In silico binding analysis and SAR elucidations of newly designed benzopyrazine analogs as potent inhibitors of thymidine phosphorylase

Article abstract of DOI:10.1016/j.bioorg.2016.07.010

Thymidine phosphorylase (TP) is up regulated in wide variety of solid tumors and therefore presents a remarkable target for drug discovery in cancer. A novel class of extremely potent TPase inhibitors based on benzopyrazine (1–28) has been developed and e

Full text of DOI:10.1016/j.bioorg.2016.07.010

Bioorganic Chemistry 68 (2016) 80–89
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
In silico binding analysis and SAR elucidations of newly designed
benzopyrazine analogs as potent inhibitors of thymidine phosphorylase
Muhammad Taha a,b, , Nor Hadiani Ismail a,b, Syahrul Imran , Fazal Rahim , Abdul Wadood ,
⇑
a,b
d
c
a,b
e
c
Laode Muhammad Ramadhan Al Muqarrabun , Khalid Mohammed Khan , Mehreen Ghufran ,
f
Muhammad Ali
a
Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia
Faculty of Applied Science, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia
Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul Wali Khan University Mardan, Pakistan
b
c
d
Department of Chemistry, Hazara University, Mansehra 21300, Khyber Pukhtunkhwa, Pakistan
e
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
Department of Chemistry, COMSATS Institute of Information Technology, University Road, Abbottabad 22060, KPK, Pakistan
f
a r t i c l e i n f o
a b s t r a c t
Article history:
Thymidine phosphorylase (TP) is up regulated in wide variety of solid tumors and therefore presents a
remarkable target for drug discovery in cancer. A novel class of extremely potent TPase inhibitors based
on benzopyrazine (1–28) has been developed and evaluated against thymidine phosphorylase enzyme.
Out of these twenty-eight analogs eleven (11) compounds 1, 4, 14, 15, 16, 17, 18, 19, 20, 24 and 28 showed
potent thymidine phosphorylase inhibitory potentials with IC50 values ranged between 3.20 ± 0.30 and
Received 4 May 2016
Revised 21 July 2016
Accepted 25 July 2016
Available online 25 July 2016
3
7.60 ± 1.15 lM when compared with the standard 7-Deazaxanthine (IC50 = 38.68 ± 4.42 lM). Structure-
Keywords:
Benzopyrazine
Synthesis
Thymidine phosphorylase inhibition
Molecular docking
SAR
activity relationship was established and molecular docking studies were performed to determine the
binding interactions of these newly synthesized compounds. Current studies have revealed that these
compounds established stronger hydrogen bonding networks with active site residues as compare to the
standard compound 7DX.
Ó 2016 Elsevier Inc. All rights reserved.
1
. Introduction
Thymidine phosphorylase (EC 2.4.2.4) is an angiogenic factor
carcinoma of stomach, colon, and bladder, atherosclerosis and
numerous inflammatory diseases [10]. The inhibition of TP may
result in the reduction of tumor growth and metastasis [11–14].
Physically, the most potent TP inhibitors are pyrimidine deriva-
tives, which were planned to interact with the binding site of
thymidine. The most potent inhibitor of TP established so far is
the 5-chloro-6-[1-(2-iminopyrrolidinyl)methyl] uracil hydrochlo-
ride (TPI) [15] that showed hefty inhibitory potential with IC50
value of 0.035 mM [16]. Further some purine derivatives like 7-
deazaxanthine (7DX) [9] (Fig. 1) have also been found to exhibit
inhibitory potentials against TP. However, 7DX (IC50
value = 40 mM [17]) is not as potent as TPI, however, additional
structural variations may lead to more potent purine-like TP inhi-
bitors. In addition, some multi-substrate inhibitors designed for
interacting with both the thymidine and phosphate-binding site
in the literature has been reported [18–20].
that exerts its effect by stimulating the endothelial cell migration.
Thymidine phosphorylase (TP) enzyme similar to platelet derived
endothelial cell growth factor [1–5], catalyzes the reversible
phosphorolysis of thymidine to thymine and 2-deoxy-a-D-
ribofuranosylphosphate. In contrast to non-neoplastic tissues, high
level of TP have been found in ovarian, colorectal, breast, and pan-
creatic tumors [6,7] and in other hyperproliferative disease states
such as rheumatoid arthritis [8] and psoriasis [9]. Abnormal level
of thymidine phosphorylase is communicated with various patho-
logical disorders including gastric carcinoma, pancreatic, uterine
sarcoma, colon carcinoma, renal carcinoma, uterine leiomyoma,
breast and lung cancers, astrocytic tumors, gastric carcinoma,
Benzopyrazine (quinoxaline) and its analogs are significant
nitrogen possessing heterocyclic compounds with numerous phar-
maceutical applications. Substituted benzopyrazine derivatives
possess broad range of biological potentials that include antibacte-
rial [21,22], anticancer [23], antifungal [24,25], antileishmanial
⇑
Corresponding author at: Atta-ur-Rahman Institute for Natural Product
Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar
Puncak Alam, Selangor, Malaysia.
E-mail addresses: taha_hej@yahoo.com, muhamm9000@puncakalam.uitm.edu.
my (M. Taha).
http://dx.doi.org/10.1016/j.bioorg.2016.07.010
0
045-2068/Ó 2016 Elsevier Inc. All rights reserved.

M. Taha et al. / Bioorganic Chemistry 68 (2016) 80–89
81
potentials against thymidine phosphorylase and angiogenesis as
compared to 7DX. To the best of our knowledge, benzopyrazine
analogs are first one to be recognized as efficient inhibitors of
the pyrimidine nucleoside phosphorylase, i.e. TPase, while these
analogs possess the strong and convincing potential in the future
design of new TPase inhibitors. The inhibitory potentials noted
for benzopyrazine analogs against TPase demonstrated to be at
much higher levels in magnitude when compared with 7DX. All
synthesized compounds 1–28 were evaluated against thymidine
phosphorylase inhibition according to a literature known proce-
dure [51]. The inhibitory potential of compounds were shown in
terms of IC50 values and were compared with the reference inhibi-
(
a) 7DX
(b) TPI
Fig. 1. (a) 7DX. (b) TPI.
[
26], antitubercular [27], antidepressant activities [28,29] and anti-
malarial [30,31]. Some of these analogs have also been reported to
show antimicrobial [32,33] potent antithrombotic [34], and anti-
inflammatory potential [35,36]. The quinoxaline or benzopyrazine
is also described as a bioisoster of quinoline, benzothiophene,
naphthalene and other aromatic rings such as pyrazine and pyri-
dine. Benzopyrazines have also found application in the field of
agriculture as herbicide, fungicide and insecticide [37]. Quinoxa-
line scaffolds are further present in the structure of several antibi-
otics such as levomycin, echinomycin, and actinoleutin that inhibit
the growth of gram positive bacteria and active against various
transplantable tumors [38]. Some of benzopyrazine derivatives
have also certain applications in dyes [39].
Literature has revealed that the addition of second ring, i.e. pyr-
role, in the design of standard inhibitor 7DX creates extra stabiliz-
ing interactions by filling the apparent space near the ring of
residue Phe210. This modification lead 7DX a novel prototype inhi-
bitor of TPase [40]. Current study involves the designing of new
and novel inhibitors based on benzopyrazine nucleus. Benzopy-
razine nucleus has replaced 5-membered pyrrole moiety of 7DX
with a 6-membered pyrazine heterocyclic moiety; the fact that
tor, 7-Deazaxanthine, 7DX, (IC50 = 38.68 ± 4.42
twenty-eight analogs eleven (11) compounds such as 1, 4, 14, 15,
6, 17, 18, 19, 20, 24 and 28 showed potent thymidine phosphory-
lM). Out of these
1
lase inhibition when compared with the standard. Compound 13
and 21 showed good thymidine phosphorylase inhibition. While
compound 2, 3, 5, 6, 12 and 27 exhibit good to moderate inhibition
(Table 1).
Structure-activity relationship (SAR) suggested that TP inhibi-
tory potential mainly based on substituent pattern on phenyl ring.
We observed during this study that all those analogs having hydro-
xyl group on phenyl ring showed potent inhibition. Among the
hydroxyl containing analogs, those analogs having trihydroxyl
functionality such as 18 and 19 are superior to dihydroxy analogs
i.e. 14, 15, and 16 except analog 17. Further decline in inhibitory
potential was observed in monohydroxyl analogs such as 12, 13,
2
0, 21, 24 and 28. The greater potential showed by all these ana-
logs might be due to the hydroxyl groups, which may interact with
the enzyme through hydrogen bonding. We observed here that the
number and position of hydroxyl group on phenyl ring also affect
the inhibitory potential of compounds. The greater potential
shown by compound 18 than 17, which is >19 on the basis of
7
DX contains a second (pyrrole) ring entity in the molecule may
open interesting perspectives in the design of new more potent
inhibitors. Second, pyrimidine ring in 7DX has been replaced with
an unsubstituted benzene ring just to provide a space or cavity fill-
ing in the enzyme pocket site. In the newly developed scaffolds;
however, the stress has been given on more nitrogen atoms to cre-
ate extra hydrogen bonding sites. We are currently heterocyclic
hybrid molecules [41] and we found that the hybrid compounds
are more potent than their individual molecules. We believe that
introduction of unsubstituted aromatic moiety together with
another fused 6-mebered heterocyclic system instead of 5-
membered pyrrole ring should create extra stabilizing interactions
by filling the apparent space near the ring of residue Phe210 more
effectively. Herein, we report novel benzopyrazine analogs as new
leads against TPase and their comparison with the standard inhibi-
tor 7DX with particular reference to molecular docking analysis.
0
0
0
hydroxyl position 2 < 4 < 3 . Similarly compound 14
,3-dihydroxy analog, 15 a 2,4-dihydroxy analog and 16 a 2,5-
dihydroxy analog with IC50 value 15.40 ± 1.04, 33.40 ± 1.20 and
5.50 ± 1.25 M respectively showed potent inhibition. The slight
a
2
3
l
activity difference among these analogs showed that position of
hydroxyl group on aromatic ring also affects the inhibition. In
mono hydroxylated analogs like 12, a meta hydroxyl analog, 13, a
para hydroxyl analog, and 28 a ortho hydroxyl analog with IC50
value 63.20 ± 2.2.1, 48.60 ± 1.17 and 37.16 ± 1.16, it was observed
that the position of hydroxyl group also play an important role in
this inhibition.
We have also check the effect of other substituents like flouro
(
(
analogs 1, 2, 3) the order of activity was 2-F > 4-F > 3-F. The chloro
analogs 4, 5, 6) inhibition pattern is observed similar as for flouro
analogs 2-Cl > 4-Cl > 3-Cl. The nitro and methyl substituted
analogs were found to completely inactive. For the confirmation
of the binding interaction, molecular docking analysis was carried
out.
2
. Results and discussion
2
.1. Chemistry
2.3. Molecular modeling and docking studies
In the continuation of our work on heterocyclic hydrazones
[
42–50]; in this study benzopyrazine derivatives 1–28 (Table 1)
The synthesized compounds were docked into the active site of
were synthesized by reacting benzopyrazine hydrazide (a) with
various aromatic aldehydes in the presence of catalytic amount
of acetic acid (Scheme 1). The structures of the benzopyrazine
hydrazones 1–28 were fully characterized by using various spec-
troscopic techniques such as NMR, MS and were further confirmed
using CHN analysis.
the target enzyme using MOE with the default parameters i.e.
Placement: Triangle Matcher, Rescoring 1: London dG, Refinement:
Forcefield, Rescoring 2: London dG. In order to validate the docking
protocol, the 3D crystal structure of the thymidine phosphorylase
complex with analog of DIDEOXYURIDINE was retrieved from the
protein databank PDB ID: 4EAD and redocked in the active site of
the enzyme. The ligand binding site for docking was defined as a
collection of amino acids enclosed within a sphere of 4.5 Å radius
around the coordinates of the ligand, which is the inhibitor
molecule present in the binding site of thymidine phosphorylase.
For each ligand ten conformations were generated and the top
2.2. Biological activity
The benzopyrazine class of heterocycles should be considered
as new lead inhibitors with much more pronounced inhibitory

8
2
M. Taha et al. / Bioorganic Chemistry 68 (2016) 80–89
Table 1
Synthesis of various analogs of quinoxaline derivatives (1–28) and its thymidine phosphorylase inhibition.
S. No.
1
R
IC50
(
l
M ± SEM
a
)
S. No.
15
R
a
IC50 (lM ± SEM )
33.60 ± 1.01
33.40 ± 1.20
2
66.12 ± 2.22
16
35.50 ± 1.25
3
4
58.10 ± 1.10
37.60 ± 1.15
17
18
8.60 ± 0.42
3.20 ± 0.30
5
6
93.50 ± 2.30
73.40 ± 2.28
19
20
9.10 ± 0.44
34.40 ± 1.26
7
8
N.A.
N.A.
21
22
46.90 ± 1.14
N.A.
9
N.A.
N.A.
23
24
N.A.
1
0
36.26 ± 1.30
1
1
1
2
N.A.
25
26
N.A.
N.A.
63.20 ± 2.2.1

M. Taha et al. / Bioorganic Chemistry 68 (2016) 80–89
83
Table 1 (continued)
S. No.
R
IC50
(
l
M ± SEM
a
)
S. No.
27
R
a
IC50 (lM ± SEM )
1
3
48.60 ± 1.17
84.26 ± 3.20
1
4
15.40 ± 1.04
28
37.16 ± 1.16
7
-Deazaxanthine (7DX)
38.68 ± 4.42 lM
Scheme 1. Synthesis of quinoxaline derivatives (1–28).
ranked conformation on the basis of docking score was selected for
further studies in molecular docking. The RMSD between docked
and co-crystallized ligand was found to be 1.89 Å indicates that
the docking method is reliable [41]. The superposition of docked
and co-crystallized ligands is shown in Fig. 2. After the molecular
of ꢀ7.5093 and biological activity with IC50 of 38.68 ± 4.42. The
three hydroxyl moieties present at the phenyl ring of the com-
pound formed five hydrogen bonds with His 85, Tyr 168, Arg
171, Ser 186 and Lys 190 respectively. Furthermore, nitrogen atom
of the quinoxaline ring formed hydrogen bond with active site resi-
due Gly 88 whereas the phenyl ring of the compound formed aren-
arene interaction with phenyl ring of the Tyr 168 (Fig. 3b). This
strong bonding network might be one of the reasons for this com-
pound to show good biological activity. The docking conformation
of the second most active compound, compound 17
(IC50 = 8.60 ± 0.42), showed that this compound formed five hydro-
gen bonds with the active site residues Arg115, Thr 120, Thr 168,
Ser 186 and Lys 190 respectively (Fig. 3c) and good docking score
(ꢀ10.0389). If we compare the structure of compound 17 with
the structure of compound 18 the only difference is the presence
of one more hydroxyl moiety in compound 18. The extra hydroxyl
moiety might be one the reasons for the good biological activity of
compound 18 as compare to compound 17. The docking results
showed that comparatively good interactions were observed for
compound 18 than compound 17 (Fig. 3b and c). In case of com-
pounds having only one hydroxyl group (compound 12 and 13)
on phenyl ring showed less biological activities and poor interac-
tions with active site residues. For example, compound 12 has
IC50 = 63.20 ± 2.21 and docking score (ꢀ8.8330), showed only
two hydrogen bonds with active site residues (Fig. 3d). Like the
number of hydroxyl moiety the position of hydroxyl moiety also
play a role in the activity of these compounds. For example,
although compound 19, the third most active compound in the ser-
ies has good inhibitory activity with IC50 value 9.10 ± 0.44 and
docking score i.e. ꢀ9.7813, have three hydroxyl groups but at dif-
ferent positions as in compound 18. This different positions of
hydroxyl moieties in compounds 19 (Fig. 4a), made this compound
to established poor interactions with the active site residues and
showed less biological activity as compare to compound 18. About
similar experimental and docking results were observed for com-
pounds 20, 21 and 24.
docking we analyzed the best poses having polar and
tions by Pymol software.
p-p interac-
In order to explore the binding mode of these newly synthe-
sized compounds, molecular docking was performed. The docking
results showed that all the compounds well accommodated in the
active site of thymidine phosphorylase (Fig. 3a). The predicted
docking scores and interactions of these compounds are well cor-
related with the observed experimental results (Tables 1 and 2)
as the most active compounds showed good docking score and
good interaction as compare to less active compounds. From the
docking conformation of the most active compound, compound
1
8 (IC50 = 3.20 ± 0.30), it was observed that this compound estab-
lished six hydrogen bonds and one arene-arene interactions with
active site residues and good docking score (ꢀ13.8359) as compare
to the standard inhibitor (7-Deazaxanthine) having docking score
In case of compounds having fluoro or chloro phenyl ring, it was
observed that when fluorine or chlorine present at ortho position of
phenyl ring (compound 1 and compound 4) showed good interac-
tions as compare to compounds having fluorine or chlorine moiety
Fig. 2. Superposition of docked and co-crystallized ligand, green represents the co
crystallized ligand and yellow the re-docked conformation of the ligand.

8
4
M. Taha et al. / Bioorganic Chemistry 68 (2016) 80–89
Fig. 3. (a) Docking conformations of different ligands, (b) docking conformation of compound 18, (c) docking conformation of compound 17 and (d) docking conformation of
compound 12 in the active site of thymidine phosphorylase.
Table 2
Predicted docking scores and interactions of the compounds 1–28.
Compound
Docking scores
Number of HBs
No. of arene-arene interactions
No. of arene-cation interactions
1
2
3
4
5
6
1
1
1
1
1
1
1
1
2
2
2
2
2
ꢀ8.6575
ꢀ7.9256
ꢀ7.9994
ꢀ8.0696
ꢀ6.0817
ꢀ7.0607
ꢀ8.8330
ꢀ7.5155
ꢀ9.0315
ꢀ9.2134
ꢀ8.1358
ꢀ10.0389
ꢀ13.8359
ꢀ9.7813
ꢀ8.1737
ꢀ8.0450
ꢀ8.1296
ꢀ6.6362
ꢀ8.0315
2 (Tyr168)
–
–
1 (Lys190)
–
–
2 (Tyr168, Arg171)
–
2 (Ser86, Tyr168,)
1 (Tyr168)
–
–
–
1 (Lys190)
1 (Lys190)
1 (Lys190)
1 (Lys190)
1 (Lys190)
2 (Lys190)
–
1 (Lys190)
–
1 (Lys190)
–
–
–
–
1 (Phe210)
–
–
–
2
3
4
5
6
7
8
9
0
1
4
7
8
–
1 (Phe210)
–
–
–
1 (Tyr168)
1 (Tyr168)
1 (Tyr168)
1 (Phe210)
–
1 (Arg115)
5 (Arg115, Thr120, Tyr168, Ser186, Lys 190)
6 (His85, Tyr168, Ser186, Arg171, Lys190)
3 (Gln156, Arg171, (Lys190))
–
1 (Tyr168)
1 (Ser186)
–
–
1 (Lys190)
1 (Lys190)
–
–
1 (Tyr168)
–
1 (Lys190)
at meta or para position of phenyl ring (compound 2, 3, 5 and 6).
For example, compound 1 has IC50 = 33.60 ± 1.01 and docking
score of ꢀ8.6575, showed good interactions with active site
residues (Fig. 4d) and poor docking score. The steric hindrance or
low polarizability might be one the reasons for these compounds
to show no biological activity.
The binding mode of the standard compound (7-
Deazaxanthine; 7DX) showed that this compound established
three polar and one arene-arene interaction with active site resi-
dues of the enzyme (Fig. 5). If we compare the binding interactions
of this compound with most active compounds of the series (17,
18, and 19, etc.) it was observed that these compounds established
residues (Fig. 4b) as compare to compound
2
with IC50 of
6
6.12 ± 2.22 and docking score of ꢀ7.9256 (Fig. 4c). About similar
docking results were observed for compound 4, 5 and 6. From
the docking results of the inactive compounds e.g. compound 22
having no biological activity (Table 1), it was observed that these
compounds showed very poor interactions with active site

M. Taha et al. / Bioorganic Chemistry 68 (2016) 80–89
85
Fig. 4. Docking conformation of compound 19 (a), compound 1 (b), 2 (c) and 22 (d) in the active site of thymidine phosphorylase.
Fig. 5. Docking conformation of standard inhibitor (7-Deazaxanthine) in the active site of thymidine phosphorylase.
strong hydrogen bonding network with active site residues as
compare to the standard compound (Figs. 3b, 3c, 4a and 5). These
strong bonding interactions might be one of the reasons for these
compounds to be more active as compare to the standard
compound.
Over all the docking results showed that the number as well as
positions of hydroxyl moiety on phenyl ring of these newly synthe-
sized compounds plays a key role in their biological activities.
3. Conclusion
In conclusion we have synthesized benzopyrazine analogs
1–28, characterized by EI-MS and H NMR and checked for thymi-
dine phosphorylase inhibitory potentials. Eleven compounds were
identified as potent thymidine phosphorylase inhibitor. Com-
pounds 1, 4, 14, 15, 16, 17, 18, 19, 20, 24 and 28 showed potent
thymidine phosphorylase inhibition. Subsequent molecular
1

8
6
M. Taha et al. / Bioorganic Chemistry 68 (2016) 80–89
docking studies were performed to determine the binding interac-
tion of the compounds. These analogs offer the possibility of conve-
nient further modifications that could give rise to further
optimized lead structures with improved pharmacokinetic proper-
ties and selectivity towards the enzyme.
4.4. General procedure for the synthesis of compounds 1–28
Equimolar quantities (1 mmol) of compound 5 and substituted
benzaldehydes (1 mmol) in methanol (25 mL) were refluxed for
3 h, in the presence of catalytic amount of glacial acetic acid. The
resulting solid was filtered and recrystallized from methanol in
good yields.
4
. Material and methods
NMR experiments were performed on Avance Bruker AM
00 MHz machine. CHN Analysis was performed on a Carlo Erba
4
.4.1. (E)-2-(2-(2-fluorobenzylidene)hydrazinyl)quinoxaline (1)
Yield: 94%. m.p.: 239–241 °C. H NMR (500 MHz, DMSO-d
3
1
6
): d
Strumentazion-Mod-1106, Italy. Ultraviolet (UV) spectra were
recorded on Perkin–Elmer Lambda-5 UV/vis spectrophotometer
in MeOH. Infrared (IR) spectra were recorded on JASCO IR-A-302
spectrometer as KBr (disc). Electron impact mass spectra (EI MS)
were recorded on a Finnigan MAT-311A (Germany) mass spec-
trometer. Thin layer chromatography (TLC) was performed on
pre-coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck,
Germany). Chromatograms were visualized by UV at 254 and
1
7
7
1
1
C
C
1.83 (s, 1H), 9.13 (s, 1H), 8.35 (s, 1H), 8.09 (t, J = 7.6 Hz, 1H),
.94 (d, J = 8.2 Hz, 1H), 7.76–7.64 (m, 2H), 7.59–7.40 (m, 2H),
13
.35–7.24 (m, 2H).
6
C NMR (125 MHz, DMSO-d ): d 163.7;
61.3; 150.2; 140.8; 138.0; 137.3; 136.4; 130.8; 130.3; 128.8;
26.2; 125.3; 122.9; 115.9; 112.4. HREI-MS: m/z calcd for
15
H
11FN
11FN
4
[M]+ 266.0968; Found 266.0972; Anal. Calcd for
15
H
4
, C, 67.66; H, 4.16; N, 21.04; Found C, 67.67; H, 4.15;
ꢀ1
N, 21.05. IR (cm , KBr): 3460 (NH); 1582 (C@N).
3
65 nm.
4
.4.2. (E)-2-(2-(3-fluorobenzylidene)hydrazinyl)quinoxaline (2)
Yield: 71%. m.p.: 239–240 °C. H NMR (500 MHz, DMSO-d
4.1. Preparation of ligands
1
6
): d
1
7
1.81 (s, 1H), 9.16 (s, 1H), 8.14 (s, 1H), 7.93 (d, J = 8.1 Hz, 1H),
.70 (dt, J = 6.6, 3.9 Hz, 2H), 7.65 (d, J = 10.2 Hz, 1H), 7.59 (d,
All the twenty-eight synthesized inhibitors of the thymidine
phosphorylase were built in MOE (Molecular Operating Environ-
ment) software. The synthesized ligands were energy minimized
by using the default parameters of the MOE i.e. gradient: 0.05,
Force Field: MMFF94X and saved all the ligands in mdb file for fur-
ther assessment in molecular docking.
J = 7.7 Hz, 1H), 7.54–7.46 (m, 2H), 7.22 (td, J = 8.3, 2.3 Hz, 1H);
7
0
0
0
13
.28 (2H, m, H2 ) and (H4 or H6 ). C NMR (125 MHz, DMSO-
d
6
): d 161.2; 159.2; 150.1; 140.8; 138.0; 136.3; 134.3; 131.0;
30.4; 128.8; 126.2; 125.4; 124.9; 122.2; 115.8 ppm. HREI-MS:
1
m/z calcd for C15
H11FN
4
[M]+ 266.0968; Found 266.0961; Anal.
Calcd for C15 11FN
H, 4.17; N, 21.03. IR (cm , KBr): 3443 (NH); 1580 (C@N).
H
4
, C, 67.66; H, 4.16; N, 21.04; Found C, 67.64;
4.2. Preparation of protein
ꢀ1
The 3D crystal structure of the thymidine phosphorylase was
retrieved from the protein databank PDB ID: 4EAD. All the water
molecules and ions from the target protein were removed. The
energy minimization was carried out after the 3D protonation by
the default parameters of the MOE for the stability of the protein.
4.4.3. (E)-2-(2-(4-fluorobenzylidene)hydrazinyl)quinoxaline (3)
Yield: 87%. m.p.: 217–219 °C. H NMR (500 MHz, DMSO-d ): d
11.68 (s, 1H), 9.11 (s, 1H), 8.15 (s, 1H), 7.92 (d, J = 8.2 Hz, 1H),
1
6
7.84 (dd, J = 8.4, 5.8 Hz, 2H), 7.76–7.63 (m, 2H), 7.61–7.46
1
3
(
m, 1H), 7.28 (t, J = 8.8 Hz, 2H). C NMR (125 MHz, DMSO-d
162.35, 146.39, 143.94, 143.79, 141.92, 139.04, 131.58, 130.53,
30.53, 130.10, 129.21, 127.32, 115.42, 115.42, 115.12. HREI-MS:
6
): d
4.3. Thymidine phosphorylase assay
1
Since human TP is not easily to get, we used commercially
m/z calcd for C15H11FN4 [M]+ 266.0968; Found 266.0974; Anal.
available recombinant E. coli TP. Main sequence of TP is frequently
preserved throughout evolution as mammalian TP is reported to
share 39% sequence resemblance with the TP of E. coli. The
mammalian enzyme as well shared up to 70% resemblance with
the active site residues, and three dimensional structure of E. coli
TP enzyme. The Thymidine phosphorylase/PD-ECGF (E. coli)
activity was determined by measuring the absorbance at 290 nm
Calcd for C15H11FN , C, 67.66; H, 4.16; N, 21.04; Found C, 67.64;
H, 4.18; N, 21.06. IR (cm , KBr): 1583 (C@N).
4
ꢀ1
4
.4.4. (E)-2-(2-(2-chlorobenzylidene)hydrazinyl)quinoxaline (4)
Yield: 79%. m.p.: 210–211 °C. H NMR (500 MHz, DMSO-d
1
6
): d
1
1
2
1
1
C
C
1.92 (s, 1H), 9.13 (s, 1H), 8.53 (s, 1H), 8.17 (dd, J = 7.5, 1.8 Hz,
H), 7.94 (d, J = 8.2 Hz, 1H), 7.76–7.65 (m, 2H), 7.60–7.48 (m,
spectrophotometrically. In brief, total reaction mixture of 200
contained 145 L of potassium phosphate buffer (pH 7.4), 30
l
l
L
L
13
6
H), 7.48–7.37 (m, 2H). C NMR (125 MHz, DMSO-d ): d 150.1;
l
40.8; 138.0; 137.6; 136.4; 132.2; 131.9; 130.5; 130.3; 129.8;
28.8; 127.5; 126.6; 126.3; 125.4 ppm. HREI-MS: m/z calcd for
of enzyme (E. coli) at concentration 0.05 and 0.002 U, respectively,
were incubated with 5 L of test materials for 10 min at 25 °C in
microplate reader. After incubation, pre read at 290 nm was taken
to deduce the absorbance of substrate particles. Substrate (20 L,
.5 mM), was dissolved in potassium phosphate buffer, was imme-
diately added to plate and continuously read after 10, 20, and
0 min in microplate reader (spectra max, molecular devices, CA,
USA). All assays were performed in triplicate.
l
15
H
11ClN
11ClN
4
[M]+ 282.0672; Found 282.0678; Anal. Calcd for
15
H
4
, C, 63.72; H, 3.92; N, 19.82; Found C, 63.74; H, 3.91;
l
ꢀ1
N, 19.83. IR (cm , KBr): 1584 (C@N).
1
4
.4.5. (E)-2-(2-(3-chlorobenzylidene)hydrazinyl)quinoxaline (5)
Yield: 82%. m.p.: 235 °C. H NMR (500 MHz, DMSO-d ): d 11.87
6
3
1
(
(
(
1
1
1
2
s, 1H), 9.16 (s, 1H), 8.92 (s, 1H), 8.57 (d, J = 4.4 Hz, 1H), 8.23
d, J = 7.9 Hz, 1H), 8.18 (s, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.70
q, J = 8.4 Hz, 2H), 7.52 (t, J = 6.8 Hz, 1H), 7.48 (dd, J = 7.9, 4.8 Hz,
4.3.1. Calculations
Reactions for above mentioned biological activities were carried
1
3
6
H). C NMR (125 MHz, DMSO-d ): d 150.2; 140.8; 140.2; 138.0;
out in triplicate. Results were then processed using SoftMax Pro 4.8
software (Molecular Devices, CA, USA) and then by Microsoft Excel.
Percent inhibition for above mentioned biological activities was
calculated by following formula:
37.0; 136.5; 133.7; 130.6; 130.3; 128.8; 127.1; 126.2; 125.7;
25.3; 125.2 ppm. HREI-MS: m/z calcd for
C
15
4
H11ClN [M]+
82.0672; Found 282.0679; Anal. Calcd for C15
H
11ClN , C, 63.72;
4
ꢀ
1
H, 3.92; N, 19.82; Found C, 63.73; H, 3.93; N, 19.80. IR (cm
,
Percent Inhibition ¼ 100 ꢀ ðODtest compound=ODcontrolÞ ꢁ 100
KBr): 1587 (C@N).

M. Taha et al. / Bioorganic Chemistry 68 (2016) 80–89
87
4
.4.6. (E)-2-(2-(3-chlorobenzylidene)hydrazinyl)quinoxaline (6)
Yield: 89%. m.p.: 249–250 °C. H NMR (500 MHz, DMSO-d
C
16
H
14
N
4
, C, 73.26; H, 5.38; N, 21.36; Found C, 73.27; H, 5.39; N,
1
ꢀ1
6
): d
21.34. IR (cm , KBr): 1581 (C@N).
1
7
1.76 (s, 1H), 9.11 (s, 1H), 8.13 (s, 1H), 7.92 (d, J = 8.1 Hz, 1H),
1
3
.80 (d, J = 8.5 Hz, 2H), 7.73–7.65 (m, 2H), 7.55–7.47 (m, 3H).
): d 150.2; 140.8; 140.4; 138.0; 136.4;
33.6; 133.5; 130.3; 128.8; 128.7; 128.1; 126.2; 125.2 ppm.
C
4
.4.12. (E)-3-((2-(quinoxalin-2-yl)hydrazono)methyl)phenol (12)
Yield: 86%. m.p.: 269–270 °C. H NMR (500 MHz, DMSO-d
NMR (125 MHz, DMSO-d
1
6
1
6
): d
1
1.64 (s, 1H), 9.58 (s, 1H), 9.05 (s, 1H), 8.07 (s, 1H), 7.92
HREI-MS: m/z calcd for
C
15
H
11ClN [M]+ 282.0672; Found
4
(
2
d, J = 7.9 Hz, 1H), 7.73–7.63 (m, 2H), 7.49 (ddd, J = 8.3, 6.3,
.0 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H), 7.21–7.19 (m, 1H), 7.15 (d,
2
1
82.06774; Anal. Calcd for C15 11ClN , C, 63.72; H, 3.92; N,
9.82; Found C, 63.74; H, 3.91; N, 19.81. IR (cm , KBr): 1578
H
4
ꢀ1
13
J = 7.7 Hz, 1H), 6.81 (dd, J = 8.0, 2.4 Hz, 1H). C NMR (125 MHz,
DMSO-d ): d 157.7; 150.3; 142.1; 141.0; 137.9; 136.3; 136.0;
30.3; 129.9; 128.8; 126.2; 125.1; 118.0; 116.6; 112.3. HREI-MS:
m/z calcd for C15 O [M]+ 264.1011; Found 264.1018; Anal.
Calcd for C15
(
C@N).
6
1
4.4.7. (E)-2-(2-(2-nitrobenzylidene)hydrazinyl)quinoxaline (7)
12 4
H N
1
Yield: 81%. m.p.: 220–221 °C. H NMR (500 MHz, DMSO-d
6
): d
H
12
N
4
O, C, 68.17; H, 4.58; N, 21.20; Found C, 68.18;
12.05 (s, 1H), 9.09 (s, 1H), 8.52 (s, 1H), 8.26 (d, J = 7.7 Hz, 1H),
8.03 (d, J = 8.1 Hz, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.78 (t, J = 7.6 Hz,
1H), 7.72 (dd, J = 7.8, 7.2 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.61 (t,
ꢀ1
H, 4.56; N, 21.18. IR (cm , KBr): 1574 (C@N).
13
4.4.13. (E)-4-((2-(quinoxalin-2-yl)hydrazono)methyl)phenol (13)
J = 7.5 Hz, 1H), 7.52 (t, J = 7.4 Hz, 1H). C NMR (125 MHz, DMSO-
): d 150.0; 147.6; 140.7; 138.2; 136.5; 136.3; 133.3; 130.4;
29.6; 128.8; 128.7; 127.9; 126.4; 125.6; 124.5 ppm. HREI-MS:
m/z calcd for C15 [M]+ 293.0913; Found 293.0919; Anal.
Calcd for C15 , C, 61.43; H, 3.78; N, 23.88; Found C, 61.44;
H, 3.76; N, 23.86. IR (cm , KBr): 3489 (NH); 1580 (C@N); 1512
and 1344 (NO ).
1
Yield: 95%. m.p.: 257–258 °C. H NMR (500 MHz, DMSO-d
6
): d
d
6
1
1.46 (s, 1H), 9.82 (s, 1H), 9.05 (s, 1H), 8.06 (s, 1H), 7.90
1
(
(
d, J = 8.3 Hz, 1H), 7.68–7.63 (m, 2H), 7.60 (d, J = 8.5 Hz, 2H), 7.46
11 5 2
H N O
ddd, J = 8.2, 5.6, 2.7 Hz, 1H), 6.84 (d, J = 8.5 Hz, 2H). 13C NMR
11 5 2
H N O
ꢀ
1
6
(125 MHz, DMSO-d ): d 158.7; 150.4; 142.4; 141.1; 137.7; 136.4;
1
30.2; 128.7; 128.2; 128.2; 126.0; 125.7; 124.7; 115.7; 115.7.
HREI-MS: m/z calcd for [M]+ 264.1011; Found
64.1017; Anal. Calcd for C15 O, C, 68.17; H, 4.58; N, 21.20;
Found C, 68.18; H, 4.59; N, 21.18. IR (cm , KBr): 3489 (OH);
2
C
15
H
12
4
N O
2
H
12
N
4
4.4.8. (E)-2-(2-(4-nitrobenzylidene)hydrazinyl)quinoxaline (8)
ꢀ1
1
6
Yield: 85%. m.p.: 270–271 °C. H NMR (500 MHz, DMSO-d ): d
1
599 (C@N).
1
8
7
1
1
+
2.08 (s, 1H), 9.18 (s, 1H), 8.28 (d, J = 8.3 Hz, 2H), 8.23 (s, 1H),
.04 (d, J = 8.4 Hz, 2H), 7.96 (d, J = 8.2 Hz, 1H), 7.78–7.66 (m, 2H),
.54 (t, J = 7.3 Hz, 1H). 13C NMR (125 MHz, DMSO-d
): d 150.0;
47.1; 141.2; 140.7; 139.1; 138.2; 136.4; 130.4; 128.8; 127.2;
6
4.4.14. (E)-3-((2-(quinoxalin-2-yl)hydrazono)methyl)benzene-1,2-
diol (14)
26.4; 125.7; 124.0 ppm. HREI-MS: m/z calcd for C15
293.0913; Found 293.0906; Anal. Calcd for
H
11
N
5
O
2
[M]
Yield: 82%. m.p.: 240–241 °C. 1H NMR (500 MHz, DMSO-d
6
): d
1.70 (s, 1H), 10.08 (s, 1H), 9.35 (s, 1H), 8.85 (s, 1H), 8.43 (s, 1H),
C
15
11
H N
5 2
O ,
1
7
5
1
1
1
C
C
C, 61.43; H, 3.78; N, 23.88; Found C, 61.42; H, 3.76; N, 23.85.
.91 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 2.7 Hz, 2H), 7.49 (ddd, J = 8.2,
.3, 3.0 Hz, 1H), 7.16 (d, J = 7.6 Hz, 1H), 6.82 (dd, J = 7.8, 1.3 Hz,
ꢀ1
2
IR (cm , KBr): 3412 (NH); 1578 (C@N); 1510 and 1340 (NO ).
1
3
6
H), 6.73 (t, J = 7.8 Hz, 1H). C NMR (125 MHz, DMSO-d ): d
4.4.9. (E)-2-(2-(2-methylbenzylidene)hydrazinyl)quinoxaline (9)
49.5; 145.6; 145.0; 142.2; 141.1; 137.8; 136.7; 130.3; 128.7;
26.2; 125.0; 120.4; 119.2; 117.9; 116.3. HREI-MS: m/z calcd for
1
6
Yield: 91%. m.p.: 162–163 °C. H NMR (500 MHz, DMSO-d ): d
1
7
2
1
1
C
C
2
1.65 (s, 1H), 9.08 (s, 1H), 8.42 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H),
15
H
H
12
N
N
4
O
O
2
[M]+ 280.0960; Found 280.0953; Anal. Calcd for
.68 (q, J = 8.0 Hz, 2H), 7.49 (t, J = 7.0 Hz, 1H), 7.39–7.12 (m, 3H),
15
12
4
2
, C, 64.28; H, 4.32; N, 19.99; Found C, 64.29; H, 4.30;
1
3
.47 (s, 3H).
C NMR (125 MHz, DMSO-d
6
): d 150.3; 140.9;
ꢀ1
N, 20.01. IR (cm , KBr): 1584 (C@N).
40.9; 137.9; 136.4; 136.0; 132.6; 130.9; 130.3; 128.9; 128.8;
26.5; 126.2; 126.0; 125.1; 19.4. HREI-MS: m/z calcd for
4
.4.15. (E)-4-((2-(quinoxalin-2-yl)hydrazono)methyl)benzene-1,3-
diol (15)
Yield: 95%. m.p.: 280–281 °C. 1H NMR (500 MHz, DMSO-d
1.49 (s, 1H), 10.70 (s, 1H), 9.80 (s, 1H), 8.79 (s, 1H), 8.32 (s, 1H),
.88 (d, J = 8.1 Hz, 1H), 7.70–7.59 (m, 2H), 7.55–7.40 (m, 2H),
16
H
H
14
N
N
4
[M]+ 262.1218; Found 262.1225; Anal. Calcd for
, C, 73.26; H, 5.38; N, 21.36; Found C, 73.27; H, 5.36; N,
16
14
4
ꢀ1
6
): d
1.34. IR (cm , KBr): 1591 (C@N).
1
7
4.4.10. (E)-2-(2-(3-methylbenzylidene)hydrazinyl)quinoxaline (10)
1
3
1
6.36 (dd, J = 6.6, 2.1 Hz, 2H). C NMR (125 MHz, DMSO-d
1
1
for C15H N O
12 4 2
C
6
): d
60.0; 158.2; 149.5; 142.7; 141.2; 137.6; 136.8; 130.2; 129.1;
28.7; 126.1; 124.7; 111.7; 107.7; 102.5 ppm. HREI-MS: m/z calcd
[M]+ 280.0960; Found 280.0968; Anal. Calcd for
, C, 64.28; H, 4.32; N, 19.99; Found C, 64.26; H, 4.30;
Yield: 78%. M.P.: 194–195 °C. H NMR (500 MHz, DMSO-d
6
):
d 11.67 (s, 1H), 9.11 (s, 1H), 8.12 (s, 1H), 7.92 (d, J = 8.2 Hz, 1H),
.74–7.64 (m, 2H), 7.60 (s, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.49 (t,
J = 7.2 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 7.21 (d, J = 7.4 Hz, 1H), 2.38
7
1
3
15
12
H N
4
O
2
(
1
1
s, 3H). C NMR (125 MHz, DMSO-d
6
): d 150.3; 142.0; 140.9;
38.0; 137.9; 136.4; 134.6; 130.2; 129.9; 128.7; 128.6; 126.9;
26.1; 125.0; 123.8; 20.9 ppm. HREI-MS: m/z calcd for C16
ꢀ1
N, 19.98. IR (cm , KBr): 3435 (NH); 1584 (C@N).
14 4
H N
[
M]+ 262.1218; Found 262.1223; Anal. Calcd for C16
14
H N
4
, C,
4.4.16. (E)-2-((2-(quinoxalin-2-yl)hydrazono)methyl)benzene-1,4-
7
3.26; H, 5.38; N, 21.36; Found C, 73.27; H, 5.36; N, 21.35. IR
diol (16)
ꢀ1
Yield: 91%. m.p.: 271–272 °C. 1H NMR (500 MHz, DMSO-d
(
cm , KBr): 3445 (NH); 1595 (C@N).
6
): d
1.64 (s, 1H), 9.72 (s, 1H), 8.90 (d, J = 9.9 Hz, 2H), 8.37 (s, 1H),
1
4
.4.11. (E)-2-(2-(4-methylbenzylidene)hydrazinyl)quinoxaline (11)
Yield: 81%. m.p.: 220–221 °C. H NMR (500 MHz, DMSO-d ): d
6
1.61 (s, 1H), 9.09 (s, 1H), 8.12 (s, 1H), 7.92 (d, J = 8.1 Hz, 1H),
7.91 (d, J = 8.1 Hz, 1H), 7.78–7.55 (m, 2H), 7.55–7.38 (m, 1H),
7.17 (d, J = 2.5 Hz, 1H), 6.75 (d, J = 8.6 Hz, 1H), 6.68 (dd, J = 8.6,
1
1
3
1
2.3 Hz, 1H).
6
C NMR (125 MHz, DMSO-d ): d 150.0; 149.8;
7
.74–7.59 (m, 4H), 7.49 (ddd, J = 8.2, 6.1, 2.2 Hz, 1H), 7.27 (d,
149.2; 141.1; 140.8; 137.8; 136.5; 130.3; 128.7; 126.2; 125.0;
120.5; 118.0; 116.9; 111.8 ppm. HREI-MS: m/z calcd for
1
3
J = 7.9 Hz, 2H). C NMR (125 MHz, DMSO-d
6
): d 150.3; 142.0;
41.0; 138.9; 137.8; 136.4; 132.0; 130.3; 129.4; 129.4; 128.8;
26.5; 126.5; 126.1; 125.0; 21.0. HREI-MS: m/z calcd for
[M]+ 262.1218; Found 262.1213; Anal. Calcd for
1
1
C
C
C
15
H
H
12
N
N
4
O
O
2
[M]+ 280.0960; Found 280.0951; Anal. Calcd for
, C, 64.28; H, 4.32; N, 19.99; Found C, 64.31; H, 4.31;
15
12
4
2
ꢀ1
16
H
14
N
4
N, 20.00. IR (cm , KBr): 3454 (O-H); 1589 (C@N).

8
8
M. Taha et al. / Bioorganic Chemistry 68 (2016) 80–89
4
.4.17. (E)-4-((2-(quinoxalin-2-yl)hydrazono)methyl)benzene-1,2-
diol (17)
Yield: 85%. m.p.: 263–264 °C. 1H NMR (500 MHz, DMSO-d
1.43 (s, 1H), 9.33 (s, 1H), 9.18 (s, 1H), 8.99 (s, 1H), 7.98 (s, 1H),
.89 (d, J = 8.3 Hz, 1H), 7.66 (d, J = 3.3 Hz, 2H), 7.63–7.40 (m, 1H),
7.73–7.47 (m, 3H), 7.41 (dd, J = 55.1, 4.5 Hz, 1H), 7.32 (dd, J = 9.7,
1
3
2.2 Hz, 2H), 6.99–6.82 (m, 1H), 3.82 (s, 3H). C NMR (125 MHz,
DMSO-d ): d 159.6; 150.3; 141.7; 140.9; 137.9; 136.4; 136.1;
130.3; 129.8; 128.8; 126.1; 125.1; 119.2; 115.3; 110.9; 55.2 ppm.
HREI-MS: m/z calcd for [M]+ 278.1168; Found
278.1172; Anal. Calcd for C16 O, C, 69.05; H, 5.07; N, 20.13;
6
): d
6
1
7
7
C
16
H
14
4
N O
1
3
.24 (s, 1H), 6.97 (d, J = 8.1 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H).
): d 150.3; 147.3; 145.7; 142.8; 141.1;
37.7; 136.4; 130.2; 128.7; 126.2; 126.0; 124.7; 119.5; 115.6;
12.5 ppm. HREI-MS: m/z calcd for C15 [M]+ 280.0960;
, C, 64.28; H, 4.32; N,
C
H
14
N
4
ꢀ
1
NMR (125 MHz, DMSO-d
1
1
6
Found C, 69.07; H, 5.05; N, 20.12. IR (cm , KBr): 3453 (NH);
1582 (C@N).
12 4 2
H N O
Found 280.0964; Anal. Calcd for C15
9.99; Found C, 64.30; H, 4.35; N, 19.97. IR (cm , KBr): 3500
OH); 3464 (NH); 1591 (C@N).
H
12
N
4
O
2
4.4.23. (E)-2-(2-(4-methoxybenzylidene)hydrazinyl)quinoxaline (23)
ꢀ1
1
1
(
Yield: 89%. m.p.: 220–222 °C. H NMR (400 MHz, DMSO-d
6
): d
11.55 (s, 6H), 9.07 (s, 6H), 8.10 (s, 6H), 7.90 (d, J = 8.1 Hz, 7H),
.71 (d, J = 8.7 Hz, 12H), 7.66 (d, J = 6.4 Hz, 9H), 7.47 (s, 5H), 7.01
7
1
3
4
.4.18. (E)-5-((2-(quinoxalin-2-yl)hydrazono)methyl)benzene-1,2,4-
triol (18)
Yield: 87%. m.p.: 263–264 °C. 1H NMR (500 MHz, DMSO-d
1.41 (s, 1H), 9.87 (s, 1H), 9.37 (s, 1H), 8.80 (s, 1H), 8.50 (s, 1H),
.28 (s, 1H), 7.88 (d, J = 7.7 Hz, 1H), 7.64 (s, 2H), 7.45 (s, 1H), 7.07
6
(d, J = 8.6 Hz, 13H), 3.81 (s, 17H). C NMR (125 MHz, DMSO-d ):
d 160.2; 150.3; 141.8; 141.0; 137.8; 136.4; 130.2; 128.7; 128.0;
127.3; 126.0; 124.8; 114.3; 55.2 ppm. HREI-MS: m/z calcd for
6
): d
1
8
C
C
16
H
H
14
N
N
4
O [M]+ 278.1168; Found 278.1161; Anal. Calcd for
16
14
4
O, C, 69.05; H, 5.07; N, 20.13; Found C, 69.03; H, 5.04;
1
3
ꢀ1
(
s, 1H), 6.36 (s, 1H). C NMR (125 MHz, DMSO-d
6
): d 153.88,
50.83, 145.73, 143.97, 143.89, 141.98, 140.21, 139.09, 131.88,
29.32, 129.27, 127.36, 117.27, 112.91, 102.80. HREI-MS: m/z calcd
[M]+ 296.0909; Found 296.0916; Anal. Calcd for
, C, 60.81; H, 4.08; N, 18.91; Found C, 60.82; H, 4.09;
N, 20.14. IR (cm , KBr): 3451 (NH); 1612 (C@N).
1
1
4.4.24. (E)-4-methoxy-2-((2-(quinoxalin-2-yl)hydrazono)methyl)
phenol (24)
12 4 3
for C15H N O
C
H
12
N
4
O
3
Yield: 91%. m.p.: 258–261 °C. 1H NMR (500 MHz, DMSO-d
6
): d
15
ꢀ
1
N, 18.89. IR (cm , KBr): 3500 (OH); 3464 (NH); 1591 (C@N).
11.69 (s, 1H), 9.98 (s, 1H), 8.97 (s, 1H), 8.42 (s, 1H), 7.91 (d,
J = 8.2 Hz, 1H), 7.71–7.65 (m, 2H), 7.49 (ddd, J = 8.2, 6.2, 2.1 Hz,
1H), 7.31 (s, 1H), 6.86 (d, J = 1.6 Hz, 2H), 3.77 (s, 3H). 13C NMR
4
.4.19. (E)-5-((2-(quinoxalin-2-yl)hydrazono)methyl)benzene-2,4,6-
triol (19)
Yield: 93%. m.p.: 274–276 °C. 1H NMR (500 MHz, DMSO-d
1.53 (s, 1H), 10.93 (s, 2H), 9.76 (s, 1H), 8.58 (s, 1H), 8.50 (s, 1H),
.87 (d, J = 8.1 Hz, 1H), 7.65 (d, J = 3.8 Hz, 2H), 7.46 (dt, J = 8.2,
.0 Hz, 1H), 5.88 (s, 2H). 13C NMR (125 MHz, DMSO-d
): d 163.29,
(125 MHz, DMSO-d
141.98, 139.09, 131.88, 129.32, 129.27, 127.36, 121.70, 116.57,
116.33, 112.80, 56.03; HREI-MS: m/z calcd for C16 [M]+
294.1117; Found 294.1117; Anal. Calcd for C16
6
): d 154.41, 154.30, 145.73, 143.97, 143.89,
6
): d
1
7
4
1
1
14 4 2
H N O
H
14
N
4
O
2
, C, 65.30;
ꢀ1
6
H, 4.79; N, 19.04; Found C, 65.29; H, 4.77; N, 19.06. IR (cm
,
61.49, 161.49, 143.87, 143.92, 142.94, 141.92, 139.89, 131.58,
29.31, 129.25, 127.16, 106.63, 95.78, 95.78. HREI-MS: m/z calcd
KBr): 1589 (C@N).
for C15
H
12
N
4
O
3
[M]+ 296.0909; Found 296.0901; Anal. Calcd for
4.4.25. (E)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)quinoxaline (25)
1
C
15
H
12
N
4
3
O , C, 60.81; H, 4.08; N, 18.91; Found C, 60.82; H, 4.09;
Yield: 86%. m.p.: 236–238 °C. H NMR (400 MHz, DMSO-d
6
): d
ꢀ1
N, 18.90. IR (cm , KBr): 3467 (OH); 3264 (NH); 1592 (C@N).
11.97 (s, 1H), 9.13 (s, 1H), 8.47 (s, 1H), 8.18 (d, J = 8.6 Hz, 1H),
7
.94 (d, J = 8.0 Hz, 1H), 7.74–7.67 (m, 3H), 7.53 (dd, J = 8.1, 1.6 Hz,
1
3
4
.4.20. (E)-5-methoxy-2-((2-(quinoxalin-2-yl)hydrazono)methyl)
phenol (20)
Yield: 91%. m.p.: 252–255 °C. 1H NMR (500 MHz, DMSO-d
1.57 (s, 1H), 10.87 (s, 1H), 8.82 (s, 1H), 8.36 (s, 1H), 7.89 (d,
J = 7.9 Hz, 1H), 7.67 (s, 2H), 7.61 (d, J = 8.5 Hz, 1H), 7.47 (s, 1H),
6
1H), 7.50 (dd, J = 8.6, 1.8 Hz, 1H). C NMR (125 MHz, DMSO-d ):
d 149.85, 146.31, 143.97, 143.89, 141.98, 141.65, 139.09, 135.66,
133.80, 131.88, 129.32, 129.27, 127.36, 124.41. HREI-MS: m/z calcd
6
): d
1
for C14
H
11
N
5
[M]+ 249.1014; Found 249.1019; Anal. Calcd for
C H N , C, 67.46; H, 4.45; N, 28.10; Found C, 67.47; H, 4.43; N,
14 11 5
ꢀ1
6
.53 (dd, J = 8.6, 2.3 Hz, 1H), 6.49 (d, J = 2.3 Hz, 1H), 3.77 (s, 4H).
28.11. IR (cm , KBr): 3524 (NH); 1621 (C@N).
1
3
C NMR (125 MHz, DMSO-d
43.89, 141.98, 139.09, 131.88, 130.82, 129.32, 129.27, 127.36,
13.95, 107.29, 102.05, 56.03. HREI-MS: m/z calcd for C16
6
): d 162.00, 161.23, 147.11, 143.97,
1
1
4.4.26. (E)-2-(2-(pyridin-4-ylmethylene)hydrazinyl)quinoxaline (26)
1
H
14
N
4
O
2
Yield: 92%. m.p.: 256–257 °C. H NMR (500 MHz, DMSO-d
6
): d
[
6
(
M]+ 294.1117; Found 294.1125; Anal. Calcd for C16
H
14
N
4
O
2
, C,
12.02 (s, 1H), 9.18 (s, 1H), 8.63 (d, J = 6.0 Hz, 2H), 8.11 (s, 1H),
7.96 (d, J = 7.9 Hz, 1H), 7.77–7.68 (m, 4H), 7.57–7.52 (m, 1H). 13
NMR (125 MHz, DMSO-d ): d 150.19, 146.30, 143.94, 143.82,
41.92, 140.73, 139.19, 131.85, 129.39, 129.17, 127.48, 122.25,
122.25; HREI-MS: m/z calcd for C14 [M]+ 249.1014; Found
249.1019; Anal. Calcd for C14
5.30; H, 4.79; N, 19.04; Found C, 65.31; H, 4.81; N, 19.06. IR
C
ꢀ1
cm , KBr): 3515 (OH); 3354 (NH); 1583 (C@N).
6
1
4
.4.21. (E)-2-methoxy-5-((2-(quinoxalin-2-yl)hydrazono)methyl)
phenol (21)
Yield: 81%. m.p.: 243–244 °C. 1H NMR (500 MHz, DMSO-d
1.50 (s, 1H), 9.22 (s, 1H), 9.02 (s, 1H), 8.01 (s, 1H), 7.90 (d,
J = 8.1 Hz, 1H), 7.74–7.63 (m, 2H), 7.47 (ddd, J = 8.3, 5.7, 2.6 Hz,
H), 7.30 (d, J = 2.0 Hz, 1H), 7.08 (dd, J = 8.3, 2.0 Hz, 1H), 6.97 (d,
11 5
H N
H
11
N
5
, C, 67.46; H, 4.45; N, 28.10;
ꢀ1
6
): d
Found C, 67.47; H, 4.43; N, 28.08; IR (cm , KBr): 1578 (C@N).
1
4.4.27. (E)-2-(2-((1H-indol-3-yl)methylene)hydrazinyl)quinoxaline
1
(27)
1
3
Yield: 96%. m.p.: 256–257 °C. 1H NMR (500 MHz, DMSO-d
J = 8.3 Hz, 1H). C NMR (125 MHz, DMSO-d
1
1
for C16
C
6
): d 149.90, 146.65,
45.38, 143.97, 143.89, 141.98, 139.09, 131.88, 129.56, 129.32,
29.27, 127.36, 120.35, 115.24, 115.11, 56.78. HREI-MS: m/z calcd
[M]+ 294.1117; Found 294.1126; Anal. Calcd for
, C, 65.30; H, 4.79; N, 19.04; Found C, 65.28; H, 4.77;
6
): d
11.52 (s, 1H), 11.33 (s, 1H), 9.10 (s, 1H), 8.37 (s, 1H), 8.33–8.27
(m, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.80 (s, 1H), 7.65 (d, J = 3.7 Hz,
1
3
H
14
N
4
O
2
2H), 7.50–7.42 (m, 2H), 7.27–7.20 (m, 2H). C NMR (125 MHz,
DMSO-d ): d 143.97, 143.89, 141.98, 139.09, 134.84, 134.81,
134.47, 131.88, 129.32, 129.27, 127.36, 127.11, 121.32, 120.74,
18.38, 113.20, 105.66. HREI-MS: m/z calcd for C17 [M]+
16
H
14
N
4
O
2
6
ꢀ
1
N, 19.01. IR (cm , KBr): 3428 (OH); 3451 (NH); 1624 (C@N).
1
13 5
H N
4
.4.22. (E)-2-(2-(3-methoxybenzylidene)hydrazinyl)quinoxaline (22)
287.1171; Found 287.1178; Anal. Calcd for C17
4.56; N, 24.37; Found C, 71.07; H, 4.54; N, 24.35. IR (cm , KBr):
H
13
N
5
, C, 71.06; H,
1
ꢀ1
Yield: 75%. m.p.: 179–180 °C. H NMR (400 MHz, DMSO-d
6
): d
1
1.71 (s, 1H), 9.13 (s, 1H), 8.12 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H),
1618 (C@N).

M. Taha et al. / Bioorganic Chemistry 68 (2016) 80–89
89
4
.4.28. (E)-2-((2-(quinoxalin-2-yl)hydrazono)methyl)phenol (28)
[20] S.A. Diab, S.C. De, M. Muzard, R. Plantier-Royon, E. Pfund, T. Lequeux, J. Med.
Chem. 55 (2012) 2758.
1
Yield: 95%. m.p.: 256–257 °C. H NMR (500 MHz, DMSO-d
6
): d
[
21] H.M. Refaat, A.A. Moneer, O.M. Khalil, Arch. Phram. Res. 27 (2004) 1093.
22] S.A. El-Hawash, N.S. Habib, N.H. Fanaki, Pharmazie 54 (1999) 808.
1
1.69 (s, 1H), 10.54 (s, 1H), 8.91 (s, 1H), 8.45 (s, 1H), 7.91 (d,
[
J = 8.2 Hz, 1H), 7.74 (d, J = 7.5 Hz, 1H), 7.68 (q, J = 8.3 Hz, 2H),
[23] A. Monge, F.J. Martinez-Crespo, A.L. Cerain, J.A. Palop, S. Narro, V. Senador, A.
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7
2
1
1
.57–7.45 (m, 1H), 7.30–7.21 (m, 1H), 6.92 (dd, J = 16.1, 7.9 Hz,
4488.
1
3
H). C NMR (125 MHz, DMSO-d
37.8; 136.7; 130.5; 130.3; 128.8; 127.1; 126.2; 125.1; 120.2;
19.4; 116.2. HREI-MS: m/z calcd for C15 O [M]+ 264.1011;
O, C, 68.17; H, 4.58; N,
6
): d 156.3; 149.7; 141.1; 141.0;
[
[
[
24] V.K. Tandon, D.B. Yadav, H.K. Maurya, A.K. Chaturvedi, P.K. Shukla, Bioorg.
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12 4
H N
1169.
Found 264.1002; Anal. Calcd for C15
1.20; Found C, 68.18; H, 4.56; N, 21.18. IR (cm , KBr): 3491
OH); 1580 (C@N).
12 4
H N
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2
(
[
[
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The author (Dr. M. Taha) would like to acknowledge Universiti
Teknologi MARA – Malaysia for the financial support under the
Research Intensive Faculty grant scheme with reference number
UiTM 600-RMI/DANA 5/3/LESTARI (54/2015) and The author (Dr.
M.A.) is grateful to Higher Education Commission of Pakistan for
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