Bioorganic & Medicinal Chemistry Letters

Design, synthesis and biological evaluation of benzamide derivatives as novel NTCP inhibitors that induce apoptosis in HepG2 cells

Shuangmei Zhaoa, Yongqi Zhena, Leilei Fu, Feng Gao, Xianli Zhou, Shuai Huang⁎, Lan Zhang⁎
School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, PR China

A R T I C L E I N F O

Keywords:
NTCP inhibitors Apoptosis
Anti-proliferative activity Hepatocellular carcinoma

A B S T R A C T

Sodium taurocholate cotransport polypeptide (NTCP) plays an important role in the development of hepatitis and acts as a switch to allow hepatitis virus to enter hepatic cells. As the entry receptor protein of hepatitis virus, NTCP is also an effective target for the treatment of hepatocellular carcinoma. Herein, twenty-five benzamide
analogues were synthesized based on the virtual screening design and their anti-proliferative activities against HepG2 cells were evaluated in vitro. Compound 35 was found to be promising, with an IC50 value of 2.8 μM. The apoptosis induced by 35 was characterized by the regulation of markers, including an increase in Bax, cleaved- caspase 3, and cleaved-PARP proteins, and a decrease in Bcl-2 protein. Molecular docking and molecular dy- namics (MD) simulation confirmed that compound 35 can bind tightly to NTCP. Western blot analysis also showed that NTCP was inhibited. Altogether, these results indicate that compound 35 acts as a novel NTCP inhibitor to induce apoptosis in HepG2 cells.

Hepatocellular carcinoma (HCC) is the fifth most common malig- nancy and the third leading cause of cancer-related death worldwide.1,2 To date, various treatments have been used in HCC, including mainly radiotherapy, chemotherapy, biological immunotherapy, surgical re- section and liver transplantation.3,4 However, poor prognosis and high recurrence rates are still the main causes of death in HCC patients.5 The occurrence and development of HCC are related to various factors, in- cluding aflatoXin intake and smoking,6 alcoholic liver disease and non- alcoholic fatty liver disease,7 hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, or other causes of cirrhosis.8,9 Studies have shown that there is a close relationship between hepatitis virus infection and HCC, and in approXimately 40% of patients, HCC was caused by viral hepa- titis C.10 Therefore, development and application of hepatitis virus-re- lated targeted drugs may be an effective means for HCC treatment.

The sodium taurocholate cotransport polypeptide (NTCP) is the cardinal liver transporter that binds bile acids and is a functional re- ceptor for human HBV and hepatitis D viruses (HDVs).11 Jia and Xie et al. found that NTCP could mediate the uptake of bile acids in he- patoma cells, and the bile acid-microbial axis is formed when the bile acid ingested by NTCP increases, which regulates the occurrence and development of HCC.12 When the S267F mutation occurs in NTCP, the HBV infection capacity is decreased, and the incidence of HBV infec- tion-associated cirrhosis and HCC is reduced.13 Taken together, NTCP is

an important target for the treatment of HCC.
Currently, many old drugs have been identified as NTCP inhibitors and applied to the study of NTCP (Fig. 1). For instance, rosiglitazone, zafirlukast, TRIAC and sulfasalazine have shown good inhibitory ac- tivity against NTCP in 1280 screenings of clinically approved drugs.11 Fluvastatin is an NTCP inhibitor, which interacts with NTCP at the level of the bile acid binding pocket.14 Macitentan15, bosentan16 and irbe- sartan17 were also shown to act as NTCP inhibitors.18 Studies have indicated that fasiglifam could inhibit NTCP and affect HBV infection,17 which also has severe hepatotoXicity.19 Despite their NTCP inhibitory activities and anti-neoplastic properties in HCC, these inhibitors are associated with many side effects.20–22 Thus, the development of effi- cient NTCP inhibitors is the primary goal for the treatment of HCC.
In this study, we discovered a novel NTCP inhibitor (compound 35) that inhibits the expression of NTCP. Compound 35 exhibited excellent anti-proliferative activity and induced apoptosis in HepG2 cells. Our study shows that compound 35, as an NTCP inhibitor, could be used as a candidate drug for future treatment of HCC.
To discover novel NTCP inhibitors, homology modeling was per- formed to obtain the structure of NTCP. We first screened for leading candidate compounds from the ZINC database according to Lipinski’s Rule of Five (Fig. 2). Then, 10,000 compounds were selected using the LibDock protocol. Subsequently, the top 200 hits were further screened

Corresponding authors at: School of Life Science and Engineering, Southwest Jiaotong University, NO. 111, North Second Ring Road, Chengdu 610031, Sichuan, PR China.
E-mail addresses: [email protected] (S. Huang), [email protected] (L. Zhang).
a These authors contributed equally to this work and should be considered co-first authors.

https://doi.org/10.1016/j.bmcl.2019.126623

Received 8 April 2019; Received in revised form 22 July 2019; Accepted 13 August 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.

Fig. 1. The structures and activities of several NTCP inhibitors.

Fig. 2. Virtual screening protocol for identifying NTCP inhibitors.

Fig. 3. Structure-activity relationships of the compounds.

using the CDOCKER protocol. As a result, we discovered 10 hits bearing better CDOCKER interaction energies and LibDock scores (Table S1). The anti-proliferative activities against HepG2 cells of these 10 com- pounds were determined by MTT assays (Fig. S1). Because of its ex- cellent activity, AO-081/40926746 was selected as a leading com- pound with significant potential for further optimization.
Based on the leading compound, we focused our attention on varying parts A and B and retained the chain of N-(2-(2-benzylindenyl- 2-oXoethyl) acetamide (Fig. 3). The electron donating group and an electron withdrawing group (R1, R2, R3) were initially introduced into parts A and B, and the derivatives 18–29 were synthesized (Scheme 1). Subsequently, part A was replaced by meta and para, and derivatives 31 and 33 were synthesized (Scheme 2–3). Introducing oXygen hetero- cycles in part A and substituting R3, the derivatives 35–37 were syn- thesized (Scheme 4), and the para-substituted derivatives 39 and 40 were synthesized (Scheme 5). 2-Chloropyridine was substituted for part

B, and derivative 43 was synthesized (Scheme 6). Finally, parts A and B were replaced at the same time, and derivatives 44–47 were synthe- sized (Scheme 7–10). According to the biological activity results (Table 1), we discovered compounds 23, 36, 39, 43 and 47 exhibited
moderate anti-proliferative activity, while compounds 35, 44 and 45 exhibited superior biological activity. The remaining derivatives ex- hibited lower anti-proliferative activity. The anti-HBs and anti-HBe antigen activities of all compounds were tested in HepG2.2.15 cells at 5 μM, and Tenofovir was used as a positive control. These compounds had similar anti-HBV potency in terms of their anti-proliferative ac- tivities, among which compounds 35, 44 and 45 showed the most po- tent anti-HBV activities (Table 1). To further evaluate the HBV in- hibitory effects of compounds 35, 44 and 45, we detected the expression of HBV DNA in cells by using real-time PCR and found that HBV DNA levels in HepG2.2.15 cells were also markedly reduced after treatment with compounds 35, 44 and 45 (Fig. 4).

Scheme 1. General synthesis of compounds 18–29. Reagents and conditions: (i) ethanol, K2CO3, KI, 85 °C, refluX, 4–8 h; (ii) acetonitrile; K2CO3, r.t, overnight; (iii) methanol, 80% hydrazine hydrate solution, 85 °C, refluX, overnight; (iv) ethanol, acetic acid, 85 °C, refluX, 6–8 h.

Scheme 2. General synthesis of compounds 31. Reagents and conditions: (i) ethanol, K2CO3, KI, 85 °C, refluX, 4–8 h; (ii) ethanol, acetic acid, 85 °C, refluX, 6–8 h.

Scheme 3. General synthesis of compounds 33. Reagents and conditions: (i) ethanol, K2CO3, KI, 85 °C, refluX, 4–8 h; (ii) ethanol, acetic acid, 85 °C, refluX, 6–8 h.

Scheme 4. General synthesis of compounds 35–37. Reagents and conditions: (i) ethanol, K2CO3, KI, 85 °C, refluX, 4–8 h; (ii) ethanol, acetic acid, 85 °C, refluX, 6–8 h.

Scheme 5. General synthesis of compounds 39 and 40. Reagents and conditions: (i) ethanol, K2CO3, KI, 85 °C, refluX, 4–8 h; (ii) ethanol, acetic acid, 85 °C, refluX, 6–8 h.

Scheme 6. General synthesis of compounds 43. Reagents and conditions: (i) acetonitrile; K2CO3, r.t, overnight; (ii) methanol, 80% hydrazine hydrate solution, 85 °C, refluX, overnight; (iii) ethanol, acetic acid, 85 °C, refluX, 6–8 h.
Scheme 7. General synthesis of com- pounds 44. Reagents and conditions:
(i) ethanol, acetic acid, 85 °C, refluX, 6–8 h.
Scheme 8. General synthesis of com- pounds 45. Reagents and conditions:
(i) ethanol, acetic acid, 85 °C, refluX, 6–8 h.

 

Scheme 9. General synthesis of compounds 46. Reagents and conditions: (i) ethanol, acetic acid, 85 °C, refluX, 6–8 h.

Table 1

Anti-proliferative activities and anti-HBV activities of the compounds.

Scheme 10. General synthesis of com- pounds 47. Reagents and conditions: (i) ethanol, acetic acid, 85 °C, refluX, 6–8 h.

No. Anti-proliferative activity (IC50, μM)a

HBsAg (ng/ml)b HBeAg (ng/ml)b
Control –d 8.41 ± 0.54 25.13 ± 1.93
Tenofovir –d 1.72 ± 0.27 5.60 ± 0.58
Cisplatin(DDP)c 3.8 ± 1.1 –d –d

AO-081/
40926746

13.4 ± 1.6 3.95 ± 0.43 15.01 ± 0.97

18 24.9 ± 0.5 5.03 ± 0.58 14.53 ± 2.20
19 > 50 8.33 ± 0.71 25.22 ± 1.39
20 > 50 8.37 ± 0.62 24.55 ± 2.56
21 > 50 7.94 ± 0.80 23.53 ± 2.48
22 > 50 8.04 ± 0.55 24.34 ± 2.23
23 15.7 ± 3.8 4.23 ± 0.59 13.07 ± 1.53
24 > 50 8.17 ± 0.69 24.86 ± 1.75
25 > 50 8.29 ± 0.60 25.47 ± 2.32
26 > 50 8.25 ± 0.77 25.25 ± 1.94
27 > 50 7.66 ± 0.71 23.04 ± 1.69
28 > 50 7.79 ± 0.87 22.99 ± 2.03
29 > 50 8.31 ± 0.73 24.88 ± 2.16
31 47.5 ± 2.5 5.74 ± 0.68 18.09 ± 2.12
33 28.1 ± 1.8 4.83 ± 0.57 14.29 ± 2.09
35 2.8 ± 2.3 1.93 ± 0.18 7.43 ± 0.85
36 17.1 ± 1.0 3.98 ± 0.44 11.84 ± 1.42
37 > 50 7.72 ± 0.75 24.07 ± 2.86
39 14.9 ± 2.4 3.91 ± 0.30 11.47 ± 1.10
40 > 50 7.66 ± 0.68 23.80 ± 1.66
43 16.9 ± 3.2 4.08 ± 0.37 12.55 ± 1.55
44 3.1 ± 0.6 2.05 ± 0.20 6.43 ± 1.33
45 4.7 ± 1.3 2.48 ± 0.23 7.36 ± 1.01
46 > 50 8.39 ± 0.43 25.47 ± 2.23
47 10.1 ± 3.1 3.71 ± 0.26 11.45 ± 0.97
a Each compound was tested in triplicate.
b Each compound was tested at 5 μM for HBV antigen activity by ELISA.
c Cisplatin was used as a positive control for anti-proliferative activity.
d Data were not determined.

Fig. 4. HBV DNA inhibitory efficacies of NTCP inhibitors. HepG2.2.15 cells were treated with or without 10 μM Tenofovir or 10 μM 35, 44, or 45, and HBV DNA in the supernatants were detected using real-time PCR.
To explore the binding mode of compound 35 to NTCP (Fig. 5A), a molecular docking simulation was performed using Discovery Studio
3.5 (Fig. 5B). Compound 35 could form three hydrogen bonds with Ser109, Asn54 and Tyr108. Additionally, the benzene ring of compound 35 could interact with Phe44 to form π-π interaction. Whereas the benzyl group at the other end of compound 35 could penetrate the hydrophobic cavity of NTCP. To further demonstrate the stability of the compound 35-NTCP complex, a molecular dynamics simulation of 100 ns was established. The low root-mean-square deviation (RMSD) and the low binding free energy indicated that the system was stable (Fig. 5C and D). These results indicate that compound 35 could stably bind to NTCP.
Next, we investigated the effect of compound 35 on HepG2 pro- liferation, and with cisplatin as the positive control. It was found that
Fig. 5. Molecular docking and simulation of molecular dynamics. (A) Chemical structure of compound 35. (B) Molecular docking of compound 35 and NTCP. (C) Molecular dynamics (MD) simulation of the compound 35-NTCP complex. (D) The binding free energy of complex.

compound 35 inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner (Fig. 6A and B). When the cells were treated with compound 35, condensed chromatin and bright fluorescence was observed by fluorescence microscopy after Hoechst 33258 staining (Fig. 6C). In addition, apoptosis was further evaluated by Annexin-V/PI double staining. Compound 35 significantly increased the rate of apoptosis in HepG2 cells (Fig. 6D). Meanwhile, western blot analysis showed compound 35 inhibited NTCP expression, upregulated Bax, downregulated Bcl-2, and increased the cleavage of caspase 3 and PARP (Fig. 6E). These results indicate that compound 35 acts as an NTCP inhibitor to induce HepG2 cells apoptosis.
As a major cause of hepatocellular carcinoma induced by hepatitis,

NTCP is one of the main targets for the treatment of HCC. In conclusion, we synthesized twenty-five structural analogs as candidate compounds. Molecular docking and MD simulation show that compound 35-NTCP is a stable complex system. The MTT assay demonstrated that compound 35 caused time-dependent and concentration-dependent inhibition of the proliferation of HepG2 cells, and this anti-proliferative effect ap- peared to be due to its ability to induce apoptotic cells. In addition, the induction of apoptosis by compound 35 was confirmed using fluores- cence microscopy, flow cytometry and western blot analysis. These findings will provide valuable guidelines for the development of tar- geted NTCP small molecule drugs for the treatment of HCC.
Fig. 6. Compound 35 induced apoptosis in HepG2 cells. (A, B) The cells were treated with different concentrations of compound 35 for indicated times, and then the cell inhibition rates were measured using MTT assay; (C) The cells were treated with different concentrations of compound 35 for 24 h. Morphologic changes were observed by fluorescence microscopy after Hoechst 33258 staining. Scale bar = 50 μm; (D) The cells were treated with different concentrations of compound 35 for 24 h, and the apoptosis ratios were determined by flow cytometry analysis after Annexin-V/PI double staining; (E) The cells were treated with different con- centrations of compound 35 for 24 h and then the expressions of Caspase Inhibitor VI NTCP, PARP, Bax, Bcl-2 and caspase-3 were detected by western blot analysis.
Acknowledgements
We are grateful to Prof. Bo Liu and Liang Ouyang (Sichuan University) for providing critical suggestions for this study. This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81602953 and 81602627).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmcl.2019.126623.
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