1000008960863.Pdf

Fitoterapia 95 (2014) 34–41

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Fitoterapia

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Anti-HIV-1 tigliane diterpenoids from Excoecaria acertiﬂia Didr

Sheng-Zhuo Huang a,b, Xuan Zhang c, Qing-Yun Ma a, Hua Peng b, Yong-Tang Zheng c, Jiang-Miao Hu b, Hao-Fu Dai a, Jun Zhou b,⁎, You-Xing Zhao a,⁎ a Key Laboratory of Biology and Genetic Resources of Tropical Crops, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, PR China b State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, PR China c Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650204, PR China article info abstract

Article history: Three tigliane-type diterpenoids named excoecafolins A–C and two daphnane-type diterpenoids Received 22 January 2014 named excoecafolins D and E, together with 13 known compounds, were isolated from the EtOAc Accepted in revised form 21 February 2014 extract of Excoecaria acerifolia Didr. Their structures were elucidated through the analysis of the Available online 5 March 2014 spectroscopic data. The anti-HIV-1 activity evaluation of five of these compounds showed that

four possessed moderate anti-HIV-1 activities with EC50 0.258, 0.036, 0.046, and 0.978 μM, Chemical compounds studied in this article: SI N1,836.9, 431.1, 298.7, and N503.7, respectively. Additionally, the chemotaxonomic issue 4-O-methyl-TPA (PubChem CID 72292) of the affinity correlation between Thymelaeceae and Euphorbiaceae is discussed based on excoecariatoxin (PubChem CID 6442008) the isolates. schisanwilsonene A (PubChem CID © 2014 Elsevier B.V. All rights reserved. 56671370) caryolanel,9β-diol (PubChem CID 44559977) dapneolon (PubChem CID 5316300) daphneticin (PubChem CID 158341) catechin (PubChem CID 9064)

Keywords: Excoecaria acerifolia Euphorbiaceae Excoecafolin Anti-HIV-1 Chemotaxonomy

1. Introduction to clearly define their relationships, other natural products from the two family species and the biosynthesis of The relationship between the Thymelaeceae and Euphor- daphnane diterpenoids need to be investigated. Excoecaria biaceae is controversial with both families categorized into species (Euphorbiaceae), well known for living in mangrove different orders in the APG III [1]. Some systematists claimed and tropical rain forests, are used as traditional medicines that the two families (or elevated to be orders) were sister in the south of China and Thailand as a uterotonic [7]. groups in the Malvales [2–4]. Nevertheless, characteristic Previous studies focused on the common species of tigliane and daphnane type diterpenoids isolated in both this genus such as Excoecaria agallocha and reported a Thymelaeceae and Euphorbiaceae families serve as strong great number of compounds including tigliane and chemotaxonomic evidence for their affinity [5,6].Inorder daphnane-type diterpenoids [8–12]. Nowadays, daphnane and analogous diterpenoids are found to have remarkable biological activities, especially anti-HIV activity [9,13–15]. ⁎ Corresponding authors. Tel./fax: +86 898 66989095. E-mail addresses: [email protected] (J. Zhou), Taken together, the study of the Excoecaria species can be [email protected] (Y.-X. Zhao). very promising.

Excoecaria acerifolia Didr. is widely distributed in dry hot with petroleum ether (3 × 5 L) and EtOAc (3 × 5 L), respec- valleys of Southwest China as an epibiotic species in this genus tively. The EtOAc extract (305 g) was separated by silica gel

[4]. It is usually used as the ethnodrug “Gua-jing-ban” by CC using a gradient of CHCl3/MeOH (9:1–3:1, 10 L) to give minority nationalities in Yunnan Province for its antiphlogistic, fractions A–C. Fraction A (101 g) was separated by silica gel antidote, antitussive, anti-malaria, laxative, and anti-virus CC (ϕ 16 × 160 cm) using a gradient solvent petroleum properties [16]. Diterpenoids were the main type of natural ether/EtOAc (10:1–1:2) to afford fractions A1–A7. Fraction products of E. acerifolia [17] and some pimaranes were isolated A1 (1.4 g) was subjected to repeated RP-18 (MeOH/H2O, 3:2, from this plant in our previous studies [18,19]. In a search for 4:1, 9:1, 1:0 v/v) and semi-preparative HPLC (MeOH/H2O, new bioactive tigliane and daphnane-type diterpenoids, our 85:25 or 90:10 v/v) to yield 2 (11.5 mg) and 3 (9.7 mg); continuative investigation on natural products from E. acerifolia Fraction A2 (1.4 g) was subjected to repeated RP-18 (MeOH/ was carried out and five new tigliane and daphnane-type H2O, 3:2, 4:1, 9:1, 1:0 v/v, respectively) and silica gel CC diterpenoids named excoecafolins A–E(1–5), along with 13 (CHCl3/MeOH, 20:1) to give 5 (12.0 mg), respectively; known compounds 6–18, were isolated from the EtOAc extract Fraction A3 (2.1 g) was subjected to repeated RP-18 of E. acerifolia. The anti-HIV-1 activities of five isolates (1–4 (MeOH/H2O, 3:2, 4:1, 9:1, 1:0 v/v) and silica gel CC (CHCl3/ and 6) were tested. Additionally, the relationship between MeOH, 15:1 v/v) to obtain 9 (5.7 mg), 11 (7.2 mg), 12 Thymelaeceae and Euphorbiaceae is discussed on the basis of (6.9 mg), and 13 (13.5 mg), respectively; Fraction A4 the compounds isolated, and the possible biogenetic pathways (420 mg) was subjected to repeated RP-18 (MeOH/H2O, to daphnane diterpenoids. Herein, the isolation, structural 3:2, 4:1, 9:1, 1:0 v/v) and semi-preparative HPLC (MeOH/ elucidation, anti-HIV-1 activities, as well as chemotaxonomy H2O, 85:25 or 90:10 v/v) to yield 1 (35.9 mg), 4 (11.4 mg), discussion, are described. and 14 (9.7 mg), respectively; Fraction A5 (210 mg) was

subjected to repeated RP-18 (MeOH/H2O, 3:2, 4:1, 9:1, 1:0 v/v) 2. Experimental and semi-preparative HPLC (MeOH/H2O, 53:47 or 1:1 v/v)to afford 7 (6.9 mg), 8 (7.7 mg), and 10 (4.3 mg), respectively; 2.1. General experimental procedures. Fraction A6 (1.7 g) was subjected to repeated RP-18 (MeOH/

H2O, 3:2, 4:1, 9:1, 1:0 v/v) and semi-preparative HPLC (MeOH/ Optical rotations were measured on a Horiba SEPA-300 H2O, 53:47 or 1:1 v/v) to yield 16 (35.3 mg), 6 (43.2 mg), and polarimeter, whereas UV spectra were obtained on a 10 (6.6 mg), respectively; Fraction A7 (54.4 g) was subjected

Shimadzu double-beam 210A spectrometer. IR spectra were to repeated silica gel column (CHCl3/MeOH, 15:1–5:1 v/v), obtained on a Tensor 27 spectrometer with KBr pellets. NMR RP-18 (MeOH/H2O, 3:2, 4:1, 9:1, 1:0 v/v, respectively) and spectra were acquired using a Bruker AV-400, a DRX-500, or silica gel column (CHCl3/MeOH, 10:1 v/v)togive16 (15.2 mg) AVANCE III-600 spectrometer with TMS as an internal 17 (24.8 mg), and 18 (24.5 g), respectively. standard. ESIMS and HRESIMS were obtained using an API QSTAR Pulsar 1 spectrometer, whereas EIMS and HREIMS were obtained using a Bruker HCT/Esquire and API Qstar Table 1 Pulsar, respectively. Silica gel (200–300 mesh, Qingdao 1H and 13C NMR spectroscopic data of compounds 1 and 4. Marine Chemical Inc., People's Republic of China), RP-18 No. 1 (in CD OD) 4 (in CDCl ) (40–70 μm, Fuji Silysia Chemical Ltd., Japan) and Sephadex 3 3

LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used δH multi, J (Hz) δC δH multi, J (Hz) δC for column chromatography (CC). Semipreparative HPLC was 1 7.62 (1H, d, 1.8) 162.9 7.48 (1H, d, 1.1) 161.6 performed on an Agilent 1100 liquid chromatograph with a 2 135.8 134.2

Zorbax SB-C18, 9.4 mm × 25 cm, column. Fractions were 3 210.2 209.1 monitored by TLC and spots were visualized by heating 4 74.8 78.2 5 4.11 (1H, s) 70.6 4.08 (1H, s) 68.9 after spraying with 5% H SO in EtOH (B.p. 77–79 °C). 2 4 6 64.4 62.5 7 3.20 (1H, d, 1.3) 66.1 3.18 (1H, d, 2.1) 64.2 2.2. Plant material. 8 1.15 (1H,dd, 1.3, 7.6) 32.8 3.11 (1H, dd, 2.1, 2.2) 37.6 9 64.9 73.9 Stems of E. acerifolia were collected in Dali city Yunnan 10 3.93 (1H, d, 1.8) 51.4 3.85 (1H, d, 1.1) 49.4 11 1.86 (1H, ddq, 6.9, 39.5 2.03 (1H, ddq, 2.0, 38.0 Province, People's Republic of China, and identified by Prof. H. 9.1, 6.3) 9.8, 6.8) Peng, and Dr. Y. Niu (Kunming Institute of Botany, Chinese 12β 1.86 (1H, dd, 6.9, 17.0) 33.2 1.71 (1H, dd, 2.0, 11.0) 37.9 Academy of Sciences). A voucher specimen (HUANG0006) α 1.54 (1H, dd, 9.1, 17.0) 1.78 (1H, dd, 9.8, 11.0) was deposited at the State Key Laboratory of Phytochemistry 13 76.9 72.8 14 2.85 (1H, d, 7.6) 37.0 3.93 (1H, d, 2.2) 77.8 and Plant Resources in West China, Kunming Institute of 15 25.2 144.9 Botany, Chinese Academy of Sciences, People's Republic of 16 1.17 (3H, s) 23.0 5.00 (1H, d, 1.0) 114.4 China. 4.96 (1H, d, 1.0) 17 1.06 (3H, s) 16.2 1.71 (3H, s) 18.7 2.3. Extraction and isolation 18 0.90 (3H, d, 6.3) 19.4 0.86 (3H, d, 6.8) 17.8 19 1.73 (3H, s) 10.0 1.65 (3H, s) 9.6 20 3.97 (1H, d, 12.5) 64.9 3.86 (1H, d, 13.1) 64.2 Dried and powdered stems of E. acerifolia (19 kg) were 3.58 (1H, d, 12.5) 3.53 (1H, d, 13.1) extracted with EtOH-H2O (95:5, 3 × 50 L) under water bath O-Ac 174.6 79 °C reflux for 2 h. The combined extract was concentrated 2.05 (3H, s) 21.0 1 13 and suspended in H2O (3 L) followed by successive partition H and C NMR data measured at 400 and 100 MHz, respectively. 36 S.-Z. Huang et al. / Fitoterapia 95 (2014) 34–41

Table 2 1 13 H and C NMR spectroscopic data of compounds 2, 3, and 5 (in CDCl3).

No. 2a 3a 5b

δH multi, J (Hz) δC δH multi, J (Hz) δC δH multi, J (Hz) δC 1 7.67 (1H, d, 1.8) 162.3 7.59 (1H, d, 1.6) 161.3 7.61 (1H, d, 1.5) 161.1 2 134.0 132.8 – 136.7 3 209.9 209.3 – 209.8 4 73.5 73.7 – 79.7 5 4.39 (1H, s) 71.2 2.50 (2H, m) 38.5 4.24 (1H, s) 71.9 6 139.8 140.0 60.4 7 5.70 (1H, d, 5.8) 133.9 5.68 (1H, d, 5.2) 130.4 3.44 (1H, d, 1.3) 64.1 8 3.22 (1H, dd, 4.7, 5.8) 39.5 3.03 (1H, dd, 4.7, 5.2) 39.1 3.00 (1H, m) 36.7 9 63.3 63.3 72.1 10 3.93 (1H, d, 1.8) 53.3 3.27 (1H, d, 1.6) 53.7 3.78 (1H, d, 1.5) 47.9 11 1.95 (1H, ddq, 6.9, 9.1, 6.3) 32.7 1.99 (1H, m) 36.2 3.76 (1H, m) 48.0 12β 2.13 (1H, dd, 6.9, 17.0) 31.9 2.09 (1H, dd, 6.8, 17.0) 32.6 4.95 (1H, d, 1.2) 78.2 α 1.60 (1H, dd, 9.1, 17.0) 1.62 (1H, dd, 9.0, 17.0) 13 75.0 76.2 84.5 14 0.88 (1H, d, 4.7) 32.8 0.89 (1H, d, 4.7) 32.6 4.44 (1H, d, 1.7) 81.9 15 25.2 22.9 – 145.8 16 1.07 (3H, s) 15.3 1.06 (3H, s) 15.4 5.02 (1H, brs) 111.5 4.92 (1H, brs) 17 1.21 (3H, s) 23.0 1.26 (3H, s) 23.2 1.80 (3H, s) 20.4 18 0.94 (3H, d, 6.3) 19.0 0.89 (3H, d, 6.4) 18.6 0.88 (1H, d, 7.2) 18.9 19 1.79 (3H, s) 10.0 1.76 (3H, s) 10.1 1.76 (3H, s) 10.4 20 3.93 (1H, d, 12.5) 67.6 4.03 (1H, d, 17.2) 68.2 3.86 (1H, d, 14.3) 65.2 3.84 (1H, d, 12.5) 3.95 (1H, d, 17.2) 3.79 (1H, d, 14.3)

1′ 168.8 168.8 115.6 2′ 5.86 (1H, d, 15.3) 120.5 5.84 (1H, d, 15.3) 120.6 5.77 (1H, m) 126.6 3′ 7.33 (1H, dd, 11.1, 15.3) 145.7 7.30 (1H, dd, 11.1, 15.3) 145.5 6.73 (1H, m) 132.9 4′ 6.38 (1H, dd, 11.1, 15.4) 127.0 6.37 (1H, dd, 11.1, 15.4) 127.0 6.29 (1H, m) 127.1 5′ 6.18 (1H, dd, 5.9, 15.4) 146.7 6.17 (1H, dd, 5.9, 15.4) 146.4 6.35 (1H, m) 132.2 6′ 4.24 (1H, m) 71.9 4.25 (1H, m) 71.9 2.23 (2H, m) 31.8 7′ 1.93 (2H, m) 37.0 1.94 (2H, m) 37.0 2.08 (2H, m) 22.7 8′–13′/ 1.23–1.62 31.9 1.23–1.62 31.8 1.21–1.54 28.9 8′–16′ (12H, overlap) – (12H, overlap) – (18H, overlap) – 22.6 22.6 29.5 14′/17′ 0.88 (3H, t, 7.4) 14.1 0.86 (3H, t, 7.4) 14.1 1.16 (1H, m) 22.3 1′′ 161.5 2′′ 133.0 3′′/7′′ 5.90 (2H, m) 125.3 4′′/6′′ 5.94 (2H, m) 128.9 5′′ 6.34 (1H, m) 128.8 a1H and 13C NMR data measured at 400 and 100 MHz, respectively. b1H and 13C NMR data measured at 400 and 150 MHz, respectively.

2.3.1. Excoecafolin A (1) 13α-acetyl-6α,7α- epoxy-phorbol 2.3.3. Excoecafolin C (3) 13α-(2′E,4′E,diene-6-hydroxyl- 32 White amorphous powder; [α]D −8.4 (c 3.23, MeOH); UV tetradecanoicoate)-5-dehydroxyl-phorbol 28 (MeOH) λmax (log ε) 308 (2.70), 247 (3.78); IR (KBr) νmax 3422, Colorless amorphous powder; [α]D +11.42 (c 0.15, 2959, 2928, 2875, 1725, 1703, 1629, 1454 1377, 1327, 1266, MeOH); UV (MeOH) λmax (log ε) 263 (4.50), 210 (3.95), 196 1 13 1248, 1121, 1080, 997, 936; for Hand CNMRspectroscopic (4.10); IR (KBr) νmax 3423, 2958, 2927, 2872, 2856, 1725, 1696, data, see Table 1;ESIMSpositivem/z [M + Na]+ 445(10); 1638, 1463, 1380, 1333, 1285, 1134, 1076, 1005; For 1Hand13C + HRESIMS m/z [M + Na] 445.1831 (calcd for C22H30O8Na, NMR spectroscopic data, see Table 2; ESIMS positive m/z 445.1838). [M + Na]+ 593(100); HRESIMS m/z [M + Na]+ 593.3459

(calcd for C34H50O7Na, 593.3454).

2.3.2. Excoecafolin B (2)13α-(2′E,4′E,diene-6-hydroxyl- 2.3.4. Excoecafolin D (4) 2,3,9,13,14-quinthydroxyl-6,7-epoxyl- tetradecanoic fatty acid)-phorbol ester 1(2)Z,15-diene-daphnan-3-one 28 28 Colorless amorphous powder; [α]D −15.4 (c 0.19, Colorless amorphous powder; [α]D −17.0 (c 2.31, MeOH); UV (MeOH) λmax (log ε) 198 (4.03), 262 (4.36); IR MeOH); UV (MeOH) λmax (log ε) 243 (3.79), 206 (3.73); IR (KBr) νmax 3418, 2956, 2927, 2856, 1697, 1637, 1460, 1379, (KBr) νmax 3421, 2961, 2930, 2878, 1697, 1628, 1515, 1453, 1333, 1279, 1243, 1134, 1078, 1004; For 1H and 13C NMR 1382, 1333, 1285, 1160, 1120, 1030, 1010, 925, 905, 810, 779; spectroscopic data see, Table 2; ESIMS positive m/z for 1H and 13C NMR spectroscopic data, see Table 1; ESIMS [M + Na]+ 609(100); HRESIMS m/z [M + Na]+ 609.3402 positive m/z [M + Na]+ 419 (80); HRESIMS m/z [M + Na]+

(calcd for C34H50O8Na, 609.3403). 419.1674 (calcd for C20H28O8Na, 419.1681). S.-Z. Huang et al. / Fitoterapia 95 (2014) 34–41 37

2.3.5. Excoecafolin E (5) 12-benzoate-daphanol-(2′E,3′E-daturic)- to afford five new diterpenoids excoecafolins A–E(1–5) 9,13,14-othor ester (Fig. 1), along with 13 known compounds, daphnopsis factor 17 Colorless amorphous powder; [α]D +22.17 (c 0.375, R2 (6) [21], 4-O-methyl-TPA (7) [22], 13-benzoate-phorbol (8) MeOH); UV (MeOH) λmax (log ε) 238 (4.06), 203 (3.92); IR [22,23], irritant factor M3 (9)[24], daphnediterp A (10) [25], (KBr) νmax 3444, 2926, 2855, 1699, 1631, 1453, 1380, 1315, excoecaria factor A1 (11) [7], excoecariatoxin (12) [13], 1285, 1253, 1074, 1036, 1011, 930, 797; For 1H and 13C NMR schisanwilsonene A (13) [26], caryolanel,9β-diol (14) [27], spectroscopic data, see Table 2; ESIMS positive m/z dapneolon (15) [28], daphneticin (16) [29], malloapelin (17) [M + Na]+ 769 (70); HREIMS m/z [M + Na]+ 769.4033 [30], and catechin (18) [31], respectively.

(calcd for C44H58O10Na, 769.4030). Compound 1 was obtained as a white amorphous powder, and its molecular formula was assigned to be

2.4. Anti-HIV assays C22H30O8 with 8° of unsaturation according to its positive + HRESIMS (m/z 445.1831 [M + Na] ,calcd.forC22H30O8Na, Anti-HIV activity was evaluated on the compounds by 445.1838) and NMR spectroscopic data (Table 1). The IR − the inhibition assay for the cytopathic effects of HIV-1 spectrum displayed the presence of hydroxyls (3422 cm 1), −1 −1 (EC50) and the cytotoxicity assay against the C8166 cell carbonyls (1725, 1703 cm ), and double bond (1629 cm ) 1 δ line (IC50)withMTTmethodsasdescribedinthe absorptions. The H NMR spectra showed three methyls [ C literature [13]. Briefly, cells were seeded in the absence 0.90 (3H, d, J = 6.3 Hz), 1.73 (3H, s), and 2.05 (3H, s)] and one or presence of various concentrations of compounds in olefinic proton [7.62 (1H, d, J = 1.8 Hz)]. Analysis of its 13C triplicate for 3–7days.Thepercentageofviablecellswas NMR and DEPT spectra showed 22 carbon resonances, quantified at 595/630 nm by an ELISA reader. The including five methyls, two methylenes (one oxygenated), cytopathic effect was measured by counting the number seven methines (two oxygenated, and one olefinic), and eight of syncytia (multinucleated giant cell) in each well under an quaternary carbons (two carbonyls, four oxygenated, and one inverted microscope. AZT (3′-azido-3′-deoxythymidine) was olefinic). The 1Hand13C NMR spectroscopic data (Table 1)of used as a positive control. The concentration of the compound 1 were similar to those of daphnopsis factor R2 (6) antiviral sample reducing HIV-1 replication by 50% (EC50) [21], a tigliane diterpenoid, having two oxygenated carbon was determined from the dose–response curve and calcu- signals at δC 64.4 (s, C-6), 66.1 (d, C-7) in 1 instead of two lated with the Reed and Muench method [20].The olefinic resonances at δC 139.8 (s, C-6), 133.7 (d, C-7) in 6, selectivity index (SI) was calculated from the ratio of IC50/ suggesting that the double bond between C-6 and C-7 was EC50. oxygenated into an epoxy group in compound 1 based on the analysis of its molecular formula. The HMBC (Fig. 2)

3. Results and discussion correlations of 1 from H-5 [δH 4.11 (1H, s)], H-8 [δH 1.15 (1H, dd, J = 1.3, 7.6 Hz)], and H-20 [3.97 (1H, d, J =12.5Hz),3.58 The 95% EtOH extract prepared from the stems of (1H, d, J = 12.5 Hz)] to C-6 confirmed this hypothesis. Other E. acerifolia was suspended in water and then partitioned by correlations in the HMBC and 1H–1H COSY spectrum (Fig. 2) petroleum ether and EtOAc, respectively. The EtOAc extract further supported the atom connectivities in compound 1.The was subjected to repeated column chromatography over silica configuration of the tigliane diterpene skeleton in compound 1 gel, Sephadex LH-20, and RP-18, and further purified by HPLC, was elucidated by ROESY experiment (Fig. 3)anddetermined

Fig. 1. Structures of compounds 1–6. 38 S.-Z. Huang et al. / Fitoterapia 95 (2014) 34–41

Fig. 2. Key 1H–1H COSY and HMBC correlations of 1–5 (1H–1H COSY ▬, ROESY →).

to be the same as those of daphnopsis factor R2 (6)with (one oxygenated), twelve methines (two oxygenated and α-orientations of H-10, H-14, and 13-OAc and β-orientation of six olefinic), and eight quaternary carbons (two carbonyls H-8. The α-orientation 9-OH and β-orientation of 4-OH were and three oxygenated). Comparison of its 13C NMR spectro- accordingly elucidated by comparison of its NMR spectroscopic scopic data with those of daphnopsis factor R2 (6) [21], data with 6 (Table 1). The β-orientation of CH3-17 was showed that 2 had additional signals at δC 168.8 (s, C-1′), 120.5 assigned from NOE of H-8/H-17 [δH 1.06 (3H, s)] shown in (d, C-2′), 145.7 (d, C-3′), 127.0 (d, C-4′), 145.7 (d, C-5′), 71.9 (d, Fig. 2.Thea-configuration of the 6,7-epoxy group was proposed C-6′), 37.0 (t, C-7′), 31.9, 29.5, 29.4, 29.2, 25.2, 22.6 (t, C-8′-13′), by the key NOE of H-8/H-20. The α-orientations of H-5 and and 14.1 (q, C-14′) for an (2E,4E)-6-hydroxytetradeca-

CH3-18 were deduced by the NOE analysis of H-5/H-10 [δH 3.93 2,4-dienoic group [32] and was absent for the acetyl signals (1H, d, J =1.8Hz)],H-14[δH 2.85 (1H, d, J =7.6Hz)]/CH3-18 (δC 173.4 and 21.2) in 6, suggesting that 2 had the same basic [δH 0.9 (3H, d, J =6.3Hz)],andH-14/H-16[δH 1.17 (3H, s)], skeleton as 6 except for the acetyl group at C-13 being replaced respectively. Thus, compound 1 was assigned as shown in Fig. 1, by a 6-hydroxytetradeca-2(E),4(E)-dienoic group in 2.The and named excoecafolin A. length of fatty acid in compound 2 was determined by the Compound 2 was isolated as a colorless amorphous molecular formula established by HRESIMS. The HMBC (Fig. 2) powder, and its molecular formula was determined to be correlations of 2 from H-11 [δH 1.95 (1H, ddq, J = 6.5, 9.1, C34H50O8 from its positive HRESIMS (m/z 609.3402 6.3 Hz)], and H-14 [δH 0.88 (1H, d, J = 4.7 Hz, H-14)] to C-13 + [M + Na] ,calcd.forC34H50O8Na, 609.3403). Its IR spec- (δC 75.0) further confirmed the hypothesis. Other HMBC −1 trum showed the presence of hydroxyls (3418 cm ), correlations from H-10 [δH 3.93 (1H, d, J = 1.8 Hz)] to C-2 −1 −1 carbonyls (1697 cm ), and double bonds (1637 cm ). (δC 134.0) and from H-5 [δH 4.39 (1H, s)] to C-3 (δC 209.9) and 13 The C NMR and DEPT spectra (Table 2)of2 displayed 34 COSY cross-peaks of H-11/H-18 [δH 0.94 (3H, d, J = 6.3 Hz)], carbon resonances, including five methyls, nine methylenes H-11/H-12 [δH 2.13 (1H, dd, J = 6.9, 17.0 Hz) and 1.60 (1H, dd, S.-Z. Huang et al. / Fitoterapia 95 (2014) 34–41 39

Fig. 3. Key ROESY correlations of 1–4.

J =9.1,17.0Hz)],H-8[δH 3.22 (1H, dd, J = 4.7, 5.8 Hz)]/H-7 [δH Compound 4 was given the molecular formula of C20H28O8, + 5.70 (1H, d, J = 5.8 Hz)] and H-8/H-14 [δH 0.88 (1H, d, J = based on the positive HRESIMS (m/z 419.1674 [M + Na] , 4.7 Hz)] (Fig. 2) further confirmed the atom connectivities and calcd. for C20H28O8Na, 419.1681) and NMR (Table 2)spectro- the group position in compound 2. The configuration of the scopic analysis. The 13CNMRandDEPT(Table 1)spectraof tigliane diterpene skeleton in compound 2 was determined to compound 4 were similar to those of daphnediterp A (10), a be the same as those of daphnopsis factor R2 (6)bycomparison daphnane-type diterpene skeleton, except for the absence of of their NMR data and confirmed by the ROESY experiment angeloyl group signals [δC 166.8 (s), 128.2 (s), 138.2 (d), 20.5 such as NOE of H-18/H-10, H-10/H-5, and H-8/H-17 [δH 1.21 (q), and 15.9 (q)] at C-14 in 10. This difference suggested that (3H, s)] (Fig. 3). The linkage of the hydroxyl group to C-6′ was compound 4 was possibly derived from compound 10 with the 1 1 proposed by the key H– HCOSY(Fig. 2) correlation of H-5′ [δH hydroxyl replaced by an angeloyl group. The correlations in 1 1 6.18 (1H, dd, J = 5.9, 15.4 Hz)]/H-6′ [δH 4.24 (1H, m)] and HMBC and H– HCOSYspectrum(Fig. 2) further verified the HMBC correlations from H-4′ [δH 6.38 (1H, dd, J =11.1, structural assignment of 4. The configuration of daphnane-type 15.4 Hz)] and H-5′ [δH 6.18 (1H, dd, J =5.9,15.4Hz)]toC-6′ diterpene skeleton [33] in compound 4 was deduced to be the [δC 71.9 (d)]. Thus, compound 2 wasassignedasshownin same as that of daphnediterp A (10) by their comparison of Fig. 1, and named excoecafolin B. NMR data and the similar NOE in ROESY experiment (Fig. 3).

Compound 3 was assigned the molecular formula C34H50O7 Thus, compound 4 was elucidated as shown, and named by analyses of its positive HRESIMS (m/z 593.3459 [M + Na]+, excoecafolin D. calcd. for C34H50O7Na, 593.3454) and NMR spectroscopic data Compound 5 was assigned to be C44H58O10 according to (Table 2). 3 had the similar IR absorptions as 2 for hydroxyls the positive HRESIMS (m/z 769.4033 [M + Na]+,calcd.for −1 −1 (3423 cm ), carbonyls (1725 and 1696 cm ), and double C44H58O10Na, 769.4030) and NMR spectroscopic data (Table 2). bonds (1638 cm−1). The NMR data (Table 2) of compound 3 Its 13C NMR and HSQC (Table 2) data were similar to those of were extremely similar to those of 2 except that methylene yunhuajine [13,34], suggesting an orthoester skeleton of 5.The signals [δC 38.5 and δH 2.50 (2H, m)] in 3 replaced the methine differences were the absence of signals for one double bond at C-5 [δC 71.2 and δH 4.39 (1H, s)] in 2, indicating that 3 was carbon and the appearance of additional signals for serial derived from 2 by loss of the hydroxyl group at C-5. This was saturated methylenes at δC 28.9–29.5 (d, C-8′-16′)andδH 1.26– confirmed by key HMBC correlations from H-5 [δH 2.50 (2H, m)] 1.81 (18H, m, H-8′-16′) indicating that the (2E,4E,6E)- to C-3 (δC 209.3), and from H-7 [δH 5.68 (1H, d, J =5.2Hz)] deca-2,4,6-trienoic moiety in yunhuajine was replaced by a and H-20 [δH 4.30 (1H, d, J = 17.2 Hz) and 3.95 (1H, d, J = (2E,4E)-heptadeca-2,4-dienoic moiety [35,36] in compound 5. 17.2 Hz)] to C-5 (δC 38.5). The structure of compound 3 was The assignment of compound 5 was further confirmed by the further defined by other 2D correlations in HMBC and 1H–1H analysis of correlations in HMBC and 1H–1H COSY spectrum COSY (Fig. 2). The configuration of compound 3 was established (Fig. 2). The daphnane-type diterpene skeleton configuration to be as 2 by comparison of their NMR data and the similar NOE [33] in compound 5 was established to be the same as that of in a ROESY experiment (Fig. 3). Finally, compound 3 was yunhuajine based on their similar NMR data. Finally, com- assigned as shown in Fig. 1, and named excoecafolin C. pound 5 was determined and named excoecafolin E. 40 S.-Z. Huang et al. / Fitoterapia 95 (2014) 34–41

Table 3 The characteristic tigliane and dapnane diterpenoids in Summary of anti-HIV-1 of compounds 1–4 and 6. Thymelaeceae and Euphorbiaceae as chemotaxonomic

No. Cytotoxity Anti-HIV-1 Selectivity evidence proved the affinity theory of the two families proposed

IC50 (μM) activity index SI by previous pioneers [5,6]. The compounds isolated from EC50 (μM) (IC50/EC50) Thymelaeceae mainly consist of rare tigliane diterpenoids, 1 N473.93 0.258 N1836.9 abundant daphnane diterpenoids, lignans (including special 2 15.52 0.036 431.1 C6–C5–C6 norlignan type), coumarins (including special 3 13.74 0.046 298.7 coumarinolignoids), flavans, and sesquiterpenoids [15,37,38]. 4 286.82 32.616 8.8 Also in addition to tigliane diterpenoids, daphnane diterpenoids N N 6 492.61 0.978 503.7 – – – 3′-Azido-3′- 3678.96 0.00951 386,851.7 [7,39 41], lignans (including C6 C5 C6 norlignan type), couma- deoxythymidine rins (including special coumarinolignoids) [30], flavans, and sesquiterpenoids were identified from Euphorbiaceae in our study as well as in previous reports. These chemical studies strongly confirmed the affinity theory. Additionally, the The anti-HIV activity of the five compounds 1–4 and 6 was plausible biosynthetic pathway (Fig. 4)fromtigliane(I)to evaluated by assaying inhibition of cytopathic effects of the daphnane (V) [14,42,43] was previously described according to HIV-1 (EC ) and cytotoxicity against the C8166 cell line by 50 literature report and the isolates in our study. Moreover, MTT methods. Moderate anti-HIV-1 activities occurred with plentiful precursors of daphnane diterpenoid such as tigliane the four tigliane diterpenoids tested, among which tigliane and other macrocycloditerpenoids (including cembrene, diterpenoids 2 and 3 exhibited the highest anti-HIV-1 casbene, and lathyrene) [44] were also isolated from Euphor- activities (EC levels of 0.036 μM and 0.046 μM, respective- 50 biaceae plants. All these chemical information indicated that ly) than those of compounds 1 and 6 without a fatty acyl Thymelaeceae and Euphorbiaceae bear chemotaxonomic group (EC 0.258, 0.978 μM, respectively) (Table 3). This 50 affinity correlation. suggested that the fatty acyl group at C-13 in tigliane diterpenoid might be responsible for the moderate enhanced anti-HIV activity and cytotoxicity. Daphnane diterpenoid as 4 Conﬂict of interest without a fatty acyl ortho-ester did not, however, exhibit significant anti-HIV-1 activity, while the daphnane diterpene We declare that we have no financial and personal ortho ester did as reported in previous literature [13] relationships with other people or organizations that can (Table 3). This suggests that the fatty acyl orther ester moiety inappropriately influence our work, and there is no profes- is also important for the anti-HIV-1 activity in daphnane sional or other personal interest of any nature or kind in any diterpenoids. product, service and/or company that could be construed as

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