20(R)- and 20(S)-Simarolide Epimers Isolated from Simaba cuneata: Chemical Shifts Assignment of Carbon and Hydrogen Atoms

Article

20( R )- and 20( S )-Simarolide Epimers Isolated from Simaba cuneata - Chemical Shifts Assignment of Carbon and Hydrogen Atoms

Ivo José Curcino Vieira^a, Raimundo Braz Filho^a, Edson Rodrigues Filho^b, Paulo Cezar Vieira^b, Maria Fátima G.F. da Silva^b, and João Batista Fernandes*^b

^a Setor de Produtos Naturais-CCTA/CCT, Universidade Estadual do Norte Fluminense, 28015-620 Campos dos Goytacazes - RJ, Brazil

^b Departamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luiz km 235, 13565-905 São Carlos - SP, Brazil

Os quassinóides C₂₅ 20(R)- e 20(S)-simarolídeo foram isolados de Simaba cuneata, Simaroubaceae. As estruturas destas substâncias foram caracterizadas por dados espectrais, incluindo espectros de RMN 1D e 2D que foram também usados para a completa atribuição dos deslocamentos químicos dos átomos de carbono e hidrogênio.

The C₂₅-quassinoids 20(R)- and 20(S)-simarolide were isolated from Simaba cuneata, Simaroubaceae. The structures of these two compounds were characterized by spectral data, including 1D and 2D NMR spectra which were also used for the complete assignment of the carbon and hydrogen atom chemical shifts.

Keywords: Simaba cuneata, Simaroubaceae, C25-quassinoids

Introduction

20(R)-simarolide (1a), a C-25 quassinoid was first isolated from Simarouba amara. Its structural elucidation, including absolute stereochemistry has already been described in the literature^1,2. The phytochemical study of a Simaba cuneata specimen has allowed the isolation of (1a) and its C-20 epimer (1b). The characterization of these two quassinoids was based on spectral data analysis, obtained from the diacetylated derivatives 2a and 2b, especially NMR [(1D: ¹H-NMR, ¹³C-NMR-PND and ¹³C-NMR-DEPT) and (2D: ¹H x ¹H-COSY, ¹H x ¹H-NOESY, HMQC and HMBC)]. The NMR data comparison of both compounds isolated from Simaba cuneata allowed the localization of an acetoxyl group at C-11 (1a and 1b, Table 1), and the correct assignment of the chemical shifts of all hydrogen (d_H) and 13-carbon (d_C) atoms of both diacetyl derivatives (2), 20(R)-2-O-acetylsimarolide (2a) and 20(S)-2-O-acetylsimarolide (2b).

The presence of a carbonyl group at C-17 led us to discuss the possibility of 1b being the result of a C-20 epimerization (a carbon to the carbonyl group) of the major natural compound 20(R)-simarolide (1a), during the extraction and isolation processes. However, the bioproduction of 20(S)-simarolide still can not be discarded.

Results and Discussion

The ¹³C-NMR spectra signals of 2a and 2b (Table 1) corresponding to quaternary carbons [(C)₉: two ketone carbonyls, four ester and lactone carbonyls and three Csp³ in C-C bonds], methines [(CH)₈: three Csp³ attached to oxygen and five Csp³ involved in C-C bonds], methylenes [(CH₂)₆: one Csp³ attached to oxygen and five Csp³ involved in C-C bonds] and methyls [(CH₃)₆: four involved in C-C bonds and two attached to Csp² carbonyl carbon] were identified by the comparative analysis of the hydrogen decoupling spectra (PND = proton noise decoupling) and by the DEPT technique (Distortionless Enhancement by Polarization Transfer, q = 90°: only CH signals; q = 135°: CH₂ signals in opposite phase to CH and CH₃; after the removal of these signals from the PND spectra, the quaternary carbon signals were obtained)³. Based on small chemical shift differences observed between 2a and 2b we suggested that they were diastereomers.

The number of functional groups {two carbonyl ketones (2a: d_C 211.90, C-17 and 206.60, C-1; 2b: d_C 211.96, C-17 and 206.66, C-1), two acetoxyls [2a: d_C 169.94 and 20.58; d_H 2.13 (AcO-2); d_C 171.66 and 21.03; d_H 2.04 (AcO-11); 2b: d_C 170.01 and 20.64; d_H 2.13 (AcO-2); d_C 171.73 and 21.09; d_H 2.04 (AcO-11)], one g-lactone [2a: d_C 174.45 (C-23); 2b: d_C 174.50 (C-23)], one d-lactone [2a: d_C 169.25 (C-16); 2b: d_C 169.31 (C-16)] and the remaining quaternaries carbons [2a: (C)₃; 2b (C)₃], methine [2a: (CH)₈; 2b: (CH)₈], methylene [2a: (CH₂)₆; 2b: (CH₂)₆] and methyls remaining [2a: (CH₃)₄; 2b: (CH₃)₄]}, combined with the d_C and d_H and the multiplicity of the hydrogen atom signals observed in the ¹H-NMR spectra (1D and 2D ¹H x ¹H-COSY), have allowed the structure of simarolide (1a), a quassinoid with twenty five carbon atoms (C₂₅) isolated from Simarouba amara¹ to be proposed. The relative stereochemistry of 1a was defined on the basis of X-ray analysis^1,2. This is the first report of the complete NMR carbon and hydrogen assignment of 1a.

The ¹H-NMR (Table 2) and ¹H x ¹H-COSY of 2a and 2b revealed small significant differences for the protons H-20 [2a: d_H 4.31 (m); 2b: d_H 4.30 (m)] and CH₂-21 [2a: d_H 4.59 (t, J = 7.2 Hz) and 4.30 (m); 2b: d_H 4.60 (t, J = 7.6 Hz) and 4.34 (m)], clearly showing different spectral patterns (Figure 1 and 2). These differences were confirmed by the comparison (Figure 3 and 4) of the heteronuclear correlation spectra (HMQC)^4,5 of 2a and 2b (two epimers at C-20), which revealed the d_C CH-20 [2a: d_C/ d_H 41.50 (CH-20)/4.31 (m, H-20); 2b: d_C/ d_H 41.58 (CH-20)/4.30 (m, H-20)] and CH₂-21 [2a: d_C/ d_H 70.26 (CH₂-21)/4.59 (t, H-21) and 4.30 (m, H-21b); 2b: d_C/ d_H 70.22 (CH-21)/4.60 (t, H-21a) and 4.34 (m, H-21b)] and have permitted the observation of distinct chemical shifts for H-20 (d_H 4.31) in 2a and (d_H 4.30) in 2b, and for H-21 (d_H 4.30) in 2a and (d_H 4.34) in 2b. Therefore, it was possible to deduce that the two acetylated derivatives 2a and 2b differ only in the stereochemistry of the chiral carbon C-20. From HMQC spectra of 2a and 2b it was possible to assign all chemical shifts for both, hydrogen and hydrogenated carbon atoms (Tables 1 and 2), also establishing the d_H observed for CH-20 (2a: d_H 4.31 (m); 2b: d_H 4.30), which is surprising for a methine hydrogen occupying the a position in relation to the carbonyl group, since the d_C [2a: d_C 41.50 (CH-20); 2b: d_C 41.58 (CH-20)] revealed to be compatible with the parameters expected for d_C in these structural conditions³.

The unequivocal assignment of the d_C of all 2a carbon atoms (Table 1) was assured by the analysis of the heteronuclear correlation bidimensional spectrum, HMBC. Therefore, for example, the spin-spin interactions of C-1 (d_C 206.60) with the 3H-19 [d_H 1.24 (Table 2), ³J_CH] and C-17 (d_C 211.90) with the H-12ax [d_H 1.69 (Table 2), ³J_CH] and the 3H-18 [d_H 0.98 (Table 2), ³J_CH] have allowed the correct assignment of the chemical shifts for C-1 and C-17. Other long range correlations can be found in Table 1. Based on the correct assignment of C-1 (d_C 206.60) and C-17 (d_C 211.90), it was possible to reassign the chemical shifts for this structure and correct some data from the literature. So, the assignment of the d_C of C-1 (d_C 214.17) and C-17 (numbered as C-22 in reference 6: d_C 200.55) of the quassinoid (3) isolated from Picrasma javanica⁶ should be corrected to C-1 (d_C 200.55) and C-22 = C-17 (d_C 214.17), as observed in the quassinoid (4) isolated from Picrasma ailanthoides⁷, since a solvent (pyridine-d₅) effect should not be responsible for such a modification. The comparison of the ¹³C-NMR chemical shift for 2a and 2b also permitted the correct assignment of the 2b quaternary carbon d_C (Table 1).

With the unequivocal assignment of the d_H for 2a and 2b through analysis of the HMQC spectrum (Tables 1 and 2), the homonuclear correlation ¹H x ¹H-COSY has allowed the recognition of all spin-spin interactions of the hydrogen atoms, especially CH-20, CH₂-21 and CH₂-22 (Fig. 1). The spectrum of 2a revealed, not only the vicinal interactions, but also the coupling of H-21a [d_H 4.59 (t)] in W or M (zig-zag)⁵ with H-22 [d_H 2.75 (m)], which was not observed in the spectrum of 2b (Fig. 1). This comparative analysis allowed the suggestion that the g-lactone ring in 2a adopts a conformation in which the spin-spin coupling through syn periplanar four bonds with H-21a and H-22 is possible in a W or M geometry (⁴J_w-H,H), not observed in 2b. The conformational behavior differences of the g-lactone ring on both epimers (2a and 2b) can only be justified by a steric interference involving the C-ring and the CH₃-18 and CH₃-30 methyl groups. In fact, the most significant differences in the interatomic distances (Table 3), measured at the minimum energy conformation (2a: 102.95 kcal/mol; 2b: 100.49 kcal/mol) using the molecular modeling program - PCMODEL (version 3.0), involve H-20 and 3H-18 hydrogen atoms (2a: 3.867, 4.230, 5.120 Å, with a mean approximated value = 4.406 Å; 2b: 2.363, 3.583, 3.746 Å, with a mean approximated value = 3.231 Å); DÅ = 4.406 - 3.231 = 1.175 Å) and 2H-12 hydrogen atoms (2a: 3.804 and 5.000 Å, with a mean approximated value = 4.402 Å; 2b: 1.880 and 3.447 Å, with a mean approximated value = 2.663 Å; DÅ = 4.402 - 2.663 = 1.739 Å). These data were employed to show, as foreseen, that the epimerization of the CH-20 chiral carbon produces diastereoisomers with different energies and interatomic differences between H-20 and the hydrogen atoms bonded to carbons placed close to the chiral center.

The ¹H x ¹H-NOESY dipolar interaction bidimensional spectra of 2a and 2b (Table 4) were used to confirm the trans junctions of the A/B and B/C rings and cis junctions of the B/d-lactone and C/d-lactone rings and their corresponding conformations. The interpretation difficulties observed for the spatial interactions (dipolar-dipolar coupling) due especially to the proximity of H-20 and H-21b absorption positions, involving CH-20, CH-21 and 2H-22 hydrogen atoms were attenuated by the calculation of the interatomic distances (Table 3). Therefore, it was possible to assume the existence of NOE’s between H-20 (d_H 4.30) and H-12eq (d_H 2.49) and 3H-18 (d_H 0.98) of 2b, which were not observed in 2a (Table 3). Furthermore, in the ¹H x ¹H-NOESY spectrum of 2b, a dipolar interaction was observed between H-11ax (d_H 4.92) and H-22 (d_H 2.75), indicating spatial proximity of these two hydrogen atoms. As in the spectrum of 2a, the spatial proximity of H-21b (d_H 4.30) with H-12eq (d_H 2.49), 3H-18 (d_H 0.98) and 3H-30 (d_H 0.90) could also be assumed. These data were used to postulate the configuration 20(R)-2-O-acetylsimarolide (2a) for the acetylated derivative of the major natural product (1a) and 20(S)-2-O-acetylsimarolide (2b) of the minor natural product (1b). This proposal is in agreement with the specific rotation {2a: [a]_D = -14.0° (c 0.8, CHCl₃); 2b [a]_D = -19.2° (c 1.48, CHCl₃)} and with biogenetic considerations, since Simarouba amara¹, Picrasma javanica⁶, Picrasma ailanthoides⁷, are bioproducers of C₂₅ quassinoids with 20(R) carbon, and Simaba cuneata belongs to the same Simaroubaceae family. This biogenetic consideration reinforces the possibility of 1b being a product formed by an epimerization reaction that may have occurred during the extraction and isolation process, but still does not definitely rule out its biogenetic origin.

Finally, the mass spectrum of 2a and 2b, obtained by electronic impact at 70 eV, has revealed the peak corresponding to the molecular ion (m/z 546, 1%), and the peaks with m/z 486 (29 %) as well as the peaks m/z at 427 (7 %) and 426 (16 %) Daltons (M-AcOH – AcO• and/or M-AcO• - AcOH, and M-AcOH-AcOH respectively). Other peaks classified as being important for the structural information can be found summarized in Scheme 1. The analysis of the mass spectrum can also be useful for investigations involving the study of crude extracts by GC/MS⁸.

Experimental

General

The melting points were determined in a Microquimica MQRPF-301 digital model equipment with heating plate. The IR spectra were registered on KBr pellets in a Pelkin-Elmer spectrometer. The Bruker ARX-400 model spectrometer (¹H: 400 MHz; ¹³C: 100 MHz) was used to obtain the ¹H-NMR and ¹³C-NMR spectra, using CDCl₃ as solvent and TMS as internal reference. The low resolution mass spectra were obtained in a Fisons/Plataform DI/MS 2000 model operating at 70 eV. The specific rotation measurements [a]_D were done in a Perkin-Elmer model 241 digital polarimeter, using 20 s intervals for each reading, averaging a total of ten measurements. For the chromatographic analysis a drop countercurrent chromatograph Eyela DCC-300 model, equipped with 300 columns (42.0 x 0.3 cm) and also with Merck silica gel columns (40-60 mm) were used.

Plant material

The aerial parts of Simaba cuneata St.Hill. were collected in January of 1993, in highway of Sol, Guarapari - ES, and were identified by Prof. Dr. José Rubens Pirani. The respective voucher can be found deposited in the Herbarium of the São Paulo University, Biosciences Institute - SP. The leaves, bark and branches were dried separately in a stove with a circulation of air at 30 °C and afterwards were milled in a Willey mill.

Extraction and isolation of the chemical constituents

The branches (680 g) were extracted consecutively with hexane and methanol at room temperature. After distillation of the solvents were obtained, respectively, 2.1 g and 6.4 g of solid residues. The residue obtained from the MeOH extraction (~5.5 g) was submitted to droplet countercurrent chromatography in descendent mode, using CHCl₃:MeOH:H₂O (5:5:3 v/v) as solvent. The organic phase was mobile and the aqueous phase was stationary. After 56 h of analysis, 259 fractions, 13 mL each, were collected and divided into 8 groups based on the results from analytical TLC. The fraction 3 (789 mg) was submitted to chromatography on a silica gel column, under pressure, using CHCl₃:MeOH (95:5 initial v/v) as eluent, with an eluting gradient till MeOH (100%). 20 fractions, 20 mL each, were obtained. The fractions 3 (420 mg) and 4 (120 mg) have supplied, respectively, the impure quassinoids 1a and 1b.

Acetylated derivations

The fractions 3 (420 mg) and 4 (120 mg), previously described, were acetylated with acetic anhydride/pyridine (2:1), as usual, during 12 h. The acetylated derivatives 2a (395 mg) and 2b (8 mg) were obtained and purified by re-crystallization in MeOH.

20(R)-2-O-acetylsimarolide (2a). m.p. > 280 °C. [a]_D = -14.0° (c 0.8, CHCl₃). ¹³C-NMR (100 MHz): Table 1 and ¹H-NMR (400 MHz): Table 2. MS m/z (relative intensity, %): M^+• 546 (1.4 %), 486 (37), 427 (7), 426 (20), 373 (21), 177 (41), 135 (37), 133 (69), 119 (100), 113 (70), 107 (98), 105 (93).

20(S)-2-O-acetylsimarolide (2b). m.p. > 280 °C. [a]_D = -19.2° (c 1.48, CHCl₃). ¹³C-NMR (100 MHz): Table 1 and ¹H-NMR (400 MHz): Table 2. MS, m/z (relative intensity, %): 546 (1 %), 486 (29), 427 (5), 426 (16), 373 (16), 177 (37), 135 (37), 133 (65), 119 (99), 113 (67), 107 (100), 105 (96).

Acknowledgment

The authors would like to thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for scholarships and the financial support as well as the Coordenação de Pessoal de Nível Superior (CAPES), Financiadora de Estudos e Projetos (FINEP), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Fundação Estadual do Norte Fluminense (FENORTE).

Received: March 20, 1998

FAPESP helped in meeting the publication costs of this article

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Publication Dates

• Publication in this collection
  07 Oct 2002
• Date of issue
  Jan 1999