Two clerodane diterpenes and flavonoids from Croton brasiliensis

ARTICLE

Two clerodane diterpenes and flavonoids from Croton brasiliensis

Sebastião F. Palmeira Jr.^I; Lucia M. Conserva^{*, I}; Edilberto R. Silveira^II

^IDepartamento de Química, Universidade Federal de Alagoas, 57072-970 Maceió-AL, Brazil

^IIDepartamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60021-970 Fortaleza-CE, Brazil

ABSTRACT

Two new clerodane diterpenes, crotobrasilin A and crotobrasilin B, were isolated in addition to four known 3-methoxyflavones: casticin, penduletin, chrysosplenol-D and artemetin from leaves and stems of Croton brasiliensis. The structural elucidation of these compounds was made on the basis of spectroscopic data analyses, especially NMR, including 2D techniques (COSY, HMQC, HMBC and NOESY).

Keywords:Croton brasiliensis, clerodane diterpenes, 3-methoxyflavones

RESUMO

Dois novos diterpenos do tipo clerodano, crotobrasilina A e crotobrasilina B, foram isolados juntamente com quatro 3-metoxiflavonas conhecidas: casticina, penduletina, crisosplenol-D e artemetina, das folhas e caule de Croton brasiliensis. A elucidação estrutural destes compostos foi feita com base na análise dos dados espectroscópicos, especialmente RMN, incluindo técnicas bidimensionais (COSY, HMQC, HMBC e NOESY).

Introduction

The genus Croton (Euphorbiaceae), which comprises about 700 species, is widely distributed in all regions of Brazil.¹ Many of these species are known in the Brazilian Northeast region as 'capixingui' and 'marmeleiro'¹ and have been used in folk medicine for a large number of applications.² Previous phytochemical investigations show that this genus possess alkaloids,² flavonoids,^3-6 triterpenoids,^4,7,8 and a large number of diterpenoids.^5,6,9-11 In this paper, we report the isolation and structural elucidation of two new clerodane diterpenes and four known 3-methoxyflavones from leaves and stems of C. brasiliensis Mull. Arg.

Results and Discussion

The dichloromethane extracts from leaves and stems of C. brasiliensis after chromatography fractionation and recrystallizations afforded two clerodane diterpenes (1-2) in addition to four known 3-methoxyflavones (3-6).

Compound 1, isolated as a white amorphous powder, mp 198.5-201.4 °C, by EIMS showed a molecular ion at m/z 416. This information, along with NMR data, suggested C₂₃H₂₈O₇ as the molecular formula, in accordance with the observed molecular weight. The FT–IR spectrum indicated the presence of ester and lactone carbonyls (1734 cm^-1), b-substituted furan ring (1490 and 816 cm^-1), and double bond (1665 cm^-1). The ¹³C NMR spectra (CPD, DEPT and HMQC) showed 23 carbon signals (four methyls, four methylenes, eight methines, and seven non-hydrogenated), consistent with substituted diterpene skeleton. The NMR spectra, including ¹H-¹H COSY, NOESY, HMQC and HMBC, showed signals for a cis-disubstituted double bond [d_H 5.81 (m, H-2) and 6.40 (d, J 10.3 Hz, H-3); d_C 131.1 (C-2) and 125.9 (C-3)], a b-substituted furan ring [d_H 6.21 (br s, H-14), 7.15 (br s, H-16) and 7.16 (br s, H-15); d_C 109.4 (C-14), 126.7 (C-13), 139.9 (C-16) and 144.0 (C-15)], two angular methyl groups [d_H 0.80 (s, Me-20) and 1.13 (s, Me-19); d_C 15.6 (Me-20) and 16.8 (Me-19)], a carbonyl methyl ester group [d_H 3.49 (s, OMe); d_C 52.4 (OMe) and 170.6 (C-18)], and signals for an acetyl group [d_H 1.80 (s); d_C 21.4 and 170.3], whose correlations observed in the HMBC spectrum (Figure 1), specially for H-6_ax (d 1.37), H-10 (d 1.94), and Me-19 (d 1.13) with C-4 (d 87.8) defined its position in this carbon. Moreover, also were observed signals for sp³ oxymethine carbon [d_H 5.06 (dd, J 5.1 and 11.6 Hz, H-12); d_C 71.7 (C-12)], which chemical shifts and multiplicities are characteristic of hydrogen attached to a carbon involved in a d-lactone moiety [d_C 171.0 (C-17)]. Correlations observed in the HMBC spectrum (Figure 1) between H-12 (d 5.06) and C-13 (d 126.7), C-14 (d 109.4), and C-16 (d 139.9) evidenced the relationship between the furan ring and d-lactone moiety. The remaining NMR data showed characteristic chemical shifts for two methines, four methylenes, and three non-hydrogenated carbons (Table 1). Thus, on the basis in the evidence of the spectral data and comparison with those reported for cordatin (7) and epi-cordatine (8), two b-furan clerodane diterpenes isolated from Aparisthmium cordatum¹² and Burasia madagascarensis,¹³ respectively, structure of a b-furan clerodane diterpene was proposed.

NOESY experiment deduced the relative configurations at asymmetric centers (Figure 2). Dipolar-dipolar correlations between H-12 (d 5.06) and Me-20 (d 0.80), and this later with Me-19 (d 1.13), as well as for H-6_ax (d 1.37) and H-8 (d 1.73) with H-10 (d 1.94) demonstrated a 1,3-cis relationship among these hydrogens and suggested that 1 have A/B and B/C trans ring junctions. These relative configurations were also supported by comparison of the NMR data of 1, exactly when recorded also in CDCl₃, with those of 7, whose structure was determined by X-rays diffraction analysis and possess A/B trans and B/C cis ring junctions. In particular, compound 1 exhibited significant differences in the chemical shifts of C-8, C-11, and C-17 and showed signals for H-8, H-10 and H-11 at upfield (Table 1). These differences in the chemical shifts these hydrogens can be explained if ring C to adopt a skew-boat conformation, with the ring furan in b-equatorial. Thus, compound 1, named here crotobrasilin A, was proposed to be the (5R,8R,9R,10R)-4R-acetoxy-15,16-epoxy-neo-cleroda-2,13(16),14-trien-17,12S-olide-18 -oate methyl.

Compound 2 was isolated as yellowish oil, in a mixture with 1. Due to the small quantity of the mixture, we have not attempted to separate both compounds in order to avoid any loss of material. However, its NMR data were discernable since furnished different intensities of the signals for both compounds [ratio 2.5:1 (2): (1)]. So, all assignments of the chemical shifts for 2 were inferred (Table 1). In a similar way that compound 1, the NMR spectral data of 2 also showed signals for a b-substituted furan ring [d_H 6.48 (br s, H-14), 7.47 (br s, H-15) and 7.53 (br s, H-16); d_C 109.9 (C-14), 125.2 (C-13), 141.0 (C-16) and 144.9 (C-15)], a cis-disubstituted double bond [d_H 5.99 (m, H-2) and 5.37 (d, J 9.4 Hz, H-3); d_C 131.4 (C-2) and 129.7 (C-3)], two angular methyl groups [d_H 1.16 (s, Me-19) and 1.39 (s, Me-20); d_C 16.1 (Me-19) and 25.8 (Me-20)], a carbonyl methyl ester [d_H 3.85 (s); d_C 54.8 (OMe) and 177.3 (C-18)], a d-lactone [d_H 5.31 (dd, J 5.5 and 13.5 Hz); d_C 71.2 and 175.3], besides signals for two methines, four methylenes, and three non-hydrogenated carbons. The ¹³C NMR spectra (CPD, DEPT and HMQC) displayed 21 carbon signals assignable to three methyls, four methylenes, eight methines, and five non-hydrogenated carbons. In the general mode, the NMR spectral data of 2 were closely similar to those of 1, except for the absence of the signals for an acetyl group, which was replaced by a hydroxyl group [1: d_C 87.8 (C-4) and 2: d_C 82.1 (C-4)], suggesting that 2 may be the 4-deacetylderivative. In fact, the deacetylation to yield the hydroxyl group caused the expected upfield shift of the C-4 (Dd = -5.7 ppm). Extensive use of NMR techniques, including ¹H-¹H COSY, HMQC, and HMBC (Figure 1), supported the unambiguous assignments of all ¹H and ¹³C shift values (Table 1).

Although some of the NMR assignments of cordatin (7)¹² must be revised, comparison of the ¹³C NMR data of 2 with those of 7 were very similar, except for the chemical shifts of the C-3 (2: d_C 129.7; 7: d_C 123.2, value interchanged in the paper with C-2) and C-18 (2: d_C 177.3; 7: d_C 173.7), suggesting that two compounds are epimers at C-4. However, when compared with those of epi-cordatine (8),¹³ which possess A/B and B/C cis ring junctions, revealed that the two compounds are not identical since differences in the chemical shifts of the several carbons were observed (Table 1). Therefore, compound 2 was proposed to be the (5R,8S,9R,10R)-4S-hydroxy-15,16-epoxy-neo-cleroda-2,13(16),14-trien -17,12S-olide-18-oate methyl, a clerodane diterpene with A/B trans and B/C cis ring junctions, named here crotobrasilin B.

Several structurally distinct clerodane-type diterpenes have been found in Croton species, but to our knowledge, this is the first report of the occurrence of crotobrasilin A and crotobrasilin B from natural sources.

The IR, ¹H and ¹³C NMR spectral data for casticin (3),^14,15 peduletin (4),¹⁴ artemetin (5)¹⁶ and chrysosplenol-D (6)¹⁶ were in accordance with those reported in the literature. Several structural types of flavonoids have been reported from Croton species.^3-6 However, all the 3-methoxyflavones related in this work are described for the first time in this genus.

Experimental

Instrumental and chromatography materials

The melting point was measured using an MQAPF-302 apparatus and is uncorrected. Optical rotation was measured on a Perkin-Elmer 341 polarimeter. The NMR spectra were recorded either on a Bruker DRX-500 or Mercury-Varian 200 operating at 500 and 200 MHz, respectively, for ¹H and 125 and 50.3 MHz, respectively, for ¹³C, in C₆D₆ or CDCl₃, and DMSO-d₆ solutions with TMS as internal standard. IR spectra were obtained on a FT-IR 1750 Perkin-Elmer spectrometer. EIMS were measured at 70 eV on a HP 5990/5988A spectrometer. Silica gel (70-230 and 230-400 mesh, Merck) and Sephadex LH-20 (Pharmacia) were used for column chromatography separations.

Plant material

The leaves and stems of C. brasiliensis Mull. Arg. (syn. C. polyandrus Spreng) were collected in November 1999, in the Área de Proteção Ambiental de Santa Rita (Mucuri), Marechal Deodoro, Alagoas, Brazil, and was identified by Rosangela P. de Lyra Lemos of the Departamento de Botânica do Instituto do Meio Ambiente do Estado de Alagoas (IMA-AL), where a voucher specimen was deposited (MAC-10752).

Extraction and isolation of the constituents

The air-dried and powdered leaves (1300 g) and stems (900 g) were extracted with acetone followed by 90% EtOH at room temperature. The crude extracts [leaves: acetone (142.8 g); stems: acetone (23.9 g) and EtOH (16.2 g)] were suspended in MeOH-H₂O (3:2) solution and extracted successively with C₆H₁₄, CH₂Cl₂ and EtOAc. The CH₂Cl₂ residue of the acetone extract from leaves (16.7 g) was filtrated on silica gel with C₆H₁₄-CH₂Cl₂ 1:1 (7.3 g, A), CH₂Cl₂ (0.9 g, B), CH₂Cl₂-EtOAc 1:1 (2.9 g, C), EtOAc (1.06 g, D) and MeOH (1.51 g, E). Fractions A (7.3 g), B (0.9 g) and C (2.9 g) were chromatographed on silica gel column using C₆H₁₄ with increasing amounts of CH₂Cl₂. The subfractions obtained after gel permeation on Sephadex LH-20 (MeOH) and successive recrystallizations from MeOH afforded 1 (33 mg), 3 (21 mg), 4 (40 mg), and 5 (108 mg).

The CH₂Cl₂ residues from stems (5.0 g) were submitted to silica gel filtration with C₆H₁₄-CH₂Cl₂ 1:1 (1.2 g, A), CH₂Cl₂ (1.21 g, B), CH₂Cl₂-EtOAc 9:1 (0.94 g, C), EtOAc (1.35 g, D) and MeOH (0.08 g, E). Fraction A (1.2 g), after gel permeation on Sephadex LH-20 (MeOH) and successive recrystallizations from MeOH, afforded a mixture 2.5:1 containing 1 and 2 (16 mg), and 6 (18 mg).

(5R,8R,9R,10R)-4R-Acetoxy-15,16-epoxy-neo-cleroda -2,13(16),14-trien-17,12S-olide-18-oate methyl (Crotobrasilin A, 1)

White amorphous powder, mp 198.5-201.4 °C; [a]_D²⁰ + 25.1° (CHCl₃, c 0.0052). IR (KBr) n_max/cm^-1: 2953, 1734, 1665, 1490, 1450, 1374, 1267, 1134, 1076, 1020, 876, 816, 785; EIMS, 70 eV m/z (rel. int.): 416 [M] (2), 401 (M-CH₃, 2), 357 (M-OAc, 10), 374 (M-C₂H₂O, 3), 342 (M-C₃H₆O₂, 2), 315 (100), 297 (5), 247 (3), 203 (10), 153 (20), 121 (10), 105 (21), 91 (22); ¹H (500 MHz) and ¹³C (125 MHz, C₆D₆) NMR spectra (see Table 1).

(5R,8S,9R,10R)-4S-Hydroxy-15,16-epoxy-neo-cleroda -2,13(16),14-trien-17,12S-olide-18-oate methyl (Crotobrasilin B, 2).

Yellowish oil. ¹H (500 MHz) and ¹³C (125 MHz, CDCl₃) NMR data are given in Table 1.

Acknowledgments

The authors are grateful to FAPEAL, CNPq, and Ministério da Ciência e Tecnologia-Instituto do Milênio do Semi-Árido (MCT-IMSEAR) for financial support and research fellowships, to Vicente Carlos C. de Oliveira (LTF, Universidade Federal da Paraíba), Daniel Esdras de Andrade Uchoa (CENAUREMN, Universidade Federal do Ceará), and Dr Daniela F. Navarro (Depto Química, Universidade Federal de Pernambuco) for the NMR and MS spectra.

Received: August 15, 2005

Published on the web: October 11, 2005

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Publication Dates

• Publication in this collection
  20 Jan 2006
• Date of issue
  Nov 2005

History

• Received
  15 Aug 2005