Bioreversible derivatives of 5-fluorouracil (5FU)

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Title:
Bioreversible derivatives of 5-fluorouracil (5FU) improving dermal and transdermal delivery with prodrugs
Alternate title:
Improving dermal and transdermal delivery with prodrugs
Physical Description:
xiii, 160 leaves : ill. ; 29 cm.
Language:
English
Creator:
Beall, Howard D., 1954-
Publication Date:

Subjects

Subjects / Keywords:
Fluorouracil -- analogs & derivatives   ( mesh )
Prodrugs -- chemical synthesis   ( mesh )
Prodrugs -- pharmacokinetics   ( mesh )
Prodrugs -- chemistry   ( mesh )
Antineoplastic Agents -- chemical synthesis   ( mesh )
Antineoplastic Agents -- pharmacokinetics   ( mesh )
Antineoplastic Agents -- chemistry   ( mesh )
Administration, Cutaneous   ( mesh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1991.
Bibliography:
Includes bibliographical references (leaves 153-159).
Statement of Responsibility:
by Howard D. Beall.
General Note:
Typescript.
General Note:
Vita.

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Source Institution:
University of Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
oclc - 49645371
ocm49645371
System ID:
AA00011216:00001

Full Text












BIOREVERSIBLE DERIVATIVES OF 5-FLUOROURACIL (5FU):
IMPROVING DERMAL AND TRANSDERMAL DELIVERY
WITH PRODRUGS















BY


HOWARD D. BEALL


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

1991




























Copyright 1991

by

Howard D. Beall















ACKNOWLEDGEMENTS


I would like to thank the members of my supervisory

committee, Dr. Kenneth Sloan, Dr Margaret James, Dr. Koppaka

Rao, Dr. Richard Prankerd, and Dr. John Zoltewicz for their

guidance and expert advice over the past four years. My

sincerest thanks go to my research advisor and committee

chairman, Dr. Sloan, for sharing his enthusiasm for teaching

and science. I am especially grateful for his patience and

understanding during my seemingly endless questions and

interruptions.

I would also like to acknowledge the enthusiastic

support of Dr. Noel Meltzer of Hoffmann-La Roche. This

project was partially funded by a grant from Hoffmann-La

Roche.

My special thanks go to my parents for their love and

support throughout my life and to my two-year-old son,

Michael, who could make me laugh when it was the last thing I

felt like doing. But most of all, I want to thank my wife,

Donna, whose love, support, and countless sacrifices made my

return to school and the completion of this project possible.















TABLE OF CONTENTS


ACKNOWLEDGEMENTS ................. ........... ............. iii

LIST OF TABLES .. .......................... ..............vi

LIST OF FIGURES ............................................ viii

KEY TO ABBREVIATIONS ............. ......................... ix

ABSTRACT ............ .......................... ..............x

INTRODUCTION ................ .................... .............. 1

Fluorouracil.. .............................................1
Fluorouracil Derivatives................................... 2
Fluorouracil Metabolism .................................... 3
Structure of the Skin.....................................6
Mechanisms of Transdermal Penetration ..................... 8
Passive Diffusion........................................ 10
Enhancement of Skin Penetration. ..........................11
Prodrugs and Skin Penetration............................13
Dermal versus Transdermal Delivery.......................16
Cutaneous Metabolism......................................17
Hairless Mouse Skin as a Model Membrane ..................18
Research Proposal... ......................................20

1-ALKYLOXYCARBONYL DERIVATIVES ............................ 23

Introduction.. .................................... ........ 23
Materials and Methods. .....................................25
Results and Discussion. ....................................35
Summary ................................................... 51

1-ACYL DERIVATIVES ........................................... 53

Introduction.. .................................... ........53
Materials and Methods. .....................................53
Results and Discussion ...................................64
Summary .......................... .........................80

1,3-BIS-ACYL DERIVATIVES ............. ..................... 82

Introduction.. .................................... ........ 82
Materials and Methods. .....................................84
Results and Discussion ...................................93
Summary ................................................... 107











3-ACYL DERIVATIVES ........................................ 109

Introduction ............................................. 109
Materials and Methods.................................... 111
Results and Discussion................................... 120
Summary .................................... ............. 146

SUMMARY AND CONCLUSIONS ................................... 148

LIST OF REFERENCES .........................................153

BIOGRAPHICAL SKETCH .............. ...................... ..160















LIST OF TABLES


Table 1-1. Structures of 5FU and prodrug derivatives
of 5FU .................... .. ........ ................... 21

Table 2-1. Structures of 1-alkyloxycarbonyl
derivatives................... .......................... 24

Table 2-2. Melting points (MP), lipid solubilities
(SIPM), and aqueous solubilities (SAQ) for
1-alkyloxycarbonyl derivatives............................39

Table 2-3. Solubility ratios (SR), partition
coefficients (PC), and hydrophobicity parameters (9)
for 1-alkyloxycarbonyl derivatives...................... 40

Table 2-4. Pseudo-first-order rate constants (k) and
half-lives (tl/2) for hydrolysis of 1-methyloxy-
carbonyl-5FU in 0.05 M phosphate buffer (pH=7.1,
I=0.12) with and without formaldehyde at 32 OC.......... 44

Table 2-5. Fluxes (J), lag times (tL), and skin
accumulation (SA) values for 1-alkyloxycarbonyl
derivatives ............... ............................... 48

Table 2-6. Second application fluxes (J) and lag times
(tL) for 1-alkyloxycarbonyl derivatives................. 49

Table 3-1. Structures of 1-acyl derivatives. .............. 54

Table 3-2. Melting points (MP), lipid solubilities
(SIPM), and aqueous solubilities (SAQ) for 1-acyl
derivatives ............... ..... .......................... 68

Table 3-3. Partition coefficients (PC) and hydropho-
bicity parameters (c) for 1-acyl derivatives ........... 69

Table 3-4. Pseudo-first-order rate constants (k) and
half-lives (tl/2) for hydrolysis of 1-acyl derivatives
in 0.05 M phosphate buffer (pH=7.1, I=0.12) at 32 C .... 71

Table 3-5. Pseudo-first-order rate constants (k) and
half-lives (ti/2) for hydrolysis of l-acetyl-5FU in
0.05 M phosphate buffer (pH=7.1, 1-0.12) with and
without formaldehyde at 32 C. ........................... 72










Table 3-6. Fluxes (J), lag times (tL), and skin
accumulation (SA) values for 1-acyl derivatives .........77

Table 3-7. Second application fluxes (J) and lag times
(tL) for 1-acyl derivatives.............................. 78

Table 4-1. Structures of 1,3-bis-acyl derivatives. ........ 83

Table 4-2. Melting points (MP), lipid solubilities
(SIPM), and aqueous solubilities (SAQ) for 1,3-bis-
acyl derivatives ........................................ 98

Table 4-3. Pseudo-first-order rate constants (k) and
half-lives (tl/2) for hydrolysis of 1,3-bis-acetyl-5FU
in 0.05 M phosphate buffer (pH=7.1, I=0.12) with and
without formaldehyde at 32 C ......................... .. 100

Table 4-4. Fluxes (J), lag times (tL), and skin
accumulation (SA) values for 1,3-bis-acyl
derivatives............................................. 104

Table 4-5. Second application fluxes (J) and lag times
(tL) for 1,3-bis-acyl derivatives........................ 105

Table 5-1. Structures of 3-acyl derivatives. .............. 110

Table 5-2. Melting points (MP), lipid solubilities
(SIPM), and aqueous solubilities (SAQ) for 3-acyl
derivatives............................................. 127

Table 5-3. Solubility ratios (SR), partition
coefficients (PC), and hydrophobicity parameters (E)
for 3-acyl derivatives.................................. 128

Table 5-4. Pseudo-first-order rate constants (k) and
half-lives (t1/2) for hydrolysis of 3-acyl derivatives
in 0.05 M phosphate buffer (pH=7.1, 1=0.12) with and
without formaldehyde at 32 C ............................. 134

Table 5-5. Reaction products formed during hydrolysis of
3-acetyl-5FU in 0.05 M phosphate buffer (pH=7.1,
I=0.12) with formaldehyde at 32 C.. ..................... 135

Table 5-6. Fluxes (J), lag times (tL), and skin
accumulation (SA) values for 3-acyl derivatives......... 142

Table 5-7. Second application fluxes (J) and lag times
(tL) for 3-acyl derivatives............................. 143















LIST OF FIGURES


Figure 2-1. Plots of ln(C) versus time (min) for
hydrolysis of l-methyloxycarbonyl-5FU in 0.05 M
phosphate buffer (pH=7.1, I=0.12) with and without
formaldehyde at 32 OC....................................43

Figure 2-2. Plots of cumulative amount of total 5FU
species that diffused (Lmol) versus time (h) for
compounds 1, 2, 3, and 5FU. ............................ 46

Figure 2-3. Plots of cumulative amount of total 5FU
species that diffused (Imol) versus time (h) for
compounds 4, 5, 6, and 5FU. ............................ 47

Figure 3-1. X-ray structure of l-acetyl-5FU unprimedd). ... 66

Figure 3-2. X-ray structure of l-acetyl-5FU (primed). ..... 67

Figure 3-3. Plot of pseudo-first-order rate constant (k)
versus formaldehyde concentration (M) for hydrolysis
of l-acetyl-5FU in 0.05 M phosphate buffer (pH=7.1,
1-0.12) at 32 OC........................................ 73

Figure 3-4. Plots of cumulative amount of total 5FU
species that diffused (Jnol) versus time (h) for
compounds 7, 8, 9, and 5FU. ............................ 75

Figure 3-5. Plots of cumulative amount of total 5FU
species that diffused (pmol) versus time (h) for
compounds 10, 11, 12, and 5FU ..........................76

Figure 4-1. X-ray structure of 1,3-bis-acetyl-5FU. ........ 95

Figure 4-2. Plots of In(At-Ao) versus time (min) for
hydrolysis of 1,3-bis-acetyl-5FU in 0.05 M phosphate
buffer (pH=7.1, I=0.12) with and without formaldehyde
at 32 OC.............................................. 99

Figure 4-3. Plots of cumulative amount of total 5FU
species that diffused (pmol) versus time (h) for
compounds 13, 14, and 5FU. ............................. 102

Figure 4-4. Plots of cumulative amount of total 5FU
species that diffused (lnmol) versus time (h) for
compounds 15, 16, and 5FU. ............................. 103










Figure 5-1. Possible scheme for thermal decomposition of
3-acetyl-5FU............................................ 123

Figure 5-2. Possible scheme for thermal intramolecular
rearrangement for 3-acetyl-5FU to l-acetyl-5FU...........124

Figure 5-3. Plot of ln(At-A-) versus time (min) for
hydrolysis of 3-acetyl-SFU in 0.05 M phosphate buffer
(pH=7.1, I=0.12) at 32 oC................................130

Figure 5-4. Plot of ln(At-Ao) versus time (min) for
hydrolysis of 3-propionyl-5FU in 0.05 M phosphate
buffer (pH=7.1, I=0.12) at 32 C ........................ 131

Figure 5-5. Plots of ln(C) versus time (min) for
hydrolysis of 3-acetyl-5FU in 0.05 M phosphate buffer
(pH=7.1, I=0.12) with (n=2) and without (n=3)
formaldehyde at 32 OC................................... 132

Figure 5-6. Plots of ln(C) versus time (min) for
hydrolysis of 3-acetyl-5FU in 0.05 M phosphate buffer
(pH=7.1, 1-0.12) at 32 C using actual concentration
(Ct) and concentration corrected for secondary
degradation (Ccorr) .... ...... ... .... ................. 133

Figure 5-7. Possible scheme for reaction of 3-acetyl-5FU
with formaldehyde to form l-acetyloxymethyl-5FU and
3-acetyloxymethyl-5FU ................................... 136

Figure 5-8. Plots of cumulative amount of total 5FU
species that diffused (IUmol) versus time (h) for
compounds 17, 18, and 5FU.............................. 140

Figure 5-9. Plots of cumulative amount of total 5FU
species that diffused (lmol) versus time (h) for
compounds 19, 20, and 5FU.............................. 141















KEY TO ABBREVIATIONS


bs broad singlet

CDC13 chloroform-d

(CD3)2SO dimethylsulfoxide-d6

CH3CN acetonitrile

d doublet

dec decomposition

dist t distorted triplet

m multiple

q quartet

Rf retention factor

t triplet















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


BIOREVERSIBLE DERIVATIVES OF 5-FLUOROURACIL (5FU):
IMPROVING DERMAL AND TRANSDERMAL DELIVERY
WITH PRODRUGS

By

Howard D. Beall

December, 1991




Chairman: Kenneth B. Sloan
Major Department: Medicinal Chemistry


Fluorouracil (5FU) is an antineoplastic agent used

topically for treatment of actinic keratoses, superficial

basal cell carcinomas, psoriasis, and other skin conditions,

but treatment is often ineffective due to its poor skin

penetration properties. Four homologous series of

bioreversible derivatives (prodrugs) of 5FU were synthesized,

characterized, and their ability to penetrate through

transdermall) and accumulate in dermall) the skin was

evaluated. Six 1-alkyloxycarbonyl, six 1-acyl, four 1,3-bis-

acyl, and four 3-acyl derivatives were studied.

Lipid solubility was a minimum of 40 times greater for

the derivatives than for 5FU, and aqueous solubility was

actually higher than 5FU for five derivatives. The










partitioning-based method for determining aqueous solubility

gave reliable values for relative solubility in each

homologous series, and overall, the values correlated well

with conventionally determined solubilities. This was the

first known successful attempt to obtain aqueous solubilities

for chemically unstable prodrugs (e.g., 1-acyl and 1,3-bis-

acyl derivatives).

Hydrolysis rates for N1-substituents followed pseudo-

first-order kinetics, but plots for N3-acyl hydrolysis were

biexponential. An 02-acyl intermediate was proposed to

account for the unusual hydrolysis and thermal decomposition

of the N3-acyl group. X-ray crystal analysis showed that the

N3-acyl group was oriented perpendicular to the 5FU ring and

was sterically and electronically hindered from nucleophilic

attack.

Skin penetration was 1.2 to nearly 40 times greater for

the prodrugs than for 5FU. The highest flux was recorded for

l-acetyl-5FU, whereas 3-propionyl-5FU, 1-ethyloxycarbonyl-

5FU, and 1,3-bis-acetyl-5FU exhibited the highest rates of

delivery for their respective series. These derivatives were

also the most aqueous soluble members of each series. This

demonstrates the importance of biphasic solubility for

achieving optimal transdermal delivery of 5FU from a

homologous series of prodrugs.

Skin accumulation was highest for l-acetyl-5FU and

l-propionyl-5FU (>18 times more than 5FU). Rapid hydrolysis

of the 1-acyl series upon partitioning into the skin may


xii










cause retention of highly polar 5FU in the more lipophilic

regions of the epidermis.

Skin damage was only slightly increased with some

prodrugs, all derivatives were stable in formulation, and

overall, l-acetyl-5FU was the best candidate for improving

dermal or transdermal delivery of 5FU.















CHAPTER 1
INTRODUCTION


Fluorouracil


Fluorouracil (5FU) is one of the most widely used and

studied2 anticancer agents. It is used in the palliative

treatment of solid tumors and is most effective in

combination with other antineoplastic agents3 and in

conjunction with radiation therapy.2 It has been used to

treat carcinoma of the colon, rectum, breast, and stomach

and, with less effectiveness, carcinoma of the ovary, cervix,

urinary bladder, liver, and pancreas.4

Fluorouracil (5FU) is used as a single agent in topical

preparations and is effective in treating actinic (solar)

keratoses,5-8 superficial basal cell carcinomas,9-10 and

psoriasis.11-13 Topical 5FU has also been used to treat a

variety of other precancerous conditions, malignant and

benign tumors, and dermatoses.14 Commercial 5FU creams and

solutions may be adequate for treating lesions on the face,

but other areas, especially the forearms and hands, respond

poorly probably due to lack of penetration by 5FU.15 Some

conditions, such as psoriasis, respond to topical 5FU therapy

when the drug is applied under an occlusive dressing.13









However, this is neither the most convenient nor comfortable

treatment option.

The toxicity of 5FU is related to its effect on rapidly

proliferating host cells especially those of the bone marrow

and gastrointestinal lining. Toxicity following systemic

therapy is common while adverse effects from topical therapy

appear to be minimal, other than those associated with the

local inflammatory reactions usually necessary for a

therapeutic response.4


Fluorouracil Derivatives


Since 5FU was first synthesized16 and tested17 as an

antitumor antimetabolite in 1957, numerous attempts have been

made to improve its efficacy and reduce its toxicity.

Derivatives of 5FU and its nucleosides, 5-fluorouridine (FUR)

and 5-fluoro-2'-deoxyuridine (FdUR), have been developed with

this in mind. The majority of these derivatives have been

designed with the expectation that 5FU will be released in

vivo, so they are essentially prodrugs.

A prodrug has been defined as "an agent which must

undergo chemical or enzymatic transformation to the active or

parent drug after administration, so that the metabolic

product or parent drug can subsequently exhibit the desired

pharmacological response" (p. 1275).18 Prodrugs, or

bioreversible derivatives, of 5FU have included hydroxy-

methyl,19 alkyloxyalkyl,20 acyloxyalkyl,21-24 acyl,25-28










alkyloxycarbonyl,25,29-30 alkyloxycarbonyloxyalkyl,31-33 alkyl-

carbamoyl,34-37 aminoalkyl (Mannich base),38-39 phthalid-

yl,40-41 and derivatives containing sulfur rather than oxygen

in some of the above functional groups.42-43 Polymer44-45 and

peptide46 derivatives have also been proposed as sources of

5FU in vivo. Currently, at least two bioreversible

derivatives of 5FU, 1-(tetrahydrofuran-2-yl)-5-fluorouracil47

and l-hexylcarbamoyl-5-fluorouracil,48 have been marketed in

Japan.

The above is just a small sample of the synthetic

literature involving 5FU. In addition, a large number of

studies have been devoted to synthesizing analogs of FUR and

FdUR containing an N1-glycosidic linkage. FdUR, or

floxuridine, is available in the United States, but is only

approved for intraarterial infusion.4 It appears to offer no

advantage over intravenous 5FU, and this therapy is both

hazardous and expensive.3 The nucleosides and their analogs

are converted to 5FU in vivo by a pyrimidine phosphorylase.

Since phosphorylase activity is higher in tumor tissue, it is

believed that development of compounds that are good

substrates for phosphorylase may lead to greater tumor

specificity and decreased toxicity.49


Fluorouracil Metabolism


Fluorouracil (5FU) undergoes extensive catabolic and

anabolic metabolism. Approximately 15% of a single










intravenous dose of 5FU is excreted unchanged in the urine

within six hours, and 90% of that is detected within the

first hour.4 Approximately 80% of the dose is metabolized by

the liver and extrahepatic tissues.1 The principal

catabolite of 5FU is a-fluoro-P-alanine (FBAL), which

accounts for over 95% of the catabolic products found in the

urine. Bile acid conjugates of FBAL represent the major

biliary catabolites of 5FU.1

A number of anabolic pathways have been characterized

for 5FU, and one or more of them may be responsible for the

activation and subsequent cytotoxicity of the drug. The most

direct activation pathway utilizes orotidine monophosphate

phosphoribosyl transferase (OMPT) to form 5-fluorouridine-5'-

monophosphate (FUMP) from 5FU and 5-phosphoribosyl-l-pyro-

phosphate. FUMP can also be generated in two steps with

uridine phosphorylase and uridine kinase. With two more

kinases 5-fluorouridine-5'-triphosphate (FUTP) is formed

which can be incorporated into ribonucleic acid (RNA) leading

to RNA dysfunction.1 Some cell lines appear to favor the

OMPT pathway, while others favor the uridine phosphorylase

pathway, and the FUTP formed from each is added to different

fractions of RNA.50

Some of the 5-fluorouridine-5'-diphosphate (FUDP) that

is generated above as an intermediate can become a substrate

for ribonucleotide reductase. The resulting deoxynucleotide,

5-fluoro-2'-deoxyuridine-5'-diphosphate (FdUDP), can form

5-fluoro-2'-deoxyuridine-5'-triphosphate (FdUTP) using










another kinase. At this point FdUTP can be incorporated into

deoxyribonucleic acid (DNA), but the contribution to 5FU

cytotoxicity by this mechanism is unclear. DNA protective

enzymes, dUTPase and uracil-DNA glycosylase, which are

responsible for keeping uracil residues out of DNA, target

5FU residues as well.50

A diphosphohydrolase converts FdUTP to 5-fluoro-2'-

deoxyuridine-5'-monophosphate (FdUMP). FdUMP can also be

formed from 5FU by thymidine phosphorylase and thymidine

kinase, although this may be quantitatively less important

than other activation steps.1 FdUMP binds covalently to

thymidylate synthase and its cofactor, 5,10-methylene

tetrahydrofolate, thereby preventing synthesis of thymidine-

5'-monophosphate (dTMP) and subsequently, thymidine-5'-

triphosphate (dTTP), an essential component in DNA synthesis.

FdUMP has a greater affinity for thymidylate synthase than

the normal substrate, 2'-deoxyuridine-5'-monophosphate

(dUMP).1

Either inhibition of thymidylate synthase by FdUMP or

incorporation of FUTP into RNA can lead to cell death. The

mechanism that is most important depends on the cell line

being studied.51

Fluorouracil (5FU) may also affect glycoprotein and

glycolipid metabolism, since it is known that FUDP sugars can

be formed.50 The possibility that membrane effects from

altered glycoprotein synthesis could lead to cytotoxicity has

been noted, but has not been well studied.1











Structure of the Skin


Fluorouracil (5FU) is typical of polar, high-melting,

heterocyclic compounds which exhibit poor skin permeability.38

Since the goal of this project is to improve dermal and

transdermal delivery of 5FU, it is necessary to examine the

structure of the barrier to be penetrated, the skin.

The skin is composed of two major tissue layers, the

epidermis and dermis. The epidermis is a continuous, elastic

sheet that is interrupted only by glandular pores and hair

follicles. It consists of four definable sublayers and

averages 75 to 150 gm in thickness. The basal cell layer,

which borders the dermis, is a single layer of keratinocytes.

This is the germinal layer, and all epidermal cells are

initially formed here before moving outward to the next

layer, the stratum spinosum. The basal cells are cuboidal or

columnar in shape, while the stratum spinosum cells are

polyhedral. By the time the cells have migrated to the next

layer, the stratum granulosum, they have become flattened and

contain characteristic keratohyalin granules. The stratum

granulosum marks the transition between nucleated cells and

the anucleated stratum corneum. The stratum corneum cells in

the last layer are highly keratinized, markedly flattened

cells in which cellular components, such as mitochondria and

ribosomes, have degraded along with the nucleus. Stratum

corneum normally consists of about 15 to 20 layers, but each










layer is only about 0.5 pm in thickness. The outer layers

are continuously desquamated and replaced from below. The

entire transit time from basal layer to desquamation is 26 to

42 days. The cells of the epidermis are attached to each

other by desmosomes, which degrade just prior to

desquamation.52

Below the epidermis lies the dermis which constitutes

the bulk of the approximately 2 mm thickness of human skin.

The two layers are anchored to the basal lamina by various

fibrils and microfibrils. The attachment is enhanced by the

interlocking nature of the junction.52

The dermis consists of two regions, the papillary dermis

and reticular dermis. The papillary dermis is the thinner,

outermost region that is molded against the ridges and

grooves of the epidermis. It contains small, loosely

distributed fibrils and encloses the microcirculatory blood

and lymph vessels. In contrast, the collagen bundles and

elastin fibers of the reticular dermis are more densely

packed, and this region is relatively acellular and

avascular. Collagen is the major component of the dermis,

and it gives the skin its tensile strength. The dermis also

contains nerves, excretory and secretary glands, and hair

follicles.52











Mechanisms of Transdermal Penetration


For most substances, the stratum corneum provides the

rate-limiting barrier to skin penetration.53-55 The stratum

corneum is 75-85% protein (dry weight),54 mostly intracellular

keratin, while the intercellular space is a lipid-enriched

region. During the outward migration of the keratinocytes

through the epidermis, lamellar bodies are synthesized, which

contain lipids, polysaccharides, and hydrolytic enzymes. As

the granular cells prepare to enter the stratum corneum, the

lamellar bodies move to the cell periphery and empty their

contents into the spaces between the cells.56 The corneocytes

and intercellular lipids are arranged in a brick and mortar

fashion,57 and the result is a very dense (1.4 g/cm3 in the

dry state),58 highly efficient moisture barrier.

In order for a substance to penetrate the stratum

corneum, it must either (1) cross through the densely packed

corneocytes, (2) traverse through the intercellular region,

thereby avoiding the keratinized cells, or (3) bypass the

stratum corneum completely by diffusing through shunt

pathways such as sweat ducts and hair follicles.53 The shunt

pathways are not considered to be significant, especially

when steady state diffusion has been attained,59 and these

pathways are generally disregarded in discussions of

penetration mechanisms.










Until recently, it was thought that the transcellular

pathway was the primary route through the stratum corneum.

This was based, at least in part, on a gross underestimation

of the volume of the intercellular space.60 The current

belief is that passive diffusion occurs predominantly through

the intercellular channels.61-62 While this seems logical for

lipophilic compounds, this pathway has also been shown to be

the dominant route for mercuric chloride, an ionic compound.

Bodd6 et al.63 showed that mercuric sulfide could be

precipitated in the stratum corneum after topical application

of mercuric chloride. They found that mercuric cation

accumulates initially in the intercellular spaces throughout

the stratum corneum. Intracellular uptake was observed

later, but only in the apical corneocytes by way of

disintegrating desmosomal attachments. Since the

intercellular lipids are arranged in multiple lamellar

bilayers,64 the mercuric cation, and possibly other ions and

hydrophilic molecules, may diffuse through the interlamellar,

hydrophilic channels that are associated with the polar

headgroups of the lipids.63

While most of the current research is directed toward

understanding the intercellular region, Barry59 cautions that

the transcellular route should not be dismissed as

unimportant, especially in penetration enhancer research.











Passive Diffusion


Although the skin is not a homogeneous tissue, it has

the characteristics of a passive diffusion barrier. Fick's

first law of diffusion defines flux as the amount of material

flowing through a unit cross section of a barrier in a unit

time.65 Flux is also proportional to the concentration

gradient, and the first law can be written:

J = -D-dC/dx (1)

where J is the flux, D is the diffusion coefficient of the

penetrant in the barrier, and dC/dx is the concentration

gradient. While the first law is used to describe steady-

state diffusion, Fick's second law describes the change in

concentration with respect to time at a specific location.65

Our interest is in steady-state diffusion, and the second law

will not be discussed further.

The concentration gradient term (dC/dx) from Fick's

first law can be approximated by (C1-C2)/h, where C1 is the

concentration in the barrier on the donor side of the

barrier, while C2 is the concentration in the barrier on the

receptor side, and h is the thickness of the diffusional

barrier. Therefore, the expression becomes

J = D-(CI-C2)/h (2)

For skin penetration studies, "sink" conditions can be

assumed which means that C20O, and

J = D-C1/h (3)










Since C1 is essentially the concentration in the first

layer(s) of skin, C1 will now be referred to as Cs, so

J = D-Cs/h (4)

While Cs is not usually a known quantity, it can be

represented instead by the product of the penetrant

concentration in the donor vehicle (Cv) and the

membrane-vehicle partition coefficient (Km = Cs/Cv), and the

expression now becomes

J = D-Km-Cv/h (5)

This expanded form of Fick's first law is often cited and has

been verified by data from numerous skin penetration

studies.66

Another useful measure of skin penetration is the

permeability coefficient (P). This is simply the

concentration-normalized flux which is expressed:

P = J/Cv = D-Km/h (6)

Permeability coefficients are often used when comparing a

single penetrant in a series of vehicles or when studying an

homologous series of penetrants.


Enhancement of Skin Penetration


The driving force for skin penetration is the membrane-

vehicle partition coefficient (Km). If a penetrant is

delivered in a saturated solution in the presence of excess

solid, then the chemical potential, or escaping tendency, is

maximized, and the actual concentration in the vehicle (Cv)










is irrelevant. Since the membrane thickness (h) and

diffusion coefficient (D) remain relatively constant (D is

inversely proportional to the cube root of the molar volume

according to the Stokes-Einstein equation), Km is the only

variable that can substantially influence the flux.

There are several general approaches for enhancing skin

penetration. Four of these--occlusion, use of penetration

enhancers, iontophoresis, and sonophoresis--produce their

enhancement effects by changing the barrier properties of the

skin. Occlusion involves covering the application site,

impeding transepidermal water loss, and increasing the

hydration state of the skin.67 With penetration enhancers,

accelerants, or promoters in topical formulations, the

reversible reduction of barrier resistance in the stratum

corneum is the goal, and ideally, incorporation of the

enhancer into the skin will not result in cell damage.59

Iontophoresis is a technique in which electroosmotic volume

flow from an applied electric field leads to increases in

mass transfer in excess of passive diffusion.68 Sonophoresis

uses ultrasonic frequencies to increase skin penetration.

The other two methods, formulation (without penetration

enhancers) and the use of prodrugs, do not disrupt the

barrier layer. Essentially, the formulation approach

involves changing the penetrants solubility in the vehicle by

changing the vehicle. The effect on flux of this approach is

indeterminate according to equation (5). If the solubility

in the vehicle (Cv) is increased, then the partition










coefficient (Km) decreases and vice versa. In a series of

papers,69-72 Sloan and coworkers suggested that a parabolic

relationship exists for log Km and log P when they are

plotted against vehicle polarity. Log Km and log P reach a

minimum where vehicle polarity is equal to penetrant polarity

or in other words, where penetrant solubility in the vehicle

is the greatest. While this relationship might be expected,

a more interesting finding was that in most instances fluxes

are also lower from those vehicles in which the penetrants

are most soluble. While a formulation approach may permit

some improvement in skin penetration values, the maximum

achievable levels appear to be limited.

The final method for enhancing skin penetration is the

prodrug approach. This is the only method in which a

substantial increase in Km can be realized, and according to

equation (5), this should translate into substantially

improved skin penetration. It was this knowledge that led to

adoption of the prodrug method for the current project.


Prodrugs and Skin Penetration


The term, prodrug, was defined earlier in this chapter.

Prodrugs, bioreversible derivatives, or latentiated drugs are

simply compounds which undergo biotransformation before

producing their pharmacological effects.73

Generally, prodrugs are designed to overcome some kind

of barrier to a drug's usefulness. These barriers can










include (1) premature metabolism prior to reaching the active

site, (2) too rapid absorption and distribution when

prolonged action is needed, (3) toxicity associated with

(a) local irritation or (b) distribution to tissues other

than the target site, (4) poor site specificity leading to

subtherapeutic levels at the target site, and (5) generally

poor physical chemical properties resulting in (a) solubility

problems in the dosage form or (b) poor absorption across

biological membranes such as the blood brain barrier,

gastrointestinal lining, or skin.74 Obviously, it is the

latter problem that is addressed in the current project.

The idea that improving skin penetration is a solubility

problem has been documented in this chapter, and the

importance of biphasic (lipid and aqueous) solubility is well

recognized.66,75-76 In a recent review of prodrug approaches

for improving dermal delivery, numerous literature examples

are cited in which both solubility and skin penetration data

are presented.77 The evidence strongly suggests that:

although an increase in lipid solubility due to
transient masking of a polar functional group almost
always results in enhanced dermal delivery of the parent
drug, in order to optimize delivery, it is necessary to
use the members of the (homologous) series (of prodrugs)
that are more water soluble than the parent drug or that
are the more water-soluble members) of the series
(p. 68).77

Derivatization to improve solubility characteristics of

polar heterocycles with amide and/or imide functional groups

is relatively easy to accomplish. For example, successive

methylation of uracil at the N1- and N3-positions, while










obviously improving lipid solubility, also increases water

solubility from 3 mg/mL for uracil to 500 mg/mL for 1,3-di-

methyluracil even though the methyl group is hydrophobic

itself. Melting points are also decreased as the two

intermolecular hydrogen-bonding N-H sites are masked.78 Since

alkyl groups such as methyl are stable and therefore not

bioreversible, they are not candidates to function as prodrug

promoieties. However, many other derivatives, such as those

cited earlier, when linked to the N1- or N3-site of 5FU, do

qualify as promoieties and could potentially give 5FU the

improved solubility characteristics necessary for enhanced

skin penetration.

Of the many studies devoted to making bioreversible

derivatives of 5FU, only a few have been directed at

improving its topical delivery. Mollgaard et al.21 looked at

two 1-acyloxymethyl derivatives of 5FU. One of the compounds

delivered 5FU more readily than 5FU itself through excised

human skin. Both compounds showed greatly increased lipid

solubilities with only slightly reduced water solubilities.

Hydrolysis of these derivatives was attributed to cutaneous

metabolism by hydrolytic enzymes.

Three 1,3-bis-aminomethyl (Mannich base) derivatives of

5FU were prepared by Sloan and coworkers,38-39 and their

topical delivery was studied using hairless mouse skin.

Solubilities in lipid and aqueous phases were substantially

increased as were the rates of delivery through the skin of

5FU from these prodrugs. Due to the instability of these










compounds in water, no enzymatic activation was necessary to

release the parent compound. In a later report,79 one of

these prodrugs was compared with a number of 5FU formulations

including four commercially available creams and solutions.

The prodrug outperformed the formulations by a minimum of

four times in terms of 5FU delivered through hairless mouse

skin.

Sasaki et al.37 studied the delivery of 1-alkylcarbamoyl

derivatives of 5FU through rat skin. All three derivatives

(butyl, hexyl, and octyl) were more effective in delivering

5FU than 5FU itself. The lipid solubilities of the three

derivatives were comparable with each other and much higher

than 5FU. The aqueous solubilities, while less than 5FU,

showed an order of magnitude decrease between each derivative

beginning with the butyl derivative. Interestingly, the best

performing compound was the least lipid-soluble and most

water-soluble derivative, the butyl derivative.


Dermal versus Transdermal Delivery


It is important to make a distinction between dermal and

transdermal delivery. Most in vitro skin penetration testing

is done with excised skin to which a drug in a formulation is

applied on the donor side and samples are removed from the

receptor side. These experiments give information on

transdermal rates of delivery.










Topical 5FU works on afflicted cells in the epidermal

region of the skin. This is considered dermal delivery.

Transdermal techniques provide valuable information for

dermally targeted drugs since they indicate how effectively

the drugs penetrate the barrier layer of the skin. Other

experiments can be done to augment the transdermal results

such as measuring accumulation of the drug in the skin.

The correlation between transdermal delivery rates and

epidermal uptake was studied by Sloan et al.79-80 Incorpora-

tion of 3H-deoxyuridine into epidermal DNA of live hairless

mice was quantitated by scintillation counting following

application of various 5FU formulations and a 5FU prodrug.

The prodrug, which had the highest in vitro transdermal flux,

was also the most effective at inhibiting epidermal DNA

synthesis in vivo. The correlation was also good among the

formulations.


Cutaneous Metabolism


When a prodrug is applied topically and targeted for a

dermal site, it is essential that the parent drug is released

before the prodrug leaves the epidermal region. If a prodrug

has a relatively short half-life under physiological

conditions, such as the aforementioned Mannich-base prodrugs

of 5FU, then release of the parent drug will probably occur

either prior to or during its transit through the viable

epidermis. However, if a compound depends on enzymatic











rather than chemical activation, then an appropriate

cutaneous enzyme must be present.

The ability of the skin to metabolize drugs and other

foreign compounds is well known.81-82 Phase I reactions

(oxidation, reduction, and hydrolysis) and phase II

conjugations are known to occur. Drug-metabolizing enzymes

are distributed in all layers of the skin and the appendages.

Of particular interest in prodrug chemistry is the

presence of nonspecific esterases in the skin.82 The

predictable metabolism of esters by these hydrolytic enzymes

makes this functional group a popular choice for prodrug

synthesis.


Hairless Mouse Skin as a Model Membrane


A number of animal skins have been suggested as model

membranes for skin penetration studies. The two most common

models for in vitro diffusion studies are human and hairless

mouse skin. While the advantages of human skin are obvious,

there are also disadvantages. Human skin can be difficult to

obtain and store,83 it can be expensive,83 and it is known to

have high barrier variability.83-84 Factors such as age,

diet, and disease state may not be well controlled with human

skin.85 Hairless mouse skin, on the other hand, is easily

obtained and prepared, and other factors can ordinarily be

controlled.









The importance of hairless mouse skin for in vitro

experimentation has been noted86 despite its generally greater

permeability when compared to human skin. It has been

suggested that this difference may actually be an asset in

that small changes in permeability will be amplified in the

mouse skin model.86

A major criticism of hairless mouse skin involves its

ability to withstand the effects of hydration,87-88 a

necessary condition for controlled in vitro diffusion

experiments. Permeability increases as a function of

hydration time have been attributed to breakdown of the

stratum corneum barrier in these studies. A recent report,89

however, suggested that the absence of an adequate

preservative in the receptor phase may actually be

responsible for breakdown of the skin. Skin penetration data

was collected for delivery of theophylline from a propylene

glycol vehicle following skin hydration periods ranging from

4 to 120 hours. It was found that increased theophylline

flux and loss of barrier function corresponded to the

presence of microbial growth in the receptor phase. When

microbial growth was completely inhibited, fluxes were

essentially constant for all hydration periods.89 Finally,

with regard to hydration, Scheuplein and Ross noted that

"even well-hydrated stratum corneum preferentially dissolves

lipid-soluble molecules, so that the selective permeability

of these molecules is preserved" (p. 353).90










Another difference between human and hairless mouse skin

could be significant in prodrug design. Rat and mouse skins

apparently have higher levels of enzymatic activity for drug

metabolism than human skin. Esterase activity, specifically,

appears to be low in human skin.54


Research Proposal


The overall goal of the present research is to develop

prodrug derivatives of 5FU with solubility characteristics

and skin penetration properties superior to 5FU itself. The

derivatives should be stable in formulation, but should

readily convert to 5FU in the skin since the purpose of

topical 5FU therapy is to deliver 5FU dermally, not

transdermally. In pursuit of this goal, it is hoped that

experimental data will be obtained which supports a

solubility-based method for designing prodrugs with optimized

topical delivery characteristics. The objectives for meeting

this goal are to:

1) synthesize several homologous series of acylated
derivatives of 5FU,

2) verify structures by melting point, elemental
analysis (novel compounds), and standard spectral
techniques,

3) determine physical chemical properties such as lipid
and aqueous solubilities, partition coefficients, and
rates of hydrolysis, and

4) determine skin penetration parameters and quantitate
skin accumulation using hairless mouse skin as the model
membrane.











Table 1-1. Structures of 5FU and prodrug derivatives of 5FU.






O O
HF R F





H Ri






Series R1 R2

l-alkyloxycarbonyl-5FU (I) -(C=O)O(CH2)nCH3 -H
l-acyl-5FU (II) -(C=O) (CH2)nCH3 -H
1,3-bis-acyl-5FU (III) -(C=0) (CH2)nCH3 -(C=0) (CH2)nCH3
3-acyl-5FU (IV) -H -(C=0)(CH2)nCH3










Four series of compounds have been selected as potential

prodrugs of 5FU. They are the l-alkyloxycarbonyl (I), 1-acyl

(II), 1,3-bis-acyl (III), and 3-acyl (IV) derivatives of 5FU

(Table 1-1). Fluorouracil (5FU) has two acidic pKa values,

8.0 and 13.0.91 Spectral studies have shown that the

monoanion is actually a mixture of N1- and N3-anions.30 A

comparison of ionization constants for 5FU derivatives that

are identically substituted at the N1- or N3-positions22,30

suggests that the N3-position is probably the most acidic.

However, the N1-position is the most reactive site for both

synthesis and hydrolysis of acylated derivatives of 5FU.

The three series of acyl derivatives were chosen for two

reasons; they exhibit high lipid and aqueous solubilities,

and they readily hydrolyze without enzymatic activation.26

While rapid hydrolysis is seen as a drawback by some

investigators,26,42 it may actually be an advantage when

developing prodrugs for dermal delivery.38-39,92 The

l-alkyloxycarbonyl series was selected for comparison with

the 1-acyl series since it shows good chemical stability, but

rapid enzymatic hydrolysis.30 Various members of each series

have been synthesized previously,25-27,30,93 but homologous

series have not been examined, and nobody has studied their

applicability for dermal delivery.















CHAPTER 2
1-ALKYLOXYCARBONYL DERIVATIVES


Introduction


Alkyloxycarbonyl derivatives of 5-fluorouracil (5FU)

have previously been studied as potential sources of 5FU in

viva.25,29-30 When substitution is at the N3-position of 5FU,

the derivatives are chemically stable. Their hydrolyses in

human plasma and liver homogenate are also slow enough to

raise questions about their usefulness as prodrugs for the

oral or rectal delivery of 5FU.29 Thus, there is no doubt

that they are too stable to serve as prodrugs for dermal

delivery. On the other hand, substitution at the N1-position

produces compounds that are relatively stable chemically,30

but which are sufficiently labile in the presence of enzymes30

to justify consideration as dermal prodrugs.

Six straight-chain 1-alkyloxycarbonyl derivatives were

selected for study. The derivatives and their structures are

shown in Table 2-1.











Table 2-1. Structures of 1-alkyloxycarbonyl derivatives.







O
H F








RO 0






Compound R

l-methyloxycarbonyl-5FU (1) -CH3
l-ethyloxycarbonyl-5FU (2) -CH2CH3
l-propyloxycarbonyl-5FU (3) -(CH2)2CH3
l-butyloxycarbonyl-5FU (4) -(CH2)3CH3
l-hexyloxycarbonyl-5FU (5) -(CH2)5CH3
l-octyloxycarbonyl-5FU (6) -(CH2)7CH3











Materials and Methods



Synthesis


Melting points (mp) were determined with a Thomas-Hoover

capillary melting point apparatus and are uncorrected.

Elemental microanalyses were obtained for all novel compounds

through Atlantic Microlab, Incorporated in Norcross, Georgia.

Proton nuclear magnetic resonance (1H NMR) spectra were

obtained at 90 MHz on a Varian EM-390 spectrometer. Chemical

shifts (6) are reported in parts per million (ppm) from the

internal standard, tetramethylsilane (TMS). Coupling

constants (J) are expressed in cycles per second (Hz).

Infrared (IR) spectra were recorded with a Perkin-Elmer 1420

spectrophotometer and absorbances are reported in cm-.

Ultraviolet (UV) spectra were obtained with a Cary 210 or

Shimadzu UV-265 spectrophotometer. Maximum absorbances are

reported in nm along with the molar absorptivities (E) in

L/mol.

l-Alkyloxvcarbonyl-5-fluorouracil (general procedure)

To 0.66 g (0.01 mol) of 85% potassium hydroxide

dissolved in methanol (20-50 mL) was added 1.33 g of 5FU

(0.0101 mol). Slightly more than an equivalent of 5FU was

used to prevent excess base from catalyzing the aldol

condensation of acetone. When present, condensation products

complicated product isolation. The methanol suspension was









stirred for 30 minutes, and the methanol was evaporated under

reduced pressure. The potassium salt was suspended in

acetone (25-50 mL) which was evaporated under reduced

pressure to remove residual methanol. The salt was

resuspended in acetone (25-50 mL), and the suspension was

added dropwise over a three minute period to a well stirred

acetone (20 mL) solution containing 1.0 to 1.2 equivalents of

the appropriate alkyl chloroformate. The mixture was stirred

at room temperature for 60 minutes. The mixture was

filtered, and the residue was washed with acetone (20 mL).

The combined acetone solutions were evaporated under reduced

pressure, and the solid residue was crystallized from an

appropriate solvent or solvent combination.1

l-Methyloxycarbonyl-5-fluorouracil (1)

Crystallization from acetone gave 1.36 g of 1 (72%):

mp 158-60 *C (lit.30 mp 159-60 OC); IR (KBr) 1695, 1710, 1740,

and 1760 cm-1 (C=O); 1H NMR [(CD3)2SO] 6 3.86 (s, 3H, C13) and

8.16 (d, J=7 Hz, 1H, C6-f); UVax (CH3CN) 254 nm (E=9.63x103).

l-Ethyloxycarbonyl-5-fluorouracil (2)

Crystallization from acetone/ether gave 1.31 g of 2

(65%): mp 127-8 OC (lit.30 mp 126-8 OC); IR (KBr) 1690, 1730,

and 1750 cm-1 (C=O); 1H NMR [(CD3)2SO] 8 1.31 (t, J=7 Hz, 3H,

Ca3), 4.31 (q, J=7 Hz, 2H, OCH2), and 8.16 (d, J=7 Hz, 1H,
C6-H); UVmax (CH3CN) 254 nm (e=9.86x103).




1Several compounds in this series were provided by Kenneth B.
Sloan, Ph. D. based on the author's procedure.









l-Propyloxycarbonyl-5-fluorouracil (3)

Crystallization from acetone/ether gave 1.37 g of 3

(64%): mp 124-6 oC; IR (KBr) 1690, 1730, and 1755 cm-1 (C=O);

1H NMR [(CD3)2SO] 8 0.95 (t, J=7 Hz, 3H, Cfl3), 1.70 (m, 2H,

OCH2Cf2), 4.23 (t, J=7 Hz, 2H, OCl2), and 8.15 (d, J=7 Hz, 1H,

C6-) ; UV max (CH3CN) 254 nm (E=1.001x104).

Anal. Calc. for C8H9FN204: C, 44.45; H, 4.20; N, 12.96.

Found: C, 44.53; H, 4.23; N, 12.89.

l-Butyloxycarbonyl-5-fluorouracil (4)

Crystallization from dichloromethane/hexane gave 1.33 g

of 4 (58%): mp 97-8 oC (lit.30 mp 102-4 oC); IR (KBr) 1695,

1735, and 1765 cm-1 (C=O); 1H NMR [(CD3)2SO] 8 0.91 (t,

J=7 Hz, 3H, Cl13), 1.3-1.8 (m, 4H, OCH2Cl2Cfl2), 4.27 (t, J=6

Hz, 2H, OCfI2), and 8.13 (d, J=7 Hz, 1H, C6-Ij); UVmax (CH3CN)

254 nm (e=9.93x103).

l-Hexyloxycarbonyl-5-fluorouracil (5)

Crystallization from dichloromethane/hexane gave 1.27 g

of 5 (49%): mp 66-7 C (lit.93 mp 68-9 OC); IR (KBr) 1690,

1730, and 1750 cm-1 (C-O); 1H NMR [(CD3)2SO] 8 0.87 (distd t,

3H, Ca3), 1.1-1.8 (m, 8H, OCH2CIH2C2CHi2CH22), 4.26 (t, J=6 Hz,

2H, OCIH2), and 8.13 (d, J=7 Hz, 1H, C6-Hi); UVmax (CH3CN) 254

nm (e=1.004x104).

l-Octyloxvcarbonyl-5-fluorouracil (6)

Crystallization from dichloromethane/hexane gave 1.80 g

of 6 (61%): mp 97-8 oC; IR (KBr) 1690, 1730, and 1750 cm-1

(C=O); 1H NMR [(CD3)2SO] 8 0.87 (distd t, 3H, CJ3), 1.2-1.9









(m, 12H, OCH2CIj2Ca2Cni2CL2Ci2Ca2), 4.27 (t, J=6 Hz, 2H, OCf2),

and 8.15 (d, J=7 Hz, 1H, C6-i) ; UVmax (CH3CN) 254 nm

(e=1.009x104).

Anal. Calc. for C13H19FN204: C, 54.53; H, 6.69; N, 9.79.

Found: C, 54.46; H, 6.73; N, 9.77.


Lipid Solubility


Lipid solubilities were determined using isopropyl

myristate (IPM), a commercial vehicle used in cosmetics and

topical medicinals,94 as the lipid solvent. The use of IPM as

a model lipophilic vehicle in skin penetration studies is

well established.77,95

Three suspensions of each derivative were stirred at

221 OC for 48 hours. The suspensions were filtered through

0.45 gm nylon filters, and the saturated solutions were

diluted in acetonitrile and analyzed by UV spectroscopy.

Solubilities were calculated using Beer's Law:

A = E-C-d (1)

where A is the absorbance, E is the molar absorptivity, C is

the concentration in mol/L, and d is the path length of the

cuvette in cm. Molar absorptivities were predetermined in

triplicate in acetonitrile at 254 nm.


Aqueous Solubility


For direct measurement of aqueous solubilities, three

suspensions of each derivative were vigorously stirred in










0.05 M acetate buffer (pH=4.0) at 221 oC for 60 minutes.

The suspensions were filtered through 0.45 pLm nylon filters,

and the saturated solutions were diluted in acetonitrile and

analyzed by UV spectroscopy. Solubilities were calculated

using Beer's Law as previously described.


Partition Coefficients


The partitioning-based method for determining aqueous

solubility utilized the saturated IPM solutions from the

lipid solubility study. For most compounds, equal volumes

(1 mL) of saturated IPM solution and 0.05 M acetate buffer

(pH=4.0) were used. The use of equal or near-equal phase

volumes is known to facilitate rapid equilibrium.96 The two

phases were mixed thoroughly for ten seconds and allowed to

separate for 60 seconds. A preliminary study showed that

there was virtually no difference in partition coefficient

(PC) values when partitioning was carried out for 10, 20, or

30 seconds (see Chapter 3). The IPM layers were diluted in

acetonitrile and analyzed by UV spectroscopy. The IPM-buffer

partition coefficients were calculated as follows:

PC = Aafter/(Abefore-Aafter) VAQ/VIPM (2)

where Aafter is the absorbance from the IPM layer after

partitioning, Abefore is the absorbance from the IPM layer

before partitioning, VAQ is the volume of the aqueous phase,

and VIPM is the volume of the IPM phase. Estimated aqueous









solubilities (SAQ) were calculated from the IPM solubility

(SIpM) and the partition coefficient:

SAQ = SIPM/PC (3)

Partitioning was carried out in triplicate for a fixed volume

ratio for each derivative. For those compounds with large

differences in solubility in one phase relative to the other,

volume ratios (IPM:buffer) other than 1:1 were necessary, but

the ratio never exceeded 10:1 or 1:10.


Hvdrolysis Kinetics


Hydrolysis rates have previously been reported for

several members of this homologous series.30,93 In the

present study, hydrolysis rates were determined at 32 C for

l-methyloxycarbonyl-5FU (1) in 0.05 M phosphate buffer

(pH=7.1, 1=0.12) and in the same buffer with 0.11%

formaldehyde (3.6x10-2 M). The rate in the presence of

formaldehyde was determined for comparison with the rate in

plain buffer since formaldehyde was used as a preservative in

the diffusion cell experiments described in the following

section.

The hydrolyses were followed by high performance liquid

chromatography (HPLC). The HPLC system consisted of a

Beckman model 110A pump with a model 153 UV detector, a

Rheodyne 7125 injector with a 20 Im loop, and a Hewlett-

Packard 3392A integrator. The column was a Lichrosorb RP-8

10 pm reversed-phase column, 250 mm x 4.6 mm (inside










diameter). The mobile phase contained 10% methanol and 90%

0.025 M acetate buffer (pH=5.0) and was run at 1.0 mL/min.

The column effluent was monitored at 254 nm, and quantitation

was based on peak areas. Standards chromatographed under the

same conditions were used for calibration.

Hydrolysis was initiated by adding 0.4 mL of a stock

solution of compound 1 in acetonitrile to 25 mL of buffer

prewarmed to 32 OC in a constant temperature water bath to

give final concentrations of ~1.8x10-4 M. Aliquots were

removed at appropriate intervals and chromatographed

immediately. Pseudo-first-order rate constants were

determined from the expression:

In(Ct) = In(Co)-kt (4)

where Ct is the concentration at some time=t, Co is the

concentration at t-0, k is the pseudo-first-order rate

constant, and t is the time. The slopes, -k, of linear plots

of ln(Ct) versus t were determined by linear regression. The

half-lives (tl/2) were calculated from

tl/2 = 0.693/k (5)

Each hydrolysis reaction was run in triplicate and was

followed for a minimum of three half-lives. The correlation

coefficients were >0.999.


Skin Penetration Studies


Diffusion cell experiments were performed to measure the

transdermal delivery of 5FU and the 5FU prodrugs. Franz-type









diffusion cells from Crown Glass in Somerville, NJ with

4.9 cm2 donor surface areas and 20 mL receptor phase volumes

were used for this purpose. The full-thickness skins were

obtained from female hairless mice (SKH-hr-1) from Temple

University Skin and Cancer Hospital.

The mice were killed by cervical dislocation, their

skins were removed immediately by blunt dissection, and

dorsal sections were mounted in the diffusion cells. The

dermal sides of the skins were placed in contact with

receptor phase which contained 0.05 M phosphate buffer

(pH=7.1, I=0.12) with 0.11% formaldehyde as a preservative.

The effectiveness of formaldehyde for this purpose has

recently been documented.89 The receptor phases were stirred

continuously and kept at constant temperature (32 oC) by a

circulating water bath. A preapplication period of 48 hours

was established to uniformly condition the skins and to

remove water-soluble UV-absorbing materials. The receptor

phases were changed three times during this period, and

control experiments from earlier studies have shown that this

procedure effectively removes those materials.97 The

epidermal sides of the skins were exposed to the air and were

left untreated during this period.

After the preapplication period, 0.5 mL aliquots from

suspensions of the prodrugs in IPM were applied to the

epidermal sides of the skins. The IPM suspensions were

stirred at 221 C for 48 hours prior to application to

ensure that saturation was attained. Total concentrations of









the IPM suspensions ranged from 0.3 M to 0.8 M with enough

excess solid present to maintain saturation for the duration

of the application period (see below). Each drug-vehicle

combination was run in triplicate.

Samples were taken from the receptor phases at 4, 8, 12,

21, 24, 27, 30, 33, 36, 45, and 48 hours after donor phase

application. The receptor phases were changed following

removal of each sample so that "sink" conditions were

maintained. Samples were analyzed for total 5FU species that

had diffused by UV spectroscopy (e=7.13x103 at 266 nm) after

allowing at least 72 hours for complete prodrug hydrolysis.

Cumulative amounts of total 5FU species that diffused (pmol)

were plotted against time (h), and the slopes of the linear,

"steady-state" regions were calculated using linear

regression. The slopes, when divided by 4.9 (the area of the

donor surface in cm2), gave the "steady-state" fluxes

(gmol/cm2/h). In a separate experiment, HPLC was used to

determine intact prodrug content in the receptor phases at

each sampling time. Mobile phase containing 18-50% methanol

in 0.025 M acetate buffer (pH=5.0) was used with the system

described earlier. Aliquots were removed and chromatographed

immediately after the samples were taken. Prodrug fluxes

were calculated in the same manner as fluxes for total 5FU.

Donor phases were changed every twelve hours and were

set aside for 1H NMR analysis. Stability of the prodrugs in

IPM was determined from the chemical shift of C6-H. In

dimethylsulfoxide-d6, the C6-H signal for 5FU appears at










8-7.73 ppm. For each of the 1-alkyloxycarbonyl derivatives,

the same signal in dimethylsulfoxide-d6 appears at 8>8.10 ppm.

Since this area of the spectrum is free from interference by

IPM absorbances, the two signals can be identified and

quantified if necessary.

Following removal of the donor phases after the 48-hour

application period, the epidermal sides of the skins were

washed three times with 5 mL portions of methanol to remove

all remnants of prodrug and vehicle from the skin surfaces.

This was accomplished quickly (<3 min) to minimize contact

time between the skins and methanol. The receptor phases

were changed again, and the dermal sides were kept in contact

with the fresh buffer for 23 hours while the epidermal sides

were again left exposed to the air. After this "leaching"

period, another sample was taken from each cell to measure

the skin accumulation of total 5FU species.

Second applications to the epidermal sides of the skins

were made after the "leaching" period with a standard drug-

vehicle suspension Theophylline in propylene glycol

(0.4 M) was applied to assess the damage to the skins from

application of the initial drug-vehicle combinations.

Samples were taken at 1, 2, 3, 5, 7, 9, and 11 or 12 hours

after application. The samples were analyzed for

theophylline by UV spectroscopy (E=1.02x104 at 271 nm) and

second application fluxes were determined as described above.










Results and Discussion



Synthesis and Structure Determination


The known l-alkyloxycarbonyl-5FU derivatives have

melting points25,30,93 and spectral properties (UV30 and 1H

NMR25) in agreement with those reported in the literature.

The structures of the novel compounds were assigned by

comparison of their spectral properties with those of the

known homologs. Elemental microanalyses were obtained for

the novel compounds and were within acceptable limits

(0.4%).

Acylation on the N1- or N3-position can be distinguished

by UV and 1H NMR analysis. Anions of N3-substituted

derivatives undergo a substantial shift of their UVmax to

longer wavelength while anions of N1-substituted compounds do

not.30,98 This is reportedly due to the extended conjugation

possible for the N1-anion.20 Differences in 1H NMR spectra

are also well defined. In chloroform-d, the C6-H signal for

N1-substituted derivatives is a sharp doublet indicating

coupling with C5-F. The same signal in chloroform-d for

N3-substituted derivatives appears as a broad singlet or

triplet-like doublet of doublets from additional coupling of

C6-H with N1-H.20 When the substituents contain a carbonyl

group attached to the N1-position, as they do in the

1-alkyloxycarbonyl series, an anisotropic effect is observed










in which the C6-H signal is shifted downfield relative to 5FU

or the N3-substituted derivatives.25 For example, the C6-H

chemical shift for l-ethyloxycarbonyl-5FU (8=8.00) is 0.77

ppm downfield when compared with 3-ethyloxycarbonyl-5FU

(8=7.23) in chloroform-d.


Solubility


Solubility determinations are generally accomplished by

stirring excess solute in a solvent until saturation is

attained. The excess solid is removed and the saturated

solution is assayed for solute content. This approach is

suitable for stable solutes and for unstable solutes in

aprotic solvents, but another method is needed for measuring

aqueous solubilities of chemically unstable compounds.

An alternative to the direct method for determining

aqueous solubilities is the partitioning-based method. The

advantage of this method is that contact time between the

unstable compound and the aqueous phase can be minimized.

However, several points regarding this procedure require

clarification.

First, partition coefficients are concentration

dependent except when compounds with low associating

tendencies are present in dilute solutions (<10-1 M).96

Solubilities based on partition coefficients can only be

reported as estimates since activity coefficients become more

important at higher solute concentrations.99










Second, the partitioning and phase separation times that

were used in the partitioning-based method for estimating

aqueous solubility were chosen empirically. The times had to

be sufficiently long to allow equilibrium distribution of the

compounds between the phases to occur and to allow subsequent

separation of the phases to occur without substantial

hydrolysis of the prodrugs. Longer times than those chosen

could have been used for these more stable 1-alkyloxycarbonyl

derivatives, but in order to validate the procedure for all

four series, shorter times that were more appropriate for the

less stable derivatives were used.

Finally, due to the experimental design and the liability

of the prodrugs, mutual saturation of the phases prior to

partitioning could not be accomplished. Since the ester

(IPM) that was used as the lipid phase in these experiments

is practically insoluble in water,94 changes in the phase

volumes during partitioning are probably insignificant. This

potential volume change is a common source of error when more

water-soluble lipid solvents are not presaturated with their

corresponding aqueous phases.100

The conventional, direct method was used for determining

lipid solubilities. Since the 1-alkyloxycarbonyl derivatives

are the most chemically stable compounds of the four series,

both the direct and the partitioning-based methods were used

for determining their aqueous solubilities. Thus, the two

methods for determining aqueous solubility were compared

using the 1-alkyloxycarbonyl derivatives as a model series.










Lipid (SIpM) and aqueous (SAQ) solubilities for the

l-alkyloxycarbonyl derivatives are presented in Table 2-2

along with their melting points. Lipid solubilities are

greatly enhanced by making derivatives. Solubility values

range from over 40 times greater than 5FU for 1-methyloxy-

carbonyl-5FU (1) to more than 3000 times greater than 5FU for

l-hexyloxycarbonyl-5FU (5). Increases in lipid solubility

with increasing chain length are accompanied by decreases in

melting point. A change in that trend is observed for

l-octyloxycarbonyl-5FU (6), but this is not unexpected.

Since melting point and solubility depend in part on crystal

lattice energies,101 this result indicates that the crystal

structure is dominated by the 5FU nucleus for lower homologs

and by the aliphatic side chain for higher homologs.

Aqueous solubilities are reported for both the direct

and partitioning methods. The results show that aqueous

solubility peaks for l-ethyloxycarbonyl-5FU (2) and then

decreases. Compounds 1 and 2 have aqueous solubilities

greater than 5FU even though a hydrogen-bonding group (NI-H)

has been masked. Again, this is reflected in the lowered

melting points for the derivatives when compared to 5FU.

Aqueous solubilities determined by the partitioning-

based method underestimated the direct solubilities by 7% for

compounds 5 and 6, 8% for compound 1, 21% for compound 4, 22%

for compound 3, and 34% for compound 2. Relative aqueous

solubility among members of the series is the same with













Table 2-2. Melting points (MP), lipid solubilities (SIPM), and
aqueous solubilities (SAQ) for 1-alkyloxycarbonyl derivatives.


MP
Compound (OC)


SIpMa
(mM)


5FU 280-2 0.049 96 -85

1 158-60 2.1 120 111 124
2 127-8 13 263 174 34
3 124-6 15 55 43
4 97-8 34 29 23 26
5 66-7 153 5.4 5.0 5.8
6 97-8 36 0.14 0.13


aStandard deviations
solubilities.
bStandard deviations


from the mean were within 3% for IPM

from the mean were within 8% for


aqueous solubilities determined by direct method.
cStandard deviations from the mean were within 6% for
experimental values used to calculate partition coefficients
and estimated aqueous solubilities (11% for compound 2).
dLiterature values from references 30 and 93.












Table 2-3. Solubility ratios (SR), partition coefficients
(PC), and hydrophobicity parameters (w) for
l-alkyloxycarbonyl derivatives.


Compound SRa log(SR) Xb


PCc log(PC) Kd


1 0.018 -1.75 0.019 -1.72 0.03
2 0.050 -1.30 0.45 0.075 -1.12 0.60 0.18
3 0.28 -0.56 0.74 0.36 -0.45 0.67 0.11
4 1.2 0.07 0.63 1.4 0.16 0.61 0.09
5 29 1.46 0.70 31 1.48 0.66 0.02
6 257 2.41 0.48 285 2.45 0.49 0.04


log (PC) -
log (SR)


aSolubility ratio calculated from SIPM/SAQ.
bAlog(SR) for compound and preceding compound.
cExperimental partition coefficient (CIPM/CAQ).
dAlog(PC) for compound and preceding compound.










either procedure, and overall, agreement between the two

methods is good.

The direct aqueous solubility values for compounds 1, 4,

and 5 agree with the corresponding literature values included

in Table 2-2. However, the value for compound 2 is much

higher than the literature value. The basis for this

discrepancy is not clear.

In Table 2-3, the solubility ratios (SR) and

experimentally determined partition coefficients (PC) are

compared for the 1-alkyloxycarbonyl series. The values for

log(PC)-log(SR) indicate that the partition coefficients are

somewhat higher than the corresponding solubility ratios.

Generally, the more polar derivatives (log[PC]<0) show the

greatest difference with the exception of compound 1.

Yalkowsky et al.102 studied solubility ratios and

octanol-water partition coefficients for a broad range of

solutes. They concluded that self-association of polar

solutes in octanol increases the ability of octanol to

accommodate the solute which increases the partition

coefficient. Conversely, a nonpolar solute causes a decrease

in the partition coefficient by self-associating in the

aqueous phase.102 The present results can be explained on the

same basis. The lower than expected log(PC)-log(SR) value

for compound 1 could be due to the low concentration in the

IPM phase during partitioning of this derivative. Since the

aqueous solubility of compound 1 is nearly 60 times its lipid

solubility, the concentration in the IPM phase is reduced










well below its already low concentration at saturation.

Therefore, the solute-solute interactions that lead to higher

partition coefficients would also be reduced.

The hydrophobicity parameters100 (t) in Table 2-3 were

calculated from the relationship:103

log(PC)n = log(PC)0+ln (6)

where n is the number of methylene units in a homologous

series using both log(PC) and log(SR) values. Both

calculations yield an average I value equal to 0.60. Values

for I from the literature include 0.54 for silicone oil-water

and 0.66 for hexane-waterl01 indicating that 0.60 is a

reasonable value for the IPM-buffer partitioning system.


Hydrolysis Kinetics


Hydrolysis of l-methyloxycarbonyl-5FU (1) to 5FU in

0.05 M phosphate buffer (pH=7.1, 1=0.12) with and without

0.11% formaldehyde (3.6x10-2 M) was followed by HPLC at 32 OC.

Disappearance of compound 1, indicated by ln(C), is plotted

versus time (min) in Figure 2-1. The linearity of the plots

suggests that hydrolysis of 1 follows first-order kinetics in

the presence and absence of formaldehyde. Pseudo-first-order

rate constants (k) and half-lives (t1/2) from the linear plots

are presented in Table 2-4.

Hydrolysis of compound 1 is clearly faster in the

presence of formaldehyde indicating that general base

catalysis by formaldehyde hydrate may be involved (see
















B Plain Buffer

Formaldehyde Buffer
-9E





B
0 -10.



*
B

-11 0






-12
0 200 400 600 800

Time (min)




Figure 2-1. Plots of In(C) versus time (min) for hydrolysis
of l-methyloxycarbonyl-5FU in 0.05 M phosphate buffer
(pH=7.1, I=0.12) with and without formaldehyde
at 32 oC.













Table 2-4. Pseudo-first-order rate constants (k) and half-
lives (tl/2) for hydrolysis of l-methyloxycarbonyl-5FU
in 0.05 M phosphate buffer (pH=7.1, I=0.12)
with and without formaldehyde at 32 OC.


Compound


Formaldehyde
(M)


k(SD)a
(min-1)


tl/2
(min)


1 0 3.33x10-3(0.04x10-3) 208
1 3.6x10-2 3.78x10-3(0.05x10-3) 183


aMean standard deviation for n=3 values.










Chapter 3). Significant buffer catalysis by phosphate has

also been demonstrated for compounds in this series, and the

reader is referred to the work of Buur and Bundgaard30 for

complete pH-rate profiles and probable hydrolysis mechanisms.

In any case, release of the parent drug from compound 1

by chemical means is too slow for dermal delivery purposes.

Other members of the series would also appear to be poor

candidates, since they hydrolyze chemically over two times

slower than compound 1.30,93 In 80% human plasma, however,

hydrolysis rates are much faster (ti/2=2-3 min)30,93

suggesting enzyme catalysis of the 1-alkyloxycarbonyl series.

Since the skin is metabolically active, further study of

these compounds in diffusion cells was warranted.


Skin Penetration


Skin penetration data from the diffusion cells are

plotted as cumulative amount of total 5FU species that

diffused (JLmol) versus time (h). In Figure 2-2, results for

l-methyloxycarbonyl-5FU (1), l-ethyloxycarbonyl-5FU (2), and

l-propyloxycarbonyl-5FU (3) are compared to 5FU itself. In

Figure 2-3, results for l-butyloxycarbonyl-5FU (4),

l-hexyloxycarbonyl-5FU (5), and l-octyloxycarbonyl-5FU (6)

are compared to 5FU. Error bars correspond to the standard

deviation from the mean for n=3 values.

Fluxes (J), lag times (tL), and skin accumulation (SA)

values for each compound are reported in Table 2-5. Lag time












1000


0 Cpd1

800 Cpd2

Cpd3

E O 5FU
- 600 1

0
E

400 .





200 -




I I

0 10 20 30 40

Time (h)




Figure 2-2. Plots of cumulative amount of total 5FU species
that diffused (Imol) versus time (h) for
compounds 1, 2, 3, and 5FU.





47




300

I Cpd4

Cpds

SI Cpd

t "
E 200 5FU


o









o0 ,I I i II


0 10 20 30 40

Time (h)



Figure 2-3. Plots of cumulative amount of total 5FU species
that diffused (pol) versus time (h) for
compounds 4, 5, 6, and 5FU.












Table 2-5. Fluxes (J), lag times (tL), and skin accumulation
(SA) values for 1-alkyloxycarbonyl derivatives.


J(SD)a
Compound (Lmol/cm2/h)


Prodrug
S-Sb(11 h)c
(%)


tL SA(SD)a
(h) (pmol)


5FU 0.24(0.09) 13 3.7(0.9)

1 2.6(0.6) 42(16) 14 8.3(0.1)
2 5.9(1.3) 90(75) 13 18(4)
3 2.3(0.2) 78(43) 11 5.0(1.4)
4 2.2(0.1) 73(32) 10 4.2(0.5)
5 1.5(0.1) 79(14) 6.2 11(0)
6 0.29(0.02) 8.7 3.2(0.5)


aMean standard deviation for n=3 values.
percent of total 5FU as intact prodrug during "steady-state"
phase in separate experiment (n=l).
CPercent of total 5FU as intact prodrug from 11 h sample in
separate experiment (n=l).













Table 2-6. Second application fluxes (J) and lag times (tL)
for 1-alkyloxycarbonyl derivatives.


Ja(SD) b
(lomol/cm2/h)


Compound


5FU 1.2(0.2) 1.2

1 1.9(0.1) 0.8
2 1.8(0.4) 0.9
3 1.7(0.3) 1.0
4 1.8(0.2) 1.0
5 1.8(0.1) 0.8
6 1.8(0.2) 0.9


aFlux of 0.4 M theopylline from propylene glycol.
bMean standard deviation for n=3 values.










refers to the intersection of the linear, or "steady-state,"

region of each graph with the time (x) axis, and it is the

time required for establishing a uniform concentration

gradient within the skin.65 The percent of total 5FU present

as intact prodrug in the receptor phase is also reported in

Table 2-5. These values were calculated from samples taken

during the "steady-state" phase and from an earlier sample

(11 h) in a separate experiment using HPLC analysis (n=l).

The improvement in skin penetration of 5FU from the

1-alkyloxycarbonyl derivatives is significant except for

l-octyloxycarbonyl-5FU (6). Increases in flux are generally

about one order of magnitude, and l-ethyloxycarbonyl-5FU (2)

with nearly a 25-fold improvement is easily the best

derivative.

The presence of large amounts of intact prodrugs in the

receptor phases is a matter of interest. The high

percentages indicate that the hydrolytic enzymes of the skin

are not effectively converting the prodrugs to 5FU. It is

interesting to note, however, that percentages calculated

from a sample removed prior to "steady-state" are much lower

than the "steady-state" values. It is possible that the

large amounts of diffusant present at "steady-state" are

simply too much for the enzymes to handle. Another

possibility is that the continuous changing of the receptor

phase with each sample eventually depletes the enzymatic

activity.










The trend in skin accumulation is similar to the trend

in flux with the exception of l-hexyloxycarbonyl-5FU (5).

The large skin accumulation value and short lag time for

compound 5 may indicate a high affinity for the lipid regions

of the skin but less affinity for the hydrated regions and

the receptor phase.

Second application fluxes and lag times are presented in

Table 2-6. Skin penetration by theophylline from propylene

glycol, the standard drug-vehicle combination, is

approximately one and one-half times higher for the skins

treated with the 1-alkyloxycarbonyl derivatives than for

those treated with 5FU, and it is consistent throughout the

series. Therefore, skin damage is greater with the prodrugs,

but the difference is small when compared with the general

improvement in delivery of 5FU from the prodrugs.

The stability of the prodrugs in the IPM formulations

was assessed by 1H NMR analysis of the donor phases. After a

minimum of five days from the time the suspensions were

prepared until their 1H NMR spectra were recorded, including

at least twelve hours during which the formulations were in

contact with the skins, the 1-alkyloxycarbonyl derivatives

were found to be intact with no evidence of 5FU formation.


Summary


The 1-alkyloxycarbonyl derivatives of 5FU exhibited

decreased melting points and increased lipid solubilities










when compared to 5FU. Aqueous solubility reached a maximum

for l-ethyloxycarbonyl-5FU (2) and decreased from there with

increasing chain length. Skin penetration and skin

accumulation were also maximized for compound 2 suggesting

that both lipid and aqueous solubilities are important for

predicting transdermal and dermal delivery of these 5FU

prodrugs. The presence of high percentages of prodrugs in

the receptor phases, indicating insufficient release of the

parent drugs in the hairless mouse skin model, may limit the

potential of this series of prodrugs at least for dermal

delivery purposes. Finally, the partitioning-based method

for determining aqueous solubility appears to be a useful

method particularly for determining relative solubilities in

an homologous series.















CHAPTER 3
1-ACYL DERIVATIVES


Introduction


The 1-acyl derivatives of 5-fluorouracil (5FU) are

chemically unstable in aqueous solutions at all pH values.

In fact, their liability has been cited as a limitation to

their usefulness as drugs or prodrugs.26,42 If properly

formulated in an aprotic vehicle, however, this series of 5FU

derivatives may have potential for use as prodrugs for dermal

delivery.

Six straight-chain 1-acyl derivatives were selected for

study. The derivatives and their structures are shown in

Table 3-1.


Materials and Methods



Synthesis


Melting points (mp) were determined with a Thomas-Hoover

capillary melting point apparatus and are uncorrected.

Elemental microanalyses were obtained for all novel compounds

through Atlantic Microlab, Incorporated in Norcross, Georgia.

Proton nuclear magnetic resonance (1H NMR) spectra were

obtained at 90 MHz on a Varian EM-390 spectrometer. Chemical











Table 3-1. Structures of 1-acyl derivatives.






O
H F








R/O 0






Compound R

l-acetyl-5FU (7) -CH3
1-propionyl-5FU (8) -CH2CH3
1-butyryl-5FU (9) -(CH2)2CH3
1-valeryl-5FU (10) -(CH2)3CH3
l-hexanoyl-5FU (11) -(CH2)4CH3
l-octanoyl-5FU (12) -(CH2)6CH3









shifts (6) are reported in parts per million (ppm) from the

internal standard, tetramethylsilane (TMS). Coupling

constants (J) are expressed in cycles per second (Hz).

Infrared (IR) spectra were recorded with a Perkin-Elmer 1420

spectrophotometer and absorbances are reported in cm-1.

Ultraviolet (UV) spectra were obtained with a Cary 210 or

Shimadzu UV-265 spectrophotometer. Maximum absorbances are

reported in nm along with the molar absorptivities (E) in

L/mol. Single-crystal X-ray analysis was obtained for

l-acetyl-5FU through Hoffmann-La Roche in Nutley, NJ.

l-Acyl-5-fluorouracil (general procedure)

To 0.66 g (0.01 mol) of 85% potassium hydroxide

dissolved in methanol (20-50 mL) was added 1.31 g of

5-fluorouracil (0.01 mol). The methanol suspension was

stirred for 30 minutes, and the methanol was evaporated under

reduced pressure. The potassium salt was suspended in

acetonitrile (25-50 mL) which was evaporated under reduced

pressure to remove residual methanol. The salt was

resuspended in acetonitrile (25-50 mL), and the suspension

was added dropwise over 15 to 30 minutes to a well stirred

acetonitrile (25 mL) solution in an ice bath containing 1.0

to 1.2 equivalents of the appropriate acid chloride. The

mixture was stirred at 0 C for 60 minutes. The mixture was

filtered, and the residue was washed with acetonitrile

(25 mL). The combined acetonitrile solutions were evaporated

under reduced pressure, and the solid residue was










crystallized from an appropriate solvent or solvent

combination.

l-Acetvl-5-fluorouracil (7)

Crystallization from dichloromethane gave 0.98 g of 7

(57%): mp 129-30 OC (lit.27 mp 126-7 oC); IR (KBr) 1670, 1695,

1725, and 1770 cm-1 (C=O); 1H NMR (CDC13) 8 2.73 (s, 3H, CH3)

and 8.23 (d, J=7 Hz, 1H, C6-H); UVmax (CH3CN) 261 nm

(E=1.125x104).

l-Propionyl-5-fluorouracil (8)

Crystallization from dichloromethane/hexane gave 1.32 g

of 8 (71%): mp 130-1 OC (lit.27 mp 124-5 oC); IR (KBr) 1695,

1710, and 1740 cm-1 (C=O); 1H NMR (CDC13) 5 1.25 (t, J=7 Hz,

3H, CH13), 3.14 (q, J=7 Hz, 2H, COCH2), and 8.27 (d, J=7 Hz,

1H, C6-H); UVmax (CH3CN) 261 nm (E=1.141x104).

Anal. Calc. for C7H7FN203: C, 45.17; H, 3.79; N, 15.05.

Found: C, 45.26; H, 3.83; N, 14.97.

l-Butyryl-5-fluorouracil (9)

Crystallization from dichlormethane/hexane gave 0.86 g

of 9 (43%): mp 145-6 OC; IR (KBr) 1690, 1710, and 1740 cm-1

(C=O); 1H NMR (CDC13) 8 1.01 (t, J=7 Hz, 3H, CH3), 1.77 (m,

2H, COCH2CH2), 3.09 (t, J=7 Hz, 2H, COCH2), and 8.25 (d,

J=6 Hz, 1H, C6-j); UV max (CH3CN) 261 nm (E=l.168x104).

Anal. Calc. for C8H9FN203: C, 48.00; H, 4.53; N, 14.00.

Found: C, 48.12; H, 4.58; N, 13.91.










l-Valeryl-5-fluorouracil (10)

Crystallization from dichloromethane/hexane gave 1.35 g

of 10 (63%): mp 120-1 C; IR (KBr) 1695, 1715, and 1740 cm-1

(C=O); 1H NMR (CDC13) 8 0.95 (t, J=7 Hz, 3H, CH3), 1.3-1.8 (m,

4H, COCH2CH2CBI2), 3.11 (t, J=7 Hz, 2H, COCH2), and 8.24 (d,

J=7 Hz, 1H, C6-H); UVmax (CH3CN) 261 nm (E=1.175x104).

Anal. Calc. for C9H11FN203: C, 50.47; H, 5.18; N, 13.08.

Found: C, 50.52; H, 5.23; N, 13.03.

l-Hexanoyl-5-fluorouracil (11)

Crystallization from dichloromethane/hexane gave 1.71 g

of 11 (75%): mp 101-2 OC; IR (KBr) 1690, 1715, and 1745 cm-1

(C=O); 1H NMR (CDC13) 8 0.92 (distd t, 3H, Cf3), 1.2-1.9 (m,

6H, COCH2Cl2CH2CH2), 3.09 (t, J=7 Hz, 2H, COCE2), and 8.24 (d,

J=7 Hz, 1H, C6-K) ; UVmax (CH3CN) 261 nm (E=1.158x104) .

Anal. Calc. for Co1H13FN203: C, 52.63; H, 5.74; N, 12.27.

Found: C, 52.69; H, 5.75; N, 12.27.

l-Octanoyl-5-fluorouracil (12)

Crystallization from dichloromethane/hexane gave 1.28 g

of 12 (50%): mp 83-4 oC; IR (KBr) 1685, 1710, and 1745 cm-1

(C=O); 1H NMR (CDC13) 5 0.90 (distd t, 3H, CJ3), 1.2-1.8 (m,

10H, COCH2CHi2C Ii2Ck22C2Ci), 3.10 (t, J=7 Hz, 2H, COCI2), and

8.23 (d, J=7 Hz, 1H, C6-H); UVmax (CH3CN) 261 nm

(E=1.155x104).

Anal. Calc. for C12H17FN203: C, 56.24; H, 6.69; N, 10.93.

Found: C, 56.22; H, 6.73; N, 10.96.











Lipid solubility


Lipid solubilities were determined using isopropyl

myristate (IPM), a commercial vehicle used in cosmetics and

topical medicinals,94 as the lipid solvent. The use of IPM as

a model lipophilic vehicle in skin penetration studies is

well established.77,95

Three suspensions of each derivative were stirred at

221 OC for 48 hours. The suspensions were filtered through

0.45 pm nylon filters, and the saturated solutions were

diluted in acetonitrile and analyzed by UV spectroscopy.

Solubilities were calculated using Beer's Law:

A = E-C-d (1)

where A is the absorbance, E is the molar absorptivity, C is

the concentration in mol/L, and d is the path length of the

cuvette in cm. Molar absorptivities were predetermined in

triplicate in acetonitrile at 261 nm.


Aqueous Solubility


Because of the chemical instability of the 1-acyl

derivatives, direct measurement of aqueous solubilities for

these prodrugs was not attempted. A comparison of the direct

and partitioning-based methods for determining aqueous

solubility was presented in Chapter 2 for the 1-alkyloxy-

carbonyl derivatives.










Partition Coefficients


The partitioning-based method for determining aqueous

solubility utilized the saturated IPM solutions from the

lipid solubility study. For most compounds, equal volumes

(1 mL) of saturated IPM solution and 0.05 M acetate buffer

(pH=4.0) were used. The use of equal or near-equal phase

volumes is known to facilitate rapid equilibrium.96 The two

phases were mixed thoroughly for ten seconds and allowed to

separate for 60 seconds. A preliminary study with 1-acetyl-

5FU (7) showed that there was virtually no difference in

partition coefficient (PC) values when partitioning was

carried out for 10, 20, or 30 seconds (PC=0.1830.006, a

standard deviation of only 3%). The IPM layers were diluted

in acetonitrile and analyzed by UV spectroscopy. The IPM-

buffer partition coefficients were calculated as follows:

PC = Aafter/ (Abefore-Aafter) VAQ/VIPM (2)

where Aafter is the absorbance from the IPM layer after

partitioning, Abefore is the absorbance from the IPM layer

before partitioning, VAQ is the volume of the aqueous phase,

and VIpM is the volume of the IPM phase. Estimated aqueous

solubilities (SAQ) were calculated from the IPM solubility

(SIPM) and the partition coefficient:

SAQ = SIPM/PC (3)

Partitioning was carried out in triplicate for a fixed volume

ratio for each derivative. For those compounds with large










differences in solubility in one phase relative to the other,

volume ratios (IPM:buffer) other than 1:1 were necessary, but

the ratio never exceeded 10:1 or 1:10.


Hydrolysis Kinetics


Hydrolysis rates have previously been reported for one

member of this homologous series.26 In the present study,

hydrolysis rates were determined at 32 OC for all six 1-acyl

derivatives in 0.05 M phosphate buffer (pH=7.1, I=0.12) and

for 1-acetyl-5FU (7) in the same buffer with 0.11%

formaldehyde (3.6x10-2 M). The rate in the presence of

formaldehyde was determined for comparison with the rate in

plain buffer since formaldehyde was used as a preservative in

the diffusion cell experiments described in the following

section. Two other concentrations of formaldehyde (1.8x10-1 M

and 3.6x10-1 M) were studied to assess the catalytic role of

formaldehyde in the hydrolysis of compound 7.

The hydrolyses were followed by UV spectroscopy at

266 nm where the absorbance decrease accompanying hydrolysis

of the 1-acyl derivatives was maximized. Hydrolysis was

initiated by adding 60 to 75 gL of stock solutions of the

derivatives in acetonitrile to 3 mL of buffer prewarmed to

32 OC in a thermostated quartz cuvette to give final

concentrations of 1-2x10-4 M. Absorbances were recorded at

appropriate intervals and pseudo-first-order rate constants










were determined from the expression:

ln(At-A-) = In(Ao-A-)-kt (4)

where At is the absorbance at some time=t, A- is the

absorbance at t=-, Ao is the absorbance at t=0, k is the

pseudo-first-order rate constant, and t is the time. The

hydrolyses were sufficiently fast to allow experimental

determination of A.. The slopes, -k, of linear plots of

In(At-Ao) versus time were determined by linear regression.

The half-lives (t1/2) were calculated from

tl/2 = 0.693/k (5)

Each hydrolysis reaction was run in triplicate and was

followed for a minimum of three half-lives. The correlation

coefficients were 20.999.


Skin Penetration Studies


Diffusion cell experiments were performed to measure the

transdermal delivery of 5FU and the 5FU prodrugs. Franz-type

diffusion cells from Crown Glass in Somerville, NJ with 4.9

cm2 donor surface area and 20 mL receptor phase volume were

used for this purpose. The full-thickness skins were

obtained from female hairless mice (SKH-hr-1) from Temple

University Skin and Cancer Hospital.

The mice were killed by cervical dislocation, their

skins were removed immediately by blunt dissection, and

dorsal sections were mounted in the diffusion cells. The

dermal sides of the skins were placed in contact with










receptor phase which contained 0.05 M phosphate buffer

(pH-7.1, I=0.12) with 0.11% formaldehyde as a preservative.

The effectiveness of formaldehyde for this purpose has

recently been documented.89 The receptor phases were stirred

continuously and kept at constant temperature (32 OC) by a

circulating water bath. A preapplication period of 48 hours

was established to uniformly condition the skins and to

remove water-soluble UV-absorbing materials. The receptor

phases were changed three times during this period, and

control experiments from earlier studies have shown that this

procedure effectively removes those materials.97 The

epidermal sides of the skins were exposed to the air and were

left untreated during this period.

After the preapplication period, 0.5 mL aliquots from

suspensions of the prodrugs in IPM were applied to the

epidermal sides of the skins. The IPM suspensions were

stirred at 221 OC for 48 hours prior to application to

ensure that saturation was attained. Total concentrations of

the IPM suspensions ranged from 0.6 M to 1.0 M with enough

excess solid present to maintain saturation for the duration

of the application period (see below). Each drug-vehicle

combination was run in triplicate.

Samples were taken from the receptor phases at 4, 8, 12,

21, 24, 27, 30, 33, 36, 45, and 48 hours after donor phase

application. The receptor phases were changed following

removal of each sample so that "sink" conditions were

maintained. Samples were analyzed for total 5FU species that









had diffused by UV spectroscopy (E=7.13x103 at 266 nm) after

allowing at least 24 hours for complete prodrug hydrolysis.

Cumulative amounts of total 5FU species that diffused (Imol)

were plotted against time (h), and the slopes of the linear,

"steady-state" regions were calculated using linear

regression. The slopes, when divided by 4.9 (the area of the

donor surface in cm2), gave the "steady-state" fluxes

(pmol/cm2/h). Because of the rapid chemical hydrolysis of

the 1-acyl derivatives, no attempt was made to analyze the

receptor phases for prodrug content.

Donor phases were changed every twelve hours and were

set aside for 1H NMR analysis. Stability of the prodrugs in

IPM was determined from the chemical shift of C6-H. In

dimethylsulfoxide-d6, the C6-H signal for 5FU appears at

5=7.73 ppm. For each of the 1-acyl derivatives, the same

signal in dimethylsulfoxide-d6 appears at 8>8.20 ppm. Since

this area of the spectrum is free from interference by IPM

absorbances, the two signals can be identified and quantified

if necessary.

Following removal of the donor phases after the 48-hour

application period, the epidermal sides of the skins were

washed three times with 5 mL portions of methanol to remove

all remnants of prodrug and vehicle from the skin surfaces.

This was accomplished quickly (<3 min) to minimize contact

time between the skins and methanol. The receptor phases

were changed again, and the dermal sides were kept in contact

with the fresh buffer for 23 hours while the epidermal sides










were again left exposed to the air. After this "leaching"

period, another sample was taken from each cell to measure

the skin accumulation of total 5FU species.

Second applications to the epidermal sides of the skins

were made after the "leaching" period with a standard drug-

vehicle suspension Theophylline in propylene glycol

(0.4 M) was applied to assess the damage to the skins from

application of the initial drug-vehicle combinations.

Samples were taken at 1, 2, 3, 5, 7, 9, and 11 or 12 hours

after application. The samples were analyzed for

theophylline by UV spectroscopy (E=1.02x104 at 271 nm) and

second application fluxes were determined as described above.


Results and Discussion



Synthesis and Structure Determination


The known l-acyl-5FU derivatives have melting points27

and 1H NMR spectra25 in agreement with those reported in the

literature. The structures of novel compounds were assigned

by comparison of their 1H NMR spectra with those of the known

homologs. Elemental microanalyses were obtained for the

novel compounds and were within acceptable limits (0.4%).

The differences in spectral properties between N1- and

N3-acylated derivatives were discussed in detail in

Chapter 2. Ultraviolet (UV) spectra were not obtained for

the 1-acyl derivatives under basic conditions because of










their instability. However, differences in 1H NMR spectra

are very clear. In chloroform-d, the C6-H signal for

l-acetyl-5FU is a sharp doublet at 8=8.23, while the same

signal for 3-acetyl-5FU is a broad singlet at 8=7.23, a

difference of 1.00 ppm.

Results from the single-crystal X-ray analysis show that

the N1-assignment for l-acetyl-5FU is correct. The unit cell

was found to contain two independent molecules (Figure 3-1

and Figure 3-2). Both conformations show that the carbonyl

group from the 1-acyl group is positioned cis to the C6-H.

The circulating K electrons of carbonyl bonds are known to

deshield ortho protons in structurally similar compounds such

as acetophenone.105 Thus, the X-ray results support the

explanation that an anisotropic effect is responsible for the

downfield shift of C6-H in 1H NMR spectra of Nl-acylated

derivatives.


Solubility


Lipid (SIPM) and aqueous (SAQ) solubilities for the

1-acyl derivatives are presented in Table 3-2 along with

their melting points. In general, melting points decrease

and lipid solubilities increase with increasing chain length.

An exception to both of these trends is seen for 1-butyryl-

5FU (9). The basis for this variance from the observed

trends is not clear.























































Figure 3-1. X-ray structure of l-acetyl-5FU unprimedd).
Source: Hoffman-La Roche, Nutley, NJ.























































Figure 3-2. X-ray structure of l-acetyl-5FU (primed).
Source: Hoffmann-La Roche, Nutley, NJ.













Table 3-2. Melting points (MP), lipid solubilities (SipM), and
aqueous solubilities (SAQ) for 1-acyl derivatives.


SIPMa
(mM)


Compound


5FU 280-2 0.049 96

7 129-30 22 119
8 130-1 36 48
9 145-6 17 6.5
10 120-1 39 3.5
11 101-2 112 3.0
12 83-4 111 0.15


aStandard deviations from the mean
solubilities.


were within 5% for IPM


bSolubility determined by direct method.
CStandard deviations from the mean were within 5% for
experimental values used to calculate partition coefficients
and estimated aqueous solubilities.













Table 3-3. Partition coefficients (PC) and hydrophobicity
parameters (t) for 1-acyl derivatives.


Compound


log (PC)


7 0.19 -0.73
8 0.76 -0.12 0.61
9 2.7 0.43 0.55
10 11 1.05 0.62
11 38 1.58 0.53
12 759 2.88 0.65


aExperimental partition coefficient (CIPM/CAQ).
bAlog(PC) for compound and preceding compound.










Aqueous solubilities were determined by the

partitioning-based method for which a theoretical discussion

was presented in Chapter 2. For the 1-acyl series, aqueous

solubility decreases with increasing chain length, and only

l-acetyl-5FU (7) is more water soluble than 5FU.

In Table 3-3, partition coefficients (PC) and

hydrophobicity parameters (t) are listed for the 1-acyl

derivatives. The average E value is 0.60, the same value

that was calculated for the 1-alkyloxycarbonyl series.

Individual values for the 1-acyl derivatives are all within

0.07 of the average. These results further support use of

the partitioning-based method for determining relative

aqueous solubility in a homologous series.


Hydrolysis Kinetics


Hydrolysis of the 1-acyl derivatives was studied in

0.05 M phosphate buffer (pH=7.1, 1=0.12) at 32 OC. Pseudo-

first-order rate constants (k) and half-lives (tl/2) are

presented in Table 3-4. Half-lives are relatively consistent

throughout the series ranging from 3.1 minutes for

l-propionyl-5FU (8) to 4.8 minutes for l-acetyl-5FU (7).

Hydrolysis of compound 7 was also studied in the same

buffer with increasing concentrations of formaldehyde added

to assess the catalytic role of formaldehyde hydrate. These

results are shown in Table 3.5. It is clear that the

hydrolysis rate for compound 7 is faster in the presence of













Table 3-4. Pseudo-first-order rate constants (k) and half-
lives (tl/2) for hydrolysis of 1-acyl derivatives in
0.05 M phosphate buffer (pH=7.1, 1-0.12) at 32 OC.


k(SD)a
(min-1)


Compound


tl/2
(min)


7 0.143(0.002) 4.8
8 0.222(0.004) 3.1
9 0.163(0.003) 4.3
10 0.169(0.006) 4.1
11 0.173(0.003) 4.0
12 0.183(0.003) 3.8


aMean standard deviation for n=3 values.













Table 3-5. Pseudo-first-order rate constants (k) and half-
lives (t1/2) for hydrolysis of l-acetyl-5FU in 0.05 M
phosphate buffer (pH=7.1, I=0.12) with and without
formaldehyde at 32 OC.


Compound


Formaldehyde
(M)


k(SD)
(min-1)


tl/2
(min)


7 0 0.143(0.002)a 4.8
7 3.6x10-2 0.161(0.008)b 4.3
7 1.8x10-1 0.219(0.004)a 3.2
7 3.6x10-1 0.279(0.006)a 2.5


aMean standard deviation for n=3 values.
bMean standard deviation for n=5 values.












0.3

y= 0.14644 + 0.37545x RA2 = 0.996











0.2








a Cpd7




0.1

0.0 0.1 0.2 0.3 0.4

Formaldehyde (M)




Figure 3-3. Plot of pseudo-first-order rate constant (k)
versus formaldehyde concentration (M) for hydrolysis of
l-acetyl-5FU in 0.05 M phosphate buffer
(pH=7.1, 1=0.12) at 32 OC.










formaldehyde and the rate is concentration dependent. In

Figure 3-3, the pseudo-first-order rate constants are plotted

against formaldehyde concentration. The slope of the linear

plot gives the catalytic rate constant (kcat=0.375) for

formaldehyde catalysis of compound 7. Hydrolysis rates for

the 1-acyl derivatives have previously been shown to be

independent of pH at acidic pH values.26 Therefore,

formaldehyde catalysis is likely due to general base rather

than general acid catalysis.


Skin Penetration


Skin penetration data from the diffusion cells are

plotted as cumulative amount of total 5FU species that

diffused (gmol) versus time (h). In Figure 3-4, results for

l-acetyl-5FU (7), l-propionyl-5FU (8), and l-butyryl-5FU (9)

are compared to 5FU itself. In Figure 3-5, results for

l-valeryl-5FU (10), l-hexanoyl-5FU (11), and l-octanoyl-5FU

(12) are compared to 5FU. Error bars correspond to the

standard deviation from the mean for n=3 values.

Fluxes (J), lag times (tL), and skin accumulation (SA)

values for each compound are reported in Table 3-6. Lag time

refers to the intersection of the linear, or "steady-state,"

region of each graph with the time (x) axis, and it is the

time required for establishing a uniform concentration

gradient within the skin.65 The percentages of total 5FU











1200

a Cpd7

1000- Cpd

SCpd 9

O 5FU

0 600-





S 400-
E *


200 -

a iI!!

o gI, I o a -
0 10 20 30 40

Time (h)




Figure 3-4. Plots of cumulative amount of total 5FU species
that diffused (gmol) versus time (h) for
compounds 7, 8, 9, and 5FU.











200




1 Cpd10
Cpd11

E Cpd 12
S 5FU
- 0
E 100



E






0
S' I I



I r i 1 I


0 10 20 30 40
Time (h)



Figure 3-5. Plots of cumulative amount of total 5FU species
that diffused (.mol) versus time (h) for
compounds 10, 11, 12, and 5FU.













Table 3-6. Fluxes (J), lag times (tL), and skin accumulation
(SA) values for 1-acyl derivatives.


Compound


J(SD) a
(imol/cm2/h)


SA(SD)a
(pmol)


5FU 0.24(0.09) 13 3.7(0.9)

7 9.3(0.3) 10 68(10)
8 4.3(0.1) 13 69(10)
9 1.3(0.2) 12 8.2(2.7)
10 1.0(0.1) 9.0 16(4)
11 1.1(0.0) 5.6 11(3)
12 0.60(0.01) 6.5 12(3)


aMean standard deviation for n=3 values.













Table 3-7. Second application fluxes (J) and lag times (tL)
for 1-acyl derivatives.


Ja (SD)b
(Ilmol/cm2/h)


Compound


tL(h)


5FU 1.2(0.2) 1.2

7 1.6(0.0) 0.6
8 1.2(0.2) 0.1
9 1.0(0.0) 0.6
10 0.80(0.03) 0.8
11 0.47(0.02) 0.2
12 0.72(0.11) 0.4


aFlux of 0.4 M theopylline from propylene glycol.
bMean standard deviation for n=3 values.










present as prodrug are presumed to be zero since the half-

lives of these prodrugs under diffusion cell conditions are

only three to five minutes.

The improvement in skin penetration of 5FU from the

1-acyl derivatives is substantial. The best compound,

l-acetyl-5FU (7), shows an increase in flux of nearly 40

times when compared to 5FU. As chain length increases, the

fluxes decrease, but even l-octanoyl-5FU (12) improves

transdermal delivery of 5FU by two and one-half times.

Skin accumulation values are also much higher for the

1-acyl derivatives than for 5FU. They show a decrease with

increasing chain length, but the value for l-butyryl-5FU (9)

is lower than expected. As noted earlier, the lipid

solubility of compound 9 was also less than expected when

compared to the compounds around it in the 1-acyl series.

Therefore, the low skin accumulation value for compound 9 may

be due to its low affinity for the lipid regions of the skin.

Overall, skin accumulation is higher for this series than for

the 1-alkyloxycarbonyl series while skin penetration data for

the two series are similar. Rapid hydrolysis of the more

lipid-soluble 1-acyl derivatives to highly polar 5FU as they

partition into the skin may indicate that only 5FU is

diffusing through the remaining lipid regions of the skin.

This may effectively "lock in" large amounts of 5FU leading

to high skin accumulation values.

Second application fluxes and lag times are reported in

Table 3-7. Skin penetration by theophylline from propylene










glycol, the standard drug-vehicle combination, after

treatment with the 1-acyl derivatives is similar to that

following treatment with 5FU. In fact, the longer-chain

derivatives in this series actually appear to have a

protective effect on the skin since they cause less damage

than 5FU itself.

The stability of the prodrugs in the IPM formulations

was assessed by 1H NMR analysis of the donor phases. After a

minimum of five days from the time the suspensions were

prepared until their 1H NMR spectra were recorded, including

at least twelve hours during which the formulations were in

contact with the skins, the 1-acyl derivatives were found to

be intact with no evidence of 5FU formation.


Summary


The 1-acyl derivatives of 5FU exhibited decreased

melting points and increased lipid solubilities when compared

to 5FU. Aqueous solubility reached a maximum for 1-acetyl-

5FU (7) and decreased from there with increasing chain

length. Skin penetration was also maximized for compound 7

while skin accumulation values were highest and essentially

the same for compound 7 and l-propionyl-5FU (8). As was also

the case for the 1-alkyloxycarbonyl series, this demonstrates

that both lipid and aqueous solubilities are important for

predicting transdermal and dermal delivery of 5FU prodrugs.

The 1-acyl derivatives may be better candidates for dermal






81



delivery purposes than the 1-alkyloxycarbonyl derivatives

because of their rapid hydrolysis and higher skin

accumulation values. The partitioning-based method for

determining aqueous solubilities of chemically unstable

prodrugs was used to estimate the aqueous solubilities of the

1-acyl derivatives since this could not be accomplished by

other methods.















CHAPTER 4
1,3-BIS-ACYL DERIVATIVES


Introduction


The 1,3-bis-acyl derivatives of 5FU are double prodrugs.

The acyl groups at the N1-position are rapidly hydrolyzed

leaving the 3-acyl derivatives. The 3-acyl derivatives will

be discussed in Chapter 5. The rate of N1-deacylation for

the 1,3-bis-acyl derivatives is even faster than hydrolysis

of the 1-acyl derivatives. This can best be explained by

comparing pKa values for the 3-acyl derivatives and 5FU. The

3-acyl derivatives have reported pKa values of 7.1 to 7.226

while the pKa for the first ionization of 5FU is 8.0. Thus,

the leaving group potential of the N3-acyl anions is greater

than the 5FU anion, and the rates of N1-deacylation are

faster for the 1,3-bis-acyl derivatives.

The 1,3-bis-acyl derivatives were chosen as prodrug

candidates in order to study a series in which both hydrogen-

bonding groups were masked. It was anticipated that these

compounds would be more lipid soluble and less water soluble

than the other series and would be useful for comparison with

the other series.











Table 4-1. Structures of 1,3-bis-acyl derivatives.


R2


A


RO/


Compound RI,R2


1,3-bis-acetyl-5FU (13)
1,3-bis-propionyl-5FU (14)
1,3-bis-butyryl-5FU (15)
1,3-bis-valeryl-5FU (16)


-CH3
-CH2CH3
-(CH2)2CH3
-(CH2)3CH3










Four straight-chain 1,3-bis-acyl derivatives were

selected for study. The derivatives and their structures are

shown in Table 4-1.


Materials and Methods



Synthesis


Melting points (mp) were determined with a Thomas-Hoover

capillary melting point apparatus and are uncorrected.

Elemental microanalyses were obtained for all novel compounds

through Atlantic Microlab, Incorporated in Norcross, Georgia.

Proton nuclear magnetic resonance (1H NMR) spectra were

obtained at 90 MHz on a Varian EM-390 spectrometer. Chemical

shifts (6) are reported in parts per million (ppm) from the

internal standard, tetramethylsilane (TMS). Coupling

constants (J) are expressed in cycles per second (Hz).

Infrared (IR) spectra were recorded with a Perkin-Elmer 1420

spectrophotometer and absorbances are reported in cm-1.

Ultraviolet (UV) spectra were obtained with a Cary 210 or

Shimadzu UV-265 spectrophotometer. Maximum absorbances are

reported in nm along with the molar absorptivities (e) in

L/mol. Single-crystal X-ray analysis was obtained for

1,3-bis-acetyl-5FU through Hoffmann-La Roche in Nutley, NJ.

1.3-bis-Acyl-5-fluorouracil (general procedure)

To 1.31 g (0.01 mol) of 5FU suspended in acetonitrile

(20 mL) was added 1.01 g of triethylamine (0.01 mol) in









acetonitrile (5 mL). The mixture was stirred continuously at

0 *C while 0.011 mol of the appropriate acid chloride in

acetonitrile (5 mL) was added dropwise over 5-10 minutes.

The above sequence was repeated until 0.03 mol of

triethylamine and 0.033 mol of acid chloride were added, then

the mixture was stirred for an additional 30 minutes at 0 C.

Alternate addition of base and acylating agent was found to

increase yield and decrease formation of colored side

products that were difficult to remove. The mixture was

filtered, and the residue was washed with acetonitrile

(25 mL). The combined acetonitrile solutions were evaporated

under reduced pressure, and the solid residue was

crystallized from an appropriate solvent or solvent

combination.

1.3-bis-Acetyl-5-fluorouracil (131

Crystallization from ether gave 1.69 g of 13 (79%): mp

112-3 OC (lit.25 mp 111-3 oC); IR (KBr) 1680, 1695, 1740,

1750, and 1795 cm-1 (C=0); 1H NMR (CDC13) 8 2.58 (s, 3H,

3-CH3), 2.72 (s, 3H, 1-Cl13), and 8.23 (d, J=7 Hz, 1H, C6-1j);

UVmax (CH3CN) 262 nm (E=9.75x103).

1.3-bis-Propionyl-5-fluorouracil (14)

Crystallization from ether gave 1.82 g of 14 (75%): mp

100-1 OC; IR (KBr) 1690, 1725, and 1795 cm-1 (C=0); 1H NMR

(CDC13) 8 1.25 (t, J=7 Hz, 3H, 3-CH3), 1.28 (t, J=7 Hz, 3H,

1-CH3), 2.85 (q, J=7 Hz, 2H, 3-COCf2), 3.11 (q, J=7 Hz, 2H,










1-COCfl2), and 8.25 (d, J=7 Hz, 1H, C6-j); UVmax (CH3CN) 262 nm

(e=9.65x103).

Anal. Calc. for C10H11FN204: C, 49.59; H, 4.58; N, 11.57.

Found: C, 49.44; H, 4.59; N, 11.53.

1.3-bis-Butyryl-5-fluorouracil (151

Extraction of the residue with hot low-boiling petroleum

ether gave a cloudy solution which cleared upon cooling to

0 oC and produced a yellow resinous sediment. The mixture

was allowed to warm to room temperature, and the clear

solution was decanted from the sediment. The solution was

cooled again to 0 oC and crystallization gave 1.70 g of 15

(63%): mp 48-9 OC (lit.26 mp 47.5-48.5 OC); IR (KBr) 1685,

1710, 1735, and 1795 cm-1 (C=O); 1H NMR (CDCl3) 8 1.00 (t,

J=7 Hz, 3H, 3-Cl3), 1.03 (t, J=7 Hz, 3H, l-Cl3), 1.6-1.9 (m,

4H, 3-COCH2CH2 and 1-COCH2CIi2), 2.80 (t, J=7 Hz, 2H, 3-COCfl2),

3.07 (t, J=7 Hz, 2H, 1-COCH2), and 8.23 (d, J=6 Hz, 1H, C6-a);

UV max (CH3CN) 262 nm (E=1.051x104).

1.3-bis-Valeryl-5-fluorouracil (16)

Extraction of the residue with hot low-boiling petroleum

ether gave a cloudy solution which cleared upon cooling to

0 C and produced a brown resinous sediment. The mixture was

allowed to warm to room temperature, and the clear solution

was decanted from the sediment. The solution was cooled

again to 0 oC and crystallization gave 2.24 g of 16 (75%):

mp 47-8 *C; IR (KBr) 1685, 1710, 1735, and 1795 cm-1 (C=O);

1H NMR (CDC13) 8 0.95 (t, J=7 Hz, 6H, 3-CH3 and 1-Ca3),










1.3-1.8 (m, 8H, 3-COCH2CHi2CI2 and 1-COCH2CZCH2), 2.83 (t,

J=7 Hz, 2H, 3-COCj2), 3.08 (t, J=7 Hz, 2H, 1-COCH2), and 8.22

(d, J=7 Hz, 1H, C6-H); UVmax (CH3CN) 262 nm (e=1.071xl04).

Anal. Calc. for C14H19FN204: C, 56.37; H, 6.42; N, 9.39.

Found: C, 56.29; H, 6.48; N, 9.33.


Lipid solubility


Lipid solubilities were determined using isopropyl

myristate (IPM), a commercial vehicle used in cosmetics and

topical medicinals,94 as the lipid solvent. The use of IPM as

a model lipophilic vehicle in skin penetration studies is

well established.77,95

Three suspensions of each derivative were stirred at

221 OC for 48 hours. The suspensions were filtered through

0.45 pm nylon filters, and the saturated solutions were

diluted in acetonitrile and analyzed by UV spectroscopy.

Solubilities were calculated using Beer's Law:

A = E-C-d (1)

where A is the absorbance, E is the molar absorptivity, C is

the concentration in mol/L,and d is the path length of the

cuvette in cm. Molar absorptivities were predetermined in

triplicate in acetonitrile at 262 nm.


Aqueous Solubility


Because of the chemical instability of the 1,3-bis-acyl

derivatives, direct measurement of aqueous solubilities for