Discovery and molecular docking of quinolyl-thienyl chalcones as anti-angiogenic agents targeting VEGFR-2 tyrosine kinase

Article abstract of DOI:10.1016/j.bmcl.2011.12.017

Vascular endothelial growth factor Receptor-2 (VEGFR-2) kinase inhibition is one of the well established strategies to promptly tackle tumor growth by suppression of angiogenesis. In the current study, structure-based virtual screening methodology of a series of quinolyl-thienyl chalcones indicated their strong potential as VEGFR-2 kinase inhibitors. In vitro VEGFR-2 kinase inhibitory activity was found to be significant (compound 19, IC₅₀: 73.41 nM). All compounds showed significant inhibition of human umbilical vein endothelial cells (HUVEC) proliferation (compound 19, IC₅₀: 21.78 nM). Molecular interactions of the compounds were studied using molecular docking studies.

Full text of DOI:10.1016/j.bmcl.2011.12.017

Bioorganic & Medicinal Chemistry Letters 22 (2012) 942–944
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and molecular docking of quinolyl-thienyl chalcones as
anti-angiogenic agents targeting VEGFR-2 tyrosine kinase
Syed Umar Farooq Rizvi a, Hamid Latif Siddiqui a, , Muhammad Nisar b, Nematullah Khan c,d
,
⇑
Inamullah Khan c,
⇑
a Institute of Chemistry, University of the Punjab, Lahore 54590, Pakistan
b Institute of Chemical Sciences, University of Peshawar, Peshawar, Pakistan
c Department of Pharmacy, University of Peshawar, 25120, Pakistan
d School of Pharmacy, University of Queensland, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Vascular endothelial growth factor Receptor-2 (VEGFR-2) kinase inhibition is one of the well established
strategies to promptly tackle tumor growth by suppression of angiogenesis. In the current study,
structure-based virtual screening methodology of a series of quinolyl-thienyl chalcones indicated their
strong potential as VEGFR-2 kinase inhibitors. In vitro VEGFR-2 kinase inhibitory activity was found to
be significant (compound 19, IC50: 73.41 nM). All compounds showed significant inhibition of human
Received 4 September 2011
Revised 2 November 2011
Accepted 3 December 2011
Available online 8 December 2011
umbilical vein endothelial cells (HUVEC) proliferation (compound 19, IC50: 21.78 nM). Molecular
interactions of the compounds were studied using molecular docking studies.
Keywords:
Quinolyl-thienyl chalcones
Angiogenesis
Ó 2011 Elsevier Ltd. All rights reserved.
VEGFR-2 kinase
Docking
Cancer is one of challenging field for medicinal chemists to dis-
cover effective yet safer chemotherapeutic agents targeting various
biochemical processes involved in progression of cancers.1 Among
these targets, angiogenesis is one of the critical processes affecting
growth and development of cancerous cells. Angiogenesis refers to
generation of new blood vessels from existing vasculature. It is the
key factor in development and progression of various human dis-
eases, including cancer, where it is necessary for the growth,
spread and survival of tumors.2 Angiogenesis is involved in metas-
tasis (uncontrolled spread of tumor cells) by supplying oxygen,
nutrients, and related growth factors to small tumors and remov-
ing the waste products of metabolism. Tumors that lack an ade-
quate vasculature become necrotic or apoptotic leading to their
limited growth. Thus, new means to retard angiogenesis have
shown promise as potential cancer therapies and inhibition of
the vascular endothelial growth factor (VEGF) signaling pathway
has emerged as one of the most promising new approaches for che-
motherapy of cancers.3,4
growth.5 There is much evidence that direct inhibition of the kinase
activity of VEGFR-2 will result in the reduction of angiogenesis and
the suppression of tumor growth.1 In literature, several structural
classes of small molecule inhibitors for example, indolin-2-ones,6
phthalazines,7 quinolinones,8 imidazopyridines,9 benzimidazoles,10
quinoline amides1 and pyridines,11 and quinazolines,12 have been
reported as potent inhibitors of VEGFR-2 and angiogenesis. In the
O
(ii)
(i)
NH
NH
CH
3
2
1
1
R
R
O
O
2
R
(iii)
VEGF is secreted by tumors and induces a mitogenic response
through its binding to one of three tyrosine kinase receptors
(VEGFR-1–3) on nearby endothelial cells. Thus inhibition of this
signaling pathway should block angiogenesis and subsequent tumor
O
N
Cl
N
Cl
2
1
1
R
H
C
3
R
R
1-33
⇑
Corresponding authors. Tel.: +92 42 3583 0503; fax: +92 42 99230998 (H.L.S.);
tel.: +92 91 9216750; fax: +92 91 9218131(I.K.).
Scheme 1. Reagents and conditions: Reaction protocol for the synthesis of
chalcones (1–33): (i) AcOH, H3PO4, reflux, 4–6 h; (ii) POCl3, DMF, 80 °C; (iii)
acetylthiophene, NaOH, rt, 2 h.
E-mail
addresses:
drhamidlatif@yahoo.com
(H.L.
Siddiqui),
inam_marwat333@yahoo.com (I. Khan).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.017

S. U. F. Rizvi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 942–944
943
current investigation, we have discovered quinolyl-thienyl chal-
O
cones as a new class of small-molecule inhibitors of VEGFR-2.
The synthesis of compounds 1–11 and 12–33 (Scheme 1) has
been already described.13,14 In order to evaluate the synthesized
compounds for their in vitro VEGFR-2 kinase inhibitory profile15
and anti-angiogenic effects via cellular proliferation assays using
HUVEC. Cell proliferation assay was determined employing 5-
bromo-2-deoxyuridine (BrdU) method based on commercially
available kits (Roche Diagnostics, USA). HUVEC were cultured in
a medium comprising 5% FBS in Collagen-coated 96-well plates
(type 1) and were incubated overnight at 37 °C and 5% CO2. The
medium was aspirated from the cells, and various concentrations
of the compounds (inhibitors) in serum-free medium were added
to each well. After 30 min, VEGF (10 ng mlÀ1) was added. Cells
were further incubated for an additional 72 h and BrdU (10 M)
was added during the last 18–24 h of incubation. Data were fitted
with a curve described by the equation, y = Vmax (1 À (x/(K + x))),
where K is equal to the IC50 valve (Dev et al., 2004).
R
H3C
S
N
Cl
Figure 1. General structure representing series of synthesized quinolyl-thienyl
chalcones.
All compounds of the series were subjected to in vitro VEGFR-2
kinase inhibitory assay followed by cell proliferation assay using
HUVEC. Interestingly, compound 19 showed most potent activity
against VEGFR-2 kinase (IC50: 73.41 nM) and HUVEC (IC50:
21.78 nM) among the series (Table 1). Detailed analysis showed
the fact that derivatives having 2,5-dichloro thienyl ring were the
most active especially when methyl group is located at position 7
of quinoline ring. However, derivatives 2,4-dimethylthienyl groups
were least active because of less polar and more bulkier methyl
groups in comparison to dichloro groups. Among other derivatives,
compounds having 3-halothien-2-yl groups especially in the case of
Figure 2. Binding mode of the most active compound (19) docked inside ATP-
binding pocket of VEGFR-2 tyrosine kinase. Hydrogen bonds are visible (blue dotted
lines). Aromatic nitrogen of quinolyl ring and both chlorine atoms are interacting
with Cys 919, Asp 1046 and Lys 868, respectively.
Table 1
Inhibitory effects as IC50 values of quinolyl-thienyl chalcones for VEGFR-2 kinase
inhibition assay and HUVEC proliferation assay
R1
R2
IC50 (nM)
a
Compound
VEGFR-2
HUVEC
1
2
3
4
5
6
7
8
9
6-Methyl
6-Methyl
6-Methyl
6-Methyl
6-Methyl
6-Methyl
6-Methyl
6-Methyl
6-Methyl
6-Methyl
6-Methyl
7-Methyl
7-Methyl
7-Methyl
7-Methyl
7-Methyl
7-Methyl
7-Methyl
7-Methyl
7-Methyl
7-Methyl
7-Methyl
8-Methyl
8-Methyl
8-Methyl
8-Methyl
8-Methyl
8-Methyl
8-Methyl
8-Methyl
8-Methyl
8-Methyl
8-Methyl
Thien-3-yl
287.28
184.53
169.17
176.24
453.41
87.11
106.94
127.39
106.41
127.59
228.57
37.49
59.41
26.19
61.91
88.43
129.28.
106.94
124.11
98.93
102.29
293.78
34.02
49.51
21.78
61.91
83.61
3-Methylthien-2-yl
4-Methylthien-2-yl
5-Methylthien-2-yl
2,5-Dimethylthien-3-yl
3-Chlorothien-2-yl
5-Chlorothien-2-yl
2,5-Dichlorothien-3-yl
3-Bromothien-2-yl
5-Bromothien-2-yl
5-Iodothien-2-yl
107.23
81.43
129.26
112.52
154.27
207.51
157.21
139.36
129.54
561.29
82.71
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Thien-3-yl
3-Methylthien-2-yl
4-Methylthien-2-yl
5-Methylthien-2-yl
2,5-Dimethylthien-3-yl
3-Chlorothien-2-yl
5-Chlorothien-2-yl
2,5-Dichlorothien-3-yl
3-Bromothien-2-yl
5-Bromothien-2-yl
5-Iodothien-2-yl
92.23
73.41
Figure 3. A 3D cut-view surface of ATP-binding pocket of VEGFR-2 tyrosine kinase
showing electrostatic and steric interactions of the most active compound (19).
[Color encoding (White area: hydrophobic region, red area: area with aggregated
negative electrostatic potential, and blue area: area with aggregated positive
electrostatic potential). Hydrogen atoms 9 except polar ones) were omitted for
clarity.
112.26
103.82
148.31
389.16
267.27
232.78
261.88
>1000
129.56
189.07
357.81
312.98
367.32
481.97
134.76
291.21
188.96
152.63
144.72
NDb
Thien-3-yl
3-Methylthien-2-yl
4-Methylthien-2-yl
5-Methylthien-2-yl
2,5-Dimethylthien-3-yl
3-Chlorothien-2-yl
5-Chlorothien-2-yl
2,5-Dichlorothien-3-yl
3-Bromothien-2-yl
5-Bromothien-2-yl
5-Iodothien-2-yl
3-chlorothien-2-yl group were also active but lesser than com-
pounds with 2,4-dichlorothienyl ring. Compounds having 5-iodo
thienyl groups were least active among halogen, which could be
attributed to larger atomic size of iodine as in comparison to the
size of deep pocket inside ATP-binding site of VEGFR-2 kinase.
Structural coordinates of VEGFR-2 kinase shows remarkable
conformational changes between hydrophobic ATP-binding pocket
of active and inactive enzyme states.1 In the current investigation,
91.49
116.49
294.56
187.21
243.08
283.57.
a
IC50 values were averaged values determined by at least three experiments.
ND: not determined.
b
molecular docking studies were conducted using
a complex

944
S. U. F. Rizvi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 942–944
Laudeman, C. P.; Luttrell, D. K.; Mook, R. A.; Nolte, R. T.; Rudolph, S. K.;
Szewczyk, J. R.; Truesdale, A. T.; Veal, J. M.; Wang, L. P.; Stafford, J. A. J. Med.
Chem. 2008, 51, 4632.
structure (PDB: 2XIR) at a resolution of 1.5 Å. ATP-binding pocket of
VEGFR-2 kinase posses the conserved Asp-Phe-Gly (DFG) loop
which plays a crucial role in designing and/or virtual screening of
new compounds targeting VEGFR-2 tyrosine kinase domain.Figures
2 and 3 shows binding mode of the most potent compound (19).
Molecular insights based on molecular docking16 indicated
favorable binding interactions of quinolyl-thienyl chalcones with
ATP-binding pocket of VEGFR-2 kinase including Asp-Phe-Gly
(DFG). Structural analysis revealed the fact that quinolyl moiety
was favorably penetrated into flat but slightly polar pocket sur-
rounded by Cys 919, Phe 918, Leu 840, and Lys 920 (Fig. 1). Com-
pound 19 showed significant binding interaction with amino
group of Cys 919 via hydrogen bonding, which was further rein-
forced by favorable electrostatic interaction of Chlorine atom at po-
sition 2 of quinoline moiety with aggregated positively charged
pocket (light blue color in Fig. 3). On the other hand, 5-chloro moi-
ety of thienyl ring was identified to be involved in hydrogen bond-
ing with amino group of Asp 1046. Furthermore, 2-chloro group of
the same ring was found to be interacting with Lys 868 thus fur-
ther supporting the favorable binding interactions of compound
19 with ATP-binding pocket of tyrosine kinase domain of VEGFR-
2 kinase. Thienyl moiety along 2,5-dichloro moiety penetrated dee-
ply with sling turning owing to its better potency. Deeper pocket
favorably matched thienyl ring based on its shape, electrostatic
environment (Fig. 2) and hydrogen bonding. However, all com-
pounds with methyl group at position 8 of quinoline ring showed
very low activity which could be attributed to its minor steric clash
with outer pocket of the ATP-binding pocket of VEGFR-2 kinase.
In conclusion, a series of quinolyl-thienyl chalcones showed sig-
nificant anti-angiogenic potential. These compounds have strong
potential to be further developed as a new class of VEGFR-2 kinase
inhibitory with promising anti-angiogenic potential. Further chem-
ical modification via fragment modifications guided by structure
and ligand-based computational methodologies can lead to dis-
cover better agents as potential clinical candidates.
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15. Briefly, VEGFR kinase protein (Upstate Biotechnology, Lake Placid, USA). The
enzyme selectivity screen was performed with a tyrosine kinase assay kit
(Invitrogen, PanVera Co., USA) based on fluorescence polarization detection.
Reactions were performed in 96-well polystyrene round-bottomed plates in a
final volume of 100 mL. Reaction mixtures contained 20 mM HEPES (pH 7.4),
5 mMMgCl2, 2 mM MnCl2, 50 mM Na3VO4, 200 ng/mL enzyme, 20 mM ATP,
and 1 ng/mL poly(Glu,Tyr) 4:1. One hundred compounds at concentrations of
10 and 1 mM were tested. After 1 h of incubation, the reactions were
terminated by adding a 6 mM EDTA solution; anti-phosphotyrosine antibody,
PTK green tracer, and FP dilution buffer mixtures were then added. The
fluorescence polarization values were measured after 30 min at room
temperature, using
a fluorescence reader (BioTek, USA). Kinase inhibition
analysis was performed by using Prism 5.0 (GraphPad Software Inc., USA).
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