J. Chil. Chi. Soc., 51, N°.4 (2006), p.1057-1058

DITERPENOIDS FROM Calceolaria filicaulis SSP LUXURIANS.*

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

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

ABSTRACT

From the aerial parts of Calceolaria filicaulis ssp luxurians, two new diterpenes, 13,18-dihydroxy-thyrsiflorane and 13-isovaleroyl-18-hydroxy-thyrsiflorane were isolated, purified and identified as their acetylderivatives, besides the ß-sitosterol. Their structures were determined by spectroscopic means.

Key words: Calceolaria filicaulis ssp luxurians, Scrophulariaceae, diterpenes, thyrsiflorame derivatives.

INTRODUCTION

Genus Calceolaria (Scrophulariaceae) in Chile is represented by approximately fifty species [2], some of which are used as stomachic drugs, sweetening agents and against bacterial infections [3, 4]. This genus is very rich in diterpenes, specially of the pimarane [5], stemarane [6] and thyrsiflorane [7] type. From a biogenetic point of view, the enzymatic system for the formation of the diterpenes, present in this genus, is different because they conduce to an abnormal cyclization of the geranyl-geranyl-pirophosphate to a chair-boat, instead of the normal chair-chair in the construction of the bicyclic intermediate. This mechanism leads to an epimerization of C-9 and consequently H-9 is found in the same orientation as the methyl group on C-10 [8], originating the 9-epi-pimaranes and the other skeletons present in this genus, like the stemaranes or the thyrsifloranes [9].

Continuing with our work on the terpenoids occurring in Calceolaria species, we have examined the aerial parts of Calceolaria filicaulis ssp luxurians. From this plant we have isolated the diterpenes 13-isovaleroyl-18-hydroxy-thyrsiflorane (1) and 13,18-dihydroxy-thyrsiflorane (2), together with -sitosterol.

EXPERIMENTAL

Materials and Methods

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. Chemical shifts are in ppm (d scale) and the coupling constants are in Hertz. Low mass spectra were taken at 70 eV (probe) in a Shimadzu QP-2000. For analytical TLC Merck silica gel 60 in 0,25 mm thick layers were used. Cromatographic separations were carried out by conventional column on Merck silica gel 60 (0,040-0,063 mm) using petrol (60-80°C)-ethyl acetate mixtures of increasing polarity. Spots were detected on TLC by heating after spraying with 25% H₂SO₄ in H₂O.

Plant Material

The aerial parts of Calceolaria filicaulis ssp luxurians, were collected in Portillo, V Region, Chile , in January 2002, 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 (# 2008).

Extraction and isolation: The aerial parts of Calceolaria filicaulis ssp luxurians (1500 g) were extracted at room temp. successively with petrol and CH₂Cl₂ for 48 h each. The solvents were removed in vacuo to yield 7.0 g (petrol extract) and 1.0g (CH₂Cl₂ extract) of a syrup residue. The petrol extract was subjected to chromatography over silica gel column (400 g) and eluted with mixtures of petrol and EtOAc of increasing polarity. (10:1 - 0:1), affording 40 fractions (125 ml). These fractions were combined based on TLC monitoring and the combined frs. purified by repeated CC on silica gel and silica gel impregnated with AgNO3 (10%). Fractions 19-21 and 30-35 prior to their purification were acetylated with acetic anhydride in pyridine, due that in the ¹H NMR of these fractions appeared signals corresponding to carbinol protons.

13-isovaleroyl-18-hydroxy-thyrsiflorane (1): Fraction 19-21 contained compound 1 impure. This fraction was treated with pyridine (1 ml) and acetic anhydride (3 ml) at room temperature for 6 hours. The mixture was concentrated under reduced pressure. The residue was purified with silica gel column chromatography [petrol-EtOAc (10:1)] to afford 1a: colorless oil; Rf = 0.72 (petrol-EtOAc 10:1 ). IR_?KBR_max cm^-1: 2950, 2880, 1750, 1460, 1380, 1250, 1190, 1040. ¹H NMR (400 MHz, CDCl₃) d: 4.58 (¹H, dd, J = 5.0,10.0 Hz , H-13), 3.81 (¹H, d, J = 10.8 Hz, H-18), 3.59 (¹H, d, J = 10,8 Hz, H-18'), 2.15 (2H, d, J = 6.9 Hz, CH₂-isoval), 2.03 (3H, s, OAc), 1.13 (¹H, m, CH-isoval), 1,00 (3H, s, Me-19), 0,93 (6H, d, J = 6.4 Hz, 2 Me-isoval), 0,90 (3H, s, Me-17), 0,80 (3H, s, Me-20). ¹³C NMR: See Table 1. EM (70eV) m/z (%): 432 [M⁺](0.1), 372 [M⁺-HOCOCH₃] (23.5), 330 [M⁺-HOCOCH₂CH(CH₃)₂] (29.9), 270 (1.0), 257 (76.3), 229 (18.3), 187 (15.3), 175 (16.8), 123 (10.8), 121 (10.7), 119 (10.9), 109 (12.8), 107 (17.5), 105 (13.9), 86 (37.9), 84 (77.8), 69 (14.5), 49 (100). Therefore, the compound present in C. filicaulis ssp. luxurians is 13-isovaleroyl-18hydroxy-thyrsiflorane 1.

13,18-dihydroxy-thyrsiflorane (2): Fraction 30-35 contained compound 2 impure. This fraction was treated with pyridine (1 ml) and acetic anhydride (5 ml) at room temperature for 12 h. After usual work, the residue was purified with silica gel column chromatography and eluted with petrol-EtOAc affording compound 2a.

Colourless oil; Rf = 0.47 (petrol-EtOAc 10:1 )

IR_?KBR_maxcm^-1: 2930-2880, 1740, 1460, 1380, 1245, 1150, 1030, 980. ¹H NMR (400MHz, CDCl₃) : 4.59 (¹H, dd, J = 5.5, 10.5 Hz, H-13), 3.80 (¹H, d, J =10.8 Hz, H-18), 3.60 (¹H, d, J = 10.8 Hz, H-18'), 2.05 y 2.02 (3H each, s, OAc), 1.00 (3H, s, Me-17), 0.91 (3H, s, Me-19), 0.83 (3H, s, Me-20), ¹³C NMR: See Table 1. EM (70eV) m/z (%): 390 [M⁺] (0.2), 330 [M⁺-CH₃COOH] (66.1), 317 (13.9), 270 [M⁺-2 CH₃COOH] (19.1), 258 (34.8), 257 (80.9), 229 (46.8), 215 (12.6), 201 (13.9), 187 (28.5), 175 (25.6) ,161 (26.7), 147 (23.6), 145 (20.0), 135 (15.8), 133 (21.7), 131 (25.4), 123 (26.6), 121 (20.9), 119 (38.4), 109 (30.6), 107 (40.8), 105 (56.7), 95 (47.2), 91 (55.1), 83 (86.8), 81 (80.0), 69 (49.6), 55 (60.7), 43 (100.0).

After usual work up of CH₂Cl₂ extract, there were several mixtures of minor compounds. That were unresolved.

RESULTS AND DISCUSION

Compound 1 was isolated from the petrol extract of C. filicaulis ssp luxurians and was identified as its acetate derivative 1a. In its MS, the molecular ion appeared at m/z 432 (C₂₇H₄₄O₄). The IR spectrum showed signals for carbonyl functions. The NMR spectral data for compound 1a (See Experimental) assigned by a combination of 1D and 2D NMR, spectroscopy techniques (DEPT, HSQC, HMBC) revealed the presence of four quaternary methyl groups, singlets at H 2.03(OCOCH₃),1.00 (Me-19), 0.90 (Me-17) and 0.80 ppm (Me-20), a primary ester group with signals at d 3.81 and 3.59 ppm, a proton geminal to another ester group. This together with the MS indicated that 1a must be a tetracyclic diterpene of the thyrsiflorane type with two ester functions. The stereochemistry of the thyrsiflorane skeleton was determined by R-X diffraction [7].

Furthermore, the signals in the ¹H NMR at a d 2,15 (2H, d, J = 6.9 Hz, H-2'), 1.13(¹H, m, H-3') and at 0,93 (6H, d, J = 6.4 Hz, H₃-4' y H₃-5') correspond to an isovaleric moeity, whose signals in the de ¹³C NMR spectrum appeared at 172.9 (C-1'), 43.9 (C-2'), 25.7 (C-3'), 22.4 (C-4') ppm [6]. These fact is confirmed by a loss of 102 u.m.a. in the MS.

The correlations in the HMBC spectrum of the signal at d 172.9 ppm with d 4.58 (H-13) and with 2.15 ppm (CH₂ of isovaleric group) indicated that the isovaleroyl moiety is on C-13, and it must be equatorial due to the coupling constant of C-13. The correlations between 171.2 with 2.03 ppm (OAc) and with ? 3.81 and 3.59 ppm confirms that the acetate group is in C-18, and it must be equatorially orientated (C-19 appeared at 17.8 ppm) [10]. Other correlations are shown in Fig. 1. Therefore, compound 1a is 13-isovaleryl-18-acetoxy-thyrsiflorane.

Fig1. HMBC correlations of 1a.

Compound 2 was purified as its diacetyl derivative 2a. It displayed a m/z at 390 corresponding to a molecular formula of C₂₄H₃₈O₄

The ¹H NMR spectrum of 2a showed the presence of a dd at 4.59 ppm corresponding to a geminal proton to an acetate group, two doublets at 3.8 and 3.6 ppm corresponding to a primary ester group, and three quaternary methyl groups.

Comparing the spectra of 2a and of 1a, showed minor differences for the skeletal signals; in particular the signals for the isovaleric group were missing and appeared the signal corresponding to an acetate group. Therefore, compound 2a is 13,18-diacetoxy-thyrsiflorane.

ACKNOWLEGMENTS

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

REFERENCES

1. María Cristina Chamy, Marisa Piovano, Juan A. Garbarino and Luis Espinoza, (2006), "Diterpenoids from Calceolaria paralia" J. of Chilean Chemical Soc. 50, 793.

2. Ehrhart, Christine (2000), "Die Gattung Calceolaria (Scrophulariaceae) in Chile" Biblioteca Botanica. Heft 153.

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

4. Muñoz, M. Barrera, M., Mez, I., (1981), "El uso medicinal de plantas nativas y naturalizadas en Chile, publicación ocasional Nº 23, Museo Historia Natural, Santiago de Chile, p.48.

5. Chamy, M.C., Piovano, M., Garbarino, J.A. Miranda, C. and Gambaro,V.; (1990) Phytochemistry, 29, 2943.

6. Chamy, M.C., Piovano, M., Garbarino, J.A. y Vargas, C.; (1995), Phytochemistry, 40, 1751.

7. Chamy, M.C., Piovano, M., Garbarino, J.A., Miranda, C., Gambaro, V., Rodríguez, M.L., Ruíz-Pérez, C. y Brito, I.; (1991), Phytochemistry, 30, 589.

8. Nishiya,K., Kimura,T., Takeya,H., Kawazu,K., Koshimizu,K., Mitsui,Y. (1990) Phytochemistry, 30, 2410

9. Kempe De, N., Schamp, N., VanPuyvelde,L., Dobé, S., Chagnon-Dibé M., Borremans, F., Anteunis, M.J.O., Declerq, J.P., Germain, G. y Van Meersche, M., (1982), Journal Organic Chemistry, 47, 3828.

10. Ashraf, K., Gedara, S. R., Lahloub, M. F., Halim, A.F. y Voehler, M., (1997), Phytochemistry, 44, 475.

To whom correspondence should be addressed. Tel: 56-32-654225. Fax: 56-32-654782. E-Mail: juan.garbarino@usm.cl

* Part 25 in the series "Diterpenoids of Calceolaria species". For part 24 see ref.[1]