Bioorganic & Medicinal Chemistry Letters 24 (2014) 2159–2162
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Three new cucurbitane triterpenoids from Hemsleya penxianensis and their cytotoxic activities Xiaoting Xu a,b, Hong Bai a, Ling Zhou a, Zhipeng Deng a, Hao Zhong a, Zhongyu Wu a, Qingqiang Yao a,⇑ a b
Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan, Shandong 250062, PR China School of Medicine and Life Sciences, University of Jinan, Shandong Academy of Medical Sciences, Jinan, Shandong 250062, PR China
a r t i c l e
i n f o
Article history: Received 14 December 2013 Revised 25 February 2014 Accepted 11 March 2014 Available online 20 March 2014 Keywords: Hemsleya penxianensis Cucurbitane triterpenoids Cytotoxic activities
a b s t r a c t Two new cucurbitane glycosides, hemslepenside A (1) and 16,25-O-diacetyl-cucurbitacin F-2-O-b-Dglucopyranoside (3), one new cucurbitacin, 16-O-acetyl-cucurbitacin F (2), along with three known cucurbitane compounds, were isolated from the roots of Hemsleya penxianensis. The structures of 1–6 were established on the basis of extensive spectroscopic and chemical methods. The isolated compounds were evaluated for their cytotoxic activities against different three human cancer cell lines, with IC50 values in the low microgram range. Ó 2014 Elsevier Ltd. All rights reserved.
The genus Hemsleya (Cucurbitaceae) is consist of more than thirty species. The roots of Hemsleya penxianensis, known as ‘Xue Dan’ in Chinese, are widely distributed in southwestern region of China, such as Yunnan, Sichuan and Guizhou provinces.1 H. penxianensis is listed in the Chinese Pharmacopoeia as a traditional herbal medicine for the treatment of bronchitis, bacillary dysentery, tuberculosis, diabetes, whooping cough and bile duct infection.2 The main chemical constituents of this species are triterpenoid saponins, including oleanane glycosides and cucurbitane-type glucosides.1 Cucurbitane-type compounds were also reported to demonstrate cytotoxicity,3 anti-cancer activity,4 antiinflammatory,5 purgative and anti-fertility activities. Considering the extensive interests and biological activities of cucurbitane-type compounds, we carried out a phytochemical and bioactive investigation on H. penxianensis collected from Chongqing city, China, which resulted in the isolation of three new cucurbitane triterpenoids, hemslepenside A (1), 16-O-acetyl-cucurbitacin F (2) and 16,25-O-diacetyl-cucurbitacin F-2-O-b-D-glucopyranoside (3), together with three known analogues 25-O-acetyl-23,24-dihydrocucurbitacin F (4),2 23,24-dihydrocucurbitacin F (5)2 and jinfushanoside A (6)2 (Fig. 1). This Letter deals with the isolation, structural elucidation and cytotoxic activities of these compounds. The air-dried roots of H. penxianensis6 (9 kg) were extracted with 95% EtOH under refluxing three times and filtered. The EtOH extract was partitioned between H2O and EtOAc. The portion EtOAc extract (300 g/700 g) was subjected to silica gel column ⇑ Corresponding author. Tel.: +86 531 82919960. E-mail address: [email protected] (Q. Yao). http://dx.doi.org/10.1016/j.bmcl.2014.03.027 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
chromatography (CC) with a gradient system of CHCl3-MeOH to give fourteen fractions (Fr. A-Fr. N). Compound 4 (4.8 g) was obtained by repeated recrystallization from Fr. D (55.0 g). Further isolation of Fr. E (40.9 g), Fr. F (11.0 g) and Fr. N (11.0 g) by repeated Sephadex LH-20 CC, ODS CC and pre-HPLC to give compounds 1 (15.0 mg), 2 (15.7 mg), 3 (5.5 mg), 5 (188.0 mg) and 6 (24.0 mg), respectively. Compound 17 was obtained as white amorphous powder with the molecular formula of C36H56O8, in agreement with the positive ion (HRESIMS m/z: 639.4613 [M+Na]+, calcd for C36H56O8Na 639.4613). IR absorptions at 3375, 1720 and 1688 cm1 indicated the presence of hydroxyl and carbonyl groups. Enzymatic hydrolysis8,9 of 1 afforded D-glucose as the composition sugar, which was determined by HPLC analysis of its 1-[(S)-N-acetyl-a-methylbenzylamino]-1-deoxyglucitol acetate derivatives.10 The b-anomeric configuration for glucose was determined by its large 3JH-1, 13 C NMR spectrum with H-2 coupling constant value (7.8 Hz). The DEPT experiments showed 36 carbon signals due to seven methyls (dC 9.3, 17.4, 18.9, 19.0, 20.4, 26.1, 28.9), nine methylenes, twelve methines and eight quaternary carbons (including four olefinic carbon at dC 119.8, 140.5, 142.2, 157.5 and two carbonyl carbons at dC 197.3, 215.9), of which 30 were assigned to the aglycon part and the remaining 6 were ascribed to the sugar moiety (Table 1). The 1H NMR spectrum of 1 displayed signals corresponding to seven methyl signals at dH 0.73 (3H, s), 0.95 (3H, d, J = 6.6 Hz), 1.03 (3H 2, s), 1.07 (3H, s), 1.21 (3H, s), 1.69 (3H, s), two olefinic protons at dH 5.59 (1H, br s), 6.60 (1H, t, J = 7.2 Hz) and an aldehyde proton at dH 9.33 (1H, s), which suggested that 1 possess a tetracycliccucurbitane skeleton.11 The NMR data of 1 were similar to those
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X. Xu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2159–2162
Figure 1. Structures of compounds 1–6.
Table 1 H (600 MHz) and
1
13
C (150 MHz) data for compounds 1–3 in CD3OD
Position
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 16-OOCCH3 25-OOCCH3 b-D-glc 10 20 30 40 50 60 a
1
2
3
dH
dC
dH
dC
dH
dC
1.55a, 1.01a 2.41a, 2.01 (m) 3.39 (br s)
22.7 29.1 88.2 42.7 142.2 119.8 25.0 45.4 50.6 37.0 217.8 49.6
1.03 (m), 1.80 (m) 3.54 (ddd, 12.0, 9.6, 3.6) 2.85 (d, 9.6)
34.7 71.6 81.9 43.4 142.7 119.9 24.7 43.9 49.7 34.9 215.4 49.6
1.94a, 1.41a 3.62 (m) 3.01 (d, 7.8)
34.1 83.3 81.0 43.3 142.5 120.0 24.8 44.0 49.9 35.0 216.0 49.8
5.59 (br s) 2.32a, 1.95 (m) 1.92 (m) 2.41a 2.36 (d, 14.4) 3.06 (d, 14.4)
(s) (s) (s) (s) (s)
50.2 50.4 35.6 28.9 50.9 17.4 19.0 37.2 18.9 35.8 27.0 157.5 140.5 197.3 9.3 26.1 20.4 28.9
4.25 (d, 7.8) 3.14 3.28 3.24 3.19 3.79, 3.61
106.8 75.7 78.4 71.8 77.8 62.9
1.734 (m), 1.30a 1.99a, 1.38a 1.79 (m) 0.73 (s) 1.03 (s) 1.47 (d, 6.6) 0.95 (d, 6.6) 1.61a, 1.38 (m) 1.92a, 2.36a 6.60 (t, 7.2) 9.33 1.69 1.21 1.03 1.07
5.73 (br s) 2.41 (m), 1.94 (m) 1.98 (m) 2.48 (d, 12.6) 3.38 (d, 15.0) 2.60 (d, 15.0)
1.94a, 1.34a 5.28 (t, 8.4) 2.83 (d, 7.8) 0.97 (s) 1.08 (s) 1.40 (s) 6.82 (d, 15.0) 7.06 (d, 15.0) 1.33 1.33 0.96 1.16 1.26 1.84
(s) (s) (s) (s) (s) (s)
49.6 51.5 44.3 75.4 55.7 20.5 20.5 79.4 24.5 204.1 120.5 156.4 71.6 29.4 29.4 22.3 25.3 19.6 172.2, 21.1
5.70 (m) 2.36a, 2.02a 1.94 (m) 2.47 (d, 13.2) 3.30a, 2.55a
1.95a, 1.32a 5.30 (t, 7.8) 2.75 (d, 7.2) 0.92 (s) 1.03 (s) 1.35 (s) 6.74 (d, 15.0) 7.06 (d, 15.0) 1.53 1.52 0.95 1.16 1.21 1.84 1.98
(s) (s) (s) (s) (s) (s) (s)
4.44 (d, 7.8) 3.17 3.31 3.23 3.18 3.85, 3.67
49.1 51.6 44.5 75.4 56.1 20.6 20.7 79.8 24.8 204.1 121.9 153.0 81.0 27.0 27.1 22.4 25.4 19.6 172.6, 21.9 171.8, 21.3 106.0 75.8 78.2 71.6 78.2 62.9
Overlapped signals.
of jinfushanoside E,12 except for the presence of an aldehyde proton [dH 9.33 (1H, s)] at C-26 in 1 instead of a hydroxymethyl in jinfushanoside E, which was confirmed by the HMBC correlations from
dH 9.33 (H-26) to dC 140.5 (C-25) and 9.3 (C-27) (Fig. 2). Furthermore, HMBC correlation between dH 4.25 (Glc-H-1) and dC 88.2 (C-3) suggested that b-D-glucopyranosyl was linked at C-3 (Fig. 2).
X. Xu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2159–2162
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Figure 2. Key HMBC and 1H–1H COSY correlations of compounds 1–3.
The relative stereochemistry of compound 1 was determined by the coupling constants and NOESY experiment (Fig. 3). A broad singlet of an oxygenated methine proton at dH 3.39 (H-3) suggested an equatorial configuration (J3eq, 2ax ffi J3eq, 2eq) of H-3.13 Assume an a orientation for H-10,14,15 a orientations of H-3, H-10, H-17, Me-28 and Me-30 were established by the NOESY correlations of H-3/Me-28, Me-28/H-10, H-10/Me-30 and Me-30/H-17. In addition, the cross-peaks of H-8/Me-18 and H-8/Me-19 indicated the b-orientation of these protons. Thus, the structure of 1 was determined to be 3b-hydroxycucurbita-5,24(E)-diene-11-one-26al-3-O-b-D-glucopyranoside. Compound 216 was obtained as white amorphous powder with the molecular formula of C32H48O8, in agreement with the positive ion HRESIMS (positive) m/z: 583.3309 [M+Na]+ calcd for C32H48O8Na 583.3309). In the 1H NMR spectrum, it’s easy to figure out eight methyls at dH 0.96 (3H, s), 0.97 (3H, s), 1.08 (3H, s), 1.16 (3H, s), 1.26 (3H, s), 1.33 (3H 2, s), 1.40 (3H, s) and three olefinic protons at dH 5.73 (1H, br s), 7.06 (1H, d, 15.0 Hz) and 6.82 (1H, d, 15.0 Hz). The 1H and 13C NMR data of 2 were almost superimposable with those of 23,24-dihydrocucurbitacin F (5) isolated from the same extract, expect the appearance of a double bond and an acetyl group. Two methylenes at dC 34.9 (C-23) and 38.1 (C-24) in 5 was replaced by the double bond at dC 120.5 (C-23) and 156.4 (C-24) in 2. The deduction is consistent with the HMBC correlations from dH 6.82 (H-23) and 7.06 (H-24) to dC 204.1 (C-22), 7.06 (H-24) to 71.6 (C-25), and 1.33 (CH3-27) to 156.4 (C-24). In addition, the acetyl group at C-16 in 2 was implied by the HMBC correlation from dH 5.28 (H-16) to dC 172.2 (acetyl). The relative stereochemistry of compound 2 was also determined by the coupling constants and NOESY experiment (Fig. 3). The signal ascribed to H-3 was observed at dH (2.85, 1H, d, J = 9.6 Hz), suggesting 2a and 3b configurations,12 respectively. The NOESY correlations of
H-10/Me-28, Me-28/H-2, H-10/Me-30, Me-30/H-17 indicated the a-orientation of above protons. Further cross-peaks between H-8/Me-18 and H-8/Me-19 indicated the b-orientation of them. Thus, the structure of 2 was determined to be 2b,3a,20,25-tetrahydroxycucurbita-5,23(E)-diene-11, 22-dione-16-acetate. Compound 317 was obtained as white amorphous powder with the molecular formula of C40H60O14, in agreement with the positive ion HRESIMS (positive) m/z: 787.4234 [M+Na]+ calcd for C40H60O14Na 787.4234). Enzymatic hydrolysis8 of 3 afforded D-glucose as the composition sugar. The signal at dH 4.44 (1H, d, J = 7.8 Hz) suggesting the b-orientation of anomeric proton. Considering that the molecular weight of 3 was 204 mass units than that of 2, except a glucose moiety, another acetyl group in 3 was suggested. Careful comparison of the NMR data of the aglycon of 3 with that of 2 suggested that they were almost superimposable, except for C-2 and C-25. It was apparent that the signal of C-2 in 3 being shifted downfield by 11.7 ppm than that in 2, which indicated that D-glucose was linked at C-2. Above deduction was also confirmed by HMBC correlation between dH 4.44 (Glc-H-1) and dC 83.3 (C-2). Furthermore, an acetyl group at C-25 was suggested by the shifted downfield by 9.4 ppm at C-25 in 3 than that of 2.18,19 Thus, the structure of 3 was determined to be 2b,3a, 20-trihydroxycucurbita-5,23(E)-diene-11,22-dione-16,25-diacetyl2-O-b-D-glucopyranoside. Since cucurbitane-type compounds are reported for their cytotoxic behavior3 and were a hot topic of drug discovery community from an anti-cancer drug development perspective,5 the cytotoxic activities of the isolated compounds (1–6) against human lung adenocarcinoma cells (H460), human colon cancer cells (SW-620) and human colon cancer cells (COLO205) cancer cells in vitro were evaluated20,21 The results were listed in Table 2, and used Cisplatin as the positive control. As can be observed in Table 2, most of
Figure 3. Key NOESY correlations of compounds 1–2.
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X. Xu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2159–2162
Table 2 In vitro cytotoxicities of 1–6 against three different human cancer cell lines IC50a (lg/mL)
Compound
1 2 3 4 5 6 Cisplatinb a b
H460
SW-620
COLO205
0.46 ± 0.87 3.43 ± 0.30 8.55 ± 0.35 11.53 ± 0.30 7.09 ± 0.22 2.96 ± 0.11 0.66 ± 0.15
0.64 ± 0.21 0.42 ± 0.09 6.63 ± 0.05 1.11 ± 0.11 1.03 ± 0.10 1.99 ± 0.05 2.85 ± 0.07
0.78 ± 0.77 0.44 ± 0.33 24.94 ± 0.90 1.09 ± 0.21 0.64 ± 0.60 3.84 ± 0.20 1.57 ± 0.13
Means ± S.D. From three independent experiments (n = 3). Positive control.
compounds displayed cytotoxic activities against the cancer cell lines. Compound 1 revealed the highest cytotoxicities, even more active than Cisplatin in all three assays. The strong cytotoxicities of 1 may be related to the aldehyde function at C-26, which is the unique structural difference compared to its analogues. Further, compound 2 was more active than compound 3, which suggested that the glycosidation and acetylation may reduce cytotoxic activities. On the basis of these promising results, compounds 1–6 could serve as potential anti-cancer drug for future cancer chemotherapy.
9. 10. 11. 12. 13. 14. 15. 16.
Acknowledgment 17.
This project was supported by the Key Laboratory for Rare and Uncommon Diseases of Shandong Province, PR China. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03. 027.
18. 19. 20.
References and notes 1. Nie, R. L. Acta Bot. Yunnan. 1994, 16, 201. 2. Chen, J. C.; Niu, X. M.; Li, Z. R.; Qiu, M. H. Planta Med. 2005, 71, 983. 3. Fuller, R. W.; Cardellina, J. H.; Cragg, G. M.; Boyd, M. R. J. Nat. Prod. 1994, 57, 1442. 4. Boykin, C.; Zhang, G.; Chen, Y. H.; Zhang, R. W.; Fan, X. E.; Yang, W. M.; Lu, Q. Br. J. Cancer 2011, 104, 781. 5. Alghasham, A. A. Int. J. Health Sci. 2013, 7, 77. 6. Plant material: The roots of H. penxianensis were collected from Chongqing, People’s Republic of China, in September 2012, and identified by Dr. Si-rong Yi, at Chongqing Institute of Pharmaceutical Plant. A voucher specimen (HA201209) of the plant has been kept in the Institute of Materia Medica, Shandong Academy of Medical Science. 7. Hemslepenside A (1): white amorphous powder; mp 208–209 °C; [a]D 20 +83.5 (c 1.07, MeOH); IR (KBr) mmax 3375, 2952, 1720, 1688, 1463, 1384, 1074, 1032 1 1 cm ; UV (MeOH) kmax (log e): 230 (3.16), 203 (2.98) nm; H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Table 1; positive-ion HRESIMS m/z: 639.4613 [M+Na]+ (calcd for C36H56O8Na 639.4613). 8. Enzymatic hydrolysis and determination of the absolute configuration of the monosaccharides: 20 mg cellulase (Worthington, USA) was added to the
21.
solution of 1 and 3 (4 mg dissolved in 4 mL H2O respectively) and then the mixture was stirred at 40 °C for 120 h. The reaction mixture was extracted with EtOAc. The organic layers were combined and evaporated to afford the aglycone and the aqueous layers were evaporated to dryness to get the sugar fractions. The sugar residue and authentic sample of D-(+)-b-glucose and L-()b-glucose were dissolved in 1 mL H2O and mixed with 1 mL EtOH which contains (S)-()-a-methylbenzylamine (17 ll) and NaBH3CN (8 mg) respectively. The mixture was agitated at 40 °C for 4 h, then added 0.2 mL of glacial acetia acid to it and concentrated to dry. Added 0.3 mL acetic anhydride and 0.3 mL pyridine to the residue and acetylated at room temperature for 24 h. After the pyridine was removed, the aqueous solution of the reaction mixture was subjected to a Cleanert C18-N column (Agela) and used H2O, 20% and 50% CH3CN (15, 15 and 10 mL) as the eluent. The 50% CH3CN fraction was analyzed by HPLC under the following conditions: Agilent SB-C18 column (4.6 250 mm, 5 lm); mobile phase, 40% CH3CN; flow rate, 0.8 mL/min; DAD detection, 230 nm. The absolute configuration of the monosaccharide was determined by comparing the retention time tR (min) of their derivatives with those of authentic samples: 22.36 (derivative of D-glucose), 20.80 (derivative of L-glucose). The sugars in compounds 1 and 3 were determined to be D-glucose. Zhao, H. X.; Nie, T. T.; Guo, H. J.; Li, J.; Bai, H. Phytochem. Lett. 2012, 5, 240. Oshima, R.; Yamauchi, Y.; Kumanotani, J. Carbohydr. Res. 1982, 107, 169. Chen, J. C.; Chiu, M. H.; Nie, R. L.; Cordell, G. A.; Qiu, S. X. Nat. Prod. Rep. 2005, 22, 386. Chen, J. C.; Zhou, L.; Wang, Y. H.; Tian, R. R.; Yan, Y. X.; Nian, Y.; Sun, Y.; Zheng, Y. T.; Qiu, M. H. Nat. Prod. Bioprospect. 2012, 2, 138. Ramalhete, C.; Lopes, D.; Mulhovo, S.; Molnár, J.; Rosário, V. E.; Ferreira, M.-J. U. Bioorg. Med. Chem. 2010, 18, 5254. Xu, R.; Fazio, G. C.; Matsuda, S. Phytochemistry 2004, 65, 261. Ramalhete, C.; da Cruz, F. P.; Lopes, D.; Mulhovo, S.; Rosário, V. E.; Prudêncio, M.; Ferreira, M.-J. U. Bioorg. Med. Chem. 2011, 19, 7474. 16-O-Acetyl-cucurbitacin F (2): white amorphous powder; mp 152–153 °C; [a]D 20 +21.2 (c 0.33, MeOH); IR (KBr) mmax 3387, 2932, 1735, 1686, 1374, 1251, 1027 cm1; UV (MeOH) kmax (log e): 231 (2.95), 204 (3.06) nm; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Table 1; positive-ion HRESIMS m/z: 583.3309 [M+Na]+ (calcd for C32H48O8Na 583.3309). 16,25-O-Diacetyl-cucurbitacin F-2-O-b-D-glucopyranoside (3): white amorphous powder; mp 157–158 °C; [a]D 20 +16.1 (c 0.93, MeOH); IR (KBr) mmax 3423, 2972, 1720, 1690 1374, 1252, 1028 cm1; UV (MeOH) kmax (log e): 231 (2.98), 204 (2.97) nm; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Table 1; positive-ion HRESIMS m/z: 787.4234 [M+Na]+ (calcd for C40H60O14Na 787.4234). Chiu, M. H.; Gao, J. Chin. Chem. Lett. 2003, 14, 389. Kasai, R.; Matsumoto, K.; Nie, R. L.; Zhou, J.; Tanaka, O. Chem. Pharm. Bull. 1988, 36, 234. Bioassays for cytotoxic activity: The human lung adenocarcinoma cells (H460), human colon cancer cells (SW-620), human colon cancer cells (COLO205) were obtained from the Institute of Materia Medica, Chinese Academy of Medical Sciences. The cytotoxic assays were evaluated using MTT method and Cisplatin was selected as the positive control. These three cell lines were cultured in RPMI1640 medium (Gibgo, USA) which contains 15% fetal bovine serum. The human lung adenocarcinoma cells (H460), human colon cancer cells (SW-620) were washed and digested with 0.25% trypsin, resuspended in the above medium to 1 105/mL; and the human colon cancer cells (COLO205) were resuspended in the above medium to 2 105/mL. Then, an aliquot (100 lL) of the cell suspension was placed in each cell of 96-well flat-bottom plates and were incubated in 5% CO2 at 37 °C for 24 h. After incubation, added the each agent at final concentrations of 50, 10, 2, 0.4, 0.08 lg/mL to the medium with further incubated for 48 h. When the incubation was end, added 10 lL 0.5% MTT (Sigma, USA) to each well and re-incubated for 4 h at 37 °C. Then removed the supernatant from each well and added 200 lL DMSO, respectively. Mixed the plates on a microshaker to make the formazan be sufficiently dissolved and the optical density (OD) was measured on Victor1420 multifunctional-counter instrument at 570 nm. The inhibition (%) was calculated as (OD0 ODS)/ OD0 100%, the OD0 and ODS are the values of the cells without and with test agent. The results (IC50) were defined as the concentration of samples that inhibit 50% control growth after the incubation period. All the data are the three experiments’ mean values which were carried out in triplicate (n = 3). Bai, H.; Zhong, Y.; Xie, Y. Y.; Wang, Y. S.; Liu, L.; Zhou, L.; Wang, J.; Mu, Y. L.; Zuo, C. X. Chem. Biodivers. 2007, 4, 955.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Three new cucurbitane triterpenoids from Hemsleya penxianensis and their cytotoxic activities Xiaoting Xu a,b, Hong Bai a, Ling Zhou a, Zhipeng Deng a, Hao Zhong a, Zhongyu Wu a, Qingqiang Yao a,⇑ a b
Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan, Shandong 250062, PR China School of Medicine and Life Sciences, University of Jinan, Shandong Academy of Medical Sciences, Jinan, Shandong 250062, PR China
a r t i c l e
i n f o
Article history: Received 14 December 2013 Revised 25 February 2014 Accepted 11 March 2014 Available online 20 March 2014 Keywords: Hemsleya penxianensis Cucurbitane triterpenoids Cytotoxic activities
a b s t r a c t Two new cucurbitane glycosides, hemslepenside A (1) and 16,25-O-diacetyl-cucurbitacin F-2-O-b-Dglucopyranoside (3), one new cucurbitacin, 16-O-acetyl-cucurbitacin F (2), along with three known cucurbitane compounds, were isolated from the roots of Hemsleya penxianensis. The structures of 1–6 were established on the basis of extensive spectroscopic and chemical methods. The isolated compounds were evaluated for their cytotoxic activities against different three human cancer cell lines, with IC50 values in the low microgram range. Ó 2014 Elsevier Ltd. All rights reserved.
The genus Hemsleya (Cucurbitaceae) is consist of more than thirty species. The roots of Hemsleya penxianensis, known as ‘Xue Dan’ in Chinese, are widely distributed in southwestern region of China, such as Yunnan, Sichuan and Guizhou provinces.1 H. penxianensis is listed in the Chinese Pharmacopoeia as a traditional herbal medicine for the treatment of bronchitis, bacillary dysentery, tuberculosis, diabetes, whooping cough and bile duct infection.2 The main chemical constituents of this species are triterpenoid saponins, including oleanane glycosides and cucurbitane-type glucosides.1 Cucurbitane-type compounds were also reported to demonstrate cytotoxicity,3 anti-cancer activity,4 antiinflammatory,5 purgative and anti-fertility activities. Considering the extensive interests and biological activities of cucurbitane-type compounds, we carried out a phytochemical and bioactive investigation on H. penxianensis collected from Chongqing city, China, which resulted in the isolation of three new cucurbitane triterpenoids, hemslepenside A (1), 16-O-acetyl-cucurbitacin F (2) and 16,25-O-diacetyl-cucurbitacin F-2-O-b-D-glucopyranoside (3), together with three known analogues 25-O-acetyl-23,24-dihydrocucurbitacin F (4),2 23,24-dihydrocucurbitacin F (5)2 and jinfushanoside A (6)2 (Fig. 1). This Letter deals with the isolation, structural elucidation and cytotoxic activities of these compounds. The air-dried roots of H. penxianensis6 (9 kg) were extracted with 95% EtOH under refluxing three times and filtered. The EtOH extract was partitioned between H2O and EtOAc. The portion EtOAc extract (300 g/700 g) was subjected to silica gel column ⇑ Corresponding author. Tel.: +86 531 82919960. E-mail address: [email protected] (Q. Yao). http://dx.doi.org/10.1016/j.bmcl.2014.03.027 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
chromatography (CC) with a gradient system of CHCl3-MeOH to give fourteen fractions (Fr. A-Fr. N). Compound 4 (4.8 g) was obtained by repeated recrystallization from Fr. D (55.0 g). Further isolation of Fr. E (40.9 g), Fr. F (11.0 g) and Fr. N (11.0 g) by repeated Sephadex LH-20 CC, ODS CC and pre-HPLC to give compounds 1 (15.0 mg), 2 (15.7 mg), 3 (5.5 mg), 5 (188.0 mg) and 6 (24.0 mg), respectively. Compound 17 was obtained as white amorphous powder with the molecular formula of C36H56O8, in agreement with the positive ion (HRESIMS m/z: 639.4613 [M+Na]+, calcd for C36H56O8Na 639.4613). IR absorptions at 3375, 1720 and 1688 cm1 indicated the presence of hydroxyl and carbonyl groups. Enzymatic hydrolysis8,9 of 1 afforded D-glucose as the composition sugar, which was determined by HPLC analysis of its 1-[(S)-N-acetyl-a-methylbenzylamino]-1-deoxyglucitol acetate derivatives.10 The b-anomeric configuration for glucose was determined by its large 3JH-1, 13 C NMR spectrum with H-2 coupling constant value (7.8 Hz). The DEPT experiments showed 36 carbon signals due to seven methyls (dC 9.3, 17.4, 18.9, 19.0, 20.4, 26.1, 28.9), nine methylenes, twelve methines and eight quaternary carbons (including four olefinic carbon at dC 119.8, 140.5, 142.2, 157.5 and two carbonyl carbons at dC 197.3, 215.9), of which 30 were assigned to the aglycon part and the remaining 6 were ascribed to the sugar moiety (Table 1). The 1H NMR spectrum of 1 displayed signals corresponding to seven methyl signals at dH 0.73 (3H, s), 0.95 (3H, d, J = 6.6 Hz), 1.03 (3H 2, s), 1.07 (3H, s), 1.21 (3H, s), 1.69 (3H, s), two olefinic protons at dH 5.59 (1H, br s), 6.60 (1H, t, J = 7.2 Hz) and an aldehyde proton at dH 9.33 (1H, s), which suggested that 1 possess a tetracycliccucurbitane skeleton.11 The NMR data of 1 were similar to those
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Figure 1. Structures of compounds 1–6.
Table 1 H (600 MHz) and
1
13
C (150 MHz) data for compounds 1–3 in CD3OD
Position
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 16-OOCCH3 25-OOCCH3 b-D-glc 10 20 30 40 50 60 a
1
2
3
dH
dC
dH
dC
dH
dC
1.55a, 1.01a 2.41a, 2.01 (m) 3.39 (br s)
22.7 29.1 88.2 42.7 142.2 119.8 25.0 45.4 50.6 37.0 217.8 49.6
1.03 (m), 1.80 (m) 3.54 (ddd, 12.0, 9.6, 3.6) 2.85 (d, 9.6)
34.7 71.6 81.9 43.4 142.7 119.9 24.7 43.9 49.7 34.9 215.4 49.6
1.94a, 1.41a 3.62 (m) 3.01 (d, 7.8)
34.1 83.3 81.0 43.3 142.5 120.0 24.8 44.0 49.9 35.0 216.0 49.8
5.59 (br s) 2.32a, 1.95 (m) 1.92 (m) 2.41a 2.36 (d, 14.4) 3.06 (d, 14.4)
(s) (s) (s) (s) (s)
50.2 50.4 35.6 28.9 50.9 17.4 19.0 37.2 18.9 35.8 27.0 157.5 140.5 197.3 9.3 26.1 20.4 28.9
4.25 (d, 7.8) 3.14 3.28 3.24 3.19 3.79, 3.61
106.8 75.7 78.4 71.8 77.8 62.9
1.734 (m), 1.30a 1.99a, 1.38a 1.79 (m) 0.73 (s) 1.03 (s) 1.47 (d, 6.6) 0.95 (d, 6.6) 1.61a, 1.38 (m) 1.92a, 2.36a 6.60 (t, 7.2) 9.33 1.69 1.21 1.03 1.07
5.73 (br s) 2.41 (m), 1.94 (m) 1.98 (m) 2.48 (d, 12.6) 3.38 (d, 15.0) 2.60 (d, 15.0)
1.94a, 1.34a 5.28 (t, 8.4) 2.83 (d, 7.8) 0.97 (s) 1.08 (s) 1.40 (s) 6.82 (d, 15.0) 7.06 (d, 15.0) 1.33 1.33 0.96 1.16 1.26 1.84
(s) (s) (s) (s) (s) (s)
49.6 51.5 44.3 75.4 55.7 20.5 20.5 79.4 24.5 204.1 120.5 156.4 71.6 29.4 29.4 22.3 25.3 19.6 172.2, 21.1
5.70 (m) 2.36a, 2.02a 1.94 (m) 2.47 (d, 13.2) 3.30a, 2.55a
1.95a, 1.32a 5.30 (t, 7.8) 2.75 (d, 7.2) 0.92 (s) 1.03 (s) 1.35 (s) 6.74 (d, 15.0) 7.06 (d, 15.0) 1.53 1.52 0.95 1.16 1.21 1.84 1.98
(s) (s) (s) (s) (s) (s) (s)
4.44 (d, 7.8) 3.17 3.31 3.23 3.18 3.85, 3.67
49.1 51.6 44.5 75.4 56.1 20.6 20.7 79.8 24.8 204.1 121.9 153.0 81.0 27.0 27.1 22.4 25.4 19.6 172.6, 21.9 171.8, 21.3 106.0 75.8 78.2 71.6 78.2 62.9
Overlapped signals.
of jinfushanoside E,12 except for the presence of an aldehyde proton [dH 9.33 (1H, s)] at C-26 in 1 instead of a hydroxymethyl in jinfushanoside E, which was confirmed by the HMBC correlations from
dH 9.33 (H-26) to dC 140.5 (C-25) and 9.3 (C-27) (Fig. 2). Furthermore, HMBC correlation between dH 4.25 (Glc-H-1) and dC 88.2 (C-3) suggested that b-D-glucopyranosyl was linked at C-3 (Fig. 2).
X. Xu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2159–2162
2161
Figure 2. Key HMBC and 1H–1H COSY correlations of compounds 1–3.
The relative stereochemistry of compound 1 was determined by the coupling constants and NOESY experiment (Fig. 3). A broad singlet of an oxygenated methine proton at dH 3.39 (H-3) suggested an equatorial configuration (J3eq, 2ax ffi J3eq, 2eq) of H-3.13 Assume an a orientation for H-10,14,15 a orientations of H-3, H-10, H-17, Me-28 and Me-30 were established by the NOESY correlations of H-3/Me-28, Me-28/H-10, H-10/Me-30 and Me-30/H-17. In addition, the cross-peaks of H-8/Me-18 and H-8/Me-19 indicated the b-orientation of these protons. Thus, the structure of 1 was determined to be 3b-hydroxycucurbita-5,24(E)-diene-11-one-26al-3-O-b-D-glucopyranoside. Compound 216 was obtained as white amorphous powder with the molecular formula of C32H48O8, in agreement with the positive ion HRESIMS (positive) m/z: 583.3309 [M+Na]+ calcd for C32H48O8Na 583.3309). In the 1H NMR spectrum, it’s easy to figure out eight methyls at dH 0.96 (3H, s), 0.97 (3H, s), 1.08 (3H, s), 1.16 (3H, s), 1.26 (3H, s), 1.33 (3H 2, s), 1.40 (3H, s) and three olefinic protons at dH 5.73 (1H, br s), 7.06 (1H, d, 15.0 Hz) and 6.82 (1H, d, 15.0 Hz). The 1H and 13C NMR data of 2 were almost superimposable with those of 23,24-dihydrocucurbitacin F (5) isolated from the same extract, expect the appearance of a double bond and an acetyl group. Two methylenes at dC 34.9 (C-23) and 38.1 (C-24) in 5 was replaced by the double bond at dC 120.5 (C-23) and 156.4 (C-24) in 2. The deduction is consistent with the HMBC correlations from dH 6.82 (H-23) and 7.06 (H-24) to dC 204.1 (C-22), 7.06 (H-24) to 71.6 (C-25), and 1.33 (CH3-27) to 156.4 (C-24). In addition, the acetyl group at C-16 in 2 was implied by the HMBC correlation from dH 5.28 (H-16) to dC 172.2 (acetyl). The relative stereochemistry of compound 2 was also determined by the coupling constants and NOESY experiment (Fig. 3). The signal ascribed to H-3 was observed at dH (2.85, 1H, d, J = 9.6 Hz), suggesting 2a and 3b configurations,12 respectively. The NOESY correlations of
H-10/Me-28, Me-28/H-2, H-10/Me-30, Me-30/H-17 indicated the a-orientation of above protons. Further cross-peaks between H-8/Me-18 and H-8/Me-19 indicated the b-orientation of them. Thus, the structure of 2 was determined to be 2b,3a,20,25-tetrahydroxycucurbita-5,23(E)-diene-11, 22-dione-16-acetate. Compound 317 was obtained as white amorphous powder with the molecular formula of C40H60O14, in agreement with the positive ion HRESIMS (positive) m/z: 787.4234 [M+Na]+ calcd for C40H60O14Na 787.4234). Enzymatic hydrolysis8 of 3 afforded D-glucose as the composition sugar. The signal at dH 4.44 (1H, d, J = 7.8 Hz) suggesting the b-orientation of anomeric proton. Considering that the molecular weight of 3 was 204 mass units than that of 2, except a glucose moiety, another acetyl group in 3 was suggested. Careful comparison of the NMR data of the aglycon of 3 with that of 2 suggested that they were almost superimposable, except for C-2 and C-25. It was apparent that the signal of C-2 in 3 being shifted downfield by 11.7 ppm than that in 2, which indicated that D-glucose was linked at C-2. Above deduction was also confirmed by HMBC correlation between dH 4.44 (Glc-H-1) and dC 83.3 (C-2). Furthermore, an acetyl group at C-25 was suggested by the shifted downfield by 9.4 ppm at C-25 in 3 than that of 2.18,19 Thus, the structure of 3 was determined to be 2b,3a, 20-trihydroxycucurbita-5,23(E)-diene-11,22-dione-16,25-diacetyl2-O-b-D-glucopyranoside. Since cucurbitane-type compounds are reported for their cytotoxic behavior3 and were a hot topic of drug discovery community from an anti-cancer drug development perspective,5 the cytotoxic activities of the isolated compounds (1–6) against human lung adenocarcinoma cells (H460), human colon cancer cells (SW-620) and human colon cancer cells (COLO205) cancer cells in vitro were evaluated20,21 The results were listed in Table 2, and used Cisplatin as the positive control. As can be observed in Table 2, most of
Figure 3. Key NOESY correlations of compounds 1–2.
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X. Xu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2159–2162
Table 2 In vitro cytotoxicities of 1–6 against three different human cancer cell lines IC50a (lg/mL)
Compound
1 2 3 4 5 6 Cisplatinb a b
H460
SW-620
COLO205
0.46 ± 0.87 3.43 ± 0.30 8.55 ± 0.35 11.53 ± 0.30 7.09 ± 0.22 2.96 ± 0.11 0.66 ± 0.15
0.64 ± 0.21 0.42 ± 0.09 6.63 ± 0.05 1.11 ± 0.11 1.03 ± 0.10 1.99 ± 0.05 2.85 ± 0.07
0.78 ± 0.77 0.44 ± 0.33 24.94 ± 0.90 1.09 ± 0.21 0.64 ± 0.60 3.84 ± 0.20 1.57 ± 0.13
Means ± S.D. From three independent experiments (n = 3). Positive control.
compounds displayed cytotoxic activities against the cancer cell lines. Compound 1 revealed the highest cytotoxicities, even more active than Cisplatin in all three assays. The strong cytotoxicities of 1 may be related to the aldehyde function at C-26, which is the unique structural difference compared to its analogues. Further, compound 2 was more active than compound 3, which suggested that the glycosidation and acetylation may reduce cytotoxic activities. On the basis of these promising results, compounds 1–6 could serve as potential anti-cancer drug for future cancer chemotherapy.
9. 10. 11. 12. 13. 14. 15. 16.
Acknowledgment 17.
This project was supported by the Key Laboratory for Rare and Uncommon Diseases of Shandong Province, PR China. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03. 027.
18. 19. 20.
References and notes 1. Nie, R. L. Acta Bot. Yunnan. 1994, 16, 201. 2. Chen, J. C.; Niu, X. M.; Li, Z. R.; Qiu, M. H. Planta Med. 2005, 71, 983. 3. Fuller, R. W.; Cardellina, J. H.; Cragg, G. M.; Boyd, M. R. J. Nat. Prod. 1994, 57, 1442. 4. Boykin, C.; Zhang, G.; Chen, Y. H.; Zhang, R. W.; Fan, X. E.; Yang, W. M.; Lu, Q. Br. J. Cancer 2011, 104, 781. 5. Alghasham, A. A. Int. J. Health Sci. 2013, 7, 77. 6. Plant material: The roots of H. penxianensis were collected from Chongqing, People’s Republic of China, in September 2012, and identified by Dr. Si-rong Yi, at Chongqing Institute of Pharmaceutical Plant. A voucher specimen (HA201209) of the plant has been kept in the Institute of Materia Medica, Shandong Academy of Medical Science. 7. Hemslepenside A (1): white amorphous powder; mp 208–209 °C; [a]D 20 +83.5 (c 1.07, MeOH); IR (KBr) mmax 3375, 2952, 1720, 1688, 1463, 1384, 1074, 1032 1 1 cm ; UV (MeOH) kmax (log e): 230 (3.16), 203 (2.98) nm; H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Table 1; positive-ion HRESIMS m/z: 639.4613 [M+Na]+ (calcd for C36H56O8Na 639.4613). 8. Enzymatic hydrolysis and determination of the absolute configuration of the monosaccharides: 20 mg cellulase (Worthington, USA) was added to the
21.
solution of 1 and 3 (4 mg dissolved in 4 mL H2O respectively) and then the mixture was stirred at 40 °C for 120 h. The reaction mixture was extracted with EtOAc. The organic layers were combined and evaporated to afford the aglycone and the aqueous layers were evaporated to dryness to get the sugar fractions. The sugar residue and authentic sample of D-(+)-b-glucose and L-()b-glucose were dissolved in 1 mL H2O and mixed with 1 mL EtOH which contains (S)-()-a-methylbenzylamine (17 ll) and NaBH3CN (8 mg) respectively. The mixture was agitated at 40 °C for 4 h, then added 0.2 mL of glacial acetia acid to it and concentrated to dry. Added 0.3 mL acetic anhydride and 0.3 mL pyridine to the residue and acetylated at room temperature for 24 h. After the pyridine was removed, the aqueous solution of the reaction mixture was subjected to a Cleanert C18-N column (Agela) and used H2O, 20% and 50% CH3CN (15, 15 and 10 mL) as the eluent. The 50% CH3CN fraction was analyzed by HPLC under the following conditions: Agilent SB-C18 column (4.6 250 mm, 5 lm); mobile phase, 40% CH3CN; flow rate, 0.8 mL/min; DAD detection, 230 nm. The absolute configuration of the monosaccharide was determined by comparing the retention time tR (min) of their derivatives with those of authentic samples: 22.36 (derivative of D-glucose), 20.80 (derivative of L-glucose). The sugars in compounds 1 and 3 were determined to be D-glucose. Zhao, H. X.; Nie, T. T.; Guo, H. J.; Li, J.; Bai, H. Phytochem. Lett. 2012, 5, 240. Oshima, R.; Yamauchi, Y.; Kumanotani, J. Carbohydr. Res. 1982, 107, 169. Chen, J. C.; Chiu, M. H.; Nie, R. L.; Cordell, G. A.; Qiu, S. X. Nat. Prod. Rep. 2005, 22, 386. Chen, J. C.; Zhou, L.; Wang, Y. H.; Tian, R. R.; Yan, Y. X.; Nian, Y.; Sun, Y.; Zheng, Y. T.; Qiu, M. H. Nat. Prod. Bioprospect. 2012, 2, 138. Ramalhete, C.; Lopes, D.; Mulhovo, S.; Molnár, J.; Rosário, V. E.; Ferreira, M.-J. U. Bioorg. Med. Chem. 2010, 18, 5254. Xu, R.; Fazio, G. C.; Matsuda, S. Phytochemistry 2004, 65, 261. Ramalhete, C.; da Cruz, F. P.; Lopes, D.; Mulhovo, S.; Rosário, V. E.; Prudêncio, M.; Ferreira, M.-J. U. Bioorg. Med. Chem. 2011, 19, 7474. 16-O-Acetyl-cucurbitacin F (2): white amorphous powder; mp 152–153 °C; [a]D 20 +21.2 (c 0.33, MeOH); IR (KBr) mmax 3387, 2932, 1735, 1686, 1374, 1251, 1027 cm1; UV (MeOH) kmax (log e): 231 (2.95), 204 (3.06) nm; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Table 1; positive-ion HRESIMS m/z: 583.3309 [M+Na]+ (calcd for C32H48O8Na 583.3309). 16,25-O-Diacetyl-cucurbitacin F-2-O-b-D-glucopyranoside (3): white amorphous powder; mp 157–158 °C; [a]D 20 +16.1 (c 0.93, MeOH); IR (KBr) mmax 3423, 2972, 1720, 1690 1374, 1252, 1028 cm1; UV (MeOH) kmax (log e): 231 (2.98), 204 (2.97) nm; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Table 1; positive-ion HRESIMS m/z: 787.4234 [M+Na]+ (calcd for C40H60O14Na 787.4234). Chiu, M. H.; Gao, J. Chin. Chem. Lett. 2003, 14, 389. Kasai, R.; Matsumoto, K.; Nie, R. L.; Zhou, J.; Tanaka, O. Chem. Pharm. Bull. 1988, 36, 234. Bioassays for cytotoxic activity: The human lung adenocarcinoma cells (H460), human colon cancer cells (SW-620), human colon cancer cells (COLO205) were obtained from the Institute of Materia Medica, Chinese Academy of Medical Sciences. The cytotoxic assays were evaluated using MTT method and Cisplatin was selected as the positive control. These three cell lines were cultured in RPMI1640 medium (Gibgo, USA) which contains 15% fetal bovine serum. The human lung adenocarcinoma cells (H460), human colon cancer cells (SW-620) were washed and digested with 0.25% trypsin, resuspended in the above medium to 1 105/mL; and the human colon cancer cells (COLO205) were resuspended in the above medium to 2 105/mL. Then, an aliquot (100 lL) of the cell suspension was placed in each cell of 96-well flat-bottom plates and were incubated in 5% CO2 at 37 °C for 24 h. After incubation, added the each agent at final concentrations of 50, 10, 2, 0.4, 0.08 lg/mL to the medium with further incubated for 48 h. When the incubation was end, added 10 lL 0.5% MTT (Sigma, USA) to each well and re-incubated for 4 h at 37 °C. Then removed the supernatant from each well and added 200 lL DMSO, respectively. Mixed the plates on a microshaker to make the formazan be sufficiently dissolved and the optical density (OD) was measured on Victor1420 multifunctional-counter instrument at 570 nm. The inhibition (%) was calculated as (OD0 ODS)/ OD0 100%, the OD0 and ODS are the values of the cells without and with test agent. The results (IC50) were defined as the concentration of samples that inhibit 50% control growth after the incubation period. All the data are the three experiments’ mean values which were carried out in triplicate (n = 3). Bai, H.; Zhong, Y.; Xie, Y. Y.; Wang, Y. S.; Liu, L.; Zhou, L.; Wang, J.; Mu, Y. L.; Zuo, C. X. Chem. Biodivers. 2007, 4, 955.
