Discovery and optimization of selective FGFR4 inhibitors via scaffold hopping

Article abstract of DOI:10.1016/j.bmcl.2017.04.014

Introduction of a Michael acceptor on a flexible scaffold derived from pan-FGFR inhibitors has successfully yielded a novel series of highly potent FGFR4 inhibitors with selectivity over FGFR1. Due to reduced lipophilicity and aromatic ring count, this series demonstrated improved solubility and permeability. However, plasma instability and fast metabolism limited its potential for in vivo studies. Efforts have been made to address these problems, which led to the discovery of compound (?)-11 with improved stability, CYP inhibition, and good activity/selectivity for further optimization.

Full text of DOI:10.1016/j.bmcl.2017.04.014

Accepted Manuscript
Discovery and Optimization of Selective FGFR4 Inhibitors via Scaffold Hop-
ping
Yikai Wang, Zhengxia Chen, Meibi Dai, Peipei Sun, Chunqiu Wang, Yang Gao,
Haixia Zhao, Wenqin Zeng, Liang Shen, Weifeng Mao, Tian Wang, Guoping
Hu, Jian Li, Shuhui Chen, Chaofeng Long, Xiaoxin Chen, Junhua Liu, Yang
Zhang
PII:
S0960-894X(17)30376-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.014
BMCL 24867
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 January 2017
16 March 2017
4 April 2017
Please cite this article as: Wang, Y., Chen, Z., Dai, M., Sun, P., Wang, C., Gao, Y., Zhao, H., Zeng, W., Shen, L.,
Mao, W., Wang, T., Hu, G., Li, J., Chen, S., Long, C., Chen, X., Liu, J., Zhang, Y., Discovery and Optimization of
Selective FGFR4 Inhibitors via Scaffold Hopping, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://
dx.doi.org/10.1016/j.bmcl.2017.04.014
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Bioorganic & Medicinal Chemistry Letters
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Discovery and Optimization of Selective FGFR4 Inhibitors via Scaffold Hopping
Yikai Wanga∗, Zhengxia Chena, Meibi Daia, Peipei Suna, Chunqiu Wanga, Yang Gaoa, Haixia Zhaoa,
Wenqin Zenga, Liang Shena, Weifeng Maoa, Tian Wanga, Guoping Hua, Jian Lia, Shuhui Chena, Chaofeng
Longb, Xiaoxin Chenb, Junhua Liub, and Yang Zhanga∗
a WuXi AppTec (Shanghai) Co., Ltd., 288 FuTe Zhong Road, Shanghai 200131, P. R. China
bGuangdong Zhongsheng Pharmaceutical Co., Ltd., Dongguan 523325, P.R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Introduction of a Michael acceptor on a flexible scaffold derived from pan-FGFR inhibitors has
successfully yielded a novel series of highly potent FGFR4 inhibitors with selectivity over
FGFR1. Due to reduced lipophilicity and aromatic ring count, this series demonstrated improved
solubility and permeability. However, plasma instability and fast metabolism limited its
potential for in vivo studies. Efforts have been made to address these problems, which led to the
discovery of compound (-)-11 with improved stability, CYP inhibition, and good
activity/selectivity for further optimization.
Revised
Accepted
Available online
Keywords:
hepatocellular carcinoma
fibroblast growth factor 19
fibroblast growth factor receptor 4
covalent inhibitor
2009 Elsevier Ltd. All rights reserved
.
isoform selective
———
∗ Corresponding author. Tel.: +86-21-50172812; e-mail: wang_yikai@wuxiapptec.com, zhang_yang@wuxiapptec.com

Hepatocellular carcinoma (HCC) is the second most lethal
cancer with very limited targeted therapies due to its complexity
resistance.22, 23 One of the common approaches to develop such
an inhibitor is to introduce a reactive electrophile on a known
noncovalent inhibitor at a position in close proximity to a
reactive cysteine residue.24, 25 In the case of BLU9931, via adding
an acrylamide moiety on a pan-FGFR inhibitor PD173034, Ex.
5226 with less than 10 nM activity against FGFR4 and larger than
100-fold selectivity over FGFR1 was achieved (Figure 1).
Subsequent optimization involved replacement of tertiary
acrylamide with o-phenylenediamine linked secondary
acrylamide, which maintained the spatial proximity of the
Michael acceptor and cysteine thiol. This strategy was also
applied by Celgene27 and Eisai28 independently to obtain FGFR4-
selective inhibitors (e.g. Ex. 2 and Ex. 108, Figure 1).
2
and heterogeneity.1, Sorafenib, standard care for almost ten
years, only provided minimal survival benefit for patients with
advanced HCC.3 Recently, regorafenib demonstrated improved
overall survival in a phase III trial for patients who progressed on
sorafenib therapy, which may become an option for second-line
treatment.4, 5 However, their mechanisms of action are expected
to be similar given the structure similarity of the two multi-kinase
inhibitors. Therefore there is still an urgent need for more
effective HCC therapies.
Recent advances in genomic sequencing and analysis have
shed light on aberrant signaling pathways involved in this
complex disease, among which the FGF19-FGFR4 axis was
found to be frequently altered in a subset of HCC patients.6-8
Fibroblast growth factor 19 (FGF19) is a tightly controlled
endocrine hormone that is involved in liver homeostasis through
its interaction with fibroblast growth factor receptor 4 (FGFR4)
under normal physiological context.9 It was found that HCCs
harboring FGF19 amplification are able to hijack this signaling
cascade leading to uncontrolled cell growth.10 Proof of concept
studies in mice using FGFR4 or FGF19 antibodies to block this
pathway have demonstrated anti-tumor effects, which make it an
appealing target for therapeutic intervention.11, 12
Potential problems associated with the introduction of
acrylamides to make covalent drugs include the increase in
molecular weight, hydrogen-bond donor/acceptor count, and
metabolic liability, which all negatively impact drug-like
properties. It is noteworthy that previous searches for FGFR4
selective inhibitors started with bicyclic or pseudo-bicyclic cores
from pan-FGFR inhibitors.26-34 The resulting covalent inhibitors
usually have high molecular weight (> 500 Da) and high
lipophilicity. With this observation in mind, we turned our
interest to start with a smaller and less lipophilic core.
O
a
F
At the time we launched our project to develop FGFR4
inhibitors for HCC indication, there were more than 20 FGFR
inhibitors in phase I to II clinical studies to treat various types of
tumors.13, 14 However, the potential of most candidates to block
the FGF19-FGFR4 pathway is limited due to lack of potency
against FGFR4 or dose limiting toxicity arising from FGFR1
inhibition and off-target effects such as VEGFR blockage.15, 16
The only two selective FGFR4 inhibitors to treat HCC patients
with FGF19 amplification or FGFR4 overexpression that drew
our attention were BLU9931 (BLU554 as the clinical candidate,
Blueprint Medicines) and FGF401 (Novartis). Although little was
disclosed on FGF401 except for a patent describing a series of
substituted picolinaldehyde,17 detailed study of the former was
well documented.18 The high selectivity of BLU9931 against
FGFR4 was achieved by forming a covalent bond with the side
chain of Cysteine 552 (Cys552) located at the hinge region of
FGFR4 which is not present in the other FGFRs.18
O
N
N
O
HO
N
F
N
H
N
ASP5878
Figure 2. (a) Structure of pan-FGFR inhibitor ASP5878 with a flexible
linker. (b) Docking result of ASP5878 with BLU9931 (4XCU used as
docking template). (c) Docking result of ASP5878 with Eisai-108 (4QQ5
used as docking template). BLU9931, Eisai-108 and ASP5878 were colored
light blue, purple and brown, respectively. The magenta line represents a
hydrogen bond. For clarity, only important residues were shown with line
model and colored green.
O
O
O
Cl
N
O
O
N
O
N
O
Cl
HN
N
N
NH
NH
N
N
N
NH
HN
N
H
N
O
O
NH
N
N
Et
O
Et
Et
Et
PD173034
Blueprint
Ex. 52
BLU9931
A pan-FGFR inhibitor that drew our attention was ASP587835,
(Figure 2a). It possesses a monocyclic hinge binding motif
O
Cl
N
36
N
O
with a two-atom chain linked with a substituted phenyl group.
Modeling results against FGFR4 suggest that binding mode of
ASP5878 is very similar to BLU9931 (Figure 2b) and Ex. 108
(Figure 2c). They all form two hydrogen bonds with the same
hinge residues. The 3,5-dimethoxy phenyl groups are all nestled
in a back hydrophobic pocket where the O atom is in close
contact with the NH of Asp630, suggestive of an additional
hydrogen bond. To fit into this exact geometry, the length and
conformation of the linker are very important. In BLU9931, the
quinazoline ring itself serves as a rigid linker pointing the phenyl
group in the right direction, whereas in ASP5878 the flexible
methlyene-ether linker adopts an extended conformation.
Interestingly, the urea moiety in Ex. 108 together with the
pyrimidine ring forms a pseudo-bicyclic structure via an
intramolecular hydrogen bond, which in turn poses the phenyl
group in the same position as BLU9931 and ASP5878. Our
Cl
HN
N
N
O
H
N
O
Celgene
Ex. 2
O
O
Et
Cl
Et
Cl
N
N
N
N
N
N
HN
N
N
HN
O
O
Cl
Cl
O
N
H
N
O
N
H
N
O
NH
BGJ-398
Eisai
Ex. 108
Figure 1. Strategy to develop FGFR4 selective inhibitors via introduction of
Michael acceptors on pan-FGFR inhibitors.
There is a resurgent interest of developing covalent drugs over
the past few years, as is evidenced by the successful approval of
ibrutinib, afatinib, and osimertinib.19-21 Irreversible inhibitors
have a number of potential advantages including high potency,
extended pharmacodynamics, high selectivity, and delaying

aLipophilic ligand efficiency (LLE) was calculated by substraction of cLogP
from pIC50
observation was in good accordance with the literature report on
conformational analysis of BGJ398,37 the pan-FGFR inhibitor
from which Ex. 108 was derived.
.
Next, physicochemical properties of compound 1 were
profiled and compared with BLU9931 (Table 2). With reduced
molecular weight, aromatic ring count and cLogP, compound 1
showed much higher kinetic solubility than BLU9931 at pH 7.4
(39 µg/mL versus <0.5 µg/mL) and decreased LogD by about 1.5
log unit. The permeability was also improved from 3.5 nm/s to
86 nm/s. Moreover, CYP inhibition of compound 1 was slightly
improved across all isoforms. Overall, by simply switching to a
flexible scaffold, we were able to achieve a compound with better
potency, improved selectivity and physicochemical properties.
Table 1. Activity and selectivity of covalent inhibitors based on
flexible cores.
IC50 (nM)
Compound No.
R1
R2
R3
FGFR4
FGFR1
8,600
An obvious limitation associated with this new scaffold was
its fast metabolism (Table 3). Although human microsomal
metabolic stability (MMS) was within the acceptable range for
covalent inhibitors, compound 1 was found to be extremely
unstable (Clint(liver) = 9643 mL/min/kg) in mouse microsomes.
Another problem that concerns us was its plasma instability.
When 1 and BLU9931 were both incubated with mouse and
human plasma for 2 hours, 13% and 48% of parent compound 1
remained respectively, which is less stable than BLU9931.
Initially, it was suspected that compound 1 was a better substrate
for esterases. However, metabolite identification (MetID) study
in human plasma failed to identify any detectable amount of
hydrolytic product or any other metabolite after in vitro
incubation for 2 hours. When analyzing the plasma MetID data
more carefully, it was found that the peak area corresponding to
the parent compound 1 was decreased by about 30% after 2
hours.39 The fact that parent compound disappeared without
generation of any metabolites indicated a potential covalent
addition to plasma protein. Although esterase hydrolysis cannot
be ruled out especially in mouse plasma, the hydrolytic product
o-phenylenediamine might be oxidized to 1,2-quinone-type
intermediate that still bears the risk of addition with cysteine
residue. Protein covalent modification has been recognized as a
potential liability for the development of irreversible inhibitors
that should be mitigated.40-43 In an analysis published by
researchers at Celgene, greater than 50% remaining after 60 min
incubation in whole blood was proposed as a cutoff for filtering
out compounds with high risk of covalent addition.43 In our case,
plasma stability of compound 1 obviously required further
optimization.
1
2
3
Me
Me
Me
H
H
H
F
Cl
H
3
4
4,340
188
> 10,000
4
Me Me Cl > 10,000 > 10,000
5
H
-
H
-
F
-
8
60
BLU9931
49
2,700
Based on the structural analysis, we hypothesized that adding
the acrylamide moiety to the core of ASP5878 would result in a
covalent FGFR4-selective inhibitor. Compound 1 was thus
synthesized and tested against both FGFR4 and FGFR1, which
showed an IC50 of 3 nM against FGFR4 and > 8 µM against
FGFR1 (Table 1). Encouraged by this discovery, several
analogues were made to probe the structure-activity relationship
(SAR) around the initial hit. Fluorine atoms can be substituted
with chlorine yielding compound 2 without obvious change in
activity (4 nM versus 3 nM). Analogous to the SAR around
BGJ398,37 halogen atoms were also required for maintaining
activity in this case, since the non-halogenated version was
around 50 fold less potent than the original compounds (3 versus
1 and 2). Compound 4 with an extra methyl group on the linker
was completely inactive against FGFR4. The 1-(2,6-
dichlorophenyl)ethoxy moiety in compound 4 was also found in
crizotinib, a c-Met and ALK inhibitor. From the reported
structure information of crizotinib binding with c-Met,38 we
realized that the methyl group could bias the whole molecule
toward a folded rather than extended conformation, yet the latter
was required for binding with FGFR4. Lastly, the role of the tolyl
methyl group in controlling selectivity over FGFR1 was
rediscovered in our case as was seen that selectivity of compound
5, the des-methyl analogue of compound 1, was significantly
eroded.
O
O
O
F
F
F
O
O
O
N
O
N
O
N
O
F
F
F
HN
N
HN
N
HN
N
H
H
H
N
N
N
O
O
O
N
N
N
N
N
N
6
7
8
O
Table 2. Physicochemical properties, permeability and CYP
inhibition profile of compound 1 and BLU9931.
O
F
O
F
F
F
O
O
N
O
O
N
O
Properties
1
BLU9931
HN
N
HN
N
H
N
H
N
O
O
Molecular weight (Da)
cLogP
456.4
4.6
509.4
6.3
9
(
)-10
O
F
O
F
F
F
Lipophilic ligand efficiencya
Kinetic solubility pH=7.4 (µg/mL)
LogD 7.4 (octanol/buffer)
Papp A to B (nm/s), (efflux ratio)
3.9
1
O
O
N
O
N
O
HN
N
HN
N
H
H
N
39
<0.5
N
O
O
O
O
3.0
> 4.5
3.5, (1.5)
(+)-11
(-)-11
Figure 4. Analogues based on o-phenylenediamine and saturated cyclic
86, (1.0)
diamines.
CYP (IC50, µM) 1A2, 2C9, 2C19,
2D6, 3A4
>50, 3.7, 1.5,
35.0, 6.9
15.9, 0.9, 1.7,
12.5, 3.0
To explore the electronic and steric effects of the
phenylacrylamide on plasma stability, several analogues were

evaluated (Figure 4). Adding electron donating piperazine
substituents to the phenyl ring or increasing the steric hindrance
of the Michael acceptor were able to maintain good activity and
increase microsomal metabolic stability, but failed to improve
mouse plasma stability (Table 3). Instead of making more
derivatives around the o-phenylenediamine moiety that seems to
be intrinsically problematic, saturated rings were explored as
bioisosteres. It was quite satisfying to find that all these
analogues (compound 9-11) were very stable after incubation for
2 hours at 37 °C in human or mouse plasma. Compound 9 and (-
)-11 were also found to be metabolically more stable than
compound 1 and 10. Activity against FGFR4 was measured, and
the eutomer (-)-11 was 4 fold less potent than compound 1 but
300 fold more potent than its enantiomer (+)-11.44 To our
surprise, compound 9 and 10 suffered notable activity loss
against FGFR4. The steep SAR trend implies the importance of
the acrylamide trajectory that greatly affects the rate of covalent
bonding formation, which happens to be the origin of both
potency and selectivity in this case.
distribution (0.58 L/kg) and high plasma clearance (Cl = 65
mL/min/kg). PO dosing of (-)-11 at 1 mg/kg failed to deliver any
calculable exposure, most likely resulting from notable first pass
elimination due to high microsomal instability. Although
optimization on mouse MMS was not our primary focuses, the
problem needs to be addressed in order to demonstrate in vivo
efficacy in mice models.
Table 4. Physicochemical properties and in vitro ADME profile
of compound (-)-11
Properties
(-)-11
436.4
2.5
Molecular weight (Da)
cLogP
Lipophilic ligand efficiency
Kinetic solubility pH=7.4 (µg/mL)
LogD 7.4 (octanol/buffer)
Papp A to B (nm/s), (efflux ratio)
PPB (Unbound %, H, M)
5.4
78
1.8
73, (3.8)
35.4/30.9
Table 3. Activity, selectivity, plasma and microsomal metabolic
stability of analogues around compound 1
Plasma metabolic
CYP (IC50, µM) 1A2, 2C9, 2C19, 2D6, 3A4 >50, 29.1, 19.8, >50, 13.3
Clint(liver)
(mL/min/kg)
stability
Human Mouse
%left at 2h
IC50
Compound
No.
Around the same time we were preparing this manuscript, a
patent from Eisai was published covering some efforts on this
particular flexible scaffold including the racemate of 11.45
However no detailed profiling of this particular compound as
well as others was disclosed.
FGFR4
FGFR1
2,700
8,600
4,340
2,900
9,500
4,800
>10,000
3,300
7,400
3,500
H/ M
41/97
BLU9931
49
3
71
48
63
13
1
165/9643
N/A
In summary, via introduction of acrylamide moieties on a
scaffold from a pan-FGFR inhibitor, a series of selective FGFR4
inhibitors were discovered and characterized. The impact of the
flexible ether linker on improvement of physicochemical
properties was observed. Yet the initial hit compound suffered
from very poor plasma and microsomal stability. Efforts to
address this issue led to the discovery of (-)-11 with decent
activity and drug-like properties. Further optimization around this
compound is currently underway.
2
4
N/A
N/A
N/A
N/A
90
0.2
0.1
0.3
0.4
100
96
6
7
4
120/180
265/220
N/A
6
8
27
9
148
1,200
3,500
13
46/564
154/2032
N/A
10
96
(+)-11
(-)-11
N/A
92
N/A
97
18/474
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Graphical Abstract
Discovery and Optimization of Selective
FGFR4 Inhibitors via Scaffold Hopping
Leave this area blank for abstract info.
Yikai Wang,* Zhengxia Chen, Meibi Dai, Peipei Sun, Chunqiu Wang, Yang Gao, Haixia Zhao, Wenqin Zeng,
Liang Shen, Weifeng Mao, Tian Wang, Guoping Hu, Jian Li, Shuhui Chen, Chaofeng Long, Xiaoxin Chen,
Junhua Liu, and Yang Zhang*