Terpenoids and Steroids from Trichilia Species

O trabalho descreve o isolamento de três novos sesquiterpenóides do extrato metanólico dos galhos de Trichilia claussenii: 14-hidroxielemol, germacra-10(14)-en-9,11,15-triol e germacra3,10(14)-dien-9,11-diol-4-carbaldeído. Também foram isolados os sesquiterpenos β-eudesmol, criptomeridiol e o triterpeno 22,25-diidroxi-9β,19-ciclolanost-23-en-3-ona. Do extrato diclorometânico das folhas de T. lepidota foram isolados mistura de hidrocarbonetos (C 29 H 60 , C 31 H 64 e C 33 H 68 ), mistura dos sesquiterpenos epóxido de cariofileno e epóxido de humuleno, espatulenol e os esteróides 24-metileno-12β-hidroxicolesterol, 24-metil-12β-hidroxicolest-4-en-3ona, 3-palmitato de 24-metil-12β-hidroxicolest-5-eno, mistura de 24-metilenocolesterol, campesterol, estigmasterol e β-sitosterol, além de α-tocoferol e fitol. O extrato metanólico forneceu a N-metil-4hidroxiprolina. As estruturas das substâncias isoladas foram estabelecidas com base em dados espectroscópicos.


Introduction
The Meliaceae family has shown to be of much interest among phytochemists because either it contains plants which produce very complex chemical structures, or because of their biological activity, mainly against insects.As part of our interest in the chemistry of this family, we have already reported the isolation of γ-lactones from the fruits, 1 androstane and pregnane steroids from the steams 2 and cycloartane triterpenoids and a mixture of ω-phenyl alkanoic and alkenoic acids from the leaves 3 of Trichilia claussenii collected in Brazil.In continuation of our study of Trichilia species, we now report the isolation of five sesquiterpenoids and a cycloartane triterpenoid from the steams of T. claussenii and steroids from the leaves of T. lepidota Martius ssp schumanniana Harms, which has never been investigated before.

Experimental
General experimental procedures 1 H and 13 C NMR spectra were recorded at 400. 13 and 100.62 MHz, respectively, in a Bruker DRX spectrometer.
The spectra were run in CDCl 3 with TMS as internal standard, pyridine-d 5 and D 2 O. DCCC was performed in an Eyela Droplet Counter Current Chromatograph D.C.C. -300.The GC-MS analyses were carried out with a Hewlett Packard System 5995 fitted with a dimethylpolysiloxane HP-1 (25 m x 0.20 mm i.d.x 0.30 film tickness) capillary column, with hydrogen as the carrier gas.The temperature was programmed initially at 120 o C for 2 min., then increased with a rate of 8 o C min -1 to 280 o C and kept for 20 min.The injection volume was 1 µL in a split mode and temperature of the injector was 280 o C. (Ac 2 O-pyridine) and the mixture obtained was purified on silica gel column (CH 2 Cl 2 :MeOH 19:1 v/v) to yield 4a (4.7 mg), 4b (3.4 mg) and 4c (1.9 mg).
The data reported for β-eudesmol 4 and cryptomeridiol 5,6 confirmed the structures for 1 and 2. Other possible isomers for 2 were ruled out through comparison with authentic compounds reported in the literature. 6,7ompound 3 was established from 1 H and 13 C NMR spectral data.The IR spectrum suggested the presence of hydroxyl groups (3401 cm -1 ).The 13 C NMR spectrum showed 15 carbon signals.The multiplicities of the carbons In the MS the parent ion observed, m/z 220, does not represent the molecular ion which was expected to be m/z 238, due to the loss of one molecule of water.The PENDANT spectrum showed four olefinic carbons at δ 148.0 (d), 147.7 (s), 113.2 (t) and 112.5 (d).The remaining unsaturations suggested that 3 was a monocyclic sesquiterpenoid.The proton signals at δ 1.39 (s) and 1.40 (s) in the 1 H NMR spectrum and at δ 27.4 (q), 27.8 (q) and 71.6 (s) in the PENDANT spectrum suggested the presence of a 1-hydroxy-1-methylethyl group, which was confirmed in the mass spectrum by the fragment m/z 59.In the 1 H-1 H COSY experiment of 3, H-1 (δ 6.10, dd) was coupled to two H-2 (δ 5.20, dd and 5.36, dd).Both H-2 were coupled to each other.These olefinic signals suggested a vinyl group connected to a quaternary carbon.The olefinic protons H-3 (δ 4.95 and 4.79) were not coupled to other protons and the signals for C-3 (δ 112.5, t) and C-4 (δ 147.7, s) suggested an exomethylene group, where a methyl group H-15 (δ 1.78, s) is attached to C-4.These data together with consideration of isoprene rule led us to propose a sesquiterpene elemane derivative for 3.The 1 H NMR spectrum of 3 also showed an AX coupling system (δ 4.29, 4.03, both d, J 10.8 Hz) and the 13 C NMR showed a carbinolic carbon at δ 61.5 (t), which led us to suggest that methyl group (C-14) was replaced by a hydroxymethylene group.Other carbon data are described in Table 1.The attribution of the carbon signals was based on related elemanes from the literature. 8,9Acetylation of 3 with acetic anhydride in pyridine provided the monoacetate of the primary alcohol (3a).The relative configurations of 3 were proposed on basis on the biogenesis of the elemane derivatives. 10ompound 4 was also found to be a monocyclic sesquiterpenoid.The IR spectrum suggested the presence of hydroxyl groups (3396 cm -1 ).The 13  s, H-14a) and 5.27 (br.s, H-14b).The 1 H-1 H COSY spectrum showed that both carbinolic protons at δ 3.77 (dd, H-15a) and 3.66 (dd, H-15b) were coupled to each other and coupled to another proton at δ 2.25 (m, H-4).The HMBC experiment showed the correlation of the carbinolic proton at δ 4.59 (br.t, H-9) with the olefinic carbons C-10 and C-14, therefore H-9 should be carbinolic and allylic.The 1 H-1 H COSY experiment showed that H-9 was coupled with two protons at δ 2.36 (dt, H-8a) and 1.97 (dt, H-8b), which are both coupled to each other and coupled to another proton at δ 2.22 (m, H-7).The HMBC experiment showed that H-9 was also correlated with C-7 (δ 41.8) and C-1 (32.9).The presence of a 1-hydroxy-1-methylethyl group was also characterized by the correlation of the methyl proton signals at δ 1.30 (s, H-12) and 1.40 (s, H-13) with the quaternary carbinolic carbon at δ 72.2 (C-11) in the HMBC experiment and the fragment m/z 59 in the mass spectrum.The possible bisabolane structure for compound 4 was ruled out by these correlations in addition to the correlations of C-11 with H-7 and C-7 with methyl protons (H-12 and H-13), which confirmed a germacrane structure for the sesquiterpenoid 4. The chemical shifts for C-12 and C-13 are very close for compounds 1 (∆δ 0.1), 2 (∆δ 0.6) and 3 (∆δ 0.4), and more distinct for 4 (∆δ 5.1).This difference may be due to the hydrogen bond between the hydroxyl groups at C-9 and C-11, which might fix a preferential conformation.The chemical shifts observed for C-12 and C-13 for acetates 4a (∆δ 1.5), 4b (∆δ 2.1), and 4c (∆δ 1.3) suggested that the hydrogen bond is stronger when hydroxy group at C-9 is not esterified.
The IR spectrum of 5 suggested the presence of hydroxyl (3417 cm -1 ) and α, β-unsaturated carbonyl (1684 cm -1 ) groups.The 1 H NMR spectrum showed two methyl groups at δ 1.14 (s, H-12) and 1.08 (s, H-13), one carbinolic and allylic proton at δ 4.26 (t, H-9) and three olefinic protons at δ 6.47 (dd, H-3), 5.12 (br.s, H-14), and 5.06 (br.s, H-14).A signal at δ 9.38 (d, H-15) suggested the presence of an aldehyde group.The 13 C NMR spectrum of 5 showed 15 carbon signals.The multiplicities of the carbons determined by DEPT led to the attribution of: 3 C, 4 CH, 6 CH 2 and 2 CH 3 .Four olefinic carbons were observed at δ 150.0 (d, C-3), 142.0 (s, C-4), 148.9 (s, C-10), and 115.5 (t, C-14), indicating the presence of two double bonds, including one exomethylene group.The signal at δ 195.2 (d, C-15) confirmed the presence of an aldehyde group.Two carbinolic carbons were observed at δ 72.8 (d, C-9) and 73.0 (s, C-11).The molecular formula proposed for 5 was C 15 H 24 O 3 .In the MS the parent ion observed, m/z 234, does not represent the molecular ion which was expected to be m/z 252, due to the loss of one molecule of water.Compound 5 was also found to be a germacrane sesquiterpenoid and the spectral data suggested the presence of one 1-hydroxy-1-methylethyl group, similar to the other sesquiterpenoids described before.The chemical shifts for the olefinic proton (δ 6.47) and olefinic C NMR showed 15 carbon signals.The multiplicities of the carbons determined by PENDANT led to the attribution of 2 C, 3 CH, 8 CH 2 and 2 CH 3 , of which 3 were carbinolic carbons: δ 72.8 (d, C-9), 72.2 (s, C-11) and 67.4 (t, C-15).The carbon signals at δ 153.2 (s, C-10) and 111.2 (t, C-14) suggested the presence of an exomethylene group.The molecular formula proposed for 4 was C 15 H 28 O 3 .The 1 H NMR spectrum showed the olefinic proton signals at δ 5.66 (br. Isolation of the constituents from T. claussenii Stems (2.4 kg) of T. claussenii were collected in Rio Claro, SP, Brazil, and a voucher is deposited in the Herbarium of Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP, Brazil.The stems were dried, powdered and extracted with MeOH.The MeOH extract (45.7 g) was suspended in MeOH:H 2 O (1:3 v/v) and partitioned with CH 2 Cl 2 , EtOAc and n-BuOH.The CH 2 Cl 2 fraction was concentrated and then partitioned with hexane and MeOH.The MeOH fraction (10.35 g) afforded 9 fractions after dry silica gel column using as eluent hexane:CH 2 Cl 2 :MeOH (10:10:1 v/v/v).

Table 1 .
13C NMR data for sesquiterpenes and their acetates (δ, 100 c Signals may be interchangeable.

Table 2 .
HMBC correlations for 4 and 5 by PENDANT led to the attribution of 3 C, 3 CH, 6 CH 2 and 3 CH 3 .The signals at δ 71.6 (s) and δ 61.5 (d) were attributed to carbinolic carbons, allowing us to propose the molecular formula C 15 H 26 O 2 for compound 3. determined