T R Govindachari’s natural products chemistry

GENERAL  ARTICLE

T R Govindachari’s Natural Products Chemistry K Nagarajan Prof. Govindachari’s research career, which spanned more than five decades, was almost completely devoted to the chemistry of Indian plants wherein he made seminal contributions. His work was notable for two major reasons: Firstly, it focused on the isolation and structure elucidation of plant constituents, occasionally supported by total or partial synthesis. Secondly his interests were copiously distributed among different classes, alkaloids, terpenes, oxygen heterocycles and other systems. It is impossible to do full justice to his work and this article provides only a selection which will give a flavor of his contributions.

K Nagarajan was among the first batch of PhD students of Prof. TRG. Currently he is advisor to Hikal Ltd., Alkem Laboratories Ltd and Research Centre. His interests are in natural products, heterocyclic synthesis, NMR, medicinal chemistry and crop protection chemicals.

Alkaloids Tylophorine and other Phenanthraindolizidines Tylophora asthmatica Wight et Arn. (synonym Tylophora indica, Sanskrit – Latakshiri) belongs to the family Asclepiadaceae and has many reported uses in Ayurveda, two of them being in the treatment of asthma and dysentery. In 1935 two alkaloids, tylophorine and tylophorinine, had been isolated from the plant. The structural elucidation of these alkaloids was undertaken by TRG and his colleagues at the Chemistry Department of Presidency College, Madras around 1951. The work spread over nearly 20 years resulted in the isolation of tylophorine, and many of its congeners, which were found to have the unique phenanthraindolizidine skeleton shown in structure 1 for tylophorine.

OMe MeO H N MeO OMe 1

1

Tylophorine has the molecular formula, C24H27NO4 and []D– 11.6o (CHCl3). After successive Hofmann degradations1 it gives a nitrogen-free product, indicating that it has a nitrogen atom common to two rings (Scheme 1).

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Quaternization with methyl iodide, conversion of the salt to the methohydroxide by stirring with an aqueous suspension of siver oxide, filtration and evaporation of the filtrate to dryness and pyrolysis of the residue.

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GENERAL  ARTICLE

OMe

OMe MeO

MeO (i) CH 3I (ii) Ag2O N

(iii) 

N

(Hofmann degradation)

Me MeO

MeO

OMe

OMe

(Hofmann degradation)

OMe

OMe

MeO

MeO (Hofmann degradation) N Me Me

-Me 3N MeO

MeO OMe

OMe 2

( or 4+2 cyclization product)

Scheme 1. 2

Reduction in water with amalgam. 3

Ehrlich test – heating with pdimethylaminobenzaldehyde and acid to give a purple colour.

Keywords Tylophorine, ancistrocladine, tiliacorine, ishwarone, polyalthic acid, litsomentol, azadirachtins, wedelolactone.

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Emde degradation2 of the methochloride of tylophorine gave a product which underwent catalytic dehydrogenation to afford a pyrrole as observed by Ehrlich test3 indicating that one of the nitrogen containing rings was five membered (Scheme 2). Tylophorine and many degradation products displayed the UV spectra of substituted phenanthrenes (UV spectrophotometer was the only available instrument at that time). Oxidation of the second stage Hofmann elimination product 2 gave 2,3,6,7tetramethoxyphenanthrene-9,10-dicarboxylimide 3, while tylophorine itself was oxidized to m-hemipinic acid 4 (Scheme 3). These results were put together to arrive at structure 1 for tylophorine.

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GENERAL  ARTICLE

OMe

OMe

MeO

MeO Na/Hg, water N+ Me

MeO

Cl

( Emde degradation)

-

Me

N Me

MeO

OMe

OMe

Pd/C, 

OMe MeO

Me

Scheme 2.

N Me

MeO OMe

OMe MeO

COOH COOH

KMnO 4 N

MeO OMe

MeO OMe 1

4

OMe

OMe

MeO

MeO O KMnO 4 NH

N Me Me

O

MeO

MeO OMe 2

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Scheme 3.

OMe 3

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GENERAL  ARTICLE

OMe

OMe

MeO

MeO Cl

+

N

N H

MgBr MeO

MeO OMe

OMe OMe

OMe

MeO

MeO (i) [H]

POCl3

(ii) HCOOH

N

N OHC

Cl

MeO

MeO

+

-

OMe

OMe

OMe

OMe MeO

MeO H NaBH 4

H resolution N

N MeO

MeO OMe dl-tylophorine

Scheme 4.

OMe 1 (-)-tylophorine

Elaborate chromatography of the total alkaloids of the plant gave related compounds – tylophorinine, tylophorinidine, septicine and isotylocrebrine. Tylophorine was synthesized by the route shown in Scheme 4, exploiting the alkylation of N-pyrrolyl magnesium bromide with an alkyl halide at position 2 followed by a few more steps. It was finally resolved to give (–)-tylophorine identical with the natural product. Derivation of S-homoproline (pyrrolidine-2-acetic acid) from tylophorine by exhaustive ozonolysis led to the assignment of the same absolute configuration to the alkaloid. The observation of anticancer activity for tylophorine and some

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related alkaloids led to several syntheses of the former, two of them enantiospecific for the S isomer and one for the R. These indicated that S-tylophorine was dextrorotatory and the (–)-isomer had the R configuration. Interestingly, there was a wide variation in the rotatory values reported in the three syntheses, +15o,+ 73o, and –74.5o. The problem was complicated by an observation of instability of the alkaloid in chloroform solution. Application of HPLC methods using chiral columns which became available years later showed that natural tylophorine isolated from Tylophora asthmatica was probably a mixture of the two enantiomers in the ratio of 2:3, indicating that it was largely racemic. The conflict between the absolute configuration assigned by degradation studies and by resolution of the synthetic racemic alkaloid on the one hand and on the other by enantiospecific syntheses was a matter of much concern to TRG and remains to be solved. It will be appropriate if the matter is clinched by a chemist of this country where Tylophora is readily available. Ancistrocladin TRG’s work on the alkaloids of Ancistrocladus heyneanus Wall. is an apt illustration of the value of random examination of plants as well as the combination of painstaking efforts and ingenuity that he and his associates brought to the structural elucidation of natural products. From the roots of A. leyneanus, the only species of the family Ancistrocladaceae occurring in India, four novel alkaloids, ancistrocladine, ancistrocladinine, ancistrocladisine and ancistrocladidine were isolated. They represented at that time a totally new class of alkaloids having a naphthylisoquinoline framework illustrated in structure 5 which was arrived at using extensive degradation studies and the liberal application of spectroscopic data, particularly of NMR.

OMe OMe 5'

4' 2' 1'

Me Me

HO 5

3

1

NH

OMe Me

Ancistrocladine is an optically active, cryptophenolic (hindered phenol) alkaloid with a molecular formula, C25H29NO4, []D =

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5

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OMe OMe

Me CO2Me 6

025.5o (MeOH). It formed a N, O-diacetyl derivative (1630, 1738 cm–1) giving, on hydrolysis, N-acetylancistrocladine, indicating the presence of OH and NH groups. Ancistrocladine was poorly soluble in NMR solvents for getting proper signals but its HCl salt was soluble in DMSO-d6. The proton NMR spectrum showed two secondary C-Me and three O-Me groups and also an aromatic methyl group in a shielded position (s, 2.05 ). Thus all the hetero-atoms were accounted for. The UV spectra of the alkaloid and several of its degradation products were similar to that of 1,8-dimethoxy-3-methylnaphthalene. Mild oxidation of the hydrochloride with KMNO4 in aqueous acetone at room temperature followed by isolation of the acidic products, conversion to the methyl esters and exhaustive column chromatography gave a methyl naphthoate identified as 6 by comparison with a synthetic sample. The chemical shift of the aromatic methyl group in 6 was normal (s, 2.42) signifying the attachment of a shielding aromatic ring at position 1 of naphthalene in ancistrocladine which provided the CO2H in the oxidation product. Ancistrocladine was N-formylated and converted to O-methyl-Nformylancistrocladine which was reduced by lithium aluminium hydride to O,N-dimethylancistrocladine. Systematic Hofmann degradation, oxidation reactions and NMR studies showed the presence of a 1,3-dimethyltetrahydroisoquinoline 7.

Me NH Me 7

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The free phenolic group in ancistrocladine was present in the isoquinoline part and not in the naphthyl moiety for the following reasons. In the NMR spectra of N-formyl, N-methyl and N-acetylancistrocladine, the signals due to the phenolic OH appeared as singlets at  5.27, 4.90 and 5.25 ppm, respectively. If they were present at position 4 or 5 of the naphthalene ring, H bonding with a peri-O-Me group would require the signals to appear considerably downfield. Further the significant shielding of the O-Me (s, 3.57 ) in the NMR spectrum of O-methylancistrocladine would require the placement of the O-Me at position 6 of the tetrhydroisoquinoline ring and its attachment to the naphthalene ring via position 5. The third O-Me group in ancistrocladine was

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GENERAL  ARTICLE

located at position 8 of the isoquinoline as was demonstrated by Claisen rearrangement of the O-allyl derivative which migrated to a free ortho position (C-7). Thus the gross structure of ancistrocladine was deduced to be 8. Dehydrogentaion of O-methyl-ancistrocladine with 10% Pd-C in refluxing decalin gave the naphthyl-isoquinoline 9, which exhibited optical activity, []D= + 58.57o (CHCl3), signifying that in the ancistrocladine series, restricted rotation around the naphthyl and hydrogenated ring contributed to the overall optical activity.

OMe OMe

Me Me

HO

NH OMe Me 8 OMe OMe

In the 100 MHz proton NMR spectrum of O-methylancistrocladine, decoupling of the multiplet for C-1 H from the methyl group showed no long range coupling with C-4 H, requiring it to be assigned an equatorial configuration. A similar decoupling of C-3 H from the appended methyl group showed diaxial and axialequatorial coupling with C-4 protons, thus locating C-3 H in the axial orientation. The studies thus demonstrated that the two Me groups were trans to each other with C-1 Me in the axial and C-3 Me in the equatorial positions as in 10. Single crystal X-ray studies on ancistrocladine hydrobromide confirmed the gross structure deduced so far and the relative stereochemistry of the two Me groups in the isoquinoline ring as being trans. It further showed that the planes of the naphthalene and the isoquinoinme rings were at an angle of 87o. The relative stereochemistry of the methyl groups at C-1 and C-2 was also revealed leading to structure 10 without the absolute stereochemistry not yet becoming evident. This was derived by a powerful technique called Exciton Chirality method which had been developed around that time by Prof. K Nakanishi. Application of this to the naphthylisoquinoline 9, wherein the optical activity was solely due to the atropisomerism, demonstrated that the absolute configuration of ancistrocladine is as shown in 5 with napthaleneisoquinoline axis being as indicated. Typical of TRG, the matter was not allowed to rest there. Clinching chemical proof for the absolute configuration at C-3

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Me Me

MeO N OMe Me 9

OMe OMe

Me Me

MeO

NH OMe Me 10

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GENERAL  ARTICLE

H N

MeO2C H

Me O 11

N COCF3

and hence of 5 was provided by exhaustive ozonolysis of ancistrocladine and isolation of (+)-L- -aminobutyric acid, []D= +28.86o (H2O), which was further confirmed by studying a dipeptide 11 derived from it. The structures of the other three alkaloids from the plant were easily deduced by chemical correlations.

It is noteworthy that despite being an isoquinoline alkaloid, ancistrocladine has a different biogenetic origin, which is via the polyketide route. Tiliacorine Investigation of the alkaloids of Tiliacora racemosa Colebr. (Menispermaceae), synonymous with Tiliacora acuminata (Lam.) Miers proved to be an arduous but rewarding exercise since the alkaloids belonging to the bisbenzylisoquinoline group had the unusual dibenzodioxine system and also a unique (at that time) diphenyl moiety instead of the common diphenyl ether fragment.

O O 12

526

Using a combination of chromatography and counter-current distribution (kind of a last resort those days, now largely replaced by preparative HPLC), four alkaloids were isolated – tiliacorine, tiliacorinine and the latter’s N-desmethyl derivatives, nortiliacorinine A and nortiliacorinine B. Tenacious retention of solvents of recrystallization by tiliacorine made it difficult to assign a molecular formula which was eventually found to be C36H36N2O5. NMR spectrum revealed two N-Me (2.30, 2.66) and two O-Me (3.83, 3.93) groups besides nine aromatic protons. Another oxygen atom was found to be in the form of a cryptophenol, not methylated by diazomethane. The UV spectrum of tiliacorine was similar to those of trilobine and menisarine containing a dibenzodioxine template 12, thus accounting for the two remaining oxygen atoms. This was confirmed by the characteristic formation of a blue colour when the alkaloid was heated with a mixture of concentrated sulphuric and nitric acids. Alkaline permanganate oxidation of O-methyl-tiliacorine

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GENERAL  ARTICLE

13

OMe

MeO 2 C

O

CO2 Me

O

CO2 Me

CO 2 Me 13

MeO 2 C

C O 2 Me

OMe

OMe

14

CO2H

HO2C OMe 15

dimethiodide followed by methylation of the acidic products and chromatography gave a tetracaraboxylic and a dicarboxylic ester. The former was identified as the dibenzodioxin derivative 13 by analytical and NMR data and by comparison with a standard sample made by unambiguous and painstaking efforts. The dicarboxylic ester was similarly recognized to be 14. The formation of a diphenyldicarboxylic acid 15 showed that in tiliacorine the two benzyl groups were connected directly through the phenyl moieties uninterrupted by an oxygen atom. The formation of 4methoxyisophthalic acid and the methoxydibenzodioxin tetracarboxylic acid in the oxidation of tiliacorine dimethiodide established that the cryptophenolic OH was located in the diphenyl residue. A likely gross structure could now be written for tiliacorine as 16 or 17 with a monomethoxy-dibenzodioine unit as in trilobine. Tiliacorinine, the second most abundant alkaloid in the plant with the molecular formula C36H36N2O5 was found to be isomeric with tiliacorine and with a similar UV spectrum indicative of a dibenzodioxine chromophore. It also had two O-Me and two N-Me and one crypto-phenolic groups. The two-stage Hofmann degradation of O-methyltiliacorine dimethiodide and O-methyltiliacorinine dimethiodide gave an identical nitrogen-free compound 18 in confirmation of the presence of two benzylisoquinoline moieties. Similar transformations of O-ethyltiliacorine through its dimethiodide gave an identical nitrogen-free product 19 thus showing that the free phenolic OH was in the same benzylisoquinoline residue in both alkaloids. Thus tiliacorine and tiliacorinine were demonstrated to be diastereoisomers of structure 16 or 17. To cap the successful elucidation of structure 16 or

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GENERAL  ARTICLE

OMe

OMe

O

O N

O

R1

O

N R

OR 2

OR 3

16 R= R 1= R 2= Me; R 3 = H 17 R= R 1= R 3= Me; R 2 = H 20

R = R 1 = Me, H or vice versa R 2 = R 3= Me, H or vice versa

OR

OR 1

18 R= R 1= Me 19 R=Et, R 1 = Me or vice versa

17 for tiliacorine, O-methyltiliacorine was synthesized by standard but very demanding exercises. Two minor alkaloids, nortiliacorinine A and nortiliacorinine B were easily recognized as being des-N-methyltiliacorinines because both gave tiliacorinine on N-methylation. They can thus be represented as isomeric des-N-methyltiliacorinines of structure 20.

4

Shift of an absorption peak to longer wavelength.

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Collaborative work with Prof. M Shamma allowed the gross structures of tiliacorine and tiliacorinine to be narrowed down to 16, leaving only the absolute configurations at positions 1 and 1 to be deciphered. The ingenious experiment which led to the assignement of structure 16 involved selective scission of one of the two benzylic links in O-acetyltiliacorine and deacetylation to the lactam 21. Proton NMR spectrum showed H* to be significantly deshielded relative to its location in the spectrum of tiliacorine. UV spectrum in alkali exhibited a strong bathochromic shift4 caused by conjugation of the phenolate ion with an aldehyde.

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GENERAL  ARTICLE

O OMe

MeO

O N

O

N

HO

*

O

H*

N

Me

Me

Me R or S

1' N

O

Me

1 N Me

CHO

HO 22 OMe

OH

21

OMe

OH

16 Tiliacorine 1-R, 1'-S Tiliacorinine 1-S, 1'-S

The relative sterecochemistry of tiliacorine and tiliacorinine at positions 1 and 1 in the two isoquinoline rings in 16 and their absolute configuration were solved by Bhakuni and Kapil through strategically planned biosynthetic experiments by feeding the putative precursor, N-methylcoclaurine 22 of both S and R absolute configurations carrying tritium labels. These experiments established that tiliacorinine had the S,S absolute configuration at positions 1 and 1 while tiliacorine had 1-R and 1-S configuration. Terpenes Ishwarone The ketone, ishwarone isolated from Aristolochia indica of Aristolochiaceae called Ishwaramuli in Kannada was proved to belong to the rare class of tetracyclic sesquiterpenes (three isoprenoids). The work on structure elucidation started around 1956 at Presidency College, Madras. Progress was slow due to limited spectroscopic facilities but the work picked up momentum in 1963 at Ciba Research Centre established in Bombay, resulting in a satisfactory solution in 1969 with the help of a 100 MHz NMR facility available at Ciba, Basel. Ishwarone turned out to be another example of cracking a structural enigma through systematic, multipronged degradation studies and insightful application of NMR spectroscopy.

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GENERAL  ARTICLE

5

Formation of deeply coloured products by treatment with mdinitrobenzene in alkali.

Ishwarone, C15H22O, had an intense band in the IR at 1706 cm–1 characteristic of a cyclohexanone. It was found that the resistance of ishwarone to hydrogenation coupled with the absence of ethylenic unsaturation in IR, NMR and Raman spectra suggested that it was tetracyclic. A band at 1418 cm–1 in the IR spectrum and a positive Zimmerman Reaction5 indicated the presence of an active CH2 adjacent to a ketone. Ishwarone exchanged only two atoms of deuterium under base catalysis; hence the C=O group was flanked by a CH2 on one side and a quaternary carbon on the other. The NMR spectrum of ishwarone showed two tertiary methyl and one secondary methyl groups. A cyclopropane ring was present as shown by a one proton multiplet at 0.55 ppm while another proton peak lay hidden in the methyl region.

6

Exposure to oxygen in the presence of potassium t-butoxide in t-butanol.

A series of experiments starting with the Barton oxidation6, leading to a 1,2-diketone in the form of an enol (dio-sphenol), oxidation with hydrogen peroxide to a dicarboxylic acid, C15H22O4, pyrolysis to norishwarone (cyclopentanone, IR 1728 cm–1), a second Barton oxidation to a new diosphenol and treatment of this with alkaline hydrogen peroxide gave norishwaric acid. This was shown to be a glutaric acid by the facile formation of an anhydride with IR bands at 1760 and 1800 cm–1, typical of a glutaric anhydride. These results coupled to NMR and deuteration studies were interpreted as shown in Scheme 5. The 100 MHz NMR spectrum of norishwaric acid was particularly interesting in that it revealed uniquely the two cyclopropane protons ( 0.58, 0.96 ppm) suspected to be present in ishwarone. Equally important was the fact that all the signals in the proton spectrum could be analyzed. The architecture of the remaining tricyclic system was unraveled by taking advantage of the acid lability of the cyclopropane ring present therein. Treatment of ishwarone in dry HCl in ether at 0 oC followed by a brief exposure to pyridine gave a mixture of two isomeric unsaturated ketones, one having an exocyclic

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O

O

CO 2 H CO 2 H

OH

Me

Me

Me

Me

Me

O

O

CO 2 H Me

Me

CO 2H Me

Me

OH Me

Me

Me

O O Me

O

Scheme 5.

Me

methylene group, 23 and the other 24, having an endocyclic – CH= C(CH3) group. Both had the same molecular formula as ishwarone and the latter came to be called isoishwarone. The former isomerised to the latter under acid catalysis. Ozonolysis of 24 gave a diketo-aldehyde and a diketone, C14H22O2 which was identified as 25 by comparison with a sample made from valerianol, 26 via the ketone 27 (Scheme 6). Compound 23 on treatment with osmium tetroxide followed by periodate gave a diketone with IR bands at 1702 (6-membered ketone) and 1726 cm–1 [bicyclo(2,2,2) octanone]. The correlations bolstered by similar studies on isoishwarane obtained by Wolff–Kischner reduction of isoishwarone led to the delineation of absolute structures of isoishwarane and isoishwarone as 29 and 30, respectively and of ishwarone itself as having the novel tetracyclic structure 31 in its full stereochemical splendour.

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GENERAL  ARTICLE

O

O

CHO O3 O

O 24

O

O3 O

O H 23

28

H

HO

O

26

O

O 25

27

Scheme 6. O

29

30

O

31

The full paper [1] gives a detailed account of the extensive proton–proton decoupling studies carried out on a 100 MHz NMR instrument. While such studies are machine programmed nowadays in even more powerful instruments (300 MHz or more) and computer-delivered, it required back-breaking efforts of specialists in those days. Polyalthic Acid The extraction of Polyalthia fragrance Bth. (Anonaceae), a large tree common to the west coast of India, with cold petroleum ether gave a new crystalline diterpene (four units of isoprene) acid in a

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yield of nearly 1% (generous for a natural product). Degradation studies and correlations with transformation products of other terpenes were deployed to arrive at the structure and absolute stereochemistry as 32.

O

CO 2 H

Polyalthic acid was purified by chromatography over silica gel to give pure material, m.p. 102 oC. Elementary analysis of the acid and its salts with cyclohexyl amine and diethyl amine (for confirmation) agreed with the formula, C20H28O3 (mol.wt 316) which was corroborated by titration (mol. wt. 317) and by the Rast method (depression of m.p. of camphor; mol. wt. 305). Polyalthic acid has two C-Me groups as shown by Kuhn–Roth oxidation7. Zerewitinoff analysis8 indicated the presence of one active hydrogen attributable to a carboxyl group. Titration with iodine monochloride showed the presence of three double bonds confirmed by reduction of the acid to a hexahydro derivative. Polyalthic acid was not esterified by the Fischer procedure (MeOH–HCl) but it gave the methyl ester with diazomethane. This ester was fairly resistant to alkaline hydrolysis showing the acid to be tertiary.

32

7

Drastic oxidation with chromic acid, distilling of acetic acid and titrimetry. 8

Treatment with MeMgBr and measurement of the methane gas produced.

Polyalthic acid gave a purple colour in the Ehrlich test indicative of the presence of a furan ring which was confirmed by its end absorption in the UV in the 210 nm region, comparable to those of furanoid terpenes. Structure elucidation of polyalthic acid illustrates the transition from hoary, classical techniques to modern ones. The 60 MHz NMR spectrum of polyalthic acid in CDCl39 revealed clearly two tertary Me groups, one being more deshielded than the other due to the proximity of a carboxyl group, fourteen cyclic methine and methylene protons, an exocyclic methylene group and two  and one  furan protons. The presence of a  -substituted furan ring was proved by doing an Alder–Rickert degradation on polyalthic alcohol (obtained by LAH reduction of methyl polyalthate) as shown in Scheme 7 (part structures).

9 The original paper [2] quotes chemical shifts in values which are derived by subtracting delta values from 10 – a practice that has disappeared over the years.

Selenium dehydrogenation of polyalthic acid gave 1,2,5-

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GENERAL  ARTICLE

CO2Et

+

[H]

 O

O CO2Et

O

EtO2C

CO2Et

EtO2C

CO2Et 

O

EtO2C

Scheme 7.

10

Cleavage of furan ring and scission of the exocyclic methylene group.

CO2H O

H

HO2C

CO2Et

trimethylnaphthalene, revealing the skeleton. Ozonolysis of polyalthic acid10, followed by oxidation of the resultant ketoacid with alkaline hydrogen peroxide afforded the ketotricarboxylic acid 33. This was established to be the optical antipode (enantiomer) of ketodiacid 34 obtained from neoabeitic acid 35 of established structure and absolute configuration by drastic ozonolysis followed by treatment with alkaline hydrogen peroxide. Polyalthic acid thus possesses the ‘wrong’ absolute configuration with respect to steroids like farnesiferol A and andrographalide.

33

Litsomentol CO2 H O

HO2 C

H

Litsomentol, isolated from the bark of Litsea tomentosa Heyne (Lauraceae), was found to have have the mol. formula C30H52O2. It was shown to be a triterpene (six isoprene units) of structure 36

34 11

H

1

H

10

HO2C

H 35

534

HO

OH 36

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GENERAL  ARTICLE

and the simplest member of the complex cucurbitacin group. It was unique in lacking an oxygen function at C-11, making it the sole member at that time of the Lauracea family. In spite of the presence of multiple centres of chirality in this natural product, its specific rotation was found to be 0o. The rotation could have been also measured at a different wavelength from sodium D line generally used.

H HO O 37

Litsomentol had one secondary OH group (acetate, oxidation to a ketone), one tertiary OH group (dehydration with anhydrous potassium bisulphate to give the anhydro derivative) and a trisubstituted double bond (reduction, epoxidation). The double bond was situated on the side chain since ozonolysis gave acetone. The presence of a hydrogen atom at C-10 was shown by Barton oxidation of anhydrodihydrolitsomentol acetate 38 to the diosphenol 37 in which the proton at C-1 was seen as doublet at 6.12 ppm (J 2.5 Hz) due to coupling with C-10 H. Circular dichroism studies on litsomentone (by oxidation of litsomentol 36) and anhydrodihydrolitsomentone pointed to their having 3-oxocucurbitacin framework. The mass spectral fragmentation of litsomentol and its derivatives conformed to structure 36 for the former. This structure and stereochemistry were confirmed by the sequence of reactions shown in Scheme 8 on 38 involving a meth-yl migration to dihydroagnosterol 39 of established architecture. Azadirachtins Azadirachtins are tetranortriterpenopids isolated from the neem tree (Azadirachata indica A. Juss. of family Meliaceae), having a decalin segment and a modified furan residue connected by a single bond between C-8 and C-14. Azadirachtin A (40), isolated from neem kernel, has earned international reputation as a nontoxic pest control agent, being an antifeedant and an ecdysis inhibitor. The complex structure was unraveled by the collective effort of several laboratories over seventeen years and using mainly 1H and 13C NMR spectroscopy. TRG spent the last lap of

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GENERAL  ARTICLE

H

H

H

H

mCPBA AcO

AcO

O

38

H

MeSO2Cl, Pyridine

BF3.Et2O AcO

H

OH

H

Li/Ethylenediamine HO

AcO

H 39

Scheme 8.

TigO

O C O 2 Me OH 8

AcO MeO 2 C

14

OH

H

OH

O

O H

O

O

40

Tig=

O H OH

TigO

AcO H 3C

OH

H

O H

O

O

OH

O

O

41

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GENERAL  ARTICLE

his scientific career in a thorough investigation of all parts of the neem tree. Using preparative HPLC, azadirachtin A was separated in >98% purity and crystals were obtained for structure determination by X-ray diffraction, confirming the earlier conclusion. HPLC could demonstrate the presence of at least 12 congeners besides 40. Many of these were obtained pure and structures deduced by NMR spectroscopy and chemical correlations. The structure of azadirachtin I (41) is given as just one example. Oxygen Heterocycles Wedelolactone Wedelia calendulaceae Less (Compositae) and Eclipta alba (L.) Hassk. (Compositae) are plants with reputed hepatoprotective properties used in Ayurveda and folk medicine. In one of the earliest forays into natural products chemistry by TRG, wedelolactone was isolated from these two plants.. It has the molecular formula, C16H10O7,with one of the oxygen atoms being present as a methoxyl group as determined by the Zeisel method11. It had an IR band at 1707 cm–1 characetristic of an unsaturated lactone and it forms a triacetate, tribenzoate and trimethyl ether. Its structure was derived as 42 by a remarkably textbook-like degradation exercise (Scheme 9) when even elementary facilities like IR spectrophotometer was not available. The degradation ended in the isolation of 6-hydroxyveratraldehyde 43 and 2,4,6trimethoxy-benzoic acid 44. A similar sequence of reactions starting with tri-O-ethylwedelolactone with a slight modification towards the end helped in locating the lone methoxyl group in wedelolactone as shown in 42.

11

Boiling with hydriodic acid, distilling off methyl iodide and argentimetry.

Tri-O-methylwedelolactone 45 was synthesized as shown in Scheme 10. Wedelolactone happened to be the first benzofurobenzopyranone reported at that time. Many congeners have become known since then and the basic framework is named as coumestan. There is continuing fascination for the multiple biological activities of

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GENERAL  ARTICLE

O HO

O O

HO

O HO

Me2SO4

MeO

K2CO 3

MeO

O O

OMe

MeO

OMe

42 - + OH, H

CO2H

MeO MeO

OH

(i) CH2N2

O

(ii) OH, H +

MeO

CO2H

MeO

OMe

MeO

O

OMe

MeO

OMe



MeO MeO

OMe

CHO O

MeO

O

O3

O MeO

MeO

OMe

MeO

OMe

OMe

OH, H +

OMe

Scheme 9.

Acknowledgements The author thanks Dr A Murugan for help in the preparation of this manuscript.

538

MeO

CHO

MeO

OH

+ 43

HO2C MeO

OMe 44

wedelolactone and it is being offered by some catalogue chemical companies as a biochemical tool for studies of inhibition of some important enzymes.

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GENERAL  ARTICLE

OH

HO

CH2CN

MeO

ZnCl2, HCl

+ MeO

OH Hoesch reaction

HO

OMe

OH

MeO O OMe

MeO

OH

Me2SO4 K2CO3 acetone MeO

O MeO MeO

O

CO(OEt)2

HO OMe

OMe

MeO

Na

O OMe

MeO

MeO

OH

OMe

Py.HCl  O HO HO

O O

O HO

Me2SO4

MeO

K2CO3 acetone

MeO

OH

O O MeO

OMe

45 (Tri-O-methylwedelolactone)

Norwedelolactone Scheme 10.

Suggested Reading [1] H Fuhrer, A K Ganguly, K W Gopinath, T R Govindachari, K Nagarajan, B R Pai and P C Parthsarathy, Tetrahedron, Vol.26, p.2371, 1970. [2] K W Gopinath, T R Govindachari, P C Parthasarathy and N Viswanathan, Helv. Chim. Acta, Vol.44, p.1040, 1961. [3] T R Govindachari, Five decades in the study of natural products, Proc. Indian Acad. Sci. (Chem. Sci.), Vol.114, No.3, pp.175–195, 2002. [4] N R Krishnaswamy, Resonance, Vol 1, No.5, pp.17–18, 1996.

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Address for Correspondence K Nagarajan Hikal R and D Centre Bannerghatta Road Bangalore 560 076, India. Email: [email protected]

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Potential for Research on Natural Products India is home to bounteous varieties of plants. However, very few of them have been studied, a vast majority being untouched. The scope for research in this area is enormous, particularly because they include plants of medicinal value employed in the traditional system of Ayurveda. They have also been a source of many commonly used important allopathic medicines as well as lead-drugs that have inspired much of drug discovery efforts in the past and present days. Unfortunately, research in natural products is considered unappealing these days and very few chemists take it up as their research career. The rapidly expanding pharmaceutical industry may turn the tide in its favour. If more vigorous effort is put in, India can become a leader in this area of research. G Nagendrappa

Medicinal Plants

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