New Triterpene and Antibacterial Labdenoic Acid Derivatives from Moldenhawera nutans

Um novo triterpeno (22β-hidroxilupeol) foi isolado do extrato em metanol do caule de Moldenhawera nutans (Leguminosae) além de diterpenos derivados do ácido labdenóico de ocorrência comum nesta espécie. A partir do ácido labd-8(17)-en-15-óico foram preparados derivados com atividade in vitro discreta frente à Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella cholerasuis e Vibrio parahaemolyticus.


Introduction
Genus Moldenhawera Schrard.(Leguminosae: Caesalpinoideae), endemic to Northeast Brazil, is represented by approximately ten species. 1 Previous phytochemical study of M. nutans resulted in the isolation of four known labdene diterpenes besides a new bisditerpene named moldenin. 2The present work describes the results of the fractionation of the hexane phase obtained from the MeOH extract of M. nutans.Besides the known diterpenes previously isolated, it was also obtained the 3oxo-labd-8(17)-en-15-oic acid (1) as a methyl derivative (1a), and three triterpenes, lupeol, betulin, and the new lupane derivative (2).The labd-8(17)-en-15-oic acid (3)  was the predominant compound in this extract.From this compound, derivatives (4-8) were prepared and some of them were submitted to in vitro antibacterial assays.

Results and Discussion
The structural elucidation of 1a (Figure 1) was based on MS, IR, and NMR data analyses.Comparison of NMR data of the methyl ester derivative with moldenin, 2 methyl ent-3-oxo-labd-8(17)-en-15-oate 3 and literature data 4 allowed establishing the labdanic structure.The normal series of this compound was confirmed by positive optical rotation.
The HREIMS of 2 exhibited a molecular ion signal at m/z 442.3829, indicating the molecular formula C 30 H 50 O 2 (requires 442.3811).The 1 H NMR data (Experimental section) showed characteristic signals of lupane triterpene, seven methyl groups, one isopropenyl (δ 4.61, 4.71, and 1.70) and two signals of oxymethine hydrogens (δ 3.68 and 3.20).The 13 C NMR spectra (BB and DEPT 135 o ) displayed 30 signals and confirmed the data above through the resonances displayed at δ 19.3, 109.8,150.3, as well as at δ 78.0 and 79.7 for the isopropenyl and two oxymethine groups, respectively.The presence of an additional oxymethine signal indicated that 2 is a hydroxylated lupeol derivative.The localization of the hydroxyl group at C-22 of the lupane framework was proposed by comparison with 13 C NMR data (Table 1) of 16-hydroxylupeol 5 and the correlations observed in the long range HETCOR of H-22 (δ 3.68) and C-28 (δ 12.2), C-19 (δ 45.9) and C-18 (δ 45.1) as well as by the carbon shifts observed in the diacetyl derivative (2a) (Table 1).Thus, the NMR spectra of 2a showed a shielding effect at C-21 (Δδ = -3.5 ppm), which is indicative that C-22 bears a hydroxyl group.The insignificant effect observed at C-19 in the NMR spectra of 2a together with the coupling constants observed in the 1 H NMR suggested that the hydroxyl group is in equatorial position.The 1 HNMR nOe difference spectra of this compound permitted to corroborate the proposition.When the H-22 was irradiated it was possible to assign increments in Hα-21 (25%), H-29 (3%) and, H-19 (10%).These findings are indicative that all the affected protons were in the same plane.The fragmentation pattern observed in the MS of 2 was also indicative of a hydroxyl group in C-22 of the cyclopentyl ring, especially by mass fragments of 2 at m/z 374 and 2a at m/z 458 (Figure 2).
Since labd-8(17)-en-15-oic acid (3) showed antibacterial activity (Table 2), some of its simple chemical derivatives were prepared and also evaluated.Derivative 3 was refluxed with MeOH/HCl to obtain the isomeric methyl esters 4a and 5. Next, 4a was submitted to allylic oxidation by t-butyl chromate 6 and furnished 6a.It was also prepared the epoxy derivatives 7a and 8a from 4a by reaction with MCPBA.The derivatives 7a and 8a are new and they were characterized by spectrometric analyses data.Compounds 1a, 2, 4a, and 6a-8a were submitted to antibacterial evaluation and none of them was active; while the acid derivatives (1, 4, 6-8) obtained after saponification, together with 9, a labdane previously obtained from this plant, showed moderate antibacterial activity at 90 μg per disc (Table 2).These results indicate the need of the presence of an acid group for the antibacterial activity and also that structural features contribute to the inhibition area as well.

General procedures
The 1 H and 13 C NMR, DEPT, COSY, and HETEROCOSY (J 140 and 9 Hz) spectra were obtained  on a Varian Gemini 2000 instrument employing CDCl 3 as both solvent and reference.The FTIR spectrum was recorded on a JASCO spectrophotometer Mod.Valor III.MS was recorded on a Micromass Autospec spectrometer (HRMS) and an HP model 5973 spectrometer (EIMS).Melting points were measured on a Microquímica MIAPF 301 apparatus and are uncorrected.Column chromatography was carried out on silica gel 60 and, silica gel TLC was used to monitor the fraction employing iodine fumes, Liberman-Buchard spray reagent, and UV light (254/366 nm).

Plant material
The plant material of M. nutans were collected at sandy soil of Reserva do Parque da Lagoa do Abaeté, Salvador, BA, Brazil in the spring of 1997 and identified by Prof. Maria L. S. Guedes of Herbarium Alexandre Leal Costa, where a voucher (#029057) is deposited.

Preparation of derivatives Acetylation of 22β-hydroxylupeol (2)
Compound 2 (15.0 mg) was added to a solution of pyridine (0.5 mL) and acetic anhydride (2.0 mL) and the mixture was left at room temperature for 24 h.Cold H 2 O was added and the diacetyl derivative (2a, 14.3 mg) was extracted with CHCl 3 .

Figure 1 .
Figure 1.Isolated compounds from Moldenhawera nutans, their derivatives and mass fragments of 2 and 2a.

Table 2 .
Diameter of inhibition zones of compounds 1, 3, 4, 6-9 from M. nutans in agar diffusion test in mg per disk