Design, synthesis and evaluation of novel thienopyrimidine-based agents bearing diaryl urea functionality as potential inhibitors of angiogenesis

Article abstract of DOI:10.1016/j.ejmech.2020.112942

Inhibition of angiogenesis is a promising strategy for the treatment of cancer. Herein, we describe the design and synthesis of thieno[2,3-d]pyrimidine-1,3,4-thiadiazole-aryl urea derivatives 11a-m to evaluate their efficacy in the chick chorioallantoic membrane (CAM) assay. Among target agents, 11i had a considerable activity against prostate cancer cell line, PC3 (IC₅₀ = 3.6 μM). Moreover, induction of apoptosis, good inhibitory activity against the growth of capillary blood vessels, and inhibition of VEGFR-2 phosphorylation were noticeable parameters which convinced us that 11i could be considered as a promising candidate for the discovery of novel drugs to treat tumors, particularly prostate cancer.

Full text of DOI:10.1016/j.ejmech.2020.112942

Journal Pre-proof
Design, synthesis and evaluation of novel thienopyrimidine-based agents bearing
diaryl urea functionality as potential inhibitors of angiogenesis
Aram Faraji, Tayebeh Oghabi Bakhshaiesh, Zaman Hasanvand, Rasoul Motahari,
Elahe Nazeri, Mohammad Amin Boshagh, Loghman Firoozpour, Hossein Mehrabi, Ali
Khalaj, Rezvan Esmaeili, Alireza Foroumadi
PII:
S0223-5234(20)30914-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112942
EJMECH 112942
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 June 2020
Revised Date: 10 October 2020
Accepted Date: 11 October 2020
Please cite this article as: A. Faraji, T.O. Bakhshaiesh, Z. Hasanvand, R. Motahari, E. Nazeri, M.A.
Boshagh, L. Firoozpour, H. Mehrabi, A. Khalaj, R. Esmaeili, A. Foroumadi, Design, synthesis and
evaluation of novel thienopyrimidine-based agents bearing diaryl urea functionality as potential
inhibitors of angiogenesis, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112942.
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Design, synthesis and evaluation of novel thienopyrimidine-based agents
bearing diaryl urea functionality as potential inhibitors of angiogenesis
a
b
a
a
Aram Faraji , Tayebeh Oghabi Bakhshaiesh , Zaman Hasanvand , Rasoul Motahari , Elahe
Nazeri b, Mohammad Amin Boshagh b, Loghman Firoozpour c, Hossein Mehrabi d, Ali Khalaj a,
Rezvan Esmaeili b,*, Alireza Foroumadi a, c, **
a Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
b Genetics Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
c
Drug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran
University of Medical Sciences, Tehran, Iran
d Department of Chemistry, Vali-e-Asr University of Rafsanjan, 77176 Rafsanjan, Iran
Cl
Cl
O
N
S
N
N
N
NH
NH
S
N
11i
S
Inhibition
Cancerous cells
VEGF
11i
VEGF-R
VEGF release
Angiogenesis
Endothelial cell
Metastasis

Design, synthesis and evaluation of novel thienopyrimidine-based agents
bearing diaryl urea functionality as potential inhibitors of angiogenesis
a
b
a
a
Aram Faraji , Tayebeh Oghabi Bakhshaiesh , Zaman Hasanvand , Rasoul Motahari , Elahe
Nazeri b, Mohammad Amin Boshagh b, Loghman Firoozpour c, Hossein Mehrabi d, Ali Khalaj a,
Rezvan Esmaeili b,*, Alireza Foroumadi a, c, **
a Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
b Genetics Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
c
Drug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran
University of Medical Sciences, Tehran, Iran
d Department of Chemistry, Vali-e-Asr University of Rafsanjan, 77176 Rafsanjan, Iran
Abstract:
Inhibition of angiogenesis is a promising strategy for the treatment of cancer. Herein, we
describe the design and synthesis of thieno[2,3-d]pyrimidine-1,3,4-thiadiazole-aryl urea
derivatives 11a-m to evaluate their efficacy in the chick chorioallantoic membrane (CAM) assay.
Among target agents, 11i had a considerable activity against prostate cancer cell line, PC3 (IC50
= 3.6 μM). Moreover, induction of apoptosis, good inhibitory activity against the growth of
capillary blood vessels, and inhibition of VEGFR-2 phosphorylation were noticeable parameters
which convinced us that 11i could be considered as a promising candidate for the discovery of
novel drugs to treat tumors, particularly prostate cancer.
Keywords: Angiogenesis, Thieno[2,3-d]pyrimidine, 1,3,4-Thiadiazole, CAM assay, PC3,
Apoptosis, VEGFR-2
* Corresponding author.
Tel: 98 21 88796003; Fax: 98 21 88796003. E-mail: esmaeili.rezvan@gmail.com (R. Esmaeili)
** Corresponding author.
Tel.: 98 21 66406757; Fax: 98 21 66461178. E-mail: aforoumadi@yahoo.com (A. Foroumadi)

1. Introduction
Cancer is the second lethal disease all around the world. According to World Health
Organization (WHO) reports, 9.6 million cancer deaths occurred in 2018. Regarding the
increasing rate of cancer prevalence, a great deal of effort in medicinal chemistry has been taken
toward design and synthesis of potential anti-cancer agents [1]. A considerable part of ongoing
research has been focused on providing novel targets controlling certain signaling pathways
attributed to cancer cell proliferation and propagation. Angiogenesis, process of new blood
vessel formation, is vital for cell development and reproduction. Considering the similar function
in cancerous cells, angiogenesis plays a significant role in tumor progression and metastasis.
Therefore, efficient anti-angiogenesis agents could be considered as promising strategy for
cancer treatment [2].
Growth factors, including vascular endothelial growth factors (VEGFs) and their receptors
(VEGFRs) regulate angiogenesis. Among three subtypes of VEGFRs, VEGFR-2 plays the most
substantial role in the tumor angiogenesis. VEGF-VEGFR-2 interaction leads dimerization and
subsequently autophosphorylation of tyrosine (Tyr1054 and Tyr1059) in receptor resulted into
angiogenesis signaling pathways in tumor environment [3]. Therefore, antitumor agents
interfering with tyrosine phosphorylation have inhibitory activity in angiogenesis. In addition to
VEGFR-2, tyrosine-protein kinase receptor (Tie-2) and erythropoietin producing hepatocyte
receptor B4 (EphB4) taking participation in maturation of blood vessels have recently
received interest in medicinal chemistry [4].
Over decades, different scaffolds showing VEGFR-2 inhibitory activity have been reported.
Among them, compounds having quinazolines, thienopyrimidines, indolines, urea derivatives, as
well as pyridine and pyrimidine derivatives have been more prevalent [5-13]. Thieno[2,3-
d]pyrimidines, in particular, have been found in many natural products and drugs, including
Relugolix, PRX-08066, and DDP-225. Moreover, they have shown wide spectrum of biological
activities, for example anticancer, kinase inhibition (like VEGFR-2 inhibition, FGFR1 inhibition,
B-Raf inhibition, FLT3 inhibition, and EGFR kinase inhibition), antioxidant, anti-inflammatory,
antimicrobial, antiviral, antituberculosis, and CNS protection activities [14, 15].
Some drugs containing diaryl urea, including sorafenib, regorafenib, and tivozanib have been
approved and marketed as VEGFR-2 inhibitors (Fig. 1). Diaryl ureas have played important role

in VEGFR-2 inhibitory potency; however, they have been modified to evaluate their activities.
For example, urea functional group has been replaced by thiourea moiety, as well as oxadiazole,
1,3,4thiadiazole, and 1,2,3-triazole rings to achieve more binding affinity with receptor [16-18].
Additionally, quinazolinones, indazolines, and thieno[2,3-d]pyrimidines have been utilized as
aryl ring substituent [19-21].
SKLB1002, quinazoline bearing 1,3,4-thiadiazole, has been identified as potent VEGFR-2
inhibitor with minimum toxicity [22-23]. In present study, SKLB1002 and sorafinib have been
employed to design a novel series of thieno[2,3-d]pyrimidine-1,3,4-thiadiazole-aryl urea
derivatives 11a-m (Fig. 2). Two major modifications have been introduced based on known
VEGFR-2 inhibitors: quinazoline ring of SKLB1002 has been replaced with its bioisoster,
thieno[2,3-d]pyrimidine. Moreover, aromatic ring linker in Sorafinib has been replaced with
1,3,4-thiadiazole moiety.
2. Chemistry
The synthetic approach toward target products 11a-m has been outlined in Scheme 1. Gewald
reaction using 4-acetylpyridine 1, ethyl cyanoacetate 2, and sulfur 3 was applied to prepare
multi-substituted thiophene 4 [24,25] which subsequently underwent cyclization by formamidine
acetate to form thieno[2,3-d]pyrimidin skeleton 5. The chlorination of carbonyl group in this
moiety with phosphoryl chloride was carried out to obtain compound 6. On the other hand, the
reaction between thiosemicarbazide 7 and carbon disulfide 8 afforded 5-amino-1,3,4-thiadiazole-
2-thiol 9 followed by condensation with substituted phenyl isocyanates to obtain compounds 10.
Finally, nucleophilic aromatic substitution between chlorine moiety compound 6 and
corresponding diaryl urea 10 was performed to synthesize desirable products 11a-m.
3. Results and Discussion
3.1. The cytotoxic activity
Synthesized thieno[2,3-d]pyrimidine-1,3,4-thiadiazole-aryl urea derivatives 11a-m were
evaluated for their anti-proliferative activities against PC3, HepG2, T47D, and HUVEC. The
results were summarized in Table 1. The IC50 values against the PC3 cell line revealed that the
target compounds showed significant cytotoxic activity at concentrations less than 13 μM. In
other words, all final products, without exception, were more potent than the positive control.
Compound 11h, bearing a fluorine substituent at the para position had the best anti-proliferative

activity against PC3. Furthermore, the other three derivatives (11e, 11g, and 11i) were effective
against PC3 and their cytotoxicity should not be ignored. Given that compounds 11e and 11g
could favorably inhibit the proliferation of PC3 cells, the placement of methyl substituent at the
para position contributes presumably to the optimal cytotoxicity against this cancer cell line.
To evaluate the anti-proliferative activity against liver cancer, the HepG2 cell line was selected.
What Table 1 does imply is that 11d, 11e and 11f were the best compounds against HepG2. In
comparison to the positive control, the final derivatives had a moderate cytotoxic effect against
HepG2. Besides, disubstituted derivatives bearing the chlorine atom at the para position (11i and
11k) were the weakest ones against HepG2.
The comparison of IC50 values against T47D cells revealed that most of the compounds had
minimal potency. Neither para nor meta-methoxy substituted derivatives (11f and 11l) had good
anti-proliferative activity against T47D and as Table 1 implies, the IC50 values of these
derivatives were more than 50 μM. HUVEC (human umbilical vein endothelial cell) cells are
exposed to VEGF and involved in the process of angiogenesis. Thus, through the evaluation of
cytotoxic effects of the corresponding compounds against HUVEC, their anti-angiogenic activity
would be predictable to some extent. The presence of four derivatives (11b, 11i, 11j, and 11m)
owning IC50 values less than 15 μM is an indication of how structures bearing the diaryl urea
functionality can affectionately inhibit the proliferation of HUVEC cells and presumably
angiogenesis. Compound 11i was the most potent one with IC50 = 7.8 μM.
Preclinical data have revealed that VEGF has a stimulating effect on the proliferation of prostate
cancer cells and angiogenesis [26]. In other words, VEGF as an angiogenic factor is involved in
the process of neovascularization observed in prostate cancer [27]. Moreover, in present study,
results revealed that PC3 cells were more sensitive to compounds 11a-m in comparison to other
three cell lines (HepG2, T47D, and HUVEC). Comparing to sorafinib, compound 11i was
efficiently inhibit PC3, T47D, and HUVEC cell proliferation; however, it did not show good
cytotoxic activity against HepG2 cells (IC50 = 61.9 μM). Additionally, the cytotoxic effect of
compound 11i against normal fibroblast Hu02 cell line (IC50 = 34.3 ± 0.3 μM) was comparable to
that of sorafenib (IC50 = 40.0 ± 0.5 μM) (Table 2).
3.2. Apoptosis-inducing activity

Apoptosis, the programmed cell death, causes the damaged cells to eliminate. Any perturbation
in this physiological process leads to various diseases including cancer. In a general sense, the
cell death pathway conducted by anticancer agents is divided into two more pathways: apoptosis
and necrosis. In an attempt to figure out which pathway, apoptosis or necrosis, is responsible for
killing the cells, a double staining flow cytometry assay using Annexin V-FITC/propidium
iodide was carried out. Dimethyl sulfoxide was used as solvent and sorafenib as positive control.
Derivatives selected for this purpose included 11h and 11i and they were evaluated at their IC50
on the PC3 cell line. PC3 cells were affected moderately by compound 11h so that the
population of apoptotic cells was 24.47%. It should be mentioned that one-third of cells (32.2%)
were affected by the necrosis. On the other hand, the treatment of PC3 cells with compound 11i
gave rise to severe apoptosis so that nearly 44% of cells were influenced by the programmed cell
death. Dimethyl sulfoxide and sorafenib resulted in 10.88% and 18.52% cell apoptosis. As
shown in Fig. 3, compound 11i provoked cell death in an apoptotic pathway.
Given the role of endothelial cells in vascular homeostasis and cancer pathogenesis, the HUVEC
cell line was selected to assess the apoptosis-inducing activity of 11i. As illustrated in Fig. 4, the
apoptotic elimination of HUVEC cells was occurred moderately by 11i (28.45%) which was
more than apoptotic cells provoked by sorafenib (19.09%). The percentage of HUVEC cells
apprehended in the necrosis phase treated with 11i was comparatively lower than that of
sorafinib (21.0% in comparison with 16.4%).
3.3. Cell cycle analysis
In accordance with the IC50 values obtained from Table 1, compounds 11h and 11i were selected
to be further examined in respect of their effect on the cell cycle progression (Figs.5 and 6).
Interference with the normal cell cycle distribution of the PC3 cell line caused by 11h was noted.
A glimpse of the chart as demonstrated in Fig. 6, reveals that compound 11h could increase the
percentage of cells in the G1-phase by 1.26-fold, respectively compared to the control (55.92%
vs 44.11%). Of course, in comparison with the control (44.12%), the percentage of cells in the S-
phase was lower (29.76%). Simultaneously, an increase in the number of cancerous cells in the
G1-phase and a decrease in the S-phase is usually indicative of the cell cycle arrest in the G1-
phase. Thus, a quantity of PC3 cells treated with 11h were imprisoned in the G1-phase and
couldn’t enter the next one. The cell cycle distribution of PC3 cells exposed to sorafenib showed

similar results so that, it could tremendously increase the number of G1-cells (66.19%) and
decrease S-cells (22.32%) as compared with the control (G1= 44.11%, S= 44.12%). However, no
meaningful difference was observed between the control (G1= 44.11%, S= 44.12%, G2=
15.97%) and 11i (G1= 45.23%, S= 40.58%, G2= 12.85%) categories.
In terms of the cell cycle analysis, we often pose the question, whether the cells imprisoned in
the subG1-phase are apoptotic or not?
An elevated subG1 cell count has a low predictive value for apoptosis and there is no direct link
between the percentage of cells halted in the subG1-phase and the number of apoptotic cells.
Analyzing the inducing-apoptosis activity (Fig. 3) and the cell cycle assay (Figs. 5 and 6)
indicated the interesting outcomes. As Fig. 7 implies, compound 11i induced the more significant
apoptosis (44%) as compared with 11h (24.5%). However, the percentage of PC3 cells arrested
in the subG1-phase by 11i (4.38%) was fewer than 11h (9.73%). Also, in conformity with
findings obtained from Fig. 3, it is understandable that in comparison with 11i (12.6%), the
induction of the necrosis phase by 11h (32%) was more significant. Thus, there is no direct
relation necessarily on every occasion between the cell population arrested in the subG1-phase
and the amount of induced apoptosis.
The corresponding analysis was performed to evaluate the extent of DNA damage and the
percentage of HUVEC cells in various phases (subG1, G1, S, and G2/M) of the cell cycle.
Interestingly, the results of the HUVEC cell cycle analysis were the same one observed in the
PC3 cell line. As shown in Fig. 8, sorafenib increased tremendously the number of G1-cells
(58.69%) and decreased S-cells (27.1%) as compared with the control (G1= 40.93%, S=
41.49%). However, no meaningful difference was observed between the control (G1= 40.93%,
S= 41.49%, G2= 6.25%) and 11i (G1= 48.3%, S= 49.8%, G2= 5.53%) categories. Although the
apoptosis was induced by 11i in PC3 and HUVEC cell lines, the subG1-peak was not observable
in the corresponding diagrams of the cell cycle analysis (Figs. 5c and 8b). On the other hand, as a
result of the treatment of PC3 and HUVEC cells with 11i, the cell cycle distribution was not
altered as compared with the control. In this manner, the apoptosis is induced probably in all cell
subpopulations (G1, S, and G2/M) equally.
3.4. Anti-vascular effects

With regard to the anti-proliferative activity of target compounds against the HUVEC cell line
and the analysis of IC50 values, superior derivatives (11b, 11h, 11i, 11j, and 11m) were selected
to survey the vascular disrupting effect. The chick chorioallantoic membrane (CAM) assay was
utilized to assess two aspects of the capillary blood vessels; the number and the length of vessels.
As illustrated in Fig. 9, compound 11i could tremendously reduce the number of capillary blood
vessels (65%), which was comparable to sorafenib as the positive control (64%). The inhibitory
activity of 11m was partially satisfactory (35%), even though a substantial reduction was not
observed when CAM was exposed to 11b (29%), 11h (27%), and 11j (21%) (Fig. 10a).
The effects of corresponding compounds on the length of the capillary blood vessels were nearly
identical to those illustrated in Fig. 10a. As Fig. 10b does imply, 11i could reduce the length of
the vessels to a degree (46%) not seen with other evaluated derivatives. In this context, the effect
of sorafenib was almost similar to 11i.
3.5. Effect on VEGFR-2 phosphorylation
Disruption in the VEGF signaling pathway is a key prerequisite for the inhibition of
angiogenesis. On the other hand, as demonstrated through the CAM assay, it was confirmed that
11i is capable of inhibiting angiogenesis. Thus, the Western blot analysis was implemented to
identify the mechanism of action by which 11i prevents the growth of the capillary blood vessels.
As Fig. 11 does imply, in a time-dependent manner, 11i could reduce massively the levels of
phosphorylated form of VEGFR-2. The nearly same effect was achieved with sorafenib.
However, after 48 h, the reduced level of p-VEGFR-2 by 11i was a little more than sorafenib.
The Western blotting was performed also in a concentration-dependent manner. The PC3 cells
were exposed to three different concentrations of 11i (2.4 μM, 3.6 μM, and 5.4 μM) and
incubated for 48 h. Cell lysates were blotted against VEGFR-2 and p-VEGFR-2. As reflected in
Fig. 12, the phosphorylation of VEGFR-2 was disturbed in a concentration-dependent manner so
that a perceptible reduction of band size has occurred in the term of 5.4 μM concentration.
These outcomes give an explanation to the sensational anti-angiogenic activity of 11i and show a
logical connection between the inhibition of angiogenesis and disruption of the VEGF signaling.
Ideally, the normalization control (ꢀ-actin) was present at constant levels in every sample.
3.6. Molecular docking study

Docking runs were carried out by AutoDock 4.2 with Lamarckian genetic algorithm (LGA) and
each Lamarckian job compromised of 50 runs. The co-crystal structure of VEGFR-2 and
sorafenib (3WZE) with 1.90 Å resolution was selected and compound 11i was docked into the
active site of the receptor.
The scientific results reported by Okamoto et al. provide an admirable overview of the binding
mode of sorafenib and lenvatinib. Across the sorafenib and lenvatinib molecules, two hydrogen
bonding interactions can be observed between the urea carbonyl and Asp1046 and the urea NH
and Glu885 [28]. Molecular dynamics (MD) simulation suggests that the hydrogen bond
engaging Asp1046 is sufficiently stable and is almost preserved throughout the MD simulation
time. However, the bifurcated hydrogen bonds involving Glu885 occur occasionally [29]. As
illustrated in Fig. 13, the same interactions are observable between 11i and the receptor. The
MM2 methodology was used to determine the free energy of binding. The calculated free energy
of binding for 11i and sorafinib were -8.49 and -8.57 kcal/mol, respectively. It confirmed
compound 11i would form greater interactions with active site of the receptor.
4. Conclusion
In conclusion, a novel series of thienopyrimidine-based agents possessing 1,3,4-thiadiazole-aryl
urea derivatives 11a-m were designed, synthesized, and evaluated as potential inhibitors of cell
proliferation and angiogenesis. The MTT assay demonstrated that all synthesized compounds
had more potent anti-proliferative activity than sorafenib against the PC3 cell line. Compound
11i (IC50= 3.6 μM) was efficiently induced apoptosis (44% in PC3 cells and 28.45% in HUVEC
cells) comparing to standard drug sorafenib. Moreover, compound 11i showed the noticeable
angiogenesis and VEGFR-2 inhibitory activity. Regarding our results, compound 11i can be
considered as a promising candidate for the treatment of metastatic prostate cancer. However, the
tumor xenograft model can be helpful to obtain more accurate information.
5. Experimental section
5.1. General chemistry
All starting materials, reagents and solvents were purchased from Merck and Aldrich companies
without any purification. The reaction progress and the purity of synthesized compounds were
monitored by thin-layer chromatography (TLC) on silica gel 250-micron F254 plastic sheets.
Flash chromatography was performed for more purification of chlorine intermediate (6) by using

230-400 mesh silica gel and the indicated solvent system. The melting points were determined
by Electrothermal IA9100 apparatus and are uncorrected. IR spectra were obtained on Perkin-
Elmer Spectrum Version 10.03.06 (potassium bromide disks). Nuclear magnetic resonance
(NMR) spectra were performed on Varian 500, Bruker 400, and Bruker 300 (TMS as IS).
Chemical shifts were reported in parts per million (ppm), downfield from tetramethylsilane.
Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), and broad (b). The mass spectra were run on a Finigan TSQ-70 spectrometer
(Finigan, USA) at 70 eV. The elemental analysis for C, H, and N was carried out with an
elemental analyzer GmbH VarioEL.
5.1.1. Ethyl 2-amino-4-(pyridin-4-yl)thiophene-3-carboxylate (4)
A mixture of 4-acetylpyridine (1) (14.5 ml, 130.2 mmol), ethyl cyanoacetate (2) (15.3 ml, 143.2
o
mmol) and morpholine (2.25 ml, 26 mmol) was stirred in EtOH at 60-70 C for 30 min. Then,
sulfur (3) (5 g, 156.25 mmol) was added portion by portion over 5 min. The resulting mixture
was refluxed under an argon atmosphere overnight. After the consumption of starting materials,
cool water was added and the precipitated solid was filtered, washed with n-hexane and allowed
o
to dry to acquire compound 4, 22.6 g in 70% yield. Yellow solid; mp: 200 C; IR (KBr, cm-1):
1
3396, 3039, 1660, 1607, 1499,1474, 1363; H NMR (500 MHz, DMSO‑d6) ꢁ: 8.49 (d, 2H, H-
2,6 pyridine, J = 4.5 Hz), 7.48 (bs, 2H, NH2), 7.25 (d, 2H, H-3,5 pyridine, J = 4.5 Hz), 6.37 (s,
1H, H-5 thiophene), 3.98 (q, 2H, CH2, J = 7.1 Hz), 0.92 (t, 3H, CH3, J = 7.1 Hz); 13C NMR (125
MHz, DMSO-d6) ꢁ: 165.89, 164.73, 150.28, 149.01 (2C), 146.10, 138.33, 124.07 (2C), 118.49,
107.60, 102.75, 59.33, 14.11; Anal. Calcd. For C12H12N2O2S: C, 58.05; H, 4.87; N, 11.28.
Found: C, 58.22; H, 5.05; N, 11.41.
5.1.2. 5-(Pyridin-4-yl)thieno[2,3-d]pyrimidin-4(3H)-one (5)
o
As compound 4 (8.26 g, 33.3 mmol) was dissolved in DMF (90 ml) at 100 C, formamidine
acetate (24.26 g, 233.1 mmol) was added over 60 min and the mixture was stirred for 16 h. A
yellow solid was precipitated, when cold water was added to the reaction mixture slowly. The
precipitated solid was separated by filtration, washed with ether and allowed to be dry to give
o
desired compound 5, 5.66 g in 74% yield. Off-white solid; mp: 304-306 C; IR (KBr, cm-1):
1
3048, 1692, 1451, 1442, 1420,1359; H NMR (400 MHz, DMSO‑d6) ꢁ: 12.62 (bs, 1H, NH),
8.58 (d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.19 (s, 1H, H-2 thienopyrimidine), 7.77 (s, 1H, H-6

thienopyrimidine), 7.56 (d, 2H, H-3,5 pyridine, J = 4.5 Hz); 13C NMR (125 MHz, DMSO-d6) ꢁ:
166.43, 157.73, 149.37 (2C), 146.60, 142.86, 136.19, 124.45 (2C), 123.61, 121.25; Anal. Calcd.
For C11H7N3OS: C, 57.63; H, 3.08; N, 18.33. Found: C, 57.51; H, 3.22; N, 18.50.
5.1.3. 4-Chloro-5-(pyridin-4-yl)thieno[2,3-d]pyrimidine (6)
Under the ice-bath condition, POCl3 (8 ml) was added dropwise to a solution of
compound 5 (920 mg, 4 mmol) and DIPEA (3 ml). After completion of the addition, the reaction
mixture was warmed to room temperature and stirred for 30 min. Subsequently, the mixture was
o
heated to 45 C for 2 h. When the starting material was consumed, the reaction was cooled to
room temperature. It was neutralized with saturated sodium bicarbonate solution and taken up in
ethyl acetate and water. The organic layer was separated, dried over anhydrous Na2SO4,
concentrated under vacuo and chromatographed on a flash silica gel column (EtOAc/petroleum
ether = 1:1) to obtain the desired product 6, 420 mg in 42% yield. Pale yellow solid; mp: 173-
175 oC; IR (KBr, cm-1): 3031, 1602, 1545, 1494; 1H NMR (400 MHz, DMSO‑d6) ꢁ: 9.01 (s, 1H,
H-2 thienopyrimidine), 8.68 (d, 2H, H-2,6 pyridine, J = 4.4 Hz), 8.19 (s, 1H, H-6
thienopyrimidine), 7.56 (d, 2H, H-3,5 pyridine, J = 4.4 Hz); MS (m/z, %): 249 (M+ +2, 33), 247
(M+, 100), 212 (56); Anal. Calcd. For C11H6ClN3S: C, 53.34; H, 2.44; N, 16.96. Found: C,
53.48; H, 2.58; N, 16.84.
5.1.4. 5-Amino-1,3,4-thiadiazole-2-thiol (9)
Thiosemicarbazide (7) (5 g, 54.94 mmol) and Na2CO3 (5.76 g, 54.94 mmol) were dissolved in
dry ethanol and the mixture was heated to 60 oC for 30 min. Carbon disulfide (8) (8.35 g, 109.88
mmol) in dry ethanol was added dropwise and the resultant mixture was refluxed overnight.
When the completion of the reaction was detected by TLC, the solvent was evaporated under
reduced pressure. The residue was diluted through the addition of 100 ml water and then
concentrated HCl solution was added carefully to obtain yellow precipitate known as 5-amino-
o
1,3,4-thiadiazole-2-thiol or compound 9, 6.2 g in 84% yield. Yellow solid; mp: 233-235 C; IR
1
(KBr, cm-1): 3401, 3273, 1595, 1533, 1496; H NMR (300 MHz, DMSO‑d6) ꢁ: 13.11 (s, 1H,
NH), 7.05 (s, 2H, NH2); 13C NMR (75 MHz, DMSO-d6) ꢁ: 180.93 (C-2, Thiadiazole), 161.48
(C-5, Thiadiazole); MS (m/z, %): 133 (M+, 100), 74 (37.5), 57 (50); Anal. Calcd. For C2H3N3S2:
C, 18.04; H, 2.27; N, 31.55. Found: C, 18.16; H, 2.36; N, 31.50.

5.1.5. General procedure for synthesis of intermediates 10a-m
Compound 9 (200 mg, 1.5 mmol) was dissolved in dry DCM and stirred at room temperature for
30 min. Then, appropriate phenyl isocyanate derivative (1.5 mmol) in dry DCM was added and
the resulting yellowish mixture was allowed to stir under an argon atmosphere at room
temperature overnight. The final white precipitate was kept in a vacuum oven for 2 h to give
corresponding urea (10a-m). The product was used for the next step without any further
purification.
5.1.6. General procedure for synthesis of final compounds 11a-m
A mixture of appropriate urea derivative (10a-m) (0.60 mmol) and KOH (33.6 mg, 0.60 mmol)
in EtOH was stirred at 60oC for 45 min, and then compound 6 (150 mg, 0.60 mmol) was added
portion by portion over 5 min. The resulting mixture was refluxed overnight. The precipitated
solid was filtered, washed with water and dried in a vacuum oven for 24 h to obtain the target
compound (11a-m).
5.1.6.1.
1-Phenyl-3-(5-((5-(pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)thio)-1,3,4-thiadiazol-2-
yl)urea (11a)
Cream solid; yield: 63%; mp: 237-239 oC; IR (KBr, cm-1): 3037, 1711, 1620, 1598, 1543, 1499;
1H NMR (500 MHz, DMSO‑d6) ꢁ: 9.12 (bs, 1H, NH urea), 8.94 (s, 1H, H-2 thienopyrimidine),
8.75 (d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.10 (s, 1H, H-6 thienopyrimidine), 7.64 (d, 2H, H-3,5
pyridine, J = 4.5 Hz), 7.48 (d, 2H, H-2,6 phenyl, J = 7.95 Hz), 7.33 (t, 2H, H-3,5 phenyl, J =
7.95 Hz), 7.07 (t, 1H, H-4 phenyl, J = 8.05 Hz); 13C NMR (125 MHz, DMSO-d6) ꢁ: 167.67,
160.19, 152.39, 150.02 (2C), 142.94, 138.58, 132.43, 129.37 (2C), 128.72, 125.72, 125.46,
123.72, 119.50 (2C); Anal. Calcd. For C20H13N7OS3: C, 51.82; H, 2.83; N, 21.15. Found: C,
51.13; H, 2.73; N, 20.97.
5.1.6.2.
1-(5-((5-(Pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)thio)-1,3,4-thiadiazol-2-yl)-3-(m-
tolyl)urea (11b)
Cream solid; yield: 68%; mp: 231-236 oC; IR (KBr, cm-1): 3381, 2917, 1712, 1634, 1600, 1495;
1H NMR (500 MHz, DMSO‑d6) ꢁ: 11.20 (bs, 1H, NH urea), 9.03 (bs, 1H, NH urea), 8.94 (s, 1H,
H-2 thienopyrimidine), 8.74 (d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.09 (s, 1H, H-6
thienopyrimidine), 7.64 (d, 2H, H-3,5 pyridine, J = 4.5 Hz), 7.31 (s, 1H, H-2 m-tolyl), 7.25 (dd,

1H, H-6 m-tolyl, J = 8.2, 1.15 Hz), 7.19 (t, 1H, H-5 m-tolyl, J = 7.55 Hz), 6.87 (d, 1H, H-4 m-
tolyl, J = 7.35 Hz), 2.28 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) ꢁ: 167.11, 163.96, 159.58,
151.85, 151.37, 149.50 (2C), 148.75, 142.41, 138.18, 137.99, 131.87, 128.70, 128.23, 125.12,
124.95 (2C), 123.89, 119.32, 116.02, 21.07; Anal. Calcd. For C21H15N7OS3: C, 52.81; H, 3.17;
N, 20.53. Found: C, 52.64; H, 3.42; N, 20.31.
5.1.6.3.
1-(3-Chlorophenyl)-3-(5-((5-(pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)thio)-1,3,4-
thiadiazol-2-yl)urea (11c)
o
1
Cream solid; yield: 64%; mp: 236-239 C; IR (KBr, cm-1): 3375, 1731, 1596, 1498, 1454; H
NMR (500 MHz, DMSO‑d6) ꢁ: 9.37 (bs, 1H, NH urea), 8.96 (s, 1H, H-2 thienopyrimidine), 8.75
(d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.10 (s, 1H, H-6 thienopyrimidine), 7.70 (s, 1H), 7.65 (d,
2H, H-3,5 pyridine, J = 4.5 Hz), 7.35 (d, 2H), 7.12 (d, 1H); 13C NMR (125 MHz, DMSO-d6) ꢁ:
167.65, 164.47, 159.97, 152.35, 150.00 (2C), 149.47, 142.94, 140.24, 133.73, 132.39, 130.97,
128.76, 125.67, 125.45 (2C), 123.32, 118.86, 117.95; Anal. Calcd. For C20H12ClN7OS3: C,
48.24; H, 2.43; N, 19.69. Found: C, 48.42; H, 2.63; N, 19.52.
5.1.6.4.
1-(5-((5-(Pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)thio)-1,3,4-thiadiazol-2-yl)-3-(3-
(trifluoromethyl)phenyl)urea (11d)
o
Cream solid; yield: 67%; mp: 238-240 C; IR (KBr, cm-1): 3380, 1728, 1599, 1497, 1240, 1205,
1165, 1108, 1071; 1H NMR (500 MHz, DMSO‑d6) ꢁ: 11.54 (bs, 1H, NH urea), 9.58 (bs, 1H, NH
urea), 8.95 (s, 1H, H-2 thienopyrimidine), 8.75 (d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.10 (s, 1H,
H-6 thienopyrimidine), 7.99 (s, 1H, H-2 (3-trifluoromethyl)phenyl), 7.69 (d, 1H, H-6 (3-
trifluoromethyl)phenyl, J = 7.25 Hz), 7.64 (d, 2H, H-3,5 pyridine, J = 4.5 Hz), 7.55 (t, 1H, H-5
(3-trifluoromethyl)phenyl, J = 7.95 Hz), 7.39 (d, 1H, H-4 (3-trifluoromethyl)phenyl, J = 7.25
Hz); 13C NMR (125 MHz, DMSO-d6) ꢁ: 167.67, 152.42, 150.03 (2C), 142.93, 132.42, 130.54,
128.76 (q, JC-F = 2.31 Hz), 125.46 (2C), 123.16, 121.69 (q, JC-F = 7.4 Hz), 120.14 (q, JC-F = 16.15
Hz), 115.41 (q, JC-F = 4.81 Hz); MS (m/z, %): 531 (M+, 0.6), 187 (50), 161 (100); Anal. Calcd.
For C21H12F3N7OS3: C, 47.45; H, 2.28; N, 18.45. Found: C, 47.60; H, 2.10; N, 18.73.
5.1.6.5. 1-(3-Chloro-4-methylphenyl)-3-(5-((5-(pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)thio)-
1,3,4-thiadiazol-2-yl)urea (11e)

Yellow solid; yield: 68%; mp: 231-133 oC; IR (KBr, cm-1): 3257, 3183, 3041, 2976, 2920, 1606,
1
1498; H NMR (500 MHz, DMSO‑d6) ꢁ: 9.28 (bs, 1H, NH urea), 8.95 (s, 1H, H-2
thienopyrimidine), 8.75 (d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.10 (s, 1H, H-6 thienopyrimidine),
7.68 (s, 1H, H-2 (3-chloro-4-methyl)phenyl), 7.63 (d, 2H, H-3,5 pyridine, J = 4.5 Hz), 7.27 (s,
2H, H-5,6 (3-chloro-4-methyl)phenyl), 2.25 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) ꢁ:
167.67, 160.06, 152.38, 150.02 (2C), 142.94, 139.16, 137.86, 133.65, 132.42, 131.74, 131.57,
130.29, 128.75, 125.70, 125.46 (2C), 119.42, 118.24, 19.29; Anal. Calcd. For C21H14ClN7OS3:
C, 49.26; H, 2.76; N, 19.15. Found: C, 49.10; H, 2.64; N, 18.97.
5.1.6.6.
1-(4-Methoxyphenyl)-3-(5-((5-(pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)thio)-1,3,4-
thiadiazol-2-yl)urea (11f)
o
White solid; yield: 65%; mp: 235-238 C; IR (KBr, cm-1): 3381, 3322, 2918, 1713, 1601, 1511;
1H NMR (400 MHz, DMSO‑d6) ꢁ: 11.24 (bs, 1H, NH urea), 9.00 (bs, 1H, NH urea), 8.97 (s, 1H,
H-2 thienopyrimidine), 8.78 (d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.13 (s, 1H, H-6
thienopyrimidine), 7.67 (d, 2H, H-3,5 pyridine, J = 4.5 Hz), 7.40 (d, 2H, H-2,6 methoxyphenyl, J
= 8.8 Hz), 6.93 (d, 2H, H-3,5 methoxyphenyl, J = 9.2 Hz), 3.74 (s, 3H, OCH3); Anal. Calcd. For
C21H15N7O2S3: C, 51.10; H, 3.06; N, 19.86. Found: C, 50.93; H, 2.81; N, 19.68.
5.1.6.7.
1-(5-((5-(Pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)thio)-1,3,4-thiadiazol-2-yl)-3-(p-
tolyl)urea (11g)
o
1
Cream solid; yield: 67%; mp: 234-237 C; IR (KBr, cm-1): 3396, 1707, 1597, 1420, 1315; H
NMR (500 MHz, DMSO‑d6) ꢁ: 11.20 (bs, 1H, NH urea), 9.04 (bs, 1H, NH urea), 8.93 (s, 1H, H-
2 thienopyrimidine), 8.75 (d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.09 (s, 1H, H-6
thienopyrimidine), 7.63 (d, 2H, H-3,5 pyridine, J = 4.5 Hz), 7.35 (d, 2H, H-2,6 p-tolyl, J = 8.2
Hz), 7.12 (d, 2H, H-3,5 p-tolyl, J = 8.2 Hz), 2.24 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-
d6) ꢁ: 167.67, 160.22, 152.39, 150.02 (2C), 142.95, 136.02, 132.74, 132.43, 129.76 (2C), 129.56,
128.71, 125.72, 125.45 (2C), 119.55 (2C), 20.81; Anal. Calcd. For C21H15N7OS3: C, 52.81; H,
3.17; N, 20.53. Found: C, 52.99; H, 3.42; N, 20.43.
5.1.6.8.
1-(4-Fluorophenyl)-3-(5-((5-(pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)thio)-1,3,4-
thiadiazol-2-yl)urea (11h)

o
1
White solid; yield: 60%; mp: 235-237 C; IR (KBr, cm-1): 3393, 1717, 1599, 1509, 1459; H
NMR (500 MHz, DMSO‑d6) ꢁ: 11.25 (bs, 1H, NH urea), 9.19 (bs, 1H, NH urea), 8.93 (s, 1H, H-
2 thienopyrimidine), 8.75 (d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.09 (s, 1H, H-6
thienopyrimidine), 7.63 (d, 2H, H-3,5 pyridine, J = 4.5 Hz), 7.48-7.50 (m, 2H, H-2,6
fluorophenyl), 7.15 (t, 2H, H-3,5 fluorophenyl, J = 8.7 Hz); 13C NMR (75 MHz, DMSO-d6) ꢁ:
167.14, 164.42, 159.52, 152.33, 151.89, 149.51 (2C), 148.80, 142.43, 139.29, 131.89, 130.02,
129.77, 129.35, 128.26, 125.82, 125.15, 124.95 (2C), 122.59, 119.30 (d, 2C, JC-F = 7.86 Hz ),
114.87 (d, 2C, JC-F = 14.41 Hz); MS (m/z, %): 481 (M+, 3), 137 (67), 111 (100), 83 (42), 57 (30);
Anal. Calcd. For C20H12FN7OS3: C, 49.89; H, 2.51; N, 20.36. Found: C, 50.10; H, 2.70; N,
20.18.
5.1.6.9. 1-(3,4-Dichlorophenyl)-3-(5-((5-(pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)thio)-1,3,4-
thiadiazol-2-yl)urea (11i)
Cream solid; yield: 64%; mp: 233-238 oC; IR (KBr, cm-1): 3361, 1719, 1599, 1585, 1531, 1494;
1H NMR (500 MHz, DMSO‑d6) ꢁ: 11.55 (bs, 1H, NH urea), 9.44 (bs, 1H, NH urea), 8.95 (s, 1H,
H-2 thienopyrimidine), 8.74 (d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.10 (s, 1H, H-6
thienopyrimidine), 7.87 (d, 1H, H-2 dichlorophenyl, J = 2 Hz), 7.63 (d, 2H, H-3,5 pyridine, J =
4.5 Hz), 7.55 (d, 1H, H-5 dichlorophenyl, J = 8.65 Hz), 7.43 (dd, 1H, H-6 dichlorophenyl, J =
8.85, 1.85 Hz); 13C NMR (75 MHz, DMSO-d6) ꢁ: 167.13, 164.03, 159.36, 152.04, 151.82,
149.47 (2C), 148.97, 142.43, 138.49, 131.84, 131.08, 130.61, 128.29, 125.12, 124.94 (2C),
124.55, 120.11, 119.08; MS (m/z, %): 531 (M+, 0.2), 233 (17), 191 (4), 189 (24), 187 (40), 165
(10), 163 (60), 161 (100); Anal. Calcd. For C20H11Cl2N7OS3: C, 45.12; H, 2.08; N, 18.42. Found:
C, 44.84; H, 2.87; N, 18.26.
5.1.6.10.
1-(4-Chlorophenyl)-3-(5-((5-(pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)thio)-1,3,4-
thiadiazol-2-yl)urea (11j)
o
1
White solid; yield: 67%; mp: 236-238 C; IR (KBr, cm-1): 3389, 1720, 1597, 1493, 1076; H
NMR (500 MHz, DMSO‑d6) ꢁ: 11.39 (bs, 1H, NH urea), 9.36 (bs, 1H, NH urea), 8.94 (s, 1H, H-
2 thienopyrimidine), 8.75 (d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.09 (s, 1H, H-6
thienopyrimidine), 7.63 (d, 2H, H-3,5 pyridine, J = 4.5 Hz), 7.51 (d, 2H, H-2,6 chlorophenyl, J =
7.6 Hz), 7.37 (d, 2H, H-3,5 chlorophenyl, J = 7.8 Hz); 13C NMR (125 MHz, DMSO-d6) ꢁ:

167.67, 164.50, 160.11, 152.37, 150.01 (2C), 142.93, 137.70, 132.42, 129.21 (2C), 129.03,
128.72, 127.32, 125.70, 125.44 (2C), 121.03 (2C), 120.27; Anal. Calcd. For C20H12ClN7OS3: C,
48.24; H, 2.43; N, 19.69. Found: C, 48.07; H, 2.66; N, 19.42.
5.1.6.11. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(5-((5-(pyridin-4-yl)thieno[2,3-d]pyrimidin-
4-yl)thio)-1,3,4-thiadiazol-2-yl)urea (11k)
o
White solid; yield: 67%; mp: 239-240 C; IR (KBr, cm-1): 3389, 1720, 1597, 1546, 1493, 1458,
1
1340, 1310, 1093, 1076; H NMR (300 MHz, DMSO‑d6) ꢁ: 11.89 (bs, 1H, NH urea), 9.99 (s,
1H, NH urea), 8.92 (s, 1H, H-2 thienopyrimidine), 8.74 (d, 2H, H-2,6 pyridine, J = 4.2 Hz), 8.07
(s, 2H), 7.75 (d, 1H, J = 10.1 Hz), 7.62 (d, 3H); 13C NMR (75 MHz, DMSO-d6) ꢁ: 167.14,
164.40, 159.45, 152.50, 151.87, 149.51 (2C), 148.86, 142.42, 138.11, 132.08, 131.87, 128.29,
125.51 (q, JC-F = 158.85 Hz), 125.13, 124.95 (2C), 123.72, 123.63, 117.50 (q, JC-F = 5.49 Hz);
MS (m/z, %): 565 (M+, 0.5), 456 (18), 244 (24), 223 (23), 221 (69), 197 (30), 195 (100); Anal.
Calcd. For C21H11ClF3N7OS3: C, 44.56; H, 1.96; N, 17.32. Found: C, 44.74; H, 2.13; N, 17.51.
5.1.6.12.
1-(3-Methoxyphenyl)-3-(5-((5-(pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)thio)-1,3,4-
thiadiazol-2-yl)urea (11l)
o
White solid; yield: 62%; mp: 238-240 C; IR (KBr, cm-1): 3373, 1725, 1597, 1545, 1494, 1300,
1237, 1216, 1163; 1H NMR (500 MHz, DMSO‑d6) ꢁ: 11.20 (bs, 1H, NH urea), 9.14 (bs, 1H, NH
urea), 8.94 (s, 1H, H-2 thienopyrimidine), 8.75 (d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.09 (s, 1H,
H-6 thienopyrimidine), 7.64 (d, 2H, H-3,5 pyridine, J = 4.5 Hz), 7.22 (t, 1H, H-5
methoxyphenyl, J = 8.2 Hz), 7.14 (s, 1H, H-2 methoxyphenyl), 6.99 (dd, 1H, H-6
methoxyphenyl, J = 7.2, 1.25 Hz), 6.63 (dd, 1H, H-4 methoxyphenyl, J = 8.2, 2.1 Hz), 3.73 (s,
3H, OCH3); 13C NMR (125 MHz, DMSO-d6) ꢁ: 167.68, 160.21, 160.16, 152.39, 150.01 (2C),
142.94, 139.78, 132.43, 130.19, 128.71, 125.71, 125.45 (2C), 111.75, 109.17, 105.33, 55.55;
Anal. Calcd. For C21H15N7O2S3: C, 51.10; H, 3.06; N, 19.86. Found: C, 51.27; H, 3.24; N, 19.69.
5.1.6.13.
1-(3-Fluorophenyl)-3-(5-((5-(pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl)thio)-1,3,4-
thiadiazol-2-yl)urea (11m)
o
1
Cream solid; yield: 65%; mp: 234-236 C; IR (KBr, cm-1): 3390, 1716, 1599, 1547; H NMR
(300 MHz, DMSO‑d6) ꢁ: 11.42 (bs, 1H, NH urea), 9.38 (s, 1H, NH urea), 8.90 (s, 1H, H-2

thienopyrimidine), 8.73 (d, 2H, H-2,6 pyridine, J = 4.5 Hz), 8.05 (s, 1H, H-6 thienopyrimidine),
7.60 (d, 2H, H-3,5 pyridine, J = 4.5 Hz), 7.45 (d, 1H, H-6 fluorophenyl, J = 10.95 Hz), 7.28-7.33
(m, 1H, H-5 fluorophenyl), 7.20 (d, 1H, H-2 fluorophenyl, J = 7.11 Hz), 6.84 (bs, 1H, H-4
fluorophenyl); 13C NMR (75 MHz, DMSO-d6) ꢁ: 167.13, 163.93, 161.7 (d, JC-F = 240.74 Hz),
159.47, 152.04, 151.83, 151.74, 149.50 (2C), 148.99, 142.45, 140.01 (d, JC-F = 10.85 Hz),
131.87, 130.46 (d, JC-F = 9.24 Hz), 128.78, 128.28, 124.97 (2C), 114.70, 109.49 (d, JC-F = 21.06
Hz), 105.69 (d, JC-F = 26.24 Hz); Anal. Calcd. For C20H12FN7OS3: C, 49.89; H, 2.51; N, 20.36.
Found: C, 50.01; H, 2.74; N, 20.18.
5.2. MTT assay
Cytotoxicity of synthesized target compounds (11a-m) was examined with MTT assay. For this
purpose, concerning similar studies, four appropriate cancer cell lines (PC3, HepG2, T47D, and
HUVEC) were selected provided by the National Cell Bank of Iran (Pastor Institute, Tehran,
Iran). Contents of each well in a 96-well microtiter plate included 200 μl of complete medium
o
containing 8000 cells, incubated at 37 C in a humidified 5% CO2 incubator overnight. Cells
were exposed to various concentrations of test compounds prepared earlier (in DMSO) and
further incubated at 37 oC for 48 h. After incubation, the solution containing 5% MTT was added
to all wells and incubated for another 4 h. The absorbance was measured with a microplate
reader (Bio-Rad microplate reader, Model 680) at a test wavelength of 570 nm. The absorbance
measured is directly proportional to the percentage of live cells. A simple calculation known as
half-maximal inhibitory concentration (IC50) was used to gauge whether target compounds are
effective or not. As a consequence, particular curves indicating cell survival percentage versus
different concentrations of the compounds were drawn and analyzed. Herein, sorafenib and 0.1%
DMSO were regarded as positive and negative controls, respectively.
5.3. Analysis of cellular apoptosis
Annexin V-FITC/propidium iodide dual staining assay was implemented to analyze the
induction of apoptosis by 11h and 11i (tested at IC50). PC3 cells were seeded into 6-well plates
o
and incubated overnight at 37 C under 5% CO2. Cells were exposed to the IC50 of target
compounds and then incubated for 48 h. Treated cells were trypsinized, washed with PBS twice,
and centrifuged at 1200 rpm. After that, 500 ml of binding buffer and 5 ml of Annexin V-FITC
and PI were added to suspended cells. Finally, to examine the cell apoptosis with the assistance

of flow cytometry (FACS Calibur Bectone-Dickinson), suspended cells were incubated at room
temperature in darkness for 5-15 min. The potential proapoptotic activity of 11i was surveyed in
a similar way against the HUVEC cell line. In these experiments, sorafenib was utilized as the
positive control [30].
5.4. Cell cycle analysis
Through flow cytometry analysis, propidium iodide (PI) staining assay was implemented as a
challenge to explore the cell cycle distribution in PC3 cells. PI attaches to the DNA molecule and
emits a fluorescent, the intensity of which is directly proportional to the DNA content. The cells
were exposed to IC50 of test compounds (11h and 11i), after 48 h, the treated cells were
trypsinized, washed with PBS, and centrifuged at 1200 rpm for 5 min. As soon as the incubation
o
with PBS was carried out, prepared PC3 cells were fixed with 70% cold ethanol (-20 C). The
fixed cells were washed with PBS. After rinsing, in an orderly fashion, they were treated with
RNase A (0.1 mg/ml), incubated for half an hour, and exposed to 50 mg/ml of PI, continuously
incubated for 15 min more. To calculate the cell cycle distribution, the familiar equipment,
Novocyte flow cytometer (ACEA Biosciences) was employed, and the data were analyzed by
NovoExpress 1.1.0 software. All the experiments were performed on three samples in parallel.
The cell cycle analysis of HUVEC was performed similarly.
5.5. Chick chorioallantoic membrane (CAM) assay
Since the allantois of the chick embryo appears at about 3.5 days of incubation, the
corresponding eggs were incubated (37.5 oC, relative humidity: 55-65%). To decrease the risk of
infection, the particular regions of the egg, e.g., the front side of the embryo were cleaned and
sterilized with 70% EtOH. On the 6th incubation day, a false air sac was created directly over the
CAM, permitting its detachment from the shell membrane. The procedure was continued through
a square incision over the CAM. The prepared window in a square form (1×1 cm), was covered
by a flexible film and then transported to the incubator. On the 8th incubation day, the particular
paper discs (Whatman) were coated with target agents and placed in the central area of the
corresponding window. The surface of CAM was visually evaluated on the 12th incubation day
using the stereomicroscope. We decided to use the number and the length of capillary blood
vessels as the index of anti-angiogenic activity. Consequently, the prepared images were
analyzed by the advantageous software, Image J [31].

5.6. Western blot analysis
When exposed to IC50 of corresponding compounds, 11i and sorafenib, PC3 cells were incubated
for 24, 48, and 72 h. PC3 cells were thawed in a 100 ml lysis buffer and immediately lysed.
o
Then, they were centrifuged at 1200 rpm at 4 C for 10 min. The obtained total proteins were
loaded and electrophoresed on 12% SDS-PAGE and transferred to a nitrocellulose membrane
(0.45 mm) for 1.5 h at 100 V and blocked with 5% blocking buffer. The next step in the western
o
blotting was the incubation of prepared antibodies at 4 C for 12 h. The housekeeping protein
utilized in this procedure was beta-actin. Immunoblots were uncovered using the enhanced
chemiluminescence (ECL) method.
5.7. Molecular docking study
The analysis of data prepared by Western blotting has challenged us to explore the best poses
and interactions of compound 11i in the active site of the receptor and for this purpose, the
AutoDock 4.2 program was implemented. The crystallographic structure of the VEGFR-2
receptor in complex with ligand sorafenib (3WZE) was taken from PDB. To prepare 2D
structures, MarvinSketch 15.10.12 was utilized and using Chem3D ultra 8.0, the corresponding
structures were minimized and converted to pdb format. The next step involved the preparation
of the corresponding macromolecule; VEGFR-2, so that extra molecules of the protein were
removed by the discovery studio software and subsequently polar hydrogens and electric charges
added by AutoDockTools version 1.5.6. All maps were provided with 0.375 Å spacing between
grid points and through the selection of appropriate coordinates 81 Å (x), 61 Å (y), and 64 Å (z),
the proper location of the grid box was determined [32]. Discovery Studio Visualizer (Ver. 17.2)
and Pymol (Ver. 1.level) were used to visualize possible interactions between the receptor and
the ligand. Finally, to validate the docking protocol, the RMSD was calculated (0.46 Å) which
was demonstrating a successfully docked procedure (Fig. 14).
Acknowledgement
This work was supported and funded by Tehran University of Medical Sciences and Health
Services Grant no. 98-3-104-45990; and National Institute for Medical Research Development
Grant no. 962567.

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Captions:
Table 1. In vitro anti-proliferative effects (IC50, μM) of compounds 11a-m against PC3, HepG2,
T47D and HUVEC cell lines.
Table 2. In vitro cytotoxic effects (IC50, μM) of compound 11i against Hu02 cell line.
Figure 1. Diaryl urea containing anticancer agents in clinical use or in clinical trials.
Figure 2. Design strategy of final compounds 11a-m.
Figure 3. Flow cytometry analysis of PC3 cells treated with compounds 11h and 11i. (a)
Nontreated cells as negative control group; (b) treated with sorafenib as positive control at its
IC50; (c) treated with 11h at its IC50; (d) treated with 11i at its IC50; (e) treated with dimethyl
sulfoxide as solvent. Early apoptotic cells (Annexin V-positive, propidium iodide-negative)
appear in the lower right quadrant and late apoptotic cells (positive for both markers) in the
upper right quadrant.
Figure 4. Flow cytometry analysis of HUVEC cells treated with compound 11i. (a) Nontreated
cells as negative control group; (b) treated with sorafenib as positive control at its IC50; (c)
treated with 11i at its IC50; (d) treated with dimethyl sulfoxide as solvent. Early apoptotic cells
(Annexin V-positive, propidium iodide-negative) appear in the lower right quadrant and late
apoptotic cells (positive for both markers) in the upper right quadrant.
Figure 5. Effect of compounds 11h and 11i on the cell cycle distribution of PC3 cells by flow
cytometry analysis. (a) Non-treated cells as the control group; (b) treated with 11h at its IC50; (c)
treated with 11i at its IC50; (d) treated with sorafenib at its IC50.
Figure 6. Quantitative measurement of the cell cycle analysis.
Figure 7. The amount of apoptosis mediated by 11h, 11i, and sorafenib versus the cell
population arrested in the subG1-phase. The graph was presented by separated symbols, trying to
make a logical connection between the dependent variables.
Figure 8. Effect of compound 11i on the cell cycle distribution of HUVEC cells by flow
cytometry analysis. (a) Non-treated cells as the control group; (b) treated with 11i at its IC50; (c)
treated with sorafenib at its IC50.

Figure 9. Screening compounds by chick chorioallantoic membrane (CAM) assay. (a) Negative
control; (b) sorafenib; (c) 11i.
Fig. 10. a) Quantification of the effects of 11b, 11h, 11i, 11j, and 11m on the number of CAM
blood vessels. The bars show the mean ± SD (n=5). Statistical significance: **P < 0.01. b)
Quantification of the effects of 11b, 11h, 11i, 11j, and 11m on the length of CAM blood vessels.
The bars show the mean ± SD (n=5). Statistical significance: **P < 0.01, ***P <0.001. 11i
concentration: 3.6 ꢂM; sorafenib concentration: 15.5 ꢂM.
Figure 11. Western blot analysis of 11i and sorafenib on the VEGFR-2 phosphorylation in the
PC3 cell line after 24, 48, and 72 h.
Figure 12. Expression of VEGFR-2 and p-VEGFR-2 in PC3 cells treated with different
concentrations of 11i for 48 h. Control: treated with no drug; 11i-L: treated with 2.4 ꢂM 11i; 11i-
N: treated with 3.6 ꢂM 11i; 11i-H: treated with 5.4 ꢂM 11i.
Figure 13. a) 2D illustration of compound 11i and the corresponding interactions with residues
in the active site of VEGFR-2; b) 3D illustration of compound 11i and the corresponding
interactions with residues in the active site of VEGFR-2.
Figure 14. Superimposed representation of redocking lowest energy poses into the co-crystalized
ligand sorafenib over 3WZE.
Scheme 1. Reagents and conditions: a) morpholine, reflux, 14 h, 70%; b) formamidine acetate,
DMF, 100 oC, 16 h, 74%; c) POCl3, DIPEA, 45 oC, 42%; d) sodium carbonate, dry EtOH, reflux,
8 h, 84%; e) various substituted phenyl isocyanate, dry DCM, rt, 24 h, 45-55%; f) KOH/EtOH,
reflux, 6h, 60-68%.

Fig. 1. Approved VEGFR-2 inhibitors containing diaryl ureas
Fig. 2. Design strategy of final compounds 11a-m.

Table 1: In vitro anti-proliferative effects (IC50, ꢃM) of compounds 11a-m against PC3, HepG2, T47D and HUVEC cell
linesa.
Compound
R1
R2
PC3
HepG2
T47D
HUVEC
H
H
5.7 ± 0.8
19.4 ± 0.5
23.3 ± 0.5
19.3 ± 0.5
11a
CH3
Cl
H
5.2 ± 0.8
8.0 ± 0.7
11.5 ± 0.6
4.7 ± 0.8
9.9 ± 0.7
2.1 ± 0.8
0.97 ± 0.8
3.6 ± 0.9
7.7 ± 0.8
12.1 ± 0.6
6.8 ± 0.6
20.7 ± 0.4
22.9 ± 0.4
15.3 ± 0.5
16.0 ± 0.6
14.6 ± 0.5
52.0 ± 0.4
31.7 ± 0.2
61.9 ± 0.4
28.9 ± 0.2
45.3 ± 0.2
28.4 ± 0.4
27.2 ± 0.4
43.9 ± 0.2
45.1 ± 0.1
34.8 ± 0.4
>100
12.2 ± 0.5
21.0 ± 0.4
22.4 ± 0.3
23.2 ± 0.5
16.9 ± 0.5
19.3 ± 0.5
28.3 ± 0.4
7.8 ± 0.8
11b
11c
11d
11e
11f
11g
11h
11i
H
CF3
Cl
H
CH3
OCH3
CH3
F
H
H
17.8 ± 0.6
18.9 ± 0.5
14.6 ± 0.7
23.3 ± 0.4
28.0 ± 0.2
56.9 ± 0.3
H
Cl
Cl
H
Cl
11.6 ± 0.5
18.4 ± 0.5
67.7 ± 0.2
11j
11k
11l
CF3
OCH3
Cl
H

F
-
H
-
6.6 ± 0.9
64.1 ± 0.1
29.6 ± 0.3
16.7 ± 0.6
6.4 ± 0.6
13.7 ± 0.5
2.8 ± 0.8
11m
15.5 ± 0.4
Sorafenib
a Values were the means of three replicates ± standard deviation (SD).
Table 2. In vitro cytotoxic effects (IC50, ꢂM) of compound 11i against
Hu02 cell linea
Compound
Hu02
34.3 ± 0.3
40.0 ± 0.5
11i
Sorafenib
a Values were the means of three replicates ± standard deviation (SD).

a.
b.
c.
d.
e.
Fig. 3. Flow cytometry analysis of PC3 cells treated with compounds 11h and 11i. (a) Nontreated cells as negative
control group; (b) treated with sorafenib as positive control at its IC50; (c) treated with 11h at its IC50; (d) treated
with 11i at its IC50; (e) treated with dimethyl sulfoxide as solvent. Early apoptotic cells (Annexin V-positive,
propidium iodide-negative) appear in the lower right quadrant and late apoptotic cells (positive for both markers) in
the upper right quadrant.

Fig. 4. Flow cytometry analysis of HUVEC cells treated with compound 11i. (a) Nontreated cells as negative control
group; (b) treated with sorafenib as positive control at its IC50; (c) treated with 11i at its IC50; (d) treated with
dimethyl sulfoxide as solvent. Early apoptotic cells (Annexin V-positive, propidium iodide-negative) appear in the
lower right quadrant and late apoptotic cells (positive for both markers) in the upper right quadrant.

Fig. 5. Effect of compounds 11h and 11i on the cell cycle distribution of PC3 cells by flow cytometry analysis. (a)
Non-treated cells as the control group; (b) treated with 11h at its IC50; (c) treated with 11i at its IC50; (d) treated with
sorafenib at its IC50.