Dammarane-Type Triterpenoids from The Stembark of Aglaia argentea (Meliaceae)

Two dammarane-type triterpenoids, 20S,24S-epoxy-3α,25-dihydroxydammarane (1) and 3α-acetyl-20S,24Sepoxy-3α,25-dihydroxydammarane (2), have been isolated from the stembark of Aglaia argentea. The chemical structure of compounds (1 and 2) were identified by spectroscopic evidences including UV, IR, 1D-NMR, 2DNMR and MS as well as by comparing with previously reported spectral data. Those compounds were isolated from this plant for first time. Compounds (1 and 2) showed cytotoxic activity against P-388 murine leukemia cells with IC50 values of 23.96 and 8.14 M, respectively.

The genus Aglaia is the largest genus of the family of Meliaceae comprises more than 100 species distributed mainly in India, Indonesia, Malaysia, and parts of the Western Pacific region (Leong et al., 2016).Some species of Aglaia have been phytochemically investigated previously with major constituents of dammarane-type triterpenoids (Zhang et al., 2010;Harneti et al., 2012) and cycloartanetype triterpenoids (Awang et al., 2012;Leong et al., 2016) and glabretal-type triterpenoids (Su et al., 2006).In our continous search for novel secondary metabolites from Indonesian Aglaia plants, we isolated and described triterpenoids, aglinone and aglinin E, from the bark of A. smithii (Harneti et al., 2012), and protolimonoid from the stembark of A. argentea (Farabi et al., 2017).In the further screening for novel triterpenoid compounds from Indonesia Aglaia plants, we found that the n-hexane of A. argentea exhibited the presence of triterpenoids.We report herein the isolation, structural elucidation of dammaranetype triterpenoid compounds (1-2).

General Experimental Prosedure
Melting points were measured on an electrothermal melting point apparatus and are uncorrected.The IR spectra were recorded on a Perkin-Elmer spectrum-100 FT-IR in KBr.Mass spectra were obtained with a Synapt G2 mass spectrometer instrument.NMR data were recorded on a JEOL ECZ-600 spectrometer at 600 MHz for 1 H and 150 MHz for 13 C and JEOL JNM A-500 spectrometer at 500 MHz for 1 H and 150 MHz for 13 C, chemical shifts are given on a  (ppm) scale and tetramethylsilane (TMS) as an internal standard.Column chromatography was conducted on silica gel 60 (Kanto Chemical Co., Inc., Japan).TLC plates were precoated with silica gel GF 254 (Merck, 0.25 mm) and detection was achieved by spraying with 80% H 2 SO 4 in water, followed by heating.

Plant Material
The stembark of A. argentea were collected in Bogor Botanical Garden, Bogor, West Java Province, Indonesia in June 2015.The plant was identified by the staff of the Bogoriense Herbarium, Bogor, Indonesia and a voucher specimen (No.Bo-1288718) was deposited at the Herbarium.

RESULTS AND DISCUSSION
The methanolic extract from the dried stembark of A. argentea was concentrated and extracted successively with n-hexane, ethyl acetate, and n-butanol.The n-hexane exhibited the presence of triterpenoid compounds.By using triterpenoid test to guide separations, the n-hexane fraction was separated by combination of column chromatography on silica gel and preparative TLC on silica gel GF 254 to afford two dammarane-type triterpenoids (1-2).
In order to clarify the position of functional groups in compound (1), 1 H-1 H COSY and HMBC experiments were carried out and the results was shown in Figure 1.The 1 H-1 H COSY spectrum of 1 displayed the correlations in C 1 -C 2 -C 3 , C 5 -C 6 -C 7 , C 9 -C 11 -C 12 -C 13 , C 14 -C 15 -C 16 -C 17 , and C 22 -C 23 -C 24 , supporting the presence of dammaran-type triterpenoid structure in (1).In the HMBC spectrum, the correlations arising from the tertiary methyl protons to their neighboring carbons enabled the assignment of the eight singlet methyls at C-4 (2), C-8, C-10, C-14, C-20, C-26, and C-27, respectively.A methylene protons at  H 1.55 and methyl protons at  H 0.82 (CH 3 -29) were correlated to oxygenated carbon at  C 76.4 (C-3), indicated that a secondary hydroxyl group was attached at C-3. Methyl protons at  H 1.17 and 1.09, as well as an oxygenated methine at  H 3.62 were correlated to oxygenated carbon at  C 70.3 (C-25), indicated that a tertiary alcohol and an isopropyl group were attached at C-25 and C-24, respectively.A methine proton at  H 1.44 was correlated to C-20 ( C 86.7), whereas the methyl proton at  H 1.13 was correlated to C-20 ( C 86.7), C-17 ( C 49.8), and C-22 ( C 35.3), indicated that a tetrahydrofuran ring was attached at C-17.The presence of a tetrahydrofuran ring at C-17 was supported also by correlation between a methylene proton at  H 1.85 and C-24 ( C 86.3).Relative stereochemistry of compound (1) was determined on the basis of coupling constant ( 3 J) and chemical shift in the 1 H and 13 C-NMR spectra.A methine proton at C-3 has a 3 J 3.0 Hz, indicating that H-2 and H-3 has axial-equatorial orientation, consequently 3-OH has -orientation (Zhang et al., 2010;Farabi et al., 2017).A detail analysis of NMR spectra with side chain of 20,34-epoxy-25hydroxy, indicated that  C values can be used for determining of 24R and 24S isomer, where  C 83.2 for R isomer and 86.5 for S isomer.In addition, the chemical shift and coupling constant of H-2 can be used also for determining 24R and 24S isomer with chemical shift  H 3.7 (1H, t, J=7.0 Hz) and  H 3.6 (1H, dd, J=5.5, 10.0 Hz), respectively (Roux et al., 1998;Harneti et al., 2012;Harneti et al., 2014).Compound (1) showed the chemical shift for dd,J=4.8,10.2 Hz), as well as  C 86.7 for C-20, consequently configuration for C-20 and C-24 are S orientation.
A comparison of the NMR data of (1) with those of 20S,24R-epoxy-25hydroxydammarane isolated from A. foveolata (Roux et al., 1998) revealed that the structures of the two compounds are closely related, the main differences is the chemical shift of C-24 ( C 83.3), whereas compound (1) was  C 86.3, consequently compound (1) was identified as 20S,24S-epoxy-25-hydroxydammarane, which showed from this plant for the first time.
Compound (2) was isolated as a solid amorphous powder.The molecular formula of (2) was established to be C 32 H 54 O 4 based on of ESI-HRTOFMS spectra (m/z 502.4022 [M+H] + , calcd.for C 30 H 52 O 3 m/z 502.4022) together with NMR data (Tabel 1), thus requiring six degrees of unsaturation.The UV spectrum showed no conjugated double based on the absorption maximum above 200 nm.IR spectrum of (2) showed the presence of a hydroxyl group (3200 cm -1 ), an aliphatic bands (2949 cm -1 ), a gem-dimethyl (1457 and 1380 cm -1 ) and an ether group (1080 cm -1 ).
A NMR spectra of (2) was very similar to those of (1), the main differences are the absence one of the hydroxyl group and the presence of an acetyl group at [ C 171.1 (s), 21.5 (q) and  H 2.08 (3H, s)].In order to determine the position of newly acetyl group, HMBC experiment was carried, as the results was shown in Figure 1.In the HMBC spectrum, a methyl proton at  H 2.08 was correlated to carbonyl ester at  C 171.1, whereas the oxygenated methine at  H 4.61 was correlated also to carbonyl ester at  C 171.1, indicating that an acetyl group was attached at C-3.
A detailed comparison of the NMR spectra of (2) to those of 3α-acetil-20S,24Sepoxy-25-hydroxydammarane isolated from A. foveolata (Roux et al., 1998) revealed that the structures of the two compounds are very similar, consequently compound (2) was identified as 3α-asetil-20S,24S-epoxy-25hydroxydammarane, which showed from this plant for the first time.