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LIAO, Wan-tzu, 1947- THE ISOLATION AND CHEMICAL CHARACTERIZATION OF HYPOTENSIVE ALKALOIDS HRCM THE ROOT OF THALICTKLM MINUS L. RACE B.

The Ohio State University, Ph.D., 1976 Health Sciences, pharmacy

Xerox University Microfilms, Ann Arbor, Michigan 48106 THE ISOLATION AND CHEMICAL CHARACTERIZATION OP

HYPOTENSIVE ALKALOIDS FROM THE ROOT OF

THALICTRUM MINUS L. RACE B

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By Wan-tzu Liao, B.Sc. *****

The Ohio State University 1976

Reading Committee: Approved by Dr. Jack L. Beal Dr. Raymond W. Doskotch Dr. Duane D. Miller Dr. Larry W. Robertson "/ Adviser ollege of Pharmacy ACK NO WLE DGMEN TS

Through the years in The Ohio State University I have been very fortunate to have the help and guidance of many people, to whom I wish to express my thanks.

First, I wish to express my sincere appreciation to my adviser, Professor Jack L. Beal for his guidance, support, patience, and constant encouragement.

I also wish to express my gratitude to Professor Raymond W. Doskotch for his invaluable suggestions and discussion of the chemistry of alkaloids. I would like to thank my fellow graduate students and the postdoctoral fellows for their suggestions, dis cussions, and assistance. I am especially grateful to Dr. Wu-Nan Wu, who provided most of the authentic samples and whose expert knowledge in Thalictrum was particularly helpful. I wish to acknowledge the assistance of the following individuals: Mr. A. Weisenberger of the Chemistry Depart ment, The Ohio State University and Dr. Roger Foltz of Battelle Memorial Institute who provided part of the mass spectra, Mr. Edward Fairchild who determined the 90MHz NMR spectra and part of the mass spectra, Dr. Kadhim N. Salman who performed the hypotensive studies in dogs and rabbits, ii and Mr. Ruey-Ping Leu who carried out microbiological assays. I also wish to acknowledge the National Institutes of Health for their financial support throughout this study. I want to thank Dr. M. V. Lakshmikantham of the

Department of Chemistry, University of Pennsylvania for providing the authentic sample of (3)-0-methylarmepavine

I also wish to express my gratitude to my family, especially my parents, sisters, and brother, for their constant encouragement and love. At last, I wish to thank my fianc£, Dr. Wen-Shin Liu for his understanding, patience, and faithful love through countless letters and long distance calls.

ill VITA

October 22, 1947...... Born - Taipei, Taiwan 19 70...... B. Sc., Department of Pharmacy, School of Medicine, National Taiwan University

19 70-19 72 ...... Graduate Research Associate, College of Pharmacy, The Ohio State University, Columbus, Ohio 1972-1974 ...... Graduate Teaching Associate, College of Pharmacy, The Ohio State University, Columbus, Ohio

1974-1976 ...... Graduate Research Associate, College of Pharmacy, The Ohio State University, Columbus, Ohio

FIELDS OF STUDY

Major Field: Pharmacognosy and Natural Product Chemistry Professor Jack L. Beal, Adviser

iv TABLE OF CONTENTS Page ACKNOWLEDGMENTS...... ii VITA AND FIELDS OF S T U D Y ...... iv LIST OF T A B L E S ...... * LIST OF F I G U R E S ...... xii INTRODUCTION ...... I I. Introduction of Thalictrum minus L. race B. . . . X (A) Identification of Thalictrum minus

L. race B ...... 1 (B) Pharmacological tests of Thalictrum

minus L. race B ...... 2 (C) Isolation and identification of compounds from Thalictrum minus L. race B. . 2 (D) Search for hypotensive alkaloids from Thalictrum minus L. race B ...... 5 II. Literature survey of Thalictrum alkaloids (1970- summer 1975) 9 (A) Pharmacological studies of Thalictrum

species and alkaloids...... 9 (B) New alkaloids isolated from Thalictrum

species (1970- summer 1975)...... 13 (1) Isoquinolones...... 14

(2) Benzylisoquinolines ...... 16

v (3) Aporphines...... 17 (4) Phenanthrene alkaloids ...... 20 (5) Protoberberines...... 23

(6 ) Bisbenzylisoquinolines ...... 24 (7) Aporphine-benzylisoquinoline dimers. . 32

(8 ) Aporphine-pavine dimers...... 37 (C) Known alkaloids isolated from Thalictrum species (1970- summer 1975) 50 EXPERIMENTAL...... 53 (A) Material...... 53 (B) Methodology-chemical analyses and physical analyses...... 53 (C) Extraction and fractionation of the alkaloids from the roots of Thalictrum minus race B. . . . 59 (1) Extraction of total alkaloids ...... 59 (2) Fractionation of the ethanolic extract. . . 59 (3) Fractionation of tertiary alkaloid ether fraction...... 63 (D) Separation and isolation of the hypotensive alkaloids, alkaloid VI and alkaloid VII, from chloroform extract...... 65 (1) Column chromatography of chloroform

ex t r a c t ...... 65 (2) Column chromatography of alkaloid VI fraction...... 67

vi (3) Isolation of thalirabine (alkaloid VI). . . 67 (4) Preparation of methothalirabine diiodide. . 70 (5) Preparation of O-methylthalirabine. .... 71

(6 ) Preparation of O-methylthalirabine

monomethodiiodide ...... 7 4

(7) Preparation of O-ethylthalirabine ...... 75

(8 ) Sodium-liquid ammonia cleavage of

O-ethylthalirabine...... 76 (9) Column chromatography of alkaloid VII

fraction...... 81 (10) Isolation of alkaloid VIIB...... 83 (E) Separation and isolation of the tertiary, phenolic alkaloids from the tertiary, phenolic alkaloid fraction...... 84

(1) Column chromatography of the tertiary, phenolic alkaloid fraction...... 84

(2) Isolation of (S)-reticuline ...... 8 6

(F) Separation and isolation of the tertiary, non- phenolic alkaloids from the ether soluble, tertiary, non-phenolic alkaloid fraction .... 87 (1) Column chromatography of the ether

soluble, tertiary, non-phenolic alkaloid fraction ...... 87

(2) Isolation of compound XVI ...... 90

(3) Isolation of thalfine ...... 91

vii f (4) Transformation of thalfine to thalfinine and isothalfinine ...... 93

(5) Isolation of thalidasine...... 97

(6 ) Isolation of adiantifoline...... 97 (7) Column chromatography of adiantifoline

mother-liquor ...... 98

<*> Isolation of thaliadine ...... 100 (9) Synthesis of thaliadine...... 101

( 1 0 ) Preparative thin layer chromatography of fractions 63-110, obtained from column

chromatography of the adiantifoline mother-liquor ...... 10 3

( 1 1 ) Isolation of thalfinine...... 104

(1 2 ) Sodium-liquid ammonia cleavage of thalfinine...... 106

( 1 3 ) Isolation of adiantifoline...... 110

( 1 4 ) Isolation of thaliglucinone ...... 110

(15) Isolation of thaliracebine...... Ill

( 1 4 ) Preparation of thaliracebine dimethiodide . 113

( 1 7 ) Sodium-liquid ammonia cleavage of thaliracebine ...... 114

( 1 8 ) Isolation of thalrugosaminine ...... 117

( 1 8 ) Isolation of obaberine...... 118

(2 0 ) Isolation of thaliadanine ...... 120

( 2 1 ) Preparation of O-methylthaliadanine .... 122

viii (22) Preparation of O-ethylthaliadanine ...... 124

(23) Oxidation of O-ethylthaliadanine ...... 124

(24) Synthesis of dehydrothaliadine from

adiantifoline...... 128

(25) Synthesis of O-ethylthalifoline...... 131

(G) Results of Pharmacological tests of alkaloids isolated from T. minus race B ...... 132 (1) Effects of thalirabine on blood pressure in dogs...... 132

(2) Effects of alkaloids isolated from T. minus race B on blood pressure

in rabbits ...... 134 (3) Results of antimicrobial tests ...... 139

DISCUSSION...... 141

SUMMARY...... i 9 2

APPENDIX ...... 1 9 5

BIBLIOGRAPHY...... 2 i 3

ix LIST OF TABLES Page 1. New alkaloids isolated from Thalictrum species

(1970-summer 1975)...... 38 2. Known alkaloids isolated from Thalictrum species (1970- summer 1975) 50 3. Results of the column chromatography of the

chloroform extract...... 66 4. Results of the column chromatography of the

alkaloid VI fraction ...... 6 8

5. Results of the column chromatography of the

alkaloid VII fraction ...... 82

6 . Results of the column chromatography of the tertiary, phenolic alkaloid fraction ...... 85 7. Results of the column chromatography of the

ether soluble, tertiary, non-phenolic

alkaloid fraction ...... 88

8 . Results of the column chromatography of the adiantifoline mother-liquor ...... 99 9. Effects of thalirabine on blood pressure

in d o g s ...... 133 10. Effects of alkaloids isolated from T. minus race B on blood pressure in r a bbits...... 135

x 11. Results of antimicrobial tests of the

alkaloids isolated from T. minus race B . . . . 140

12. Chemical shift data (6 values) for thalira

cebine dimethiodide and methothalistyline d i i o d i d e ...... 148

13. Chemical shift data (6 values) for thalfine, thalfinine, and isothalfinine from different sources ...... 171

14. Chemical shift data (6 value) of adianti foline and its derivatives ...... 182

xi LIST OF FIGURES

Fractionation scheme of ethanolic extract of powdered roots of Thalictrum minus race B . 6

Fractionation of methanol eluent from cellulose column ...... 7 Flow sheet for the fractionation of the ethanolic extract ...... 62 Flow sheet for the fractionation of the tertiary alkaloid ether fraction ...... 64

NMR spectrum of thaliracebine ...... 196

IR spectrum of thaliracebine ...... 197

CD and UV spectra of thaliracebine ...... 198

El mass spectrum of thaliracebine ...... 199

NMR spectrum of thalirabine...... 200

IR spectrum of thalirabine ...... 201

CD and UV spectra of thalirabine ...... 202

Cl mass spectrum of thalirabine ...... 203

El mass spectrum of thalirabine ...... 204

NMR spectrum of thaliadine ...... 205

IR spectrum of thaliadine ...... 206

CD and UV spectra of thaliadine...... 207

El mass spectrum of thaliadine ...... 208

NMR spectrum of thaliadanine ...... 209

xii 19. IR spectrum of thaliadanine...... 210 20. CD and UV spectra of thaliadanine...... 211 21. El mass spectrum of thaliadanine ...... 212

xiii I. INTRODUCTION

I. Introduction of Thalictrum minus L . race B

(A) Identification of Thalictrum minus L. race B The seeds of this plant were originally obtained

from the Royal Botanical Gardens of Edinburgh, Scotland,

through the University of Washington, College of Pharmacy, Medicinal Plant Garden. The seeds were labeled as Thalic

trum rhynchocarpum Dill, et Rich. However, after the

seeds were planted in the medicinal Plant Garden of The Ohio State University, College of Pharmacy, it was found

that the taxonomy of the plants, developed from the seeds, did not fit the taxonomic description of T. rhynchocarpum. The plant was later identified as Tha]ictrum minus L. by

Dr. Bernard Boivin, botanist of Central Experimental Farm, Plant Research Institute, Department of Agriculture,

Ottawa, Ontario, Canada. Morphologically, the plant was

identical with Thalictrum minus L, obtained from the Ohio

State University, Department of Horticulture. However, there was a definite qualitative difference in the alkaloid content and quantitative difference in pharmacological ac tivity of the plants. Dr. Boivin suggested that the T. minus L. obtained from the O.S.U. Department of Horticulture be designated race A and the plant originating from the 3 Royal Botanical Gardens of Edinburgh be designated race B. 1 2

(B) Pharmacological tests of Thalictrum minus L. race B

The results of pharmacological screening study of eleven species of Thalictrum carried out in these labora tories were reported in 1965.^ The non-quaternary alkaloid fractions were tested for their ability to affect the blood pressure in normotensive dogs, to relax the intes tinal smooth muscle of the rabbit, and to depress the heart rate and amplitude of contraction of the isolated rabbit heart. An extract of T. minus race B produced the greatest hypotensive activity of the species tested. It also showed the ability to relax the intestinal smooth muscle of the rabbit as well as to decrease the heart rate and contraction of the isolated rabbit heart. On the basis of its marked pharmacological test results, Thalictrum minus L. race B was, therefore, chosen for a more extensive study of the hypotensive alkaloids.

Thalictrum minus L. race B In the process of searching for hypotensive alkaloids from T. minus race B, the ethanolic extract of the powdered roots was fractionated into non-phenolic tertiary, phenolic tertiary, and quaternary alkaloid fractions. Four alkaloids, namely, adiantifoline(I), thalfine(II), palmatine(III), and berlambine(IV), were isolated from the non-phenolic tertiary alkaloid fraction. Berberine(V) and magnoflorine(VI), 3 were isolated from the quaternary alkaloid fraction. None of the alkaloids isolated from the non-phenolic tertiary 4 alkaloid fraction was shown to have hypotensive activity.

The glycoside fraction of the aerial part of T. minus race B was also investigated in a collaborative study be tween our laboratory and the laboratory of Dr. K. Wagner,

University of Munich. Three flavone-C-glycosides, saponaretin(VII), orientin(VIII), and iso-orientin(IX)

were isolated chromatographically. 5 4

c h 3o

OCH, (III)

CH 3 0 9

'1 "2 * 3 (VII) H Glucosyl H (VIII) Glucosyl H OH (IX) H Glucosyl OH (D) Search for hypotensive alkaloids from Thalictrum minus L. race B Although the hypotensive effect of the non-quaternary alkaloid fraction of T. minus L. race B has been known since 1965,1 the alkaloids, which were responsible for the 2 hypotensive effect, were not isolated until 1972. The isolation of the active alkaloids resulted from the frac tionation and column chromatography of the alkaloid ex tract, which were monitored by measuring the blood pressure

in normotensive dogs. The fractionation scheme of the ethanolic extract of

the powdered root is summarized in Fig. 1. The ethanolic extract of the powdered root was dissolved in 5% acetic acid solution and extracted with ether to remove the neutral and acidic compounds. The aqueous solution was then made alkaline with ammonium hydroxide and extracted with chloroform. The chloroform extract (non-quaternary alkaloid fraction) was the active fraction.

This active fraction was transformed to chloride salts by dissolving in methanolic HC1. After crystalline berberine chloride was removed, the residual alkaloidal chlorides were chromatographed on a dry-packed cellulose column. The alkaloids were eluted with methyl ethyl ketone satu

rated with water and then methanol. The active fraction was shown to be in the methanol eluent. 6

T. minus race B powdered roots 95% ethanol

ethanolic extract

1) 5% acetic acid 2 ) ether extraction I------ether extract acidic solution 1) NH4OH

2) CHCI3 extraction 1 CHCl^ extract (active) alkaline solution

1) 0.5% HC1 in MeOH 2 ) crystallization ------

berberine chloride alkaloidal hydrochlorides (active)

cellulose column chromatography

Fig. 1. Fractionation scheme of ethanolic extract of powdered roots of Thalictrum minus race B

The methanol eluent from cellulose column was further fractionated as shown in Fig. 2. This methanol eluent was partitioned between water and chloroform. The aqueous layer was made alkaline with sodium hydroxide to pH 10 and extracted with ether. The remaining aqueous solution was acidified with hydrochloric acid and made alkaline again with ammonium hydroxide and then extracted with chloroform. The hypotensive activity was shown to be in this chloroform extract.

methanol eluent from cellulose column

H^O/CHCl^ partition

CHC1^ layer aqueous layer 1) NaOH to PH10 2 ) ether extraction 1 ether extract alkaline solution

1) HC1 2) NH4OH 3} CHCI3 extrac tion

CHCl3 extract (active) aqueous solution

Fig. 2. Fractionation of methanol eluent from cellulose column

The active chloroform extract, obtained from the fractionation as described in Fig. 2, was chromatographed on a silicic acid column. The alkaloids were eluted successively with benzene-methanol-water (60:37:3), (50:47:3), (30:67:3), methanol, methanol-water (1:1), and finally, with methanol-ammonium hydroxide (9:1). The only fraction that showed strong hypotensive activity was the methanol-ammonium hydroxide eluent which contained two major alkaloids. These two alkaloids were separated by column chromatography on neutral alumina, activity I. The column was eluted with chloroform-methanol (9:1), chloroform-methanol (4:1), and methanol. The chloroform-methanol

(4:1) eluent contained a major alkaloid, which was desig nated as alkaloid VII, while the methanol eluent contained another major alkaloid, which was designated as alkaloid VI. The Rf values of alkaloid VI and alkaloid VII on sili ca gel G t.l.c. plate were 0.18 and 0.06 respectively.

Methanol-water - ammonium hydroxide ( 8:1:1 ) was

used as the solvent system. Alkaloid VI and alkaloid VII both showed strong hypotensive effect (1.2 mg/kg). The total yield of alkaloid VI and alkaloid VII through this isolation procedure was very small being only

6 mg/kg of powdered roots. In order to improve the yield of these two alkaloids the fractionation and isolation scheme was modified. The modified procedure is detailed in the experimental part. 9

II. Literature survey of Thalictrum alkaloids (1970- summer 1975 The literature of Thalictrum alkaloids has been 6 ~i 3 reviewed by several researchers; but none of the re views have covered the literature since 19 70. Therefore, the literature of 1970-summer 1975 period will be reviewed in this dissertation in regard to pharmacological studies, as well as the new alkaloids and known alkaloids isolated.

(A) Pharmacological studies of Thalictrum species and alkaloids Folkloric uses of Thalictrum species have been known for many years. It is evident from the literature that there is still significant interest in the pharmacological aspect of the alkaloids isolated from species of this genus. In our laboratories, T. aquilegifolium, T. minus race B, T. polygamum, and T. rugosum were reported to show 14 antibacterial and antifungal activity. T. dasycarpum 14 was also shown to have antibacterial activity. Leifer- tova et al. also reported antifungal activity of T. aquilegifolium, T. flavum, T. foetidum, T. qlaucum (T. rugosum),

T. minus, and T. sparsiflarum. ^ The CNS stimulant and antitumor activities of T. dioicum were shown in a biological and phytochemical screening 17 AU of plants. In folklore medicine, T. flavum ssp. flavum, T. morisonii, and T. aquilegifolium were used as antipyretics.

T. flavum ssp. flavum was also used as an antidiarrheal agent. T. hernandezii has been reported to be used against cancer.19 Because of the demonstrated pharmacological activity of extracts of the various species of Thalictrum, the alkaloids isolated from such species have been subjected to pharmacological studies. Such studies have placed emphasis on antibacterial, antifungal, anticancer, anti inflammatory, and hypotensive activity. In our laboratories, the search for alkaloids from

Thalictrum species possessing antimicrobial activity has been carried out. Thalicarpine(X) and berberine(V) chloride, isolated from T. polygamum had antibacterial activity. Thaliglucinone(XI), which was isolated from the 20 same plant, showed antibacterial and antifungal activity. Fractionation of extracts of T. rugosum led to the isola tion of berberine(V), obamegine(XII), thalidasine(XIII), thalrugosine(XIV), thalrugosidine(XV), and alkaloid D as 21 active antibacterial agents. The alkaloid D was later shown to be identical to thaliglucinone(XI).20

Veronamine(XVI)^ and fetidine(XVII)^ were reported to have hypotensive activity. 11

Thalmine(XVIII) ,2 4 O-methylthalicberine(XIX) 2 4 and 2 5 thalfoetidine(XX) were found to possess antiinflammatory activity. Although the study of the tumor-inhibitory alkaloids, thalicarpine (X) and thalidasine(XIII) began prior to 1970, interest in these compounds and their activity has extended 26 into the period of 1970-summer 1975.

n" C H

CH 0 C H-0

L-Rhamnose-0 (XVI) (IIAX)

H=H (AX)

CHD=a (IIIX)

HOO

03 H

eHD=a (AIX) H=H (IIX)

ZT 13

(XVIII)

H CO

(XX) R=H (XIII) r=ch3

(B) New alkaloids isolated from Thalictrum species (1970- summer 1975) The new alkaloids isolated from Thalictrum species in the period of 1970-sununer 1975 can be classified into eight groups. Each new alkaloid will be discussed under the group to which it belongs. The new alkaloids are 14 also listed alphabetically in Table l(p. 38), where their physical data, sources, and references are given.

(1 ) Isoquinolones Three new isoquinolones were found in Thalictrum 27 species, namely, thalflavine(XXI) , N-methylcorydaldine 28 (XXII), and N-methylthalidaldine(XXIII). Thalflavine(XXI) was isolated from the roots of T. flavum L. by Umarov et al. in 1970. According to the ir, nmr, and mass spectral data, structure(XXI) was proposed 27 as the most probable structure for thalflavine. N-methylcorydaldine(XXII) and N-methylthalidaldine

(XXIII) were isolated from T. fendleri Engelm. ex Gray as oils by Shamma and Podczasy in 1971. From spectral data of N-methylthalidaldine two structures (XXIII) and (XXIV) were considered. The correct structure was proven by chemical means to be (XXIII). The oxidation product of (-)-thalifendlerine(XXV) with potassium permanganate in acetone was identical with N-methylthalidaldine. The final structural proof of N-methylcorydaldine as (XXII) was achieved by comparison of the alkaloid with the oxidative product of the bisbenzylisoquinoline alkaloid cissampar- 2 8 eine(XXVI). frCJUWX

(AIXX)

0 CH 0 0 HO\

(IIIXX)

0 HO

HDO (ixx)

HOO 16

(2) Benzylisoquinolines Only one benzylisoquinoline was reported to be new.

The quaternary alkaloid N-methylpalaudinium chloride

(XXVII) was isolated from T. polygamum Muhl. by shamma and Moniot in 1972. Sodium borohydride reduction of N- methyl palaudinium chloride(XXVII) yielded the known alka

loid (±)-laudanine(XXVIII). Treatment of a sample of palaudine(XXIX) with methyl iodide followed by ion exchange

afforded a compound which was identical with the natural N-methyl palaudinium chloride(XXVII), thus the structure 29 of the new alkaloid was proven to be (XXVII).

CH 0 Na8H4 3 DMel 2) ion exchange

(XXVII) CH-0 CH-0 CH_0 CH 0

CH-0 CH.0

(XXVIII) (XXIX) 17

(3) Aporphines Of the five new aporphines isolated, two were

N-oxides, thalicmidine N-oxide(XXX) and preocoteine N-

oxide(XXXI), 30 one was a dehydroaporphine base, dehydro- 31 thalicmine(XXXII), two were novel aporphine alkaloids 32 with a methylenoxy bridge, thalphenine(XXXIII) and bis-

northalphenine (XXXIV) Khozhdaev et^ al^ isolated thalicmidine N-oxide(XXX)

and preocoteine N-oxide(XXXI) from the roots of T. minus

L.^The uv, ir, and nmr spectra of thalicmidine N-oxide (XXX) and of thalicmidine(XXXV) were very similar. How

ever, their mass, solubility, and Rf values in various solvent systems were different. Thalicmidine N-oxide(XXX) was soluble in water. Its molecular weight was 16 units

higher than thalicmidine(XXXV) and its mass spectrum

showed a strong peak at M-16 due to the ejection of oxygen.

These properties led to the assumption that thalicmidine N-oxide(XXX) was the N-oxide of thalicmidine. The final structural proof was made by chemical interconversion of thalicmidine(XXXV) and thalicmidine N-oxide(XXX). The

oxidation of thalicmidine(XXXV) with ^ 2^2 in ethanol gave thalicmidine N-oxide(XXX). The reduction of thalicmidine oxide(XXX) with Zn/HCl yielded thalicmidine(XXXV). The structure of preocoteine N-oxide(XXXI) was also established ' 30 by a similar procedure. 3 (XXX) OCH_ (XXXV)

ch3o

(XXXI) Dehydrothalicmine(XXXII) was isolated from T. iso- pyroides by Maekh et al.-^The new alkaloid contained the same substituents as thalicmine(XXXVI) but differed from it by two mass units. The oxidation of thalicmine(XXXVI) with potassium permanganate in acetone gave dehydrothalic mine (XXXII) . 3 1 19 OCH

< CH 3 K M n0 4 3 <------

CH3° OCH 3 0 C H 3 (XXXII) (XXXVI)

Thalphenine(XXXIII) chloride, isolated from T. polygamum Muhl. by Shamma et al^. was the first aporphine alka loid with a methylenoxy-bridge. Its structure was estab lished by spectral studies and X-ray analysis of thalphen ine iodide. Hofmann degradation of thalphenine yielded thaliglucine(thalphenine methine) (XXXVII) which was also 32 a new alkaloid. The second aporphine alkaloid with a methylenoxy- bridge, bisnorthalphenine(XXXIV), was also isolated from T. polygamum Muhl. by Shamma and Moniot. N-methylation of bisnorthalphenine(XXXIV) with Mel gave (+)-thalphenine

(XXXIII) iodide. 3 3 20

CH,0 V CH

V - 0 (XXXIII) {XXXVII)

CH , 0

{XXXIV) (4) Phenanthrene alkaloids Six new phenanthrene alkaloids, thaliglucine (XXXVII),34 thaliglucine methochloride(XXXVIII),^ thaliglucinone(XI) thaliglucinone methochloride(XXXIX),^ thalicsine (XL) and thalflavidine(LXXVI) were isolated. The structures of thaliglucine{XXXVII) and thaliglucinone(XI) were firstly established by Mollov et. al.34 The two alkaloids were isolated from the above-ground parts of T. ruqosum Ait. The phenanthrene aromatic system of thaliglucine(XXXVII) was indicated by its uv spectrum, which contained maxima at 263my(e=102,000), 289my(e=93,000) and 321 m y (£=65,000). The presence of the dimethylamino- 21 ethyl group in thaliglucine(XXXVII) was shown by the nmr spectrum and Hofmann degradation of its quaternary ammon ium salt to des-N-methine(XLI). The oxidation of thalig lucine (XXXVI I) with potassium bichromate in an acetic acid medium gave thaliglucinone(XI) which was shown to have an a-pyrone system. Thus, the position of the methylenoxy group in thaliglucine(XXXVII) was clarified. 34

CH 3

(XXXVII) Hofmann 1) M«l Degradation 2) I on Excha 1)Me I CH3 2) Ion Exchange

(XLI) (XXXIX)

(XXXVIII) 22

32 Independently, Shamma et^ al^ established the struc ture of thaliglucine(XXXVII), which was isolated from T. polygamum Muhl., by spectral means as well as by a direct chemical correlation with (+)-thalphenine(XXXIII), Thalig lucine methochloride(XXXVIII), which could be obtained by quaternarization of thaliglucine(XXXVII) with Mel, followed by resin anion exchange to chloride, was also reported to 32 be isolated from T. polygamum Muhl. by Shamma and Moniot. 20 In our laboratories, Garbo et al. also reported the isolation of thaliglucinone(XI) from T. polygamum. They also noted the isolation of thaliglucinone(XI) from

T. rochebrunianum, T. minus var, adiantifolium, and T. rugosum. However, the alkaloid was not identified at the time of the initial isolation. 20

Thaliglucinone methochloride(XXXIX) was also isolated 33 from T. polygamum Muhl. by Shamma and Moniot. The structure of thalicsine(XL), isolated from the above-ground part of T. longipedunculatum, was similar to that of thaliglucinone(XI) except the position of the methoxy group. The methoxy group in the structure of thalicsine ( XL ) was at C-3 instead of C-2 as in thalig- 35 lucinone(XI). Thalflavidine(LXXVI) was isolated from the roots of 66 T. flavum L. by Umarov et al^ The structure of thalflavidine (LXXVI) was proposed by spectral means. This 23 alkaloid had an a-pyrone system and two methoxy groups at

C-2 and C-3.

OCH

(XL) R=H (LXXVI) R=OCH.

(5) Protoberberines One new protoberberine alkaloid was isolated. The alkaloid was tetrahydrothalifendine(XLII) which was iso lated from T. fendleri Engelm. ex Gray by Shamma and Podczasy. The structure of tetrahydrothalifendine(XLII) was established by spectral studies and chemical conversion from thalifendine(XLIII). When thalifendine(XLIII) was reduced with Adams catalyst, racemic tetrahydrothalifen- 28 dine(XLII) was yielded. 24 < X~

OCH OCH 3

OH OH (XLII) (XLIII)

(6 ) Bisbenzylisoquinolines Five new bisbenzylisoquinolines were found in Thalic- trum species, namely, thalrugosidine(XV), 3 6 thalrugosamine n is ip 39 (XLIV), thalrugosine (thaligine) (XIV), thalfine(II), and thalfinine(XLV). The alkaloid thalibrunine(XLVI),

isolated from T. rochebrunianum in our laboratories, was originally characterized only xn a prelimxnary, . . manner. 40 Its structure was finally established by Cava et al^. in

1974.^ The structure of thalfoetidine(XX), which was 42 previously proposed as (XLVII), was revised by Georgxev and Mollov in 1971.^ Thalrugosidine(XV), thalrugosamine(XLIV), thalrugo sine (XIV) were isolated from T. rugosum Ait. in our labora- 36 37 tories by Mitscher et al^. ' All three of these alka loids were cryptophenolic bases and were converted to the known methyl ether derivatives, thalidasine(XIII) , O-methyi- oxyacanthine (XLVIII) , and isotetrandrine(XLIX), respec tively. The location of the phenolic hydroxyl group in thalrugosine(XIV) was apparent from the mass spectrum (XLIV) R=H

(XLVIII) R= c h 3

0CH3 CH3°

CH,

0CH3 (XIV) R=H (XLIX) R=CH Na/NH. (LII) R-C„H c h 3 c .h 3o

N CH,

o c h 3 h o 26

[formulae of the most significant peaks (L) and (LI)] and the nmr spectrum of isotetrandrine(XLIX), indicated that the phenolic group was at C-7. Reductive cleavage of O- ethyl ether of thalrugosine(LII) further confirmed the location of the phenolic hydroxyl group and also confirmed 36 the absolute configuration of thalrugosine(XIV). The 36 structures of thalrugosidine(XV) and thalrugosamine 37 (XLIV) were also confirmed in a similar manner.

Independently, Shamma and Yao also isolated thal rugosine (XIV), which they named thaligine, from T. poly- 38 gamum Muhl. Thalfine(II) and thalfinine(XLV) were isolated from 64 T. foetidum L. by Abdizhabbarova et^ al^ m 1968. Later, the structures of thalfine(II) and thalfinine(XLV) were proposed on the basis of the results of chemical degrada- 39 tions. Two repetitions of the Hofmann degradation of thalfine (II) gave trimethylamine and a nitrogen containing product. Thalfine dimethiodide(LIII) was reduced with zinc in 20% H2 S0 4. The product, N-methyltetrahydrothalfine methiodide(LIV), was treated with ethanolamine to yield

N-methyltetrahydrothalfine which was identical to thalfinine(XLV). When thalfine(II) was oxidized with KMnO^ (II)

3 (LIII) iZn/20% <

'3 (LIV) ^ H 0 CH 2 CH 2 .NH 2

(XLV) 28

in acetone, and methylated with diazomethane, an ester was

formed. The ester was identified as the dimethyl ester of

2-methoxy-{diphenyl oxide)-5,4'-dicarboxylic acid(LV).

Reductive cleavage of thalfine(II) afforded laudanidine(LVI) 39 and O-methylarmepavine(LVII).

OjCH, I) KMnO. H3C02C ^ (II) 2 )0 ^ 2 Nd/NH. (LV)

CH _ 0

CHj + CH.

CH XX (LVI) (LVII)

The structure of thalibrunine(XLVI) was recently established by means of chemical and spectroscopic studies.4 1 Thalibrunine(XLVI) had a highly hindered phenolic hydroxyl group, and could not be derivatized by reaction with either acetic anhydride or diazomethane. Sodium-ammonia reduction of thalibrunine(XLVI) gave a complex mixture of products, from which dihydrothalibrunine(LVIII) and (S)-N-methyl- coclaurine(LIX) were isolated. Photoxidation of thalib- OCH

{XLVI)

N o /NH3

OCH

(LVIII) (LIX)

I) hV,02 2)Zn/HCI

(LX)

(LXI) 30

runine(XLVI) by Bick's method, followed by Zn-HCl reduction, afforded a complex mixture, from which a head-to-head frag

ment (LX) was isolated. The structure of the fragment(LX) was confirmed by synthesis. The mass spectrum of thalib

runine (XLVI) showed two very strong peaks at m/e 561(M- CyH^O) and 515(M-CgHgO^) which corresponded to the loss of

fragments c+H and d+OH, respectively, thus indicating that

the methoxy group of thalibrunine(XLVI) as well as the phenolic hydroxyl group are present in ring E. The sub stitution pattern of ring E was proposed as (XLVI) based on the biogenetic consideration, the nmr spectrum of thalibrunine, a positive Gibbs test, as well as the hin dered nature of the phenolic function. The similarity of

the cd curves of thalibrunine(XLVI) and hernandezine(LXI) allowed the S,S-configuration to be assigned to thalib runine (XLVI) . 4 1

The structure of thalfoetidine was originally pro- 42 posed as (XLVII) by Mollov et al^ In 1971, Geogriev and Mollov revised the structure of thalfoetidine as (XX) by means of sodium in liquid ammonia cleavage of O-ethylthal- foetidine(LXII). The non-phenolic base of the degradation was identified as d-O-ethylpavine(LXIII). The structure of the phenolic base(LXIV) was established by synthesis. The position of the ether bridges in the molecule of thal- 31 foetidine was thus established by the two hydroxyl groups of the phenolic base (LXIV). The absolute configuration of thalfoetidine was established as S,S by establishment 43 of the configuration of bases (LXIII) and (LXIV).

OCH. ChLO CH. H3C

OH (XLVII)

(XX) R=H (LXII)) R=C, R=C2 H5

Na/NH. I

OCH.

OCH. +

0 C & > (LXIII) (LXIV) 32

(7) Aporphine-benzylisoquinoline dimers

Eight new aporphine-benzylisoquinoline dimers were 44 isolated. These are thalmineline{LXI) , thalmelatidine

(LXII)4^, O-desmethyladiantifoline(LXIII)^, thalidoxine 4 0 4q 4 Q (LXIV) , thalictropine(LXV) , thalictrogamine(LXVI) , pennsylvanine(LXVII)^, and pennsylvanamine (LXVIII) .

The structure of fetidine was revised as (XVII).51 Kaniewska and Borkowski isolated a phenolic base, thalmineline(LXI), from the roots of T. minus L. var. 44 elatum Koch. From nmr and mass spectral data, thalmine- 45 line was proposed as (LXI)

(LXI)

Thalmelatidine(LXII) was found by Mollov and Thaun 46 in the roots of T. minus ssp. elatum. Treatment of thalmelatidine(LXII) with potassium permanganate in an acetone medium yielded the aldehyde(LXIX) and isoquinolone(XXI).

The aldehyde(LXIX) was identical to the one obtained during 33 the oxidation of adiantifoline(I). The structure of the isoquinolone(XXI) was proved by synthesis. The absolute configuration was determined by optical rotation.4®

OCH non CH 0 o

(LXII)

KMnO, I OCH CH,0

O 3 °

(XXI)

(LXIX)

O-desmethyladiantifoline(LXIII) was also isolated from the roots of T. minus ssp. elatum by Mollov et al.

Methylation of O-desmethyladiantifoline(LXIII) with diazomethane afforded adiantifoline(I). The position of 34 the phenolic group was proved by the oxidation of the O-ethyl derivative of O-desmethyladiantifoline(LXX) with potassium permanganate in acetone. Its oxidation products were two known compounds, the aldehyde(LXIX) and l-oxy-2- methyl-6-methoxy-7-ethyoxy-l,2,3,4-tetrahydroisoquinoline

(LXXI) , 47

CH OCH

OR CH x H.C

CH_0 OCH (LXIII) (I) R=CH. (LXX) R=C 2 Hs

KMnOa 1

+ H C O C f s CH, 0 3 O CH,

(LXXI) ^ y A o C H 3 OCH. (LXIX) 35

A new phenolic analog of thalicarpine(X), thalidoxine(LXIV), was isolated from T. dioicum L. by Shamma et^ al.

O-methylation of thalidoxine(LXIV) with diazomethane afforded tharlicarpine(X). The phenolic hydroxyl group was proposed at C-4" instead of at C-5 " from the study of the 48 nmr spectrum of thalidoxine acetate(LXXII). Four new aporphine-benzylisoquinoline dimers# thalictropine(LXV), thalictrogamine(LXVI), pennsylvanine(LXVII), and pennsylvanamine(LXVIII), were isolated from T. poly gamum Muhl. by Shamma et^ slI. The four new alkaloids were all the phenolic analogs of (+)-thalicarpine(X). The structural assignment for these new alkaloids were derived from the combination of uv, nmr, and mass spectral data for the alkaloids and their acetates. Diazomethane O-methyl ation of thalictrogamine(LXVI) yielded thalictropine(LXV) and thalicarpine(X). Similarly, when pennsylvanamine (LXVIII) was treated with diazomethane, thalictrogamine

(LXVI) and thalicarpine

O-methylation of pennsylvanine(LXVII) afforded thalicarpine

(X). Therefore, the absolute configuration of these four 49 50 new alkaloids were the same as thalicarpine(X). ' 36

R1 R 2 R 3 R4 (LXIV) CH, CH , H CH (X) CH^ CH, CH, CH (LXV) CH, H CH, c:l (LXVI) H H CH , CH (LXVII) c h 3 CH, CH, H (LXVIII) CH, CH3 H (LXXII) c h 3 c h 3 c h 3c o CH

The alkaloid, fetidine(XVII), was proposed as (LXXIII) 52 by Ismailov and Yunusov in 1966. Cava and Waikisaka re vised the structure of fetidine as (XVII) based on the nmr 51 spectrum at 220 MHz.

(LXXIII) (8 ) Aporphine-pavine dimers From T. polygamum Muhl., Shamma et al. isolated two new alkaloids, pennsylpavine(LXXIV) and pennsylpavoline

(LXXV), which were the first two examples of a new dimeric alkaloid group, the aporphine-pavines. The two alkaloids were phenolic. Their structures were proposed mainly on the basis of the uv, nmr, and mass spectra of the alkaloids and their acetates. Their absolute configuration was determined by the aromatic chirality method. 6 5

OCH3

(LXXIV) H CH 3

(LXXV) H H Table 1* Kc« «lk«loiu M »fr 1975 38 T*fcU 1. (C M llk w i) 39 o

IOO Ot'I'Off'U)ts-» (OS)

'(tissfti-f ‘trm I l f ()« (*!« • OOt (OS) (ixna)

)(■(•(«€ ■lOT'tiitt J!!Tt ItW H •t»t*a*T0 .ho-*0™ IK) 'ti t'oi t ’ot t *cioo IOO (to n ««n t (Of) WV*»

(M*H‘f f 0 at (is) (•*»*>*0t‘*K t f i ' i f t i' jo* '(Ti*nvaitl '(iT ’ftm (iaitt*)iOT-iOT>(ia«tta M liurt)

(gs) MIMVUIWI 1 m i ♦ (Jl») 'tn * m i *«»'(*»•»• MianvMiE .•aotwwttZI-IOT MtMau|li ua|

(to (aiimiM) (Kto»-r

*WI I 0’»’lf»!«iatito

fl'i'tlttf'1

'M't'StfH't'BM

KOI 1f’[ '( CH30t> (to (fftlMMlt l-)m *(*tm

(to

(auiicioa) (sdag.r

'PPHO'fOft'aiaxio

ICfM't'JS-f

■ im fi'tc*'*™ It ft (troaaott IC C 'f it ’ |f IOOl) •(Kflaagot i-uoi'i-im w (to llfltf'tff Moo ft»« lf*t tiaaiat (tit (.Mtaw'fo a) 'Slt'iES'ttt'OM •tlfOvaOK ttl-fOC a tn *" • o V V » » u ti h h im t^ *i '(a««l>m'(tWIOI» *13031 •“ - I t 1*1 '( If f t o il ISO (laatailJWK ' ( M m ) a»T*»

t«»to tt-tJttop uf a/a m.4j»bl H h paiau dvjAiatito aanalai amaadg ■IKK Of 'unljajJi uui I _a> 'M tN fl ai 1 t«I 'Ml 'IDHllI M 3, da gw* »a*n»*)j*o Ftomtf

(rOMJttMO) *1 *14*1 Ti ll* !• IC w tlw w il 41 MU 1. (CVftttMMd) 42 T*kt( 1. ICwtilHHdt 43 M ll l> (tMtlmd) 44 Tabl« 1. tc n tliw a d t 45 Tab)* I. (Conti mad)

A1UU1I Mrlutint »p *C UV l i v t l l l ! M ( )<>S < l* npoctra, cia** NKK ipfetrm , (9 W f t , IfW ldc IMItlM *ml*« otherwise M ti4 K«tfl B ptetnm V o In tofiwi riant fhoiiflttClM MthO* H I - I H OMN- ‘ ■m " i n c i j 7)tbOH) (13) I- n lw -a (11) CfcloridoUUVIin, • t b t n t » t 240(4.42),272(4.4(1, MCftjl 3.37(3WCHj)

W n V 212(4.4)) OCHji 3.** 124(1,«2).140(1.21). Urttt 3.«*U)o7.IS(l)t )))(].12),110'1.12) 7,41 (M qulrtet, (11) OCH^Oi 4,0* ArCHjOi i . l J (3»)

* (XI) , 124-1 (athaz) ()4> vl?4l

' a i 1! * * * 110-112 (attar) (20) tM ) V C tty i 2-40 I. polya.asu (10) 110-1*1 (aatftansl I (20) OCM^t 4,00 S, anghahrua-

ehlorlda lt(-»M fciht 7.20(7) ,7.10 h 4 U n w (M l (HtkMWlI (20) 7. 30 (AB q u i t f t , X. ■ *"". o a r. J-0CT4.2M) adlantifolliw OCN^Oi 4.30 (20) othcrvt 7.10-3.50 (ti.hjhj) IJ4)

IktlitllKlM M 224-22) (H*0«) (21) 27S.M4.1S), *1731 O i> *coc)^ !• “"1 ~ (11)

•aUkoefclariOa 211(4.)4).25?a)i(4.»l, X C H y }»)7{tN) (sail) 24X4.51)], 210(1.1)), OCll^i 4.10 cai,ai",»ei )I)I). **),lD ihtl. 11), AfUi 7 .7 S (1 ),7. 400 0.41) ID)

OCHjOi 1,34 (13) Table 1, |Cwtt««a

UUltK KrlMtlm nd op *C (TV (peetra, no.09 1 < 1* apoctre, ca'x OKS •reetm o. *# J'S*. Specific rotation or,let* otherviee notedon** ipiclTio o/o in O eorat* P la n t

ttalM U iiU M lU tt). 12«-122(StOK) (4*1 w fM (0 , 0,1 iC D C lj |a)x* * 47(c. 1*, £. olftu* lap. (4 *) OCH,, 2 .* 0, 2 .4 * CHCljl (4*1 elatoa (44) C4JM<»l' j 010 OCItj, l.lt (lO CIIjl.l.lO .

l.»(l(JOn(}),l.T»,2.*2

AlHi t.04(1),S.*4(1),

*, 11( 2),(.* 011)

CTtjOji S .44 (44)

*4-41(a ther-heptane) , , IB r )440h , I. olou* L. »ar. ItolainaliaoUM ), Xm 5* itm .**1 usi HI lOOhlll.tCUClj 742(0*,undo* 14). U l” + MC* *,». IM -llt (ethanol) 2*70*.1420*. MCH}I 2.41.2.SO Ill,SltiSlO,*21 NaOU) U S ) Ilatuo Sock

« $ ) 15*0v,lS2*», OCly 2.47, 1.7*,),12, (41) (4(1 I4IOi,141*«, 1.74,7.1),).**

1 4 0 * ., 1110a , M il: S.71,(.41,(.41,

117*..1210a, 4 .4 4 ,7 .4 1

12) 10. 1100a , Otheror 7.1-4.)<14R,

112*0 . m o w , CS,,CT) I4JI

1«*1h .M 0h .

IIOh (4 *)

D w lp s a e la a 145-1*4 (0*041- I * 10*1. 1 2 1 (4 .1 2 ), * (COj) jSO J. oolraaooa (11) Ml 151(14-1)*,2*1,2*0 w » • «» t n x u t ) . U M 4 M ) (11) 23C>M4.71l.2IOeti().«*) m C H jlji 1 .05, 7 ,4 * (») (o i.j,non) i»)

• I l K i M i iorJida 144-14* 1** 0 . 1 ) ) , 11111. * 4 ) . O C ly 1 .7 *

(■ater-acetonc) (])) HUSO,*7) Ol) h is 1 4.02(11,*.7*(1)

othcrti 5.00(M4,,20,ai}0

J>14|.c.*.I4tp*)

(.•Kd.ifl.ncO jO ,

J-2,*ep.) (12) ?**)« 1- (CMllMMd)

-1 MUltU Oar Ira U n a and np *C tv apactra. im. lo* c IH apoctra, e» IK apection. (ft mi, Sprcltie rotation wilea* nlw tvlu netad aami i i w t n n ■ / • in <«i i h i r l n t

Tfcalropoaaatna OCUv), 1 1 M » ( « > (17) (CDC1, 10* (K ,11),1*1(111, talj® » 1B0IKC3M] (171 VioVi 1*1(70,1(1(17). (17) OCMji 1.(0,1.71,1.11 10K13) ,10111*1. aiKi *,1-7.s (it) (171 linto) ,i»; ibAici, Lino0”* 110(11) ,17033) , [ I ] , , ..*10 171(1*1. 17(11*1,

ltiliai,i«*[*oi ta|J10*l»,ooo (171 (17

llalrip iK taa(KT). 171-17*(aathanol)(II) ijjj' 111(1.1*) (>«) *CDC1 j (M l (alp -ltKMnOM) I- raocar* (10) WA 171(1.11) PCTIji 1.11,1,(0 (Ml p j i : CD(naOii) >

1.17 ArP 1 1.1-7.7 (I) (Ml

(»)

•U 00 Oi T f

( i t ) 0 M 'i m t« j

* nat I*#'!*'*1!*! (K)

(n o i h ' c r « K * t (nw 'tit-g 3)no * lf w i o i ‘t

IK) (M ori- ;t*» « lt'(*-*” I*) M t)#f» '«** oot'it-*'*!!! M 't'lC t'K ’l ‘'loo

80*1+***1*1 l i t ) m > i i t li l'tC I ilIO* **r 1 * 1 <» ( T30J)

l i t ) ‘(ICIilt’IH )llt («) (onct't-ort (N0*K'(t'« 0) rt't'tffti-t '*100 (If) liiflltl wv*» (It) w aiuiua *1 L* * ‘iim »'ut*t¥iip» o t't’tf i ''no* IK) '(tt'tltlt IK ) (HO*MltfT IMKI |H t* t1 « U ) (M l i i i W l *1 ( M M l I t t * 0j M 1*0 (tX)l»ll'IO» ‘ to o o t 1*0 (tftltlt (ill < i wool f »i r - m KtpMtfelTWU

4 » » U t » i ) 4 p U} pnM » |u a|]6 n>[itA M tlllM OfjpOMlt *»lM « t '«*>1 J30d» HUM ^ • 3 « > *01 *tW 'fllMhlE M) 3. *1 pin tMiiniigg I )l«miV

(|*«i<|1oa3) *1 »!W 50

(I970-summer 1975) The known alkaloids, isolated from Thalictrum species in the period of 1970-summer 1975, are listed alphabetically in Table 2. Their sources and references are given.

TABLE 2

KNOWN ALKALOIDS ISOLATED FROM THALICTRUM SPECIES (1970-SUMMER 1975)

Alkaloid Plant Reference

Adiantifoline T. minus f. elatum 47 T. minus race B 4

6 -Alloc ryptopine T. simplex 57

Berberine T. minus 44,58,61 T. minus race B 4

T. polygamum 20,29

Berberrubine T. polyqamum 2 0

Berlambine(Oxyberberine) T. minus race B 4

Corydine T. dioicum 59

Deoxythalidastine T. polvaamum 2 0

Hernandezine T. simplex 57 Hydroxy-N-norhydrastinine T. minus f. elatum 47

Isocorydine T. aauileaifolium 60

Jatrorrhizine T. minus 61 51 TABLE 2 Continued

Alkaloid Plant Reference

Magnoflorine T. minus 61

T. ruqosum 2 1 T. minus race B 4

T. polygamum 20,29 T. simplex 57

O-Methylthalicberine T, minus 60,58

O-Methy1 1 halme thine T. minus 58

Obamegine T. ruqosum 2 1

Pallidine T. dioicum 59 Palmatine T. minus race B 4 Thalactamine T. minus 58

Thalicarpine T. flavum 27

T . polygamum 2 0

Thalicberine T. minus 58 Thalicmidine T. minus 30

Thalicmine T. minus 30 T. simplex 57

Thaiicminine T. minus 30 T. simplex 57

Thalidasine T . ruqosum 2 1 Thalidezine T. ruqosum 60

Thalifendine T. minus 61 T. polygamum 20,29 52

TABLE 2 Continued

Alkaloid Plant Reference

Thalmethine T. minus 62,58,63

Thalsimine T. rugosum 60 Veronamine T. fendleri 28 EXPERIMENTAL

(A) Material The plant material used in this investigation was the

root of Thalictrum minus L. race B (Ranunculaceae), which was cultivated in the medicinal plant garden of The Ohio State University, College of Pharmacy. A herbarium speci men is on file in the herbarium of the Division of Pharma cognosy and Natural Products chemistry. The roots were washed with water, and then oven dried at 40°C. The dried roots were ground to a fine particle size (80-100 mesh) by means of a Wiley mill.

(B) Methodology Chemical analyses 73 1. Valser's reagent This reagent was prepared by slowly adding 15g. of mercuric iodide with stirring to a solution of potassium iodide (lOg in 100ml of water). The mixture was then filtered to remove excess potassium iodide. The presence of alkaloids was indicated by a white or yellow precipitate formed when one or two drops of

Valser's reagent was added to about one ml of aqueous acidic solution of the sample. 73 2. Mayer's reagent The reagent was prepared by pouring a mercuric chlor- 53 54 ide solution (14g in 60ml of water) into a solution of potassium iodide (5g. in 10ml of water). The solution was then diluted to 1 0 0 ml with water. The presence of alkaloids was shown by the formation of a white or yellow precipitate when one or two drops of Mayer's reagent was added to about one ml of aqueous acidic solution of the sample. 74 3. Dragendorff's reagent Stock solution- The stock solution was prepared by boiling 2.6g of bismuth subcarbonate and 7.0g of dry sodium iodide in 25ml of glacial acetic acid for a few minutes.

The solution was kept at room temperature overnight, and filtered. The filtrate was mixed with four times its volume of ethyl acetate. Spray reagent- The spray reagent was prepared by mixing 10ml of the stock solution with 50ml of glacial acetic acid and 120ml of ethyl acetate. To this solution

1 0 ml of water was slowly added while stirring. An orange to red color at alkaline spots on paper or thin layer chromatograms was shown when sprayed with

Dragendorff1s reagent. The orange background was decolor ized by spraying with 2 % aqueous acetic solution.

4. Phosphomolybdic acid reagent75

This reagent was prepared by dissolving phosphomolyb dic acid (2 g) in 1 0 0 ml of acetone-water mixture (1 :1 ) and filtered. 55

The spots on paper or thin layer chromatograms were

sprayed with phosphomolybdic acid reagent and exposed to ammonium hydroxide fumes. The spots of phenolic compounds

showed a dark blue color. 5. Chromatropic acid test for methylenedioxy groups 76

This test solution was prepared by dissolving chroma-

tropic acid (0.5g) in 50ml of water and 200ml of 12.5 M

sulfuric acid. A small amount of compound to be tested was dissolved

in 0.5ml of chromatropic acid test solution and heated in

a steam bath for 30 minutes. A pink color of the solution indicated the presence of a methylenedioxy group in the

compound.

6 . Gibbs t e s t ^ A small amount (about lmg) of the compound to be

tested was placed in a 1 0 ml volumetric flask and dissolved

in 1 ml of saturated sodium bicarbonate aqueous solution. To the solution was added a freshly prepared suspension

of Gibbs reagent (2,6 -dichlorobenzoquinone chloroimide)

(30mg in 25ml of water) and the mixture was diluted to 10ml with saturated sodium bicarbonate aqueous solution. The absorption spectrum was taken 20 minutes after mixing. A precisely similar mixture of the Gibbs reagent in a satur ated sodium bicarbonate solution was used in the solvent cell of the spectrophotometer. The appearance of a blue color (absorption at the 500-700nm region) indicated the 56

presence of an unsubstituted CH para to a phenolic group*

7. Thin layer chromatography Unless otherwise specified, thin layer chromatography

was carried out on silica gel G using either 20X20 cm or 5X20 cm glass plates with a thickness of 0.25mm of adsorb

ent. The alkaloidal spots were detected by spraying with

Dragendorff's reagent. In some cases, silica gel HF-254

was used as adsorbent.

8 . Column chromatography The adsorbents used for column chromatography were neutral alumina, silica gel PF-254, silica gel 60 (70-230 mesh) and refined silica gel 60 (finer than 230 mesh). The refined silica gel 60 was obtained by treatment of silica gel 60 (finer than 230 mesh) with a stirring- 82 settling-decanting technique and was used for the separa tion of the rather polar alkaloids, alkaloid VI and alka loid VII. Five four-liter beakers were placed in a series numbered from No. 1 to No. 5. Silica gel 60 (750g) was placed in the No. 1 beaker and suspended in four liters of water. The suspension was stirred with a wooden rod back and forth to avoid vortex. After the stirring, the sus pension was allowed to settle for one minute and the top one-third of the volume was poured into the No. 2 beaker. To the No. 1 beaker was added water to 4 liters and the stirring-settling-decanting procedure was repeated. When the suspension in No. 2 beaker reached a volume of 4 literst it was stirred, allowed to settle for two minutes, and the one-third volume poured into the No. 3 beaker. This stirring-settling-decanting procedure was continued through

the No. 5 beaker with a settling time of 4 minutes, 8 min

utes, and 16 minutes for No. 3,4, and 5 beakers respec

tively. This procedure was repeated until the top one- third volume of suspension showed no adsorbent particles

while decanting. The adsorbent in each beaker was then

filtered, activated at 110°C for six hours and deactivated by adding 13% (w/w) of water. Each fraction of adsorbent

was numbered from 1 to 5 according to the number of the beaker. For example, silica gel 60, No. 5 represents the adsorbent collected from the No. 5 beaker. Silica gel 60,

No. 2 and 3 were used for crude separation, while No. 4 and 82 5 were for fine separation.

Physical analyses

1. Infrared spectrophotometrie analysis The infrared spectra were taken in either chloroform

solution or a potassium bromide pellet using Beckman IR 4230 or Beckman 33 infrared spectrophotometers.

2. Ultraviolet spectrophotometrie analysis. The ultraviolet spectra were taken in methanol or

chloroform on a Cary Model 15 recording spectrophotometer. All compounds were analyzed also in 0.01 N methanolic

sodium hydroxide solution and 0.01 N methanolic hydrochlo- 58

ride solution. 3. Nuclear magnetic resonance spectrometric analysis

Unless otherwise specified, the nuclear magnetic resonance spectra were taken in deuterochloroform with

tetramethylsilane as an internal standard on a Varian

Model A-6 QA instrument and were reported in 5 (ppm) units. In some cases, the nuclear magnetic resonance spectra were taken on a Bruker HX-90E by Mr. Edward Fairchild of the College of Pharmacy, The Ohio State University.

4. Mass spectrometric analysis The mass spectra were determined on a MS-9 mass spectrometer in part by Mr. A.Weisenberger of the Chemistry

Department, The Ohio State University, on a Du Pont Model 21-491 by Mr. Edward Fairchild of the College of Pharmacy, The Ohio State University, and on a Finnigan 1015 quadru- pole instrument and A.E.I. MS-902 double focusing mass spectrometer by Dr. Roger Foltz of Battelle Memorial

Institute, Columbus, Ohio.

5. Microanalysis The microanalyses were determined by Scandinavian

Microanalytical Laboratory, Box 2i>, Herley, Denmark.

6 . Optical rotation measurement.

The optical rotation measurements were taken in methanol or chloroform on a Perkin-Elmer Model 241 polarimeter. 59

7- Circular dichroism and optical rotation dispersion measurement

The circular dichroism and optical rotation disper sion spectra were taken in methanol or chloroform on a

Durram-Jasco ORD/UV-5 spectropolarimeter with the Sproul

Scientific SS-20 modification.

8 . Melting point measurement The melting points were determined with Thomas- Hoover Apparatus. The values were not corrected.

C. Extraction and fractionation of the alkaloids from the roots of Thalictrum minus race B (1) Extraction of total alkaloids

A quantity of 14.2kg of powdered roots of T. minus race B was extracted in percolators at room temperature with 95% ethanol. The plant material was macerated in the solvent for 24 hours before percolation. The percolate was then collected and concentrated to a thick syrup ijn vacuo at 4 0°C. The recovered solvent was returned to the plant material. The percolation was carried out until a residue of 50ml of percolate showed a negligible positive result to Valser's reagent. An amount of l,592g of a dark, brown, syrupy ethanolic extract was obtained. (2) Fractionation of the ethanolic extract

The fractionation of the ethanolic extract was modi fied from the previous procedure mentioned in the introduc- 60

tion of this dissertation under the subtitle (D) Search for hypotensive alkaloids from T. minus race B (p. 5 ).

The fractionation scheme is illustrated in Fig. .3. To the ethanolic extract was added a small amount of

95% ethanol to make the extract flow freely. The extract

was poured slowly into a 2 % citric acid aqueous solution#

and was stirred with a mechanical stirrer. The citric acid solution was chilled in the refrigerator overnight# and then filtered. The residue was redissolved in fresh

2% citric acid solution, cooled, and filtered. The pro cedure was repeated until the filtrate showed a negligible

positive result to Valser's reagent. The citric acid solutions were combined to give a total volume of 14 1.

The citric acid solution was extracted with one half volume of ethyl acetate twice. The ethyl acetate solutions were washed with small amount of distilled water# dried over anhydrous sodium sulfate# filtered, and the solvent

removed ill vacuo at 40°C to leave a dark syrupy extract

(44.6 g). This extract was designated as the acid-neutral

fraction.

The citric acid solutions were cooled, refrigerated overnight# and then made alkaline with ammonium hydroxide to pH 8-9. Upon basifying the citric acid solutions# pre cipitation occurred. The basic solution was filtered to yield a dark brown solid (3.7g). This fraction was desig nated as precipitate A. 61

The filtrate was extracted with one half volume of ethyl ether three times. The ethyl ether extracts were washed with a small amount of distilled water, dried over anhydrous sodium sulfate, filtered, and the solvent removed in vacuo at 40°C to yield a brown solid (79.5g). This fraction was designated as tertiary alkaloid ether extract. The alkaline solution was subsequently extracted with one-half volume of chloroform three times. During the extraction a precipitate was formed Detween the aqueous and chloroform layers. This precipitate was removed by filtration to yield a brown solid (7.0g) which was desig nated as precipitate B. The chloroform extracts were washed with a small amount of distilled water, dried over anhydrous sodium sulfate, filtered, and the solvent removed in vacuo at 40°C to yield a brown solid (75.lg). This fraction, which was designated as chloroform extract, con tained the desired hypotensive alkaloids, alkaloid VI and alkaloid VII. The alkaline solutions were pooled, acidified with saturated citric acid solution to pH4, and then Mayer’s reagent (eleven liters) was added until precipitation was complete. The solution was placed in a refrigerator over night and then filtered to yield a light brown precipitate (857g). This fraction was designated as the quaternary alkaloid precipitate. 62

Thalictrum minus L. race B

powdered roots (14.2kg)

95% ethanol

ethanol extract residue (l,592g) 1 ) 2 % citric acid

2 ) ethyl acetate extraction I------ethyl acetate extract acidic solution (44.6 g)(acidic neutral 1 ) ammonium hydroxide fraction) 2 ) ether extraction 1 ether extract (79.5g) precipitate alkaline solution (tertiary alkaloid A (3.7g) chloroform ether extract) extraction chloroform extract precipitate alkaline solution (75.lg)(active B (7. Og) 1 ) sat.citric acid fraction) I 2) Mayer's reagent thalirabine alkaloid VII B quaternary alkaloid aqueous solution precipitate (857g)

Fig. 3. H o w sheet for the fractionation of the ethanolic extract 63

(3) Fractionation of tertiary alkaloid ether fraction

The tertiary alkaloid ether fraction was fractionated

into phenolic and non-phenolic alkaloid fractions as fol

lows. The fractionation scheme is illustrated in Fig. 4. A quantity of 33.Ig of the tertiary alkaloid ether fraction was dissolved in 200ml of 2% HC1 aqueous solution.

The acidic solution was filtered to give an acid-insoluble residue (0.8g). The filtrate was basified to pHlO with 5% NaOH solution. The basic solution was extracted with ether (500ml X4). The ether extracts were combined, washed

with a small amount of distilled water, dried over anhy drous sodium sulfate, filtered, and the solvent removed

in vacuo at 40°C to yield a light brown solid (24.Og).

This fraction was designated as ether soluble, tertiary,

non-phenolic alkaloid fraction. The remaining aqueous solution was subsequently ex

tracted with chloroform (500ml X4). The chloroform ex

tracts were combined, washed with a small amount of dis tilled water, dried over anhydrous sodium sulfate, filtered,

and the solvent removed iji vacuo at 4Q°C to give a dark brown solid (4.5g). This fraction was designated as the chloroform soluble ,tertiary, non-phenolic alkaloid fraction.

The remaining alkaline solution was acidified to pH3 with 20% HCl aqueous solution, and then made alka line to pHlO with ammonium hydroxide. The alkaline solution was extracted with chloroform (500ml x4). The chloroform extracts were combined, washed with a small amount of distilled water, dried over anhydrous sodium sulfate, and the solvent removed ijri vacuo at 40°C to yield a dark brown solid (l.lg). This fraction was designated as the tertiary phenolic alkaloid fraction. The remaining alkaline solution was discarded.

Tertiary alkaloid ether fraction (33.lg) 1. 2% HCl 2 . filtered Acid-insoluble residue Acidic solution (0 .8 g) 1. 5% solution NaOH 2 . ether extraction ether soluble, tertiary, non- alkaline solution phenolic alkaloid fraction CHC1.J extraction (24.Og), Compound XVI thalfine chloroform soluble, thalidasine tertiary, non-phenolic adiantifoline alkaloid fraction thaliadine thalfinine (4.5g) thaliglucinone alkaline solution thaliracebine 1. 20% HCl thalrugosaminine 2. NH4OH obaberine 3. CHC1^ extraction thaliadanine

Tertiary, phenolic alkaloid alkaline solution fraction (l.lg) (discarded)

(S)-reticuline

Fig. 4. Flow sheet for the fractionation of the tertiary alkaloid ether fraction 65

(D) Separation and isolation of the hypotensive alkaloids,

alkaloid VI and alkaloids VII, from chloroform extract

(1) Column chromatography of chloroform extract 2 According to the results of previous experiments , neutral alumina, activity IV,was shown to be the suitable adsorbent for separation of alkaloid VI and alkaloid VII from the chloroform extract. In order to avoid possible decomposition of alkaloid VI and alkaloid VII on a neutral alumina column due to the prolonged contact of the alka loids with the alumina, the chloroform extract was divided into small protions {lg each), and eluted on neutral alum ina columns separately. Table 3 shows the results of col umn chromatography of the chloroform extract (lg) on neut ral alumina column. The elution of the column was started with 2 0 % methanol in ethyl acetate, and then the polarity of eluent was increased by raising the percentage of meth anol in ethyl acetate. Alkaloid VII came off the column with the 20% methanol in ethyl acetate eluent. Alkaloid VI was obtained from the 40% methanol in ethyl acetate eluent.

The chromatographic fractions were monitored by tic and were combined on the basis of the tic results. The thin layer chromatography was carried out on silica gel G using methanol-water-ammonium hydroxide (8 :1 :1 ) as the developing solvent. The Rf of alkaloid VI and alkaloid VII was 0.18 and 0.06, respectively. Berberine, which also appeared in the chloroform extract, had the same Rf value as that 66

of alkaloid VI. However, berberine showed a black color instead of an orange color, when the developed chromatogram

was sprayed with Dragendorff's reagent. Furthermore, ber

berine showed a strong yellow fluorescence under uv light, while alkaloid VI showed a slight blue fluorescence. There

fore, it was possible to distinguish alkaloid VI from ber berine on a thin layer chromatogram when they appeared in

separate fraction. The combined fractions which contained a mixture of

alkaloid VI and alkaloid VII were further subjected to column chromatography on neutral alumina to separate the

two alkaloids. After repeated column chromatography, three major fractions were obtained, namely, berberine

fraction (1.5g), alkaloid VII fraction (0.5g), and alka

loid VI fractions{5.Og).

Table 3. Results of the column chromatography of the chloroform extract

Chloroform extract used: lg Column: neutral alumina, activity IV : lOOg

Fractions Eluent Weight of {1 0 ml) (% methanol in Major alkaloids fractions ethyl acetate) (mg)

1-26 2 0 less polar alkaloids 255 27-54 it berberine 67.5

55-84 It alkaloid VII 19 85-134 40 alkaloid VI 288 135-234 methanol trace of alkaloid VI 50 67

(2) Column chromatography of alkaloid VI fraction

A column (2.4X68 cm) was packed in methanol-ammonium hydroxide-benzene (90:10:1) with a slurry of silica gel 60* No. 5 (80g) to a height of 51cm, Elution was started with methanol-ammonium hydroxide-benzene (90:10:1). The column was first eluted with methanol ammonium hydroxide-benzene

(90:10:1) and then with methanol-water-ammonium hydroxide- benzene (65:25:10:1)* methanol-water-ammonium hydroxide- benzene (40:50:10:1), and 1% HCl in methanol. The fractions were combined according to the results of monitoring by tic with methanol-water-ammonium hydroxide (8 :1 :1 ) as develop ing solvent. The results of the column chromatography are shown in Table 4. Fractions 21-27 contained alkaloid VI. Fractions 15-20 contained alkaloid VI as the major alkaloid along with some minor alkaloids. They all had similar Rf but one was able to discern several alkaloids by study of the nmr spectra.

(3) Isolation of thalirabine (alkaloid VI)(LXXXII)

The residue of fractions 21-27 from the column chroma tography of the alkaloid VI fraction (see Table 4) was purified by column chromatography on neutral alumina* activity IV. The elution was carried out beginning with ethyl acetate* followed by ethyl acetate with an increasing percentage of methanol. From the 20% methanol in ethyl acetate eluent was obtained alkaloid VI (937mg) as an 68

amorphous solid. The alkaloid VI was named thalirabine.

Attempts of crystallization of thalirabine as a free base from various solvents failed. The alkaloid was dissolved

Table 4. Results of the column chromatography of the alkaloid VI fraction

Fractions Eluent Alkaloids Weight (50ml) composition* (mg)

1 - 1 2 A less polar alkaloids 325 13-14 IV less polar alkaloids, 29 alkaloid VI

15-20 11 alkaloid VI(major) 1,114 21-27 W alkaloid VI 1,076

28-43 ii alkaloid VII 50

44-49 B II 69 50-55 C II 25

56-75 D It 15

* A: methanol-ammonium hydroxide-benzene (90:10:1)

B: methanol-water-ammonium hydroxide-benzene (65:25:10:1)

C: methanol-water-ammonium hydroxide-benzene (40:50:10:1)

D: 1% HCl in methanol 69

in a minimum amount of nitromethane and to the solution was added excess ether to precipitate the alkaloid as a 2 6 light tan amorphous powder, m.p. 131-132°C; [cxJD + 142° (c 0.548, MeOH). An attempt was also made to crystallize

the alkaloid as the monoiodide salt. The residue of thal irabine (158mg) was dissolved in methanol. To the solution

was added methanolic solution of potassium iodide (70mg). The solvent was removed and the residue was redissolved in

minimum amount of methanol. Water was added to this methanolic solution until turbidity started to form. The solution was then kept at room temperature for one week.

Light tan rosettes (lOmg) were formed and collected. The m.p. of thalirabine monoiodide was 205-206°C. The physical data of thalirabine were taken as its free base. The ir spectrum in chloroform showed a phenolic hydroxyl absorption at 3510cm The uv spectrum showed

xMeOH 283nrn (i0g E 3 .ao), 276(3.82), and 207(4.99); KlclX X0.1N methanolic NaOH 283nm (io g e 3.91), 276(3.90); max ^

x 0.01N methanolic HCl 280nm (io g e 3.78), 273(3.81), and max ^ 209(4.77). The alkaloid was negative to phosphomolybdic acid reagent and gave a strong greenish blue color

( A ~697nm) with Gibbs reagent. The cd spectrum in max methanol showed maxima at (0 )2 7 5 +6 ,2 6 0 , (0 ]2 2 o+^®'*

The nmr spectrum (6 , CDC13) showed the signals of one tertiary N-methyl group at 2.53(s, lXNCH^)* four O-methyl 70 groups at 3.63(s,lXOCH-j), 3.77(s,3XOCH3), one methylene-

dioxy group at 5.92 (s,0CH2 0 ) , and nine aromatic protons at 5.57(s,lXArH), 5.80(s,lXArH). 6.30-7.07(m,7XArH). The nmr spectrum also showed a quaternary N-methyl singlet at

6 3.43. Due to the overlap of the proton signals, the in tegration of this peak for either one or two quaternary

N-methyl groups was not readily observable. The alkaloid

gave a positive result to the chromatopic acid test for a methylenedioxv group.

The Cl mass spectrum showed the base peak at m/e 669.

The other intense peaks were at m/e 683(28%), 670(45), 448(15), 257 (26), 255(34), 222 (38),220 (40), 208(26),

206(42), 60(66).

The high resolution El mass spectrum showed the base peak at m/e 220.09 70. The other intense peaks were found at m/e 223.1167(8%),222.1133(57),221.1009(14),219.0878{3),

218.0820(8) ,207.086 2 (2),206.1172(7) ,206.0800(3),205.0721(3),

204.0663(2).

(4) Preparation of methothalirabine diiodide (LXXXV)

Thalirabine (293mg) was dissolved in 20ml of nitromethane. To the solution was added 10ml of methyl iodide. The solution was kept at room temperature overnight after which the nitromethane and excess methyl iodide were re moved in vacuo to yield 311mg of residue, which was crystallized from methanol to give light tan rosettes 71

(11 ling) . m.p. 198-200°C (decomp.); + 106.1° (c 0.013, MeOH). This crystalline methothalirabine diiodide showed a single spot (Rf 0.27) on tic with methanol-water-ammonium

hydroxide (4:5:1) as solvent system. The compound gave a strong blue color ( X ~'654nm) with the Gibbs reagent. J max The ir spectrum in KBr indicated a broad band at

3380cm-'*'. The uv spectrum showed 283nm{loge 3.80), u l a X 275(3.84), 212(5.01); X°*°lN methanolic NaOH 28Q nm(loge mdx 3.87), 275(3.86); a°*01n methanol;LC H C 1 280nm(loge 3.83), mdx 2 72(3.88), 212(4.96). The nmr spectrum (6,CD3 N02) showed

four quaternary N-methyl groups at 3.30,3.35,3.60,3.65 (s,4X+NCH_3) , four O-methyl groups at 3. 51 (s , lXOCH-j) ,

3.53(s,1XOCH3), 3. 8 2 (s,2XOCH3), one methylenedioxy group at 6.05(s,0CH20) , and nine aromatic protons at 5.59

(s,lXArH), 5.68(s,lXArH), 6.63-7.20(m,7XArH). The cd spec trum in methanol revealed [0]2^8+8,1000,[0]22g+135,000.

Anal. Calcd for Cij^Hg8 N 2 0gI2 : C,51.69; H,5.29; N,2.94;

1,26.64. Calcd for (c 4 1 H 5 {}N 2 0 8 I2* 3CH3 OH) : C ,50.39; H,5.96; N,2.67; 1,24.20. Found: C,50.28; H ,5.70; N ,2.70; 1,24.28.

(5) Preparation of O-methylthalirabine (LXXX1II)

Thalirabine (40mg) was dissolved in methanol(50ml).

To this solution was added an ethereal solution of diazomethane, which was generated from 2.14g of Diazald. After standing at 0°C for one week, the solvents and excess diazomethane were removed in vacuo to yield 60mg of residue, which showed three spots (Rf 0,95,0.18,0.06) on tic with methanol-water-ammonium hydroxide (8 :1 :1 ) as solvent system.

The alkaloid at Rf 0.06 was the major component which was the desired O-methylthalirabine. The alkaloid at Rf 0.18 was the unreacted thalirabine. This residue was chromato graphed on a small column of silica gel 60,No.5 (2g). The column was eluted first with methanol-water-ammonium hydroxide(8 :1:1) (90ml) and then with methanol-water- ammonium hydroxide(13:5:2)(100ml). From the eluent of methanol-water-ammonium hydroxide(13:5:2) O-methylthalira bine was obtained as an amorphous solid (34mg), which was dissolved in minimum amount of chloroform. To this solu tion was added excess ether to precipitate the alkaloid as a light tan powder(26mg).

The ir spectrum in chloroform showed a broad hydroxyl band at 334 0cm However, the sharp phenolic hydroxyl band at 3510cm 1 disappeared. This spectrum was superimposable with that of thalistyline isolated from T. longistylum DC, and T. podocarpum H.B.K. in our labora- tones. 78

The uv spectrum showed Ida X 320nm shfloge 3.23), 284(3.81),278(3.83),230sh(4.51),208(4.93); A°*01N methan- max olic NaOH 320nin sh (log e 3.32) ,284(3.80) ,278(3.82) ,230sh 73

(4.45); x°*0lN methanolic HCl 320nm sh (loge 3.23),280 m3 x (3. 83),273{3.85) ,230sh(4.77),208 (5.04) . The cd spectrum

in methanol indicated[8 ] 285+^#610,[6]22g+®®'800;

(a)^4 +122°(c 0.082,MeOH). The nmr spectrum(S,CDCl3) showed signals correspond

ing to one tertiary N-methyl group at 2.47(s,lXNCH^), one quaternary N-methyl group at 3.45(s,1X+NCH3), five O-methyl

groups at 3 . 63 (s , 1X0CH3> , 3.77 (s, lXOCH-j) , 3.78

and nine aromatic protons at 5 . 6 8 (s,lXArH) ,5.74(s,lXArH),

6.31(s,lXArH),6.54-7.01 (m,6 XArH). The other quaternary N-methyl signal was not observable due to the overlap with

the other proton signals.

The Cl mass spectrum showed a base peak at m/e 683.

The other intense peaks were at m/e 698(25%), 697(53),

684(45), 258(34), 255(34), 229(31), 220(31), 61(51), 60(31).

The high resolution El spectrum showed a base peak at

m/e 220.0941. The other intense peaks were at m/e 238.1325 (2%),237.1302(14),236.1252(91),234.1115(5),222.1092(6),

221.1001(15),219.0869(3),218.0799^9),206.1161(4). The ir, uv, cd, nmr, mass spectral data, and tic behavior of O-methylthalirabine were identical to those of thalistyline isolated from T. longistylum DC. and T. podo- 78 carpum H.B.K. » . 74

(6) Preparation of O-methylthalirabine monomethodiiodide

fLXXXVI) O-methylthalirabine (2 3mg) was dissolved in 10ml of nitromethane and to the solution was added 5ml of methyl iodide. The solution was kept at room temperature over night after which the nitromethane and excess methyl iodide were removed in vacuo to yield 33mg of residue. The compound was crystallized from methanol-ether and recrys tallized once from methanol-ether to give 18.5mg of ro settes, m.p. 255-257°C (decomp.). The compound showed a single spot (Rf 0.13) on tic with methanol-water-ammonium hydroxide(4:5:1 ) as solvent system. The ir spectrum in KBr indicated a broad band at

3540cm ^ and was identical to that of methothalistyline diiodide. The compound gave a negative test to the Gibbs reagent.

The nmr spectrum (6,CD3 N02) showed signals corres ponding to five O-methyl groups at 3.50, 3.53, 3.81(2XOCH2),

3.90, one methylenedioxy group at 6.02, and nine aromatic protons at 5.59(s, lXArH), 5.81(s, lXArH), 6.67-7.13 (m, 7XArH). Four quaternary N-methyl groups were also observed, two being at 3.26 and 3.28, the other two overlapping with the two O-methyl groups at 3.50 and 3.53. The pattern of the nmr spectrum was identical to that of methothalistyline diiodide.

The cd spectrum in methanol revealed [0]28q+14,000, 75 and [9]2 2 5 +150'000. This alkaloid was identical to metho thalistyline diiodide in terms of tic behavior, ir, nmr, and cd spectral data.

(7) Preparation of O-ethylthalirabine (LXXXIV) 1 Thalirabine (210mg) was dissolved in methanol(100ml). To the solution was added ethereal diazoethane generated from 12g of nitrosoethylurea. The solution was kept at

0°C for one week at which time the solvents were removed to yield 271mg of residue. The residue was dissolved in minimum amount of methanol-water-ammonium hydroxide(8 :1 :1 ) and passed through a column of silica gel 60, No.5 (4g). The column was eluted with methanol-water-ammonium hydrox ide (8:1:1)(50ml) and methanol-water-ammonium hydroxide

(13:5:2)(20ml). From the eluent of methanol-water- ammonium hydroxide(13:5:2), O-ethylthalirabine was iso lated as an amorphous solid(145mg). The ir spectrum in chloroform showed a broad band at 32 80cm However, the sharp phenolic hydroxyl band at 3510cm 1 observed in the ir spectrum of thalirabine disappeared in that of O-ethylthalirabine. The nmr spectrum (6,CDC13) showed signals corresponding to one tertiary N-methyl group at 2.48(s, 1XNCH3), one quaternary

N-methyl group at 3.47 (s, 1X+NCH3>, four O-methyl groups at 3.63 (s, 1X0CH3), 3.76 (s, 3XOCH3), one methyl group at 1.31 (t,J=7cps,OCH2 CH3), one methylene group centered 76 at 4.06 (q,J=7 cps,OCH.2 CHj) , one methylenedioxy group at

5.91 (s,OCH2 0 ) , nine aromatic protons at 5.75 (s,2XArH),

6.28(s,IXArH), 6.53-7.45 (m,6 XArH). The other quaternary N-methyl group was not easily observable due to the overlap of the proton signals.

The Cl mass spectrum revealed a base peak at m/e 697.

The other intense peaks were at m/e 711(26%), 698(47), 520(22), 492(32), 331(77), 250(34), 234(31), 220(43),

60 (61). The high resolution El mass spectrum indicated the base peak at m/e 220.0829. The other intense peaks were a t m / e 264.1578(22%), 251.1452(32), 250.1260(94),- 248.1242(16), 221.1023(32), 219.0883(11), 218.0833(26), 206.1200(19), 206.0832(14).

(8 ) Sodium-liquid ammonia cleavage of O-ethylthalirabine

(LXXXIV) A 50ml three-necked round bottom flask equipped with an equilibrated dropping funnel, a nitrogen gas inlet, and a nitrogen gas outlet on a Dewar-type condenser, was placed in a dry ice-acetone bath (-30°C). Dry liquid ammonia(25ml) was collected and a magnetic stirrer was added to the flask. A suitable amount of freshly cut metallic sodium (30mg) was added until the blue color of the solution was maintained for at least half an hour. O-ethylthalirabine(78mg) was dissolved in 10ml of 77 tetrahydrofuran and the solution was added to the sodium-

liquid ammonia mixture drop by drop in one hour. The blue color solution was maintained at a temperature of -30°C with stirring for another three hours. Ammonia was then allowed to evaporate by raising the temperature of the flask to room temperature. Methanol(5ml) was added to con sume excess sodium and the solvents were then removed in vacuo at 4 0°C. The residue was partitioned between ether

(75ml) and 5% NaOH aqueous solution (75ml). The alkaline solution was extracted with another 75ml of ether. The ether solutions were combined, washed with water, and dried over anhydrous sodium sulfate. The solvent was removed to give an oily residue (31mg) of non-phenolic bases, which was designated as the non-phenolic fraction. This residue revealed one major (Rf 0.51) and one minor base (Rf 0.28) on tic with benzene-acetone ammonium hydroxide (32:16:1) as solvent system. The alkaline solution was acidified with glacial acetic acid and then made alkaline with ammonium hydroxide. The alkaline solution was extracted twice with ether (100ml each time). The ether solutions were combined, washed with water, and dried over anhydrous sodium sulfate. The sol vent was removed to give a solid residue (2 2 mg), which was designated as the phenolic fraction. This phenolic frac tion revealed one major base (Rf 0.21) and two minor bases (Rf 0.51 and 0.28) on tic with the same solvent system used for the non-phenolic fraction. 78

a. non-phenolic fraction The two bases in the non-phenolic fraction were separated using preparative tic with silica gel HP-254 as adsorbent. The plate was developed with benzene-

acetone-ammonium hydroxide(32:16:1). Two zones which contained the bases, were scraped from the plate and extracted with chloroform-methanol mixture(1:1). The

solvents were then removed to give two fractions, one of which contained the major base, the other contained the minor base. The major base(15mg) isolated from this

preparative tic was finally purified on a silica gel

PF-254 column (3g> with 1% methanol in chloroform as eluent. A yellow oil (14mg) was obtained. This compound

was designated as the non-phenolic cleavage product A of

O-ethylthalirabine. The ir spectrum of the non-phenolic cleavage product A of O-ethylthalirabine was taken in chloroform. No band

for a phenolic hydroxyl group was observed. The uv spec

trum showed max 284nm {loge 3. 69) , 278 (3. 72) , 225 (4 . 55) , and 206(4.84). The mass spectrum showed a molecular ion at m/e 357(8%) and a base peak at m/e 58. The other sig nificant peaks were at m/e 358(2%),299 (1),185(3),183(5), 178(2) ,121(8) ,91(1) ,59(5) . The nmr spectrum ( 5,CDC13) showed signals corresponded to two N-methyl groups at

2.38 (s,N(CH3)2) two O-methyl groups at 3.74,3.78 (s,2XOCH3), one methyl group at 1.42 (t,J=7cps,OCH2 CH3), one methylene 79

group centered at 4.00 (q,J-7cpsjOCH^CH^)/ two methylene

groups at 4.50 (s,ArCH2 CH2 Ar), six aromatic protons at

6.2 9 (s,2XArH),6 . 8 8 and 7.13(AB quartet,J=9cps,4XArH). The compound was optically inactive. The cd spectrum in methanol revealed no Cotton effect . The minor base(2mg) isolated from preparative tic was purified by dissolving the compound in 2% HCl aqueous solution{50ml). The acidic solution was extracted twice with ether (100ml each time). The acidic solution was then made alkaline with 5% NaOH aqueous solution and then extracted twice with ether {100ml each time). The ether solutions were combined, washed with water, and dried over anhydrous sodium sulfate. The solvent was removed in vacuo to give 2 mg of oily substance which was designated as non-phenolic cleavage product B of O-ethylthalirabine.

The ir spectrum in chloroform showed a band for a phenolic hydroxyl group at 3530cm The uv spectrum in methanol showed 285nm{log e 2.98) ,277 (3.30) ,225sh(4.06) , and

205(4.42); a£ t21N methanollc Na0H 297nm sh(loge 3.00), max 285(3.34),277(3.36),255sh(3.40), and 230sh(4.05). The uv spectrum in 0.01N methanol HCl showed no observable shift, comparing to that in methanol. The compound showed negative result to Gibbs reagent. The mass spectrum indicated a molecular ion at 373(4%) and a base peak at 58.

The other intense peaks were at m/e 121(7%), 77(2),59(4). 80

The 90MHz nmr spectrum (6 ,CDCl3) showed signals at 2.43 (s,N(CH3)2) ,2.81{s,ArCH2CH2Ar), 2.78(s,1XOCH3) , 2 . 81 (s, lXOCH-j) ,

1.41 (t,J=7cps,OCH2 CH3) ,4.10 (q,J=7cps,OCH2 CH3) ,6.40

(s,lXArH),6.80 and 7.07 (two doublets,J=8 .5cps,4XArH) , and

5.04 (s, broad, ArOH).

b. phenolic fraction The major base of the phenolic fraction was crystal lized from methanol as colorless prisms(9mg), which was designated as the phenolic cleavage product of O-ethyl

thalirabine, m.p. 221-222°C. Its mixture melting points with the phenolic cleavage products of thaliracebine and

thalistyline were not depressed. The compound showed a single spot(Rf 0.21) on tic with benzene-acetone-ammonium

hydroxide(32:16:1) as solvent system. The ir spectrum in ICBr showed a phenolic hydroxyl band at 34 00 cm ^ and was identical to that of the phenolic

cleavage products of thaliracebine (see p. 116 ) and thal- 78 istyline.

The uv spectrum revealed max 285nm(loge 3.59), cqi nnAtA ca\ ->0.01N methanolic NaOH 279(3.59),225sh(4.33),204(4.68): a max 295nm (log e 3.57),2 86(3.62),245sh(4.05). The nmr spectrum

(6 , pyridine-dj) showed the presence of one N-methyl group at 2.50 (s,1XNCH-), one O-methyl group at 3.63 (s,1XOCH3),

two phenolic hydroxyl groups at 4.83 (s, broad, 2XArOH), one methine group at 3.92 (t,lXCH), and six aromatic 81 protons at 6.33 (d,lXArH), 6.73 (d,lXArH), 7.09 and 7.29 (AB quartet,4XArH). The cd spectrum in methanol exhibited

[9]287-3,610, [0]2 7 1 +2,O6O, [0]228+42,8OO, [a] £ 6 + 103.4° (0.029, MeOH). The ir,uv,nmr, and cd spectral data, as

well as melting point, tic behavior of this compound were identical to those of the phenolic cleavage products of thaliracebibe and thalistyline. 7 8 In addition, the mix ture m.p. of this compound and the phenolic cleavage product of thaliracebine was not depressed.

(9) Column chromatography of Alkaloid VII fraction The alkaloid VII fraction, obtained from repeated column chromatography of the chloroform extract (see p. 6 6 ), showed one spot (Rf 0.06) on tic with methanol-water- ammonium hydroxide (8:1:1) as solvent system. However, when the tic plate was developed five times with this solvent system, the alkaloid VII fraction showed a long alkaloidal streak instead of a single spot. This indicated the presence of several alkaloids, in this fraction. This fraction was, therefore, chromatographed on a column (1.4X25cm) of silica gel 60, No. 5,(20g) to isolate the individual alkaloids. The column was eluted with methanol- water-ammonium hydroxide (8:1:1), (13:5:2), and (4:5:1) The results of the column chromatography are shown in

Table 5. The combining of eluents was monitored by tic. The tic plates were developed with methanol-water-ammonium 82

hydroxide (8 :1 :1 ) two or five times according to the

polarity of the alkaloids.

Table 5. Results of the column chromatography of the alkaloid VII fraction

Fractions Eluent * * Weight (50ml) Composition Rf values of alkaloids (mg)

1 A 0.97 75.5

2 I* 0. 73 14. 5

3 11 0.69,0.61 1 2 i 4 II 0.61,0.53,0.44 10.5

5-6 11 0.53,0.44 18. 5

7-8 II 0.36 139.5 9-13 •1 0.42,0.26 125.5

14-21 B 0.42,0.26,0.35(tailing) 117 22-24 11 0.31 37 25-33 C 0.31,0.22 42.5

*A: methanol-water-ammonium hydroxide (8:1:1) B: methanol-water-ammonium hydroxide (13:5:2)

C: methanol-water-ammonium hydroxide (4:5:1) ** The tic plates of fractions 1-21 were developed twice with methanol-water-ammonium hydroxide (8 :1 :1 ) and those of fractions 22-33 were developed five times. 83

(10) Isolation of alkaloid VI1B) Alkaloid VIIB was obtained from the fractions 7-8 of

the column chromatography of the alkaloid VII fraction

(see Table 5). The fractions 7-8 was chromatographed on a column of

neutral alumina (activity IV, 4g). The column was eluted with ethyl acetate. The residue of the ethyl acetate eluent (141mg) was dissolved in minimum amount of chloro

form and excess ether was added to the chloroform solution to precipitate the alkaloid. The precipitate (60.5mg) was crystallized from water to yield a crystalline substance, which was recrystallized once from methanol-water to give brown microcrystals of alkaloid VIIB(8 mg), m.p. 182-184°C,

[a] £ 7 +158.3°(c 0.058,MeOH). In order to compare the relative polarities of alka loid VIIB with thalirabine and O-methylthalirabine, tic of these alkaloids were done with methanol-water-ammonium hydroxide(13:5:2) as solvent system. The Rfs of alkaloid

VIIB, thalirabine, and O-methylthalirabine were 0.24, 0.45, and 0.25 respectively. The ir spectrum showed phenolic hydroxyl absorption at 3520cm The uv spectrum revealed A 279nm a -irtcfji oa\. >0-0lN methanolic NaOH (logs 3. 74), 240sh(4.37), 205 (4. 8 8 ); a IUuX 302nm sh(loge 3.64), 280(3.82), 235sh(4.34); X0.01N methanolic HCl 281nm (loge3.71>, 240sh<4.33), max ' -a 205(4.82). 84

The nmr spectrum {6 , CDCl^) showed one tertiary

N-methyl group at 2.60, (s,1XNCH3), eight aromatic protons at 5.45 (s,broad, lXArH), 5.75 (s,lXArH), 6.37-7.20

(m,6 XArH). The signals at 3.38, 3.45, 3.75, 3.78, 3.92 corresponded to two quaternary N-methyl and five O-methyl groups. The Cl mass spectrum showed a base peak at m/e 669.

The other intense peaks were at m/e 684(30%), 683(65), 670(44), 667(16), 450(18), 255(21), 222(16), 116(26), 102(17), 98(36). The high resolution El mass spectrum showed a base peak at m/e 333.1027. The other intense peaks were at 404.1637(16%), 349,1335(54), 348.1226(23),

347.1068(26), 334.1080(32), 205.0726(23), 191.0574(22),

59.0736(28), 58.0679(22).

The cd spectrum in methanol exhibited [ 0 ] 2 9 5 "^ * ^10 •

(0]2 3 3 +75,200, and [0]2Q8+259,000.

(E) Separation and isolation of the tertiary, phenolic alkaloids from the tertiary, phenolic alkaloid

fraction. (1) Column chromatography of the tertiary, phenolic

alkaloid fraction A column (2.8 x 80 cm.) was packed in chloroform with silica gel PF-254 (90g) to a height of 4 3cm. The 85 tertiary, phenolic alkaloid fraction (1.05g) was dissolved in chloroform and applied to the column. The elution was carried out beginning with chloroform followed by chloroform with an increasing percentage of methanol. Each fraction collected was 100ml. Thin layer chroma tography was employed using benzene-acetone-ammonium hydroxide (32:16:1) as a developing solvent system. The results of the column chromatography are shown in Table 6 .

Table 6 . Results of the column chromatography of the tertiary, phenolic alkaloid fraction

Fractions Eluent (%methanol in Alkaloid Rf values Weights chloroform) (mg)

1-4 chloroform non-alkaloid 1 2 II 5-16 1 75 ll 17-23 2 39

24-30 5 0.55 337 31-35 M 0.61,0.55,0.33 62

36-41 II 0.61,0.55,0.47,0.39 217 0.24

42-48 1 0 0.47,0.32,0.24 229

49-52 2 0 0.47,0.32,0.24 6 6

53-56 40 0.04 116

57-66 1 0 0 0 . 0 0 57

67-76 1% HCl in 0 . 0 0 15 methanol 86

(2) Isolation of (SHreticuline (CXVXI) The residue of fractions 24-30 was dissolved in ethyl

ether. Upon the addition of a saturated methanolic oxalic

acid solution to this ethereal solution, the white oxalate

salt was precipitated from the solution. It was recrys tallized twice from methanol-ether to yield white rosettes

(74mg), m.p. 156.5-158°C. The spectral measurements made using the alkaloid

base were [a]*5u + 138.9° (c 0.190,MeOH>; max 283nm i \ ^ oo\ i 0.01N Methanolic NaOH {logs 3.91), 230(4.36), 205 (4. 98); a max 290nm (loge 3.92). The infrared spectrum of the compound

in chloroform showed a phenolic hydroxyl absorption at 3510 cm ^ and was superimposable with that of authentic (S)-reticuline. The nuclear magnetic resonance spectrum

(6,CDC13) indicated the presence of one N-methyl group at 2.44 ( s ,lXNCH^), two 0-methyl groups at 3.83 (S,2XOCH3)#

five aromatic protons at 6.40-6.82 (m,5H), and two

phenolic hydroxy protons at 4.88(s, broad, 2H) which

disappeared after deuterium exchange. An additional seven protons (methylene and methine) were found as

multipletes between 2.5 and 4.0. The cd spectrum in

methanol showed t® 3 2 S6 + 1 ^,0 0 0 , + 2 1 ,0 0 0 , and

iej208 + 88,000. This compound was identified as S-reticuline by comparison of its tic behavior as well as its ir, uv, nmr, 6 7 79 and cd spectral data with authentic (S) -reticuline. ' 87

(F) Separation and isolation of the tertiary, non-phenolic alkaloids from the ether soluble, tertiary, non- phenolic alkaloid fraction

(1 ) column chromatography of the ether soluble, tertiary, non-phenolic alkaloid fraction

A column (6x110cm.) was packed in chloroform with silica gel PF-254 (1 kg.) to a height of 77cm. The ether soluble, tertiary, non-phenolic alkaloid fraction (2 0 .0 0 g) was dissolved in chloroform and applied to the column. The elution was started with chloroform. The polarity of the eluent was then increased by increasing the percentage of methanol in chloroform. Each fraction collected was 900ml. Thin layer chromatography was employed for mon itoring using benzene-acetone-ammonium hydroxide (32:8:0.5) and (32:16:0.5) as developing solvent systems. The results of the column chromatography are shown in Table 7. 88

Table 7. Results of the column chromatography of the ether soluble, tertiary non-phenolic alkaloid fraction

Fractions Eluent Alkaloid Rf Developing (900 ml) (% methanol in values solvent Weights chloroform) system (mg) for TLC*

1 - 2 chci3 non-alkaloid A 2 0

3-4 11 0.97 IV 298 5-7 •1 non-alkaloid tt 28

11 II 8 0.92 181

9 H 0.61,0.50 It 17 10-14 0.5 0.61,0.50 11 76

•1 15-17 1 0 .8 8 ,0 . 6 8 50 0.47 n 18 n 0 .8 8 ,0 .75 2 0 0.68,0.47

19-20 it 0.43 ti 64

ti 2 1 - 2 2 ii 0.41 327

23-24 N 0. 36 if 195

25-28 It 0.66,0.36 282

29 2 0.93,0.70 B 325

30-33 II 0.79,0.70 It 4,256 0. 55

34 If 0.79,0.70,0.6 0 1 , 0 1 0

35 II 0.70 11 1,754 36-38 n 0.70,0.49 •1 1,921

39-42 Vf 0.70,0.60 f! 1,858 0.49,0.36 89

Table 7. (Continued)

Fractions Eluent Alkaloid Rf Developing (900 ml) (% methanol in values solvent Weights chloroform) system (mg) for TLC*

43-45 5 0.70,0.60, B 490 0.49,0.36

46 11 0.70,0.49 •1 1,252 0. 36 II 47 H 0.70,0.60, 1,995 0.55,0.49, 0.36,0.29

48-52 0.60,0.55, 11 2,134 0.49,0.36, 0.29

11 53-55 1 0 0.60,0.55, 406 0. 49,0.29 II 56-58 II 0.60,0.55, 999 0.49,0.36 0.29

59-61 H 0.52,0.45 II 607 II 62-70 2 0 0.52,0.45 1,023 II 71-74 40 0.52,0.45 193 II 75-76 1 0 0 0.42 124 77-80 2% HC1 in 0.42 II 654 methanol

*A: benzene-acetone-ammonium hydroxide (32:8:0.5) B: benzene-acetone-ammonium chloroxide (32:16:0.5) 90

(2) Isolation of compound XVI The residue from the fractions 3-4 of the column chromatography of the ether soluble tertiary, non-phenolic

alkaloid fraction {see Table 7) was dissolved in benzene and chromatographed on a column of silica gel 60, No.5, (3g). The column was eluted with benzene and each fraction collected was 5 ml. From fraction 2, a colorless oil (160mg) was obtained. This fraction showed a single spot on tic with benzene as solvent system. The Rf was 0.53 and was identical to that of base A isolated from Phe1lodendron wilsonii Hay. et Kane.tRutaceae)81 in our laboratories. This compound showed a pale orange color with Dragendorff*s reagent and turned a purple color with sulfuric acid spray reagent on tic plate. It gave negative results to

Valser's and Mayer's reagents. The compound was optically inactive and showed no Cotton effect in cd spectrum. The ir spectrum (neat) showed a very strong carbonyl band at 1740cm ^ and was superimposable to that of base A. No hydroxyl group was observed in the ir spectrum. The nmr spectrum ^/CDCl^) showed a symmetric multiplet centered at 7.58 {about 4H's), two singlets at 4.28 and 4.18 (about 2H's each), and a multiplet at 0.66-2.17 {about 33 H's), which were identical

to those of base A. The uv spectrum showed max 281nm (loge 3.02),274(3.08), and 225(4.02). The spectra in 91

0.01N methanolic NaOH and 0.01N methanolic HC1 showed no apparent shifts. The mass spectrum showed a base peak at m/e 149. The other intense peaks were at m/e 279(33%), 167(39), 150(12), 113(12), 71(20), 70(18), 57(31), 55(15), 43(22), 41(20). According to the ir, uv, nmr, cd and mass

spectral data, as well as tic behavior of this compound, compound XVI was shown to be identical to base A isolated 81 from P. wilsonii Hay.et Kane. The structural determina

tion of base A is still under investigation.

(3) Isolation of thalfine (XCIX)

The residue of fractions 21-22, which was obtained

from the column chromatography of the ether soluble, tertiary, non-phenolic alkaloid fraction (see Table 7),

was crystallized from methanol to yield 15 7mg of yellow

prisms, m.p. 150-151°C; taj£ 6 + 84.9°(c 0.297,MeOH). These crystals showed a single spot (Rf 0.41) on tic with benzene-acetone-ammonium hydroxide (32:8:0.5) as solvent system. The compound showed a strong green fluorescence under uv light on tic plate and had the same Rf as that

of thalfine isolated previously from this plant. 3 ' 4 The ir spectrum in chloroform showed a peak at

164 0 cm”*, which indicated the presence of an imino func tion, and was superimposable with that of authentic

thalfine. The uv spectrum showed 350nm(loge 3.76), u l a X 288sh (3.58), 261 (4.54), 239sh (4.55), 206 (4.93),and 92 was unaffected by base. However, the spectrum gave a bathochromic shift in 0.01N methanolic HCl solution;

X0.01N methanolic HCl 410nm{loge 3 .64),284(4.47),237sh max (4.52), and 208(4.84); In]^6 +84.9°(c 0.350,MeOH). The nmr spectrum (6,CDC13) indicated one N-methyl group at2.28 (s,lXNCH3) , four O-methyl groups at 3.48, 3. 58, 3. 7 3, 3. 86 (s,4J3XH3), one methylenedioxy group at

6.12 (s,0CH20), ten aromatic protons at 6.03{s,lXArH), 6.46(s,lXArH),7.43(d,J=6cps,lXArH),8.38(d,J=6cps,lXArH),

6 . 53-7.17(m,6XArH), and two benzylic protons at 4.53 and 4.85(d,J=14cps,ArCH^). The cd spectrum in methanol showed [0 ] ^^ - l , 390,

I01328+694' [6]289“28,000, (9]263+82,200, [0]233+67,100, and [0]2O8+134,OOO. The ir, uv, and nmr spectral data as well as tic behavior of this compound were identical to those of thal- 3 4 fine previously isolated from this plant. ' 93

(4) Transformation of thalfine (XCIX) to thalfinine(Q and isothalfinine (Cl) Thalfine{52mg) was dissolved in 20ml of 5% aqueous

HCl and gave a bright yellow solution. Zinc dust(556mg) was added and the solution was boiled for two hours

during which time the bright color of the solution turned

to pale yellow. The solution was cooled and filtered. The filtrate was made alkaline with 5% NaOH aqueous solution and extracted twice with ether (100ml each time).

The ether solutions were pooled, washed with water, and dried over anhydrous sodium sulfate. The solvent was re moved to give 70mg of residue. This residue showed a spot (Rf 0.20) having a blue fluorescence under uv light on

tic plate. The solvent system used was benzene-acetone- ammonium hydroxide (48:16:1). Thalfine had Rf 0.56 with this solvent system and showed a green fluorescence under uv light. The residue from this Zn-HCl reduction was not purified further and was subjected directly to N-methylation. To the methanolic solution (5ml) of this residue was added 5ml of formaldehyde solution (37% HCHO in 100ml methanol). The solution was stirred for one and half hours and then sodium borohydride (200mg) was added and the solution was stirred for an additional hour. Acetone was added to consume the excess sodium borohydride. The solution was then evaporated to dryness. The residue was dissolved in 5% NaOH aqueous solution (50ml) and the

alkaline solution was extracted three times with ether

(100ml each time). The ether solutions were pooled, washed with water, dried over anhydrous sodium sulfate. The solvent was removed to yield 74mg of residue. This

residue showed one spot (Rf 0.40) on tic with benzene- acetone-ammonium hydroxide (48:16:1) as solvent system and

the Rf was identical to that of thalfinine which was an alkaloid isolated from this plant and described on p.104 .

However, when the tic plate was developed with 10% methanol in chloroform, two alkaloidal spots appeared having Rf

values of 0.38 and 0.28. Thalfinine had an Rf of 0.28 with

this solvent .system. The residue was chromatographed on a

column of silica gel PF-254 (lOg). The column was eluted successively with chloroform (80ml), 1% methanol in chloro form (200ml), 2% methanol in chloroform (200ml), and 5%

methanol in chloroform (200ml).

a. isothalfinine (Cl) From the 1% methanol in chloroform eluent was ob

tained the residue (25mg) of a compound having Rf 0.38 on tic with 10% methanol in chloroform as the developing

solvent system. This compound was designated isothalfinine.

The residue of isothalfinine was crystallized from methanol to yield light reddish prisms(13mg), m.p. 204-205®C;

[a]p6 +45.5® (c 0.073,MeOH). 95

The ir spectrum in chloroform showed a slight differ

ence from that of thalfinine. The uv spectrum gave

AMeOHin&x 283nm(loge 3.73), 276 (3.72), 238sh(4.49), and

209 (4.96). The nmr spectrum (6 ,CDCl.j) revealed the signals

corresponding to two N-methyl groups at 2.44,2.67(s,2XNCH^),

four 0 -methyl groups at 3.21, 3.48, 3.73, 3.8 6 (s,4XOCH^), one methylenedioxy group at 5.87 (s, OCH^O), and eight

aromatic protons at 5.87 (s,lXArH, the singlet of this proton overlapped with that of methylenedioxy group),

6.39-7.55 (m,7XArH). The mass spectrum showed a molecular ion at m/e

6 6 6 ( 6 6 %) and a base peak at m/e 220. The other intense peaks were at 667(28%), 665(14), 440(8), 439(24), 425(22),

408(10), 220.5 (28), 204 (14) , 197(22), 190 (10).

The cd spectrum in methanol showed [0]2 84”41'

[01236 + 38' 900' t9l 220"2G,40°' and C 61206+14S' 000 * b. thalfinine (C) The compound of Rf 0.28 was obtained from the resi due (27mg) of the 5% methanol in chloroform fraction. The residue was dissolved in 1 0 ml of methanol and the solution acidified with 5% methanolic HI solution. Excess ether was added to the solution to precipitate the compound as the dihydroiodide salt. The precipitate was then crystal

lized from methanol-water to yield light yellow rosettes 96

(Img), m.p. 234-236°C. The mixture melting point of this compound and the dihydroiodide salt of thalfinine, iso lated from this plant (see p.105), was not depressed. The physical data of the free base of this compound were as follows: The uv spectrum gave max 312 nm sh (loge 2.68), 280(3.67), and 206(4.87). The ir spectrum in chloroform was superimposable to that of thalfinine isolated from this plant. The nmr spectrum (<5,CDC12) exhibited signals corresponding to two N-methyl groups at

2.42, 2.64 (s, 2 XNCH3 ), one methylenedioxy group at 5.89 (s, OCH^O), eight aromatic protons at 5.98 (s, lXArH),

6.41(s, lXArH), 6.80 (s, 2XArH), 6 . 6 8 and 7.19 (AB quartet, J=9cps, 4XArH). The cd spectrum in methanol revealed

(0]2 8 8 -ll,5OO, (0)2 5 5 +21,8OO, [9]227+48,700, IQ)2q7+ 164,000; Cot 1 +98.7° (c 0.075, MeOH) . 97

(5) Isolation of thalidasine (XIII) The residue from fractions 25-28 of the column chromatography of the ether soluble, tertiary, non-phenolic alkaloid fraction (see Table 7) was dissolved in benzene

and chromatographed over a column of neutral alumina (activity I, 15g). The chloroform-benzene (1:1) eluent

contained a major compound. Upon removal of the solvents, a colorless amorphous solid (30mg) was obtained. Trials

of crystallization of the compound from various solvents

failed. fa] ^ 6 -60.5° (c 0.190,MeOH); UV: 270nm (loge 3.80),281(3.75);NMR(6,CDC13): 2.28,2.62(s,2XNCH3),

3.27,3.50,3.75,3.87,3.90 (s, SXOCH-j) , 6 . 32-7 . 60 (m,9XArH);

CD spectrum (MeOH) : [ 9]3gg“24,900, [0]269+3'170,

(9]2 4 4 +4 6 ,200. The ir, uv, nmr, cd spectra, and tic of this compound were identical to those of thalidasine 21 (XIII) isolated from T. ruqosum in our laboratories.

(6 ) Isolation of adiantifoline (I) Adiantifoline was crystallized as yellow needles

from the ethanol solution of the fractions 30-33, which were obtained from the column chromatography of the ether

soluble, tertiary, non-phenolic alkaloid fraction (see Table 7). The crystals were recrystallized from ethanol

twice to yield 1.621g of yellow needles, m.p. 145°C.

The mother liquor of adiantifoline was designated as the adiantifoline mother-liquor and was chromatographed on 98 a silica gel PF-254 column as mentioned elsewhere in this dissertation, (see p. 98 ). The Rf of adiantifoline was 0.70 on tic with benzene-acetone-ammonium hydroxide (32:16:0.5) as solvent system. The alkaloid fluorescenced blue under uv light. The ir spectrum in chloroform was superimposable with 4 that of adiantifoline previously isolated from this plant.

The uv spectrum showed max 312 nm(loge 4.22)/ 302 (4.28), and 282 (4.39). The cd spectrum gave the following molecular ellipticities : [ 0 ] 3Q5-22 , 300, [0 ] 2 7 -7-2 1 ,600,

[6 ] 2 3 9 + 210' 000* Tlle nmr spectrum (6 /CDCl^) showed two N-methyl groups at 2.43,2.48, eight O-methyl groups at 3.59(3H) , 3.78{9H), 3.82<3H), 3.90 (3H), 3.94(3H), and 3.96(3H), and six aromatic protons at 6.24(1H), 6.56(2H), 6.60(1H), 6.63(1H), and 8.06(lH). The ir, uv, cd and nmr spectral data, as well as the melting point and tic beha vior of this compound were identical to those of adianti- 4 foline isolated from this plant and T. minus var. 9 6 8 6 9 adiantifoline Hort. ' * in our laboratories.

(7) Column chromatography of adiantifoline mother-liquor

The residue of adiantifoline mother-liquor was dis solved in chloroform and applied to a column packed in chloroform with 200g of silica gel PF-254 (3.8x 48cm).

Elution was started using chloroform as eluent. The polar ity of the eluent was increased by raising the percentage of methanol in the eluent. 99

Table 8 . Results of the column chromatography of the adiantifoline mother-liquor

Fractions Eluent Alkaloid Rf Weights (% methanol in values* (mg) chloroform)

1 - 2 chci3 non-alkaloid 3

3-20 0.5 ri 70

ai 21-38 1 3

39 11 0.96,0.87 34

40-45 VI 0.95,0.87 29 46-48 II 0.74 23 49-51 1.5 0.74 23 52-55 11 non-alkaloid 46 56-59 •a 0.92,0.35,0.27 24 60-62 M 0.44,0.27 26 63-65 tl 0.77,0.44,0.28 134

6 6 2 0.77,0.28 131

67-70 tl 0.61,0.46,0.28 313 71-110 It 0.61,0.46,0.28, 1,355 0 . 1 1 111-115 •1 0.46,0.27,0.19 106

116-125 3 0.46,0.27,0.19 139

126-145 n 0.46,0.27,0.19 270

146-165 5 0.19 42

* tic solvent system: benzene-acetone-ammonium hydroxide (32:8:1) 1 0 0

Each fraction collected was 60ml. Thin layer chroma tography was employed using benzene-acetone-anunonium hydrox ide (32:8:1) as the developing solvent system. The results of the column chromatography are shown in Table 8.

(8 ) Isolation of thaliadine(CIX)

The residue of the fraction 39 from the column chromatography of the adiantifoline mother-liquor (see

Table 8) was dissolved in methanol. Thaliadine crystal

lized as yellow needles from this solution. Recrystalliza

tion of thaliadine from methanol resulted in yellow rosettes (13.5mg), m.p. 143.5-144.5°C, which gave a single spot (Rf 0.79) on tic with benzene-acetone-ammonium

hydroxide (24:8:0.5) as solvent system. The uv spectrum showed 337nm sh (logs 4.01), m a X 312(4.30), 300(4.30), 277(4.50), 237sh(4.48), and 220(4.62);

a0.01N methanolic HCl n 2 nm(loge 4 ,1 2 ), 300(4.19), 280 max * » (4.30), and 221(4.61). The uv spectrum in 0.01N methanolic NaOH showed no shift . The ir spectrum in chloroform re

vealed a conjugated carbonyl absorption at 1670cm The nmr spectrum (5,CDCl.j) showed one N-methyl group at 2.50, six 0-methyl groups at 3.79{3H), 3.81(3H), 3.91

(6H) , 3.93 (3H), 3.97{3H), four aromatic protons at 6.46,

6.77, 7.40, 8.08(s, 4H) andone aldehyde proton at 10.38.

The mass spectrum gave a base peak at m/e 535, which was also the molecular ion peak. The other intense peaks 1 0 1

were atm/e 536(32%), 534(48), 533(11), 521(16), 520(42), 505(13), 504(36), 492(8), 46(8), 58(27),

The specific rotation [ot]p^ was 0 ° (c 0.223, CHCl^)-

The cd spectrum in chloroform revealed maxima at (0 ) 3 0 0 -23,600, [6]278-37,500, and [9)24Q+214 ,000.

(9) Synthesis of thaliadine (C1X) To a solution of adiantifoline (205mg) was added

a solution of potassium permanganate (51mg in 1 0 ml of acetone). The solution was stirred for five minutes and

the methanol (1 0 ml) was added to destroy the excess potas

sium permanganate. The solution was stirred until the

purple color of the solution disappeared(1/2 hr.). The

solvents were removed and the resulting residue was dis

solved in acetone and filtered. The filtrate was evapo

rated to dryness in vacuo to yield 2 1 0 mg of residue.

This residue showed five alkaloidal spots (Rf 0.95,0.79, 0.71,0.60,0.43) on tic with benzene-acetone-ammonium

hydroxide as solvent system. The spot at Rf 0.43 was adiantifoline. The residue was chromatographed on a column of silica gel PF-254 (20g,1.4Xl9cm). The column was eluted with chloroform(120ml), 0.5% methanol in chloro

form (60ml) , 1 % methanol in chloroform (80ml), 2 % methanol

in chloroform (120ml), and 4% methanol in chloroform(60ml). From the chloroform eluent was obtained the alkaloid with 102

Rf 0.79 (18mg). This alkaloidal residue was designated as adiantifoline oxidation product A. From the 2% methanol

in chloroform eluent, adiantifoline (155mg) was recovered. The recovered adiantifoline was subjected to oxida tion again. The procedure of oxidation and purification was the same as mentioned above. After column chromato graphy, the alkaloid with Rf 0.79 {12mg)was obtained. This alkaloidal residue was designated as adiantifoline oxidation product B. An amount of 70mg of adiantifoline was recovered. The adiantifoline oxidation products A and B were combined. The combined residue was crystallized from methanol to yield yellow rosettes, which was recrystal lized once from methanol to give pure crystals(13mg), m.p.

143-144t>C. The mixture melting point of this crystalline compound and thaliadine was not depressed. The ir spectrum in chloroform showed a band at 16 70cm corresponding to a conjugated carbonyl group, and was superimposable with that of thaliadine. The uv spectrum exhibited

AMeOH 337nm sh (loge 3.91), 312(4.22), 300(4.23), 277(4.42), max 237sh (4.44),ia aa\ 220(4.59);coi. i 0.01N methanolic HCl 312 „nm lU a X (loge 4.13 ), 300(4.18) , 280(4.25), 221(4.59). Theuv spectrum in 0.01N methanolic NaOH showed no uv shift. 103

The nmr spectrum (S,CDC13) showed the signals corresponding to one N-methyl group at 2.51, six O-methyl

groups at 3.81(6H), 3.92(6H), 3.93(3H), 3.97(3H), four

aromatic protons at 6.46,6.78,7.40,8.09(s,4H), and one aldehyde proton at 10.39. The cd spectrum in chloroform showed [0]2gg-25,100,

[9]278“39,000, and [0]24Q+223,000. The specific rotation

[a]p6 was 0°(c 0.213, CHCl-j) . The specific rotation at other wavelengths were taken from the ord curve of this 2 6 compound. They were [a]5 QQ-5.90r [a ] ^^ - l l . 8 ° ,

[al400-32*4°(c °*085' CHC13). This compound and thaliadine were identical based on the data of their ir, uv, nmr, and cd spectra, as well as their melting points, mixture melting point, and tic behavior.

(10) Preparative thin layer chromatography of fractions 6 3-110, obtained from column chromatography of the adiantifoline mother-liquor

The alkaloidal mixture of the fractions 63-110 from column chromatography of the adiantifoline mother-liquor

(see Table 8 ) contained three major alkaloids. Thin layer chromatography, using benzene-acetone-ammonium hydroxide (32:8:0.5) as the developing solvent system, showed three alkaloidal spots at Rf 0.61,0.46, and 0.28.

The alkaloid having Rf 0.28 showed strong green fluores- 104

cence under uv light, while the alkaloid having Rf 0.61 and 0.46 showed blue fluorescence. Since repeated

column chromatography on silica gel PF-254 did not

separate these three alkaloids, preparative thin layer

chromatography was utilized for separation of the alka

loids. The alkaloid mixture was dissolved in chloroform,

streaked onto ten preparative thin layer silica gel G

plates (2 0 x2 0 cm, 0 .6 mm thick), and the plates developed with benzene-acetone-ammonium hydroxide (32:16:1) twice.

Three zones, two with blue fluorescence and one with green, were scraped from the plates and extracted with

chloroform-methanol (1:1), The solvents were then re moved to yield three fractions which were designated as

fractions A, B, and C(427mg, 1.584g, and 149mg). Silica gel tic, with a solvent system benzene-acetone-ammonium hydroxide (32:8:1) demonstrated only one major alkaloid

in each fraction, with Rf 0.61, 0.46, and 0.28 for

fractions A, B, and C respectively.

(11) Isolation of thalfinine(C) The residue of the fraction A from preparative tic

(see p. 1 0 4 ) was dissolved in benzene and chromatographed on a column of neutral alumina(activity I,20g). The colhmn 105 was eluted first with benzene followed by benzene with in creasing amount of chloroform. Thalfinine was obtained from

the 2 0 % chloroform in benzene fraction as an amorphous base (35 7mg), m.p. 117-119°C. The attempts to crystallize the free base, dihydrochloride salt,and dihydroperchlorate salt from various solvents failed. However, the dihydroiodide salt of the compound crystallized as rosettes{m.p.234-236°C, decomp.) from methanol-water. Anal. Calcd for C 39H44N2°8I2 39H42N2° 8 ^ : C,50.77;H,4.81;N,3.04;I,27.51. Calcd for C 39H48N2Oi0I2 (C39H42N208«2HI*2H20):C,48.86;H,5.05;N,2.92;I,26.48.

Found:C,48.98;H,5.11;N ,3.12;I ,26.59. The spectral measurements of this compound were made by using the free base. ta]p^+ 141.2°(c 0.250,MeOH); MeOH 312nm sh(loge 2.74), 280{3.76), and 206(4.94), and was ulaX unaffected by acid or base. The ir spectrum in chloroform did not indicate any hydroxyl group absorption. The nmr spectrum (S,CDC13) revealed two N-methyl groups at 2.36,2.60{s,2XNCH.j), four 0-methyl groups at 3.43, 3.50, 3.73, 3.87(s, 4XOCH^), one methylenedioxy group at 5.86

(s,0CH2 0) , eight aromatic protons at 6 .02(s,lXArH), 6.4 2

(s,lXArH), 6 .6 8 (d,J=9cps, 2XArH), 6 .77(s,2XArH), 7.18(d, J=9cps, 2XArH), and two methine protons at 4.45(t, J=3.5cps, 2XCH). The alkaloid gave positive result to the chromatropic acid test for methylenedioxy groups. The mass spectrum gave a molecular ion peak at m/e

6 6 6 (95%) and a base peak at m/e 220. The other intense X06 peaks were at m/e 667(40%), 665(22), 440(14), 439(50), 425(26), 408(18), 333(2), 220.5(25), 204(16), 190(12). The cd spectrum in methanol indicated the maximum at [0]2 8 8 -12,8OO, 16]255 + 36,200, [0]2 2 7 +5 7 ,100, and

[0]2O7+195,OOO.

(12) Sodium-liquid ammonia cleavage of thalfinine(C) Cleavage of thalfinine was carried out in the same manner as the cleavage of 0-ethylthalirabine. Liquid ammonia(25ml) was collected in a 50ml three-necked flask and finely cut sodium (50mg) was added. A solution of

130mg of thalfinine in 10ml of dry tetrahydrofuran was added dropwise within one hour. At the end of the hour, the reaction mixture turned yellow. Sodium (50mg) was added again to maintain the blue color of the solution.

The reaction was kept for another three hours and then the excess ammonia was allowed to evaporate. The reaction mixture was carefully treated with methanol(5ml) to consume the unreacted sodium. The solution was then evap orated to a few milliliters to which 100ml of 5% NaOH aque ous solution was added and then extracted twice with ether (100ml each). The ether extracts were combined, washed with water, dried over anhydrous sodium sulfate, and evaporated to dryness to yield 28mg of residue, which was designated as the non-phenolic fraction. This fraction 107

showed two alkaloidal spots (Rf 0.51 and 0.10) on tic

with benzene-acetone-ammonium hydroxide (32:16:1) as

solvent system.

The remaining alkaline solution was acidified

with glacial acetic acid, made alkaline again with

ammonium hydroxide, and extracted twice with ether (1 0 0 ml each time). The ether extracts were combined, washed with water, dried over anhydrous sodium sulfate, and evaporated to dryness to give 40mg of residue. This fraction was designated as the phenolic fraction, and

revealed two alkaloidal spots (Rf 0.53 and 0.40) on tic with benzene-acetone-ammonium hydroxide (16:16:1) as

solvent system.

a. non-phenolic fraction

The major base (Rf 0.51) in the non-phenolic

fraction was isolated by preparative tic on silica gel

PF-254 with benzene-acetone-ammonium hydroxide (32:16:1) as developing solvent system. The residue of the major base, obtained by preparative tic, was finally purified by dissolving in 1% HCl aqueous solution (20ml) and extracting twice with ether (100ml each time). The aqueous solution was then made alkaline with 2 0 ml of 1 0 % NaOH aqueous solution and extracted twice with ether

(100ml each time). The ether extracts were combined, 108 washed with water, dried over anhydrous sodium sulfate, and evaporated to dryness to give 18mg of oily base.

This base was designated as the non-phenolic cleavage product of thalfinine. Its oxalate salt was prepared by dissolving the residue in methanol and adding the meth anolic oxalic acid (8mg) to the solution. The solvent was then removed and the residue was crystallized from acetone to give white plates (18mg), m.p. 106-108°C. Its mixture m.p. with the oxalate salt of the non-phenolic cleavage product of thaliracebine was not depressed. The physical data of the non-phenolic cleavage product of thalfinine were taken with its free base. The ir spectrum in chloroform was superimposable with that of 8 0 authentic (S)-0 -methylarmepavine. The uv spectrum re vealed XMe0H 283 nm(loge 3.74), 225(4.37), 204 (4.74). nicix The spectrum was not changed in acid or base. The nmr spectrum (S,CDCl3) showed one N-methyl group at 2.53

(s, lXNCH^), three O-methyl groups at 3.58,3.78,3.83 (s, 3XOCH^), six aromatic protons at 6.05(s, lXArH),

6.55(s, lXArH), and an AB quartet centered at 6.78 and

7.03 (J=9cps, 4XArH). The cd spectrum in methanol showed

[el288+10'100' tei271-l,570, [9]232+45,600, [6]208+69,200 .

[alp6 +45.3° (c 0.095, MeOH).

The non-phenolic cleavage product of thalfinine was identified as {S)-0-methylarmepavine by comparison of its ir, uv, nmr, and cd spectra as well as tic behavior with 80 authentic (S)-0-methylarmepavine. b. phenolic fraction Attempts to isolate the two phenolic bases from the phenolic fraction by column chromatography and preparative tic were not successful. The sodium-liquid ammonia cleavage of thalfinine was repeated again with 99mg of thalfinine to yield 25mg of the non-phenolic fraction and 45mg of the phenolic fraction. The phenolic fraction was dissolved in 2ml of methanol. To the solution was added ethereal diazomethane generated from 2g of Diazald. The solution was then stored at 5°C for 3 days. The solvents and excess diazo methane were removed to give 27mg of residue. This residue showed four alkaloidal spots (Rf 0.75, 0.38; 0.30, 0.26) on tic with benzene-acetone-ammonium hydroxide

(32:16:1) as solvent system. Attempts to isolate the O- methylated compounds by column chromatography and prepara tive tic were not successful either. 110

(13) Isolation of adiantifoline (I) Adiantifoline was isolated from the fraction B of

the preparative tic (see p . 104). The crystals obtained

from fraction B dissolved in ethanol were recrystalli2 ed from ethanol to yield yellow rosettes (379mg), m.p. 145°C.

The ir spectrum and tic behavior were identical to those of adiantifoline previously isolated in this work

(see p. 97).

(14) Isolation of thaliglucinone (XI)

The crude residue of the fraction C from the

preparative tic (see p.104) was dissolved in chloroform and chromatographed on a column of neutral alumina,

(activity I, 5g)* From the chloroform eluent, 6 6 mg of residue of thaliglucinone was obtained. This residue was crystallized from methanol to yield yellow needle crystals(5mg). The compound showed strong green fluores

cence under uv light and had the same Rf (0.2 8 ) as that of authentic thaliglucinone 20 with benzene-acetone-

ammonium hydroxide (32:8:1) as solvent system. The ir spectrum in chloroform showed a lactone car

bonyl group at 1730cm ^ and was identical to that of authentic thaliglucinone isolated in our laboratories. 20

The uv spectrum showed 390nm (logs 3.83), 311(4.19), m a X 284(4.02), 264(4.72), 255(4.57), and 236(4.48) and no

shift was observed in acid or base. The nmr spectrum I l l

(6 ,CDCl^) exhibited one N-methyl signal at 2.39(s,N(CH^)2),

one O-methyl signal at 4.09 {S/IXOCH^}, and a methylenedioxy signal at 6.35 (s.OCH^O). On the basis of its ir, uv, nmr spectral data, and tic behavior, this compound

was identified as thaliglucmone.20

(15) Isolation of thaliracebine (LXXVI) The residue of the fraction 34, which was obtained

from the column chromatography of the ether soluble, ter

tiary, non-phenolic alkaloid fraction (see Table 7), was

dissolved in chloroform-benzene (4:1) and chromatographed

on a column of neutral alumina, (activity I, 45g) with

chloroform-benzene (4:1) as eluent. The first 150ml of the eluent contained three alkaloids with Rf 0.79,0.70, and 0.60 on tic with benzene-acetone-ammonium hydroxide

(32:16:1) as solvent system. The residue of the second 150ml eluent (192mg) contained only one alkaloid with Rf 0.70. This alkaloid was designated as thaliracebine. The three alkaloids in the residue of the first 150ml eluent were separated by preparative tic. The alkaloid mixture was dissolved in chloroform and streaked onto

five preparative thin layer silica gel HF-254 plates (20x20cm, 0.6mm thick). The plates were developed with benzene-acetone-ammonium hydroxide(48:16:1) twice. Three

zones, showing blue fluorescence under uv light, were observed on the developed plates. These zones were 1 1 2

collected and extracted with chloroform-methanol(1 :1 ). The solvents were then removed to yield three fractions,

which were designated as fractions A, B and C (16mg,360mg, and 137mg) with Rf 0.79,0.70, and 0.60, respectively.

The fraction B was dissolved in benzene and chroma

tographed on a column of neutral alumina (activity I, 15g). The polarity of the eluent was increased by increasing the

percentage of chloroform in benzene. Thaliracebine

(195mg) was obtained from the eluent of 40%, 60%, 80%

chloroform in benzene (400ml, 100ml, 200ml) and chloroform

(200ml). This residue of thaliracebine was combined with

that obtained previously from the column chromatography on neutral alumina. 26 This compound was amorphous, m.p. 83-84°C; falD + 121.4° (c 0.280, MeOH). The compound showed a negative p’nosphomolybdic acid test result and a positive chromatropic acid test result. The nmr spectrum (6 ,CDCl3) showed two N-methyl groups at 2.48 (s,2XNCH^)» four O-methyl groups at 3.63(s, 2XOCH3) , 3.76 (s ,1XOCH3), 3.78 (s ,1X0CH3), one methylenedioxy group at 5.89 (SjOCH^O), and ten aromatic protons at 5.78 (s,lXArH), 6.16 (s,lXArH),

6.52 (s,lXArH), and 6 .68-7.11(m,7XArH). The ir spectrum in chloroform showed no hydroxyl group. The uv spectrum indicated X**e0H 278 nm (logs 3.90) and showed no shift in max acid or base. The mass spectrum showed a molecular ion peak at m/e 652(0.05%) and a base peak at m/e 206. 113

The other intense peaks were at m/e 221(16%),

220(95), 218(10), 207(14), 204(13), 85(38), 83(60),

69(24), 57(10), 50(12). The cd spectrum in methanol showed

[0l31O~509' [ 61281+11'100' [0 12 38+103'000- (16) Preparation of thaliracebine dimethiodide (LXXX1) Thaliracebine(28mg) was dissolved in 5ml of acetone.

To the solution was added 3ml of methyl iodide. The

solution was left at room temperature overnight after

which the acetone and excess methyl iodide were removed

in vacuo to yield 36mg of residue. This residue was

chromatographed on a neutral alumina, column (activity I,

lOg, 1X23cm). The column was eluted with 5% methanol in chloroform (100ml) and 10% methanol in chloroform(lOOml).

From the 10% methanol in chloroform eluent, thaliracebine dimethiodide(2 0 mg) was obtained as an amorphous solid.

Attempts of crystallization of thaliracebine methiodide from various solvents failed. The compound was obtained as light tan powder, m.p. 203-205°C, by dissolving in minimum amount of methanol and then adding excess ether to the methanolic solution to precipitate out the compound.

The nmr spectrum (6 , CF-jCOOH) showed four quaternary N-methyl signals at 3.28(s,2X+NCH3), 3.56(s,2X+NCH3), four 0-methyl signals at 3.63, 3.72, 3.96, 4 . 00 (s,4XOCH3) , one methylenedioxy signal at 6 .1 0 (s^CH^O), and ten aromatic proton signals at 5.69(s,IXArH), 5.96(s,lXArH), 6.59-7.28 114

(m,8XArH).

The cd spectrum in methanol indicated (0 ] 2 7 -7+1 3 ,000,

fG)2 2 6 + 1 5 6 '0 0 °*

(17) Sodium-liquid ammonia cleavage of thaliracebine (LXXVI) Cleavage of thaliracebine was performed in the same manner as the cleavage of o-methylthalirabine. Twenty-five

milliliters of liquid ammonia was collected in a 50ml

three-necked flask and finely divided sodium (40mg) was

added. A solution of 104mg of thaliracebine in 20ml of

dry toluene was added dropwise within one hour. The reac

tion was kept for another three hours, then the excess ammonia was allowed to evaporate and methanol (5ml) was

carefully added to consume the excess metallic sodium. The reaction mixture was evaporated to a few milliliters. One hundred milliliters of 5% NaOH aqueous solution was added and the resulting solution was extracted with ether (100ml). The ether extract was washed with water, and dried over anhydrous sodium sulfate. The solvent was re moved to give 4 5mg of yellow oily residue which was designated as non-phenolic fraction. This non-phenolic

fraction showed two bases (Rf 0.50,0.35) on tic with benzene-acetone-ammonium hydroxide (32:16:1) as solvent system. The base with Rf 0.50 was the major spot. The remaining alkaline solution was acidified with glacial 115 acetic acid, made alkaline with ammonium hydroxide, and extracted with two 5 0ml portions of ether. The ether extracts were combined, washed with water, and dried over anhydrous sodium sulfate. The solvent was then removed to yield 47mg of solid residue, which was designated as phenolic fraction. This phenolic fraction showed two bases (Rf 0.21, 0.10) on tic with the same solvent system for the non-phenolic fraction. a. non-phenolic fraction Two bases in the non-phenolic fraction were separated by preparative tic with silica gel HF-254 as adsorbent.

The plate was developed with benzene-acetone-ammonium hydroxide (20:8:1). The major base (37mg), isolated from this preparative tic was purified by column chromatography on silica gel PF-254 (lg) . The column was eluted with chloroform(1 2 ml) and 1 % methanol in chloroform(1 0 0 ml). From 1% methanol in chloroform eluent was obtained a yellow oily residue(24mg), which was designated as non- phenolic cleavage product of thaliracebine. This base 80 had the same Rf as authentic (S)-o-methylarmepavine. This base was crystallized as the oxalate salt from acetone, m.p. 106-108°C. The physical data of this compound were taken as its free base. The ir spectrum in chloroform was superimposable with that of authentic (S)-O-methylarmepavine; 116 UV: A Me0H 283 nmfloge 3.73), 225(4.33), 204(4.73);CD (in max methanol) : [6 1 2 8 8 +8'800' ^270- 9 4 3 232+38/600' ^ 2 0 7 +

37,700; (a]p6 +6 8 .3°(c 0.120,MeOH); NMRU ,CDC13) , 2.52

(s ,1XNCH3), 3.57,3.77,3.82 (s,3XOCH3), 6.05(s,lXArH), 6.55

(lXArH), and an AB quartet centered at 6.76 and 7.02(4XArH, J=9cps). The mass spectrum showed a base peak at m/e 206 and a molecular ion at m/e 327(0.05%). The other intense peaks were at m/e 208(1%), 207(20), 204(1), 192(1), 191(4),

190(8), 162(2), 161(1), 135(1), 121(2), 77(1). The non-phenolic cleavage product of thaliracebine was identified as (S)-O-methylarmepavine by comparison of its tic behavior as well as its ir, uv, nmr, and cd spec- 8 0 tral data with the authentic sample. The melting point of the oxalate was in agreement with the value reported in 84 the literature (106-108°C). b. Phenolic fraction The phenolic fraction was chromatographed on a column of silica gel PF-254 (3g). The major base was obtained from the 4% methanol in chloroform eluent as a solid residue (31mg), which was crystallized from methanol to yield white prisms (15.5mg), m.p. 221-222°C; [alp^+98.1°(0.0 78, MeOH).

The mixture m.p. with the phenolic cleavage product of O- ethylthalirabine was not depressed. * * The ir spectrum in KBr showed a phenolic hydroxyl band at 3400cm-1 and was superimposable with that of the phenolic cleavage product of O-ethylthalirabine. UV: 117

xMeOH 2S5nm (i0 g E 3 .5 9 )# 280(3.60), 225sh(4.21), 204(4.61): max X0.01N methanolic NaOH 295nm (i0ge3.64), 287(3.69), 245sh max (4.17); NMR(6 , pyridine-d,.) ; 2.50(s,lXNCH^),3.64(s,lXOCH^),

3.92(t,J=6cps, CH), 4.94(s,broad,2XArOH), 6 .34(d,J=2.5cps, lXArH), 6 .73(d,J=2.5cps,lXArH), 7.08(d,J=9cps,2XArH), 7.29 (d,J=9cps,2XArH): Mass spectrum: 299(M+ , 0,05%), 194(1),

193(13),192(100),190(4),171(1),148(1); CD(in methanol):

[0]2 8 8 -l,2 7O, [0]271+2,28O, [9]227+41,100.

The compound was identified as(S)-l-(41-hydroxybenzyl)- 2-methyl-5-methoxy-7-hydroxy-l,2,3,4-tetrahydroisoquinoline by comparison of its m.p. and tic behavior, as well as its ir, uv, nmr, cd, and mass spectral data with those of the phenolic cleavage products of O-ethylthalirabine (see p. 80 ) and thalistyline. 7 8 In addition, the mixture m.p.’s of the compound with the phenolic cleavage products of O- ethylthalirabine and thalistyline were not depressed.

(18) Isolation of thalruqosaminine(CXIX) The residue of the fraction C from the preparative tic (see p.1 1 2 ) was dissolved in benzene and chromato graphed on a neutral alumina column (activity I, lOg) in the usual way. The residue of the alkaloid was obtained from the eluent of 20%,40%,60%, 80% chloroform in benzene (40ml, 100ml, 100ml, 100ml) and chloroform(100ml). The

Rf value of this alkaloid was 0.60 and was the same as 118

that of authentic thalrugosaminine isolated from T. 72 revolutum in our laboratories.

The alkaloid was amorphous and could not be crystal- 2 6 lized from various solvents, [<*1D ”80.0°{c 0.30, MeOH) . The cd spectrum in methanol showed the molecular ellip-

licities at [0 ]2 8 g + 6 ,1 1 0 , (0 ]2 7 Q-1 2 ,2 0 0 , [ 8 ]241~60,000, and [8 ]2 2 4 +112,000. The uv spectrum revealed the maxima at *Me0H 310 nm sh(loge 3.15), 282(3.91), 232 sh(4.65), max and 206(5.09). The nmr spectrum(6 ,CDC1^) indicated two N-methyl groups at 2.53,2.57 (s,2XNCH3), five 0-methyl groups at 3.09,3.40,3.81,3.85,3.95(s,SXOCH^), and nine aromatic protons at 6.42-7.20 (m,9XArH). The ir spectrum in chloroform was identical to that of authentic thalrugo- 72 saminine. The cd, uv, nmr, ir spectra and tic behavior of this alkaloid were identical to those of authentic thalrugosaminine.^

(19) Isolation of obaberine (CXV1II)

The alkaloid was isolated as the dihydrochloride salt from the fraction 35 and fractions 36-38 of the column chromatography of the ether soluble, tertiary, non- phenolic alkaloid fraction (see Table 7). The procedures of isolation are described as follows. The residue of the fraction 35 from the column chromatography of the ether soluble, tertiary, non-phenolic alkaloid fraction (see Table 7) was dissolved in 20ml of 119 methanol. A 10% HC1 solution in methanol was added to the methanolic solution of the alkaloidal residue until the pH of the solution was 3- The solvent was removed and the residue dissolved in ethanol. Concentration of the solution resulted in white needle crystalline material

(4 9 mg), which was designated as the obaberine dihydro chloride fraction A. The residue of the fractions 36-38 was chromato graphed on a column of neutral alumina (activity I, 55g) in the usual way. From the first 140 ml of chloroform eluent, obaberine was obtained as an amorphous solid (809 mg), which was transformed to the dihydrochloride salt as described above. From the ethanolic solution of the residue of the dihydrochloride salt, a compound crys tallized as white needles (645mg) was obtained and was designated obaberine dihydrochloride fraction B. The second 410 ml of chloroform eluent contained thaliadanine, which had Rf 0.49 on tic with benzene-acetone-ammonium hydroxide (32:16:0.5) as solvent system. This fraction was further purified to isolate thaliadanine, as described on p. 120. Obaberine dihydrochloride fractions A and B were combined and recrystallized from methanol twice to yield 306mg of white needles, m.p.255°C. The spectral measurements were made using the alka loid base. + i6 8«9°(c 0.147,MeOH). The uv spectrum gave 283nm (loge 3.82), 235sh<4.43), and 207(4.89). 120

The nmr spectrum (<5,CDCl3) showed two N-methyl groups at

2.56, 2, 6 5 {s, 2XNCH 3)# four O-methyl groups at 3.19,3.62,

3.78,3.88(s,4XOCH3), ten aromatic protons at 5.48(s,lXArH), 6.31-7.47(m,9XArH). The ir spectrum in chloroform was

superimposable with that of authentic obaberine isolated from T. lucidum 15 in our laboratories. The cd spectrum in methanol showed [ 0]2g3+6,440, [9J2g4+6,150, [Q] 2 35 +108,000, and [0]2^g+237,000. The ir, uv, nmr, cd spectral data, and tic behavior of this alkaloid were identical to those of authentic obaberine. 15

(20) Isolation of thaliadanine (CVII)

The alkaloid thaliadanine was obtained from two sources. One was from fractions 36-38 of the column chromatography of the ether soluble, tertiary, non-phenolic alkaloid fraction (see Table 7). The other was from fractions 39-4 5 of the same column chromatography. Their isolation procedures will be mentioned separately as follows. The residue of fractions 36-38 was chromatographed on a neutral alumina column as mentioned on p.119- Obaberine was obtained from the first 14 0ml of chloroform eluent. The second 410ml of chloroform eluent contained thaliadanine and was purified by preparative tic. The residue was dissolved in chloroform and streaked on six silica gel G plates (20X20cm, 0.6mm thick). The plates 1 2 1 were developed with benzene-acetone-ammonium hydroxide

(48:16:1) twice. The zones, which contained thaliadanirie were collected and extracted with chloroform-methanol (1 :1 ). The solvents were removed to yield the amorphous solid

(267mg), which was designated as thaliadanine fraction A. The residue from the fractions 39-45 was chromato graphed on a column of silica gel PF-254 (220g,4X49cm). The column was eluted with 1%(1,000ml), 2%(700ml), 3%(800ml), 5%(1,000ml), 10%(2,000ml) methanol in chloro form. From the 3% methanol in chloroform eluent was obtained an amorphous solid (799mg). This amorphous solid was then further purified by preparative tic. The pro cedure was the same as that described above. An amorphous solid of thaliadanine (367mg) was obtained. This was designated as thaliadanine fraction B. The residue (300mg) of thaliadanine fraction B was purified again with column chromatography on silica gel PF-254 (30g, 1.4X30 cm). From the 2% methanol in chloroform (170ml) was obtained pure amorphous thaliadanine (165mg). Attempts to crystal lize this alkaloid with various solvents failed. The ir spectrum in chloroform revealed a hydroxyl group absorption at 3540cm~*. The alkaloid showed negative result to phosphomolybdic acid test. The uv spectrum showed 312nm (loge 4.11), 302(4.18), and 281(4.33). u ia X 26 The spectrum gave no uv shift in acid or base.[a]D +80.7°

(c 0.408, MeOH). The cd spectrum in methanol indicated 122

101 308~15'900/ 1 e1276_17'600' [0]2 4i+166'°00* The nmr sPec_ trum (6,CDC13) showed signals corresponding to two N-methyl

groups at 2.38, 2.48(s,2XNCH3), seven 0-methyl groups at

3.78 (s ,4XOCH3>, 3.88(s ,1XOCH3>, 3.97(s,2XOCH3, six aromatic

protons at 6.45,6.50,6.55,6.58,6.67,8.05(s,6XArH), and a deutetrium exchangeable phenolic proton at 4.83{s,broad, lXArOH). The mass spectrum showed a very weak molecular ion nt

m/e 712(0.1%) and a base peak at m/e 192. The other intense peaks were at 520(13), 370(7), 354(3), 206(66), 149(22),

69(28), 57(35), 55(40), 45(22), 44(52), 43(28), 41(48).

(21) Preparation of 0-methylthaliadanine (I)(Adiantifoline)

Thaliadanine(62mg) was dissolved in 20ml of methanol.

To the solution was added ethereal diazomethane generated

from lg of Diazald. It was left at 0°C for three days and then the solvents and excess diazomethane were removed in vacuo to yield 70mg of residue, which was crystallized

from ethanol to yield light yellow crystals(6 mg), m.p. 142°C. Its mixture melting point with authentic adianti

foline showed no depression. The ir spectrum in chloroform was identical to that of authentic adiantifoline. UV: 312nm (log e 4.17),

300(4.26),285(4.40); NMR (6,CDC13): two N-methyl groups at 2.43, 2.4 7(s,2XNCH3), eight 0-methyl groups at 3.58

(s,1XOCH3), 3.77 (s,3XOCH3) , 3.82(s,lXOCH-j) , 3.8 8 (s,1X0CH3), 123

3.92 {s,lXOCH^), 3.95(s,lXOCH^), and six aromatic protons at 6.20, 6.50, 6.53, 6.57, 6.60, 8. 02 (s, 6XArH) ; {ct ]

+83.0°{0.190,MeOH); CD(in methanol): [9]^q^-17,400,

[0]277-15,8OO, [9]23g+156,000.

This compound was shown to be identical to adianti

foline by the comparison of its melting point, tic behav

ior, as well as its ir, uv, nmr, and cd spectral data with authentic adiantifoline. 124 (22) Preparation of O-ethylthaliadanine(CVII1) Thaliadanine (153mg) was dissolved in 30ml of methanol. To the solution was added ethereal diazoethane generated from 6 g of nitrosoethylurea. The solution was stored at 0°C for two days. The solvents and excess diazoethane were removed to yield 239mg of residue. The residue was crystallized from methanol to yield yellow rosettes(103mg), m.p. 161-162°C. The ir spectrum in chloroform was very similar to that of adiantifoline and showed no hydroxyl absorption.

The nmr spectrum (5.- CDC13) showed signals corresponding to two N-methyl groups at 2.43, 2.48(s, 2XNCH3), seven O- methyl groups at 3.78(s,3XOCH3), 3.81,3.90,3.94, and

3.96, one methyl group at 1. 29

The mass spectrum showed a base peak at m/e 220 and a very weak molecular ion at m/e 740(0.1%) . The other intense peaks were at m/e 521(1%), 520(1),222(1),221(23),

219 (1),218(1),192(1),191(1) ,190(1) .

Anal. Calcd for C4 3H52N 209 :C, 69 . 71 :H, 7. 08; N, 3. 78. Found: C,69.57;H,7.11;N,3.88. (23) Oxidation of O-ethylthaliadanine(CV1I1)

O-ethylthaliadanine (165mg) was dissolved in acetone (20ml). Potassium permanganate (200mg) was added over a period of four hours while stirring and then the mixture was stirred for an additional four hours. The excess of potassium permanganate was destroyed by adding 1 0 ml of 125

methanol and stirring for half an hour. The solvents were removed and the residue was redissolved in acetone. The solution was then filtered. The filtrate was evapo rated to dryness to give 177mg of solid residue. The residue was dissolved in 10% HCl aqueous solu

tion (50ml) and extracted twice with ether (100ml each time). The ether extracts were combined, washed with water, and dried over anhydrous sodium sulfate. The sol vent was then removed to give 126mg of residue which was designated as neutral fraction. This fraction contained

three alkaloidal compounds (Rf 0.79,0.70, and 0.41) on

tic with benzene-acetone-ammonium hydroxide (40:10:1) as

solvent system. The residue of the neutral fraction was chromato graphed on a column of silica gel PF-254 (5g). From the first 16ml of the 1 % methanol in chloroform eluent was obtained the residue of a compound (71mg) with Rf 0.79. This compound was designated as the O-ethylthaliadanine oxidation product A. From the next 8 ml of 1% methanol*, in chloroform eluent was obtained the residue of a compound

(27mg) having an Rf of 0.41. This compound was designated as the O-ethylthaliadanine oxidation product B.

After the neutral fraction was removed, the acidic aqueous solution, mentioned above, was made alkaline with ammonium hydroxide and extracted three times with ether

(100ml each time). The ether extracts were combined, 126 washed with water, and dried over anhydrous sodium sul fate. The solvent was then removed to yield 7 2mg of residue, which was designated as the basic fraction. This fraction was chromatographed on a column of silica gel PF-254 (lOg). From the eluent, 5mg of the O-ethylthaliadanine oxidation product A and 6 mg of O-ethylthalia danine oxidation product B were obtained. These two alkaloidal fractions were combined with those obtained from the column chromatography of the neutral fraction. a. O-ethylthaliadanine oxidation product A (CXI).

The combined fraction of the O-ethylthaliadanine oxidation product A from the column chromatography was purified with neutral alumina, column (activity I, lOg). From the 10% chloroform in benzene eluent (50ml) was ob tained 69mg of the residue of O-ethylthaliadanine oxida tion product A, which was crystallized from acetone to give 30mg of yellow prisms, m.p. 147°C. Its mixture melting point with authentic dehydrothaliadine (CXI), obtained from oxidation of adiantifoline (see p.129), was not depressed. The ir spectrum in chloroform gave a strong con jugated carbonyl absorption at 1675cm 1 and was superimposable with that of authentic dehydrothaliadine. The uv spectrum showed XM®°Hmax 330nm (loge 4.24), 272(4.67), 257 (4.68); A0-01N methanoli c HCl 3 5 4 (3 ,4 4 ), 347(3.47), 319 127

(4.01), 305sh(4.04), 270(4.86), 235sh(4.37), 222sh(4.40), 202(4.63). The spectrum in base was identical to that in

methanol. The nmr spectrum (6 , CDCl^) showed the signals corresponded to one N-methyl group at 3.02(s,lXNCH^), six

0 -methyl groups at 3.76, 3.93, 3.95, 4.00, 4.02, 4.08

(s,6 XOCH^), two methylene groups at 3.27(s,2 XCH2 ), five

aromatic protons at 6 . 50(s,2XArH) , 7.08(s,lXArH), 7.43

(s,lXArH), 9.15(s,lXArH), and one aldehyde proton at 10.40

(1XCHO). This compound was identified as dehydrothaliadine on the basis of its tic behavior, melting point, ir, uv,

nmr spectral comparison with authentic dehydrothaliadine (see p.128) and no depression of the mixture m.p.

b. O-ethylthaliadanine oxidation product B(CX) The combined fraction of the O-ethylthaliadanine

oxidation product B from the column chromatography was

finally purified by preparative tic on silica gel HF-254

with 2 % methanol in chloroform as developing solvent system. The residue (30mg) obtained from the preparative

tic was crystallized from methanol to yield 9mg of crys talline plates, m.p. 120.5°C. Its mixture melting point with O-ethylthalifoline(CX) was not depressed.

The ir spectrum in chloroform showed a strong car bonyl absorption at 1640cm * and was superimposable with that of O-ethylthalifoline. The uv spectrum revealed 128

X in ci x 296nm{l0g e 3.79), 270(3-87), 261(3-91), 220(4-55), 208sh(4.50). The spectrum showed no change in acid or

base. The nmr spectrum (6 , CDCl^) showed one N-methyl

group at 3.13(s/lXNCH^), one O-methyl group at 3-89

(s, 1XOCH^), one methyl group at 1.46(t, J=7cps, OCI^CH^) ,

three methylene groups at 4.16(q, J-7cps, OCH2 CH3), 2.92

and 3.55(t, J=6.5cps, CH^CH^), and two aromatic protons at 6.62 and 7.60(s,2XArH).

The mass spectrum showed a molecular ion at m/e 2 35,

which was also the base peak. The other intense peaks were at m/e 236(15%), 220(10), 207(10), 206(10), 192(56), 164(82), 163(18), 136(33), 42(10).

This compound was identified as O-ethylthalifoline on the basis of its melting point, tic behavior, ir, uv, nmr, mass spectral comparison with synthetic O-ethylthalifoline (see p- 131) and no depression of the mixture m.p.

(24) Synthesis of dehydrothaliadine (CXI) from adiantifoline(1 ) Adiantifoline (200mg) was dissolved in 20ml of acetone. Potassium permanganate (5 30mg) was added over a

4 hour period while stirring. The solution was stirred 129

for an additional two hours. The excess potassium per manganate was destroyed by sodium bisulfite. The mixture was filtered and the residue was washed with acetone. The

filtrate was evaporated to dryness to give 134.5mg of residue, which showed two alkaloidal spots (Rf 0.75,0.44) on tic with benzene-acetone-ammonium hydroxide (32:16:1) as solvent system. This residue was chromatographed on a

column of silica gel 60 (20g). The column was eluted with benzene (100ml), 0.5% acetone in benzene (400ml),

1% acetone in benzene (1.51). From the 1% acetone in benzene eluent, dehydrothaliadine (64mg, Rf 0.75) was obtained first. N-methylcorydaldine(9mg, Rf 0.44) was obtained from the later fractions of the same eluent.

dehydrothaliadine The residue of dehydrothaliadine was crystallized from acetone methanol to give yellow needle crystals, which were recrystallized once from acetone-methanol to give 36mg of pure compound, m.p. 147-148°C. The ir spectrum in chloroform showed a strong con jugated carbonyl absorption at 16 75cm The uv spectrum revealed AMe0H 3 3 0 nm (logs 4.28), 272(4.71), 257(4.71); max

X0.01N methanolic HC1 3 6 4 0 .5 0 ), 347(3.54), 320(4.06), max 307sh(4.11), 270(4.90), 236sh(4.41), 222sh(4.44), 204 (4.62). The spectrum in base was identical to that in methanol. The nmr spectrum (fi^DCl^) showed one N-methyl 130 group at 3.00(s,lXNCfTj), six O-methyl groups at 3.75, 3.92,

3.94, 3.99, 4.01, 4.07 (s,6 XOCH3), two methylene groups at

3.2 5(s,CH2 CH2), five aromatic protons at 6 .49(s,2XArH),

7.0 8 (s,lXArH), 7.42 (s,lXArH), 9.13(s,lXArH), and one alde hyde proton at 10.40(s,CHO). The mass spectrum showed a molecular ion peak at m/e 533, which was also the base peak. The other intense peaks were at m/e 535 {5%) ,

534(22), 530(3), 519(3), 518(6), 503(3), 502(3), 222(3),

44(3) . b. N-methylcorydaldine(CX) The residue of N-methylcorydaldine, obtained from the column chromatography, was purified by preparative tic on silica gel HF-254. The tic plate was developed with

2% methanol in chloroform. The compound isolated from this preparative tic was 6 .5mg and was crystallized from ethanol to give 3mg of crystalline plates, m.p. 120.S^C.

The ir spectrum showed a strong carbonyl absorption at 1640cm The uv spectrum showed X 295nm(log e 3.83),

269(3.90), 261(3.92), 221(4.51), and 208sh(4.38). The spectrum revealed no change in acid or base. The nmr spectrum showed one N-methyl group at 3.12 (s,1XNCH3), two 0-methyl groups at 3.89, 3.91(s,2XOCH3), two methylene groups at 2.92, 3.54(t, J=6 cps,CH2CH2), and two aromatic protons at 6.64, 7.61(s,2XArH). 131

(25) Synthesis of O-ethylthalifoline (XCII) Synthetic thalifoline (14mg)67'79 was dissolved in methanol (1 0 ml) and to the solution was added ethereal diazoethane, generated from lg of nitrosoethylurea. It was left at 0°C for three days and then the solvents and excess diazoethane were removed in vacuo to yield 19mg of residue, which was crystallized from ethanol to yield 5mg of crystalline plates, m.p. 120.5°C. The ir spectrum in chloroform showed a strong car bonyl absorption at 1640cm *. The uv spectrum revealed AMeOH 2g6nm (logc 3.78), 270 ( 3.86),261(3.91), 220 (4.52), max and 208sh(4.43). The nmr spectrum (6 ,CDCl.j) showed one N-methyl group at 3.13(s,lXNCH^) one O-methyl group at 3. 89 (s, 1XOCH.J , one methyl group at 1.46(t,J=7cps,0CH_CHo), * " j 6 " J and two aromatic protons at 6.62, 7.60(s,2XArH).

The mass spectrum showed a molecular ion peak at m/e 2 35, which was also the base peak. The other intense peaks were at m/e 236(12%), 220(8), 207(7), 206(7), 192(52), 165(7), 164(74), 163(15), 136(26), 135(7), 42(8). 132

(C) Results of pharmacological tests of alkaloids isolated from T. minus race B (1) Effects of thalirabine on blood pressure in dogs

Dogs of either sexes weighing 7 .5-12.5kg were utilized in this study. The dogs were anesthetized with 35mgAg sodium pentobarbital, i.v. Trachea was cannulated and in some experiments bilateral vagotomy was performed. The right carotid artery was cannulated and the arterial blood pressure was recorded via a linear core transducer (Narco

Bio-System, Inc.) connected to a physiograph. The femoral vein was isolated and cannulated for the administration of the alkaloid. The solutions of the alkaloid were prepared in 0.1N HC1 aqueous solution. Injection volumes never exceeded 2 ml/injection.

The mean blood pressure was determined as follows;

Mean blood pressure (MBP) = diastolic blood pressure (DBP) + 1/3 pulse pressure (PP). Pulse pressure (PP) = systolic pressure-diastolic pressure.

The effects of thalirabine on blood pressure of dogs are summarized in Table 9. Table 9. Effects of thalirabine on blood pressure in dogs

Alkaloid Dose No. of Change in mean Duration Remarks (mg/kg) exp. b.p.(mmHg) (Min.)*

thalirabine 0 . 1 1 - - No apparent changes were observed

* 0 . 2 1 -56 - The duration of the effect was not determined.

1 -117,-116 40,- Two doses of thalirabine were administered to the animal. The response to the second dose was a biphasic one: an initial pressor response (1 / 2 min.), equivalent to +18mmHg was fol lowed by a sustained fall in blood pressure. The duration of the fall in blood pressure of the second dose was not deter mined .

thalirabine 0 . 1 1 — — No apparent changes were observed monohydrodi- 0 . 2 -40 - The duration of the effect was iodide not determined.

0,5 1 -151 12 Animal died 12min. after adminis tration of the alkaloid

1.5 1 -170 3 Animal died 3min. after adminis tration of the alkaloid * The duration was calculated from the initial change in blood pressure to the complete w recovery in blood pressure to preinjection level. w 134

(2) Effects of alkaloids isolated from T. minus race B on blood pressure in rabbits

The effects of thalirabine and other alkaloids isolated from this plant on the blood pressure of anesthe tized rabbits were studied. Rabbits of either sex of weight ranging between 2.4 and 3.2 kg were anesthetized with 7ml/kg of a 25% aqueous solution of urethane# i.v. Blood pressure was determined in a manner similar to that described in the experiments of dogs. In some experi ments an electrocardiogram was used to monitor the experi ment. The effects of the alkaloids on blood pressure of rabbits are summarized in Table 10. Table 10. Effects of alkaloids isolated from T. minus race B on blood pressure in rabbits

Alkaloid Dose No. of Change in mean Duration Remarks (mg/kg) exp. b.p.(mmHg) (min.)*

thalirabine 0 . 1 3 +24, + 27,+28 3,3,3

0 . 2 2 +13,+13 3.3

1 +15 3 Initial increase in blood pres sure followed by a drop to fatal level in 4 min.

0.4 1 +14 0.75

0.5 1 +14 0.75 Initial increase in blood pres sure followed by a drop to fatal level in 4 min. Gradual decrease in heart rate was observed.

1 1 + 28 2.5 Initial increase in blood pres sure followed by a drop to fatal level in 4 min. Gradual decrease in heart rate was observed.

methothali- 0 . 1 1 -- No apparent changes were rabine observed. diiodide 0 . 2 + 26 1.25 Gradual increase in blood pres sure followed by a drop to fatal level in 8 min.

thalirac 0 . 1 2 -2 0 , - 2 2 0.5,0.5 These changes occurred not immed ebine iately but rather 1 - 2 min. after 0 . 2 -10,-13 0.5,0.5 administration of the alkaloid. 0.4 -14,-8 0.5,0.5

1 -16 0.5 * The duration was calculated from the initial change in blood pressure to the complete £ recovery in blood pressure to preinjection level 01 Table 10. (Continued)

Alkaloid Dose No. of Change in mean Duration Remarks (mg/kg) exp. b.p.(mmHg) (min.)

thalirac- 0 . 2 1 + 1 0 0.5 In contrast to thaliracebine, ebine thaliracebine dimethiodide pro dimethio- duced a pressor response. dide 0.4 +18 1.75

Alkaloid 0 . 1 1 —- No apparent changes were

VIIB - - observed. 0 . 2

0.4 — —

thalfine 0 . 1 1 —- No apparent changes were observed. 0 . 2 — -

1 — -

thalfinine 0 . 1 2 - - No immediate changes were observed. However, a decrease 0 . 2 1 - - of mean blood pressure (10 to 23 mmHg) was observed 2 to 3 min, 0.4 2 - - after the administration of the alkaloid. The drop in blood 1 - - pressure lasted for 0.5 min.

thaliadan- 0 . 1 2 -15,-16 1.5,1.5 ine 0 . 2 -15,-19 1.5,1.5

0.4 -22,-17 2 , 2

1 1 -13 2 Ul Ci Table 10. (Continued)

--- = --- Alkaloid Dose No. Of Change in mean Duration Remarks (mg/kg) exp. b.p.(mmHg) (min.)

thaliadine 0 . 1 2 -27,-22 3.5,2

0 . 2 -35,-23 4,2.5

0.4,1 1 , 1 -25,-30 " I " The duration of the effect was not determined

(S)- reticul- 1 1 - - No apparent changes were ine observed. 2 - —

4 — — thalidas- 1 1 - 2 0.5 ine 2 - 1 1 2 4 -18 3

adiantifol- 1 1 -25 2 ine

thaligluc- 0.5 1 —— No apparent changes were inone* observed. 1 . 0 -7 1 1.75 -32 4

thalrugos- 1 1 -18 2 aminine * This alkaloid was injected into the jugular vein. Table 10. (Continued)

Alkaloid Dose NO. Of Change in mean Duration Remarks (mg/kg) exp. b.p,(mmHg) (min.) obaberine 1 1 -45 3

4 -45 2.5 139

(3} Results of antimicrobial tests Although the major goal of this work was to isolate

the hypotensive alkaloid(s) , the alkaloids isolated were

also subjected to antimicrobial tests, due to the reported

presence of antimicrobial activities of the ethanolic 14 extract of this plant. The method of testing was the agar dilution-streak technique. 14 Six microorganisms were

used, namely, 1. Staphylococcus aureus, Smith strain (ATCC no. 13709); 2. Escherichia coli (ATCC no. 9637); 3. Salmonella gallinarum (ATCC no. 9184): 4. Klebsiella pneumoniae AD (ATCC no. 10031); 5. Mycobacterium smegmatis

607B (ATCC no. 607); 6 . Candida albicans (ATCC no. 10231).

The results of the antimicrobial tests are summarized in

Table 11. 140

Table 11. Results of antimicrobial tests of the alkaloids isolated from T. minus race B.

* Alkaloid Minimum inhibitory Bacterial no. concentration(yg/ml) 1 2 3 4 5 6

** thalirabine 1 0 0 i i i i a i

thaliracebine 1 0 0 i i i i a i

* thalfine 1 0 0 i i i l a i

thalfinine 50 i i i i a i

thaliadanine 1 0 0 i i i i a i

thaliadine 1 0 0 i i i i i i

adiantifoline 1 0 0 i i i i i i

* The bacterial no. refers to the microorganisms mentioned on p. 139. ** i=inactive, a=active DISCUSSION

In a pharmacological screening of eleven species of Thalictrum growing in the medicinal plant garden of The Ohio

State University, College of Pharmacy, the non-quaternary alkaloid fraction of Tj_ minus race B was found to produce a prolonged hypotensive activity in normotensive dogs.^ Two minor alkaloids, alkaloid VI and alkaloid VII, from the plant, were responsible for a major portion of the hypo- 2 tensive activity. The fractionation scheme was designed to isolate these hypotensive alkaloids. The active fraction, chloroform extract, was subjected to repeated column chro matography resulting in the isolation of alkaloid VI which was designated thalirabine. In this study the structural elucidation of thalirabine was carried out. The effect of thalirabine on the blood pressure of the anesthetized dog and rabbit was determined. The alkaloid VII was found to be a mixture of alkaloids. Only one of the alkaloids (alkaloid VII B) from this frac tion was isolated. However, its structure elucidation could not be carried out due to its low yield. The tertiary alkaloid ether extract (see Fig. 3) was also investigated. The alkaloids isolated from this frac tion were subjected to antimicrobial tests, due to the re- 141 142 ported presence of antimicrobial activities of the ethanolic extract of this plant. 14 Also it may be added, thalirabine obtained from the chloroform extract was a partially quater- narized bisbenzylisoquinoline. Hopefully, the investigation of the tertiary alkaloid ether extract would lead to the iso lation of the structurally related tertiary bisbenzyliso quinoline of thalirabine which might serve as additional evidence to support the structural assignment of thalira bine. Of the twelve alkaloids so far isolated from the tertiary alkaloid ether extract, (s )-reticuline was obtained from the fractions that normally contained phenolic alka loids, and the remainder were obtained from the fractions which generally contain non-phenolic alkaloids (compound XVI, thalfine, thalidasine, adiantifoline, thaliadine, thalfinine, thaliglucinone, thaliracebine, thalrugosaminine, obaberine, thaliadanine). Thalfine and adiantifoline have previously been iso- 4 lated from this plant. (S)-reticulrne, thalidasine, adianti foline, thaliglucinone, thalrugosaminine, and obaberine are completely characterized known compounds. The isolation of thalfine and thalfinine have been reported in the literature and they have been partially characterized. 3 ^ ' 6 4 Compound XVI, thaliadine, thaliracebine, and thaliadanine have been isolated for the first time.

Structural assignments of thaliracebine, thalirabine, thalfinine, thalfine, thaliadine, and thaliadanine will be discussed. The remainder of the alkaloids isolated from 143

T. minus race B will also be discussed in turn.

A. Thaliracebine (LXXVI) This alkaloid was named thaliracebine after the race of the plant. In the rabbit, 0.1 mg/kg of the alkaloid produced lowering of the mean blood pressure (20-22 mmHg).

In addition, a weak antimicrobial activity (lOOgg/ml) against smegmatis was also observed. The uv spectrum of this alkaloid was characteristic of a benzylisoquinoline or a bisbenzylisoquinoline alkaloid.

The absence of a bathochromic uv shift in base, a phenolic hydroxyl absorption in the ir spectrum, and a positive result when tested with the phosphomolybdic acid test reagent indicated that the alkaloid was non-phenolic. The nmr spectrum showed the presence of two overlapping

N-methyl groups at 62.48(s, 2XNCH^), and four O-methyl groups at 63.63(s, 2X0CH.j), 3.76 (s, lXOCH^), and 3.78 (s, lXOCH^) . A two-proton singlet at <55. 89 suggested the presence of a methylenedioxy group, which was also indicated by the positive test result with chromatropic acid test reagent. Ten aromatic protons were observed in the nmr spectrum with two high field aromatic protons at 65.78 and

6.16 (C- 8 and C-8 ' or C- 8 * and C-8 ), and eight other aro matic protons at 66.52(s, 1H), 6.68-7.11 (m, 7H).

Assuming that thaliracebine was a bisbenzylisoquinoline alkaloid containing two N-methyl groups, four O-methyl 144 groups, one methylenedioxy group, and ten aromatic protons the alkaloid would have one diphenyl ether bridge and the molecular formula C39H44N2<*>7 wou-*-^ appropriate.

0 CH

(LXXVI)

V ?C H 3

(IXXVII) m/e 206 (U£X7III)m/« 220

The low resolution El mass spectrum gave a very weak molecular ion peak at 652 (0.05%), which fit the suggested molecular formula, obtained from the interpretation of the nmr spectrum. The two most intense peaks at m/e 206 (100%) and 220 (95%) indicated the presence of fragments (LXXVII) and (LXXVIII), which resulted from the highly favored benzylic cleavages. The very weak molecular ion and the intense ions due to the two isoquinoline nuclei (LXXVII) and (LXXVIII) suggested thaliracebine to be a bisbenzyliso- OC quinoline with a tail to tail diphenyl ether linkage. 145

From the nmr and mass spectral data mentioned above, three of the four O-methyl groups could be assigned to the

ring B and C. The fourth O-methyl group, therefore, would

be in ring E or F. In order to establish the substitution pattern and the absolute configuration, a sodium-liquid ammonia cleavage of thaliracebine was performed. The reaction yielded two major

bases, one of which was non-phenolic and the other was phe nolic. The tic behavior and the nmr, ir, and cd spectra of the non-phenolic cleavage product were identical to 80 those obtained from authentic (S)-O-methylarmepavine (LXXIX). The fragmentation pattern of the low resolution El mass

spectrum of the non-phenolic cleavage product also was in agreement with the same data for (S)-0-methylarmepavine. The spectrum showed a low intensity molecular ion at m/e 327

(0.05%, C20H25NO3^ and a 1 3 3 3 6 Pealt at m/e correspond ing to the fragment (LXXVII). The melting point of its oxalate salt, 106-108°C, was also identical to the re- 84 ported value in the literature (lit. 106-108°C). The phenolic cleavage product of thaliracebine was identified as (S)-1-(4'-hydroxybenzyl)“2-methyl-5-methoxy-7- hydroxy-1,2,3,4-tetrahydroisoquinoline (LXXX) on the basis of its identical m.p. and identical ir, uv, nmr, cd and mass spectral data with the same data obtained from the phenolic cleavage products of O-ethylthalirabine and thalistyline. The structure of the phenolic cleavage product of 146 thalistyline was established as structure (LXXX) by

Wu et al. 7 8 in our laboratories.

(LXXVI)

Ng /NH.

OCH. OGH.

OCH. CH- +

OCH 3 (LXXIX) (LXXX)

With the structural proof of these two sodium-liquid ammonia cleavage products in hand# the position of four

O-methyl groups of thaliracebine was established at C-6 , C-7, C-5' and C4". The methylenedioxy group was assigned at C-6 ' and C-7*. Furthermore# the fact that two cleavage products possessed S-configuration allowed the S^-configura tions to be assigned to thaliracebine. Assuming bisbenzylisoquinolines are formed in nature through the phenolic oxidative coupling of simple benzyliso- 97 quinolines, C-3", C-4"' diphenyl ether linkage of two benzylisoquinoline units in thaliracebine would be in favor over a C-2", C-4'" linkage. The location of the ether link age could be confirmed with permanganate oxidation of 147

thaliracebine to yield a substituted diphenyl ether dicar-

boxylic acid. However, the oxidation was not performed

due to the extremely low yield of this reaction and the small quantity of thaliracebine in hand. From the above spectroscopic evidence, the nature of the cleavage products and biogenetic consideration, the structure of thaliracebine was tentatively proposed as

(LXXVI). Preparation of thaliracebine dimethiodide (LXXXI) was

also carried out. Quaternarization of both nitrogens was

confirmed by the presence of two quaternary N-methyl sing lets at 63.28(2X+NCH3) and 3.56 (2X+NCH3) in the nmr spectrum (in CF-jCOOH). The nmr spectrum of this dimethi odide salt was very similar to that of methothalistyline

diiodide (LXXXVI), only being different in one less O-methyl group and one more aromatic proton. (See Table 12) The cd spectrum of this dimethiodide was taken and was used for the assignment of the absolute configuration of thalirabine and thalistyline which will be discussed later in this dissertation.

(LXXXI) 148

Table 12. Chemical shift data ( 6 values) for thaliracebine dimethiodide and methothalistyline diiodide*

Alkaloid +n c h 3 o c h 3 ArH o c h 2o (C- 8 & C- 8 ' )

thaliracebine 3.28, 3.28, 3.63, 3.72 5.69, 5.96 6.10 dimethiodide 3.56, 3.56 3.96, 4.00, (LXXXI)

methothalistyline 3.27, 3.27, 3.63, 3.73, 5.75, 5.88 6.08 diiodide 3.53, 3.53 3.98, 4.03, (LXXXVI) 4.06

*The nmr spectra were taken in CF^COOH. 149

Thaliracebine dimethiodide was also tested for hypo

tensive activity in the rabbit. However, in contrast to thaliracebine, this compound produced a pressore response,

(see Table 10)

B. Thalirabine (LXXXII) This alkaloid was originally designated as alkaloid VI

and later was named Thalirabine after the race of the plant. The administration of 0.2mg/kg of thalirabine to the

anesthetized vagotamized dog produced a hypotensive effect with change in mean blood pressure ranging from -37 to -

120mmHg. The doses of 0.5mg/kg and 1.5mg/kg caused very marked fall in mean blood pressure, followed by death of the

animal. In contrast, this compound produced a pressor effect when administered in equivalent doses to rabbits. In

some cases the death of the animal was apparently due to myocardial depression, as evidenced in the ECG. Thalirabine also showed weak antimicrobial activity (lOOpg/ml) against

M. smegmatis. Thalirabine was a very polar alkaloid. In previous 2 experiments , when the active chloroform extract (see Fig.2) was chromatographed on silicic acid, the affinity of silicic acid for the active alkaloids (alkaloid VI and alkaloid VII) was so strong, that even methano1 -water (1 :1 ) was not polar enough to elute the active alkaloids off the column. These active alkaloids were finally eluted off the column with 2 1 0 % ammonium hydroxide in methanol. In this work, the 150 separation of alkaloid VI and VII was done on a neutral

alumina column (see p. 65). The alkaloid VI fraction obtained from the neutral alumina column was then purified

with a silica gel 60 column (see p. 67). Fractions 16-20 showed a single spot in the tic but the nmr spectrum indi

cated the fractions to be impure. However, fractions 21-27 appeared to be pure according to tic examination and study

of the nmr spectrum. Thalirabine was isolated from fractions 21-27 as amorphous solid. Its crystalline monoiodide salt was pre pared in low yield (lOmg from 158mg of amorphous thalira bine) . The structural elucidation studies were carried out with the amorphous thalirabine. The uv spectrum of thalirabine was typical of a benzylisoquinoline or a bisbenzylisoquinoline alkaloid. Its ir spectrum contained a phenolic hydroxyl absorption at

3510cm There was no bathochromic uv shift in base and the phosphomolybdic acid test was negative. However, the alkaloid reacted with diazomethane to yield an O-methylated product. The nmr spectrum of thalirabine indicated the presence of one tertiary N-methyl group at 6 2.53 (lXNCH^) and four

O-methyl groups at 63.6StixoCH^) and 3.77 OXOCH^). A two- proton singlet at 6 5.92 indicated the presence of a methylenedioxy group, which was verified by a positive result of the chromatropic acid test. Nine aromatic protons were also 151 observed in the nmr spectrum with two high field one-proton

singlets at 65.57 and 5.80 (C- 8 and C-8 * or C- 8 ' and C- 8 ) as

well as seven other aromatic protons at 6 6 . 3 0 - 7 . 0 7(multip-

let). The number of O-methyl groups, methylenedioxy group and aromatic protons indicated that thalirabine was a bis benzylisoquinoline instead of a benzylisoquinoline. Since

only one tertiary N-methyl group appeared in the nmr spec trum and the alkaloid was very polar, the presence of N,N- dimethyl quaternary nitrogen center (e.g. (+)-tubocurarine

(XCVII) 8 6 or an N-oxide (e.g. thalicmidine N-oxide (XXX))"*8 was possible. Indeed, a quaternary N-methyl singlet at 6 3.43 was observed in the nmr spectrum of thalirabine* However, due to the overlap of proton signals, the integration of this signal for either one or two quaternary N-methyl groups was not readily observable. The possibility of N- oxide was excluded because attempts to reduce this alkaloid 30 8 7 88 with Zn/HCl , FeSO^ in conc. ammonia , FeSO^ aq. solution, and triphenylphosphine 89 failed. These reactions had been used by others for the reduction of N-oxides. Assuming that thalirabine contained a positively charged, N,N-dimethyl quaternary nitrogen center, there must be a counter ion. If one examines the isolation procedure (extraction with chloroform from alkaline aqueous solution, column chromatography on neutral alumina with methanol-ethyl acetate mixture as eluent, followed by column chromatography on silica gel 60 with methanol-water-ammonium hydroxide 152 mixture as eluent), it is most likely that the counter ion was hydroxide (X=OH in (LXXXII)).

3 (XCVXI)

The Cl and high resolution El mass spectra of thalira bine were very informative and will be discussed as follows.

The Cl mass spectrum showed a base peak at 669 which pre sumably was the (M+l) peak. However, one has to keep in mind that the apparent molecular weight obtained from the mass spectrum is that of the volatilized sample. Therefore, a sample of a quaternary salt will thermally fragment before volatilizing, normally with elimination of HX or CH^X 90 Assuming Lhe molecular formula of thalirabine is M+X , the alkaloid shall eliminate CH^X prior to volatilization and show an apparent molecular ion at (M-15)+ . Therefore, the actual molecular weight of thalirabine is 683, calculated for ^ 4 oH47N2°8)+ * A peak at m/e 683 (28%) in the Cl mass spectrum of thalirabine was formed via transmethylation 153 after the sample was thermally fragmented and volatilized.

The high resolution El mass spectrum of thalirabine showed the base peak at m/e 220 (measured 220.0970 and cal culated at 220.0974 for C ^2H14N°3^ w^ich indicated the presence of fragment (LXXVIII), Another intense peak was at m/e 222 (measured 222.1133 and calculated as 222.1130 for C. _H, ,NO.j) / which corresponded to fragment (LXXXVII) . 1^ lb j These two fragments, (LXXVIII) and (LXXXVII), resulted from the highly favored benzylic cleavage. The apparent molecular ion (M-15) was not observed. The fragmentation pattern of thalirabine in this mass spectrum indicated that thalira bine is a bisbenzylisoquinoline with a tail to tail diphenyl ether linkage.

OCH

OCH.

R m/e m/e 220 (IXXXVII) H 222 (LXX Vill) (LXXXVIII) CH. 236 (LXXXIX) CjL 250 * 5

From the spectral data mentioned above, one could pro pose the structure of thalirabine as a monoquaternary bis- + benzylisoquinoline, ^ 4 0 ^4 7 ^ 2 ^8 ^ * W1th four O-methyl groups, one methylenedioxy group, and one tail to tail diphenyl linkage. Since seven of the eight oxygen atoms in 154 thalirabine has been assigned, the alkaloid could only con tain one phenolic group. This was later proved by study of the nmr and mass spectral data of its O-methylated product. Thalirabine gave a positive Gibbs test result, which sug gested the phenolic group was para to an aromatic proton.

(LXXXII) R=H (IXXXIII) R=CH3 (LXXXIV) R=

Reaction of Thalirabine with methyl iodide afforded a crystalline compound, m.p. 198-200°C(decomp. MeOH), which was designated as methothalirabine diiodide (LXXXV). According to its molecular formula, * 3CH^OH, obtained from the microanalysis, this compound had an additional N-methyl group when compared to the molecular formula of thalirabine.

The nmr spectrum of this compound (in CD3 N02) also indicated the presence of an additional N-methyl group. The tertiary N-methyl signal that had been in the nmr spec trum of thalirabine was not present; instead, four quater nary N-methyl groups were observed in the nmr spectrum of 155 methothalirabine diiodide at 6 3.30,3.35, 3.60, and 3.65

(s, 4X+NCH^>. Furthermore, four O-methyl groups at 6 3.51 (1X0CH3), 3.53 (1X0CH3), 3.82(s,2XOCH3), one methylenedi-

oxy group at 6 6.05 (s,OCH30) and nine aromatic protons at

65.59 (s, 1H, C- 8 or C-8 '), 5.68 (s, 1H, C-8 ' or C-8 ), 6.63-7.20 (m, 7H) were also observed.

(LXXXV) R=H (LXXXVI) R=CH3

Methothalirabine diiodide gave a positive Gibbs test which supported the assignment of the phenolic group in thalirabine at the para position of an aromatic proton. Thalirabine was O-methylated with diazomethane, to obtain further confirmation about the number and location of phenolic groups in thalirabine. The O-methylated product was named O-methylthalirabine (LXXXIII). The phenolic hydroxyl absorption at 3510 cm - 1 observed in the ir spectrum of thalirabine was not present in the spectrum of O-methylthalirabine. This indicated that the 156 phenolic group(s) of thalirabine was methylated completely. Its nmr spectrum was very similar to that of thalirabine# with the exception that O-methylthalirabine had one addi tional O-methyl signal at 6 3.83 (s, lXOCH^)• This suggested that only one phenolic group was present in thalirabine.

The presence of one phenolic group was also confirmed by the study of Cl and high resolution El mass spectra of

O-methylthalirabine* The base peak (M-15+l)+ at m/e 683 in the Cl mass spectrum of O-methylthalirabine was 14 mass units higher than that in thalirabine. Its high resolution

El mass spectrum showed the same base peak at m/e 220 (frag ment (LXXVIII)) as thalirabine. However, the next most in tense peak (m/e 2 36) was 14 mass units higher than that of thalirabine. These data not only indicated the presence of one phenolic group in thalirabine but also showed that the phenolic group was in ring B. Since the phenolic group of thalirabine is para to an aromatic proton, according to the positive Gibbs test, then C-5 is the only available position in ring B. Furthermore, the physical data of O-methylthalirabine

(ir, uv, nmr, cd, and mass spectral data, as well as tic behavior) was found to be identical to a new hypotensive alkaloid, thalistyline (LXXXIII), isolated from T_^ longisty- 78 lum and T^ podocarpurn in our laboratories. The O-methylthalirabine monomethodiiodide (LXXXVI) was also prepared and compared with methothalistyline diiodide which had been 157 prepared from reaction of thalistyline with methyl iodide.'7 S These two dimethiodides were also identical in respect to their tic behavior, ir, uv, cd, and nmr spectra. While both dimethiodides were pure there was a difference in melting point (O-methylthalirabine monomethodiiodide, m.p. 255-257°C decomp., MeOH-ether; methothalistyline diio dide m.p. 266-268°C decomp., MeOH). The difference in melting point is believed to be due to the fact that the two compounds were crystallized from different solvent systems. It is interesting to note that methothalistyline was also isolated from T\_ podocarpum.7 8 Identity of O-methylthalirabine and thalistyline is very helpful in the structural study of thalirabine. The location of the diphenyl ether linkage (C-3", C-4"') and the methylenedioxy group {C-6 *, C-7’) of thalistyline was confirmed by isolation of two oxidation products, (XC) and (XCI), with potassium permanganate in acetone. 78 Therefore, the location of the diphenyl ether linkage and the methyl enedioxy group of thalirabine may be assigned to C-3*',

C-4' '' and C-6 ' , C-7'. The location of the quaternary nitrogen center of thalistyline was confirmed to be in ring A on the basis of study of its sodium-liquid ammonia cleavage products,

(LXXX) and (XCII), which were isolated and characterized.

Conversion of the phenolic cleavage product to the known 91 structure (XCIII) with diazomethane, led to the assignment 158 of structure (LXXX) to this phenolic cleavage product.

The optically inactive product (XCII) must be derived from 86 Emde type degradation of the quaternary half of thalisty line. In consequence, the quaternary nitrogen center of thalirabine must be in ring A.

KMnO (lxxxiii) . r** > X l || *t-

Na/N 0CH3 3 h3c (LXXX) .^OOc3 H3C

i OCH. (XCII)

CH-,0

No/NH,

(XCIII)

(XCIV) 159

The sodium-liquid ammonia cleavage of O-ethylthalirabine (LXXXIV) was carried out to confirm the structure of thalirabine. O-ethyl ether of thalirabine was used in the reaction so that the phenolic group of thalirabine was labelled. The nmr and mass spectral data of O-ethylthalirabine fit the assignment of one phenolic group in ring B for thalirabine. Its nmr spectrum showed the presence of one O-ethyl ether at 61.31 (t, J=7cps, OC^CH^) and 4.06

(q, J=7cps, OCH^CHj). The base peak of the Cl mass spectrum at m/e 697 was 28 mass units higher than that of thalira bine. Its high resolution mass spectrum showed the two most intense peaks at m/e 220 (100%) and 250 (94%) . The peak at m/e 220 corresponding to (LXXVIII) remained the same as in thalirabine. However, the peak at m/e 250 correspond ing to (LXXXIX) was 28 mass units higher. Three sodium-liquid ammonia cleavage products were isolated, two,(XCV) and (XCVI), being isolated from the non- phenolic fraction and one, (LXXX) from the phenolic frac tion .

(lxxxiv) N a / N H ^ ^ (l x x x ) _j_ — ------^ T

(XCV) R=H (XCVI) R=OH 160

The phenolic cleavage product was identified as (LXXX) on the basis of its identical m.p., tic behavior, as well as comparison of the uv, ir, nmr, cd, and mass spectral data with the same physical data of phenolic cleavage products 78 of thaliracebine and thalistyline. The mixture m.p. of this compound with the phenolic cleavage product of thalira cebine was not depressed. The isolation and identification of this phenolic cleavage product (LXXX) confirmed the substitution pattern and stereochemistry of the tertiary half of thalirabine to be as shown in (LXXXII). The two bases isolated from the non-phenolic fraction were the optically inactive, Emde type degradation products of the quaternary half of O-ethylthalirabine. The assign ment of structure (XCV) to the major base was decided by studying its nmr and mass spectra. The nmr spectrum showed two N-methyl groups at 6 2.38 (s, NtCH^^)/ two O-methyl groups at 6 3.74, 3.78 (s, 2XOCH^) , and one O-ethyl group at

6 1.42{t, J=7cps, 1X0CH2 CH3) and 4.00 (q, J=7cps,lXOCH2 CH3>.

The four-proton singlet at 5 4.50 corresponded to the two methylene groups (C-l' and C-2')- The AB quartet of the four aromatic protons in ring B was centered at 6 6 . 8 8 and 7.13 (J=9cps). The other two aromatic protons appeared as a singlet at 6 6.29 . These two aromatic protons are most likely to be meta to each other, namely at C-4 and C-6 , because hindered O-methyl groups of bisbenzylisoquinolines tend to cleave upon sodium-liquid ammonia reduction, e.g. 161 91 the cleavage product (XCIII) from hernandezine (XCIV). The O-ethyl group was assigned to C-3 position due to the previous assignment of the phenolic group of thalirabine at the C-5 position. The fragmentation of the low resolution El mass spectrum of this compound fit the structure (XCV).

It showed a molecular ion peak at m/e 357 (C22H3lN03^ an<* other significant peaks at m/e 299/ 121, and 58 (base peak) due to the fragments (M-a)+, b+ , and a+, respectively.

The structure of the minor base isolated from the non- phenolic fraction was assigned structure (XCVI) on the basis of interpretation of the spectral data. The 90 MHz nmr spectrum of the compound was very similar to the nmr spec trum of the major base (XCV) with the exception that this compound has one less aromatic proton at <56.40 (s, lXArH) and one additional phenolic group at 65.04 (s, broad,lXArOH).

The ir spectrum also showed the presence of a phenolic hydroxyl absorption at 3530cm ^ . Upon consideration of the tendency of cleavage of hindered 0 -methyl group(s) in sodium-liquid ammonia reduction 91 and the isolation of this compound from the non-phenolic fraction, the phenolic group would be most possibly at the hindered C-4 position. The negative result of Gibbs test of this compound also ruled out the C-3 position for the phenolic group. The fragmenta tion pattern of the mass spectrum also fit structure (XCVI).

It showed a molecular ion at 373 {C2 2 H 3 ^NO^) and the other intense peak at 121 and 58 (base peak) corresponding to frag- 162 ments b+and a+( respectively. With the characterization of these two cleavage products, (XCV) and (XCVI), the location of the quaternary nitrogen center of thalirabine was further confirmed to be on ring A. Since the asymmetric center at C-l of thalira bine was destroyed upon sodium-liquid ammonia cleavage, the assignment of stereochemistry at C-l had to rely on the comparison of the cd spectrum of thalirabine with that of a related compound of which the stereochemistry was known.

A comparison of the cd curves for methothalirabine diiodide (LXXXV) and thaliracebine dimethiodide (LXXXI) suggested they had the same stereochemistry. Both exhibited two positive Cotton effects: methothalirabine diiodide with

[6 ]2 7 g+8 ,1 0 0 , [9]225+135,000, and thaliracebine dimethio dide with (0]277+13,100, [ 0],000. Since the stereo chemistry of thaliracebine (LXXVI) was proved to be S_,S as mentioned in the discussion of thaliracebine, one could assign the S,S configurations to thalirabine as well as thalistyline and methothalistyline. After examining all of the above evidences, structure

(LXXXII) was postulated as the structure of thalirabine. Structurewise, thalirabine has three interesting fea- tures. Firstly, this compound and thalistyline 78 are the first two monoquaternary bisbenzylisoquinoline alkaloids to be isolated from plants of Thalictrum species. The only other such type alkaloids ever found in nature are (+)- 163 gc 92 tubocurine (XCVII) and cycleahomine (XCVIII).

X~ 0 CH

OCH (XCVIII)

Both were isolated from the Menispermaceae family. Secondly, in Mollov and Georgiev's review of Thalictrum alkaloids 13 , they noted that none of the bisbenzylisoquinoline alkaloids containing a methylenedioxy group had been found in plants of Thalictrum species, although such alkaloids were known to occur in nature. Isolation of thalfine and thalfinine 39 marked the first example of the existence of such alkaloids in plants of Thalictrum species. Isolation of thalirabine 78 7 8 and thaliracebine, thalistyline , methothalistyline added four new members to this type of alkaloid. Thirdly, thalir abine contains an unusually high number of oxygens (eight oxygens) for a bisbenzylisoquinoline alkaloid. The first such example is base B(C 3 5 H 3 5 N 2 °g' m *P* 230°C {decomp.)) 40 isolated from Chondodendron limaciifolium Diels. Thalib- 40 41 93 39 39 runine * , thalibrunimine , thalfine , and thalfinine 78 also contain eight oxygens. Thalirabine, thalistyline 7 8 and methothalistyline added three new alkaloids to this type of highly oxygenated bisbenzylisoquinoline alkaloids. 164

C. Thalfinine (C) This alkaloid gave no immediate changes on blood pressure in rabbits. However, a transient fall of mean blood pressure (10-23mmHg) was observed 2 to 3 min. after the administration of the alkaloid. The alkaloid also showed antimicrobial activity at 50 ug/ml against M_^ smegmatis. This alkaloid was isolated as an amorphous free base and crystallized as the dihydroiodide salt, m.p. 234-236°C (decomp., water-methanol). The ir spectrum indicated no M g OH hydroxyl absorption. The uv spectrum showed A max 312nm sh (loge 2.74), 280(3.76), and 206(4.94), and was unaffected by base. These suggested that the alkaloid was a non-phenolic benzylisoquinoline or bisbenzylisoquinoline alkaloid.

The nmr spectrum showed signals that corresponded to two N-methyl groups at 62.36, 2.60 , four 0-methyl groups at

63.43,3.50, 3.73, 3.87 , one methylenedioxy group at 6 5.86, and eight aromatic protons at 66.02(s, 1H, C-8 ), 6.42 (s, 1H) ,

6.77 (s, 2H), and an AB quartet centered at 6 6 . 6 8 and 7.18

(J=9cps, 4H). The positive result of chromatropic acid test also suggested the presence of a methylenedioxy group. From the number of aromatic protons and the substituents indicated by the nmr spectral data, the alkaloid should be a bisbenzylisoquinoline with two diphenyl ether linkages which was also indicated by its mass spectrum. An intense molecular ion at m/e 666(95%) was calculated for C 39H42N 2°8' 165 which was also supported by the elemental analysis data of its dihydroiodide salt. The double benzylic cleavage of the singly and doubly charged molecular ions at 440(14%,

(C24H28N2°8)^ )and 2 2 0 {100%' (C24H28N 2°3)++)Were most re“ vealing and corresponded to fragments (CII) and (CIII). These fragments also suggested that a methylenedioxy group, three 0 -methyl groups and one aromatic proton were in the rings B and C.

(CII) m/e 440 OCH.

H f <

(CIII) m/e 220 166

In order to obtain further structural information of

this compound/ the sodium-liquid ammonia reduction was carried out to confirm the substitution pattern as well as the stereochemistry of the compound by studying the cleavage

products. Unfortunately, only a non-phenolic cleavage product was isolated from the reduction. Attempts to iso

late the phenolic cleavage products or their O-methylated

derivatives were not successful. The non-phenolic cleavage product was identified as (SJ-O-methylarmepavine (LXXIX) by the comparison of its tic behavior, ir, cd, and nmr spectral 8 0 data with the authentic sample. This established the

location of three O-methyl groups to be at C-6 , C-7, and C-4" and the S-configuration at C-l. In addition, since the nmr spectrum of the alkaloid indicated the presence of one

C-8 aromatic proton,the diphenyl ether linkage must reside at C-5.

N o/NH

3 (LXXIX) 167

There are three possible patterns of ring c as shown in structures (C), (CIV), and (CV). However, the presence of thalirabine (LXXXII) and thaliracebine (LXXVI) in this plant makes this alkaloid most likely to have a substitution pattern of ring c as in the structure (C) if they share the common biogenetic pathway.

(CV) (CIV) rest of molecule as in (C) rest of molecule as in (C)

On the basis of the above spectroscopic evidence, the nature of the cleavage product from sodium-liquid ammonia reduction, and biogenetic consideration, the structure of this alkaloid was postulated as (C).

The structure (C) was identical to that of a new alkaloid, thalfinine, isolated from T^_ foetidum by Russian researchers3^ ' 64 with the exception that the absolute con figuration of thalfinine at C-l was not reported by the Russian researchers. The spectral data of this alkaloid were in agreement with those reported for thalfinine^' . 168

(xeix)

DZn/HCI.boil 2)HCH0,NqBH4

0 CH

OCH <

fci) or (C) 169

(For the reported physical data of thalfinine see Table 1, for the comparison of the nmr spectral data see Table 13) . Unfortunately, a suitable sample was not available for us to make direct comparison. The possibility that thalfinine from this plant was the diastereoisomer of thalfinine from T_^ foetidum was ex cluded by studying two diastereoisomers, which was prepared from thalfine (XCIX). The imino function of thalfine was first reduced with Zn/HCl to form a tetrahydroisoquinoline nucleus 9 4 , which was then N-methylated with • formalde-

8 3 hyde and sodium borohydride to yield two diastereoisomers in 1 : 1 ratio. One of them was identified as thalfinine (C) by com parison of its tic behavior and m.p. as well as its uv, ir, nmr, and cd spectra with those of thalfinine isolated from this plant. The mixture m.p. of the dihydroiodide salts of this compound and thalfinine was not depressed. The other diastereoisomer showed very similar uv, ir, and mass spectral data with thalfinine. However, its nmr spectrum, optical rotation, cd curve, and crystal form were clearly different from those of thalfinine. This diastereo isomer was named isothalfinine and isolated as prisms, m.p. 204-205°C (methanol). The chemical shifts of the two N- methyl groups (62.44, 2.66), the one upfield O-methyl group

(63.21), and the C- 8 aromatic proton (65.87) were very different from those of thalfinine isolated from this plant 170

and from foetidum. (See Table 13 for the comparison of 2 6 chemical shifts). Its optical rotation, ([a]D +45.5° (c 0.073, MeOH)),was much less positive than the reported

value of thalfinine from T_^ foetidum ([g]p6 +115°(c 0.95,

ethanol) ) 64 and the value of thalfinine from this plant

([a] ^6+141. 2° (c 0.250, MeOH)). The stereochemistry of isothalfinine was different

from that of thalfinine at C-l', because the imino function

of thalfine (XCIX) was assigned to ring D. Therefore, the two reductive products of thalfine should have different

stereochemistry at C-l'. Although the cd curves of thalfinine (C), isothalfin-

ine (Cl), and thalfine (XCIX) were taken, the assignment of the absolute configuration at C-l' of thalfinine and iso-

thalfinine from the cd curves could not be done due to the

lack of suitable model compounds for comparison. Table 13. Chemical shift data (6 values) for thalfine, thalfinine, and isothalfinine from different sources

Alkaloid ?ich3 och3 o c h 2o ArH Source (C-8 )

thalfine* 2 .2 0 , - 3.40, 3.50, 3.61, 3.76 6.04 5.93 T. foetidum

thalfine** 2.28, - 3.47, 3.57, 3.73, 3.87 6.13 6.04 T. minus race B

thalfinine* 2.30, 2.54 3.36, 3.43, 3.66, 3.80 5.80 5.92 T. foetidum

thalfinine** 2.36, 2.60 3.43, 3.50, 3.73, 3.87 5.86 6 . 0 2 T. minus race B

isothalfinine* * 2.44, 2 . 6 6 3.21, 3.48, 3.73, 3.86 5.87 5.87 Reaction product of thalfine

*The nmr spectrum was taken on a JNM-lOQ/lOOMHz instrument in deuterochloroform?®'^

**The nmr spectrum was taken on a Varian Model A-60A instrument in deuterochloroform. 172

D. Thaifine(XCIX) The isolation of this alkaloid from T. minus race B

was previously reported by Gharbo et al. in our labora-

tories. 3 ' 4 It was also isolated from T. minus var. adiantifolium in our laboratories and designated as alka- g loid A at that time. Isolations of thalfine and thal

finine from T. foetidum were first reported by Russian researchers. 64 The structure of thalfine was later postu- 39 lated as structure (II) by chemical degradation study which was mentioned early in the introduction of this dissertation (see p. 26 ). However, the absolute configura

tion at C-l was not mentioned. Unfortunately, a sample of

thalfine,isolated by Russian researchers, was not available

to us to enable a direct comparison to be made. This alkaloid showed no hypotensive effect in the

rabbit up to the dose of Img/kg. However, it gave weak

antimicrobial activity at 100 yg/ml against M. smegmatis.

Thalfine, which was isolated in the present study

showed the same tic behavior, nmr, uv, ir spectral data as those of thalfine isolated previously from this plant. However, its optical rotation, +84 . 9° (c , 0 . 297, MeOH), taken on a Perkin-Elmer Model 241 polarimeter was much 4 more positive than that reported by Gharbo and close to the value reported by the Russian researchers. The value 25 taken by Gharbo ej: al. was [ci]D +18.3° (c 0.49, EtOH) and 4 was taken on a Jasco Model ORD/UV-5 spectropolarimeter. 173 15 The value reported by the Russian group was [a]D +69° 64 C 1.0, EtOH). Although it was reported that the mass spectrum of thalfine,isolated from T. minus race B,showed a molecular 4 ion at m/e 648, the detail fragmentation pattern was not discussed. Since an unpublished high resolution El mass spectrum of thalfine isolated from T. minus var. adiantifolium was available, a rationalization of fragmentation pattern was carried out to support the identity of thalfine isolated in our laboratories and that isolated by the Russian group.

(XCIX)

The spectrum showed a molecular ion at m/e 648 (measured 648.2415 and calculated 648.2472 for C^gH^gN^Og), which was also the base peak. The double charged molecular ion was also observed at m/e 324(44%,measured 324.1247 and calculated 324.1236 for Mc^H^gl^Og)) . The peak at m/e 174 633(91%, measured 633.2216 and calculated 633.2237 for

C ^ H ^ ) was formed due to the loss of CH 3 at C-4"

from the molecular ion presumable occurring after benzylic 8 5 cleavage at C-l as in structure (XCIX). In addition, the peaks of m/e 442(8%, measured 442.1632 and calculated

422.1654 for C27H24N<">5^, m/e 220(15%, measured 220.0966 and calculated 220.0974 for C^H-^NO^) , and m/e 204 (23%, measured 204.1019 and calculated 204.209 for C ^ H ^ qNO^) were very informative. They corresponded to fragments

(M-a-l)+ , (b-l)+ , and (b-l-CH^)+ as in structure (XCIX), respectively, and indicated that the N-methyl group resided in ring A instead of ring D. In other words, the C-l of thalfine was an asymmetric carbon. This evidence also supports that thalfinine(C) and isothalfinine(Cl) possessed different absolute configuration at C-l' and the same absolute configuration at C-l. Since the absolute configu ration of C-l in thalfinine(C) has been determined to be S, thalfine should have S configuration at C-l also. Prom the interpretation of physical data and chemical transformation of this alkaloid, it was believed that this alkaloid was identical to thalfine originally isolated from T. foetidum. Furthermore, configuration was assigned to C-l of thalfine as shown in structure (XCIX). 175

E. Thaliadine tCIX) This alkaloid was assigned the name, thaliadine,

which refers to its structural relationship with adianti-

foline(I). The administration of graded doses of thalia

dine (0 .1 -1 . 0 mg/kg) to rabbit produced a dose-related decrease in mean blood pressure in the range of 22 to 35 mmHg* However, in the antimicrobial tests the alkaloid was inactive against the organisms, tested at 1 0 0 pg/ml. The ir spectrum of thaliadine showed a conjugated carbonyl absorption at 1675 cm ^. The nmr spectrum indica

ted the presence of one N-methyl group at 62.50, six 0- methyl groups at 63.79,3.81, 3.91, 3.91, 3.93, 3.97, four aromatic protons at 66.46,6.77, 7.40, 8.08, and one aldehyde proton at 610.38. The down field aromatic proton at 68.08 suggested that thaliadine was an aporphine type alkaloid with a C-ll hydrogen. Hernandaline(CXIV), an elaborated aporphine, very closely resembled thaliadine 95 in its uv and nmr spectral properties. The only differ ence was that thaliadine contained one additional O-methyl group and one less aromatic proton. Isolation of hernanda line (CXIV) and thalicarpine(X) from the same plant, Hernandia ovigera L. (Hernandiaceae), suggested that thaliadine may be an elaborated aporphine with an 0 -methyl group at C-3 as shown in structure (CIX), since adiantifoline(I) was also isolated from T. minus race B. The 176 mass spectrum of thaliadine showed a molecular ion at m/e

535(100%, calculated for C^qH ^ N O q ) , which fit the proposed structure(CIX) for thaliadine.

HO CH.

C H,Q

(CIX) R = OCH.

(CXIV) R = H

The proposed structure of thaliadine was confirmed by its synthesis from adiantifoline(I). On the oxidation of adiantifoline(I) with potassium permanganate in acetone by

9 6 a short oxidation time (5 min.) , a crystalline compound was obtained. This compound was identical with the natural material, thaliadine(CIX).

OCH K M n 04> (I) Acoton« OCH + (CIX) H 5 Min. 3

(CX) 177

Thaliadine(CIX) is the second example of a naturally

occurring elaborated aporphine, which is an intermediate to the aporphine-benzylisoquinoline dimer type. The 95 first example is hernandaline (CXIV). Furthermore, thaliadine is the first example of this type alkaloid isolated from Thalictrum species.

F. Thaliadanine(CVII) This alkaloid was named thaliadanine after adianti

foline (I), because it is a phenolic analog of adiantifoline.

The hypotensive effect of thaliadanine was observed after

the administration of graded doses(0 . 1 to 1 . 0 mg/kg) of

the alkaloid in rabbits. The drop of the mean blood pressure ranged between 13 and 22 mmHg. An antimicrobial activity of 100 ug/rnl of thaliadanine against M. smegmatis was also observed. Tfaliadanine was isolated as an amorphous base. Its uv spectrum was reminiscent of that for adiantifoline(I)•

Although a bathochromic shift was not observed in basic medium and the phosphomolybdic acid test of the alkaloid was negative, the ir spectrum showed a phenolic absorption at 3540 cm ^ and the nmr spectrum showed a deuterium exchangeable phenolic proton at 54.83. The nmr spectrum showed two N-methyl groups at 62.38,2.48 seven O-methyl groups at63.78(4XOCH3), 3.8 8 (1X0CH3), 3.97 (2XOCH3), and six aromatic protons at 66.45,6.50, 6.55, 6.58, 6.67, and 178

R 1 R 2 R3

(I) ch3 ch3 OCH 3

(CVI) H ch3 0CH3

(CVII) CH3 H 0CH3

3 och3 (CVIII) CH C2H5

(CXV) ch3 HH

8.05 . The nmr spectrum very closely resembled that of adiantifoline {see Table 14) with exception that the C-7' O-methyl group was absent in the nmr spectrum of thalia danine and its C- 8 ’ proton was shifted downfield. This suggested that thaliadanine was a phenolic analog of adiantifoline. Indeed, diazomethane O-methylation of thaliadanine afforded a crystalline compound identical with adiantifoline in terms of m.p., mixture m.p. tic behavior, as well as ir, uv, nmr, and cd spectra. The mass spectrum of thaliadanine m/e 712(0.1%, M+),

520 ((M-a)+), 370 ((M-b)+) ,354( (M-c)+) ,192 (a+, base) indicated that 179

thaliadanine contained a phenolic group in ring B located

either at C-6 ' or C-7’ as shown in structures (CVI) or

(CVII).

(CXVII) H C2 H 5

(cxviii) c 2h5 H

The mass spectrum of 0 —ethyl thaliadanine also indi

cated that the phenolic group of thaliadanine was in

ring B, because its base peak m/e 220, corresponding to

fragment (CXVII) or (CXVIII), was 28 mass units higher

than that of thaliadanine. In addition, the formula C 43H52N 2°9 740) for O-ethylthaliadanine was supported by its elemental analysis data and its mass spectrum.

In order to locate the phenolic group of thalia danine, the potassium permanganate oxidation of O-ethylthaliadanine was carried out in acetone to yield two oxida tion products. One product was identical with authentic dehydrothaliadine (CXI) in terms of tic behavior, m.p., as well as uv, ir, and nmr spectra. In addition,there was no depression in mixture m.p. The authentic dehydrothalia dine (CXI) was prepared by potassium permanganate oxida tion of adiantifoline in acetone by a long reaction time 180

KMnO* f^Y^ir°CH3 *N. !!«,. u +

0 (CXVI) (CXIII)

(6 hr.). The other oxidation product was identical with authentic O-ethylthalifoline (CXII) in terms of uv, ir, nmr, and mass spectra, as well as tic behavior, and m.p.

In addition, there was no depression in mixture m.p. Authentic O-ethylthalifoline (CXII) was prepared by diazoethane O-ethylation of thalifoline (CXIII), which was

6 7 79 synthesized by Chen et al. in our laboratories. r

From the above physical and chemical data, thalia danine was postulated as structure (CVII). In 1970, A *? Mollov et al. reported the isolation of a phenolic analog of adiantifoline from T. minus ssp. elatum. The alkaloid was named O-desmethyladiantifoline and structure

(CVII) was assigned to this alkaloid on the basis of the isolation of two products from potassium permanganate 181 oxidation of its O-ethyl ether in acetone. The two oxidation products was identified as (CXI) and (CXII). A direct comparison of thaliadanine and O-desmethyladiantifoline was not possible due to the unavailability of

O-desmethyladantifoline. However, the two alkaloids may not be identical because they showed very distinct differ ence in their nmr spectra (see Table 14).

KMnCh (1) Acetone A ' , > 6 hr.

(CXI)

In studying the nmr spectra of six thalicarpine 59 phenolic analogs, Shamma and Moniot made some useful generalizations for specfiically locating the phenolic functions(s). They pointed out that if a phenolic group is at C-6 ' of isoquinoline nucleus, the nmr spectrum of the alkaloid shows an upfield C-7' O-methyl signal at about 63.58 and a C-8 * aromatic proton signal at about 66.23. If the phenolic group is at C-7* of the isoquino line nucleus, the nmr spectrum of the alkaloid does not show a upfield C-7' O-methyl signal at about 6 3.58 and the Table 14. Chemical shift data (5 value) of adiantifoline and its derivatives

Alkaloid n -c h 3 OCH 3 ArH

C-7f C-8 1 C-ll

69 adiantifoline 2.44,2.47 3.59 3.78,3.78,3.78,3.82,3.89,3.94,3.96 6.24 6.55,6.55,6.60,6.60 8.08

O-desmethylad- 2.45,2.50 3.56 3.77,- 3.80,3.83,3.90,3.96,3.96 5.78 6.43,6.50,6.60,6.60 8.05 iantifoline^

thaliadanine 2.38,2.48 - 3.78,3.78,3.78,3.78,3.88,3.97,3.97 6.45 6.50,6.55,6.58,6.67 8.05

O-ethylthali- 2.43,2.48 - 3.78,3.78,3.78,3.81,3.90,3.94,3.96 6.04 6.51,6.55,6.60,6.60 8.06 adanine 183

C-8 ' aromatic proton signal shifts downfield to about 66.4 rather than remaining at 5 6 .2 . With the application of this generalization, thalia danine is more likely to have a phenolic group at C-7' as in structure {CVII) since the absence of upfield C-7' 0- methyl signal and the presence of a downfield C- 8 * aromatic proton at 5 6.45 were observed in the nmr spectrum of thalia danine. O-desmethyladiantifoline should have a phenolic group at C-6 1 as in structure (CVI) because its nmr spectrum showed the presence of the upfield C-7' O-methyl signal and a upfield C-8 ' aromatic proton at 55.78. The assignment of structure (CXII) to the oxidation product of O-desmethyladiantifoline ethyl ether could be erroneous since the identification was based on the identical m.p.'s, chromatographic behaviors, and ir spectra of the oxidation product and authentic isoquinolone (CXII), as well as no depression of the mixture m.p. The authentic isoquinolone (CXII) was obtained from the similar oxidation of thalmela- tine(CXV). The isoquinolines (CXII) and (CXVI) could have very similar physical properties. Thaliadanine is the second phenolic analog of adiantifoline to be found in nature. 184

G. Alkaloid VIIB This alkaloid was isolated in an extremely minor amount. After the administration of alkaloid VIIB up to

the dose of 0.4mg/kg, no apparent change of mean blood

pressure in rabbit was observed. The effect on blood pressure in the dog and the antimicrobial tests were not

determined due to the scarcity of the alkaloid. The uv spectrum of this alkaloid was characteristic of a benzylisoquinoline or a bisbenzylisoquinoline alkaloid. No bathochromic uv shift was observed although its ir spectrum revealed a phenolic hydroxyl absorption at 3520cm The nmr spectrum indicated the presence of one

tertiary N-methyl group at 6 2.60. It also showed the possibile presence of eight aromatic protons, five O-methyl groups, and two quaternary N-methyl groups. The presence

of a quaternary nitrogen center could explain the high

polarity of this alkaloid.

The Cl mass spectrum of this alkaloid showed a base peak at m/e 669 (M'+l) , Therefore, its apparent molecular

weight would be 6 6 8 , which was calculated for C39H44N2°8* if the alkaloid contained a quaternary nitrogen center, it would thermally fragment with the elimination of CH^X 90 before volatilizing. Thus, its base peak should corres pond to the (M-CH^+l)+ fragment and the empirical formula

^C40H47N2°8*+X” would be suitable for alkaloid VIIB. 185

Lack of material prevented further work on this alka

loid. Therefore, its structural elucidation was not

carried out.

H. (S)-reticuline (CXVI1) This alkaloid was the major and the only compound

isolated from the tertiary, phenolic alkaloid fraction.

The alkaloid was identified as (S)-re ticuline by comparison of its tic behavior, and uv, ir, nmr, cd spectra with authentic (S)-reticuline isolated from Doryphora sassafras 6 7 79 in our laboratories. ' The alkaloid showed no hypo

tensive effect in the rabbit after the administration of doses up to 4mg/kg. The alkaloid was also reported to 6 7 have no antimicrobial activity at 1 0 0 yg/ml.

HO CH

(CXVII)

I. Compound XVI This compound was isolated as a colorless oil. It showed a very faint orange color with Dragendorff's spray reagent, and gave negative results to Valser's reagent and

Mayer's reagent. 186

The compound was optically inactive. The ir spectrum showed a very strong carbonyl absorption at 1740cm ^ and

revealed no hydroxyl absorption. The uv spectrum, xMeOH 281nm (loge 3.02), 2.74(3.08), and 225(4.02), showed max no shift in acid and base. The mass spectrum gave a base peak at m/e 149. The highest intense peaks observed were

280(5%) and 279 (33%) . In the nmr spectrum, most of proton signals were at the aliphatic proton region. In addition, two singlets at 64.18 and 4.28 (about 2H's each) and one very symmetric multiplet centered at 67.58 (about 4Hfs) were observed. The tic behavior as well as the ir, uv, nmr, and mass spectral data of this compound were identical to the data obtained for base A isolated from Phellodendron 81 wilsonii Hay. et Kane. (Rutaceae) by Wu et al^ The investigation of this structure is still underway in our laboratories using the base A isolated from P. wilsonii. While it is a minor compound in T. minus race B, it is present in a large quantity in P. wilsonii (3.5 8 g from

280g of bark). 187

J. Thalidasine (XIII) This alkaloid was isolated as an amorphous solid in a minor amount. It was identified as thalidasine on the basis of its identical Rf as well as its identical ir, uv, nmr and cd spectral data with those of authentic thalida- 21 sine isolated from T. rugosum in our laboratories.

After the administration of the doses ranging from

1 to 4 mg/kg of thalidasine, a drop in mean blood pressure in the rabbit, with the change ranging from 2 to 18 mmHg, was observed. Thalidasine has been shown to be a tumor inhibitor84 and a weak antimicrobial agent.^

K. Adiantifoline (I)

Adiantifoline has been isolated previously from T. 4 minus race B. It was isolated as a major alkaloid in the ether soluble, tertiary, non-phenolic alkaloid fraction. Its tic behavior and ir spectrum were identical with those of an authentic sample. The m.p. as well as the uv, nmr, and cd spectral data agreed with the reported data. 69

Adiantifoline caused a drop in mean blood pressure (2 5mmHg) in the rabbit with a dose of lmg/kg. In the antimicrobial test, it showed no activity at 1 0 0 . 188

L . Thaliglucinone (XI) This alkaloid was isolated in extremely low yield.

Its ir spectrum showed a lactone carbonyl group at 17 30cm ^ and was superimposable with that of authentic thaligluci— none. 20 The nmr spectrum indicated the signals corres ponding to one N-methyl group at 62.39, one 0-methyl group at 64.09, and a methylenedioxy group at 66.35. The uv spectrum also was identical to that of authentic thaliglucinone. 20,70 The ir, uv, and nmr spectral data and the Rf value of this alkaloid agreed with those reported in the literature for thaliglucinone.^ . 20,70 The dose, 1.75mg/kg, of thaliglucinone caused a fall in mean blood pressure (32mmHg) in the rabbit. However, the administration of a lower dose (0.5mg/kg) did not show any apparent change in mean blood pressure. In the anti microbial test, thaliglucinone was reported to have anti microbial activity against M. smegmatis (25 pg/ml),

C . albicans (50 pg/ml) , 13. coli (100 yg/ml) , S_. gallinarum

(100 pg/ml), 1C. pneumoniae (100 yg/ml) , and S.. aureus 70 ( 2 00 ug/ml). 189

M. Thalrugosaminine (CX1X) This alkaloid was isolated as an amorphous base.

Its identification was based on its identical uv, ir, nmr, and cd spectral data, as well as its identical Rf value 72 with those of authentic thalrugosaminine (CXIX). Thalrugosamine is a new bisbenzylisoquinoline alka loid, and its isolation from T. rugosum was first reported by Wu et ad. in our laboratories.^ ^ Its structure was originally proposed as structure (CXIX) without the assignment of the stereochemistry. 70 ' 71 Later, this alkaloid 72 was isolated from T. revolutum by Wu et al. The isola tion of two phenolic cleavage products, (CXX) and (CXXI), from the sodium-liquid ammonia reduction of this alkaloid 72 confirmed the proposed structure and its stereochemistry. Thalrugosamine showed a hypotensive effect (18nunHg) after the administration of a dose of lmg/kg, in the rabbit. It was also reported to be active against M. 72 smegmatis at 50 (Jg/ml. 1 9 0

H C 3 H

CH,0 (CXIX)

| N0/NH3

OCH, OCH. +

(CXX) (CXXX) N. Obaberine (CXVIII) This alkaloid was isolated as the dihydrochloride salt, m.p. 255°C (MeOH)(lit.9 8 m.p. 260-261°C). The uv, ir, nmr, and cd spectral data as well as the Rf of its

free base were identical to those of authentic obaberine isolated from T. lucidum in our laboratories.^

Doses of lmg/kg and 4mg/kg of obaberine were observed to produce a drop in mean blood pressure (45mmHg) in rabbit. In the antimicrobial tests, the alkaloid was reported to be active against J>. aureus, M. smeqmatis, 15 and C. albicans at 1,000 ug/ml.

OCH CH_0

OCH 192

SUMMARY

A literature survey of Thalictrum minus L. race B and the Thalictrum alkaloids isolated in the period of 1970- summer 19 75 was presented.

The fractionation scheme of the ethanolic extract from the roots of T. minus race B was designed to isolate the hypotensive alkaloid(s). Fractionation and chromato graphy led to the isolation of fourteen compounds. Two were isolated from the chloroform extract, one from the tertiary, phenolic alkaloid fraction, and eleven from the ether soluble, tertiary, non-phenolic alkaloid fraction. Column chromatographies on neutral alumina and silica gel 60 of the chloroform extract led to the isola tion of thalirabine and alkaloid VIIB. The structure of thalirabine, a monoquaternary bisbenzylisoquinoline, was proposed on the basis of its spectroscopic and chemical evidence. The physical data of alkaloid VIIB was reported and no further study was made due to the scarcity of the material. Column chromatography on silica gel PF-254 of the tertiary, phenolic alkaloid fraction resulted in the isolation and identification of (S)-reticuline.

Column chromatographies on silica gel PF-254 and 193 nautral alumina and preparative tic on silica gel G and silica gel HF-254 of the ether soluble, tertiary, non- phenolic alkaloid fraction resulted in the isolation of eleven compounds: thaliadine, thaliadanine, thaliracebine, and compound XVI were new compounds; thalfine and thalfinine were partially characterized known alkaloids; thalidasine, adiantifoline, thaliglucinone, thalrugosaminine, and obaberine were completely characterized known alkaloids. The structure of thaliadine, an elaborated aporphine, was confirmed by synthesis. The structures of thalia danine, a phenolic aporphine-benzylisoquinoline dimer, and thaliracebine, a bisbenzylisoquinoline, were proposed by spectroscopic and degradative experiments. The physical data of compound XVI were reported and its structural elucidation is still under investigation. Thalfine and thalfinine were identified on the basis of their spectro scopic data and chemical evidence. The stereochemistry of thalfine and the absolute configuration at one of the two asymmetric centers in thalfinine were determined. Thalidasine, adiantifoline, thaliglucinone, thalrugosaminine, and obaberine were identified by studies of their physical data. In the pharmacological studies in dogs and rabbits, thalirabine showed a marked hypotensive effect in dogs and a pressor effect in rabbits. Thaliracebine, thaliadanine, thaliadine, thalidasine, adiantifoline, thaliglucinone, thalrugosaminine, and obaberine showed hypotensive effects in rabbits. However, thaliadine, thaliadanine, and thaliracebine seemed to be more active than the rest of alkaloids which showed hypotensive effects. In the antimicrobial studies, thalfinine, thalirabine, thaliracebine, thalfine and thaliadanine were shown to be active against M. smegmatis: Thalfinine was active at

50 pg/ml; the rest were active at 1 0 0 pg/ml. APPENDIX

195 4.0 so to 7 0 S.0

■M— r

5.0 PPM(J)' 4.0 3.0 3.0

Fig. 5 NMR spectrum of thaliracebine (CDClj) n*om i. . R pcrmo taiaeie (CHCl^) thaliracebine of spectrum IR 6. Pig. W A V E W M i i ! : *

O 197 o o b Z t e *10 i. . D n U seta f hlrcbn (MeOH) thaliracebine of spectra UV and CD 7. Fig. 100 0 4 30 0 2 10 50 200 200 X(nm) X(nm) 0 5 2 0 5 2 0 0 3 0 0 3 198 Relative Intensity %50l 10 (H 0 70 0 6 50 IlL LuJU 80 i . Elspectrummass thaliracebine of 8Fig. 1 0 0 TO 120 TIO 100 90 nLjiJnj 1 , 1 1 3 10 5 160 150 140 130 m/e ] h i t 7 10 9 20 1 220 210 200 190 180 170 ‘ i ‘ * “ ki_Ji ------" • r • 1"- 206 220

230 199 4 .0 PPM It ) 6.0 7.0 80 9.0

m too m

7 A 200

Fig. 9. NMR spectrum of thalirabine ECDCl^) I i tot N'T U* l.tt.lS ftO ’l i. 0 I pcrmo hlrbn (CHCl^) IRspectrumthalirabine of 10.Fig.

WAV WAV l KLIM OV* t M 201 202

100

2 50 K a?

200 2 5 0 3 0 0 X(nm)

100

8 0

r - 6 0

4 0

200 2 5 0 3 0 0 X(nm)

Fig. 11. CD and UV spectra of thalirabine(MeOH) Relative intensity i t S Relative Intensity 3 Fig. 12. Cl mass spectrum of thalirabine zoz RELATIVE INTENSITY i. 3 Elspectrumthalirabine ofmass 13Fig.

m/e 204 JUL SO PPMlri 6.0 7.0 8.0 *0 “TT- X TT T y r n I 1 da x*> l i j ( , f , f j ' I f i I -j : i 150 cps ; sweep (offset

. I~- __L I I L

1 ‘“'Vi f *tY r‘'W T ^ v r 'w “m , '‘ Ti^,7n'f>r'r _ I P - f H - r R - ! » f 1 ~ h -i-lJ I I \ : l_ ■ A

- F „ . i TV-fu'L - TT TT To To muTj Slot 3.0 3.0 1.0

Fig. 14. n m r spectrum of thaliadine (CDC13) 205 H'Vm* i. 5 I pcrmo hlaie (CHC13) IRspectrumthaliadine of 15.Fig. : j ■n- WAVltF'v j'K

t 206 t X(nm)

in MeOH in 0.01 N methanolic HC1 40

210 0 3 0 025 350

A(nm) Fig. 16. CD and UV spectra of thaliadine (CD in CHCI3 ; UV in MeOH) Relative Intensity 100 w M- M M TO- Fig. 17. El mass spectrum Elthaliadineof mass 17.Fig.

m/e 208 2.0 PPM IT) 6.0 7.0

kfi.

7.0 6.0 S.0 PPM[ i 3.0 1.0

Fig. 18. NMR spectrum of thaliadanine (CDC13) 209 : v

7000

Fig. 19. IR spectrum of thaliadanine (CHCl^} 210 e « 1 0 -20 140 120 160 25 0 4 20 15 10 60 20 5 0 i. 0 C n J pcr f hlaaie (MeOH)CJV and CD thaliadanine of spectra 20.Fig. 0 5 2 0 5 2 A ( ) m \(n nm) m (n 0 0 3 0 0 3 0 5 3 350 1 1 2 Relative Intensity Relative Intensity

Fig. 21. El mass spectrum of thaliadanine ZTZ 213

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