Discovery of [5-Amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-(1H-indol-2-yl)methanone (CH5183284/Debio 1347), An Orally Available and Selective Fibroblast Growth Factor Receptor (FGFR) Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.6b01156

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases regulates multiple biological processes, such as cell proliferation, migration, apoptosis, and differentiation. Various genetic alterations that drive activation of the recep

Full text of DOI:10.1021/acs.jmedchem.6b01156

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Article
Discovery of [5-amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-
(1H-indol-2-yl)methanone (CH5183284/Debio 1347), an orally available
and selective #broblast growth factor receptor (FGFR) inhibitor.
Hirosato Ebiike, Naoki Taka, Masayuki Matsushita, Masayuki Ohmori, Kyoko Takami, Ikumi
Hyohdoh, Masami Kohchi, Tadakatsu Hayase, Hiroki Nishii, Kenji Morikami, Yoshito Nakanishi,
Nukinori Akiyama, Hidetoshi Shindoh, Nobuya Ishii, Takehito Isobe, and Hiroharu Matsuoka
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01156 • Publication Date (Web): 07 Nov 2016
Downloaded from http://pubs.acs.org on November 7, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Discovery of [5ꢀaminoꢀ1ꢀ(2ꢀmethylꢀ3Hꢀ
benzimidazolꢀ5ꢀyl)pyrazolꢀ4ꢀyl]ꢀ(1Hꢀindolꢀ2ꢀ
yl)methanone (CH5183284/Debio 1347), an orally
available and selective fibroblast growth factor
receptor (FGFR) inhibitor.
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Hirosato Ebiike, Naoki Taka, Masayuki Matsushita, Masayuki Ohmori, Kyoko Takami,
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Ikumi Hyohdoh, Masami Kohchi, Tadakatsu Hayase, Hiroki Nishii, Kenji Morikami, Yoshito
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Nakanishi, Nukinori Akiyama, Hidetoshi Shindoh, Nobuya Ishii, Takehito Isobe, and
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Hiroharu Matsuoka
AUTHORS’ ADDRESS
1
Research Division, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa 247ꢀ
8530, Japan
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Research Division, Chugai Pharmaceutical Co., Ltd., 1ꢀ135 Komakado, Gotemba, Shizuoka
412ꢀ8513, Japan
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KEYWORDS
Fibroblast growth factor receptor (FGFR), aminopyrazole, SAR
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ABSTRACT
The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases regulates
multiple biological processes, such as cell proliferation, migration, apoptosis, and differentiation.
Various genetic alterations that drive activation of the receptors and the pathway are associated
with tumor growth and survival; therefore, the FGFR family represents an attractive therapeutic
target for treating cancer. Here, we report the discovery and the pharmacological profiles of 8
(CH5183284/Debio 1347), an orally available and selective inhibitor of FGFR1, FGFR2, and
FGFR3. The chemical modifications, which were guided by 3Dꢀmodeling analyses of the
inhibitor and FGFRs, led to identifying an inhibitor that is selective to FGFR1, FGFR2, and
FGFR3. In in vitro studies and xenograft models in mice, 8 shows antitumor activity against
cancer cell lines that harbor genetically altered FGFRs. These results support the potential
therapeutic use of 8 as a new anticancer agent.
INTRODUCTION
Tyrosine phosphorylation is a critical event in the signal transduction that is mediated by
growth factor, and the protein tyrosine kinases are key components of the phosphorylation
process. Several molecular targeted kinase inhibitors have been launched, such as epidermal
1
growth factor receptor (EGFR) inhibitors (erlotinib and gefitinib), erbꢀb2 receptor tyrosine
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kinase 2 (HER2) inhibitor (lapatinib), breakpoint cluster region abelson tyrosine kinase (BCRꢀ
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ABL) inhibitors (imatinib and dasatinib), BꢀRAF inhibitors (vemurafenib and dabrafenib), and
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anaplastic lymphoma kinase (ALK) inhibitors (crizotinib and alectinib). While these kinase
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inhibitors that can treat patients according to their genetic status have contributed remarkable
progress to cancer therapy, these reagents cannot cover all tumors, and the need to treat tumors
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harboring other genetic alterations still remains. Thus, the potential therapeutic strategy using a
molecular targeted agent to satisfy the unmet medical needs of a variety of cancers harboring
3
novel genetic alterations has attracted attention over the years.
In cancer, genetic alterations that constitutively activate FGFR, such as amplification and
mutation, drive activation of the receptors and the pathway in several tumor types, and are
known to be associated with cell growth, angiogenesis, cell migration, invasion, and metastasis
4,5
(
Table 1). Several FGFR inhibitors targeting multiple kinases have been reported and are
currently under clinical trial. While these drugs show some clinical benefits in patients that show
6
a
positive for FGFR genetic alterations, some of the drugs, such as dovitinib (TKI258 , Fig. 1)
6b
and cediranib (AZD2171 , Fig. 1), also target kinase insert domain receptor (KDR), which
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causes grade 3/4 hypertension, and dose limiting toxicity has been observed in a clinical trial.
Since compounds that are selective to one target kinase can potentially claim a more favorable
toxicity profile than multiꢀtargeted compounds, selective and potent FGFR inhibitors are
8
required. Currently, several FGFRꢀselective inhibitors, such as 21 (AZD4547) and 22 (NVPꢀ
9
BGJ398) (Fig. 1), are under clinical trials targeting patients who have FGFR genetic alterations.
1
0
These inhibitors, as well as the FGFRꢀselective inhibitor 20 (PD173074), share a common 3,5ꢀ
dimethoxyꢀphenyl ring that interacts with the backpocket region of the FGFR, according to Xꢀ
8, 9, 11
ray structures of the binary complex of FGFR with each inhibitor.
Since it is possible that a
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common single mutation, such as the gatekeeper mutation, is causing resistance to these
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inhibitors, a potential strategy to overcome this issue is the rational design of a novel inhibitor
with a different chemical scaffold.
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This report describes the optimization program that led to identifying the novel inhibitor (8,
(CH5183284/Debio 1347)), which potently and selectively inhibits the FGFR family of receptor
1
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tyrosine kinases and which is currently in Phase I clinical trial.
4
,5
Table 1. Genetic alteration and amplification of FGFR’s and related cancers.
Squamous NSCLC (20%), Breast (10%), Ovarian
FGFR1
FGFR2
Amplification
(~5%), Bladder (3%)
Amplification Gastric (10%), breast (4% of tripleꢀnegative cases)
Mutation
Mutation
Endometrial (12%), Squamous NSLC (5%)
Bladder (50ꢀ60% nonꢀmuscle invasive type, 10ꢀ15%
invasive type)
FGFR3
Translocation Myeloma (15ꢀ20%)
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Figure 1. FGFR inhibitors.
CHEMISTRY
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The synthesis of aminopyrazole derivatives 1ꢀ3 is outlined in Scheme 1. Cyanomethylation of
commercially available esters (Intermediate b) with acetonitrile anion, which had been generated
by treating acetonitrile (MeCN) with lithium hexamethyldisilazide (LHMDS) in THF at ꢀ78 °C,
provided cyanomethyl compounds (Intermediate c). The intermediate c that were available
commercially or obtained above were treated with dimethylformamide dimethyl acetal (DMFꢀ
DMA) in THF and yielded enamines (Intermediate d). The aminopyrazoles were formed by
treating the intermediate d with aryl hydrazine hydrochlorides at 100 °C. The synthesis of indole
derivatives is outlined in Scheme 2. Because a protective group at 1ꢀposition of indole gave
more selective cyclization to aminopyrazole, a benzenesulfonyl (Bs) group or a tosyl (Ts) group
was introduced in the synthesis of indole derivatives (Scheme 2). The functional groups at 5ꢀ
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position or 6ꢀposition of indoles were introduced in advance to form the aminopyrazole ring,
except for the sulfone compound (16, Scheme 3). The methanesulfonyl group in 16 was
introduced to the aminopyrazole derivative (16c) by Cuꢀcatalyzed reaction with methanesulfinic
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acid sodium salt in DMSO. Aryl hydrazines were obtained by reducing diazonium salts with
SnCl (procedure C, Scheme 4) or introducing the hydrazine moiety to heteroaryl bromide
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derivatives by Pdꢀcatalyzed coupling reaction of diꢀtertꢀbutyl hydrazodiformate (BocNHNHBoc)
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as a hydrazine source (procedure D, Scheme 4). The aryl hydrazines, especially heteroaryl
hydrazines, were unstable in air. The aryl hydrazines were each isolated as a salt of hydrochloric
acid, and subjected to the aminopyrazole formation steps using inꢀsitu neutralization protocols
with a neutralizing reagent.
a
Scheme 1. Syntheses of aminopyrazole derivatives.
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Scheme 2. Syntheses of indole aminopyrazole derivatives (4-15, 17-19).
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Scheme 3. Synthesis of 16.
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Reagents and conditions: (a) LHMDS, MeCN, THF ꢀ78 °C; (b) DMFꢀDMA, THF; (c)
ArNHNH HCl, 100 °C (d) sodium methanesulfinate, CuI, N1,N2ꢀdimethylcyclohexaneꢀ1,2ꢀ
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Scheme 4. Syntheses of aryl hydrazines (7a, 9c, 10c, 11c and 12a).
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Reagents and conditions: (a) H , PdꢀC, H OꢀEtOH; (b) NaNO , dil. HCl then SnCl , 0 C;
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(c) Pd dba , tꢀBu XPhos, BocNHNHBoc, Cs CO , toluene, 100 C; (d) 4 M HClꢀAcOEt.
2 3 2 3
MOLECULAR DESIGN
In order to obtain an FGFRꢀselective inhibitor, we performed a highꢀthroughput screening
against our chemical library and identified a screening hit (1) (Table 3) that inhibited a relatively
broad range of kinases but had a different chemical scaffold from the other known FGFR
inhibitors (Fig. 1). The chemical modifications strategy guided by 3D modeling of inhibitors and
FGFR1 is outlined in Figure 2. A 3D model structure of 1 and FGFR1 suggested that compound
1
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1
was located in the ATPꢀbinding site of FGFR1 (Fig. 2). Analysis of the 3D modeling
structure also suggested an interaction of the amino group and the carbonyl group of 1 with the
backbone of the hinge region (E562 and A564) of FGFR1. Additionally, the phenolic hydroxyl
group of 1 was supposed to interact with D641 through a hydrogen bond in the backpocket
region of FGFR1. In addition to these interactions, we assumed that a further functional group
could be introduced to 1 to gain additional interaction with FGFRs. In 1, an mꢀtolyl group was
located near the tyrosine residue (Y563) in the hinge region, and replacing the mꢀtolyl moiety
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was expected to strengthen the πꢀπ interaction between Y563 and the inhibitors. Addition of a
hydrogen donor to 1 in the direction of the carbonyl group of A564 was predicted to enhance the
interaction at the hinge region of FGFRs. Replacement of the phenol moiety by an isostere of
phenol was considered to be essential to avoid metabolic instability. We envisioned that we
could improve the specificity to FGFRs by implementing either a favorable interaction between
the inhibitor and amino acid residues common in FGFRs or an unfavorable interaction between
the inhibitor and amino acid residues in offꢀtarget kinases. Interaction of inhibitors with both the
methionine residue (M535 in FGFR1) and the valine residue (V561 in FGFR1) could improve
both kinase inhibitory activity and specificity to FGFRs (Table 2). Introducing a solubilizing
group at the mꢀtolyl moiety was considered to be beneficial if the solubility of the inhibitors was
insufficient.
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To confirm the selective antitumor efficacy of the inhibitors, we tested them in a kinase
inhibitory assay and an in vitro antiproliferative activity assay. Because compound 1 inhibited a
relatively broad range of kinases such as KDR and protoꢀoncogene (SRC) which were
considered to potentially be offꢀtarget kinases, the inhibitory activity against FGFRs, KDR, and
1
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SRC were measured in cellꢀfree system to evaluate selectivity of kinase inhibition. The
sequence identity between FGFR1 and FGFR2 at the kinase domain was 87.6%, and the pivotal
interactions between 1 and the amino acid residues of FGFR1 were expected to agree with those
of FGFR2. Because the inhibitors had the same binding mode with FGFR1 and FGFR2, the
inhibitory activity against FGFR1 correlated well with that against FGFR2. We concluded that
the antiproliferative activity of the inhibitors in SNUꢀ16 (gastric cancer, FGFR2 amplification)
was appropriate for the in vitro assay, even though the 3D modeling of the inhibitors was
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performed in FGFR1. The antiproliferative activity against HCT116 (colorectal cancer, WT
FGFR) was evaluated as a negative control cell lines.
As an entry to the lead optimization process, we prepared a series of compounds bearing an
aminopyrazole scaffold to explore their structureꢀactivity relationships (SARs) by replacing the
mꢀtolyl group in the solventꢀexposed region of 1.
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Modification strategy
(1) Addition of aromatic ring (πꢀπ interaction)
(2) Addition of hydrogen donor (interaction of
hinge region)
(3) Replacement to isostere of phenol
(4) Addition of suitable group to achieve
kinase selectivity
Figure 2. Structure analysis of 3D modeling of 1 (magenta) and FGFR1. The side chains of
amino acid residues important for ligand recognition of FGFR1 are highlighted as sticks (green).
Hydrogen bonds at hinge region are indicated in blue dotted lines.
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Table 2. Amino acid sequences of FGFR1, KDR, and SRC.
Backpocket region
Gatekeeper residue Hinge region
DFG motif
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31 532 533 534 535 559 560 561 562 563 564 641
642
F
643
G
FGFR1
KDR
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D
85 886 887 888 889 914 915 916 917 918 919 1046 1047 1048
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13 314 315 316 317 339 340 341 342 343 344 407
SRC
E
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V
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M
RESULTS AND DISCUSSION
Hit-to-lead
The mꢀtolyl group was replaced into bicyclic aromatics in order to direct the aromatic ring of
inhibitors in the solventꢀexposed region near Y563 in FGFR1. As we expected, the bicyclic
compounds 2 and 3 showed better kinase inhibitory activity than the monoꢀcyclic compound (1)
because the πꢀπ interaction between Y563 of FGFR1 and inhibitors was enhanced (Table 3).
Interestingly, compound 4 showed the most potent kinase inhibitory activity out of the bicyclic
compounds. The 3D modeling analysis of 4 with FGFR1 suggested that the phenyl moiety of the
indole was forced into parallel geometry with Y563, and the hydrogen of the NH in the indole
additionally interacted with the backbone of A564. We concluded that the indole was the optimal
bicyclic heteroaromatic and explored modification of the backpocket region and the solventꢀ
exposed region in next step, using 4 as the lead compound.
Compared with the lead compound (4), a drop in inhibitory activity of one order of magnitude
was observed in 5 by removing the phenolic hydroxyl group in 4. The hydroxyl group in 4
probably interacted with the carboxylic acid at D641 in FGFR1 by a hydrogen bond. This
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interaction was supposed to be essential to keep the inhibitory activity against FGFRs, because
the aspartic acids corresponding to D641 in FGFR1 were well conserved in FGFRs. On the other
hand, the aspartic acids corresponding to D641 in FGFR1 were also conserved in several kinases
including KDR and SRC (Table 2). As described earlier, the inhibition of KDR is known to
induce hypertension. We were also concerned about inhibition of SRC which is reported to lead
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
8
to osteopetrosis in animal study. In addition to the interaction between inhibitors and kinases, a
phenolic hydroxyl group was generally known to form a conjugate metabolite. To avoid a both
broad kinase inhibition spectrum and drug metabolism, it was necessary to replace the phenol to
its isostere. We analyzed the 3D structure of the backpocket region and the hinge region, and
hypothesized that compounds in FGFR1 interacting with M535, V559, and V561, which are the
commonly conserved amino acid residues in FGFRs, could improve the specificity to FGFRs
over SRC and KDR (Table 2 and Fig. 3). The discrimination between FGFR1 and SRC would
be considered to be due to the different characteristic of V561 (in FGFR1) versus T341 (in SRC)
and of V559 (in FGFR1) versus I339 (in SRC). Though the only amino acid residues that were
different at the ATPꢀbinding site in FGFR1 and KDR were M535 (FGFR1) and L889 (KDR), a
steric repulsion between the inhibitors and L889 (KDR) could be expected at this site. When a
solubilizing group was introduced in the solventꢀexposed indole moiety of 4, the kinase
inhibitory activity of 6 was improved (Table 3). On the other hand, the selectivity to FGFRs over
SRC and KDR was insufficient due to the high sequence similarity of FGFRs, SCR, and KDR at
the solventꢀexposed regions. Thus, we tried to modify the backpocket region to improve the
specificity to FGFRs.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. SAR of Hitꢀtoꢀlead.
Enzyme IC50 (nM)
In vitro IC50 (nM)
Cmpd
Structure
FGFR1 FGFR2 FGFR3 FGFR4 KDR SRC
SNUꢀ16 HCT116
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1900
270
940
140
7400
940
4100
730
2900
170
250
4.3
15000
3300
>20000
8200
(Hit)
2
3
450
230
720
740
1600
31
8900
9300
4
130
30
25
200
28
0.18
11
360
2600
2700
(Lead)
5
1100
920
4800
3100
410
2900
6
1.2
4.2
4.0
160
2.4
0.43
550
3600
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Figure 3. (A) (B) Neighboring amino acid residues of FGFR1 (green), KDR (magenta), and
SRC (cyan) around 4 (orange). The binding models were created by overlapping Xꢀray structures
of FGFR1 (2FGI), KDR (3WZD), and SRC (3G5D). (C) (D) Superimposed structures of 4
(purple) and 8 (green) in FGFR1. The hydrogen bonds between 8 and FGFR1 (E531 and D641)
are indicated in blue dotted lines. The benzimidazole in 8 interacts better with V561, I545, and
F641 by CHꢀπ interaction (orange dotted lines) than phenoxy group in 4.
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1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Modification of the backpocket region
By analyzing a 3D structure of the backpocket region of FGFR1, we designed several bicyclic
heteroaromatics with a hydrogen donor that could form a hydrogen bond to D641 to fit the cavity
formed by E531, M535, V561, A640, and D641 in the binding pocket (Fig. 2 and Table 4). A
screening of the heteroaromatics revealed that the benzimidazole derivatives (7, 8, and 9)
showed greater specificity to FGFRs than the low specificity to FGFRs of indole derivatives (10
and 11, Table 4). According to 3D modeling of 10 and 11 with FGFR1, the indole derivatives
(10 and 11) still showed inhibitory activity against FGFRs probably due to the hydrogen bond
between the NH group of indole and E531 in FGFRs. Interestingly, compound 11 showed better
inhibitory activity against FGFRs than 10 probably because of CHꢀπ interaction between the 2ꢀ
methyl group of the indole in 11 and F642 in FGFR1, but the specificity of these indole
derivatives to FGFRs needed to be improved to avoid the potential risk of drug related toxicity
from having a broad spectrum of kinase inhibition.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We explored 2ꢀposition of the benzimidazole of 7, and it was revealed that 2ꢀmethylꢀ
benzimidazole derivative (8) was optimal in terms of kinase selectivity. In addition to kinase
selectivity, the profiles of 8 were well balanced in terms of in vitro cellular antiproliferative
activity against SNUꢀ16 and stability in human liver microsome. While the 2ꢀethyl
benzimidazole derivative (9) showed equivalent kinase inhibition and cellular growth inhibition
profiles as 8, its stability in human liver microsome did not meet our criteria (Table 5). The low
kinase inhibitory activity of the benzimidazolinone derivative (12) against all the tested kinases
suggested the importance of both the hydrogen acceptor and the hydrogen donor of the
benzimidazole group. We speculated that the nitrogen of the benzimidazole group of 8 formed a
hydrogen bond with the backbone of A640, and the NH group of the benzimidazole formed
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another hydrogen bond with the side chain of E531. In addition to these hydrogen bonds, the
benzimidazole ring was located in the hydrophobic pocket formed by M535, V561, and F642,
according to an Xꢀray structure of the binary complex of 8 and FGFR1 (Fig. 5A). Furthermore,
an Sꢀπ interaction between the sulfur atom of M535 and the benzimidazole ring was suggested.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
9
The selectivity of 8 to inhibit FGFR over KDR was suggested to be caused by the difference in
the interaction with M535 in FGFR1 and L889 in KDR, because the leucine residue in KDR
would disrupt the formation of the Sꢀπ interaction with the benzimidazole moiety of 8 (Fig. 5B).
The selectivity to FGFR over SRC was also suggested to be the result of a disruption of a crucial
interaction between 8 and the lipophilic binding pocket formed by V561 and V559 in FGFR1,
which were T314 and I339 in SRC (Fig. 5B). On the other hand, compound 4 showed a broad
kinase inhibitory activity against the tested kinases because the chloroꢀphenyl moiety of 4 had
effective interactions with amino acid residues other than those in FGFR1. The 3D modeling of 4
and 8 (Fig. 3) suggested that the chloroꢀphenyl ring of 4 and the benzimidazole ring of 8 had
different geometry, and the chloroꢀphenyl moiety of 4 had no repulsive interaction against either
FGFR1, KDR, or SRC (Fig. 3A and Fig. 3B). Furthermore, the benzimidazole derivatives were
much soluble in fasted state simulated intestinal fluid (FaSSIF) than indole derivatives (Table 5).
As described above, we concluded that the 2ꢀmethylꢀbenzimidazole group was optimal in the
backpocket region of an inhibitor and planned to modify the solventꢀexposed region of
compounds that had 2ꢀmethylꢀbenzimidazole group in 1ꢀposition of the aminopyrazole.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. SAR in modification of backpocket region.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Enzyme IC (nM)
In vitro IC (nM)
50
50
2
Cmpd
Ar
FGFR1
33
FGFR2
150
FGFR3
FGFR4
KDR
340
SRC
2000
SNUꢀ16 HCT116
7
120
220
380
17
17000
5900
1500
8
(
CH5183284
9.3
2.9
7.6
65
22
79
290
250
2100
1700
5900
/
Debio 1347)
9
16000
77
1
1
1
0
1
2
160
69
450
32
270
18
110
85
52
6.5
230
330
190
53
470
5300
33000
>50000
870
>50000
>50000
2700
33000
N.T.
N.T.: Not tested.
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Table 5. Solubility in FaSSIF and stability (CL ) in human liver microsome.
int
Cmpd
Solubility (ꢀg/mL)
CL (ꢀL/min/mg)
int
4
6
7
8
9
20
40
37
29
42
4.0
10
15
20
4.2
13
23
4.4
10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
0
11
Modification of the solvent-exposed region to reduce cardiovascular effects
+
In spite of 8’s high specificity to FGFRs, an inhibition of cardiac K channel encoded by the
human etherꢀaꢀgoꢀgoꢀrelated gene (hERG) (IC 6.9 ꢀM, 69% inhibition at 10 ꢀM) was
5
0
2
0
observed in an early safety study. In addition to hERG inhibition, the inhibitory activity of 8 on
KDR (IC 2.1 ꢀM) was also suggested to cause hypertension. Cardiovascular side effects,
5
0
especially QTc prolongation, are associated with inhibition of the hERG channel, which has
resulted in several drugs being withdrawn from the market, and which is still one of the potential
21
risks in the R&D process of new molecular entities. As we mentioned above, hypertension
(grade 3 and 4) caused by inhibiting KDR was reported to be the doseꢀlimiting toxicity of KDR
inhibitors in clinical trials. We hypothesized that modifying the solventꢀexposed region of 8
might improve the hERG inhibition without losing the inhibitory activity against FGFRs. Both
lipophilic and hydrophilic functionalities were introduced to 8 to reduce hERG inhibition by
2
2
controlling log P.
First, amino groups with low pKa, such as morpholine or 4ꢀdifluoropiperidine, were introduced
at the 5ꢀ or 6ꢀposition of indole in 8. The inhibitory activities against FGFRs and SNUꢀ16 were
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
maintained by introducing amino groups (Table 6). Because introducing an electronegative
oxygen at βꢀposition or an electronꢀwithdrawing fluorine atom at γꢀposition of basic nitrogen
reduced pKa value of amino groups, the reduction of the hERG binding affinity by lowering the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
basicity of nitrogen in amino groups was expected. The pKa value of nitrogen of morpholine in
13 and 14 was reduced from 9.23 (piperidine) to 7.11 (13) and 7.24 (14) by replacing piperidine
to morpholine, and the pKa value of nitrogen in 4ꢀdifluoropiperidine derivative (15) was also
reduced to 6.39 (Fig. 4). The strategy to reduce inhibition of hERG by polar groups worked well
in 13 and 14, but not in 15 (Table 7). The solubility of these amino derivatives (13, 14, and 15)
was improved by introducing hydrophilic groups, but the permeability in a Caco2 study was
reduced, compared to 8 (Table 7). The low permeability of these compounds resulted in low oral
bioavailability (BA) in a monkey pharmacokinetic (PK) study (Table 7). Next, lipophilic groups
were introduced in 6ꢀposition of indole, and the in vitro profiles were summarized in Table 6.
The 6ꢀbromoꢀindole derivative (17) and the 6ꢀmethylꢀindole derivative (18) showed better
permeability in the Caco2 study among lipophilic derivatives, and compound 17 exhibited 20%
BA in the monkey PK study (Table 7). Unfortunately, a weaker inhibition of hERG was
observed in 17 than 8, but another cardiovascular sideꢀeffect was suggested by a safety study
(data not shown). The 6ꢀphenylꢀindole derivative (19) showed the same in vitro properties as 8
and a weak hERG inhibition, but the BA of 19 was low (3% in monkey). We concluded that oral
BA could be predicted by the permeability in Caco2, and that hERG inhibition and oral BA
could not be improved at the same time by modifying the solventꢀexposed region.
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9
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18
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45
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Figure 4. pKa values of amines in the solvent exposed region.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 6. SAR in optimization of solvent exposed region.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Enzyme IC50 (nM)
In vitro IC50 (nM)
1
2
Cmpd
R
R
FGFR1 FGFR2 FGFR3
FGFR4
KDR
2100
SRC
5900
SNUꢀ16 HCT116
8
H
H
H
9.3
2.9
1.5
3.0
56
7.6
5.2
3.6
9.4
21
22
4.4
3.5
130
68
290
17
17
2.6
6.0
82
52
18
84
5900
8900
4100
3200
37000
11000
9700
3800
1
1
3
4
26
210
120
280
1000
290
370
510
300
H
H
H
H
H
H
13
15
130
240
490
340
720
390
1
6
MeSO
2
7000
>50000
3400
17
Br
49
19
150
180
230
1
1
8
9
Me
18
10
Ph
50
41
3400 >50000
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Table 7. Physicochemical properties, suppression of hERG, and BA in monkey.
CLint
Caco2
(10 cm/sec)
hERG IC50
hERG %inhibition
Monkey BA
(%)
Cmpd
(ꢀL/min/mg)
ꢀ6
(ꢀM)
@10 ꢀM
Human
8
13
15
28
0.35
1.2
6.9
>10
>10
8.6
69
7.0
15
58
4.0
31
64
15
30
N.T.
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
1
1
3
4
5
9.6
14
19
5.0
N.T.
20
16
5.4
4.2
140
3.2
0.075
2.2 *
32
>10
>10
6.9
1
1
1
7
8
9
N.T.
3.0
0.070
>10
*
Caco2 assay at 100 ꢀM except for 17 (10 ꢀM).
N.T.: Not tested.
Assessment of cardiovascular effect of 8
We tried to evaluate the profiles of 8 in detail, because there was some concern around possible
cardiovascular risk due to inhibitory activity against KDR and hERG inhibition as described
earlier. We examined a safety assessment study to evaluate a potential risk of cardiovascular
effect of 8. Compound 8 did not lead to either QTc prolongation in Langendorff’s isolated heart
perfusion model in guinea pig or to significant changes in blood pressure compared with vehicleꢀ
dosed rats and monkeys (data not shown). The effect of 8 on blood pressure in telemetryꢀ
instrumented rats at a dose of 30 mg/kg did not lead to significant changes in blood pressure
2
4
compared with vehicleꢀdosed animals. Of note, the plasma exposure of 8 in rats (AUC: 102
ꢀ1
µg·h·mL ) at a dose of 30 mg/kg was 4ꢀfold higher than the exposure inducing a greater than
ꢀ1
100% tumor growth inhibition in the SNUꢀ16 mouse xenograft model (AUC: 26 µg·h·mL at a
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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dose of 30 mg/kg). Therefore, compound 8 was predicted to have no biologically significant
effect on blood pressure at multiples of the efficacious dose.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Efficacy in tumor xenograft studies
The in vivo efficacy was evaluated in mice bearing an SNUꢀ16 xenograft. Compound 8 was
orally administered onceꢀdaily for 11 days, and the body weight of mice and the volume of the
tumors were measured twice a week. A doseꢀdependent tumor regression (tumor growth
inhibition (TGI) = 106% at 30 mg/kg and TGI = 147% at 100 mg/kg) was observed without
2
4
apparent body weight loss. Compound 8 also showed significant in vivo efficacy in xenograft
mice models with FGFR genetic alterations, such as KG1 (leukemia, FGFR1OPꢀFGFR1 fusion),
MFEꢀ280 (endometrial cancer, FGFR2 S252W mutation), UMꢀUCꢀ14 (bladder cancer, FGFR3
2
4
S249C mutation) and RT112/84 (bladder cancer, FGFR3ꢀTACC3 fusion). These results
suggested 8’s therapeutic potential of cancers harboring FGFR genetic alterations.
CONCLUSION
We identified 8 as a potent and selective FGFR inhibitor with a novel chemical scaffold by
exploiting crystallographic structure information on FGFRs with their inhibitors. Compound 8
only bound to five kinases, including FGFR1, FGFR2 and FGFR3, out of 442 WT and mutant
24
kinases at 100 nM in KINOMEscan panel (DiscoveRx). The benzimidazole moiety of 8 was
located at the backpocket region of FGFR1 according to the Xꢀray crystal structure of binary
complex of FGFR1 and 8 (Fig. 5A). The recognition of the different amino acid residues in
FGFRs, KDR, and SRC was achieved by introducing 2ꢀmethylꢀbenzimidazole in the backpocket
region of 8 (Fig. 5B). The benzimidazole moiety was positioned in the same region as the 3,5ꢀ
dimethoxyꢀphenyl groups in 21 and 22 (Fig. 5C). It may be possible to overcome acquired
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resistance, such as a gatekeeper mutation, because the binding mode of 8 is different from that of
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1 and 22 at both the backpocket region and the hinge region of FGFR1. A preclinical PK
evaluation in mice, rats, and monkeys revealed that the compound was orally bioavailable in
both rodents and nonꢀrodents. When the anticancer efficacy was evaluated, compound 8 potently
inhibited the proliferation of gastric cancer cell lines with amplified FGFR2 and exhibited dose
dependent antitumor efficacy in SNUꢀ16 xenograft models. Compound 8 will provide
therapeutic opportunities for patients who have FGFR genetic alterations and patients with
acquired resistance to existing FGFRꢀselective inhibitors.
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Figure 5. 3D structures of 8, 21, and 22. (A) The Xꢀray crystal structure (PDB code: 5B7V) of 8
(purple) and FGFR1. The side chains of the amino acid residues important for ligand recognition
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of FGFR1 are highlighted as sticks (green). Hydrogen bonds were indicated in blue dotted lines.
The CHꢀπ and Sꢀπ interactions were indicated in orange dotted lines. (B) The overlapped
structure of 8 (green), FGFR1 (green), KDR (magenta), and SRC (light blue). The side chains of
the amino acid residues around 8 in the backpocket regions were reported as sticks. (C) The
overlapped structure of 8 (magenta), 21 (light blue), and 22 (green) in FGFR1. The side chains of
the amino acid residues around 8, 21 and 22 in the backpocket regions were reported as sticks.
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EXPERIMENTAL SECTION
General Synthetic and Analytical Chemistry Methods
1
All reagents and solvents were purchased from commercial sources and used as received. H and
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C NMR spectra were recorded on a JEOL JNMꢀECA400 (400 MHz), a Varian MR400 (400
MHz), a Bruker AVANCE III HD600 (600 MHz) or a Bruker AVANCE III 600 CryoꢀTCI (600
MHz). The chemical shifts are expressed as δ units using tetramethylsilane as an internal
standard. The following abbreviations are used: singlet (s), doublet (d), double doublets (dd),
triplet (t), quartet (q), multiplet (m), broad peak (br) and broad singlet (brs). LCꢀMS analyses
were performed on a Waters Micromass SQD instrument coupled to a Waters Acuity UPLC and
PDA instruments. Analytical reverseꢀphase ultra high performance liquid chromatography
(UPLC) experiment was carried out on an Ascentis Express C column (2.1 x 50 mm, 2.7 ꢁm)
18
or Acquity UPLC BEH Phenyl column (2.1 x 50 mm, 1.7 ꢁm). Eluents in UPLC used were
solvent A (H O with 0.1% formic acid) and solvent B (MeCN with 0.1% formic acid), or solvent
2
C (H O with 0.03% TFA) and solvent D (MeCN with 0.03% TFA) and eluted by a linear
2
gradient at 1.0 mL/min. High resolution mass spectra (HRMS) were measured with a Xevo G2ꢀ
SL (Waters) MS spectrometer using an ESI source coupled to a Waters HPLC system operating
in reversed phase with an ACQUITY UPLC BEH C column (2.1 x 50 mm, 1.7 ꢁm). Normal
18
phase and reverseꢀphase flash column chromatography were performed with Biotage SNAP
cartridges equipped with SP1 or Isolera One (Biotage). The purities of all tested compounds are
>95%. The purity was determined by LCꢀMS analysis.
General procedure A for synthesis of the aminopyrazoles compound 1-3 (Scheme 1).
[5-Amino-1-(2-chloro-5-hydroxy-phenyl)pyrazol-4-yl]-(m-tolyl)methanone (1).
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(
E)ꢀ3ꢀ(Dimethylamino)ꢀ2ꢀ(3ꢀmethylbenzoyl)propꢀ2ꢀenenitrile (1a, 107 mg, 0.500 mmol) and the
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ꢀchloroꢀ3ꢀhydrazinoꢀphenol hydrochloride (146 mg, 0.750 mmol) were placed in a screwꢀ
capped vial, and then EtOH (1.0 mL) was added to the vial. Nitrogen gas was bubbled into the
reaction mixture, and then the mixture was stirred at 78 °C for 10 hours under nitrogen
atmosphere. The solution was diluted with DMSO (2 mL), and then the solution was subjected to
a C medium pressure liquid chromatography (MPLC) column. The column was eluted with
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.1% formic acidꢀH OꢀMeCN by a linear gradient. The fractions containing the target were
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collected, and then concentrated under reduced pressure to give the titled compound as a light
yellow powder (26 mg, 15%).
1
HꢀNMR (400 MHz, DMSOꢀD ) δ: 10.13 (1H, s), 7.76 (1H, s), 7.56ꢀ7.54 (2H, m), 7.46 (1H, d, J
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= 8.8 Hz), 7.41ꢀ7.39 (2H, m), 7.04 (2H, s), 6.96 (1H, dd, J = 8.8, 2.7 Hz), 6.87 (1H, d, J = 2.7
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Hz), 2.40 (3H, s); CꢀNMR (100 MHz, DMSOꢀD ) δ: 187.74, 157.14, 152.15, 141.40, 139.71,
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37.89, 134.79, 131.84, 130.90, 128.42, 128.31, 125.07, 120.75, 118.36, 116.64, 102.50, 20.97.
+
+
HRMS (ESI): m/z calculated for C H ClN O + H [M + H] : 328.0853, found: 328.0852.
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[5-Amino-1-(2-chloro-5-hydroxy-phenyl)pyrazol-4-yl]-(benzofuran-2-yl)methanone (2).
Prepared by following the general procedure A.
1
HꢀNMR (400 MHz, DMSOꢀD ) δ: 10.20 (1H, s), 8.47 (1H, s), 7.83 (1H, dd, J = 2.7, 1.1 Hz),
6
7.81 (1H, dd, J = 3.3, 1.1 Hz), 7.77 (1H, d, J = 1.1 Hz), 7.55ꢀ7.51 (1H, m), 7.49 (1H, d, J = 8.8
Hz), 7.38 (1H, td, J = 7.7, 0.9 Hz), 7.21 (2H, s), 6.99 (1H, dd, J = 8.8, 2.7 Hz), 6.92 (1H, d, J =
13
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.7 Hz); CꢀNMR (100 MHz, DMSOꢀD ) δ: 174.34, 157.12, 154.75, 153.52, 152.64, 140.87,
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34.63, 130.92, 127.43, 126.94, 123.86, 123.03, 120.84, 118.43, 116.69, 112.34, 111.38, 101.95.
+
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HRMS (ESI): m/z calculated for C H ClN O + H [M + H] : 354.0645, found: 354.0645.
1
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