Design and synthesis of 2-Substituted-4-benzyl-5-methylimidazoles as new potential Anti-breast cancer agents to inhibit oncogenic STAT3 functions

Article abstract of DOI:10.1016/j.bioorg.2021.105033

STAT3 signaling is known to be associated with tumorigenesis and further cancer cell-intrinsic activation of STAT3 leads to altered regulation of several oncogenic processes. Given the importance of STAT3 in cancer development and progression particularly breast cancer, it is crucial to discover new chemical entities of STAT3 inhibitor to develop anti-breast cancer drug candidates. Herein, 4-benzyl-2-benzylthio-5-methyl-1H-imidazole (2a) and 4-benzyl-5-methyl-2-[(2,6-difluorobenzyl)thio]-1H-imidazole (2d) from a group of thirty imidazole-bearing compounds showed greater STAT3 inhibition than their lead compounds VS1 and the oxadiazole derivative MD77. Within all tested compounds, ten derivatives effectively inhibited the growth of the two tested breast cancer cells with IC₅₀ values ranging from 6.66 to 26.02 μM. In addition, the most potent derivatives 2a and 2d inhibited the oncogenic function of STAT3 as seen in the inhibition of colony formation and IL-6 production of breast cancer cell lines. Modeling studies provided evidence for the possible interactions of the synthesized compounds with the key residues of the STAT3-SH2 domain. Collectively, our present study suggests 2-substituted-4-benzyl-5-methylimidazoles are a new class of anti-cancer drug candidates to inhibit oncogenic STAT3 function.

Full text of DOI:10.1016/j.bioorg.2021.105033

Bioorganic Chemistry 113 (2021) 105033
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and synthesis of 2-Substituted-4-benzyl-5-methylimidazoles as new
potential Anti-breast cancer agents to inhibit oncogenic STAT3 functions
,
,
Botros Y. Beshaya 1, Amira A. Abdellatefb 1, Yasser M. Lokshac, Salwa M. Fahmyd,
Nargues S. Habibd, Alaa El-Din A. Bekhite, Paris E. Georghiouf, Yoshihiro Hayakawab
,
,
*
,g,h,*
Adnan A. Bekhitd
a Department of Pharmaceutical Chemistry, College of Pharmacy, Arab Academy for Science, Technology and Maritime Transport, Alexandria, Egypt
b Section of Host Defences, Institute of Natural Medicine, University of Toyama, Toyama, Japan
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sinai University, Al-Arish, North Sinai, Egypt
d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, 2152 Alexandria, Egypt
e Department of Food Science, University of Otago, Dunedin, New Zealand
f Department of Chemistry, Memorial University of Newfoundland, St. John’s, NL, Canada
g Pharmacy Program, Allied Health Department, College of Health and Sport Sciences, University of Bahrain, Bahrain
h Cancer Nanotechnology Research Laboratory (CNRL), Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Imidazole
STAT3
STAT3 signaling is known to be associated with tumorigenesis and further cancer cell-intrinsic activation of
STAT3 leads to altered regulation of several oncogenic processes. Given the importance of STAT3 in cancer
development and progression particularly breast cancer, it is crucial to discover new chemical entities of STAT3
inhibitor to develop anti-breast cancer drug candidates. Herein, 4-benzyl-2-benzylthio-5-methyl-1H-imidazole
(2a) and 4-benzyl-5-methyl-2-[(2,6-difluorobenzyl)thio]-1H-imidazole (2d) from a group of thirty imidazole-
bearing compounds showed greater STAT3 inhibition than their lead compounds VS1 and the oxadiazole de-
rivative MD77. Within all tested compounds, ten derivatives effectively inhibited the growth of the two tested
breast cancer cells with IC50 values ranging from 6.66 to 26.02 µM. In addition, the most potent derivatives 2a
and 2d inhibited the oncogenic function of STAT3 as seen in the inhibition of colony formation and IL-6 pro-
duction of breast cancer cell lines. Modeling studies provided evidence for the possible interactions of the
synthesized compounds with the key residues of the STAT3-SH2 domain. Collectively, our present study suggests
2-substituted-4-benzyl-5-methylimidazoles are a new class of anti-cancer drug candidates to inhibit oncogenic
STAT3 function.
Anti-cancer
IL-6
In silico docking
1. Introduction
transcriptional activities [5]. To date, seven members of the STAT family
have been identified in mammals [6-8]. The activation of STATs in
Signal transducers and activators of transcriptions (STATs) are a
group of latent cytoplasmic proteins that play a crucial physiological
role to be activated by extracellular ligands such as cytokines, growth
factors, and hormones. Several recent reviews on STATs comprehen-
sively highlight their diverse roles [1-5] and the importance of the Janus
tyrosine kinases (JAKs) for their activation [6,7]. In general, phos-
phorylated STATs form homo- or hetero-dimers are translocated to the
nucleus where they regulate the target genes expression through their
normal cells is tightly regulated in order to maintain homeostatic
regulation of their target gene expression, however, the aberrant acti-
vation of STATs in cancer cells has been widely known. Among various
STAT family members, JAK-STAT3 signaling is known to associate with
tumorigenesis [6,7] and further cancer cell-intrinsic activation of STAT3
leads to altered regulation of several oncogenic processes such as pro-
liferation, cell cycle progression, apoptosis, angiogenesis, metastasis,
and immune evasion [9-11]. The cell-intrinsic activation of STAT3 has
* Corresponding authors at: Pharm. Chem. Dept., Faculty of Pharmacy, Alexandria University, Alexandria 215211, Egypt (Adnan A. Bekhit) and Section of Host
Defences, Institute of Natural Medicine, University of Toyama, Sugitani 2630, Toyama, Toyama Japan 930-0194. (Yoshihiro Hayakawa).
E-mail addresses: haya@inm.u-toyama.ac.jp (Y. Hayakawa), adnbekhit@pharmacy.alexu.edu.eg (A.A. Bekhit).
1
These authors are equally contributed to this study.
https://doi.org/10.1016/j.bioorg.2021.105033
Received 28 January 2021; Received in revised form 17 April 2021; Accepted 24 May 2021
Available online 27 May 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

B.Y. Beshay et al.
Bioorganic Chemistry 113 (2021) 105033
been found in many different types of malignancies, such as head, neck,
breast, prostate, skin, and pancreatic cancers [12-16].
with the bioisosteric replacement of the 1,2,5-oxadiazole scaffold which
is present in MD77, with an imidazole ring, the new target compounds
A-F were conceived. Furthermore, the present work also aimed to study
the effect of having different spacers between the imidazole scaffold and
the hydrophobic moiety at C2. The proposed candidates which were
designed and synthesized (Fig. 2) comprised the key four pharmaco-
phore features established by the receptor-based pharmacophore model
constructed by Poli et al. [28]. These newly synthesized compounds
were screened for their anti-proliferative activity against triple-negative
breast cancer cell lines.
Given the importance of STAT3 in cancer, many studies have been
conducted to discover new chemical entities of STAT3 inhibitor by un-
derstanding of the binding to the active site(s) of STAT3 protein using
computational simulations and structural modification [17-20]. While
many approaches have considered different protein domains on STAT3
to design high potent and selective inhibitors of STAT3, the majority of
STAT3 inhibitors were designed to target the SH2 domain of STAT3
which regulates the phosphorylation-induced protein dimerization step
[21-25]. Upon STAT3 dimerization, the molecular interface is formed
between the SH2 domains and the two carboxyl transcription activation
domains to stabilize the dimer and lead to the phosphorylation of
Tyr705 in a specific pocket of the SH2 domain. The conserved arginine
residue in all known SH2 domains (Arg609 in STAT3) are located in the
interior of the SH2 domain and represents the key element for the p-
Tyr705 recognition [20]. Considering this arginine residue stabilizes the
p-Tyr705 binding by forming an energetically-favorable electrostatic
interaction between the negatively charged phosphate and the positive
NH-phosphorylated STAT3, the competitive compounds for the binding
of p-Tyr705 to the SH2 domain of STAT3 has been structurally opti-
mized to synthesize potential STAT3 inhibitors [24,26,27].
2. Results and discussion
2.1. Synthesis of the target compounds
The Dakin-West reaction was used to prepare the imidazole-2-thione
1 (Scheme 1) from d,l-phenylalanine [30-32]. This imidazole-2-thione
derivative 1 is the key precursor compound for the synthesis of the
target imidazole derivatives 2a-e, 3–5, 6a-b, and 7a-h (Schemes 1-3).
As shown in Scheme 1, the desired 2-arylmethylthio-4-benzyl-5-
methyl-1H-imidazoles (2a-e) were obtained by the potassium
carbonate-mediated coupling of 4-benzyl-5-methyl-1,3-dihydro-2H-
imidazole-2-thione 1 and the respective benzylbromide derivatives in
acetone. Previously, Loksha et al. [31] were able to synthesize 2a in 26%
yield by the coupling of 1 with benzyl bromide in the presence of po-
tassium carbonate in N,N-dimethylformamide (DMF). In the present
investigation, 2a was easily obtained in a much higher yield (90%) using
acetone as the solvent.
A systematic STAT3–SH2 binding site analysis was later conducted
by Poli and coworkers [28] who developed a structure-based pharma-
cophore model by considering the protein–protein interactions previ-
ously identified in the STAT3 dimer involving the pTyr705 and Leu706
residues of the phosphopeptide of one monomers with the residues of
the of the SH2 domain of the other monomer. The resulting pharma-
cophore model which they constructed included the following four
components: (i) an electron-withdrawing region to represent the
pTyr705 phosphate group; (ii) an aromatic component representing the
pTyr705 aromatic ring; (iii) a hydrogen-bonding component repre-
It is worth noting that compound 2b showed a doubling of the signals
of both hydrogen and carbon atoms in its 1H NMR and 13C NMR spectra.
This can be explained by the imidazole ring tautomerism, (Fig. 3) that
has been shown to cause the disappearance of some carbon signals of the
imidazole and aliphatic carbons in several other reported compounds
[31].
–
senting the interaction of the amide N H moiety of Leu706 with
Ser636, a second phosphorylation site in the C-terminal domain of the
STAT structure; and (iv) a hydrophobic region representing the Leu706
lateral chain. Their study resulted in the finding of a non-peptide thio-
urea-based STAT3-SH2 inhibitor, VS1 (Fig. 1) [28]. This molecule
exhibited good STAT3 inhibitory activity and was compared with the
more highly potent STAT3 inhibitor lead compound, MD77 (Fig. 1)
[26,27]. Besides, several studies have introduced compounds bearing-
imidazole ring as an important scaffold in drug discovery, particularly
as anti-cancer agents. Recently, certain number of imidazole-based
compounds including dacarbazine, temozolomide, zoledronic acid,
and mercaptopurine are being used in clinical trials for cancer patient
treatment [29].
The coupling reactions of thiols with aryl halides generally require
harsh reaction conditions using solvents such as ethanol heated under
reflux, or DMSO at 90 ◦C [33,34]. Rout et al. [35] reported that CuI
–
could efficiently catalyze the C S cross-coupling of thiols with aryl
halides in the presence of tetra-n-butylammonium bromide (TBAB) in
water. In the current work, the high reactivity of the nitroaryl halide
–
derivatives facilitated the C S cross-coupling reaction due to the strong
electron-withdrawing nitro group substituent(s) in the ortho and/or para
position(s). Thus, the coupling reaction of 4-benzyl-5-methylimidazole-
2-thione 1 (Scheme 2) with 1-chloro-2,4-dinitrobenzene was achieved
using potassium hydroxide in methanol at rt. However, the synthesis of
4-benzyl-5-methyl-2-((2-nitrophenyl)thio)-1H-imidazole 4 required the
use of a strong base, sodium hydride, in DMF.
The objective of the present work was to optimize the affinity of the
structurally different lead compounds VS1 and MD77 with the insertion
of imidazole scaffold, through the application and synthesis of different
drug design strategies. Ring-closure of the thiourea moiety of VS1
afforded a series of more rigid imidazole derivatives which, together
Direct coupling of 1 with phenacyl bromide derivatives in ethanol
using the reported procedure [36] did not afford the desired 2-aroyl-
methythio-4-benzyl-5-methyl-1H-imidazoles 6a, b in good yields, even
Cl
OH
Cl
O
S
O
N
NH
O
N
H
N
H
N
CF3
O
VS1
MD77
Fig. 1. Chemical structures of lead compounds VS1 and MD77.
2

B.Y. Beshay et al.
Bioorganic Chemistry 113 (2021) 105033
Fig. 2. Design of 2,4,5-trisusbstituted imid-
azole derivatives A-F connected to aryl moi-
eties with different small electron-
withdrawing groups via S-methylene, sulfur,
S-hydrazone or S-acetyl linkers. The red cir-
cles or ovals refer to electron withdrawing
features. The blue ovals refer to H-bond
donor features and the cyan squares refer to
the aromatic and hydrophobic features.
Different spacers are highlighted by different
colors. (For interpretation of the references
to colour in this figure legend, the reader is
referred to the web version of this article.)
R1
Me
R
3 = H, CN, Br, CH3
R1,R2 = H, F
N
Me
N
S
O
NH
S
R2
N
H
R3
NH
R2 = H, CH3,Cl, Br, OCH3, CN
A
2,4,5-Trisusbstituted imidazoles
with
R2
B
S-methylene linker
2.4.5- Imidazoles with
S-acetamide linker
OH
Cl
O
S
O
R
1 = H,Cl
N
H
N
H
Me
N
Me
N
S
VS1
N
R4
O
R4 = NO2
R5= H, NO2
H
S
N
H
Cl
R5
C
R1
F
N
O
NH
2,4,5-Trisusbstituted imidazole derivatives
N
CF3
with
S-linker
O
2,4,5-Imidazoles
with S-acetyl linker
MD77
Me
Me
N
O
N
O
S
S
N
HN
N
H
H
N
R5
N
R6
R4
R3
R5 , R6 = different aliphatic chains or ring
R
3 = H, Cl, Br, OCH3
R4 = H, CH3
E
D
2,4,5-Imidazoles with
2,4,5-Imidazoles with
S-hydrazone linker
S-acetamide linker
Me
NH
Me
NH
S
N
R1
a
S
N
H
R2
R3
2a-e
1
b
Me
R1 R2 R3
2
a
b
c
d
e
NH
O
H
H
H
F
H
H
H
F
H
Br
S
N
O
CN
H
H
H
CH3
3
Scheme 1. Reagents and conditions: (a) ArCH2Br, K2CO3, acetone, rt, 8 h (b) 2a, H2O2, EtOH, 60 ◦C, 24 h.
3

B.Y. Beshay et al.
Bioorganic Chemistry 113 (2021) 105033
Me
Me
NH
NH
S
N
NO2
S
N
NO2
4
5
NO2
a
b
Me
NH
O
c
S
N
1
d
R1
6a,b
R1 = a, H; b, Cl
Me
NH
O
NH
S
N
R2 = a, H; b, 4-Cl; c, 3-Cl; d, 4-CH3; e, 3-CH3;
f, 4-OCH3; g, 4-CN; h, 4-Br
R2
7a-h
Scheme 2. Reagents and conditions: (a) 2-nitrofluorobenzene, NaH, DMF, rt, 12 h (b) 2,4-dinitrochlorobenzene, MeOH, KOH, rt, 6 h (c) ArCOCH2Cl, NaH, DMF, rt,
3 h (d) ArNHCOCH2Cl, NaH, DMF, rt, 3 h.
using different conditions including the use of potassium carbonate in
DMF; potassium carbonate in acetone, triethylamine in DMF, or sodium
hydride in DMF. The most convenient method that also gave the highest
yields of 6a (92%) and 6b (89%) within a short time (2 h) was the use of
sodium hydride in DMF under anhydrous conditions, at rt.
corresponding N,N-dimethylamide 9, cyclohexylamide 10a, and 2-N,N-
dimethyl)-N-ethylamide 10b were synthesized cleanly from the corre-
sponding primary and secondary aliphatic amines using a modification
of a reported procedure [38]. A higher boiling point solvent such as
dioxane, and varying reaction times from 24 to 48 h, according to the
reactivity of the amines was required for these amides.
The substituted chloroacetanilide derivatives, which were required
for the preparation of compounds 7a-h, were prepared from the corre-
sponding aniline derivatives and chloroacetyl chloride [37] at rt under
basic anhydrous DMF conditions with trimethylamine. S-alkylation of
these chloroacetanilide derivatives with 1 to form the corresponding
products 7a-h was achieved in excellent and reproducible yields
(85–94%) using sodium hydride in DMF at rt for short reaction times (2
h). Other researchers [38] described S-alkylation reactions of thiols with
chloroacetanilide derivatives in 70% yields using DMF containing tri-
methylamine under reflux conditions, but here we found the use of so-
dium hydride was optimal.
The corresponding acid hydrazide 11 was obtained by heating ester
8 with an excess amount of hydrazine in ethanol under reflux using the
procedure described by Salman et al. [38]. Despite, the target hydrazone
derivatives 12a-g were prepared from the acid hydrazide by a conden-
sation reaction of the appropriate aldehydes and ketones in ethanol in
the presence of a few drops of glacial acetic acid [39]. All desired
hydrazones precipitated out from the reaction mixture after few hours,
while compound 12e precipitated out only after pouring the reaction
mixture onto ice-cold water.
The synthesis of ethyl 2-[(4-benzyl-5-methyl-1H-imidazol-2-yl) thio]
acetate 8 (Scheme 3) was accomplished in a pure and crystalline state by
the S-alkylation reaction of 1 with ethyl chloroacetate in anhydrous
acetone and potassium carbonate. With compound 8 in hand, the
4

B.Y. Beshay et al.
Bioorganic Chemistry 113 (2021) 105033
Scheme 3. Reagents and conditions: (a) Cl-CH2COOEt, K2CO3, acetone, rt, 6 h (b) Me2NH, 1,4-dioxane, reflux, 36 h (c) R-NH2, 1,4-dioxane, reflux, 24–48 h (d)
N2H4⋅H2O, ethanol, 60 ◦C, 6 h (e) aromatic aldehydes or ketones, acetic acid, ethanol, rt, 6–24 h.
Me
Me
N
NH
S
S
N
N
H
Br
Br
2b
2b
Fig. 3. The imidazole ring tautomers of 2b.
2.2. Biological screening
Table 1
In vitro anti-tumor activity of tested imidazole derivatives on breast cancer cell
2.2.1. Anti-tumor activities of synthesized imidazole derivatives against
breast cancer cells.
lines.
Compound ID
IC50 values (µM)
MD77 and VS1 are previously reported as STAT3 inhibitors directly
targeting its SH2 domain [26-28], however, their potency needs to be
improved. Therefore, we conducted the screening of the newly synthe-
sized imidazole derivatives for their anti-tumor effect on breast cancer
cell lines, 4 T1, and MDA-MB-231 in which STAT3 is over activated. The
summary of IC50 values is shown in Table1. Among thirty tested imid-
azole derivatives, a group of ten compounds showed varying degrees of
anti-cancer activities. While there are compounds with low (2e, 7e) or
moderate (4, 5, 7a, 7c, 12a, 12e) activity, the two compounds (2a, 2d)
with the S-methylene linkers of unsubstituted phenyl ring (2a), and 2,6-
4 T1-Luc2
MDA-MB-231
2a
2d
2e
4
8.03
8.26
6.66
9.50
17.98
8.73
10.83
13.88
7.70
5
7.04
7a
7c
7e
12a
12e
10.44
15.99
26.02
10.42
10.86
13.47
8.11
10.43
14.38
10.54
5

B.Y. Beshay et al.
Bioorganic Chemistry 113 (2021) 105033
greater activity than the mono-substituted derivative 4. Based on these
results, a clear structure and activity relation could be concluded
wherein the insertion of small linkers such as a sulfur atom or a S-
methylene group, directly attached to an aromatic ring with small-sized
electronegative substituents in the target compounds 2a and 2d seem to
be necessary for the potent activity.
2a (µM)
2d (µM)
8 10
2
4
8
10
2
4
p-STAT3
Replacing the S-methylene linker with the longer S-acetamide spacer
decreased the inhibitory activity (8.11–26.02 µM). Among the S-acet-
amide linker-diaryl-substituted imidazole derivatives, 7c showed high
anti-tumor activity on MDA-MB-231 cells, with IC50 = 8.11 µM. The
substitution of the phenyl ring, which is attached to the S-acetamide
linker, with small electron-donating or withdrawing groups at the meta-
positions slightly decreased the activity as shown in compound 7c.
However, the substitution of the phenyl ring at the para-positions
abolished the activity of the other compounds (7b, 7d, 7f-h) in this
series. It is clear that the presence of nitro or fluoro substituents plays a
pivotal role in enhancing the potent activity of the tested cells. The
introduction of S-hydrazone spacers led to a remarkable decline in the
anti-tumor activity of imidazole derivatives 12a-g, except for candidates
12a and 12e which maintained their anti-tumor activity. While 12a
exhibited preferential cytotoxic activity on 4 T1 cells with IC50 = 10.42
STAT3
p-STAT1
STAT1
β-actin
Fig. 4. Inhibition of phosphorylated-STAT3 and STAT1 by the target
compounds 2a and 2d. 4 T1 cells (1x106 cells/well) were pre-treated with 2a
or 2d (DMSO vehicle, 2, 4, 8, 10 μM) for 6 h. Whole-cell lysates were immu-
noblotted with anti-STAT3, p-STAT3 (Tyr705), STAT1, p-STAT1 (Tyr701) an-
tibodies. β-actin was used as the loading control.
µM, 12e showed the highest potency on MDA-MB-231 cells with IC50
10.54 µM.
=
difluorophenyl ring (2d) showed the potent anti-tumor effect on the
tested cell lines.
It is also clear that the replacement of the arylhydrazone moiety with
different open-chain and cyclic aliphatic fragments markedly abolished
the anticancer potency of imidazole derivatives 8, 9, 10a-b, and 11.
These results highlight the importance of the aromatic ring, which is
attached to C2 of the imidazole ring, for maintaining the inhibitory
activity against breast cancer cells.
2.2.2. Imidazole derivatives 2a and 2d suppressed STAT3 phosphorylation
and its oncogenic functions.
To examine whether 2a and 2d inhibit STAT3 activity, the effects of
2a and 2d on the phosphorylation of STAT3 in 4 T1 cells were evaluated.
As shown in Fig. 4, both 2a and 2d treatment inhibited the expression of
phosphorylated STAT3 at Tyr705 in a dose-dependent manner without
affecting the total expression of STAT3. Considering the expressions of
both STAT1 and phosphorylated STAT1 were not affected by 2a or 2d
treatment, the primary target of 2a and 2d should be specific to STAT3.
Such specificity of 2a or 2d to STAT3 inhibition is contrary to MD77,
which is known to inhibit both STAT3 and STAT1 with IC50 17.7 and 7.2
µM, respectively [28]. Furthermore, in silico docking studies revealed
that a number of compounds with STAT3 inhibitory activity often show
STAT1 inhibition [40,41]; therefore, the selectivity of 2a and 2d on
STAT3 might be an advantage to the other known compounds.
To further determine the significance of 2a and 2d on their inhibition
on the oncogenic STAT3 function, we tested 2a and 2d on the in vitro
colony formation of 4 T1 and MDA-MB-231 breast cancer cell lines. As
shown in Fig. 5, both 2a or 2d pre-treatment reduced the colony for-
mation of 4 T1 and MDA-MB-231 cells. Furthermore, 2a and 2d also
inhibited the production of IL-6 from 4 T1 cells, which is known to be a
pro-tumorigenic cytokine to activate STAT3. Collectively, these data
strongly suggest those 2a and 2d as 2-substituted-4-benzyl-5-methylimi-
dazoles are novel class of synthetic anti-breast cancer compounds by
inhibiting oncogenic STAT3 activity (see Fig. 6).
2.3. Molecular Modeling
A docking study was performed into the binding site of STAT3-SH2
(PDB ID: 1BG1), to compare the mechanism of action of their inhibi-
tory activities and their binding affinities to the protein active site
relative to the lead compounds VS1 and MD77 [42]. The docking study
was performed using Discovery Studio (DS) 5.0 client (Accelrys) [43].
The selection of the docking poses generated with CDOCKER, which is a
grid-based docking program within the DS software, was based upon the
conformation with the highest score and the best binding interactions. In
addition, binding energy scores (CDOCKER energy), and the formation
of hydrogen bonds with the conserved amino acid residues were the
factors determining the binding affinities to the binding pockets of the
selected proteins.
The most active compounds (2a and 2d) were examined for their top-
scored binding with the best binding affinities and they were found to
bind at the active site with better scoring than lead compound VS1 and
were comparable to MD77, with hydrophilic and hydrophobic types of
interactions comparable to those shown with the lead compounds
(Table 2) [26-28].
Examination of the best-docked poses of imidazole derivative 2a
(Fig. 7) revealed that it was perfectly positioned in the active site of
STAT3-SH2 with a scoring energy of ꢀ 20.12 kcal/mol. The nitrogen
atom of the imidazole fragment of compound 2a displayed the corner-
stone hydrogen bonds with the amine functionalities of Arg609. This
amino acid contributed markedly to STAT3 and SH2 peptide binding, as
the mutation of Arg609 has been shown to abolish the peptide-binding
ability of this domain [44,45]. Furthermore, the hydrogen bonds with
Ser613 (two hydrogen bonds), Glu612, and other electrostatic forces
with Lys591 (ð-cation), Pro639 (ð-alkyl) also enhanced the blocking of
the STAT3-SH2 binding site.
2.2.3. Structure-activity relationship of synthesized compounds.
The obtained results indicated that an imidazole scaffold with
different spacer groups resulted in diverse effects on the in vitro anti-
tumor activity. The results can be summarized as follows: (i) The
imidazole derivatives 2a and 2d with S-methylene linkers showed the
strongest activities. (ii) The presence of the small electron-donating
methyl group on the para-position of the benzyl moiety in 2e
appeared to slightly decrease the anti-tumor activity. It was evident also
that the presence of the large electron-withdrawing groups (bromo or
cyano groups) on the benzyl moiety abolished the activity of compounds
2b and 2c. The presence of a sulphonyl-methylene linker in 3 formed by
the oxidation reaction of 2a (Scheme 1) led to the loss of the inhibitory
activity against the tested cell lines. (iii) The imidazole candidates
having only a sulfur atom linker showed good inhibitory activity on the
tested cell lines, with the dinitro-substituted derivative 5 exhibiting
Additionally, the sulfur atom of the S-methylene linker forms two
hydrogen bonds with the NH back-bone of Glu612 and Ser611. The
imidazole scaffold NH forms a strong hydrogen bond with Ser613.
Additionally, strong electrostatic interactions of the ð-cation type are
formed between the ð-cloud of the imidazole ring and the NH+3 group of
6

B.Y. Beshay et al.
Bioorganic Chemistry 113 (2021) 105033
Fig. 5. Inhibition of the colony formation by the
target compounds 2a and 2d. 4 T1 and MDA-MB-
231 cells were treated with 2a or 2d (10 µM) or with
the vehicle for 24 h, then the number of formed cell
colonies was counted 10 days after seeding. Data are
expressed as colony-forming efficiency calculated as
a percentage with respect to control cells. The
representative images of colonies formed (upper
panel) and the summaries of number of formed cell
colonies (lower panel) are presented. The data are
presented as the mean ± SD.
Concentration 10 µM
2a 2d
Control
MD
A-
MB-
231
4T1
-
Luc
2
*
*
Lys591. The binding mode of 2a is comparable to p-Tyr705 because it
involves the same pocket into which p-Tyr705 is inserted when the two
STAT3 subunits are assembled in the dimer. This pocket is placed on the
protein surface and is surrounded by hydrophilic and polar amino acids
to better interact with the negatively-charged side-chain of the phos-
phorylated tyrosine, which is mimicked by the benzylth-
iomethylimidazole moiety.
2.4. Optimization measures
2.4.1. Ligand Efficiency (LE) and (Ligand Lipophilic Efficiency) LLE
As shown in Table 3, the most active compounds fall within the
acceptable Ligand Efficiency (LE) and (Ligand Lipophilic Efficiency) LLE
ranges [46-48]. Compounds 2a, 2d, and 5 showed LE values ranged
between 0.74 and 0.95. In terms of lipophilicity, compounds 2a and 2d
exhibited LLE values of 9.82 and 7.47, respectively. Amongst all tested
compounds, 2a and 2d presented the optimum values (LE > 0.3, LLE >
5), which possessed the best drug-like criteria along with significant
potency as promising anti-cancer agents.
The docking results obtained for 2d revealed a good binding pattern
in the active site of STAT3-SH2 with a binding energy score of ꢀ 17.22
kcal/mol. The binding mode of 2d showed that the two nitrogen atoms
of the imidazole conserved the crucial hydrogen bonds with Arg609 and
Ser613. Additionally, one of the fluorine atoms at an ortho position of
the 2,6-diflurobenzyl fragment forms an intramolecular hydrogen bond
with the NH of the imidazole scaffold, which also forms a strong
hydrogen bond with Ser613. We can presume that these bridged
hydrogen bonds are also crucial for maintaining the STAT3 inhibitory
activity of 2d. Additionally, the decreased STAT3 inhibitory activity of
2d in comparison with 2a could be explained by the loss of the addi-
tional hydrogen bonds formed with Ser613, Glu612, and the electro-
static interactions with Pro639 (Fig. 8).
3. Conclusions
Our approach in designing imidazole derivatives as potential new
anti-cancer agents was successfully accomplished and the results can be
summarized as follows: All newly synthesized compounds revealed
moderate-to-potent anti-tumor activity against breast cancer cells.
Imidazole derivatives with S-methylene linker 2a and 2d exhibited the
most potent anti-tumor activity. Specifically, compound 2d had IC50
values of 6.66 and 9.50 µM against 4 T1 and MDA-MB-231 cells,
7

B.Y. Beshay et al.
Bioorganic Chemistry 113 (2021) 105033
1.4
0.7
0
1.4
0.7
0
2d
2a
0
4
8
10
0
4
8
10
Concentration (µM)
Concentration (µM)
Fig. 6. Suppression of the pro-inflammatory cytokine IL-6 by the target compounds 2a and 2d. 4 T1-Luc2 cells (2x105 cells/well) were incubated with 2a or 2d
(DMSO vehicle, 4, 8, 10
μ
M) for 24 h at 37 ◦C, and the level of IL-6 were then determined in the cell-free culture supernatants by ELISA. The data are presented as the
mean ± SD.
the remarkable anticancer activity against breast tumors.
Table 2
Binding score and the detailed interactions of the target compounds 2a and 2d in
the active site of STAT3-SH2.
4. Experimental details
Comp.
No.
Binding
score
H-bonding
Electrostatic
Interactions
Hydrophobic
Interaction
4.1. Chemistry
Interactions
(kcal/
mol)
4.1.1. Materials and methods
Starting materials, reagents and solvents were purchased from
Sigma-Aldrich, Merck, Acros, Alfa Aeser, and Gomhoria Co. Melting
points were determined in open-glass capillaries using a Graffin melting
point apparatus and are all uncorrected. The synthetic reactions were
monitored by Merck silica gel thin-layer chromatography (TLC) sheets
VS1
ꢀ 12.3
Ser611,
Ser612,
Ser613,
Ser636
Thr620, Trp623,
Gln635, Glu638,
Pro639, Val637,
Tyr657, Thr714,
Phe716
MD77
ꢀ 18.56
Arg595,
Lys591,
Arg609,
Gln635
Arg609,
Ser613,
Glu612
Arg609,
Ser613
Arg595, Gln635
¨
and the spots were visualized with a UV lamp at e 254 nm. Infrared
spectra (IR) were recorded, using KBr discs, ʋ (cmꢀ 1), on a Perkin-Elmer
1430 infrared spectrophotometer, Central Laboratory, Faculty of Phar-
macy, Alexandria University. 1H NMR (300 MHz) and 13C NMR (75
MHz) spectra were performed in CDCl3 or DMSO‑d6, on a Bruker
AVANCE III instrument at the Department of Chemistry, Memorial
University of Newfoundland, St. John’s, Newfoundland and Labrador,
Canada, as were the HRMS-ESI which were run on an Agilent 1260 In-
finity LC-6230 TOF LC/MS spectrometer (LCMS). Microanalyses were
performed on Perkin-Elmer 2400 elemental analyzer, and the values
found were within ± 0.3% of the theoretical values. Compounds 1 and
imidazole derivative 2a were prepared according to the procedures
described by Dakin & West [30] and Loksha et al. [31,32].
2a
2d
ꢀ 20.12
ꢀ 17.22
Lys591,
Pro639
Lys557, Lys591,
Ser611, Ser636,
Val637, Glu638,
Lys557, Lys591,
Glu594, Ser611,
Glu612, Ser613,
Thr620, Ser636,
Val637, Glu638
Lys591
respectively, and compound 2a showed IC50 values of 8.03 and 8.26 µM
against the same cell lines. Compounds 2a and 2d displayed excellent p-
STAT3 inhibitory activity compared to the lead compounds VS1 and
MD77. The most potent compounds were further examined in vitro for
suppressing the oncogenic functions of STAT3 which were recognized by
inhibiting the pro-inflammatory cytokine IL-6 and suppressing the col-
ony formation of breast cancer cells.
4.1.2. 4-Benzyl-5-methyl-1H-imidazole-2(3H)-thione (1).
A mixture of 3 (6.50 g, 32.5 mmol) and potassium thiocyanate (3.10
g, 32.5 mmol) in water (100 mL) was heated under reflux for 3 h. The
reaction mixture was cooled and the solid product was isolated by
filtration and recrystallized from ethanol/water to give 4.64 g of 1 as
pale-yellow crystals; yield 70%; mp 275–279 ◦C (lit. [31,32]
The docked models obtained for these active molecules revealed
excellent binding profiles in the active site of STAT3-SH2 (PDB: 1BG1).
Good scoring values in addition to the similarities shown with the lead
compounds VS1 and MD77 in the types of interactions have been
observed. In addition, the optimization measurement tools as LE and
LLE revealed that the imidazole derivatives 2a and 2d had optimum
results (LE > 0.3, LLE > 5) as promising anti-cancer agents. It is worth-
mentioning, structures of these two compounds suggest that their diaryl-
substituted imidazole structure has a small S-methylene group linker
and which have strong electron-withdrawing groups i.e. fluorine or nitro
on the S-methylene-linked aromatic ring are essential for maintaining
1
◦
¨
270–273 C. H NMR (DMSO‑d6) a [ppm]: 1.98 (s, 3H, CH3), 3.66 (s, 2H,
CH2-C4), 7.19–7.31 (m, 5H, Harom.), 11.65, 11.69 (s, 2H, 2NH). 13C NMR
¨
(DMSO‑d6) [a ppm]: 9.03 (CH3), 29.48 (CH2-C4), 120.63, 123.35,
126.72, 128.65, 128.88, 139.56, 159.32 (Carom, C5, C4 and C2). HRMS-
ESI: m/z = 205.0793 (C11H13N2S, [M + H+]); requires 205.0799.
4.1.3. 2-(Arylmethylthio)-4-benzyl-5-methyl-1H-imidazoles (2a-e).
To a suspension of 1 (0.41 g, 2.0 mmol) in acetone (25 mL), appro-
priate benzyl bromide derivatives (2.0 mmol) and potassium carbonate
8

B.Y. Beshay et al.
Bioorganic Chemistry 113 (2021) 105033
Fig. 7. Docking and binding pattern of compound 2a into the active site of STAT3-SH2. Left and right panels represent the 2D and 3D plots of PDB:1BG1,
respectively. Hydrogen bonds are shown with green dotted lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
Fig. 8. Docking and binding pattern of compound 2d into the active site of STAT3-SH2. Left and right panels represent the 2D and 3D plots of PDB:1BG1,
respectively. Hydrogen bonds are shown with green dotted lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
128.51, 128.55, 128.83, 131.05, 134.48, 135.85, 138.09, 141.34 (Carom
Table 3
C5, C4 and C2). HRMS-ESI: m/z = 295.1248 (C18H19N2S, [M + H+]);
LE and LLE values for the active compounds.
requires 295.1269.
¨
Comp. No.
NHA
Log P
A G
LE
LLE
2a
2d
5
21
23
26
4.86
5.09
4.64
20.12
‘17.22
9.78
0.95
0.74
0.76
9.82
7.47
2.50
4.1.3.2. 4-Benzyl-2-[(4-bromobenzyl)thio]-5-methyl-1H-imidazole (2b)..
Colorless crystals; yield 0.65 g (88%); mp 160–163 ◦C. 1H NMR
¨
(DMSO‑d6) a [ppm]: 2.05, 2.08 (2 s, 3H, CH3, tautomeric), 3.72, 3.79 (2
*Bold numbers represent the top LE and LEE values of the tested compounds.
s, 2H, CH2-C4, tautomeric), 4.12 (s, 2H, S-CH2), 7.11–7.22 (m, 3H, Harom
at C4), 7.15 (d, J = 8.3 Hz, 2H, Harom at C2), 7.27 (t, J = 7.70 Hz, 2H,
H3′’ & H5′’, Harom at C4), 7.41 (d, J = 8.3 Hz, 2H, H3′ & H5′, Harom at
(0.28 g, 2 mmol) were added. The reaction mixture was stirred for 8 h at
rt, and then the solvent was removed under reduced pressure. Ice-cold
water was added to the residue and the solid thus obtained was
filtered, washed with water and crystallized from acetone.
13
¨
C2), 11.84, 11.88 (2 s, 1H, NH, tautomeric). C NMR (DMSO‑d6) [a
ppm]: 9.69, 12.88 (CH3, tautomeric), 30.17, 33.03 (CH2-C4, tauto-
meric), 37.52, 37.67 (S-CH2, tautomeric), 120.53, 126.02, 126.51,
128.50, 128.55, 128.81,128.86, 131.32, 131.41, 131.55, 131.65, 138.39
79
(Carom). HRMS-ESI: m/z = 373.0380 and 375.0380 (C18
H
BrN2S,
18
4.1.3.1. 4-Benzyl-2-benzylthio-5-methyl-1H-imidazole (2a).. Colorless
crystals; yield 0.53 g (90%); mp 103–105 ◦C (lit. [31] 105–107 ◦C). 1H
C
H
BrN2S [M + H+]); requires 373.0374 and 375.0354.
81
18 18
¨
NMR (CDCl3) a [ppm]: 2.15 (s, 3H, CH3), 3.85 (s, 2H, CH2-C4), 4.08 (s,
13
4.1.3.3. 4-{[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]methyl}benzoni-
¨
2H, S-CH2), 7.12–7.33 (m, 10H, Harom.). C NMR (CDCl3) [a ppm]:
trile (2c).. Colorless crystals; yield 0.59 g (92%); mp 158–160 ◦C. 1H
11.18 (CH3), 31.79 (CH2-C4), 40.28 (S-CH2), 126.31, 127.27, 128.38,
9

B.Y. Beshay et al.
Bioorganic Chemistry 113 (2021) 105033
¨
NMR (CDCl3) a [ppm]: 2,14 (s, 3H, CH3), 3.82 (s, 2H, CH2-C4), 4.04 (s,
solvent was removed under reduced pressure, water (25 mL) was added
to the residual material. The solid product was filtered off and recrys-
2H, S-CH2), 7.09–7.31 (m, 5H, Harom at C4), 7.15 (d, J = 8.0 Hz, 2H,
Harom at C2), 7.40 (d, J = 8.0 Hz, 2H, Harom at C2), 8.55, 9.22 (2 s, 1H,
tallized from ethanol to give 0.315 g of compounds 5 as yellow-orange
13
1
NH, tautomeric). C NMR (CDCl3) [a ppm]: 39.65 (S-CH2), 110.95,
crystals; yield 85%; mp 105–106 ◦C. H NMR (DMSO‑d6) a [ppm]:
¨
¨
118.69 (CN), 126.71, 128.34, 128.65, 129.49, 132.20, 134.67, 143.81
(Carom). HRMS-ESI: m/z = 320.1206 (C19H18N3S, [M + H+]); requires
320.1221.
2.20 (s, 3H, CH3), 3.91 (s, 2H, CH2-C4), 6.97 (d, J = 9.0 Hz, 1H, Harom at
C2), 7.17–7.33 (m, 5H, Harom at C4), 8.43 (dd, J = 2.6, 9.0 Hz. 1H, Harom
at C2), 8.99 (d, J = 2.6 Hz, 1H, Harom at C2), 12.68 (s, 1H, NH). 13C NMR
¨
(DMSO‑d6) [a ppm]: 9.84 (CH3), 33.10 (CH2-C4), 121.82, 126.22,
128.63, 128.76, 129.41, 140.56, 141.06, 144.05, 145.24, 146.07 (Carom
,
4.1.3.4. 4-Benzyl-5-methyl-2-[(2,6-difluorobenzyl)thio]-1H-imidazole
C5, C4 and C2). HRMS-ESI: m/z = 371.0727 (C17H14N4O4S, [M + H+]);
(2d).. Colorless crystals; yield 0.56 g (85%); mp 120–122 ◦C. 1H NMR
requires 371.0814.
¨
(CDCl3) a [ppm]: 2.14 (s, 3H, CH3), 3.85 (s, 2H, CH2-C4), 4.06 (s, 2H, S-
CH2), 6.73, 6.76 (t, J = 7.9 Hz, 2H, Harom at C2), 7.08–7.29 (m, 6H,
arom at C2 and C4), 8.98 (s, 1H, NH). 13C NMR (CDCl3) [a ppm]: 27.38
4.1.7. 2-Aroylmethylthio-4-benzyl-5-methyl-1H-imidazoles (6a, b).
Under anhydrous conditions, a mixture of 1 (0.20 g, 1 mmol), the
appropriate phenacyl bromide derivatives (1.0 mmol) and sodium hy-
dride (0.048 g, of a 55% suspension in paraffin oil, 2.0 mmol) in
dimethylformamide (5.0 mL) was stirred for 2 h at rt. The reaction
mixture was treated with ice-cold water (100 mL) and left to stand at rt
for 3 h. The solid product was isolated by filtration and recrystallized
from acetone/water to give compounds 6a,b.
¨
H
(S-CH2), 111.00–111.14, 114.78, 126.27, 128.40, (Carom), 128.90 (t, JCF
= 10.5 Hz, C’4, Carom at C2), 129.07, 134.94 (Carom), 161.61 (dd, JCF
=
7.7, 249.8 Hz, C2′ & C6′, Carom at C2). HRMS-ESI: m/z = 331.1069
(C18H17F2N2S, [M + H+]); requires 331.1081.
4.1.3.5. 4-Benzyl-2-[(4-methylbenzyl)thio]-5-methyl-1H-imidazole
(2e).. Colourless crystals; yield 0.54 g (88%); mp 155–157 ◦C. 1H NMR
¨
(DMSO‑d6) a [ppm]: 2.06 (s, 3H, CH3-C5), 2.25 (s, 3H, (p-CH3-Bz), 3.32
4.1.7.1. 4-Benzyl-2-benzoylmethylthio-5-methyl-1H-imidazole
(6a)..
(s, 2H, CH2-C4), 4.11 (s, 2H, S-CH2), 7.04 (d, J = 8.3 Hz, 2H, Harom at
C2), 7.09 (d, J = 8.3 Hz, 2H, Harom at C2), 7.15–7.22 (m, 3H, Harom at
C4), 7.27 (t, J = 8.3 Hz, 2H, Harom at C4) 11.82 (1H, NH). 13C NMR
Colorless crystals; yield 0.95 g (92%); mp 156–157 ◦C. 1H NMR (CDCl3)
¨
a [ppm]: 2.15 (s, 3H, CH3), 3.85 (s, 2H, CH2-C4), 4.31 (s, 2H, S-CH2),
7.15–7.29 (m, 5H, Harom at C4), 7.44 (t, J = 7.4 Hz, 2H, Harom at C2),
7.58 (t, J = 7.4 Hz, 1H, Harom at C2), 7.90 (d, J = 7.4 Hz, 2H, Harom at
¨
(DMSO‑d6) [a ppm]: 21.16 (p-CH3-Bz), 38.18 (S-CH2), 126.20, 128.67,
129.09, 129.34, 135.50, 136.64 (Carom). HRMS-ESI: m/z = 309.1467
C2). 13C NMR (CDCl3) [a ppm]: 41.33 (S-CH2), 126.28, 128.41, 128.55,
(C19H21N2S, [M + H+]); requires 309.1425.
¨
128.62, 128.80, 133.89, 135.31 (Carom). HRMS-ESI: m/z = 323.1190
(C19H19N2OS, [M + H+]); requires 323.1218.
4.1.4. 4-Benzyl-2-benzylsulfonyl-5-methyl-1H-imidazole (3).
A solution of 2a (0.59 g, 2.0 mmol) in ethanol was treated with
hydrogen peroxide (3.0 mL). The reaction mixture was heated at 60 ◦C
for 24 h, and then cooled to rt. The solid was obtained by removal of the
solvent followed by crystallization from ethanol to give 3 (0.45 g) as
colorless crystals; yield 70%; mp 193–195 ◦C. IR (KBr) í [cmꢀ 1]: 3085
4.1.7.2. 4-Benzyl-2-(4-chlorobenzoylmethylthio)-5-methyl-1H-imidazole
(6b).. Colourless crystals; yield 0.31 g (87%); mp 165–168 ◦C. 1H NMR
¨
(DMSO‑d6) a [ppm]: 2.04 (s, 3H, CH3), 3.72 (s, 2H, CH2-C4), 4.53 (s, 2H,
S-CH2), 7.14–7.27 (m, 5H, Harom at C4), 7.55 (d, J = 8.5 Hz, 2H, Harom at
–
–
–
(NH); 1636 (C N); 1602, 1495 (C C); 1319, 1121 (SO ); 1070, 1111
C2), 7.93 (d, J = 8.5 Hz, 2H, Harom at C2), 11.89 (s, 1H, NH). 13C NMR
–
2
1
– –
S
¨
¨
(C
C). H NMR (DMSO‑d6) a [ppm]: 2.12 (s, 3H, CH3), 3.87 (s, 2H,
(DMSO‑d6) [a ppm]: 128.64, 128.67, 129.21, f130.85, 134.55, 136.19,
–
CH2-C4), 4.68 (s, 2H, SO2-CH2), 7.07–7.31 (m, 10H, Harom), 13.07,
138.79 (Carom), 193.94 (C O). HRMS-ESI: m/z = 357.0812 and
–
13
35
37
18
¨
13.17 (2 s, 1H, NH, tautomeric). C NMR (DMSO‑d6) [a ppm]: 9.55
359.0790 (C
H
ClN2OS, C19
H
ClN2OS [M + H+]); requires 357.0828
19 18
(CH3), 32.97 (CH2-C4), 60.89 (SO2-CH2), 126.35, 126.37, 128.60,
and 358.0799.
128.77, 128.79, 128.87, 131.35, 136.65, 138.86, 139.49, 140.76 (Carom
,
C5, C4 and C2). HRMS-ESI: m/z = 327.1146 (C18H19N2O2S, [M + H+]);
4.1.8. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N-(aryl)acetamides
(7a-h).
requires 327.1167.
Under anhydrous condition, compound 1 (0.204 g, 1 mmol) was
dissolved in dimethylformamide (5 mL), sodium hydride (0.048 g, of its
55% suspension in paraffin oil, 2 mmol) was added to the solution
portion-wise under ice cooling. After stirring the mixture for 0.5 h,
appropriate chloroacetanilide derivatives (1 mmol) was added portion-
wise to the reaction mixture under ice cooling stirred at rt for 4 h. The
reaction mixture was treated with ice/cold water (100 mL) and left to
stand at rt for 3 h. The solid product was isolated by filtration and
recrystallized from ethanol/water to give compounds 7a-h.
4.1.5. 4-benzyl-5-methyl-2-[(2-nitrophenyl)thio]-1H-imidazole (4).
Under anhydrous conditions, a mixture of 1 (0.20 g, 1.0 mmol), 1-
flouro-2-nitrobenzene (0.10 mL, 1.0 mmol) and sodium hydride
(0.048 g, of a 55% suspension in paraffin oil, 2.0 mmol) in dime-
thylformamide (5.0 mL) was stirred for 12 h at rt. The reaction mixture
was treated with ice-cold water (100 mL) and left to stand at rt for 3 h.
The solid product was isolated by filtration and crystallized from ethanol
to give 4 (0.26 g) as pale orange crystals; yield 80%; mp 181–182 ◦C. 1H
¨
NMR (DMSO‑d6) a [ppm]: 2.18 (s, 3H, CH3), 3.89 (s, 2H, CH2-C4), 6.77
(d, J = 7.6 Hz, 1H, Harom at C2), 7.21–7.30 (m, 5H, Harom at C4), 7.43 (t,
J = 7.6 Hz, 1H, Harom at C2), 7.63 (t, J = 7.6 Hz, 1H, Harom at C2), 8.25
(d, J = 7.6 Hz, 1H, Harom at C2), 12.68 (s, 1H, NH). 13C NMR (DMSO‑d6)
4.1.8.1. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N-phenyl-
acetamide (7a).. Colorless crystals; yield 0.315 g (93%); mp 156–157 ◦C.
1
¨
H NMR (DMSO‑d6) a [ppm]: 2.09, 2.12 (2 s, 3H, CH3, tautomeric), 3.77
¨
[a ppm]: 9.81, 12.93 (CH3, tautomeric), 31.15, 33.03 (CH2-C4, tauto-
(s, 2H, CH2-C4), 3.81, 3.84 (2 s, 2H, S-CH2, tautomeric), 7.02–7.60 (m,
10H, Harom.), 10.57, 10.62 (2 s, 1H, NHCO, tautomeric), 12.04 (s, 1H,
meric) 126.29, 126.90, 128.26, 128.71, 130.26, 135.10, 137.75, 139.94,
141.08 and 144.83 (Carom, C5, C4 and C2). HRMS-ESI: m/z = 326.0952
(C17H15N3O2S, [M + H+]); requires 326.0963.
NH of N1). 13C NMR (DMSO‑d6) [a ppm]: 9.66 (CH3), 32.99 (CH2-C4),
¨
38.53 (S-CH2), 119.45, 123.84, 126.11, 128.64, 128.80, 129.23, 134.38,
–
–
136.98, 137.07, 139.40, 141.44 (Carom, C5, C4 and C2), 167.51 (C O).
HRMS-ESI: m/z = 338.1303 (C19H20N3OS, [M + H+]); requires
4.1.6. 4-Benzyl-2-[(2,4-dinitrophenyl)thio]-5-methyl-1H-imidazole (5).
To a solution of potassium hydroxide (0.056 g, 1.0 mmol) in meth-
anol (15 mL), 1 (0.20 g, 1.0 mmol) was added and the mixture was
stirred for 0.5 h. 1-chloro-2,4-dinitrobenzene (0.20 g, 1.0 mmol) was
added to the reaction mixture and stirred at rt for an additional 6 h. The
338.1327.
4.1.8.2. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N-(4-chlor-
ophenyl)acetamide (7b).. Colorless crystals; yield 0.34 g (92%); mp
10

B.Y. Beshay et al.
Bioorganic Chemistry 113 (2021) 105033
1
◦
¨
199–202 C. H NMR (DMSO‑d6) a [ppm]: 2.10 (s, 3H, CH3), 3.79 (s, 2H,
CH2-C4), 3.82 (s, 2H, S-CH2), 7.16–7.24 (m, 5H, Harom at C4), 7.33 (d, J
= 8.4 Hz, 2H, Harom at C2), 7.51–7.53 (m, 2H, Harom at C2), 10.73 (s, 1H,
4.1.8.8. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N-(4-bromo-
phenyl)acetamide (7 h).. Colorless crystals; yield 0.39 g (94%); mp
1
◦
¨
205–207 C. H NMR (DMSO‑d6) a [ppm]: 2.09 (s, 3H, CH3), 3.78 (s, 2H,
NHCO), 12.06 (s, 1H, NH of N1). 13C NMR (DMSO‑d6) [a ppm]: 10.39
CH2-C4), 3.81 (s, 2H, S-CH2), 7.15–7.23 (m, 5H, Harom at C4), 7.42–7.47
¨
(CH3), 32.75 (CH2-C4), 38.51 (S-CH2), 121.00, 126.25, 126.34, 127.39,
(m, 4H, Harom at C2), 10.71 (s, 1H, NHCO), 12.03 (s, 1H, NH of N1). 13
C
–
–
¨
128.72, 129.15, 136.09, 138.36 (Carom), 167.65 (C O). HRMS-ESI: m/z
NMR (DMSO‑d6) [a ppm]: 38.38 (S-CH2), 115.38, 121.36, 126.28,
= 372.0915 and 374.0894 (C19H19N3OS35Cl, C19H19N3OS37Cl [M +
128.70, 128.71, 132.04, 136.06, 138.76 (Carom), 167.65 (C O). Anal.
–
–
H+]); requires 372.0937 and 374.0908.
calcd. For C19H18BrN3OS (415.03): C, 54.81; H, 4.36; Br, 19.19; N,
10.09; S, 7.70. Found: C, 54.74; H, 4.52; Br, 19.02; N, 10.25; S, 7.57%
4.1.8.3. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N-(3-chlor-
4.1.9. Ethyl 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]acetate (8).
To a stirred solution of 1 (4.08 g, 20.0 mmol) in acetone (100 mL)
was added potassium carbonate (2.76 g, 20.0 mmol) and the reaction
mixture was stirred at rt for 1 h. Ethyl chloroacetate (2.15 mL, 20.0
mmol) was added dropwise to the reaction mixture and stirred for
additional 6 h at rt. The solvent was removed under reduced pressure
and the residue was treated with water (20 mL), filtered off and crys-
ophenyl)acetamide (7c). Colorless crystals; yield 0.33 g (90%); mp
1
◦
¨
135–137 C. H NMR (DMSO‑d6) a [ppm]: 2.09 (s, 3H, CH3), 3.77 (s, 2H,
CH2-C4), 3.80 (s, 2H, S-CH2), 7.09–7.21 (m, 6H, Harom at C4 and C2),
7.31 (d, J = 5.3 Hz, 2H, Harom at C4), 7.80 (s, 1H, Harom at C2). 13C NMR
¨
(DMSO‑d6) [a ppm]: 11.03 (CH3), 31.99 (CH2-C4), 38.57 (S-CH2),
117.84, 118.97, 123.48, 126.20, 128.68, 130.92, 133.57, 136.20,
–
–
140.97 (Carom), 168.08 (C O). Anal. calcd. for C19H18ClN3OS (371.08):
tallized from ethanol/water to afford 4.6 g of compound 8 as colorless
C, 61.36; H, 4.88; Cl, 9.53; N, 11.30; S, 8.62. Found: C, 61.02; H, 5.06;
1
crystals; yield 80%; mp 155–156 ◦C. H NMR (CDCl3) a [ppm]: 1.21,
¨
Cl, 9.30; N, 11.50; S, 8.69%.
1.27 (2 t, J = 7.4 Hz, 3H, CH2CH3, tautomeric), 2.13, 2.19 (2 s, 3H, CH3-
C5, tautomeric), 3.56 (s, 3H, CH2-C4), 3.85, 3.88 (2 s, 2H, S-CH2,
tautomeric), 4.13, 4.21 (2q, J = 7.4 Hz, 2H, CH2CH3), 7.15 (m, 1H, Harom
at C4), 7.22–7.30 (m, 4H, Harom at C4), 9.69, 9.92 (2 s, 1H, NH, tauto-
4.1.8.4. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N-(p-tolyl)acet-
amide (7d).. Colorless crystals; yield 0.32 g (91%); mp 211–212 ◦C. 1H
¨
NMR (DMSO‑d6) a [ppm]: 2.11 (s, 3H, CH3), 2.25 (s, 3H, CH3-Ph), 3.79
meric). 13C NMR (CDCl3) [a ppm]: 9.78, 11.37 (CH3, at C5), 12.47, 14.03
¨
(s, 4H, CH2-C4 and S-CH2), 7.09 (t, J = 7.7 Hz, 2H, Harom at C4),
7.17–7.23 (m, 5H, Harom at C4 and C2), 7.35 (d, J = 7.9 Hz, 1H, Harom at
C2), 7.45 (d, J = 7.0 Hz, 1H, Harom at C2), 10.47, 10.51 (2 s, 1H, NHCO,
(CH2CH3), 30.70 (CH2-C4), 33.46, 36.71 (S-CH2), 61.98, 62.09
(CH2CH3), 125.84, 126.68, 128.29, 128.32, 128.51, 128.78, 130.65,
tautomeric), 12.03 (s, 1H, NH of N1). 13C NMR (DMSO‑d6) [a ppm]: 9.67
130.79, 134.80, 135.49, 137.95, 138.48, 140.60, 141.35 (Carom, C5, C4
¨
–
–
and C2), 171.60 (C O). HRMS-ESI: m/z = 291.1165 (C15H19N2O2S, [M
(CH3), 20.92 (CH3-Ph), 33.01 (CH2-C4), 38.50 (S-CH2), 119.46, 126.13,
+ H+]); requires 291.1167. Anal. calcd. for C15H18N2O2S (290.10): C,
62.04; H, 6.25; N, 9.65; S, 11.04. Found: C62.22; H, 610; N, 9.81; S,
10.96%.
128.64, 128.80, 129.61, 132.78, 136.63, 136.91, 137.08, 141.45 (Carom
,
–
–
C5, C4 and C2), 167.21 (C O). HRMS-ESI: m/z
= 352.1448
(C20H22N3OS, [M + H+]); requires 352.1484.
4.1.10. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N-(2-
4.1.8.5. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N-(m-tolyl)acet-
amide (7e).. Colorless crystals; yield 0.315 g (90%); mp 145–147 ◦C. 1H
(dimethylamino)ethyl-acetamide (9).
A mixture of 8 (0.58 g, 2.0 mmol) and N,N-dimethylethylenediamine
(3.0 mL, 10 mmol) in 1,4-dioxane (25 mL) was heated under reflux for
36 h. The solvent was evaporated under reduced pressure. The residue
was treated with water, filtered off, recrystallized from ethanol to afford
9 as colorless crystals; yield 0.33 g (50%); mp 165–166 ◦C. 1H NMR
¨
NMR (DMSO‑d6) a [ppm]: 2.09 (s, 3H, CH3), 2.25 (s, 3H, CH3-Ph), 3.80
(s, 4H, CH2-C4 and S-CH2), 6.88 (d, J = 7.1 Hz, 1H, Harom at C2),
7.14–7.39 (m, 8H, Harom.), 10.47, 10.54 (2 s, 1H, NHCO, tautomeric),
13
¨
12.01 (s, 1H, NH of N1). C NMR (DMSO‑d6) [a ppm]: 12.78 (CH3),
21.64 (CH3-Ph), 32.99 (CH2-C4), 38.55 (S-CH2), 116.69, 119.99,
¨
(CDCl3) a [ppm]: 2.07 (s, 3H, CH3-C5), 2.10 (s, 6H, 2CH3, N(CH3)2), 2.22
124.25, 126.08, 128.61, 128.77, 129.09, 135.57, 138.42, 139.34
(t, J = 6.6, 2H, NHCH2CH2N(CH3)2), 3.11 (q, J = 6.6, 2H, NHCH2CH2N
(CH3)2), 3.32 (s, 2H, CH2-C4), 3.75 (s, 2H, S-CH2), 7.13–7.20 (m, 3H,
–
–
(Carom), 167.38 (C O). HRMS-ESI: m/z = 352.1477 (C20H22N3OS, [M
+ H+]); requires 352.1484.
H’2, H’4 & H’6, Harom at C4), 7.21–7.28 (m, 2H, H’3 & H’5, Harom), 8.17
13
¨
(s, 1H, NHCO), 11.90 (s, 1H, NH of N1). C NMR (CDCl3) [a ppm]:
4.1.8.6. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N-(4-methox-
10.21 (CH3), 37.34 (CH2-C4), 37.51(S-CH2), 45.58, 58.40 (2CH3, N-
yphenyl)acetamide (7f).. Colorless crystals; yield 0.31 g (85%); mp
–
(CH3)2), 126.22, 128.70 (Carom), 168.41 (C O). HRMS-ESI: m/z =
–
1
◦
¨
199–202 C. H NMR (DMSO‑d6) a [ppm]: 2.11 (s, 3H, CH3), 3.72 (s, 3H,
CH3-O), 3.78 (s, 4H, CH2-C4 and S-CH2), 6.88 (t, J = 7.9 Hz, 2H, Harom at
C4), 7.17–7.23 (m, 5H, Harom at C4 and C2), 7.38 (d, J = 8.44 Hz, 1H,
Harom at C2), 7.48 (d, J = 5.87 Hz, 1H, Harom at C2), 10.40, 10.43 (2 s,
333.1740 (C17H25N4OS, [M + H+]); requires 333.1749.
4.1.11. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N,N-
dimethylacetamide (10a).
1H, NHCO, tautomeric), 12.03 (s, 1H, NH of N1). 13C NMR (DMSO‑d6) [a
¨
As described for 9, using dimethylamine (10 mL), the reaction
mixture was heated under reflux for 24 h to give 10a (0.35 g) as col-
ppm]: 9.65 (CH3), 32.98 (CH2-C4), 38.45 (S-CH2), 55.64 (CH3-O),
114.37, 120.98, 126.11, 128.66, 128.79, 132.58, 155.76 (Carom), 166.93
1
◦
¨
ourless crystals; yield 60%; mp 168–170 C. H NMR (CDCl3) a [ppm]:
(C O). HRMS-ESI: m/z = 368.1402 (C20H22N3O2S, [M + H+]); requires
–
–
2.17 (s, 3H, CH3), 2.99, 3.03 (2 s, 6H, 2CH3, N(CH3)2), 3.68 (s, 2H, CH2-
368.1433.
C4), 3.87 (s, 2H, S-CH2), 7.16–7.30 (m, 5H, Harom). 13C NMR (CDCl3) [a
¨
ppm]: 10.95 (CH3), 32.22 (CH2-C4), 35.01(S-CH2), 36.23, 37.89 (2CH3,
4.1.8.7. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N-(4-cyano-
–
–
N(CH3)2), 126.07, 128.40, 128.43, 136.23 (Carom), 170.12 (C O). Anal.
phenyl)acetamide (7 g).. Colorless crystals; yield 0.34 g (94%); mp
calcd. for C15H19N3OS (289.10): C, 62.25; H, 6.62; N, 14.52; S, 11.08.
1
◦
¨
190–191 C. H NMR (DMSO‑d6) a [ppm]: 2.10 (s, 3H, CH3), 3.77 (s, 2H,
Found: C, 62.5; H, 6.6; N, 14.3; S, 11.0%.
CH2-C4), 3.86 (s, 2H, S-CH2), 7.15–7.20 (m, 5H, Harom at C4), 7.65–7.75
(m, 4H, Harom at C2), 11.03 (s, 1H, NHCO), 12.06 (s, 1H, NH of N1). 13
C
4.1.12. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N-
cyclohexylacetamide (10b).
¨
NMR (DMSO‑d6) [a ppm]: 9.60 (CH3), 32.91 (CH2-C4), 38.60 (S-CH2),
105.52 (Carom), 119.48 (CN), 124.62, 126.06, 128.69, 128.73, 133.79,
As described for 9 using cyclohexylamine (5.0 mL), the reaction
mixture was heated under reflux for 48 h to give 10b (0.36 g) as
–
–
135.93, 143.58 (Carom), 168.35 (C O). HRMS-ESI: m/z = 363.1256
(C20H19N4OS, [M + H+]); requires 363.1280.
1
◦
¨
colorless crystals; yield 53%; mp 166–168 C. H NMR (CDCl3) a [ppm]:
1.06–1.83 (m, 10H, 5CH2, C6H11), 2.18 (s, 3H, CH3), 3.44 (s, 2H, CH2-
11

B.Y. Beshay et al.
Bioorganic Chemistry 113 (2021) 105033
–
C4), 3.68–3.72 (m, 1H, CH, cC6H11), 3.84 (s, 2H, S-CH2), 7.14–7.30 (m,
142.46, 146.90 (N = CH), 170.42 (C O). Anal. calcd. for C20H19BrN4OS
–
5H, Harom), 7.86, 7.88 (s, 1H, NHCO, tautomeric). 13C NMR (CDCl3) [a
(442.04) C, 54.18; H, 4.32; Br, 18.02; N, 12.64 S, 7.23. Found: C, 54.4;
¨
ppm]: 9.40 (CH3), 23.96, 24.61, 25.47, 30.36 (cC6H11), 32.43 (CH2-C4),
37.27 (S-CH2), 48.73 (cC6H11), 126.21, 128.39, 128.49, 136.99 (Carom),
H, 4.46; Br, 17.9; N, 12.7; S, 7.36%.
–
169.33 (C O). Anal. calcd. for C19H25N3OS (343.17): C, 66.44; H,7.34;
–
4.1.14.4. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N’-(4-methox-
ybenzylidene)aceto-hydrazide (12d).. Colorless crystals; yield 0.63 g
N, 12.23; S, 9.34. Found: C, 66.2; H, 7.50; N, 12.4; S, 9.51%.
1
◦
¨
(80%); mp 192–194 C. H NMR (DMSO‑d6) a [ppm]: 2.09, 2.10 (2 s, 3H,
CH3, tautomeric), 3.71 (s, 2H, CH2-C4), 3.79 (s, OCH3), 4.09 (s, 2H, S-
CH2), 6.95–7.00 (m, 2H, Harom at C2), 7.11–7.23 (m, 5H, Harom at C4),
7.55–7.59 (m, 2H, Harom at C2), 7.90 (s, 1H, N = CH), 11.36, 11.719 (2 s,
1H, NH = N), 11.92, 12.01 (2 s, 1H, NH of N1, tautomeric). 13C NMR
4.1.13. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]acetohydrazide
(11).
A mixture of 8 (5.80 g, 20.0 mmol) and hydrazine hydrate (10 mL)
was heated in ethanol (100 mL) at 60 ◦C for 6 h. The solvent was
evaporated under reduced pressure. The residue was treated with water,
filtered off, crystallized from ethanol to afford 11 (3.3 g) as colorless
¨
(DMSO‑d6) [a ppm]: 55.77 (O-CH3), 114.70, 114.76, 127.15, 128.60,
128.87, 129.16 (Carom), 143.57, 147.11, (N = CH), 161.09, 161.32
1
◦
–
–
¨
crystals; yield 60%; mp 180–184 C. H NMR (DMSO‑d6) a [ppm]: 2.08
(s, 3H, CH3), 3.58 (s, 2H, CH2-C4), 3.74 (s, 2H, S-CH2), 4.28 (s, 2H,
NHNH2), 7.18–7.26 (m, 5H, Harom at C4), 9.35 (s, H, CONHNH2), 11.93
(Carom), 170.05 (C O). Anal. calcd. for C21H22N4O2S (394.14): C,
63.94; H, 5.62; N, 14.20; S, 8.13. Found: C, 64.2; H, 5.82; N, 14.5; S,
8.36%.
(s, 1H, NH of N1). 13C NMR (DMSO‑d6) [a ppm]: 9.75 (CH3), 31.58 (CH2-
¨
–
–
C4), 34.06 (S-CH2), 126.62, 128.70, 128.81 (Carom), 167.83 (C O).
4.1.14.5. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N’-(2,4-dime-
Anal. calcd. for C13H16N4O4S (276.10): C, 56.50; H, 5.84; N, 20.27; S,
thoxybenzylidene)-acetohydrazide (12e).. Colorless crystals; yield 0.65 g
1
(79%); mp 170–171 ◦C. H NMR (DMSO‑d6) a [ppm]: 2.00, 2.09 (2 s,
11.60. Found: C, 56.4; H, 6.04; N, 20.1; S, 11.8%.
¨
3H, CH3, tautomeric), 3.72 (s, 2H, CH2-C4), 3.79, 3.83 (2 s, 6H, 2
(OCH3), 4.12 (s, 2H, S-CH2), 7.00 (t, J = 9.1 Hz, 1H, Harom at C4),
7.15–7.40 (m, 7H, Harom at C4 and C2), 7.78, 8.00 (2 s, 1H, N = CH),
11.40, 11.71 (2 s, 1H, NH = N), 11.92,11.99 (s, 1H, NH of N1, tauto-
4.1.14. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N’-
arylideneacetohydrazides (12a-e).
To a solution of 11 (0.55 g, 2.0 mmol) in ethanol (25 mL), appro-
priate aromatic aldehyde (2.0 mmol) and 2 drops of glacial acetic acid
were added. The reaction mixture was stirred for 6 h with12a or 12b; for
12 h with 12c and for 24 h with 12d or 12e) at rt. The solid formed was
filtered, washed with water, and crystallized from ethanol to afford
products 12a-e.
meric). 13C NMR (DMSO‑d6) [a ppm]: 8.99 (CH3), 36.55 (S-CH2), 55,91,
¨
56.04 (2(O-CH3), 108.77, 109.01, 111.92, 127.22, 127.33, 128.69,
128.70, 134.08, 149.43 (N = CH), 151.21, 164.81 (Carom), 171.07
–
–
(C O). Anal. calcd. for C22H24N4O3S (424.15): C, 62.24; H, 5.70; N,
13.20; S, 7.50. Found: C, 62.4; H, 5.58; N, 13.3; S, 7.33%.
4.1.14.1. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N’-benzylidenea-
4.1.15. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N’-(1-
arylethylidene)acetohydrazide (12f, g).
cetohydrazide (12a).. Colorless crystals; yield 0.64
g (88%); mp
1
194–195 ◦C. H NMR (DMSO‑d6) a [ppm]: 2.01, 2.10 (2 s, 3H, CH3,
tautomeric), 3.68, 3.75, (2 s, 2H, CH2-C4, tautomeric), 3.78, 4.14 (2 s,
2H, S-CH2, tautomeric), 7.15–7.26 (m, 5H, Harom at C4), 7.42–7.46 (m,
3H, H’3, H’4 & H’5, Harom at C2), 7.64–7.67 (m, 2H, H’2 & H’6, Harom at
C2), 7.96 (s, 1H, N = CH), 11.50, 11.87 (2 s, 1H, NH = N, tautomeric),
¨
To a solution of 11 (0.55 g, 2.0 mmol) in ethanol (25 mL), the
appropriate aromatic ketones (2.0 mmol) and 2 drops of glacial acetic
acid were added. The reaction mixture was stirred for 12 h (12f) or for
24 h (12 g) hours at rt. The solid formed was filtered, washed with
water, crystallized from ethanol to afford compounds 12f or 12 g.
11.95, 12.03 (2 s, 1H, NH of N1, tautomeric). 13C NMR (DMSO‑d6) [a
¨
ppm]: 9.63 (CH3), 35.71 (CH2-C4), 35.89 (S-CH2), 127.30, 127.57,
4.1.15.1. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N’-(1-phenyl-
ethylidene)aceto-hydrazide (12f).. Colorless crystals; yield 0.67 g (89%);
128.55, 128.82, 129.23, 129.27, 130.31, 130.58, 132.51, 134.55
–
–
1
◦
(Carom), 143.67, 147.22 (N = CH). 160.61, 161.62 (C O). Anal. calcd.
¨
mp 164–166 C. H NMR (DMSO‑d6) a [ppm]: 2.00, 2.10 (2 s, 3H, CH3-
–
–
for C20H20N4OS (364.13): C, 65.91; H, 5.53; N, 15.37; S, 8.80. Found: C,
C5 tautomeric), 2.23 (s, 3H, N C(CH ) 3.33 (2 s, 2H, CH -C4), 4.16 (s,
2H, S-CH2), 7.14–7.25 (m, 5H, Harom at C4), 7.38–7.41 (m2, 3H, Harom at
,
3 ,
66.2; H, 5.67; N, 15.2; S, 8.64%.
C2), 7.75–7.77 (m, 2H, Harom at C2), 10.71, 11.00 (2 s, 1H, NH = N),
11.94, 12.09 (2 s, 1H, NH of N1, tautomeric). 13C NMR (DMSO‑d6) [a
4.1.14.2. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N’-(4-chlor-
obenzylidene)aceto-hydrazide (12b).. Colorless crystals; yield 0.66 g
¨
–
ppm]: 14.12 (N C(CH ) 126.56, 126.79, 128.78, 129.53, 129.75,
–
3 ,
1
◦
–
–
¨
(83%); mp 189–190 C. H NMR (DMSO‑d6) a [ppm]: 1.99, 2.09 (2 s, 3H,
CH3, tautomeric), 3.66, 3.74 (2 s, 2H, CH2-C4, tautomeric), 4.08, 4.12 (s,
2H, S-CH2, tautomeric), 7.11–7.23 (m, 5H, Harom at C4), 7.50–7.69 (m,
4H, Harom at C2) 7.89, 7.92 (2 s, 1H,N = CH, tautomeric), 11.54, 11.92
130.61, 138.46 (Carom), 148.27 (N = CH), 171.15 (C O). HRMS-ESI: m/
z = 379.1547 (C21H23N4OS, [M + H+]); requires 379.1593.
4.1.15.2. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N’-[1-(4-bromo-
phenyl)ethylidene] acetohydrazide (12 g).. Colorless crystals; yield 0.81
(2 s, 1H, NH = N, tautomeric), 11.92, 12.01 (2 s, 1H, NH of N1, tauto-
13
1
◦
¨
meric). C NMR (DMSO‑d6) [a ppm]: 8.51 (CH3), 35.71 (CH2-C4),
¨
g (89%); mp 177–179 C. H NMR (DMSO‑d6) a [ppm]: 2.00, 2.10 (2 s,
–
–
38.09 (S-CH2), 124.61, 128.54, 128.79, 128.91, 129.28, 129.58, 130.50,
3H, CH -C5 tautomeric), 2.21, 2.27 (s, 3H, N C(CH ) tautomeric),
3
,
3 ,
–
–
133.51, 134.99 (Carom) 145.90, 146.33 (N = CH), 161.07 (C O). Anal.
3.78, 380 (2 s, 2H, CH2-C4), 4.13 (s, 2H, S-CH2), 7.14–7.25 (m, 5H,
calcd. for C20H19ClN4OS (398.09): C, 60.22; H, 4.80; Cl, 8.89; N, 14.05;
H
arom at C4), 7.56–7.88 (m,4H, Harom at C2), 10.77, 11.06, 11.35 (3 s,
1H, NH = N), 11.94, 12.09 (2 s, 1H, NH of N1, tautomeric). 13C NMR
S, 8.04. Found: C, 59.9; H, 4.58; Cl, 8.78; N, 13.9; S, 8.25%.
–
–
¨
(DMSO‑d ) [a ppm]: 15.12 (N C(CH ) 125.42, 126.93, 128.55,
128.80, 129.04, 131.71, 131.73, 132.713(C,arom), 140.49, 145.46 (N =
6
4.1.14.3. 2-[(4-Benzyl-5-methyl-1H-imidazol-2-yl)thio]-N’-(4-bromo-
–
benzylidene)aceto-hydrazide (12c).. Colorless crystals; yield 0.70
g
CH), 160.20 (C O). HRMS-ESI: m/z = 457.0733 and 459.0715
–
1
(80%); mp 180–182 ◦C. H NMR (DMSO‑d6) a [ppm]: 2.00, 2.09 (2 s,
3H, CH3, tautomeric), 3.72 (s, 2H, CH2-C4), 4.30 (s, 2H, S-CH2),
7.17–7.22 (m, 5H, Harom at C4), 7.59–7.75 (m, 4H, Harom at C2), 7.92 (s,
(C
H
BrN4OS, C21
H
BrN4OS [M + H+]); requires 457.0698 and
79
81
¨
21 22
22
459.0677.
1H, N = CH), 11.55 (s, 1H, NH = N), 11.92, 12.01 (2 s, 1H, NH of N1,
13
¨
tautomeric). C NMR (DMSO‑d6) [a ppm]: 123.46, 123.79, 127.96,
128.54, 128.81, 129.14, 130.68, 132.80, 132.71, 133.83 (Carom),
12

B.Y. Beshay et al.
Bioorganic Chemistry 113 (2021) 105033
4.2. Biological activity
were generated and energetically minimized using the “Generate Con-
formations” tool in Discovery Studio (DS) 5.0 client (Accelrys). The
lowest energetic conformation thus obtained was subjected to the
“Prepare Ligands” module to generate its isomers at physiological pH.
The CHARMM force field was employed to develop the partial atomic
charges on each atom of the isomer. The isomer with the lowest
CHARMM energy was used for the docking study.
4.2.1. Cells and reagents
All tested imidazole derivatives were dissolved in DMSO to for-
m100µM stock solutions and were stored at ꢀ 20 ◦C. Murine 4 T1-Luc2,
and human MDA-MB-231 breast cancer cells, were maintained in Ros-
well Park Memorial Institute (RPMI)-1640 medium (Nissui, Tokyo,
Japan) supplemented with 10% bovine serum and 20 U/mL penicillin,
and 20 ìg/mL streptomycin, and the cells incubated at 37 ◦C in a hu-
midified incubator in an atmosphere containing 5% CO2 in the air. The
primary antibodies against STAT3, p-STAT3, STAT1, and p-STAT1 were
purchased from Cell Signaling Technology (Beverly, MA, USA) and the
4.3.2. Protein preparation and docking process.
The X-ray coordinates of STAT3 (pdb ID: 1BG1, resolution 2.25 Å)
was retrieved from the protein data bank (www.rcsb.org). The “Prepare
Protein” tool in DS was used to add missing atoms/chains and remove
water molecules in the protein structure. The “Prepare Protein” algo-
rithm was employed to protonate amino acid residues according to the
physiological conditions. Determination of the binding site is accom-
plished by choosing the binding sphere covering the SH2 domain of
STAT3. CDOCKER, a grid-based docking program, was used to dock the
active compounds in the SH2 domain, considering the default parame-
ters. The most favorable pose of the docked compounds was identified
based on the CDOCKER energy (-CDE).
ˆ
antibody against a-actin was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA, USA).
4.2.2. In vitro cell viability assay
The anti-tumor activity of tested compounds was quantified using
WST-8 assay (Wako Pure Chemical Industries, Tokyo, Japan). The cells
in the exponential growth were placed at a final concentration (104
cells/well) in a 96-well plate. After 24 h of incubation, the target com-
pounds were added. Controls received DMSO vehicle at a concentration
equal to that in drug-treated cells. After the incubation with test com-
pounds for 72 h, WST-8 reagent was added and the absorbance at 450
nm/620 nm was measured using a microplate reader and cell viability
was calculated. The inhibitory concentration-50 was obtained from the
concentrations-inhibition response curve (n = 3).
4.4. Optimization parameters LE and LLE.
The most active compounds were subjected to the prediction of drug-
likeness and ligand-likeness scoring through some reliable measuring
tools as LE and LLE. Molinspiration chemoinformatic server was used for
the logP calculations [49].
4.2.3. Western blotting
4 T1-Luc2 cells (106 cells/well) were treated with tested compounds
for 6 h, then collected using Trypsin-EDTA and centrifuged for 10 min at
2000 rpm, 4 ◦C. The supernatant was discarded and the cells were lysed
in whole-cell lysis buffer (25 mmol/L HEPES, pH 7.7, 300 mmol/L NaCl,
1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.1% Triton X-100, 20 mmol/L
glycerophosphate, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl
fluoride, 1 mmol/L dithiothreitol, 10 mg/mL aprotinin, 10 mg/mL
leupeptin). Cell lysates were subjected to electrophoresis in 10% SDS-
PAGE, and electrophoretically transferred to Immobilon-P nylon mem-
brane (Millipore, Bedford, MA, USA). The membranes were treated with
Block Ace (Dainippon Pharmaceutical, Co. Ltd., Osaka, Japan) for at
least 2 h, and probed with the indicated primary antibodies overnight,
followed by horseradish peroxidase-conjugated secondary antibodies
(1:1000 dilutions). Bands were visualized using ECL reagents (Amer-
sham Bioscience, Piscataway, NJ, USA).
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105033.
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