Biogenetic relationship of polyoxygenated diterpenes in Coleus forskohlii

Phytochemistry,Vol. 29, No. 3, pp. 821-824, 1990. Printedin Great Britain.

BIOGENETIC

0

RELATIONSHIP OF POLYOXYGENATED COLEUS FORSKOHLIl ANAND

Central

AKHILA, KUMKUM

Institute

of Medicinal

RANI

and

and Aromatic

RAGHUNATH

Plants,

Key Word Index-Coleus forskohlii; Labiatae;

diterpenes;

forskolin;

DITERPENES

IN

S. THAKUR

Lucknow

(Received in revised form 18 September

003 l-9422/90 $3.00 + 0.00 1990 PergamonPressplc

226 016, India

1989)

biosynthesis.

Abstract-The isotopic and atomic 14C/3H ratios obtained for polyoxygenated diterpenes isolated from the roots of Coleusforskohlii, that had been fed with [2-‘4C,2-3HJ-, [2-14C,4R-3H,]-, [2-14C,5-3H,]MVA, revealed that 8,13epoxy-labd-14-en-11-one is the first mono-oxygenated labdane-type diterpene to be formed on the biosynthetic pathway leading from the labdane-diterpene skeleton. Subsequent sequential addition of oxygen gives 19-dideoxyforskolin, 9-deoxyforskolin and forskolin, along with many other diterpenes. Forskolin is the last compound to be formed in the biogenetic sequence.

INTRODUCTION

Between 1974 and 1986, researchers at the CDRI, Lucknow isolated a group of diterpenoids (11-17) possessing the basic skeleton of 1 I-oxo-manoyl oxide [l-9] from the medicinal plant Coleusforskohlii Briq. The most active diterpene was characterized and named coleonol (11). Thereafter a group of researchers at Hoechst India Limited, Bombay, named the same compound as forskolin (1) with pharmaceutical activities similar to 11. The only difference between the structures of coleonol and forskolin was the configuration of the 7-acetoxy group. The Hoechst group assigned the /3-configuration to the 7acetoxy group of forskolin [2], whereas the CDRI group assigned cc-stereochemistry to the group at that position [l]. Later on both compounds were assigned the same structure and stereochemistry, the 7-acetoxy group having the /?-configuration as in forskolin [S, 91. Recently, a series of compounds have been reported (l-9) with fi-

configuration of the 7-oxygenated group, i.e. deacetyl forskolin (2) [2], 9-deoxyforskolin (3) [4, 71, 1,9-dideoxyforskolin (4) [2, 61, 1,9-dideoxy-7-deacetyl forskolin (5) [Z], coleol (6) [S], 7p-acetoxy-6/?,9a-dihydroxy-8,13epoxy-labd- 14-en- 1 l-one (7) [lo], 6P,7b,9a-trihydroxy8,13-epoxy-labd-14-en-11-one (8) [lo], 6/Ghydroxy-8,13epoxy-labd-14-en-1 l-one (9) [lo] and 8,13-epoxy-labd14-en-11-one (10) [lo]. Earlier a series of compounds were reported other than coleonol with a-configuration of the 7-oxygenated group. These were coleonol B and C (15, 16) [4], deoxycoleonol (13) [4], coleonol E (14) [6], coleonol D (17) [S] and coleonol F (12) [6]. Compounds in which C-l 1 is not oxygenated have also been reported such as coleonone [S]. With ca 10 diterpenoids possessing different oxygenated functions in the 11-oxo-manoyl oxide moiety, the plant has become remarkably interesting as far as the biosynthesis and sequential introduction of oxygen into the 11-oxo-manoyl oxide skeleton is concerned. There are

16

RI0 =

LI’ I, 3“,,,& s - 18 9 ” I IOf- 8 0 ‘4 2

R4 311 4 6 "0,, 8 R2

4 5 6 I 8 9

R' OH OH OH H H H H H H

10

H

I 2

3

CIMAP

Publication

R' OH OH OH OH OH H on OH OH H

R" R’

R3 OAc OH OAc OAc OH H OAC OH H H

R4 OH OH H H H OH OH OH H H

II 12

13 14

R' OH H

R' OH UAc

OH

OH

R' OAc OH OAc OAc OH

R4 OH OH H H

OH

OH

15

H OH OAc OH (C-16methylis p-oriented)

16

OH

OAc

on

(C-16 methylisa-oriented) 17

no. 6/89. 821

H

OH

OAc

OH

A. AKHILA et al.

822

five oxygenated carbons in forskolin (l), C-l, C-6, C-7, C9 and C-l 1, and similarly two, three, four or five carbons are oxygenated in various related compounds (2-9). It is a challenging problem to establish which of these compounds is formed first. The two most obvious possibilities are (i) that it is the mono- or dioxygenated compounds such as 10,6 and 9 which are formed first and are then oxygenated at positions C-l, C-7 or C-9 and (ii) that 1 is formed first and is then deoxygenated to form 7 and 8 which in turn give rise to 6 and 10 (see Scheme 2). Two different routes (A and B) have been proposed (Scheme 2) for the formation of these compounds.

200:293 suggesting that at least two 3H atoms were lost and these two must be lost from C-l and C-7, the sites which are labelled from [2-‘H,]MVA along with C-12 and C-20. However, C-12 and C-20 are unsubstituted. This atomic ratio of ca 2: 3 will be found if 1 is formed via route A as shown in Scheme 2 or directly from 10. A

18

RESULTS AND DlSCtiSSlON

In order to find out the mode of introudction of oxygen in the diterpenes 1-17, MVA labelled with 3H at C-2, C-4 or C-5 (18) was fed to the roots of C.forskohlii in different sets of experiments. It is very well established that C-2 of MVA gives rise to C-l, C-7, C-12 and C-20 of the diterpenes, C-4 to C-3, C-5, C-9 and C-14, and C-5 to C-2, C-6, C-l 1 and C-15 [l l-131. The same is expected for the 1 I-oxomanoyl oxide skeleton (see Scheme 1,21). All these diterpenes may be formed directly from a mono-oxygenated compound such as 10 (lO-+l, route A, Scheme 2) or, in the reverse direction, from a tri-oxygenated compound such as 1 (l-+10, route B). In both cases the number of 3H present (expected atomic ratio) in various compounds will be different (Table 1). Forskolin (l), isolated after the plant was fed with [214C, 2-3H,]MVA, possessed an atomic 14C: 3H ratio of

19

I

,9&3_ &gpp Scheme 1. Suggested biogenetic route to labdane-type diterpene skeleton from MVA. 0, denotes 14C from [2-‘%ZJMVA and 3H from [2-3H,]MVA; A, denotes ‘H from [,!G3H,]MVA; *, denotes -‘H from [4R-“H,]MVA.

Two compounds with oxygen at C-6, C-9,

I maybepresent. Sofar compounds with oxygens

C-l 1 and at C-7, C-9 C-l

at these positions

have not been tsolated.

II

I’ 8.13

- Epoxy-labd-14.en-1

I-one

A G== n

( IO)

(C-11)

COI~OI (

6)

A

Coleonol F ( 12 ) Coleonol D ( 17 )

z===

ti

(C-9, C-l I)

Compounds

7 and 8

(C-h, c-7. c-9,

n\

C-I

I)

A

A Forskolin

li

/

(2)

Coleonol

(15.16)

(C-l

I %Dideoxyforskohn

(4)

I,9-Dideoxy-7.deacetylforskolin Coleonol

E (

(C-6. C-7, C-l

14 )

1)

A (5)

e n

9-Dcoxyforskolm Deoxycoleonol (C-l,

( (

( 1)

Deacetylforskolin BandC

, C-6, C-7, C-9, C-

I I)

3)

13 )

C-6. C-7, C-l I)

Scheme 2. Possible routes for the biosynthesis of various diterpenes from the mono-oxygenated compound IO. Route A represents a step-by-step addition of oxygen to a labdane-type diterpene skeleton, the final product being forskolin (1). Whereas route B shows de-oxidation process from 1 to IO through various stages. The oxygenated carbon atom of the diterpene skeleton has been numbered in parenthesis. Out of all the diterpenes mentioned in this scheme; only 1,3,4,6,7,9 and 10 were isolated in sufficient amounts to be assayed for radioactivity. Broken arrows represent pathways to some more diterpenes which may be present in this plant.

Polyoxygenated Table

1. Isotopic

and atomic

r4C/sH

diterpenes

in Coleusforskohlii

823

ratios in various diterpenes obtained from C.forskohlii after being fed with doubly

labelled

MVA Observed Substrate

Experiment

Diterpenes isolated [2-L4C,2-3H,]MVA Forskolin (1) 9-Deoxyforskolin (3) 1,9-Dideoxyforskolin (4) 8,13-Epoxy-labd-14-en-l l-one (10) Coleol (6) 1-Deoxyforskolin (7) 6b-Hydroxy-8,13-epoxylabd-14-en-l l-one (9) [2-‘4C,4R-3H,]MVA Forskolin (1) 9-Deoxyforskolin (3) 1,9-Dideoxyforskolin (4) 8,13-Epoxy-labd-14-en-l l-one (10) Coleol (6) I-Deoxyforskolin (7) 6/%Hydroxy-8,13-epoxylabd-14-en-11-one (9) [2-14C,5-3HJMVA Forskolin (1) 9-Deoxyforskolin (3) 1,9-Dideoxyforskolin (4) 8,13-Epoxy-labd-l4-en-11-one (10) Coleol (6) I-Deoxyforskolin (7) 6/%Hydroxy-8,13-epoxylabd- 14-en- 11-one (9)

(dpm)*

Isotopic (W: 3H) ratio

Atomic (W: 3H) ratio

476 445 354 446 450 464

1~4.92 1: 3.60 1: 3.62 1: 4.26 1:4.82 1:4.75 1:4.19

1:2 4: 5.86 4: 5.88 4:6.92 4:7.84 4~7.72 4:6.82

1:4.82 1 : 5.01 1: 3.66 1:4.78 1 z4.73 1:4.86 1:3.61 1:3.63

4:7.84 1:l 4:2.92 4: 3.82 4: 3.78 4: 3.88 4:2.88 4:2.90

693 614 494 355 559 628

1:4.81 1: 4.86 1: 2.95 1: 2.99 1: 2.94 1 : 3.57 1: 3.46 1: 2.94

4: 3.84 1:2 4:4.86 4:4.92 4:4.84 4:5.88 4:5.78 4: 4.84

585

1:2.96

4:4.88

14C

262 445 446 252 443 455 454 442

Expected

atomic

ratio?

Route A

Route B

4:6 416 417 4:8 4:8 411

4~6 4:6 4:6 4~6 4:6 4:6

4:8

4~6

4:3 4:4 4:4 4:4 4:3 4:3

4:3 4:3 4:3 4:3 4:3 4:3

4:4

4:3

4:5 4:5 4:5 4~6 416 415

4:5 4:5 4:5 4:5 4:5 4:5

4:5

4:5

*MVA was incorporated into diterpenes in C.forskohlii in 0.002~.003% yield. tThe expected atomic ratios in Route A are calculated by assuming that the diterpenes (1,3,4,6,7,9 and 10) are biosynthesized from mono-oxygenated compound IO via di-, tri, tetra- and penta-oxygenated compounds; whereas the atomic ratios in Route B are calculated considering the biogenetic pathway operated in the reverse direction to that shown in Route A.

similar result was obtained for 9-deoxyforskolin (3) (Table 1). This also suggests loss of two 3H atoms from Cl and C-7, whether formed via route A or route B (Scheme 2). Compounds 4, 6, 7, 9 and 10 isolated after the plant was fed with the same precursor showed atomic ratios which were suggestive of one 3H loss in 4 and 7 and no 3H loss in 10,6 and 9. All these ratios confirm route A for the biosynthesis of diterpenes 4, 6, 7, 9 and 10. In order to confirm these findings and to establish the route for 1 and 3, two more experiments were conducted by feeding [2-‘4C,4R-3H,]MVA and [2-‘4C,53H,]MVA to different plants. The atomic ratios obtained for 1, 6 and 7 (Table 1) with [2-14C,4R-3H,]MVA revealed loss of one 3H whereas the atomic ratios for 3,4, 10 and 9 suggested no loss of 3H from these diterpenes. The results tabulated in Table 1 from Experiment 2 clearly indicate that route A is operative for the biosynthesis of 3,4, 10 and 9. The atomic ratio in diterpenes isolated after feeding [2-r4C, 5-‘HJMVA to C.,forskohlii suggested loss of three 3H from 1, 3, 4, 7 and 9 (Table 1) and two 3H from 10 and 6 confirming route A for 10 and 6. The results obtained from the three different feeding

experiments establish that the biogenesis of all these diterpenes proceeds via route A as shown in Scheme 1. The expected atomic ratio in 1 via route A and B in each of the three experiments is the same indicating that it may be formed via route A or even directly. However, as all of the other diterpenes are formed via route A it can be safely suggested that it is the last polyoxygenated diterpene to be formed via route A. The biogenetic pathways in Scheme 2 also suggest the presence of a few more diterpenes which have not yet been isolated from C. forskohlii. These may be di-oxygenated compounds with oxygens at C-11 and C-7 or tri-oxygenated compounds with oxygens at C-6, C-9 and C-l 1 or at C-7, C-9 and C- 11. Efforts are being made to isolate these compounds from this plant species.

EXPERIMENTAL Materiaf. Coleus forskohlii Briq. was grown on the experiLucknow, India. [2-i4C]-, [4R-3H,]-, [4S3H,]-, [2-3H,]- and [5-3H,]MVA lactone were purchased

mental farms of CIMAP,

824

A.

AKHILA

from the Radiochemical Centre, Amersham, U.K. and the Bhabha Atomic Research Centre, Bombay, India. Feeding methods and isolation of products. Labelled MVA lactone ([2-3H,]MVA 1.28 Ci/mmol, [2-‘4C]MVA 53 mCi/ mmol, [4R, or 4S-4-3H,]MVA 1.36 Ci/mmol and [5-3H,]MVA 2.52 Cijmmol) was hydrolysed to its free acid with aq. NaHCO, by known methods [I4161. The shoots of C.forskohlii were ca 3O4Ocm long with profuse branching and a rhizome/root system and weighed ca 100-150 g at the time of feeding. The plants were dug out of the ground, the rhizomes washed free of soil and immersed in a soln containing “C/3H-labelled MVA and left under natural conditions to allow the uptake of the substrate on sterile and bacteria free medium [16]. In all, 9 plants were taken and their rhizomes/root system weighed 850 g. 10 &i of 14C and ca 50 &i of 3H as a mixture of [Z-‘“Cl- and [4R-‘H,]-, [S-‘H,]or [2-3H,]MVA was fed to a set of three plants. After 60 hr the rhizome/roots were washed free of substrate, excised from the rest of the plant, air-dried at room temp. and extracted with EtOAc at room temp. The isolation of compounds was carried out following the experimental method reported earlier [IO]. All the experiments were carried out in duplicate. During the preliminary experiments, all the above diterpenes were isolated and identified by spectral data available in the literature [IO]. The diterpenes were used as carriers while isolating the radioactive compounds. The EtOAc extract was coned and the residue taken up with MeOH and filtered. The mother liquors were ‘&apd and the residue subjected to CC on silica gel, cluting with cyclohexane-EtOAc (4: I). Three fractions were collected (A-C). Fraction A was subjected to CC (silica gel, cyclohexane and gradient of CH,Cl,) and the following compounds were sequentially eluted. Coleol (6), R, 0.91, oil, [a]:’ +4.6” (c 0.3); ldeoxyforskolin (7), R,0.82, mp 136-137” (n-hexane), [E];” -23.1” (c 0.10) and 1,9-dideoxyforskolin (4), R, 0.63, mp 148-149” (nhexane) [%]A” - 105.1” (c 0.3). Fraction B on CC (silica gel, CH,CI,) yielded the following compounds: 6,!?-hydroxy-8,13epoxy-labd- 14-en- 1 I -one (9), R, 0.56, mp 119-120’ (cyclohexan&EtOAc), [XI;’ - 132.0” (c 0.10) and 8,13-epoxy-labd-14en-l l-one (lo), R, 0.89, mp 96-97” (i-PrOH), [n];;” - 103.0” (c 0.30). Fraction C was crystallized from methyl-t-butylether to yield forskolin (1) identical with an authentic sample, mp 220-221 L’,[a]hO - 16‘. After the removal of 1 the mother liquor on CC (silica gel, CH,Cl,-EtOAc 9: 1) yielded 9-deoxyforskolin (3), Rf 0.23, mp 187-188” (n-hexane-methyl-t-butyl ether), [g];” -98.0” (c 0.30). Radiochemical methods. These have been described previously [15, 171. The samples for assay by liquid scintillation spectro-

et

al.

metry contained 200-500 dpm as 14C and upto 2000 dpm as ‘H. 4OOQO dpm were accumulated to make sure that 20 was & 1%. Radioactive compounds were purified by recrystallization to constant sp. radioactivity. All the experiments were carried out in duplicate. Acknowledyements-The authors wish to thank Dr B. R. Tyagi of this institute for providing plants of C..forskohlii containing a high percentage of forskolin. One of us (KR) is grateful to CSIR for financial support.

REFERENCES

1. Tandon, J. S., Dhar, M. M., Ramkumar, S. and Venkatesan, K. (1977) Indian J. Chm. ISB, 880. 2. Bhat, S. V., Bajwa, B, S., Dornauer, H., De Souza. N. J. and Fehlhaber, H. W. (1977) Tetrahedron Letters 1669. 3. Jauhari, P. K., Katti, S. B., Tandon. J. S. and Dhar, M. M. (1978) Indian J. Chem. 16B, 1055. 4. Tandon, J. S. Jauhari, P. K., Singh. R. S. and Dhar. M. M. (1978) Indian J. Chem. 16B, 341. 5. Katti, S. B., Jauhari, P. K. and Tandon. J. S. (1979) Indian J. Chem. 178, 321. 6. Painuly, P., Katti. S. B. and Tandon, J. S. (1979) Indian J. Chem. ISB, 214. 7. Bhat. S. V., Dohadwalla, A. N., Bajwa, B. S., Dadkar, N. K., Dornauer. H. and De Souza, N. J. (1983) J. Med. Chem. 26, 486. 8. Saxena, A. K., Green, M. J., Shue, H. J., Wang, J. K. and McPhail, A. T. (1985) Tetrahedron Letters 26, 551. 9. Prakash, O., Roy, R. and Dhar. M. M. (1986) J. Chem. Sot. Perkin Trans II 1779. 10. Gabetta, B., Zini, G. and Danieli, B. (1989) Phytochemistry 28, 859. 11. Birch, A. J., Richards, R. W., Smith, H., Harris, A., Whalley, W. B. (1959) Tetrahedron 7, 241. 12. Hanson, J. R. (1972) Proy. Phptochem. 3, 231. 13. Hanson, J. R. (ed.) (1977) Terpenoids, Steroids: Specialist Periodical Reporl Vol. VII. The Chemical Society, London. 14. Al1en.K. G., Banthorpe,D. V.,Charlwood, B. V., Ekundayo, 0. and Mann, J. (1976) Phyrochrmistry 15. 101. 15. Banthorpe. D. V., Modawi, B. M.. Poots. I. and Rowan, M. G. (1978) Phgrochemistry 17, 1 I 15. 16. Banthorpe, D. V., Mann, J. and Turnbull, K. W. (1970) J. Chem. Sot. C, 2689. 17. Allen, K. G., Banthorpe. D. V.. Charlwood. B. V. and Ekundayo, 0. (1980) Phyrochemistry 19. 1429.