Exploration of carbamide derived pyrimidine-thioindole conjugates as potential VEGFR-2 inhibitors with anti-angiogenesis effect

Article abstract of DOI:10.1016/j.ejmech.2020.112457

The development of new small molecules from known structural motifs through molecular hybridization is one of the trends in drug discovery. In this connection, we have combined the two pharmacophoric units (pyrimidine and thioindole) in a single entity via molecular hybridization strategy along with introduction of urea functionality at C2 position of pyrimidine to increase the efficiency of H-bonding interactions. Among the synthesized conjugates 12a-aa, compound 12k was found to exhibit significant IC₅₀ values 5.85, 7.87, 6.41 and 10.43 μM against MDA-MB-231 (breast), HepG2 (liver), A549 (lung) and PC-3 (prostate) cancer cell lines, respectively. All these compounds were further evaluated for their inhibitory activities against VEGFR-2 protein. The results specified that among the tested compounds, 12d, 12e, 12k, 12l, 12p, 12q, 12t and 12u prominently suppressed VEGFR-2, with IC₅₀ values of 310–920 nM in association to the positive control (210 nM). Angiogenesis inhibition was evident by tube formation assay in HUVECs and cell-invasion by transwell assay. The mechanism of cellular toxicity on MDA-MB-231 was found through depolarisation of mitochondrial membrane potential, increased ROS production and subsequent DNA damage resulting in apoptosis induction. Moreover, clonogenic and wound healing assays designated the inhibition of colony formation and cell migration by 12k in a dose-dependent manner. Molecular docking studies also shown that compound 12k capably intermingled with catalytically active residues GLU-885, ASP-1046 of the VEGFR-2 through hydrogen-bonding interactions.

Full text of DOI:10.1016/j.ejmech.2020.112457

Journal Pre-proof
Exploration of carbamide derived pyrimidine-thioindole conjugates as potential
VEGFR-2 inhibitors with anti-angiogenesis effect
Sravani Sana, Velma Ganga Reddy, Sonal Bhandari, T. Srinivasa Reddy, Ramya
Tokala, Akash P. Sakla, Suresh K. Bhargava, Nagula Shankaraiah
PII:
S0223-5234(20)30428-1
DOI:
https://doi.org/10.1016/j.ejmech.2020.112457
EJMECH 112457
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 March 2020
Revised Date: 28 April 2020
Accepted Date: 10 May 2020
Please cite this article as: S. Sana, V.G. Reddy, S. Bhandari, T.S. Reddy, R. Tokala, A.P. Sakla, S.K.
Bhargava, N. Shankaraiah, Exploration of carbamide derived pyrimidine-thioindole conjugates as
potential VEGFR-2 inhibitors with anti-angiogenesis effect, European Journal of Medicinal Chemistry
(2020), doi: https://doi.org/10.1016/j.ejmech.2020.112457.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
©
2020 Published by Elsevier Masson SAS.

Graphical Abstract
Exploration of Carbamide Derived Pyrimidine-Thioindole Conjugates as Potential
VEGFR-2 Inhibitors with Anti-Angiogenesis Effect
a
b
a
b
a
Sravani Sana , Velma Ganga Reddy *, Sonal Bhandari , T. Srinivasa Reddy , Ramya Tokala ,
a
b
a
Akash P. Sakla , Suresh K. Bhargava , Nagula Shankaraiah *
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad - 500 037, India
a
b
Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science,
RMIT University, GPO Box 2476, Melbourne 3001, Australia
E-mail.: shankar.niperhyd@gov.in

Exploration of Carbamide Derived Pyrimidine-Thioindole Conjugates as Potential
VEGFR-2 Inhibitors with Anti-Angiogenesis Effect
a
b
a
b
a
Sravani Sana , Velma Ganga Reddy *, Sonal Bhandari , T. Srinivasa Reddy , Ramya Tokala ,
a
b
a
Akash P. Sakla , Suresh K. Bhargava , Nagula Shankaraiah *
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad - 500 037, India
a
b
Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science,
RMIT University, GPO Box 2476, Melbourne 3001, Australia
Abstract: The development of new small molecules from known structural motifs through
molecular hybridization is one of the trends in drug discovery. In this connection, we have
combined the two pharmacophoric units (pyrimidine and thioindole) in a single entity via
molecular hybridization strategy along with introduction of urea functionality at C2 position of
pyrimidine to increase the efficiency of H-bonding interactions. Among the synthesized
conjugates 12a-aa, compound 12k was found to exhibit significant IC values 5.85, 7.87, 6.41
5
0
and 10.43 µM against MDA-MB-231 (breast), HepG2 (liver), A549 (lung) and PC-3 (prostate)
cancer cell lines, respectively. All these compounds were further evaluated for their inhibitory
activities against VEGFR-2 protein. The results specified that among the tested compounds, 12d,
1
2e, 12k, 12l, 12p, 12q, 12t and 12u prominently suppressed VEGFR-2, with IC50 values of 310
to 920 nM in association to the positive control (210 nM). Angiogenesis inhibition was evident
by tube formation assay in HUVECs and cell-invasion by transwell assay. The mechanism of
cellular toxicity on MDA-MB-231 was found through depolarisation of mitochondrial membrane
potential, increased ROS production and subsequent DNA damage resulting in apoptosis
induction. Moreover, clonogenic and wound healing assays designated the inhibition of colony
formation and cell migration by 12k in a dose-dependent manner. Molecular docking studies also
shown that compound 12k capably intermingled with catalytically active residues GLU-885,
ASP-1046 of the VEGFR-2 through hydrogen-bonding interactions.
Keywords: Angiogenesis, VEGFR-2, pyrimidine-thioindole, urea, cytotoxicity, apoptosis.
Corresponding author: Dr. Nagula Shankaraiah, E-mail: shankar@niperhyd.ac.in;
shankarnbs@gmail.com; Dr. Velma Ganga Reddy, E-mail: ganga.reddy.velma@rmit.edu.au.
1

1
.0 Introduction
In drug discovery, lead generation always starts with the identification of chemical starting
points with the consumption of known literature such as fast-follower or knowledge-based
programs [1]. According to the scrutiny of Hit-to-Clinical Pairs stated in 2016-2017, a slight
fewer than half (43%) of the clinical candidates defined were resulted from recognized starting
points [2]. Among the 66 hit-to-clinical pairs, 30% of disease area was led by oncology,
particularly targeting kinase receptors [3] highlighting cancer as a focus of interest for the
expansion of new anticancer agents to overcome multidrug resisitance with restored clinical
assistance [4].
Angiogenesis is a multitier strategy, wherein endothelial cells become stimulated,
dissociate, migrate and proliferate to form new sprouts from the pre-existing blood vessels and
observed to be pivotal during the embryo development, wound healing and reproduction [5].
Metastatic growth of primary tumors is an angiogenesis-dependent process, regulated by
numerous stimulatory and inhibitory pathways, of which, growth factors such as VEGF, EGF,
FGF, and PIGF have been identified as critical regulators [6]. Among these, Vascular
Endothelial Growth Factor (VEGF) is a potent angiogenic tyrosine kinase receptor,
overexpressed in majority of the solid tumors. VEGFR-2 is the prime mediator of VEGF-induced
angiogenic signaling, which makes the target attract toward certain advanced cancers for
involvement in the multitier strategies of angiogenesis, including vascular permeability,
endothelial cell proliferation, migration, and survival [7]. Moreover, a significant number of
small molecules of VEGFR-2 inhibitors namely sorafenib, sunitinib, axitinib (A, Figure 1),
vatalanib and regorafenib have been approved for clinical use in cancer therapy. Taking these
into consideration, targeting VEGFR-2 represents as a hopeful strategy for quantifiable benefit in
the treatment of cancer [8].
Clinical success rate and presence of pyrimidine ring in DNA and RNA explicates its
medicinal attributes and contemporary interest of researchers towards the development of
potential therapeutic candidates in drug discovery [9]. Existence of pyrimidine in small molecule
inhibitors as an essential fragment has been evidenced to hold diverse therapeutic aptitudes such
as anticancer [10], anti-HIV, antidiabetic [11], antihypertensive, analgesic, antimicrobial,
antiinflammatory [12], antitubercular, A2B adenosine receptor antagonists and antibacterial [13].
In addition, the tumor cell-killing potential of pyrimidine based frameworks has been witnessed
2

through diverse mechanisms, taking an account of inhibition of tyrosine kinase [14],
serine/threonine protein kinase, histone deacetylase [15], autotaxin, heat shock protein, inositol
kinase [16], etc. Pazopanib (B, Figure 1) is a pyrimidine centered small molecule identified with
antiangiogenetic effect, approved by FDA in 2009 as multi-targeted receptor tyrosine kinase
inhibitor [17]. Incessantly, CEP-11981 (C, Figure 1), a potent multi-targeted cytotoxic
pyrimidine derivative was recognized with antiangiogenetic activity [18].
Besides, indole is a significant basis for biologically active compounds, which may
possibly be acquired from organic synthesis or isolated from natural products. Indole as a core
nucleus has been explored for various therapeutic activities such as anticancer [19], anti-
inflammatory, antioxidant, anti-HIV [20], antimalarial, anti-tubercular, anticonvulsant,
antidiabetic [21], antimicrobial, antifungal, antidyslipidemic and so on [22]. Cediranib (D,
Figure 1) is an indole fused trioxyquinazoline derivative and observed to retain better
discrimination over a panel of tyrosine and serine/threonine kinases along with VEGFR
inhibitory activity. Brivanib (E, Figure 1) is also an indole based multi-targeted tyrosine kinase
inhibitor with anti-tumorigenic effects towards hepatocellular carcinoma [23].
<
Insert Figure 1 here>
As well, the evolution in the development of urea and its derivatives amplified the drug
th
discovery since the latter half of the 20 century, exhibiting broad range of medicinal
applications like anticancer, antibacterial, anticonvulsive, anti-HIV and anti-diabetic [24]. In
precise, antitumor activity of urea functionality is emphasized by targeting protein kinase or
microtubules with the ability of establishing multiple stable hydrogen bonds with protein and
receptor targets by improving drug properties such as potency and selectivity [25]. Sorafenib (F,
Figure 1) is a diaryl urea multikinase inhibitor approved in 2007 by FDA, underlining the
significance of protein-ligand interaction with the formation of critical hydrogen-bonding with
main-chain atoms of Asp1046 and the side-chain atoms of Glu885 vascular endothelial growth
factor receptor 2 (VEGFR-2) [26]. In the recent years, considerable amount of aryl urea
substructures has been introduced clinically, especially in the fields of kinase inhibitors i.e.,
regorafenib (G, Figure 1), linifanib and lenvatinib as VEGFR inhibitors [27], tandutinib as type
III receptor tyrosine kinase inhibitor, gedatolisib as dual inhibitor of PI3K/mTOR [28].
3

Incorporation of molecular docking into the drug design, reveals that the binding site of
VEGFR-2 has some common pharmacophoric features for most of the VEGFR-2 inhibitors i.e. i)
typically a hetero aromatic ring binds to the hinge region, ii) hydrogen bond-rich region indicates
where hydrogen donors or acceptors like urea or amide are favorable, iii) hydrophobic I & II
regions, which can put up monocyclic or bicyclic ring structures [29]. Moreover, the association
of molecular assortment that combines two or more pharmacophores in a single entity through
molecular hybridization might be an effective way to aid the factors like synergism, drug
resistance for better clinical upshot [30].
In the way of our obligation headed for the advance of bioactive molecules [31] and in sight of
aforementioned distinctions regarding the pharmacophoric features and molecular hybridization
by means of known VEGFR-2 inhibitors, a library of 600 molecules were designed and docked
in to the active site of VEGR-2. Based on the results, a series of 27 carbamide derived
pyrimidine-thioindole conjugates were synthesized based on the synthestic feasibility and
predicted as encouraging molecules of VEGFR-2 inhibition. In the current work, all the defined
molecules were armed with the crucial structural features like i) indole at C4 of pyrimidine as
hinge-binding region, ii) urea functionality at C2 of pyrimidine as hydrogen-bond rich region, iii)
1
˚/2˚ nitrogen-containing heterocycles at C6 of pyrimidine is designed to occupy hydrophobic I
region, iv) pyrimidine ring is required to anchor hydrophobic II region.
<
Insert Figure 2 here>
2
2
.0 Results and Discussion
.1 Chemistry
As depicted in Scheme 1, the carbamide derivatives of thioether-linked indole-pyrimidine
conjugates 12a–aa were synthesized in a merging slant utilizing the nucleophilic reactivity of
thioindole substituted pyrimidin-2-amines 10a–i with substituted aryl isocyanates 11a–f as
electrophiles. The key intermediate 1H-indole-3-thiol was synthesized by electrophilic attack of
thiourea at C3-position of indole via alkaline treatment of thiourea intermediate. Next, a variety
of 6-amino-pyrimidin-2-amines 9a–i was synthesized according to the reported procedures [32].
The condensation reaction between diethyl malonate and guanidine hydrochloride in the
presence of a base with elimination of ethanol leads to the formation of 2-aminopyrimidine-4,6-
4

3
diol. The alcoholic functionalities were then chlorinated using POCl to form an easy leaving
group i.e., chlorophosphite and subsequently chlorine group attacks the carbon of this
intermediate to provide 4,6-dichloropyrimidin-2-amine. Next, one of the chlorine groups of 4,6-
dichloropyrimidin-2-amine derivative undergoes nucleophilic substitution selectively with
different 1˚ aromatic amines and cyclic 2˚ non-aromatic amines using acidic as well as basic
conditions in protic solvents to afford 6-amino-pyrimidin-2-amines 9a–i. The other chlorine was
then substituted with indole-3-thiol as nucleophile using KI and Et N to afford 4-((1H-indol-3-
3
yl)thio)-6-(1˚/2˚ amino)pyrimidin-2-amine 10a–i. The nucleophilic amine group at C2 position
of these derivatives was successively reacted with substituted aryl isocyanates to afford the titled
carbamide derivatives of thioether-linked indole-pyrimidine conjugates 12a–aa.
<
Insert Scheme 1 here>
1
The most potent compound 12e was characterized by H NMR and showed a characteristic
sharp singlet at δ 10.04 and 9.15 ppm which indicates the proton ensuing carbamide formation.
Proton of –NH– corresponding to thioindole resonated as a singlet at δ 11.44 ppm. The doublet
of one proton at δ 7.37 ppm belongs to the –CH– proton adjacent to the thioindole –NH. The
signal at δ 6.06 ppm resembles the proton of –CH– at C5 position of pyrimidine. The multiplet of
six protons at δ 1.44 and 1.58 ppm corresponds to –CH – protons of C3, C5 and C4 protons of
2
2
piperidine ring, respectively. The four protons of –CH – at C2 and C6 which is adjacent to N–
atom of piperidine ring resonated as a multiplet at δ 3.43 ppm. Distinctive methyl protons of
phenyl urea seemed as a singlet at δ 1.89 ppm and the left over protons lie in the aromatic region
1
3
at δ 7.68–6.96 ppm. Likewise, C NMR interpretation of compound 12e indicates the presence
of carbonyl carbon of urea functionality at δ 152.7 ppm. The carbon in between the thioether
bridge and N-1 atom of pyrimidine was resonated at δ 169.1 ppm. The C4-carbon of pyrimidine
ring attached to the piperidine was appeared at δ 161.3 ppm. The C2-carbon of pyrimidine ring
showed a characteristic peak at δ 157.4 ppm and all the remaining aromatic carbons resonates
between δ 136.8–91.3 ppm. Distinctive methyl carbons of phenyl urea were resonated at δ 18.3
ppm. The signals at δ 24.4 and 25.5 ppm accounts for the C3, C–5 and C4 carbons of piperidine
ring respectively. The two carbons of piperidine ring adjacent to N-atom resonated at δ 45.0
1
13
ppm. Similarly, all the synthesized derivatives were scrutinized by using HRMS, H, C NMR
and FT-IR and are in agreement with the relevant structures as showed in the experimental
5

+
+
section. The HRMS (ESI) of all the compounds showed an [M + H] or [M + 2] peaks
conforming their particular molecular formula.
2
2
.2 Biological evaluation
.2.1 In vitro anticancer screening
The synthesized pyrimidine-thioindole conjugates 12a–aa were assessed for their in vitro
cytotoxicity employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay [33] against certain human cancer cell lines like A549 (human lung cancer), PC-3 (human
prostate cancer), MDA-MB-231(human breast cancer) and HepG2 (human liver cancer). The
IC (μM) values from the in vitro anticancer screening of the derivatives 12a–aa along with
5
0
standard drug sorafenib were inferred in Table 1. Interestingly, the verified derivatives exhibited
moderate to significant antiproliferative activity against the selected cancer cell lines.
Remarkably, primary results directed that most of the synthesized derivatives 12a–aa showed
significant cytotoxicity against triple-negative breast cancer (TNBC) cell line such as MDA-MB-
2
31. For instance, compounds 12b–i, 12k, 12l, 12q, 12t, 12v, 12z and 12aa exhibited notable
antiproliferative activities with IC50 values 5.85 to 10.26 µM. Moreover, compounds also
displayed good to moderate antiproliferative activities against A549 (lung) and PC-3 (prostate)
cancer cell lines with IC50 values 6.41 to 48.43 µM and above.
From the detailed study of IC50 values, it was inferred that the derivatives holding five-
membered nitrogen heterocyclic ring such as pyrrolidine influenced the activity on all tested
cancer cell lines. Note worthily, one of the tested compounds 12k of carbamide derivatives
disclosed effective in vitro cytotoxicity against MDA-MB-231 breast cancer cell with IC value
5
0
5
7
.85 μM. This derivative also observed to be active with corresponding IC values 6.41 μM,
50
.87 μM and 10.42 µM against lung (A549), liver (HepG2) and prostate (PC-3). It was also
observed that compound 12l exposed to be active in liver (HepG2), lung (A549), breast (MDA-
MB-231), and prostate (PC-3) with 50% inhibition at 7.15 μM, 8.93 μM, 9.44 μM and 12.86 μM
respectively. It is obvious from IC50 values that the presence of thioindole attached to C4-
pyrimidine ring was well tolerated for cytotoxicity. Nonetheless, it could be witnessed from the
in vitro cytotoxicity results that the presence of non-aromatic nitrogen heterocycles significantly
inclined the cytotoxic profile especially towards MDA-MB-231 breast cancer cell line which
assumes that these results may support in identification of new anti-breast cancer agents with
superior activity. Based on the cytotoxicity, the most potent compounds 12e and 12k were
6

selected for additional studies in the direction of identification of mechanism for cancer cell
growth inhibition. Furthermore, all the titled compounds were screened for their VEGFR-2
inhibitory potency to realize the concealed mechanism of the anticancer effects.
<
Insert Table 1 here>
2
.3 In-vitro VEGFR-2 inhibition assay
VEGFR-2 inhibitory potency of indole-pyrimidine conjugates were tested for their in
vitro activities with the inclusion of sorafenib (VEGFR-2 inhibitor) as a known positive control.
The results were outlined in Table 2. In accordance with the obtained results, all the compounds
exhibited from moderate (2.14 - 5.22 μM) to strong (0.31 - 1.92 μM) inhibitory activities when
compared with sorafenib (210 nM). In specific, among the 27 molecules, 8 compounds potently
inhibited VEGFR-2 with IC value of 12d (IC = 350 nM), 12e (IC = 310 nM), 12k (IC =
5
0
50
50
50
3
30 nM), 12l (IC = 430 nM), 12p (IC = 920 nM), 12q (IC = 670 nM), 12t (IC = 840 nM)
50 50 50 50
and 12u (IC50 = 440 nM). Also, 9 compounds 12a, 12b, 12c, 12f, 12g, 12n, 12w, 12z, 12aa
exhibited good VEGFR-2 inhibitory activity at low micromolar IC values (1.13 to 1.92 μM).
5
0
However, the other 10 compounds exhibited moderate inhibition to VEGFR-2 (2.14 to 5.22 μM).
Insert Table 2 here>
<
3
3
.0 In silico Studies
.1 Molecular Docking
The higher inhibition pattern displayed by the title carbamide derivatives stimulated us to
explore their feasible binding modes within VEGFR-2 kinase domain using molecular docking
study, so that to afford an agreeable clarification for the consistency of biological activity. For
this protocol, we selected PDB ID: 3WZE [34] cocrystallized with sorafenib and then the
authentication of the docking procedure was achieved by re-docking of the co-crystallized ligand
(sorafenib) in the VEGFR-2 active site, in which sorafenib reproduced all the key interactions
with critical amino acids in the active site (Cys919, Glu885, and Asp1046). This point outs the
aptness of the used protocol for intended docking study, in which the most potent carbamide
derivatives 12d, 12e, 12k, 12l, 12p, 12q, 12t and 12u were docked into the active site of
VEGFR-2 kinase (Figure 3a).
<
Insert Figure 3 here>
7

Molecular insights of the lead carbamide derivatives 12k and 12e (Figure 3b & c)
indicated that both the amino hydrogen atoms of urea linker were able to forms H-bond with the
…
…
…
carbonyl oxygen atom of Glu885 (12e: N2-H O: 1.67 Å, N1-H O: 2.31 Å; 12k: N2-H O: 1.66
…
Å, N1-H O: 2.33 Å). Whereas the other H-bond was recognised between carbonyl oxygen atom
…
…
of urea and the amino hydrogen atom of Asp1046 (12e: O H-N: 2.30 Å; 12k: O H-N: 2.34 Å)
in an extended channel situated straight next to the ATP binding site. On the other hand, the
binding pattern of urea functionality for the most active compounds accomplishes a similar
binding pattern (hydrogen bonds with Glu885 and Asp1046) as that of co-crystal (sorafenib) by
augmenting the hypothesis, with an exception that compound 12t interacts with Glu815.
Consequently, the cyclic secondary amine motifs of 12e and 12k (piperidine and pyrrolidine)
along with phenyl group of urea moiety achieves a hydrophobic interaction with hydrophobic
side chains of Phe1047, Cys1045, Ile1044, Leu1019, Cys1024, Ile1025, Val 899, Ile 888, Leu
8
89 and Val 914 amino acids of hydrophobic pocket. Apart from this, the thioindole group of
other compounds 12p, 12d and 12t shared hydrophobic cleft of Cys1024, Ile1025, Leu1019,
Cys1024, Ile892, Ile888 and Leu889, justifying their biological activity. Furthermore, the amino
cation of Lys868 forms a pi-cation interaction with the phenyl ring of compounds 12e and 12k.
From the docking study, it was observed that the most potent compounds in study were well
accommodated inside the binding site of the VEGFR-2. To make deeper understanding of our
outcomes, we tried the superimposition of the potent ligands 12e and 12k (Figure 4a) and the
resultant pose reveals that all the structural motifs were superimposed identically with one
another suggesting the probable binding mode. The subsequent superimposition poses of
sorafenib with the potent ligand 12k recommend that urea functionality of ligand 12k occupied
identical position with the urea functionality of the sorafenib as noticed in Figure 4b. The H-
bonding interactions for docking poses along with docking scores were presented in Table 3 and
2
D diagrams were included in the supplementary data.
Insert Figure 4 here>
Insert Table 3 here>
<
<
3
.2 In silico ADME/T studies
A set of pharmacokinetic properties for 8 potent molecules were calculated through
QikProp program (Qikprop, version 6.5, Schrödinger, LLC, New York, NY, 2014) to examine
8

drug-likeliness in terms of ADME/T properties [35]. The computed ADME/T parameters of
investigated compounds are within their prescribed range as mentioned in Table 4. This in silico
study revealed that all the calculated compounds exhibited adequate values of molecular weight
(mol. Wt.), hydrogen bond donors (donor HB), hydrogen bond acceptors (acceptor HB), partition
coefficient (QPlogPo/w), and ensuing Lipinski’s rule of 5. Comparison of the ADME/T
properties of the potent molecules with that of known drug depicts that these conjugates show no
violation in the recommended ranges of physico-chemical descriptors, stating that the
compounds are not toxic and obeys Lipinski’s rule of 5.
<
Insert Table 4 here>
3
.3 3D-QSAR study
In order to further explore the SARs of pyrimidine-thioindole conjugates, the in vitro VEGFR-2
inhibition data was selected to concept the 3D-quantitative structure activity relationship (3D-
QSAR) models. Based on the comprehensive procedure manual described in the literature,[36]
the IC values of all derivatives against VEGFR-2 were picked and transformed to pIC values.
5
0
50
<
Insert Table 5 here>
3
.3.1 Field and Atom-based 3D-QSAR analysis
The accurate PLS statistics outcomes of the Field and Atom based 3D-QSAR models were
disclosed in Table 5. The experimental and the predicted pIC50 values of the target compounds
against VEGFR-2 are listed in Table 6. The PLS study elucidates the predictive aptitude and the
2
self-consistence of the model in which, the cross-validation correlation coefficient (q ) and
2
correlation coefficient (r ) are two essential measures to figure out the strength of PLS analysis.
The cross-validation correlation coefficient of more than 0.3 will be advised statistically as the
2
2
chance of noticeable correlation being <95%. The q and r of the field based model were 0.689
2
2
and 0.43, respectively, and the atom based 3D-QSAR model q and r were 0.481 and 0.43
respectively as tabulated in Table 5. The ideal sum of components used to produce field and
atom based models were in the acceptable range. The Fisher test results of models were found to
be 66.6 and 71.2 respectively. Furthermore, the predicted standard errors of the field and atom-
based models were found to be 0.163 and 0.179 respectively. The input of the steric,
electrostatic, hydrophobic, hydrogen bond donor and acceptor fields of the field-based model
were 37.3%, 11.6%, 24.0%, 16.5% and 10.4%, respectively and the input of the electrostatic,
hydrophobic, hydrogen bond donor fields of the atom-based model were 0.7%, 68.9% and 5.0%,
9

respectively. The obtained results suggested that the field and atom-based models constructed
were consistent and supportable modification of the target molecules to pick-up the better
activity. The detailed PLS data were shown in Table 6.
<
Insert Table 6 here>
The linear relationship results in Figure 5 shown that the calculated pIC and the experimental
5
0
pIC50 values of the field and atom-based models had a worthy linear relationship. Besides, due to
structural variations, a small amplitude instabilities were detected among the calculated pIC50
and the experimental pIC values.
5
0
<
Insert Figure 5 here>
3
.3.2 Countour analysis
In order to understand the impact of various fields on the target property, by Schrodinger
software, Field and Atom contour maps were produced to recognise the impression of different
fields (the steric field, electrostatic field, hydrophobic field and hydrogen bond donor and
acceptor fields) on the activity data of compounds. StDev*Coeff mapping route was used to
transform Field and atom-based models into visual results. All contour maps represented 80%
level contributions for favored and 20% level contributions for disfavored. As indicated in
Figure 6 and Figure 7, the template compound 12e (with the highest pIC50 value) and the
compound 12p were chose to disclose the 3D-QSAR information of field and atom-based
models. These results can support to understand the connection between structure and biological
activity.
<
Insert Figure 6 here>
3
.3.2.1 Field-based contour analysis:
According to the field contour maps shown in Figure 6, it can be obviously realized that the
thioindole ring, benzene ring of urea was covered with a green contour. Thus, it is sensible to
introduce the thioindole structure on the pyrimidine ring promoting the VEGFR-2 inhibition.
o
Also, the occupancy of green contours around the 2 amine rings showed the importance of the
ring expansion strategy in the designing of titled molecules. Similarly, the results of red contours
o
around the N-atom of 2 amine revealed that antiproliferative activity of the target compound
o
decreased as the volume of the 2 amine ring increased (12d, 12l > 12p, 12s). In addition, the
o
presence of a minor amount of yellow contours around the 2 amine ring directed that the
o
hydrophobicity of the 2 amine ring was favorable to uphold the proliferative activity of the
1
0

compounds against VEGFR-2 protein. A big cyan contour appeared around the N-H atoms and
gray contour around the O-atom of urea functionality indicated that the urea functionality plays
an important role in preserving the VEGFR-2 activity of the target compounds.
3
.3.2.2 Atom-based contour analysis
<
Insert Figure 7 here>
Figure 7 showed the hydrophobic and electron withdrawing cubes of the atom-based model. A
group of light-green cubes overlapped around the benzene ring of the urea functionality. It
revealed that the electron withdrawing substitutions on the benzene ring is beneficial to increase
3
the VEGFR-2 inhibition (R = 4-Cl > 2-CH -4-Cl > H > 4-OMe > 3,5-diOMe). In the atom-based
model, we observed that the orange-yellow cubes appeared around the pyrimidine ring,
indicating that this region would be conducive to maintain the proliferative activity of the
compounds against VEGFR-2 protein.
4
.0 Structure Activity Relationships (SARs)
Structure activity relationship (SAR) of the synthesized compounds was shaped (Figure 8)
to study the impact of substitution on pyrimidine ring in comparison with the inhibitory activity
against human cancer cell lines as well as VEGFR-2.
Comparing the activity of compounds 12a–aa in relation to cytotoxicity revealed that the
presence of non-aromatic nitrogen heterocycles (12b-i, 12k, 12l) and strained cyclopropyl ring
(12u & 12v) at C6-position of pyrimidine were active particularly towards breast cancer cell
lines (MDA-MB-231). Primary aromatic amines (12m, 12w-aa) have deviated the cytotoxicity
profile against all the tested cancer cell lines, except that, the compounds 12y, 12z & 12aa
bearing 3,5-dimethoxy and 4-chloro-phenyl exhibited good activity against PC-3 and MDA-MB-
2
31 cell lines respectively. In specific, the presence of five-membered pyrrolidine ring (12k)
favoured the good anticancer effects against all the selected cancer cell lines. Both N-substituted
12n, 12p, 12q) as well as saturated nitrogen heterocycles (12a, 12d, 12e, 12k and 12l) were
(
found to be endurable towards liver cancer cell lines (HepG2).
Relating the inhibitory concentration of compounds 12b–i and 12k, 12l, 12q, 12t, 12v, 12z,
1
2aa indicated that both the introduction of electron donating (4-methoxy, 2,5-dimethyl, 2,5-
1
1

dimethoxy) as well as electron withdrawing (4-chloro, 2-methyl-4-chloro) groups were found to
be tolerable on aryl urea.
<
Insert Figure 8 here>
Conversely, in relation to VEGFR-2 inhibition, the introduction of non-aromatic nitrogen
heterocycles such as morpholine, pyrrolidine, piperidine 12a–l and N-substituted piperazines
1
2n–t (N-benzyl, N-pyridyl) at C6-position of pyrimidine resulted in significant to moderate
VEGFR-2 inhibition (IC ranging from 0.31 to 4.86 μM). Compounds with primary aromatic
5
0
amines (12m and 12w–aa) exhibited the moderate inhibitory action on VEGFR-2 (IC ranging
5
0
from 1.33 to 3.51 μM). Similarly, introduction of strained functionality like cyclopropyl amine
2u also afforded the most active VEGFR-2 inhibition (IC 0.44 μM).
1
5
0
Finally, we focused the effect of substitution on phenyl urea at C2-position of pyrimidine.
Introduction of chloro group at para position of phenyl urea 12k (IC50 0.33 μM) resulted in
about 8 and 10-fold rise in enzyme inhibition relative to the electron donating 12j and
unsubstituted analogue 12i (IC 2.61 μM and 3.23 μM) respectively. Also, with compounds 12c
5
0
and 12f bearing chloro group was shown substantial inhibition (IC50 1.44 μM, 1.82 μM
respectively). On the other hand, 2-methyl-4-chloro substitution (12d, 12l and 12p) displayed
similar levels of enzyme inhibition (IC50 ranging from 0.35 to 0.92 μM) with all the non-
aromatic nitrogen heterocycles, highlighting the impact of this group for VEGFR-2 inhibition.
The same trend was observed with comparison of 2-methyl-4-chloro and unsubstituted phenyl
urea with cyclopropyl (IC50 0.44 μM > 5.22 μM; 12u was 12-fold > 12v) and phenylamino rings
(
IC50 1.33 μM > 2.74 μM; 12w > 12m) respectively. Similarly, incorporation of 2-methyl-4-
chloro along with bulky benzyl piperazine 12s (IC50 4.86 μM) led to non-significant activity. The
unsubstituted phenyl group 12a, 12h, 12n and 12q also led to significant to reasonable VEGFR-2
suppressive activity (IC ranging from 0.67 to 4.55 μM). Concerning the influence of electron
5
0
donating substitutions like 2,5 dimethyl 12e, 12r, 12x, 12t (IC50 ranging from 0.31 to 2.82 μM)
4-methoxy 12b, 12g, 12j, 12o, 12z (IC ranging from 1.13 to 4.15 μM) > 2,5 dimethoxy 12y
>
5
0
(
IC50 3.51 μM) exhibited potent to moderate VEGFR-2 suppressive activity. Grafting of
mercapto indole at C4-position of pyrimidine was well tolerated for both in vitro anticancer
effects on human cell lines and VEGFR-2 inhibition.
1
2

5
.0 HUVECs tube formation assay
Tube formation assay is the most widely used in vitro assay in which capacity of
endothelial cells, overlaid at sub-confluent densities will be measured to model the
reorganization stage of angiogenesis. Typically, this assay is employed to measure the ability of
the anti-angiogenic compounds to promote or inhibit capillary-like structures (tubes) which
could be useful to study the underlying mechanisms in cancer [37]. To study the anti-angiogenic
properties of the potent compound 12k, matrigel tube formation assay of HUVECs was
performed. As shown in Figure 9, treatment given with compound 12k at 2.5 and 5 μM was
significantly inhibited the networks of HUVECs in wells where elongated, robust networks
clearly seen in wells seeded with untreated. The results of the matrigel assay demonstrated that
compound 12k inhibited the formation of HUVEC tube like structures in a dose dependant
manner where similar effects were noticed with standard drug sorafenib. In addition,
quantification of tube formation assay was analysed using WimTube analysis [38] which are
highlighting the remarkable antiangiogenic properties of the compound 12k with sorafenib
(Figure S2) where all aspects of network formation were significantly affected in compound
treated wells, as indicated by a decrease of cell-covered area, tube length, branching points and
total loops (Figure S3).
<
Insert Figure 9 here>
5
.1 Cell invasion/transwell assay
Cell migration and invasion are the two main essential steps for the formation of new blood
vessels during the angiogenesis processes which lead to angiogenesis and metastasis [39].
Herein, we proceeded with Boyden chamber (Transwell) assay in HUVECs to determine the
ability of cancer cells to pass through the Matrigel and membrane barriers with 2.5 and 5 µM of
compound 12k where sorafenib used as a standard and results were shown in Figure 10. In
control, majority of the cells migrated to the bottom layer of the Boyden chamber where invaded
cells were significantly reduced by 12k. In the results, it was clearly indicating that compound
1
2k preventing the cell invasion in a dose dependant manner.
Insert Figure 10 here>
<
1
3

5
.2 Colony forming assay
The proliferative potential and ability of adherent cells to form distinct colonies from a
single cell can be assessed using the colony formation assay. Hence, it is the method of choice to
define the effectiveness of cytotoxic agents based on cell survival and cell reproductive death
[40]. In this assay, the colony forming ability of MDA-MB-231 cells was measured by treating
with media containing different concentrations (1, 3 and 5 µM) of compound 12k over a period
of 7 days to form colonies. The cells were then stained with crystal violet to visualise colonies
and from Figure 11, it clearly directs the potential of 12k in inhibition of colony formation as
compared to the control and so it could be one of the mechanisms reflected in prompting
cytotoxic activity in MDA-MB-231 cancer cells.
<
Insert Figure 11 here>
5
.3 Wound healing assay (migration assay)
Cell migration is a vital possession involved in the dissemination of cancerous cells mostly
during cancer metastasis. The cell culture wound-closure is of particular interest to examine the
inhibition of migration for effective cancer treatment [41]. A wound healing assay was
performed on MDA-MB-231 cells to study the inhibitory influence of compound 12k and
sorafenib on migration ability. In this protocol, wounds were made on highly metastases
confluent cell monolayers of MDA-MB-231 by scratching with a sterile pipette tip and picturing
the response using phase contrast microscopy. As shown in Figure 12, after 24 h, the cells
treated with 2.5 µM of compound 12k and sorafenib displayed considerable inhibition of cell
migration compared to the control cells, while migration was totally significant after treatment
with a 5 µM solutions.
<
Insert Figure 12 here>
6
6
.0 Apoptosis detection studies
.1 Identification of apoptotic cells
Conception of cell death, principally apoptosis is one of the most-widely studied scenarios
in cancer. Effect of chemotherapeutic agents on morphological hallmarks of apoptosis like
chromatin condensation, nuclear fragmentation and pyknosis is of high value to examine using
fluorescent staining techniques. Hoechst is a cell-permeate nuclear counter stain, binds
1
4

immutably to the DNA minor groove at A–T-rich clusters with specific staining of living or
fixed nuclei. Binding of Hoechst to ds DNA emits blue fluorescence, stains the condensed
chromatin in apoptotic cells further deeply than the chromatin in normal cells [42]. Hence,
apoptotic cells come out brighter reasonably than live cells. As exposed in Figure 13, the control
(a), cell nuclei preserve intact showing usual morphology whereas, the MDA-MB-231 cells
treated with 2.5 µM (b & d) and 5 µM (c & e) of compounds 12e and 12k respectively exhibited
the typical apoptotic features together with nuclear shrinkage, pyknotic or fragmented bright
nuclei, and chromatin condensation in a dose-dependent manner.
<
Insert Figure 13 here>
6
.2 Quantification of apoptosis induction
Annexin V-FITC/Propidium iodide dual staining assay assists the discrepancy between
necrotic cells (Q1-UL; AV-/PI+), late apoptotic cells (Q1- UR; AV+/PI+), early apoptotic cells
Q3-LR; AV+/PI-) and live cells (Q2-LL; AV-/PI-) using flow cytometry. In view of high
(
affinity towards phosphatidylserine (PS), Annexin V (Annexin V-FITC) identifies the apoptotic
cells, in which exteriorization of PS occurs as a normal process of apoptosis [43]. MDA-MB-231
breast cancer cells were treated with compound 12k at different concentrations of 2.5 and 5 µM.
It can be observed from Figure 14 that the percentage of late and early apoptotic cells
significantly improved from 3.8 to 11.2% and 19.4 to 36.6% respectively with increase in the
concentration from 2.5 to 5 µM concentration, which signifies the dose-dependent apoptosis
induction on MDA-MB-231 breast cancer cells.
<
Insert Figure 14 here>
6
.3 Effect on mitochondrial membrane potential (DΨm)
Distinct aspects of mitochondria function is a key indicator of cell health, chiefly associates
with the generation of mitochondrial membrane potential (MMP) via ATP synthesis through a
series of redox reactions. Variations in the redox equilibration of the mitochondrial membrane
presumed to enhance the generation of reactive oxygen species (ROS) and thereby loss of
mitochondrial membrane potential (DΨm) [44]. Hence, it is an influential undertaking to
consider the effect of compound 12k on mitochondrial membrane potential (DΨm). The
membrane-permeate JC-1 dye was used for defining DΨm, in which J-monomers in depolarized
1
5

mitochondria, therefore, stains green in color whereas healthy mitochondria consist of J-
aggregates, thus stains red in color. MDA-MB-231 cells were treated with 2.5 µM (b & d) and 5
µM (c &e) of compounds 12e and 12k respectively where proportionality was observed in
depolarized cell population with rise in the concentration. Loss of membrane integrity was
observed in a dose-dependent manner as shown in Figure 15.
<
Insert Figure 15 here>
6
.4 Effect on intracellular ROS generation
Cells are supplied with a controlling antioxidant defense structure to battle extreme
production of ROS enhanced free radical generation, crushes the cells’ natural antioxidant
defenses, leads to the destruction of mitochondrial potential thereby causes apoptosis [45].
Therefore, to observe the degree of ROS generation by compound 12k, the intracellular ROS
generation was driven using the Carboxy-H DCFDA staining. MDA-MB-231 cells were treated
2
with different doses of compound 12e and 12k, in which DCFDA is reacted to form 2',7'-
dichlorofluorescein (DCF) in the existence of ROS generation, led to increase in green
fluorescence with increase in the concentration. From Figure 16, it certainly indicates dose
dependency in inducing ROS generation as compared to the control and thus it might be one of
the reflected mechanisms in instigation of apoptosis in MDA-MB-231 breast cancer cells.
<
Insert Figure 16 here>
7
.0 Conclusion
In summary, the present study organically highlights the design, synthesis and biological
evaluation of carbamide derived pyrimidine-thioindole conjugates, in which a series of 27
molecules were synthesized by using molecular hybridization approach from the known
VEGFR-2 inhibitors. Justification of anticancer effects by means of in vitro VEGFR-2 inhibition
sets the origin towards the identification of possible mechanism for cytotoxicity at molecular
level. From the initial screening, it was inferred that among the synthesized compounds, 8
compounds i.e. 12d, 12e, 12k, 12l, 12p, 12q, 12t and 12u prominently suppressed VEGFR-2,
with IC values of 310 to 920 nM in contrast to the positive control, sorafenib 210 nM. The
5
0
enzymatic inhibition results also evidenced that all the synthesized compounds were found to
unveil substantial cytotoxicity against VEGFR-2 protein with IC50 0.31 ± 0.07 to 5.22 ± 0.42
1
6

µM. Later, in vitro antiproliferative effects of these derivatives were also evaluated against a
certain human cancer cell lines. The Structure Activity Relationship (SAR) studies pointed that,
substitution of cyclic secondary amines with 3-methyl-4-chloro group on aryl urea seems to be
the central aspect for affecting the VEGFR-2 activity of the proposed structural motifs. The
mechanism of cellular toxicity in MDAMB-231 was established through apoptosis induction by
depolarization of mitochondrial membrane potential, increased ROS production and subsequent
DNA damage. In addition, clonogenic and wound healing assays indicated the inhibition of
colony formation and cell migration by 12k in a dose-dependent manner. Angiogenesis
inhibition was evident by HUVECs tube formation assay and cell invasion by transwell assay.
Molecular modeling studies supported the possible binding mode of 8 potent molecules to the
catalytic cleft of VEGFR-2 protein. All the potent molecules exhibited pleasing ADMET
property results (obeys the Lipinski rule of 5). Inclusively, the newly synthesized
pyrimidylphenylurea derivatives can be developed as prospective anticancer agents through
VEGFR-2 inhibition and apt structural reforms may succeed in breeding promising anticancer
agents.
8
8
.0 Experimental section
.1 Chemistry
General Methods. All the reagents and solvents were obtained from commercial suppliers and
were used without further purification. Analytical thin layer chromatography (TLC) was
performed on MERCK pre-coated silica gel 60-F254 (0.5 mm) aluminum plates. Visualization of
1
13
the spots on TLC plates was achieved by UV light. H and C NMR spectra were recorded on
Bruker 500 MHz by making a solution of samples in the DMSO-d as solvent using tetramethyl
6
1
13
silane (TMS) as the internal standard. Chemical shifts for H and C are reported in parts per
million (ppm) downfield from tetra methyl silane. Spin multiplicities are described as s (singlet),
brs (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Coupling constant (J)
values are reported in hertz (Hz). HRMS were determined with Agilent QTOF mass
spectrometer 6540 series instrument. Wherever required, column chromatography was
performed using silica gel (60-120; 100-200). The reactions wherever anhydrous conditions
required are carried under nitrogen positive pressure using freshly distilled solvents. All
evaporation of solvents was carried out under reduced pressure using rotary evaporator below 45
1
7

o
C. Melting points were determined with an electro thermal digital melting point apparatus
IA9100 and are uncorrected. The names of all the compounds given in the experimental section
were taken from ChemBioDraw Ultra, Version 12.0.
4
8
.1.1 6-((1H-Indol-3-yl)thio)-N -substituted amino pyrimidine-2,4-diamine (10a-i)
To a mixture of amino derivatives of pyrimidine (9a–i, 1 equiv.), indole-3-thiol (3, 2.5
o
equiv.), potassium iodide (1.5 equiv.) in ethanol, under 80 C was added triethyl amine and
stirred under reflux till complete consumption of the starting materials as determined by TLC.
The solvent was then removed using rotary evaporator and extracted using ethyl acetate (25 mL
x 3) and water. The organic layer was concentrated under in vacuo and the residue obtained was
chromatographed on silica gel (elution with hexane/EtOAc = 7:3-5:5) to provide the 6-((1H-
4
indol-3-yl)thio)-N -substituted amino pyrimidine-2,4-diamine derivatives 10a-i in good yields.
8
.1.2 1-(4-((1H-Indol-3-yl)thio)-6-aminopyrimidin-2-yl)-3-arylurea (12a-aa)
To a mixture of 4-((1H-indol-3-yl)thio)-6-aminopyrimidin-2-amine (10a-i, 1 equiv.) in
o
dioxane, substituted aryl isocyanates (11a-f, 1 equiv.) were added under 100 C and stirred till
complete consumption of the starting materials as determined by TLC. The reaction mixture was
then quenched with water and extracted using ethyl acetate (25 mL x 3). The organic layer was
concentrated under in vacuo and the residue obtained was chromatographed on silica gel (elution
with hexane/EtOAc = 8:2-4:6) to provide the 1-(4-((1H-indol-3-yl)thio)-6-aminopyrimidin-2-yl)-
3
-arylurea 12a-aa in moderate to good yields.
8
.1.2.1 1-(4-((1H-Indol-3-yl)thio)-6-morpholinopyrimidin-2-yl)-3-phenylurea (12a)
o
-1
White solid; yield 83%; mp: 185-189 C; FT-IR (cm ): 3365, 3116, 2922, 2862, 1897,
1
1
7
710, 1370, 738; H NMR (500 MHz, DMSO-d ): δ 11.80 (s, 1H), 11.16 (s, 1H), 9.50 (s, 1H),
6
.84 (s, 1H), 7.49–7.44 (m, 2H), 7.24 (dd, J = 23.6, 16.0 Hz, 3H), 7.18 (d, J = 7.9 Hz, 1H), 7.10
1
3
(
dd, J = 13.4, 7.7 Hz, 3H), 5.92 (s, 1H), 3.57 (s, 8H); C NMR (125 MHz, DMSO-d
6
): δ 165.2,
56.3, 152.4, 146.1, 144.5, 134.5, 129.7, 129.2, 128.9, 128.0, 128.0, 127.2, 126.9, 126.8, 124.3,
19.2, 107.5, 102.0, 64.3, 60.3, 54.5, 14.3; HRMS (ESI): m/z calculated for C H N O S
1
1
4
2
3
23
6
2
+
47.1603 found 447.1628 [M+H] .
.1.2.2 1-(4-((1H-Indol-3-yl)thio)-6-morpholinopyrimidin-2-yl)-3-(3-methoxyphenyl)urea (12b)
8
1
8

o
-1
White solid; yield 86%; mp: 183-186 C; FT-IR (cm ): 3373, 3116, 2922, 2876, 1834,
1
1
1
1
3
1
715, 1693, 1487, 754; H NMR (500 MHz, DMSO-d ): δ 11.78 (s, 1H), 10.93 (s, 1H), 9.42 (s,
6
H), 7.83 (s, 1H), 7.47 (dd, J = 13.6, 5.4 Hz, 2H), 7.20 (d, J = 7.3 Hz, 1H), 7.11 (t, J = 7.2 Hz,
H), 6.94 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 5.97 (s, 1H), 3.73 (s, 3H), 3.59 (s, 4H),
1
3
.48 (s, 2H), 3.16 (s, 2H); C NMR (125 MHz, DMSO-d ): δ 169.8, 161.8, 157.1, 155.5, 152.1,
6
37.1, 133.1, 131.7, 122.8, 121.3, 120.8, 118.7, 114.3, 113.0, 96.5, 91.7, 66.0, 55.6, 44.2; HRMS
+
(
ESI): m/z calculated for C H N O S 477.1709 found 477.1724 [M+H] .
2
4
25
6
3
8
.1.2.3 1-(4-((1H-Indol-3-yl)thio)-6-morpholinopyrimidin-2-yl)-3-(3-chlorophenyl)urea (12c)
o
-1
Yellow solid; yield 85%; mp:178-182 C; FT-IR (cm ): 3368, 3119, 2927, 2868, 1839,
1
1
7
788, 1367, 758; H NMR (500 MHz, DMSO-d ): δ 11.80 (s, 1H), 11.19 (s, 1H), 9.59 (s, 1H),
6
.84 (s, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 7.9 Hz, 1H), 7.29 (d, J = 8.8 Hz, 2H), 7.18 (t,
J = 7.1 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 6.99 (d, J = 8.8 Hz, 2H), 6.03 (s, 1H), 3.60 (s, 4H), 3.41
1
3
(
s, 4H); C NMR (125 MHz, DMSO-d ): δ 169.4, 161.8, 157.0, 152.0, 137.7, 137.1, 133.0,
6
1
29.4, 128.9, 126.8, 122.8, 121.1, 120.8, 118.7, 113.0, 96.5, 92.0, 66.0, 44.2; HRMS (ESI): m/z
+
calculated for C H ClN O S 481.1213 found 481.1240 [M+H] .
2
3
22
6
2
8
.1.2.4 1-(4-((1H-Indol-3-yl)thio)-6-morpholinopyrimidin-2-yl)-3-(4-chloro-2-methylphenyl)urea
(12d)
o
-1
White solid; yield 76%; mp:183-185 C; FT-IR (cm ): 3372, 3112, 2927, 2856, 1839,
1
1
7
1
788, 1367, 748; H NMR (500 MHz, DMSO-d
6
): δ 11.79 (s, 1H), 11.22 (s, 1H), 9.61 (s, 1H),
.84 (d, J = 2.6 Hz, 1H), 7.57 (d, J = 1.9 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.44 (d, J = 7.8 Hz,
H), 7.20 (dd, J = 14.1, 7.7 Hz, 2H), 7.11 (t, J = 7.4 Hz, 1H), 6.56 (dd, J = 8.2, 1.8 Hz, 1H), 5.93
1
3
(
s, 1H), 3.62–3.55 (m, 4H), 3.38 (s, 4H), 2.27 (s, 3H); C NMR (125 MHz, DMSO-d ): δ 169.8,
6
1
1
61.5, 158.9, 157.0, 152.0, 148.0, 137.9, 133.5, 133.1, 131.5, 128.8, 122.8, 119.7, 118.3, 113.6,
6 2
13.0, 96.5, 91.8, 44.1, 43.5, 19.3; HRMS (ESI): m/z calculated for C24H24ClN O S 495.1370
+
found 495.1397 [M+H] .
8
.1.2.5 1-(4-((1H-Indol-3-yl)thio)-6-(piperidin-1-yl)pyrimidin-2-yl)-3-(2,5-dimethylphenyl)urea
(12e)
o
-1
White solid; yield 86%; mp:179-184 C; FT-IR (cm ): 3376, 3115, 2949, 2832, 1837, 1768,
1
1
347, 774; H NMR (500 MHz, DMSO-d ): δ 11.44 (s, 1H), 10.04 (s, 1H), 9.15 (s, 1H), 7.68 (s,
6
1
9

1
1
1
1
H), 7.37 (s, 1H), 7.05 (s, 3H), 6.96 (d, J = 7.0 Hz, 3H), 6.06 (s, 1H), 3.43 (s, 4H), 1.89 (s, 6H),
1
3
.58 (s, 2H), 1.43 (s, 4H); C NMR (125 MHz, DMSO-d ): δ 169.1, 161.3, 157.4, 152.7, 136.8,
6
35.6, 134.9, 132.8, 128.7, 127.8, 126.4, 122.4, 120.5, 118.2, 112.5, 96.5, 91.3, 45.0, 25.5, 24.4,
+
8.5, 18.3; HRMS (ESI): m/z calculated for C26
H
29
N
6
OS 473.2124 found 473.2163 [M+H] .
8
.1.2.6 1-(4-((1H-Indol-3-yl)thio)-6-(piperidin-1-yl)pyrimidin-2-yl)-3-(4-chlorophenyl)urea (12f)
o
-1
White solid; yield 89%; mp:189-192 C; FT-IR (cm ): 3372, 3118, 2949, 2822, 1827,
1
1
7
2
799, 1370, 747; H NMR (500 MHz, DMSO-d ): δ 11.79 (s, 1H), 11.32 (s, 1H), 9.53 (s, 1H),
6
.84 (d, J = 2.5 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.30 (d, J = 8.7 Hz,
H), 7.19 (t, J = 7.5 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 7.05 (d, J = 8.7 Hz, 2H), 5.93 (s, 1H), 3.37
1
3
(
d, J = 26.1 Hz, 4H), 1.56 (d, J = 4.2 Hz, 2H), 1.43 (s, 4H); C NMR (125 MHz, DMSO-d
6
): δ
69.3, 161.7, 159.0, 157.4, 148.0, 138.0, 136.8, 135.6, 135.0, 128.7, 127.8, 126.3, 122.4, 120.5,
18.2, 113.6, 107.6, 96.3, 44.4, 18.6, 18.4; HRMS (ESI): m/z calculated for C H ClN OS
1
1
4
24
24
6
+
79.1421 found 479.1452 [M+H] .
8
.1.2.7 1-(4-((1H-Indol-3-yl)thio)-6-(piperidin-1-yl)pyrimidin-2-yl)-3-(4-methoxyphenyl)urea
(12g)
o
-1
White solid; yield 87%; mp:187-190 C; FT-IR (cm ): 3366, 3111, 2922, 2853, 1822,
1
1
7
2
733, 1398, 762; H NMR (500 MHz, DMSO-d ): δ 11.77 (s, 1H), 11.06 (s, 1H), 9.34 (s, 1H),
6
.82 (s, 1H), 7.45–7.33 (m, 2H), 7.22–7.16 (m, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.01 (d, J = 9.0 Hz,
H), 6.85 (d, J = 15.4 Hz, 2H), 5.85 (s, 1H), 3.76–3.72 (m, 3H), 3.37 (s, 4H), 1.56 (s, 2H), 1.43
1
3
(
s, 4H); C NMR (125 MHz, DMSO-d
6
): δ 169.7, 161.1, 157.2, 155.5, 152.1, 137.1, 133.1,
1
31.8, 128.8, 122.7, 121.2, 120.7, 120.4, 118.7, 114.4, 113.0, 96.8, 91.4, 55.6, 45.1, 25.3, 24.3;
+
HRMS (ESI): m/z calculated for C H N O S 475.1916 found 475.1949 [M+H] .
2
5
27
6
2
8
.1.2.8 1-(4-((1H-Indol-3-yl)thio)-6-(piperidin-1-yl)pyrimidin-2-yl)-3-phenylurea (12h)
o
-1
Yellow solid; yield 85%; mp:190-196 C; FT-IR (cm ): 3369, 3119, 2982, 2843, 1838,
1
1
7
892, 1489, 742; H NMR (500 MHz, DMSO-d
6
): δ 11.81 (s, 1H), 11.29 (s, 1H), 9.46 (s, 1H),
.84 (s, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.27 (t, J = 7.9 Hz, 2H), 7.20 (t, J
=
7.2 Hz, 1H), 7.15 (d, J = 7.8 Hz, 2H), 7.11 (t, J = 7.4 Hz, 1H), 7.01 (t, J = 7.3 Hz, 1H), 5.82 (s,
1
3
1
1
H), 3.37 (s, 4H), 1.56 (s, 2H), 1.42 (s, 4H); C NMR (125 MHz, DMSO-d
6
): δ 169.7, 161.1,
57.2, 155.5, 152.1, 137.1, 133.1, 131.8, 128.8, 122.7, 121.2, 120.7, 120.4, 118.7, 114.4, 113.0,
2
0

9
4
25 6
6.8, 91.4, 55.6, 45.1, 25.3, 24.3; HRMS (ESI): m/z calculated for C24H N OS 445.1811 found
+
45.1835 [M+H] .
8
.1.2.9 1-(4-((1H-Indol-3-yl)thio)-6-(pyrrolidin-1-yl)pyrimidin-2-yl)-3-phenylurea (12i)
o
-1
White solid; yield 83%; mp:198-202 C; FT-IR (cm ): 3372, 3118, 2922, 2853, 1832,
1
1
7
793, 1389, 752; H NMR (500 MHz, DMSO-d ): δ 11.79 (s, 1H), 11.53 (s, 1H), 9.49 (s, 1H),
6
.85 (s, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 7.9 Hz, 2H), 7.30 (t,
J = 7.8 Hz, 2H), 7.22 (t, J = 7.5 Hz, 1H), 7.12 (t, J = 7.4 Hz, 1H), 7.03 (t, J = 7.3 Hz, 1H), 5.37
1
3
(
s, 1H), 3.51 (s, 2H), 2.80 (s, 2H), 1.83 (s, 4H); C NMR (125 MHz, DMSO-d ): δ 170.2, 159.1,
6
1
9
56.8, 152.2, 139.0, 137.1, 133.1, 129.3, 128.7, 123.2, 122.8, 120.7, 119.5, 118.7, 112.9, 96.9,
+
1.7, 46.6, 24.8; HRMS (ESI): C H N OS 431.1654 found 431.1683 [M+H] .
2
3
23
6
8
.1.2.10 1-(4-((1H-Indol-3-yl)thio)-6-(pyrrolidin-1-yl)pyrimidin-2-yl)-3-(4-methoxyphenyl)urea
(12j)
o
-1
White solid; yield 90%; mp:172-176 C; FT-IR (cm ): 3370, 3116, 2983, 2868, 1849,
1
1
7
765, 1368, 748; H NMR (500 MHz, DMSO-d
6
): δ 11.79 (s, 1H), 11.35 (s, 1H), 9.41 (s, 1H),
.82 (s, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.26 (d, J = 8.6 Hz, 2H), 7.22 (t,
J = 7.5 Hz, 1H), 7.12 (t, J = 7.3 Hz, 1H), 6.88 (d, J = 8.7 Hz, 2H), 5.38 (s, 1H), 3.73 (s, 3H), 3.50
1
3
(
s, 2H), 2.81 (s, 2H), 1.82 (s, 4H); C NMR (125 MHz, DMSO-d ): δ 170.2, 159.1, 156.9, 155.5,
6
1
2
52.2, 137.1, 133.1, 132.0, 128.7, 122.8, 121.2, 120.7, 118.7, 114.5, 112.9, 96.9, 91.6, 55.6, 46.0,
+
4.79; HRMS (ESI): m/z calculated for C24
H
25
N
6
O
2
S 461.1760 found 461.1794 [M+H] .
8
.1.2.11
1-(4-((1H-Indol-3-yl)thio)-6-(pyrrolidin-1-yl)pyrimidin-2-yl)-3-(4-chlorophenyl)urea
(12k)
o
-1
White solid; yield 89%; mp:170-175 C; FT-IR (cm ): 3378, 3119, 2973, 2853, 1839,
1
1
7
714, 1398, 748; H NMR (500 MHz, DMSO-d ): δ 11.79 (s, 1H), 11.53 (s, 1H), 9.49 (s, 1H),
6
.85 (s, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 7.9 Hz, 2H), 7.30 (t,
J = 7.8 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H), 7.12 (t, J = 7.4 Hz, 1H), 7.03 (t, J = 7.3 Hz, 1H), 5.37
1
3
(
s, 1H), 3.51 (s, 2H), 2.80 (s, 2H), 1.83 (s, 4H); C NMR (125 MHz, DMSO-d
6
): δ 168.8, 160.3,
56.3, 154.6, 151.3, 136.3, 132.2, 131.0, 127.9, 121.9, 120.3, 119.9, 119.5, 117.8, 113.5, 112.1,
5.9, 90.5, 44.2, 24.5; HRMS (ESI): m/z calculated for C H ClN OS 465.1264 found 465.1286
1
9
2
3
22
6
+
[
M+H] .
2
1

8
.1.2.12
1-(4-((1H-Iindol-3-yl)thio)-6-(pyrrolidin-1-yl)pyrimidin-2-yl)-3-(4-chloro-2-methyl
phenyl)urea (12l)
o
-1
Off-white solid, yield 85%; mp:185-189 C; FT-IR (cm ): 3348, 3012, 2972, 2853, 1864,
1
1
7
710, 1388, 753; H NMR (500 MHz, DMSO-d ): δ 11.37 (s, 1H), 9.97 (s, 1H), 9.08 (s, 1H),
6
.61 (s, 1H), 7.30 (s, 1H), 6.98 (s, 3H), 6.89 (d, J = 7.0 Hz, 3H), 5.99 (s, 1H), 3.37 (s, 4H), 2.18
1
3
(
s, 3H), 1.36 (s, 4H); C NMR (125 MHz, DMSO-d ): δ 167.4, 159.6, 155.7, 151.0, 135.1,
6
1
1
33.9, 133.2, 131.1, 127.0, 126.1, 124.7, 120.8, 118.8, 116.5, 110.8, 94.8, 89.7, 43.4, 23.8, 22.7,
+
6.8; HRMS (ESI): m/z calculated for C H ClN OS 479.1421 found 479.1452 [M+H] .
2
4
24
6
8
.1.2.13 1-(4-((1H-Indol-3-yl)thio)-6-(phenylamino)pyrimidin-2-yl)-3-phenylurea (12m)
o
-1
Yellow solid; yield 87%; mp: 193-197 C; FT-IR (cm ): 3358, 3212, 2976, 2876, 1862,
1
1
1
7
7
712, 1609, 1395, 755; H NMR (500 MHz, DMSO-d ): δ 11.91 (s,1H), 11.58 (s, 1H), 9.38 (s,
6
H), 8.94 (s, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 7.5 Hz, 1H), 7.77 (d, J = 7.0 Hz, 1H),
.72 (d, J = 8.1 Hz, 2H), 7.62 (s, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.44–7.40 (m, 2H), 7.34 (d, J =
13
.3 Hz, 2H), 7.27 (d, J = 6.4 Hz, 2H), 6.98 (t, J = 7.7 Hz, 1H), 6.90 (t, J = 7.4 Hz, 1H); C NMR
(
125 MHz, DMSO-d ): δ 169.8, 161.5, 158.9, 157.0, 152.0, 148.0, 137.9, 137.1, 133.5, 133.1,
6
1
31.5, 129.8, 128.8, 122.8, 120.8, 119.7, 118.7, 118.3, 113.6, 113.0, 107.5, 96.5, 91.8; HRMS
+
(ESI): m/z calculated for C25
H
21
N
6
OS 453.1498 found 453.1506 [M+H] .
8
.1.2.14
1-(4-((1H-Indol-3-yl)thio)-6-(4-(pyridin-2-yl)piperazin-1-yl)pyrimidin-2-yl)-3-
phenylurea (12n)
o
-1
Light brown solid; yield 76%; mp: 195-198 C; FT-IR (cm ): 3368, 3112, 2967, 2886,
1
1
9
7
1
878, 1705, 1619, 1385, 765; H NMR (500 MHz, DMSO-d ): δ 11.82 (s, 1H), 11.21 (s, 1H),
6
.53 (s, 1H), 8.11 (s, 1H), 7.85 (s, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.48 (dd, J = 15.8, 8.1 Hz, 2H),
.27 (t, J = 7.9 Hz, 2H), 7.21 (t, J = 7.6 Hz, 1H), 7.12 (t, J = 6.9 Hz, 3H), 7.02 (t, J = 7.3 Hz,
1
3
H), 6.82 (d, J = 8.6 Hz, 1H), 6.68–6.63 (m, 1H), 5.96 (s, 1H), 3.54 (s, 8H); C NMR (125
MHz, DMSO-d ): δ 169.9, 161.6, 159.0, 157.1, 152.0, 148.0, 138.8, 138.0, 137.2, 133.2, 129.2,
6
1
28.8, 123.2, 122.8, 120.8, 119.6, 118.7, 113.6, 113.0, 107.6, 96.5, 91.7, 44.2, 43.5; HRMS
+
(ESI): m/z calculated for C28
H
27
N
8
OS 523.2029 found 523.2052 [M+H] .
8
.1.2.15
1-(4-((1H-Indol-3-yl)thio)-6-(4-(pyridin-2-yl)piperazin-1-yl)pyrimidin-2-yl)-3-(4-
methoxyphenyl)urea (12o)
2
2

o
-1
White solid; yield 79%; mp: 188-189 C; FT-IR (cm ): 3370, 3113, 2967, 2881, 1896,
1
1
1
702, 1609, 1395, 755; H NMR (500 MHz, DMSO-d ): δ 11.80 (s, 1H), 11.18 (s, 1H), 9.48 (s,
6
H), 7.83 (s, 1H), 7.48–7.43 (m, 2H), 7.32 (dd, J = 14.4, 7.3 Hz, 4H), 7.26–7.22 (m, 2H), 7.18 (t,
J = 7.5 Hz, 1H), 7.11–7.06 (m, 2H), 7.00 (t, J = 7.3 Hz, 1H), 5.92 (s, 1H), 3.47 (s, 3H), 3.41 (s,
1
3
4
1
9
H), 2.34 (s, 4H); C NMR (125 MHz, DMSO-d ): δ 169.7, 161.5, 157.1, 152.0, 138.7, 138.0,
6
37.1, 133.0, 129.3, 129.2, 128.7, 128.7, 127.5, 123.3, 122.8, 120.8, 119.6, 118.7, 113.0, 96.6,
1.8, 62.2, 52.3 44.0; HRMS (ESI): m/z calculated for C H N O S 553.2134 found 553.2152
2
9
29
8
2
+
[
M+H] .
8
.1.2.16
1-(4-((1H-Indol-3-yl)thio)-6-(4-(pyridin-2-yl)piperazin-1-yl)pyrimidin-2-yl)-3-(4-
chloro-2-methylphenyl)urea (12p)
o
-1
Off-white solid; yield 73%; mp:187-191 C; FT-IR (cm ): 3368, 3212, 2931, 2611, 1869,
1
1
9
739, 1738, 1675, 1385, 795; H NMR (500 MHz, DMSO-d ): δ 11.80 (s, 1H), 11.26 (s, 1H),
6
.63 (s, 1H), 8.11 (d, J = 6.1 Hz, 1H), 7.85 (s, 1H), 7.59 (s, 1H), 7.53 (d, J = 6.9 Hz, 1H), 7.49
(d, J = 8.2 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.21 (dd, J = 14.9, 7.8 Hz, 2H), 7.11 (t, J = 7.8 Hz,
1
8
1
1
H), 6.81 (d, J = 8.6 Hz, 1H), 6.68–6.63 (m, 1H), 6.58 (d, J = 6.2 Hz, 1H), 5.96 (s, 1H), 3.54 (s,
1
3
6
H), 2.28 (s, 3H); C NMR (125 MHz, DMSO-d ): 169.8, 161.5, 158.9, 157.0, 152.0, 148.0,
37.9, 137.1, 133.5, 133.1, 131.5, 129.8, 128.8, 122.8, 120.8, 119.7, 118.7, 118.3, 113.6, 113.0,
07.5, 96.5, 91.8, 44.1, 43.5, 19.3; HRMS (ESI): m/z calculated for C H ClN OS 571.1795
2
9
28
8
+
found 571.1799 [M+H] .
8
.1.2.17
1-(4-((1H-Indol-3-yl)thio)-6-(4-benzylpiperazin-1-yl)pyrimidin-2-yl)-3-phenylurea
(12q)
o
-1
Off-white solid; yield 73%; mp:189-193 C; FT-IR (cm ): 3358, 3102, 2986, 2611, 1879,
1
1
1
7
3
1
1
563, 1384, 1175, 785; H NMR (500 MHz, DMSO-d ): δ 11.79 (s, 1H), 11.19 (s, 1H), 9.55 (s,
6
H), 8.89 (s, 1H), 7.83 (d, J = 2.1 Hz, 1H), 7.46 (dd, J = 15.9, 7.6 Hz, 3H), 7.34–7.27 (m, 6H),
.18 (t, J = 7.4 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H), 6.96 (d, J = 8.5 Hz, 2H), 6.03 (s, 1H), 3.61–
1
3
6
.33 (m, 6H), 2.35 (s, 4H); C NMR (125 MHz, DMSO-d ): δ 169.3, 161.1, 158.5, 156.5, 151.5,
47.5, 137.5, 136.7, 133.0, 132.6, 131.1, 129.3, 128.3, 122.3, 120.3, 119.2, 118.3, 117.8, 113.1,
12.6, 107.1, 96.0, 91.3, 43.7, 43.0; HRMS (ESI): m/z calculated for C H N OS 536.2233
30
30
7
+
found 536.2251 [M+H] .
2
3

8
.1.2.18
1-(4-((1H-Indol-3-yl)thio)-6-(4-benzylpiperazin-1-yl)pyrimidin-2-yl)-3-(3,5-
dimethylphenyl)urea (12r)
o
-1
Light brown solid; yield 78%; mp: 188-191 C; FT-IR (cm ): 3367, 3114, 2898, 2609,
1
1
9
859, 1563, 1394, 1275, 785; H NMR (500 MHz, DMSO-d ): δ 11.44 (s, 1H), 9.93 (s, 1H),
6
.28 (s, 1H), 8.12 (s, 1H), 7.69 (s, 1H), 7.55 (t, J = 6.9 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.04 (d,
J = 10.7 Hz, 2H), 7.02–6.90 (m, 5H), 6.85 (d, J = 8.6 Hz, 1H), 6.70–6.63 (m, 1H), 6.22 (s, 1H),
1
3
3
1
1
6
.58 (d, J = 33.6 Hz, 10H), 1.86 (s, 6H); C NMR (125 MHz, DMSO-d ): δ 169.8, 161.5, 158.9,
57.0, 152.0, 148.0, 137.9, 137.1, 133.5, 133.1, 131.5, 129.8, 128.8, 122.8, 120.8, 119.7, 118.7,
18.3, 113.6, 113.0, 107.6, 96.5, 91.8, 44.1, 43.5, 19.3; HRMS (ESI): m/z calculated for
+
C H N OS 564.2546 found 564.2556 [M+H] .
32
34
7
8
.1.2.19
1-(4-((1H-Indol-3-yl)thio)-6-(4-benzylpiperazin-1-yl)pyrimidin-2-yl)-3-(4-chloro-2-
methylphenyl)urea (12s)
o
-1
Light brown solid; yield 72%; mp: 183-187 C; FT-IR (cm ): 3368, 3112, 2920, 2651,
1
1
9
7
1
1
1
851, 1556, 1389, 1285, 760; H NMR (500 MHz, DMSO-d ): δ 11.78 (s, 1H), 11.23 (s, 1H),
6
.58 (s, 1H), 7.83 (s, 1H), 7.54 (s, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 7.9 Hz, 1H), 7.34–
.25 (m, 5H), 7.18 (t, J = 8.9 Hz, 2H), 7.10 (t, J = 7.4 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 5.91 (s,
1
3
6
H), 3.47 (s, 2H), 3.40 (s, 4H), 2.33 (s, 4H), 2.26 (s, 3H); C NMR (125 MHz, DMSO-d ): δ
69.7, 161.4, 157.0, 152.0, 138.1, 137.9, 137.1, 133.5, 133.0, 131.5, 129.8, 129.3, 128.7, 127.4,
22.8, 120.8, 119.6, 118.7, 118.2, 113.0, 96.5, 91.8, 62.2, 52.3, 43.9, 19.3; HRMS (ESI): m/z
+
calculated for C H ClN OS 583.1999 found 583.2011 [M+H] .
3
1
31
7
8
.1.2.20
1-(4-((1H-Indol-3-yl)thio)-6-(4-benzylpiperazin-1-yl)pyrimidin-2-yl)-3-(4-
methoxyphenyl)urea (12t)
o
-1
White solid; yield 88%; mp: 192-196 C; FT-IR (cm ): 3346, 3119, 2986, 1689, 1654,
1
1
9
527, 1203, 875; H NMR (500 MHz, DMSO-d ): δ 11.77 (d, J = 1.9 Hz, 1H), 10.91 (s, 1H),
6
.37 (s, 1H), 7.81 (d, J = 2.7 Hz, 1H), 7.44 (dd, J = 12.8, 8.0 Hz, 2H), 7.37–7.24 (m, 5H), 7.17
(dd, J = 11.0, 3.9 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 6.88 (dd, J = 23.0, 7.2 Hz, 2H), 6.81 (d, J =
1
3
9
.0 Hz, 2H), 5.97 (s, 1H), 3.72 (d, J = 5.4 Hz, 3H), 3.57–3.34 (m, 6H), 2.34 (s, 4H); C NMR
(
125 MHz, DMSO-d ): δ 169.7, 161.1, 157.2, 155.5, 154.8, 152.1, 137.1, 133.4, 133.1, 131.8,
6
2
4

1
28.8, 122.7, 121.2, 120.7, 120.4, 118.7, 114.4, 113.0, 96.8, 91.4, 66.8, 55.6, 45.1; HRMS (ESI):
+
m/z calculated for C H N O S 566.2338 found 566.2342 [M+H] .
3
1
32
7
2
8
.1.2.21
1-(4-((1H-Indol-3-yl)thio)-6-(cyclopropylamino)pyrimidin-2-yl)-3-(4-chloro-2-
methylphenyl)urea (12u)
o
-1
Off-white solid; yield 85%; mp: 190-195 C; FT-IR (cm ): 3369, 3285, 2981, 1695,
1
1
1
1
1
656, 1635, 1253, 831; H NMR (500 MHz, DMSO-d
6
): δ 12.61 (s, 1H), 12.31 (s, 1H), 10.43 (s,
H), 8.58 (d, J = 46.6 Hz, 3H), 8.29 (d, J = 27.4 Hz, 3H), 8.04 (s, 2H), 7.94 (s, 2H), 3.53 (m,
1
3
H), 3.08 (s, 3H), 1.50–0.84 (m, 4H); C NMR (125 MHz, DMSO-d ): δ 163.0, 156.9, 152.3,
6
38.3, 137.2, 133.5, 131.6, 129.8, 122.7, 120.8, 120.0, 118.7, 112.9, 19.3, 6.6; HRMS (ESI): m/z
+
calculated for C H ClN OS 465.1264 found 465.1282 [M+H] .
2
3
22
6
8
.1.2.22 1-(4-((1H-Indol-3-yl)thio)-6-(cyclopropylamino)pyrimidin-2-yl)-3-phenylurea (12v)
o
-1
Off-white solid; yield 78%; mp: 198-202 C; FT-IR (cm ): 3367, 3151, 2891, 1692,
1
1
1
1
0
1
675, 1643, 1425, 955; H NMR (500 MHz, DMSO-d ): δ 12.12 (s, 2H), 9.84 (s, 1H), 8.13 (s,
6
H), 7.93 (s, 1H), 7.80 (dd, J = 23.9, 7.7 Hz, 4H), 7.61 (t, J = 7.0 Hz, 2H), 7.53 (t, J = 7.1 Hz,
H), 7.45 (t, J = 7.0 Hz, 1H), 7.34 (t, J = 6.7 Hz, 1H), 5.80 (d, J = 145.1 Hz, 1H), 3.05 (s, 1H),
1
3
6
.96–0.43 (m, 4H); C NMR (125 MHz, DMSO-d ): δ 163.0, 157.0, 152.3, 139.0, 137.2, 129.1,
28.8, 123.3, 122.7, 120.8, 120.0, 112.9, 96.9, 14.5, 6.7; HRMS (ESI): m/z calculated for
+
C H N OS 417.1498 found 417.1521 [M+H] .
22
21
6
8
.1.2.23 1-(4-((1H-Indol-3-yl)thio)-6-(phenylamino)pyrimidin-2-yl)-3-(p-tolyl)urea (12w)
o
-1
White solid; yield 86%; mp: 196-199 C; FT-IR (cm ): 3368, 3112, 2889, 1698, 1675,
1
1
9
638, 1412, 890; H NMR (500 MHz, DMSO-d
6
): δ 11.87 (s, 1H), 11.44 (s, 1H), 9.90 (s, 1H),
.46 (s, 1H), 7.87 (s, 1H), 7.59–7.39 (m, 5H), 7.38–7.07 (m, 6H), 7.01 (t, J = 6.8 Hz, 1H), 6.90
13
(
s, 1H), 5.70 (s, 1H), 2.27 (s, 3H); C NMR (125 MHz, DMSO-d ): δ 160.3, 156.9, 152.1,
6
1
1
39.4, 138.1, 137.2, 133.5, 131.5, 129.8, 129.3, 128.8, 123.7, 122.9, 121.4, 121.0, 119.5, 118.7,
18.3, 113.0, 96.4, 19.3; HRMS (ESI): m/z calculated for C H N OS 467.1654 found 467.1663
2
6
23
6
8
.1.2.24
1-(4-((1H-Indol-3-yl)thio)-6-(phenylamino)pyrimidin-2-yl)-3-(4-chloro-2-
methylphenyl)urea (12x)
2
5

o
-1
White solid; yield 82%; mp: 198-205 C; FT-IR (cm ): 3366, 3207, 2926, 1696, 1685,
1
1
9
1
587, 1253, 875; H NMR (500 MHz, DMSO-d ): δ 11.78 (s, 1H), 11.35 (s, 1H), 9.81 (s, 1H),
6
.37 (s, 1H), 7.78 (s, 1H), 7.45 (t, J = 8.2 Hz, 3H), 7.35 (s, 2H), 7.19–7.12 (m, 2H), 7.11 (dd, J =
1
3
6.9, 8.0 Hz, 4H), 6.92 (t, J = 6.8 Hz, 1H), 5.61 (s, 1H), 2.19 (s, 3H); C NMR (125 MHz,
DMSO-d ): δ 159.5, 156.1, 151.3, 138.6, 137.3, 136.5, 132.8, 130.7, 129.0, 128.6, 128.1, 122.9,
6
1
22.1, 120.7, 120.2, 118.7, 117.9, 117.5, 112.2, 95.6, 18.5; HRMS (ESI): m/z calculated for
+
C H ClN OS 501.1264 found 501.1276 [M+H] .
26
22
6
8
.1.2.25
1-(4-((1H-Indol-3-yl)thio)-6-((3,5-dimethoxyphenyl)amino)pyrimidin-2-yl)-3-(3,5-
dimethoxyphenyl)urea (12y)
o
-1
White solid; yield 85%; mp: 199-202 C; FT-IR (cm ): 3274, 2967, 2881, 1732, 1702,
1
1
8
1
=
609, 1495, 755; H NMR (500 MHz, DMSO-d
6
): δ 11.78 (s, 1H), 10.93 (s, 1H), 9.42 (s, 1H),
.36 (s, 1H), 7.83 (s, 1H), 7.46 (d, J = 3.8 Hz, 1H), 7.35 (d, J = 8.5 Hz, 2H), 7.19 (t, J = 7.4 Hz,
H), 7.11 (t, J = 7.3 Hz, 1H), 6.94 (d, J = 8.5 Hz, 1H), 6.87–6.83 (m, 4H), 5.97 (s, 1H), 3.73 (d, J
1
3
7.3 Hz, 6H), 3.59 (s, 3H), 3.39 (s, 3H); C NMR (125 MHz, DMSO-d
6
): δ 169.7, 161.8, 157.1,
1
1
5
55.5, 154.7, 153.4, 152.1, 137.1, 133.4, 133.1, 131.7, 128.8, 122.8, 121.3, 120.8, 120.3, 118.7,
29 6 5
14.3, 113.0, 96.5, 91.6, 55.6; HRMS (ESI): m/z calculated for C29H N O S 573.1920 found
+
73.1936 [M+H] .
8
.1.2.26
1-(4-((1H-Indol-3-yl)thio)-6-((3,5-dimethoxyphenyl)amino)pyrimidin-2-yl)-3-(4-
methoxyphenyl)urea (12z)
o
-1
White solid; yield 79%; mp: 200-206 C; FT-IR (cm ): 3364, 3151, 2881, 1685, 1646,
1
1
8
7
1
1
1
527, 1418, 813; H NMR (500 MHz, DMSO-d
6
): δ 11.71 (s, 1H), 10.86 (s, 1H), 9.35 (s, 1H),
.30 (s, 1H), 7.76 (s, 1H), 7.39 (dd, J = 12.1, 8.2 Hz, 2H), 7.28 (d, J = 8.5 Hz, 3H), 7.13 (t, J =
.4 Hz, 1H), 7.04 (t, J = 7.3 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 6.78 (d, J = 7.3 Hz, 3H), 5.90 (s,
1
3
H), 3.66 (d, J = 7.3 Hz, 6H), 3.52 (s, 3H); C NMR (125 MHz, DMSO-d
6
): δ 168.7, 160.7,
56.0, 154.4, 153.7, 152.3, 151.0, 136.1, 132.3, 132.1, 130.6, 127.8, 121.7, 120.2, 119.7, 119.3,
27 6 4
17.6, 113.3, 111.9, 95.4, 90.6, 54.5; HRMS (ESI): m/z calculated for C28H N O S 543.1814
+
found 543.1826 [M+H] .
8
.1.2.27
1-(4-((1H-Indol-3-yl)thio)-6-((4-chloro-phenyl)amino)pyrimidin-2-yl)-3-phenylurea
(12aa)
2
6

o
-1
White solid; yield 76%; mp: 200-206 C; FT-IR (cm ): 3364, 3151, 2881, 1685, 1646,
1
1
1
2
1
1
1
527, 1418, 813; H NMR (500 MHz, DMSO-d ): δ 11.88 (d, J = 1.8 Hz, 1H), 11.42 (s, 1H),
6
0.04 (s, 1H), 9.55 (s, 1H), 7.94 (d, J = 4.7 Hz, 1H), 7.88 (d, J = 2.7 Hz, 1H), 7.54 (t, J = 7.9 Hz,
H), 7.48 (d, J = 8.1 Hz, 1H), 7.34–7.24 (m, 4H), 7.30–7.13 (m, 4H), 7.05 (dd, J = 8.5, 5.7 Hz,
1
3
H), 5.70 (s, 1H); C NMR (125 MHz, DMSO-d ): δ 169.8, 161.5, 158.9, 157.0, 152.0, 148.0,
6
37.9, 137.1, 133.5, 133.1, 131.5, 129.8, 128.8, 122.8, 120.8, 119.7, 118.7, 118.3, 113.6, 113.0,
07.5, 96.5, 91.8; HRMS (ESI): m/z calculated for C H ClN OS 487.1108 found 487.1116
2
5
20
6
+
[
M+H] .
8
.2 Pharmacology
8
.2.1 Cell culture
Prostate (PC-3), lung (A549), Breast (MDAMB-231) and Liver (HepG2) cells were
purchased from ATCC. PC-3 and A549 cells were grown in RPMI medium whereas MDAMB-
2
1
31, HepG2 were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and
% penicillin-streptomycin (PS). All the cell lines were grown in an incubator with 75%
humidity and 5% CO at 37 ºC. 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA, Life
2
Technologies) was used for harvesting the cells. For all the assays, stock solutions of the
compounds were prepared in DMSO (10 mM).
8
.2.2 Evaluation of in vitro cytotoxic effects
In this assay, Prostate (PC-3), lung (A549), Breast (MDA-MB-231) and Liver (HepG2)
cells were seeded in 96 well plates depending on their doubling time and were grown overnight.
The cells were exposed to different concentrations of carbamide derivatives of indole-pyrimidine
conjugates 12a-aa (100, 10, 1, 0.1 and 0.01 μM) for 72 h. Then, the medium containing
compounds was removed and replaced with 100 μL of MTT solution (5 mg/mL) and the cells
were further incubated for 4 h in dark at 37 ºC. The unreacted MTT solution was removed and
1
00 μL DMSO was added to each well to solubilize the produced formazan crystals. The
absorbance of the purple formazan solution was recorded using a plate reader (SpectraMax) at
70 nm and the IC50 values for each compound were calculated. All the experiments were
repeated three times and the standard deviations are reported in Table 1.
5
8
.2.3 In-Vitro VEGFR-2 inhibition assay
2
7

The VEGFR-2 tyrosine kinase activity of the compounds was performed according to
BPS Bioscience Corporation, San Diego, CA, USA (www.bpsbioscience.com) protocol, where
VEGFR-2 (KDR) (BPS#40301) served as the enzyme source and Poly (Glu, Tyr) sodium salt,
(
4:1, Glu:Tyr) (Sigma#P7244) served as the standardized substrate and Kinase Glo Plus
Luminescence kinase assay kit (Promega#V3772) [46]. In this assay, 5 μL of the compounds
2a-aa (1 nM, 10 nM, 100 nM, 1 μM, 10 μM) was added to a 45 μL of reaction mixture (40 mM
1
Tris, pH 7.4, 10 mM MgCl , 0.1 mg/mL BSA, 1 mM DTT, 10 μM ATP, Kinase substrate and
2
VEGFR-2) and incubated at 30 ºC for 45 min. After the enzymatic reaction, 50 μL of Kinase-Glo
Plus Luminescence kinase assay solution (Promega) was added to each reaction well and the
plate was further incubated for 15 min at room temperature in dark [47]. The luminescent signal
was measured using a microplate reader (SpectraMax). The intensity of the ATP luminescence is
inversely proportional to the amount of kinase activity. The assays were performed in triplicate
for each concentration and the IC values were determined from non-linear regression analysis
5
0
of the sigmoidal dose-response curve generated in Graph pad prism.
.2.4 Molecular docking
8
The molecular docking studies were performed at the AXI binding site of VEGFR-2
PDB ID: 3WZE). The coordinates of the crystal structure were obtained from RCSB-Protein
(
Data Bank and suitable corrections were made using Protein Preparation Wizard from the
Schrodinger package. Regarding the ligands, molecules were constructed using ChemBio3D
Ultra 12.0 and their geometries were optimized using molecular mechanics. Finally, docking
studies were performed according to the standard protocol implemented in maestro software,
version 9.9 on the most active molecules [48]. The ligand-protein complex was analyzed for
interactions and 3D pose of most active compound 12k was imaged using Schrödinger. In this
research, in order to further investigate the antiproliferative activity of target compounds against
VEGFR-2, the in vitro antiproliferative activity data of VEGFR-2 were chose to construct the
3
D-Quantitative Structure Activity Relationship (3D-QSAR) models. Table 6 listed the
structures of all target compounds and the IC values against VEGFR-2. Field and atom-based
5
0
analysis were performed using IC50 values for all target compounds against VEGFR-2. Then, the
IC50 values were converted to pIC50 values according to the following formula: pIC50 = Ig
1
/IC50. Construction of all target compounds, structural optimization, and 3D-QSAR modeling
were all performed on maestro software, version 9.9.
2
8