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Bioorganic & Medicinal Chemistry Letters 25 (2015) 38643866

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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
Department of Pharmacy Engineering, Tianjin University of Technology, Tianjin 300384, PR China
b
Xinjiang Institute of Materia Medica, rmuqi 830001, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Four phenolic compounds, including two new ones, Nigephenol A and B (12), and a new natural product,
Received 5 May 2015 Nigephenol C (3), were isolated from the seeds of Nigella glandulifera. Their structures were elucidated on
Revised 11 July 2015 the basis of spectroscopic analyses using HR-ESI-MS, 1D and 2D NMR spectra. All compounds were eval-
Accepted 18 July 2015
uated by MTT method for in vitro cytotoxicity against four human cancer cell lines (Bel7402, HepG2, HCT-
Available online 26 July 2015
8 and A549). The results revealed that Compounds 14 showed more selective activities against HepG2
cells, and that Compound 2 showed signicant inhibitory effects against four human tumor cell lines with
Keywords:
IC50 values comparable to those of 5-uorouracil.
Nigella
Nigella glandulifera
2015 Elsevier Ltd. All rights reserved.
Ranunculaceae
Phenolic compounds
Cytotoxic activity

The genus Nigella (Ranunculaceae) contains about 20 species, Nigephenol C (3), and a known compound Salfredin B11 (4) from
distributed mainly in the Mediterranean region and west Asia.1 N. glandulifera (Fig. 1).20 The structures of these compounds were
The chemical investigations on the genus Nigella have led to the identied by extensive spectroscopic analyses. The cytotoxic activ-
discovery of a series of antitumor products, including triterpenoid ities against four human tumor cell lines in vitro of all compounds
saponins,2,3 benzoquinone compound thymoquinone and its were also evaluated by MTT assay.
derivatives.46 Nigella glandulifera Freyn et Sint. is one of the two Compound 121 was obtained as a white solid. Its molecular for-
species growing in China, together with Nigella damascene L. N. mula was determined to be C12H18O4 on the basis of a pseudmolec-
glandulifera seeds are commonly eaten in many food preparations ular ion peak at m/z 225.1138 [MH] (calcd for C12H17O4,
by the Uyghur people. The seeds are believed to have diuretic, 225.1127) in the HR-ESI-MS, indicating that the molecule has four
analgesic, spasmolytic, galactagogue and bronchodilator proper- degrees of unsaturation. Strong absorption bands accounting for
ties, and to cure edema, urinary calculus and bronchial asthma.7 hydroxyl group (3444 cm1), aromatic ring (1629 cm1) and gem-
Phytochemical studies on N. glandulifera have reported the pres- inal dimethyl group (1400 cm1) could be observed in its IR
ence of indazole alkaloids, aporphine alkaloids, dolabellane-type spectrum.
diterpene alkaloids, norditerpenoid alkaloids, pyrroloquinoline In its 1H NMR spectrum (Table 1), two aromatic protons with
alkaloids, avonol glycosides, phthalide derivatives, oleanane-type the same chemical shift at d 6.30 (2H, s) indicated a symmetrical
triterpenoid saponins, with multiple biological activities including unit of tetra-substituted benzene ring, which was also evidenced
antitubercular activity, PTP1B inhibitory activity, tyrosinase inhibi- by displaying only four aromatic carbons (dC 157.2, 116.2, 106.5,
tory activity and anti-tumor effects, etc.819 As some of the 141.1) in its 13C NMR spectrum. The NOESY interactions of H-4,6
metabolites from our investigation of N. glandulifera have been (dH 6.30, s) and H-7 (dH 4.39, s), along with the HMBC correlations
found to possess attracting cytotoxic activities,14 we thus contin- from H-4,6 (dH 6.30, s) to C-7 (dC 65.3) and from H-7 (dH 4.39, s) to
ued our chemical studies of this medicinal plant with the aim of C-4,6 (dC 106.5) (Fig. 2), established a benzyl alcohol substructure
discovering new bioactive natural products. In the present study with the aromatic protons (dH 6.30, s) vicinal to its two sides.
we further isolated four phenolic metabolites, including two new The 1H NMR spectrum (Table 1) also exhibited the characteristic
ones, Nigephenol A and B (12), a new natural product, 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
Corresponding author. Tel.: +86 22 60214259; fax: +86 22 60214252. 2.63, t, J = 8.3 Hz) to C-1,3 (dC 157.2) and C-2 (dC 116.2) conrmed
E-mail address: coumarin1968@hotmail.com (Y.-M. Liu).

http://dx.doi.org/10.1016/j.bmcl.2015.07.055
0960-894X/ 2015 Elsevier Ltd. All rights reserved.
L. Sun et al. / Bioorg. Med. Chem. Lett. 25 (2015) 38643866 3865

HO HO HO 8
7 7 9 O
5 5 7 6
8a
6 4 6 4 6 8 O
5a 9
1 3 1 3 5 8a 5 9a
HO 2 OH HO 2 OH HO 4a O HO 4a O
10 10
1' 1' 4
2' 2' 2 11 4
2 11
3 3
3' 3'
4' 5' 4' 5'
OH

1 2 3 4

Figure 1. Chemical structures of compounds 14.

Table 1 (3427 cm1), aromatic ring (1629 cm1) and olen group
1
H and 13C NMR data of compounds 13 (d in ppm and J in Hz) (1602 cm1).
No. 1 2 3 In its 1H NMR spectrum (Table 1), two aromatic protons with
dH dC dH dC dH dC
the same chemical shift at d 6.23 (2H, s) indicated a symmetrical
unit of tetra-substituted benzene ring, which was also evidenced
1 157.2 155.0
2 116.2 113.5 76.7
by displaying only four aromatic carbons (dC 155.0, 113.5, 104.7,
3 157.2 155.0 5.52 (1H, d, 129.1 138.9) in its 13C NMR spectrum. A benzyl alcohol substructure,
9.9) with the aromatic protons (dH 6.23, s) vicinal to its two sides,
4 6.30 (1H, s) 106.5 6.23 (1H, s) 104.7 6.60 (1H, d, 118.2 was constructed by the NOESY interactions between H-4,6 (dH
9.9)
6.23, s) and H-7 (dH 4.34, s) and by the HMBC correlations from
4a 109.8
5 141.1 138.9 154.3 H-4,6 (dH 6.23, s) to C-7 (dC 63.5) and from H-7 (dH 4.34, s) to
6 6.30 (1H, s) 106.5 6.23 (1H, s) 104.7 6.33 (1H, s) 107.1 C-4,6 (dC 104.7) (Fig. 2). The 1H NMR spectrum (Table 1) also exhib-
7 4.39 (2H, s) 65.3 4.34 (2H, s) 63.5 144.0 ited the characteristic signals for a 3-methyl-2-butenyl group (dH
8 6.25 (1H, s) 107.3 5.13 t, 3.21 d, 1.66 s, 1.55 s). The location of 3-methyl-2-butenyl
8a 155.2
group was assigned at C-2 of the benzene ring according to the
9 4.41 (2H, s) 65.1
10 1.34 (3H, s) 28.0 HMBC correlations (Fig. 2) from H-10 (dH 3.21, d, J = 6.8 Hz) to
11 1.34 (3H, s) 28.0 C-1,3 (dC 155.0) and C-2 (dC 113.5). On the base of further analyses
10 2.63 (2H, t, 19.3 3.21 (2H, d, 21.4 of its DEPT, 1H1H COSY, HSQC, HMBC and NOESY spectra, com-
8.3) 6.8)
pound 2 was elucidated to be 5-(hydroxymethyl)-2-(3-methyl-
20 1.64 (2H, t, 43.6 5.13 (1H, t, 122.4
8.3) 6.8) but-2-enyl)benzene-1,3-diol (Fig. 1) and named as Nigephenol B.
30
72.0 130.1 Compound 323 was afforded to be a white solid. HR-ESI-MS of 3
40 1.22 (3H, s) 29.2 1.66 (3H, s) 16.5 displayed the pseudmolecular ion peak at m/z 207.1010 [M+H]+
50 1.22 (3H, s) 29.2 1.55 (3H, s) 24.5 (calcd for C12H15O3, 207.1021) corresponding to the molecular for-
mula C12H14O3 with six degrees of unsaturation. The IR spectrum
of 3 exhibited characteristic absorption bands for hydroxyl
HO (3428 cm1), aromatic ring (1626 cm1) and olen group
HO HO
(1602 cm1).
The 1H NMR spectrum of 3 (Table 1) showed two aromatic pro-
tons 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) ]. Signicant NOE interactions
HO OH HO OH HO O
(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 corre-
lations (Fig. 2) were found from the hydroxymethyl protons at dH
4.41 (s, H-9), which correlated with carbon resonance at dC 65.1
OH in the HSQC spectrum, to C-6 (dC 107.1) and C-8 (dC 107.3), and
1 2 3 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
H C H 4.41) was attached to position 5 (at C-7, dC 144.0) of the 1,2,3,5-te-
HMBC NOESY H
trasubstituted benzene ring, and the symmetrical tetra-substituted
Figure 2. Key HMBC and NOESY correlations of compounds 13. benzene ring as 1 and 2 was absent in 3. Analysis of the 1H NMR
data (Table 1), aided by the 1H1H COSY and NOE experiments, dis-
closed the presence of two olenic protons [ dH 5.52 (1H, d, H-3)
that the 3-hydroxy-3-methylbutyl group was attached to C-2 of and dH 6.60 (1H, d, H-4) ], and the small coupling constant of
the benzene ring. On the base of further analyses of its DEPT, 9.9 Hz between H-3 and H-4 implied a Z-conguration for this ole-
1
H1H COSY, HSQC, HMBC and NOESY spectra, compound 1 was nic bond. The HMBC correlations (Fig. 2) from the olenic proton
identied as 2-(3-hydroxy-3-methylbutyl)-5-(hydroxymethyl) H-3 (dH 5.52, d) to C-4a (dC 109.8), C-2 (dC 76.7), C-10 (dC 28.0) and
benzene-1,3-diol (Fig. 1) and was named Nigephenol A. C-11 (dC 28.0) showed a (Z)-3-methylbutenyl unit, which was
Compound 222 was isolated as a white solid. Its molecular for- located at C-4a (dC 109.8) of the tetrasubstituted benzene ring.
mula was established as C12H16O3 from a pseudmolecular ion peak The 13C NMR and DEPT spectra of 3 indicated the presence of
at m/z 207.1040 [MH] (calcd for C12H15O3, 207.1021) in the HR- two oxygenated sp3 quaternary carbons at C-2 (dC 76.7) and C-8a
ESI-MS, indicating that the molecule has ve degrees of unsatura- (dC 155.2), and their chemical shifts were quite closed to those of
tion. The IR spectrum of 2 revealed the presence of hydroxyl group C-2 and C-9a in salfredin B11.8 In view of the asymmetry in 3 again,
3866 L. Sun et al. / Bioorg. Med. Chem. Lett. 25 (2015) 38643866

Table 2 3. Cheng, L.; Xia, T.-S.; Wang, Y.-F.; Zhou, W.; Liang, X.-Q.; Xue, J.-Q.; Shi, L.; Wang,
Cytotoxity data IC50 (lM) of compounds 14 against four human tumor cells 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.
Compound Cell lines (IC50 lM) 5. Yusu, 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,
Bel7402 HepG2 HCT-8 A549
3101.
1 >40 17.79 0.31 >40 >40 6. Banerjee, S.; Azmi, A. S.; Padhye, S.; Singh, M. W.; Baruah, J. B.; Philip, P. A.;
2 7.69 0.22 15.83 0.54 11.39 0.25 20.06 0.31 Sarkar, F. H.; Mohammad, R. M. Pharm. Res. 2010, 27, 1146.
3 >40 17.32 0.27 >40 36.31 0.10 7. State Administration of Traditional Chinese Medicine Chinese Medicinal
4 >40 36.95 0.42 24.130.16 >40 Herbs In ; Shanghai Science and Technology Publishing House: Shanghai,
5-Fluorouracil 10.07 0.08 16.42 0.19 6.30 0.32 14.15 0.23 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.
it was inferred that an oxygen bridge should be formed between
2012, 66, 645.
C-8a and C-2. On the base of further analyses of its DEPT, 1H1H 11. Nguyen, D. H.; Lee, J.-E.; Kim, E.-K. Korean J. Chem. Eng. 2011, 1070, 28.
COSY, HSQC, HMBC and NOESY spectra, compound 3 was eluci- 12. Nguyen, D. M.; Nguyen, D. H.; Lyun, H.-L.; Lee, H.-B.; Shin, J.-H.; Kim, E.-K. J.
dated to be 7-(hydroxymethyl)-2,2-dimethyl-2H-chromen-5-ol 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.
(Fig. 1) and named as Nigephenol C. Although it was previously 14. Tian, Z.; Liu, Y.-M.; Chen, S.-B.; Yang, J. S.; Xiao, P. G.; Wang, L.; Wu, E. Molecules
reported in two organic synthetic studies,24,25 this is the rst report 2006, 11, 693.
of its occurrence in nature. 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.
The known compound salfredin B11 (4) was conrmed by com- 17. Feng, Y.; Liu, Y.-M.; Liu, Q. H.; Lei, Y.-J. Heterocycles 2012, 85, 3015.
paring its spectroscopic data with the corresponding literature 18. Liu, Y.-M.; Sun, L.; Liu, Q.-H.; Lu, S.-R.; Chen, B.-Q. Biochem. Syst. Ecol. 2013, 49,
values.8,26 43.
19. Liu, Y.-M.; Jiang, Y.-H.; Liu, Q.-H.; Chen, B.-Q. Phytochem. Lett. 2013, 6, 556.
Compounds 14 were evaluated for cytotoxic activities against 20. The oil-free seeds (18 kg) of N. glandulifera were extracted three times with
four human tumor cell lines Bel7402 (liver carcinoma), HepG2 95% EtOH for 2 h under reux and then extracted three times with 50% EtOH
(liver carcinoma), HCT-8 (colon adenocarcinoma) and A549 (non- for 2 h under reux. After combination and removal of the solvent in vacuo, the
EtOH extract was then suspended in distilled water and partitioned
small cell lung carcinoma) by using MTT assay.27 5-Fluorouracil
successively with petroleum ether, EtOAc and n-BuOH. The n-BuOH fraction
was used as a positive control, for compounds 14 and 5-uo- (375 g) was chromatographed over silica gel and eluted with CHCl3MeOH
rouracil could be all classied into simple 1,3-benzenediol deriva- gradient solvent (20:11:1). Combination of similar fractions on the basis of
TLC analysis afforded 13 fractions. Fraction F2 (85.5 g) was subjected to silica
tives. As can be seen in the Table 2, compounds 14 showed more
gel column chromatography and eluted with CHCl3acetone (20:15:1) to
selective activities against HepG2 cells, and the activities of 13 yield six subfractions AF. Subfraction E was isolated by column
stronger than 4 were observed as well. It seemed that the hydrox- chromatography on silica-gel (CHCl3acetone, 5:1) and by MPLC on
ymethyl moiety might be responsible for the increased cytotoxic- 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
ity. Also showed is it in the Table 2 that compound 3 exhibited (CHCl3acetone, 10:1) to give compound 2 (10.0 mg). Subfraction C was
weak cytotoxicity to A549 cells. However, some chromene com- isolated by column chromatography on silica-gel (CHCl3acetone, 15:1) and on
pounds synthesized from 3 had been found to have higher cyto- Sephadex LH-20 (70% MeOH in H2O) to yield compound 3 (6.4 mg). Subfraction
B was isolated by column chromatography on silica-gel (CHCl3acetone, 20:1)
toxic potency in three tumor cell lines A549, HCT29 and to afford compound 4 (7.9 mg).
L1210.24,25 At last, among the tested compounds, compound 2 21. Compound 1: White solid; UV (MeOH) kmax (log e) 238 (4.00), 281 (3.56) nm; IR
showed signicantly better inhibitory effects against four human (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)
tumor cell lines with IC50 values similar to those of 5-uorouracil. and 13C NMR (100 MHz, CD3OD) spectral data see Table 1.
The fact that only compound 2 has the prenyl group encouraged us 22. Compound 2: White solid; UV (MeOH) kmax (log e) 239 (4.20), 280 (3.81) nm; IR
to suggest that the prenyl group plays a positive role in the medi- (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
ating the cytotoxicity against four human tumor cell lines. Quite a
NMR (400 MHz, CD3OD+CDCl3) and 13C NMR (100 MHz, CD3OD+CDCl3)
number of ortho-prenylated phenols have been reported to possess spectral data see Table 1.
anti-tumor activity in recent studies.2831 Owing to its low molec- 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
ular weight and its privileged structure, compound 2 could be a
(positive mode) m/z 207.1010 [M+H]+ (calcd 207.1021 for C12H15O3); 1H
promising candidate for future development of anti-tumor agents. NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectral data see
Table 1.
Acknowledgments 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.;
This work was supported by the National Natural Science Pratsinis, H. Bioorg. Med. Chem. Lett. 2002, 12, 1443.
Foundation of Peoples Republic of China, under Grant no. 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
81073153. cultured at 37 C, with 5% CO2 in incubator for 24 h. Each cancer cell line was
exposed to the test compound at ve 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
Supplementary data
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
Supplementary data associated with this article can be found, in dissolve the crystals, then the optical density values were read on the
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.07. microplate reader (BioTek Epoch) at 544 nm. All tests and analyses were
carried out in triplicate. DMSO and 5-uorouracil were applied as the blank
055. 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.;
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