Soulamarin, a New Coumarin from Stem Bark of Calophyllum soulattri

The extracts of the stem bark of Calophyllum soulattri gave a new pyranocoumarin, soulamarin (1), together with five other xanthones caloxanthone B (2), caloxanthone C (3), macluraxanthone (4), trapezifolixanthone (5) and brasixanthone B (6) one common triterpene, friedelin (7), and the steroidal triterpene stigmasterol (8). The structures of these compounds were established based on spectral evidence (1D and 2D NMR).


Introduction
The genus Calophyllum comprises over 200 species of trees and shrubs native to tropical Asia, East Africa, India and Australia. Calophyllum species have been used in traditional Chinese folk medicine for the treatment of wounds, inflammation and rheumatism. Phytochemical studies on Calophyllum species have revealed the presence of xanthones [1][2][3], coumarins [4], triterpenoids [5] and flavonoids [6]. The coumarins have been reported to have displayed anti-HIV biological effects [7] and are used as cancer chemo-preventive agents [8]. Various xanthone derivatives show antifungal [9], antimicrobial [10] and molluscicidal [11] effects. We report here the isolation and characterization of a new pyranocoumarin, soulamarin (1), from the stem bark of Calophyllum soulattri.

Results and Discussion
Soulamarin (1) was isolated as yellowish oil from the hexane extract of the stem bark of Calophyllum soulattri. The HRESIMS displayed a negative molecular ion peak at m/z 387.1818 [M−H] − indicating a molecular formula C 22 H 28 O 6 . Compound 1 gave typical coumarin IR absorptions at 3,296 (OH), 2,930 (sp 2 and sp 3 CH), 1,706 (C=O) and 1,621 (C=C) cm −1 . Maximum absorptions were observed at 314, 300, 274 and 268 nm in the UV spectrum [12].
The 1 H-NMR spectrum revealed the presence of one chelated hydroxyl proton signal at δ 12.46 (s, 1H), two vinylic proton signals at The structure was further elucidated by HMBC spectral analysis after the assignment of the protons to their direct bonding carbons by the HMQC spectrum. The low field chelated proton at δ 12.46 was due to hydrogen-bonding with the carbonyl group (δ 199.4, C=O) which resulted in the deshielding effect. The 3 J and 2 J connectivity of the chelated hydroxyl proton at δ 12.46 with δ 102.1 (C-5), 102.9 (C-7) and 157.0 (C-6) confirmed its location at C-6 (see Figure 1). The HMBC experiment also demonstrated long-range 2 J and 3 J correlations between the two doublet protons at δ 5.46 (H-10, J = 10.1 Hz) and 6.60 (H-9, J = 10.1 Hz) with the carbon signal at δ 78.2 (C-11) respectively. The linkages between the two aliphatic methyls at δ 1.42 (H-12) and δ 1.44 (H-13) to the two carbon signals at δ 78.2 (C-11) and 125.8 (C-10) were also seen. These data together with the COSY spectrum analysis suggested the existence of a pyran ring. The pyran ring was clearly fused onto the non-oxygenated carbon C-7 and an oxygenated carbon C-8, as confirmed by the long range ( 3 J) correlations of H-9 (δ 6.60) with C-8 (δ 159.6) and H-10 (δ 5.46) with C-7 (δ 102.9). Meanwhile

Plant Material
The stem bark of Calophyllum soulattri was collected from the Sri Aman district in Sarawak, Malaysia. This plant was identified by Dr. Rusea Go from the Department of Biology, Faculty of Science, Universiti Putra Malaysia.

General
EIMS were recorded on a Shimadzu GC-MS model QP2010 Plus spectrometer. NMR spectra were obtained using a JEOL 500 MHz FT-NMR spectrometer using tetramethylsilane (TMS) as an internal standard. Ultraviolet spectra were recorded in EtOH on a Shimadzu UV-160A, UV-Visible Recording Spectrophotometer. Infrared spectra were measured using the universal attenuated total reflection (UATR) technique on a Perkin-Elmer 100 Series FT-IR spectrometer.

Extraction and Isolation
Approximately 1 kg of air-dried stem bark of Calophyllum soulattri was ground to a fine powder and extracted successively in a Soxhlet apparatus with n-hexane (68-72 °C, 3 × 2 L) and dichloromethane (40 °C, 3 × 2 L) for 24 hours. The extracts were evaporated to dryness under vacuum to give 101.2 g of n-hexane extract and 15.3 g of dichloromethane extract. Part of each extract was subjected to column chromatography over silica gel and eluted with a stepwise gradient system of nhexane, dichloromethane, ethyl acetate and methanol. Further purification of the n-hexane extract afforded the new coumarin soulamarin (1, 8 mg) and the triterpene friedelin (7, 450 mg). Caloxanthone B (2). Yellow needles; M.P. 157-158 °C (lit. 160.5 °C [13]); Spectral data were consistent with published data [13].