Design, synthesis, and molecular docking of novel indole scaffold-based VEGFR-2 inhibitors as targeted anticancer agents

Article abstract of DOI:10.1002/ardp.201700299

A series of new indole derivatives 1–18 was synthesized and tested for their cytotoxic activity on a panel of 60 tumor cell lines. Additionally, molecular docking was carried out to study their binding pattern and binding affinity in the VEGFR-2 active site using sorafenib as a reference VEGFR-2 inhibitor. Based on the molecular docking results, compounds 5a, 5b, 6, 7, 14b, 18b, and 18c were selected to be evaluated for their VEGFR-2 inhibitory activity. Compound 18b exhibited a broad-spectrum antiproliferative activity on 47 cell lines, with GI % ranging from 31 to 82.5%. Moreover, compound 18b was the most potent VEGFR-2 inhibitor with an IC₅₀ value of 0.07 μM, which is more potent than that of sorafenib (0.09 μM). A molecular docking study attributed the promising activity of this series to their hydrophobic interaction with the VEGFR-2 binding site hydrophobic side chains and their hydrogen bonding interaction with the key amino acids Glu885 and/or Asp1046.

Full text of DOI:10.1002/ardp.201700299

Received: 23 September 2017
DOI: 10.1002/ardp.201700299
Revised: 3 December 2017
Accepted: 13 December 2017
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FULL PAPER
Design, synthesis, and molecular docking of novel indole
scaffold-based VEGFR-2 inhibitors as targeted
anticancer agents
1
1
1
|
|
|
Hanaa M. Roaiah
Iman A. Y. Ghannam
Islam H. Ali
2
,3
4
2
|
|
|
Ahmed M. El Kerdawy
Mamdouh M. Ali
Safinaz E-S. Abbas
1
Sally S. El-Nakkady
1
National Research Centre, Pharmaceutical
and Drug Industries Research Division,
Chemistry of Natural and Microbial Products
Department, Cairo, Egypt
Abstract
A series of new indole derivatives 1–18 was synthesized and tested for their cytotoxic
activity on a panel of 60 tumor cell lines. Additionally, molecular docking was carried
out to study their binding pattern and binding affinity in the VEGFR-2 active site using
sorafenib as a reference VEGFR-2 inhibitor. Based on the molecular docking results,
compounds 5a, 5b, 6, 7, 14b, 18b, and 18c were selected to be evaluated for their
VEGFR-2 inhibitory activity. Compound 18b exhibited a broad-spectrum antiprolifer-
ative activity on 47 cell lines, with GI %rangingfrom 31 to 82.5%. Moreover, compound
2
Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, Cairo University,
Cairo, Egypt
3
Molecular Modeling Unit, Faculty of
Pharmacy, Cairo University, Cairo, Egypt
4
National Research Centre, Genetic
Engineering and Biotechnology Division,
Department of Biochemistry, Cairo, Egypt
1
8b was the most potent VEGFR-2 inhibitor with an IC50 value of 0.07 μM, which is
Correspondence
more potent than that of sorafenib (0.09 μM). A molecular docking study attributed the
promising activity of this series to their hydrophobic interaction with the VEGFR-2
binding site hydrophobic side chains and their hydrogen bonding interaction with the
key amino acids Glu885 and/or Asp1046.
Dr. Iman A. Y. Ghannam, National Research
Centre, Pharmaceutical and Drug Industries
Research Division, Chemistry of Natural and
Microbial Products Department, El-Bohouth
Street, Dokki, Cairo 12622, Egypt.
Email: iman.youssef.ghannam@gmail.com
K E Y W O R D S
Funding information
anticancer agents, indole, molecular docking, VEGFR-2 inhibitors
National Research Centre – Cairo – Egypt,
Grant number: P100220
1
|
INTRODUCTION
origin, to enter into circulation and enhance metastasis
[
5]
elsewhere.
Cancer remains one of the most life-threatening diseases
The vascular endothelial growth factor receptor-2 (VEGFR-2), one
of the tyrosine kinase receptors, is considered as the most important
transducer of VEGF-dependent angiogenesis. VEGFR-2 is highly up-
regulated in several solid tumors and plays a critical role in the process
of tumor angiogenesis, hence VEGFR-2 inhibition emerged as a prime
approach for discovering new therapies for many human angiogenesis-
[
1]
worldwide.
Moreover, most of cancer patients suffer from
serious adverse toxicities upon conventional chemotherapy treat-
ment. Consequently, there is a necessity for designing new
chemotherapeutic agents targeting cancer cells with minimum
[
2,3]
side effects associated with the normal cells collateral damage.
[6]
Angiogenesis, or formation of new blood vessels, has a central role
dependent malignancies.
[
4]
®
in the cancer development. The new blood vessels formation
allows local tumors growth and feeds their growth in different
sites, hence enables malignant cells to escape from their primary
Sunitinib (Sutent ) (I), an FDA approved oxindole multi-targeted
kinase inhibitor, simultaneously blocks VEGFRs, platelet derived
growth factor receptors (PDGFRs), and stem cell factor (c-kit) receptor
|
Arch Pharm Chem Life Sci. 2018;e1700299.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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ROAIAH ET AL.
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and is used for the treatment of gastrointestinal stromal tumor (GIST)
and advanced renal cell carcinoma (RCC); it has VEGFR-2 inhibition
[7–11]
with IC50 value of 38 nM.
PubChem 47037197 (II), 2-aryl
benzimidazole derivative, is also a multi-targeted kinase inhibitor. It
[12,13]
inhibits epidermal growth factor receptor (EGFR) and VEGFRs
Figure 1).
Among the wide range of tested compounds as potential
(
anticancer agents, the indole derivatives have been reported to
exhibit remarkable antitumor activities; as an example, compound III
showed an IC50 value of 5.7 nM against MCF-7 breast carcinoma cell
[
14]
line.
in vitro anticancer activity against MCF-7 cancer cell line.
Additionally, some bicyclic systems, such as the chromone derivatives
V, VI), have been reported to possess anticancer activities against
MCF-7 and A549 lung cancer cell lines with IC50 values of 8.91, 2.23,
CJM 126 (IV), a 2-aryl benzothiazole derivative, shows also an
[15]
(
[16]
1
1.5, and 1.73 μM, respectively.
Motivated by the VEGFR-2 inhibitory and anticancer activities of
I–VI, it seemed of interest to design and synthesize the target
compounds 1–18. 2-Phenylindoles 2–5 (class A) are derived from
PubChem 47037197 (II) and the 3-substituted-2-phenylindoles 6–9
(
class B) from the lead compound III.
Indole thiazolinones 14a–e (class C) are structural hybrids from the
lead compounds I, V, while indole imidazolones 17a–d and 18a–c (class
D) are structural hybrids from the lead compounds I, VI (Figure 2).
The designed modifications aimed to highlight the effect of
lipophilicity, electronic nature and steric parameters of the newly
synthesized indole derivatives on the VEGFR-2 inhibitory and
anticancer activities.
FIGURE 2 Structural comparison of the target compounds
classes (A–D) with sunitunib (I), PubChem 47037197 (II) and the
anticancer compounds: indole derivative (III) and chromone
derivatives (V, VI)
Herein, we report the synthesis, anticancer activity of the newly
synthesized compounds 1–18. A docking study was also performed.
Based on the scoring energy and binding mode, some compounds were
selected toinvestigate theirmechanismofactionasVEGFR-2 inhibitors.
3-aminoacetophenone to give the hydrazone which upon cyclization
with polyphosphoric acid produced compound 1.
Reaction of the amino functionality of 1 with the appropriate acid
anhydride afforded compounds 2a,b. IR spectra of both compounds
revealed the appearance of two new carbonyl absorption bands at 1710–
2
2
|
RESULTS AND DISCUSSION
Chemistry
−1
1
713 cm , whereas mass spectra disclosed molecular ion peaks at m/z
1
2
88 and 338, respectively. H-NMR spectrum of compound 2a showed
.1
|
two new doublet signals at δ 7.41 and 7.90 ppm corresponding to the two
13
In Scheme 1, 3-(1H-indol-2-yl)aniline 1 was synthesized via Fischer
protons of the pyrrole dione ring. C-NMR spectrum of 2b revealed a
signal at δ 167.6 ppm corresponding to the two carbonyl carbons.
[17–19]
indole method.
Phenyl hydrazine was allowed to react with
FIGURE 1 Chemical structures of selected VEGFR-2 inhibitors: sunitunib (I) and PubChem 47037197 (II)

ROAIAH ET AL.
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morpholine carbons in addition to a signal at δ 62.6 ppm attributed
to CH
2
carbon.
In Scheme 2, N-(3-(1H-indol-2-yl)phenyl)acetamide 6 was previ-
[20]
ously synthesized.
In our work, we synthesized it in a much simpler
method; less reaction time and better product yield via refluxing of 1 in
glacial acetic acid.
Vilsmeir Haack formylation reaction catalyzed by phosphorous
[21,22]
oxychloride and N,N-dimethylformamide
was used to formylate
compound 6 giving N-(3-(3-formyl-1H-indol-2-yl)phenyl)acetamide 7.
SCHEME 1 Synthesis of target compounds 1–5. Reagents and
reaction conditions: (i) Acid anhydrides, glacial acetic acid, reflux, 2–3 h,
yield: 40–45%. (ii) Acid chlorides, DMF, stirring, rt, 2 h, yield: 42–50%.
(iii) Chloroacetyl chloride, DMF, stirring, rt, 1 h, yield: 52%. (iv)
Secondary amines, DMF, K CO , stirring, rt, 1 h, yield: 55–60%
2
3
The N-substitued benzamide and 4-chlorobenzamide derivatives
a,b were obtained upon stirring 1 with the appropriate acid chloride in
3
dimethylformamide at room temperature. IR spectra showed the
appearance of a new amidic carbonyl absorption band in the region of
−1
1
650–1686 cm . Furthermore, the mass spectrum of 3b revealed a
1
molecular ion peak at m/z 346 as a base peak. H-NMR spectra of
compounds 3a,b showed a new exchangeable singlet signal at δ
1
0.36 ppm corresponding to HN-CO and an increase in the
1
3
integration of aromatic protons was also noticed. C-NMR spectrum
of 3b revealed the appearance of a signal at δ 166.9 ppm attributed to
the carbonyl carbon.
N-(3-(1H-Indol-2-yl)phenyl)-2-chloroacetamide 4 was formed via
treating 1 with chloroacetyl chloride in dimethylformamide at room
temperature. IR spectrum showed the appearance of a new amidic
−
1
1
carbonyl band at 1677 cm , whereas H-NMR spectrum revealed a
new singlet signal at δ 4.29 ppm corresponding to CH
-CO protons
in addition to a new exchangeable singlet signal at δ 10.39 ppm
2
1
3
corresponding to HN-CO. C-NMR spectrum showed the appear-
2
ance of signals at δ 44.0 and 165.4 ppm corresponding to CH and
carbonyl carbons, respectively.
Reaction of 4 with the appropriate secondary amine in dimethyl-
1
formamide at room temperature gave 5a–c. H-NMR spectra disclosed
the appearance of two multiplets at δ 1.43–1.61 and 2.45–2.47 ppm
corresponding to piperidine protons, two multiplets at δ 2.46–2.50 and
2
.60–2.68 ppm attributed to piperazine protons and two other
SCHEME 2 Synthesis of target compounds 6–9. Reagents and
reaction conditions: (i) Glacial acetic acid, reflux, 3 h, yield: 64%. (ii)
multiplets at δ 2.47–2.48 and 3.66–3.69 ppm related to morpholine
protons, respectively. Mass spectrum of 5b illustrated a molecular ion
3
POCl , DMF, 1.5 h, yield: 35%. (iii) Semicarbazide HCl, isonicotinic
13
peak at m/z 334. C-NMR spectrum of 5b revealed the appearance of
piperazine carbon signals at δ 46.0 and 53.3 ppm whereas compound
acid hydrazide o-chlorophenylhydrazine HCl, EtOH, acetic acid,
reflux, 2 h, yield: 30–37%. (iv) Active methylenes, EtOH, TEA, reflux,
3 h, yield: 42–49%
5
c showed signals at δ 53.8 and 66.7 ppm corresponding to

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ROAIAH ET AL.
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IR spectrum exhibited the appearance of an absorption band at
−
1
1
676 cm attributed to (CO aldehydic) and mass spectrum revealed
1
a molecular ion peak at m/z 278. H-NMR spectrum of 7 illustrated the
appearance of the aldehydic proton (HCO) at δ 10.00 ppm in
addition to lack of a singlet at δ 6.73 ppm that corresponds to H-3
13
indole. C-NMR spectrum disclosed a signal at δ 186.0 ppm attributed
to aldehydic carbon.
Refluxing the formyl derivative 7 with semicarbazide hydrochlo-
ride, isonicotinic acid hydrazide, and o-chlorophenylhydrazine hydro-
chloride in ethanol using a catalytic amount of glacial acetic acid gave
1
compounds 8a–c, respectively. H-NMR spectra revealed the
appearance of an azomethine (HCN) as a singlet signal in the region
δ 8.2–8.8 ppm in addition to lack of the aldehydic proton singlet signal
δ 10.00 ppm. An extra exchangeable singlet signal at δ 9.55–
13
1
1.76 ppm had also appeared corresponding to (N-NH). C-NMR
spectrum of 8a revealed the absence of the aldehydic carbonyl carbon
signal at δ 186.0 ppm.
Reaction of 7 with active methylene derivatives as malononitrile or
ethyl cyanoacetate was performed in refluxing ethanol in presence of
triethylamine to produce compounds 9a,b. IR spectra showed the
absence of the carbonyl absorption bands of the aldehydic group, and
showed the appearance of an absorption band of the cyano group in
SCHEME 3 Synthesis of target compounds 10–14. Reagents and
reaction conditions: (i) Chloroacetyl chloride, DMF, stirring, rt, 2–4 h
4
yield: 60–80%. (ii) NH SCN, EtOH, reflux, 3–12 h, yield: 73–90%.
(
iii) POCl , DMF, 1.5 h, yield: 81%. (iv) Compound 13, glacial acetic
3
acid, sodium acetate, reflux, 5–21 h, yield: 40–60%
−1
the region of 2214–2219 cm and the ester absorption band at
−1
1
1
683 cm for 9b. H-NMR spectrum of 9b demonstrated a triplet-
quartet pattern of the ethyl protons at 1.27–1.29 and
.22–4.29 ppm, respectively, and a singlet at δ 7.90 ppm related to
In Scheme 4, N-benzoyl/chlorobenzoylglycine intermediates 15a,
b were synthesized as reported by reaction of glycine with benzoyl/p-
δ
[32,33]
4
chlorobenzoyl chloride via stirring in sodium hydroxide solution.
the methine proton (CHC). Mass spectra denoted the appearance of
their molecular ion peaks at m/z 326 and 373, respectively.
Reaction of 13 with 15a,b in acetic anhydride in the presence of
anhydrous sodium acetate gave the indole oxazolone intermediates
[
32,34]
In Scheme 3, a series of intermediates 10a–e, 11a–e, and 13 was
first synthesized to produce 14a–e. N-Chloroacetyl sulfonamide
intermediates 10a–e were prepared as previously reported by stirring
a mixture of the appropriate sulfonamide and chloroacetyl chloride in
16a,b.
IR spectrum of the new compound 16b illustrated the
−
1
appearance of absorption bands at 1800 and 1685 cm correspond-
ing to the oxazolone carbonyl and the amidic carbonyl groups,
respectively, as well as the disappearance of NH indole absorption
[
23,24]
1
dimethylformamide,
were also prepared according to the reported procedures by reacting
the intermediate thiazolidinones 11a–e
band. H-NMR spectrum of 16b revealed two new singlets at δ 2.77
and 8.89 ppm corresponding to the CH
respectively.
3
and methine CHC protons,
1
0a–e with ammonium thiocyanate and refluxing in absolute
[
16]
ethanol.
Additionally, 1H-indole-3-carbaldehyde 13 was prepared
In order to synthesize the target indole imidazolones 17a–d and
18a–c, indole oxazolones 16a,b were reacted with the appropriate
sulfonamide derivative in glacial acetic acid and in the presence of
anhydrous sodium acetate. Upon monitoring the reaction rate by thin
layer chromatography, it was noticed that compounds 17a–d were
synthesized within 1–2 h, while compounds 18a–c were produced
after 26–30 h. The difference in reaction time resulted in two different
series of acetylated and deacetylated products 17a–d and 18a–c,
respectively.
as reported via Vilsmeir Haack formylation of the commercially
available 1H-indole 12 using phosphorous oxychloride (POCl ) and
3
[25–29]
dimethylformamide (DMF).
The target indole thiazolinones 14a–e were synthesized by the
Knoevenagel condensation reaction where indole-3-carbaldehyde 13
was reacted with thiazolidinones 11a–e in glacial acetic acid in
[16,30] 1
presence of sodium acetate.
H-NMR spectra of 14a–e lacked
the singlet signal at δ 4.00 ppm corresponding to the two protons of
thiazolinone ring and displayed the appearance of a new singlet signal
of the methine proton (─CH) which appeared as a singlet signal at δ
IR spectra of 17a–d and 18a–c revealed the appearance of
−
1
imidazolone carbonyl absorption band at the region 1704–1715 cm
7
.93 ppm for 14a or included within a multiplet in the aromatic region
and the disappearance of the oxazolone carbonyl absorption band at
−1 1
1
3
at δ 7.87–8.52 ppm for 14b–e. On the other hand, C-NMR spectrum
of 14a showed the disappearance of CH carbon signal of
1800 cm . H-NMR spectra of 17a–d showed the appearance of the
singlet signal for OC-CH
disappeared in the H-NMR spectra of 18a–c. C-NMR spectra of
17a–d confirmed that the acetyl group was retained due to the
protons at δ 2.77 ppm, while this signal
13
2
3
1
thiazolidinone intermediate 11a at δ 37.6 ppm and the appearance
of a new methine carbon (─CHC) signal at δ 152.8 ppm. Compound
[31]
1
4a was previously synthesized ; however, its physical properties
3 3
presence of two signals of acetyl carbons (N-CO-CH and N-CO-CH )
and spectral data are reported in the present study for the first time.
at δ 24.4 and 169.0 ppm, respectively.

ROAIAH ET AL.
| 5 of 17
validation step efficiently reproduced the experimental binding
pattern of the co-crystallized ligand indicating that the adapted
docking protocol is suitable for the current docking study. The
suitability of the docking setup is demonstrated by the small RMSD of
0
.470 Å between the docking pose obtained in the validation step and
the co-crystalized ligand and the ability of the docking pose to
reproduce all the key interactions with the active site hotspots
(
Glu885, Cys919, and Asp1046). The scoring energy of the synthesized
compounds (1–18) ranged from −8.61 to −17.4 kcal/mol compared
with sorafenib calculated as −15.19 kcal/mol (for more details, see the
Supporting Information).
For compound 1, the molecular docking study showed a unique
predicted binding pattern involving the fitting of the 2-phenylindole
moiety in the middle of the active site at the interface between the ATP
binding site and the hydrophobic allosteric back pocket directing its
aminophenyl moiety toward the hydrophobic back pocket. In addition,
the indole ring interacts by its NH as hydrogen bond donor through
hydrogen bonding with the side chain carboxylate of Glu885 of the αC
helix (Figure 3).
For the 3′-amido derivatives 2a–b, 3a–b, 4, 5a–c, 6, and 7, the
general binding pattern involves the accommodation of the 2-phenyl
indole moiety in the vicinity of the hydrophobic side chains of Val848,
Lys868, Leu889, Val916, and Phe1047 amino acids to be involved in a
hydrophobic interaction with these hydrophobic side chains. Further-
more, the amido extension at 3′-position of the phenyl ring is involved
in hydrogen bonding interactions by its NH and CO functional groups
with side chain carboxylate of Glu885 of the αC helix and DFG motif
Asp1046, respectively. The hydrophobic moiety at the end of this
extension is accommodated in the hydrophobic allosteric back pocket
and so is involved in a hydrophobic interaction that increases the
SCHEME 4 Synthesis of target compounds 15–18. Reagents and
reaction conditions: (i) NaOH (10%), DMF, stirring, rt, 2 h, yield:
9
0–95%. (ii) Compound 13, acetic anhydride, sodium acetate, reflux,
h, yield: 35–46%. (iii) Appropriate sulfonamide, glacial acetic acid,
4
sodium acetate, reflux, 1–30 h, yield: 30–60%
2.2
|
Molecular docking and structure–activity
[36]
compounds' binding affinity
Information).
(for more details, see the Supporting
relationship
[
35]
In the present study, we used PDB ID: 4ASD
which has VEGFR-2 in
Figure 4 shows compound 5a adapting this aforementioned
binding pattern with its piperidyl moiety accommodated in the VEGFR-
2 hydrophobic allosteric back pocket and its amido moiety is involved
in hydrogen bonding interactions with side chain carboxylate of
Glu885 of the αC helix and DFG motif Asp1046. This binding pattern is
the inactive conformation (DFG-out) co-crystallized with sorafenib as
inhibitor.
Initially, the docking protocol was validated by docking of the co-
crystalized ligand sorafenib in the VEGFR-2 active site. The re-docking
FIGURE 3 2D diagram (a) and 3D representation (b) of compound 1 showing its interaction with the VEGFR-2 receptor active site
distances in Å)
(

6
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ROAIAH ET AL.
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FIGURE 4 2D diagram (a) and 3D representation (b) of compound 5a showing its interaction with the VEGFR-2 receptor active site
distances in Å)
(
responsible for the promising VEGFR-2 inhibitory activity of
compound 5a as indicated by its high in vitro VEGFR-2 level reducing
effect in MCF-7 cell line of 79% and its IC50 in VEGFR-2 inhibition
assay of 1.14 µM.
the benzene sulfonamide moiety in the hydrophobic allosteric back
pocket of the VEGFR-2 binding site forming hydrophobic interaction
through its phenyl moiety with the hydrophobic side chains of the
amino acids lining this back pocket and hydrogen bond interaction
through its sulfonamide moiety NH with the His1026 imidazole ring π
electrons (for more details, see the Supporting Information).
Figure 6 shows that compound 14b adapts this binding pattern
rationalizing its promising VEGFR-2 inhibitory activity as indicated by
its high in vitro VEGFR-2 level reducing effect in MCF-7 cell line of 61%
and its IC50 in VEGFR-2 inhibition assay of 1.87 µM.
For N-(3-(3-formyl-1H-indol-2-yl)phenyl)acetamide derivatives
8
a–c, 9a–b, the general binding pattern involves the accommodation
of the hydrophobic indole phenyl acetamide moiety in the hydrophobic
back pocket of the active site lined with the hydrophobic side chains of
amino acids Ile888, Leu889, Ile892, Val898, Val899, Leu1019
achieving hydrophobic interaction with these side chains. In addition,
these compounds are involved in hydrogen bonding with the key
amino acids Glu885 or/and Asp1046 through the substituent on the
Compounds 17a–d and 18a–c show a binding pattern which is
consistent with that of these series of compounds with their indolyl
moiety involved in a hydrophobic interaction with the hydrophobic
side chains of Val848, Lys868, Leu889, Val916, and Phe1047 amino
acids. Phenyl substituents at positions 2 and 3 of the imidazolyl moiety
are accommodated in the hydrophobic allosteric back pocket and are
involved in a hydrophobic interaction which is responsible for the
pronounced VEGFR-2 inhibitory activity of this series as reflected in
their docking scores (Supporting Information Table S1) and the
experimental results (see below).
3
-position of the indole ring (Figure 5) (for more details, see the
Supporting Information).
Sulfonamide derivatives 14a–e adapted similar binding pattern to
that of 3′-amido derivatives with their indolyl moiety accommodated
in the hydrophobic vicinity of Val848, Lys868, Leu889, Val916, and
Phe1047 side chains and involved in a hydrophobic interaction with
them. In addition, the 2-amino-4-oxo-4,5-dihydrothiazolyl moiety
accomplishes two hydrogen bond interactions through its ring N and
NH at position 2 of the thiazolyl moiety with DFG motif Asp1046 and
side chain carboxylate of Glu885, respectively. This binding pattern fits
Figure 7 shows compound 18b adapting this binding pattern
which not only achieves the previously mentioned hydrophobic
FIGURE 5 2D diagram (a) and 3D representation (b) of compound 8b showing its interaction with the VEGFR-2receptor active site
distances in Å)
(

ROAIAH ET AL.
| 7 of 17
FIGURE 6 2D diagram (a) and 3D representation (b) of compound 14b showing its interaction with the VEGFR-2 receptor active site
distances in Å)
(
interactions but also it forms hydrogen bonding interaction through its
carbonyl oxygen at position 5 of the imidazolyl moiety with Lys868.
These interactions ensure that compound 18b possesses potential
VEGFR-2 inhibitory activity as indicated by its promising IC50 in
VEGFR-2 inhibition assay of 0.07 µM and this is reflected in its docking
score of −15.08 kcal/mol which is comparable to that of sorafenib
leukemia and lung, brain, colon, kidney, ovary, prostate, and breast
cancers. A protocol of 48 h drug exposure and sulforhodamine B (SRB)
protein assay was used to determine the cell viability and growth.
The antiproliferative activity was presented by the NCI as percent
growth of the treated cells, and is presented in Table 1 as growth
inhibition percentage (GI %) achieved by the tested compounds. The
tested compounds exhibited diverse antiproliferative activities.
Compound 18b (Table 1) exhibited antiproliferative activity
against 47 cancer cell lines with GI % ranging from 31 to 82.5%. It
showed potent growth inhibitory effects with GI % from 62 to 82.5%
against leukemia (CCRF-CEM, HL-60(TB), MOLT-4, RPMI-8226 and
SR), non-small cell lung (A549/ATTC and NCI-H460), colon (COLO
205, HCT-116, HCT-15, HT29 and KM12), CNS (U251, melanoma
LOX IM VI, M 14 and MDA-MB-435), ovarian (OVCAR-8), renal
(786-0 and UO-31), prostate (PC-3), and breast (MCF7 and T-47D)
cell lines.
(
−15.19 kcal/mol).
In summary, the promising VEGFR-2 inhibitory activity of the newly
synthesized compounds can be attributed to their hydrophobic
interactions with the hydrophobic side chains of Val848, Lys868,
Leu889, Val916, and Phe1047 amino acids and the hydrophobic allosteric
back pocket of the active site lined with the hydrophobic side chains of
amino acids Ile888, Leu889, Ile892, Val898, Val899, Leu1019. Besides,
the ability of most of them to accomplish hydrogen bonding interactions
with the active site key amino acids Glu885 and/or Asp1046.
Regarding sensitivity to individual cell lines, it was noticed that the
newly synthesized compounds show selectivity to nearly all leukemia
cells, non-small cell lung (HOP-92), renal (A498 and UO-31), and breast
2
.3
|
Biological studies
In vitro antiproliferative activity
2
.3.1
|
(
MDA-MB-231/ATTC) cell lines.
Twenty-three of the newly synthesized compounds were selected and
tested for their in vitro antiproliferative activity by the National Cancer
2
.3.2 In vitro determination of VEGFR-2 level for
|
[37]
Institute (NCI), Bethesda, Maryland, USA,
under the Developmental
selected compounds in MCF-7 cell line
[38–42]
Therapeutic Program (DTP).
The selected compounds were evaluated at a concentration of
Recent studies showed the up-regulation of VEGFR-2 in MCF-7 cell
[43,44]
1
0 μM on a panel of 60 tumor cell lines representing melanoma,
line.
Based on the docking study results represented in the
FIGURE 7 2D diagram (a) and 3D representation (b) of compound 18b showing its interaction with the VEGFR-2 receptor active site
distances in Å)
(

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TABLE 1 Growth inhibition % (GI %) of the selected compounds in vitro against a panel of tumor cell lines at 10 μM
Compound ID
Subpanel
Leukemia
CCRF-CEM
HL-60(TB)
K-562
3b
5a
5c
7
9a
9b
14c
14e
17a
17c
17d
18a
18b
a
41
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
44
–
35
–
52
–
–
–
–
–
–
–
78
32
–
31
–
33
–
73.5
–
60
85
41
–
–
32
40
39
31
40
49
38
39
MOLT-4
62
–
–
–
82.5
76
PRMI-8226
SR
40
–
–
58
–
47.5
45
–
–
80.5
Non-small cell lung cancer
A549/ATTC
EKVX
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
61
50
41
44
39
–
35
–
–
–
–
–
–
HOP-62
–
–
–
–
–
HOP-92
50
–
39
–
35
–
31
–
30
–
–
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
–
32.5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
81
50
42
–
–
–
–
40
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
63
35
70
75
80
76
43
–
–
–
52
35
33
–
–
–
49
74
30
–
–
HT29
–
KM12
30
–
SW-620
–
CNS cancer
SF-268
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
53
SF-295
–
–
–
50
SF-539
–
–
–
44.5
45
SNB-19
–
–
32
–
SNB-75
34
–
45
U251
33
44
65
Melanoma
LOX IMVI
MALME-3M
M14
32.5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
66
–
–
–
–
62
63
–
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
–
35.5
–
–
–
–
35
N.D.
–
–
–
–
–
39
–
35
(
Continues)

ROAIAH ET AL.
| 9 of 17
TABLE 1 (Continued)
Compound ID
Subpanel
3b
5a
5c
7
9a
9b
14c
14e
17a
17c
17d
18a
18b
Ovarian cancer
IGROV1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
40
51
53
–
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
–
–
–
38
–
–
–
–
–
–
38
–
34
–
62
–
–
–
–
–
–
Renal cancer
7
86-0
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
67.5
39
A498
32
–
32
–
–
–
–
–
39
–
33
–
RXF 393
SN 12C
–
–
–
–
51
–
–
–
–
–
–
–
–
41.5
–
TK-10
–
–
–
–
–
–
–
–
UO-31
38
–
37
33
31
33
–
–
70
Prostate cancer
PC-3
47
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
73.5
46
DU-145
–
Breast cancer
MCF7
32
50
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
50
–
–
–
–
–
–
–
75
MDA-MB-231/ATTC
HS 578T
BT-549
54
–
30
–
30
–
50
–
43.5
50
–
–
–
–
–
T-47D
37.5
54
–
30
32.5
–
44
–
62
MDA-MB-468
–
–
31
a
Growth inhibition % produced by the compound is below 30%.
compounds scoring energy and binding mode in the VEGFR-2 active
site, 10 of the newly synthesized compounds were selected for the
evaluation of their effect on VEGFR-2 level in human breast cancer
MCF-7 cell line. The effect of the tested compounds on VEGFR-2 as
a marker for angiogenesis was determined as triplicate determi-
nations (Table 2).
2 inhibitory activity using the VEGFR-2 kinase inhibitory kit assay. IC50
of the selected tested compounds are given in Table 3 and compared
with sorafenib as a reference standard.
As seen in Table 3, the seven tested compounds displayed
moderate to potent VEGFR-2 inhibitory activities. Compounds 5a and
6 exhibited good potency with IC50 values of 1.14 and 1.11 μM,
respectively. Compound 18b was the most potent showing an IC50
value of 0.07 μM as compared to sorafenib IC50 (0.09 μM).
Compounds 5a, 6, 14b, and 18c showed potent inhibitory
effect of the VEGFR-2 level in (MCF-7) cell line as compared to the
untreated cancer cells with percentage inhibition values of 79, 68,
6
1, and 85%, respectively. While sorafenib, the reference
3
|
CONCLUSION
compound used in this test, showed a percentage reduction value
of 84%.
A series of indole based analogs 1–18 was synthesized. Most of the
synthesized compounds were chosen by the NCI DTP in Bethesda
(
Rockville, MD) for testing their antiproliferative action on a panel of 60
2
.3.3 In vitro VEGFR-2 kinase inhibitory assay
|
cell lines andcompound 18bexhibited a broad antiproliferative activity on
numerous cell lines. It was noticed that there was selectivity to nearly all
leukemia cell lines, non-small cell lung cancer (HOP-92), renal cancer
Based on their docking results in the VEGFR-2 active site and testing
the effect on cellular VEGFR-2 level, seven compounds (5a, 5b, 6, 7,
1
4b, 18b, and 18c) were chosen for further evaluation of their VEGFR-
(A498 and UO-31), and breast cancer (MDA-MB-231/ATTC).

10 of 17
ROAIAH ET AL.
|
TABLE 2 In vitro determination of VEGFR-2 level for selected
compounds in MCF-7 cell line
accomplish hydrogen bonding interactions with the active site key
amino acids Glu885 and/or Asp1046.
Compound
ID
IC50 MCF-
VEGFR-2 level
VEGFR-2
a
b
c
7 (µM)
(ng/mL)
inhibition (%)
4
4
4
|
EXPERIMENTAL
Chemistry
DMSO
–
1978.76 ± 236.00
1790.48 ± 188.00
410.10 ± 45.13
–
3
5
5
6
8
1
1
1
1
1
b
a
b
34.7
13
9.5
79
9
.1
|
53.3
16
1800.11 ± 195.00
632.85 ± 77.11
.1.1 General
|
68
0.4
6
All chemicals and solvents used were purchased from commercial
suppliers. Thin layer chromatography (TLC) was performed on
precoated silica gel 60 F245 aluminium sheets (Merck) and visualized
using Vilber Lourmet ultraviolet lamp at λ = 254 nm. Melting points
were recorded by open capillary tube method using an Electrothermal
melting point apparatus and are uncorrected. Spectral data and
elemental analysis were performed at the Central Services Unit –
National Research Centre (NRC) and Microanalytical Units – Faculty of
Science and Faculty of Pharmacy – Cairo University – Cairo – Egypt.
Infra-red spectra were recorded using KBr pellets on a Jasco FT/IR
b
90.6
54.3
17.3
87.4
83.2
11.4
6.12
1970.00 ± 220.47
1860.40 ± 190.34
766.81 ± 80.16
4a
4b
4d
4e
8c
61
0.4
0.65
85
84
1970.80 ± 223.76
1965.80 ± 211.70
291.88 ± 30.00
Sorafenib
320.60 ± 36.50
a
The concentration of each compound required to produce 50% inhibition
of cell growth.
1
13
b
6100 spectrophotometer. H- and C-NMR spectra were performed
at 400 (100) MHz recorded on a Bruker AVANCE III Nano Bay FT-NMR
Data were recorded as mean of three independent experiments.
c
Percentage inhibition as compared with control untreated cells.
6
spectrophotometer using dimethylsulfoxide (DMSO-d ) as a solvent.
Chemical shifts (δ) are given in parts per million (ppm) relative to
tetramethylsilane (TMS) as an internal standard. Coupling constants
are reported in Hertz (Hz). Mass spectra were recorded on FINNIGAN
MAT SSQ 7000 digital DEC 3000 and JEOL JMS-AX 500 mass
spectrometers. Elemental analysis was performed on a Vario EL
Elementar.
Compounds 5a, 6, 14b, and 18c showed potent reducing effect
on VEGFR-2 level in human breast cancer cell line (MCF-7) as
compared to the untreated cancer cells with percentage inhibition
values of 79, 68, 61, and 85%, respectively. The synthesized
compounds were docked into sorafenib VEGFR-2 active site and
based on the docking study results, seven compounds (5a, 5b, 6, 7,
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
1
4b, 18b, and 18c) were selected for further VEGFR-2 inhibitory
study. Compound 18b proved to be more potent than sorafenib with
IC50 values of 0.07 and 0.09 μM, respectively. Molecular docking
study attributed the remarkable activity of this series to their
hydrophobic interactions with the hydrophobic side chains of
Val848, Lys868, Leu889, Val916, and Phe1047 amino acids and
the hydrophobic allosteric back pocket of the active site lined with
the hydrophobic side chains of amino acids Ile888, Leu889, Ile892,
Val898, Val899, Leu1019. Besides, the ability of most of them to
[
17–19]
3
-(1H-Indol-2-yl)aniline (1)
It was reported to be synthesized by Fischer indole method. In our
work, we reported its synthesis for the first time via polyphosphoric
[19]
acid (PPA) as a cyclizing agent. Yield (90%); m.p. 133°C, (Lit. 136°C).
4
(
.1.2
1H-indol-2yl)phenyl)pyrroledione and isoindoline
derivatives (2a,b)
|
General procedure for the synthesis of 2-(3-
3
-(1H-indol-2-yl)aniline 1 (0.5 g, 2.4 mmol) and the appropriate acid
TABLE 3 IC50 (μM) values of selected compounds on VEGFR-2
anhydride (2.4 mmol) were refluxed in least amount of glacial acetic
acid (5 mL) for 2–3 h. The solution was left to concentrate in air, water
was added and the precipitate was collected, washed with water and
crystallized from acetone for compound 2b while compound 2a was
purified by column chromatography using ethyl acetate/petroleum
ether (80:100) in a ratio of 3:2.
a
Compound ID
VEGFR-2 IC50 (µM)
5
5
6
7
1
1
1
a
1.14
3.18
1.11
2.13
1.87
0.07
1.61
0.09
b
4b
8b
8c
1
-(3-(1H-Indol-2-yl)phenyl)-1H-pyrrole-2,5-dione (2a)
White crystals (yield 40%); m.p. 157–159°C; reaction time 2 h; IR
−
1
1
(
KBr) cm : 3352 (NH), 1708 (CO); H-NMR (DMSO-d
.93 (s, 1H, H-3 indole); 6.99–7.03 (m, 1H, Ar-H); 7.10–7.14 (m, 1H,
Ar-H); 7.24–7.28 (m, 3H, Ar-H); 7.41 (d, 1H, CH pyrrole dione,
6
) (δ, ppm):
Sorafenib
6
a
The concentration recorded to produce 50% inhibition of VEGFR-2.

ROAIAH ET AL.
11 of 17
|
J = 8 Hz); 7.54–7.60 (m, 2H, Ar-H); 7.82–7.83 (m, 1H, Ar-H); 7.90 (d,
4.36; N, 8.08; O, 4.61; Cl, 10.22. Found: C, 72.38; H, 3.98; N, 8.45; O,
4.22; Cl, 10.45.
1
H, CH pyrrole dione, J = 8 Hz); 11.60 (s, 1H, NH indole,
+
D
2
O
exchangeable); EIMS, m/z: 288 (M ); Anal. calcd. for
C
18
H
12
N
2
O
2
(288.09): C, 74.99; H, 4.20; N, 9.72; O, 11.10. Found:
4
.1.4 Synthesis of N-(3-(1H-indol-2-yl)phenyl)-2-
|
C, 74.38; H, 3.98; N, 9.45; O, 11.22.
chloroacetamide (4)
2
-(3-(1H-Indol-2-yl)phenyl)isoindoline-1,3-dione (2b)
To a well stirred solution of 3-(1H-indol-2-yl)aniline 1 (0.5 g, 2.4 mmol)
in dry dimethylformamide (5 mL), chloroacetyl chloride (0.3 mL,
2.4 mmol) was dropwisely added with continuous stirring on cold for
1 h. The solution was poured onto ice/water, the obtained precipitate
was collected, washed with water and purified by column chromatog-
raphy using ethyl acetate/petroleum ether (80:100) in a ratio of 3:2.
Green crystals (yield 45%); m.p. 95–97°C; reaction time 3 h; IR
−
1
(
1
KBr) cm
:
3368 (NH), 3054 (CH aromatic), 1713 (2CO);
H-NMR (DMSO-d ) (δ, ppm): 6.73 (s, 1H, H-3 indole); 6.90–7.08
m, 3H, Ar-H); 7.35–7.63 (m, 4H, Ar-H); 7.91–8.14 (m, 5H, Ar-H);
6
(
1
3
1
2 6
1.61 (s, 1H, NH indole, D O exchangeable); C-NMR (DMSO-d )
−
1
(
δ, ppm): 99.9, 111.9, 116.0, 117.3, 120.4, 120.7, 121.0, 122.3,
Yield 52%; m.p. 209–211°C; IR (KBr) cm : 3406, 3278 (NH indole,
1
1
1
23.0, 124.0, 124.5, 126.9, 129.8, 131.0, 132.0, 133.1, 135.3,
NH-CO), 1677 (CO amidic); H-NMR (DMSO-d
6
) (δ, ppm): 4.29 (s,
+
67.5; EIMS, m/z: 338 (M ); Anal. calcd. for C22
H
14
N
2
O
2
(338.11):
2Hs, OC-CH
7. 40–7.60 (m, 5H, Ar-H); 8.01–8.02 (m, 1H, Ar-H); 10.39 (s, 1H,
NHCO,
exchangeable); 11.53 (s, 1H, NH indole,
O exchangeable);
2
-Cl); 6.80 (s, 1H, H-3 indole); 6.95–7.13 (m, 2H, Ar-H);
C, 78.09; H, 4.17; N, 8.28; O, 9.46. Found: C, 77.81; H, 4.64; N,
.31; O, 9.36.
8
2
D O
1
3
D
2
6
C-NMR (DMSO-d ) (δ, ppm): 44.0, 99.3,
1
1
11.9, 116.8, 119.3, 119.9, 120.6, 121.2, 122.2, 129.0, 129.9,
33.4, 137.7, 138.0, 139.4, 165.3; Anal. calcd. for
4
(
.1.3
|
General procedure for the synthesis of N-(3-
1H-indol-2-yl)phenyl)benzamide/4-chlorobenzamide
derivatives (3a,b)
C
16
2
H13ClN O (284.07): C, 67.49; H, 4.60; N, 9.84; O, 5.62; Cl,
1
2.45. Found: C, 67.79; H, 4.80; N, 10.14; O, 5.22; Cl, 12.25.
To a well stirred solution of 3-(1H-indol-2-yl)aniline 1 (0.5 g, 2.4 mmol)
in dry dimethylformamide (3 mL), the appropriate acid chloride
4
(
.1.5
|
General procedure for the synthesis of N-(3-
(
2.4 mmol) was dropwisely added with continuous stirring on cold
1H-indol-2-yl)phenyl)-2-(piperidine/piperazin/
morpholine-1-yl)acetamide (5a–c)
for 2 h. The solution was poured onto ice/water, the formed
precipitate was collected, washed with water and crystallized from
ethanol for compound 3b while compound 3a was purified by column
chromatography using ethyl acetate/petroleum ether (80:100) in a
ratio of 3:2.
To a well stirred solution of N-(3-(1H-indol-2-yl)phenyl)-2-chloroace-
tamide 4 (0.5 g, 1.8 mmol) in dry dimethylformamide (3 mL), the
appropriate secondary amine (2 mmol) was added in the presence of
(
0.2 g, 2.2 mmol) anhydrous potassium carbonate with continuous
N-(3-(1H-Indol-2-yl)phenyl)benzamide (3a)
stirring on cold for 1 h. The solution was poured onto ice/water to give
a precipitate which was collected, washed with water and crystallized
from acetone for compound 5a and ethanol for compounds 5b and 5c.
Commercially available, CAS Registry Number: 1115499-93-8; whitish
−1
yellow crystals (yield 42%); m.p. 222–224°C; IR (KBr) cm : 3429, 3394
1
(
NH), 1650 (CO amidic); H-NMR (DMSO-d
6
) (δ, ppm): 6.8 (s, 1H,
H-3 indole); 6.99–7.03 (m, 1H, Ar-H); 7.09–7.13 (m, 1H, Ar-H); 7.4–7.5
N-(3-(1H-Indol-2-yl)phenyl)-2-(piperidin-1-yl)acetamide (5a)
−
1
(
m, 2H, Ar-H); 7.55–7.7 (m, 5H, Ar-H); 8–8.03 (m, 2H, Ar-H); 8.25–8.3
m, 1H, Ar-H); 10.36 (s, 1H, NHCO, D O exchangeable); 11.55 (s, 1H,
O exchangeable); Anal. calcd. for C21 O (312.13):
Green crystals (yield 55%); m.p. 112–114°C; IR (KBr) cm : 3409, 3283
1
(
2
(NH), 1673 (CO amidic); H-NMR (DMSO-d
6
) (δ, ppm): 1.43–1.61 (m,
NH indole, D
2
H
16
N
2
6H, piperidine H); 2.45–2.47 (m, 4H, piperidine H) 3.07 (s, 2H, CH
2
);
C, 80.75; H, 5.16; N, 8.97; O, 5.12. Found: C, 80.81; H, 5.64; N, 8.31;
O, 5.36.
6.83 (s, 1H, H-3 indole); 6.97–7.12 (m, 2H, Ar-H); 7.37–7.62 (m, 5H,
Ar-H); 8.05–8.06 (m, 1H, Ar-H); 9.68 (s, 1H, NHCO,
2
D O exchangeable); 11.48 (s, 1H, NH indole, D
2
O exchangeable);
N-(3-(1H-Indol-2-yl)phenyl)-4-chlorobenzamide (3b)
Anal. calcd. for C21 O (333.18): C, 75.65; H, 6.95; N, 12.60; O,
23 3
H N
−
1
Green crystals (yield 50%); m.p. 172–174°C; IR (KBr) cm : 3413 (NH),
4.80. Found: C, 75.38; H, 6.38; N, 12.85; O, 4.22.
1
1
686 (CO amidic); H-NMR (DMSO-d
6
) (δ, ppm): 6.83 (s, 1H, H-3
indole); 7.01–7.02 (m, 1H, Ar-H); 7.09–7.13 (m, 1H, Ar-H); 7.41–7.48
m, 2H, Ar-H); 7.57 (d, 2H, Ar-H, J = 8.4 Hz); 7.61–7.67 (m, 2H, Ar-H);
.95 (d, 2H, Ar-H, J = 8.4 Hz); 8.05 (d, 1H, Ar-H, J = 8.4 Hz); 8.29 (s, 1H,
Ar-H); 10.43 (s, 1H, NH-CO, D
O exchangeable); 11.56 (s, 1H, NH
O exchangeable); C-NMR (DMSO-d ) (δ, ppm): 99.2, 111.9,
18.0, 119.9, 120.4, 121.2, 122.1, 129.0, 129.2, 129.7, 130.1, 131.6,
33.2, 134.0, 137.7, 138.1, 138.3, 139.9, 166.9; EIMS, m/z: 348
N-(3-(1H-Indol-2-yl)phenyl)-2-(piperazin-1-yl)acetamide (5b)
−1
(
Green crystals (yield 60%); m.p. 125–127°C; IR (KBr) cm : 3318 (NH),
1
7
1703 (CO amidic); H-NMR (DMSO-d
6
) (δ, ppm): 2.46–2.50 (m, 4H,
); 3.36 (s,
O exchangeable); 6.83 (s, 1H, H-3 indole);
6.97–7.20 (m, 2H, Ar-H); 7.40–7.61 (m, 5Hs, Ar-H); 8.07–8.09 (m, 1H,
2
piprazine H); 2.60–2.68 (m, 4H, piprazine H); 3.30 (s, 2H, CH
2
1
3
indole, D
2
6
1H, NH piprazine, D
2
1
1
Ar-H); 9.75 (s, 1H, NHCO, D
13
2
O exchangeable); 11.50 (s, 1H, NH indole,
+
+
(
M +2), 346 (M ); Anal. calcd. for C21
H15ClN
2
O (346.09): C, 72.73; H,
2 6
D O exchangeable); C-NMR (DMSO-d ) (δ, ppm): 46.0, 53.2, 62.3,

12 of 17
ROAIAH ET AL.
|
9
1
9.3, 111.9, 116.9, 119.2, 119.9, 120.6, 120.8, 122.1, 129.0, 129.8,
4.1.6
|
General procedure for the synthesis of 2-((2-
+
33.3, 137.6, 138.0, 139.56, 169.0 EIMS, m/z: 334 (M ); Anal. calcd.
O (334.41): C, 71.83; H, 6.63; N, 16.75; O, 4.78. Found: C,
1.61; H, 6.94; N, 16.31; O, 4.96.
(3-acetamidophenyl)-1H-indol-3-yl)methylene)-
hydrazinecarboxamide (8a) and N-(3-(3-((2-
isonicotinyl/2-chlorophenylhydrazono)methyl)-1H-
indol-2-yl)phenyl)acetamide (8b,c)
22 4
for C20H N
7
N-(3-(1H-Indol-2-yl)phenyl)-2-morpholinoacetamide (5c)
N-(3-(3-Formyl-1H-indol-2-yl)phenyl)acetamide 7 (0.5 g, 1.8 mmol)
and semicarbazide hydrochloride or isonicotinic acid hydrazide or
o-chlorophenylhydrazine hydrochloride, respectively (1.9 mmol), were
refluxed in absolute ethanol (20 mL) in the presence of 2–3 drops of
glacial acetic acid for 2 h. The solution was concentrated to half its
volume and diluted with water. A precipitate was formed, filtered and
crystallized from ethanol.
−1
Green crystals (yield 57%); m.p. 193–195°C; IR (KBr) cm : 3412, 3281
1
(
NH), 1669 (CO amidic); H-NMR (DMSO-d
4
6
) (δ, ppm): 2.47–2.48 (m,
-CO); 3.66–3.69 (m, 4H,
morpholine H); 6.82 (s, 1H, H-3 indole); 6.99–7.20 (m, 2H, Ar-H);
.40–7.59 (m, 5H, Ar-H); 8.07–8.08 (m, 1H, Ar-H); 9.77 (s, 1H, NHCO,
O exchangeable); 11.48 (s, 1H, NH indole, D O exchangeable);
) (δ, ppm): 53.7, 62.6, 66.6, 99.3, 111.9, 116.9,
19.3, 119.9, 120.6, 120.8, 122.1, 129.0, 129.7, 133.2, 137.6, 138.0,
39.5, 168.8; Anal. calcd. for C20 (335.40): C, 71.62; H, 6.31;N,
2.53; O, 9.54. Found: C, 71.08; H, 6.98; N, 12.85; O, 9.22.
H, morpholine H); 3.14 (s, 2H, CH
2
7
D
2
2
1
3
C-NMR (DMSO-d
6
1
1
1
2-((2-(3-Acetamidophenyl)-1H-indol-3-yl)methylene)hydrazine
21 3 2
H N O
carboxamide (8a)
Yellowish brown crystals (yield 37%); m.p. 130–132°C; IR (KBr)
−
1
[
20]
cm : 3435, 3318, & 3142 (NH indole, NH-C

O, N-NH), 1676
6
) (δ, ppm): 2.10 (s,
N-(3-(1H-Indol-2-yl)phenyl)acetamide (6)
1
(
CO amidic), 1585 (CN); H-NMR (DMSO-d
3
-(1H-Indol-2-yl)aniline 1 (40 g, 192 mmol) was refluxed with glacial
3
H, CH
s, 2H, NH
2
exchangeable); 10.34 (s,1H, N-NH, D O
3
); 7.14–7.99 (m, 8H, Ar-H); 8.19 (s, 1H, CHN); 9.45
exchangeable); 9.86 (s, 1H, NHCO,
exchangeable);
O exchangeable); C-NMR (DMSO-d
(δ, ppm): 24.5, 108.3, 112.0, 119.6, 120.2, 121.1, 122.7, 124.1,
acetic acid (100 mL) for 3 h, the reaction mixture was concentrated to
half its volume and water was added to give the acetylated product
which was filtered and crystallized from ethanol. Green precipitate
(
2 2
, D O
D
2
O
1
3
−
1
11.78 (s,1H, NH indole, D
2
6
)
(
yield 64%); m.p. 194–196°C; IR (KBr) cm : 3404 (NH indole, NH-
1
CO), 2923 (CH aliphatic), 1663 (CO amidic); H-NMR (DMSO-d
)
6
1
1
25.6, 125.7, 129.7, 130.1, 132.1, 136.9, 138.7, 140.2, 158.3,
(
δ, ppm): 2.09 (s, 3H, CH
3
); 6.73 (s, 1H, H-3 indole); 6.97–6.99 (m, 1H,
+
69.2; EIMS, m/z: 335 (M ); Anal. calcd. for C18
H
17
N
5
O
2
(335.14):
Ar-H); 7.11–7.21 (m, 1H, Ar-H); 7.35–7.73 (m, 5H, Ar-H); 8.01–8.05
C, 64.47; H, 5.11; N, 20.88; O, 9.54. Found: C, 64.08; H, 5.62; N,
20.67; O, 9.77.
(
m, 1H, Ar-H); 10.00 (s, 1H, NHCO, D
2
O exchangeable); 11.48 (s, 1H,
+
NH indole, D
2
O exchangeable); EIMS, m/z: 250 (M ); Anal. calcd. for
C
16
H
14
N
2
O (250.11): C, 76.78; H, 5.64; N, 11.19; O: 6.39. Found: C,
N-(3-(3-((2-Isonicotinoylhydrazono)methyl)-1H-indol-2-yl)-
7
6.28; H, 5.33; N, 10.89; O, 6.77.
phenyl)acetamide (8b)
−
1
Green crystals (yield 35%); m.p. 257–259°C; IR (KBr) cm : 3392,
N-(3-(3-Formyl-1H-indol-2-yl)phenyl)acetamide (7)
3
1
260 (NH indole, NH-CO), 1665 (CO amidic), 1597 (CN);
H-NMR (DMSO-d ) (δ, ppm): 2.09 (s, 3H, CH ); 7.20–7.83 (m, 8H,
6 3
Phosphorous oxychloride (12 mL, 80 mmol) was dropwisely added to
dimethylformamide (18 mL, 240 mmol) while cooling in an ice bath for
Ar-H); 8.44–8.71 (m, 4H, Ar-H); 8.76 (s, 1H, CHN); 10.16 (s,
H, NHCO,
exchangeable); 11.75 (s, 1H, N-NH,
O exchangeable); 11.91 (s, 1H, NH indole, D O exchangeable);
(397.15):
C,69.51; H, 4.82; N, 17.62; O, 8.05. Found C, 69.28; H, 5.02; N,
7.89, O, 8.77.
1
4
h. A solution of N-(3-(1H-indol-2-yl)phenyl)acetamide 6 (10 g,
1
2
D O
0 mmol) in dimethylformamide (20 mL) was portionwisely added to
D
2
2
the formylating mixture at 0–10°C and the temperature was raised and
kept at 35°C for 1.5 h. The mixture was then poured onto ice/cold
water and neutralized with sodium hydroxide solution to give a yellow
precipitate which was collected by vacuum filtration and purified by
column chromatography using ethyl acetate/petroleum ether (60:80)/
chloroform in a ratio of 3:2:1. Yield 35%; m.p. 250–252°C; IR (KBr)
+
19 5 2
EIMS m/z (%): 397 (M ), 7.7%; Anal. calcd. for C23H N O
1
N-(3-(3-((2-(2-Chlorophenyl)hydrazono)methyl)-1H-indol-2-yl)-
−
1
cm : 3435, 3262 (NH indole, NH-CO), 2929 (CH aliphatic), 1676
phenyl)acetamide (8c)
1
−1
(
CO aldehydic), 1640 (CO amidic); H-NMR (DMSO-d
6
) (δ, ppm):
Light brown crystals (yield 30%); m.p. 137–139°C; IR (KBr) cm
:
2
7
1
.09 (s, 3H, CH
3
); 7.21–7.32 (m, 2H, Ar-H); 7.39–7.41 (m, 1H, Ar-H);
3396, 3250 (NH indole, NH-CO, N-NH), 2919 (CH aliphatic),
1
.45–7.55 (m, 2H, Ar-H); 7.66 (d, 1H, Ar-H, J = 7.8 Hz); 7.94–7.96 (m,
H, Ar-H); 8.20 (d, 1H, Ar-H, J = 9 Hz); 10.00 (s, 1H, CHO); 10.16 (s, 1H,
1667 (CO amidic), 1596 (CN); H-NMR (DMSO-d
6
) (δ, ppm):
2.09 (s, 3H, CH
CHN); 9.55 (s, 1H, N-NH, D
NHCO,
exchangeable); 11.66 (s, 1H, NH indole,
3
); 6.74–8.35 (m, 12H, Ar-H); 8.61 (s, 1H,
NHCO,
D
D
2
O
exchangeable); 12.38 (s, 1H, NH indole,
O exchangeable); 10.16 (s, 1H,
2
13
2
O exchangeable); C-NMR (DMSO-d
6
) (δ, ppm): 24.5, 112.5,
2
D O
+
+
1
1
14.0, 120.6, 120.7, 121.5, 122.9, 124.2, 124.6, 126.1, 129.9, 130.5,
D
2
O exchangeable); EIMS, m/z: 404 (M +2), 403 (M +1), 402
+
+
36.3, 140.2, 149.4, 169.1, 186.0. EIMS, m/z: 278 (M ); Anal. calcd. for
(M ); Anal. calcd. for C23
H
19ClN
4
O (402.12): C, 68.57; H, 4.75;
C
17
H
14
N
2
O
2
(278.11): C, 73.37; H, 5.07; N, 10.07; O, 11.50. Found: C,
Cl, 8.80; N, 13.91; O, 3.97. Found: C, 69.08; H, 4.02; Cl, 8.20; N,
13.67; O, 3.57.
7
3.28; H, 5.63; N, 10.89; O, 11.77.

ROAIAH ET AL.
13 of 17
|
4.1.7
|
General procedure for the synthesis of (2-(3-
4.1.11 General procedure for the synthesis of 4-((5-
|
acetamidophenyl)-1H-indol-3-yl)methylene
derivatives (9a,b)
((1H-indol-3-yl)methylene)-4-oxo-4,5-dihydrothiazol-
2-yl)-amino)benzenesulfonamide derivatives (14a–e)
N-(3-(3-Formyl-1H-indol-2-yl)phenyl)acetamide 7 (0.5 g, 1.8 mmol)
and the appropriate active methylene derivative (1.8 mmol) were
refluxed in absolute ethanol (15 mL) in the presence of few drops of
triethylamine for 3h. The reaction mixture was concentrated to half its
volume and diluted with cold water where a precipitate was formed,
filtered, and crystallized from ethanol.
A mixture of indole-3-carbaldehyde 13 (1.45 g, 10 mmol), the
appropriate thiazolidinone 11a–e (10 mmol) and anhydrous sodium
acetate (0.82 g, 10 mmol) in glacial acetic acid (15 mL) was refluxed for
5–18 h. The separated solid was filtered while hot, washed with hot
water and crystallized from dimethylformamide/water, except com-
pounds 14b,d were crystallized from ethanol.
N-(3-(3-(2,2-Dicyanovinyl)-1H-indol-2-yl)phenyl)acetamide (9a)
4-((5-((1H-Indol-3-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-yl)-
−1
[31]
Yellow crystals (yield 49%); m.p. 160–162°C; IR (KBr) cm : 3250 (NH),
amino)benzene sulfonamide (14a)
1
2
919 (CH aliphatic), 2217 (CN), 1564 (CC). H-NMR (DMSO-d
6
) (δ,
Green crystals (yield 60%); m.p. >300°C; reaction time 5 h; IR (KBr)
−
1
ppm): 2.10 (s, 3H, CH
3
); 7.02–8.08 (m, 8H, Ar-H); 8.15 (s, 1H, CHC);
cm : 3396, 3310, 3240 (NH, NH ), 3046 (CH aromatic), 1667 (CO
2
1
1
0.23 (s, 1H, NHCO, D
2
O exchangeable); 13.08 (s, 1H, NH indole,
amidic), 1319 (SO2 asymmetric), 1155 (SO2 symmetric); H-NMR
+
D
2
O
exchangeable). EIMS, m/z: 326 (M ); Anal. calcd. for
(DMSO-d ) (δ, ppm): 7.04–7.20 (m, 4H, Ar-H); 7.32 (s, 2H, NH ,
6
2
C
20
H
14
N
4
O (326.12): C, 73.61; H, 4.32; N, 17.17; O, 4.90. Found: C,
2
D O exchangeable); 7.49 (d, 1H, Ar-H, J = 8 Hz); 7.70 (s, 1H, NH,
7
3.18; H, 4.21; N, 17.45; O, 5.22.
2
D O exchangeable); 7.83–7.86 (m, 4H, 3Ar-H, H-2 indole); 7.93 (s, 1H,
13
2
methine H); 11.88 (s, 1H, NH indole, D O exchangeable); C-NMR
Ethyl 3-(2-(3-acetamidophenyl)-1H-indol-3-yl)-2-cyanoacrylate
9b)
Yellow crystals (yield 42.5%); m.p. 149–151°C; IR (KBr) cm
(DMSO-d ) (δ, ppm): 111.0, 112.9, 116.0, 118.7, 121.3, 122.1, 123.4,
6
(
127.3, 127.7, 128.4, 136.70, 140.1, 151.8, 152.8, 164.2, 172.7. EIMS,
−
1
+
:
m/z: 398 (M ); Anal. calcd. for C18H14N O S (398.46): C, 54.26; H,
4
3 2
3
1
262 (NH), 2921 (CH aliphatic), 2214 (CN), 1683 (CO ester),
3.54; N, 14.06; O, 12.05; S, 16.09. Found: C, 54.01; H, 3.14; N, 14.31;
O, 12.66; S, 16.56.
1
575 (CC); H-NMR (DMSO-d
) (δ, ppm): 1.24–1.29 (t, 3H,
); 4.22–4.29 (q, 2H, CH -CH );
.12–7.29 (m, 3H, Ar-H); 7.44–7.57 (m, 2H, Ar-H); 7.79 (d, 1H,
Ar-H, J = 7.5 Hz); 7.85–7.90 (m, 2H, Ar-H, CHC); 8.18 (d, 1H,
Ar-H, J = 6 Hz); 10.20 (s, 1H, NH-CO, D
O exchangeable);
exchangeable); EIMS, m/z:
(373.14): C, 70.76; H,
6
CH
2
-CH
3
); 2.09 (s, 3H, CH
3
2
3
7
4-((5-((1H-Indol-3-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-yl)-
amino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (14b)
Brown crystals (yield 50%); m.p. 268–270°C; reaction time 18 h;
−1
2
1
3
5
1
2.70 (s, 1H, NH indole,
D
2
O
IR (KBr) cm : 3332 (NH), 3062 (CH aromatic), 2927 (CH aliphatic),
+
73 (M ); Anal. calcd. for C22
H
19
N
3
O
3
1
654 (CO amidic), 1222 (SO
2
asymmetric), 1161 (SO
2
symmet-
.13; N, 11.25; O, 12.85. Found: C, 71.01; H, 4.64; N, 11.31; O,
2.36.
1
ric); H-NMR (DMSO-d
6
) (δ, ppm): 2.31 (s, 3H, CH
3
); 6.18 (s, 1H,
CH isoxazole); 7.16–7.26 (m, 4H, Ar-H); 7.49 (d, 1H,
Ar-H, J = 7.9 Hz); 7.66 (s, 1H, NH, exchangeable);
.87–7.96 (m, 5H; 3Ar-Hs, methine H, & H-2 indole); 11.45 (s,
1H, NH, exchangeable); 11.94 (s, 1H, NH indole,
O exchangeable); Anal. calcd. for C22 (479.53): C,
2
D O
7
4
.1.8
|
Synthesis of N-chloroacetyl sulfonamide
[
23,24]
2
D O
derivatives (10a–e)
D
2
17 5 4 2
H N O S
Compounds 10a–e were prepared according to the procedure cited in
literature. Yield: 60–80%; m.p. are 216–218, 146–148, 235–237,
55.10; H, 3.57; N, 14.60; O, 13.35; S, 13.37. Found: C, 55.38; H,
3.38; N, 14.85; O, 13.11; S, 13.66.
1
92–194, 228–230°C, respectively.
4-((5-((1H-Indol-3-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-yl)-
amino)-N-(pyrimidin-2-yl)benzenesulfonamide (14c)
4
.1.9
|
Synthesis of 4-((4-oxo-4,5-dihydrothiazol-2-
[
16]
Green crystals (yield 40%); m.p. >300°C; reaction time 18 h; IR
yl)-amino)benzene sulfonamide derivatives (11a–e)
−
1
(
KBr) cm : 3327, 3250 (NH), 3064 (CH aromatic), 1682 (CO
1
Compounds 11a–e were synthesized according to the reported
procedure. Yield: 73–90%; m.p. are 244–246, 259–261, 297°C decom-
posed, 240–242, >300°C, respectively.
amidic), 1291 (SO2 asymmetric), 1152 (SO2 symmetric); H-NMR
(DMSO-d ) (δ, ppm): 7.02–7.03 (m, 1H, Ar-H); 7.14–7.25 (m, 4H,
6
Ar-H); 7.50 (d, 1H, Ar-H, J = 7.8 Hz); 7.64 (s, 1H, NH,
D
2
O exchangeable); 7.85 (d, 1H, Ar-H, J = 7.5 Hz); 7.96–8.03 (m,
5
H, 3Ar-H, H-2 indole & SO NH); 8.50–8.52 (m, 2H, Ar-H &
2
4
.1.10 Synthesis of 1H-indole-3-carbaldehyde
|
methine H); 11.97 (s, 1H, NH indole, D
2
O exchangeable). EIMS, m/
(476.53): C, 55.45; H,
[
25–29]
(
13)
+
z: 476 (M ); Anal. calcd. for C22
H
16
N
6
O
3
S
2
It was synthesized from 1H-indole 12 according to the reported
3.38; N, 17.64; O, 10.07; S, 13.46. Found: C, 55.38; H, 3.78; N,
17.35; O, 10.51; S, 13.66.
[25–29]
procedure. Yield: 81%; m.p. 194°C (Lit., 194–196°C).

14 of 17
ROAIAH ET AL.
|
4
-((5-((1H-Indol-3-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-yl)-
1H, Ar-H); 7.53 (d, 2H, Ar-H, J = 8.5 Hz); 7.70–7.75 (m, 1H, Ar-H); 7.93
(d, 2H, Ar-H, J = 8.5 Hz); 8.15–8.19 (m, 1H, Ar-H); 8.36–8.44 (m, 1H,
Ar-H); 8.89 (s, 1H, methine H); 10.10 (s, 1H, H-2 indole; Anal. calcd. for
amino)-N-(pyridin-2-yl)benzenesulfonamide (14d)
Green crystals (yield 45%); m.p. >300°C; reaction time 10 h; IR (KBr)
−
1
cm : 3389 (NH), 1630 (CO amidic), 1292 (SO
assymetric), 1133
C
H
13ClN
2 3
O (364.06): C, 65.85; H, 3.59; N, 7.68; O, 13.16; Cl, 9.72.
2
20
1
(
SO
2
symetric); H-NMR (DMSO-d
6
) (δ, ppm): 6.87–6.91 (m, 1H, Ar-H);
Found: C, 65.55; H, 3.79; N, 7.48; O, 13.66; Cl, 9.92.
7
.15–7.40 (m, 4H, Ar-H); 7.49 (d, 1H, Ar-H, J = 7.5 Hz); 7.63 (s, 1H, NH,
D
2
O exchangeable); 7.74–8.04 (m, 9H; 6 Ar-Hs, H-2 indole, SO
2
NH &
O exchangeable). EIMS, m/z:
(475.54): C, 58.09; H, 3.60; N,
4
(
.1.14
|
General procedure for the synthesis of (Z)-4-
methine H); 11.97 (s, 1H, NH indole, D
2
4-((1-acetyl/1H-indol-3-yl)methylene)-5-oxo-2-
phenyl/4-chlorophenyl-4,5-dihydro-1H-imidazol-1-
yl)-benzene sulfonamide/N-substituted
+
4
1
75 (M ); Anal. calcd. for C23
H
17
N
5
O
3
S
2
4.73; O, 10.09; S, 13.49. Found: C, 58.38; H, 3.78; N, 14.35; O, 9.88;
S, 13.66.
benzenesulfonamide (17a–d and 18a–c)
4
-((5-((1H-Indol-3-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-yl)-
A mixture of the oxazolone derivative 16a,b (10 mmol), the appropriate
sulfonamide (10 mmol), and anhydrous sodium acetate (0.5 g, 6 mmol)
in glacial acetic acid (20 mL) was heated in a boiling water bath for
1–30 h. Compounds 17a–d were precipitated on hot, filtered, washed
with hot water, and crystallized from dimethylformamide/water.
Compounds 18a–c were precipitated after pouring onto ice/cold
water, filtered, washed with hot water, and purified with column
chromatography using ethyl acetate/petroleum ether/chloroform
3:2:1.
amino)-N-(thiazol-2-yl)benzenesulfonamide (14e)
Yellow crystals (yield 54%); m.p. 253–255°C; reaction time 21 h; IR
−1
(
KBr) cm : 3429, 3263 (NH), 1645 (CO amidic), 1222 (SO
2
1
asymmetric), 1138 (SO
2
symmetric); H-NMR (DMSO-d
6
) (δ, ppm):
6
.84 (d, 1H, CH-5 thiazole, J = 4.5 Hz); 7.17–7.23 (m, 4H, Ar-H); 7.27
d, 1H, CH-4 thiazole, J = 4.5 Hz); 7.49 (d, 1H, Ar-H, J = 7.7 Hz); 7.66 (s,
H, NH, D O exchangeable); 7.81–7.94 (m, 5H; 3Ar-H, H-2 indole &
methine H); 11.90 (s, 1H, SO NH, D O exchangeable); 12.25 (s, 1H,
NH indole, D O exchangeable); Anal. calcd. for C21
481.57): C, 52.38; H, 3.14 N, 14.54; O, 9.97; S, 19.98. Found: C,
(
1
2
2
2
2
15 5 3 3
H N O S
(
(Z)-4-(4-((1-Acetyl-1H-indol-3-yl)methylene)-5-oxo-2-phenyl-
4,5-dihydro-1H-imidazol-1-yl)-N-(pyridin-2-yl)-
benzenesulfonamide (17a)
5
2.48; H, 3.66; N, 14.35; O, 10.22; S, 19.66.
Yellow crystals (yield 30%); m.p. 278–280°C; reaction time 2 h; IR (KBr)
4
.1.12
|
Synthesis of N-benzoyl/N-(4-chlorobenzoyl)-
−1
cm : 3429 (NH), 2925 (CH aliphatic); 1705 (CO imidazolone), 1631
CO amidic); 1375 (SO
asymmetric), 1134 (SO symmetric);
) (δ, ppm): 2.77 (s, 3H, CH ); 7.14–7.22 (m, 2H,
glycine (15a,b)
(
2
2
1
Compounds 15a,b were synthesized according to the reported
H-NMR (DMSO-d
6
3
[32,33]
procedure.
respectively.
Yield: 90–95%; m.p. are 187–189C, 144–146°C,
Ar-H); 7.38–7.49 (m, 3H, Ar-H); 7.50–8.07 (m, 10H, 9Ar-H & CH
methine); 8.34 (d, 1H, Ar-H, J = 8 Hz); 8.39 (d, 1H, Ar-H, J = 8 Hz); 8.49
(
d, 1H, Ar-H, J = 8 Hz); 8.90 (s, 1H, H-2 indole); 10.58 (s, 1H, SO
2
NH,
1
2 6
3
D
O exchangeable); C-NMR (DMSO-d ) (δ, ppm): 24.2, 115.1, 116.4,
4
(
.1.13
(1-acetyl-1H-indol-3-yl)methylene)-2-phenyl/4-
chlorophenyl)oxazol-5(4H)-one (16a,b)
|
General procedure for the synthesis of (Z)-4-
1
1
1
16.5, 116.7, 119.8, 119.9, 120.4, 121.2, 124.2, 124.7, 125.9, 126.2,
27.7, 128.0, 128.4, 128.5, 128.9, 129.3, 129.7, 131.25, 131.4, 133.7,
35.0, 135.8, 137.5, 143.3, 159.1, 164.8, 169.0, 170.2. EIMS, m/z: 561
+
A
mixture of indole-3-carbaldehyde 13 (1.45 g, 10 mmol), the
(M ); Anal. calcd. for C31
H
23
N
5
O
4
S (561.61): C, 66.30; H, 4.13; N,
appropriate N-benzoyl glycine/(hippuric acid) derivative 15a,b
12.47; O, 11.40; S, 5.71. Found: C, 66.56; H, 4.18; N, 12.18; O, 11.99;
S, 5.32.
(
(
12 mmol) and fused sodium acetate (0.5 g, 6 mmol) in acetic anhydride
20 mL) was heated in a boiling water bath for 4 h. The mixture was
cooled and ethanol was slowly added and allowed to stand overnight at
°C (in refrigerator). A crystalline product was formed, filtered, washed
with hot water and crystallized from petroleum ether (40:60).
(Z)-4-(4-((1-Acetyl-1H-indol-3-yl)methylene)-5-oxo-2-phenyl-
4,5-dihydro-1H-imidazol-1-yl)-N-(thiazol-2-yl)-
benzenesulfonamide (17b)
8
Yellow crystals (yield 40%); m.p. >300°C; reaction time 1h; IR (KBr)
−
1
(
Z)-4-((1-Acetyl-1H-indol-3-yl)methylene)-2-phenyloxazol-
cm : 3430 (NH), 2894 (CH aliphatic); 1705 (CO imidazolone), 1631
[
32,34]
5
(4H)-one (16a)
(CO amidic); 1376 (SO
H-NMR (DMSO-d ) (δ, ppm): 2.77 (s, 3H, CH ); 6.88 (d, 1H, CH-5
6 3
2
asymmetric), 1146 (SO
2
symmetric);
[34]
1
Yield: 46%; m.p. 203–205°C as reported in the literature.
thiazole, J = 4.5 Hz); 7.29 (d, 1H, CH-4 thiazole, J = 4.5 Hz); 7.42–7.46
(m, 5H, Ar-H); 7.52–7.56 (m, 4H, Ar-H); 7.58 (s, 1H, methine H); 7.88 (d,
2H, Ar-H, J = 8.3 Hz); 8.39 (d, 1H, Ar-H, J = 7.7 Hz); 8.49 (d, 1H, Ar-H,
(
Z)-4-((1-Acetyl-1H-indol-3-yl)methylene)-2-(4-chlorophenyl)-
oxazol-5(4H)-one (16b)
−
1
Yellow crystals (yield 35%); m.p. 142–144°C; IR (KBr) cm : 2928 (CH
J = 7.2 Hz); 8.90 (s, 1H, H-2 indole); 12.84 (s, 1H, SO
2
NH,
) (δ, ppm): 24.4, 109.1,
116.5, 116.8, 120.4, 121.2, 124.5, 125.2, 125.8, 126.2, 127.4, 128.6,
1
3
aliphatic), 1800 (O-CO oxazolone), 1685 (CO amidic), 1591
2 6
D O exchangeable); C-NMR (DMSO-d
1
(
CC); H-NMR (DMSO-d
6
) (δ, ppm): 2.77 (s, 3H, CH
3
); 7.41–7.48 (m,

ROAIAH ET AL.
15 of 17
|
1
1
29.1, 129.4, 132.1, 133.7, 135.8, 137.7, 138.0, 141.7, 142.4, 159.0,
69.0, 169.5, 170.2; Anal. calcd. for C29 (567.64): C, 61.36;
1631 (CO amidic), 1373 (SO
H-NMR (DMSO-d ) (δ, ppm): 2.32 (s, 3H, CH ); 6.16 (s, 1H, CH
6 3
2
asymmetric), 1162 (SO
2
symmetric;
1
H
21
N
5
O
4
S
2
H, 3.73; N, 12.34; O, 11.27; S, 11.30. Found: C, 61.88; H, 3.38; N,
2.85; O, 11.11; S, 11.66.
isoxazole); 7.22–7.26 (m, 3H, Ar-H); 7.375–7.52 (m, 5H, Ar-H); 7.67 (s,
1H, methine H); 7.91–8.08 (m, 2H, Ar-H); 8.10–8.15 (m, 1H, Ar-H);
1
8
.41–8.45 (m, 1H, Ar-H); 8.61 (s, 1H, H-2 indole); 11.56 (s, 1H, SO
O exchangeable), 12.18 (s, 1H, NH indole, D O exchangeable); Anal.
calcd. for C28 S (523.56): C, 64.23; H, 4.04; N, 13.38; O, 12.22;
2
NH,
(
Z)-4-(4-((1-Acetyl-1H-indol-3-yl)methylene)-2-(4-
chlorophenyl)-5-oxo-4,5-dihydro-1H-imidazol-1-yl)-N-(pyridin-
-yl)benzenesulfonamide (17c)
Yellow crystals (yield 50%); m.p. 258–260°C; reaction time 2 h; IR (KBr)
D
2
2
21 5 4
H N O
2
S, 6.12. Found: C, 64.77; H, 4.13; N, 13.87; O, 11.88; Cl, 5.97.
−
1
cm : 3429 (NH), 2920 (CH aliphatic); 1714 (CO imidazolone), 1634
(Z)-4-(4-((1H-Indol-3-yl)methylene)-2-(4-chlorophenyl)-5-oxo-
4,5-dihydro-1H-imidazol-1-yl)benzenesulfonamide (18c)
Orange crystals (yield 60%); m.p. 158–160°C; reaction time 26 h;
1
(
CO amidic); 1379 (SO
DMSO-d )(δ, ppm): 2.78 (s, 3H, CH
m, 1H, Ar-H); 7.42–7.51 (m, 9H, Ar-H); 7.61 (s, 1H, methine H); 7.76–7.78
m, 1H, Ar-H); 7.95 (d, 2H, Ar-H, J = 7.8 Hz); 8.39 (d, 1H, Ar-H, J = 8 Hz);
.50 (d, 1H, Ar-H, J = 7 Hz); 8.91 (s, 1H, H-2 indole); 12.20 (s, 1H, SO NH,
O exchangeable); Anal. calcd. for C31 22ClN S (596.06): C, 62.47; H,
2
asymmetric), 1141 (SO
2
symmetric); H-NMR
(
6
3
); 6.88–6.89 (m, 1H, Ar-H); 7.19–7.21
−
1
(
IR (KBr) cm : 3423, 3264 (NH, NH
1379 (SO asymmetric), 1160 (SO
2
), 1706 (CO imidazolone);
symmetric); H-NMR (DMSO-
1
(
2
2
8
2
d
6
) (δ, ppm): 7.21–7.28 (m, 2H, Ar-H); 7.45–7.58 (m, 9H, 7Ar-H &
NH ); 7.69 (s, 1H, methine H); 7.90 (dd, 2H, Ar-H, J = 4.2,
4.42 Hz); 8.40 (d, 1H, Ar-H, J = 6.5 Hz); 8.61 (s, 1H, H-2 indole);
12.20 (s, 1H, NH indole, D O exchangeable); Anal. calcd. for
17ClN S (476.93): C, 60.44; H, 3.59; N, 11.75; O, 10.06;
D
2
H
O
5 4
2
3
.72; N, 11.75; O, 10.74; S, 5.38, Cl, 5.95. Found: C, 62.38; H, 3.98; N,
1.35; O, 10.88; S, 5.66; Cl, 5.68.
1
2
C
24
H
4 3
O
(
Z)-4-(4-((1-Acetyl-1H-indol-3-yl)methylene)-2-(4-
chlorophenyl)-5-oxo-4,5-dihydro-1H-imidazol-1-yl)-N-(thiazol-
-yl)benzenesulfonamide (17d)
Yellow crystals (yield 60%); m.p. 280–282°C; reaction time 2 h; IR (KBr)
S, 6.72, Cl, 7.43. Found: C, 60.38; H, 3.78; N, 11.35; O, 10.51; S,
6.66; Cl, 7.89.
2
4.2
|
Molecular docking study
−
1
cm : 3430 (NH), 2913 (CH aliphatic); 1715 (CO imidazolone), 1636
CO amidic); 1376 (SO
asymmetric), 1148 (SO symmetric);
) (δ, ppm): 2.78 (s, 3H, CH ); 6.87 (d, 1H, CH-5
thiazole, J = 4.5 Hz); 7.30 (d, 1H, CH-4 thiazole, J = 4.5 Hz); 7.40–7.47
m, 5H, Ar-H); 7.52–7.58 (m, 4H, Ar-H); 7.62 (s, 1H, methine H); 7.90 (d,
(
1
2
2
All the molecular modeling studies were carried out using Molecular
Operating Environment (MOE, 10.2008) software. All minimizations
were performed with MOE until an RMSD gradient of
H-NMR (DMSO-d
6
3
−1
−1
(
0.05 kcal · mol
Å
with MMFF94x force field and the partial charges
2
H, Ar-H, J = 8.3 Hz); 8.40 (d, 1H, Ar-H, J = 7.6 Hz); 8.50 (d, 1H, Ar-H,
were automatically calculated. The X-ray crystallographic structure of
VEGFR-2 co-crystallized with sorafenib as inhibitor (PDB ID: 4ASD)
13
2
J = 7.3 Hz); 8.91 (s, 1H, H-2 indole); 12.83 (s, 1H, SO NH); C-NMR
[45]
(
DMSO-d
6
) (δ, ppm): 24.4, 116.4, 116.6, 120.8, 121.2, 124.7, 126.2,
was downloaded from the Protein Data Bank.
The protein was
1
1
27.7, 128.0, 128.5, 128.9, 129.1, 131.1, 133.89, 135.8, 136.8, 137.3,
prepared for docking study using Protonate 3D protocol in MOE with
default options followed by water molecules removal. The
co-crystalized ligand was used to define the active site for docking.
Triangle Matcher placement method and London dG scoring function
were used for docking. Docking setup was first validated by re-docking
of the co-crystallized ligand (sorafenib) in the vicinity of the active site
of the protein with energy score (S) = −15.19 kcal/mol and RMSD of
0.470 Å. The validated setup was then used in predicting the ligand
receptor interactions at the active site for the newly synthesized
compounds.
+
37.6, 142.6, 149.3, 154.2, 158.1, 168.8, 170.2. EIMS, m/z: 601 (M );
Anal. calcd. for C29
O, 10.63; S, 10.65; Cl, 5.89. Found: C, 57.48; H, 3.66; N, 11.35; O,
0.22; S, 10.36; Cl, 6.23.
5 4 2
H20ClN O S (601.06): C, 57.85; H, 3.35; N, 11.63;
1
(Z)-4-(4-((1H-Indol-3-yl)methylene)-5-oxo-2-phenyl-4,5-
dihydro-1H-imidazol-1-yl)benzenesulfonamide (18a)
Yellow crystals (yield 45%); m.p. 140–142°C; reaction time 28 h; IR
−
1
(
KBr) cm : 3422 & 3264 (NH
2
), 1707 (CO imidazolone), 1373 (SO
2
1
asymmetric), 1160 (SO
2
symmetric); H-NMR (DMSO-d
6
) (δ, ppm):
); 7.67 (s, 1H,
methine H); 7.89 (d, 3H, Ar-H, J = 8 Hz); 8.40 (d, 1H, Ar-H, J = 8 Hz);
.61 (s, 1H, H-2 indole); 12.17 (s, 1H, NH indole, D O exchangeable);
Anal. calcd. for C24 S (442.49): C, 65.14; H, 4.10; N, 12.66; O,
0.85; S, 7.25. Found: C, 65.01; H, 4.54; N, 12.31; O, 10.66; Cl, 7.56.
7
.23–7.28 (m, 3H, Ar-H); 7.40–7.56 (m, 8H, 6 Ar-H & NH
2
4
.3
|
Biological studies
In vitro antiproliferative activity
8
2
4
.3.1
|
18 4 3
H N O
1
The cytotoxicity assays for the synthesized compounds were
performed against 60 cell line panel at the National Cancer Institute
(NCI), Bethesda, Maryland, USA under the Developmental Therapeutic
(Z)-4-(4-((1H-Indol-3-yl)methylene)-5-oxo-2-phenyl-4,5-
[
37]
dihydro-1H-imidazol-1-yl)-N-(5-methylisoxazol-3-yl)-
benzenesulfonamide (18b)
Program (DTP).
Compounds with drug-like mode of action, based
on computer-aided design, are to be prioritized in the NCI screening
service. Selection of the compounds for anticancer screening is based
on the ability of the submitted compounds to add diversity to the NCI
Orange crystals (yield 35%); m.p. 120–122°C; reaction time 30 h; IR
−1
(
KBr) cm : 3404 (NH), 2922 (CH aliphatic), 1704 (CO imidazolone),

16 of 17
ROAIAH ET AL.
|
small molecule compound libraries. The operation of this screening
panel utilizes 60 different human tumor cell lines. The experimental for
the single dose screen is reported as a mean graph at a single dose of
ORCID
Iman A. Y. Ghannam
http://orcid.org/0000-0003-3176-0187
1
0 µM in detail (Supporting Information).
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4
.3.2
|
In vitro determination of VEGFR-2 level for
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1
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ACKNOWLEDGMENTS
[
[
The authors thank the “National Research Centre (NRC)” for financially
supporting the manuscript through the fund of the NRC project no.
P100220. The authors are grateful to the National Cancer Institute
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CONFLICTS OF INTEREST
The authors have declared no conflict of interest.

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SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
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How to cite this article: Roaiah HM, Ghannam IAY, Ali IH,
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