Fitoterapia 108 (2016) 81–86
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New clerodane diterpenoids from Croton crassifolius☆ Maosong Qiu b, Di Cao b, Youheng Gao b, Shuhua Li b, Jinping Zhu b, Bao Yang b, Lian Zhou b, Yuan Zhou b, Jing Jin a,⁎, Zhongxiang Zhao b,⁎⁎ a b
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
a r t i c l e
i n f o
Article history: Received 5 October 2015 Received in revised form 14 November 2015 Accepted 19 November 2015 Available online 21 November 2015 Keywords: Croton crassifolius Clerodane diterpenoids Alkaloid MTT method
a b s t r a c t Two new clerodane diterpenoids (1–2), one new clerodane diterpenoid alkaloid (3), as well as thirteen known compounds were isolated from Croton crassifolius. The structures of new compounds were established by a combination of spectroscopic methods, including HRMS, 1H NMR, 13C NMR, 1H 1H COSY, HSQC, HMBC, NOESY and Xray crystallographic analysis. Compound 3 is firstly reported as the clerodane-type diterpenoid alkaloid in natural products. All of the compounds were evaluated for in vitro cytotoxic activities against CT26.WT cell using the MTT method. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The genus Croton (family Euphorbiaceae) contains about 800 species, widely distributed throughout tropical areas in the world and south region of China, and the plants of this genus are rich in biological active diterpenoids, including clerodanes, kauranes, labdanes and trachylobanes [1]. Croton crassifolius Geisel is mainly distributed in south China and other areas in Southeast Asia [2], and has been traditionally used for the treatment of stomach pain, sore throat and cancer [3].Previous phytochemical investigations have revealed that C. crassifolius is abundant with terpenoids, including sesquiterpenes, diterpenoids and triterpenoids, in which clerodane-type diterpenoids are considered as the main and characteristic components [4–8]. Up to now, more than 16 clerodane-type diterpenoids have been isolated from different parts of this plant, including chettaphanin I [3], 9-[2(2(5H)-furanone-4-yl)ethyl]-4,8,9-trimethyl-1,2,3,4,5,6,7,8-octahydronaphthalene-4-carboxylic acid, methyl 9-[2-(2(5H)-furanone-4yl)ethyl]-4,8,9-trimethyl-1,2,3,4,5,6,7,8-octahydro-naphthalene-4-carboxylic ester [5], crassifolin A–G, spiro[furan-3-(2 H),1′(2′H)-naphthalene]-5′-carboxylic acid, penduliflaworosin, 1,4-methano-3-benzoxepin-2(1 H)-one, isoteucvin, teucvin [2], mallotucin B [9], and crassifolin H-I [6]. In addition, two novel sesquiterpenoids (Crocrassins A and B
☆ The authors declare no competing financial interest. ⁎ Correspondence to: J. Jin, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, Guangdong Province 510006, China. ⁎⁎ Correspondence to: Z. Zhao, School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province 510006, China. E-mail addresses: [email protected] (J. Jin), [email protected] (Z. Zhao).
http://dx.doi.org/10.1016/j.fitote.2015.11.016 0367-326X/© 2015 Elsevier B.V. All rights reserved.
[7]), a halimane diterpenoid (crassifoliusin A [8]), and several other constituents [10] have been reported. Pharmacological investigations have demonstrated that clerodane-type diterpenoids are a rich source of biological activities [11–14]. A phytochemical investigation of the roots of C. crassifolius was thus performed as part of our continuous work on the discovery of new or bioactive compounds from Chinese herbal medicines. This led to isolation of two new clerodane diterpenoids, cracrosons A–B (1–2), one new clerodane diterpenoid alkaloid, cracroson C (3), along with thirteen known compounds. In this paper, the isolation and structure elucidation of these compounds are described, with their inhibition activities on CT26.WT cell proliferation also being assayed. 2. Experimental 2.1. General procedures Melting points were obtained on an X-4 micro-melting point apparatus. Optical rotations were measured with a Perkin-Elmer precisely model 341 polarimeter (Perkin-Elmer, Inc., Waltham, MA). UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. IR spectra were recorded on a Nicolet Nexus 300 FTIR spectrometer (Thermo Nicolet Corporation, Madison, USA). 1D and 2D-NMR were performed on a Bruker AVANCE DRX-400 NMR spectrometer (Bruker Biospin Gmbh, Rheistetten, Germany) in CDCl3 using tetramethylsilane (TMS) as internal standard. HREIMS and HRESIMS were determined on a MAT 95 XP spectrometer (Thermo Finnigan, Bremen, Germany) and an LTQ-Orbitrap Elite spectrometer (Thermo Fisher Scientific, San Jose, CA), respectively. Column chromatography was carried out with silica
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gel (100–200, 200–300 and 300–400 mesh, Qingdao Marine Chemical Co., Qingdao, China), C18 reversed-phase silica gel (YMC CO., LTD, Kyoto, Japan) and Sephadex LH-20 (GE-Healthcare Bio-Sciences AB, Uppsala, Sweden). Further purifications were performed by semipreparative HPLC on a RP-C18 column (5 μm, 250 × 10 mm, Kromasil Co., Bohus, Sweden) with a Wufeng LC-100 apparatus (Shanghai Wufeng Technical Instrument Co., Shanghai, China), at the flow rate of 3 mL/min, and the temperature of 30 °C. 2.2. Plant material The roots of C. crassifolius were collected in Fujian Province, People's Republic of China, in June of 2009, and were authenticated by Prof. Zhong-Xiang Zhao of Guangzhou University of Chinese Medicine, China. A voucher specimen (No. 200906) was deposited in the School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, China. 2.3. Extraction and isolation The dried and powdered roots of C. crassifolius (4.5 kg) were percolated with 80% MeOH (40 L) at room temperature. The extracts were combined and concentrated under reduced pressure to leave a residue, which was then suspended in H2O (2.5 L) and partitioned in petroleum ether (2.5 L × 3), CHCl3 (2.5 L × 3), and EtOAc (2.5 L × 3). The CHCl3 extract (210 g) was chromatographed on silica gel column with gradient mixtures of petroleum ether–acetone (100:1 → 5:1) to afford nine fractions, Fr. A to Fr. I. Fr. A was crystallized from CHCl3-acetone (3:1) to yield 14 (20 g). 15 (500 mg) was provided from Fr. B by Sephadex LH-20 using CHCl3-MeOH (1:1). Fr. C was subjected to Sephadex LH20, eluting with CHCl3-MeOH (1:1), to provide 16 (450 mg). Fr. D was fractionated by Sephadex LH-20 (CHCl3-MeOH, 1:1) and then chromatographed on silica gel column eluted with petroleum etherEtOAc (20:1) to afford 13 (150 mg). Fr. F was separated by Sephadex LH-20 (CHCl3-MeOH, 1:1) and further subjected to silica gel column (petroleum ether-EtOAc, 7:1) to obtain 7 (54 mg). Fr. G was fractionated by Sephadex LH-20 (CHCl3-MeOH, 1:1), then separated over silica gel column eluted with petroleum ether-EtOAc (3:1), and 11 (337 mg) and 4 (10 mg) were provided. Fr. H was separated over silica gel column
(petroleum ether-EtOAc, 3:1) to give seven subfractions, H1-H7. Subfraction H2 was chromatographed over silica gel column (petroleum ether-acetone, 3:1) and then purified by silica gel column (CHCl3-EtOAc, 20:1) to obtain 8 (211 mg). 6 (215 mg) was isolated from subfraction H5 by Sephadex LH-20 (CHCl3-MeOH, 1:1) and then fractionated by silica gel column (petroleum ether–acetone, 5:1). Fr. I was separated over silica gel column eluted with petroleum etherEtOAc (5:2) to give eleven subfractions, I1 to I11. Subfraction I1 was purified by silica gel column (petroleum ether-EtOAc, 5:1) to yield 12 (17 mg). Subfraction I2 was further fractionated by silica gel column using petroleum ether-EtOAc (5:2) to provide five subfractions, I2-1 to I2-5. 2 (10 mg) was obtained from subfraction I2-2 by Sephadex LH20 (CHCl3-MeOH, 1:1). Subfraction I3 was subject to silica gel column, eluting with petroleum ether-EtOAc (3:1), to afford 9 (320 mg). Subfraction I6 was chromatographed on silica gel column using petroleum ether-EtOAc (3:2) to give 10 (77 mg). Subfraction I11 was subjected to repeated column chromatography and purified by Sephadex LH20 and semipreparative HPLC (MeOH-H2O, 50:50) to afford 5 (20 mg, tR 12.1 min), 1 (12 mg, tR 22.4 min), 3 (11 mg, tR 27.8 min). Cracroson A (1): colorless crystals (MeOH); mp 262–265 °C; [α]D25 + 27 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 209 (1.47) nm; IR (KBr) max 3416, 3150, 1762, 1475, 1384, 1344, 1212, 1185, 1018, 980, 873, 599 cm− 1; 1H and 13C NMR data, see Table 1; HRESIMS [M]+ m/z 345.1335 (calcd for C19H21O6, 345.1332). Cracroson B (2): colorless crystals (MeOH); mp 194–195 °C; [α]D25 − 30.2 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 215 (2.61) nm; IR (KBr) max 3442, 3153, 2965, 1754, 1708, 1653, 1455, 1225, 876, 782, 599 cm−1; 1H and 13C NMR data, see Table 1; HREIMS [M]+ m/z 358.1411 (calcd for C20H22O6, 358.1411). Cracroson C (3): colorless crystals (MeOH); mp 217–219 °C; [α]D25 − 67 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 278 (3.18) nm; IR (KBr) max 3424, 2910, 1761, 1685, 1384, 1183, 1162, 1007 cm− 1; 1 H and 13 C NMR data, see Table 1; HREIMS [M] + m/z 325.1303 (calcd for C19H19O4N, 325.1309). 2.4. X-ray crystal structure analysis of compounds 1 and 4 Colorless crystals of 1 and 4 were obtained from MeOH. The intensity data were collected on an Xcalibur Onyx Nova diffractometer with Cu
Table 1 1 H and 13C NMR spectroscopic data of compounds 1–3 [400 MHz, CDCl3, δ (ppm), (J in Hz)]. Position
1
2
3
δC
δH
δC
δH
δC
δH
1
25.1
21.2
29.9
3
17.3
1.64 m 1.93 dd (14.1, 6.9) 2.06 m 1.41 dddd (14.1, 10.6, 6.9, 3.5) 2.24 m 2.31 dd (14.1, 2.3)
22.0
2
2.00 m 2.37 dd (14.0, 9.2) 1.48 dt (14.0, 2.8) 2.08 m 1.95 m 2.21 m
1.74 m 2.15 m 1.35 m 1.91 m 2.25 m 2.43 m
4 5 6 7
69.2 137.4 77.0 32.6
8 9 10 11
35.1 52.7 133.1 39.7
12 13 14 15 16 17 18 20
72.7 124.6 108.1 144.5 139.9 16.2 176.1 176.2 OH
5.14 t (7.6) 2.12 m 1.82 m
2.29 dd (14.0, 9.2) 2.73 dd (14.0, 8.4) 5.49 t (8.4) 6.42 s 7.46 s 7.49 s 1.14 d (7.6)
3.48 s
22.9 19.9 169.6 131.7 104.5 37.9 38.4 53.4 33.9 38.4 71.7 125.8 107.8 144.4 139.1 15.2 158.9 177.0 50.2/OMe
1.92 m 2.58 dd (13.9, 8.9) 2.10 m 3.21 m 2.34 m 1.83 dd (14.1, 5.9) 5.39 dd (8.9, 5.9) 6.33 dd (1.7, 0.8) 7.43 t (1.7) 7.41 m 1.32 d (7.4)
3.16 s
22.9 19.6 143.1 129.8 135.0 110.6 40.4 51.2 33.7 37.9 71.5 125.8 107.9 144.1 138.9 16.2 171.7 177.1 NH
5.44 d (6.4) 2.61 m 3.20 m 2.00 dd (13.8, 6.0) 2.62 m 5.38 dd (8.3, 6.0) 6.34 s 7.43 s 7.40 s 1.29 d (7.0)
7.70 s
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Kα radiation. The crystal structures of 1 and 4 were solved by the direct method (SHLXS-97), expanded using difference Fourier technique, and refined by the program and the full-matrix least-squares calculations. The nonhydrogen atoms were refined anisotropically, and hydrogen atoms were fixed at calculated positions. Crystallographic data for the structures of 1 and 4 have been deposited in the Cambridge Crystallographic Data Centre (deposition numbers: CCDC 1402275 and 1402261). Copies of these data can be obtained free of charge via
83
www.ccdc.cam.ac.uk (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+ 44) 1223336-033; or [email protected]). Crystal data of 1: C19H20O6·CH4O,Mr. = 376.39, monoclinic, space group, P21, a = 7.76825 (7) Å, b = 12.07112 (12) Å, c = 9.55484 (9) Å; α = 90.00°, β = 99.5128 (9)°, γ = 90.00°, V = 883.65 (1) Å3, Z = 2, μ(Cu Kα) = 0.89 mm−1, DX = 1.415 Mg/m3, F(000) = 400, 3419 reflections independent and 3394 reflections observed [I N 2σ(I)].
Fig. 1. Structures of compounds 1–16.
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The final R[F2 N 2σ(F2)] = 0.029, wR(F2) = 0.074, Flack parameter = 0.06 (11), S = 1.07. Crystal data of 4: C19H20O5, Mr. = 328.4, Monoclinic, space group, P21, a = 10.3857 (2) Å, b = 6.15709 (13) Å, c = 12.9966 (2) Å; α = 90.00°, β = 100.2672 (18) °, γ = 90.00°, V = 817.77 (3) Å3, Z = 2, μ(Cu Kα) = 0.79 mm−1, DX = 1.333 Mg/m3, F(000) = 348, 3113 reflections independent and 2988 reflections observed [I N 2σ(I)]. The final R[F2 N 2σ(F2)] = 0.036, wR(F2) = 0.098, flack parameter = 0.07(17), S = 1.04. 2.5. Cytotoxicity assay against CT26.WT cell The cytotoxicity of the compounds against CT26.WT cell line was assessed using the MTT method. Cells were plated in 96-well plates 24 h before treatment and continuously exposed to different concentrations of compounds. After 20 h, MTT(10 μL, 5 mg/mL) was added to each well, which were incubated for another 24 h. Then DMSO(100 μL) was added to each well. Finally, the OD value of each well was recorded at 570 nm. The results were expressed as the percentage of inhibition that produced a reduction in the absorbance by the treatment of the compounds compared to the controls. 3. Results and discussion Air-dried roots of C. crassifolius were percolated with 80% MeOH, and then the extract was concentrated under reduced pressure to leave a residue, which was suspended in H2O and successively extracted with petroleum ether, CHCl3, and EtOAc. The CHCl3 fraction was subjected repeatedly to column chromatography over silica gel, Sephadex LH20, and semipreparative HPLC to afford two new clerodane diterpenoids (1–2), one new clerodane diterpenoid alkaloid (3), and thirteen known (4–16) compounds. The known compounds were identified as crassifolin H (4) [6], crassifolin I (5) [6], crassifolin B (6) [2], crassifolin A (7) [2], isoteufin (8) [15], teucvidin (9) [16], teucvin (10) [16], chettaphanin I (11) [17], (12S)-15,16-epoxy-6β-methyoxy-19-norneoclerodane4,13(16),14-triene-18,6α,20,12-diolide (12) [18], cyperenol (13) [9], cyperenoic acid (14) [9], acetyl aleuritolic acid (15) [19], lupeol (16) [9], respectively, by comparison of observed and reported spectroscopic and mass spectrum data. Compound 1 was isolated as colorless crystals. Its molecular formula C19H21O6 was determined by the HRESIMS at m/z 345.1335, [M]+, corresponding to ten degrees of unsaturation. The IR absorption bands of 1 revealed the presence of characteristic hydroxyl (3416 cm−1), lactone (1762, 1212 cm− 1) and a furan ring [20] (3150, 1475, 873 cm−1)
absorptions. The 1H NMR spectrum of 1 (Table 1) showed the existence of a methyl (δH 1.14, d, J = 7.6 Hz), and a typical β-substituted furan ring [21] [δH 6.42, 7.46, and 7.49 (1 H each, H-14, H-15, and H-16)]. The 13C NMR spectrum (Table 1) displayed 19 carbon signals, with the aid of DEPT and HSQC spectra, were assigned to one methyl (δC 16.2), five aliphatic methylene carbons (δC 17.3, 25.1, 29.9, 32.6, and 39.7), three aliphatic methine carbons (δC 35.1, 72.7, and 77.0), two aliphatic quaternary carbons (δC 52.7 and 69.2), three olefinic methine carbons (δC 108.1, 139.9 and 144.5), three olefinic quaternary carbons (δC 124.6, 133.1 and 137.4), and two ester carbonyl signals (δC 176.1 and 176.2). The HMBC correlations (Fig. 2) from H-14 to C-13, C-15, and C-16 further proved the existence of the furan ring, and from H-12 to C-13, C14 and C-16 indicated the presence of the furan ring at C-12. The COSY correlations (Fig. 2) of H-11/H-12 and HMBC correlations of H-11/C-8, C-9, C-13, C-20 and H-8/C-11, C-20 suggested the presence of lactonic ring (ring D), supported by the chemical shifts of C-20 (δC 176.2). The A and B rings (Fig. 2) were affirmed by the COSY correlations of H-1/H-2, H-6/H-7/H-8 and HMBC correlations of H-1/C-3, C-5, C-10, H-2/C-10, H-3/C-5, H-6/C-5, C-10 and H-7/C-6, C-9, C-10. The chemical shifts of C-4 (δC 69.2), C-6 (δC 77.0) and C-18 (δC 176.1) indicated the location of lactone (ring C) at C-4 and C-6. With the aid of COSY correlations of H-17/H-8 and HMBC correlations of H-17/C-7, C-8, the location of the C-17 was confirmed at C-8. The aforementioned spectroscopic analysis, in conjunction with biogenetic considerations, 1 was considered to be a clerodane diterpenoid. Detailed analysis of the NMR spectroscopic data of 1 with 4, a previously known clerodane diterpenoid, indicated their structural similarity. The main differences between them were the presence of an additional hydroxyl in 1 and the chemical shifts of C-4 (δC 69.2 in 1; δC 39.7 in 4), suggesting that the hydroxyl group was located at C-4. The signal assignments were completed by analysis of 1H–1H COSY, HSQC, HMBC and NOESY correlations. To further verify the configurations, 1 was crystallized and this was submitted for X-ray structural analysis (Fig. 4). The results affirmed our NMR assignments and established the relative stereochemistry of 1. The absolute configurations of C-4, C-6, C-8, C-9 and C-12 were thus assigned as S, S, R, R, and S respectively. The structure of 1 was therefore established and named as cracroson A. Compound 2 was obtained as colorless crystals. Its molecular formula was deduced to be C20H22O6 from the molecular ion at m/z 358.1411 [M]+ in its HREIMS. The IR spectrum showed absorptions indicating an ester lactone (1754, 1225 cm−1), carbonyl (1708 cm−1), olefinic (1653 cm−1), and a furan ring [16] (3138, 1504, 876 cm−1). The 1H NMR date of 2 (Table 1) revealed a methyl (δH 1.32, d, J = 7.4 Hz), a methoxyl (δH 3.16, s) and a β-substituted furan ring [21] [δH 6.33 (dd,
Fig. 2. Key COSY and HMBC correlations of compounds 1–3.
M. Qiu et al. / Fitoterapia 108 (2016) 81–86
85
Fig. 3. Key NOESY correlations of compounds 1–3.
J = 1.7, 0.8 Hz), 7.41 (m), 7.43 (t, J = 1.7 Hz)]. The 13C NMR and DEPT spectrum (Table 1) showed 20 carbon signals for one methyl (δC 15.2), one methoxyl (δC 50.2), six olefinic carbons (δC 107.8, 125.8, 131.7, 139.1, 144.4 and 169.6) and two carbonyl signals (δC 158.9 and 177.0). The NMR data of 2 were similar to those of teucvidin (compound 9) [16], suggesting a close analog of teucvidin. The differences between them were chemical shift for C-6 (δC 104.5 in 2; δC 76.0 in teucvidin) and an additional methoxyl in 2. The additional methoxyl group was presumed to be attached to C-6.This was confirmed by NOESY (Fig. 3) correlations of H3-19 (δ3.16, s)/H3-17 (δ1.32, d, J = 7.4 Hz). From the above evidences and combined with biogenetic considerations, Me-17 and OMe-19 both possessed α-orientations. On the basis of the above analysis, the structure of 2 was finally established and named cracroson B. Compound 3 was isolated as colorless crystals, and had a molecular formula of C19H19O4N as deduced from its HREIMS spectrum (m/z 325.1303, [M]+) and 13C NMR spectra, indicating eleven degrees of unsaturation. The IR bands showed the presence of carbonyl (1761 cm−1), and conjugated lactam [22] (1685 cm−1) group. The 1H
NMR data of 3 (Table 1) revealed a methyl (δH 1.29, d, J = 7.0 Hz), and four olefinic protons [δH 5.44 (d, J = 6.4), 6.34 (s), 7.40 (s), 7.43 (s) Hz)]. The 13C NMR (Table 1) and DEPT spectra showed 19 carbon signals, including one methyl (δC 16.2), four aliphatic methylene (δC 19.6, 22.0, 22.9 and 37.9), three methine (δC 33.7, 40.4, 71.5), one aliphatic quaternary carbon (δC 51.2), four olefinic methine (δC 107.9, 110.6, 138.9, 144.1), four olefinic quaternary carbon (δC 125.8, 129.8, 135.0, 143.1), one carbonyl (δC 177.1) and an amide carbonyl signal (δC 171.7). In the HSQC spectrum, the proton signal as δH 7.70 (s) was not correlated to any carbon signal suggesting the proton (δH 7.70) belongs to NH group. The aforementioned spectroscopic analysis suggested 3 was a clerodane diterpenoid alkaloid, whose NMR data were very similar to those of teucvidin (compound 9) [16]. The obvious difference was the appearance of two extra olefinic carbons (δC 110.6 and 135.0) in 3 and the absence of C-6 (δC 76.0) and C-7 (δC 35.7) in teucvidin, indicated a double bond was located at C-6 and C-7. The other difference was the presence of a conjugated lactam located at C4 and C-6 in 3, instead of the ester lactone at C-4 and C-6 in teucvidin.
Fig. 4. X-ray crystallographic structure of compounds 1 and 4.
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This was verified by the HMBC (Fig. 3) correlations of H (δH 7.70)/C-4, C5, C-6, C-18. Moreover, the α-orientation of H-10 and CH3-17 were confirmed by the NOESY correlations of H-3α (δH 1.91, m)/H-10 (δH 3.20, m)/H3-17 (δH 1.29, d, J = 7.0 Hz) (Fig. 3). The signal assignments were completed by the analysis of 1H–1H COSY, HSQC, HMBC and NOESY correlations, and the structure of 3 was established as shown in Fig. 1, and named cracroson C. Compound 4 was isolated as colorless crystals. Its molecular formula C19H20O5 was determined by the HREIMS at m/z 328.1301 [M]+. The signal assignments were completed by 1D and 2D NMR, and the results showed it was a previously reported diterpenoid, crassifolin H [6]. Although 4 is a known compound, its absolute configurations have not been fully demonstrated. Therefore, a single crystal X-ray diffraction study of 4 was performed, and the absolute configurations of C-4, C-6, C-8, C-9 and C-12 were thus assigned as R, S, R, R and S, respectively (Fig. 4). C19-diterpenoid alkaloids comprise a large class of structurally complex natural products, and were classified as aconitines, lycoctonines, pyro-type, lactone type, 7,17-seco type and rearranged type [23]. However, the clerodane C19-diterpenoid alkaloid has not been reported in natural products. To the best of our knowledge, compound 3 represents the first example of a clerodane diterpenoid alkaloid, which is of particular significance for the phytochemical investigation of C19-diterpenoid alkaloids. From the perspective of biogenetic relationship, the nitrogen atom in compound 3 does not seem to be derived from an amino acid, and it is probably originated from ammonia. Therefore, compound 3 is likely to be a diterpene pseudo-alkaloid, and the possible biosynthetic process of the conjugated lactam in compound 3 is one of the problems which needs to be further studied. All compounds were tested for their cytotoxicity against CT26.WT cell line using the MTT method as previously reported [24]. Cisplatin was included as a positive control and for comparison purposes. The results showed that compound 6 exhibited weak cytotoxicity against the CT26.WT cell. The IC50 values of cisplatin and 6 were 33.3 ± 2.6 and 96.6 ± 17.3 μM, respectively. The other compounds were inactive (IC50 N 100 μM).
Acknowledgments This work was supported by the program for Outstanding Young Teachers in Higher Education Institutions of Guangdong Province (Yq2013045).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2015.11.016.
References [1] Y. W, Z. Z, P. C, Study on the diterpenes and their bioactivities in croton plant[J], Int. J. Tradit. Chin. Med. 28 (1) (2006) 17–26. [2] G.C. Wang, J.G. Li, G.Q. Li, J.J. Xu, X. Wu, W.C. Ye, et al., Clerodane diterpenoids from Croton crassifolius[J], J. Nat. Prod. 75 (12) (2012) 2188–2192. [3] L. Boonyarathanakornkit, C.T. Che, H.H. Fong, N.R. Farnsworth, Constituents of Croton crassifolius roots [J], Planta Med. 54 (1) (1988) 61–63. [4] S. Boonyaratavej, S. Roengsumran, R.B. Bates, R.B. Ortega, A. Petsom, X-ray structure of cyperenoic acid from Croton crassifolius[J], J. Nat. Prod. 51 (4) (1988) 769–770. [5] Y. Hu, L. Zhang, X.Q. Wen, X.J. Zeng, W. Rui, Y.Z. Cen, Two new diterpenoids from Croton crassifolius[J], J. Asian Nat. Prod. Res. 14 (8) (2012) 785–788. [6] W.H. Huang, G.Q. Li, J.G. Li, X. Wu, G. Wei, Two new clerodane diterpenoids from Croton crassifolius[J], ChemInform 89 (2014) 1585–1593. [7] Z.X. Zhang, H.H. Li, F.M. Qi, L.L. Dong, Y. Hai, G.X. Fan, et al., Crocrassins a and B: two novel sesquiterpenoids with an unprecedented carbon skeleton from Croton crassifolius[J], RSC Adv. 4 (57) (2014) 30059. [8] Z.X. Zhang, H.H. Li, F.M. Qi, H.Y. Xiong, L.L. Dong, G.X. Fan, et al., A new halimane diterpenoid from Croton crassifolius[J], Bull. Kor. Chem. Soc. 35 (5) (2014) 1556–1558. [9] X.H. Yang, S.W. Chen, S.M. Deng, Study on the chemical constituents of Croton crassifolius[J], Lishizhen Med. Mater. Med. Res. 3 (2009) 001. [10] H.H. Li, F.M. Qi, L.L. Dong, G.X. Fan, J.M. Che, D.D. Guo, et al., Cytotoxic and antibacterial pyran-2-one derivatives from Croton crassifolius[J], Phytochem. Lett. 10 (2014) 304–308. [11] C.P. Liu, J.B. Xu, J.X. Zhao, C.H. Xu, L. Dong, J. Ding, et al., Diterpenoids from Croton laui and their cytotoxic and antimicrobial activities[J], J. Nat. Prod. 77 (4) (2014) 1013–1020. [12] M.A. Salah, E. Bedir, N.J. Toyang, I.A. Khan, M.D. Harries, D.E. Wedge, Antifungal clerodane diterpenes from Macaranga monandra (L) Muell. et Arg. (Euphorbiaceae)[J], J. Agric. Food Chem. 51 (26) (2003) 7607–7610. [13] Y. Sun, M. Wang, Q. Ren, S. Li, J. Xu, Y. Ohizumi, et al., Two novel clerodane diterpenenes with NGF-potentiating activities from the twigs of Croton yanhuii[J], Fitoterapia 95 (2014) 229–233. [14] J. Xu, Q. Zhang, M. Wang, Q. Ren, Y. Sun, D.Q. Jin, et al., Bioactive clerodane diterpenoids from the twigs of Casearia balansae[J], J. Nat. Prod. 77 (10) (2014) 2182–2189. [15] M. Bruno, F. Piozzi, G. Savona, C. Maria, B. Rodriguez, Neo-clerodane diterpenoids from Teucrium canadense[J], Phytochemistry 28 (12) (1989) 3539–3541. [16] B. Rodriguez, M.L. Jimeno, 1H and 13C NMR spectral assignments and conformational analysis of 14 19-nor-neoclerodane diterpenoids[J], Magn. Reson. Chem. 42 (7) (2004) 605–616. [17] I.S. Marcos, F.A. Hernandez, M.J. Sexmero, D. Diez, P. Basabe, A.B. Pedrero, et al., Synthesis and absolute configuration of (−)-chettaphanin I and (−)-chettaphanin II[J], Tetrahedron 59 (5) (2003) 685–694. [18] G. Dominguez, M.C. De La Torre, B. Rodriguez, Transformation of neoclerodane diterpenoids into 19-norneoclerodane derivatives[J], J. Org. Chem. 56 (23) (1991) 6595–6600. [19] Y.F. Xie, Z.M. Tao, H.B. Wang, G.W. Qin, Chemical constituents from the roots of Vernicia fordii[J], Chin. J. Nat. Med. 8 (4) (2010) 264–266. [20] H.W. Lv, J.G. Luo, M.D. Zhu, S.M. Shan, L.Y. Kong, Teucvisins A–E, five new neoclerodane diterpenes from Teucrium viscidum[J], Chem. Pharm. Bull. (Tokyo) 62 (5) (2014) 472–476. [21] Z. Pan, D. Ning, X. Wu, S. Huang, D. Li, S. Lv, New clerodane diterpenoids from the twigs and leaves of Croton euryphyllus[J], Bioorg. Med. Chem. Lett. 25 (6) (2015) 1329–1332. [22] Y.C. Shen, Y.B. Cheng, J. Kobayashi, T. Kubota, Y. Takahashi, Y. Mikami, et al., Nitrogen-containing verticillene diterpenoids from the Taiwanese soft coral Cespitularia taeniata[J], J. Nat. Prod. 70 (12) (2007) 1961–1965. [23] F.P. Wang, Q.H. Chen, X.Y. Liu, Diterpenoid alkaloids[J], Nat. Prod. Rep. 27 (4) (2010) 529–570. [24] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[J], J. Immunol. Methods 65 (1–2) (1983) 55–63.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
New clerodane diterpenoids from Croton crassifolius☆ Maosong Qiu b, Di Cao b, Youheng Gao b, Shuhua Li b, Jinping Zhu b, Bao Yang b, Lian Zhou b, Yuan Zhou b, Jing Jin a,⁎, Zhongxiang Zhao b,⁎⁎ a b
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
a r t i c l e
i n f o
Article history: Received 5 October 2015 Received in revised form 14 November 2015 Accepted 19 November 2015 Available online 21 November 2015 Keywords: Croton crassifolius Clerodane diterpenoids Alkaloid MTT method
a b s t r a c t Two new clerodane diterpenoids (1–2), one new clerodane diterpenoid alkaloid (3), as well as thirteen known compounds were isolated from Croton crassifolius. The structures of new compounds were established by a combination of spectroscopic methods, including HRMS, 1H NMR, 13C NMR, 1H 1H COSY, HSQC, HMBC, NOESY and Xray crystallographic analysis. Compound 3 is firstly reported as the clerodane-type diterpenoid alkaloid in natural products. All of the compounds were evaluated for in vitro cytotoxic activities against CT26.WT cell using the MTT method. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The genus Croton (family Euphorbiaceae) contains about 800 species, widely distributed throughout tropical areas in the world and south region of China, and the plants of this genus are rich in biological active diterpenoids, including clerodanes, kauranes, labdanes and trachylobanes [1]. Croton crassifolius Geisel is mainly distributed in south China and other areas in Southeast Asia [2], and has been traditionally used for the treatment of stomach pain, sore throat and cancer [3].Previous phytochemical investigations have revealed that C. crassifolius is abundant with terpenoids, including sesquiterpenes, diterpenoids and triterpenoids, in which clerodane-type diterpenoids are considered as the main and characteristic components [4–8]. Up to now, more than 16 clerodane-type diterpenoids have been isolated from different parts of this plant, including chettaphanin I [3], 9-[2(2(5H)-furanone-4-yl)ethyl]-4,8,9-trimethyl-1,2,3,4,5,6,7,8-octahydronaphthalene-4-carboxylic acid, methyl 9-[2-(2(5H)-furanone-4yl)ethyl]-4,8,9-trimethyl-1,2,3,4,5,6,7,8-octahydro-naphthalene-4-carboxylic ester [5], crassifolin A–G, spiro[furan-3-(2 H),1′(2′H)-naphthalene]-5′-carboxylic acid, penduliflaworosin, 1,4-methano-3-benzoxepin-2(1 H)-one, isoteucvin, teucvin [2], mallotucin B [9], and crassifolin H-I [6]. In addition, two novel sesquiterpenoids (Crocrassins A and B
☆ The authors declare no competing financial interest. ⁎ Correspondence to: J. Jin, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, Guangdong Province 510006, China. ⁎⁎ Correspondence to: Z. Zhao, School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province 510006, China. E-mail addresses: [email protected] (J. Jin), [email protected] (Z. Zhao).
http://dx.doi.org/10.1016/j.fitote.2015.11.016 0367-326X/© 2015 Elsevier B.V. All rights reserved.
[7]), a halimane diterpenoid (crassifoliusin A [8]), and several other constituents [10] have been reported. Pharmacological investigations have demonstrated that clerodane-type diterpenoids are a rich source of biological activities [11–14]. A phytochemical investigation of the roots of C. crassifolius was thus performed as part of our continuous work on the discovery of new or bioactive compounds from Chinese herbal medicines. This led to isolation of two new clerodane diterpenoids, cracrosons A–B (1–2), one new clerodane diterpenoid alkaloid, cracroson C (3), along with thirteen known compounds. In this paper, the isolation and structure elucidation of these compounds are described, with their inhibition activities on CT26.WT cell proliferation also being assayed. 2. Experimental 2.1. General procedures Melting points were obtained on an X-4 micro-melting point apparatus. Optical rotations were measured with a Perkin-Elmer precisely model 341 polarimeter (Perkin-Elmer, Inc., Waltham, MA). UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. IR spectra were recorded on a Nicolet Nexus 300 FTIR spectrometer (Thermo Nicolet Corporation, Madison, USA). 1D and 2D-NMR were performed on a Bruker AVANCE DRX-400 NMR spectrometer (Bruker Biospin Gmbh, Rheistetten, Germany) in CDCl3 using tetramethylsilane (TMS) as internal standard. HREIMS and HRESIMS were determined on a MAT 95 XP spectrometer (Thermo Finnigan, Bremen, Germany) and an LTQ-Orbitrap Elite spectrometer (Thermo Fisher Scientific, San Jose, CA), respectively. Column chromatography was carried out with silica
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gel (100–200, 200–300 and 300–400 mesh, Qingdao Marine Chemical Co., Qingdao, China), C18 reversed-phase silica gel (YMC CO., LTD, Kyoto, Japan) and Sephadex LH-20 (GE-Healthcare Bio-Sciences AB, Uppsala, Sweden). Further purifications were performed by semipreparative HPLC on a RP-C18 column (5 μm, 250 × 10 mm, Kromasil Co., Bohus, Sweden) with a Wufeng LC-100 apparatus (Shanghai Wufeng Technical Instrument Co., Shanghai, China), at the flow rate of 3 mL/min, and the temperature of 30 °C. 2.2. Plant material The roots of C. crassifolius were collected in Fujian Province, People's Republic of China, in June of 2009, and were authenticated by Prof. Zhong-Xiang Zhao of Guangzhou University of Chinese Medicine, China. A voucher specimen (No. 200906) was deposited in the School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, China. 2.3. Extraction and isolation The dried and powdered roots of C. crassifolius (4.5 kg) were percolated with 80% MeOH (40 L) at room temperature. The extracts were combined and concentrated under reduced pressure to leave a residue, which was then suspended in H2O (2.5 L) and partitioned in petroleum ether (2.5 L × 3), CHCl3 (2.5 L × 3), and EtOAc (2.5 L × 3). The CHCl3 extract (210 g) was chromatographed on silica gel column with gradient mixtures of petroleum ether–acetone (100:1 → 5:1) to afford nine fractions, Fr. A to Fr. I. Fr. A was crystallized from CHCl3-acetone (3:1) to yield 14 (20 g). 15 (500 mg) was provided from Fr. B by Sephadex LH-20 using CHCl3-MeOH (1:1). Fr. C was subjected to Sephadex LH20, eluting with CHCl3-MeOH (1:1), to provide 16 (450 mg). Fr. D was fractionated by Sephadex LH-20 (CHCl3-MeOH, 1:1) and then chromatographed on silica gel column eluted with petroleum etherEtOAc (20:1) to afford 13 (150 mg). Fr. F was separated by Sephadex LH-20 (CHCl3-MeOH, 1:1) and further subjected to silica gel column (petroleum ether-EtOAc, 7:1) to obtain 7 (54 mg). Fr. G was fractionated by Sephadex LH-20 (CHCl3-MeOH, 1:1), then separated over silica gel column eluted with petroleum ether-EtOAc (3:1), and 11 (337 mg) and 4 (10 mg) were provided. Fr. H was separated over silica gel column
(petroleum ether-EtOAc, 3:1) to give seven subfractions, H1-H7. Subfraction H2 was chromatographed over silica gel column (petroleum ether-acetone, 3:1) and then purified by silica gel column (CHCl3-EtOAc, 20:1) to obtain 8 (211 mg). 6 (215 mg) was isolated from subfraction H5 by Sephadex LH-20 (CHCl3-MeOH, 1:1) and then fractionated by silica gel column (petroleum ether–acetone, 5:1). Fr. I was separated over silica gel column eluted with petroleum etherEtOAc (5:2) to give eleven subfractions, I1 to I11. Subfraction I1 was purified by silica gel column (petroleum ether-EtOAc, 5:1) to yield 12 (17 mg). Subfraction I2 was further fractionated by silica gel column using petroleum ether-EtOAc (5:2) to provide five subfractions, I2-1 to I2-5. 2 (10 mg) was obtained from subfraction I2-2 by Sephadex LH20 (CHCl3-MeOH, 1:1). Subfraction I3 was subject to silica gel column, eluting with petroleum ether-EtOAc (3:1), to afford 9 (320 mg). Subfraction I6 was chromatographed on silica gel column using petroleum ether-EtOAc (3:2) to give 10 (77 mg). Subfraction I11 was subjected to repeated column chromatography and purified by Sephadex LH20 and semipreparative HPLC (MeOH-H2O, 50:50) to afford 5 (20 mg, tR 12.1 min), 1 (12 mg, tR 22.4 min), 3 (11 mg, tR 27.8 min). Cracroson A (1): colorless crystals (MeOH); mp 262–265 °C; [α]D25 + 27 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 209 (1.47) nm; IR (KBr) max 3416, 3150, 1762, 1475, 1384, 1344, 1212, 1185, 1018, 980, 873, 599 cm− 1; 1H and 13C NMR data, see Table 1; HRESIMS [M]+ m/z 345.1335 (calcd for C19H21O6, 345.1332). Cracroson B (2): colorless crystals (MeOH); mp 194–195 °C; [α]D25 − 30.2 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 215 (2.61) nm; IR (KBr) max 3442, 3153, 2965, 1754, 1708, 1653, 1455, 1225, 876, 782, 599 cm−1; 1H and 13C NMR data, see Table 1; HREIMS [M]+ m/z 358.1411 (calcd for C20H22O6, 358.1411). Cracroson C (3): colorless crystals (MeOH); mp 217–219 °C; [α]D25 − 67 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 278 (3.18) nm; IR (KBr) max 3424, 2910, 1761, 1685, 1384, 1183, 1162, 1007 cm− 1; 1 H and 13 C NMR data, see Table 1; HREIMS [M] + m/z 325.1303 (calcd for C19H19O4N, 325.1309). 2.4. X-ray crystal structure analysis of compounds 1 and 4 Colorless crystals of 1 and 4 were obtained from MeOH. The intensity data were collected on an Xcalibur Onyx Nova diffractometer with Cu
Table 1 1 H and 13C NMR spectroscopic data of compounds 1–3 [400 MHz, CDCl3, δ (ppm), (J in Hz)]. Position
1
2
3
δC
δH
δC
δH
δC
δH
1
25.1
21.2
29.9
3
17.3
1.64 m 1.93 dd (14.1, 6.9) 2.06 m 1.41 dddd (14.1, 10.6, 6.9, 3.5) 2.24 m 2.31 dd (14.1, 2.3)
22.0
2
2.00 m 2.37 dd (14.0, 9.2) 1.48 dt (14.0, 2.8) 2.08 m 1.95 m 2.21 m
1.74 m 2.15 m 1.35 m 1.91 m 2.25 m 2.43 m
4 5 6 7
69.2 137.4 77.0 32.6
8 9 10 11
35.1 52.7 133.1 39.7
12 13 14 15 16 17 18 20
72.7 124.6 108.1 144.5 139.9 16.2 176.1 176.2 OH
5.14 t (7.6) 2.12 m 1.82 m
2.29 dd (14.0, 9.2) 2.73 dd (14.0, 8.4) 5.49 t (8.4) 6.42 s 7.46 s 7.49 s 1.14 d (7.6)
3.48 s
22.9 19.9 169.6 131.7 104.5 37.9 38.4 53.4 33.9 38.4 71.7 125.8 107.8 144.4 139.1 15.2 158.9 177.0 50.2/OMe
1.92 m 2.58 dd (13.9, 8.9) 2.10 m 3.21 m 2.34 m 1.83 dd (14.1, 5.9) 5.39 dd (8.9, 5.9) 6.33 dd (1.7, 0.8) 7.43 t (1.7) 7.41 m 1.32 d (7.4)
3.16 s
22.9 19.6 143.1 129.8 135.0 110.6 40.4 51.2 33.7 37.9 71.5 125.8 107.9 144.1 138.9 16.2 171.7 177.1 NH
5.44 d (6.4) 2.61 m 3.20 m 2.00 dd (13.8, 6.0) 2.62 m 5.38 dd (8.3, 6.0) 6.34 s 7.43 s 7.40 s 1.29 d (7.0)
7.70 s
M. Qiu et al. / Fitoterapia 108 (2016) 81–86
Kα radiation. The crystal structures of 1 and 4 were solved by the direct method (SHLXS-97), expanded using difference Fourier technique, and refined by the program and the full-matrix least-squares calculations. The nonhydrogen atoms were refined anisotropically, and hydrogen atoms were fixed at calculated positions. Crystallographic data for the structures of 1 and 4 have been deposited in the Cambridge Crystallographic Data Centre (deposition numbers: CCDC 1402275 and 1402261). Copies of these data can be obtained free of charge via
83
www.ccdc.cam.ac.uk (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+ 44) 1223336-033; or [email protected]). Crystal data of 1: C19H20O6·CH4O,Mr. = 376.39, monoclinic, space group, P21, a = 7.76825 (7) Å, b = 12.07112 (12) Å, c = 9.55484 (9) Å; α = 90.00°, β = 99.5128 (9)°, γ = 90.00°, V = 883.65 (1) Å3, Z = 2, μ(Cu Kα) = 0.89 mm−1, DX = 1.415 Mg/m3, F(000) = 400, 3419 reflections independent and 3394 reflections observed [I N 2σ(I)].
Fig. 1. Structures of compounds 1–16.
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The final R[F2 N 2σ(F2)] = 0.029, wR(F2) = 0.074, Flack parameter = 0.06 (11), S = 1.07. Crystal data of 4: C19H20O5, Mr. = 328.4, Monoclinic, space group, P21, a = 10.3857 (2) Å, b = 6.15709 (13) Å, c = 12.9966 (2) Å; α = 90.00°, β = 100.2672 (18) °, γ = 90.00°, V = 817.77 (3) Å3, Z = 2, μ(Cu Kα) = 0.79 mm−1, DX = 1.333 Mg/m3, F(000) = 348, 3113 reflections independent and 2988 reflections observed [I N 2σ(I)]. The final R[F2 N 2σ(F2)] = 0.036, wR(F2) = 0.098, flack parameter = 0.07(17), S = 1.04. 2.5. Cytotoxicity assay against CT26.WT cell The cytotoxicity of the compounds against CT26.WT cell line was assessed using the MTT method. Cells were plated in 96-well plates 24 h before treatment and continuously exposed to different concentrations of compounds. After 20 h, MTT(10 μL, 5 mg/mL) was added to each well, which were incubated for another 24 h. Then DMSO(100 μL) was added to each well. Finally, the OD value of each well was recorded at 570 nm. The results were expressed as the percentage of inhibition that produced a reduction in the absorbance by the treatment of the compounds compared to the controls. 3. Results and discussion Air-dried roots of C. crassifolius were percolated with 80% MeOH, and then the extract was concentrated under reduced pressure to leave a residue, which was suspended in H2O and successively extracted with petroleum ether, CHCl3, and EtOAc. The CHCl3 fraction was subjected repeatedly to column chromatography over silica gel, Sephadex LH20, and semipreparative HPLC to afford two new clerodane diterpenoids (1–2), one new clerodane diterpenoid alkaloid (3), and thirteen known (4–16) compounds. The known compounds were identified as crassifolin H (4) [6], crassifolin I (5) [6], crassifolin B (6) [2], crassifolin A (7) [2], isoteufin (8) [15], teucvidin (9) [16], teucvin (10) [16], chettaphanin I (11) [17], (12S)-15,16-epoxy-6β-methyoxy-19-norneoclerodane4,13(16),14-triene-18,6α,20,12-diolide (12) [18], cyperenol (13) [9], cyperenoic acid (14) [9], acetyl aleuritolic acid (15) [19], lupeol (16) [9], respectively, by comparison of observed and reported spectroscopic and mass spectrum data. Compound 1 was isolated as colorless crystals. Its molecular formula C19H21O6 was determined by the HRESIMS at m/z 345.1335, [M]+, corresponding to ten degrees of unsaturation. The IR absorption bands of 1 revealed the presence of characteristic hydroxyl (3416 cm−1), lactone (1762, 1212 cm− 1) and a furan ring [20] (3150, 1475, 873 cm−1)
absorptions. The 1H NMR spectrum of 1 (Table 1) showed the existence of a methyl (δH 1.14, d, J = 7.6 Hz), and a typical β-substituted furan ring [21] [δH 6.42, 7.46, and 7.49 (1 H each, H-14, H-15, and H-16)]. The 13C NMR spectrum (Table 1) displayed 19 carbon signals, with the aid of DEPT and HSQC spectra, were assigned to one methyl (δC 16.2), five aliphatic methylene carbons (δC 17.3, 25.1, 29.9, 32.6, and 39.7), three aliphatic methine carbons (δC 35.1, 72.7, and 77.0), two aliphatic quaternary carbons (δC 52.7 and 69.2), three olefinic methine carbons (δC 108.1, 139.9 and 144.5), three olefinic quaternary carbons (δC 124.6, 133.1 and 137.4), and two ester carbonyl signals (δC 176.1 and 176.2). The HMBC correlations (Fig. 2) from H-14 to C-13, C-15, and C-16 further proved the existence of the furan ring, and from H-12 to C-13, C14 and C-16 indicated the presence of the furan ring at C-12. The COSY correlations (Fig. 2) of H-11/H-12 and HMBC correlations of H-11/C-8, C-9, C-13, C-20 and H-8/C-11, C-20 suggested the presence of lactonic ring (ring D), supported by the chemical shifts of C-20 (δC 176.2). The A and B rings (Fig. 2) were affirmed by the COSY correlations of H-1/H-2, H-6/H-7/H-8 and HMBC correlations of H-1/C-3, C-5, C-10, H-2/C-10, H-3/C-5, H-6/C-5, C-10 and H-7/C-6, C-9, C-10. The chemical shifts of C-4 (δC 69.2), C-6 (δC 77.0) and C-18 (δC 176.1) indicated the location of lactone (ring C) at C-4 and C-6. With the aid of COSY correlations of H-17/H-8 and HMBC correlations of H-17/C-7, C-8, the location of the C-17 was confirmed at C-8. The aforementioned spectroscopic analysis, in conjunction with biogenetic considerations, 1 was considered to be a clerodane diterpenoid. Detailed analysis of the NMR spectroscopic data of 1 with 4, a previously known clerodane diterpenoid, indicated their structural similarity. The main differences between them were the presence of an additional hydroxyl in 1 and the chemical shifts of C-4 (δC 69.2 in 1; δC 39.7 in 4), suggesting that the hydroxyl group was located at C-4. The signal assignments were completed by analysis of 1H–1H COSY, HSQC, HMBC and NOESY correlations. To further verify the configurations, 1 was crystallized and this was submitted for X-ray structural analysis (Fig. 4). The results affirmed our NMR assignments and established the relative stereochemistry of 1. The absolute configurations of C-4, C-6, C-8, C-9 and C-12 were thus assigned as S, S, R, R, and S respectively. The structure of 1 was therefore established and named as cracroson A. Compound 2 was obtained as colorless crystals. Its molecular formula was deduced to be C20H22O6 from the molecular ion at m/z 358.1411 [M]+ in its HREIMS. The IR spectrum showed absorptions indicating an ester lactone (1754, 1225 cm−1), carbonyl (1708 cm−1), olefinic (1653 cm−1), and a furan ring [16] (3138, 1504, 876 cm−1). The 1H NMR date of 2 (Table 1) revealed a methyl (δH 1.32, d, J = 7.4 Hz), a methoxyl (δH 3.16, s) and a β-substituted furan ring [21] [δH 6.33 (dd,
Fig. 2. Key COSY and HMBC correlations of compounds 1–3.
M. Qiu et al. / Fitoterapia 108 (2016) 81–86
85
Fig. 3. Key NOESY correlations of compounds 1–3.
J = 1.7, 0.8 Hz), 7.41 (m), 7.43 (t, J = 1.7 Hz)]. The 13C NMR and DEPT spectrum (Table 1) showed 20 carbon signals for one methyl (δC 15.2), one methoxyl (δC 50.2), six olefinic carbons (δC 107.8, 125.8, 131.7, 139.1, 144.4 and 169.6) and two carbonyl signals (δC 158.9 and 177.0). The NMR data of 2 were similar to those of teucvidin (compound 9) [16], suggesting a close analog of teucvidin. The differences between them were chemical shift for C-6 (δC 104.5 in 2; δC 76.0 in teucvidin) and an additional methoxyl in 2. The additional methoxyl group was presumed to be attached to C-6.This was confirmed by NOESY (Fig. 3) correlations of H3-19 (δ3.16, s)/H3-17 (δ1.32, d, J = 7.4 Hz). From the above evidences and combined with biogenetic considerations, Me-17 and OMe-19 both possessed α-orientations. On the basis of the above analysis, the structure of 2 was finally established and named cracroson B. Compound 3 was isolated as colorless crystals, and had a molecular formula of C19H19O4N as deduced from its HREIMS spectrum (m/z 325.1303, [M]+) and 13C NMR spectra, indicating eleven degrees of unsaturation. The IR bands showed the presence of carbonyl (1761 cm−1), and conjugated lactam [22] (1685 cm−1) group. The 1H
NMR data of 3 (Table 1) revealed a methyl (δH 1.29, d, J = 7.0 Hz), and four olefinic protons [δH 5.44 (d, J = 6.4), 6.34 (s), 7.40 (s), 7.43 (s) Hz)]. The 13C NMR (Table 1) and DEPT spectra showed 19 carbon signals, including one methyl (δC 16.2), four aliphatic methylene (δC 19.6, 22.0, 22.9 and 37.9), three methine (δC 33.7, 40.4, 71.5), one aliphatic quaternary carbon (δC 51.2), four olefinic methine (δC 107.9, 110.6, 138.9, 144.1), four olefinic quaternary carbon (δC 125.8, 129.8, 135.0, 143.1), one carbonyl (δC 177.1) and an amide carbonyl signal (δC 171.7). In the HSQC spectrum, the proton signal as δH 7.70 (s) was not correlated to any carbon signal suggesting the proton (δH 7.70) belongs to NH group. The aforementioned spectroscopic analysis suggested 3 was a clerodane diterpenoid alkaloid, whose NMR data were very similar to those of teucvidin (compound 9) [16]. The obvious difference was the appearance of two extra olefinic carbons (δC 110.6 and 135.0) in 3 and the absence of C-6 (δC 76.0) and C-7 (δC 35.7) in teucvidin, indicated a double bond was located at C-6 and C-7. The other difference was the presence of a conjugated lactam located at C4 and C-6 in 3, instead of the ester lactone at C-4 and C-6 in teucvidin.
Fig. 4. X-ray crystallographic structure of compounds 1 and 4.
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This was verified by the HMBC (Fig. 3) correlations of H (δH 7.70)/C-4, C5, C-6, C-18. Moreover, the α-orientation of H-10 and CH3-17 were confirmed by the NOESY correlations of H-3α (δH 1.91, m)/H-10 (δH 3.20, m)/H3-17 (δH 1.29, d, J = 7.0 Hz) (Fig. 3). The signal assignments were completed by the analysis of 1H–1H COSY, HSQC, HMBC and NOESY correlations, and the structure of 3 was established as shown in Fig. 1, and named cracroson C. Compound 4 was isolated as colorless crystals. Its molecular formula C19H20O5 was determined by the HREIMS at m/z 328.1301 [M]+. The signal assignments were completed by 1D and 2D NMR, and the results showed it was a previously reported diterpenoid, crassifolin H [6]. Although 4 is a known compound, its absolute configurations have not been fully demonstrated. Therefore, a single crystal X-ray diffraction study of 4 was performed, and the absolute configurations of C-4, C-6, C-8, C-9 and C-12 were thus assigned as R, S, R, R and S, respectively (Fig. 4). C19-diterpenoid alkaloids comprise a large class of structurally complex natural products, and were classified as aconitines, lycoctonines, pyro-type, lactone type, 7,17-seco type and rearranged type [23]. However, the clerodane C19-diterpenoid alkaloid has not been reported in natural products. To the best of our knowledge, compound 3 represents the first example of a clerodane diterpenoid alkaloid, which is of particular significance for the phytochemical investigation of C19-diterpenoid alkaloids. From the perspective of biogenetic relationship, the nitrogen atom in compound 3 does not seem to be derived from an amino acid, and it is probably originated from ammonia. Therefore, compound 3 is likely to be a diterpene pseudo-alkaloid, and the possible biosynthetic process of the conjugated lactam in compound 3 is one of the problems which needs to be further studied. All compounds were tested for their cytotoxicity against CT26.WT cell line using the MTT method as previously reported [24]. Cisplatin was included as a positive control and for comparison purposes. The results showed that compound 6 exhibited weak cytotoxicity against the CT26.WT cell. The IC50 values of cisplatin and 6 were 33.3 ± 2.6 and 96.6 ± 17.3 μM, respectively. The other compounds were inactive (IC50 N 100 μM).
Acknowledgments This work was supported by the program for Outstanding Young Teachers in Higher Education Institutions of Guangdong Province (Yq2013045).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2015.11.016.
References [1] Y. W, Z. Z, P. C, Study on the diterpenes and their bioactivities in croton plant[J], Int. J. Tradit. Chin. Med. 28 (1) (2006) 17–26. [2] G.C. Wang, J.G. Li, G.Q. Li, J.J. Xu, X. Wu, W.C. Ye, et al., Clerodane diterpenoids from Croton crassifolius[J], J. Nat. Prod. 75 (12) (2012) 2188–2192. [3] L. Boonyarathanakornkit, C.T. Che, H.H. Fong, N.R. Farnsworth, Constituents of Croton crassifolius roots [J], Planta Med. 54 (1) (1988) 61–63. [4] S. Boonyaratavej, S. Roengsumran, R.B. Bates, R.B. Ortega, A. Petsom, X-ray structure of cyperenoic acid from Croton crassifolius[J], J. Nat. Prod. 51 (4) (1988) 769–770. [5] Y. Hu, L. Zhang, X.Q. Wen, X.J. Zeng, W. Rui, Y.Z. Cen, Two new diterpenoids from Croton crassifolius[J], J. Asian Nat. Prod. Res. 14 (8) (2012) 785–788. [6] W.H. Huang, G.Q. Li, J.G. Li, X. Wu, G. Wei, Two new clerodane diterpenoids from Croton crassifolius[J], ChemInform 89 (2014) 1585–1593. [7] Z.X. Zhang, H.H. Li, F.M. Qi, L.L. Dong, Y. Hai, G.X. Fan, et al., Crocrassins a and B: two novel sesquiterpenoids with an unprecedented carbon skeleton from Croton crassifolius[J], RSC Adv. 4 (57) (2014) 30059. [8] Z.X. Zhang, H.H. Li, F.M. Qi, H.Y. Xiong, L.L. Dong, G.X. Fan, et al., A new halimane diterpenoid from Croton crassifolius[J], Bull. Kor. Chem. Soc. 35 (5) (2014) 1556–1558. [9] X.H. Yang, S.W. Chen, S.M. Deng, Study on the chemical constituents of Croton crassifolius[J], Lishizhen Med. Mater. Med. Res. 3 (2009) 001. [10] H.H. Li, F.M. Qi, L.L. Dong, G.X. Fan, J.M. Che, D.D. Guo, et al., Cytotoxic and antibacterial pyran-2-one derivatives from Croton crassifolius[J], Phytochem. Lett. 10 (2014) 304–308. [11] C.P. Liu, J.B. Xu, J.X. Zhao, C.H. Xu, L. Dong, J. Ding, et al., Diterpenoids from Croton laui and their cytotoxic and antimicrobial activities[J], J. Nat. Prod. 77 (4) (2014) 1013–1020. [12] M.A. Salah, E. Bedir, N.J. Toyang, I.A. Khan, M.D. Harries, D.E. Wedge, Antifungal clerodane diterpenes from Macaranga monandra (L) Muell. et Arg. (Euphorbiaceae)[J], J. Agric. Food Chem. 51 (26) (2003) 7607–7610. [13] Y. Sun, M. Wang, Q. Ren, S. Li, J. Xu, Y. Ohizumi, et al., Two novel clerodane diterpenenes with NGF-potentiating activities from the twigs of Croton yanhuii[J], Fitoterapia 95 (2014) 229–233. [14] J. Xu, Q. Zhang, M. Wang, Q. Ren, Y. Sun, D.Q. Jin, et al., Bioactive clerodane diterpenoids from the twigs of Casearia balansae[J], J. Nat. Prod. 77 (10) (2014) 2182–2189. [15] M. Bruno, F. Piozzi, G. Savona, C. Maria, B. Rodriguez, Neo-clerodane diterpenoids from Teucrium canadense[J], Phytochemistry 28 (12) (1989) 3539–3541. [16] B. Rodriguez, M.L. Jimeno, 1H and 13C NMR spectral assignments and conformational analysis of 14 19-nor-neoclerodane diterpenoids[J], Magn. Reson. Chem. 42 (7) (2004) 605–616. [17] I.S. Marcos, F.A. Hernandez, M.J. Sexmero, D. Diez, P. Basabe, A.B. Pedrero, et al., Synthesis and absolute configuration of (−)-chettaphanin I and (−)-chettaphanin II[J], Tetrahedron 59 (5) (2003) 685–694. [18] G. Dominguez, M.C. De La Torre, B. Rodriguez, Transformation of neoclerodane diterpenoids into 19-norneoclerodane derivatives[J], J. Org. Chem. 56 (23) (1991) 6595–6600. [19] Y.F. Xie, Z.M. Tao, H.B. Wang, G.W. Qin, Chemical constituents from the roots of Vernicia fordii[J], Chin. J. Nat. Med. 8 (4) (2010) 264–266. [20] H.W. Lv, J.G. Luo, M.D. Zhu, S.M. Shan, L.Y. Kong, Teucvisins A–E, five new neoclerodane diterpenes from Teucrium viscidum[J], Chem. Pharm. Bull. (Tokyo) 62 (5) (2014) 472–476. [21] Z. Pan, D. Ning, X. Wu, S. Huang, D. Li, S. Lv, New clerodane diterpenoids from the twigs and leaves of Croton euryphyllus[J], Bioorg. Med. Chem. Lett. 25 (6) (2015) 1329–1332. [22] Y.C. Shen, Y.B. Cheng, J. Kobayashi, T. Kubota, Y. Takahashi, Y. Mikami, et al., Nitrogen-containing verticillene diterpenoids from the Taiwanese soft coral Cespitularia taeniata[J], J. Nat. Prod. 70 (12) (2007) 1961–1965. [23] F.P. Wang, Q.H. Chen, X.Y. Liu, Diterpenoid alkaloids[J], Nat. Prod. Rep. 27 (4) (2010) 529–570. [24] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[J], J. Immunol. Methods 65 (1–2) (1983) 55–63.
