Megastimanes and Ergostane Type Steroid from Leaves Cratylia mollis (Leguminosae)

Do extrato metanólico das folhas de Cratylia mollis, foram isolados através de técnicas cromatográficas, (3S,5S,6S,9R)-3,6-di-hidróxi-5,6-diidro-β-ionol (1) e um novo nor-isoprenóide identificado como (4S*, 6S*)-4-but-1E-enil-4,6-diidróxi-3,5,5-trimetil-ciclo-hex-2-enona (2) além do 5α,8α-epidioxiergosta-6,22-dien-3-β-ol. As estruturas foram elucidadas por meio da análise dos dados de EM, IV, RMN H e C.


Introduction
Cratylia is one of 670 genera belonging to Leguminosae family, 1 it is included in the Phaseolae tribe (subtribe Diocleinae) 2 and this position is maintained until the most recent classifications. 3 This genus comprises only five species, C. argentea (Desvaux) O. Kuntze, C. bahiensis L. P. de Queiroz, C. hypargyrea Martius ex Benth., C. intermedia (Hassler) L. P. de Queiroz and C. mollis Martius ex Benth. From chemical point of view the subtribe genera can be characterized by the presence of the non-proteic aminoacids, especially canavanine. 4 However, there are no data of the occurrence of this aminoacid in Cratylia species. On the other hand, other chemical characteristic of Cratylia is the presence of lectins in their seeds which shows great similarity with the lectins isolated from seeds of other species of same tribe. 5 Cratilya mollis is a legume shrub native to the Northeast semi-arid region of Brazil, especially in "caatinga". This species is popularly known as "camaratuba" or "camaratu" and is highly resistant to desiccation. The leaves have been an alternative source of nutrition for cattle, being recommended to be employed by locals as forage to improve cattle's nutrition, especially during the dry seasons, contributing to regional development of the semi-arid. 6 However, in spite of studies the about of the biological activities of this and related species 7 to date there are no phytochemical studies regarding C. mollis.

Results and Discussion
The C l3 -norisoprenoid (1) is known as 3,6-dihydroxy-5,6-dihydro-b-ionol. It was identified by analysis of ESIMS, IR, optical rotation, 1 H and 13 C NMR and comparison with data previously published in the literature. 8 Moreover, HMQC, HMBC and COSY spectra permitted to attribute unequivocally all the NMR signals. The 13 C NMR data of 1 are compatible with that previously described for (3S,5R,6S,9R)-3,6-dihydroxy-5,6-dihydro-b-ionol obtained from Apollonias barbujana (Lauraceae) 8 except for C-1. However, the HMQC and HMBC of 1 permitted to attribute the peak at d 16.4 to C-13 by the observed correlations of one doublet at d 0.79 and this carbon in the HMQC and C-4 (d 39.9) and C-6 (d 78.1) observed in the HMBC experiment. This findings permitted to determinate an axial position of CH 3 -13 instead of equatorial as previously proposed and, concluded compound 1 is (3S,5S,6S,9R)-3,6-dihydroxy-5,6dihydro-b-ionol. Thus, it is a must a revision of 13 C NMR data and structure of the megastimane isolated from A. barbujana.
The 13 C NMR spectra (including DEPT experiments) of 2 showed signals were assigned to four methyls, four methines, one methylene and four non-hydrogenated carbons. These findings besides the protoned molecular ion [M-H] + observed at m/z 223.1330 in the negative HRESIMS permitted to propose molecular formula C 13 H 20 O 3 (requires 223.1334) which was indicative of a C 13 -norisoprenoid derivative. The signals at d 198.5, 126.7 and 163.4 observed in the 13 C NMR spectra, and the singlet at d 5.95 in the 1 H NMR spectra were indicative of presence of a a,b-unsaturated ketone group. These findings together with the additional chemical shifts indicated the structural similarity of 2 and the known megastigmane glycosides 4 and 5. 9 The HMQC spectrum was elucidative once it permitted to identify the correlations of closer hydrogen signals with respective carbon resonances. However, the analysis of HMBC spectral data was conclusive to confirm the structure of compound 2. Briefly, the correlations of H-4 (d 5.95), H-7 (d 5.79) and the C-6 (d 78.9) and, H-10 (d 1.22) and C-9 (d 49.6) and C-8 (d 135.6) were indicative the E-butenyl group is attached at C-6 ( Table 1).   The relative configuration of compound 2 was determinate by phase-sensitive NOESY, once the spatial interactions of H-7 and H-2 were indicative the butenyl group and H-2 were in same face (Figure 2). The proposed relative stereochemistry was also confirmed by analysis of coupling constant of derivative 2a, obtained as main product of reduction of 2 by NaBH 4 /MeOH. The 1 H NMR spectrum of 2a showed the peak of H-3 as a double doublet at d 3.4 (J 12.9 6.5 Hz) revealing pseudodiaxial coupling of H-3 and H-2, which allowed to confirm the relative configuration of compound 2.
The APCI-MS, 1 H NMR and 13 C NMR spectra of compound 3 and comparison with data previously describe in literature 10 permitted to identify this steroid. However correlations observed in COSY, HMQC and HMBC experiments indicated the values of 13 C NMR data previously established for C-6 and C-7 must be changed. This is the first occurrence of the megastimane 4-but-1-enyl-4,6-dihydroxy-3,5,5-trimethyl-cyclohex-2-enone (2). Compound 1 was previously isolated from Apollonias barbujana (Lauraceae) 8 but this is the first time it is being reported in Leguminosae family. Compound 3 was previously isolated from fungus Lactarium volemus, Schinopsis brasiliensis and Typha latifolia. 10 However the detailed analysis of correlations observed in HMQC and HMBC experiments permitted to attribute unequivocally the C-13 NMR data for this steroid.

Experimental
General procedures 1 H (300 MHz); 13 C NMR and DEPT (75 MHz) experiments were carried out in a Varian mod. Gemini 2000. HMQC, NOESY and HMBC were run on a Varian INOVA 500: chemical shifts were recorded in d (ppm) from the solvent peak relative to TMS; APCI and ESIMS were obtained on Shimadzu LCMS-2010; HRESIMS was recorded on Bruker micrOTOF II, IR spectra were taken on a Varian mod. 640-IR spectrophotometer and optical rotations were measured with a Perkin Elmer polarimeter mod. 341.

Plant material
Botanical material of C. mollis was collected at Jacobina, Bahia State, a region where "caatinga" vegetation is prevalent. A voucher is deposited at Herbarium of Universidade Estadual de Feira de Santana under number LP5119.