Diterpenes from Calceolaria latifolia

Short Reports REFERENCES

1. Hayashi, T., Kawasaki, M., Kishi, M., Arisawa, M., Shimuzu, M., Morita, N., Tezuka, Y., Kikuchi, T. and Berganza, L. H. (1987) Tennen Yuki Kagobutsu Toronkai Koem Yoshishu 29, 544. 2. Hayashi, T., Kishi, M., Kawasaki, M., Arisawa, M., Shimuzu, M., Suzuki, S., Yoshizaki, M., Morita, N. and Tezuka, Y.

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(1987) Tetrahedron Letters 28, 3693. 3. Li, J., Li, Y., Nie, R. and Zhou, J. (1981) Yun Nan Zhi Wu Yan Jui, 3, 475. 4. Ramesh, P., Nair, A. G. R. and Subramanian, S. (1979) Curr. Sci. 48, 67. 5. Bohlmann, F., Zdero, C., King, R. M. and Robinson, H. (1984) Liebigs Ann. Chem. 250.

Phytochemistry,Vol. 29, No. 9, pp. 3037-3039,1990.

0031-9422/!&l $3.00+0.00 0 1990Pergamon Press plc

Printed in Great Britain.

DITERPENES FROM CALCEOLARIA LA TIFOLIA * JUAN A. GARBARINO~ and AURORA MOLINARI$ Departamento de Quimica, Facultad de Ciencia, Universidad TBcnica Federico Santa Maria, Casilla 110-V, Valparaiso, Chile; $Instituto de Quimica, Facultad de Ciencias Bgsicas y MatemLtica, Universidad Cat6lica de Valparaiso, Casilla 4059, Valparaiso, Chile (Receioed 31 January 1990)

Key Word Index-Calceolaria

Intifolia; Scrophulariaceae; pimarane diterpenes; stemarane diterpenes.

Abstract-Four new diterpene compounds have been isolated from the aerial parts of Calceolaria latijolia, and their structures elucidated by spectroscopic methods. Two of them present a pimaradiene type skeleton and the other two, a stemarane skeleton.

INTRODUaION

Following research on diterpene compounds from Calspecies [l, 23, we have examined C. latifolia Benth., a species which grows on hills of north and central Chile [3]. Four new diterpenes were isolated and their structures elucidated. They were identified as entisopimara-8(9),15-diene-18-01 (l), ent-isopimara-8(9),15diene-19-01 (2), ent-stemara-13(14)-en-19-01 (3), and entstemara-13(14)-en-18-01 (4). ceolaria

2 2a

R=H R=Ac

RESULTS ANDDISCUSSION

The petrol extract of the fresh aerial parts of C. latifolia Benth., was subjected to column chromatography on silica gel, using increasing proportions of ethyl acetate in petrol to afford compounds l-4. Compound 1, C,,H,,O (CM]+ at m/z 288), showed bands for hydroxy and olefinic groups in the IR spectrum, and its ‘HNMR and *Part 6 in the series ‘Diterpenoids from Calceolaria species’. For Part 5 see ref. [2]. TAuthor to whom correspondence should be addressed.

3 3a

R.H R.Ac

4

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Short Reports

‘%NMR spectra exhibited characteristic signals of a diterpene with pimaradiene skeleton. The ‘H NMR spectrum showed three methyl groups as singlets at 60.80,0.93, and 1.00, and also exhibited an AB pattern corresponding to the methylene protons of a hydroxy methyl groups equatorially oriented [4] at 6 3.15 ( 1H, d, H- 18) and 6 3.45 ( 1H, d, H- 18’). These signals were in agreement with the value 672.09 for C-18 [S]. The r3C NMR spectrum showed two signals at 6 124.10 and 136.50, attributed to a tetrasubstituted double bond at A*(‘) [6]. The ‘H NMR spectrum also revealed the presence of an ABX pattern, characteristic of a vinyl group linked to a quaternary carbon. This signal appeared at 65.83 (lH, dd, H-15), 4.92 (lH, dd, H-16t) and 4.89 (lH, dd, H-16~). The values at 6149.25 and 109.35 were assigned respectively, to C-15 and C-16. The multiplicities observed for H-15, besides the values attributed to C-15, C-16 and C-17, are characteristic for a equatorially oriented vinyl group at C-13, in A’(‘), 15-pimaradiene [6]. The remaining 13CNMR signals (Table l), were consistent with those that indicated 1 to be entisopimara-8(9),15-diene-18-01. Compound 2, was isolated as its acetyl derivative 2a. This last compound showed a molecular formula of C22H3402 by mass spectrometry ([Ml’ at m/z 330). The spectral data were similar to 1, confirming the same A* (‘),15-pimaradiene skeleton. Furthermore, the ‘H NMR spectrum showed the AB pattern corresponding to an acetoxymethyl group at 63.91 (lH, d, H-19) and 64.24 (lH, d, H-19’), and the value of 667.19 observed in the r3CNMR spectrum, is attributed to this group. All these data indicated the axial orientation for the acetoxymethyl group [4, 51. This stereochemistry caused a deshielding on C-5 (852.52) of ca 7 ppm related to the observed value for this carbon in compounds having an equatorial hydroxymethyl or acetoxymethyl group. This is in accordance with the signals observed for similar structures [7,8]. The remaining 13C NMR signals of 2a. (Table 1) were in good agreement with the ent-isopimara8(9),15-diene-19-acetoxy structure, so that compound 2 is a C-4 epimer of 1. Compound 3, was purified and characterized as its acetylated derivative 3a. The mass spectrum showed a molecular formula of Cz2HS402 ([Ml’ at m/z 330). The ‘H and r3C NMR spectra showed characteristic signals of a tetracyclic diterpene with a stemarane skeleton [2,9,1(-U. The ‘HNMR of 3a showed two methyl groups as singlets at 60.93 and 1.02, in addition to a signal at 6 1.63 (d, H-17), attributed to an olefinic methyl group. This spectrum also showed an unusual upfield signal due to a methinic olefinic proton at 64.77 (brs, H-14). The r3C NMR spectrum exhibited two signals at 6 138.41 and 123.16 corresponding, respectively, to C-13 and C-14 of a trisubstituted double bond. No signal of a vinyl group was observed. The ‘HNMR spectrum also showed the signals corresponding to an AB pattern of an axially oriented acetoxymethyl group at 6 3.84 (1H, d, H-19) and 4.28 (lH, d, H-19’). This compound was deacetylated by treatment with K&O,-MeOH, obtaining the natural product 3. The spectral data of compound 3 were very similar to 3a, but the acetate methyl signal was missing, and the methylene-19 signal was shifted upfield at 63.38 (lH, d, H-19) and 63.78 (lH, d, H-19’) confirming the axial orientation of this group [4].

Table

1. r3C NMR spectral data of compounds 4 (CDCl,, TMS)

C

1

1 2 3

36.30 18.72 35.12 31.68”

36.59 18.59 36.09 37.34”

31.78 18.28 36.31 38.47”

31.69 18.37 36.10 36.79’

31.45 18.15 35.55 36.24”

45.20 20.75 35.22 124.10 136.35 37.34”

52.50 20.97 32.87 124.30 136.11 36.98”

49.37 21.53 35.34 42.25 51.42 38.25”

49.44 21.66 36.32 42.20 51.38 38.47=

41.74 21.55 35.55 42.20 51.39 38.30”

18.41 34.00 34.79 42.24 149.25 109.35 23.25 72.09 17.53 19.88

19.12 33.86 34.67 42.15 149.24 109.49 23.08 27.11 67.19 19.87 171.35 20.66

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Meg00 &COO

2a

3

1,2a, 3,3a and

24.55 41.71 138.64 123.42 34.16 28.06 21.85 26.70 65.02 18.09

3a

24.61 41.76 138.41 123.16 34.18 28.06 21.97 27.29 66.8 1 18.01 171.09 20.97

4

24.43 41.74 138.41 123.37 34.08 27.63 22.04 72.16 17.65 17.38

“These values may be interchanged within the same column.

On the basis of these data and by comparison with similar structures, compound 3 was shown to be entstemara-13(14)-en-19-01 and 3a to be ent-stemara-13 (14)en-19-acetoxy. This compound was also identified in another species of this genus, C. l&da [2]. Compound 4, presents a molecular formula C,,H,,O by mass spectrometry (CM]’ at m/z 288), and the spectral data were very similar to compound 3, confirming the same 13(14)-stemarane skeleton for 4. The differences were observed in the values of the signals corresponding to the AB pattern of the hydroxymethyl group, which was shifted upfield at 63.12 (lH, d, H-18) and 63.38 (lH, d, H18’), and the 13CNMR showed at 672.16 the signal attributed to this group. All of these signals were indicative of an equatorially oriented hydroxymethyl group. Therefore, compound 4 is shown to be ent-stemara13(14)-en-18-01, and is a C-4 epimer of 3. Finally, the assignment of configurations for all proposed structures also relied heavily on biogenetical considerations observed in the Calceolaria genus [l. 21.

EXPERIMENTAL

Mps: uncorr. ‘H NMR: 100 and 250 MHz in CDCI,, with TMS as int. standard. Assignments of “C NMR chemical shifts were made with the aid of APT; IR: film on NaCl and CHCl, soln. MS: direct inlet, 70 eV. Calceolaria kztijiilia Benth. was collected between La Serena and Los Vilos, IV Region, Chile, in September 1987. A voucher specimen is deposited at Universidad Tecnica Federico Santa Maria.

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Short Reports The fresh aerial parts of C. latifolia (1.1 kg) were extracted at room temp. with petrol for 72 hr, affording 21 g of a syrup. This crude material was chromatographed on a silica gel column (600 g, for TLC) and eluted with mixts of petrol and EtOAc of increasing polarity. Fractions of 125 ml were taken and combined based upon TLC and ‘H NMR (60 MHz) monitoring. Fractions 13-17 contained a mixture of 1 and 4, and fractions 19-26 contained a mixture of 2 and 3. The mixture containing 1 and 4 was subjected to silica gel CC (80 g for TLC) impregnated with AgNO, (10%) using petrol-EtOAc (8: 1), affording 1 (60mg) and 2 (35mg). The mixture containing 2 and 3 was acetylated by treatment with AczO-pyridine overnight at room temp.; the acetylated mixture was subjected to silica gel CC (80 g for TLC) impregnated with AgNO, (10%) using petrol-EtOAc (30: l), and afforded 2~ (80 mg) and 3a (90 mg). ent-lsopimara-8(9),15-diene-18-01 (1). Viscous oil, [a];’ +40.5” (CHCI,; c 2.01). IR v:!!” cm- I: 3360,3090,2980-2840, 1830,1670,1640,1460-1450,1420,1385,1155,1050,1035,915. ‘HNMR (100 MHz): 65.83 (lH, dd, J=ll.O, 17.8 Hz, H-15), 4.92 (lH, dd, J= 1.0, 17.8 Hz, H-16t), 4.89 (lH, dd, J= 1.0, 11.0 Hz, H-16c), 3.45 (lH, d, J= 11.0 Hz, H-18), 3.15 (lH, d, J =ll.O, H-18’), 1.00, 0.93, 0.80 (3H, each s, H-17, 19, 20). 13CNMR: see Table 1; MS m/z (rel. int.): 288 [C,,H,,O]+ (98), 273 [M -Me]+, (58), 270 [M-H,O]+ (54), 257 [M -CH,OH]+ (55), 255 [M-Me-H,O)+ (48). ent-lsopimara-8(9),15-diene-19-ncetoxy (Za). Amorphous crystals, [a];’ +75.0” (CHCI,; c 1.0). IR vkyp3 cm-‘: 3090, 2980-2840, 1730,1640, 1470-1450, 1400, 1380, 1240, 1035,990, 910. ‘HNMR(250 MHz): 65.78 (lH,dd,J= 11.0, 17.8 Hz, H-15), 4.88 (lH, dd, J=l.O, 17.8 Hz), 4.85 (lH, dd, J=l.O, 11.0 Hz, H16c), 4.24 (lH, d, J= 11.0 Hz, H-19), 3.91 (lH, d, J= 11.0 Hz, H19’),2.02 (3H, s, OAc), 0.98, 0.96,0.92 (3H each, s, H-17, 18, 20). MS m/z (rel. int.): 330 [CzzH,,O,]+ (42), 315 [M-Me]+ (67), 288 [CzOHBzO]+ (14), 270 [M-HOAc]+ (14), 255 [M-Me -HOAc]+ (100). 13CNMR: see Table 1. ent-Stemara-13(14)-en-19-acetoxy (3a). Crystals. Mp 96-97”, [a];’ - 17.02” (CHCl,; ~2.0). IR v:!:‘~ cm-‘: 2980-2840, 1720, 1710, 1460-1450, 1385, 1365, 1240, 1030, 980. ‘HNMR (250MHz):S4.77(1H,6rs,H-14),4.28(1H,d,5=11.0Hz,H-19), 3.84 (lH, d, J= 11.0 Hz, H-19’), 2.03 (3H, s, OAc), 1.63 (3H, d, J = 1.0 Hz, H-17), 1.02, 0.93 (3H each, s, H-18, 20). MS m/z (rel. int.): 330 [C,,Ha,O,]+ (52), 315 [M-Me]+ (54), 287 [M -MeCO]‘(8),270[M-HOAc]+ (8),255 [M-Me-HOAc]+ (82). 13CNMR: see Table 1.

ent-Stemara-13(14)-en-19-o/ (3). Viscous oil. ‘H NMR (250 MHz): 64.77 (lH, brs, H-t4), 3.78 (lH, d, J= 11.0 Hz, H-19), 3.38 (lH, d, .!=ll.OHz, H-19’), 1.62 (lH, d, J=l.OHz, H-17), 0.97,0.93 (3 each, s, H-18,20). MS m/z (rel. int.): 288 [C,,H,,O] + (65), 273 [M-Me]+ (82), 270 [M-H,O]+ (23), 257 [M -CH,OH] (lOO), 255 [M-Me-H,O]’ (34). “CNMR: see Table 1. ent-Stemara-13(14)-en-18-01 (4). Viscous oil. [a];’ + 17.4” (CHCl,; c 1.4). IRvEF” cm-‘: 3350, 2980-2840, 2800, 1710, 1440-1460,1380,1250. ‘HNMR (100 MHz): 64.80 (lH, brs, H14), 3.38(1H,d,J=ll.O,H-18), 3.12(1H,d,5=11.0Hz,H-18’), 1.62 (3H, d, J = 1.0 Hz, H-17), 1.04, 0.78 (3H each, s, H-19, 20). MS m/z (rel. int.): 288 [Cz0HJ20]+ (15), 273 [M-Me]+ (84), 257 [M-CH,OH]+ (67), 255 [M-CHzOH-Me]+ (75). 13CNMR: see Table 1. Acknowledgements-We are grateful to Professor E. G. Gros (Universidad de Buenos Aires, Argentina) and Professor P. Manitto (UniversitB degli Studi di Milano, Italia) for recording ‘H and 13CNMR spectra, and to Professor 0. Zoellner (Universidad Cat6lica de Valparaiso, Chile) for identification of the plant material. This research was supported by grant # 891303 from DGDCYT, Universidad T6cnica Federico Santa Maria. REFERENCES 1. Piovano, M., Chamy, M. C., Garbarino, J. A. and Gambaro, V. (1989) Phytochemistry 28, 2844. 2. Chamy, M. C., Piovano, M., Garbarino, J. A., Miranda, C. and Gambaro, V. (1990) Phytochemistry 29. 3. Marticorena, C. and Quezada, M. (1988) Gayana (Botdnica) 42, 1. 4. Gaudemer, A., Polonsky, J. and Wenkert, E. (1964) Bull. hoc. Chim. Fr. 407. 5. Combie, R. C. (1975) J. Org. Chem. 40, 3789. 6. Wenkert, E. and Buckwalter, B. L. (1972) J. Am. C:.~X Sot. 94, 4367. 7. Ceccherelli, P., Curini, M. and Marcatullio, M. C. (1985) J. Chem. Sot. Perkin Trans I 2173.

8. Wehrli, F. W. and Nishida, T. (1979) Prog. Chem. Org. Nat. Prod. 36,60.

9. Bohlmann, F., Zdero, C., King, R. and Robinson, H. (1984) Lieb. Ann. Chem. 250.

10. Taran, M. and Delmond, D. (1986) Tetrahedron 42,4795.