Indoline ureas as potential anti-hepatocellular carcinoma agents targeting VEGFR-2: Synthesis, in vitro biological evaluation and molecular docking

Article abstract of DOI:10.1016/j.ejmech.2015.05.040

In our effort to develop potent and effective agents with anti-proliferative activity towards HepG2 hepatocellular carcinoma cells with potential inhibitory activity against VEGFR-2, a novel series of 1-(4-((2-oxoindolin-3-ylidene)amino)phenyl)-3-arylurea

Full text of DOI:10.1016/j.ejmech.2015.05.040

Accepted Manuscript
Indoline ureas as potential anti-hepatocellular carcinoma agents targeting VEGFR-2:
Synthesis, in vitro biological evaluation and molecular docking
Wagdy M. Eldehna, Mohamed Fares, Hany S. Ibrahim, Mohamed H. Aly, Suher Zada,
Mamdouh M. Ali, Sahar M. Abou-Seri, Hatem A. Abdel-Aziz, Dalal A. Abou El Ella
PII:
S0223-5234(15)30067-2
10.1016/j.ejmech.2015.05.040
EJMECH 7919
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 January 2015
Revised Date: 23 May 2015
Accepted Date: 25 May 2015
Please cite this article as: W.M. Eldehna, M. Fares, H.S. Ibrahim, M.H. Aly, S. Zada, M.M. Ali, S.M.
Abou-Seri, H.A. Abdel-Aziz, D.A. Abou El Ella, Indoline ureas as potential anti-hepatocellular carcinoma
agents targeting VEGFR-2: Synthesis, in vitro biological evaluation and molecular docking, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.05.040.
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ACCEPTED MANUSCRIPT
Graphical abstract
Indoline ureas as potential anti-hepatocellular carcinoma agents targeting
VEGFR-2: Synthesis, in vitro biological evaluation and molecular docking
a
a
a
b,c
Wagdy M. Eldehna ,*, Mohamed Fares , Hany S. Ibrahim , Mohamed H. Aly , Suher
c
d
e
f,g
Zada , Mamdouh M. Ali , Sahar M. Abou-Seri , Hatem A. Abdel-Aziz *, Dalal A. Abou El
h
Ella
A novel series of indoline ureas 9a-x was designed and synthesized and evaluated for their anti-
proliferative activity against HepG2 cancer cell line and for their inhibitory activity against VEGFR-2.

ACCEPTED MANUSCRIPT
Indoline ureas as potential anti-hepatocellular carcinoma agents
targeting VEGFR-2: Synthesis, in vitro biological evaluation and
molecular docking
a
a
a
b,c
Wagdy M. Eldehna ,*, Mohamed Fares , Hany S. Ibrahim , Mohamed H. Aly ,
c
d
e
f,g
Suher Zada , Mamdouh M. Ali , Sahar M. Abou-Seri , Hatem A. Abdel-Aziz *,
h
Dalal A. Abou El Ella
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr
City, Cairo, P.O. Box 11829, Egypt
b
Department of Pharmacology and Toxicology, Faculty of Pharmacy, British University in Egypt,
Cairo, Egypt
c
Department of Biology, The American University in Cairo, P.O. Box 11835, New Cairo, Egypt
Biochemistry Department, Division of Genetic Engineering and Biotechnology, National Research
d
Centre, Dokki 12622, Giza, Egypt
e
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini
Street, Cairo, P.O. Box 11562, Egypt.
f
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box
2
457, Riyadh 11451, Saudi Arabia
Department of Applied Organic Chemistry, National Research Center, Dokki, Giza, P.O. Box 12622,
g
Egypt.
h
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Cairo,
Abbassia, P.O. Box 11566, Egypt.
Abstract
In our effort to develop potent and effective agents with anti-proliferative activity
towards HepG2 hepatocellular carcinoma cells with potential inhibitory activity
against VEGFR-2, a novel series of 1-(4-((2-oxoindolin-3-ylidene)amino)phenyl)-3-
arylureas was designed and synthesized. All the newly prepared ureas 9a-x were in
vitro evaluated for their anti-proliferative activity against HepG2 hepatocellular
carcinoma cell line. Compounds 9a-c, 9e, 9f, 9j, 9m-o, 9t-v and 9x exhibited good
activity against HepG2 cancer cells (IC = 1.22±0.11
̶
8.37±0.85 µM) comparable to
5
0
that of doxorubicin and sorafinib (IC = 2.90±0.36 and 3.40±0.25 µM, respectively).
5
0
These thirteen compounds were further evaluated for their inhibitory activity against
VEGFR-2. Compound 9x emerged as the most active counterpart against VEGFR-2
with IC value of 0.31±0.04 µM. Furthermore, a molecular docking of the tested
5
0
compounds was carried out in order to investigate their binding pattern with the
prospective target, VEGFR-2 (PDB-code: 4ASD).
_
_________
Keywords: Design; HepG2; Urea; VEGFR-2; Docking
1

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*
Corresponding author. Tel.: +2-02-2864-3342; fax: +2-02-2864-3332; e-mail:
wagdy2000@gmail.com
Corresponding author. Tel.: +966-146-73764; fax: +966-146-76220; e-mail:
hatem_741@yahoo.com
∗
1
-
Introduction
The term angiogenesis is generally applied to the process of sprouting or splitting
of novel capillary blood vessels from the pre-existing vasculature. This process
involves the migration, growth, and differentiation of endothelial cells, which line the
inside wall of the blood vessels [1, 2]. Normally, angiogenesis occurs during
embryogenesis, reproduction, wound healing and ovulation [3]. Angiogenesis is a
complex process, in both physiological and pathophysiological conditions, and it is
orchestrated via the production of a range of pro-angiogenic and anti-angiogenic
factors [4]. However, aberrant equilibrium between these factors that regulate the
angiogenesis is implicated in the etiology of different pathologies, such as rheumatoid
arthritis, age-related macular degeneration, atherosclerosis, diabetic retinopathy and
cancer [5]. Indeed, to grow beyond a size of 1−2 mm, tumors demand new blood
capillaries to guarantee their own oxygen and nutrient supply, remove metabolic
waste and facilitate metastasis formation [6]. These requirements can be achieved by
means of the expression of pro-angiogenic growth factors, including members of the
vascular endothelial growth factor (VEGF) family of ligands. The subtype VEGFR-2
exists predominately in vascular endothelial cells and hematopoietic stem cells and
appears to mediate almost all of the known cellular responses to VEGF, which
stimulate multiplication of vascular endothelial cells and growth of blood vessels [7].
The discovery of small molecule inhibitors that block the autophosphorylation of
VEGFR-2 has represented a major area of therapeutic intervention for the treatment of
different cancers [8].
Hepatocellular carcinoma (HCC), one of the vascular solid tumors, accounts for
7
0% - 85% of all malignant neoplasms of the liver burden worldwide [9]. HCC in
men is the fifth most frequently diagnosed cancer worldwide but the second-
commonest cause of cancer-related death. In women, it is the seventh most commonly
diagnosed cancer and the sixth leading cause of cancer death [9]. An estimated
7
82,000 new liver cancer cases and 746,000 cancer deaths occurred worldwide in
2

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2
012 [10]. Pro-angiogenic signaling pathways are rational therapeutic targets for this
disease. HCC is characterized by vascular recruitment as well as invasion and high
microvessel density predicts early recurrence after potentially curative surgery. The
VEGF signaling pathway in particular is involved in HCC angiogenesis and seems to
play a crucial role in disease pathogenesis [11]. In addition several HCC studies
correlated the high VEGF levels with the biological behavior of the disease and
clinical outcomes. Elevated VEGF levels have been detected in the serum and
cancerous tissue of patients with HCC [12-14]. While sorafenib is the only current
standard of care for HCC, the development of anti-angiogenic agents for HCC
treatment has been saddled with higher attrition rate due to narrow therapeutic
windows or suboptimal efficacy. Therefore, there is an urgent necessity to pay much
attention to update and modify drug leads from the point of view of medicinal
chemistry and drug design to fulfill more potent and effective therapies.
Figure 1
Sorafenib, 1 (BAY-439006) (Figure 1), trade name Nexavar, is a potent
diphenylurea VEGFR-2 inhibitor. Moreover, it inhibits c-Kit, PDGFRβ, and Raf
kinases [15]. It was approved by U.S. FDA for the treatment of advanced renal cell
carcinoma (RCC) and for HCC [16]. Regorafenib, 2 (BAY 73-4506) (Figure 1), a
fluoro derivative of sorafenib developed by Bayer [17], inhibits angiogenic kinases
VEGFR-1/3, PDGFRβ, FGFR1, and Tie-2. Furthermore, it showed anti-proliferative
activities on different cancer cell lines [18]. On September 27, 2012, the FDA
approved regorafenib for the previously treated metastatic colorectal cancer (mCRC)
and then in February 2013, FDA expanded the approved use of regorafenib to treat
patients with advanced gastrointestinal stromal tumors (GIST) [19]. A phase III study
is currently underway to evaluate the efficacy and safety of regorafenib in patients
with HCC whose disease has progressed after treatment with sorafenib [10].
Isatin (1H-indole-2,3-dione) is a privileged scaffold and one of the most
promising class of heterocycles that possesses diverse interesting activity profiles and
well-tolerated in humans [20-22]. Sunitinib, 3 (Figure 1), trade name Sutent, is a
small-molecule indol-2-one multikinase inhibitor active on VEGFR, PDGFR, FLT3
and c-Kit. In 2006, it was approved by the FDA for the treatment of advanced renal
3

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cell carcinoma RCC and GIST [23-25]. Moreover, the efficacy of sunitinib in patients
with advanced HCC was evaluated in four phase II studies [26-29].
Based on the previous findings, we have inspired to design and synthesize a novel
series of indolines incorporating a diphenylurea moiety, the main pharmacophoric
feature in sorafenib, with the prime aim of developing agents with potential anti-
proliferative activity towards HepG2 hepatocellular carcinoma cells targeting
VEGFR-2. Of note, many studies reported useful data about the binding pattern of
sorafenib with the active site of VEGFR-2 [30-32]. Utilizing these data; we will carry
out molecular docking to justify the inhibitory action of our target compounds
towards VEGFR-2.
2
2
-Results and Discussion
.1. Chemistry
The route adopted for the preparation of compounds 9a-x is depicted in Scheme 1.
Synthesis was initiated by reacting appropriate anilines 5a-h with 4-nitrophenyl
isocyanate 4 in a refluxing acetonitrile to furnish the 1-(4-nitrophenyl)-3-phenylureas
6
a-h. Next, reduction of the nitro derivatives 6a-h was achieved via hydrogenation
with Pd/C in methanol to afford the corresponding 1-(4-aminophenyl)-3-phenylurea
derivatives 7a-h. Finally, the condensation of amines 7a-h with indoline-2,3-diones
8
a-c in ethanol in the presence of a catalytic amount of glacial acetic acid furnished
the target ureas 9a-x.
Scheme 1
The IR spectra of the target compounds 9a-x revealed the presence of the two
characteristic carbonyl absorption bands of isatin and urea moieties around 1699-1749
-1
cm , in addition to absorption bands due to the NH groups of isatin and urea moieties
-1
around 3196-3333 cm . Also, the IR spectra of 9c, 9k and 9s showed the sharp
-1
absorption band of carbonitrile group in the region 2220-2227 cm . On the other
1
hand, the H NMR spectra of 9a-x showed three singlet D O-exchangeable signals
2
attributable to NH protons of the isatin and the urea function (NH-CO-NH) in the
1
regions δ 10.85-11.12, 8.30-9.13 and 8.67-9.29 ppm, respectively. Moreover, the H
NMR spectra of 9h, 9p and 9x revealed the D O exchangeable singlet signals of the
2
4

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1
sulfonamido group in the range δ 7.19-7.22 ppm. In addition, the H NMR spectra of
9
g, 9o and 9w exhibited the signals of the methoxy group protons in the range δ 3.72-
.73 ppm.
3
2
.2. Biological Evaluation
2
.2.1. In vitro anti-proliferative activity against HepG2
The in vitro anti-hepatocellular carcinoma activity of the newly prepared compounds
a-x was examined against HepG2 cell line using sulforhodamine B (SRB)
9
colorimetric assay as described by Skehan et al. [33]. Doxorubicin which is one of the
most effective anticancer agents and the lead drug sorafenib were included in the
experiment as positive control. The results were expressed as growth inhibitory
concentration (IC ) values, which represent the compounds concentrations required
5
0
to produce a 50% inhibition of cell growth after 72 hours of incubation, compared to
untreated controls (Table 1).
Table 1
From the obtained results, it was obvious that most of the prepared compounds
displayed excellent to modest anti-proliferative activity against HepG2 cell line. In
particular, compound 9n (IC = 1.81±0.14 µM) was found to be the most potent
5
0
counterpart as it was 1.6 times more active than doxorubicin (IC = 2.90±0.36 µM)
5
0
and 1.9 times than sorafenib (IC = 3.40±0.25 µM). Besides, compounds 9j, 9m, 9o
5
0
possessed excellent anti-proliferative activities against HepG2 cells (IC = 2.67±0.28,
5
0
2
.29±0.38 and 2.79±0.29 µM, respectively) which are better than the used reference
drugs. Several compounds showed anti-proliferative activity less than doxorubicin
and better than sorafenib as 9b, 9e-f, 9t, 9u and 9x with IC values ranging from
5
0
2
.96
̶ 3.20 µM. Also, compounds 9a, 9c and 9v displayed good activities against
HepG2 cell line with IC of 3.46±0.29, 7.73±0.23 and 8.37±0.85 µM, respectively.
5
0
On the other hand, compounds 9d, 9h, 9i, 9l, 9p, 9q, 9s and 9w were moderately
active with IC values ranging from 10.11-
̶
30.70 µM.
5
0
2
.2.2. In vitro VEGFR-2 kinase assay
5

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The most active anti-proliferative derivatives 9a-c, 9e-f, 9j, 9m-o, 9t-v and 9x were
selected to evaluate their inhibitory activities against VEGFR-2 by use of an anti-
phosphotyrosine antibody with the Alpha Screen system (PerkinElmer, USA). The
results were reported as a 50% inhibition concentration value (IC ) which determined
5
0
as triplicate determinations from the standard curve and summarized in Table 1.
Sorafenib was used as positive control in this assay. The tested compounds displayed
good to moderate inhibitory activity with IC values ranging from 0.31 to 5.61 µM.
5
0
Among them, compound 9x potently inhibited VEGFR-2 at low IC value (0.31±0.04
5
0
µM). Also, compounds 9u and 9v possessed sub-micromolar IC values (0.75±0.05
5
0
and 0.71±0.08 µM, respectively).
Observing the results, we could deduce valuable data about the structure activity
relationships. Firstly, we explored the effect of the substitution of the indoline moiety.
The decreased IC50 values of compounds 9b, 9e and 9f, with incorporated
unsubstituted indoline moiety, (4.26±0.31, 2.58±0.18 and 2.51±0.09, respectively)
than that of their corresponding members 9j, 9m and 9n, with 5-flouro substituted
indoline moiety, (2.12±0.33, 1.99±0.11 and 1.93±0.07, respectively) indicated that
substitution with halogens is advantageous. Moreover, introduction of chlorine atom
(more bulky than fluorine) at the 5-position resulted in remarkable increase of activity
against VEGFR-2, as for compounds 9u and 9v (IC = 0.75±0.05 and 0.71±0.08) in
5
0
comparison to 9m and 9n.
We then investigated the impact of the substitution of the pendant phenyl group
on the prepared indoline ureas activities. Concerning the activities of derivatives with
unsubstituted indoline moiety 9a-f, incorporation of unsubstituted phenyl group led to
compound 9a with the least inhibitory action on VEGFR-2 relative to the substituted
analogues. With regard to the effect of substitution in these members, the activities
were decreased in the order of 4-Cl > 3-Cl > 3-CF > 4-CN. Also, for the 5-flouro
3
substituted indoline counterparts, the activities were decreased in the order of 4-Cl >
3
-Cl > 3-CF > 4-OCH . Moreover, 4-cholrophenyl substituted derivative 9v, entailed
3 3
with 5-chloroindolines showed better activity than the 4-flouro analogue 9t and is
almost equipotent to the 3-chlorophenyl derivative 9u, indicating that grafting a large
electron withdrawing substituent, as chlorine, at the 4- or 3-positions is beneficial for
the activity. Interestingly, the introduction of 4-sulfonamido functionality afforded the
most potent member 9x with superior inhibitory activity (IC50 = 0.31±0.04 µM),
6

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highlighting the weight of sulfonamido group in the preferable binding of some kinase
inhibitors, as Pazopanib, to their biotargets [34].
3
. Molecular docking
We next analyzed the binding pattern between the tested compounds and VEGFR-
to understand how they can inhibit the activation of VEGFR-2 kinase and its
2
downstream signaling pathways. Sorafenib would be considered as a reference
compound in docking study as it is used as a reference in the biological activity.
Consequently, the distinctive binding pattern of sorafenib to VEGFR-2 kinase
active site would be discussed and compared to the tested compounds. The 3-chloro-
4
-trifluoromethyphenyl group shares as a hydrophobic moiety embedded in the
allosteric site. The phenyl group between urea and oxygen acts as hydrophobic moiety
and interacts with the hydrophobic site in the hinge region - the segment that connects
the amino- and carboxy-terminal kinase domains of the active site. On the urea linker,
there are two important hydrogen bonds with Glu885 and Asp1046, which they were
included in an extended channel located directly adjacent to the ATP binding site. N-
methylpicolinamide connects to Cys919 by two hydrogen bonds (supplementary
data). All these data were found as reported [30-32].
On the other hand, docking simulations of our target compounds revealed that the
urea linker in all compounds –including the most active compound 9x– could interact
with the two key amino acids Glu885 and Asp1046 with three hydrogen bonds in
addition to another hydrogen bond by NH of isatin with Glu 917. The previous note
beside their CDOCKER interaction scores (Table 2) – relative to sorafenib – gave us
precious results that justify their biological activity. The 2D diagrams for docking
poses are presented in the supplementary data.
Table 2
Considering the most active compound 9x, the presence of sulfonamido group
added an important connection by a hydrogen bond with Ile1025 (Figure 2). This pose
also confirmed by the docking poses of the analogous compounds containing
sulfonamide group as 9h and 9p (supplementary data). Moreover, docking score of
compound 9x was the highest score amongst all the synthesized compounds and near
to the score of sorafenib. These additional features in the docking pose of 9x represent
7

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the reason for its superiority over other synthesized derivatives in the biological
activity.
Figure 2
4
. Conclusion
In summary, we have designed and synthesized twenty four novel isatin-based
derivatives incorporating the substantial diphenylurea moiety and evaluated their anti-
proliferative activities against HepG2 cancer cell line. Of these derivatives, compound
9
n (IC = 1.81±0.14 µM) emerged as the most active member against HepG2, as it
50
was 1.6 times more active than doxorubicin (IC = 2.90±0.36 µM) and 1.9 times
5
0
more active than sorafenib (IC = 3.40±0.25 µM). Also, compounds 9a, 9b, 9e, 9f,
5
0
9
j, 9m, 9o, 9t-v and 9x displayed good activity against HepG2. The inhibitory
activities of the most active thirteen derivatives against VEGFR-2 were further
evaluated. Compound 9x was found to be the most active counterpart against
VEGFR-2 with IC50 value of 0.31±0.04 µM. Docking studies were performed to
justify the biological activities of the synthesized compounds. All of these compounds
could interact by three hydrogen bonds to Glu885 and Asp1046 via the urea linker
and with Glu 917 via NH of isatin group. Compound 9x, as the most active
compound, could interact by extra hydrogen bond with Ile1025 and it had the highest
energy score. Further structural optimization of the most active candidates is in
progress in our laboratories and will be reported in due course.
5
5
5
. Experimental
.1. Chemistry
.1.1. General
Melting points were measured with a Stuart apparatus and were uncorrected. IR
spectra were recorded on Schimadzu FT-IR 8400S spectrophotometer and expressed
-1
in wave number (cm ). The NMR spectra were recorded on Bruker
spectrophotometer at 400 MHz or Varian Gemini-300BB at 300 MHz (Varian Inc.,
1
13
Palo Alto, CA). All H NMR spectra were run at 400 MHz, while C NMR spectra
were run at 75 or 100 MHz in deuterated dimethylsulphoxide (DMSO-d ).Chemical
6
shifts (δ ) are reported relative to TMS as internal standard. All coupling constant (J)
H
8

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values are given in hertz. The abbreviations used are as follows: s, singlet; d, doublet;
m, multiplet. Mass spectra were measured on a GCMS-QP1000 EX spectrometer at
7
0 e.V. Analytical thin layer chromatography (TLC) on silica gel plates containing
UV indicator was employed routinely to follow the course of reactions and to check
the purity of products. All reagents and solvents were purified and dried by standard
techniques. Compounds 7a [35], 7b [36], 7e [37], 7f [38], 7g [38] are previously
reported.
5
.1.2. 1-(4-Nitrophenyl)-3-phenyl/substituted phenylurea (6a-h).
Compounds 6a-h were prepared according to the literature procedure [37].
5
5
.1.3. 1-(4-Aminophenyl)-3-phenyl/substituted phenylurea (7a-h).
Compounds 7a-h were prepared according to the literature procedure [36].
.1.3.1. 1-(4-Cyanophenyl)-3-(4-aminophenyl)urea (7c). White crystals (yield 67%);
-1
m.p. 217-219 °C (decompose); IR (KBr, ν cm ): 3448, 3356, 3311 (NH, NH ), 1639
2
1
(
C=O); H NMR (DMSO-d )
δ
ppm: 4.76 (s, 2H, D O exchangable, -NH ), 6.50 (d,
6
2
2
J= 9.0 Hz, 2H, Ar-H ), 7.04 (d, J= 9.0 Hz, 2H, Ar-H), 7.56 – 7.61 (m, 4H, Ar-H),
13
8
.24 (s, 1H, D O exchangable, -NH), 8.94 (s, 1H, D O exchangable, -NH); C NMR
2
2
(
DMSO-d , 75 MHz)
δ
(ppm): 114.02, 119.51, 119.73, 120.51, 120.72, 128.73,
6
1
33.19, 143.84, 153.08, 154.09.
5
.1.3.2. 1-(4-Aminophenyl)-3-(4-fluorophenyl)urea (7d). White crystals (yield 78 %),
-1
1
m.p. 273-275 °C. IR (KBr, ν cm ): 3481, 3379, 3305 (NH, NH ), 1637 (C=O); H
2
NMR (DMSO-d6)
δ (ppm): 4.68 (s, 2H, D O exchangeable, -NH ), 6.51 (d, J = 8.7
2 2
Hz, 2H, Ar-H), 6.96 (m, 4H, Ar-H), 7.40 (d, 2H, J = 4.8, Ar-H), 8.04 (s, 1H, D O
2
1
3
exchangeable, -NH), 8.43 (s, 1H, D O exchangeable, -NH); C NMR (DMSO-d , 75
2
6
2
3
MHz)
δ (ppm): 114.03, 115.02 ( J = 21.3 Hz), 119.54 ( J = 7.56 Hz), 120.73,
F-C F-C
1
1
28.43, 136.49, 144.04, 152.95, 155.43 ( JF-C = 236.4 Hz).
5
.1.3.3. 4-(3-(4-Aminophenyl)ureido)benzenesulfonamide (7h). White crystals (yield
-1
8
3 %), m.p. > 300 °C. IR (KBr, ν cm ): 3452, 3371, 3313 (NH, NH ), 1635 (C=O);
2
1
H NMR (DMSO-d6)
δ
(ppm): 6.53 (d, J = 8.7 Hz, 2H, Ar-H), 7.08 (d, J = 8.7 Hz,
2
7
H, Ar-H), 7.14 (s, 2H, D O exchangeable, -SO NH ), 7.55 (d, J = 8.7 Hz, 2H, Ar-H),
2
2
2
.68 (d, J = 8.7 Hz, 2H, Ar-H), 8.26 (s, 1H, D O exchangeable, -NH), 8.86 (s, 1H,
2
9

ACCEPTED MANUSCRIPT
1
3
D O exchangeable, -NH); C NMR (DMSO-d , 75 MHz)
δ (ppm): 117.61, 117.80,
2
6
1
25.08, 126.78, 137.51, 141.25, 142.09, 145.93, 151.76.
5
.1.4. General procedure for preparation of target compounds (9a-x)
To a stirred solution of 1-(4-aminophenyl)-3-phenyl/substituted phenylurea 7a-h
(0.002 mol), in absolute ethyl alcohol, a solution of the appropriate indoline-2,3-dione
8
a-c (0.002 mol) in absolute ethyl alcohol and catalytic amount of glacial acetic acid
were added. The mixture was heated under reflux for 3 h, filtered while hot and the
precipitate was washed with ethanol. The solid product was collected and crystallized
from an ethanol/DMF mixture (4:1).
5
.1.4.1. (Z/E)1-(4-((2-Oxoindolin-3-ylidene)amino)phenyl)-3-phenylurea (9a). Yellow
-1
powder (yield 78 %), m.p. 291-293 °C. IR (KBr, ν cm ): 3277 (NH), 1739, 1722
1
(
C=O); H NMR (DMSO-d )
δ
(ppm): 6.70 (d, J = 7.6 Hz, 1H, Ar-H), 6.77 (t, J = 7.6
6
Hz, 1H, Ar-H), 6.90 (d, J = 8.0 Hz, 1H, Ar-H), 6.98 (d, J = 6.8 Hz, 2H, Ar-H), 7.27-
7
8
.44 (m, 4H, Ar-H), 7.47 (d, J = 8.6 Hz, 2H, Ar-H), 7.56 (d, J = 8.6 Hz, 2H, Ar-H),
.73 and 8.75 (s, 1H, D O exchangeable, NH urea), 8.78 and 8.84 (s, 1H, D O
2
2
13
exchangeable, NH urea), 10.92 and 10.95 (s, 1H, D O exchangeable, NH isatin); C
2
NMR (DMSO-d , 75 MHz) δ: 111.33, 115.76, 118.08, 119.09, 121.71, 125.06,
6
1
1
28.62, 134.08, 137.33, 139.64, 144.15, 145.03, 146.77, 152.44, 154.41, 158.61,
+
63.61; MS m/z [%] 356 [M , 33], 282 [100].
5
.1.4.2.
(Z/E)1-(4-((2-Oxoindolin-3-ylidene)amino)phenyl)-3-(3-
(trifluoromethyl)phenyl) urea (9b). Yellow powder (yield 83 %), m.p. 287-290 °C. IR
-1
1
(
KBr, ν cm ): 3277 (NH), 1739, 1724 (C=O); H NMR (DMSO-d ) δ (ppm): 6.68 (d,
6
J = 7.2 Hz, 1H, Ar-H), 6.76 (t, J = 8.4 Hz, 1H, Ar-H), 6.90 (d, J = 8.0 Hz, 1H, Ar-H),
.98 (d, J = 8.8 Hz, 2H, Ar-H), 7.31-7.55 (m, 4H, Ar-H), 7.57 (d, J = 8.8 Hz, 2H, Ar-
H), 8.04 (s, 1 H, Ar-H), 8.85 and 8.90 (s, 1H, D O exchangeable, NH urea), 9.04 and
6
2
9
.08 (s, 1H, D O exchangeable, NH urea), 10.92 and 10.95 (s, 1H, D O exchangeable,
2
2
1
3
NH isatin); C NMR (DMSO-d , 75 MHz) δ: 111.00, 114.12, 115.74, 118.53,
6
1
1
18.71, 119.35, 119.52, 121.73, 125.01, 126.00, 129.28, 129.69, 134.31, 136.87,
+
40.54, 144.52, 146.80, 152.52, 154.52, 163.58; MS m/z [%]:424 [M , 0.1], 105
[
100].
1
0

ACCEPTED MANUSCRIPT
5
.1.4.3.
(Z/E)1-(4-Cyanophenyl)-3-(4-((2-oxoindolin-3-ylidene)amino)phenyl)urea
-1
(
9c). Red powder (yield 69 %), m.p. > 300 °C. IR (KBr, ν cm ): 3354 (NH), 2227
1
(
CN), 1728, 1705 (C=O); H NMR (DMSO-d ) δ (ppm): 6.67 (d, J = 7.6 Hz, 1H, Ar-
6
H), 6.90 (d, J = 7.6 Hz, 1H, Ar-H), 6.99 (d, J = 8.8 Hz, 2H, Ar-H), 7.34-7.76 (m, 6H,
Ar-H), 7.80 (d, J = 8.8 Hz, 2H, Ar-H), 8.88 and 8.99 (s, 1H, D O exchangeable, NH
2
urea), 9.15 and 9.25 (s, 1H, D O exchangeable, NH urea), 10.86 and 10.97 (s, 1H,
2
1
3
D O exchangeable, NH isatin), C NMR (DMSO-d , 100 MHz) δ: 111.95, 117.42,
2
6
1
1
17.56, 118.53, 119.16, 119.61, 119.73, 119.96, 122.21, 128.98, 133.78, 134.78,
+
37.16, 147.31, 152.61, 152.84, 163.11; MS m/z [%]: 382 [M , 2.1], 184 [100].
5
.1.4.4.
(Z/E)1-(4-Fluorophenyl)-3-(4-((2-oxoindolin-3-ylidene)amino)phenyl)urea
-1
(
9d). Orange powder (yield 80 %), m.p. > 300 °C. IR (KBr, ν cm ): 3277 (NH), 1728
1
(
C=O); H NMR (DMSO-d )
δ (ppm): 6.69 (d, J = 7.6 Hz, 1H, Ar-H), 6.76 (t, J = 7.6
6
Hz, 1H, Ar-H), 6.90 (d, J = 8.0 Hz, 1H, Ar-H), 6.97 (d, J = 8.8 Hz, 2H, Ar-H), 7.11 (t,
J = 9.0 Hz, 1H, Ar-H), 7.33-7.50 (m, 4H, Ar-H), 7.55 (d, J = 8.8 Hz, 2H, Ar-H), 8.72
and 8.30 (s, 1H, D O exchangeable, NH urea), 8.78 (s, 1H, D O exchangeable, NH
2
2
1
3
urea), 10.85 and 10.94 (s, 1H, D O exchangeable, NH isatin); C NMR (DMSO-d ,
2
6
2
3
7
1
5 MHz) δ: 111.32, 114.98, 115.15 ( J = 24.5 Hz), 118.58 ( J = 13.7 Hz),
F-C F-C
1
19.15, 119.83, 120.09, 121.58, 125.06, 134.08, 134.26, 135.94, 137.27, 144.18 ( JF-C
+
=
193.5 Hz), 152.62, 154.43, 163.59; MS m/z [%]: 374 [M , 100].
5
.1.4.5.
(Z/E)1-(3-Chlorophenyl)-3-(4-((2-oxoindolin-3-ylidene)amino)phenyl)urea
-1
(
9e). Yellow powder (yield 79 %), m.p. > 300 °C. IR (KBr, ν cm ): 3275 (NH), 1739,
1
1
724 (C=O); H NMR (DMSO-d )
δ
(ppm): 6.67 (d, J = 7.6 Hz, 1H, Ar-H), 6.76 (t, J
6
=
7.6 Hz, 1H, Ar-H), 6.90 (d, J = 7.6 Hz, 1H, Ar-H), 6.98 (d, J = 6.6 Hz, 2H, Ar-H),
.01-7.43 (m, 4H, Ar-H), 7.55 (d, J = 6.6 Hz, 2H, Ar-H), 7.73 and 7.31 (s, 1 H, Ar-
H), 8.85 and 8.87 (s, 1H, D O exchangeable, NH urea), 8.93 (s, 1H, D O
7
2
2
1
3
exchangeable, NH urea), 10.85 and 10.96 (s, 1H, D O exchangeable, NH isatin), C
2
NMR (DMSO-d , 100 MHz) δ: 111.97, 116.26, 117.01, 117.93, 118.07, 119.17,
6
1
1
21.95, 122.14, 122.88, 125.52, 130.88, 133.68, 137.46, 141.73, 144.95, 147.30,
+
+
52.91, 155.03, 164.12; MS m/z [%]: 392 [(M+2) , 19],390 [M , 43.3], 215 [100].
1
1

ACCEPTED MANUSCRIPT
5
.1.4.6.
(Z/E)1-(4-Chlorophenyl)-3-(4-((2-oxoindolin-3-ylidene)amino)phenyl)urea
-1
(
9f). Orange powder (yield 90 %), m.p. > 300 °C. IR (KBr, ν cm ): 3275 (NH), 1728
1
(
C=O); H NMR (DMSO-d )
δ (ppm): 6.69 (d, J = 7.6 Hz, 1H, Ar-H), 6.78 (t, J = 7.6
6
Hz, 1H, Ar-H), 6.90 (d, J = 7.6 Hz, 1H, Ar-H), 6.97 (d, J = 8.8 Hz, 2H, Ar-H), 7.33-
7
.47 (m, 3H, Ar-H), 7.49 (d, J = 8.8 Hz, 2H, Ar-H), 7.55 (d, J = 8.8 Hz, 2H, Ar-H),
8
.30 (s, 1H, D O exchangeable, NH urea), 8.86 (s, 1H, D O exchangeable, NH urea),
2
2
13
1
0.85 and 10.96 (s, 1H, D O exchangeable, NH isatin); C NMR (DMSO-d , 100
2 6
MHz) δ: 111.96, 116.26, 118.48, 119.19, 119.69, 120.24, 122.19, 125.51, 125.84,
1
29.11, 134.74, 137.61, 139.17, 144.84, 147.29, 152.96, 154.99, 164.12. ; MS m/z
+
+
[
%]: 392 [(M+2) , 8.8], 390 [M , 11.7], 111 [100].
5
.1.4.7. (Z/E)1-(4-Methoxyphenyl)-3-(4-((2-oxoindolin-3-ylidene)amino)phenyl)urea
-1
(
9g). Yellow powder (yield 71 %), m.p. > 300 °C. IR (KBr, ν cm ): 3273 (NH), 1739,
1
1
1
8
8
720 (C=O); H NMR (DMSO-d )
δ
(ppm): 3.73 (s, 3 H, OCH ), 6.70 (d, J = 8.0 Hz,
6
3
H, Ar-H), 6.76 (t, J = 7.6 Hz, 1H, Ar-H), 6.87 (d, J = 9.2 Hz, 2H, Ar-H), 6.96 (d, J =
.8 Hz, 2H, Ar-H), 7.34-7.39 (m, 4H, Ar-H), 7.54 (d, J = 8.8 Hz, 2H, Ar-H), 8.49 and
.50 (s, 1H, D O exchangeable, NH urea), 8.67 and 8.70 (s, 1H, D O exchangeable,
2
2
1
3
NH urea), 10.85 and 10.94 (s, 1H, D O exchangeable, NH isatin); C NMR (DMSO-
2
d6, 75 MHz)
δ: 63.21, 115.73, 116.98, 118.07, 119.25, 119.52, 121.67, 128.52,
1
33.28, 133.96, 136.64, 142.58, 144.16, 146.81, 152.09, 154.56, 163.59, 167.42; MS
+
m/z [%]: 386 [M , 41.2], 275 [100].
5
.1.4.8.
(Z/E)4-(3-(4-((2-Oxoindolin-3-
ylidene)amino)phenyl)ureido)benzenesulfonamide (9h). Yellow powder (yield 88 %),
-1
m.p > 300 °C. IR (KBr, ν cm ): 3333, 3275, 3197 (NH, NH ), 1722, 1703 (C=O)
2
1
1
312, 1152 (SO ); H NMR (DMSO-d )
δ
(ppm): 6.67 (d, J = 7.6 Hz, 1H, Ar-H), 6.71
2
6
(t, J = 7.6 Hz, 1H, Ar-H), 6.90 (d, J = 7.6 Hz, 1H, Ar-H), 6.99 (d, J = 8.4 Hz, 2H, Ar-
H), 7.22 (s, 2H, D O exchangeable, - SO NH ), 7.38 (t, J = 8.2 Hz, 1H, Ar-H), 7.57
2
2
2
(d, J = 8.4 Hz, 2H, Ar-H), 7.63 (d, J = 8.8 Hz, 2H, Ar-H), 7.74 (d, J = 8.8 Hz, 2H, Ar-
H), 8.87 and 8.92 (s, 1H, D O exchangeable, NH urea), 9.11(s, 1H, D O
2
2
exchangeable, NH urea), 10.86 and 10.96 (s, 1H, D O exchangeable, NH isatin), );
2
1
3
C NMR (DMSO-d , 75 MHz) δ: 111.35, 115.74, 117.35, 118.55, 119.24, 121.66,
6
1
2

ACCEPTED MANUSCRIPT
1
1
25.06, 126.77, 134.15, 134.31, 136.84, 142.78, 144.54, 146.79, 152.26, 154.54,
+
+
63.58; MS m/z [%]: 436 [(M+1) , 28.9], 435 [M , 100].
5
.1.4.9.
(Z/E)1-(4-((5-Fluoro-2-oxoindolin-3-ylidene)amino)phenyl)-3-phenylurea
-1
(
9i). Orange powder (yield 71 %), m.p. > 300 °C. IR (KBr, ν cm ): 3283 (NH), 1740,
1
1
724 (C=O); H NMR (DMSO-d )
δ
(ppm): 6.38 (dd, J = 6Hz, 3Hz, 1H, Ar-H), 6.91
6
(dd, J = 4.4Hz, 4Hz, 1H, Ar-H), 6.97-7.02 (m, 2H, Ar-H), 7.21-7.49 (m, 6H, Ar-H),
7
.58 (dd, J = 4.8 Hz, 2.4 Hz, 2H, Ar-H), 8.71 and 8.73 (s, 1H, D O exchangeable, NH
2
urea), 8.80 and 8.84 (s, 1H, D O exchangeable, NH urea), 10.88 (s, 1H, D O
2
2
1
3
3
exchangeable, NH isatin); C NMR (DMSO-d , 75 MHz)
δ
: 111.50, 116.04 ( J
=
6
F-C
2
8
1
2
.0 Hz), 118.10, 118.32 ( J = 31.9 Hz), 119.12, 121.74 ,121.92, 128.63, 137.71,
F-C
2
1
38.19, 139.60, 143.16 ( J = 31.1 Hz), 152.40, 152.51, 153.91, 155.15 ( J
=
F-C
F-C
+
36.3 Hz), 163.64; MS m/z [%]: 374 [M , 18.6], 194 [100].
5
.1.4.10.
(Z/E)1-(4-((5-Fluoro-2-oxoindolin-3-ylidene)amino)phenyl)-3-(3-
(trifluoromethyl) phenyl) urea (9j). Orange powder (yield 78 %), m.p. 289-291 °C. IR
-1
1
(
KBr, ν cm ) 3275 (NH), 1736 (C=O); H NMR (DMSO-d ) δ (ppm): 6.39 (m, 1H,
6
Ar-H), 6.91 (m, 1H, Ar-H), 7.01 (d, J = 8.8 Hz, 2H, Ar-H), 7.20-7.53 (m, 4H, Ar-H),
.60 (d, J = 8.8 Hz, 2H, Ar-H), 8.05 (s, 1H, Ar-H), 9.13 (s, 1H, D O exchangeable,
7
2
NH urea), 9.29 (s, 1H, D O exchangeable, NH urea), 10.98 and 10.99 (s, 1H, D O
2
2
+
exchangeable, NH isatin); MS m/z [%]: 424 [M , 12.5], 107 [100].
5
.1.4.15.
(Z/E)1-(4-Cyanophenyl)-3-(4-((5-fluoro-2-oxoindolin-3-
ylidene)amino)phenyl)urea (9k). Red powder (yield 70 %), m.p. > 300 °C. IR (KBr, ν
-1
1
cm ) 3356 (NH), 2220 (CN), 1724, 1701 (C=O); H NMR (DMSO-d )
δ
(ppm): 6.36
s, 1H, Ar-H), 7.01 (d, J = 8.4 Hz, 1H, Ar-H), 7.52 (d, J = 8.8 Hz, 2H, Ar-H), 7.59 (d,
J = 8.8 Hz, 2H, Ar-H), 7.65 (d, J = 8.8 Hz, 2H, Ar-H), 7.73 (d, J = 8.8 Hz, 2H, Ar-H),
6
(
7
9
.82 (d, J = 8.4 Hz, 1H, Ar-H), 8.93 and 8.99 (s, 1H, D O exchangeable, NH urea),
2
.26 (s, 1H, D O exchangeable, NH urea), 11.01 (s, 1H, D O exchangeable, NH
2
2
1
3
2
isatin); C NMR (DMSO-d , 100 MHz)
δ
: 103.75, 111.97 ( J = 21.3 Hz), 113.03,
6
F-C
3
3
2
1
16.55 ( J = 8.0 Hz), 117.59, 118.42 ( J = 9.0 Hz), 118.55, 119.75 ( J = 24.0
F-C
F-C
F-C
1
Hz), 121.07, 127.86, 129.00, 133.77, 137.55, 142.92, 143.70, 152.61, 154.58 ( JF-C
=
+
2
36.0 Hz), 167.96; MS m/z [%]: 399 [M , 41.4], 312 [100].
1
3

ACCEPTED MANUSCRIPT
5
.1.4.12.
(Z/E)1-(4-((5-Fluoro-2-oxoindolin-3-ylidene)amino)phenyl)-3-(4-
-1
fluorophenyl)urea (9l). Orange powder (yield 82 %), m.p. > 300 °C. IR (KBr, ν cm )
1
3
2
7
277 (NH), 1738, 1724 (C=O); H NMR (DMSO-d )
δ
(ppm) 6.37 (dd, J = 5.6 Hz,
6
.8 Hz, 1H, Ar-H), 6.90-6.94 (m, 1H, Ar-H), 7.00 (d, J = 8.8 Hz, 2H, Ar-H), 7.11-
.50 (m, 5H, Ar-H), 7.57 (d, J = 8.8 Hz, 2H, Ar-H), 8.73 and 8.75 (s, 1H, D O
2
exchangeable, NH urea), 8.77 and 8.81 (s, 1H, D O exchangeable, NH urea), 10.86
2
13
and 11.00 (s, 1H, D O exchangeable, NH isatin); C NMR (DMSO-d , 75 MHz) δ:
2
6
3
3
1
1
1
1
11.73, 115.01 ( J = 11.3 Hz), 115.49, 116.12, 118.75, 118.96 ( J = 11.9 Hz),
F-C F-C
2
19.82, 122.00, 134.00, 135.91, 137.66, 138.11, 143.14 ( JF-C = 32.1 Hz), 152.60,
1
+
+
55.13, 155.71 ( J = 237.0 Hz), 163.64; MS m/z [%]: 393 [(M+1) , 28.3], 392 [M ,
F-C
00].
5
.1.4.13.
(Z/E)1-(3-Chlorophenyl)-3-(4-((5-fluoro-2-oxoindolin-3-
ylidene)amino)phenyl)urea (9m). Orange powder (yield 85 %), m.p. > 300 °C. IR
-1
1
(
KBr, ν cm ) 3273 (NH), 1738, 1721 (C=O); H NMR (DMSO-d )
δ
(ppm): 6.40 (m,
6
1
H, Ar-H), 6.93 (m, 1H, Ar-H), 7.01 (d, J = 8.6 Hz, 2H, Ar-H), 7.25-7.50 (m, 4H, Ar-
H), 7.58 (d, J = 8.6 Hz, 2H, Ar-H), 7.73 (s, 1H, Ar-H), 8.90 (s, 1H, D O
2
exchangeable, NH urea), 8.93 (s, 1H, D O exchangeable, NH urea), 10.99 (s, 1H, D O
2
2
13
exchangeable, NH isatin); C NMR (DMSO-d , 75 MHz)
δ
: 112.00, 113.00, 117.67,
6
1
1
18.69, 119.23, 119.41, 122.00, 131.00, 133.15, 137.34, 141.19, 143.19, 143.86,
+
52.38, 154.00, 158.50, 163.62; MS m/z [%]: 409 [M , 2.6], 297 [100].
5
.1.4.14.
(Z/E)1-(4-Chlorophenyl)-3-(4-((5-fluoro-2-oxoindolin-3-
ylidene)amino)phenyl)urea (9n). Orange powder (yield 87 %), m.p. > 300 °C. IR
-1
1
(
KBr, ν cm ) 3273 (NH), 1740 (C=O); H NMR (DMSO-d )
δ
(ppm): 6.36 (m, 1H,
6
Ar-H), 6.92 (m, 1H, Ar-H), 7.00 (d, J = 8.8 Hz, 2H, Ar-H), 7.20-7.30 (m, 1H, Ar-H),
7
2
.33 (d, J = 8.4 Hz, 2H, Ar-H), 7.50 (d, J = 8.8 Hz, 2H, Ar-H), 7.57 (d, J = 8.4 Hz,
H, Ar-H), 8.86 (s, 1H, D O exchangeable, NH urea), 8.87 (s, 1H, D O exchangeable,
2
2
1
3
NH urea), 10.99 (s, 1H, D O exchangeable, NH isatin); C NMR (DMSO-d , 75
2
6
3
2
MHz)
δ: 111.41, 116.02 ( J = 8.0 Hz), 118.69, 118.90 ( J = 29.3 Hz), 119.09,
F-C F-C
2
1
20.61, 125.35, 128.46, 128.70, 137.48 ( JF-C = 33.0 Hz), 138.62, 141.83, 143.17,
1
4

ACCEPTED MANUSCRIPT
3
1
1
52.30 ( J = 8.6 Hz), 153.97, 155.15 ( J = 253.7 Hz), 163.62; MS m/z [%]: 409
F-C
F-C
+
[
M , 17.1], 96 [100].
5
.1.4.15.
(Z/E)1-(4-((5-Fluoro-2-oxoindolin-3-ylidene)amino)phenyl)-3-(4-
methoxyphenyl)urea (9o). Orange powder (yield 70 %), m.p. > 300 °C. IR (KBr, ν
-1
1
cm ) 3273 (NH), 1740, 1724 (C=O); H NMR (DMSO-d )
δ
(ppm): 3.72 (s, 3H,
6
OCH ), 6.37 (dd, J = 5.6 Hz, 2.8 Hz, 1H, Ar-H), 6.87 (d, J = 8.8 Hz, 2H, Ar-H), 6.93-
3
6
.94 (m, 1H, Ar-H), 6.98 (d, J = 8.4 Hz, 2H, Ar-H), 7.22-7.25 (m, 1H, Ar-H), 7.35 (d,
J = 8.8 Hz, 2H, Ar-H), 7.55 (d, J = 8.8 Hz, 2H, Ar-H), 8.50 (s, 1H, D O
2
exchangeable, NH urea), 8.74 (s, 1H, D O exchangeable, NH urea), 10.86 and 11.00
2
1
3
(
s, 1H, D O exchangeable, NH isatin); C NMR (DMSO-d , 75 MHz)
δ
: 63.50,
2
6
2
3
3
1
1
1
3
11.41 ( J = 26.2 Hz), 112.56, 116.02 ( J = 8.0 Hz), 116.98 ( J = 10.9 Hz),
F-C
F-C
F-C
2
18.65, 119.15, 120.34, 122.12, 127.37, 128.40, 133.23, 137.03 ( JF-C = 27.7Hz),
1
+
43.82, 152.09, 155.15 ( JF-C = 236.3 Hz), 163.61, 167.42; MS m/z [%]: 404 [M ,
.6], 43 [100].
5
.1.4.16.
(Z/E)4-(3-(4-((5-Fluoro-2-oxoindolin-3-
ylidene)amino)phenyl)ureido)benzenesulfonamide (9p). Orange powder (yield 84 %),
-1
m.p. > 300 °C. IR (KBr, ν cm ) 3271, 3196 (NH, NH ), 1728 (C=O), 1312, 1157
2
1
(
SO ); H NMR (DMSO-d )
δ
(ppm): 6.35 (m, 1H, Ar-H), 6.91 (m, 1H, Ar-H), 7.01
2
6
(
d, J =8.8 Hz, 1H, Ar-H), 7.22 (s, 2H, D O exchangeable, -SO NH ), 7.25-7.46 (m,
2 2 2
2
H, Ar-H), 7.57 (d, J = 8.8 Hz, 2H, Ar-H), 7.63 (d, J = 9.2 Hz, 2H, Ar-H), 7.72 (d, J =
9
.2 Hz, 2H, Ar-H), 8.91 and 8.96 (s, 1H, D O exchangeable, NH urea), 9.11 and 9.13
2
(
s, 1H, D O exchangeable, NH urea), 10.88 and 11.00 (s, 1H, D O exchangeable, NH
2
2
1
3
3
isatin); C NMR (DMSO-d , 75 MHz)
δ
: 111.71, 112.52, 116.02 ( J = 8.3 Hz),
6
F-C
2
2
1
17.57, 118.68, 118.88 ( JF-C = 29.3 Hz), 119.44, 120.95, 126.77, 136.85 ( J = 28.7
F-C
3
1
Hz), 142.07, 142.75, 143.20, 152.15 ( JF-C = 8.6 Hz), 154.04, 155.15 ( J = 236.0
F-C
+
Hz), 163.62; MS m/z [%]: 453 [M , 26.2], 91 [100].
5
.1.4.17. (Z/E)1-(4-((5-Chloro-2-oxoindolin-3-ylidene)amino)phenyl)-3-phenylurea
-1
(
9q). Orange powder (yield 81 %), m.p. > 300 °C. IR (KBr, ν cm ) 3281 (NH), 1742,
1
1
1
726 (C=O); H NMR (DMSO-d )
δ
(ppm): 6.66 (s, 1H, Ar-H), 6.93 (d, J = 8.4 Hz,
6
H, Ar-H), 7.01 (d, J = 8.8 Hz, 2H, Ar-H), 7.28 (d, J = 7.6 Hz, 1H, Ar-H), 7.30 (t, J =
1
5

ACCEPTED MANUSCRIPT
7
.6 Hz, 2H, Ar-H), 7.41-7.49 (m, 3H, Ar-H), 7.58 (d, J = 8.8 Hz, 2H, Ar-H), 8.72 (s,
1
H, D O exchangeable, NH urea), 8.84 (s, 1H, D O exchangeable, NH urea), 11.00
2
2
13
and 11.10 (s, 1H, D O exchangeable, NH isatin); C NMR (DMSO-d , 75 MHz)
δ
:
2
6
1
1
1
12.92, 116.92, 118.37, 119.04, 121.97, 125.19, 128.64, 128.89, 133.40, 133.63,
+
37.77, 139.59, 143.59, 145.53, 152.50, 153.32, 163.32; MS m/z [%]: 392 [(M+2) ,
+
9], 390 [M , 43.3], 215 [100].
5
.1.4.18.
(Z/E)1-(4-((5-Chloro-2-oxoindolin-3-ylidene)amino)phenyl)-3-(3-
(trifluoromethyl)phenyl)urea (9r). Orange powder (yield 79 %), m.p. > 300 °C. IR
-1
1
(
KBr, ν cm ) 3263 (NH), 1740, 1724 (C=O); H NMR (DMSO-d )
δ
(ppm): 6.63 (s,
H, Ar-H), 6.93 (d, J = 8.4 Hz, 1H, Ar-H), 7.02 (d, J = 8.8 Hz, 2H, Ar-H), 7.22 (d, J =
.4 Hz, 1H, Ar-H), 7.32 (d, J = 8.4 Hz, 1H, Ar-H), 7.41-7.53 (m, 2H, Ar-H), 7.59 (d,
6
1
8
J = 8.8 Hz, 2H, Ar-H), 8.04 (s, 1H, Ar-H), 8.97 (s, 1H, D O exchangeable, NH urea),
2
9
.11 (s, 1H, D O exchangeable, NH urea), 11.11 (s, 1H, D O exchangeable, NH
2
2
13
isatin); C NMR (DMSO-d , 100 MHz)
δ
: 113.58, 117.42, 118.59, 119.33, 119.86,
6
1
1
22.33, 122.82, 124.80, 125.71, 126.04, 126.81, 130.40, 134.06, 137.84, 141.02,
+
44.45, 146.08, 153.01, 153.97, 163.83; MS m/z [%]:458 [M , 1.3], 398 [100].
5
.1.4.19.
(Z/E)1-(4-((5-Chloro-2-oxoindolin-3-ylidene)amino)phenyl)-3-(4-
-1
cyanophenyl)urea (9s). Red powder (yield 74 %), m.p. > 300 °C. IR (KBr, ν cm )
1
3
1
8
7
9
356 (NH), 2226 (CN), 1730, 1705 (C=O); H NMR (DMSO-d )
δ
(ppm): 6.61 (s,
6
H, Ar-H), 6.93 (d, J = 8.4 Hz, 1H, Ar-H), 7.02 (d, J = 8.8 Hz, 1H, Ar-H), 7.59 (d, J =
.8 Hz, 2H, Ar-H), 7.65 (d, J = 8.8 Hz, 2H, Ar-H), 7.74 (d, J = 8.8 Hz, 2H, Ar-H),
.82 (d, J = 8.4 Hz, 2H, Ar-H), 8.99 and 9.04 (s, 1H, D O exchangeable, NH urea),
2
.25 and 9.27 (s, 1H, D O exchangeable, NH urea), 11.00 and 11.12 (s, 1H, D O
2
2
1
3
exchangeable, NH isatin); C NMR (DMSO-d , 100 MHz)
δ
: 103.75, 113.58, 117.40,
6
1
1
18.57, 119.33, 119.90, 122.5, 125.71, 127.5, 129.00, 133.77, 137.59, 140.00, 144.63,
+
+
46.08, 152.60, 154.03, 163.82; MS m/z [%]: 417 [(M+2) , 8.5], 415 [M , 23.8], 139
[
100].
5
.1.4.20.
(Z/E)1-(4-((5-Chloro-2-oxoindolin-3-ylidene)amino)phenyl)-3-(4-
-1
fluorophenyl)urea (9t). Orange powder (yield 88 %), m.p. > 300 °C. IR (KBr, ν cm )
1
3
283 (NH), 1740, 1728 (C=O); H NMR (DMSO-d )
δ
(ppm): 6.65 (s, 1H, Ar-H),
6
1
6

ACCEPTED MANUSCRIPT
6
.87 (d, J = 8.4 Hz, 1H, Ar-H), 7.01 (d, J = 8.8 Hz, 2H, Ar-H), 7.11 (t, J = 9.2 Hz, 1H,
Ar-H), 7.22 (d, J = 8.8 Hz, 1H, Ar-H), 7.41-7.55 (m, 3H, Ar-H), 7.58 (d, J = 8.8 Hz,
H, Ar-H), 8.73 and 8.75 (s, 1H, D O exchangeable, NH urea), 8.78 and 8.83 (s, 1H,
2
2
D O exchangeable, NH urea), 10.98 and 11.10 (s, 1H, D O exchangeable, NH isatin);
2
2
1
3
3
2
C NMR (DMSO-d , 75 MHz)
δ
: 112.94 ( J = 13.7 Hz), 114.98 ( J = 21.4 Hz),
6
F-C
F-C
3
2
1
1
1
17.62, 118.96 ( J = 9.7 Hz), 122.41, 123.81 ( J = 27.3 Hz), 125.18, 126.28,
F-C
F-C
1
33.37, 135.93, 137.72, 138.28, 143.61, 145.53, 153.33, 155.75 ( JF-C = 237 Hz),
+
63.31; MS m/z [%]: 408 [M , 1.9], 139 [100].
5
.1.4.21.
(Z/E)1-(4-((5-Chloro-2-oxoindolin-3-ylidene)amino)phenyl)-3-(3-
-1
chlorophenyl)urea (9u). Orange powder (yield 85%), m.p. > 300 °C. IR (KBr, ν cm )
1
3
6
267 (NH), 1740, 1722 (C=O); H NMR (DMSO-d )
δ
(ppm): 6.63 (s, 1H, Ar-H),
6
.92 (dd, J = 6.0, 2.4 Hz, 1H, Ar-H), 7.01 (d, J = 8.8 Hz, 2H, Ar-H), 7.21 (t, J = 8.8
Hz, 1H, Ar-H), 7.29 (d, J = 6.0 Hz, 1H, Ar-H), 7.41 (d, J = 2.4 Hz, 1H, Ar-H), 7.43
d, J = 8.8 Hz, 1H, Ar-H), 7.58 (d, J = 8.8 Hz, 2H, Ar-H), 7.73 (s, 1H, Ar-H), 8.93 (s,
H, D O exchangeable, NH urea), 8.95 (s, 1H, D O exchangeable, NH urea), 10.98
(
1
2
2
13
and 11.11 (s, 1H, D O exchangeable, NH isatin); C NMR (DMSO-d , 100 MHz) δ:
2
6
1
1
13.57, 114.30, 117.19, 118.11, 119.36, 121.98, 122.88, 124.60, 124.79, 125.72,
30.88, 133.67, 137.72, 137.92, 141.69, 144.36, 146.06, 152.88, 153.94, 163.84; MS
+
m/z [%]: 425 [M , 1.48], 424 [100].
5
.1.4.22.
(Z/E)1-(4-((5-Chloro-2-oxoindolin-3-ylidene)amino)phenyl)-3-(4-
-1
chlorophenyl)urea (9v). Orange powder (yield 90 %), m.p. > 300 °C. IR (KBr, ν cm )
1
3
6
2
8
271 (NH), 1749, 1738 (C=O); H NMR (DMSO-d )
δ
(ppm): 6.63 (s, 1H, Ar-H),
6
.93 (d, J = 8.4 Hz, 1H, Ar-H), 7.00 (d, J = 8.8 Hz, 2H, Ar-H), 7.32 (d, J = 8.6 Hz,
H, Ar-H), 7.41 (d, J = 8.4 Hz, 1H, Ar-H), 7.48 (d, J = 8.6 Hz, 2H, Ar-H), 7.57 (d, J =
.8 Hz, 2H, Ar-H), 8.82 and 8.85 (s, 1H, D O exchangeable, NH urea), 8.83 and 8.87
2
(
s, 1H, D O exchangeable, NH urea), 11.10 (s, 1H, D O exchangeable, NH isatin);
2
2
1
3
C NMR (DMSO-d , 75 MHz)
δ
: 113.00, 116.91, 118.76, 119.00, 119.18, 119.79,
6
1
1
25.18, 125.36, 128.68, 133.50, 137.54, 138.62, 143.75, 145.55, 152.40, 153.38,
+
63.31; MS m/z [%]: 426 [(M+1) , 100], 425 [M+, 11.5].
1
7

ACCEPTED MANUSCRIPT
5
.1.4.23.
(Z/E)1-(4-((5-Chloro-2-oxoindolin-3-ylidene)amino)phenyl)-3-(4-
methoxyphenyl)urea (9w). Orange powder (yield 70 %), m.p. > 300 °C. IR (KBr, ν
-1
1
cm ) 3275 (NH), 1740, 1722 (C=O); H NMR (DMSO-d )
δ (ppm): 3.73 (s, 3H, -
6
OCH ), 6.67 (s, 1H, Ar-H), 6.88 (d, J = 9.0 Hz, 2H, Ar-H), 6.93 (d, J = 8.4 Hz, 1H,
3
Ar-H), 7.00 (d, J = 8.8 Hz, 2H, Ar-H), 7.37 (d, J = 9.0 Hz, 2H, Ar-H), 7.41 (d, J = 8.4
Hz, 1H, Ar-H), 7.57 (d, J = 8.8 Hz, 2H, Ar-H), 8.52 (s, 1H, D O exchangeable, NH
2
urea), 8.76 (s, 1H, D O exchangeable, NH urea), 11.09 (s, 1H, D O exchangeable, NH
2
2
13
isatin); C NMR (DMSO-d , 75 MHz)
δ
: 55.00, 113.87, 114.06, 116.05, 118.75,
6
1
1
18.98, 119.97, 120.18, 124.56, 125.07, 131.95, 132.59, 137.95, 143.13, 143.38,
+
52.71, 154.48, 163.64; MS m/z [%]: 421 [M , 1.2], 382 [100].
5
.1.4.24.
(Z/E)4-(3-(4-((5-Chloro-2-oxoindolin-3-
ylidene)amino)phenyl)ureido)benzenesulfonamide (9x). Red powder (yield 89 %),
-1
m.p. > 300 °C. IR (KBr, ν cm ) 3271 (NH, NH ), 1726, 1699 (C=O) 1312, 1157
2
1
(
SO ); H NMR (DMSO-d )
δ
(ppm): 6.62 (s, 1H, Ar-H), 6.93 (d, J = 8.8 Hz, 1H, Ar-
2
6
H), 7.02 (d, J = 8.8 Hz, 2H, Ar-H), 7.19 and 7.21 (s, 2H, D O exchangeable, -
2
SO NH ), 7.41 (d, J = 8.4 Hz, 2H, Ar-H), 7.44 (d, J = 8.8 Hz, 1H, Ar-H), 7.60 (d, J =
2
2
8
.4 Hz, 2H, Ar-H), 7.74 (d, J = 8.8 Hz, 2H, Ar-H), 8.89 and 8.98 (s, 1H, D O
2
exchangeable, NH urea), 8.95 and 9.14 (s, 1H, D O exchangeable, NH urea), 11.00
2
13
and 11.12 (s, 1H, D O exchangeable, NH isatin); C NMR (DMSO-d , 100 MHz) δ:
2
6
1
1
3
14.29, 117.42, 118.00, 119.36, 121.41, 122.84, 124.80, 125.71, 127.30, 134.07,
+
37.35, 137.71, 143.26, 146.07, 152.76, 153.98, 163.83; MS m/z [%]:470 [M , 1.4],
89 [100].
4
.2 Biological evaluation
4
.2.1. In vitro cytotoxic activity:
HepG2 liver cancer cell line was obtained from the National Cancer Institute
(Cairo, Egypt). HepG2 cells were grown in DMEM. Media were supplemented with
1
0% heat inactivated FBS, 50 units/mL of penicillin and 50 g/mL of streptomycin and
maintained at 37°C in a humidified atmosphere containing 5% CO . The cells were
2
maintained as “monolayer culture” by serial sub-culturing. Cytotoxicity was
36
determined using SRB method as previously described by Skehan et al.
1
8

ACCEPTED MANUSCRIPT
Exponentially growing cells were collected using 0.25% Trypsin-EDTA and seeded
in 96-well plates at 1000-2000 cells/well in DMEM supplemented medium. After 24
h, cells were incubated for 72 h with various concentrations of the tested compounds
as well as sorafenib and doxurubicin as reference drugs. Following 72 h of treatment,
the cells will be fixed with 10% trichloroacetic acid for 1 h at 4 ºC. Wells were
stained for 10 min at room temperature with 0.4% SRB dissolved in 1% acetic acid.
The plates were air dried for 24 h and the dye was solubilized with Tris-HCl for 5 min
on a shaker at 1600 rpm. The optical density (OD) of each well was measured
spectrophotometrically at 564 nm with an ELISA microplate reader (ChroMate-4300,
FL, USA). The IC values were calculated according to the equation for Boltzman
5
0
sigmoidal concentration–response curve using the nonlinear regression fitting models
Graph Pad, Prism Version 5). The results reported are means of at least three separate
(
experiments. Statistical differences were analyzed according to one way ANOVA test
wherein the differences were considered to be significant at p < 0.05.
4
.2.2. Measurement of inhibitory activity against VEGFR-2
The kinase activity of VEGFR-2 was measured by use of an anti-phosphotyrosine
antibody with the Alpha Screen system (PerkinElmer, USA) according to
manufacturer’s instructions. Enzyme reactions were performed in 50 mM Tris–HCl
pH 7.5, 5 mM MnCl , 5 mM MgCl , 0.01% Tween-20 and 2 mM DTT, containing 10
2
2
µM ATP, 0.1 µg/mL biotinylated poly-GluTyr (4:1) and 0.1 nM of VEGFR-2
Millipore, UK). Prior to catalytic initiation with ATP, the tested compounds at final
(
concentrations ranging from 0-100 µg/mL and enzyme were incubated for 5 min at
room temperature. The reactions were quenched by the addition of 25 µL of 100 mM
EDTA, 10 µg/mL Alpha Screen streptavidine donor beads and 10 µg/mL acceptor
beads in 62.5 mM HEPES pH 7.4, 250 mM NaCl, and 0.1% BSA. Plate was
incubated in the dark overnight and then read by ELISA Reader (PerkinElmer, USA).
Wells containing the substrate and the enzyme without compounds were used as
reaction control. Wells containing biotinylated poly-GluTyr (4:1) and enzyme without
ATP were used as basal control. Percent inhibition was calculated by the comparison
of compounds treated to control incubations. The concentration of the test compound
causing 50% inhibition (IC ) was calculated from the concentration–inhibition
5
0
1
9

ACCEPTED MANUSCRIPT
response curve (triplicate determinations) and the data were compared with sorafenib
as standard VEGFR-2 inhibitor.
4
.3. Molecular Docking
The molecular docking of the tested compounds was performed using Discovery
Studio 4 /CDOCKER protocol (Accelrys Software Inc.). The protein crystallographic
structure, VEGFR-2 (PDB: 4ASD) was downloaded from the Protein Data Bank
(PDB). The protein was prepared for docking process according to the standard
protein preparation procedure integrated in Accelry’s discovery studio 4 and prepared
by prepare protein protocol. Sorafenib and the selected compounds were drawn as a
database and prepared by prepare ligand protocol to generate 3D structure and refine
using CHARMM force field with full potential. Docking simulations were run using
CDOCKER protocol where maximum bad orientations was 800 and orientation vdW
energy threshold was 300. Simulated annealing simulation would be then carried out
consisting of a heating phase 700 K with 2,000 steps and a cooling phase back to
5
,000 steps. The binding energy was calculated as a score to rank the docking poses.
The top 10 docking poses would be finally saved. Docking poses were ranked
according to their –CDOCKER interaction energy, and the top pose was chosen for
analysis of interactions for each compound.
Acknowledgements
The authors would like to extend their sincere appreciation to the Deanship of
Scientific Research at King Saud University for its funding of this research through
the Research Group Project no. PRG-1436-05. Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Egyptian Russian University, Cairo, Egypt, is highly
appreciated for supporting this research.
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Captions page
Figure 1. Structures of VEGFR-2 inhibitors currently approved or in clinical trials for
treatment of HCC 1-5 and the designed target indoline ureas 9a-x.
Figure 2. Binding mode of 9x in the active site of VEGFR2 (PDB: 4ASD) showing
three hydrogen bonds with Asp1046, Glu885 and Ile1025. Atoms of 9x are
represented by atom type and hydrogen bonds are represented by blue dots. All
amino acids are colored in grey except the three amino acids that are connected
by hydrogen bonds are colored in red. Pi stack of isatin group are represented by
orange lines. Polar hydrogens are displayed only for simplification.
Figure 3. Molecular docking view of 9x showing 5-chloroisatin moiety embedded in
the hydrophobic pocket of Cys919, Phe918, Gly922, Glu 850 and Leu 840. The
receptor is represented in the ribbon mode. Hydrophobic surface that surrounds
9x colored as Blue-White-Brown range from hydrophilic to hydrophobic
respectively.
Scheme 1. Reagents and conditions: i, CH CN, reflux 2h; ii, H , Pd/C, MeOH, rt; iii,
3
2
EtOH, AcOH (catalytic), reflux 3 h.
Table 1. Anti-proliferative activity towards HepG2 hepatocellular carcinoma cells
and enzymatic inhibition of VEGFR-2.
Table 2. Docking scores of the tested compounds and sorafenib.
2
3

ACCEPTED MANUSCRIPT
Table 1. Anti-proliferative activity towards HepG2 hepatocellular carcinoma cells and enzymatic
inhibition of VEGFR-2.
a
IC (µM)
5
0
Compound
X
R
HepG2
VEGFR2
5.61±0.65
4.26±0.31
5.09±1.05
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
H
H
H
H
H
H
H
H
F
F
F
F
F
H
3-CF
4-CN
4-F
3-Cl
4-Cl
4-OCH
4-SO
H
3-CF
4-CN
4-F
3-Cl
4-Cl
4-OCH
4-SO
H
3-CF
4-CN
4-F
3-Cl
4-Cl
4-OCH
4-SO NH
3.46±0.29
3.05±0.23
7.73±0.23
10.11±3.75
3.20±0.36
3.12±0.18
3
c
NT
2.58±0.18
2.51±0.09
f
b
c
g
h
i
j
k
l
m
n
o
p
q
r
s
t
u
v
w
x
3
NA
NT
NT
NT
c
NH
11.68±1.56
22.54±1.25
2.67±0.28
2
2
2
2
c
2.12±0.33
3
b
c
NA
NT
NT
c
30.70±2.97
2.29±0.38
1.81±0.14
2.79±0.29
13.83±1.45
21.37±2.58
1.99±0.11
1.93±0.07
2.67±0.22
F
F
F
3
c
NH
NT
NT
2
c
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
b
c
NA
NT
3
c
10.26±1.39
3.19±0.97
2.96±0.35
8.37±0.85
22.22±3.09
3.15±0.36
2.90±0.36
3.40±0.25
NT
3.92±0.46
0.75±0.05
0.71±0.08
c
NT
3
0.31±0.04
-
0.10±0.02
2
Dox.
Sorafenib
a
b
c
IC values are the mean ± S.D. of three separate experiments.
5
0
NA: Compounds having IC value > 50 µM.
5
0
NT: Compounds not tested for their VEGFR-2 inhibitory activity.
1

ACCEPTED MANUSCRIPT
Table 2. Docking scores of the tested compounds and sorafenib.
Compound
CDOCKER
Energy (Kcal/mol)
-23.403
CDOCKER Interaction
Energy (Kcal/mol)
-55.267
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
-27.677
-24.683
-26.344
-26.236
-35.727
-27.513
-24.295
-25.9772
-25.359
-35.473
-24.578
-26.819
-26.486
-36.032
-44.691
-61.454
-58.335
-59.443
-59.236
-64.842
-26.046
-57.964
-59.718
-60.725
-65.1928
-58.8145
-61.4614
-60.942
-66.467
-68.502
e
f
h
j
m
n
o
p
t
u
v
x
Sorafenib
2

ACCEPTED MANUSCRIPT
Figure 1. Structures of VEGFR-2 inhibitors currently approved or in clinical trials for
treatment of HCC 1-3 and the designed target indoline ureas 9a-x.
Figure 2. Binding mode of 9x in the active site of VEGFR-2 (PDB: 4ASD) showing
five hydrogen bonds with Asp1046, Glu885, Glu 917 and Ile1025. It shows also Pi
stack of aromatic rings of 9x with Phe1047, His 1026 and Phe 918. Atoms of 9x are
represented as stick and colored by atom type. Hydrogen bonds are represented by
green dots and Pi stacks are represented by purple dots. All amino acids are
represented in line formula and amino acids in contact with 9x are labeled. Polar
hydrogens are displayed only for simplification.

ACCEPTED MANUSCRIPT
Scheme 1. Reagents and conditions: i, CH CN, reflux 2h; ii, H , Pd/C, MeOH, rt; iii,
3
2
EtOH, AcOH (catalytic), reflux 3 h.

ACCEPTED MANUSCRIPT
Highlights
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Novel series of Indoline ureas (9a-x) was designed and synthesized.
Anti-proliferative activity of 9a-x was evaluated against HepG2.
9n (IC = 1.81±0.14 µM) is the most potent derivative against HepG2.
5
0
Inhibitory activity against VEGFR-2 was evaluated for thirteen derivatives.
Molecular docking was carried out to analyze the binding pattern with VEGFR-2.