Design, synthesis and anti-HBV activity of NVR3-778 derivatives

Journal Pre-proofs Design, Synthesis and anti-HBV activity of NVR3-778 derivatives Kai Lv, Shuo Wu, Wenyan Li, Yunhe Geng, Meng Wu, Jinming Zhou, Yuhuan Li, Qiang Gao, Mingliang Liu PII: DOI: Reference:

S0045-2068(19)31254-4 https://doi.org/10.1016/j.bioorg.2019.103363 YBIOO 103363

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Bioorganic Chemistry

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1 August 2019 6 October 2019 12 October 2019

Please cite this article as: K. Lv, S. Wu, W. Li, Y. Geng, M. Wu, J. Zhou, Y. Li, Q. Gao, M. Liu, Design, Synthesis and anti-HBV activity of NVR3-778 derivatives, Bioorganic Chemistry (2019), doi: https://doi.org/ 10.1016/j.bioorg.2019.103363

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Design, Synthesis and anti-HBV activity of NVR3-778 derivatives Kai Lv a, 1, Shuo Wu a, 1, Wenyan Li b, Yunhe Geng a, b, Meng Wu,a Jinming Zhou,c Yuhuan Li a, *, Qiang Gao a, Mingliang Liu a, * a Institute

of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union

Medical College, Beijing 100050, China b College

of Chemistry & Material Science, Hebei Normal University, Shijiazhuang 050024, China

c

Key Laboratory of the Ministry of Education for Advanced Catalysis Materials, Department of Chemistry, Zhejiang Normal University, Jinhua, 321004, China * Corresponding author. E-mail addresses: [email protected] (M. Liu), [email protected] (Y. Li). 1 These authors contributed equally to this work.

Abstract NVR3-778, one of the most advanced capsid assembly modulators (CAMs), is currently

in phase II clinical trial for the treatment of HBV infection. In this study, we reported the first structure optimization of NVR3-778. Compound 2d was found to exhibit more potent anti-HBV activity (IC50: 0.25 µM), lower cytotoxicity (CC50, 10.68 µM) and higher selectivity index (SI, 40.72) than NVR3-778 (IC50, 0.33 µM; CC50, 5.14 µM; SI: 18.36) in vitro, and also display similar inhibitory effect on the assembly of HBV capsids as NVR3778. Molecular docking further suggested that compound 2d might form a stronger interaction with core protein. Moreover, compound 2d also showed acceptable pharmacokinetic profiles. Currently compound 2d was selected as a new lead for further modifications, and studies to determine the in vivo anti-HBV studies of 2d will begin soon. Keywords: NVR3-778; structure modification; sulfamoylbenzamides; anti-HBV activity; capsid assembly modulators.

1. Introduction The safe and effective prophylactic Hepatatis B virus (HBV) vaccine has been available since 1982, and vaccination dramatically reduced new HBV infections among children[1]. However, HBV infection remains a major health problem. The World Health Organization (WHO) estimated that approximately 257 million people are living with

chronic HBV, and 0.88 million died from HBV related complications (including cirrhosis and hepatocellular carcinoma) worldwide in 2015 [2]. Currently, the treatment of chronic HBV mainly relies on pegylated interferon-alpha (PEG-INFα) and nucleos(t)ide analogues, such as lamivudine, tenofovir, and entecavir [3]. These treatments can slow the progression of cirrhosis, reduce incidence of liver cancer, and improve long-term survival, but seldom result in cure as they do not eradicate the covalently closed circular DNA (cccDNA), which provides for viral persistence [4]. Therefore, at present, long-term (potentially lifelong) therapy is required for the majority of patients. As such, there is an urgent need for discovery of new drugs which could suppress HBV viral load and decrease cccDNA formation. F H N

F A F

O

F

F B

O S N O H

H N

F A F

DVR-23

B O

O S N O H

H N

F A F F

AB-423 (phase I)

B O

O S N O C OH

NVR3-778 (phase II)

Figure 1. Structures of DVR-23, AB-423 and NVR3-778 In recent years, sulfamoylbenzamides (SBAs), a novel class of HBV agents as capsid assembly modulators (CAMs) are expected to significantly decrease the formation of cccDNA and cure HBV infection [5]. Since the discovery of the early SBA lead DVR-23 in 2013 by Blumberg Institute [6], various optimization efforts were conducted by industrial and academia [7]. Currently, multi-SBAs have entered clinical trials such as AB423 (phase I, Arbutus Biopharma Inc and Blumberg Institute) [8], NVR3-778 (phase II, Novira Therapeutics and Janssen Pharmaceutical, Figure 1) [9, 10] and JNJ6379 (phase II, structure not disclosed, Janssen Pharmaceutical) [11]. In fact, although NVR3-778 [12] and AB423 [8] have advanced into clinical trials for years, their mystical structures were just uncovered in 2018. Inspired by the promising anti-HBV activity and disclosed structures [13, 14], we intend to design, and synthesis new SBAs based on the structure feature of NVR3-778, which were expected to explore structure-activity relationships (SAR) of SBAs, and identify alternative candidate as anti-HBV agents. 2. Results and discussion 2.1. Structure modification and SAR

Although the structure of NVR3-778 was just disclosed in 2018 [9], the SBAs have been a hot spot in the anti-HBV research area since the discovery of DVR-23 in 2013. A lot of SBAs related patents have been applied [15-19], mainly revolving around the modification of C segment of NVR3-778 (seen in figure 1). In addition, two papers from Vandyck [11] and Schinazi [12] also reported the SAR of the SBAs, suggesting that the ring A and B (seen in figure 1) might be important to the anti-HBV activity of these SBA analogues. Moreover, based on the recently reported crystal structure of NVR3-778 and core protein (PDB code: 5T2P), the benzamide oxygen of NVR3-778 forms essential hydrogen bonds with the side chains of Trp102 (Chain B) and Thr128 (Chain C), and the 4-hydoxyl-piperidyl group is fully accessible to solvent [8, 20]. Thus, we focused on the modification of 4-hydoxyl-piperidine moiety of NVR3-778 in this research. As shown in Table 1, the hydroxyl group on the piperidine ring of NVR3-778 was replaced with various functional groups such as amine, ester, oxime et al. They were synthesized and evaluated for their anti-HBV activity and cytotoxicity against HepAD38 cell line. Compared to NVR3-778, most of them show decreased anti-HBV activity (1a-f, IC50s: 0.75 - >2). More precisely, the introduction of amine or ester functional groups to the piperidine ring diminished the anti-HBV activity significantly (1a-c, IC50 > 2 µM). To our delight, compounds 1g-h with ketone and hydroxylamine functional group displayed comparable anti-HBV activity (IC50s: 0.21-0.30 µM) to NVR3-778 (IC50: 0.28 µM). Additionally, compounds 1g-h also showed slightly less cytotoxic to HepAD38 cells (CC50s: 7.41 µM) and higher selectivity index (SIs, 24.70 - 35.29) than NVR3-778 (CC50: 5.77 µM; SI, 18.36). Table 1. Structures and activity of new SBAs (Series 1) F H N

F

O S W O

O

F F

Compd.

W group

1a

N

1b

N

NH2

O O

IC50 CC50 (µM) (µM)

SI

>2

96.15

-

>2

138.67

-

O

1c

N

1d

N

O O

OH

O

>2

>200

-

1.02

12.83

12.58

0.75

66.67

88.89

1.05

>200

>190.48

0.21

7.41

35.29

1e

N

1f

N

NH2 O N

1g

N

OH N

1h

N

O

0.30

7.41

24.70

NVR3-778

N

OH

0.28

5.14

18.36

Changes were subsequently focused on the piperidine ring of NVR3-778, whereas the hydroxyl functional group was maintained. As shown in table 2, the piperidine ring of NVR3-778 was exchanged by fused/bridged/spiro-heterocycle, amino-cyclohexyl, phenylamino or opening rings. Among them, compounds 2a-c with fused/bridged/spiroheterocycle exhibited good anti-HBV activity (IC50s, 0.25-0.48 µM) and also significantly decreased cytotoxicity (CC50s, 22.22-155.21 µM). Interestingly, the cis-aminocyclohexan1-ol substituted analogue 2d (IC50, 0.25 µM) was significantly more active than its transdiastereomer 2e (IC50 > 2 µM). N-methylation of the amide of 2d, as in 2f, resulted in reduced anti-HBV activity. Introduction of opening rings or phenol also led to lose potency (2g-i, IC50 > 2 µM). Table 2. Structures and activity of new SBAs (Series 2) F H N

F

O

F

O S W O

F

Compd.

W group

2a

N

2b

N

2c

N

2d

HN

OH OH

OH OH

IC50 (µM) 0.30

CC50 (µM) 22.22

SI

0.35

10.68

30.51

0.48

155.21

323.35

0.25

10.68

42.72

74.07

2e

HN

2f

OH

N

OH

>2

>200

-

1.46

22.22

15.21

>2

12.83

-

>2

66.67

>2

10.68

0.33

5.14

Me

2g

HN

OH OH

2h

HN OH

2i

NH OH

NVR3-778

15.58

According the SAR findings discussed above, the hydroxyl group on piperidine of NVR3-778 might be replaced by ketone or hydroxylamine; the fused/ spiro/ bridged heterocycle ring could be an alternative selection of piperidine. We therefore installed the ketone or oximes groups to the hydroxyl position of 2a-c, resulting in SBAs 3a-e as shown in table 3. In accordance with the above SAR, introduction of ketone and hydroxylamine maintained the potent anti-HBV activity (3a-b and 3d-e, IC50s: 0.22-0.54 µM). In contrast, compound 3c with methoxylamine showed decreased anti-HBV activity with IC50 of > 2 µM. In addition, the cytotoxicity of 3a-b and 3d-e (CC50, 7.41-18.74) were all lower than that of NVR3-778. Table 3. Structures and activity of new SBAs (Series 3) F H N

F

O

F

O S W O

F

Compd. 3a 3b 3c

W group

SI

0.39

CC50 (µM) 18.74

48.05

N

O

N

OH N

0.54

7.41

13.72

N

O N

>2

>200

-

0.44

9.54

21.68

0.22

7.41

33.68

0.31

5.14

16.58

3d

N

3e

N

NVR3-778

IC50 (µM)

O O

2.2 Detection of the effect of compounds on capsids assembly by a particle gel assay We and others have demonstrated that type I CAMs such as Bay 41-4109 can abolish capsid formation while type II CAMs may not change the amounts of capsids but induce the assembly of capsids with faster electrophoresis mobility [21-22]. Thus, capsid electrophoresis mobility shift can used as a diagnostic marker of compounds that target core protein assembly and predicts sensitivity of HBV strains to specific type II CAMs [2122]. To further determine the anti-HBV activity of the novel compounds, the effects of these compounds on capsids at a concentration of 1 μM were then examined by a particle gel assay. As shown in Figure 2, NVR3-778 treatment reduced the amounts of slowmigrating capsids but significantly increased the amounts of fast-migrating capsids, which is consistent with the results of other SBAs [21-22]. Compounds with relatively good antiHBV activities (1g-h, 2a-b, 2d, 3a-3b and 3d-3e) showed similar capsids electrophoresis mobility alteration characteristics, while compounds with IC50 more than 2 µM (1a-c, 2e, 2g-2i, and 3c) exhibited similar capsids electrophoresis migration profile to the mock treated (NC) group. These results indicated that the synthesized novel compounds with good antiviral activities also have inhibitory effect on the assembly of HBV capsids.

Figure 2. Particle gel assay of capsids assembled in cells treated with the indicated compounds. HepAD38 cells were culture in the absence of tetracycline and mock treated (NC) or treated with the indicated compounds at a concentration of 1 μM for 6 days. The total amounts of capsids were separated by a particle gel assay in a 1.5% agarose gel

electrophoresis, transferred onto a nylon membrane, and detected by a rabbit polyclonal antibody against core protein. 2.3. Pharmacokinetic profiles of selected SBAs SBAs 1h, 2a-d, 3a and 3d with potent anti-HBV activity were selected and further evaluated for their in vivo pharmacokinetic (PK) profiles in ICR mouse after a single oral administration of 50 mg/kg. As shown in table 4, except for compound 2d, the AUC0-ts of these SBAs were more than 10 times lower than that of NVR3-778. The introduction of ketone (1h, 3a, 3d) resulted in dramatically decreased AUC0-t probably due to the ketalization of ketone in vivo. Compound 2d showed acceptable PK profiles with AUC0-t of 232025 h.ng/mL and T1/2 of 7.79 h although the AUC0-t of 2d was lower than that of NVR3-778. Currently, studies to determine the in vivo anti-HBV activity of 2d will begin soon. Table 4. In vivo PK profiles of selected SBAs in mice at 50 mg/Kg Compd. 1h 2a 2b 2c 2d 3a 3d NVR3-778

T1/2 (h) 4.97±1.59 3.54 ±1.77 2.18±0.65 1.71±0.59 7.79±0.77 10.9±5.04 0.62±0.24 8.14±0.95

Tmax (h) 1.50±2.17 0.33±0.14 0.33±0.14 0.25±0 0.83±0.28 0.25±0 0.25±0 1.08±0.88

Cmax (ng/mL) 70.1±5.8 5406 ± 2203 1226±167 846±270 38247±10310 819±301 110±42 66090±17219

AUC0-t (h.ng/mL) 311±44.7 10132± 3448 3051±224 503±81 232025±46728 1425±246 80.6±31.0 455931±56864

MRT (h) 7.68±1.91 2.65±0.34 2.58±0.53 1.43±0.73 9.78±3.19 13.01±5.93 0.88±0.23 10.71±3.71

2.4. Molecular docking study of compound 2d The capsid inhibitors bound to the core protein dimer-dimer interface were widely reported [8, 20, 23-24], which indicted that compound 2d might also bound to the same site. Thus, we predicted the binding mode of compound 2d in the core protein dimer-dimer interface (PDB code: 5T2P) through molecular docking with CDOCK module of Discovery Studio 3.5. The docked model of the compound 2d chemical structure was compared with NVR3-778 which was an intrinsic ligand from the X-ray structure (PDB code: 5T2P). As the chemical structure of compound 2d was similar to that of NVR3-778, the binding model of compound 2d also showed a closed interaction pattern with NVR3-

778 (Figure 3A). The ligand-receptor interaction analysis showed that the central amide of NVR3-778 forms a crucial hydrogen bond with the Trp102 of core protein (Figure 3B), while compound 2d forms a hydrogen bond with Trp102 through its central amide as well as an additional hydrogen bond with Leu140 through its sulfanilamido radical (Figure 3C), which indicates compound 2d forms a stronger interaction with core protein. What is more, comparing to NVR3-778, the more flexible sulfanilamido radical of compound 2d is supposed to form a more suitable binding mode with the hydrophobic pocket around Leu140 at dimer-dimer interface of core protein, thus strengthen its interaction with core protein.

Figure 3 Binding mode of compound 2d and X-ray structure of core protein: (A) comparison of the predicted binding model of compound 2d and NVR3-778 in the core protein X-ray structure (PDB code: 5T2P) , the surface of the capsid protein is in yellow, the NVR3-778 is in purple stick and compound 2d is in blue stick; (B) The detailed interactions between NVR3-778 and core protein at the dimer-dimer interface site; (C) The

predicted binding mode of compound 2d and core protein at the dimer-dimer interface site. 3. Chemistry Detailed synthetic pathways to target compounds 1-3 are outlined in scheme 1. Electrophilic substitution of 4-flurobenzoic acid with chlorsulfonic acid gave sulfuryl chloride 5, which was refluxed with thionyl chloride to yield compound 6. Refluxing compound 6 and 3,4,5-trifluoroaniline in toluene formed the key intermediate 7 as the reported procedure. [17] Coupling compound 7 with various amines afforded target compounds 1-3. For the preparation of 1a, the Boc-group on piperdin-4-amine has to be subsequently removed by TFA in DCM. F

Cl

a

O O S F b

Cl

O O S F

O F

c

F F

HOOC

HOOC

4 F

d

5 O

ClOC O W S O

6

O

Cl S O

NH F

7

F F

NH F 1-3

Regents and conditions: a) ClSO3H, reflux, 3h, 71% yield; b) SOCl2, 80 oC, 85% yield; c) toluene, 3,4,5-trifluoroaniline, reflux, 83% yield; d) i) Et3N, CH3CN, rt, 40-89% yields; ii) For compound 1a, then TFA, DCM, 75% yield. Scheme 1. Synthesis of the target compounds 1-3 4. Conclusion In summary, multi-series of NVR3-778 analogues were designed and synthesized as new anti-HBV agents in this study. Structural modifications and SAR studies focused on the 4-hydoxyl-piperidine moiety of NVR3-778. The alternative replacement of hydroxyl group and piperidine ring were investigated respectively. In addition, a series of SBAs was further designed and synthesized according the SAR findings above. Compared to NVR3778, SBAs 1g-h, 2a-d, 3a and 3d-e were found to have comparable anti-HBV activity (IC50: 0.25-0.48 µM), lower cytotoxicity and higher selectivity index. Moreover, the particle gel assay proved that these compounds have inhibitory effect on the assembly of HBV capsids.

However, most of them displayed decreased AUC0-t in PK evaluation. Among them, compound 2d exhibited acceptable PK profiles with AUC0-t of 232025 h.ng/mL, and T1/2 of 7.79 h. In addition, molecular docking further suggested that compound 2d might forms a stronger interaction with core protein. Based on above results, compound 2d was selected as a new lead for further modifications, and studies to determine the in vivo anti-HBV studies of 2d will begin soon. Funding Sources This work is supported by CAMS Innovation Fund for Medical Science (CAMS-2018I2M-3-004), the National Mega-project for Innovative Drugs (2018ZX09711001-007-002, 2018ZX09101003-003-003), National Natural Science Foundation of China (81621064) and the Fundamental Research Funds for the Central Universities (3332019007). References [1] Y. Pei, C. Wang, S.F. Yan, G. Liu, Past, Current, and Future Developments of Therapeutic Agents for Treatment of Chronic Hepatitis B Virus Infection, J. Med. Chem., 60 (2017) 6461-6479. Doi:10.1021/acs.jmedchem.6b01442. [2]

WHO.

Hepatitis

B:

World

Health

Organization

Fact

Sheet

204.

http://www.who.int/mediacentre/factsheets/fs204/en/ (accessed Jan 17, 2018). [3] Z. Qiu, X. Lin, M. Zhou, Y. Liu, W. Zhu, W. Chen, W. Zhang, L. Guo, H. Liu, G. Wu, M. Huang, M. Jiang, Z. Xu, Z. Zhou, N. Qin, S. Ren, H. Qiu, S. Zhong, Y. Zhang, Y. Zhang, X. Wu, L. Shi, F. Shen, Y. Mao, X. Zhou, W. Yang, J.Z. Wu, G. Yang, A.V. Mayweg, H.C. Shen, G. Tang, Design and Synthesis of Orally Bioavailable 4-Methyl Heteroaryldihydropyrimidine Based Hepatitis B Virus (HBV) Capsid Inhibitors, J. Med. Chem.,

59 (2016) 7651-7666. doi:

10.1021/acs.jmedchem.6b00879. [4] X. Zhang, J. Cheng, J. Ma, Z. Hu, S. Wu, N. Hwang, J. Kulp, Y. Du, J.-T. Guo, J. Chang, Discovery of Novel Hepatitis B Virus Nucleocapsid Assembly Inhibitors, ACS Infect. Dis., 5 (2019) 759-768. doi: 10.1021/acsinfecdis.8b00269. [5] J.M. Berke, P. Dehertogh, K. Vergauwen, E. Van Damme, W. Mostmans, K. Vandyck, F. Pauwels, Capsid Assembly Modulators Have a Dual Mechanism of Action in Primary Human Hepatocytes Infected with Hepatitis B Virus, Antimicrob. Agents Chemother., 61 (2017) e0056000517. Doi: 10.1128/aac.00560-17.

[6] M.R. Campagna, F. Liu, R. Mao, C. Mills, D. Cai, F. Guo, X. Zhao, H. Ye, A. Cuconati, H. Guo, J. Chang, X. Xu, T.M. Block, J.-T. Guo, Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids, J. Virol., 87 (2013) 6931-6942. Doi: 10.1128/jvi.00582-13. [7] B. Nijampatnam, D.C. Liotta, Recent advances in the development of HBV capsid assembly modulators, Curr. Opin. Chem. Biol., 50 (2019) 73-79. Doi: 10.1016/j.cbpa.2019.02.009. [8] N. Mani, A.G. Cole, J.R. Phelps, A. Ardzinski, K.D. Cobarrubias, A. Cuconati, B.D. Dorsey, E. Evangelista, K. Fan, F. Guo, H. Guo, J.-T. Guo, T.O. Harasym, S. Kadhim, S.G. Kultgen, A.C.H. Lee, A.H.L. Li, Q. Long, S.A. Majeski, R. Mao, K.D. McClintock, S.P. Reid, R. Rijnbrand, N.M. Snead, H.M. Micolochick Steuer, K. Stever, S. Tang, X. Wang, Q. Zhao, M.J. Sofia, Preclinical Profile of AB-423, an Inhibitor of Hepatitis B Virus Pregenomic RNA Encapsidation, Antimicrob. Agents Chemother., 62 (2018) e00082-00018. Doi: 10.1128/aac.00082-18. [9] A.M. Lam, C. Espiritu, R. Vogel, S. Ren, V. Lau, M. Kelly, S.D. Kuduk, G.D. Hartman, O.A. Flores, K. Klumpp, Preclinical Characterization of NVR 3-778, a First-in-Class Capsid Assembly Modulator against Hepatitis B Virus, Antimicrob. Agents Chemother., 63 (2019) e01734-01718. Doi: 10.1128/aac.01734-18. [10] O. Sari, S. Boucle, B.D. Cox, T. Ozturk, O.O. Russell, L. Bassit, F. Amblard, R.F. Schinazi, Synthesis of sulfamoylbenzamide derivatives as HBV capsid assembly effector, Eur. J. Med. Chem., 138 (2017) 407-421. Doi: 10.1016/j.ejmech.2017.06.062. [11] K. Vandyck, G. Rombouts, B. Stoops, A. Tahri, A. Vos, W. Verschueren, Y. Wu, J. Yang, F. Hou, B. Huang, K. Vergauwen, P. Dehertogh, J.M. Berke, P. Raboisson, Synthesis and Evaluation of N-Phenyl-3-sulfamoyl-benzamide Derivatives as Capsid Assembly Modulators Inhibiting Hepatitis

B

Virus

(HBV),

J.

Med.

Chem.,

61

(2018)

6247-6260.

Doi:

10.1021/acs.jmedchem.8b00654. [12] NCT02112799 (single-center) and NCT02401737 (multicenter), Clinicaltrials.gov. [13] K. Klumpp, T. Shimada, L. Allweiss, T. Volz, M. Lütgehetmann, G. Hartman, O.A. Flores, A.M. Lam, M. Dandri, Efficacy of NVR 3-778, Alone and In Combination With Pegylated Interferon, vs Entecavir In uPA/SCID Mice With Humanized Livers and HBV Infection, Gastroenterology, 154 (2018) 652-662. Doi: 10.1053/j.gastro.2017.10.017. [14] M.F. Yuen, E.J. Gane, D.J. Kim, F. Weilert, H.L. Yuen Chan, J. Lalezari, S.G. Hwang, T. Nguyen, O. Flores, G. Hartman, S. Liaw, O. Lenz, T.N. Kakuda, W. Talloen, C. Schwabe, K. Klumpp, N. Brown, Antiviral Activity, Safety, and Pharmacokinetics of Capsid Assembly Modulator NVR 3-778 in Patients with Chronic HBV Infection, Gastroenterology, 156 (2019) 1392-1403. Doi: 10.1053/j.gastro.2018.12.023.

[15] G. D. Hartman, Combination therapy for treatment of HBV infections, 2017, US20170182021 A1. [16] G. D. Hartman, Hepatitis B antiviral agents, 2016, US20160158214 A1. [17] G. D. Hartman, Crystalline forms of a hepatitis B antiviral agent, 2017, US20170114018A1. [18] K. Vandyck, J. S. Last, P. J. Bernerd, Sulfamoyl-arylmides and the use thereof as medicaments for treatment of hepatitis B, 2019, US20190077749. [19] J.T. Guo, X.D. Xu, T. M. Block, Sulfamolylbenzamide derivatives as antiviral agents against HBV infection, 2013, WO2013006394. [20] Z. Zhou, T. Hu, X. Zhou, S. Wildum, F. Garcia-Alcalde, Z. Xu, D. Wu, Y. Mao, X. Tian, Y. Zhou, F. Shen, Z. Zhang, G. Tang, I. Najera, G. Yang, H.C. Shen, J.A.T. Young, N. Qin, Heteroaryldihydropyrimidine (HAP) and Sulfamoylbenzamide (SBA) Inhibit Hepatitis B Virus Replication by Different Molecular Mechanisms, Sci. Rep., 7 (2017) 42374. Doi: 10.1038/srep42374. [21] S. Wu, Y. Luo, U. Viswanathan, J. Kulp, J. Cheng, Z. Hu, Q. Xu, Y. Zhou, G.-Z. Gong, J. Chang, Y. Li, J.-T. Guo, CpAMs induce assembly of HBV capsids with altered electrophoresis mobility: Implications for mechanism of inhibiting pgRNA packaging, Antiviral Res., 159 (2018) 1-12. Doi: 10.1016/j.antiviral.2018.09.001. [22] S. Wu, Q. Zhao, P. Zhang, J. Kulp, L. Hu, N. Hwang, J. Zhang, T.M. Block, X. Xu, Y. Du, J. Chang, J.-T. Guo, Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly, J. Virol., 91 (2017), e00519-17. Doi: 10.1128/JVI.00519-17. [23] Sarah P. Katen, Z. Tan, Srinivas R. Chirapu, M.G. Finn, A. Zlotnick, Assembly-Directed Antivirals Differentially Bind Quasiequivalent Pockets to Modify Hepatitis B Virus Capsid Tertiary and Quaternary Structure, Structure, 21 (2013) 1406-1416. Doi: 10.1016/j.str.2013.06.013. [24] K. Klumpp, A.M. Lam, C. Lukacs, R. Vogel, S. Ren, C. Espiritu, R. Baydo, K. Atkins, J. Abendroth, G. Liao, A. Efimov, G. Hartman, O.A. Flores, High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein, Proc. Natl. Acad. Sci. USA, 112 (2015) 15196-15201. Doi: 10.1073/pnas.1513803112.

Hight Lights 1. 2. 3. 4. 5.

2d exhibited potent anti-HBV activity as NVR3-778. 2d displayed lower cytotoxicity and higher selectivity index than NVR3-778. 2d showed acceptable PK properties. 2d displayed similar inhibitory effect on the assembly of HBV capsids as NVR3-778. Docking suggested that 2d might form a stronger interaction with core protein.

Graphical Abstract Title: Design, Synthesis and anti-HBV activity of NVR3-778 derivatives Kai Lv a, 1, Shuo Wu a, 1, Wenyan Li b, Yunhe Geng a, b, Meng Wu,a Jinming Zhou,c Yuhuan Li a, *, Qiang Gao a, Mingliang Liu a, * a Institute

of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China b College

of Chemistry & Material Science, Hebei Normal University, Shijiazhuang 050024, China

Key Laboratory of the Ministry of Education for Advanced Catalysis Materials, Department of Chemistry, Zhejiang Normal University, Jinhua, 321004, China c

* Corresponding author. E-mail addresses: [email protected] (M. Liu), [email protected] (Y. Li). 1

These authors contributed equally to this work.

F H N

F

O

F F

F

O S N O

H N

F OH

O

F

NVR3-778 (phase II)

O S N O H

F

Anti-HBV activity IC50: 0.33 µM CC50: 5.14 µM SI: 15.58

OH

2d

Anti-HBV activity IC50: 0.25 µM CC50: 10.68 µM SI:

40.72 T1/2: 8.14 h AUC0-t: 455931 h.ng/mL

T1/2: 7.79 h AUC0-t: 232025

h.ng/mL We report herein the design, synthesis and anti-HBV activity of NVR3-778 derivatives. Results revealed that 2d not only exhibited potent anti-HBV activity (IC50: 0.25 µM), but also low cytotoxicity (CC50: 10.68 µM), good selectivity index (SI: 40.72), and acceptable PK profiles. Further modifications and in vivo antiHBV studies of 2d will begin soon.