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Chemistry of Magnolia grandiflora L.

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Title:
Chemistry of Magnolia grandiflora L.
Creator:
Davis, Terry Lee, 1945-
Publication Date:
Language:
English
Physical Description:
vii, 117 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Acetates ( jstor )
Alkaloids ( jstor )
Aporphines ( jstor )
Chromatography ( jstor )
Ethers ( jstor )
Iodides ( jstor )
Ketones ( jstor )
Lignans ( jstor )
Protons ( jstor )
Silica gel ( jstor )
Chemistry, Agricultural ( mesh )
Dissertations, Academic -- medicinal chemistry -- UF ( mesh )
Medicinal Chemistry Thesis Ph.D ( mesh )
Sesquiterpenes ( mesh )
Trees -- analysis ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

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

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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023160522 ( ALEPH )
25174106 ( OCLC )
AEK1521 ( NOTIS )

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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




CHEMISTRY OF Magno!i a 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


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, University 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
Pag;e
ACKNOWLEDGMENTS iii
ABSTRACT v
CHAPTER
1 SECONDARY METABOLISM AND THE CHEMISTRY OF
Maqnolia; PHARMACOLOGICAL ACTIVITY OF Maqnolia
EXTRACTS AND COMPONENTS 1
Biogenetic Classification of Secondary
Metabolisms 1
Terpenoids of Genus Maqnolia 1
Alkaloids of Genus Maqnolia 5
Compounds Derived from Shikimic Acid 13
Pharmacological Activity of Maqnolia
Preparations and Maqnolia Compounds 21
2 SESQUITERPENOIDS OF Maqnolia grandiflora L 27
Ketone from the Bark Extract 27
Antibiotic Principle of the Leaves 41
Experimental 44
3 PHENYLPROPANOIDS OF Maqnolia grandiflora L 50
Bis-allylphenol from the Bark 50
Lignan from the Wood 60
Experimental 71
4 ALKALOIDS OF Maqnolia grandiflora L 81
Toxic Alkaloidal Fraction 81
Non-toxic Alkaloidal Fraction 94
Experimental 97
General Experimental 104
LIST OF REFERENCES 105
BIOGRAPHICAL SKETCH 117
iv


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 Magno!ia grandiflora L.
By
Terry Lee Davis
June 1981
Chairman: K. V. Rao
Major Department: Medicinal Chemistry
Magno!ia 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. Magno!ia 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 Magno1ia
spp. Another sesquiterpene, a lactone, identified as parthenolide was 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 1 ignoid
v


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.
vi


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 reprsented 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.
vi 1


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 Magno!iaceae, a family
which has been variously classified under the order Ranales or Mag
no! ialesJ The chemistry of this very primitive plant has been
studied extensively, particularly the alkaloids. Magno!ia 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.^
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
3
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
Magno!ia to be described in this dissertation.
Terpenoids of Genus Magno!ia
The terpenoids are compounds arising from mevalonic acid-
derived isoprenes, such as isopentenyl pyrophosphate, by catenation
1


2
Table 1.1 Species of Magnolia reported in the chemical literature
Subgenus Magnolia
Sect. Gwi11imia (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. officinal is Rehd. et Wils.
M. rostrata W.W. Smith
M. triptala L. (syn. M. umbrel1 a Desr.)
Sect. Oyama (Asia)
M. parviflora (syn. M. Sieboldii K. Koch)
M. Sieboldi i K. Koch (syn. M. parviflora Sieb. et Zucc.)
M. sinensis Stapf. (syn. M. globosa var. sinensis Rehd. et Wils.)
Sect. Magno!iastrum (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 PIeurochasma
Sect. Yulania (Asia)
M. Campbel1i Hook f. et Thoms.
M. denudata Desr. (syn. M. Yulan Desf.)
Sect. Buergeria (Asia)
M. Kobus DC.
M. salicifolia Maxim.
M. stellata Maxim.


3
Table 1.l--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-
gesii (syn. Michelia Fargesii Andr.), Magnolia fuscata Andr. (syn.
Michel i a fuscata Blume, M. Figo Spreng.), M. LenneiTopf. (syn. M. Soulan-
geana var. Lennei Rehd.), and M. mutabilis Rege!. M. X Soulangeana Soul,
is M. denudata x M. liliflora.


4
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 Magno!ia 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.


5
In the present work the first isolation from any Magno!ia 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 Michel i a champaca L., Michelia
5 6
lanuginosa, and is known to occur also in Magnolia grandiflora.
In Table 1.2 are presented the terpenoid compounds of various
7-13
types which have been isolated from genus Magnolia.
Alkaloids of Genus Maonolia
Studies on the alkaloids of Magnolia have accounted for the
bulk of the chemical and pharmacological literature of the genus and have
14
been reviewed by Tomita and Nakano.
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
15-46
throughout the Magnoliaceae are detailed in Figure 1.1.


Table 1.2 Compounds of mevalonate origin reported in the genus Magno!ia
Compound
Class
Species
citral
monocyc1ic terpene
M. obovata (lvs)3,7
M. Kobus (lvs, stms)8
M. salicifolia (lvs, stms)^>9
1imonene
monocyclic terpene
M. obovata (lvs)7
M. Kobus (bds)10
cineole
monocarbocyclic terpenes
M. Kobus (lvs, stms)8 (bds)10
M. salicifolia (lvs, stms)
a- and 3-pinenes
bicyclic terpenes
M. obovata (lvs)7
M. Kobus (lvs, stms)^
bornyl acetate
bicyclic terpene
M. obovata (lvs)?
camphene
bicyclic terpene
M. obovata (lvs)7
camphor
bicyclic terpene
M. Kobus (bds)10
caryophyl1 ene
bicyclic sesquiterpene
M. obovata (lvs)7
caryophyllene epoxide
bicarbocyclic sesquiterpene
M. obovata (lvs)7
a-, 3- and y-eudesmols
bicyclic sesquiterpene
(eudesmane type)
M. obovata (lvs)7
cyclocolorenone
tricyclic sesquiterpene
(aromadendrane type)
M. grandiflora^


Table 1.2--continued
Compound
Class
Species
costunolide
monocarbocyclic sesquiterpene
lactone (germacranolide)
M. acuminata (rt-bk)^
parthenolide
monocarbocyclic sesquiterpene
lactone (germacranolide)
M. grandiflora (lvs)b,b (stms)b
crytomeridiol
bicyclic sesquiterpene
M. obovata (bk)^
campesterol
tetracyclic triterpene (sterol)
M. obovata (lvs)7
cholesterol
tetracyclic triterpene (sterol)
M. obovata (lvs)7
M. grandiflora (bk)c
0-sitosterol
tetracyclic triterpene (sterol)
M. acuminata (rt-bk)^
M. Campbelli (bk)13
M. grandiflora (bk)c
stigmasterol
tetracyclic triterpene (sterol)
M. obovata (lvs)7
M. grandiflora (bk)c
a-amyrin
pentacyclic triterpene
M. obovata (lvs)7
lupeol
pentacyclic triterpene
M. ovovata (lvs)7
abk = bark, lvs leaves, htwd = heartwood, rts = roots, stms = stems, bds = buds, wd = wood, flwr =
flowers, sds = seeds.
bThis work,
c
In the course of this work, materials with the same tic behavior and glc retention times (on one column)
as commercial samples of the above were observed.


8
magnoflorine
1.8
Figure 1.1 Probable derivation of Magno1ia alkaloids magnoflorine
and reticulene from tyrosine


9
Further oxidative processes can convert these alkaloids to
oxoaporphines such as liriodenine 1.9 and todimeric alkaloids such as
magnoline 1.10. The known alkaloids of Magno1ia 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
40
isolated from this plant, 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
40
M. grandiflora, and, in the course of this work, it was also observed
in the bark.
1.11
1.12


10
Table 1.3 Alkaloids isolated from Magnolia spp.
Compound
phenyl ethyl amine
candicine
M.
salicifoline
M.
M.
M.
M.
M.
M.
M.
M.
benzylisoquinoline
magnococline
M.
N-norarmepavine
M.
reticuline
M.
benzylisoquinoline
(quaternary)
magnocurarine
M.
M.
M.
M.
M.
M.
M.
M.
bisbenzylisoquino!ine
magnolamine
M.
magnoline
M.
aporphine
anonaine
M.
M.
Occurrence
grandiflora (bk)15 (rts)^
acuminata (stms)^
coco (stmsy8
denudata (bk)19
grandiflora (rts)16 (bk)a,2
Kobus (bk)22>23
liliflora (bk, rts, wd)24
salicifolia (bk)25
stellata (bk)25
coco (stms)2'7
kachirachirai (htwd)28,29
obovata (rts)30
acuminata (stms)]?
denudata (bk)'^>20
fuscatab (lvs, bk)33
liliflora (bk)2^
obovata (rts)2Q (bk)2^25 (htwd)35
officinal is (htwd)36
parviflora (bk)37
salicifolia (bk)22
fuscatab (lvs)33,38,39 (bk)33
fuscatab (lvs)38
grandiflora (wd)c^0 (lvs)40
obovata (lvs)30 (rts)30 (htwd)35,41


11
Table 1.3--continued
Compound Occurrence
aporphine (continued)
anonaine acetamide
anolobine
asimilobine
glaucine
N-norglaucine
N-nornuciferine
obovanine
aporphine (quaternary)
N ,N-dimethyl1indcarpine
iodide
magnoflorine
menisperine
proaporphine
stepharine
7-hydroxyaporphine
michelalbine
(norushinsunine)
M. obovata (htwd)^l
M. acuminata (rt-bk)9
M. coco (stms)27,42 (rt-bk)
M. grandiflora (wd)40
M. obovata (Ivs, rts)30
M. kachirachirai (htwd)18,29
M. obovata (lvs)30
M. kachirachirai (htwd)29
M. grandiflora (wd)40
M. obovata (Ivs)30
M. acuminata (rt-bk)^
M. acuminata (Ivs, stms)e
M. coco (stms)^
M. denudata (rts)^
M. fuscatab (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)^^ (rt-bk)^3
M. obovata (htwd)35


12
Table 1.3--continued
Compound
Occurrence
7-oxotetradehydroaporphi ne
lanuginosine
M. Campbelli (bk)^
liriodenine
(oxoushinsunine)
M. Campbelli (bk)^
M, COCO (stms)18 4n 46 40
M. grandiflora (bk)c (wd)C> (lvs)
M. mutabilis (lvs)'l oc
M. obovata (lvs, rts)JU (htwd)"33
(bk)35
oxoglaucine
29
M. kachirachirai (htwd)
oxolaureline
46
M. X Soulangeana
aAbsence of salicifoline in the bark of M. grandiflora var. lanceolata
Ait. has been noted by Tomita et al.20
^Now considered by most authors to be Michelia fuscata Blume,^ or
Michelia Figo Spreng3132
cThis work
dNot isolated; identified by paper chromatography^7


13
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 Magno!iaceae
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-
8_10,47-i
derived compounds isolated from Magno!ia are listed in Table 1.4.
Oxidative juncture of cinnamyl alcohol and its derivatives at
the 8-positions of the 2-propenyl sidechains results in a class of
compounds Haworth has termed the lignans.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


Table 1.4 Shikimic acid-derived compounds of Magnolia spp.
Compound
Structural Type
Occurrence
anisaldehyde
benzaldehyde
M. salicifolia (lvs, stms)8
eugenol
allylphenol
M. Kobus (lvs, stms)8
methylchavicol
allylphenol
M. Kobus (lvs, stms)8
methyleugenol
allylphenol
M. salicifolia (bds)^
safrole
allylphenol
M. salicifolia (bds)10
trans-anethole
propenylphenol
M. salicifolia (lvs, stms)8>9
trans-asarone
propenylphenol
M. salicifolia (bds)10
syringin (syringoside)
3-hydroxypropenylphenol
glycoside
(lvs, stm-bk) of:^8
M. acuminata
M. Campbelli
M. denudata
M. grandiflora
M. Kobus
M. lili flora
M. macrophylla
M. obovata
M. parviflora
M. salicifoTTa
M. X Soulangeana
M. stellata
M. triptala
M. Wilsonii
M. grandiflora (wd)49


Table 1.4--continued
Compound
Structural Type
Occurrence
magnolidin
a-tocopherol and 4 uniden
tified chromanols
magnolioside
acuminatin
honokiol
magnolol
mehonokiol
calopiptin
glycosidic ester of a
cinnamic acid
chromanol
coumarin glycoside3
neolignan--bis(allylphenol)
neolignan--bis(allylphenol)
neolignan--bis(allylphenol)
neolignan--bis(allylphenol)
neoliqnan--galgravin type
neolignan--galgravin type
M. grandiflora^ 5,49,50
M. X Soulangeana (lvs)5^
M. macrophylla (bk)52
M. acuminata (rt-bk)9>53
M. obovata (bk)12,54,55
M. officinalis (bk)4,55
M. triptala (bk)55
M. grandiflora (sds)5
M. rostrata (bk)53
M. obovata (bk)12,54,55
M. officinalis (bk)54,55
M. triptala (bk)
M. virginiana (bk)^5
M. grandiflora (sds)56
M. rostrata (bk)57
M. grandiflora (bk) (sds)
M. acuminata (rt-bk)9>53
M. acuminata (rt-bk)^
galgravin


Table 1.4--continued
Compound
Structural Type
Occurrence
veraguensin
neolignan--galgravin type
M. acuminata (rt-bk)^33
sesamin
1ignan--pinoresinol type
13
M. mutabilis (lvs, stms)
eudesmin
1ignan--pinoresinol type
M. Fargesii (flwr-bds)^8
magnolin
1ignan--pinoresinol type
M. Fargesii (flwr-bds)58
syringaresinol
1ignan--pinoresinol type
M. grandiflora (wd)a
syringaresinol dimethyl
ether
1 ignan--pinoresinol type
M. Fargesii (flwr-bds)33
M. Kobus (sds)59
aThis work


17
2,5-diarylfurans
(olivil type)
(pinoresinol type)
OH
1-ary1 naptha 1 ene 1-arylnapthalenes
lactones (podophyllotoxins)
Figure 1.2 Notional representation of lignan biogenesis and struc
tural types


18
6i 62
designated "neolignans." The neolignans are more restricted in
their distribution than the lignans proper, having been isolated only
rp
from two subclasses, the Magno!iidae and the Rosidae. Those large
groups encompass, however, a number of major families, including the
Magno!jales 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 Magno!ia species (cf.
Table 1.4), while the latter, isolated from Litsea turfosa Kosterm. (Laur-
63
aceae ) is the only bis-allylphenol-type neolignan occurring outside
the genus Magnolia.


19
t\-j 2
R-j = R2 = R^ = Rg = OCH3, R0 = Rg = H
R-| = ^2 = ^4 = ^5 = ^6 = R3 = H
R1 = R3 = r4 = r6 = 0CH3, r2 = r5 = OH
R1 = R2 = r3 = R4 = R5 = Rg = OCH3
sesamin
magnolin
eudesmin
syringaresinol 1.13
syringaresinol
dimethyl ether
R1 = R3 = = Rg = OCH3, r2 = OGlu, Rg = OH acanthoside B
R = (CH2) calopiptin
Figure 1.3 Known lignans of Genus Magnolia


20
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.3 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 Zeyheria^ montana
Mart, and 1. tuberculosa (Veil.) Bur. ex Verlot (Bignoniaceae)^4 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
M ,. 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 *H- and 13c_nmr spectroscopy.36
^Zeyheria is spelled Zehyera by the author.


21
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,^ American
71 72
Indian medicine, and even listed in American pharmacopoeiae and
pharmacognosy texts as bitter tonics, antimalarials, and diapho-
73 74 75
reties. The use of Magnolia extract in medicine has been pro-
7 c
posed even as recently as 1953.
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.^ An extract of M. Kobus has shown some antiviral
activity in mice at subtoxic levels. The extract of M. grandiflora
80
was found to have acaricidal activity, and to lower blood pressure
without effect on heart action or breathing.^
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
81
extract, at the same dose, showed prompt respiratory paralysis.
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
82
(Ci7HigNOs) with curare-like action from the same drug.


22
Several of the active principles of neuroactive Magno!ia
extracts have been isolated and identified. The bis-benzylisoquinoline,
magnoline, 1.10, has a hypotensive effect and shows anticholinesterase
83
activity. Compounds associated with the observed "curare-like"
effects of Magno!ia extracts are magnocurarine 1.20, magnoflorine 1.8,
84
and salicifoline 1.21. Magnocurarine, the most active of these
84
quaternary alkaloids, has a ganglionic-blocking activity comparable
85
in strength to hexamethonium bitartrate. It exerts upon frogs an
action similar to that of d-tubocurarine (d-TC) 1.22,a but of longer
87
duration and only one-tenth as active.
H3C0'
HO'
00<5
/ CH3
CH3
HO'
HO*^
^ CH3
1.20
1.21
Previously thought to be fujly quaternized, the structure of
d-TC has recently been revised.86


23
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 (j_n vitro), except in the case
85
of salicifoline, which caused contraction of the muscle.
The noraporphine anonaine 1.11, isolated in the course of this
work, has antibiotic activity at 100 mg/ml against Staphylococcus
88
aureus, Mycobacterium smegmatis, and Candida albicans. It also
89
exerts a hypotensive effect in mice and rabbits and has been shown to be
an inhibitor of dopaminergic response, an activity associated with a
90
number of analogs of apomorphine 1,23. Its specific effect in rats is
as an antagonist of dopamine-sensitive adenylate cyclase in the caudate
i 90
nucleus.
In these laboratories, anonaine (as the hydrochloride) showed
no acute toxicity on injection in mice at a dose of 200 mg/kg (IP).
The Maqnolia alkaloid liriodenine 1.9, also observed in the
course of this work, has an antibiotic activity similar to that of
88 91
anonaine. In addition, it is an inhibitor of human carcinoma of
92
the nasopharynx in_ v i tro.


24
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-blocking^9^"6 and ganglionic-blocking activities'^69^596 of
which are well established.9 Menisperine, then known as "chakranine,"
was originally described as exerting ". . ganglionic blockade selec
tively exercised against the autonomous nerve pathways of the respiratory
93
system." Erhart and Soine, using the cat tongue/hypoglossal nerve
system in vivo, have effectively characterized the nmj-blocking activity
94
of this drug as very similar to that of d-TC itself.
The LDgg of the pure iodide was determined in this work to be
10 mg/kg (21 ymol/kg, IP, mouse). For comparison, Kamat et al_. obtained
93
a value of 2.2 mg/kg (5.9 ymol/kg, IV, mouse) for the chloride.
The results of a pharmacological screening procedure routinely
employed in these laboratories are presented in Table 1.5.6 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
contractibi1itv and excitability in a manner resembling tetraethyl am
monium salts."97 This same author observed meiosis upon intra-lymphatic
injection in frogs.98
6After the method of Campbell and Richter.99


25
Table 1.5 Pharmacological evaluation of Magnolia toxic alkaloid
Samp 1 e Magnolia^Bark Date 9/19/78
Dose 1_2 mg/kg, 0.6 mq/ml Time 3:08 p.m.
Animal Mice
Animals
1
2
3
4
5
Final
Paw Temp b NT
raw lemp. after
Rndv Tmp before
37.
5
37.5
37.5
37
.5
37.5
oooy lemp. after

35
36
-

32
Average Wt. (g)
25
Sign: 1.
Paw Temp., t



-


2.
Body Temp., +
-
-
-
3.
Body T emp., +
-
-
-
4.
Ptosis
-
-
-
5.
Salivation
-
-
-
6.
Lachrymation
-
-
-
7.
Mydriasis
+
+
-

Miosis
C/>
0)
4->
-
-

0)
*->
-
8.
Piloerection
c
E
-
-
c
*E
-
9.
Locomotor Activity, +
m
-
-
m
C\J
-
10.
Straub tail phenom.
0>
V*-
-
-
07
4->
4-
-
11.
Righting reflex
(r.r.) abolished
Dead
-
-
Dead
12.
Head drop
(with r.r. present)
-
-
-
13.
Pos. Haffner
(with r.r. present)
-
-
-
14.
Locomotion Activity, +
-
-
-
15.
Abduced hindlegs
(with r.r. present)
-
-
-
16.
Unsteady gait
1 -
-
-
17. Others: Onset of effects ca. 5 min., with marked depression of activity 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 LD50, death
occurs in less than 5 minutes. At LD50 deaths occur at 15-20 min., with an occasional
mouse succumbing more promptly.
18. CMTD (mg/kg)
Animals remain capable of locomotion when disturbed.


26
99
(head-drop, ptosis, strabismus) other than apparent respiratory
arrest is evident. However, such activity is not observed under these
qq
conditions for d-TC itself.
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
93
0.3 mg/ml, respectively.
The sesquiterpene lactones parthenolide 1.6 and costunolide
1.24^ 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.
The lignans sesamin, eudesmin, and syringaresinol 1.13, all of
which occur in various species of Magno!ia (cf. Table 1.4) have some
anti tubercular activity in vitro. ^
Finally, compounds related to the diallylbiphenols found in
103
Magno!ia have been synthesized for medicinal and other applications,
and several have been patented for use as anticoccidial agents in
104
poultry feed.


CHAPTER 2
SESQUITERPENOIDS OF Magno1ia 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
15
described elsewhere. Fractionation oftiie 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 nonphenol ic. This com
ponent was isolated by column chromatography on silica gel-cellulose
(1:1) by elution with 51 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
27


28
Ethanolic extract of
the bark
concentrate and
partition between
water and ethyl acetate
Lipophi
ic
fraction
Silica gel
chromatography
cyclocolorenone >
phytosterols
Mehonokiol Syringaresinol
Sephadex
chromatography
Hydrophilic fraction
Alkaloid Magnolenins Magnolidins
fraction A,B,C A,B
Ion-exchange
chromatography
Magnoflorine Candi cine
Figure 2.1 Fractionation of Magnolia grandiflora extract


29
samples of 3-sitosterol, stigmasterol and cholesterol, all of which
have been observed in the extracts of Magnolia obovata by Fujita et
alJ 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 FI ori sil 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 a molecular formula
of 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 and unsaturation (1600 cm^). Its nmr spectrum
gave evidence for the presence of three different types of methyl
groups: 0.72 ppm, d, CH-CH^; 1-00 ppm> s> anc* 1-23 ppm, s, F^C-iCH^;
and 1.70 ppm, d, -C^-CH^; along with a considerable overlap of signals
due to a CH^- 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.


30
Its uv-absorption maximum of 390 nm indicated that it was derived from
an a,8-unsaturated ketone, although the Amax was higher than that
expected for one double bond conjugated with the keto group.^,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 acid^ gave a ketonic product which, how
ever, was different from the original enone (higher and higher Amax).
In the absence of mineral acid, no exchange took place. Thus, altera
tion of the structure in the presence of acid was apparent.
The A of 260 nm observed for the ketone is intermediate in
max
value between that of an enone (220-250 nm) and that of a dienone
1 Qg
(280 nm). The literature suggested as a model for this chromophore
the cyclopropyl-conjugated enone 2.1, which was described by Bchi et
109
al., as an intermediate in their synthesis of maaliol 2.2. The
extent of overlap of the ir-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.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


31
spectral fragment at m/e 175 (M-C^H^). 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.
On the basis of the available information, a search of the
literature was conducted which revealed that the properties of the
Magno!ia enone and those of its 2,4-dinitrophenylhydrazone derivative
corresponded with those described for the sesquiterpene ketone, cyclocolor-
enone 2.3, first isolated from Pseudowintera colorata (Raoul)
112 113
Dandy. 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


32
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 ally! alcohol 2.4 from which the starting enone could be
regenerated by oxidation with manganese dioxide. This alcohol appears
113
to correspond with cyclocolorenol 2,4 described by Corbett and Speden
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 38-40 was also prepared from a-gurjunene and characterized
114
as 4a,5a-cyclocoloranone which agrees with the properties of the
present ketone. However, the melting point of the dinitrophenylhydrazone
of the saturated ketone from Magno!ia 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 Magno!ia enone contained a 5-membered ring.


33
The nmr spectrum of the Magno1ia 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-l (J-| i2 = Hz). The signal centered at 2.84 ppm, a doublet of
doublets, represents the a-proton of C-2 coupled to the C-2B and C-l
protons with J2a of 18 Hz and ^ ^ f 6.5 Hz. The C-l proton
appears as a multiplet at 2.94 ppm with H-2a at 2.29 ppm, ^ 2a = 2 Hz.
All of these signals show general agreement with those
115
described by Bchi et al_., for cyclocolorenone. The spectrum of the


34
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.^^ This model compound shows ir
spectral bands at 1700 and 1630 cm ^ for C=0 and C=C, respectively,^
in comparison with the reported values of 1690 and 1630 cm-^ for the
respective groups of cyclocolorenone.
H-4 2.85 ppm
H-5o 2.37 ppm
H-5a 1.95 ppm
J
J
J
gem
4,5(cis)
4,5(trans)
19.5 Hz
6.5 Hz
2.5 Hz
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-l proton multiplet 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
118
that observed in the spectrum of a-gurjunene, photosantonic acid
119
lactone and a-santonin derivatives. In a-santonin derivatives, the


35
homoallylic coupling is of the order of 1.3-1.4 Hz, with a critical bond
118
angle of 115. The angle measured for a stereomodel of cyclocoloren-
one was found to be 115 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
118
Ourisson 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.
115
Epimerization of cyclocolorenone was observed Biichi et al.,
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-
120
duced the reverse reaction. Corbett and Young 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


36
product showed the expected shift in A w toward 250 nm, thus indicating
iilaX
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 tic 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 Magno!ia
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,^^ 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, and of the Magno!ia
enone are compared in Figures 2.3 and 2.4 (an impurity appearing on the
carbonyl band diminishes as the sample is purified).


(a) (b)
Figure 2.3 Comparison of nmr spectra of (a) Magnolia enone and (b) synthetic cyclocolorenone


M 1 Jl It J 5 4 J / 1 $ t 10 M t| II U
CO
00
Figure 2.4 Comparison of ir spectra of (a) Magnolia enone and (b) synthetic cyclocolorenone


39
Cyclocolorenone was first isolated from Pseudowintera colorata,
a plant which the authors ascribe to Magno!iaceae, although it is
123-]26
usually assigned to Winteraceae. 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
Japnica (Hepaticae). In addition to these, the compound was found
1 op
to be present in Boronia ledifolia Gay (Rubiaceae) and in the golden-
129
rod, Solidago canadensis (Compositae).
Although the basic structure of cyclocolorenone was determined
by the authors who isolated it originally, its stereochemistry was
115
determined largely by Bchi et al_., through their synthesis of
epicyclocolorenone, and from its relationship to a-gurjunene.
Cyclocolorenone itself was finally synthesized by Ingwalson and Caine
121 122
starting from (-)-maalione.
Cyclocolorene is a member of what has recently been described
130
as . the rare class of aromadendrane sesquiterpenes." 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,53 while
alloaromadendrene 2J3 is 1B,5B;131,132 a-gurjunene 2.9118,133-135 anc|


40
1 2g
6-spathulene 2.10 represent the prototypes with a 4,5-double bond
and 1$ hydrogen: cyclocolorenone is of this type. This relationship
between cyclocolorenone and a-gurjunene is accentuated further by the
137
fact that the two occur together in Pseudowintera colorata, and
there has been speculation that cyclocolorenone might have been an
artifact arising from a-gurjunene on long storage through air/photo-
118
oxidation. A number of alcohols derived from either aromadendrane
131 138
(e.g., globulol 2.11 and spathulenol 2.12) or alloaromadendrene
118 118
(e.g., ledol 2.13, viridiflorol 2.14 ) have been described. An
139
unusual isonitrile, axisonitrile II 2.15 and derivatives 2.16 and
2.17^0 have been isolated from the sponge, Axinella canabina (Porifera).
2.11, R = CH-,, R' = OH
2.13, R = CH0, R'
= OH
2.15,
R = -N=C
2.12, R = R'"= CH2
2.14, R = OH, R1
= ch3
2.16,
2.17,
R = -N=C=S
R = -NHCH0


41
Antibiotic Principle of the Leaves
The extract of yellowed leaves of Magnolia grandiflora was
found to possess antibiotic activity when assayed with Bacillus subtil is
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 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 FI ori sil, 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


42
an oil and triturated with ethanol to give a solid. Crystallization
from ether gave large, colorless crystals, 210 mg, mp 112-114C, and
MIC 3 ug/ml.
The mass spectrum (M+ 248) and elemental analysis are consistent
with the molecular formula C-i^H^qO^. The ir spectrum (K3r) 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, CH£ 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 aj_. and a comparison
of their nmr spectrum with that of the Magno!ia antibiotic is shown
in Figure 2.5.
2.16


43
_! I 1 1 ' '' I | 1 1
To ?-06*0 5-0 4-0 3-0 2-0 i-0
J. ppm
Figure 2.5 Comparison of nmr spectra of (a), parthenolide and
(b) Hagnolia antibiotic (DgDfISO)


44
The melting point and ir spectra are in agreement with the
4
published data of Govindachari et aj_., 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
141 142
parthenium (1.) Bernh. and subsequently from Ambrosia dumosa
143 144
Gray, and Ambrosia confertiflora DC., all Compositae. Indeed, the
Compositae, and particularly Ambrosia and their relatives, elaborate a
144
large variety of sesquiterpene lactones. Such compounds are much
less prevalent in other families.
Within the Magnoliaceae, in addition to M. grandiflora,^
parthenolide has been isolated from Michelia campaca L. by Govindachari
4
et al_., who revised the original structure proposed by Herout et
141 142 145
al. and Soucek et al_, and from Michelia lanuginosa L. The
antibiotic activity of this compound has not been previously described.
The stereochemistry of parthenolide was determined by Bawdekar
146
et al_., who correlated it with the known stereochemistry of
costunol ide.
Parthenolide and costunol ide are the only sesquiterpene lactones
reported from Magno!ia. Interestingly, a number of related costunolide
derivatives are known from the magnoliaceous tree, Liriodendron tulipi-
147 148 147
fera. 5 These, like parthenolide, have antitumor activity.
Experimental
Extraction of Magnolia bark: Cyclocolorenone 2.3
Ground bark (41 g) of Magnolia grandiflora, collected in
Gainesville, Florida, was extracted with ethanol (10 £) at 25 C for two


45
days. Three such extracts were combined, concentrated at 20 mm pressure
to a syrup which was partitioned between water and ethyl acetate (2 i
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
eluate contained besides carotenoid and other highly lipid-soluble
components, the phenyl propanoid component of which is described in Chapter
3. The column was then eluted with 5% acetone in benzene which eluted
cyclocolorenone, recognizable by its tic behavior and the uv-maxima
at 260 nm, as well as phytosterols which moved in tic at siightly 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 5C
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 FI ori sil (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 tic and gc (T 175, retention
time on 3% 0V-17, 6 ft.; 5 min.; on 3% 0V-1: 3 min.); uv: X 264 nm;
max
loge4.12 (lit. 264/4.12)^^ mass spectrum: m/e 218 (M+ 100%), 203,
175, 162, 161, 149, 147, 134, 133, 119, 105, 93 and 91.


46
Analysis of the Phytosterol Fraction
The column consisted of 3% OV-17 on Gaschrom Q, 6 ft., programmed
from 150-275C at 10 per minute. The mixture showed components with
retention characteristics similar to those of cholesterol (Aldrich),
lanosterol (Mann Research) and 6-sitosterol (Fisher). Under isothermal
conditions at 250C, the retention times are as follows:
cholesterol
13.5 min.
lanosterol peak 1
19.0 min.
peak 2
20.5 min.
6-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 217.5C (lit. 218);^ uv A
max
400 nm, log 4.51 (lit. 404, log 4.40); ^ 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.


47
After five minutes at 50C, it was cooled, neutralized with IN 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 ^.
The sample in hexane (10 ml) was stirred at 25C with activated
manganese dioxide (Baker and Adamson) for two hours. Filtration and
concentration of the filtrate gave an oil, identical with 2.3 in tic,
and uv spectra.
Reduction of Cyclocolorenone to Cyclocoloranone 2.5
The procedure was an adaptation of the one described by Corbett
113
and Speden: 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 Ci^H^O^O: c, 75.69; H, 10.92. Found: C,
76.48; H, 10.79; ir: 1745 cm^; 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 (Mf), 177, 149, 136, 135, 122, 109.


48
Cyclocoloranone 2,4-dinitrophenylhydrazone
This was prepared as described under the cyclocolorenone
dinitrophenylhydrazone. It is a light orange crystalline solid,
mp 207-210 from ethyl acetate; uv: Amax 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 A
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 A^ax 250 nm, with nmr indicating 40% 2.3, yield
150 mg.
120
According to the procedure of Corbett and Young, 2.3 (200 mg)
was heated under reflux in 50 ml of ethanolic K0H (0.5 N) for two hours,
cooled, neutralized (H^SO^), 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 (^SO^) and concentrated to give an
oil, 190 mg, which could not be induced to crystallize. The product
was homogeneous (tic) with 2.3, A 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.


49
Epieye1ocolorenone 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, 188-189C
1 ?f)
(lit. 189), homogeneous with 2.3-DNP on tic, uv 400 nm, log 4.48/
chloroform (lit. 397.5 nm/4.49).^
Parthenolide 2.16
Yellowed leaves of M. grandiflora (500 g) were cut into small
pieces and extracted with ethanol for one day at 25C. 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
1 AR
from ether-hexane; yield, 0.5 g; mp 112-4 (lit. 115C) ; ir: 1730
and 1655 cm ^; mass spectrum: m/e 248 (M+), 233, 230, 190 and 43.
Anal. calc, for C-i^qO^: C, 72.55; H, 8.12. Found: 72.33:
H, 8.17.


CHAPTER 3
PHENYLPROPANOIDS OF Magno!ia grandiflora L.
Bis-allylphenol from the Bark
The distribution of diallylbiphenol derivatives in Magno!ia
spp. was discussed in Chapter 1. So far, three members of this group
12 53-54
have been isolated from Magno!ia spp., magnolol 3.1, hono-.. .
i? 54-57 q 53
kiol 3.2 and acuminatin 3.3. A search for these or related
55
compounds in Magnolia grandiflora was made but with negative results.
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.
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 Amflx of 290 nm. The phenolic nature of the compound
was demonstrated by the shift in A to 320 nm in the presence of
J max K
50


51
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 C-jgH^gO^ on the basis of elemental analysis and mass spectrum
(M+ 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~^ (phenolic OH), 1620 cm^
(aromatic, 985, 905 cm ^ (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 CHgCH-CHgj
4.86 ppm, s, exchangeable, ArOH/, 3.85 ppm, ArOCH^, 3.28, 3.40 ppm,
overlapping doublets, J = 6 Hz, 2 ArCH^2CH=CH2--clearly indicated that
there are two non-equivalent ally! groups on aromatic rings comprising
1,2,4 substitution patterns.


52
The ally! groups were readily reducible to afford a tetrahydro
compound. Elemental analysis and the mass spectrum (M+ 284) were
consistent with the molecular formula C^H^O^ The nmr spectrum showed
two non-equivalent benzylic n-propyl groups: 2.40 and 2.62 ppm, over
lapping triplets, J 3 5.2 Hz, 2 ArCH^HpCH^-, 1.63 and 1.67 ppm, over
lapping sextets, J = 8 Hz 2 ArCHpCHpCHg, 0.94 and 0.97 ppm, overlapping
triplets, J 3 8 Hz, 2 ArCH2CH2CH3.
Acetylation of the phenolic component gave a monoacetate
^21H22^3 322). The ir spectrum of the acetate (1755 cm^) and the
nmr signal (2.02 ppm, s, 3H) showed that the compound was a phenolic
acetate.
Methylation provided a monomethyl ether C2qH2202 (M+ 294) which
showed no hydroxyl 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-O-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-
149 150
able using ferric ion, hydrogen peroxide, hydrogen peroxide and


53
151 152
ferrous sulfate, and enzymic catalysis. However, not only are
the yields in thse 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
12
Ullmann coupling procedure employed by Fujita et aj_. 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 £-propyl phenol 3.11 and
the o-allyl phenol 3.12.
3.1, R1 = R2 = H
r3 = ch2ch=ch2
3.5,R-| = R2 = CH^
r3 = ch2ch2ch3
3.8, R1 = R2 = H
r3 = ch2 ch2ch3
3.2, R-j = R2 = H
r3 = ch2ch=ch2
3.6,R-j = R2 = CH3
r3 = ch2ch2ch3
^9, R1 = R2 = H
R3 = CH2CH2CH3
M. Rt = r2 3 H
R3 = CH2CH=CH2
3.7,R-| = R2 = CH3
r3 = CH2CH2CH3
3.10, R-j = r2 = H
r3 ch2ch2ch3


54
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, 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
153
of Fujita et a]_. using copper powder activated by the procedure of
154
Kleidner, and heating without solvent at 260C. 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
12
as recommended by Fujita et al.
Linder these conditions, dimethyl tetrahydromagnolol 3.5 was
obtained in a 30% yield from 3.16. Coupling of 3.17 provided


55
3,3'-di-n-propyl-4,4'-biDhenol dimethyl ether 3.17 in a 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 r
3.18, R = CH2CH=CH2
3.20, R = CH2CH2CH3
3.19, R = CH2CH=CH2
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


56
spectral and complexing 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_J5 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 values, they were designated as
ether-A (higher R^) and ether-B (lower R^). 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


57
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.
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 anisle, used as model compounds, in a
neutral medium, phenol was degraded rapidly in a basic medium while
anisle 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 appropriatehydroxybenzoic acids, followed by Clemmensen reduction of


58
th keto groups. With regard to acid 3.22, 5-propionyl salicylic acid
3.25 has been prepared by the Fries rearrangement of the propionate
1 55
ester 3.24. It was methylated to the ether but not reduced to the
2-methoxy-5-n-propylbenzoic acid 3.22. Cox also carried out a Fries
156
rearrangement of the propionate of methyl salicylate. Although no
assignment was made for the product, the properties appear to correspond
to the ortho-migration product, 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
157 158 159
Claisen rearrangement of the corresponding ally! ether 3.27.
Methylation gave the ether 3.30, which on hydrolysis gave the acid
157
3.31. 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 £-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^ but this com
pound was not reduced.


59
l
3.27, R = CH2CH = CH2
*' = c2h5
3.32, R = COCH2CH3
R' = H
3.28, R = H, R' = C2H5
3.30, R = CH3, R = C2H5
3.33, R CH3, R1 H
3,29, R = R' = H
3.31, R = R' = CH3
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)
3.35
aThe melting point of 5-n-propylsalicylic acid 3.25 obtained by
hydrolysis of this sample is much greater than obtained by Cox^56
(210.5-213 vs. 177-9C), supporting the contention that Cox has ob
tained the wrong isomer.


60
For the synthesis of the ester of 3.23, the ally! ether of
methyl £-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.
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 R^), which was identical
with the tetrahydro derivative of the natural product, gave as
product 3.31 while ether-B (lower R^.) 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 180-82C
[a]p = +4.0 (c 1.12,CHC13). Its characteristic uv spectrum, Amax


61
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
(Mf 418) and elemental analysis agreed with the molecular formula
^22^26^8* "^he 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"^; 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:
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.


62
Confirmation for the furofuran type structure was obtained
through the analysis of the mass spectrum along the lines described by
Pelter for pinoresinol.^ 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 Magno!ia lignan is one of the diastereomeric 1irioresinols and it
remains to determine which of the three stereochemical representatations
shown in Figure 3.2 is the correct one for the compound. (Relative
1 fi?
stereochemistries were determined by Briggs, Cambie, and Couch.)
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 aj_., who first isolated two of these
lirioresinols (A and B) by the acid hydrolysis of 1iriodendrin, a
glycoside from Liriodendron tulipifera L. (Magno!iaceae) as well as
1 fid
from commercial beechwood sulfite liquors (Populus spp., Salicaceae).
I fiO
Of these, syringaresinol corresponds to dl_-lirioresinol B. Emulsin-
hydrolysis of liriodendrin gave an aglycone which was first assigned the
163
structure of lirioresenil C but was later shown to be a mixture
*1 go
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
1 fi?
excel sum (Forst. f.) Miq. (Piperaceae).
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


63
Phenol and Phenol Ether Processes M+-29(CHO) m/e 389 (1%P)
M-t 15(CH3 ) m/e 403 (2SP) M+-30(CH20) m/e 388 (4%P)
M+-28(CsO) m/e 390 (0%P)
aIntensity of peak from Magnolia 1 ignan/intensity of corresponding peak
of pinoresinol
Figure 3.1 Comparison of mass-spectral fragmentation patterns of
Magno!ia lignan and pinoresinol


G4
different enantiomeric compositions.^,164 phyS-¡ca] pr0perties of
the three 1irioresinols 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
Ar
Ar. ^0'
0^ ''
H
Ar
3.41
3.42
3.43
Lirioresinol A
(axial/equatorial)
Lirioresinol B
(diequatorial)
(Lirioresinol C)
(diaxial)
Figure 3.2 Relative stereochemistries of the lirioresinols
The infrared spectra (KBr pellets) of the Magno!ia lignan and
of synthetic syringaresinol (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


65
Table 3.1 Properties of the lirioresinols and their derivatives
Compound
mp, C
wD
Reference
Lirioresinol A
210-211
+ 127
163
180-181

164
177
-90
165
Lirioresinol A
dimethyl ether
118-120
+ 119
163
diacetate
188

164
Lirioresinol B
177-183
-34.8
166
175-179a

167
172-179
+62.2
163
170
-21.5
165
dimethyl ether
126.5-127

59
122-123
+46.2
163
122-123
+45.8
168
121-122
+46.0
166
116-118a

167
115-119a

169
diacetate
188

163
Syringaresinol
179-185.5
0
170
(dl-lirioresinol B)
175
+1.93
171
174
0
172
170-171
0
173
168-172
+3.93
164
dimethyl ether
107_9b
-4.14
171
107-8
0
172
diacetate
188-189
-4.73
171
181-182

164
181-182
0
173
Lirioresinol Cc



dimethyl ether
145-147
+284
162
Magnolia lignan
180-182
+4.0
this work
dimethyl ether
107.5-108

this work
diacetate
185-186
0
this work
Assignment unclear
^Synthetic
cGenuine--many reports of lirioresinal C are incorrect


WvtNUM Cm'
Figure 3.3 Infrared spectra (KBr) of (a) Magnolia lignan and (b) synthetic syringaresinol


67
may alter the infrared spectra according to the enantiomer con-
tent.'74
The nmr spectra of lirioresinols A and B are not available,
175
although the corresponding isomers in the pinoresinol series are.
The two spectra are presented in Figure 3.4 and the spectrum of the
Magno!ia 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),^ 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
1 go
of the axial protons in the presence of an endo-aryl group. 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 $-ray studies.^ Assignments of these
resonances based upon J are in error: it has been stated that in
lignan spectra, overdependence on coupling constants, based upon bond


68
(a)Mmr spectrum of (+)-epipinoresinol (stereochemistry of
lirioresinol A)
IMkl
(b)Nmr spectrum of (+)-pinoresinol (stereochemistry of lirioresinol
B)
(5Ha 4.98 ppm (d, J = 5 Hz)
6HB 3.25 ppm
6Hyax 3.72 ppm
(c)Spectral assignments for lirioresinol C dimethyl ether (R = OCH^)
Figure 3.4 Nmr spectra of the three pinoresinol lirioresinol skeleta


I .... I .... I .... I .... I .... I ... I .... I .... I .... I .... I
To To sTo PPM () 4.0 3.0 2.0
Figure 3.5 Nmr spectrum of Magnolia lignan (CDCl^)


70
angles is undependable.^,179 ^ore emphas-¡s -¡s piaced 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.
59
The dimethyl ether has been isolated from Magnolia Kobus 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
172 181
sinapyl alcohol. Subsequently, it was synthesized by nonenzymic
182
oxidative coupling of sinapyl alcohol and sinapic acid. In the
latter synthesis, the dilactone initially produced was reduced and
pyrolyzed to yield syringaresinol.
Syringaresinol glycosides occur in a number of plants, including
Magnol ia grandiflora and Liriodendron tulipifera L.^ (Magnol iaceae).
Syringaresinol itself, as well as the optically active form lirioresinol
B, has been obtained from Liriodendron tulipifera^169 (^agnoljaceae)
164 173 182
Populus spp., Fagus spp. (Betulaceae), Picea excelsa
183 171
Engelm. (Pinaceae), Sinomenium acutum Rehd. et Wils. (Menisperma-
ceae), and Xanthoxylum inerme Koidz.^0 (Rutaceae). The dimethyl ether
has been obtained from Liriodendron tulipifera |_J67,169 (f/|agnoi jaceae),
168
Eremophila glabra (Myoporaceae), Macropiper excel sum (Forst. f.)
MiqJ^ (Piperaceae), and Aspidosperma Marcgravianum Woodson^
(Apocynaceae), in addition to the Magnolias previously cited.


71
Experimental
Isolation of Mehonokiol 3.19
Ground bark (4 kg) of Magnolia grandiflora, collected in Gaines
ville, Florida, was extracted with ethanol (3 £) at 25C for two days.
Three such extracts were combined and concentrated to a syrup which
was partitioned between water and ethyl acetate (1 £ 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 (tic: 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 benzenejiiexane (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: Am,v 255 nm (log e 4.1) and
290 nm (log e 3.8); with base, A 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, OCH^;
3.40 and 3.28, overlapping doublets, J = 6 Hz, 2 ArCH2CH=CH2; ms:
280.1456; calc, for C10H2Q02: 280.1464.


72
Mehonokiol Acetate
A mixture of 3.19 (0.05 g), acetic anhydride (1 ml) and pyridine
(two drops) was heated at 70C 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^; nmr: 2.01 ppm, s, 3H;
ms: Mf 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 ^20H222: 294.1621.
Tetrahydromehonokiol 3.21
A solution of i> (0.1 g) in ethanol (5 ml) was hydrogenated in the
presence of Pt02 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"^; nmr
(ppm); 240 and 2.62 ppm, overlapping triplets, J = 5.2 Hz, 2 ArCHpCHoCH.,
1.63 and 1.67 ppm, overlapping sextets, J = 8 Hz, 2 ArCHpCHgCH.,, 0.94
and 0.97 ppm, overlapping triplets, J = 8 Hz, 2 ArCH2CH2CH3; ms:Mf 284,
m/e 255, 223, 205.


73
Anal. Calc, for 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 anisle (10 g) was
-153
iodinated by the method of Hata and Sato using mercuric oxide-
catalyzed iodination by in ethanol. The resulting product was
purified by distillation (1.5 mm, 105-115C) 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, 100C); yield 8 g (88%).
Tetrahydromagnolol dimethyl ether 3.5
The coupling of 3.16 (10 g) was carried out by a modification of
55
the procedure of Fujita et al_., in which the copper powder (22 g)
was gradually added to 3.16 at 240C. 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),


74
256 nm (log e 4.0); nmr (ppm): 6.70-7.20, m, 6 ArH; 3.68, s, 2 OCH^;
2.50, 1.60 and 0.95, triplet/sextet/triplet, respectively, 2 CHgC^CH^.
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. 143C).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. 114C).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 113-114C (lit. 112.5C).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 25C
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 79-80C;
nmr: (ppm) 3.4-2.6, m, 6-ArH; 4.88, s, Ar-OH; 6.23, s, OCH^; ms: Mf 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% 0V-17, T 180C,
3.5, tr 11 min., 25%; 3.6, tr 15.8 min., 55%; and 3.7, t 20.8 min.,
19%) was subjected to chromatography on FI ori sil for preliminary
purification. Direct crystallization gave 3.7 (0.9 g), the mother
liquors were purified by chromatography (silica gel, cyclohexane with


75
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: 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 117-117.5C (lit.
118C).53
Mono-O-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 25C 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, 200C; 3.21 t 5.5 min.; 3.22 t 8.3 min., tic: 0.90,
0.60, respectively, silica benzene). The monoether of higher R^ was
identical with the natural tetrahydromehonokiol by tic, gc and spec
tral comparison. The mixture was separated by chromatography (silica
gel, cyclohexane with a benzene gradient). The high R^ ether 3.21
was obtained as a colorless oil: ms: 284.1790 (calc, for C-jgi^O^:
284.1775); m/e 269 (100%), 256 (17%) and 255 (80%).
Anal. Calc, for 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 H2: 77.85; H, 8.59.
Found: C, 77.69: H, 8.65.


76
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 -30C 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 (tic: 0.60, silica, benzene; gc: 3% SE-30, 130C,
t 7 min.).


77
Similar degradation of 3.21 gave, after esterification, a product
identical with methyl-3-n-propyl £-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 (tic: 0.25, silica, benzene; gc: 3% SE-30, 130C, t^,
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 0C. After 16 hours at 5C it was diluted
with benzene and washed successively with IN 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^; 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; ArCh^CH^CH^.
Saponification (IN K0H, reflux 5 min.) gave, on workup, the
acid 3.25, mp 210-211.5C from water (lit. 177-9C) 0 (cf. p. 59).


78
Methyl-3-n-propyl-£-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"^; 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 220C 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~^; 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 25C. The
mixture was filtered and the product recovered by concentration of the
filtrate and crystallization; mp 122.5-125C. 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-£-anisate 3.31 as a
colorless crystalline solid; mp 36-37C. It was identical with the
methylated ozonolytic degradation product of tetrahydromehonokiol, 3.21
Saponification (IN K0H, refluxed 5 min.) gave on workup the acid,
3.29, mp 115-17C from water (lit. 116.8C).^


79
(di )-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 (Na^SO^),
concentrated, the phenolic lignan extracted and isolated by chromatogra
phy (silica gel, 20 g, benzene with ethyl acetate solvent gradient).
The lignan, (tic: 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]p
+4.0 (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) ^ -¡rj nmr: mp cf. Figures 3.3, 3.5,
and Table 3.1.
Anal. Calc, for £2282503^ 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 C24HgQ0g: C, 64.56; H, 6.77. Found: C, 64.41;
H, 6.67.


80
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 0.0 (c 0.50, CHC13); nmr: 2.32 ppm, 6H, s, CH3C02Ar;
ir: 1764 cm^.
Anal. Calc, for C26^30^10: 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 Magno1ia 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 Magno!ia 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 Hgl^" 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 Hgl^" ion by Cl" 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
81


82
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_.g_., 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 Magno1ia
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


83
Wood Extract
Figure 4.1 Fractionation scheme for Magnolia wood alkaloids


84
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
15
magnoflorine, which is the major alkaloid of Magnolia grandiflora bark.
The quaternary alkaloid was next converted to the iodide salt
by ion-exchange using Dowex-1 iodide and was obtained as a crystalline
solid, mp 222-25 (dec.); [a]g+212 (c 1.27, ethanol) representing a


Optical density at 270 nm
85
Figure 4.2 Cellulose partition chromatography of BuOH fraction


86
yield of 0.02%. It was also toxic to mice with an LD^q value of
10 mg/kg.
Elemental analysis of the toxic alkaloid agreed with the molecu
lar formula C20H 2gN0^I. Its uv spectrum showed Amax 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\
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 CH^X, or a Hofmann elimination of HX. The actual course
184
depends on the alkaloid and the nature of X: with Magno!ia 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 (Mf -CH2=NCH3), m/e 326 (M+ -CH^and m/e 310
(M+ -0CH3).
The data suggested that the Magno!ia 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(0CH3); 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
to an iminium ion such as 4.3 derived by the ot-fission of a
185 186 187
benzyltetrahydroisoquinolinesystem and the uv spectrum estab
lished that the alkaloid has an aporphine skeleton 4.1.
The presence of a phenolic hydroxyl
tion of a monomethyl ether, nmr: 3.71 ppm,
ppm, 3H. Acetylation with acetic anhydride
ceed, possibly due to steric hindrance, but
gave an acetate, ir: 1770 cm"^ and nmr: 2
was verified by the forma-
6H; 3.90 ppm, 3H and 3.92
and pyridine did not pro
acid-catalyzed acetylation
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
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 Magno1ia 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
186 187
nm. 5 This difference has been said to reflect the greater inter
ference with coplanarity of the biphenyl system in a 1,2,10,11-
187
oxygenation pattern. The mass spectra of several 1,2,9,10-oxygenated
185 186 188
aporphines have been published 5 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 (Mf -1), (b) subsequent formation
of systems such as 4.8 or 4.9 with the loss of either OCH^ or CH^,
respectively, and, (c) retro Diels-Alder cleavage to 4.10 which can
1gg 1gg
undergo further loss of OCH^ or CH^. In contrast, the mass
spectun of the Magnolia alkaloid showed no M+ -1 peak, low intensity


89
Figure 4.3 Mass spectral behavior of a typical aporphine


90
M+ -43 peak (retro Diels-Alder process) but a relatively high intensity
M+, M+ -OCH^ peaks. Such behavior is peculiar to those aporphines with
189
1,2,10,11-oxygenation pattern.
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
Magno!ia alkaloid by comparison of its tic and spectral behavior.
magnoflorine 4.10 = H
dimethyl magnoflorine 4.11 R^ = R^ 3 CH,
For the Magno1ia 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


91
N-methyl-10-0-methylhernovine methiodide
N-methyl corydinium iodide
N-methyl isocorydinium iodide
4.12 R1 Hr R2 = R3 = R1 = CH3
4.13 R2 = H1, R] = R3 = R4 = CH3
4.14 R3 = H], R] = R2 = R4 CH3
4.15 R4 = R4 = H1, R1 =R2 = R4 = CH3
Figure 4.4 Isomeric hydroxy-trimethoxyaporphinium methiodides
them by Hofmann elimination, with free hydroxyl at C-l or C-11, are
known to show hydroxyl frequencies below 3300 cm"\^ 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-l and C-ll do so at 3.4-3.7 ppm. 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
189
of 4.13 and 4.14 were somewhat different, the spectrum of the Magno!ia
alkaloid was not sufficiently similar to either of the literature spectra
to allow an assignment. Neither were the reported nmr spectra of use:


92
H-8 and H-9 of 4.13 have been reported as singlets at 7.27 and 7.33 ppm,
193
with H-3 at 7.05 ppm while 4,14 showed H-8and H-9as a singlet at
194
6.96 ppm, and H-3 at 7.02 ppm. As the chemical shift differences for
H-8 and H-9 are small, the outer peaks of the AB-quartet are small and,
therefore, often overlooked. Furthermore, variations from these values
195 196
are also found in the literature. The spectra also vary
depending on the solvent used (DgDMSO, CD30D, CF^COOH or DgO). The
observation of an AB-quartet for H-8 and 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 Magno!ia 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 Magno!ia
toxic alkaloid and its ether
H3, ppm
H8
and
H9
alkaloid
6.96
6.98d
or
7.lid
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 an unsubtituted posi
tion para to the phenol ate function which is very reactive toward
electrophilic reagents, whereas N-methylcorydine 4.13 has none. This
195
difference has been exploited in a number of ways: deuteration,
197
and the Gibbs test (reaction with 2,6-dibromoquinone chlorimide)


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UNIVERSITY OF FLORIDA
3 1262 08554 3634


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INGEST IEID E2E5PRADX_7WIBP6 INGEST_TIME 2011-09-29T20:56:13Z PACKAGE AA00004903_00001
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CHEMISTRY OF Magno!i a 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

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, University 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
Pag;e
ACKNOWLEDGMENTS iii
ABSTRACT v
CHAPTER
1 SECONDARY METABOLISM AND THE CHEMISTRY OF
Maqnolia; PHARMACOLOGICAL ACTIVITY OF Maqnolia
EXTRACTS AND COMPONENTS 1
Biogenetic Classification of Secondary
Metabolisms 1
Terpenoids of Genus Maqnolia 1
Alkaloids of Genus Maqnolia 5
Compounds Derived from Shikimic Acid 13
Pharmacological Activity of Maqnolia
Preparations and Maqnolia Compounds 21
2 SESQUITERPENOIDS OF Maqnolia grandiflora L 27
Ketone from the Bark Extract 27
Antibiotic Principle of the Leaves 41
Experimental 44
3 PHENYLPROPANOIDS OF Maqnolia grandiflora L 50
Bis-allylphenol from the Bark 50
Lignan from the Wood 60
Experimental 71
4 ALKALOIDS OF Maqnolia grandiflora L 81
Toxic Alkaloidal Fraction 81
Non-toxic Alkaloidal Fraction 94
Experimental 97
General Experimental 104
LIST OF REFERENCES 105
BIOGRAPHICAL SKETCH 117
iv

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 Magno!ia grandiflora L.
By
Terry Lee Davis
June 1981
Chairman: K. V. Rao
Major Department: Medicinal Chemistry
Magno!ia 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. Magno!ia 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 Magno1ia
spp. Another sesquiterpene, a lactone, identified as parthenolide was 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 1 ignoid
v

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.
vi

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 reprsented 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.
vi 1

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 Magno!iaceae, a family
which has been variously classified under the order Ranales or Mag¬
no! ialesJ The chemistry of this very primitive plant has been
studied extensively, particularly the alkaloids. Magno!ia 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.^
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
3
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
Magno!ia to be described in this dissertation.
Terpenoids of Genus Magno!ia
The terpenoids are compounds arising from mevalonic acid-
derived isoprenes, such as isopentenyl pyrophosphate, by catenation
1

2
Table 1.1 Species of Magnolia reported in the chemical literature
Subgenus Magnolia
Sect. Gwi11imia (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. officinal is Rehd. et Wils.
M. rostrata W.W. Smith
M. tripétala L. (syn. M. umbrel1 a Desr.)
Sect. Oyama (Asia)
M. parviflora (syn. M. Sieboldii K. Koch)
M. Sieboldi i K. Koch (syn. M. parviflora Sieb. et Zucc.)
M. sinensis Stapf. (syn. M. globosa var. sinensis Rehd. et Wils.)
Sect. Magno!iastrum (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 PIeurochasma
Sect. Yulania (Asia)
M. Campbel1i Hook f. et Thoms.
M. denudata Desr. (syn. M. Yulan Desf.)
Sect. Buergeria (Asia)
M. Kobus DC.
M. salicifolia Maxim.
M. stellata Maxim.

3
Table 1.l--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-
gesii (syn. Michelia Fargesii Andr.), Magno1ia fuscata Andr. (syn.
Michel i a fuscata Blume, M. Figo Spreng.), M. LenneiTopf. (syn. M. Soulan-
geana var. Lennei Rehd.), and M. mutabilis Rege!. M. X Soulangeana Soul,
is M. denudata x M. liliflora.

4
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 Magno!ia 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.

5
In the present work the first isolation from any Magno!ia 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 Michel i a champaca L., Michelia
5 6
lanuginosa, and is known to occur also in Magnolia grandiflora.
In Table 1.2 are presented the terpenoid compounds of various
7-13
types which have been isolated from genus Magnolia.
Alkaloids of Genus Maonolia
Studies on the alkaloids of Magnolia have accounted for the
bulk of the chemical and pharmacological literature of the genus and have
14
been reviewed by Tomita and Nakano.
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
15-46
throughout the Magnoliaceae are detailed in Figure 1.1.

Table 1.2 Compounds of mevalonate origin reported in the genus Magno!ia
Compound
Class
Species
citral
monocyclic terpene
M. obovata (lvs)3’^
M. Kobus (lvs, stms)8
M. salicifolia (lvs, stms)^>9
1imonene
monocyclic terpene
M. obovata (lvs)7
M. Kobus (bds)10
cineole
monocarbocycl ic terpenes
M. Kobus (lvs, stms)8 (bds)10
M. salicifolia (lvs, stms)°
a- and 3-pinenes
bicyclic terpenes
M. obovata (lvs)7
M. Kobus (lvs, stms)8
bornyl acetate
bicyclic terpene
M. obovata (lvs)?
camphene
bicyclic terpene
M. obovata (lvs)7
camphor
bicyclic terpene
M. Kobus (bds)10
caryophyl1 ene
bicyclic sesquiterpene
M. obovata (lvs)7
caryophyllene epoxide
bicarbocyclic sesquiterpene
M. obovata (lvs)7
a-, 3- and y-eudesmols
bicyclic sesquiterpene
(eudesmane type)
M. obovata (lvs)7
cyclocolorenone
tricyclic sesquiterpene
(aromadendrane type)
M. grandiflora^

Table 1.2--continued
Compound
Class
Species
costunolide
monocarbocyclic sesquiterpene
lactone (germacranolide)
M. acuminata (rt-bk)^
parthenolide
monocarbocyclic sesquiterpene
lactone (germacranolide)
M. grandiflora (lvs)b,b (stms)b
crytomeridiol
bicyclic sesquiterpene
M. obovata (bk)^
campesterol
tetracyclic triterpene (sterol)
M. obovata (lvs)7
cholesterol
tetracyclic triterpene (sterol)
M. obovata (lvs)7
M. grandiflora (bk)c
0-sitosterol
tetracyclic triterpene (sterol)
M. acuminata (rt-bk)^
M. Campbelli (bk)^3
M. grandiflora (bk)c
stigmasterol
tetracyclic triterpene (sterol)
M. obovata (lvs)7
M. grandiflora (bk)c
ct-amyrin
pentacyclic triterpene
M. obovata (lvs)7
lupeol
pentacyclic triterpene
M. ovovata (lvs)7
abk = bark, lvs * leaves, htwd = heartwood, rts = roots, stms = stems, bds = buds, wd = wood, flwr =
flowers, sds = seeds.
bThis work,
c
In the course of this work, materials with the same tic behavior and glc retention times (on one column)
as commercial samples of the above were observed.

8
magnoflorine
1.8
Figure 1.1 Probable derivation of Magno!ia alkaloids magnoflorine
and reticulene from tyrosine

9
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 Magno1ia 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
40
isolated from this plant, 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
40
M. grandiflora, and, in the course of this work, it was also observed
in the bark.
1.11
1.12

10
Table 1.3 Alkaloids isolated from Magnolia spp.
Compound
phenyl ethyl amine
candicine
M.
salicifoline
M.
M.
M.
M.
M.
M.
M.
M.
benzylisoquinoline
magnococline
M.
N-norarmepavine
M.
reticuline
M.
benzylisoquinoline
(quaternary)
magnocurarine
M.
M.
M.
M.
M.
M.
M.
M.
bisbenzylisoquino!ine
magnolamine
M.
magnoline
M.
aporphine
anonaine
M.
M.
Occurrence
grandiflora (bk)15 (rts)"^
acuminata (stms)l?
coco (stmsy8
denudata (bk)19
grandiflora (rts)16 (bk)a,2‘
Kobus (bk)22>23
lilif1 ora (bk, rts, wd)24
salicifolia (bk)25
stellata (bk)25
coco (stms)2?
kachirachirai (htwd)28,29
obovata (rts)30
acuminata (stms)]?
denudata (bk)'9>20
fuscatab (lvs, bk)33
liliflora (bk)2^
obovata (rts)2Q (bk)2^’25 (htwd)35
officinal is (htwd)36
parviflora (bk)2?
salicifolia (bk)22
fuscatab (lvs)33,38,39 (bk)33
fuscata5 (lvs)38
grandiflora (wd)c>40 (lvs)40
obovata (lvs)30 (rts)30 (htwd)35,41

11
Table 1.3--continued
Compound Occurrence
aporphine (continued)
anonaine acetamide
anolobine
asimilobine
glaucine
N-norglaucine
N-nornuciferine
obovanine
aporphine (quaternary)
N ,N-dimethyl1indcarpine
iodide
magnoflorine
menisperine
proaporphine
stepharine
7-hydroxyaporphine
michelalbine
(norushinsunine)
M. obovata (htwd)^l
M. acuminata (rt-bk)9
M. coco (stms)27,42 (rt-bk)
M. grandiflora (wd)40
M. obovata (Ivs, rts)30
M. kachirachirai (htwd)18,29
M. obovata (lvs)30
M. kachirachirai (htwd)29
M. grandiflora (wd)40
M. obovata (Ivs)30
M. acuminata (rt-bk)^
M. acuminata (Ivs, stms)e
M. coco (stms)^
M. denudata (rts)^
M. fuscatab (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)^7»42 (rt-bk)^3
M. obovata (htwd)35

12
Table 1.3--continued
Compound
Occurrence
7-oxotetradehydroaporphi ne
lanuginosine
M. Campbelli (bk)^
liriodenine
(oxoushinsunine)
M. Campbelli (bk)^
M, COCO (stms)18 4n 46 40
M. grandiflora (bk)c (wd) ’ ’ (lvs)
M. mutabilis (lvs)'l oc
M. obovata (lvs, rts)JU (htwd)"33’
(bk)35
oxoglaucine
29
M. kachirachirai (htwd)
oxolaureline
46
M. X Soulangeana
aAbsence of salicifoline in the bark of M. grandiflora var. lanceolata
Ait. has been noted by Tomita et al.20
^Now considered by most authors to be Michelia fuscata Blume,^ or
Michel i a Figo Spreng31«32
cThis work
dNot isolated; identified by paper chromatography^7

13
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 Magno!iaceae
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-
8_10,47-i
derived compounds isolated from Magno!ia are listed in Table 1.4.
Oxidative juncture of cinnamyl alcohol and its derivatives at
the 8-positions of the 2-propenyl sidechains results in a class of
compounds Haworth has termed the lignans.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

Table 1.4 Shikimic acid-derived compounds of Magnolia spp.
Compound
Structural Type
Occurrence
anisaldehyde
benzaldehyde
M. salicifolia (lvs, stms)8
eugenol
allylphenol
M. Kobus (lvs, stms)8
methylchavicol
allylphenol
M. Kobus (lvs, stms)8
methyleugenol
allylphenol
M. salicifolia (bds)^°
safrole
allylphenol
M. salicifolia (bds)10
trans-anethole
propenylphenol
M. salicifolia (lvs, stms)8>9
trans-asarone
propenylphenol
M. salicifolia (bds)10
syringin (syringoside)
3-hydroxypropenylphenol
glycoside
(lvs, stm-bk) of:^8
M. acuminata
M. Campbelli
M. denudata
M. grandiflora
M. Kobus
M. liliflora
M. macrophylla
M. obovata
M. parviflora
M. salicifoTTa
M. X Soulangeana
M. stellata
M. tripétala
M. Wilsonii
M. grandiflora (wd)49

Table 1.4--continued
Compound
Structural Type
Occurrence
magnolidin
a-tocopherol and 4 uniden¬
tified chromanols
magnolioside
acuminatin
honokiol
magnolol
mehonokiol
calopiptin
glycosidic ester of a
cinnamic acid
chromanol
coumarin glycoside3
neolignan--bis(allylphenol)
neolignan--bis(allylphenol)
neolignan--bis(allylphenol)
neolignan--bis(allylphenol)
neoliqnan--galgravin type
neolignan--galgravin type
M. grandiflora^ 5,49,50
M. X Soulangeana (lvs)5^
M. macrophylla (bk)52
M. acuminata (rt-bk)9>53
M. obovata (bk)12,54,55
M. officinalis (bk)54,55
M. tripétala (bk)55
M. grandiflora (sds)56
M. rostrata (bk)53
M. obovata (bk)^2,54,55
M. officinalis (bk)54,55
M. tripétala (bk)55
M. virginiana (bk)^5
M. grandiflora (sds)56
M. rostrata (bk)57
M. grandiflora (bk)6 (sds)^6
M. acuminata (rt-bk)9>53
M. acuminata (rt-bk)^
galgravin

Table 1.4--continued
Compound
Structural Type
Occurrence
veraguensin
neolignan--galgravin type
M. acuminata (rt-bk)^’33
sesamin
1ignan--pinoresinol type
13
M. mutabilis (lvs, stms)
eudesmin
1ignan--pinoresinol type
M. Fargesii (flwr-bds)^8
magnolin
1ignan--pinoresinol type
M. Fargesii (flwr-bds)58
syringaresinol
1ignan--pinoresinol type
M. grandiflora (wd)a
syringaresinol dimethyl
ether
1 ignan--pinoresinol type
M. Fargesii (flwr-bds)33
M. Kobus (sds)59
aThis work

17
2,5-diarylfurans
(olivil type)
(pinoresinol type)
OH
1-ary1 naptha 1 ene 1-arylnapthalenes
lactones (podophyllotoxins)
Figure 1.2 Notional representation of lignan biogenesis and struc¬
tural types

18
6i 62
designated "neolignans." ’ The neolignans are more restricted in
their distribution than the lignans proper, having been isolated only
rp
from two subclasses, the Magno!iidae and the Rosidae. Those large
groups encompass, however, a number of major families, including the
Magno!jales 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 Magno!ia species (cf.
Table 1.4), while the latter, isolated from Litsea turfosa Kosterm. (Laur-
63
aceae ) is the only bis-allylphenol-type neolignan occurring outside
the genus Magnolia.

19
r\-| j ^2
R-j = R2 = R^ = Rg = OCH^, R0 = Rg = H
R-| = ^2 = R4 = R5 = R6 = OCH^j R^ = H
R] = FI3 = R4 = r6 = 0CH3, R2 = r5 = OH
R1 = R2 3 R3 = R4 = R5 = R6 0CH3
sesamin
magnolin
eudesmin
syringaresinol 1.13
syringaresinol
dimethyl ether
R1 = R3 = R4 = Re = R2 = OGlu, R^ = OH acanthoside B
R = (CH2) calopiptin
Figure 1.3 Known lignans of Genus Magnolia

20
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.3 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 Zeyheria^ montana
Mart, and 1. tuberculosa (Veil.) Bur. ex Verlot (Bignoniaceae)^4 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
m t • 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 *H- and 13c_nmr spectroscopy.56
^Zeyheria is spelled Zehyera by the author.

21
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,^ American
71 72
Indian medicine, ’ and even listed in American pharmacopoeiae and
pharmacognosy texts as bitter tonics, antimalarials, and diapho-
73 74 75
reties. ’ ’ The use of Magnolia extract in medicine has been pro-
7 f)
posed even as recently as 1953.
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.^ An extract of M. Kobus has shown some antiviral
activity in mice at subtoxic levels. The extract of M. grandiflora
80
was found to have acaricidal activity, and to lower blood pressure
without effect on heart action or breathing.^
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
81
extract, at the same dose, showed prompt respiratory paralysis.
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
82
(Ci7HigNOs) with curare-like action from the same drug.

22
Several of the active principles of neuroactive Magno!ia
extracts have been isolated and identified. The bis-benzylisoquinoline,
magnoline, 1.10, has a hypotensive effect and shows anticholinesterase
83
activity. Compounds associated with the observed "curare-like"
effects of Magno!ia extracts are magnocurarine 1.20, magnoflorine 1.8,
84
and salicifoline 1.21. Magnocurarine, the most active of these
84
quaternary alkaloids, has a ganglionic-blocking activity comparable
85
in strength to hexamethonium bitartrate. It exerts upon frogs an
action similar to that of d-tubocurarine (d-TC) 1.22,a but of longer
87
duration and only one-tenth as active.
H3C0'
HO'
00<5
/ CH3
— CH3
HO'
H0^^
^ CH3
1.20
1.21
Previously thought to be fujly quaternized, the structure of
d-TC has recently been revised.86

23
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 (j_n vitro), except in the case
85
of salicifoline, which caused contraction of the muscle.
The noraporphine anonaine 1.11, isolated in the course of this
work, has antibiotic activity at 100 mg/ml against Staphylococcus
88
aureus, Mycobacterium smegmatis, and Candida albicans. It also
89
exerts a hypotensive effect in mice and rabbits and has been shown to be
an inhibitor of dopaminergic response, an activity associated with a
90
number of analogs of apomorphine 1,23. Its specific effect in rats is
as an antagonist of dopamine-sensitive adenylate cyclase in the caudate
i 90
nucleus.
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
88 91
anonaine. ’ In addition, it is an inhibitor of human carcinoma of
92
the nasopharynx j_n vitro.

24
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-blocking^’9^"® and ganglionic-blocking activities'^®’9^5’9® of
which are well established.9 Menisperine, then known as "chakranine,"
was originally described as exerting ". . . ganglionic blockade selec¬
tively exercised against the autonomous nerve pathways of the respiratory
93
system." Erhart and Soine, using the cat tongue/hypoglossal nerve
system in vivo, have effectively characterized the nmj-blocking activity
94
of this drug as very similar to that of d-TC itself.
The LDgg of the pure iodide was determined in this work to be
10 mg/kg (21 ymol/kg, IP, mouse). For comparison, Kamat et al_. obtained
93
a value of 2.2 mg/kg (5.9 ymol/kg, IV, mouse) for the chloride.
The results of a pharmacological screening procedure routinely
employed in these laboratories are presented in Table 1.5.® 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
contractibi1itv and excitability in a manner resembling tetraethyl am¬
monium salts."97 This same author observed meiosis upon intra-lymphatic
injection in frogs.98
®After the method of Campbell and Richter.99

25
Table 1.5 Pharmacological evaluation of Magnolia toxic alkaloid
Sample Wa9no1ia Bark Date 9/19/78
Dose 1_2 mg/kg, 0.6 mq/ml Time 3:08 p.m.
Animal Mice
Animals
1
2
3
4
5
Final
Paw Temo b NT
raw lemp. after
Body T»mp before
37.
5
37.5
37.5
37
.5
37.5
Dooy lemp. after
—
35
36
-
--
32
Average Wt. (g)
25
Sign: 1.
Paw Temp., t
—
—
—
-
—
—
2.
Body Temp., +
-
-
-
3.
Body T emp., +
-
-
-
4.
Ptosis
-
-
-
5.
Salivation
-
-
-
6.
Lachrymation
-
-
-
7.
Mydriasis
+
+
-
♦
Miosis
(/)
0)
4->
-
-
«✓>
0)
4->
-
8.
Piloerection
c
E
-
-
c
*E
-
9.
Locomotor Activity, +
m
-
-
m
ro
-
10.
Straub tail phenom.
0>
-
-
07
4->
v*-
-
11.
Righting reflex
(r.r.) abolished
Dead
-
-
Dead
12.
Head drop
(with r.r. present)
-
-
-
13.
Pos. Haffner
(with r.r. present)
-
-
-
14.
Locomotion Activity, +
-
-
-
15.
Abduced hindlegs
(with r.r. present)
-
-
-
16.
Unsteady gait
1 -
-
-
17. Others: Onset of effects ca. 5 min., with marked depression of activity® 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 LD5Q, death
occurs in less than 5 minutes. At LD50 deaths occur at 15-20 min., with an occasional
mouse succumbing more promptly.
18. CMTD (mg/kg)
®Animals remain capable of locomotion when disturbed.

26
99
(head-drop, ptosis, strabismus) other than apparent respiratory
arrest is evident. However, such activity is not observed under these
conditions for d-TC itself.
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
93
0.3 mg/ml, respectively.
The sesquiterpene lactones parthenolide 1.6 and costunolide
1.24^ 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.
The lignans sesamin, eudesmin, and syringaresinol 1.13, all of
which occur in various species of Magno!ia (cf. Table 1.4) have some
anti tubercular activity in vitro. ^
Finally, compounds related to the diallylbiphenols found in
103
Magno!ia have been synthesized for medicinal and other applications,
and several have been patented for use as anticoccidial agents in
104
poultry feed.

CHAPTER 2
SESQUITERPENOIDS OF Magno1ia 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
15
described elsewhere. Fractionation oftiie 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 nonphenol ic. 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 5°C. Examination of the steroid
fraction by temperature-programmed and isothermal gas-chromatography
showed the presence of peaks which were identifiable with authentic
27

28
Ethanolic extract of
the bark
concentrate and
partition between
water and ethyl acetate
Lipophi
ic
fraction
Silica gel
chromatography
cyclocolorenone >
phytosterols
Mehonokiol Syringaresinol
Sephadex
chromatography
Hydrophilic fraction
Alkaloid Magnolenins Magnolidins
fraction A,B,C A,B
Ion-exchange
chromatography
Magnoflorine Candi cine
Figure 2.1 Fractionation of Magnolia grandiflora extract

29
samples of 3-sitosterol, stigmasterol and cholesterol, all of which
have been observed in the extracts of Magnolia obovata by Fujita et
al? 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 FI ori sil 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 a molecular formula
of C-i^H^O. 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 and unsaturation (1600 cm”^). Its nmr spectrum
gave evidence for the presence of three different types of methyl
groups: 0.72 ppm, d, CH-CH^; 1-00 ppm> s> anc* 1-23 ppm, s, F^C-iCH^;
and 1.70 ppm, d, -C^-CH^; along with a considerable overlap of signals
due to a CH^- 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.

30
Its uv-absorption maximum of 390 nm indicated that it was derived from
an a,8-unsaturated ketone, although the Amax was higher than that
expected for one double bond conjugated with the keto group.^,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 acid^ gave a ketonic product which, how¬
ever, was different from the original enone (higher and higher Amax).
In the absence of mineral acid, no exchange took place. Thus, altera¬
tion of the structure in the presence of acid was apparent.
The A of 260 nm observed for the ketone is intermediate in
max
value between that of an enone (220-250 nm) and that of a dienone
1 Qg
(280 nm). The literature suggested as a model for this chromophore
the cyclopropyl-conjugated enone 2.1, which was described by Büchi et
109
al., as an intermediate in their synthesis of maaliol 2.2. The
extent of overlap of the Tr-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.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

31
spectral fragment at m/e 175 (M-C^Hy). 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.
On the basis of the available information, a search of the
literature was conducted which revealed that the properties of the
Magno!ia enone and those of its 2,4-dinitrophenylhydrazone derivative
corresponded with those described for the sesquiterpene ketone, cyclocolor-
enone 2.3, first isolated from Pseudowintera colorata (Raoul)
112 113
Dandy. ’ 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

32
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 ally! alcohol 2.4 from which the starting enone could be
regenerated by oxidation with manganese dioxide. This alcohol appears
113
to correspond with cyclocolorenol 2,4 described by Corbett and Speden
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 38-40° was also prepared from a-gurjunene and characterized
114
as 4a,5a-cyclocoloranone which agrees with the properties of the
present ketone. However, the melting point of the dinitrophenylhydrazone
of the saturated ketone from Magno!ia 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 Magno!ia enone contained a 5-membered ring.

33
The nmr spectrum of the Magno1ia 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-l (J-| i2 = Hz). The signal centered at 2.84 ppm, a doublet of
doublets, represents the a-proton of C-2 coupled to the C-2B and C-l
protons with J2a of 18 Hz and ^ ^ °f 6.5 Hz. The C-l proton
appears as a multiplet at 2.94 ppm with H-2a at 2.29 ppm, ^ 2a = 2 Hz.
All of these signals show general agreement with those
115
described by Büchi et al_., for cyclocolorenone. The spectrum of the

34
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.^’^ This model compound shows ir
spectral bands at 1700 and 1630 cm ^ for C=0 and C=C, respectively,^
in comparison with the reported values of 1690 and 1630 cm-^ for the
respective groups of cyclocolorenone.
H-4 2.85 ppm
H-5o 2.37 ppm
H-5a 1.95 ppm
J
J
J
gem
4,5(cis)
4,5(trans)
19.5 Hz
6.5 Hz
2.5 Hz
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-l proton multiplet 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
118
that observed in the spectrum of a-gurjunene, photosantonic acid
119
lactone and a-santonin derivatives. In a-santonin derivatives, the

35
homoallylic coupling is of the order of 1.3-1.4 Hz, with a critical bond
118
angle of 115°. The angle measured for a stereomodel of cyclocoloren-
one was found to be 115° 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
118
Ourisson 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.
115
Epimerization of cyclocolorenone was observed Biichi et al.,
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-
120
duced the reverse reaction. Corbett and Young 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

36
product showed the expected shift in X toward 250 nm, thus indicating
iilaX
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 tic 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 Magno!ia
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,^’^ 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, ’ and of the Magno!ia
enone are compared in Figures 2.3 and 2.4 (an impurity appearing on the
carbonyl band diminishes as the sample is purified).

(a) (b)
Figure 2.3 Comparison of nmr spectra of (a) Magnolia enone and (b) synthetic cyclocolorenone

M * i Ji « it j s' « • j / ; > i t io ii ii ii u
CO
00
Figure 2.4 Comparison of ir spectra of (a) Magnolia enone and (b) synthetic cyclocolorenone

39
Cyclocolorenone was first isolated from Pseudowintera colorata,
a plant which the authors ascribe to Magno!iaceae, although it is
123-]26
usually assigned to Winteraceae. 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
Japónica (Hepaticae). In addition to these, the compound was found
1 op
to be present in Boronia ledifolia Gay (Rubiaceae) and in the golden-
129
rod, Solidago canadensis (Compositae).
Although the basic structure of cyclocolorenone was determined
by the authors who isolated it originally, its stereochemistry was
115
determined largely by Büchi et al_., through their synthesis of
epicyclocolorenone, and from its relationship to a-gurjuneneJ
Cyclocolorenone itself was finally synthesized by Ingwalson and Caine
121 122
starting from (-)-maalione. ’
Cyclocolorene is a member of what has recently been described
130
as . . the rare class of aromadendrane sesquiterpenes." 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,53 while
alloaromadendrene 2J3 is 1 B,5B;131,132 a-gurjunene 2.9118,133-135 anc|

40
1 2g
6-spathulene 2.10 represent the prototypes with a 4,5-double bond
and 1$ hydrogen: cyclocolorenone is of this type. This relationship
between cyclocolorenone and a-gurjunene is accentuated further by the
137
fact that the two occur together in Pseudowintera colorata, and
there has been speculation that cyclocolorenone might have been an
artifact arising from a-gurjunene on long storage through air/photo-
118
oxidation. A number of alcohols derived from either aromadendrane
131 138
(e.g., globulol 2.11 and spathulenol 2.12) or alloaromadendrene
118 118
(e.g., ledol 2.13, viridiflorol 2.14 ) have been described. An
139
unusual isonitrile, axisonitrile II 2.15 and derivatives 2.16 and
2.17^ have been isolated from the sponge, Axinella canabina (Porifera).
2.11, R = CH0, R' = OH
2.13, R = CH0, R'
= OH
2.15,
R = -N=C
2.12, R = R'"= CH2
2.14, R = OH, R1
= ch3
2.16,
2.17,
R = -N=C=S
R = -NHCH0

41
Antibiotic Principle of the Leaves
The extract of yellowed leaves of Magnolia grandiflora was
found to possess antibiotic activity when assayed with Bacillus subtil is
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 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 FI ori sil, 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

42
an oil and triturated with ethanol to give a solid. Crystallization
from ether gave large, colorless crystals, 210 mg, mp 112-114°C, and
MIC 3 ug/ml.
The mass spectrum (M+ 248) and elemental analysis are consistent
with the molecular formula C-i^H^qO^. The ir spectrum (K3r) 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, CH£ 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 aj_. and a comparison
of their nmr spectrum with that of the Magno!ia antibiotic is shown
in Figure 2.5.
2.16

43
Figure 2.5 Comparison of nmr spectra of (a), parthenolide and
(b) Hagnolia antibiotic (DgDMSO)

44
The melting point and ir spectra are in agreement with the
4
published data of Govindachari et aj_., 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
141 142
parthenium (1.) Bernh. * and subsequently from Ambrosia dumosa
143 144
Gray, and Ambrosia confertiflora DC., all Compositae. Indeed, the
Compositae, and particularly Ambrosia and their relatives, elaborate a
144
large variety of sesquiterpene lactones. Such compounds are much
less prevalent in other families.
Within the Magnoliaceae, in addition to M. grandiflora,^
parthenolide has been isolated from Michelia campaca L. by Govindachari
4
et al_., who revised the original structure proposed by Herout et
141 142 145
al. and Soucek et al_, and from Michelia lanuginosa L. The
antibiotic activity of this compound has not been previously described.
The stereochemistry of parthenolide was determined by Bawdekar
146
et al_., who correlated it with the known stereochemistry of
costunol ide.
Parthenolide and costunol ide are the only sesquiterpene lactones
reported from Magno!ia. Interestingly, a number of related costunolide
derivatives are known from the magnoliaceous tree, Liriodendron tulipi-
147 148 147
fera. 5 These, like parthenolide, have antitumor activity.
Experimental
Extraction of Magnolia bark: Cyclocolorenone 2.3
Ground bark (41 g) of Magnolia grandiflora, collected in
Gainesville, Florida, was extracted with ethanol (10 l) at 25° C for two

45
days. Three such extracts were combined, concentrated at 20 mm pressure
to a syrup which was partitioned between water and ethyl acetate (2 i
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
eluate contained besides carotenoid and other highly lipid-soluble
components, the phenylpropanoid component of which is described in Chapter
3. The column was then eluted with 5% acetone in benzene which eluted
cyclocolorenone, recognizable by its tic behavior and the uv-maxima
at 260 nm, as well as phytosterols which moved in tic at siightly 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 5°C
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 FI ori sil (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 tic and gc (T 175°, retention
time on 3% 0V-17, 6 ft.; 5 min.; on 3% 0V-1: 3 min.); uv: X 264 nm;
max
log e 4.12 (lit. 264/4.12)^ mass spectrum: m/e 218 (M+ 100%), 203,
175, 162, 161 , 149, 147, 134, 133, 119, 105, 93 and 91.

46
Analysis of the Phytosterol Fraction
The column consisted of 3% OV-17 on Gaschrom Q, 6 ft., programmed
from 150-275°C at 10° per minute. The mixture showed components with
retention characteristics similar to those of cholesterol (Aldrich),
lanosterol (Mann Research) and 6-sitosterol (Fisher). Under isothermal
conditions at 250°C, the retention times are as follows:
cholesterol
13.5 min.
lanosterol peak 1
19.0 min.
peak 2
20.5 min.
6-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
50°C 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 217.5°C (lit. 218°);^ uv A
max
400 nm, log 4.51 (lit. 404, log 4.40); ^ 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.

47
After five minutes at 50°C, it was cooled, neutralized with IN 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” ^.
The sample in hexane (10 ml) was stirred at 25°C with activated
manganese dioxide (Baker and Adamson) for two hours. Filtration and
concentration of the filtrate gave an oil, identical with 2.3 in tic,
and uv spectra.
Reduction of Cyclocolorenone to Cyclocoloranone 2.5
The procedure was an adaptation of the one described by Corbett
113
and Speden: 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 Ci^H^O^O: c, 75.69; H, 10.92. Found: C,
76.48; H, 10.79; ir: 1745 cm”^; 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 (Mf), 177, 149, 136, 135, 122, 109.

48
Cyclocoloranone 2,4-dinitrophenylhydrazone
This was prepared as described under the cyclocolorenone
dinitrophenylhydrazone. It is a light orange crystalline solid,
mp 207-210° from ethyl acetate; uv: Amax 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 A
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 A^ax 250 nm, with nmr indicating 40% 2.3, yield
150 mg.
120
According to the procedure of Corbett and Young, 2.3 (200 mg)
was heated under reflux in 50 ml of ethanolic K0H (0.5 N) for two hours,
cooled, neutralized (H^SO^), 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 (^SO^) and concentrated to give an
oil, 190 mg, which could not be induced to crystallize. The product
was homogeneous (tic) with 2.3, A 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.

49
Epieye1ocolorenone 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, 188-189°C
1 ?f)
(lit. 189), homogeneous with 2.3-DNP on tic, uv 400 nm, log 4.48/
chloroform (lit. 397.5 nm/4.49).^
Parthenolide 2.16
Yellowed leaves of M. grandiflora (500 g) were cut into small
pieces and extracted with ethanol for one day at 25°C. 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
1 AR
from ether-hexane; yield, 0.5 g; mp 112-4° (lit. 115°C) ; ir: 1730
and 1655 cm \ mass spectrum: m/e 248 (M+), 233, 230, 190 and 43.
Anal. calc, for C-i^gOg: C, 72.55; H, 8.12. Found: 72.33:
H, 8.17.

CHAPTER 3
PHENYLPROPANOIDS OF Magno!ia grandiflora L.
Bis-allylphenol from the Bark
The distribution of diallylbiphenol derivatives in Magno!ia
spp. was discussed in Chapter 1. So far, three members of this group
12 53-54
have been isolated from Magno!ia spp., magnolol 3.1, ’ hono-_. _
i? 54-57 q 53
kiol 3.2 ’ and acuminatin 3.3. * A search for these or related
55
compounds in Magnolia grandiflora was made but with negative results.
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.
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 Amflx of 290 nm. The phenolic nature of the compound
was demonstrated by the shift in A to 320 nm in the presence of
J max K
50

51
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 C-jgH^gO^ on the basis of elemental analysis and mass spectrum
(M+ 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~^ (phenolic OH), 1620 cm”^
(aromatic, 985, 905 cm ^ (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 CHgCH-CHgj
4.86 ppm, s, exchangeable, ArOH/, 3.85 ppm, ArOCIH^, 3.28, 3.40 ppm,
overlapping doublets, J = 6 Hz, 2 ArCH^2CH=CH2--clearly indicated that
there are two non-equivalent ally! groups on aromatic rings comprising
1,2,4 substitution patterns.

52
The ally] groups were readily reducible to afford a tetrahydro
compound. Elemental analysis and the mass spectrum (M+ 284) were
consistent with the molecular formula C-j9^2482* nmr sPectrum showed
two non-equivalent benzylic n-propyl groups: 2.40 and 2.62 ppm, over¬
lapping triplets, J 3 5.2 Hz, 2 ArQ^CHpCH^-, 1.63 and 1.67 ppm, over¬
lapping sextets, J = 8 Hz 2 ArCH2CH2CH3, 0.94 and 0.97 ppm, overlapping
triplets, J 3 8 Hz, 2 ArCH2CH2CH3.
Acetylation of the phenolic component gave a monoacetate
^21H22^3 322). The ir spectrum of the acetate (1755 cm”^) and the
nmr signal (2.02 ppm, s, 3H) showed that the compound was a phenolic
acetate.
Methylation provided a monomethyl ether C2qH2202 (M+ 294) which
showed no hydroxyl 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-O-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-
149 150
able using ferric ion, hydrogen peroxide, hydrogen peroxide and

53
151 152
ferrous sulfate, and enzymic catalysis. However, not only are
the yields in thse 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
12
Ullmann coupling procedure employed by Fujita et aj_. 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 £-propyl phenol 3.11 and
the o-allyl phenol 3.12.
3.1, R1 = R2 = H
r3 = ch2ch=ch2
3.5,R-| = R2 = CH^
r3 = ch2ch2ch3
3.8, R1 = R2 = H
r3 = ch2 ch2ch3
lii, Ri = R2 - H
r3 = CH2CH=CH2
3.6,R-j = R2 = CH3
r3 = ch2ch2ch3
^9, R1 = R2 = H
R3 = CH2CH2CH3
M. Rt = r2 3 H
R3 = CH2CH=CH2
3.7,R-| = R2 = CH3
r3 = CH2CH2CH3
3.10, R-j = r2 = H
r3 » ch2ch2ch3

54
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, 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
153
of Fujita et al_. using copper powder activated by the procedure of
154
Kleidner, and heating without solvent at 260°C. 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
12
as recommended by Fujita et al.
Linder these conditions, dimethyl tetrahydromagnolol 3.5 was
obtained in a 30% yield from 3.16. Coupling of 3.17 provided

55
3,3,-di-n-propyl-4,4'-biohenol dimethyl ether 3.17 in a 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 r
3.18, R = CH2CH=CH2
3.20, R = CH2CH2CH3
3.19, R = CH2CH=CH2
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

56
spectral and complexing 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_J5 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 values, they were designated as
ether-A (higher R^) and ether-B (lower R^). 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

57
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.
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 anisóle, used as model compounds, in a
neutral medium, phenol was degraded rapidly in a basic medium while
anisóle 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 appropriatehydroxybenzoic acids, followed by Clemmensen reduction of

58
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
1 55
ester 3,24. It was methylated to the ether but not reduced to the
2-methoxy-5-n-propylbenzoic acid 3.22. Cox also carried out a Fries
156
rearrangement of the propionate of methyl salicylate. Although no
assignment was made for the product, the properties appear to correspond
to the ortho-migration product, 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
157 158 159
Claisen rearrangement of the corresponding ally! ether 3.27. ’ ’
Methylation gave the ether 3.30, which on hydrolysis gave the acid
157
3.31. 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 £-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’^ but this com¬
pound was not reduced.

59
l
3.27, R = CH2CH = CH2
*' = c2h5
3.32, R = COCH2CH3
R' = H
3.28, R = H, R' = C2H5
3.30, R = CH3, R‘ = C2H5
3.33, R - CH3, R1 * H
3,29, R = R' = H
3.31, R = R' = CH3
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)
3.35
aThe melting point of 5-n-propylsalicylic acid 3.25 obtained by
hydrolysis of this sample is much greater than obtained by Cox^56
(210.5-213 vs. 177-9°C), supporting the contention that Cox has ob¬
tained the wrong isomer.

60
For the synthesis of the ester of 3.23, the ally! ether of
methyl £-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.
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 R^), which was identical
with the tetrahydro derivative of the natural product, gave as
product 3.31 while ether-B (lower R^.) 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 180-82°C
[a]p = +4.0° (c 1.12,CHC13). Its characteristic uv spectrum, Amax

61
270 run, 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
(Mf 418) and elemental analysis agreed with the molecular formula
^22^26^8* "^he 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"^; 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:
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.

62
Confirmation for the furofuran type structure was obtained
through the analysis of the mass spectrum along the lines described by
Pelter for pinoresinol.^ 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 Magno!ia lignan is one of the diastereomeric 1irioresinols and it
remains to determine which of the three stereochemical representatations
shown in Figure 3.2 is the correct one for the compound. (Relative
1 F9
stereochemistries were determined by Briggs, Cambie, and Couch.)
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 aj_., who first isolated two of these
lirioresinols (A and B) by the acid hydrolysis of 1iriodendrin, a
glycoside from Liriodendron tulipifera L. (Magno!iaceae) as well as
1 FA
from commercial beechwood sulfite liquors (Populus spp., Salicaceae).
I ¿TO
Of these, syringaresinol corresponds to dl_-lirioresinol B. Emulsin-
hydrolysis of liriodendrin gave an aglycone which was first assigned the
163
structure of lirioresenil C but was later shown to be a mixture
*1 go
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
1 6?
excel sum (Forst. f.) Miq. (Piperaceae).
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

63
Phenol and Phenol Ether Processes M+-29(CHO) — m/e 389 (1%P)
M+-15(CH3 ) — m/e 403 (2SP) M+-30(CH20) — m/e 388 (4%P)
M+-28(CsO) — m/e 390 (0%P)
aIntensity of peak from Magno!ia 1ignan/intensity of corresponding peak
of pinoresinol
Figure 3.1 Comparison of mass-spectral fragmentation patterns of
Magno!ia lignan and pinoresinol

G4
different enantiomeric compositions.^,164 phyS-¡ca] pr0perties of
the three 1irioresinols 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
Ar
Ar. ^0'
0^ ''
H
Ar
3.41
3.42
3.43
Lirioresinol A
(axial/equatorial)
Lirioresinol B
(diequatorial)
(Lirioresinol C)
(diaxial)
Figure 3.2 Relative stereochemistries of the lirioresinols
The infrared spectra (KBr pellets) of the Magno!ia lignan and
of synthetic syringaresinol (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

65
Table 3.1 Properties of the lirioresinols and their derivatives
Compound
mp, °C
wD°
Reference
Lirioresinol A
210-211
+ 127
163
180-181
—
164
177
-90
165
Lirioresinol A
dimethyl ether
118-120
+ 119
163
diacetate
188
—
164
Lirioresinol B
177-183
-34.8
166
175-179a
—
167
172-179
+62.2
163
170
-21.5
165
dimethyl ether
126.5-127
—
59
122-123
+46.2
163
122-123
+45.8
168
121-122
+46.0
166
116-118a
—
167
115-119a
—
169
diacetate
188
—
163
Syringaresinol
179-185.5
0
170
(dl’-lirioresinol B)
175
+1.93
171
174
0
172
170-171
0
173
168-172
+3.93
164
dimethyl ether
107_9b
-4.14
171
107-8°
0
172
diacetate
188-189
-4.73
171
181-182
—
164
181-182
0
173
Lirioresinol Cc
—
—
—
dimethyl ether
145-147
+284
162
Magnolia lignan
180-182
+4.0
this work
dimethyl ether
107.5-108
—
this work
diacetate
185-186
0
this work
Assignment unclear
^Synthetic
cGenuine--many reports of lirioresinal C are incorrect

W«vtNU«M« Cm'
Figure 3.3 Infrared spectra (KBr) of (a) Magnolia lignan and (b) synthetic syringaresinol

67
may alter the infrared spectra according to the enantiomer con-
tent.'74
The nmr spectra of lirioresinols A and B are not available,
175
although the corresponding isomers in the pinoresinol series are.
The two spectra are presented in Figure 3.4 and the spectrum of the
Magno!ia 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),^ 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. 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 $-ray studies.^ Assignments of these
resonances based upon J „ are in error: it has been stated that in
lignan spectra, overdependence on coupling constants, based upon bond

68
(a)Mmr spectrum of (+)-epipinoresinol (stereochemistry of
lirioresinol A)
IMkl
(b)Nmr spectrum of (+)-pinoresinol (stereochemistry of lirioresinol
B)
(5Ha 4.98 ppm (d, J = 5 Hz)
6HB 3.25 ppm
6Hyeq 3.63 ppm
6Hyax 3.72 ppm
(c)Spectral assignments for lirioresinol C dimethyl ether (R = OCH^)
Figure 3.4 Nmr spectra of the three pinoresinol lirioresinol skeleta

I .... I .... I .... I .... I .... I ... I .... I .... I .... I .... I
To To sTo PPM (Í) 4.0 • 3.0 2.0
Figure 3.5 Nmr spectrum of Magnolia lignan (CDCl^)

70
angles is undependable.^,179 ^ore emphas-¡s -¡s piaced 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.
59
The dimethyl ether has been isolated from Magnolia Kobus 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
172 181
sinapyl alcohol. * Subsequently, it was synthesized by nonenzymic
182
oxidative coupling of sinapyl alcohol and sinapic acid. In the
latter synthesis, the dilactone initially produced was reduced and
pyrolyzed to yield syringaresinol.
Syringaresinol glycosides occur in a number of plants, including
Magnol ia grandiflora and Liriodendron tulipifera L.^ (Magnol iaceae).
Syringaresinol itself, as well as the optically active form lirioresinol
B, has been obtained from Liriodendron tulipifera^»169 (^agnoljaceae)
164 173 182
Populus spp., Fagus spp. ’ (Betulaceae), Picea excelsa
183 171
Engelm. (Pinaceae), Sinomenium acutum Rehd. et Wils. (Menisperma-
ceae), and Xanthoxylum inerme Koidz.^0 (Rutaceae). The dimethyl ether
has been obtained from Liriodendron tulipifera |_J67,169 (fj|agn0-| jaceae),
168
Eremophila glabra (Myoporaceae), Macropiper excel sum (Forst. f.)
MiqJ^ (Piperaceae), and Aspidosperma Marcgravianum Woodson^
(Apocynaceae), in addition to the Magnolias previously cited.

71
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 25°C for two days.
Three such extracts were combined and concentrated to a syrup which
was partitioned between water and ethyl acetate (1 £ 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 (tic: 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 benz.ene.:iiexane (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: A v 255 nm (log e 4.1) and
290 nm (log e 3.8); with base, A „ 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, OCH^;
3.40 and 3.28, overlapping doublets, J = 6 Hz, 2 ArCH2CH=CH2; ms:
280.1456; calc, for C10H2Q02: 280.1464.

72
Mehonokiol Acetate
A mixture of 3.19 (0.05 g), acetic anhydride (1 ml) and pyridine
(two drops) was heated at 70°C 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”^; nmr: 2.01 ppm, s, 3H;
ms: Mf 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 ^20H22°2: 294.1621.
Tetrahydromehonokiol 3.21
A solution of i> (0.1 g) in ethanol (5 ml) was hydrogenated in the
presence of Pt02 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"^; nmr
(ppm); 240 and 2.62 ppm, overlapping triplets, J = 5.2 Hz, 2 ArCHpCHoCH.,
1.63 and 1.67 ppm, overlapping sextets, J = 8 Hz, 2 ArCHpCHgCH^, 0.94
and 0.97 ppm, overlapping triplets, J = 8 Hz, 2 ArCH2CH2CH3; ms:Mf 284,
m/e 255, 223, 205.

73
Anal. Calc, for C-jg^C^; 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 anisóle (10 g) was
-153
iodinated by the method of Hata and Sato using mercuric oxide-
catalyzed iodination by in ethanol. The resulting product was
purified by distillation (1.5 mm, 105-115°C) 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, 100°C); yield 8 g (88%).
Tetrahydromagnolol dimethyl ether 3.5
The coupling of 3.16 (10 g) was carried out by a modification of
55
the procedure of Fujita et al_., in which the copper powder (22 g)
was gradually added to 3.16 at 240°C. After completion of the
addition, the mixture was maintained at this temperature for four to
six hours instead of heating to 285°C. 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),

74
256 nm (log e 4.0); nmr (ppm): 6.70-7.20, m, 6 ArH; 3.68, s, 2 OCH^;
2.50, 1.60 and 0.95, triplet/sextet/triplet, respectively, 2 CHgC^CHg.
Demethylation of 3.5 (1 g) with acetic anhydride (10 ml) and
hydriodic acid (8 ml) gave tetrahydromagnolol, yield 0.5 g (64%); mp
142-142.5°C (lit. 143°C).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 113-14°C
(lit. 114°C).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 113-114°C (lit. 112.5°C).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 25°C
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 79-80°C;
nmr: (ppm) 3.4-2.6, m, 6-ArH; 4.88, s, Ar-OH; 6.23, s, OCH^; ms: Mf 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% 0V-17, T 180°C,
3.5, tr 11 min., 25%; 3.6, tr 15.8 min., 55%; and 3.7, t 20.8 min.,
19%) was subjected to chromatography on FI ori sil for preliminary
purification. Direct crystallization gave 3.7 (0.9 g), the mother
liquors were purified by chromatography (silica gel, cyclohexane with

75
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: 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 117-117.5°C (lit.
118°C).53
Mono-O-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 25°C 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, 200°C; 3.21 t 5.5 min.; 3.22 t 8.3 min., tic: 0.90,
0.60, respectively, silica benzene). The monoether of higher R^ was
identical with the natural tetrahydromehonokiol by tic, gc and spec¬
tral comparison. The mixture was separated by chromatography (silica
gel, cyclohexane with a benzene gradient). The high R^ ether 3.21
was obtained as a colorless oil: ms: 284.1790 (calc, for C-jgi^O^:
284.1775); m/e 269 (100%), 256 (17%) and 255 (80%).
Anal. Calc, for 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 H2®: 77.85; H, 8.59.
Found: C, 77.69: H, 8.65.

76
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 -30°C 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 35-37°C; underpressed when mixed with an authentic sample of methyl
3-n-propyl-p-anisate 3.31. The spectral (ir, nmr) and chromatographic
were identical (tic: 0.60, silica, benzene; gc: 3% SE-30, 130°C,
t 7 min.).

77
Similar degradation of 3.21 gave, after esterification, a product
identical with methyl-3-n-propyl £-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 (tic: 0.25, silica, benzene; gc: 3% SE-30, 130°C, t^,
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 0°C. After 16 hours at 5°C it was diluted
with benzene and washed successively with IN 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 61-62°C; 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”^; 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; ArCh^CH^CH^.
Saponification (IN K0H, reflux 5 min.) gave, on workup, the
acid 3.25, mp 210-211.5°C from water (lit. 177-9°C) 0 (cf. p. 59).

78
Methyl-3-n-propyl-£-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"^; 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 220°C 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~^; 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 25°C. The
mixture was filtered and the product recovered by concentration of the
filtrate and crystallization; mp 122.5-125°C. 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-£-anisate 3.31 as a
colorless crystalline solid; mp 36-37°C. It was identical with the
methylated ozonolytic degradation product of tetrahydromehonokiol, 3.21
Saponification (IN K0H, refluxed 5 min.) gave on workup the acid,
3.29, mp 115-17°C from water (lit. 116.8°C).^

79
(di )-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 (Na^SO^),
concentrated, the phenolic lignan extracted and isolated by chromatogra¬
phy (silica gel, 20 g, benzene with ethyl acetate solvent gradient).
The lignan, (tic: 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 180-182°C; [a]p
+4.0° (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) , ^ -¡rj nmr: mp cf. Figures 3.3, 3.5,
and Table 3.1.
Anal. Calc, for ^2Z^25^8: 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-
108°C; ms: cf. Figure 3.1; uv: 270 nm, log e 3.40; 240 nm, log e 4.21.
Anal. Calc, for C24^30^8: C, 64.56; H, 6.77. Found: C, 64.41;
H, 6.67.

80
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
185-186°C, [a]D 0.0° (c 0.50, CHC13); nmr: 2.32 ppm, 6H, s, CH3C02Ar;
ir: 1764 cm”^.
Anal. Calc, for C26^30^10: 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 Magno1ia 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 Magno!ia 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 Hgl^" 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 Hgl^" ion by Cl" 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
81

82
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_.g_., 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 Magno1ia
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

83
Wood Extract
Figure 4.1 Fractionation scheme for Magnolia wood alkaloids

84
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
15
magnoflorine, which is the major alkaloid of Magnolia grandiflora bark.
The quaternary alkaloid was next converted to the iodide salt
by ion-exchange using Dowex-1 iodide and was obtained as a crystalline
solid, mp 222-25° (dec.); [a]g+212° (c 1.27, ethanol) representing a

Optical density at 270 nm
85
Figure 4.2 Cellulose partition chromatography of BuOH fraction

86
yield of 0.02%. It was also toxic to mice with an LD^q value of
10 mg/kg.
Elemental analysis of the toxic alkaloid agreed with the molecu¬
lar formula C20H 2gN0^I. Its uv spectrum showed Amax 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”\
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 CH^X, or a Hofmann elimination of HX. The actual course
184
depends on the alkaloid and the nature of X: with Magno!ia 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 (Mf -CH2=NCH3), m/e 326 (M+ -CH^and m/e 310
(M+ -0CH3).
The data suggested that the Magno!ia 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(0CH3); 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
to an iminium ion such as 4.3 derived by the ot-fission of a
185 186 187
benzyltetrahydroisoquinolinesystem and the uv spectrum ’ estab¬
lished that the alkaloid has an aporphine skeleton 4.1.
The presence of a phenolic hydroxyl
tion of a monomethyl ether, nmr: 3.71 ppm,
ppm, 3H. Acetylation with acetic anhydride
ceed, possibly due to steric hindrance, but
gave an acetate, ir: 1770 cm"^ and nmr: 2
was verified by the forma-
6H; 3.90 ppm, 3H and 3.92
and pyridine did not pro¬
acid-catalyzed acetylation
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
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 Magno1ia 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
186 187
nm. 5 This difference has been said to reflect the greater inter¬
ference with coplanarity of the biphenyl system in a 1,2,10,11-
187
oxygenation pattern. The mass spectra of several 1,2,9,10-oxygenated
185 186 188
aporphines have been published ’ 5 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 (Mf -1), (b) subsequent formation
of systems such as 4.8 or 4.9 with the loss of either «OCH^ or ‘CH^,
respectively, and, (c) retro Diels-Alder cleavage to 4.10 which can
185 188
undergo further loss of ‘OCH^ or «CH^. ’ In contrast, the mass
spectun of the Magnolia alkaloid showed no M+ -1 peak, low intensity

89
Figure 4.3 Mass spectral behavior of a typical aporphine

90
M+ -43 peak (retro Diels-Alder process) but a relatively high intensity
M+, M+ -OCH^ peaks. Such behavior is peculiar to those aporphines with
189
1,2,10,11-oxygenation pattern.
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
Magno!ia alkaloid by comparison of its tic and spectral behavior.
magnoflorine 4.10 3 = H
dimethyl magnoflorine 4.11 R^ = R^ 3 CH,
For the Magno1ia 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

91
N-methyl-10-0-methylhernovine methiodide
N-methyl corydinium iodide
N-methyl isocorydinium iodide
4.12 R1 »Hr R2 = R3 = R1 = CH3
4.13 R2 = H1, R] = R3 = R4 = CH3
4.14 R3 = H1, R] = R2 = R4 - CH3
4.15 R4 = R4 = H1, R1 =R2 = R4 = CH3
Figure 4.4 Isomeric hydroxy-trimethoxyaporphinium methiodides
them by Hofmann elimination, with free hydroxyl at C-l or C-11, are
known to show hydroxyl frequencies below 3300 cm"\^ 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-l and C-ll do so at 3.4-3.7 ppm. ’ 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
189
of 4.13 and 4.14 were somewhat different, the spectrum of the Magno!ia
alkaloid was not sufficiently similar to either of the literature spectra
to allow an assignment. Neither were the reported nmr spectra of use:

92
H-8 and H-9 of 4.13 have been reported as singlets at 7.27 and 7.33 ppm,
193
with H-3 at 7.05 ppm while 4,14 showed H-8and H-9as a singlet at
194
6.96 ppm, and H-3 at 7.02 ppm. As the chemical shift differences for
H-8 and H-9 are small, the outer peaks of the AB-quartet are small and,
therefore, often overlooked. Furthermore, variations from these values
195 196
are also found in the literature. ’ The spectra also vary
depending on the solvent used (DgDMSO, CD30D, CF^COOH or DgO). The
observation of an AB-quartet for H-8 and 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 Magno!ia 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 Magno!ia
toxic alkaloid and its ether
H3, ppm
H8
and
H9
alkaloid
6.96
6.98d
or
7.lid
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 an unsubtituted posi¬
tion para to the phenol ate function which is very reactive toward
electrophilic reagents, whereas N-methylcorydine 4.13 has none. This
195
difference has been exploited in a number of ways: deuteration,
197
and the Gibbs test (reaction with 2,6-dibromoquinone chlorimide)

93
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 M NaHCOg.
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 Amax 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 aj_., who also determined
its structure. An alkaloid originally called "Chakranine" isolated
from Bragantia wallichii R. Br. (Sanskrit: "Chakrani," Aristolochia-
93
ceae) has been found to be identical to N-methyl isocorydinium
190
iodide. The alkaloid is of widespread occurrence, having been
isolated from the Annonaceae, Lauraceae, Menispermaceae, Papaveraceae,
191-92
and Berberidaceae. 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

94
moiety, followed by Pshorr synthesis of the aporphine (closure via the
diazonium ion)J^’^
Non-toxic Alkaloidal Fraction
The second alkaloid isolated mainly from the chloroform
extract and described earlier, was obtained as the hydrochloride, a
colorless, crystalline solid, mp 265-275°C (dec), [a]D~73° (c 1.1,EtOH).
The free base could not be induced to crystallize.
Elemental analysis of the hydrochloride and the mass spectrum
(M+ 265) of the base were consistent with the molecular formula
C.|^H.|gN02 for the base. The uv spectrum Amax 235 nm, log e 4.21;
271 nm, log e 4.21; 315 nm, log e 3.75, unchanged in base is charac-
186 187
teristic of the aporphine nucleus. ’ The nmr spectrum of the
free base indicated the presence of a methylenedioxy group (6.00 ppm,
1H, d and 5.87 ppm, 1H d), five aromatic protons: 8.00 ppm, 1H, m,
7.02-7.13, 3H, m, 6.50, 1H, s and other signals characteristic of an
194
aporphine system: 3.7-4.1 ppm, 1H, m (H-6a) and 2.4-3.5 ppm, 6H, m.
The presence of a one proton singlet at 2.87 ppm which was shifted by
acid, and the lack of a N-methyl function indicated that the nitrogen
is secondary. This was verified by the formation of an acetamide,
mp 229-231°C.
The mass spectrum showed the expected intense molecular ion
(m/e 265, 63%) and even stronger M+ -1 (m/e 264, 100%) characteristic
185 "188
of aporphines. ’ Lacking any other functionality except the
methylenedioxy group it could not undergo the methoxyl group processes
outlined earlier. The only other meaningful ions in the spectra might

95
be attributed to loss of *HC=0 and (m/e 236, 24% and m/e 235,
14%); and M-31, which might be expected to arise from loss of methylene*
imine (retro Diels-Alder) as shown in Figure 4.4.
The nonequivalency of the methylenedioxy signals, which origi¬
nates from anisotropy due to the twisted biphenyl system, is observed
in aporphines bearing the group at the 1,2- or 9,10-positions,^ i_._e.,
in a position where the methylenes are subject to differential deshield¬
ing by the adjacent ring of the biphenyl system.
The only structure which was considered likely for this alkaloid
on a biogenetic basis is anonaine 4.16, with a 1,2-methylenedioxy
group. A 2,3-methylenedioxy group can be firmly excluded on the basis
of the nmr spectrum. Such a group would show a single resonance for the
reasons discussed above. The nmr signal at 6.50 ppm was assignable to
H-3 and the multiplet at 8.00, deshielded by the adjacent aromatic
ring, was assigned to H-ll.
From this information it was concluded that the alkaloid was
identical to the norapoephine,(-)-anonaine 4.16. This identification was
confirmed by the melting point of the acetamide, 229-30°C (lit. 229-
4,16

96
Anonaine was first isolated by Santos from Annona reticulata
202 201
L., but was not identified until 1939 by Barger and Weitnauer.
(The difference in spelling is due to the use of the native Philippine
name by Santos.) It occurs in Magnolia obovata^ and M. grandiflora^
(cf. Table 1.3). Its acetamide occurs in Magnolia obovata^ and
203
Liriodendron tulipifera L. (Magno!iaceae), a plant which produces a
203 204
number of acetylated noraporphines. ’ It occurs in most species
of Annona, and is of very frequent occurrence within the Annonaceae in
191 192
general. ’ Outside this family, it has been reported only from
88 205
Doryphora sassafrass Endlicher (Monimiaceae), Roemeria refracta DC.
(Papaveraceae), Colubrina faralolatra H. Perrier (Rhamnaceae), and
207 208
Nelumbo nucifera Gaertn. ’ (Nymphaceae). In the latter, it is
208
accompanied by 4,5-dehydroanonaine.
Anonaine was accompanied in this fraction by two minor
alkaloids. The nmr spectra suggested that they were the epimeric
7-hydroxyanonaines, but insufficient quantities were available for
complete characterization.
The fluorescent, yellow alkaloid occurring in the same fraction
was separated from anonaine by extraction and alumina chromatography.
On crystallization, 100 mg/5 kg of wood of ochre-colored crystals,
mp 280-282°C (dec.) were obtained. The mass spectrum indicated a
molecular weight of 275, corresponding to C-jyHgNO^- The ir spectrum
showed a carbonyl group at 1660 cm"1 indicating that the compound was
a 7-oxoaporphine. The nmr spectrum showed a methylenedioxy group
6.72, 2H, s, and aromatic protons at 7.63 ppm, 1H, s, and 7.7-8.9, 6H,

97
m. The uv spectrum showed A 246 nm, 265 nm, 280 (sh) nm, and 300
nm. On the basis of the methylenedioxy signal, the unusual chromophore,
and the ketonic function, the ir frequency of which is the region
associated with a diaryl ketone or amide, the compound was readily
identified as liriodenine, 4.17.
Liriodenine occurs in a number of Magnolia spp. (cf. Table 1.3),
and, like anonaine, is of widespread occurrence throughout the
191 192
Annonaceae. ’ Generally, liriodenine is present in plants con¬
taining anonaine. Also known as oxoushinsunine, it often occurs along
with ushinsunine 4.18, which may be regarded as an oxidation state
intermediate between the oxoaporphines and the aporphines. A second
oxoaporphine, oxoglaucine, also occurs in Magnolia (cf. Table 1.3).
Experimental
Isolation of Alkaloid Fractions from Magnolia Wood
Shavings from Magnolia trunk wood (4.9 kg) were extracted with
ethanol (12 ¿) at 20°C for two days. A combination of three such
extracts was concentrated to a syrupy consistency (23 g), at which
stage it showed an LD^g value 700 mg/kg (for details of the biological

98
assay: cf: Table 1.5). The concentrate was partitioned between ethyl
acetate and HCL (0.01N, 800 ml each). The aqueous layer, which
contained 28.8 g of total solids with an LDj-g of 350 mg/kg, was washed
once more with ethyl acetate, filtered through cotton to remove any
tarry residue. It was concentrated to approximately 250 ml and treated
with Mayer's reagent until precipitation was complete. The precipitate
was filtered on a Celite pad and the cake washed with HC1 (0.01N). The
filter cake was stirred with acetone-water (1:1) and filtered. The
filtrate and wash were passed through a column of Dowex-1 (20 g, chlor¬
ide). The column was washed with 1:1 acetone-water until the washings
gave a negative test with Mayer's reagent (after removal of the acetone
from the aliquot). The effluent and wash, containing an alkaloidal
material (7.65 g), LDj-g 70 mg/kg, was concentrated to remove the acetone,
adjusted to pH 8 with ammonia and extracted twice with chloroform.
The chloroform extract was concentrated to 5-10 ml, diluted with
an equal volume of chloroform and treated with methanolic HC1 (IN). The
alkaloid hydrochloride which precipitated out was filtered and washed
with ether; yield 1 g (tic: R^ 0.6, silica gel, 10% methanol¡chloroform);
and gave a positive reaction with acidic iodoplatinate reagent. The
mother liquors from the hydrochloride were set aside for the separation
of the trace components.
The aqueous layer, after the chloroform extraction, was read¬
justed to pH 7 and extracted twice with n-butanol. The butanol layer
yielded 3.3 g of a solid, LD^g, 80 mg/kg. Thin-layer chromatography
showed that it contained a major alkaloidal component (tic: 0.8, silica

99
gel, chloroform:methonol:water:acetic acid, 14:6:1:1), along with a
minor amount of the alkaloid seen in the chloroform extract. The aqueous
layer from n-butanol extraction was concentrated to near dryness to
yield a solid, 3.4 g, LD^g 40 mg/kg and containing the same alkaloid
as the major component of n-butanol extract.
N-methyl isocorydinium iodide 4.14
The butanol-extractable fraction (3.3 g) was chromatographed on
Sephadex LH-20 (200 g). Elution with 10% ethanol:ethyl acetate gave a
fraction containing anonaine 4.16, corresponding to 50 mg of the hy¬
drochloride. Elution with 20% ethanol:ethyl acetate gave the toxic
fraction, 530 mg, LD^g 17 mg/kg. The alkaloid was converted to the
iodide by passage through a Dowex-1 (iodide) column, and evaporated to
dryness. The product was triturated with ether, and crystallized from
methanol, giving 4.14, a colorless, crystalline solid, mp 222-25°C, dec.
(lit. 225-30°C);194 LD5q 10 mg/kg; [a]Q +212° (c 1.27, EtOH); uv: 220 nm,
log e 4.76; 270 nm, log e 4.21; 302 nm, log e 3.82, shifting to 250 and
354 nm in base (lit. 222/4.09, 271/4.09, 304/3.65),194 ms (m/e):
341 (Mf 100%), 376 (M+ -CHg, 25%), 310 (M+ -OCHg, 20%), 298 (Mf
-CH2=NCH3/21%), 283 (m/e 298-CH3/43%), 270 (m/e 298-0^07/96%); 142
(CH3I, 74%), 127 (I+, 52%), 56 (20%); nmr (ppm): 8.67, m, ArOH; 7.00,
1H, aromatic; 6.98 and 7.11, AB-quartet, J = 8 Hz, aromatic; 3.72,
3.85, and 3.93, 3H each, s, (0CH3); 3.44 and 2.98 ppm, 3H each, s,
N(CH3)3+; ir (KBr): 3200 cm”^ (ArOH), 1600 cm-"', aromatic.
Chromatography of the non-extractable fraction, eluted by 20%
ethanol: ethyl acetate, afforded 819 mg of 4.14 on workup as before.

100
Chromatography by partition on a cellulose column, gave much
better elution patterns and was easier to carry out: Cellulose (Solka
Floe, 200 g) was taken up in ethyl acetate: n-butanol (500 ml, 3:1)
equilibrated with water (100 ml), and the mixture stirred until the
aqueous phase was no longer apparent (30 min.). The slurry was packed
in a column and charged with 10 ml of slurry containing the non-
extractable alkaloid fraction (2.3 g), and eluted with the ethyl
acetate:butanol system, affording 660 mg of 4.14 iodide on workup.
N-methyl isocorydine methyl ether
A sample of 4.14 (100 mg) was stirred with dimethyl sulfate
(0.8 ml) and anhydrous potassium carbonate (0.57 g) in acetone (50 ml)
for 48 hours. Tic showed a single band, 0.50,a silica, chloroform:
methanol: water, 6:4:1; uv: no shift in base. The product was filtered,
the cake washed with ether, the product leached out with methanol, and
converted to the iodide as before. Crystallization from methanol gave
the methyl ether iodide 4.14, 100 mg, mp 255°C, dec. (lit. 258°C,
197 -1
dec.); uv: similar to 4.14, but not shifted in base; ir: 3200 cm no
longer present; nmr (ppm): 3.71, 6H, 3.90, 3H, 3.92, 3H; ms: Mf 369).
Magnoflorine dimethyl ether 4.11
A sample of 4.10 (100 mg), dimethyl sulfate (1.6 ml) and anhydrous
potassium carbonate (1.2 g) was methylated and worked up as described
for 4.14 methyl ether, given 4.11, 100 mg, identical to 4.14 methyl ether
by tic, ir, and nmr spectral comparison.
Starting material, 0 in this system.

101
0-Acetyl-N-methyl isocorydine
A sample of 4.14 (100 mg) in acetic anhydride (5 m) was stirred
overnight with pyridine (0.1 ml). Tic showed no reaction, so more
pyridine (5 ml) was added. After 48 hours, no change was noted (tic).
Heating at 50°C for two hours also failed to cause acetylation.
In acetic anhydride (5 ml) containing one drop of phosphoric acid,
4.14 (100 mg) was successfully acetylated by heating two hours at 100°C
(tic: Rf 0.8, silica, chloroformrmethanolrwater 14:16:1). The product
was cooled, dilutes with methanol and passed through a Dowex-1 (acetate)
column to remove phosphoric acid. The product was concentrated to dryness
and chromatographed (Sephadex LH-20 g, eluted by 10% ethanol: ethyl
acetate) then converted to the iodide as before. Crystallization from
methanol gave 0-acetyl 4.14 iodide, 70 mg, mp 265°C, dec. (lit. 260-265°C,
dec.):^ ir: 1770 cirf^ (CH^^Ar) n0 3200 cm~^ band; nmr: 2.10 ppm.
Anonaine Hydrochloride 4.14
The chlorofom extractable alkaloids and the chromatographic frac¬
tion from the toxic, butanol fraction, were taken up in chloroform (5 ml)
and 4.16 hydrochloride precipitated by careful addition of methanolic
HC1 (IN). The mother liquors were chromatographed on alumina (20 g)
with 5% methanol:chloroform, to afford a total of 1 g of 4.16 on conver¬
sion to the salt. Several recrystallizations from methanol gave mp 265-
275°C, dec. (lit. 277.5°C, dec.);202 [a]D-73° (c l.l.EtOH) (1 it.-69°)208
uv: 235 nm, log 4.21; 271 nm, log 4.21; 315 nm, log 3.75 (lit.
234 nm/4.24, 269 nm/4.24, 312 nm/3.49);202 nmr (ppm, free base in CDCl^):
6.00 and 5.87 ppm, two 1H d, J = 1.5 Hz, methylenedioxy, five aromatic

102
protons: 8.00, 1H, m; 7.02-7.13, 3H, m; 6.50, 1H, s; in addition to
3.7-4.1, 1H, m (H-6a); 2.4-3.5 ppm, 6H, m; and 2.87 ppm, 1H, s, NH; ms
(m/e): 265 (Mf 63%), 264 (M+ -1, 100%), 236 (M+ -29), 234 (M+ -31).
Anal. Calc, for C]6N02C1-h H20: C, 65.70; H, 5.51; N, 4.51;
Cl, 11.41. Found: C, 66.61; H, 5.23; N, 4.57; Cl, 11.40.
Anonaine acetamide
In acetic anhydride:acetic acid (5 ml, 1:1) 4.16 (100 mg) was
stirred overnight, then heated at 70°C for 30 min. The reaction mixture
was poured over ice and the product filtered to give 4.16 acetamide
(100 mg). Additional material was obtained by adjusting the filtrate
to pH 9, and extracting with chloroform. The chloroform extracts were
washed with 10% sodium bicarbonate, and dried (Na2S04). Concentration
gave 10 mg 4.14, tic: 4.16, 0.60; amide 0.80, silica, 10% methanol:
chloroform, the amide giving no reaction with acidic iodoplatinate
reagent. Crystallization from methanol gave 100 mg, mp 229-31°C (lit.
229-30°C);203 ir: 1645 cm"1 (lit. 164);203 nmr (CDC13): 2.21, 3H, s
(ch3con).
Liriodenine 4.17
Weakly basic 4.17 was isolated during chromatography of 4.16 (tic:
4.16, R^0.60, 4,17, R^ 0.95, si 1 ica, 10%methanol (chloroform) and from
liquors of crystallization of 4,16. After extracting 4.16 and trace alka¬
loids with 9.01 N HC1, 4.17 was extracted into IN HC1, neutralized, and
reextracted into chloroform. On concentration 4.17 was obtained as an
ochre powder, recrystallization from chloroform giving mp 280-2°C, dec.

103
(lit. 285-6°C);^' ir: 1655 cm ^ (lit. 1660 cm nmr (ppm): 6.72,
2H, s, methylenedioxy, aromatic protons: 7.63, 1H, s, and 7.7-8.9, 6H, nr,
uv: 246 nm, 265 nm, 280 nm (sh), and 300 nm (lit. 245 nm; 265 nm, 280
General Experimental
Melting points were obtained with a Fisher-Johns (hot-stage)
apparatus and are uncorrected. Infrared (ir) spectra were recorded with
a Beckman Acculab 3 spectrophotometer as KBr pellets unless otherwise
indicated. Ultraviolet (uv) spectra were obtained with a Beckman DB
UV/visible spectrophotometer using a Sargent Model SRL recorder. Nuclear
magnetic resonance (nmr) spectra were obtained with a Varian A60-A or
T60-A spectrometers, with a tetramethyl silane (TMS) internal standard.
High-resolution nmr spectra were determined by Dr. Wallace Brey of the
Department of Chemistry of the University of Florida, employing a Varian
HA-100 spectrometer. Alkaloid spectra are in DgDMSO unless otherwise
specified. Mass spectra (ms) were obtained using an Hitachi-Perkin-
Elmer RMU-6E, single-focusing, electron impact spectrometer, with a
Perkin-Elmer MS-30 double-focusing spectrometer being used for high-
resolution work. Thin-layer chromatography (tic) plates were hand-cast
on microscope slides with E. Merck silica gel PF 254+366 without binder.
Gas chromatography (gc) was carried out using a Varian series 2100 gas
chromatograph with flame-ionization detector. Silanized glass columns,
six feet in length and 3/16 inch i.d. were routinely employed. Integra¬
tion of peak areas was carried out by triangulation, and quantitative
figures are uncorrected for flame-sensitivity factors. Standard flow

104
rates were: hydrogen, 40 ml/min.; air, 300 ml/min.; and helium, 60 ml/
min. Molecular rotations were obtained with a Perkin-Elmer model 141
polarimeter.
Antibiotic assays were carried out using the filter-paper disk
method on agar seeded with Bacillus subtil is spore. The agar medium
consisted of 1% (w/v) dextrose, 1.8% agar (Difco Bacteriological), 0.3%
peptone (Difco, Technical), and 0.3% beef extract (Difco, Technical).
The agar was autoclaved and cooled in a bath to cji. 55°C before inocula¬
tion with 0.5 ml/200 ml of medium of B_. subtil is spore suspension pre-
210
pared according to the method of Grove and Randall. The agar was
poured in 10 ml aliquots into Petri dishes and stored under refrigeration.
Filter-paper disks (Schleicher and Schuell No. 740-E) were impregnated
with test solutions and plated. The petri dishes were incubated 24 hrs.
at 37°C and the clear zone of inhibition read. Minimal inhibitory
concentrations (MIC) were determined by serial dilution, plotting log
concentration vs. diameter of the zone of inhibition, and extrapolating
to the origin.

LIST OF REFERENCES
1. J. E. Dandy, "A Survey of the Genus Maqnolia Together with
Manglietia and Michel i a," in "Camellias and Magnolias, Rep. Conf.
Royal Hort. Soc.," 1950. Spottiswode, Ballantyne and Co., Ltd.,
London, Eng., p. 65.
2. J. E. Dandy, ibid., pp. 64-81.
3. D. H. G. Crout and T. A. Geissman, "Organic Chemistry of Secondary
Plant Metabolism," Freeman, Cooper and Co., San Francisco, Cal.,
1969, pp. 315-7.
4. T. R. Govindachari, B. S. Joshi, and V. N. Kamat, Tetrahedron, 21,
1509 (1965).
5. S. K. Talapatra and B. Talapatra, Phytochemistry, 1217, 1827 (1973)
6. R. M. Wiedhopf, M. Young, E. Bianchi, and J. R. Cole, J_. Pharm. Sci
62, 345 (1973).
7. M. Fujita, H. Itokawa, and Y. Sashida, J_. Pharm. Soc. Japan, 93,
415 (1973); Chem. Abstr., 79, 23501j (1973TI
8. Y. Fujita, J. Japan Botan., 30, 188 (1955); Chem. Abstr., 52, 7620g
(1958).
9. T. Nagasawa, M. Shiga, and K. Umemoto, Koryo, 98, 35 (1971); Chem.
Abstr., 75, 67378w (1971).
10. M. Nagasawa, T. Murakami, K. Ikeda, and Y. Hisada, Pharm. Soc.
Japan, 89, 454 (1969); Chem. Abstr., 7T_, 42156b (1969).
11. M. Flom, Doctoral Dissertation, Ohio State University, Columbus,
Ohio, 1971; Piss. Abstr. Inti. B, 32_, 2312 (1971 ).
12.M. Fujita, H. Itokawa, and Y. Sashida, J_. Pharm. Soc. Japan, 93,
422 (1973); Chem. Abstr., 79, 35031u (1973^
13. B. Talapatra, P. Mukhopadhyay, and L. Dutta, Phytochemistry, 14,
589 (1975).
14. M. Tomita and T. Nakano, Planta Med., 33 (1957).
15.K. V. Rao, Planta Med., 27, 31 (1975).
105

106
16. T. Nakano, Pharm. Bull., 2, 321 (1954); Chem. Abstr., 50, 6475e
(1956).
17. G. J. Kapaida, H. H. Baldwin, and N. J. Shah, J. Pharm. Pharmacol.,
16, 285 (1964).
18. H. Yang, S. Lu, and C. Hsiao, J. Pharm. Soc. Japan, 82, 816 (1962);
Chem. Abstr., 58, 7991h (1963).
19.M. Tomita and T. Nakano, J. Pharm. Soc. Japan, 72, 1260 (1952);
Chem. Abstr., 47, 12288h Tl 953)7“
20.M. Tomita, Y. Watanabe, and H. Furukawa, J. Pharm. Soc. Japan, 81,
144 (1961); Chem. Abstr., 55, 13772Í (1961).
21. T. Nakano, Pharm. Bull., 2 ,-326 (1954); Chem. Abstr., 50, 6475Í (1956).
22. M. Tomita and T. Nakano, J. Pharm. Soc. Japan, 72, 727 (1952);
Chem. Abstr., 48, 2639b (19547!
23. T. Nakano and M. Uchiyama, Pharm. Bull., 4, 409 (1956); Chem. Abstr.,
57 10548f (1957).
24. T. Nakano, Pharm. Bull., 7 29 (1953); Chem. Abstr., 48, 955i (1954).
25.M. Tomita and T. Nakano, J_. Pharm. Soc. Japan, 72, 197 (1952);
Chem. Abstr., 47, 1627i (19577
26.M. Tomita and T. Nakano, J. Pharm. Soc. Japan, 72, 766 (1952);
Chem. Abstr., 47, 12409f 119577“
27.T. Yang and S. Lu, J_. Chin. Chem. Soc. (Taipei), US, 91 (1971);
Chem. Abstr., 75, 148521q (1971).
28. T. Yang and S. Lu, J. Pharm. Soc. Japan, 83, 22 (1963); Chem. Abstr.,
59, 3974d (1963).
29. M. Tomita, S. Lu, S. Wang, C. Lee, and H. Shih, J. Pharm. Soc.
Japan, 88, 1143 (1968); Chem. Abstr., 70, 44850b (196977
30. K. Ito and S. Asai, J. Pharm. Soc. Japan, 94, 729 (1974); Chem.
Abstr., 87 166344n Tl 974)7“
37 J. E. Dandy, "A Survey of the Genus Magnolia Together with
Manglietia and Michelia," in "Camellias and Magnolias, Rep. Conf.
Royal Hort. Soc.," 1950. Spottiswode, Ballantyne and Co., Ltd.,
London, Eng., p. 77.
"Hortus Third, A Concise Dictionary of Plants Cultivated in the
United States and Canada," compiled by: L. H. Bailey and E. Z.
Bailey, revised and expanded by the Staff of the Liberty Hyde
Bailey Hortorium, Cornell Univ., Macmillan Publishing Co., Inc.,
New York, N. Y., 1976, p. 737
32.

107
33.K. Ito and I. Uchida, J. Pharm. Soc. Japan, 79, 1108 (1959);
Chem. Abstr., 53, 2274b (T959T.
34. M. Tomita, Y. Inubushi, and M. Yamaqata, J. Pharm. Soc. Japan, 71,
1069 (1951); Chem. Abstr., 46, 5059h (1952).
35. K. Ito and A. Yoshida, J. Pharm. Soc. Japan, 86, 124 (1966);
Chem. Abstr., 64, 16285f (19661.
36. T. Nakano, Pharm. Bull., 3, 234 (1955); Chem. Abstr., 50, 10748d
(1956).
37. T. Nakano and M. Uchiyama, Pharm. Bull., 4, 408 (1956); Chem. Abstr.,
51_, 19548d (1957).
38. N. F. Proskurnina and A. P. Orechov, J. Gen. Chem. U.S.S.R. (Eng.
Translation), 9, 126 (1936); Chem. Abstr., 33, 1439 (6) (1939).
39. K. Ito and T. Aoki, J. Pharm. Soc. Japan, 79, 325 (1959); Chem.
Abstr., 53, 14132f (1959^
40. M. Tomita and M. Kozuka, J. Pharm. Soc. Japan, 87, 1134 (1967);
Chem. Abstr., 68, 10233w XT963).
41. Y. Sashida, R. Sugiyama, S. Iwasaki, H. Shimomura, H. Itokawa, and
M. Fujita, J. Pharm. Soc. Japan, 96, 659 (1976); Chem. Abstr., 88,
3043u (1978T.
42. T. Yang and S. Lu, Tai-Wan Ko Hsueh, 24, 94 (1970); Chem. Abstr.,
75; 31299g (1971).
43. T. Yang and S. Lu, Pei I Hsueh Pao, 3, 121 (1973); Chem. Abstr.,
81_, 60844k (1974).
44. T. Nakano, Pharm. Bull., 4, 67 (1956); Chem. Abstr., 51, 2823f
(1957).
45. K. V. Rao, unpublished results.
46. R. Ziyaev, A. Abdusamatov, and S. Yu. Yunusov, Khim. Prir. Soedin.,
TJ_, 528 (1975); Chem. Abstr., 84, 44478a (1976).
47.S. Fujita and Y. Fujita, Chem. Pharm. Bull., 22, 707 (1974).
48. V. Plouvier, C. R. Acad. Sci. (D) (Paris), 254, 4196 (1962).
49. K. V. Rao and W. Wu, Lloydia, 41_, 56 (1978).
50. R. Juneau, Doctoral Dissertation, Univ. of Fla., Gainesville,
Fla., 1972; Piss. Abstr. Int., B, 33, 3013 (1972); K. V. Rao and
R. Juneau, L1oydia, 38, 339 (1975).

108
51. H. K. Lichtenhaler, 1. Pflanzenphysiol., 53, 388 (1965).
52. V. Plouvier, C. R. Acad. Sci. (D) (Paris), 266, 1526 (1968).
53. R. W. Doskotch and M. S. Flom, Tetrahedron, 28, 4711 (1972).
54. Y. Suqii, J. Pharm. Soc. Japan, 50, 183 (1930); Chem. Abstr., 24,
3505 (1930"). ~
55. M. Fujita, H. Itokawa, and Y. Sashida, J. Pharm. Soc. Japan, 93,
429 (1973); Chem. Abstr., 79, 52926g (1973T
56. F. S. El-Feraly and W. Li, Lloydia, 41, 442 (1978).
57. W. Yan, Chih. Wu Hsueh Pao, 21, 54 (1979); Chem. Abstr., 90,
200353n(1979). —
58. H. Kakisawa, K. Takenori, H. Y. Hsu, and Y. P. Chen, Bull. Chem.
Soc. Japan, 43, 3631 (1970); Chem. Abstr., 74, 50498s (1971)7
59. M. Hirose, Y. Sat5, and A. Haqitani, Nippon Kaqaku Zasshi, 89, 889
(1968); Chem. Abstr., 70, 48779h (1969)7
60. R. D. Haworth, "Natural Resins," in "Ann. Rep. Prog. Chem.," 33,
270 (1936). —
61. 0. R. Gottlieb, "Neolignans," in Fortschr. Chem. Org. Naturst.,
35, 1 (1978).
62. 0. R. Gottlieb, Rev. Latinoamer. Quim., 5, 1 (1974); Chem. Abstr.,
81, 27379g (1974)7”
63. D. M. Holloway and F. Scheinemann, Phytochemistry, 12, 1503
(1973). —
64. 0. R. Gottlieb, J. Chagas da Silveira, G. G. de Oliveira, and M.
de L. D. Weinberg, unpublished results, cited in 0. R. Gottlieb,
Rev. Latinoamer. Quim., 5, 1 (1974); Chem. Abstr., 81, 27379q
TT974T!
65. T. Nakaoki, N. Morita, A. Hiraki, and Y. Kurokawa, JL Pharm. Soc.
Japan, 76, 347 (1956); Chem. Abstr., 50, 9688a (1956).
66. F. S. Santamour, Jr., Morris Arbor. Bull., 16, 63 (1965).
67. F. J. Francis and J. B. Harborne, Proc. Amer. Soc. Hort. Sci.,
89, 657 (1966).
68. F. S. Santamour, Jr., Morris Arbor. Bull., 17, 13 (1966).
69.
F. S. Santamour, Jr., Morris Arbor. Bull., 17, 65 (1966).

109
70. B. E. Read, "Chinese Medical Plants," 3rd ed., Peking Natural
History Bulletin, Peking, China, 1936, p. 161.
71. F. G. Speck, Jr., Primitive Man, 14, 49 (1941); cited in D. E.
Moerman, "American Ethnobotany," Garland Publishing Co., New York,
N.Y., 1977, p. 113.
72. D. I. Bushnell, !S.I_.-B.A.E_. Bulletin, 48, 1909, cited in D. E.
Moerman, "American Ethnobotany," Garland Publishing Co., New York,
N.Y., 1977, p. 113.
73. "Pharmacopoeia of the United States of America," (6th Decennial
Revision), W. W. Wood, Ed., New York, N.Y., 1883, p. 215.
74. "Dispensatory of the United States of America," 20th ed., J. P.
Remington and H. C. Wood, Eds., Lippincott, Philadelpha, Pa.,
1918, p. 1479.
75. E. P. Claus, "Pharmacognosy," Lea and Febiger, Philadelphia, Pa.,
1956, p. 222.
76. 0. I. Belova, Ya. Kh. Nolle Aptechnoe, 2, 65 (1953); Chem. Abstr,
47, 8319i (19537.
77. H. Yang, H. H. Chang, and T. C. Weng, J. Formosan Med. Assoc.,
52, 109 (1953); Chem. Abstr., 47, 8175d (1953).
78.E. Furusawa and W. Cutting, Proc. Soc. Exptl. Biol. Med., 122,
280 (1966); Chem. Abstr., 65, 4494bTl 966)T
79. E. Furusawa, S. Ramanathan, S. Furusawa, Y. K. Woo, and W. Cutting,
Proc. Soc. Exptl. Biol. Med., 125, 234 (1967); Chem. Abstr., 67,
31387u~(T967).
80. N. V. Tsitsin, V. F. Kovtunenko, D. K. Polyatov, and K. M.
Khaidarov, Zasch. Rast. Vred. Bolez, 2, 167 (1973); Chem. Abstr.,
81_, 146847a-(T97477
81. K. Watanabe, Y. Goto, and K. Yoshitomi, Chem. Pharm. Bull., 21,
1700 (1973).
82. M. Kimura, M. Yoshizaki, I. Muro, and D. Shiho, J. Pharm. Soc.
Japan, 85, 570 (1965); Chem. Abstr., 63, 15397b T196FT”
83. E. F. Aleshinskaya, Tr. Krym. Med. Inst., 18, 675 (1957); Chem.
Abstr., 53, 5505b (1959).
84. K. Inoue, Nippon Yakurigaku Zasshi, 53, 797 (1957); Chem. Abstr.,
52, 18870b (1958).
85. K. Shimamoto, K. Inoue, and K. Ogui, Japan J. Pharmacol., 7, 135
(1958); Chem. Abstr., 52, 188961 i (1958^

no
86. A. J. Everett, L. A. Lowe, and S. Wilkinson, Chem. Commun., 1020
(1970).
87. K. Ogui and M. Morita, Japan J. Pharmacol., 2, 89 (1953); Chem.
Abstr., 47, 10128b (195TK
88. C. R. Chen, J. L. Beal, R. W. Doskotch, L. A. Mitscher, and G. H.
Svoboda, Lloydia, 37, 493 (1973).
89. S. F. Fakhrutdinov and M. B. Sultanov, Farmakol. A1kaloidov
Serdechnykh Glikozidov, 207 (1971); Chem. Abstr., 77, 109453r
(1972). “
90. H. Sheppard and C. R. Burghardt, Ex. Med. Int. Congr. Ser. 359
(Neuropsychopharmacology), J. R. Boissier, H. Hippius, and P.
Pichot, Eds., American Elsevier Publishing Co., Inc., New York,
N. Y., 1975, p. 866.
91. C. D. Hufford, M. J. Funderbunk, J. M. Morgan, and L. W. Robertson,
JL Pharm. Sci., 64, 789 (1975).
92. D. Warthen, E. L. Gooden, and M. Jacobsen, J. Pharm. Sci., 58,
637 (1969). —
93. V. N. Kamat, A. Vaz, P. V. Divekar, F. Fernandes, and S. S.
Bhatnagar, Indian J. Med. Research, 46, 418 (1958).
94.P. W. Erhardt and T. 0. Soine, J_. Pharm. Sci., 64, 53 (1975).
95.S. F. Fakhrutdinov, Farmakol. Alkaloidov Serdechnykh Glikozidov,
141 (1971); Chem. Abstr., 78, 76333a (19737:
96. S. Oyaizu, Nippon Yakurigaku Zasshi, 54, 1106 (1958); Chem. Abstr.,
54, 3728c (1960).
97. F. Moisset de Espanes, Rev. Soc. Argentina Biol., 32, 108 (1956);
Chem. Abstr., 51_, 5992gTT‘957]T
98. F. Moisset de Espanes, Rev. Soc. Argentina Biol., 31, 253 (1955);
Compt. Rend. Soc. Biol., 149, 1791 (1955); Chem. Abstr., 50,
11515h TT956).
99. D. E. S. Campbell and W. Richter, Acta Pharmacol. Toxicol., 25,
345 (1967). —
100.R. W. Doskotch and F. S. El-Feraly, JL Pharm. Sci., 58, 877 (1969).
101.A. S. Ramaswamy and M. Sirsi, Naturwissenschaften, 44, 380 (1957).
102.
A. S. Ramaswamy and M. Sirsi, Indian J. Pharm., 22, 34 (1960).

Ill
103. E. C. Britton and J. F. Livak, U.S. Patent 2,229,010 (1941); Chem.
Abstr., 35, P2910 (4) (1941).
104. J. E. Johnson, Jr., and D. R. Mussel!, U.S. Patent 2,532,233
(1950); Chem. Abstr., 45, P3132a (1950).
105. J. P. Phillips, J_. Org. Chem., 27, 1443 (1962).
106. A. I. Scott, "Interpretation of the Ultraviolet Spectra of Natural
Products," Pergamon Press, New York, N.Y., 1964, pp. 75-8.
107. M. Keeney, Anal. Chem., 1489 (1959).
108. A. I. Scott, "Interpretation of the Ultraviolet Spectra of Natural
Products," Pergamon Press, New York, N.Y., 1964, p. 78.
109. R. B. Bates, G. Büchi, T. Matsura, J. Amer. Chem. Soc., 82, 2327
(1960).
110. R. Hoffmann, Tetrahedron Letters, 3819 (1965).
111. E. M. Kosower and M. Ito, Proc. Chem. Soc., 25 (1962).
112. R. E. Corbett and P. K. Grant, J_. Sci. Food Agrie., 9_, 733 (1958).
113. R. E. Corbett and R. N. Speden, Chem. Soc., 3710 (1958).
114. P. Pesnelle and G. Ourisson, J_. Org. Chem., 30, 1744 (1965).
115. G. Büchi, J. M. Kauffman, and H. J. E. Lowenthal, J. Amer. Chem.
Soc., 88, 3403 (1966).
116. J. Grimaldi and M. Bertrand, Bul 1. Soc. Chim. Fr., 13, 957 (1971 ).
117. R. J. Abraham, K. Parry, and W. A. Thomas, J. Chem. Soc. (B), 446
0971).
118. J. Streith and G. Ourisson, Bul 1. Soc. Chim. Fr., 8, 1950 (1963).
119. J. T. Finley, Tetrahedron Letters, 275 (1963).
120. R. E. Corbett and H. Young, Aust. J_. Chem., 16, 250 (1963).
121. P. F. Ingwalson, Doctoral Dissertation, Georgia Instit. Technol.,
Atlanta, Ga., 1973; Piss. Abstr. Int., 34, B, 1425 (1973).
122. P. F. Ingwalson and D. Caine, J_. Org. Chem., 37, 3751 (1972).
123. J. Hutchinson, Kew Bul 1., 185 (1921 ).
124. G. Bentham and J. D. Hooker, "Genera Plantarum," vol. I, Verlag
Von J. Cramer, Weinheim, Ger., 1965, p. 17.

112
125.T. M. Whittaker, J_. Arn. Arboretum, 14, 376 (1933).
126. W. Wink, "Blumen," 18, 225 (1970); Biol. Abstr., 52, 64864 (1970).
127. A. Matsuo, M. Nakayama, S. Sato, T. Nakamoto, S. Uto, and S.
Hayashi, Experientia, 30, 321 (1974).
128. R. 0. Hellyer and E. V. Lassak, Aust. J_. Chem., 20, 2297 (1967).
129. J. Krepinsky and V. Herout, Coll. Czech. Chem. Commun., 27, 459
(1962). ~
130. R. C. Pettersen, D. L. Cullen, T. D. Spittler, and D. G. I.
Kingston, Acta Crystallogr., 1331 , 1124 (1975).
131. G. Biichi, S. W. Chow, T. Matsuura, T. L. Popper, H. H. Rennhard,
and M. Schach von Wittenau, Tetrahedron Letters, 14 (1959).
132. G. Büchi, W. Hofheinz, and J. V. Paukstelis, J. Amer. Chem. Soc.,
91, 6473 (1969).
133. M. Palmade and G. Ourisson, Bull. Soc. Chim. Fr., 886 (1958).
134. J. Streith, P. Pesnelle, and G. Oirisson, Tetrahedron
Letters, 677 (1962).
135. J. Streith and G. Ourisson, Bull. Soc. Chim. Fr., 8, 1960 (1963).
136. S. J. Terhune, J. W. Hogg, and B. M. Lawrence, Phytochemistry,
13, 865 (1974).
137. R. Corbett, priv. commun., cited in J. Streith and G. Ourisson,
Bull. Soc. Chim. Fr., 8, 1950 (1963).
138. R. C. Bowyer and P. R. Jeffries, J. Chem. Ind., 30, 1245
(1963).
139. E. Fattorusso, S. Magno, L. Mayol, C. Santacroce, and D. Sica,
Tetrahedron, 30, 3911 (1974).
140. E. Fattorusso, S. Magno, L. Mayol, C. Santacroce, and D. Sica,
Tetrahedron, 31, 269 (1975).
141. V. Herout, M. Soucek, and F. Sorm, Chem. Ind. (London), 1069
(1965).
142. M. Soucek, V. Herout, and F. Sorm, Coll. Czech. Chem. Commun.,
26, 803 (1960).
143.
T. A. Geissman and S. Matsuda, Phytochemistry, 7_, 1613 (1968).

113
144. H. Yoshioka, W. Reynold, N. H. Fisher, A. Higo and T. J. Mabry,
Phytochemistry, 9_, 823 (1970).
145. S. K. Talapatra and B. Talapatra, Phytochemistry, 12, 1827 (1973).
146. A. S. Bawdekar, G. R. Kelkar and S. C. Bhattacharya, Tetrahedron
Letters, U_, 1225 (1966).
147. R. W. Doskotch and F. S. E1-Feraly, J. Pharm. Sci., 58, 877
(1969). ~
148. R. W. Doskotch and F. S. El-Feraly, J. Org. Chem., 35, 1928
(1970). —
149. H. Erdtman and J. Runeberg, Acta Chem. Scand., 11 , 1060 (1957).
150. Y. Fumita and J. Shigenori, Nippon Kagaku Zasshi, 87, 1002 (1966);
Chem. Abstr., 65, 18450g(1960)7
151. T. Ohta and Y. Mori, Ann. Rept. Tokyo Col 1. Pharm., 3, 206 (1953);
Chem. Abstr., 49, 7514a (1955]".
152. J. C. Pew, W. J. Connors and A. Kunishi, "Chim., Biochim., Lignine,
Cellulose, Hemicelluloses," Actes Symp. Intern., Grenoble, France,
1964, pp. 229-45; Chem. Abstr., 65, 2462c (1966).
153. Hata and T. Sato, "Yuki_Kagobutsu Gosei Ho," vol. 14, Yüki
Gosei Kagaku Kyóhen, Gihodo, Tokyo, Japan, 1962, pp. 48-50; cited
in M. Fujita, H. Itokawa and Y. Sashida, J. Pharm. Soc. Japan, 93,
422 (1973); Chem. Abstr., 79, 35031u (1973).
154.E. C. Kleidner, J. Amer. Chem. Soc., 55, 4219 (1933).
155. Z. Budesinsky and A. Svab, Chem. Listy, 48, 421 (1954); Chem.
Abstr., 49, 3879g (1955).
156. E. H. Cox, J. Amer. Chem. Soc., 52, 352 (1930).
157. J. M. Vander Zanden and G. deVries, Rec. Trav. Chim., 70, 647
(1951). ~
158. L. Claisen and 0. Eisleb, Ann., 401, 21 (1913).
159.L. Canónica, A. Bonat, and C. Tedeshi, Ann. Chim. (Rome), 46,
465 (1956); Chem. Abstr., 51, 13813f (1957]".
160. P. Da Re, A. Colleoni, and L. Verlicchi, Ann. Chim. (Rome), 48,
762 (1958), Chem. Abstr., 53, 21923h (1959)7
161. A. Pelter, J_. Chem. Soc., (C) 1376 (1967).

114
162. L. H. Briags, R. C. Cambie, and R. A. F. Couch, J. Chem. Soc.
(C), 3042 (1968). ~
163. E. E. Dickey, J. 0r£. Chem., 23, 179 (1958).
164. I. A. Pearl, D. C. Beyer, and E. E. Dickey, J. Org. Chem., 23,
705 (1958). ~ —
165. L. A. Elyakova, A. K. Dzizenko, and G. B. Elyakov, Dokl. Akad.
Nauk S.S.S.R., 165, 562 (1965); Chem. Abstr., 64, 8290a (1966).
166. R. R. Arndt, S. H. Brown, N. C. Ling, P. Roller and C. Djerassi;
J. M. Ferreira, F. B. Gilbert, E. C. Miranda, and S. E. Flores;
A. P. Duarte; and E. P. Carrazzoni; Phytochemistry, 6, 1653
(1967).
167. C. L. Chen, H. M. Chang, and E. B. Cowling, Phytochemistry, 15,
547 (1976). —
168. P. R. Jeffries, J. R. Knox, and D. E. White, Aust. J. Chem.,
T4, 175 (1961). ~
169. C. Chen, H. Chang, E. B. Cowling, C. H. Hsu, and R. P. Gates,
Phytochemistry, 15, 1161 (1976).
170. H. Ishi, H. Ohida, and J. Haainawa, J. Pharm. Soc. Japan, 92,
118 (1972); Chem. Abstr., 77, 16530y (1972T7
171. Y. Sasaki and K. Matoba, J. Pharm. Soc., Japan, 87, 284 (1967);
Chem. Abstr., 67, 32607c T1967J-
172.K. Freudenberg and H. Dietrich, Chem. Ber., 86, 4 (1953).
173.H. Nimz and H. Gaber, Chem. Ber., 98, 538 (1965).
174. E. E. Eliel, "Stereochemistry of Carbon Compounds," McGraw-Hill
Book Co., Inc., New York, N.Y., 1962, pp. 43-47.
175. C. H. Ludwig, B, J. Nist, and J. L. McCarthy, J. Amer. Chem.
Soc., 86, 1186 (1964).
176. K. Freudenberg and G. S. Sidhu, Tetrahedron Letters, 20, 3
(1960). —
177. E. N. Maslen, C. Nockolds, and M. Patón, Aust. J. Chem., 15,
161 (1962). ~~
178. A. J. Birch, P. L. Macdonald and A. Pelter, J. Chem. Soc. (C),
1968 (1967). ~
179. E. D. Becker and M. Beroza, Tetrahedron Letters, 157 (1962).

115
180. C. K. Atal, K. L. Dahr, and A. Pelter, J. Chem. Soc. (C), 2228
(1967).
181. K. Freudenberg, R. Kraft, and W. Heimberger, Chem. Ber., 84,
472 (1951). —
182. K. Freudenberg, Chem. Ber., 88, 16 (1955).
183.K. Freudenberg, C. L. Chen, J. M. Jarkin, H. Nimz, and H. Renner,
Chem. Commun., 11, 224 (1965).
184. M. Hesse, W. Vetter, and H. Schmid, Helv. Chim. Acta, 48, 674
(1965).
185. H. Budzikiewicz, C. Djerassi, and D. H. Williams, "Structure Elu¬
cidation of Natural Products by Mass Spectrometry," vol. 1,
Holden-Day, Inc., San Francisco, Cal., 1964, pp. 174-177.
186. M. Shamma and W. Slusarchyk, "The Aporphine Alkaloids," Chem.
Rev., 64, 59 (1964).
187. J. C. Craig and S. K. Roy, Tetrahedron, 21, 395 (1965).
188. M. Ohaishi, J. M. Wilson, H. Budziekiewicz, M. Shamma, W. A.
Slusarchyk, and C. Djerassi, J_. Amer. Chem. Soc., 85, 2807 (1963).
189. A. H. Jackson and J. A. Martin, J_. Chem. Soc. (Cj, 2181 (1966)
190. A. R. Katritzky, R. A. Y. Jones and S. S. Bhatnagar, J. Chem.
Soc., 1950 (1960).
191. H. Guinaudeau, M. Leboeuf, and A. Cavé, Lloydia, 38, 275 (1975).
192. H. Guinaudeau, M. Leboeuf, and A. Cavé, J. Nat. Prod., 42, 325
(1979).
193. A. M. Kuck, Chem. Ind. (London), 118 (1966).
194.A. Groebel, D. Lenoir, and R. Pernet, Planta Med., 18, 66 (1970).
195. D. S. Bhakuni, S. Tewari, and M. M. Dhar, Phytochemistry, 11,
1819 (1972).
196. S. A. Johns and J. A. Lamberton, Aust. vL Chem., 20, 1277 (1967).
197.J. R. Cannon, G. K. Hughes, E. Ritchie, and W. C. Taylor, Aust.
J. Chem., 6, 86 (1953).
198.I. Kikkawa, J. Pham. Soc. Japan, 78, 1006 (1958); Chem. Abstr.,
53, 3260h (1959T

116
199. I. Kikkawa, Japanese Patent 7335 ('62) (1958); Chem. Abstr., 59,
1698b (1959).
200. M. Shamma, "The Isoquinoline Alkaloids," Academic Press, Inc.,
New York, N.Y. 1972, p. 195.
201. G. Barger and G. Weitnauer, Helv. Chim. Acta, 22, 1036 (1939).
202. A. C. Santos, Philippine J. Sci., 43, 561 (1930).
203. C. D. Hufford, Phytochemistry, 15, 1169 (1976).
204. C. D. Hufford and M. J. Funderbunk, J. Pharm. Sci., 63, 1338
(1974).
205. M. S. Yunosov, S. T. Akramov and S. Yu.Yunosov, Dokl. Akad.
Nauk Uzb. S.S.S.R., 23, 38 (1965); Chem. Abstr., 65, 13781a
TÍ965TT
206. H. Guinaudeau, M. Leboeuf, A. Cavé, S. Duret and R. R. Paris,
Planta Med., 30, 54 (1976).
207. J. Kunitomo, Y. Nagai, Y. Okamoto and H. Furukawa, J. Pharm. Soc.
Japan, 90, 1165 (1970); Chem. Abstr., 74, 1110a (1971).
208. J. Kunitomo, Y. Yoshikawa, S. Tanaka, Y. Imori and K. Isoi,
Phytochemistry, 12, 699 (1973).
209. S. R. Johns, J. A. Lamberton, C. S. Li and A. A. Sioumis, Aust.
J. Chem., 23, 423 (1970).
210.D. C. Grove and W. A. Randall, "Assay Methods of Antibiotics,"
Medical Encyclopedia, Inc., New York, N.Y., 1955, p. 35.

BIOGRAPHICAL SKETCH
Terry Lee Davis was born in Oak Ridge, Tennessee, on October 31,
1945, the oldest of three sons. His family moved to Louisville, Ken¬
tucky, in 1950. He graduated from Seneca High School in 1963.
While attending the University of Kentucky, he was a member of
Alpha Tau Omega social fraternity and in 1966 was president of the
University of Kentucky chapter of Alpha Chi Sigma, chemical fraternity.
He received his B.S. degree from the University of Kentucky in 1967.
He married his wife, Sandi, in June of 1969. Sandi is a teacher,
with a master's degree in early childhood education. They have one
daughter, Tiffany, born in December 1976.
While attending the University of Florida, he received a
teaching award from the department of chemistry in 1970. He received the
master's degree in chemistry in June 1971.
Terry's interests are bonsai and exotic plants. He has pub¬
lished a number of articles on bonsai.
117

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
David N. Silverman
Professor of Pharmacology and Therapeutics
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
0
ÍA
es
i A. Zoltewicz Q
Jc
Pr
Zoltewicz
essor of Chemistry
This dissertation was submitted to the Graduate Faculty of the College
of Pharmacy and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
June 1981
Dean for Graduate Studies and Research

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
/0üM^Í?7
David N. Silverman
Professor of Pharmacology and Therapeutics
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
This dissertation was submitted to the Graduate Faculty of the College
of Pharmacy and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
June 1981
Dean, College of Pharmacy
Dean for Graduate Studies and Research

I certify that
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
K. V. Rao, Chairman
Professor of Medicinal Chemistry
I have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Assistant Professor of Medicinal Chemistry
I have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for„the degree of
Stephen G. Schulman 7”
Professor^of Medicinal Chemistry

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
K-
K. V. Rao, Chairman
Professor of Medicinal Chemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy. 4
Rfennetn B. Sloan
Assistant Professor of Medicinal Chemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
A
St'ephen^G.) Schulman /
Profess&rof Pharmacy

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