Synthesis and Biological Evaluation of Some N-Substituted Quinoxaline Derivatives as Antitumor Agents

Article abstract of DOI:10.1134/S1068162020030097

Abstract: A new hybrid molecules containing 1-(N-substituted)-quinoxaline derivatives were synthesized from condensation of 3-hydroxy-2-oxo quinoxaline with 2,3-unsaturated carbonyl compounds under different conditions in order to yield ester and amide derivatives. The structure of the prepared compounds was elucidated on the bases of IR, ¹H NMR, ¹³C NMR, mass and elemental analyses. All the prepared compounds were evaluated for their anticancer activity against two cancer cell line (MCF-7 and Hela). Cell cycle analysis of 3-(p-methoxyphenyl)-3-(3-hydroxy-2-oxoquinoxalin-1-yl)-2-cyanoacrylic acid hydrazides compound demonstrated cell cycle arrest at S phase and Pre-G1 apoptosis. DNA synthesis inhibitory percentage revealed that this mentioned compound showed equipotent activity to Doxorubicin. Additionally, apoptosis was confirmed by increase the percentage of caspase 3/7 higher than Cisplatin.

Full text of DOI:10.1134/S1068162020030097

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2020, Vol. 46, No. 3, pp. 409–416. © Pleiades Publishing, Ltd., 2020.
Synthesis and Biological Evaluation of Some N-Substituted
Quinoxaline Derivatives as Antitumor Agents
Adil A. Gobouri1
Department of Chemistry, Faculty of Science, Taif University, Al-Haweiah, P.O. Box 888, Zip Code, Taif, 21974 Saudi Arabia
Received December 2, 2019; revised December 11, 2019; accepted December 26, 2019
Abstract—A new hybrid molecules containing 1-(N-substituted)-quinoxaline derivatives were synthesized
from condensation of 3-hydroxy-2-oxo quinoxaline with 2,3-unsaturated carbonyl compounds under differ-
ent conditions in order to yield ester and amide derivatives. The structure of the prepared compounds was elu-
cidated on the bases of IR, 1H NMR, 13C NMR, mass and elemental analyses. All the prepared compounds
were evaluated for their anticancer activity against two cancer cell line (MCF-7 and Hela). Cell cycle analysis
of 3-(p-methoxyphenyl)-3-(3-hydroxy-2-oxoquinoxalin-1-yl)-2-cyanoacrylic acid hydrazides compound
demonstrated cell cycle arrest at S phase and Pre-G1 apoptosis. DNA synthesis inhibitory percentage
revealed that this mentioned compound showed equipotent activity to Doxorubicin. Additionally, apoptosis
was confirmed by increase the percentage of caspase 3/7 higher than Cisplatin.
Keywords: quinoxaline, anti-proliferative, cell cycle analysis, caspase 3/7, cisplatin
DOI: 10.1134/S1068162020030097
INTRODUCTION
the building block of many useful intermediate in
organic synthesis and useful dyes [1]. Quinoxaline
derivatives are of great interest to researchers for their
significant role in pharmaceutical industry [2, 3]. The
importance of these compounds was developed due to
their antiviral, kinase inhibitors, anticancer, antibac-
terial and anti-inflammatory activities [4–8]. Some
quinoxaline derivatives such as structures 1, 2 and 3
are known to biologically active as antiviral, anticancer
2-Quinolinone derivatives were known to possess a
wide range of pharmacological activities [1–7]. For
example, dynemicin and streptonigrin are naturally
occurring members of the class of antitumor antibiot-
ics [8, 9]. Quinoline derivatives have displayed potent
anticancer activity targeting different sites like
Topoisomerase, telomerase, farnasyltransferase, tyro-
sine kinase and protein kinase CK-11. Quinoxaline
derivatives are very important class of heterocyclic
compounds containing two nitrogen atoms, which is and antibacterial activities.
N
N
N
N
N
N
O
O
Structure 1
Structure 2
Br
Br
F3C
N
N
Structure 3
1 Corresponding author: e-mail: gobouria@gmail.com.
409

410
ADIL A. GOBOURI
N
OH
O
N
H
(I)
COOEt
CN
R−Ph
DMF/Et3N
N
N
OH
O
COOEt
R−Ph
CN
(IIa, b)
N2H4/EtOH
N
N
OH
O
CONHNH2
R−Ph
CN
(IIIa, b)
Ac2O
N
N
OH
O
N
N
OH
O
OH
CONHNHCOCH3
R−Ph
R−Ph
CN
N
N
CH3
NC
HO
(IVa, b)
Scheme 1. Synthesis of quinoxaline derivatives (II)–(IV) (IIa), (IIIa) and (IVa) R = OCH3, (IIb), (IIIb), and (IVb) R = Cl.
Development of many strategy for the preparation ity of the synthesized compounds against two cancer
breast cancer cell line and cervix cancer cell line.
of substituted quinoxaline has been reported [9, 10].
The most common method are the condensation of an
2,3-aryl diamine with 2,3-dicarbonyl compounds in
refluxing acetic acid, 4 N hydrochloric acid and/or
ethanol. Considering the importance of quinoxaline
derivatives, the aim of the present study was to prepare
some N-substitutedquinoxaline derivatives by using
RESULTS AND DISCUSSION
Chemistry
The synthetic pathway to obtain the N-substituted
quinolxaline derivatives are outlined in Schemes 1 and 2.
quinoxaline-2,3-dione and examine the cytotoxic activ- The starting material quinoxaline-2,3-diones (I) was
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SYNTHESIS AND BIOLOGICAL EVALUATION
411
obtained by refluxing 1-phenylenediamine with oxalic 11.94 ppm confirming the formation of the com-
acid in 4 N hydrochloric acid according to literature
reported [11, 12]. Addition of quinoxaline (I) to 2,3-
dicarbonyl compounds (namely ethyl 3-(p-methoxy-
phenyl)-2-cynaoacrylate and ethyl 3-(4-chlorophe-
nyl)-2-cynaoacrylate) in dimethyl formamide in the
presence of triethylamine as catalyst under reflux led
to the formation of ethyl 3-(3-hydroxy-2-oxoquinox-
alin-1-yl)-3-aryl-2-cyanoacrylate (IIa, b). The struc-
ture of compounds (IIa) was confirmed by its
1H NMR spectrum which showed two signals at δ 1.30
and 4.31 ppm as triplet and quartet for the protons of
pound. In addition, the aromatic protons were
observed in the region δ 7.08–8.31 ppm as multiplet
signals. The 13C-NMR spectrum of compounds (IIa)
showed two signals at δ 164.02 and δ 162.84 ppm
assigned to two carbons of carbonyl groups of quinox-
aline and ester. The three signals due to the two car-
bons at δ 62.56 and 14.50 ppm of ethoxy function and
at δ 56.24 ppm of methoxy group. Also, two signals
due to the two carbons at δ 155.64 ppm and δ
154.93 ppm attributed to two C–O groups. Aromatic,
ethoxy group (CH3CH2O–). A two singlet signals for cyano and olefinic carbons were observed within the
methoxy and hydroxy protons appeared at δ 3.87 and expected chemical shift region at δ 134–98.99 ppm.
N
OH
O
N
H
(I)
COOH
Pyridine
Ph
X
N
N
OH
N
N
OH
O
O
COOH
Ph
O
Ph
(V)
(VI)
Scheme 2. Synthesis of quinoxaline derivative (VI).
Treatment of ester (IIa, b) with hydrazine in etha- The carbons of carbonyl and C–O of the com-
nol afforded the corresponding 3-(aryl)-3-(3- pound (IVa) appeared in the region at δ 160.93–
hydroxy-2-oxoquinoxalin-1-yl)-2-cyanoacrylic acid 169.57 ppm, which elucidate the presence of com-
hydrazides (IIIa, b). The structure of compounds pound (IVa) in enol-keto form.
(IIIa, b) was supported via acetylation of acid hydra-
zide (III) with acetic anhydride under reflux to give
N-acetyl-3-(aryl)-3-(3-hydroxy-2-oxoquinoxalin-1-yl)-
Condensation of 3-hydroxy-2-oxoquinoxaline (I)
with cinnamic acid in pyridine under reflux was
expected to give 3-phenyl-3-(3-hydroxy-2-oxoqui-
noxalin-1-yl) acrylic acid (V), but only 1-(cinnam-
oyl)-2-oxo-3-hydroxyquinoxaline (VI) was afforded.
The 1H NMR spectrum of compound (VI) showed the
protons of hydroxyl group in position 3 of quinoxaline
resonated as a broad singlet signal at δ 11.95 ppm. The
protons of aromatic rings and olefinic were resonated
as multiplet signals in the region at δ 6.52–7.67 ppm.
The 13C NMR spectrum of compound (VI) showed
three signals for the carbons at δ 168.58, 168.40 and
δ 155.66 ppm assigned to the two carbonyl (C=O) and
C–O groups. In addition, a characteristic carbon sig-
1
2-cyanoacrylic acid hydrazides (IVa, b). H NMR
spectrum of acid hydrazide derivatives (IVa) as exam-
ple showed characteristic five singlet signals at δ 11.49,
11.38, 11.33, 3.85 and 3.74 ppm due to the protons of
three hydroxyl (3OH), methoxy (OCH3) and methyl
(CH3) groups protons of the aromatic ring and NH at
δ 6.91–8.64 ppm as multiplet signal were observed.
The 13C NMR spectrum of compound (IVa) showed
two signals at δ 55.75 and δ 41.60 ppm assigned to two
carbons of methoxy (OCH3) and methyl (CH3)
groups.
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ADIL A. GOBOURI
nals in the region at δ 144.00–115.00 ppm for aromatic was carried out using DNA flow cytometry analysis in
rings and olefinic carbons.
Evaluation of Biological Activity
MCF-7 cells. MCF-7 cells was incubated with IC50
concentration of compound (IIIa) and cisplatin for
24 h and then subjected to DNA flow cytometry anal-
ysis. As shown in Figs. 1 and 2, exposure of MCF-7
cells to compound (IIIa) resulted in interference of the
normal cell distribution in the cell cycle profile of
MCF-7 cells. Compound (IIIa) could enhance the S
phase by 20.12% compared with the untreated control.
This effect was accompanied by increase in cell per-
centage in G1 and G2/M phase of the cell cycle. These
results suggested that compound (IIIa) induce cancer
cell death via S phase arrest with apoptosis inducing
activity marked by the presence pre-G1 peak in the cell
cycle distribution profile of MCF-7 cells.
DNA synthesis inhibition assay. In order to investi-
gate the DNA inhibition activity induced by com-
pound (IIIa) on MCF-7 cell line. The DNA inhibition
percentage (%) of compound (IIIa) and cisplatin at
their IC50 concentration values was investigated using
BrdU assay to detect the proliferation of MCF-7 cells
for 24 h. As shown in (Fig. 3) compound (IIIa) was
found to have 81.82% DNA synthesis inhibition per-
centagein comparison with cisplatin which had
88.05% DNA synthesis inhibition percentage. The
results in this experiment confirmed that, the mecha-
nism of compound (IIIa) induced MCF-7 cell death
was inhibition of DNA synthesis and it is in line with-
the previously discussed data.
Activated caspase 3/7 as execution factor for apop-
tosis. Caspase 3/7 are member of the cysteine aspartic
acid-specific protease family and play a key effector
roles in apoptosis [13]. The activation of caspase 3/7
was determined in compound (IIIa) treated MCF-7
cells at IC50 concentration dose value using green flow
cytometry assay for 24 h. The result in (Fig. 4) showed
an increase in the level of caspase 3/7 percentage by
11.71-fold more than untreated control. Additionally,
compound (IIIa) revealedcaspase 3/7 activity more than
cisplatin as reference compound. In conclusion, com-
pound (IIIa) can induce apoptosis in compound (IIIa)
treated MCF-7 cells and increase caspase 3/7 percent-
In vitro cytotoxic activity against two cancer cell
line. The effect of quinoxaline derivatives (IIa, b),
(IIIa, b), (IVa, b) and (VI) on the viability of two can-
cer cell lines were studied using MTT assay. The cyto-
toxicity was assessed using cisplatin as positive control.
The two cancer cell line are MCF-7 (breast cancer cell
line) and Hela (cervix cancer cell line). Treatment of
MCF-7 and Hela cell lines with different concentra-
tion of quinoxaline derivatives revealed that some of
the tested compounds showed promising cytotoxic
activity against MCF-7 cells, and the compounds
showed weak cytotoxic activity against Hela cells as
concluded from their IC50 values as shown in Table 1.
Structurally,
3-(aryl)-3-(3-hydroxy-2-oxoquinox-
alin-1-yl)-2-cyanoacrylic acid hydrazides (IIIa, b)
showed the highest cytotoxic activity against MCF-7
cells which were more potent than ester precursor
(IIa, b), acetylated derivatives (IVa, b) and 1-(cinnam-
oyl)-2-oxo-3-hydroxyquinoxaline (VI). Considering
compounds (IIIa) and (IIIb), compound with 4-methoxy
phenyl moiety showed increased activity in compari-
son with compounds containing 4-chloro phenyl sug-
gesting that electron donating group is better than
electron withdrawing group. In conclusion, (IIIa) and
(IIIb) were the most potent antitumor compound over
MCF-7 cell line, since both of the two compounds
possessed the highest cytotoxic activity. The highest
cytotoxic compound was (IIIa) which had IC50 value
of 2.61 μM which was comparable of 2.12 μM of cis-
platin.
DNA Flow Cytometry Analysis
Cell cycle analysis. To study the mechanism of anti-
cancer activity of compound (IIIa), cell cycle analysis
Table 1. In vitro antitumor activity ofquinoxaline deriva- age higher than cisplatin.
tives II, III, IV and VI over MCF-7 and Hela cell lines. Data
are expressed as the mean three experiments
EXPERIMENTAL
IC50 values, μM
Chemistry
Compound no.
MCF-7
Hela
Melting point were measured using open capillary
tubes on a melt-temp melting point apparatus and are
uncorrected. FT-IR spectra were recorded on a Ther-
mofisher Nicolete IS 10 spectrophotometer using KBr
pellets. 1H and 13C NMR (400 MHz) spectra recorded
on a Brucker Avance-400 spectrometer using DMSO-
d6 as solvent- chemical shifts are reported on ppm
downfield from internal tetramethylsilane and are
given in the scale. The elemental analysis was carried
out on a Perkin-Elmer 2400 series II CHN analyzer.
All synthesized quinoxaline derivatives give satisfac-
(IIa)
(IIb)
89 2.23
23.41 0.27
2.61 0.32
7.37 0.42
6.15 2.21
>100
>100
63.05 0.97
8.92 0.41
25.05 0.63
>100
(IIIa)
(IIIb)
(IVa)
(IVb)
(VI)
>100
23.34 2.58
2.12 0.09
19.06 1.91
4.62 0.14
Cisplatin
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SYNTHESIS AND BIOLOGICAL EVALUATION
(b)
413
(a)
(c)
G1: 28.33%
S: 59.59%
G2/M: 55.72%
G1: 29.61%
G1: 50.70%
S: 57.98%
S: 37.86%
800
600
400
200
100
800
600
400
200
100
800
600
400
200
100
G2/M: 7.61%
G2/M: 9.65%
0
20 40 60 80 100 120
FL2A
0
20 40 60 80 100 120
FL2A
0
20 40 60 80 100 120
FL2A
Fig. 1. Effect of compound (IIIa) in MCF-7 cell distribution after treatment for 24 h. (a) Control group, (b) compound (IIIa)
and (c) cisplatin.
tory elemental analysis within 0.4% of the theoretical 62.56 (OCH2), 56.24 (OCH3), 14.50 (CH3) ppm. MS:
m/z (%) = 391 (M+, 16.30). Anal. calcd. for
C21H17N3O5 (391): C, 64.45; H, 4.35; N, 10.74.
Found: C, 64.24; H, 4.13; N, 10.47.
values. Chemicals and solvents were purchased from
commercial sources an analytical grade purity.
Ethyl 3-aryl-3-(3-hydroxy-2-oxoquinoxalin-1-yl-
2-cyanoacrylate (IIa, b). A mixture of equimolar
Ethyl 3-(4-chlorophenyl)-3-(3-hydroxy-2-oxoqui-
noxalin-1-yl)-2-cyanoacrylate (IIb). Yellow crystals,
yield 77%, mp 82–84°C. IR (KBr): 3421 (OH), 2224
(CN), 1718 (C=O), 1608, 1595 (C=C), 1171, 1118,
quantity
of
3-hydroxy-2-oxoquinoxaline
(I)
(0.01 mol) and 2,3-unsaturated ester (0.01 mol) in
dimethyl formamide in the presence of triethyl amine
(3 mL) was heated under reflux for 4 h. The reaction
mixture was cooled and poured into water and neu-
tralized with dilute hydrochloric acid (2%). The solid
product was filtered off, washed with water, dried and
crystallized from ethanol to give (II).
1
1017 (C–O) cm–1. H NMR (DMSO-d6) δ: 1.31 (t,
3H, CH3), 4.21 (q, 2H, OCH2), 7.21–8.45 (m, 8H,
Ar-H), 11.92 (br. s, 1H, OH) ppm. MS: m/z (%) = 397
(M+ + 2, 3.30), 395 (M+, 10.30). Anal. calcd. for
C20H14N3ClO4 (395): C, 60.76; H, 3.54; N, 10.63.
Found: C, 60.46; H, 3.33; N, 10.41.
Ethyl 3-(4-methoxyphenyl)-3-(3-hydroxy-2-oxo-
quinoxalin-1-yl)-2-cyanoacrylate (IIa). Pale green
crystals, yield 76%, mp 75–77°C. IR (KBr): 3415
(OH), 2214 (CN), 1716 (C=O), 1605, 1585 (C=C),
3-(Aryl)-3-(3-hydroxy-2-oxoquinoxalin-1-yl)-2-
cyanoacrylic acid hydrazides (IIIa, b). A mixture of
ester derivatives (II) (0.10 mol) and hydrazine hydrate
(0.03 mol) in ethanol (70 mL) was heated under reflux
for 4 h. The reaction mixture was cooled, then poured
into water and neutralized with dilute hydrochloric
1
1182, 1124, 1015 (C–O) cm–1. H NMR (DMSO-d6)
δ: 1.30 (t, 3H, CH3), 3.87 (s, 3H, OCH3), 4.31 (q, 2H,
OCH2), 7.08–8.31 (m, 8H, Ar-H), 11.94 (s, 1H, OH)
ppm. 13C NMR (DMSO-d6) δ: 164.02, 162.84 (C=O), acid (2%). The solid formed was filtered off, washed
with water, dried and the product was crystallized from
ethanol to give (III).
155.64, 154.85 (C–O), 134.00, 126.05, 124.42, 123.48,
116.74, 115.58, 115.44, 98.99 (C aromatic and CN),
100
80
60
40
20
0
110
G0
100
90
80
70
60
50
40
30
20
10
0
G2/M
S
G1
CTRL
(IIIa) (2.61 µM)
Cisplatin (2.12 µM)
(IIIa) (2.61 µM)
Cisplatin (2.12 µM)
Fig. 2. Graphical presentation of the effect of compound
(IIIa) in cell distribution after treatment for 24 h compared
with cisplatin.
Fig. 3. DNA synthesis inhibition percentage after treat-
ment with (IIIa) and cisplatin at their IC50 (μM) for 24 h.
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ADIL A. GOBOURI
(b)
(a)
(с)
D
L
D
N
D
104
103
102
101
104
104
103
102
101
N
N
103
102
101
L
L
A
A
A
0.82%
9.61%
8.89%
100
101 102 103 104
100
101 102 103 104
100
101 102 103 104
Caspase 3/7 green flourescence (530/30 BP)
FL1A
Fig. 4. Green flow cytomertic assay of caspase 3/7 (%) of the tested compounds (IIIa) compared to cisplatin at their IC50 (μM).
(a) Control group, (b) compound (IIIa) and (c) cisplatin.
3-(4-Methoxyphenyl)-3-(3-hydroxy-2-oxoquinox-
1693 (br, C=O), 1605, 1595 (C=C), 1128, 1087 (C–O)
cm–1. 1H NMR (DMSO-d6) δ: 3.59 (s, 3H, CH3), 3.83
(s, 3H, OCH3), 3.39 (s, 2H, NH2), 6.86–8.17 (m, 8H,
Ar-H), 8.64 (s, 2H, 2CONH), 11.33 (s, 1H, OH) ,
11.38 (s, 1H, OH), 11.49 (s, 1H, OH) ppm.13C NMR
(DMSO-d6) δ: 169.57, 169.14, 163.44 (C=O), 162.14,
161.32, 160.93 (C–O), 147.14, 143.20, 142.87 (C=N),
130.45, 129.18, 128.88, 128.79, 127.39, 127.31, 127.24,
127.16, 127.05, 114.86, 114.76, 114.61 (C aromatic, ole-
finic and CN), 55.75 (OCH3) ppm. MS: m/z (%) =
alin-1-yl)-2-cyanoacrylic acid hydrazides (IIIa). Col-
orless crystals, yield 68%, mp 135–137°C. IR (KBr):
3408 (OH), 3218, 3152 (NH2, NH), 2320 (CN), 1693
(C=O), 1605, 1580 (C=C), 1062 (C–O) cm–1.
1H NMR (DMSO-d6) δ: 3.83 (s, 3H, OCH3), 4.19 (s,
2H, NH2), 6.92–7.95 (m, 8H, Ar-H), 8.64 (s, 1H,
CONH), 11.93 (s, 1H, OH) ppm. 13C NMR (DMSO-d6)
δ: 162.14, 161.02 (C=O), 155.86, 154.65 (C–O),
130.46, 129.11, 127.97, 127.01, 126.06, 123.49, 115.58,
114.87, 114.74, 114.63, 114.51 (C aromatic and CN), 419 (M+, 3.20). Anal. calcd. for C21H17N5O5 (419): C,
55.85 (OCH3) ppm. MS: m/z (%) = 377 (M+, 15.20). 60.14; H, 4.06; N, 16.71. Found: C, 59.98; H, 3.83; N,
16.43.
N-Acetyl 3-(4-chlorophenyl)-3-(3-hydroxy-2-oxo-
Anal. calcd. for C19H15N5O4 (377): C, 60.48; H, 3.98;
N, 18.57. Found: C, 60.40; H, 3.63; N, 18.33.
quinoxalin-1-yl)-2-cyanoacrylic acid hydrazides (IVb).
Orange crystals, yield 63%, mp 205–2077°C. IR
(KBr): 3430 (OH), 3227 (NH), 2223 (CN), 1699–
1693 (br, C=O), 1607, 1585 (C=C), 1078, 1023 (C–O)
cm–1. 1H NMR (DMSO-d6) δ: 3.43 (s, 3H, CH3), 4.23
(s, 2H, NH2), 7.12–8.24 (m, 8H, Ar-H), 8.71 (s, 1H,
CONH), 11.34 (s, 1H, OH), 11.39 (s, 1H, OH), 11.78
(s, 1H, OH) ppm. MS: m/z (%) = 425 (M+ + 2, 2.57),
423 (M+, 9.63). Anal. calcd. for C20H14N5ClO4 (423):
C, 56.68; H, 3.33; N, 16.52. Found: C, 56.45; H, 3.08;
N, 16.63.
3-(4-Chlorophenyl)-3-(3-hydroxy-2-oxoquinoxalin-
1-yl)-2-cyanoacrylic acid hydrazides (IIIb). Pale yel-
low crystals, yield 63%, mp 127–129°C. IR (KBr):
3445 (OH), 3320, 3215 (NH2, NH), 2221 (CN), 1696
(C=O), 1605, 1585 (C=C), 1019 (C–O) cm–1.
1H NMR (DMSO-d6) δ: 4.23 (s, 2H, NH2), 6.95–
8.23 (m, 8H, Ar-H), 8.81 (s, 1H, CONH), 11.96 (br. s,
1H, OH) ppm. MS: m/z (%) = 383 (M+ + 2, 2.30), 381
(M+, 7.20). Anal. calcd. for C18H12N5ClO3 (381): C,
56.63; H, 3.17; N, 18.34. Found: C, 56.36; H, 3.03; N,
18.52.
1-(Cinnamoyl)-2-oxo-3-hydroxyquinoxaline (VI).
mixture of 3-hydroxy-2-oxoquinoxaline (I)
N-Acetyl 3-(aryl)-3-(3-hydroxy-2-oxoquinoxalin-
1-yl)-2-cyanoacrylic acid hydrazides (IVa, b). A solu-
tion of acrylic acid hydrazide derivatives (III)
(0.01 mol) in acetic acid anhydride (25 mL) was
heated under reflux for 2 h, then cooled and poured
into ice-water. The reaction mixture was left 24 h and
the solid formed was filtered off, washed with water
and dried. Finally, the product was crystallized from
ethanol to give compound (IV).
A
(0.01 mol) and cinnamic acid (0.01 mol) in pyridine
(30 mL) was heated under reflux for 6 h, then cooled
and poured into water. The reaction mixture was neu-
tralized with dilute hydrochloric acid (2%) and the
resulting solid was filtered off, washed with water and
dried. Finally, the product was crystallized from buta-
nol to give compound (VI) as colorless crystals.
Yield 73%, m.p. 197–199°C. IR (KBr): 3446 (br,
OH), 1682 (C=O), 1630 (C=N), 1605, 1583 (C=C),
1230 (C–O) cm–1. 1H NMR (DMSO-d6) δ: 6.52–7.67
N-Acetyl 3-(4-methoxyphenyl)-3-(3-hydroxy-2-
oxoquinoxalin-1-yl)-2-cyano acrylic acid hydrazides
(IVa). Orange crystals, yield 67%, mp 221–223°C. IR
(KBr): 3425 (OH), 3225 (NH), 2225 (CN), 1698– (m, 11H, Ar-H and olefinic H), 11.95 (br. s, 1H, OH)
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SYNTHESIS AND BIOLOGICAL EVALUATION
Caspase 3/7 Assay of Compound (IIIa)
415
ppm. 13C NMR (DMSO-d6) δ: 168.58, 168.40 (C=O),
155.66 (C–O), 143.89, 143.68, 134.88, 130.55, 129.35,
128.58, 126.06, 123.48, 120.65, 120.36, 115.59 (C aro-
matic and olefinic) ppm. MS: m/z (%) = 292 (M+,
32.35). Anal. calcd. for C17H12N2O3 (292): C, 69.86;
H, 4.11; N, 9.59. Found: C, 69.59; H, 3.89; N, 9.35.
The enzymatic activities of caspase 3/7 were
assayed in cell lysates (100 mg protein in 50 mL lysis
buffer) using caspase 3/7 green flow cytometry assay
kit as per the manufacturer’s instructions. Briefly,
control and compound (IIIa) treated MCF-7 cells
(2.5 × 105/mL) were washed with ice cold PBS, cell
lysates. were prepared and subsequently protein con-
centration was estimated. Lysates were combined with
reaction buffer and incubated with specific colorimet-
ric substrates (Caspase 3/7 Detection Reagent) at
37°C for 6 h. The samples were measured at 488 nm in
BD FACS Calibur flow cytometer. All experiments
were done in triplicates.
Biological Evaluation
Anti-tumor activity against two cancer cell line
(MCF-7 and Hela). The cytotoxic activity was mea-
sured in vitro for the newly synthesized compounds
using the MTT assay. Cells were plated in 96-multi-
wall plate (105 cells/well) for 24 h before treatment
with the compounds. Test compounds were dissolved
in dimethyl sulfoxide. Different concentrations of the
compound under test (10, 25, 50, and 100 μM) were
added to the cell’s monolayer. Triplicate wells were
prepared for each individual concentration. Mono-
layer cells were incubated with the compound(s) for 48
h at 37°C and in atmosphere of 5% CO2. After 48 h,
cells were fixed, washed and stained with 40 μL of
MTT solution (5 mg/mL of MTT in 0.9% NaCl) in
each well was added and incubated for an additional
4 h. MTT crystals were solubilized by adding 180 μL of
acidified isopropanol/well and the plate was shaken at
room temperature, followed by photometric determi-
nation of the absorbance at 570 nm using ELISA
reader. The molar concentration required to inhibit
50% of cell viability (IC50) was calculated and com-
pared with the reference drug doxorubicin. The sur-
viving fractions were expressed as means S.E.M.
CONCLUSION
In summary, hybrid molecules having 1-(N-substi-
tuted) quinoxaline derivatives (II–VI) were prepared
from 3-hydroxy-2-oxo quinoxaline (I) with 2,3-unsat-
urated carbonyl compounds and cinnamic acid under
different conditions. The structure of the prepared
1
compound were confirmed via IR, H NMR, 13C
NMR, MS and elemental analyses. All the prepared
compounds were evaluated for their anticancer activity
against two cancer cell line (MCF-7 and Hela). The
most promising compound in this series was 3-(4-
methoxyphenyl)-3-(3-hydroxy-2-oxoquinoxalin-1-yl)-
2-cyanoacrylicacid hydrazides (IIIa) which signifi-
cantly inhibited MCF-7 cells with IC50 value of
2.61 μM. Moreover cell cycle analysis of compound (IIIa)
demonstrated cell cycle arrest at S phase and Pre-G1
Cell cycle analysis of compound (IIIa). MCF-7 apoptosis. DNA synthesis inhibitory percentage
cells, (3.0 × 105 cells/well) and incubated at 37°C for
12 h. The target cells were then treated with the com-
pound (IIIa) at its IC50 concentration for 24 h. After
treatment, cells were collected and fixed with 75% eth-
anol at 20°C overnight, then, cells were washed with
PBS followed by centrifugation and incubated with
(10 mg/mL) RNase (Sigma, USA) and (5 mg/mL)
propidium iodide (PI, Sigma) before flow cytometry
analysis (FACSCalibur cytometer using Cellquest
software, BD Bioscience, USA).
revealed that compound (IIIa) showed equipotent
activity to doxorubicin. Additionally, Apoptosis was
confirmed by increase the percentage of caspase 3/7
higher than cisplatin.
COMPLIANCE WITH ETHICAL STANDARDS
This article does not contain any studies involvinghu-
man participants performed by any of the authors anddoes
not contain any studies involving animals performedby any
of the authors.
DNA synthesis inhibition assay. MCF-7 cells (2 ×
104/well) were treated with compounds (IIIa) and
Cisplatinat their IC50 values for 24 h. DNA inhibition
activity were determined using BrdU Kit according to
the manufacturer’s instruction. Briefly, cytotoxicity
test were performed using the cell proliferation
ELISA. After that cells were treated with BrdU solu-
tion for 2 h at 37°C, then treated with specific antibody
and incubated with further 90 min. Then, the cells
were washed with PBS until the color development
was adequate. Optical density of the samples were
measured in an ELISA reader. Finally, the percentage
of cell growth was measured versus positive control.
Conflict of Interests
The authors report no conflicts of interest.
SUPPLEMENTARY MATERIALS
Supplementary materials are available for this article at
https://doi.org/10.1134/S1068162020030097 and are accessi-
ble for authorized users.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 46 No. 3 2020

416
ADIL A. GOBOURI
7. Lee, S.H., Kim, N., Kim, S.J., Song, J., Gong, Y.D.,
REFERENCES
1. Brock, E., Lewis, D., Yousaf, T., and Harper, H., The
Procter and Gamble Company, USA Wo 9951688, AI,
1999.
and Kim, S.U., J. Cancer Res. Clin. Oncol., 2013,
vol. 139, pp. 1279–1294.
8. Pradeep, K., Kotra, V., Priyadarshimi, R.I., and Pra-
tap, V., Int. J. Pharm. Sci., 2015, vol. 7, pp. 243–246.
2. Abu-Hashem, A.A., Am. J. Org. Chem., 2015, vol. 5,
pp. 14–56.
9. Woo, G.H., Snyder, J.K., and Wan, Z.K., Prog. Hetero-
cycl. Chem., 2002, vol. 14, pp. 279–284.
10. Porter, A., Katritzky, A., and Rees, C., Comprehensive
Heterocyclic Chemistry, Katritsky, C.W., Ed., Oxford:
Pergamon Press, 1984, vol. 3.
3. Ingle, R.G., and Marathe, R.P., Pharmacophore, 2012,
vol. 3, pp. 109–116.
4. Loriga, M., Piras, S., Sanna, P., and Pagliette, G., Far-
maco Sci., 1997, vol. 52, pp. 157–166.
5. He, W., Myers, M.R., Hanney, B., Spade, A.P., Bilder, G.,
Galzcinski, H., Amin, D., Needle, S., Page, K., and
Jayyosi, Z., Bioorg. Med. Chem. Lett., 2003, vol. 13,
pp. 3097–3100.
11. El-Deen, I.M., and Mahmoud, F.F., Phosphorus, Sul-
fur, Silicon Relat. Elem., 2000, vol. 165, pp. 205–212.
12. El-Deen, I.M., and El-Fattah, M.E.A., J. Serb. Chem.
Soc., 2000, vol. 65, pp. 95–102.
6. Lindskey, C.W., Zhao, Z., Leister, W.H., Robinson, R.G.,
Barnett, S.E., Defeo-Jones, D., Jones, R.E., Hart- 13. Zaki, I., Abdelhameid, M.K., El-Deen, I.M.,
man, G.D., Huff, J.R., and Huber, H.E., Bioorg. Med.
Chem. Lett., 2005, vol. 15, pp. 761–764.
Wahab, A.H.A.A., Ashmawy, A.M., and Mohamed, K.O.,
Eur. J. Med. Chem., 2018, vol. 156, pp. 563–579.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 46
No. 3
2020