Bioorganic & Medicinal Chemistry Letters 25 (2015) 1986–1989
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Five new phorbol esters with cytotoxic and selective anti-inflammatory activities from Croton tiglium Jun-Feng Wang a,b, , Sheng-Hui Yang a, , Yan-Qun Liu a, Din-Xiang Li c, Wei-Jun He a, Xiao-Xiao Zhang a, Yong-Hong Liu b,⇑, Xiao-Jiang Zhou a,⇑ a
College of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, PR China CAS Key Laboratory of Tropical Marine Bio-resources and Ecology/Guangdong Key Laboratory of Marine Materia Medica/RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China c Key Laboratory of Hunan Province for Integrated Traditional Chinese and Western Medicine on Prevention and Treatment of Cardio-Cerebral Diseases, Hunan University of Chinese Medicine, Changsha 410208, PR China b
a r t i c l e
i n f o
Article history: Received 13 October 2014 Revised 13 February 2015 Accepted 7 March 2015 Available online 14 March 2015 Keywords: Croton tiglium Phorbol esters Cytotoxicity Anti-inflammatory activity
a b s t r a c t Five new phorbol esters, (four phorbol diesters, 1–4, and one 4-deoxy-4a-phorbol diester, 5), as well as four known phorbol esters analogues (6–9) were isolated and identified from the branches and leaves of Croton tiglium. Their structures were elucidated mainly by extensive NMR spectroscopic, and mass spectrometric analysis. Among them, compound (1) was the first example of a naturally occurring phorbol ester with the 20-aldehyde group. Compounds 2–5, and 7–9 showed potent cytotoxicity against the K562, A549, DU145, H1975, MCF-7, U937, SGC-7901, HL60, Hela, and MOLT-4 cell lines, with IC50 values ranging from 1.0 to 43 lM, while none of the compounds exhibited cytotoxic effects on normal human cell lines 293T and LX-2, respectively. In addition, compound 3 exhibited moderate COX-1 and COX-2 inhibition, with IC50 values of 0.14 and 8.5 lM, respectively. Ó 2015 Elsevier Ltd. All rights reserved.
Phorbol esters (PEs) are the tetracyclic diterpenoids generally known for their unusual structure features and exhibited a wide range of biological activities including inhibition of HIV-1 protease, activation of protein kinase C (PKC), as well as anti-CHIKV, cytotoxic, platelet aggregation, cell differentiation, and COX-1 and COX-2 inhibitory activities.1–5 In addition, PEs and their derivatives are reported to be potent tumor promoters at exceptionally low concentration.6,7 Up to now, more than 50 naturally occurring PEs have been isolated from many plants of the family Euphorbiaceae and Thymelaeaceae. Producers belong mainly to the species Croton tiglium, Croton ciliatoglandulifer, Croton spareiflorus, Euphorbia frankiana, Euphorbia cocrulescence, Euphorbia ticulli, Excoecaria agallocha, Homalanthus nutans, Jatropha curcas, Sapium indicum, and Schistosoma japonicum.8 The complex structures and the wide range of biological activities of PEs have stimulated many synthesis programmes.9–11 Owing to the considerable interest in structurally diverse tigliane-type diterpenes from Croton species and their bioactivities,
⇑ Corresponding authors. Tel./fax: +86 020 8902 3244 (Y.-H.L.); tel.: +86 731 88458234; fax: +86 731 88458227 (X.-J.Z.). E-mail addresses: [email protected] (Y.-H. Liu), [email protected] (X.-J. Zhou). Both authors contributed equally to this Letter. http://dx.doi.org/10.1016/j.bmcl.2015.03.017 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
we studied the chemical constituents of the branches and leaves of C. tiglium. Five new tigliane-type diterpenes (1–5), and four known analogues, 12-O-tiglylporbol-13-propionate (6),3 12-O-tiglylphorbol-13-isobutyrate (7),12 12-O-tiglylphorbol-13-(2-methyl)butyrate (8),1 tiglin A (9)13 were isolated from the ethanol extract of C. tiglium (Fig. 1). Herein, we report the isolation,14 structural elucidation of these compounds, and the cytotoxic and anti-inflammatory activities of compounds 2–5, 7–9 were also evaluated in our study. Compound (1)15 was obtained as colorless oil. Its molecular formula was established as C30H40O8 (eleven degrees of unsaturation) by HRESIMS, combined with 1H and 13C NMR spectroscopic data (Table 1). The 1H NMR spectrum recorded in MeOD intuitively revealed one formyl proton [dH 9.46, s, H-20], three olefinic protons [dH 7.59, s, H-1; dH 6.91, qd, J = 7.0, 1.1 Hz, H-30 ; dH 6.90, dd, J = 5.7, 2.5 Hz, H-7], one oxygenated methane [dH 5.57, d, J = 10.3 Hz, H12], five methine protons, two methylene units, along with eight methyl groups (Table 1). The 1H–1H COSY correlations (Fig. 2) indicated the presence of C-1–C-10, C-7–C-8–C-14, C-12–C-11–C-18, C-30 –C-40 , and C-40 –C-30 –C-20 –C-50 moieties. These features characteristically revealed the structure of 1 as possessing a tigliane (phorbol) backbone, consistent with the known compounds 6–9.1,3,12,13 The 1H and 13C NMR spectra of 1 were closely related to those of 12-O-tiglylphorbol-13-(2-methyl)butyrate
1987
J.-F. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1986–1989
4'
O
O
5'
2' 3'
1'
O
18
H
1 2 19
3
O
O
OH 1 O
OH H
H
R2 O
R1
O
R2 O
O
4''
H
H
H
H
OH
O
O
O
H
R1
2'' 3''
12 13 15 16 11 14 H 9 8 H 10 OH 7 4 5 6 20
HO
O
5''
1'' 17
Compd 2 3 6 7 8
H
OH
OH OH
R1 acetyl acylbenzene tiglyl tiglyl tiglyl
R2 isobutyryl (2-methyl)butyryl propionyl isobutyryl (2-methyl)butyryl
O
H H O
OH
OH
Compd R 1 R2 4 tiglyl (2-methyl)butyryl 9 acetyl (2-methyl)butyryl
OH
5
Figure 1. Structures of compounds 1–9.
Table 1 H and 13C NMR data for 1–5 (600, 150 MHz, CD3OD, TMS, d ppm)
1
Position
1 2 3 4 5
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0
1 20 30 40 0
5
100 200 300 400 500
1
2
3
4
5
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
160.1, CH 135.2, C 209.6, C 73.7, C 34.2, CH2
7.59, s
160.6, CH 134.6, C 210.4, C 74.7, C 38.5, CH2
7.51, s
160.5, CH 134.7, C 210.3, C 74.8, C 38.5, CH2
7.61, s
7.56, s
159.4, CH 143.9, C 214.5, C 50.3, CH 27.2, CH2
7.27, s
2.61, d (19.0) 2.55, d (19.0)
159.0, CH 136.8, C 205.8, C 74.3, C 138.3, CH
5.70, d (4.9)
149.7, C 202.1, C
145.3, C 158.1, CH 42.2, CH 80.4, C 57.3, CH 45.0, CH 78.0, CH 66.6, C 36.6, CH 27.7, C 17.4, CH3 24.0, CH3 14.5, CH3 10.2, CH3 195.6, CH 169.3, C 129.6, C 139.3, CH 12.4, CH3 14.9, CH3 180.6, C 42.6, CH 27.5, CH2 12.0, CH3 16.8, CH3
2.91, d (19.4) 2.50, d (19.4)
6.90, dd (5.7, 2.5) 3.76, t (5.6) 2.99, m 2.33, m 5.57, d (10.3) 1.41, d (5.3) 1.35, s 1.30, s 0.93, d (6.5) 1.79, dd (2.8, 1.3) 9.46, s
142.9, C 129.3, CH 40.0, CH 79.8, C 57.3, CH 44.4, CH 78.5, CH 66.8, C 37.3, CH 27.6, C 17.3, CH3 24.2, CH3 14.8, CH3 10.3, CH3 68.0, CH2 172.9, C 21.0, CH3
2.47, s 2.45, s
5.57, dd (5.7, 1.1) 3.25, t (5.5) 3.11, m 2.16, m 5.34, d (10.4) 1.05, d (5.3) 1.21, s 1.18, s 0.85, d (6.5) 1.70, dd (2.9, 1.4) 3.91, d (13.0) 3.87, d (13.0) 2.02, s
6.91, qd (7.1, 1.1) 1.87, d (7.1) 1.88, s
2.45, m 1.76, m; 1.52, m 1.00, t (7.4) 1.19, d (7.0)
180.8, C 35.5, CH 19.0, CH3 19.0, CH3
2.51, m 1.12, d (7.0) 1.11, d (7.0)
143.0, C 129.3, CH 40.1, CH 79.9, C 57.4, CH 44.8, CH 79.2, CH 66.9, C 37.7, CH 27.7, C 17.7, CH3 24.2, CH3 14.9, CH3 10.3, CH3 68.0, CH2 167.8, C 131.3, C 130.6, CH 129.8, CH 134.5, CH 180.6, C 42.6, CH 27.5, CH2 12.0, CH3 16.8, CH3
3.43, t (5.3) 3.24, m 2.43, m 5.73, d (10.4) 1.20, overlap 1.45, s 1.27, s 0.98, d (6.5) 1.78, dd (2.9, 1.2) 4.01, d (12.9) 3.98, d (12.9)
8.04, d (8.5) 7.54, t (7.9) 7.66, t (7.4)
2.45, m 1.77, m; 1.52, m 1.00, t (7.7) 1.20, d (7.0)
55.8, CH 77.3, C 60.3, CH 46.2, CH 77.8, CH 67.0, C 31.2, CH 27.1, C 17.4, CH3 23.9, CH3 14.9, CH3 10.4, CH3 62.4, CH2 169.1, C 129.5, C 139.3, CH 12.3, CH3 14.5, CH3 180.6, C 42.6, C 27.5, CH2 12.0, CH3 16.8, CH3
6.91, t (1.4)
3.80, d (5.4) 3.07, m 2.28, m 5.47, d (10.3) 1.71, d (2.6) 1.22, s 1.14, s 0.87, d (6.6) 1.75, dd (2.9, 1.4) 4.21, m
137.3, C 125.2, CH 42.4, CH 79.4, C 48.5, CH 46.3, CH 75.7, CH 68.5, C 37.6, CH 26.4, C 16.7, CH3 24.6, CH3 12.6, CH3 10.3, CH3 69.1, CH2
2.67, dt (6.4, 4.3) 3.28, m 2.19, dd (15.2, 4.3) 5.07, (s) 1.88, m 3.44, m 1.61, m 3.85, d (10.0) 0.70, d (5.2) 1.18, s 1.18, s 1.17, d (6.7) 1.68, s 3.81, d (9.5)
6.82, qd (7.1, 1.4) 1.78, d (7.1) 1.79, s
2.36, m 1.68, m; 1.44, m 0.91, t (7.5) 1.11, d (7.0)
180.8, C 42.5, CH 27.6, CH2 12.0, CH3 16.8, CH3
2.37, m 1.65, m; 1.44, m 0.88, t (7.4) 1.08, d (7.0)
1988
J.-F. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1986–1989 Table 2 Cytotoxicities against ten different tumor cells for 2–5, and 7–9
1' 2' 3'
4'
O
18 1
19
O
O
5'
10
O
12 13 11 14 9 8
OH 2 3
O
1''
4
OH
17
15 16
2'' 3'' 4''
18
K562 MOLT-4 U937 MCF-7 Hela DU145 A549 SGC-7091 H1975 HL60
12 11
1
7
17 8
14
10
6 20
7
O 1
Figure 2. The key 1H–1H COSY (—), HMBC ( compound 1.
IC50a (lM)
Cell lines
5''
), and ROESY (
) correlations of a b
(8).1 The difference was the appearance of a carbonyl group (dC/H 195.6/9.46, C-20) in 1, instead of the presence of the hydroxymethyl group in 8. This was confirmed by the key HMBC correlations from H-20 to C-5 (dC 34.2) and C-6 (dC 145.3) (Fig. 2). The relative configuration of compound 1 was deduced from ROESY correlations and comparison with data reported in the literature. The ROESY correlations of H-8/H-11, H-11/H-17, and H-17/H-8 indicated that they are all cofacial, arbitrarily assigned as b-oriented. The correlation between H3-18 and H-12 suggested that the tiglyl moiety is also b-oriented (Fig. 2). Although no other ROESY correlation could support the trans A/B ring junction with H-10a and OH-4b, and 13-(2-methyl)butyryl group in an a-position, their orientations were based on biogenetic considerations from all phorbol-type diterpenes characterized so far and because the carbon chemical shift values of 1 were in good agreement with those of structurally related compounds.1,3,12,13 Consequently, the structure of 1 was determined to be 20-deoxy-20-oxophorbol 12tiglate 13-(2-methyl)butyrate. Compound (2)16 was also obtained as colorless oil. Its molecular formula was determined as C26H36O8 on the basis of HRESIMS, with nine degrees of unsaturation. The IR spectrum indicated the presence of OH (3430 cm1) and CO (1742 cm1) groups. Detailed analysis of the 1D- and 2D-NMR spectra data revealed that 2 were very similar to those of 12-O-tiglylphorbol-13-isobutyrate (7)12, indicating that they shared the same skeleton. However, signals for a tiglyl group in 7 were replaced by one acetyl moiety [dC 172.9, C-10 ; dC/H 21.0/2.02, C-20 ] in 2. The HMBC spectrum showed long-range correlations between H-12 (dH 5.34, d, J = 10.4 Hz) and the carbonyl carbon of the acetyl moiety (C-10 ). Thus, compound 2 was assigned as 12-O-acetylphorbol-13isobutyrate. Compound (3)17 was isolated as colorless oil. Its molecular formula was established as C32H40O8 (thirteen degrees of unsaturation) by HREIMS, combined with 1H and 13C NMR spectroscopic data (Table 1). The general features of its NMR spectroscopic data (Table 1) were markedly similar to those of 12-Otiglylporbol-13-propionate (6).12 Detailed comparison of NMR data of these two compounds suggested that they had the same tigliane (phorbol) backbone. By analysis of the COSY and HMBC spectra of 3, the aromatic protons at dH 8.04 (2H, d, J = 8.5 Hz), 7.54 (2H, d, J = 7.9 Hz), 7.66 (1H, t, J = 7.4 Hz) represented a mono-substituted benzene ring. The only significant difference was the presence of the mono-substituted benzene ring in 3, instead of the tiglyl group in 6. This deduction was further supported by the HMBC correlations of H-30 /70 (dH 8.04) with C-10 (dC 167.8) (Fig. 2). Hence, compound 3 was assigned as 12-O-benzoylphorbol-13-(2methyl)butyrate. Compound (4)18 was isolated as colorless oil. Its molecular formula was established as C30H40O9 (eleven degrees of unsaturation) by HRESIMS, combined with 1H and 13C NMR spectroscopic data
2
3
4
5
7
8
9
Taxol (nM)
4.0 2.4 6.8 13 3.9 7.2 5.8 13 10 12
15 12 17 20 4.6 4.3 6.9 10 3.3 6.8
17 4.8 21 20 5.0 10 19 23 10 10
8.0 9.9 18 24 10 10 4.5 5.4 3.3 9.8
4.4 1.1 5.5 >50 9.2 1.1 32 43 10 1.2
2.2 1.0 2.6 NTb 10 5.0 NT NT 10 1.2
7.2 10 14 >50 10 11 >50 >50 10 9.9
5.2 2.2 2.5 6.2 3.2 3.4 4.4 5.1 7.7 1.8
Compounds with IC50 >50 (lM) were considered inactive. NT = not tested.
Table 3 Cytotoxicities against 293T and LX-2 cells for 2–5, and 7–9 IC50 (lM)
Cell lines
293T LX-2
2
3
4
5
7
8
9
Taxol
291.6 >500
420.4 >500
455.3 >500
191.0 >500
171.4 >500
157.2 >500
146.6 351.6
2.4 3.3
(Table 1). The general features of its NMR spectroscopic data closely resembled those of tiglin A (9).13 Detailed comparison of NMR data of these two compounds suggested that compound 4 had the same tigliane (phorbol) skeleton except for the substitution of a tiglyl group in 4 instead of the acetyl group in 9. On the basis of the HMBC spectrum, the tiglyl group was assigned to C-12. Therefore, compound 4 was identified as 12-O-tiglyl-7oxo-5-ene-phorbol-13-(2-methylbutyrate). Compound (5)19 was also isolated as colorless oil. The molecular formula was established to be C25H36O6 (eight degrees of unsaturation) by HRESIMS, together with analysis of the 1H and 13C NMR spectroscopic data. Comparison of the 1H and 13C NMR data indicated that compound 5 possesses the same 4-deoxy-4a-phorbol unit as the known compound 13-O-acetyl-4-deoxy-4a-phorbol-20-linoleate and 13-O-acetyl-4-deoxy-4a-phorbol-20-oleate.3 In addition, signals for a 2-methylbutyryl group in 5 were observed which was supported by COSY correlations of H3-500 /H-200 /H2-300 / H3-400 and by HMBC correlations of H-200 , H2-300 , and H3-500 to C100 . Comparison of the chemical shifts of H-12, C-12, and C-13 in 5 with those in 13-O-acetyl-4-deoxy-4a-phorbol-20-linoleate and 13-O-acetyl-4-deoxy-4a-phorbol-20-oleate indicated that it was C-13 rather than C-12 attached to the 2-methylbutyryl group.3 Therefore, the structure of compound 5 was assigned as 13-O-(2metyl)butyryl-4-deoxy-4a-phorbol. In summary, five new phorbol esters (1–5), as well as four known phorbol esters analogues (6–9) were isolated and identified from the branches and leaves of Croton tiglium. To the best of our knowledge, compound (1) was the first example of a naturally occurring phorbol ester with the 20-aldehyde group. The cytotoxicity of the new compounds (2–5), and three known phorbol esters analogues (7–9) against the K562, A549, DU145, H1975, MCF-7, U937, SGC-7901, HL60, Hela, and MOLT-4 cell lines were assayed with the CCK8 (Dojindo, Japan) method.20 All the tested compounds (2–5, and 7–9) had significant in vitro cytotoxicities against the K562, A549, DU145, H1975, MCF-7, U937, SGC-7901, HL60, Hela, and MOLT-4 cell lines (Table 2), while none of the compounds exhibited cytotoxic effects on normal human cell lines 293T and LX-2, respectively (Table 3).21 Furthermore, compound 3 exhibited moderate cyclooxygenases-1 and -2 inhibition, with IC50 values of 0.14 and 8.5 lM, respectively, while none of the
J.-F. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1986–1989
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31170336), the Hunan Provincial Natural Science Foundation, China (No. 14JJ2108) and the Hunan Province University Innovation Platform Open Foundation, China (No. 12K085).
17.
18.
Supplementary data
19.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.03. 017.
20.
”
16.
”
15.
divided by semi-preparative HPLC (54% aqueous Acetonitrile) to give compound 1 (1.3 mg), compound 3 (5.7 mg) and compound 4 (5.1 mg). Compound (1): colorless oil; [a]25 D +34.9 (c 0.16, MeOH); UV (MeOH) kmax (log ”) 218 (4.03), 336 (3.13) nm; IR (KBr) mmax 3449, 1709, 1637, 1458, 1384, 1277, 1252, 1195, 1128, 1094, 1046 cm1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 551.2617 [M+Na]+ (calcd for C30H40NaO8, 551.2621). Compound (2): colorless oil; [a]25 D +43.3 (c 0.23, MeOH); UV (MeOH) kmax (log ) 204 (3.97), 230 (3.70) nm; IR (KBr) mmax 3430, 1742, 1711, 1631, 1461, 1426, 1383, 1334, 1273, 1236, 1195, 1158, 1145, 1096, 1022 cm1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 499.2304 [M+Na]+ (calcd for C26H36NaO8, 499.2308). Compound (3): Colorless oil; [a]25 D +35.8 (c 0.25, MeOH); UV (MeOH) kmax (log ) 241 (3.99) nm; IR (KBr) mmax 3410, 1716, 1629, 1457, 1379, 1317, 1271, 1193, 1178, 1152, 1096, 1071, 1026, 1011 cm1; 1H NMR and 13C NMR data, see Table 1; HREIMS m/z 552.2721 [M]+ (calcd for C32H40O8, 552.2723). Compound (4): Colorless oil; [a]25 D +7.9 (c 0.32, MeOH); UV (MeOH) kmax (log ) 218 (4.30) nm; IR (KBr) mmax 3444, 1713, 1631, 1458, 1417, 1384, 1255, 1165, 1154, 1130, 1112, 1092, 1045 cm1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 567.2569 [M+Na]+ (calcd for C30H40NaO9, 567.2570). Compound (5): Colorless oil; [a]25 D 47.6 (c 0.24, MeOH); UV (MeOH) kmax (log ) 201 (3.69), 233 (3.78) nm; IR (KBr) mmax 3427, 1709, 1635, 1458, 1384, 1328, 1288, 1245, 1193, 1153, 1126, 1087, 1046, 1026 cm1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 455.2414 [M+Na]+ (calcd for C25H36NaO6, 455.2410). Cytotoxicity was assayed with the CCK8 (Dojindo, Japan) method. Cell lines, K562, A549, DU145, H1975, MCF-7, U937, SGC-7901, HL60, Hela, and MOLT-4 were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cells were routinely grown and maintained in mediums RPMI or DMEM with 10% FBS and with 1% penicillin/streptomycin. All cell lines were incubated in a Thermo/Forma Scientific CO2 Water Jacketed Incubator with 5% CO2 in air at 37 °C. Cell viability assay was determined by the CCK8 (Dojindo, Japan) assay. Cells were seeded at a density of 400-800 cells/well in 384 well plates and treated with various concentration of compounds or solvent control. After 72 h incubation, CCK8 reagent was added, and absorbance was measured at 450 nm using Envision 2104 multi-label Reader (Perkin Elmer, USA). Dose response curves were plotted to determine the IC50 values using Prism 5.0 (GraphPad Software Inc., USA). Taxol was used as the positive control. Inhibitory effects of compounds on 293T and LX-2 cell lines. 293T and LX-2 (human hepatic stellate cells) cell lines were kindly provided by Modern Analysis and Testing Center of Central South University, Hunan Province. Cells were routinely cultured in growth medium supplemented with 88% Dulbecco’s modified eagle’s medium (DMEM, Hyclone), 12% fetal bovine serum(FBS, GIBCO), 100 units of penicillin and streptomycin(Sigma) per mL. Compounds isolated from C. tiglium were diluted using growth medium. Taxol was purchased from Sigma-Aldrich, dissolved with DMSO and diluted by growth medium. Fifty-percent inhibitory concentration (IC50) of compounds on 293T and LX-2 cell lines were determined on the MTT assay. Cell lines (2 105 cells/ mL) were seeded in 96-well plates (100 lL/well) and incubated in 5% CO2 atmosphere at 37 °C for 24 h. Afterwards, cell lines supernatants were discarded and complete growth medium with different concentrations of compounds (80 lM, 40 lM, 20 lM, 10 lM and 5 lM) was added into wells and continually cultured. Cell lines without addition of compounds were administered as negative control, and cell lines with addition of Taxol were used as positive control. After 44 h of incubation, each well was added with 20 lL of MTT solution (5 mg/mL) and then incubated for another 4 h. The absorbance was measured at 490 nm in an ELISA reader (BioTech, USA). Compounds were evaluated for COX inhibitory activity in vitro by using Cayman’s COX Fluorescent Inhibitor Screening Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA). Ovine COX-1 and human recombinant COX-2 enzymes were pre-incubated with serially diluted test compounds for 15 min at rt, the heme and fluorometric substrate were added and incubated for another 15 min at rt. The reaction was started by the addition of arachidonic acid and allowed to proceed for 2 min. The fluorescence was measured at a 530 nm excitation wavelength and 595 nm emission wavelength using a micro plate reader (Envision, PerkinElmer), the data were analyzed using Graphpad Prism5 (GraphpadSoftware, Inc.). Celecoxib was used as the positive control, with IC50 values of 78.4 and 0.019 lM, respectively. ”
other compounds exhibited anti-inflammatory activities against COX-1 and COX-2 (IC50 > 50 lM).22 The potent inhibition effects against COX-1 and COX-2 shown by compound 3 may be attributable to the presence of an acyl benzene group in the phorbol ester molecule.
”
References and notes 1. EI-Mekkawy, S.; Meselhy, M. R.; Nakamura, N.; Hattori, M.; Kawahata, T.; Otake, T. Phytochemistry 2000, 53, 457. 2. Rios, M. Y.; Aguilar-Guadarrama, A. B. J. Nat. Prod. 2006, 69, 887. 3. Zhang, X. L.; Wang, L.; Li, F.; Yu, K.; Wang, M. K. J. Nat. Prod. 2013, 76, 858. 4. Goel, G.; Makkar, H. P. S.; Francis, G.; Becker, K. Int. J. Toxicol. 2007, 26, 279. 5. Bourjot, M.; Delang, L.; Nguyen, V. H.; Neyts, J.; Gueritte, F.; Leyssen, P.; Litaudon, M. J. Nat. Prod. 2012, 75, 2183. 6. Hecker, E. Cancer Res. 1968, 28, 2338. 7. Li, C. Y.; Devappa, R. K.; Liu, J. X.; Lv, J. M.; Makkar, H. P. S.; Becker, K. Food Chem. Toxicol. 2010, 48, 620. 8. Beutler, J. A.; Alvarado, A. B.; Mccloud, T. G.; Cragg, G. M. Phytother. Res. 1989, 3, 188. 9. Bertolini, T. M.; Giorgione, J.; Harvey, D. F.; Newton, A. C. J. Org. Chem. 2003, 68, 5028. 10. Wada, R.; Suto, Y.; Kanai, M.; Shibasaki, M. J. Am. Chem. Soc. 2002, 124, 10658. 11. Carroll, G. L.; Little, R. D. Org. Lett. 2000, 2, 2873. 12. Marshall, G. T.; Kinghorn, A. D. J. Am. Oil Chem. Soc. 1984, 61, 1220. 13. Zhao, Y. M.; Wu, X. N.; Ren, F. X. CN Patent, 1872066A, 2006. 14. Plant material: The branches and leaves of C. tiglium (10 kg) were purchased from the herbal medicine market of Chengdu Hehuachi, Sichun Province of China, in September 2010, and authenticated by corresponding author (X. J. Zhou). A voucher specimen (ZHXJ-006) was deposited at our laboratory in Hunan University of Chinese Medicine. Extraction and isolation: The dried stem and leaf of C. tiglium (10 kg) were extracted with ethanol (2 60 L) to give an extract (910 g), which was suspended in water and partitioned by petroleum ether and EtOAc (each 4 3 L), respectively. The EtOAc extracts (135 g) were fractionated by a silica gel column and eluted with CHCl3 with increasing amounts of methanol to afford 6 fractions (Frs. 1-6). Fr. 2 (41 g) was divided into four parts (Frs. 2-1-2-4) by a MCI gel CHP 20P column eluting with gradient aqueous MeOH. Frs. 2-2 (8.6 g) was submitted by RP-18 gel column (MeOH–H2O 2:8–1:0) to lead four parts (Frs. 2-2-1-2-2-4). Frs. 2-2-2 (1.922 g) separated by semi-preparative HPLC eluting with 70% aqueous MeOH to produce compound 6 (1.8 mg), compound 7 (74.7 mg), compound 8 (177.5 mg), part A and part B. part A (123.8 mg) further purified by semipreparative HPLC eluting with 60% aqueous MeOH to yield compound 9 (38.2 mg). part B (27.9 mg) further divided by semi-preparative HPLC (50% aqueous acetonitrile) to afford compound 2 (7.6 mg) and compound 5 (4.0 mg). Frs. 2-2-3 (0.754 g) was first submitted by Sephadex LH-20 (MeOH), and finally
21.
22.
1989
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Five new phorbol esters with cytotoxic and selective anti-inflammatory activities from Croton tiglium Jun-Feng Wang a,b, , Sheng-Hui Yang a, , Yan-Qun Liu a, Din-Xiang Li c, Wei-Jun He a, Xiao-Xiao Zhang a, Yong-Hong Liu b,⇑, Xiao-Jiang Zhou a,⇑ a
College of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, PR China CAS Key Laboratory of Tropical Marine Bio-resources and Ecology/Guangdong Key Laboratory of Marine Materia Medica/RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China c Key Laboratory of Hunan Province for Integrated Traditional Chinese and Western Medicine on Prevention and Treatment of Cardio-Cerebral Diseases, Hunan University of Chinese Medicine, Changsha 410208, PR China b
a r t i c l e
i n f o
Article history: Received 13 October 2014 Revised 13 February 2015 Accepted 7 March 2015 Available online 14 March 2015 Keywords: Croton tiglium Phorbol esters Cytotoxicity Anti-inflammatory activity
a b s t r a c t Five new phorbol esters, (four phorbol diesters, 1–4, and one 4-deoxy-4a-phorbol diester, 5), as well as four known phorbol esters analogues (6–9) were isolated and identified from the branches and leaves of Croton tiglium. Their structures were elucidated mainly by extensive NMR spectroscopic, and mass spectrometric analysis. Among them, compound (1) was the first example of a naturally occurring phorbol ester with the 20-aldehyde group. Compounds 2–5, and 7–9 showed potent cytotoxicity against the K562, A549, DU145, H1975, MCF-7, U937, SGC-7901, HL60, Hela, and MOLT-4 cell lines, with IC50 values ranging from 1.0 to 43 lM, while none of the compounds exhibited cytotoxic effects on normal human cell lines 293T and LX-2, respectively. In addition, compound 3 exhibited moderate COX-1 and COX-2 inhibition, with IC50 values of 0.14 and 8.5 lM, respectively. Ó 2015 Elsevier Ltd. All rights reserved.
Phorbol esters (PEs) are the tetracyclic diterpenoids generally known for their unusual structure features and exhibited a wide range of biological activities including inhibition of HIV-1 protease, activation of protein kinase C (PKC), as well as anti-CHIKV, cytotoxic, platelet aggregation, cell differentiation, and COX-1 and COX-2 inhibitory activities.1–5 In addition, PEs and their derivatives are reported to be potent tumor promoters at exceptionally low concentration.6,7 Up to now, more than 50 naturally occurring PEs have been isolated from many plants of the family Euphorbiaceae and Thymelaeaceae. Producers belong mainly to the species Croton tiglium, Croton ciliatoglandulifer, Croton spareiflorus, Euphorbia frankiana, Euphorbia cocrulescence, Euphorbia ticulli, Excoecaria agallocha, Homalanthus nutans, Jatropha curcas, Sapium indicum, and Schistosoma japonicum.8 The complex structures and the wide range of biological activities of PEs have stimulated many synthesis programmes.9–11 Owing to the considerable interest in structurally diverse tigliane-type diterpenes from Croton species and their bioactivities,
⇑ Corresponding authors. Tel./fax: +86 020 8902 3244 (Y.-H.L.); tel.: +86 731 88458234; fax: +86 731 88458227 (X.-J.Z.). E-mail addresses: [email protected] (Y.-H. Liu), [email protected] (X.-J. Zhou). Both authors contributed equally to this Letter. http://dx.doi.org/10.1016/j.bmcl.2015.03.017 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
we studied the chemical constituents of the branches and leaves of C. tiglium. Five new tigliane-type diterpenes (1–5), and four known analogues, 12-O-tiglylporbol-13-propionate (6),3 12-O-tiglylphorbol-13-isobutyrate (7),12 12-O-tiglylphorbol-13-(2-methyl)butyrate (8),1 tiglin A (9)13 were isolated from the ethanol extract of C. tiglium (Fig. 1). Herein, we report the isolation,14 structural elucidation of these compounds, and the cytotoxic and anti-inflammatory activities of compounds 2–5, 7–9 were also evaluated in our study. Compound (1)15 was obtained as colorless oil. Its molecular formula was established as C30H40O8 (eleven degrees of unsaturation) by HRESIMS, combined with 1H and 13C NMR spectroscopic data (Table 1). The 1H NMR spectrum recorded in MeOD intuitively revealed one formyl proton [dH 9.46, s, H-20], three olefinic protons [dH 7.59, s, H-1; dH 6.91, qd, J = 7.0, 1.1 Hz, H-30 ; dH 6.90, dd, J = 5.7, 2.5 Hz, H-7], one oxygenated methane [dH 5.57, d, J = 10.3 Hz, H12], five methine protons, two methylene units, along with eight methyl groups (Table 1). The 1H–1H COSY correlations (Fig. 2) indicated the presence of C-1–C-10, C-7–C-8–C-14, C-12–C-11–C-18, C-30 –C-40 , and C-40 –C-30 –C-20 –C-50 moieties. These features characteristically revealed the structure of 1 as possessing a tigliane (phorbol) backbone, consistent with the known compounds 6–9.1,3,12,13 The 1H and 13C NMR spectra of 1 were closely related to those of 12-O-tiglylphorbol-13-(2-methyl)butyrate
1987
J.-F. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1986–1989
4'
O
O
5'
2' 3'
1'
O
18
H
1 2 19
3
O
O
OH 1 O
OH H
H
R2 O
R1
O
R2 O
O
4''
H
H
H
H
OH
O
O
O
H
R1
2'' 3''
12 13 15 16 11 14 H 9 8 H 10 OH 7 4 5 6 20
HO
O
5''
1'' 17
Compd 2 3 6 7 8
H
OH
OH OH
R1 acetyl acylbenzene tiglyl tiglyl tiglyl
R2 isobutyryl (2-methyl)butyryl propionyl isobutyryl (2-methyl)butyryl
O
H H O
OH
OH
Compd R 1 R2 4 tiglyl (2-methyl)butyryl 9 acetyl (2-methyl)butyryl
OH
5
Figure 1. Structures of compounds 1–9.
Table 1 H and 13C NMR data for 1–5 (600, 150 MHz, CD3OD, TMS, d ppm)
1
Position
1 2 3 4 5
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0
1 20 30 40 0
5
100 200 300 400 500
1
2
3
4
5
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
160.1, CH 135.2, C 209.6, C 73.7, C 34.2, CH2
7.59, s
160.6, CH 134.6, C 210.4, C 74.7, C 38.5, CH2
7.51, s
160.5, CH 134.7, C 210.3, C 74.8, C 38.5, CH2
7.61, s
7.56, s
159.4, CH 143.9, C 214.5, C 50.3, CH 27.2, CH2
7.27, s
2.61, d (19.0) 2.55, d (19.0)
159.0, CH 136.8, C 205.8, C 74.3, C 138.3, CH
5.70, d (4.9)
149.7, C 202.1, C
145.3, C 158.1, CH 42.2, CH 80.4, C 57.3, CH 45.0, CH 78.0, CH 66.6, C 36.6, CH 27.7, C 17.4, CH3 24.0, CH3 14.5, CH3 10.2, CH3 195.6, CH 169.3, C 129.6, C 139.3, CH 12.4, CH3 14.9, CH3 180.6, C 42.6, CH 27.5, CH2 12.0, CH3 16.8, CH3
2.91, d (19.4) 2.50, d (19.4)
6.90, dd (5.7, 2.5) 3.76, t (5.6) 2.99, m 2.33, m 5.57, d (10.3) 1.41, d (5.3) 1.35, s 1.30, s 0.93, d (6.5) 1.79, dd (2.8, 1.3) 9.46, s
142.9, C 129.3, CH 40.0, CH 79.8, C 57.3, CH 44.4, CH 78.5, CH 66.8, C 37.3, CH 27.6, C 17.3, CH3 24.2, CH3 14.8, CH3 10.3, CH3 68.0, CH2 172.9, C 21.0, CH3
2.47, s 2.45, s
5.57, dd (5.7, 1.1) 3.25, t (5.5) 3.11, m 2.16, m 5.34, d (10.4) 1.05, d (5.3) 1.21, s 1.18, s 0.85, d (6.5) 1.70, dd (2.9, 1.4) 3.91, d (13.0) 3.87, d (13.0) 2.02, s
6.91, qd (7.1, 1.1) 1.87, d (7.1) 1.88, s
2.45, m 1.76, m; 1.52, m 1.00, t (7.4) 1.19, d (7.0)
180.8, C 35.5, CH 19.0, CH3 19.0, CH3
2.51, m 1.12, d (7.0) 1.11, d (7.0)
143.0, C 129.3, CH 40.1, CH 79.9, C 57.4, CH 44.8, CH 79.2, CH 66.9, C 37.7, CH 27.7, C 17.7, CH3 24.2, CH3 14.9, CH3 10.3, CH3 68.0, CH2 167.8, C 131.3, C 130.6, CH 129.8, CH 134.5, CH 180.6, C 42.6, CH 27.5, CH2 12.0, CH3 16.8, CH3
3.43, t (5.3) 3.24, m 2.43, m 5.73, d (10.4) 1.20, overlap 1.45, s 1.27, s 0.98, d (6.5) 1.78, dd (2.9, 1.2) 4.01, d (12.9) 3.98, d (12.9)
8.04, d (8.5) 7.54, t (7.9) 7.66, t (7.4)
2.45, m 1.77, m; 1.52, m 1.00, t (7.7) 1.20, d (7.0)
55.8, CH 77.3, C 60.3, CH 46.2, CH 77.8, CH 67.0, C 31.2, CH 27.1, C 17.4, CH3 23.9, CH3 14.9, CH3 10.4, CH3 62.4, CH2 169.1, C 129.5, C 139.3, CH 12.3, CH3 14.5, CH3 180.6, C 42.6, C 27.5, CH2 12.0, CH3 16.8, CH3
6.91, t (1.4)
3.80, d (5.4) 3.07, m 2.28, m 5.47, d (10.3) 1.71, d (2.6) 1.22, s 1.14, s 0.87, d (6.6) 1.75, dd (2.9, 1.4) 4.21, m
137.3, C 125.2, CH 42.4, CH 79.4, C 48.5, CH 46.3, CH 75.7, CH 68.5, C 37.6, CH 26.4, C 16.7, CH3 24.6, CH3 12.6, CH3 10.3, CH3 69.1, CH2
2.67, dt (6.4, 4.3) 3.28, m 2.19, dd (15.2, 4.3) 5.07, (s) 1.88, m 3.44, m 1.61, m 3.85, d (10.0) 0.70, d (5.2) 1.18, s 1.18, s 1.17, d (6.7) 1.68, s 3.81, d (9.5)
6.82, qd (7.1, 1.4) 1.78, d (7.1) 1.79, s
2.36, m 1.68, m; 1.44, m 0.91, t (7.5) 1.11, d (7.0)
180.8, C 42.5, CH 27.6, CH2 12.0, CH3 16.8, CH3
2.37, m 1.65, m; 1.44, m 0.88, t (7.4) 1.08, d (7.0)
1988
J.-F. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1986–1989 Table 2 Cytotoxicities against ten different tumor cells for 2–5, and 7–9
1' 2' 3'
4'
O
18 1
19
O
O
5'
10
O
12 13 11 14 9 8
OH 2 3
O
1''
4
OH
17
15 16
2'' 3'' 4''
18
K562 MOLT-4 U937 MCF-7 Hela DU145 A549 SGC-7091 H1975 HL60
12 11
1
7
17 8
14
10
6 20
7
O 1
Figure 2. The key 1H–1H COSY (—), HMBC ( compound 1.
IC50a (lM)
Cell lines
5''
), and ROESY (
) correlations of a b
(8).1 The difference was the appearance of a carbonyl group (dC/H 195.6/9.46, C-20) in 1, instead of the presence of the hydroxymethyl group in 8. This was confirmed by the key HMBC correlations from H-20 to C-5 (dC 34.2) and C-6 (dC 145.3) (Fig. 2). The relative configuration of compound 1 was deduced from ROESY correlations and comparison with data reported in the literature. The ROESY correlations of H-8/H-11, H-11/H-17, and H-17/H-8 indicated that they are all cofacial, arbitrarily assigned as b-oriented. The correlation between H3-18 and H-12 suggested that the tiglyl moiety is also b-oriented (Fig. 2). Although no other ROESY correlation could support the trans A/B ring junction with H-10a and OH-4b, and 13-(2-methyl)butyryl group in an a-position, their orientations were based on biogenetic considerations from all phorbol-type diterpenes characterized so far and because the carbon chemical shift values of 1 were in good agreement with those of structurally related compounds.1,3,12,13 Consequently, the structure of 1 was determined to be 20-deoxy-20-oxophorbol 12tiglate 13-(2-methyl)butyrate. Compound (2)16 was also obtained as colorless oil. Its molecular formula was determined as C26H36O8 on the basis of HRESIMS, with nine degrees of unsaturation. The IR spectrum indicated the presence of OH (3430 cm1) and CO (1742 cm1) groups. Detailed analysis of the 1D- and 2D-NMR spectra data revealed that 2 were very similar to those of 12-O-tiglylphorbol-13-isobutyrate (7)12, indicating that they shared the same skeleton. However, signals for a tiglyl group in 7 were replaced by one acetyl moiety [dC 172.9, C-10 ; dC/H 21.0/2.02, C-20 ] in 2. The HMBC spectrum showed long-range correlations between H-12 (dH 5.34, d, J = 10.4 Hz) and the carbonyl carbon of the acetyl moiety (C-10 ). Thus, compound 2 was assigned as 12-O-acetylphorbol-13isobutyrate. Compound (3)17 was isolated as colorless oil. Its molecular formula was established as C32H40O8 (thirteen degrees of unsaturation) by HREIMS, combined with 1H and 13C NMR spectroscopic data (Table 1). The general features of its NMR spectroscopic data (Table 1) were markedly similar to those of 12-Otiglylporbol-13-propionate (6).12 Detailed comparison of NMR data of these two compounds suggested that they had the same tigliane (phorbol) backbone. By analysis of the COSY and HMBC spectra of 3, the aromatic protons at dH 8.04 (2H, d, J = 8.5 Hz), 7.54 (2H, d, J = 7.9 Hz), 7.66 (1H, t, J = 7.4 Hz) represented a mono-substituted benzene ring. The only significant difference was the presence of the mono-substituted benzene ring in 3, instead of the tiglyl group in 6. This deduction was further supported by the HMBC correlations of H-30 /70 (dH 8.04) with C-10 (dC 167.8) (Fig. 2). Hence, compound 3 was assigned as 12-O-benzoylphorbol-13-(2methyl)butyrate. Compound (4)18 was isolated as colorless oil. Its molecular formula was established as C30H40O9 (eleven degrees of unsaturation) by HRESIMS, combined with 1H and 13C NMR spectroscopic data
2
3
4
5
7
8
9
Taxol (nM)
4.0 2.4 6.8 13 3.9 7.2 5.8 13 10 12
15 12 17 20 4.6 4.3 6.9 10 3.3 6.8
17 4.8 21 20 5.0 10 19 23 10 10
8.0 9.9 18 24 10 10 4.5 5.4 3.3 9.8
4.4 1.1 5.5 >50 9.2 1.1 32 43 10 1.2
2.2 1.0 2.6 NTb 10 5.0 NT NT 10 1.2
7.2 10 14 >50 10 11 >50 >50 10 9.9
5.2 2.2 2.5 6.2 3.2 3.4 4.4 5.1 7.7 1.8
Compounds with IC50 >50 (lM) were considered inactive. NT = not tested.
Table 3 Cytotoxicities against 293T and LX-2 cells for 2–5, and 7–9 IC50 (lM)
Cell lines
293T LX-2
2
3
4
5
7
8
9
Taxol
291.6 >500
420.4 >500
455.3 >500
191.0 >500
171.4 >500
157.2 >500
146.6 351.6
2.4 3.3
(Table 1). The general features of its NMR spectroscopic data closely resembled those of tiglin A (9).13 Detailed comparison of NMR data of these two compounds suggested that compound 4 had the same tigliane (phorbol) skeleton except for the substitution of a tiglyl group in 4 instead of the acetyl group in 9. On the basis of the HMBC spectrum, the tiglyl group was assigned to C-12. Therefore, compound 4 was identified as 12-O-tiglyl-7oxo-5-ene-phorbol-13-(2-methylbutyrate). Compound (5)19 was also isolated as colorless oil. The molecular formula was established to be C25H36O6 (eight degrees of unsaturation) by HRESIMS, together with analysis of the 1H and 13C NMR spectroscopic data. Comparison of the 1H and 13C NMR data indicated that compound 5 possesses the same 4-deoxy-4a-phorbol unit as the known compound 13-O-acetyl-4-deoxy-4a-phorbol-20-linoleate and 13-O-acetyl-4-deoxy-4a-phorbol-20-oleate.3 In addition, signals for a 2-methylbutyryl group in 5 were observed which was supported by COSY correlations of H3-500 /H-200 /H2-300 / H3-400 and by HMBC correlations of H-200 , H2-300 , and H3-500 to C100 . Comparison of the chemical shifts of H-12, C-12, and C-13 in 5 with those in 13-O-acetyl-4-deoxy-4a-phorbol-20-linoleate and 13-O-acetyl-4-deoxy-4a-phorbol-20-oleate indicated that it was C-13 rather than C-12 attached to the 2-methylbutyryl group.3 Therefore, the structure of compound 5 was assigned as 13-O-(2metyl)butyryl-4-deoxy-4a-phorbol. In summary, five new phorbol esters (1–5), as well as four known phorbol esters analogues (6–9) were isolated and identified from the branches and leaves of Croton tiglium. To the best of our knowledge, compound (1) was the first example of a naturally occurring phorbol ester with the 20-aldehyde group. The cytotoxicity of the new compounds (2–5), and three known phorbol esters analogues (7–9) against the K562, A549, DU145, H1975, MCF-7, U937, SGC-7901, HL60, Hela, and MOLT-4 cell lines were assayed with the CCK8 (Dojindo, Japan) method.20 All the tested compounds (2–5, and 7–9) had significant in vitro cytotoxicities against the K562, A549, DU145, H1975, MCF-7, U937, SGC-7901, HL60, Hela, and MOLT-4 cell lines (Table 2), while none of the compounds exhibited cytotoxic effects on normal human cell lines 293T and LX-2, respectively (Table 3).21 Furthermore, compound 3 exhibited moderate cyclooxygenases-1 and -2 inhibition, with IC50 values of 0.14 and 8.5 lM, respectively, while none of the
J.-F. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1986–1989
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31170336), the Hunan Provincial Natural Science Foundation, China (No. 14JJ2108) and the Hunan Province University Innovation Platform Open Foundation, China (No. 12K085).
17.
18.
Supplementary data
19.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.03. 017.
20.
”
16.
”
15.
divided by semi-preparative HPLC (54% aqueous Acetonitrile) to give compound 1 (1.3 mg), compound 3 (5.7 mg) and compound 4 (5.1 mg). Compound (1): colorless oil; [a]25 D +34.9 (c 0.16, MeOH); UV (MeOH) kmax (log ”) 218 (4.03), 336 (3.13) nm; IR (KBr) mmax 3449, 1709, 1637, 1458, 1384, 1277, 1252, 1195, 1128, 1094, 1046 cm1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 551.2617 [M+Na]+ (calcd for C30H40NaO8, 551.2621). Compound (2): colorless oil; [a]25 D +43.3 (c 0.23, MeOH); UV (MeOH) kmax (log ) 204 (3.97), 230 (3.70) nm; IR (KBr) mmax 3430, 1742, 1711, 1631, 1461, 1426, 1383, 1334, 1273, 1236, 1195, 1158, 1145, 1096, 1022 cm1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 499.2304 [M+Na]+ (calcd for C26H36NaO8, 499.2308). Compound (3): Colorless oil; [a]25 D +35.8 (c 0.25, MeOH); UV (MeOH) kmax (log ) 241 (3.99) nm; IR (KBr) mmax 3410, 1716, 1629, 1457, 1379, 1317, 1271, 1193, 1178, 1152, 1096, 1071, 1026, 1011 cm1; 1H NMR and 13C NMR data, see Table 1; HREIMS m/z 552.2721 [M]+ (calcd for C32H40O8, 552.2723). Compound (4): Colorless oil; [a]25 D +7.9 (c 0.32, MeOH); UV (MeOH) kmax (log ) 218 (4.30) nm; IR (KBr) mmax 3444, 1713, 1631, 1458, 1417, 1384, 1255, 1165, 1154, 1130, 1112, 1092, 1045 cm1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 567.2569 [M+Na]+ (calcd for C30H40NaO9, 567.2570). Compound (5): Colorless oil; [a]25 D 47.6 (c 0.24, MeOH); UV (MeOH) kmax (log ) 201 (3.69), 233 (3.78) nm; IR (KBr) mmax 3427, 1709, 1635, 1458, 1384, 1328, 1288, 1245, 1193, 1153, 1126, 1087, 1046, 1026 cm1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 455.2414 [M+Na]+ (calcd for C25H36NaO6, 455.2410). Cytotoxicity was assayed with the CCK8 (Dojindo, Japan) method. Cell lines, K562, A549, DU145, H1975, MCF-7, U937, SGC-7901, HL60, Hela, and MOLT-4 were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cells were routinely grown and maintained in mediums RPMI or DMEM with 10% FBS and with 1% penicillin/streptomycin. All cell lines were incubated in a Thermo/Forma Scientific CO2 Water Jacketed Incubator with 5% CO2 in air at 37 °C. Cell viability assay was determined by the CCK8 (Dojindo, Japan) assay. Cells were seeded at a density of 400-800 cells/well in 384 well plates and treated with various concentration of compounds or solvent control. After 72 h incubation, CCK8 reagent was added, and absorbance was measured at 450 nm using Envision 2104 multi-label Reader (Perkin Elmer, USA). Dose response curves were plotted to determine the IC50 values using Prism 5.0 (GraphPad Software Inc., USA). Taxol was used as the positive control. Inhibitory effects of compounds on 293T and LX-2 cell lines. 293T and LX-2 (human hepatic stellate cells) cell lines were kindly provided by Modern Analysis and Testing Center of Central South University, Hunan Province. Cells were routinely cultured in growth medium supplemented with 88% Dulbecco’s modified eagle’s medium (DMEM, Hyclone), 12% fetal bovine serum(FBS, GIBCO), 100 units of penicillin and streptomycin(Sigma) per mL. Compounds isolated from C. tiglium were diluted using growth medium. Taxol was purchased from Sigma-Aldrich, dissolved with DMSO and diluted by growth medium. Fifty-percent inhibitory concentration (IC50) of compounds on 293T and LX-2 cell lines were determined on the MTT assay. Cell lines (2 105 cells/ mL) were seeded in 96-well plates (100 lL/well) and incubated in 5% CO2 atmosphere at 37 °C for 24 h. Afterwards, cell lines supernatants were discarded and complete growth medium with different concentrations of compounds (80 lM, 40 lM, 20 lM, 10 lM and 5 lM) was added into wells and continually cultured. Cell lines without addition of compounds were administered as negative control, and cell lines with addition of Taxol were used as positive control. After 44 h of incubation, each well was added with 20 lL of MTT solution (5 mg/mL) and then incubated for another 4 h. The absorbance was measured at 490 nm in an ELISA reader (BioTech, USA). Compounds were evaluated for COX inhibitory activity in vitro by using Cayman’s COX Fluorescent Inhibitor Screening Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA). Ovine COX-1 and human recombinant COX-2 enzymes were pre-incubated with serially diluted test compounds for 15 min at rt, the heme and fluorometric substrate were added and incubated for another 15 min at rt. The reaction was started by the addition of arachidonic acid and allowed to proceed for 2 min. The fluorescence was measured at a 530 nm excitation wavelength and 595 nm emission wavelength using a micro plate reader (Envision, PerkinElmer), the data were analyzed using Graphpad Prism5 (GraphpadSoftware, Inc.). Celecoxib was used as the positive control, with IC50 values of 78.4 and 0.019 lM, respectively. ”
other compounds exhibited anti-inflammatory activities against COX-1 and COX-2 (IC50 > 50 lM).22 The potent inhibition effects against COX-1 and COX-2 shown by compound 3 may be attributable to the presence of an acyl benzene group in the phorbol ester molecule.
”
References and notes 1. EI-Mekkawy, S.; Meselhy, M. R.; Nakamura, N.; Hattori, M.; Kawahata, T.; Otake, T. Phytochemistry 2000, 53, 457. 2. Rios, M. Y.; Aguilar-Guadarrama, A. B. J. Nat. Prod. 2006, 69, 887. 3. Zhang, X. L.; Wang, L.; Li, F.; Yu, K.; Wang, M. K. J. Nat. Prod. 2013, 76, 858. 4. Goel, G.; Makkar, H. P. S.; Francis, G.; Becker, K. Int. J. Toxicol. 2007, 26, 279. 5. Bourjot, M.; Delang, L.; Nguyen, V. H.; Neyts, J.; Gueritte, F.; Leyssen, P.; Litaudon, M. J. Nat. Prod. 2012, 75, 2183. 6. Hecker, E. Cancer Res. 1968, 28, 2338. 7. Li, C. Y.; Devappa, R. K.; Liu, J. X.; Lv, J. M.; Makkar, H. P. S.; Becker, K. Food Chem. Toxicol. 2010, 48, 620. 8. Beutler, J. A.; Alvarado, A. B.; Mccloud, T. G.; Cragg, G. M. Phytother. Res. 1989, 3, 188. 9. Bertolini, T. M.; Giorgione, J.; Harvey, D. F.; Newton, A. C. J. Org. Chem. 2003, 68, 5028. 10. Wada, R.; Suto, Y.; Kanai, M.; Shibasaki, M. J. Am. Chem. Soc. 2002, 124, 10658. 11. Carroll, G. L.; Little, R. D. Org. Lett. 2000, 2, 2873. 12. Marshall, G. T.; Kinghorn, A. D. J. Am. Oil Chem. Soc. 1984, 61, 1220. 13. Zhao, Y. M.; Wu, X. N.; Ren, F. X. CN Patent, 1872066A, 2006. 14. Plant material: The branches and leaves of C. tiglium (10 kg) were purchased from the herbal medicine market of Chengdu Hehuachi, Sichun Province of China, in September 2010, and authenticated by corresponding author (X. J. Zhou). A voucher specimen (ZHXJ-006) was deposited at our laboratory in Hunan University of Chinese Medicine. Extraction and isolation: The dried stem and leaf of C. tiglium (10 kg) were extracted with ethanol (2 60 L) to give an extract (910 g), which was suspended in water and partitioned by petroleum ether and EtOAc (each 4 3 L), respectively. The EtOAc extracts (135 g) were fractionated by a silica gel column and eluted with CHCl3 with increasing amounts of methanol to afford 6 fractions (Frs. 1-6). Fr. 2 (41 g) was divided into four parts (Frs. 2-1-2-4) by a MCI gel CHP 20P column eluting with gradient aqueous MeOH. Frs. 2-2 (8.6 g) was submitted by RP-18 gel column (MeOH–H2O 2:8–1:0) to lead four parts (Frs. 2-2-1-2-2-4). Frs. 2-2-2 (1.922 g) separated by semi-preparative HPLC eluting with 70% aqueous MeOH to produce compound 6 (1.8 mg), compound 7 (74.7 mg), compound 8 (177.5 mg), part A and part B. part A (123.8 mg) further purified by semipreparative HPLC eluting with 60% aqueous MeOH to yield compound 9 (38.2 mg). part B (27.9 mg) further divided by semi-preparative HPLC (50% aqueous acetonitrile) to afford compound 2 (7.6 mg) and compound 5 (4.0 mg). Frs. 2-2-3 (0.754 g) was first submitted by Sephadex LH-20 (MeOH), and finally
21.
22.
1989
