Indian Journal of Chemistry Vol. 408, March 200 I, pp. 228-231

A new chiratane triterpenoid from Swertia chirata

Ajit K Chakravarty"·* , Tapas Sarkar", Binayak Das", Kazuo Masudah & Kenji Shiojimah

"Indian Inst itute of Chemical Biology, Calcutta 700 032, Indi a

hShowa College of Pharmaceutical Sciences, Machida, Tokyo 194-8543, Japan

Received 4 February 2000; accepTed (revised) 29 AugusT 2000

Reinvestigation of Swertia chirata resulted in the isolation of a new chiratane triterpenoid, chirat- 16-en-3~ .

24-diol, besides known 24-ethylcholest-4-en-3-one and I, 5, 8-trihydroxy-3-methoxy-xanthone-8-0-B-o-gluco­pyranoside. The structure of the new triterpenoid has been elucidated on the basis of 2D NMR spectral analyses.

Swertia chirata, the well-known Indian medicinal plant 1

, was earlier found by us to be the only higher plant elaborating rearranged gammacerane and hopane triterpenoids belonging to novel skeleta2

·6

, viz. swertane, kairatane and chiratane. The only chiratane triterpenoid obtained was chiratenol 1 which was presumed to be formed biogeneticall/ from isohopane cation (at C-22) by rearrangement of its C20-C21 bond followed by the elimination of a proton from C-16. A re-examination of the plant now yielded a new chiratane triterpenoid, characterized as chirat-

16-en-3~, 24-diol 2, besides known 24-ethylcholest-4-en-3-one 3 and a xanthone-0-glucoside, identified as 1, 5, 8-trihydroxy-3-methoxyxanthone-8-0-~-D­glucopyranoside 47

. We report herein the isolation and structure elucidation of 2 mainly on the basis of 20 NMR spectral analyses.

Compound 2, m.p.288-90°C, [a] 0 +52.6° (pyridine), showed in its HR-MS, the M+ at m/z 442.3793 corresponding to the molecular formula C30 H500 2. It showed IR absorption band for OH group(s). On acetylation with Ac20-pyridine, 2 yielded a diacetate 5, m.p. 214-15° C, (a]0 +65 .0°

1 Rt = OH, R2 = CH3 2 R1 = OH, R2 = CH20H 5 R1 = OAc, R2 = CH20Ac 6 R1 = OAc, R2 = CH3

7

(CHCI3), M+ at m/z 526.4004 (C34H 540 4). The 11-1 NMR spectrum of 2 (Table I) exhibited signals for a CH20H and a CHOH groups, a tri substituted vinyl proton and seven tertiary methyl groups. The coupling constant (10 .5 Hz) of the one-proton broad doublet at 8 3.675, shifted down-field to 8 4.572dd (J = 10.8, 6.0 Hz) in the spectrum of the diacetate (5), indicated that the secondary OH group of 2 is equatorial in nature. Mass spectral fragmentation of 2 generated8 ions at mlz 223 (a, 7%), 205 (b, 48%), 203 (c, 37%), 204 (d, 53%), 187 (e, 57%) and 189 (j, 100%) (Chart 1). The formation of species a and b clearly indicated that

Table I- 1H Chemical shifts" (8, 500 MHz) of 2 and 5.

Compd H3-23 H3-25 H,-26 H3-27 Hr28 H3-29 Hr30 H-3a H-16 H~-24

2h 1.574 0.874 0.934 1.021 0.979 0.952 0.835 3.675br.d 5.33ldd 3.725dd( 11 .0,5.3) (10.5) ( 1.8,3. 7) 4.527d( 11 .0)

s< 1.006 0.889 0.965 0.965 0.943 0.919 0.763 4.572dd 5.212br.d 4. 11 2d( ll.6) ( I 0.8.6.0) (5.1) 4.344d( 11.6)

"Assignments were done on the basis of 20 NMR spectral analyses. Values in the parentheses are the coupling constan ts in Hz. bSpectrum recorded in pyridine-d5.

<spectrum recorded in CDCI .1 .

CHAKRAVARTY eta/.: A NEW CHIRATANE TRITERPENOID FROM SWETIA CHIRATA 229

HO~• CH20H

a, mlz 223 (7%)

d. mlz 204 (53%)

Qj CH20H

b, mlz 205 (48%)

e. mlz 187 (57%)

Chart 1

c. mlz 203 (37%)

+

f, mlz 189(100'/o)

both the OH groups are located in the A/B part of the n\olecule. 13C NMR spectrum (Table II) of the diacetate 5 was found to be close to that of chiratenyl acetate 6 except the signals for C-4, C-6, C-23 and C-24 indicating that either of the gem-dimethyl groups of ring A of 1 has been oxidised to CH20H group in 2.

In order to unambiguously elucidate the structure of 2, the compound was subjected to 20 NMR spectral analyses. A detailed analysis of the two- and three-bond correlations of the methyl proton and the vinyl proton signals with those of the neighbouring carbons (Table III) in the HMBC spectrum of 2 revealed the presence of the part structure shown by heavy lines 111 structure 7 indicating that the compound belongs to chiratane skeleton. The methylene carbon (C-22) resonating at 8 46.4 was found to be correlated through three bonds with the vinyl proton (8 5.331) on the one hand, and two

methyl proton (H3-29 and Hr30) signals at 8 0.835 and 0.952 on the other hand, conclusively proved that the trisubstituted double bond is located at ~ 16-position and the gem-dimethyl group is located at C-21 rather than C-20 or C-22. As expected, the vinyl

Table II-13C Chemical shifts" (8, 125 MHz) of 1, 2, 5 and 6

Carbon

2 3 4 5 6 7

8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 COCH3

COCH3

1 b. c 2d

38.8 39.0 27.4 28.6 79.0 80. 1 38.8 43.3 55.3 56.3 18.2 19.0 33.7 34.4 41.0 (40.4) 41.2 50.7 51.0 37.1 (36.8) 37.1 21.4 21.8 23.3 23 .5 45.2 45.5 40.4 40.6 32.1 32.3 120.0 120.5 139.2 139.3 36.8 (37 . 1) 37.0 38.3 38.5 35.2 35.4 32.5 32.6 46.2 46.4 28.1 23.6 15.4 64.5 16.3 (16.2) 17.0 16.2 (16.8) 16.8 16.8 ( 17.5) 16.3 17.5 (16.3) 17.7 32.4 32.4 24.6 24.7

sc 38.6 23.7 80.3 41.1 55.9 19.2 34.1 41.0 50.7 36.9 21.6 23 .3 45.2 40.3 32.0 11 9.9 139.2 36.8 38.2 35.2 32.5

6b.c

38.4 23.7 8 1.0 37.8 55.3 18. 1 33.6 41.0 (40.4) 50.6 37.0 (36.8) 21.4 23.3 45 .1 40.4 32.1 120.0 139.2 36.8 (37.0) 38.2 35.2 32.5

46.2 46.2 22.5 28.0 65.4 16.6 16.2 16.3 (16.1) 16.0 16. 1 (16.8) 16.6 16.8 (17.5) 17.5 17.5 ( 16.3 ) 32.3 32.4 24.6 24.6 21.1, 2 1.2 21.3 170.6, 171.0 171.0

"Assignments were done on the basis of anal yses of 20 NMR spectra, viz. 1H- 1H COSY, HSQC and HMBC spectra. bRevised assignments based on 20 NMR spectral analyses. Values in the parentheses are the assignments reported earlier in ref. 3. <spectra recorded in CDCI3•

dSpectrum recorded in pyridine-ds.

Table III--One-bond and multiple-bond 1H- 13C correlation data of 2

81-1ppm One-bond correlation Multiple-bond correla tions

8cppm be Ill

1.574 (Hr23) 23.6 (C-23) 43.3 (C-4) 56.3 (C-5) 64.5 (C-24) 80.1 (C-3) 3.725, 4.527 (H 2-24) 64.5 (C-24) 23.6 (C-23) 43.3 (C-4) 80. 1 (C-3) 0.874 (H3-25) 17.0 (C-25) 37. 1 (C-10) 39.0 (C-1) 51.0 (C-9) 56.3 (C-5) 0.934 (Hr26) 16.8 (C-26) 34.4 (C-7) 40.6 (C-14) 41 .2 (C-8) 51.0 (C-9) 1.021 (Hr27) 16.3 (C-27) 32.3 (C- 15) 40.6 (C-14) 41.2 (C-8) 45 .5 (C- 13) 0.979 (Hr28) 17.7 (C-28) 37.0 (C-18) 38.5 (C-19) 45.5 (C-13) 139.3 (C-17) 0.952 (H3-29) 32.4 (C-29) 24.7 (C-30) 32.6 (C-21) 35.4 (C-20) 46.4 (C-22) 0.835 (H3-30) 24.7 (C-30) 32.4 (C-29) 32.6 (C-21) 35.4 (C-20) 46.4 (C-22) 5.33 1 (H3-16) 120.5 (C-20) 32.3 (C-15 ) 37.0 (C-18) 40.6 (C- 14) 46.4 (C-22)

230 INDIAN J. CHEM., SEC. B, MARCH 200 1

proton also showed three-bond correlations with the quaternary carbons C-14 (8 40.6) and C-18 (8 37 .0). Again, both the methyl proton signals at 8 1.574 and the methylene proton signals of the CH20H group at 8 3.725 and 4.527 were correlated through three bonds to the hydroxymeth ine carbon resonating at 8 80. 1 clearly demonstrating that either of the carbons C-23 and C-24 is hydroxylated. That the axial carbon (C-24) is hydroxylated was evident from the fact that the methylene protons of the CH20H group showed NOE interactions with H3-25 in the NOESY spectrum of 2. Moreover, the relati ve stereochemistry at the ring juncture chiral centres could also be establi shed from the NOE interacti ons observed in the NOESY spectra of both 2 and its diacetate 5 as depicted in Figure 1.

Based on the above evidence, the structure of compound 2 was establi shed as chirat-16-en-3~ , 24-diol. lncindentally, 2 happens to be the second member belonging to the chiratane skeleton .

It may be mentioned here that the structure elucidation of chiratenol 1 described in our earlier paper3 was based on the analysis of only the HMBC spectrum of its 3-oxo derivati ve, and 1 H- 13C COSY or HSQC spectrum could not be recorded due to the non­avail ability of the facility with us. As a result, some earlier reported 13C chemical shift ass ignments, particularly for some methyl and quaternary carbons of 1, have been found to be erroneous. However, based on the present study and reinvestigation of the 20 NMR spectra of chiratenyl acetate 6, these 13C chemical shift assignments have been revised (Table II) .

Compounds 3 and 4 were independently identified as 24-ethylcholest-4-en-3-one9 and I ,5,8-trihydroxy-3-methoxy-xanthone-8-0-~-D-glucopyranoside10 res­pectively on the bas is of their 20 NMR spectral analyses. In the process, their 1 H and 13C chemical shifts were unambiguously assigned and included in the Experimental Section .

Experimental Section General. Melting points are uncorrected. NMR

spectra were recorded on a 500 MHz instrument with TMS as internal standard. ElMS were recorded at 30 eY.

Isolation. Air-dried and milled aerial parts of S. chirata (2.5 kg) were successively extracted with petroleum ether (60-80° C) and MeOH in a Soxhlet apparatus. The extracts were evaporated to dryness under reduced pressure to obtain petroleum ether

1-10

2

AcO

Figure 1-NOE interactions observed in the NOESY spectra of 2 and 5

extract (50 g) and MeOH extract (96 g). The MeOH extract was fractionated into CHCI 3 soluble part, n­BuOH soluble part and water soluble part.

Purification of the petroleum ether extract by repeated column chromatography over Silica gel and neutral alumina yielded 2 (51 mg) and 3 (20 mg) besides swertanone2 and chiratenol3 1 already reported by us, while the 11-BuOH soluble part of the MeOH extract on purification through column chromatography over Silica gel afforded 4 (5 mg).

Chirat-16-en-3~, 24-diol 2. Recrystallised from MeOH-CHCI3, m.p. 288-290° C, [a]0 +52.6° (c, 0.6, pyridine). IR : 3510, 3340, 1048, 1020 cm-1. El-MS m/z (rei. int.) : 442.3793 (M+, 30), 427 (18), 424 (57), 409 (37), 394 (32), 229 (24), 223 (7) , 205 (48), 204 (53), 203 (37), 191 (64),189 (100), 187 (57) . 1H NMR: Table I. 13C NMR: Table II.

24-Ethylcholest-4-en-3-one 3. Recrystallised from acetone, m.p. 60-62° C ( lit.9 m.p.92-94° C ). IR: 1680, 1620 cm-1. EI-MS m/z (rei. int.): 412 (M+, I 00), 397 (18), 271 (18), 229 (56). I H NMR (CDCIJ): 8 0.740 (s, H3-18), 1.182 (s , Hr 19), 0.917 (d, J = 6.4 Hz, Hr 21), 0.814 (d, J = 7.0 Hz, Hr26), 0.836 (d, J = 7.0 Hz, H3-27), 0.843 (t, J = 7.0 Hz, H3-29), 5.726 (br. s, H-4). 13C NMR (CDCI3): 8 35.7 (C-1), 33.9 (C-2), 199.9 (C-3), 123.9 (C-4), 171.8 (C-5), 33.0 (C-6), 32.1 (C-7), 35.6 (C-8), 53.8 (C-9), 38.6 (C-10), 21.0 (C-11), 39.6 (C-12), 42.4 (C-13), 56.0 (C-14), 24.2 (C-15), 28.2 (C-16), 55.9 (C-17), 12.0 (C-18), 17.4 (C-19), 36.1 (C-20), 18.7 (C-2 1 ), 33.9 (C-22), 26.1 (C-23), 45.8 (C-24), 29.1 (C-25), 19.8 (C-26), 19.0 (C-27), 23.1 (C-28), 12.0 (C-29).

CHAKRAVARTY eta/.: A NEW CHIRATANE TRITERPENOID FROM SWETIA CHIRATA 231

Chirat-16-en-3~, 24-diyl acetate 5. Compound 2 (10 mg) was acetylated with Ac20-pyridine at 100° C for 2 hr. Purification of the product through chroma­tography and crystaiiisation from MeOH-CHC13

yielded 5 as flakes, m.p. 214-15°C, [a] 0 +65.0° (c, 0.4, CHCb). IR: 1742, 1725, 1250 em·'. HR-MS: M+ at m/z 528.4050. 1H NMR: Table I. 13C NMR: Table II.

1, 5, 8-Trihydroxy-3-methoxyxanthone-8-0-~­D-glucopyranoside 4. Recrystaiiised from MeOH as globules, m.p. 234-35° C (lit.7 mp 215-l7°C); [a]0

-68.4° (c, 0.1, pyridine). FAB-MS: m/z 437 [M+Ht. 1H NMR (pyridine-d5): 8 6.531 (d, J = 2.5 Hz, H-2), 6.224 (d, J = 2.5 Hz, H-4), 7.408 (d, J = 8.9 Hz, H-6), 7.632 (d, J = 8.9 Hz, H-7), 5.507 (d, J = 7.6 Hz, H­I'), 4.480 (m, H-2') , 4.428 (m, H-3') , 4.380 (dd, J = 8.8, 8.8 Hz, H-4'), 4.214 (m, H-5'), 4.480 (m, Ha-6') , 4.665 (dd, J = 11.9, 2.1 Hz, Hb-6') , 3.644 (s, OMe). 13C NMR (pyridine-d5): 8 164.1 (C-1 ), 97.9 (C-2), 166.9 (C-3), 92.2 (C-4), 157.3 (C-4a), 146.4 (C-4b), 142.9 (C-5), 122.1 (C-6), 113.7 (C-7), 151.0 (C-8),

113.2 (C-8a), 104.8 (C-8b), 182.4 (C=O), 105.9 (C-1'), 75.4 (C-2'), 77.9 (C-3'), 71.4 (C-4'), 79.5 (C-5'), 62.7 (C-6'), 55.9 (OMe).

References I Chopra R N, Nayar S L & Chopra I C, Glossary of Indian

Medicinal Plallls (Council of Scientific and Industrial Research, New Delhi) 1956, p 237.

2 Chakravarty A K, Das B, Pakrashi S C, McPhail D R & McPhail AT, J Chem Soc Chem Commun, 1989, 438.

3 Chakravarty A K, Das B, Masuda K & Ageta H, Tetrah edron Lett., 31, 1990, 7649.

4 Chakravarty A K, Mukhopadhyay S & Das B, Phytochemistry, 30, 1991,4087.

5 Chakravarty A K, Mukhopadhyay S, Masuda K & Agela H, Indian J Chem, 31 B. 1992, 70.

6 Chakravarty A K, Mukhopadhyay S, Masuda K, & Ageta H, Tetrahedron Lett., 33. 1992, 125.

7 Dhoubhadel S P, Wagley P P & Pradhan S D, Indian J Chem. 19B, 1980,929.

8 Shiojima K, Masuda K, Suzuki H, Lin T, Ooishi Y & Ageta H, Chem Pharm Bull. 43, 1995, 1634.

9 Achari B, Ali E, Ghosh Dastidar P P & Pakrashi S C. J Indian Chem Soc, 5 I, 1974, 419.