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Journal of the Chilean Chemical Society

On-line version ISSN 0717-9707

J. Chil. Chem. Soc. vol.51 no.1 Concepción Mar. 2006

http://dx.doi.org/10.4067/S0717-97072006000100005 

J. Chil. Chem. Soc., 51, Nº 1 (2006)

DITERPENOIDS FROM CALCEOLARIA PARALIA.*

MARÍA CRISTINA CHAMY, MARISA PIOVANO, JUAN A. GARBARINO¡ AND LUIS ESPINOZA.

Departamento de Química, Universidad Técnica Federico Santa María, Casilla 110-V, Valparaíso, Chile.

* Part 24 in the series "Diterpenoids of Calceolaria species". For part 23 see ref.[1]

E-mail: juan.garbarino@usm.cl


ABSTRACT

From the aerial parts of Calceolaria paralia, a new diterpene ,19-butyloxy-9a-hydroxy-13-epi-ent-pimara-15-ene (1), together with 13-epi-ent-pimara-9(11)15-diene-19-oic acid (2), 9a,19-dihydroxy-13-epi-ent-pimara-15-ene(3) , 19-acetoxy-13-epi-ent-pimara-9(11)15-diene (4), bis malonate(13-epi-ent-pimara-9(11),15-diene-19-yl) (5) and ent-stemar-13(14)en-19-oic acid (6) . Their structures were elucidated by spectroscopic means.


 

INTRODUCTION

The genus Calceolaria (Scrophulariaceae), native of South America, includes numerous plant species used in local folk medicine, particularly in Chile2. Only the aerial parts of the plant are used for their digestive, diuretic and antibacterial properties2,3.

We began a phytochemical investigations on plants in this genus that has resulted so far in the isolation of diterpenes of the labdane 4,5, pimarane 6,7, abietane8 and stemarane 6,9 skeletons. In this paper we report the isolation of 19-butyloxy-9a-hydroxy-13-epi-ent-pimara-15-ene (1) as a new diterpene from the aerial parts of C. paralia collected in Termas de Catillo, VII Región, Chile, together with the known diterpenes, 13-epi-ent-pimara-9(11)15-diene-19-oic acid6 (2), 9a,19-dihydroxy-13-epi-ent-pimara-15-ene 10(3), 19-acetoxy-13-epi-ent-pimara-9(11),15-diene 10(4), bis malonate(13epi-ent-pimara-9(11),15-diene-19-yl) 6(5) and ent-stemar-13(14)-en-19-oic acid10(6). The previously known diterpenoids isolated from other species of Calceolaria6,10, were identified by their physical and spectroscopic data and by comparison TLC with authentic samples.

EXPERIMENTAL

Materials and Methods

melting points were determined on a Kofler hot-stage apparatus and are uncorrected. IR spectra were recorded on a Nicolet Impact 420 spectrophotometer. 1H and 13C NMR were recorded in CDCl3 solutions with a Bruker Avance 400, with TMS as int. stand. and CDCl3 as solvent. Low mass spectra were taken at 70 eV (probe) in a Shimadzu QP-2000. Silica Gel (200-300 mesh) was used for CC and silica HF-254 for TLC. Spots were detected on TLC by heating after spraying with 25% H2SO4 in H2O.

Plant Material

The aerial parts of Calceolaria paralia Cav were collected in Termas de Catillo, VII Region, Chile , in November 2000, and authenticated by Professor Melica Muñoz, Museo de Historia Natural, Santiago. A voucher specimen is deposited at the Herbarium of the Natural Product Laboratory of Universidad Técnica Federico Santa María (# 20007).

Extraction and isolation:The aerial parts of C. paralia (1500 g) were extracted at room temp. successively with petrol and CH2Cl2 for 48 h each. The solvents were removed in vacuo to yield 10 g (petrol extract) and 15g (CH2 Cl2) extract of a syrup residue. The extracts were subjected separately to chromatography over silica gel column (400 g) and eluted with mixtures of petrol and EtOAc of increasing polarity. Frs (125 ml) were combined based on TLC monitoring, and purified by repeated CC on silica gel or silica gel impregnated with AgNO3 (10%). Some fractions prior to their purification were acetylated with acetic anhydride in pyridine or methylated with ethereal diazomethane.

19- butyloxy- 9a-hydroxy-ent-pimara-15-ene (1): Mp: 96-97 ºC, Rf = 0.71 (petrol-EtOAc 8:2 ).IR nKBr max cm-1: 3565, 2980, 1720, 1630, 1450, 1200, 1050.

1H NMR ( 400MHz, CDCl3) d(ppm): 5,79 (1H, dd, J = 10,5; 17,5Hz , H-15), 4,94 (1H, d, J = 17,5 Hz, H-16t), 4,88 (1H, d, J = 10,5 Hz, H-16c), 4,27 (1H, d, J = 10,8 Hz, H-19), 3,85 (1H, d, J = 10,8 Hz, H-19'), 2,25 (1H, t, J = 7,5 Hz, H-22), 1,00 (3H, s, Me-17), 0,91 (1H, t, J = 7,0 Hz, H-24), 0,91 (3H, s, Me-18), 0,87 (3H, s, Me-20).

13C NMR: 31.56 (C-1), 18.23 (C-2), 36.33(C-3), 36.95 (C-4), 49.11 (C-5), 21.86 (C-6), 30.06 (C-7), 38.46 (C-8), 76.10 (C-9), 42.24 (C-10), 23.42 (C-11), 30.28 (C-12), 34.94 (C-13), 40.55 (C-14), 149.25 (C-15), 109.23 (C-16), 33.49 (C-17), 27.70 (C-18), 66.77 (C-19), 15.54 (C-20), 173.84 (C-21), 36.3 (C-22), 18.2(C-23), 13.7(C-24).

MS m/z(rel. int.): 376[ M+,C24H40O3 ](5), 288 [M-HOCOCH2CH2CH3] (30), 275 (27), 270 (26), 257 (73), 254 (23), 187 (26), 175 (17), 173 (18), 164 (27), 161 (20), 151 (27), 149 (60), 145 (15), 138 (63), 135 (56), 133 (31), 131 (17), 123 (65), 120 (56), 119 (29), 109 (59), 107 (56), 105 (46), 97 (27), 93 (99), 88 (5), 79 (99), 67 (99), 57 (50), 55 (58), 41 (100).

The structures of compounds 2 - 6 were established by comparison of their spectral data and by TLC with authentic samples previously isolated by us from others members of this genus6,10.

RESULTS AND DISCUSSION

Compound 1, obtained as yellow crystals, displayed a molecular ion at m/z 376 corresponding to the formula C24 H40O3. The Rf value was 0.71 by TLC (petrol ether-EtOAc, 8 : 2). The IR spectrum of (1) displayed absorptions due to an ester carbonyl (1720 cm-1), a hydroxyl (3565 cm-1) and an olefinic group (1630 and 900 cm-1). The NMR spectral data for compound 1 (See experimental) assigned by a combination of 1D and 2DNMR, spectroscopy techniques (DEPT, HSQC, HMBC, TOCSY and NOESY) revealed the presence of three tertiaryl methyl groups, singlets at dH 1.00, 0.91 and 0.87, a vinyl group at d 5.79 (1H, dd, J = 10.8, 17.4Hz, H-15), 4.94 (1H, d, J=17.4 Hz, H-16t) and 4.88 (1H, d, J = 10.8 Hz, H-16c) a primary ester group (d 4.27 d and 3.85 ppm, d). The remaining oxygen atom in the molecule is part of a tertiary hydroxyl group, because the 1HNMR spectrum of 1, lacked the signals attributed to geminal proton of hydroxyl group. The above evidence suggested that 1 possessed an ent-pimarane type structure11,12, and the other carbons (C4) must be part of the acyl moiety. That the ester side as a butyrate was deduced from the MS spectra m/z at 288 corresponding to M+ - HOCOCH2CH2CH3 and by the signals in the 1HNMR at dH 2.25 (2H, t, H-22) and 0.91 (3H, t, C-24). The 13CNMR confirmed this residue by signals at d 173.8 (C-21), 36.3 (C-22),18.2 (C-23) and 13.7 (C-24), it also established that the ester group was axially orientated (C-19 at d 66.8 ppm). The location of the terciary hydroxyl group at C-9 was discerned from the 13CNMR spectrum, which clearly showed a g effect on C-12 and the expected deshielding effect on C-10. The a orientation of this group and of H-8 were deduced from the pronounced shielding effect on C-7 and the deshielding effect on C-8. The configuration of C-13 was established by the chemical shift of C-17 (near d 33.5 ppm) which was only compatible with an axial methyl group. On the basis of these data, compound 1 is shown to be 19-butyloxy-9a-hydroxy-ent-pimar-15-ene.

ACKNOWLEDGMENTS

We are grateful to Professor Melica Muñoz (Museo Historia Natural, Santiago, Chile) for identification of the plant material, to Karen Catalan, for technical assistance. This research was supported by a Grant (# 1020070) from FONDECYT.

REFERENCES

1. Chamy, M.C., Piovano, M., Garbarino, J.A. (2005) Nat. Prod. Research accepted for publication.

2. Navas L.E.; "Flora de la Cuenca de Santiago de Chile", Tomo III. Ediciones U. de Chile. Ed. A. Bello (1976), p 105-106 and 111-112.         [ Links ]

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4. Garbarino, J.A., Molinari, A.; (1992), J. Nat. Prod. 55, 744-747.         [ Links ]

5. Garbarino, J.A., Molinari, A.; . (1993), J. Nat. Prod. 56, 624-626.         [ Links ]

6. Chamy, M.C., Piovano, M., Garbarino, J.A. and Gambaro, V.; (1991), Phytochemistry, 30, 3365-3368.         [ Links ]

7. Chamy, M.C., Piovano, M., Garbarino, J.A. and Hernandez, C.; (1998), Bol Soc. Chil. Quim., 43, 241-245.         [ Links ]

8. Chamy, M.C., Piovano, M., Garbarino, J.A.; (1992), Phytochemistry, 31, 4233-4235         [ Links ]

9. Chamy, M.C., Piovano, M., Garbarino, J.A. and Vargas, C.; (1995), Phytochemistry, 40, 1751-1754.         [ Links ]

10. Chamy, M.C., Piovano, M., Garbarino, J.A., C. Miranda and Gambaro, V.; (1990), Phytochemistry, 29, 2943-2946.         [ Links ]

11. Piovano, M., Chamy, M.C., Garbarino, J.A. and Gambaro, V.; (1989), Phytochemistry, 28, 2844-2845.         [ Links ]

12. Chamy, M.C., Piovano, M., Garbarino, J.A., Gambaro, V. and Miranda, C.; (1989), Phytochemistry, 28, 571-574.         [ Links ]

13. Bretmaier, E. and Voelter, W. (1978) 13CNMR Spectroscopy, 2nd Edn.Verlag Chemie, Werheim.         [ Links ]

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