Org. 549

Viridin. Part V1.l Evidence for a Steroidal Pathway in the Biogenesis of Viridin from Mevalonic Acid

By John Freder ick G r o v e , t Tropical Products Institute, London, W.C.l

[l*C]Viridin, derived from [2J4C] mevalonate, has a labelling pattern consistent with its biogenesis by tail-to-tail condensation of two farnesyl residues. The carbon a t position 4 is apparently derived from the methyl group of mevalonate. The absolute configuration (V) is deduced for viridin.

THE structure (V) (Scheme 1) of viridin strongly sug- gested 1 a steroidal biogenetic pathway from (R)- mevalonic acid lactone (I) by way of farnesyl pyrophos- phate, squalene epoxide (11), and lanosterol (111). Although the skeleton of viridin contains only nineteen carbon atoms, a similar loss occurs in the formation from cholestane of the C,, skeleton of oestrone. Neverthe- less, the aromatic ring c and the oxygenation pattern of ring A of viridin are unique among naturally occurring steroids, and the possibility that the compound might be derived from mevalonic acid via digeranyl pyro- phosphate and a tricyclic diterpenoid skeleton (XIV) of the cassaic acid type could not be excluded.

[14C]Viridin derived from [2-14C]mevalonate by way of such a diterpenoid intermediate would be expected to have the labelling pattern (XV) (Scheme 2) and to give benzene-l,2,3,4-tetracarboxylic acid (XVI ; R = H) labelled in the aromatic ring on oxidation with per- manganate. On the other hand, [14C]viridin derived from [2-14C]mevalonate by tail-to-tail condensation of two f arnesyl residues to give a steroidal intermediate would be expected to have the labelling pattern (V) (Scheme 1) and to give benzene-1,2,3,4-tetracarboxylic acid (VI; R = H) labelled only in the carboxy-groups.

The labelling pattern observed in the [14C]benzene- tetracarboxylic acid so obtained is consistent only with the biogenesis of viridin by tail-to-tail condensation of two f arnesyl residues to give a steroidal precursor.2 The derivation of the C,, skeleton of the Simarubaceae bitter principles related to glaucarubin presents a

Present address: University Chemical Laboratory, Lensfield

Part V, J. F. Grove, P. McCloskey, and J. S. Moffat, J .

2 M. M. Blight, J. J. W. Coppen, and J. F. Grove, Chem.

Road, Cambridge.

C h e w SOC. (C), 1966, 743.

Comm., 1968, 1117.

similar problem, and a similar conclusion, biogenesis from mevalonate by way of a triterpenoid intermediate, has been r e a ~ h e d . ~ , ~

Radioactivity of degradation products of viridin r.m.a. x

Sodium formate (IV) ..................... 52 Formamidine hydrochloride (VIII) ... 50

Sodium formate (X) ..................... 0 Tetra-ester (VI; R = Me) ............ 320 Silver salt (IX) ........................... 336 Bromo-ester (XII) ..................... 0 Barium carbonate (XIII) ............ 163

Viridin (V) .............................. 502

Keto-ester (XI) ........................... 336

Before the oxidative degradation of the mevalonate labelled [14C]viridin was studied it was important to know which of the two carbon atoms, labelled or un- labelled, from the gem-dimethyl substituent of the hypo- theticd terpenoid precursors (XIV) or (111) was re- tained at position 4 in viridin (for nomenclature see ref. 1). This position has hitherto1 been regarded as the likely source of the formic acid resulting from the alka- line hydrolysis of ~ i r i d i n . ~ The sodium formate (IV), and derived NN’-diphenylformamidine hydrochloride (VIII), obtained by this reaction from the labelled viridin were radioactive but the relative molar activities (r.m.a.) (see Table) were not a reasonable fraction of the total. Since partial randomisation of label in the gem-di- methyl group of a viridin precursor was unlikely, it was concluded that two carbon centres in the viridin mole- cule must give rise to formic acid. It has been observed that a second mol. of volatile acid is slowly liberated on

3 J. Moron, J. Rondest, and J. PoIonsky, Exfierientia, 1966, 22, 511.

4 J. Moron and J. Polonsky, Tetrahedron Letters, 1963, 385. J. F. Grove, J. S. Moffat, and E. B. Vischer, J . Chem. SOL,

1965, 3803.

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550 J. Chem. SOC. (C), 1969

distillation from acid medium of the products of alkaline hydrolysis.

The keto-ester (XI), obtained by oxidation of the labelled viridin with chromic oxide, had two-thirds, not three-quarters, of the total radioactivity, and the sodium

mevalonate. The methyl substituent at position 4 in the Simarubaceae bitter principles is similarly d e r i ~ e d . ~ . ~

Radioactivity in the sodium formate (IV) must there- fore come from the carbon at position 1 of viridin, possibly by a competitive reaction sequence in which

Y LA

SCHEME 1 Degradation of [I4C]viridin and its biogenesis from mevalonic acid via a hypothetical steroidal precursor

0

* *C02H otvl) (20)

I I

1 - - - _ _ - -L - - - - - V Y

O 0 2 M e + BaEOs

(0.5 C *) MeOzC \ Br Br

(XVll) (IC.) SCHEME 2 Hypothetical biogenesis of viridin from a tricyclic [14C]diterpenoid precursor and labelling pattern of the

degradation products

formate (X) resulting from alkaline hydrolysis of this product was unlabelled. The carbon at position 4 in viridin obtained from [2-14C]mevalonate is therefore unlabelled and, unless some more complex biogenetic scheme is involved, is derived froin the methyl group of

retro-aldol fission of ring A is followed by the loss of formic acid from the activated llb-position in the A- seco-product (XVIII).

A decision between the two possible modes of viridin biogenesis was made possible by decarboxylation of the

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Org. 551 diester, labelling pattern (VII) or (XVI). An attempt to obtain this compound directly, by partial methylation of the crude product of the permanganate oxidation of viridin, was unsuccessful; but it was readily obtained

0

by partial esterification of the pure tetracarboxylic acid (VI; R = H) obtained by hydrolysis of the tetra- methyl ester (VI; R = Me), which is easily isolated and p~r i f i ed .~ As expected from the activity of the keto- ester (XI), two-thirds of the radioactivity of the labelled viridin was also found in the tetra-ester (VI; R = Me). The attempted decarboxylation of the diester (VII) with copper chromite in quinoline gave unsatisfactory products; however, treatment of the silver salt (IX) with bromine under the conditions of the Hunsdiecker reaction 6 gave carbon dioxide, isolated as barium carb- onate (XI1 I), and methyl 2,3-dibromot erepht halate (XII) in acceptable yield. This dibromoester was found to be unlabelled; all of the radioactivity present in the silver salt (IX) appeared in the barium carbonate (XIII). These results are consistent only with the labelling patterns (XII) for the dibromoester and (V) for viridin, and exclude (XVII) and (XV) respectively.

With the steroidal nature of viridin now firmly established the p-absolute configuration of the llb- methyl substituent is clearly indicated. Since the conformation and relative configuration of the sub- stituents at positions 1 and 2 are known,l the absolute configuration (V) for viridin is deduced.

EXPERIMENTAL

Melting points were corrected. 1.r. spectra were determined for Nujol mulls. Radioactivity of solid samples a t ' infinite thickness ' was measured by end-window counting with Panax D 657 equipment by use of standard procedure^,*^^ and is recorded in the Table as relative molar activity (r.m,a.).

[K] Viridin.-[W]Viridin,l from Gliocladiunz virens grown in the presence of ( +)-[2-14C]mevalonic acid lactone, was diluted with the unlabelled compound to give material with r.ni.a. 502 x 103. The [14C]~xidation products (XI), m.p. 300" (decomp.) and (VI; R = Me), m.p. 128", were prepared from [l4C]viridin as previously de~cribed.~ The intermediates in the conversion of the [14C] tetra-ester (VI; R = Me) into the dibromo-ester (XII) are described in the following paper.6

A l ka l iw Hydrolysis of [14C] Vi~idin.-[~~C]Viridin (1 75 mg.) was heated under reflux for 2 hr. with 0-1N-sodium hydroxide (20 ml.) under n i t r ~ g e n . ~ The cooled solution was acidified with 3~-sulphuric acid (3 ml.) and steam-

M. M. Blight, J. J. W. Coppen, and J. F. Grove, following

M. M. Blight, unpublished results. paper.

distilled. The distillate (300 ml.) was titrated potentio- metrically with 0-1N-sodium hydroxide (4.84 ml.) to pH 7 and evaporated to dryness in vacuo. The resulting an- hydrous solid (39 mg.; r.m.a. 52 X lo3), aniline (40 mg.), and aniline hydrochloride (200 mg.) were heated under reflux for 15 min. Excess of 2~-hydrochloric acid was added to the cooled mixture and the [14C]-NN'-diphenyl- formamidine hydrochloride (VIII) which separated was crystallised from 2~-hydrochloric acid to give needles (12 mg.), m.p. 251" (decomp.) (sealed tube).

Alkaline Hydrolysis of the [14C]Keto-ester (XI) .-The keto-ester (20 mg.) was heated under reflux for 30 min. with N-sodium hydroxide (2 ml.) as described above. The solid (4.0 mg. ; inactive) obtained on evaporation of the neutralised [O. 1N-sodium hydroxide (0.45 ml.) J steam- distillate gave (after reduction) a positive chromotropic acid test for formate5 and showed the i.r. bands at 2710, 1600, and 775 cm.-l characteristic of anhydrous sodium formate.

Degradation of the [14CJ Tetramethyl Ester (VI ; R = Me) .- The ester (30 mg.) was heated under reflux for 4 hr. with N-methanolic potassium hydroxide (4 ml.) and the solution was then concentrated to 1 ml. by distillation. Water (3 ml.) was added, the residual methanol was distilled off, and the solution was adjusted to pH 10 with 2~-hydrochloric acid (2 ml.). To the hot solution was added 10% barium chloride and, after 1 hr. a t O", the precipitated barium salt was filtered off. It was dissolved in 2~-hydrochloric acid (2 ml.) and 2~-sulphuric acid was added dropwise until there was no further precipitate of barium sulphate. This was filtered off and the filtrate was evaporated almost to dryness. The recovered [14C]tetracarboxylic acid (VI; R = H) was washed with 2~-hydrochloric acid and dried irt vacuo (16 mg.). It was heated under reflux for 4 hr. with methanolic hydrogen chloride (10%; 1 ml.), the solution was concen- trated to 0.6 ml., and water (2 ml.) was added. Extraction with ethyl acetate furnished the diester (VII) (18 mg.), m.p. 165-172" (from ethyl acetate), identified by its i.r. spectrum. The diester was neutralised with B~-ammonium hydroxide (0.1 ml.). Addition to this solution of a slight excess of silver nitrate precipitated the [l4C]silver salt (IX) (20 mg.), which was filtered off after 30 min. at 0" and dried in vawo at 20".

The powdered silver salt, suspended in carbon tetra- chloride (3 ml.), was heated under reflux in a slow stream of carbon dioxide-free nitrogen during 4 hr. while bromine (40 mg.) in carbon tetrachloride (0.125 ml.) was added at hourly intervals. The apparatus was connected, through a trap cooled at -7O", to an absorption train charged with saturated barium hydroxide. The barium carbonate (XIII) (6 mg.) formed was collected, washed, and dried.

The mixture was filtered hot, and the precipitate of silver bromide was washed with carbon tetrachloride, The combined filtrate and washings were shaken with sodium hydrogen carbonate and the recovered neutral product was sublimed a t 100°/10-2 mm. to give methyl 2,3-dibromo- terephthalate (XII) (5 mg. ; inactive), m.p. 85-88', identified by its i.r. spectrum.6

I thank Mr. A. Elmer for technical assistance.

[8/1460 Received, October 9th, 19681

A. J. Birch, R. A. Massy-Westropp, R. W. Rickards, and

G. Popjak, Biochem. J., 1950, 46, 560. 13. Smith, J . Chern. Suc., 1958, 360.

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