mg (93%);
XH NMR 5 1.18 (d, 3H), 1.61 (m, 6H), 3.39 (m, 8H), 4.08-4.66 (br, 3H),
7.23 (s, 15H), 7.98 (m, 3H).
Anal. cal. for C32H38N405: C, 68.80; H, 6.86; N, 10.02. Found:
C, 68.57; H, 6.91; N, 9.84.
Agrobactin A (via scheme II)
A mixture of (D,L)-M4-threonyl-N1,N8-bis(2,3-dihydroxybenzoyl)sper-
midine-HBr73 (100 mg, 0.17 mmol) and triethylamine (24 yl, 0.17 mmol)
in dry DMF (20 ml) was cooled to 0C under N2. A solution of N-hydroxy-
succinimido-2,3-dihydroxybenzoate [52) (43 mg, 0.07 mmol) in DMF (20 ml)
was added dropwise over 15 min. After 12 hours, the solvent was removed
in vacuo and the residue chromatographed on LH-20 (10-^30% EtOH/benzene)
to afford 82 mg (75%) of product as a white solid. The TLC and 300 MHz
!H NMR spectral characteristics of this product were identical to that
of agrobactin A in Scheme 1.

132
High field XH NMR spectroscopy
High resolution FT *H NMR spectra were recorded on a Nicolet NT-300
spectrometer equipped with a Nicolet 1280 computer. Samples were run at
room temperature (23 1C) unless otherwise noted. Generally, when
CDC13 or CDC13/DMS0-d6 were employed as the solvent, 1-4 mg of sample were
dissolved in approximately 500 yl of solvent. Chemical shift and cou
pling constant values, when obtainable, are given in Table 6-1 in the
solvents indicated. All chemical shifts are in ppm downfield from an
internal TMS standard. Samples for the temperature dependence studies
were prepared by dissolving 5-10 mg of sample in 500 ul DMS0-d6 or DMF-dy
as indicated in Table 6-2. Activation energies were measured by observ
ing the coalescence temperature of the resulting y-methyl singlets pro
duced upon irradiation of the adjacent e-methine. The coalescence tem
peratures were measured both on heating and cooling cycles and activation
energies subsequently calculated using the rate equations as described
by Gutowsky and Cheng.77 Additionally, the results were found to be in
good agreement with those calculated from a total line shape analysis
using a program contained in the NT-300 software for unequally populated
two-site exchange. The results indicated that the experimental 5v's were
within 1 Hz of the simulated values. Therefore, assigning a maximum
error of 1.0C in Tc, and an error of 1.0 Hz in measurement of the
5v, the range of error in determining the Ea is 0.15 kcal/mole.
Results and Discussion
Synthesis
The 'inside-out1 synthesis of agrobactin A employs the reagent
N1 ,N8-bis(t-butoxycarbonyl )spermidine (1_3). Originally developed as a
reagent to selectively generate N4-modified spermidine derivatives for

133
the delivery of antineoplastics to leukemia cells,75 it is stable
and easily accessible in high yields. Furthermore, both the homo- and
norspermidine homologues are also available, allowing for the synthesis
of the homo- and noragrobactin A homologues.
The synthesis of these reagents is easily effected by reacting the
appropriate secondary N-benzyl polyamine with t-butoxycarbonyloxyimino-
2-phenylacetonitrile, followed by debenzylation to afford the terminally
t-BOC protected polyamine in 90% overall yield/5 Agrobactin A and its
homospermidine homologue are subsequently synthesized in three high yield
steps from this point, as illustrated for agrobactin A in Figure 6-1.
The procedure first calls for reacting the succinimido ester of
t-BOC threonine, previously generated by reacting t-BOC threonine (51),
N-hydroxysuccinimide and DCC in THF, with a solution of N1,N8-bis(t-
butoxycarbonyl )spermidine hydrochloride (1_3) or its homospermidine ana
logue and triethylamine in aqueous CH3CN for 48 hours. The condensation
proceeds smoothly and cleanly to afford the t-BOC triamide (J33) in 90%
crude yield. This product can be easily purified via silica gel chroma
tography eluting with CHCl3/EtOAc (1:1); however, further purification
was found to be unnecessary as the impurities can be removed after the
next step. Hence, (53j is deprotected by brief exposure to trifluoro-
acetic acid. The residue is dissolved in water, and the impurities from
the preceding condensation extracted into CHC13 prior to lyophilization
of the aqueous layer to afford N4-threonyl spermidine (54) or N5-threonyl
homospermidine (_57) as the trifluoroacetate salt in 80% overall yield
from N-t-BOC-threonine.
The final and most crucial step of the synthesis is the addition
of the three 2,3-dihydroxybenzoyl moieties to the N4-threony1 triamine.

134
13
Figure 6-1. "Inside out" synthesis of Agrobactin A via
Nl-N8-bis(t-butoxycarbonyl)spermidine (13)
(Scheme I).

135
Previous studies suggested it would be necessary first to protect the
catechol groups of 2,3-dihydroxybenzoic acid prior to condensation. How
ever, because of the known acid-catalyzed N- to O-migration of acyl
groups fixed to threonine's nitrogen1"1>102 only protecting groups capa
ble of being removed under neutral or basic conditions could be con
sidered. Under acid conditions threonine's N-acyl carbonyl becomes more
electrophilic, promoting intramolecular transacylation, resulting in the
formation of the threonyl ester and the amine salt. The migration can
be reversed, in some cases, under basic conditions.131 During our initial
synthesis of compound II, we had developed the reagent 2,3-diacetoxyben-
zoyl chloride which could efficiently acylate spermidine's primary amines,
and the acetoxy protecting groups could then be cleanly removed by treat
ment with methanolic sodium methoxide."
When triamine (54) was reacted with three equivalents of 2,3-di-
acetoxybenzoyl chloride, the acetoxy protecting groups removed under
basic conditions, and the products worked up in weak acid, two products
were repeatedly obtained in low yields. The major product was determined
to be the N- to O-migrated ester of agrobactin A, as indicated by its
300 MHz XH NMR spectra characterized by the chemical shifts of the a-,
6-, and y-methyl protons of 6 5.2, 5.5, and 1.4, respectively. This
migration was likely the result of protonating the catechols during
acidic workup. Even when cold pH 6.5 phosphate buffer was employed to
protonate the catechols, substantial migration still occurred. Fur
thermore, the minor product, although identical on TLC to agrobactin A,
demonstrated a XH NMR with the a- and g-methines shifted 0.2 ppm upfield
to those values previously reported. In addition, obvious differences
in the aromatic region were also noted. In light of these problems,
this method was abandoned.

136
Alternatively, the possibility of attaching the 2,3-dihydroxyben-
zoyl groups directly to the N4-threonyl triamine without catechol pro
tecting groups was considered. Recently, Van Brussel and Van Sumere
have shown it possible to generate the succimido active esters of a num
ber of mono- and dihydroxybenzoic acids in the presence of the unpro
tected phenols.76 This approach, besides being the most direct, would
generate agrobactin A under essentially neutral conditions. This is de
sirable as it would eliminate using acid to protonate the catechols, re
ducing or eliminating the amount of migrated ester formed.
Therefore, succinimide-2,3-dihydroxybenzoate (52_) was prepared by
reacting 2,3-dihydroxybenzoic acid and N-hydroxysuccimide in the presence
of DCC to afford (52J upon recrystallization from methanol/H20. The
final step of the synthesis was successfully accomplished by reacting
three equivalents of the active ester (52_) with M4-threonyl spermidine
in aqueous THF for 48 hours. Thin layer chromatography of the products
indicated the reaction proceeds with little or no formation of the un
desired migrated ester. The reaction mixture was concentrated and the
crude agrobactin A easily purified by chromatography on Seph-adex LH-20
to afford agrobactin A (55_) in 75% yield. The symmetrical homologue
of agrobactin A, N5-[N-(2,3-dihydroxybenzoyl)]-Nx,N8-bis(2,3-dihydroxy-
ben zoyl)homospermidine (58) was prepared by reacting (52) with N5-threonyl
spermidine (57_) and purified in a similar manner. Additionally, the
hexamethyl derivative of agrobactin A, N4-[N-(2,3-dimethoxybenzoyl)-
threonyl-N1,N8-bis(2,3-dimethoxybenzoyl)spermidine (59) was also syn
thesized using these procedures. The succimide active ester of 2,3-
dimethoxybenzoic acid was generated as previously for (52) and reacted
with the triamine (54) to produce hexamethyl agrobactin A (59J in 95%

137
i
55
Figure 6-2.
Synthesis of Agrobactin A via N1,N8-bis(2,3-di
methoxybenzoy1jspermidine (Scheme II).

yield. The synthesis of (59j, in addition to providing us with a nec-
cessary derivative for our *H NMR studies, illustrates that the number
of compounds which can be generated by this method are limited only by
the acylating or alkylating agent employed.
Finally, as proof of structure, agrobactin A was synthesized by an
alternate route using the versatile reagent N1,N8-bis(2,3-dimethoxyben-
zoyl)spermidine85 as illustrated in Figure 6-2. As previously described
in our synthesis of parabactin,73 N1,N8-bis(2,3-dimethoxybenzoyl)sper-
midine was condensed with N-CBZ threonine, again using the coupling agents
DCC and N-hydroxysuccimide to produce N4-[N-CBZ threonyl1-N1,N8-bis-2,3-
(dimethoxybenzoyl)spermidine. The CBZ protecting group is then removed
by hydrogenolysis in methanolic HC1 over PdCl2 catalyst, followed by
the removal of the methoxy groups with BBr3 to afford N4-threonyl-N1,N8-
bis(2,3-dihydroxybenzoyl)spermidine. The final product was once again
produced by reacting the succinimido ester of 2,3-dihydroxybenzoic acid
(52) with N4-threonyl-N1,N8-bis(2,3-dihydroxybenzoyl)spermidine. Although
this second method also represents an effective means of generating
agrobactin A, the preceding 'inside out' synthesis is preferred prepara-
tively as it contains one less step and requires only one chromatographic
separation instead of three.
Chemical shifts and coupling constants of agrobactin A and derivatives
at 300 MHz
The 300 MHz XH NMR spectrum of agrobactin A in CDC13/DMS0-d6 (10:1)
at 23C is shown in Figure 6-3. This solvent system was employed for
the purpose of comparison with earlier published spectra of agrobactin
A as well as with agrobactin and parabactin. The choice of solvent is
critical as the chemical shifts of these compounds are extremely sen
sitive to changes in solvent. The <5 values observed are identical to

'iL.r;
o 6 6 4 2 0 PPM
Figure 6-3. 300 MHz ]H NMR Spectrum of Agrobactin A in CDCL3/DMS0-d6 (10:1) at 23 C
The downfield phenolic region at -45 is shown in the insert.
1 39

140
the chemical shifts previously reported for agrobactin A,75 and the
overall appearance of the spectra is similar to the previously pub
lished NMR spectra of agrobactin75 and parabactin73 noting, however,
the differences in the a, 8, and y threonine resonances between the open
and closed forms.
For purposes of analysis, the spectra can be divided into two re
gions, the upfield portion above 6 6.00 containing the resonances from
the spermidine backbone and the threonine residue, and the downfield
portion containing the aromatic, phenolic, and amido protons. The high
field end of the spectrum is characterized by a pair of doublets cen
tered at 5 1.20 which integrates to three protons. These protons are
assigned to the y-methyl of threonine, each doublet equally coupled to
the 8-methine (J^ = 6.3 Hz) as confirmed by decoupling experiments.
The envelope between 6 1.5 and 2.1 integrates to six protons, and ori
ginates from the three internal methylene groups of the spermidine back
bone, while the four external methylene groups adjacent to the amides
are responsible for the eight proton envelope observed from 6 3.15 to
3.75. The final set of peaks in the upfield region of the spectra in
clude an extensively split multiplet at 6 4.18 and an apparent doublet
of doublets at <$ 5.02, both integrating to one proton each. These have
been assigned as the 8- and a-methines, respectively, by the appropriate
decoupling experiments. Additionally, a broad hump is observed at about
6 4.7 resulting from the threonine hydroxyl which disappears on exchange
with D20. Furthermore, as expected the hydroxyl's intensity and loca
tion will vary with concentration and temperature.
The downfield portion of the spectra consists of the aromatic pro
tons, amido protons and the phenolic hydroxyls. The aromatic region

141
lies between 6 6.6 and 7.4 and integrates to nine protons. Their
assignment, although similar to the aromatics of N1,N8-bis(2,3-dihydroxy-
benzoyl )spermi dine reported earlier,lc:3is further complicated by the
third 2,3-dihydroxybenzoyl moiety. The upfield overlapping multiplets
centered at 6 6.70 integrate to three protons and can be assigned to
the meta protons. Downfield to this are four lines centered at 6 7.00,
which correspond to the three para protons. Finally, the ortho protons
appear the furthest downfield as two apparent triplets centered at <5 7.19
and 7.25, the upfield triplet integrating to two protons versus one for
the downfield triplet. The NH protons make up the next group of signals
downfield from the aromatics; those resulting from spermidine's terminal
amides are located at 6 8.03 and 8.24 while the threonyl N-H is located
between them at 6 8.09. The phenolic protons lie the furthest downfield
and, like the threonine hydroxyl, undergo rapid exchange at 23C- This
exchange results in broad, undefined signals whose location is, again,
dependent upon such factors as temperature and amount of water present.
At -45C, however, the rate of exchange is considerably slowed, result
ing in sharper signals at approximately 5 12.0 and 13.0 as shown in the
insert in Figure 6-3.
Of particular interest, however, is the duplicity of signals ori
ginating from the threonyl moiety. The threonine coupling pattern, in
the absence of any conformational effects, represents a simple spin sys
tem. The a-methine should be split once by the amide resulting in a
doublet with a J => 6.9 Hz. This doublet should then be further split
by the S producing a doublet of doublets. The B-methine is coupled to
the three y-methyl protons, resulting in a quartet which is split again
by the a-methine producing eight lines which would be expected to give

142
rise to a complex multiplet. Finally, the y-methyl should be split
principally by the single 8-methine proton resulting in a doublet with
an expected coupling in the order of 5.5-6.5 Hz, as long range a-y cou
pling is likely to be small.
Inspection of Figure 6-4b and 6-5b reveals that these predicted
coupling patterns do exist; however, they do exist in duplicate. For
example, the y-methyl group exists as a pair of doublets instead of a
single doublet, Figure 6-4b. These doublets are observed in a ratio
of approximately 2:1 in CDC13/DMS0-d6 and are both equally coupled to
the B-methine (J = 6.3 Hz). Likewise, the B-methine also exhibits an
additional set of signals. This is more clearly seen when the y-methyl
is decoupled, Figure 6-5b. Again, in the absence of any conformational
effects, irradiation of the y-methyl should result in the B-methine
collapsing to a doublet arising from the coupling with the a-methine
(J = 2.6 Hz). However, in agrobactin A irradiation of the y-methyl re
sults in a pair of doublets centered at 5 4.2, Figure 6-5b, also in
approximately a 2:1 ratio. Surprisingly enough, the a-methine, however,
does not show an additional set of lines, giving rise to the expected
doublet of doublets as one might anticipate i.e., split once by the
amide (J = 3.4 Hz) and once by the B-methine (J = 2.6 Hz).
The XH NMR data are consistent with the threonine substituent exist
ing in at least two distinct magnetic environments. Because the B-methine
and y-methyl protons are located further out on the threonyl side chain
i.e., closer to the terminal 2,3-dihydroxybenzoyl groups than the a-
methine, on rotation about the central amide bond they see more signifi
cant changes in magnetic environment. The a-methine, having a more
'internal' location, moves through a relatively smaller area upon

143
Figura 2
1.2 1.1 PPM 1.2 1.1 P
Figure 6-4. 300 MHz NMR spectra of y-methyl region in
solvents indicated: a) homoagrobacti n a 5J3 (C D C L 3/
DMSO-dg); b) agrobactin A 5_5 (CDC13/DMSO-dg); c)
53 (CDCI3); d) 56 (CDC13).

144
Figure 6-5. 300 MHz NMR spectra of the a- and B-methine~
region of a) Homoargrobactin A; b) agrobactin A;
c) 5_3 ; d) 56 in the solvents indicated in figure
6-4. (y-metTvyls decoupled to simplify spectra)

145
rotation and at greater distances from the aromatic rings. Therefore,
it sees little or no change in its environment resulting in only one
observable signal. This idea is illustrated in Figure 6-6. The inner
and outer cylinders represent the sweep volumes of the a-methine and
Y-methyl group, respectively. The a-methyl group and 8-methine can lie
in close proximity to the terminal aromatic rings and can easily be
influenced by the anisotropic effects. However, since these effects
decrease rapidly with increased distances, the a-methine is likely to
be too distant from the aromatic rings to be affected by their aniso
tropy.
Furthermore, if the cylinders are cut in half by a plane passing
through the central nitrogen perpendicular to the spermidine backbone,
it can easily be seen that corresponding points in either half are at
different intramolecular distances from the aromatic rings due to the
asymmetry of the spermidine chain. Accordingly, these points will also
experience different magnetic fields. Therefore, a proton existing in
a conformation lying in the left half will likely result in a different
signal as compared to its counterpart that lies in the right half of
the cylinder. Although this model does not consider all of the possibl
orientations of the spermidine backbone and aromatic rings, a recent
x-ray crystallographic study of agrobactin104 does suggest a conforma
tion with a nearly linear spermidine backbone that is similar to that
depicted in the model.
Based on this model, a symmetrical analogue should equalize the in
tramolecular distances on either half, eliminating the duplicity of sig
nals observed for the 8- and y-protons of agrobactin A.
Neilands has also attributed the appearance of the XH NMR spectrum
of agrobactin A to the asymmetrical nature of the spermidine chain and,

CONHCH2CR2CH2NCH2CH2CH2CH2NHCO
Figure 6-6. Diagram illustrating the approximate sweep volumes of the a- and
7-protons of agrobactin A on rotation about the central amide.
CT>

147
in an attempt to resolve the problem, synthesized a symmetrical analogue
to agrobactin A, containing a glycine residue instead of the threonine
residue. As we have noted, the a-methine of agrobactin A does not appear
to 'see' a difference in conformation; hence, a model lacking the threo
nine side chain may not be the appropriate model to verify the symmetry
(or asyrcmetry) of the polyamine backbone in the *H NMR spectrum.
Accordingly, in order to verify the glycine work, the symmetrical
analogue of agrobactin A incorporating homospermidine as the backbone
was synthesized. As expected, the symmetrical homoagrobactin A result
ed in a simplified spectrum. The y-methyl of homoagrobactin A now ex
hibits only a single doublet, Figure 6-4a, as does its uncoupled e-
methine located at 5 4.2, Figure 6-5a. The appearance of the a-methine,
<5 5.0, is similar in both homoagrobactin A and agrobactin A, Figure
6-5, a and b, respectively, although the doublet structure of the latter
appears to be broader indicating that, perhaps, minor conformational
effect is being felt by the a-methine in agrobactin A. Similar results
are observed in the 300 MHz XH NMR of the asymmetrical t-butoxycarbonyl
precursor (53) and its symmetrical homologue (56). The y-methyl protons
of (53) are observed as two overlapping doublets centered at 5 1.18,
Figure 6-4c. However, in the symmetrical precursor (56), only one doub
let is observed for the y-methyl, <5 1.17, Figure 6-4d. Additionally,
the threonyl NH (6 5.48) is simplified in (56) and is observed as a
doublet, Figure 6-5d, as compared to a doublet of doublets for (53),
Figure 6-5c. On the other hand, the a- and 6-methines (<5 4.41 and 4.04,
respectively) of (5J3) experience little change in their magnetic envi
ronment as a result of conformation, Figure 6-5c.
It is interesting that the g-methine of (53) lacks the duplicity
of its counterpart in agrobactin A; however, this might be explained by

148
the differences in anisotropy generated by the t-butoxycarbonyl and 2,3-
dihydroxybenzoyl groups. Additionally, the conformation of the threo
nine residue may be different between agrobactin A and (52). This is
supported by the observation that no coupling is seen between the a-
and 8-methines of (53) compared to the J = 2.6 Hz in agrobactin A
(Table 6-1), indicative of a difference in dihedral angle between the
a- and e-methines in agrobactin A and (53).
It was mentioned earlier that the chemical shifts of agrobactin A
and its related siderophores are extremely sensitive to the XH NMR
solvents employed. Similarly, the conformer population of agrobactin A
was found to be effected by solvent. As previously illustrated by agro
bactin A's y-methyl and B-methine signals in Figures 6-4b and 6-5b,
the conformers exist in approximately a 2 to 1 ratio in CDC13/DMS0-dg
(10:1). Upon switching to a more polar hydrogen bond acceptor solvent,
such as DMSO or DMF, the conformer population appears to even out, ap
proaching a 1:1 ratio as illustrated in Figure 6-7 for DMF. This sol
vent dependency suggests that hydrogen bonding may play a role in de
termining the conformer population. It is suspected that DMSO or DMF
is interacting intermolecularly with agrobactin A, reducing the intra
molecular interactions responsible for the conformer preference seen in
the less polar CDC13.
Determination of activation energies using NMR temperature coales
cence experiment
Since agrobactin A, agrobactin and N4-[N-t-butoxycarbonyl)threonyl]-
N1,N8-bis(t-butoxycarbonyl)spermidine (53) all exhibit duplicity in
their XH NMR spectra, the energy of activation (Ea) for the intercon
version between the conformers can be estimated by measuring the coal
escence temperature (Tc) of these compounds. A qualitative comparison

Figure 6-7, 300 MHz ^ H NMR spectra of agrobactin A in DMF-d7
a) Y-methyl decoupled 8-methine region; b) y- '
methyl region.

150
of the estimated Ea's might, therefore, provide additional information
regarding the factors affecting conformation in these siderophores.
Although the two y-signals corresponding to the two polyamide conformers
do not quite represent an equally populated spin system, the Ea for
their interconversion can be estimated using methods developed for equal
ly populated, noncoupled spin systems.77 It is generally acknowledged
that computer total line-shape analysis should be employed for highly
accurate values of Ea; however, highly accurate values are not required
for the qualitative comparisons necessary in these studies. In this
regard the relative differences in Ea's between the compounds are more
important than their absolute values.
Of all the barriers involved in control!ing the interconversion be
tween the conformers in these polyamides, hydrogen bonding and steric
factors are likely to be the dominant ones. The question is how to de
termine the contribution of each of these components in the interconver
sion. The answer is to carefully remove or introduce hydrogen bonding
and/or steric interactions in the systems of interest and then evaluate
the effect on Ea for the process. In this study we have chosen to con
sider the Ea of agrobactin, agrobactin A, the precursor (53) and methy
lated agrobactin A (59). The structural differences and similarities
between these four compounds should be sufficient to help assign the
role of steric and hydrogen bonding factors controlling the conformer
populations of the spermidine siderophores.
The coalescence temperatures of agrobactin A, hexamethyl-agrobactin
(59), and precursor (53) were all determined in DMS0-d6 as described in
the experimental section and listed in Table 6-2. Irradiation of the
e-methine of agrobactin A, for example, results in the collapse of the

Figure 6-8. Effects of Temperature upon y-methyl region of agrobactin A in DMSO-d
(e-methine decoupled).

152
Y-methyl resonance into two lines at room temperature. Upon heating,
the two lines at first gradually broaden, then move rapidly as the coal
escence temperature is approached, Figure 6-8. Finally, the lines are
observed to coalesce at approximately 74C and, with a further increase
in temperature, the line width of the resultant single line now decreases
as its intensity increases.
The observed Tc of 74C for agrobactinA corresponds to activation
energy of approximately 18.2 .15 kcal/mole. This is quite similar to
the value reported for agrobactin by Neilands and was unexpected in view
of the difference between the relatively rigid oxazoline ring versus the
more flexible open agrobactin A. A second unexpected result was the high
coalescence temperature of the methylated derivative (59_). It was ini
tially anticipated that methylation of the catechols would lower the co
alescence temperature of the various conformers by destroying the stabi
lizing hydrogen bonding network. On the contrary, (59) coalesced at a
much higher temperature than agrobactin A. In fact, the Tc of (59) ex
ceeded the operating limit of our probe, preventing the precise measure
ments of (59^)1 s Ea. At 130C, the highest temperature that could be
measured, the decoupled y-methyl signals of (59j were nearing coales
cence. The Ea of (59) was, therefore, estimated based on a Tc of at
least 130C and 5v of 9.4 Hz, to be greater than 21 kcal/mole. Since
the Ea varies directly with temperature, the further increase in tem
perature above 130C can only increase the Ea. Therefore, 21 kcal/mole
represents the minimal Ea for (59_). This large increase in Ea for (59)
over agrobactin A can be explained by an increase in steric interactions
that are preventing interconversion between conformers. It does not,
however, necessarily mean steric factors are more important than hydrogen

153
bonding as the solvent, DMS0-d6, used in the measurements, is certainly
capable of competing for intramolecular hydrogen bonds. This implies
that the initial measurement of Ea for agrobactin A made in DMSO is
largely a measurement of steric control of the conformer population as
intramolecular hydrogen bonding is likely minimized by intermolecular
competition with DMSO.
The coalescence temperature of the tri-t-BOC precursor (53) further
supports the idea that DMSO prevents intramolecular hydrogen bonding in
the system studied. As shown in Figure 6-4c, the y-methyl of (53) exists
as an overlapping set of doublets in CDCI3 at room temperature. In
DMS0-d6, however, even at room temperature only one doublet is observed.
Therefore, the change from the less polar CDC13 to DMS0-d6 is sufficient
alone to allow rapid interconversion between conformers of (53).
The high freezing point of DMSO precluded its further use in deter
mining the Tc value of (53); therefore, an alternate solvent was chosen.
However, in order to make a comparison of the Ea's between (53) in an
alternate solvent to those of agrobactin and its homologues in DMS0-d6,
it is important that the solvent itself does not change the Ea signifi
cantly. Dimethylformamide (DMF) has a similar dielectric to DMSO, but
a much lower freezing point necessary for the measurement, making it an
appropriate choice. To determine if DMF would have any effect on the
Ea, the Tc and Ea of agrobactin A was subsequently measured in DMF.
No significant difference was noted between the Ea and Tc of agrobactin
A determined in DMF versus DMSO suggesting the data of (53) in DMF can
be compared to those in DMSO.
The ¡H NMR of (53) in DMF-d7 at room temperature also lacked the
duplicity seen in CDC13. However, upon cooling the decoupled y-methyl

154
signal broadened, and eventually displayed two signals that coalesced
at 18C, corresponding to a Ea of 15.2 0.15 kcal/mole. This low Ea
observed for (53) as compared to agrobactin was anticipated, at least
in direction if not in magnitude. The reason for this may not lie in
a difference in overall size between the t-butyl versus the 2,3-dihy-
droxybenzoyl group, as molecular models suggest that planar 2,3-dihy-
droxybenzoyl group to be only slightly bulkier. However, it may indi
cate that a small role is played by catechol hydrogen bonding in the
conformer populations of agrobactin A versus the precursor (53). Alter
natively, the ether linkage present in the t-butoxycarbonyl group may
provide the t-butyl group an additional degree of freedom, making it
easier for it to avoid the centerpiece as it rotates.
The coalescence data suggest, then, that in solvents such as DMSO
which can compete for intramolecular hydrogen bonds, steric factors pri
marily influence interconversion between conformers in the systems
studied. It further implies that the molecular hydrogen bonding in
agrobactin A is not unusually strong. It is certain, however, that
hydrogen bonding must play a role in conformer population in nonpolar
solvents. This was illustrated by the effect of solvent upon duplicity
of XH NMR signals in agrobactin A and, most dramatically, in the pre
cursor (53) in which DMS0-d6 spectra lacked any sign of the conformers
present in CDC13 at 23C. This effect on the *H NMR of (53), on chang
ing between DMSO and CDC13, is of additional interest in view of the
fact that the t-butoxy group is not able to form as many hydrogen bonds
as the 2,3-dihydroxybenzoyl group. This suggests that an even greater
solvent effect may be experienced by agrobactin A and its related si-
derophores, producing much higher Tc and Ea values in nonpolar solvents

155
for these compounds. However, due to
in all but the most polar solvents, a
Ta's in other solvents is essentially
agrobactin A's poor solubility
comprehensive determination of
not possible.

156
Table 6-1. 300 MHz XH NMR Chemical Shifts and Coupling Constants
of Polyamides (23C).
Agrobactin A
(CDC13/DMS0-d6)
Homoagrobactin A
(CDCl3/DMS0-d6)
(53)
(CDC13)
(56)
(CDC13)
Threonine
(JaNH)
5.02
5.03
4.39
4.39
(8.4 Hz)
(8.4 Hz)
(9.6 Hz)
(9.6 Hz)
0 (Jag)
4.18
4.21
4.02
4.02
(2.7 Hz)
(3.0 Hz)
Y (JSy)
1.20
1.19
1.18
1.17
(6.3 Hz)
(6.6 Hz)
(6.1 Hz)
(6.3 Hz)
OH
4.7
4.7
5.2
4.2
NH
8.09
8.10
5.47
5.48
t-Butoxy


1.45
1.44
cch2c
1.5-2.1
1.5-1.85
1.4-1.9
1.36-1.7
nch2c
3.15-3.75
3.15-3.65
2.96-3.6
3.12,3.3,
Aromatics
6.70,7.00,7.22
6.68,6.98,7.22
NH
8.03,8.24
8.0,8.15
4.70,4.97
4.70,5.00
Phenols
12.0,13.0
12.0,13.0

157
Table 6-2. Coalescence Temperatures and Activation Energies
of Agrobactin A and Homologues in DMSO-dg.
Tc(C)
Ea(kcalmole-^)
Tri(tBOC) compound 5_3
1 8a
15.2 t 0.15
Agrobactin A (55)
74
18.2 t 0.15
Agrobactin A-OMe (59)
>130
>21
Tri(benzoyl) cmpd. 6£
67 2C
17.9 0.20
Agrobactin^
n.7 5
^1 8
adetermined in DMF-dy
^From reference 72
cEstimated due to poor resolution

n "i i ~j i i
,3 7
Figure 6-?
a \
l i ] T~> 1 f | i i --"r -j ~r i T-T I i~ i | iir ( || i r ~t i
' :.S *1 h 7 i
. 300 MHz NMR spectrum of N^-[N-(t-butoxycarbony1)threony1]-N^ ,N^-
bis(t-butoxycarbonyl)spermidine(5J3).

:zzlizii: zztz z zzzzzzzziziiiiii x> nzzju.
-jii-
11 i i ~ i
| 0
ill
so
M 0
3 0
20
1.0
Figure 6-10. 60 MHz NMR spectrum of N^-threonylspermidine trif1uoroacetate (54).

Figure 6-1 1, 300 MHz ]H NMR spectrum of N4-[N-(2,3-dimethoxybenzoy1)threony1]- g
Nl,N8-bis(2,3-dimethoxybenzoyl)spermidine (59).

. -7 |
T
iir
n
Figure 6-12.
300 MHz NMR spectrum of
(benzoyl)spermidine(60).
3 2 1 u
[N-(benzoylJthreonyll-N1 ,N8-bis-

CHAPTER SEVEN
CONCLUSIONS
Selective and efficient syntheses of polyamine derivatives were
described. Using the key reagents N4-benzylspermidine, N4-benzylnorsper-
midine, and N5-benzylhomospermidine, terminally bis-acylated or alkylated
polyamine derivatives were easily generated in two and three high yield
steps, respectively.
In an extension of this synthetic scheme, reagents for the selective
secondary N-acylation of spermidine and its homologues were developed.
The benzylated spermidines were terminally bis-acylated with t-butoxy-
carbonyloxyimino-2-phenylacetonitrile to afford the terminally t-BOC
protected polyamine reagents upon debenzylation. These reagents were used
for the high yield synthesis of N4-acylated or alkylated spermidine de
rivatives.
The uptake characteristics and LD50 toxicities of the terminally or
secondary N-modified polyamines thusly generated were also determined.
Terminally bis-acylated spermidine derivatives, regardless of size, were
shown to be unable to compete with 3H-spermidine for uptake in LI210
leukemia cells. Bis-alkylated derivatives, on the other hand, were fair
competitors in inhibiting labelled spermidine uptake, but the size of the
alkyl substituent was found to be restrictive.
162

163
Secondary N-modified polyamines, however, were found to be less
restrictive than their terminally N-modified counterparts in competing
for uptake. Although the N4-alkylated spermidines were the best inhibitors
of spermidine uptake, the N4-acyl derivatives were also found to be capable
to competing for uptake.
The structural requirements for polyamine uptake as defined by these
data, therefore, indicated that the free primary amines of spermidine are
critical in inferring uptake specificity. On the other hand those poly
amine derivatives modified at the secondary amine were less limiting upon
uptake, suggesting only a minor role of the secondary amine in recognition
by the receptor for uptake. Based on these findings, two anti neoplastic
spermidine conjugates, N4-chlorambucil spermidine and N4-[4-(2,3-dihydro-
lH-imidazo]l,2-b]pyrazolo)carboxamido)butyryl]spermidine were synthesized.
Preliminary in vitro findings indicate a preference for these conjugates
over the native antineoplastic for uptake, and tin's specificity may be
responsible for the increased cytotoxicity seen in the N4-IMPY spermidine
conjugate over the glutaryl IMPY precursor. Those studies, although in
the early stages, suggest the feasibility of utilizing spermidine as a
carrier to target the delivery of anti neoplasties to tumor cells.
Of the polaymine derivatives tested for acute toxicity, an interest
ing relationship between the compounds LD50 and cellular uptake was
noticed. The terminally N-modified derivatives, which were the least
effective in competing for uptake, were also the least toxic, having
LD50S in excess of 800 mg/Kg. Likewise, the best inhibitors of spermidine
uptake, the N4-substituted derivatives, were also the more toxic of the
group, suggesting a link between uptake and toxicity.

164
Additionally, the synthesis, a third reagent for the selective
acylation of spermidine, was described. The new reagent, N4-benzyl-N1(t-
butoxycarbonyl)-N8-(trifluoroacetoxy)spermidine employs three different
protecting groups, each removable under different conditions. According
ly, each one of spermidine's three nitrogens can now be functionalized in
any order. As an illustrative example, N8-acetyl-N4-benzoyl-N1-(2,3-
dimethoxybenzoyl)spermidine was successfully prepared using this reagent.
Finally, the natural product agrobactin A and a number of its deriva
tives were synthesized. Employing the reagent N1,N8-bis(t-butoxycarbonyl )-
spermidine, agrobactin A was generated in three high yield steps. The
scheme highlighted an "inside-out" approach, which attached the three
2,3-dihydroxybenzoyl groups in the last step, requiring no catechol pro
tecting groups.
High field *H NMR spectroscopy of agrobactin A revealed that the com
pound existed in a series of conformers as determined by the duplicity in
the threonyl signals. These conformers were attributed to the asymmetri
cal nature of the spermidine backbone and subsequently the symmetrical
homologue of agrobactin A incorporating homospermidine as the backbone was
synthesized. The 300 MHz XH NMR spectra of this symmetrical derivative,
homoagrobactin A, confirming the role of the polyamine backbone.
The coalescence temperatures and activation energies between con
formers of agrobactin A and several closely related analogues were deter
mined using 1H NMR. A comparison of the activation energies suggested
that steric factors were the predominant influence in controlling con-
former interconversion. However, the role of hydrogen bonding could not
be as easily assessed due to the hydrogen bonding capability of the sol
vent used.

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43. Thiele, J. and Dralle, E. (1898) Ann. 302, 275.
44. Williams-Ashman, H.G. and Schenone, A. (1972) Biochem. Biophys.
Res. Comm. 46_, 288.
45. Freedlander, B.L. and French, F.A. (1958) Cancer Res. 1_8, 360.
46. Krokan, H. and Eriksen, A. (1977) Eur. J. Biochem. 72^, 501.
47. Oettgen, H.F. and Burcheral, J.H. (1959) Medizinische 29/30, 1362.
48. Regelson, W. and Holland, J.R. (1961) Cancer Chemother. Rep. 11,
81.
49. Mihich, E. (1963) Cancer Res. 23, 1375.
50. Milkes-Robertson, F., Feurstein, B., Dave, C. and Porter, C.W.
(1979) Cancer Res. 39^, 1919.
51. Pleshkewych, A., Kramer, D.L., Kelly, E. and Porter, C.W. (1980)
Cancer Res. 40, 4533.
52. Porter, C.W., Dave, C. and Mihich, E. (1981) in Perpsectives and
Issues in Polyamine Research (Morris, D. and Morton, L., eds.)
pp. 407-436, Marcel Dekker, New York.
53. Bloch, J.B., Field, M. and Oliverio, V.T. (1964) Cancer Res. 24,
1947.
54. Field, M., Bloch, J.B., Oliverio, V.T. and Rail, D.P. (1964)
Cancer Res. 24, 1939.
55. Seppanen, P., Alhonen-Hongiston, L., Poso, H. and Janne, J. (1980)
FEBS Letters 111, 99.

163
56. Dave, C. and Caballes, L. (1973) Fed. Proc. 32, 736.
57. Seppnen, P., Alhonen-Hongiston, L. and Janne, J. (1980) Eur. J.
Biochem. 110, 7.
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70, 956.
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7, 710.
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192, 941.
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L.V.C. (1979) FEBS Lett. 102, 325.
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Comm. 74, 1626.
71. Neilands, J.B. and Peterson, T. (1979). Tetrahedron Lett., 4805.
72. Neilands, J.B., Peterson, T., Falk, K.E., Leong, S.A. and Klein,
M.E. (1980) J. Am. Chem. Soc. 102, 7715.
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254, 1860.
75. Bergeron, R.J., Stolowich, N.J. and Porter, C.W. (1982) Synthesis,
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76. Van Brussel, W. and Van Sumere, C.F. (1978) Bull. Chim. Belg. 87,
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169
77. Gutowsky, H.S. and Cheng, H.N. (1975) J. Chem. Phys. 63, 2439.
78. Ganem, B. (1982) Acc. Chem. Res. 1_5, 290.
79. Eugster, C.H. and Walchi-Schaer, E. (1978) Helv. Chim. Acta
61_, 928.
80. Humora, M. and Quick, J.J. (1979) J. Org. Chem. 44, 1166.
81. Ganem, B. and McManis, J.J. (1980) 45, 2042.
82. Bergeron, R.J., Burton, P.S., McGovern, K.A. and Kline, S.J.
(1981) Synthesis, 732.
83. Umino, N., Inakuma, T. and Itoh, N. (1976) Tetrahedron Lett., 763.
84. Itoh, M., Hagiwara, D. and Kamiya, T. (1975) Tetrahedron Lett.,
4393.
85. Bergeron, R.J., Kline, S.J., Stolowich, N.J., McGovern, K.A. and
Burton, P.S. (1981) J. Org. Chem. 46, 4524.
86. Krimen, L.I. (1970) Organic Syn. 50^, 1.
87. Borch, R.F. and Hassid, A.I. (1972) J. Org. Chem. 37, 1673.
88. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951)
J. Biol. Chem. 193, 265.
89. Greco, W.R., Priore, R.L., Sharma, M. and Korytnyk, W. (1982)
Comp. Biomed. Res. 1_5, 39.
90. Rodichok, L.D. and Friedman, A.H. (1978) Life Sci. 23^, 2137.
91. Porter, C.W. and Bergeron, R.J. (1983) Science 219, 1083.
92. Cory, J.G. and Fleischer, A.E. (1980) Cancer Res. 40^, 3891.
93. Burton, P.S. (1981) "Synthesis and Evaluation of Spermidine Sidero-
phores for Iron Chelation Therapy and the Role of Cyclohexaamylose
in Caralytic Hydrolysis," Ph.D. dissertation, University of Florida.
94. Porter, C.W. Private Communication.
95. Newman, H.J. (1965) J. Org. Chem. 30, 1287.
96. Flouret, G., Morgan, R., Gendrich, R., Wilber, J. and Seibel, M.
(1973) J. Med. Chem. 16, 1137.
97. Bergeron, R.J. and Garlich, J.R., submitted.

170
98. Curran, W.V. and Angier, R.B. (1966) J. Org. Chem. 31_, 3867.
99. Bergeron, R.B., McGovern, K.A., Channing, M.A. and Burton, P.S.
(1980) J. Org. Chem. 45, 1589.
100. Bergeron, R.B., Stolowich, N.J. and Garlich, J.R., manuscript
in preparation.
101. Elliot, D.F. (1948) Nature 162, 657.
102. Pfister, K., III, Robinson, C.A., Shabica, A.C. and Tishler, M.J.
(1949) J. Amer. Chem. Soc. 71_, 1101.
103. Bergeron, R.B., Burton, P.S., Kline, S.J. and McGovern, K.A. (1981)
J. Org. Chem. 46, 3712.
104. Eng-Wilmot, D.L. and van der Helm, D.J. (1980) J. Amer. Chem. Soc.
102, 7719.

BIOGRAPHICAL SKETCH
Neal James Stolowich was born in Rockville Centre, New York, on
May 3, 1957. After graduating from Baldwin High School in 1975, he en
rolled at the State University College at Oswego, New York, for one year.
He then transferred to the State University of New York at StonyBrook,
where he earned a B.S. in biochemistry in 1979. The author then entered
graduate school at the University of Florida, working under the direction
of Or. Raymond Bergeron. He then married Barbara Shallcross in 1981,
and two years later a son, Daniel, was born. After receiving his Ph.D.
in late 1983, the author accepted a postdoctoral position at Yale Uni
versity.
171

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Chairman
Raymond J. Bergeron, Chfairman
Associate Professor of Medicinal Chemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Margaret J James
Assistant Professor of Medicinal Chemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
\vt ¡7 C
Federico A. yilalloriga
Professor of Pharmacy
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy,
(JL£J A ;/
Richard R. Streiff
Professor of Medicinal Cfiemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Cc-/* >*7
Cemal Kemal, Assistant Professor of
Chemistry

This dissertation was submitted
of Pharmacy and to the Graduate
fulfillment of the requirements
December 1983
to the Graduate Faculty of the College
School, and was accepted as partial
for/the degree of Doctor of Philosophy.
/ CX.
Dean, College of Pharmacy
Dean for Graduate Studies and Research

UNIVERSITY OF FLORIDA
3 1262 08554 7882



169
77. Gutowsky, H.S. and Cheng, H.N. (1975) J. Chem. Phys. 63, 2439.
78. Ganem, B. (1982) Acc. Chem. Res. 1_5, 290.
79. Eugster, C.H. and Walchi-Schaer, E. (1978) Helv. Chim. Acta
61_, 928.
80. Humora, M. and Quick, J.J. (1979) J. Org. Chem. 44, 1166.
81. Ganem, B. and McManis, J.J. (1980) 45, 2042.
82. Bergeron, R.J., Burton, P.S., McGovern, K.A. and Kline, S.J.
(1981) Synthesis, 732.
83. Umino, N., Inakuma, T. and Itoh, N. (1976) Tetrahedron Lett., 763.
84. Itoh, M., Hagiwara, D. and Kamiya, T. (1975) Tetrahedron Lett.,
4393.
85. Bergeron, R.J., Kline, S.J., Stolowich, N.J., McGovern, K.A. and
Burton, P.S. (1981) J. Org. Chem. 46, 4524.
86. Krimen, L.I. (1970) Organic Syn. 50^, 1.
87. Borch, R.F. and Hassid, A.I. (1972) J. Org. Chem. 37, 1673.
88. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951)
J. Biol. Chem. 193, 265.
89. Greco, W.R., Priore, R.L., Sharma, M. and Korytnyk, W. (1982)
Comp. Biomed. Res. 1_5, 39.
90. Rodichok, L.D. and Friedman, A.H. (1978) Life Sci. 23^, 2137.
91. Porter, C.W. and Bergeron, R.J. (1983) Science 219, 1083.
92. Cory, J.G. and Fleischer, A.E. (1980) Cancer Res. 40^, 3891.
93. Burton, P.S. (1981) "Synthesis and Evaluation of Spermidine Sidero-
phores for Iron Chelation Therapy and the Role of Cyclohexaamylose
in Caralytic Hydrolysis," Ph.D. dissertation, University of Florida.
94. Porter, C.W. Private Communication.
95. Newman, H.J. (1965) J. Org. Chem. 30, 1287.
96. Flouret, G., Morgan, R., Gendrich, R., Wilber, J. and Seibel, M.
(1973) J. Med. Chem. 16, 1137.
97. Bergeron, R.J. and Garlich, J.R., submitted.


1- 1 lit 1.1 I .1... 1 .11IIl1- 1 I t I I I ~ I I 1 I 1
TO iO 50 *4.0 3.0 i.O 1.0 O
Figure 2-17. 60 MHz Hi NMR spectrum of N^-methyl-n\N-bis(t-butoxycarbony1 )-
spermidine(24j ,


63
synthesis of a variety of N-acylated and N-alkylated spermidines was
described and illustrates that the number of polyamine derivatives that
can be generated by these methods are virtually limitless. Additionally,
these reagents can and have been used toward the synthesis of other sys
tems, such as the siderophores, an example of which is described in
Chapter Five.


Figure 5-5. 60 MHz ^ H NMR spectrum of N-[N-t-butoxycarbonyl-3-aminopropyl]-
benzylamine (33).


. -7 |
T
iir
n
Figure 6-12.
300 MHz NMR spectrum of
(benzoyl)spermidine(60).
3 2 1 u
[N-(benzoylJthreonyll-N1 ,N8-bis-


BIOGRAPHICAL SKETCH
Neal James Stolowich was born in Rockville Centre, New York, on
May 3, 1957. After graduating from Baldwin High School in 1975, he en
rolled at the State University College at Oswego, New York, for one year.
He then transferred to the State University of New York at StonyBrook,
where he earned a B.S. in biochemistry in 1979. The author then entered
graduate school at the University of Florida, working under the direction
of Or. Raymond Bergeron. He then married Barbara Shallcross in 1981,
and two years later a son, Daniel, was born. After receiving his Ph.D.
in late 1983, the author accepted a postdoctoral position at Yale Uni
versity.
171


27
R] COiMH (CHZ) M (CHo ) b NHCGR,
VIII
I
R,CCNH (CH2)aNH (CH-,) NHCCR,
IX
'I
R]CH,NH CH2)a- NH (CH2)b NhC'rUR,
X
Figure 2-2. General scheme for the synthesis of terminally
N-modified polyamine derivatives.


89
may produce a less effective chelator, thereby decreasing its activity.
Accordingly, the synthesis of an analogue in which this amide is reduced
to an amine is currently being investigated.
Finally, although the in vitro data at this point are limited, it
indicates that these conjugates are indeed taken up via the spermidine car
rier and represent a potential drug delivery system. Accordingly, further
testing and synthesis of other systems are currently being evaluated at
this time.


99
N4-Benzy1-N1-(t-butoxycarbony1 )spermidine (35)
Raney nickel (3.0 g) was added to a solution of (34) (13.6 g, 0.0409
mole) and NaOH (4.0 g, 0.10 mole) in 100 ml 95% EtOH and the resulting
suspension was hydrogenated under 40 psi for 28 hours. The catalysts
were then filtered, washed well with 95% EtOH, and the filtrates concen
trated. The residue was taken up in H20 (250 ml), and the product ex
tracted into CH2C12 (4 x 50 ml), dried and concentrated to afford 13.4 g
(97%) of the desired product as a nondistillable oil. Thin layer chroma
tography and NMR analysis indicated the purity of the product was in
excess of 95% and could be used without further purification.
An analytical sample was prepared by chromatography on silica gel
eluting with 30% MeOH/CHCl3: NMR 6 1.50 (m, 17H), 2.47 (m, 6H), 3.10
(quar., 2H), 3.47 (s, 2H), 5.33 (br, 1H), 7.17 (s, 5H); IR (neat) 2940
(s), 1710 (s), 1510 (m), 1170 (s), 740 (m) cm-1.
Anal. cal. for C19H33N302: C, 68.02; H, 9.91; N, 12.53. Found:
C, 67.79; H, 9.96; N, 12.43.
N4-Benzyl-N1-(t-butoxycarbonyl)-N8-trifluoroacetylspermidine (36)
A solution of trifluoroacetic anhydride (2.3 g, 11 mmol) in 10 ml
dry CH2C12 was slowly added to a cooled solution of (35J, (3.5 g, 10
mmol) and triethylamine (1.4 ml, 10 mmol) in 20 ml dry CH^Cl9 under N2>
The reaction was allowed to warm to room temperature and stirred for
16 h, at which time additional CH2C12 was added (50 ml). The organic
layer was washed with cold 3% HC1 (2 x 15 ml), H20 (1 x 25 ml), 5%
NaHC03 (2 x 25 ml), dried and concentrated to afford 3.9 g (91%) of
the desired product: NMR 6 1.42 (s, 9H), 1.58 (m, 6H), 2.43 (m, 4H),
3.15 (m, 4H), 3.50 (s, 2H), 5.22 (br, 1H), 7.25 (s, 5H), 7.58 (br, 1H);
IR (CHC13) 3300 (m), 3020 (m), 1710 (s), 1620 (s), 1170 (s), 750 (s) cm'1.


167
37. Mamont, P.S., Bohlen, P., McCann, P.P., Bey, P., Schuber, F. and
Tardif.C. (1976) Prod. Nat. Acad. Sci. 73, 1626.
38. Metcalf, B.W., Bey, P., Dazin, C., Jung, M.J., Casara, P. and
Vevent, J.P. (1978) J. Am. Chem. Soc. 100, 2551.
39. Seiler, N., Danzin, C., Prakash, N.J., Koch-Weser, J. (1978)
in Enzyme-Activated Irreversible Inhibitors, pp. 55-71, Elsevier/
North Holland.
40. Bacchi, C.J., Nathan, H.C., Hunter, S.H., McCann, P.P. and Sjoerdsma,
A. (1980) Science 210, 332.
41. Mamont, P.S., Duchesne, M.C., Grove, J. and Bey, P. (1978) Biochim.
Biophys. Res. Comm. 81_, 58.
42. Porter, C.W., Bergeron, R.J. and Stolowich, N.J. (1982) Cancer Res.
42, 4072.
43. Thiele, J. and Dralle, E. (1898) Ann. 302, 275.
44. Williams-Ashman, H.G. and Schenone, A. (1972) Biochem. Biophys.
Res. Comm. 46_, 288.
45. Freedlander, B.L. and French, F.A. (1958) Cancer Res. 1_8, 360.
46. Krokan, H. and Eriksen, A. (1977) Eur. J. Biochem. 72^, 501.
47. Oettgen, H.F. and Burcheral, J.H. (1959) Medizinische 29/30, 1362.
48. Regelson, W. and Holland, J.R. (1961) Cancer Chemother. Rep. 11,
81.
49. Mihich, E. (1963) Cancer Res. 23, 1375.
50. Milkes-Robertson, F., Feurstein, B., Dave, C. and Porter, C.W.
(1979) Cancer Res. 39^, 1919.
51. Pleshkewych, A., Kramer, D.L., Kelly, E. and Porter, C.W. (1980)
Cancer Res. 40, 4533.
52. Porter, C.W., Dave, C. and Mihich, E. (1981) in Perpsectives and
Issues in Polyamine Research (Morris, D. and Morton, L., eds.)
pp. 407-436, Marcel Dekker, New York.
53. Bloch, J.B., Field, M. and Oliverio, V.T. (1964) Cancer Res. 24,
1947.
54. Field, M., Bloch, J.B., Oliverio, V.T. and Rail, D.P. (1964)
Cancer Res. 24, 1939.
55. Seppanen, P., Alhonen-Hongiston, L., Poso, H. and Janne, J. (1980)
FEBS Letters 111, 99.


SPCNT TRANSMISSION
WAVtttNOIH III MICRO! (S
45 S<
3000
WMtM moimiwC incur chari mo iosa
2000 |000
WAVlNUMBCR CM*
60 *
1600 1400 1200
mcirran 45IRUWIMIS c. rimrttcHi. cmvoinm, u s a.
1000
M4I0 KUI
Figure 5-19. IR spectrum of N-(N-t-butoxycarbony 1 -4-ami nobutyl ) benzy 1 ami ne (4_]J
ro
O
PS*r-


\
~&/ ,.i. Hi1 i -1, i i 1.. iii .-I-- i i -1 i i 1 i i 1. i 1 i -1
1-0 0 5.0 M.O 3 0 J.O 1.0 O
0
Figure 2- 20. 60 MHz NMR spectrum of homospermidine trihydrochi oride (27 ).


30
Figure 2
HC1
tBOC-NH (CH2)a NH (CH2)b NH-tBOC
XII
Y
I2
co
tBOC-NH (CH2)a N (CH2)b NH-tBOC
XIII
I
I2
CO
H2N (CH2)a N (CH2)b NH2
XIV
*
H2N (CH2)a N (CH2}b NH
2
XV
4. Synthesis of secondary N-modified polyamine
deriva ti ves .


15
R,CONH (CH2)a NH (CH2)b NHCOR,]
R]CH2NH (CH2)a NH (CH2)b NHCH2Rj
CO
H2N (CH2)a N (CH2)b NH2
CH-
I
H2N (CH2)a N (CH2)b NH2
Figure 1-4. Generalized structures of polyamine derivatives
synthesized,


Figure 2-12. 60 MHz Hi NMR spectrum of N^-hexanoy 1-NHfi^-bi s (t-butoxycarbony 1 )-
spermidine (17).


105
for (46). Yield: 240 mg (98%); XH NMR 6 1.43 (s, 9H), 1.70 (m, 6H),
1.93 (s, 3H), 3.27 (m, 8h), 5.38 (br, 1H), 6.44 (br, 1H), 7.37 (s, 5H),
IR (2980) m, 1710 (s), 1630 (br, s), 1180 (m) cm'1.
Anal. cal. for C21H33N304: C, 64.43; H, 8.50; N, 10.73. Found:
C, 64.17; H, 8.5a N, 10.51.
N8-Acetyl-N4-benzoylspermidine (4)
Trifluoroacetic acid (10 ml) was added to a flask containing 48
(320 mg, 0.82 mmol) and the resulting solution allowed to stir for 20
min. The solvent was then quickly evaporated, the residue dissolved
in 25 ml MeOH and concentrated (twice). The crude product was then
treated with 15% Na2C03 (10 ml), extracted with CH2C12 (3 x 25 ml), dried
and concentrated to afford 180 mg (75%) product as a light yellow oil:
NMR 6 1.66 (m, 8H), 1.94 (s, 3H), 2.61 (m, 2H), 3.24 (m, 6H), 6.41
(br, lh), 7.35 (s, 5H); IR (CHC13) 2990 (m), 1650 (s), 1620 (s), 740 (s)
cm'1.
Anal. cal. for C1gH25N302: C, 65.95; H, 8.65; N, 14.42.
N8-Acety1-N4-benzoyl-N1-2,3-dimethoxybenzoylspermidine (50.)
A solution of 2,3-dimethoxybenzoyl acid chloride (95 mg, 0.47
mmol) in 10 ml dry CH^Cl2 was slowly added to a cooled solution of (49)
(125 mg, 0.43 mmol) and trimethyl amine (70 yl, 0.5 mmol) in 10 ml dry
CH2C12. The reaction was allowed to stir for 18 hours, and purified as
described for (46). Yield: 180 mg (93%); NMR 6 1.68 (m, 6H), 1.95
(s, 3H), 3.24 (m, 8H), 3.87 (s, 6H), 6.45 (br, 1H), 6.93-8.12 (m, 9H);
IR (CHC13) 3310 (m), 2980 (m), 1650 (br, s), 1530 (s), 740 (m) cm"1.
Anal. cal. for C9 H N 0 : C, 65.91; H, 7.30; N, 9.22. Found:
C, 65.88; H, 7.46; N, 9.05.


10
Recently, a difluoro analog of a-methylornithine, difluoromethyl-
ornithine (DFMO), has been synthesized which has been found to be a po
tent irreversible inhibitor of ODC.38 Difl uoromethylornithine, which
is nontoxic in mammals,39 has been shown to block multiplication of the
parasite Trypanosoma b. brucei, and can cure mice infected with the para
site simply by administration in their drinking water.40 Furthermore,
the cure can be reversed by injection of small doses of polyamines.
Difluoromethylomithine has also been shown to inhibit growth of LI210
leukemia cells, again, its antiproliferative effects prevented by spermi
dine and putrescine.41 In fact, LI210 cells depleted in polyamines by
pretreatment with DFMO show up to a 3-fold increase in polyamine uptake
as compared to untreated cells,42 evidently in an attempt to restore nor
mal levels. This observation will have additional importance in follow
ing discussions.
Up to this point, all the inhibitors of polyamine synthesis discussed,
with the exception of ethionine,are inhibitors of ODC. However, an im
portant group of SAM-DC inhibitors has been recently discovered and
warrants further discussion. The first of this group, methylglyoxal
bis(guanylhydrazone) (MGBG), was first synthesized in 1898,43 but only
as recently as 1972 shown to be a potent and specific inhibitor of SAM-
DC by Williams-Ashman and Schenone.44 The discovery of MGBG as an inhi
bitor of SAM-DC grew out of an investigation of its known antitumor ef
fects. Methylglyoxal bis(guanylhydrazone) was first shown to be active
against LI210 leukemia,45 but has since been tested against a wide range
of experimental tumors.4647 However, leukemias exhibit the greatest
sensitivity to MGBG and, accordingly, MGBG recently has been used to
treat several forms of human leukemia clinically.48


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13. Russell, D.H. and Lombardini, J.B. (1971) Biochim. Biophys. Acta
240, 273-286.
14. Sturman, J.A. and Gaul 1, G.E. (1974) Ped. Res. 8, 231-237.
15. Jnne, J., Hbltt, E. and Guha, S.K. (1976) in Progress in Liver
Diseases (Popper, H. and Schaffner, F., eds.) Vol. 5, pp 100-124, Grue
and Stratton, New York.
16. Jnne, J. (1962) Acta Physiol. Scand. supp. 300, 1-71.
17. Raina, A., Jnne, J. and Siimes, M. (1966) Biochim. Biophys. Acta
123, 197-201.
18. Jnne, J. and Raina, A. (1968) Acta Chem. Scand. 22, 1349.
165


132
High field XH NMR spectroscopy
High resolution FT *H NMR spectra were recorded on a Nicolet NT-300
spectrometer equipped with a Nicolet 1280 computer. Samples were run at
room temperature (23 1C) unless otherwise noted. Generally, when
CDC13 or CDC13/DMS0-d6 were employed as the solvent, 1-4 mg of sample were
dissolved in approximately 500 yl of solvent. Chemical shift and cou
pling constant values, when obtainable, are given in Table 6-1 in the
solvents indicated. All chemical shifts are in ppm downfield from an
internal TMS standard. Samples for the temperature dependence studies
were prepared by dissolving 5-10 mg of sample in 500 ul DMS0-d6 or DMF-dy
as indicated in Table 6-2. Activation energies were measured by observ
ing the coalescence temperature of the resulting y-methyl singlets pro
duced upon irradiation of the adjacent e-methine. The coalescence tem
peratures were measured both on heating and cooling cycles and activation
energies subsequently calculated using the rate equations as described
by Gutowsky and Cheng.77 Additionally, the results were found to be in
good agreement with those calculated from a total line shape analysis
using a program contained in the NT-300 software for unequally populated
two-site exchange. The results indicated that the experimental 5v's were
within 1 Hz of the simulated values. Therefore, assigning a maximum
error of 1.0C in Tc, and an error of 1.0 Hz in measurement of the
5v, the range of error in determining the Ea is 0.15 kcal/mole.
Results and Discussion
Synthesis
The 'inside-out1 synthesis of agrobactin A employs the reagent
N1 ,N8-bis(t-butoxycarbonyl )spermidine (1_3). Originally developed as a
reagent to selectively generate N4-modified spermidine derivatives for


145
rotation and at greater distances from the aromatic rings. Therefore,
it sees little or no change in its environment resulting in only one
observable signal. This idea is illustrated in Figure 6-6. The inner
and outer cylinders represent the sweep volumes of the a-methine and
Y-methyl group, respectively. The a-methyl group and 8-methine can lie
in close proximity to the terminal aromatic rings and can easily be
influenced by the anisotropic effects. However, since these effects
decrease rapidly with increased distances, the a-methine is likely to
be too distant from the aromatic rings to be affected by their aniso
tropy.
Furthermore, if the cylinders are cut in half by a plane passing
through the central nitrogen perpendicular to the spermidine backbone,
it can easily be seen that corresponding points in either half are at
different intramolecular distances from the aromatic rings due to the
asymmetry of the spermidine chain. Accordingly, these points will also
experience different magnetic fields. Therefore, a proton existing in
a conformation lying in the left half will likely result in a different
signal as compared to its counterpart that lies in the right half of
the cylinder. Although this model does not consider all of the possibl
orientations of the spermidine backbone and aromatic rings, a recent
x-ray crystallographic study of agrobactin104 does suggest a conforma
tion with a nearly linear spermidine backbone that is similar to that
depicted in the model.
Based on this model, a symmetrical analogue should equalize the in
tramolecular distances on either half, eliminating the duplicity of sig
nals observed for the 8- and y-protons of agrobactin A.
Neilands has also attributed the appearance of the XH NMR spectrum
of agrobactin A to the asymmetrical nature of the spermidine chain and,


104
solid. Thin layer chromatography and NMR analysis indicated product's
purity was in excess of 95%, and could be used without further purifica
tion.
An analytical sample was prepared by preparative TLC eluting with
10% MeOH/CHCl3: lH NMR 6 1.37 (s, 9H), 1.68 (m, 6H), 3.27 (m, 8H), 5.18
(br, 1H), 7.28 (s, 5H), 7.62 (br, 1H); IR (CHC13) 3310 (m), 3010 (m),
1710 (s), 1620 (s), 1170 (s), 750 (s) cm'1.
Anal. cal. for C21H30N304F3: C, 56.62; H, 6.79; N, 9.43. Found:
C, 56.50; H, 6.82; N, 9.20.
N^Benzoyl-N^t-butoxycarbonylspermidine (47)
Potassium carbonate (660 mg, 4.6 mmol) was added to a solution of
(46) (515 mg, 1.15 mmol) in 30 ml MeOH and 2 ml H20. The reaction mix
ture was refluxed for 2 hours, cooled, and the solvent evaporated. The
residue was dissolved in 50 ml CH2C12 washed with H20 (2 x 10 ml),
dried, and concentrated to afford the crude product. Further purifica
tion was effected by chromatography of silica gel eluting with 20% MeOH/
CHC13 to afford 325 mg (81%) product: XH NMR 5 1.20-1.94 (m, 17H), 256
(m, 2H), 3.30 (m, 7H), 5.26 (br, 1H), 7.33 (s, 5H); IR (neat) 3000 (m),
1710 (s), 1625 (s), 1510 (s), 1175 (s), 760 (s) cm1.
Anal. cal. for C19H31N303'H20: C, 62.10; H, 8.94; N, 11.43. Found:
C, 62.67; H, 8.69; N, 10.89.
N8-Acetyl-N4-benzoyl-N1-t-butoxycarbonylspermidine (4B.)
A solution of acetyl chloride (85 mg, 0.71 mmol) in 5 ml dry CH2C12
was added to a cooled solution of 47 (215 mg, 0.62 mmol) and triethyl-
amine (150 pi, 1 mmol) in 10 my dry CH2C12 under N2. The reaction was
allowed to stir for 18 hours, and then worked up and purified as described


67
Results
Uptake of Polyamine Derivatives
The inhibition of 3H-Spd uptake in ascites L1210 cells by Spd deriva
tives, polyamines, and MGBG are summarized in Tables 3-1, 3-2, and 3-3.
For the control experiments, ascites LI210 cells exposed to 10 uM 3H-Spd
alone for 20 min at 37C take up approximately 56 pmol Spd/107 cells/min,
Table 3-1. However, at 4C this uptake is reduced to 10% of that at
37C and may represent the fraction of nonspecific binding to the cell
surface. Also shown in Table 3-1, with the exception of putrescine,
all polyamines are quite effective in preventing 3H-Spd uptake. The
polyamines Spm and hSpd are equally effective as Spd itself in competing
for uptake, inhibiting 90% of 3H uptake.
The uptake characteristics of terminally bis-modified Spd derivat
ives are reported in Table 3-2. In general, modification of Spd primary
amines produces derivatives that are either weak inhibitors or unable
to inhibit 3H-Spd uptake at all. All the bis-acylated derivatives tested
could be considered noncompetitive in preventing Spd uptake, allowing
90% of the control uptake of 3H-Spd. Only Bis(ethyl)Spd was capable of
substantially inhibiting Spd uptake, having a Ki of 62 uM.
On the other hand, modification of Spd's secondary amine appears
less restrictive. All of the secondary N-alkylated derivatives, Table
3-3, are quite competitive in inhibiting 3H-Spd uptake. Additionally,
a preference in backbone chain length of the benzyl derivatives is shown.
For example, BhSpd is more effective than BSpd in preventing uptake,
whereas BnSpd is less effective. Of the secondary N-acylated Spd deriva
tives, only N4-AcetylSpd is capable of weakly inhibiting Spd uptake.


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103
N5-Benzyl-N1-t-butoxycarbony1 -N9-trifluoroacetylhomospermidine (44)
A solution of (43) and trifluoroacetic anhydride was reacted and
purified as previously described for (36). Yield: 1.8 g (76%); lH NMR
1.52 (m, 17H), 2.38 (m, 4H), 3.18 (m, 4H), 3.43 (s, 2H), 4.70 (br, 1H),
7.18 (s, 5H), 7.62 (br, 1H); IR (neat) 3320 (m), 2975 (s), 1710 (s),
1520 (s), 1170 (s), 740 (m) cm*1.
Anal. cal. for C22H34N3O3F3: C, 59.31; H, 7.69; N, 9.43. Found:
C, 59.12; H, 7.69; N, 9.41.
Nl-t-Butoxycarbonyl-N8-trif1uoroacetylspermidine (45)
Palladium chloride (90 mg) was added to a solution of (36) (1.0 g,
2.3 mmol) in 25 ml MeOH containing six drops concentrated HC1. The res-
sulting suspension was stirred under a H2 atmosphere for 12 hours at
which time the catalysts were filtered off, washed with MeOH, and the
filtrates evaporated. The crude product was recrystallized from EtOH/
ether to afford 710 mg (82%) of pure (45j: mp 140-141; NMR (D20) 6
1.54 (s, 9H), 1.80 (m, 6H), 3.30 (m, 8H); IR (KBr) 3380 (m), 2960 (m),
2800 (m), 1705 (s), 1530 (m), 1175 (s) cm"1.
Anal. cal. for C14H27N3C1 F303-H20; C, 42.50; H, 7.38; N, 10.62.
Found: C, 42.55; H, 7.32; N, 10.64.
N4-Benzoyl-N^t-butoxycarbonyl -N8-trifluoroacetyl spermidine (46j
A solution of benzoyl chloride (240 mg, 1.7 mmol) was slowly added
to a cooled solution of (45) 560 mg, 1.5 mmol) and trimethylamine (280
pi, 2.0 mmol) in 25 ml dry CH2C12 under N2. The reaction was allowed
to warm to room temperature and stirred for 18 hours. Additional CH2C12
was added (50 ml) and the organic layer washed with 3% HC1 (3 x 15 ml),
H20 (2 x 15 ml), 5% NaHC03 (3 x 15 ml), H20 (2 x 15 ml), dried, and
concentrated to afford 620 mg (92%) of the desired product as a puffy


126
be acylated with the appropriate acylating agent, in this case acetyl
chloride. The synthesis of (50j would subsequently be accomplished by
debenzylation, acylation with benzoyl chloride, removal of the t-BOC
group, and acylation with 2,3-dimethoxybenzoyl chloride.
In addition to the preparation of triacylated spermidines as
described by the example here, a limitless number of mono- and bis-
acylated spermidines can be generated using these new reagents as well.
Moreover, the ability to remove the protecting groups in any order im
parts enormous flexibility into the types of substituents to be added.
This feature, for example, can 'custom tailor1 the synthesis so as to
add a particularly labile group last.


ACKNOWLEDGEMENTS
I would like to express my sincere appreciation to my research ad
visor, Dr. Raymond Bergeron, under whose guidance this work was accom
plished. I would also like to express my gratitude to the rest of my
supervisory committee, Dr. Richard Streiff, Dr. Margaret James, Dr.
Federico Vilallonga, and Dr. Cemal Kemal.
The uptake determinations of the polyamine derivatives were performed
by Dr. Carl Porter and his group at Roswell Park Memorial Institute,
Buffalo, New York. I wish to extend my appreciation to them for these
determinations, as the data were a significant part of this work.
Finally, I would like to thank all my family and friends that have
helped make this work possible: first, to my parents, Frank and Helen
Stolowich, for their guidance to further pursue my academic career;
secondly, to all my friends, past and present, in medicinal chemistry for
their friendship and assistance. Special thanks to Dr. Kathy McGovern
for those technical discussions at Joe's and for going through this at
the same time. And, last but certainly not least, to my wife Barbara
for her continual support and motivation to carry me through graduate
school.
ii i


79
Figure 4-2. Synthesis of N4-G IMPY-spermi di ne (_32 )


33
ice bath was removed and the mixture allowed to stir for a total of 18
hours. Additional CH2C12 (50 ml) was then added and the reaction mix
ture washed with cold 3% HC1 (3 x 20 ml), H20 (2 x 20 ml), 5% NaHC03
(3 x 20 ml), H20 (2 x 20 ml), dried, and evaporated to afford 1.3 g
(98%) of the desired product as an oil: NMR <$ 1.50 (m, 6H), 1.84 (d,
6H), 2.40 (m, 4H), 3.17 (m, 4H), 3.42 (s, 2H), 6.26 (br, 2H), 7.20 (s,
5H); IR (CHClg) 3310 (s), 2975 (s), 1650 (s), 1550 (s), 760 (s) cm'1.
An analytical sample was prepared by preparative TLC eluting with
10% methanol/CH2Cl2. Analysis calculated for Ci8H29N302-H20: C, 65.82;
H, 9.21; N, 12.79. Found: C, 65.67; H, 9.24; N, 12.73.
N4-Benzyl-N1 ,N8-bis(propionyl )spermidine (2_)
A solution of propionyl chloride and N -benzylspermidine was react
ed and purified in the same manner as for (1_). Yield: 1.35 g (93%);
:H NMR 6 1.10 (dt, 6H), 1.56 (m, 6H), 2.05 (quar., 4H), 2.38 (m, 4H),
3.2 (m, 4H), 3.46 (s, 2H), 6.14 (br, 2H), 7.23 (s, 5H); IR (CHC13)
3320 (s), 2980 (s), 1660 (s), 1550 (s), 770 (s) cm'1.
Anal. cal. for C2oH33N30-tH20: C, 67.38; H, 9.61; N, 11.79. Found:
C, 67.61; H, 9.60; N, 11.75.
N4-Benzyl-N1,N8-bis(benzoyl)spermidine (2)
A solution of benzoyl chloride and N4-benzylspermidine was reacted
and purified in the same manner as for (1_). Yield: 3.4 g (91%); XH NMR
<5 1.65 (m, 6H), 2.50 (m, 4H), 3.45 (m, 4H), 6.57 (br, 2H), 6.96-7.85
(m, 15H); IR (CHC13) 3340 (s), 1640 (s), 1520 (s), 1310 (m), 690 (m) cm'1.
Anal. cal. for C28H33N302: C, 75.81; H, 7.50; N, 9.47. Found:
C, 75.73; H, 7.46; N, 9.37.


136
Alternatively, the possibility of attaching the 2,3-dihydroxyben-
zoyl groups directly to the N4-threonyl triamine without catechol pro
tecting groups was considered. Recently, Van Brussel and Van Sumere
have shown it possible to generate the succimido active esters of a num
ber of mono- and dihydroxybenzoic acids in the presence of the unpro
tected phenols.76 This approach, besides being the most direct, would
generate agrobactin A under essentially neutral conditions. This is de
sirable as it would eliminate using acid to protonate the catechols, re
ducing or eliminating the amount of migrated ester formed.
Therefore, succinimide-2,3-dihydroxybenzoate (52_) was prepared by
reacting 2,3-dihydroxybenzoic acid and N-hydroxysuccimide in the presence
of DCC to afford (52J upon recrystallization from methanol/H20. The
final step of the synthesis was successfully accomplished by reacting
three equivalents of the active ester (52_) with M4-threonyl spermidine
in aqueous THF for 48 hours. Thin layer chromatography of the products
indicated the reaction proceeds with little or no formation of the un
desired migrated ester. The reaction mixture was concentrated and the
crude agrobactin A easily purified by chromatography on Seph-adex LH-20
to afford agrobactin A (55_) in 75% yield. The symmetrical homologue
of agrobactin A, N5-[N-(2,3-dihydroxybenzoyl)]-Nx,N8-bis(2,3-dihydroxy-
ben zoyl)homospermidine (58) was prepared by reacting (52) with N5-threonyl
spermidine (57_) and purified in a similar manner. Additionally, the
hexamethyl derivative of agrobactin A, N4-[N-(2,3-dimethoxybenzoyl)-
threonyl-N1,N8-bis(2,3-dimethoxybenzoyl)spermidine (59) was also syn
thesized using these procedures. The succimide active ester of 2,3-
dimethoxybenzoic acid was generated as previously for (52) and reacted
with the triamine (54) to produce hexamethyl agrobactin A (59J in 95%


4
unlike most mammalian enzymes, ODC is strikingly inducible2 and has a
very short half life10-30 min.3 Its basal activity is the lowest of
the four; thus, it is considered to be the rate-controlling enzyme in
polyamine biosynthesis.4
The other decarboxylase, S-adenosyl-methionine decarboxylase, de-
carboxylates the methionine residue that is covalently bound to the aden
osine. It too has a short half life of about one hour5 but, unlike ODC
and most other decarboxylases, does not need pyridoxal phosphate for
catalytic activity. Instead, SAM-DC contains pyruvate residues within
its peptide chain that serve as the carbonyl cofactor.6
The spermidine and spermine synthases catalyze the transfer of pro
pylamine groups from decarboxylated SAM to putrescine and spermidine,
respectively. These synthases have been only partially purified at the
present time, but have been shown to be relatively stable enzymes with
long half lives.7 The differences between the decarboxylases' and syn
thases' half lives are of significant importance in considering the in
hibition of polyamine biosynthesis, as will be discussed later.
As mentioned earlier, polyamines have gained considerable interest
in the past few decades, and this has been mainly on two fronts. First,
polyamines have been implicated as being directly involved in cellular
function and, secondly, the polyamine spermidine serves as a backbone
to a number of naturally occurring siderophores or iron chelators.
The latter will be briefly dealt with later, while some of the aspects
of polyamine involvement in cellular function will be discussed now.
Polyamines and Growth
As illustrated in Figure 1-3, there have been many suggested in vivo
and in vitro functions of polyamines. Of all of these, this thesis is


PERCENT TRANSMISSION
WAVUNGIH IN MICRONS
3000
WMH IIOIMIMO VKtl (Hill HO. lomi
3000 ieoo
WAVENUMBER CM*
1600 1100 1300 1000
mccmam ti&iauMHiis **c. imita ion. CMifoaHM. u a a.
600
HHIIUHUIA.
Figure 2-15. IR spectrum of N^-hexanoy1spermidine (20).
cn
-P*
PERCENT TRANSMISSION


10 40 so *0 3 0 2.0 1.0 a
Figure 5-16. 60 MHz ^H NMR spectrum of N8-acetyl-N4-benzoyl-N1-2,3-dimethoxybenzoyl-spermidine (50).


18
spermidine (parabactin A, lb) by Tait63 in 1975, Figure 1-6. For the
most part, this is due to the lack of a satisfactory therapeutic device
for the treatment of various iron overload syndromes,64-66 and the poten
tial that these compounds have shown for clearing iron.6768
For example, both compound II and parabactin A have been shown to
be more effective at removing iron from transferrin than desferrioxime,6970
the drug which is currently being used clinically for chelation therapy.
Since the initial isolation of parabactin A, it has been revealed that
the product isolated may have resulted from the hydrolysis of the sidero-
phore N4-[N-(2-hydroxyphenyl)-4-carboxyl-5-methyl-2-oxazolinej-N1 ,N8-bis-
(2,3-dihydroxybenzoyl)spermidine (parabactin, 111 b) actually produced by
the microorganism. Based on Neilands' and Peterson's study,71 it is
likely that, during the original Tait isolation of parabactin A, the acidic
workup employed would have been sufficient to hydrolyze parabactin's oxa-
zoline ring to produce the open form lb isolated. Both the open threonyl
form and the closed oxazoline form can be easily differentiated, among
other means, by high field proton nuclear magnetic resonance ^H-NMR) spec
troscopy due to characteristic chemical shifts and coupling patterns of
the threonyl residue versus the oxazoline residue.7273 Furthermore,
it has been recently observed that parabactin exists in at least two dis
tinct conformers as determined by 3H-NMR spectroscopy.73 The existence
of these conformers is supported by the threonine oxazoline proton signals,
which exist in duplicate.
A similar system to the parabactin/parabactin A system has been
recently isolated from Agrobacterium tumifaciens B6 cultures, given the
trivial names of agrobactin A (la) and agrobactin (Ilia),74 Figure 1-6.
Like parabactin, agrobactin A is a spermidine-derived siderophore;


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
SYNTHESIS AND PROPERTIES OF N-ACYLATED AND N-ALKYLATED
POLYAMINE DERIVATIVES AND OF AGROBACTIN A,
A NATURALLY OCCURRING SIDEROPHORE
BY
NEAL J. STOLOWICH
December 1983
Chairman: Raymond J. Bergeron
Major Department: Medicinal Chemistry
The synthesis of reagents for the selective functionalization of
the polyamines spermidine, norspennidine, and homospermidine is described.
With these reagents various N-acylated and N-alkylated polyamine deriva
tives are generated and their uptake characteristics studied in Murine
LI210 leukemia cells. In general those polyamine derivatives having their
terminal amines modified were unable to compete with 3H-spermidine for
uptake into those cells. Polyamine derivatives in which the central ni
trogen is modified are found to compete with 3H-spermidine for uptake.
This suggests that the primary amines are critical for inferring uptake
specificity while the secondary amine is less important. The synthesis
of two antineoplastic spermidine conjugates is also described as a pre
liminary investigation into the potential of spermidine as a drug delivery
device.
In a related synthetic sequence, Agrobactin A and a number of ana
logues are prepared. High field proton nuclear magnetic resonance
VI


46
(t, 3H), 1.12-1.95 (m, 30H), 2.31 (quar., 2H), 3.18 (m, 8H), 5.00 (br,
2H); IR (CHC13) 3330 (m), 2970 (s), 1680 (s), 1625 (m), 1175 (s) cm1.
Anal. cal. for C23H45N305: C, 62.27; H, 10.22; N, 9.47. Found:
C, 62.48; H, 10.25; N, 9.23.
N^Benzoyl-N1 ,N8-bis(t-butoxycarbonyl)spermidine (18)
A solution of benzoyl chloride and (1_3) was reacted and purified in
the same manner as described for (16). Yield: 470 mg (95%); !H NMR 5
1.42-1.92 (m, 24H), 2.83-3.58 (m, 8H), 5.23 (br, 2H), 7.19 (s, 5H); IR
(CHC13) 1710 (s), 1630 (m), 1515 (m), 1225 (s), 770 (s) cm-1.
Anal. cal. for C24H39N305-jH20: C, 62.86; H, 8.79; N, 9.16. Found:
C, 62.98; H, 8.99; N, 8.85.
N4-Acetylspermidine hydrochloride (19)
To a cooled flask containing (16) (600 mg, 1.55 mmol) was added
20 ml trifluoroacetic acid. The reaction was allowed to warm to room
temperature and stirred for 20 minutes at which time the solvent was
quickly evaporated. The residue was dissolved in methanol and evaporated
(twice). The residue was then dissolved in ethanol/ether, cooled, and
HC1 gas bubbled through. The solid was collected by filtration, and
dried in vacuo over P205 to afford 360 mg (89%) of the desired product:
XH NMR (D20) 6 1.30-1.88 (m, 24H), 2.06 (s, 3H), 3.18 (m, 8H), 5.00 (br,
2H); IR (KBr) 3300 (br), 1690 (s), 1520 (m) cm"1.
Anal. cal. for C9H23N30C12: C, 41.54; H, 8.91; N, 16.15. Found:
C, 41.18; H, 9.04; N, 15.95.
N4-Hexanoy1spermidine dihydrochloride (20)
A solution of (17) was deprotected and purified in a similar manner
as described for (19). Yield: 500 mg (89%); XH NMR (TFA) 6 0.96 (m, 3H),
1.40 (m, 6H), 1.60-2.36 (m, 6H), 2.66 (m, 2H), 2.96-3.90 (m, 8H), 6.92
(br, 6H); IR (KBr) 3300 (br), 1680 (s), 1530 (m) cm"1.


6
primarily concerned with the polyamines' role in cellular growth in both
the normal and diseased states. Several excellent reviews8-10 are avail
able for information concerning the other biological effects and will
not be discussed here.
The initial observations linking polyamines to cell growth were es
tablished in several different rapidly growing tissue systems during the
mid-sixties. In studies involving chick embryos, marked increases in
polyamine levels along with increased ODC and SAM-DC activities were ob
served during development relative to those of the mature animal.11-13
In the human fetal liver, putrescine concentration displayed a distinct
peak in the fifth and sixth month of gestation.1415 Although the em
bryonic data are limited, the higher polyamine levels were all attributed
to higher ornithine and adenosylmethionine decarboxylase activities as
sociated with rapid growth.
The regenerating rat liver has been a more widely used system to
study the effect of growth upon polyamine biosynthesis. Experimental
evidence from several groups has shown that enhanced accumulation of
putrescine reaches a maximum approximately four hours after partial
hepatectomy,16 resulting in a moderate enhancement of spermidine levels
in 2-3 days.17 This early increase in putrescine coincides with enhanced
ODC activity, which can be as great as 100- to 500-fold.1218 This in
crease is extraordinarily high as most enzymes in vivo only fluctuate
2-, 3-fold. The enhanced ODC activity, along with recent data reveal
ing that this enzyme is closely regulated by transcriptional and trans
lational mechanisms,1920 suggests that this early dramatic increase is
the primary event responsible for triggering polyamine biosynthesis.
The increased levels of putrescine and spermidine in the regenerating


CONHCH2CR2CH2NCH2CH2CH2CH2NHCO
Figure 6-6. Diagram illustrating the approximate sweep volumes of the a- and
7-protons of agrobactin A on rotation about the central amide.
CT>


26
VI Ib
Figure 2-1. Synthesis of secondary N-benzylated polyamines.


13
but not putrescine, effectively block MGBG's uptake. Accordingly, it
is postulated that MGBG competes with the carrier or receptor site re
sponsible for spermidine uptake. Additional evidence which supports this
is that, in DFMO pretreated LI210 cells, which respond by increasing
polyamine uptake, uptake of MGBG is also increased.42
Structural Requirements for Polyamine Uptake
Whatever MGBG's mechanism of action may be, whether it acts by in
hibiting SAM-DC and diminishing spermidine levels, or by replacing sper
midine at some intracellular site, perhaps in the mitochondria, it is
of significant importance that MGBG and spermidine both compete for
energy-dependent uptake. Moreover, little is known as to what structural
requirements are necessary for a molecule to be actively transported into
the cell via this carrier. Indeed, up until the present time very little
has been done to investigate the effects upon cell growth and uptake by
derivatizing or modifying the polyamines. Several N-modified spermidine
and spermine compounds have been synthesized,as in the case of the mono-
and bis-acridyl-spermidine and spermines; however, these compounds were
initially synthesized in order to investigate their ability to inter
calate with DNA.58 Likewise, several novel branched spermidine and sper
mine homologues have been recently synthesized and screened for their
antineoplastic activity^9,5but the uptake characteristics of these com
pounds have not yet been investigated. From the studies discussed thus
far, it is apparent that certain structural parameters are necessary for
polyamine uptake; consequently, it would be desirable to further define
the structural requirements for uptake.
Recent evidence exists that of the polyamines, spermidine, sper
mine, and putrescine, the carrier is more specific for spermidine.


44
Anal. cal. for C23H3gN30l+: C, 65.53; H, 9.32; N, 9.97. Found: C,
65.49; H, 9.35; N, 9.9$.
N5-Benz,yl-N1>N9-bis(t-butox,ycarbony1) homos pe rmi dine (12)
A solution of N5-benzylhomospermidine and BOC-ON was reacted and
purified as described previously for (10): Yield: 1.35 g (93%); XH NMR
5 1.38-1.87 (m, 8H), 1.47 (s, 18H), 2.13-2.71 (m, 4H), 2.78-3.33 (m, 4H),
3.51 (s, 2H), 4.80-5.65 (br, 2H), 7.19 (s, 5H); IR (CHC13) 3420 (m),
1690 (s), 1520 (s), 1180 (s), 750 (m) cm"1.
Anal. cal. for C25H43N304: C, 66.78; H, 9.64; N, 9.35. Found:
C, 66.52; H, 9.69; N, 9.25.
N1,M8-Bis(t-butoxycarbonyl)spermidine-hydrochloride M31
Palladium chloride (350 mg) was added to a solution of (1_0) (3.9 g,
9.0 mmol) in 50 ml methanol containing concentrated hydrochloric acid
(0.75 ml, 9.0 mmol). The resulting suspension was stirred under a hydro
gen atmosphere overnight (18 h). The catalysts were then filtered, washed
with MeOH, and the filtrates evaporated. The resulting crude solid was
recrystallized from ethanol/ether to afford 3.4 g (94%) of the desired
product: mp 149-150C; lH NMR (D20) 1.50 (s, 18H), 1.66 (m, 6H), 3.14
(m, 8H); IR (KBr) 3380 (s), 1690 (s), 1520 (s), 1190 (m) cm1
Anal. cal. for C17H36N304C1: C, 53.46; H, 9.50; N, 11.00. Found:
C, 53.41; H, 9.54; N, 10.99.
N1,N7-Bis(t-butoxycarbonyl)norspermidine-hydrochloride (14)
A solution of (1J_) was hydrogenated and purified in a similar man
ner as described for (1_3). Yield: 1.2 g (89%); mp 173-174C; NMR
(D20) 6 1.48 (s, 18H), 1.78 (m, 4H), 3.16 (m, 8H); IR (KBr) 3400 (s),
1700 (s), 1515 (s), 1170 (m) cm-1.


21
IV
V
VI
la
HO NH-1 BO C
*
Figure 1-7. Synthesis of agrobactin A.


CHAPTER SIX
SYNTHESIS AND SOLUTION DYNAMICS OF AGROBACTIN A
Experimental Section
Materials
All reagents, with the exception of t-butoxycarbonyloxyimino-2-
phenylacetonitrile (BOC-ON, Sigma Chemical Co., St. Louis, MO) were pur
chased from Aldrich Chemical Co., Metuchen, NJ and, unless noted, were
used without further purification. The reagents N1,N8-bis(t-butoxycar-
bonyl)spermidine-HCl (13) and N1,N9-bis(t-butoxycarbonyl)homospermidine
(15) were prepared as previously described in Chapter Two. Sodium sul
fate (Na2S04) was used as the drying agent. Melting points were taken on
a Fischer-Johns apparatus and are uncorrected. Routine *H NMR spectra
were recorded on a Varian T-60 and prepared in DCC13 or DMS0-d6 with
chemical shifts given in parts per million () from an internal Me4Si
standard. The infrared spectra were recorded on a Beckman 4210 spectro
photometer. Elemental analyses were performed by Atlantic Microlabs,
Atlanta, GA.
N-(t-Butoxycarbonyl)threonine (511
This compound was prepared by reacting D,L-threonine with t-butoxy-
carbonyloxyimino-2-phenylacetonitrile:8t* mp 75-76C (Et20/Pet ether)
(lit. 74-77).
Succinimide-2,3-dihydroxybenzoate (52)
To a solution of 2,3-dihydroxybenzoic acid (1.16 g, 7.53 nmol)
and N-hydroxysuccinimide (1.04 g, 9.04 mmol) in dry dioxane (30 ml) was
127


To Barb and Dan,
for their unending love and patience


Figure 2 6. 60 MHz Vi NMR spectra of NVN-bi?(acety1)spermidine(4).


164
Additionally, the synthesis, a third reagent for the selective
acylation of spermidine, was described. The new reagent, N4-benzyl-N1(t-
butoxycarbonyl)-N8-(trifluoroacetoxy)spermidine employs three different
protecting groups, each removable under different conditions. According
ly, each one of spermidine's three nitrogens can now be functionalized in
any order. As an illustrative example, N8-acetyl-N4-benzoyl-N1-(2,3-
dimethoxybenzoyl)spermidine was successfully prepared using this reagent.
Finally, the natural product agrobactin A and a number of its deriva
tives were synthesized. Employing the reagent N1,N8-bis(t-butoxycarbonyl )-
spermidine, agrobactin A was generated in three high yield steps. The
scheme highlighted an "inside-out" approach, which attached the three
2,3-dihydroxybenzoyl groups in the last step, requiring no catechol pro
tecting groups.
High field *H NMR spectroscopy of agrobactin A revealed that the com
pound existed in a series of conformers as determined by the duplicity in
the threonyl signals. These conformers were attributed to the asymmetri
cal nature of the spermidine backbone and subsequently the symmetrical
homologue of agrobactin A incorporating homospermidine as the backbone was
synthesized. The 300 MHz XH NMR spectra of this symmetrical derivative,
homoagrobactin A, confirming the role of the polyamine backbone.
The coalescence temperatures and activation energies between con
formers of agrobactin A and several closely related analogues were deter
mined using 1H NMR. A comparison of the activation energies suggested
that steric factors were the predominant influence in controlling con-
former interconversion. However, the role of hydrogen bonding could not
be as easily assessed due to the hydrogen bonding capability of the sol
vent used.


yield. The synthesis of (59j, in addition to providing us with a nec-
cessary derivative for our *H NMR studies, illustrates that the number
of compounds which can be generated by this method are limited only by
the acylating or alkylating agent employed.
Finally, as proof of structure, agrobactin A was synthesized by an
alternate route using the versatile reagent N1,N8-bis(2,3-dimethoxyben-
zoyl)spermidine85 as illustrated in Figure 6-2. As previously described
in our synthesis of parabactin,73 N1,N8-bis(2,3-dimethoxybenzoyl)sper-
midine was condensed with N-CBZ threonine, again using the coupling agents
DCC and N-hydroxysuccimide to produce N4-[N-CBZ threonyl1-N1,N8-bis-2,3-
(dimethoxybenzoyl)spermidine. The CBZ protecting group is then removed
by hydrogenolysis in methanolic HC1 over PdCl2 catalyst, followed by
the removal of the methoxy groups with BBr3 to afford N4-threonyl-N1,N8-
bis(2,3-dihydroxybenzoyl)spermidine. The final product was once again
produced by reacting the succinimido ester of 2,3-dihydroxybenzoic acid
(52) with N4-threonyl-N1,N8-bis(2,3-dihydroxybenzoyl)spermidine. Although
this second method also represents an effective means of generating
agrobactin A, the preceding 'inside out' synthesis is preferred prepara-
tively as it contains one less step and requires only one chromatographic
separation instead of three.
Chemical shifts and coupling constants of agrobactin A and derivatives
at 300 MHz
The 300 MHz XH NMR spectrum of agrobactin A in CDC13/DMS0-d6 (10:1)
at 23C is shown in Figure 6-3. This solvent system was employed for
the purpose of comparison with earlier published spectra of agrobactin
A as well as with agrobactin and parabactin. The choice of solvent is
critical as the chemical shifts of these compounds are extremely sen
sitive to changes in solvent. The <5 values observed are identical to


86
0.80 mmol) in 50 ml dry pyridine. The resulting suspension was allowed
to stir for 36 hours at which time a solution of (1_3) (300 mg, 0.75 mnol)
in 10 ml CH2C12 was added and the reaction allowed to proceed an addi
tional 36 hours. The solvent was then evaporated and the residue dis
solved up in 100 CH2Cl2 filtered, washed with ice cold 3% HC1 (2 x 20
ml), H20 (2 x 20 ml), 5% NaHC03 (3 x 20 ml), H20 (3 x 20 ml), dried and
concentrated to afford 310 mg (83%) as a white crystalline solid; mp
155-157C (CHC13/cydohexane); NMR 5 1.20-1.82 (m, 6H), 1.39 (s, 18H),
2.12-2.67 (m, 2H), 2.75-3.44 (overlapping m, 12H), 4.32 (s, 4H), 5.61-
6.28 (m, 3H), 7.16 (s, 1H); IR (CHC13).
Anal. cal. for C27H46Ng06; C, 68.89; H, 8.42; N, 15.26. Found:
C, 59.17; H, 8.40; N, 15.07.
N4-[4-(2,3-Dihydro-lH-imidazo[1,2-b]pyrazo1o)carboxamido)butyrylJsper-
midine trifluoroacetate (N4-GIMPY Spd) (32)
A solution of (31_) and trifluoroacetic acid was reacted and puri
fied as described previously for 29; 400 mg (91%); XH NMR (TFA) 6 1.57-
1.96 (m, 6H), 2.10-2.62 (m, 2H), 2.74-3.57 (overlapping, 12H), 4.37
(s, 4H), 6.30 (s, 1H), 7.53 (s, 1H), 7.85 (br s, 6H).
Anal. cal. for C23H32FgN608: C, 39.89; H, 4.80; N, 12.19. Found:
C, 39.76; H, 5.11; N, 12.03.
Results and Discussion
The antineoplastic Spd conjugates N4-Chlorambucil Spd (29j and N4-
GIMPY Spd (32) were successfully synthesized in a two-step fashion em
ploying the bis(t-BOC)-protected reagent (1_3). Chlorambucil or GIMPY
(30) were condensed with (1_3) using the condensing reagents DCC and
N-hydroxysuccinimide in 70-80% yields. The yields were lower than the
corresponding secondary N-acylations illustrated in Chapter Two. This
was probably due to the less active acylating agent employed (succinimide


133
the delivery of antineoplastics to leukemia cells,75 it is stable
and easily accessible in high yields. Furthermore, both the homo- and
norspermidine homologues are also available, allowing for the synthesis
of the homo- and noragrobactin A homologues.
The synthesis of these reagents is easily effected by reacting the
appropriate secondary N-benzyl polyamine with t-butoxycarbonyloxyimino-
2-phenylacetonitrile, followed by debenzylation to afford the terminally
t-BOC protected polyamine in 90% overall yield/5 Agrobactin A and its
homospermidine homologue are subsequently synthesized in three high yield
steps from this point, as illustrated for agrobactin A in Figure 6-1.
The procedure first calls for reacting the succinimido ester of
t-BOC threonine, previously generated by reacting t-BOC threonine (51),
N-hydroxysuccinimide and DCC in THF, with a solution of N1,N8-bis(t-
butoxycarbonyl )spermidine hydrochloride (1_3) or its homospermidine ana
logue and triethylamine in aqueous CH3CN for 48 hours. The condensation
proceeds smoothly and cleanly to afford the t-BOC triamide (J33) in 90%
crude yield. This product can be easily purified via silica gel chroma
tography eluting with CHCl3/EtOAc (1:1); however, further purification
was found to be unnecessary as the impurities can be removed after the
next step. Hence, (53j is deprotected by brief exposure to trifluoro-
acetic acid. The residue is dissolved in water, and the impurities from
the preceding condensation extracted into CHC13 prior to lyophilization
of the aqueous layer to afford N4-threonyl spermidine (54) or N5-threonyl
homospermidine (_57) as the trifluoroacetate salt in 80% overall yield
from N-t-BOC-threonine.
The final and most crucial step of the synthesis is the addition
of the three 2,3-dihydroxybenzoyl moieties to the N4-threony1 triamine.


Figure 5-7. 60 MHz ^NMR spectrum of N^-benzy1 -N1 -(t-butoxycarbony1)spermidine (35).
6


135
Previous studies suggested it would be necessary first to protect the
catechol groups of 2,3-dihydroxybenzoic acid prior to condensation. How
ever, because of the known acid-catalyzed N- to O-migration of acyl
groups fixed to threonine's nitrogen1"1>102 only protecting groups capa
ble of being removed under neutral or basic conditions could be con
sidered. Under acid conditions threonine's N-acyl carbonyl becomes more
electrophilic, promoting intramolecular transacylation, resulting in the
formation of the threonyl ester and the amine salt. The migration can
be reversed, in some cases, under basic conditions.131 During our initial
synthesis of compound II, we had developed the reagent 2,3-diacetoxyben-
zoyl chloride which could efficiently acylate spermidine's primary amines,
and the acetoxy protecting groups could then be cleanly removed by treat
ment with methanolic sodium methoxide."
When triamine (54) was reacted with three equivalents of 2,3-di-
acetoxybenzoyl chloride, the acetoxy protecting groups removed under
basic conditions, and the products worked up in weak acid, two products
were repeatedly obtained in low yields. The major product was determined
to be the N- to O-migrated ester of agrobactin A, as indicated by its
300 MHz XH NMR spectra characterized by the chemical shifts of the a-,
6-, and y-methyl protons of 6 5.2, 5.5, and 1.4, respectively. This
migration was likely the result of protonating the catechols during
acidic workup. Even when cold pH 6.5 phosphate buffer was employed to
protonate the catechols, substantial migration still occurred. Fur
thermore, the minor product, although identical on TLC to agrobactin A,
demonstrated a XH NMR with the a- and g-methines shifted 0.2 ppm upfield
to those values previously reported. In addition, obvious differences
in the aromatic region were also noted. In light of these problems,
this method was abandoned.


19
1 I i
conhch2ch2ch2nch2ch2ch2ch2i\jhco
la R=OH
Ib R= H
HO
HO
conhch2ch2ch,nch2ch2ch
WH
II
Ilia R=0H
111b R= H
Figure 1-6. Structures of the siderophores Agrobactin A and
Agrobactin (la and Ilia), compound II, and Para-
bactin A and Parabactin (I b and 111 b).


137
i
55
Figure 6-2.
Synthesis of Agrobactin A via N1,N8-bis(2,3-di
methoxybenzoy1jspermidine (Scheme II).


156
Table 6-1. 300 MHz XH NMR Chemical Shifts and Coupling Constants
of Polyamides (23C).
Agrobactin A
(CDC13/DMS0-d6)
Homoagrobactin A
(CDCl3/DMS0-d6)
(53)
(CDC13)
(56)
(CDC13)
Threonine
(JaNH)
5.02
5.03
4.39
4.39
(8.4 Hz)
(8.4 Hz)
(9.6 Hz)
(9.6 Hz)
0 (Jag)
4.18
4.21
4.02
4.02
(2.7 Hz)
(3.0 Hz)
Y (JSy)
1.20
1.19
1.18
1.17
(6.3 Hz)
(6.6 Hz)
(6.1 Hz)
(6.3 Hz)
OH
4.7
4.7
5.2
4.2
NH
8.09
8.10
5.47
5.48
t-Butoxy


1.45
1.44
cch2c
1.5-2.1
1.5-1.85
1.4-1.9
1.36-1.7
nch2c
3.15-3.75
3.15-3.65
2.96-3.6
3.12,3.3,
Aromatics
6.70,7.00,7.22
6.68,6.98,7.22
NH
8.03,8.24
8.0,8.15
4.70,4.97
4.70,5.00
Phenols
12.0,13.0
12.0,13.0


2-10. IR spectrum of N1,N8-bis(ethy1)spermidine (7).
PERCENT TRANSMISSION


157
Table 6-2. Coalescence Temperatures and Activation Energies
of Agrobactin A and Homologues in DMSO-dg.
Tc(C)
Ea(kcalmole-^)
Tri(tBOC) compound 5_3
1 8a
15.2 t 0.15
Agrobactin A (55)
74
18.2 t 0.15
Agrobactin A-OMe (59)
>130
>21
Tri(benzoyl) cmpd. 6£
67 2C
17.9 0.20
Agrobactin^
n.7 5
^1 8
adetermined in DMF-dy
^From reference 72
cEstimated due to poor resolution


77
to usual peptide condensation techniques. The reaction scheme is shown
in Figure 4-1. The succinimide active ester of chlorambucil is first
generated using dicyclohexylcarbodiimide and N-hydroxysuccinimide and
then reacted with N1,N8-bis(t-butoxycarbonyl)spermidine producing N4-
chlorambucil-N1,H8-bis(t-butoxycarbonyl )spermidine (28). The desired
N4-chlorambucil spermidine conjugate (29j is prepared by brief exposure
of intermediate (28) to trifluoroacetic acid.
Although IMPY does not contain a carboxylic acid with which to
directly condense with the protected spermidine reagent, a short con
necting bridge, such as glutaric acid, could be used. Subsequently,
IMPY can be reacted with glutaric anhydride, thereby producing an inter-
mediate (30) with a free carboxylic acid. This is then condensed with
N1,N8-bis(t-butoxycarbonyl)spermidine in the same manner as described
above. The entire synthesis of N4-[4-(2,3-dihydro-lH-imidazo[l,2-b]-
pyrazolo)carboxamido)butyryl]spermidine (32_) is shown in Figure 4-2.
Once these antineoplastic-Spd conjugates are synthesized, they will
be tested in vitro for their ability to compete with spermidine for
uptake as well as their antileukemic activity. The former assay is per
formed in the same manner as for the polyamine derivatives in the pre
ceding chapter. The antineoplastic-Spd conjugate will be incubated
in the presence of 3H-Spd in ascites LI210 cells, and in inhibition of
3H uptake determined as before. Additionally, the uptake of the native
antineoplastics themselves will also be determined.
The antileukemic activity of the antineoplastic-Spd conjugates
and the native anti neoplasties will be ascertained by determining the
dose necessary to prevent 50% of cell growth (ID50) of LI210 cell cul
tures. It is anticipated that if conjugation of spermidine to the


en
ro
Figure 2-13. 60 MHz ^ H NMR spectrum of N^-hexanoylspermidine tris(trifluoro-
a ce ta te)(20).


129
N4-Threon,y1 spermidine-trifluoroacetate (54)
Trifl uoroacetic acid (25 ml) was slowly added to a cooled flask con
taining (53J (650 mg, 1.2 mmol) and the resulting solution stirred for
25 min while warming to room temperature. The solvent was then quickly
evaporated, the residue dissolved in MeOH (25 nl), and evaporated (twice).
The product was then dissolved in 50 ml H20, washed with cold CHC13
(3 x 10 ml), and the aqueous layer lyophilized to afford 690 mg (97%)
as a light tan hygroscopic solid.
An analytical sample was prepared by chromatography on Sephadex LH-20
eluting with 20% MeOH/EtOAc: lH NMR (DMS0-d6) 6 1.15 (d, 3H), 1.36-2.08
(m, 6H), 2.80-3.32 (m, 8H), 4.52 (m, 3H), 7.94 (m, 9H); IR (KBr) 1685
(s), 1190 (s) cm-1.
Anal. cal. for C17H2gFgN40p: C, 34.70; H, 4.97; N, 9.52. Found:
C, 34.50; H, 5.03; N, 9.43.
N4-[N-(2,3-Dihydroxybenzoyl)threonylj-N1,N8-bis(2,3-dihydroxybenzoyl)-
spermidine (Agrobactin A) (5,I.)
A solution of succinimide 2,3-dihydroxybenzoate (52J (350 mg, 1.4
mmol) in 15 ml THF was slowly added dropwise to a solution of (54.) (275
mg, 0.46 mmol) and Et3N (210 ml, 1.5 mmol) in 5% aqueous THF (40 ml) un
der N2. After 36 hours the solvent was evaporated to dryness and the
residue preabsorbed on Sephadex LH-20, and eluted with an ethanol/ben
zene gradient (5-25% v/v) to yield 225 mg (75%) of the desired product.
The spectral characteristics were identical to those reported in the
literature.72
Anal. cal. for C32H38N4011*H20: C, 57.13; H, 5.99; N, 8.33. Found:
C, 57.10; H, 6.09; N, 8.26.


75
as 1,8-diaminooctane, in which the central nitrogen is replaced by a car
bon, are still very good inhibitors of 3H-Spd uptake having a Ki of 22.1 uM91.
Nevertheless, the central nitrogen may be the appropriate site to con
jugate antineoplastics to.


9
2 yM.33 In vivo, the presence of 0.5 mM ct-hydrazinoornithine added to
the medium of hepatoma cells could prevent the normal rapid accumulation
of putrescine and, likewise, partially prevent the increased accumulation
of putrescine due to partial hepatectomy.34 In both systems, however,
hydrazinoomithine did not disturb the enhanced synthesis of RNA, a nega
tive observation as far as indicating the need of polyamines for cell
proliferation or RNA stabilization.
A close analog to hydrazinoomi thine, D,L-a-hydrazino-6-aminova1eric
acid was an even more effective inhibitor of ornithine decarboxylase
than the former, having a Ki of 0.5 yM.35 Administration of hydrazino-
aminovaleric acid blocked the increase in DNA synthesis, the accumulation
of putrescine, as well as the weight gain usually seen in mouse sarcomas.36
Similar results were also obtained from cultures derived from hamster
tumors.36 In all these cases spermidine and spermine levels remained
unchanged, and the effects of hydrazinoamino valeric acid could be re
versed by the addition of putrescine, but not spermidine35 or cadaverine.36
Perhaps the simplest of ornithine analogs, a-methylornithine, syn
thesized in 1974, is also a potent competitive inhibitor (Ki value 20
yM) of ODC. Addition of a-methyl ornithine to cultures of rat hepatoma
cells completely prevented accumulation of putrescine, and likewise pre
vented increases in cellular spermidine.37 Incorporation of thymidine
in DNA greatly declined after the first doubling time in its presence.
Furthermore, a-methylornithine's growth inhibition could be immediately
reversed by concentrations of spermidine, spermine, and putrescine lower
than that of ornithine, offering good evidence that the intracellular

polyamine levels are casually related to proliferation.


88
Table 4-1. Uptake and Cytotoxicity of Antineoplastics
and Antineoplastic-Spermidine Conjugates in
L1210 Leukemia Cells in vitro.
Ki(uM)
ID50(uM)
Ch1orambuci1
350
15
N4-Chlorambuci1Spd (29)
6
15
GIMPY (30)
*
>1000*
N4-GIMPY Spd (32)
150
350
insolubility at higher concentrations prevented determina
tion


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M.O
3 0
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1.0
Figure 2-18. 60 MHz ^ H nmr spectrum of N^-methylspermidine (25).


29
Figure 2-3. Synthesis of n"* ,N-bis (t-Butoxycarbonyl)spermi-
dine.


7
liver are also usually accompanied by a decrease in spermine17 as sper
mine synthetase has been shown to be inhibited by high concentrations of
putrescine.21
More recently, a third type of rapidly proliferating cell and its
corresponding high polyamine levels have drawn much attention the cancer
cell. This was first shown in Ehrlich ascite carcinoma cells grown in
mice by two separate labs.2223 Both groups found that in the early
stages of tumor growth, a remarkably high putrescine content was found
that rapidly decreased after the first week following inoculation, where
as the concentration of spermidine peaked near the second week after
inoculation. These data are comparable to that found in the regenerat
ing rat liver system mentioned earlier. Wi11iams-Ashman et al. were further
able to show, by using hepatomas of vastly different growth rates, that
the accumulation of putrescine was proportional to the rate of tumor
growth.2425 Putrescine levels in the most rapidly growing hepatomas
were ten times as high as levels in the normal rat liver. Furthermore,
ornithine decarboxylase activity also coincided well with rate of tumor
growth.
Similar results have also been found for other types of cancer.
For example, markedly elevated levels of spermidine as well as ornithine
decarboxylase activity were found in d/3 sarcoma as compared to normal
muscle tissue.26 Ornithine decarboxylase activity was found to be six
times higher in mouse LI210 leukemia tumor cells, as well as containing
more than double their normal values of putrescine and spermidine.27
Additionally, some rapidly growing tumors produce enough polyamines that
they are actually excreted into the surrounding interstitial fluid caus
ing elevated polyamine serum levels.2829 Accordingly, clinical researchers


12
Mefhyigiyoxal-Bis (Guanylhydrczone)
Spermidine
Figure 1-3. Structures of a) Methylglyoxal-bis-(guanyl-
hydrazone), b) S-adenosyl-L-methionine, and
c) Spermidine.


97
protecting group. The remainder of the scheme lies on the key reduction
of (XXII) to N-[N-(t-butoxycarbony1)-4-aminobutyl]benzylamine (XXIII).
It has been recently reported in the literature on its ability to selec
tively reduce amides in the presence of urethane linkages using borane-
THF complexes.98 An approach such as this, if applicable to this system,
would generate (XXIII) from benzylamine in just two steps compared to
the three required for synthesis of (XIX).
The remainder of the scheme, Figure 5-4, is identical to that of
the 3,4-homologue. Intermediate (XXIII) is reacted with 4-chlorobutyryl-
nitrile, followed by Raney nickel reduction producing (XXV). Reaction
of amine (XXV) with trifluoroacetic anhydride would afford the desired
tris-protected homospermidine (XXVI) in five steps from benzylamine.
Finally, an example of the use of the tris-protected reagent is
given. The spermidine derivative N8-acetyl-N4-benzoyl-N1-(2,3-dimethoxy-
benzoyl)spermidine is synthesized via selective removal and acylation of
the spermidine reagent.
Experimental
Materials
The starting material N-(3-ami nopropyl)benzyl amine was prepared
as previously described.99 All other reagents were purchased from Aldrich
Chemical Company, Metuchen, NJ and, except where noted, used without
further purification. Dry THF was obtained by freshly distilling from
calcium hydride. The physical measurements were performed as described
in the materials section of Chapter Two.
N-[N-t-Butox,ycarbonyl )-3-aminopropyl jbenzylamine (33.)
A solution of BOC-ON (6.9 g, 0.028 mole) in 20 ml THF was slowly
added dropwise to a solution of N-(3-aminopropyl)benzylamine (5.0 g,


98
0.030 mole) in 30 ml at 0C. After the addition was completed, the
ice bath was removed, and the reaction stirred for 8 hours. The solvent
was then evaporated, the residue dissolved in 100 ml ether, washed with
5% NaOH (4 x 20 ml), dried, and concentrated to afford 3.2 g crude prod
uct. The product was further purified by distillation to afford 6.6 g
(89%) pure (33): bp 150-151C (0.13 nm); *H NMR 6 1.53 (s, 9H), 1.77
(m, 3H), 2.72 (t, 2H), 3.16 (quar., 2H), 3.70 (s, 2H), 5.26 (br, 1H),
7.23 (s, 5H); IR (CHC13) 2950 (m), 1690 (s), 1490 (m), 1160 (s), 750
(s) cm-1.
Anal. cal. for C15H24N202: C, 68.15; H, 9.15; N, 10.60. Found:
C, 68.14; H, 9.19; N, 10.55.
N-[N-(t-Butox,ycarbonyl)-3-aminopropyl ]-N-(3-cyanopropyl )benzylamine (34)
A solution of 4-chlorobutyrylnitrile (4.7 g, 0.095 mole) in 100
ml BuOH was slowly added to a suspension of (3J3) (10.0 g, 0.038 mole),
Na2C03 (4.4 g, 0.042 mole) and KI (1.4 g, 0.0095 mole) in 100 ml BuOH.
The reaction mixture was then gently refluxed for 48 hours, cooled,
and diluted with an equal volume of ether. The salts are filtered,
washed with ether, and the filtrates evaporated. The crude product was
chromatographed on silica gel, eluted with 5% Me0H/CHCl3 to afford 1.2 g
(95%) of the desired product as an oil: XH NMR 6 1.43 (s, 9H), 1.73
(m, 4H), 2.43 (m, 4H), 3.12 (quat., 2H), 3.47 (s, 2H), 4.83 (br, 1H),
7.23 (s, 5H); IR (neat) 2990 (s), 2250 (w), 1710 (s), 1520 (s), 1180
(s), 740 (m) cm-1.
Anal. cal. for C1?H2gH302: C, 68.85; H, 8.82; N, 12.68. Found:
C, 68.57; H, 8.85; N, 12.59.


n
If one were to look at MGBG's structure (Figure 1-3), one might
assume that the mode of action of inhibiting SAM-DC is by mimicking SAM's
structure, and that this inhibition of polyamine synthesis might be re
sponsible for the antileukemic activity. However, there appears to be
no simple correlation between the ability to inhibit SAM-DC and the anti
leukemic activity. In general, minor modifications of MGBG, such as the
dimethyl and ethylglyoxal analogues, do not have any pronounced effect
against its ability to inhibit SAM-DC, but lack antileukemic activity.49
Recently, an alternative mechanism for MGBG's antiproliferative
activity has been suggested. Several recent studies have shown that
cultured LI210 cells treated with micromolar concentrations of MGBG de
velop excessively swollen mitochondria several hours before detectable
changes in polyamine levels or cell growth.5051 This suggested MGBG
may act as a mitochondrial poison. The damage in most instances is re
versible and normal appearance can be restored by the addition of sper
midine. However, the irreversible inhibitor 1,1](methylethane diylidene
dinitrilo]bis(3-aminoguanidine) (MGAG) which is equally effective as
MGBG in inhibiting cell growth, is much slower to produce mitochondrial
damage.52
Of additional interest, MGBG is concentrated in a variety of cells,53
apparently through a saturable energy-dependent transport mechanism.54
In fact, in Ehrlich ascite carcinoma cells, MGBG is concentrated so
effectively that a concentration gradient as high as 1000-fold across
the cell membrane has been observed, producing minimolar intracellular
MGBG levels.55 Furthermore, the accumulation of MGBG could be blocked
competitively by polyamines and, to a lesser extent, some diamines.5657
For example, only micromolar concentrations of spermidine or spermine,


CHAPTER SEVEN
CONCLUSIONS
Selective and efficient syntheses of polyamine derivatives were
described. Using the key reagents N4-benzylspermidine, N4-benzylnorsper-
midine, and N5-benzylhomospermidine, terminally bis-acylated or alkylated
polyamine derivatives were easily generated in two and three high yield
steps, respectively.
In an extension of this synthetic scheme, reagents for the selective
secondary N-acylation of spermidine and its homologues were developed.
The benzylated spermidines were terminally bis-acylated with t-butoxy-
carbonyloxyimino-2-phenylacetonitrile to afford the terminally t-BOC
protected polyamine reagents upon debenzylation. These reagents were used
for the high yield synthesis of N4-acylated or alkylated spermidine de
rivatives.
The uptake characteristics and LD50 toxicities of the terminally or
secondary N-modified polyamines thusly generated were also determined.
Terminally bis-acylated spermidine derivatives, regardless of size, were
shown to be unable to compete with 3H-spermidine for uptake in LI210
leukemia cells. Bis-alkylated derivatives, on the other hand, were fair
competitors in inhibiting labelled spermidine uptake, but the size of the
alkyl substituent was found to be restrictive.
162


31
employing an appropriate reducing agent. Likewise, the secondary N-
acylated compounds (XIV) could be converted to the alkyl derivatives
(XV), Figure 2-4, in the same manner.
Experimental
Materials
The reagents N4-benzylspermidine, N4-benzylnorspermidine, and N5-
benzylhomospermidine were prepared as previously described.82 All other
reagents were purchased from Aldrich Chemical Company and, except where
indicated, used without further purification. Dry methylene chloride
O
(CH2C12) was obtained by distillation followed by storage over 3 A mole
cular sieves. Dry dioxane was obtained by distillation from sodium metal
immediately before use. Sodium sulfate was used as the drying agent.
Sephadex LH-20 was obtained from Pharmacia Fine Chemicals. Preparative
thin layer chromatography (TLC) was performed on Anal tech 20 x 20 cm
silica gel GF plates.
For the physical measurements, melting points were taken on a Fisher-
Johns apparatus and are uncorrected. Proton nuclear magnetic resonance
^H-NMR) spectra were recorded on a Varian T-60 and, unless otherwise
noted, prepared in deuterated chloroform (DCC13) with chemical shifts
() given in parts per million relative to an internal (CH3)4Si stan
dard. The infrared (IR) spectra were recorded on a BeckmanAcculab 1 spec
trophotometer. Elemental analyses were performed by Galbraith Labora
tories, Knoxville, TN, or Atlantic Microlabs, Atlanta, GA.
Synthesis of Terminally N-modified Spermidine Derivatives
General methods
The bis-acylated polyamines were prepared from the appropriate
secondary benzylated polyamine as described for the following synthesis


71
Acute LD5Q Toxicity Studies of Polyamine Derivatives
The acute toxicity of some of the polyamine derivatives synthesized
was investigated in vivo in white mice. For purposes of comparison,
the LD50 of Spd was also measured. The results of this study are pre
sented in Table 3-4. The LD50 for Spd was determined to be about 400
mg/Kg. This value is somewhat lower than a previously reported value of
470 mg/Kg;90 however, this difference can be attributed to the difference
in duration between the two studies. In the earlier study, deaths were
recorded only up to five hours while, in this study, deaths were recorded
up to 36 hours. In fact, excluding the very toxic doses, the greatest
percentage of animals given Spd died between 18 and 24 hours. The most
likely cause of death appeared to be respiratory failure.
The toxicity behavior of MeSpd was virtually identical to Spd, both
in LD50 and time course of death. However, the toxicity of BSpd and
BenzoylSpd was very much different. Death usually resulted in five minutes
or less marked by convulsions. With all four compounds, in almost every
instance, if the animals survived the first minutes no ill effects were
seen afterwards. Interestingly enough, the LD50 of the three benzyl
homologues also showed a dependency on chain length with BhSpd being the
most toxic of all compounds tested. Finally, all of the terminally N-
modified compounds were shown to be relatively nontoxic, the three bis(acyl)-
Spd having LD50's exceeding 800 mg/Kg.
Piscussion
Several lines of evidence support the existence of an energy depen
dent transport carrier for the uptake of polyamines across the cell mem
brane. The anti cancer agent MGBG has been shown to actively compete with
spermidine for uptake and is concentrated intracellularly via this carrier.56


143
Figura 2
1.2 1.1 PPM 1.2 1.1 P
Figure 6-4. 300 MHz NMR spectra of y-methyl region in
solvents indicated: a) homoagrobacti n a 5J3 (C D C L 3/
DMSO-dg); b) agrobactin A 5_5 (CDC13/DMSO-dg); c)
53 (CDCI3); d) 56 (CDC13).


CHAPTER THREE
BIOLOGICAL EVALUATION OF POLYAMINE DERIVATIVES
Materials and Methods
Polyamine Derivatives
The following compounds were synthesized as described in Chapter
Two and the following abbreviations will be used: N4-benzylspermidine
(BSpd), N4-benzylnorspermidine (BnSpd), N5-benzylhomospermidine (BhSpd),
N4-benzoylspermidine (Benzoyl Spd), N4-methylspermidine (MeSpd), N4-
acetylspermidine (AcetylSpd), N4-Ethylspermidine (EtSpd), N4-hexanoyl-
spermidine (HexanoylSpd), N4-hexylspermidine (HexylSpd), N-(2-Cyano-
ethyl)-N-(3-cyanopropyl)benzylamine (BSpdN), N1,N8-bis(t-butoxycarbonyl)-
spermidine (BisBOCSpd), N1,N8-bis(acetyl)spermidine (BisAcetylSpd),
N1,N8-bis(ethyl)spermidine (BisEtSpd), M1,N8-bis(propionyl)spermidine
(BisPropionylSpd), N1,N8-bis(propyl)spermidine (BisPropylSpd), norsper-
midine (nSpd), and homospermidine (hSpd). The following were purchased
from Aldrich Chemical Co., Metuchen, NJ: spermidine (Spd), spermine
(Spm), and putrescine (Put). Available in this lab already was N^N8-
bis(2,3-dihydroxybenzoyl)spermidine (DHBSpd).
Ascites Leukemia Cells
Murine LI210 leukemia cells were maintained by weekly i.p. trans
plantation in female DBA/2J mice. For in vitro studies 106 leukemic
cells were inoculated i.p., four days prior to use. Cells were col
lected by peritoneal lavage with RPMI-1640. The cells were washed
twice, counted electronically, and adjusted to a density of 107 cell s/ml
for uptake studies. The effects of DMFO on Spd uptake were studied by
64


pkcenuransmission
Figure 5-18. IR spectrum of N-(N-t-butoxycarbonyl-4-aminobutyryl)benzylamine (40).
RRCENT TRANSMISSION


I
Figure 4-3. 300 MHz NMR spectrum of N^-Chlorambuci1-N^,N^-bis(t-butoxy-
carbonyl)spermidine (28).


95
selectivity is expected based on the known preference in reactivity of
primary over secondary amines seen with t-BOC forming reagents.8096
Once (XIX) is obtained, the remainder of the synthetic sequence is essen
tially identical to the corresponding benzyl spermidine scheme. Deriva
tive (XIX) can either be reacted with 4-chlorobutyrylnitrile producing
the 3,4-precursor, or with acrylonitrile to produce the 3,3-precursor.
The nitriles (XX) are subsequently reduced with Raney nickel97 to generate
the intermediates (XVII a & b) which,when reacted with trifluoroacetic
anhydride, form the tris-protected reagents of spermidine and norsper-
midine.
The synthesis of the corresponding homospermidine reagent would
proceed along the same lines, requiring the synthon N-(4-aminobutyl)-
benzyl amine (XXI) in place of N-(3-ami nopropyl)benzyl amine (XVIII). The
preparation of (XXI), however, is not a trivial matter as compared to
the synthesis of (XVIII) from acrylonitrile and benzylamine. For in
stance, alkylation of excess benzylamine with 4-chlorobutyrylnitrile
affords very little of the nitrile precursor to (XXI), the major product
resulting from bis-alkylation instead.
Therefore, it seemed obvious that the synthesis of (XXI) should be
effected by a higher yield acylation of benzylamine, instead of alkyla
tion, followed by reduction. The requisite 3-cyanopropionic acid for
this acylation is not commercially available; however, N-(t-butoxycar-
bonyl)-4-aminobutyric acid (t-BOC-GABA) is available and offers an
attractive scheme as outlined in Figure 5-4.
Benzylamine can be reacted with the succinimide ester of t-BOC-GABA,
previously generated with DCC and N-hydroxysuccinimide, to afford the
N-benzyl butanamide derivative (XXII), which already contains the t-BOC


32
of N1,N8-bis(acetyl)spermidine. The procedure is the same regardless of
the benzylated polyamine or acylating agent employed.
A solution containing 2.2 equivalents of acetyl chloride was react
ed with N4-benzylspermidine in the presence of triethyl amine as base to
cleanly afford N4-benzyl-N1,N8-bis(acetyl)spermidine in 98% crude yield.
The crude products are usually of sufficient purity that they can be
debenzylated without further purification. Accordingly, N4-benzyl-N1,N8-
bis(acetyl)spermidine was hydrogenated overnight over palladium chloride
catalyst in methanol/HCl to afford pure N1,N8-bis(acetyl)spermidine as
the hydrochloride salt upon recrystallization. The corresponding amine
was prepared by reduction of this bis(amide) with a suitable reducing
agent.
Of the many reducing agents available, sodium borohydride-trifluoro-
acetoxy complex83 was chosen due to its relative ease and mildness of
reduction. Other reagents, such as lithium aluminum hydride, are often
less selective and may cleave tertiary amides, an important consideration
for the preparation of the N4-alkyl derivatives. Therefore, N2,N8-bis-
(ethyl)spermidine was prepared by refluxing N1,N8-bis(acetyl)spermidine
and sodium trifluoroacetoxyborohydride in dry dioxane for eight hours to
afford the desired product in 78% yield after distillation. The result
ing amines are then usually converted to their hydrochloride salts to
prevent oxidation and improve handling.
N4-Benzyl-N1,N8-bis(acetyl)spermidine (1)
A solution of acetyl chloride (725 mg, 9.2 mmol) in 10 dry CH2Cl2
was added dropwise to a cooled solution (ice bath) of N4-benzylspermidine
(1.0 g, 4.2 mmol) and triethylamine (1.3 ml, 9.2 mmol) in 20 ml dry
CH2C12 under a N2 atmosphere. After the addition was completed, the


Figure 6-1 1, 300 MHz ]H NMR spectrum of N4-[N-(2,3-dimethoxybenzoy1)threony1]- g
Nl,N8-bis(2,3-dimethoxybenzoyl)spermidine (59).


TCI
Anal. cal. for C20H30N303F3: C, 57.54; H, 7.24; N, 10.06. Found:
C, 57.62; H, 7.27; N, 9.95.
N-(N-t-Cutoxycarbonyl -4-aminobutyryl) benzyl amine (40)
A solution of DCC (2.47 g, 12 mmol) in 20 ml THF was slowly added
to a cooled solution of N-t-BOC-GABA (2.0 g, 10 mmol) and N-hydroxysuc-
cinimide (1.4 g, 12 mmol) in 20 ml THF. The ice bath was removed and
the reaction allowed to stir for 16 hours during which time a thick
white precipitate ensued. The DCU precipitates were filtered, washed
with THF, and the filtrates concentrated to a volume of 20 ml.
The above solution was then added to a solution of benzylamine (1.6
g, 15 imol) in 20 ml THF, and a white precipitate immediately resulted.
The suspension was stirred for 18 hours at room temperature at which
time the solvent was evaporated. The residue was dissolved up in 100
ml ether, and the insoluble matter filtered. The ether layer was washed
with 3% HC1 (2 x 20 ml), H20 (2 x 20 ml), 5% NaHC03 (2 x 20 ml), dried
and concentrated to afford the crude product. Recrystallization from
ether/cyclohexane afforded 2.5 g (87%) pure (40) as a white solid:
mp 110-111C; lH NMR 6 1.37 (s, 9H), 1.8 (quar., 2H), 2.18 (t, 2H),
3.06 (quar., 2H), 4.30 (d, 2H), 4.80 (br, 1H), 6.47 (br, 1H), 7.12 (s,
5H); IR (CHC13) 3010 (m), 1700 (s), 1660 (s), 1515 (s), 760 (s) cm'1.
Anal. cal. for C16H24N203: C, 65.73; H, 8.27; N, 9.58. Found:
C, 65.70; H, 8.30; N, 9.56.
N-(N-t-Butoxycarbony1-4-aminobutyl)benzylamine (H )
A solution of 1 M diborane-THF complex (13 ml) was slowly added to
a cooled suspenson of (40) (1.9 g, 6.5 mmol) in 20 ml dry THF under N2.
Once the addition was completed, the ice bath was removed, and the now
clear solution brought to reflux for 20 hours. The reaction mixture


48
N4-Hexylspermidine trihydrochloride (23)
A solution of (20) was reduced and purified in a similar manner as
described for (22). Yield: 180 mg (82%); *H NMR 5 0.85 (m, 3H), 1.04-
1.82 (overlapping m, 18H), 2.12-2.85 (m, 1 OH); IR (CHC1 3) 3300 (m),
2970 (s), 1425 (m) cm-1.
Anal. cal. for C:3H31+N3C13: C, 46.09; H, 10.12; N, 12.40. Found:
C, 46.37; H, 10.04; N, 12.17.
N4-Methy1-NI>N8-bis(t-butoxycarbonyl)spermidine (24)
Three hundred fifty milligrams of N1,N8-bis(t-butoxycarbonyl)sper-
midine hydrochloride (1_3) were added to a 15% aqueous solution of Na2C03.
The solution was extracted with ether (3 x 30 ml), the organic layer
dried, and concentrated to afford 300 mg of the free amine as an oil.
To a solution of N1,N8-bis(t-butoxycarbonyl)spermidine (300 mg, 0.9
mmol) in 3 ml acetonitrile was added a 37% aqueous formaldehyde solution
(0.4 ml, 5 mmol) and sodium cyanoborohydride (100 mg, 1.6 mmol). A mild
ly exothermic reaction ensued which subsided after a minute or so. After
15 min, the pH of the solution was checked, adjusted to pH 7.0 with acetic
acid, and the reaction allowed to stir for an additional 1.5 h.
The solvent was then reduced in vacuo, the residue dissolved in
2N K0H (5 ml), and the product extracted with ether (3 x 15 ml). The
ether layer was dried and concentrated to afford 400 mg of the crude
product.
Further purification was effected by chromatography on silica gel
(Merck 7734) eluting with Me0H/CHCl3 (5% -* 15% MeOH) to afford 210 mg
(65%) pure (24): lH NMR 6 1.43 (m, 24H), 2.24 (s, 3H), 2.42 (m, 4H),
3.06 (m, 4H), 5.14 (br, 2H); IR (CHC13) 3390 (m), 1700 (s), 1510 (s),
1180 (s) cm-1.


150
of the estimated Ea's might, therefore, provide additional information
regarding the factors affecting conformation in these siderophores.
Although the two y-signals corresponding to the two polyamide conformers
do not quite represent an equally populated spin system, the Ea for
their interconversion can be estimated using methods developed for equal
ly populated, noncoupled spin systems.77 It is generally acknowledged
that computer total line-shape analysis should be employed for highly
accurate values of Ea; however, highly accurate values are not required
for the qualitative comparisons necessary in these studies. In this
regard the relative differences in Ea's between the compounds are more
important than their absolute values.
Of all the barriers involved in control!ing the interconversion be
tween the conformers in these polyamides, hydrogen bonding and steric
factors are likely to be the dominant ones. The question is how to de
termine the contribution of each of these components in the interconver
sion. The answer is to carefully remove or introduce hydrogen bonding
and/or steric interactions in the systems of interest and then evaluate
the effect on Ea for the process. In this study we have chosen to con
sider the Ea of agrobactin, agrobactin A, the precursor (53) and methy
lated agrobactin A (59). The structural differences and similarities
between these four compounds should be sufficient to help assign the
role of steric and hydrogen bonding factors controlling the conformer
populations of the spermidine siderophores.
The coalescence temperatures of agrobactin A, hexamethyl-agrobactin
(59), and precursor (53) were all determined in DMS0-d6 as described in
the experimental section and listed in Table 6-2. Irradiation of the
e-methine of agrobactin A, for example, results in the collapse of the


TI
tQ
c
5
0>
en
i
tO l-H
O 70
fD
-5
3 "O
0)
Q. O
3 5
0> C
U> O
en -h
8LL
SNOH7IW NI MIOMIIIAVM


22
by attaching three 2,3-dihydroxybenzoyl groups to the triamine (VI).
Recently, Van Brussel and Van Sumere have shown it to be possible to
generate the succinimide esters of a number of mono- and dihydrobenzoic
acids in the presence of the unprotected phenols.76 Thus, when N^-threonyl-
spermidine was reacted with excess succinimide 2,3-dihydroxybenzoate in
the presence of triethylamine in aqueous THF, agrobactin A was obtained
in 75% yield after chromatography.
Unlike previous synthesis of catechol ami des which usually attached
the "centerpiece" in the final stages of the synthesis, this synthesis
of agrobactin A reverses this order, building the molecule from the
"inside out" and attaching the catechols in the final step. This is im
portant as any number of groups can now be attached to the triamine
(VI), making it possible to easily synthesize additional agrobactin A
derivatives. Examples of some derivatives that were synthesized in this
manner included the 2,3-dimethoxybenzoyl derivative of agrobactin A as
well as an unsubstituted benzoyl derivative. Likewise, symmetrical ana
logues of agrobactin A can be generated as both nor- and homospermidine
homologues of N1,N8-bis(t-B0C)spermidine are available.75
With these compounds at hand, their XH NMR spectra were subsequently
investigated. Not surprisingly, agrobactin A also exhibited conformers
in its NMR spectrum as determined by the duplicity in the signals
originating from the threonine residue. If, indeed, these duplicate
signals are a result of an equilibrium between two interconverting iso
mers and not two distinctly separate compounds, the activation energy
for this interconversion can be measured using XH NMR. The rate of
interconversion or exchange between conformer population can be estimated
by determining the coalescence temperature of the duplicate signals.


43
The resulting trifluoroacetic salts are very hydroscopic and therefore,
conmonly converted to the hydrochloride salts. Finally, N4-hexanoyl-
spermidine dihydrochloride can be converted to the corresponding amine,
again utilizing sodium trifluoroacetoxyborohydride as the reducing agent.
N4-Benzyl-N1,N8-bis(t-butoxycarbony1)spermidine (10)
A solution of BOC-ON (5.4 g, 0.022 mol) in 50 ml of distilled tetra-
hydrofuran (THF) was slowly added dropwise with stirring to a cooled solu
tion (ice bath) of N4-benzylspermidine (2.35 g, 0.010 mol) in 75 ml THF
under N2. After the addition was completed, the ice bath was removed,
and the reaction mixture allowed to stir for eight hours at which time
the solvent was evaporated. The residue was dissolved in 150 ml of ether
and washed with 5% NaOH (4 x 25 ml), water (3 x 25 ml), dried, and evap
orated to afford 4.3 g (99%) of the desired product, as a viscous light
yellow oil. Thin layer chromatography and XH NMR analysis indicated
the crude oil purity was in excess of 95%, and subsequently used without
further purification.
An analytical sample was prepared by preparative TLC, eluting with
10% Me0H/CH2Cl2: XH NMR 5 1.45 (s, 18H), 1.40-1.88 (m, 6H), 2.18-2.70
(m, 4H), 2.82-3.35 (m, 4H), 3.48 (s, 2H), 4.79-5.63 (br, 2H), 7.17
(s, 5H); IR (CHC13) 3390 (m) 1705 (s), 1510 (s), 1190 (s), 760 (m) cm'1.
Anal. cal. for C24H41N304: C, 66.18; H, 9.49; N, 9.65. Found: C,
65.93; H, 9.79; N, 9.38.
N4-Benzy1-NI,N7-bis(t-butoxycarbonyl)norspermidine (11)
A solution of N4-benzylnorspermidine and B0C-0N was reacted and
purified as described previously for (JO): yield: 2.0 g (98%); XH NMR
6 1.41-1.83 (m, 4H), 1.44 (s, 18H), 2.11-2.68 (m, 4H), 2.77-3.35 (m, 4H),
3.50 (s, 2H), 4.75-5.68 (br, 2H), 7.21 (s, 5H); IR (CHC13) 3400 (m), 1700
(s), 1510 (s), 1175 (s), 755 (m) cm*1.


CHAPTER FIVE
SYNTHESIS OF TRIS-PROTECTED SPERMIDINES
The reagents described in Chapter Two, N4-benzylspermidine and
N1,N8-bis(t-butoxycarbonyl)spermidine, have demonstrated themselves as
important reagents for the selective functionalization of spermidine's
primary and secondary nitrogens. Using these methods, derivatives can
be obtained that 1) contain only one substituent on the secondary ni
trogen, 2) contain the two same substituents on both primary nitrogens,
or 3) a combination of both; two identical substituents on the primary
nitrogens and a third different substituent on the secondary nitrogen.
However, it is not possible to place three different groups on spermi
dine or, for that matter, only one substituent on only one primary ni
trogen via these methods. The latter group of derivatives, those modi
fied at only one terminal nitrogen, pose an additional interesting ques
tion concerning uptake --how effective would these derivatives be in com
peting for uptake?
The answer to this question, based upon the uptake data presented
in Chapter Three, is yes. The N1,N8-bis(alkyl)spermidine derivatives
were able to compete with Spd for uptake, and it is expected that modi
fication of only one terminal amine should accentuate this uptake.
This may offer another potential avenue in conjugating antineoplastics
to spermidine. Accordingly, the synthesis of such a reagent is desired.
The ideal reagent would be one in which each of spermidine's ni
trogens is protected with three different protecting groups, each remov
able separately under different conditions as illustrated in Figure 5-1.
90


5-10. 60 MHz NMR spectrum of N
amine (41).
(N-t-butoxycarbonyl-4-ami nobutyl)benzyl-
O


o?
Additionally, the boundary conditions of earlier synthesis must be still
met in that the reagent should be generated in as few steps as possible,
and the protecting groups removed cleanly and efficiently. The benzyl
and t-butoxycarbonyl protecting groups employed earlier have already proven
themselves as easily removable in the presence of one another and, ac
cordingly, their use will be maintained. The choice of the third pro
tecting group should therefore be one which is removable under basic
conditions as the t-BOC and benzyl groups are removed under acid and neu
tral conditions, respectively. A prime candidate for the base removable
protecting group is the trifluoroacetoxy protecting group.9- It can be
easily attached via either the anhydride or the acid chloride, and re
moved under relatively mild basic conditions employing sodium bicarbonate.
Synthesis
In the simplest terms, the synthesis of the target tris-protected
reagent would be nothing more than the synthesis of N4-benzyl-N1,N8-
bis(t-butoxycarbonyl)spermidine however, replacing one of the t-BOC
groups with the trifluoroacetoxy group. However, in reality the removal
of one t-BOC protecting group would likely be impossible. If, on the
other hand, this intermediate (XVI) figure 5-2, could be generated by
some other means, the target compound (XVII) could be realized.
Recalling the synthesis of the benzylated polyamines in Figure 2-1,
a synthetic scheme for intermediate (XVI) can be envisioned by incor
porating a t-BOC protecting group early on in the synthesis. Such a
scheme is outlined in Figure 5-3.
The key to the whole scheme lies in the ability to selectively react
the terminal amine of N-(3-ami nopropyl)benzyl amine with BOC-ON to pro
duce fl-[N-(t-butoxycarbonyl)3-ami nopropyl]benzylamine (XIX). The desired


124
ethoxycarbonyl group which is not susceptible to acid cleavage. For
tunately, an alternate method to prepare N-(4-ami nobutyl)benzylamine in
large quantities has recently been devised by our group.100
The remainder of the synthesis of the tris-protected homospermidine
reagent proceeds in an identical fashion to the spermidine reagent (36).
The t-BOC-protected intermediate (4]_) was reacted with excess 4-chloro-
butyrylnitrile, thusly producing the nitrile (42), once again in high
yields. Finally, N5-benzyl-N1-t-butoxycarbonyl-N8-trifluroracetoxyhomo-
spermidine (44) is produced by reduction of nitrile (42), followed by
acylation with trifluoroacetic anhydride.
Synthesis of N8-acetyl-Ntl-benzoyl-N1-2,3-dimethox,ybenzoyl spermidine (501
As an illustrative example of the use of the tris-protected spermi
dine reagents, the synthesis of (50) was demonstrated. The choice of
the acyl groups affixed: acetyl, benzoyl, 2,3-dimethoxybenzoyl, were
determined solely on their relative ease of identification in the NMR.
However, any other groups could have been used as well. Likewise, the
order and position in which these groups were attached was entirely random.
The synthesis of (50) was accomplished by three series of deprotec
tions and acylations as follows. The benzyl protecting group was first
removed by hydrogenolysis employing the same conditions described earlier.
Hence, a solution of the spermidine reagent, N4-benzyl-N8-t-butoxycar-
bonyl-N4-trifluoroacetyl spermidine (36) in methanol/HC1 was hydrogenated
overnight over PdCl2 catalysts to afford the debenzylated adduct (45) as
the hydrochloride salt in 85% yield after recrystallization. The reac
tion proceeded cleanly without any ill effects to the two remaining pro
tecting groups.


73
Furthermore, in cells having their polyamine pools depleted by pretreat
ment with DFMO, both polyamine and MGBG uptake is enhanced several-fold.42
Accordingly, it is hypothesized that several structural parameters must
be recognized by this receptor or carrier for uptake.
In an effort to further define these necessary parameters for up
take, a wide array of spermidine derivatives were synthesized and assayed
for their ability to compete with 3H-Spd for uptake. These studies sug
gest that the primary amines of spermidine are critical for recognition
by the carrier for uptake. Acylation of both primary amines produces
derivatives that are very poor inhibitors of 3H-Spd uptake. Reduction
of these amides to the corresponding amines offers some improvement in
the derivatives' ability to compete for uptake; however, this uptake
appears severely restricted by the size of the alkyl substituent. For
example, only BisEtSpd is as effective as MGBG in preventing Spd uptake.
Modification of spermidine's secondary amine appears less critical
in conferring uptake specificity, as both N-alkylated and N-acylated de
rivatives inhibited 3H-Spd uptake. However, there is an obvious prefer
ence for the alkylated derivatives. Moreover, there is less restriction
upon the size of the alkyl substituent as all four alkyl derivatives
ranging up to hexyl and benzyl all have Ki's comparable or better than
MGBG.
With respect to chain length, quite unexpectedly, the hSpd backbone
competed more effectively than the Spd backbone as demonstrated by the
order of preference BhSpd>BSpd>BnSpd. This finding is interesting since
hSpd is not found in mammalian systems. Whether this effect will be
seen in other derivatives is currently under investigation.


35
N1,N8-Bis(ethy1)spermidine trihydrochloride ()
A suspension of N1,N8-bis(acetyl)spermidine hydrochloride (330 mg,
1.24 mmol) and sodium borohydride (500 mg, 13 mmol) in 20 ml freshly dis
tilled dioxane was cooled to 10-15C under a N2 atmosphere. A solution
of trifluoroacetic acid (1.5 g, 13 mmol) in 10 ml dry dioxane was slowly
added dropwise with stirring. After the addition was completed, the
suspension was slowly brought to a gentle reflux and the reaction con
tinued for ten hours.
The reaction was then cooled, the excess reducing agent destroyed
by careful addition of water (2 ml), and the solvent reduced under high
vacuum. The residue was treated with 2N K0H (5-10 ml), the product ex
tracted into CH2C10 (4 x 20 ml), dried, and concentrated to afford 240
mg crude product as a semi sol id. The product was further purified by
distillation to afford 190 mg (78%): bp 133-135, 0.25 mm; NMR <5 0.88-
1.82 (overlapping multiplets, 15H), 2.60 (m, 12H); IR (CHC13) 2980 (s),
1465 (m), 1120 (m), 750 (s) cm-1.
The distilled amine was converted to the hydrochloride salt by dis
solving in a solution of ethanol and ether (1:1), cooling, and then bub
bling HC1 gas through.
Anal. cal. for C11H30N3C13: C, 42.52; H, 9.73; N, 13.52. Found:
C, 42.37; H, 9.68; N, 13.19.
N1 ,N8-Bis(propy1)spermidine trihydrochloride (&)
A suspension of N1,N8-bis(propionyl)spermidine hydrochloride was
reduced and purified as described for (7J. Yield: 210 mg (71%); bp
144-147, 0.10 mm; *H NMR 0.90 (t, 6H), 1.16-1.80 (m, 13H), 2.55 (m,
12H); IR (CHC13) 2975 (s), 1460 (m), 1120 (m), 760 (s) cm-1.


155
for these compounds. However, due to
in all but the most polar solvents, a
Ta's in other solvents is essentially
agrobactin A's poor solubility
comprehensive determination of
not possible.


45
Anal. cal. for C16H34N304C1: C, 52.23; H, 9.31; N, 11.42. Found:
C, 52.29; H, 9.31; N, 11.37.
N1>N9-Bis(t-butoxycarfaon.yl) hornos pe rmi dine hydrochloride (15)
A solution of (12) was hydrogenated and purified in a similar manner
as described for (13). Yield: 770 mg (92%); mp 187-188C; XH NMR (D20)
1.47 (m, 26H), 3.19 (m, 8H); IR (KBr) 3380 (s), 1690 (s), 1510 (s),
1175 (m) cm-1.
Anal. cal. for C18H38N3O4CI: C, 54.60; H, 9.67; N, 10.61. Found:
C, 54.58; H, 9.67; N, 10.59.
N4-Acetyl-NI>N8-bis(t-butoxycarbonyl )spermidine (16.)
A solution of acetyl chloride (180 mg, 2.2 mmol) in 10 ml dry CH2CI2
was slowly added to a cooled solution of (1_3) (760 mg, 2.0 mmol) and
triethylamine (600 yl, 4.4 mmol) in 30 ml CH2C12 under N2. The solution
was allowed to warm to room temperature and stirred overnight (18 h)
at which time additional CH2C12 (25 ml) was added. The organic layer
was washed with 3% HC1 (3 x 15 ml), H20 (2 x 15 ml), 5% NaHC03 (3 x 15
ml), H20 (2 x 15 ml), dried and concentrated to afford 720 mg (93%)
of the product as a colorless oil: NMR 6 1.42 (s, 18H), 1.64 (m, 6H),
2.06 (s, 3H), 3.18 (m, 8H), 5.02 (br, 2H); IR (CHC13) 3320 (m), 2960 (s),
1690 (s), 1620 (m), 1170 (s) cm-1.
An analytical sample was prepared by chromatography of silica gel
(70-230 mesh) eluting with EtOAc/CHCl3 (1:1). Anal. cal. for ClgH37N305-r
H20: C, 57.55; H, 9.66; N, 10.60. Found: C, 57.46; H, 9.30; N, 10.22.
N4-Hexanoyl-N1,N8-bis(t-butoxycarbony1)spermidine (17)
A solution of hexanoyl chloride and (1_3) were reacted and purified
in the same manner as for (1_6). Yield: 620 mg (95%); NMR 6 0.90


134
13
Figure 6-1. "Inside out" synthesis of Agrobactin A via
Nl-N8-bis(t-butoxycarbonyl)spermidine (13)
(Scheme I).


8
are currently developing diagnostic tools to follow tumor regression and
perhaps detect early tumor growth.30
From the above data it can be suggested that all cells, whether
normal or neoplastic, undergoing rapid proliferation contain elevated
levels of polyamines, notably putrescine and spermidine.
If polyamines are so intimately involved and required for rapid
cell growth as implied so far, then the selective inhibition of their
biosynthesis should have a pronounced detrimental effect upon cell growth.
This concept of inhibiting cell growth by inhibiting polyamine synthesis
would not only be valuable in determining the actual physiological sig
nificance of polyamines but could also represent a possible method of
arresting cancer growth. As mentioned earlier, the short half lives of
both ornithine and SAM decarboxylases are significant in that they should
be the easiest enzymes to inhibit. This is in fact the case as most of
the research in inhibiting polyamine synthesis in vitro and in vivo has
been centered on finding selective inhibitors of these two enzymes uti
lizing various tumor cell lines.
A great majority of the inhibitors that have been developed in the
last twenty years are congeners of ornithine and methionine. Ethionine,
the ethyl analog of methionine, was probably the first compound used to
inhibit polyamine synthesis. Although injections of it led to an initial
decrease in spermidine concentration, chronic treatment eventually led to
an overall increase in spermidine concentration,31 and its use as an
inhibitor subsequently was abandoned.
In 1973, a-hydrazinoomithine, a potent reversible inhibitor of or
nithine decarboxylase (ODC), was synthesized.32 Synthesis of a-hydra-
zinoornithine found it to be a competitive inhibitor with a Ki value of


t
1.0
to
so
M.0
Figure 2-7. 60 MHz NMR spectrum of N1
bis(ethyl )spermidine(_7).


Figure 2-5. 60 MHz ^ H NMR spectrrum of N4-benzy1-N1,N8-bis(acety1)spermi di ne (1).
co
en


153
bonding as the solvent, DMS0-d6, used in the measurements, is certainly
capable of competing for intramolecular hydrogen bonds. This implies
that the initial measurement of Ea for agrobactin A made in DMSO is
largely a measurement of steric control of the conformer population as
intramolecular hydrogen bonding is likely minimized by intermolecular
competition with DMSO.
The coalescence temperature of the tri-t-BOC precursor (53) further
supports the idea that DMSO prevents intramolecular hydrogen bonding in
the system studied. As shown in Figure 6-4c, the y-methyl of (53) exists
as an overlapping set of doublets in CDCI3 at room temperature. In
DMS0-d6, however, even at room temperature only one doublet is observed.
Therefore, the change from the less polar CDC13 to DMS0-d6 is sufficient
alone to allow rapid interconversion between conformers of (53).
The high freezing point of DMSO precluded its further use in deter
mining the Tc value of (53); therefore, an alternate solvent was chosen.
However, in order to make a comparison of the Ea's between (53) in an
alternate solvent to those of agrobactin and its homologues in DMS0-d6,
it is important that the solvent itself does not change the Ea signifi
cantly. Dimethylformamide (DMF) has a similar dielectric to DMSO, but
a much lower freezing point necessary for the measurement, making it an
appropriate choice. To determine if DMF would have any effect on the
Ea, the Tc and Ea of agrobactin A was subsequently measured in DMF.
No significant difference was noted between the Ea and Tc of agrobactin
A determined in DMF versus DMSO suggesting the data of (53) in DMF can
be compared to those in DMSO.
The ¡H NMR of (53) in DMF-d7 at room temperature also lacked the
duplicity seen in CDC13. However, upon cooling the decoupled y-methyl


Figure 5-11. 60 MHz ^ H NMR spectrum of fP-t-butoxycarbonyl-N^-trif1uoroacetyl-
spermidine Hydrochloride (45 ).


28
In turning our attention towards a reagent for the selective secon
dary N-acylation of spermidine and its homologues, the lack of a suitable
reagent in the literature necessitated that a new approach be developed.
Starting with the already available benzylated reagents (VII), this should
easily be accomplished by first blocking the terminal amines followed by
debenzylation. The only requirement in such a synthesis would be an easily
attachable/removable protecting group which is stable to the hydrogenation
conditions employed. The t-butoxycarbonyl protecting group meets this
requirement.
Hence, (VII) is reacted with two equivalents of t-butoxycarbonyl-
oxyimine 2-phenylacetonitrile (BOC-ON) to form the terminally bis-t-
butoxycarbonyl compounds in high yield, Figure 2-3. The benzyl group is
then removed via hydrogenolysis as before producing the reagents (XII)
capable of selectively secondary N-acylation. These t-BOC protected re
agents are then reacted with a variety of acylating agents, again of vary
ing size such as acetyl, benzoyl, and hexanoyl acid chlorides. The t-BOC
protecting groups are then removed quantitatively with trifluoroacetic
acid generating the secondary N-acylated spermidine derivatives (XIV),
as illustrated in Figure 2-4.
In considering the synthesis of N-alkylatod spermidine derivatives,
although the reagents N4-benzylspermidine and the symmetrical homologues
are available, direct alkylation of these compounds would presumably
result in over alkylation and poor yields. Therefore, it would seem
more practical to reduce the N-acylated derivatives already on hand via
the above two procedures directly to the N-alkylated compounds. Accord
ingly, the terminally bis-acylated derivatives (IX) could be converted
directly to the bis-alkylated derivatives (X) as shown in Figure 2-2


1 7
Figure 1-5. Structures of a) N4-C h1orambuci1spermidine ,and
b) N -[4-(2,3-dihydro-1H-i midazo]l,2-b]pyrazolo)
carboxamido)butyryl]spermidine.