Optimization and Synthesis of Pyridazinone Derivatives as Novel

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

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, Jian-Ping Zuo, and Youhong Hu ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.6b00159 • Publication Date (Web): 18 Dec 2016 Downloaded from http://pubs.acs.org on December 19, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Infectious Diseases is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Infectious Diseases

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,*, † Youhong Hu*,§

§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

* To whom all correspondences should be addressed:

Youhong Hu, Ph.D, Fax: +86-2150805896. E-mail: [email protected];

Jianping Zuo, Ph.D, Fax: +86-2150806701. E-mail: [email protected];

Li Yang, Ph.D, Fax: +86-2150806701. E-mail: [email protected];

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: The capsid of Hepatitis B virus (HBV) plays a vital role in the virus DNA replication. Targeting nucleocapsid function has been demonstrated as an effective approach for anti-HBV drugs development. The high-throughput screening and mechanism study revealed the hit compound 4a as an HBV assembly effectors (AEfs), which could inhibit HBV replication by inducing the formation of HBV DNA-free capsids. The subsequent SARs 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-analog resistant HBV mutants and synergistic effect with nucleoside-analogs in HepG2.2.15 cell. KEYWORDS: Pyridazinone, HBV DNA-free capsids, drug-like optimization, SARs study, lead candidate INTRODUCTION

Hepatitis B virus (HBV), the prototype of the Hepadnaviridae family, is a small, circular, enveloped, and partially double-stranded DNA virus harboring only four overlapping reading frames that encode for precore/core, polymerase, envelop and X proteins.1-3 Chronic HBV infection remains a common public health issue worldwidely, which is strongly associated with cirrhosis, liver failure, and hepatocellular carcinoma. 4-10

Indeed, approximately 240 million people around the world are chronically infected

and contributes to 300,000 deaths annually.11 Currently, treatments for HBV included interferon (interferon-α and pegylated


Page 2 of 25

Page 3 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 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 the nucleoside analogues beside tenofovir.19-23 More recently, non-nucleos(t)ide inhibitors are demonstrated equally effective against wild-type and nucleos(t)ide inhibitors-resistant viruses.24-26 Thus, new non-nucleos(t)ide agents targeting different stages in the viral life cycle are expected and drug combinations may contribute to treatment of HBV infection.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 25

Figure 1. Structures of approved HBV inhibitors. 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 last decade, assembly effectors (AEfs), involving heteroaryldihydropyrimidines31-35

and

phenylpropenamides29,

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 icosahedral capsid formation in vitro. Phenylpropenamides, such as AT-130, increase capsid assembly rate with disrupting the capsid tertiary and quaternary structure.29 Importantly, previous research has demonstrated that either of them is active against the main lamivudine- and adefovir-resistant mutants,24

,40

which suggest 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-(4-chlorobenzyl)-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 ( Table S1-S2 in SI). Moreover, the subsequent mechanism exploration indicated that compound 4a is an HBV assembly effector (AEf), which inhibits HBV replication by


Page 5 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.

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

synthetic

route

of

compounds

4a-4z

was

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 at the presence of cesium carbonate in DMF afforded the key intermediates 3. Then, coupling of 3 with the corresponding boronic acids or esters by a Suzuki reaction produced the target compounds 4a-4z. Scheme 1. Synthesis of compounds 4a-4za

a

Reagents and conditions: (a) AcOH, reflux; (b) 1-chloro-4-(chloromethyl)benzene, Cs2CO3, DMF, 50 0C; (c) the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

corresponding boronic acids or esters, Pd(dppf)2Cl2, K3PO4, 1,4-dioxane/H2O=4/1, 90 0C.

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 AT-130, were evaluated in HepG2.2.15 cells. The results are summarized in Tables 1-3. 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 the loss of activity (Table S1-S2). Thus, the 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 were summarized in Table 1. The pyridine analogues 4b and 4c, with IC50 vales of 6.31±0.34 µM and 7.35± 1.28 µM respectively, exhibited comparable anti-HBV activities and cytotoxicity (CC50 >100 µM), indicating that pyridine ring was an ideal moiety to replace the 6-phenyl of pyridazinone. Oppositely, replacement of 6-phenyl with pyrimidyl, thiazolyl or pyrazinyl (4d-4f) led to the loss of antiviral activity. The derivatives with quinoline or 1H-pyrrolo[2,3-b]pyridine moiety (4g-4h) possessed the moderate activities. However, they had pronounced cytotoxicity (CC50 = 47.6±3.2 and 11.1±0.9 µM, respectively),


Page 6 of 25

Page 7 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


resulting in a relatively narrow selectivity index (SI =7.0 and 1.1, respectively). It is noteworthy that Table 1: SAR Study on A ring of pyridazinone core

IC50 (µM)a

CC50 (µM)b

SIc

cLogPd

4a

1.14±0.21

>100

>90.9

3.50

4b

6.31±0.34

>100

>15.6

2.86

4c

7.35±1.28

>100

>13.8

2.86

4d

45.48±7.33

>100

>2.2

1.88

4e

NAe

>100

--

2.88

4f

26.72±2.55

>100

>3.7

2.09

4g

6.85±0.76

47.6±3.2

7.0

4.25

4h

7.11±0.42

11.1±0.9

1.6

3.40

3TC

0.36±0.08

>1000

>2778

--

AT-130

0.91±0.41

>20

>22.0

--

compd

a

A ring

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

concentration of CC50.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 25

When the fluorine atom was introduced to 2 position of pyridine ring (4i), the anti-HBV activity increased dramatically with 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 formed 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 compound was lost when the fluoro group was moved to 4 position (4j). Moreover, two analogues 4k and 4l, with fluorine atom at 5 or 6 positions of pyridine ring, showed good anti-HBV activity with their 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 electron-donating group (4m, 4n) or strong electron-withdrawing group (4q) at 2 position 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 2 position of the pyridine ring is optimal for anti-HBV activity. Table 2: SAR Study of substituents on pyridine moiety.

R

IC50 (µM)a

CC50 (µM)b

SIc

cLogPd

4i

2-F

0.45±0.07

95.4±5.31

212

3.05

4j

4-F

NAe

10.4±0.29

--

3.05

compd


Page 9 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4k

5-F

4l

6-F

4m

2-NH2

1.80±1.56

>100

>55.5

3.05

>100

>52.6

3.05

NAe

>100

--

2.55

1.90

±0.57

4n

2-OMe

NAe

46.7±15.1

--

3.08

4o

2-Me

4.88±0.39

57.3±26.3

11.7

3.06

4p

2-CN

4.64±0.65

>100

>21.6

2.66

4q

2-CF3

NAe

>100

--

3.86

3TC

0.36±0.08

>1000

>2778

--

AT-130

0.91±0.41

>20

>22.0

--

a

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

at concentration of CC50.

The further optimization and SAR exploration based on compound 4i were carried out to explore the effects of substituents at 6 position of the 2,6-disubstituted derivatives, which retained the F at 2 position. Encouragingly, all the 2,6-disubstituted derivatives exhibited significant potency against HBV DNA replication, except for the analogues 4y and 4z, with bulky group at 6 position, which decreased or lost the activities. 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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 25

introduction of a methoxyl group, resulted in a remarkable decreasing 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. Table 3: SAR Study of 2,6-disubstituted derivatives

compd

R

IC50 (µM)a

CC50 (µM)b

SIc

cLogPd

4r

F

0.087±0.002

90.6±2.06

1041

3.21

4s

OMe

1.22±0.03

70.7±14.4

58.0

3.82

4t

NH2

0.30±0.14

44.1±13.7

147

2.77

4u

Me

0.54±0.33

84.5±7.97

92.0

3.55

4v

0.22±0.01

28.4±2.66

129

3.58

4w

0.27±0.02

6.67±0.05

24.7

2.93

4x

0.94±0.05

20.9±4.81

22.2

3.76

4y

2.10±0.13

21.9±3.08

10.4

4.82

4z

NAe

2.96±0.22

--

3.04

3TC

0.36±0.08

>1000

>2778

--

AT-130

0.91±0.41

>20

>22.0

--


Page 11 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a

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

at concentration of CC50.

The anti-HBV activity and cytotoxicity of 4r were furtherly examined by using various HepG2 hepatoma-derived cell lines that stably replicate HBV: HepG2.2.15 (ayw serotype, GenBank accession number: U95551), HepG2.117 (ayw serotype, GenBank accession number: U95551), and HepGTo2.45 (adw serotype, GenBank accession number: X02763.1). The result showed that 4r also inhibit HBV DNA synthesis effectively in other HBV-producing cell lines, with IC50 values similar to those observed in HepG2.2.15 cells (Table 4). Table 4: Anti-HBV activity and cytotoxicity of 4r, AT-130 and 3TCa Cells 4r AT-130 3TC a

HepG2.2.15(ayw) IC50 (µM) CC50(µM) 0.087±0.002 90.6±2.06 0.91±0.41 >20 0.36±0.08 >1000

HepG2.117(ayw) HepGTo2.45(adw2) IC50(µM) CC50(µM) IC50(µM) CC50(µM) 0.082±0.011 >100 0.034±0.005 >100 0.63±0.02 >20 0.015±0.003 >20 0.029±0.011 >1000 0.20±0.03 >1000

HepG2.2.15 and HepG2.117 cells carry genotype D HBV (ayw serotype, GenBank accession number: U95551), and

HepGTo2.45 cells contain HBV genotype A2 genome (adw2 subtype; GenBank accession number: X02763.1).

Subsequently, the study on the mechanism revealed that compound 4r remained displaying the same mode of action as compound 4a, which could induce the formation of genome-free capsids (Figure 3A, 1st 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, 2nd



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

panel). Moreover, the active pyridazinones to accumulate DNA-free capsids were correlated with their antiviral activities. Compound 4a, 4i, 4r, which exhibited potent anti-HBV activities, could interfere with nucleocapsid assembly correspondingly. Oppositely, compound 4n with no HBV activity had no effect on capsid assembly (Figure 3B). A:

B:

Figure 3: (A) 4r induced the accumulation of HBV DNA-free capsids and inhibited


Page 12 of 25

Page 13 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


intracellular HBV DNA replication intermediates. (B) The assembly effects of a set of pyridazinones. The emergency 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 strain, such as 3TC/ETV-dual-resistant mutant (rtL180M/M204V). 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-analog resistant HBV mutant in a dose-dependent manner, which confirmed the possible utility in patients who have developed drug resistant strains (Figure 4).

Figure 4: 4r dose-dependently inhibited the replication of 3TC/ETV-resistant HBV mutant. Finally, we evaluated the multidrug treatment of compound 4r with nucleoside analogs entecavir (ETV). 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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 25

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, the compound 4r exhibited synergistic effect (CI

Optimization and Synthesis of Pyridazinone Derivatives as Novel

Recommend Documents