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Design, synthesis and biological evaluation of matrine derivatives as potential anticancer agents
Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of matrine derivatives as potential anticancer agents Zheng Li a,d, Mengyang Luo a,d, Bin Cai c, Lichuan Wu b, Mengtian Huang a, Haroon-Ur-Rashid a, Jun Jiang a,⇑, Lisheng Wang b,⇑ a b c
School of Chemistry and Chemical Engineering, Guangxi University, Nanning, Guangxi 530004, PR China Medical College of Guangxi University, Guangxi 530004, PR China Suzhou Galaxy biopharma, CO., LTD., Suzhou, Jiangsu 215000, PR China
a r t i c l e
i n f o
Article history: Received 15 November 2017 Revised 7 January 2018 Accepted 12 January 2018 Available online xxxx Keywords: Matrine Derivatives Pyrrole Indole Anticancer Structure-activity relationship Apoptosis Cell cycle arrest
a b s t r a c t Using matrine (1) as the lead compound, a series of new 14-(N-substituted-2-pyrrolemethylene) matrine and 14-(N-substituted-indolemethylene) matrine derivatives was designed and synthesized for their potential application as anticancer agents. The structure of these compounds was characterized by 1H NMR, 13C NMR and ESI-MS spectral analyses. The target compounds were evaluated for their in vitro cytotoxicity against three human cancer cell lines (SMMC-7721, A549 and CNE2). The results revealed that compound A6 and B21 displayed the most significant anticancer activity against three cancer cell lines with IC50 values in range of 3.42–8.05 lM, which showed better activity than the parent compound (Matrine) and positive control Cisplatin. Furthermore, the Annexin V-FITC/PI dual staining assay revealed that compound A6 and B21 could significantly induce the apoptosis of SMMC-7721 and CNE2 cells in a dose-dependent manner. The cell cycle analysis also revealed that compound A6 could cause cell cycle arrest of SMMC-7721 and CNE2 cells at G2/M phase. Ó 2018 Elsevier Ltd. All rights reserved.
Matrine (Fig. 1) is one of the main active ingredients in FufangKushen injection. In combination with other anticancer agents such as vinorelbine, taxol and cisplatin, matrine was also approved by CFDA in 1995 as an adjuvant to treat non-small cell lung cancer (NSCLC), liver cancer and gastric cancer.1–8 It has been found that matrine displayed potent anticancer activity and induced apoptosis in cancer cells via downregulation of Bcl-2, upregulation of Bax and suppression of b-catenin/survivin pathway.9–14 Owing to its multiple drug like properties, such as special scaffold, simple structure and high solubility, matrine has been considered as an ideal lead compound for further structural modifications and optimizations.15–17 In our previous study, we selected matrine as a lead compound and successfully identified that introduction of double bond and aromatic ring at 14 site of matrine skeleton could largely increase its anticancer activity.18–20 The representative compound YF18 (Fig. 1), 14-napthylmethylene matrine, exhibited a significantly enhanced anticancer activity in three human lung cancer cell lines i.e. A549, H1975 and 95D, with IC50 ranging from 10 to 15 lM. ⇑ Corresponding authors. d
E-mail addresses: [email protected] (J. Jiang), [email protected] (L. Wang). Contributed equally.
Thus, compound YF18 showed much better anticancer activity than matrine.20 Moreover, the introduction of nitrogen-containing heterocycle is a ubiquitous strategy towards the structure modification of natural products as nitrogen atom can influence the interaction between the molecule and its target.21 It is also reported that an N-substituted pyrrole scaffold (Fig. 1) is essential to enhance anticancer activity of several natural products, such as lukianol A and lamellarin O.22 And it is well-known that a N-benzyl indole scaffolds possess various biological activities and continues to be a pervasive structural feature of many pharmacologically active compounds, such as a newly discovered anticancer agent Indibulin.23 Based on this idea, it provoked our strong interest to introduce N-substituted pyrrole and indole group into 14 site of matrine in order to discover a novel class of anticancer candidates (Fig. 1). Therefore, a variety of N-substituted pyrroles and indole were introduced on the 14 site of matrine skeleton which resulted in designing two series of new matrine derivatives. These derivatives were synthesized, and biologically evaluated for their anticancer activities. In this study, structure–activity relationship (SAR) was first conducted to focus on the variations of the N-substitution of pyrrole, while keeping the 14-(2-pyrrolemethylene) matrine
https://doi.org/10.1016/j.bmcl.2018.01.017 0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Chemical structure of matrine, 14-(1-naphthalenylmethylene)matrine (YF18), lukianol A, lamellarin O, indidulin and design strategy for matrine derivatives.
scaffold intact. Similarly, N-benzyl indole group (Fig. 1), as a functional group for antitumor activity, was also added on the 14 site of matrine and then another series of 35 new 14-(N-substituted-indolemethylene) matrine derivatives were designed, synthesized, and examined for their anticancer activities. Moreover, cell death and cell cycle of the representative compounds were explored. As described in Scheme 1, twenty-three target compounds were prepared using commercially available 1 as the original material. Compounds of N-substituted pyrrole-2-carboxyaldehyde a1–a23 and N-substituted indole-carboxyaldehyde b1–b35 were obtained by the reaction of pyrrole-2-carboxyaldehyde using alkyl halides in the presence of NaH through a simple after-treatment and were then directly applied in the next step without further purification. The 14-pyrrolemethylene matrine A1–A23 (Scheme 1) and 14indolemethylene matrine B1–B35 (Scheme 2) were obtained through further aldol reaction of various aromatic aldehyde with 1 in 19%–54% yields. All the synthesized compounds were purified by silica gel column chromatography using ethyl acetate and petroleum ether as gradient eluents and their structures were characterized by 1H NMR, 13C NMR and ESI-MS. As illustrated in Table 1, all the desired compounds were evaluated for their cytotoxic activities in three human cancer cell lines utilizing MTT assay with matrine being tested as a comparative compound and Cisplatin as the positive control.24 Replacing 14-H of 1 with a 1-methyl-2-pyrrolemethylene group offered compound A1, which showed a poor activity (IC50 > 100 lM), while compound A2 possessing a 1-benzyl-2-pyrrolemethylene group at the same position exhibited a greatly improved anticancer activity with an IC50 of 38.42 ± 3.05, 42.88 ± 5.08, 45.67 ± 4.35 lM against SMMC-7721, A549 and CNE2 cells respectively, and was about 115–172 times lower than that of matrine. It could be inferred that the introduction of a benzyl moiety on the nitrogen atom of pyrrole could significantly improve the anticancer activity indicating that benzyl might interact with a large pocket within the binding site.
Scheme 1. Synthetic route for matrine derivatives. Reaction conditions: (a) NaH, DMF, alkyl halide, 0 °C ? RT, 30 min; (b) NaH, dry THF, reflux, 24 h.
Encouraged by these results, we further introduced a variety of benzylic substituents upon 1-nitrogen atom, resulting in 21 new 14-(N-substituted-2-pyrrolemethylene)matrine (A3–A23) derivatives. They were tested to explore the structure–activity relationship (SAR) of the substituent group variation. Among the benzyl-substituted derivatives, it was found that compounds
Please cite this article in press as: Li Z., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.017
Z. Li et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
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Scheme 2. Synthetic route for matrine derivatives. Reaction conditions: (i) NaH, DMF, alkyl halide, 0 °C ? RT, 30 min; (ii) NaH, dry THF, reflux, 36 h.
A3, A6 and A7 showed prominent cytotoxic activities against at least one cancer cell line (IC50 < 10 lM), suggesting that methoxybenzyl at N-position is benefit to anticancer activity improvement. A5, A12, A17, A18 and A22 also showed moderate anticancer activity against the three cancer cells with IC50 < 20 lM. Bearing methoxy group at m-position of phenyl ring (compounds A3, A5, A6, A7) has increased the cytotoxicity against all the three cell lines. Among them, compound A6 decorated with 3,5-dimethoxy units on phenyl ring exhibited the most potent cytotoxic activity against SMMC-7721, A549 and CNE2 cells with IC50 values of 4.65 ± 0.23, 8.05 ± 0.56 and 3.55 ± 0.18 lM, respectively, stronger than those of positive control. The compounds A2, A4, A8, A10, A14, A15, A16, A19, A20, A21 and A23 have shown cytotoxicity in the range of 20–40 mM against all the three cell lines. Then, SAR analysis was focused on the substituents at the 1nitrogen atom of the 14-[(indol-3-yl)methylene]-matrine moiety. In this part, a variety of benzyl were introduced into the 1-nitrogen atom, by which 16 new 14-(N-substituted-3-indolemethylene) matrine (compounds B1–B16) were generated and tested. All the
substituents were globally well tolerated and the compound B7, B8, B9, B11, B14 and B16 is active against all the cancer cell lines with IC50 < 10 lM, suggesting that fluorobenzyl, methylbenzyl, tert-butylbenzyl, chlorobenzyl, trifluoromethoxybenzyl and 2naphthalenylmethyl at N position is beneficial for the activity. Next, we retained the fluorobenzyl, methylbenzyl, tert-butylbenzyl, chlorobenzyl, trifluoromethoxybenzyl and 2-Naphthalenylmethyl at N-position of indolemethylene as a pharmacophore for activity, and changed the substituents of indole or the attachment position to the matrine core to explore the SAR. Therefore, 19 new 14-(N-substituted-3-indolemethylene) matrine were made and tested. We first evaluated the effects of substitutents at the positions of the indole ring. Replacing the halogen atom of compound B17 and B18 with a lipophilic and electron-releasing methoxyl in situ afforded derivative B21 and B23, resulting in an dramatically increased cytotoxicity. Changing the position of the methoxyl group of compound B22–B23 from C-5 to C-6 of the indole ring conferred decreased cytotoxicity on isomer B28–29.
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Table 1 The anti-proliferative activities of matrine derivatives A1-A23 and B1-B35. Compd.
IC50 (lM) ± SDa SMMC-7721
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 B29 B30 B31 B32 B33 B34 B35 Matrine Cisplatin
>100 38.42 ± 3.05 13.41 ± 1.42 41.61 ± 2.69 12.25 ± 1.05 4.65 ± 0.23 13.26 ± 1.51 22.42 ± 2.03 30.68 ± 1.23 27.91 ± 2.67 49.36 ± 5.59 16.00 ± 1.15 >40 25.97 ± 2.32 20.93 ± 1.56 21.56 ± 2.41 15.12 ± 1.26 16.21 ± 1.23 17.58 ± 3.08 34.03 ± 4.25 36.57 ± 2.68 12.93 ± 1.20 20.30 ± 2.12 11.94 ± 1.23 13.68 ± 1.11 8.009 ± 0.67 11.81 ± 1.09 15.45 ± 1.25 11.95 ± 0.84 8.52 ± 0.54 9.12 ± 0.86 9.55 ± 0.45 17.56 ± 1.76 7.58 ± 0.63 11.21 ± 1.05 16.81 ± 2.01 6.82 ± 0.38 9.76 ± 0.81 5.65 ± 0.32 9.81 ± 0.67 8.65 ± 1.02 11.45 ± 0.65 6.21 ± 0.43 3.95 ± 0.34 5.65 ± 0.64 6.89 ± 0.81 8.35 ± 0.56 3.26 ± 0.21 23.68 ± 2.65 9.73 ± 0.78 8.73 ± 0.76 9.06 ± 0.59 6.83 ± 0.73 28.21 ± 3.01 13.78 ± 1.23 25.78 ± 3.11 10.74 ± 1.23 27.03 ± 3.21 6591 ± 521 6.08 ± 0.43
b
Clog Pc b
b
A549
CNE2
>100 42.88 ± 5.08 29.83 ± 0.95 39.11 ± 2.11 14.52 ± 2.12 8.05 ± 0.56 18.93 ± 1.64 29.32 ± 1.92 44.33 ± 3.99 24.39 ± 3.03 >50 15.83 ± 0.88 >40 26.22 ± 2.84 22.33 ± 2.41 21.55 ± 2.06 12.01 ± 1.05 13.8 ± 2.01 22.95 ± 1.58 31.57 ± 3.95 47.06 ± 5.21 13.09 ± 1.51 23.91 ± 1.96 13.89 ± 1.31 15.32 ± 1.43 11.20 ± 0.98 12.51 ± 1.54 >50 12.06 ± 1.16 9.24 ± 0.85 9.84 ± 0.94 9.68 ± 0.67 18.34 ± 1.54 7.53 ± 0.47 12.13 ± 1.54 18.08 ± 1.45 7.03 ± 0.65 12.56 ± 1.43 8.53 ± 0.19 16.84 ± 1.54 9.74 ± 1.21 9.04 ± 0.89 6.33 ± 0.63 4.96 ± 0.54 7.12 ± 0.56 7.45 ± 0.68 9.13 ± 0.78 5.04 ± 0.43 26.32 ± 2.76 5.24 ± 0.89 10.06 ± 1.11 13.21 ± 1.23 7.07 ± 0.32 28.45 ± 2.43 15.45 ± 1.12 29.87 ± 2.71 10.89 ± 1.31 30.22 ± 2.61 5725 ± 602 8.56 ± 0.58
>100 45.67 ± 4.35 7.05 ± 5.21 28.81 ± 2.23 11.59 ± 0.71 3.55 ± 0.18 7.83 ± 0.93 28.42 ± 1.58 24.46 ± 1.91 21.94 ± 1.08 32.11 ± 4.21 13.85 ± 2.01 >40 26.32 ± 3.26 16.33 ± 1.84 15.17 ± 1.84 17.34 ± 1.62 14.97 ± 0.98 17.84 ± 1.42 26.43 ± 2.15 23.91 ± 2.64 10.84 ± 0.65 14.71 ± 1.68 10.83 ± 1.02 9.31 ± 0.87 8.43 ± 0.75 11.59 ± 0.97 19.98 ± 2.03 10.83 ± 0.79 7.91 ± 0.47 8.91 ± 0.67 7.73 ± 0.68 17.43 ± 0.98 6.36 ± 0.56 10.98 ± 1.23 14.74 ± 1.23 6.64 ± 0.54 11.72 ± 1.53 4.56 ± 0.28 8.50 ± 0.97 12.5 ± 1.01 6.04 ± 0.54 6.06 ± 0.45 3.42 ± 0.23 4.65 ± 0.39 6.72 ± 0.48 11.8 ± 1.02 3.53 ± 0.21 23.74 ± 1.96 3.52 ± 0.41 6.84 ± 0.71 5.42 ± 0.34 6.64 ± 0.48 15.53 ± 1.65 9.75 ± 1.04 14.56 ± 1.21 8.99 ± 1.02 14.32 ± 1.31 5278 ± 498 3.89 ± 0.23
2.802 4.320 4.239 4.239 5.242 4.328 3.620 4.769 4.819 4.819 5.318 6.146 7.972 5.033 5.033 5.033 5.183 4.178 5.183 4.463 5.348 6.007 5.494 5.704 5.623 5.623 5.712 5.004 6.153 6.203 6.203 7.530 6.417 6.418 6.417 6.567 6.732 7.391 6.878 7.322 7.783 7.637 6.474 6.474 6.474 6.935 6.916 7.378 7.172 7.487 6.474 6.935 6.417 5.623 7.530 6.417 7.530 6.878 1.361
The bold values represent the IC50 values of the representative compounds which were further explored for their cell death and cell cycle. a Cytotoxicity (as IC50 for each cell line) is the concentration of compound which reduced by 50% the optical density of treated cells with respect to untreated cells using the MTT assay. IC50 values are taken as a mean from three experiments. Mean ± SD. b Cell lines include human hepato carcinoma (SMMC-7721), lung carcinoma (A549) and nasopharyngeal carcinoma (CNE2). c Clog P values produced by Chemdraw software.
Moreover, after changing the linking position of matrine attached to the indole from 3-position to 4-position or 5-position, respectively, compounds B30–B35 showed decreased activities compared with their analogues respectively.
It was further revealed that compound 1 and A1 with low Clog P values of 1.361, 2.802, respectively, exhibited weaker antiproliferative potency. However, compounds having slightly high Clog P values showed potent antiproliferative effect. These results suggested
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that the Clog P values of all molecules seemed to be a very important determinant factor of the antiproliferative properties. Interestingly, most derivatives possessed the strongest cytotoxicity against CNE2 cells, followed by SMMC-7721 cells, and were least cytotoxic to A549 cells. Thus, we subsequently selected A6
5
and B21 for further investigation to explore its anti-cancer mechanisms on CNE2 and SMMC-7721 cancer cells. In order to investigate whether compound A6 and B21 could induce cell apoptosis, CNE2 and SMMC-7721 cells treated with 0, 5, 10, 30 lM of A6 and 0, 10, 20, 50 lM of B21, were subjected
Fig. 2. Compound A6 could induce apoptosis in CNE2 and SMMC-7721 cells in a dose-dependent manner.
Fig. 3. Compound B21 could induce apoptosis in CNE2 and SMMC-7721 cells in a dose-dependent manner.
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to AnnexinV-FITC/propidium iodide (PI) dual staining and quantified by flow cytometry.25 As shown in Fig. 2, the percentage of apoptotic cells (Lower right quadrant, AV+/PI and Upper right quadrant, AV+/PI+) in CNE2 significantly increased from 4.93% (control) to 11.96% (5 lM), 34.04% (10 lM) and 50.50% (30 lM) respectively after treatment with different concentrations of compound A6 for 24 h. And the percentage of apoptotic cells in SMMC7721 increased from 2.60% (control) to 19.93% (5 lM), 53.58% (10 lM) and 61.50% (30 lM), respectively. Consistently, as shown in Fig. 3, the percentage of apoptotic cells of in CNE2 significantly increased from 5.29% (control) to 17.52% (10 lM), 38.60% (20 lM) and 74.20% (50 lM) respectively after treatment with different concentrations of compound B21 for 24 h. And the percentage of apoptotic cells in SMMC-7721 increased from 2.60% (control) to 39.00% (10 lM), 58.80% (20 lM) and 95.00% (50 lM), respectively. The results revealed that compound A6 and B21 could induce the apoptosis of CNE2 and SMMC-7721 cells in a dose-dependent manner. To reveal whether the inhibition of cancer cells growth by compound A6 and B21 was in associate with cell cycle arrest, CNE2 and SMMC-7721 cells were treated with different concentrations of compound A6 (0, 5, 10 and 30 lM) and B21 (0, 10, 20 and 50
lM) for 24 h. Cell cycle distribution was investigated by flow cytometric analysis using propidium iodide staining method.26 Compound B21 did not show significant cell cycle arrest (data not shown), while compound A6 significantly cell cycle arrest as shown in Fig. 4. The percentage of G2/M phase in CNE2 gradually increased from 19.8% (control) to 29.5% (5 lM), 89.4% (10 lM) and 81.1% (30 lM). Consistently, the percentage of G2/M phase in SMMC-7721 cells increased from 18.4% (control) to 24.1% (5 lM), 78.9% (10 lM) to 79.1% (30 lM). These results confirmed that compound A6 could arrest the cell cycle of CNE2 and SMMC-7721 cells at G2/M phase. In summary, 58 new matrine derivatives A1–A23 and B1–B35 were synthesized, and their anti-proliferative activities against human cancer cell lines such as SMMC-7721, A549 and CNE2 were investigated. SAR analysis revealed that suitable nitrogen-containing heterocycle groups on 14 site of matrine were benefit to anticancer activity improvement. In MTT assay, compound A6 and B21 exhibited the most significant growth inhibition of cancer cells. Further studies indicated that A6 exerted its anti-cancer activity by inducing the apoptosis of CNE2 and SMMC-7721 cells and G2/M cell cycle arrest in a dose dependent manner. B21 exerted its anti-cancer activity by inducing the apoptosis of CNE2
Fig. 4. Compound A6 could induce G2/M cell cycle arrest in CNE2 and SMMC-7721 cancer cells. (A) A6 induces G2/M cell cycle arrest in CNE2 and SMMC-7721 cells in a dose-dependent manner. (B) Distributions of G1, S and G2/M in CNE2 and SMMC-7721 cells under A6 treatment.
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and SMMC-7721 cells. More studies are needed to explore the precise antitumor mechanism. Acknowledgments This work was supported by the National Natural Science Foundation of China (21402032, 21262005), the high level innovation team and outstanding scholar project of Guangxi institutions of higher education (guijiaoren [2014] 49 hao), Guangxi Natural Science Foundation (2014GXNSFBA118031, 2017GXNSFBA198240) and the State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University (CMEMR2017-B14). We also thank Professor Cai Bin of Suzhou Galaxy biopharma, CO., LTD. for the experiment support. A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bmcl.2018.01.017. References 1. Zhong LY, Wu H. Zhongguo Zhong Yao Za Zhi. 2006;31:1561. 2. Sun M, Cao H, Sun L, et al. Evid Based Complement Alternat Med. 2012;2012:373219.
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3. Sun Q, Ma W, Gao Y, Zheng W, Zhang B, Peng Y. Afr J Tradit Complement Altern Med Ajtcam. 2011;9:178. 4. Wang C-Y, Bai X-Y, Wang C-H. Am J Chin Med. 2014;42:543. 5. Wei R, Yang DY, Jiang WZ, Dai YY, Wan LY, Yang Z. Zhonghua Zhong Liu Za Zhi. 2011;33:391. 6. Liu J, Xue M, Huang X, Wang S, Jiang Z, Zhang L. Zhongguo Zhong yao za zhi. 2012;37:1845. 7. Qi L, Zhang J, Zhang Z. Chin J Chromatogr. 2013;31:249. 8. Tian J, Wang WH, Gao HM, Wang ZM. Zhongguo Zhong Yao Za Zhi. 2007;32:222. 9. Yong J, Wu X, Lu C. Curr Pharm Des. 2015;21:3673. 10. Liu TY, Yan S, Hua C, Pan SH, Sun XY. Biol Pharm Bull. 2010;33:1740. 11. Li H, Li X, Bai M, Suo Y, Zhang G, Cao X. Int J Clin Exp Path. 2015;8:14793. 12. Li H, Tan G, Jiang X, et al. Am J Chin Med. 2010;38:1115. 13. Zhou W, Xu X, Gao J, et al. Int J Clin Exp Med. 2015;8:9004. 14. Wang HQ, Jin JJ, Wang J. Oncol Rep. 2015;33:2561. 15. Peng-Yi HU, Qin Z, Hui C, Zhen-Feng WU, Yue PF, Ming Y. Chin J New Drugs. 2012;21:2662. 16. Li Y, Min G, Xue Q, Chen L, Liu W, Chen H. Biomed Chromatogr BMC. 2004;18:619. 17. Ye G, Zhu HY, Li ZX, et al. Biomed Chromatogr. 2007;21:655. 18. Wang L, You Y, Wang S, et al. Bioorg Med Chem Lett. 2012;22:4100. 19. Xie M, Yi X, Wang R, et al. Cell Physiol Biochem. 2014;33:1475. 20. Wu L, Wang G, Wei J, et al. Oncotarget. 2016;8. 21. Xu H, Tang H, Feng H, Li Y. Eur J Med Chem. 2014;73:46. 22. Boger DL, Boyce CW, Labroli MA, Jin CASQ. J Am Chem Soc. 1999;121. 23. Yu PF, Chen H, Wang J, et al. Cheminform. 2008;39:831. 24. Wu LC, Wen ZS, Qiu YT, et al. ACS Med Chem Lett. 2013;4:921. 25. Browne LJ, Gude C, Rodriguez H, Steele RE, Bhatnager A. J Med Chem. 1991;34:725. 26. Rajan S, Indu S, Manish Kumar J, Naidu VGM, Wahid K, Ramakrishna S. Int J Pharm. 2015;486:287.
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Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of matrine derivatives as potential anticancer agents Zheng Li a,d, Mengyang Luo a,d, Bin Cai c, Lichuan Wu b, Mengtian Huang a, Haroon-Ur-Rashid a, Jun Jiang a,⇑, Lisheng Wang b,⇑ a b c
School of Chemistry and Chemical Engineering, Guangxi University, Nanning, Guangxi 530004, PR China Medical College of Guangxi University, Guangxi 530004, PR China Suzhou Galaxy biopharma, CO., LTD., Suzhou, Jiangsu 215000, PR China
a r t i c l e
i n f o
Article history: Received 15 November 2017 Revised 7 January 2018 Accepted 12 January 2018 Available online xxxx Keywords: Matrine Derivatives Pyrrole Indole Anticancer Structure-activity relationship Apoptosis Cell cycle arrest
a b s t r a c t Using matrine (1) as the lead compound, a series of new 14-(N-substituted-2-pyrrolemethylene) matrine and 14-(N-substituted-indolemethylene) matrine derivatives was designed and synthesized for their potential application as anticancer agents. The structure of these compounds was characterized by 1H NMR, 13C NMR and ESI-MS spectral analyses. The target compounds were evaluated for their in vitro cytotoxicity against three human cancer cell lines (SMMC-7721, A549 and CNE2). The results revealed that compound A6 and B21 displayed the most significant anticancer activity against three cancer cell lines with IC50 values in range of 3.42–8.05 lM, which showed better activity than the parent compound (Matrine) and positive control Cisplatin. Furthermore, the Annexin V-FITC/PI dual staining assay revealed that compound A6 and B21 could significantly induce the apoptosis of SMMC-7721 and CNE2 cells in a dose-dependent manner. The cell cycle analysis also revealed that compound A6 could cause cell cycle arrest of SMMC-7721 and CNE2 cells at G2/M phase. Ó 2018 Elsevier Ltd. All rights reserved.
Matrine (Fig. 1) is one of the main active ingredients in FufangKushen injection. In combination with other anticancer agents such as vinorelbine, taxol and cisplatin, matrine was also approved by CFDA in 1995 as an adjuvant to treat non-small cell lung cancer (NSCLC), liver cancer and gastric cancer.1–8 It has been found that matrine displayed potent anticancer activity and induced apoptosis in cancer cells via downregulation of Bcl-2, upregulation of Bax and suppression of b-catenin/survivin pathway.9–14 Owing to its multiple drug like properties, such as special scaffold, simple structure and high solubility, matrine has been considered as an ideal lead compound for further structural modifications and optimizations.15–17 In our previous study, we selected matrine as a lead compound and successfully identified that introduction of double bond and aromatic ring at 14 site of matrine skeleton could largely increase its anticancer activity.18–20 The representative compound YF18 (Fig. 1), 14-napthylmethylene matrine, exhibited a significantly enhanced anticancer activity in three human lung cancer cell lines i.e. A549, H1975 and 95D, with IC50 ranging from 10 to 15 lM. ⇑ Corresponding authors. d
E-mail addresses: [email protected] (J. Jiang), [email protected] (L. Wang). Contributed equally.
Thus, compound YF18 showed much better anticancer activity than matrine.20 Moreover, the introduction of nitrogen-containing heterocycle is a ubiquitous strategy towards the structure modification of natural products as nitrogen atom can influence the interaction between the molecule and its target.21 It is also reported that an N-substituted pyrrole scaffold (Fig. 1) is essential to enhance anticancer activity of several natural products, such as lukianol A and lamellarin O.22 And it is well-known that a N-benzyl indole scaffolds possess various biological activities and continues to be a pervasive structural feature of many pharmacologically active compounds, such as a newly discovered anticancer agent Indibulin.23 Based on this idea, it provoked our strong interest to introduce N-substituted pyrrole and indole group into 14 site of matrine in order to discover a novel class of anticancer candidates (Fig. 1). Therefore, a variety of N-substituted pyrroles and indole were introduced on the 14 site of matrine skeleton which resulted in designing two series of new matrine derivatives. These derivatives were synthesized, and biologically evaluated for their anticancer activities. In this study, structure–activity relationship (SAR) was first conducted to focus on the variations of the N-substitution of pyrrole, while keeping the 14-(2-pyrrolemethylene) matrine
https://doi.org/10.1016/j.bmcl.2018.01.017 0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Chemical structure of matrine, 14-(1-naphthalenylmethylene)matrine (YF18), lukianol A, lamellarin O, indidulin and design strategy for matrine derivatives.
scaffold intact. Similarly, N-benzyl indole group (Fig. 1), as a functional group for antitumor activity, was also added on the 14 site of matrine and then another series of 35 new 14-(N-substituted-indolemethylene) matrine derivatives were designed, synthesized, and examined for their anticancer activities. Moreover, cell death and cell cycle of the representative compounds were explored. As described in Scheme 1, twenty-three target compounds were prepared using commercially available 1 as the original material. Compounds of N-substituted pyrrole-2-carboxyaldehyde a1–a23 and N-substituted indole-carboxyaldehyde b1–b35 were obtained by the reaction of pyrrole-2-carboxyaldehyde using alkyl halides in the presence of NaH through a simple after-treatment and were then directly applied in the next step without further purification. The 14-pyrrolemethylene matrine A1–A23 (Scheme 1) and 14indolemethylene matrine B1–B35 (Scheme 2) were obtained through further aldol reaction of various aromatic aldehyde with 1 in 19%–54% yields. All the synthesized compounds were purified by silica gel column chromatography using ethyl acetate and petroleum ether as gradient eluents and their structures were characterized by 1H NMR, 13C NMR and ESI-MS. As illustrated in Table 1, all the desired compounds were evaluated for their cytotoxic activities in three human cancer cell lines utilizing MTT assay with matrine being tested as a comparative compound and Cisplatin as the positive control.24 Replacing 14-H of 1 with a 1-methyl-2-pyrrolemethylene group offered compound A1, which showed a poor activity (IC50 > 100 lM), while compound A2 possessing a 1-benzyl-2-pyrrolemethylene group at the same position exhibited a greatly improved anticancer activity with an IC50 of 38.42 ± 3.05, 42.88 ± 5.08, 45.67 ± 4.35 lM against SMMC-7721, A549 and CNE2 cells respectively, and was about 115–172 times lower than that of matrine. It could be inferred that the introduction of a benzyl moiety on the nitrogen atom of pyrrole could significantly improve the anticancer activity indicating that benzyl might interact with a large pocket within the binding site.
Scheme 1. Synthetic route for matrine derivatives. Reaction conditions: (a) NaH, DMF, alkyl halide, 0 °C ? RT, 30 min; (b) NaH, dry THF, reflux, 24 h.
Encouraged by these results, we further introduced a variety of benzylic substituents upon 1-nitrogen atom, resulting in 21 new 14-(N-substituted-2-pyrrolemethylene)matrine (A3–A23) derivatives. They were tested to explore the structure–activity relationship (SAR) of the substituent group variation. Among the benzyl-substituted derivatives, it was found that compounds
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Scheme 2. Synthetic route for matrine derivatives. Reaction conditions: (i) NaH, DMF, alkyl halide, 0 °C ? RT, 30 min; (ii) NaH, dry THF, reflux, 36 h.
A3, A6 and A7 showed prominent cytotoxic activities against at least one cancer cell line (IC50 < 10 lM), suggesting that methoxybenzyl at N-position is benefit to anticancer activity improvement. A5, A12, A17, A18 and A22 also showed moderate anticancer activity against the three cancer cells with IC50 < 20 lM. Bearing methoxy group at m-position of phenyl ring (compounds A3, A5, A6, A7) has increased the cytotoxicity against all the three cell lines. Among them, compound A6 decorated with 3,5-dimethoxy units on phenyl ring exhibited the most potent cytotoxic activity against SMMC-7721, A549 and CNE2 cells with IC50 values of 4.65 ± 0.23, 8.05 ± 0.56 and 3.55 ± 0.18 lM, respectively, stronger than those of positive control. The compounds A2, A4, A8, A10, A14, A15, A16, A19, A20, A21 and A23 have shown cytotoxicity in the range of 20–40 mM against all the three cell lines. Then, SAR analysis was focused on the substituents at the 1nitrogen atom of the 14-[(indol-3-yl)methylene]-matrine moiety. In this part, a variety of benzyl were introduced into the 1-nitrogen atom, by which 16 new 14-(N-substituted-3-indolemethylene) matrine (compounds B1–B16) were generated and tested. All the
substituents were globally well tolerated and the compound B7, B8, B9, B11, B14 and B16 is active against all the cancer cell lines with IC50 < 10 lM, suggesting that fluorobenzyl, methylbenzyl, tert-butylbenzyl, chlorobenzyl, trifluoromethoxybenzyl and 2naphthalenylmethyl at N position is beneficial for the activity. Next, we retained the fluorobenzyl, methylbenzyl, tert-butylbenzyl, chlorobenzyl, trifluoromethoxybenzyl and 2-Naphthalenylmethyl at N-position of indolemethylene as a pharmacophore for activity, and changed the substituents of indole or the attachment position to the matrine core to explore the SAR. Therefore, 19 new 14-(N-substituted-3-indolemethylene) matrine were made and tested. We first evaluated the effects of substitutents at the positions of the indole ring. Replacing the halogen atom of compound B17 and B18 with a lipophilic and electron-releasing methoxyl in situ afforded derivative B21 and B23, resulting in an dramatically increased cytotoxicity. Changing the position of the methoxyl group of compound B22–B23 from C-5 to C-6 of the indole ring conferred decreased cytotoxicity on isomer B28–29.
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Table 1 The anti-proliferative activities of matrine derivatives A1-A23 and B1-B35. Compd.
IC50 (lM) ± SDa SMMC-7721
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 B29 B30 B31 B32 B33 B34 B35 Matrine Cisplatin
>100 38.42 ± 3.05 13.41 ± 1.42 41.61 ± 2.69 12.25 ± 1.05 4.65 ± 0.23 13.26 ± 1.51 22.42 ± 2.03 30.68 ± 1.23 27.91 ± 2.67 49.36 ± 5.59 16.00 ± 1.15 >40 25.97 ± 2.32 20.93 ± 1.56 21.56 ± 2.41 15.12 ± 1.26 16.21 ± 1.23 17.58 ± 3.08 34.03 ± 4.25 36.57 ± 2.68 12.93 ± 1.20 20.30 ± 2.12 11.94 ± 1.23 13.68 ± 1.11 8.009 ± 0.67 11.81 ± 1.09 15.45 ± 1.25 11.95 ± 0.84 8.52 ± 0.54 9.12 ± 0.86 9.55 ± 0.45 17.56 ± 1.76 7.58 ± 0.63 11.21 ± 1.05 16.81 ± 2.01 6.82 ± 0.38 9.76 ± 0.81 5.65 ± 0.32 9.81 ± 0.67 8.65 ± 1.02 11.45 ± 0.65 6.21 ± 0.43 3.95 ± 0.34 5.65 ± 0.64 6.89 ± 0.81 8.35 ± 0.56 3.26 ± 0.21 23.68 ± 2.65 9.73 ± 0.78 8.73 ± 0.76 9.06 ± 0.59 6.83 ± 0.73 28.21 ± 3.01 13.78 ± 1.23 25.78 ± 3.11 10.74 ± 1.23 27.03 ± 3.21 6591 ± 521 6.08 ± 0.43
b
Clog Pc b
b
A549
CNE2
>100 42.88 ± 5.08 29.83 ± 0.95 39.11 ± 2.11 14.52 ± 2.12 8.05 ± 0.56 18.93 ± 1.64 29.32 ± 1.92 44.33 ± 3.99 24.39 ± 3.03 >50 15.83 ± 0.88 >40 26.22 ± 2.84 22.33 ± 2.41 21.55 ± 2.06 12.01 ± 1.05 13.8 ± 2.01 22.95 ± 1.58 31.57 ± 3.95 47.06 ± 5.21 13.09 ± 1.51 23.91 ± 1.96 13.89 ± 1.31 15.32 ± 1.43 11.20 ± 0.98 12.51 ± 1.54 >50 12.06 ± 1.16 9.24 ± 0.85 9.84 ± 0.94 9.68 ± 0.67 18.34 ± 1.54 7.53 ± 0.47 12.13 ± 1.54 18.08 ± 1.45 7.03 ± 0.65 12.56 ± 1.43 8.53 ± 0.19 16.84 ± 1.54 9.74 ± 1.21 9.04 ± 0.89 6.33 ± 0.63 4.96 ± 0.54 7.12 ± 0.56 7.45 ± 0.68 9.13 ± 0.78 5.04 ± 0.43 26.32 ± 2.76 5.24 ± 0.89 10.06 ± 1.11 13.21 ± 1.23 7.07 ± 0.32 28.45 ± 2.43 15.45 ± 1.12 29.87 ± 2.71 10.89 ± 1.31 30.22 ± 2.61 5725 ± 602 8.56 ± 0.58
>100 45.67 ± 4.35 7.05 ± 5.21 28.81 ± 2.23 11.59 ± 0.71 3.55 ± 0.18 7.83 ± 0.93 28.42 ± 1.58 24.46 ± 1.91 21.94 ± 1.08 32.11 ± 4.21 13.85 ± 2.01 >40 26.32 ± 3.26 16.33 ± 1.84 15.17 ± 1.84 17.34 ± 1.62 14.97 ± 0.98 17.84 ± 1.42 26.43 ± 2.15 23.91 ± 2.64 10.84 ± 0.65 14.71 ± 1.68 10.83 ± 1.02 9.31 ± 0.87 8.43 ± 0.75 11.59 ± 0.97 19.98 ± 2.03 10.83 ± 0.79 7.91 ± 0.47 8.91 ± 0.67 7.73 ± 0.68 17.43 ± 0.98 6.36 ± 0.56 10.98 ± 1.23 14.74 ± 1.23 6.64 ± 0.54 11.72 ± 1.53 4.56 ± 0.28 8.50 ± 0.97 12.5 ± 1.01 6.04 ± 0.54 6.06 ± 0.45 3.42 ± 0.23 4.65 ± 0.39 6.72 ± 0.48 11.8 ± 1.02 3.53 ± 0.21 23.74 ± 1.96 3.52 ± 0.41 6.84 ± 0.71 5.42 ± 0.34 6.64 ± 0.48 15.53 ± 1.65 9.75 ± 1.04 14.56 ± 1.21 8.99 ± 1.02 14.32 ± 1.31 5278 ± 498 3.89 ± 0.23
2.802 4.320 4.239 4.239 5.242 4.328 3.620 4.769 4.819 4.819 5.318 6.146 7.972 5.033 5.033 5.033 5.183 4.178 5.183 4.463 5.348 6.007 5.494 5.704 5.623 5.623 5.712 5.004 6.153 6.203 6.203 7.530 6.417 6.418 6.417 6.567 6.732 7.391 6.878 7.322 7.783 7.637 6.474 6.474 6.474 6.935 6.916 7.378 7.172 7.487 6.474 6.935 6.417 5.623 7.530 6.417 7.530 6.878 1.361
The bold values represent the IC50 values of the representative compounds which were further explored for their cell death and cell cycle. a Cytotoxicity (as IC50 for each cell line) is the concentration of compound which reduced by 50% the optical density of treated cells with respect to untreated cells using the MTT assay. IC50 values are taken as a mean from three experiments. Mean ± SD. b Cell lines include human hepato carcinoma (SMMC-7721), lung carcinoma (A549) and nasopharyngeal carcinoma (CNE2). c Clog P values produced by Chemdraw software.
Moreover, after changing the linking position of matrine attached to the indole from 3-position to 4-position or 5-position, respectively, compounds B30–B35 showed decreased activities compared with their analogues respectively.
It was further revealed that compound 1 and A1 with low Clog P values of 1.361, 2.802, respectively, exhibited weaker antiproliferative potency. However, compounds having slightly high Clog P values showed potent antiproliferative effect. These results suggested
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that the Clog P values of all molecules seemed to be a very important determinant factor of the antiproliferative properties. Interestingly, most derivatives possessed the strongest cytotoxicity against CNE2 cells, followed by SMMC-7721 cells, and were least cytotoxic to A549 cells. Thus, we subsequently selected A6
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and B21 for further investigation to explore its anti-cancer mechanisms on CNE2 and SMMC-7721 cancer cells. In order to investigate whether compound A6 and B21 could induce cell apoptosis, CNE2 and SMMC-7721 cells treated with 0, 5, 10, 30 lM of A6 and 0, 10, 20, 50 lM of B21, were subjected
Fig. 2. Compound A6 could induce apoptosis in CNE2 and SMMC-7721 cells in a dose-dependent manner.
Fig. 3. Compound B21 could induce apoptosis in CNE2 and SMMC-7721 cells in a dose-dependent manner.
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to AnnexinV-FITC/propidium iodide (PI) dual staining and quantified by flow cytometry.25 As shown in Fig. 2, the percentage of apoptotic cells (Lower right quadrant, AV+/PI and Upper right quadrant, AV+/PI+) in CNE2 significantly increased from 4.93% (control) to 11.96% (5 lM), 34.04% (10 lM) and 50.50% (30 lM) respectively after treatment with different concentrations of compound A6 for 24 h. And the percentage of apoptotic cells in SMMC7721 increased from 2.60% (control) to 19.93% (5 lM), 53.58% (10 lM) and 61.50% (30 lM), respectively. Consistently, as shown in Fig. 3, the percentage of apoptotic cells of in CNE2 significantly increased from 5.29% (control) to 17.52% (10 lM), 38.60% (20 lM) and 74.20% (50 lM) respectively after treatment with different concentrations of compound B21 for 24 h. And the percentage of apoptotic cells in SMMC-7721 increased from 2.60% (control) to 39.00% (10 lM), 58.80% (20 lM) and 95.00% (50 lM), respectively. The results revealed that compound A6 and B21 could induce the apoptosis of CNE2 and SMMC-7721 cells in a dose-dependent manner. To reveal whether the inhibition of cancer cells growth by compound A6 and B21 was in associate with cell cycle arrest, CNE2 and SMMC-7721 cells were treated with different concentrations of compound A6 (0, 5, 10 and 30 lM) and B21 (0, 10, 20 and 50
lM) for 24 h. Cell cycle distribution was investigated by flow cytometric analysis using propidium iodide staining method.26 Compound B21 did not show significant cell cycle arrest (data not shown), while compound A6 significantly cell cycle arrest as shown in Fig. 4. The percentage of G2/M phase in CNE2 gradually increased from 19.8% (control) to 29.5% (5 lM), 89.4% (10 lM) and 81.1% (30 lM). Consistently, the percentage of G2/M phase in SMMC-7721 cells increased from 18.4% (control) to 24.1% (5 lM), 78.9% (10 lM) to 79.1% (30 lM). These results confirmed that compound A6 could arrest the cell cycle of CNE2 and SMMC-7721 cells at G2/M phase. In summary, 58 new matrine derivatives A1–A23 and B1–B35 were synthesized, and their anti-proliferative activities against human cancer cell lines such as SMMC-7721, A549 and CNE2 were investigated. SAR analysis revealed that suitable nitrogen-containing heterocycle groups on 14 site of matrine were benefit to anticancer activity improvement. In MTT assay, compound A6 and B21 exhibited the most significant growth inhibition of cancer cells. Further studies indicated that A6 exerted its anti-cancer activity by inducing the apoptosis of CNE2 and SMMC-7721 cells and G2/M cell cycle arrest in a dose dependent manner. B21 exerted its anti-cancer activity by inducing the apoptosis of CNE2
Fig. 4. Compound A6 could induce G2/M cell cycle arrest in CNE2 and SMMC-7721 cancer cells. (A) A6 induces G2/M cell cycle arrest in CNE2 and SMMC-7721 cells in a dose-dependent manner. (B) Distributions of G1, S and G2/M in CNE2 and SMMC-7721 cells under A6 treatment.
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and SMMC-7721 cells. More studies are needed to explore the precise antitumor mechanism. Acknowledgments This work was supported by the National Natural Science Foundation of China (21402032, 21262005), the high level innovation team and outstanding scholar project of Guangxi institutions of higher education (guijiaoren [2014] 49 hao), Guangxi Natural Science Foundation (2014GXNSFBA118031, 2017GXNSFBA198240) and the State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University (CMEMR2017-B14). We also thank Professor Cai Bin of Suzhou Galaxy biopharma, CO., LTD. for the experiment support. A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bmcl.2018.01.017. References 1. Zhong LY, Wu H. Zhongguo Zhong Yao Za Zhi. 2006;31:1561. 2. Sun M, Cao H, Sun L, et al. Evid Based Complement Alternat Med. 2012;2012:373219.
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