Optimization and Synthesis of Pyridazinone Derivatives as Novel

Article pubs.acs.org/journal/aidcbc

Optimization and Synthesis of Pyridazinone Derivatives as Novel Inhibitors of Hepatitis B Virus by Inducing Genome-free Capsid Formation Dong Lu,∥,†,# Feifei Liu,∥,‡,# Weiqiang Xing,† Xiankun Tong,‡ Lang Wang,† Yajuan Wang,‡,# Limin Zeng,† Chunlan Feng,‡ Li Yang,*,‡ Jianping Zuo,*,‡ and Youhong Hu*,†

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†

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 ZuChongZhi Road, Shanghai 201203, China ‡ Laboratory of Immunopharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 ZuChongZhi Road, Shanghai 201203, China # University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China S Supporting Information *

ABSTRACT: The capsid of hepatitis B virus (HBV) plays a vital role in virus DNA replication. Targeting nucleocapsid function has been demonstrated as an effective approach for anti-HBV drug development. A high-throughput screening and mechanism study revealed the hit compound 4a as an HBV assembly effector (AEf), which could inhibit HBV replication by inducing the formation of HBV DNA-free capsids. The subsequent SAR study and drug-like optimization resulted in the discovery of the lead candidate 4r, with potent antiviral activity (IC50 = 0.087 ± 0.002 μM), low cytotoxicity (CC50 = 90.6 ± 2.06 μM), sensitivity to nucleoside analogue-resistant HBV mutants, and synergistic effect with nucleoside analogues in HepG2.2.15 cells. KEYWORDS: pyridazinone, HBV DNA-free capsids, drug-like optimization, SAR study, lead candidate

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INTRODUCTION

Hepatitis B virus (HBV), the prototype of the Hepadnaviridae family, is a small, circular, enveloped, and partially doublestranded DNA virus harboring only four overlapping reading frames that encode for precore/core, polymerase, envelope, and

Figure 2. Structures of Bay41-4109, AT-130, and compound 4a as assembly effectors (AEfs).

X proteins.1−3 Chronic HBV infection remains a common public health issue worldwide and is strongly associated with cirrhosis, liver failure, and hepatocellular carcinoma.4−10 Indeed, approximately 240 million people around the world are chronically infected, contributing to 300,000 deaths annually.11 Currently, treatments for HBV include interferon (interferonα and pegylated interferon-α) and nucleoside analogues (i.e., lamivudine, telbivudine, adefovir, tenofovir, entecavir, and clevudine (Figure 1)).2 Interferon-α enhances the innate immune response by triggering a complex intracellular cascade Received: September 8, 2016 Published: December 18, 2016

Figure 1. Structures of approved HBV inhibitors. © 2016 American Chemical Society

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DOI: 10.1021/acsinfecdis.6b00159 ACS Infect. Dis. 2017, 3, 199−205

ACS Infectious Diseases

Article

Scheme 1. Synthesis of Compounds 4a−4za

a Reagents and conditions: (a) AcOH, reflux; (b) 1-chloro-4-(chloromethyl)benzene, Cs2CO3, DMF, 50 °C; (c) corresponding boronic acids or esters, Pd(dppf)2Cl2, K3PO4, 1,4-dioxane/H2O = 4:1, 90 °C.

Table 1. SAR Study on the A-Ring of the Pyridazinone Core

a

IC50 is 50% inhibitory concentration of cytoplasmic HBV-DNA replication. bCC50 is 50% cytotoxicity concentration in HepG2 2.2.15 cells. Selectivity index (SI = CC50/IC50). dcLogP is the calculated logarithm of the partition coefficient in octanol/water using the ChemBioDraw Ultra 12.0 program. eNA, not active at concentration of CC50. c

different stages in the viral life cycle are expected, and drug combinations may contribute to treatment of HBV infection. During the life cycle of HBV, the capsid plays vital roles in pregenomic RNA (pgRNA) packaging and reverse transcription progress.8,27−29 The process of nucleocapsid formation is exquisitely timed and regulated, and any disruption could have significant effects on virus replication.30 In the past decade, assembly effectors (AEfs), involving heteroaryldihydropyrimidines31−35 and phenylpropenamides,29,36−39 targeting the encapsidation step have been developed (Figure 2). Heteroaryldihydropyrimidines, including Bay41-4109, prevent the proper formation of viral core particles and inhibit normal

from the JAK-STAT pathway, which restricts viral dissemination.7,12−14 Due to the low response rate (20−30%), the need for subcutaneous injections, and the high frequency of adverse effects, the application of interferon is limited.15−18 Nucleoside analogues (Figure 1) are orally effective and well tolerated through directly inhibiting HBV by targeting viral DNA polymerase. However, emergence of drug resistance and relapse are frequent after long-term therapy with all of the nucleoside analogues besides tenofovir.19−23 More recently, non-nucleos(t)ide inhibitors have been demonstrated equally effective against wild-type and nucleos(t)ide inhibitor-resistant viruses.24−26 Thus, new non-nucleos(t)ide agents targeting 200



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Initially, the screening for the in-house libraries result showed that 4-chlorobenzyl attached to the N atom of pyridazinone core and 4-methyl group of pyridazinone are crucial for anti-HBV activity; any modification on these two groups led to a loss of activity (Tables S1 and S2). Thus, further structural optimization should be achieved on the 6-phenyl (A-ring) of pyridazinone. Considering the low hydrophilicity of compound 4a, the further modification on the 6-phenyl (A-ring) of pyridazinone was achieved by introducing various hydrophilic heterocycles to increase the solubility and investigate the SARs. The anti-HBV activity and cytotoxicity of analogues 4b−4h are summarized in Table 1. The pyridine analogues 4b and 4c, with IC50 vales of 6.31 ± 0.34 and 7.35 ± 1.28 μM, respectively, exhibited comparable anti-HBV activities and cytotoxicity (CC50 > 100 μM), indicating that the pyridine ring was an ideal moiety to replace the 6-phenyl of pyridazinone. In contrast, replacement of 6-phenyl with pyrimidyl, thiazolyl, or pyrazinyl (4d−4f) led to a loss of antiviral activity. The derivatives with quinoline or 1Hpyrrolo[2,3-b]pyridine moiety (4g, 4h) possessed moderate activities. However, they had pronounced cytotoxicity (CC50 = 47.6 ± 3.2 and 11.1 ± 0.9 μM, respectively), resulting in a relatively narrow selectivity index (SI = 7.0 and 1.1, respectively). When the fluorine atom was introduced to the 2-position of the pyridine ring (4i), the anti-HBV activity increased dramatically with an IC50 value of 0.45 ± 0.07 μM against HBV DNA replication. The computerized docking result (Figure S1) showed that the F atom of 4i could form two hydrogen bonds with Trp102 and Ser106 residues of capsid protein, which may contribute to the improvement of the activity. However, the anti-HBV activity of the compound was lost when the fluoro group was moved to the 4-position (4j). Moreover, two analogues 4k and 4l, with a fluorine atom at the 5- or 6-position of pyridine ring, showed good anti-HBV activity with IC50 values of 1.80 ± 1.56 and 1.90 ± 0.57 μM, respectively, which is slightly more potent than compound 4b with no substituent on the pyridine ring. Incorporation of an electron-donating group (4m, 4n) or a strong electron-withdrawing group (4q) at the 2position of pyridine led to the loss of anti-HBV activity. In addition, compound 4o, with a methyl group, or compound 4p, with a cyano group, also exhibited moderate anti-HBV activity. The above results indicated that the substituent with F at the 2position of the pyridine ring is optimal for anti-HBV activity. Further optimization and SAR exploration based on compound 4i were carried out to explore the effects of substituents at the 6-position of the 2,6-disubstituted derivatives, which retained the F at the 2-position. Encouragingly, all of the 2,6-disubstituted derivatives exhibited significant potency against HBV DNA replication, except for analogues 4y and 4z, with bulky groups at the 6-position, which decreased or eliminated the activities. The 2,6-difluoro analogue (4r), combined with the substitution of 4i and 4l, showed the most potent anti-HBV activity, with an IC50 value of 0.087 ± 0.002 μM against HBV DNA replication and excellent SI data = 1041 (CC50 = 90.6 ± 2.06 μM). However, compound 4s, with the introduction of a methoxyl group, resulted in a remarkable decrease in anti-HBV potency. Although the analogues 4t−4w showed comparable anti-HBV activities, the cytotoxicity also increased dramatically compared to 4r. In addition, when the amino group of compound 4t was disubstituted, the activity of compound 4x was also decreased. The anti-HBV activity and cytotoxicity of 4r were further examined by using various HepG2 hepatoma-derived cell lines that stably replicate HBV: HepG2.2.15 (ayw serotype, GenBank

icosahedral capsid formation in vitro. Phenylpropenamides, such as AT-130, increase capsid assembly rate with disruption of the capsid tertiary and quaternary structures.29 Importantly, previous research has demonstrated that either of these is active against the main lamivudine- and adefovir-resistant mutants,24,40 which suggests that targeting nucleocapsid functions may represent an efficient approach to the development of novel HBV inhibitors to prevent and combat drug resistance. In the high-throughput screening of our in-house collection of compounds for anti-HBV activity, compound N-(4-(1-(4chlorobenzyl)-5-methyl-6-oxo-1,6-dihydropyridazin-3-yl)phenyl)acetamide (4a, Figure 2) was identified as a modestly potent HBV inhibitor with the primary SARs (Tables S1 and S2 in the Supporting Information). Moreover, the subsequent mechanism exploration indicated that compound 4a is an HBV AEf, which inhibits HBV replication by inducing genome-free capsid formation.41 In consideration of its novel structural scaffold, differing from those of all reported HBV inhibitors,42−50 here we studied further the structure−activity relationships (SARs) and drug-like optimization of the related class of compounds to find the lead candidate.

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CHEMISTRY The synthetic route of compounds 4a−4z is depicted in Scheme 1. 6-Chloro-4-methylpyridazin-3(2H)-one (2) was synthesized by the reported procedure.51 N-benzylation of pyridazinone 2 with the 1-chloro-4-(chloromethyl)benzene in the presence of cesium carbonate in DMF afforded the key intermediate 3. Then, coupling of 3 with the corresponding boronic acids or esters by a Suzuki reaction produced the target compounds 4a−4z.

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RESULTS AND DISCUSSION As described previously,52 the potential anti-HBV activity and cytotoxicity of the synthesized pyridazinone analogues, together with the reference anti-HBV agent lamivudine (3TC) and AT130, were evaluated in HepG2.2.15 cells. The results are summarized in Tables 1−3. Table 2. SAR Study of Substituents on the Pyridine Moiety

compd

R

IC50 (μM)a

CC50 (μM)b

SIc

cLogPd

4i 4j 4k 4l 4m 4n 4o 4p 4q 3TC AT-130

2-F 4-F 5-F 6-F 2-NH2 2-OMe 2-Me 2-CN 2-CF3

0.45 ± 0.07 NAe 1.80 ± 1.56 1.90 ± 0.57 NA NA 4.88 ± 0.39 4.64 ± 0.65 NA 0.36 ± 0.08 0.91 ± 0.41

95.4 ± 5.31 10.4 ± 0.29 >100 >100 >100 46.7 ± 15.1 57.3 ± 26.3 >100 >100 >1000 >20

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3.05 3.05 3.05 3.05 2.55 3.08 3.06 2.66 3.86

>55.5 >52.6

11.7 >21.6 >2778 >22.0

a

IC50 is 50% inhibitory concentration of cytoplasmic HBV-DNA replication. bCC50 is 50% cytotoxicity concentration in HepG2 2.2.15 cells. cSelectivity index (SI = CC50/IC50). dcLogP is the calculated logarithm of the partition coefficient in octanol/water using the ChemBioDraw Ultra 12.0 program. eNA, not active at concentration of CC50. 201



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Table 3. SAR Study of 2,6-Disubstituted Derivatives

a

IC50 is 50% inhibitory concentration of cytoplasmic HBV-DNA replication. bCC50 is 50% cytotoxicity concentration in HepG2 2.2.15 cells. Selectivity index (SI = CC50/IC50). dcLogP is the calculated logarithm of the partition coefficient in octanol/water using the ChemBioDraw Ultra 12.0 program. eNA, not active at concentration of CC50. c

Table 4. Anti-HBV Activity and Cytotoxicity of 4r, AT-130, and 3TCa HepG2.2.15(ayw) 4r AT-130 3TC

HepG2.117(ayw)

HepGTo2.45(adw2)

IC50 (μM)

CC50 (μM)

IC50 (μM)

CC50 (μM)

IC50 (μM)

CC50 (μM)

0.087 ± 0.002 0.91 ± 0.41 0.36 ± 0.08

90.6 ± 2.06 >20 >1000

0.082 ± 0.011 0.63 ± 0.02 0.029 ± 0.011

>100 >20 >1000

0.034 ± 0.005 0.015 ± 0.003 0.20 ± 0.03

>100 >20 >1000

a

HepG2.2.15 and HepG2.117 cells carry genotype D HBV (ayw serotype, GenBank accession no. U95551), and HepGTo2.45 cells contain HBV genotype A2 genome (adw2 subtype; GenBank accession no. X02763.1).

accession no. U95551), HepG2.117 (ayw serotype, GenBank accession no. U95551), and HepGTo2.45 (adw serotype, GenBank accession no. X02763.1). The result showed that 4r also inhibited HBV DNA synthesis effectively in other HBVproducing cell lines, with IC50 values similar to those observed in HepG2.2.15 cells (Table 4). Subsequently, the study on the mechanism revealed that compound 4r displayed the same mode of action as compound 4a, which could induce the formation of genome-free capsids (Figure 3A, first panel). Southern blotting analysis of 4r

demonstrated that the compound could inhibit the intracellular HBV DNA replication intermediates (RC and SS HBV DNA) in a dose-dependent manner (Figure 3A, second panel). Moreover, the active pyridazinones accumulating DNA-free capsids were correlated with their antiviral activities. Compounds 4a, 4i, and 4r, which exhibited potent anti-HBV activities, could interfere with nucleocapsid assembly correspondingly. In contrast, compound 4n with no HBV activity had no effect on capsid assembly (Figure 3B). 202



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Table 5. Dose−Effect Relationship Parameters and Mean Combination Index (CI) Values of 4r and Entecavir (ETV) two-chemical combination (nM)

CI50 valuea

4r (D)1

ETV (D)2

2CI50 = [(D)1/D50)1] + [(D)2/D50)2]

0.24 0.98 3.91 15.6 62.5

10.4 8.68 3.02 2.56 2.52

0.98 0.83 0.33 0.42 0.96

a

CI50 is the combination index for two chemicals (4r and ETV) at 50% inhibition; D is the dose of chemical, D50 is the effective dose of a chemical that caused a 50% inhibition of HBV replication ((D50)1 = 86.9 nM and (D50)2 = 10.6 nM).

Figure 3. (A) 4r induced the accumulation of HBV DNA-free capsids and inhibited intracellular HBV DNA replication intermediates. (B) Assembly effects of a set of pyridazinones.

The emergence of drug-resistant strains is the main problem associated with the current small-molecule antiviral HBV medications. It is thus extremely necessary that new anti-HBV inhibitors should be evaluated for activity against drug-resistant HBV mutations, as this would be a desirable feature. Compound 4r was tested against the major class of drug-resistant HBV strains, such as 3TC/ETV-dual-resistant mutant (rtL180M/ M204 V). The result showed that the 3TC/ETV-resistant HBV mutants showed resistance to 3TC and ETV but remained sensitive to 4r treatment. Compound 4r could inhibit the various forms (RC and SS HBV DNA) of nucleoside analogue-resistant HBV mutant in a dose-dependent manner, which confirmed the possible utility in patients who have developed drug-resistant strains (Figure 4). Finally, we evaluated the multidrug treatment of compound 4r with nucleoside analogue entecavir (ETV). HepG2.2.15 cells

Figure 5. 4r showed obvious synergistic effect (CI < 1) by cotreatment with entecavir (ETV) in HepG2.2.15 cells.

were cultured in 96-well plates and treated with 4r and/or ETV at various concentrations for 8 days. HBV replication intermediates were detected by qPCR. The results were analyzed using the median effect/combination index (CI) isobologram equation.53 For each effect level, combination index (CI) values were then calculated according to the general combination index equation for n chemical combination at x% inhibition, where n(CI)x is the combination index for n chemicals at x% inhibition.54 As shown in Table 5 and Figure 5, compound 4r exhibited a synergistic

Figure 4. 4r dose-dependently inhibited the replication of 3TC/ETV-resistant HBV mutant. 203



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(dppf)2Cl2, 1,1′-bis(diphenylphosphino)ferrocene−palladium(II) dichloride dichloromethane complex; K3PO4, potassium phosphate

effect (CI < 1) by cotreatment with entecavir (ETV) in HepG2.2.15 cells.

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CONCLUSIONS In conclusion, considering the low hydrophilicity of compound 4a, the further drug-like modification on the 6-phenyl (A-ring) of pyridazinone was achieved by introducing various hydrophilic heterocycles to increase the solubility and investigate the SARs. A series of novel pyridazinone derivatives based on hit 4a were synthesized and evaluated for their in vitro anti-HBV DNA replication and cytotoxicity. Most of the compounds exhibited moderate to good anti-HBV activity. The most promising compound, 4r, displayed potent anti-HBV activity, low cytotoxicity, sensitivity to nucleoside analogue-resistant HBV mutants, and synergistic effect with nucleoside analogues. Moreover, the subsequent mechanistic study presented that compound 4r displayed the same mode of action as compound 4a, which could induce the formation of genome-free capsids. Overall, the potent activity, the ease of synthesis, and the unique mode of action for 4r make it an attractive lead candidate worthy of further investigation in the treatment of HBV infection.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.6b00159. Initial SAR study of pyridazinone derivatives; docking study of 4i with capsid protein; synthetic procedures and characterization data of target compounds; biological assay methods (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*(L.Y.) Fax: +86-2150806701. E-mail: [email protected]. *(J.Z.) Fax: +86-2150806701. E-mail: [email protected]. *(Y.H.) Fax: +86-2150805896. E-mail: [email protected]. ORCID

Youhong Hu: 0000-0003-1770-6272 Author Contributions ∥

D.L. and F.L. contributed equally and are considered co-first authors of this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a grant from the National Science Fund (81225022, 81322049, and 31570166), the National Program on Key Basic Research Project (973 Program) (2013CB911104), and Institutes for Drug Discovery and Development, Chinese Academy of Sciences (No. CASIMM0120162013). We thank Dr. Michael Nassal (Internal Medicine II/Molecular Biology, University Hospital Freiburg, Germany) and Prof. Dr. Mengji Lu (Institute of Virology, University Hospital Essen, University Duisburg Essen, Essen, Germany) for providing HepG2.117 cell line.

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ABBREVIATIONS HBV, hepatitis B virus; 3TC, Lamivudine; ETV, entecavir; NA, not active; SI, selectivity index; RC, relaxed circular HBV DNA; SS, single-stranded HBV DNA; CI, combination index; DMF, N,N-dimethylformamide; Cs2CO3, cesium carbonate; Pd204



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