Discovery of the Irreversible Covalent FGFR Inhibitor 8-(3-(4

Drug Annotation pubs.acs.org/jmc

Discovery of the Irreversible Covalent FGFR Inhibitor 8‑(3-(4Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)2-(methylamino)pyrido[2,3‑d]pyrimidin-7(8H)‑one (PRN1371) for the Treatment of Solid Tumors Ken A. Brameld,* Timothy D. Owens, Erik Verner,† Eleni Venetsanakos, J. Michael Bradshaw, Vernon T. Phan,‡ Danny Tam,§ Kwan Leung,∥ Jin Shu, Jacob LaStant, David G. Loughhead, Tony Ton, Dane E. Karr, Mary E. Gerritsen, David M. Goldstein, and Jens Oliver Funk Principia Biopharma, Inc., 400 East Jamie Court, South San Francisco, California 94080, United States S Supporting Information *

ABSTRACT: Aberrant signaling of the FGF/FGFR pathway occurs frequently in cancers and is an oncogenic driver in many solid tumors. Clinical validation of FGFR as a therapeutic target has been demonstrated in bladder, liver, lung, breast, and gastric cancers. Our goal was to develop an irreversible covalent inhibitor of FGFR1−4 for use in oncology indications. An irreversible covalent binding mechanism imparts many desirable pharmacological benefits including high potency, selectivity, and prolonged target inhibition. Herein we report the structure-based design, medicinal chemistry optimization, and unique ADME assays of our irreversible covalent drug discovery program which culminated in the discovery of compound 34 (PRN1371), a highly selective and potent FGFR1−4 inhibitor.

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INTRODUCTION Next generation sequencing of a broad array of solid tumors identified aberrant signaling of fibroblast growth factor receptor (FGFR) 1, 2, 3, and 4 in 7% of cancers, implicating the pathway in oncogenesis and as a potential point of therapeutic intervention.1 FGFR signaling is initiated by binding of an extracellular FGF ligand which leads to receptor dimerization and cross-phosphorylation of the tyrosine kinase domains. Upon activation, the phosphorylated kinase domains proceed to phosphorylate intracellular substrates such as PLCγ, Gab1, FRS2, and STAT1. Subsequent downstream signaling is complex and includes activation of the PI3K-Akt and the Ras/Raf/Mek/Erk pathways. In normal cells, FGFR signaling is important for diverse functions, some of which are cell differentiation, wound healing, and tissue homeostasis.2 A wide range of oncogenic alterations involving activation of the FGFR pathway have been identified including ligand or receptor amplification or overexpression, receptor mutations, and receptor translocations/fusion.3 Small molecule FGFR tyrosine kinase inhibitors (TKIs) have shown clinical activity in the treatment of a variety of solid tumors such as metastatic urothelial,4 squamous cell lung,5 intrahepatic cholangiocarcinoma,6 and breast cancers.5 First generation FGFR TKIs are not selective for the FGFR family, often inhibiting a broad range of additional kinases, commonly VEGFR1−3, PDGFR-α, and PDGFR-β. Several of these agents, such as dovitinib7 and lucitanib,8 are now in advanced clinical trials and have dose-limiting toxicities largely attributed to VEGFR inhibition. Next generation FGFR TKIs with improved © 2017 American Chemical Society

selectivity are in early stage clinical trials (Chart 1), and promising clinical responses have been reported for 1 Chart 1. Chemical Structures of Second-Generation Clinical FGFR Inhibitors

(AZD4547)9 and 2 (infigratinib).10 While not entirely selective for FGFR1−3 and of moderate potency, 1 and 2 do not significantly inhibit VEGFR activity, thereby further validating FGFR inhibition alone as therapeutic modality. Single agent treatment with 2 provided objective response rates (ORR) of 22% and 36% in cholangiocarcinoma harboring FGFR2 translocations6 and in metastatic urothelial cancer harboring Received: March 7, 2017 Published: June 30, 2017 6516

DOI: 10.1021/acs.jmedchem.7b00360 J. Med. Chem. 2017, 60, 6516−6527

Journal of Medicinal Chemistry

Drug Annotation

FGFR3 mutations or translocations,4a respectively. Small molecule inhibitors that bind their target irreversibly through covalent bond formation are showing clinical benefits over their reversible comparators, particularly for oncology indications. First generation reversible inhibitors such as bortezomib,11 targeting the proteasome, or erlotinib,12 an EGFR TKI, are being displaced by irreversible inhibitors carfilzomib13 and afatinib,14 respectively, due to superior efficacy and relative safety profiles. An irreversible binding mechanism is especially well suited for TKIs that are required to compete with high intracellular ATP concentrations while maintaining selectively among the 500+ kinase family members.15 Engaging a nonconserved cysteine residue for covalent binding within the small molecule binding site can impart substantial selectivity benefits over a reversible noncovalent inhibitor. The resulting sustained inhibition profile of an irreversible inhibitor requires new protein synthesis to restore pathway activation. Depending upon the rate of target protein turnover, it is possible to discover drugs that maintain a prolonged high level of target inhibition yet have rapid systemic clearance and a low body burden. Such a profile minimizes any reversible off-target mediated side effects and is ideal for use in combination therapies which are common in oncology. We set out to discover highly selective irreversible inhibitors of FGFR1−4 as a targeted therapy for the treatment of solid tumors having FGFR alterations. The heterogeneity of FGFR isoforms and types of pathway alterations reported in a wide range of tumor types suggested that an inhibitor of all FGFR isoforms would provide the broadest therapeutic utility. For example, FGFR1/2 alterations are found in squamous nonsmall-cell lung cancer, FGFR2 alterations are found in gastric and cholangiocarcinoma, FGFR3 alterations are common in urothelial cancer, and FGFR4 alterations are found in hepatocellular carcinoma.3 Targeting the kinase activity of these FGFR isoforms ensures pathway inhibition regardless of the mechanism of activation which may be due to ligand or receptor amplification or overexpression, receptor mutations, and receptor translocations/fusion. We sought compounds with high cellular potency capable of achieving complete target inhibition at low exposures followed by rapid systemic clearance. Herein we describe the design, structure−activity relationship (SAR), pharmacokinetics, and in vivo efficacy of a series of irreversible covalent FGFR1−4 inhibitors culminating in the selection of compound 34 (PRN1371) for advancement into clinical trials.

Figure 1. Structural assessment of cysteine residues for covalent binding to FGFR1−4. (A) Several cysteine residues are proximal to the ATP binding pocket as illustrated by the spheres superimposed on the 2FGI X-ray structure. Listed for each highlighted cysteine sequence/structure location are the other kinases which share a cysteine at that position. (B) Structural alignment and overlay of three X-ray crystal structures (PDB codes 2FGI, 3GQL, 3C4F) of FGFR1 with different ligands occupying the ATP binding site. The highlighted cysteine residue (Cys488 in FGFR1) targeted for covalent binding by the compounds described in this work is located in the flexible glycinerich loop and adopts a different conformation in each structure.

closely related off-targets, VEGFR2, PDGFRα, or PDGFRβ, for which high selectivity is desired. Using multiple published X-ray crystal structures of reversible noncovalent small molecule ligands bound to FGFR1 (Protein Data Bank (PDB) codes 2FGI, 3GQL, 3C4F), it was apparent that Cys488 resides in a flexible region of the glycine-rich loop (Figure 1b). Structure-based ligand design suggested the need to sample a range of linker lengths and trajectories spanning the region between the core molecular scaffold that recognizes the ATP binding pocket and the electrophile destined to covalently bond to Cys488. At the onset of the program, a number of diverse scaffolds were evaluated, and upon the basis of the available vectors toward Cys488, we quickly focused upon a series of 6-phenylpyridopyrimidines 3 and 417 first reported by researchers at Parke-Davis (Chart 2). SAR from this work established a preference for 2,6-dichloro or 3,5-dimethoxy substitutions at the 6-phenyl region to impart selectivity and potency. Inspection of the X-ray crystal structure of 4 bound to FGFR1 (PDB code 2FGI)18 revealed that the t-Bu-urea at the C-7 position could be used to approach the cysteine, but taking into consideration properties of druglikeness, we wanted to avoid a biarylurea functionality in the lead. In a subsequent publication from Parke-Davis, isosteric 6-phenylpyridopyrimidinones such as 519 were reported to maintain FGFR activity. The N-8 position of the pyridopyrimidinone provided

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RESULTS AND DISCUSSION Starting with a focus on the design of compounds that covalently bind to cysteine residues, our lead generation strategy entailed identifying a suitable cysteine residue within the ATP binding pocket and applying structure-based design to generate small focused libraries of possible lead molecules. Within 8 Å of the ATP binding pocket of FGFR, there are three possible cysteine residues to target for covalent binding (Figure 1A).16 One of these cysteine residues is only found in FGFR4 and was not pursued further due to the desire to discover an FGFR1−4 inhibitor. A second cysteine residue that would require a DFG-out ligand binding conformation is present in FGFR1−4; however it is also conserved in 16 additional kinases, presenting a possible selectivity challenge. On this basis, we focused upon covalent binding to Cys488 (FGFR1 residue numbering) which is conserved in only six additional kinases. Importantly, this cysteine is not present in any of the 6517



Drug Annotation

The p-phenethyl linker of compound 10 imparted reasonable potency of 38 nM against FGFR1 while maintaining a desirable >25-fold selectivity window over VEGFR2. Inhibition profiling at 1 μM 10 against a subset of kinases (FGR, c-Src, TNK1, and YES) with a conserved cysteine residue in the same location as Cys488 of FGFR1 identified none with >85% inhibition. In contrast, the original lead 5 was reported to be >10-fold more potent against c-Src than FGFR in a cellular assay.19 With this selectivity and potency profile, compound 10 was considered to be a viable lead and further optimization was initiated. Several assays were used to evaluate the SAR and guide lead optimization. In addition to traditional biochemical and cellular potency measurements, we relied extensively on an FGFR1 biochemical off-rate assay which has been described previously.21 Briefly, preincubation of inhibitor with FGFR1 is followed by dilution and addition of a fluorescent competitive tracer. Time-dependent binding by the fluorescent tracer to FGFR1 is detected using time-resolved fluorescence resonance energy transfer (TR-FRET) and can only occur upon dissociation of the preincubated inhibitor. Our goal was to discover inhibitors that would rapidly and completely form an irreversible covalent bond upon binding to the receptor. Inhibitors with such a profile have durable and high target occupancy (e.g., >90%) at all time points of the experiment (15 min to 24 h). Tight binding inhibitors that do not form a covalent bond or only partially bond to a receptor have low target occupancy at all time points or a reduction in target occupancy over time. This assay does not replace a traditional kinetics analysis measuring kinact/Ki which defines the second order rate constant for covalent binding to a target protein.22 While measurements of kinact and Ki are of great utility, they are generally resource intensive, although a high throughput method was recently disclosed.23 For the purposes of guiding our SAR optimization, the occupancy assay was adapted to a moderate throughput format and provided invaluable kinetics data. The cellular activity of compounds was routinely evaluated in two assays. Pathway inhibition in human umbilical vein endothelial cells (HUVEC) was determined by measuring bFGF induced ERK phosphorylation using Alphascreen technology.24 Inhibition of cell proliferation was evaluated in the SNU16 gastric cancer cell line which overexpresses FGFR2. Two regions of the lead compound 10 were the focus of initial SAR studies. Modifications at the C-2 and N-8 positions were investigated while maintaining a 2-chloro-3,4-dimethoxy substitution pattern on the 6-phenyl substituent. Table 2 depicts the SAR observed when varying the linker between N-8 of the pyridopyrimidinone scaffold and the electrophilic acrylamide. The m-benzyl linker (11), which is reportedly optimal for the dihydropyrimidopyrimidinone scaffold (6), had only 65% FGFR1 occupancy at 15 min, indicative of incomplete covalent bond formation. Extending the linker by one carbon to yield the phenethyl analogs 12 and 16 improved the 15 min occupancy to 82% and 90%, respectively. Little difference in biochemical potency or occupancy was measured between the meta (12) and para (16) acrylamide regioisomers. However, the cellular potencies in both the HUVEC and SNU16 assays were 2- to 3-fold improved for the p-phenethyl linker (e.g., 16, SNU16 IC50 = 1.5 nM) when compared to the m-phenethyl analog (e.g., 12, SNU16 IC50 = 5.3 nM) perhaps indicative of a weak preference for the p-phenethyl linker. Introduction of nitrogen in the phenyl ring ortho or para to the acrylamide (e.g., 14, 15, 18, 19, and 20) had a pronounced

Chart 2. Chemical Structures of Reported FGFR Inhibitors and Irreversible Covalent Inhibitor Design Hypothesis

a good vector in the direction of Cys488 and was synthetically tractable for linker exploration. Furthermore, a covalent inhibitor of FGFR1−4 had recently been reported (6,20 Chart 2) for a dihydropyrimidopyrimidinone core scaffold. The design hypothesis used for initial lead generation is depicted in Chart 2. Within a small series of compounds that differed in the region linking the pyridopyrimidinone scaffold to the electrophile (e.g., 7−10, Table 1) a sharp SAR emerged. Table 1. FGFR1-4 and VEGFR2 Activity of Representative 8-Pyridopyrimidinone Derivatives

a Biochemical potency against FGFR1−4 and selected off-target VEGFR2. bEstimated from single concentration inhibition of 66% at 1 μM. cEstimated from single concentration inhibition of 58% at 1 μM.

6518



Drug Annotation

upon published SAR,19,26 a series of alkylamino side chains were introduced at the C-2 amino position (Table 3). The

Table 2. FGFR1 Biochemical Activity and Occupancy and Cellular Activity of Representative 8-Pyridopyrimidinone Derivatives

Table 3. FGFR1 Biochemical Activity, Cellular Activity, and hERG Potency of Representative 2-Amino Derivatives

a Biochemical potency against FGFR1. bFGFR1 biochemical % target occupancy as determined by competition with fluorescent tracer following washout at 15 min or 24 h. cInhibition of FGFb-mediated ERK phosphorylation in HUVEC primary cells. dInhibition of proliferation of SNU16 gastric cancer cell line overexpressing FGFR2.

a Biochemical potency against FGFR1. bFGFR1 biochemical % target occupancy as determined by competition with fluorescent tracer following washout at 15 min or 24 h. cInhibition of FGFb-mediated ERK phosphorylation in HUVEC primary cells. dInhibition of proliferation of SNU16 gastric cancer cell line overexpressing FGFR2. eIn vitro inhibition at 1 μM compound on the human ether-a-go-go-related gene (hERG) potassium channel current as measured using QPatch HT patch clamp system.

impact on the FGFR1 occupancy with sustained 100% occupancy observed at both the 15 min and 24 h time points for all the compounds. This improved FGFR1 occupancy occurred independent of the acrylamide regioisomer. Methylation of the acrylamide nitrogen reduced the biochemical potency and occupancy (e.g., 13 and 17). In retrospect, it is apparent that the biochemical occupancy data for compounds 12−20 reflect a combination of the geometric preferences of the linker region and the intrinsic chemical reactivity of the acrylamide functional group. Reaction rate measurements between glutathione (GSH) and a diverse set of acrylamides have illuminated a broad spectrum of intrinsic reactivity that is particularly sensitive to substitutions proximal to the acrylamide. In the study reported by Flanagan et al., pyridoacrylamide, the electrophilic functional group in 14, 15, 18, and 19, is most reactive and had the shortest measured half-life of 0.13 h, whereas the N-methylacrylamide in 13 and 17 had a much longer half-life of 3.5 h.25 We observed that the more reactive pyridoacrylamides suffered from poor whole blood stability (e.g., 18, mouse whole blood t1/2 = 51 min) and made the decision to focus exclusively upon a pphenethyl linker in subsequent SAR optimization (e.g., 16). Next we sought to improve upon the poor aqueous solubility of compound 16 (100% bioavailability (F) suggested good absorption and partial saturation of clearance mechanisms at the 20 mg/kg dose. Unique to the rat, there is a large difference in half-life between the iv (t1/2 = 0.8 h) and po (t1/2 = 3.8 h) routes of administration, also indicative of possible saturation of a clearance mechanism upon oral dosing. In the dog, the same methylcellulose suspension formulation used for the rat gave low oral absorption and bioavailability (F < 15%). We hypothesized that the less acidic gastric and intestinal pH of the dog may be contributing to low absorption of the free base of compound 34.31 Coadministration of a molar equivalent of citric acid improved the oral absorption of a 10 mg/kg dose (e.g., Cmax = 1103 ng/mL, AUC = 1134 ng·h/mL, F = 94%) and brought it in line with the rat PK. Exceptionally low oral exposure in the monkey (e.g., Cmax = 96 ng/mL, AUC = 84 ng·

Table 5. Comparison between Electrophile Reactivity toward β-Mercaptoethanol and Selected ADME Properties

a Chemical reactivity assay measuring apparent Kd of compounds toward β-mercaptoethanol (BME) after 2 h incubation. bPercent compound remaining after 2 h incubation with human plasma or human whole blood. cMetabolic stability in pooled human liver S9 fraction with (w/) and without (w/o) NADPH cofactor.

Compound 34 presents a unique profile of high biochemical and cellular potency (FGFR1 IC50 = 0.6 nM, SNU16 IC50 = 2.6 nM), prolonged target engagement (FGFR1 occupancy 24 h = 96%), < 30% hERG inhibition at 1 μM, and good predicted ADME stability with BME reactivity Kd > 100 μM. A rat iv (2 mg/kg) PK study of compound 34 showed rapid clearance (Cl = 160 mL min −1 kg −1 ), yet dosing po (20 mg/kg) demonstrated high oral exposure (AUC = 4348 h·ng/mL) and a reasonable half-life (t1/2 = 3.8 h). Broader kinome-wide biochemical profiling of 34 against 251 kinases identified only FGFR1−4 and CSF1R as being potently inhibited (e.g., IC50 < 20 nM) (Table 6 and Supporting Information Table S1). The ATP binding site of CSF1R does not have a proximal cysteine residue, and compound 34 binds noncovalently, as determined by recovery of kinase activity upon dialysis. Consistent with reversible binding, there is a large shift between the biochemical potency (8.1 nM IC50) and the cellular potency of CSF1R Table 6. Kinase Selectivity Profile of Compound 34 kinasea

IC50 ± Std Dev (nM)b

kinasea

IC50 ± Std Dev (nM)b

FGFR1 FGFR2 FGFR3 FGFR4 CSF1R BMX BLK

0.6 ± 0.1 (n = 5) 1.3 ± 0.2 (n = 5) 4.1 ± 0.7 (n = 5) 19.3 ± 4.7 (n = 5) 8.1 ± 0.6 (n = 4) 272 ± 7 (n = 2) 371 ± 12 (n = 2)

FLT-4 JAK3 LYNB LYNA KDR PERK TAOK2

410 ± 9 (n = 2) 556 ± 6 (n = 2) 631 ± 1 (n = 2) 639 ± 5 (n = 2) 705 ± 63 (n = 5) 792 ± 50 (n = 2) 1175 ± 43 (n = 2)

a

Potency of biochemical inhibition by compound 34 of kinases that demonstrated >50% inhibition at 1 μM from a panel of 251 different kinases (see Supporting Information for inhibition data for full kinase panel). bIC50 values for kinase inhibition as determined using the Caliper electrophoresis method. Uncertainties are the standard deviation (Std Dev) for multiple independent determinations (n). 6521



Drug Annotation

Scheme 1. Chemical Synthesis of Compound 34

Several in vivo studies of compound 34 were conducted to understand the relationship between PK and FGFR pathway inhibition. Exploratory mouse studies identified the chemokine CCL2 as elevated following tail vein injection of bFGF. Specifically, upon stimulation with 10 μg of bFGF, it was found that CCL2 increased from a basal level of 301 ± 101 pg/mL to a level of 1487 ± 537 pg/mL at 4 h poststimulation. We evaluated the percent inhibition of bFGF-induced CCL2 at 1, 5, and 12 h after oral administration of a 10 mg/kg dose of compound 34 in rats. The levels of compound 34 in circulation were measured at the same time points to assess whether CCL2 inhibition was driven by long-lasting binding of compound 34 to FGFR or by compound in circulation. The plasma concentration of compound 34 peaked at 538 ± 88 ng/mL 1 h after dosing and fell to a nearly undetectable level after 12 h (Figure 2b). Strong inhibition of CCL2 was sustained from 99.5% ± 3.8% at 1 h to 93.5% ± 4.7% 12 h after dosing, suggesting long-lasting FGFR target inhibition by compound 34. The effect of noncovalent FGFR inhibitor 2 on CCL2 inhibition was also investigated. In contrast to compound 34, the level of CCL2 inhibition significantly declined from 98.2%

Table 7. Kinetics Values of Compound 34 Binding to FGFR1-4 target

kinact (s−1)

Ki (nM)

kinact/Ki (μM−1 s−1)

FGFR1 FGFR2 FGFR3 FGFR4

0.0019 ± 0.0015 0.00092 ± 0.00016 0.00097 ± 0.00015 0.00066 ± 0.00007

1.6 ± 1.1 1.3 ± 0.6 2.2 ± 0.5 73 ± 22

1.2 ± 0.1 0.75 ± 0.17 0.46 ± 0.06 0.010 ± 0.003

h/mL) was initially a cause for concern. We were able to attribute this to intestinal Cyp3A4 mediated metabolism. It has been reported that the bioavailability in monkey of compounds that undergo intestinal metabolism is much lower than that of rat or human.32 For neratinb and ibrutinib, two covalent kinase inhibitors with acrylamide Michael acceptors that undergo extensive Cyp3A4 mediated metabolism, monkey PK grossly overestimates clearance and underestimates absorption, making monkey an inappropriate species for the prediction of human absorption.29 On this basis, we were comfortable with the preclinical PK of compound 34 as it predicted our desired profile of high oral absorption followed by rapid clearance. Table 8. Pharmacokinetic Parameters of Compound 34 rata

dogb

monkeyc

parameter

iv

po

iv

po

iv

po

Cl (mL min−1 kg−1) Vss (L/kg) t1/2 (h) Cmax (ng/mL) AUC (ng·h/mL) F (%)

160 7.20 0.764 596 208 NA

NA NA 3.77 1785 4348 208

117 2.90 0.33 1087 284 NA

NA NA 0.58 1103 1344 94

42.9 1.14 0.349 999 472 NA

NA NA 0.486 95.8 84.4 2.3

a

Female Sprague-Dawley rats dosed at 2 mg/kg iv and 20 mg/kg po. bMale beagle dogs dosed at 2 mg/kg iv and 10 mg/kg po. cMale cynomolgus monkeys dosed at 1.23 mg/kg iv and 10 mg/kg po. 6522



Drug Annotation

Figure 2. Rat in vivo inhibition of bFGF-induced CCL2 production (black line, right y-axis) by a 10 mg/kg dose of compound 2 or 34 and associated compound plasma levels (gray line, left y-axis) at 1, 5, and 12 h postdose (n = 5 per group): (A) reversible compound 2; (B) irreversible covalent compound 34.

± 4.1% at 5 h to 59.5% ± 11.6% at 12 h for compound 2 (Figure 2A), indicating that the covalent binding mechanism of compound 34 to FGFR is likely responsible for the long lasting inhibition of CCL2. Compound 34 was evaluated in pharmacodynamics and efficacy studies using a SNU16 gastric cancer xenograft mouse model with high overexpression of FGFR2. In nude mice implanted with subcutaneous SNU16 tumors, pFGFR2 levels in the tumor were measured via Western blotting at 8 h following a 10 mg/kg oral dose (Figure 3A). Low levels of pFGFR2 confirmed the ability of compound 34 to block FGFR2 activity in tumor tissue. Efficacy was determined by measuring tumor growth inhibition in the same SNU16 xenograft model (Figure 3B). Compound 34 induced a dose-dependent reduction in tumor volume and up to 68% tumor growth inhibition at the highest dose of 10 mg/kg b.i.d. following 27 days of treatment. All doses were well tolerated with no significant body weight loss. With good in vivo efficacy and PK, compound 34 was progressed into preclinical safety evaluations including 28 day GLP toxicology studies in rats and dogs. The toxicological findings were consistent with those reported for other FGFR inhibitors, predominantly phosphorus dysregulation and concomitant soft tissue mineralization.33 Phosphorus homeostasis is dependent upon FGF23 signaling in the kidney. As a consequence, clinical on-target mediated effects of FGFR blockade include elevated serum FGF23, phosphate, and vitamin D.4b,34 The combination of a clean preclinical safety profile, favorable human PK projections, and efficacy in the xenograft models gave us confidence to advance compound 34 into human clinical trials. A phase 1 dose escalation study is being conducted in patients with advanced solid tumors and metastatic disease to assess pharmacokinetics, tolerability, and objective response

Figure 3. PK/PD and efficacy of compound 34 in a SNU16 mouse xenograft model. (A) Western blots showing blockade of pFGFR within the tumor 8 h postdose. Lanes 1−4 are individual mice dosed with vehicle, and lanes 5−8 are individual mice dosed with compound 34 at 10 mg/kg q.d. (B) Tumor growth inhibition (TGI) dose response upon oral administration of compound 34 (n = 10 per dose group).

rate, among other end points (ClinicalTrials.gov identifier NCT02608125). Compound 34 free base has been administered orally once daily as powder in a capsule on a 28-day continuous schedule. Human plasma concentrations for doses ranging from 15 to 35 mg (Figure 4A) confirm good oral exposure, rapid systemic clearance, no accumulation from day 1 to day 15, and a dose-dependent increase in AUC.35 Serum phosphate, a pharmacodynamic marker of FGFR inhibition, is increased for all doses studied and shows a dose-dependent increase between 20 and 35 mg, despite the administration of prophylactic phosphate binders (Figure 4B). Additional cohorts and dosing regimens are being explored in ongoing clinical studies and will be reported in due course.

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CONCLUSIONS Structure-based design and medicinal chemistry optimization that was focused on maximizing FGFR target occupancy led to the discovery of compound 34, an irreversible nanomolar inhibitor of FGFR1−4. Several specific assays were critical in guiding the lead optimization process including measures of FGFR1 occupancy, human plasma stability, and Michael acceptor chemical reactivity. Initial progress toward optimization of a potent lead molecule (compound 10) having a pphenethyl linker between N-8 of the pyridopyrimidinone scaffold and the electrophilic acrylamide was unsuccessful in identifying a clinical candidate primarily due to poor solubility 6523



Drug Annotation

Kd Determination Using β-Mercaptoethanol. Solutions were prepared containing 0, 1.5, 15, 150, and 1500 mM β-mercaptoethanol (BME) in a 1:1 mixture of ethanol and phosphate buffered saline (pH 7.4, PBS). Aliquots of a 10 mM DMSO stock solution of test compound (10 μL) were separately added to 90 μL aliquots of the above-described ethanol/PBS solutions containing 0−1500 mM BME. After these solutions had been allowed to stand at room temperature for 2 h, they were analyzed using an Agilent 1200 LCMS system equipped with a 50 mm × 2 mm Phenomenex Luna 5 μm C18 100A column. Samples were eluted using a gradient of acetonitrile and water, with both solvents containing 0.1% formic acid. Peaks corresponding to parent and BME adduct were identified by their masses, and the percent parent in each sample was determined by measuring the area under the curve for the extracted mass peaks from the positive ion trace corresponding to parent and BME adduct. Percent parent was plotted versus the log of the BME concentration using GraphPad Prism to determine an apparent Kd for the reaction. Kinase Assays. Enzyme inhibition was determined using a Caliper capillary electrophoresis system that separates phosphorylated and nonphosphorylated peptides on the basis of charge. Different concentrations of inhibitor were first preincubated with enzyme for 15 min. The reaction was initiated with addition of peptide substrate, ATP, and Mg2+ and incubated at 25 °C for 3 h. To stop the reaction, the mixture was quenched with EDTA. The buffer was 100 mM HEPES, pH 7.5, 0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl2, 10 mM sodium orthovanadate, 10 μM β-glycerophosphate, and 1% DMSO. The ATP concentration of the reaction was at the predetermined value of the Km for ATP. All enzyme inhibition data were acquired by Nanosyn, Inc. (Santa Clara, CA; www.nanosyn.com). Kinase selectivity was also evaluated using the Nanosyn 250 kinase panel. ERK Phosphorylation in HUVECs. Human umbilical vein endothelial cells (HUVECs) were incubated in media supplemented with 10% FBS and seeded at 30 000 cells per well in a 96-well plate overnight. HUVECs were then transferred into serum free media 1 h before compound treatment. A compound concentration series was added to cells and incubated for 1 h at 37 °C. Cells were then stimulated with either 50 ng/mL of FGF2 (R&D Systems catalog no. 233-FG-025) or 50 ng/mL of VEGF (R&D Systems catalog no. 293VE-050) for 10 min. Ice cold PBS was added to stop the reaction, and cells were washed three times to remove media. A pERK SureFire kit (PerkinElmer) was utilized to determine ERK phosphorylation using an Envision multilabel plate reader (PerkinElmer). FGFR1 Residence Time Using Fluorescence Competition. Using an assay buffer of 50 mM Hepes pH 7.5, 10 mM MgCl2, 0.01% Triton-X 100, and 1 mM EGTA, 1 μL of 15 μM compound was added to 9 μL of 0.5 μM FGFR1 (Invitrogen PV4105) in a 96-well polypropylene plate. Following 60 min of incubation, the mixture was diluted in assay buffer 100-fold. An amount of 10 μL of diluted mixture was transferred to a Greiner 384-well black plate. Europium-coupled Anti-6XHis Ab (PerkinElmer AD0205) and Cy5-labeled pyridopyrimidinone tracer were added to a final concentration of 15 nM and 0.75 μM, respectively, in 20 μL volume. Data were acquired using a PerkinElmer Envision plate reader (model 2101) containing LANCE TR-FRET compatible excitation and emission filters. Fluorescence at 665 nM and 615 nM wavelengths was collected at various times. In each experiment, a condition that provides the maximum signal (max) was acquired consisting of the signal from enzyme, europium-coupled Anti-6XHis Ab, and tracer in the absence of test compound. A background signal (bkg) was also acquired where a 1 μM concentration of PP-ir was added to completely block tracer binding. Data for each test compound were reported as % occupancy, which is calculated as 100 × (1 − (compd − bkg)/(max − bkg)). FGFR1 Progress Curve Analysis. Progress curves of FGFR1 peptide (5-FAM-KKKKEEIYFFF-NH2) phosphorylation were acquired at six concentrations. The real-time curves were obtained for a total of 5 h using the climate controlled Caliper LabChip instrument. The obtained curves were fit using XLfit4 software to the time dependent inhibition equation: [P] = Vst + ((Vi − Vs)/Kobs)(1 − exp(−Kobst)). In the equation, Vi is the initial velocity, Vs is the steady

Figure 4. Phase I clinical data for once daily oral administration of compound 34. (A) Total plasma concentration over time. (B) Percent change at day 8 of serum phosphate, a pharmacodynamic marker of FGFR inhibition; the asterisk (∗) indicates prophylactic administration of phosphate binders.

and a potential hERG liability. Subsequent incorporation of an aliphatic amine in place of the p-phenethyl linker led to compound 34 which maintained high FGFR1 occupancy with improved solubility and exceptional oral bioavailability. A low dose, high clearance, irreversible covalent drug has the potential for a large therapeutic window due to durable target inhibition that continues after the drug has cleared systemic circulation. Compound 34 has this ideal PK profile and, as a consequence of its irreversible binding mechanism, demonstrates extended FGFR inhibition in vivo after plasma concentrations are below the limits of quantitation. For irreversible inhibitors, the rate of new protein synthesis largely determines the duration of target inhibition. The protein half-life of FGFR3 has been reported to be approximately 4 h and is context dependent.36 PK/PD studies in preclinical xenograft models confirmed blockade of FGFR signaling and potent tumor growth inhibition. Initial cohorts of a phase 1 dose escalation trial support the predicted PK profile, were well tolerated, and warrant further clinical investigation. Expanded trials in a targeted patient population will be required to demonstrate the clinical benefits of an irreversible covalent binding mechanism for FGFR inhibition.

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EXPERIMENTAL SECTION

Cell Lines and Reagents. The gastric cancer cell line SNU16 was purchased from American Tissue Culture Collection (ATCC, Manassas, VA) and cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS, Gibco catalog no. 26140-079). HUVECs (Genlantis catalog no. PH20005N) were maintained in endothelial cell growth medium (Genlantis catalog no. PM211500) containing 10% FBS. Cells were maintained in standard culture conditions of 37 °C, 5% CO2, and 95% humidity and were kept in culture for up to 15− 20 passages. 6524



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state velocity, and Kobs reflects the rate of inactivation. For time dependent inhibitors, the obtained Kobs values were plotted against compound concentration using either a hyperbolic or a linear fit. From these plots, kinact and Ki were determined. Progress curve data were acquired by Nanosyn, Inc. (Santa Clara, CA; www.nanosyn.com). SNU16 Cell Proliferation. SNU16 cells were first seeded into 384well plates and compounds added such that the highest final compound concentration is 5 μM. Cells were incubated with compound for 72 h at 37 °C. To detect viability, the Presto-Blue cell viability reagent (Life Technologies catalog no. A13261) was added per manufacturer’s instructions. Plates were read using an Analyst HT with a fluorescent mode employing 530 nm excitation and 590 nm emission. Xenograft Mouse Studies. For xenograft studies with SNU16 cells, a suspension of 1 × 107 cells were injected at the upper right back of 7 week old female nude mice (Crown Bioscience, Taichang, P. R. China). The care and treatment of experimental animals were in accordance with institutional guidelines. Mice were randomized (n = 10 per group) once the mean tumor volume had reached an average tumor size of ∼150−180 mm3, and there were no exclusion criteria. Compound 34 was suspended in 0.5% methylcellulose w/w in deionized water. Tumor volumes were measured three times weekly using a caliper, and the volume was expressed in mm3 using the formula V = 0.5ab2 where a and b are the long and short diameters of the tumor, respectively. Tumor weight was measured at study termination. SNU16 tumor cell lysates were evaluated for pFGFR by SDS−PAGE and immunoblotting using a rabbit anti-pFGFR2 antibody (Cellular Signaling Technology catalog no. 3471S) and a mouse anti-FGFR2 antibody (R&D Systems catalog no. MAB6841). General Synthetic Methods. Synthetic methods for compounds 7−31 and 32−39 are described in patent applications WO201418282937 and WO2015120049,38 respectively. Unless otherwise noted, all materials were obtained from commercial suppliers and used as obtained. Anhydrous organic solvents were purchased from Aldrich packaged under N2 in Sure/Seal bottles and used directly. 1H NMR spectra were recorded on a Bruker AV-400 (400 MHz) spectrometer at ambient temperature. Chemical shifts are reported in parts per million (δ) and are calibrated using residual undeuterated solvent as an internal reference. Data for 1H NMR spectra are reported as follows: chemical shift (δ ppm) (multiplicity, coupling constant (Hz), integration). Multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, or combinations thereof. Purity was assayed by HPLC (XBRIDGE C18 column, 3.5 μm × 50 mm) with a gradient of 10−100% acetonitrile (containing 0.05% TFA) in 0.05% aqueous TFA with UV detection at λ= 210 and 254 nm. All final compounds were obtained with ≥95% purity. 8-(3-(4-Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin7(8H)-one, 34. 1H NMR (400 MHz, DMSO-d6): 8.62 (s, 1H), 7.88 (d, J = 4.5 Hz, 1H), 7.68 (s, 1H), 6.98 (S, 1H), 6.77 (dd, J = 10.5, 16.7 Hz, 1H), 6.08 (dd, J = 2.4, 16.7 Hz), 5.65 (dd, J = 2.4, 10.5 Hz, 1H), 4.41−4.25 (m, 2H), 3.96 (s, 6H), 3.49−3.43 (m, 4H), 2.92 (d, J = 4.9 Hz, 3H), 2.42- 2.25 (m, 6H), 1.90−1.78 (m, 2H). MS (ESI, pos ion) m/z: 561.1 (M+ 1). 13C NMR (100 MHz, CDCl3) 165.3, 162.2, 161.1, 158.8, 155.8, 154.6, 136.3, 136.0, 127.7, 127.5, 125.0, 115.3, 97.5, 56.6, 56.0, 55.9, 53.3, 52.7, 45.8, 41.9, 39.6, 39.5, 28.5, 24.9.

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Drug Annotation

AUTHOR INFORMATION

Corresponding Author

*Phone: 650-416-7700. E-mail: ken.brameld@principiabio. com. ORCID

Ken A. Brameld: 0000-0001-8643-3218 Present Addresses †

E. Verner: Corvus Pharmaceuticals, South San Francisco, California, U.S. ‡ V. T. Phan: Medivation, San Francisco, California, U.S. § D. Tam: Gilead Sciences, Foster City, California, U.S. ∥ K. Leung: Cytokinetics, South San Francisco, California, U.S. Notes

The authors declare no competing financial interest.

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DEDICATION This article is dedicated to the memory of our colleague JensOliver Funk. ABBREVIATIONS USED F, % bioavailability; ADME, absorption, distribution, metabolism, and excretion; ATP, adenosine triphosphate; AUC, area under the plasma−concentration time curve; b.i.d., two times a day; BQL, below quantitation limit; BME, β-mercaptoethanol; C0, concentration extrapolated to time zero following iv administration; CL, total body clearance; Cmax, maximal plasma concentration; CSF1R, colony stimulating factor 1 receptor; CYP, cytochrome P450; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; GAPDH, glyceraldehyde 3phosphate dehydrogenase; GSH, glutathione; hERG, human ether-a-go-go-related gene; HUVEC, human umbilical vein endothelial cell; IC50, inhibitory concentration 50% (concentration causing half-maximal inhibition); iv, intravenous administration; KDR, kinase insert domain receptor, also known as VEGFR2; Ki, inhibition constant; kinact, maximal inactivation; LYN, Lck-Yes-related novel protein tyrosine kinase; ORR, objective response rate; PK, pharmacokinetics; po, oral administration; RTK, receptor tyrosine kinase; t1/2, elimination half-life; TGI, tumor growth inhibition; TKI, tyrosine kinase inhibitor; Tmax, time to maximum concentration; TR-FRET, fluorescence resonance energy transfer; Vd, volume of distribution; VEGFR, vascular endothelial growth factor receptor; Vss, volume of distribution at steady state

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00360. Table S1 showing biochemical kinase inhibition profiling of compound 34 against 251 kinases (PDF) Molecular formula strings with FGFR1 enzyme and cellular inhibition data for compounds 7−39 (CSV) 6525



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Discovery of the Irreversible Covalent FGFR Inhibitor 8-(3-(4

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