Fitoterapia 91 (2013) 224–230 Contents lists available at ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote Terpenoids and their anti-HIV-1 activities from Excoecaria acerifolia Sheng-Zhuo Huang a,d, Xuan Zhang c, Qing-Yun Ma a,d, Yong-Tang Zheng c, Feng-Qing Xu b, Hua Peng b, Hao-Fu Dai a,d, Jun Zhou b,⁎, You-Xing Zhao a,d,⁎⁎ a Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, People's Republic of China b State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People's Republic of China c Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Science & Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, People's Republic of China d Hainan Key Laboratory for Research and Development of Natural Products from Li Folk Medicine, Haikou 571101, People's Republic of China a r t i c l e i n f o Article history: Received 6 August 2013 Accepted in revised form 10 September 2013 Available online 21 September 2013 Chemical compounds studied in this article: Caulophyllogenin (PubChem CID: 104361) Keywords: Excoecaria acerifolia Euphorbiaceae Anti-HIV Diterpenoid Triterpenoid a b s t r a c t Five new diterpenoids named excocarinols A–E (1–5) including three pimaranes, one cleistanthane, and one nor-beyerane, together with nine known compounds, were isolated from the EtOAc extract of the Chinese ethnodrug Gua-jing-ban (Excoecaria acerifolia Didr.). Their structures were elucidated by the analysis of spectroscopic data including 1D, 2D NMR and HR-MS. The anti-HIV-1 bioassay on the diterpenoids showed that excocarinol A (1) exhibited moderate anti-HIV-1 activity with EC50 5.58 μM and SI (Selection Index) over 112.71. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. 1. Introduction Some Excoecaria species (Euphorbiaceae), the well-known mangroves-living irritant latex [1], are widely used in Southeast Asia as a uterotonic by the Tai herbalist [2]. Excoecaria acerifolia Didr., one of the Euphorbiaceae species, widely distributed in the dry hot valleys in Yunnan and Sichuan provinces, is epibiotic [1]. This feature is unique among these ⁎ Corresponding author. Tel./fax: +86 898 66989095. ⁎⁎ Correspondence to: Y.-X. Zhao, Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, People's Republic of China. Tel./fax: + 86 898 66989095. E-mail addresses: junzhou3264@126.com (J. Zhou), zhaoyouxing@itbb.org.cn (Y.-X. Zhao). genera. Also E. acerifolia is used as an ethnodrug Gua-jing-ban by minority nationalities in Southwest China for antidote, antiphlogistic, laxative, antitussive, anti-malaria, and anti-virus purposes [3]. Previous studies focused on the common species of this genus such as Excoecaria agallocha and Excoecaria oppositifolia, and reported different types of compounds including phenols [4–6], triterpenoids [7–9], and diterpenoids [2,10,11]. Until now there were few diterpenoids and phenols that had been found in E. acerifolia and few anti-virus researches on this species [12,13]. To search for new bioactive compounds, our continuous investigation on chemical compositions of E. acerifolia was carried out. Five new diterpenoids named excocarinols A–E (1–5), including three pimaranes, one cleistanthane, and one nor-beyerane, were isolated together with nine known compounds agallochin J (6) [14], agallochin K (7) [14], 14α,18-dihydroxy-7,15-isopimaradiene (8) [15], 0367-326X/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.09.007 S.-Z. Huang et al. / Fitoterapia 91 (2013) 224–230 7β,12β-dihydroxypimara-8,15-dien-14-one (9) [16], ribenone (10) [17], 15-O-acetylspiraminol (11) [18], angustanoic acid B (12) [19], caulophyllogenin (13) [20], and rubusic acid (14) [21]. Additionally, the compounds 1, 3, 4, and 5 have been tested for anti-HIV-1 activities. Excocarinol A (1) exhibited moderate anti-HIV-1 activity at the levels of EC50 5.58 μM and SI over 112.71. Herein, the isolation process, the structural elucidation of the new diterpenoids excocarinols A–E (1–5), and the anti-HIV-1 activities of four new compounds were described. 2. Experimental 2.1. Generals Optical rotations were measured on a Jasco P-1020 polarimeter. UV spectra were obtained on a Shimadzu double-beam 210A spectrometer. IR spectra were obtained on a Tensor 27 spectrometer with KBr pellets. NMR spectra were recorded on a Bruker AV-400, a DRX-500, or AVANCE III-600 spectrometer with TMS as an internal standard. ESIMS and HRESIMS were recorded with a Bruker HCT/Esquire HPLC-Iron Trap spectrometer and an API QSTAR Pulsar 1 spectrometer. EIMS and HREIMS were recorded with a Waters Autospec Premier. Silica gel (200– 300 mesh, Qingdao Marine Chemical Inc., People's Republic of China), RP-18 (40–70 μm, Fuji Silysia Chemical Ltd., Japan) and Sephadex LH-20 (GE Healthcare, USA) were used for column chromatography (CC). Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18, 9.4 mm × 25 cm, column. Fractions were monitored by TLC and spots were visualized by heating after spraying with 5% H2SO4 in ethanol (b. p. 77–79 °C). 2.2. Plant The stems of E. acerifolia were collected in Dali, Yunnan Province, People's Republic of China, and identified by Prof. H. Peng, and Dr. Y. Niu (Kunming Institute of Botany, Chinese Academy of Sciences). Voucher specimen (HUANG0006) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, People's Republic of China. 2.3. Extraction and isolation The dried and powdered stems of E. acerifolia (19 kg) were extracted with 95% EtOH under reflux for three times (3 × 30 L). The extract (1.5 kg) was then concentrated and suspended in water followed by successive partition with petroleum ether (3 × 5 L) and EtOAc (3 × 5 L), respectively. The EtOAc extract (305 g) was separated by silica gel CC (ϕ 16 × 160 cm) using a gradient solvent CHCl3/MeOH (9:1, 7:1, 5:1, 3:1, v/v, each 10 L) to afford fractions A–C. Fraction B (101 g) was separated by silica gel CC (ϕ 16 × 160 cm) using a gradient solvent petroleum ether/EtOAc (10:1, 8:1, 5:1, 3:1, 1:2, v/v, each 4 L) to afford fractions B1–B7. Fractions B1–B7 were purified by Sephadex LH-20 CC (ϕ 3.6 × 160 cm, CHCl3/MeOH 1:1, v/v, 1500 mL) to give subfractions B1a–B7a, respectively. Subfraction B1a (3.4 g) was subjected to repeated RP-18 (ϕ 3.6 × 40 cm, MeOH/H2O 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 v/v, each 1 L) and semi-preparative HPLC (MeOH/H2O 45:55 or 50:50, v/v, 2.0 mL/min) to yield 3 225 (18.9 mg) and 9 (25.4 mg); subfraction B2a (2.1 g) was subjected to repeated RP-18 CC (ϕ 3.6 × 40 cm, MeOH/H2O 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 v/v, each 1 L) and Sephadex LH-20 (ϕ 1.4 × 50 cm, MeOH, 100 mL) to yield 5 (75.6 mg) and 6 (28.6 mg), and 7 (6.5 mg); subfraction B3a (2.6 g) was subjected to repeated RP-18 (ϕ 3.6 × 40 cm, MeOH/H2O 3:7, 4:6, 5:5, 6:4, 7:3, v/v, each 1 L) and silica gel CC (ϕ 2.5 × 30 cm, petroleum ether/EtOAc 3:1, v/v, 400 mL) to yield 4 (5.3 mg) and 8 (11.5 mg); subfraction B4a (4.7 g) was subjected to repeated RP-18 (ϕ 3.6 × 40 cm, MeOH/H2O 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 v/v, each 1 L) and semi-preparative HPLC (MeOH/H2O, 50:50, v/v, 1.5 mL/min) to yield 1 (6.5 mg) and 10 (7.8 mg); subfraction B5a (1.8 g) was subjected to repeated RP-18 (ϕ 3.6 × 40 cm, MeOH/H2O 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 v/v, each 1 L) and silica gel CC (ϕ 2.5 × 30 cm, petroleum ether/EtOAc 2:1, v/v, 700 mL) to yield 2 (17.9 mg), 11 (12.5 mg), and 12 (9.8 mg); subfraction B6a (794 mg) was subjected to repeated RP-18 (ϕ 3.6 × 40 cm, MeOH/H2O 3:7, 4:6, 5:5, 6:4, 7:3, v/v, each 1 L) and semi-preparative HPLC (MeOH/H2O 45:55, v/v, 1.7 mL/min) to yield 7 (5.6 mg); and subfraction B7a (530 mg) was subjected to RP-18 (ϕ 3.6 × 40 cm, MeOH/H2O 3:7, 4:6, 5:5, 6:4, 7:3, v/v, each 1 L) and silica gel column CC (ϕ 2.5 × 30 cm, CHCl3/MeOH 15:1, v/v, 500 mL) to yield 13 (28.2 mg) and 14 (19.6 mg). 2.3.1. Excocarinol A (12α,14β-dihydroxyl-3-oxo-pimara-8(9), 15-diene) (1) White powder; [α]18 D − 50.92 (c 0.80, MeOH); UV (MeOH) λmax (logε) 196 (2.31), 215 (2.77), 248 (2.49), 291 (1.919) nm; IR (KBr) νmax 3435, 2966, 2935, 2873, 1704, 1638, 1460, 1383, 1273, 1117, 1017, 1002, 919 cm−1; 1H and 13C NMR see Tables 1 and 2; ESIMS positive m/z [M + Na]+ 341 (20); HREIMS m/z [M + Na]+ 341.2101 (calcd. for C20H30O3Na, 341.2092). 2.3.2. Excocarinol B (3α,12α,14β-trihydroxyl-pimara-8(9), 15-diene) (2) White powder; [α]32 D −41.2 (c 1.13, MeOH); UV (MeOH) λmax (logε) 243 (3.33), 209 (3.85) nm; IR (KBr) νmax 3432, 2936, 2872, 1725, 1633, 1454, 1384, 1288, 1124, 1065, 1021 cm−1; 1H and 13C NMR see Tables 1 and 2; ESIMS positive m/z [M + Na]+ 343 (10); HRESIMS m/z [M + Na]+ 343.2246 (calcd. for C20H32O2Na, 343.2249). 2.3.3. Excocarinol C (3α,12α,14α-trihydroxyl-isopimara-8(9), 15-diene) (3) White powder; [α]32 D − 32.7 (c 1.33, MeOH); UV (MeOH) λmax (logε) 242 (3.52), 208 (3.74) nm; IR (KBr) νmax 3431, 2960, 2934, 2872, 1724, 1639, 1458, 1414, 1380, 1287, 1122, 1075, 1031, 1016 cm−1; 1H and 13C NMR see Tables 1 and 2; ESIMS negative m/z [M + Cl]− 355 (30); HRESIMS m/z [M + Cl]− 355.2042 (calcd. for C20H32O3Cl, 355.2039). 2.3.4. Excocarinol D (cleistantha-8,11,13,15-tetraene-3β-ol) (4) White powder; [α]27 D − 24.93 (c 0.05, MeOH); UV (MeOH) λmax (logε) 209 (3.66), 245 (3.03) nm; IR (KBr) νmax 3441, 2966, 2939, 2857, 1725, 1630, 1455, 1376, 1287, 1122, 1098, 1072, 1037, 1004, 936, 921, 812 cm−1; 1H and 13C NMR see Tables 1 and 2; EIMS m/z [M]+ 284 (100); HREIMS m/z [M]+ 284.2140 (calcd. for C20H28O, 284.2140). 226 S.-Z. Huang et al. / Fitoterapia 91 (2013) 224–230 Table 1 1 H NMR data of compounds 1–5 (δ in ppm, J in Hz). Position 1a 2a 3b 4 5 1α 1.99 m 1.57 m 1.78 m 2.32 ddd (3.5, 3.6, 13.1) 1.53 ddd (4.1, 11.5, 13.1) 1.41 m 2.08 m β 2β 1.68 m 2.56 m 1.51 m 1.96 m 1.25 m 1.75 m 1.82 m 1.77 m 1.56 m 1.88 m α 3 4 5 6α 2.52 m 1.65 m 3.47 dd (2.7, 2.9) 1.62 m 3.27 dd (4.5, 11.8) 3.30 dd (4.7, 11.5) 1.76 dd (4.6, 12.4) 1.72 m 1.65 dd (2.0, 11.8) 1.65 m 1.17 dd (1.2, 12.0) 1.77 m 4.35 2.19 1.12 3.40 β 7α 1.59 m 2.46 m 1.46 m 1.98 m 1.49 m 2.38 m β 9 11β 2.09 m 2.38 m 2.02 m 2.36 dd (5.3, 16.5) 2.32 m α 12 2.07 dd (7.3, 16.5) 3.73 dd (5.3, 7.3) 14 15 16 17 18 19 20 a b 1.29 dd (2.0, 12.5) 1.90 m 1.70 m ddd (2.0, 2.8, 3.4) m dd (2.1, 11.2) ddd (4.0, 11.0, 11.2) 2.66 ddd (7.7, 11.2, 17.7) 2.87 ddd (5.3, 6.0, 17.7) 1.41 dd (4.0, 17.7) 1.12 dd (11.0, 17.7) 2.32 dd (5.6, 16.0) 7.10 d (7.8) 1.85 dd (4.0, 12.8) 1.94 m 2.12 m 2.06 m 3.67 dd (5.5, 7.8) 2.06 dd (7.4, 16.0) 3.69 d (5.6, 7.4) 7.03 d (7.8) 3.76 s 6.05 dd (11.1, 17.7) 5.42 dd (1.0, 11.1) 3.73 s 6.05 dd (11.1, 17.8) 5.41 dd (1.1, 11.1) 3.74 s 6.04 dd (11.0, 17.8) 5.41 dd (1.2, 11.0) 5.33 1.16 1.10 1.12 1.08 5.33 1.17 1.02 1.01 0.91 5.32 1.14 1.00 1.02 0.83 dd (1.0, 17.7) s s s s dd (1.1, 17.8) s s s s dd (1.2, 17.6) s s s s 1.24 1.39 1.85 5.71 5.58 6.60 dd (11.5, 18.0) 5.51 dd (1.0, 11.5) 5.22 dd (1.0, 18.0) 2.26 0.88 1.07 1.20 s s s s m m dd (4.0, 12.8) d (5.6) d (5.6) 1.06 s 1.15 d (7.1) Recorded in CDCl3 at 400 MHz. Recorded in CDCl3 at 600 MHz. 2.3.5. Excocarinol E (6-hydroxyl-15-ene-mononorbeyeran19(3)-lide) (5) White powder; [α]27 D − 34.43 (c 0.07, MeOH); UV (MeOH) λmax (logε) 206(3.38) nm; IR (KBr) νmax 3438, 2957, 2926, 2872, 1729, 1455, 1380, 1366, 1289, 1116, 10, 1098, 1060, 1019, 746 cm−1; 1H and 13C NMR see Tables 1 and 2; ESIMS positive m/z [M + Na]+ 325 (40), 303 (10); HRESIMS m/z [M + H]+ 303.1960 (calcd. for C19H27O3, 303.1958). 2.4. Anti-HIV assays The anti-HIV test was evaluated by the inhibition assay for the cytopathic effects of HIV-1 (EC50) and cytotoxicity assay against C8166 cell line (IC50) as described in the literature [22,23]. AZT (3′-azido-3′-deoxythymidine, Sigma Aldrich, ≥ 98%) was used as positive control. The concentration of the antiviral sample reducing HIV-1 replication by 50% (EC50) was determined from the dose response curve and calculated with Reed and Muench method [24]. The selectivity index (SI) was calculated from the ratio of IC50/EC50. 3. Results and discussion 95% EtOH concentrated extract prepared from the stems of E. acerifolia was suspended in water and then partitioned into petroleum ether and EtOAc extracts, respectively. The EtOAc extract was subjected to repeated CC over silica gel, Sephadex LH-20, and RP-18 and purified further by HPLC to give five new diterpenoids excocarinols A–E (1–5), along with the nine known compounds (Fig. 1). The new ones were identified by comparing literature and spectroscopic means including 1D and 2D NMR. Table 2 13 C NMR data of compounds 1–5 (CDCl3, δ in ppm). Carbon 1a 2a 3c 4b 5c 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 35.1 t 34.2 t 217.3 s 47.1 s 50.7 d 19.4 t 29.0 t 129.0 s 134.6 s 37.1 s 30.5 t 72.8 d 45.9 s 77.9 d 138.1 d 118.8 t 20.3 q 21.1 q 26.7 q 19.0 q 29.4 t 25.5 t 75.7 d 37.5 s 44.2 d 18.0 t 28.6 t 127.9 s 136.7 s 37.2 s 30.6 t 73.1 d 46.1 s 78.0 d 138.2 d 118.8 t 20.1 q 19.2 q 28.0 q 22.1 q 34.8 t 27.5 t 78.7 d 47.2 s 50.9 d 18.2 t 29.1 t 128.0 s 136.3 s 38.8 s 30.6 t 73.0 d 45.9 s 77.9 d 138.2 d 118.7 t 20.2 q 19.5 q 27.8 q 15.5 q 37.3 t 28.0 t 78.7 d 38.9 s 49.3 d 18.9 t 29.7 t 133.0 s 147.0 s 37.6 s 123.1 d 127.8 d 132.6 s 137.7 s 135.5 d 119.1 t 20.5 q 15.3 q 28.1 q 24.9 q 29.2 t 20.8 t 79.1 d 36.5 d 55.8 d 72.2 d 45.5 t 49.3 s 45.4 d 45.2 s 20.7 t 33.8 t 43.6 s 61.0 t 135.2 d 136.1 t 19.4 q 24.9 q 174.9 s a b c Recorded at 100 MHz. Recorded at 120 MHz. Recorded at 150 MHz. S.-Z. Huang et al. / Fitoterapia 91 (2013) 224–230 Fig. 1. Structures of 1–14. Fig. 2. Key 1H–1H COSY ( ) and HMBC (H → C) correlations of 1–5. 227 228 S.-Z. Huang et al. / Fitoterapia 91 (2013) 224–230 Excocarinol A (1) was isolated as white powder and its molecular formula, C20H30O3 requiring six degrees of unsaturation, was determined by the HRESIMS positive (m/z 341.2101 [M + Na]+, calcd. for C20H30O3Na, 341.2092) and NMR spectroscopic data (Tables 1 and 2). The IR absorption also indicated the presence of hydroxyls (3435 cm−1), carbonyl (1704 cm−1), and double bonds (1638 cm−1). The analysis of the 13C NMR and DEPT spectra (Table 2) showed 20 carbon resonances, including four methyls, six methylenes (one olefinic), four methines (two oxygenated and one olefinic), and six quaternary carbons (three aliphatic, one carbonyl and two olefinic). These characteristics were reminiscent of the presence of pimarane diterpenoid as 7β,12β-dihydroxypimara8,15-dien-14-one (9) [16]. The differences between 1 and 9 were the positions of the hydroxyl and carbonyl. The location of carbonyl group at C-3 in 1 was determined by the HMBC correlations from H-19 [δH 1.12 (3 H, s)] and H-20 [δH 1.08 (3 H, s)] to C-3 [δC 217.3 (s)]. The linkage of the hydroxyl to C-14 in 1 was elucidated from the HMBC correlations of H-17 [δH 1.16 (3H, s)] and H-12 [δH 3.73 (1 H, dd, J = 5.3, 7.3 Hz)] with C-14 [δC 77.9 (d)]. The other correlations in the HMBC and 1H–1H COSY spectrum (Fig. 2) further confirmed the atom connectivities in compound 1. Compound 1 had the same configurations as 9 with β-orientation of 18-Me and α-orientation of H-5 based on the comparison of their NMR data and the ROESY experiment of 1 (Fig. 3). The β-orientation of 14-OH was determined by NOE of H-14 [δH 3.76 (1H, s)]/H-17 [δH 1.16 (s)], H-14/H-11α [δH 2.07 (1H, dd, J = 7.3, 16.5 Hz)] and H-18 [δH 1.10 (3H, s)]/H-11β [δH 2.36 (1H, dd, J = 5.3, 16.5 Hz)]. The α-orientation of 12-OH was deduced by NOE of H-12/H-16 [δH 5.33 (1H, dd, J = 1.0, 17.7 Hz)]. Thus, compound 1 was assigned as shown with pimarane skeleton, and named excocarinol A. Excocarinols B (2) and C (3) were both obtained as white powder. They were respectively assigned the same molecular formula C20H32O3 with five degrees of unsaturation according to the HRESIMS positive m/z [M + Na]+ 343.2246 (calcd. for C20H32O2Na, 343.2249) for 2 and negative m/z 355.2042 [M + Cl]− (calcd. for C20H32O3Cl, 355.2039) for 3. Their 1H and 13C NMR data (Tables 1 and 2) were similar to those of compound 1, indicative of the same planar construction of 2 and 3 as 1. The major difference was the absence of the carbonyl at C-3 (δC 217.3) in 1 and the presence of an oxygenated methine signal at δC 75.7 (C-3) in 2 and δC 78.7 (C-3) in 3, which was further confirmed by the key HMBC correlation from H-20 [δH 0.91, (3 H, s)] to C-3 [δC 75.7 (d)] in 2 and from H-20 [δH 0.83 (3 H, s)] with C-3 [δC 78.7 (d)] in 3. On the basis of the ROESY correlations (Fig. 3), the relative configuration of 2 was determined to be the same as those of 1 and H-3 was assigned to be β-orientated by the key NOE of H-3 [δH 3.47 (1H, dd, J = 2.7, 2.9 Hz)]/H-20. Compound 3 has similar configuration as compound 2 except for the hydroxyl group at C-3. The β-configuration of 3-OH in 3 was established by the key NOE of H-3 [δH 3.27 (1H, dd, J = 4.5, 11.8 Hz)]/H-19 [δH 1.02 (1H, s)]. Thus, compounds 2 and 3 were assigned as shown, and named excocarinols B and C, respectively. Excocarinol D (4) was given the molecular formula C20H28O with 7° of unsaturation based on its HREIMS m/z 284.2140 [M]+ (calcd. 284.2140) and NMR spectroscopic data (Tables 1 and 2). Analysis of its 13C NMR and DEPT spectra (Table 2) showed the presence of 20 carbon resonances, including four methyls, five methylenes (one olefinic), five methines (one oxygenated, two aromatic, and one olefinic), and six quaternary carbons. The NMR data (Tables 1 and 2) of 4 were similar to those of (4R,5S,10S)-cleistantha-8,11,13-trien-19-ol [25], a cleistanthane diterpenoid, except for the additional signal at δC 78.7 (d, C-3) in 4 instead of δC 65.7 (t, C-19) in (4R,5S,10S)cleistantha-8,11,13-trien-19-ol, indicating that the hydroxyl group at C-19 in (4R,5S,10S)-cleistantha-8,11,13-trien-19-ol was moved to C-3 in 4. This was also confirmed by the 1H–1H COSY (Fig. 2) correlations of H-2 [δH 1.82 (1H, m) and 1.77 (1H, m)] with H-3 [δH 3.30 (1H, dd, J = 4.7, 11.5 Hz)] and the HMBC correlations from H-3 to C-4 [δC 38.9 (s)] and from H-18 (δH 0.88, 3H, s) to C-3. The α-orientation of H-5 and β-orientation of 20-Me in 4 were determined to be the same as those in (4R,5S,10S)-cleistantha-8,11,13-trien-19-ol [25]. The βorientation of 3-OH was elucidated by the correlations of H-3 with H-5 [δH 1.29 (1 H, dd, J = 2.0, 12.5 Hz)] and H-19 [δH 1.07 Fig. 3. Key ROESY (↔) correlations of 1–5. 229 S.-Z. Huang et al. / Fitoterapia 91 (2013) 224–230 Table 3 Summary of anti-HIV-1 of compounds 1, 3, 4, and 5. No. Cytotoxicity IC50 (μM) Anti-HIV-1activity EC50 (μM) Selectivity index SI (IC50/EC50) 1 3 4 5 3′-Azido-3′-deoxythymidine N628.93 163.75 280.21 110.66 3676.20 5.58 60.13 57.73 11.14 0.00951 N112.71 2.72 4.85 9.93 386561.51 (3H, s)] in the ROESY experiment (Fig. 3). Thus, compound 4 was assigned as shown, and named excocarinol D. Excocarinol E (5) was obtained as white powder as well and its molecular formula C19H26O3 representing 7° of unsaturation was assigned according to the HRESIMS positive m/z 303.1960 [M + H]+ (calcd. for C19H27O3, 303.1958) and NMR spectroscopic data (Tables 2 and 3). The 13C NMR and DEPT spectra (Table 2) of 5 showed 19 carbon resonances, including two methyls, six methylenes, seven methines (two oxygenated and two olefinic), and four quaternary carbons (one carbonyl), which were similar to those of agallochin I [26], a nor-beyerane diterpenoid, except for the additional signals at δC 79.1 (d, C-3) and 174.9 (s, C-19) in 5 instead of δC 98.3 (s, C-3) and 68.5 (t, C-19) in agallochin I. This was reminiscent of the presence of lactone formed between C-19 and C-3 in 5 replaced for the ether structure of C-19-O-C-3 in agallochin I, which was further confirmed by the HMBC correlations from H-3 [δH 4.35 (1H, ddd, J = 2.0, 2.8, 3.4 Hz)] and H-5 [δH 1.12 (1H, dd, J = 2.1, 11.2 Hz)] to C-19 [δC 174.9 (s)]. The other correlations in the HMBC and 1H–1H COSY spectrum (Fig. 2) also supported the atom connectivities in compound 5. The relative configuration of 5 was established from the ROESY experiment (Fig. 3). The α-orientation of H-5 in 5 was hypothetically assigned to be the same as that of agallochin I. The α-orientations of H-4, H-6, and H-9 were elucidated by NOE of H-5/H-4 [δH 2.19 (1H, m)], H-4/ H-6 [δH 3.40 (1 H, ddd, J = 4.0, 11.0, 11.2 Hz)] and H-6/H-9 [δH 1.85 (1H, dd, J = 4.0, 12.8 Hz)], respectively. The α-orientation of H-3 was deduced by the key NOE of H-5/H-3 [δH 4.35 (1H, ddd, J = 2.0, 2.8, 3.4 Hz)], which accordingly assigned the β-orientation of C-19. The α-orientations of C-15 and C-16 were determined by the key NOE of H-6/H-15 [δH 5.71 (1H, d, J = 5.6 Hz)]. Thus, compound 5 was assigned as shown, and named excocarinol E. Four compounds 1, 3, 4, and 5 isolated here were evaluated for their in vitro anti-HIV-1 activity using previously-described method [22,23] (Table 3). Compound 1 exhibited moderate anti-HIV-1 activity at the levels of EC50 5.58 μM and SI over 112.71, though its homologue 3 and other two isolates showed low activity. This may propel the research of pimarane type compounds to inhibit HIV. Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments This work was financially supported by the Special Fund for Agro-scientific Research in the Public Interest (201303117), the National Support Science and Technology Subject (2013BAI11B04), and the Fundamental Scientific Research Funds for CATAS (1630052013001; ITBB110301). The authors thank Dr Y. L. Huang and Prof. Z. H. 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