Discovery of novel diarylpyrazolylquinoline derivatives as potent anti-dengue virus agents

Article abstract of DOI:10.1016/j.ejmech.2017.10.001

A number of diarylpyrazolylquinoline derivatives were synthesized and evaluated for their anti-dengue virus (DENV) activity. Among them, 6-fluoro-2-(1-(4-fluorophenyl)-3- (4-methoxyphenyl)-1H-pyrazol-5-yl)quinoline (11c), 2-[1,3-bis(4-methoxyphenyl)-1H-pyrazol- 5-yl]-6-fluoroquinoline (12c), and 4-[5-(6-fluoroquinolin-2-yl)-3-(4-methoxyphenyl)-1H-pyrazol- 1-yl]benzenesulfonamide (13c) exhibited approximately 10-folds more active anti-DENV-2 activity (IC₅₀ of 1.36, 1.09 and 0.81 μM, respectively) than that of ribavirin (IC₅₀ = 12.61 μM). Compound 13c was also potent inhibited other sero-types of DENV. It reduced DENV replication in both viral protein and mRNA levels, and no significant cell cytotoxicity was detected, with greater than 50% viability of Huh-7-DV-Fluc cells at a concentration of 200 μM. Furthermore, compound 13c can effectively protect mice from DENV infection by reducing disease symptoms and mortality of DENV-infected mice. It represents a potential antiviral agent to block DENV replication in vitro and in vivo. Structural optimization of the initial lead compound, 13c, and the detailed molecular mechanism of action are ongoing.

Full text of DOI:10.1016/j.ejmech.2017.10.001

Accepted Manuscript
Discovery of novel diarylpyrazolylquinoline derivatives as potent anti-dengue virus
agents
Jin-Ching Lee, Chin-Kai Tseng, Chun-Kuang Lin, Chih-Hua Tseng
PII:
S0223-5234(17)30793-6
10.1016/j.ejmech.2017.10.001
EJMECH 9793
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 July 2017
Revised Date: 29 September 2017
Accepted Date: 1 October 2017
Please cite this article as: J.-C. Lee, C.-K. Tseng, C.-K. Lin, C.-H. Tseng, Discovery of novel
diarylpyrazolylquinoline derivatives as potent anti-dengue virus agents, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.001.
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ACCEPTED MANUSCRIPT
Graphical abstract
New diarylpyrazolylquinolines with anti-DENV activity were designed and
synthesized. Among them, compound 13c was potent inhibited all four sero-types of
DENV. It can effectively protect mice from DENV infection by reducing disease
symptoms and mortality of DENV-infected mice.

ACCEPTED MANUSCRIPT
Discovery of novel diarylpyrazolylquinoline derivatives as potent anti-dengue virus agents
a,b,c
d,e
f,g
c,h,i,j,k*
Jin-Ching Lee , Chin-Kai Tseng , Chun-Kuang Lin , and Chih-Hua Tseng
a
Department of Biotechnology, College of Life Science, Kaohsiung Medical University,
Kaohsiung City 807, Taiwan
b
Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University,
Kaohsiung, Taiwan
c
Research Center for Natural Products and Drug Development, Kaohsiung Medical University,
Kaohsiung, Taiwan
d
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University,
Tainan, Taiwan
e
Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung
University, Tainan, Taiwan
f
Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University, Kaohsiung,
Taiwan
g
Doctoral Degree Program in Marine Biotechnology, Academia Sinica, Taipei, Taiwan
h
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan
i
Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical
University, Kaohsiung City 807, Taiwan
j
Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung
City 807, Taiwan
k
Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung City 807,
Taiwan
*Corresponding author. Tel.: +886 7 3121101 x2163; fax: +886 7 3125339.
E-mail addresses: chihhua@kmu.edu.tw
1

ACCEPTED MANUSCRIPT
Abstract
A number of diarylpyrazolylquinoline derivatives were synthesized and evaluated for their
anti-dengue virus (DENV) activity. Among them, 6-fluoro-2-(1-(4-fluorophenyl)-3-
(4-methoxyphenyl)-1H-pyrazol-5-yl)quinoline (11c), 2-[1,3-bis(4-methoxyphenyl)-1H-pyrazol-
5
-yl]-6-fluoroquinoline (12c), and 4-[5-(6-fluoroquinolin-2-yl)-3-(4-methoxyphenyl)-1H-pyrazol-
-yl]benzenesulfonamide (13c) exhibited approximately 10-folds more active anti-DENV-2 activity
1
(
IC of 1.36, 1.09 and 0.81 µM, respectively) than that of ribavirin (IC = 12.61 µM). Compound
50 50
13c was also potent inhibited other sero-types of DENV. It reduced DENV replication in both viral
protein and mRNA levels, and no significant cell cytotoxicity was detected, with greater than 50%
viability of Huh-7-DV-Fluc cells at a concentration of 200 µM. Furthermore, compound 13c can
effectively protect mice from DENV infection by reducing disease symptoms and mortality of
DENV-infected mice. It represents a potential antiviral agent to block DENV replication in vitro
and in vivo. Structural optimization of the initial lead compound, 13c, and the detailed molecular
mechanism of action are ongoing.
Keywords: Diarylpyrazolylquinoline, Ribavirin, Dengue virus, Anti-dengue virus activity
2

ACCEPTED MANUSCRIPT
1. Introduction
Dengue is a mosquito-borne viral disease that has become a major public health concern
worldwide in recent years. Annually, 100 million cases of dengue fever and 500,000 cases of
dengue hemorrhagic fever occur, particularly in tropical Asia, Latin America, and the Caribbean [1].
At present, dengue is endemic in 112 countries around the world [2]. However, there is no vaccine
or treatment other than vector control and supportive medical care. The development of safe and
effective therapeutics is therefore urgently needed [3-6].
The dengue virus (DENV) belongs to the family Flaviviridae and is closely related to the
West Nile virus (WNV), the Yellow Fever virus (YFV) and the Hepatitis C virus (HCV) [7].
DENV are transmitted by mosquitos of the species Aedes aegypti and cause clinical symptoms
ranging from mild fever to Dengue hemorrhagic fever (DHS) and Dengue shock syndrome (DSS)
[
8, 9]. However, there are no specific approved drugs or vaccines for the treatment or prevention of
DHS and DSS.
Quinoline and pyrazole are isosteric with purine and pyrimidine nuclei respectively, which are
present in a number of fundamental cellular components and bioactive compounds [10-16]. This
heterocycle may represent a kind of privileged substructure, which may interact with different
proteins and enzymes. Indeed, a number of important drugs used in different therapeutic areas
contain the quinoline ring, as well as several other kinds of still investigational therapeutic agents,
including antitumorals [17-20] and antivirals [21-25]. Among these known antiviral quinoline and
pyrazole derivatives, compound 1 (Figure 1) exhibited inhibitory activities against a DENV
serotype 2 (DENV-2) sub-genomic replicon cell line with an IC value of 5 µM and against a
5
0
WNV sub-genomic replicon cell line with an IC of 1.9 µM [26]. Compound 2 inhibited the
50
growth of Hepatitis C virus with an IC value of 7.0 µM [27]. Recently, we have synthesized a
5
0
number of 2-aroyl-3-arylquinoline derivatives and evaluated for their anti-DENV activity [28].
Compounds 3 and 4 were found to significantly inhibit the DENV-2 RNA expression in
3

ACCEPTED MANUSCRIPT
Huh-7-DV-Fluc cells with potencies approximately equal to that of ribavirin. Both compounds
were capable of reducing DENV-2 replication in both viral protein and mRNA levels. Compound 5
is a triarylpyrazoline derivative which has been discovered to inhibit flavivirus infection in cell
culture [29]. In continuation of our studies to identify potential antiviral agents with a novel type of
structures, we describe herein the preparation of hybrid pyrazolylquinoline derivatives (target
compounds, Figure 1) and evaluation of their anti-DENV activity in vitro and in vivo.
<
Insert Figure 1 here >
2
. Chemistry
Oxidation
-fluoro-2-formylquinoline (7) which was then condensed with acetophenone to afford exclusively
of
6-fluoro-2-methylquinoline
(6)
with
selenium
dioxide
gave
6
trans conjugated carbonyl product, (E)-3-(6-fluoroquinolin-2-yl)-1-phenylprop-2-en-1-one (8a) as
depicted in Scheme 1. Accordingly, compounds 8b and 8c were obtained from 4-fluoroactophenone
and 4-methoxyacetophenone respectively with compound 7 under the same reaction conditions.
Treatment of 8a with phenylhydrazine followed by DDQ oxidation gave a mixture of
2-(2,3-diphenyl-2H-pyrazol-5-yl)-6-fluoroquinoline
(9a)
and
2-(2,5-diphenyl-2H-
pyrazol-3-yl)-6-fluoroquinoline (10a) in 21% and 36% yield respectively as outlined in Scheme 2.
The mixture of 9a and 10a was separated by silica gel column chromatography eluting with solvent
mixtures of CH Cl -MeOH (100:1, 50:1, and 20:1).
2
2
Accordingly, a mixture of compounds 9b and 10b was obtained from compound 8b with
phenylhydrazine and the mixture of compounds 9c and 10c was obtained from compound 8c under
the same reaction conditions. However, reflux of 8a with 4-fluorophenylhydrazine hydrochloride
gave exclusively 6-fluoro-2-[1-(4-fluorophenyl)-3-phenyl-1H-pyrazol-5-yl]quinoline (11a) as a
sole product. Compound 11b and 11c was obtained from 4-fluorophenylhydrazine hydrochloride
and compound 8b and 8c respectively. Reflux of 8a with 4-methoxyphenylhydrazine hydrochloride
gave exclusively 6-fluoro-2-[1-(4-methoxyphenyl)-3-phenyl-1H-pyrazol-5-yl]quinoline (12a) as a
sole product. Compound 12b and 12c was obtained from 4-methoxyphenylhydrazine hydrochloride
4

ACCEPTED MANUSCRIPT
and compound 8b and 8c respectively under the same reaction conditions. Accordingly, reflux of
8a with 4-hydrazinobenzenesulfonamide hydrochloride gave exclusively 4-[5-(6-fluoroquinolin-2-
yl)-3-phenyl-1H-pyrazol-1-yl]benzenesulfonamide (13a) as a sole product. Compound 13b and 13c
was obtained from 4-hydrazinobenzenesulfonamide hydrochloride and compound 8b and 8c
respectively under the same reaction conditions. It is of interest to note that when 14 was reacted
with 4-hydrazinobenzenesulfonamide hydrochloride the product was identified as the 13c and not
the expected regioisomer 15 as depicted in Scheme 3.
<
Insert Scheme 1-3 here >
Structure of 9c and 10c were unambiguously determined by X-ray crystallographical analysis.
The structure was solved and refined by direct methods Shelx 97 suite of programs [30].
Compounds 9c (R = 0.52, (CH Cl ：hexane = 4/1)) and 10c (R = 0.51, (CH Cl ：hexane = 4/1))
f
2
2
f
2
2
were obtained by slow evaporation from MeOH/CH Cl (30/70) solution. The crystal structure and
2
2
crystal packing diagram of compounds 9c and 10c were shown in Figure 2, respectively, while the
crystallographic data and structure refinement details were given in Table 1. Complete
crystallographic data for the structural analysis have been deposited with the Cambridge
Crystallographic Data Centre, CCDC 1054761 for compound 9c, and CCDC 1054760 for
compound 10c. Copies of this information may be obtained free of charge from the Director,
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge, CB2 1EZ, UK. (fax:
+44-1223-336033, e-mail: deposit@ccdc.cam.ac.uk or via www.ccdc.cam.ac.uk).
The X-ray analysis proved unambiguously the regio-chemistry of the synthesized compounds
1
and was in good agreement with the results by the H NMR analysis. As shown in Figure 2,
compounds 9c and 10c with the same formula were clearly assigned as the N-2-phenyl and
N-3-phenyl isomers, respectively. Compounds 9c and 10c had a singlet signal appearing at δ=
7
.30, 7.18 ppm in CDCl andδ= 7.29, 7.49 ppm in DMSO-d were assigned as pyrazole 5-H,
3
6
respectively (Figure 3). The structures of the N-2 (9a-c) and N-3 (10a-b, 11a-c, and 12a-c)
1
substituted isomers in Table 2 were assigned according to the methods of X-ray and H NMR for
5

ACCEPTED MANUSCRIPT
9c and 10c. Consistent with previous observations, the N-2 substituted isomers (e.g., 9a-c) had
chemical shifts at the range of 7.30-7.38 ppm (lower fields) for the pyrazole 5-H as a singlet signal
1
in their individual H-NMR spectra in CDCl , while the N-3 substituted isomers (e.g., 10a-c, 11a-c,
3
and 12a-c) had chemical shifts at the range of of 7.16-7.25 ppm (higher fields) for the pyrazole 5-H
as a singlet signal in CDCl . The compounds 13a-c had chemical shifts at the range of of 7.58-7.68
3
1
ppm (lower fields) for the pyrazole 5-H as a singlet signal in their individual H-NMR spectra in
DMSO-d , which was similar to the N-3 substituted isomers 10c with a singlet signal appearing at
6
δ= 7.49 ppm (lower fields) in DMSO-d In conclusion, all the structures of the regioisomers in
6
.
1
this series could be definitely assigned by the H NMR combined with single-crystal X-ray
diffraction.
<
Insert Figure 2,3 and Table 1,2 here >
3. Biological Results and Discussion
3
.1. Anti-DENV-2 and Antiproliferative Activities
The anti-DENV-2 activities and cytotoxicities of diarylpyrazolylquinoline derivatives are
summarized in Table 3. Huh-7-DV-Fluc cells were treated with compounds 9 – 13 or the positive
ribavirin respectively at a concentration of 1 or 10 µM for three days. Cells were then analyzed
through the firefly luciferase activity. Compounds which exhibited >32% inhibition of DENV-2 at
a concentration of 10 µM were considered as active. Results from Table 3 indicated that
2-(1,3-diphenyl-1H-pyrazol-5-yl)-6-fluoroquinoline (9a), which exhibited 15% inhibition of
DENV-2 at a concentration of 10 µM, was more active than its fluoro-substituted counterpart, 9b,
and methoxy-substituted counterpart 9c. Compound 9a was also more active than its positional
isomer, 10a. However, 6-fluoro-2-[5-(4-fluorophenyl)-1-phenyl-1H-pyrazol-3-yl]quinoline (10b),
which exhibited 25% inhibition of DENV-2 at a concentration of 10 µM, was more active than its
positional isomer, 9b. Introduction of 4-methoxy or 4-aminosulfonyl at N-3 phenyl ring improved
anti-DENV2 in which compounds 12a and 13a were more active than 10a while compounds 12c
and 13c were more active than 10c. However, compound 10b was exceptionally active against
6

ACCEPTED MANUSCRIPT
DENV-2 and more active than that of 12a and 13a. Among these pyrazolylquinoline derivatives,
6
-fluoro-2-(1-(4-fluorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-5-yl)quinoline (11c), 2-[1,3-bis
4-methoxyphenyl)-1H-pyrazol-5-yl]-6-fluoroquinoline (12c), and 4-[5-(6-fluoroquinolin-2-yl)-
-(4-methoxyphenyl)-1H-pyrazol-1-yl]benzenesulfonamide (13c) are three of the most active
(
3
which exhibited 55%, 66% and 85% inhibition of DENV-2 replication in the Huh-7-DV-Fluc cells
at a concentration of 10 µM compared to 33% inhibition exhibited by the positive ribavirin. The
anti-DENV2 activity decreased in an order of 13c (R = OMe, R = SO NH ; 85% inhibition) > 12c
1
2
2
2
(
R = OMe, R = OMe; 66% inhibition) > 11c (R = OMe, R = F; 55% inhibition) indicated that
1 2 1 2
R -substitution is the most prefer to be a 4-aminosulfonyl group. With the same 4-aminosulfonyl
2
group substituted at R -position, the methoxy group substituted at R -position is the most favorable
2
1
in which the anti-DENV2 activity decreased in an order of 13c (R = OMe; 85% inhibition) > 13b
1
(
R = F; 13% inhibition), 13a (R = H; 12% inhibition).
1
1
The cell cytotoxicity of was determined by XTT assay in the Huh-7 cells after 3 days
treatment with 20 and 200 µM of diarylpyrazolylquinoline derivatives. Compound 11c, 12c and 13c
exhibited low cytotoxicity, with greater than 50% viability at a concentration of 200 µM. The 50%
inhibitory concentration of DENV-2 replication (IC ), the 50% cytotoxic concentration for cell
5
0
growth (CC ), and the selective index (SI : CC /IC ) of compounds 11c, 12c and 13c were
50
50
50
determined as shown in Table 4. Results indicated that these compounds exhibited approximately
0-folds more active anti-DENV-2 activity (IC of 1.36, 1.09 and 0.81 µM, respectively) than that
1
5
0
of ribavirin (IC = 12.61 µM). These compounds have also demonstrated a good selectivity with SI
5
0
value of greater than 147, 183 and 247 respectively which was higher than that of ribavirin (SI =
.47). Compound 13c was also tested for other sero-types of DENV, and results shown in Table 5
indicated that it exhibited potent inhibition of all sero-types of DENV.
Insert Table 3-5 here >
4
<
3.2. Compound 13c reduced DENV-2 replication in DENV-2-infected Huh-7 cells
To confirm the results of dose-dependent decrease luciferase activity representing the
7

ACCEPTED MANUSCRIPT
DENV-2 replication in the Huh-7-DV-Fluc cells, we performed western blotting and RT-qPCR
using a specific antibody against DENV-2 NS2B protein and specific primers targeting DENV-2
NS5 gene, respectively, in which cellular GAPDH was served as a loading control. Both results of
western blotting and RT-qPCR revealed that 13c consistently reduced DENV-2 replication in the
Huh-7-DV-Fluc cells at the indication concentrations after 3 days treatment. Treatment of 0.1%
DMSO were served as a mock control on inhibition of DENV-2 replication (Figures 4 and 5).
<
Insert Figure 4,5 here >
3.3. Compound 13c decreases the mortality of DENV-infected ICR suckling mice.
To investigate whether 13c exerted protective effects against DENV infection in vivo,
-day-old ICR suckling mice were injected with DENV-2 intracerebrally and with 13c (1, 5, 10 and
0 mg/kg) intraperitoneally at 1, 3, and 5 days post-infection (dpi). Mice injected with heat-inactive
6
2
DENV-2 (iDENV) were used as mock controls. Survival rates and clinical scores of
DENV-infected mice treated with or without 13c were measured daily for 6 days. As shown in
Figure 6A, DENV-infected mice that were not treated with 13c developed severe sickness leading
to death within 4–6 dpi compared with iDENV-infected control mice. In contrast, 13c at various
concentrations shielded the mice from the life-threatening effects of the DENV-2 infection when
compared to the non-13c-treated mice. DENV-infected mice that did not receive 13c developed
severe paralysis, anorexia, and asthenia and lost of their body weight compared with
iDENV-treated mice at 6 dpi (Figure 6B and 6C). In contrast, DENV-infected mice treated with
13c showed slight paralysis, anorexia, and asthenia compared with iDENV-infected mice at 6 dpi
(Figure 6B and 6C).
<
Insert Figure 6 here >
4. Conclusion
We have identified 4-[5-(6-fluoroquinolin-2-yl)-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]-
benzenesulfonamide (13c) as a potential anti-DENV agent which exhibited a potent inhibitory
activity against DENV-2 (IC = 0.81 µM, SI > 246.91) and was more active than the positive
5
0
8

ACCEPTED MANUSCRIPT
ribavirin (IC = 12.61 µM, SI = 4.47). It was also potent inhibited other sero-types of DENV.
5
0
Furthermore, it can effectively protect mice from DENV infection by reducing disease symptoms
and mortality of DENV-infected mice. It represents a potential antiviral agent to block DENV
replication in vitro and in vivo. Further structural optimization of compound 13c is ongoing.
5
5
. Experimental
.1. General
Melting points were determined on an Electrothermal IA9100 melting point apparatus and are
uncorrected. IR spectra were measured using a Perkin Elmer System-2000 spectrometer. UV
spectra (λmax in nm) were recorded in spectroscopic grade MeOH on a Shimadzu UV-160A
1
13
UV-vis spectrophptometer. Nuclear magnetic resonance ( H and C) spectra were recorded on a
Varian Gemini 200 spectrometer or Varian-Unity-400 spectrometer. Chemical shifts were
expressed in parts per million (δ) with tetramethylsilane (TMS) as an internal standard. Thin-layer
chromatography was performed on silica gel 60 F-254 plates purchased from E. Merck and Co..
Mass spectra were recorded on Bruker APEX II (ESI) mass spectrometer. The elemental analyses
were performed in the Instrument Center of National Science Council at National Cheng-Kung
University and National Taiwan University using Heraeus CHN-O Rapid EA, and all values are
within ± 0.4% of the theoretical compositions.
5.2. General procedure for the preparation of quinolinyl chalcones 8a-c
A mixture 6 (0.48 g, 3.0 mmol) and selenium dioxide (0.66 g, 6.0 mmol) in 1,4-dioxane (50
o
mL) was heated at 100 C for 2 h (TLC monitoring). After cooling, the mixture was treated with
5
% NaHCO aqueous (80 mL), extracted with CH Cl (50 mL X 3), the organic layer was collected,
3 2 2
dried over MgSO , and evaporated. The crude product was crystallized with EtOH to give 7 (0.43 g,
4
81%) as a white solid. Compound 7 (0.36 g, 2.0 mmol) and appropriate acetophenone (2.0 mmol)
o
were stirred at 0 C for 15 min. Aqueous solution of KOH (6 equiv) was added and the mixture was
stirred at room temperature for 12 h (TLC monitoring). After the reaction reached completion, the
resulting mixture was added 1M HCl until pH 3 resulted and extracted with ethyl acetate (50 mL X
9

ACCEPTED MANUSCRIPT
3
). The organic layer was collected, dried over MgSO and concentrated in vacuo. The crude
4
product was purified and crystallized with EtOH to give quinolinyl chalcones 8a-c.
.2.1. (E)-3-(6-Fluoroquinolin-2-yl)-1-phenylprop-2-en-1-one (8a). Yield: 50% as a yellow solid.
5
o
1
Mp. 156.7-157.7 C. UV λ nm (logε): 327 (4.23), 264 (4.67), 218 (4.69) in MeOH. H NMR (400
max
MHz, CDCl ): 7.45 (dd, 1H, J = 8.4, 2.8 Hz, Ar-H), 7.51-7.56 (m, 3H, Ar-H), 7.60-7.64 (m, 1H,
3
Ar-H), 7.68 (d, 1H, J = 8.4 Hz, 3-H), 7.93 (d, 1H, J = 15.6 Hz, trans-CH=), 8.10-8.17 (m, 5H,
1
3
Ar-H). C NMR (100 MHz, CDCl ): 110.61 (d, J = 22.0 Hz), 120.50 (d, J = 25.8 Hz), 122.20,
3
CF
CF
127.02, 128.71 (2C), 128.77 (2C), 128.83 (d, J = 9.7 Hz), 132.43 (d, J = 9.1 Hz), 133.12,
CF CF
136.14 (d, JCF = 5.3 Hz), 137.78, 143.18, 145.50, 152.87 (d, JCF = 3.0 Hz), 160.95 (d, JCF = 248.7
+
Hz), 190.58 (C=O). ESIMS [M+H] : 278. Anal. calcd for C H FNO·0.5 H O: C 75.51, H 4.58, N
1
8
12
2
4.89; found C 75.50, H 4.38, N 4.65.
5.2.2. (E)-1-(4-Fluorophenyl)-3-(6-fluoroquinolin-2-yl)prop-2-en-1-one (8b). Yield: 69% as a
o
yellow solid. Mp. 194.7-195.4 C. UV λ nm (logε): 327 (4.07), 263 (4.65), 218 (4.70) in MeOH.
max
1
H NMR (400 MHz, CDCl ): 7.18-7.24 (m, 2H, Ar-H), 7.45 (dd, 1H, J = 8.4, 2.8 Hz, Ar-H), 7.54
3
(ddd, 1H, J = 9.2, 8.4, 2.8 Hz, Ar-H), 7.67 (d, 1H, J = 8.4 Hz, 3-H), 7.93 (d, 1H, J = 15.6 Hz,
1
3
trans-CH=) , 8.12-8.18 (m, 5H, Ar-H). C NMR (100 MHz, CDCl ): 110.66 (d, J = 21.9 Hz),
3
CF
1
1
6
2
3
5
15.86 (2C, d, J = 21.3 Hz), 120.58 (d, J = 25.7 Hz), 122.37, 126.49, 128.91 (d, J = 9.8 Hz),
CF CF CF
31.41 (2C, d, JCF = 9.8 Hz), 132.42 (d, JCF = 9.1 Hz), 134.12 (d, JCF = 2.8 Hz), 136.20 (d, JCF
=
.1 Hz), 143.28, 145.49, 152.66 (d, JCF = 3.1 Hz), 160.97 (d, JCF = 248.6 Hz), 165.82 (d, J =
CF
+
53.1 Hz), 188.84 (C=O). ESIMS [M+H] : 296. Anal. calcd for C H F NO·0.4 H O: C 71.47, H
1
8
11
2
2
.93, N 4.63; found C 71.57, H 3.96, N 4.65.
.2.3. (E)-3-(6-Fluoroquinolin-2-yl)-1-(4-methoxyphenyl)prop-2-en-1-one (8c). Yield: 79% as a
o
yellow solid. Mp. 157.7-158.6 C. UV λ nm (logε): 333 (4.43), 264 (4.71), 218 (4.74) in MeOH.
max
1
H NMR (400 MHz, CDCl ): 3.91 (s, 3H, OCH ), 7.01 (d, 2H, J = 8.8 Hz, Ar-H), 7.44 (dd, 1H, J =
3
3
8.8, 2.8 Hz, Ar-H), 7.52 (ddd, 1H, J = 9.2, 8.4, 2.8 Hz, Ar-H) , 7.67 (d, 1H, J = 8.4 Hz, 3-H), 7.92
1
3
(
d, 1H, J = 15.6 Hz, trans-CH=) , 8.12-8.19 (m, 5H, Ar-H). C NMR (100 MHz, CDCl ): 55.51,
3
1
0

ACCEPTED MANUSCRIPT
1
9
1
10.62 (d, J = 21.9 Hz), 113.92 (2C), 120.43 (d, J = 25.7 Hz), 122.29, 126.92, 128.80 (d, J =
CF
CF
CF
.8 Hz), 130.76, 131.14 (2C), 132.37 (d, JCF = 9.1 Hz), 136.09 (d, JCF = 5.3 Hz), 142.30, 145.48,
+
53.05 (d, J = 3.0 Hz), 160.87 (d, J = 248.7 Hz), 163.70, 188.66 (C=O). ESIMS [M+H] : 308.
CF
CF
Anal. calcd for C H FNO ·0.5 H O: C 72.14, H 4.78, N 4.43; found C 72.15, H 4.51, N 4.18.
1
9
14
2
2
5.3. General procedure for the preparation of 1,3,5-trisubstituted pyrazole derivatives 9a-10c
via cyclocondensation of quinolinyl chalcones 8a-c and phenylhydrazines.
To a solution of quinolinyl chalcones 8a, 8b or 8c (2 mmol) in EtOH (10 mL) was added
phenylhydrazine (2.1 mmol). The resulting solution was refluxed until the reaction was completed
as monitored by TLC (ca. 18 h). The solvent was evaporated in vacuo, and then added
2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) in 1,4-dioxane (10 mL). The reaction mixture was
refluxed for 12 h (TLC monitoring) and then concetntrated under vacuum. The residue was
extracted with CH Cl (50 mL X 3). The organic layer was collected, dried over MgSO and
2
2
4
concentrated in vacuo. The crude product was purified by silica gel column chromatography eluting
with solvent mixtures of CH Cl -MeOH (100:1, 50:1, and 20:1) to afford the pyrazole
2
2
products 9a-10c.
.3.1. 2-(2,3-Diphenyl-1H-pyrazol-5-yl)-6-fluoroquinoline (9a) and 2-(2,5-diphenyl-1H-
5
pyrazol-3-yl)-6-fluoroquinoline (10a). Compound 9a was obtained in 21% yield (0.16 g) as a
o
yellow solid. Mp. 153.4-154.4 C. R = 0.52, (CH Cl ：hexane = 3/1). UV λ nm (logε): 331 (4.10),
f
2
2
max
1
2
8
1
1
60 (4.78), 218 (4.78) in MeOH. H NMR (400 MHz, CDCl ): 7.33-7.51 (m, 13H, Ar-H),
3
1
3
.14-8.17 (m, 2H, Ar-H), 8.30 (d, 1H, J = 8.8 Hz, 4-H). C NMR (100 MHz, CDCl ): 106.83,
3
10.68 (d, J = 21.2 Hz), 119.55 (d, J = 25.0 Hz), 119.56, 125.41 (2C), 127.75, 128.34 (d, J =
CF
CF
CF
0.6 Hz), 128.39, 128.48 (2C), 128.77 (2C), 128.98 (2C), 130.33, 131.88 (d, J = 9.1 Hz), 135.77
CF
(d, JCF = 5.3 Hz), 140.07, 144.87, 145.20, 151.62, 152.30, 160.30 (d, JCF = 246.3 Hz). ESIMS
+
[
M+H] : 366. Anal. calcd for C H FN ·0.1 H O: C 78.50, H 4.45, N 11.44; found C 78.37, H
2
4
16
3
2
4.52, N 11.45.
o
Compound 10a was obtained in 36% yield (0.15 g) as a pink solid. Mp. 114.5-115.2 C. R = 0.42,
f
1
1

ACCEPTED MANUSCRIPT
1
(
CH Cl ：hexane = 3/1). UV λ nm (logε): 258 (4.78), 219 (4.78) in MeOH. H NMR (400 MHz,
2 2 max
1
3
CDCl ): 7.25-7.30 (m, 2H, Ar-H), 7.34-7.51 (m, 10H, Ar-H), 7.96-8.01 (m, 4H, Ar-H). C NMR
3
(
100 MHz, CDCl ): 106.62, 110.57 (d, JCF = 22.0 Hz), 120.26 (d, JCF = 25.8 Hz), 121.87, 125.54
3
(2C), 125.88 (2C), 127.68 (d, JCF = 9.8 Hz), 127.80, 128.11, 128.65 (2C), 128.93 (2C), 133.14 (d,
JCF = 9.1 Hz), 132.82, 135.55 (d, JCF = 5.3 Hz), 140.48, 143.56, 144.99, 148.81 (d, JCF = 3.0 Hz),
+
1
7
5
6
52.23, 160.77 (d, JCF = 247.8 Hz). ESIMS [M+H] : 366. Anal. calcd for C H FN ·0.1 H O: C
24
16
3
2
8.50, H 4.45, N 11.44; found C 78.22, H 4.54, N 11.37.
.3.2. 6-Fluoro-2-[5-(4-fluorophenyl)-1-phenyl-1H-pyrazol-3-yl]quinoline
(9b)
and
-fluoro-2-[3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-5-yl]quinoline (10b) Compound 9b was
o
obtained in 22% yield (0.17 g) as a yellow solid. Mp. 178.0-178.9 C. R = 0.63, (CH Cl ：hexane =
f
2
2
1
4
/1). UV λmax nm (logε): 330 (4.12), 259 (4.80), 219 (4.81) in MeOH. H NMR (400 MHz, CDCl ):
3
7.01-7.06 (m, 2H, Ar-H), 7.27-7.32 (m, 2H, Ar-H), 7.34 (s, 1H, pyrazolyl-H), 7.35-7.51 (m, 7H,
1
3
Ar-H), 8.13-8.17 (m, 2H, Ar-H), 8.29 (d, 1H, J = 9.2 Hz, 4-H). C NMR (100 MHz, CDCl ):
3
106.81, 110.69 (d, J = 22.0 Hz), 115.65 (2C, d, J = 21.2 Hz), 119.50, 119.60 (d, J = 25.0 Hz),
CF CF CF
1
25.41 (2C), 126.45 (d, J = 2.8 Hz), 127.89, 128.36 (d, J = 9.8 Hz), 129.07 (2C), 130.59 (2C, d,
CF
CF
J = 7.6 Hz), 131.86 (d, J = 9.1 Hz), 135.80 (d, J = 5.3 Hz), 139.87, 143.84, 145.18, 151.47 (d,
CF
CF
CF
+
J = 3.0 Hz), 152.34, 160.26 (d, J = 234.0 Hz), 162.33 (d, J = 234.1 Hz). ESIMS [M+H] : 384.
CF
CF
CF
Anal. calcd for Anal. calcd for C H F N ·0.1 H O: C 74.83, H 3.98, N 10.91; found C 74.68, H
2
4
15
2
3
2
4.16, N 10.87.
o
Compound 10b was obtained in 35% yield (0.19 g) as a yellow solid. Mp. 68.8-69.8 C. R = 0.58,
f
1
(
CH Cl ：hexane = 4/1). UV λ nm (logε): 323 (4.07), 260 (4.81), 219 (4.81) in MeOH. H NMR
2
2
max
(
400 MHz, CDCl ): 7.11-7.15 (m, 2H, Ar-H), 7.20 (s, 1H, pyrazolyl-H), 7.34-7.52 (m, 8H, Ar-H),
3
1
3
7
1
.92-8.01 (m, 4H, Ar-H) , 8.00 (d, 2H, J = 8.4 Hz, 4-H). C NMR (100 MHz, CDCl ): 106.38,
3
10.59 (d, J = 22.0 Hz), 115.59 (2C, d, J = 21.2 Hz), 120.33 (d, J = 25.0 Hz), 121.82, 125.51
CF
CF
CF
(
2C), 127.57 (d, J = 9.8 Hz), 127.70 (2C, d, J = 9.9 Hz), 127.89, 128.98 (2C), 132.13 (d, J =
CF CF CF
9
.1 Hz), 135.59 (d, J = 6.0 Hz), 135.82 (d, J = 2.8 Hz), 140.36, 143.69, 144.99, 148.69 (d, J =
CF CF CF
1
2

ACCEPTED MANUSCRIPT
+
2
.8 Hz), 151.36, 160.78 (d, J = 247.8 Hz), 162.82 (d, J = 244.7 Hz). ESIMS [M+H] : 384. Anal.
CF CF
calcd for C H F N ·0.1 H O: C 74.83, H 3.98, N 10.91; found C 74.99, H 4.33, N 10.61.
2
4
15
2
3
2
5.3.3.
6-Fluoro-2-[5-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl]quinoline
(9c)
and
6-fluoro-2-[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-5-yl]quinoline (10c). Compound 9c was
o
obtained in 23% yield (0.18 g) as a white solid. Mp. 175.3-176.0 C. R = 0.52, (CH Cl ：hexane =
f
2
2
1
4
/1). UV λmax nm (logε )： 331 (4.12), 261 (4.81), 219 (4.81) in MeOH. H NMR (400 MHz, CDCl ):
3
3
.82 (s, 3H, OCH ), 6.86 (d, 2H, J = 8.8 Hz, ArH), 7.25 (d, 2H, J = 8.8 Hz, Ar-H), 7.30 (s, 1H,
3
1
3
pyrazolyl-H), 7.32-7.51 (m, 7H, Ar-H), 8.13-8.17 (m, 2H, Ar-H), 8.29 (d, 1H, J = 8.4 Hz, 4-H). C
NMR (100 MHz, CDCl ): 55.25, 106.28, 110.67 (d, J = 22.0 Hz), 113.93 (2C), 119.52 (d, J =
3
CF
CF
2
1
1
7
5.7 Hz), 119.57, 122.73, 125.41 (2C), 127.66, 128.32 (d, J = 9.1 Hz), 128.96 (2C), 130.07 (2C),
CF
31.86 (d, J = 9.1 Hz), 135.73 (d, J = 5.3 Hz), 140.18, 144.73, 145.19, 151.71 (d, J = 2.3 Hz),
CF
CF
CF
+
52.19, 159.63, 160.28 (d, JCF = 246.2 Hz). ESIMS [M+H] : 396. Anal. calcd for C H FN O: C
25
18
3
5.93, H 4.59, N 10.63; found C 75.63, H 4.61, N 10.96.
o
Compound 10c was obtained in 40% yield (0.32 g) as a white solid. Mp. 142.8-143.5 C. R = 0.51,
f
1
(
CH Cl ：hexane = 4/1). UV λ nm (logε)：260 (4.81), 219 (4.82) in MeOH. H NMR (400 MHz,
2
2
max
CDCl ): 3.86 (s, 3H, OCH ), 6.98 (d, 2H, J = 8.8 Hz, Ar-H), 7.18 (s, 1H, pyrazolyl-H), 7.27 (d, 1H,
3
3
J = 9.2 Hz, Ar-H), 7.33-7.51 (m, 7H, Ar-H), 7.89 (d, 2H, J = 8.8 Hz, Ar-H) , 7.98-8.01 (m, 2H,
1
3
ArH). C NMR (100 MHz, CDCl ): 55.31, 106.22, 110.57 (d, J = 21.9 Hz), 114.06 (2C), 120.24
3
CF
(
d, J = 25.7 Hz), 121.90, 125.51 (2C), 125.59, 127.16 (2C), 127.61, 127.70, 128.92 (2C), 132.13
CF
(
d, J = 9.1 Hz), 135.51 (d, J = 5.3 Hz), 140.49, 143.46, 144.99, 148.90 (d, J = 3.1 Hz), 152.08,
CF
CF
CF
+
1
4
5
1
59.67, 160.74 (d, J = 247.8 Hz). ESIMS [M+H] : 396. Anal. calcd for C H FN O: C 75.93, H
CF 25 18 3
.59, N 10.63; found C 75.95, H 4.61, N 10.83.
.4. General procedure for the preparation of 1,3,5-trisubstituted pyrazole derivatives
1a-13c via cyclocondensation of quinolinyl chalcones 8a-c and 4-substituted
phenylhydrazines.
To a solution of quinolinyl chalcones 8a, 8b or 8c (2 mmol) in EtOH (10 mL) was added
1
3

ACCEPTED MANUSCRIPT
4-substituted phenylhydrazine hydrochloride (2.1 mmol). The resulting solution was refluxed until
the reaction was completed as monitored by TLC (ca.12 h). The solvent was evaporated in vacuo,
and purified by flash chromatography on silica gel, using CH Cl / MeOH(20:1) as eluant and
2
2
recrystallized with EtOH to afford the pyrazole products (11a-13c).
5.4.1. 6-Fluoro-2-[1-(4-fluorophenyl)-3-phenyl-1H-pyrazol-5-yl]quinoline (11a). Yield 54% as
o
a yellow solid. Mp. 151.2-152.8 C. UV λ nm (logε )： 323 (4.17), 246 (4.59) 222 (4.52) in MeOH.
max
1
H NMR (400 MHz, CDCl ): 7.05-7.10 (m, 2H, Ar-H), 7.23 (s, 1H, pyrazolyl-H), 7.35-7.51 (m, 8H,
3
1
3
Ar-H), 7.92-7.96 (m, 3H, Ar-H), 8.04 (d, 1H, J = 8.4 Hz, 4-H). C NMR (100 MHz, CDCl ):
3
1
1
06.45, 110.60 (d, J = 22.0 Hz), 115.73 (2C, d, J = 22.7 Hz), 120.39 (d, J = 25.8 Hz), 121.66,
CF CF CF
25.88 (2C), 127.47 (d, J = 8.3 Hz), 127.69 (d, J = 9.9 Hz), 128.21, 128.70 (2C), 132.12 (2C, d,
CF
CF
J = 9.1 Hz), 132.69, 135.76, 136.87, 143.56, 144.92, 148.48, 152.26, 160.83 (d, J = 247.9 Hz),
CF
CF
+
1
1
5
61.92 (d, JCF = 245.7 Hz). ESIMS [M+H] : 384. Anal. calcd for C H F N : C 75.19, H 3.94, N
24 15 2 3
0.96; found C 75.30, H 3.63, N 10.87.
.4.2. 2-[1,3-Bis(4-fluorophenyl)-1H-pyrazol-5-yl]-6-fluoroquinoline (11b). Yield 57% as a
o
yellow solid. Mp. 188.2-189.8 C. UV λ nm (logε)：326 (4.08), 262 (4.61) 220 (4.51) in MeOH.
max
1
H NMR (400 MHz, CDCl ): 7.06-7.16 (m, 4H, ArH), 7.18 (s, 1H, pyrazolyl-H), 7.35 (d, 1H, J =
3
1
3
8
.8 Hz, ArH), 7.40-7.52 (m, 4H, ArH), 7.90-7.96 (m, 3H, Ar-H), 8.04 (d, 1H, J = 8.8 Hz, 4-H). C
NMR (100 MHz, CDCl ): 106.23, 110.61 (d, J = 21.9 Hz), 115.64 (2C, d, J = 21.3 Hz), 115.78
3
CF
CF
(
2C, d, J = 22.7 Hz), 120.45 (d, J = 25.7 Hz), 121.61, 127.39 (2C, d, J = 9.1 Hz), 127.55 (2C,
CF CF CF
d, JCF = 8.4 Hz), 127.69 (d, JCF = 10.6 Hz), 128.87 (d, JCF = 3.1 Hz), 132.08 (d, JCF = 9.1 Hz),
1
1
4
5
35.83 (d, J = 5.3 Hz), 136.67 (d, J = 3.0 Hz), 143.65, 144.88, 148.29 (d, J = 3.1 Hz), 151.35,
CF CF CF
+
60.82 (d, J = 247.8 Hz), 161.91 (d, J = 246.3 Hz), 162.84 (d, J = 245.6 Hz). ESIMS [M+H] :
CF
CF
CF
02. Anal. calcd for C H F N : C 71.82, H 3.52, N 10.47; found C 71.98, H 3.21, N 10.40.
2
4
14
3
3
.4.3. 6-Fluoro-2-(1-(4-fluorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-5-yl)quinoline (11c).
o
Yield 59% as a yellow solid. Mp. 180.4-181.6 C. UV λ nm (logε)：326 (4.17), 262 (4.62), 221
max
1
(
4.51) in MeOH. H NMR (400 MHz, CDCl ): 3.86 (s, 3H, OCH ), 6.96-7.10 (m, 4H, Ar-H), 7.16
3
3
1
4

ACCEPTED MANUSCRIPT
(s, 1H, pyrazolyl-H), 7.35 (d, 1H, J = 8.8 Hz, Ar-H), 7.39-7.51 (m, 4H, Ar-H), 7.86-7.89 (m, 2H,
1
3
Ar-H), 7.94 (dd, 1H, J = 9.2, 5.2 Hz, Ar-H), 8.03 (d, 1H, J = 8.4 Hz, 4-H). C NMR (100 MHz,
CDCl ): 55.31, 106.05, 110.59 (d, J = 21.2 Hz), 114.07 (2C), 115.72 (2C, d, JCF = 22.7 Hz),
3
CF
1
20.36 (d, J = 25.9 Hz), 121.68, 125.39, 127.13 (2C), 127.39 (2C, d, J = 9.1 Hz), 127.65 (d, J
CF CF CF
=
9.9 Hz), 132.08 (d, JCF = 9.1 Hz), 135.74 (d, JCF = 5.3 Hz), 136.80 (d, JCF = 3.1 Hz), 143.44,
1
44.88, 148.51 (d, J = 3.0 Hz), 152.07, 159.71, 160.77 (d, J = 247.8 Hz), 161.82 (d, J = 246.3
CF
CF
CF
+
Hz). ESIMS [M+H] : 414. Anal. calcd for C H F N O: C 72.63, H 4.14, N 10.16; found C 72.86,
2
5
17
2
3
H 4.23, N 9.94.
.4.4. 6-Fluoro-2-[1-(4-methoxyphenyl)-3-phenyl-1H-pyrazol-5-yl]quinoline (12a). Yield: 58%
5
o
1
as a brown solid. Mp. 203.6-204.5 C. UV λ
nm (logε)：258 (4.72), 215 (4.81) in MeOH. H
max
NMR (400 MHz, CDCl ): 3.83 (s, 3H, OCH ), 6.88-6.91 (m, 2H, Ar-H), 7.25 (s, 1H, pyrazolyl-H),
3
3
1
3
7
.26-7.27 (m, 1H, Ar-H), 7.33-7.53 (m, 7H, Ar-H), 7.95-8.05 (m, 4H, Ar-H). C NMR (100 MHz,
CDCl ): 55.52, 106.17, 110.55 (d, JCF = 21.9 Hz), 114.11 (2C), 120.24 (d, JCF = 25.0 Hz), 121.80,
3
1
1
1
4
5
25.84 (2C), 126.95 (2C), 127.63 (d, J = 9.9 Hz), 128.01, 128.63 (2C), 132.12 (d, J = 9.1 Hz),
CF CF
32.89, 133.67, 135.51 (d, J = 5.3 Hz), 143.56, 144.98, 148.83 (d, J = 3.0 Hz), 151.91, 159.15,
CF
CF
+
60.73 (d, J = 247.8 Hz). ESIMS [M+H] : 396. Anal. calcd for C H FN O·0.1 H O: C 75.59, H
CF
25 18
3
2
.62, N 10.58; found: C 75.49, H 4.47, N 10.54.
.4.5. 6-Fluoro-2-[3-(4-fluorophenyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl]quinoline (12b).
o
Yield: 59% as a brown solid. Mp. 167.5-168.3 C. UV λ
nm (logε)：259 (4.84), 219 (4.84) in
max
1
MeOH. H NMR (400 MHz, CDCl ): 3.83 (s, 3H, OCH ), 6.88-6.92 (m, 2H, Ar-H), 7.10-7.15 (m,
3
3
2H, Ar-H), 7.19 (s, 1H, pyrazolyl-H), 7.24 (d, 1H, J = 8.8 Hz, Ar-H), 7.33-7.36 (m, 2H, Ar-H), 7.40
(dd, 1H, J = 8.8, 2.8 Hz, Ar-H), 7.47-7.52 (m, 1H, Ar-H) , 7.90-7.94 (m, 2H, Ar-H) , 7.98 (d, 1H, J
1
3
=
8.8 Hz, 4-H) , 8.03 (dd, 1H, J = 9.2, 5.6 Hz, Ar-H). C NMR (100 MHz, CDCl ): 55.53, 105.91
3
(
d, JCF = 22.0 Hz), 114.15 (2C), 114.19, 115.55 (d, JCF = 22.0 Hz), 120.28 (d, JCF = 25.7 Hz),
21.74, 126.91 (2C), 127.52 (2C, d, JCF = 8.4 Hz), 127.65 (d, JCF = 9.9 Hz), 129.13 (d, JCF = 3.0
Hz), 132.13 (d, J = 9.1 Hz), 133.55, 135.53 (d, J = 5.3 Hz), 143.72, 144.99, 148.71 (d, J = 3.0
1
CF
CF
CF
1
5

ACCEPTED MANUSCRIPT
+
Hz), 151.03, 159.20, 160.75 (d, J = 247.8 Hz), 162.76 (d, J = 245.5 Hz). ESIMS [M+H] : 414.
CF
CF
Anal. calcd for C H F N O·0.2 H O: C 72.00, H 4.21, N 10.08; found C 71.90, H 4.33, N 9.79.
2
5
17
2
3
2
5.4.6. 2-[1,3-Bis(4-methoxyphenyl)-1H-pyrazol-5-yl]-6-fluoroquinoline (12c). Yield: 62% as a
o
1
brown solid. Mp. 63.5-63.9 C. UV λ nm (logε)：261 (4.85), 218 (4.86) in MeOH. H NMR (400
max
MHz, CDCl ): 3.83 (s, 3H, OCH ), 3.86 (s, 3H, OCH ), 6.88-6.90 (m, 2H, Ar-H), 6.96-6.99 (m, 2H,
3
3
3
Ar-H), 7.17 (s, 1H, pyrazolyl-H), 7.25 (d, 1H, J = 8.8 Hz, Ar-H), 7.334-7.37 (m, 2H, Ar-H), 7.40
(dd, 1H, J = 8.8, 2.8 Hz, Ar-H), 7.47-7.52 (m, 1H, Ar-H), 7.88-7.90 (m, 2H, Ar-H), 7.98 (d, 1H, J =
1
3
8
1
1
1
.8 Hz, 4-H) , 8.03 (dd, 1H, J = 8.8, 5.2 Hz, Ar-H). C NMR (100 MHz, CDCl ): 55.30, 55.53,
3
05.71, 110.54 (d, JCF = 22.0 Hz), 114.04 (2C), 114.11 (2C), 120.19 (d, JCF = 25.0 Hz), 121.82,
25.71, 126.94 (2C), 127.12 (2C), 127.58 (d, JCF = 9.8 Hz), 132.15 (d, JCF = 9.1 Hz), 133.75,
35.45 (d, JCF = 5.3 Hz), 143.53, 145.03, 148.96, 151.78, 159.10, 159.60, 160.72 (d, JCF = 247.9
+
Hz). ESIMS [M+H] : 426. Anal. calcd for C H FN O : C 73.40, H 4.74, N 9.88; found C 73.29,
2
6
20
3
2
H 4.95, N 9.84.
5.4.7. 4-[5-(6-Fluoroquinolin-2-yl)-3-phenyl-1H-pyrazol-1-yl]benzenesulfonamide (13a). Yield:
o
1
5
5% as a brown solid. Mp. 244.5~245.3 C. UV λ nm (logε)：260 (4.86), 219 (4.86) in MeOH. H
max
NMR (400 MHz, DMSO-d ): 7.39-7.44 (m, 1H, Ar-H), 7.49-7.52 (m, 4H, Ar-H), 7.68 (s, 1H,
6
pyrazolyl-H), 7.61-7.75 (m, 4H, Ar-H), 7.82-7.89 (m, 4H, Ar-H), 7.99-8.01 (m, 2H, Ar-H), 8.46 (d,
1
3
1
H, J = 8.4 Hz, 4-H). C NMR (100 MHz, DMSO-d ): 107.58, 111.11 (d, J = 22.0 Hz), 120.37
6 CF
(
d, J = 25.8 Hz), 121.94, 125.53 (2C), 125.70 (2C), 126.27 (2C), 127.67 (d, J = 9.8 Hz), 128.45,
C
F
C
F
128.88 (2C), 131.62 (d, JCF = 9.1 Hz), 132.12, 136.74 (d, JCF = 5.3 Hz), 142.90 , 142.95, 143.29,
+
1
44.02, 147.97 (d, JCF = 2.3 Hz), 151.65, 160.24 (d, JCF = 244.8 Hz). ESIMS [M+H] : 445. Anal.
calcd for C H FN O S: C 64.85, H 3.86, N 12.60; found C 64.60, H 3.94, N 12.45.
2
4
17
4
2
5.4.8.
4-[3-(4-Fluorophenyl)-5-(6-fluoroquinolin-2-yl)-1H-pyrazol-1-yl]benzenesulfonamide
o
(
13b). Yield: 48% as a green solid. Mp. 236.5-237.7 C. UV λmax nm (logε): 332 (4.10), 260 (4.61),
1
2
24 (4.52) in MeOH. H NMR (400 MHz, DMSO-d ): 7.32-7.37 (m, 2H, Ar-H), 7.49 (s, 2H, NH ),
6
2
7.67 (s, 1H, pyrazolyl-H), 7.60-7.75 (m, 4H, Ar-H), 7.82-7.88 (m, 4H, Ar-H), 8.02-8.05 (m, 2H,
1
6

ACCEPTED MANUSCRIPT
1
3
Ar-H), 8.46 (d, 1H, J = 8.8 Hz, 4-H). C NMR (100 MHz, DMSO-d ): 107.50, 111.10 (d, J
=
6
CF
22.0 Hz), 115.79 (2C, d, JCF = 21.3 Hz), 120.38 (d, JCF = 25.8 Hz), 121.90, 125.68 (2C), 126.27
(
2C), 127.59 (2C, d, J = 8.3 Hz), 127.67 (d, J = 9.1 Hz), 128.70 (d, J = 3.0 Hz), 131.61 (d, J
CF CF CF
CF
CF
=
=
9.8 Hz), 136.74 (d, J = 5.3 Hz), 142.87, 142.93, 143.37, 144.01, 147.92, 150.76, 160.04 (d, J
CF
+
244.8 Hz), 162.25 (d, J = 244.0 Hz). ESIMS [M+H] : 463. Anal. calcd for C H F N O S·0.5
CF
24 16
2
4
2
H O: C 61.14, H 3.63, N 11.88; found C 61.43, H 3.40, N 11.76.
2
5.4.9. 4-[5-(6-Fluoroquinolin-2-yl)-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]benzenesulfonamide
o
(
13c). Yield 53% as a yellow solid. Mp. 233.1-234 C. UV λmax nm (logε)：262(4.89), 219 (4.89) in
1
MeOH. H NMR (400 MHz, DMSO-d ): 3.37 (s, 3H, OCH ), 7.05-7.07 (m, 2H, Ar-H), 7.49 (s, 2H,
6
3
NH ), 7.58 (s, 1H, pyrazolyl-H), 7.59-7.76 (m, 5H, Ar-H), 7.82-7.93 (m, 6H, Ar-H), 8.46 (d, 1H, J
2
1
3
=
8.8Hz, 4-H). C NMR (100 MHz, DMSO-d ): 55.20, 107.21, 111.11 (d, J = 22.0 Hz), 114.27
6 CF
(
2C), 120.36 (d, J = 25.7 Hz), 121.95, 124.70, 125.58 (2C), 126.27 (2C), 126.92 (2C), 127.66 (d,
CF
J = 11.3 Hz), 131.63 (d, J = 9.9 Hz), 136.71 (d, J = 5.3 Hz), 142.73, 143.00, 143.18, 144.05,
CF
CF
CF
+
1
48.09 (d, JCF = 3.0 Hz), 151.59, 159.52, 160.04 (d, JCF = 244.8 Hz). ESIMS [M+H] : 475. Anal.
calcd for C H FN O S·0.5 H O: C 62.10, H 4.17, N 11.59; found C 61.72, H 3.85, N 11.51.
2
5
19
4
3
2
5
5
.5. Cytotoxicity and antiviral activity assays
.5.1. Compounds
Compounds were dissolved in DMSO at 10 mM and then diluted in culture medium.
.5.2. Cells and virus
5
Human hepatoma Huh-7 cells were purchased from Bioresources Collection and Research Center,
Taiwan. Huh-7-DV-Fluc cells and C6/36 cells were kindly provided by Dr Huey Nan Wu, Institute
of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC. The DENV-2 strain 16681 was
isolated from a patient with dengue hemorrhage fever (DHF) from Thailand [31], and was
amplified in C6/36 mosquito cells.
5.5.3. Cytotoxicity assays
3
For cytotoxicity tests, run in parallel with antiviral assays, plates at an initial density of (5 x 10
1
7

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cells/well), in maintenance medium, with or without test compounds at two concentrations (20 and
o
2
00 µM) or 0.1% DMSO as control. Cell viability was determined after 3 days at 37 C in a
humidified CO (5%) atmosphere by the (2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-
2
tetrazolium-5-carboxanilide) (XTT) method [32].
5.5.4. Analysis of luciferase activity
4
Huh-7-DV-Fluc cells were seeded in 24-well plates at a density of 2 × 10 cells per well, and
treated with the diarylpyrazolylquinoline compounds at two concentrations (1 and 10 µM) or 0.1%
DMSO as control. After 3 days of incubation, the luciferase activity assay was performed using the
Bright-Glo Luciferase assay system (Promega) according to the manufacturer’s instructions.
5.5.5. Immunoblot analysis
The standard procedure was followed to perform western blotting as described previously [33]. The
membranes were incubated overnight with rabbit polyclonal antibodies against NS2B (1：3000,
Genetex, Irvine, CA, USA) and GAPDH (1：10000, Genetex, Irvine, CA, USA) serving as loading
control.
5.5.6. Quantification of DENV RNA
Total cellular RNA was extracted after 3 days compounds treatment through Total RNA Miniprep
Purification Kit (GMbiolab, Taiwan) according to the manufacturer’s instructions. The expression
of DENV NS5 mRNA level was determine through quantification real-time RT-PCR (RT-PCR)
with specific primers corresponding to the DENV NS5 gene：Forward primer, 5-AAG GTG AGA
AGC AAT GCA GC-3；reverse primer, 5-CCA CTC AGG GAG TTC TCT CT-3. The copy number
of NS5 in each sample was normalized to cellular endogenous reference gene
glyceraldehydes-3-phosphate dehydrogenase (gapdh)；Forward primer: 5-GTC TTC ACC ACC
ATG GAG AA-3 and Reverse primer: 5-ATG GCA TGG ACT GTG GTC AT-3. The CT value of
each sample was determined by the ABI Step One Real-Time PCR-System (ABI Warrington, UK).
5.5.7. DENV infection assay
4
Huh-7 cells were seeded at the density of 4 X 10 cells/well in the 24 well culture plate for 16-20
1
8

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o
hours and then infected with DENV-2 (16681 strain) at an MOI of 0.1 for 2 hours at 37 C. Cells
were washed with PBS once and then re-filled with DMEM-2% FBS medium containing various
indicating concentration of 13c.
5.5.8. Anti-DENV activity assay in vivo
Six-days-old ICR suckling mice were randomly divided into 3 groups (n = 5/group): Group 1:
5
intracerebrally injected 2.5x10 pfu 60°C heat-inactive DENV (iDENV). Group 2: intracerebrally
5
injected 2.5x10 pfu DENV + intraperitoneally injected saline (DENV). Group 3; intracerebrally
5
injected 2.5x10 pfu DENV virus+ intraperitoneally injected 1, 5, 10 and 20 mg/kg 13c. Mice were
given saline, 1, 5, 10 and 20 mg/kg 13c by intraperitoneal injection at 1, 3, 5 days post-infection
(D.P.I). The survival rate, body weight and clinical score were measured every day after DENV
injection. Illness symptom were scored ad follow; 0 for no symptom; 1 for slight losing weight and
ruffled hair; 2 for slowing of activity; 3 for asthenia; 4 for paralysis and mortally ill; 5 for death
[
34].
.5.9. Statistical analysis
5
The results were expressed as means±S.D. Differences in mean values between groups were
analyzed by a one-way analysis of variance (ANOVA) and Student's t-test. For the septic shock
assay, we used the log-rank test. Statistical significance was assessed as p<0.05 [*p<0.05;
**p<0.01].
Acknowledgment. Financial supports of this work by the Minister of Science and Technology of
the Republic of China (Most 106-2320-B-037-015, MOST 105-2320-B-037-007, MOST
105-2320-B-037-011, MOST 103-2320-B-037-011-MY3), and Kaohsiung Medical University
(KMU-TP104E16, KMU-TP104E42, KMU-TP104H03, KMU-TP104H07, KMU-TP104H08,
KMU-TP105E16, KMU-TP105E33, KMU-TP105H07, KMU-TP105H08) are gratefully
acknowledged. In part by a grant from the National Sun Yat-Sen University-KMU Joint Research
Project (NSYSU-KMU 102-I004, NSYSU-KMU 104-I011) are gratefully acknowledged. We also
thank Center for Research Resources and Development at Kaohsiung Medical University for the
1
9

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instrumentation and equipment support.
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Figure captions:
Figure 1. The structure of quinoline and pyrazole derivatives.
Figure 2. ORTEP diagram of 6-fluoro-2-[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-5-yl]
quinoline (9c) (A) and 6-fluoro-2-[5-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl]quinoline (10c)
(B).
Figure 3. The chemical shift differences for pyrazole 5-H of regioisomers, 9c (N substituted) and
2
1
0c (N substituted) in both CDCl and in DMSO-d .
3 3 6
Figure 4. Inhibition of DENV-2 RNA expression in DENV-infected Huh-7 cells by 13c.
DENV-infected Huh-7 cells were treated with 0.5, 1, 2.5 and 5 µM of 13c for 3 days. Total RNA
was extracted and quantified DENV2 RNA levels by RT-qPCR. DENV2 RNA expression was
normalized by cellular GAPDH mRNA. Treatment with 0.1% DMSO served as a mock control.
The results are expressed as the means ± standard deviations (SD) of triplicate experiments.
Figure 5. Inhibition of DENV-2 protein expression in DENV-infected Huh-7 cells by 13c.
DENV-infected Huh-7 cells were treated with 0.5, 1, 2.5 and 5 µM of 13c for 3 days. Total cell
lysate was collected for performing western blotting to analyze DENV protein synthesis. Levels of
GAPDH were used as equal loading control.
Figure 6. 13c shows therapeutic efficacy in DENV-infected ICR suckling mice. Six-day-old ICR
5
suckling mice were injected with 2.5 × 10 pfu DENV-2 intracerebrally and with 1, 5, 10 and 20
mg/kg of 13c intraperitoneally at 1, 3 and 5 dpi. (A) Survival rate, (B) clinical score, and (C) body
weight of the mice were measured every day after the DENV injection. Illness symptoms were
scored as follow: 0, no symptom; 1, slight weight loss and ruffled hair; 2, slow activity; 3, asthenia
and anorexia; 4, paralysis and fatal illness; and 5, death.
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Table 1. The crystallographic data and structure refinement details of 9c and 10c.
Identification code
Empirical formula
Formula weight
9c
10c
C H FN O
C H FN O
25 18
25
18
3
3
395.42
395.42
Temperature
100(2) K
0.71073 Å
100(2) K
0.71073 Å
Wavelength
Crystal system, space group
Triclinic, P -1
Monoclinic, P 1 21/c 1
a = 12.8551(14) Å α= 90°
a = 6.262(3) Åα= 68.380(11)°
Unit cell dimensions
b = 11.961(5) Å
c = 13.502(6) Å
β= 87.617(13)° b = 14.1552(15) Å β= 98.879(2)°
γ = 83.998(12)° c = 10.8351(13) Å γ = 90°
3
3
Volume
Z, Calculated density
Absorption coefficient
F(000)
935.0(8) Å
1948.0(4) Å
3
3
2, 1.405 Mg/m
4, 1.348 Mg/m
-1
-1
0.094 mm
412
0.091 mm
824
3
3
Crystal size
0.20 x 0.05 x 0.05 mm
0.30 x 0.30 x 0.08 mm
Theta range for data
collection
1
.62 to 26.51°
1.60 to 26.41°
-
4<=h<=7, -14<=k<=14,
-16<=h<=15, -15<=k<=17,
-13<=l<=13
Limiting indices
-16<=l<=16
Reflections collected/unique
Completeness to
13276/3808 [R(int) = 0.0258]
θ = 26.51, 98.3 %
16028/3984 [R(int) = 0.0338]
θ = 26.41, 99.6 %
Absorption correction
Max. and min. transmission
Refinement method
Semi-empirical from equivalents
0.9486 and 0.8995
Semi-empirical from equivalents
0.9486 and 0.8609
2
2
Full-matrix least-squares on F
Full-matrix least-squares on F
Data/restraints/parameters
Goodness-of-fit on F2
3808 / 0 / 272
3984 / 0 / 272
1.006
1.019
2
5

ACCEPTED MANUSCRIPT
Final R indices [I >2σ (I)]
R indices (all data)
R1 = 0.0370, wR2 = 0.0865
R1 = 0.0536, wR2 = 0.0952
R1 = 0.0358, wR2 = 0.0834
R1 = 0.0495, wR2 = 0.0904
-3
-3
Largest diff. peak and hole
0.215 and -0.280 e.Å
0.222 and -0.228 e.Å
2
6

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1
Table 2. Comparative studies on the chemical shifts for H NMR signals of the pyrrazoyl part of
diarylpyrazolylquinoline derivatives.
Pyrazole-5-H chemical shift (ppm)
Compounds
R1
R2
N-2-Aryl
N-3-Aryl
a
H
F
--
--
7.38
9
a
--
--
--
a
7.34
9
b
a
b
OMe
H
--
7.30 (7.29)
9
c
a
H
--
--
--
--
--
--
--
--
--
--
--
--
1
0a
0b
0c
1a
1b
1c
2a
2b
2c
3a
3b
3c
7.25
a
F
H
1
7.20
a
b
OMe
H
H
1
7.18 (7.49)
a
F
1
7.23
a
F
F
1
7.18
a
OMe
H
F
1
7.16
a
OMe
OMe
OMe
1
7.25
a
F
1
7.19
a
OMe
H
1
7.17
b
SO NH
1
2
2
2
2
7.68
b
F
SO NH
2
1
7.67
b
OMe SO NH
1
2
7.58
a
b
CDCl ; DMSO-d
3
6
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Table 3. Anti-DENV-2 activities and cytotoxicities of diarylpyrazolylquinoline derivatives.
DENV-2
Huh-7 cell
compounds
% Inhibition at 1
% Inhibition at 10 % viability at 20 % viability at 200
µM
µM
µM
µM
5
3
4
3
.21 ± 3.1
.58 ± 1.7
.14 ± 2.3
.02 ± 1.8
15.31 ± 2.3
6.14 ± 2.1
7.15 ± 1.9
5.13 ± 1.4
25.39 ± 3.4
8.09 ± 2.3
8.31 ± 1.5
22.35 ± 1.7
55.32 ± 1.8
20.68 ± 3.7
15.83 ± 3.2
65.71 ± 4.1
11.81 ± 1.9
12.75 ± 3.8
85.34 ± 6.7
32.53 ± 2.3
124.10 ± 7.48
88.26 ± 9.46
95.56 ± 5.07
125.54 ± 6.35
88.13 ± 1.84
103.99 ± 3.75
97.82 ± 3.68
95.32 ± 2.61
98.75 ± 2.87
115.73 ± 3.28
89.08 ± 8.16
95.81 ± 4.82
89.22 ± 4.03
91.12 ± 2.04
83.71 ± 2.52
71.37 ± 1.31
115.31 ± 11.43
89.72 ± 1.71
93.68 ± 5.75
98.29 ± 7.59
87.49 ± 7.61
98.90 ± 9.26
91.56 ± 3.85
86.51 ± 4.51
95.37 ± 3.64
97.35 ± 8.21
52.55 ± 1.89
98.40 ± 16.44
83.64 ± 2.68
42.97 ± 2.47
89.53 ± 2.41
26.78 ± 1.59
9
a
9
b
9
c
1
0a
0b
0c
1a
1b
1c
2a
2b
2c
3a
3b
3c
ribavirin
1
1.23 ± 2.8
1
5
.31 ± 1.6
.36 ± 1.7
.91 ± 1.3
1
4
1
8
1
4
1.42 ± 2.4
1
7
.81 ± 2.5
.12 ± 2.1
6.87 ± 5.3
1
6
1
4
1
4
.52 ± 2.7
.71 ± 1.7
8.96 ± 2.8
1
5
1
5
1
10.14 ± 1.98
2
8