Discovery and optimization of selective FGFR4 inhibitors via scaffold hopping

Bioorganic & Medicinal Chemistry Letters 27 (2017) 2420–2423

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Discovery and optimization of selective FGFR4 inhibitors via scaffold hopping Yikai Wang a,⇑, Zhengxia Chen a, Meibi Dai a, Peipei Sun a, Chunqiu Wang a, Yang Gao a, Haixia Zhao a, Wenqin Zeng a, Liang Shen a, Weifeng Mao a, Tian Wang a, Guoping Hu a, Jian Li a, Shuhui Chen a, Chaofeng Long b, Xiaoxin Chen b, Junhua Liu b, Yang Zhang a,⇑ a b

WuXi AppTec (Shanghai) Co., Ltd., 288 FuTe Zhong Road, Shanghai 200131, PR China Guangdong Zhongsheng Pharmaceutical Co., Ltd., Dongguan 523325, PR China

a r t i c l e

i n f o

Article history: Received 11 January 2017 Revised 16 March 2017 Accepted 4 April 2017 Available online 5 April 2017 Keywords: Hepatocellular carcinoma Fibroblast growth factor 19 Fibroblast growth factor receptor 4 Covalent inhibitor Isoform selective

a b s t r a c t 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. Ó 2017 Elsevier Ltd. All rights reserved.

Hepatocellular carcinoma (HCC) is the second most lethal cancer with very limited targeted therapies due to its complexity and heterogeneity1,2 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 ⇑ Corresponding authors. E-mail addresses: [email protected] (Y. Wang), zhang_yang@ wuxiapptec.com (Y. Zhang). http://dx.doi.org/10.1016/j.bmcl.2017.04.014 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

antibodies to block this pathway have demonstrated anti-tumor effects, which make it an appealing target for therapeutic intervention.11,12 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 FGF19FGFR4 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 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

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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 100fold selectivity over FGFR1 was achieved (Fig. 1). Subsequent optimization involved replacement of tertiary acrylamide with ophenylenediamine 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, Fig. 1). 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. A pan-FGFR inhibitor that drew our attention was ASP587835,36 (Fig. 2a). It possesses a monocyclic hinge binding motif with a twoatom chain linked with a substituted phenyl group. Modeling results against FGFR4 suggest that binding mode of ASP5878 is very similar to BLU9931 (Fig. 2b) and Ex. 108 (Fig. 2c). They form two hydrogen bonds with the same hinge residues in common. 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 the BLU9931 case, the quinazoline ring itself serves as a rigid linker pointing the phenyl group in the right direction, whereas the flexible methlyene-ether linker of ASP5878 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 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. Based on the structural analysis, we hypothesized that adding the acrylamide moiety to the core of ASP5878 would result in a

O

O

O Cl

N HN

O N

N O

Et

N

Et

N

O

NH

O N

N

N

NH

O Et

PD173034

N

N

NH NH

O Cl

N

HN

H N O

Et

Blueprint Ex. 52

BLU9931 O Cl N

N H N

HN

N

N

O O

Cl

O Celgene Ex.2 O Et

O

Cl

N N

N

N

N H BGJ-398

Et

HN N

O O

Cl

N N

N

Cl O

NH

N H

N

HN N

O O

Cl

Eisai Ex.108

Fig. 1. Strategy to develop FGFR4 selective inhibitors via introduction of Michael acceptors on pan-FGFR inhibitors.

a

O F HO

N N

N N N H ASP5878

O

O F

Fig. 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.

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 lM 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. 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 lg/mL versus <0.5 lg/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. 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 h, 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,

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Table 1 Activity and selectivity of covalent inhibitors based on flexible cores.

R3 O

N H N

HN

O

N R1

R2

R3

O

O

R1

Compound No.

1 2 3 4 5 BLU9931

R2

Me Me Me Me H –

R3

H H H Me H –

Table 2 Physicochemical properties, permeability and CYP inhibition profile of compound 1 and BLU9931. Properties

1

BLU9931

Molecular weight (Da) cLogP Lipophilic ligand efficiencya Kinetic solubility pH = 7.4 (lg/mL) LogD 7.4 (octanol/buffer) Papp A to B (nm/s), (efflux ratio) CYP (IC50, mM) 1A2, 2C9, 2C19, 2D6, 3A4

456.4 4.6 3.9 39 3.0 86, (1.0) >50, 3.7, 1.5, 35.0, 6.9

509.4 6.3 1 <0.5 >4.5 3.5, (1.5) 15.9, 0.9, 1.7, 12.5, 3.0

a Lipophilic ligand efficiency (LLE) was calculated by substraction of cLogP from pIC50.

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 h. 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 h.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

IC50 (nM)

F Cl H Cl F –

FGFR4

FGFR1

3 4 188 >10,000 8 49

8600 4340 >10,000 >10,000 60 2700

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. To explore the electronic and steric effects of the phenylacrylamide on plasma stability, several analogues were evaluated (Fig. 3). 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 (Fig. 3). It was quite satisfying to find that all these analogues (compound 9–11) were very stable after incubation for 2 h 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. In addition to FGFR1, compound 1 and ( )-11 were profiled against a small panel of kinases (including VEGFR2, EGFR, BTK, FMS, ITK, JAK3, and TEC) together with BLU9931 to evaluate the

Table 3 Activity, selectivity, plasma and microsomal metabolic stability of analogues around compound 1. Compound No.

IC50

Plasma metabolic stability Human

BLU9931 1 2 6 7 8 9 10 (+)-11 ( )-11

FGFR4

FGFR1

%left at 2 h

49 3 4 4 6 27 148 1200 3500 13

2700 8600 4340 2900 9500 4800 >10,000 3300 7400 3500

71 48 N/A N/A N/A N/A 90 96 N/A 92

Clint(liver) (mL/min/kg) Mouse H/M 63 13 0.2 0.1 0.3 0.4 100 96 N/A 97

41/97 165/9643 N/A 120/180 265/220 N/A 46/564 154/2032 N/A 18/474

Y. Wang et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2420–2423 O

O

F O

N H N

HN

O

F

H N

O

O

N

O F

N

HN

F

H N

O N

N

N 6

O

8 O

O

F

O F

H N

O

O

N

O

N

O F

N

HN

(±)-10 O

O

O

F

O

N N

A. Supplementary data

O

F

H N

particular flexible scaffold including the racemate of 11.45 However no detailed profiling of this particular compound as well as others was disclosed. 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.

O 9

HN

O F

N 7

F

HN

N

N

O

H N

HN

O

N

N

O

N

O F

N

2423

O

O

N

F

H N O

(+)-11

HN O

N

O F

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.04. 014.

(-)-11

Fig. 3. Analogues based on o-phenylenediamine and saturated cyclic diamines.

Table 4 Physicochemical properties and in vitro ADME profile of compound ( )-11. Properties

( )-11

Molecular weight (Da) cLogP Lipophilic ligand efficiency Kinetic solubility pH = 7.4 (lg/mL) LogD 7.4 (octanol/buffer) Papp A to B (nm/s), (efflux ratio) PPB (Unbound %, H, M) CYP (IC50, mM) 1A2, 2C9, 2C19, 2D6, 3A4

436.4 2.5 5.4 78 1.8 73, (3.8) 35.4/30.9 >50, 29.1, 19.8, >50, 13.3

influence of the flexible linker on the selectivity profile (Table S1 in Supporting Information). We are particularly interested in these kinases because they all belong to tyrosine kinase family and bear accessible cysteines in the active site.24 It turned out that BLU9931 is only a moderate inhibitor of FMS, which is in consistence with the literature report.18 Either compound 1 or ( )-11 showed less than 20% inhibitory activity at 10 lM against the kinases tested. At this point it was quite satisfied to observe this level of selectivity. A more comprehensive evaluation against a larger panel would be performed in due course. Although less potent than compound 1, LLE of ( )-11 was actually increased by 1.5 log unit. The impact of decreasing CLogP was further reflected by improved solubility and decreased LogD summarized in Table 4. Besides, ( )-11 was found to have minimal inhibition against the major CYP isoforms. Permeability was high with slight efflux. Plasma protein binding of ( )-11 was reduced to around 30% unbound. The in vitro profile of ( )-11 was quite promising so that it was subjected to in vivo PK characterization. After IV dosing at 0.2 mg/kg in mice, the compound was found to have a relatively small volume of 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. Around the same time we were preparing this manuscript, a patent from Eisai was published covering some efforts on this

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40. 41. 42. 43. 44. 45.

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