Synthesis of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole derivatives as potent antiproliferative agents via a hybrid pharmacophore approach

Article abstract of DOI:10.1016/j.bioorg.2020.104293

Imiquimod (1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine) is efficacious in topical therapy for certain types of skin cancers. Structurally similar EAPB0203 (N-methyl-1-(2-phenethyl)imidazo[1,2-a]quinoxalin-4-amine) has been shown higher in vitro potency than imiquimod. Besides, triazole, oxadiazole, and thiadiazole rings are privileged building blocks in drug design. A series of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole and [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-thiadiazole derivatives were therefore synthesized by incorporation of these rings into the structure of EAPB0203 and assessed their antiproliferative effects against various cancer cell lines. The 1,3,4-oxadiazole derivatives demonstrated the superior effectiveness compared to imiquimod and EAPB0203. Our findings highlight the excellent potential of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole derivatives as anticancer agents.

Full text of DOI:10.1016/j.bioorg.2020.104293

BioorganicChemistry104(2020)104293
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Bioorganic Chemistry
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Short communication
Synthesis of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole derivatives
as potent antiproliferative agents via a hybrid pharmacophore approach
Daiki Kanekoa, Masayuki Ninomiyaa, Rina Yoshikawaa, Yukari Onoa, Amol D. Sonawanea,
⁎
Kaori Tanakab,c, Atsuyoshi Nishinad, Mamoru Koketsua,
a Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
b Division of Anaerobe Research, Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan
c United Graduate School of Drug Discovery and Medicinal Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan
d College of Science and Technology, Nihon University, Chiyoda, Tokyo 101-0062, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Imiquimod (1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine) is efficacious in topical therapy for certain types of
skin cancers. Structurally similar EAPB0203 (N-methyl-1-(2-phenethyl)imidazo[1,2-a]quinoxalin-4-amine) has
been shown higher in vitro potency than imiquimod. Besides, triazole, oxadiazole, and thiadiazole rings are
privileged building blocks in drug design. A series of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole and
[1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-thiadiazole derivatives were therefore synthesized by incorporation of
these rings into the structure of EAPB0203 and assessed their antiproliferative effects against various cancer cell
lines. The 1,3,4-oxadiazole derivatives demonstrated the superior effectiveness compared to imiquimod and
EAPB0203. Our findings highlight the excellent potential of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole
derivatives as anticancer agents.
Imiquimod
EAPB0203
[1,2,4]Triazolo[4,3-a]quinoxaline-1,3,4-
oxadiazole derivative
Antiproliferative activity
Hybrid pharmacophore approach
1. Introduction
molecules as chemotherapeutic agents has attracted substantial atten-
tion worldwide. Triazoles are five-membered heterocycles containing
Imiquimod (1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine, Fig. 1)
belonging to the imidazoquinoline family, is a prescription drug ap-
proved initially for the topical treatment of external genital and peri-
anal warts by the US Food and Drug Administration (FDA) in 1997
[1–3]. Then, it was consecutively approved to treat actinic keratosis
(AK) and superficial basal cell carcinoma (BCC). Extensive studies have
continued to provide evidence that imiquimod is also efficacious in the
treatment of skin cancers including squamous cell carcinoma (SCC),
malignant melanoma, cutaneous Kaposi’s sarcoma, and breast cancer
skin metastasis. Additional evidence in vitro has suggested its usefulness
in other cancer types [4–7].
three nitrogen atoms and considered to be privileged fragments pos-
sessing diverse biological activities [11,12]. Anastrozole containing a
1,2,4-triazole ring is used in the treatment of postmenopausal women
with estrogen-responsive breast cancer [13]. Oxadiazoles are five-
membered heterocycles containing two nitrogen atoms and one oxygen
atom. 1,3,4-Oxadiazoles show better physicochemical and pharmaco-
kinetic properties compared to other isomeric oxadiazoles [14,15]. An
anticancer drug candidate, Zibotentan (ZD4054) containing a 1,3,4-
oxadiazole ring has also been investigated in preclinical and clinical
trials for treatment of several cancer types [16]. Thiadiazoles are five-
membered heterocycles containing two nitrogen atoms and one sulfur
atom [17]. In phase I and II trials, Filanesib containing a 1,3,4-thia-
diazole ring recently demonstrated encouraging activity in relapsed/
refractory multiple myeloma with a manageable safety profile [18].
Cancer is one of the leading causes of human death (7–10 million
deaths per year in the world). However, it requires immeasurable costs
and times to develop innovative therapeutic anticancer drugs. A hybrid
pharmacophore approach consists of combination of several pharma-
cophoric units from different existing approved drugs and clinically
validated candidates into a single molecule [19–21]. This often
EAPB0203 (N-methyl-1-(2-phenethyl)imidazo[1,2-a]quinoxalin-4-
amine, Fig. 1) belonging to the imidazoquinoxaline family (nick-named
imiqualine), is a structurally analogue of imiquimod developed by C.
Deleuze-Masquéfa and P.-A. Bonnet group [8]. They reported that
EAPB0203 exerted 110- and 45-times higher potency than fotemustine
and imiquimod in human melanoma A375 cells, respectively [9].
Moreover, EAPB0203 showed cytotoxicity against several human leu-
kemic cell lines [10].
Exploration of triazole-, oxadiazole-, and thiadiazole-fused
⁎ Corresponding author.
E-mail address: koketsu@gifu-u.ac.jp (M. Koketsu).
https://doi.org/10.1016/j.bioorg.2020.104293
Received 8 July 2020; Received in revised form 8 September 2020; Accepted 15 September 2020
Availableonline19September2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

D. Kaneko, et al.
BioorganicChemistry104(2020)104293
in EtOH afforded the key material 2-chloro-3-hydrazinelyquinoxaline
(2) in 87% yield. Refluxing 2 with 1.5 equiv of aromatic carboxylic
acids in POCl3 in the presence of 2.0 equiv of Li2CO3 allowed access to
4-chloro-[1,2,4]triazolo[4,3-a]quinoxalines (3a–3d) in 11–62% yields
via carboxyl hydrazidation/intramolecular cyclization cascade. To in-
troduce a nitrogen atom into the carbon chain linkage, 2 was reacted
with 1.0 equiv of benzyl isothiocyanate (BnNCS) in pyridine at reflux
referencing a previous report [23], unexpectedly giving N-benzyl-1H-
[1,3,4]thiadiazino[5,6-b]quinoxaline-3-amine in 18% yield. There were
two causes that can be attributed to the failure; the first was basicity of
pyridine and the second was nucleophilicity of a sulfur atom. Therefore,
we planned to prepare the desired 4-chloro-[1,2,4]triazolo[4,3-a]qui-
noxalines (3e–3 g) via Hg(OAc)2-mediated desulfurizative cyclization
in base-free-mild conditions [24]. Treatment of 2 with 1.5 equiv of
R2NCS in DCM proceeded at room temperature to form thiosemi-
carbazide intermediates, which were immediately cyclized using 1.5
equiv of Hg(OAc)2 in dioxane at 50 °C to 3e–3g in 52–55% yields.
Methylamination of 3 by 40% NH2Me/MeOH solution smoothly furn-
ished the corresponding derivatives 4a–4g in 58%–quantitative yields.
In an attempt to explore the effects of chemical structures upon
antiproliferative potential, we assessed the synthetic derivatives 4 for in
vitro effects against human acute myelocytic leukemia HL-60 cells using
the CCK-8 assay method [21,25]. Imiquimod (purchased from FUJIF-
ILM Wako Pure Chemical Corporation) and EAPB0203 (prepared from
1 over four steps [8]) were used as reference drugs. The cells were
treated with increasing concentrations of each compound for 48 h, with
DMSO as control. As shown in Fig. 2, the derivatives 4a–4d, 4f, and 4g
showed relatively weak inhibition; particularly, over 90% of the cul-
tured cells survived even when 4b, 4c, and 4d were treated at both
concentrations of 20 μM and 40 μM. It was apparent that influence of
the linkage length and flexibility is minimal. A similar tendency was
Fig. 1. Structures of imiquimod and EAPB0203.
improves the efficacy of existing drugs and overcomes their dis-
advantages such as low solubility, adverse effects, and multidrug re-
sistance. Herein, we report our initial results of the hybridization of
EAPB0203 with privileged heterocyclic fragments and structure-ac-
tivity relationship analysis of the hybrid derivatives as potent antic-
ancer agents.
2. Results and discussion
Imiquimod and EAPB0203 contain the imidazole ring system in the
fundamental core and the later has an ethyl linkage (C2H4) between the
imidazole and benzene rings. Based on their chemical structures, we
commenced to replace the imidazole ring with triazole ring.
Furthermore, adjustments of its linkage (carbon chain length, flex-
ibility, and nitrogen atom insertion) were performed (Scheme 1). 2,3-
Dichloroquinoxaline (1) is a valuable synthetic precursor for carbon-
heteroatom bond formation at the C2 and/or C3 positions on qui-
noxaline [22]. Treatment of 1 with 2.2 equiv of hydrazine monohydrate
Scheme 1. Synthesis of triazole-fused derivatives 4.
2

D. Kaneko, et al.
BioorganicChemistry104(2020)104293
R3 = OH), and 7f (X = O, R3 = NH2) significantly inhibited pro-
liferation of the cells. It is noteworthy that incorporation of 1,3,4-ox-
adiazole and 1,3,4-thiadiazole units improved the efficacy of 4e.
Additionally, there was a trend that the antiproliferative effects of
1,3,4-oxadiazole-containing derivatives are higher than those of 1,3,4-
thiadiazole counterparts.
20
M
40
M
100
80
60
40
20
0
In addition to them, 5 and 6 having Me, OH, and NH2 groups were
integrated into the fundamental core of imiquimod [28], as shown in
Scheme 3. The first step was nitration of 2,4-dihydroxyquinoline which
was effected in HNO3 at 70 °C, giving a mononitrated 2,4-dihydrox-
yquinoline 8 in 93% yield. Without purification by silica gel column
chromatography, chlorination of 8 by POCl3 furnished a dichloro de-
rivative, which was substituted by isobutylamine in the presence of
K2CO3 in EtOH, to provide 9 in 46% yield over two steps. Bechamp
reduction in the Fe powder/NH4Cl system succeeded in 95% yield for
10. Upon treatment with triethyl orthoformate CH(OEt)3 at 60 °C, 4-
chloro-1-isobutyl-1H-imidazo[4,5-c]quinoline (11) was obtained in
76% yield. 1,3,4-Oxadiazole and 1,3,4-thiadiazole derivatives (5 and 6)
were incorporated into 10 under similar reaction conditions described
above, affording 12a–12f in 52–95% yields.
EAPB
0203
4a
4b
4c
4d
4e
4f
4g
IMQ
Fig. 2. Antiproliferative effects of triazole-fused derivatives (4). HL-60 cells
were treated with the seven derivatives (4), EAPB0203, and imiquimod (IMQ)
for 48 h. CCK-8 assay was performed to determine their antiproliferative effects
and the results were quantified relative to untreated controls (mean
SEM,
n = 3).
observed in the investigation of EAPB0203 and its related compounds
[26]. Interestingly, the antiproliferative effect of triazolo derivative 4b
was considerably poor relative to that of intact EAPB0203. On the other
hand, the aniline-substituted triazolo derivative 4e markedly inhibited
cell proliferation compared to imiquimod. Its inhibition was the same
degree with EAPB0203. From these results, 4e can be emerged as a
promising lead compound for additional modifications.
When treated at a concentration of 10 μM of imiquimod-1,3,4-ox-
adiazole and imiquimod-1,3,4-thiadiazole derivatives (12), the cell
viability ranged from approximately 80% to 90% (12a: 87.3
2.2%;
1.9%; 12e:
2.4%). Unfortunately, their effects were
12b: 82.5
1.6%; 12c: 90.6
1.6%; 12d: 84.5
80.5 1.7%; 12f: 90.2
the same level as imiquimod (cell viability: 90.8
1.8%). As con-
With 3e in hand, 1,3,4-oxadiazole and 1,3,4-thiadiazole units were
incorporated (Scheme 2). 5-Phenyl-1,3,4-oxadiazole-2-thiols (5) were
prepared by the reaction of benzhydrazides with CS2 in heated DMF
[21]. 5-Phenyl-1,3,4-thiadiazole-2-thiols (6) were prepared as follows:
treatment of benzhydrazides with CS2 in the presence of KOH in EtOH
gave the respective potassium 3-aroyl dithiocarbazates, which upon
acidic cyclization using concentrated H2SO4 at 0 °C afforded 6 [27].
Compound 3e was substituted by 5 and 6 (1.5 equiv) using Et3N (3.0
equiv) in DMF at 60 °C, successfully affording the desired [1,2,4]tria-
zolo[4,3-a]quinoxaline-1,3,4-oxadiazole and [1,2,4]triazolo[4,3-a]qui-
noxaline-1,3,4-thiadiazole derivatives 7a–7l in 6–83% yields.
trasted to the above results, incorporation of 1,3,4-oxadiazole and
1,3,4-thiadiazole units contributed little to the antiproliferative effect of
imiquimod.
To validate the efficacy of 7b, 7e, and 7f, other cancer cell lines
including U937 (histiocytic lymphoma), B16 (melanoma), HepG2 (he-
patoma) were used in addition to HL-60. The derivatives 7b, 7e, and 7f
displayed the dose-dependent inhibition and their IC50 values are
summarized in Table 1. They possessed intense antiproliferative effects
against leukemia cell lines (HL-60 and U937) at the same level as
EAPB0203. Most importantly, 7b, 7e, and 7f demonstrated the re-
markable IC50 values (7b: 5.35
3.84
0.22 μM; 7e: 4.86
0.25 μM; 7f:
To gain insights into the structure–activity relationships, the syn-
thetic derivatives (7) were assayed their in vitro antiproliferative effects
on HL-60 cells. At a concentration of 10 μM, most derivatives exhibited
0.13 μM) against HepG2 cells compared to EAPB0203 and
imiquimod. Moderate effects were achieved for 7b, 7e, and 7f against
B16 cells. Moreover, their cytotoxic effects on human non-malignant
keratinocyte HaCaT cells were evaluated. When 7b, 7e, and 7f were
treated at a concentration of 5 μM for 48 h, over 80% of the cultured
high potency with cell viability of less than 50% (7a: 24.5
0.6%; 7b:
0.2%; 7f:
1.2%;
7.8
0.8%; 7c: 20.7
0.9%; 7g: 19.9
3.8%; 7k: 32.0
0.3%; 7d: 14.5
0.7%; 7h: 53.9
1.0%; 7l: 36.4
1.5%; 7e: 8.9
10.1
1.3%; 7i: 37.4
cells survived (7b: 102.7
87.7
proliferation with cell viability of 69.1
4.0%; 7e: 80.6
3.3%; 7f:
7j: 42.4
(64.8
0.9%) compared to 4e
1.9%). EAPB0203 showed weak inhibition of HaCaT cell
3.0%.
1.1%). In particular, 7b (X = O, R3 = Me), 7e (X = O,
The derivatives 7b, 7e, and 7f accomplished efficacious results
against HepG2 cells. C. Deleuze-Masquéfa and P.-A. Bonnet group
confirmed the main metabolized structures (hydroxylations and N-de-
methylation) of EAPB0203 in rat and dog liver microsomes by LC-MS/
MS and NMR analyses [29,30]. EAPB0203 has three hydroxylable po-
sitions on benzene and imidazole rings, whereas the derivatives 7b, 7e,
and 7f have the two positions as indicated in Fig. 3. Similar to
EAPB0203, the benzene rings of 7b, 7e, and 7f might be hydroxylated
(two positions). By contrast, the triazole ring of them should not accept
further hydroxylation. There might be a small possibility that the 1,3,4-
oxadiazole units of 7b, 7e, and 7f are released (S-dealkylation [31,32]).
Even if they were eliminated, the both degraded fragments would ex-
hibit anticancer properties [33–35]. As described, we presume that the
1,3,4-oxadiazole derivatives 7b, 7e, and 7f show distinct metabolic
profiles in vivo.
3. Conclusions
A series of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole and
[1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-thiadiazole derivatives (7)
were synthesized and evaluated for their antiproliferative effects on
Scheme 2. Synthesis of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole and
[1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-thiadiazole derivatives 7.
3

D. Kaneko, et al.
BioorganicChemistry104(2020)104293
Scheme 3. Incorporation of 1,3,4-oxadiazole and 1,3,4-thiadiazole units into imiquimod.
spectrometers with tetramethylsilane as an internal standard. Silica gel
column chromatography (CC) was performed on silica gel 60 N
(Wakogel, 38–100 μm). Thin-layer chromatography (TLC) spots on
plates pre-coated with silica gel 70F254 were detected with a UV lamp
(254 nm). Fractionations for all CCs were based on TLC analyses.
Table 1
IC50 values of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole derivatives
(7b, 7e, and 7f) against cancer cell lines.
IC50 (μM
SEM)
HL-60
U937
B16
HepG2
4.1.2. Synthetic methods
7b
3.23
3.37
2.72
2.07
46.7
0.17
0.10
0.27
0.06
1.3
1.56
3.73
2.65
0.82
40.2
0.25
0.32
0.13
0.15
1.3
11.3
0.2
0.1
0.1
5.35
4.86
3.84
60.9
> 80
0.22
0.25
0.13
1.2
Detailed synthetic conditions and spectroscopic data of compounds
are given in the Supplementary data. The physical data of 7a–7f are
shown here.
7e
10.6
7f
26.2
EAPB0203
Imiquimod
> 80
> 80
N-Phenyl-4-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)-[1,2,4]triazolo[4,3-
a]quinoxalin-1-amine (7a): IR (ATR): νmax 2230, 1693, 1578, 1543,
1466, 1397, 1351, 1240, 1142, 946, 856, 839, 775, 636, 594, 496,
1
468 cm−1; H NMR (400 MHz, DMSO‑d6): δ 9.41 (1H, s, –NH-), 8.33
(1H, d, J = 8.2 Hz, H-6), 8.10–8.08 (2H, m, H-2″ and H-6″), 7.70–7.53
(6H, m, H-2′, H-4″, H-6′, H-7, H-8, and H-9), 7.25 (2H, t, J = 8.0 Hz, H-
3′ and H-5′), 7.10–7.08 (2H, m, H-3″ and H-5″), 6.93 (1H, t, J = 7.3 Hz,
H-4′); 13C NMR (100 MHz, DMSO‑d6): δ 168.4, 156.7, 149.4, 148.6,
143.2, 136.0, 133.3, 130.2 (3C), 129.7 (2C), 129.5, 129.2, 128.1, 127.4
(2C), 125.9, 123.4, 121.7, 117.2 (2C), 116.9; HRESITOFMS: m/z
438.1126 [M + H]+ (calcd. for C23H16N7OS, 438.1137).
N-Phenyl-4-((5-(p-tolyl)-1,3,4-oxadiazol-2-yl)thio)-[1,2,4]triazolo
[4,3-a]quinoxalin-1-amine (7b): IR (ATR): νmax 1598, 1544, 1493, 1456,
1410, 1354, 1248, 1145, 1102, 1080, 944, 824, 743, 733, 690, 657,
1
500, 463 cm−1; H NMR (400 MHz, DMSO‑d6): δ 9.42 (1H, s, –NH-),
Fig. 3. Proposed hydroxylable positions of EAPB0203 and [1,2,4]triazolo[4,3-
a]quinoxaline-1,3,4-oxadiazole derivatives (7b, 7e, and 7f) in hepatic meta-
bolism. Red arrows point hydroxylable positions.
8.31 (1H, d, J = 8.2 Hz, H-6), 7.97 (2H, d, J = 8.2 Hz, H-2″ and H-6″),
7.67 (1H, dd, J = 7.8 Hz and 1.4 Hz, H-9), 7.61–7.58 (1H, m, H-7 or H-
8), 7.53 (1H, t, J = 7.1 Hz, H-7 or H-8), 7.45 (2H, d, J = 8.2 Hz, H-2′
and H-6′), 7.25 (2H, t, J = 7.8 Hz, H-3′ and H-5′), 7.10 (2H, d,
J = 7.3 Hz, H-3″ and H-5″), 6.93 (1H, t, J = 7.6 Hz, H-4′), 2.42 (3H, s,
–CH3); 13C NMR (100 MHz, DMSO‑d6): δ 168.5, 156.3, 149.3, 148.7,
143.6, 143.2, 141.2, 136.0, 130.7 (2C), 129.7 (2C), 129.5, 129.1,
128.0, 127.3 (2C), 125.9, 121.7, 120.6, 117.2 (2C), 116.9, 21.7; HRE-
SITOFMS: m/z 452.1291 [M + H]+ (calcd. for C24H18N7OS, 452.1291).
4-((5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-N-phenyl-[1,2,4]
triazolo[4,3-a]quinoxalin-1-amine (7c): IR (ATR): νmax 2997, 1602,
1577, 1556, 1502, 1414, 1305, 1253, 1176, 1147, 1032, 954, 829, 749,
692, 666, 627, 503, 479 cm−1; 1H NMR (500 MHz, DMSO‑d6): δ 9.41
(1H, s, –NH-), 8.32–8.30 (1H, m, H-6), 8.03 (2H, d, J = 8.6 Hz, H-2″
and H-6″), 7.67 (1H, d, J = 8.0 Hz, H-9), 7.60 (1H, t, J = 7.2 Hz, H-7 or
H-8), 7.53 (1H, t, J = 7.2 Hz, H-7 or H-8), 7.26 (2H, t, J = 8.0 Hz, H-3′
and H-5′), 7.18 (2H, d, J = 9.2 Hz, H-2′ and H-6′), 7.10 (2H, d,
J = 8.0 Hz, H-3″ and H-5″), 6.93 (1H, t, J = 7.4 Hz, H-4′), 3.87 (3H, s,
-OMe); 13C NMR (125 MHz, DMSO‑d6): δ 168.3, 163.2, 155.8, 149.3,
148.7, 146.8, 143.2, 141.2, 136.1, 129.7 (2C), 129.5, 129.3 (2C),
cancer cell lines. The 1,3,4-oxadiazole derivatives 7b, 7e, and 7f
bearing Me, OH, and NH2 groups on the phenyl ring respectively, were
found to be the most beneficial compounds when compared to imi-
quimod and EAPB0203. The new structural combination of [1,2,4]
triazolo[4,3-a]quinoxaline and 1,3,4-oxadiazole units is valuable for
accelerating anticancer drug development.
4. Experimental section
4.1. Chemistry
4.1.1. General
All solvents and reagents were purchased from the suppliers and
used without further purification. IR spectra were recorded on a
PerkinElmer FT-IR/FIR Spectrometer 400. MS spectra were obtained
using the Waters UPLC-MS system (Aquity UPLC XevoQTof). 1H and 13
C
NMR spectra were recorded with JEOL ECX-400P and ECA-500
4

D. Kaneko, et al.
BioorganicChemistry104(2020)104293
129.2, 128.1, 125.9, 121.7, 117.2 (2C), 116.9, 115.7 (2C), 56.2; HRE-
incubation, CCK-8 solution (10 μL) was added, and the plates were
incubated for an additional 3 h [21,25]. Visible absorption (490 nm)
was measured using a microplate reader (Emax precision microplate
reader, Molecular Devices Japan, Tokyo, Japan).
SITOFMS: m/z 468.1250 [M
+
H]+ (calcd. for C24H18N7O2S,
468.1243).
4-((5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)-N-phenyl-[1,2,4]
triazolo[4,3-a]quinoxalin-1-amine (7d): IR (ATR): νmax 2194, 1599,
1538, 1499, 1479, 1455, 1408, 1243, 1144, 1092, 1079, 1013, 946,
837, 748, 735, 691, 981, 656, 619, 555, 527 cm−1; 1H NMR (500 MHz,
DMSO‑d6): δ 9.42 (1H, s, –NH-), 8.32 (1H, d, J = 6.4 Hz, H-6), 8.10
(2H, d, J = 8.7 Hz, H-2′ and H-6′), 7.73 (2H, d, J = 8.7 Hz, H-2″ and H-
6″), 7.70–7.68 (1H, m, H-9), 7.61 (1H, t, J = 7.1 Hz, H-7 or H-8), 7.53
(1H, t, J = 7.1 Hz, H-7 or H-8), 7.26 (2H, t, J = 7.8 Hz, H-3′ and H-5′),
7.10 (2H, d, J = 7.8 Hz, H-3″ and H-5″), 6.93 (1H, t, J = 7.3 Hz, H-4′);
13C NMR (125 MHz, DMSO‑d6): δ 167.6, 157.0, 149.3, 148.5, 143.2,
141.2, 138.1, 136.1, 130.4 (2C), 129.7 (2C), 129.5, 129.2 (3C), 128.1,
125.9, 122.3, 121.7, 117.2 (2C), 116.9; HRESITOFMS: m/z 472.0748
[M + H]+ (calcd. for C23H15N7OSCl, 472.0747).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The authors express our sincere thanks to Assistant Prof. Taro
Udagawa (Gifu University) for his helpful assistance.
4-(5-((1-(Phenylamino)-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)thio)-
1,3,4-oxadiazol-2-yl)phenol (7e): IR (ATR): νmax 3335, 2851, 1663,
1600, 1542, 1483, 1455, 1406, 1220, 1165, 772, 744, 690, 500,
454 cm−1; 1H NMR (500 MHz, DMSO‑d6): δ 10.47 (1H, s, –OH), 9.41
(1H, s, –NH-), 8.31 (1H, d, J = 8.0 Hz, H-6), 7.92 (2H, d, J = 8.6 Hz, H-
2″ and H-6″), 7.67 (1H, d, J = 8.0 Hz, H-9), 7.59 (1H, t, J = 7.2 Hz, H-7
or H-8), 7.53 (1H, t, J = 7.5 Hz, H-7 or H-8), 7.25 (2H, t, J = 8.0 Hz, H-
3′ and H-5′), 7.10 (2H, d, J = 8.0 Hz, H-2′ and H-6′), 6.99 (2H, d,
J = 8.6 Hz, H-3″ and H-5″), 6.93 (1H, t, J = 7.4 Hz, H-4′); 13C NMR
(125 MHz, DMSO‑d6): δ 168.6, 163.9, 162.0, 155.4, 149.3, 148.8,
143.2, 141.2, 136.1, 129.7 (2C), 129.5 (2C), 129.2, 128.0, 125.9,
121.7, 117.2 (2C), 117.0 (2C), 116.9, 114.1; HRESITOFMS: m/z
454.1074 [M + H]+ (calcd. for C23H16N7O2S, 454.1086).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104293.
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