New N-(1,3,4-thiadiazol-2-yl)furan-2-carboxamide derivatives as potential inhibitors of the VEGFR-2

Article abstract of DOI:10.1016/j.bioorg.2021.105176

The present study reports the synthesis and biological evaluation of a new series of novel N-(1,3,4-thiadiazol-2-yl)furan-2-carboxamide derivatives. The reactions were executed under both conventional and microwave irradiation conditions. An enhancement i

Full text of DOI:10.1016/j.bioorg.2021.105176

Bioorganic Chemistry 115 (2021) 105176
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Bioorganic Chemistry
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New N-(1,3,4-thiadiazol-2-yl)furan-2-carboxamide derivatives as potential
inhibitors of the VEGFR-2
,
,*
Mohamed H. Hekala *, Paula S. Faraga, Magdy M. Hemdana, Wael M. El-Sayedb
a Department of Chemistry, Faculty of Science, Ain Shams University, Abbassia 11566, Cairo, Egypt
b Department of Zoology, Faculty of Science, Ain Shams University, Abbassia 11566, Cairo, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
The present study reports the synthesis and biological evaluation of a new series of novel N-(1,3,4-thiadiazol-2-
yl)furan-2-carboxamide derivatives. The reactions were executed under both conventional and microwave
irradiation conditions. An enhancement in the synthetic yields and rates was observed when the reactions were
carried out under the microwave compared with the classical conditions. The structures of the products were
ascertained by different analytical and spectral analyses. The antiproliferative activities were evaluated against
three human epithelial cell lines; breast (MCF-7), colon (HCT-116), and prostate (PC-3) using MTT assay tech-
nique and doxorubicin was utilized as a reference drug. Besides, molecular docking studies were also performed
and the vascular endothelial growth factor recptor-2 (VEGFR-2) was identified as a potential molecular target.
Compounds 6, 7, 11a, 11b, 12, 14, and 16 showed promising antiproliferative activity against the three cancer
cell lines investigated. Compounds 2 and 15b had significant antiproliferative activities against only colon and
breast cells but not against the prostate cells. All the active antiproliferative compounds were highly selective. All
the active antiproliferative compounds were good inhibitors of the VEGFR-2 at 7.4–11.5 nM compared with
Pazopanib. Compound 7 with the most favorable orientation to the VEGFR-2 from the docking studies, was also
the best inhibitor of the receptor. The antiproliferative activity of these compounds is in partial caused by their
ability to inhibit the VEGFR-2 and since other molecular targets were not examined, other possibilities cannot be
ruled out.
Thiadiazole derivatives
Anticancer agents
VEGFR-2 inhibitors
Molecular docking
1. Introduction
reactions have acquired special concern in the recent years.[14–16]
Moreover, it is considered environmentally friendly and usually pro-
1,3,4-Thiadiazoles, a unique heterocyclic scaffold containing sulfur
and nitrogen atoms, have diverse pharmacological applications
including antituberculosis, antimicrobial,[1] antioxidant, [2] anti-in-
flammatory,[3] antidepressant,[4] anxiolytic,[5] anticonvulsant,[6]
antihypertensive,[7] antifungal,[8] and anti-cancer [9,10] activities.
The existence of sulfur atom in the 1,3,4-thiadiazole scaffold impacts the
physicochemical and pharmacokinetic properties as well as the biolog-
ical activity. Compared to other isomeric thiadiazoles, 1,3,4-derivatives
showed better metabolic stability and water solubility.[11] The 1,3,4-
thiadiazole moiety also acts as a bioisostere for carbonyl containing
compounds such as esters, amides, and carbamates.[12] Many drugs
such as Acetazolamide, Methazolamide, Megazol, Cefazolin and Sulfa-
methizole contain thiadiazole moiety (Fig. 1).[13]
vides high yields compared to the classical condition techniques.[17] In
the microwave irradiation process, the reactions occur safely, rapidly,
and often better than the conventional techniques which require pro-
longed reaction times and greater amounts of solvents and reagents, and
cause health hazards and environmental pollution.[18,19] This study
displays a simple and efficient method to synthesize novel N-(1,3,4-
thiadiazol-2-yl)furan-2-carboxamide derivatives via microwave-assisted
organic synthesis (MAOS) under lower solvent conditions and short re-
action times aiming at investigating these new derivatives as anticancer
agents and potential inhibitors of the vascular endothelial growth factor
receptor-2 (VEGFR-2).
In spite of the significant progress in the diagnosis, treatment, and
prevention of cancer, it remains one of the most common causes of
mortality throughout the world and therefore, the development of
potent and more effective anticancer agents still represents real unmet
Microwave irradiation technique has been applied for rapid prepa-
ration of a diversity of heterocyclic compounds and solvent-free organic
* Corresponding authors.
E-mail addresses: mohamed.hekal@sci.asu.edu.eg (M.H. Hekal), wael_farag@sci.asu.edu.eg (W.M. El-Sayed).
https://doi.org/10.1016/j.bioorg.2021.105176
Received 9 April 2021; Received in revised form 21 May 2021; Accepted 11 July 2021
Available online 16 July 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

M.H. Hekal et al.
Bioorganic Chemistry 115 (2021) 105176
medical challenges in the therapeutics due to the predictable emergence
of resistance among cancer cells.[20] Traditional methods for cancer
treatment including radiotherapy, chemotherapy, and immunotherapy
are of own limitations due to the toxicities of these therapies.[21]
Suppression of angiogenesis which is the key process involved in both
cancer and cardiovascular diseases, has become a prime target in cancer
therapies. The VEGF produced by carcinoma cells and its receptor
VEGFR-2 are essential for cancer development and progression. [22]
Therefore, inhibition of the VEGFR-2 signaling pathway is regarded as
an attractive therapeutic target for inhibition of the angiogenesis and
consequently the tumor growth.[23]
Reaction of furoyl chloride with ammonium thiocyanate in dry
acetonitrile under stirring at room temperature gave the furoyl iso-
thiocyanate (1) which in situ reacted with 2-cyanoacetohydrazide
yielding the corresponding 1-(2-cyanoacetyl)-4-(furan-2-carbonyl) thi-
osemicarbazide (2) [39] (Scheme 1). The chemical structure of com-
pound 2 was confirmed by its spectral and elemental analyses. The 1
H
NMR spectrum revealed signals for methylene, NH, and furan protons
that confirmed the assigned structure 2. Chemical cyclization of the
thiosemicarbazide derivative 2 into different heterocyclic compounds
containing furan scaffold under different reaction conditions has been
performed as shown in Scheme 1. Refluxing of compound 2 with etha-
nolic KOH solution or exposing it to MW irradiation afforded a product
which was identified as pyrazolo[1,5-a]-s-triazine derivative 3 in a
reasonably good yield (74%). The structure of compound 3 was sug-
gested from its elemental analysis, IR and NMR spectral data. The IR
spectrum showed strong absorption bands at 3160 cmꢀ 1 correlated for
NH group. Moreover, the absence of the stretching absorption band for
the cyano group indicated its corporation in cyclization process. Com-
pound 3 was previously prepared in low yield via refluxing of compound
2 in absolute ethanol in the presence of sodium ethoxide as a catalyst.
[39]
Therefore in the current study and guided by preliminary docking
studies, a molecular hybridization of furan-2-carboxamide moiety and
1,3,4-thiadiazole scaffold was carried out in an endeavor to obtain new
biological hybrid compounds with the main pharmacophoric features
substantial for the VEGFR-2 inhibitors. In addition, the anti-proliferative
activity against human cancer cell lines and molecular docking studies
of the new series of 1,3,4-thiadiazole derivatives were performed in
order to predict the affinity and orientation of these compounds at the
active site of the VEGFR-2. The selectivity of the new derivatives has also
been assessed. This was followed by investigating the ability of the new
derivatives as inhibitors of the VEGFR-2 as compared to the standard
inhibitor pazopanib.
In addition, refluxing of compound 2 with triethyl amine in ethanol
for four hrs or subjecting it to the MW irradiation yielded the sulphur
free product which was identified as 6-(furan-2-yl)-4-iminopyrazolo
[3,4-d][1,3]oxazin-3(4H)-one (4) (Scheme 1). Adequate evidence for
the chemical structure of compound 4 was substantiated from the cor-
rect elemental analysis and the spectroscopic data (c.f. experimental).
Moreover, N-(5-imino-3-oxopyrazolidine-1-carbonothioyl)furan-2-
carboxamide (5) was obtained as a sole product when compound 2 was
treated with concentrated sulfuric acid at 0 ◦C. The IR spectrum of
compound 5 showed absorption bands corresponding to carbonyl group
as well as NH groups and lacked the stretching absorption band for the
cyano group which supported the assigned structure 5. On the other
hand, 1,3,4-oxadiazole derivative 6 was easily obtained by cyclo-
desulfurization of the thiosemicarbazide 2 upon refluxing in ethanolic
mercuric oxide solution for four hrs or under the MW irradiation
(Scheme 1). Intramolecular cyclization reaction of compound 2 with
boiling glacial acetic acid or under the MW irradiation furnished a
bioisostere of compound 6 which was identified as 1,3,4-thiadiazole
2. Results and discussion
2.1. Chemistry
As a part of our concern in synthesizing a broad scope of heterocyclic
systems with biological applications [24–35] and in a continuation of
utilizing green chemistry tools in heterocyclic synthesis, [36–38] the
current study reported the use of furoyl isothiocyanate in the synthesis
of several new substituted 1,3,4-thiadiazole derivatives incorporating
furan moiety under both conventional and microwave (MW) irradiation
conditions and evaluated their anti-proliferative activity. Also, based on
the pharmacophoric features of the VEGFR-2 inhibitors that are indis-
pensable for the anti-proliferative activity, we designed 1,3,4-thiadia-
zole scaffold incorporating furan moiety followed by cyclization
process with a variety of heterocyclic moieties.
Fig. 1. Thiadiazole-containing Drugs.
2

M.H. Hekal et al.
Bioorganic Chemistry 115 (2021) 105176
O
Cl
O
O
S
a
C
N
b
O
(1)
O
S
H
O
N
O
O
N
e
c
N
N
O
H
HN
N
N
O
S
HN
H
H
N
S
O
(5)
N
N
H
H
N
(3)
O
(2)
O
d
N
O
f
g
N
CN
O
O
N
HN
(6)
O
N
N
O
O
O
NH
(4)
S
CN
HN
(7)
N
N
Scheme 1. Reagents and conditions: (a) NH4SCN, CH3CN, rt; (b) 2-cyanoacetohydrazide, CH3CN, rt, 90%. (c) KOH, EtOH, reflux, 74%, MW irradiation, 83%; (d)
TEA, EtOH, reflux, 35%, MW irradiation, 47%; (e) Conc. H2SO4, 0 ◦C, 90%; (f) HgO, EtOH, reflux, 25%, MW irradiation, 40%; (g) Glacial AcOH, reflux, 55%, MW
irradiation, 76%
Scheme 2. Reagents and conditions: (a) Salicylaldehyde, EtOH, piperidine, reflux, 78%, MW irradiation, 85%; (b) 3-aminopyridine and/or p-toluidine, glacial acetic
acid, rt, 87%; (c) Thiophene-2-carbaldehyde or p-chlorobenzaldehyde, EtOH, piperidine, reflux, 87–88%, MW irradiation, 93–94%; (d) 11a, thioglycolic acid,
pyridine, reflux, 60%, MW irradiation, 83%; (e) cyclohexanone, EtOH, piperidine, reflux, 74%, MW irradiation, 86%; (f) 2-(4-chlorobenzyl-idene)malononitrile,
EtOH, piperidine, reflux, 73%, MW irradiation, 84%.
3

M.H. Hekal et al.
Bioorganic Chemistry 115 (2021) 105176
derivative 7 [39] (Scheme 1). The structures of compounds 6 and 7 were
deduced from their IR and NMR spectra. The 1H NMR spectra
[DMSO‑d6] of both compounds displayed signals corresponding to furan
protons and methylene protons as well as NH proton in the down field
region that were exchangeable with D2O. In addition, the 13C NMR
spectrum of compound 6 exhibited signals in concurrence with the
proposed structure (c.f. experimental). The nucleophilic reactivity of the
key starting intermediate 7 towards different carbon electrophiles has
been studied. Treatment of compound 7 with salicylaldehyde in etha-
nolic piperidine solution under Knoevenagel reaction gave 2-iminocou-
marin derivative 8 which was also obtained by repeating the reaction
using the MW irradiation (Scheme 2). The structure of iminocoumarin
derivative 8 was deduced on the basis of analytical and spectroscopic
conditions yielded the corresponding condensation product 13 (Scheme
2).
On the other hand, subjecting 1,3,4-thiadiazole derivative 7 to react
with 2-(4-chloro-benzylidene)malononitrile in the presence of a cata-
lytic amount of piperidine afforded a yellow crystalline product upon
heating which was identified as the arylidene derivative 11a (minor
product), while acidification of the reaction filtrate with cold dilute HCl
furnished a red precipitate that was detected as the 1,3,4-thiadiazolo
[3,2-a]pyridine derivative, compound 14 (major product). Compounds
11a and 14 were also synthesized under the MW irradiation conditions
(Scheme 2).
Furthermore, the refluxing of 1,3,4-thiadiazole derivative 7 with
glycolic acid, lactic acid and thioglycolic acid in pyridine afforded
oxazol-4(5H)-ones 15a, 15b, and thiazol-4(5H)-one derivative 15c,
respectively. Compounds 15a-c were also obtained by the MW irradia-
tion at 600 W for five min. These chemical structures were elucidated by
the analytical and spectroscopic methods (c.f. experimental). The exis-
tence of these compounds as E-isomers rather than Z-isomers may be
attributed to the high stability of the former one as a result of the
extension of conjugation and existence of intra-molecular H-bonding
interaction. On the other side, treatment of thiazol-4(5H)-one derivative
15c with p-chlorobenzldehyde afforded the corresponding condensation
product which surprisingly in situ undergoes nucleophilic substitution
reaction by the excess piperidine giving 1,3,4-thiadiazole derivative 16
as the sole product (one spot in TLC) [41] that was also obtained by the
MW irradiation under solvent free conditions (Scheme 3). The IR spec-
trum of compound 16 showed the appearance of conjugated carbonyl
group at lower υC=O broad at 1683 cmꢀ 1 than that was observed with
compound 15c confirming that the carbanion was derived from the
active methylene group of the thiazolidinone ring. Moreover, the
structure of compound 16 gets a further support from the 1H NMR
spectrum [DMSO‑d6] which revealed signals for aromatic protons,
vinylic proton as well as pipredine protons and in addition to NH proton
in the downfield region that was exchangeable with D2O. Furthermore,
the 13C NMR spectrum of compound 16 is completely in agreement with
the proposed structure (c.f. Experimental).
data. The IR spectrum of 8 displayed υNH at 3302 and 3131 cmꢀ 1
, υC=O
at 1673 cmꢀ 1 and was devoid of the stretching absorption band for
nitrile group. It was supposed that the reaction of iminocoumarin system
like compound 8 with aromatic amines would afford the corresponding
arylimino derivative 10 according to the reported procedure.[40]
However, the reaction of compound 8 with p-toluidine, p-anisidine,
and/or 3-aminopyridine in the presence of glacial acetic acid afforded
the unexpected coumarin derivative 9 rather than the arylimino deriv-
ative 10 which could be due to undesirable side reaction such as acid
hydrolysis of iminocoumarin to the corresponding coumarin (Scheme
ꢀ 1
–
2). The appearances of a high frequency C O stretching at 1713 cm
–
cited for the coumarin oxo function in the IR spectrum beside the
absence of NH proton of imino group at 9.03 ppm in the 1H NMR
spectrum of compound 9 agree well with the proposed structure.
Furthermore, the structure of compound 9 was supported by the 13C
NMR spectrum (c.f. experimental).
Further, the Knoevenagel condensation of 1,3,4-thiadiazole deriva-
tive 7 with thiophene-2-carbaldehyde and/or p-chlorobenzaldehyde
was refluxed in ethanol containing a catalytic amount of piperidine for
three hours or subjected to the MW irradiation at 600 W for four minutes
afforded the corresponding arylidene derivatives 11a and 11b, respec-
tively. In addition, cyclocondensation of the arylidene derivative 11a
with thioglycolic acid in the presence of boiling pyridine or under the
MW irradiation furnished the thiazolinone derivative 12. Similarly,
refluxing of compound 7 with cyclohexanone under the same reaction
Thiophene derivative 17 was obtained from the reaction of 1,3,4-
thiadiazole derivative 7 with ethyl cyanoacetate in the presence of
Scheme 3. Reagents and conditions: (a) Glycolic acid, lactic acid, or thioglycolic acid, pyridine, reflux, 75–88%, MW irradiation, 82–90%; (b) 15c, p-chlor-
obenzaldehyde, EtOH, piperidine, reflux, 87%, MW irradiation, 93%; (c) Sulfur metal, ethylcyanoacetate, EtOH, TEA, reflux, 87%, MW irradiation, 92%; (d) KOH,
DMF, CS2, rt; (e) 1,2-Dibromoethane, rt, 87%; (f) Chloroacetylchloride, rt, 88%; (g) Ethyl chloroacetate, rt, 89%.
4

M.H. Hekal et al.
Bioorganic Chemistry 115 (2021) 105176
elemental sulfur, ethanol and TEA or by the MW irradiation without
using solvent (Scheme 3). Compound 7 is active enough to react with
electrophiles due to chemical reactivity of the methylene moiety at the
5-position of thiadiazole ring. Thus, it readily adds to one carbon donor
such as carbon disulfide in DMF-containing potassium hydroxide at
room temperature giving the non-isolable dipotassium sulphide salt 18
which in situ underwent heterocyclization upon treatment with 1,2-
dibromoethane and chloroacetyl chloride giving the corresponding
1,3-dithiolane 19 and 1,3-dithiolone 20 derivatives (Scheme 3). The
structures of compounds 19 and 20 were clarified based on the
elemental analysis and spectral data (c.f. Exp.). Treatment of the dipo-
tassium sulphide salt 18 with ethyl chloroacetate gave the uncyclized S-
alkylation product 21 and no evidence was recorded for the cyclized
product 22 (Scheme 3). The appearance of strong absorption band for
the nitrile group at 2196 cmꢀ 1 and the carbonyl ester group at 1717
cmꢀ 1 in the IR spectrum of the product was completely in agreement
with the assigned structure 21 and ruled out the cyclized product 22.
Moreover, the 1H NMR spectrum of compound 21 revealed the existence
of ethyl protons of ester group and a singlet signal at δ 4.58 ppm for CH-
CN proton as well as methylene protons and furan protons and in
addition to NH proton in the down field region which was exchangeable
with D2O.
the prostate cells. All the active antiproliferative compounds were
highly selective with selectivity indices ~7.5–14.7 µM. Compound 2 was
moderate in selectivity.
2.2.2. Molecular docking
To predict how the new synthesized compounds could act as in-
hibitors of the VEGFR-2, molecular docking studies were carried out
using AutoDock 4.2 program. Therefore, the crystal structure of VEGFR-
2 receptor was retrieved from the protein data bank (PDBID, 4ASE). The
reference ligand (pazopanib) displayed several hydrogen bonding in-
teractions with key amino acid residues in the VEGFR-2 active site;
Glu885, Cys919, and Asp1046 in addition to their hydrophobic in-
teractions. The sulfonamide moiety of pazopanib is involved in water-
mediated interactions with Lys868, Asp1046 and the backbone
carbonyl group of His1026. The amino group is hydrogen bonded to key
residues, Glu885 and Lys868 which are responsible for increasing the
binding affinity of pazopanib. The nitrogen atom at 1-position of inda-
zole moiety is stabilized by a hydrogen bonding interaction with back-
bone amino group of the key residue, Cys919. Furthermore, the phenyl
moiety of the pazopanib is located in the hydrophobic pocket formed by
Leu889, Ile892, Val898, Leu1019, His1026 and Ile1044. The pyrimidine
moiety is anchored by hydrophobic interactions with Val899, Val914,
Val916, and Cys1045. Moreover, the central indazole moiety is stabi-
lized by an aromatic stacking interaction with Phe1047, cationic-
π
2.2. Biological evaluation
interaction with Phe918 and located in lipophilic cage made up of
Leu840, Val848, Ala866 and Leu1035 (Fig. 2).
2.2.1. In vitro cytotoxic activity
The obtained docking results confirmed that all the synthesized
compounds have similar position and orientation inside the putative
binding site of the VEGFR-2. Moreover, the obtained results of the
computed free energy of binding (ΔG) indicated that most of the syn-
thesized compounds have good binding affinity towards the receptor
(Table 2).
The newly synthesized compounds have been evaluated for their
anti-proliferative activities against three human cancer cell lines; breast
adenocarcinoma (MCF-7), colon cancer (HCT-116), and prostate cancer
(PC-3) cells and compared to the activity of doxorubicin (Table 1).
Doxorubicin had its antiprolifertaive activity against the three cancer
cell lines at ~4–5 µM. Doxorubicin was also cytotoxic to the normal
fibroblast cells (WI-38) with ZERO selectivity (SI = 1). Based on the cut-
off limits set by the National Cancer Institute, compounds that have IC50
at ≤ 10 µM are considered hits. Compounds 6, 7, 11a, 11b, 12, 14, and
16 showed promising antiproliferative activity against the three cancer
cell lines investigated. Compounds 2 and 15b had significant anti-
proliferative activities against only colon and breast cells but not against
To avoid the cumbersome discussion of all the synthesized com-
pounds, we offered the detailed docking studies for only some active
compounds (6, 7, 11a, and 11b). The obtained binding mode of com-
pound 11a showed that the unprotonated nitrogen atom of the thia-
diazole moiety of compound 11a is involved in a hydrogen bonding
interaction with the backbone amino group of Asp1046. The amino
group of carboxamide moiety is stabilized by the hydrogen bonding
interaction with Glu885. Furthermore, the furan ring of compound 11a
is anchored by an aromatic stacking interaction with His1026 and
located in hydrophobic pocket formed by lipophobic part of Glu885,
Ile888, Leu889, Ile892, Val898, and Cys1024. In addition, the thiophene
vinyl moiety of 11a is stabilized by hydrophobic interactions with
Leu840, Val848, Ala866, Cys919 and Leu1035 (Fig. 2). The desirable
interactions and the size of hydrophobic pocket of thiophene moiety
could be responsible for increasing the affinity towards the VEGFR-2
where, the size of this hydrophobic pocket is large enough to accom-
modate compound 11a which could contribute to increased affinity of
compound 11a. As for compound 11b, the substitution of p-chlor-
ophenyl moiety instead of thiophene ring in the same predominant
hydrophobic pocket leads to reduction in the affinity of compound 11b
owing to the polarity induced by the halogen atom which may shift the
hydrophobic interaction balance.
Table 1
The cytotoxicity of the new thiadiazole derivatives against human breast
adenocarcinoma (MCF-7), colon cancer (HCT-116), and prostate cancer (PC-3)
cells as well as normal fibroblasts (WI-38).
Compound
IC50 (µM)a
MCF-7
HCT-116
PC3
WI-38
SIb
Doxorubicin
4.17 ± 0.2
7.73 ± 0.6
17.02 ± 1.4
55.25 ± 3.5
6.58 ± 0.7
5.51 ± 0.4
48.36 ± 3.0
78.11 ± 4.0
6.41 ± 0.4
9.48 ± 0.8
9.58 ± 0.6
51.09 ± 3.2
6.70 ± 0.9
18.12 ± 1.4
9.27 ± 3.0
84.81 ± 4.4
8.13 ± 0.5
32.74 ± 2.1
23.81 ± 1.9
14.13 ± 1.2
5.23 ± 0.3
6.18 ± 0.4
34.14 ± 2.6
43.84 ± 3.2
4.19 ± 0.3
3.97 ± 0.2
37.26 ± 2.8
82.39 ± 4.1
5.01 ± 0.9
8.15 ± 0.7
9.62 ± 0.7
59.45 ± 3.7
6.12 ± 0.8
13.78 ± 1.8
8.65 ± 3.7
91.27 ± 4.6
5.63 ± 0.7
17.03 ± 1.4
25.72 ± 1.9
12.78 ± 1.1
5.22 ± 0.6
11.86 ± 1.1
81.12 ± 4.7
49.97 ± 3.3
7.76 ± 0.6
9.10 ± 0.8
42.01 ± 3.1
75.22 ± 4.7
8.47 ± 0.8
7.41 ± 0.6
4.80 ± 0.3
73.56 ± 4.3
7.94 ± 0.4
11.30 ± 1.4
15.59 ± 3.3
>100
4.93 ± 0.8
26.33 ± 3.9
53.05 ± 8.5
61.20 ± 9.1
81.17 ± 4.2
68.67 ± 3.0
47.33 ± 8.1
>100
1.0
2
3.1
1.2
3
5
1.2
6
13.1
11.1
1.1
7
8
9
1.3
11a
11b
12
13
14
15a
15b
15c
16
19
20
21
92.00 ± 6.4
89.26 ± 5.3
64.10 ± 4.0
>100
13.9
10.7
8.0
The most favorable orientation found was for compound 7. The
amino group of the carboxamide moiety of compound 7 is involved in a
hydrogen bonding interaction with the side-chain of Glu885 and located
within 4.3 Å from the amino group side-chain of Lys868 thus unable to
connect to this amino acid residue. The carbonyl group of carboxamide
moiety is hydrogen bonded to the backbone amino group of Asp1046
which is believed to be crucial for binding and contributes to an increase
in the affinity of compound 7. Furthermore, furan moiety is stabilized by
an aromatic stacking interaction with His1026 and located in the hy-
drophobic pocket formed by Ile888, Leu889, Ile892, Val898, Leu1019,
Cys1024, and Ile1044. The thiadiazole cyanomethyl moiety is anchored
by an aromatic stacking interaction with Phe1047 and hydrophobic
1.7
>100
14.7
1.4
20.01 ± 4.3
83.37 ± 9.1
>100
7.5
1.1
6.30 ± 0.6
24.49 ± 1.9
39.23 ± 2.8
15.38 ± 1.3
84.07 ± 8.7
20.13 ± 4.2
27.00 ± 4.5
15.57 ± 2.4
12.6
0.8
0.9
1.1
a
IC50 values are the mean ± standard deviation (SD) of three separate
experiments.
b
SI: selectivity index = IC50 against WI-38 /median IC50 against cancer cells.
5

M.H. Hekal et al.
Bioorganic Chemistry 115 (2021) 105176
Pazopanib
Compound 11a
Compound 7
Compound 6
Fig. 2. Predicted binding mode of pazopanib, 11a, 7, and 6 with VEGFR-2 receptor. The hydrogen bonding interactions are presented by yellow dashed lines.
Residues contacting ligand are demonstrated as lines. H-bonds (yellow dotted lines), Hydrogen (white), nitrogen (blue), oxygen (red) and sulfur (yellow). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
interactions with Val848, Ala866, Val899, Val914, Val916, Leu1035
and Cys1045. The presence of the furan ring, carboxamide moiety, and
thiadiazole moiety are probably essential for increasing the affinity for
the VEGFR-2 due to the existence of additional hydrogen bonding and
hydrophobic interactions (Fig. 2). The molecular docking study of
compound 6 inside the binding site of VEGFR-2 is similar to that of
compound 7 where the interactions between the amino group and
carbonyl group of carboxamide moiety with Glu885 and Asp1046,
respectively are maintained. Additionally, the oxygen atom of oxadia-
zole moiety is anchored by an additional hydrogen bonding interaction
with the backbone amino group of Asp1046. The unprotonated nitrogen
atom of oxadiazole moiety is involved in a hydrogen bonding interaction
with key residue, Lys868. As expected, the VEGFR-2 inhibition and the
binding free energy of thiadiazole moiety of compound 7 are more than
the oxadiazole moiety of compound 6. This could be attributed to the
fact that the thiadiazole moiety is more aromatic and electron rich than
the oxadiazole moiety and thus more involved in hydrophobic
interactions.
Asp1046. An active methylene linker substituted with heterocyclic ring
at the 5-position of 1,3,4-oxadiazole and 1,3,4-thiadiazole scaffolds 6
and 7 were more potent than those possessing vinylic hydrogen linker
11a. This is due to that the active methylene linker can nicely fit and
accommodate into the binding site with favorable hydrophobic in-
teractions especially with the key residue, Cys919 (Fig. 3). Amide and
thioamide linker groups show distinct orientation due to sulfur atom of
the thioamide group acting as a hydrogen bond acceptor and more
flexible (the degree of conformational flexibility) which was indicated
by the difference in distances among the hydrogen bonding acceptor and
donor in ligand receptor affinity. The distance between the amide oxy-
gen atom of compound 6 and the backbone amino group of the key
residue, Asp1046 is 2.9 Å, and the distance between the amide nitrogen
atom of compound 6 and the backbone carbonyl group of the key res-
idue, Glu885 is 3.1 Å (Fig. 3).
2.2.3. The VEGFR-2 kinase inhibitory assay
The compounds with significant anti-proliferative activities (2, 6, 7,
11a, 11b, 12, 14, 15b, and 16) were further selected and evaluated for
their inhibitory action against the VEGFR-2 kinase Pazopanib [42] as
one of the most potent inhibitors of the VEGFR-2 was used as a positive
control (Table 3). All compounds exhibited inhibitory action against the
VEGFR-2 at ~7.5–11.5 nM. Compounds 11a, 11b, 14 and 7 exhibited
the best inhibitory action which was better than or as equipotent as
pazopanib. At 7.4 nM, compound 7 with the most favorable orientation
The outcome of docking results with VEGFR-2 indicated that the
interactions with Glu885, Cys919 and Asp1046 are crucial for the
VEGFR-2 inhibitors. Moreover, the hydrophobic interaction domains
located at the binding pocket seem to be responsible for the affinity of
VEGFR-2. Furthermore, the amide moiety is important for receptor af-
finity in particular, the amide moiety is involved in hydrogen bonding
interactions with key amino acid residues; Glu885, Lys868, and
6

M.H. Hekal et al.
Bioorganic Chemistry 115 (2021) 105176
to the VEGFR-2 from the docking studies, was also the best inhibitor of
the receptor. The reduced inhibitory effect of compounds 2 and 15b on
the VEGFR-2 could be due to the absence of suitable size of the hydro-
phobic head as a prerequisite pharmacophoric feature existing in the
reference drug. Many cancer types overexpress the VEGFR-2. This
overexpression is responsible for the growth, angiogenesis, invasion,
and metastasis of many tumors. [43] Many drugs are developed as in-
hibitors of this critical tyrosine kinase pathway. Sorafenib, Sunitinib,
Bevacizumab, Lenvatinib, Vandetanib, Pazopanib, Axitinib, and Cabo-
zantinib are some effective examples in use for treating some of the most
resistant tumors with bad median life expectancy. [44–47]
Table 2
The calculated free energy of binding of the synthesized compounds with
VEGFR-2 receptor, forming hydrogen bonding and
CompoundNo.
π
-π
interactions.
ΔG
No. of hydrogen
No. of aromatic
kcal/
mol
bonding interactions
stacking interactions
2
3
5
6
ꢀ 60.43
ꢀ 50.27
ꢀ 40.44
ꢀ 55.52
(Glu885, Asp1046)
(Glu885, Asp1046)
(Glu885, Asp1046)
(Glu885, Lys868,
Asp1046)
(Phe918, Phe1047)
(Phe1047)
(Phe1047)
(His1026, Phe1047)
7
ꢀ 55.90
(Glu885, Lys868,
Asp1046)
(His1026, Phe1047)
8
ꢀ 49.72
ꢀ 48.87
ꢀ 67.65
(Glu885, Asp1046)
(Glu885, Asp1046)
(Lys868, Glu885,
Asp1046)
(Phe918, Phe1047)
(Phe918, Phe1047)
(Phe918, His1026,
Phe1047)
9
2.3. Structure–activity relationships
11a
11b
12
ꢀ 60.04
ꢀ 58.21
ꢀ 38.87
ꢀ 60.33
ꢀ 67.42
(Glu885, Asp1046)
(Asp1046)
(Phe918, Phe1047)
(Phe918, Phe1047)
(Phe1047)
By comparing the reported cytotoxicity of the synthesized com-
pounds in this study to their structures, the following structure-activity
relationships were postulated;
13
(Asp1046)
14
(Glu885)
(Phe918, Phe1047)
(Phe918, Phe1047)
15a
(Glu885, Cys919,
Asp1046)
▪ Compound 2 displayed high anti-proliferative activity probably
due to the presence of three NH groups which may react or
make hydrogen bonds with the DNA.
15b
15c
16
ꢀ 58–73
ꢀ 50.87
ꢀ 62.80
(Glu885, Asp1046))
(Glu885, Asp1046))
(Asp1046)
(Phe918, Phe1047)
(Phe918, Phe1047)
(Phe918, His1026,
Phe1047)
▪ Compound 6 with oxadiazole moiety has the same potency as
the bioisosteric thiadiazole moiety 7 although compound 7 has
more aromatic character due to the presence of sulfur atom.
The carbonyl group of carboxamide moiety is believed to be
crucial for binding and contributes to an increase in the affinity
of compound 7.
19
20
ꢀ 50.39
ꢀ 52.22
–
(His1026, Phe1047)
(Phe918, Phe1047)
(Glu885, Cys919,
Asp1046)
21
ꢀ 58.01
ꢀ 67.65
(Cys919, Asp1046)
(Lys868, Glu885,
Cys919)
(Phe918, Phe1047)
(Phe918, Phe1047)
Pazopanib
▪ In addition, integrating active vinylic group to 1,3,4-thiadia-
zole motif would be lucrative to the cytotoxic activity of com-
pound 11a. However, incorporating
a double bond in
compounds 13, 19 and 20 led to a significant reduction in their
cytotoxic activity.
▪ The substitution with dithiolane moiety (non-aromatic char-
acter) in compounds 19 and 20 instead of thiophene and ben-
zene moiety (aromatic character) in compounds 11a and 11b,
respectively, may be unfavorable to cytotoxicity, indicating
that the aromatic character of compounds 11a or 11b was
likely required for the anticancer activity by involving the ar-
omatic moiety in aromatic stacking interactions.
▪ Further, the incorporation of electron-withdrawing substituent
such as chloride atom at the phenyl moiety connected with
vinylic hydrogen linker did not affect the antitumor activity of
11b compared with compound 11a.
▪ Transformation of cyano group (compound 11a) into thiazoli-
none ring (compound 12) led to decreasing the cytotoxic ac-
tivity against both the breast and colon cells by ~50% but
enhancing the anti-proliferative activity against the prostate
cells by 50% for unknown reasons.
Fig. 3. Superimposed of compound 6 and compound 11a with the VEGFR-2.
Table 3
▪ The 1,3,4-thiadiazolo[3,2-a]pyridine derivative 14 had high
cytotoxic activity, which may be attributed to the presence of
pyridine ring bridgehead with 1,3,4-thiadiazole motif and one
NH2 group as well as 4-chlorophenyl ring attached to the pyr-
idine ring.
The inhibitory activity of the new thiadiazole de-
rivatives on the VEGFR-2.
Compound
IC50 (nM)a
Pazopanib
9.7 ± 0.4
11.5 ± 0.4
10.3 ± 0.7
7.4 ± 0.8
7.6 ± 0.4
8.2 ± 0.4
10.5 ± 0.2
8.3 ± 0.2
11.5 ± 0.1
10.3 ± 0.3
2
▪ Moreover, incorporation of active vinylic group linked with
oxazol-4(5H)-one ring (15b) to 1,3,4-thiadiazole moiety
showed higher anti-proliferative activity than substitution with
thiazol-4(5H)-one ring (compound 15c). Also, the substitution
of CH3 group at the 5-position of the attached oxazol-4(5H)-one
ring (compound 15b) enhanced the anti-proliferative activity
compared to the non-substituted oxazol-4(5H)-one derivative
15a.
6
7
11a
11b
12
14
15b
16
a
The data are expressed as the mean ± standard
▪ It is noteworthy that the 1,3,4-thiadiazole derivative 16 has
higher antiproliferative efficiency as compared to the non-
substituted thiazol-4(5H)-one derivative 15c via introducing
of 4-(piperidin-1-yl)benzylidene moiety at the 5-position of
deviation (SD) of three independent experiments.
7

M.H. Hekal et al.
Bioorganic Chemistry 115 (2021) 105176
thiazol-4(5H)-one ring which enhanced the anti-proliferative
acetonitrile was stirred at room temperature with 2-cyanoacetohydra-
zide (0.99 g, 10 mmol) for 30 min, then poured into cold water. The
resulting precipitate was separated by filtration and recrystallized from
ethanol affording the pure compound 2 as colorless crystals (2.27 g,
90%); m.p 188–190 ◦C; IR (KBr) (υmax, cmꢀ 1): 3227 (NH), 3029 (CH
activity.
2.4. Conclusions
aromatic), 2974, 2932 (CH aliphatic), 2259 (C N), 1669 (C O). 1H
–
–
–
–
–
Novel 19 N-(1,3,4-thiadiazol-2-yl)furan-2-carboxamide derivatives
were synthesized under both conventional and microwave irradiation
conditions, and their structures were confirmed by different spectro-
scopic analyses. The antiproliferative activity of these derivatives was
evaluated against three human epithelial cell lines; breast (MCF-7),
colon (HCT-116), and prostate (PC-3). Compounds 6, 7, 11a, 11b, 12,
14, and 16 showed promising antiproliferative activity against the
cancer cell lines investigated. Compounds 2 and 15b had significant
antiproliferative activities against only colon and breast cells but not
against the prostate cells. The ineffective cytotoxicity of compounds 2
and 15b against the prostate cells could reflect that these compounds
may need prerequisite metabolism for the activity that could not be
provided by these cells. But this remains an assumption that needs
further investigation. All the active antiproliferative compounds were
highly selective. Besides, molecular docking studies were also per-
formed and the vascular endothelial growth factor recptor-2 (VEGFR-2)
was identified as a potential molecular target. The presence of the furan
ring, carboxamide moiety, and thiadiazole moiety are essential for
increasing the affinity for the VEGFR-2 due to the existence of additional
hydrogen bonding and hydrophobic interactions. The interactions with
Glu885, Cys919 and Asp1046 are crucial for the VEGFR-2 inhibitors.
Moreover, the hydrophobic interaction domains located at the binding
pocket seem to be responsible for the affinity of VEGFR-2. Compound 7
with the most favorable orientation to the VEGFR-2 from the docking
studies, was also the best inhibitor of the receptor. The reduced inhibi-
tory effect of compounds 2 and 15b on the VEGFR-2 could be due to the
absence of suitable size of the hydrophobic head as a prerequisite
pharmacophoric feature existing in the reference drug. Although the
docking study was able to predict the target protein (VEGFR-2) of the
newly synthesized thiadiazole derivatives but it failed to identify the
right inhibitors of the receptor. Only nine out of 19 were truly inhibitors
of the VEGFR-2 at the nanomolar concentration. The antiproliferative
activity of the thiadiazole derivatives was positively correlated to the
inhibitory activity of these derivatives against the VEGFR-2. All the
active antiproliferative compounds were good inhibitors of the VEGFR-2
at 7.4–11.5 nM compared with Pazopanib. However, since other mo-
lecular targets were not examined, other possibilities cannot be ruled
out.
NMR (300 MHz, DMSO‑d6) δppm: 3.84 (s, 2H, CH2), 6.74 (dd, J = 1.8,
1.5 Hz, 1H), 7.83 (d, J = 3.6 Hz, 1H), 8.05 (d, J = 1.8 Hz, 1H), 11.16 (br
s, 1H, CSNH, exchangeable with D2O), 11.53 (br s, 1H, NHCO,
exchangeable with D2O), 12.50 (br s, 1H, CSNHCO, exchangeable with
D2O). Anal. calcd for C9H8N4O3S (252.25)%: C, 42.85; H, 3.20; N, 22.21;
found: C, 42.73; H, 3.15; N, 22.16.
2-(Furan-2-yl)-4-thioxo-3,4-dihydropyrazolo[1,5-a][1,3,5]tri-
azin-7(6H)-one (3)
Method 1 (conventional) A mixture of compound 2 (1 g, 4 mmol)
and 10 mL of 10% ethanolic KOH was refluxed for 6 hrs. The progress of
the reaction was monitored by TLC. The solid product was obtained
upon pouring onto ice-water containing few drops of hydrochloric acid,
and then collected by filtration, dried, and recrystallized from ethanol/
dioxane (1:3) affording the pure compound 3 as gray crystals (0.68 g,
74%); m.p. > 300 ◦C; IR (KBr) (υmax, cmꢀ 1): 3180, 3160 (NH), 3001 (CH
aromatic), 2967, 2900 (CH aliphatic), 1731 (C O), 1626 (C N). 1H
–
–
–
–
NMR (400 MHz, DMSO‑d6) δppm: 5.99 (s, 1H, CH olefinic), 6.77 (dd, J =
1.68, 1.7 Hz, 1H), 7.80 (d, J = 3.4 Hz, 1H), 8.04 (d, J = 3.32 Hz, 1H),
11.78 (br s, 1H, CSNH, exchangeable with D2O), 13.63 (br s,1H, CONH,
exchangeable with D2O). 13C NMR (400 MHz, DMSO‑d6) δppm: 86.76,
113.34, 116.93, 141.45, 144.81, 145.40, 148.00, 167.20, 168.23. Anal.
Calcd. for C9H6N4O2S (234.23)%: C, 46.15; H, 2.58; N, 23.92; Found: C,
46.18; H, 2.61; N, 23.95.
Method 2 (MW) A mixture of compound 2 (1 g, 4 mmol) and 5 mL of
10% ethanolic KOH was subjected to MW at 600 W for 5 min. The sol-
vent was evaporated under reduced pressure and the remaining solid
was treated with ethanol. The solid product was filtered off, washed
with absolute ethanol, dried, and recrystallized from ethanol/dioxane
(1:3) affording the pure compound 3 as gray crystals (0.77 g, 83%).
(identity mp, TLC, and IR).
6-(Furan-2-yl)-4-iminopyrazolo[3,4-d][1,3]oxazin-3(4H)-one (4)
Method 1 (conventional) Few drops of TEA were added to an
ethanolic solution of compound 2 (1 g, 4 mmol), then the mixture was
refluxed for 4 hrs and then acidified by cold dil. HCl. The progress of the
reaction was monitored by TLC. The resulting precipitate was separated
by filtration, dried and recrystallized from ethanol/dioxane (1:2)
affording the pure compound 4 as brown crystals (0.3 g, 35%); m.p
230–232 ◦C; IR (KBr) (υmax, cmꢀ 1): 3130 (NH), 3050 (CH aromatic),
2953, 2918, 2849 (CH aliphatic), 1669 (C O). 1H NMR (500 MHz,
–
–
3. Experimental
DMSO‑d6) δppm: 6.74 (dd, J = 2, 2.5 Hz, 1H), 7.67 (d, J = 3.5 Hz, 1H),
–
8.02 (d, J = 3.4 Hz, 1H), 12.67 (br s, 1H, C NH, exchangeable with
–
3.1. Chemistry
D2O). 13C NMR (500 MHz, DMSO‑d6) δppm: 112.46, 117.11, 145.36,
147.54 (2), 155.01(2), 155.89, 165. Anal. calcd for C9H4N4O3
(216.16)%: C, 50.01; H, 1.87; N, 25.92; found C, 50.22; H, 1.96; N,
26.01.
All melting points were taken on a Griffin and George melting-point
apparatus (Griffin & George Ltd., Wembley, Middlesex, UK) and were
uncorrected. The IR spectra were recorded on Pye Unicam SP1200
spectrophotometer (Pye Unicam Ltd., Cambridge, UK) using the KBr
wafer technique. The 1H NMR spectra were determined on a Varian
Gemini 300 MHz, 400 MHz, and 500 MHz on Bruker Avance III using
tetramethylsilane as an internal standard (chemical shifts in δ scale),
while the 13C NMR spectra were run at 100 MHz. TMS was used as an
internal standard in deuterated dimethylsulphoxide (DMSO‑d6).
Chemical shifts were quoted as δ. Elemental analyses were carried out at
the Microanalytical Unit, Faculty of Science, Ain Shams University
(Cairo, Egypt), using a Perkin-Elmer 2400 CHN elemental analyzer, and
satisfactory analytical data (±0.4) were obtained for all compounds. The
purity of the synthesized compounds was confirmed by the thin layer
chromatography (TLC) using aluminum sheet silica gel F254 (Merck).
N-(2-(2-Cyanoacetyl)hydrazine-1-carbonothioyl)furan-2-carbox-
amide (2)
Method 2 (MW) Few drops of TEA were added to a solution of
compound 2 (1 g, 4 mmol) in ethanol (5 mL) and the mixture was
subjected to MW at 600 W for 3 min. After cooling the reaction mixture
was acidified by cold dil. HCl. The deposited solid was filtrated, dried,
and recrystallized from ethanol/dioxane (1:2) giving the pure com-
pound 4 as brown crystals (0.4 g, 47%). (identity mp, TLC, and IR).
N-(5-Imino-3-oxopyrazolidine-1-carbonothioyl)furan-2-carbox-
amide (5)
A mixture of compound 2 (1 g, 4 mmol) and sulfuric acid (10 mL)
kept in refrigerator overnight, and then poured onto ice-water. The
precipitated solid was separated by filtration, dried and recrystallized
from DMF giving the pure compound 5 as pale yellow crystals (0.9 g,
90%); m.p > 300 ◦C; IR (KBr) (υmax, cmꢀ 1): 3133, 3124 (NH), 3008 (CH
1
–
aromatic), 2906, 2817 (CH aliphatic), 1752, 1669 (C O). H NMR (500
–
MHz, DMSO‑d6) δppm: 3.92 (s, 2H, CH2), 6.74 (dd, J = 1.5, 2 Hz, 1H),
A solution of 2-furoyl isothiocyanate (1) (1.53 g, 10 mmol) in dry
8

M.H. Hekal et al.
Bioorganic Chemistry 115 (2021) 105176
–
7.26 (br s, 1H, C NH, exchangeable with D O), 7.68 (d, J = 3.5 Hz, 1H),
The deposited solid was filtered off, dried, and recrystallized from
dioxane affording the pure compound 8 as orange crystals (1.39 g, 85%).
(identity mp, TLC, and IR).
–
7.77 (br s, 1H, NHCO, exchangeable with D22O), 8.02 (d, J = 3.5 Hz, 1H),
12.91 (br s, 1H, CSNHCO, exchangeable with D2O). 13C NMR (500 MHz,
DMSO‑d6) δppm: 36.33, 76.04, 112.44, 117.18, 131.66, 147.61, 156.09,
158.63, 169.38. Anal. calcd for C9H8N4O3S (252.25)%: C, 42.85; H,
3.20; N, 22.21; Found: C, 42.90; H, 3.18; N, 22.19.
N-(5-(2-Oxo-2H-chromen-3-yl)-1,3,4-thiadiazol-2-yl)furan-2-
carboxamide (9)
A mixture of compound 8 (1 g, 4 mmol) and 3-aminopyridine and/or
p-toluidine (10 mmol) in glacial acetic acid (10 mL) was stirred for 3 hrs,
then kept standing overnight at room temperature. The resulting pre-
cipitate was separated by filtration, washed by cold water, dried and
recrystallized from ethanol/dioxane (1:5) affording the pure compound
9 as green crystal (0.87 g, 87%); m.p 298–300 ◦C; IR (KBr) (υmax, cmꢀ 1):
N-(5-(Cyanomethyl)-1,3,4-oxadiazol-2-yl)furan-2-carboxamide
(6)
Method 1 (conventional) A mixture of compound 2 (1 g, 4 mmol)
and mercuric oxide (2 g, 1.5 mmol) in absolute ethanol (10 mL) was
refluxed for 4 hrs. The reaction mixture was cooled to room temperature
until the black mercuric sulfide precipitated then filtered off and the
mercuric sulfide was washed with boiling ethanol. The filtrate was
treated with water until a turbidity formed, then kept standing overnight
at room temperature. The product was separated by filtration, dried, and
recrystallized from ethanol giving the pure compound 6 as pale yellow
crystals (0.21 g, 25%); m.p 210–211 ◦C. IR (KBr) (υmax, cmꢀ 1): 3127
(NH), 3005 (CH aromatic), 2955, 2925, 2852 (CH aliphatic), 2261
3168 (NH), 3021 (CH aromatic), 1713, 1681 (C O), 1604 (C N). 1H
–
–
–
–
NMR (500 MHz, DMSO‑d6) δppm: 6.76 (dd, J = 1, 1.5 Hz, 1H), 7.46–7.55
(m, 5H, ArH), 8.06 (d, J = 3 Hz, 1H), 9.15 (s, 1H, C = CH),11.97 (br s,
1H, NHCO, exchangeable with D2O). 13C NMR (400 MHz, DMSO‑d6)
δ
ppm: 112.54, 116.36, 117.51, 117.91, 118.86, 125.29, 129.86, 133.35,
140.10, 145.20, 147.87, 153.23, 154.73, 159.47, 172.05. Anal. calcd for
C16H9N3O4S (339.33)%: C, 56.63; H, 2.67; N, 12.38; Found: C, 56.60; H,
2.70; N, 12.41.
1
–
–
–
–
–
(C N), 1696 (C O), 1637 (C N). H NMR (500 MHz, DMSO‑d ) δ :
–
–
6
ppm
4.60 (s, 2H, CH2), 6.73 (dd, J = 1.5, 2 Hz, 1H), 7.54 (d, J = 3.3 Hz, 1H),
General procedure for the synthesis of compounds (11a,b)
Method 1 (conventional) A mixture of compound 7 (1 g, 4 mmol)
and aldehyde derivatives; thiophene-2-carbaldehyde (0.45 mL, 4 mmol)
or p-chlorobenzaldehyde (0.6 g, 4 mmol) in absolute ethanol (20 mL)
with a catalytic amount of pieridine was refluxed for 2–3 hrs. The
progress of the reaction was monitored by TLC. The precipitated crystals
while heating were separated by filtration and washed by boiling
ethanol giving compounds 11a and 11b, respectively.
8.04 (d, J = 2.4 Hz, 1H), 12.15 (br s, 1H, exchangeable with D2O). 13
C
NMR (500 MHz, DMSO‑d6) δppm: 15.56, 88.80, 112.50, 114.49, 117.40,
147.60, 158.09. Anal. Calcd. for C9H6N4O3 (218.17)%: C, 49.55; H,
2.77; N, 25.68; Found: C, 49.52; H, 2.71; N, 25.73.
Method 2 (MW) A mixture of compound 2 (1 g, 4 mmol) and mer-
curic oxide (2 g, 1.5 mmol) in absolute ethanol (10 mL) was exposed to
MW at 400 W for 4 min. After cooling, the mercuric sulfide was filtrated
off and the filtrate was treated by cold water giving yellow precipitate,
then the product was separated by filtration, dried and recrystallized
from ethanol affording the pure compound 6 as pale yellow powder
(0.34 g, 40%). (identity mp, TLC, and IR).
Method 2 (MW) A mixture of compound 7 (1 g, 4 mmol) and alde-
hyde derivatives; thiophene-2-carbaldehyde (0.45 mL, 4 mmol) or p-
chlorobenzaldehyde (0.6 g, 4 mmol) with a catalytic amount of pieridine
was exposed to MW at 600 W for 2.5 and 4 min, respectively. After
cooling, the reaction mixture was acidified with cold dil. HCl. The
deposited solid was filtered off, dried and recrystallized from ethanol/
dioxane (1:5) affording the pure compounds 11a and 11b, respectively
(identity mp, TLC, and IR).
N-(5-(Cyanomethyl)-1,3,4-thiadiazol-2-yl)furan-2-carboxamide
(7)
Method A (conventional) A solution of compound 2 (1 g, 4 mmol) in
glacial acetic acid (10 mL) was refluxed for 90 min. The deposited solid
while heating was filtered off and washed by boiling glacial acetic acid
affording the pure compound 7 as yellow crystals (0.51 g, 55%); m.p
298–300 ◦C; IR (KBr) (υmax, cmꢀ 1): 3130, 3101 (NH), 3001 (CH aro-
N-(5-(1-Cyano-2-(thiophen-2-yl)vinyl)-1,3,4-thiadiazol-2-yl)
furan-2-carboxamide (11a)
Pale green crystals (1.21 g, 87%); m.p > 300 ◦C; IR (KBr) (υmax
,
1
matic), 2944, 2908 (CH aliphatic), 2255 (C N), 1657 (C O). H NMR
cmꢀ 1): 3125 (NH), 3097, 3006 (CH aromatic), 2906, 2817 (CH
–
–
–
–
–
aliphatic), 2225 (C N), 1680 (C O). 1H NMR (400 MHz, DMSO‑d6)
–
–
–
–
–
(300 MHz, DMSO‑d6) δppm: 4.59 (s, 2H, CH2), 6.76 (dd, J = 1.5, 1.5 Hz,
1H), 7.73 (d, J = 3.3 Hz, 1H), 8.04 (d, J = 1.4 Hz, 1H), 13.15 (br s, 1H,
CONH, exchangeable with D2O). Anal. calcd for C9H6N4O2S (234.23)%:
C, 46.15; H, 2.58; N, 23.92; found: C, 46.18; H, 2.63; N, 23.89.
Method B (MW) A solution of compound 2 (1 g, 4 mmol) in glacial
acetic acid (10 mL) was exposed to MW at 600 W for 2 min. The solvent
was evaporated under reduced pressure and the residue was washed
with boiling glacial acetic acid giving compound 7 as pure yellow
crystals (0.70 g, 76%). (identity mp mixed mp, TLC, and IR).
δ
ppm: 6.78 (dd, J = 1.68, 1.24 Hz, 1H), 7.33 (dd, J = 3.84, 3.80 Hz, 1H),
7.68 (d, J = 3.12 Hz, 1H), 7.77 (d, J = 2.8 Hz, 1H), 7.94–8.09 (m, 2H,
ArH), 8.55 (s, 1H olefinic), 13.33 (br s, 1H, NH, exchangeable with D2O).
13C NMR (400 MHz, DMSO‑d6) δppm: 98.76, 113.03, 116.20, 118.26,
128.94, 134.88, 136.99, 137.88, 141.22, 142.30, 147.06, 148.49,
157.60, 159.46; Anal. calcd for C14H8N4O2S2 (328.37)%: C, 51.21; H,
2.46; N, 17.06; Found: C, 51.18; H, 2.33; N, 16.92.
N-(5-(2-(4-Chlorophenyl)-1-cyanovinyl)-1,3,4-thiadiazol-2-yl)
furan-2-carboxamide (11b).
N-(5-(2-Imino-2H-chromen-3-yl)-1,3,4-thiadiazol-2-yl)furan-2-
carboxamide (8).Method 1 (conventional) An ethanolic solution of
compound 7 (1 g, 4 mmol) and salicylaldehyde (0.5 mL, 4 mmol) with a
catalytic amount of pieridine was refluxed for 3 hrs. The progress of the
reaction was followed up by TLC. The obtained product on heating was
filtered off and recrystallized from dioxane affording compound 8 as
orange crystals (1.27 g, 78%); m.p > 300 ◦C; IR (KBr) (υmax, cmꢀ 1): 3302
Yellow crystals (1.34 g, 88%); m.p > 300 ◦C; IR (KBr) (υmax, cmꢀ 1):
3150, 3129 (NH), 3096 (CH aromatic), 2920, 2850 (CH aliphatic), 2216
1
–
–
–
(C N), 1673 (C O). H NMR (400 MHz, DMSO‑d ) δppm: 6.76 (dd, J =
–
–
6
1.60, 1.64 Hz, 1H), 7.64–7.71 (m, 3H, ArH), 8.04–8.06 (m, 3H, ArH),
8.25 (s, 1H olefinic), 13.37 (br s, 1H, NH, exchangeable with D2O). 13
C
NMR (400 MHz, DMSO‑d6) δppm: 103.93, 112.94, 115.85, 117.93,
129.74, 131.93, 132.04, 136.85, 146.12, 146.39, 148.22, 156.91,
159.10, 160.49. Anal. calcd for C19H9ClN4O2S (356.79)%: C, 53.86; H,
2.54; Cl, 9.94; N, 15.70; Found: C, 53.90; H, 2.61; Cl, 10.02; N, 15.79.
N-(5-(1-(4-Oxo-4,5-dihydrothiazol-2-yl)-2-(thiophen-2-yl)vinyl)-
1,3,4-thiadiazol-2-yl)furan-2-carboxamide (12)
–
–
(NH), 3131 (CH aromatic), 2952, 2913 (CH aliphatic), 1673 (C O),
1
–
–
1658 (C N). H NMR (300 MHz, DMSO‑d ) δppm: 6.75 (dd, J = 9, 6 Hz,
6
1H), 7.21–7.73 (m, 5H, ArH), 8.03 (d, J = 9 Hz, 1H), 8.60 (s, 1H, C =
CH), 9.03 (br s, 1H, C = NH, exchangeable with D2O), 12.98 (br s, 1H,
NHCO, exchangeable with D2O). Anal. calcd for C16H10N4O3S
(338.34)%: C, 56.80; H, 2.98; N, 16.56; Found: C, 56.77; H, 3.01; N,
16.60.
Method 1 (conventional) A solution of compound 11a (1 g, 3 mmol)
and thioglycolic acid (3 mmol) in pyridine (10 mL) was refluxed for 2
hrs. The progress of the reaction was monitored by TLC. The formed
crystals while heating was filtered off and washed with boiling DMF
giving the pure compound 12 as colorless crystals (0.73 g, 60%); m.p
292–294 ◦C; IR (KBr) (υmax, cmꢀ 1): 3267, 3130 (NH), 3092 (CH
Method 2 (MW) An ethanolic solution of compound 7 (1 g, 4 mmol)
and salicylaldehyde (0.5 mL, 4 mmol) with a catalytic amount of pier-
idine was subjected to MW at 600 W for 3 min. The solvent was evap-
orated under reduced pressure and the residue was treated with ethanol.
9

M.H. Hekal et al.
Bioorganic Chemistry 115 (2021) 105176
1
–
–
aromatic), 2963, 2923 (CH aliphatic), 1721, 1697 (C O). H NMR (400
Method 1 (conventional) A solution of compound 7 (1 g, 4 mmol) in
pyridine with glycolic acid (0.31 mL, 4 mmol), lactic acid (0.36 mL, 4
mmol), or thioglycolic acid (0.40 mL, 4 mmol) was refluxed for 3–4 hrs.
The progress of the reactions was followed up by TLC. The formed
crystals while heating was filtrated and recrystallized from dioxane/
DMF affording the compounds 15 (a-c), respectively.
–
–
MHz, DMSO‑d6) δ : 3.94 (s, 2H, CH ), 6.34 (s, 1H, C CH), 6.75–6.76
(m, 2H), 7.68–7.69 (m, 2H), 8.034–28.036 (m, 2H), 11.48 (br s, 1H,
ppm
NHCO, exchangeable with D2O). 13C NMR (400 MHz, DMSO‑d6) δ
:
39.7, 98.7, 113.03, 116.20, 118.26, 128.94, 134.88, 136.99, 137p.8pm8,
141.22, 148.49, 159.46. Anal. calcd for C16H10N4O3S3 (402.47)%: C,
47.75; H, 2.50; N, 13.92; Found: C, 47.77; H, 2.55; N, 13.99.
Method 2 (MW) A solution of compound 11a (1 g, 3 mmol) and
thioglycolic acid (3 mmol) in pyridine (5 mL) was exposed to MW at 600
W for 3 min. After cooling, the reaction mixture was treated with DMF.
The deposited solid was filtered off and washed well by boiling DMF
giving the pure compound 12 as colorless crystals (1.01 g, 83%) (iden-
tity mp, TLC, and IR).
Method 2 (MW) A solution of compound 7 (1 g, 4 mmol) in pyridine
with glycolic acid (0.31 mL, 4 mmol), lactic acid (0.36 mL, 4 mmol), or
thioglycolic acid (0.40 mL, 4 mmol) was exposed to MW at 600 W for
5–7 min. The solvent was evaporated, and the residue was treated with
dioxane. The solid product was filtered off, recrystallized from dioxane/
DMF affording the compounds 15 (a-c), respectively. (identity mp, TLC,
and IR).
N-(5-(Cyano(cyclohexylidene)methyl)-1,3,4-thiadiazol-2-yl)
furan-2-carboxamide (13)
(E)-N-(5-((4-Oxo-4,5-dihydrooxazol-2-yl)methyl)-1,3,4-thiadia-
zol-2-yl)furan-2-carboxamide (15a)
Method 1 (conventional) A mixture of compound 7 (1 g, 4 mmol)
and cyclohexanone (0.4 mL, 4 mmol) in ethanol in the presence of a
catalytic amount of piperidine was refluxed for 3 hrs. The progress of the
reaction was checked by TLC, and then pouring onto crushed ice con-
taining HCl. The resulting precipitate was separated by filtration, dried
and recrystallized from ethanol/dioxane (1:3) affording the pure com-
pound 13 as pale yellow crystals (0.99 g, 74%); m.p 234–236 ◦C; IR
(KBr) (υmax, cmꢀ 1): 3216 (NH), 3129, 3004 (CH aromatic), 2920, 2850
Colorless crystals (1.09 g, 88%); m.p > 300 ◦C; IR (KBr) (υmax, cmꢀ 1):
3266 (NH), 3091, 3006 (CH aromatic), 2962, 2924, 2849 (CH aliphatic),
1719, 1696 (C O). 1H NMR (400 MHz, DMSO‑d6) δppm: 3.94 (s, 2H,
–
–
CH2), 6.34 (s, 1H, =CH olefinic), 6.75 (dd, J = 2.5, 1 Hz, 1H), 7.83 (d, J
= 2.5 Hz, 1H), 8.05 (d, J = 3 Hz, 1H), 11.44 (br s, 1H, NH, exchangeable
with D2O), 12.92 (br s, 1H, NHCO, exchangeable with D2O). 13C NMR
(400 MHz, DMSO‑d6) δppm: 60.33, 88.70, 90.22, 112.73, 114.60,
117.15, 146.53, 148.11, 159.85, 173.88. Anal. calcd for C11H8N4O4S
(292.27)%: C, 45.20; H, 2.76; N, 19.17; Found: C, 45.09; H, 2.63; N,
19.02.
(CH aliphatic), 2211 (C N), 1686 (C O). 1H NMR (500 MHz,
–
–
–
–
–
DMSO‑d6) δppm: 1.61–1.66 (m, 4H), 1.75–1.76 (m, 2H), 2.7 (t, 4H), 6.77
(dd, J = 2.5, 1 Hz, 1H), 7.74 (d, J = 3.5 Hz, 1H), 8.06 (d, J = 3.4 Hz, 1H),
13.28 (br s, 1H, NH, exchangeable with D2O). 13C NMR (500 MHz,
DMSO‑d6) δppm: 18.33, 24.92, 27.43, 27.85, 32.06, 35.55, 66.36, 98.11,
112.60, 116.48, 117.65, 117.85, 144.99, 148.05, 155.18, 155.77,
159.60, 170.26. Anal. calcd for C15H14N4O2S (314.36)%: C, 57.31; H,
4.49; N, 17.82; Found: C, 57.29; H, 4.53; N, 17.85.
(E)-N-(5-((5-Methyl-4-oxo-4,5-dihydrooxazol-2-yl)methyl)-
1,3,4-thiadiazol-2-yl)furan-2-carbox-amide (15b)
Red crystals (1.08 g, 83%); m.p 284–286 ◦C; IR (KBr) (υmax, cmꢀ 1):
3266, 3210 (NH), 3091, 3006 (CH aromatic), 2962, 2924, 2849 (CH
1
–
aliphatic), 1719, 1696 (C O). H NMR (400 MHz, DMSO‑d ) δppm: 1.59
–
6
–
–
–
–
(d, J = 4.7 Hz, 3H, CH CH3), 4.04 (q, 1H, CH CH ), 6.35 (s, 1H, CH
3
Method 2 (MW) A mixture of compound 7 (1 g, 4 mmol) and
cyclohexanone (0.4 mL, 4 mmol) with catalytic amount of pieridine was
exposed to MW at 600 W for 4 min. The residue was treated with
dioxane. The solid product was filtered off, washed with dioxane, dried,
and recrystallized from dioxane giving the pure compound 13 as pale
yellow crystals (1.15 g, 86%). (identity mp mixed mp, TLC, and IR).
N-(5-Amino-6,8-dicyano-7-(4-(piperidin-1-yl)phenyl)-7H-[1,3,4]
thiadiazolo[3,2-a]pyridin-2-yl)furan-2-carboxamide (14)
olefinic), 6.75 (dd, J = 2.5, 2 Hz, 1H), 7.68 (d, J = 3.4 Hz, 1H), 8.02 (d, J
= 1.04 Hz, 1H), 11.56 (br s, 1H, NH, exchangeable with D2O), 12.92 (br
s, 1H, NHCO, exchangeable with D2O). 13C NMR (400 MHz, DMSO‑d6)
δ
ppm: 15.36, 65.33, 87.70, 90.18, 112.89, 114.56, 117.61, 146.50,
148.02, 159.65, 173.72. Anal. calcd for C12H10N4O4S (306.30)%: C,
47.06; H, 3.29; N, 18.29; Found: C, 47.26; H, 3.47; N, 18.33.
(E)-N-(5-((4-Oxo-4,5-dihydrothiazol-2-yl)methyl)-1,3,4-thiadia-
zol-2-yl)furan-2-carboxamide (15c)
Method 1 (conventional) A mixture of compound 7 (1 g, 4 mmol)
and 2-(4-chloro benzyl- idene)malononitrile “arylidene” (0.8 g, 4 mmol)
in ethanol (20 mL) with a catalytic amount of pieridine was refluxed for
5 hrs. The progress of the reaction was monitored by TLC, and the
crystals that precipitated while heating were filtered off and recrystal-
lized from dioxane giving the pure compound 14 as red crystals (1.31 g,
73%); m.p 262–264 ◦C; IR (KBr) (υmax, cmꢀ 1): 3358, 3222 (NH2), 3130,
Yellow crystals (0.98 g, 75%); m.p 290–292 ◦C; IR (KBr) (υmax
,
cmꢀ 1): 3266, 3210 (NH), 3091, 3006 (CH aromatic), 2962, 2924, 2849
1
–
(CH aliphatic), 1719, 1696 (C O). H NMR (400 MHz, DMSO‑d ) δ
:
ppm
–
6
–
3.94 (s, 2H, CH ), 6.34 (s, 1H, CH olefinic), 6.75 (dd, J = 3, 2.4 Hz,
–
2
1H), 7.68 (d, J = 2.5 Hz, 1H), 8.05 (d, J = 3.24 Hz, 1H), 11.44 (br s, 1H,
NH, exchangeable with D2O), 12.91 (br s, 1H, NHCO, exchangeable with
D2O). 13C NMR (400 MHz, DMSO‑d6) δppm: 34.30, 90.18, 108.55,
112.89,114.50, 117.61, 146.50, 148.02, 159.65, 173.72. Anal. calcd for
–
–
3100 (CH aromatic), 2925, 2854 (CH aliphatic), 2213 (C N), br.1660
–
1
–
–
(C O). H NMR (400 MHz, DMSO‑d ) δppm: 1.58–1.68 (m, 6H), 3.08 (t,
C11H8N4O3S2 (308.34)%: C, 42.85; H, 2.62; N, 18.17; Found: C, 42.80;
4H), 4.58 (s, 1H), 6.76 (br s, 2H6, NH2, exchangeable with D2O),
7.58–7.86 (m, 7H, ArH), 8.04 (br s, 1H, NHCO, exchangeable with D2O).
13C NMR (400 MHz, DMSO‑d6) δppm: 18.82, 22.14, 22.73, 44.30, 71.47,
72.75, 92.30, 98.35, 112.94, 113.29, 116.98, 117.83, 129.28, 129.52,
130.28, 131.05, 131.25, 134.62, 135.78, 148.16, 154.35, 160.56,
161.47. Anal. calcd for C19H11ClN6O2S (422.85)%: C, 53.97; H, 2.62; Cl,
8.38; N, 19.87; Found: C, 53.82; H, 2.70; Cl, 8.43; N, 19.93.
The filtrate was acidified by cold dilute hydrochloric acid. The
deposited solid was filtered off, dried and recrystallized from dioxane
afford the pure compound 11b.
H, 2.57; N, 18.14.
N-(5-((4-Oxo-5-(4-(piperidin-1-yl)benzylidene)-4,5-dihy-
drothiazol-2-yl)methyl)-1,3,4-thiadiazol-2-yl)furan-2-carboxamide
(16)
Method 1 (conventional) A mixture of compound 15c (1 g, 3 mmol)
and p-chlorobenzaldehyde (0.45 g, 3 mmol) in absolute ethanol (15 mL)
with a catalytic amount of pieridine was refluxed for 3 hrs. The progress
of the reaction was controlled by TLC. The obtained product upon
heating was filtered off, dried and recrystallized from dioxane giving the
compound 16 as orange crystals (1.35 g, 87%); m.p 278–280 ◦C; IR
Method 2 (MW) A mixture of compound 7 (1 g, 4 mmol) and 2-(4-
chlorobenzylidene) malononitrile “arylidene” (0.8 g, 4 mmol) with
catalytic amount of pieridine was subjected to MW at 600 W for 5.5 min.
After cooling, the obtained residue was treated with ethanol. The
deposited solid was filtered off, dried and recrystallized from dioxane
affording the pure compound 14 as red crystals (1.51 g, 84%). (identity
mp, TLC, and IR).
(KBr) (υmax, cmꢀ 1): 3145 (NH), 3005 (CH aromatic), 2925, 2849 (CH
1
–
aliphatic), 1683 (C O). H NMR (400 MHz, DMSO‑d ) δppm: 1.05–1.67
–
6
(m, 6H, 3 CH2 of piperidinyl ring), 2.98 (t, 4H, 2 CH2 of piperidinyl
ring), 6.75 (s, 1H, CH olefinic), 7.28–8.05 (m, 7H, ArH + 1H olefinic),
12.27 (br s, 1H, NH, exchangeable with D2O), 12.89 (br s, 1H, NHCO,
exchangeable with D2O).13C NMR (400 MHz, DMSO‑d6) δppm: 15.46,
22.14, 22.57, 44.02, 112.95, 117.88, 127.71, 128.31, 128.61, 128.73,
128.90, 128.21, 129.69, 129.84, 131.61, 131.65, 131.75, 131.89,
General procedure for the synthesis of compounds (15a-c)
10

M.H. Hekal et al.
Bioorganic Chemistry 115 (2021) 105176
131.98, 133.43, 134.31, 168.75. Anal. calcd for C23H21N5O3S2
(479.57)%: C, 57.60; H, 4.41; N, 14.60; Found: C, 57.54; H, 4.45; N,
14.66.
Dark green crystals (1.50 g, 89%); m.p 296–298 ◦C; IR (KBr) (υmax
,
cmꢀ 1): 3128 (NH), 3098 (CH aromatic), 2976, 2943 (CH aliphatic),
–
–
–
–
–
–
2196 (C N), 1717 (C
O
), 1663 (C O
amide). 1H NMR (500 MHz,
DMSO‑d6) δppm: 1.19 (t, 3eHst,erCH2CH3), 4.08 (q, 2H, CH2CH3), 4.11 (s,
–
Method 2 (MW) A mixture of compound 15c (1 g, 3 mmol) and p-
chlorobenzaldehyde (0.45 g, 3 mmol) with catalytic drops of pieridine
was exposed to MW at 600 W for 4 min. After cooling, the obtained
product was treated with ethanol and recrystallized from dioxane givi-
ing the compound 16 as orange crystals (1.44 g, 93%). (identity mp,
TLC, and IR).
–
2H, SCH2), 4.58 (s, 1H, CH CN), 6.74 (dd, J = 4, 2.8 Hz, 1H), 7.72 (d, J
= 4 Hz, 1H), 8.04 (d, J = 7 Hz, 1H,), 13.10 (br s, 1H, NHCO,
exchangeable with D2O). Anal. calcd for C14H12N4O4S3 (396.46)%: C,
42.41; H, 3.05; N, 14.13; Found: C, 42.39; H, 3.00; N, 14.11.
N-(5-(3-Amino-4-cyano-5-oxo-2,5-dihydrothiophen-2-yl)-1,3,4-
thiadiazol-2-yl)furan-2-carboxamide (17)
3.2. Biological evaluation
Method 1 (conventional) A mixture of compound 7 (1 g, 4 mmol),
sulfur metal (0.32 g, 10 mmol) and ethylcyanoacetate (0.5 mL, 4 mmol)
in absolute ethanol with few drops of TEA was refluxed for 15 hrs. The
progress of the reaction was followed up by TLC, then pouring onto
crushed ice-water containing hydrochloric acid. The resulting precipi-
tate was separated by filtration, dried and washed by boiling xylene (to
dissolve the excess sulfur metal), then recrystallized from ethanol/
dioxane (1:5) affording the pure compound 17 as gray crystals (1.23 g,
87%); m.p over 300 ◦C; IR (KBr) (υmax, cmꢀ 1): 3293, 3145 (NH), 3005
3.2.1. In vitro cytotoxic activity
The cytotoxicity assays were performed using three different human
cancer cell lines; breast adenocarcinoma (MCF-7), human colon cancer
(HCT-116) and human prostate cancer (PC-3) cells as well as the normal
fibroblasts (WI-38), which were purchased from the American Type Cell
Culture Collection (ATCC; Manassas, VA, USA) and grown on the suit-
able growth medium (RPMI 1640) supplemented with 100 mg/ml of
streptomycin, 100 u/ml of penicillin and 10% of heat-inactivated fetal
bovine serum in a humidified, 5% (v/v) CO2 atmosphere at 37 ◦C. The
cytotoxicity was performed using the MTT assay. The antiproliferative
activities were performed at the Micro analytical Center of Mansoura
University (Mansoura, Egypt).
–
–
(CH aromatic), 2920, 2850 (CH aliphatic), 2204 (C N), 1736, 1674
–
1
–
–
(C O). H NMR (400 MHz, DMSO‑d ) δppm: 3.95 (s, 1H), 6.75 (dd, J =
1.52, 1.44 Hz, 1H), 7.27 (br s, 1H, NH6, exchangeable with D2O), 7.78 (br
–
s, 1H, C
–
C OH, exchangeable with D2O), 7.70 (d, J = 3.36 Hz, 1H),
–
The exponentially growing cells from the cancer cell lines were
trypsinized, counted, and seeded at the appropriate densities (~2000
cells/well) into 96-well microtiter plates. The cells were then incubated
in a humidified atmosphere at 37 ◦C for 24 hrs. Then, the cells were
exposed to different concentrations of the compounds (0.5–100 µM) for
48 hrs. Then the viability of the treated cells was determined using the
MTT technique. Briefly, the media were removed, cells were incubated
with 200 µl of 5% MTT solution/well (5 mg mLꢀ 1) (Sigma-Aldrich, MO)
and were allowed to metabolize the dye into colored-insoluble formazan
crystal for four hrs at 37 ◦C. The remaining MTT solution was discarded
and the formazan crystals were dissolved in DMSO for 30 mins, covered
with aluminum foil with continuous shaking using MaxQ 2000 plate
shaker (Thermo Fischer Scientific Inc, Ml) at room temperature.
Absorbance was measured at 570 nm using ELISA plate reader. The cell
viability was expressed as percentage of control and the concentration
that induces 50% of maximum inhibition of cell proliferation (IC50).
8.03 (d, J = 3.20 Hz, 1H), 12.84 (br s, 1H, NHCO, exchangeable with
D2O). 13C NMR (400 MHz, DMSO‑d6) δppm: 36.79, 112.88, 117.61,
118.02, 145.87, 148.04, 156.27, 159.08, 160.02, 160.28, 169.81. Anal.
calcd for C12H7N5O3S2 (333.35)%: C, 43.24; H, 2.12; N, 21.01; Found: C,
43.30; H, 2.16; N, 20.98.
Method 2 (MW) A mixture of 7 (1 g, 4 mmol), sulfur metal (0.32 g,
10 mmol) and ethylcyanoacetate (0.5 mL, 4 mmol) with catalytic
amount of TEA was exposed to MW at 600 W for 7 min. After cooling, the
obtained product was treated with ethanol, dried and then recrystallized
from ethanol/dioxane affording compound 17 as white crystals (1.31 g,
92%) (identity mp, TLC, and IR).
General procedure for synthesis of compounds (19–21)
1,3,4-Thiadiazole derivative 7 (1 g, 4 mmol) was added to a cold
suspension of finely divided potassium hydroxide (0.25 g, 4 mmol) in
DMF (15 mL) with stirring for 15 min. Carbon disulphide (0.5 mL, 4
mmol) was added dropwise and then the mixture was allowed to stand
overnight. 1,2-Dibromoethane, chloro acetylchloride, or ethyl chlor-
oacetate (4 mmol) was added dropwise, stirred at room temperature for
3 hrs and then allowed to stand overnight. The reaction mixture was
poured onto ice-cold water with stirring. The separated solid was filtered
off, washed with water, dried and crystallized from dioxane giving
compounds 19–21, respectively.
3.2.2. Molecular docking
All the synthesized compounds were subjected to molecular docking
in order to explore their binding modes towards the VEGFR-2 receptor.
AutoDock program was used.[48] AutoDock is a suit of automated
docking tools which allows flexible ligand docking and freely available
under the GNU general public license.[49] AutoDock predicts how small
molecules such as substrates or drug candidates bind to a receptor of
known 3D structure. AutoDock suit includes two main programs; the
AutoGrid, which pre-calculates the grids describing the target protein
and the AutoDock, which performs the docking of the ligand to the
target protein.
N-(5-(Cyano(1,3-dithiolan-2-ylidene)methyl)-1,3,4-thiadiazol-2-
yl)furan-2-carboxamide (19).
Brown crystals (1.25 g, 87%); m.p > 300 ◦C; IR (KBr) (υmax, cmꢀ 1):
–
–
3128 (NH), 3005 (CH aromatic), 2921 (CH aliphatic), 2204 (C N),
–
1669 (C O). 1H NMR (500 MHz, DMSO‑d6) δppm: 3.85 (t, 4H, 2CH2),
–
–
6.75 (dd, J = 4.5, 5 Hz, 1H), 7.83 (d, J = 1.5 Hz, 1H), 8.05 (d, J = 1.5 Hz,
The scoring function used is empirically derived for empirical
binding free energy force field that allows the prediction of binding free
energies for docked ligands. AutoDock is based on the United Atom
force-filed of AMBER which uses only polar hydrogen. This helps to
reduce the number of atoms that must be modelled explicitly during the
docking, thus speeding up the calculations.
1H), 13.20 (br s, 1H, NHCO, exchangeable with D2O). Anal. calcd for
C
12H8N4O2S3 (336.41)%: C, 42.84; H, 2.40; N, 16.66; Found: C, 42.90;
H, 2.34; N, 16.61.
N-(5-(Cyano(4-oxo-1,3-dithiolan-2-ylidene)methyl)-1,3,4-thia-
diazol-2-yl)furan-2-carboxamide (20)
Dark red crystals (1.31 g, 88%); m.p > 300 ◦C; IR (KBr) (υmax, cmꢀ 1):
3131 (NH), 3098 (CH aromatic), 2963, 2918 (CH aliphatic), 2195
3.2.3. The VEGFR-2 kinase inhibitory assay
1
–
–
–
–
(C N), 1718, 1665 (C O). H NMR (400 MHz, DMSO‑d ) δppm: 4.1 (s,
The kinase activity of VEGFR-2 was carried out using an anti-
phosphotyrosine antibody with the Alpha Screen system (PerkinElmer)
based on the methodology described elsewhere. [50,51] The reaction
was performed in 50 mM Tris-HCl, pH 7.5, 5 mM MnCl2, 5 mM MgCl2,
0.01% Tween-20, and 2 mM DTT, containing 10 µM adenosine
triphosphate (ATP), 0.1 µg/ml biotinylated poly-GluTyr (4:1) and 0.1
nM of VEGFR-2 (Millipore, UK). Before catalytic initiation with ATP, the
2H, CH2), 6.76 (dd, J = 4.0, 2.8 Hz, 1H), 7.73 (d, J = 4.06Hz, 1H), 8.09
–
(d, 1H, J = 5.2 Hz), 12.84 (br s, 1H, NHCO, exchangeable with D2O).
Anal. calcd for C12H6N4O3S3 (350.40)%: C, 41.13; H, 1.73; N, 15.99;
Found: C, 41.21; H, 1.69; N, 16.07.
Ethyl2-((2-cyano-2-(5-(furan-2-carboxamido)-1,3,4-thiadiazol-
2-yl)ethanethioyl)thio)acetate (21)
11

M.H. Hekal et al.
Bioorganic Chemistry 115 (2021) 105176
[16] K. Bougrin, A. Loupy, M. Soufiaoui, Microwave-Assisted Solvent-Free Heterocyclic
Synthesis, J. Photochem. Photobiol. C. 6 (2005) 139–167.
tested compounds at final concentrations 0–100 nM and the protein
were incubated for 5 mins at room temperature. The reactions were
quenched by the addition of 25 µl of 100 mM EDTA, 10 µg/ml
AlphaScreen streptavidin cytotoxicity evaluation donor beads and 10
µg/ml acceptor beads in 62.5 mM 4-(2-hydroxymethyl)-1-piprazinee-
thanesulfonic acid, pH 7.4, 250 mM NaCl, and 0.1% bovine serum al-
bumin. The plate was incubated in the dark overnight and then read by
ELISA reader (PerkinElMer). Wells containing the substrate and the
enzyme without compounds were used as control. Wells containing
biotinylated poly-GluTyr (4:1) and enzyme without ATP were used as
basal control. The percent inhibition (IC50) was calculated from the
concentration-inhibition response curve (triplicate determinations) and
the data were compared with pazopanib (Sigma-Aldrich) as a standard
inhibitor of the VEGFR-2.
[17] M.A. Surati, S. Jauhari, K.R. Desai, A Brief Review: Microwave Assisted Organic
Reaction, Arch. Appl. Sci. Res. 4 (2012) 645–661.
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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.
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105176.
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