www.mdpi.org/molecules Relative Stereochemistry of a Diterpene from Salvia cinnabarina

The relative stereochemistry of 3,4-secoisopimara-4(18),7,15-triene-3-oic acid, a diterpenoid with antispasmodic, hypotensive and antibacterial activities isolated from Salvia cinnabarina, was determined by an X-ray diffraction analysis of a single crystal of a suitable crystalline derivative.


Introduction
The genus Salvia (family Lamiaceae) includes over 900 species growing in the temperate and tropical zones of the world; and various taxa of this genus are commonly used in traditional medicine [1].Interesting bioactive compounds isolated from Salvia species are flavonoids, essential oils, diterpenes, and triterpenes with antifeedant, antibacterial, antifungal [2,3], hallucinogenic [3] and antioxidant activities [4].
In previous papers we have described the antispasmodic, hypotensive and antibacterial activities of a new secoisopimarane diterpenoid isolated from Salvia cinnabarina M. Martens and Galeotti, whose relative stereochemistry could be only partially determined on the basis of NMR spectroscopic techniques [5][6][7][8][9].We now report the complete relative stereochemistry of this compound, determined by an X-ray diffraction analysis of a single crystal of a suitable crystalline derivative.

Results and Discussion
In a previous paper, and on the basis of NMR spectroscopic techniques, we were able to report only the relative configuration at C-9 and C-13 of 3,4-secoisopimara-4(18),7,15-triene-3-oic acid (1), as the compound did not give crystals suitable for X-ray diffraction analysis.In order to confirm these partial results and to obtain the complete relative stereochemistry of 1 we have now prepared derivatives of the carboxylate group since this group was the most suitable for synthesis of derivatives without changes in the configuration of the rings (Scheme 1).

Scheme 1. Chemical structures of compounds 1 -4.
We prepared the methyl ester 2 (an oil) from the sodium salt of 1 and methyl iodide and then the hydrazide 3 by treatment of 2 with hydrazine hydrate.Since compound 3 did not give suitable crystals either, we prepared the acetone derivative 4, which finally provided crystals suitable for X-ray diffraction analysis.Since compounds 1, 2, 3 and 4 showed in the 13 C-NMR practically identical signals for the 19 carbons of the diterpenoid moiety and only the substituted C-3 showed different δvalues (Table 1), it may be inferred that the same relative stereochemistry is maintained in all compounds.In the crystal of 4 the asymmetric unit is formed by two independent molecules.A perspective view of one molecule is shown in Figure 1, together with the atomic labelling scheme.The two molecules are related by a non-crystallographic pseudo-twofold axis and are stabilized by means of two intermolecular hydrogen bonds: N1•••O1'= 2.894 (3) Å, and N1'•••O1= 2.953 (4) Å (Figure 2).They present geometric and conformational similarities, the only difference being observed for the C16 atom of the ethylenic group, which is rotated about 70° around the C13-C15 bond in one structure compared with the other.Upon best-fit superposition, the r.m.s.deviation of corresponding atoms, excluding the side substituents, is only 0.044 Å.Bond lengths and angles are within the expected ranges and generally agree well with the values reported in the literature for correlated molecules [16][17][18][19].In the absence of atoms with strong anomalous scattering, the absolute configuration was not determined and the configuration shown was chosen arbitrarily.On this basis, the relative configurations at the chiral centers are fixed as C5S*, C9S*, C10S* and C13S*.
Thus, taking into account the X-ray structure of derivative 4, in the original natural compound 1 H-5 and H-9 are axial and on the opposite side of the 20 axial methyl group; moreover, the vinyl group at C-13 is equatorial and the C-17 axial methyl group is on the same side of the C-20 axial methyl group.These results are in agreement with the partial stereochemistry previously determined with NMR spectroscopic techniques and with the signal of the H-5 at δ 2.18 in the 1 H-NMR spectrum of 1, which showed one axial/axial (J 5ax/6ax = 12.0 Hz) and one axial/equatorial (J 5ax/6eq = 3.0 Hz) coupling, typical of an axial configuration [5].

General
Silica gel 60 (Merck 230-400 mesh) was used for column chromatography; aluminium sheets of silica gel 60 F 254 (Merck 0.2 mm thick) with CHCl 3 /MeOH (12:05) as eluent were used and the spots were detected by spraying 5% H 2 SO 4 , followed by heating.IR spectra were recorded on a Perkin-Elmer 1310 spectrophotometer.NMR spectra were recorded in CDCl 3 on a BRUKER DRX 600 spectrometer (operating at 600 MHz for 1 H and 150 MHz for 13 C) using TMS as internal standard.The optical rotation was recorded on a Perkin-Elmer 241MC polarimeter.Melting points are uncorrected and were measured on a Tottoli melting point apparatus (Büchi).

X-ray structure determination of 4
A selected crystal was mounted on the glass fiber and the diffraction intensity data were collected at room temperature by Nonius KappaCCD diffractometer with graphite monochromatized Mo-Kα radiation (λ = 0.71073 Å).Accurate cell parameters were obtained by least-squares refined of the setting angles of 421 reflections at medium θ using DIRAX software [10].Data collection were carried out with φ and ω scans, using COLLECT software [11].Data reduction and absorption correction were performed using SADABS [12].Structure solution was solved using direct method (SIR97) [13] and refinement was performed using SHELXL97 software package [14].ORTEP-3 software was employed for molecular graphics [15].All H atoms were found in difference Fourier maps and were included in the final refinement assuming idealized geometry, with C-H distances = 0.98, 0.97, 0.96 and 0.93 Å for tertiary CH, secondary CH 2 , methyl CH 3 and sp 2 CH atoms, respectively, and with amide N-H distance of 0.86 Å.They were refining with U iso values equal to 1.2U eq parent atoms.
All the crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (Accession No. CCDC 650071).Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).

a
Reference [5]; b this C showed an HMBC correlation with the NH proton at δ 8.53; c this C showed HMBC correlations with the NH proton and the Me protons of the acetone moiety at δ 1.95 and 1.79.

Figure 2 .
Figure 2. A perspective view of the two independent molecules of 4. Dotted lines indicate the two intermolecular hydrogen bonds.