Discovery of 3-arylquinoxaline derivatives as potential anti-dengue virus agents

Article abstract of DOI:10.3390/ijms20194786

We designed and synthesized a series of novel 3-arylquinoxaline derivatives and evaluated their biological activities as potential dengue virus (DENV) replication inhibitors. Among them, [3-(4-methoxyphenyl)quinoxalin-2-yl](phenyl)methanol (19a), [6,7-dic

Full text of DOI:10.3390/ijms20194786

International Journal of
Molecular Sciences
Article
Discovery of 3-Arylquinoxaline Derivatives as
Potential Anti-Dengue Virus Agents
Chih-Hua Tseng 1,2,3,4,5,
*
, Cheng-Ruei Han 1 and Kai-Wei Tang 1
1
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan;
st88036942017@gmail.com (C.-R.H.); dadaking1107@gmail.com (K.-W.T.)
Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
Drug Development and Value Creation Research Center, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan
Department of Pharmacy, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung 801, Taiwan
Correspondence: chihhua@kmu.edu.tw; Tel.: +886-7-312-1101 (ext. 2163)
2
3
4
5
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 2 September 2019; Accepted: 25 September 2019; Published: 26 September 2019
Abstract: We designed and synthesized a series of novel 3-arylquinoxaline derivatives and evaluated
their biological activities as potential dengue virus (DENV) replication inhibitors. Among them,
[3-(4-methoxyphenyl)quinoxalin-2-yl](phenyl)methanol (19a), [6,7-dichloro-3-(4-methoxyphenyl)quinoxalin-
2-yl](phenyl)methanol (20a), and (4-methoxyphenyl)(3-phenylquinoxalin-2-yl)methanone (21b) were found
to significantly inhibit the DENV RNA expression in Huh-7-DV-Fluc cells with a potency better than that of
ribavirin. Compound 19a reduced DENV replication in both viral protein and messenger RNA (mRNA)
levels in a dose-dependent manner and exhibited no significant cell cytotoxicity. Notably, compound 19a
exhibited a half maximal effective concentration (EC50) value at 1.29
±
0.74 µM. We further observed that the
inhibitory effect of 19a on DENV replication was due to suppression of DENV-induced cyclooxygenase-2
(COX-2) expression. Docking studies also showed that 19a caused hydrophobic interactions at the active
sites with Arg29, Glu31, Tyr116, Leu138, Pro139, Lys454, Arg455, and Gln529. The calculated lowest binding
energy between the 19a and COX-2 was 9.10 kcal/mol. In conclusion, compound 19a might be a potential
−
lead compound for developing an anti-DENV agent.
Keywords: 3-arylquinoxaline; dengue virus; ribavirin; cyclooxygenase-2
1. Introduction
Dengue virus (DENV) infections significantly increased in the past decades and, with more than
half the world’s population living in areas at risk of infection, the World Health Organization estimated
the true number of cases to exceed 50 million annually [1]. There are four serotypes of dengue, DENV-1,
DENV-2, DENV-3, and DENV-4, and the infection severity ranges from the self-limiting dengue fever
(DF) to the more serious dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), with
reported ranges of 1–5% to 10–30% of cases resulting in death, respectively [2–4]. There is no antiviral
drug currently available for the treatment of dengue, although potential anti-DENV drugs being
tested in human clinical trials are at different stages of development. Some drugs entered into such
trials include chloroquine, celgosivir, balapiravir, prednisolone, lovastatin, ivermectin, and ribavirin;
however, none of these are yet approved for treatment against the virus [5]. Therefore, the development
of supplemental agents or more effective and safer agents is required for DV therapy.
Cyclooxygenase-2 (COX-2) is one of the important mediators of inflammation in response to
viral infection, and it contributes to viral replication, for example, cytomegalovirus or hepatitis C
Int. J. Mol. Sci. 2019, 20, 4786; doi:10.3390/ijms20194786
www.mdpi.com/journal/ijms

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virus replication [6–10]. More recently, Lin et al. revealed that COX-2 is an important factor for
DENV replication and can serve as a potential target for developing therapeutic agents against DENV
infection [11].
Certain heterocyclic compounds were discovered to possess anti-DENV activities. For example,
Saudi et al. identified an imidazole 4,5-dicarboxamide derivative as a novel anti-DENV agent through
high-throughput screening (HTS) and proceeded to synthesize a series of 29 compounds, from which
pyrazine-based compound
was identified [12]. Venkatesham et al. structurally modified the molecule and discovered quinazoline
derivative with an EC50 value of 2.6 M against DENV [13]. We also prepared certain quinoline derivatives
for anti-DENV evaluation. Among them, 2-(hydroxyphenylmethyl)-3-(4-methoxyphenyl)quinoline ( ) and
2-(4-hydroxybenzoyl)-3-(4-hydroxyphenyl)quinoline ( ) were found to inhibit the DENV2 RNA expression
in Huh-7-DV-Fluc cells with a higher potency than that of ribavirin [14]. More recently, we further
identified 4-[5-(6-fluoroquinolin-2-yl)-3-(4-methoxy phenyl)-1H-pyrazol-1-yl]benzenesulfonamide ( ),
which exhibited approximately 10-fold more active anti-DENV-2 activity (EC50 = 0.81 M) than that
of ribavirin (EC50 = 12.61 M) [15]. Quinoxaline is a versatile heterocyclic compound and belongs to
1, with a significant potency against DENV2 in VERO-B cells (EC50 = 0.93 µM),
2
µ
3
4
5
µ
µ
the isosteric isomer of quinoline. Several quinoxaline-containing compounds possess a wide variety of
biological activities including antitubercular, antibacterial, anti-trypanosomal, anticancer, anti-inflammatory,
antimalarial, anti-human immunodeficiency virus (HIV), antidepressant, and anti-hyperglycemic
effects [16–22]. The present report describes the synthesis of 3-arylquinoxaline derivatives (target compounds,
Figure 1), whose structures are considered as the 4-aza isosteric isomers of compounds
3
and . Quinoxaline
4
is more hydrophilic than quinoline due to an additional nitrogen atom which bears a pair of non-pair
electrons to form hydrogen bonding with water. A reasonable high water solubility is important for in vivo
and pre-clinical evaluations. Our aim was to discover potential anti-DENV drug candidates with higher
potency and less cytotoxicity than our initial lead compounds 3 and 4.
Figure 1. Structures of N2-(4-fluorophenyl)-N3-[4-(pyridin-4-yl)benzyl]pyrazine-2,3-dicarboxamide (
4-[4-methyl-2-(3-methyl-1H-pyrazol-5-yl)benzyl)-2-(4-methylbenzyl)quinazoline (
methyl)-3-(4-methoxyphenyl)quinoline ( ), 2-(4-hydroxy benzoyl)-3-(4-hydroxyphenyl)quinoline (
1
),
2
), 2-(hydroxyphenyl
3
4
),
4-[5-(6-fluoroquinolin-2-yl)-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]benzenesulfonamide (
compounds (15a–26b; isosteric isomers of compounds 3 and 4).
5), and target

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2. Results and Discussion
2.1. Chemistry
The known 2-methyl-3-phenylquinoxaline (9a) was prepared via the condensation of 1-phenylpropane-
1,2-dione ( ) and benzene-1,2-diamine according to a previous study [23]. Accordingly, compounds
9b 11b were obtained from the starting materials , respectively, under the same reaction conditions in a
yield of 73–96%. Oxidation of 9a 11b with selenium oxide afforded 3-phenylquinoxaline-2-carbaldehyde
12a) or its analogs 12b 14b, respectively, in a yield of 58–88% [24]. Treatment of 12a with phenyl
6
–
6–8
–
(
–
magnesium bromide (Grignard reagent) afforded phenyl(3-phenylquinoxalin-2-yl)methanol (15a),
which was then oxidized with MnO2 to give phenyl(3-phenylquinoxalin-2-yl)methanone (21a) as
described in Scheme 1. Accordingly, Grignard reaction of compounds 12b
15b 20b, respectively, which were then oxidized with MnO2 to give their respective carbonyl
products 21b 26b under the same reaction conditions. Demethylation of 15b with 48% HBr gave
–14b afforded compounds
–
–
(4-hydroxyphenyl)(3-phenylquinoxalin-2-yl)methanone (27) as described in Scheme 2. However, reaction of
19b with 48% HBr did not give (4-hydroxyphenyl)(3-phenylquinoxalin-2-yl)methanone (29) nor the
expected 4-[3-(4-hydroxy benzyl)quinoxalin-2-yl]phenol (28). Oxidation of intermediate (28) with
SeO2 gave (4-hydroxyphenyl) [3-(4-hydroxyphenyl)quinoxalin-2-yl)methanone (29), as described in
Scheme 3. The structure of 15a–
29 was determined by NMR (1H and 13C) (spectra data can be found in
Supplementary Materials) and further confirmed by elemental analysis.
Scheme 1. Synthesis of 3-arylquinoxaline derivatives 21a–26b.

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Scheme 2. Synthesis of (4-hydroxyphenyl)(3-phenylquinoxalin-2-yl)methanone (27).
Scheme 3. Synthesis of (4-hydroxyphenyl)(3-(4-hydroxyphenyl)quinoxalin-2-yl)methanone (29).
2.2. Biological Evaluation
2.2.1. Anti-DENV Activities and Cytotoxicities
The anti-DENV activities and cytotoxicities of 3-arylquinoxaline derivatives are summarized in
Table 1. Huh-7-DV-Fluc cells were treated with compounds , and 15 28 for three days. Anti-DENV
activities were determined by firefly luciferase activity. Compounds which exhibited >32% inhibition
of DENV-2 at a concentration of 10 M were considered as active [15]. For the 2-hydroxymethyl
3,
4
–
µ
derivatives, 2-(hydroxyphenyl methyl)-3-phenylquinoline (15a) was inactive, while its methoxy
derivative (15b, R3 = OMe) was moderately active and exhibited 48% inhibition of DENV-2 at a
concentration of 10
antiviral activity, indicating the electron-donating group at R3 position was crucial. The same
trend was observed in which 16b was more active than 16a, and 17b was more active than 17a
Although compound 15b was less active than the initial lead of , its cytotoxicity was also lower than
compound
. Substitution of Cl group at R2 position decreased anti-DENV activity, in which 16b
was inactive (9% inhibition) at a concentration of 10 M, while the unsubstituted 15b exhibited 48%
µ
M. Introduction of MeO group at R3 position (15a vs. 15b) strongly enhanced
.
3
3
µ
inhibition of DENV-2 at the same concentration. Substitution of F group at R1 position decreased
anti-DENV activity in which 17b (22% inhibition) was much less active than the unsubstituted 15b,
indicating the electron-withdrawing group at R1 position was unfavorable. Contrarily, introduction
of the electron-donating MeO group at R1 position (15a vs. 19a) strongly enhanced antiviral activity.
[3-(4-Methoxyphenyl)quinoxalin-2-yl](phenyl)methanol (19a), whose structure belongs to the 4-aza
analog of
derivatives. It is worth mentioning that compound 19a was non-cytotoxic to Huh-7 cells, exhibiting
85% cell viability at a concentration of 200
M. Further substitution of the MeO group at R3 position
19b vs. 19a) or Cl group at R2 position (20a vs. 19a) decreased antiviral activity. Compound 20b was
3, was the most active (82% inhibition) among these newly synthesized 3-arylquinoxaline
µ
(
inactive, which bears MeO groups at both R1 and R3 positions and also a Cl group at R2 position. These
results indicated that the optimal electron environment is important, in which the electron-donating
MeO group may be substituted at either R1 or R3 but not both positions. For the 2-carbonyl derivatives,
phenyl(3-phenylquinoxalin-2-yl)methanone (21a) was inactive. Introduction of the electron-donating

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MeO group at R3 position (21b vs. 21a) strongly enhanced anti-DENV activity. Compound 21a
(R3 = H) was inactive, while 21b (R3 = OMe) was highly active, exhibited 51% inhibition of DENV-2
at a concentration of 10 µM, and was non-cytotoxic against Huh-7 cells at 200 µM. Introduction of
Cl group at R2 position (21b vs. 22b) or F group at R1 position (21b vs. 23b) decreased antiviral
activity. Introduction of the MeO group at R1 position (21a vs. 25a) strongly enhanced antiviral activity,
indicating the electron-donating group at R1 position was favorable. Compound 26b was inactive,
which bears MeO groups at both R1 and R3 positions and also a Cl group at R2 position.
Table 1. Antiviral activities and cytotoxicities of 3-arylquinoxaline derivatives.
DENV2
Huh-7 Cell
Compounds
a % Inhibition at 1 µM % Inhibition at 10 µM % viability at 20 µM
% viability at 200 µM
3
4
12.37 ± 2.67
11.21 ± 2.31
2.13 ± 1.12
24.34 ± 2.51
1.97 ± 0.72
2.41 ± 1.48
2.58 ± 0.85
19.17 ± 3.5
3.41 ± 1.11
36.74 ± 3.71
35.47 ± 4.82
16.31 ± 3.42
2.75 ± 1.69
2.76 ± 1.55
42.78 ± 5.13
12.74 ± 3.91
5.19 ± 2.78
34.17 ± 5.12
16.44 ± 3.11
2.54 ± 1.21
40.44 ± 2.22
41.51 ± 3.25
1.95 ± 0.82
1.17 ± 0.41
44.85 ± 3.68
2.36 ± 1.11
10.14 ± 1.98
50.14 ± 3.47
41.24 ± 2.88
5.31 ± 1.73
48.67 ± 4.71
6.42 ± 2.97
9.46 ± 2.88
4.78 ± 1.93
22.89 ± 4.54
6.74 ± 1.57
82.71 ± 2.88
47.69 ± 5.41
58.71 ± 2.86
4.39 ± 1.52
7.98 ± 2.14
51.85 ± 3.52
19.74 ± 4.17
24.68 ± 1.89
40.78 ± 2.58
33.58 ± 4.21
23.47 ± 3.71
47.53 ± 3.51
49.74 ± 4.38
14.47 ± 2.55
19.28 ± 3.85
48.69 ± 3.48
4.25 ± 1.39
32.53 ± 2.30
90.28 ± 2.62
96.62 ± 8.78
104.71 ± 5.66
103.29 ± 1.79
108.06 ± 8.91
104.05 ± 4.18
96.35 ± 3.52
102.33 ± 1.98
102.04 ± 3.96
99.66 ± 1.65
96.43 ± 1.24
99.42 ± 3.31
95.47 ± 3.96
107.34 ± 2.30
106.46 ± 3.12
108.76 ± 1.36
103.09 ± 8.30
98.61 ± 5.68
104.26 ± 1.85
106.06 ± 2.12
101.13 ± 2.39
106.59 ± 3.03
99.39 ± 6.50
104.35 ± 4.22
101.58 ± 2.84
103.62 ± 2.91
71.37 ± 1.31
21.87 ± 3.55
41.38 ± 5.71
59.94 ± 8.33
61.42 ± 2.67
20.62 ± 5.37
72.96 ± 9.05
73.26 ± 6.57
100.73 ± 1.27
21.50 ± 1.60
85.26 ± 5.63
21.34 ± 1.55
27.12 ± 1.24
34.21 ± 4.97
79.45 ± 7.07
95.13 ± 4.32
103.05 ± 2.67
92.02 ± 3.08
87.59 ± 5.75
103.32 ± 1.03
91.94 ± 2.83
100.30 ± 4.59
99.42 ± 2.14
35.29 ± 4.95
99.11 ± 2.40
18.43 ± 2.33
51.91 ± 1.62
14.36 ± 2.14
15a
15b
16a
16b
17a
17b
18a
19a
19b
20a
20b
21a
21b
22a
22b
23a
23b
24a
25a
25b
26a
26b
27
29
Ribavirin b
DENV—dengue virus; a Values are means
±
standard deviations (SD) of at three separate experiments. Ribavirin: reference [15].
b
In general, the 2-hydroxymethyl derivatives were less active than their respective 2-carbonyl
counterparts (15b 21b 16a 22a 16b 22b 17a 23a 17b 23b 19b 25b) with an exception of
19a, which was more active than 25a. Compounds 19a 20a, and 21b were three of the most active
antiviral agents, exhibiting >50% inhibition of DENV-2 at a concentration of 10 M, and were selected
<
;
<
;
<
;
<
;
<
;
<
,
µ
for further evaluation. The effective concentration that inhibited 50% DENV replication (EC50), the
concentration that inhibited 50% cell growth (CC50), and the selective index (SI: CC50/EC50) of these
compounds were determined with our initial leads
indicated that compounds 19a, 20a, and 21b were superior to compounds
ribavirin (Table 2). Among them, compound 19a was the most active (EC50 of 1.29
approximately 10-fold more anti-DENV activity than that of ribavirin (EC50 = 13.16
3
and
4
, using ribavirin as a positive control. Results
and and more active than
M), exhibiting
M). In addition,
3
4
µ
µ
compound 19a was less cytotoxic than ribavirin. The selective index (SI) of 19a was approximately
35-fold higher than that of ribavirin (155.04 vs. 4.47). Based on the above results, compound 19a was
selected as a lead compound for further pharmacological studies.

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Table 2. Antiviral activities ((EC50) (µM)) a of the compounds tested.
a
SI c
b
Compounds
EC50
CC50
3
4
19a
20a
21b
12.57 ± 2.16
13.16 ± 1.23
1.29 ± 0.74
5.68 ± 0.81
3.25 ± 0.89
12.61 ± 1.17
139.11 ± 2.32
163.67 ± 2.32
>200
>200
>200
56.31 ± 2.32
11.07
12.44
>155.04
>35.21
>61.54
4.47
Ribavirin
a
b
EC50 is the concentration of the compound resulting in a 50% inhibition in virus production. CC50 is the concentration of the
c
compound causing a 50% growth inhibition of uninfected Huh-7 cells. SI: selectivity index. SI = CC50/EC50. Values are means
± standard deviations (SD) of at three separate experiments.
2.2.2. Compound 19a Reduced DENV Replication in Huh-7 cells
To further confirm the anti-DENV effect of compound 19a, we treated compound 19a at indicated
concentrations in Huh-7 cells for three days. Both Western blotting and RT-qPCR were performed
to determine the activity of compound 19a against DENV replication, and the results showed that
compound 19a dose-dependently reduced DENV protein synthesis and RNA replication in Huh-7
cells (Figures 2 and 3).
Figure 2. Inhibition of dengue virus (DENV) RNA expression in DENV-infected Huh-7 cells by 19a
.
Huh-7 cells were treated with 0.5, 1, 5, and 10 19a for three days. Total RNA was extracted
µM
and quantified DENV RNA levels by RT-qPCR. DENV RNA expression was normalized by cellular
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Treatment with 0.1% dimethyl sulfoxide
(DMSO) served as a mock control. The results are expressed as the means
of triplicate experiments.
±
standard deviations (SD)
Figure 3. Inhibition of DENV protein synthesis in Huh-7 cells by 19a. Huh-7 cells were treated with
0.5, 1, 2.5, and 5 19a for three days. Total cell lysate was collected for performing Western blotting
to analyze DENV protein synthesis. Levels of GAPDH were used as equal loading control.
µM

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2.2.3. Compound 19a Reduced DENV Replication through Inhibiting COX-2 Expression
In a previous study, Lin et al. reported that induction of COX-2 could suppress DENV
replication [11]. To determine whether compound 19a had impact on COX-2 protein and mRNA
expression in Huh-7 cells, we treated Huh-7 cells with 0.5 to 5 µM of 19a. The results showed
that compound 19a could inhibit COX-2 protein and mRNA level in Huh-7 cells, compared
with the DMSO-treated Huh-7 cells (Figure 4A,B). To characterize whether the 19a-mediated
downregulation of COX-2 expression and its catalytic activity was involved in the suppression of DENV
replication, we overexpressed exogenous COX-2 to evaluate the inhibitory activity of 19a on DENV
replication. Huh-7 cells were transfected with vehicle or various concentrations of cytomegalovirus
(pCMV)-COX-2-Myc vector encoding the cox-2 gene, and the plasmid-transfected cells were incubated
with 1 µM 19a or 0.1% DMSO, as a negative control. As shown in Figure 4C, the 19a-reduced viral RNA
level was gradually rescued following the increasing expression of exogenous COX-2-Myc compared
with that in the vehicle-transfected Huh-7 cells incubated with DMSO. Consistently, the exogenous
expression of COX-2 attenuated the inhibitory effect of 19a on the non-structural 2B (NS2B) protein
levels, as analyzed by Western blotting.
Figure 4. Inhibitory effect of 19a on DENV-induced cyclooxygenase-2 (COX-2) expression in Huh-7
cells. Concentration-dependent reduction of DENV-induced COX-2 RNA replication (
expression ( ) by 19a. Huh-7 cells were treated with 19a at the indicated concentrations for three
days. Cell lysates were subjected to Western blotting and RT-qPCR. ( ) Concentration-dependent
restoration of the DENV protein level. Cell lysates were subjected to Western blotting with an antibody
against NS2B, Myc, or GAPDH. ( ) Concentration-dependent reduction of DENV-induced COX-2
A) and protein
B
C
D
gene promoter activity by 19a in Huh-7 cells. Huh-7 cells were transfected with pCOX-2-Luc reporter
plasmid encoding firefly luciferase under the control of the COX-2 promoter. After treatment with 19a
at the indicated concentrations for three days, cell lysates were subjected to a luciferase activity assay.
The results are expressed as the means ± standard deviations (SD) of triplicate experiments.

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These results clearly indicated that 19a inhibited DENV replication by downregulating the
virus-induced COX-2 expression. We also used a COX-2 promoter-based reporter assay to evaluate
the inhibitory effect of 19a on COX-2 at the transcriptional level. The pCOX-2-Luc, a plasmid
encoding firefly luciferase under the control of the COX-2 promoter, was transfected into Huh-7 cells
or DENV-infected Huh-7 cells and, then, these plasmid-transfected cells were incubated with 19a at
increasing concentrations for three days. As shown in Figure 4D, 19a dose-dependently decreased the
DENV-elevated COX-2 promoter activity in DENV-infected cells.
2.3. Molecular Docking
In order to explain the binding mode and interaction, molecule docking of 19a was performed
against COX-2 (Protein Data Bank (PDB) code 3LN1) enzyme. Figure 5 shows the three-dimensional
(3D) molecular interaction of 19a against COX-2. The binding between 19a and COX-2 was stabilized
through hydrophobic interactions, hydrogen binding, and π–cation interactions. The amino acids
involved including 19a caused hydrophobic interactions at the active sites with Arg29, Glu31, Tyr116,
Leu138, Pro139, Lys454, Arg455, and Gln529. Arg29, located in the binding pocket, played a vital role
in the conformation of 19a, causing three kinds of interaction. The calculated lowest binding energy
between 19a and COX-2 was −9.10 kcal/mol.
Figure 5. Docking poses of compound 19a at COX-2 (PDB code 3LN1) enzyme. The interactions
illustrated by green lines represent hydrophobic interactions, blue lines represent hydrogen bonds, and
red lines represent π–cation interactions.
3. Materials and Methods
3.1. Chemistry Section
Melting points were determined on an Electrothermal IA9100 melting point apparatus
(Electrothermal, Staordshire, UK). Nuclear magnetic resonance (1H) spectra were recorded on a
Varian-Unity-400 spectrometer (LabX, Canada). Chemical shifts were expressed in parts per million (δ)
with tetramethylsilane (TMS) as an internal standard. Thin-layer chromatography (TLC) was performed
on silica gel 60 F-254 plates purchased from E. Merck KG (Darmstadt, Germany). The elemental
analyses were performed in the Instrument Center of National Science Council at National Cheng-Kung

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University and National Chung-Hsing University using Heraeus CHN-O Rapid EA (Labexchange,
Burladingen, Germany), and all values were within ±0.4% of the theoretical compositions.
3.1.1. General Procedure for the Preparation of 2-Methyl-3-phenylquinoxalines 9a–11b
Compound 6 (2.0 mmol) and appropriate benzene-1,2-diamine (2.0 mmol) were refluxed under
(H2O:EtOH = 1:1) for 3 h (TLC monitoring). After cooling, the solvent was removed in vacuo to provide
the crude product, which was purified by flash column chromatography on silica gel (CH2Cl2/MeOH
100/1) and recrystallized with EtOH to give 2-methyl-3-phenylquinoxalines 9a and 9b. Compounds
10a
–11b were prepared from 7 or 8 (2.0 mmol) and appropriate benzene-1,2-diamine (2.0 mmol) under
the same reaction procedures [23].
3.1.2. General Procedure for the Preparation of 3-Phenylquinoxaline-2-carbaldehydes 12a–14b
A mixture of 9a (3.0 mmol) and selenium dioxide (0.66 g, 6.0 mmol) in 1,4-dioxane (50 mL) was
refluxed for 3 h (TLC monitoring) and then cooled to room temperature. The reaction mixture was
filtered through celite to remove the black residue. Evaporation of the solvent afforded a residue which
was dissolved in ethyl acetate (200 mL), washed with brine (100 mL), H2O (100 mL), and saturated
sodium bicarbonate solution (100 mL), and dried (MgSO4). The crude product was recrystallized with
EtOH to give 3-phenylquinoxaline-2-carbaldehydes 12a and 12b. Compounds 13a
from 10a–11b (3.0 mmol) under the same reaction procedures [24].
–14b were prepared
3.1.3. General Procedure for the Preparation of Phenyl(3-phenylquinoxalin-2-yl)methanols 15a–20b
A mixture of 3-phenylquinoxaline-2-carbaldehyde 12a (1.0 mmol), appropriate phenyl magnesium
bromide (3 mmol, 3 mL of a 1 M solution in tetrahydrofuran (THF)), and dry THF (30 mL) was stirred
◦
at 0 C for 12 h (TLC monitoring). The reaction was quenched by addition of water (3 mL) and
partitioned between H2O (50 mL) and CH2Cl2 (50 mL). The organic layer was washed with brine and
dried over MgSO4, and removal of the volatiles in vacuo provided a residue, which was purified by
flash chromatography on silica gel (n-hexane: ethyl acetate = 3/2) and recrystallized from EtOH to give
compounds 15a or 15b. Accordingly, compounds 16a–20b were obtained in the same manner.
Phenyl(3-phenylquinoxalin-2-yl)methanol (15a)
Yield 40%, Melting point (Mp): 146.0–147.0 ◦C. 1H NMR (400 MHz, CDCl3)
), 6.13 (d, 1H, J = 7.2 Hz, C OH), 6.80 (dd, 2H, J1 = 8.0 Hz, J2 = 1.6 Hz), 7.10 (m, 3H), 7.27 (m, 2H),
7.42 (m, 3H), 7.82 (m, 2H), 8.16 (m, 1H), 8.22 (m, 1H). 13C NMR (100 MHz, CDCl3)
72.87, 127.56
δ 5.58 (d, 1H, J = 7.2 Hz,
O
H
H
δ
(2C), 127.73, 128.23 (2C), 128.53, 128.55 (2C), 128.76 (2C), 129.13, 129.33, 130.21, 130.32, 137.57, 139.52,
141.19, 141.61, 154.16, 154.32. Analysis calculated for C21H16N2O: C 80.75, H 5.16, N 8.97; found C
80.55, H 5.10, N 8.90.
(4-Methoxyphenyl)(3-phenylquinoxalin-2-yl)methanol (15b)
Yield 59%, Mp: 166.0–168.6 ◦C. 1H NMR (400 MHz, CDCl3)
), 6.07 (s, 1H, C OH), 6.62 (d, 2H, J = 8.8 Hz), 6.72 (d, 2H, J = 8.8 Hz), 7.26 (d, 2H, J = 8.4 Hz), 7.44
(m, 3H), 7.82 (m, 2H), 8.16 (m, 1H), 8.22 (m, 1H). 13C NMR (100 MHz, CDCl3)
55.18, 72.37, 113.69
(2C), 128.52 (3C), 128.74 (2C), 128.84 (2C), 129.09, 129.32, 130.13, 130.28, 133.51, 137.63, 139.52, 141.56,
δ 3.71 (s, 3H, OCH3), 5.51 (br s, 1H,
O
H
H
δ
154.19, 154.59, 159.12. Analysis calculated for C22H18N2O2
76.39, H 5.46, N 8.27.
·
0.1H2O: C 76.76, H 5.34, N 8.14; found C
(6,7-Dichloro-3-phenylquinoxalin-2-yl)(phenyl)methanol (16a)
◦
1
Yield 40%, Mp: 136.4–136.9 C. H NMR (400 MHz, CDCl3)
6.13 (d, 1H, J = 6.8 Hz, C OH), 6.78 (m, 2H), 7.11 (m, 3H), 7.27 (m, 2H), 7.42 (m, 3H), 8.27 (s, 1H),
8.35 (s, 1H). 13C NMR (100 MHz, CDCl3)
72.95, 127.46 (2C), 127.98, 128.37 (2C), 128.65 (2C), 128.67
δ 5.24 (d, 1H, J = 7.2 Hz, OH),
H
δ

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(2C), 129.18, 129.54, 129.96, 134.92, 135.03, 136.90, 138.33, 140.35, 140.64, 155.21, 155.79. Analysis
calculated for C21H14Cl2N2O·0.1H2O: C 65.82, H 3.74, N 7.31; found C 65.67, H 3.80, N 7.52.
(6,7-Dichloro-3-phenylquinoxalin-2-yl)(4-methoxyphenyl)methanol (16b)
Yield 37%, Mp: 157.2–157.6 ◦C. 1H NMR (400 MHz, CDCl3)
J = 7.2 Hz, ), 6.07 (d, 1H, J = 7.2 Hz, C OH), 6.63 (d, 2H, J = 8.8 Hz), 6.70 (d, 2H, J = 8.8 Hz), 7.26
(d, 2H, J = 8.4 Hz), 7.44 (m, 3H), 8.26 (s, 1H), 8.35 (s, 1H). 13C NMR (100 MHz, CDCl3)
55.19, 72.45,
δ 3.72 (s, 3H, OCH3), 5.16 (d, 1H,
O
H
H
δ
113.80 (2C), 128.62 (2C), 128.65 (2C), 128.77 (2C), 129.16, 129.50, 129.95, 132.89, 134.8, 134.97, 136.96,
138.34, 140.30, 155.25, 156.05, 159.26. Analysis calculated for C22H16Cl2N2O2: C 64.22, H 3.92, N 6.81;
found C 63.84, H 3.88, N 6.76.
[3-(4-Fluorophenyl)quinoxalin-2-yl](phenyl)methanol (17a)
◦
1
Yield 41%, Mp: 157.4–158.0 C. H NMR (400 MHz, CDCl3)
6.07 (d, 1H, J = 6.4 Hz, C OH), 6.82 (m, 2H), 7.11 (m, 5H), 7.25 (m, 2H), 7.84 (m, 2H), 8.15 (m, 1H), 8.23
(m, 1H). 13C NMR (100 MHz, CDCl3)
72.98, 115.63 (d, JCF = 22 Hz, 2C), 127.58 (2C), 127.91, 128.37
(2C), 128.55, 129.27, 130.37, 130.50, 130.74 (d, JCF = 8.3 Hz, 2C), 133.68 (d, JCF = 3.8 Hz), 139.52, 141.07,
δ 5.79 (d, 1H, J = 6.8 Hz, OH),
H
δ
141.56, 153.16, 154.18, 163.30 (d, JCF = 267.1 Hz). Analysis calculated for C21H15FN2O 0.1H2O: C 75.93,
·
H 4.62, N 8.44; found C 75.64, H 4.54, N 8.45.
[3-(4-Fluorophenyl)quinoxalin-2-yl](4-methoxyphenyl)methanol (17b)
Yield 78%, Mp: 173.0–174.7 ◦C. 1H NMR (400 MHz, CDCl3)
6.65 (d, 2H, J = 8.8 Hz), 6.75 (d, 2H, J = 8.8 Hz), 7.10 (m, 2H), 7.25 (m, 2H), 7.85 (m, 2H), 8.14 (m, 1H), 8.23
(m, 1H). 13C NMR (100 MHz, CDCl3)
55.20, 72.44, 113.77 (2C), 115.60 (d, JCF = 21.7 Hz, 2C), 128.52,
128.87 (2C), 129.24, 130.29, 130.47, 130.70 (d, JCF = 8.4 Hz), 133.30, 133.71 (d, JCF = 3.4 Hz, 2C), 139.49,
δ 3.72 (s, 3H, OCH3), 6.02 (s, 1H, CHOH),
δ
141.48, 153.17, 154.41, 159.20, 163.24 (d, JCF = 247.8 Hz). Analysis calculated for C22H17FN2O2 0.2H2O:
·
C 72.59, H 4.83, N 7.70; found C 72.43, H 4.73, N 7.73.
[6,7-Dichloro-3-(4-fluorophenyl)quinoxalin-2-yl](phenyl)methanol (18a)
Yield 52%, Mp: 150.0–150.2 ◦C. 1H NMR (400 MHz, CDCl3)
H
OH), 6.81 (m, 2H), 7.13 (m, 5H), 7.25 (m, 2H), 8.12 (s, 1H), 8.36 (s, 1H). 13C NMR (100 MHz, CDCl3):
δ 5.22 (br s, 1H, OH), 6.07 (s, 1H,
C
73.06, 115.79 (d, JCF = 21.8 Hz, 2C), 127.49 (2C), 128.15, 128.51 (2C), 129.18, 129.90, 130.73 (d, JCF = 8.4,
2C), 133.02 (d, JCF = 3.4), 135.11, 135.25, 138.33, 140.28, 140.54, 154.20, 155.65, 163.49 (d, JCF = 248.5 Hz).
Analysis calculated for C21H13Cl2FN2O: C 63.18, H 3.28, N 7.02; found C 63.29, H 3.06, N 7.07.
[3-(4-Methoxyphenyl)quinoxalin-2-yl](phenyl)methanol (19a)
Yield 48%, Mp: 160.4–161.9 ◦C. 1H NMR (400 MHz, CDCl3)
J = 7.2 Hz, ), 6.16 (d, 1H, J = 7.2 Hz, C OH), 6.86 (m, 2H), 6.93 (m, 2H), 7.12 (m, 3H), 7.25 (m, 2H),
7.80 (m, 2H), 8.14 (m, 1H), 8.21 (m, 1H). 13C NMR (100 MHz, CDCl3)
55.46, 72.91, 114.14 (2C), 127.42
δ 3.87 (s, 3H, OCH3), 5.55 (d, 1H,
O
H
H
δ
(2C), 127.99, 128.40 (2C), 129.12, 129.30, 129.86, 130.28 (2C), 134.69, 134.79, 138.17, 140.43, 140.88, 154.90,
155.95, 160.75. Analysis calculated for C22H18N2O2: C 77.17, H 5.30, N 8.18; found C 77.02, H 5.23,
N 8.10.
(4-Methoxyphenyl)[3-(4-methoxyphenyl)quinoxalin-2-yl]methanol (19b)
Yield 78%, Mp: 132.4–136.0 ◦C. 1H NMR (400 MHz, CDCl3)
3), 5.45 (d, 1H, J = 7.6 Hz, O ), 6.10 (d, 1H, J = 7.2 Hz, C OH), 6.65 (d, 2H, J = 8.8 Hz), 6.89 (d, 2H,
J = 8.8 Hz), 6.93 (d, 2H, J = 8.8 Hz), 7.25 (d, 2H, J = 8.8 Hz), 7.80 (m, 2H), 8.14 (m, 1H), 8.19 (m, 1H).
13C NMR (100 MHz, CDCl3)
55.19, 55.42, 72.34, 113.71 (2C), 114.01 (2C), 128.46, 128.80 (2C), 129.23,
δ 3.72 (s, 3H, OCH3), 3.87 (s, 3H,
OC
H
H
H
δ
130.03 (2C), 130.09, 130.26 (2C), 133.73, 139.39, 141.63, 153.89, 154.74, 159.12, 160.41. Analysis calculated
for C23H20N2O3: C 74.18, H 5.41, N 7.52; found C 73.92, H 5.33, N 7.47.

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[6,7-Dichloro-3-(4-methoxyphenyl)quinoxalin-2-yl](phenyl)methanol (20a)
Yield 55%, Mp: 156.8–157.1 ◦C. 1H NMR (400 MHz, CDCl3)
J = 7.2 Hz, ), 6.16 (d, 1H, J = 6.0 Hz, C OH), 6.85 (dd, 2H, J1 = 8.8 Hz, J2 = 2.0 Hz), 6.94 (d, 2H,
J = 8.8 Hz), 7.15 (m, 3H), 7.28 (d, 2H, J = 8.8 Hz), 8.25 (s, 1H), 8.33 (s, 1H). 13C NMR (100 MHz, CDCl3)
55.46, 72.91, 114.14 (2C), 127.42 (2C), 127.99, 128.40 (2C), 129.12, 129.30, 129.86, 130.28 (2C), 134.69,
δ 3.88 (s, 3H, OCH3), 5.22 (d, 1H,
OH
H
δ
134.79, 138.17, 140.43, 140.88, 154.90, 155.95, 160.75. Analysis calculated for C22H16Cl2N2O2
C 63.94, H 3.96, N 6.78; found C 63.75, H 3.88, N 6.77.
·0.1H2O:
[6,7-Dichloro-3-(4-methoxyphenyl)quinoxalin-2-yl](4-methoxyphenyl)methanol (20b)
Yield 56%, Mp: 144.1–144.5 ◦C. 1H NMR (400 MHz, CDCl3)
OCH3), 5.13 (d, 1H, J = 7.6 Hz, O ), 6.11 (d, 1H, J = 6.8 Hz, C OH), 6.66 (d, 2H, J = 8.8 Hz), 6.78
(d, 2H, J = 8.8 Hz), 6.94 (d, 2H, J = 8.8 Hz), 7.26 (d, 2H, J = 8.8 Hz), 8.24 (s, 1H), 8.32 (s, 1H). 13C NMR
(100 MHz, CDCl3) 55.20, 55.45, 72.42, 113.83 (2C), 114.10 (2C), 128.74 (2C), 129.11, 129.36, 129.85,
δ 3.72 (s, 3H, OCH3), 3.88 (s, 3H,
H
H
δ
130.28 (2C), 133.14, 134.62, 134.67, 138.18, 140.38, 154.91, 156.19, 159.26, 160.71. Analysis calculated for
C23H18Cl2N2O3: C 62.60, H 4.11, N 6.35; found C 62.40, H 4.00, N 6.42.
3.1.4. General procedure for the Preparation of Phenyl(3-phenylquinoxalin-2-yl)methanones (21a–26b)
A mixture of 15a (1.0 mmol) and MnO2 (10.0 mmol) in CH2Cl2 (20 mL) was stirred at room
temperature for 12 h (TLC monitoring). The reaction mixture was partitioned between H2O (50 mL)
and CH2Cl2 (50 mL). The organic layer was washed with brine and dried over MgSO4, and removal of
the volatiles in vacuo provided a residue, which was crystallized from MeOH to give compound 21a
Accordingly, compounds 21b–26b were obtained in this manner.
.
Phenyl(3-phenylquinoxalin-2-yl)methanone (21a)
Yield 89%, Mp: 155.2–156.7 ◦C. 1H NMR (400 MHz, CDCl3)
(tt, 1H, J1 = 7.6 Hz, J2 = 1.6 Hz), 7.70 (m, 2H), 7.82 (m, 1H), 7.88(m, 1H), 7.94 (m, 2H), 8.16 (m, 1H),
8.24 (m, 1H). 13C NMR (100 MHz, CDCl3)
128.70 (4C), 129.11 (2C), 129.46, 129.49, 129.59, 130.44,
δ 7.38 (m, 3H), 7.47 (m, 2H), 7.461
δ
130.52 (2C), 131.40, 134.05, 135.62, 137.27, 139.65, 142.14, 151.31, 152.81, 193.98. Analysis calculated for
C21H14N2O 0.3H2O: C 79.87, H 4.67, N 8.87; found C 79.75, H 4.46, N 8.92.
(4-Methoxyphenyl)(3-phenylquinoxalin-2-yl)methanone (21b)
Yield 70%, Mp: 130.0–132.4 ◦C. 1H NMR (400 MHz, CDCl3)
7.39 (m, 3H), 7.72 (dd, 2H, J1 = 6.0 Hz, J2 = 2.4 Hz), 7.81 (m, 1H), 7.87 (m, 1H), 7.92 (m, 2H), 8.16 (m, 1H),
8.23 (m, 1H). 13C NMR (100 MHz, CDCl3)
55.56, 114.05 (2C), 128.67 (2C), 128.72, 129.06 (2C), 129.40,
δ 3.88 (s, 3H, OCH3), 6.94 (m, 2H),
δ
129.46, 129.56, 130.35, 131.22, 132.95 (2C), 137.36, 139.66, 142.08, 151.64, 152.75, 164.36, 192.58 Analysis
calculated for C22H16N2O2·0.2H2O: C 76.81, H 4.82, N 8.15; found C 76.76, H 4.72, N 8.08.
(6,7-Dichloro-3-phenylquinoxalin-2-yl)(phenyl)methanone (22a)
Yield 66%, Mp: 154.3–156.8 ◦C. 1H NMR (400 MHz, CDCl3)
(tt, 1H, J1 = 7.6 Hz, J2 = 1.2 Hz), 7.68 (m, 2H), 7.93 (m, 2H), 8.28 (s, 1H), 8.36 (s, 1H). 13C NMR (100 MHz,
CDCl3) 128.81 (4C), 129.09 (2C), 129.96, 130.02, 130.09, 130.48 (2C), 134.33, 135.17, 135.25, 136.23,
δ 7.40 (m, 3H), 7.49 (m, 2H), 7.64
δ
136.52, 138.27, 140.84, 152.18, 153.84, 193.31. Analysis calculated for C21H12Cl2N2O: C 66.51, H 3.19, N
7.39; found C 66.61, H 3.00, N 7.38.
(6,7-Dichloro-3-phenylquinoxalin-2-yl)(4-methoxyphenyl)methanone (22b)
Yield 79%, Mp: 171.9–172.3 ◦C. 1H NMR (400 MHz, CDCl3)
δ 3.89 (s, 3H, OCH3), 6.96 (m, 2H), 7.41
(m, 3H), 7.70 (dd, 2H, J1 = 8.0 Hz, J2 = 1.6 Hz), 7.91 (m, 2H), 8.28 (s, 1H), 8.35 (s, 1H). 13C NMR (100 MHz,
CDCl3) δ 55.62, 114.17 (2C), 128.31, 128.77 (2C), 129.04 (2C), 129.91, 129.99, 130.06, 132.94 (2C), 135.03,

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136.01, 136.61, 138.30, 140.77, 152.52, 153.80, 164.57, 191.90 Analysis calculated for C22H14Cl2N2O2: C
64.56, H 3.45, N 6.84; found C 64.19, H 3.46, N 6.85.
[3-(4-Fluorophenyl)quinoxalin-2-yl](phenyl)methanone (23a)
Yield 85%, Mp: 94.1–96.6 ◦C. 1H NMR (400 MHz, CDCl3)
J = 8, 1.6 Hz), 7.64 (tt, 1H, J = 7.6, 1.6 Hz), 7.66 (m, 2H), 7.90 (br s, 1H), 7.93 (m, 2H), 7.96 (br s, 1H).
13C NMR (100 MHz, CDCl3)
115.87 (d, JCF = 22 Hz, 2C), 128.79 (2C), 129.23 (d, JCF = 6.8 Hz, 2C),
δ 7.08 (tt, 2H, J = 8.4, 2.0 Hz), 7.49 (tt, 2H,
δ
130.53 (2C), 130.57, 131.07, 131.16, 131.56, 133.37 (d, JCF = 3.0 Hz), 134.23, 135.46, 139.62, 142.06, 151.02,
151.66, 163.67 (d, JCF = 248.7 Hz), 193.95. Analysis calculated for C21H13FN2O: C 76.82, H 3.99, N 8.53;
found C 76.44, H 3.86, N 8.55.
[3-(4-Fluorophenyl)quinoxalin-2-yl](4-methoxyphenyl)methanone (23b)
Yield 83%, Mp: 147.6–147.9 ◦C. 1H NMR (400 MHz, CDCl3)
J = 9.2 Hz), 7.08 (m, 2H), 7.72 (m, 2H), 7.83 (m, 1H), 7.88 (m, 1H), 7.92 (m, 2H), 8.16 (m, 1H), 8.21
(m, 1H). 13C NMR (100 MHz, CDCl3)
55.59, 114.13 (2C), 115.84 (d, JCF = 21.8 Hz, 2C), 128.51, 129.37,
129.40, 130.49, 131.06 (d, JCF = 8.7 Hz, 2C), 131.38, 132.98 (2C), 133.46 (d, JCF = 3.5 Hz), 139.63, 141.98,
δ 3.89 (s, 3H, OCH3), 6.95 (d, 2H,
δ
151.37, 151.60, 163.65 (d, JCF = 248.2 Hz), 164.48, 192.55. Analysis calculated for C22H15FN2O2 0.1H2O:
·
C 73.35, H 4.26, N 7.78; found C 73.06, H 4.27, N 7.77.
(6,7-Dichloro-3-(4-fluorophenyl)quinoxalin-2-yl)(phenyl)methanone (24a)
Yield 75%, Mp: 184.3–186.4 ◦C. 1H NMR (400 MHz, CDCl3)
J = 8 Hz), 7.64-7.70 (m, 3H), 7.92 (dd, 2H, J1 = 8.0 Hz, J2 = 1.6 Hz), 8.27 (s, 1H), 8.33 (s, 1H). 13C
NMR (100 MHz, CDCl3) 116.03 (d, JCF = 22 Hz, 2C), 128.90 (2C), 129.93, 129.95, 130.48 (2C), 131.17
δ 7.08 (t, 2H, J = 8.8 Hz), 7.50 (t, 2H,
δ
(d, JCF = 8.8 Hz, 2C), 132.63 (d, JCF = 3.0 Hz), 134.51, 135.09, 135.29, 136.40, 138.23, 140.74, 151.87, 152.65,
163.93 (d, JCF = 249.7 Hz), 193.28. Analysis calculated for C21H11Cl2FN2O: C 63.50, H 2.79, N 7.05;
found C 63.37, H 2.68, N 6.96.
[3-(4-Methoxyphenyl)quinoxalin-2-yl](phenyl)methanone (25a)
Yield 91%, Mp: 93.9–98.2 ◦C. 1H NMR (400 MHz, CDCl3)
(m, 2H), 7.61 (tt, 1H, J1 = 7.6 Hz, J2 = 1.6 Hz), 7.68 (m, 2H), 7.79 (m, 1H), 7.86 (m, 1H), 7.94 (m, 2H),
8.14 (m, 1H), 8.21 (m, 1H). 13C NMR (100 MHz, CDCl3)
55.30, 114.29 (2C), 128.72 (2C), 129.33, 129.39,
129.64, 130.05, 130.52 (2C), 130.65 (2C), 131.28, 134.06, 135.61, 139.41, 142.20, 151.20, 152.19, 160.88,
δ 3.80 (s, 3H, OCH3), 6.90 (m, 2H), 7.47
δ
194.26. Analysis calculated for C22H16N2O2
N 7.97.
·
0.5H2O: C 75.62, H 4.91, N 8.02; found C 75.23, H 4.87,
(4-Methoxyphenyl)[3-(4-methoxyphenyl)quinoxalin-2-yl]methanone (25b)
Yield 76%, Mp: 144.5–146.2 ◦C. 1H NMR (400 MHz, CDCl3)
3.80 (s, 3H, OCH3), 3.87 (s, 3H,
δ
OCH3), 6.90 (d, J = 9.2 Hz), 6.94 (d, J = 9.2 Hz), 7.70 (d, J = 8.8 Hz), 7.78 (td, J1 = 6.8 Hz, J2 = 1.2 Hz,
1H), 7.84 (td, J1 = 6.8 Hz, J2 = 1.2 Hz, 1H), 7.92 (d, J = 8.8 Hz), 8.13 (dd, J1 = 8.0 Hz, J2 = 1.2 Hz, 1H),
8.20 (dd, J1 = 8.0 Hz, J2 = 1.2 Hz, 1H). 13C NMR (100 MHz, CDCl3)
δ 55.27, 55.55, 114.06 (2C), 114.21
(2C), 128.64, 129.26, 129.30, 129.68, 129.97, 130.57 (2C), 131.11, 132.93 (2C), 139.38, 142.09, 151.50, 152.10,
160.80, 164.34, 192.88. Analysis calculated for C23H18N2O3: C 74.58, H 4.90, N 7.56; found C 74.54,
H 4.86, N 7.60.
[6,7-Dichloro-3-(4-methoxyphenyl)quinoxalin-2-yl](phenyl)methanone (26a)
Yield 41%, Mp: 173.9–174.3 ◦C. 1H NMR (400 MHz, CDCl3)
J = 8.8 Hz), 7.50 (t, 2H, J = 7.6 Hz), 7.66 (m, 3H), 7.93 (dd, 2H, J1 = 8.4 Hz, J2 = 1.2 Hz), 8.25 (s, 1H), 8.32
(s, 1H). 13C NMR (100 MHz, CDCl3)
55.36, 114.39 (2C), 128.85 (2C), 129.85, 129.89, 130.49 (2C), 130.73
δ 3.81 (s, 3H, OCH3), 6.91 (d, 2H,
δ

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(2C), 134.35, 134.63, 135.22, 136.04, 138.01, 140.90, 152.05, 153.16, 161.28, 193.62. Analysis calculated for
C22H14Cl2N2O2·0.3H2O: C 63.70, H 3.56, N 6.76; found C 63.58, H 3.45, N 6.75.
[6,7-Dichloro-3-(4-methoxyphenyl)quinoxalin-2-yl](4-methoxyphenyl)methanone (26b)
Yield 78%, Mp: 178.8–179.0 ◦C. 1H NMR (400 MHz, CDCl3)
OCH3), 6.90 (m, 2H), 6.95 (m, 2H), 7.68 (m, 2H), 7.90 (m, 2H), 8.24 (s, 1H), 8.31 (s, 1H). 13C NMR
(100 MHz, CDCl3) 55.32, 55.61, 114.20 (2C), 114.33 (2C), 128.29, 128.91, 129.82, 129.84, 130.68 (2C),
δ 3.81 (s, 3H, OCH3), 3.89 (s, 3H,
δ
132.93 (2C), 134.49, 135.80, 138.04, 140.84, 152.40, 153.11, 161.23, 164.57, 192.22. Analysis calculated for
C23H16Cl2N2O3·0.2H2O: C 62.64, H 3.30, N 6.35; found C 62.45, H 3.67, N 6.26.
3.1.5. (4-Hydroxyphenyl)(3-phenylquinoxalin-2-yl)methanone (27)
A solution of 15b (0.33 g, 1.0 mmol) in 48% HBr (5 mL) was heated at reflux for 48 h. The mixture
was cooled and evaporated in vacuo to give a residue which was treated with H2O (50 mL). The crude
product was collected and crystallized from MeOH to give 27 (0.29 g, 87%) as a white solid, Mp:
196.5–199.3 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ
6.88 (d, J = 8.8 Hz, 2H, 3”–H, 5”–H), 7.42–7.46 (m, 3H),
7.65–7.68 (m, 2H), 7.65 (d, J = 8.8 Hz, 2H, 2”–H, 6”–H), 7.92–8.01 (m, 2H), 8.15 (dd, J1 = 8.0 Hz,
J2 = 1.6 Hz), 8.23 (dd, J1 = 8.0 Hz, J2 = 1.6 Hz), 10.78 (br s, 1H, Ph-O
). 13C NMR (100 MHz, DMSO-d6)
115.77 (2C), 126.87, 128.56 (2C), 128.84 (2C), 128.97, 129.12, 129.57, 130.94, 131.66, 133.16 (2C), 137.09,
H
δ
138.99, 141.37, 151.77, 151.87, 163.35, 191.99. Analysis calculated for C21H14N2O2
H 4.31, N 8.12; found C 73.23, H 4.69, N 8.14.
·
1.0H2O: C 73.05,
3.1.6. 4-[3-(4-Hydroxybenzyl)quinoxalin-2-yl]phenol (28)
Compound 28 was obtained from 19b as described for 27 in 37% yield, Mp: 184.8–185.9 ◦C. 1H
NMR (400 MHz, DMSO-d6) 4.30 (s, 2H, CH2), 6.58 (d, J = 10.8 Hz, 2H), 6.75 (d, J = 8.8 Hz, 2H), 7.87 (d,
J = 8.4 Hz, 2H), 7.38 (dd, J1 = 1.6 Hz, J2 = 8.4 Hz, 2H), 7.75–7.79 (m, 2H), 8.01–8.06 (m, 2H), 9.12 (br s,
1H, Ph-O ), 9.74 (br s, 1H, Ph-O 40.75, 114.91 (2C), 114.99 (2C),
). 13C NMR (100 MHz, DMSO-d6)
δ
H
H
δ
128.23, 128.36, 128.53, 129.19, 129.26, 129.36 (2C), 130.36 (2C), 140.34, 154.77 (2C), 155.56 (2C), 158.07
(2C). Analysis calculated for C21H16N2O2: C 76.81, H 4.91, N 8.53; found C 76.48, H 4.69, N 8.23.
3.1.7. (4-Hydroxyphenyl)(3-(4-hydroxyphenyl)quinoxalin-2-yl)methanone (29)
A mixture of 28 (0.33 g, 1.0 mmol) and selenium dioxide (0.22 g, 3.0 mmol) in 1,4-dioxane (20 mL)
was refluxed for 6 h (TLC monitoring) and then cooled to room temperature. The reaction mixture was
filtered through celite to remove the black residue. Evaporation of the solvent afforded a residue that
was dissolved in ethyl acetate (200 mL), washed with brine (100 mL), H2O (100 mL), and saturated
sodium bicarbonate solution (100 mL), and dried (MgSO4). The crude product was recrystallized with
EtOH to give 29 (0.31 g, 90%) as a white solid, Mp: 254.0–254.6 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ
6.80 (d, J = 8.8 Hz, 2H, 3”–H, 5”–H), 6.88 (d, J = 8.8 Hz 2H), 7.54 (d, J = 8.8 Hz 2H), 7.77 (d, J = 8.8 Hz,
2H, 2”–H, 6”–H), 7.86–7.96 (m, 2H), 8.11 (dd, J1 = 8.4 Hz, J2 = 1.2 Hz), 8.19 (dd, J1 = 8.4 Hz, J2 = 1.2 Hz),
9.85 (br s, 1H, Ph-O ), 10.78 (br s, 1H, Ph-O 115.52 (2C), 115.81
). 13C NMR (100 MHz, DMSO-d6)
(2C), 126.86, 127.65, 128.88, 128.91, 130.33, 130.51 (2C), 131.46, 133.05 (2C), 138.65, 141.44, 151.44, 151.66,
H
H
δ
159.02, 163.32, 192.30. Analysis calculated for C21H14N2O2
69.02, H 4.39, N 7.51.
·
1.2H2O: C 69.29, H 4.55, N 7.69; found C
3.2. Biological Activity
3.2.1. Compounds
Compounds were dissolved in DMSO at 10 mM and then diluted in culture medium.

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3.2.2. Cell
Human hepatoma Huh-7 cells were purchased from Bioresources Collection and Research Center,
Taiwan. Huh-7-DV-Fluc cells, C6/36 cells, and DENV-2 strain 16,681 were kindly provided by Dr. Huey
Nan Wu, Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.
3.2.3. Cytotoxicity Assays
For cytotoxicity tests, run in parallel with antiviral assays, plates at an initial density of
5 × 103 cells/well were treated with or without serial dilutions of test compounds. Cell viability
was determined after 72 h at 37 ◦C in a humidified CO2 (5%) atmosphere by the (2,3-bis[2-methyloxy-
4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) (XTT) method [25].
3.2.4. Transfection and Luciferase Activity Assay
Huh-7-DV-Fluc cells were seeded in 24-well plates at a density of 2
treated with the 3-arylquinoxaline compounds at two concentrations (1 and 10
×
104 cells per well, and
M) or 0.1% DMSO as
µ
control. After three days of incubation, the luciferase activity assay was performed using the Bright-Glo
Luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
To determine the regulation of COX-2 promoter activation during DENV-2 infection, Huh-7 cells
were transfected with 1 µg of the reporter plasmid pCOX-2-Luc (BD Biosciences Clontech, Palo Alto,
CA, USA) by using T-pro reagent (Ji-Feng Biotechnology CO., Ltd., Taipei, Taiwan) following the
instruction of the manufacturer. After 6 h of transfection, the transfection reagents were changed with
fresh medium containing 19a at different concentrations. After three days of incubation, cell extracts
were subjected to luciferase activity assay by using the Bright-Glo Luciferase Assay System (Promega)
in accordance with manufacturer’s instruction. To determine exogenous gene expression, Huh-7 cells
were transfected with either a vehicle vector or COX-2 expression vector pCMV-COX-2-Myc with
several concentrations (0.25 and 0.5
medium containing 19a at 1 M. In the knockdown of COX-2 gene expression, Huh-7 cells were
transfected with either a control vector -galactosidase (LacZ) short hairpin RNA (shRNA) or COX-2
µg) for 6 h. The transfection reagents were replaced with fresh
µ
β
shRNA expression vector (National RNAi Core Facility, Academia Sinica, Taipei, Taiwan) for 6 h. After
three days, protein and RNA levels were analyzed by Western blotting with a specific antibody as
previously described respectively [11].
3.2.5. Immunoblot Analysis
Huh-7 cells were seeded in 24-well plates at a density of 5
×
104 cells per well overnight and
treated with indicated reagent at proper concentrations for three days. Cells were washed with cold
phosphate-buffered saline (PBS) and lysed by radioimmunoprecipitation assay (RIPA) lysis buffer
(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM ethylenediaminetetraacetic acid
(EDTA), 1 mM ethylene glycol bis(2-aminoethyl)tetraacetic acid (EGTA), 1 mM NaVO3, 10 mM NaF,
1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 25
g/mL leupeptin) and stored at
20 ◦C. The protein concentration was determined by the Bradford
method. Then, 10- g protein samples were separated by 10% SDS-PAGE and transferred onto a
µg/mL aprotinin, and 25
µ
−
µ
polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat dried milk
and incubated with specific antibodies against GAPDH (1:10000, Genetex, Irvine, CA, USA) and COX-2
(1:1000; Cayman Chemical, Ann Arbor, ML, USA). Antibodies were diluted in 5% milk containing
Tris-buffered saline (TBS) and 0.5% Tween. The blotting signal was developed using an enhanced
chemiluminescence (ECL) detection kit (PerkinElmer, Waltham, MA, USA) and was counted by the
software Quantity One (Bio-Rad, Hercules, CA, USA).

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3.2.6. Quantification of DENV RNA
Total cellular RNA samples were extracted after three days of compound treatment through a Total
RNA Miniprep Purification Kit (GeneMark Biolab, Taiwan) following the manufacturer’s instructions,
and then transcribed to complementary DNA (cDNA) with Moloney murine leukemia virus (M-MLV)
reverse transcriptase (Promega, USA). The levels of DENV-2 replication and COX-2 were analyzed
by RT-PCR as described previously with the following specific primers: a forward primer, 50–AAG
GTG AGA AGC AAT GC AGC–30, and a reverse primer, 50–CCA CTC AGG GAG TTC TCT CT–30,
targeting the DENV-2 NS5 gene 44. DENV-2 and COX-2 RNA levels were normalized to the cellular
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA level of each sample.
3.2.7. Molecular Docking Study
The crystal structure of COX-2 (PDB ID: 3LN1) was acquired from the RCSB Protein Date Bank
(https://www.rcsb.org/). The 3D conformation of target compound 19a was produced by ChemBio
3D Ultra 14.0 (PerkinElmer, Waltham, MA, USA). The molecular docking was performed by Achilles
Blind Docking Server (http://bio-hpc.ucam.edu/achilles/). The “blind docking” approach was used for
the docking of the small molecule to the targets, which was done without a priori knowledge of the
location of the binding site by the system [26]. Visual representation of molecules was created with
3Dmol by Nicholas Rego and David Koes [27].
3.2.8. Statistical Analysis
The results were expressed as means
analyzed by a one-way analysis of variance (ANOVA) and Student’s t-test.
±
SD. Differences in mean values between groups were
4. Conclusions
In this study, we synthesized certain 3-arylquinoxaline derivatives in order to evaluate their
inhibitory activities of anti-DENV replication. Among them, compound 19a exhibited the most
potential activity against DENV replication, with an EC50 value of 1.29
of an antiviral mechanism, the results indicated that compound 19a reduced DENV replication through
±
0.74 µM. By the determination
COX-2 inhibition. Molecular docking results also showed that the lowest binding energy between
19a and COX-2 enzyme was 9.10 kcal/mol. Further studies are still needed in order to improve our
−
understanding of how these compounds elicit their anti-DENV activity.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/19/
4786/s1.
Author Contributions: C.-H.T. suggested the research idea, participated in the interpretation of the results, and
was responsible for manuscript writing; C.-R.H. participated in the synthesis, purification, and characterization of
the chemical compounds; K.-W.T. participated in molecular docking.
Funding: Financial support of this work by the Minister of Science and Technology of the Republic of China
(MOST 107-2320-B-037-015, MOST 108-2320-B-037-026-MY2) is gratefully acknowledged.
Acknowledgments: We thank the Scientific Integration Design Service Corporation for performing all biological
assays, and the Center for Research Resources and Development at Kaohsiung Medical University for
instrumentation and equipment support.
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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