Design and synthesis of new quinoxaline derivatives as anticancer agents and apoptotic inducers

Article abstract of DOI:10.3390/molecules24061175

The quinoxaline scaffold is a promising platform for the discovery of active chemotherapeutic agents. Three series of quinoxaline derivatives were synthesized and biologically evaluated against three tumor cell lines (HCT116 human colon carcinoma, HepG2, liver hepatocellular carcinoma and MCF-7, human breast adenocarcinoma cell line), in addition to VEGFR-2 enzyme inhibition activity. Compounds VIId, VIIIa, VIIIc, VIIIe and XVa exhibited promising activity against the tested cell lines and weak activity against VEGFR-2. Compound VIIIc induced a significant disruption in the cell cycle profile and cell cycle arrest at the G2/M phase boundary. In further assays, the cytotoxic effect of the highly active compounds was determined using a normal Caucasian fibroblast-like fetal lung cell line (WI-38). Compound VIIIc could be considered as a lead compound that merits further optimization and development as an anti-cancer and an apoptotic inducing candidate against the HCT116 cell line.

Full text of DOI:10.3390/molecules24061175

molecules
Article
Design and Synthesis of New Quinoxaline
Derivatives as Anticancer Agents and
Apoptotic Inducers
Aliya M. S. El Newahie , Yassin M. Nissan , Nasser S. M. Ismail , Dalal A. Abou El Ella 5,6,
1
2,3
4
Sohair M. Khojah 7 and Khaled A.M. Abouzid
5,8,
*
1
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, October University for Modern
Science and Arts (MSA), Cairo 12611, Egypt; anewahie@msa.eun.eg
2
3
4
5
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt;
yassin.nissan@hotmail.com
Pharmaceutical Chemistry Department, Faculty of Pharmacy, October University for Modern Science and
Arts (MSA), Cairo 12611, Egypt
Pharmaceutical Chemistry Department, Faculty of Pharmaceutical Sciences and Pharmaceutical Industries,
Future University in Egypt, Cairo 12311, Egypt; saadnasser2003@yahoo.com
Pharmaceutical Chemistry Department, Faculty of Pharmacy Ain Shams University, Abbassia,
Cairo 11566, Egypt; dalal999@hotmail.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Nahda University, Beni Suef 62513, Egypt
Biochemistry Department, Faculty of Science, King Abdulaziz University,
Jeddah 21589, Kingdom of Saudi Arabia; smkhojah@yahoo.com
6
7
8
Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City,
Menoufia 32897, Egypt
*
Correspondence: Khaled.abouzid@pharma.asu.edu.eg; Tel.: +20-122-2165-624; Fax: +20-225-0807-28
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 4 March 2019; Accepted: 22 March 2019; Published: 25 March 2019
Abstract: The quinoxaline scaffold is a promising platform for the discovery of active chemotherapeutic
agents. Three series of quinoxaline derivatives were synthesized and biologically evaluated against
three tumor cell lines (HCT116 human colon carcinoma, HepG2, liver hepatocellular carcinoma and
MCF-7, human breast adenocarcinoma cell line), in addition to VEGFR-2 enzyme inhibition activity.
Compounds VIId, VIIIa, VIIIc, VIIIe and XVa exhibited promising activity against the tested cell
lines and weak activity against VEGFR-2. Compound VIIIc induced a significant disruption in the
cell cycle profile and cell cycle arrest at the G2/M phase boundary. In further assays, the cytotoxic
effect of the highly active compounds was determined using a normal Caucasian fibroblast-like fetal
lung cell line (WI-38). Compound VIIIc could be considered as a lead compound that merits further
optimization and development as an anti-cancer and an apoptotic inducing candidate against the
HCT116 cell line.
Keywords: quinoxaline; synthesis; anti-cancer activity; cell cycle
1
. Introduction
Cancer is a major human health problem that remains the second highest cause of mortality
worldwide [
in the United States in 2017 [
human carcinogenesis [ ], whereas cell cycle progression, cell division and proliferation are viewed
as a sequence of events controlled by a cascade of those protein kinases, so PTKs have emerged
as promising new cancer therapy targets [ ]. Quinoxalines are considered as an important basis
for anti-cancer drugs as they are proved to be selective adenosine triphosphate (ATP) competitive
1], where 1,688,780 new cancer cases and 600,920 cancer deaths were projected to occur
2]. Human protein tyrosine kinases (PTKs) play a central role in
3
4
Molecules 2019, 24, 1175; doi:10.3390/molecules24061175
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1175
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inhibitors in many kinases [
5
] for example: vascular endothelial growth factor receptor (VEGFR),
platelet-derived growth factor receptor (PDGFR), proto-oncogene non-receptor tyrosine-protein
kinase (Src), c-Met proto-oncogene (c-Met kinase), epidermal growth factor receptor human
epidermal growth factor receptor (EGFR/HER-2), Janus kinase receptor (JAK-2), FMs-related tyrosine
kinase 3 (FLT-3) and cyclin dependent kinase (CDK1,2,4,6) [ ]. In 2016, Zghaib et al. reported
that imidazo[1,2-a]quinoxaline derivatives were major microtubule-interfering agents with potent
anticancer activity [ ]. Quinoxalines with amide and sulphonamide moieties have been reported to
inhibit the growth of human tumor cell lines [8].
Ghorab et al. [ ] designed (quinoxalin-2-yl)benzene sulphonamide derivative
a potent anti-cancer activity against human liver cancer cell line (Hep G2). Quinoxaline-bisarylurea
(Figure 1) has been reported by Göring et al. [10] to exhibit anti-tumor activity. Additionally,
quinoxalines were found to induce apoptosis as a mechanism for their anti-cancer activity [11 12].
\
6
7
9
1 (Figure 1) with
2
,
In 2014, Shahin et al. [13] designed a series of new quinoxaline-based scaffolds bearing amide,
sulphonamide and urea moieties which were biologically evaluated for their inhibitory activity against
VEGFR-2. In that study, compound
compound (Figure 2) displayed the best inhibition percentage against VEGFR-2 which was 69% [
Furthermore, quinoxaline having an amide group (Figure 2) has been also reported by Ramurthy et
3
(Figure 1) displayed the best IC50 value of 10.27
µM, while
4
6].
5
al. as a potent Rapidly Accelerated Fibrosarcoma (Raf) kinase inhibitor [14]. Finally, quinoxalines were
recently reported to exhibit inhibition against MCF-7 (83.3%) and HCT116 (70%) cell lines [15].
Figure 1. Design of the synthesized compounds IXa,b), (XVIIIa,b, VIII a–e, XVII a–f and XXV.
The antitumor activity of kinase inhibitors containing a diaryl urea scaffold has been gaining great
attention as they possess a unique binding mode and obvious kinase inhibition profile [16]. Sorafenib

Molecules 2019, 24, 1175
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is a well-known diarylurea multi-targeted inhibitor of several kinases including Raf, VEGFR, c-Kit and
PDGFR [17].
Based on the abovementioned anti-proliferative quinoxaline compounds 1–5 and sorafenib as
a urea derivative [18], we aimed to design a new series of quinoxaline-based compounds with amide,
urea, thiourea and sulphonamide moieties. Hence, linking pharmacophoric functionalities to different
aryl moieties promises to have anti-proliferative and apoptotic inducing activity. The design of the
target compounds is presented in Figures 1 and 2.
Figure 2. Design of the synthesized compounds VII a–d, XVI a–d, VI a–c, (XV a–c and XXIV a,b).
2
. Results and Discussion
2
.1. Chemistry
Quinoxalines are prepared by the reaction of o-phenylenediamine and
α
-ketocarboxylic acids [19
]
through different methods. The synthesis of the starting compounds
Schemes 1–3, respectively.
I, X and XIX is depicted in
3
-Methylquinoxalin-2(1H)-one (
reaction of o-phenylenediamine with ethyl pyruvate (Westphal et al. [20]) for
acid (for XIX) in n-butanol [21 22]. The treatment of substituted o-phenylenediamines with oxalic
acid in 4N hydrochloric acid under reflux quickly afforded quinoxaline-2,3(1H,4H)-dione ( ) [23 24].
The starting compounds were then subjected to chlorination by treatment with POCl3 [20 21 25] alone
compounds ) or with PCl (compound XIX) [26] affording compounds II, XI and XX in a pure
I
) and 2(1H)-quinoxalinone (XIX) could be obtained by the
I
or with glyoxylic
,
X
,
,
,
(
I, X
5
form with melting points as reported.
The chloroquinoxalines were further refluxed with m-aminobenzoic acid in n-butanol [27],
the solution was left to cool and excess m-aminobenzoic acid was removed by dissolving in 5% NaOH,
then intermediate compounds III
hydrochloric acid. In our present study, we planned to synthesize target amide compounds VIa
XVa and XXIVa . For the preparation of such compounds, the reaction of the three intermediates III
XII and XXI with the corresponding aromatic amine seemed to be most convenient approach.
, XII and XXI were reprecipitated by dropwise addition of concentrated
–c,
,
–c
,b
In order to achieve such a reaction, the three intermediates were firstly activated as acid chloride
derivatives by refluxing with excess thionyl chloride (Wang et al. [28]), then reacting the freshly
prepared acid chlorides with the appropriate aromatic amines (aniline, p-chloroaniline, p-methoxy
aniline) in dichloromethane yielding the final products VIa–c
Kakuta et al. [29]).
, XVa–c and XXIVa,b in moderate yields
(

Molecules 2019, 24, 1175
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(
a)
(
b)
benzene-
ethyl Pyruvate
1,2-diamine
(
c)
(d)
(
e)
(
f)
VIa : X=H
VIb :X= Cl
VIc:X= OCH3
(
g)
(h)
(i)
VIIIa:X=S,Y=H
VIIIb:X=O,Y=H
VIIIc:X=O,Y=4-Cl
VIIId:X=O,Y=3-CH3
VIIa:X=H
VIIb:X=4-Cl
VIIc:X=4-OCH3
IXa: X=CH3
IXb:X=NO2
VIIIe:X=O,Y=3-OCH3
VIId:X=4-CH3
Scheme 1. Synthetic pathways for compounds VI–IX. Conditions and Reagents: (a): n-BuOH reflux
For 1 h, 97%, (b): POCl 1.5 h, 70%, (c): m-amino benzoic acid in BuOH, conc HCl reflux 4 h, 75%,
3
◦
(
(
(
d): p-phenylene diamine reflux at 110 C, 5 h, 70%, (e): SOCl in dry benzene reflux 5 h, 60%,
2
f): aromatic amines, TEA in dry DCM, reflux 4 h, (g): aromatic acid chlorides in dry toluene reflux 2 h,
h): phenyl isocyanates/isothiocyanates in dry toluene, reflux 5 h, (i): benzene sulphonyl chlorides in
dry pyridine reflux 2 h.
In another route, the respective chloroquinoxalines II
p-phenylenediamine in n-butanol at 110 C [30] to give high yields of another three important
,
XI and XX were refluxed with
◦
intermediate compounds IV
amides VIIa XVIa , thioureas VIIIa
IXa–b, XVIIIa–b.
The amide derivatives were synthesized from the reaction of intermediate compounds IV
and XXII with different aromatic acid chlorides (viz; benzoyl chloride, p-chlorobenzoyl chloride,
p-methoxybenzoyl chloride, p-tolylbenzoyl chloride) in diethyl ether (compounds VIIa XVIa) or
toluene (compounds VIIb XVIb (Kakuta et al. [29]). The thiourea and urea derivatives VIIIa
XVIIa and XXV were obtained by the reaction of intermediate compounds IV XIII and XXII with
,
XIII and XXII, which were further utilized to obtain different target
–
d,
–
d
,
XVIIa, ureas VIIIb XVIIb and XXV and sulphonamides
–
e
,
–e
,
XIII
,
–d
,
–d
–e,
–
e
,
phenyl isothiocyanate, phenyl isocyanate, p-chloro, m-methyl and m-methoxyphenyl isocyanates
in dry toluene [31]. As for the urea compound XVIIf, 4-methoxybenzoyl azide was used as the

Molecules 2019, 24, 1175
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starting material for the synthesis of p-methoxyphenyl isocyanate (via a Curtius rearrangement [32]),
which was further reacted with XIII in dry toluene to yield the desired compound. Finally, the
synthesis of the desired sulphonamides derivatives IXa
of the key intermediate compounds IV XIII and XXII with a number of arylsulphonyl chlorides
p-methylbenzenesulfonyl chloride, p-nitrobenzenesulfonyl chloride) in dry pyridine as a solvent [33].
,b, XVIIIa,b was carried out by the reaction
,
(
Scheme 2. Synthetic pathways for compounds XV
, XVIIf, XVI–XVIII. Reagents and Conditions:
(
a): Aq. HCl, reflux 2 h, 97.3%, (b): POCl3, reflux 12 h, 73.7%, (c): m-aminobenzoic acid in n-BuOH,
◦
conc HCl, reflux 5 h, 75%, (d): p-phenylenediamine, reflux at 110 C, 5 h, 65%, (e): SOCl in dry
2
benzene, reflux 5 h, 65%, (f): aromatic amines, TEA in dry DCM, reflux 4 h, (g): aromatic acid
chlorides in dry toluene, reflux 2 h, (h): phenyl isocyanates/isothiocyanates in dry toluene, reflux 5 h,
◦
(
i): benzenesulphonyl chlorides in dry pyridine, reflux 2 h, (j): Toluene, 60 C 30 min, (k): Ar-NH , dry
2
toluene, reflux 5 h.
The structures of the newly synthesized compounds were confirmed by elemental analyses and
1
spectral data. H-NMR spectra for the synthesized amides VIa
–c
,
XVa–c and XXIVa,b showed new
signals that appeared downfield in the range
δ (11.93–10.58), (11.90–10.47) and (10.53–10.50) ppm
assignable to the protons of the newly formed amide, respectively, while the carboxylic acid protons
signals disappeared and new aromatic protons appeared in the aromatic range. The spectrum of XVa

Molecules 2019, 24, 1175
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showed one doublet at
δ
7.80 ppm and two additional triplet signals at
δ
7.40, 7.15 ppm. The spectrum
of compound XVb displayed two doublets signals at
doublet signals at 7.70, 6.96 ppm. The data of compound XXIVa exhibited two doublet signals at
7.07, 6.65 ppm. In addition, an extra singlet signal attributed to the methoxy protons appeared for
δ 7.84, 7.45 ppm. Compound XVc showed two
δ
δ
compounds VIc
compound XVa showed one signal at
the amidic carbonyl group (C=O) at 1651–1681 cm in lower frequency confirming the conversion of
,
XVc and XXIVb at
δ
3.77, 3.70 and 3.78 ppm respectively. The 13C-NMR spectrum of
δ 164.92 related to the amide group. The FT-IR spectra displayed
−
1
the acidic carboxylic acid (COOH) carbonyls into amides. Compound XXIVb revealed a new band
−
1
corresponding to aliphatic CH groups at 2920 cm
.
Scheme 3. Synthetic pathways for compounds XXIV and XXV. Conditions and Reagents: (a): Glyoxalic
acid in n-BuOH reflux for 5 h, 76%, (b): POCl , PCl , reflux 4 h, 46%, (c): m-amino- benzoic acid in
3
5
◦
n-BuOH, conc. HCl, reflux 5 h, 75%, (d): p-phenylenediamine, reflux at 110 C, 5 h, 65%, (e): SOCl in
2
dry benzene, reflux, 5 h, 64%, (f): aromatic amines, TEA in dry DCM, reflux, 4 h, (g) m-methoxyphenyl
isocyanate in dry toluene, reflux, 5 h, 61%.
1
On the other hand, the H-NMR spectra of the corresponding amides VIIa
–
d
and XVIa
–
d
showed
an increase in the integration of aromatic protons corresponding to the additional unsubstituted and
substituted phenyl rings. The spectrum of compound VIIb showed additional deshielded aromatic
protons as two doublets at
additional deshielded aromatic protons as two doublets at
respectively, in addition to a new single signal at 3.80 and 3.88 ppm corresponding to methoxy
protons. The spectra of compounds VIId and XVId showed an additional singlet signal at
δ
8.02 and 7.95 ppm. Compounds VIIc and XVIc spectrum showed
δ
(8.00, 7.08 ppm) and (8.20, 7.09 ppm)
δ
δ
1
2.42 and
3
2.40 ppm, corresponding to the three protons of the newly formed methyl group. Also, the C-NMR
spectruma of VIId showed two signals at δ 165.00 and 21.49 ppm related to the newly formed amide
and methyl groups, respectively. The FT-IR spectra displayed the amidic carbonyl group (C=O) at
−
636–1650 cm
1.
1

Molecules 2019, 24, 1175
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Concerning the urea and thiourea final compounds VIIIa
–
e
,
XVIIa
–
f
and XXV, the corresponding
1
H-NMR spectra showed two extra signals of equal integration that appeared downfield corresponding
to two D O exchangeable protons of the urea group in the range of (10.36–8.57), (11.00–8.63) and
2.27, 2.29 ppm appeared corresponding to
the methyl protons for VIIId and XVIId, in addition to a singlet signal at 3.74, 3.68 and 3.86 ppm
attributed to the methoxy protons for VIIIe XVIIe and XVIIf respectively. Elucidation of the structures
of the target sulfonamide derivatives IXa and XVIIIa was also proved by the increase in the
integration of aromatic protons. The spectra of compounds IXa and XVIIIa showed new singlet signals
equivalent to the methyl protons that appeared at 2.32 and 2.37 ppm, respectively. The spectrum
δ
2
(
9.67–9.57) ppm, respectively. An extra singlet signal at δ
δ
,
,
b
,b
δ
of compound IXb showed additional aromatic protons as two doublets at
δ
8.22 and 7.95 ppm.
154.00 and 160.00 ppm,
55.00 ppm related
to the methoxy group of VIIIe. The IR spectra of the sulphonamide compounds showed the two
Interestingly, the 1 C-NMR spectra of VIIIc
3
,
VIIIe showed two signals at
δ
respectively, corresponding to the newly formed urea, in addition to a signal at
δ
−
1
characteristic SO bands at around 1332–1347 and 1159–1165 cm
.
2
2
.2. Biological Evaluation
2
.2.1. In Vitro Cell Proliferation Assay
The targeted compounds were tested for their in-vitro anti-cancer activity against three tumor
cell lines (HCT116, Hep G2 and MCF-7). Doxorubicin was included in the experiments as a reference
cytotoxic compound. Exponentially growing cells from different cancer cell lines were trypsinized,
counted and seeded at the appropriate densities into 96-well microtiter plates. Cells then were
◦
incubated in a humidified atmosphere at 37 C for 24 h. Then, cells were exposed to different
concentrations of compounds for 72 h. Then the viability of treated cells was determined using MTT
technique (described latter).
As shown in Tables 1–4, the different series of synthesized compounds displayed different
anticancer activity with the IC50 values as low as 2.5
VIa XVa and XXIVa bearing a N-(phenyl)-3-(quinoxalin-2-ylamino) benzamide moiety towards
the three cancer cell lines varies significantly. Compound XVa exhibited good anti-cancer activity
against tested cell lines but its activity against HCT116 and MCF-7 was more prominent (IC50
.4, 5.3 M, respectively). Compound XVb exhibited moderate to weak activities against the tested
µM. The anti-proliferative activity for compounds
–
c,
–
c
,b
=
4
µ
cell lines, whereas, the rest of the compounds (VIa, VIb, VIc, XVc, XXIVa and XXIVb) showed no
significant inhibitory activity.
SAR studies for this series of amide derivatives revealed that having a quinoxaline (compounds
XXIVa,b) or methyl quinoxaline (compounds VIa ) as ring A as diminished the activity towards the
–
c
three cell lines. The introduction of a small electron withdrawing group such as a chloro (compounds
XVa,b) into ring A slightly improved the activity. The highest activity was observed, within this series,
when ring B was a phenyl group (compound XVa). The presence of an electron-withdrawing group
in ring B (XVb) slightly decreased the activity, while an electron donating group (compound XVc
)
resulted in an inactive compound, as shown in Table 1.
As for compounds VIIa
moiety, the following results were observed: compound VIId exhibited its optimum inhibitory activity
against the HCT-116 cell line (IC50 = 7.8 M), while it showed moderate activity against the Hep G2
–d and XVIa–d bearing a N-(4-(quinoxalin-2-yl)amino)phenyl) benzamide
µ
cell line and the least activity was observed against the MCF-7 cell line. Compounds VIIa and VIIc
showed moderate to weak activity against the tested cell lines. Compound VIIb showed weak activity
towards the HCT116 cell line and no inhibitory activity towards the Hep G2 and MCF-7 cell lines.
Finally, compounds XVIa–d showed no inhibitory activity.

Molecules 2019, 24, 1175
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Table 1. Results of in-vitro anti-cancer activity of N-(phenyl)-3-(quinoxalin-2-ylamino) benzamide
derivatives screened against three tumor cell lines.
Ring A
R)
Ring B
(X)
HCT116 Cell Line
Hep G2 Cell Line
MCF-7 Cell Line
Compound
(
IC50 (µM)
IC50 (µM)
IC50 (µM)
VIa
VIb
VIc
CH3
CH3
CH3
Cl
H
Cl
OCH3
H
602.5
537
275
4.4
>1000
955
479
692
692
288
5.3
XVa
10
XVb
XVc
XXIVa
XXIVb
Doxorubicin
Cl
Cl
H
H
Cl
OCH3
Cl
15.5
182
219
302
0.62
27.5
912
436.5
490
21.4
380
182
219
0.9
OCH3
1.2
On the contrary to the previously mentioned SAR studies, the second series of amides XVIa
having an electron withdrawing group (chloro) as R showed no significant activity towards the three
cell lines, while an electron donating group such as methyl (compounds VIIa ) exhibited moderate
activity. It was found that among the methyl quinoxaline derivatives the activity increased when ring
B was substituted by an electron donating group (compounds VIIc ) and decreased when substituted
–c
–d
,
d
by an electron withdrawing group (compound VIIb) as shown in Table 2.
Table 2. Results of in-vitro anti-cancer activity of N-(4-(quinoxalin-2-yl)amino)phenyl)benzamide
derivatives screened against three tumor cell lines.
Ring A
R)
Ring B
(X)
HCT116 Cell Line
Hep G2 Cell Line
MCF-7 Cell Line
Compound
(
IC50 (µM)
IC50 (µM)
IC50 (µM)
VIIa
VIIb
VIIc
VIId
XVIa
XVIb
XVIc
CH3
CH3
CH3
CH3
Cl
H
Cl
OCH3
CH3
H
21.9
27.5
33.9
27.5
114.8
19
25.7
138
24
60.3
323.6
>1000
>1000
0.9
7.8
25.7
346.7
>1000
>1000
0.62
512.9
>1000
>1000
1.2
Cl
Cl
Cl
4-OCH3
Doxorubicin
Concerning compounds VIIIa
-ylamino)phenyl)urea and thiourea moieties. Compound VIIIc showed the best anti-cancer activity
against the HCT116 and MCF-7 cell lines (IC50 = 2.5, 9 M, respectively), but it exhibited moderate
activity towards the Hep G2 cell line. Compound VIIIa exhibited good anti-cancer activity against the
–e, XVIIa–f and XXV bearing a 1-(phenyl)-3-(4-(quinoxalin-
2
µ

Molecules 2019, 24, 1175
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Hep G2 cell line (IC50 = 9.8
µM), while it showed moderate activity towards the HCT116 and MCF-7
cell lines. Compound VIIIe showed good anti-cancer activity against the HCT116 cell line (IC50
=
8
.4 µM), but moderate activity towards the Hep G2 and MCF-7cell lines. Compound VIIId possessed
moderate anti-cancer activity against the HCT-116 cell line but weak activity towards the Hep G2
and MCF-7 cell lines. Compound VIIIb showed moderate activity against the MCF-7 cell line, weak
activity against the HCT116 cell line and no significant inhibitory activity towards the Hep G2 cell line.
Compounds XVIIe and XXV showed weak activity towards the tested cell lines. Finally, compounds
XVIIa–d and XVIIf showed no inhibitory activity against the tested cell lines.
Concerning SAR studies for urea and thiourea derivatives, when ring A was a quinoxaline or
chloroquinoxaline the activity ranged from weak to no activity towards the three cell lines, whereas,
the methylquinoxaline derivatives were found to be more active. A methylquinoxaline with a thiourea
derivative and a benzene ring as ring B (compound VIIIa) was the most potent towards Hep G2 and it
lost its activity by 10-fold when converted into the corresponding urea derivative VIIIb. However,
replacement of the benzene ring in ring B of that urea derivative with a p-chlorobenzene (compound
VIIIc) resulted in an improvement of its activity. Interestingly, adding a meta electron- donating group
(compounds VIIId, VIIIe) instead of a para electron withdrawing group (compound VIIIc) slightly
decreased the activity as shown in Table 3.
Table 3. Results of in-vitro anti-cancer activity of 1-phenyl-3-(4-(quinoxalin-2-yl)amino)phenyl)
thiourea and urea derivatives screened against three tumor cell lines.
Ring A
R
Ring B
Y
HCT116 Cell Line
Hep G2 Cell Line
MCF-7 Cell Line
Compound
X
IC50 (µM)
IC50 (µM)
IC50 (µM)
VIIIa
VIIIb
VIIIc
VIIId
VIIIe
XVIIa
XVIIb
XVIIc
XVIId
XVIIe
XVIIf
CH3
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
Cl
S
H
H
4-Cl
3-CH3
3-OCH3
H
H
4-Cl
3-CH3
3-OCH3
4-OCH3
3-OCH3
12.3
38
2.5
11
8.4
309
170
257
190.5
40.7
457
31
9.8
98
22
15.5
13.5
9
O
O
O
O
S
O
O
O
O
O
O
36
30
21.4
575
513
616.5
513
55
724
64.5
1.2
24.5
170
148
692
64.5
47.9
436.5
32
Cl
H
XXV
Doxorubicin
0.62
0.9
Finally, within compounds bearing a N-(4-(quinoxalin-2-yl)amino)phenyl)-substituted benzene
sulfonamide moiety (compounds IXa and XVIIIa ), the methylquinoxaline benzene sulfonamide
derivatives IXa were more potent than that of chloroquinoxalines (compounds XVIIIa ).
,b
,b
,b
,b
Substitution of ring B with a para electron withdrawing group (compound IXb) was more favorable
than substitution with a para electron donating group (compound IXa) as shown in Table 4.
The overall results from the cell lines’ point of view revealed that the three 3-(methylquinoxalin-
2
-yl)amino derivatives VIId
,
VIIIc and VIIIe were active as anti-cancer agents against the HCT116
M) respectively, while only one compound belonging to the
-(chloroquinoxalin-2-yl)amino group (compound XVa) showed marked anti-cancer activity against
cell line (IC50 = 7.8, 2.5 and 8.4
3
µ
the HCT116 cell line (IC50 = 4.4 µM). Derivatives belonging to the unsubstituted quinioxalin-2-yl-amino

Molecules 2019, 24, 1175
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group didn’t exhibit any anti-cancer activity. Regarding the Hep G2 and MCF-7 cell lines, the majority
of the screened compounds did not show any remarkable anti-cancer activity against both cell lines
except compound VIIIa against the Hep G2 cell line (IC50 = 9.8
µM) and both compounds VIIIc and
XVa against the MCF-7 cell line (IC50 = 9.0, 5.3 µM), respectively.
Table 4. Results of in-vitro anti-cancer activity of N-(4-(quinoxalin-2-yl)amino)phenyl)substituted
benzene sulfonamide derivatives screened against three tumor cell lines.
HCT116 Cell Line
Hep G2 Cell Line
MCF-7 Cell Line
Compound
R
X
IC50 (µM)
IC50 (µM)
IC50 (µM)
IXa
IXb
XVIIIa
CH3
CH3
Cl
CH3
NO2
CH3
NO2
21.9
12
302
>1000
0.62
22.9
24.5
489.8
>1000
1.2
22.9
10.23
537
55
XVIIIb
Doxorubicin
Cl
0.9
2
.2.2. In Vitro VEGFR-2 Inhibition Assay
In an attempt to investigate the possible mechanism of action of the synthesized compounds
based on structural similarity with sorafenib, all of them were tested for VEGFR-2 inhibitory activity
at Bio Science Corporation (BPS, San Diego, CA, USA). The assay was performed using the Kinase-Glo
Plus luminescence kinase assay kit (Promega, San Diego, CA, USA). It measures kinase activity by
quantitating the amount of ATP remaining in solution following a kinase reaction. The percent
inhibition of the enzymatic activities caused by our compounds against VEGFR-2 was evaluated
against a reference kinase inhibitor (staurosporine) at a single concentration of 10 µM. Unfortunately,
our compounds showed weak activity against VEGFR-2, where most of the compound’s activity was
between 2% and 21% as illustrated in Table 5.
Table 5. Percentage of inhibition of VEGFR-2 with 10 µM of the synthesized compounds.
%
of VEGFR-2
Inhibition
% of VEGFR-2
Inhibition
% of VEGFR-2
Inhibition
Compounds
Compounds
Compounds
VIIa
XVIa
VIIb
XVIb
VIIc
XVIc
VIId
XVId
IXa
2
8
4
4
6
4
3
3
3
4
3
9
VIIIa
XVIIa
VIIIb
XVIIb
XVIIf
VIIIc
XVIIc
VIIId
XVIId
VIIIe
XVIIe
XVa
7
10
4
10
6
7
6
5
5
VIa
XVb
XVc
7
7
5
5
9
3
21
100
VIc
XXIVb
XXIVa
XXV
Staurosporine
XVIIIa
IXb
XVIIIb
6
8
6
2
.2.3. Cell Cycle Analysis
In another attempt to study the effect of the synthesized quinoxalines that exhibited potent
antiproliferative activity against the three NCI cell lines (HCT116, Hep G2 and MCF-7), on cell cycle

Molecules 2019, 24, 1175
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progression and induction of apoptosis, compound VIIIc was selected as being the most active
compound for this study on the human colon carcinoma cell line HCT116. The effect of compound
VIIIc on the normal cell cycle progression was characterized using flow cytometric analysis of the
DNA ploidy in HCT116 cells Figure 3. Exposure of HCT116 cells to VIIIc at IC50 concentration (2.5 µM)
for 24 h resulted in an increase in G1 phase and after 48 h a significant disruption in cell cycle profile
and cell cycle arrest at G2/M phase boundary with concurrent time dependent increase in pre-G cell
population Figure 4. The observed increase in pre-G cell population may imply DNA fragmentation
and apoptosis as a potential mechanism for VIIIc-induced cancer cell death.
Figure 3. The effect of compound VIIIc on the normal cell cycle progression in HCT116 cells.
Figure 4. Exposure of HCT116 cells to VIIIc at GI50 concentration (0.36 µM) for 24 h and 48 h.
2
.2.4. Apoptosis Determination
For further investigation of the ability of compound VIIIc to induce apoptosis, an Annexin V
(
conjugated to FITC) apoptosis detection kit was employed. This assay detects phosphatidylserine (PS)
expressed on the surface of apoptotic cells and fluoresces green after interacting with the labeled
annexin V. During early apoptosis, membrane asymmetry is lost, and PS translocates from the
cytoplasmic side of the membrane to the external leaflet. Propidium iodide (PI), the counterstain
used in this assay, has the ability to cross only compromised membranes to intercalate into the DNA.
Therefore, PI is used to detect the late stages of apoptosis by presence of red fluorescence. Exposure
of HCT116 cells to VIIIc at its IC50 (2.5 µM) for 24 h and 48 h increased the percentage of annexin-V
positive cells indicating an early (lower right quadrant) or late (upper right quadrant) apoptosis in
a time dependent manner compared to dimethylsulphoxide (DMSO) treated cells Figure 5.

Molecules 2019, 24, 1175
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Figure 5. Exposure of HCT116 cells to VIIIc at its IC50 (0.36 µM) for 24 h and 48 h increased the
percentage of annexin-V positive cells indicating an early (lower right quadrant) or late (upper
right quadrant) apoptosis in a time dependent manner compared to dimethylsulphoxide (DMSO)
treated cells.
2
.2.5. In Vitro Cytotoxic Assay
In an attempt to investigate the cytotoxic potential of our designed compounds, MTT cytotoxicity
assay was performed for the most active compounds VIId
fibroblast-like fetal lung cell line (WI-38) using staurosporine as a reference compound. The results
revealed that compounds XVa VIIIa were highly selective anti-cancer agents having IC50 of (163.6,
26.19 M) respectively. Compounds VIIIc and VIIIe showed moderate selectivity having IC50 of
97.2, 96.4 M) respectively. Lastly, compound VIId exhibited low selectivity having IC50 of (40.13 M)
as shown in Table 6.
, VIIIa, VIIIc, VIIIe and XVa on Caucasian
,
1
µ
(
µ
µ
Table 6. Values of IC50 for the most active compounds on WI-38 cell line.
Compound
IC50 (µM)
XVa
VIIIa
VIIIc
VIId
VIIIe
163.6 ± 9.2
126.19 ± 6.81
97.2 ± 5.66
40.13 ± 3.42
96.4 ± 5.72
staurosporine
101.86 ± 6.34
3
. Materials and Methods
3
.1. General Information
The reactions were monitored and the purities of the compounds were checked by ascending thin
layer chromatography (TLC) on silica gel-coated aluminum plates (60 F254, 0.25 mm, Merck, Darmstadt,
Germany) using mixture of chloroform and methanol (1:9) and the spots were visualized under ultra
violet light at 254 and 366 nm. Melting points were determined in open capillaries using a Stuart
(Biocote, Staffordshire, ST15 OSA, UK) scientific melting point apparatus without correction. EI-MS
spectra were recorded on a Finnigan Mat SSQ 7000 (70 ev) mass spectrometer (Ringoes, NJ, USA)
1
at Regional Center for Mycology and Biotechnology at El Azhar University. H-NMR spectra were
recorded on the
δ
scale given in ppm on a Bruker 400 MHz spectrophotometer (Billerica, MA, USA)
) were expressed in parts per million
ppm) and coupling constants (J) were expressed in Hertz (Hz). The signals were designated as follows:
s, singlet; d, doublet; t, triplet; m, multiplet at Microanalytical Center unit FOPCU at Cairo University.
referred to TMS using DMSO-d as solvent. Chemical shifts (
δ
6
(

Molecules 2019, 24, 1175
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FT-IR spectra were determined (KBr) using a IR-435 (Shimadzu, Kyoto, Japan) or FT-IR 1650 (Perkin
Elmer, Waltham, MA, USA) spectrometer at the Faculty of Pharmacy, Cairo University. Elemental
analyses were performed at Regional Center for Mycology and Biotechnology at El-Azhar University.
The synthesis of the starting compounds
method [20–24].
I, X and XIX according to the previously reported
3
.2. Chemistry
3
.2.1. General Procedures for the Synthesis of Compounds VIa–c, XVa–c and XXIVa,b
A mixture of intermediates III XII and XXI (1.8 mmol) and thionyl chloride (SOCl , 15 mL) was
stirred under reflux for 5 h. The mixture was then evaporated under reduced pressure, yielding acid
chlorides XIV and XXIII 28]. The title amides were afforded by drop wise addition of a solution of
,
2
V
,
[
the corresponding acid chloride (1.5 mmol) in methylene chloride (5 mL) to a hot solution of excess
aromatic amines (aniline, p-chloroaniline, p-methoxyaniline) in DCM (5 mL). The mixture was left to
reflux for 4–6 h till precipitation occurs. The precipitate was filtered while hot, washed with DCM and
crystallized from EtOH to afford the title compounds [29].
3
.2.2. 3-((3-Methylquinoxalin-2-yl)amino)-N-phenylbenzamide (VIa)
◦
1
Brown solid (yield = 65%); m.p. 250–252 C; H-NMR (DMSO-d ) δ: 11.93 (s, 1H, NH amide),
6
−
0.53 (s, 1H, NH), 8.39–7.19 (m, 13H, Ar-H), 2.50 (s, 3H, CH ). FT-IR (ύ max, cm ): 3356 (2 NH), 2993
3
CH aliphatic), 1666 (C=O amide). MS (MWt: 354.4): m/z, 354.4 [M , (8.16%)], 45.68 (100%); Anal.
1
1
(
+
Calcd for C H N O: C, 74.56; H, 5.12; N, 15.81. Found: C, 71.82; H, 5.18; N, 15.97.
22
18
4
3
.2.3. N-(4-Chlorophenyl)-3-((3-methylquinoxalin-2-yl)amino)benzamide (VIb)
◦
1
Dark green (yield = 60%); m.p. 240–241 C; H-NMR (DMSO-d ) δ: 11.19 (s, 1H, NH amide), 10.47
6
−
s, 1H, NH), 8.54–7.41 (m, 12H, Ar-H), 2.59 (s, 3H, CH ). FT-IR (ύ max, cm ): 3421 (2 NH), 2923 (CH
3
aliphatic), 1652 (C=O amide). MS (MWt: 388.85): m/z, 390 [M +2, (4.45%)], 388 [M , (0.97%)], 264.03,
1
(
+
+
(
100%)]. Anal. Calcd for C H ClN O: C, 67.95; H, 4.41; N, 14.41. Found: C, 68.09; H, 4.45; N, 14.63.
22 17 4
3
.2.4. N-(4-Methoxyphenyl)-3-((3-methylquinoxalin-2-yl)amino)benzamide (VIc)
◦
1
Brown solid (yield = 69%); m.p. 255–256 C; H-NMR (DMSO-d )
δ: 10.58 (s, 1H, NH amide), 9.96
6
(
(
3
s, 1H, NH), 8.38 (s, 1H, Ar-H), 8.31-6.82 (m, 11H, Ar-H), 3.77 (s, 3H, OCH ), 2.51 (s, 3H, CH ). FT-IR
3
3
−
1
ύ
max, cm ): 3394 (2 NH), 3055 (CH aromatic), 2997 (CH aliphatic), 1651 (C=O amide). MS (MWt:
+
84.43): m/z, 384.07 [M , (5.51%)], 320.13, (100%)]; Anal calcd for C H N O : C, 71.86; H, 5.24; N,
23
20
4
2
1
4.57. Found: C, 72.04; H, 5.32; N, 14.72.
3
.2.5. 3-((3-Chloroquinoxalin-2-yl)amino)-N-phenylbenzamide (XVa)
◦
1
Creamy white solid (yield = 71%); m.p. > 300 C; H-NMR (DMSO-d )
δ: 10.47 (s, 1H, NH amide),
6
9.43 (s, 1H, NH), 8.42 (m, 1H, Ar-H), 8.32 (s, 1H, Ar-H), 8.18 (d, J = 8 Hz, 1H, Ar-H), 8.02 (d, J = 8 Hz,
1
H, Ar-H), 7.87 (t, 1H, Ar-H), 7.80 (d, J = 8 Hz, 2H, Ar-H), 7.70 (d, J = 8 Hz, 1H, Ar-H), 7.62–7.55 (m,
13
1H, Ar-H), 7.40 (t, 1H, Ar-H), 7.34 (m, 2H, Ar-H), 7.15 (t, 1H, Ar-H); C-NMR (DMSO-d6)
δ: 164.92,
1
42.04, 139.37, 137.40, 134.05, 131.02, 130.76, 129.16, 128.58, 127.69, 127.45, 127.12, 124.45, 121.01, 120.91;
−
1
+
FT-IR (
ύ
max, cm ): 3433,3414 (2 NH), 1678 (C=O amide); MS (MWt: 374.82): m/z, 376.47 [M + 2,
+
(1.64%)], 374.99 [M , (2.28%)], 113. (100%); Anal. Calcd for C H ClN O: C, 67.29; H, 4.03; N, 14.95.
21
15
4
Found: C, 67.48; H, 4.02; N, 15.13.
3
.2.6. N-(4-Chlorophenyl)-3-((3-chloroquinoxalin-2-yl)amino)benzamide (XVb)
◦
1
Creamy white solid (yield = 60%); m.p. 283–285 C; H-NMR (DMSO-d )
δ
: 10.60 (s, 1H, NH
amide), 10.50 (s, 1H, NH), 8.31 (s, 1H, Ar-H), 8.18 (d, J = 8 Hz, 1H, Ar-H), 8.03 (d, J = 8 Hz, 1H, Ar-H),
.87–7.85 (m, 1H, Ar-H), 7.84 (d, J = 8 Hz, 2H, Ar-H), 7.80–7.78 (m, 2H, Ar-H), 7.57–7.55 (m, 2H, Ar-H),
6
7

Molecules 2019, 24, 1175
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max, cm 1): 3336 (2 NH), 3062 (CH aromatic), 1681 (C=O amide);
−
7
.45 (d, J = 8 Hz, 2H, Ar-H); FT-IR (
ύ
+
+
MS (MWt: 409.27): m/z, 413.85 [M + 4, (0.53%)], 409.20 [M , (27.06%)], 98.03(100%); Anal. Calcd for
C H Cl N O: C, 61.63; H, 3.45; N, 13.69. Found: C, 61.58; H, 3.42; N, 13.87.
21
14
2
4
3
.2.7. 3-((3-Chloroquinoxalin-2-yl)amino)-N-(4-methoxyphenyl)benzamide (XVc)
◦
1
Creamy white (yield = 70%); m.p. 240–241 C; H-NMR (DMSO-d )
δ
: 11.90 (s, 1H, NH amide),
0.38 (s, 1H, NH), 8.30 (s, 1H, Ar-H), 8.10 (d, J = 8 Hz, 1H, Ar-H), 7.90 (d, J = 8 Hz, 1H, Ar-H), 7.83 (d,
J = 8 Hz, 1H, Ar-H), 7.80 (m, 1H, Ar-H), 7.70 (d, J = 8 Hz, 2H, Ar-H), 7.50 (m, 1H, Ar-H), 7.20–7.00 (m,
6
1
−
1
2
H, Ar-H), 6.96 (d, J = 8 Hz, 2H, Ar-H), 3.70 (s, 3H, OCH ); FT-IR (
CH aromatic), 2935 (CH aliphatic), 1678 (C=O amide); MS (MWt: 404.85): m/z, 404.36 [M , (5%)] 44
100%); Anal. Calcd for C H ClN O : C, 65.27; H, 4.23; N, 13.84. Found: C, 65.54; H, 4.28; N, 14.01.
ύ
max, cm ): 3356 (2 NH), 3055
3
+
(
(
22
17
4
2
3
.2.8. N-(4-chlorophenyl)-3-(quinoxalin-2-ylamino)benzamide (XXIVa)
◦
1
Buff solid (yield = 75%); m.p. > 300 C; H-NMR (DMSO-d )
δ: 10.53 (s, 1H, NH amide), 10.40 (s,
6
1
H, NH), 8.38 (s, 1H, Ar-H), 8.04 (d, J = 8 Hz, 1H, Ar-H), 7.74–7.15 (m, 5H, Ar-H), 7.07 (d, J = 8 Hz, 2H,
−
1
Ar-H), 6.86 (m, 2H, Ar-H), 6.65 (d, J = 8 Hz, 2H, Ar-H); FT-IR (
amide); MS (MWt:374.8): m/z, 374.21 [M , (7.82%)], 84.22 (100%); Anal. Calcd for C H ClN O: C,
ύ
max, cm ): 3282 (2 NH), 1681 (C=O
+
21
15
4
6
7.29; H, 4.03; N, 14.95. Found: C, 67.44; H, 4.09; N, 15.08.
3
.2.9. N-(4-Methoxyphenyl)-3-(quinoxalin-2-ylamino)benzamide (XXIVb)
◦
1
Dark brown solid (yield = 79%); m.p. > 300 C; H-NMR (DMSO-d )
δ
: 10.50 (s, 1H, NH amide),
6
9
6
.92 (s, 1H, NH), 8.38 (s, 1H, Ar-H), 8.30 (t, 1H, Ar-H), 7.74-7.51 (m, 5H, Ar-H), 7.13–6.72 (m, 4H, Ar-H),
−
1
.65 (d, J = 8 Hz, 1H, Ar-H), 6.52 (d, J = 8 Hz, 1H, Ar-H), 3.78 (s, 3H, OCH ); FT-IR (
ύ
max, cm ): 3367
3
+
(
2 NH), 3066 (CH aromatic), 2920 (CH aliphatic), 1651 (C=O amide); MS (MWt: 370.4): m/z, 371 [M +
+
H, (1.16%)], 370 [M , (2%)], 119.98 (100%); Anal. Calcd for C H N O : C, 71.34; H, 4.90; N, 15.13.
22
18
4
2
Found: C, 71.48; H, 4.97; N, 15.36.
3
.2.10. General Procedures for the Synthesis of Compounds VIIa–d and XVIa–d
The title compounds were afforded by dropwise addition of the appropriate benzoyl chlorides
(
1.2 mmol; 1 eq) (viz; benzoyl chloride, p-chlorobenzoyl chloride, p-methoxybenzoyl chloride,
p-tolyl-benzoyl chloride) to a hot solution of IV or XIII (1.2 mmol) in 5 mL of dry diethyl ether
compound VIIa XVIa) or 5 mL of dry toluene (compounds VIIb XVIb ). The mixture was left
(
,
–d
,
–d
to reflux for 2–4 h till precipitation occured, then the precipitate was filtered while hot, washed with
toluene and crystallized from EtOH to afford the title compounds [29].
3
.2.11. N-(4-((3-Methylquinoxalin-2-yl)amino)phenyl)benzamide (VIIa)
◦
1
Dark brown solid (yield = 70%); m.p. 210–212 C; H-NMR (DMSO-d ) δ: 10.32 (s, 1H, NH
6
−
1
amide), 8.97 (s, 1H, NH), 8.07–7.39 (m, 13H, Ar-H), 2.75 (s, 3H, CH ); FT-IR (
057 (CH aromatic), 2995 (CH aliphatic), 1645 (C=O amide); MS (MWt.: 354.4): m/z, 355.12 [M + H,
13.88%)], 354.12 [M , (57.34%)], 105.05 (100%); Anal. calcd for C H N O: C, 74.56; H, 5.12; N, 15.81.
ύ
max, cm ): 3334 (2 NH),
3
+
3
(
+
22 18 4
Found: C, 74.58; H, 5.19; N, 16.02.
3
.2.12. 4-Chloro-N-(4-((3-methylquinoxalin-2-yl)amino)phenyl)benzamide (VIIb)
◦
1
Golden brown solid (yield = 64%); m.p. 234–235 C; H-NMR (DMSO-d ) δ: 10.31 (s, 1H, NH
6
amide), 8.66 (s, 1H, NH), 8.02 (d, J = 8 Hz, 2H, Ar-H), 7.95 (d, J = 8 Hz, 2H, Ar-H), 7.80 (d, J = 8 Hz, 1H,
Ar-H), 7.77 (d, J = 8 Hz, 2H, Ar-H), 7.68 (d, J = 8 Hz, 1H, Ar-H), 7.63 (d, J = 8 Hz, 2H, Ar-H), 7.58 (t,
−
1
1
1
H, Ar-H), 7.44 (t, 1H, Ar-H), 2.73 (s, 3H, CH ); FT-IR (
ύ
+
max, cm ): 3441 (2 NH), 2991 (CH aliphatic),
3
+
647 (C=O amide); MS (MWt: 388.8): m/z, 390.18 [M + 2, (18.13%)], 388.17 [M , (52%)], 139 (100%);
Anal. calcd for C H ClN O: C, 67.95; H, 4.41; N, 14.41. Found: C, 68.12; H, 4.46; N, 14.59.
22
17
4

Molecules 2019, 24, 1175
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3
.2.13. 4-Methoxy-N-(4-((3-methylquinoxalin-2-yl)amino)phenyl)benzamide (VIIc)
◦
1
Greyish-brown solid (yield = 76%); m.p. 230–231 C; H-NMR (DMSO-d )
δ: 10.58 (s, 1H, NH
6
amide), 8.99 (s, 1H, NH), 8.00 (d, J = 8 Hz, 2H, Ar-H), 7.84 (d, J = 8 Hz, 2H, Ar-H), 7.80 (d, J = 8 Hz, 1H,
Ar-H), 7.73 (d, J = 8 Hz, 2H, Ar-H), 7.61 (t, 1H, Ar-H), 7.48 (t, 1H, Ar-H), 7.08 (d, J = 8 Hz, 2H, Ar-H),
−
1
7
3
(
.00 (d, J = 8 Hz, 1H, Ar-H), 3.80 (s, 3H, OCH ), 2.76 (s, 3H, CH ); FT-IR (
ύ
max, cm ): 3392 (2 NH),
3
3
+
032 (CH aromatic), 2935 (CH aliphatic), 1639 (C=O amide); MS (MWt: 384.4): m/z, 385.19 [M + H,
3.74%)], 384.18 [M , (14.7%)], 40.16 (100%); Anal. Calcd for C H N O : C, 71.86; H, 5.24; N, 14.57.
+
23
20
4
2
Found: C, 72.04; H, 5.31; N, 14.71.
3
.2.14. 4-Methyl-N-(4-((3-methylquinoxalin-2-yl)amino)phenyl)benzamide (VIId)
◦
1
Black solid (yield = 68%); m.p. 220–222 C; H-NMR (DMSO-d )
δ: 10.15 (s, 1H, NH amide),
6
9
.90 (s, 1H, NH), 8.05 (m, 2H, Ar-H), 7.89 (d, J = 8 Hz, 2H, Ar-H), 7.64–7.52 (m, 2H, Ar-H), 7.46–7.43
13
(
m, 1H, Ar-H), 7.41–7.30 (m, 5H, Ar-H), 2.50 (s, 3H, CH ), 2.42 (s, 3H, CH ); C-NMR (DMSO-d6) δ:
3 3
1
65.55, 148.10, 141.94, 136.00, 135.00, 132.00, 129.72, 129.37, 128.15, 127.67, 122.39, 121.17, 22.10, 21.49;
−
1
FT-IR (
ύ
max, cm ): 3340 (2 NH), 3032 (CH aromatic), 2916 (CH aliphatic), 1647 (C=O amide); MS:
+
+
(MWt: 368.43): m/z, 369.16 [M + H, (3.17%)], 368.15 [M , (10.27%)], 118.98 (100%); Anal. calcd for
C H N O: C, 74.98; H, 5.47; N, 15.21. Found: C, 75.19; H, 5.55; N, 15.34.
23
20
4
3
.2.15. N-(4-((3-Chloroquinoxalin-2-yl)amino)phenyl) benzamide (XVIa)
◦
1
Light brown solid (yield = 72%); m.p. 270–273 C; H-NMR (DMSO-d )
δ
: 10.29 (s, 1H, NH
6
−
1
amide), 9.37 (s, 1H, NH), 8.44–7.24 (m, 13H, Ar-H); FT-IR (
amide); MS (MWt: 374.82): m/z, 374.25 [M , (4.60%)], 57.07 (100%); Anal. Calcd for C H ClN O: C,
ύ max, cm ): 3421 (2 NH), 1636 (C=O
+
21
15
4
6
7.29; H, 4.03; N, 14.95. Found: C, 67.47; H, 4.11; N, 15.23.
3
.2.16. 4-Chloro-N-(4-((3-chloroquinoxalin-2-yl)amino)phenyl) benzamide (XVIb)
◦
1
Orange solid (yield = 66%); m.p. 233–235 C; H-NMR (DMSO-d )
δ: 10.35 (s, 1H, NH amide),
6
9
7
.61 (s, 1H, NH), 8.04 (d, J = 8 Hz, 4H, Ar-H), 7.81 (d, J = 8 Hz, 2H, Ar-H), 7.64 (d, J = 8 Hz, 2H, Ar-H),
−
1
.60–7.56 (m, 2H, Ar-H), 7.33 (m, 2H, Ar-H); FT-IR (ύ max, cm ): 3320 (2 NH), 1650 (C=O amide); MS
+
+
(MWt: 409.3): m/z, 410 [M + H, (2%)], 409.26[M , (2.8%)], 135 (100%); Anal. Calcd for C H C N O:
21 14 l2 4
C, 61.63; H, 3.45; N, 13.69. Found: C, 61.94; H, 3.5; N,13.78.
3
.2.17. N-(4-((3-Chloroquinoxalin-2-yl)amino)phenyl)-4-methoxybenzamide (XVIc)
◦
1
Orange solid (yield = 66%); m.p. 235–236 C; H-NMR (DMSO-d ) δ: 10.15 (s, 1H, NH amide),
6
9
7
.72 (s, 1H, NH), 8.20 (d, J = 7 Hz, 2H, Ar-H), 7.94–7.92 (m, 2H, Ar-H), 7.84 (d, J = 8 Hz, 2H, Ar-H),
.64–7.57 (m, 2H, Ar-H), 7.37–7.31 (m, 2H, Ar-H),7.09 (d, J = 8 Hz, 2H, Ar-H), 3.88 (s, 3H, OCH ); FT-IR
3
−
1
(ύ
max, cm ): 3275 (2 NH), 3062 (CH aromatic), 2958 (CH aliphatic), 1643 (C=O amide); MS (MWt:
+
4
04.85): m/z, 406.01 [M + 2, (2.45%)], 43.96 (100%); Anal. Calcd for C H ClN O : C, 65.27; H, 4.23;
22
17
4
2
N, 13.84. Found: C, 65.42; H, 4.27; N, 13.96.
3
.2.18. N-(4-((3-Chloroquinoxalin-2-yl)amino)phenyl)-4-methylbenzamide (XVId)
◦
1
Orange solid (yield = 69%); m.p. 245–246 C; H-NMR (DMSO-d )
δ: 10.21 (s, 1H, NH amide),
6
9
.89 (s, 1H, NH), 8.02–7.83 (m, 6H, Ar-H), 7.64–7.57 (m, 2H, Ar-H), 7.36 (d, J = 8 Hz, 4H, Ar-H), 2.40
−
s, 3H, CH ); FT-IR (ύ max, cm ): 3307,3228 (2NH), 3059 (CH aromatic), 2954 (CH aliphatic), 1637
3
1
(
(
+
+
C=O amide); MS (MWt: 388.8): m/z, 89.14 [M + H, (3.9%)], 388.15 [M , (5.79%)], 277.13 (100%); Anal.
Calcd for C H ClN O: C, 67.95; H, 4.41; N, 14.41. Found: C, 68.17; H, 4.49; N, 14.68.
22
17
4
3
.2.19. General Procedures for the Synthesis of Compounds VIIIa–e, XVIIa–f and XXV
The title compounds were afforded by dropwise addition of phenyl isothiocyanate (compounds
VIIIa and XVIIa) or the appropriate phenyl isocyanate (compounds VIIIb–e and XVIIb–e, 1.2 mmol;

Molecules 2019, 24, 1175
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1
eq) (viz; phenyl isocyanate, p-chloro-, m-methyl- and m-methoxyphenyl isocyanate) to a hot solution
of IV, XIII or XXII (1.2 mmol) in dry toluene (5 mL). The mixture was refluxed for 3–5 h till precipitation
occurs. The precipitate was filtered while hot, washed with toluene and crystallized from EtOH to
afford the title compounds [31].
3
.2.20. 1-(4-((3-Methylquinoxalin-2-yl)amino)phenyl)-3-phenylthiourea (VIIIa)
◦
1
Olive green solid (yield = 76%); m.p. 175–176 C; H-NMR (DMSO-d )
δ: 10.36 (s, 1H, NH
6
thiourea), 10.03 (s, 1H, NH thiourea), 8.90 (s, 1H, NH), 8.00–6.82 (m, 13H, Ar-H), 2.70 (s, 3H, CH );
3
−
1
FT-IR (
(
ύ
max, cm ): 3354 (3 NH), 3053 (CH aromatic), 2950 (CH aliphatic), 1219 (C=S amide); MS
MWt: 385.5): m/z, 385 [M , (1%)], 93.05 (100%); Anal. calcd for C H N S: C, 68.55; H, 4.97; N, 18.17.
+
22
19
5
Found: C, 68.69; H, 5.03; N, 18.42.
3
.2.21. 1-(4-((3-Methylquinoxalin-2-yl)amino)phenyl)-3-phenylurea (VIIIb)
◦
1
Brown solid (yield = 68%); m.p. > 300 C; H-NMR (DMSO-d ) δ: 9.78 (s, 1H, NH urea), 9.68 (s,
6
−
1
1
H, NH urea), 8.65 (s, 1H, NH), 7.95–7.11 (m, 13H, Ar-H), 2.72 (s, 3H, CH ); FT-IR (
ύ
max, cm ): 3356
3
(
[
3 NH), 3055 (CH aromatic), 2993 (CH aliphatic), 1640 (C=O amide); MS (MWt.: 369.42): m/z, 370.28
M + H, (5.17)],369.29 [M , (7.41%) ], 40.36 (100%); Anal. calcd for C H N O: C, 71.53; H, 5.18; N,
22 19 5
+
+
1
8.96. Found: C, 71.80; H, 5.26; N, 19.18.
.2.22. 1-(4-Chlorophenyl)-3-(4-((3-methylquinoxalin-2-yl)amino)phenyl)urea (VIIIc)
3
8
◦
1
Greyish-brown solid (yield = 79%); m.p. 248–249 C; H-NMR (DMSO-d )
δ: 8.85 (s, 1H, NH urea),
6
.65 (s, 1H, NH urea), 8.57 (s, 1H, NH), 7.86 (d, J = 8 Hz, 2H, Ar-H), 7.79 (d, J = 8 Hz, 1H, Ar-H), 7.64 (d,
J = 8 Hz, 1H, Ar-H), 7.56 (t, 1H, Ar-H), 7.53 (d, J = 3.2 Hz, 2H, Ar-H), 7.49 (d, J = 8 Hz, 2H, Ar-H), 7.43
t, 1H, Ar-H), 7.34 (d, J = 8 Hz, 2H, Ar-H), 2.70 (s, 3H, CH3); 1 C-NMR (DMSO-d6)
3
δ: 154.00, 149.00,
(
1
1
4
47.50, 139.00, 138.80, 137.00, 135.00, 129.50, 129.00, 128.00, 127.50, 127.00, 126.00, 125.00, 123.00, 120.00,
−
1
17.70, 22.00; FT-IR (
03.86): m/z, 405.04 [M + 2,(1.28%) ], 403.89 [M , (0.9%)], 89.99 (100%); Anal. calcd for C H ClN O:
ύ max, cm ): 3464 (3 NH), 3059 (CH aromatic), 1635 (C=O amide); MS (MWt.:
+
+
22
18
5
C, 65.43; H, 4.49; N, 17.3. Found: C, 65.58; H, 4.57; N, 17.51.
3
.2.23. 1-(4-((3-Methylquinoxalin-2-yl)amino)phenyl)-3-(m-tolyl)urea (VIIId)
◦
1
Black solid (yield = 65%); m.p. 280–281 C; H-NMR (DMSO-d )
δ
: 9.18 (s, 1H, NH urea), 8.57
s, 1H, NH urea), 8.56 (s, 1H, NH), 7.85 (d, J = 8 Hz, 1H, Ar-H), 7.71–7.46 (m, 4H, Ar-H), 7.35 (s, 1H,
Ar-H), 7.31 (m, 1H, Ar-H), 7.23 (d, J = 8 Hz, 2H, Ar-H), 7.14 (t, 1H, Ar-H), 6.78 (d, J = 8 Hz, 2H, Ar-H),
6
(
−
.49 (s, 3H, CH ), 2.27 (s, 3H, CH ); FT-IR (ύ max, cm ): 3456 (3 NH), 3035 (CH aromatic), 2958 (CH
3 3
aliphatic), 1639 (C=O amide); MS (MWt: 383.45): m/z, 384.12 [M + H, (3.19%)], 383.12 [M , (12.44%)],
1
2
+
+
2
49.08 (100%); Anal. calcd for C H N O: C, 72.04; H, 5.52; N, 18.26. Found: C, 72.23; H, 5.61; N, 18.42.
23 21 5
3
.2.24. 1-(3-Methoxyphenyl)-3-(4-((3-methylquinoxalin-2-yl)amino)phenyl)urea (VIIIe)
◦
1
Black solid (yield = 79%); m.p. 228–230 C; H-NMR (DMSO-d )
δ: 8.68 (s, 1H, NH urea), 8.60
6
(s, 1H, NH urea), 8.57 (s, 1H, NH), 7.86 (d, J = 8 Hz, 2H, Ar-H), 7.79 (d, J = 8 Hz, 1H, Ar-H), 7.64 (d,
J = 8 Hz, 1H, Ar-H),7.53 (t, 1H, Ar-H), 7.47 (d, J = 8 Hz, 2H, Ar-H), 7.42 (t, 1H, Ar-H), 7.38 (s, 1H,
Ar-H), 7.22–7.20 (m, 1H, Ar-H), 7.18 (d, J = 8 Hz, 1H, Ar-H), 6.95 (d, J = 8 Hz, 1H, Ar-H), 3.74 (s, 3H,
13
OCH ), 2.71 (s, 3H, CH ); C-NMR (DMSO-d6)
δ: 160.00, 153.00, 149.00, 146.00, 142.00, 141.00, 137.00,
3
3
1
36.00, 135.00, 130.00, 129.00, 127.00, 126.00, 125.00, 123.00, 118.00, 111.00, 109.00, 105.00, 55.00, 22.00;
−
1
FT-IR (
ύ
max, cm ): 3298 (3 NH), 3059 (CH aromatic), 2935 (CH aliphatic),1639 (C=O amide); MS
+
+
(MWt: 399.45): m/z, 400.85 [M + H, (2.88%) ], 399.24 [M , (11.49%) ], 54.07 (100%); Anal. calcd for
C H N O : C, 69.16; H, 5.30; N, 17.53. Found: C, 69.41; H, 5.37; N, 17.65.
23
21
5
2

Molecules 2019, 24, 1175
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3
.2.25. 1-(4-((3-Chloroquinoxalin-2-yl)amino)phenyl)-3-phenylthiourea (XVIIa)
◦
1
Brown solid (yield = 62%); m.p. 190–191 C; H-NMR (DMSO-d )
δ
: 11.00 (s, 1H, NH thiourea),
6
1
0.60 (s, 1H, NH thiourea), 9.90 (s, 1H, NH), 8.04–7.90 (m, 3H, Ar-H), 7.60–7.50 (m, 4H, Ar-H), 7.40–7.30
−
1
(
m, 5H, Ar-H), 7.29–7.15 (m, 1H, Ar-H); FT-IR (
ύ
max, cm ): 3371 (3 NH), 3059 (CH aromatic), 1238
+
+
(
C=S amide); MS (MWt: 405.9): m/z, 407.11 [M + 2, (3.9%)], 405.07 [M , (4.2%)], 45.04 (100%); Anal.
Calcd for C H ClN S: C, 62.14; H, 3.97; N, 17.25. Found: C, 62.37; H, 4.03; N, 17.47.
21
16
5
3
.2.26. 1-(4-((3-Chloroquinoxalin-2-yl)amino)phenyl)-3-phenylurea (XVIIb)
◦
1
Brown solid (yield = 58%); m.p. 280–281 C; H-NMR (DMSO-d )
δ: 11.00 (s, 1H, NH urea), 10.60
6
(
7
1
s, 1H, NH urea), 9.90 (s, 1H, NH), 8.10 (m, 1H, Ar-H), 8.03–7.94 (m, 3H, Ar-H), 7.60–7.50 (m, 4H, Ar-H),
−
1
.42–7.30 (m, 4H, Ar-H), 7.15 (m, 1H, Ar-H). FT-IR (
ύ
max, cm ): 3302 (3 NH), 3059 (CH aromatic),
+
+
635 (C=O amide); MS (MWt: 389.84): m/z, 391.25 [M + 2, (4.94%)], 389.46 [M , (6.21%)], 55.19 (100%);
Anal. Calcd for: C H ClN O: C, 64.70; H, 4.14; N, 17.96. Found: C, 64.86; H, 4.17; N, 18.13.
21
16
5
3
.2.27. 1-(4-Chlorophenyl)-3-(4-((3-chloroquinoxalin-2-yl)amino)phenyl)urea (XVIIc)
◦
1
Olive green solid (yield = 63%); m.p.> 300 C; H-NMR (DMSO-d )
δ: 9.70 (s, 1H, NH urea), 9.50
6
(
s, 1H, NH urea), 9.01 (s, 1H, NH), 8.00–7.90 (m, 1H, Ar-H), 7.59–7.47 (m, 5H, Ar-H), 7.39–7.31 (m, 6H,
−
1
Ar-H); FT-IR (
ύ
max, cm ): 3294 (3 NH), 3066 (CH aromatic), 1635 (C=O amide); MS (MWt: 424.28):
+
+
m/z, 428.32 [M + 4, (0.7%)], 424.14 [M , (2.59%)], 60.07 (100%); Anal. Calcd for C H C N O: C,
21
15 l2
5
5
9.45; H, 3.56; N, 16.51. Found: C, 59.68; H, 3.54; N, 16.79.
3
.2.28. 1-(4-((3-Chloroquinoxalin-2-yl)amino)phenyl)-3-(m-tolyl) urea (XVIId)
◦
1
Dark brown solid (yield = 77%); m.p. > 300 C; H-NMR (DMSO-d )
δ
: 9.05 (s, 1H, NH urea),
6
8.63 (s, 1H, NH urea), 8.58 (s, 1H, NH), 7.96 (m, 2H, Ar-H), 7.84–7.79 (m, 2H, Ar-H), 7.58–7.53 (m, 1H,
Ar-H), 7.51 (d, J = 8 Hz, 2H, Ar-H), 7.32 (s, 1H, Ar-H), 7.27 (d, J = 8 Hz, 2H, Ar-H), 7.17 (t, 1H, Ar-H),
−
.80 (d, J = 8 Hz, 1H, Ar-H), 2.29 (s, 3H, CH ); FT-IR (ύ max, cm ): 3298 and 3194 (3 NH), 2954 (CH
3
aliphatic), 1635 (C=O amide); MS (MWt: 403.86): m/z, 405.33 [M + 2, (1.15%)], 403.2 [M , (3.24%)], 63
1
6
+
+
(
100%); Anal. Calcd for C H ClN O: C, 65.43; H, 4.49; N, 17.34. Found: C, 65.64; H, 4.58; N, 17.46.
22 18 5
3
.2.29. 1-(4-((3-Chloroquinoxalin-2-yl)amino)phenyl)-3-(3-methoxyphenyl) urea (XVIIe)
◦
1
Olive green solid (yield = 76%); m.p. >300 C; H-NMR (DMSO-d )
δ: 9.57 (s, 1H, NH urea), 9.16
6
(
s, 1H, NH urea), 8.74 (s, 1H, NH), 8.01–7.90 (m, 3H, Ar-H), 7.80 (d, J = 8 Hz, 1H, Ar-H), 7.52–7.42 (m,
−
H, Ar-H), 7.27–7.25 (m, 3H, Ar-H), 7.16–7.08 (m, 2H, Ar-H), 3.68 (s, 3H, OCH ); FT-IR (ύ max, cm ):
3
1
3
3
4
310 (3 NH), 3030 (CH aromatic), 2920 (CH aliphatic), 1639 (C=O amide), 1215 (OCH ); MS (MWt:
3
+
19.86): m/z, 419.27 [M , (2.52%)], 83.98 (100%); Anal. Calcd for C H ClN O : C, 62.93; H, 4.32; N,
22
18
5
2
1
6.68. Found: C, 63.14; H, 4.36; N, 16.89.
3
.2.30. 1-(3-Methoxyphenyl)-3-(4-(quinoxalin-2-ylamino)phenyl)urea (XXV)
◦
1
Brown solid (yield = 61%); m.p. > 300 C; H-NMR (DMSO-d ):
δ: 9.67 (s, 1H, NH urea), 9.57
6
(
4
s, 1H, NH urea), 8.64 (s, 1H, NH), 7.38–7.12 (m, 5H, Ar-H), 7.02–6.91 (m, 4H, Ar-H), 6.67–6.54 (m,
−
1
H, Ar-H), 3.79 (s, 3H, OCH ). FT-IR (
ύ
max, cm ): 3286 (3 NH), 3066 (CH aromatic), 2924 (CH
3
+
aliphatic), 1716 (C=O amide); MS (MWt: 385.42): m/z, 385.4[M , (1.6%)], 118.98 (100%); Anal. Calcd
for C H N O : C, 68.56; H, 4.97; N, 18.17. Found: C, 68.79; H, 5.12; N, 18.43.
22
19
5
2
3
.2.31. 1-(4-((3-Chloroquinoxalin-2-yl)amino)phenyl)-3-(4-methoxyphenyl)urea (XVIIf)
◦
p-Methoxy- benzoyl azide (10 mmol) was heated in dry toluene (100 mL) at 60 C for half an
hour, then intermediate XIII (10 mmol) in dry toluene (5 mL) was added dropwise to the solution and
the reaction was refluxed for additional 5 h [32]. The solid formed was filtered, washed with toluene
◦
1
and dried to give the urea derivative XVIIf. Light green solid (yield = 71%); m.p. > 300 C; H-NMR

Molecules 2019, 24, 1175
18 of 22
(DMSO-d6)
δ: 10.12 (s, 1H, NH urea), 9.58 (s, 1H, NH urea), 8.80 (s, 1H, NH), 8.58 (d, J = 12 Hz, 1H,
Ar-H), 8.05 (d, J = 16 Hz, 1H, Ar-H), 7.88 (m, 1H, Ar-H), 7.51 (m, 2H, Ar-H), 7.37 (m, 4H, Ar-H), 6.89
−
m, 3H, Ar-H), 3.86 (s, 3H, OCH ); FT-IR (ύ max, cm ): 3309 (3 NH), 3066 (CH aromatic), 2927 (CH
3
aliphatic), 1639 (C=O amide); MS (MWt: 419.86): m/z, 420 [M + H, (0.7%)], 419.42 [M , (0.96%)], 139
1
(
+
+
(
100%); Anal. Calcd for C H ClN O : C, 62.93; H, 4.32; N, 16.68. Found: C, 63.12; H, 4.37; N, 16.85.
22 18 5 2
3
.2.32. General Procedures for the Synthesis of Compounds IXa,b and XVIIIa,b
To a solution of IV or XIII (0.35 mmol) in dry pyridine (10 mL) the appropriate sulfonyl chloride
(1.05 mmol; 3 eq.) namely (4-methylbenzenesulfonyl chloride, 4-nitrobenzenesulfonyl chloride) was
added and the mixture was refluxed for 2 h, poured onto cold water (30 mL) and stirred for 30 min [33].
The resulted solid was filtered, washed with water, dried and recrystallized from ethanol to afford the
title compounds IXa,b or XVIIIa,b, respectively.
3
.2.33. 4-Methyl-N-(4-((3-methylquinoxalin-2-yl)amino)phenyl)benzenesulphonamide (IXa)
◦
1
Dark green solid (yield = 75%); m.p. 198–199 C; H-NMR (DMSO-d )
δ: 10.70 (s, 1H, NH
6
sulfonamide), 8.63 (s, 1H, NH), 8.54–6.70 (m, 12H, Ar-H), 2.72 (s, 3H, CH ), 2.32 (s, 3H, CH ); FT-IR (
ύ
3
3
−
1
+
max, cm ): 3421 (2 NH), 1580 (NH bending), 1335 and 1160 (SO ); MS (MWt.: 404.5): m/z, 405 [M +
2
+
H, (2.74%)], 404.08 [M , (9.05%)], 249 (100%); Anal. calcd for C H N O S: C, 65.33; H, 4.98; N, 13.85.
22
20
4
2
Found: C, 65.49; H, 5.06; N, 13.97.
3
.2.34. N-(4-((3-Methylquinoxalin-2-yl)amino)phenyl)-4-nitrobenzenesulfonamide (IXb)
◦
1
Black solid (yield = 70%); m.p. 180–182 C; H-NMR (DMSO-d ) δ: 8.63 (s,1H, NH sulfonamide),
6
8
7
.32 (s, 1H, NH), 8.22 (d, J = 8 Hz, 2H,Ar-H), 7.95 (d, J = 4 Hz, 2H, Ar-H), 7.85 (d, J = 8 Hz, 2H, Ar-H),
.78 (d, J = 8 Hz, 2H, Ar-H), 7.71 (d, J = 8 Hz, 1H, Ar-H), 7.58 (m, 1H, Ar-H), 7.44 (t, 1H, Ar-H), 7.36
−
1
(t, 1H, Ar-H), 2.73 (s, 3H, CH ); FT-IR (
ύ
max, cm ): 3414 (2 NH), 3035 (CH aromatic), 2924 (CH
3
+
aliphatic), 1604 (NH bending), 1346 and 1165 (SO ); MS (MWt: 435.46): m/z, 435.42 [M , (10.24%)],
2
4
4.88 (100%); Anal. calcd for C H N O S: C, 57.92; H, 3.93; N, 16.08. Found: C, 58.09; H, 3.97;
21 17 5 4
N, 16.23.
3
.2.35. N-(4-((3-Chloroquinoxalin-2-yl)amino)phenyl)-4-methylbenzenesulfonamide (XVIIIa)
◦
1
Light green solid (yield = 75%); m.p. 250–252 C; H-NMR (DMSO-d )
δ: 10.21 (s, 1H, NH
6
sulfonamide), 9.89 (s, 1H, NH), 8.02–7.91 (m, 4H, Ar-H), 7.85 (d, J = 8 Hz, 2H, Ar-H), 7.64–7.57 (m, 2H,
−
1
Ar-H), 7.36 (d, J = 8 Hz, 4H, Ar-H), 2.37 (s, 3H, CH ); FT-IR (ύ max, cm ): 3386 (2 NH), 3083 (CH
3
+
+
aromatic), 2921 (CH aliphatic), 1332–1159 (SO ); MS (MWt: 424.9): m/z, 425 [M + H, (1%)], 424.7 [M ,
2
(
1.58%)], 261 (100%); Anal. Calcd for C H ClN O S: C, 59.36; H, 4.03; N, 13.19. Found: C, 59.48; H,
21 17 4 2
4
.12; N, 13.34.
.2.36. N-(4-((3-Chloroquinoxalin-2-yl)amino)phenyl)-4-nitrobenzenesulfonamide (XVIIIb)
3
◦
1
Dark brown solid (yield = 76%); m.p. > 300 C, H-NMR (DMSO-d )
δ: 10.46 (s, 1H, NH
6
sulfonamide), 9.40 (s, 1H, NH), 8.42–8.19 (m, 2H, Ar-H), 8.00–7.83 (m, 5H, Ar-H), 7.60-7.46 (m, 2H,
−
1
Ar-H), 7.35–7.13 (m, 3H, Ar-H); FT-IR (ύ max, cm ): 3385 (3 NH), 3053 (CH aromatic), 1347–1165
+
(
SO ); MS (MWt.: 455.9): m/z, 455.68 [M , (0.77%)], 107 (100%); Anal. Calcd for C H ClN O S: C,
2
20 14
5
4
5
2.69; H, 3.10; N, 15.36. Found: C, 52.85; H, 3.07; N, 15.48.
3
.3. Biological Studies
3
.3.1. In-Vitro Anti-Cancer Activity
Three cancer cell lines including human colon carcinoma (HCT116), human breast
adenocarcinoma (MCF-7) and human hepatocellular carcinoma (Hep G2), were purchased from
American Type Cell Culture Collection (ATCC, Manassas, VA, USA) and grown on the appropriate

Molecules 2019, 24, 1175
19 of 22
growth medium Dulbecco’s modified Eagle’s medium (DMEM) or Roswell Park Memorial Institute
medium (RPMI 1640) supplemented with 100 mg/mL of streptomycin, 100 units/mL of penicillin and
◦
1
0% of heat-inactivated fetal bovine serum in a humidified, 5% (v/v) CO atmosphere at 37 C [34].
2
Cytotoxicity assay by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT).
Exponentially growing cells from different cancer cell lines were trypsinized, counted and seeded
2
at the appropriate densities (2000–1000 cells/0.33 cm well) into 96-well microtiter plates. Cells then
◦
were incubated in a humidified atmosphere at 37 C for 24 h. Then, cells were exposed to different
concentrations of compounds (0.1, 10, 100, 1000
determined using MTT technique as follow. Media were removed; cells were incubated with 200
% MTT solution/well (Sigma Aldrich, Darmstadt, Germany) and were allowed to metabolize the dye
into a colored-insoluble formazan crystal for 2 h. The remaining MTT solution were discarded from
the wells and the formazan crystals were dissolved in 200 l/well acidified isopropanol for 30 min,
µM) for 72 h. Then the viability of treated cells was
µL of
5
µ
covered with aluminum foil and with continuous shaking using a MaxQ 2000 plate shaker (Thermo
Fisher Scientific Inc, Waltham, MA, USA) at room temperature. Absorbance were measured at 570 nm
R
using a Stat Fax 4200 plate reader (Awareness Technology, Inc., Awareness Technology, FL, USA).
The cell viability was expressed as percentage of control and the concentration that induces 50% of
maximum inhibition of cell proliferation (IC ) were determined using Graph Pad Prism version 5
50
software (Graph Pad Software Inc, San Diego, CA, USA) (1,2) [35].
3
.3.2. In-Vitro VEGFR-2 Inhibition Assay
The in-vitro enzyme inhibition determination for the synthesized compounds was carried out
at Bio Science Corporation (BPS)-San Diego, CA, USA. The assay was performed using Kinase-Glo
Plus luminescence kinase assay kit (Promega), where the kinase activity was measured by quantitating
the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from
the assay is correlated with the amount of ATP present and is inversely correlated with the amount of
kinase activity. Our compounds were diluted with 10% DMSO and 5
a 50 L reaction so that the final concentration of DMSO is 1% in all of reactions. All of the enzymatic
reactions were conducted at 30 C for 40 min. The 50
.4, 10 mM MgCl , 0.1 mg/mL BSA, 1 mM DTT, 10 M ATP, Kinase substrate and the enzyme. After
L of Kinase-Glo Plus Luminescence kinase assay solution (Promega San
µL of the dilution was added to
µ
◦
µL reaction mixture contains 40 mM Tris, pH
7
µ
2
the enzymatic reaction, 50
µ
Diego, CA, USA) was added to each reaction and incubate the plate for 5 min at room temperature.
Luminescence signal was measured using a BioTek Synergy 2 microplate reader.
Kinase activity assays were performed in duplicate at each concentration. The luminescence data
were analyzed using the computer software, Graphpad Prism. The difference between luminescence
intensities in the absence of Kinase (Lu
t
c
) and in the presence of Kinase (Lu ) was defined as 100%
activity (Lut − Lu
c
). Using luminescence signal (Lu) in the presence of the compound, % activity was
calculated as:
%
activity = {(Lut − Lu)(Lut − Luc)} × 100%
where Lu = the luminescence intensity in the presence of the compound.
3
.3.3. Cell Cycle Analysis
6
HCT116 cells at a density of 4
×
10 cell/T 75 flask were exposed to 1-(4-Chlorophenyl)-3-(4-
(
(3-methylquinoxalin-2-yl)amino)phenyl)urea VIIIc at its IC50 concentration for 24 and 48 h. The cells
then were collected by trypsinization, washed with phosphate buffered saline (PBS), and fixed in
TM
ice-cold absolute alcohol. Thereafter, cells were stained, using Cycle test
Plus DNA Reagent Kit (BD
Biosciences, San Jose, CA, USA), according to the manufacturer’s instructions. Cell cycle distribution
was determined using a FACS Calibur flow cytometer (BD Biosciences).

Molecules 2019, 24, 1175
20 of 22
3
.3.4. Apoptosis Determination
Apoptosis was determined by staining cells with Annexin V–fluorescein isothiocyanate (FITC)
and counterstaining with propidium iodide (PI) using the Annexin V–FITC/PI apoptosis detection
kit (BD PharMingen, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly,
6
4
× 10 cell/T 75 flask were exposed to compound VIIIc at its IC50 concentration for 24 and 48 h.
6
The cells then were collected by trypsinization and 0.5
×
10 cells were washed twice with
phosphate-buffered saline (PBS) and stained with 5
µ
L Annexin V–FITC and 5
buffer (BD PharMingen) for 15 min at room temperature in the dark. Analyses were performed using
FACS Calibur flow cytometer (BD Biosciences).
µL PI in 1
×
binding
3
.3.5. In Vitro Cytotoxic Assay
Cell Line cells were obtained from American Type Culture Collection, cells were cultured using DMEM
(
Invitrogen/Life Technologies, Waltham, MA, USA) supplemented with 10% FBS (Hyclone, Waltham,
MA, USA), 10 g/mL of insulin (Sigma), and 1% penicillin-streptomycin. All of the other chemicals and
reagents were from Sigma, or Invitrogen. Plate cells (cells density 1.2–1.8 10,000 cells/well) in a volume
of 100 L complete growth medium + 100 L of the tested compound per well in a 96-well plate for 24 h
before the MTT assay.
µ
×
µ
µ
4
. Conclusions
The present paper aimed to design some quinoxaline scaffolds and screened them for their
cytotoxicity against three tumor cell lines and as well as measuring their selectivity. Compound VIIIc
showed promising activity against human colon carcinoma (HCT116) cell line (IC50 = 2.5 M), also
µ
it induced a significant disruption in cell cycle profile and cell cycle arrest at G2/M phase boundary
with concurrent time dependent increase in pre-G cell population. Owing to the good anti-cancer
activity of compound VIIIc toward (HCT116) cells and its selectivity, it could be considered as a lead
compound that merits further optimization and development as an anti-cancer and an apoptotic
inducing candidate against (HCT116) cells.
Author Contributions: K.A.M.A. created the whole idea of designing and synthesis of compounds, in addition to
revising the manuscript. A.M.S.E.N. performed the practical work, analyzed the data and wrote the manuscript.
S.M.K. performed the biological experiments. Y.M.N. and N.S.M.I. supervised the practical work and manuscript
writing. D.A.A.E.E. co-senior author supervised the study. All authors read and approved the final manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).