Design, docking and synthesis of novel bromo isatin incorporated isoxazole derivatives as vegfr-2 inhibitors

Article abstract of DOI:10.22159/ijpps.2019v11i4.31933

Objective: To design, synthesize, in vitro Vascular Endothelial Growth Factor Receptor (VEGFR-2) assay, antiproliferative activity an Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) studies of some novel bromoisatin incorporated isoxa

Full text of DOI:10.22159/ijpps.2019v11i4.31933

International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491
Vol 11, Issue 4, 2019
Original Article
DESIGN, DOCKING AND SYNTHESIS OF NOVEL BROMO ISATIN INCORPORATED ISOXAZOLE
DERIVATIVES AS VEGFR-2 INHIBITORS
RADHIKA T.*, SAISREE K., HARINADHA BABU V.
*Department of Pharmaceutical Chemistry, G. Pulla Reddy College of Pharmacy, Osmania University, Hyderabad 500028, India,
Department of Pharmacy, OUCT, Osmania University, Hyderabad. India
Email: radhikavanam25@gmail.com
Received: 08 Jan 2019 Revised and Accepted: 15 Feb 2019
ABSTRACT
Objective: To design, synthesize, in vitro Vascular Endothelial Growth Factor Receptor (VEGFR-2) assay, antiproliferative activity an Absorption,
Distribution, Metabolism, Excretion and Toxicity (ADMET) studies of some novel bromoisatin incorporated isoxazole derivatives.
Methods: Designed compounds were synthesized by the condensation of different 3-aryl-5-methylisoxazole-4-carbohydrazides (5a-h) with 5-bromoisatin
to give the target molecules. To predict the affinity and activity of the ligand molecule the docking program GOLD 3.1 was employed to generate different
bioactive binding poses of designing molecules at the active site of protein VEGFR-2. All the synthesized compounds were characterized based on the spectral
and elemental analysis data. Antiproliferative activity performed against Human Umbilical vein endothelial cells (HUVEC cell line).
1
Results: All the synthesized compounds showed the characteristic peaks in FTIR, H, C13NMR and Mass spectral analysis. In molecular docking, all
the synthesized compounds (6a-j) exhibited high fitness scores with minimum three bonding interaction with the active site VEGFR-2 kinase. In in-
vitro, VEGFR-2 kinase assay, compounds 6a, 6b, 6d and 6e exhibited more than 70% inhibition at a single dose concentration of 5μM. In
antiproliferative assay against HUVEC cell lines, compounds 6d and 6e exhibited potent activity with IC50 values in nanomolar concentrations.
ADMET results of 6a, 6b, 6d and 6e are quite promising with least hepatotoxicity and good bioavailability.
Conclusion: The derivatives were synthesized in quantitative yields. New derivatives posses antiproliferative activity, least hepatotoxicity and good
bioavailability.
Keywords: Bromo isatin, Isoxazole hydrazides, Molecular Docking, VEGFR-2 Kinase enzyme assay, In vitro antiproliferative assay, ADMET study
© 2019 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2019v11i4.31933
INTRODUCTION
represents an important target since it plays a central role in
angiogenesis. Thus, inhibition of VEGFR-2 signaling pathway is
considered to be an attractive target while designing new anti-cancer
molecules [17]. In the present study, while designing target molecules
we have followed hybridization approach where isoxazole heterocycle
is conjugated with isatin scaffold in order to obtain new hybrid
molecules with potent VEGFR-2 inhibitor activity. The design of target
molecules is presented in fig 1. Further, the designed molecules were
computationally docked into VEGFR-2 kinase enzyme (PDB: 4AG8)
using GOLD 3.1 software in order to gain some structural insights into
the binding mode of designing molecules. The compounds that
demonstrated a high fitness score in comparison with the reference
drug, semaxanib and are further planned to screen for in vitro VEGFR-
2 kinase assay [18] and anti-proliferation study against Human
Umbilical Vein Endothelial Cells (HUVEC) [19].
Cancer is the leading cause of the deaths worldwide. Hence there is a
need to develop new drugs to treat this life threatening disease
without side effects. In recent years, isoxazoles derivatives have
acquired significance due to their wide spectrum of biological
activities such as anticancer [1], antibacterial [2], anti-inflammatory
[3], anticonvulsant [4], anti-tubercular [5], antiviral [6], antioxidant
[7], hypoglycemic [8] and antimicrobial activities [9] on the other hand
isatin derivatives has attained much attention due to considerable
pharmacological actions such as antimicrobial [10], antiviral [11],
anticonvulsant [12] and anticancer [13] activities and Alzheimer's
disease [14]. Tyrosine kinase inhibitors [15] are an important class of
anti-cancer drugs that act by interfering with specific cell signaling
pathways. Among the tyrosine kinases, VEGFR-2 Kinase [16]
Fig. 1: Design of target molecules

Radhika et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 1-7
MATERIALS AND METHODS
Chemistry
uncorrected. FT-IR spectra were recorded on a Shimadzu FTIR
spectrophotometer. 1H NMR spectra were recorded on AVANCE 300
MHz spectrometer using DMSO-d6 as solvent and tetramethyl silane
(TMS) as an internal standard. All the 1H NMR Chemical shift values
All the solvents and chemicals used in synthesis were AR and
synthetic grade obtained from SD fine chemicals, E. Merck (India)
and Aldrich Chemicals (India). The reactions were monitored by
analytical thin layer chromatography (TLC) using E. Merck 0.25 mm
silica gel plates. Melting points were determined in one end open
capillary tubes using ANALAB melting point apparatus and were
are recorded in
δ scale. [13]C NMR spectra of synthesized
compounds were recorded on Varian Gemini 100 MHz
spectrophotometer. Mass spectra of the compounds were recorded
on Agilent 6430 mass spectrophotometer. Elemental analysis was
performed at Central University, Hyderabad, India.
Scheme 1: The total synthetic pathway, reagents and conditions: (i) NH2OH. HCl, NaOH/CH3COONa, CH3OH, reflux 1-2 h; (ii) N-
Chlorosuccinamide, DMF, stirring 12 h; (iii) CH3COOC2H5, CH3OH, NaOH, stirring 1-2 h; (iv) 99 % NH2NH2. H2O, reflux 10-12 h; (v) 5-
bromoisatin, DMF, reflux 2-3 h
Table 1: Derivatives of scheme 1
Compound
R
OCH3
Cl
F
Cl
OCH3
OH
NO2
Br
CH3
CH3
R1
H
H
R2
H
H
H
H
H
H
H
H
R3
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
Cl
OCH3
H
H
H
H
H
H
CH3
6j
General procedure for synthesis of Ethyl 3-aryl-5-methyl-
isoxazole-4-carboxylates (4a-h)
crushed ice drop by drop with constant stirring and the solid
separated was filtered, dried and recrystallized from 90% ethanol.
A methanolic solution of arylhydroxymoyl chloride (0.01 mol) was
added in small portions to a solution of sodium salt of ethyl
General procedure for synthesis of Ethyl (Z)-3-(3-aryl)-N'-(2-
oxoindolin-3-ylidene) isoxazole-4-carbohydrazides (6a-j)
acetoacetete (0.02 mol) over
a period of 1 h at 0-5 °C and
To a solution of ethyl 3-aryl-5-methyl isoxazolehydrazide (0.01 mol)
in dimethylformamide (25 ml), 5-bromo isatin (0.01 mol) was added
heated to reflux for 2-3 h. The progress of the reaction was
monitored by TLC. The reaction mixture was then poured into
crushed ice drop by drop with constant stirring. The solid separated
was filtered, dried and recrystallized from 90% ethanol.
subsequently stirred for 1h at room temperature by maintaining the
pH 10 with aq. NaOH. The reaction was monitored by TLC. After
completion of reaction, the mixture was poured into ice cold water
and the solid separated was filtered and recrystallized from 90%
ethanol.
General procedure for synthesis of Ethyl 3-aryl-5-methyl-
isoxazolehydrazides (5a-h)
Spectral data
(Z)-N'-(6-bromo-2-oxoindolin-3-ylidene)-3-(4-methoxyphenyl)-
5-methylisoxazole-4-carbohydrazide (6a)
To a solution of ethyl 3-aryl-5-methyl-isoxazole-4-carboxylate (0.01
mol) in ethanol, was added hydrazine hydrate (99%, 0.04 mol) and
heated to reflux for 10-12 h. The progress of the reaction was
monitored by TLC. The reaction mixture was then poured into
Yield: 56 %. Red solid. mp: 190-196 °C. IR (KBr, cm-1): 3249, 3148
(NH), 1689, 1663 (C=O), 1601 (CN). 1H NMR (300 MHz, CDCl3) δ:
2

Radhika et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 1-7
12.6 (s, 1H, NH of isatin), 6.9-7.3 (7H, Ar), 6.0 (s, 1H, NH,
exchangeable with D2O), 3.5 (s, 3H, OCH3), 2.9 (s, 3H, CH3). [13]C
NMR: 175.7, 163.4, 160.2, 140.4, 136.4, 134.7, 132.6, 128.3, 121.6,
119.6, 118.5, 118.2, 114.5, 111.1, 55.6, 13.0. MS (ESI): m/z 455
[M+1]. HRMS calcd for C20H15BrN4O4 454.0276. Found454.0271.
12.9 (s, 1H, NH of isatin), 6.8-7.8 (7H, Ar), 6.9 (s, 1H, NH,
exchangeable with D2O), 2.8 (s, 3H, CH3). [13]C NMR: 175.4, 167.8,
163.5, 140.2, 136.5, 134.7, 132.6, 132.4, 128.8, 128.0, 123.4, 119.5,
118.6, 118.0, 111.9, 14.0. MS (ESI): m/z 503 [M+1]. HRMS calcd
forC19H12Br2N4O3501.9276. Found 501.9271.
(Z)-N'-(6-bromo-2-oxoindolin-3-ylidene)-3-(4-chlorophenyl)-5-
methylisoxazole-4-carbohydrazide(6b)
(Z)-N'-(6-bromo-2-oxoindolin-3-ylidene)-5-methyl-3-(p-tolyl)
isoxazole-4-carbohydrazide (6i)
Yield: 67 %. Yellow solid. mp: 202-208 °C. IR (KBr, cm-1): 3486, 3239
(NH), 1678, 1626 (C=O), 1600 (C=N), 752 (C-Cl). 1H NMR (300 MHz,
CDCl3) δ: 12.9 (s, 1H, NH of isatin), 6.8-7.2 (7H, Ar), 6.8 (s, 1H, NH,
exchangeable with D2O), 2.6 (s, 3H, CH3).). [13]C NMR: 175.4, 168.4,
163.6, 162.8, 140.6, 136.7, 134.8, 134.0, 132.5, 129.4, 128.6, 127.4,
119.2, 118.6, 118.0, 111.0, 13.0. MS (ESI): m/z 459 [M+1]. HRMS
calcd for C19H12BrClN4O3 457.9781. Found 457.9784.
Yield: 82 %. Red solid. mp: 201-205 °C. IR (KBr, cm-1): 3348, 3238
(NH), 1699, 1670 (C=O), 1600 (CN). H NMR (300 MHz, CDCl3): 12.7
1
(s, 1H, NH of isatin), 6.9-7.7 (7H, Ar), 6.0 (s, 1H, NH, exchangeable
with D2O),, 2.6 (s, 6H, CH3). [13]C NMR: 175.6, 168.2, 163.6, 162.4,
141.5, 133.9, 131.9, 131.0, 129.7, 129.2, 126.0, 125.4, 123.8, 119.2,
117.4, 111.4, 20.8, 13.0. MS (ESI): m/z 440 [M+1]. HRMS calcd for
C20H15 BrN4O3 438.127. Found 438.1272.
(Z)-N'-(6-bromo-2-oxoindolin-3-ylidene)-3-(4-fluorophenyl)-5-
methylisoxazole-4-carbohydrazide(6c)
(Z)-N'-(6-bromo-2-oxoindolin-3-ylidene)-3-(2,4-
dimethylphenyl)-5-methylisoxazole-4-carbohydrazide(6j)
Yield: 86 %. Yellow solid. mp: 169-174 °C. IR (KBr, cm-1): 3380, 3293
(NH), 1683, 1616 (C=O), 1600 (CN). 1H NMR (300 MHz, CDCl3) δ:
12.6 (s, 1H, NH of isatin), 6.8-7.2 (7H, Ar), 6.4 (s, 1H, NH,
exchangeable with D2O), 2.8 (s, 3H, CH3). [13]C NMR: 175.4, 168.4,
162.2, 163.6, 162.6, 140.4, 136.7, 134.5, 132.5, 130.4, 124.3, 119.4,
118.4, 118.0, 116.2, 111.4, 13.0. MS (ESI): m/z 443 [M+1]. HRMS
calcd forC19H12BrFN4O3442.0076. Found 442.0070.
Yield: 82 %. Red solid. mp: 220-225 °C. IR (KBr, cm-1): 3348, 3238
(NH), 1699, 1670 (C=O), 1600 (CN). 1H NMR (300 MHz, CDCl3) δ: 12.7
(s, 1H, NH of isatin), 6.9-7.7 (6H, Ar), 6.0 (s, 1H, NH, exchangeable with
D2O), 2.8 (s, 3H, CH3), 2.6 (s, 6H, CH3). [13]C NMR: 174.4, 168.2, 163.6,
161.9, 150.6, 149.2, 141.6, 134.8, 131.8, 129.6, 126.0, 124.8, 120.4,
119.6, 117.5, 111.8, 111.0, 108.7, 56.4, 13.0. MS (ESI): m/z 407 [M+1].
HRMS calcd for C21H17BrN4O3 452.1277. Found 452.1272.
(Z)-N'-(6-bromo-2-oxoindolin-3-ylidene)-3-(3,4-
dichlorophenyl)-5-methylisoxazole-4-carbohydrazide (6d)
2.3 In vitro VEGFR-2 kinase activity
The in vitro enzyme inhibition assay for five selected compounds was
carried out in Radiant Research Services Pvt. Ltd, Bangalore, India
(http://www.radiantresearch.in/) at a single dose concentration of 5
μM. VEGFR-2 (KDR, Lot # 061716 0713) (Ray BioR) has served as the
enzyme source and Poly (Glu, Tyr) sodium salt, (4:1, Glu: Tyr) (Sigma
Aldrich) served as the standardized substrate and Kinase-Glo Plus
Yield: 73 %. Pale yellow solid. mp: 186-192 °C. IR (KBr, cm-1): 3220,
3196 (NH), 1700, 1626 (C=O), 1600 (CN), 755 (C-Cl). 1H NMR (300
MHz, CDCl3) δ: 12.4 (s, 1H, NH of isatin), 7.0-7.6 (6H, Ar), 6.6 (s, 1H,
NH, exchangeable with D2O), 2.9 (s, 3H, CH3). [13]C NMR:
173.9,167.5,
163.4,
162.0,
140.6,
136.2,
Luminescence kinase assay kit (PromegaTM
)
134.3,133.6,132.8,132.6,132.4,130.8,128.2,127.4,119.5,118.5,118.1,
111.8,13.0. MS (ESI): m/z 493 [M+1]. HRMS calcd for
In vitro HUVEC anti-proliferative assay
C
19H11BrCl2N4O3 491.9391. Found 491.9396.
The In vitro HUVEC proliferative assay for four selected compounds
was carried out by Radiant Research Pvt Ltd, Bangalore, India
(http://www. radiantresearch. in/) at concentration of 500 nm, 1μM,
2μM and 5μM. HUVEC (human umbilical vein endothelial cells) served
as the cell source, in Medium 200 (Hi-Media), with large vessel
endothelial supplement (LVES) (Life Technologies) and Pen-step (Hi-
Media). Alamar Blue (Hi-Media) was used as the fluorescent reagent.
(Z)-N'-(6-bromo-2-oxoindolin-3-ylidene)-3-(3,4-
dimethoxyphenyl)-5-methylisoxazole-4-carbohydrazide (6e)
Yield: 72 %. Red solid. mp: 193-197 °C. IR (KBr, cm-1): 3336, 3241
(NH), 1689, 1666 (C=O), 1600 (CN). 1H NMR (300 MHz, CDCl3) δ:
12.8 (s, 1H, NH of isatin), 6.9-7.7 (6H, Ar), 6.0 (s, 1H, NH,
exchangeable with D2O), 3.5 (s, 6H, OCH3), 2.4 (s, 3H, CH3). [13]C
NMR:
175.2,
168.8,
163.6,
162.4,150.6,
149.6,
ADME and toxicity studies
140.4,137.2,134.6,132.8,126.6,120.1,119.4, 118.7, 118.2,111.8,111.2,
108.8, 56.5, 13.0. MS (ESI): m/z 485 [M+1]. HRMS calcd
forC21H17BrN4O5 484.0382. Found 484.0380.
Compounds 6a, 6b, 6d and 6e are further studied for their
pharmacokinetic and toxicity studies using ADMET descriptor
analysis protocol in Discovery Studio. Using the standards provided
by Discovery Studio, the results are analyzed. The calculated
parameters are tabulated in the table 6.
(Z)-N'-(6-bromo-2-oxoindolin-3-ylidene)-3-(4-hydroxyphenyl)-
5-methylisoxazole-4-carbohydrazide (6f)
Yield: 80 %. Yellow solid. Mp: 170-175 °C. IR (KBr, cm-1): 3380, 3530
(NH), 1680, 1662 (C=O), 1620 (CN). 1HNMR (300 MHz, CDCl3) δ:
12.7 (s, 1H, NH of isatin), 6.7-8.1 (7H, Ar), 6.4 (s, 1H, NH,
exchangeable with D2O), 4.5 (s, br, OH, exchangeable with D2O), 2.8
(s, 3H, CH3). [13]C NMR: 174.9,168.2,163.6,162.4,158.8, 140.6,136.6,
134.7,132.6, 128.6, 121.8, 119.5,118.6,118.0,116.2, 114.6, 111.0,
13.0. MS (ESI): m/z 441 [M+1]. HRMS calcd for
C19H13BrN4O4440.0120. Found 440.0122.
Molecular docking study
Molecular docking is an effective technique to predict the preferred
orientation of ligand molecules with a macromolecular target (protein
receptor) when bound to each other to form a stable complex. The
primary objective in molecular docking is its ability to estimate the
scoring function and evaluate protein-ligand interactions in order to
predict the affinity and activity of the ligand molecule. The docking
program GOLD 3.1 was employed to generate different bioactive
binding poses of designed molecules at the active site of protein
VEGFR-2. Protein coordinates from the crystal structure were used to
define the active site. Docking calculations were performed using the
default GOLD fitness function and default GOLD parameters to
produce the set of optimal conformations of both the ligand and the
protein. The default parameters used are: population size ¼ 100;
selection pressure ¼ 1.1; # operations ¼ 100,000; islands ¼ 5; inches
size ¼ 2; migration ¼ 10; mutation ¼ 95; crossover ¼ 95. Each
simulation is performed 10 times; yielding 10 docked conformations
unless three of the 10 poses were within 1.5 A °RMSD of each other.
The lowest energy conformations were regarded as the binding
conformations between ligands and the receptor protein. The scoring
function was used to reach optimal accuracy for candidate compound
selection. The greater the GOLD fitness score, the better is the binding
(Z)-N'-(6-bromo-2-oxoindolin-3-ylidene)-5-methyl-3-(4-
nitrophenyl)isoxazole-4-carbohydrazide (6g)
Yield: 72 %. Yellow solid. mp: 195-197 °C. IR (KBr, cm-1): 3438, 3196
(NH), 1680, 1662 (C=O), 1602 (C=N), 1535, 1344 (NO2).1H NMR
(300 MHz, CDCl3) δ: 12.8 (s, 1H, NH of isatin), 6.6-8.1 (7H, Ar), 6.4 (s,
1H, NH, exchangeable with D2O), 2.9 (s, 3H, CH3).[13]C NMR: 175.4,
168.1,
163.6,
162.0,147.2,140.6,136.8,
135.2,134.8,
MS(ESI): m/z
132.6,126.6,124.0,119.4,118.9,118.2,111.2,13.0.
470[M+1]. HRMS calcd for C19H12BrN5O5 469.0021. Found 469.0027.
(Z)-N'-(6-bromo-2-oxoindolin-3-ylidene)-3-(4-bromophenyl)-5-
methylisoxazole-4-carbohydrazide (6h)
Yield: 67 %. Yellow solid. mp: 180-184 °C. IR (KBr, cm-1): 3220, 3195
(NH), 1699, 1622 (C=O), 1602 (C=N). 1H NMR (300 MHz, CDCl3) δ:
3

Radhika et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 1-7
affinity. Hit molecules which showed the expected interactions with
the critical amino acids present in the active site of the protein, were
selected as potent inhibitors of VEGFR-2. Using Define and Edit
Binding Site tools in Accelrys Discovery Studio 2.1, the binding site of
the VEGFR-2 domain (PDB: 4AG8) was predicted based on the
occupied volume of the known ligand, Axitinib in the active site. The
co-crystallized ligand, Axitinib molecule was first selected and a
sphere was created around the molecule using define sphere for the
selected option within the DS. The binding site containsVal914,
Val916, Glu885, Ala866, Phe1047, Gly922, Leu1035, Glu917, Lys920,
Cys1045, Cys919, Lys920. A sphere is defined around the residues
comprising binding site at a radius of 10A °.
j). The characterization of intermediates and final compounds was
done on the basis of FTIR, MASS, 1H NMR and [13]C NMR spectral data.
In IR spectra, the final compounds (6a-j) showed the presence of two
carbonyl absorption peaks around 1700 cm-1 and 1640 cm-1due to the
carbonyl group of isatin and the carbonyl of hydrazide. Moreover, a
sharp absorption band around 1600 cm-1was observed in all the
spectra due to C=N stretching. In 1H NMR, the NH protons of isatin and
amide appeared as singlets around δ 12.9 and δ 6.8. The three protons
of methyl group appeared as singlet around δ 2.9 and as usual
aromatic protons appeared in the range of δ 7.0-8.0. The appearance
of molecular ion peaks corresponding to their molecular weights in
mass spectra further confirmed the structures. Moreover, the different
carbons present in the synthesized molecules were observed at the
expected chemical shifts and integral values in 13C NMR spectra.
Molecular docking
Molecular docking study was carried out with the synthesized
compounds into the ATP binding site of VEGFR-2 kinase enzyme
(PDB: 4AG8) (Based on the occupied volume of known ligand
Axitinib into the active site). The study was performed using GOLD
3.1 software. Among sixteen compounds, compound 6d showed high
fitness score of 56.19 with target protein VEGFR-2 through six
bonding interactions involving five amino acids i. e, Asp 1046, Glu
885, Lys 868, Phe 1047 and val 914 at the active site (fig. 4) through
hydrogen bonding and vanderwall interactions, while the reference
drug, semaxinib showed only two hydrogen bond interactions (Asp
1046 and Phe 1047) with fitness score of 50.02 (fig. 3). The binding
configuration of compound 6d shows that the substituted phenyl
ring and isoxazole scaffold are very well lodged into the receptor
pocket, while 4-chloro group is in hydrogen bonding interaction
with Phe 1046. The C-21 of isoxazole showed interaction with Val
914. The NH motif of amide moiety formed two hydrogen bonds
with Asp 1046 and Glu 885. The nitrogen adjacent to the amide
moiety also showed hydrogen bond interaction with Asp 1046.
Compounds 6a,6b, 6d and 6e also exhibited good fitness scores of
54.47, 52.38,56.19 and 52.07 and with 3 to 6 bonding interactions at
the active site (fig. 5-7). The docking score results of all synthesized
compounds (6a-j) are presented in table 2.
Fig. 2: Ligand molecule
RESULTS AND DISCUSSION
Chemistry
The synthetic route for the synthesis of final compounds is described
under Scheme-1. Reaction of different aromatic aldehydes with
hydroxylamine hydrochloride afforded corresponding oximes (1)
which on treatment with N-chlorosuccinamide in DMF gave aryl
hydroximoyl chlorides (2). The cyclization of compounds (2) with
ethyl acetoacetate in methanol provided 3-aryl-5-methyl-isoxazole-4-
carboxylates (3) in reasonable yields. Conversion of compounds (3)
into hydrazides was achieved by treating with hydrazine hydrate
(99%) in methanol. Further, the coupling of hydrazides (5a-j) with
isatin and 5-bromoisatinin in DMF afforded the final compounds (6a-
Fig. 3–4: 3D images of molecular docking poses of semaxanib and compounds (6d)
Table 2: Gold docking results of all synthesized compounds
Compounds
6a
6b
6c
6d
6e
6f
6g
Fitness score
54.47
52.38
48.44
56.19
52.07
47.28
50.40
51.51
50.51
S(hb_ext)
1.51
1.49
0.86
1.69
1.61
1.57
3.31
2.00
S(vdw_ext)
45.28
41.14
38.15
44.44
45.05
39.84
43.80
41.66
45.22
41.90
38.28
S(hb_int)
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
S(int)
-9.29
-7.67
-4.87
-6.60
-11.48
-9.07
-9.64
-7.78
-11.78
-9.41
-2.61
6h
6i
6j
0.10
2.55
0.00
0.00
0.00
0.00
50.75
50.02
Semaxinib
4

Radhika et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 1-7
In vitro VEGFR-2 kinase assay
performed at Radiant Research Services Pvt. Ltd, Bangalore, India
(http://www.radiantresearch.in/). The results revealed that the
compound 6d has potent activity against VEGFR-2 kinase at 5 μM
concentration with 86 % inhibition while the other compounds 6a,
6e showed more than 75 %. However, Compound 6h exhibited 52 %
inhibition and the results are presented in table 3.
In molecular docking, compounds 6a, 6b, 6d, 6e and 6h
demonstrated high fitness scores relative to the reference drug,
semaxinib and were considered for in vitro VEGFR-2 kinase enzyme
assay using a single dose concentration of 5 μM. The study was
Table 3: Percent inhibition of VEGFR-2 enzyme activity
R1 R2 R3
Compounds
R
% inhibition
6a
6b
6d
6e
6h
OCH3
Cl
Cl
OCH3
Br
H
H
Cl
OCH3
H
H
Br
75
72
86
78
52
H
H
H
H
Br
Br
Br
Br
Based on (table 3) results, four compounds (6a, 6b, 6d and 6e)which
exhibited above 70 % VEGFR-2 inhibition were selected for further dose-
related VEGFR-2 enzymatic inhibition at 500 nM, 1 μM, 2 μM and 4 μM
concentrations in order to calculate their IC50 values (table 3). Two
compounds 6d and 6a exhibited potent VEGFR-2 inhibitor activity with
IC50 values of 1.33 μM and 1.75 μM while the reference drug semaxinib
[20] showed IC50 value of 1.24 μM. However, compounds 6a and 6d
exhibited reasonable activity. A good correlation was observed among
the studies of molecular docking, in vitro VEGFR-2 kinase enzyme assay
and in vitro IC50 values. The IC50 values are presented in the table 4.
Table 4: The IC50 values of selected compounds based on VEGFR-2 inhibition
VEGFR-2 (% inhibition) VEGFR-2 (IC50
Compounds
)
6a
6b
6d
6e
78 1.75
72
86
70
98
2.18
1.33
2.33
1.24
semaxinib
In vitro antiproliferative assay
the compounds exhibited varied antiproliferative activity.
Compounds 6d and 6e showed good antiproliferative activity with
percentage inhibition of 78 % and 98 %, while the compounds 6a
and 6b exhibited 72 % and 70 % and the reference semaxinib
showed 100% inhibition. These values are in good correlation with
the in vitro VEGFR-2 enzyme inhibitory assay. The results are shown
in table 5.
For antiproliferative assay, HUVEC cell line was selected since they
play a major role in angiogenesis or new blood vessel formation.
Four compounds (6a, 6b, 6d and 6e) that demonstrated more than
70 % VEGFR-2 inhibition were selected for their activity against
HUVEC cell line at a single dose concentration of 10 µM. In screening,
Table 5: The effect of compounds on HUVEC cell line at 10 µM
Compounds
6a
6b
6d
%Cell growth
28.69
50.06
30.02
0.65
%Cell inhibition
72
70
78
98
100
6e
semaxinib
0.29
Fig. 5: Plot of PSA versus Log P for candidate compounds showing the 95% and 99% confidence limit ellipses corresponding to the blood–
brain barrier and intestinal absorption models ADME and toxicity studies
5

Radhika et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 1-7
DISCUSSION
values represent high penetration of the compounds. All the
compounds exhibited in silco cytochrome P450 2D6 inhibition. Similarly,
all the compounds are satisfactory with respect to CYP2D6 value is
near to 0, suggesting that these compounds should be non-inhibitors
of CYP2D6. The plasma protein binding property prediction denotes
that all of them have binding ≥95% indicating that most of the
compounds have good bioavailability and are not likely to be highly
bound to carrier proteins in the blood. Further, all the compounds
have been predicted to have the probable hepatotoxic levels less than
1. Of all the compounds, compound 6a showed least probability value
of 0.894 suggesting that this compound is least toxic compared to all
the compounds. The results are shown in table 6.
The ADMET studies deal with in silico prediction of the adverse effects
of the synthesized compounds. The properties such as absorption,
distribution, metabolism, excretion and toxicity (ADMET) are
important in order to determine the success of the compound for
human therapeutic use. The results were compared to the reference
Level values of Discovery Studio to analyse the properties of our
compounds. The absorption levels (human intestinal absorption) of all
the compounds are predicted to be having good absorption. The
solubility levels of the compounds were in the range of 1–2, indicating
good solubility. BBB penetration of all the compounds are 3 and 4, the
Table 6: Absorption, distribution, metabolism, excretion and toxicity (ADMET) of synthetic derivatives
Name
BBB_
level
Absorption_ level
Solubility_ level
Hepatotoxicity_
probability
PPB_
level
CYP2D6_
probability
Alogp98
Axitinb
1
4
4
3
4
0
0
0
0
0
2
2
1
2
2
0.96
1
2
2
2
2
0.613
0.366
0.198
0.297
0.336
4.492
3.026
4.388
3.639
3.043
Comp. 6a
Comp. 6b
Comp. 6d
Comp. 6e
0.894
0.927
0.933
0.913
CONCLUSION
5. Chikkula KSR. Isoxazole –A potent pharmacophore. Int J
Pharm Pharma Sci 2017;9:13-4.
Ten novel isatin incorporated isoxazole derivatives were designed
and synthesized by the condensation of different 3-aryl-5
methylisoxazole-4-carbohydrazides (5a-j) with 5-bromoisatin in
order to give the target compounds that act as VEGFR-2 inhibitors.
The synthesized compounds were characterized on the basis of
spectral and elemental analysis data. In molecular docking, all the
designed compounds (6a-j) exhibited high fitness scores with
minimum three bonding interactions with the active site of VEGFR-2
kinase. In in vitro VEGFR-2 kinase enzyme assay, compounds 6a, 6b,
6d and 6e exhibited more than 70% inhibition at a single dose
concentration of 5 µM. In antiproliferative assay against HUVEC cell
line, compounds 6d and 6e exhibited potent activity with IC50 values
in nanomolar (μm) concentrations. ADMET results of 6a, 6b, 6d and
6e are quite promising with least hepatotoxicity and good
bioavailability.
6. Yang Z, Li P, Gan X. Novel pyrazole-hydrazone derivatives
containing an isoxazole moiety: design, synthesis, and antiviral
activity. Mol 2018;23:1798.
7. Kalirajan R, Rafick MH, Sankar S, Jubie S. Docking studies,
synthesis, characterization and evaluation of their antioxidant
and cytotoxic activities of some novel isoxazole-substituted 9-
anilinoacridine derivatives. Sci World J 2012;2012:165258.
8. Kumar A, Maurya RA, Sharma S, Ahmad P, Singh AB, Tamrakar
AK, et al. Design and synthesis of 3, 5-diarylisoxazole
derivatives as novel class of anti-hyperglycemic and lipid
lowering agents. Bio Med Chem 2009;17:5285-92.
9. Basha SS, Divya K, Padmaja A, Padmavathi V. Synthesis and
antimicrobial activity of thiazolyl pyrazoles and isoxazoles. Res
Chem Int 2015;41:10067-83.
10. Meenakshi K, Gopal N, Sarangapani M. Synthesis,
characterization and antimicrobial activity of some novel schiff
and mannich bases of isatin. Int J Pharm Pharm Sci 2014;6:318-
22.
11. Debnath B, Ganguly S. Molecular docking studies and ADME
prediction of novel isatin analogs as hiv-1-rt inhibitors with
broad spectrum chemo therapeutic properties. Asian J Pharm
Clin Res 2014;7:186-9.
12. Smitha S, Pandeya S, Stables J, Ganapathy S. Anticonvulsant and
sedative-hypnotic activities of N-acetyl/methyl isatin
derivatives. Sci Pharm 2008;76:621-36.
13. El-Faham A, Farooq M, Khattab SN, Abutaha N, Wadaan MA,
Ghabbour HA, et al. Synthesis, characterization, and anti-cancer
activity of some new N′-(2-Oxoindolin-3-ylidene)-2-
propylpentane hydrazide-hydrazones derivatives. Mole
2015;13:14638-55.
ACKNOWLEDGEMENT
The authors are thankful to the management of G. Pulla Reddy
College of Pharmacy, Hyderabad, India for providing facilities. The
authors are also thankful to Central University, Hyderabad for
providing Mass and 1H NMR spectral data.
AUTHORS CONTRIBUTIONS
All the authors have contributed equally
CONFLICT OF INTERESTS
The authors declare that there is no conflict of interest
REFERENCES
1. Kamal A, Reddy JS, Ramaiah MJ, Dastagiri D, Bharathi EV, Azhar
MA, et al. Design, synthesis and biological evaluation of 3, 5-
diaryl-isoxazoline/isoxazole-pyrrolobenzodiazepine
conjugates as potential anticancer agents. Eur J Med Chem
2010;45:3924-37.
14. Chandra PM, Venkateshwar J. Biological evaluation of schiff
bases of new isatin derivatives for anti alzheimer’s activity.
Asian J Pharm Clin Res 2014;7:114-7.
15. Hoff PM, Wolff RA, Bogaard K, Waldrum S, Abbruzzese JL. A
phase
I study of escalating doses of the tyrosine kinase
2. Rajanarendar E, Reddy MN, Krishna SR, Reddy KG, Reddy YN,
Rajam MV. Design, synthesis, in vitro antimicrobial and
anticancer activity of novel methylenebis-isoxazolo [4, 5-b]
azepines derivatives. Eur J Med Chem 2012;50:344-9.
3. Radhika T, Sravanthi S, Babu VH, Reddy BM. Synthesis,
biological evaluation and molecular docking studies of
isoxazole synchronized quinazolinone derivatives. J Pharm Res
2017;11:895-2.
inhibitor semaxanib (SU5416) in combination with irinotecan
in patients with advanced colorectal carcinoma. Japan J Clin
Oncol 2006;1:100-3.
16. Shibuya M. Vascular endothelial growth factor (VEGF) and its
receptor (VEGFR) signaling in angiogenesis: a crucial target for
anti-and pro-angiogenic therapies. Gen Canc 2011;2:1097-5.
17. SB B, Adhikari S, Surana SJ. Tyrosine kinase receptor inhibitors:
A new target for anticancer drug development. J Pharma Sci
Tech 2012;1:36-45.
4. Edafiogho IO, Hinko CN, Chang H, Moore JA, Mulzac D,
Nicholson JM, et al. Synthesis and anticonvulsant activity of
enaminones. J Med Chem 1992;35:2798-5.
18. Li Z, Wang B, Tang L, Chen S, Li J. Quinazoline derivative
compound (11d) as a novel angiogenesis inhibitor inhibiting
6

Radhika et al.
Int J Pharm Pharm Sci, Vol 11, Issue 4, 1-7
VEGFR-2 and blocking VEGFR2-mediated Akt/mTOR/p70s6k
signaling pathway. Iran J B Med Scien 2016;19:411.
19. Corbacho AM, Macotela Y, Nava G, Torner L, Duenas Z, Noris G,
et al. Human umbilical vein endothelial cells express multiple
prolactin isoforms. J End 2000;166:53-62.
20. Haddad JJ. The immunopharmacologic potential of Semaxanib
and new generation directed therapeutic drugs: receptor
tyrosine
kinase
regulation
with
anti-
tumorigenensis/angiogenesis properties. Sau Pharma
2012;30:103-23.
J
7