DITERPENOIDS FROM CALCEOLARIA GLABRATA.*

MARÍA CRISTINA CHAMY, MARISA PIOVANO, JUAN A. GARBARINO° and JAMILET MENDOZA.

Departamento de Química, Universidad Técnica Federico Santa María, Casilla 110-V,
Valparaíso, Chile. e.mail: jgarbari@qui.utfsm.cl
(Received: December 20, 2000 - Accepted: April 12, 2001)
° To whom correspondence should be addressed.

ABSTRACT

From the aerial parts of Calceolaria glabrata, a new diterpene ,13-methylene-7-acetoxy-stemarane (1), together with 13-isovaleroyl-7-acetoxy-thyrsiflorane (2) and 13-isovaleroyl-7-malonyloxy-thyrsiflorane (3) were isolated. Their structures were elucidated by spectroscopic means.

KEY WORDS: Calceolaria, Scrophulariaceae, diterpenes, stemaranes, thyrsifloranes

RESUMEN

De las partes aéreas de Calceolaria glabrata, se aisló un nuevo diterpeno, 13-metilen-7-acetoxi-estemarano (1), junto con los diterpenos 13-isovaleroil-7-acetoxi-tirsiflorano (2) y 13-isovaleroil-7-malonil-tirsiflorano (3). Sus estructuras fueron determinadas por métodos espectroscópicos

PALABRAS CLAVES: Calceolaria, Scrophulariaceae, diterpenos, estemaranos, tirsifloranos.

INTRODUCTION

The genus Calceolaria (Scrophulariaceae) comprises 500 species with a distribution in Central and South America and New Zealand. In Chile, there are about 85 species of this genus [2]. Calceolaria glabrata is a shrub, common in the southern part of Chile.

Over a decade ago, we began a phytochemical investigation on plants belonging to this genus that has resulted so far in the isolation of diterpenes of the abietane [3], labdane [4], pimarane [5], and stemarane [6] skeletons. In this paper we report the isolation of 13-methylene-7-acetoxy-stemarane (1), a new diterpene, from the aerial parts of C. glabrata collected in Chillán,VIII Región, Chile, together with the known diterpenes,13-isovaleroyl-7-acetoxy-thyrsiflorane (2) and 13-isovaleroyl-7-malonyloxy-thyrsiflorane (3). The structures of these natural products were established by spectroscopic means.

EXPERIMENTAL

General Methods

Melting points were determined on a Stuart Scientific SMP3 apparatus. Optical rotations were measured in CHCl₃ with a Schmidt-Haensch polarimeter. IR spectra were recorded in KBr pellets (FT-IR Nicolet Impact 420). NMR: Bruker AC-200 and AM-500, with TMS as int. stand. and CDCl₃ as solvent. MS: 70 eV, Shimadzu QP-2000. Silica gel (200-300 mesh) was used for CC and silica gel HF-254 for TLC, eluting with mixts of petrol (60-80)- ethyl acetate of different polarities. Spots were detected on TLC by heating after spraying with 25% H₂SO₄ in H₂O.

Extraction and Isolation of the Products

The aerial parts of Calceolaria glabrata Phil. var. glabrata were collected in Chillan, VIII Region, Chile , in January 1998, and authenticated by professor Melica Muñoz, Museo de Historia Natural, Santiago. A voucher specimen is deposited in the Herbarium at the Natural Product Laboratory of Universidad Técnica Federico Santa María (# 98007). The air-dried aerial parts of C. glabrata (1500 g) were extracted at room temp. successively with petrol and CH₂Cl₂ for 48 h each. The solvents were removed in vacuo to yield 30 g (petrol extract) and 50g (CH₂Cl₂ extract) of syrupy residues. The extracts were subjected separately to chromatography over silica gel columns (400 g) and eluted with mixts of petrol (60- 80C) and EtOAc of increasing polarity. Frs (125 ml) were combined based on TLC and ¹H-NMR (60 MHz) monitoring, and the combined frs. purified by repeated CC on silica gel or silica gel impregnated with AgNO₃ (10%).

13-Methylene-7-acetoxy-stemarane (1) : Crystals mp 89-91C, [a]_D²⁵ = + 63 (c 0.35,CHCl₃). IR(KBr) n_max (cm^-1) : 2920 - 2880 , 1720, 1630, 1460, 1370, 1230, 1120, 1100, 1020, 980, 890. ¹H-NMR d(ppm): 4.73 (1H, dt, J= 5.4, 10.8 Hz), 4.46 (1H, brd, J= 2.0 Hz, H-17) ,4.39 (1H, brd, J= 2.0 Hz, H-17'), 2.01 (3H, s, OAc) 1.00 (3H, s, Me-20) 0.82 and 0.79 (3H each, s, Me-18 and Me-19). ¹³C-NMR d(ppm) : 170.95 (s , COCH₃), 152,35 (s, C-13), 104.08 (d, C-17), 76.80 (d, C-7), 53.03 (s, C-9), 46.26 (d, C-5), 44.11 (d, C-12), 43.80 (d, C-8), 41.90 (t, C-3), 39.61 (t, C-11), 38.2 (s, C-10), 33.49 (q, C-19), 33.14 (s, C-4), 33.01 (t, C-15), 32.07 (t, C-1), 30.70 (t, C-14), 27.98 (t, C-6), 24.44 (t, C-16), 21.92 (q, C-18), 21.31 (q, COCH₃), 18.62 (t, C-2), 17.40 (q, C-20). MS [70 eV] (%) : 330 [M⁺] (C₂₂H₃₄O_2, 0.6), 287 [M⁺ - 43] (0.3), 270 [M⁺ - HOCOCH₃], (24.3), 255 [270 - Me] (40.3), 185 (11.4), 159 (14.1), 157 (10.6), 149 (9.2), 148 (24.8), 147 (18.8), 145 (14.7), 143 (12.1), 133 (11.4), 131 (21.9), 129 (10.8), 121 (11.3), 119 (19.3), 117 (21.3), 109 (21.0), 107 (24.1), 105 (41.9), 104 (18.4), 95 (31.4), 91 (54.3), 83 (14.5), 79 (52.5), 77 (30.7), 69 (54.3), 67 (37.6), 55 (51.5), 43 (75.0), 41 (53.5), 32 (49.6), 27 (100).

Compounds ( 2) and (3) were both identified by comparison of their spectral data and by TLC with authentic samples previously isolated by us from another member of this genus (C. dentata) [6,14].

Full details of isolation, identification of the compounds and copies of the original spectra are available on request to the senior author⁽°⁾

RESULTS AND DISCUSSION

Compound 1, obtained as white crystals, displayed a molecular ion at m/z 330 corresponding to the formula C₂₂ H₃₄O_2. The R_f value was 0.70 by TLC ( petrol ether-EtOAc, 8 : 2 ). The IR spectrum of (1) displayed absorptions due to an ester carbonyl ( 1720 cm^-1 ) and an exomethylene group (1630 and 890 cm^-1). The ¹H-NMR spectrum of compound (1) indicated the existence of an acetoxy group at d 2.01 (3H, s), attached to a carbocyclic secondary carbon at d 4.73 (1H, dt, J= 5.4, 10.8 Hz,), two olefin proton signals at d 4.46 and 4.39 ppm were assigned to an exomethylene group (1H each, brd,) and three tertiary methyl groups at d 0.79, 0. 82 and 1.00 ppm. The orientation of the acetoxy group must be axial due to the chemical shift of H-7 (d_H 4.73 ppm and d_C 76.8 ppm). In the case of an axial H-7, the value of d_C should be near 68 ppm [7].

The ¹³C-NMR and DEPT spectra showed the presence of three methyl , nine methylene, four methine and four quaternary carbon atoms, (See Experimental) besides the signal of the acetate moiety. Therefore, a tetracyclic structure incorporating the bicyclo [3,2,1] octane moiety constituting the C/D ring system relates compound (1) with stemarane skeletons [8,9] having an acetate group at C-7. Comparison with the spectral data for similar stemarane diterpenoids [9-13], showed unambiguosly that (1) had to be 13-methylene -7-acetoxy-stemarane.

As a confirmation of the proposed structure, de-acetylation of (1) afforded (1a), whose spectral and physical data were in full agreement with those of 13-methylene-7-hydroxy-stemarane isolated from Calceolaria dentata [9].

ACKNOWLEDGMENTS

The authors are grateful to Dr. Melchor G. Hernández, (CSIC, La Laguna, España), for recording the ¹H and ¹³C-NMR spectra (200 and 500 MHz ¹H, 50 and 125 MHz ¹³C), and professor Melica Muñoz (Museo de Historia Natural, Santiago, Chile) for identification of the plant material. This research was supported by a Grant (# 19720) from FONDECYT.

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* Part 20 in the series "Diterpenoids of Calceolaria species". For part 19 see ref.[1]

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14. Note: The structure shown in Fig.4 of reference [6] must not have a OR₁ group at C-2 and the value for C-5 in compound 4a must be d 46.3 ppm and not 36.4 ppm.