Design, synthesis and biological evaluation of O-linked indoles as VEGFR-2 kinase inhibitors (I)

Article abstract of DOI:10.1016/j.cclet.2015.07.016

Inhibition of VEGFR-2 signaling pathway has already become one of the most promising approaches for the treatment of cancer. In this study, we describe the design, synthesis, and biological evaluation of a series of O-linked indoles as potent inhibitors of VEGFR-2. Among these compounds, 18 showed significant anti-angiogenesis activities via VEGFR-2 in enzymatic proliferation assays, with IC₅₀ value of 3.8 nmol/L. Kinase selectivity profiling revealed that 18 was a multitargeted inhibitor, and it also exhibited good potency against VEGFR-1, PDGFR-α and β.

Full text of DOI:10.1016/j.cclet.2015.07.016

Accepted Manuscript
Title: Design, synthesis and biological evaluation of O-linked
indoles as VEGFR-2 kinase inhibitors (I)
Author: Guo-Rui Gao Meng-Yuan Li Lin-Jiang Tong Li-Xin
Wei Jian Ding Hua Xie Wen-Hu Duan
PII:
S1001-8417(15)00302-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2015.07.016
CCLET 3393
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
19-5-2015
20-6-2015
6-7-2015
Please cite this article as: <doi>http://dx.doi.org/10.1016/j.cclet.2015.07.016</doi>
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Graphical Abstract
Design, synthesis and biological evaluation of O-linked indoles as VEGFR-2 kinase inhibitors (I)
Guo-Rui Gao ‡,a , Meng-Yuan Li ‡,b,c, Lin-Jiang Tongb, Li-Xin Weic, Jian Ding b,*, Hua Xie b,*, Wen-Hu Duan a, d,
a School of Pharmacy, East China University of Science & Technology, Shanghai 200237, China
b Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China
c Pharmacology and Safety Evaluation Key Laboratory of Tibetan Medicine in Qinghai Province, Northwest Institute of Plateau
Biology, Chinese Academy of Sciences, Xining 810008, China
d Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
China
H
N
Brivanib
O
H
H
F
N
N
N
O
Hybrid design
N
SAR exploring
O
O
N
O
N
N
N
O
F
F
N
N
N
O
NH2
N
N
S
N
SO2NH2
N
O
H
H
N
1, VEGFR-2 IC50: 39 nmol/L
18, VEGFR-2 IC50: 3.8 nmol/L
N
H
SO2NH2
Pazopanib
In an effort to discover potent VEGFR-2 inhibitors, a series of 2,4 or 4,6-disubstitued O-linked indoles derivatives were designed and
synthesized. The structural activity relationships led to identification of a potential VEGFR-2 inhibitor compound 18.
Corresponding author
E-mail addresses: whduan@simm.ac.cn (W-H. Duan); hxie@simm.ac.cn (H. Xie); jding@simm.ac.cn (J. Ding)
‡These authors contributed equally to this work.
Page 1 of 6

Original article
Design, synthesis and biological evaluation of O-linked indoles as VEGFR-2 kinase
inhibitors (I)
b,
b,
Guo-Rui Gao ‡,a, Meng-Yuan Li ‡,b,c, Lin-Jiang Tongb, Li-Xin Weic, Jian Ding
Duan a, d,
*, Hua Xie *, Wen-Hu
a School of Pharmacy, East China University of Science & Technology, Shanghai 200237, China
b Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China
c Pharmacology and Safety Evaluation Key Laboratory of Tibetan Medicine in Qinghai Province, Northwest Institute of Plateau Biology, Chinese Academy of
Sciences, Xining 810008, China
d Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
ARTICLE INFO
ABSTRACT
Inhibition of VEGFR-2 signaling pathway has already become one of the most promising
approaches for the treatment of cancer. In this study, we describe the design, synthesis, and
biological evaluation of a series of O-linked indoles as potent inhibitors of VEGFR-2. Among
these compounds, 18 showed significant anti-angiogenesis activities via VEGFR-2 in
enzymatic proliferation assays, with IC50 value of 3.8 nmol/L. Kinase selectivity profiling
revealed that 18 was a multitargeted inhibitor, and it also exhibited good potency against
VEGFR-1, PDGFR-α and β.
Article history:
Received 19 May 2015
Received in revised form 20 June 2015
Accepted 6 July 2015
Available online
Keywords:
VEGFR-2
Inhibitor
Indole
1. Introduction
Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a normal and vital process in growth and
development, as well as in wound healing and female reproductive cycling[1,2]. Pathological angiogenesis has been correlated with a
variety of diseases, such as retinopathies, diabetes, rheumatoid arthritis, psoriasis and cancer [3,4]. Tumor angiogenesis is a
prerequisite for tumor growth as it is a fundamental step in the growth and metastasis of cancer [5]. This makes angiogenesis inhibition
a promising therapeutic strategy against cancer.
Tumor angiogenesis is initiated by many factors, produced by both host and tumor cells, including vascular endothelial growth
factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and other cytokines [6]. VEGFs and receptor
(VEGFR-2) is very important in the direct regulation of angiogenesis, mitogenic signaling, and permeability-enhancing effects [7].
VEGFR-2 is expressed at high level in many kinds of cancers [8]. Disruption of VEGF signaling pathway by either specific binding of
circulating VEGF or inhibiting receptor tyrosine kinases with small molecules has been found to inhibit angiogenesis, tumor
progression and dissemination. Some VEGFR-2 inhibitors have been approved by FDA, such as Sunitinib [9], Sorafenib [10],
Vandetanib [11], Pazopanib [12] (Fig. 1).
Corresponding author
E-mail addresses: whduan@simm.ac.cn (W.-H. Duan); hxie@simm.ac.cn (H. Xie); jding@simm.ac.cn (J. Ding)
‡These authors contributed equally to this work.
Page 2 of 6

Fig. 1. Some reported VEGFR-2 inhibitors.
Brivanib (Fig. 2) was reported as an ATP competitive VEGFR-2 inhibitor with an IC50 of 25 nmol/L [13]. By analyzing the structure
of Pazopanib and Brivanib, we speculated that the indole segment in Brivanib and indazole segment in Pazopanib may have similar
interaction patterns with VEGFR-2. On the basis of this assumption, compound 1 (Fig.2) was designed and synthesized. As expected,
compound 1 potently inhibited VEGFR-2 with enzymatic IC50 values of 39 nmol/L. Encouraged by this promising result, we
investigated the structure-activity relationship (SAR) of compound 1 by exploring changes on the pyrimidine core structure and the
side chains. In this regard, a series of O-linked indole analogues were synthesized and tested in enzymatic level.
Fig. 2. Design of hybrid compound 1.
2. Experimental
The synthesis of compounds 1, and 8-31was shown in Scheme 1. 2,3,4-Trifluornitrobenzene was chosen as starting material. Key
intermediate (compound 7) was obtained according to the reported method [14]. 2,4-Dicholoropyrimidine or 4,6- dicholoropyrimidine
was coupled with compound 7 to give 8a and 8b. All the analogues (1, 9-32) were prepared through an acid-catalyzed SNAr reaction
of O-linked indole intermediates with substituted anilines. The structure of the new analogues was characterized by 1H NMR and MS.
All analogues depicted in Tables 1 were evaluated for their enzymatic activities against VEGFR-2.
Scheme 1. Synthesis of compounds 1, and 9-32. Reagents and conditions: (a) NaH, ethyl acetoacetate, THF, 5 oC –r.t., 12 h; (b) HCl, HOAc, reflux, 12 h; (c)
NaOAc, DMF, 100 oC, 10 h; (d) H2, Pd/C, EtOH-DMF, 40 oC, 10 h; (e) aq.NaHCO3, MeOH, r.t., 10 h; (f) 2,4-dicholoropyrimidine or 4,6- dicholoropyrimidine,
aq. NaOH, acetone, 0-80 oC; (g) substituted anilines, 36% HCl, i-PrOH, sealed tube, 100 oC.
3. Results and discussion
We firstly investigated the effect of substitution of the aniline at pyrimidine ring on the activity. Removal of methyl group led to
compound 9, which exhibited higher potency on VEGFR-2 than compound 1 with an IC50 of 9 nmol/L. The result showed that the
p-methyl group on the ring was not crucial to enzymatic activity. The para-sulfonamide analogue (compound 10) showed a drastically
decreased potency by 3.4-fold (IC50=31 nmol/L) than compound 9, which was indicated that the influence of the position of substituent
was important. Compound 11, with trimethoxy group on the ring, showed similar potency with compound 9 (IC50=31 nmol/L).
Next, 4,6-disubstitued compound 12 was designed and synthesized. To our delight, this compound showed an improved VEGFR-2
inhibitory potency by 6-fold (IC50=6.5 nmol/L) than 2,4-disubstitued compound 1. Then a series of 4, 6-substitued indole analogues
were obtained as new scaffold for further research. Similarly, compound 13 with removal of p-methyl group increased activity by
1.3-fold (IC50=5 nmol/L) than compound 12. By variation of the position of substituent, compound 14 reduced potency with an IC50 of
40 nmol/L. compound 15 (IC50=6.8 nmol/L) and 16 (IC50=10.3 nmol/L) indicated that bulky group at the ortho-position of ring was not
tolerable. Then a set of compounds (17-32) was obtained to investigate the effect of substitution pattern of the aniline at pyrimidine
ring on the activity. As shown in Table 1, most of analogues showed good VEGFR-2 inhibitory activity. Analogues (compound 17~26)
with electron-withdrawing group displayed potent VEGFR-2 inhibitory activities. Among these analogues, compound 18 exhibited
high inhibitory activity with an IC50 value of 3.8 nmol/L. The meta-nitro substituted compound 26 exhibited weak activity (IC50=21.3
nmol/L) compared with other analogues. Then analogues (compound 27~30) with methoxy group as electron donor were screened.
Page 3 of 6

Compound 29 exhibited potent activity in enzymatic assay (IC50 = 4.9 nmol/L). Compound 31, with no substituent on the ring also
showed good activity with an IC50 value of 12.1 nmol/L. Interestingly, compound 32 with alkyl ring could inhibit VEGFR-2 with an
IC50 value of 35.1 nmol/L. The SARs study revealed that this place can tolerate different substituent on the 4-position of pyrimidine.
Table 1
VEGFR-2 inhibitory activity of compounds1 and 9-32.
VEGFR-2
IC50 (µmol/L)
Compound
X
C
C
Y
N
N
R
1
9
0.039±0.001
0.009±0.004
0.031±0.001
10
11
C
C
N
N
0.009±0.001
12
13
N
N
C
C
0.0065±0.0011
0.005±0.001
14
15
16
N
N
N
C
C
C
0.0404±0.0055
0.0068±0.0004
0.0103±0.0008
17
N
C
0.0077±0.0018
18
19
20
N
N
N
C
C
C
0.0038±0.0033
0.0071±0.0008
0.0838±0.0084
21
22
23
N
N
N
C
C
C
0.0073±0.0007
0.0084±0.0018
0.0060±0.0004
24
25
N
N
C
C
0.0046±0.0023
0.0107±0.0062
26
27
28
N
N
N
C
C
C
0.0213±0.0033
0.0088±0.0019
0.0221±0.0077
29
N
C
0.0049±0.0006
30
31
N
N
C
C
0.0065±0.0011
0.0121±0.0017
Page 4 of 6

32
N
C
0.0351±0.0027
0.0022±0.0005
Su11248
-
-
-
To assess the selectivity of this class of derivatives, compound 18 was further evaluated on a panel of tyrosine kinases. As shown in
Table 2, compound 18 demonstrated good potency against VEGFR-1, PDGFR-α, and PDGFR-β, with IC50 values of 40.4, 24.1, and
33.6 nmol/L, respectively. However, it showed high selectivity over VEGFR-3 (135-fold, IC50 = 515.8 nmol/L) and excellent
selectivity (>2530-fold) over ErbB2, ErbB4, EGFR, EGFR (T790M/L858R), ABL, EPH-A2, FGFR-1, FGFR-2 and IGF-1R.
Table 2
Kinase-selectivity profiling of compound 18.a
enzyme
IC50 (nmol/L) enzyme
IC50 (nmol/L)
>10000
VEGFR-1
VEGFR-2
VEGFR-3
PDGFR-α
PDGFR-β
ErbB2
40.4
3.8
EGFR
EGFR(T790M/L858R) >10000
515.8
24.1
ABL
>10000
>10000
>10000
>10000
>10000
EPH-A2
FGFR-1
FGFR-2
IGF-1R
33.6
>10000
>10000
ErbB4
a The inhibitory activity of compounds against 14 tyrosine kinases were measured with ELISA at a concentration of 10 µmol/L.
Finally, to further analyze the SAR of this series of analogues with O-linked indole scaffold, the docking model of compound 18
with VEGFR-2 was investigated (Fig. 3). Hydrogen bond was formed between the amide NH of indole with Glu885. Backbone amide
NH and carbonyl oxygen of Cys919 formed hydrogen bonds with nitrogen of the pyrimidine and adjacent N-H of amine in the hinge
region. The model study revealed the binding mode of VEGFR-2 to its inhibitor and helps to interpret the SAR of the O-linked indoles
analogues.
Fig. 3. Docking model of compound 18 with VEGFR-2 (PDB: 4ASD).
4. Conclusion
In summary, a series of O-linked indoles analogues were designed and synthesized, and they were identified as potential VEGFR-2
inhibitors. Therefore, our results indicated that this class of compounds could be served as lead compound for development of more
selective anticancer medication.
Acknowledgment
We thank the National Natural Science Foundation (Nos. 81273365, 81173080 and 81321092), National Science & Technology
Major Project ‘‘Key New Drug Creation and Manufacturing Program’’ (Nos. 2012ZX09103101-024 and 2014ZX09304002-008-001),
Chinese National Programs for High Technology Research and Development (No. 2012AA020302) and the Shanghai Science and
Technology Commission (No.12DZ1930802) for their financial support.
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