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

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. 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: H-NMR, C-NMR-PND and C-NMRDEPT) and (2D: H x H-COSY, H x H-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 (δH) and 13-carbon (δC) atoms of both diacetyl derivat ives (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 (α 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.

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 (α 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 13 C-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 Csp  3 .Based on small chemical shift differences observed between 2a and 2b we suggested that they were diastereomers.]}, combined with the δC and δH and the multiplicity of the hydrogen atom signals observed in the 1 H-NMR spectra (1D and 2D 1 H x 1 H-COSY), have allowed the structure of simarolide (1a), a quassinoid with twenty five carbon atoms (C 25 ) isolated from Simarouba amara 1 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 1 H-NMR (Table 2) and  1 and 2).These differences were confirmed by the comparison (Figure 3 and 4) of the heteronuclear correlation spectra (HMQC) 4,5   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 δH observed for CH-20 (2a: δH 4.31 (m); 2b: δH 4.30), which is surprising for a methine hydrogen occupying the α position in relation to the carbonyl group, since the δC [2a: δC 41.50 (CH-20); 2b: δC 41.58 (CH-20)] revealed to be compatible with the parameters expected for δC in these structural conditions 3 .The unequivocal assignment of the δC 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 (δ C 206.60) with the 3H-19 [δ H 1.24 (Table 2), 3 J CH ] and C-17 (δ C 211.90) with the H-12ax [δ H 1.69 (Table 2), 3 J CH ] and the 3H-18 [δ H 0.98 (Table 2), 3 J 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 (δ C 206.60) and C-17 (δ 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 δC of C-1 (δ C 214.17) and C-17 (numbered as C-22 in reference 6: δC 200.55) of the quassinoid (3) isolated from Picrasma javanica 6 should be corrected to C-1 (δ C 200.55) and C-22 = C-17 (δ C 214.17), as observed in the quassinoid (4) isolated from Picrasma ailanthoides 7 , since a solvent (pyridine-d 5 ) effect should not be responsible for such a modification.The comparison of the 13 C-NMR chemical shift for 2a and 2b also permitted the correct assignment of the 2b quaternary carbon δC (Table 1).
With the unequivocal assignment of the δH for 2a and 2b through analysis of the HMQC spectrum (Tables 1 and  2), the homonuclear correlation 1 H x 1 H-COSY has allowed the recognition of all spin-spin interactions of the hydrogen atoms, especially CH-20, CH 2 -21 and CH 2 -22 (Fig. 1).The spectrum of 2a revealed, not only the vicinal interactions, but also the coupling of H-21a [δ H 4.59 (t)] in W or M (zig-zag) 5 with H-22 [δ H 2.75 (m)], which was not observed in the spectrum of 2b (Fig. 1).This comparative analysis allowed the suggestion that the γ-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 ( 4 J w-H,H ), not observed in 2b.The conformational behavior differences of the γ-lactone ring on both epimers (2a and 2b) can only be justified by a steric interference involving the C-ring and the CH 3 -18 and CH 3 -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: 100.49kcal/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); ∆Å = 4.406 -3.231 = 1.175Å) 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; ∆Å = 4.402 -2.663 = 1.739Å).These data were employed to show, as foreseen, that the epimerization of the CH-20 * The data obtained from HMBC spectra was also used in the assignment of these δC.The hydrogen atoms δH corresponding to the hydrogenated carbons (CH, CH2 and CH3) are described in Table 2.
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.
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/MS 8 .

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 ( 1 H: 400 MHz; 13 C: 100 MHz) was used to obtain the 1 H-NMR and 13 C-NMR spectra, using CDCl 3 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 [α] 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-  Table 3. Interatomic distances (Å) of H-20 and other neighbouring hydrogen, measured at the minimum energy conformation of 2a (102.95kcal/mol) and of 2b (100.49kcal/mol) using the molecular modeling software -PCMODEL.

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 CHCl 3 :MeOH:H 2 O (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 CHCl 3 :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.
[α] D = -14.0°(c 0.8, CHCl 3 ). 13C-NMR (100 MHz): Table 1 3 in C-C bonds], methines [(CH) 8 : three Csp 3 attached to oxygen and five Csp 3 involved in C-C bonds], methylenes [(CH 2 ) 6 : one Csp 3 attached to oxygen and five Csp 3 involved in C-C bonds] and methyls [(CH 3 ) 6 : four involved in C-C bonds and two attached to Csp 2 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, θ = 90°: only CH signals; θ = 135°: CH 2 signals in opposite phase to CH and CH 3 ; after the removal of these signals from the PND spectra, the quaternary carbon signals were obtained)
* The unequivocal assignment of the hydrogen atoms δH were based on the HMBC spectra of 2a and 2b.