Design, diversity-oriented synthesis and biological evaluation of novel heterocycle derivatives as non-nucleoside HBV capsid protein inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112495

The capsid assembly is a significant phase for the hepatitis B virus (HBV) lifespan and is an essential target for anti-HBV drug discovery and development. Herein, we used scaffold hopping, bioisosterism, and pharmacophore hybrid-based strategies to design and synthesize six series of various heterocycle derivatives (pyrazole, thiazole, pyrazine, pyrimidine, and pyridine) and screened for in vitro anti-HBV non-nucleoside activity. Drug candidate NZ-4 and AT-130 were used as lead compounds. Several compounds exhibited prominent anti-HBV activity compared to lead compound NZ-4 and positive drug Lamivudine, especially compound II-8b, showed the most prominent anti-HBV DNA replication activity (IC₅₀ = 2.2 ± 1.1 μM). Also compounds IV-8e and VII-5b showed the best in vitro anti-HBsAg secretion (IC₅₀ = 3.8 ± 0.7 μM, CC₅₀ > 100 μM) and anti-HBeAg secretion (IC₅₀ = 9.7 ± 2.8 μM, CC₅₀ > 100 μM) respectively. Besides, II-8b can interact HBV capsid protein with good affinity constants (K_D = 60.0 μM), which is equivalent to lead compound NZ-4 ((K_D = 50.6 μM). The preliminary structure-activity relationships (SARs) of the newly synthesized compounds were summarized, which may help researchers to discover more potent anti-HBV agents.

Full text of DOI:10.1016/j.ejmech.2020.112495

Journal Pre-proof
Design, Diversity-Oriented Synthesis and Biological Evaluation of Novel Heterocycle
Derivatives as Non-nucleoside HBV Capsid Protein Inhibitors
Haiyong Jia, Ji Yu, Xianhong Du, Srinivasulu Cherukupalli, Peng Zhan, Xinyong Liu
PII:
S0223-5234(20)30467-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.112495
EJMECH 112495
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 March 2020
Revised Date: 16 May 2020
Accepted Date: 18 May 2020
Please cite this article as: H. Jia, J. Yu, X. Du, S. Cherukupalli, P. Zhan, X. Liu, Design, Diversity-
Oriented Synthesis and Biological Evaluation of Novel Heterocycle Derivatives as Non-nucleoside
HBV Capsid Protein Inhibitors, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112495.
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Graphical abstract
Five skeletons of heterocycle derivatives (pyrazole, thiazole, pyrazine,
pyrimidine and pyridine analogs) were designed as potential HBV non-nucleoside
inhibitors. And seven synthetic routes were employed to acquire target compounds by
diversity-oriented synthesis.

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Design, Diversity-Oriented Synthesis and Biological Evaluation
of Novel Heterocycle Derivatives as Non-nucleoside HBV Capsid
Protein Inhibitors
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Haiyong Jia1,2, Ji Yu1, Xianhong Du2,3, Srinivasulu Cherukupalli1, Peng Zhan1,*
and Xinyong Liu1,*
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1 Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry
of Education), School of Pharmaceutical Sciences, Shandong University, 44 West
Culture Road, 250012, Jinan, Shandong, PR China
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2 School of Pharmacy, Weifang Medical University, 261053 Weifang, Shandong, PR
China.
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3Department of Immunology, Key Laboratory for Experimental, Teratology of
Ministry of Education, Shandong Provincial Key Laboratory of Infection and
Immunology, Shandong University School of Medicine, Jinan 250012, Shandong
Province, China.
*Corresponding author. Tel.: +86 531 88380270; fax: +86 531 88382731.
E-mail address: zhanpeng1982@sdu.edu.cn, xinyongl@sdu.edu.cn
Abstract:
The capsid assembly is a significant phase for the hepatitis B virus (HBV)
lifespan and is an essential target for anti-HBV drug discovery and development.
Herein, we used scaffold hopping, bioisosterism, and pharmacophore hybrid-based
strategies to design and synthesize six series of various heterocycle derivatives
(pyrazole, thiazole, pyrazine, pyrimidine, and pyridine) and screened for in vitro
anti-HBV non-nucleoside activity. Drug candidate NZ-4 and AT-130 were used as
lead compounds. Several compounds exhibited prominent anti-HBV activity
compared to lead compound NZ-4 and positive drug Lamivudine, especially
compound II-8b, showed the most prominent anti-HBV DNA replication activity

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(IC50 = 2.2 ± 1.1 µM). Also compounds IV-8e and VII-5b showed the best in vitro
anti-HBsAg secretion (IC50 = 3.8±0.7 µM, CC50 > 100 µM) and anti-HBeAg secretion
(IC50 = 9.7±2.8 µM, CC50 > 100 µM) respectively. Besides, II-8b can interact HBV
capsid protein with good affinity constants (KD = 60.0 μM), which is equivalent to
lead compound NZ-4 ((KD = 50.6 μM). The preliminary structure-activity relationships
(SARs) of the newly synthesized compounds were summarized, which may help
researchers to discover more potent anti-HBV agents.
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Keywords: HBV, Capsid protein, Diversity-oriented synthesis, Heterocycle
derivatives.
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11 1. Introduction
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Hepatitis B is a serious infectious disease caused by the hepatitis B virus (HBV).
Long-term development of hepatitis B frequently causes acute or chronic viral
hepatitis, severe hepatitis, liver cirrhosis, and hepatocellular carcinoma. The recent
World Health Organization (WHO) statistics indicated nearly 2 billion people
worldwide had been infected with HBV, of which about 350 million people with
chronic HBV infection. An average of about 60 million people die every year due to
acute or chronic viral hepatitis and related concurrency disease. China is a high
endemic area of hepatitis B with about 93 million hepatitis B virus (HBV) carriers;an
average of about 300,000 patients die each year due to infection with the hepatitis B
virus[1, 2]. Due to the high incidence, long course and difficulty in curing, hepatitis B
has become a major disease that seriously affects people's health and social
development. Therefore research on effective drugs of HBV has become the top
priority[3].
HBV is a member of hepadnaviridae, consisting partially double-stranded
circular genome. HBV replication process possesses adsorption and fusion to
hepatocyte, DNA repair and transcription of HBV covalently closed circular DNA
(cccDNA), translation and reverse transcription of progenome RNA (PgRNA). It also
possesses capsid assembly and DNA replication, viral particle recirculation and

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release, etc. [4]. Few drugs have been developed for the treatment of chronic hepatitis
B (CHB) by an in-depth understanding of the HBV life cycle and molecular biology.
At present, only interferon (interferon-α and pegylated interferon-α) and six
nucleoside or nucleotide drugs such as Lamivudine (3TC), Adefovir dipivoxil (ADV),
Entecavir (ETV), Telbivudine (LdT), Tenofovirdisoproxil fumarate (TDF) and
Tenofovir alafenamide (TAF) have been approved by the U.S. Food and Drug
Administration (FDA) for HBV treatment. Though they profoundly suppress HBV
replication, the disadvantage of high price, low cure rate, serious side effects,
resistance and high recurrence is undoubtedly essential to combat the HBV
infection[5, 6].
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The capsid is a T=4 icosahedral complex assembled from 120 copies of core
protein homodimer, which is the most populous species found in HBV virions. It
consists of the N-terminal assembly domain (Cp149, residues 1-149) and the
C-terminal binding domain (protamine domain, residues 150-183) [7]. As capsids
provide the structural background for encasement of the RNA pregenome, assembly
of a viral core protein and RNA pregenome reverse transcription, interfering assembly
of capsid formation is assumed to be less prone to developing drug resistance [8]. The
HBV capsid protein and its assembly process have no human analogs, making HBV
assembly has become an attractive target for new antiviral therapies [1, 9-11].
NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA
and HBcAg in the capsid assembly process, thus increasing the replication-deficient
HBV capsids. NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells
with an IC50 value of 1.33 μmol/L, whereas the compound inhibited the cell viability
with a CC50 value of 50.4 μmol/L [12]. AT-130, a phenylpropanamide derivative, has
more potent anti-HBV DNA replication activity with an IC50 value of 0.13 μmol/L
and CC50 value of more than 51 μmol/L[6, 13-15]. NZ-4 and AT-130 also have four
various fragments within the structure (yellow, aqua, pink and spring green), as
illustrated in Figure 1. Herein, six small series of heterocycle derivatives (pyrazole,

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thiazole, pyrazine, pyrimidine, and pyridine analogs) were designed as potential HBV
non-nucleoside inhibitors through scaffold hopping, bioisosterism, and
pharmacophore hybrid-based strategies.
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Figure 1. The structural modifications of six small series of heterocycle derivatives as potent
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non-nucleoside HBV inhibitors.
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2. Results and discussion
2.1 Chemistry
The synthetic route for the target compounds I-4a(1-5), I-4b, I-6a, and I-6b(1-2)

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is illustrated in Scheme 1. The key intermediates I-2a and I-2b were prepared from
the commercially available starting material ethyl(E)-2-cyano-3-ethoxyacrylate (I-1)
by condensation reaction with aromatic hydrazine at 120 [16]. Further, the
intermediate I-3 was achieved from I-2a by allowing diazotization reaction.
Intermediates I-2a, I-2b and I-3 were converted to target compounds I-4a(1-5) and
I-4b by treating with various substituted alcohol, amines, and halo hydrocarbon
through nucleophilic substitution reaction. Further, I-4a and I-4b were hydrolyzed to
obtain intermediates I-5a and I-5b respectively by hydrolysis reaction in the solution
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of sodium hydroxide, water, ethanol and tetrahydrofuran under 50
. The
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intermediates I-5a and I-5b were allowed to react with corresponding substituted
amines via an amide condensation reaction to achieve the final target compounds I-6a
and I-6b(1-2)
[17]. Both analytical and spectral data of all the synthesized compounds are in
full agreement with the proposed structures.
F
R1
N
O
NH2
N
iii
ii
i
O
O
N
N
N
COOEt
CN
I-1
I-2a: R1 = 3-fluorophenyl
I-2b: R1 = thiazol-2-yl
I-3
R1
COOEt
NH
iv
R1
N
R1
R2
N
vi
N
v
N
N
NH
X
COOH
R3
COOEt
I-6a: R1 = 3-fluorophenyl
I-6b(1-2): R1 = thiazol-2-yl
I-5a: R1 = 3-fluorophenyl
I-5b: R1 = thiazol-2-yl
I-4a(1-5): R1 = 3-fluorophenyl; X: NH, S
I-4b: R1 = thiazol-2-yl; X: NH
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Scheme 1. Reagents and conditions: (i) CH3COONa, CH3COOH, H2O, reflux; (ii) CuBr2,
CH3CN, 50 ; (iii) DMF, NaH, R2-X; (iv) DMF, NaH, R2OH/ R2NH2; (v) NaOH, C2H5OH,
H2O, THF, 50 ; (vi) DCM, EDC, HOBt.
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The synthetic route for the target compounds II-8(a-c) was conducted as
illustrated in Scheme 2. The initial intermediate II-2 was achieved by allowing the

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2
substitution reaction between commercially available diethyl carbonate (1) and
4-methylpentan-2-one.
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4
The key intermediate II-5 was prepared from intermediate II-2 through oxime
synthesis, reduction and amide condensation reaction, which was further converted to
intermediate II-6 by condensation cyclization reaction with Lawesson's reagent at
120 . Then, II-6 was hydrolyzed to obtain intermediate II-7 by hydrolysis reaction in
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the solution of sodium hydroxide, water, ethanol and tetrahydrofuran under 50
.
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Treatment of intermediate II-7 with corresponding substituted amines via amide
condensation reaction gave the target compounds II-8(a-c) [18].
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Scheme 2. Reagents and conditions: (i) 4-methylpentan-2-one, NaH, AcOH, THF; (ii)
NaNO2, CH3COOH, H2O, -5 ℃; (iii) Pd/C, HCl/EtOH, RT; (iv) HATU, DMF, DIPEA, RT;
(v) Lawesson's reagent, toluene, reflux; (vi) NaOH, THF, EtOH, H2O, RT; (vii) HATU, DMF,
DIPEA, RT.
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The synthetic route of the target compounds III-7(a-c) is demonstrated in
Scheme 3. Intially, compound III-3 was achieved by substitution reaction between
intermediate (II-2) and N-bromo succinimide.
The key intermediate ethyl
2-amino-4-isobutylthiazole-5-carboxylate (III-4) was obtained by treating ethyl
2-bromo-5-methyl-3-oxohexanoate (III-3) with thiourea by condensation reaction at

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3
4
5
80
.
Then, intermediate III-4 was hydrolyzed to intermediate
2-amino-4-isobutylthiazole-5-carboxylic acid (III-5) by hydrolysis reaction as
followed in Scheme 2. Treatment of intermediate III-5 with 4-fluorbenzylamine by
the condensation reaction gave the intermediate III-6, which was further modified to
obtain target compounds III-7(a-c)[19].
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Scheme 3. Reagents and conditions: (i) DCM, NBS, RT; (ii) EtOH, thiourea, 80 ℃; (iii)
NaOH, THF, EtOH, H2O, 50 ℃; (iv) DMF, HATU, DIPEA, RT; (v) a: THF, DMAP, 80 ℃;
b: HATU, DIPEA, RT.
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The synthetic route of the target compounds IV-8(a-e) is displayed in Scheme 4.
The key intermediate ethyl 2-bromo-4-isobutylthiazole-5-carboxylate (IV-5) was
prepared from ethyl 2-amino-4-isobutylthiazole-5-carboxylate (III-4) through diazo
substitution reaction. Then, intermediate IV-5 was hydrolyzed to obtain
2-bromo-4-isobutylthiazole-5-carboxylic acid (IV-6) via hydrolysis reaction in the
solution of sodium hydroxide, water, ethanol and tetrahydrofuran at 50
℃. Treatment
of intermediate IV-6 with 4-fluorbenzylamine by the condensation reaction gave the
intermediate IV-7, which was further modified by Suzuki reaction to obtain the target
compounds IV-8(a-e).

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Scheme 4 Reagents and conditions: (i) CH3CN, CuBr, tert-Butyl nitrite, RT; (ii) NaOH,
THF, EtOH, H2O, 50 ℃; (iii) DMF, HATU, DIPEA, RT; (iv) Pd(PPh3)4, K2CO3,
1,4-Dioxane, H2O, 100 ℃.
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The synthetic route of the target compound V-5 is illustrated in Scheme 5.
Initially, compound V-2 was achieved by allowing the reaction between acetophenone
(V-1) and 1,1-dimethoxy-N,N-dimethylmethanamine 4-methylpentan-2-one. The key
intermediate ethyl-2,6-diphenylnicotinate (V-3) was achieved by condensation
reaction of (E)-3-(dimethylamino)-1-phenylprop-2-en-1-one (V-2) withethyl
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benzoylacetate at 120
2,6-diphenylnicotinic acid (V-4) by hydrolysis reaction in the solution of sodium
hydroxide, water, ethanol and tetrahydrofuran under 80 . Treatment of intermediate
V-4 with different substituted amines leads to the target compound V-5[20].
℃. Then, intermediate V-3 was hydrolyzed to obtain
℃
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Scheme
1,1-dimethoxy-N,N-dimethylmethanamine, 120
120 ; (iii) NaOH, THF, EtOH, H2O, 80 ; (iv) DMF, HATU, triethylamine, RT.
The synthetic route of the target compound VI-3 is displayed in Scheme 6. The
5.
Reagents
and
conditions:
(i)
℃; (ii) CH3COOH, CH3COONH4,
℃
℃
target compound VI-3 was achieved from the commercially available methyl
3-amino-6-bromopyrazine-2-carboxylate and 2-methoxybenzeneboronic acid after
Suzuki and acylation reactions[21, 22].

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Scheme 6. Reagents and conditions: (i) Pd(dppf)Cl2, Cs2CO3, 1,4-Dioxane, H2O,
; (ii) DMF, triethylamine, 100
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4
100
℃
℃.
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The synthetic route of the target compounds VII-4(a-b) and VII-6(a-b) is
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presented
in
Scheme
7.
The
key
intermediate
(VII-3)
ethyl
was
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(E)-2-((dimethylamino)methylene)-5-methyl-3-oxohexanoate
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prepared from ethyl 5-methyl-3-oxohexanoate (II-2) by condensation reaction
with 1,1-dimethoxy-N,N-dimethylmethanamine at 100 . And the target
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compounds VII-4(a-b) were achieved from compound VII-3 by allowing
condensation reaction with benzamidine hydrochloride or 3-fluoro-benzamidine
hydrochloride. Then, VII-4(a-b) were hydrolyzed to obtain the desired
intermediates VII-5a and VII-5b correspondingly by hydrolysis reaction in the
solution of sodium hydroxide, water, ethanol, and tetrahydrofuran under 50
.
Treatment of intermediates VII-5a or VII-5b with different substituted amines
leads to the target compounds VII-6a and VII-6b, respectively[23].
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Scheme 7. Reagents and conditions: (i)1,1-dimethoxy-N,N-dimethylmethanamine,
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100 ℃; (ii) C2H5ONa, C2H5OH, 80 ℃; (iii) NaOH, THF, EtOH, H2O, 80 ℃; (iv) DMF,
HATU, triethylamine, RT.
22 2.2. Biological activity and the SAR studies

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All the synthesized compounds were preliminary evaluated for (in vitro) their
anti-HBV DNA replication activity and cytotoxicity in HepG2.2.15 cells (human
HBV transgenic hepatocellular carcinoma cells) using standard PCR and cell counting
kit-8(CCK-8) methods. Then the advanced target compounds were further evaluated
for their cytotoxicity, inhibitory effect on HBV DNA replication and HBsAg, and
HBeAg secretion in HepG2.2.15 cells via CCK-8, PCR and standard ELISA methods,
respectively. The concentration of compound required for 50% inhibition of HBeAg,
HBsAg secretion or DNA replication was defined as IC50 and the concentration of
compound that induced the death of the HepG2.2.15 cell cultures by 50% was defined
as CC50. Selectivity index (SI) was determined as the CC50/IC50 value. Lamivudine
and lead compound NZ-4 was used as positive standards.
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Primary anti-HBV DNA replication activity, cytotoxicity in HepG2.2.15 cell for
series I-II target compounds and lead compound (NZ-4) and positive drug
Lamivudine under 100 μM concentration were shown in table 1. Most of these
pyrazole analogs exhibited low toxicity as compared to positive drug Lamivudine
except compound I-4a5. It was observed that the electron-withdrawing group of R2
might improve the toxicity of the inhibitors. However, most of these pyrazole analogs
also exhibited low or none anti-HBV DNA replication activity. To our encouragement,
compounds with amide substitution in pyrazole moiety (I-6a, I-6b1and I-6b2)
exhibited better anti-HBV DNA replication activity than ester substitution. Besides, to
our delight, series II target compounds with thiazole moiety exhibited advanced
anti-HBV activity. Among thiazole series, compounds II-8a and II-8b showed potent
anti-HBV DNA replication (83.8±3.5% and 89.8±3.3%, respectively) with 24.9±2.3%
and none cytotoxicity under 100 μM, respectively. To the best, target compounds II
-8a and II-8b have similar anti-HBV activity with positive drug Lamivudine
(87.9±2.4 %) and they are better than the lead compound (NZ-4, II-8c), and II-8c
possessed high cytotoxicity (98.8±0.2 %), which can be further evaluated.

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Table 1 Primary anti-HBV DNA replication activity and cytotoxicity in HepG2.2.15
cell of series I-II target compounds, NZ-4, and Lamivudine under 100 μM
concentration.
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Inhibition percentages (%)
Compounds
X
R2/R3/R4
DNA replication
HepG2.2.15 Cell
I-4a1
I-4a2
NH
NH
NH
S
benzyl
ethyl
14.6
39.9±8.7
NI
NIa
10.9±9.7
5.7±1.7
18.1±7.5
51.3±9.2
30.4±2.2
40.8±4.6
16.9±2.9
29.2±6.1
24.9±2.3
NI
I-4a3
methyl
I-4a4
cyclohexane
NI
I-4a5
NH
NH
4-nitrobenzoyl
benzyl
49.5±2.0
NI
I-4b
I-6a
cyclopropyl
38.8±7.1
32.9±22.9
41.6±1.3
83.8±3.5
89.8±3.3
97.3±0.2
87.9±2.4
I-6b1
2,4-difluorobenzyl
2-chloro-4-fluorobenzyl
2,4-difluorobenzyl
NH-(4-fluorophenyl)
4-fluorobenzyl
I-6b2
II -8a
II-8b
II-8c(NZ-4)
Lamivudine
98.8±0.2
12.7 ±3.7
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a NI: None inhibition under 100 μM
Primary anti-HBV DNA replication activity and cytotoxicity in HepG2.2.15 cell
for series III-IV target compounds under 20 μM concentration were shown in table 2.

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To our surprise, most of these thiazole compounds showed low toxicity and anti-HBV
DNA replication activity. Unfortunately, a significant reduction in anti-HBV DNA
replication activity was observed by small changes in thiazole moiety and fragments
of thiazole. However, we found that compound IV-8e exhibited moderate anti-HBV
DNA replication activity with 57.8±13.2 %, which can be further evaluated.
Table 2 Primary anti-HBV DNA replication activity and cytotoxicity in HepG2.2.15
cell of series III-IV target compounds under 20 μM concentration.
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Inhibition percentages (%)
Compouds
R1/R2
DNA replication
HepG2.2.15 Cell
15.2
III-7a
III-7b
III-7c
IV-8a
IV-8b
IV-8c
IV-8d
IV-8e
2-thienylcarbonyl
4-fluorobenzenesulfonyl
4-fluorobenzoyl
2-thienyl
NIa
NI
17.5±20.2
20.7±18.6
17.8±8.6
NI
29.2±5.0
NI
5-methylthiophen-2-yl
3-thienyl
NI
NI
6.7±4.6
NI
benzo[b]thiophen-2-yl
4-pyridinyl
21.1±14.2
57.8±13.2
35.8±5.9
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13
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a NI: None inhibition under 20 μM
Primary anti-HBV DNA replication activity and cytotoxicity in HepG2.2.15 cell
for series V-VII target compounds under 50 μM concentration were shown in table 3.
From the results, it was noticed that pyridine compound V-5 and pyrazine derivative
VI-3 showed low toxicity and none anti-HBV DNA replication activity. However,

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most pyrimidine derivatives exhibited moderate anti-HBV DNA replication activity
with none cytotoxicity in HepG2.2.15 cell. Among them, compound VII-5b presented
the best anti-HBV DNA replication activity with 54.2±12.8 %. The preliminary
structure-activity relationship (SAR) of pyrimidine derivatives was investigated and
revealed that 3-fluorinated phenyl substitution is superior to the benzene group in the
position of R1 (VII-4b > VII-4a, VII-6b > VII-6a). The obtained anti-HBV DNA
replication activity results showed for 5 position derivatives showed as sequence:
carboxyl group > ester group > amide group (VII-5b > VII-4b > VII-6b).
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Table 3 Primary anti-HBV DNA replication activity and cytotoxicity in HepG2.2.15
cell of Series V-VII target compounds under 50 μM concentration.
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Inhibition percentages (%)
Compounds
R1/R2 /R3/R4
DNA replication
NIa
HepG2.2.15 Cell
V-5
4-fluorobenzyl
benzoyl
4.4±0.3
5.7±7.8
NI
VI-3
NI
VII-4a
VII-4b
VII-5b
VII-6a
VII-6b
phenyl
37.3±0.9
46.3±4.1
54.2±12.8
NI
3-fluorophenyl
NI
NI
benzyl
NI
4-fluorobenzyl
35.6
NI
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13
14
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a NI: None inhibition under 50 μM
The activity of compounds II-8(a-c), IV-8e, VII-5b, and Lamivudine were
further evaluated in HepG2.2.15 cells in a dose-dependent manner (100 µM, 20 µM, 4
µM, 0.8 µM, 0.16 µM) to analyze their IC50 and CC50 values. Among them,

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3
4
5
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7
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compound II-8b showed the best in vitro anti-HBV DNA replication activity (IC50 =
2.2 ± 1.1 µM, CC50 = 80.8±14.4 µM), which was better than that of lead compound
II-8c (IC50 = 2.3 ± 0.5 µM, CC50 = 59.2 ±2.4 µM). Compound IV-8e and VII-5b showed
the best in vitro activity for anti-HBsAg secretion (IC50 = 3.8±0.7 µM, CC50 > 100 µM)
and anti-HBeAg secretion (IC50 = 9.7±2.8 µM, CC50 > 100 µM), respectively, which
were better than lead compound and positive drug Lamivudine.
Table 4. Further evaluation for cytotoxicity, inhibitory effect on HBV DNA
replication and HBsAg/HBeAg secretion of compounds II-8(a-c), IV-8e, VII-5b and
Lamivudine.
10
DNA
HBsAg
HBeAg
a
CC50
b
b
b
R
Compounds
IC50
IC50
IC50
(µmol/L)
SIc
SIc
SIc
(µmol/L)
(µmol/L)
(µmol/L)
30.1±8.3
>100
II-8a
2,4-difluorobenzyl
45.4±2.2 2.7±3.0
80.8±14.4 2.2±1.1
16.8 21.0±1.7
2.2
1.5
II-8b
IV-8e
VII-5b
II-8c d
NH-(4-fluorophenyl)
36.7
>2.5
>100
< 0.8
< 0.8
>100
>100
40.5±34.6
9.9±3.7
3.8±0.7 >26.3 62.3±32.5 >1.6
>10.1
>100
—
1.9
—
9.7±2.8
31.7±4.1
>100
>10.3
1.9
4-fluorobenzyl
59.2 ±2.4 2.3±0.5
>100 0.6±0.4
25.7 31.7±7.8
>167 >100
Lamivudine
—
11
12
13
14
15
16
aCC50: Concentration required to reduce the viability of mock-infected cells by 50%.
bIC50: Concentration of compound required for 50% inhibition of HBV DNA replication or
HBsAg /HBeAg secretion.
cSI: Selectivity index, the ratio of CC50/IC50.
d IV-8c：The lead compound NZ-4.

1
2
2.3 Molecular modeling
II-8b, the best compound in cell-based HBV infection assay, was docked into the
HBV capsid (PDB ID: 5gmz) using Sybyl-X 2.0 to study its binding mode. Default
parameters were used as described in the Sybyl-X 2.0 manual unless otherwise
specified, the result was displayed by PyMOL (Figure 2). It was shown that the
conformation of II-8b (green) can be well overlapped with the ligand (NVR10-001E2,
yellow) in the core protein crystal structure. Thiazole ring region of II-8b occupied a
hydrophobic pocket, surrounded by Pro-130 and Pro-129. And 2-fluorine phenyl
region occupied another hydrophobic pocket, surrounded by Pro25, Asp29 and Leu30,
while F atoms can form a hydrogen bond with Thr-33. Besides, the amide of II-8b
form a hydrogen bond with Thr128, which might contribute to the anti-HBV activity.
3
4
5
6
7
8
9
10
11
12
13
Figure 2. The molecular modeling of target compound II-8b with core protein crystal
structure (PDB：5gmz)
14
15 2.4 Surface plasmon resonance (SPR)
16
17
18
19
20
21
To acquire better understand of the hydrogen bond interactions, we evaluated
compound II-8b the binding affinity with HBV capsid protein by surface plasmon
resonance (Biacore T200). The study was shown that compound II-8b can steady-state
bind to HBcAg protein in a dose-dependent manner (22.2 µM, 14.8 µM, 14.8 µM, 9.9
µM, 6.6 µM and 0 µM) with response value (RU) of 26.38, 21.33, 20.60, 18.83, 15.36
and 11.31, respectively. While the lead compound NZ-4 bound to HBcAg protein in a

1
2
3
dose-dependent manner (100 µM, 50 µM, 25 µM, 25 µM, 12.5 µM, 6.25 µM and 0
µM) with response value (RU) of 63.55, 41.35, 38.93, 38.35, 23.85, 17.72 and 14.56),
respectively. Therefore, fitted by 1:1 binding model the affinity constant KD value of
4
5
compound II-8b and NZ-4 was 60.0 µM and 50.6 µM, respectively.
3. Conclusion
6
7
In conclusion, six small series of heterocycle derivatives (pyrazole, thiazole,
pyrazine, pyrimidine and pyridine analogs) were designed as potential HBV
non-nucleoside inhibitors through scaffold hopping, bioisosterism and pharmacophore
hybrid-based strategies. All the designed compounds were evaluated for their
anti-HBV activity. Among them, compound II-8b displayed the most potent
anti-HBV DNA replication activity (IC50 = 2.2 ± 1.1 µM). And compound IV-8e and
VII-5b showed the best in vitro activity for anti-HBsAg secretion (IC50 = 3.8±0.7 µM,
CC50 > 100 µM) and anti-HBeAg secretion (IC50 = 9.7±2.8 µM, CC50 > 100 µM),
respectively. The surface plasmon resonance study revealed that the best anti-HBV
DNA replication compound II-8b can interact HBV capsid protein with good affinity
constants (KD = 60.0 μM), which was equivalent with lead compound NZ-4 ((KD =
50.6 μM). The preliminary structure-activity relationships (SARs) of the new
compounds were summarized, which may help in discovering more potent anti-HBV
agents.
8
9
10
11
12
13
14
15
16
17
18
19
20 4. Experimental section
21
4.1. Chemistry
22
23
24
25
26
27
28
All melting points were determined on a micro melting point apparatus. 1H NMR
spectra were obtained on a Brucker Avance-400/300 NMR spectrometer in the
indicated solvents. Chemical shifts are expressed in δ units and TMS as internal
reference. Mass spectra were taken on a LC Autosampler Device: Standard G1313A
instrument. TLC was performed on Silica Gel GF254 for TLC (Merck) and spots
were visualized by irradiation with UV light (254 nm). Flash column chromatography
was performed on column packed with Silica Gel 60 (200–300 mesh). Solvents were

1
2
3
4
5
6
reagent grade and, when necessary, were purified and dried by standard methods.
Concentration of the reaction solutions involved the use of rotary evaporator at
reduced pressure. All solvents of 1,4-dioxane, tetrahydrofuran, DCM, CH3COOH,
DMF and ethanol were obtained from Sinopharm Chemical Reagent Co.,Ltd(SCRC),
which were of AR grade. Chemicals and reagents of N-bromosuccinimide,
Pd(dppf)Cl2, Cs2CO3, Pd/C, HOBt, HATU, DIPEA, EDC etc. were obtained from
7
8
beijing innochem science & technology Co.,Ltd, which were of CP grade.
4.1.1. General procedure for preparation of compounds I-2a and I-2b
9
10
11
12
13
14
15
16
17
To the mixture solution of water (1 mL) and sodium acetate (286 mg, 3.49 mmol)
in acetic acid (3 mL) was added 3-fluorophenyl hydrazine hydrochloride (1.23 mmol)
or thiazol-2-yl hydrazine hydrochloride (1.23 mmol). The reaction mixture was stirred
at reflux temperature for 4 hours. Upon completion of the reaction, the solvent was
cooled to room temperature and 20 mL cooled water was added and was extracted
with ethyl acetate (15 mL × 3) and washed with saturated sodium chloride (30 mL).
The organic layer was dried over anhydrous sodium sulfate, and then the solvent was
removed under vacuum. The residue was chromatographed on silica gel using ethyl
acetate and petroleum ether. Pure fractions were collected and concentrated, giving
18
19
the desired compounds I-2a and I-2b in good yield.
Ethyl 3-amino-1-(3-fluorophenyl)-1H-pyrazole-4-carboxylate (I-2a). Pale yellow
1
20
21
22
23
24
25
solid, yield 91%, m.p.130-131 ; H NMR(400 MHz, CDCl3): δ 7.79 (s, 1H),
7.51-7.45 (m, 1H), 7.37-7.30 (m, 2H ), 7.13-7.08 (m, 1H ), 5.36 (s, 1H), 4.31 (q, 2H,
J=7.1 Hz), 1.37 (t, 3H, J=7.1 Hz ); 13C NMR (100 MHz, CDCl3): δ 164.39, 163.20 (d,
J = 255 Hz), 149.15, 140.97, 139.10 (d, J = 9 Hz), 131.05(d, J = 9 Hz), 118.87(d, J =
3 Hz), 115.0(d, J = 21 Hz), 111.29(d, J = 24 Hz), 96.57, 59.78, 14.49; ESI-MS: 250.4
[M+H]+.
26
27
28
29
Ethyl 3-amino-1-(thiazol-2-yl)-1H-pyrazole-4-carboxylate (I-2b). white solid,
1
yield 35%, m.p.99-101 ; H NMR (400 MHz, CDCl3): δ 7.75 (s, 1H), 7.53 (d, 1H,
J=3.6 Hz), 7.14 (s, 2H), 7.05 (d, 2H, J=3.6 Hz), 4.30 (q, 2H, J=7.2 Hz), 1.36 (t, 3H,
13
J=7.2 Hz); C NMR (100 MHz, CDCl3): δ 164.00, 162.38, 150.38, 142.70, 139.70,

1
2
114.54, 95.46, 77.35, 77.24, 77.04, 76.72, 59.82, 14.52; ESI-MS: 239.0 [M+H]+.
4.1.2. General procedure for preparation of compound Ethyl
3-bromo-1-(3-fluorophenyl)-1H-pyrazole-4-carboxylate (I-3)
3
4
To the mixture of Ethyl 3-amino-1-(3-fluorophenyl)-1H-pyrazole-4-carboxylate
(I-2a) (200 mg, 0.8 mmol) in acetonitrile (5 mL) was added cuprous bromide. Then
the reaction mixture was cooled to 0 and the butyl nitrite (223 mg, 2.17 mmol) was
5
6
7
added, which was stirred at 50 ℃for overnight. Upon completion of the reaction, the
8
solvent was cooled to room temperature and 50 mL water was added. It was extracted
with ethyl acetate (25 mL × 3) and washed with saturated sodium chloride (50 mL).
The organic layer was dried over anhydrous sodium sulfate, and then the solvent was
removed under vacuum. The residue was chromatographed on silica gel using ethyl
acetate and petroleum ether. Pure fractions were collected and concentrated, giving
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1
the desired compound I-3 as a yellow solid 195 mg, yield: 72%. m.p.76-79 ℃; H
NMR(400 MHz, CDCl3): δ 8.14 (s, 1H), 7.52-7.4 (m, 1H), 7.36 (dd, 1H, J1=2.0 Hz,
J2=7.6 Hz), 7.32-7.28 (m, 1H ), 7.23-7.18 (m, 1H ), 4.37 (q, 2H, J=7.2 Hz), 1.37 (t,
3H, J=7.2 Hz ) , 1.40 (t, 3H, J=7.2 Hz ); 13C NMR (100 MHz, CDCl3): δ 162.46 (d, J
= 248.7 Hz), 161.62, 143.43, 139.55 (d, J = 10.1 Hz), 130.35 (d, J = 8.9 Hz), 121.83
(d, J = 3.4 Hz), 117.78, 116.39 (d, J = 21.0 Hz), 115.55, 113.86 (d, J = 24.8 Hz),
60.71, 14.33, ESI-MS: 313.3 [M+H]+.
4.1.3. General procedure for preparation of compounds I-4a (1-3) and I-4b
To the solution of intermediate I-2a or I-2b (1.12 mmol) in DMF (5 mL) was
added potassium carbonate, cesium carbonate or sodium hydride. The mixture
solution was stirred for 5 min, added different substituent haloalkane (1.35 mmol) and
then was further stirred under room temperature for overnight. Upon completion of
the reaction, the solvent was added 50 mL water, extracted with ethyl acetate (25 mL
× 3) and washed with saturated sodium chloride (50 mL × 3). The organic layer was
dried over anhydrous sodium sulfate, and then the solvent was removed under
vacuum. The residue was chromatographed on silica gel using ethyl acetate and

1
2
petroleum ether. Pure fractions were collected and concentrated, giving the desired
compounds I-4a (1-3) and I-4b.
3
General
procedure
for
preparation
of
compound
Ethyl
4
3-(benzylamino)-1-(3-fluorophenyl)-1H-pyrazole-4-carboxylate(I-4a1). Light yellow
1
5
oil, yield:73%; H NMR(400 MHz, CDCl3): δ 7.81 (s, 1H), 7.43-7.4536 (m, 2H),
6
7.32-7.27 (m, 1H), 7.25-7.22 (m, 3H), 7.12-7.07 (m, 1H) 7.04 (dd, 2H, J1=2.0 Hz,
J2=7.6 Hz) , 6.52 (s, 1H), 4.29 (q, 2H, J=7.2 Hz), 4.03 (s, 1H), 1.34 (t, 3H, J=7.2 Hz );
13C NMR (100 MHz, CDCl3): 164.72, 162.76 (d, J = 248.3 Hz), 151.71, 141.21,
140.82 (d, J = 10.0 Hz), 137.81, 130.38 (d, J = 9.0 Hz), 128.66, 127.66, 127.19,
120.36 (d, J = 3.3 Hz), 115.18 (d, J = 21.1 Hz), 112.39 (d, J = 24.4 Hz), 99.11, 59.84,
49.79, 14.45; ESI-MS: 340.5 [M+H]+.
7
8
9
10
11
12
General
procedure
for
preparation
of
compound
Ethyl
13
14
15
16
17
18
19
20
21
22
23
24
25
26
3-(ethylamino)-1-(3-fluorophenyl)-1H-pyrazole-4-carboxylate(I-4a2). Colorless oil,
yield: 72%; 1H NMR (400 MHz, CDCl3): δ 7.81 (s, 1H), 7.46-7.39 (m, 2H), 7.38-7.34
(m, 1H ), 7.11-7.06 (m, 1H ), 6.00 (s, 1H), 4.30 (q, 2H, J=7.2 Hz), 2.87 (q, 2H, J=7.2
Hz), 1.37 (t, 3H, J=7.1 Hz ) , 1.08 (t, 3H, J=7.1 Hz ); 13C NMR (100 MHz, CDCl3): δ
164.87, 162.76 (d, J = 248.0 Hz), 152.33, 141.21, 141.06 (d, J = 10.0 Hz), 130.33 (d,
J = 9.0 Hz), 120.06 (d, J = 3.3 Hz), 114.94 (d, J = 21.0 Hz), 112.04 (d, J = 24.5 Hz),
98.58, 59.76, 40.92, 15.55, 14.50; ESI-MS: 278.2 [M+H]+.
General
procedure
for
preparation
of
compound
Ethyl
1-(3-fluorophenyl)-3-(methylamino)-1H-pyrazole-4-carboxylate(I-4a3). Colorless oil,
yield: 38%; 1H NMR (400 MHz, CDCl3): δ 7.80 (s, 1H), 7.47-7.32 (m, 3H), 7.12-7.07
(m, 1H ), 4.30 (q, 2H, J=7.2 Hz), 2.95 (s, 3H), 1.36 (t, 3H, J=7.2 Hz ); 13C NMR (100
MHz, CDCl3): δ 164.88, 163.96, 153.23, 141.10, 140.85, 131(d, J = 9.0 Hz), 120.51(d,
J = 3.0 Hz), 115.11(d, J = 21 Hz), 112.47(d, J = 24 Hz), 97.75, 59.78, 32.74, 14.49;
ESI-MS: 264.3 [M+H]+.
27
28
29
30
31
General
procedure
for
preparation
of
compound
Ethyl
3-(benzylamino)-1-(thiazol-2-yl)-1H-pyrazole-4-carboxylate(I-4b). White solid, yield:
35%, m.p.90-92 ; 1H NMR (400 MHz, CDCl3): δ 8.77 (s, 1H), 7.84 (s, 1H), 7.46 (d,
1H, J=3.6 Hz ), 7.35-7.24 (m, 5H), 7.03 (d, 1H, J=3.6 Hz ), 5.08 (d, 2H, J=6.0 Hz ),
4.25 (q, 2H, J=7.2 Hz), 1.32 (t, 3H, J=7.2 Hz ); 13C NMR (100 MHz, CDCl3): δ

1
2
163.10, 162.71, 150.23, 145.53, 139.32, 139.10, 128.63, 127.43, 127.33, 114.68,
97.00, 59.96, 49.40, 14.46; ESI-MS: 657.2 [2M+H]+.
3
General
procedure
for
preparation
of
compound
Ethyl
4
3-(cyclohexylthio)-1-(3-fluorophenyl)-1H-pyrazole-4-carboxylate(I-4a7). To the
mixture solution of intermediate I-3 (200 mg, 0.64 mmol) in DMF (5 mL) was added
with 60% NaH (31 mg, 0.77 mmol). The mixture solution was stirred for 5 min,
added cyclohexane thiol and then was further stirred under room temperature for
overnight. Upon completion of the reaction, to the solvent was added 50 mL water,
extracted with ethyl acetate (25 mL × 3) and washed with saturated sodium chloride
(50 mL × 3). The organic layer was dried over anhydrous sodium sulfate, and then the
solvent was removed under vacuum. The residue was chromatographed on silica gel
using ethyl acetate and petroleum ether. Pure fractions were collected and
concentrated, giving the desired compound I-4a7. Colorless oil, yield: 38%; 1H NMR
(400 MHz, CDCl3): δ 8.14 (s, 1H), 7.49-7.43 (m, 1H), 7.33-7.27 (m, 2H ), 7.19-7.15
(m, 1H ), 4.37 (q, 2H, J=7.2 Hz), 4.13-4.03 (m, 2H ), 3.30-3.28 (m, 1H ), 1.73-1.50
(m, 5H ), 1.40 (t, 3H, J=7.2 Hz ) , 1.21-1.15 (m, 5H ); 13C NMR (100 MHz, CDCl3): δ
162.49, 162.30 (d, J = 247.8 Hz), 143.09, 140.33 (d, J = 10.0 Hz), 138.71, 129.90 (d,
J = 8.9 Hz), 122.34 (d, J = 3.3 Hz), 118.63, 115.74 (d, J = 21.0 Hz), 114.18 (d, J =
24.6 Hz), 67.11, 60.45, 48.95, 32.99, 25.59, 25.44, 14.39; ESI-MS: 349.5 [M+H]+.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
General
procedure
for
preparation
of
compound
21
22
23
24
25
26
27
28
29
30
1-(3-fluorophenyl)-3-(4-nitrobenzamido)-1H-pyrazole-4-carboxylate (I-4a8). To the
mixture of intermediate I-2a (100 mg, 0.4 mmol) in DMF (5 mL) was
added triethylamine (122 mg, 1.2 mmol). The mixture solution was cooled to 0
slowly added 4-nitrobenzoyl chloride and then was further stirred at 60
,
for
overnight. Upon completion of the reaction, to the solvent was added 30 mL water,
extracted with ethyl acetate (15 mL × 3) and washed with saturated sodium chloride
(20 mL). The organic layer was dried over anhydrous sodium sulfate, and then the
solvent was removed under vacuum. The residue was chromatographed on silica gel
using ethyl acetate and petroleum ether. Pure fractions were collected and
concentrated, giving the desired compound I-4a8.White solid, yield: 74%,

1
2
3
4
5
6
m.p.151-153 ; 1H NMR (400 MHz, CDCl3): δ 9.59 (s, 1H), 8.32 (d, 2H, J=8.4 Hz),
8.04 (d, 3H, J=6.8 Hz), 7.42-7.27(m, 3H), 7.07(t, 1H, J=8.0 Hz), 7.35 (q, 2H, J=7.2
13
Hz), 1.38 (t, 3H, J=7.2 Hz); C NMR (100 MHz, CDCl3): δ 164.03, 163.02, 162.75
(d, J = 248.0 Hz), 150.40, 141.24 (d, J = 10.2 Hz), 140.66, 139.71, 137.77, 130.47 (d,
J = 9.0 Hz), 128.90, 124.15, 118.51 (d, J = 3.2 Hz), 115.33 (d, J = 21.0 Hz), 110.75 (d,
J = 25.2 Hz), 105.62, 60.96, 14.33; ESI-MS: 399.3 [M+H]+.
7
8
4.1.4. General procedure for preparation of compounds I-5a and I-5b
To the mixture of intermediate I-4a (0.74 mmol) or I-4b (0.74 mmol) in ethanol
(2 mL), water (2 mL) and THF (2 mL) was added along with NaOH (3.7 mmol). The
mixture solution was stirred at 50 for overnight. Upon completion of the reaction,
the solvent ethanol and THF were removed under vacuum. Then the mixture was
added to 30 mL ammonium chloride solution, extracted with ethyl acetate (15 mL ×
3) and washed with saturated sodium chloride (20 mL). The organic layer was dried
over anhydrous sodium sulfate, and then the solvent was removed under vacuum. The
residue was recrystallized to obtain the desired compounds I-5a and I-5b.
9
10
11
12
13
14
15
16
17
General
procedure
for
preparation
of
compound
3-(benzylamino)-1-(3-fluorophenyl)-1H-pyrazole-4-carboxylic acid (I-5a). White
1
18
19
20
21
22
23
solid, yield: 44%; H NMR (400 MHz, CDCl3): δ 7.88 (s, 1H), 7.42 (td, J = 8.1, 6.1
Hz, 1H), 7.37 – 7.31 (m, 1H), 7.30 – 7.21 (m, 5H), 7.12 (tdd, J = 8.3, 2.5, 0.9 Hz, 1H),
7.02 (dd, J = 7.6, 1.8 Hz, 2H), 6.57 (s, 1H), 4.07 (s, 2H); 13C NMR (100 MHz,
CDCl3): δ 169.57, 162.72 (d, J = 248.8 Hz), 152.18, 141.89, 140.54 (d, J = 10.0 Hz),
137.58, 130.42 (d, J = 9.0 Hz), 128.74, 127.77, 127.04, 120.74 (d, J = 3.3 Hz), 115.55
(d, J = 21.0 Hz), 112.78 (d, J = 24.3 Hz), 97.91, 49.50; ESI-MS: 312.4 [M+H]+.
24
25
26
27
28
29
30
General
procedure
for
preparation
of
compound
3-(benzylamino)-1-(thiazol-2-yl)-1H-pyrazole-4-carboxylic acid (I-5b). White solid,
1
yield: 87%; H NMR (400 MHz, DMSO): δ 12.19 (s, 1H), 8.67 (t, J = 6.5 Hz, 1H),
7.84 (s, 1H), 7.67 (d, J = 3.6 Hz, 1H), 7.54 (d, J = 3.6 Hz, 1H), 7.45 – 7.11 (m, 6H),
5.05 (d, J = 6.5 Hz, 2H); 13C NMR (100 MHz, DMSO): δ 163.86, 162.81, 150.02,
146.01, 139.99, 139.87, 129.02, 127.66, 127.63, 117.11, 97.64, 48.39; ESI-MS: 299.5
[M-H]-.

1
2
4.1.5. General procedure for preparation of compounds I-6a and I-6b(1-2)
To the mixture of intermediate I-5a (0.19 mmol) or I-5b (0.19 mmol) in DCM (5
mL) was slowly added EDCl (43 mg, 0.25 mmol) and HOBt (39 mg, 0.25 mmol)
under low temperature. The mixture solution was stirred for 10 min, added different
substituted amine and then was further stirred at room temperature for overnight.
Upon completion of the reaction, the mixture was added to 30 mL water, extracted
with ethyl acetate (15 mL × 3) and washed with saturated sodium chloride (20 mL).
The organic layer was dried over anhydrous sodium sulfate, and then the solvent was
removed under vacuum. The residue was chromatographed on silica gel using ethyl
acetate and petroleum ether. Pure fractions were collected and concentrated, giving
the desired compounds I-6a and I-6b(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
General
procedure
for
preparation
of
compound
3-(benzylamino)-N-cyclopropyl-1-(3-fluorophenyl)-1H-pyrazole-4-carboxamide
(I-6a). White solid, yield: 32%, m.p.87-89 ; 1H NMR (400 MHz, CDCl3): δ 7.89 (s,
1H), 7.45-7.40 (m, 1H), 7.35-7.33 (m, 1H ), 7.33-7.24 (m, 4H ), 7.14-7.09 (m, 1H ),
7.02 (dd, 2H, J1=2.0 Hz, J2=7.6 Hz) , 6.57 (s, 1H), 4.07 (s, 1H); 13C NMR (100 MHz,
CDCl3): δ 162.76 (d, J = 248.2 Hz), 151.35, 140.87 (d, J = 10.0 Hz), 137.97, 137.59,
130.35 (d, J = 9.0 Hz), 129.17, 128.53, 127.46, 127.26, 120.22 (d, J = 3.3 Hz), 115.09
(d, J = 21.1 Hz), 112.25 (d, J = 24.5 Hz), 100.82, 49.66, 29.70, 6.82; ESI-MS: 312.4
[M+H]+.
General
procedure
for
preparation
of
compound
3-(benzylamino)-N-(2,4-difluorobenzyl)-1-(thiazol-2-yl)-1H-pyrazole-4-carboxamide
(I-6b1). White solid, yield: 70%; 1H NMR (400 MHz, CDCl3): δ 8.37 (s, 1H), 7.61 (s,
1H), 7.49 (d, 1H, J=3.6 Hz ), 7.34-7.23 (m, 6H), 7.05 (d, 1H, J=3.2 Hz), 6.84-6.77 (m,
2H), 6.14 (s, 1H), 4.79 (s, 2H), 4.52 (d, 2H, J=6.4 Hz ); 13C NMR (100 MHz, CDCl3):
δ 162.83, 162.38 (d, J = 236.7 Hz), 162.28, 148.76, 142.24, 139.47, 138.72, 131.25 (d,
J = 6.0 Hz), 131.17 (d, J = 9.6 Hz), 128.56, 127.37, 127.31, 121.62, 114.90, 111.37
(dd, J = 21.1, 3.7 Hz), 103.87 (t, J = 25.4 Hz), 101.05, 49.36, 37.04; ESI-MS: 426.2

1
2
[M+H]+.
General
procedure
for
preparation
of
compound
3
3-(benzylamino)-N-(2-chloro-4-fluorobenzyl)-1-(thiazol-2-yl)-1H-pyrazole-4-carboxa
1
4
mide (I-6b2). White solid, yield: 45%, m.p.154-156 ; H NMR (400 MHz, CDCl3):
5
δ 8.37 (s, 1H), 7.63 (s, 1H), 7.48 (d, 1H, J=3.6 Hz ), 7.36 (dd, 1H, J1=6.0 Hz, J2=8.4
Hz ), 7.21-7.20 (m, 5H), 7.11 (dd, 1H, J1=2.4 Hz, J2=8.4 Hz ), 7.04 (d, 1H, J=3.6 Hz ),
6.95 (dt, 1H, J1=3.6 Hz, J2=8.4 Hz ), 6.23 (t, 1H, J=5.6 Hz ), 4.78 (s, 2H), 4.56 (d, 2H,
J=6.0 Hz ); 13C NMR (100 MHz, CDCl3): δ 162.83, 162.76, 161.87 (d, J = 249.6 Hz),
148.76, 142.28, 139.47, 138.71, 134.20 (d, J = 10.3 Hz), 131.94 (d, J = 3.5 Hz),
131.37 (d, J = 8.7 Hz), 128.57, 127.36, 127.31, 116.92 (d, J = 24.8 Hz), 114.90,
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
114.21 (d, J = 20.9 Hz), 101.07, 49.37, 40.97; ESI-MS: 442.1 [M+H]+.
4.1.6. General procedure for preparation of compound Ethyl
2-(hydroxyimino)-5-methyl-3-oxohexanoate (II-3)
The commercially available 4-methylpentan-2-one was slowly added to the
solution of 60% NaH (480 mg, 11.98 mmol) in anhydrous tetrahydrofuran (15 mL) at
0
with stirring. After 30 min. diethyl carbonate (II-1) (1.77 g, 14.98 mmol) was
added to the mixture solution and was stirred at 60 for 4 h. The reaction mixture
was added to the 50 mL ice cold water and neutralized with 1.5 mL CH3COOH at
about 5 . The mixture was extracted with ethyl acetate for three time and the
combined organic layer was washed by 10% Na2CO3 and H2O. The product ethyl
5-methyl-3-oxohexanoate (II-2) was obtained as brown oil by removing ethyl acetate
solvent under reduced pressure. To the mixture solution of intermediate II-2 (1 g, 5.81
mmol) in acetic acid (5 mL) was slowly added sodium nitrite (1 g, 14.52 mmol)
dissolved in water (4 mL) under -5 . The mixture solution was stirred for 2 h and
further stirred for 3 h at room temperature. Upon completion of the reaction, the
mixture was added to 40 mL water, extracted with ethyl acetate (30 mL × 3), washed
with sodium bicarbonate solution (50 mL), and washed with saturated sodium
chloride (50 mL). The organic layer was dried over anhydrous sodium sulfate, and
1
then the solvent was removed under vacuum to afford oil with yield 54%; H NMR
(400 MHz, CDCl3): δ 9.24 (s, 1H), 4.39 (q, 2H, J=7.2 Hz), 2.67 (d, 1H, J=7.2 Hz),
2.27-2.18 (m,1H), 1.36 (t, 3H, J=7.2 Hz), 0.95 (d, 6H, J=6.0 Hz); ESI-MS: 202.3
[M+H]+.

1
2
4.1.7. General procedure for preparation of compound Ethyl
2-amino-5-methyl-3-oxohexanoate (II-4)
3
4
To the mixture of intermediate II-3 (0.6 g, 2.98 mmol) in hydrochloric acid
saturated solution of ethanol (10 mL) was added 10% Pd/C. The solution was stirred
at room temperature under H2 atmosphere for overnight. Upon completion of the
reaction, the mixture was filtrated with diatomite and the residue was washed by
ethanol (30 mL × 3). Then the solvent was removed under vacuum and the residue
5
6
7
1
8
was recrystallized to obtain the desired compound II-4. White solid, yield: 65%; H
9
NMR (400 MHz, CD3OD) δ: 4.38 (q, 2H, J=7.2 Hz), 2.82-2.67 (m, 2H), 2.25-2.15
13
10
11
(m,1H), 1.36 (t, 3H, J=6.8 Hz), 0.99 (d, 3H, J=6.8 Hz), 0.94 (d,3H, J=6.4 Hz); C
NMR (100 MHz, CD3OD): 197.72, 163.28, 63.43, 48.78, 23.93, 21.31, 21.12, 12.89;
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
ESI-MS: 375.4 [2M+H]+.
4.1.8. General procedure for preparation of compound Ethyl
5-methyl-3-oxo-2-(thiazole-2-carboxamido) hexanoate (II-5)
To the solution of intermediate II-4 (346 mg, 1.55 mmol) in DMF (10 mL) was
slowly added HATU (706 mg, 1.86 mmol) under lower temperature. The mixture
solution was stirred for 10 mins, slowly added 2-carboxylthiazole (200 mg, 1.55
mmol) and DIPEA (600 mg, 4.64 mmol), then was further stirred at room temperature
for overnight. Upon completion of the reaction, the solvent was added to 30 mL water,
extracted with ethyl acetate (15 mL × 3) and washed with saturated sodium chloride
(20 mL). The organic layer was dried over anhydrous sodium sulfate, and then the
solvent was removed under vacuum. The residue was chromatographed on silica gel
using ethyl acetate and petroleum ether. Pure fractions were collected and
1
concentrated, giving the desired compound II-5. White solid, yield: 59%; H NMR
(400 MHz, CDCl3): δ 8.33(d, 1H, J=6.4 Hz), 7.94(d, 1H, J=2.4 Hz), 7.62(d, 1H, J=2.8
Hz), 5.38(d, 1H, J=6.8 Hz), 4.31(q, 2H, J=7.2 Hz), 2.67(d, 1H, J=2.0 Hz), 2.65 (d, 1H,
J=0.8 Hz), 2.29-2.22 (m,1H), 1.33 (t, 3H, J=7.2 Hz), 0.97 (d, 3H, J=6.8 Hz), 0.93 (d,
3H, J=6.8 Hz); 13C NMR (100 MHz, CDCl3): δ 199.82, 165.61, 162.01, 159.20,
144.00, 125.05, 62.80, 49.56, 24.39, 22.51, 22.24, 14.09; ESI-MS: 299.3 [M+H]+.
30
31
32
4.1.9.
Ethyl-5-isobutyl-[2,2'-bithiazole]-4-carboxylate (II-6)
To the solution of intermediate II-5 (360 mg, 1.21 mmol) in toluene (40 mL) was
General
procedure
for
preparation
of
compound

1
2
slowly added Lawesson's reagent (732 mg, 1.81 mmol). The mixture was stirred
under reflux temperature for 5 h. Upon completion of the reaction, toluene was
removed under vacuum. The residue was added to ethyl acetate (60 mL), washed with
sodium bicarbonate saturated solution (30 mL × 3) and washed with saturated sodium
chloride (20 mL). The organic layer was dried over anhydrous sodium sulfate, and
then the solvent was removed under vacuum. The residue was chromatographed on
silica gel using ethyl acetate and petroleum ether. Pure fractions were collected and
3
4
5
6
7
1
8
concentrated, giving the desired compound II-5. White solid, yield: 36%; H NMR
9
(400 MHz, CDCl3): δ 7.88(d, 1H, J=3.2 Hz), 7.47(d, 1H, J=3.2 Hz), 4.44(q, 2H, J=7.2
Hz),3.16(d, 2H, J=7.2 Hz), 2.06-1.96 (m,1H), 1.45 (t, 3H, J=7.2 Hz), 1.01 (d, 6H,
10
11
12
13
14
13
J=6.4 Hz); C NMR (100 MHz, CDCl3): δ 162.18, 160.99, 157.66, 150.96, 143.80,
142.41, 121.46, 61.38, 36.25, 31.04, 22.31, 14.34; ESI-MS: 297.5 [M+H]+.
4.1.10.
General
procedure
for
preparation
of
compound
5-isobutyl-[2,2'-bithiazole]-4-carboxylic acid (II-7)
15
16
17
18
19
20
21
22
23
To the mixture of intermediate II-6 (129 mg, 0.44 mmol) in ethanol (5 mL),
water (5 mL) and THF (5 mL) was added along with NaOH (87 mg, 2.18 mmol). The
solution was stirred under 30 for overnight. Upon completion of the reaction, the
solvent ethanol and THF were removed under vacuum. Then the mixture was added
to 30 mL ammonium chloride solution, extracted with ethyl acetate (15 mL × 3) and
washed with saturated sodium chloride (20 mL). The organic layer was dried over
anhydrous sodium sulfate, and then the solvent was removed under vacuum. The
residue was recrystallized to obtain the desired compound II-7. White solid, yield:
1
85%; H NMR (400 MHz, CDCl3): δ 13.22 (s, 1H), 7.99 (dd, 1H, J = 9.6, 3.2 Hz),
24
25
3.15 (d, 2H, J=7.2 Hz), 1.98-1.91 (m,1H), 0.94 (d, 6H, J=6.4 Hz).
4.1.11. General procedure for preparation of compounds II-8(a-c)
26
27
28
29
30
31
To the mixture of intermediate II-7 (0.15 mmol) in DMF (5 mL) was slowly
added HATU (68 mg, 0.16 mmol) under low temperature. The solution was stirred for
10 min, added different substituted amine, DIPEA (39 mg, 0.3 mmol), and then was
further stirred under room temperature for overnight. Upon completion of the
reaction, the mixture was added 30 mL water, extracted with ethyl acetate (15 mL ×
3) and washed with saturated sodium chloride (20 mL). The organic layer was dried

1
2
over anhydrous sodium sulfate, and then the solvent was removed under vacuum. The
residue was chromatographed on silica gel using ethyl acetate and petroleum ether.
Pure fractions were collected and concentrated, gave the desired compounds II-8(a-c).
3
4
General
procedure
for
preparation
of
compound
5
6
N-(2,4-difluorobenzyl)-5-isobutyl-[2,2'-bithiazole]-4-carboxamide (II-8a). White solid,
1
yield: 60%; H NMR (400 MHz, CDCl3): δ 7.89(d, 1H, J=2.8 Hz), 7.80 (s, 1H),
7
7.46(d, 1H, J=2.8 Hz), 7.41 (dd, 1H, J1=6.4 Hz, J2=2.0 Hz), 6.89-6.81 (m,2H), 4.65(d,
2H, J=6.4 Hz), 3.30(d, 2H, J=7.2 Hz), 2.06-2.00 (m, 1H), 1.01 (d, 6H, J=6.8 Hz; 13C
NMR (100 MHz, CDCl3): δ 162.36 (dd, J = 248.2, 11.9 Hz), 162.01, 160.95 (dd, J=
248.8, 11.9 Hz), 160.83, 156.73, 148.32, 144.00, 143.45, 130.92 (dd, J= 9.7, 5.9 Hz),
121.44 (dd, J= 15.0, 3.7 Hz), 121.23, 111.36 (dd, J= 21.1, 3.7 Hz), 103.88 (t, J= 25.4
Hz), 36.48 (d, J= 3.7 Hz), 35.77, 31.07, 22.31; ESI-MS:394.3 [M+H]+.
8
9
10
11
12
13
14
15
16
17
18
19
20
General
procedure
for
preparation
of
compound
N'-(4-fluorophenyl)-5-isobutyl-[2,2'-bithiazole]-4-carbohydrazide (II-8b). White solid,
1
yield: 63%; H NMR (400 MHz, CDCl3): δ 9.05 (s, 1H), 7.91(d, 1H, J=3.2 Hz),
7.49(d, 1H, J=3.2 Hz), 6.98-6.89 (m, 4H), 6.23(d, 1H, J=17.2 Hz), 3.25(d, 2H, J=7.2
Hz), 2.06-1.96 (m,1H), 0.99 (d, 6H, J=6.8 Hz); 13C NMR (100 MHz, CDCl3): δ
162.07, 160.67, 158.04 (d, J = 238.7 Hz), 157.51, 149.40, 144.24 (d, J = 2.2 Hz),
144.12, 141.87, 121.43, 115.80 (d, J = 22.8 Hz), 115.05 (d, J = 7.8 Hz), 35.65, 31.06,
22.24; ESI-MS:377.3 [M+H]+.
21
22
23
24
25
26
27
General
procedure
for
preparation
of
compound
N-(4-fluorobenzyl)-5-isobutyl-[2,2'-bithiazole]-4-carboxamide (II-8c). White solid,
1
yield: 54%; H NMR (400 MHz, CDCl3): δ 7.88(d, 1H, J=3.2 Hz), 7.80 (s, 1H),
7.44(d, 1H, J=3.2 Hz), 7.35 (dd, 1H, J1=5.6 Hz, J2=3.2 Hz), 7.04(t, 2H, J=8.8 Hz),
4.62(d, 2H, J=6.0 Hz), 3.32(d, 2H, J=7.2 Hz), 2.08-2.01 (m, 1H), 1.02 (d, 6H, J=6.8
Hz); 13C NMR (100 MHz, CDCl3): δ 162.18 (d, J = 245.4 Hz), 161.97, 160.86,
156.70, 148.28, 144.01, 143.56, 134.26 (d, J = 3.2 Hz), 129.39 (d, J = 8.1 Hz), 121.18,
115.55 (d, J = 21.5 Hz), 42.37, 35.82, 31.10, 22.34; ESI-MS:376.3 [M+H]+.
28
29
30
4.1.12.
General
procedure
for
preparation
of
compound
Ethyl-2-amino-4-isobutylthiazole-5-carboxylate (III-4)
31
To the mixture of intermediate II-2 (4 g, 23.2 mmol) in DCM (150 mL) was

1
2
added NBS (6.74 g, 0.038 mol) under ice-bath. The solution was stirred at room
temperature for 4 hours. Upon completion of the reaction, the mixture was washed
with water (60 mL × 3) and washed with saturated sodium chloride (40 mL). The
organic layer was dried over anhydrous sodium sulfate, and then the solvent was
removed under vacuum to obtain the desired compound III-3 as yellow oil.
Intermediate III-3 (5.34 g, 21.3 mmol) and thiourea (1.62 g, 21.3 mmol) were
dissolved in ethanol, and the mixture solution was stirred at 80 for 4 hours. Upon
completion of the reaction, ethanol was removed under vacuum. The residue was
added water (100 mL), extracted with ethyl acetate (45 mL × 3), washed with
saturated sodium chloride (60 mL). The organic layer was dried over anhydrous
sodium sulfate, and then the solvent was removed under vacuum. The residue was
chromatographed on silica gel using ethyl acetate and petroleum ether. Pure fractions
were collected and concentrated, giving the desired compound III-4. White solid,
yield: 70%, m.p.148-149 ; 1H NMR (400 MHz, CDCl3): δ 5.68 (s, 2H), 4.28 (q, 2H,
J=7.2 Hz), 4.23 (d, 2H, J=7.2 Hz), 2.09-1.98 (m,1H), 1.32 (t, 3H, J=7.2 Hz), 0.93 (d,
6H, J=6.8 Hz); 13C NMR (100 MHz, CDCl3): δ 169.46, 162.67, 162.43, 111.83, 60.54,
39.32, 28.98, 22.47, 14.37; ESI-MS: 229.0 [M+H]+, 457.7 [2M+H]+.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
4.1.13.
General
procedure
for
preparation
of
compound
2-amino-4-isobutylthiazole-5-carboxylic acid (III-5)
20
21
22
23
24
25
26
27
28
29
To the mixture of intermediate III-4 (500 mg, 2.19 mmol) in ethanol (5 mL) and
THF (5 mL) was added sodium hydroxide solution (4 mL, 1mol/L). The solution was
stirred under 50 for 3 hours. Upon completion of the reaction, the solvent ethanol
and THF were removed under vacuum. Then the mixture was adjusted the pH to less
than 7 with HCl (1mol/L), added 60 mL water, extracted with ethyl acetate (30 mL ×
3) and washed with saturated sodium chloride (50 mL). The organic layer was dried
over anhydrous sodium sulfate, and then the solvent was removed under vacuum. The
residue was recrystallized to obtain the desired compound III-5. Light yellow solid,
1
yield: 57％, m,p.148-149
℃
; H NMR (400 MHz, DMSO): δ 12.20 (s, 1H), 7.58 (s,
2H), 2.72 (d, 2H, J=7.2 Hz), 2.03-1.97 (m,1H), 0.86 (d, 6H, J=6.4 Hz); 13C NMR

1
(100 MHz, DMSO): δ 169.26, 162.79, 161.41, 108.87, 37.96, 27.51, 21.77; ESI-MS:
2
3
4
199.2 [M-H]-.
4.1.14.
General
procedure
for
preparation
of
compound
2-amino-N-(4-fluorobenzyl)-4-isobutylthiazole-5-carboxamide (III-6)
5
6
To the mixture of intermediate III-5 (102 mg, 0.51 mmol) in DMF (5 mL) was
slowly added HATU (232 mg, 0.61 mmol) under low temperature. The mixture
solution was stirred for 10 min, added 4-fluorobenzylamine (70 mg, 0.56 mmol),
DIPEA (131 mg, 1.02 mmol), and then was further stirred at room temperature for 2h.
Upon completion of the reaction, the mixture was added to 80 mL water, extracted
with ethyl acetate (20 mL × 3) and washed with saturated sodium chloride (40 mL).
The organic layer was dried over anhydrous sodium sulfate, and then the solvent was
removed under vacuum. The residue was chromatographed on silica gel using ethyl
acetate and petroleum ether. Pure fractions were collected and concentrated, giving
the desired compound III-6. White solid, yield: 70%, m.p.123-124 ℃; 1H NMR (400
MHz, CDCl3) δ: 7.30-7.27 (m,2H), 7.05-7.00 (m,2H), 5.78 (s, 1H), 5.47 (s, 2H), 4.51
(d, 2H, J=6.0 Hz), 2.75 (d, 2H, J=7.2 Hz), 2.08-2.01 (m,1H), 0.92 (d, 6H, J=6.8 Hz);
13C NMR (100 MHz, CDCl3): 166.58, 163.47, 162.08, 161.02, 157.78, 134.01 (d, J =
3.2 Hz), 129.45 (d, J = 8.1 Hz), 115.62 (d, J = 21.6 Hz), 43.26, 39.60, 28.96, 22.49;
ESI-MS: 308.0 [M+H]+, 330.4 [M+Na]+.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
4.1.15.
General
procedure
for
preparation
of
compound
N-(4-fluorobenzyl)-4-isobutyl-2-(thiophene-2-carboxamido)
thiazole-5-carboxamide (III-7a)
23
24
25
26
27
28
29
To the mixture of intermediate III-6 (100 mg, 0.33 mmol) in DMF (5 mL) was
slowly added HATU (148 mg, 0.39 mmol) under low temperature. The solution was
stirred for 5 min, added 2-thiazole carboxylic acid (42 mg, 0.33 mmol), DIPEA (84
mg, 0.65 mmol), and then was further stirred under room temperature for 2h. Upon
completion of the reaction, the mixture was added to 30 mL water, extracted with
ethyl acetate (15 mL × 3) and washed with saturated sodium chloride (30 mL). The
organic layer was dried over anhydrous sodium sulfate, and then the solvent was