Clerodane diterpenes from leaves of Casearia sylvestris Swartz

ARTIGO

Clerodane diterpenes from leaves of Casearia sylvestris Swartz

André G. dos Santos; Carla C. Perez; Aristeu G. Tininis; Vanderlan da S. Bolzani and Alberto J. Cavalheiro^*

Instituto de Química de Araraquara, Universidade Estadual Paulista "Júlio de Mesquita Filho", R. Francisco Degni, s/n, 14800-900 Araraquara - SP, Brasil

ABSTRACT

Ethanolic extracts of the leaves of Casearia sylvestris yielded a novel clerodane diterpene, 15-hydroxy-3-cleroden-2-one, together with the known diterpenes (-)-hardwickiic acid, reported for the first time from this species, and casearins B and G, previously isolated from C. sylvestris. The structures of all four compounds were determined by spectrometric analysis. The new clerodane diterpene and (-)-hardwickiic acid contain structural features that are completely different from the highly oxygenated casearins and casearvestrins isolated from C. sylvestris.

Keywords:Casearia sylvestris; clerodane diterpenes; 15-hydroxy-3-cleroden-2-one.

INTRODUCTION

Casearia sylvestris Swartz (Flacourtiaceae) is a tree that is widely distributed within various ecosystems of South America, such as the Cerrado and the Atlantic and Amazon forests¹. In the popular medicine of Brazil, the use of the plant¹ is correlated with its pharmacological properties including anti-inflammatory², anti-ophidian^2,3 and anti-ulcer^1,4 activities. A number of phytochemical investigations of species of Casearia^5-9 have revealed the occurrence within the genus of oxygenated tricyclic clerodane diterpenes exhibiting a cis configuration between rings A and B and a characteristic diacetal system in ring C at positions C-18 and C-19. Several compounds of this type have been isolated from C. sylvestris, specifically casearins and casearvestrins, and they exhibited cytotoxic activity^5,7-9.

This paper describes the isolation and structure elucidation of a novel clerodane diterpene, 15-hydroxy-3-cleroden-2-one (1), from the leaves of C. sylvestris, together with (-)-hardiwickiic acid (2), reported for the first time from this species. In addition, casearin B (3) and G (4) were isolated, diterpenes that have been previously detected in the species^5,7,8. The structures of compounds 1-4 (Figure 1) were deduced on the basis of their spectral data and comparison with appropriate values reported in the literature.

RESULTS AND DISCUSSION

Ethanol extract of the leaves of C. sylvestris was submitted to chromatographic fractionation and yielded compounds 1-4. In the positive-ion mode, the HRTOF-ESIMS of 1 exhibited an [M+Na]⁺ ion at m/z 329.2481 that was compatible with a molecular formula of C₂₀H₃₄O₂. Analysis of the PND and DEPT 135º ¹³C-NMR spectra of 1 revealed twenty signals similar to those of the clerodane diterpene skeleton^6-11, being five of them attributed to methyl groups: C-16 (d 19.7), C-17 (d 16.0), C-18 (d 20.5), C-19 (d 32.2), C-20 (d 19.3). The IR and UV absorptions at 1653 cm^-1 and at 218 nm (l_max) and the NMR signals at d 199.3 (C-2), 128.6 (C-3), 168.5 (C-4) and 5.84 br s (H-3) indicated the presence of an a,b-unsaturated ketone group. In addition, the IR absorption at 3448 cm^-1 and the NMR signals observed at d 61.2 (C-15) and 3.68 m (H-15) are characteristic of a hydroxymethine group. The ¹H NMR and ¹³C assignments (Table 1) together with the results of HMQC, COSY and HMBC (Figure 2) experiments led to structure of compound 1. The relative configuration assignment at C-5, C-8, C-9 and C-10 was firstly proposed based on comparison of chemical shifts of methyl groups C-17, C-19 and C-20 of compound 1 with literature data, as follow. The cis stereochemistry at the junction of rings A and B in compound 1 could be deduced from the chemical shift of C-19 (d 32.2) when compared with C-19 chemical shifts of cis-clerodane diterpenes, for example floridiolic acid¹² and 13-hydroxy-cis-ent-cleroda-3,14-diene ¹³ (d 33.6 and 33.0, respectively). In contrast, the trans-clerodane diterpenes 2-oxokolavenic acid¹⁰ and eremone¹¹ presented chemical shifts for this methyl carbon at d 19.5 and 18.8, respectively. In clerodane diterpenes with a cis configuration between methyl groups C-17 and C-20, such as hautriwaic acid¹¹, 2-oxokolavenic acid¹⁰ and floridiolic acid¹², the chemical shift of C-20 was observed at approximately d 18.0, whilst clerodane diterpenes with a trans configuration between these methyl groups, as in the casearins and casearvestrins^5-9, show chemical shift for C-20 at approximately d 26.0. So, the chemical shifts attributed to C-20 in compound 1 (19.3) is indicative of a cis relationship between these methyl groups. These propositions were confirmed by the NOE enhancements arising from the dipolar interactions between H-19 (1.22 s) and H-10 (1.87 br d; 6.5 Hz) and between H-17 (0.77 d; 7.0 Hz) and H-20 (0.57 s). In addition, correlations observed in the NOE experiments (Figure 3) between H-1a (d 2.71) and H-19 and between H-1b and H-20, revealed the trans relationship between C-17/C-20 and C-19 methyl groups. A diastereomer of compound 1 was previously isolated from Cistus populifolius L. (Cistaceae)¹⁴, but the ¹³C NMR data of this compound were not included in the article to a complete comparison.

In the positive-ion mode, the HRTOF-ESIMS of compound 2 exhibited an [M+H]⁺ ion at m/z 317.2117, compatible with a molecular formula of C₂₀H₂₈O₃, which was confirmed by the finding of [M+Na]⁺ ion at m/z 339.1963. Analysis of the PND ¹³C-NMR spectra revealed twenty signals similar to those of the clerodane diterpene skeleton^6-11 and showed the presence of three methyl groups: C-17 (d 16.0), C-19 (d 20.6) and C-20 (d 18.3). IR absorptions presented at 3397 cm^-1 and 1684 cm^-1, UV absorption (l_max) at 210 nm and the signals observed in the ¹³C-NMR spectra at d 172.5 (C-18), 140.2 (C-3) and 141.5 (C-4) were indicative of an a,b-unsaturated carboxylic group. In addition, the ¹H and ¹³C NMR spectra showed signals that could be ascribed to a b-mono-substituted furan ring (C-13, C-14, C-15 and C-16) in the lateral chain of the diterpene. The complete attribution of the NMR signals (table 1) was based on the correlations observed in the HMQC, HMBC and COSY contour plots and this attribution is in agreement with the NMR data from literature for hardiwickiic acid¹⁵. The negative value of [a]_D²⁵ confirmed the absolute configuration as (-)-hardiwickiic acid¹⁶. Compound 2 or its enantiomer were previously isolated from other species of different families, as Copaifera duckei, C. guianensis, Croton californicus, C. aromaticus, Hardwickia pinnata, Salvia divinorum and Sindora sumatrana^15,17, and now we found it for the first time in a Casearia species.

The ¹H- and ¹³C-NMR data of 3 (C₃₁H₄₄O₁₀) and 4 (C₂₉H₄₂O₈) showed signals identical of the compounds previously reported in C. sylvestris^5,7,8, casearins B and G, respectively.

EXPERIMENTAL

General

Column chromatography (CC) and solid phase extraction (SPE) were performed over activated charcoal (Synth), silica gel G (40-63 µm; Merck) or RP-C18 (40-63 µm; Merck) and the resulting fractions were monitored by TLC, HPLC-UV and ¹H-NMR. Comparative TLC was carried out on silica gel G60 layers (0.25 mm thickness; Merck). HPLC-UV analyses were conducted using a Supelcosil LC-18 column (250 x 4.6 mm; i.d. 5 µm) connected to a Varian chromatographic system consisting of a ProStar 240 solvent delivery module, a ProStar 330 photodiode array detector, a ProStar 410 autosampler and a Star chromatography workstation. For preparative HPLC, a Supelcosil PLC-18 column (250 x 21.2 mm; i.d. 12 µm) was coupled to a Varian system consisting of Dynamax model Sd-1 solvent delivery system and a ProStar 320 detector with Star integrator software.

IR spectra (KBr disc) were obtained using a Perkin Elmer FTIR 1600 spectrometer, UV spectra were measured on a Cary 13E instrument and a Perkin Elmer 341 polarimeter was employed to determine optical activities. NMR spectra of 1 and 2 were recorded at 500 and 125 MHz for ¹H- and ¹³C-NMR, respectively, using a Varian INOVA 500 spectrometer with CHCl₃ as internal standard. The NMR spectra of 3 and 4 were measured at 200 and 50 MHz for ¹H- and ¹³C-NMR, respectively, on a Bruker AC-200 instrument using the same internal standard. Accurate-mass measurements were performed on an ESI-quadrupole-time of flight instrument (UltrOTOF_Q, Bruker Daltonics, Billerica, MA).

Plant material

Leaves of Casearia sylvestris Swartz (Flacourtiaceae) were collected at the Carlos Botelho State Park (São Paulo State, Brazil) and in Araraquara (São Paulo State, Brazil) between August and November 1999. Voucher specimens (AGS13 and AGS45) are deposited at the State Herbarium "Maria Eneida P. Kaufmann" of the Botanic Institute (São Paulo State, Brazil).

Extraction and isolation of 1, 3 and 4

Dried and powdered leaves of C. sylvestris (2.1 kg) were extracted by sonication with EtOH (3 x 6.0 L; 20 min per extraction) and concentrated under reduced pressure to yield 139.0 g of residue. The crude extract was partitioned between MeOH:water (7:3, v/v, 0.8 L) and CH₂Cl₂ (1.5 L) and the CH₂Cl₂ layer separated, dried and concentrated under reduced pressure to yield 71.0 g of a residue that was subjected to SPE over silica gel eluted sequentially with CH₂Cl₂, CH₂Cl₂:MeOH (9:1) and MeOH, yielding 8 fractions. SPE-fraction 5 (13.5 g) eluted with CH₂Cl₂:MeOH (9:1) was submitted to CC over silica gel eluted sequentially with hexane:EtOAc (8:2, 6:4 and 4:6), EtOAc, EtOAc:MeOH (9:1) and MeOH, yielding 12 CC-fractions. CC-fraction 8 (1.45 g) eluted with hexane:EtOAc 4:6 was submitted to RP-CC over octadecyl silane eluted sequentially with MeOH:water (6:4, 8:2 and 9.5:0.5), MeOH and CH₂Cl₂. Sub-fraction 3 (120.0 mg) eluted with MeOH:water 8:2 was submitted to preparative RP-HPLC (C-18, 65% MeOH, isocratic mode, flow rate 13.0 mL/min, l 235 nm) to yield 1 (5.0 mg). Further, CC-fraction 7 (2.4 g) eluted with hexane:EtOAc 6:4 was submitted to preparative RP-HPLC (C-18, 75% MeOH, isocratic mode, flow rate 10.0 mL/min, l 235 nm), yielding compounds 3 (86.2 mg) and 4 (30.9 mg).

Extraction and isolation of 2

Dried and powdered leaves of C. sylvestris (2.0 kg) were extracted by maceration under agitation with EtOH (3 x 3.0 L; 2 h per extraction) and concentrated under reduced pressure to yield 190.0 g of residue. The crude extract was partitioned between EtOH/Water (6:4, v/v, 1.5 L) and hexane (1.5 L), and the hexane layer separated, dried and concentrated under reduced pressure to yield 120.2 g of a residue that was subjected to SPE over silica gel:activated charcoal (1:1) eluted sequentially with hexane:EtOAc (7:3 and 3:7) and MeOH. The fraction eluted with hexane/EtOAc 3:7 (28.4 g) was submitted to CC over silica gel eluted sequentially with hexane:EtOAc (7:3, 6:4, 1:1, 4:6 and 3:7) and EtOAc, yielding 24 fractions. Fraction 11 (34.0 mg) eluted with hexane:EtOAc 1:1 was submitted to SPE over RP-C18 eluted with MeOH:water (98:2) to yield 2 (29.0 mg).

15-hydroxy-3-cleroden-2-one (1)

White powder (5.0 mg). [a]_D²⁰ +42º (c 0.14; MeOH). UV l_max nm (MeOH) 218 (e = 10.5 x 10³ cm³ mol^-1 cm^-1). IR n_max cm^-1 3448, 2958-2853, 1653, 1037 (KBr). ¹H- and ¹³C-NMR see Table 1. HRTOF-ESIMS m/z 329.2481 [M+Na]⁺ (calcd for C₂₀H₃₄O₂Na⁺ , 329.2451).

(-)-Hardiwickiic acid (2)

White powder (29.0 mg). [a]_D²⁵ -35º (c 1.0; MeOH). UV l_max nm (MeOH) 210 (e = 7.2 x 10³ cm³ mol^-1 cm^-1). IR n_max cm^-1 3397, 2958-2864, 1684, 1640, 1257, 1078 (KBr). ¹H- and ¹³C-NMR see Table 1. HRTOF-ESIMS m/z 317.2117 [M+H]⁺ (calcd for C₂₀H₂₉O₃, 317.2117) and m/z 339.1963 [M+Na]⁺ (calcd for C₂₀H₂₈O₃Na⁺ , 339.1930).

CONCLUSIONS

The new compound (+)-15-hydroxy-3-cleroden-2-one (1) and (-)-hardwickiic acid (2) are the first diterpenes reported in C. sylvestris without the typical highly oxygenated backbone. Casearins and casearvestrins, previously isolated from this species, have common stereochemical features of a cis-clerodane diterpene, notably a cis configuration at the junction of rings A and B and, in addition, trans configuration between the methyl groups at C-17 and C-20^5,7-9. However, whilst the newly described (+)-15-hydroxy-3-cleroden-2-one (1) is a typical cis-cledorane, (-)-hardiwickiic acid (2) possesses a trans-clerodane configuration. In conclusion, the occurrence of compounds 1, 2, 3 and 4 in C. sylvestris reveals the ability of this species in biosynthesize both cis and trans-clerodanes as it is common in other plant species as Adelanthus lindenbergianus (Adelhantaceae)¹³ and Grangea maderaspatana (Asteraceae)¹⁸.

ACKNOWLEDGMENTS

The authors are grateful to the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Instituto Florestal da Secretaria do Meio Ambiente do Estado de São Paulo for support of the project; Laboratório de Espectrometria de Massas da Faculdade de Ciências Farmacêuticas da Universidade de São Paulo, campus de Ribeirão Preto for MS determination.

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Recebido em 18/4/06; aceito em 10/11/06; publicado na web em 17/7/07

Publication Dates

• Publication in this collection
  28 Sept 2007
• Date of issue
  Oct 2007

History

• Accepted
  10 Nov 2006
• Received
  18 Apr 2006