Chemistry of Magnolia grandiflora L.

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Title:
Chemistry of Magnolia grandiflora L.
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vii, 117 leaves : ill. ; 29 cm.
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English
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Davis, Terry Lee, 1945-
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Subjects

Subjects / Keywords:
Chemistry, Agricultural   ( mesh )
Trees -- analysis   ( mesh )
Sesquiterpenes   ( mesh )
Medicinal Chemistry Thesis Ph.D   ( mesh )
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non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1981.
Bibliography:
Bibliography: leaves 105-116.
Statement of Responsibility:
by Terry Lee Davis.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
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oclc - 25174106
notis - AEK1521
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Full Text














CHEMISTRY OF Magnolia grandiflora L.










By

TERRY LEE DAVIS














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





UNIVERSITY OF FLORIDA

1981
































This paper is dedicated to my wife, Sandi, and my parents with

thanks for their love and support.
















ACKNOWLEDGMENTS


Special thanks are due to Dr. K. V. Rao, for his professional

guidance, and to Dr. Bill Kline, for his friendship and counsel.

Thanks also are due to Dr. Roy King and Dr. Wallace Brey,

Department of Chemistry, Universi'ty of Florida, for performing mass

spectral work and high-resolution nmr studies, respectively, and to

Dr. Drury Caine of the University of Georgia for providing spectra of

cyclocolorenone.















TABLE OF CONTENTS


ACKNOWLEDGMENTS . . .

ABSTRACT . . . .

CHAPTER


1 SECONDARY METABOLISM AND THE CHEMISTRY OF
Magnolia; PHARMACOLOGICAL ACTIVITY OF Magnolia
EXTRACTS AND COMPONENTS . .

Biogenetic Classification of Secondary
Metabolisms . .
Terpenoids of Genus Magnolia .
Alkaloids of Genus Magnolia .
Compounds Derived from Shikimic Acid .
Pharmacological Activity of Magnolia
Preparations and Magnolia Compounds .

2 SESQUITERPENOIDS OF Magnolia grandiflora L. .

Ketone from the Bark Extract .
Antibiotic Principle of the Leaves .
Experimental . .

3 PHENYLPROPANOIDS OF Magnolia grandiflora L. .

Bis-allylphenol from the Bark .
Lignan from the Wood . .
Experimental . .

4 ALKALOIDS OF Magnolia grandiflora L .


Toxic Alkaloidal Fraction .
Non-toxic Alkaloidal Fraction .
Experimental . .
General Experimental .


LIST OF REFERENCES . .

BIOGRAPHICAL SKETCH . .


Page
ini


. 13

. 21

. 27


I I r I


. 117














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



CHEMISTRY OF Magnolia grandiflora L.

By

Terry Lee Davis

June 1981
Chairman: K. V. Rao
Major Department: Medicinal Chemistry

Magnolia spp. have been investigated extensively in the past,

especially for the purpose of characterizing the alkaloidal constituents

which have various pharmacological activities. The chemistry and per-

tinent activities of Magnolia spp. were reviewed to set a background for

the work to be described here. Magnolia spp. are a rich source of a

variety of natural products besides alkaloids.

In the present work, a study was made of the bark, wood and

leaves of M. grandiflora L. with special reference to the presence of

sesquiterpene and lignan constituents. From the bark was isolated the

sesquiterpene ketone, cyclocolorenone, for the first time from a Magnolia

spp. Another sesquiterpene, a lactone, identified as parthenolidewas iso-

lated from the leaves and shown to be the active principle responsible

for the antibacterial activity found in the leaves. The bark also

yielded a novel neolignan, which was named mehonokiol, the structure of

which was established by unequivocal degradative methods. Another lignoid











component, syringaresinol, was isolated for the first time from the

wood of a Magnolia spp. In addition, the extract of the wood was found

to be toxic to mice when administered intraperitoneally, and the active

principle responsible was isolated. It was characterized as menisperine,

isolated for the first time from a Magnolia spp. Two other alkaloids,

anonaine and liriodenine, were also found to be present in the extract

of the wood.

Cyclocolorenone, a sesquiterpene ketone with an unusual chromo-

phoric system, a cyclopropyl-conjugated enone, is a member of the

aromadendrane group. An analysis of the nmr spectral data was provided

to substantiate this characterization. In support of the structure, a

number of derivatives have been described.

The isolation of the sesquiterpene lactone, parthenolide, from

the leaves was described. It was characterized on the basis of its

spectral data as a member of the germacranolide type. This is the

first report of its antibiotic activity.

A new, highly lipophilic, phenolic neolignan was isolated from

the bark. It was shown to be a monomethyl ether of a bis-allylphenol

known as honokiol and hence named mehonokiol. Of the two possible

isomeric structures, the correct structure was deduced by degration

of the tetrahydro derivatives of both to the respective n-propyl anisic

acids and identification of each of these acids through unambiguous

synthesis.

Syringaresinol, a lignan of the pinoresinol type was isolated

from the wood of Magnolia and its structure and stereochemistry deter-

mined from spectral data.










The toxicity of the extract of Magnolia wood was described

for the first time in the present work. Fractionation showed that the

toxicity was due to an alkaloid. This fraction was separated into a

phenolic quaternary alkaloid, which represented the toxic principle, and

two other non-toxic alkaloids anonaine and liriodenine. Analytical and

spectral data of the quaternary alkaloid showed that it was identical

with the N-methyl isocorydinium cation, isolated for the first time from

a Magnolia spp.
















CHAPTER 1
SECONDARY METABOLISM AND THE CHEMISTRY OF Magnolia;
PHARMACOLOGICAL ACTIVITY OF Magnolia
EXTRACTS AND COMPONENTS


The Southern Magnolia, Magnolia grandiflora L., in itself prac-

tically a symbol of the Old South, belongs to the Magnoliaceae, a family

which has been variously classified under the order Ranales or Mag-

noliales.1 The chemistry of this very primitive plant has been

studied extensively, particularly the alkaloids. Magnolia species

which have undergone chemical investigation in this and other studies

are summarized in Table 1.1 according to the classification of J. E.

Dandy.2


Biogenetic Classification of Secondary Metabolites

Within the last half-century, fundamental studies of secondary

metabolism have led to a biogenetic classification of secondary

metabolites. This has led to such concepts as the biogenetic isoprene

rule, the acetate-malonate hypothesis, and the shikimate pathway which,

in their present form, are of great predictive value in ascertaining

the structure of a metabolite produced by an organism. Some discussion

of this classification is of value in structuring the chemistry of

Magnolia to be described in this dissertation.


Terpenoids of Genus Magnolia

The terpenoids are compounds arising from mevalonic acid-

derived isoprenes, such as isopentenyl pyrophosphate, by catenation











Table 1.1 Species of Magnolia reported in the chemical literature


Subgenus Magnolia

Sect. Gwillimia (Asia)
M. coco DC.

Sect. Lirianthe (Asia)


Sect. Rytidospermum (Asia and America)
M. Ashei Weatherby
M. macrophylla Michx.
M. obovata Thunb. (syn. M. Hypoleuca Sieb. et Zucc.)
M. officinalis Rehd. et Wils.
M. rostrata W.W. Smith
M. tripetala L. (syn. M. umbrella Desr.)

Sect. Oyama (Asia)
M. parviflora (syn. M. Sieboldii K. Koch)
M. Sieboldii K. Koch (syn. M. parviflora Sieb. et Zucc.)
M. sinensis Stapf. (syn. M. globosa var. sinensis Rehd. et Wils.)

Sect. Magnoliastrum (America)
M. virginiana L. (M. glauca Thunb.)

Sect. Theorhodon (America)
M. grandiflora L.
M. Schiediana Schlect.

Sect. Gymnopodium (Asia)
M. kachirachirai Dandy (syn. Michelia kachirachirai Dandy)

Sect. Maingola (Asia)


Subgenus Pleurochasma

Sect. Yulania (Asia)
M. Campbelli Hook f. et Thoms.
M. denudata Desr. (syn. M. Yulan Desf.)

Sect. Buergeria (Asia)
M. Kobus DC.
M. salicifolia Maxim.
M. stellata Maxim.











Table 1.1--continued


Subgenus Pleurochasma (continued)

Sect. Tulipastrum (Asia and America)
M. acuminata L.
M. cordata Michx.
M. liliflora Desr.


Note: Species cited in this work but not classified by Dandy: M. Far-
gii (syn. Michelia Fargesii Andr.), Magnolia fuscata Andr. (syn.
Michelia fuscata Blume, M. Figo Spreng.), M. LenneiTopf. (syn. M. Soulan-
geana var. Lennei Rehd.), and M. mutabilis Regel. M. X Soulangeana So-ul.
is M. denudata x M. liliflora.











and pursuant cyclizations. The carbon skeletons thus obtained may be

further modified by rearrangements: the structures of the resultant

molecules are in many cases determined by the conformations of the

immediate precursors and by the stereochemical requirements of the

cyclizations and subsequent rearrangements.

Depending on the number of isoprenoid units involved, they are

classified as mono-, sesqui-, di-, tri-, and sesterterpenes with 10,

15, 20, 30, and 40 carbon atoms, respectively.

Chemical investigation of the terpenoids of Magnolia has not

been extensive, as most of the emphasis has been placed upon isolation

and identification of the alkaloids. A number of more or less ubiqui-

tous compounds and several sesquiterpenes of more restricted occurrence

have been isolated. These sesquiterpenes are of the caryophyllane 1.1,

eudesmane 1.2, aromadendrane 1.3, and germacrane 1.4 skeletal types.


1.2


1.4


1.5











In the present work the first isolation from any Magnolia of the

sesquiterpene ketone, cyclocolorenone 1.5 is reported. This cyclopropyl-

conjugated enone is a member of a class of tricyclic compounds having

the aromadendrane skeleton 1.3. In addition, it was found in the course

of this work that the extract of the leaves possessed antibiotic activ-

ity. The compound responsible for this activity was isolated and found

to be parthenolide 1.6, a cytotoxic sesquiterpene of the germacranolide

class, based upon the germacrane skeleton 1.4. This compound has
4
previously been isolated from the shrub Michelia champaca L., Michelia

lanuginosa,5 and is known to occur also in Magnolia grandiflora.6
In Table 1.2 are presented the terpenoid compounds of various
7-13
types which have been isolated from genus Magnolia.1


Alkaloids of Genus Magnolia

Studies on the alkaloids of Magnolia have accounted for the

bulk of the chemical and pharmacological literature of the genus and have

been reviewed by Tomita and Nakano.14

A biogenetic definition of the alkaloids might state that they

are nitrogeneous secondary metabolites derived from amino acids. The

benzylisoquinoline and aporphine alkaloids characteristic of Magnolia

spp. are derived from phenylalanine. The formation of reticuline 1.7

and magnoflorine 1.8, examples of these classes of general distribution

throughout the Magnoliaceae are detailed in Figure 1.1.15-46





















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Figure 1.1


magnoflorine

1.8




Probable derivation of Magnolia alkaloids magnoflorine
and reticulene from tyrosine


H3CO











0 1 C i TT D' 1133"TT 1
0 HO CHH HO NCH

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1.9 1.10

Further oxidative processes can convert these alkaloids to
oxoaporphines such as liriodenine 1.9 and to-dimeric alkaloids such as
magnoline 1.10. The known alkaloids of Magnolia are presented in
Table 1.3.
In this work are reported the isolations of anonaine 1.11;
menisperine (N-methylisocorydine, 1.12), and lirodenine 1.9 from the
wood of Magnolia grandiflora. Although anonaine has previously been
isolated from this plant,40 this is the first reported isolation of
menisperine from any of the Magnoliaceae. This quaternary aporphine was
also found to account for the toxicity of the wood extract. Liriodenine
has previously been reported to be present in the leaves and wood of
M. grandiflora,40 and, in the course of this work, it was also observed
in the bark.

H3CO
CO 0 N O CH
SH3CO 'CH3
0 HO
H3CO
1.11, 1.1


1.11


1.12











Table 1.3 Alkaloids isolated from Magnolia spp.


Compound


Occurrence


phenylethylamine


candicine


salicifoline


benzylisoquinoline

magnococline

N-norarmepavine

reticuline

benzylisoquinoline
(quaternary)


magnocurarine


bisbenzylisoquinoline


magnolamine

magnoline


aporphine


anonaine


M. grandiflora (bk)15 (rts)16

M. acuminata (stms)17
M. coco (stms)18
M. denudata (bk)19
M. grandiflora (rts)16 (bk)a,21
M. Kobus (bk)22,23
M. liliflora (bk, r wd)24
M. salicifolia (bk)25
M. stellata (bk)26


M. coco (stms)27

M. kachirachirai (htwd)28,29

M. obovata (rts)30


M. acuminata (sts )17
M. denudata (bk)19,20
M. fuscatab (Ivs, bk)33
M. liliflora (bk)24
M. obovata (rts)30 (bk)34,35 (htwd)35
M. officinalis (htwd)36
M. parviflora (bk)37
M. salicifolia (bk)25


M. fuscatab (lvs)33,38,39 (bk)33

M. fuscatab (lvs)38


M. randiflora (wd)c,40 (Ivs)40
M. obovata (Ivs)30 (rts)30 (htwd)35,41










Table 1.3--continued


Occurrence


aporphine (continued)

anonaine acetamide


anolobine


asimilobine

glaucine


N-norglaucine

N-nornuciferine

obovanine


aporphine (quaternary)

N,N-dimethyllindcarpine
iodide

magnoflorine


menisperine

proaporphine

stepharine


7-hydroxyaporphine

michelalbine
(norushinsunine)


obovata (htwd)41

acuminata (rt-bk)9
coco (stms)27,42 (rt-bk)43
grandiflora (wd)40

obovata (Ivs, rts)30

kachirachirai htwd)18,29
obovata (Ivs)3u

kachirachirai (htwd)29

grandiflora (wd)40

obovata (lvs)30


M. acuminata (rt-bk)9

M. acuminata (Ivs, stms)e
Mi. coco (stms)18
M. denudat (rts)44
M. fuscata (Ivs, bk)33
M. grandiflora (bk)15,20,21
M. kachirachirai (htwd)18,29
M. Kobus (bk)22
M. parviflora (bk)37

M. grandiflora (wd)c


M. coco (stms)27,42 (rt-bk)43


M. obovata (htwd)35


Compound












Table 1.3--continued


Occurrence


7-oxotetradehydroaporphine

lanuginosine

liriodenine
(oxoushinsunine)


oxoglaucine

oxolaureline


Campbelli (bk)11

Campbelli (bk)11
coco (stms)18 4
grandiflora (bk)c (wd)c'40'45 (lvs)
mutabilis (lvs)11 30 ,41
obovata (Ivs, rts) (htwd)3
(bk)Jb

kachirachirai (htwd)29

X Soulangeana46


aAbsence of salicifoline in the bark of M. grandiflora var. lanceolata
Ait. has been noted by Tomita et al.20 -
bNow considered by most authors to be Michelia fuscata Blume,3 or
Michelia Figo Spreng31,32
CThis work
dNot isolated; identified by paper chromatography17


Compound











Liriodenine is of especially frequent occurrence in these

plants, anonaine also being common, especially in those plants contain-

ing liriodenine. The quaternary alkaloid menisperine, however, is

reported in only one of the Annonaceae, and in none of the Magnoliaceae

prior to the present work.


Compounds Derived from Shikimic Acid

A major group of secondary metabolites of plants consists of

phenylpropanoid derivatives, ultimately arising via shikimic acid,

with the amino acids phenylalanine and/or tyrosine as immediate pre-

cursors. The members often bear as the stigma of their origin a 4-,

3,4- or 3,4,5-oxygenation pattern on the aromatic ring. Shikimic acid-

derived compounds isolated from Magnolia are listed in Table 1.4.10,47

Oxidative juncture of cinnamyl alcohol and its derivatives at

the 6-positions of the 2-propenyl sidechains results in a class of

compounds Haworth has termed the lignans.661 Oxidatively coupled

phenolic compounds also appear in lignins, the polymeric phenylpropanoids

which constitute the "glue" that binds wood cellulose.

To date, several hundred lignans have been isolated from the

Gymnospermae and class Dicotyledonae of the Angiospermae. A summary of

skeletal types of the classical lignans is presented in Figure 1.2.

More recently, the collective name "lignoids" has been proposed

for the dimeric phenylpropanoids. Those derived from two units of

cinnamic acid, two units of cinnamyl alcohol, or one of each,retain

the "lignan" designation, whereas those formed from two units of

allylbenzene, two units of propenylbenzene, or one unit of each are







14









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*4) C I I I I I

tJ* 0 a c C c c

(n 0 cm S- S- a)













0 U tu 0) 0) 0-





0 3 0 0 (a







I- > U) <) E U) U1


















HOG H






2,5-diarylfurans
(olivil type)


diarylfurofurans
(pinoresinol type)


? HO= OH


OH

1-arylnapthalene 1-arylnapthalenes
lactones (podophyllotoxins)


0 O -H+





H OH


butanolldes
(ntairesinol


OH OH






0


dlbenzyl THF
type)


2C
,------,l


o+ o0-1





HO HO





Figure 1.2 No
tu


OH






1,4-diarylbutanes
(guaiuretic acid type)


tional representation of lignan biogenesis and struc-
ral types


H+
OH +0





PH (19










designated "neolignans." 6162 The neolignans are more restricted in

their distribution than the lignans proper, having been isolated only

from two subclasses, the Magnoliidae and the Rosidae.62 Those large

groups encompass, however, a number of major families, including the

Magnoliales and the Laurales.

The known lignans of Magnolia grandiflora are presented in

Figure 1.3. To this list one may now add the isolation of the lignan

syringaresinol 1.13, described in this work--the first reported occur-

rence of this compound from a member of the genus Magnolia.

One class of compounds which would now be considered as neolig-

nans is that of the bis-allylbiphenols, which include magnolol 1.14,

honokiol 1.15, acuminatin 1.16, and dehydrodieugenol 1.17. Of these,

the former three have been found only in various Magnolia species (cf.

Table 1.4), while the latter, isolated from Litsea turfosa Kosterm. (Laur-

aceae )63 is the only bis-allylphenol-type neolignan occurring outside

the genus Magnolia.



OH rH 3 CH3



Ho //R 0 H3CO

1.14 1.15 1.16

H3CO OH



HO OCH3


1.17





















= OCH20, R3 = R6 = H

R5 = OCH3, R3 = R6 = H

R5 = R6 = OCH3, R3 = H

R6 = OCH3, R2 = R5 = OH

R4 = R5 = R6 = OCH3


R1 = R3 = R4 = R6 = OCH3, R2 = OGlu, R5 = OH


sesamin

magnolin

eudesmin

syringaresinol 1.13

syringaresinol
dimethyl ether

acanthoside B


R10

R10


OCH3

OGlu


OCH3


magnolenin C


fargesin


R = CH3

R = (CH2)


veraguensin

calopiptin


R = CH3


S OR
galgravin


Figure 1.3 Known lignans of Genus Magnolia


R1 ,R2 =

R1 R2

R1 = R2
R1 R3

R1 R2


R4, R5


= R4 =

SR4 =
= R4 =

= R3 =


HO


H3CO

















OH
H H3CO OH

O O^0 OCH3
0 HO OCH3g
1.18 1.19


To this class, a fifth compound, mehonokiol 1.18, may now be
added, having been isolated in the course of this work from the bark of

M. grandiflora.a The structure was unequivocally established by spec-
troscopic and degradative methods, followed by syntheses of the degrada-
tion products.

A sixth biphenyl, zehyerol 1.19, obtained from Zeyheriab montana
Mart. and Z. tuberculosa (Vell.) Bur. ex Verlot (Bignoniaceae)64 has

been reported recently, although, strictly speaking, it is not a neolig-

nan but a lignan.

A number of compounds of mixed shikimic acid-acetogenin origin
such as flavonols and floral anthocyanins, have also been isolated from
65-69
Magnolia species.

aRecently El-Feraly and Li have obtained 1.18 from the seeds of
this plant, in addition to magnolol and honokiol. The structure was
determined by 1H- and 13C-nmr spectroscopy.56
bZeyheria is spelled Zehyera by the author.











Pharmacological Activity of Magnolia Preparations
and Magnolia Compounds
Much of the impetus for research upon Magnolia has been its
long history of use in medicinal preparations. Historically, a number

of species of Magnolia have been employed in Chinese medicine,70 American
Indian medicine,71,72 and even listed in American pharmacopoeiae and

pharmacognosy texts as bitter tonics, antimalarials, and diapho-
retics.73,74,75
retics.73, 5 The use of Magnolia extract in medicine has been pro-

posed even as recently as 1953.76

A number of these crude preparations do display some pharma-

cological activity. A preparation from Cortex Magnoliae has been found

to be bacteriostatic against Staphylococcus aureus, but not against

Eschirechia coli.77 An extract of M. Kobus has shown some antiviral

activity in mice at subtoxic levels.78,79 The extract of M. grandiflora

was found to have acaricidal activity,80 and to lower blood pressure

without effect on heart action or breathing.76

Many of the Magnolia preparations show pronounced effect on

the nervous system; some contain anticholinesterase, neuromuscular-

junction (nmj) blocking or ganglionic blocking agents. The ether

extract of M. obovata, at a dose of 1 g/kg intraperitoneally (IP) in

mice afforded a depression of spontaneous activity and muscular weak-

ness, characterized as a central nervous system effect. The aqueous

extract, at the same dose, showed prompt respiratory paralysis.81

The aqueous extract of the Chinese herbal Shin-I (bark of Magnolia

Fargiesii) displays marked acetylcholine-like action on the frog rectus-

abdominus. In addition, the authors isolated an unidentified alkaloid

(C17H19NO3) with curare-like action from the same drug.82










Several of the active principles of neuroactive Magnolia
extracts have been isolated and identified. The bis-benzylisoquinoline,
magnoline, 1.10, has a hypotensive effect and shows anticholinesterase
activity.83 Compounds associated with the observed "curare-like"
effects of Magnolia extracts are magnocurarine 1.20, magnoflorine 1.8,
and salicifoline 1.21.84 Magnocurarine, the most active of these
84
quaternary alkaloids,4 has a ganglionic-blocking activity comparable
in strength to hexamethonium bitartrate.85 It exerts upon frogs an
action similar to that of d-tubocurarine (d-TC) 1.22abut of longer
duration and only one-tenth as active.87


H3CO c CH3
HO CH3 H CO N CH3

0O CH3
HO

1.20 1.21


H3C H3CN/CH3

0 % 3 H N CH3

0
HO

O CH2
1.22a



previously thought to be fully quaternized, the structure of
d-TC has recently been revised.86










In other work, on rat sciatic-skeletal muscle preparation in
situ, these alkaloids exerted a curare-like action. The same blockade

was observed with frog rectus abdominus (in vitro), except in the case

of salicifoline, which caused contraction of the muscle.85

The noraporphine anonaine 1.11, isolated in the course of this
work, has antibiotic activity at 100 mg/ml against Staphylococcus

aureus, Mycobacterium smegmatis, and Candida albicans.88 It also

exerts a hypotensiveeffect in mice and rabbits89 and has been shown to be

an inhibitor of dopaminergic response, an activity associated with a

number of analogs of apomorphine 1.23.90 Its specific effect in rats is

as an antagonist of dopamine-sensitive adenylate cyclase in the caudate

nucleus.90





HO '' C

HO

1.23



In these laboratories, anonaine (as the hydrochloride) showed
no acute toxicity on injection in mice at a dose of 200 mg/kg (IP).

The Magnolia alkaloid liriodenine 1.9, also observed in the
course of this work, has an antibiotic activity similar to that of
anonaine.88'91 In addition, it is an inhibitor of human carcinoma of

the nasopharynx in vitro.92











The toxicity of M. grandiflora extracts has not previously been

reported. In the course of this work, it was found that the aqueous

fraction resulting from distribution of the extract between water and

ethyl acetate is toxic at a level of 250-350 mg/kg (IP) in mice. The

action is overtly curare-like, resulting in prompt respiratory arrest.

The alkaloid responsible for this activity was isolated and found to be

identical with menisperine 1.12 (N-methyl isocorydinium iodide), the

nmj-blocking76'93-6 and ganglionic-blocking activities76'93'95'96 of

which are well established.a Menisperine, then known as "chakranine,"

was originally described as exerting ". ganglionic blockade selec-

tively exercised against the autonomous nerve pathways of the respiratory

system."93 Erhart and Soine, using the cat tongue/hypoglossal nerve

system in vivo, have effectively characterized the nmj-blocking activity

of this drug as very similar to that of d-TC itself.94

The LD50 of the pure iodide was determined in this work to be

10 mg/kg (21 ,mol/kg, IP, mouse). For comparison, Kamat et al. obtained

a value of 2.2 mg/kg (5.9 pmol/kg, IV, mouse) for the chloride.93

The results of a pharmacological screening procedure routinely

employed in these laboratories are presented in Table 1.5.b These data

do not indicate any definitive pattern of pharmacological effects for

this compound: although the mydriasis observed is indicative of gangli-

onic blockade, no positive indication for nmj-blocking activity

aMoisset des Espanes has reported, however, that menisperine does
not have a curarizing effect, but ". diminishes direct and indirect
contractibility and excitability in a manner resembling tetraethylam-
monium salts."97 This same author observed meiosis upon intra-lymphatic
injection in frogs.98
bAfter the method of Campbell and Richter.99













Table 1.5 Pharmacological evaluation of Magnolia toxic alkaloid


Sample Magnolia Bark Date 9/19/78

Dose 12 mg/kg, 0.6 mg/ml Time 3:08 p.m.

Animal Mice
Animals

1 2 3 4 5 Final

Paw Temp. re NT

before 37.5 37.5 37.5 37.5 37.5
Body T after _--- 36 36 ---

Average Wt. (g) 25

Paw Temp., + --- --- ---

Body Temp., + -

Body Temp., + -

Ptoss -

Salivation -

Lachrymation -

Mydriasis + + +

Miosis -

Ploerection *'

Locomotor Activity, + -
I-I
Straub tail phenom. -

Righting reflex
(r.r.) abolished

Head drop
(with r.r. present)

Pos. Haffner
(with r.r. present)

Locomotion Activity, +

Abduced hindlegs
(with r.r. present)

Unsteady gait


Sign: 1.

2.

3.

4.

5.

6.

7.


8.

9.

10.

11.


12.


13.


14.

15.


16.

17.


18. CMTD (mg/kg)


aAnimals remain capable of locomotion when disturbed.


Others: Onset of effects ca. 5 min., with marked depression of activitya and labored
breathing. Moribund animals become increasingly cyanotic, and breathing more con-
vulsively. Just prior to death, animal usually rears and/or scampers frantically.
Animals not receiving a lethal dose usually recover within 30 min. At 2-3 x LDSO, death
occurs in less than 5 minutes. At LDSO deaths occur at 15-20 min., with an occasional
mouse succumbing more promptly.











(head-drop, ptosis, strabismus)99 other than apparent respiratory

arrest is evident. However, such activity is not observed under these

conditions for d-TC itself.99

Antibiotic activity of a very low order has been reported for

menisperine (chloride): it is active against Mycobacterium pyogenes

var. aureus and Streptococcus pyogenes at concentrations of 0.5 and

0.3 mg/ml, respectively.93

The sesquiterpene lactones parthenolide 1.6 and costunolide

1.24100 have been shown to be inhibitors of human epidermoid carcinoma

in the nasopharynx test system. In the course of this work it was

established that parthenolide is responsible for the antibiotic activ-

ity observed in extracts of old, yellowed leaves of M. grandiflora.




,H


0
0

1.24



The lignans sesamin, eudesmin, and syringaresinol 1.13, all of

which occur in various species of Magnolia (cf. Table 1.4) have some

antitubercular activity in vitro.101,102

Finally, compounds related to the diallylbiphenols found in

Magnolia have been synthesized for medicinal and other applications,103

and several have been patented for use as anticoccidial agents in

poultry feed.104
















CHAPTER 2
SESQUITERPENOIDS OF Magnolia grandiflora L.


Ketone from the Bark Extract
Partition of the concentrated ethanolic extract between water

and ethyl acetate separated two fractions of grossly different polari-

ties. The hydrophilic fraction contained highly polar compounds such

as glycosides and quaternary alkaloids, the isolation of which has been

described elsewhere.15 Fractionation of the lipophilic components and

characterization of the major principles form the subject matter for

the bulk of this dissertation. A summary of the various constituents

found in the bark of Magnolia grandiflora is given in Figure 2.1.

Thin-layer chromatographic examination of the lipophilic fraction

revealed the presence of significant amounts of an ultraviolet-absorbing

component which gave a positive reaction with 2,4-dinitrophenylhydrazine

spray. The sample had a uv-maximum of 260 nm which was unaffected by

the addition of base, thus showing that it was nonphenolic. This com-

ponent was isolated by column chromatography on silica gel-cellulose

(1:1) by elution with 5% acetone in benzene. The sample so obtained

was, however, contaminated with phytosterols which were partially remov-

able by precipitation from methanol at 5C. Examination of the steroid

fraction by temperature-programmed and isothermal gas-chromatography

showed the presence of peaks which were identifiable with authentic














Ethanolic


extract of
the bark
concentrate and
partition between
water and ethyl acetate


- I


Lipophilic fraction


ccloco orenone,
phytosterols


Silica gel
chromatography


Mehonokiol



Sephadex
chromatography
I


Syringaresinol


Hydrophilic fraction


Alkaloid
fraction


Ion-exchange
chromatography


Magnoforne Candcne
Magnoflorine Candicine


I
Magnolenins
A,B,C


I
Magnolidins
A,B


Figure 2.1 Fractionation of Magnolia grandiflora extract











samples of 8-sitosterol, stigmasterol and cholesterol, all of which

have been observed in the extracts of Magnolia obovata by Fujita et

al.7 Further purification of the desired compound by a repetition of

the silica gel chromatography using 2.5% acetone in benzene as eluent

was not satisfactory because of extensive tailing of the peak and

inefficient separation from the remaining phytosterols. Substitution

of Florisil as adsorbent gave, however, a sharper elution pattern and

a pure sample.

The 260 nm absorbing component was obtained as a colorless heavy

oil which, however, could not be induced to crystallize. Elemental

analysis and mass spectral data (M+ 218) indicated molecular formula

of C15H220. It showed a characteristic uv-maximum at 260 nm with

log e = 4.10. The ir spectrum showed no hydroxyl but a strong carbonyl

absorption (1690 cm-1) and unsaturation (1600 cm-1). Its nmr spectrum

gave evidence for the presence of three different types of methyl

groups: 0.72 ppm, d, CH-CH3; 1.00 ppm, s, and 1.23 ppm, s, R2C-(CH3)2

and 1.70 ppm, d, -C=C-CH3; along with a considerable overlap of signals

due to a CH2- envelope. The mass spectrum, besides providing a strong

molecular ion was not very helpful in giving useful structural informa-

tion because of the absence of favorable, structurally meaningful frag-

mentation pathways. The analytical and spectral data suggested, however,

that the compound might be a member of the class of sesquiterpene

ketones.

A crystalline 2,4-dinitrophenylhydrazone derivative was prepared

and its elemental analysis confirmed the molecular formula of the ketone.











Its uv-absorption maximum of 390 nm indicated that it was derived from

an a,c-unsaturated ketone, although the max was higher than that

expected for one double bond conjugated with the keto group.105'106

Efforts made to regenerate the ketone, possibly in purer form, by the

hydrolytic exchange of the crystalline dinitrophenylhydrazone with

levulinic acid and a mineral acid107 gave a ketonic product which, how-

ever, was different from the original enone (higher Rf and higher Xmax).

In the absence of mineral acid, no exchange took place. Thus, altera-

tion of the structure in the presence of acid was apparent.

The max of 260 nm observed for the ketone is intermediate in

value between that of an enone (220-250 nm) and that of a dienone

(280 nm).108 The literature suggested as a model for this chromophore

the cyclopropyl-conjugated enone 2.1, which was described by BUchi et

al., as an intermediate in their synthesis of maaliol 2.2.109 The

extent of overlap of the i-orbitals of the enone with the orbitals of

the cyclopropyl ring will determine the energy of the associated elec-

tronic transition which, in turn, will be determined by the conformation

of the enone and the cyclopropyl group.110,111 In accordance with this

idea, the Magnolia enone system might also possess a stereochemical

relationship similar to that of the model compound 2.1. This was

supported by the observed base-catalyzed epimerization of the enone

from Magnolia to a product with a Amax of 250 nm, apparently as a result

of greater deviation from periplanarity of the enone and the cyclopropyl

systems which are suspected to be present. Other evidence, also con-

sistent with the presence of a cyclopropyl moiety, is the prominent mass











spectral fragment at m/e 175 (M-C3H7). The epimerization alluded to

here, which will be discussed in more detail later, also indicated the

presence of a proton either alpha to the carbonyl or at the allylic

position, which can also be a ring junction.


S11
10
3 1 8

H H H 12 _A,
2.1 2.2 2.3



On the basis of the available information, a search of the

literature was conducted which revealed that the properties of the

Magnolia enone and those of its 2,4-dinitrophenylhydrazone derivative

correspondediwith those described for the sesquiterpene ketone, cyclocolor-

enone 2.3, first isolated from Pseudowintera colorata (Raoul)

Dandy.1213 However, in the absence of an authentic sample for com-
parison (the compound is unstable and polymerizes on standing) and in

view of much of the confusion in the literature concerning the possible

presence of its epimer in the samples reported in the earlier literature,

it was deemed necessary to proceed with proper structural elucidation

to establish its identity.

In order to establish the ring size of the enone through the

infrared spectral frequency of the corresponding saturated ketone,

catalytic hydrogenation by platinum in ethanol was carried out but with











no reaction. In acidic ethanol, a product was obtained which, however,

showed neither a carbonyl nor a hydroxyl function.

Reduction of the enone with sodium borohydride gave the cor-

responding allyl alcohol 2.4 from which the starting enone could be

regenerated by oxidation with manganese dioxide. This alcohol appears

to correspond with cyclocolorenol 2.4 described by Corbett and Speden113

by the action of lithium aluminum hydride on cyclocolorenone. These

authors also prepared the saturated ketone 2.5 from cyclocolorenone by

reduction with lithium and ammonia. This procedure was followed in the

present case and the saturated ketone was obtained, except that it was

a crystalline solid, mp 38-40 in contrast to Corbett and Speden's

description of it as an oil. A ketone of this same structure and a melt-

ing point of 38-400 was also prepared from a-gurjunene and characterized

as 4a,5a-cyclocoloranonell4 which agrees with the properties of the

present ketone. However, the melting point of the dinitrophenylhydrazone

of the saturated ketone from Magnolia enone differed from that of the

cyclocoloranone dinitrophenylhydrazone described in the literature by

either group. All these suggested that either during the reduction

process or, during the formation of the 2,4-dinitrophenylhydrazone,

epimerization was possible and that the melting point of the derivative

might reflect the degree of epimerization. The frequency of the car-

bonyl band (1745 cm- ) indicated, however, that the saturated ketone

from Magnolia enone contained a 5-membered ring.












H H

H 0

HH
H H
H

2.4 2.5




The nmr spectrum of the Magnolia enone was reported briefly

earlier and a more detailed analysis will now be presented with a view

to find support for its possible identity with cyclocolorenone.

The doublet at 0.72 ppm (J = 7 Hz) can be assigned to the

methyl group at C-10, split by the proton at C-10, with some overlap

from signals due to the methine protons of the cyclopropyl ring. The

singlet at 1.00 ppm is assignable to the exo methyl and the singlet at

1.23 ppm to the endo methyl group of the gem dimethyl function. The

absence of any signal due to an olefinic proton indicates that the sys-

tem is fully substituted. The presence of a methyl group on the enone

system is evidenced by the doublet at 1.70 ppm, and decoupling studies

showed that the methyl protons are coupled to the methine proton at

C-1 (J1,12 = 1.4 Hz). The signal centered at 2.84 ppm, a doublet of

doublets, represents the a-proton of C-2 coupled to the C-28 and C-1

protons with J2a,2U of 18 Hz and J ,2 of 6.5 Hz. The C-1 proton

appears as a multiple at 2.94 ppm with H-2aat 2.29 ppm, J1,2a = 2 Hz.

All of these signals show general agreement with those

described by BUchi et al.,115 for cyclocolorenone. The spectrum of the











cyclopentenone portion of the molecule may also be compared with that

of 2,4-dimethylcyclopent-2-en-l-one 2.6, the pertinent parameters of

which are shown in Figure 2.2.116,117 This model compound shows ir

spectral bands at 1700 and 1630 cm1 for C=0 and C=C, respectively,116

in comparison with the reported values of 1690 and 1630 cm-1 for the

respective groups of cyclocolorenone.



4 H

0 1 -CH3
SCCHH
0 1

6 3
2.6


H-4 2.85 ppm J 19.5 Hz
H-5 2.37 ppm J94e(cis) 6.5 Hz
H- 1.95 ppm 4,5(trans) 2.5 Hz
4,5(trans)

Figure 2.2 Nmr spectral parameters for 2,4-dimethylcyclopent-2-en-l-one


Based on the information from the model compound, additional

data on cyclocolorenone were obtained by the use of double-resonance

techniques. Irradiation of C-1 proton multiple at 2.94 ppm resulted

in the collapse of the C-12 allylic methyl doublet at 1.70 ppm, thus

implying a long-range homoallylic coupling of these protons similar to

that observed in thespectrum of a-gurjunene,ll8 photosantonic acid

lactone and a-santonin derivatives.119 In a-santonin derivatives, the











homoallylic coupling is of the order of 1.3-1.4 Hz, with a critical bond

angle of 1150.118 The angle measured for a stereomodel of cyclocoloren-

one was found to be 1150 and a coupling constant of 1.4 Hz was observed.

In contrast, homoallylic coupling with the C-6 proton seems to be

neglible in cyclocolorenone as might be anticipated from a 0 bond

angle. This suggests that cyclocolorenone assumes a preferred confor-

mation in which the orbital overlap of the enone and cyclopropyl systems

is maximal, which occurs when the cyclopropane ring is orthogonal to

the cyclopentenone ring.

The assignment of the 1.23 ppm singlet to the endo methyl of

the gem-dimethyl function is based on the arguments of Streith and

Ourisson118 who state that the endo methyl group is in a relatively

more deshielding region of the enone system. The carbonyl group

itself must make a substantial contribution to this effect as relative

deshielding is still significant in the spectrum of cyclocoloranone 2.5.

Epimerization of cyclocolorenone was observed BUchi et al.,115

somewhat by accident, during chromatography on alumina. They tried to

use this reaction in their attempted synthesis of cyclocolorenone by the

epimerization of epicyclocolorenone as the last step. However, epicyclo-

colorenone did not produce cyclocolorenone under conditions which pro-

duced the reverse reaction. Corbett and Young120 use boiling ethanolic

potassium hydroxide for epimerization. The enone from Magnolia was

subjected to epimerization both by passage through by an alumina column

as well as by boiling in alcoholic potassium hydroxide. Although the











product showed the expected shift in Amax toward 250 nm, thus indicating

epimerization had taken place, its nmr spectrum showed the reaction

to have proceeded only to the extent of 50-75%. (The mixtures show two

pairs of gem-dimethyl signals, the ratios of which vary with the

extent of epimerization.) The mixture was inseparable by tlc or by gas

chromatography. Also, the reaction with alcoholic base was rather

drastic and a significant extent of degradation accompanied epimerization.

The mixture resulting from the best epimerization reaction of the Magnolia

enone was converted to the 2,4-dinitrophenylhydrazone, which was

recrystallized to a constant melting point. The melting point and

spectral data of this product were consistent with those described for

the epicyclocolorenone,115,120 although this derivative was also unre-

solvable chromatographically from the dinitrophenylhydrazone of

cyclocolorenone. Thus, the situation with the epimerization is far from

clear, either in the present work or in the descriptions that appear in

the literature. Another reason to suspect this is that the melting

point of the cyclocoloranone 2,4-dinitrophenylhydrazone varies over a

range, again attributable to an undetermined degree of epimerization.

The discrepancies observed, reflecting the proclivity of the

molecule to undergo epimerization, might readily have been anticipated.

It was felt that, given the analytical and nmr data, and the unusual

chromophore, the identity of this compound is no less certain. As

final proof, however, copies of the ir and nmr spectra of synthetic
121 ,122
cyclocolorenone, obtained from Dr. D. Caine,121122 and of the Magnolia

enone are compared in Figures 2.3 and 2.4 (an impurity appearing on the

carbonyl band diminishes as the sample is purified).
























0
c-
O


S-
o
0


u

q)





r-
-U




















0
0


--












5-
cn











































'a C
0
cS.






























O







* 2
01
*r-

















C.
0)













L.
<4-
0r










*r




*OT
ll-





































7-~C.: --L---- ---i-- I:







--4-4-



A ai
-4---!-- -~t '


7-

"rr-....... ..........;


L1 -









77'








-J7.
rn1











77-~
It. -. 4L.PXf -
















































It-Q
If:II











Cyclocolorenone was first isolated from Pseudowintera colorata,

a plant which the authors ascribe to Magnoliaceae, although it is

usually assigned to Winteraceae.123126 These two families are con-

sidered by many authors to be among the most primitive of the Angiosper-

mae. Recently, cyclocolorenone has been isolated from an even more

primitive plant, the liverwort, Plagiochila acanthophylla, subspecies

Japonica (Hepaticae).127 In addition to these, the compound was found

to be present in Boronia ledifolia Gay (Rubiaceae)128 and in the golden-

rod, Solidago canadensis (Compositae).129

Although the basic structure of cyclocolorenone was determined

by the authors who isolated it originally, its stereochemistry was

determined largely by BUchi et al.,115 through their synthesis of

epicyclocolorenone, and from its relationship to a-gurjunene.114,118

Cyclocolorenone itself was finally synthesized by Ingwalson and Caine

starting from (-)-maalione.121,122

Cyclocolorene is a member of what has recently been described

as ". the rare class of aromadendrane sesquiterpenes."130 The

class appears to be neither so rare nor so restricted in its membership

as has been previously thought, numbering at present some fifteen

compounds, some of which are widely distributed. Nearly all of the

members contain the cyclopropyl ring in the 6S,7S-configuration of

(-)-cyclorocolorenone. The fusion of the 5- and 7-membered rings can

be either cis or trans, aromadendrene 2.7 itself being la,5B while

alloaromadendrene 2.8 is 1B,5B;131,132 a-gurjunene 2.9118,133-135 and











0-spathulene 2.10136 represent the prototypes with a 4,5-double bond

and 1B hydrogen: cyclocolorenone is of this type. This relationship

between cyclocolorenone and a-gurjunene is accentuated further by the

fact that the two occur together in Pseudowintera colorata,137 and

there has been speculation that cyclocolorenone might have been an

artifact arising from a-gurjunene on long storage through air/photo-

oxidation.118 A number of alcohols derived from either aromadendrane

(e.g., globulol 2.11131 and spathulenol 2.12)138 or alloaromadendrene

(e.g., ledol 2.13,118 viridiflorol 2.14118) have been described. An

unusual isonitrile, axisonitrile II 2.15139 and derivatives 2.16 and

2.17140 have been isolated from the sponge, Axinella canabina (Porifera).


2.7, R = aH
2.8, R = BH


2.9, R = H, R' = CH3
2.10, R = R = >CH2
2


2.11, R = CH3, R' = OH
2.12, R = R' = CH2


2.13, R = CH3, R' = OH
2.14, R = OH, R' = CH3


2.15, R = -N=C
2.16, R = -N=C=S
2.17, R = -NHCHO











Antibiotic Principle of the Leaves

The extract of yellowed leaves of Magnolia grandiflora was

found to possess antibiotic activity when assayed with Bacillus subtilis

using the disk-plate method. Partitions between ethyl acetate and

water at pH 9 and pH 2 indicated that the antibiotic principle is a

neutral molecule, readily extractable in this solvent.

The extract was therefore partitioned between ethyl acetate and

dilute aqueous ammonium hydroxide (pH 10) to remove phenolic materials,

and the combined solvent phases chromatographed on silica gel-cellulose.

The activity was found in the 5% acetone/benzene eluate, associated

wtih a component of Rf 0.38 (5% acetone/benzene), which turned bright

red when sprayed with sulfuric acid reagent and heated. The component

was also active in the antibiotic assay.

Although the antibiotic was also found to be present in fresh

green leaves in approximately the same amounts, purification was more

difficult because of the chlorophyll, which appeared in the same

chromatographic fractions. Despite the fact that the chlorophyll was

removable by chromatography on Florisil, it was found to be more con-

venient to use the yellow leaves.

With this method of purification, the extract of 500 g of

leaves afforded 41 g of material on concentration, with a minimum

inhibitory concentration (MIC) of 400 g/ml. Upon partition, the solvent

phase yielded 5 g, MIC 55 g/ml. Chromatography on silica gel-cellulose

yielded an antibiotic-containing fraction, 672 mg of an oily liquid,

which was extracted with petroleum ether, the extract concentrated to











an oil and triturated with ethanol to give a solid. Crystallization

from ether gave large, colorless crystals, 210 mg, mp 112-1140C, and

MIC 3 jg/ml.

The mass spectrum (M+ 248) and elemental analysis are consistent

with the molecular formula C15H2003. The ir spectrum (KBr) showed

an ester carbonyl (1738 cm- ), C=C (1655 cm- ), and a possible terminal

methylene group (890 cm" ). The nmr spectrum confirmed the presence of

the latter through signals at 5.72 and 6.15 ppm, 2d, J = 3.6 Hz,

and showed the following peaks: 5.25 ppm, m, 1H, vinylic; 3.98 ppm, 1H,

t, J = 18.4 Hz, R2CHOR; 2.87 ppm, 1H, d, J = 18.4 Hz, epoxide methine;

1.85-2.40 ppm, 8H, CH2 envelope: 1.70, singlet with fine structure,

allylic methyl group; and 1.23 ppm, 1H, s, methine. The uv spectrum

showed only end absorption.

The melting point and spectral data are in agreement with those
4
reported for parthenolide 2.16 by Govindachari et al. and a comparison
of their nmr spectrum with that of the Magnolia antibiotic is shown

in Figure 2.5.


2.16





43










(a) 0


0 L L
8-0 60 5.0 4-0 3-0 20 0
A. ppm













(b)
y^--
















.. '- "- .. ... ., "



Figure 2.5 Comparison of nmr spectra of (a).parthenolide and
(b) Magnolia antibiotic (D6DMSO)











The melting point and ir spectra are in agreement with the
4
published data of Govindachari et al., though the carbonyl band is

distorted to a lower frequency, probably a result of having been deter-

mined in the solid state (KBr).

This compound was originally isolated from Chrysanthemum

parthenium (1.) Bernh.141,142 and subsequently from Ambrosia dumosa

Gray,143 and Ambrosia confertiflora DC.,144 all Compositae. Indeed, the

Compositae, and particularly Ambrosia and their relatives, elaborate a

large variety of sesquiterpene lactones.144 Such compounds are much

less prevalent in other families.

Within the Magnoliaceae, in addition to M. grandiflora,6

parthenolide has been isolated from Michelia campaca L. by Govindachari
4
et al., who revised the original structure proposed by Herout et

al.141 and Soucek et al,142 and from Michelia lanuginosa L.145 The

antibiotic activity of this compound has not been previously described.

The stereochemistry of parthenolide was determined by Bawdekar

et al.,46 who correlated it with the known stereochemistry of

costunolide.

Parthenolide and costunolide are the only sesquiterpene lactones

reported from Magnolia. Interestingly, a number of related costunolide

derivatives are known from the magnoliaceous tree, Liriodendron tulipi-

fera.147,148 These, like parthenolide, have antitumor activity.147


Experimental

Extraction of Magnolia bark: Cyclocolorenone 2.3

Ground bark (41 g) of Magnolia grandiflora, collected in

Gainesville, Florida, was extracted with ethanol (10 Z) at 250 C for two










days. Three such extracts were combined, concentrated at 20 mm pressure

to a syrup which was partitioned between water and ethyl acetate (2 Z

each). Concentration of the solvent layer gave a heavy oil (120 g).

An aliquot of the oil (20 g) was chromatographed on silica gel
cellulose (500 g) in benzene. Fractions (10-12 ml) were collected and

tested by absorbance (260 nm) and thin layer chromatography. The benzene

eluatecontained besides carotenoid and other highly lipid-soluble

components, the phenylpropanoid component ofwhich is described in Chapter

3. The column was then eluted with 5% acetone in benzene which eluted

cyclocolorenone, recognizable by its tlc behavior and the uv-maxima

at 260 nm, as well as phytosterols which moved in tlcatslightly lower

Rf and produced a purplish-brown color when sprayed with 1% sulfuric

acid/acetic acid and heated gently. The mixture of the cyclocolorenone

and phytosterols was dissolved in methanol (10 ml) and kept at 50C

overnight. The precipitate was filtered off and set aside for gas-

chromatographic study.

The filtrate and wash were concentrated to an oil, dissolved in

benzene (20 ml) and applied to a column of Florisil (100 g) in the same

solvent. The fractions which contained the ketonic component were

combined and concentrated to yield a colorless, heavy oil; yield, 0.5%

of the bark by weight; homogeneous in tlc and gc (T 1750, retention

time on 3% OV-17, 6 ft.; 5 min.; on 3% OV-1: 3 min.); uv: Xmax 264 nm;

logc44.12 (lit. 264/4.12)113 mass spectrum: m/e 218 (M+ 100%), 203,

175, 162, 161, 149, 147, 134, 133, 119, 105, 93 and 91.











Analysis of the Phytosterol Fraction

The column consisted of 3% OV-17 on Gaschrom Q, 6 ft., programmed

from 150-275C at 100 per minute. The mixture showed components with

retention characteristics similar to those of cholesterol (Aldrich),

lanosterol (Mann Research) and B-sitosterol (Fisher). Under isothermal

conditions at 2500C, the retention times are as follows:

cholesterol 13.5 min.
lanosterol peak 1 19.0 min.
peak 2 20.5 min.

B-sitosterol peak 1 22.0 min.
peak 2 24.5 min.
peak 3 27.0 min.

Each of these peaks was homogeneous with the corresponding peaks ob-

tained from the mixture.


Cyclocolorenone 2,4-dinitrophenylhydrazone

A solution of the 2.3 (0.4 g) in methanol (20 ml) containing

2,4-dinitrophenylhydrazine (0.4 g) and 6N HC1 (0.5 ml) was heated at

50C for 15 minutes. After cooling for one hour, the dark red solid

was filtered and washed with cold methanol. It was recrystallized from

ethyl acetate; yield, 0.5 g (70%), mp 217.50C (lit. 218);113 uv max

400 nm, log 4.51 (lit. 404, log 4.40);113 mass spectrum: m/e 398, 356,

105, 91, 77, 55, 43 and 41.

Anal. calc. for C21H2604N4*H20: C, 60.56, H, 6.78; N, 13.45.

Found: C, 60.62; H, 7.31; N, 13.19.


Reduction of Cyclocolorenone to Cyclocolorenol 2.4

A solution of 2.3 (0.1 g) in absolute ethanol (5 ml) was treated

with sodium borohydride (0.05 g) and the mixture stirred for 30 minutes.











After five minutes at 50C, it was cooled, neutralized with 1N HC1 and

concentrated to a small volume. Extraction with ether, drying of the

extract and concentration gave 2.4 a colorless oil; yield, 0.07 g; uv:

no absorption above 220 nm; ir: 3200-3600 cm-1

The sample in hexane (10 ml) was stirred at 250C with activated

manganese dioxide (Baker and Adamson) for two hours. Filtration and

concentration of the filtrate gave an oil, identical with 2.3 in tlc,

and uv 'spectra.


Reduction of Cyclocolorenone to Cyclocoloranone 2.5

The procedure was an adaptation of the one described by Corbett

and Speden:113 in a flask equipped with an acetone dry-ice condenser

and containing anhydrous ammonia (500 ml) was dissolved lithium (1.89 g).

The ketone (5 g) in anhydrous ether (100 ml) was added dropwise with

stirring over a period of one hour. After another 30 minutes, ammonium

chloride (1.5 g) was added in small portions and the ammonia allowed

to evaporate overnight. The solid mass was stirred with ether, filtered,

the filtrate washed with salt solution, dried and concentrated to

dryness. It was dissolved in 1:1 benzene/hexane and chromatographed

on alumina (100 g) in the same solvent. Fractions from the major band

on concentration gave 2.5, a crystalline solid, recrystallized from

hexane; mp 38-40; yield 1 g (20%).

Anal. calc. for C15H240*H20: C, 75.69; H, 10.92. Found: C,

76.48; H, 10.79; ir: 1745 cm-1; nmr (ppm): 1.7-2.3, 6H; 1.26, s, 3H;

1.05, s, 3H; 1.11, d, 3H, J = 8.5 Hz; 0.90, d, 3H, J = 7.8 Hz and 0.75-

1.60, m, 5H; mass specrum; m/e 220 (Mt), 177, 149, 136, 135, 122, 109.











Cyclocoloranone 2,4-dinitrophenylhydrazone

This was prepared as described under the cyclocolorenone

dinitrophenylhydrazone. It is a light orange crystalline solid,

mp 207-2100 from ethyl acetate; uv: Xmax 360 nm, log 4.33.


Epicyclocolorenone

A 200 mg sample of 2.3 was chromatographed on a 50 g column

of neutral alumina (Woelm, activity grade 1) in benzene. Elution with

2% acetone/benzene gave a product, identical with 2.3 but with a Xm
max
of 255 nm instead of 264 nm. The nmr spectrum showed duplicate signals

for the geminal methyls at 1.25, 1.23 ppm, and 1.04, 1.01 ppm, indicating

a mixture containing 70% of 2.3. Repetition of this procedure with

the same sample gave lax 250 nm, with nmr indicating 40% 2.3, yield

150 mg.
120
According to the procedure of Corbett and Young,120 2.3 (200 mg)

was heated under reflux in 50 ml of ethanolic KOH (0.5 N) for two hours,

cooled, neutralized (H2S04), and reduced in volume to 10 ml. Unlike

these authors, chilling overnight did not afford a solid product. The

solution was diluted to 50 ml with water and extracted three times

with ether. The ether was dried (Na2SO4) and concentrated to give an

oil, 190 mg, which could not be induced to crystallize. The product

was homogeneous (tlc) with 2.3, xmax 255 nm. The product was isolated

by chromatography on silica gel (5% acetone in benzene) to afford 70 mg

of a ketone, ir 1610 cm- homogeneous with 2.3 on gc under the condi-

tions cited for cyclocolorenone, nmr indicated 40% epimerization.











Epicyclocolorenone 2,4-dinitrophenylhydrazone

The product of alumina-induced epimerization, 150 mg, was

converted to the DNP derivative as described for 2.3. Four recrystal-

lizations from ethyl acetate gave a product of constant mp, 188-1890C

(lit. 189),120 homogeneous with 2.3-DNP on tlc, uv 400 nm, log 4.48/

chloroform (lit. 397.5nm/4.49).120


Parthenolide 2.16

Yellowed leaves of M. grandiflora (500 g) were cut into small

pieces and extracted with ethanol for one day at 250C. Three such

extracts were combined, concentrated to a syrup which was then parti-

tioned between 0.1 N ammonium hydroxide and ethyl acetate (250 ml each).

The solvent layer was concentrated to dryness, the residue dissolved

in benzene (10 ml) and applied to a column of Florisil (50 g) in the

same solvent. Elution with 5% acetone in benzene gave the antibiotic

fraction which was recovered by concentration. The resulting oil was

stirred with hexane, filtered and the solid washed with hexane. The

filtrate was again concentrated to an oil which was triturated with

ethanol (1 ml). The crystalline solid was filtered and recrystallized

from ether-hexane; yield, 0.5 g; mp 112-4 (lit. 1150C)145; ir: 1730

and 1655 cm-1; mass spectrum: m/e 248 (M+), 233, 230, 190 and 43.

Anal. calc. for C15H2003: C, 72.55; H, 8.12. Found: 72.33:

H, 8.17.














CHAPTER 3
PHENYLPROPANOIDS OF Magnolia grandiflora L.

Bis-allylphenol from the Bark
The distribution of diallylbiphenol derivatives in Magnolia
spp. was discussed in Chapter 1. So far, three members of this group
12 53-54
have been isolated from Magnolia spp., magnolol 3.12,5354 hono-
kiol 322,54-57 9,53
kiol 3.212'5 7 and acuminatin 3.3.9, A search for these or related
compounds in Magnolia grandiflora was made but with negative results.55
In the present study, a new member of this group, a derivative of
honokiol has been obtained from M. grandiflora and its isolation and
elucidation of its structure from the subject of this chapter.



H H H3C CH3

OO OOH COHO

.H H3CO
3.1 3.2 3.3


The lipophilic fraction of the ethanolic extract of the bark
of Magnolia grandiflora when subjected to chromatography on silica
gel-cellulose (1:1) yielded a component with a characteristic uv
absorption with a Amax of 290 nm. The phenolic nature of the compound
was demonstrated by the shift in Xmax to 320 nm in the presence of
max











base. However, the compound was still contaminated with other non-

acidic components and was too lipophilic to be extracted into aqueous

base from solvents such as chloroform, benzene or ether, which might

explain why the previous workers did not recognize its presence. It

was, however, possible to separate this phenolic component from the

neutral compounds by partition between hexane and aqueous methanolic

(1:1) base. Further chromatography on silica gel gave the pure phenolic

component in a yield of 0.005-0.01% which was homogeneous by thin-layer

chromatography in two different systems and by gas chromatography.

With the help of this solvent-partition scheme and gas chromatography,

the extract of the wood of M. grandiflora was examined for the presence

of this phenolic component but it was found to be absent.

The phenolic component is a colorless oil with the molecular

formula C19H2002 on the basis of elemental analysis and mass spectrum

(Mt 280). Its uv spectrum showed maxima at 255 and 290 nm with log E

of 4.10 and 3.8, respectively, which shifted to 320 nm on the addition

of base. Its ir spectrum--3200-3600 cm-1 (phenolic OH), 1620 cm-1

(aromatic, 985, 905 cm-1 (CH=CH2)--indicated the presence of phenolic

and olefinic functions. The nmr spectrum--6.65-7.22 ppm, m, 6 aromatic

H; 5.55-6.32 ppm, m, 2 CH2CH=CH2; 4.92 ppm, m, and 5.11 ppm, m, 2 CH2CH=CH2;

4.86 ppm, s, exchangeable, ArOH; 3.85 ppm, ArOCH3, 3.28, 3.40 ppm,
43
overlapping doublets, J = 6 Hz, 2 ArCH2CH=CH2--clearly indicated that

there are two non-equivalent allyl groups on aromatic rings comprising

1,2,4 substitution patterns.











The allyl groups were readily reducible to afford a tetrahydro

compound. Elemental analysis and the mass spectrum (M+ 284) were

consistent with the molecular formula C19H2402. The nmr spectrum showed

two non-equivalent benzylic n-propyl groups: 2.40 and 2.62 ppm, over-

lapping triplets, J = 5.2 Hz, 2 ArCH2CH2CH3; 1.63 and 1.67 ppm, over-

lapping sextets, J = 8 Hz 2 ArCH2CH2CH3, 0.94 and 0.97 ppm, overlapping

triplets, J = 8 Hz, 2 ArCH CH CH3.

Acetylation of the phenolic component gave a monoacetate

C21H2203 (M+ 322). The ir spectrum of the acetate (1755 cm-1) and the
nmr signal (2.02 ppm, s, 3H) showed that the compound was a phenolic

acetate.

Methylation provided a monomethyl ether C20H2202 (Mt 294) which

showed nohydroxyl signal in its ir spectrum and two singlets 3.68 and

3.71 ppm, each representing a methoxyl group, in its nmr spectrum.

The preceding data suggested the possibility that the lipophilic

phenolic component of Magnolia might be one of the monomethyl ethers

of the diallylbiphenols: magnolol 3.1, honokiol 3.2, or 3,3'-diallyl-

4,4'-dihydroxy-biphenyl 3.4 (which has not yet been isolated from a

natural source). To confirm this possibility and to determine which

of the three substitution patterns corresponds to the phenol in ques-

tion, the respective di-0-methyl tetrahydro-derivatives 3.5, 3.6 and

3.7, respectively, of the three were synthesized for comparison with the

methylated, reduced phenol from Magnolia.

For the synthesis of the dipropyl biphenols 3.8, 3.9 and 3.10,

there are several methods based on oxidative coupling of phenols avail-

able using ferric ion,149 hydrogen peroxide,150 hydrogen peroxide and











ferrous sulfate,51 and enzymic catalysis.52 However, not only are

the yields in those reactions low ( 10%) but with multiplicity of

sites for coupling, unequivocal assignment of structures becomes diffi-

cult, esepcially when unsymmetrical coupling is involved. Hence, the

Ullmann coupling procedure employed by Fujita et al.12 was selected as

a better synthetic route because of the greater certainty of the course

of reaction and relatively higher yields. The necessary iodo compounds

are accessible from the commercially available p-propyl phenol 3.11 and

the o-allyl phenol 3.12.


OR1 R3



R3 OR2


SR1i R3

O R2
R3


3.1, R1 = R2 = H
R3 = CH2CH=CH2

3.5, R1 = R2 = CH3
R3 = CH2CH2CH3

3.8, R1 = R2 = H
R3 = CH2 CH2CH3


3.2,
R3

3.6,

R3 =
^3-
3.9,
R3


R1 = R2 = H
CH2CH=CH2

R1 = R2 = CH3

CH2CH2CH3

R1 = R2 = H
CH2CH2CH3


3.4, R1 = R2 = H
R3 = CH2CH=CH2

3.7, R1 = R2 = CH3

R3 = CH2CH2CH3

3.10, R1 = R2 = H
R3 = CH2CH2CH3



















3.11 3.12 3.13 3.14 3.15
3.13, R = H 3.15, R = H
3.16, R = CH3 3.17, R = CH3

For the preparation of the iodo compound 3.13, iodination of

3.11 in aqueous base was employed. The yield of the monoiodo compound

3.13 did not go beyond 50%, due to competing formation of the diiodo

compound 3.14, as determined by gas chromatography. Because of the

limited yield and the difficulty of large-scale separation of 3.13 and

3.14, an alternative procedure described by Hata and Sato,153 using

iodine and mercurous oxide, was found to be preferable. The monoiodo

compounds 3.14 and 3.15 were readily prepared by this procedure with no

competing iodination of the iodophenols,and converted to their respec-

tive methyl ethers 3.16 and 3.17, suitable for the coupling reaction.

The method adopted for the Ullmann reaction is essentially that

of Fujita et al.153 using copper powder activated by the procedure of

Kleidner,154 and heating without solvent at 2600C. Maintaining the

reaction at this temperature for six hours after completion of addition

of the copper was found to be preferable to increasing the temperature

as recommended by Fujita et al.12

Under these conditions, dimethyl tetrahydromagnolol 3.5 was

obtained in a 30% yield from 3.16. Coupling of 3.17 provided











3,3'-di-n-propyl-4,4'-biohenol dimethylether 3.17 ina 60% yield as a

colorless, crystalline solid. Crossed coupling of 3.16 and 3.17 in

equimolar proportions yielded a mixture of the three expected products,

3.5, 3.7, and dimethyl tetrahydrohonokiol 3.6 in a ratio of 1:1:2 as

determined by gas chromatography. The dimethyl ethers were converted

to the corresponding biphenols 3.8, 3.9, and 3.10 by hydrolysis with HI.

The methylated, hydrogenated phenolic component from Magnolia

was found to be identical with dimethyl tetrahydrohonokiol 3.6 by

spectral, thin-layer, and gas-chromatographic comparison. Since the

natural product is a monomethyl ether, it still remained to determine

which of the two monomethyl ethers of honokiol, 3.18 or 3.19, actually

represents the correct structure for the phenolic component from Magnolia.




CH3 H

QHO" Qch3


3.18, R = CH2CH=CH2 3.19, R = CH2CH=CH2

3.20, R = CH2CH2CH3 3.21, R = CH2CH2CH3


A number of alternatives was considered for providing an un-

equivocal choice between 3.18 and 3.19. For example, if the natural

product had the structure 3.18, an acid-catalyzed cyclization to a

furan or a chroman might be possible, or oxidation of the allyl func-

tions to carboxyls can result in the formation of a salicylic acid

derivative. Either the spectral properties of the cyclic ether or the











spectral and completing properties of the salicylic acid derivative

would permit a choice to be made. However, if 3.19 were the correct

structure, both of these would be negative and it is not prudent to

rely on negative data. Since the supply of the natural product was

also very low, the selected method must be based more on a more readily

available synthetic compound such as 3.8. If the isomeric monomethyl

ethers 3.20 and 3.21 of tetrahydrohonokiol were synthesized and their

respective structures established, identity of the tetrahydro derivative

of the natural product with one of these isomers of known structure

would provide the structure for the natural product.

Accordingly, with the expectation that possible influence of

the n-propyl group on the ortho-methoxyl of 3.6 might result in selec-

tive demethylation, reaction of 3.6 with anhydrous aluminum chloride

was studied. The reaction, however, yielded both monomethyl ethers 3.20

and 3.21 in equal amounts, along with the fully demethylated 3.9.

Alternatively, complete demethylation of 3.6 to 3.9 and careful partial

methylation gave the mixture of 3.20 and 3.21 in a better yield. Based

on the thin-layer chromatographic Rf values, they were designated as

ether-A (higher Rf) and ether-B (lower Rf). Of these, A was found to

be identical with the tetrahydro derivative of the natural product,

and it remained to associate the ether-A with 3.20 or 3.21 to establish

the choice between 3.20 and 3.21.

For a definitive assignment of the structures of the ethers

A and B (3.20 and 3.21), a method based on degradation of the aromatic

ring which carries the phenolic hydroxyl to yield an n-propyl anisic











acid, followed by establishment of its identity by synthesis was found

to be the most conclusive. Thus, 3.20 and 3.21 under conditions of

such degradation will yield the acids 3.22 and 3.23, respectively.





OCH3 O OCH3 CH3

COH HOC
OH II
0 0
3.22 3.23 3.29

For the degradation of the phenolic ring, ozonolytic cleavage

was selected. Although no difference was noted in the rate of degrada-

tion by ozone of phenol and anisole, used as model compounds, in a

neutral medium, phenol was degraded rapidly in a basic medium while

anisole was relatively unaffected. The monomethyl ether 3.29 prepared

from 3.10, upon ozonolysis at pH 9, followed by a brief treatment with

potassium permanganate, readily yielded 3.23 whose analytical and spec-

tral data indicated that it was an n-propyl anisic acid. In a similar

manner, the ether-A (3.21) gave the acid 3.23 and the ether-B (3.20),

the acid 3.22. The identity of these two acids remained to be estab-

lished through synthesis.

A search of the literature revealed that although related

compounds were described, neither of the acids 3.22 of 3.23 are known.

It was proposed to synthesize these acids by Friedel-Crafts acylation of

the appropriate hydroxybenzoic acids, followed by Clemmensen reduction of











the keto groups. With regard to acid 3.22, 5-propionyl salicylic acid

3.25 has been prepared by the Fries rearrangement of the propionate

ester 3.24.155 Itwasmethylated to the ether but not reduced to the

2-methoxy-5-n-propylbenzoic acid 3.22. Cox also carried out a Fries

rearrangement of the propionate of methyl salicylate.156 Although no

assignment was made for the product, the properties appear to correspond

to the ortho-migration product, 3.26.


OR 0 R OR


O\ '^( Q or Q


3.24 3.25, R = H 3.26



Analogs of the acid 3.23 have been prepared by two routes. In

one of these, ethyl 3-allyl-4-hydroxybenzoate 3.28 was obtained by

Claisen rearrangement of the corresponding allyl ether 3.27.157,158,159

Methylation gave the ether 3.30, which on hydrolysis gave the acid

3.31.157 Hydrolysis and reduction of 3.28 gave the propylanisic acid
159
3.29. However, neither the methoxy derivative 3.23 nor its ester

3.31 was prepared.

The second route requires acylation of p-hydroxybenzoic acid to
give the ketone 3.34, followed by reduction of the side chain. Fries

rearrangement of the propionyl ester 3.32 gave 3.34'160 but this com-

pound was not reduced.
















ROOR'


3.27, R = CH2CH = CH2
R' = C2H5
3.32, R = COCH2CH3
R' = H


3.28, R = H, R' = C2H5
3.30, R = CH3, R' = C2H5
3.33, R CH3, R' = H


3.29, R = R' = H
3.31, R = R' = CH3


3.34

In the present work, Friedel-Crafts reaction of methyl

salicylate with propionyl chloride gave methyl 5-propionyl salicylate

(3.35).a This was reduced by the Clemmensen method to methyl

5-n-propylsalicylate and methylated to methyl 2-methoxy-5-n-propyl

benzoate (3.36)






0

3.35



aThe melting point of 5-n-propylsalicylic acid 3.25 obtained by
hydrolysis of this sample is much greater than obtained by Cox156
(210.5-213 vs. 177-90C), supporting the contention that Cox has ob-
tained the wrong isomer.





60





For the synthesis of the ester of 3.23, the allyl ether of

methyl p-hydroxybenzoate 3.37 was subjected to Claisen rearrangement

to methyl 3-allyl-4-hydroxybenzoate 3.38. This was reduced to the

n-propyl derivative 3.36 and methylated to methyl 4-methoxy-3-n-

propyl benzoate 3.31.


OCH3 CH3 OCH

O O O


0 OH OH

3.37 3.38 3.39


With the availability of the authentic samples of the two

esters 3.36 and 3.31 which correspond to the acids 3.22 and 3.23, a

comparison was made of the product of ozonolysis, brief permanganate

treatment and esterification with diazomethane of the tetrahydro

honokiol monomethyl ethers. Ether-A (higher Rf), which was identical

with the tetrahydro derivative of the natural product, gave as

product 3.31 while ether-B (lower Rf) gave 3.36. On the basis of

these results, the structure of the natural product can be repre-

sented as 3.19.


Lignan from the Wood

From the lipophilic phase of the wood extract of Magnolia

grandiflora was isolated a crystalline solid, melting point 180-820C

[a]D = +4.00 (c 1.12,CHC13). Its characteristic uv spectrum, Xmax











270 nm, log E 3.41; 240 nm, log E 4.21, and a base-induced shift of the

maximum indicated that it was a phenolic substance. The mass spectrum

(Mt 418) and elemental analysis agreed with the molecular formula

C22H2608. The nmr spectrum showed the presence of four equivalent
methoxyl groups and four equivalent aromatic protons. The presence of

two phenolic hydroxyls was deduced from the formation of a diacetate

(ir: 1763 cm-1; nmr 2.32 ppm, 6H, s) and a dimethyl ether, which showed

nmr signals for six methoxyl groups. The aromatic portion of the nmr

spectrum with two equivalent protons on each ring may be ascribed to

one of the two alternative structures:




3C CH3

H Q HO

H3C HCH3
3.39 3.40


On the basis of the molecular formula and other nmr spectral

characteristics which suggest a relationship to the lignoids, the

syringyl residue 3.39 is much more consistent with the biogenetic origin

of this class of compounds. Two syringyl residues are therefore con-

sidered to be present in the structure. The nmr spectrum, an analysis

of which will be presented later, also suggested that the compound

might possess a furofuranoid skeleton as found in lignans of the pinore-

sinol type.











Confirmation for the furofuran type structure was obtained

through the analysis of the mass spectrum along the lines described by

Pelter for pinoresinol.161 A list of the major peaks and their probable

origins and structures is shown in Figure 3.1. The close correspondence

of the observed peaks with those expected for such a system showed that

the Magnolia lignan is one of the diastereomeric lirioresinols and it

remains to determine which of the three stereochemical representatations

shown in Figure 3.2 is the correct one for the compound. (Relative

stereochemistries were determined by Briggs, Cambie, and Couch.)162

Although three more isomers with a trans fusion of the furofuran

ring system are theoretically possible, no examples of such a lignan

have been isolated to date. The representation of the three known

lirioresinols is due to Dickey et al., who first isolated two of these

lirioresinols (A and B) by the acid hydrolysis of liriodendrin, a

glycoside from Liriodendron tulipifera L. (Magnoliaceae) as well as

from commercial beechwood sulfite liquors (Populus spp., Salicaceae).164

Of these, syringaresinol corresponds to dl-lirioresinol B.162 Emulsin-

hydrolysis of liriodendrin gave an aglycone which was first assigned the

structure of lirioresenil C163 but was later shown to be a mixture
162
containing lirioresinol B as the major component. A compound of the

structure of lirioresinol C has not yet been isolated from a natural

source, although its dimethyl ether has been obtained from Macropiper

excelsum (Forst. f.) Miq. (Piperaceae).162

The confusion that exists with lirioresinols is partially due

to the lack of reliable chromatographic and.spectroscopic information

together with the variation in melting points probably arising from















M+ (P)
m/e 418 (100/100%P)a

"ArCH2 ArH
m/e 167 m/e 154
(30-50/58%P)(10-15/13%P)





+ /

A 0 'Ar 0



Ar
m/e 251 m/e 388
(3/7%P) (2/4%P)


0 Ar


Ar
Ar


(20-30/32%P)
ArCEO+
m/e 181
(50-60/68%P)



\ IA
B C


/ I
A Ar


(Ar ,OH)
m/e 210
(16/10%P)


+











H OCH3
m/e 235
(15/10%)


Ar
B I__

+to
m/e 209
(6/11%P)

C


Ar ~~+
m/e 193
(50-60%)


Phenol and Phenol Ether Processes
M+-15(CH3 ) m/e 403 (2%P)
M+-28(CEO) m/e 390 (0%P)


M+-29(CHO.) -
M+-30(CH20) -


m/e 389 (1%P)
m/e 388 (4%P)


alntensity of peak from Magnolia lignan/intensity of corresponding peak
of pinoresinol


Figure 3.1


Comparison of mass-spectral fragmentation patterns of
Magnolia lignan and pinoresinol











different enantiomeric compositions.162'164 The physical properties of

the three lirioresinols and their derivatives as reported in the liter-

ature are summarized in Table 3.1. Although the data from the table

indicate that the lignan in question is indeed identical with syringa-

resinol B, there is sufficient variation in the literature data that

such a conclusion may not be unequivocal, and additional evidence is

desirable.



Ar 1 Ar Ar 0

H H H H How

Ar 0 r "'Ar

3.41 3.42 3.43
Lirioresinol A Lirioresinol B (Lirioresinol C)
(axial/equatorial) (diequatorial) (diaxial)

H3C

Ar = HO

H3C

Figure 3.2 Relative stereochemistries of the lirioresinols


The infrared spectra (KBr pellets) of the Magnolia lignan and

of synthetic syringaresinol163 (cf. Figure 3.3) are practically identi-

cal and differ sufficiently from that of lirioresinol A that the latter

may be excluded from consideration. However, such solid state spectra

may not be completely defendable, as the formation of a racemic compound









Table 3.1 Properties of the lirioresinols and their derivatives


Compound mp, OC [ia]D Reference


Lirioresinol A



Lirioresinol A
dimethyl ether
diacetate

Lirioresinol B



dimethyl ether





diacetate

Syringaresinol
(dT-lirioresinol B)



dimethyl ether

diacetate



Lirioresinol Cc
dimethyl ether

Magnolia lignan
dimethyl ether
diacetate


210-211
180-181
177


118-120
188


177-183
175-179a
172-179
170
126.5-127
122-123
122-123
121-122
116-118a
115-119a
188

179-185.5
175
174
170-171
168-172
107-9,
107-8"
188-189
181-182
181-182


145-147

180-182
107.5-108
185-186


+127

-90


+119


-34.8

+62.2
-21.5

+46.2
+45.8
+46.0




0
+1.93
0
0
+3.93
-4.14
0
-4.73

0


+284

+4.0


170
171
172
173
164
171
172
171
164
173


this work
this work
this work


aAssignment unclear
Synthetic
cGenuine--many reports of lirioresinal C are incorrect


















1i






F I c


:1 it1] -



F -. "I


i F~'-
11+'i









a Ii


-I it~ I

I I(

-if ~ -a C


C--.













-le










J-











may alter the infrared spectra according to the enantiomer con-

tent.174

The nmr spectra of lirioresinols A and B are not available,

although the corresponding isomers in the pinoresinol series are.1

The two spectra are presented in Figure 3.4 and the spectrum of the

Magnolia lignan is shown in Figure 3.5. General similarities between

the spectrum of Magnolia lignan and that of (+) pinoresinol (the

stereochemistry of which is firmly established),176 are clearly apparent

as are differences from that of (+)-epipinoresinol (same stereochemistry

as lirioresinol A). Additional evidence that the Magnolia lignan

possesses equatorially disposed aryl groups is obtained from assignment

of these resonances.

In lirioresinol B, where both a-protons are axially disposed,

the resonance appears at 4.75 ppm. In lirioresinol C, where both

protons are oriented equatorially, they absorb at 4.98 ppm, while in

lirioresinol A, the axial proton absorbs at 4.41 ppm, and the equatorial

proton resonance appears at 4.83 ppm. This reflects greater shielding
162
of the axial protons in the presence of an endo-aryl group.162 In the

case of the Magnolia lignan, the a-protons show a single resonance

at 4.75 ppm, indicating that both protons are axially disposed; thus

the Magnolia lignan has the same relative stereochemistry as liriores-

inol B, which has been definitively shown to have a diequatorial dis-

position of the aryl groups by x-ray studies.177 Assignments of these

resonances based upon JaB are in error: it has been stated that in

lignan spectra, overdependence on coupling constants, based upon bond






















(a) Nmr spectrum of (+)-epipinoresinol (stereochemistry of
lirioresinol A)


ac Ye


(b) Nmr spectrum of (+)-pinoresinol (stereochemistry of lirioresinol
B)


6Ha 4.98 ppm (d, J =5 Hz) H 0
6HB 3.25 ppm H0 N
MHYeq 3.63 ppmR
6Hyax 3.72 ppm O R R


(c) Spectral assignments for lirioresinol C dimethyl ether (R = OCH3)

Figure 3.4 Nmr spectra of the three pinoresinol lirioresinol skeleta










69




























0


























r-
0 -<
























0













*r-

o
r-










E




\ U)
c-











J*

I)
La


u-











angles is undependable.178179 More emphasis is placed upon such

shielding effects in making assignments.162'178'180

The isolation of syringaresinol from Magnolia grandiflora

constitutes the first report of its presence in any species of Magnolia.

The dimethyl ether has been isolated from Magnolia Kobus59 and Magnolia

(or Michelia) Fargesii. All lignoids isolated from Magnolia spp. to

date are of three types: pinoresinol type, the galgravin type, (cf.

Figure 1.3) and the bisallylphenol neolignans. As a chemical entity,

syringaresinol was known long before its isolation from a natural

source, having been obtained through oxidative, enzymic coupling of

sinapyl alcohol.172181 Subsequently, it was synthesized by nonenzymic

oxidative coupling of sinapyl alcohol and sinapic acid.182 In the

latter synthesis, the dilactone initially produced was reduced and

pyrolyzed to yield syringaresinol.

Syringaresinol glycosides occur in a number of plants, including

Magnolia grandiflora,49 and Liriodendron tulipifera L.163 (Magnoliaceae).

Syringaresinol itself, as well as the optically active form lirioresinol

B, has been obtained from Liriodendron tulipifera167'169 (Magnoliaceae)

Populus spp.,164 Fagus spp.173182 (Betulaceae), Picea excelsa

Engelm.183 (Pinaceae), Sinomenium acutum Rehd. et Wils.171 (Menisperma-

ceae), and Xanthoxylum inerme Koidz.170 (Rutaceae). The dimethyl ether

has been obtained from Liriodendron tulipifera L167,169 (Magnoliaceae),
168
Eremophila glabra68 (Myoporaceae), Macropiper excelsum (Forst. f.)

Miq.162 (Piperaceae), and Aspidosperma Marcgravianum Woodson166

(Apocynaceae), in addition to the Magnolias previously cited.











Experimental
Isolation of Mehonokiol 3.19

Ground bark (4 kg) of Magnolia grandiflora, collected in Gaines-

ville, Florida, was extracted with ethanol (3 Z) at 250C for two days.

Three such extracts were combined and concentrated to a syrup which

was partitioned between water and ethyl acetate (1 Z each). Concentra-

tion of the solvent extract gave a heavy oil (120 g).

Ten-gram portions of the oil were chromatographed on silica gel

(250 g) in benzene. The benzene eluates contained, besides carotenoid

pigments, a phenolic component (tlc: Rf 0.66 in silica benzene; positive

diazo coupling). Fractions containing this were combined, concentrated

and partitioned between hexane and NaOH (0.1N) in methanol:water (1:1).

The aqueous alcoholic layer was concentrated, acidified (pH 2) and

extracted with ether. The product from the ether was rechromatographed

on silica gel using benzine:Ji.exane (1:1). Fractions of the major

band on concentration gave a colorless, heavy oil which was homogeneous

on tic and gc; yield, 0.2 g, 0.005%; uv: Xmax 255 nm (log E 4.1) and
-1
290 nm (log e 3.8); with base, mA 320 nm; ir (neat, cm- ) 3560,

3400-3200, 3090, 3010, 3000-2900, 2860, 1630, 1600, 1490, 1480, 1455,

1430, 1425, 1275, 1240, 1170, 1130, 1115, 1045, 1025, 987, 905, 810,

780, 725: nmr (ppm): 7.22-6.65, m, 6 ArH; 6.32-5.55, m, 2 CH2CH=CH2;

5.81, m, 4.92, m, 2 CH2CH=CH2; 4.86, s, exchangeable ArOH; 3.85, s, OCH3;

3.40 and 3.28, overlapping doublets, J = 6 Hz, 2 ArCH2CH=CH2; ms:

280.1456; calc. for C10H2002: 280.1464.











Mehonokiol Acetate

A mixture of 3.19 (0.05 g), acetic anhydride (1 ml) and pyridine

(two drops) was heated at 700C for ten minutes. It was cooled and

diluted with water. After 15 minutes, it was extracted with ether,

the extract washed successively with dilute acid and aqueous sodium

bicarbonate, concentrated to dryness and purified by a short silica

gel column (5 g) in 2:1 hexane/benzene. The major fraction was

obtained as a colorless, heavy oil: ir: 1755 cm-1; nmr: 2.01 ppm, s, 3H;

ms: M+ 322.1566, calc. for C21H2203: 322.1570.

Dimethylhonokiol

A mixture of 3.19 (0.1 g), methyl sulfate (0.1 ml) and anhydrous

potassium carbonate (0.5 g) in acetone (5 ml) was stirred for 15 hours

and filtered. The filtrate and wash were concentrated to dryness and

purified by chromatography on silica gel (5 g) in 1:1 benzene/hexane

to give the methyl ether as a colorless oil, ir: no OH; nmr (ppm); 3.71,

s, 3H; 3.68, s, 3H; ms: M+ 294.1616; calc. for C20H2202: 294.1621.

Tetrahydromehonokiol 3.21

A solution of 5 (0.1 g) in ethanol (5 ml) was hydrogenated in the

presence of PtO2 in a Parr apparatus at 30 psi for one hour. The mix-

ture was filtered and the filtrate concentrated to dryness to yield

3.21 as a colorless oil; ir: no bands at 1620, 985 or 905 cm-1; nmr

(ppm); 240 and 2.62 ppm, overlapping triplets, J = 5.2 Hz, 2 ArCH2CH2CH3;

1.63 and 1.67 ppm, overlapping sextets, J = 8 Hz, 2 ArCH2CH2CH3, 0.94

and 0.97 ppm, overlapping triplets, J = 8 Hz, 2 ArCH2CH2CH 3; ms:M+ 284,

m/e 255, 223, 205.











Anal. Calc. for C19H2402: C, 80.25; H, 8.52. Found: C, 80.24,

H, 8.80.


4-Iodo-2-propylanisole 3.17

o-Allylphenol (Aldrich Chemical Co., 6 g) was hydrogenated as

described for 3.21 and methylated in acetone (200 ml) with methyl

sulfate (10.5 ml) and potassium carbonate (15 g), stirring at room

temperature for 36 hours. The product, 2-propyl anisole (10 g) was

iodinated by the method of Hata and Sato153 using mercuric oxide-

catalyzed iodination by 12 in ethanol. The resulting product was

purified by distillation (1.5 mm, 105-1150C) to give 3.13; yield 10 g

(90%).


2-Iodo-4-propylanisole 3.16

Anethole (Aldrich Chemical Co., 10 g) was hydrogenated and the

product iodinated as above. The iodinated product was purified by

distillation (1.5 mm, 1000C); yield 8 g (88%).

Tetrahydromagnolol dimethyl ether 3.5

The coupling of 3.16 (10 g) was carried out by a modification of

the procedure of Fujita et al.,55 in which the copper powder (22 g)

was gradually added to 3.16 at 2400C. After completion of the

addition, the mixture was maintained at this temperature for four to

six hours instead of heating to 285C. This change gave a better

quality product and a higher yield. Purification was by chromatography

on silica gel. Using benzene:hexane (1:1) as the eluent 3.5 was ob-

tained as a colorless oil, 3 g; yield, 56%; uv: 287 nm (log e 3.7),











256 nm (log E 4.0); nmr (ppm): 6.70-7.20, m, 6 ArH; 3.68, s, 2 OCH3;
2.50, 1.60 and 0.95, triplet/sextet/triplet, respectively, 2 CH2CH2CH3.

Demethylation of 3.5 (1 g) with acetic anhydride (10 ml) and
hydriodic acid (8 ml) gave tetrahydromagnolol, yield 0.5 g (64%); mp
142-142.5C (lit. 1430C).55

4,4'-Bis(2-propyl)anisole 3.7
Coupling of 3.17 (8 g), carried out as described under 3.5, gave
3.7 as a colorless, crystalline solid; yield, 4.3 g (95%); mp 113-14C
(lit. 1140C).55
Demethylation of 3.7 (1.2 g) with hydroidic acid (7 ml) gave
4,4'-bis(2-propyl)phenol, yield, 1 g (97%); mp 113-1140C (lit. 112.50C).55
Methylation of 4,4'-bis(2-propyl)phenol (0.75 g) in acetone (20
ml) with methyl sulfate (0.36 g) and potassium carbonate (1 g) at 250C
for 24 hours gave a mixture of products from which the monomethyl ether
3.24 was separated by chromatography (silica gel, hexane with benzene
gradient) and crystallized from hexane; yield, 0.4 g (50%); mp 79-800C;
nmr: (ppm) 3.4-2.6, m, 6-ArH; 4.88, s, Ar-OH; 6.23, s, OCH3; ms: Mt 284.

Dimethyltetrahydrohonokiol 3.6
Crossed Ullmann coupling of 3.16 and 3.14 was carried out using
6 g of each as described under 3.5. The product (gc: 3% OV-17, T 1800C,
3.5, tr 11 min., 25%; 3.6, tr 15.8 min., 55%; and 3.7, tr 20.8 min.,
19%) was subjected to chromatography on Florisil for preliminary
purification. Direct crystallization gave 3.7 (0.9 g), the mother
liquors were purified by chromatography (silica gel, cyclohexane with











a benzene gradient, 25-100%). The first two product bands contained

3.5 and 3.7 and the third band the desired 3.6, identical with the

natural 0-methyl tetrahydromehonokiol (gc as above; tic: Rf 0.70,

silica, benzene:hexane, 1:1); yield, 1.3 g. A total yield of 67% of the

three coupled products (99% conversion) was obtained.

Demethylation of 3.6 (1 g) by hydriodic acid (10 ml) gave

tetrahydrohonokiol 3.9; yield, 0.74 g (83%); mp 117-117.50C (lit.

118oC).53

Mono-0-methyltetrahydrohonokiols 3.21 and 3.22

A solution of 3.9 (1.1 g) in acetone (25 ml) was stirred with

methyl sulfate (0.64 g) and potassium carbonate (2 g) at 250C for 48

hours. In addition to the dimethyl ether (40%), and the unreacted

diol (20%), the two monoethers were formed in a yield of 20% each

(gc: 3% SE-30, 2000C; 3.21 t 5.5 min.; 3.22 t 8.3min., tic: Rf 0.90,

0.60, respectively, silica benzene). The monoether of higher Rf was

identical with the natural tetrahydromehonokiol by tec, gc and spec-

tral comparison. The mixture was separated by chromatography (silica

gel, cyclohexane with a benzene gradient). The high Rf ether 3.21

was obtained as a colorless oil: ms: 284.1790 (calc. for C19H2402

284.1775); m/e 269 (100%),256 (17%) and 255 (80%).

Anal. Calc. for C19H2402: C, 80.24, H, 8.50. Found: C, 80.32,

H, 8.71.

The low Rf ether 3.22 was obtained as a colorless oil; (spectral
data similar); Anal. Calc. for C19H2402* H20: C, 77.85; H, 8.59.

Found: C, 77.69: H, 8.65.











Partial Demethylation of Tetrahydrohonokiol Dimethyl Ether

A mixture of 3.6 (50 mg) and aluminum chloride (50 mg) in

nitrobenzene (50 mg) in nitrobenzene (5 ml) was stirred overnight,

diluted to 20 ml (benzene) and washed three times with HC1 (IN),

extracted twice with brine. The phenols were extracted into 30%

methanol/KOH (0.5), the extract neutralized and examined by gc as

above: 3.20 and 3.21 were formed in equal amounts.

Ozonolytic Degradations

Ozone from a generator (Ozone Research and Equipment Co.) was

bubbled into a solution of 3.24 (0.1 g) in N ethanolic potassium

hydroxide (0.02N) at -300C until the abosrbance value at 290 nm reached

a stable value at approximately 10% of the original. The reaction

mixture was maintained basic during this time with additional base when

needed. The mixture was concentrated to remove the ethanol, the resi-

due dissolved in water (10 ml) and treated with 5% aqueous potassium

permanganate dropwise until the reaction became slugglish. It was

then acidified (pH 1-2) and treated with sodium bisulfite until a

clear, colorless solution was obtained, which was extracted twice with

ether. The acidic product was recovered from this by washing with

aqueous bicarbonate, followed by acidification of the aqueous layer

and reextraction with ether. The product from the extract was esteri-

fied with diazomethane to the crystalline methyl ester, 35 mg,

mp 35-37C; underpressed when mixed with an authentic sample of methyl

3-n-propyl-p-anisate 3.31. The spectral (ir, nmr) and chromatographic

were identical (tlc: Rf 0.60, silica, benzene; gc: 3% SE-30, 1300C,

tr 7 min.).











Similar degradation of 3.21 gave, after esterification, a product

identical with methyl-3-n-propyl e-anisate 3.31 by mp, spectral, and

chromatographic comparison.

Degradation of 3.22 and esterification gave a product identical

with methyl-3-n-propylsalicylate 3.36 by spectral and chromatographic

comparison (tlc: Rf 0.25, silica, benzene; gc: 3% SE-30, 1300C, tr,

7.3 min.)

Methyl-3-n-propylsalicylate 3.36
Methyl salicylate (2 g) in nitrobenzene (10 ml) was treated with

anhydrous aluminum chloride (1.7 g) and then with propionyl chloride

(1.3 g), added dropwise at 00C. After 16 hours at 50C it was diluted

with benzene and washed successively with 1N hydrochloric acid, water

and 0.1N sodium hydroxide. The basic phase was acidified, extracted

with ether and the product purified by chromatography (silica gel,

1:1 benzene:hexane (1:1)). Methyl-5-propionyl-salicylate 3.35 was

obtained as a colorless crystalline solid, mp 61-62C; yield, 0.5 g.

The sample was boiled under reflux with zinc amalgam (5 g) and

6-N hydrochloric acid (50 ml) for five hours. The cooled mixture

was extracted with ether and the extract concentrated. Chromatography

of the residue gave methyl-3-n-propylsalicylate 3.36 as a colorless

oil; yield, 0.35 g; ir: 1730, 1610, 1255 cm-1; nmr (ppm); 7.60, 72.6,

6.86, pattern of 1,2,4-protons on benzene, 3H; 3.86, s, 6H; 2.56, 1.62,

0.91, triplet/sextet/triplet; ArCH2CH3CH3.

Saponification (1N KOH, reflux 5 min.) gave, on workup, the

acid 3.25, mp 210-211.5C from water (lit. 177-90C)156 (cf. p. 59).











Methyl-3-n-propyl-p-anisate 3.31

A mixture of methyl p-hydroxybenzoate (6 g) allyl bromide (5.2 g)

and anhydrous potassium carbonate (8 g) in acetone was boiled under

reflux for six hours. The cooled, filtered reaction mixture was con-

centrated to yield 4-carbomethoxy phenylallyl ether 3.37 as a colorless

oil; yield, 6.8 g (90%); ir: 1720, 1610, 1000, 930 cm-l; nmr (ppm)

7.96, 6.89, AB-pattern, 4H; 6.40-5.10, 4.60, m, CH2CH=CH2, 3.85, s,

OCH3'

A sample of 3.31 (5 g) was rearranged by heating (N2) in phenyl

ether (20 ml) at 2200C for five hours. After cooling, the phenolic

product was separated by extraction with sodium hydroxide (0.1N) and

recovered by acidification of the basic extract followed by extraction

with ether. Concentration of the extract gave methyl 3-allyl-4-hydroxy*

benzoate 3.38 as a colorless oil, yield 4.5 g (90%); ir: 3600-3200,

1685, 1640, 1610, 990, 910 cm-l; uv: 262 nm, log e 4.2, shifted to 306

nm with base.

The sample was methylated in acetone by stirring with methyl

sulfate (5 g) and potassium carbonate (8 g) for 15 hours at 250C. The

mixture was filtered and the product recovered by concentration of the

filtrate and crystallization; mp 122.5-1250C. When subjected to

catalytic hydrogenation in ethanol in the presence of Pt at 40 psi in

a Parr apparatus, it gave methyl-3-n-propyl-p-anisate 3.31 as a

colorless crystalline solid; mp 36-370C. It was identical with the

methylated ozonolytic degradation product of tetrahydromehonokiol, 3.21.

Saponification (lN KOH, refluxed 5 min.) gave on workup the acid,

3.29, mp 115-17C from water (lit. 116.80C).159











(dl)-Syringaresinol 3.43

The ethyl acetate phase of the wood extract (10 g) was taken up
in ethyl acetate (50 ml) and extracted three times with KOH (0.01 N,

30 ml). The combined extracts were neutralized and reextracted with

130 ml chloroform. The combined chloroform phases were dried (Na2SO4),

concentrated, the phenolic lignan extracted and isolated by chromatogra-

phy (silica gel, 20 g, benzene with ethyl acetate solvent gradient).
The lignan, (tlc: Rf 0.30, 5% methanol:chloroform), was eluted by

25% ethyl acetate, the lignan-containing fractions concentrated and

crystallized from ether, affording 3.43; 60 mg, mp 180-182C; [a]D

+4.00 (c 1.12 CHC13); uv: 220 nm, log E 3.41; 240 nm, log e 4.21

(lit. 270 nm/3.46, 237 nm/4.66),164 ir, nmr: mp cf. Figures 3.3, 3.5,

and Table 3.1.

Anal. Calc. for C22H2508: C, 63.14; H, 6.26. Found: C, 62.83;
H, 6.34.

Syringaresinol Dimethyl Ether

The lignan 3.43 (100 mg) was methylated with dimethyl sulfate
(0.5 ml) and anhydrous potassium carbonate (0.79) in acetone (15 ml),

with vigorous stirring for 48 hours. The reaction mixture was filtered,

the filtrate concentrated and the residual oil chromatographed on a 50 g

silica-gel column (50 g). It was eluted with 2.5% acetone in benzene.
The fractions of the band, on concentration, gave 100 mg (92% yield)

of a colorless, crystalline solid, recrystallized from ether: mp 107.5-

108C; ms: cf. Figure 3.1; uv: 270 nm, log E 3.40; 240 nm, log E 4.21.

Anal. Calc. for C24H3008: C, 64.56; H, 6.77. Found: C, 64.41;
H, 6.67.











Syringaresinol Diacetate

The lignan 3.43 (300 mg) was treated at room temperature with

3 ml of acetic anhydride and two drops of pyridine. After 48 hours, the

solution was diluted to 20 ml with water. After one hour, the derivative

was extracted into chloroform, the extract washed with 10% sodium

bicarbonate, followed by brine, dried over sodium sulfate, and concen-

trated to dryness. The resulting oil was crystallized from ether,

affording 3.43 diacetate, 320 mg, a colorless, crystalline solid, mp

185-186C, [a]D 0.00 (c 0.50, CHC13); nmr: 2.32 ppm, 6H, s, CH3CO2Ar;

ir: 1764 cm-1

Anal. Calc. for C26H30010: C, 62.14; H, 6.02. Found: C, 61.87:

H, 6.10.
















CHAPTER 4
ALKALOIDS OF Magnolia grandiflora L.


Toxic Alkaloidal Fraction

The present study started with the observation that the extract

of the wood of Magnolia was toxic to mice when administered by the

intraperitoneal route. The major symptom of toxicity appeared to be

respiratory paralysis. Since no prior reports existed regarding this

activity, in spite of extensive studies on Magnolia spp., isolation of

the active principle was justified.

Partition of the concentrate of the alcoholic extract between

water at pH 2 or 9 and ethyl acetate showed the activity in the aqueous

phase, thus showing that the active principle was water soluble. Since

the aqueous layer gave a positive test for alkaloids, an aliquot of the

concentrate was treated with Mayer's reagent, the alkaloidal principle

separated from the nonalkaloidal components and both samples freed from

the Hgl4 ion and tested. The alkaloidal fraction was found to be toxic.

Since extraction at pH 9 did not transfer the activity into the solvent

layer, the alkaloid was considered to be quaternary.

For the isolation of the active alkaloid, precipitation with

Mayer's reagent, followed by exchange of the HglI4 ion by C1- ion using

a weak or strong base type ion-exchange resin was found to be the most

convenient. At this point, extraction with a solvent such as chloro-

form was again attempted at pH 9 and found that one of the alkaloids was











extractable, although the activity still remained in the aqueous layer.

The bulk of the extractable alkaloid was therefore removed by this

method and purified as the crystalline hydrochloride. It was further

observed that the toxic quaternary alkaloid(s) could be extracted par-

tially into n-butanol. The n-butanol extract contained approximately

40% of the toxic fraction, the remainder being left behind in the

aqueous layer. The recovery and purification methods employed are

summarized in Figure 4.1.

Methods available in the literature for the purification of

quaternary alkaloids are very few. They are generally processed via

precipitation, regeneration to a salt such as Cl- or I-,and crystallized.

When this is not possible, chromatographic methods have been used with

silica gel or alumina. However, because of the necessity for the use

of solvents of high polarity (e..., 10-30% methanol in chloroform) the

resolution is very poor. The situation is even more difficult when the

quaternary alkaloid is also phenolic which results in an even stronger

affinity for the adsorbent. The alkaloid in question from Magnolia

appeared to be phenolic in nature as judged by the base-induced uv spec-

tral shifts and very strongly adsorbed on silica in thin-layer chroma-

tography unless very polar solvents were used.

A method based on chromatography on Sephadex LH-20 is employed

in these laboratories for the purification of relatively water-soluble

compounds such as glycosides, quaternary alkaloids and peptides. A

combination of adsorption/partition processes is involved when a solvent

such as ethyl acetate is used in combination with varying proportions










Wood Extract

0.01N HC1


Mayer's Reagent
I


Filtrate


1N HC1

Liquors


Liquors




Alumina
Chromatography
HC
HC1


Weakly
Basic Alkaloid


Alkaloid Complex
Dowex-l Cl" Column


CHC13


Non-Toxic
Fraction

I


Cl -


Aqueous


I
Toxic
Fraction


BuOH


Sephadex Adsorption
or
Cellulose Partition
Chromatography


Dowex-l I-
Column





Toxic Alkaloid I-


Fractionation scheme for Magnolia wood alkaloids


EtOAc


Figure 4.1











of ethanol and water. Unlike the conditions which exist in gel-

permeation chromatography, the solutes are not eluted on the basis of

their molecular size but are adsorbed on the basis of their polarity,

aromaticity and such characteristics, and are eluted by the appropriate

solvent. Also, unlike adsorption chromatography with silica gel or

alumina, there is virtually no danger of loss of compounds due to

irreversible adsorption.

By this procedure, the crude alkaloidal fraction from the

n-butanol extract was purified using the solvent mixture 10% ethanol in

ethyl acetate on Sephadex LH-20. Because of the low solubility of the

sample in the solvent mixture, the initial chromatography gave broad

elution peaks which appeared to contain different alkaloidal components.

However, when these were rechromatographed on a second column, the

elution profile was much sharper and more reproducible. Alternatively,

partition chromatography on cellulose using the system ethyl acetate;

n-butanol (3:1) and water was also satisfactory for the purification.

Several alkaloidal fractions were recognized in the elution profile as

shown in Figure 4.2. The first contained a compound which was the

same as the one extractable by chloroform. The second and major peak

represented the toxic, quaternary alkaloid. The final peak contained

a small amount of a quaternary alkaloid which was characterized as

magnoflorine, which is the major alkaloid of Magnolia grandiflora bark.15

The quaternary alkaloid was next converted to the iodide salt

by ion-exchange using Dowex-l iodide and was obtained as a crystalline

solid, mp 222-25 (dec.); [a]D+2120 (c 1.27, ethanol) representing a




















Fraction A: 20 alkaloid
Fraction B: 2 alkaloid +
trace alkaloids
Fraction C: toxic alkaloid
Fraction D: magnoflorine


Figure 4.2


Elution volume, ml


Cellulose partition chromatography of BuOH fraction











yield of 0.02%. It was also toxic to mice with an LD50 value of

10 mg/kg.

Elemental analysis of the toxic alkaloid agreed with the molecu-

lar formula C20H26N041. Its uv spectrum showed Xmax at 220 nm, log 4.76;

270 nm, log 4.21 and 302 nm, log 3.82, with base-induced shifts to

250 and 354 nm. The ir spectrum showed bands for a phenolic.hydroxyl,

3200 cm-, 1230 cm" and for an aromatic system, 1600 cm-1

Quaternary alkaloids generally undergo some form of dequater-

nization in the mass spectrometer by one of the following processes:

nucleophilic attack by the counterion on the methyl group which leads

to a loss of CH3X, or a Hofmann elimination of HX. The actual course

depends on the alkaloid and the nature of X:184 with Magnolia alkaloids

and X = I-, the former predominates to give a spectrum identical to that

of the norbase. The alkaloid in question gave a molecular ion m/e 341

corresponding to that of the norbase,and methyl iodide m/e 142, with

lesser peaks at m/e 298 (M+ -CH2=NCH3),m/e 326 (M+ -CH and m/e 310

(M+ -OCH3).

The data suggested that the Magnolia alkaloid might possess

either an aporphine system or a benzyltetrahydroisoquinoline system with

a methylene dioxy group as shown in 4.1 or 4.2. The nmr spectrum which

showed signals at 8.67 ppm, broad, exchangeable, Ar-OH; 7.00 ppm, 1H;

6.98 and 7.11 ppm, AB-q, J = 8 Hz, 3.72, 3.85 and 3.93, singlets each

equal to 3H(OCH3); 3.44 and 2.98 singlet each equal to CH3(NCH3)

clearly eliminated a structure such as 4.2, and indicated the presence

of 3 methoxyl and 1 hydroxyl groups. Other lines of evidence such as

the mass spectrum, which did not show significant peaks to correspond





87








0
o0J + N3 CH +
CH 3
3-- (OCH3) 3 OCH3

S-OH -- OH

4.1 4.2

to an iminium ion such as 4.3 derived by the a-fission of a
benzyltetrahydroisoquinolinesystem85 and the uv spectrum186,187 estab-
lished that the alkaloid has an aporphine skeleton 4.1.






CH3

4.3



The presence of a phenolic hydroxyl was verified by the forma-
tion of a monomethyl ether, nmr: 3.71 ppm, 6H; 3.90 ppm, 3H and 3.92

ppm, 3H. Acetylation with acetic anhydride and pyridine did not pro-
ceed, possibly due to steric hindrance, but acid-catalyzed acetylation
gave an acetate, ir: 1770 cm-1 and nmr: 2.10 ppm. Thus, the alkaloid
is a hydroxytrimethoxyaporphine with the oxygenation pattern yet to be
determined. On a biogenetic basis, only two oxidation patterns are
likely: 1,2,9,10 (4.4) or 1,2,10,11 (4.5).






88




3 4- -
0 50
2 CH3 + -CH3
0 6 H3 0 CH3
11 H 0 H
0O1 (CH3O
S10 09 (CH)3 0 (CH3)3
0

4.4 4.5



The former may be eliminated by spectroscopic evidence from

several sources. The aromatic region of the nmr spectrum shows an

AB-quartet consistent with the presence of two ortho-protons which can

only be explained by 4.5. The uv spectrum of the Magnolia alkaloid:

220, 270 and 302 nm is typical of a 1,2,10,11-oxygenated aporphine and

not of a 1,2,9,10 system, which has maxima at 280-283 nm and 302-310

nm.186,187 This difference has been said to reflect the greater inter-

ference with coplanarity of the biphenyl system in a 1,2,10,11-

oxygenation pattern.187 The mass spectra of several 1,2,9,10-oxygenated

aporphines have been published185'186'188 and the characteristic

processes are summarized in Figure 4.3. Chiefly, these include (a) a-

cleavage to an iminium ion such as 4.7 which appears as the molecular

ion, or through the loss of H- to 4.6 (Mt -1), (b) subsequent formation
of systems such as 4.8 or 4.9 with the loss of either *OCH3 or -CH3,
respectively, and, (c) retro Diels-Alder cleavage to 4.10 which can

undergo further loss of .OCH3 or *CH3.185'188 In contrast, the mass

spectun of the Magnolia alkaloid showed no M+ -1 peak, low intensity







89












H3CO
H3CO CH3
ge 'H3
ge Mt -1


\_O

4.6



H3CO
H3CO


O



4.10 (M+ -43)
-CH3.* -CH3.


(MF~ -58) (M+ -74)


4.9 (MF -15)


Mass spectral behavior of a typical aporphine


Figure 4.3











M+ -43 peak (retro Diels-Alder process) but a relatively high intensity
M+, M+ -OCH3 peaks. Such behavior is peculiar to those aporphines with
1,2,10,11-oxygenation pattern.189

Final proof for the 1,2,10,11-oxygenation pattern was obtained
through methylation of magnoflorine 4.10. The resulting dimethylmagno-
florine 4.11 was found to be identical with the methyl ether of the
Magnolia alkaloid by comparison of its tlc and spectral behavior.

H3CO
RI O CH3
R10 3

R20Y
H3CO

magnoflorine 4.10 R1 R2 = H
dimethyl magnoflorine 4.11 R1 = R2 = CH3




For the Magnolia alkaloid, four structural possibilities exist
for the location of the phenolic hydroxyl, as shown in Figure 4.4.
Of these, neither 4.12 nor 4.15 has been isolated from natural sources
so far, while 4.13, N-methyl corydinium iodide and 4.14, N-methyl
isocorydinium iodide are known.
The difficulty with acetylation of the phenolic hydroxyl might
suggest that it be present at C-l or C-ll. This is further supported
by the hydroxyl frequency of 3200 cm- in the ir spectrum. Aporphines
of this substitution pattern, as well as the phenanthrenes derived from












1 n CH
R O H3
R20 CH3
'H 3
R30 H

R40

N-methyl-10-O-methylhernovine methiodide 4.12 R =H1, R2 R3R1 =CH3

N-methyl corydinium iodide 4.13 R2= H1, R1 =R = R4=CH3

N-methyl isocorydinium iodide 4.14 R3=H1, R =R2=R4=CH3
-----------------4.15 R =R =H,R1 =R2= R4=CH3


Figure 4.4 Isomeric hydroxy-trimethoxyaporphinium methiodides






them by Hofmann elimination, with free hydroxyl at C-1 or C-ll, are

known to show hydroxyl frequencies below 3300 cm-190 Alternatively,

location of a methoxyl at C-2 and/or C-10 was supported by the nmr

spectral data. Methoxyls at C-2 and C-10 resonate at 3.85-390 ppm
191,192
whereas methoxyls at C-1 and C-11 do so at 3.4-3.7 ppm.91'192 The methoxyl

signals of the Magnolia alkaloid (3.70, 3.90, 3.92 ppm) indicated that

the C-2 and C-10 positions had the methoxyl groups.

To discriminate between the structures 4.13 and 4.14 a more

detailed comparison of the spectra was made. Although the mass spectra

of 4.13 and 4.14 weresomewhat different,189 the spectrum of the Magnolia

alkaloid was not sufficiently similar to either of the literature spectra

to allow an assignment. Neither were the reported nmr spectra of use:











H-8 and H-9 of 4.13 have been reported as singlets at 7.27 and 7.33 ppm,

with H-3at 7.05 ppm193 while 4.14 showed H-8and H-9as a singlet at
194
6.96 ppm, and H-3at 7.02 ppm.194 As the chemical shift differences for

H-8and H-9are small, the outer peaks of the AB-quartet are small and,

therefore, often overlooked. Furthermore, variations from these values

are also found in the literature.195,196 The spectra also vary

depending on the solvent used (D6DMSO, CD300, CF3COOH or D20). The

observation of an AB-quartet for H-8and H-9 is the key to assignment of

all the resonances, whereas, if only singlets appear, the assignments

are questionable.

The assignments proposed for the Magnolia alkaloid and its

methyl ether based upon the AB-quartets are shown in Table 4.1.


Table 4.1 Spectral assignments of the aromatic protons of Magnolia
toxic alkaloid and its ether


H3, ppm H8 and H9

alkaloid 6.96 6.98d or 7.11d

methyl ether 6.96 6.97d or 7.15d



The discrimination between 4.13 and 4.14 was ultimately made

chemically: in base, N-methylisocorydine 4.14 has anunsubtituted posi-

tion para to the phenolate function which is very reactive toward

electrophilic reagents, whereas N-methylcorydine 4.13 has none. This

difference has been exploited in a number of ways: deuteration,195

and the Gibbs test (reaction with 2,6-dibromoquinone chlorimide)197











having been used for this purpose. In this work, diazonium coupling

was employed. The thin-layer chromatogram of magnoflorine 4.10, the

Magnolia alkaloid, and its methyl ether was sprayed with a solution of

4-carbethoxybenzenediazonium fluoborate, followed by 1% NaHCO3.

Magnoflorine and the Magnolia alkaloid both formed highly colored azo

dyes,.whereas the methyl ether, which has no free-phenolate activating

group, did not. Therefore, the Magnolia alkaloid corresponds to 4.14,

N-methylisocorydinium iodide, which has a reactive para-position.

Further verification was obtained by carrying out the coupling in solu-

tion. The presence of a phenolic function conjugated with the azo

group was verified by demonstration of a shift in Xmax upon alkalini-

zation (375 nm -+ 516 nm). Therefore, it may be concluded that the

toxic alkaloid is identical to (+)-N-methyl isocorydinium iodide 4.14.

The present work describes the first reported isolation of

(+)-N-methyl isocorydine from any species of Magnolia. N-methyl

isocorydine, also known as menisperine, was first isolated from

Xanthoxylum brachyanthum F. Muell. and X. veneficum F. M. Bail., two
197
members of Rutaceae by Cannon et al.,197 who also determined

its structure. An alkaloid originally called "Chakranine" isolated

from Bragantia wallichii R. Br. (Sanskrit: "Chakrani," Aristolochia-

ceae)93 has been found to be identical to N-methyl isocorydinium
190
iodide.190 The alkaloid is of widespread occurrence, having been

isolated from the Annonaceae, Lauraceae, Menispermaceae, Papaveraceae,
191-92
and Berberidaceae. 191 It has recently been synthesized de noveau

by a route involving preparation of the corresponding benzyltetrahydro-

isoquinoline with an amino function at the ortho position of the benzyl