Design, synthesis and biological evaluation of pyrazolylaminoquinazoline derivatives as highly potent pan-fibroblast growth factor receptor inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.04.028

Fibroblast growth factor receptors (FGFRs) are important oncology targets due to the dysregulation of this signaling pathway in a wide variety of human cancers. We identified a series of pyrazolylaminoquinazoline derivatives as potent FGFR inhibitors with low nanomolar potency. The representative compound 29 strongly inhibited FGFR1-3 kinase activity and suppressed FGFR signaling transduction in FGFR-addicted cancer cells; FGFRs-driven cell proliferation was also strongly inhibited regardless of mechanistic complexity implicated in FGFR activation, which further confirmed that 29 was a potent pan-FGFR inhibitor. The flexibility of our structure offered the potential to preserve good affinity for mutant FGFR, which is important for developing TKIs with long-term efficacy.

Full text of DOI:10.1016/j.bmcl.2016.04.028

Accepted Manuscript
Design, synthesis and biological evaluation of pyrazolylaminoquinazoline de-
rivatives as highly potent pan-fibroblast growth factor receptor inhibitors
Jun Fan, Yang Dai, Jingwei Shao, Xia Peng, Chen Wang, Sufen Cao, Bin Zhao,
Jing Ai, Meiyu Geng, Wenhu Duan
PII:
S0960-894X(16)30401-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.04.028
BMCL 23790
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 January 2016
25 March 2016
12 April 2016
Please cite this article as: Fan, J., Dai, Y., Shao, J., Peng, X., Wang, C., Cao, S., Zhao, B., Ai, J., Geng, M., Duan,
W., Design, synthesis and biological evaluation of pyrazolylaminoquinazoline derivatives as highly potent pan-
fibroblast growth factor receptor inhibitors, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.04.028
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Graphical Abstract
Design, synthesis and biological evaluation of
pyrazolylaminoquinazoline derivatives as
highly potent pan-fibroblast growth factor
receptor inhibitors
Leave this area blank for abstract info.
Jun Fana,#, Yang Daib,#, Jingwei Shaoa, Xia Pengb, Chen Wangc, Sufen Caod, Bin Zhaoa, Jing Aib,,
Meiyu Gengb,, Wenhu Duana,

Bioorganic & Medicinal Chemistry Letters
Design, synthesis and biological evaluation of pyrazolylaminoquinazoline
derivatives as highly potent pan-fibroblast growth factor receptor inhibitors
Jun Fana,#, Yang Daib,#, Jingwei Shaoa, Xia Pengb, Chen Wangc, Sufen Caod, Bin Zhaoa, Jing Aib,, Meiyu
Gengb,, Wenhu Duana,
aDepartment of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), 555 Zu Chong Zhi Road,
Shanghai 201203, China
bDivision of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), 555
Zu Chong Zhi Road, Shanghai 201203, China
cDrug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), 555
Zu Chong Zhi Road, Shanghai 201203, China
dSchool of Pharmacy, East China University of Science & Technology, 130 Mei Long Road, Shanghai 200237, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Fibroblast growth factor receptors (FGFRs) are important oncology targets due to the
dysregulation of this signaling pathway in a wide variety of human cancers. We identified a
series of pyrazolylaminoquinazoline derivatives as potent FGFR inhibitors with low nanomolar
potency. The representative compound 29 strongly inhibited FGFR1–3 kinase activity and
suppressed FGFR signaling transduction in FGFR-addicted cancer cells; FGFRs-driven cell
proliferation was also strongly inhibited regardless of mechanistic complexity implicated in
FGFR activation, which further confirmed that 29 was a potent pan-FGFR inhibitor. The
flexibility of our structure offered the potential to preserve good affinity for mutant FGFR,
which is important for developing TKIs with long-term efficacy.
Keywords:
Antitumor
FGFR inhibitor
Pyrazolylaminoquinazoline
2016 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +86-21-50806032; e-mail: whduan@simm.ac.cn (W. Duan)
 Corresponding author. Tel.: +86-21-50806072; e-mail: mygeng@simm.ac.cn (M. Geng)
 Corresponding author. Tel.: +86-21-50806600 ext. 2413; e-mail: jai@simm.ac.cn (J. Ai)
# These authors contributed equally to this work.

Fibroblast growth factor receptors (FGFRs) are
transmembrane receptor tyrosine kinases (RTKs).1 Binding of
FGFRs to their cognate ligands fibroblast growth factors (FGFs)
in concert with heparin sulfate proteoglycans (HPSG) activates
FGF/FGFR signaling pathway, leading ultimately to the
upregulation of several downstream signaling pathways including
MEK1/2-ERK1/2, PLC, etc.1 FGF/FGFR signaling plays a
ubiquitous role in cell proliferation, differentiation, migration and
survival.1-6 In cancer, constitutive FGFR signaling is activated by
point mutations, gene amplification, or chromosomal
translocations/rearrangements.6 Dysregulation of FGFR signaling
has been documented in clinical samples of bladder, breast, and
gastric cancers and so on.1, 6-11 To date many methods have been
adopted to generate agents to disrupt FGF-ligand/receptor
activity, including monoclonal antibodies, FGF-ligand traps, and
small-molecule tyrosine kinase inhibitors (TKIs).12 The most
clinically advanced of these agents are mainly mixed kinase
inhibitors such as lucitanib,13 dovitinib,14-15 brivanib,16-17 and
ponatinib;18 they exert anti-FGFR activity but the predominant
activity is inhibition of vascular endothelial growth factor
receptor 2 (VEGFR2), which compromises the FGFR selectivity
and may also bring about toxicity issues. Selective FGFR TKIs
have begun to emerge in recent years, and several inhibitors have
progressed robustly into clinical trials, such as NVP-BGJ398,19
AZD4547,20 JNJ-42756493, TAS-120, CH-5183284,21 and LY-
2874455.22
VEGFR2,20 and it is currently in phase II clinical trials.
Compound 1 (Figure 1) was another potent and selective FGFR
inhibitor developed by AstraZeneca.23 The crystal structure of 1
with FGFR1 revealed that 1 formed a U-shaped configuration to
bind to the ATP binding pocket of FGFR1 (PDB ID: 4NK9)24:
the phenethyl pyrazolamine moiety anchored the hinge region
and interacted with the ATP back pocket of FGFR1 in the same
manner as AZD4547 (PDB ID: 4WUN)25, but the other
substituent groups oriented oppositely. Based on these
observations, we designed and synthesized compound 2 with
quinazoline scaffold by scaffold hopping and molecular
hybridization strategies, hoping to develop a new class of FGFR
inhibitors. Ethyl piperazine derived from BGJ398 was used in
place of dimethyl piperazine owing to its easier accessibility and
the overall synthetic convenience. Bioassay showed that 2 was
highly active at the enzyme level (FGFR2 IC50 < 10 nM), but the
cellular potency was poor (SNU16 cell proliferation inhibition
IC50 > 100 nM), we speculated that 2 was too bulky to permeate
the cell membrane and the relevant subcellular compartments. To
this end, we tried to reduce the size of 2 by removing its
isoxazole moiety to form compound 3 (Figure 1), because phenyl
ring occupied the ATP back pocket of FGFR which was a well-
established potency and selectivity handle,19 and solubilizing
groups were beneficial to optimize the physicochemical
properties of TKIs. To our delight, compound 3 exhibited low
nanomolar potency at both enzymatic and cellular levels against
FGFR2. Thereby structural optimization of 3 was carried out to
gain further insight into the potential of this series of compounds.
AZD4547 inhibits recombinant FGFR kinase activity in vitro
at the low nanomolar level, along with favorable selectivity over
Figure 1. Design of pyrazolylaminoquinazolines.
The synthesis of compounds 3, 12–44, 46 and 50 were shown
in Scheme 1 (refer to supporting information for the preparation
of the starting materials). Condensation of 4 with 2,2-dimethyl-
phosphorus oxychloride. Displacement of the 4-Cl in 45 was
performed in a sealed tube to afford analogue 46. CpRuCl(PPh3)2
catalyzed cyclization of 47 yielded 48, which was then
transformed to chloride 49 in refluxing POCl3, nucleophilic
attack of 49 by 11 under harsh microwave condition generated 50.
1,3-dioxane-4,6-dione and
triethyl
orthoformate gave
intermediate 5, which was subsequently cyclized to produce the
4-hydroxyquinoline 6. Quinazolinones 8 were prepared by
treatment of diverse anthranilic acid esters 7 with formamidine
acetate. 2-Substituted quinazolinones 9 were prepared by reaction
of 7 with different acyl chlorides to generate intermediate amides
which then underwent cyclization with ammonium hydroxide in
a pressure tube. Compounds 6, 8, and 9 were converted to their
corresponding chlorides 10 with phosphorus oxychloride. Finally,
an acid-catalyzed nucleophilic substitution of 10 with another
key intermediate 11 under microwave (MW) irradiation afforded
the final products 3 and 12–44 in good yields. Dichloro
compound 45 was synthesized by a two-step sequence:
cyclization of 7 with urea and subsequent chlorination with
Guagnano and colleagues reported some important points
about SAR of phenyl ring moiety.19 First, the presence of two
methoxy groups at the C3 and C5 positions of the phenyl ring is
necessary to maintain the high affinity and selectivity for
inhibitors of FGFR, so we kept the 3,5-dimethoxy-phenyl moiety
unchanged in all new analogues. Second, in some rigid
structures, introducing chlorine atoms in the ortho positions of
the dimethoxy-phenyl ring would enhance selectivity versus
VEGFR2, therefore we prepared the 2,6-dichloro compound 12
(Table 1) which was proven to be highly potent at both
enzymatic and cellular levels as expected.

Scheme 1. Reagents and conditions: (a) triethyl orthoformate, 2,2-dimethyl-1,3-dioxane-4,6-dione, 90 °C, 35%; (b) diphenyl ether, 230 °C, 60%–68%; (c)
formamidine acetate, 2-methoxyethanol, reflux, 80%–95%; (d) R2COCl, CH2Cl2; (e) ammonium hydroxide, 120 °C, sealed tube, 70%–75% for two steps; (f)
POCl3, DIPEA, toluene, 90 °C, 20%–86%; (g) 11, 1 M HCl, THF, MW, 50 °C, 80%–90%; (h) urea, 200 °C; (i) POCl3, DIPEA, reflux, 58% for 2 steps; (j) 11,
AcONa, THF/H2O, 90 °C, sealed tube, 51%; (k) CpRuCl(PPh3)2, pyridine, 90 °C, 99%; (l) POCl3 , reflux, 65%; (m) 11, K2CO3, DMF, MW, 190 °C, 46%.
form intramolecular hydrogen bonds, which would stabilize the
compound in the planar conformation to form critical hydrogen
bonds with the hinge strand in the ATP binding site; in contrast,
quinoline moiety did not have such N-3 nitrogen to provide such
CH···N H-bonds, which could explain the low efficacy for 13.
While the N-3 nitrogen played an important role in preserving
affinity, the N-1 was not indispensable, compound 50 exhibited
great potency at both the enzymatic and cellular levels. Then our
further SARs investigation focused on substitution on
quinazoline moiety.
Table 1
Biological activity of compounds 3, 12, 13 and 50
IC50 (nM)a
FGFR2
C2-substitution was first explored. Straight-chain aliphatic
groups and chlorine atom were incorporated at the quinazoline
C2 to maintain the molecule size smaller. The resulting
compounds 14–16 and 46 displayed subnanomolar potency on
enzyme, however they were less potent on SNU16 cells (Table 2).
These data revealed that C2-substitution was detrimental to
cellular activity.
SNU16 cells
proliferation
inhibition
Compound
X
Y
R3
kinase
inhibition
3
N
N
N
H
0.8 ± 0.2
1.2 ± 0.0
1.4 ± 0.3
NDb
12
13
N
2,6-di-Cl
H
0.3 ± 0.1
The next modification was investigated on the effect of the
substitution at quinazoline C5 and C8 (Table 3). Compound 18
with chlorine substitution at C5 was five-fold less potent than
unsubstituted 17, and methoxy-substitution displayed an even
worse effect (19 vs 17), which indicated that quinazoline C5 was
not tolerable to any substitution. Substitution on quinazoline C8
was also proven not to be beneficial to the activity (compounds
20–22).
CH
N
29.9% @ 100 nM
50
N
-
CH
H
-
1.2 ± 0.2
1.2 ± 0.2
1.8 ± 0.4
3.6 ± 1.2
AZD4547
aValues are the mean ± SD of two independent assays.
bND, data not determined.
Then a large diversity of substituents, rigid or flexible, cyclic
or linear, basic or neutral, were explored at C7 (Table 4). The
piperazine substituents (3, 23–30) generally led to more potent
cellular proliferation inhibition than did other heterocyclic (31–
33) or flexible substituents (37–39). Bulky benzenesulfonyl
substitution (27) led to a degradation of potency. Eliminating the
proximal piperazine nitrogen atom led to a slight but consistent
reduction in potency in the cellular proliferation assay (34 vs 23);
A quinoline analogue 13 and isoquinoline 50 (Table 1) were
prepared to investigate the effect of the isosteric replacement.
Unfortunately, the replacement of quinazoline with quinoline led
to a dramatic loss of activity. Pierce reported that there may exist
aromatic CH···X (X can be O or N) H-bonds in quinazoline-
containing structures.26 In our pyrazolylaminoquinazoline
scaffolds, the pyrazole CH and quinazoline N-3 nitrogen may

the removal of the distal nitrogen dramatically deteriorated the
cellular potency (35); replacement of the distal methylene of 35
with an oxygen retrieved cellular proliferation inhibition (36).
Compound 40 with no polar groups retained a good level of
enzyme inhibition, but the cellular potency was significantly
reduced, which may be attributed to decreased cell permeability.
These data suggested that substitution at C7 was well tolerated by
the enzyme with compounds showing low nanomolar inhibition
of FGFR2. The good tolerability of substitution at C7 could be
ascribed to the fact that this moiety of the series of compounds
were exposed to the solvent region, and therefore it was ideal to
anchor various side chains bearing solubilizing groups to not
only enhance the in vitro potencies but also optimize the
physicochemical properties of the compounds. Translocating the
C7 side chain to C6 led to significant decrease in potencies
(Table 5) (41 vs 39, 42 vs 3), suggesting that substitution at C6
was not tolerable.
22
H
-
4.3 ± 0.1
1.2 ± 0.2
13.8 ± 0.2
3.6 ± 1.2
AZD4547
-
aValues are the mean ± SD of two independent assays.
Table 4
Biological activity of compounds 3, 23–40
Compound
7
IC50 (nM)a
FGFR2
SNU16 cells
proliferation
inhibition
Table 2
kinase
Biological activity of compounds 14–16 and 46
inhibition
1.0 ± 0.2
0.7 ± 0.1
23
3
0.8 ± 0.2
1.2 ± 0.0
0.6 ± 0.0
< 0.1
24
25
26
27
28
0.6 ± 0.1
< 0.1
IC50 (nM)a
0.7 ± 0.2
49.6% @ 4 nM
21.2% @ 100 nM
63.9% @ 20 nM
FGFR2
kinase
SNU16 cells
proliferation
inhibition
Compound
R2
41.8% @ 10 nM
0.9 ± 0.1
inhibition
14
methyl
ethyl
propyl
chloro
-
0.6 ± 0.2
0.5 ± 0.1
0.7 ± 0.2
0.6 ± 0.2
1.2 ± 0.2
105.7 ± 12.8
24.7 ± 0.2
15
0.2 ± 0.1
0.2 ± 0.1
0.8 ± 0.1
< 0.1
29
30
16
90.0 ± 30.0
108.2 ± 36.9
3.6 ± 1.2
46
AZD4547
0.7 ± 0.2
10.8 ± 0.6
31
aValues are the mean ± SD of two independent assays.
0.3 ± 0.1
0.4 ± 0.2
17.5 ± 3.6
10.0 ± 0.6
32
33
Table 3
Biological activity of compounds 17–22
0.8 ± 0.1
9.0 ± 0.5
34
35
36
37
87.4% @ 1 nM
0.4 ± 0.1
36.8% @ 100 nM
3.9 ± 0.9
IC50 (nM)a
FGFR2
SNU16 cells
1.3 ± 0.3
13.2 ± 1.9
Compound
5
8
kinase
proliferation
inhibition
> 200
inhibition
3.2 ± 0.5
16.9 ± 0.2
100.9 ± 0.2
4.8 ± 0.9
3.9 ± 0.1
2.7 ± 0.2
10.3 ± 1.2
38
17
18
19
20
21
H
H
0.9 ± 0.2
54.1 @ 20 nM
39
40
Cl
OMe
H
H
> 200
H
> 200
16.9% @ 1 nM
22.3% @ 100 nM
Cl
OMe
> 200
73.3% @ 10 nM
1.2 ± 0.2
H
> 200
3.6 ± 1.2
-
AZD4547

aValues are the mean ± SD of two independent assays.
effectors, PLC and ERK, was also strongly inhibited in
accordance with FGFR phosphorylation. This result suggested
that 29 exhibited an effective inhibition of FGFR activation and
its downstream signaling. We then set to investigate the
inhibitory effects of compound 29 on cell proliferation in FGFR-
addicted human cancer cell lines with different mechanisms of
FGFR activation. As shown in Table 8, 29 strongly inhibited the
cell proliferation of these cell lines, with IC50 values of
subnanomolar levels, confirming that 29 was a potent pan-FGFR
inhibitor.
Table 5
Biological activity of compounds 41–44
IC50 (nM)a
Table 7
Inhibitory activity of 29 on FGFR family kinases
SNU16
cells
FGFR2
kinase
IC50 (nM)a
Compound
7
6
Kinase
29
AZD4547
proliferation
inhibition
inhibition
FGFR1
FGFR2
FGFR3
FGFR4
1.0 ± 0.1
0.2 ± 0.1
5.0 ± 1.3
52.7 ± 0.6
0.8 ± 0.3
1.2 ± 0.2
5.6 ± 1.8
45.7 ± 11.2
43.9% @
100 nM
H
H
NDb
ND
ND
41
42
43
29.9% @
100 nM
aValues are the mean ± SD of two independent assays.
Table 8
58.8% @
100 nM
Anti-proliferative activity of 29 on FGFR addicted cell lines
IC50 (nM)a
Cancer cell lines
66.7% @
100 nM
9.7% @
100 nM
44
29
AZD4547
H1581 (FGFR1 amplification)
KG-1 (FGFR1 translocation)
KATOIII (FGFR2 amplification)
RT112 (FGFR3 amplification)
< 0.5
32.9 ± 3.0
1.2 ± 0.2
3.6 ± 1.2
-
-
AZD4547
aValues are the mean ± SD of two independent assays.
bND, data not determined.
< 0.2
< 0.4
< 0.2
< 0.2
20.2 ± 0.5
0.6 ± 0.3
Five compounds with good potencies in both enzyme and
cancer cells were selected for evaluation of their selectivity over
VEGFR2. The result showed that they were at least 50 times
more potent against FGFR2 than VEGFR2 (Table 6). Given
robust enzymatic and cellular potency, as well as favorable
selectivity, the representative compound 29 was selected for
further evaluation.
aValues are the mean ± SD of two independent assays.
Table 6
VEGFR2 selectivity of selected compounds
FGFR2
VEGFR2
(IC50, nM)a
21.5 ± 4.9
> 100
VEGFR2/FGFR2
ratio
Compound
(IC50, nM)a
0.2 ± 0.1
1.3 ± 0.3
0.9 ± 0.2
0.3 ± 0.1
0.5 ± 0.1
1.2 ± 0.2
29
> 100
> 70
> 100
> 50
> 70
>20
37
39
> 100
Figure 2. Compound 29 effectively inhibited the phosphorylation of FGFR1
and the downstream effectors PLCand Erk in KG-1cells, the specific
phosphorylation sites tested of p-FGFR1, p-PLC, and p-ERK were indicated.
12
17.5 ± 5.0
35.8 ± 6.1
29.5±8.6
15
AZD4547
aValues are the mean ± SD of two independent assays.
Compound 29 was evaluated for its activity against other
FGFR isoforms, including FGFR1, FGFR3, and FGFR4.
Compared with its high potency against FGFR2, it showed
almost similar potency against FGFR1 and FGFR3, 50-fold less
potency against FGFR4, with IC50 values of 1.0, 5.0, 52.7 nM,
respectively (Table 7), indicating that it was a potent pan-FGFR
inhibitor. Further, the cellular targeting activity of compound 29
was analyzed in representative FGFR1-fusion protein driven
human myeloid leukemia cancer cell line KG-1. As shown in
Figure 2, 29 inhibited the phosphorylation of FGFR in a dose
dependent manner. The phosphorylation of FGFR downstream
Figure 3. Compound 29 (white) was docked into the ATP binding site of a
published crystallographic structure of FGFR 1 (PDB ID: 4WUN).
Intermolecular hydrogen bonds between 29 and FGFR1 were shown in black
dashed lines.

To clarify its binding mode, we carried out a docking study
with 29 in FGFR1 kinase domain. The proposed binding mode of
29 in the FGFR1 catalytic site were illustrated in Figure 3. The
pyrazolylaminoquinazoline core was in a planar conformation to
form three critical H-bonds to the hinge region: the pyrazolamine
NH and the adjacent pyrazole nitrogen of 29 formed two H-
bonds with the carbonyl and the amino group of alanine 564,
respectively; an additional H-bond occurred between the
backbone carbonyl group of glutamic acid 562 and the pyrazole
N-H. 3,5-Dimethoxylphenyl group penetrated into the back of
ATP pocket which was thought to exert selectivity,19 the phenyl
ring was in vicinity to alanine 640 (the distance between 2’-H to
the side chain of alanine 640 was approximately 4 Å ), the energy
penalty caused by a larger cysteine residue in VEGFR2 may offer
a potential rationale for the selectivity. The 5- and 6-positions of
the quinazoline were in close contact with the hinge region,
accounting for the low tolerance at these positions, while 2- and
8-positions had vectors pointing into more open space, the
piperazine moiety was protruded into the solvent region. The
binding model was consistent with the SAR described above,
which helped to rationalize the FGFR potency and selectivity.
Further structural optimization and biological evaluation of this
class of compounds are under way and will be reported in due
course.
Acknowledgments
We are grateful for financial support from the National
Natural Science Foundation of China (Grant Nos. 81273365,
81573271, 81473243, 81321092); The National Science &
Technology Major Project "Key New Drug Creation and
Manufacturing
2014ZX09304002-008-001,
Program"
of
China
(Grant
Nos.
The
2012ZX09301001-007).
Shanghai Science and Technology Commission (No.
1315431901300), SA-SIBS Scholarship Program is also
gratefully acknowledged.
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Figure 4. Compound 29 (white) was docked into the asymmetric units of the
V561M FGFR1 (PDB ID: 4RWK), superimposition of 29 and AZD4547 in
different binding ways were shown respectively. AZD4547 is shown in
yellow, 29 in white.
In summary, we have identified pyrazolylaminoquinazoline
compounds as a new class of potent and selective FGFR
inhibitors. The representative compound 29 demonstrated
subnanomolar inhibition against FGFR2 in both enzymatic and
cellular assays, as well as exquisite selectivity over VEGFR2.
Compound 29 also strongly inhibited FGFR1–3 kinase activity
and suppressed FGFR signaling transduction in FGFR-addicted
cancer cells. In turn, 29 potently inhibited FGFR-driven cell
proliferation regardless of mechanistic complexity implicated in
FGFR activation, which further confirmed that 29 was a potent
pan-FGFR inhibitor. Docking study disclosed the binding mode
of 29 with the FGFR1 kinase domain, which helped to rationalize
the SAR study and selectivity. The flexibility of our structure
offered the potential to preserve good affinity for V561M FGFR1.

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