Optimization of 1H-indazol-3-amine derivatives as potent fibroblast growth factor receptor inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.06.068

Fibroblast growth factor receptor (FGFR) is a potential target for cancer therapy because of its critical role in promoting cancer formation and progression. In a continuing effort to improve the cellular activity of hit compound 7r bearing an indazole scaffold, which was previously discovered by our group, several compounds harnessing fluorine substituents were designed, synthesized and biological evaluated. Besides, the region extended out to the ATP binding pocket toward solvent was also explored. Among them, compound 2a containing 2,6-difluoro-3-methoxyphenyl residue exhibited the most potent activities (FGFR1: less than 4.1?nM, FGFR2: 2.0?±?0.8?nM). More importantly, compound 2a showed an improved antiproliferative effect against KG1 cell lines and SNU16 cell lines with IC₅₀ values of 25.3?±?4.6?nM and 77.4?±?6.2?nM respectively.

Full text of DOI:10.1016/j.bmcl.2017.06.068

ꢀꢁꢁꢂꢃꢄꢂꢅꢆ ꢇꢈꢉꢊꢁꢋꢌꢃꢄ

Optimization of 1H-indazol-3-amine derivatives as potent
Fibroblast Growth Factor Receptor inhibitors
Jing Cuia,1, Xia Peng b,1, Dingding Gaoa,1, Yang Dai b, Jing Aib,*, Yingxia Lia,*
a School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
b Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China.
ABSTRACT
Fibroblast growth factor receptor (FGFR) is a potential target for cancer therapy because of its critical role in
promoting cancer formation and progression. In a continuing effort to improve the cellular activity of hit compound 7r
bearing an indazole scaffold, which was previously discovered by our group, several compounds harnessing fluorine
substituents were designed, synthesized and biological evaluated. Besides, the region extended out to the ATP binding
pocket toward solvent was also explored. Among them, compound 2a containing 2,6-difluoro-3-methoxyphenyl residue
exhibited the most potent activities (FGFR1: less than 4.1 nM, FGFR2: 2.0 0.8 nM). More importantly, compound 2a
showed an improved antiproliferative effect against KG1 cell lines and SNU16 cell lines with IC50 values of 25.3 4.6
nM and 77.4 6.2 nM respectively.
Keywords: FGFR, inhibitors, cellular activity, fluorine.
* Corresponding authors. Tel.: +86 21 50806600x2413 (J.A.), +86 21 51980127(Y.L.).
E-mail addresses: jai@simm.ac.cn, liyx417@fudan.edu.cn.
1 These authors contributed equally to this work.

The fibroblast growth factor receptor (FGFR) family consists of four RTKs (FGFR1-4) which bind a
diverse family of 18 FGF ligands and play a fundamental role in many physiological processes, involving
embryogenesis, tissue homeostasis, tissue repair, wound healing, and inflammation.1,2 In cancer, the
constitutive FGFR signaling is activated by gene amplification, point mutations, or chromosomal
translocations/rearrangements in several tumor types and involved in cell growth, angiogenesis, cell
migration, invasion, and metastasis.3,4 A recent analysis of 4,853 solid tumors found FGFR aberrations in
7.1% of cancers, gene amplification (66%), mutations (26%) and rearrangements (8%). FGFR1 (mostly
amplification) was affected in 3.5% of patients, FGFR2 in 1.5%, FGFR3 in 2.0%, and FGFR4 in 0.5%. The
most commonly affected cancers were urothelial (32%), breast (18%), endometrial (~13%), lung (squamous)
(~13%), and ovarian (~9%).5 Owing to their prominent roles in tumor, FGFRs have become crucial targets
for cancer therapy.6-9
Currently, several second-generation FGFR-selective inhibitors, such as AZD4547, NVP-BGJ398,
CH-5183284, and LY-2874455 are under clinical trials targeting patients who have FGFR genetic alterations
(Fig. 1).10-14 As a frontrunner in the group of pan-FGFR inhibitors, AZD4547 was demonstrated to promote
favorable therapeutic outcomes against a variety of FGFR-deregulated cancer models, including
glioblastoma, non–small cell lung cancer, gastric cancer, and multiple myeloma.10, 15
Figure 1. Structures of representative FGFR inhibitors
Based on AZD4547, scaffold hopping and molecular hybridization strategies were utilized to identify the
compound 7r as a potent FGFR inhibitor (Fig. 2). This compound showed potent enzymatic potency against
FGFR1 and modest cellular inhibition.16 Fluorine is often used to improve permeability through the
modulation of molecule’s lipophilicity, direct fluorine−protein interactions, or reduction of amine basicity.
Large amounts of case studies on the use of fluorine interactions in rational drug design have been

reported.17-20 Herein, we first focus on the incorporation of fluorine substituents on ring A、B and C(7r, Fig. 2)
to improve the cellular potency(1a-g and 2a) . Then the region extended out to the ATP binding pocket
toward solvent was also explored (2b-g).
Figure 2. Previous work and design of target compounds
The preparation of target compounds 1a-b was described in Scheme 1. Compounds 5a-b were obtained
from commercial sources. Coupling of 4-bromo-2-fluorobenzonitrile derivatives with hydrazine gave 6a-b in
good yields. Then the key intermediates 7a-b were obtained by condensation of 4a with 6a-b in the presence
of Al(CH3)3 at 120°C. Treatment of 7a-b with 2-fluoro-3-methoxyphenylboronic acid under Suzuki coupling
conditions gave target compounds 1a-b. The preparation of 1c-e was illustrated in Scheme 2 and 3.
Condensation of 11c-d, 14 with 10 afforded compounds 12c–d and 15 in the presence of EDCI, DMAP at
60 °C. Finally, coupling of 12c-d and 15 with 2-fluoro-3-methoxyphenylboronic acid yielded target
compounds 1c-e. The synthesis of compounds 2a-g and 1f-g was shown in Scheme 4. Condensation of 10
with 4a-g in the presence of Al(CH3)3 at 120°C gave intermediates 16a-g, which directly attempted to
Suzuki coupling with 2-fluoro-3-methoxyphenylboronic acid were unsuccessful. After protected the free NH
of the 1H-indazol scaffold with Boc group, we successfully got target compounds 2a-g and 1f-g via Suzuki
coupling reaction.
N
N
O
a
HN
Br
F
N
N
N
O
H
H
Br
R1
N
N
N
N
O
3
4a
N
N
d
c
R1
H
R2
HN
F
N
R2
HN
Br
R1
N
O
O
b
R1
CN
7a: R1 = F, R2 = H
7b: R1 = H, R2 = F
1a: R1 = F, R2 = H
1b: R1 = H, R2 = F
R2
NH2
R2
5a: R1 = F, R2 = H
5b: R1 = H, R2 = F
6a: R1 = F, R2 = H
6b: R1 = H, R2 = F
Scheme 1 Reagents and conditions: (a) 4-fuorobenzoate, K2CO3, DMSO, 110oC, 89.2%; (b) hydrazine hydrate, n-butanol, 120oC, 85.7% (c) Al(CH3)3, toluene, 120 oC, 15.9% (d) 2-
Fluoro-3-methoxyphenylboronic acid, Cs2CO3, Pd(dppf)Cl2, dioxane, 120 oC, 35.7%-49.6%.

The target compounds were then evaluated for inhibitory activities against the FGFR1, FGFR2 enzymes
and two representative cancer cell lines with different mechanisms of FGFR activation (FGFR1-fusion
protein driven human myeloid leukemia cancer cell line KG-1 and FGFR2-amplified dependent human
gastric cancer cell line SNU-16). As shown in Table 1, incorporation of fluorine at C-6 position in ring A
improved both enzymatic potency and cellular potency (2a, FGFR1: less than 4.1 nM, FGFR2: 2.0 0.8 nM,
KG1 cell lines: 25.3 4.6 nM, SNU16 cell lines: 77.4 6.2 nM). However, compounds 1a-e displayed a
significant decrease of activities both on enzymes and cells, which indicated that ring B and C were not
tolerable to any fluorine substitution patterns. While introducing fluorine at both C-4 and C-6 positions in
ring A led to the activity decrease (1f and 1g).

Table 1 In vitro enzymatic inhibitory activities and antiproliferative activities of designed compounds
compd.
FGFR1 IC50 a (nM)
35.2±2.2
219.6 104.4
147.6 16.0
>1000
FGFR2 IC50 (nM)
9.5 0.1
KG1 IC50 (nM)
683.9 114.9
>1000 b
SNU16 IC50 (nM)
>1000
1a
1b
132.7 19.6
108.5 11.1
>1000
>1000
1c
>1000
>1000
1d
>1000
>1000
1e
>1000
>1000
>1000
>1000
2a
<4.1
2.0 0.8
25.3 4.6
>1000
77.4 6.2
>1000
1f
49.0 4.3
99.3 18.8
6.2 0.7
35.1 2.4
91.0 21.6
4.0 0.4
1g
>1000
>1000
7r
283.9 13.4
590.8 105.8
AZD4547
1.2 0.1
0.6 0.1
3.3 0.2
3.4 0.2
a The IC50 values are shown as the mean SD (nM) or estimated values from two separate experiments.
b If a specific compound is given a value>1000, it indicates that a specific IC50 cannot be calculated from the data points collected,
meaning‘ no effect ’.
To further improve the antiproliferative activities against KG1 cell lines and SNU16 cell lines, the
modification was concentrated on ring C which extended out to the ATP binding pocket toward solvent.
Then a diversity of substituents was explored (guided by a calculated lipophilicity threshold). The procedure
was shown in Scheme 4 which was similar to the synthesis of compound 2a. The results were listed in Table
2. In light of these data, all of these tested compounds exhibited potent FGFR1 and FGFR2 enzymatic
activities. Actually, we did not found the correlation between clogP and activities in this series compounds.
Compared to 7r, compounds 2a, 2c and 2f not only displayed better enzymatic inhibitory activities, but also
exhibited superior antiproliferative activities. However, compounds 2b, 2d and 2e showed weaker cellular
activities, perhaps owing to the poor cellular penetration.
Table 2 In vitro enzymatic inhibitory activities and antiproliferative activities of designed compounds
compd.
cLog P b
5.74
5.67
5.05
4.23
5.39
5.09
5.59
5.55
4.43
FGFR1 IC50 a (nM)
<4.1
FGFR2 IC50 (nM)
2.0 0.8
KG1 IC50 (nM)
25.3 4.6
SNU16 IC50 (nM)
77.4 6.2
2a
2b
14.0 1.5
3.7 0.3
7.0 0.6
347.7 12.4
51.5 17.8
595.4 98.1
47.3 18.9
41.5 5.8
582.4 16.3
101.5 42.9
322.2 127.0
430.8 43.1
35.6 11.5
2c
2.0 0.7
2d
13.0 0.2
10.4 1.4
5.6 1.5
2.9 1.4
2e
16.6 2.0
<4.1
2f
2g
21.1 7.6
6.2 0.7
35.1 2.4
4.0 0.4
332.6 61.3
283.9 13.4
3.3 0.2
365.6 109.5
590.8 105.8
3.4 0.2
7r
AZD4547
1.2 0.1
0.6 0.1
a The IC50 values are shown as the mean SD (nM) or estimated values from two separate experiments.
b The cLog P values were calculated by ACD-Labs (Version 6.0).
To elucidate the binding mode of compound 2a to the FGFR1 protein (PDB code: 4ZSA),16 molecular
docking was performed using Glide module encoded in Schrodinger, Maestro 10.1 (Fig. 3A). For
comparison, the binding mode of hit compound 7r was also generated (Fig. 3B) and superimposed on that of
2a (Fig. 3C). As depicted in Figure 3, both compounds could tightly bind to the ATP-binding site of FGFR1.
In detail, the 3-aminoindazole fragment occupied the hinge region of the protein via three hydrogen bonds

with Glu562 and Ala564 and the phenyl ring of indazole core interacted with Phe 489 via π-π stacking.
Additionally, the fluorine substituted 3-methoxyphenyl moiety located at the hydrophobic pocket.
Interestingly, compound 2a with two fluorine atoms at phenyl ring rendered the orientation greatly changed
compared with compound 7r and gained another hydrogen bond between the methoxy oxygen and the amino
group of Asp641. Furthermore, the two fluorine atoms can form hydrophobic interactions with Ala640 and
Val492. These favorable changes could be the possible reasons that compound 2a showed a slightly stronger
activity than compound 7r.
Figure 3. (A) Predicted binding mode for compound 2a (B) Predicted binding mode for compound 7r (C) A comparison of the
binding mode of 2a (green) and compound 7r (orange) with FGFR1. The pictures were generated using Pymol.
In summary, the search to find compounds that exhibited better cell potency than hit 7r led us to
discovery many fluorine substituents with significantly improved cell potency. The basic N-ethylpiperazine
moiety which extended out to the ATP binding pocket toward solvent was also optimized. As we expected,
the most potent compound 2a exhibited increasing enzymatic and antiproliferative activities (FGFR1: less
than 4.1 nM, FGFR2: 2.0 0.8 nM, KG1 cell lines: 25.3 4.6 nM, SNU16 cell lines: 77.4 6.2 nM). Further
investigation on the mechanism and in vivo antitumor activity of compound 2a are undergoing and will be
reported in due course.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (Grant No.
81473075, 81473243), and "Personalized Medicines–Molecular Signature-based Drug Discovery and
Development" and Strategic Priority Research Program of the Chinese Academy of Sciences (No.
XDA12020103).
Supplementary data
Supplementary data is available on the publishers’web site along with the published article.
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Optimization of 1H-indazol-3-amine derivatives as potent
Fibroblast Growth Factor Receptor inhibitors
Jing Cuia,1, Xia Peng b,1, Dingding Gaoa,1, Yang Dai b, Jing Aib,*, Yingxia Lia,*
a School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
b Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China.
In a continuing effort to develop more potent cellular activity of hit compound 7r which was previously discovered by
our group, several compounds harnessing fluorine substituents were designed, synthesized and biological evaluated.
Additionally, the region extended out to the ATP binding pocket toward solvent was also explored.
*
*
*
*
* Corresponding authors. Tel.: +86 21 50806600x2413 (J.A.), +86 21 51980127(Y.L.).
E-mail addresses: jai@simm.ac.cn, liyx417@fudan.edu.cn.
1 These authors contributed equally to this work.