Design, synthesis and evaluation of pyrazole derivatives as non-nucleoside hepatitis B virus inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.07.048

In continuation of our efforts toward the discovery of potent non-nucleoside hepatitis B virus (HBV) inhibitors with novel structures, we have employed bioisosterism and hybrid pharmacophore-based strategy to explore the chemically diverse space of bioactive compounds. In this article, the original thiazole platform was replaced with pyrazole scaffold to yield the optimal pharmacophore moieties in order to generate novel non-nucleoside HBV inhibitors with desirable potency. Some of the new compounds were able to inhibit HBV activity in the low micromolar range. In particular, compound 6a3 displayed the most potent activity against the secretion of HBsAg and HBeAg with IC₅₀of 24.33?μM and 2.22?μM, respectively. The preliminary structure-activity relationship (SAR) of this new series of compounds was investigated, which may help designing more potent molecules.

Full text of DOI:10.1016/j.ejmech.2016.07.048

Accepted Manuscript
Design, synthesis and evaluation of pyrazole derivatives as non-nucleoside hepatitis
B virus inhibitors
Haiyong Jia, Fuxiang Bai, Na Liu, Xiaohong Liang, Peng Zhan, Chunhong Ma,
Xuemei Jiang, Xinyong Liu
PII:
S0223-5234(16)30609-2
10.1016/j.ejmech.2016.07.048
EJMECH 8766
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 May 2016
Revised Date: 18 July 2016
Accepted Date: 20 July 2016
Please cite this article as: H. Jia, F. Bai, N. Liu, X. Liang, P. Zhan, C. Ma, X. Jiang, X. Liu, Design,
synthesis and evaluation of pyrazole derivatives as non-nucleoside hepatitis B virus inhibitors, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.048.
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ACCEPTED MANUSCRIPT
Graphical abstract
A series of novel pyrazole derivatives were identified as non-nucleoside HBV
inhibitors via bioisosterism and pharmacophore hybrid strategy.

ACCEPTED MANUSCRIPT
Design, Synthesis and Evaluation of Pyrazole Derivatives as
Non-nucleoside Hepatitis B Virus Inhibitors
1
2
1
2
1
2
Haiyong Jia , Fuxiang Bai , Na Liu , Xiaohong Liang , Peng Zhan , Chunhong Ma ,
3
1,*
Xuemei Jiang and Xinyong Liu
1
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of
Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road,
2
50012, Jinan, Shandong, PR China
2
Department 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.
3
Department of Hepatic Diseases, Jinan Infectious Disease Hospital, Jingshi Road, 173
ꢀ250021,
Jinan, Shandong, P. R. China
*
Corresponding author. Tel.: +86 531 88380270; fax: +86 531 88382731.
E-mail address: xinyongl@sdu.edu.cn.
Abstract
In continuation of our efforts toward the discovery of potent non-nucleoside
hepatitis B virus (HBV) inhibitors with novel structures, we have employed
bioisosterism and hybrid pharmacophore-based strategy to explore the chemically
diverse space of bioactive compounds. In this article, the original thiazole platform
was replaced with pyrazole scaffold to yield the optimal pharmacophore moieties in
order to generate novel non-nucleoside HBV inhibitors with desirable potency. Some
of the new compounds were able to inhibit HBV activity in the low micromolar range.
In particular, compound 6a3 displayed the most potent activity against the secretion
of HBsAg and HBeAg with IC50 of 24.33 µM and 2.22 µM, respectively. The
preliminary structure-activity relationship (SAR) of this new series of compounds was
investigated, which may help designing more potent molecules.

ACCEPTED MANUSCRIPT
Keywords: HBV; Bioisosterism; Hybrid pharmacophore-based; Non-nucleoside;
SAR.
1
. Introduction
Viral hepatitis type B, referred to as Hepatitis B, is a serious infectious disease
caused by the hepatitis B virus (HBV). Long-term development of Hepatitis B can
lead to acute or chronic viral hepatitis, severe hepatitis, liver cirrhosis (LC) and
hepatocellular carcinoma (HCC)[1]. According to the report of World Health
Organization (WHO), about 240 million people worldwide had been involved in
chronic HBV infection[2], and more than 780,000 people die every year[3]. Due to
the high incidence, long course and difficulty to cure, hepatitis B has become a major
disease which seriously affect people's health and social development, therefore
research of effective drugs of HBV has become the top priority [4]. China is a high
prevalence country of hepatitis B with about 90 million HBV carriers and an average
2
8 million people have chronic hepatitis B (CHB).
HBV is a member of hepadnaviridae, whose genome is partially double-stranded
circular. But its replication process possesses the same characteristics as RNA
retroviruses replication process, which including: adsorption and fusion to the target
cells, DNA repair and transcription, the translation and reverse transcription of
progenome RNA, viral particle assembly and budding, etc[5]. Based on the
understanding of HBV life cycle and molecular biology, some drugs have been
developed for the treatment of CHB. Those currently used drugs mainly include
interferon, immunomodulatory drugs, and DNA polymerase inhibitors. Despite the
dual role of immunomodulation and anti-virus of α-interferon (IFN-α) [6], it is
effective only to 30%-40% of patients, moreover adverse reactions limit its clinical
application. Thymosin-α1 (Tα1) [7] and other immunomodulatory drugs can improve
the body's specific immunity to HBV but lack of pertinency, so they can be used only
as adjuvant drugs or as part of a combined treatment with other HBV drugs. Five
nucleoside/nucleotide HBV DNA polymerase inhibitors (lamivudine, adefovir
dipivoxil, entecavir, telbivudine and tenofovir) (Figure 1) are widely used in
clinic[8]. However, because of the genetic heterogeneity of HBV genome, the virus
can easily develop resistance to this kind of drugs. Therefore, discovery and

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development of novel anti-HBV inhibitors with improved potency, low toxicity, or
novel modes of actions is undoubtedly essential to combat the HBV infection[9].
NH2
O
O
N
HN
N
N
NH
O
N
H
O
N
N
NH2
H
H
O
O
S
HO
HO
OH
HO
HO
Entecavir
Lamivudine
Telbivudine
NH2
N
NH2
N
N
N
N
N
N
N
O
O
O
P
OH
O
O
O
O
O
P
HO
O
O
Tenofovir
Adefovir dipivoxil
Figure 1. Five nucleoside/nucleotide drugs approved by US FDA for HBV treatment.
Previously, Chen and co-workers first reported that leucamide A (Figure 2)
isolated from the Australian marine sponge Leucetta microraphis, a cyclic
heptapeptide containing a mixed 4,2-bisheterocycle tandem pair showed poor antiviral
9
activity . They synthesized a library of 4,2-bisheterocycle tandem derivatives, of
which compound B (IC :76.4 ꢀmol/L) (Figure 2) showed moderate activity against
5
0
HBV DNA replication. Compound B is further optimized to afford compound C
IC : 1.1 ꢀmol/L) (Figure 2) with improved activity[10]. In 2014, Li et al. reported
(
5
0
that a new leucamide A derivative isothiafludine (NZ-4) (Figure 3) bearing
bis-heterocycle tandem pairs suppressed intracellular HBV replication in HepG2.2.15
cells with an IC value of 1.33 ꢀM. NZ-4 could inhibit HBV DNA replication by
5
0
interfering with the interaction between HBcAg and pgRNA in the capsid assembly
process, and provides a new therapeutic strategy to combat HBV infection[11].
Moreover, it is noteworthy that compound D (Figure 3), a NZ-4 analogue, have more
potent activity against HBV replication[12]. Besides, phenylpropenamide AT-130

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(
Figure 3) also has potent activity against HBV DNA replication with IC value of
5
0
0
.13 ꢀM, which successfully decreased HBV production by blocking RNA packaging
and producing apparently abnormal capsids that lacked genetic material[13, 14].
Generally, the structure of these compounds have four fragments (shown in
different color, Figure 3), including the core domain (pink), a five- or six-membered
aromatic ring (blue), the hydrophobic part (green), and the different amides and esters
(
yellow). In addition, with the aim of exploring novel structural motifs and
establishing structure-activity relationships, the thiazole core of compound C was
replaced with imidazole as a potential bioisosteric moiety, resulting in moderate
anti-HBV activity[10]. Therefore, the five-membered heterocycle portion of these
inhibitors could be acting as versatile building blocks to introduce different new
functional groups and to anchor these groups into the optimal space for binding[15].
To investigate the differences in the electronic and conformational contribution
of the five-membered heterocyclic moiety to the anti-HBV potency, herein, in this
paper, two different series of pyrazole derivatives were designed via the bioisosterism
and hybrid pharmacophore-based strategy, in which the thiazole core was replaced by
its isostere pyrazole to find more potent anti-HBV inhibitors.
Figure 2. The structures of the anti-HBV natural product Leucamide A and its derivatives
compound B, compound C obtained by structural simplification.

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Figure 3. The structures of three lead compounds and the newly designed pyrazole series via
bioisosterism and pharmacophore hybrid strategy.
2
. Results and discussion
2
.1. Chemistry
The synthetic route of the target compounds 6a1-6a9 and 6k1-6k7 was shown in
Scheme 1. The key intermediate (E)-ethyl
-((dimethylamino)methylene)-5-methyl-3-oxohexanoate (3) was prepared from the
intermediate ethyl 5-methyl-3-oxohexanoate (2) by condensation reaction with
,1-dimethoxy-N,N-dimethylmethanamine under 100ꢁ.[16] The intermediate 2 was
achieved from the commercially available diethyl carbonate (1) and
-methylpentan-2-one by substitution reaction. Intermediate 3 was converted to 4a-4k
2
1
4
with different substituted hydrazines or hydrazines hydrochloride. Then, 4a and 4b
are hydrolyzed to intermediate 5a and 5b correspondingly by hydrolysis reaction in

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the solution of sodium hydroxide, water, ethanol and tetrahydrofuran under 50 ꢁ.
Treatment of intermediate 5a or 5b with different substituted amines by the reaction
gave the target compound 6a1-6a9 or 6k1-6k7, respectively. Both analytical and
spectral data of all the synthesized compounds are in full agreement with the proposed
structures.
R1
O
O
R1
N
N
R1
N
O
O
O
N
N
N
i
ii
O
iii
v
iv
O
O
O
NH
R2
N
COOEt
a-4k
O
COOH
a: R1 = thiazol-2-yl;
1
2
3
4
6a1-6a9: R1 = thiazol-2-yl;
5
6k1-6k7: R1 = 3-fluorophenyl
5
k: R1 = 3-fluorophenyl
Scheme 1. Reagents and conditions: (i) 4-methylpentan-2-one, NaH, AcOH, THF; (ii)
1
1
,1-dimethoxy-N,N-dimethylmethanamine, 80
ꢁ
(iii) EtOH, Et N, R -NHNH ; (iv) NaOH, H O,
3
2
2
2
THF, EtOH; (v) R -NH , DMF, HATU, Et N.
2
3
2
.2. Biological activity
All the synthesized compounds were evaluated for (in vitro) their anti-HBV
activity (HBeAg, HBsAg, DNA) and cytotoxicity in HepG2.2.15 cells (human HBV
transgenic hepatocellular carcinoma cells) using standard ELISA, PCR and cell
counting kit-8(CCK-8) method. The concentration of compound required for 50%
inhibition of HBeAg, HBsAg secretion or DNA replication was defined as IC and
5
0
the concentration of compound that induced the death of the HepG2.2.15 cells
cultures by 50% was defined as CC . Selectivity index (SI) was determined as the
5
0
CC /IC value. Lamivudine was used as positive standards.
50
50
2
.2.1 Cytotoxicity
The cytotoxicity of these new pyrazole derivatives (4a-4j, 6a1-6a9 and 6k1-6k7)
was measured by cell counting kit-8(CCK-8) method in HepG2.2.15 cell line. As
shown in Table 1, most of these pyrazole analogs exhibited low toxicity as positive
drug lamivudine except compound 4d and 4f. It was shown that electron-withdrawing
1
group of R may improve the toxicity of the inhibitors.
2
.2.2 Inhibitory effect on HBsAg and HBeAg secretion

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1
To identify the effects of different polarized substituents on position R , ten ethyl
3
-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxylate derivatives (4a-4j) were
synthesized. However, as listed in Table 1, most compounds showed poor antiviral
activities. The 4-methoxyphenyl, 3-methoxyphenyl and 2-fluorophenyl substituent
(4e, 4g and 4j) with less hydrophilic property indicated that lipophilic property was an
important determinant for the anti-HBV activity.
To identify the effects of the substituents in amide position, two subseries of
derivatives 6a1-6a9 and 6k1-6k7 were synthesized and assessed for anti-HBV
activity (Table 1). Compounds 6a1-6a9 with different aromatic substituents on amide
position were first prepared and the pharmaceutical profiles were tested. Compared to
compounds 6a8 and 6a9, 6a6 showed more potent activity against the secretion of
HBeAg with IC value 95.65 ꢀmol/L, which is less potent than compound 6a1. These
5
0
results suggested that hydrophobic group was important to the antiviral activity.
Besides, compound 6a4 bearing a trifluoromethyl moiety exhibited no antiviral
activity. Additionally, the fluorobenzyl derivatives 6a1, 6a2 and 6a7 displayed
comparable activities against the secretion of HBsAg with the IC of 44.48 ꢀmol/L,
5
0
5
0.12 ꢀmol/L and 58.80 ꢀmol/L, respectively. However, they demonstrated in
opposite order (6a1<6a2<6a7) for the activity against the secretion of HBeAg with
the IC of 31.43 ꢀmol/L, 26.00 ꢀmol/L and 17.25 ꢀmol/L, respectively. In particular,
5
0
compound 6a5 have more potent activity against the secretion of HBsAg and HBeAg
than compound 6a1 and 6a2, which suggested that their activity is controlled by the
presence of fluoro atom at different positions of the benzyl group.
Furthermore, 4-fluorophenyl group was better than 4-fluorobenzyl group as
compound 6a3 and 6k3 possessed more potent anti-HBV activity than compound 6a1
and 6k1, respectively. Also, compound 6a1 and 6a3 were more potent inhibitors than
compound 6k1 and 6k3, which showed that thiazol-2-yl group was better than
3
-fluorophenyl group for the activity against the secretion of HBsAg and HBeAg.
In general, the newly designed pyrazole derivatives had more potent activities
against the secretion of HBsAg and HBeAg than positive drug lamivudine, but only
compound 6k3 showed slightly activity against HBV DNA replication. These results

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would provide valuable information for further modification through bioisosterism
and pharmacophore hybrid to find more potent anti-HBV inhibitors.
Table 1. Anti-HBV activity, cytotoxicity and selectivity indices of pyrazole derivatives (4a-4j,
6
a1-6a9 and 6k1-6k7) and positive drug lamivudine.
HBsAg
HBeAg
a
1
3
CC50
b
b
R /R
Compound
IC50
IC50
c
c
(
µmol/L)
SI
SI
(
µmol/L)
>100
63.3
(µmol/L)
>100
59.4
4
4
a
thiazol-2-yl
3-nitrophenyl
4-COOH-Ph
>100
59.3
—
0.94
—
—
0.94
—
b
4
c
>100
7.1
>100
41.9
>100
47.9
4
d
3-COOH-Ph
0.17
1.12
0.10
1.11
—
0.15
1.08
>0.78
1
4
e
4-methoxyphenyl
2-nitrophenyl
3-methoxyphenyl
4-nitrophenyl
4-fluorophenyl
2-fluorophenyl
4-fluorobenzyl
2-fluorobenzyl
4-fluorophenyl
77.08
4.85
68.53
49.26
63.34
>100
>100
94.84
44.48
50.12
24.33
71.57
<6.25
70.62
>100
>100
82.32
31.43
26.00
2.22
4f
4
4
g
70.49
>100
>100
>100
>100
>100
83.67
h
—
4i
—
—
4j
1.05
>2.25
>2
1.21
>3.18
>3.85
37.69
6a1
6a2
6a3
3.44

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6
6
6
6
6
6
6
6
6
6
6
6
6
a4
a5
a6
a7
a8
a9
k1
k2
k3
k4
k5
k6
k7
4-(trifluoromethyl)benzyl
2,4-difluorobenzyl
4-methoxybenzyl
3-fluorobenzyl
>100
83.60
>100
>100
>100
>100
>100
85.89
36.21
67.94
45.44
>100
>100
41.82
>100
58.80
>100
>100
—
2
>100
8.84
—
9.46
>1.05
5.80
—
—
95.65
17.25
>100
86.15
>100
38.49
24.69
44.81
34.01
71.17
1.7
—
4-aminobenzyl
4-hydroxybenzyl
4-fluorobenzyl
—
>1.16
—
>100
38.55
25.94
57.64
51.90
>100
—
benzyl
2.23
1.4
1.18
—
2.23
1.47
1.52
1.34
1.41
4-fluorophenyl
(4-methoxybenzyl)oxy
NH-(4-fluorophenyl)
pyridin-4-ylmethyl
tert-butyl 4-methyl-
—
-piperidine-1-carboxylate
>100
>100
>100
>100
—
>100
>100
—
Lamivudine
—
—
d
NZ-4
51.47
34.69
1.48
31.56
1.63
a
CC : Concentration required to reduce the viability of mock-infected cells by 50%.
5
0
b
IC : Concentration of compound required for 50% inhibition of HBeAg or HBsAg
5
0
secretion.
c
SI: Selectivity index, the ratio of CC /IC .
5
0
50
d
NZ-4ꢂThe values for NZ-4 are from single determinations.
2
.2.3 Inhibitory effect on HBV DNA replication
The inhibitory effect on HBV DNA replication of some target compounds was
screened in the HepG2.2.15 cell line using lamivudine (LAM) as a positive control.
After the treatment of the cells with the test compounds at the concentration of 100

ACCEPTED MANUSCRIPT
ꢀ
M for 8 days, and then extra cellar HBV DNA levels were quantified by real-time
FQ-PCR. As shown in the fig. 4, most of pyrazole derivatives have relative inhibitory
activity against HBV DNA replication.
1
00
80
60
40
20
0
Pyrazole derivatives and lamivudine(Lam) with 100 µM
Fig. 4. Inhibitory effect of pyrazole derivatives on HBV DNA level. HepG2.2.15 cells were
cultured in the presence of pyrazole derivatives and lamivudine at concentrations of 100 ꢀM for 8
days, and then extra cellar HBV DNA levels were quantified by real-time FQ-PCR.
2
.3. In silico prediction of physicochemical properties[17]
Furthermore, some physicochemical properties of 6a1, 6a3, 6a5 and 6k3 were
predicted for their compliance with the Lipinski's rule of five using free online
software (http://www.molinspiration.com/). As shown in Table 2, 6a1, 6a3, 6a5 and 6k3
compounds conformed well to the Lipinski's rule of five. Overall, compound 6a3
exhibited potent anti-HBV activity with low toxicity and reasonable physicochemical
properties, and thus could be considered as a promising candidate for further
development.
a
Table 2. Prediction of physicochemical properties of 6a1, 6a3, 6a5 and 6k3
Compound nViol MW
natoms miLogP nON nOHNH Nrotb TPSA
MV
Range
<500
<5
<10 <5
<=10 <140
6
6
6
6
a1
a3
a5
k3
0
0
0
0
0
385.4 25
344.4 24
376.4 26
355.4 26
3.36
3.66
3.45
4.54
3.18
5
5
5
4
4
1
1
1
1
1
6
5
6
59.82
59.82
59.82
46.92
314.5
297.6
319.4
316.1
5
3
77.5
6
54.35 326.1
NZ-4
25

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4
88.3
6
104.47 386.0
AT-130
0
31
4.04
8
1
a
nViol: no. of violations; natoms: no. of atoms; miLogP: molinspiration
predicted LogP; MW: molecular weight; nON: no. of hydrogen bond acceptors;
nOHNH: no. of hydrogen bond donors; nrotb: no. of rotatable bonds; TPSA:
topological polar surface area; MV: molar volume.
3
. Conclusions
In summary, the present work is an extension of our ongoing efforts toward the
development and the identification of new molecules with anti-HBV activity. In the
present investigation, the bioisosterism and hybrid pharmacophore-based drug design
led to the identification of pyrazole derivatives with potency against HBV activity.
Among them, compound 6a3 was identified as the most promising candidate with
favorable inhibitory activity against the secretion of HBsAg and HBeAg with IC of
5
0
2
4.33 µmol/L and 2.22 µmol/L, respectively. Preliminary SAR results for the newly
synthesized congeners are presented, providing insights for discovery of more potent
anti-HBV inhibitors with diverse structures.
4
. Experimental
4
.1. Chemistry
All melting points were determined on a micro melting point apparatus and are
1
uncorrected. H NMR spectra were obtained on a Brucker Avance-400 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–
00 mesh). Solvents were reagent grade and, when necessary, were purified and dried
3
by standard methods. Concentration of the reaction solutions involved the use of
rotary evaporator at reduced pressure.
4
2
.1.1.
General
procedure
for
the
synthesis
of
(E)-ethyl
-((dimethylamino)methylene)-5-methyl-3-oxohexanoate (3)

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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 half an hour diethyl carbonate (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 CH COOH at
3
about 5 ꢁ. The mixture was extracted with ethyl acetate for three time and the
combined was washed by 10% Na CO and H O. The product ethyl
2
3
2
5
-methyl-3-oxohexanoate (2) was obtained as brown oil by removing ethyl acetate
layer under reduced pressure[18], which is directly added to 10 mL
,1-dimethoxy-N,N-dimethylmethanamine and stirred for overnight under 100 ꢁ.
1
Upon completion of the reaction, the solvent was evaporated, giving the desired
compound 3 as an orange oil in good yield without further purification[19].
4
.1.2. General procedure for preparation of compounds 4a–4k
To
the
mixture
solution
of
(E)-ethyl
2
-((dimethylamino)methylene)-5-methyl-3-oxohexanoate (3) (335 mgꢀ1.47 mmol)
and Et N (622 mgꢀ6.14 mmol) in ethanol (6 mL) were added with hydrazine or
3
hydrazine hydrochloride (1.23 mmol). The reaction mixture was stirred at room
temperature overnight. Upon completion of the reaction, the solvent was evaporated,
leaving a residue which was treated with ethyl acetate (30 mL) and washed with water
(3 x 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 the desired compounds (4a–4k) in good yield.
4
.1.2.1. Ethyl 3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxylate (4a)
1
White powder, yield: 25%. H NMR (400 MHz, CDCl ) δppm:8.02 (s, 1H),
3
7
.60 (d, 1H, J=3.2 Hz), 7.18 (d, 1H, J=3.6 Hz), 4.33(q, 2H, J=7.2 Hz), 3.51 (d, 2H,
13
J=7.2 Hz), 2.16~2.06 (m,1H), 1.38 (t, 3H, J=7.2 Hz), 0.94 (d, 6H, J=6.8 Hz); C
NMR (100 MHz, CDCl ): 163.14, 161.89, 148.96, 143.46, 140.40, 117.18, 115.17,
3
+
6
0.25, 33.02, 28.89, 22.13, 14.35; EI-MS: 280.3 [M+H] .
4
.1.2.2. Ethyl 3-isobutyl-1-(3-nitrophenyl)-1H-pyrazole-4-carboxylate (4b)

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1
Yellow oil, yield: 45%. H NMR (400 MHz, CDCl ) δppm: 8.35~8.33 (m, 2H),
3
8
.09 (s, 1H), 7.80 (d, 1H, J=8.0 Hz), 7.72 (t, 1H, J=8.0 Hz), 4.34 (q, 2H, J=7.2 Hz),
.98 (d, 2H, J=7.2 Hz), 1.90~1.79 (m, 1H), 1.39 (t, 3H, J=7.2 Hz), 0.77 (d, 6H, J=6.8
2
Hz). 13C NMR (100 MHz, CDCl3): 163.32, 148.58, 147.76, 142.95, 140.41, 132.02,
1
1
4
30.33, 123.52, 121.32, 113.81, 77.35, 77.24, 77.04, 76.72, 60.21, 33.36, 29.09, 22.22,
+
4.39; EI-MS: 318.5 [M+H] .
.1.2.3. 4-(4-(ethoxycarbonyl)-3-isobutyl-1H-pyrazol-1-yl)benzoic acid (4c)
1
White powder, yield: 50%. H NMR (400 MHz, CDCl ) δppm: 9.84 (s, 1H), 8.27
3
(d, 2H, J=8.8 Hz), 8.11 (s, 1H), 7.56 (d, 2H, J=8.8 Hz), 4.34 (q, 2H, J=7.2 Hz), 2.99
(d, 2H, J=7.2 Hz), 1.86~1.79 (m, 1H), 1.39 (t, 3H, J=7.2 Hz), 0.75 (d, 6H, J=6.8 Hz);
+
EI-MS: 317.5 [M+H] .
4
.1.2.4. 3-(4-(ethoxycarbonyl)-3-isobutyl-1H-pyrazol-1-yl)benzoic acid (4d)
1
White powder, yield: 52%. H NMR (400 MHz, CDCl ) δppm: 9.80 (s, 1H), 8.21
3
(dd, 1H, J =1.6 Hz, J =7.2 Hz), 8.15 (s, 1H), 8.10 (s, 1H), 4.34 (q, 2H, J=7.2 Hz), 2.94
1 2
1
3
(
d, 2H, J=7.6 Hz), 1.88~1.81 (m, 1H), 1.39 (t, 3H, J=7.2 Hz), 0.76. C NMR (100
MHz, CDCl3): 170.08, 163.55, 147.77, 142.47, 139.57, 131.49, 130.79, 130.52,
1
29.68, 127.88, 113.25, 77.35, 77.24, 77.03, 76.71, 60.12, 33.36, 28.94, 22.23, 14.40;
+
EI-MS: 317.5 [M+H] .
4
.1.2.5. Ethyl 3-isobutyl-1-(4-methoxyphenyl)-1H-pyrazole-4-carboxylate (4e)
1
Yellow oil, yield: 32%. H NMR (400 MHz, CDCl ) δppm: 8.03 (s, 1H), 7.29
3
(
dd, 2H, J =2.4 Hz, J =6.8 Hz), 6.99 (dd, 2H, J =2.4 Hz, J =6.8 Hz ), 4.32 (q, 2H,
1 2 1 2
J=7.2 Hz), 3.87 (s, 1H), 2.86 (d, 2H, J=7.2 Hz), 1.90~1.80 (m, 1H), 1.37 (t, 3H, J=7.2
+
Hz ), 0.75 (d, 6H, J=6.8 Hz), EI-MS: 303.5 [M+H] .
4
.1.2.6. Ethyl 3-isobutyl-1-(2-nitrophenyl)-1H-pyrazole-4-carboxylate (4f)
1
Yellow oil, yield: 52%. H NMR (400 MHz, CDCl ) δppm: 8.10 (dd, 1H,
3
J =0.8 Hz, J =8.0 Hz), 8.05 (s, 1H), 7.78 (dt, 1H, J =1.2 Hz, J =7.6 Hz), 7.68 (dt, 1H,
1
2
1
2
J =1.2 Hz, J =7.6 Hz), 7.54 (dd, 1H, J =0.8 Hz, J =7.6 Hz), 4.33 (q, 2H, J=7.2 Hz),
1
2
1
2
2
.83 (d, 2H, J=7.6 Hz), 1.89~1.79 (m, 1H), 1.38 (t, 3H, J=7.2 Hz), 0.81 (d, 6H, J=6.8
13
Hz). C NMR (100 MHz, CDCl ): 163.34, 148.59, 146.03, 143.16, 133.50, 132.53,
3
1
30.42, 129.77, 125.64, 113.52, 77.36, 77.05, 76.73, 60.08, 33.45, 28.43, 22.23, 14.36;

ACCEPTED MANUSCRIPT
+
EI-MS: 318.5 [M+H] .
4
.1.2.7. Ethyl 3-isobutyl-1-(3-methoxyphenyl)-1H-pyrazole-4-carboxylate (4g)
1
Yellow oil, yield: 47%. H NMR (400 MHz, CDCl ) δppm: 7.39 (t, 1H, J=8.4
3
Hz), 7.00 (dd, 1H, J =1.6 Hz, J =8.4 Hz), 6.98 (dd, 1H, J =1.6 Hz, J =8.4 Hz), 6.92 (t,
1
2
1
2
1
H, J=2.0 Hz), 4.32 (q, 2H, J=7.2 Hz), 3.85 (s, 3H), 2.92 (d, 2H, J=7.2 Hz), 1.89~1.82
13
(
m, 1H), 1.38 (t, 3H, J=7.2 Hz), 0.76 (d, 6H, J=6.8 Hz). C NMR (100 MHz, CDCl ):
3
1
7
63.69, 160.20, 147.55, 141.98, 140.27, 129.92, 118.58, 114.93, 112.81, 112.12,
7.35, 77.24, 77.03, 76.72, 59.95, 55.58, 33.38, 28.74, 22.24, 14.41; EI-MS: 303.5
+
[
M+H] .
4
.1.2.8. Ethyl 3-isobutyl-1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate (4h)
1
Yellow oil, yield: 37%. H NMR (400 MHz, CDCl ) δppm: 8.39 (dd, 2H, J =2.0
3
1
Hz, J =6.8 Hz), 8.09 (s, 1H), 7.64 (dd, 2H, J =2.0 Hz, J =6.8 Hz), 7.27 (s, 1H), 4.34
2
1
2
(q, 2H, J=7.2 Hz), 3.01 (d, 2H, J=7.2 Hz), 1.85~1.78 (m, 1H), 1.39 (t, 3H, J=7.2 H ),
1
3
0
1
1
4
.75 (d, 6H, J=6.8 Hz). C NMR (100 MHz, CDCl ): 163.28, 147.78, 147.34, 144.53,
3
43.15, 126.68, 124.82, 114.13, 77.35, 77.24, 77.04, 76.72, 60.23, 33.36, 29.09, 22.18,
+
4.38; EI-MS: 318.5 [M+H] .
.1.2.9. Ethyl 1-(4-fluorophenyl)-3-isobutyl-1H-pyrazole-4-carboxylate (4i)
1
Yellow oil, yield: 56%. H NMR (400 MHz, CDCl ) δppm:8.03 (s, 1H),
3
7
1
.38~7.35 (m, 2H), 7.21~7.17 (m, 2H), 4.32 (q, 2H, J=7.2 Hz), 2.88 (d, 2H, J=7.2 Hz),
13
.87~1.80 (m, 1H), 1.38 (t, 3H, J=7.2 Hz), 0.75 (d, 6H, J=6.8 Hz). C NMR (100
MHz, CDCl ): 163.75, 163.60, 161.27, 147.65, 142.11, 135.42, 135.39, 128.41,
3
1
1
4
28.32, 116.37, 116.14, 112.86, 77.34, 77.23, 77.02, 76.71, 59.97, 33.34, 28.76, 22.20,
+
4.39; EI-MS: 291.4 [M+H] .
.1.2.10. Ethyl 1-(2-fluorophenyl)-3-isobutyl-1H-pyrazole-4-carboxylate (4j)
1
Yellow oil, yield: 58%. H NMR (400 MHz, CDCl ) δppm: 8.09 (s, 1H),
3
7
2
.52~7.46 (m, 1H), 7.44~7.39 (m, 1H), 7.31~7.24 (m, 2H), 4.33 (q, 2H, J=7.2 Hz),
.79 (d, 2H, J=7.6 Hz), 1.90~1.80 (m, 1H), 1.38 (t, 3H, J=7.2 Hz), 0.75 (d, 6H, J=6.8
13
Hz). C NMR (100 MHz, CDCl ): 163.53, 158.33, 155.82, 149.02, 142.73, 131.29,
3
1
7
31.21, 129.52, 127.18, 127.06, 124.77, 124.73, 116.91, 116.72, 112.65, 77.36, 77.24,
+
7.04, 76.72, 59.99, 33.53, 33.51, 28.68, 22.20, 14.40; EI-MS: 291.4 [M+H] .

ACCEPTED MANUSCRIPT
4
.1.2.11. Ethyl 1-(3-fluorophenyl)-3-isobutyl-1H-pyrazole-4-carboxylate (4k)
1
Yellow oil, yield: 38%. H NMR (400 MHz, CDCl ) δppm:8.05 (s, 1H),
3
7
1
.50~7.45 (m, 1H), 7.21~7.14 (m, 3H), 4.34 (q, 2H, J=7.2 Hz), 2.94 (d, 2H, J=7.6 Hz),
13
.89~1.78 (m, 1H), 1.38 (t, 3H, J=7.2 Hz), 0.76 (d, 6H, J=6.8 Hz). C NMR (100
MHz, CDCl ): 163.90, 163.55, 161.43, 147.59, 142.37, 140.70, 140.60, 130.56,
3
1
30.47, 122.08, 122.05, 116.13, 115.92, 114.21, 113.97, 113.18, 77.35, 77.24, 77.03,
+
7
6.71, 60.04, 33.31, 28.85, 22.21, 14.40, EI-MS: 291.4 [M+H] .
4
.1.3. General procedure for preparation of compounds 5a and 5k
Ethyl 3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxylate (4a) or ethyl
-(3-fluorophenyl)-3-isobutyl-1H-pyrazole-4-carboxylate (4k) (0.69 mmol) was
1
added to the mixture solution of ethanol (3 mL), water (3 mL) and THF (3 mL) with
NaOH (1.29 g, 32.2 mmol). The reaction mixture was stirred at 50 ꢁ overnight.
Upon completion of the reaction, the ethanol and THF was evaporated, leaving a
residue which was neutralized with hydrochloric acid (1 mol/L) to PH=3, treated with
ethyl acetate (30 mL) and washed with water (3 x 30 mL). The organic layer was
dried over anhydrous sodium sulfate, and then the solvent was removed under
vacuum to give the desired compounds (5a or 5k) in good yield.
4
.1.3.1. 3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxylic acid (5a)
1
White solid, yield: 87%. H NMR (400 MHz, CDCl ) δppm: :12.79 (s, 1H), 8.11
3
(s, 1H), 7.76 (d, 1H, J=3.6 Hz), 7.67 (d, 1H, J=3.2 Hz), 3.45 (d, 2H, J=7.2 Hz),
1
3
2
1
.07~2.00 (m,1H), 0.88 (d, 6H, J=6.8 Hz); C NMR (100 MHz, CDCl ): 164.30,
3
61.71, 148.11, 144.06, 140.95, 119.37, 115.92, 32.70, 28.69, 22.34; EI-MS: 252.4
+
[
M+H] .
4
.1.3.2. 3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxylic acid (5k)
1
White solid, yield:66%. H NMR (400 MHz, CDCl ) δppm:8.02 (s, 1H), 7.60
3
(d, 1H, J=3.2 Hz), 7.18 (d, 1H, J=3.6 Hz), 4.33(q, 2H, J=7.2 Hz), 3.51 (d, 2H, J=7.2
13
Hz), 2.16~2.06 (m,1H), 1.38 (t, 3H, J=7.2 Hz), 0.94 (d, 6H, J=6.8 Hz); C NMR (100
MHz, CDCl ): 163.14, 161.89, 148.96, 143.46, 140.40, 117.18, 115.17, 60.25, 33.02,
3
+
2
8.89, 22.13, 14.35; EI-MS: 280.3 [M+H] .
4
.1.4. General procedure for preparation of compounds 6a1-6a9 and 6k1-6k8

ACCEPTED MANUSCRIPT
To the mixture solution of 3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxylic
acid (5a) or 3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxylic acid (5k) (0.38
mmol) in DMF (5 ml) 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) (174 mg, 0.46 mmol) was added under low temperature
with stirring for 10 minutes. Et N (77 mg, 0.76 mmol) and substituted amines (0.38
3
mmol) was added slowly with stirring under room temperature for 1h. Upon
completion of the reaction, the mixture solution was diluted with water (125 mL),
treated with ethyl acetate (3 x 30 mL) and washed with saturated NaCl (3 x 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
the desired compounds (6a1-9 and 6k1-8) in good yield.
4
.1.4.1.
N-(4-fluorobenzyl)-3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxamide (6a1)
1
White solid, yield: 72%. H NMR (400 MHz, CDCl ) δppm: 7.80 (s, 1H), 7.60 (d,
3
1
H, J=3.6 Hz), 7.32~7.29(m, 2H), 7.17 (d, 1H, J=3.6 Hz), 7.02 (t, 2H, J=8.4 Hz), 6.29
(s, 1H), 4.55 (d, 2H, J=5.6 Hz), 3.50 (d, 2H, J=7.2 Hz), 2.12~2.05 (m, 1H), 0.92 (d,
1
3
6
1
1
H, J=6.8 Hz); C NMR (100 MHz, CDCl ): 163.45, 162.82, 161.74, 161.01, 147.53,
3
40.43, 139.87, 134.12, 134.09, 133.82, 129.55, 129.47, 117.72, 117.17, 115.71,
15.49, 114.43, 77.37, 77.05, 76.74, 42.77, 32.92, 28.86, 22.12; EI-MS: 359.4
+
[
M+H] .
4
.1.4.2.
N-(2-fluorobenzyl)-3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxamide (6a2)
1
White solid, yield: 49%, MP: 101-102 ꢁ. H NMR (400 MHz, CDCl ) δppm:
3
7
.80 (s, 1H), 7.59 (d, 1H, J=3.2 Hz), 7.40 (t, 1H, J=7.2 Hz), 7.38~7.25(m, 1H), 7.16
(d, 1H, J=3.6 Hz), 7.14~7.04 (m, 2H), 6.24 (s, 1H), 4.65 (d, 2H, J=5.6 Hz), 3.48 (d,
1
3
2
H, J=7.2 Hz), 2.12~2.02 (m, 1H), 0.90 (d, 6H, J=7.2 Hz); C NMR (100 MHz,
CDCl ): 162.81, 162.35, 161.79, 159.90, 147.34, 140.40, 139.99, 130.39, 130.35,
3
1
3
29.45, 129.37, 125.28, 125.14, 124.40, 124.37, 117.87, 117.14, 115.55, 115.33,
+
7.59, 37.55, 32.93, 28.84, 22.06; EI-MS: 359.5 [M+H] .

ACCEPTED MANUSCRIPT
4
.1.4.3.
N-(4-fluorophenyl)-3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxamide (6a3)
1
White solid, yield: 28%, MP: 126-127 ꢁ. H NMR (400 MHz, CDCl ) δppm:
3
8
.37 (t, 1H, J=8.0 Hz), 7.95 (s, 1H), 7.71 (s, 1H), 7.62 (d, 1H, J=3.6 Hz), 7.20 (d, 1H,
J=3.6 Hz), 7.17~7.07 (m, 3H), 3.56 (d, 2H, J=7.2 Hz), 2.18~2.11 (m, 1H), 0.96 (d, 6H,
13
J=6.8 Hz); C NMR (100 MHz, CDCl ): 161.70, 160.77, 153.83, 151.42, 147.92,
3
1
1
4
3
40.45, 140.06, 126.37, 126.27, 124.70, 124.66, 124.52, 124.44, 122.05, 118.05,
+
17.39, 114.97, 114.78(d), 33.02, 28.92, 22.11; EI-MS: 345.4 [M+H] .
.1.4.4.
-isobutyl-1-(thiazol-2-yl)-N-(4-(trifluoromethyl)benzyl)-1H-pyrazole-4-carboxa
mide (6a4)
1
White solid, yield: 83%, MP: 163-164 ꢁ. H NMR (400 MHz, CDCl ) δppm:
3
7
.81 (s, 1H), 7.61 (s, 1H), 7.59 (d, 2H, J=7.2 Hz), 7.44 (d, 2H, J=8.0 Hz), 7.17 (d, 1H,
J=3.6 Hz), 6.35 (s, 1H), 4.64 (d, 2H, J=6.0 Hz), 3.51 (d, 2H, J=7.2 Hz), 2.12~2.05 (m,
1
3
1
1
7
4
H), 0.92 (d, 6H, J=7.2 Hz); C NMR (100 MHz, CDCl ): 162.97, 161.69, 147.71,
3
42.43, 140.44, 139.76, 127.88 (d), 125.70, 125.66, 117.50, 117.22, 77.34, 77.03,
+
6.71, 42.95, 32.93, 28.86, 22.09; EI-MS: 409.5 [M+H] .
.1.4.5.
N-(2,4-difluorobenzyl)-3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxamide
(6a5)
1
White solid, yield: 92%, MP: 107-108 ꢁ. H NMR (400 MHz, CDCl ) δppm:
3
7
6
2
1
1
1
7
4
3
.80 (s, 1H), 7.59 (d, 1H, J=3.2 Hz), 7.42~7.36(m, 1H), 7.16 (d, 1H, J=3.2 Hz),
.87~6.79 (m, 2H), 6.27 (s, 1H), 4.59 (d, 2H, J=5.6 Hz ), 3.48 (d, 2H, J=7.2 Hz),
1
3
.10~2.03 (m, 1H), 0.90 (d, 6H, J=6.4 Hz); C NMR (100 MHz, CDCl ): 163.77,
3
63.65, 162.87, 162.35, 162.23, 161.75, 161.30, 161.18, 159.87, 159.75, 147.39,
40.40, 139.91, 131.34, 131.28, 131.24, 131.19, 121.40, 121.36, 121.25, 121.21,
17.70, 117.16, 111.56, 111.52, 111.34, 111.31, 104.16, 103.91, 103.66, 77.34, 77.03,
+
6.71, 37.01, 36.98, 32.91, 28.83, 22.05; EI-MS: 377.5 [M+H] .
.1.4.6.
-isobutyl-N-(4-methoxybenzyl)-1-(thiazol-2-yl)-1H-pyrazole-4-carboxamide

ACCEPTED MANUSCRIPT
(6a6)
1
White solid, yield: 96%. MP: 111-113 ꢁ. H NMR (400 MHz, CDCl ) δppm:
3
7
6
.77 (s, 1H), 7.59 (d, 1H, J=3.2 Hz), 7.26 (d, 2H, J=8.4 Hz), 7.16 (d, 1H, J=2.8 Hz),
.87 (d, 2H, J=8.4 Hz), 6.12 (s, 1H), 4.52 (d, 2H, J=5.2 Hz), 3.80 (s, 3H), 3.51 (d, 2H,
13
J=7.6 Hz), 2.13~2.04 (m, 1H), 0.93 (d, 6H, J=6.8 Hz); C NMR (100 MHz, CDCl ):
3
1
1
4
62.69, 161.81, 159.16, 147.41, 140.40, 139.91, 130.30, 129.26 (d), 117.94, 117.10,
+
14.19, 77.36, 77.04, 76.72, 55.32, 43.05, 32.93, 28.86, 22.12; EI-MS: 371.5 [M+H] .
.1.4.7.
N-(3-fluorobenzyl)-3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxamide (6a7)
1
White solid, yield: 79%, MP: 117-119 ꢁ. H NMR (400 MHz, CDCl ) δppm:
3
7
7
4
.81 (s, 1H), 7.59 (d, 1H, J=3.6 Hz), 7.29 (q, 1H, J=7.6 Hz), 7.17 (d, 1H, J=3.2 Hz),
.10 (d, 1H, J=7.6 Hz), 7.03 (d, 1H, J=9.6 Hz), 6.97 (t, 1H, J=7.6 Hz), 6.28 (s, 1H),
.58 (d, 2H, J=5.6 Hz ), 3.51 (d, 2H, J=7.2 Hz), 2.12~2.06 (m, 1H), 0.92 (d, 6H, J=6.4
13
Hz); C NMR (100 MHz, CDCl ): 164.27, 162.88, 161.82, 161.73, 147.62, 140.93,
3
1
1
40.86, 140.43, 139.83, 130.32, 130.23, 123.23, 123.20, 117.64, 117.18, 114.71,
14.57, 114.49, 114.36, 77.35, 77.03, 76.72, 42.94, 32.93, 28.87, 22.11; EI-MS: 359.6
+
[
M+H] .
4
.1.4.8.
N-(4-aminobenzyl)-3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxamide (6a8)
1
White solid, yield: 79%, MP: 126-128 ꢁ. H NMR (400 MHz, CDCl ) δppm:
3
7
6
3
1
1
4
.75 (s, 1H), 7.59 (d, 1H, J=3.2 Hz), 7.15 (d, 2H, J=3.2 Hz), 7.13 (d, 1H, J=8.0 Hz),
.65 (d, 2H, J=8.0 Hz), 6.06 (s, 1H), 4.46 (d, 2H, J=5.6 Hz), 3.50 (d, 2H, J=7.2 Hz),
1
3
.42 (s, 2H), 2.14~2.06 (m, 1H), 0.92 (d, 6H, J=6.4 Hz); C NMR (100 MHz, CDCl ):
3
62.64, 161.84, 147.32, 145.97, 140.38, 139.95, 129.28, 127.97, 118.04, 117.08,
+
15.29, 77.37, 77.05, 76.73, 43.26, 32.92, 28.86, 22.13; EI-MS: 356.5 [M+H] .
.1.4.9.
N-(4-hydroxybenzyl)-3-isobutyl-1-(thiazol-2-yl)-1H-pyrazole-4-carboxamide
(6a9)
1
White solid, yield: 55%, MP: 122-124 ꢁ. H NMR (400 MHz, CDCl ) δppm:
3
9
.31 (s, 1H), 8.70 (s, 1H), 8.24 (s, 1H), 7.73 (s, 1H), 7.63 (s, 1H), 7.12 (d, 2H, J=7.6

ACCEPTED MANUSCRIPT
Hz), 6.72 (d, 2H, J=7.6 Hz), 4.33 (d, 2H, J=4.0 Hz), 3.48 (d, 2H, J=7.2 Hz), 2.01~1.98
1
3
(
m, 1H), 0.84 (d, 6H, J=6.0 Hz); C NMR (100 MHz, CDCl ): 162.28, 161.91,
3
1
4
4
56.71, 146.58, 141.59, 140.91, 130.26, 129.05, 119.07, 118.51, 115.48, 42.01, 40.62,
+
0.41, 40.20, 40.00, 39.79, 39.58, 39.37, 32.53, 28.71, 22.37; EI-MS: 357.4 [M+H] .
.1.4.10.
N-(4-fluorobenzyl)-1-(3-fluorophenyl)-3-isobutyl-1H-pyrazole-4-carboxamide
(6k1)
1
White solid, yield: 55%. H NMR (400 MHz, CDCl ) δppm: 7.79 (s, 1H),
3
7
.49~7.44 (m, 1H), 7.31 (dd, 2H, J =5.6 Hz, J =8.8 Hz ), 7.19 (dd, 2H, J =2.0 Hz,
1
2
1
J2=8.0 Hz ), 7.15~7.12 (m, 1H), 6.24 (s, 1H), 4.56 (d, 2H, J=6.0 Hz), 2.98 (d, 2H,
13
J=7.6 Hz ), 1.87~1.76 (m,1H), 0.75 (d, 6H, J=6.4 Hz); C NMR (100 MHz, CDCl ):
3
1
1
1
63.91, 163.47, 163.33, 161.44, 161.02, 146.43, 140.77, 140.67, 138.42, 134.31,
34.28, 130.56, 130.47, 129.50, 129.42, 121.95, 121.91, 116.09, 115.88, 115.73,
15.71, 115.49, 114.08, 113.84, 77.36, 77.25, 77.04, 76.73, 42.69, 33.15, 28.78, 22.19;
+
EI-MS: 370.4 [M+H] .
4
.1.4.11.
N-benzyl-1-(3-fluorophenyl)-3-isobutyl-1H-pyrazole-4-carboxamide
(6k2)
1
White solid, yield: 60%. H NMR (400 MHz, CDCl ) δppm: :7.79 (s, 1H),
3
7
.79~7.44 (m, 1H), 7.36~7.27 (m, 5H), 7.20~7.12 (m, 3H), 6.21 (s, 1H), 4.61 (d, 2H,
1
3
J=4.4 Hz), 2.99 (d, 2H, J=7.2 Hz ), 1.88~1.78 (m,1H), 0.75 (d, 6H, J=6.4 Hz); C
NMR (100 MHz, CDCl ): 163.90, 163.27, 161.42, 146.40, 140.74, 140.64, 138.40,
3
1
1
4
1
30.55, 130.46, 128.79, 127.85, 127.58, 121.96, 121.93, 116.07, 115.86, 114.09,
+
13.85, 77.35, 77.24, 77.04, 76.72, 43.45, 33.15, 28.77, 22.20; EI-MS: 352.5 [M+H] .
.1.4.12.
-(3-fluorophenyl)-N-(4-fluorophenyl)-3-isobutyl-1H-pyrazole-4-carboxamide
(6k3)
1
White solid, yield: 48%. H NMR (400 MHz, CDCl ) δppm:8.00 (s, 1H), 7.78 (s,
3
1
7
1
H), 7.56~7.53 (m, 2H), 7.51~7.45 (m, 1H), 7.21 (dd, 2H, J =2.0 Hz, J =8.0 Hz ),
1
2
.16 (dd, 1H, J =2.0 Hz, J =9.2 Hz), 7.04 (t, 2H, J=8.4 Hz), 2.99 (d, 2H, J=7.2 Hz),
1
2
1
3
.89~1.82 (m,1H), 0.75 (d, 6H, J=6.8 Hz); C NMR (100 MHz, CDCl ): 163.91,
3

ACCEPTED MANUSCRIPT
1
1
1
4
1
61.52, 161.43, 160.73, 158.30, 147.24, 140.40, 140.30, 138.37, 133.79, 133.76,
30.66, 130.57, 122.42, 122.34, 122.04, 122.00, 116.36, 116.15, 115.96, 115.81,
15.59, 114.17, 113.93, 77.36, 77.24, 77.04, 76.72, 33.19, 28.74, 22.21.
.1.4.13.
-(3-fluorophenyl)-3-isobutyl-N-((4-methoxybenzyl)oxy)-1H-pyrazole-4-carboxa
mide (6k4)
1
White solid, yield: 48%. H NMR (400 MHz, CDCl ) δppm: 8.67 (s, 1H), 7.74 (s,
3
1
H), 7.48~7.43(m, 1H), 7.35 (d, 2H, J=8.8 Hz), 7.19~7.10(m, 1H), 6.90 (d, 2H, J=8.4
Hz), 4.92 (s, 2H), 3.81 (s, 3H), 2.93 (d, 2H, J=7.6 Hz), 1.82~1.75 (m, 1H), 0.74 (d, 6H,
13
J=6.4 Hz); C NMR (100 MHz, CDCl ): 163.88, 161.40, 160.07, 146.97, 140.59,
3
1
1
40.49, 138.74, 131.04, 130.56, 130.47, 127.40, 121.92, 121.89, 116.12, 115.91,
14.04, 114.01, 113.80, 112.79, 78.19, 77.38, 77.06, 76.74, 55.29, 33.17, 28.71, 22.17;
+
EI-MS: 398.4 [M+H] .
4
1
.1.4.14.
-(3-fluorophenyl)-N'-(4-fluorophenyl)-3-isobutyl-1H-pyrazole-4-carbohydrazide
(6k5)
1
White solid, yield: 45%. H NMR (400 MHz, DMSO) δppm: 10.08 (s, 1H), 8.27
(s, 1H), 7.87 (s, 1H), 7.61 (q, 1H, J=6.8 Hz), 7.48 (d, 1H, J=9.6 Hz), 7.38 (t, 2H, J=8.0
Hz), 7.00 (t, 2H, J=8.8 Hz), 6.80~6.77(m, 2H), 2.96 (d, 2H, J=7.6 Hz), 1.65~1.58 (m,
1
3
1
1
1
4
4
1
H), 0.64 (d, 6H, J=6.4 Hz); C NMR (100 MHz, DMSO): 163.66, 163.27, 161.21,
57.48, 155.16, 146.79, 146.27, 141.13, 141.03, 139.60, 131.59, 131.50, 122.73,
16.28, 116.08, 115.71, 115.49, 114.40, 114.07, 113.88, 113.80, 40.65, 40.44, 40.24,
+
0.03, 39.82, 39.61, 39.40, 32.86, 28.40, 22.43; EI-MS: 371.4 [M+H] .
.1.4.15.
-(3-fluorophenyl)-3-isobutyl-N-(pyridin-4-ylmethyl)-1H-pyrazole-4-carboxamid
e (6k6)
1
White solid, yield: 61%. H NMR (400 MHz, CDCl ) δppm:8.53 (s, 1H), 7.89 (s,
3
1
2
H), 7.47 (q, 1H, J=6.4 Hz), 7.28~7.14(m, 5H), 6.81 (s, 1H), 4.60 (d, 2H, J=6.0 Hz),
13
.99 (t, 2H, J=7.2 Hz), 1.84~1.78 (m, 1H), 0.75 (d, 6H, J=6.8 Hz); C NMR (100
MHz, CDCl ): 163.89, 163.63, 161.42, 149.90, 147.96, 146.73, 140.67, 140.58,
3

ACCEPTED MANUSCRIPT
1
1
4
4
1
38.52, 130.60, 130.51, 122.29, 121.93, 121.90, 116.16, 115.95, 115.37, 114.07,
+
13.83, 77.37, 77.05, 76.73, 42.13, 33.12, 28.76, 22.18; EI-MS: 353.4 [M+H] .
.1.4.16.
Tert-butyl
-((1-(3-fluorophenyl)-3-isobutyl-1H-pyrazole-4-carboxamido)methyl)piperidine-
-carboxylate (6k7)
1
White solid, yield: 60%. H NMR (400 MHz, CDCl ) δppm: 7.81 (s, 1H),
3
7
3
1
.50~7.44(m, 1H), 7.20~7.13(m, 3H), 6.16 (t, 1H, J=6.0 Hz), 4.13 (d, 2H, J=12.8 Hz),
.30 (d, 2H, J=5.2 Hz), 2.97 (d, 2H, J=7.6 Hz), 2.70 (t, 2H, J=12.4 Hz),
1
3
.84~1.72(m, 4H), 1.46 (s, 9H), 1.23~1.13 (m, 2H), 0.75 (d, 6H, J=6.8 Hz); C NMR
(
100 MHz, CDCl ): 163.88, 163.61, 161.41, 154.82, 146.23, 140.77, 140.67, 138.33,
3
1
7
4
30.54, 130.45, 121.93, 121.90, 116.05, 115.96, 115.84, 114.06, 113.82, 79.41, 77.36,
7.25, 77.05, 76.73, 44.80, 43.61, 36.57, 33.11, 29.88, 28.74, 28.46, 22.17; EI-MS:
+
59.6 [M+H] .
4
.2. Biological assays
The cytotoxicity and anti-HBV activities of compounds were evaluated on the
HepG2.2.15 cell line. Cytotoxicity was assayed with cell counting kit-8 method
CCK-8; Dojindo, Tokyo, Japan). The anti-HBV antigen secretion activities were
(
assayed by enzyme linked immunosorbent assay (ELISA; Autobio Diagnostics Co.,
Ltd, China). A real-time PCR assay was used to detect the inhibiting HBV DNA
replication of the derivatives[20].
4
.2.1. Toxicity measurements[21]
Cytotoxicity induced by the tested compounds to HepG2.2.15 cells was assessed
by CCK-8 method. Briefly, HepG2.2.15 cells were cultured in triplicate of 96-well
tissue culture plates for 8 days with five different doses of tested compounds.
Untreated cells with media alone were used as controls. The CCK-8 solution was
added 0.5 h before the end of culture, and then. OD absorbance values at 450 nm were
collected by microplate reader (Bio-Rad, model 550) and the cell death percent was
calculated[22, 23].
4
.2.2. Inhibiting the secretion of HBeAg and HBsAg[24, 25]

ACCEPTED MANUSCRIPT
Aspirate cell supernatant and detect content of HBeAg and HbsAg by using
diagnostic kit (Autobio Diagnostics Co., Ltd, China). The absorbance (A) of the
sample was determined on microtiter plate ELISA reader. Drug inhibition ratio = (A
normal cell group-A experimental group) / (A blank group-A normal cell group) ×
1
00%. The concentration of compound required for 50% inhibition of HBeAg,
HBsAg secretion was defined as IC .
5
0
4
.2.3. Real time fluorescent PCR[11]
The supernatants of HepG2.2.15 cells were collected from eight days’ culture
after the compounds added. And the HBV DNA in the supernatants was quantified by
using PCR- fluorescent probing (Quantitative diagnostic kit for HBV DNA). Briefly,
1
00 ꢀL of the supernatants were added into the extraction buffer, boiled for 10 min
and centrifuged for 10 min, and then proper aliquots were used for the fluorescent
probing PCR. PCR reaction was run and results were analyzed.
Acknowledgments
The financial support from the National Natural Science Foundation of
China (NSFC Nos. 81273354), Key Project of NSFC for International
Cooperation (Nos. 81420108027, 30910103908), Major Project of Science and
Technology of Shandong Province (2015ZDJS04001), and the Science and
Technology Development Project of Shandong Province(No. 2014GSF118175) is
gratefully acknowledged.
References and notes
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Figure captions
Figure 1. Five nucleoside/nucleotide drugs approved by US FDA for HBV treatment
Figure 2. The structures of the anti-HBV natural product Leucamide A and it derivatives
compound B, compound C obtained by structural simplification.
Figure 3. The structures of three lead compounds and the newly designed pyrazole series via
bioisosterism and pharmacophore hybrid strategy.
Fig. 4. Inhibitory effect of pyrazole derivatives on HBV DNA level.
Table captions
Table 1. Anti-HBV activity, cytotoxicity and selectivity indices of pyrazole derivatives
(4a-4j, 6a1-6a9 and 6k1-6k7) and positive drug lamivudine.
a
Table 2. Prediction of physicochemical properties of 6a1, 6a3, 6a5 and 6k3.

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Schemes
R1
O
N
O
R1
N
N
R1
N
N
N
N
O
O
O
i
ii
O
iii
v
iv
O
O
O
NH
R2
COOEt
a-4k
O
COOH
a: R1 = thiazol-2-yl;
1
2
3
4
6a1-6a9: R1 = thiazol-2-yl;
5
6k1-6k7: R1 = 3-fluorophenyl
5
k: R1 = 3-fluorophenyl
Scheme 1. Reagents and conditions: (i) 4-methylpentan-2-one, NaH, AcOH, THF; (ii)
1
1
,1-dimethoxy-N,N-dimethylmethanamine, 80
ꢁ
(iii) EtOH, Et N, R -NHNH ; (iv) NaOH, H O,
3
2
2
2
THF, EtOH; (v) R -NH , DMF, HATU, Et N.
2
3