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Simplified isolation of lignans from Saururus cernuus and various biological activities of natural and synthetic lignans

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Simplified isolation of lignans from Saururus cernuus and various biological activities of natural and synthetic lignans
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Orugunty, Ravi Shankar, 1970-
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xi, 112 leaves : ill. ; 29 cm.

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Antipsychotic agents ( jstor )
Chromatography ( jstor )
Cytotoxicity ( jstor )
Diones ( jstor )
Enzymes ( jstor )
Furans ( jstor )
Lignans ( jstor )
Sodium ( jstor )
Solvents ( jstor )
Sulfates ( jstor )
Department of Medicinal Chemistry thesis Ph. D ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Medicinal Chemistry -- UF ( mesh )
Dissertations, Academic -- Medicinal Chemistry -- UF
Lignans -- chemistry ( mesh )
Lignans -- isolation & purification ( mesh )
Lignans -- pharmacology ( mesh )
Lipoxygenase Inhibitors ( mesh )
Medicinal Chemistry thesis, Ph. D
Medicine, Herbal ( mesh )
Plants, Medicinal ( mesh )
Research ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1998.
Bibliography:
Bibliography: leaves 107-112.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Ravi Shankar Orugunty.

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SIMPLIFIED ISOLATION OF LIGNANS FROM SAURURUS CERNUUS AND VARIOUS BIOLOGICAL ACTIVITIES OF NATURAL AND SYNTHETIC LIGNANS
By
RAVI SHANKAR ORUGUNTY
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1998




This work is dedicated to Dr. K. V. Rao, my advisor, whose untimely death has left a deep void. I would also like to dedicate this work to my family for their constant support and encouragement.




ACKNOWLEDGMENTS
I would like to thank my advisor Dr. K.V. Rao. Apart from being my research advisor he was a true friend, philosopher and guide whose untimely death has left a void that cannot be filled for a long time to come. He was always available with his helpful suggestions and ideas. Dr. Rao believed in doing experimental work, in order to back up theoretical ideas. He was a tireless worker. I shall always remember the time that I spent in his lab learning the art of natural product chemistry.
I would also like to thank the other members of my
committee Dr. Perrin, Dr. Schulman, Dr. Sloan, Dr. Tebbett, and Dr. Zoltewicz who were helpful throughout the course of this work. I would like to take this opportunity to thank the other faculty members of the Department of Medicinal Chemistry who were truly involved at every step of my graduate studies. I would like to thank Nancy Rosa, Jan Kalmann and my fellow graduate students for their tolerance and encouragement.
Special thanks go to my family who have constantly
supported me throughout the course of this work and every endeavor that I have taken in my life.
iii




TABLE OF CONTENTS
ACKNOWLEDGMENTS iii LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT ix CHAPTERS
1 BIOLOGICAL ACTIVITY OF LIGNANS 1
Introduction 1 Antiplatelet Activity and Platelet Activating
Factor Antagonists 2
Cytotoxic and Anticancer Activity
of Lignans 3
Lignans with Antiviral Activity 6
Lipoxygenase Inhibitory Activity
of Lignans 10
AN IMPROVED METHOD FOR THE ISOLATION OF THE
LIGNAN CONSTITUENTS OF SAURURUS CERNUUS
BY REVERSE REVERSE PHASE COLUMN
CHROMATOGRAPHY 13
Introduction 13 Materials and Methods 16 First Reverse Phase Chromatography 17 Second Chromatography 24 Result and Discussion 32
3 TOTAL ASSIGNMENT OF THE 13C NMR CHEMICAL SHIFTS
OF SOME LIGNANS ISOLATED FROM
SAURURUS CERNUUS 39
Introduction 39 Experimental 41
iv




4 LIPOXYGENASE INHIBITORY PHENOLIC LIGNANS FROM
SAURURUS CERNUUS 47
Introduction 47 Materials and Methods 58 Experimental 58 Discussion 62
5 SYNTHESIS OF STEREOISOMERS OF MANASSANTIN 68
Introduction 68 Experimental 79
6 CYTOTOXICITY OF MANASSANTIN AND ITS
SYNTHETIC ANALOGS 90
Introduction 90 Materials and Methods 90 Results and Discussion 93 Conclusions and Further areas for Research 104
LIST OF REFERENCES 107
BIOGRAPHICAL SKETCH 112
v




LIST OF TABLES
Table page
2.1: Elution sequence of Saururus Lignans ................ 34
3.1: Side Chain Carbon assignments ....................... 44
3.2: Aromatic Carbon Assignments ......................... 46
4.1: IC50 values of some of the various non-phenolic and
phenolic compounds isolated from Saururus cernuus ..... 56 6.1: L 1210 Cytotoxicity Data of Saururus Lignans ....... 103
vi




LIST OF FIGURES
Figure page
1-1: Lignans with antiplatelet activity ................... 4
1-2: Lignans with antitumor and anticancer activity ....... 5 1-3: Lignans with antiviral activity ...................... 7
Figure 1-4: Lignans with antiviral activity ............... 9
1-5: Lignans with lipoxygenase inhibitory activity ....... 12 2-1: Lignans isolated from Saururus cernuu.s .............. 20
2-2: Lignans isolated from Saururus cernuus .............. 21
2-3: New lignans isolated from Saururus cernuus .......... 22
2.4: Elution profile of the first reverse phase column ... 23 2-5: Synthesis of Machilin-D Methyl Ether ................ 33
3-1: Oxidation of Manassantin A and B .................... 45
4-1: Biosynthesis of leukotrienes ........................ 48
4-2: Lipoxygenase inhibitory activity of various fractions of Saururus cernuus ....................................... 53
Figure 4-3: LOX inhibition by various phenolic fractions 54 4-4: Lignans from Saururus cernuus with lipoxygenase
inhibitory activity ................................... 55
4-5: Kinetic plot for Machilin-D ......................... 57
4-6: Structures of Magnolidin and Furoguaiacin ........... 64
4-7: Effect of Ferric ammonium sulfate on Magnolidin ..... 65
vii




4-8: Effect of Ferric ammonium sulfate on SC-2 Diol ...... 66 4-9: Effect of Ferric ammonium sulfate on SC-6 ........... 67
5-1: Various stereoisomers of Tetrahydrofuran lignans .... 72 5-2: Synthesis of totally methylated lignans ............. 73
5-3: Synthesis of various Manassantin analogs ............ 74
5-4: Attempted synthesis of Veraguensin diol ............. 78
6-1: Saururus lignans tested for cytotoxicity ............ 94
6-2: Saururus lignans tested for cytotoxicity ............ 95
6-3: Synthetic Manassantin derivatives tested for
cytotoxicity ........................................... 96
6-4: Synthetic Manassantin analogs tested for cytotoxicity97 6-5: L1210 toxicity of S.C-1, S.C-5 and S.C-6 ............ 98
6-6: L1210 cytotoxicity of S.C-7 and S.C-8 ............... 99
6-7: L1210 cytotoxicity of S.C-7 Diketone and S.C-8
Diketone .............................................. 100
6-8: L1210 toxicity of Dialkyl Furan, Dialkyl Meso and
Dialkyl trans Tetrahydrofurans ....................... 101
6-9:L1210 toxicity of Furan SC-8, Meso SC-8 and Trans SC-8102
viii




Abstract of Dissertation Presented to the Graduate School Of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
SIMPLIFIED ISOLATION OF LIGNANS FROM SAURURUS CERNUUS AND VARIOUS BIOLOGICAL ACTIVITIES OF NATURAL AND SYNTHETIC LIGNANS
By
Ravi Shankar Orugunty
August 1998
Chairman: Dr. Koppaka V. Rao Major Department: Medicinal Chemistry
Saururus cernuus is an aquatic weed that is found
commonly throughout Florida. It was used in folk medicine as a sedative and for the reduction of pain, fever, and inflammation and for a number of other disorders. Dr. Rao systematically investigated this plant for its constituents that were toxic to mice. This led to the isolation of two new neolignans that have neuroleptic activity. The structure of these lignans is unique with regard to their stereochemistry.
One aspect of investigation in this work revealed that the phenolic fraction was a potent inhibition of the lipoxidase enzyme.
ix




The phenolic fraction was then further separated into its individual constituents and their inhibitory activity was measured. The compounds showed varying degrees of activity the most potent being Licarin, S.C-2 Diol, Dihydroguaretic acid and Machilin-D IC50 of these compounds were found to be in the range of 10-5 pM.
The earlier reported isolation scheme was protracted
and produced lignans that contained varying amounts of color pigments. In order to simplify the process of isolation of these lignans, the reverse phase column chromatography (developed by Dr. Rao) was employed in this study. Thus in two steps a dozen lignans were isolated in high purity. In addition, it was the first time that the yields of these lignans from this plant were ascertained. The total assignment of the 13C NMR of four new lignans (that have been reported so far only by our lab) was accomplished using a high field NMR spectrometer. This required the study of the HETCOR and HMBC spectra of these compounds.
In this study the non-phenolic lignans Manassantin A
and B turned out to be cytotoxic in the L-1210 cell culture bioassay. This prompted the investigation of the synthetic Manassantin-A derivatives with regard to their cytotoxic effects. Thus a number of synthetic Manassantin analogs with different stereochemistry were generated and evaluated
x




for their cytotoxicity. The all trans Manassantin A analog was comparable in its activity to the natural compound.
The furan analog was the least potent. In addition the natural and synthetic diketone compounds turned out to be 50 times less active than their reduced derivatives.
xi




CHAPTER 1
BIOLOGICAL ACTIVITY OF LIGNANS Introduction
Lignans are a class of natural products that have in common the constitutional feature of at least two C6C3 (phenylpropane) units attached at the central carbon of each side chain. Haworth' introduced the term lignan to describe such compounds. Lignans are thought to be formed by the oxidative coupling of monomeric C6C3 phenols such as eugenol, coniferyl alcohol, coniferaldehyde and ferulic acid. The mechanism postulated2 for this coupling is thought to be free radical in nature and this has been confirmed experimentally. Polymerization of the above mentioned phenols leads to the formation of lignins. Lignins are universally distributed throughout all woody tissues. Lignins3 are thought to provide strength and stability to the cell wall and thus promote the growth of plants.
Lignans are found distributed throughout the various genera in the plant kingdom. This is due to the wellestablished fact that the above-mentioned phenols arise from the shikimic acid pathway, which is one of the principal
1




2
pathways4 of secondary metabolism in plants. The earlier work done (prior to the 1980's) dealt with the isolation, characterization and synthesis of the isolated lignans. Later work dealt with isolation and the study of the wide variety of biological activities that a number of lignans have been shown to have both in vitro and in vivo.
A systematic classification of lignans shall be
presented with regard to the biological activities that have been reported in the literature.
Antiplatelet Activity and Platelet Activating Factor Antagonists
Platelet Activating Factor (PAF) is a highly potent phospholipid. PAF has been linked to various biological activities and is now considered as an important mediator of many biological processes such as aggregation and degranulation of platelets and neutrophils. It is also reported to be involved in inflammation, allergic responses, chemotaxis and hypotension.5' 6 Specific PAF receptor sites in rabbit plasma7 membranes and human platelets8 have been reported.
The 3,4-Dimethyl-2, 5-bis (3,4-dimethoxyphenyl) tetrahydrofuran groups of lignans are potent PAF antagonists. The antagonist activity was found to be dependent on the stereochemistry of the tetrahydrofuran. The most potent antagonist in this group was found to be the trans-2, 5-bis (3,4,5-trimethoxyphenyl) tetrahydrofuran (L-652, 731, Figure




3
1-1, 1) This compound had an IC50 of 0.02 pM in inhibiting the binding of PAF to receptors in rabbit platelet membranes.9 The stems of Piper puberulum (Piperaceae) has been used in Chinese folk medicine along with other Piper plants for the treatment of asthma and arthritic conditions. Bio-activity based (inhibition of PAF binding to rabbit platelet membranes) fractionation led to the isolation of three new neolignans, Pubulerins A, B, C (Figure 1-1, 2, 3, 4). Pubulerins A and C were found to be potent PAF antagonists0 with and IC50 7.3 and 5.7 pM respectively.
The whole plant of Justica procumbens (Acanthaceae)11 has been used in Chinese herbal medicine for the treatment of fever, pain and cancer. Using the antiplatelet bioassay as a guide, ten lignans were isolated. The most potent of these lignans, neojusticin A, (Figure 1-1, 5) had an IC50 of
1.1 PM.
Cytotoxic and Anticancer Activity of Lignans
Podophyllotoxin (Figure 1-2, 6) is a well known lignan that has cytotoxic activity; and its derivatives are currently used in cancer therapy. The mechanism of its activity is based on its ability to bind to microtubules12 and cause mitotic arrest in metaphase. In the course of screening tropical plants for Ras function inhibitors Ohse




4
CF630 OCKa-o AcO
~OC
1 (L-652,731) 2
cO CH3
CH30 .......AcO
CH30 0 Q H 0
3 4 OCT- 0
< 0
00
0
o-J
5
Figure 1-1: Lignans with antiplatelet activity




OH OH
0
- HH
H 0
CH30 OCH3
OCH3
6 Podophyllotoxin 7 Rocaglaol
OH 3 O I
8 Aglaiastatin 9 Dehydroaglaiastatin
Figure 1-2: Lignans with antitumor and anticancer activity




6
et al.'3 isolated three cyclopentabenzofuran lignans, from Aglaia odorata, rocaglaol, aglaiastatin and dehydroaglaiastatin (Figure 1-2, 7, 8, 9) respectively. These compounds inhibited the growth of ras expressing-cell lines. The IC50 of these compounds was on the order of 10 ng/ml. Ras4 is a member of a family of GTP-binding proteins that play an important role in various signaling pathways for cell growth, differentiation and transformation. Activation of the ras proto-oncogene is found in many human neoplasms and is especially common in pancreatic and colon carcinomas. Thus, compounds that inhibit ras functions could lead to potential lead compounds with cytotoxic or anti-cancer activity.
Lignans with Antiviral Activity
Review of recent literature on lignans shows that many lignans have shown good activity as antiviral agents. The methanolic extract of Anogeissus acuminata (Combretaceae) showed activity against HIV-l Reverse Transcriptase. Bioassay based fractionation15 led to the isolation of two new dibenzylbutadiene lignans Anolignan A and B (Figure 1-3, 10, 11) respectively. These compounds had an IC50 of 60 and 25 gg/ml respectively as HIV-RT inhibitors. Kadsura interior (Schizandraceae), a plant that has been used in




7
H
OH H
HH
HO H HO Ht
H
H
0 OH
10 Anolignan A 11 Anolignan B
o 0
CHR30 ROCH30 H3O CH30 H
0Oli 0 HO
0 0 12 R= 0 14R= c": Interiotherin A Schizantherin
13 R=
0
Interiotherin B
Figure 1-3: Lignans with antiviral activity




8
traditional Chinese medicine for the treatment of menstrual irregularities, blood deficiencies, and other feminine disorders, has been previously investigated with the isolation of two important lignans, kadsurin and interiorin. The ethanolic extract of this plant showed significant activity in inhibiting the HIV replication in H9 lymphocytes. This led to the isolation16 of two new dibenzocyclooctadiene lignans, interiotherins A and B along with schisantherin (Figure. 1-3, 12, 13, 14) respectively. Investigation of Phyllanthus myrtifolius (Euphorbiaceae) for HIV-1 Reverse Transcriptase inhibitory activity led to the isolation of two lignans Phyllamycin B and Retrojusticidin B (Figure.1-4, 15, 16) respectively. The IC50 of these compounds on HIV-I Reverse Transcriptase is in about 3-5 [M. Interestingly the IC50 of these compounds on human DNA polymerase17 was 289 and 989 pM, respectively. Thus, the selectivity shown by these compounds against the viral enzyme makes them excellent lead molecules. Recent studies with 3'-O-methyl nor-dihydroguaretic acid and nordihydroguaretic acids (Figure 1-4, 17, 18) respectively have shown that these lignans are able to inhibit HIV tatregulated transactivation in vivo. These compounds induced the protection of lymphoblastoid cells from HIV killing and were able to suppress the replication of five HIV-1 strains in mitogen stimulated




9
OCH3 0 CH30 N CH30 I 0
CH3 CH30
0
0 0
o0
o-3J o--/
15 Phyllamycin B 16 Retrojusticidin
v -OH
17 R4H Nordihydroguaretic acid
18 R= Me 3-0-Methyl nordihydroguaretic acid
Figure 1-4: Lignans with antiviral activity




10
peripheral mononuclear cells. The target of these compounds has been localized to nucleotides 87-40 of the HIV long terminal repeat. These compounds are thought to inhibit the binding of Spl to its site in the HIV long terminal repeat. It is therefore suggested that this unique mode of inhibition of proviral expression8 will be useful in inhibiting the life cycles of the wild type and RT and protease mutant viruses in HIV-infected patients.
Lipoxygenase Inhibitory Activity of Lignans
The lipoxygenase are a family of enzymes that contain non-heme iron. These enzymes convert arachidonic acid into leukotrienes. Leukotriene B4 is a potent chemotactic agent19 and is considered to be a mediator of inflammation. The peptidoleukotrienes LTC4, LTD4, LTE4 are powerful spasmogenic agents which are known to be involved in the pathology of many diseases like asthma, inflammatory bowel disease and rheumatoid arthritis. Thus, selective inhibitors of 5-LO could form a new class of therapeutic agents that could be of potential use in the treatment of such diseases.
Investigations for potent 5-LO inhibitors by Ducharme et al.2 led to the identification of naphthalenic lignan lactones (Figure 1-5, 19). Further synthetic modifications lead to certain potent compounds illustrated in Figure 1-4 20. Caffeic acid (Figure 1-5, 21), which is one the simplest lignan found widely distributed in nature, has been




11
identified to be a very potent inhibitor21 of 5-LO. Synthesis22 of analogs of caffeic acid have lead to some potent inhibitors, one of them being caffeic acid octyl amide.
In addition to the above-mentioned activities a number of lignans have been isolated23' 24 which have antimicrobial and hypolipidemic effects.
Thus it can be said with reasonable certainty that lignans which are formed by the coupling of the phenyl propanoid moiety give rise to compounds with varying structures and stereochemistry and show wide ranging pharmacological activities. The pursuit of these compounds can give rise to potentially useful therapeutic agents.




12
00 0
0 0
0 OMe 0
N0
0
19 20
OH
21
Figure 1-5; Lignans with lipoxygenase inhibitory activity




CHAPTER 2
AN IMPROVED METHOD FOR THE ISOLATION OF THE LIGNAN
CONSTITUENTS OF SAURURUS CERNUUS BY REVERSE PHASE COLUMN CHROMATOGRAPHY
Introduction
The aquatic weed, Saururus cernuus, L (N.O,
Saururaceae) which grows mainly in the eastern United States, was known and used during the 19th century in folk medicine25' 2 as a sedative, for soothing irritations and inflammations of the kidneys, bladder, prostate gland, urethra and also for its anti-inflammatory activity. A number of terpenes such as limonene and pinene were isolated27 which account for the aromatic property of the plant. A systematic study was undertaken by Rao28-30 that yielded a number of novel lignan and other constituents. The most important of these were the dineolignan type compounds Manassantins A and B31 (Figure 2-1, 22, 23, respectively) which show potent neuroleptic activity. Manassantin A (MNS-A) was evaluated by Rao32 for its central depressant effects using various behavioral parameters in mice with haloperidol as the reference compound. When administered intra-peritoneally, it caused a decrease in spontaneous motor activity and inhibited amphetamine-induced
13




14
stereotypy with an ED50 of 0.21 mg/Kg. Unlike, haloperidol it did not produce ptosis or catalepsy, which is usually considered as an indicator for the ability to produce extrapyramidal effects in humans. Unlike haloperidol, MNS-A also causes hypothermia. Thus, in this respect, MNS-A behaved as an atypical neuroleptic. The demonstration of neuroleptic activity prompted a study of its affinity for the various receptors, such as the dopamine (Dl, D2 etc.), serotonin and other receptors. Surprisingly, it did not show any strong affinity for these and other receptors. MNS-A also did not have any significant effect33 on dopamineinduced adenylate cyclase activity. These properties suggest that MNS-A may have a different mechanism of action. Thus, MNS-A requires further pharmacological evaluation. The novel dilignan structure of MNS-A is different from that of all known neuroleptic compounds currently in use. In addition, it is unique in not having any nitrogen in its structure.
The continued interest in the neuroleptic and other
activities found in this plant was one of the main reasons for the reexamination of the isolation process that was used earlier. The objective was to simplify and streamline the process for large-scale applicability. The method used earlier for the isolation of the various lignans started with the methanolic extract of the plant, which was




15
subjected first to a three-step solvent partition to separate the three fractions: 1) highly lipid-soluble components, 2) moderately lipid-soluble components, and 3) non-solvent extractable components. The neuroleptic activity was found in the moderately lipid-soluble fraction which was then subjected to chromatographic procedures in two to three stages using silica gel and/ or florisil. The close similarity of the twenty or more lignans with regards to their physical properties posed a challenge to their separation. For example, to separate the mixture of MNS-A and B, it was necessary to acetylate the mixture, separate the acetates, and then regenerate the compounds by hydrolysis.
Reverse phase column chromatography was used
successfully by Rao et al.34' 3 for the fractionation of the crude extracts of Taxus brevifolia, for the isolation of Paclitaxel and several other related taxanes. In an effort to simplify the isolation of lignans from Saururus cernuus, the methanolic extract was partitioned between benzene and water and the benzene extract was directly subjected to reverse phase column chromatography. The fractions thus obtained were purified by one normal phase silica column36 to give pure compounds.




16
Materials and Methods
Plant material
The above ground parts of the plant (which were previously identified by the University of Florida Herbarium, where a voucher specimen was submitted) are collected locally, in and around Gainesville, FL, during May through September. The plant material is dried in the sun and stored until needed for extraction. Extraction and Partition
The dried plant material was ground to a coarse mesh
(0.5-1 cm) and extracted with methanol in a stainless steel drum in 25 lb quantities. The methanolic extract was drained after 24 hours and the extraction was repeated, using absorbance at 275 nm as a guide. The extraction was complete when the absorbance at 275 nm was negligible. In order to ensure complete extraction, the plant material was extracted four times with methanol. The extracts were concentrated under reduced pressure to give a thick green syrup, which was partitioned between water (5 gallons) and benzene (5 gallons). The organic layer was separated and the aqueous layer extracted a second time with benzene (3 gallons). The combined benzene layers that contained the lignan constituents was concentrated to a dark green semi-




17
solid (5% of the weight of the dried plant). This extract was used in the further chromatographic separations.
First Reverse Phase Chromatography
A column was set up using about 800 g of C18 bonded silica gel (15-35 micron size, Phase Separations Inc., Norwalk CT) using methanol. A glass column of the MitchellMiller type (2.5" x 25", Ace Glass Co, Vineland, NJ) was used to prepare the column and was found to be suitable for low-pressure liquid chromatography. The column was equilibrated with 40% aqueous methanol and was ready for further use.
The extract (150 g) was dissolved in methanol (450 ml) by warming if necessary. Approximately 150 g of the equilibrated silica gel from the above column was added to this solution with stirring. About 400 ml of 40% aqueous methanol was added to the above slurry with stirring followed by water (600 ml). The mixture was stirred until there was no visible green precipitate or oily material. This was confirmed by taking an aliquot of the slurry in a test tube and allowing the silica to settle readily to the bottom to give a relatively clear supernatant. The slurry was then filtered using low suction; and the residue of dark green colored silica gel was then re-suspended in about 200 ml of the filtrate. The slurry thus obtained was loaded on to the top of the column. The clear filtrate was then




18
pumped onto the top of the column using a metering pump (Eldex-Fisher Scientific Co.). The column feed was checked from time to time to ensure that it was clear; if not, the solution was warmed to clarify or minimal amounts of methanol were added.
The successful loading of the sample was followed by elution of the column with a step gradient of aqueous methanol (50, 55, 60, 65, 75, 85% methanol). Fractions (200 ml) were collected and monitored by their UV abosrbance at 275 nm and by TLC. These two parameters were used in decisions to change the concentration of the methanol in the solvent. Thus, for example, when the absorbance values rose as a result of increasing the methanol concentration from 50 to 55%, the 55% solvent was continued until the absorbance values showed a tendency to reduce. Similar trends were observed on the TLC. In general, 2-4 multiples of the bed volume of the column were used for each eluant mixture. The 85% aqueous methanol eluted out the last of the lignans. This was followed by 100% methanol, which was later changed to a mixture of methanol, ethyl acetate and ligroin (2:1:1). Most of the colored pigments, including chlorophyll, were held up on the column during the run. Eluting the column with 100% methanol and the three-solvent system, consisting of methanol, ethyl acetate and ligroin, completely removed the green pigments. The column was then washed with 100%




19
methanol and equilibrated with 40% methanol for regeneration.
Considering the UV and TLC data, the fractions were
combined into small groups (3-5 fractions) and concentrated and set aside for further work. The combined fractions were examined by TLC to study the number, relative proportions and nature of the lignans present. Some of the major lignan constituents isolated earlier28, 29 from this plant, such as austrobailignan-5 (S.C-i), saucernetin (S.C-2), saucerneol (S.C-6), manassantin A and B, were used as standard markers to orient the other compounds on TLC.
The appropriate concentrates obtained from the above fractions were then partitioned using counter current distribution to separate the phenolic from the non-phenolic lignans. The solvent system used was 0.2 N potassium hydroxide in 50% aqueous methanol with benzene, ligroin (1:1) as the organic phase. The aqueous methanolic layers were partially concentrated and neutralized with 0.2 N aqueous HCI and extracted with benzene. The neutral and phenolic fractions thus separated were then subjected to a short normal phase silica column chromatography to afford the purified lignans. The structures of the known lignans isolated are provided in Figure 2-1 and Figure 2-2. In addition four new lignans were isolated and their structures are given in Figure 2-3.




20
OCH3
R20 0H0 HO OH
OCH3 OCH3 22 RI, R2 = OCH3 ; S.C-8, 23 RI, R2 = OCH2O; S.C-7
OCH3 H3C
OCH3 OCH3 OCH3 OH
24 R = OH; S.C-6 26 Guaacin
25 R= OCH3; S.C-5
CH30 9 OCH3 CH3 OCH3
OCH3 OCH3 OCH3cH 28 S.C-3
27 S.C-2
Figure 2-1: Lignans isolated from Saururus cernuus




21
<0
00
lOHO OCHH3
0 OH
o-J
29 S.C-1 30 Licarin-A
Figure 2-2: Lignans isolated from Saururus cernuus




22
....'OH
OH HO" HO 0CH3 OCH3 OcH3 OcR3
31 S.C-2 Diol 32 S.C-3 Diol
~0
HO
OCH3 N OH OH
33 Dihydroguaretic acid 34 threo -Machilin-D
Figure 2-3: New lignans isolated from Saururus cernuus




23
120000 100000 80000
U
C 60000
0
40000 200000 50 100 150 200 250 Fraction Numbers Figure 2-4: Elution profile of the first reverse phase column




24
Second Chromatography
Normal phase column chromatography was carried out using silica gel (Fisher 100-200 mesh or 235-425 mesh). Solvent sequence used was ligroin, benzene, with 5-10% acetone in benzene; alternatively mixtures of ligroin ethyl acetate were also used. All the H' and C13 NMR were obtained from either a Varian VXR-300 or a Varian Gemini 300 using CDC13 as solvent. The assignments of the carbon were made using COSY, HETCOR and HMBC spectra in the case of most compounds. The spectra were also matched with values reported elsewhere.
Characterization of the Major Lignans from S. cernuus
TLC of the first 10 fractions did not show any significant compounds. Fractions 11-20 (1.5 g) upon separation, by counter current-distribution, gave 0.6 g of phenolic lignans. Chromatography of this fraction on a silica column (25 g) and elution with 2% acetone in benzene gave 0.15 g of colorless powder identified as Saucernetin diol (S.C-2 diol Figure 2-3, 31).
'H NMR (8) S.C-2 diol: 0.68, (6 H, d, 6.6 Hz), H-9, 9', 2.24, (2H m), H-8, 8', 3.87, (6 H s, 2xOMe), 5.42, (2H d, 6 Hz), H-7, 7', 6.75-6.90, (6H, m), H-Ar.
The above compound was further characterized by
methylation with dimethyl sulfate and potassium carbonate in




25
refluxing acetone to yield the dimethyl ether, which was crystallized and found to be identical with saucernetin28
(S.C-2, Fig.2-1 27).
Fractions 21-30 (1.5 g) yielded 0.7 g of phenolic and
0.8g of non-phenolic material. The phenolic material on column chromatography gave 100 mg of a semi-solid that was identified as Veraguensin diol (S.C-3 diol, Figure.2-3, 32). Its 1H and 13C were identical to those reported by SchmediaHirschmann et al.37 and Agrawal et al.38
'H NMR (6) S.C-3 diol: 0.65ppm, (3H, d, 6.8 Hz), 1.05, (3H, d, 6.6 Hz), 1.79, (1H, m), 2.24, (1H, m), 3.83, (3H, s), 3.88, (3H, s), 4.39 (1H, d, 9 Hz), 5.11 (1H, d, 9 Hz)
6.8-7.0, (6H, m).
13C NMR (6) S.C-3 diol: 14.86, C-9, 9'; 45.89, 47.6, C8, 8'; 55.75, 2xOMe; 83.05, 87.24, C-7, 7'; 109.4, 109.7, C2, 2'; 113.9, 114.2, C-5, 5'; 119.2, 119.8, C-6, 6'; 132.6, 133.1, C-i, 1'; 144.5, 145.1, C-4, 4'; 146.1, 146.5, C-3, 3'
Fractions 36-50 (6g) upon separation by counter-current distribution yielded 3.3 g of phenolic and 2.5 g of nonphenolic fractions. The non-phenolic fractions after column chromatography on normal silica with benzene gave 1.5 g of Saucernetin (S.C-2, Figure 2-1, 27) and 100 mg of Veraguensin (S.C-3, Figure 2-1, 28).




26
'H NMR (8) S.C-2: 0.69, (6H, d, 6.6 Hz), 2.27, (2H, m) 3.89, (6H, s), 3.90, (6H, s), 5.45, (2H, d, 6.3 Hz), 6.85, (6H, s).
13C NMR (8) S.C-2: 14.73, C-9, 9'; 44.02, C-8, 8';
55.88, 2xOMe, 83.51; C-7, 7'; 109.6, 110.8, 118.4, 134.0, 147.9, 148.6, C-Ar.
'H NMR (8) S.C-339: 0.66, (3H, d, 6.6 Hz) 1.07 (3H, d,
6.0 Hz), 1.81, (1H, m) 2.25, (1H, m), 3.86, 3.88, 3.89,
3.91, (12H, s), 4.42 (1H, d, 9.3 Hz), 5.13 (1H, d, 8.4 Hz),
6.85-7.08 (6H, m).
13C NMR (8) S.C-3: 14.95, 15.00, C-9, 9'; 45.98, 47.94, C-8, 8'; 55.83, 2xOMe; 83.00, 87.23, C-7, 7'; 110.01, 110.45, 110.72, 111.09, 118.63, 119.19, 133.49, 133.79, 148.06, 148.59, 148.98.
The phenolic fractions upon column chromatography gave 115 mg of a pale yellowish semi-solid which was found to be Machilin D or threo-2- (2-methoxy-4-trans-propenylphenoxy) -l(4-hydroxy-3-methoxyphenyl)propan-l-ol, previously isolated
from Machilus thunbergii4 (Figure 2-3, 34).
'H NMR (8) Machilin D: 1.16, (3H, d, 6 Hz), 1.87, (3H, d, d, 6.6, 0.9 Hz), 3.80, (3H, s), 3.88, (3H, s), 4.11, (1H, d, q, 8.4, 6.3 Hz), 4.61, (1H, d, 8.3 Hz), 6.16, (1H, m,
6.6, 15.6 Hz), 6.35, (1H, m 0.9, 15.6 Hz), 6.8-6.9, (6H, m).




27
13C NMR (6) Machilin D: 16.9, 18.3, C-9, 9'; 78.3,
124.8, C-8, 8'; 83.9, 130.4, C-7, 7'; 120.6, 118.7, C-6, 6'; 114.1, 118.7, C-5, 5'; 145.4, 150.7, C-4, 4'; 146.6, 146.6, C-3, 3'; 109.14, 109.4, C-2, 2'; 133.3, 131.8, C-i, 1'.
The structure of Machilin-D was confirmed by
methylation and comparing it with the 13C and 'H NMR values with those reported in the literature" and by total synthesis of the methylated compound (as illustrated in Figure 2-5). The spectral data of the synthesized compound were identical to that of the methylated derivative of the natural compound.
Elution of the column with 5% acetone in benzene gave
1.6 g Saucerneol (S.C-6, Figure 2-1, 24).
'H NMR (6) S.C-6: 0.7, (3H, d, 6 Hz), 0.72, (3H, d, 6 Hz), 1.17, (3H, d, 6 Hz), 2.27, (2H, m), 3.87, 3.88, 3.90,
3.92, (12H, s, 4 OCH3's) 4.12, (1H, m); 4.65, (1H, d, 8.1 Hz); 5.44, (1H, d, 6 Hz); 5.45, (1H, d, 6 Hz), 5.60 (1H, bs, Ar-OH), 6.75-7.0, (9H m).
13C NMR (6) S.C-6: 14.8, 16.9, 44.1, 55.9, 78.4, 83.3,
83.5, 84.0, 108.8, 110.0, 110.1, 110.9, 113.9, 118.7, 119.0, 132.2, 132.6, 136.6, 144.5, 146.4, 148.9, 148.97, 150.5.
Fractions 66-75 (2 g) were separated by counter-current distribution into phenolic (ig) and non-phenolic (1 g) fractions. The phenolic fraction on column chromatography gave 400 mg of Saucerneol (S.C-6). The non-phenolic




28
fraction gave 400 mg of Saucerneol methyl ether (S.C-5,
Figure 2-1, 25).
1H NMR (6) S.C-5: 0.7, (3H, d, 6 Hz), 0.72, (3H, d, 6 Hz), 1.17, (3H, d, 6 Hz), 2.25, (2H, m), 3.87, 3.88, 3.90,
3.92 (15H, s, 5 OCH3's), 4.13, (1H, m), 4.65, (1H, d, 8.1 Hz), 5.46, (2H, d, 6 Hz), 6.7-7.0, (9H, m).
13C NMR (6) S.C-5: 14.8, 16.9, 44.0, 55.85, 55.85,
78.3, 83.3, 83.4, 83.9, 109.6, 110.0, 110.1, 110.8, 118.3, 118.7, 119.9, 132.6, 133.2, 136.6, 146.6, 147.9, 148.6, 148.8, 148.9, 150.5.
Fractions 76-85 (5.8 g) yielded about 3.3 g of nonphenolic and 1.2g of phenolic fractions after countercurrent distribution. Similarly fractions 86-95 (6 0 g) gave 4.0 g of non-phenolic and 1.5g of phenolic fractions after counter-current distribution.
The non-phenolic fractions from fractions 76-95, upon column chromatography, gave 3.1 g of S.C-8 (Figure 2-1, 22) and about 100 mg of S.C-5.
'H NMR (6) S.C-8: 0.73, (6H, d, 6 Hz), 1.17, (6H, d, 6 Hz), 2.30, (2H, m), 3.88, 3.89, 3.93, (18H, s, 6 OCH3's), 4.12 (2H, m), 4.65, (2H, d, 8.1 Hz), 5.45, (2H, d, 6 Hz),
6.78-7.00, (12H, m).
13C NMR (5) S.C-8: 14.8, 16.95, 44.13, 55.82, 78.3, 83.3, 83.9, 110.0, 110.8, 118.57, 118.6, 119.92, 132.53, 136.37, 146.4, 148.77, 148.93, 150.48.




29
The phenolic fractions on chromatography gave 200 mg of dihydroguaretic acid (Figure 2-3, 33)42 and 200 mg of Licarin-A (Figure 2-2, 30).
'H NMR (8) Dihydroguaretic acid: 0.82, (3H d, 6 Hz), 1.72, (1H m), 2.45, (2H m), 3.80, (3H s), 5.49 (1H, bs),
6.52-6.81, (3H, m).
13C NMR (6): 13.84, 37.4, 41.03, 55.74, 111.3, 113.8, 121.6, 133.54, 143.45, 146.2.
Fractions 96-104 gave 1 g each of phenolic and nonphenolic fractions. The non-phenolic fraction on chromatography gave 500 mg of S.C-7 (Figure 2-1, 23).
'H NMR (6) S.C-7: 0.72, (6H, d, 6 Hz), 1.15, (3H, d, 6 Hz), 1.17, (3H, d, 6 Hz), 2.29, (2H, m), 3.87, 3.88, 3.90,
3.92, (12H, s, 4 0CH3's), 4.11, (2H, m), 4.62, (1H, d, 9 Hz)
4.64, (1H, d, 9 Hz), 5.46, (2H, d, 6 Hz), 5.94, (2H, s),
6.78-7.00, (12H, m).
13C NMR (8) S.C-7: 14.8, 16.8, 16.9, 44.1, 55.8, 78.3, 83.3, 83.8, 83.9, 100.9, 107.5, 108.0, 110.1, 110.9, 118.6, 118.63, 118.8, 119.9, 121.0, 132.4, 134.0, 136.4, 136.5, 146.3, 146.4, 147.3, 147.66, 148.8, 148.9, 150.50, 150.52.
The phenolic fractions gave 200 mg of Guaiacin (Figure 2-1, 26).
1H NMR (8) Guaiacin: 0.84, (3H, d, 6 Hz), 1.06, (3H, d,
6 Hz), 1.50, (1H, m), 1.60, (1H, m), 2.70 (2H, m), 3.33,




30
(1H, d, 10 Hz), 3.80, (3H, s), 3.82, (3H, s), 6.22, (1H, bs, Ar-OH), 6.53, (1H, bs, Ar-OH), 6.56, (1H, d, < 1 Hz), 6.59, (1H, dd, 8.1, < 1 Hz), 6.79, (1H, d, 8.1 Hz), 7.10, (1H, s),
7.15, (1H, s).
13C NMR (6) Guaiacin: 16.75, 19.57, 35.16, 38.57,
43.22, 53.64, 55.37, 55.50, 110.13, 111.74, 114.18, 115.64, 121.83, 127.42, 132.70, 137.86, 143.40, 143.82, 144.93, 146.69.
1H NMR and 13C NMR data of the isolated Guaiacin was identical to that reported in the literature.43
Fraction 191-205 (30 g) did not contain any phenolic compounds (based on UV analysis) and was thus directly purified by column chromatography. The major compound (15.8 g) eluted with ligroin. 'H and 13C NMR spectral data confirmed the structure of this compound to be identical to that of Austerbialignan-5 (S.C-I, Figure 2-2, 29).42
'H NMR (6) S.C-I: 0.8, (3H, d, 6 Hz), 1.72, 1H, m, 2.32.57, 2H, m, 5.9, 2H, s, 6.54, (1H, d, d, 7.8, <1 Hz), 6.58, (1H, d, <1Hz), 6.69, (1H, d, 7.8 Hz).
13C NMR (6) S.C-I: 13.8, 38.17, 41.12, 100.66, 107.9, 109.28, 121.72, 133.43, 145.41, 147.39. Total synthesis of Machilin-D methyl ether
Isoeugenol (200 mg) was dissolved in 5 ml DMF and
treated with 340 mg of 2-bromo-3', 4'-Dimethoxypropiophenone




31
and 400 mg K2CO3 and stirred overnight at room temperature. The reaction mixture was treated with 50 ml of water and extracted twice with 10 ml of diethyl ether. The combined ether layers were washed with water and concentrated to dryness to obtain a colorless semi-solid (35). Yield 512 mg.
'H NMR (5) 35: 1.72, (3H, d, 6.6 Hz), 1.83, (3H, d, d,
0.9, 6.3, Hz), 3.85, 3.92, 3.94, (9H, s), 5.40, (q, 6.6 Hz), 1H; 6.0, (1H, 15.6, 6.3 Hz), 6.28, (1H, 15.6, 0.9, Hz),
6.72-7.83, 6H, m.
13C NMR (5) 35: 18.3, 19.2, 55.8, 55.9, 56.0, 78.26, 109.45, 110.04, 111.24, 115.74, 118.55, 123.59, 124.32, 127.31, 130.44, 132.44, 132.48, 145.94, 148.95, 149.81,
153.6, 197.65.
Reduction of 35 (200 mg) was accomplished by dissolving it in 5 ml THF and cooled to 00C, 100 mg of lithium aluminum hydride was added and stirred for about 1 h. The reaction mixture was warmed to room temperature acidified with IN HCl and extracted twice with ether. The ether layer was washed with water, dried over sodium sulfate and concentrated to dryness to a colorless oily product. The product which had two compounds which were separated by column chromatography using 20% ethyl acetate: ligroin as the mobile phase. The faster compound was erythro (140 mg) and the slower compound was found to be threo (45 mg).




32
'H NMR (8) (36 erythro): 1.17, (3H, d, 6.3 Hz), 1.88, (3H, d, d, 0.9, 6.6 Hz), 3.86, 3.88, 3.89, (9H, s, OCH3),
4.34, (1H m), 4.84, (1H, d, 3.3 Hz), 6.15, (1H, m, 15.9, 6.6 Hz), 6.35, (1H, m, 15.9, 0.9 Hz), 6.8-6.9, (6H, Ar-H).
13C NMR (8) (36 erythro): 13.4, 18.4, 55.8, 73.46,
82.45, 109.2, 109.4, 110.7, 118.4, 118.96, 119.3, 119.84, 124.99, 130.4, 132.45, 133.7, 145.7, 148.2, 148.9, 151.5.
IH NMR (8) (36 threo): 1.16, (3H, d, 6 Hz), 1.87, (3H, d, d, 6, 0.9 Hz), 3.87, 3.88, 3.91, 9H, s, 4.11, (1H, d q, 6, 8.4 Hz), 4.63, (1H, d, 8.4 Hz), 6.18, (1H, m, 15.6, 6.0 Hz), 6.35, (1H, m, 15.6, 0.9 Hz), 6.81- 6.94, (6H, m).
13C NMR (8) (36 threo): 16.7, 18.3, 55.8, 78.3, 84.01, 109.08, 109.9, 110.77, 118.7, 118.9, 119.92, 124.8, 130.37, 132.47, 133.38, 142.62, 148.75, 148.92, 150.69.
Result and Discussion
The utilization of reverse phase column chromatography did indeed simplify the process of isolation of these lignans as compared to the previously used methods of purification. It may be noted that a total of nearly 11 lignans were isolated of which four have been isolated for the first time from this plant. The elution of these lignans started with 50% aqueous methanol and continued up until 85 % aqueous methanol. The major components are S.C-I and S.C-8.




33
0
+
OCH3 C0Br K2CO3/ DMF
OH OCH3
Isoeugenol Dimethoxybromopropiophenone
0 0 OCH3 LA BF0COH OCH3 CH30 CH3O
OCH3 OCH3
35 36 Erythro + threo
Figure 2-5: Synthesis of Machilin-D Methyl Ether




34
Table 2.1: Elution sequence of Saururus Lignans
Compound Yield 1 S.C-2 Diol 0.15 g 2 S.C-3 Diol 0.1 g 3 S.C-2 1.5 g 4 S.C-3 0.1 g 5 Machilin-D 0.1 g 6 S.C-6 1.6 g 7 S.C-5 0.5 g 8 S.C-8 3.1 g 9 Dihydroguaretic acid 0.2 g 10 Licarin-A 0.2 g 11 S.C-7 0.5 g 12 Guaiacin 0.2 g 13 S.C-1 15 g




35
The elution profile of the reverse phase column
monitored at 275 nm is shown in Figure 2-4. The elution sequence and the yields of the various lignans isolated are given in Table 2-1. The order of elution of the compounds from the reverse phase column is interesting in that it does not seem to correlate entirely with either the polarity or molecular weight. On normal phase TLC austrobailignan-5 (S.C-I) is the most lipophilic with Rf =1 (ligroin benzene 1:1 solvent system). Licarin, guaiacin, saucernetin (S.C-2) and veraguensin (S.C-3) are compounds that have Rfs ranging from 0.8-0.6 in 5% acetone in benzene and are intermediate in their polarity. Significantly polar are the sesqui and dilignans S.C-5, S.C-6, S.C-7 and S.C-8 with Rfs of 0.6-0.3
in 10% acetone in benzene. One would have expected the most polar compounds to elute first from the reverse phase column followed by the compounds with intermediate polarity followed by the least polar compounds. However, surprisingly the compounds with intermediate polarity such as saucernetin, veraguensin and their corresponding diols eluted first these were then followed by the most polar compounds, S.C-5, S.C-6, S.C-7 and S.C-8, then followed by guaiacin, licarin and austrobailignan-5.
The reverse phase process described here clearly has some advantages over the normal phase chromatography used earlier. Firstly, it involves fewer steps than were used




36
before. Instead of having two to three solvent partitions only one partition between water and benzene was used to remove water-soluble materials. Application of the concentrated benzene extract eliminated the need for handling the lignans in three subgroups. In spite of applying the complex mixture directly to the C18 silica directly, the resolution was satisfactory. The colored pigments such as chlorophylls and carotenoids were held up by the column and did not elute with the desired compounds. Thus all the lignans were isolated as either crystalline or non-crystalline homogeneous colorless solids. It must be noted that the separation of phenolic lignans from the nonphenolic lignans was carried out at a later stage using a counter current distribution of aqueous methanolic KOH against ligroin benzene because the phenolic lignans did not partition into aqueous KOH from very polar solvents like dichloromethane. The utilization of a non-polar organic solvent system, (e.g., benzene ligroin, 1:1) lowers the solubility of the ionized phenols in the organic phase; whereas the addition of methanol in aqueous base increases the solubility of the ionized phenols in the aqueous phase.
One of the most important advantages of the new
procedure is its adaptability to larger scale operations as was shown by Rao et al., in the isolation of paclitaxel from the crude extracts of Taxus brevifolia. The solvents used




37
are methanol, aqueous methanol mixtures; that are relatively inexpensive compared to organic solvents. The high cost of the reverse phase silica is offset by the fact that it can be readily and repeatedly regenerated. Lastly, unlike the case with a normal phase silica or florisil columns, on a reverse phase column, none of the components of the extract are lost due to irreversible adsorption.
The most important part in the whole process is the
preparation of the sample for the reverse phase column. In most applications reported in the literature the preparative reverse phase chromatography is used as the penultimate or ultimate step in the purification, the sample is in a high degree of purity. The conditions employed in the operation are those that are typically found in analytical HPLC (i.e., high pressure (1000 psi) and low solvent flow rates) and the ratio of sample to the amount of silica is 1:500 or more. In the current process, the reverse phase column is the first step of the purification scheme. The crude extract is loaded directly on to the column. The ratio of crude extract to the sample is 1:5 and the pressure used for running the column is about 50 psi and rarely exceeds 100 psi. The crude mixture is highly lipid soluble in nature and hence is not soluble in 40% aqueous methanol. It must be noted that the sample cannot be directly loaded on to the column as it can block the column causing considerable




38
difficulties at later stages. However, if the sample is prepared as outlined in the Materials and Methods the C18 silica seems to act as a lipophilic solvent and absorbs the sample so that no free oily or waxy material is left after the sample preparation. This gives rise to a free flowing slurry and the column performs as though a soluble sample has been applied.




CHAPTER 3
TOTAL ASSIGNMENT OF THE 13C NMR CHEMICAL SHIFTS OF SOME
LIGNANS ISOLATED FROM SAURURUS CERNUUS.
Introduction
During the course of investigation of Saururus cernuus a number of lignans were isolated. Most of these lignans were tetrahydrofurans including S.C-5, S.C-6 and Mannasantin A and B (Figure 3-1: 37, 38, 39, 40)29 respectively that possess a unique stereochemistry. Mannasantin A and B were found32 to have a very potent neuroleptic activity. The phenolic lignans isolated from this plant showed significant activity as lipoxygenase inhibitors as will be reported in Chapter 4.
The wide variety of pharmacological activities shown by a number of different compounds led us to further investigate Saururus cernuus. In order to improve the isolation of these lignans the reverse phase column chromatographic method developed by Rao et al 34,35 was used and these lignans were isolated in good yields in a facile manner. Investigation of the literature with regards to spectroscopic data on such compounds revealed that tetrahydrofuran lignans with such unique stereochemistry has never been reported. An excellent review with regards to 39




40
the 13C NMR shifts of various types of lignans has been reported 38, 43. However, this review deals only with the 13C NMR shifts of the three side chain carbons and does not mention compounds with this stereochemistry. The paucity of spectroscopic data for these compounds led us to undertake a NMR investigation in order to provide complete carbon assignments for these compounds.
The Neuroleptic compounds Mannasantin A and B were
oxidized to their respective diketones (41, 42) with MnO2. These compounds served the twin purpose of giving related analogs and also to see whether the introduction of a carbonyl group would spread the chemical shifts of the aromatic protons. It was observed that the chemical shifts of the side chain aromatic protons that are adjacent to the carbonyl group moved downfield spreading over 6.8 ppm to
7.83 ppm. The splitting patterns were much more resolved as compared to the parent compounds that have a very poorly resolved spectrum in the aromatic region. These diketones thus aided in the assignment of the carbon chemical shifts. The proton and carbon resonances of these compounds were then assigned on the basis of 'H-'H COSY and 'H-13C HETCOR and HMBC. The 'H data is provided in the experimental section. The completely assigned 13C data is provided in Table 3-1 and Table 3-2.




41
Experimental
The isolation of S.C-5, S.C-6, S.C-7 and S.C-8 using reverse phase column chromatography has been reported36 elsewhere. NMR Spectra
All NMR spectra were obtained on Gemini 300
spectrometer with a 'H/13C multinuclear computer switchable probe (13C 75.4 Mz, pulse width 15.9 g sec). Samples contained 90-100 mg of a specific compound dissolved in approximately 1 ml of CDC13 with 0.02% of TMS as internal standard set to 0.00 ppm for the proton spectrum. The carbon spectrum was referenced using the CDC13 signal set to 77.0 ppm. Partial assignments were made with 1H and 13C spectra; complete assignments were made with the aid of COSY, HETCOR and Long Range HETCOR. The spectral windows for 1H and 13C (in the case of HETCOR and Long Range HETCOR) were set to 3000 and 15,000 Hz respectively, 2048 13C data points, Dl delay was set to 1.000s. 128-256 'H data points were collected. The total number of transients collected for the HETCOR were 128 and in the case of Long Range HETCOR it was 256. Jlxh was set at 140 Hz and JnxH was set to 0 Hz for HETCOR and in the case of LRHETCOR the JnxH was set to 6 Hz.
'H-NMR (6) S.C-5 (37): 0.7, (3H, d, 6 Hz), 0.72, (3H, d, 6 Hz), 1.17, (3H, d, 6 Hz), 2.25, (2H, m), 3.87, 3.88, 3.90,




42
3.92 (15H, s, 5 OCH3's) 4.13, (1H, m), 4.65, (1H, d, 8.1 Hz)
5.46, (2H, d, 6 Hz); 6.7-7.0, (9H, m). 'H-NMR (6) S.C-6 (38): 0.7, (3H, d, 6 Hz), 0.72, (3H, d, 6 Hz), 1.17, (3H, d, 6 Hz), 2.27, (2H, m), 3.87, 3.88, 3.90, 3.92, (12H, s, 4 OCH3's), 4.12, (1H, m), 4.65, (1H, d, 8.1 Hz), 5.44, (1H, d, 6 Hz), 5.45, (1H, d, 6 Hz), 5.60, 1H, bs, Ar-OH, 6.75-7.0, (9H, m).
'H-NMR (6) S.C-7 (39): 0.72, (6H, d, 6 Hz), 1.15, (3H, d, 6
Hz), 1.17, (3H, d, 6 Hz), 2.29, (2H, m), 3.87, 3.88, 3.90,
3.92, (12H, s, 4 OCH3's), 4.11, (2H, m), 4.62, (1H, d, 9 Hz), 4.64, (1H, d, 9 Hz), 5.46, (2H, d, 6 Hz), 5.94, (2H, s), 6.78-7.00, (12H, m).
'H-NMR (6) S.C-8 (40): 0.73, (6H, d, 6 Hz), 1.17, (6H, d, 6 Hz), 2.30, (2H, m), 3.88, 3.89, 3.93, (18H, s, 6 OCH3's), 4.12, (2H, m), 4.65, (2H, d, 8.1 Hz), 5.45, (2H, d, 6Hz),
6.78-7.00, (12H, m).
Oxidation of Mannasantin A and Mannasantin B: 100 mg of either (39) or (40) was dissolved in 10 ml benzene. To this solution was added 500 mg of activated MnO2. The mixture was refluxed for one and a half-hour. By then the reaction had gone to completion. This was observed by the disappearance of the starting material with the production of a single product on TLC. The reaction mixture was filtered through celite and concentrated to dryness. The crude diketone was eluted from a silica column and eluted




43
with 5% acetone in benzene. S.C-7 yielded 90 mg of its corresponding diketone and S.C-8 yielded 93 mg of its corresponding diketone diketone. 'H-NMR (6) S.C-7-Diketone (42): 0.62, (6H, d, 6 Hz), 1.67, (3H, d, 6 Hz), 1.71, (3H, d, 6 Hz), 2.20, (2H, m), 3.84,
3.92, 3.94, (12H, s), 5.35, (2H, d, 6 Hz); 5.39, (2H, q, 6 Hz), 6.67, (2H, d, 8.1 Hz), 6.76, (2H, d, d, 8.1, 1.5 Hz),
6.83, (4H, d, 8.4 Hz), 7.60, (1H, d, <1Hz), 7.68, (1H, d, >1 Hz), 7.78, (1H, d, d 8.4, 1 Hz), 7.83, (1H, d, d 8.4, 1 Hz). 'H-NMR (5) S.C-8 Diketone (41): 0.62, (6H, d, 6 Hz), 1.71, (6H, d, 6 Hz), 2.20, (2H, m), 3.84, 3.92, 3.94, (18H, s),
5.35, (2H, d, 6 Hz); 5.39, (2H, q, 6 Hz), 6.66, (2H, d, d,
8.4, <1 Hz), 6.77, (2H, d, 8.4 Hz), 6.82, (2H, d <1 Hz),
6.83, (2H, d, 8.4 Hz), 7.67, (2H, d, <1 Hz), 7.83, (2H, d, d
8.4,<1 Hz).




44
Table 3.1. Side Chain Carbon assignments
7(7') 8(8') 9(9') 7"(7"') 8"(8"') 9"(9') OMe Methylenedioxy
1 83.4, 44.0, 14.8, 78.3 83.9 16.9 55.9
83.3 44.0 14.8 -- -- -- -2 83.5, 44.1 14.8, 78.4 84.0 16.9 55.9
83.3 ,44.1 14.8 -- -- -- -3 83.8, 44.1 14.8, 78.3 83.3, 16.8, 55.8 100.9
83.9 ,44.1 14.8 ,78.3 83.3 16.9 -4 83.3, 44.1, 14.8, 78.3, 83.9, 16.9, 55.8
83.3 44.1 14.8 78.3 83.9 16.9 -5 83.3, 43.9, 14.6, 197.1, 78.2, 18.8, 55.8 101.7
83.3 43.9 14.6 197.6 78.3 19.0 -6 83.3, 44.0, 14.7, 197.7, 78.3, 19.2, 55.9
83.3 44.0 14.7 197.7 78.3 19.2 --




45
OCH3
QooQ
RO 0,
OcH3 OCHK
37 R=OCH3 S.C 5 38 R=OH S.C 6
OCH3
RIO OCH3
0 OH H O
R20 0H 01'HO
OCH OCHK 39 RI R2 =OCH3 S.C-8
40 RI R2 = OCH20 S.C-7
MnO2fBenzene OC1H3
RIO 0C113
R20 Cono Q
0 q 0
OCHK OCH3
41 S.C-8 Diketone 42 S.C-7 Diketone
Figure 3-1: Oxidation of Manassantin A and B




Table 3.2: Aromatic Carbon Assignments
C # 1, 2,2' 3,3' 4,4' 5,5' 6,6' 1",1" 2",2" 3",3" 49,4" 5",5" 6",6" comp
1 136.6 109.6 148.6 146.4 118.3 118.7 132.6 110.0 150.5 148.96 110.8 119.9
133.8 110.1 148.8 147.9 110.8 118.7 -- -- -- -- -- -2 132.6 108.8 146.4 144.5 113.9 118.7 133.2 110.0 150.5 148.97 110.9
136.6 110.1 148.9 146.4 119.0 118.7 -- -- -- -- -3 132.4 107.5 148.8 146.3 119.9 118.6 134.0 110.1 147.3 147.66 110.1 118.63
136.4 108.0 148.9 146.4 121.0 118.6 136.5 110.9 150.5 150.52 110.1 118.8
4 136.37 110.0 148.7 146.4 118.6 118.57 132.5 110.0 150.5 148.93 110.8 119.9
5 135.4 110.4 147.98 145.7 118.4 115.5 128.2 108.8 149.5 151.9 107.8 123.5
135.6 110.4 148.89 145.6 118.4 115.96 128.2 111.2 149.7 153.5 110.0 125.3
6 135.45 110.0 149.5 145.5 115.5 118.42 128.3 111.2 148.9 153.5 110.4 123.62




CHAPTER 4
LIPOXYGENASE INHIBITORY PHENOLIC LIGNANS FROM SAURURUS CERNUUS
Introduction
The lipoxygenases are an important class of non-heme
iron containing enzymes found in plants and animals. These enzymes catalyze the dioxygenation of polyeneoic fatty acids that contain at least a 1,4-cis pentadiene structural unit.44 They are usually classified as 5-, 12- and 15lipoxygenases based on the position of the double bond of the substrate where the oxidation takes place. The animal and plant enzymes have an overall 60% sequence similarity, and the human lipoxygenase is about 25% identical to plant (soybean) lipoxygenases.45
Oxidation of arachidonic acid by 5-lipoxygenase forms the 5-hydroperoxyeicosatetraenoic acid (5-HETE), which is then converted to leukotriene A4 (LTA4), an epoxide. LTA4 can be further metabolized by two separate enzymatic routes to yield other leukotrienes-LTB4, LTC4, LTD4, LTE4 (Figure 41). These compounds are involved in various inflammation processes like increased vascular permeability, contraction of smooth muscles in bronchioles, and edema."' 47, 48
47




48
Arachidonic acid
Lipoxygenase OOH
5-HPETE
0
COOH
C5HI
LTA4
OOH~ OH
CoH oCOO
C5HI CAH1I H SCH2
CHCONHCH2COOH LTB4 I
NHOOCH2CH2CHCOOH LTC4 NH2
Figure 4-1: Biosynthesis of leukotrienes




49
It is now well acknowledged that these compounds may be involved49-52 in a number of allergic processes such as asthma. Leukotrienes are also thought to involved in diseases states such as psoriasis53' 5' and myocardial infarction.55
Thus inhibitors of these enzymes can be promising
candidates for the treatment of some of the above mentioned pathological problems. A number of inhibitors of these enzymes and antagonists are currently being studied as antiasthmatics and anti-inflammatory agents.59
Saururus cernuus has a rich history in folk medicine. This plant was used as a sedative, for soothing irritations and inflammations of the kidneys, urinary bladder, prostate, and urethra.25' 26 Investigation of this plant by Rao28-30 lead to the isolation of potent neuroleptic lignans Manassantin A and B. The isolation and assignment of 13C-NMR has been dealt with in detail in Chapters 2 and 3. Further investigation of the Saururus extract (Chapter 2, Materials and Methods, page 16) showed that the phenolic fraction of the benzene extract inhibited the soybean lipoxygenase enzyme whereas the non-phenolic fraction did not show any significant activity. Soybean lipoxygenase was found to be inhibited by a number of well known non-steroidal antiinflammatory agents (NSAIDS). Nor-dihydroguaiaretic acid (NDGA), a lignan derivative, is one of the most potent




50
inhibitors (Sircar et al.).5 The soybean lipoxygenase has a similar action on unsaturated fatty acids and the bioassay is easy to perform. In addition this enzyme is commercially available and is inexpensive. The linoleic acid used as the substrate is converted to 13-hydroperoxylinoleic acid, which can be determined by measuring the absorbance at 234 nm. However, the plant extract of Saururus, showed a strong absorbance at 234 nm and so caused interference in the direct measurement of the hydroperoxide at 234 nm. An alternative method was employed that used the oxidation of benzoyl leucomethylene blue to methylene blue by the hydroperoxide, formed in the enzymatic reaction, and measuring the absorbance at 660 nm without any interference from the plant extract.57
The benzene extract (Chapter 2, Materials and Methods, page 16) was partitioned into its phenolic and non-phenolic fractions. The benzene extract, the phenolic and nonphenolic fractions were assayed for their activity (Figure 4-2). The phenolic fraction was then subjected to normal phase silica column chromatography and the fractions obtained were tested for their activity (Figure 4-3). TLC was used to establish the identity of the compounds present in various fractions. Compounds that were previously isolated and characterized were employed as references. The pure compounds, shown in Figure, 4-4 were tested in the




51
concentration range of 100-6.25 pg/ml using the UV method (described in the experimental section). Furoguaicin (Figure 4-6: 2) a known inhibitor of this enzyme58 was used as the standard to compare the activities of these compounds. The kinetic plot of Machilin-D, one of the more potent phenolic lignan, is given in Figure 4-5. The IC50 of the phenolic compounds that were tested are given in Table 4-1.
Most of the known lipoxygenase inhibitors are classified as antioxidants, chelators and non-redox inhibitors.59 Since most of the isolated phenolic compounds showed good activity as inhibitors it was neccesary to find out if these compounds had any antioxidant or chelating properties.
Chelation by metals is known to cause a significant change in the UV spectrum of many compounds and is well documented in the case of flavonoid type compounds.60 The iron present in lipoxygenase is thought to be chelated by certain inhibitors. It was decided that the in vitro effect of iron on these compounds should be studied. Thus increasing amounts of a standard solution of ferric ammonium sulfate was added to a solution of S.C-6 and S.C-2 diol and the UV spectrum of these compounds were recorded. It is well known that the 3,4-dihydroxyphenyl system is capable of chelating metals like iron and copper. Thus, Magnolidin (Figure 4-6: 1), a compound that is structurally similar to




52
ethyl caffeate (3,4-dihydroxy cinnamic acid ethyl ester) and NDGA, was also used as a control. Magnolidin is the major glycoside of Magnolia grandiflora isolated by Rao et al.61 Magnolidin has a strong inhibitory effect on lipoxygenase (IC50= 5 PM).




53
100
0 Benzene Cci ( non-phenolic 80 0 phenolic 70
% Inhibition 50
30n
20 -phenolic
10- non-phenolic
0O------- Fraction
1000 Benzene 500
250 Concentration (pprn) 100
Figure 4-2: Lipoxygenase inhibitory activity of various fractions of Saururus cernuns




54
m Z
In
-------------------Oll ,l % uo~llq~qul




55
HO OH HO OH
CH3 CH3 CH3 CH3
S.C-2 Diol S.C-3 Diol
CH30
09 (% HO HO 0
'?,' CH30
COH3
S.C-6 OCH3 H CH3
Dihydroguaretic acid
0 CH30
0 H0
HO... Guaaei CH3
OCH3 OCH3
Machilin D H H C Guaiacin
Licarin
Figure 4-4: Lignans from Saururus cernuus with lipoxygenase inhibitory activity




56
Table 4.1: IC5o values of some of the various non-phenolic and phenolic compounds isolated from Saururus cernuus
Compound ICro (ptMx0".2)
I Crude phenolic mixture -110
2 Saucernetin diol 5 3 Veraguensin diol 30 4 Saucernetin 17 5 Magnolidin 3 6 Dihydroguaiaretic acid 4.5 7 Guaiacin 12 8 Licarin 5 9 Machilin-D 5 10 Furoguaiacin 2.5 11 S.C-1 >50 12 Manassantin A >50 13 Manassantin B >50




1.6
--Blank
1.4 50 g/ml
A-25 g/ml
1.2- w 12.5 g/ml ..,..-.'
0 6 g/ml
1
0.8
z
I
4 0.6
0.4
0.2
0
50 100 150 200 250 300 3
-0.2
Time (sec)
Figure 4-5: Kinetic plot for Machilin-D




58
Materials and Methods
Materials: The following chemicals were purchased and used as received: linoleic acid (Sigma), dimethylformamide (Fisher), Triton X-100 (Sigma), bovine hemoglobin, lipoxygenase (ICN). Benzoyl leucomethylene blue was readily prepared by the reductive benzoylation 62 of methylene blue (1 g) using excess of sodium dithionite, sodium hydroxide and benzoyl chloride in a mixture of 1:1 tetrahydrofuran and water. The reaction mixture was stirred at room temperature for about 2 hours after which the water layer was separated. The THF layer was washed twice with water and dried over anhydrous sodium sulfate and concentrated to dryness. The product (700 mg) was crystallized from a mixture of 1:1 ligroin ether as very pale blue crystals. The 1H-NMR was obtained to ascertain the identity of the compound.
All other chemicals and solvents were purchased from Aldrich, Sigma or Fischer Co.
Experimental
Determination of Lipoxygenase Activity by UV Method
The enzymatic reaction was monitored using a Shimadzu UV-visible spectrophotmeter at room temperature. All solutions were freshly prepared prior to assays. Linoleic acid solution 100 gM was prepared by dissolving 15 mg of linoleic acid in 10 ml of a pH 9.0, 0.1 M tris buffer




59
(prepared using the procedure outlined in CRC Manual 63) to obtain a stock solution. This stock solution (2 ml, equivalent to 3 mg of linoleic acid) was diluted to 100 ml using 0.1 M tris buffer.
The lipoxygenase enzyme was prepared by dissolving 1 mg of the enzyme (137200 units/mg of protein) in 10 ml of 0.1-M tris buffer. Inhibitors were dissolved in a mixture of propylene glycol and ethanol such that an aliquot of each yielded a final concentration of 4.2% propylene glycol and 2% ethanol in each assay.
The assay was carried out by using 2 ml of 100 .M linoleic acid to which 50 il of the enzyme solution was added to obtain an easily measurable initial rate of reaction.59 The progress of the reaction was monitored at 234 nm. The effect of the inhibitors on the reaction was studied by adding the inhibitor solutions to the linoleic acid solution prior to the addition of the enzyme. The production of the hydroperoxide was compared against controls under identical conditions. The substrate concentration in each case was 100 pM. Each compound was tested four times at each concentration and the mean values are reported.




60
Determination of Lipoxygenase Activity by Benzoyl Leucomethylene Blue Method
Linoleic acid solution, (1.2 ml, 100 gM) was taken and treated with 50 pl of enzyme as in the above method. The reaction mixture was incubated at room temperature for 10 minutes and then quenched with leucomethylene blue solution57. The only change that was made in this reagent was Succinate buffer (pH 5.0) was employed instead of phosphate buffer saline solution. This change did not effect the assay. This was observed by performing control experiments that employed PBS and Succinate buffer. The absorbance of each of the solutions was then observed at 660 nm after 5 minutes. The plant extract or samples to be tested were prepared as in the UV method and incubated with the linoleic acid solution and then treated with the enzyme. The reaction mixture was then quenched with the leucomethylene blue solution and the absorbance was measured at 660 nm after a period of 5 minutes. Each experiment was carried out in triplicate and the mean values are reported (Table: 4-1).
Determination of Activity in the Phenolic Fraction
The crude benzene extract was partitioned between a
mixture of 1:1 benzene: ligroin and 0.2 N KOH in 50% aqueous MeOH. These conditions were found to be ideal to separate the phenolic from the non-phenolic lignans. The aqueous




61
methanolic layer was diluted with an equal volume of water, neutralized with 0.2 N aqueous HCI, and extracted with dichloromethane. The dichlormethane layer was washed with water dried over anhydrous sodium sulfate and concentrated to dryness to yield the phenolic layer. The benzene: ligroin layer that contained the non-phenolic compounds was washed with water and dried over anhydrous sodium sulfate and concentrated to dryness. The activity of the crude benzene extract, the phenolic and the non-phenolic extract was ascertained by the leucomethylene blue method. About 500 mg of the phenolic fraction was charged on to a silica column and eluted using 1:1 benzene ligroin, 3:1 benzene/ligroin, benzene, 2, 5 and 10% acetone in benzene and 5% methanol in benzene. The solvent was evaporated from the fractions, they were reconstituted in about 1 ml of ethanol, propylene glycol (1:1) and tested for their inhibitory activity. The activity of the various fractions is shown in Figure 4-3.
Effects of Iron on the UV Spectrum of Saucerneol and Saucernetin diol
The following solutions were prepared in water or methanol depending on the solubility of the compounds.
1) Ferric ammonium sulfate (0.002 M) solution in water.
2) Magnolidin (0.5 mg/10 ml) dissolved in water.
3) S.C-6 (0.4 mg/10 ml) dissolved in methanol.




62
4) S.C-2 diol (0.4 mg/10 ml) dissolved in methanol.
The effect of ferric ammonium sulfate was studied as follows. One ml of Magnolidin solution was carefully transferred into an UV cuvette. The UV spectrum of the solution was recorded. Ferric ammonium sulfate solutions (50, 100, 150, 200 and 250 gil of 0.002 M) were successively added to the solution of Magnolidin. The solution was shaken and the UV spectrum was recorded after each addition. The experiment was repeated in a similar manner using solutions of S.C-6 and S.C-2 Diol. All the spectra were recorded using a Shimadzu UV-Visible spectrophotometer.
Discussion
The total phenolic fraction obtained from the original extract showed modest activity whereas the original benzene extract and non-phenolic fraction did not have any activity (Figure 4-2). The above experiments have established that the phenolic fraction of Saururus cernuus is responsible for the lipoxygenase inhibitory activity. The IC50 of the phenolic compounds are comparable to that of furoguaicin a known inhibitor of this class of enzymes. It is also interesting to note that veraguensin diol is a much weaker inhibitor than S.C-2 diol. This suggests that the stereochemistry of the tetrahydrofuran lignans plays an important role in the inhibition of this enzyme. However,




63
this observation needs to be clarified by conducting further experiments.
An interesting observation is that the non-phenolic compounds (S.C-I, S.C-5, S.C-7 and S.C-8) do not have any significant activity at concentrations of 100 gg/ml. This clearly shows that the lipoxygenase inhibitory activity is exclusively associated with the phenolic lignans.
Ferric ammonium sulfate did show a shift in the UV
spectrum of Magnolidin but it did not effect those of S.C-6 and S.C-diol. The results of this experiment are shown in Figures 4-7, 4-8 and 4-9. Thus, the lipoxygenase inhibitory activity of these compounds is not mediated via chelation. Thus, these compounds become very interesting lead molecules and further investigations are warranted with regards to their mechanism of action.
Based on the above results it can be concluded that the phenolic lignans isolated from Saururus cernuus have potent soybean lipoxygenase inhibitory activity. Further experiments need to been performed using mammalian enzymes. These compounds could possibly be investigated as lead molecules for the synthesis of inhibitors of lipoxygenase class of enzymes.




64
Rh O-CH
R2- 0 OCH2CH2 QH
4R~l OH
1 Rh= Rhamnose, R 1= Caffeyl, R 2 = H
2 Furoguaiacin
Figure 4-6: Structures of Magnolidin and Furoguaiacin




I
0.8
-No FAS
-50 I 0.002M FAS
- 100 10.002MFAS 0.6 150 10.002MFAS
-200 1 0.002 M FAS
-250 1 0.002 M FAS
- 50 1.02MFAS 0.4
0
'U
0.2
0
220 240 260 80 300 320 340 360 380 400 400
-0.2
Wavelength
Figure 4-7: Effect of Ferric ammonium sulfate on Magnolidin




1.4
1.2
1 No FAS
-50 I.002M FAS
-100 I.002M FAS
-150 I .002M FAS
*0.8 200 1.002M FAS
U
0.6
0 0
220 440 wavelength
Figure 4-8: Effect of Ferric ammonium sulfate on SC-2 Diol




0.8
- No FAS
0.6 50 I.002M FAS
-100 I .002M FAS
-150 I .002M FAS
-200 I .002M FAS
C
0.4
0
-0.2
wavelength
Figure 4-9: Effect of Ferric ammonium sulfate on SC-6




CHAPTER 5
SYNTHESIS OF STEREO-ISOMERS OF MANASSANTIN Introduction
The lignan constituents of Saururus represent a variety of structures. The biologically active compounds have the tetrahydrofuran ring system. These tetra-hydrofuran lignans contain two methyl groups at positions 3 and 4, and identical aromatic substituents at 2 and 5. There can be six possible isomers, two of them being meso (Figure 5-1, 43, 44) and four, chiral tetrahydrofurans (Figure 5-1, 45, 46, 47, 48) and the aromatic furan (Figure 5-1: 49). Of these, Manassantin has the stereochemistry of 2/3-cis, 3/4trans and 4/5-cis (Figure 5-1, 47). This stereochemical pattern in tetrahydrofuran lignans was first reported by Rao2' 29 and was first observed in the Saururus lignans.
Preliminary investigation of Manassantin A and B showed that they were extremely active in inhibiting the L-1210 cell line. This opened up an area of investigation of the stereochemistry of the tetrahydrofuran ring system in Manassantin and the effect of stereochemistry on L-1210 cytotoxicity.
68




69
Analogues with the same general structure of S.C-8 but with a different stereochemistry (as shown in Figure 5-1) at the tetrahydrofuran ring were synthesized and tested on L1210 cell culture. The idea being that testing these compounds will indicate if the activity is associated with any preferred stereochemical structure for the central ring. The aromatic furan analogue was also included.
The methodology for these reactions is well known. The 2,5-(3'-methoxy, 4'-hydroxy)diphenyl tetrahydrofuranoid lignans with different configurations of the ring, as shown in Figure 5-1. The completely methylated compounds such as galbelgin, veraguensin, galgravin and furoguaiacin had been prepared earlier.64,5 The synthetic scheme is given in Figure 5-2.
The starting material 1,4-(3', 4'-dimethoxy) diphenyl2,3-dimethyl-1,4-butanediones was readily prepared by the condensation of 3', 4'-dimethoxy-propiophenone with the corresponding 2-bromo derivative in the presence of sodium amide. Reduction of these diketones followed by acid catalyzed cyclization affords various tetrahydrofurans that are completely methylated. In addition, in the present work some 4,4'-diphenols were prepared, and this required some specific changes in the general schemes, as shown in Figure 5-3.




70
The preparation of the desired diphenols, required
partial demethylation. This reaction was carried out at the diketone stage (Figure 5-3, 52), to obtain the necessary regioselectivity. We have found that the use of thiocresol and sodium hydride in DMF brings about this demethylation to yield the 4,4'-demethylated diketone in good yields. This demethylation reaction was previously reported using thiophenol and thioethanol.6'67 In this scheme p-thiocresol was used as the source for the sulfide anion as it is a solid at room temperature and easy to handle. Although the racemic diketone is used in the reaction, the extremely basic conditions employed causes equilibration to the meso diphenol. Thus the racemic and meso diphenols are obtained in good yields and can be separated by chromatography. Reduction of the carbonyl groups with lithium aluminum hydride was attempted but this reaction did not proceed to give the desired compounds and the starting material was completely recovered. This is, perhaps, due to the fact that the phenols get deprotonated to form a resonance stabilized anion, which resists reduction. The diphenols were subjected to acetylation, and reduction was attempted on the diacetates. The reaction proceeds readily. Acidcatalyzed cyclization of the reduction product of the racemic diketone affords galbelgin diol as the major product. The meso diketone, by the same procedure, afforded




71
the galgravin diol. Acid catalyzed cyclization of the diketones gave the furan diol, furoguaiacin, in quantitative yields.
The galgravin diol, galbelgin diol and furoguaiacin were alkylated with 2-bromo- (3', 4'-dimethoxy)propiophenone to afford the dialkylated derivatives of the corresponding diphenols. The alkylated derivatives were reduced with sodium borohydride to afford the desired Manassantin analogs. It must be mentioned that the relative stereochemistry of the side chain was found to be erythro, the threo compound was less than 5% of the products obtained. This was based on the 'H- and 13C-NMR spectra of the final products.
Attempts were made to synthesize the all cis tetrahydrofuran diol by catalytic hydrogenation of the furan diol or its diacetate. The reaction did not give any product and the starting material was recovered without any changes. The method developed by Rao68 for the synthesis of veraguensin (Figure 5-4) was utilized for the synthesis of veraguensin diol but it resulted in a mixture of products and thus it had to be abandoned.
Thus, the synthesis of the desired Manassantin analogs was achieved readily. This is also the first reported synthesis of various phenolic tetrahydrofuran lignans.




72
cOq H HO OH
43 44
OH H 0 OH CH3O OCH3 CH3O OCH3
45 46
0O0 OHHO- (I)( OH H9 ci.o C'O
(H30 0013 CH30 OC13
47 48
CI-130 OCH3
49
Figure 5-1: Various stereoisomers of Tetrahydrofuran lignans




73
OCH3
OCH3
)1)LAH/ THF;
2) TFA/ CH2C2 0
30
OCH3
I)HCI/McOH I)LAH/ THF; C3 t ,. OH
OC309H3OH
Pd/C/H2OH
F e St COf t t
OOCH3 OOCH3
Figure 5-2: Synthesis of totally methylated iignans




74
0
CH3 Propionic acid, PPA/ 70 C CH30
CH30,- v CH30 00
50
0
Bromopropionic acid, PPA/ 70 C H3 CH30
S1
O 0 CH3O CH3O + ~3O 0NaNH2/ Liq NH3
CH3)7 'f + OCH3 rO OCH3 p-Thiocreol, Nail, DMF / reflux CH3 oCH3 CH3 aO
52
OH OH
0 0OOo
0 OCH3 0H3 OCH3
53a
53b
Figure 5-3: Synthesis of various Manassantin analogs




75
OH
C 1 3O0-I HCI/MeOH lo 0i 0 0 HCH3
53a, b
54
O OH 1) Ac2O/Pyridine 2) LAW THF 0 C
H3 'Ot 3) TFA/Be ene \ l
OH
53a H3 55
\ f
0OH 1) Ac2O/Myfidine%
O -2) LAWHFhOC
CH 3) TFA/Benzene O0
H 0Q0 0 O rO 53b 56
Figure 5-3:




76
00 0
51, K2C03 /DMF0
OCH3 CH3O
3 57 OCH3
0 000 0 0
51, K2C03 /DMF 56
OCH3 CH30 OCH3 58 0C1I3
0 0
51, K2C03 /DMF
55
OCH3 CH3O OCH3 59 OCH3
Figure 5-3:




77
OH3
CH3 HOOCH3
NaBH4 /MeOH 0H 57 so 3
60
OCH3
CH3 0 01 HO OCH3
NaBH4 IMeOH CH0 o
9 OCH3 OCH3 61
OCH3
CH3 0.OH
58 NaBH4IM eOH H3 0 58 CH3 62
Figure 5-3:




78
BF3: Et2O Ar Ar i) NaBH4 HO so r. ,O ...... A r
52
Pd/CH2 Ar) o ...Ar me Arms"' o ...mAr Veraguensin Figure 5-4: Attempted synthesis of Veraguensin diol




79
Experimental
All reactions were monitored by TLC to ensure
completion of the reaction. All NMR spectra were obtained on either Gemini 300 or Varian 300 using CDC13 as solvent.
3,4-Dimethoxypropiophenone (propioveratrone) (50). Veratrole 10g, 25 g of polyphosphoric acid and 25 ml of propionic acid were taken in a 250 ml conical flask and heated over a hot water bath (70-80 'C) with occasional shaking. The reaction mixture turns wine red in color and is complete in 1 hour. The reaction mixture was cooled to room temperature and poured in a thin stream on to crushed ice with vigorous stirring. Cold water (200 ml) was added to this mixture and stirred for about 30 minutes and the product that was obtained as a thick precipitate was filtered and dried to give 13 g (92 %) of the product. A sample was crystallized from 1:1 ethyl acetate ligroin.
'H-NMR (8): 1.22, (3 H, t, 7.5 Hz), 2.97, (2H, q, 7.5 Hz), 3.94 (3H, s), 3.95 (3H, s), 6.89 (1H, d, 8.1 Hz), 7.55 (1H, d, 2.1 Hz), 7.59 (1H, d d, 2.1, 8.1 Hz).
13 C-NMR (8) : 8.51(C-9), 31.23(C-8), 55.9(OCH3),
109.96(C-5), 110.14(C-2), 122.45(C-6), 130.14(C-1), 149.0(C3), 153.06(C-4), 199.42(C-7).




80
2-bromo-3', 4'-dimethoxypropiophenone (51). Veratrole 10 g, 30 g of polyphosphoric acid and 20 g of abromopropionic acid were taken in a 250 ml conical flask and heated on a water bath for 30 minutes. The reaction mixture was worked up in a similar manner as that of 50. The crude product was crystallized from ligroin. Yield 13 g (65 %).
'H-NMR (8): 1.89, (3H, d, 7Hz), 3.95, (3H, s) 3.96,
(3H, s), 5.30, (1H, q, 7 Hz), 6.90, (1H, d, 8.1 Hz), 7.51, (1H, d, 2 Hz), 7.67, (1H, d d, 2.0, 8.1Hz).
13C-NMR (8): 20.29 (C-9), 41.18(C-8), 55.98(OCH3), 110.07(C-5), 111.61(C-2), 123.41(C-6), 126.96(C-i), 149.21(C-3), 153.81(C-4), 192.02(C-7).
Racemic 2, 3-Bis (3, 4-dimethoxybenzoyl)butane (52).
Freshly cut sodium metal (0.5g) was added to 50 ml of liquid ammonia and stirred with a magnetic stirrer for 30 minutes. Anhydrous Ferric chloride (50 mg) was added and stirred until all the blue color had disappeared. The solution was stirred for about 30 minutes by then the solution had acquired a grayish color. To this solution was added 2 g of 3,4-Dimethoxypropiophenone (50) in small portions with stirring. 2-bromo-3',4'-dimethoxypropiophenone (51) (3 g) was added in small portions with continuous stirring. The solution was stirred for 1 hour and the reaction mixture was




81
allowed to warm to room temperature. The ammonia was allowed to evaporate and 100 ml of water was added. The mixture was stirred and filtered. The resulting brownish colored solid was purified by column chromatography. The compound was eluted with 5% acetone in benzene as a colorless crystalline solid 3.6g (90 %).
'H-NMR (6): 1.38, (6H, d, 6.6 Hz), 3.97, 4.00, (14H,
s), 6.99, (2H, d, 8.4 Hz), 7.57, (2H, d, 2.0 Hz), 7.79, (2H, d d, 2.0, 8.4 Hz).
13C-NMR (6): 15.98(C-9), 43.20(C-8), 55.90(OCH3), 110.01(C-2), 110.61 (C-5), 123.03(C-6), 129.16(C-1), 148.97(C-3), 153.16(C-4), 202.93(C-7).
Meso and racemic 2,3-Bis(4-Hydroxy 3 methoxybenzoyl)
butane (53b), (53a). Sodium hydride 50% in oil (500mg) was suspended in 10 ml of DMF. To this suspension was added 1.8 g of thiocresol dissolved in 5 ml of DMF with stirring. The stirring was continued for 15 minutes by this time effervescence had ceased. To the resulting mixture was added 0.8 g of Racemic 2,3-Bis (3,4-dimethoxybenzoyl)butane 52 and the reaction mixturewas refluxed for 90 minutes. The reaction was monitored by TLC to ensure that the reaction had gone to completion. The mixture was allowed to cool to room temperature and was poured into 100 ml of cold water.




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The resulting solution was neutralized to pH 7 with 1 N HC1 and extracted twice with 50 ml dichloromethane. The solvent layer was washed water and dried over anhydrous sodium sulfate and concentrated to remove the solvent. A pale golden colored oily material was obtained. This material was eluted from a 20 g silica column using ligroin to remove the unreacted thiocresol and other sulfur containing compounds. Elution with 2% acetone in benzene gave two compounds, 53b (220 mg) and 53a (500 mg), both were obtained as colorless solids. Yield 97% (total phenolic compounds obtained).
'H-NMR (5) 53a: 1.10, (6H, d, 6.3Hz), 3.98, (8H, s),
6.17, (2H, b s, Ar-OH), 6.99, (2H, d, 8.1 Hz), 7.60, (2H, d,
1.8 Hz), 7.70, (2H, d d, 1.8, 8.1Hz).
13C-NMR (5) 53a: 17.65(C-9), 42.82(C-8), 56.07(OCH3), 110.16(C-2), 113.97(C-5), 123.88(C-6), 129.78(C-I), 146.78(C-4), 150.71(C-3), 202.4(C-7).
'H-NMR (6) 53b: 1.30, (6H, d, 7.0Hz), 3.88, (8H, s),
6.94, (2H, d, 8.4 Hz), 7.48, (2H, d, 1.8 Hz), 7.62, (2H, d d, 1.8, 8.4 Hz), 7.77, (2H b s).
13C-NMR (5) 53b: 16.00(C-9), 43.20(C-8), 55.86(OCH3), 110.33(C-2), 113.89(C-5), 123.68(C-6), 128.75(C-1), 146.63(C-4), 150.33(C-3), 202.99(C-7).




83
Acetylation of 53a and 53b. 53a (175 mg) was dissolved in 0.3 ml of acetic anhydride and 3 drops of pyridine. The reaction mixture was heated on a water bath for 15 min. The reaction mixture was cooled to room temperature and 5 ml of cold water was added and stirred for 20 minutes. The resulting solution was neutralized with sodium bicarbonate and extracted twice with 10 ml of dichloromethane. The dichloromethane layer was washed twice with water, dried over anhydrous sodium sulfate and concentrated to dryness. The racemic diacetate was obtained as colorless crystals.
Yield 200 mg (92.5%).
Acetylation of 200 mg of 53b under similar conditions afforded 220 mg of meso diacetate (100%).
'H-NMR (6) racemic diacetate: 1.13, (6H, d, 6.3 Hz),
2.35, (6H, s), 3.92, (6H, s), 4.01, (2H, m), 7.17, (2H, d,
8.0 Hz), 7.66, (2H, d, <1 Hz), 7.69, (2H, d d, <1, 8.0 Hz).
13C-NMR (6) racemic diacetate: 17.55(C-9), 20.65(C-8),
43.22(OC-CH3), 56.06(OCH3), 111.81, 121.81, 122.92, 135.45, 144.07, 151.54, 168.46, 202.34.
'H-NMR (8) meso diacetate: 1.30, (6H, d, 7.0 Hz), 2.33, (6H, s), 3.86, (6H, s), 3.90, (2H, m), 7.15(2H, d, 8.1 Hz),
7.56, (2H, d, 1.5 Hz), 7.67, (2H, d d, 1.5, 8.1 Hz).




84
13C-NMR (8) meso diacetate: 15.66, 20.63(C-8),
43.61(OC-CH3), 55.95(OCH3), 111.98, 121.73, 122.77, 134.71, 143.73, 151.38, 168.46, 203.05.
3, 4-Dimethyl-2, 5-bis (4-hydroxy-3-methoxyphenyl) furan
(54). A mixture of 53a/53b (200 mg) was dissolved in 5ml of methanol. Three drops of concentrated HCl were added and the resulting solution was refluxed for 10 minutes. The solvent was removed and the product crystallized as colorless crystals that slowly turn pale blue in color over a period of time. 190 mg (100%).
'H-NMR (6): 2.19, (6H, s), 3.94, (6H, s), 5.7, (2H, b s), 6.96, (2H, d, 8.4 Hz), 7.16, (2H, d d, 8.4, 1.5 Hz),
7.17, (2H, d, 1.5 Hz).
13C-NMR: 9.83, 55.98, 108.53, 114.47, 117.536, 119.22, 124.59, 144.70, 146.55, 146.95.
All trans 3,4-dimethyl-2, 5-bis(4-hydroxy-3 methoxy phenyl) tetrahydrofuran (55). Racemic diacetate (200 mg) was dissolved in 5 ml of THF and cooled to 00C. This solution was added dropwise over a period of 5 minutes into a suspension of 100 mg LAH in 5 ml of THF, previously cooled to 00C. The reaction mixture was stirred for an additional 15 minutes and allowed to warm to room temperature. The excess LAH was carefully quenched with methanol and




85
neutralized with 0.1 N H2SO4. The resulting mixture was then extracted twice with 10 ml ether. The ether layers were combined and washed with water and dried over anhydrous sodium sulfate and concentrated to dryness. The residue thus obtained was dissolved in 10 ml of dry benzene and treated with about 2 ml of a 3% solution of trifluoroacetic acid in benzene with stirring at room temperature for about 15 min. The reaction mixture was washed with 5% sodium bicarbonate and water to remove the acid. The benzene layer was then dried over anhydrous sodium sulfate and concentrated to dryness. The solid thus obtained was crystallized from ligroin ethyl acetate (1:1) to yield 150 mg (78%) of 55.
'H-NMR (8): 1.04, (6H, d, 6 Hz), 1.77, (2H, m), 3.91, (6H, s), 4.63, (2H, d, 9 Hz), 5.61, (2H, b s), 6.87-6.95 (6H, m).
13C-NMR: 13.81(C-9), 50.97(C-8), 55.92(OCH3), 88.32(C7), 108.48, 113.95, 119.34, 134.27, 145.05, 146.58.
Meso 3, 4-dimethyl-2, 5-bis (4-hydroxy-3 methoxyphenyl) tetrahydrofuran (56). This compound was synthesized by the reduction of meso diacetate with LAH followed by treatment of the reduction product with trifluoroacetic acid in benzene. The procedure was same as that for the synthesis of 55. Meso diacetate (200 mg) affords 145 mg (75.5%) of 7




86
as a colorless glassy semisolid. Attempts to crystallize this compound from various solvents were unsuccessful.
'H-NMR (6): 1.06, (6H, d, 6.6 Hz), 2.35, (2H, m), 3.87, (6H, s), 4.41, (2H, d, 6.3 Hz), 5.8, (2H, br s), 6.93-6.99, (6H, m).
13C-NMR: 12.80, 44.17, 55.753, 87.23, 109.22, 114.12, 119.16, 134.08, 144.99, 146.44.
Alkylation of 54, 55 and 56. 54, 55 and 56 (100mg)
were each dissolved in 5 ml of DMF and treated with 175 mg of 51 and 100 mg of potassium carbonate and stirred for 30 minutes at 70*C. TLC showed that the reaction had gone to completion. The reaction mixture was quenched with 20 ml of cold water and extracted twice with 10 ml of dichloromethane. The dichloromethane layer was washed with water, dried over anhydrous sodium sulfate and concentrated to dryness. The alkylated products in each case were obtained in 90-95% yield
'H-NMR (6) 57: 1.74, (6H, d, 6.6 Hz), 2.15, (6H, s),
3.89, 3.92, 3.94, (18H, s), 5.46, (2H, m, 3.3 Hz), 6.83-7.86 (12H, m).




87
13C-NMR (8) 57: 9.8, 19.21, 55.92, 56.03, 78.26,
110.13, 111.313, 115.79, 118.37, 123.60, 126.37, 127.324, 145.915, 146.78, 149.041, 149.846, 153.69, 197.514.
'H-NMR (8) 59: 1.00, (6H, d, 6Hz), 1.71, (8H, d, 6.6 Hz), 3.86, 3.91, 3.94, (18H, s), 4.56, (2H, d, 8.4 Hz),
5.39, (2H, m, 6.6 Hz), 6.76-7.83, (12H, m).
13C-NMR (8) 59: 13.75, 19.09, 50.79, 55.86, 55.97, 78.17, 87.06, 88.03, 110.054, 110.68, 111.27, 115.50, 118.52, 123.55, 127.29, 136.37, 146.28, 148.92, 149.90, 153.53, 197.58.
'H-NMR (8) 58: 0.98, (6H, d, 4.8Hz), 1.71, (6H, d,
6.6Hz), 2.25, (2H, m), 3.81, 3.92, 3.94, (18H, s), 4.43, (2H, d, 6.9Hz), 5.41, (2H, m), 6.78-7.83, (12H, m).
13C-NMR (8) 58: 12.87, 19.18, 44.20, 55.90, 56.03, 78.351, 87.12, 110.13, 110.77, 111.33, 115.64, 118.58, 123.62, 127.37, 136.25, 146.33, 149.01, 149.85, 153.63, 197.59.
Reduction of 57, 58 and 59. 57, 58 and 59 (100 mg)
were each dissolved in 5 ml of methanol and treated with 50 mg of Sodium borohydride. The reaction mixture was stirred for 15 minutes at room temperature. TLC showed that the reaction had gone to completion. The reaction mixture was concentrated to dryness and dissolved in dichloromethane.




88
The dichloromethane layer was washed with water, dried over anhydrous sodium sulfate and concentrated. The products (60 61 and 62 respectively) in each case were obtained as colorless amorphous solids. Yields were essentially quantitative.
'H-NMR (5) (60): 1.22, (6H, d, 6Hz), 2.24, (6H, s), 3.88, 3.89, 3.95, (18H, s), 4.42, (2H, m), 4.47, (2H, d, 9Hz), 4.90, (2H, s), 6.84-7.26, (12H, m).
13C-NMR (5) (60): 9.86, 13.49, 55.84, 73.77, 82.13, 109.60, 109.75, 110.86, 118.48, 118.77, 119.45, 127.20, 132.51, 145.67, 146.87, 148.22, 148.86, 151.38.
'H-NMR (5) (62): 1.09, (6H, d, 6Hz), 1.18, (6H, d,
6.3Hz), 3.86, 3.89, 3.91, (18H, s), 4.36, (2H, m), 4.69, (2H, 8.7Hz), 4.85, (2H, d), 6.8-7.02, (12H, s).
13C-NMR (5) (62): 13.33, 13.78, 50.93, 55.81, 73.59, 82.22, 88.19, 109.55, 109.85, 110.84, 118.42, 118.98, 119.25, 132.55, 137.45, 146.09, 148.15, 148.80, 151.44.
'H-NMR (6) (61): 1.06, (6H, d, 6Hz), 1.19, (6H, d,
6Hz), 2.40, (2H, m), 3.86, 3.89, (18H, s), 4.37, (2H, m), 4.53, (2H, d, 5.4Hz), 4.85, (2H, d), 6.83-7.02, (12H, m).




89
13C-NMR (8) (61): 12.90, 13.43, 44.31, 55.83, 73.62,
82.20, 87.21, 109.58, 110.50, 110.86,118.45, 119.00, 119.43, 132.55, 137.22, 146.09, 148.18, 148.83, 151.32.




Full Text

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SIMPLIFIED ISOLATION OF LIGNANS FROM SAURURUS CERNUUS AND VARIOUS BIOLOGICAL ACTIVITIES OF NATURAL AND SYNTHETIC LIGNANS By RAVISHANKAR ORUGUNTY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1998

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This work is dedicated to Dr. K. V Rao, my advisor, whose untimely death has left a deep void. I would also like to dedicate this work to my family for their constant support and encouragement.

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ACKNOWLEDGMENTS I would like to thank my advisor Dr. K.V. Rao. Apart from being my research advisor he was a true friend, philosopher and guide whose untimely death has left a void that cannot be filled for a long time to come. He was always available with his helpful suggestions and ideas. Dr. Rao believed in doing experimental work, in order to back up theoretical ideas. He was a tireless worker. I shall always remember the time that I spent in his lab learning the art of natural product chemistry. I would also like to thank the other members of my committee Dr. Perrin, Dr. Schulman, Dr. Sloan, Dr. Tebbett, and Dr. Zoltewicz who were helpful throughout the course of this work. I would like to tak e this opportunity to thank the other faculty members of the Department of Medicinal Chemistry who were truly involved at every step of my graduate studies. I would like to thank Nancy Rosa, Jan Kalmann and my fellow graduate students for their tolerance and encouragement. Special thanks go to my family who have constantly supported me throughout the course of this work and every endeavor that I have taken in my life. w

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TABLE OF CONTENTS ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES ABSTRACT CHAPTERS 1 BIOLOGICAL ACTIVITY OF LIGNANS Introduction Antiplatelet Activity and Platelet Factor Antagonists Cytotoxic and Anticancer Activity of Lignans Lignans with Antiviral Activity Lipoxygenase Inhibitory Activity of Lignans Activating 2 AN IMPROVED METHOD FOR THE ISOLATION OF THE LIGNAN CONSTITUENTS OF SAURURUS CERNUUS BY REVERSE REVERSE PHASE COLUMN CHROMATOGRAPHY Introduction Materials and Methods First Reverse Phase Chromatography Second Chromatography Result and Discussion 3 TOTAL ASSIGNMENT OF THE 13C NMR CHEMICAL SHIFTS OF SOME LIGNANS ISOLATED FROM iii vi vii ix 1 1 2 3 6 10 13 13 16 17 24 32 SAURURUS CERNUUS 39 Introduction Experimental iv 39 41

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4 LIPOXYGENASE INHIBITORY PHENOLIC LIGNANS FROM 5 6 SAURURUS CERNUUS 47 Introduction Materials and Methods Experimental Discussion SYNTHESIS OF STEREOISOMERS OF MANASSANTIN Introduction Experimental CYTOTOXICITY OF MANASSANTIN AND ITS SYNTHETIC ANALOGS 47 58 58 62 68 68 79 90 Introduction 90 Materials and Methods 90 Results and Discussion 93 Conclusions and Further areas for Research 104 LIST OF REFERENCES 107 BIOGRAPHICAL SKETCH 112 V

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LIST OF TABLES Table page 2.1: Elution sequence of Saururus Lignans ................ 34 3.1: Side Chain Carbon assignments ....................... 44 3.2: Aromatic Carbon Assignments ......................... 46 4.1: IC50 values of some of the various non-phenolic and phenolic compounds isolated from Saururus cernuus ..... 56 6.1: L 1210 Cytotoxicity Data of Saururus Lignans ....... 103 VI

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LIST OF FIGURES Figure page 1-1: Lignans with antiplatelet activity ................... 4 1-2: Lignans with antitumor and anticancer activity ....... 5 1-3: Lignans with antiviral activity ...................... 7 Figure 1-4: Lignans with antiviral activity ............... 9 1-5: Lignans with lipoxygenase inhibitory activity ....... 12 2-1: Lignans isolated from Saururus cernuus .............. 20 2-2: Lignans isolated from Saururus cernuus .............. 21 2-3: New lignans isolated from Saururus cernuus .......... 22 2.4: Elution profile of the first reverse phase column ... 23 2-5: Synthesis of Machilin-D Methyl Ether ................ 33 3-1: Oxidation of Manassantin A and B .................... 45 4-1: Biosynthesis of leukotrienes ........................ 48 4-2: Lipoxygenase inhibitory activity of various fractions of Saururus cernuus . . . . . . . . . 5 3 Figure 4-3: LOX inhibition by various phenolic fractions. 54 4-4: Lignans from Saururus cernuus with lipoxygenase inhibitory activity . . . . . . . . 5 5 4-5: Kinetic plot for Machilin-D ......................... 57 4-6: Structures of Magnolidin and Furoguaiacin ........... 64 4-7: Effect of Ferric ammonium sulfate on Magnolidin ..... 65 Vll

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4-8: Effect of Ferric ammonium sulfate on SC-2 Diol ...... 66 4-9: Effect of Ferric ammonium sulfate on SC-6 ........... 67 5-1: Various stereoisomers of Tetrahydrofuran lignans .... 72 5-2: Synthesis of totally methylated lignans ............. 73 5-3: Synthesis of various Manassantin analogs ............ 74 5-4: Attempted synthesis of Veraguensin diol ............. 78 6-1: Saururus lignans tested for cytotoxicity ............ 94 6-2: Saururus lignans tested for cytotoxicity ............ 95 6-3: Synthetic Manassantin derivatives tested for cytotoxici ty .......................................... 9 6 6-4: Synthetic Manassantin analogs tested for cytotoxicity97 6-5: L1210 toxicity of S.C-1, S.C-5 and S.C-6 ............ 98 6-6: L1210 cytotoxicity of S.C-7 and S.C-8 ............... 99 6-7: L1210 cytotoxicity of S.C-7 Diketone and S.C-8 Di ketone . . . . . . . . . . . 1 O O 6-8: L1210 toxicity of Dialkyl Furan, Dialkyl Meso and Dialkyl trans Tetrahydrofurans ....................... 101 6-9:L1210 toxicity of Furan SC-8, Meso SC-8 and Trans SC-8102 Vlll

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Abstract of Dissertation Presented to the Graduate School Of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SIMPLIFIED ISOLATION OF LIGNANS FROM SAURURUS CERNUUS AND VARIOUS BIOLOGICAL ACTIVITIES OF NATURAL AND SYNTHETIC LIGNANS By Ravi Shankar Orugunty August 1998 Chairman: Dr. Koppaka V. Rao Major Department: Medicinal Chemistry Saururus cernuus is an aquatic weed that is found commonly throughout Florida. It was used in folk medicine as a sedative and for the reduction of pain, fever, and inflammation and for a number of other disorders. Dr. Rao systematically investigated this plant for its constituents that were toxic to mice. This led to the isolation of two new neolignans that have neuroleptic activity. The structure of these lignans is unique with regard to their stereochemistry. One aspect of investigation in this work revealed that the phenolic fraction was a potent inhibition of the lipoxidase enzyme. IX

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The phenolic fraction was then further separated into its individual constituents and their inhibitory activity was measured. The compounds showed varying degrees of activity the most potent being Licarin, S.C-2 Diol, Dihydroguaretic acid and Machilin-D ICso of these compounds were found to be in the range of 10-5 M. The earlier reported isolation scheme was protracted and produced lignans that contained varying amounts of color pigments. In order to simplify the process of isolation of these lignans, the reverse phase column chromatography (developed by Dr. Rao) was employed in this study. Thus in two steps a dozen lignans were isolated in high purity. In addition, it was the first time that the yields of these lignans from this plant were ascertained. The total assignment of the 13C NMR of four new lignans (that have been reported so far only by our lab) was accomplished using a high field NMR spectrometer. This required the study of the HETCOR and HMBC spectra of these compounds. In this study the non-phenolic lignans Manassantin A and B turned out to be cytotoxic in the L-1210 cell culture bioassay. This prompted the investigation of the synthetic Manassantin-A derivatives with regard to their cytotoxic effects. Thus a number of synthetic Manassantin analogs with different stereochemistry were generated and evaluated X

PAGE 11

for their cytotoxicity. The all trans Manassantin A analog was comparable in its activity to the natural compound. The furan analog was the least potent. In addition the natural and synthetic diketone compounds turned out to be 50 times less active than their reduced derivatives.

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CHAPTER 1 BIOLOGICAL ACTIVITY OF LIGNANS Introduction Lignans are a class of natural products that have in common the constitutional feature o f at least two C 6 C 3 (phenylpropane) units attached at the central carbon of each side chain. Haworth1 introduced the term lignan to describe such compounds. Lignans are thought to be formed by the oxidative coupling of monomeric C 6 C 3 phenols such as eugenol, coniferyl alcohol, coniferaldehyde and ferulic acid. The mechanism postulated2 f o r this coupling is thought to be free radical in nature and this has been confirmed experimentally. Polymerization of the above mentioned phenols leads to the formation of lignins. Lignins are universally distributed throughout all woody tissues. Lignins3 are thought to provide strength and stability to the cell wall and thus promote the growth of plants. Lignans are found distributed throughout the various genera in the plant kingdom. This is due to the wellestablished fact that the above-mentioned phenols arise from the shikimic acid pathway, which is one of the principal 1

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2 pathways4 of secondary metabolism in plants. The earlier work done (prior to the 1980's) dealt with the isolation, characterization and synthesis of the isolated lignans. Later work dealt with isolation and the study of the wide variety of biological activities that a number of lignans have been shown to have both in vitro and in vivo. A systematic classification of lignans shall be presented with regard to the biological activities that have been reported in the literature. Antiplatelet Activity and Platelet Activating Factor Antagonists Platelet Activating Factor (PAF) is a highly potent phospholipid. PAF has been linked to various biological activities and is now considered as an important mediator of many biological processes such as aggregation and degranulation of platelets and neutrophils. It is also reported to be involved in inflammation, allergic responses, chemotaxis and hypotension. 5 6 Specific PAF receptor sites in rabbit plasma7 membranes and human platelets8 have been reported. The 3,4-Dimethyl-2, 5-bis (3,4-dimethoxyphenyl) tetrahydrofuran groups of lignans are potent PAF antagonists. The antagonist activity was found to be dependent on the stereochemistry of the tetrahydrofuran. The most potent antagonist in this group was found to be the trans-2, 5-bis (3,4,5-trimethoxyphenyl) tetrahydrofuran (L-652, 731, Figure

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3 1-1, 1). This compound had an IC50 of 0.02 Min inhibiting the binding of PAF to receptors in rabbit platelet membranes.9 The stems of Piper puberulum (Piperaceae) has been used in Chinese folk medicine along with other Piper plants for the treatment of asthma and arthritic conditions. Bio-activity based (inhibition of PAF binding to rabbit platelet membranes) fractionation led to the isolation of three new neolignans, Pubulerins A, B, C (Figure 1-1, 2, 3, 4). Pubulerins A and C were found to be potent PAF antagonists1 0 with and IC50 7.3 and 5.7 M respectively. The whole plant of Justica procumbens (Acanthaceae)11 has been used in Chinese herbal medicine for the treatment of fever, pain and cancer. Using the antiplatelet bioassay as a guide, ten lignans were isolated. The most potent of these lignans, neojusticin A, (Figure 1-1, 5) had an ICso of 1.1 M. Cytotoxic and Anticancer Activity of Lignans Podophyllotoxin (Figure 1-2, 6) is a well known lignan that has cytotoxic activity; and its derivatives are currently used in cancer therapy. The mechanism of its activity is based on its ability to bind to microtubules1 2 and cause mitotic arrest in metaphase. In the course of screening tropical plants for Ras function inhibitors Ohse

PAGE 15

1 (L-652, 731) cO"""""~CH3 "'""'"\ H 0 3 0 < 0 4 CHiO CRO 0 5 ,,, y ',,, '''"""" AcO ........... H 0 2 y 1.... ..... 11111 AcO "'"""' H 0 4 Figure 1-1: Lignans with antiplatelet activity

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5 OH H 0 < 0 6 Podophyllotoxin 7 Rocaglaol 8 Aglaiastatin 9 Dehydroaglaiastatin Figure 1-2: Lignans with antitumor and anticancer activity

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6 et al.1 3 isolated three cyclopentabenzofuran lignans, from Aglaia odorata, rocaglaol, aglaiastatin and dehydroaglaiastatin (Figure 1-2, 7, 8, 9) respectively. These compounds inhibited the growth of ras expressing-cell lines. The IC50 of these compounds was on the order of 10 ng/ml. Ras14 is a member of a family of GTP-binding proteins that play an important role in various signaling pathways for cell growth, differentiation and transformation. Activation of the ras proto-oncogene is found in many human neoplasms and is especially common in pancreatic and colon carcinomas. Thus, compounds that inhibit ras functions could lead to potential lead compounds with cytotoxic or anti-cancer activity. Lignans with Antiviral Activity Review of recent literature on lignans shows that many lignans have shown good activity as antiviral agents. The methanolic extract of Anogeissus acuminata (Combretaceae) showed activity against HIV-1 Reverse Transcriptase. Bioassay based fractionation15 led to the isolation of two new dibenzylbutadiene lignans Anolignan A and B (Figure 1-3, 10, 11) respectively. These compounds had an IC50 of 60 and 25 g/ml respectively as HIV-RT inhibitors. Kadsura interior (Schizandraceae), a plant that has been used in

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OH H H H 10 Anolignan A Interiotherin A Interiotherin B 7 H OH H H 11 Anolignan B Schizantherin Figure 1-3: Lignans with antiviral activity

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8 traditional Chinese medicine for the treatment of menstrual irregularities, blood deficiencies, and other feminine disorders, has been previously investigated with the isolation of two important lignans, kadsurin and interiorin. The ethanolic extract of this plant showed significant activity in inhibiting the HIV replication in H9 lymphocytes. This led to the isolation16 of two new dibenzocyclooctadiene lignans, interiotherins A and B along with schisantherin (Figure. 1-3, 12, 13, 14) respectively. Investigation of Phyllanthus myrtifolius (Euphorbiaceae) for HIV-1 Reverse Transcriptase inhibitory activity led to the isolation of two lignans Phyllamycin Band Retrojusticidin B (Figure.1-4, 15, 16) respectively. The IC50 of these compounds on HIV-1 Reverse Transcriptase is in about 3-5 M. Interestingly the IC50 of these compounds on human DNA polymerase17 was 289 and 989 M, respectively. Thus, the selectivity shown by these compounds against the viral enzyme makes them excellent lead molecules. Recent studies with 3'-0-methyl nor-dihydroguaretic acid and nordihydroguaretic acids (Figure 1-4, 17, 18) respectively have shown that these lignans are able to inhibit HIV tatregulated transactivation in vivo. These compounds induced the protection of lymphoblastoid cells from HIV killing and were able to suppress the replication of five HIV-1 strains in mitogen stimulated

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9 0 0 0 'o-1 0 'o-1 15 Phyllamycin B 16 Retrojusticidin OR OH 17 R=H Nordihydroguaretic acid 18 R= Me 3-0-Methyl nordihydroguaretic acid Figure 1-4: Lignans with antiviral activity

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10 peripheral mononuclear cells. The target of these compounds has been localized to nucleotides 87-40 of the HIV long terminal repeat. These compounds are thought to inhibit the binding of Spl to its site in the HIV long terminal repeat. It is therefore suggested that this unique mode of inhibition of proviral expression1 8 will be useful in inhibiting the life cycles of the wild type and RT and protease mutant viruses in HIV-infected patients. Lipoxygenase Inhibitory Activity of Lignans The lipoxygenase are a family of enzymes that contain non-heme iron. These enzymes convert arachidonic acid into leukotrienes. Leukotriene B 4 is a potent chemotactic agent1 9 and is considered to be a mediator of inflammation. The peptidoleukotrienes LTC4 LTD4 LTE4 are powerful spasmogenic agents which are known to be involved in the pathology of many diseases like asthma, inflammatory bowel disease and rheumatoid arthritis. Thus, selective inhibitors of 5-LO could form a new class of therapeutic agents that could be of potential use in the treatment o f such diseases. Investigations for potent 5-LO inhibitors by Ducharme et al.20 led to the identification of naphthalenic lignan lactones (Figure 1-5, 19). Further synthetic modifications lead to certain potent compounds illustrated in Figure 1-4 20. Caffeic acid (Figure 1-5, 21), which is one the simplest lignan found widely distributed in nature, has been

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11 identified to be a very potent inhibitor21 of 5-LO. Synthesis22 of analogs of caffeic acid have lead to some potent inhibitors, one of them being caffeic acid octyl amide. In addition to the above-mentioned activities a number of lignans have been isolated23' 24 which have antimicrobial and hypolipidemic effects. Thus it can be said with reasonable certainty that lignans which are formed by the coupling of the phenyl propanoid moiety give rise to comp ounds with varying structures and stereochemistry and show wide ranging pharmacological activities. The pursuit of these compounds can give rise to potentially useful therapeutic agents.

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12 0 19 20 HO OH 21 Figure 1-5; Lignans with lipoxygenase inhibitory activity

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CHAPTER 2 AN IMPROVED METHOD FOR THE ISOLATION OF THE LIGNAN CONSTITUENTS OF SAURURUS CERNUUS BY REVERSE PHASE COLUMN CHROMATOGRAPHY Introduction The aquatic weed, Saururus cernuus, L (N.O, Saururaceae) which grows mainly in the eastern United States, was known and used during the 19th century in folk medicine25' 2 6 as a sedative, for soothing irritations and inflammations of the kidneys, bladder, prostate gland, urethra and also for its anti-inflammatory activity. A number of terpenes such as limonene and pinene were isolated27 which account for the aromatic property of the plant. A systematic study was undertaken by Rao28-30 that yielded a number of novel lignan and other constituents. The most important of these were the dineolignan type compounds Manassantins A and B31 (Figure 2-1, 22, 23, respectively) which show potent neuroleptic activity. Manassantin A (MNS-A) was evaluated by Rao32 for its central depressant effects using various behavioral parameters in mice with haloperidol as the reference compound. When administered intra-peritoneally, it caused a decrease in spontaneous motor activity and inhibited amphetamine-induced 13

PAGE 25

14 stereotypy with an ED50 of 0.21 mg/Kg. Unlike, haloperidol it did not produce ptosis or catalepsy, which is usually considered as an indicator for the ability to produce extrapyramidal effects in humans. Unlike haloperidol, MNS-A also causes hypothermia. Thus, in this respect, MNS-A behaved as an atypical neuroleptic. The demonstration of neuroleptic activity prompted a study of its affinity for the various receptors, such as the dopamine (Dl, D2 etc.), serotonin and other receptors. Surprisingly, it did not show any strong affinity for these and other receptors. MNS-A also did not have any significant effect33 on dopamineinduced adenylate cyclase activity. These properties suggest that MNS-A may have a different mechanism of action. Thus, MNS-A requires further pharmacological evaluation. The novel dilignan structure of MNS-A is different from that of all known neuroleptic compounds currently in use. In addition, it is unique in not having any nitrogen in its structure. The continued interest in the neuroleptic and other activities found in this plant was one of the main reasons for the reexamination of the isolation process that was used earlier. The objective was to simplify and streamline the process for large-scale applicability. The method used earlier for the isolation of the various lignans started with the methanolic extract of the plant, which was

PAGE 26

15 subjected first to a three-step solvent partition to separate the three fractions: 1) highly lipid-soluble components, 2) moderately lipid-soluble components, and 3) non-solvent extractable components. The neuroleptic activity was found in the moderately lipid-soluble fraction which was then subjected to chromatographic procedures in two to three stages using silica gel and/ or florisil. The close similarity of the twenty or more lignans with regards to their physical properties posed a challenge to their separation. For example, to separate the mixture of MNS-A and B, it was necessary to acetylate the mixture, separate the acetates, and then regenerate the compounds by hydrolysis. Reverse phase column chromatography was used successfully by Rao et al. 34 35 for the fractionation of the crude extracts of Taxus brevifolia, for the isolation of Paclitaxel and several other related taxanes. In an effort to simplify the isolation of lignans from Saururus cernuus, the methanolic extract was partitioned between benzene and water and the benzene extract was directly subjected to reverse phase column chromatography. The fractions thus obtained were purified by one normal phase silica column36 to give pure compounds.

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16 Materials and Methods Plant material The above ground parts of the plant (which were previously identified by the University of Florida Herbarium, where a voucher specimen was submitted) are collected locally, in and around Gainesville, FL, during May through September. The plant material is dried in the sun and stored until needed for extraction. Extraction and Partition The dried plant material was ground to a coarse mesh (0.5-1 cm) and extracted with methanol in a stainless steel drum in 25 lb quantities. The methanolic extract was drained after 24 hours and the extraction was repeated, using absorbance at 275 nm as a guide. The extraction was complete when the absorbance at 275 nm was negligible. In order to ensure complete extraction, the plant material was extracted four times with methanol. The extracts were concentrated under reduced pressure to give a thick green syrup, which was partitioned between water (5 gallons) and benzene (5 gallons). The organic layer was separated and the aqueous layer extracted a second time with benzene (3 gallons). The combined benzene layers that contained the lignan constituents was concentrated to a dark green semi-

PAGE 28

17 solid (5% of the weight of the dried plant). This extract was used in the further chromatographic separations. First Reverse Phase Chromatography A column was set up using about 800 g of C1s bonded silica gel (15-35 micron size, Phase Separations Inc., Norwalk CT) using methanol. A glass column of the MitchellMiller type (2.5" x 25", Ace Glass Co, Vineland, NJ) was used to prepare the column and was found to be suitable for low-pressure liquid chromatography. The column was equilibrated with 40 % aqueous methanol and was ready for further use. The extract (150 g) was dissolved in methanol (450 ml) by warming if necessary. Approximately 150 g of the equilibrated silica gel from the above column was added to this solution with stirring. About 400 ml of 40% aqueous methanol was added to the above slurry with stirring followed by water (600 ml). The mixture was stirred until there was no visible green precipitate or oily material. This was confirmed by taking an aliquot of the slurry in a test tube and allowing the silica t o settle readily to the bottom to give a relatively clear supernatant. The slurry was then filtered using low suction; and the residue of dark green colored silica gel was then re-suspended in about 200 ml of the filtrate. The slurry thus obtained was loaded on to the top of the column. The clear filtrate was then

PAGE 29

18 pumped onto the top of the column using a metering pump (Eldex-Fisher Scientific Co.). The column feed was checked from time to time to ensure that it was clear; if not, the solution was warmed to clarify or minimal amounts of methanol were added. The successful loading of the sample was followed by elution of the column with a step gradient of aqueous methanol (50, 55, 60, 65, 75, 85% methanol). Fractions (200 ml) were collected and monitored by their UV abosrbance at 275 nm and by TLC. These two parameters were used in decisions to change the concentration of the methanol in the solvent. Thus, for example, when the absorbance values rose as a result of increasing the methanol concentration from 50 to 55% the 55% solvent was continued until the absorbance values showed a tendency to reduce. Similar trends were observed on the TLC. In general, 2-4 multiples of the bed volume of the column were used for each eluant mixture. The 85% aqueous methanol eluted out the last of the lignans. This was followed by 100% methanol, which was later changed to a mixture of methanol, ethyl acetate and ligroin (2:1:1). Most of the colored pigments, including chlorophyll, were held up on the column during the run. Eluting the column with 100% methanol and the three-solvent system, consisting of methanol, ethyl acetate and ligroin, completely removed the green pigments. The column was then washed with 100%

PAGE 30

19 methanol and equilibrated with 40 % methanol for regeneration. Considering the UV and TLC data, the fractions were combined into small groups (3-5 fractions) and concentrated and set aside for further work. The combined fractions were examined by TLC to study the number, relative proportions and nature of the lignans present. Some of the major lignan constituents isolated earlier28' 29 from this plant, such as austrobailignan-5 (S.C-1), saucernetin (S.C-2), saucerneol (S.C-6), manassantin A and B, were used as standard markers to orient the other compounds on TLC. The appropriate concentrates obtained from the above fractions were then partitioned using counter current distribution to separate the phenolic from the non-phenolic lignans. The solvent system used was 0.2 N potassium hydroxide in 50% aqueous methanol with benzene, ligroin (1:1) as the organic phase. The aqueous methanolic layers were partially concentrated and neutralized with 0.2 N aqueous HCl and extracted with benzene. The neutral and phenolic fractions thus separated were then subjected to a short normal phase silica column chromatography to afford the purified lignans. The structures of the known lignans isolated are provided in Figure 2-1 and Figure 2-2. In addition four new lignans were isolated and their structures are given in Figure 2-3.

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20 22 R1 R2 = OCH3; S.C-8 23 R1, R2 = OCIJiO ; S.C-7 rAr .. RO~ OCH3 24 R= OH; S C-6 25 R= OCH3; S.C-5 27 S.C-2 OH 26 Guaiacin 28 S C-3 Figure 2-1: Lignans isolated from Saururus cernuus

PAGE 32

21 0 < 0 0 HO 29 S C-1 30 Licarin-A Figure 2-2: Lignans isolated from Saururus cernuus

PAGE 33

rc)l HO~ OCH3 OCH3 31 S.C-2 Diol OH 22 OH 33 Dihydroguaretic acid rc)l HO~ OH OCH3 32 S C-3 Diol OH OCH3 34 threo -Machilin-D Figure 2-3: New lignans isolated from Saururus cernuus

PAGE 34

Q) u 100000 80000 C 60000 ca 0 en .Q c( 40000 20000 23 0 50 100 150 200 250 Fraction Numbers Figure 2-4: Elution profile of the first reverse phase column

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24 Second Chromatography Normal phase column chromatography was carried out using silica gel (Fisher 100-200 mesh or 235-425 mesh). Solvent sequence used was ligroin, benzene, with 5-10% acetone in benzene; alternatively mixtures of ligroin ethyl acetate were also used. All the H1 and C13 NMR were obtained from either a Varian VXR-300 or a Varian Gemini 300 using CDC13 as solvent. The assignments of the carbon were made using COSY, HETCOR and HMBC spectra in the case of most compounds. The spectra were also matched with values reported elsewhere. Characterization of the Major Lignans from S. cernuus TLC of the first 10 fractions did not show any significant compounds. Fractions 11-20 (1.5 g) upon separation, by counter current-distribution, gave 0.6 g of phenolic lignans. Chromatography of this fraction on a silica column (25 g) and elution with 2 % acetone in benzene gave 0.15 g of colorless powder identified as Saucernetin diol (S.C-2 diol Figure 2-3, 31). 1H NMR (8) S.C-2 diol: 0.68, (6 H, d, 6.6 Hz), H-9, 9', 2 2 4 ( 2 H m) H -8 8 3 8 7 ( 6 H s 2 x OM e ) 5 4 2 ( 2 H d, 6 Hz), H-7, 7', 6.75-6.90, (6H, m), H-Ar. The above compound was further characterized by methylation with dimethyl sulfate and potassium carbonate in

PAGE 36

25 refluxing acetone to yield the dimethyl ether, which was crystallized and found to be identical with saucernetin28 (S.C-2, F ig.2-1 27). Fractions 21-30 (1.5 g) yielded 0.7 g of phenolic and 0.8g of non-phenolic material. The phenolic material on column chromatography gave 100 mg of a semi-solid that was identified as Veraguensin diol (S.C-3 diol, Figure.2-3, 32). Its 1H and 1 3C were identical to those reported by SchmediaHirschmann et al. 3 7 and Agrawal et al. 38 1H NMR (8) S.C-3 diol: 0.65ppm, ( 3 H, d, 6.8 Hz), 1.05, (3H, d, 6.6 Hz), 1.79, (lH, m), 2.24, (lH, m), 3.83, (3H, s), 3.88, (3H, s), 4.39 (lH, d, 9 Hz), 5.11 (lH, d, 9 Hz) 6.8-7.0, (6H, m). 13C NMR (8) S.C-3 diol: 14.86 C-9, 9'; 45.89, 47.6, c-8, 8'; 55.75, 2x0Me; 83.05, 87.24, C-7, 7'; 109.4, 109.7, C -2, 2'; 113.9, 114.2, C-5, 5'; 119.2 119.8, C-6, 6'; 132.6, 133.1, C-1, 1'; 144.5, 145.1, C-4, 4'; 146.1, 146.5, C-3, 3 I Fractions 36-50 (6g) upon separation by counter-current distribution yielded 3.3 g of phenolic and 2.5 g of nonphenolic fractions. The non-phenolic fractions after column chromatography on normal silica with benzene gave 1.5 g of Saucernetin (S.C-2, Figure 2-1, 27) and 100 mg of Veraguensin (S.C-3, Figure 2-1, 28).

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26 1H NMR (0) S.C-2: 0.69, (6H, d, 6.6 Hz), 2.27, (2H, m) 3.89, (6H, s), 3.90, (6H, s), 5.45, (2H, d, 6.3 Hz), 6.85, (6H, s). 1 3C NMR (0) S.C-2: 14. 73, C-9, 9'; 44.02, C-8, 8'; 55.88, 2x0Me, 83.51; C-7, 7'; 109.6, 110.8, 118.4, 134.0, 147.9, 148.6, C-Ar. 1H NMR (0) S.C-339: 0.66, (3H, d, 6.6 Hz) 1.07 (3H, d, 6.0 Hz), 1.81, (lH, m) 2.25, (lH, m), 3.86, 3.88, 3.89, 3.91, (12H, s), 4.42 (lH, d, 9.3 Hz), 5.13 (lH, d, 8.4 Hz), 6.85-7.08 (6H, m). 13C NMR (0) S.C-3: 14.95, 15.00, C-9, 9'; 45.98, 47.94, C-8, 8'; 55. 83, 2x0Me; 83. 00, 87. 23, C-7, 7'; 110. 01, 110. 45, 110. 72, 111. 09, 118. 63, 119 .19, 133. 49, 133. 79, 148.06, 148.59, 148.98. The phenolic fractions upon c olumn chromatography gave 115 mg of a pale yellowish semi-solid which was found to be Machilin Dor threo-2-(2-methoxy-4-trans-propenylphenoxy)-1-(4-hydroxy-3-methoxyphenyl)propan-1-ol, previously isolated from Machilus thunbergii4 0 (Figure 2-3, 34). 1H NMR (0) Machilin D: 1.16, (3H, d, 6 Hz), 1.87, (3H, d, d, 6.6, 0.9 Hz), 3.80, (3H, s), 3.88, (3H, s), 4.11, (lH, d, q, 8.4, 6.3 Hz), 4.61, (lH, d, 8.3 Hz), 6.16, (lH, m, 6.6, 15.6 Hz), 6.35, (lH, m 0.9, 15.6 Hz), 6.8-6.9, (6H, m).

PAGE 38

27 13C NMR (8) Machilin D: 16.9, 18.3, C-9, 9'; 78.3, 124.8, C-8, 8'; 83.9, 130.4, C-7, 7'; 120.6, 118. 7, C-6, 6'; 114.1, 118.7, C-5, 5'; 145.4, 150.7, C-4, 4'; 146.6, 146.6, C-3, 3'; 109.14, 109.4, C-2, 2'; 133.3, 131.8, C-1, 1'. The structure of Machilin-D was confirmed by methylation and comparing it with the 1 3 C and 1 H NMR values with those reported in the literature41 and by total synthesis of the methylated compound (as illustrated in Figure 2-5). The spectral data of the synthesized compound were identical to that of the methylated derivative of the natural compound. Elution of the column with 5 % acetone in benzene gave 1.6 g Saucerneol (S.C-6, Figure 2-1, 24). 1 H NMR ( 8) S C-6 : 0 7 ( 3 H, d, 6 Hz ) 0 7 2 ( 3 H, d, 6 Hz), 1.17, (3H, d, 6 Hz), 2.27, 3.92, (12H, s, 4 OCH3's) 4.12, Hz); 5.44, (lH, d, 6 Hz); 5.45, Ar-OH), 6.75-7.0, (9H m). (2H, m), 3.87, 3.88, 3.90, (lH, m); 4.65, (lH, d, 8.1 (lH d, 6 H z), 5.60 (lH, bs, 1 3 C NMR (8) S.C-6: 14.8, 16.9, 44.1, 55.9, 78.4, 83.3, 83.5, 84.0, 108.8, 110.0, 110.1, 110.9, 113.9, 118.7, 119.0, 132.2, 132.6, 136.6, 144.5, 146.4, 148.9, 148.97, 150.5. Fractions 66-75 (2 g) were separated by counter-current distribution into phenolic (lg) and non-phenolic (1 g) fractions. The phenolic fraction on column chromatography gave 400 mg of Saucerneol (S.C-6). The non-phenolic

PAGE 39

28 fraction gave 400 mg of Saucerneol methyl ether (S.C-5, Figure 2-1, 25). 1H NMR (0) S.C-5: 0. 7, (3H, d, 6 Hz), 0. 72, (3H, d, 6 Hz), 1.17, (3H, d, 6 Hz), 2.25, (2H, m), 3.87, 3.88, 3.90, 3.92 (15H, s, 5 OCH3's), 4.13, (lH, m), 4.65, (lH, d, 8.1 Hz), 5.46, (2H, d, 6 Hz), 6.7-7.0, (9H, m). 13C NMR (0) S.C-5: 14.8, 16.9, 44.0, 55.85, 55.85, 78.3, 83.3, 83.4, 83.9, 109.6, 110.0, 110.1, 110.8, 118.3, 118.7, 119.9, 132.6, 133.2, 136.6, 146.6, 147.9, 148.6, 148.8, 148.9, 150.5. Fractions 76-85 (5.8 g) yielded about 3.3 g of nonphenolic and 1.2g of phenolic fractions after countercurrent distribution. Similarly fractions 86-95 (6 0 g) gave 4.0 g of non-phenolic and 1.5g of phenolic fractions after counter-current distribution. The non-phenolic fractions from fractions 76-95, upon column chromatography, gave 3.1 g of S.C-8 (Figure 2-1, 22) and about 100 mg of S.C-5. 1H NMR (0) S.C-8: 0.73, (6H, d, 6 Hz), 1.17, (6H, d, 6 Hz), 2.30, (2H, m), 3.88, 3.89, 3.93, (18H, s, 6 OCH3's), 4.12 (2H, m), 4.65, (2H, d, 8.1 Hz), 5.45, (2H, d, 6 Hz), 6.78-7.00, (12H, m). 13C NMR (0) S.C-8: 14.8, 16.95, 44.13, 55.82, 78.3, 83.3, 83.9, 110.0, 110.8, 118.57, 118.6, 119.92, 132.53, 136.37, 146.4, 148.77, 148.93, 150.48.

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29 The phenolic fractions on chromatography gave 200 mg of dihydroguaretic acid (Figure 2-3, 33)4 2 and 200 mg of Li car in-A ( Figure 2-2, 30) 43 1H NMR (6) Dihydroguaretic acid: 0. 82, (3H d, 6 Hz), 1.72, (lH m), 2.45, (2H m), 3.80, (3H s), 5.49 (lH, bs), 6.52-6.81, (3H, m). 13C NMR ( 6) : 13 8 4, 3 7 4, 41 0 3, 5 5 7 4, 111 3, 113 8, 121.6, 133.54, 143.45, 146.2. Fractions 96-104 gave 1 g each of phenolic and nonphenolic fractions. The non-phenolic fraction on chromatography gave 500 mg of S.C-7 (Figure 2-1, 23). 1H NMR (6) S.C-7: 0.72, (6H, d, 6 Hz), 1.15, (3H, d, 6 Hz), 1.17, (3H, d, 6 Hz), 2.29, (2H, m), 3.87, 3.88, 3.90, 3.92, (12H, s, 4 OCH3's), 4.11, (2H, m), 4.62, (lH, d, 9 Hz) 4.64, (lH, d, 9 Hz), 5.46, (2H, d, 6 Hz), 5.94, (2H, s), 6. 78-7 .00, (12H, m). 13C NMR (6) S.C-7: 14.8, 16.8, 16.9, 44.1, 55.8, 78.3, 83.3, 83.8, 83.9, 100.9, 107.5, 108.0, 110.1, 110.9, 118.6, 118.63, 118.8, 119.9, 121.0, 132.4, 134.0, 136.4, 136.5, 146.3, 146.4, 147.3, 147.66, 148.8, 148.9, 150.50, 150.52. The phenolic fractions gave 200 mg of Guaiacin (Figure 2-1, 26). 1H NMR (6) Guaiacin: 0.84, (3H, d, 6 Hz), 1.06, (3H, d, 6 Hz), 1.50, (lH, m), 1.60, (lH, m), 2.70 (2H, m), 3.33,

PAGE 41

30 (lH, d, 10 Hz), 3.80, (3H, s), 3.82, (3H, s), 6.22, (lH, bs, Ar-OH), 6.53, (lH, bs, Ar-OH), 6.56, (lH, d, < 1 Hz), 6.59, (lH, dd, 8.1, < 1 Hz), 6.79, (lH, d, 8.1 Hz), 7.10, (lH, s), 7.15, (lH, s). 13C NMR (8) Guaiacin: 16.75, 19.57, 35.16, 38.57, 43.22, 53.64, 55.37, 55.50, 110.13, 111.74, 114.18, 115.64, 121.83, 127.42, 132.70, 137.86, 143.40, 143.82, 144.93, 146.69. 1H NMR and 13C NMR data of the isolated Guaiacin was identical to that reported in the literature.43 Fraction 191-205 (30 g) did not contain any phenolic compounds (based on UV analysis) and was thus directly purified by column chromatography. The major compound (15.8 g) eluted with ligroin. 1H and 1 3C NMR spectral data confirmed the structure of this compound to be identical to that of Austerbialignan-5 (S.C-1, Figure 2-2, 29) .42 1H NMR (8) S.C-1: 0.8, (3H, d, 6 Hz), 1.72, lH, m, 2.3-2.57, 2H, m, 5.9, 2H, s, 6.54, (lH, d, d, 7.8, <1 Hz), 6.58, (lH, d,
PAGE 42

31 and 400 mg K2C03 and stirred overnight at room temperature. The reaction mixture was treated with 50 ml of water and extracted twice with 10 ml of diethyl ether. The combined ether layers were washed with water and concentrated to dryness to obtain a colorless semi-solid (35). Yield 512 mg. 1HNMR (0) 35: 1.72, (3H, d, 6.6Hz), 1.83, (3H, d, d, 0.9, 6.3, Hz), 3.85, 3.92, 3.94, (9H, s), 5.40, (q, 6.6 Hz), lH; 6.0, (lH, 15.6, 6.3 Hz), 6.28, (lH, 15.6, 0.9, Hz), 6 7 2-7 8 3, 6H, m. 13C NMR ( 8) 35 : 18 3, 19 2, 5 5 8, 5 5 9, 5 6 0, 7 8 2 6, 109. 45, 110. 04, 111. 24, 115. 74, 118. 55, 123. 59, 124. 32, 127.31, 130.44, 132.44, 132.48, 145.94, 148.95, 149.81, 153.6, 197.65. Reduction of 35 (200 mg) was accomplished by dissolving it in 5 ml THF and cooled to 0C, 100 mg of lithium aluminum hydride was added and stirred for about 1 h. The reaction mixture was warmed to room temperature acidified with lN HCl and extracted twice with ether. The ether layer was washed with water, dried over sodium sulfate and concentrated to dryness to a colorless oily product. The product which had two compounds which were separated by column chromatography using 20% ethyl acetate: ligroin as the mobile phase. The faster compound was erythro (140 mg) and the slower compound was found to be threo (45 mg).

PAGE 43

32 i H NMR (6) (36 erythro): 1.17, (3H, d, 6.3 Hz), 1.88, (3H, d, d, 0. 9, 6.6 Hz), 3. 8 6, 3.88, 3. 8 9, (9H, s, OCH3), 4.34, (lH m), 4.84, (lH, d, 3. 3 Hz) 6.15, ( lH, m, 15. 9, Hz), 6.35, ( lH, m, 15. 9, 0. 9 Hz) 6.8-6.9, (6H, A r-H). 13C NMR (6) ( 36 erythro) : 13.4, 18.4, 55.8, 73.46, 82.45, 109.2, 109.4, 110. 7, 118.4, 118.96, 119.3, 119.84, 124.99, 130.4, 132.45, 133.7, 145.7, 148.2, 148.9, 151.5. 6.6 1 H NMR (6) (36 threo): 1.16, (3H, d, 6 Hz), 1.87, (3H, d, d, 6, 0.9 Hz), 3.87, 3.88, 3.91, 9H, s, 4.11, (lH, d q, 6, 8.4 Hz), 4.63, (lH, d, 8.4 Hz), 6.18, (lH, m, 15.6, 6.0 Hz), 6.35, (lH, m, 15.6, 0.9 Hz), 6.81-6.94, (6H, m). 1 3 C NMR (6) (36 threo): 16.7, 18.3, 55.8, 78.3, 84.01, 109.08, 109.9, 110.77, 118.7, 118.9, 119.92, 124.8, 130.37, 132.47, 133.38, 142.62, 148.75, 148.92, 150.69. Result and Discussion The utilization of reverse phase column chromatography did indeed simplify the process of isolation of these lignans as compared to the previously used methods of purification. It may be noted that a total of nearly 11 lignans were isolated of which four have been isolated for the first time from this plant. The elution of these lignans started with 50% aqueous methanol and continued up until 85 % aqueous methanol. The major components are S.C-1 and S.C-8.

PAGE 44

33 OH Isoeugenol Dimethoxybromopropiophenone 0 OH LAH/THFOC 0 0 35 36 Erythro + threo Figure 2-5: Synthesis of Machilin-D Methyl Ether

PAGE 45

34 Table 2.1: Elution sequence of Saururus Lignans Compound Yield 1 S.C-2 Diol 0.15 g 2 S.C-3 Diol 0.1 g 3 S.C-2 1.5 g 4 S C-3 0.1 g 5 Machilin-D 0 1 g 6 S.C-6 1 6 g 7 S.C-5 0 5 g 8 S C-8 3 1 g 9 Dihydroguaret i c acid 0.2 g 10 Licarin-A 0.2 g 11 S.C-7 0 5 g 12 Guaiacin 0 2 g 13 S.C-1 15 g

PAGE 46

35 The elution profile of the reverse phase column monitored at 275 nm is shown in Figure 2-4. The elution sequence and the yields of the various lignans isolated are given in Table 2-1. The order of elution of the compounds from the reverse phase column is interesting in that it does not seem to correlate entirely with either the polarity or molecular weight. On normal phase TLC austrobailignan-5 (S.C-1) is the most lipophilic with R t =1 (ligroin benzene 1:1 solvent system). Licarin, guaiacin, saucernetin (S.C-2) and veraguensin (S.C-3) are compounds that have R t S ranging from 0.8-0.6 in 5 % acetone in benzene and are intermediate in their polarity. Significantly polar are the sesqui and dilignans S.C-5, S.C-6, S.C-7 and S.C-8 with R t S of 0.6-0.3 in 10% acetone in benzene. One would have expected the most polar compounds to elute first from the reverse phase column followed by the compounds with intermediate polarity followed by the least polar compounds. However, surprisingly the compounds with intermediate polarity such as saucernetin, veraguensin and their corresponding diols eluted first these were then followed by the most polar compounds, S.C-5, S.C-6, S.C-7 and S.C-8, then followed by guaiacin, licarin and austrobailignan-5. The reverse phase process described here clearly has some advantages over the normal phase chromatography used earlier. Firstly, it involves fewer steps than were used

PAGE 47

36 before. Instead of having two to three solvent partitions only one partition between water and benzene was used to remove water-soluble materials. Application of the concentrated benzene extract eliminated the need for handling the lignans in three subgroups. In spite of applying the complex mixture directly to the C1 8 silica directly, the resolution was satisfactory. The colored pigments such as chlorophylls and carotenoids were held up by the column and did not elute with the desired compounds. Thus all the lignans were isolated as either crystalline or non-crystalline homogeneous colorless solids. It must be noted that the separation of phenolic lignans from the nonphenolic lignans was carried out at a later stage using a counter current distribution of aqueous methanolic KOH against ligroin benzene because the phenolic lignans did not partition into aqueous KOH from very polar solvents like dichloromethane. The utilization of a non-polar organic solvent system, (e.g., benzene ligroin, 1:1) lowers the solubility of the ionized phenols in the organic phase; whereas the addition of methanol in aqueous base increases the solubility of the ionized phenols in the aqueous phase. One of the most important advantages of the new procedure is its adaptability to larger scale operations as was shown by Rao et al., in the isolation of paclitaxel from the crude extracts of Taxus brevifolia. The solvents used

PAGE 48

37 are methanol, aqueous methanol mixtures; that are relatively inexpensive compared to organic solvents. The high cost of the reverse phase silica is offset by the fact that it can be readily and repeatedly regenerated. Lastly, unlike the case with a normal phase silica or florisil columns, on a reverse phase column, none of the components of the extract are lost due to irreversible adsorption. The most important part in the whole process is the preparation of the sample for the reverse phase column. In most applications reported in the literature the preparative reverse phase chromatography is used as the penultimate or ultimate step in the purification, the sample is in a high degree of purity. The conditions employed in the operation are those that are typically found in analytical HPLC (i.e., high pressure (1000 psi) and low solvent flow rates) and the ratio of sample to the amount of silica is 1:500 or more. In the current process, the reverse phase column is the first step of the purification scheme. The crude extract is loaded directly on to the column. The ratio of crude extract to the sample is 1:5 and the pressure used for running the column is about 50 psi and rarely exceeds 100 psi. The crude mixture is highly lipid soluble in nature and hence is not soluble in 40 % aqueous methanol. It must be noted that the sample cannot be directly loaded on to the column as it can block the column causing considerable

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38 difficulties at later stages. However, if the sample is prepared as outlined in the Materials and Methods the C1 8 silica seems to act as a lipophilic solvent and absorbs the sample so that no free oily or wa x y material is left after the sample preparation. This gives rise to a free flowing slurry and the column performs as though a soluble sample has been applied.

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CHAPTER 3 TOTAL ASSIGNMENT OF THE 1 3C NMR CHEMICAL SHIFTS OF SOME LIGNANS ISOLATED FROM SAURURUS CERNUUS. Introduction During the course of investigation of Saururus cernuus a number of lignans were isolated. Most of these lignans were tetrahydrofurans including S.C-5, S.C-6 and Mannasantin A and B (Figure 3-1: 37, 38, 39, 40)29 respectively that possess a unique stereochemistry. Mannasantin A and B were found3 2 to have a very potent neuroleptic activity. The phenolic lignans isolated from this plant showed significant activity as lipoxygenase inhibitors as will be reported in Chapter 4. The wide variety of pharmacological activities shown by a number of different compounds led us to further investigate Saururus cernuus. In order to improve the isolation of these lignans the reverse phase column chromatographic method developed by Rao et al 34 '35 was used and these lignans were isolated in good yields in a facile manner. Investigation of the literature with regards to spectroscopic data on such compounds revealed that tetrahydrofuran lignans with such unique stereochemistry has never been reported. An excellent review with regards to 39

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40 the 13C NMR shifts of various types of lignans has been reported 3 8 43 However, this review deals only with the 13C NMR shifts of the three side chain carbons and does not mention compounds with this stereochemistry. The paucity of spectroscopic data for these compounds led us to undertake a NMR investigation in order to provide complete carbon assignments for these compounds. The Neuroleptic compounds Mannasantin A and B were oxidized to their respective diketones (41, 42) with Mn02 These compounds served the twin purpose of giving related analogs and also to see whether the introduction of a carbonyl group would spread the chemical shifts of the aromatic protons. It was observed that the chemical shifts of the side chain aromatic protons that are adjacent to the carbonyl group moved downfield spreading over 6.8 ppm to 7.83 ppm. The splitting patterns were much more resolved as compared to the parent compounds that have a very poorly resolved spectrum in the aromatic region. These diketones thus aided in the assignment of the carbon chemical shifts. The proton and carbon resonances of these compounds were then assigned on the basis of 1H-1H COSY and 1H-13C HETCOR and HMBC. The 1H data is provided in the experimental section. The completely assigned 13C data is provided in Table 3-1 and Table 3-2.

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41 Experimental The isolation of S.C-5, S.C-6, S.C-7 and S.C-8 using reverse phase column chromatography has been reported36 elsewhere. NMR Spectra All NMR spectra were obtained on Gemini 300 spectrometer with a 1H/13C multinuclear computer switchable probe (13c 75.4 Mz, pulse width 15.9 sec). Samples contained 90-100 mg of a specific compound dissolved in approximately 1 ml of CDC13 with 0.02% of TMS as internal standard set to 0.00 ppm for the proton spectrum. The carbon spectrum was referenced using the CDC13 signal set to 77.0 ppm. Partial assignments were made with 1H and 13C spectra; complete assignments were made with the aid of COSY, HETCOR and Long Range HETCOR. The spectral windows for 1H and 13C (in the case of HETCOR and Long Range HETCOR) were set to 3000 and 15,000 Hz respectively, 2048 13C data points, Dl delay was set to 1.000s. 128-256 1H data points were collected. The total number of transients collected for the HETCOR were 128 and in the case of Long Range HETCOR it was 256. Jlxh was set at 140 Hz and JnxH was set to O Hz for HETCOR and in the case of LRHETCOR the JnxH was set to 6 Hz. 1 H -NMR ( 0) S C-5 ( 3 7 ) : 0 7 ( 3 H, d, 6 Hz ) 0 7 2 ( 3 H, d, 6 Hz), 1.17, (3H, d, 6 Hz), 2.25, (2H, m), 3.87, 3.88, 3.90,

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42 3.92 (15H, s, 5 OCH3'S) 4.13, 5.46, (2H, d, 6 Hz); 6. 7-7 .O, (lH, m), 4.65, (9H,m). (lH, d, 8.1 Hz) 1H-NMR (0) S.C-6 (38): 0. 7, (3H, d, 6 Hz), 0. 72, (3H, d, 6 Hz), 1.17, (3H, d, 6 Hz), 2.27, 3.92, (12H, s, 4 OCH3's), 4.12, (2H, m), 3.87, 3.88, 3.90, (lH, m), 4.65, (lH, d, 8.1 Hz), 5.44, (lH, d, 6 Hz), 5.45, (lH, d, 6 Hz), 5. 60, lH, bs, Ar-OH, 6. 75-7 .0, (9H, m). 1H-NMR (0) S.C-7 (39): O. 72, (6H, d, 6 Hz), 1.15, (3H, d, 6 Hz), 1.17, (3H, d, 6 Hz), 2.29, 3. 92, (12H, s, 4 OCH3 s), 4 .11, Hz), 4.64, (lH, d, 9 Hz), 5.46, s), 6.78-7.00, (12H, m). (2H, m), 3.87, 3.88, 3.90, (2H, m), 4.62, (lH, d, 9 (2 H d, 6Hz), 5.94, (2H, 1H-NMR (0) S.C-8 (40): 0.73, (6H, d, 6Hz), 1.17, (6H, d, 6 Hz), 2.30, (2H, m), 3.88, 3.89, 3.93, (18H, s, 6 OCH3's), 4.12, (2H, m), 4.65, (2H, d, 8.1 Hz), 5.45, (2H, d, 6Hz), 6.78-7.00, (12H, m). Oxidation of Mannasantin A and Mannasantin B: 100 mg of either (39) or (40) was dissolved in 10 ml benzene. To this solution was added 500 mg of activated Mn0 2 The mixture was refluxed for one and a half-hour. By then the reaction had gone to completion. This was observed by the disappearance of the starting material with the production of a single product on TLC. The reaction mixture was filtered through celite and concentrated to dryness. The crude diketone was eluted from a silica column and eluted

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43 with 5 % acetone in benzene. S.C-7 yielded 90 mg of its corresponding diketone and S.C-8 yielded 93 mg of its corresponding diketone diketone. 1H-NMR (0) S.C-7-Diketone (42): 0.62, (6H, d, 6 Hz), 1.67, (3H, d, 6 Hz), 1. 71, (3H, d, 6 Hz), 2.20, (2H, m), 3.84, 3.92, 3.94, (12H, s), 5.35, (2H, d, 6 Hz); 5.39, (2H, q, 6 Hz) 6. 67, ( 2H, d, 8. 1 Hz) 6. 7 6, ( 2H, d, d, 8. 1, 1. 5 Hz) 6.83, (4H, d, 8.4 Hz), 7.60, (lH, d, 1 Hz), 7.78, (lH, d, d 8.4, 1 Hz), 7.83, (lH, d, d 8.4, 1 Hz). 1H-NMR (o) S.C-8 Diketone (41): 0.62, (6H, d, 6 Hz), 1. 71, (6H, d, 6 Hz), 2.20, (2H, m), 3.84, 3.92, 3.94, (18H, s), 5.35, (2H, d, 6 Hz); 5.39, (2H, q, 6 Hz), 6.66, (2H, d, d, 8.4,
PAGE 55

44 Table 3.1. Side Chain Carbon assignments 7 7' 8 8' 9 9' 7" 7"' 8" 8"' 9" 9"' OMe 1 83.4 44 0 14. 8 78 3 83. 9 16. 9 55. 9 83. 3 44 0 14. 8 2 83. 5 44.1 14. 8 78.4 84 0 16. 9 55. 9 83. 3 ,44 1 14. 8 3 83. 8 44 1 14. 8 78.3 83.3 16.8, 55. 8 100 9 83. 9 ,44.1 14.8 78 3 83. 3 16. 9 4 83. 3 44.1 14.8 78 3 83. 9 16. 9 55. 8 83. 3 44 1 14 8 78 3 83. 9 16. 9 5 83. 3 43.9, 14 6 197 .1, 78.2 18. 8 55. 8 101.7 83. 3 43. 9 14.6 197 6 78.3 19. 0 6 83. 3, 44 0 14.7, 197 7 78. 3 19.2, 55. 9 83. 3 44.0 14.7 197 7 78. 3 19. 2

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A o RO~ OCH.J 45 0 OCH.J 37 R=OCH3 S. C 5 38 R=OH S C 6 OHrAl ... o~"'" o OCH.J 0 OCI-1.J 39 R1 R2 = OCH3 S. C-8 40 R1, R2 = OCH20 S C-7 0 rAl ... o~"'" o OCH.J 0 0 OCI-1.J 41 S. C-8 Diketone 42 S. C-7 Diketone OCH.J OCH.J OCH.J OCH.J OCH.J OCH.J Figure 3-1: Oxidation of Manassantin A and B

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Table 3.2: Aromatic Carbon Assignments C # 1,1' 2,2' 3,3' 4,4 5,5' 6,6' 1",1" 2" ,2" 3",3" 4",4 5",5" 6",6" com 1 136 6 109 6 148 6 146.4 118 3 118.7 132.6 110 0 150 5 148 96 110 8 119 9 133. 8 110 1 148 8 147 9 110 8 118 7 2 I 132.6 108 8 146.4 144 5 113. 9 118 7 133. 2 110 0 150 5 148 97 110 9 136 6 110 1 148 9 146.4 119.0 118 7 3 I 132.4 107 5 148 8 146 3 119 9 118 6 134 0 110 1 147 3 147 66 110 1 118 .63 136.4 108 0 148 9 146.4 121.0 118 6 136 5 110 9 150 5 150 52 110 1 118 8 .i::. 4 I 136 37 110 0 148 7 146.4 118 6 118 57 132.5 110 0 150 5 1 48 .93 110 8 119 9 0\ ----5 I 135.4 110.4 147 98 145.7 118.4 115 5 128 2 108 8 149 5 151.9 107 8 123. 5 135. 6 110.4 148 89 145. 6 118.4 115. 96 128 2 111.2 149 7 153. 5 110 0 125. 3 6 I 135.45 110 0 149 5 145. 5 115. 5 118.42 128 3 111.2 148 9 153. 5 110.4 123. 62

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CHAPTER 4 LIPOXYGENASE INHIBITORY PHENOLIC LIGNANS FROM SAURURUS CERNUUS Introduction The lipoxygenases are an important class of non-heme iron containing enzymes found in plants and animals. These enzymes catalyze the dioxygenation of polyeneoic fatty acids that contain at least a 1,4-cis pentadiene structural unit.44 They are usually classified as 5-, 12-and 15-lipoxygenases based on the position of the double bond of the substrate where the oxidation takes place. The animal and plant enzymes have an overall 60 % sequence similarity, and the human lipoxygenase is about 25% identical to plant ( soybean) lipoxygenases. 45 Oxidation of arachidonic acid by 5-lipoxygenase forms the 5-hydroperoxyeicosatetraenoic acid (5-HETE), which is then converted to leukotriene (LT~), an epoxide. LT~ can be further metabolized by two separate enzymatic routes to yield other leukotrienes-LTB4 LTC4 LTD4 LTE 4 (Figure 4-1). These compounds are involved in various inflammation processes like increased vascular permeability, contraction of smooth muscles in bronchioles, and edema. 46 47 48 47

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LTB4 / COOH 48 Arachidonic acid Lipoxygenase OOH 5-HPETE LTA4 Figure 4-1: Biosynthesis of leukotrienes (X)()H

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49 It is now well acknowledged that these compounds may be invol ved49-52 in a number of allergic processes such as asthma. Leukotrienes are also thought to involved in diseases states such as psoriasis53' 54 and myocardial infarction. 55 Thus inhibitors of these enzymes can be promising candidates for the treatment of some of the above mentioned pathological problems. A number of inhibitors of these enzymes and antagonists are currently being studied as antiasthmatics and anti-inflammatory agents.59 Saururus cernuus has a rich history in folk medicine. This plant was used as a sedative, for soothing irritations and inflammations of the kidneys, urinary bladder, prostate, and urethra. 25' 26 Investigation of this plant by Rao28-30 lead to the isolation of potent neuroleptic lignans Manassantin A and B. The isolation and assignment of 13C-NMR has been dealt with in detail in Chapters 2 and 3. Further investigation of the Saururus e xtract (Chapter 2, Materials and Methods, page 16) showed that the phenolic fraction of the benzene extract inhibited the soybean lipoxygenase enzyme whereas the non-phenolic fraction did not show any significant activity. Soybean lipoxygenase was found to be inhibited by a number of well known non-steroidal antiinflammatory agents (NSAIDS). Nor-dihydroguaiaretic acid (NDGA), a lignan derivative, is one of the most potent

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50 inhibitors (Sircar et al.) .5 6 The soybean lipoxygenase has a similar action on unsaturated fatty acids and the bioassay is easy to perform. In addition this enzyme is commercially available and is inexpensive. The linoleic acid used as the substrate is converted to 13-hydroperoxylinoleic acid, which can be determined by measuring the absorbance at 234 nm. However, the plant extract of Saururus, showed a strong absorbance at 234 nm and so caused interference in the direct measurement of the hydroperoxide at 234 nm. An alternative method was employed that used the oxidation of benzoyl leucomethylene blue to methylene blue by the hydroperoxide, formed in the enzymatic reaction, and measuring the absorbance at 660 nm without any interference from the plant extract.57 The benzene extract (Chapter 2, Materials and Methods, page 16) was partitioned into its phenolic and non-phenolic fractions. The benzene extract, the phenolic and nonphenolic fractions were assayed for their activity (Figure 4-2). The phenolic fraction was then subjected to normal phase silica column chromatography and the fractions obtained were tested for their activity (Figure 4-3). TLC was used to establish the identity of the compounds present in various fractions. Compounds that were previously isolated and characterized were employed as references. The pure compounds, shown in Figure, 4-4 were tested in the

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51 concentration range of 100-6.25 g/ml using the UV method (described in the experimental section). Furoguaicin (Figure 4-6: 2) a known inhibitor of this enzyme58 was used as the standard to compare the activities of these compounds. The kinetic plot of Machilin-D, one of the more potent phenolic lignan, is given in Figure 4-5. The ICso of the phenolic compounds that were tested are given in Table 4-1. Most of the known lipoxygenase inhibitors are classified as antioxidants, chelators and non-redox inhibitors.59 Since most of the isolated phenolic compounds showed good activity as inhibitors it was neccesary to find out if these compounds had any antioxidant or chelating properties. Chelation by metals is known to cause a significant change in the UV spectrum of many compounds and is well documented in the case of flavonoid type compounds.60 The iron present in lipoxygenase is thought to be chelated by certain j _nhibitors. It was decided that the in vitro effect of iron on these compounds should be studied. Thus increasing amounts of a standard s olution of ferric ammonium sulfate was added to a solution of S.C-6 and S.C-2 diol and the UV spectrum of these compounds were recorded. It is well known that the 3,4-dihydroxyphenyl system is capable of chelating metals like iron and copper. Thus, Magnolidin (Figure 4-6: 1), a compound that is structurally similar to

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52 ethyl caffeate (3,4-dihydroxy cinnamic acid ethyl ester) and NDGA, was also used as a control. Magnolidin is the major glycoside of Magnolia grandiflora isolated by Rao et al.61 Magnolidin has a strong inhibitory effect on lipoxygenase ( ICso= 5 M)

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100 90 80 70 60 % Inhibition 50 40 30 20 1000 53 500 Concentration (ppm) 250 100 Benzene CJ non-phenolic IJphenolic phenolic non-phenolic Fraction Figure 4-2: Lipoxygenase inhibitory activity of various fractions of Saururus cernuus

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80 70 60 50 C .S! :t:: :e 40 &: .5 30 20 10 0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Fraction Numbers 19 20 21 Figure 4-3: LOX inhibition by various phenolic fractions 22 23 24 25 V,

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HO HO o ........ OH CH3 S C-2 Diol 0 .... CH3 S.C-6 0 Machilin D 55 OH S C-3 Diol CH30 HO OCH3 Dih y droguaretic acid CH30 HO Guaiacin 0 Licarin Figure 4-4: Lignans from Saururus cernuus with lipoxygenase inhibitory activity

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56 Table 4.1: IC50 values of some of the various non-phenolic and phenolic compounds isolated from Saururus cernuus Compound ICso (Mx102 ) 1 Crude phenolic mixture -110 2 Saucernetin dial 5 3 Veraguensin dial 30 4 Saucernetin 17 5 Magnolidin 3 6 Dihydroguaiaretic acid 4.5 7 Guaiacin 12 8 Licarin 5 9 Machilin-D 5 10 Furoguaiacin 2.5 11 S .C-1 >SO 12 Manassantin A >50 13 Manassantin B >SO

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" u C Ill .a ... 0 Ill .a c( 1.6 ------------------------------------+-Blank 1.4 -so g/ml -6-25 g/ml 1 2 --M-12.5 g/ml -6 g/ml -+-3 g/ml 0 8 0 6 0.4 0 2 0 t 50 100 150 200 250 300 3 -0 2 Time (sec) Figure 4-5: Kinetic plot for Machilin-D VI -....)

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58 Materials and Methods Materials: The following chemicals were purchased and used as received: linoleic acid (Sigma), dimethylformamide (Fisher), Triton X-100 (Sigma), bovine hemoglobin, lipoxygenase (ICN). Benzoyl leucomethylene blue was readily prepared by the reductive benzoylation 62 of methylene blue (1 g) using excess of sodium dithionite, sodium hydroxide and benzoyl chloride in a mixture of 1:1 tetrahydrofuran and water. The reaction mixture was stirred at room temperature for about 2 hours after which the water layer was separated. The THF layer was washed twice with water and dried over anhydrous sodium sulfate and concentrated to dryness. The product (700 mg) was crystallized from a mixture of 1:1 ligroin ether as very pale blue crystals. The 1H-NMR was obtained to ascertain the identity of the compound. All other chemicals and solvents were purchased from Aldrich, Sigma or Fischer Co. Experimental Determination of Lipoxygenase Activity by UV Method The enzymatic reaction was monitored using a Shimadzu UV-visible spectrophotmeter at room temperature. All solutions were freshly prepared prior to assays. Linoleic acid solution 100 M was prepared by dissolving 15 mg of linoleic acid in 10 ml of a pH 9.0, 0.1 M tris buffer

PAGE 70

59 (prepared using the procedure outlined in CRC Manual 63) to obtain a stock solution. This stock solution (2 ml, equivalent to 3 mg of linoleic acid) was diluted to 100 ml using 0.1 M tris buffer. The lipoxygenase enzyme was prepared by dissolving 1 mg of the enzyme (137200 units/mg of protein) in 10 ml of 0.1-M tris buffer. Inhibitors were dissolved in a mixture of propylene glycol and ethanol such that an aliquot of each yielded a final concentration of 4.2% propylene glycol and 2 % ethanol in each assay. The assay was carried out by using 2 ml of 100 M linoleic acid to which 50 l of the enzyme solution was added to obtain an easily measurable initial rate of reaction.59 The progress of the reaction was monitored at 234 nm. The effect of the inhibitors on the reaction was studied by adding the inhibitor solutions to the linoleic acid solution prior to the addition of the enzyme. The production of the hydroperoxide was compared against controls under identical conditions. The substrate concentration in each case was 100 M. Each compound was tested four times at each concentration and the mean values are reported.

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60 Determination of Lipoxygenase Activity by Benzoyl Leucomethylene Blue Method Linoleic acid solution, (1.2 ml, 100 M) was taken and treated with 50 l of enzyme as in the above method. The reaction mixture was incubated at room temperature for 10 minutes and then quenched with leucomethylene blue solution5 7 The only change that was made in this reagent was Succinate buffer (pH 5.0) was employed instead of phosphate buffer saline solution. This change did not effect the assay. This was observed by performing control experiments that employed PBS and Succinate buffer. The absorbance of each of the solutions was then observed at 660 nm after 5 minutes. The plant extract or samples to be tested were prepared as in the UV method and incubated with the linoleic acid solution and then treated with the enzyme. The reaction mixture was then quenched with the leucomethylene blue solution and the absorbance was measured at 660 nm after a period of 5 minutes. Each experiment was carried out in triplicate and the mean values are reported (Table: 4-1). Determination of Activity in the Phenolic Fraction The crude benzene extract was partitioned between a mixture of 1:1 benzene: ligroin and 0.2 N KOH in 50% aqueous MeOH. These conditions were found to be ideal to separate the phenolic from the non-phenolic lignans. The aqueous

PAGE 72

61 methanolic layer was diluted with an equal volume of water, neutralized with 0.2 N aqueous HCl, and extracted with dichloromethane. The dichlormethane layer was washed with water dried over anhydrous sodium sulfate and concentrated to dryness to yield the phenolic layer. The benzene: ligroin layer that contained the non-phenolic compounds was washed with water and dried over anhydrous sodium sulfate and concentrated to dryness. The activity of the crude benzene extract, the phenolic and the non-phenolic extract was ascertained by the leucomethylene blue method. About 500 mg of the phenolic fraction was charged on to a silica column and eluted using 1:1 benzene ligroin, 3:1 benzene/ligroin, benzene, 2, 5 and 10% acetone in benzene and 5 % methanol in benzene. The solvent was evaporated from the fractions, they were reconstituted in about 1 ml of ethanol, propylene glycol (1:1) and tested for their inhibitory activity. The activity of the various fractions is shown in Figure 4-3. Effects of Iron on the UV Spectrum of Saucerneol and Saucernetin diol The following solutions were prepared in water or methanol depending on the solubility of the compounds. 1) Ferric ammonium sulfate (0.002 M) solution in water. 2) Magnolidin (0.5 mg/10 ml) dissolved in water. 3) S.C-6 (0.4 mg/10 ml) dissolved in methanol.

PAGE 73

62 4) S.C-2 diol (0.4 mg/10 ml) dissolved in methanol. The effect of ferric ammonium sulfate was studied as follows. One ml of Magnolidin solution was carefully transferred into an UV cuvette. The UV spectrum of the solution was recorded. Ferric ammonium sulfate solutions (50, 100, 150, 200 and 250 l of 0.002 M) were successively added to the solution of Magnolidin. The solution was shaken and the UV spectrum was recorded after each addition. The experiment was repeated in a similar manner using solutions of S.C-6 and S.C-2 Diol. All the spectra were recorded using a Shimadzu UV-Visible spectrophotometer. Discussion The total phenolic fraction obtained from the original extract showed modest activity whereas the original benzene extract and non-phenolic fraction did not have any activity (Figure 4-2). The above experiments have established that the phenolic fraction of Saururus cernuus is responsible for the lipoxygenase inhibitory activity. The IC50 of the phenolic compounds are comparable to that of furoguaicin a known inhibitor of this class of enzymes. It is also interesting to note that veraguensin diol is a much weaker inhibitor than S.C-2 diol. This suggests that the stereochemistry of the tetrahydrofuran lignans plays an important role in the inhibition of this enzyme. However,

PAGE 74

63 this observation needs to be clarified by conducting further experiments. An interesting observation is that the non-phenolic compounds (S.C-1, S.C-5, S.C-7 and S.C-8) do not have any significant activity at concentrations of 100 g/ml. This clearly shows that the lipoxygenase inhibitory activity is exclusively associated with the phenolic lignans. Ferric ammonium sulfate did show a shift in the UV spectrum of Magnolidin but it did not effect those of S.C-6 and S.C-diol. The results of this experiment are shown in Figures 4-7, 4-8 and 4-9. Thus, the lipoxygenase inhibitory activity of these compounds is not mediated via chelation. Thus, these compounds become very interesting lead molecules and further investigations are warranted with regards to their mechanism of action. Based on the above results it can be concluded that the phenolic lignans isolated from Saururus cernuus have potent soybean lipoxygenase inhibitory activity. Further experiments need to been performed using mammalian enzymes. These compounds could possibly be investigated as lead molecules for the synthesis of inhibitors of lipoxygenase class of enzymes.

PAGE 75

64 OCH2CH2~H OH 1 Rh= Rhamnose, R 1 = Caffeyl, R 2 = H 2 Furoguaiacin Figure 4-6: Structures of Magnolidin and Furoguaiacin

PAGE 76

1-r-----------------------------------, 0 8 -No FAS -50 I 0.002M FAS -100 I 0 002 M FAS 0 6 -{\ \ \ \ 1-150 I 0.002 M FAS -200 I 0 002 M FAS -250 I 0.002 M FAS Cl) ~\\ \ \. I \ I / I-so 1.02 M FAS u C I'll 0.4 0 UI .Q < I \ ''-' I -____, I I ,, I ,. '" -. I O'I Vl I\ \~ //~ / "'" I 0 2 0+------,---~--+--~-----,---~-----~-----,---~---~------1 420 -0 2 -----------------------------------------' Wavelength Figure 4-7: Effect of Ferric ammonium sulfate on Magnolidin

PAGE 77

1.4 --------------------------------------, 1.2 1 I 1-NoFAS -50 1.002M FAS -100 1.002M FAS -150 1.002M FAS 0 8 I 1-200 I .002M FAS C ftl ,e 0 en .Q < 0.6 I I .I O'I O'I I \ I I 0.4 0 2 ol-----------------=~==-------------J 220 440 wavelength Figure 4-8: Effect of Ferric ammonium sulfate on SC-2 Diol

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1------------------------------------. 0 8 -No FAS 0.6 \\I 1-50 1.002M FAS -100 1 002M FAS -150 1.002M FAS CII I \I u 1-200 1 002M FAS C I'll 0.4 0 en .Q c:( I_. -._, 0\ -..J I \I //// I 0.2 oi '-' 2 0 240 260 280 300 320 3 0 3 0 -0.2 _,__ ____________________________________ wavelength Figure 4-9: Effect of Ferric ammonium sulfate on SC-6

PAGE 79

CHAPTER 5 SYNTHESIS OF STEREO-ISOMERS OF MANASSANTIN Introduction The lignan constituents of Saururus represent a variety of structures. The biologically active compounds have the tetrahydrofuran ring system. These tetra-hydrofuran lignans contain two methyl groups at positions 3 and 4, and identical aromatic substituents at 2 and 5. There can be six possible isomers, two of them being meso (Figure 5-1, 43, 44) and four, chiral tetrahydrofurans (Figure 5-1, 45, 46, 47, 48) and the aromatic furan (Figure 5-1: 49). Of these, Manassantin has the stereochemistry of 2/3-cis, 3/4-trans and 4/5-cis (Figure 5-1, 47). This stereochemical pattern in tetrahydrofuran lignans was first reported by Rao28' 29 and was first observed in the Saururus lignans. Preliminary investigation of Manassantin A and B showed that they were extremely active in inhibiting the L-1210 cell line. This opened up an area of investigation of the stereochemistry of the tetrahydrofuran ring system in Manassantin and the effect of stereochemistry on L-1210 cytotoxicity. 68

PAGE 80

69 Analogues with the same general structure of S.C-8 but with a different stereochemistry (as shown in Figure 5-1) at the tetrahydrofuran ring were synthesized and tested on L-1210 cell culture. The idea being that testing these compounds will indicate if the activity is associated with any preferred stereochemical structure for the central ring. The aromatic furan analogue was also included. The methodology for these reactions is well known. The 2,5-(3'-methoxy, 4'-hydroxy)diphenyl tetrahydrofuranoid lignans with different configurations of the ring, as shown in Figure 5-1. The completely methylated compounds such as galbelgin, veraguensin, galgravin and furoguaiacin had been prepared earlier. 64 '65 The synthetic scheme is given in Figure 5-2. The starting material 1,4-(3', 4'-dimethoxy) diphenyl-2,3-dimethyl-1,4-butanediones was readily prepared by the condensation of 3', 4'-dimethoxy-propiophenone with the corresponding 2-bromo derivative in the presence of sodium amide. Reduction of these diketones followed by acid catalyzed cyclization affords various tetrahydrofurans that are completely methylated. In addition, in the present work some 4,4'-diphenols were prepared, and this required some specific changes in the general schemes, as shown in Figure 5-3.

PAGE 81

70 The preparation of the desired diphenols, required partial demethylation. This reaction was carried out at the diketone stage (Figure 5-3, 52), to obtain the necessary regioselectivity. We have found that the use of thiocresol and sodium hydride in DMF brings about this demethylation to yield the 4,4'-demethylated diketone in good yields. This demethylation reaction was previously reported using thiophenol and thioethanol.66' 67 In this scheme p-thiocresol was used as the source for the sulfide anion as it is a solid at room temperature and easy to handle. Although the racemic diketone is used in the reaction, the extremely basic conditions employed causes equilibration to the mesa diphenol. Thus the racemic and mesa diphenols are obtained in good yields and can be separated by chromatography. Reduction of the carbonyl groups with lithium aluminum hydride was attempted but this reaction did not proceed to give the desired compounds and the starting material was completely recovered. This is, perhaps, due to the fact that the phenols get deprotonated to form a resonance stabilized anion, which resists reduction. The diphenols were subjected to acetylation, and reduction was attempted on the diacetates. The reaction proceeds readily. Acidcatalyzed cyclization of the reduction product of the racemic diketone affords galbelgin dial as the major product. The mesa diketone, by the same procedure, afforded

PAGE 82

71 the galgravin diol. Acid catalyzed cyclization of the diketones gave the furan diol, furoguaiacin, in quantitative yields. The galgravin diol, galbelgin diol and furoguaiacin were alkylated with 2-bromo-(3', 4'-dimethoxy)propiophenone to afford the dialkylated derivatives of the corresponding diphenols. The alkylated derivatives were reduced with sodium borohydride to afford the desired Manassantin analogs. It must be mentioned that the relative stereochemistry of the side chain was found to be erythro, the three compound was less than 5 % of the products obtained. This was based on the 1H-and 13C-NMR spectra of the final products. Attempts were made to synthesize the all cis tetrahydrofuran diol by catalytic hydrogenation of the furan diol or its diacetate. The reaction did not give any product and the starting material was recovered without any changes. The method developed by Rao68 for the synthesis of veraguensin (Figure 5-4) was utilized for the synthesis of veraguensin diol but it resulted in a mixture of products and thus it had to be abandoned. Thus, the synthesis of the desired Manassantin analogs was achieved readily. This is also the first reported synthesis of various phenolic tetrahydrofuran lignans.

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72 OH 43 OH 45 46 OH 47 48 OH 49 Figure 5-1: Various stereoisomers of Tetrahydrofuran lignans

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OCH3 l)HCI/MeOH Pd/C/H2 73 l)LAH / THF ; 2) TF A/ CH2Cl2 rAl"''"' 0 CH30~ OCH3 Figure 5-2: Synthesis of totally methylated lignans OCH3

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74 Propionic acid, PP N 70 C 50 Bromopropionic acid, PP N 70 C 51 + OCH3 plbiocreol NaH, DMF / reflux CH 3 OCH 3 CH 3 52 OH OH CH 3 0 OCH 3 OCH 3 HO 53a 53b Figure 5-3: Synthesis of various Manassantin analogs

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75 OH HCVMeOH OCH3 OH OCH3 53a, b 54 1) Ac20/Pyridine 2) LAH/ 1BF O C 3) TF A/Benzene 0 --~00 53a 55 OCH3 / 1) Ac20/Pyridine .. 2) LAH/ 1BF O C OCiIJ 3) TF A/Benzene OH Gl30 53b 56 Figure 5-3:

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76 54 56 0 "'"( 55 59 Figure 5-3:

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77 NaBH4 /MeOH '51 60 rn,~ '\. I OCH3 .: 0 OH NaBH4 /MeOH CH3 0 0 '59 OCH3 61 NaBH4/MeOH r61HO 0 ""'1::0 '58 62 Figure 5-3:

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78 Ar Ar i) NaBH4 52 \r-( Pd/C H2 ArA0>Ar __ __ Veraguensin Figure 5-4: Attempted synthesis of Veraguensin diol

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79 Experimental All reactions were monitored by TLC to ensure completion of the reaction. All NMR spectra were obtained on either Gemini 300 or Varian 300 using CDC13 as solvent. 3, 4-Dimethoxypropiophenone (propioveratrone) (50). Veratrole 10g, 25 g of polyphosphoric acid and 25 ml of propionic acid were taken in a 250 ml conical flask and heated over a hot water bath (70-80 C) with occasional shaking. The reaction mixture turns wine red in color and is complete in 1 hour. The reaction mixture was cooled to room temperature and poured in a thin stream on to crushed ice with vigorous stirring. Cold water (200 ml) was added to this mixture and stirred for about 30 minutes and the product that was obtained as a thick precipitate was filtered and dried to give 13 g (92 %) of the product. A sample was crystallized from 1:1 ethyl acetate ligroin. 1 H -NMR ( o ) : 1 2 2 ( 3 H, t 7 5 Hz ) 2 9 7 ( 2 H, q, 7 5 Hz) 3. 94 ( 3H, s) 3. 95 ( 3H, s) 6. 8 9 ( lH, d, 8. 1 Hz) 7. 55 ( lH, d, 2 .1 Hz) 7. 59 ( lH, d d, 2 .1, 8. 1 Hz) 13C-NMR (o): 8.51(C-9), 31.23(C-8), 55.9(0CH3), 109.96(C-5), 110.14(C-2), 122.45(C-6), 130.14(C-1), 149.0(C-3), 153.06(C-4), 199.42(C-7).

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80 2-bromo-3', 4'-dimethoxypropiophenone (51). Veratrole 10 g, 30 g of polyphosphoric acid and 20 g of abromopropionic acid were taken in a 250 ml conical flask and heated on a water bath for 30 minutes. The reaction mixture was worked up in a similar manner as that of 50. The crude product was crystallized from ligroin. Yield 13 g (65 %) 1H-NMR (0): 1. 89, (3H, d, 7Hz), 3. 95, (3H, s) 3. 96, (3H, s), 5.30, (lH, q, 7 Hz), 6.90, (lH, d, 8.1 Hz), 7.51, ( lH, d, 2 Hz) 7. 67, ( lH, d d, 2. 0, 8. lHz) 13C-NMR (o): 20.29 (C-9), 41.18(C-8), 55.98(0CH3), 110.07(C-5), 111.61(C-2), 123.41(C-6), 126.96(C-1), 149.21 (C-3), 153.81 (C-4), 192.02 (C-7). Racemic 2,3-Bis (3,4-dimethoxybenzoyl)butane (52). Freshly cut sodium metal (0.5g) was added to 50 ml of liquid ammonia and stirred with a magnetic stirrer for 30 minutes. Anhydrous Ferric chloride (50 mg) was added and stirred until all the blue color had disappeared. The solution was stirred for about 30 minutes by then the solution had acquired a grayish color. To this solution was added 2 g of 3,4-Dimethoxypropiophenone (50) in small portions with stirring. 2-bromo-3' ,4'-dimethoxypropiophenone (51) (3 g) was added in small portions with continuous stirring. The solution was stirred for 1 hour and the reaction mixture was

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81 allowed to warm to room temperature. The ammonia was allowed to evaporate and 100 ml of water was added. The mixture was stirred and filtered. The resulting brownish colored solid was purified by column chromatography. The compound was eluted with 5 % acetone in benzene as a colorless crystalline solid 3.6g (90 % ) 1H-NMR (O): 1.38, (6H, d, 6.6 Hz), 3.97, 4.00, (14H, s), 6.99, (2H, d, 8.4 Hz), 7.57, (2H, d, 2.0 Hz), 7.79, (2H, d d, 2.0, 8.4 Hz). 13C-NMR (0): 15.98(C-9), 43.20(C-8), 55.90(0CH3), 110.0l(C-2), 110.61 (C-5), 123.03(C-6), 129.16(C-1), 148.97(C-3), 153.16(C-4), 202.93(C-7). Meso and racemic 2,3-Bis(4-Hydroxy 3 methoxybenzoyl) butane (53b), (53a). Sodium hydride 50% in oil (500mg) was suspended in 10 ml of DMF. To this suspension was added 1.8 g of thiocresol dissolved in 5 ml of DMF with stirring. The stirring was continued for 15 minutes by this time effervescence had ceased. To the resulting mixture was added 0.8 g of Racemic 2,3-Bis (3,4-dimethoxybenzoyl)butane 52 and the reaction mixturewas refluxed for 90 minutes. The reaction was monitored by TLC to ensure that the reaction had gone to completion. The mixture was allowed to cool to room temperature and was poured into 100 ml of cold water.

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82 The resulting solution was neutralized to pH 7 with 1 N HCl and extracted twice with 50 ml dichloromethane. The solvent layer was washed water and dried over anhydrous sodium sulfate and concentrated to remove the solvent. A pale golden colored oily material was obtained. This material was eluted from a 20 g silica column using ligroin to remove the unreacted thiocresol and other sulfur containing compounds. Elution with 2 % acetone in benzene gave two compounds, 53b (220 mg) and 53a (500 mg), both were obtained as colorless solids. Yield 97 % (total phenolic compounds obtained) 1H-NMR (0) 53a: 1.10, (6H, d, 6.3Hz), 3.98, (8H, s), 6.17, (2H, b s, Ar-OH), 6.99, (2H, d, 8.1 Hz), 7.60, (2H, d, 1. 8 Hz) 7. 7 0, ( 2H, d d, 1. 8, 8. lHz) 13C-NMR (0) 53a: 17.65(C-9), 42.82(C-8), 56.07(0CH3), 110.16(C-2), 113.97(C-5), 123.88(C-6), 129.78(C-1), 146. 78 (C-4), 150. 71 (C-3), 202.4 (C-7). 1H-NMR (0) 53b: 1.30, (6H, d, 7.0Hz), 3.88, (8H, s), 6.94, (2H, d, 8.4 Hz), 7.48, (2H, d, 1.8 Hz), 7.62, (2H, d d, 1.8, 8.4 Hz), 7.77, (2H b s). 13C-NMR (0) 53b: 16.00(C-9), 43.20(C-8), 55.86(0CH3), 110.33(C-2), 113.89(C-5), 123.68(C-6), 128.75(C-1), 146.63(C-4), 150.33(C-3), 202.99(C-7).

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83 Acetylation of 53a and 53b. 53a (175 mg) was dissolved in 0.3 ml of acetic anhydride and 3 drops of pyridine. The reaction mixture was heated on a water bath for 15 min. The reaction mixture was cooled to room temperature and 5 ml of cold water was added and stirred for 20 minutes. The resulting solution was neutralized with sodium bicarbonate and extracted twice with 10 ml of dichloromethane. The dichloromethane layer was washed twice with water, dried over anhydrous sodium sulfate and concentrated to dryness. The racemic diacetate was obtained as colorless crystals. Yield 200 mg (92.5%). Acetylation of 200 mg of 53b under similar conditions afforded 220 mg of mesa diacetate (100%). 1H-NMR (O) racemic diacetate: 1.13, (6H, d, 6.3 Hz), 2.35, (6H, s), 3.92, (6H, s), 4.01, (2H, m), 7.17, (2H, d, 8.0 Hz), 7.66, (2H, d, <1 Hz), 7.69, (2H, d d, <1, 8.0 Hz). 1 3C-NMR (O) racemic diacetate: 17.55(C-9), 20.65(C-8), 43.22(0C-CH3), 56.06(0CH3), 111.81, 121.81, 122.92, 135.45, 144.07, 151.54, 168.46, 202.34. 1H-NMR (O) mesa diacetate: 1.30, (6H, d, 7.0 Hz), 2.33, (6H, s), 3.86, (6H, s), 3.90, (2H, m), 7.15(2H, d, 8.1 Hz), 7.56, (2H, d, 1.5 Hz), 7.67, (2H, d d, 1.5, 8.1 Hz).

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84 13C-NMR (8) meso diacetate: 15.66, 20.63(C-8), 43.6l(OC-CH3), 55.95(0CH3), 111.98, 121.73, 122.77, 134.71, 143.73, 151.38, 168.46, 203.05. 3,4-Dimethyl-2, 5-bis (4-hydroxy-3-methoxyphenyl)furan (54). A mixture of 53a/53b (200 mg) was dissolved in 5ml of methanol. Three drops of concentrated HCl were added and the resulting solution was refluxed for 10 minutes. The solvent was removed and the product crystallized as colorless crystals that slowly turn pale blue in color over a period of time. 190 mg (100%). 1H-NMR (8): 2.19, (6H, s), 3.94, (6H, s), 5.7, (2H, b s), 6.96, (2H, d, 8.4 Hz), 7.16, (2H, d d, 8.4, 1.5 Hz), 7. 1 7, ( 2H, d, 1. 5 Hz) 1 3 C-NMR: 9.83, 55.98, 108.53, 114.47, 117.536, 119.22, 124.59, 144.70, 146.55, 146.95. All trans 3,4-dimethyl-2, 5-bis(4-hydroxy-3 methoxy phenyl) tetrahydrofuran (55). Racemic diacetate (200 mg) was dissolved in 5 ml of THF and cooled to 0C. This solution was added dropwise over a period of 5 minutes into a suspension of 100 mg LAH in 5 ml of THF, previously cooled to 0C. The reaction mixture was stirred for an additional 15 minutes and allowed to warm to room temperature. The excess LAH was carefully quenched with methanol and

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85 neutralized with 0.1 N H2S04 The resulting mixture was then extracted twice with 10 ml ether. The ether layers were combined and washed with water and dried over anhydrous sodium sulfate and concentrated to dryness. The residue thus obtained was dissolved in 10 ml of dry benzene and treated with about 2 ml of a 3 % solution of trifluoroacetic acid in benzene with stirring at room temperature for about 15 min. The reaction mixture was washed with 5 % sodium bicarbonate and water to remove the acid. The benzene layer was then dried over anhydrous sodium sulfate and concentrated to dryness. The solid thus obtained was crystallized from ligroin ethyl acetate (1:1) to yield 150 mg (78% ) of 55. 1H-NMR (0): 1.04, (6H, d, 6 Hz), 1. 77, (2H, m), 3.91, (6H, s), 4.63, (2H, d, 9 Hz), 5.61, (2H, b s), 6.87-6.95 (6H,m). 13C-NMR: 13.81(C-9), 50.97(C-8), 55.92(0CH3), 88.32(C-7), 108.48, 113.95, 119.34, 134.27, 145.05, 146.58. Meso 3,4-dimethyl-2, 5-bis(4-hydroxy-3 methoxyphenyl) tetrahydrofuran (56). This compound was synthesized by the reduction of meso diacetate with LAH followed by treatment of the reduction product with trifluoroacetic acid in benzene. The procedure was same as that for the synthesis of 55. Meso diacetate (200 mg) affords 145 mg (75.5%) of 7

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86 as a colorless glassy semisolid. Attempts to crystallize this compound from various solvents were unsuccessful. 1 H NMR ( 0) : 1. 0 6 ( 6 H, d, 6 6 Hz ) 2 3 5 ( 2 H, m) 3 8 7 (6H, s), 4.41, (2H, d, 6.3 Hz), 5.8, (2H, br s), 6.93-6.99, (6H,m). 13C-NMR: 12.80, 44.17, 55.753, 87.23, 109.22, 114.12, 119.16, 134.08, 144.99, 146.44. Alkylation of 54, 55 and 56. 54, 55 and 56 (100mg) were each dissolved in 5 ml of DMF and treated with 175 mg of 51 and 100 mg of potassium carbonate and stirred for 30 minutes at 70C. TLC showed that the reaction had gone to completion. The reaction mixture was quenched with 20 ml of cold water and extracted twice with 10 ml of dichloromethane. The dichloromethane layer was washed with water, dried over anhydrous sodium sulfate and concentrated to dryness. The alkylated products in each case were obtained in 90-95% yield 1H-NMR (0) 57: 1.74, (6H, d, 6.6 Hz), 2.15, (6H, s), 3.89, 3.92, 3.94, (18H, s), 5.46, (2H, m, 3.3 Hz), 6.83-7.86 (12H, m).

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87 13C-NMR (0) 57: 9.8, 19.21, 55.92, 56.03, 78.26, 110.13, 111.313, 115.79, 118.37, 123.60, 126.37, 127.324, 145.915, 146.78, 149.041, 149.846, 153.69, 197.514. 1H-NMR (0) 59: 1.00, (6H, d, 6Hz), 1. 71, (8H, d, 6.6 Hz), 3.86, 3.91, 3.94, (18H, s), 4.56, (2H, d, 8.4 Hz), 5.39, (2H, m, 6.6 Hz), 6.76-7.83, (12H, m). 1 3C-NMR (0) 59: 13.75, 19.09, 50.79, 55.86, 55.97, 78.17, 87.06, 88.03, 110.054, 110.68, 111.27, 115.50, 118.52, 123.55, 127.29, 136.37, 146.28, 148.92, 149.90, 153.53, 197.58. 1H-NMR (0) 58: 0.98, (6H, d, 4.8Hz), 1.71, (6H, d, 6.6Hz), 2.25, (2H, m), 3.81, 3.92, 3.94, (2H, d, 6.9Hz), 5.41, (2H, m), 6.78-7.83, (18H, s), 4.43, (12H,m). 13C-NMR (0) 58: 12.87, 19.18, 44.20, 55.90, 56.03, 78. 351, 87 .12, 110 .13, 110. 77, 111. 33, 115. 64, 118. 58, 123.62, 127.37, 136.25, 146.33, 149.01, 149.85, 153.63, 197.59. Reduction of 57, 58 and 59. 57, 58 and 59 (100 mg) were each dissolved in 5 ml of methanol and treated with 50 mg of Sodium borohydride. The reaction mixture was stirred for 15 minutes at room temperature. TLC showed that the reaction had gone to completion. The reaction mixture was concentrated to dryness and dissolved in dichloromethane.

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88 The dichloromethane layer was washed with water, dried over anhydrous sodium sulfate and concentrated. The products (60 61 and 62 respectively) in each case were obtained as colorless amorphous solids. Yields were essentially quantitative. 1H-NMR (0) (60): 1.22, (6H, d, 6Hz), 2.24, (6H, s), 3.88, 3.89, 3.95, (18H, s), 4.42, (2H, m), 4.47, (2H, d, 9Hz), 4.90, (2H, s), 6.84-7.26, (12H, m). 13C-NMR (0) (60): 9.86, 13.49, 55.84, 73.77, 82.13, 109.60, 109. 75, 110.86, 118.48, 118. 77, 119.45, 127.20, 132.51, 145.67, 146.87, 148.22, 148.86, 151.38. 1H-NMR (0) (62): 1.09, (6H, d, 6Hz), 1.18, (6H, d, 6.3Hz), 3.86, 3.89, 3.91, (18H, s), 4.36, (2H, m), 4.69, (2H, 8.7Hz), 4.85, (2H, d), 6.8-7.02, (12H, s). 13C-NMR (0) (62): 13.33, 13.78, 50.93, 55.81, 73.59, 82.22, 88.19, 109.55, 109.85, 110.84, 118.42, 118.98, 119.25, 132.55, 137.45, 146.09, 148.15, 148.80, 151.44. 1H-NMR (0) (61): 1.06, (6H, d, 6Hz), 1.19, (6H, d, 6Hz), 2.40, (2H, m), 3.86, 3.89, (18H, s), 4.37, (2H, m), 4.53, (2H, d, 5.4Hz), 4.85, (2H, d), 6.83-7.02, (12H, m).

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89 1 3C-NMR (B) (61): 12.90, 13.43, 44.31, 55.83, 73.62, 82.20, 87.21, 109.58, 110.50, 110.86,118.45, 119.00, 119.43, 132.55, 137.22, 146.09, 148.18, 148.83, 151.32.

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CHAPTER 6 CYTOTOXICITY OF MANASSANTIN AND ITS SYNTHETIC ANALOGS Introduction The target lignans were isolated from the methanolic extract of Saururus cernuus and several synthetic analogs were synthesized as described in the preceding chapter. The degree of cytotoxicity was determined using mouse leukemia L 1210 cells by the method of Thayer69 and the IC50 was determined for each compound. The compounds selected for the assessment of activity are shown in Figure 6-1 through Figure 6-4. Fifteen compounds were tested for their cytotoxicity. These included seven lignans that were isolated from the methanolic extract of Saururus, six that were totally synthesized analogs of Manassantin and two compounds that were the semi synthetic diketones of Manassantin A and B. The activities of these compounds were compared to that of taxol and Manassantin B (S.C-8). Materials and Methods L1210 Cytotoxicity Assay This assay is commonly used to test compounds for their cytotoxicity and as a screen for compounds with anticancer 90

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91 activity. These mouse leukemia cells have a very rapid doubling time of 24 hours. Thus, they provide a convenient and relatively reliable means for the determination of the cytotoxicity of many compounds. The cells are maintained and subcultured in RPMI medium that was prepared in a sterile manner in the laboratory. The cell population was maintained between 150,000 and 600,000 cells/ ml. At the time of the assay, the cell suspension was diluted to contain 150,000 cells/ ml. This solution (2 ml) was placed in each well of a BectonDickinson deep-well plate (24 wells per plate). The test compounds were weighed and dissolved in sufficient dimethyl sulfoxide (DMSO) to make 20 mg/ ml. Then several dilutions of these stock solutions were made and tested. The compounds were tested over a ten-fold range of concentration. To the wells that contained the L 1210 cells (2 ml/ well) were added 10 l aliquots of the DMSO solutions so that the final concentration of each compound was known in parts per million. Each compound was tested at five different concentrations and each concentration was tested in quadruplicate (4 wells). After the addition of the compounds the plates were incubated for 48 hours. The plates were then removed and the cells in each well were counted to determine the number

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92 of cells per ml. The contents of each well was thoroughly mixed using a sterile 2 ml pipette, then, 1 ml of the cell suspension was transferred to a clean test tube and diluted with 1 ml of trypan blue dye (this dye stains dead cells). The viable cells (unstained) were then counted by shaking and placing 0.1 ml of this suspension on a Fisher hemacytometer and counting the cells found in the five gridded areas of the hemacytometer. This number was then multiplied by 4000 (dilution factor derived from the amount of solution and volume in the gridded area) gave the number of cells/ ml. The test also contained controls with and without 10 l DMSO, and an active standard, which in this case was taxol. The ICso for each case was determined from the plot of log [concentration] versus percent inhibition. The percent inhibition for each concentration was determined by the following equation: % Inhibition = [1(Td T a / T c Ta.)] 100 Where Td is the number of cells per ml of the drug treated wells, T o is the number of cells at the start of the test, and T c is the average number of cells per ml in the control wells. The average of four readings for each concentration was used to calculate the IC5 0 for each compound.

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93 Results and Discussion The results of the L1210 cytotoxicity testing are shown in Figures 6-5, through 6-9. The IC so for the tested compounds is given in Table 6-1. The values reported ineach case are the average values of four readings. It can be seen that the compounds with completely reduced side chain containing the secondary benzylic hydroxyl side chain, S.C-7 and S.C-8, are fifty times more potent than the corresponding diketones. It is interesting to note that S.C-5 and S.C-6 that have only one side chain are also almost as active as S.C-7 and S.C-8. However, the compounds, S.C-2 and S.C-3, that did not contain any side chain but did have the tetrahydrofuran ring system did not show any activity at concentrations as high as 100 ppm. It is also important to note that S.C-1, that has neither the tetrahydrofuran ring system nor neuroleptic activity, has significant activity against this cell line and is almost equal to S.C-7 in its potency. In the case of the synthetic compounds a similar trend is observed in that the completely reduced synthetic analogs of S.C-8 are 50 to 25 times more active than the corresponding diketones. However none of the compounds are significantly more active than S.C-8 isolated from the natural product.

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94 S.C-2 S.C-1 OCH3 R1 R2 = CH2, S C-7 ~ R1, R2 = Me, S C-8 Figure 6-1: Saururus lignans tested for cytotoxicity

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95 OCH3 R=Me, S .C-5; R= H, S.C-6 J\O OOl3 R 1 R2 = CH2, S.C-7 Diketone; R1, R2 = Me, S C-8 Diketone Figure 6-2: Saururus lignans tested for cytotoxicity

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96 OCH3 OI30 0 OI3 0 OCH3 0 OI30 Dialkyl Furan OCH3 Dialky Trans THF 0 0 Dialkyl Meso THF Figure 6-3: Synthetic Manassantin derivatives tested for cytotoxicity

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97 OCH3 Ol30 Ol30 OCH3 0 Ol30 Furan S C-8 OCH3 00:13 Trans S.C-8 Mesa S.C-8 Figure 6-4: Synthetic Manassantin analogs tested for cytotoxicity

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98 110 100 -+-S.C-1 -S. C-5 90 -+-S.C-6 80 70 .~ 60 "" ..0 :c E 50 '# 40 30 20 10 0 .+---11e:::.....-~-~--~-~----,c----,-----,----r----, -2 5 -2 -1. 5 1 -0. 5 0 0.5 1 5 2 2 5 log(conc(ppm)J Figure 6-5: L1210 toxicity of S.C-1, S.C-5 and S.C-6

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99 110 100 -+-S. C-7 -s.C-8 90 80 70 C 60 2 :t:: .Q :c .5 50 '# 40 30 20 10 0 -2 5 -2 -1. 5 1 -0. 5 0 0 5 1 5 2 2 5 log(conc(ppm)J Figure 6-6: L1210 cytotoxicity of S.C-7 and S.C-8

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100 80 70 60 -+-S.C-7 Diketone 50 -S.C-8 Diketone 20 10 ~ t 5~--~2---1T.5~---~-0~.=~~0~~0 ~ 5 ~ ~--1~.5~-~2----,2. 5 -10 log [conc(ppm)] Figure 6-7: L1210 cytotoxicity of S.C-7 Diketone and S.C-8 Diketone

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101 120 100 -+Dialkyt Furan Dialky Meso THF 80 ......... Dialkyt Trans THF C 0 60 ie .Q :c .5 '# 40 20 o L_J~~!'!!'!!!!!'!~~:::::;~==:!!::=::::::=-~-~-~-~ 5 -2 -1.5 -1 -0 5 0 0.5 1 5 2 2 5 -20 log[conc(ppm)] Figure 6-8: L1210 toxicity ofDialkyl Foran, Dialkyl Meso and Dialkyl trans Tetrahydrofurans

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102 120 -+Furan S .C-8 100 -MesoS.C-8 ---6-Trans S .C-8 80 C .2 :t: .Q 60 :c .5 40 20 Q4-----W'---~------~-~--~-~--~-~ -2 5 -2 -1. 5 -1 -0. 5 0 0 5 1 5 2 2 5 log(conc(ppm)J Figure 6-9:L1210 toxicity of Foran S.C-8, Meso S.C-8 and Trans S.C-8

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103 Table 6.1: L 1210 Cytotoxicity Data of Saururus Lignans Compound ICso ppm M S.C-1 0 8 00245 S C-2 >100 -S C-3 >100 -S.C-5 1.77 0032 S.C-6 0 3 0 00055 S.C-7 0.5 0 00070 S C-8 0.1 0 00014 S C7 Diketone 40 0.056 S.C-8 Diketone 17. 8 0 025 Dialkyl Furan 32.0 0 044 Dialky Meso THF 12.6 0 017 Dialky Trans THF 12. 6 0 017 Furan S C-8 1.3 0 0017 Meso S.C-8 0 .25 0 00034 Trans S.C-8 0 .25 0.00034

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104 Conclusions and Further Areas for Research When one examines Table 6-1, it is clear that for the Saururus lignans to be cytotoxic they need to resemble S.C-5 or S.C-6 to show activity. These compounds do not have any neuroleptic activity. The neuroleptic activity as reported by Rao et al3 2 is associated with the Manassantins A and B, which are S.C-8 and S.C-7 respectively. The variation in the stereochemistry of the tetrahydrofuran did not produce any remarkable change in the activity; however, the furan analog was considerably less active. S.C-1 seems to be almost as active as S.C-8 on weight by weight basis but when one observes its IC50 activity on the molar scale S.C-1 is about 15-20 times less active than S.C-6 or S.C-8. The fact that it is completely devoid of neuroleptic activity and toxicity in mice makes it an interesting compound for further investigation. One can come to a similar conclusion with regards to the cytotoxic activity of S.C-5 and S.C-6 as they do not have any neuroleptic or toxic effect on mice and hence are interesting compounds that need to be further investigated with regards to their cytotoxic activity. The synthetic analogs of S.C-8 showed similar trends with regard to their cytotoxic activity in that the diketone analogs were at least 10 times less active than the corresponding totally reduced compounds.

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105 The above results open up several fascinating areas that can be explored with regards to the structure activity relationships of the lignans obtained from Saururus cernuus. The neuroleptic activity of the synthetic compounds and the semi-synthetic diketones need to be examined and this might give us an idea with regards to the stereochemical requirements for the compounds to have neuroleptic activity. The synthesis of the Meso and Trans tetrahydrofuran diphenols is very straightforward hence derivatives of these compounds with different side chains should be synthesized and their cytotoxicity and neuroleptic activity could be evaluated. Similarly, one may alkylate the phenolic lignan S.C-6 to produce compounds with unsymmetrical side chains and evaluate those compounds for activity. It was found that if one were to react S.C-8 diketone with magnesium in methanol one can obtain S.C-2 diphenol. The S.C-2 diphenol can be alkylated with different side chains and one can study their activity. The simplest lignan, S.C-1, holds great promise as an antitumor lead hence its analogs should be synthesized and tested against a variety of other cell lines. Thus it can be concluded that the lignans of Saururus cernuus indeed possess variety of pharmacological activities and further work needs to be done in order to understand

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106 their mechanism of action and their structure activity relationships.

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BIOGRAPIIlCALSKETCH The author was born on 14th March 1970,in Calcutta, West Bengal, India. He is the second of the four children born to Orugunty Krishna Rao and Orugunty Prabha Vathi. It was the author's father, a chemist/pharmacist by profession, who introduced him to the world of science at a very tender age of five. This included very elaborate lectures in chemistry, physics and mathematics at home; these lectures also included demonstrations whenever possible. The author had his preliminary education in St. Mary's Convent School in Calcutta. He later went on to graduate from Delhi Tamil Education Association Higher Secondary School, New Delhi, in 1987. He joined the Dr. M.G.R. University, Madras, and graduated with a degree in pharmacy in 1992. He joined the graduate program at the University of Florida and did his doctoral work under the guidance of Dr. K.V. Rao. 112

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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. Koppaka V. Rao, Chair 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 Docto of Phil phy. II~ Jo Perrin, Cochair Pr essor 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 ~z:hy. Kenneth B. Sloan 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 n cope and G7ali a dissertation for the degree of

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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. -~JbK Ian R. Tebbett Associate Professor of Medicinal Chemistry I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. ohn A. zoltewicz Professor of Chemistry This dissertation was submitted to the Graduate Faculty of the College of Pharmacy and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August, 1998 ;$w,J~ De ~Colleofpharmacy Dean, Graduate School