Design, synthesis and biological evaluation of quinoline amide derivatives as novel VEGFR-2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.09.014

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in the process of cancer angiogenesis. A series of quinoline amide derivatives were prepared and found to be good inhibitors of VEGFR-2. The inhibitory activities were investigated against VEGFR-2 kinase and human umbilical vein endothelial cells (HUVEC) in vitro. Compound 6 (5-chloro-2-hydroxy-N-(quinolin-8-yl)benzamide) exhibited the most potent inhibitory activity (IC₅₀ = 3.8 and 5.5 nM for VEGFR-2 kinase and HUVEC, respectively). Docking simulation supported the initial pharmacophoric hypothesis and suggested a common mode of interaction at the ATP-binding site of VEGFR-2, which demonstrates that compound 6 is a potential agent for cancer therapy deserving further researching.

Full text of DOI:10.1016/j.bmcl.2010.09.014

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6653–6656
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of quinoline amide derivatives
as novel VEGFR-2 inhibitors
⇑
Ying Yang , Lei Shi , Yang Zhou, Huan-Qiu Li, Zhen-Wei Zhu, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
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) plays a crucial role in the process of cancer
angiogenesis. A series of quinoline amide derivatives were prepared and found to be good inhibitors of
VEGFR-2. The inhibitory activities were investigated against VEGFR-2 kinase and human umbilical vein
Received 27 March 2010
Revised 14 August 2010
Accepted 3 September 2010
Available online 11 October 2010
endothelial cells (HUVEC) in vitro. Compound
6 (5-chloro-2-hydroxy-N-(quinolin-8-yl)benzamide)
exhibited the most potent inhibitory activity (IC50 = 3.8 and 5.5 nM for VEGFR-2 kinase and HUVEC,
respectively). Docking simulation supported the initial pharmacophoric hypothesis and suggested a com-
mon mode of interaction at the ATP-binding site of VEGFR-2, which demonstrates that compound 6 is a
potential agent for cancer therapy deserving further researching.
Keywords:
Quinoline amide
SAR
Ó 2010 Elsevier Ltd. All rights reserved.
Docking
VEGFR-2 inhibitor
Angiogenesis, the formation of new blood vessels from existing
vasculature, plays a major role in the progression of many human
diseases, including cancer, where it is essential for the growth and
survival of solid tumors.1 The growth of new vessels supplies oxy-
gen, nutrients, and growth factors to small tumors and removes the
waste products of metabolism. Tumors that lack an adequate vas-
culature become necrotic or apoptotic and do not grow beyond a
limited size. 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 cancer therapy.2,3
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 sig-
naling pathway should block angiogenesis and subsequent tumor
growth. There is much evidence that direct inhibition of the kinase
activity of VEGFR-2 (also referred to as the kinase insert domain con-
taining receptor (KDR)) will result in the reduction of angiogenesis
and the suppression of tumor growth.4,5 A number of small molecule
structural classes, for example, indolin-2-ones,6 phthalazines,7
quinolinones,8 imidazopyridines,9 benzimidazoles,10 pyridines,11
and quinazolines,12 have been disclosed as potent inhibitors of
VEGFR-2 in vitro or have demonstrated antiangiogenic.
the catalytic site of kinases with ATP-competitive inhibitors is a
more promising approach for drug intervention.13 In view of this,
our program has targeted the development of small-molecule
inhibitors of VEGFR-2, based on molecular modeling and the inves-
tigation of SAR between new inhibitors and VEGFR-2. The designed
quinoline amides could be interacting with the catalytic site of
VEGFR-2 well. The functional groups of these amides could form
hydrogen bonds with the catalytic site of VEGFR-2 as hydrogen do-
nor or acceptor. All synthesized compounds were evaluated for
their inhibitory activities against HUVEC and VEGFR-2 kinase.14
Compound 6 displayed the most potential inhibitory activities
against HUVEC and VEGFR-2 kinase. Docking simulations were
performed using the X-ray crystallographic structure of the
VEGFR-2 to explore the binding modes of compound 6 at the
active site.15,16
The synthesis of quinoline amide derivatives followed the gen-
eral reaction pathway outlined in Scheme 1. The compounds 1–24
(Table 1) were synthesized by coupling substituted quinoline
amines with equimolar quantities of substituted benzoic acids,
salicylic acids and niconacids, respectively, using 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDCÁHCl) as
condensing agent. The mixture was refluxed in anhydrous CH2Cl2
for 8–10 h.
Because of the difficulties associated with the competitive inhi-
bition of protein–protein interactions by small molecular weight
compounds, modulating the activity of kinases by interfering
either with ligand or substrate binding is difficult and targeting
The synthesized quinoline amides were evaluated for their
abilities to inhibit the proliferation of HUVEC induced by either
VEGF or basic fibroblast growth factor (bFGF) and VEGFR-2 kinase.
Stimulation of HUVEC proliferation with bFGF was utilized as a
measure of selectivity because this should be unaffected by a
VEGFR-2 inhibitor. The effect of kinase inhibitors on cell prolifera-
tion was measured using 5-bromo-2-deoxyuridine (BrdU) incorpo-
ration method using commercially available kits. HUVEC was
⇑
Corresponding author. Tel./fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
Both authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.014

6654
Y. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6653–6656
Scheme 1. General synthesis of compounds 1–24. Reagents and conditions: R: CH2Cl2; T: 40 °C; EDC 200 mg; reflux.
Table 1
Table 2
Chemical structures of compounds 1–24
Inhibition (IC50/nM) of VEGFR-2 kinase and inhibition (IC50/nM) of HUVEC
proliferation
Compd
R1
R2
R3
R4
R5
X
Compd
VEGFR-2a
HUVECb (VEGF)
HUVECb (bFGF)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
NO2
H
H
CH3
H
H
H
H
H
CH3
CH3
Br
Cl
CH3
CH3
H
C
C
C
C
C
C
C
C
N
N
N
N
C
C
C
C
C
C
C
N
N
N
C
N
NO2
Cl
H
H
H
CH3
H
H
H
Cl
H
H
H
H
H
H
H
H
H
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
134.6
112.7
124.6
23.6
16.5
3.8
9.6
26.5
5.5
13.5
84.9
208.8
107.2
28.4
325.6
32.4
72.8
12.4
25.5
23.4
102.1
88.5
112.6
32.7
72.4
46.7
15.2
122.7
15.8
5.5
1160
450
220
1150
210
110
45
1140
560
1150
560
346
68
660
560
240
340
470
780
1240
885
1140
720
460
H
OH
OH
OH
OH
OH
H
H
H
Br
H
OH
OH
OH
OH
Br
Cl
H
H
OH
H
19.8
26.3
12.9
13.6
140.8
210.7
78.9
11.6
76.3
18.9
28.4
18.9
35.7
58.9
77.6
59.5
89.6
14.5
Br
CH3
H
Br
H
H
Cl
Br
H
Br
Br
Cl
CH3
Br
Br
Cl
H
H
H
OH
H
H
CH3
H
a
IC50 values were averaged values determined by at least two independent
experiments.
b
Human umbilical vein endothelial cells. ND: not determined.
seeded in medium containing 5% fetal bovine serum (FBS) in type 1
collagen coated 96-well plates and incubated overnight at 37 °C,
5% CO2. The medium was aspirated from the cells, and various
concentrations of the inhibitor in serum-free medium were added
to each well. After 30 min, either VEGF (10 ng/mL) or bFGF
(0.3 ng/mL) was added to the wells. Cells were incubated for an
which was prepared by condensation of 5-chloro-2-hydroxybenzo-
ic acid with quinolin-8-amine. Most of the synthesized compounds
demonstrated significant selectivity profile against bFGF-induced
proliferation of HUVEC. The IC50 against VEGF-induced HUVEC rep-
resented significant selectivity compared to bFGF.
As showed in Table 2, the IC50 values of all compounds against
VEGFR-2 ranged broadly from 3.8 to 2010 nM. Compounds 1–12
were prepared by 8-amino quinoline, while compounds 13–24
were 3-amino quinoline derivatives. In general, compounds 1–12
showed greater inhibitory activities than compounds 13–24. Com-
pounds with hydroxyl or chlorine substituents at R1 position
showed much better inhibitory activity (with their IC50s lower
additional 72 h and BrdU (10 lM) was added during the last
18–24 h of incubation. At the end of incubation, BrdU incorpora-
tion in cells was measured by ELISA according to manufacturer’s
instructions. Data were fitted with a curve described by the
equation, y = Vmax(1 À (x/(K + x))), where K is equal to the IC50
.
The results were summarized in Table 2. A number of given com-
pounds displayed potent HUVEC and VEGFR-2 inhibitory activities.
Compound 6 showed the greatest activities against HUVEC and
VEGFR-2 kinase with IC50 of 3.8 nM and 5.5 nM, respectively,

Y. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6653–6656
6655
than 100 nM) than the other compounds with hydrogen substitu-
ent at R1 position. Furthermore, compounds 6, 9, 16 and 21 with
chloro or bromo substituent at R3 position exhibited better activity
than compounds with hydrogen or methyl substituent at R3 posi-
tion. Structure–activity relationships in these quinoline amide
derivatives demonstrated that compounds with hydroxyl or halo-
gen groups displayed better inhibitory activities than those with
hydrogen or methyl groups, which can be inferred from the IC50s
of compound 6. We can infer that the hydroxyl group at R1 and
chlorine atom at R3 may be responsible for the inhibitory activities.
Molecular docking of the most potent inhibitor 6 into ATP-bind-
ing site of VEGFR-2 kinase was performed on the binding model
based on the VEGFR-2 complex structure (3B8R: PDB17). Figure 1
shows the binding modes of compound 6 into the ATP-binding cav-
ity of VEGFR-2.
1046, Glu 885 and Lys 868, which made the 3D structure more
stable. The binding energy for compound 6 in the ATP-site is
À10.54 kcal/mol with the estimated inhibition constant,
Ki = 18.94 nM.
In summary, two series of quinoline amide derivatives were
synthesized and evaluated for their inhibitory activities against
VEGFR-2 and HUVEC. Some of the prepared compounds displayed
potent activities. The introduction of electron-withdrawing groups
(like halogeno and hydroxyl group) at R1 and R3 position was favor-
able to the inhibitory activity. Compound 6 (5-chloro-2-hydroxy-
N-(quinolin-8-yl)benzamide) exhibited the most potential activity
with the IC50 = 3.8 nM for VEGFR-2 kinase and IC50 = 5.5 nM for
HUVEC induced by VEGF. Docking simulation was performed to
position compound 6 to the active site of VEGFR-2 kinase to deter-
mine the probable binding model. The results indicated that
compound 6 was nicely bonded to VEGFR-2 with a hydrogen bond
As illustrated in Figure 1, a large conformational change in the
highly conserved Asp-Phe-Gly (DFG) loop, appeared in the docking
mode with VEGFR-2. Inspection of these crystallographic struc-
tures, however, revealed a large conformational change in the
highly conserved Asp-Phe-Gly (DFG) loop. This conformational
change, opened up this extended hydrophobic pocket which can-
not be accessed when the enzyme is in the ‘active’ conformation.
Visual inspection of the pose of 6 into the ATP-site revealed that
compound 6 appears to be interact with the region through the
protonated phenolic hydroxyl group projecting toward the oxygen
of carboxyl group of Leu 840, forming a more optimal H-bond
(O-HÁÁÁO: 2.067 Å, 125.2°) interaction. The modeling also suggested
and two
p–p stacking interactions, which demonstrated com-
pound 6 would be a potential antitumor agent that deserves
further research.
Acknowledgment
The work was financed by grants from National Natural Science
Foundation of China (Project 30772627) and the Jiangsu National
Science Foundation (No. BK2009239).
that there were two
ring and the benzene ring of Phe 918. The
p
–
p
stacking interactions between quinoline
interaction energies
Supplementary data
p–p
are of the same order of magnitude as hydrogen bonds and play an
important role in stabilizing the three dimensional structure of the
inhibitor–enzyme complex. The quinoline ring was accommodated
in the mostly hydrophobic ATP-binding cleft. Also the quinoline
ring projects into a hydrophobic region, which is comprised of
the side chains of Ala 866, Phe 918, Cys 919, Leu 840 and Leu
1035. These residues influenced the accessibility of the hydropho-
bic pocket that flanks the ATP-binding site, and their size can be a
key factor in controlling kinase selectivity. This potency increase
was attributed to a key predicted hydrogen bond illustrated
previously. In the other end of the ATP-binding pocket, the phenyl
group with chloro substituent interacted with the residues Asp
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.014.
References and notes
1. Brown, J. M.; Giaccia, A. J. Cancer Res. 1998, 58, 1408.
2. Harris, P. A.; Boloor, A.; Cheung, M.; Kumar, R.; Crosby, R. M.; Davis-Ward, R. G.;
Epperly, A. H.; Hinkle, K. W.; Hunter, R. N., III; Johnson, J. H.; Knick, V. B.;
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.
3. (a) Hickey, K.; Grehan, D.; Reid, I. M.; O’Briain, S.; Walsh, T. N.; Hennessy, T. P. J.
Cancer 1994, 74, 1693; (b) Kim, K. J.; Li, B.; Winer, J.; Armanini, M.; Gillett, N.;
Phillips, H. S.; Ferrara, N. Nature 1993, 362, 841.
4. (a) Ferrara, N.; Gerber, H.-P.; LeCouter, J. Nat. Med. 2003, 9, 669; (b) Cross, M. J.;
Claesson-Welsh, L. Trends Pharmacol. Sci. 2001, 22, 201; (c) McMahon, G.
Oncologist 2000, 5, 3.
5. Prewett, M.; Huber, J.; Li, Y.; Santiago, A.; O’Connor, W.; King, K.; Overholser, J.;
Hooper, A.; Pytowski, B.; Witte, L.; Bohlen, P.; Hicklin, D. J. Cancer Res. 1999, 59,
5209.
6. (a) Sun, L.; Tran, N.; Liang, C.; Tang, F.; Rice, A.; Schreck, R.; Waltz, K.; Shawver,
L. K.; McMahon, G.; Tang, C. J. Med. Chem. 1999, 42, 5120; (b) Sun, L.; Tran, N.;
Liang, C.; Hubbard, S.; Tang, F.; Lipson, K.; Schreck, R.; Zhou, Y.; McMahon, G.;
Tang, C. J. Med. Chem. 2000, 43, 2655.
7. Bold, G.; Altmann, K. H.; Frei, J.; Lang, M.; Manley, P. W.; Traxler, P.; Wietfeld,
B.; Bruggen, J.; Buchdunger, E.; Cozens, R.; Ferrari, S.; Furet, P.; Hofmann, F.;
artiny-Baron, G.; Mestan, J.; Rosel, J.; Sills, M.; Stover, D.; Acemoglu, F.; Boss, E.;
Emmenegger, R.; Lasser, L.; Masso, E.; Roth, R.; Schlachter, C.; Vetterli, W. J.
Med. Chem. 2000, 43, 2310.
8. Fraley, M. E.; Arrington, K. L.; Buser, C. A.; Ciecko, P. A.; Coll, K. E.; Fernandes, C.;
Hartman, G. D.; Hoffman, W. F.; Lynch, J. J.; McFall, R. C.; Rickert, K.; Singh, R.;
Smith, S.; Thomas, K. A.; Wong, B. K. Bioorg. Med. Chem. Lett. 2004, 14, 351.
9. Wu, Z.; Fraley, M. E.; Bilodeau, M. T.; Kaufman, M. L.; Tasber, E. S.; Balitza, A. E.;
Hartman, G. D.; Coll, K. E.; Rickert, K.; Shipman, J.; Shi, B.; Sepp-Lorenzino, L.;
Thomas, K. A. Bioorg. Med. Chem. Lett. 2004, 14, 909.
10. Bilodeau, M. T.; Cunningham, A. M.; Koester, T. J.; Ciecko, P. A.; Coll, K. E.;
Huckle, W. R.; Hungate, R. W.; Kendall, R. L.; McFall, R. C.; Mao, X.; Rutledge, R.
Z.; Thomas, K. A. Bioorg. Med. Chem. Lett. 2003, 13, 2485.
11. Bilodeau, M. T.; Rodman, L. D.; McGaughey, G. B.; Coll, K. E.; Koester, T. J.;
Hoffman, W. F.; Hungate, R. W.; Kendall, R. L.; McFall, R. C.; Rickert, K. W.;
Rutledge, R. Z.; Thomas, K. A. Bioorg. Med. Chem. Lett. 2004, 14, 2941.
12. (a) Hennequin, L. F.; Thomas, A. P.; Johnstone, C.; Stokes, E. S.; Plé, P. A.;
Lohmann, J. J.; Ogilvie, D. J.; Dukes, M.; Wedge, S. R.; Curwen, J. O.; Kendrew, J.;
Lambert-van der Brempt, C. J. Med. Chem. 1999, 42, 5369; (b) Hennequin, L. F.;
Stokes, E. S.; Thomas, A. P.; Johnstone, C.; Plé, P. A.; Ogilvie, D. J.; Dukes, M.;
Wedge, S. R.; Kendrew, J.; Curwen, J. O. J. Med. Chem. 2002, 45, 1300.
Figure 1. Molecular docking of compound 6 to the ATP-binding pocket of VEGFR-2.
The molecules are colored by the atoms (red for oxygen, blue for nitrogen, gray for
carbon and cyan for hydrogen). Hydrogen bonds are shown between the inhibitors
and VEGFR-2 (green dotted lines).

6656
Y. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6653–6656
13. (a) Cochran, A. G. Chem. Biol. 2000, 7, R85; (b) Manley, P. W.; Furet, P. In
Anticancer Agents: Frontiers in Cancer Chemotherapy; Ojima, I., Vite, G., Altmann,
K., Eds.; ACS Symposium Series 796; American Chemical Society: Washington,
DC, 2001. p 282.
14. Polverino, A.; Coxon, A.; Starnes, C.; Diaz, Z.; DeMelfi, T.; Wang, L.; Bready, J.;
Estrada, J.; Cattley, R.; Kaufman, S.; Chen, D.; Gan, Y.; Kumar, G.; Meyer, J.;
Neervannan, S.; Alva, G.; Talvenheimo, J.; Montestruque, S.; Tasker, A.; Patel,
V.; Radinsky, R.; Kendall, R. Cancer Res. 2006, 66, 8715.
16. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
17. Weiss, M. M.; Harmange, J.-C.; Polverino, A. J.; Bauer, D.; Berry, L.;
Berry, V.; Borg, G.; Bready, J.; Chen, D. L.; Choquette, D.; Coxon, A.;
DeMelfi, T.; Doerr, N.; Estrada, J.; Flynn, J.; Graceffa, R. F.; Harriman, S.
P.; Aufman, S.; La, D. S.; Long, A.; Neervannan, S.; Patel, V. F.;
Potashman, M.; Regal, K.; Roveto, P. M.; Schrag, M. L.; Starnes, C.;
Tasker, A.; Teffera, Y.; Whittington, D. A.; Zanon, R. J. Med. Chem. 2008,
51, 1668.
15. Huey, R.; Morris, G. M.; Olson, A. J.; Goodsell, D. S. J. Comput. Chem. 2007, 28,
1145.