Bioorganic & Medicinal Chemistry Letters 25 (2015) 3864–3866
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
New phenolic compounds from the seeds of Nigella glandulifera and their inhibitory activities against human cancer cells Lu Sun a, Yu-Ming Liu a,⇑, Bao-Quan Chen a, Qing-Hua Liu b a b
Department of Pharmacy Engineering, Tianjin University of Technology, Tianjin 300384, PR China Xinjiang Institute of Materia Medica, Ürümuqi 830001, PR China
a r t i c l e
i n f o
Article history: Received 5 May 2015 Revised 11 July 2015 Accepted 18 July 2015 Available online 26 July 2015 Keywords: Nigella Nigella glandulifera Ranunculaceae Phenolic compounds Cytotoxic activity
a b s t r a c t Four phenolic compounds, including two new ones, Nigephenol A and B (1–2), and a new natural product, Nigephenol C (3), were isolated from the seeds of Nigella glandulifera. Their structures were elucidated on the basis of spectroscopic analyses using HR-ESI-MS, 1D and 2D NMR spectra. All compounds were evaluated by MTT method for in vitro cytotoxicity against four human cancer cell lines (Bel7402, HepG2, HCT8 and A549). The results revealed that Compounds 1–4 showed more selective activities against HepG2 cells, and that Compound 2 showed significant inhibitory effects against four human tumor cell lines with IC50 values comparable to those of 5-fluorouracil. Ó 2015 Elsevier Ltd. All rights reserved.
The genus Nigella (Ranunculaceae) contains about 20 species, distributed mainly in the Mediterranean region and west Asia.1 The chemical investigations on the genus Nigella have led to the discovery of a series of antitumor products, including triterpenoid saponins,2,3 benzoquinone compound thymoquinone and its derivatives.4–6 Nigella glandulifera Freyn et Sint. is one of the two species growing in China, together with Nigella damascene L. N. glandulifera seeds are commonly eaten in many food preparations by the Uyghur people. The seeds are believed to have diuretic, analgesic, spasmolytic, galactagogue and bronchodilator properties, and to cure edema, urinary calculus and bronchial asthma.7 Phytochemical studies on N. glandulifera have reported the presence of indazole alkaloids, aporphine alkaloids, dolabellane-type diterpene alkaloids, norditerpenoid alkaloids, pyrroloquinoline alkaloids, flavonol glycosides, phthalide derivatives, oleanane-type triterpenoid saponins, with multiple biological activities including antitubercular activity, PTP1B inhibitory activity, tyrosinase inhibitory activity and anti-tumor effects, etc.8–19 As some of the metabolites from our investigation of N. glandulifera have been found to possess attracting cytotoxic activities,14 we thus continued our chemical studies of this medicinal plant with the aim of discovering new bioactive natural products. In the present study we further isolated four phenolic metabolites, including two new ones, Nigephenol A and B (1–2), a new natural product,
⇑ Corresponding author. Tel.: +86 22 60214259; fax: +86 22 60214252. E-mail address: [email protected] (Y.-M. Liu). http://dx.doi.org/10.1016/j.bmcl.2015.07.055 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Nigephenol C (3), and a known compound Salfredin B11 (4) from N. glandulifera (Fig. 1).20 The structures of these compounds were identified by extensive spectroscopic analyses. The cytotoxic activities against four human tumor cell lines in vitro of all compounds were also evaluated by MTT assay. Compound 121 was obtained as a white solid. Its molecular formula was determined to be C12H18O4 on the basis of a pseudmolecular ion peak at m/z 225.1138 [MH] (calcd for C12H17O4, 225.1127) in the HR-ESI-MS, indicating that the molecule has four degrees of unsaturation. Strong absorption bands accounting for hydroxyl group (3444 cm1), aromatic ring (1629 cm1) and geminal dimethyl group (1400 cm1) could be observed in its IR spectrum. In its 1H NMR spectrum (Table 1), two aromatic protons with the same chemical shift at d 6.30 (2H, s) indicated a symmetrical unit of tetra-substituted benzene ring, which was also evidenced by displaying only four aromatic carbons (dC 157.2, 116.2, 106.5, 141.1) in its 13C NMR spectrum. The NOESY interactions of H-4,6 (dH 6.30, s) and H-7 (dH 4.39, s), along with the HMBC correlations from H-4,6 (dH 6.30, s) to C-7 (dC 65.3) and from H-7 (dH 4.39, s) to C-4,6 (dC 106.5) (Fig. 2), established a benzyl alcohol substructure with the aromatic protons (dH 6.30, s) vicinal to its two sides. The 1H NMR spectrum (Table 1) also exhibited the characteristic signals for a 3-hydroxy-3-methylbutyl group [d 1.22 (6H, s, 40 -CH3 and 50 -CH3), 1.64 (2H, t, J = 8.3 Hz, H-20 ), 2.63 (2H, t, J = 8.3 Hz, H-10 )]. The HMBC correlations (Fig. 2) from H-10 (dH 2.63, t, J = 8.3 Hz) to C-1,3 (dC 157.2) and C-2 (dC 116.2) confirmed
3865
L. Sun et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3864–3866 HO
HO
7
HO
7
5
9
5
6
4
6
4
6
8
1
3
1
3
5
8a
HO
OH
2 1'
HO
2' 3'
4'
OH
2 1'
5'
4'
OH
3
2
10 11
8 8a
5a
9
5
9a
HO
O
4a 4 3
2
10 11
5'
2
1
O
O
4a 4
2' 3'
HO
O
6
7
3
4
Figure 1. Chemical structures of compounds 1–4.
Table 1 H and 13C NMR data of compounds 1–3 (d in ppm and J in Hz)
1
No.
1 dH
1 2 3 4 4a 5 6 7 8 8a 9 10 11 10 20
dH
6.30 (1H, s)
106.5
6.30 (1H, s) 4.39 (2H, s)
141.1 106.5 65.3
3 dC
157.2 116.2 157.2
155.0 113.5 155.0 6.23 (1H, s)
104.7
6.23 (1H, s) 4.34 (2H, s)
138.9 104.7 63.5
dH
dC 76.7 129.1
5.52 (1H, d, 9.9) 6.60 (1H, d, 9.9)
118.2 109.8 154.3 107.1 144.0 107.3 155.2 65.1 28.0 28.0
6.33 (1H, s) 6.25 (1H, s) 4.41 (2H, s) 1.34 (3H, s) 1.34 (3H, s)
2.63 (2H, t, 8.3) 1.64 (2H, t, 8.3)
0
3 40 50
2 dC
1.22 (3H, s) 1.22 (3H, s)
19.3
3.21 (2H, d, 6.8) 5.13 (1H, t, 6.8)
43.6 72.0 29.2 29.2
1.66 (3H, s) 1.55 (3H, s)
21.4 122.4 130.1 16.5 24.5
HO
HO
HO
HO
OH
HO
OH
HO
O
OH
3
2
1 HMBC
H
C
NOESY H
H
Figure 2. Key HMBC and NOESY correlations of compounds 1–3.
that the 3-hydroxy-3-methylbutyl group was attached to C-2 of the benzene ring. On the base of further analyses of its DEPT, 1 H–1H COSY, HSQC, HMBC and NOESY spectra, compound 1 was identified as 2-(3-hydroxy-3-methylbutyl)-5-(hydroxymethyl) benzene-1,3-diol (Fig. 1) and was named Nigephenol A. Compound 222 was isolated as a white solid. Its molecular formula was established as C12H16O3 from a pseudmolecular ion peak at m/z 207.1040 [MH] (calcd for C12H15O3, 207.1021) in the HRESI-MS, indicating that the molecule has five degrees of unsaturation. The IR spectrum of 2 revealed the presence of hydroxyl group
(3427 cm1), aromatic ring (1629 cm1) and olefin group (1602 cm1). In its 1H NMR spectrum (Table 1), two aromatic protons with the same chemical shift at d 6.23 (2H, s) indicated a symmetrical unit of tetra-substituted benzene ring, which was also evidenced by displaying only four aromatic carbons (dC 155.0, 113.5, 104.7, 138.9) in its 13C NMR spectrum. A benzyl alcohol substructure, with the aromatic protons (dH 6.23, s) vicinal to its two sides, was constructed by the NOESY interactions between H-4,6 (dH 6.23, s) and H-7 (dH 4.34, s) and by the HMBC correlations from H-4,6 (dH 6.23, s) to C-7 (dC 63.5) and from H-7 (dH 4.34, s) to C-4,6 (dC 104.7) (Fig. 2). The 1H NMR spectrum (Table 1) also exhibited the characteristic signals for a 3-methyl-2-butenyl group (dH 5.13 t, 3.21 d, 1.66 s, 1.55 s). The location of 3-methyl-2-butenyl group was assigned at C-2 of the benzene ring according to the HMBC correlations (Fig. 2) from H-10 (dH 3.21, d, J = 6.8 Hz) to C-1,3 (dC 155.0) and C-2 (dC 113.5). On the base of further analyses of its DEPT, 1H–1H COSY, HSQC, HMBC and NOESY spectra, compound 2 was elucidated to be 5-(hydroxymethyl)-2-(3-methylbut-2-enyl)benzene-1,3-diol (Fig. 1) and named as Nigephenol B. Compound 323 was afforded to be a white solid. HR-ESI-MS of 3 displayed the pseudmolecular ion peak at m/z 207.1010 [M+H]+ (calcd for C12H15O3, 207.1021) corresponding to the molecular formula C12H14O3 with six degrees of unsaturation. The IR spectrum of 3 exhibited characteristic absorption bands for hydroxyl (3428 cm1), aromatic ring (1626 cm1) and olefin group (1602 cm1). The 1H NMR spectrum of 3 (Table 1) showed two aromatic protons attributed to a 1,2,3,5-tetrasubstituted benzene ring [ dH 6.33 (1H, s, H-6) and dH 6.25 (1H, s, H-8) ]. Significant NOE interactions (Fig. 2) of H-6 (dH 6.33, s) and H-8 (dH 6.25, s) with hydroxymethyl protons at dH 4.41 (s, H-9) were observed, and crucial HMBC correlations (Fig. 2) were found from the hydroxymethyl protons at dH 4.41 (s, H-9), which correlated with carbon resonance at dC 65.1 in the HSQC spectrum, to C-6 (dC 107.1) and C-8 (dC 107.3), and from H-6 (dH 6.33, s) and H-8 (dH 6.25, s) to the hydroxymethyl (C-9, dC 65.1); therefore, the hydroxymethyl (C-9, dC 65.1; H-9, dH 4.41) was attached to position 5 (at C-7, dC 144.0) of the 1,2,3,5-tetrasubstituted benzene ring, and the symmetrical tetra-substituted benzene ring as 1 and 2 was absent in 3. Analysis of the 1H NMR data (Table 1), aided by the 1H–1H COSY and NOE experiments, disclosed the presence of two olefinic protons [ dH 5.52 (1H, d, H-3) and dH 6.60 (1H, d, H-4) ], and the small coupling constant of 9.9 Hz between H-3 and H-4 implied a Z-configuration for this olefinic bond. The HMBC correlations (Fig. 2) from the olefinic proton H-3 (dH 5.52, d) to C-4a (dC 109.8), C-2 (dC 76.7), C-10 (dC 28.0) and C-11 (dC 28.0) showed a (Z)-3-methylbutenyl unit, which was located at C-4a (dC 109.8) of the tetrasubstituted benzene ring. The 13C NMR and DEPT spectra of 3 indicated the presence of two oxygenated sp3 quaternary carbons at C-2 (dC 76.7) and C-8a (dC 155.2), and their chemical shifts were quite closed to those of C-2 and C-9a in salfredin B11.8 In view of the asymmetry in 3 again,
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L. Sun et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3864–3866
Table 2 Cytotoxity data IC50 (lM) of compounds 1–4 against four human tumor cells Cell lines (IC50 lM)
Compound
1 2 3 4 5-Fluorouracil
Bel7402
HepG2
HCT-8
A549
>40 7.69 ± 0.22 >40 >40 10.07 ± 0.08
17.79 ± 0.31 15.83 ± 0.54 17.32 ± 0.27 36.95 ± 0.42 16.42 ± 0.19
>40 11.39 ± 0.25 >40 24.13±0.16 6.30 ± 0.32
>40 20.06 ± 0.31 36.31 ± 0.10 >40 14.15 ± 0.23
it was inferred that an oxygen bridge should be formed between C-8a and C-2. On the base of further analyses of its DEPT, 1H–1H COSY, HSQC, HMBC and NOESY spectra, compound 3 was elucidated to be 7-(hydroxymethyl)-2,2-dimethyl-2H-chromen-5-ol (Fig. 1) and named as Nigephenol C. Although it was previously reported in two organic synthetic studies,24,25 this is the first report of its occurrence in nature. The known compound salfredin B11 (4) was confirmed by comparing its spectroscopic data with the corresponding literature values.8,26 Compounds 1–4 were evaluated for cytotoxic activities against four human tumor cell lines Bel7402 (liver carcinoma), HepG2 (liver carcinoma), HCT-8 (colon adenocarcinoma) and A549 (nonsmall cell lung carcinoma) by using MTT assay.27 5-Fluorouracil was used as a positive control, for compounds 1–4 and 5-fluorouracil could be all classified into simple 1,3-benzenediol derivatives. As can be seen in the Table 2, compounds 1–4 showed more selective activities against HepG2 cells, and the activities of 1–3 stronger than 4 were observed as well. It seemed that the hydroxymethyl moiety might be responsible for the increased cytotoxicity. Also showed is it in the Table 2 that compound 3 exhibited weak cytotoxicity to A549 cells. However, some chromene compounds synthesized from 3 had been found to have higher cytotoxic potency in three tumor cell lines A549, HCT29 and L1210.24,25 At last, among the tested compounds, compound 2 showed significantly better inhibitory effects against four human tumor cell lines with IC50 values similar to those of 5-fluorouracil. The fact that only compound 2 has the prenyl group encouraged us to suggest that the prenyl group plays a positive role in the mediating the cytotoxicity against four human tumor cell lines. Quite a number of ortho-prenylated phenols have been reported to possess anti-tumor activity in recent studies.28–31 Owing to its low molecular weight and its privileged structure, compound 2 could be a promising candidate for future development of anti-tumor agents. Acknowledgments This work was supported by the National Natural Science Foundation of People’s Republic of China, under Grant no. 81073153. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.07. 055. References and notes 1. Flora Compilation Committee of Chinese Academy of Science Flora Republicae Popularis Sinicae In ; Science Press: Beijing, 1979; Vol. 27, p 111. 2. Kumara, S. S. M.; Huat, B. T. K. Planta Med. 2001, 67, 29.
3. Cheng, L.; Xia, T.-S.; Wang, Y.-F.; Zhou, W.; Liang, X.-Q.; Xue, J.-Q.; Shi, L.; Wang, Y.; Ding, Q.; Wang, M. Int. J. Oncol. 2014, 45, 757. 4. Yang, J.; Kuang, X.-R.; Lv, P.-T.; Yan, X.-X. Tumor Biol. 2015, 36, 259. 5. Yusufi, M.; Banerjee, S.; Mohammad, M.; Khatal, S.; Venkateswara, S. K.; Khan, E. M.; Aboukameel, A.; Sarkar, F. H.; Padhye, S. Bioorg. Med. Chem. Lett. 2013, 23, 3101. 6. Banerjee, S.; Azmi, A. S.; Padhye, S.; Singh, M. W.; Baruah, J. B.; Philip, P. A.; Sarkar, F. H.; Mohammad, R. M. Pharm. Res. 2010, 27, 1146. 7. State Administration of Traditional Chinese Medicine Chinese Medicinal Herbs In ; Shanghai Science and Technology Publishing House: Shanghai, 1999; Vol. III, p 236. 8. Xin, X.; Yang, Y.; Zhong, J.; Aisa, H. A.; Wang, H. J. Chromatogr., A 2009, 1216, 4258. 9. Sun, L.; Luan, M.; Zhu, W.; Gao, S.; Zhang, Q.; Xu, C.; Lu, X.; Xu, X.; Tian, J.; Zhang, L. Chem. Pharm. Bull. 2013, 61, 873. 10. Zhang, Y.; Ge, D.; Chen, Q.; He, W.; Han, L.; Wei, H.; Jia, X.; Wang, T. J. Nat. Med. 2012, 66, 645. 11. Nguyen, D. H.; Lee, J.-E.; Kim, E.-K. Korean J. Chem. Eng. 2011, 1070, 28. 12. Nguyen, D. M.; Nguyen, D. H.; Lyun, H.-L.; Lee, H.-B.; Shin, J.-H.; Kim, E.-K. J. Microbiol. Biotechnol. 2007, 17, 1585. 13. Chen, Q.-B.; Xin, X.-L.; Yang, Y.; Lee, S.-S.; Aisa, H. A. J. Nat. Prod. 2014, 77, 807. 14. Tian, Z.; Liu, Y.-M.; Chen, S.-B.; Yang, J. S.; Xiao, P. G.; Wang, L.; Wu, E. Molecules 2006, 11, 693. 15. Liu, Y.-M.; Yang, J.-S.; Liu, Q.-H. Chem. Pharm. Bull. 2004, 52, 454. 16. Liu, Y.-M.; Liu, Q.-H.; Chen, B.-Q. Nat. Prod. Res. 2011, 25, 1334. 17. Feng, Y.; Liu, Y.-M.; Liu, Q. H.; Lei, Y.-J. Heterocycles 2012, 85, 3015. 18. Liu, Y.-M.; Sun, L.; Liu, Q.-H.; Lu, S.-R.; Chen, B.-Q. Biochem. Syst. Ecol. 2013, 49, 43. 19. Liu, Y.-M.; Jiang, Y.-H.; Liu, Q.-H.; Chen, B.-Q. Phytochem. Lett. 2013, 6, 556. 20. The oil-free seeds (18 kg) of N. glandulifera were extracted three times with 95% EtOH for 2 h under reflux and then extracted three times with 50% EtOH for 2 h under reflux. After combination and removal of the solvent in vacuo, the EtOH extract was then suspended in distilled water and partitioned successively with petroleum ether, EtOAc and n-BuOH. The n-BuOH fraction (375 g) was chromatographed over silica gel and eluted with CHCl3–MeOH gradient solvent (20:1–1:1). Combination of similar fractions on the basis of TLC analysis afforded 13 fractions. Fraction F2 (85.5 g) was subjected to silica gel column chromatography and eluted with CHCl3–acetone (20:1–5:1) to yield six subfractions A–F. Subfraction E was isolated by column chromatography on silica-gel (CHCl3–acetone, 5:1) and by MPLC on reversed-phase C18 silica gel (65% MeOH in H2O) to afford compound 1 (13.6 mg). Subfraction D was isolated by column chromatography on silica-gel (CHCl3–acetone, 10:1) to give compound 2 (10.0 mg). Subfraction C was isolated by column chromatography on silica-gel (CHCl3–acetone, 15:1) and on Sephadex LH-20 (70% MeOH in H2O) to yield compound 3 (6.4 mg). Subfraction B was isolated by column chromatography on silica-gel (CHCl3–acetone, 20:1) to afford compound 4 (7.9 mg). 21. Compound 1: White solid; UV (MeOH) kmax (log e) 238 (4.00), 281 (3.56) nm; IR (KBr) mmax 3444, 2926, 2855, 1629, 1400 cm-1; HR-ESI-MS (negative mode) m/z 225.1138 [MH] (calcd 225.1127 for C12H17O4); 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectral data see Table 1. 22. Compound 2: White solid; UV (MeOH) kmax (log e) 239 (4.20), 280 (3.81) nm; IR (KBr) mmax 3427, 2926, 2854, 1629, 1602, 1435, 1384 cm-1; HR-ESI-MS (negative mode) m/z 207.1040 [MH] (calcd 207.1021 for C12H15O3); 1H NMR (400 MHz, CD3OD+CDCl3) and 13C NMR (100 MHz, CD3OD+CDCl3) spectral data see Table 1. 23. Compound 3: White solid; UV (MeOH) kmax (log e) 232 (3.85), 276 (3.58) nm; IR (KBr) mmax 3428, 2926, 2857, 1626, 1602, 1436, 1383 cm1; HR-ESI-MS (positive mode) m/z 207.1010 [M+H]+ (calcd 207.1021 for C12H15O3); 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectral data see Table 1. 24. Kolokythas, G.; Pouli, N.; Marakos, P.; Pratsinis, H.; Kletsas, D. Eur. J. Med. Chem. 2006, 41, 71. 25. Kolokythas, G.; Kostakis, I. K.; Pouli, N.; Marakos, P.; Skaltsounis, A.-L.; Pratsinis, H. Bioorg. Med. Chem. Lett. 2002, 12, 1443. 26. Joshi, B. S.; Singh, K. L.; Roy, R. Magn. Reson. Chem. 2001, 39, 771. 27. The cell suspensions were distributed into 96-well cell culture plates and cultured at 37 °C, with 5% CO2 in incubator for 24 h. Each cancer cell line was exposed to the test compound at five different concentrations for 72 h. Then, 100 lL of MTT (0.5 mg/mL in PBS) was added to each well, and the plates were incubated at 37 °C for another 4 h. After incubation, the culture medium was replaced with 150 lL of DMSO, and the plates were shaken for 3 min to dissolve the crystals, then the optical density values were read on the microplate reader (BioTek Epoch) at 544 nm. All tests and analyses were carried out in triplicate. DMSO and 5-fluorouracil were applied as the blank control and positive control, respectively. 28. Han, Q.-B.; Qiao, C.-F.; Song, J.-Z.; Yang, N.-Y.; Cao, X.-W.; Peng, Y.; Yang, D.-J.; Chen, S.-L.; Xu, H.-X. Chem. Biodivers. 2007, 4, 940. 29. Lee, J. H.; Baek, N.; Kim, S.-H.; Park, H. W.; Yang, J. H.; Lee, J. J.; Kim, S. J.; Jeong, S. i.; Oh, C.-H.; Lee, K.-H.; Kim, D. K. Pharm. Res. 2007, 30, 408. 30. Dorn, C.; Weiss, T. S.; Heilmann, J.; Hellerbrand, C. Int. J. Oncol. 2010, 36, 435. 31. Sansom, C. E.; Larsen, L.; Perry, N. B.; Berridge, M. V.; Chia, E. W.; Harper, J. L.; Webb, V. L. J. Nat. Prod. 2007, 70, 2042.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
New phenolic compounds from the seeds of Nigella glandulifera and their inhibitory activities against human cancer cells Lu Sun a, Yu-Ming Liu a,⇑, Bao-Quan Chen a, Qing-Hua Liu b a b
Department of Pharmacy Engineering, Tianjin University of Technology, Tianjin 300384, PR China Xinjiang Institute of Materia Medica, Ürümuqi 830001, PR China
a r t i c l e
i n f o
Article history: Received 5 May 2015 Revised 11 July 2015 Accepted 18 July 2015 Available online 26 July 2015 Keywords: Nigella Nigella glandulifera Ranunculaceae Phenolic compounds Cytotoxic activity
a b s t r a c t Four phenolic compounds, including two new ones, Nigephenol A and B (1–2), and a new natural product, Nigephenol C (3), were isolated from the seeds of Nigella glandulifera. Their structures were elucidated on the basis of spectroscopic analyses using HR-ESI-MS, 1D and 2D NMR spectra. All compounds were evaluated by MTT method for in vitro cytotoxicity against four human cancer cell lines (Bel7402, HepG2, HCT8 and A549). The results revealed that Compounds 1–4 showed more selective activities against HepG2 cells, and that Compound 2 showed significant inhibitory effects against four human tumor cell lines with IC50 values comparable to those of 5-fluorouracil. Ó 2015 Elsevier Ltd. All rights reserved.
The genus Nigella (Ranunculaceae) contains about 20 species, distributed mainly in the Mediterranean region and west Asia.1 The chemical investigations on the genus Nigella have led to the discovery of a series of antitumor products, including triterpenoid saponins,2,3 benzoquinone compound thymoquinone and its derivatives.4–6 Nigella glandulifera Freyn et Sint. is one of the two species growing in China, together with Nigella damascene L. N. glandulifera seeds are commonly eaten in many food preparations by the Uyghur people. The seeds are believed to have diuretic, analgesic, spasmolytic, galactagogue and bronchodilator properties, and to cure edema, urinary calculus and bronchial asthma.7 Phytochemical studies on N. glandulifera have reported the presence of indazole alkaloids, aporphine alkaloids, dolabellane-type diterpene alkaloids, norditerpenoid alkaloids, pyrroloquinoline alkaloids, flavonol glycosides, phthalide derivatives, oleanane-type triterpenoid saponins, with multiple biological activities including antitubercular activity, PTP1B inhibitory activity, tyrosinase inhibitory activity and anti-tumor effects, etc.8–19 As some of the metabolites from our investigation of N. glandulifera have been found to possess attracting cytotoxic activities,14 we thus continued our chemical studies of this medicinal plant with the aim of discovering new bioactive natural products. In the present study we further isolated four phenolic metabolites, including two new ones, Nigephenol A and B (1–2), a new natural product,
⇑ Corresponding author. Tel.: +86 22 60214259; fax: +86 22 60214252. E-mail address: [email protected] (Y.-M. Liu). http://dx.doi.org/10.1016/j.bmcl.2015.07.055 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Nigephenol C (3), and a known compound Salfredin B11 (4) from N. glandulifera (Fig. 1).20 The structures of these compounds were identified by extensive spectroscopic analyses. The cytotoxic activities against four human tumor cell lines in vitro of all compounds were also evaluated by MTT assay. Compound 121 was obtained as a white solid. Its molecular formula was determined to be C12H18O4 on the basis of a pseudmolecular ion peak at m/z 225.1138 [MH] (calcd for C12H17O4, 225.1127) in the HR-ESI-MS, indicating that the molecule has four degrees of unsaturation. Strong absorption bands accounting for hydroxyl group (3444 cm1), aromatic ring (1629 cm1) and geminal dimethyl group (1400 cm1) could be observed in its IR spectrum. In its 1H NMR spectrum (Table 1), two aromatic protons with the same chemical shift at d 6.30 (2H, s) indicated a symmetrical unit of tetra-substituted benzene ring, which was also evidenced by displaying only four aromatic carbons (dC 157.2, 116.2, 106.5, 141.1) in its 13C NMR spectrum. The NOESY interactions of H-4,6 (dH 6.30, s) and H-7 (dH 4.39, s), along with the HMBC correlations from H-4,6 (dH 6.30, s) to C-7 (dC 65.3) and from H-7 (dH 4.39, s) to C-4,6 (dC 106.5) (Fig. 2), established a benzyl alcohol substructure with the aromatic protons (dH 6.30, s) vicinal to its two sides. The 1H NMR spectrum (Table 1) also exhibited the characteristic signals for a 3-hydroxy-3-methylbutyl group [d 1.22 (6H, s, 40 -CH3 and 50 -CH3), 1.64 (2H, t, J = 8.3 Hz, H-20 ), 2.63 (2H, t, J = 8.3 Hz, H-10 )]. The HMBC correlations (Fig. 2) from H-10 (dH 2.63, t, J = 8.3 Hz) to C-1,3 (dC 157.2) and C-2 (dC 116.2) confirmed
3865
L. Sun et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3864–3866 HO
HO
7
HO
7
5
9
5
6
4
6
4
6
8
1
3
1
3
5
8a
HO
OH
2 1'
HO
2' 3'
4'
OH
2 1'
5'
4'
OH
3
2
10 11
8 8a
5a
9
5
9a
HO
O
4a 4 3
2
10 11
5'
2
1
O
O
4a 4
2' 3'
HO
O
6
7
3
4
Figure 1. Chemical structures of compounds 1–4.
Table 1 H and 13C NMR data of compounds 1–3 (d in ppm and J in Hz)
1
No.
1 dH
1 2 3 4 4a 5 6 7 8 8a 9 10 11 10 20
dH
6.30 (1H, s)
106.5
6.30 (1H, s) 4.39 (2H, s)
141.1 106.5 65.3
3 dC
157.2 116.2 157.2
155.0 113.5 155.0 6.23 (1H, s)
104.7
6.23 (1H, s) 4.34 (2H, s)
138.9 104.7 63.5
dH
dC 76.7 129.1
5.52 (1H, d, 9.9) 6.60 (1H, d, 9.9)
118.2 109.8 154.3 107.1 144.0 107.3 155.2 65.1 28.0 28.0
6.33 (1H, s) 6.25 (1H, s) 4.41 (2H, s) 1.34 (3H, s) 1.34 (3H, s)
2.63 (2H, t, 8.3) 1.64 (2H, t, 8.3)
0
3 40 50
2 dC
1.22 (3H, s) 1.22 (3H, s)
19.3
3.21 (2H, d, 6.8) 5.13 (1H, t, 6.8)
43.6 72.0 29.2 29.2
1.66 (3H, s) 1.55 (3H, s)
21.4 122.4 130.1 16.5 24.5
HO
HO
HO
HO
OH
HO
OH
HO
O
OH
3
2
1 HMBC
H
C
NOESY H
H
Figure 2. Key HMBC and NOESY correlations of compounds 1–3.
that the 3-hydroxy-3-methylbutyl group was attached to C-2 of the benzene ring. On the base of further analyses of its DEPT, 1 H–1H COSY, HSQC, HMBC and NOESY spectra, compound 1 was identified as 2-(3-hydroxy-3-methylbutyl)-5-(hydroxymethyl) benzene-1,3-diol (Fig. 1) and was named Nigephenol A. Compound 222 was isolated as a white solid. Its molecular formula was established as C12H16O3 from a pseudmolecular ion peak at m/z 207.1040 [MH] (calcd for C12H15O3, 207.1021) in the HRESI-MS, indicating that the molecule has five degrees of unsaturation. The IR spectrum of 2 revealed the presence of hydroxyl group
(3427 cm1), aromatic ring (1629 cm1) and olefin group (1602 cm1). In its 1H NMR spectrum (Table 1), two aromatic protons with the same chemical shift at d 6.23 (2H, s) indicated a symmetrical unit of tetra-substituted benzene ring, which was also evidenced by displaying only four aromatic carbons (dC 155.0, 113.5, 104.7, 138.9) in its 13C NMR spectrum. A benzyl alcohol substructure, with the aromatic protons (dH 6.23, s) vicinal to its two sides, was constructed by the NOESY interactions between H-4,6 (dH 6.23, s) and H-7 (dH 4.34, s) and by the HMBC correlations from H-4,6 (dH 6.23, s) to C-7 (dC 63.5) and from H-7 (dH 4.34, s) to C-4,6 (dC 104.7) (Fig. 2). The 1H NMR spectrum (Table 1) also exhibited the characteristic signals for a 3-methyl-2-butenyl group (dH 5.13 t, 3.21 d, 1.66 s, 1.55 s). The location of 3-methyl-2-butenyl group was assigned at C-2 of the benzene ring according to the HMBC correlations (Fig. 2) from H-10 (dH 3.21, d, J = 6.8 Hz) to C-1,3 (dC 155.0) and C-2 (dC 113.5). On the base of further analyses of its DEPT, 1H–1H COSY, HSQC, HMBC and NOESY spectra, compound 2 was elucidated to be 5-(hydroxymethyl)-2-(3-methylbut-2-enyl)benzene-1,3-diol (Fig. 1) and named as Nigephenol B. Compound 323 was afforded to be a white solid. HR-ESI-MS of 3 displayed the pseudmolecular ion peak at m/z 207.1010 [M+H]+ (calcd for C12H15O3, 207.1021) corresponding to the molecular formula C12H14O3 with six degrees of unsaturation. The IR spectrum of 3 exhibited characteristic absorption bands for hydroxyl (3428 cm1), aromatic ring (1626 cm1) and olefin group (1602 cm1). The 1H NMR spectrum of 3 (Table 1) showed two aromatic protons attributed to a 1,2,3,5-tetrasubstituted benzene ring [ dH 6.33 (1H, s, H-6) and dH 6.25 (1H, s, H-8) ]. Significant NOE interactions (Fig. 2) of H-6 (dH 6.33, s) and H-8 (dH 6.25, s) with hydroxymethyl protons at dH 4.41 (s, H-9) were observed, and crucial HMBC correlations (Fig. 2) were found from the hydroxymethyl protons at dH 4.41 (s, H-9), which correlated with carbon resonance at dC 65.1 in the HSQC spectrum, to C-6 (dC 107.1) and C-8 (dC 107.3), and from H-6 (dH 6.33, s) and H-8 (dH 6.25, s) to the hydroxymethyl (C-9, dC 65.1); therefore, the hydroxymethyl (C-9, dC 65.1; H-9, dH 4.41) was attached to position 5 (at C-7, dC 144.0) of the 1,2,3,5-tetrasubstituted benzene ring, and the symmetrical tetra-substituted benzene ring as 1 and 2 was absent in 3. Analysis of the 1H NMR data (Table 1), aided by the 1H–1H COSY and NOE experiments, disclosed the presence of two olefinic protons [ dH 5.52 (1H, d, H-3) and dH 6.60 (1H, d, H-4) ], and the small coupling constant of 9.9 Hz between H-3 and H-4 implied a Z-configuration for this olefinic bond. The HMBC correlations (Fig. 2) from the olefinic proton H-3 (dH 5.52, d) to C-4a (dC 109.8), C-2 (dC 76.7), C-10 (dC 28.0) and C-11 (dC 28.0) showed a (Z)-3-methylbutenyl unit, which was located at C-4a (dC 109.8) of the tetrasubstituted benzene ring. The 13C NMR and DEPT spectra of 3 indicated the presence of two oxygenated sp3 quaternary carbons at C-2 (dC 76.7) and C-8a (dC 155.2), and their chemical shifts were quite closed to those of C-2 and C-9a in salfredin B11.8 In view of the asymmetry in 3 again,
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Table 2 Cytotoxity data IC50 (lM) of compounds 1–4 against four human tumor cells Cell lines (IC50 lM)
Compound
1 2 3 4 5-Fluorouracil
Bel7402
HepG2
HCT-8
A549
>40 7.69 ± 0.22 >40 >40 10.07 ± 0.08
17.79 ± 0.31 15.83 ± 0.54 17.32 ± 0.27 36.95 ± 0.42 16.42 ± 0.19
>40 11.39 ± 0.25 >40 24.13±0.16 6.30 ± 0.32
>40 20.06 ± 0.31 36.31 ± 0.10 >40 14.15 ± 0.23
it was inferred that an oxygen bridge should be formed between C-8a and C-2. On the base of further analyses of its DEPT, 1H–1H COSY, HSQC, HMBC and NOESY spectra, compound 3 was elucidated to be 7-(hydroxymethyl)-2,2-dimethyl-2H-chromen-5-ol (Fig. 1) and named as Nigephenol C. Although it was previously reported in two organic synthetic studies,24,25 this is the first report of its occurrence in nature. The known compound salfredin B11 (4) was confirmed by comparing its spectroscopic data with the corresponding literature values.8,26 Compounds 1–4 were evaluated for cytotoxic activities against four human tumor cell lines Bel7402 (liver carcinoma), HepG2 (liver carcinoma), HCT-8 (colon adenocarcinoma) and A549 (nonsmall cell lung carcinoma) by using MTT assay.27 5-Fluorouracil was used as a positive control, for compounds 1–4 and 5-fluorouracil could be all classified into simple 1,3-benzenediol derivatives. As can be seen in the Table 2, compounds 1–4 showed more selective activities against HepG2 cells, and the activities of 1–3 stronger than 4 were observed as well. It seemed that the hydroxymethyl moiety might be responsible for the increased cytotoxicity. Also showed is it in the Table 2 that compound 3 exhibited weak cytotoxicity to A549 cells. However, some chromene compounds synthesized from 3 had been found to have higher cytotoxic potency in three tumor cell lines A549, HCT29 and L1210.24,25 At last, among the tested compounds, compound 2 showed significantly better inhibitory effects against four human tumor cell lines with IC50 values similar to those of 5-fluorouracil. The fact that only compound 2 has the prenyl group encouraged us to suggest that the prenyl group plays a positive role in the mediating the cytotoxicity against four human tumor cell lines. Quite a number of ortho-prenylated phenols have been reported to possess anti-tumor activity in recent studies.28–31 Owing to its low molecular weight and its privileged structure, compound 2 could be a promising candidate for future development of anti-tumor agents. Acknowledgments This work was supported by the National Natural Science Foundation of People’s Republic of China, under Grant no. 81073153. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.07. 055. References and notes 1. Flora Compilation Committee of Chinese Academy of Science Flora Republicae Popularis Sinicae In ; Science Press: Beijing, 1979; Vol. 27, p 111. 2. Kumara, S. S. M.; Huat, B. T. K. Planta Med. 2001, 67, 29.
3. Cheng, L.; Xia, T.-S.; Wang, Y.-F.; Zhou, W.; Liang, X.-Q.; Xue, J.-Q.; Shi, L.; Wang, Y.; Ding, Q.; Wang, M. Int. J. Oncol. 2014, 45, 757. 4. Yang, J.; Kuang, X.-R.; Lv, P.-T.; Yan, X.-X. Tumor Biol. 2015, 36, 259. 5. Yusufi, M.; Banerjee, S.; Mohammad, M.; Khatal, S.; Venkateswara, S. K.; Khan, E. M.; Aboukameel, A.; Sarkar, F. H.; Padhye, S. Bioorg. Med. Chem. Lett. 2013, 23, 3101. 6. Banerjee, S.; Azmi, A. S.; Padhye, S.; Singh, M. W.; Baruah, J. B.; Philip, P. A.; Sarkar, F. H.; Mohammad, R. M. Pharm. Res. 2010, 27, 1146. 7. State Administration of Traditional Chinese Medicine Chinese Medicinal Herbs In ; Shanghai Science and Technology Publishing House: Shanghai, 1999; Vol. III, p 236. 8. Xin, X.; Yang, Y.; Zhong, J.; Aisa, H. A.; Wang, H. J. Chromatogr., A 2009, 1216, 4258. 9. Sun, L.; Luan, M.; Zhu, W.; Gao, S.; Zhang, Q.; Xu, C.; Lu, X.; Xu, X.; Tian, J.; Zhang, L. Chem. Pharm. Bull. 2013, 61, 873. 10. Zhang, Y.; Ge, D.; Chen, Q.; He, W.; Han, L.; Wei, H.; Jia, X.; Wang, T. J. Nat. Med. 2012, 66, 645. 11. Nguyen, D. H.; Lee, J.-E.; Kim, E.-K. Korean J. Chem. Eng. 2011, 1070, 28. 12. Nguyen, D. M.; Nguyen, D. H.; Lyun, H.-L.; Lee, H.-B.; Shin, J.-H.; Kim, E.-K. J. Microbiol. Biotechnol. 2007, 17, 1585. 13. Chen, Q.-B.; Xin, X.-L.; Yang, Y.; Lee, S.-S.; Aisa, H. A. J. Nat. Prod. 2014, 77, 807. 14. Tian, Z.; Liu, Y.-M.; Chen, S.-B.; Yang, J. S.; Xiao, P. G.; Wang, L.; Wu, E. Molecules 2006, 11, 693. 15. Liu, Y.-M.; Yang, J.-S.; Liu, Q.-H. Chem. Pharm. Bull. 2004, 52, 454. 16. Liu, Y.-M.; Liu, Q.-H.; Chen, B.-Q. Nat. Prod. Res. 2011, 25, 1334. 17. Feng, Y.; Liu, Y.-M.; Liu, Q. H.; Lei, Y.-J. Heterocycles 2012, 85, 3015. 18. Liu, Y.-M.; Sun, L.; Liu, Q.-H.; Lu, S.-R.; Chen, B.-Q. Biochem. Syst. Ecol. 2013, 49, 43. 19. Liu, Y.-M.; Jiang, Y.-H.; Liu, Q.-H.; Chen, B.-Q. Phytochem. Lett. 2013, 6, 556. 20. The oil-free seeds (18 kg) of N. glandulifera were extracted three times with 95% EtOH for 2 h under reflux and then extracted three times with 50% EtOH for 2 h under reflux. After combination and removal of the solvent in vacuo, the EtOH extract was then suspended in distilled water and partitioned successively with petroleum ether, EtOAc and n-BuOH. The n-BuOH fraction (375 g) was chromatographed over silica gel and eluted with CHCl3–MeOH gradient solvent (20:1–1:1). Combination of similar fractions on the basis of TLC analysis afforded 13 fractions. Fraction F2 (85.5 g) was subjected to silica gel column chromatography and eluted with CHCl3–acetone (20:1–5:1) to yield six subfractions A–F. Subfraction E was isolated by column chromatography on silica-gel (CHCl3–acetone, 5:1) and by MPLC on reversed-phase C18 silica gel (65% MeOH in H2O) to afford compound 1 (13.6 mg). Subfraction D was isolated by column chromatography on silica-gel (CHCl3–acetone, 10:1) to give compound 2 (10.0 mg). Subfraction C was isolated by column chromatography on silica-gel (CHCl3–acetone, 15:1) and on Sephadex LH-20 (70% MeOH in H2O) to yield compound 3 (6.4 mg). Subfraction B was isolated by column chromatography on silica-gel (CHCl3–acetone, 20:1) to afford compound 4 (7.9 mg). 21. Compound 1: White solid; UV (MeOH) kmax (log e) 238 (4.00), 281 (3.56) nm; IR (KBr) mmax 3444, 2926, 2855, 1629, 1400 cm-1; HR-ESI-MS (negative mode) m/z 225.1138 [MH] (calcd 225.1127 for C12H17O4); 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectral data see Table 1. 22. Compound 2: White solid; UV (MeOH) kmax (log e) 239 (4.20), 280 (3.81) nm; IR (KBr) mmax 3427, 2926, 2854, 1629, 1602, 1435, 1384 cm-1; HR-ESI-MS (negative mode) m/z 207.1040 [MH] (calcd 207.1021 for C12H15O3); 1H NMR (400 MHz, CD3OD+CDCl3) and 13C NMR (100 MHz, CD3OD+CDCl3) spectral data see Table 1. 23. Compound 3: White solid; UV (MeOH) kmax (log e) 232 (3.85), 276 (3.58) nm; IR (KBr) mmax 3428, 2926, 2857, 1626, 1602, 1436, 1383 cm1; HR-ESI-MS (positive mode) m/z 207.1010 [M+H]+ (calcd 207.1021 for C12H15O3); 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectral data see Table 1. 24. Kolokythas, G.; Pouli, N.; Marakos, P.; Pratsinis, H.; Kletsas, D. Eur. J. Med. Chem. 2006, 41, 71. 25. Kolokythas, G.; Kostakis, I. K.; Pouli, N.; Marakos, P.; Skaltsounis, A.-L.; Pratsinis, H. Bioorg. Med. Chem. Lett. 2002, 12, 1443. 26. Joshi, B. S.; Singh, K. L.; Roy, R. Magn. Reson. Chem. 2001, 39, 771. 27. The cell suspensions were distributed into 96-well cell culture plates and cultured at 37 °C, with 5% CO2 in incubator for 24 h. Each cancer cell line was exposed to the test compound at five different concentrations for 72 h. Then, 100 lL of MTT (0.5 mg/mL in PBS) was added to each well, and the plates were incubated at 37 °C for another 4 h. After incubation, the culture medium was replaced with 150 lL of DMSO, and the plates were shaken for 3 min to dissolve the crystals, then the optical density values were read on the microplate reader (BioTek Epoch) at 544 nm. All tests and analyses were carried out in triplicate. DMSO and 5-fluorouracil were applied as the blank control and positive control, respectively. 28. Han, Q.-B.; Qiao, C.-F.; Song, J.-Z.; Yang, N.-Y.; Cao, X.-W.; Peng, Y.; Yang, D.-J.; Chen, S.-L.; Xu, H.-X. Chem. Biodivers. 2007, 4, 940. 29. Lee, J. H.; Baek, N.; Kim, S.-H.; Park, H. W.; Yang, J. H.; Lee, J. J.; Kim, S. J.; Jeong, S. i.; Oh, C.-H.; Lee, K.-H.; Kim, D. K. Pharm. Res. 2007, 30, 408. 30. Dorn, C.; Weiss, T. S.; Heilmann, J.; Hellerbrand, C. Int. J. Oncol. 2010, 36, 435. 31. Sansom, C. E.; Larsen, L.; Perry, N. B.; Berridge, M. V.; Chia, E. W.; Harper, J. L.; Webb, V. L. J. Nat. Prod. 2007, 70, 2042.
