Labdane diterpenoids from Stevia seleriana

Phytochemiscry, Vol.30,No. 2,pp.599 602,1991 Printed inGreatBritain.

LABDANE

W31 9422/91$3.00+0.00 Pergamon Pressplc

DITERPENOIDS ELEAZAR

FROM STE VIA SELERIANA and ALFRED~

M.ESCAMILLA*

ORTEGA

Instituto de Quimica, Universidad National, Autbnoma de Mtxico, Circuit0 Exterior, Ciudad Universitaria, Coyoacln, 04510, MCxico, D.F.; *Departamento de Ingenieria Quimica, Instituto Tecnol6gico de Celaya, Celaya, Gto. C.P. 38010, MCxico (Received in revisedform 12 June 1990)

Key Word Index-Stevia

seleriana; Compositae; Eupatorieae; labdane diterpenoids.

Abstract-Stevia seleriana afforded, in addition to known terpenoids, three new labdane type diterpenes, 5B,9jIH,lOq7-labden-3-oxo-15-oic acid, 5/7,9BH,lOa,3,4-seco-labd-7-en-3,15-dioic acid and 5B,9/IH,lti,3,4-seco-labd7,13(Z)-dien-3,15-dioic acid. Their structures were established by chemical and spectroscopic methods.

INTRODC’CTION

Table 1. ‘HNMR

In a continuation of our search for new natural diterpenoids [14] in plants of the tribe Eupatorieae (Compositae) [S-S], a study of Stevia seleriana Robins afforded three new labdane type diterpenes, ent-labd-7-en-3-oxo15-oic acid (1) ent-3,4 seco-labd-7-en-3,15-dioic acid (2) and ent-3,4-seco-labda-7,13(Z)-dien-3,15-dioic acid (3). Two of these diterpenoids (1 and 2) are enantiomers of two compounds isolated previously [9]. In addition to the new diterpenoids, a mixture of lupeol and p-amirin was isolated [lo]. RESULTSANDDIWXJSSION

Compound 1, analysed for &,Hs203 and its IR spectrum showed typical absorptions for a free carboxylic acid, one carbonyl function and typical trisubstituted double bond [ 111. The acid nature of 1 was confirmed by its conversion to the methyl ester 4 on treatment with ethereal diazomethane. The ‘H NMR spectrum (Table 1)

1 4

R = CO,H R * CO,tdc

5

6 7

OH, R' = OH R" R'= OH,R'= C&H R' = Ok, R' = OAc *SC&H

2 R-H 8 R= Me

3

spectral data of compounds 4-7 (80 MHz, TMS as int. standard)

H

4 (C,D,)

5 (CDCl,)

6 (CDCl,)

7 (CDCI,)

2a 2 3 I 14 15 16 17 18 19 20 -0Me

2.44 ddd* 2.1 m 2.1 m 5.4 m 21 In

1.30 m

1.3 In

1.35 m

3.22 dd$ 5.35 m 1.32 3.66 t#

3.20 dd 5.36 m 2.1 m

O.Wl

0.91 d 1.68 sa 0.75 s 1.0 s 0.82 s 3.65 s

4.63 dd 5.33 M 1.35 4.09 t 0.84 d 1.67 sa 0.73 s 0.94 s 0.86 s

0.87 dt 1.62 s 0.19 s 1.05 s 0.91 s 3.42 s

1.67 sa 0.74 s 0.98 s 0.85 s

-AC OH

2.15 s 2.14 s 1.90 sa

4.48 sa

of 4 showed five methyl group signals of the labdane skeleton: a vinyl one at 6 1.62, three tertiary ones at 6 0.79, 0.91 and 1.05, a secondary one at 6 0.87. (J = 7 Hz) and a methyl ester group at 63.42. The multiplicity shown at 6 2.44 ( 1H, d&f) and 2.1 (1H) were assigned to the fi and a geminal protons adjacent to the carbonyl group. These chemical shifts were in good agreement with those of 3oxo-cativic acid [9]. The relative position of the oxygen functions was further supported by the mass spectrum (Scheme 1) and showed a fragment [M -CO,R] + and [3 19 - CO=C=C <] + . The facile retro-DielsAlder type reaction [12], located the trisubstituted double bond between C-7 and C-8 confirming the labdane skeleton of acid 1. The LiAlH,, reduction of 2 gave a CaOH3602 diol (5). The ‘HNMR chemical shifts observed for the hydroxymethylene protons in 5 (triplet centred at 63.66) 599

E. M. ESCAMILLA and A. ORTEGA

600

275(53)

196(2)

122(25)

81(50)

Scheme 1. Fragmentation pattern of compounds 1 and 6. and the signal (dd) assigned for the proton axially attached to C-3 are in complete agreement with a C-3 pequatorial function [12]. Partial reduction of 4 with NaBH, gave the alcohol 6 with molecular formula C,,H,,O,. The ‘HNMR spectrum of 6 showed characteristic signals for a secondary alcohol (6 3.20, lH, dd) which was assigned to the C-3 hydroxyl group. Acetylation of 5 (AczO-pyridine) gave the diacetate 7 (1740 cm-‘). The mass spectrum of the hydroxyester had the formula Cz4H404 ([Ml’ m/z 398) and showed an elimination of acetic acid which indicated the presence of an acetate group. The new compound 2, C,,H,,O, at m/z 338, was isolated as a crystalline compound. The IR spectrum showed typical absorptions for a carboxylic acid group and trisubstituted double bond. Ethereal diazomethane treatment of 2 gave the methyl diester 8 (CO,Me at 6 3.65 and 3.68, each 3H). The relative position of the oxygen functions was further supported by the mass spectrum (Scheme 2) which showed a fragment [M -CHzCHzCH(Me)CH,CO,R]+ and typical retro-Diels-Alder reaction. The latter fragment was transformed by McLafferty fragmentation to m/z 122 (C,H,,). The ‘H NMR spectrum (Table 2) of 8

also showed the five methyl group signals of the labdane skeleton. However the multiplicity observed suggested that 8 possesses a modified labdane skeleton because three of the methyl groups were attached to a secondary carbon atom and the other was attributed to a tertiary methyl group. Finally, one signal was assigned to the vinyl proton. The i %ZNMR signals of 8 (Table 3) at 6 173.2 (s), 122.2 (d) and 174.4 (s) supported the partial structure. The 13CNMR signals at 641.2 (t) and 32 (d) suggested that ring A was opened. All the above data must be accommodated in a structure such as 2. The stereochemistry of this compound was deduced from spin decoupling experiments and by comparing the i3C NMR chemical shift data of 8 with those reported for related compounds [9, 13, 141. The third new compound, 3, was isolated as a crystalline compound, C2,,H3z04. It was shown to be a labdane type with a conjugated double bond in the side chain, which showed UV absorption at 232 nm (E 22 387) and IR absorption at 1640,1600,980,890 cm-i. The presence of an r&unsaturated side chain (300&2500, 1710 cm - ’ ) was indicated by the mass spectral ion peaks at m/z 236 [M C,H,O,]+ and 223 [C,H,O,]+. The loss of the

-Me

~ -OMe

‘W+$~R

*

341(3)

c

307(70)

.

79(81)

*

37(15)

-CO>R

335(34)

-CHICH>COzR H

I - MeOH

1

-CH,CHICH(Me)CH#ZOIR

[Ml 366(15)

303(4)

RDA I

C&R 1'

9-

McL.ifferty ) 122(100)

\

/

196(37)

Scheme

2. Fragmentation

pattern

of compound

2.

Labdane diterpenoids from Steuia seleriana Table 2. ‘H NMR spectral data of compound 8 (80 MHz, TMS as int. standard). H

(CDG)

(CCl,)

2 I 14 16 18 19 20 -0Me

m m

2.20 5.38 2.20 0.95 0.752 0.92 0.85 3.65 3.68

: sit dS s s

2.15 5.30 2.15 0.95 0.74 0.90 0.86 3.6 3.58

13,16=7.0 Hz ;id,,,=7.5 Hz $J4,1s=7.5 Hz. Table 3. 13CNMR chemical shifts of compound 8 (CDCl,, TMS as int. standard) C

6

C

1 2 3 4 5 6 7 8 9 10 3’ 15l

38.8 t* 41.2 t 173.2 s 32.0 d 26.0 d 31.3 t 122.2 d 135.4 s 47.0 d 28.5 s

11 12 13 14 15 16 17 18 19 20 4Me -0Me

s 22.0 t 22.3 t 44.6 d 39.2 t 174.4 s 24.6 c 25.1 c 17.0 c 20.0 c 18.5 c 50.0 s 51.2 s

*Sford multiplicity.

C,H,02

moiety suggested the presence of a carboxylic acid in the other side chain. The ‘H NMR spectrum of 3

showed five methyl group signals of the labdane skeleton: a vinyl one at 6 1.75, one at 6 1.90 assigned to an a$unsaturated system, two attached to secondary carbon atoms at 6 0.90 and 0.83 (J= 7 Hz), and a quaternary methyl (60.7). Comparison of the spectroscopic and optical rotation data of 3 with 2 suggested a similar structure. The ‘HNMR signals at S 3.42 and 1.90 are typical for an olefinic system with Z configuration [15-17-J The relative position of the double bond was further supported by double resonance experiments. Based on the above we propose the structure 3, the stereochemistry for the new diterpene was in good agreement with those reported in the literature [18, 193. EXPERIMENTAL Stevia selerinna R. was collected in November

1984, 48 km Oaxaca-Tehuantepec road, Oaxaca State. A voucher specimen is deposited at the herbarium of the Instituto de Biologia (UNAM), MCxico. Dried leaves, flowers and stems (750 g) were packed in a glass column and extracted with hexane to afford 77.8 g crude syrup. It was then extracted with Me,CO to give 17.22 g crude extract, followed by MeOH which gave 38.6 g extract. The hexane extract crystallized spontaneously. The crystals obtained were washed with hexane and then chromatographed PWTO 30-:2-P

601

on a silica gel column (44.38 g) eluted with hexane (38 fr). The fourth frs onward gave 29.57 g crystalline solid. TLC showed that this sohd was a mixt. of two compounds. The solid was adsorbed on celite and chromatographed on a silica gel column deactived with 10% H,O and impregned with 9% AgNO,. The column was successively eluted with hexane-EtOAc mixts (9: 1, 4: 1 and 7: 3), and frs of ca 100 ml collected. The following diterpenoids were obtained, in order of elution %, l-4. ent-labd-7-en-3-oxo-15-oic acid (1) (16 g). Identified by physical (mp, [LY]~)and spectroscopic (IR, ‘H NMR, 13CNMR, MS) data. The analytical sample showed mp 78-82” (Me&O-hexane): [a];‘“+ 28.3 (CHCl,; c 0.493); IR vz’, cm-‘: 34&2500, 1775 (CO,H), 1709.9 (carbonyl of aliphatic ketone); 3030, 1670 and 910 (trisubstituted double bond); MS (70 eV, direct inlet.) m/z (rel. int.): 320 [M] + (22), 305 (8), 235 (38), 205 (lo), 196 (5), 122 (lOO), 81 (50), 59 (52); ‘H NMR, 80 MHz (CDCl,) 6:5.4 (H-7), 2.44 (ddd, H-2), 2.1 (H-2, 5), 1.62 (s, H-17), 105 (s, H-19), 0.91 (s, H-20), 0.87 (d, H-16), 0.79 (s, H-18). Methyl ester 4. Treatment of compound 1 (2.Og) with CH,N,-Et,0 soln gave 4 (1.78 g) as a syrup, IR vz: cm-‘: 1745,120O(ester), 1715(carbonyl), 3030,1670,9OO(double bond); ‘H NMR80 MHz (C,D,) 6:2.44 (ddd, lH, H-2), 2.1 (br, lH, H2), 5.4 (s, H-7), 2.1 (br, lH, H-14), 0.87 (d, 3H, (double bond); ‘H NMR 80 MHz (C,D,) 6:2.44 (ddd, lH, H-2), 2.1 (br, lH, H2), 5.4 (s, H-7), 2.1 (br, lH, H-14), 0.87 (d, 3H, H-20), 1.62 (s, 3H, H-17), 0.79 (s, 3H, H-18), 1.05 (s, 3H, H-19), 0.91 (s, 3H, H-20), 3.42 (s, 3H, -OMe); UV A:$‘” nm (log E):240 (3.5483;MS (70 eV, direct inlet) m/z (rel. int.): 334 [M]’ (25), 319, (8), 235 (5), 205 (5) 205 (lo), 196 (2), 122 (25), 81 (50), 59 (52), 41 (100). Li AlH, reduction o/4. LiAlH, reduction of 4 (50 mg) in THF soln gave the 3,15-diol (5) (45 mg), mp 92-94”, [a],+18 (MeOH;c 1.02). IR ~~~~‘~cm-‘: 3615 (OH), 3020, 1650, 900 (double bond); ‘H NMR 80 MHz (CM=&): 6 1.30 (m, 2H, H-2, H-2a), 3.2 (dd, lH, H-3a) 5.35 (s, lH, H-7), 1.32 (2H, H-14), 3.66(t, 2H,H-15)0.9O(d,3H,H-16), 1.67(s,3H,H-17),0.74(~,3H,H-18), 0.98 (s, 34, H-19), 0.85 (s, 3H, H-20); MS (70 eV, direct inlet) m/z (rel. int.): 308 [M]’ (4.6), 293 (1.7), 220 (5), 207 (18), 190 (22), 140 (8), 122 (18), 121 (55). 80 (20), 65 (6), 55 (100). NaBH, reduction ~$4. Treatment of compound 4 (20 mg) with NaBH, in MeOH soln gave 6 (18 mg), mp 96”, IR ~2:‘~ cm-‘: 3606, 10290 (secondary OH), 1745, 1360 (ester), 3030, 1650,900 (double bond); ‘H NMR, 80 MHz (CDCl,) 6: 1.31 (s, 2H, H-2, H2a), 3.2O(dd, lH, H-3a), 2.1 (br, 2H, H-14),0.91 (d, 3H, H-16), 1.68 (s, 3H, H-17), 3.65 (s, 3H, -OMe), 4.48 (s, lH, OH); MS (70eV, direct inlet) m/z (rel. int.): 338 [M]’ (6), 293 (2), 248 (30), 249 (45), 236 (22), 223 (40), 220 (20), 203 (50), 207 (20), 189 (20), 140(30), 122 (80), 80 (20), 60 (10) and 55 (100). Acetylation $5. Treatment of 5 (2Omg) with Ac,O-pyridine (0.54.2) gave 7 (17 mg) as a syrup: IR Y’ATcm-‘: 1740, 1240 (acetates); MS (70 eV, direct inlet) m/z (rel. int.): 392 [M]’ (15), 332 (50), 272 (60), 265 (28), 127 (68), 189 (100); ‘H NMR, 80 MHz (CDCI,): 6 1.35 (m, 2H, H-2, H-2a), 4.63 (dd, lH, H-3a), 5.33 (s, lH, H-7), 1.35 (2H, H-14), 4.09 (t, 2H, H-15), 0.84 (d, 3H, H-16), 1.67 (s, 3H, H-17), 0.73 (s, 3H, H-18), 0.94 (s, 3H, H-19), 0.86 (s, 3H, H-20), 2.15, 2.14 (s, 6H, 3H each-AcO). 5/3,9j?H,lOa,3,4-seco-labd-7-en-3,15-dioc-acid (2) was the second new compound isolated (13 g). Prep. TLC (silica gel) of 100 mg material gave, after development with hexane-EtOAc (19: 1 x 3), 60 mg of compound 2 mp 8549” (hexane-Me&O) [a];“- 15.824 (CHCl,;c0.450); IR ~2:‘~ cn-‘: 300&2500, 2735 (CO,H), 1650, 920, 800-840 (double bond); MS (70 eV, direct inlet) m/z (rel. int.): 338 [M]’ (15), 323 (25), 222 (15), 237 (80), 180 (37), 122 (lOO),44 (70). Methyl ester (8). Treatment of compound 2 (3OOmg) with CH,N,-Et,0 soln gave 8 (280 mg). IR v’di’cm-‘: 3020, 1650, 1010 (double bond), 1742, 1230 (ester). MS (70 eV, direct inlet)

602

E. M ESCAMILLA and A. ORTEGA

m/z(rel. int.): 366 [M]’ (15), 341(3), 335 (34), 307 (70), 303 (4), 279 @I), 237 (15), 196 (37), 122 (100); ‘HNMR 80 MHz, (CDCI,) s: 2.20(m,4H, H-2, H-6), 5.38 (s, 3H, H-17),0.752 (s, 3H, H-18), 0.92 (d, 3H, H-19), 0.85 (d, H-20) 3.65 (s, 3H, -Me), 3.68 (s, 3H, -OMe), 3.68 (s, 3H, -0Me). The Me,CO extract (17 g) was adsorbed on celite and chromatographed on silica gel G. The column was successively eluted with hexane and EtOAc mixts (O-+7:3). From the fraction eluted with hexane_EtOAc, 9: 1, the triterpenoids lupeol (80 mg) and /I-amirin (2 g) were isolated. Their structures were identified by direct comparison with authentic samples (provided by Dr L. QuiJano, Instituto de Quimica, UNAM). The frs eluted with hexane-EtOAc (7: 3) gave 50 mg 3. Compound 3. Mp 13&132”, [a]k5’-28.1 (MeOH;c0.62) UV ,I$:” nm (logs): 232 (2.365); IR I$::” cn-* : 3500-2500, 1710, 1021 (a&unsaturated acid), 1650, 1640,910,900 (double bonds); MS (70 eV, direct inlet) m/z (rel. int.): 336 [M] + (8), 265 (50), 263 (W), 237 (95), 196 (8), 195 (20), 193 (38), 13 (60), 182 (20), 180 (60), 122 (20), 81 (100); ‘HNMR 80 MHz (CDCI,) 6: 5.65 (br s, lH, H-14), 5.375(brs, lH, H-7),0.90(d,5=7 Hz, 3H,H-19),0.83(d,J = 7 Hz, H-20), 0.74 (s, 3H, H-18), 1.90 (br s, 3H, H-16), 1.75 (br s, 3H, H-17), 3.42 (m, 5H, H-l, H-12, and H-14).

REFERENCES 1. Retana, A. M., Escamilla, E. M., Calderon, J. and Rodriguez, B. (1984). Phytochemistry 23, 1329. 2. Escamilla, E. M. and Rodriguez, B. (1980) Phytochemistry 19, 463. 3. Escamilla, E. M. and Rodriguez, B. (1980) An. Quim. Ser. C 76. 189.

4. Ortega, A., Blount, J. F. and Manchand, P. S. (1982) .I. Chem. Sot., Perkin Tram I 2505. 5. Salmon, M., Diaz, E. and Ortega, A. (1973) J. Org. Chem. 38, 1759. 6. Salmon, M., Ortega, A. and Diaz, E. (1975) Rev. Latinoam. Quim. 8, 172. 7. Ortega, A., Martinez, R. and Garcia, C. L. (1980) Reu. Latinoam. Quim. 11, 45. 8. Salmon, M., Ortega, A., Garcia de la Mora, G., Angeles, E. (1983) Phytochemistry 21, 1512. 9. Bohlmann, F. and Zdero, Ch. (1976) Chem. Ber. 109, 1436. 10. Shorichin, M., Yamasaki, K., Yahara, R. M. S. and Tanaka, 0. (1980) Phytochemistry 19, 326. 11. Apsimon et al. (1962) Can. J. Chem. 40, 630. 12. Bohlmann, F., Jakupovic, J., King, R. M. and Robinson, H. (1982) Phytochemistry, 21, 2021. 13. Gontilez, A. G., Arteaga, J. M., Breton, J. L. and Fraga, B. M. (1977) Phytochemistry 16, 107. 14. Bohlmann, F., Borthakur, N., King, R. M. and Robinson, H. (1981) Phytochemistry 20, 2433. 15. Bohlmann, F., Zdero, C., King. R. M. and Robinson, H. (1981) Phytochemistry 20, 1657. 16. Tonn, C. E., Rossomando, P. C. and Giordano. 0. S. (1982) Phytochemistry 21, 2599. 17. Angeles, E., Foltan, K., Grieco, P. A., Huifman, J. C., Miranda, R. and Salmon, M. (1982) Phytochemistry 21,1804. 18. Sholican, N., Kasud, R. Miyama, Yakara, S. and Osamutanaka, (1980) Phytochemistry 19, 326. 19. Bohlmann, F., Zdero, C. and Grenz, M. (1977) Chem. Ber. 110. 1034