Design, synthesis, and molecular docking studies of new [1,2,4]triazolo[4,3-a]quinoxaline derivatives as potential A2B receptor antagonists

Article abstract of DOI:10.1007/s11030-020-10070-w

Abstract: Many shreds of evidence have recently correlated A2B receptor antagonism with anticancer activity. Hence, the search for an efficient A2B antagonist may help in the development of a new chemotherapeutic agent. In this article, 23 new derivatives of [1,2,4]triazolo[4,3-a]quinoxaline were designed and synthesized and its structures were confirmed by different spectral data and elemental analyses. The results of cytotoxic evaluation of these compounds showed six promising active derivatives with IC₅₀ values ranging from 1.9 to 6.4?μM on MDA-MB 231 cell line. Additionally, molecular docking for all synthesized compounds was performed to predict their binding affinity toward the homology model of A2B receptor as a proposed mode of their cytotoxic activity. Results of molecular docking were strongly correlated with those of the cytotoxic study. Finally, structure activity relationship analyses of the new compounds were explored. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-020-10070-w

Molecular Diversity
https://doi.org/10.1007/s11030-020-10070-w
ORIGINAL ARTICLE
Design, synthesis, and molecular docking studies of new [1,2,4]
triazolo[4,3‑a]quinoxaline derivatives as potential A2B receptor
antagonists
Hany G. Ezzat · Ashraf H. Bayoumi · Farag F. Sherbiny · Ahmed M. El‑Morsy1,3 · Adel Ghiaty · Mohamed Alswah ·
1
1
1,2
1
1
Hamada S. Abulkhair1
,4
Received: 6 July 2019 / Accepted: 4 March 2020
©
Springer Nature Switzerland AG 2020
Abstract
Many shreds of evidence have recently correlated A2B receptor antagonism with anticancer activity. Hence, the search for
an eꢀcient A2B antagonist may help in the development of a new chemotherapeutic agent. In this article, 23 new derivatives
of [1,2,4]triazolo[4,3-a]quinoxaline were designed and synthesized and its structures were conꢁrmed by diꢂerent spectral
data and elemental analyses. The results of cytotoxic evaluation of these compounds showed six promising active derivatives
with IC values ranging from 1.9 to 6.4 μM on MDA-MB 231 cell line. Additionally, molecular docking for all synthesized
5
0
compounds was performed to predict their binding aꢀnity toward the homology model of A2B receptor as a proposed mode
of their cytotoxic activity. Results of molecular docking were strongly correlated with those of the cytotoxic study. Finally,
structure activity relationship analyses of the new compounds were explored.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-020-10070-w) contains
supplementary material, which is available to authorized users.
*
Hamada S. Abulkhair
hamadaorganic@azhar.edu.eg
1
Pharmaceutical Organic Chemistry Department, Faculty
of Pharmacy, Al-Azhar University, 11884 Nasr City, Cairo,
Egypt
2
Pharmaceutical Organic Chemistry Department, College
of Pharmacy, Misr University for Science and Technology
(MUST), 6th October City, Egypt
3
4
Pharmaceutical Chemistry Department, College
of Pharmacy, The Islamic University, 54001 Najaf, Iraq
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Horus University - Egypt, International Costal Road,
New Damietta, Egypt
Vol.:(0
1
123456789)
3

Molecular Diversity
Graphic abstract
Keywords Design · Docking · Quinoxaline · A2B antagonists
Introduction
A2B receptors was reported to play a signiꢁcant rule in the
reduction in metastasis and regulation of oral squamous cell
carcinoma [9]. Medicinal chemistry approaches have been
applied to all the adenosine receptor subtypes (A1, A2A,
A2B, and A3) to create selective antagonists for each [1].
Currently, there is a signiꢁcant increase in the interest in
A2B receptors in diꢂerent therapeutic areas [10, 11]. Nev-
ertheless, the compendium of potent ligands targeting A2B
Adenosine is the metabolite of intracellular and extracellular
ADP and ATP and considered as a mediator for a wide range
of events under both normal and pathological conditions [1].
There are four subtypes of adenosine receptors known as A1,
A2A, A2B, and A3. Each type of these adenosine receptors
has its unmatched pharmacological proꢁle [2, 3]. Anticancer
activity is one of the medicinal features of adenosine recep-
tors and their four subtypes [4]. Unlike A1, A2A, and A3
adenosine receptors, the A2B subtype requires a high level
of adenosine for activation. Thus, the A2B receptors may
mediate pathophysiological conditions associated with the
cumulative level of adenosine as in the case of tumors and
ischemia [1, 5–7]. A2B receptors are expressed in the human
microvascular endothelial cells, where they may regulate the
angiogenic factors such as basic ꢁbroblast growth factor and
vascular endothelial growth factor and subsequently angio-
genesis, which is one of the major mechanisms for tumor
growth regulation [8]. Also, blockade of human adenosine
Fig. 1 Reported hit quinoxaline derivatives as potent A2B receptor
antagonist
1
3

Molecular Diversity
receptors is still rare [12]. An evidence for the expression
of A2B adenosine receptors in the human breast adenocar-
cinoma, 92020424 (MDA-MB-231) cells, has been proved
Moreover, a molecular docking study was performed to
elucidate the virtual binding mode of the synthesized com-
pounds using the homology model of the human adenosine
A2B receptor. Finally, the structure–activity relationship
of the synthesized compounds was illustrated based on the
results of cytotoxic evaluation against MDA-MB-231 cell
lines as a model for A2B adenosine receptor subtype [7].
[
7]. Consequently, virtual screening of potential ligands
using the adenosine A2B receptor homology model has been
accomplished. Some of these hits are related to quinoxaline
nucleus. (4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylamino)
phenyl)(4-(4-ꢃuorophenyl)piperazin-1-yl)methanone, com-
pound 1 (Fig. 1), has been used as a template for ongoing
research [13].
Furthermore, [1,2,4]triazolo[4,3-a]quinoxaline is an
important pharmacophore ring system, presented in a num-
ber of anticancer agents [14, 15]. Over the last few years,
our research group was interested in the design and synthe-
sis of novel anticancer heterocycles [16–20]. As an exten-
sion to our previous researches and depending on the upper
mentioned facts, we are reporting here the design, synthesis,
structural conꢁrmation, and docking studies for the novel
Results and discussion
Rational design
The core structure of hit compound 1 consists of
1,2,4-triazolo[4,3-a]quinoxaline ring system attached with
hydrophilic NH linker at C-4 followed by hydrophobic tail
[13]. The predicted binding mode of hit compound shows
that the compound targets nine non-conserved amino acid
residues, namely Asn273, Leu81, Lys170, Val256, Ala271,
Asn266, Lys269, Lys267, and Val250. The tricyclic moiety
of the compound is anchored by aromatic stacking interac-
tions with Trp247, Phe173, and His251 and hydrophobic
interaction with Val85, Leu86, Met182, Val253, and Ile276
(Fig. 3). These interactions are essential for compound aꢀn-
ity to the receptor.
[
1,2,4]triazolo[4,3-a]quinoxaline derivatives as potent inhib-
itors for A2B receptors. The protocol was designed through
construction of the [1,2,4]triazolo[4,3-a]quinoxaline ring
system of the hit compound with chloro-substituent at posi-
tion 4 for subsequent replacement with either aniline deriva-
tives or another hydrophilic spacer attached with hydropho-
bic heads. Diꢂerent linkers and terminals have been inserted
to investigate the eꢂect on the cytotoxic activity (Fig. 2).
Fig. 2 Lead compound and newly designed quinoxaline derivatives
1
3

Molecular Diversity
Fig. 3 Predicted binding mode
for hit compound 1 with the
homology model of the human
adenosine A2B receptor [13]
The main objective of the present work was the design
and synthesis of new 1,2,4-triazolo[4,3-a]quinoxaline
derivatives with the same essential structural features of
the reported hit compound and almost the same binding
mode. There are only two bioisosteric modiꢁcations in
our designed new derivatives. First is the replacement of
the hydrophilic NH linker at C-4 with another hydrophilic
one in which both the length and number of hydrogen bond
acceptors are increased. The second is the replacement of
terminal hydrophobic tail with diꢂerent hydrophobic moi-
eties including substituted phenyl, heteroaryl, and styryl
moieties. This variety of modiꢁcations enabled us to study
the SAR of these new derivatives as eꢂective anticancer
agents with potential A2B antagonistic activity which is
one of our major objectives of this work.
Materials and methods
Chemistry
Starting 4-chloro-1-methyl-[1,2,4]triazolo[4,3-a]quinoxa-
line (7) was synthesized as outlined in Scheme 1. Oxalic
acid in 4N aqueous HCl (50 mL) was allowed to react with
o-phenylenediamine [21], and the resulting quinoxaline-
2,3(1H,4H)-dione 4 was treated with thionyl chloride in
dichloroethane [22] to aꢂord 2,3-dichloroquinoxaline (5).
Scheme 1 Synthetic pro-
tocol of starting 4-chloro-
1
-methyl-[1,2,4]triazolo[4,3-a]
quinoxaline
1
3

Molecular Diversity
2
-Chloro-3-hydrazinylquinoxaline (6) was obtained through
4,5-dihydroisoxazole derivatives 12a–c. The IR spectrum of
10a as a representative example of 10a–e is characterized by
the stirring of 5 with hydrazine hydrate at room temperature
−1
[
23]. Subsequent heating compound 6 with acetic anhydride
the presence of absorption band at 3349 cm corresponding
to NH and disappearance of the strong absorption band at
produced our title compound 4-chloro-1-methyl-[1,2,4]
triazolo[4,3-a]quinoxaline [22].
−
1
1694 cm corresponding to carbonyl group of the starting
1
The obtained 4-chloro-1-methyl-[1,2,4]triazolo[4,3-a]
quinoxaline (7) was reꢃuxed with p-aminoacetophenone in
dry acetonitrile to get the acetyl derivative 8 (Scheme 2).
The IR spectrum of compound 8 showed two absorption
chalcone. The H NMR spectrum exhibited singlet peak at
9.86 ppm corresponding to NH next to phenyl ring which is
D O exchangeable, singlet peak at 5.59 ppm corresponding
2
to NH proton of pyrazole ring which is also exchangeable
−
1
bands at 3417 and 1670 cm corresponding to NH and
C=O. Claisen–Schmidt condensation [24] of 8 with p-sub-
stituted benzaldehydes in an ethanolic solution of KOH pro-
duced the chalcone derivatives 9a–e. These two consecutive
steps successfully aꢂorded the ꢁnal products 8 and 9a–e in
satisfying yields, almost over 70% and reasonable purities.
The IR spectra of these chalcones are characterized by the
with D O, triplet peak at 4.84 ppm corresponding to CH
2
proton of pyrazole nucleus, and two doublets at 3.02 and
3.31 ppm corresponding to CH proton of pyrazole ring.
2
The IR spectra of compounds 11a–c are characterized by
−
1
the presence of absorption bands at 3449 cm correspond-
ing to NH and disappearance of the C=O stretching absorp-
1
tion bands of the starting chalcones. The H NMR spectra
−1
presence of C=O stretching bands at 1594–1649 cm which
appeared at low absorption values because of extended con-
jugation with the double bond. The absolute geometry of the
α, β-unsaturated carbonyl linker was assigned to be in trans
conꢁguration based on the coupling constants of alkene
of this series exhibited two singlet signals at the range of
10.50–8.50 ppm corresponding to NH proton next to qui-
noxaline ring and the amide NH proton of pyrimidine ring.
The IR spectra of isoxazole derivatives 12a–c are also char-
acterized by the presence of the NH absorption bands at the
expected wavenumbers and disappearance of C=O stretch-
1
protons. The H NMR spectrum of compound 9c as a repre-
1
sentative example exhibited two doublets, each equivalent to
one proton: One doublet at 6.80 ppm corresponds to α proton
and another doublet at 7.94 ppm corresponds to β proton.
Both have the same coupling constant value of J=15 Hz,
which conꢁrms the trans conꢁguration.
ing bands. The H NMR spectra of these isoxazoles revealed
singlet signals at about 9.40 corresponding to NH proton
next to quinoxaline ring and triplet signals at 8.50 ppm for
CH proton of isoxazole ring.
2
Alternatively, 4-chloro-1-methyl-[1,2,4]triazolo[4,3-
a]quinoxaline (7) was converted into 1-methyl-[1,2,4]
triazolo[4,3-a]quinoxaline-4-thiol by the action of thio-
urea and subsequently to its potassium salt according to the
1,3-Cycloaddition of hydrazine, urea, and hydroxylamine
to chalcones 9a–c produced the corresponding 4,5-dihy-
dro-1H-pyrazole 10a–c, pyrimidin-2(1H)-one 11a–c, and
Scheme 2 Synthetic protocol of target compounds 8–12a–c
1
3

Molecular Diversity
directions of reported procedures [22]. Reaction of the lat-
Experimental
Chemistry
General
ter with diꢂerent derivatives of α-chloro-N-phenylacetamide
[
23] gave our target compounds 15a–g. In general, all reac-
tions proceeded smoothly and ꢁnal products 10–15 were
obtained in relatively good yields as detailed in the “Experi-
mental” part. The IR spectra of these N-phenylacetamide
derivatives are characterized by the presence of amidic
Melting points were taken on an electrothermal (Stuart
SMP30) apparatus and were uncorrected. IR spectra were
recorded on a Pye Unicam SP 1000 IR spectrophotometer
at Pharmaceutical Analytical Unit, Faculty of Pharmacy,
−
1
C=O absorption bands at 1661–1698 cm in addition to
−
1
1
the NH stretching at 3267–3479 cm region. The H NMR
spectra of these compounds showed the diagnostic NHC=O
1
13
D O exchangeable singlet signals at about 10.25 ppm as
Al-Azhar University. H NMR and C NMR spectra were
2
well as another singlet of the deshielded proton of SCH at
recorded in: (1) DMSO-d at 300/100 MHz on a Varian Mer-
2
6
4
.45–4.50 ppm (Scheme 3).
cury VXR-300 NMR spectrometer at NMR Lab, Faculty
of Science, Cairo University, (2) DMSO-d and CDCl at
6
3
4
00 MHz on a Joel ECA spectrometer at NMR Lab, Micro-
Biological evaluation
analytical Unit, Faculty of Pharmacy, Cairo University, (3)
CDCl at 400 MHz on a Joel ECA spectrometer at NMR
3
Production of adenosine is a mechanism by which tumors
suppress immune surveillance. Activation of host adenosine
A2B receptors has been found to inhibit the rejection of solid
tumors and promote metastasis. Adenosine A2B receptor
activation is expected to support tumor growth by stimulat-
ing the release of basic ꢁbroblast growth factor and vascular
endothelial growth angiogenic factors from vascular smooth
muscle, endothelial cells, and host immune cells [8]. From
this point and considering the role of adenosine as a cru-
cial factor in determining the cell progression pathway, the
cytotoxic activity has been selected for the pharmacological
evaluation of the newly synthesized A2B adenosine recep-
tor antagonists. The selective expression of high levels of
endogenous A2B receptors coupled to two signaling path-
ways makes MDA-MB-231 cells a suitable model for A2B
adenosine receptor subtype [7]. The A2B receptor in such
cancer cells may serve as a target to control cell growth and
proliferation [7].
Lab, Microanalytical Unit, Faculty of Pharmacy, Cairo Uni-
versity, and (4) DMSO-d at 400 MHz at the Main Chemi-
6
cal Warfare Laboratories, Chemical Warfare Department,
Ministry of Defense. Chemical shifts were related to that
of the solvent, and TMS was used as an internal standard.
Mass spectra and microanalyses were carried out at Regional
Center for Mycology Biotechnology, Al-Azhar University,
Cairo, Egypt. Progresses of the reactions were monitored
by TLC using TLC sheets precoated with UV ꢃuorescent
silica gel Merck 60 F254 plates and were visualized using
UV lamp and diꢂerent solvents as mobile phases. Starting
reagents, oxalic acid, o-phenylenediamine, phosphorus oxy-
chloride, hydrazine hydrate, acetic anhydride, p-aminoaceto-
phenone, p-substituted benzaldehydes, α-chloroacetyl chlo-
ride, and p-substituted aniline derivatives were purchased
from Aldrich Chemical Company and were used as received.
Compounds 5 and 6 were synthesized according to the direc-
tion of reported procedures [22].
Scheme 3 Synthetic protocol of target compounds 15a–g
1
3

Molecular Diversity
Synthesis of 1‑(4‑(1‑methyl‑[1,2,4]triazolo[4,3‑a]
quinoxalin‑4‑ylamino)phenyl)ethanone (8)
Calcd; C, 74.06; H, 4.72; N, 17.27, found: C, 74.06.98; H,
4.56; N, 17.03.
A mixture of 4-chloro-1-methyl-[1,2,4]triazolo[4,3-a]
quinoxaline (0.5 g, 0.1 mol) and p-aminoacetophenone
(2E)‑1‑(4‑(1‑methyl‑[1,2,4]triazolo[4,3‑a]quinoxa‑
lin‑4‑ylamino)phenyl)‑3‑(4‑chlorophenyl)prop‑2‑en‑1‑one
−
1
(
0.11 mol) in dry acetonitrile was heated under reꢃux for
(9b) Orange solid; yield: 78%; m.p. 251–253 °C. IR cm
3
h, set aside to room temperature, and then ꢁltered to give
as faint brown needles and pure enough to be used with-
(KBr): 3454 (NH), 3250 (C–H alkenes), 3094 (C–H aro-
8
matic), 2984 (C–H aliphatic), 1600 (C=O), 1525 (C=C).
1
out further puriꢁcation. Yield: 91%; m.p. 238–240 °C. IR
H NMR ppm (CDCl , 400 MHz): 9.91 (s, 1H, NH), 8.44
3
−
1
cm (KBr): 3417 (NH), 3132 (C–H aromatic), 2976 (C–H
(d, 1H, QX-H6), 8.07 (d, 2H, Ph-H2, H6), 8.04 (d, 1H,
COCH=CH, J=9.0), 7.91 (t, 1H, QX-H8), 7.85 (t, 1H,
QX-H7), 7.72 (d, 1H, QX-H9), 7.62 (d, 1H, COCH=CH–,
J=9.0), 7.50 (d, 2H, Ph-H3, H5), 7.33 (d, 2H, Ph-H2, H6),
1
aliphatic), 1786 (C=O). H NMR ppm (CDCl , 400 MHz):
3
9
.61 (s, 1H, NH), 8.25 (d, 2H, Ph-H , H ), 8.01 (d, 1H,
2
6
QX-H6), 7.94 (d, 1H, QX-H9), 7.86 (t, 1H, QX-H8), 7.65 (t,
1
3
1
2
3
H, QX-H7), 7.18 (d, 2H, Ph-H , H ), 4.21 (s, 3H, COCH ),
6.61 (t, 2H, Ph-H3, H5), 1.61 (s, 3H, CH ). C NMR ppm
3
5
3
3
.36 (s, 3H, CH ). MS (m/z): 317 (C H N O, 100%, M+),
(CDCl , 100 MHz): 14.70, 112.09, 120.10, 123.93, 125.90,
3
18 15
5
3
02 (C H N O, 4.21%), 275 (C H N , 36.99%), 198
128.11, 127.15, 128.44, 129.35, 130.28, 131.54, 132.33,
132.98, 135.71, 136.25, 137.89, 150.52, 155.18, 157.89,
161.59, 187.70. MS (m/z):439 (C H ClN O, 2.97%, M+),
1
7
12
5
16 12
5
(
C H N , 1.59%), 183 (C H N , 2.72%), 131 (C H N ,
1
0
8
5
10
7
4
8
7
2
7
.14%), 76.1 (C H , 8.22%), 43.06 (C H O, 7.06%). Anal.
6 4 2 3
25 18
5
Calcd; C, 68.13; H, 4.76; N, 22.07, found: C, 68.07; H, 4.66;
N, 21.93.
441 (C H ClN O, 0.89%, M+2), 425 (C H ClN O,
25 18 5 24 15 5
86.53%), 401 (C H N O, 2.35%), 318 (C H N O,
2
5
18
5
15 14
5
3
.21%), 302 (C H N O, 1.16%), 273 (C H N , 0.79%),
17 12 5 16 12 5
General procedure for synthesis
of (2E)‑1‑(4‑(1‑methyl‑[1,2,4]triazolo[4,3‑a]
quinoxalin‑4‑ylamino)phenyl)‑3‑arylprop‑2‑en‑1‑one
257 (C H ClNO, 15.05%), 138 (C H Cl, 61.99%), 131
15 12 8 6
(C H N , 9.41%), 111 (C H Cl, 14.87%), 76.08 (C H ,
8 7 2 6 4 6 4
13.65%). Anal. Calcd. C, 68.26; H, 4.12; N, 15.92, found:
C, 68.29; H, 4.15; N, 15.95.
(9a–e)
A mixture of the appropriate aromatic aldehyde (0.012 mol)
(2E)‑1‑(4‑(1‑methyl‑[1,2,4]triazolo[4,3‑a]quinox‑
a l i n ‑ 4 ‑ y l a m i n o ) p h e n y l ) ‑ 3 ‑ ( 4 ‑ m e t h o x y p h e n y l )
prop‑2‑en‑1‑one (9c) Faint yellow solid; yield: 80%; m.p.
and acetyl derivative 8 (0.01 mol) dissolved in ethanol
(
70 mL) was slowly added to the aqueous solution of potas-
−
1
sium hydroxide (0.0128 mol) in water (10 mL). The reaction
mixture was stirred on a crushed ice bath for 0.5 h. The tem-
perature is gradually elevated into 20–25 °C with continuous
stirring for further 5 h. The mixture was ꢁltered, and the
resulting solid was washed with cold water. The product was
221–223 °C. IR cm (KBr): 3441 (NH), 3215 (C–H alk-
enes), 3094 (C–H aromatic), 2984 (C–H aliphatic), 1649
1
(C=O), 1556 (C=C). H NMR ppm (DMSO, 400 MHz): 9.86
(s, 1H, NH), 8.30 (d, 1H, QX-H6), 8.18 (d, 2H, Ph-H2, H6),
8.02 (d, 1H, COCH=CH, J=12.0), 7.95 (t, 1H, QX-H8),
7.83 (t, 1H, QX-H7), 7.69 (d, 1H, QX-H9), 7.60 (d, 1H,
COCH=CH, J=12.0), 7.48 (d, 2H, Ph-H3, H5), 7.41 (d, 2H,
ꢁ
nally crystallized from ethanol to give the corresponding
chalcones 9a–e.
Ph-H2, H6), 6.70 (t, 2H, Ph-H3, H5), 3.80 (s, 3H, OCH ),
3
1
3
(
2E)‑1‑(4‑(1‑methyl‑[1,2,4]triazolo[4,3‑a]quinoxa‑
2.16 (s, 3H, CH ). C NMR ppm (DMSO, 100 MHz):
3
lin‑4‑ylamino)phenyl)‑3‑phenylprop ‑2‑ en‑1‑ one
14.70, 51.71. 110.62, 120.86, 125.18, 127.29, 128.39,
129.61, 130.23, 131.48, 134.31, 135.33, 136.56, 137.61,
142.24, 145.60, 146.46, 162.48, 189.46. MS (m/z):435.19
(C H N O , 0.87%, M+), 420 (C H N O , 11.30%),
−
1
(
(
9a) White solid; yield: 80%; m.p. 230–232 °C. IR cm
KBr): 3446 (NH), 3267 (C–H alkenes), 3109 (C–H aro-
1
matic), 2970 (C–H aliphatic), 1647 (C=O), 1530 (C=C). H
2
6
21
5
2
25 18
5
2
NMR ppm (CDCl , 400 MHz): 9.61 (s, 1H, NH), 8.45 (d, 1H,
273 (C H N , 1.73%), 252 (C H NO , 1.75%), 237
3
16 12 5 16 14 2
QX-H6), 8.09 (d, 2H, Ph-H , H6), 8.02 (d, 1H, COCH=CH,
(C H O , 6.31%), 198 (C H N , 1.45%), 183 (C H N ,
16 13 2 10 8 5 10 7 4
2
J=9.1), 7.93 (t, 1H, QX-H8), 7.87 (t, 1H, QX-H7), 7.74 (d,
3.95%), 133 (C H O, 65.72%), 131 (C H7N, 10.42%), 107
9 9 8
1
H, QX-H9), 7.66 (d, 1H, COCH=CH, J=9.1), 7.52 (d, 2H,
(C H O, 3.99%), 90 (C H , 100%), 76.09 (C H , 27.01%).
7 7 7 7 6 4
Ph-H3, H5), 7.38 (d, 2H, Ph-H2, H6), 7.28 (t, 2H, Ph-H3,
Anal. Calcd; C, 71.71; H, 4.86; N, 16.08, found: C, 71.73;
H, 4.88; N, 16.10.
1
3
H5), 6.91 (t, 1H, Ph-H4), 1.57 (s, 3H, CH ). C NMR ppm
3
(
CDCl , 100 MHz): 14.70, 112.09, 120.38, 123.65, 128.01,
3
1
1
1
4
28.12, 128.51, 128.80, 129.95, 130.05, 131.18, 132.90,
32.87, 135.28, 136.63, 137.62, 151.05, 146.30, 155.81,
57.89, 189.20. MS (m/z): 317 (C H N O, 100%, M+),
(2E)‑1‑(4‑(1‑methyl‑[1,2,4]triazolo[4,3‑a]quinoxa‑
lin‑4‑ylamino)phenyl)‑3‑(p‑tolyl)prop‑2‑en‑1‑one
(9d) Faint yellow solid; yield: 80%; m.p. 225–228 °C. IR
2
5
19
5
−
1
05 (C H N O, 14.67%), 275 (C H N , 100%), Anal.
cm (KBr): 3433 (NH), 3287 (C–H alkenes), 3235 (C–H
2
5
19
5
16 12
5
1
3

Molecular Diversity
aromatic), 2984 (C–H aliphatic), 1594 (C=O), 1538 (C=C).
133.71, 138.42, 138.41, 144.34, 149.68, 152.89, 163.82.
Anal. Calcd; C, 71.58; H, 5.05; N, 23.37, found: C, 71.73;
H, 5.12; N, 23.54.
1
H NMR ppm (CDCl , 100 MHz): 9.92 (s, 1H, NH), 8.59
3
(
d, 1H, QX-H6), 8.08 (d, 2H, Ph-H2, H6), 8.02 (d, 1H,
COCH=CH–, J=10.4), 7.99 (t, 1H, QX-H8), 7.85 (t, 1H,
QX-H7), 7.72 (d, 1H, QX-H9), 7.60 (d, 1H, COCH=CH,
J=10.4), 7.49 (d, 2H, Ph-H3, H5), 7.39 (d, 2H, Ph-H2,
N‑(4‑(5‑(4‑chlorophenyl)‑4,5‑dihydro‑1H‑pyrazol‑3‑yl)
phenyl)‑1‑methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑amine
−
1
H6), 6.97 (t, 2H, Ph-H3, H5), 2.28 (s, 3H, CH ), 1.56 (s,
(10b) White solid; yield: 58%; m.p. 265–267 °C. IR cm
3
3
4
H, CH ). MS (m/z):419.22 (C H N O, 10.32%, M+),
(KBr): 3402 (NH), 3048 (C–H aromatic), 2972 (C–H ali-
3
26 21
5
1
04 (C H N O, 66.59%), 236 (C H NO, 3.83%), 221
phatic). H NMR ppm (CDCl , 400 MHz): 9.85 (s, 1H,
2
5
18
5
16 14
3
(
C H O, 10.32%), 145 (C H O, 44.40%), 131 (C H N ,
NH), 8.54 (d, 1H, QX-H6), 8.42 (d, 1H, QX-H9), 8.01 (d,
2H, Ph-H2, H6), 7.99 (t, 1H, QX-H8), 7.88 (t, 1H, QX-H7),
7.65 (d, 2H, Ph-H3, H5), 7.48 (d, 2H, Ph-H2, H6), 7.30
(d, 2H, Ph-H3, H5), 5.29 (s, 1H, NH of pyrazole), 4.79 (t,
1
6
13
10
9
8
7
2
7
6
5
.32%), 117 (C H , 59.25%), 115 (C H , 100%), 91 (C H ,
9 9 9 7 7 7
2.99%), 76.07 (C H , 13.20%). Anal. Calcd; C, 74.44; H,
6
4
.05; N, 16.70, found: C, 74.47; H, 4.08; N, 16.37.
1
H, CH), 3.77 (dd, 1H, CH , J=9.0), 3.80 (dd, 1H, CH ,
2 2
1
3
(
2E)‑1‑(4‑(1‑methyl‑[1,2,4]triazolo[4,3‑a]quinoxa‑
J=9.0), 1.65 (s, 3H, CH ). C NMR ppm (DMSO-d ,
3 6
lin‑4‑ylamino)phenyl)‑3‑(4‑nitrophenyl)prop‑2‑en‑1‑one
100 MHz): 14.20, 43.62, 55.07, 111.38, 117.84, 119.27,
120.52, 122.54, 123.63, 124.76, 128.69, 132.01, 133.71,
138.42, 138.41, 144.34, 149.68, 152.84, 163.83. MS (m/z):
453 (C H ClN , 1.39%, M+), 455 (C H ClN , 0.49%,
(
9e) Reddish white solid; yield: 69%; m.p. 233–235 °C. IR
−1
cm (KBr): 3374 (NH), 3242 (C–H alkenes), 3195 (C–H
aromatic), 2987 (C–H aliphatic), 1596 (C=O), 1576 (C=C).
2
5
20
7
25 20
7
1
H NMR ppm (DMSO-d , 100 MHz): 9.91 (s, 1H, NH),
M+2), 439 (C H ClN , 8.39%), 404 (C H N , 1.38%),
6
24 18 5 24 18 7
8
1
.35 (d, 1H, QX-H6), 8.10 (d, 2H, Ph-H2, H6), 8.01 (d,
274 (C H N , 6.47%), 270 (C H N , 3.92%), 255
16 12 5 15 13 3
H, COCH=CH, J=9.7), 7.98 (t, 1H, QX-H8), 7.91 (t, 1H,
(C H ClN , 2.53%), 198 (C H N , 2.48%), 184(C H N ,
15 12 2 10 8 5 9 6 5
QX-H7), 7.74 (d, 1H, QX-H9), 7.58 (d, 1H, COCH=CH,
4.18%), 179 (C H ClN , 10.89%), 144 (C H N , 16.01%),
9 8 2 9 8 2
J=9.7), 7.45 (d, 2H, Ph-H3, H5), 7.32 (d, 2H, Ph-H2, H6),
131 (C H N , 40.34%), 111 (C H Cl, 40.06%), 90 (C H ,
8 7 2 6 4 7 6
7
.28 (t, 2H, Ph-H3, H5), 2.61 (s, 3H, CH ). Anal. Calcd; C,
100%), 76.07 (C H , 33.05%). Anal. Calcd; C, 65.86; H,
6 4
3
6
6.66; H, 4.03; N, 18.66, found: C, 66.82; H, 4.11; N, 18.81.
4.86; N, 21.50, found: C, 65.88; H, 4.88; N, 21.52.
General procedure for synthesis
of N‑(4‑(4,5‑dihydro‑5‑p‑tolyl‑1H‑pyrazol‑3‑yl)
aryl)‑1‑methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑amine
N‑(4‑(5‑(4‑methoxyphenyl)‑4,5‑dihydro‑1H‑pyrazol‑3‑yl)
phenyl)‑1‑methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑amine
−1
(10c) White solid; yield: 72%; m.p. 234–236 °C. IR cm
(10a–c)
(KBr): 3395 (NH), 3020 (C–H aromatic), 2984 (C–H ali-
1
phatic). H NMR ppm (CDCl , 400 MHz): 9.85 (s, 1H,
3
A mixture of the appropriate chalcones 9a–c (1.0 g,
NH), 8.65 (d, 1H, QX-H6), 8.52 (d, 1H, QX-H9), 8.31 (d,
2H, Ph-H2, H6), 7.99 (t, 1H, QX-H8), 7.84 (t, 1H, QX-H7),
7.62 (d, 2H, Ph-H3, H5), 7.42 (d, 2H, Ph-H2, H6), 6.62 (d,
2H, Ph-H3, H5), 5.31 (s, 1H, NH of pyrazole), 4.81 (t, 1H,
CH), 3.82 (s, 3H, OCH ), 3.71 (dd, 1H, CH , J=9.0), 3.77
0
.03 mol) and hydrazine hydrate (0.62 g, 0.12 mol) in etha-
nol (10 mL) was reꢃuxed for 4 h and then left to reach the
room temperature for 12 h. The resulting precipitate was
ꢁ
ltered out and washed with ethanol. The crude precipitate
3
2
1
3
was crystallized from ethanol to give the corresponding
(dd, 1H, CH , J=9.0), 1.67 (s, 3H, CH ). C NMR ppm
2
3
pyrazole derivatives 10a–c.
(DMSO-d , 100 MHz): 18.87, 56.55, 64.32, 66.15, 117.71,
6
1
18.28, 120.39, 122.95, 123.80, 125.00, 127.31, 129.37,
N‑(4‑(4,5‑dihydro‑5‑phenyl‑1H‑pyrazol‑3‑yl)phenyl)‑1‑me‑
thyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑amine (10a) White
130.45, 132.21, 134.24, 134.81, 138.89, 139.29, 144.57,
146.98, 150.30, 168.14. Anal. Calcd; C, 69.47; H, 5.16; N,
21.81, found: C, 69.49; H, 5.18; N, 21.83.
−
1
solid; yield: 65%; m.p. 270–272 °C. IR cm (KBr): 3349
1
(
NH), 3020 (C–H aromatic), 2864 (C–H aliphatic); H
NMR ppm (DMSO-d , 400 MHz): 9.86 (s, 1H, NH), 8.58
General procedure for synthesis of 6‑(4‑(1‑methyl‑[1,2,4]
triazolo[4,3‑a]quinoxalin‑4‑ylamino)
aryl)‑4‑p‑tolylpyrimidin‑2(1H)‑one (11a–c)
6
(
d, 1H, QX-H6), 8.35 (d, 1H, QX-H9), 8.05 (d, 2H, Ph-H2,
H6), 7.99 (t, 1H, QX-H8), 7.85 (t, 1H, QX-H7), 7.68 (d,
2
H, Ph-H3, H5), 7.57 (d, 2H, Ph-H2, H6), 7.42 (t, 1H,
Ph-H4), 7.24 (t, 2H, Ph-H3, H5), 5.59 (s, 1H, NH of pyra-
A mixture of the appropriate chalcones 9a–c (3.23 g,
0.01 mol) and urea (0.6 g, 0.01 mol) was stirred in ethanol
(20 mL), and then, hydrochloric acid (2 mL) was added. The
mixture was heated under reꢃux for 7 h. After complete reac-
tion, the solvent was concentrated under reduced pressure
zole), 4.84 (t, 1H, CH), 3.02 (dd, 1H, CH , J=9.2), 3.31
2
1
3
(
dd, 1H, CH , J=9.2), 1.56 (s, 3H, CH ). C NMR ppm
2
3
(
DMSO-d , 100 MHz): 12.07, 43.62, 55.07, 111.38, 117.84,
6
1
19.27, 120.52, 122.54, 123.63, 124.76, 128.69, 132.01,
1
3

Molecular Diversity
and poured onto ice water (50 mL). The obtained precipitate
was ꢁltered, washed with water, and crystallized from etha-
nol to yield the corresponding pyrimidinones 11a–c.
163.07, 168.29. Anal. Calcd; C, 68.20; H, 4.45; N, 20.62,
found: C, 68.08; H, 4.26; N, 20.43.
General procedure for synthesis
of N‑(4‑(4,5‑dihydro‑5‑arylisoxazol‑3‑yl)
phenyl)‑1‑methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑amine
(12a–c)
6
‑(4‑(1‑Methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑ylamino)
phenyl)‑4‑phenylpyrimidin‑2(1H)‑one (11a) Yellowish
−1
white solid; yield: 70%, m.p. 294–297 °C. IR cm (KBr):
3
1
382 (NH), 3222 (C–H aromatic), 2864 (C–H aliphatic),
1
678 (C=O). H NMR ppm (DMSO-d , 300 MHz): 10.18
A mixture of the appropriate chalcones 9a–c (3.23 g,
0.01 mol) and hydroxylamine hydrochloride (0.69 g,
0.01 mol) was stirred in ethanol (20 mL) and then sodium
hydroxide (0.8 g, 0.02 mol) was added. The reaction mix-
ture was heated under reꢃux for 24 h. After the reaction
was completed, the solvent was concentrated by evapora-
tion under reduced pressure and then poured onto ice water
(50 mL). The obtained precipitate was ꢁltered, washed with
copious amount of water, and recrystallized from ethanol to
aꢂord the target compounds 12a–c.
6
(
s, 1H, NH), 8.53 (s, 1H, NH pyrimidine), 8.28 (d, 2H,
Ph-H2, H6), 7.86 (d, 1H, QX-H6), 7.72 (d, 2H, Ph-H2,
H6), 7.68 (d, 1H, QX-H9), 7.54 (t, 1H, QX-H8), 7.36 (t,
1
6
H, QX-H7), 7.33 (t, 1H, Ph-H4), 7.21 (t, 2H, Ph-H3, H5),
.65 (d, 2H, Ph-H3, H5), 6.63 (s, 1H, pyrimidine-H5), 2.15
1
3
(
s, 3H, CH ). C NMR ppm (DMSO-d , 100 MHz): 18.92,
3 6
1
1
1
2
16.36, 117.64, 123.46, 123.64, 124.68, 126.13, 129.03,
29.14, 131.31, 134.85, 143.11, 146.43, 146.75, 149.21,
59.68, 171.08, 173.49. Anal. Calcd; C, 70.10; H, 4.30; N,
2.01, Found: C, 70.13; H, 4.33; N, 22.04.
N‑(4‑(4,5‑dihydro‑5‑phenylisoxazol‑3‑yl)phenyl)‑1‑me‑
thyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑amine (12a) White
6
‑(4‑(1‑Methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑ylamino)
−
1
phenyl)‑4‑(4‑chlorophenyl)pyrimidin‑2(1H)‑one (11b) Red-
solid; yield: 50%; m.p. 210–212 °C. IR cm (KBr): 3331
−
1
1
dish white solid; yield: 55%; m.p. 295–299 °C. IR cm
(NH), 3022 (C–H aromatic), 2964 (C–H aliphatic). H
(
KBr): 3403 (NH), 3083 (C–H aromatic), 2975 (C–H ali-
NMR ppm (DMSO-d , 300 MHz): 9.38 (s, 1H, NH), 7.94
6
1
phatic), 1660 (C=O). H NMR ppm (DMSO-d , 300 MHz):
(d, 1H, QX-H6), 7.87 (d, 1H, QX-H9), 7.77 (d, 2H, Ph-H2,
H6), 7.40 (t, 1H, QX-H8), 7.38 (t, 1H, QX-H7), 7.21 (d, 2H,
Ph-H3, H5), 7.15 (d, 2H, Ph-H2, H6), 7.09 (t, 1H, Ph-H4),
7.04 (t, 2H, Ph-H3, H5), 5.46 (t, 1H, CH), 3.64 (dd, 1H,
CH , J=9.0), 2.89 (dd, 1H, CH , J=9.0), 2.19 (s, 3H, CH ).
6
1
0.28 (s, 1H, NH), 8.53 (s, 1H, NH pyrimidine), 8.29
(
d, 2H, Ph-H2, H6), 7.86 (d, 1H, QX-H6), 7.83 (d, 2H,
Ph-H2, H6), 7.68 (d, 1H, QX-H9), 7.54 (t, 1H, QX-H8),
7
2
.36 (t, 1H, QX-H7), 7.32 (d, 2H, Ph-H3, H5), 6.65 (d,
H, Ph-H3, H5), 6.60 (s, 1H, pyrimidine-H5), 1.95 (s, 3H,
2
2
3
1
3
C NMR ppm (CDCl , 100 MHz): 11.88, 42.15, 84.86,
3
CH ). MS (m/z):479 (C H ClN O, 1.43%, M+), 481
111.43, 112.30, 120.41, 120.76, 124.80, 125.75, 127.10,
128.09, 128.83, 130.02, 136.05, 142.12, 142.49, 150.08,
156.20, 166.15. Anal. Calcd; C, 71.41; H, 4.79; N, 19.99,
found: C, 71.52; H, 4.88; N, 20.03.
3
26 18
7
(
C H ClN O, 0.39%, M+2), 464 (C H ClN O, 3.70%),
26 18 7 25 16 7
3
29 (C H N O, 14.41%), 296 (C H ClN O, 1.06%),
18 13 6 16 11 3
2
74 (C H N , 1.38%), 205 (C H ClN O, 12.29%), 199
1
6
12
5
10
6
2
(
C H N5, 3.02%), 182(C H N , 14.91%), 150 (C H ClN,
10 8 10 6 4 8 5
1
4
2
.51%), 131 (C H N , 23.49%), 76.09 (C H , 38.51%),
N‑(4‑(5‑(4‑chlorophenyl)‑4,5‑dihydroisoxazol‑3‑yl)
phenyl)‑1‑methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑amine
8
7
2
6
4
4.07 (CHNO, 100%). Anal. Calcd; C, 65.07; H, 3.78; N,
−
1
0.34, found: C, 65.09; H, 3.80; N, 20.36.
(12b) White solid; yield: 62%; m.p. 259–262 °C. IR cm
(
KBr): 3401 (NH), 3081 (C–H aromatic), 2983 (C–H ali-
1
6
‑(4‑(1‑methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑ylamino)
phatic). H NMR (DMSO-d , 300 MHz): 9.24 (s, 1H, NH),
6
phenyl)‑4‑(4‑methoxyphenyl)pyrimidin‑2(1H)‑one
8.02 (d, 1H, QX-H6), 7.98–7.90 (m, 7H, QX-H9- Ph-H2,
H6, QX-H8, QX-H7- Ph-H3, H5), 7.69 (d, 2H, Ph-H2,
H6), 7.62 (d, 2H, Ph-H3, H5), 5.85 (t, 1H, CH), 3.62 (dd,
1H, CH , J=9.5), 2.87 (dd, 1H, CH , J=9.5), 2.19 (s,
(
11c) Yellowish white solid; yield: 70%; m.p. 291–293 °C.
−1
IR cm (KBr): 3449 (NH), 3120 (C–H aromatic), 2932
1
(
C–H aliphatic), 1680 (C=O). H NMR ppm (DMSO-d ,
6
2
2
1
3
3
00 MHz): 10.49 (s, 1H, NH), 8.53 (s, 1H, NH pyrimi-
dine), 8.47 (d, 2H, Ph-H2, H6), 8.26 (d, 1H, QX-H6), 8.12
d, 2H, Ph-H2, H6), 7.69 (d, 1H, QX-H9), 7.54 (t, 1H,
QX-H8), 7.38 (t, 1H, QX-H7), 7.09 (d, 2H, Ph-H3, H5),
3H, CH ). C NMR ppm (DMSO-d , 100 MHz): 18.92,
3
6
84.14, 117.71, 118.28, 120.32, 122.90, 123.74, 125.02,
127.30, 129.38, 130.45, 132.21, 134.24, 134.79, 138.88,
139.27, 144.55, 146.99, 150.30, 169.46. MS (m/z): 454
(C H ClN O, 25.92%, M+), 453 (C H ClN O, 4.36%,
(
6
.59 (d, 2H, Ph-H3, H5), 6.55 (s, 1H, pyrimidine-H5), 4.14
2
5
19
5
25 19
5
1
3
(
s, 3H, OCH ), 1.99 (s, 3H, CH ). C NMR ppm (CDCl ,
M+2), 440 (C H ClN O, 0.98%), 286 (C H N ,
3
3
3
24 17 6 16 12 5
1
1
1
00 MHz): 11.92, 55.85, 106.12, 111.32, 112.48, 114.36,
20.91, 124.08, 125.11, 125.95, 127.26, 128.71, 130.28,
35.90, 140.12, 142.09, 149.60, 156.37, 161.51, 162.90,
1.27%), 256 (C H ClNO, 1.33%), 198 (C H N , 2.44%),
15 11 10 8 5
180 (C H ClNO, 4.13%), 154 (C H ClN O, 10.02%), 141
9
7
7
5
5
(C H ClO, 6.74%), 131 (C H N , 9.39%), 111(C H Cl,
7
5
8
7
2
6
4
1
3

Molecular Diversity
1
4
1.59%), 76.09 (C H , 38.51%). Anal. Calcd; C, 66.01; H,
(t, 1H, QX-H8), 7.27 (t, 1H, QX-H7), 7.21 (d, 1H, QX-H9),
7.19 (t, 2H, Ph-H3, H5), 7.12 (t, 1H, Ph-H4), 7.05 (d, 2H,
6
4
.21; N, 18.47, found: C, 66.13; H, 4.29; N, 18.51.
1
3
Ph-H2, H6), 4.48 (s, 2H, SCH ), 2.22 (s, 3H, CH ).
C
2
3
N‑(4‑(4,5‑dihydro‑5‑(4‑methoxyphenyl)isoxazol‑3‑yl)
phenyl)‑1‑methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑amine
NMR ppm (DMSO-d , 100 MHz): 12.30, 56.46, 113.93,
6
123.34, 123.43, 125.13, 127.80, 133.87, 146.27, 148.84,
−
1
(
12c) White solid; Yield: 62%; m.p. 221–224 °C. IR cm
149.96, 157.39, 170.12. MS (m/z): 349 (C H N OS,
1
8
15
5
(
KBr): 3362 (NH), 3106 (C–H aromatic), 2901 (C–H ali-
3.03%, M+), 335 (C H N OS, 7.18%), 256 (C H NOS,
17 13 5 12 8
1
phatic). H NMR ppm (DMSO-d , 300 MHz): 9.39 (s,
5.63%), 229 (C H N S, 6.80%), 134 (C H N , 5.05%), 120
11 9 4 8 7 2
6
1
H, NH), 8.01–7.88 (m, 8H, QX-H6, QX-H9, Ph-H2, H6,
(C H NO, 1.16%), 93 (C H N, 100%), 78 (C H , 27.10%),
7 6 6 7 6 6
QX-H8, QX-H7, Ph-H3, H5), 7.66 (d, 2H, Ph-H2, H6), 6.92
76 (C H4, 8.03%), 44 (CHNO, 77.17%). Anal. Calcd; C,
6
(
d, 2H, Ph-H3, H5), 5.25 (t, 1H, CH), 4.19(s, 3H, OCH ),
61.87; H, 4.33; N, 20.04, found: C, 61.99; H, 4.35; N, 20.16.
3
3
2
.66 (dd, 1H, CH , J=9.0), 2.91 (dd, 1H, CH , J=9.0),
2
2
.19 (s, 3H, CH ). Anal. Calcd; C, 69.32; H, 4.92; N, 18.60,
2‑(1‑Methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑ylthio)‑
N‑p‑tolylacetamide (15b) White solid; yield 72%; m.p. 296–
3
found: C, 69.48; H, 4.96; N, 18.73.
−
1
2
98 °C. IR cm (KBr):3279 (NH), 3132 (C–H aromatic),
1
Synthesis of 1‑methyl‑[1,2,4]triazolo[4,3‑a]
quinoxaline‑4‑thiol (13)
2953 (C–H aliphatic), 1673 (C=O). H NMR ppm (DMSO-
d , 400 MHz): 10.88 (s, 1H, NH), 7.57 (d, 1H, QX-H6), 7.43
6
(
t, 1H, QX-H8), 7.41 (t, 1H, QX-H7), 7.39 (d, 1H, QX-H9),
A mixture of 4-chloro-1-methyl-[1,2,4]triazolo[4,3-a]qui-
noxaline (5.0 g, 0.0245 mol) and thiourea (1.8 g, 0.0245 mol)
in absolute ethanol (150 mL) was reꢃuxed for 4 h, ꢁltered
while hot. After cooling, the obtained solid was reꢃuxed
with 10% NaOH for 0.5 h and acidiꢁed with 4 N HCl. The
resulting precipitate was ꢁltered, washed several times with
water, and dried to give the target compound as faint yellow-
7.32 (d, 2H, Ph-H3, H5), 7.29 (d, 2H, Ph-H2, H6), 4.62 (s,
1
3
2H, SCH ), 4.02 (s, 3H, CH ), 2.19 (s, 3H, CH ). C NMR
2
3
3
ppm (DMSO-d , 100 MHz): 12.02, 22.18, 39.15, 114.14,
6
120.12, 127.29, 129.04, 129.99, 135.41, 137.15, 142.11,
148.81, 150.50, 152.04, 169.10. Anal. Calcd; C, 62.79; H,
4.71; N, 19.27, found: C, 62.81; H, 4.73; N, 19.32.
−
1
ish white crystals. Yield: 65%; m.p. 305–308 °C. IR cm
2‑(1‑Methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑ylthio)‑
N‑(4‑methoxyphenyl)acetamide (15c) Faint violet solid;
(
KBr): 3433 (NH), 3099 (C–H aromatic), 2976 (C–H ali-
1
−1
phatic), 2580 (SH). H NMR ppm (DMSO-d , 100 MHz):
yield (70%); m.p. 260–262 °C. IR cm (KBr):3479 (NH),
6
1
1.96 (s, 1H, NH), 8.06 (d, 1H, QX-H6), 7.76 (t, 1H,
3132 (C–H aromatic), 2962 (C–H aliphatic), 1698 (C=O).
1
QX-H8), 7.65 (t, 1H, QX-H7), 7.27 (d, 1H, QX-H9), 2.96 (s,
H NMR ppm (DMSO-d , 400 MHz): 10.30 (s, 1H, NH),
6
3
H, CH ). MS (m/z): 216 (C H N S, 1.02%, M+), 201.23
7.57 (d, 1H, QX-H6), 7.55 (t, 1H, QX-H8), 7.41 (t, 1H,
3
10
8
4
(
C H N S, 0.71%), 185 (C H N , 2.61%), 131 (C H N ,
QX-H7), 7.39 (d, 1H, QX-H9), 7.35 (d, 2H, Ph-H3, H5),
9
6
4
10 10
4
8
7
2
5
.37%), 91 (C H N, 3.46%), 76.06 (C H , 12.77%), 43.12
7.28 (d, 2H, Ph-H2, H6), 4.49 (s, 2H, CH ), 4.22 (s, 3H,
6
5
6
4
2
1
3
(
CS, 100%). Anal. Calcd; C, 55.56; H, 3.73; N, 29.91, found:
OCH ), 2.79 (s, 3H, CH ). C NMR ppm (DMSO-d ,
3
3
6
C, 55.61; H, 3.79; N, 29.98.
100 MHz): 12.15, 39.02, 56.11, 114.18, 115.19, 123.08,
1
28.40, 130.61, 131.07, 142.20, 150.01, 151.17, 152.34,
General procedure for synthesis of 2‑(1‑methyl‑[1,2,4]
triazolo[4,3‑a]quinoxalin‑4‑ylthio)‑N‑arylacetamide
159.58, 169.03. Anal. Calcd; C, 60.14; H, 4.52; N, 18.46,
found: C, 60.19; H, 4.58; N, 18.49.
(15a–g)
2
‑(1‑Methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑ylthio)‑
A mixture of the potassium salt of compound 13 (1.5 g,
.01 mol) and the appropriate α-chloroacetanilide deriva-
N‑(4‑bromophenyl)acetamide (15d) Faint brown solid;
−
1
0
yield: 59%; m.p. 258–260 °C. IR cm (KBr):3305 (NH),
3122 (C–H aromatic), 2905, (C–H aliphatic), 1664 (C=O);
tive (0.01 mol) in DMF (20 mL) was heated over a water
bath for 3 h. The reaction mixture was then cooled, poured
into ice-cooled water (200 mL), and stirred well for 30 m.
The separated solid was ꢁltered, washed with water, dried,
and ꢁnally crystallized from methanol/toluene mixture (1:1).
1
H NMR ppm (DMSO-d6, 400 MHz): 10.23 (s, 1H, NH),
8.16 (d, 1H, QX-H6), 7.46 (t, 1H, QX-H8), 7.42 (t, 1H,
QX-H7), 7.39 (d, 1H, QX-H9), 7.29 (d, 2H, Ph H3, H5), 7.27
(d, 2H, Ph-H2, H6), 4.51 (s, 2H, SCH ), 2.22 (s, 3H, CH ).
2
3
1
3
C NMR ppm (DMSO-d6, 100 MHz): 12. 13, 39.23, 114.18,
2
‑(1‑Methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑ylthio)‑
121.21, 122.62, 127.29, 128.20, 129.16, 132.02, 137.72,
142.09, 149.58, 150.63, 152.26, 168.27. MS (m/z):428
(C H BrN OS, 4.06%, M+), 430 (C H BrN OS, 4.79%,
N‑phenylacetamide (15a) White solid; yield: 68%; m.p.
−
1
2
72 °C. IR cm (KBr): 3270 (NH), 3100 (C–H aromatic),
1
8
14
5
18 14
5
1
2
948 (C–H aliphatic), 1661 (C=O). H NMR ppm (DMSO-
M+2), 412 (C H BrN OS, 1.92%), 384 (C H N OS,
17 12 5 18 14 5
d , 400 MHz): 10.23 (s, 1H, NH), 7.57 (d, 1H, QX-H6), 7.35
3.20%), 257 (C H N OS, 8.00%), 243 (C H BrOS, 4.36%),
12 9 4 8 7
6
1
3

Molecular Diversity
2
29 (C H N4S, 40.25%), 197 (C H BrNO, 7.62%), 171
6 7 8 7 2 6 4
2.77%), 44 (CHNO, 93.09%). Anal. Calcd; C, 50.48; H,
.29; N, 16.35, found: C, 50.66; H, 3.26; N, 16.53.
Table 1 IC in μM of newly synthesized compounds on MDA-
1
1
9
7
5
50
MB-231 using MTT assay
(
C H BrN, 100%), 131 (C H N , 26.90%), 76 (C H ,
3
3
Compound
IC50 in μM
Compound
IC50 in μM
Doxorubicin
8
0.1
6.4
229
11c
12a
12b
12c
13
15a
15b
15c
15d
15e
15f
15g
15.5
27.5
144.5
363
363
2.9
17.4
15.5
2.7
2
‑(1‑Methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑ylthio)‑
9
a
N‑(4‑chlorophenyl)acetamide (15e) White solid; yield:
−1
9b
9c
36.3
213.8
151.4
60.3
128.8
34.7
17.4
162
6
2%); m.p. 228–230 °C. IR cm (KBr): 3267(NH), 3125
1
(
C–H aromatic), 2950 (CH aliphatic), 1669 (C=O). H
9
9
1
1
d
NMR ppm (DMSO-d6, 400 MHz): 10.43 (s, 1H, NH),
.06 (d, 1H, QX-H6), 7.46 (t, 1H, QX-H8), 7.40 (t, 1H,
QX-H7), 7.38 (d, 1H, QXH9), 7.31 (d, 2H, Ph-H3, H5),
.27 (d, 2H, Ph-H2, H6), 4.49 (s, 2H, SCH ), 2.22 (s, 3H,
e
8
0a
0b
10c
7
2
1
3
1.9
2.8
3.5
CH ). C NMR ppm (DMSO-d6, 100 MHz): 12.06, 39.61,
3
1
1
1a
1b
1
1
14.01, 120.40, 126.15, 127.12, 129.11, 133.32, 136.61,
38.9
42.27, 148.18, 150.17, 152.53, 168.37. MS (m/z): 383
(
C H ClN OS, 3.54%, M+), 385 (C H ClN OS, 0.98%,
18 14 5 18 14 5
M+2), 369 (C H ClN OS, 1.27%), 330 (C H ClN OS,
1
7
12
5
16 13
3
1
1
.01%), 228 (C H N S, 0.80%), 183 (C H N , 4.53%),
Biological evaluation
1
1
8
4
10
7
4
68 (C H ClNO, 3.27%), 154 (C H ClNO, 11.21%), 127
8
7
7
5
(
C H ClN, 100%), Anal. Calcd; C, 56.32; H, 3.68; N, 18.2,
Human breast adenocarcinoma cells (MDA-MB-231)
were allowed to grow in Dulbecco’s modified Eagle’s
medium (DMEM) instead of Rosewell Park Memorial
Institute (RPMI 1640) medium. The selected method is
3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bro-
mide (MTT) assay which has become one of the most widely
used tools in cell biology for measuring the metabolic
activity [25, 26]. Exponentially growing cells from MDA-
MB-231 cell line were trypsinized, counted, and seeded at
6
6
found: C, 56.48; H, 3.75; N, 18.37.
2
‑(1‑Methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑ylthio)‑
N‑(4‑ꢀuorophenyl)acetamide (15f) Rose red solid; yield:
−1
5
2%; m.p. 250–253 °C. IR cm (KBr): 3274 (NH), 3157
1
(
C–H aromatic), 2964 (C–H aliphatic), 1668 (C=O). H
NMR ppm (DMSO-d , 400 MHz): 10.23 (s, 1H, NH), 7.86
6
(
d, 1H, QX-H6), 7.46 (t, 1H, QX-H8), 7.42 (t, 1H, QX-H7),
2
7
.37 (d, 1H, QX-H9), 7.29 (d, 2H, Ph-H3, H5), 7.07 (d, 2H,
the appropriate densities (2000–1000 cells/0.33 cm well)
1
3
Ph-H2, H6), 4.48 (s, 2H, SCH ), 2.12 (s, 3H, CH ).
C
into 96-well microliter plates. Cells then were incubated in
a humidiꢁed atmosphere at 37 °C for 24 h. After that, cells
were exposed to diꢂerent concentrations of test compounds
(0.1, 10, 100, and 1000 μM) for 24, 48, and 72 h. The growth
media were removed; cells were incubated with 200 μl of
5% MTT solution (Sigma-Aldrich, MO) and were allowed
to metabolize the dye into colored insoluble formazan crys-
tals for 2 h. The remaining MTT solution was discarded
from the wells, and the formazan crystals were dissolved
in 200 μl acidiꢁed isopropanol for 30 min and covered with
aluminum foil with continuous shaking using a MaxQ 2000
plate shaker (Thermo Fisher Scientiꢁc Inc., MI) at room
temperature. Absorbance was measured at 570 nm using a
Stat FaxR 4200 plate reader (Awareness Technology, Inc.,
FL, USA). The cell viability was expressed as a percent-
age of control, and the concentration that induces 50% of
maximum inhibition of cell proliferation (IC ) was deter-
2
3
NMR ppm (DMSO-d , 100 MHz): 12.05, 39.14, 114.40,
6
1
1
15.13, 121.17, 127.17, 128.08, 129.20, 134.11, 142.91,
50.80, 151.06, 152.00, 163.12, 169.02. MS (m/z): 367
(
C H FN OS, 29.35%, M+), 354 (C H FN OS, 5.23%),
1
8
14
5
17 11
5
2
59 (C H N OS, 25.85%), 229 (C H N4S, 1.88%), 131
12 11 4 11 9
(
C H N , 59.90%), 76 (C H , 1.69%), 44 (CHNO, 15.32%).
8
7
2
6
4
Anal. Calcd; C, 58.84; H, 3.84; N, 19.06, found: C, 59.02;
H, 3.91; N, 19.24.
2
‑(1‑Methyl‑[1,2,4]triazolo[4,3‑a]quinoxalin‑4‑ylthio)‑
N‑(4‑nitrophenyl)acetamide (15g) Yellowish solid; yield:
−1
7
0%; m.p. 232–235 °C. IR cm (KBr): 3274 (NH), 3157
(
C–H aromatic), 2964 (CH aliphatic), 1668 (C=O), 1530 &
1
1
366 (N–O). H NMR ppm (DMSO-d , 400 MHz): 10.23
6
(
s, 1H, NH), 8.12 (d, 1H, QX-H6), 7.49 (t, 1H, QX-H8),
.44 (t, 1H, QX-H7), 7.39 (d, 1H, QX-H9), 7.67 (d, 2H,
Ph-H3, H5), 7.08 (d, 2H, Ph-H2, H6), 4.49 (s, 2H, SCH ),
7
5
0
mined using GraphPad Prism software version 5 (GraphPad
Software Inc., CA) [27]. Results of MTT assay and IC50 in
μM of newly synthesized compounds on MDA-MB-231 are
represented in Table 1.
2
1
3
2
1
1
1
5
.12 (s, 3H, CH ). C NMR ppm (DMSO-d , 100 MHz):
3 6
2.05, 38.09, 114.42, 120.02, 124.10, 127.71, 128.18,
29.52, 142.26, 143.57, 144.60, 149.69, 150.61, 151.98,
68.24. Anal. Calcd; C, 54.81; H, 3.58; N, 21.31, found: C,
4.67; H, 3.41; N, 21.19
1
3

Molecular Diversity
Structure activity relationship study
The results of biological evaluation could be summarized as:
Low activity of compound 7 reꢃects the importance of C-4
spacer on the activity. Replacement of C-4 chlorine atom in
7
with thiol group mildly improved the eꢂect. Among all
the synthesized derivatives, the best activity was observed
with the N-arylacetamides 15a–g. Electron-withdrawing
groups attached with the N-arylacetamide moiety increase
the potency as observed with 15e-g compared with 15b and
1
5c. Bulky atoms exhibit more steric eꢂects, resulting in
the reduction in the activity. Such eꢂect is clear in the com-
parison of the lower IC value of 15d with that of 15e.
5
0
In addition, shortening the spacer length at C-4 decreases
the activity. Chalcone derivatives 9a–e show variation of
activity depending on the para substituents on the aromatic
aldehyde. Electron-withdrawing groups increase the activity
of compound; e.g., compound 9b has higher activity than 9c
while unsubstituted aldehyde derivative 9a shows the lowest
activity of this series. Cyclization of chalcones with diꢂer-
ent binucleophiles results in an increase in the activity in
most cases.
Molecular docking
Fig. 4 Homology model of the human adenosine A2B receptor with
identiꢁed hit (ZINC09074482)
Molecular docking studies were carried out using the pro-
gram AutoDock Vina [28]. The homology model of A2B
receptor (Fig. 4) and the docking protocol described in an
earlier report were adapted for this purpose [13]. Water
molecules and identiꢁed hit were removed. The protein for
docking with AutoDock was prepared using ADT, which
includes adding polar hydrogen atoms to the protein atoms
and assigning Kollman charges afterward. For the ligand,
all hydrogen atoms must be present on it to calculate par-
tial atomic charges on the ligand. The protein active site
was deꢁned by placing a grid over the center of the identi-
Val253, and Ile276 a pocket that contributes to an increase
in the aꢀnity of the ligand (Fig. 5). Besides that, the amino
group of the compound formed a water-mediated interaction
with Glu174. The phenyl amino is predicted to be involved
in hydrophobic interactions with Ile67, Leu172, and Met279.
Because of the existence of additional hydrogen bonding
and desirable interactions, compound 15e and related deriva-
tives have a higher aꢀnity toward the receptor than all other
derivatives.
ꢁ
ed hit. Hydrogen atoms were also added to the ligand, and
The obtained binding mode of compound 15d with the
homology model of A2B receptor follows the general pat-
tern observed for compound 15e (Fig. 6). As before, the
hydrogen bonding, hydrophobic, and aromatic stacking
interactions are maintained. Insertion of bromine atom on
compound 15e instead of the chlorine atom is performed
to exploit the steric eꢂect of bromine as a bulky atom to
occupy the binding site, e.g., steric hindrance eꢂect is more
pronounced than chlorine atom which decreases the aꢀnity
of this compound.
Gasteiger charges were assigned. Before a protein is ready
for docking simulations, all the necessary grid maps were
calculated prior to docking. The grid maps were generated
with the help of AutoGrid, which is a program of the Auto-
Dock suite.
Results and discussion
The proposed binding mode of compound 15e with the
homology model of the human adenosine A receptor
The absence of a halogen atom in compound 15a is
likely important for decreasing the aꢀnity for the receptor
(Table 2). The obtained result of compound 15a is virtu-
ally the same as that of compound 15e. In addition, deletion
of halogen atom can decrease the aꢀnity of the compound
2
B
showed that the triazolo moiety of the compound is involved
in hydrogen bonding interactions with Asn254. Furthermore,
the tricyclic moiety of the compound is anchored by aro-
matic stacking interaction with Trp247, Phe173, and His251
and hydrophobic interaction with Val85, Leu86, Met182,
1
3

Molecular Diversity
Fig. 5 Predicted binding mode
for compound 15e with the
homology model of the human
adenosine A2B receptor.
Interactions between H-bonded
atoms are indicated by dotted
lines. Hydrogen (white), nitro-
gen (blue), oxygen (red), and
sulfur (yellow)
Fig. 6 Predicted binding mode
for compound 15 (aligned with
d
1
5 ) with the homology model
e
of the human adenosine A2B
receptor. Interactions between
H-bonded atoms are indicated
by dotted lines. Hydrogen
(
(
white), nitrogen (blue), oxygen
red), and sulfur (yellow)
because this compound cannot accommodate the binding
site (Fig. 7).
group of the compound is destabilized by hydrophobic inter-
actions Ile67 and Leu172 (Fig. 8).
Introduction of the nitro group in compound 15g is
likely important for decreasing the aꢀnity for the receptor
Tricyclic or bicyclic ring system and spacers with hydro-
gen bonding acceptor or donor with aromatic system con-
nected halogen are needed to increase the aꢀnity and selec-
tivity for the human adenosine A receptor; thus, reducing
(
Table 2). The obtained result of compound 15g is virtually
the same as that of compound 15e. In addition, the nitro
2
B
the spacer length as in compound 8 led to decreasing the
1
3

Molecular Diversity
Table 2 The docking scores and IC50 for all synthesized compounds
Compound IC50
Docking
score
Compound IC50
Docking
score
8
6.4
229
36
213.8 −6.50
151.4 −6.12
60.3
128.8 −6.78
34.7
17.4
162
38.9
15.5
−7.11
−6.36
−6.77
12a
12b
12c
13
15a
15b
15c
15d
15e
15f
15g
27.5
144.5 −6.13
−6.99
9a
9b
9c
9d
9e
363
363
2.9
17.4
15.5
2.7
1.9
2.8
3.5
−5.01
−5.88
−8.0
−6.90
−7.00
−7.81
−8.36
−7.92
−7.39
−6.15
1
1
1
1
1
1
0a
0b
0c
1a
1b
1c
−6.27
−6.81
−6.78
−7.50
−7.68
Fig. 8 Predicted binding mode for compound 15 with the homology
g
model of the human adenosine A2B receptor. As shown, there are
unfavorable interactions with hydrophobic groups
the receptor and the computed values reꢃect the overall
trend (Table 2).
Conclusion
The role of human adenosine A2B receptor as a media-
tor for pathophysiological conditions like tumors and as a
regulator for the angiogenic factors renders this receptor
of great medicinal interest in tumor chemotherapy. Due to
the absence of a 3D A2B target structure, diꢂerent ligand-
based methods were applied for the design of potent A2B
inhibitors. Herein, we report the design and synthesis of 23
new 1,2,4-triazolo[4,3-a]quinoxaline derivatives as poten-
tial inhibitors for A2B adenosine receptor type. Structures
of all the new derivatives were conꢁrmed by spectroscopic
methods and elemental analyses. The inhibitory eꢂect of
these compounds against A2B adenosine receptor was
measured depending on its cytotoxic eꢂects against MDA-
MB-231 cell lines. Results of cytotoxic evaluation showed
six promising active derivatives (8, 15a, 15d, 15e, 15f,
and 15g) with IC values ranging from 1.9 to 6.4 μM on
Fig. 7 Predicted binding mode for compound 15 with the homology
a
model of the human adenosine A2B receptor. As shown, the com-
pound cannot completely accommodate the binding site
aꢀnity where the compound cannot accommodate the bind-
ing site. Moreover, the substitution with aminophenyl group
(
9, 10, 11, and 12) instead of sulfur atom and spacer can
decrease the aꢀnity for the adenosine A receptor where
2
B
the introduction of phenyl group can delete the hydrogen
bonding interactions with Asn254 which is essential for
antagonist binding (Fig. 9).
5
0
MDA-MB 231 cell line. In addition, docking studies of
the synthesized compounds into the binding pocket of the
A2B receptor homology model and ꢁnally SAR analyses
of the new compounds were done. The observed IC val-
In summary, the obtained results indicated that all the
synthesized compounds have a similar position and ori-
entation inside the putative binding site of the homology
model of the human adenosine A receptor. In addition,
5
0
ues agreed with the obtained docking scores.
2
B
the results of the free energy of binding (∆G) explain that
some of these compounds have good binding aꢀnity to
1
3

Molecular Diversity
Fig. 9 Predicted binding mode
for compound 10c, with the
homology model of the human
adenosine A receptor. As
2B
shown, the hydrogen bond-
ing interaction with Asn254 is
absent
6
7
.
.
Illes P, Klotz K-N, Lohse MJ (2000) Signaling by extracellular
nucleotides and nucleosides. Naunyn Schmiedebergs Arch Phar-
macol 362:295–298. https://doi.org/10.1007/s002100000308
Panjehpour M, Castro M, Klotz K-N (2005) Human breast cancer
cell line MDA-MB-231 expresses endogenous A2B adenosine
Compliance with ethical standards
Conflict of interest The authors declare that they have no conꢃict of
interest.
2
+
receptors mediating a Ca signal. Br J Pharmacol 145:211–218.
https://doi.org/10.1038/sj.bjp.0706180
8
9
.
.
Feoktistov I, Goldstein AE, Ryzhov S et al (2002) Diꢂerential
expression of adenosine receptors in human endothelial cells:
role of A2B receptors in angiogenic factor regulation. Circ Res
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