Klodorone A and klodorol A: new triterpenes from Kleinia odora

Re-investigation of the EtOH extract of the aerial parts of Kleinia odora led to the isolation of two new triterpenes; klodorone A (3) and klodorol A (4), together with two known compounds: β-amyrin (1) and germanicol (2), which were reported from this plant for the first time. Their structures were determined by using extensive 1D (1H, 13C and DEPT) and 2D (1H–1H COSY, HMQC and HMBC) NMR and mass spectral measurements in addition to comparison of their data with the literature.


Introduction
The genus Kleinia (Asteraceae) comprises 40 species of which several species are known to be rich sources of oxygenated sesquiterpenoids and triterpenoids (Al Musayeib et al. 2013). Oleanane-and ursane-type triterpenoids had been extensively explored for their biological and pharmacological activities. These compounds have been found to exhibit a variety of properties. These activities include antitumour, anti-viral, anti-inflammatory, hepatoprotective, gastroprotective, antimicrobial, anti-diabetic and anti-protozoal activities (Sun et al. 2006;Dominguez-Carmona et al. 2010;Al Musayeib et al. 2013). Previous chemical investigation of Kleinia odora growing in Saudi Arabia resulted in the isolation and characterisation of lupeol, lupenone, epilupeol, lupeol acetate, ursolic acid, 3b-11-a-dihydroxy urs-12-ene, brein and ursolic acid lactone (Al Musayeib et al. 2013). Reviewing the current literature, there are minor reports concerning the chemical investigations for the constituents of K. odora growing in Saudi Arabia. Therefore, a phytochemical study of the plant was performed aiming to identify more of its constituents. This article reports the isolation and characterisation of two new triterpenes: klodorone A (3) and klodorol A (4), together with two known compounds: b-amyrin (1) (Mahato and Kundu 1994) and germanicol (2) (Gonzalez et al. 1981), which are reported for the first time from the aerial parts of K. odora (Figure 1).
Compound 4 was obtained as colourless needles. It gave a positive Liebermann-Burchard reaction, indicating its triterpenoidal nature (Reinhold 1935). The HR-EI-MS revealed a molecular ion peak at m/z 442.3815 [M] þ , which is consistent with the molecular formula C 30 H 50 O 2 , requiring six degrees of unsaturation. The IR spectrum indicated absorption bands at 3462 (OH) and 1070 (C-O) cm 21 . The 1 H and 13 C NMR spectra exhibited signals for 8 methyl groups at d H /d C 1.16 (H 3 -23)/28.4 (C-23), 1.18 (H 3 -24)/25.4 (C-24), 0.99 (H 3 -25)/16.6 (C-25), 0.95 (H 3 -26)/16.8 (C-26), 1.06 (H 3 -27)/15.6 (C-27), 1.16 (H 3 -28)/23.6 (C-28), 1.10 (H 3 -29)/27.4 (C-29) and 1.06 (H 3 -30)/21.4 (C-30), 11 methylenes, 3 methines including an oxymethine at d H 3.35 (H-3)/d C 84.8 (C-3) and 8 quaternary carbons, 2 of them are oxygen bearings at d C 72.8 (C-5) and 87.8 (C-10) (Supplementary Figures S7 and S8). The NMR data of 4 were similar to those of dendropanoxide (a hexacyclic triterpene oxide with a D:B-friedooleanane skeleton isolated from Dendropanax trifidus) except the presence of an additional oxygenated carbon at d C 72.8 (C-5) (Tori et al. 1988). Its position at C-5 was confirmed by the observed 3 J HMBC correlations of H-3, H 3 -23 and H 3 -24 to C-5 (d C 72.8) (Supplementary Figures S11 and S13). The location of the heterocycle in ring A between C-3 and C-10 was confirmed by means of the HMBC experiment which revealed the correlations H-2, H-8 and H-25 to C-10 and H-2, H 3 -23 and H 3 -24 to C-3. The correlations of H 3 -25 to C-8, C-9, C-10 and C-11 were consistent with the location of a methyl group at C-9. Furthermore, HMBC correlations from H 3 -27 to C-18 and C-13 and from H 3 -28 to C-17 and C-18 were observed. The relative configuration of 4 was determined by comparing the 1 H and 13 C NMR chemical shifts as well as coupling constant with those of related compounds in the literature (Tori et al. 1988;Estrada et al. 2002). On the basis of these findings and by comparing the NMR data of 4 with those of structurally related compounds (Tori et al. 1988;Estrada et al. 2002), 4 was identified as dendropanoxide with additional hydroxyl group at C-5 (5a-hydroxy dendropanoxide). To the best of our knowledge, this is a new compound isolated for the first time from K. odora for which we propose the name klodorol A.
The known compounds 1 and 2 were identified by analysing their spectroscopic data (1D, 2D NMR and MS) and by comparing their data with those in the literature as: b-amyrin (Mahato and Kundu 1994) and germanicol (Gonzalez et al. 1981). Compounds 1 and 2 were isolated for the first time from the plant.

General experimental procedures
Optical rotations were measured on a Perkin-Elmer Model 341 LC polarimeter (Perkin-Elmer, Waltham, MA, USA). Melting points were determined using an Electrothermal 9100 Digital Melting Point apparatus (Electrothermal Engineering Ltd, Essex, England). The IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer (Kyoto, Japan). HR-EI-MS was measured on JEOL JMS-HX 110 mass spectrometer (Joel, Peabody, MA, USA). EI-MS was recorded on JEOL JMS-SX/SX 102A mass spectrometer (Joel, Peabody, MA, USA). The 1D and 2D NMR spectra were recorded on a Bruker AMX-400 spectrometer (Bruker, Rheinstetten, Germany) with tetramethylsilane as an internal standard. NMR spectra were referenced to the solvent signals (CDCl 3 : 7.26 ppm for 1 H and 77.0 ppm for 13 C). Solvents were distilled prior to use, and spectral grade solvents were used for spectroscopic measurements. Column chromatographic separations were performed on silica gel 60 (0.04-0.063 mm, Merck, Darmstadt, Germany) and RP-18 (0.04-0.063 mm, Merck). Thin-layer chromatography (TLC) was performed on pre-coated TLC plates with silica gel 60 F254 (0.2 mm, Merck). The solvent systems used for TLC analyses were n-hexane-EtOAc (95:5, S1) and n-hexane-EtOAc (90:10, S2). The compounds were detected by UV absorption at l max 255 and 366 nm followed by spraying with anisaldehyde/H 2 SO 4 reagent and heating at 1108C for 1-2 min. All the chemicals were purchased from Sigma Chemical Company (St Louis, MO, USA).

Plant materials
The plant, K. odora, was collected from the south of Saudi Arabia in February 2011. The plant was kindly identified by taxonomist at the Pharmacognosy Department, College of Pharmacy, King Saud University. A voucher specimen has been deposited at the Pharmacognosy Department, College of Pharmacy, King Saud University under the registration number P-15129.