Discovery of Selective, Covalent FGFR4 Inhibitors with Antitumor Activity in Models of Hepatocellular Carcinoma

Article abstract of DOI:10.1021/acsmedchemlett.9b00601

Hepatocellular carcinoma (HCC) accounts for a majority of primary liver cancer and is one of the most common forms of cancer worldwide. Aberrant signaling of the FGF19-FGFR4 pathway leads to HCC in mice and is hypothesized to be a driver in FGF19 amplifie

Full text of DOI:10.1021/acsmedchemlett.9b00601

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Letter
Discovery of Selective, Covalent FGFR4 Inhibitors with
Anti-tumor Activity in Models of Hepato-cellular Carcinoma
Haibo Liu, Deqiang niu, Robert Tjin Tham Sjin, Alex Dubrovskiy, Zhendong Zhu, Joseph J.
McDonald, Kelly Fahnoe, Zhigang Wang, Mark Munson, Andrew Scholte, Matthieu Barrague, Maria
Fitzgerald, Jinyu Liu, Michael Kothe, Fangxian Sun, Joshua Murtie, Jie Ge, Jennifer Rocnik, Darren
Harvey, Beatriz Ospina, Keli Perron, Gang Zheng, Elvis Shehu, and Laura Akullian D’Agostino
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00601 • Publication Date (Web): 06 Mar 2020
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Discovery of Selective, Covalent FGFR4 Inhibitors with Anti-tumor
Activity in Models of Hepatocellular Carcinoma
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Haibo Liu†, Deqiang Niu†, Robert Tjin Tham Sjin†, Alex Dubrovskiy†, Zhendong Zhu†, Joseph J
McDonald†, Kelly Fahnoe†, Zhigang Wang†, Mark Munson‡, Andrew Scholte‡, Matthieu Barrague‡,
Maria Fitzgerald‡, Jinyu Liu‡, Michael Kothe‡, Fangxian Sun‡, Joshua Murtie‡, Jie Ge‡, Jennifer
Rocnik‡, Darren Harvey‡, Beatriz Ospina‡, Keli Perron‡, Gang Zheng‡, Elvis Shehu‡, Laura Akullian
D’Agostino†
†Departments of Chemistry, Biology, and Biochemistry, Bristol-Myers Squibb, 200 Cambridge Park Dr., Cambridge, MA
02140;
‡Departments of Chemistry, Biology, DMPK, Structural Biology, and Pharmacology, Sanofi, 153 Second Ave., Waltham,
MA 02451
KEYWORDS. Selective FGFR4 inhibition, kinase inhibitor, targeted covalent inhibitor, hepatocellular carcinoma
ABSTRACT: Hepatocellular carcinoma (HCC) accounts for 85–90% of primary liver cancer and is one of the most common forms
of cancer worldwide.1 Aberrant signaling of the FGF19-FGFR4 pathway has been shown to cause HCC in mice and is hypothesized
to be a driver in FGF19 amplified HCC in humans.2 Multiple small molecule inhibitors have been pursued as targeted therapies for
HCC in recent years including several selective FGFR4 inhibitors that are currently being evaluated in clinical trials. Herein we report
a novel series of highly selective, covalent 2-amino-6, 8-dimethyl-pyrido[2,3-d]pyrimidin-7(8H)-ones that potently and selectively
inhibit FGFR4 signaling through covalent modification of Cys552, which was confirmed by X-ray crystallography. Correlative
PK/PD and tumor regression were observed in preclinical models of orthotopic and sorafenib-resistant HCC.
Hepatocellular carcinoma (HCC) accounts for 85-90% of
primary liver cancer and is one of the most common forms of
cancer with the highest rate of cancer-related mortality
worldwide.1 Pathogenically, the causes for HCC have been
commonly associated with chronic liver stress such as viral
infection, metabolic disorders, or alcohol abuse. In 2018, there
were 841,000 incidents of liver cancer and 781,000 deaths
worldwide.2 In 2017, in the US alone, there were approximately
40,000 cases diagnosed and 29,000 deaths.3 The current
standard of care for HCC is the multikinase inhibitor sorafenib
which was approved in 2007 based on a three month
expressed in human hepatocytes and FGF19 is the primary,
albeit not exclusive, ligand for FGFR4. FGFR4 pathway
activation requires FGF19 binding as well as the tissue-resident
cofactor
β-klotho
(KLB).
The
combination
of
FGF19/FGFR4/KLB is uniquely effective in driving hepatocyte
proliferation, dysplasia, and neoplasia. These effects can be
reversed in vitro and in animal models via neutralization of
FGF19 and/or inhibition of FGFR4, supporting the hypothesis
that inhibition of FGFR4 signaling may provide therapeutic
benefit in certain cancers.8-10 Furthermore, through selective
inhibition of FGFR4 over FGFR 1–3, one could achieve
therapeutic benefit without encountering dose limiting
toxicities associated with inhibition of FGFR1 and FGFR3,
particularly hyperphosphatemia and hypocalcemia. Taken
together, these data collectively support the therapeutic
rationale for selective inhibition of FGFR4 in patients with
FGF19 amplified HCC.
improvement in median survival and time to progression.4
A
significant number of patients present intrinsic resistance to
sorafenib treatment and still many others develop acquired
resistance.5 In 2017, two second line therapies were approved:
multikinase inhibitor regorafenib in Japan and the EU, and anti-
PD1 immunotherapy nivolumab in the US. Notwithstanding
these notable successes, and despite many phase 3 attempts to
treat HCC, there remains a significant shortage of therapeutic
options.6
FGFR proteins are highly homologous with sequence
similarity of 88% on average and sequence homology of 68%.
When our program was initiated, only pan-FGFR inhibitors
were known and often exhibited weaker potency against
FGFR4 compared to FGFRs 1–3. Examples include PD173074,
BGJ-398 and AZD4547 (Figure 1). In recent years, the
discovery of potent and selective FGFR4 inhibitors has been
actively pursued by numerous groups and has been the topic of
multiple reviews.11 A diverse set of small molecule inhibitors
has been identified (Figure 1) including several that are
currently being evaluated in clinical trials (BLU554, H3B-6527
The fibroblast growth factor receptors (FGFRs) are a family
of transmembrane receptor tyrosine kinases comprised of
FGFR1, 2, 3, and 4 that interact with 18 different fibroblast
growth factor (FGF) ligands. FGF/FGFR signaling pathways
play essential roles in embryogenesis, metabolism, and tissue
homeostasis. Aberrant FGF/FGFR signaling has been
implicated in lung cancer, breast cancer, gastric cancer, and
HCC among others.7 FGFR4 is the predominant isoform
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and FGF401). In a recent Phase 1 study, BLU-554 was shown amino-6-methylpyrido[2,3-d]pyrimidin-7(8H)-one
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FGFR4
1
2
3
to elicit clinical responses in patients with advanced FGF19
positive HCC.12 Herein, we report the design, synthesis and
biological evaluation of a novel series of potent and selective 2-
inhibitors that promote tumor regression in sorafenib-resistant
and orthotopic human tumor xenograft models.
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Figure 1. Structures of representative pan-FGFR and selective FGFR4 inhibitors.
Covalent drugs have proven successful as targeted therapies
for a variety of malignancies with known oncogenic drivers,
achieving high levels of target engagement and protracted target
occupancy in vivo. In addition, covalent inhibition may provide
an advantage in cases of acquired drug resistance.1,3 FGFR4
contains a unique cysteine (Cys) residue numbered 552. FGFRs
1–3 present a tyrosine (Tyr) at this position. Furthermore,
Cys552 is conserved in just 10 human protein kinases including
MK2, MK3, S6K2, STK40 and TTK. Thus, covalently
targeting the Cys552 in FGFR4 is an appealing strategy for
achieving selective inhibition of FGFR4 both with respect to
isoform and kinome selectivity, as well as addressing
challenges related to acquired resistance.
Through structure-based design, we were able to identify an
ATP
competitive
2-amino-6,
8-dimethylpyrido[2,3-
d]pyrimidin-7(8H)-one scaffold that achieved covalent
modification of Cys552 and inhibition of the catalytic activity
of FGFR4. Optimization of cellular potency, selectivity, and
pharmacokinetic properties resulted in identification of
compound 1 which inhibits pFGFR4 signaling in cells with IC50
= 9 nM and >100 fold selectivity over FGFRs 1–3. FGFR2 was
used as a surrogate marker of FGFR 1–3 activity in cells as
potency against FGFRs 1–3 was generally similar.
Figure 2. Co-crystal structure of compound 1 in complex with
FGFR4 determined to 1.9 Å resolution. Compound 1 forms a
covalent bond to Cys552 and two H-bonds with Ala553 in the hinge
region of the ATP binding pocket. The dimethoxyphenyl group
binds in a hydrophobic pocket and forms H-bond interactions with
Asp630.
Structural alterations and a matched pair analysis with
compound 1 furthered our understanding of structure-activity
relationships (SAR). Covalency was critical for potency as well
as selectivity, demonstrated by a comparison of 1 with its
reversible propionamide analog 2. Positioning of the
acrylamide was impactful, and a reversal in the absolute
configuration of the adjacent stereocenter, as shown in
compound 3, resulted in a significant loss in potency and
selectivity. Covalent modifiers with increased cysteine
reactivity such as vinylsulfonamide 5 led to erosion of
selectivity without a gain in potency. The penta-substituted
benzene in the hydrophobic back pocket of FGFR4 could be
modified to maintain or attenuate potency and selectivity.
Removal of either chloro or methoxy groups led to reduced
potency. However, replacing either or both chlorines with
fluorine resulted in similar levels of potency and selectivity.
A co-crystal structure of compound 1 covalently bound to the
kinase domain of human FGFR4 was determined by X-ray
crystallography at 1.9 Å resolution (Figure 2, PDB 6V9C). The
structure shows that compound 1 binds to the DFG-in
conformation of the FGFR4 kinase domain. The acrylamide
forms a covalent bond with Cys552 in the hinge region of the
protein. Additionally, the acrylamide forms hydrogen bonding
interactions with Ala553 in the hinge region and Arg483 located
on the N lobe. The carbonyl of the pyridopyrimidinone core
forms a water mediated hydrogen bond with Asp630 near the
DFG region of the activation loop. The dichloro- and
dimethoxy- substituted benzene resides in a hydrophobic region
formed by Phe631, Met524, and multiple hydrophobic residues
located on the beta sheet of the N-lobe including Val481. In
addition to hydrophobic interactions, one methoxy group forms
a hydrogen bond with the main chain NH of Asp630.
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The synthesis of compound 1 is shown in Scheme 1.
Chloropyrimidine 11 was treated with methylamine to displace
the chloride in quantitative yield. The methylamine adduct was
then subjected to reduction with LiAlH4 and oxidation with
MnO2 to furnish aldehyde 12 in 40% overall yield in two steps.
Thermal condensation of 12 with 13 in the presence of K2CO3
provided 14 in 63% yield. Simultaneous oxidation and
chlorination of 14 was achieved with SO2Cl2 in 70% yield. The
sulfone moiety in 55 was converted to a chloride in sequential
hydrolysis and chlorination steps with 79% overall yield.
Coupling of 16 with amine 17 furnished 18 in 71% yield. Boc
deprotection followed by acrylamide formation provided
compound 1 in 65% yield over two steps.
1
2
3
4
5
6
7
8
Table 1. Kinase Inhibitory Activities of Compounds
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b
aValues determined using an Omnia® Kinase Assay at 250 uM ATP. pFGFR4 and pFGFR2 formation in Huh7 and KATO cells,
respectively. cInhibition of FGFR2 phosphorylation was used as a surrogate for FGFR1 and FGFR3, as potencies against these off-targets
were similar
Scheme 1. Synthesis of Compound 1a
CO2Et
OMe
OMe
Cl
MeO
OMe
O
Cl
NHMe
N
13
e
a, b, c
d
N
OMe
EtO
N
N
OMe
O
Cl
S
N
N
O
N
S
S
N
N
O
N
S
O
O
11
12
14
15
OMe
Cl
O
OMe
Cl
OMe
Cl
Cl
Cl
Cl
NH2
17
i, j
NH
h
O
O
Boc
N
OMe
N
OMe
N
OMe
f, g
Cl
N
N
O
N
N
N
O
N
H
N
N
O
O
H
NH
NH
Boc
16
18
1
aReagents and conditions: (a) MeNH2, DCM, 0 °C, 30 min, 100%; (b) LiAlH4, THF, 15 min, 53%; (c) MnO2, DCM, 24 h, 75%; (d) 10,
K2CO3, DMF, 110 °C, 4 h, 63%; (e) SO2Cl2, DCM, 0 °C, 30 min, 70%; (f) KOH, THF/H2O (1:1), 0 °C , 4 h, 83%; (g) POCl3, CH3CN, 90 °C,
6 h, 95%; (h) 14, DIPEA, NMP, 90 °C, 2 h, 71%; (i) TFA, DCM, 2 h, 97%; (j) acryloyl chloride, DCM, DIPEA, -78 °C, 10 min, 67%.
Pharmacokinetic analysis of 1 revealed a high Cmax, low
clearance, and 20, 12, and 27% oral bioavailability in mouse,
rat, and cyno, respectively (Table 2). Pharmacodynamic (PD)
effects were studied by measuring target occupancy and
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time in Huh7 tumor-bearing SCID mice after a single oral
administration (100 mpk) of 1.
inhibition of pFGFR4 signaling in Huh7 tumor-bearing mice
after single dose oral administration at 2.5, 10, 30 and 100
mg/kg. FGFR4 autophosphorylation, measured by Mesoscale
Discovery (MSD) assays, was inhibited in a dose dependent
manner over a time course of 1–8 hours. The degree of target
occupancy, determined by ratio of unbound to total FGFR4, was
in close correlation with inhibition of pFGFR4 signaling, with
max inhibition observed at Cmax. Target occupancy >80% was
sustained for approximately 10h after a single dose (Figure 3).
1
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9
Table 2. Pharmacokinetics of Compound 1 in Mouse, Rat and
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Cynomolgus Monkey
Species
Route
Mouse
Rat
Cyno
iv
3
po
10
iv
3
po
10
iv
3
po
10
Dose (mg/kg)
CL (mL/min/kg)
Vss (L/Kg)
T1/2(hr)
17.2
0.63
2.1
9.0
8.4
1.1
4.8
0.30
2.1
Figure 4. Antitumor activity of compound 1 (100 mg/kg BID)
against orthotopic Hep3B human tumors in SCID mice, on day 28
of treatment.
1.8
423
2960
20
3.2
588
2230
12
2.0
2820
5660
27
Cmax (mg/mL)
AUC (ng*hr/mL)
%F
6270
2910
9130
5610
5100
6420
The anti-tumor activity of 1 was studied in two human tumor
xenograft (HTX) models of HCC including an orthotopic model
of (Hep3B) HCC and a subcutaneous model of sorafenib
resistant Huh7 tumor growth, in SCID mice. In a Hep3B model,
100 mg/kg of inhibitor 1 administered PO daily resulted in
tumor regression and sustained growth inhibition over 28 days
(Figure 4). Sorafenib-resistant tumors were established by
orally administering sorafenib to animals bearing Huh7 tumors,
at a dose of 100 mg/kg twice daily for fourteen months until the
tumors were completely resistant to treatment. After
establishment of sorafenib-resistant Huh7 tumors, oral
administration of 1 at 10, 30 and 100 mg/kg BID for 11 days
resulted in dose-dependent growth inhibition of sorafenib-
resistant tumors. Tumor regression was observed at 30 and 100
mg/kg, with % ΔT/ΔC = -67% and -70% respectively (p <
0.0001). As expected, treatment with sorafenib at 100 mg/kg
once daily did not provide any benefit (Figure 5).
Figure 5. Antitumor activity of compound 1 against subcutaneous
sorafenib resistant Huh7 human tumors in SCID mice.
In summary, a series of covalent and selective FGFR4
inhibitors were identified. Potent inhibition of FGFR4
phosphorylation was achieved enzymatically and in cells.
Isoform and kinome selectivity were achieved, in part, through
covalent modification of the unique Cys552, which was
confirmed by X-ray crystallography. In addition, compound 1
achieved sufficient in vivo exposure to acheive high levels of
FGFR4 occupancy and pFGFR4 inhibition, in tumor bearing
mice. In orthotopic and sorafenib-resistant HCC HTX models,
oral administration of 1 resulted in notable and sustained tumor
regression. Collectively, these data support the potential utility
of 1 for treatment of FGF19/FGFR4 driven diseases including
sorafenib resistant HCC.
SUPPORTING INFORMATION
The Supporting Information is available free of charge on the ACS
Publications website.
Synthetic procedures and analytical data of selected compounds,
conditions for all biological assays, and X-ray crystallographic
methods and statistics, kinome inhibition profiling, in vivo PK/PD
protocols and tumor growth inhibition studies protocols. (PDF)
Figure 3. pFGFR4 inhibition and target occupancy as a function of
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(4) Llovet, J. M; Ricci, S.; Mazzaferro, V.; Hilgard, P.; Gane, E.;
Blanc, J.-F.; de Oliveira, A. C.; Santoro, A.; Raoul, J.-L.; Forner, A.;
AUTHOR INFORMATION
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Schwartz, M.; Porta, C.; Zeuzem, S. et. al. Sorafenib in Advanced
Corresponding Author
Hepatocellular Carcinoma.
N. Engl. J. Med., 2008, 359, 378-390.
* E-mail: ldagostino@celgene.com
(5) Niu, L.; Liu, L.; Yang, S.; Ren, J.; Lai, P. B.; Chen, G. G. New
insights into sorafenib resistance in hepatocellular carcinoma:
Responsible mechanisms and promising strategies. Biochim. Biophys.
Acta, Rev. Cancer, 2017, 1868, 564-570.
(6) Balogh, J.; Victor III, D.; Asham, E.; Burroughs, S.; Boktour, M.;
Saharia, A.; Li, X.; Ghobrial, R.; Monsour Jr, H. Hepatocellular
carcinoma: a review. J. Hepatocell. Carcinoma, 2016, 3, 41-53.
(7) Touat, M.; Ileana, E.; Postel-Vinay, S.; André, F.; Soria, J.-C.
Targeting FGFR Signaling in Cancer. Clin. Cancer Res., 2015, 21,
2684-2694.
Present Addresses
†If an author’s address is different than the one given in the
affiliation line, this information may be included here.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
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(8) Desnoyers, L. R.; Pai, R.; Ferrando, R. E.; Hötzel, K.; Le, T.;
Ross, J.; Carano, R.; D'Souza, A.; Qing, J.; Mohtashemi, I.;
Ashkenazi, A.; French, D. M. Targeting FGF19 inhibits tumor growth
in colon cancer xenograft and FGF19 transgenic hepatocellular
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Notes
The authors declare no competing financial interest.
ABBREVIATIONS
(9) French, D. M.; Lin, D. C.; Wang, M.; Adams, C.; Shek, T.;
Hotzel, K.; Bolon, B.; Ferrando, R.; Blackmore, C.; Schroeder, K.;
Rodriguez, L. A.; Hristopoulos, M.; Venook, R.; Ashkenazi, A.;
Desnoyers, L. R. Targeting FGFR4 Inhibits Hepatocellular
Carcinoma in Preclinical Mouse Models. PLoS One, 2012, 7, e36713.
(10) Sawey, E. T.; Chanrion, M.; Cai, C.; Wu, G.; Zhang, J.; Zender,
L.; Zhao, A.; Busuttil, R. W.; Yee, H.; Stein, L.; French, D. M.; Finn,
R. S.; Lowe, S. W.; Powers, S. Identification of a Therapeutic
Strategy Targeting Amplified FGF19 in Liver Cancer by
Oncogenomic Screening. Cancer Cell, 2011, 19, 347-358.
(11) Lu, X.; Chen, H.; Patterson, A. V.; Smaill, J. B.; Ding, K.
Fibroblast Growth Factor Receptor 4 (FGFR4) Selective Inhibitors as
Hepatocellular Carcinoma Therapy: Advances and Prospects. J. Med.
Chem., 2019, 62, 2905–2915.
(12) Kim, R. D.; Sarker, D.; Meyer, T.; Yau, T.; Macarulla, T.; Park,
J-W.; Choo, S. P.; Hollebecque, A.; Sung, M. W.; Lim, H-Y. ;
Mazzaferro, V.; Trojan, J.; Zhu, A. X.; Yoon, J-H.; Sharma, S.; Lin,
Z-Z.; Chan, S. L.; Faivre, S.; Feun, L. G.; Yen, C-J.; Dufour, J-F.;
Palmer, D. H.; Llovet, J. M.; Manoogian, M.; Tugnait, M.; Stransky,
N.; Hagel, M.; Kohl, N. E.; Lengauer, C.; Sherwin, C. A.; Schmidt-
Kittler, O.; Hoeflich, K. P.; Shi, H.; Wolf, B. B.; Kang, Y-K. First-in-
Human Phase I Study of Fisogatinib (BLU-554) Validates Aberrant
FGF19 Signaling as a Driver Event in Hepatocellular Carcinoma.
Cancer Discovery, 2019, 9, 1696-1707.
FGFR, fibroblast growth factor receptor; FGF, fibroblast growth
factor; DMPK, drug metabolism and pharmacokinetics; HCC,
hepatocellular carcinoma; PK, pharmacokinetics; PD,
pharmacodynamics; EU, European Union; anti-PD1, programmed
cell death protein 1 antibody; KLB, cofactor β-klotho; DFG, Asp-
Phe-Gly motif; Boc, tert-butyloxycarbonyl protecting group;
DIPEA, N,N-diisopropylethylamine; NMP, N-methyl-2-
pyrrolidone; TFA, trifluoroacetic acid; MSD, mesoscale
discovery; cyno, cynomolgus monkey; CL, clearance; Vss,
volume of distribution; T1/2, half-life; Cmax, maximum serum
concentration; AUC, area under the curve; %F, oral
bioavailability; HTX, human tumor xenograft; QD, once a day;
SCID, severe combined immunodeficiency.
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