Discovery of Potent and Selective 8-Fluorotriazolopyridine c-Met

Feb 20, 2015 - ... MatsudaMizuki WatanabeSatoshi Shuto. The Journal of Organic Chemistry 2018 83 (15), 7672-7682. Abstract | Full Text HTML | PDF | PD...
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Discovery of Potent and Selective 8‑Fluorotriazolopyridine c‑Met Inhibitors Emily A. Peterson,*,† Yohannes Teffera,† Brian K. Albrecht,† David Bauer,† Steven F. Bellon,† Alessandro Boezio,† Christiane Boezio,† Martin A. Broome,‡ Deborah Choquette,† Katrina W. Copeland,† Isabelle Dussault,‡ Richard Lewis,† Min-Hwa Jasmine Lin,† Julia Lohman,† Jingzhou Liu,† Michele Potashman,† Karen Rex,‡ Roman Shimanovich,† Douglas A. Whittington,† Karina R. Vaida,† and Jean-Christophe Harmange† †

Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States

‡

S Supporting Information *

ABSTRACT: The overexpression of c-Met and/or hepatocyte growth factor (HGF), the amplification of the MET gene, and mutations in the c-Met kinase domain can activate signaling pathways that contribute to cancer progression by enabling tumor cell proliferation, survival, invasion, and metastasis. Herein, we report the discovery of 8-fluorotriazolopyridines as inhibitors of c-Met activity. Optimization of the 8-fluorotriazolopyridine scaffold through the combination of structure-based drug design, SAR studies, and metabolite identification provided potent (cellular IC50 < 10 nM), selective inhibitors of c-Met with desirable pharmacokinetic properties that demonstrate potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.

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INTRODUCTION The regulation of intracellular signal transduction pathways is a key function of receptor tyrosine kinases (RTKs), and thus, inappropriate activation of RTKs can lead to deleterious consequences with regard to downstream signaling. As such, the receptor tyrosine kinase c-Met has been implicated in the progression of a variety of human cancers.1 In normal cells, activation of c-Met occurs through the extracellular binding of its natural ligand, hepatocyte growth factor/scatter factor (HGF/SF). It has been shown that a variety of human cancers contain aberrant HGF/SF or c-Met expression or activating cMet kinase mutations.2 Consequently, inhibition of c-Met activity is a potentially impactful approach to the treatment of cancers where c-Met is activated.3 An ATP-competitive, small-molecule inhibitor of c-Met has the potential to inhibit both ligand-dependent and ligandindependent tumors that are driven by c-Met. As a result, there has been significant interest in the discovery of small molecule c-Met inhibitors for the treatment of cancer.4 We previously reported the synthesis and activity of c-Met inhibitors 1a, which demonstrated nanomolar inhibition of c-Met phosphorylation and exquisite selectivity over other kinases.5 Concurrent with the design and testing of triazolopyridazine inhibitors such as 1a, a modification of the central ring system was investigated in which the triazolopyridazine was replaced by a triazolopyridine (Table 1).6 The removal of the N5 pyridazine nitrogen (1a → 1b) led to a measurable loss in potency; however, activity could be restored by the installation of an 8-fluoro substituent on the © XXXX American Chemical Society

triazolopyridine ring (1c, Table 1). It was later discovered that compounds of class 1a demonstrated time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4) and that the oxygen linker bridging the methoxyquinoline and imidazopyridine rings was a site of metabolic O-dealkylation.7 Considering these liabilities, we decided to investigate c-Met inhibitors bearing a more robust carbon linkage, such as previously reported phenol 2.5 Replacing the phenol with a quinoline bioisostere provided the more potent inhibitor 3a. As with inhibitor 1c, incorporation of the 8-fluorine substituent to give 8-fluorotriazolopyridine methylquinoline 3b improved the potency. Finally, installation of two additional fluorine atoms on the carbon bridging the 8-fluorotriazolopyridine and quinoline moieties gave inhibitor 4 (Table 1). This substitution was pursued to eliminate the potential for metabolic oxidation of the activated benzylic carbon while maintaining potency.8 Following this promising lead, structure−activity relationship (SAR) studies were initiated with the aim of further improving the potency on c-Met, establishing desirable pharmacokinetic properties and eliminating the inhibition of CYP3A4 observed with the previously reported scaffold 1. The three structural elements that were investigated starting from 8-fluorotriazolopyridine 4 were the C6 aryl group, the hinge-binder moiety, and the substitution of the carbon atom bridging the hingebinder and the triazolopyridine core.9 Herein we describe the Received: December 10, 2014

A

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Journal of Medicinal Chemistry Table 1. Modification of the Triazolopyridazine Scaffold

a

Compound

X

R

c-Met IC50 (nM)a

1a 1b 1c 2 3a 3b 4

N CH CH

H H F

9 31 7 2100 210 60 23

H F

Inhibition of c-Met kinase, HTRF, n ≥ 2.

Scheme 1. Synthesis of Inhibitor 4a

Reagents and conditions: (i) X-Phos, Pd(OAc)2, K3PO4, PhB(OH)2, dioxane, 100 °C, 18 h, 94%; (ii) NH2NH2, i-PrOH, 70 °C, 86%; (iii) Pd(PPh3)4, (2-tert-butoxy-2-oxoethyl)zinc(II) bromide, THF, 75 °C, 18 h, 55%; (iv) LiHMDS, (PhSO4)2NF, −78 °C → rt, 4 h, 47%; (v) (a) trifluoroacetic acid, CH2Cl2, rt, 18 h, 93%; (b) thionyl chloride, DMF, 0 °C, 1 h, then 6, i-Pr2EtN, DMAP, 18 h, 87% from 8; (vi) Cl3CCN, PS-PPh3, DCE, 100 °C, microwave, 10%. a

core gave improved potency, presumably due to an optimal configuration for π-stacking with Tyr1230 (Figure 1, vide infra).5 The replacement of the nitrogen at C5 with CH (1a → 1b) potentially introduces a steric clash that may result in a deviation from the desired planarity. Reasoning that inhibitors such as 4 would be subject to the same interaction, installation of a 2-pyridyl group at the C6 position gave 10a, which demonstrated an IC50 = 12 nM for c-Met kinase inhibition (Table 2). Further enhancement in potency could be obtained by the installation of five-membered ring heterocycles at the C6 position; crystallography revealed that heterocycles such as the isoxazole in 10b could participate in an advantageous water bridge in the kinase active site (Figure 1). The heterocycles exhibiting the best cellular potency were thiazole 10d, isoxazole 10b, and methylpyrazole 10e (Table 2). The next modification investigated in the difluoromethylquinoline series (10) was substitution of the hinge-binder quinoline moiety, which engages hinge-residue Met1160 through a hydrogen bond. Several alternative hinge-binder groups were investigated using the methylpyrazole at the C6 position. To provide for more rapid access to these analogs, the inhibitors in Table 3 were prepared without the fluorine atoms on the bridging carbon using the more synthetically viable

design, synthesis, and pharmacodynamic activity of 8fluorotriazolopyridine c-Met kinase inhibitors such as 4.

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RESULTS AND DISCUSSION The synthesis of 4 (Scheme 1) began with Suzuki (or Stille)10 coupling of phenylboronic acid and 5-chloro-2,3-difluoropyridine (5) followed by SNAr substitution with hydrazine to afford phenylpyridine 6. The difluoroquinoline ester 8 was prepared by Negishi coupling of 6-bromoquinoline and the zinc enolate of tert-butyl acetate followed by bis-fluorination. The union of hydrazine 6 with the acid chloride derived from tertbutyl ester 8 provided hydrazide 9. Dehydrative cyclization of hydrazide 9 could be achieved by microwave heating with trichloroacetonitrile and solid supported triphenylphosphine to provide inhibitor 4.11 By use of the synthesis described in Scheme 1, the effect of various aryl groups at the C6 position was investigated with the guidance of protein structure information. Analysis of the cocrystal of 1a with the c-Met kinase domain revealed that coplanarity of the C6 phenyl group with the triazolopyridazine ring was important for potency. As previously described for inhibitors 1a, it was observed that substituents at the C6 position that could achieve coplanarity with the triazolopyridine B

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Journal of Medicinal Chemistry

hydrogen-bonding basicity, weakening the key hinge-binding interaction with Met1160.12 Combination of the C6 substituents from the most potent inhibitors in Table 2 with the methoxyquinoline hinge-binder component provided compounds 12a and 12b, which demonstrated single-digit nanomolar inhibition of HGFmediated c-Met phosphorylation in PC3 cells (Table 4). These inhibitors, along with the most potent inhibitors from Table 2, were further evaluated in vitro in liver microsomes and in pharmacokinetic studies conducted in male Sprague−Dawley rats. It was found that the intrinsic clearance (CLint) obtained from rat liver microsomes (data not shown) agreed well with the observed clearance in rat and that compounds 12 demonstrated lower turnover than compounds 10 in both rat and human liver microsomes. Furthermore, compounds 10 and 12 were not potent inhibitors of cytochrome P450 enzymes CYP2D6 and CYP3A4, an improvement over previously reported scaffold 1. With the exception of 10d, the compounds in Table 4 had low clearance, small volume of distribution at steady state, reasonable oral half-life, and good oral bioavailability. Metabolic profiling revealed that the major metabolic fate of the compounds shown in Table 4 was glutathione S-transferase (GST) mediated displacement of the aromatic fluorine at the C8 position with glutathione (GSH) (Figure 2).13 Metabolism by GSH conjugation can prove problematic if a drug is given at high or repeated doses, because of potential GSH depletion leading to oxidative stress.14 In addition, if GST-mediated GSH conjugation is the predominant metabolic pathway for a compound, metabolic prediction studies could be complicated by the occurrence of varying GST isoforms within the human population.13 Considering these potential drawbacks, diminishing the extent of glutathione conjugation became the primary focus (see Figure 2 for GSH displacement studies). While removal of the C8 fluorine would eliminate this metabolic pathway, the presence of this fluorine was important for maintaining potency. It was hypothesized that the electrophilic nature of the 8-fluorotriazolopyridine could be attenuated by removal of inductively electron-withdrawing groups elsewhere in the molecule to potentially mitigate the extent of glutathione displacement. Therefore, initial efforts focused on systematically replacing the benzylic fluorines with methyl groups and/or hydrogens to give inhibitors 20−24 (Table 5). The synthesis of inhibitors 20 and 21 is described in Scheme 2; the synthesis of compounds 22−24 was performed in a similar manner from the appropriately functionalized quinoline carboxylic acids. A palladium-catalyzed acylation of the appropriately substituted quinoline bromide 13 provided quinoline ester 15, which could be fluorinated to give fluoromethylquinoline 16. The carboxylic acid 17, derived from hydrolysis of the tert-butyl ester 15 or 16, was coupled with the arylhydrazone 18 under standard amide coupling conditions to provide hydrazide 19. Subsequent dehydrative cyclization provided inhibitors 20 and 21.15 Compounds containing stereogenic centers (20, 21, and 24) could be obtained in >95% enantiomeric excess following chromatographic separation on chiral media.16 It was found that replacing one of the bridging fluorine atoms with a methyl group (20) was tolerated, and for several compounds the cellular IC50 was less than 10 nM (Table 5). The conformation of the bridging carbon stereocenter had a significant effect on potency, for example, (R)-20a demonstrated IC50 = 5 nM in the c-Met kinase inhibition assay

Table 2. Modification of the C6 Position

Inhibition of c-Met kinase activity, n ≥ 2. bInhibition of HGFmediated c-Met phosphorylation in PC3 cells, n ≥ 2.

a

Table 3. Modification of the Hinge-Binder

a

Inhibition of c-Met kinase, HTRF, n ≥ 2.

methylpyrazole at the C6 position. Substitution meta to the quinoline nitrogen atom was investigated, where substituents could project into the solvent front without interfering with the key hydrogen bonding interaction of the quinoline with Met1160. Installation of a methoxy group on the quinoline maintained the potency observed for parent 11a, whereas a hydroxyl group was detrimental. Other modifications, such as quinoxaline 11d or aminobenzthiazoles 11e and 11f were tolerated but did not improve potency (Table 3). The loss in potency observed for 11d−f is potentially a result of decreased C

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Journal of Medicinal Chemistry Table 4. PK Properties of Selected Inhibitorsh

Inhibition of c-Met kinase activity, HTRF, n ≥ 2. bInhibition of HGF-mediated c-Met phosphorylation in PC3 cells, n ≥ 2. cRat IV, 0.25 mg/kg, formulation: DMSO. dRat PO, 2.0 mg/kg, formulation: 2% (w/v) hydroxypropylmethylcellulose, 1% (w/v) Tween 80 in water, pH adjusted to 2.2 with HCl. eFormulation: 2% (w/v) hydroxypropylmethylcellulose, 1% (w/v) Tween 80 in water, pH adjusted to 2.2 with MSA. fFormulation: 10% (v/v) dimethylacetamide, 2% (w/v) hydroxypropylmethylcellulose, 1% (w/v) Tween 80 in water, pH adjusted to 2.2 with HCl. gNC = not calculated because of lack of slope to characterize the terminal disposition rate constant. hND = not determined. a

it sits perpendicular to the backbone carbonyl of Asn 1209 (not pictured), which may explain the increase in potency observed for the 8-fluorotriazolopyridine core.18 Inhibitors bearing only a methyl group on the bridging carbon (21) demonstrated potencies similar to inhibitors 20. Again, conformation of the bridging carbon stereocenter had a significant effect on potency, and the absolute stereochemical configurations of compounds 20, 21, and 24 were determined by potency correlation with (R)-20c and (S)-21f (see Supporting Information).19 The steric constraint for this position is further confirmed by the decreased potency of compounds 22−24, which contain more bulky substituents on the bridging carbon. As mentioned previously, it was discovered that the electronic properties of the substituents at the bridging carbon and the aromatic group at C6 synergistically affected the potency, metabolic stability, and PK properties of each scaffold. One of the major considerations with the 8-fluorotriazolopyridine core was GST-mediated GSH conjugation. To determine the liability of the 8-fluorotriazolopyridine moiety in each scaffold toward displacement by GSH, several of the more promising inhibitors from Tables 4 and 5 were incubated in rat liver microsomes (RLM) in the presence of GSH. After 2 h of

whereas (S)-20a had IC50 = 780 nM. Analysis of the cocrystal structure of (R)-20c in the kinase domain of c-Met provides an explanation for these results (Figure 1).17 The inhibitor (R)20c adopts a bent conformation to accommodate Met1211, which projects the methyl group on the bridging carbon back into a small pocket. The adjacent fluorine occupies a position relatively close to the protein surface, which would likely not accommodate a larger methyl group, thus potentially explaining the loss in potency for (S)-20a, gem-dimethyl-bridged compound 22, and cyclopropyl compound 23 (Table 5). Other key interactions in the kinase active site are hydrogen bonds between the quinoline and hinge residue Met1160, the imidazopyridine core and Asp1222, as well as a water bridge between the isoxazole nitrogen and Arg1086. As described previously,5 the coplanar nature of the C6 aryl substituent and the 8-fluorotriazolopyridine core is important for maintaining the π-stacking interaction with Tyr1230 as well as the bifurcated hydrogen-bonding interaction between the aromatic hydrogens of the isoxazole, the C7 position of the triazolopyridine core, and the carbonyl oxygen atom on residue Arg1208. This cocrystal structure also reveals the positioning of the 8-fluorine atom in a small cleft within the active site, where D

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Journal of Medicinal Chemistry Table 5. SAR with Respect to Modification of the Bridging Carbon Atomf,g

Inhibition of c-Met kinase activity, n ≥ 2. bInhibition of HGF-mediated c-Met phosphorylation in PC3 cells, n ≥ 2. cRat IV, 0.25 mg/kg, formulation: DMSO. dRat PO, 2.0 mg/kg, formulation: 2% (w/v) hydroxypropylmethylcellulose, 1% (w/v) Tween 80 in water, pH adjusted to 2.2 with HCl. eRat IV, 0.25 mg/kg (20% (w/v) hydroxypropyl β-cyclodextrin in water pH adjusted to 3.5 with MSA). fFor compounds 20 stereochemistry was assigned by correlation to (R)-20c based on in vitro potency. For compounds 21 and 24 stereochemistry was assigned by correlation to (S)-21f based on in vitro potency (cocrystal structure of c-Met and (S)-21f in table of contents graphic and Supporting Information).17 gND = not determined. a

incubation, the ratio of GSH conjugate to remaining parent was measured by LC−MS.20 The degree of GSH conjugation was

considerably influenced by the electronic nature of the substituents on the bridging carbon and the heterocycle at E

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Journal of Medicinal Chemistry Scheme 2. Synthesis of Inhibitors Bearing a Methyl Group on the Bridging Carbona

Reagents and conditions: (i) Pd(dba)2, (t-Bu)3P, NaHMDS, toluene, 80 °C, 24 h, 71−76%; (ii) LiHMDS, (PhSO4)2NF, THF, −78 °C → rt, 71− 76%; (iii) HCl, THF, 2 h, rt, 70−99%; (iv) HATU, i-Pr2EtN, DMF, rt, 78−90%; (v) PPh3, DEAD, TMSN3, THF, rt, 30 min, 22−88%.

a

Figure 1. Cocrystal structure of inhibitor (R)-20c in the c-Met kinase domain (PDB code 4XMO).

the C6 position. Inhibitors bearing the more inductively withdrawing isoxazole at C6 and geminal fluorine atoms on the bridging carbon formed the greatest amounts of GSH conjugate. The extent of GSH displacement was greatly decreased by removal of the fluorines present on the bridging carbon (10b → (R)-20c → (S)-21e, Figure 2). The electron donating capability of the heterocycle attached at the C6 position also significantly modulated the extent of GSH displacement, with the more electron donating thiazole and pyrazole groups showing negligible incorporation of GSH.21 As anticipated, removal of the bridging fluorine atoms significantly reduced the amount of GSH addition as demonstrated by methyl-bridged isoxazole (S)-21e (negligible GSH conjugation) and methyl-bridged C6 pyrazole (S)-21d (no GSH conjugate detected). To determine if the in vitro prediction of glutathione conjugation accurately reflected the in vivo metabolism, two compounds were chosen for rat bile duct cannulation (BDC) studies, 10b and (S)-21f. In the experiment, male rats were dosed intravenously with 2 mg of compound and the collected bile was evaluated by LC−MS to determine the metabolites. As expected, the majority of the metabolites (>90% based on MS intensity) resulting from treatment with compound 10b were

Figure 2. Effect of substituents on the extent of glutathione conjugation. Samples were incubated in rat liver microsomes (RLM) in the presence of glutathione (GSH) for 2 h at pH 7.4. The structures of the GSH adducts were confirmed by high resolution accurate MS and MS/MS.

identified as GSH conjugates. For (S)-21f, which showed negligible GSH-conjugation in the in vitro study, less than 10% of the detected metabolites (based on MS intensity) consisted of products resulting from GSH addition, thus demonstrating that the in vitro study correlated well with the metabolic outcome in vivo.22 Similar to the difluoro-bridged compounds 10 and 12, the most promising compounds contained the isoxazole and methylpyrazole groups at the C6 position of the 8fluoropyridine core (Table 5, (R)-20c, (R)-20d, (S)-21d, (S)-21e, and (S)-21f). Comparison of the compounds in Table 5 reveals that inhibitors (S)-21e and (S)-21f, which contain the isoxazole heterocycle at the C6 position, demonstrated the best combination of potency and pharmacokinetic properties.23 Since inhibitors (S)-21e and (S)-21f showed the best overall profile with respect to potency and pharmacokinetics with F

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Figure 3. Metabolite profile of (S)-21e in liver microsomes. Selected ion chromatograms of metabolites identified by accurate mass spectrometry and MS/MS were used to generate the profiles. MsLM = mouse liver microsomes, RLM = rat liver microsomes, DLM = dog liver microsomes, MLM = monkey liver microsomes, HLM = human liver microsomes.

Figure 4. Metabolite profile for (S)-21f in liver microsomes. Selected ion chromatograms of metabolites identified by accurate mass spectrometry and MS/MS were used to generate the profiles. MsLM = mouse liver microsomes, RLM = rat liver microsomes, DLM = dog liver microsomes, MLM = monkey liver microsomes, HLM = human liver microsomes.

complications for further advancement (Figure 3).24 Incubation of (S)-21e in monkey and human liver microsomes showed oxidation of the linker methyl group (M4) as the major pathway of biotransformation; however, this metabolite was below the detection limit in mouse, dog, and rat liver

negligible amounts of GST-mediated GSH conjugation, their metabolic fate was further investigated by incubation with NADPH in liver microsomes. Analysis by LC−MS revealed that (S)-21e displayed differing metabolite profiles between dog, rat, monkey, and human microsomes, which could present G

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Journal of Medicinal Chemistry microsomes. The major metabolite in mouse liver microsomes was demethylation of the methoxyquinoline (M1). This metabolite was also present in rat and dog liver microsomes in addition to metabolites resulting from oxidation of the methylisoxazole (M2) and the triazolopyridine ring (M3). For comparison, inhibitor (S)-21f, bearing the ethoxymethoxy solubilizing group on the quinoline, was also incubated with NADPH in the presence of the liver microsome panel. In this case the metabolic profile was relatively consistent across all species.25 The introduction of the ethoxymethoxy solubilizing chain provided an alternative site of metabolism that was the major site of metabolism in all species. The primary metabolites formed after incubation of (S)-21f with mouse, rat, dog, monkey, and human liver microsomes were demethylation of the ethoxymethoxy side chain (M6) and subsequent oxidation to acid M7 (Figure 4). The more consistent metabolic profile for (S)-21f led to its selection for additional in vivo studies. Before assessing the in vivo pharmacological activity of (S)-21f, it was evaluated against a broader panel of kinases to understand its selectivity profile. This effort revealed that (S)-21f demonstrated exquisite selectivity for c-Met with no significant inhibition of the 100 other kinases in the panel.26 To demonstrate that compound (S)-21f inhibits c-Met activity in vivo, a pharmacodynamic assay measuring changes in HGF-induced phosphorylation of cMet in the mouse liver was used. Mice were administered a single oral dose of compound (S)-21f at 10 mg/kg. Human recombinant HGF was injected intravenously at 1, 3, 6, 9, 12, and 24 h postdose. Liver and blood were harvested 5 min after administration of HGF. Treatment with compound (S)-21f resulted in inhibition of c-Met phosphorylation in the liver that was related to unbound plasma concentration (Figure 5).27 The in vivo efficacy of (S)-21f, which was sustained above 50% inhibition for up to 9 h, correlated well with the observed in vitro cellular IC50 of 5 nM.28

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CONCLUSIONS

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EXPERIMENTAL SECTION

By utilization of a combination of structure-based drug design as well as the structure−activity relationships between potency, pharmacokinetics, and the optimization of metabolite profiles, new c-Met inhibitors containing an 8-fluorotriazolopyridine core were discovered. Throughout these studies it was observed that metabolic liabilities could be significantly improved by changes in the substituents at positions remote from the site of reactivity and that the extent of glutathione conjugation could be mitigated by the removal of inductively electron-withdrawing groups from sites peripheral to the 8-fluorine substituent. Optimized molecules from this new scaffold demonstrated up to 99% inhibition of HGF-stimulated c-Met phosphorylation in our murine liver pharmacodynamic model at a 10 mg/kg PO dose as exemplified by inhibitor (S)-21f.

Chemistry. Unless otherwise noted, all materials were obtained from commercial suppliers and used as obtained. Anhydrous organic solvents were purchased from Aldrich packaged under nitrogen in Sure/Seal bottles and used directly. Microwave reactions were performed using a Biotage Initiator microwave. Reactions were monitored using Agilent 1100 series LCMS with UV detection at 254 and 215 nm and a low resonance electrospray mode (ESI). Medium pressure liquid chromatography (MPLC) was performed on a CombiFlash Companion (Teledyne Isco) with Redisep normal-phase silica gel (35−60 μm) columns and UV detection at 254 nm. Preparative reverse-phase HPLC was performed on a Gilson (GX-281 liquid handler), 150 mm × 30 mm i.d. column, 5 μm particle size, eluting with a binary solvent system A and B using a gradient elusion (A, water with 0.1% TFA; B, MeCN with 0.1%TFA) with UV detection at 254 nm. Purity was measured using Agilent 1100 series high performance liquid chromatography (HPLC) with UV detection at 254, 215, and 280 nm (15 min; 1.5 mL/min flow rate; eluting with a binary solvent system A and B using a gradient elusion (A, water with 0.1% TFA; B, MeCN with 0.1%TFA). Unless otherwise noted, the purity of all compounds was ≥95%. Enantiomeric excess for compounds bearing stereogenic centers was determined using analytical superfluid chromatography (SFC) on a Chiralpak AS-H column. 1H NMR spectra were recorded on a Bruker AV-400 (400 MHz) spectrometer at ambient temperature. Chemical shifts are reported in ppm from the solvent resonance (DMSO-d6 2.50 ppm). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, br = broad, m = multiplet), coupling constants, and number of protons. 3-Fluoro-2-hydrazinyl-5-phenylpyridine (6). (i) A sealable vial was charged with 5-chloro-2,3-difluoropyridine (1.01 g, 6.76 mmol), X-Phos (0.32 g, 0.68 mmol), palladium(II) acetate (76 mg, 0.34 mmol), potassium phosphate (4.31 g, 20.3 mmol), and phenylboronic acid (1.00 g, 8.11 mmol). The vial was sealed, and dioxane (27 mL) was added. The mixture was degassed by bubbling with N2 and then heated at 100 °C for 30 min. The mixture was concentrated and purified by MPLC using a gradient of 0−40% EtOAc in hexanes to afford 2,3-difluoro-5-phenylpyridine (1.21 g, 6.33 mmol, 94% yield) as a white solid. (ii) A sealable pressure vessel was charged with 2,3-difluoro-5phenylpyridine (1.20 g, 6.28 mmol) and isopropanol (7 mL) followed by hydrazine (1.20 mL, 38.2 mmol). The vessel was sealed and heated at 70 °C for 1.5 h until a gray precipitate formed. The precipitate was collected by vacuum filtration, rinsed with isopropanol (20 mL), and dried under positive air flow to yield 3-fluoro-2-hydrazinyl-5phenylpyridine (6) (1.34 g, 6.59 mmol, 86% yield) as a light gray solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.26 (dd, J = 1.81, 1.12 Hz, 1 H), 7.94 (s, 1 H), 7.73 (dd, J = 12.91, 1.96 Hz, 1 H), 7.60−7.67 (m, 2 H), 7.38−7.47 (m, 2 H), 7.32 (d, J = 7.34 Hz, 1 H), 4.21 (br s, 2 H).

Figure 5. A single dose of compound (S)-21f was administered by oral gavage (formulation: 2% (w/v) hydroxypropylmethylcellulose, 1% (w/ v) Tween 80 in water, pH adjusted to 2.2 with MSA) at 1, 3, 6, 9, 12, or 24 h prior to sacrifice. c-Met phosphorylation was induced in the livers of female Balb/c mice by injection of 12 μg of human recombinant HGF IV 5 min prior to sacrifice. Levels of c-Met phosphorylation were determined by an electrochemiluminescent immunoassay. Data represent the mean ± standard deviation (n = 3). Statistical significance was determined by ANOVA followed by Bonferroni/Dunn post hoc test: (∗) p < 0.0001 compared to HGF control. Black circles represent the mean terminal concentration of compound (S)-21f in the plasma ± standard deviation (n = 3). H

DOI: 10.1021/jm501913a J. Med. Chem. XXXX, XXX, XXX−XXX

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Journal of Medicinal Chemistry tert-Butyl 2,2-Difluoro-2-(quinolin-6-yl)acetate (8). (iii) Zn dust (26.5 g, 0.42 mol) was suspended in 300 mL of anhydrous THF under argon. Trimethylsilyl chloride (1.4 mL, 12.0 mmol) was added dropwise via syringe, and the solution was refluxed until a precipitate was observed. The mixture was allowed to cool to rt, and tert-butyl bromoacetate (56 mL, 0.37 mol) was added dropwise via syringe. The mixture was cooled to 0 °C and a solution of 6-bromoquinoline (25.0 g, 0.12 mol) in 25 mL of THF was added dropwise via syringe followed by addition of tetrakis(triphenylphosphine)palladium (1.4 g, 1.2 mmol) as a solid in a single portion under positive argon flow. The mixture was heated to reflux for 3 h. The reaction mixture was cooled to rt, and saturated aqueous ammonium chloride was added (100 mL). The mixture was filtered through Celite, partitioned between water (50 mL) and ethyl acetate (300 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (2 × 100 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The crude residue was purified by MPLC, eluting with 10% EtOAc in hexanes to obtain tert-butyl 2-(quinolin-6-yl)acetate (16.0 g, 65.7 mmol, 55% yield) as an orange waxy solid. 1H NMR (400 MHz, CD3OD), δ ppm 8.88−8.67 (m, 1H), 8.34−8.32 (d, 1H, J = 8.0 Hz), 7.99−7.96 (d, 1H, J = 8.8 Hz), 7.83 (s, 1H), 7.67−7.64 (m, 1H), 7.54−7.50 (m, 1H), 3.78 (s, 2H), 1.41 (s, 9H). (iv) A solution of tert-butyl 2-(quinolin-6-yl)acetate (1.63 g, 6.70 mmol) and N-fluorobenzenesulfonimide (6.34 g, 20.1 mmol) in THF (34 mL) was cooled to −78 °C. A THF solution of LiHMDS (23.5 mL, 23.4 mmol) was added, and the solution was maintained at −78 °C for 2 h, then allowed to warm to rt. Saturated aqueous NH4Cl (10 mL) was added, and the mixture was partitioned between EtOAc (30 mL) and additional saturated aqueous NH4Cl (10 mL). The layers were separated and the aqueous layer was extracted with EtOAc (1 × 25 mL). The combined organic extracts were dried (Na2SO4), concentrated in vacuo, and purified by MPLC using a gradient of 2− 90% EtOAc in hexanes to afford tert-butyl 2,2-difluoro-2-(quinolin-6yl)acetate (8) (0.87 g, 3.13 mmol, 47% yield) as a light-yellow oil, which solidified upon standing. 1H NMR (400 MHz, CD3OD), δ ppm 8.97 (d, J = 4.11 Hz, 1 H), 8.51 (d, J = 8.22 Hz, 1 H), 7.88−7.96 (m, 1 H), 8.12−8.29 (m, 2 H), 7.64 (dd, J = 8.31, 4.30 Hz, 1 H), 1.47 (s, 9 H). 2,2-Difluoro-N′-(3-fluoro-5-phenylpyridin-2-yl)-2-(quinolin6-yl)acetohydrazide (9). (v) To a solution of tert-butyl 2,2-difluoro2-(quinolin-6-yl)acetate (8) (0.87 g, 3.13 mmol) in CH2Cl2 (6.0 mL) was added trifluoroacetic acid (1.2 mL, 15.6 mmol). The solution was maintained at rt for 48 h. The solution concentrated and dried under reduced pressure to provide 2,2-difluoro-2-(quinolin-6-yl)acetic acid (65 mg, 93% yield) as a sticky brown solid, which was used without further purification. (vi) To a solution of 2,2-difluoro-2-(quinolin-6-yl)acetic acid (65 mg, 2.91 mmol) in DMF (6.3 mL) at 0 °C was added thionyl chloride (0.5 mL, 6.3 mmol) dropwise via syringe. The solution was maintained at rt for 1 h, and then triethylamine (0.5 mL, 3.14 mmol) was added followed by 3-fluoro-2-hydrazinyl-5-phenylpyridine (0.64 mg, 3.14 mmol) as a solid in a single portion, then dimethylaminopyridine (38 mg, 0.31 mmol). The solution was allowed to warm to rt and was maintained at rt for 18 h. Saturated aqueous NaHCO3 was added carefully to the solution over 20 min. The mixture was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed vigorously with 1 N NaOH (15 mL). The organic layer was dried (Na2SO4), concentrated in vacuo, and purified by MPLC using a gradient of 2−10% MeOH in CH2Cl2 to afford 2,2-difluoro-N′-(3fluoro-5-phenylpyridin-2-yl)-2-(quinolin-6-yl)acetohydrazide (9) (1.11 g, 2.72 mmol, 87% yield) as a brown solid. General Procedure for the Preparation of Compounds 3b, 4, 10, and 12. See Scheme 1, steps v−vi, exemplified by the synthesis of 4. 6-(Difluoro(8-fluoro-6-phenyl[1,2,4]triazolo[4,3-a]pyridin-3yl)methyl)quinoline (4). (vi) A microwave vial was charged with 2,2difluoro-N′-(3-fluoro-5-phenylpyridin-2-yl)-2-(quinolin-6-yl)acetohydrazide (1.11 g, 2.72 mmol) and PS-triphenylphosphine (PSPPh3) (3.04 g, 6.79 mmol). The vial was sealed with a septum cap, and dichloroethane (5 mL) was added followed by 2,2,2-trichloroacetoni-

trile (0.54 mL, 5.43 mmol) and diisopropylethylamine (0.95 mL, 5.43 mmol). The mixture was heated with microwave irradiation at 100 °C for 20 min. The mixture was filtered to remove PS-PPh3, washing with MeOH (10 mL) and CH2Cl2 (20 mL). The collected mother liquor was concentrated and purified by reverse-phase HPLC (10−90% CH3CN in H2O (0.1% TFA additive)) to provide 6-(difluoro(8fluoro-6-phenyl[1,2,4]triazolo[4,3-a]pyridin-3-yl)methyl)quinoline 2,2,2-trifluoroacetate. The product was free-based using an SCX-2 ion exchange column (Biotage), eluting with 7 N ammonia in MeOH (100 mL). The eluent was concentrated to give 6-(difluoro(8-fluoro-6phenyl[1,2,4]triazolo[4,3-a]pyridin-3-yl)methyl)quinoline (103 mg, 0.26 mmol, 10 % yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 9.06 (dd, J = 4.21, 1.76 Hz, 1 H), 8.56−8.59 (m, 2 H), 8.50 (d, J = 0.98 Hz, 1 H), 8.23 (d, J = 8.90 Hz, 1 H), 8.00−8.09 (m, 2 H), 7.92 (s, 1 H), 7.75−7.81 (m, 2 H), 7.65−7.71 (m, 1 H), 7.48−7.58 (m, 2 H). LRMS (ESI): m/z (M + H) 391.2. 6-(Difluoro(8-fluoro-6-(pyridin-2-yl)[1,2,4]triazolo[4,3-a]pyridin-3-yl)methyl)quinoline (10a). Prepared from 1-(3-fluoro-5(pyridin-2-yl)pyridin-2-yl)hydrazine (prepared as described for 6) and 2,2-difluoro-2-(quinolin-6-yl)acetic acid in 29% yield (over 2 steps) according to the general procedure described for 4. Data for 10a: 1H NMR (400 MHz, DMSO-d6) δ ppm 9.11 (d, J = 0.59 Hz, 1 H), 9.06 (dd, J = 4.21, 1.76 Hz, 1 H), 8.73 (dt, J = 3.86, 0.86 Hz, 1 H), 8.55− 8.62 (m, 1 H), 8.49 (s, 1 H), 8.27−8.35 (m, 1 H), 8.24 (d, J = 8.80 Hz, 1 H), 8.17 (d, J = 8.02 Hz, 1 H), 8.06 (dd, J = 8.90, 2.15 Hz, 1 H), 7.99 (td, J = 7.80, 1.81 Hz, 1 H), 7.68 (dd, J = 8.31, 4.21 Hz, 1 H), 7.49 (ddd, J = 7.51, 4.77, 0.83 Hz, 1 H). LCMS (ESI): m/z (M + H) = 392.4. 5-(3-(Difluoro(quinolin-6-yl)methyl)-8-fluoro[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisoxazole (10b). Prepared from 1-(3fluoro-5-(3-methylisoxazol-5-yl)pyridin-2-yl)hydrazine (18a) (prepared via Stille coupling route) and 2,2-difluoro-2-(quinolin-6-yl)acetic acid in 27% yield (over 2 steps) according to the general procedure described for 4. Data for 10b: 1H NMR (400 MHz, DMSO-d6) δ ppm 9.05 (dd, J = 4.21, 1.56 Hz, 1 H), 8.75 (s, 1 H), 8.57 (d, J = 0.69 Hz, 1 H), 8.47 (s, 1 H), 8.22 (d, J = 8.80 Hz, 1 H), 7.96−8.10 (m, 2 H), 7.66 (dd, J = 8.31, 4.21 Hz, 1 H), 7.20 (s, 1 H), 2.31 (s, 3 H). LCMS (ESI): m/z (M + H) = 396.3. 5-(3-(Difluoro(quinolin-6-yl)methyl)-8-fluoro[1,2,4]triazolo[4,3-a]pyridin-6-yl)-2-methylthiazole (10c). Prepared from 1-(3fluoro-5-(2-methylthiazol-5-yl)pyridin-2-yl)hydrazine (prepared by Stille method described for intermediate 18a) and 2,2-difluoro-2(quinolin-6-yl)acetic acid in 3% yield (over 2 steps) according to the general procedure described for 4. Data for 10c: 1H NMR (400 MHz, DMSO-d6) δ ppm 9.07 (dd, J = 4.21, 1.66 Hz, 1 H), 8.55−8.65 (m, 1 H), 8.49 (s, 1 H), 8.45 (d, J = 0.98 Hz, 1 H), 8.29 (s, 1 H), 8.23 (d, J = 8.80 Hz, 1 H), 7.99−8.08 (m, 2 H), 7.69 (dd, J = 8.27, 4.25 Hz, 1 H), 2.71 (s, 3 H). LCMS (ESI): m/z (M + H) = 412.2. 4-(3-(Difluoro(quinolin-6-yl)methyl)-8-fluoro[1,2,4]triazolo[4,3-a]pyridin-6-yl)thiazole (10d). Prepared from 1-(3-fluoro-5(thiazol-4-yl)pyridin-2-yl)hydrazine (prepared by Stille method described for intermediate 18a) and 2,2-difluoro-2-(quinolin-6-yl)acetic acid in 20% yield (over 2 steps) according to the general procedure described for 4. Data for 10d: 1H NMR (400 MHz, DMSOd6) δ ppm 9.24−9.36 (m, 2 H), 8.93−9.07 (m, 2 H), 8.70 (s, 1 H), 8.57 (d, J = 1.37 Hz, 1 H), 8.45 (d, J = 8.90 Hz, 1 H), 8.20−8.34 (m, 2 H), 7.96 (dd, J = 8.22, 4.69 Hz, 1 H). LCMS (ESI): m/z (M + H) = 398.1. 6-(Difluoro(8-fluoro-6-(1-methyl-1H-pyrazol-4-yl)[1,2,4]triazolo[4,3-a]pyridin-3-yl)methyl)quinoline (10e). Prepared from 1-(3-fluoro-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)hydrazine (prepared as described for 6) and 2,2-difluoro-2-(quinolin-6-yl)acetic acid in 10% yield (over 2 steps) according to the general procedure described for 4. Data for 10e: 1H NMR (400 MHz, DMSO-d6) δ ppm 9.06 (dd, J = 4.21, 1.66 Hz, 1 H), 8.55−8.62 (m, 2 H), 8.43−8.52 (m, 2 H), 8.23 (d, J = 8.80 Hz, 1 H), 8.12 (s, 1 H), 8.04 (dd, J = 8.85, 2.10 Hz, 1 H), 7.92 (dd, J = 12.03, 0.88 Hz, 1 H), 7.68 (dd, J = 8.31, 4.30 Hz, 1 H), 3.89 (s, 3 H). LCMS (ESI): m/z (M + H) = 395.4. 6-((8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)[1,2,4]triazolo[4,3a]pyridin-3-yl)methyl)quinoline (11a). Prepared from 2-(quinolinI

DOI: 10.1021/jm501913a J. Med. Chem. XXXX, XXX, XXX−XXX

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Journal of Medicinal Chemistry

4-(3-(Difluoro(3-methoxyquinolin-6-yl)methyl)-8-fluoro[1,2,4]triazolo[4,3-a]pyridin-6-yl)thiazole (12b). Prepared from 1-(3-fluoro-5-(thiazol-4-yl)pyridin-2-yl)hydrazine (prepared via Stille coupling route) and 2,2-difluoro-2-(3-methoxyquinolin-6-yl)acetic acid in 7% yield (over 2 steps) according to the general procedure described for 4. Data for 12b: 1H NMR (400 MHz, DMSO-d6) δ ppm 9.29 (s, 1H), 8.94 (s, 1 H), 8.79 (s, 1 H), 8.53 (s, 1 H), 8.22−8.35 (m, 2 H), 8.17 (d, J = 7.6, 1 H), 7.96 (s, 1H), 7.87 (d, J = 7.2, 1 H), 3.94 (s, 3 H). LCMS (ESI): m/z (M + H) = 428.1. 6-(Difluoro(8-fluoro-6-(1-methyl-1H-pyrazol-4-yl)[1,2,4]triazolo[4,3-a]pyridin-3-yl)methyl)-3-methoxyquinoline (12c). Prepared from 1-(3-fluoro-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)hydrazine (prepared as described for 6) and 2,2-difluoro-2-(3methoxyquinolin-6-yl)acetic acid in 15% yield (over 2 steps) according to the general procedure described for 4. Data for 12c: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.78 (d, J = 2.93 Hz, 1 H), 8.57 (d, J = 0.78 Hz, 1 H), 8.45 (s, 1 H), 8.32 (d, J = 1.47 Hz, 1 H), 8.15 (d, J = 8.71 Hz, 1 H), 8.12 (d, J = 0.68 Hz, 1 H), 7.96 (d, J = 2.74 Hz, 1 H), 7.92 (dd, J = 12.03, 0.98 Hz, 1 H), 7.87 (dd, J = 8.80, 2.15 Hz, 1 H), 3.94 (s, 3 H), 3.89 (s, 3 H). LCMS (ESI): m/z (M + H) = 425.4. General Procedure for the Preparation of Compounds 11, 20−24. See Scheme 2, steps i−v, exemplified by the synthesis of 21e (R = isoxazole, R1 = OMe, R2 = H). tert-Butyl 2-(3-Methoxyquinolin-6-yl)propanoate (15, R1 = OMe).29 A sealable vial was charged with bis(benzylideneacetone)palladium (60.4 mg, 105 μmol), tri-tert-butylphosphonium tetrafluoroborate (30 mg, 105 μmol), sodium bis(trimethylsilyl)amide (1.73 g, 9.45 mmol), 6-bromo-3-methoxyquinoline (13, R1 = OMe) (0.50 g, 2.10 mmol). The vial was sealed with a septum-cap, flushed with N2, and then tert-butyl propionate (14) (0.73 mL, 4.9 mmol) and toluene (6 mL) were added. The mixture was sparged with N2 for 5 min and then maintained at rt for 24 h. The mixture was poured into saturated aqueous NH4Cl (15 mL) and extracted with EtOAc (2 × 10 mL). The combined organic layers were dried (Na2SO4), concentrated, and purified by MPLC using a gradient of 2−60% EtOAc in hexanes to afford tert-butyl 2-(3-methoxyquinolin-6-yl)propanoate 15 (0.46 g, 76% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ ppm 8.62 (d, J = 2.84 Hz, 1 H), 7.94−8.02 (m, 1 H), 7.61 (d, J = 1.76 Hz, 1 H), 7.49 (dd, J = 8.71, 1.96 Hz, 1 H), 7.33 (d, J = 2.64 Hz, 1 H), 3.91 (s, 3 H), 3.73−3.85 (m, 1 H), 1.53 (d, J = 7.14 Hz, 3 H), 1.40 (s, 9 H). LCMS (ESI): m/z (M + H) = 288.3. tert-Butyl 2-Fluoro-2-(3-methoxyquinolin-6-yl)propanoate (16, R1 = OMe). To a solution of tert-butyl 2-(3-methoxyquinolin6-yl)propanoate (2.50 g, 8.7 mmol) in THF (8.7 mL, 8.7 mmol) at −78 °C was added a THF solution of LiHMDS (11.0 mL, 1 M). The solution was warmed to room temperature over 1 h, then recooled to −78 °C, and a THF solution of N-fluorobenzenesulfonimide (3.6 g, 11 mmol, 1 M) was added. The solution was allowed to warm to −10 °C over 1 h. Upon completion, the solution was filtered through a plug of Celite, washing with EtOAc (30 mL). The filtrate was concentrated, then redissolved in EtOAc (50 mL) and washed with saturated aqueous NH4Cl (20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 10 mL). The combined organic layers were dried (MgSO4), concentrated, and the residue was purified by MPLC, eluting with 40% EtOAc:hexanes to provide tert-butyl 2fluoro-2-(3-methoxyquinolin-6-yl)propanoate (16) (2.03 g, 76% yield) as a yellow oil. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.68 (d, J = 2.93 Hz, 1H), 7.97−8.05 (m, 2H), 7.89 (d, J = 2.93 Hz, 1H), 7.62 (dd, J = 2.10, 8.75 Hz, 1H), 3.93 (s, 5H), 3.33 (s, 3H), 1.38 (s, 9H). LCMS (ESI): m/z (M + H) = 306.4. 2-(3-Methoxyquinolin-6-yl)propanoic Acid (17, R1 = OMe, R2 = H). HCl gas was bubbled through an EtOAc solution of tert-butyl 2(3-methoxyquinolin-6-yl)propanoate (6.20 g, 21.6 mmol) for 5 min to produce a white precipitate. The precipitate was collected by vacuum filtration to give 2-(3-methoxyquinolin-6-yl)propanoic acid hydrochloride (5.48 g, 95% yield) as a colorless solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.77 (d, J = 2.25 Hz, 1H), 7.96−8.05 (m, 2H), 7.87 (s, 1H), 7.62 (dd, J = 1.56, 8.61 Hz, 1H), 3.94 (s, 3H), 1.47 (d, J = 7.14 Hz, 3H). LCMS (ESI): m/z (M + H) = 232.2.

6-yl)acetic acid hydrochloride and 1-(3-fluoro-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)hydrazine (prepared as described for 6) in 63% yield (over 2 steps) according to the general procedure described for (S)-21e. Data for 11a: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.86 (dd, J = 4.21, 1.76 Hz, 1 H), 8.62 (d, J = 1.17 Hz, 1 H), 8.31 (ddd, J = 8.46, 1.61, 0.59 Hz, 1 H), 8.25 (d, J = 0.39 Hz, 1 H), 7.96−8.03 (m, 2 H), 7.90 (d, J = 1.66 Hz, 1 H), 7.77 (dd, J = 8.71, 2.05 Hz, 1H), 7.64 (dd, J = 12.28, 1.12 Hz, 1 H), 7.50 (dd, J = 8.31, 4.21 Hz, 1 H), 4.80 (s, 2 H), 3.82 (s, 3H). LCMS (ESI): m/z (M + H) = 359.4. 6-((8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)[1,2,4]triazolo[4,3a]pyridin-3-yl)methyl)-3-methoxyquinoline (11b). Prepared from 2-(3-methoxyquinolin-6-yl)acetic acid and 1-(3-fluoro-5-(1methyl-1H-pyrazol-4-yl)pyridin-2-yl)hydrazine (prepared as described for 6) in 41% yield (over 2 steps) according to the general procedure described for (S)-21e. Data for 11b: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.55−8.66 (m, 2 H), 8.25 (s, 1 H), 8.00 (d, J = 0.68 Hz, 1 H), 7.93 (d, J = 8.61 Hz, 1 H), 7.68−7.75 (m, 2 H), 7.58−7.67 (m, 2 H), 4.79 (s, 2 H), 3.89 (s, 3 H), 3.87 (s, 3 H). LCMS (ESI): m/z (M + H) = 389.0. 6-((8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)[1,2,4]triazolo[4,3a]pyridin-3-yl)methyl)quinolin-3-ol (11c). Prepared as a byproduct in the synthesis of 11b. Data for 11c: 1H NMR (400 MHz, DMSO-d6) d ppm 10.59 (br s, 1 H), 8.57−8.66 (m, 2 H), 8.27 (s, 1 H), 8.01 (s, 1 H), 7.91 (d, J = 8.80 Hz, 1 H), 7.80 (s, 1 H), 7.65 (d, J = 12.32 Hz, 1 H), 7.61 (s, 1 H), 7.52−7.59 (m, 1 H), 4.76 (s, 2 H), 3.88 (s, 3 H). LCMS (ESI): m/z (M + H) = 375.1. 6-((8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)[1,2,4]triazolo[4,3a]pyridin-3-yl)methyl)quinoxaline (11d). Prepared from 1-(3fluoro-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)hydrazine (prepared as described for 6) and 2-(quinoxalin-6-yl)acetic acid hydrochloride in 28% yield (over 2 steps) according to the general procedure described for 4. Data for 11d: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.92 (q, J = 1.76 Hz, 2 H), 8.70 (s, 1 H), 8.27 (s, 1 H), 8.05−8.13 (m, 2 H), 8.02 (s, 1 H), 7.87 (dd, J = 8.66, 1.91 Hz, 1 H), 7.65 (d, J = 12.23 Hz, 1 H), 4.88 (s, 2 H), 3.88 (s, 3 H). LCMS (ESI): m/z (M + H) = 360.3. N-(6-((8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)[1,2,4]triazolo[4,3-a]pyridin-3-yl)methyl)benzo[d]thiazol-2-yl)acetamide (11e). Prepared from 2-(2-acetamidobenzo[d]thiazol-6-yl)acetic acid hydrochloride and 1-(3-fluoro-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2yl)hydrazine (prepared as described for 6) in 7% yield (over 2 steps) according to the general procedure described for (S)-21e. Data for 11e: 1H NMR (400 MHz, DMSO-d6) δ ppm 12.29 (s, 1 H), 8.58 (d, J = 1.08 Hz, 1 H), 8.26 (s, 1 H), 8.00 (d, J = 0.68 Hz, 1 H), 7.95 (d, J = 1.27 Hz, 1 H), 7.68 (d, J = 8.31 Hz, 1 H), 7.63 (dd, J = 12.28, 1.03 Hz, 1 H), 7.42 (dd, J = 8.36, 1.81 Hz, 1 H), 4.68 (s, 2 H), 3.88 (s, 3 H), 2.18 (s, 3 H). LCMS (ESI): m/z (M + H) = 422.0. 6-((8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)[1,2,4]triazolo[4,3a]pyridin-3-yl)methyl)benzo[d]thiazol-2-amine (11f). A solution of sodium hydroxide (2.8 mL, 1 N) was added to 11e (40 mg, 0.09 mmol). The resulting mixture was heated at 60 °C for 24 h. The mixture was cooled to rt and the precipitated solid was collected by vacuum filtration and dried under high vacuum to yield 6-((8-fluoro-6(1-methyl-1H-pyrazol-4-yl)[1,2,4]triazolo[4,3-a]pyridin-3-yl)methyl)benzo[d]thiazol-2-amine (11f) (10 mg, 0.03 mmol, 33% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.53 (s, 1 H), 8.26 (s, 1 H), 7.99 (s, 1 H), 7.58−7.68 (m, 2 H), 7.41 (s, 2 H), 7.24−7.32 (m, 1 H), 7.17−7.23 (m, 1 H), 4.58 (s, 2 H), 3.88 (s, 3 H). LCMS (ESI): m/z (M + H) = 380.2. 5-(3-(Difluoro(3-methoxyquinolin-6-yl)methyl)-8-fluoro[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisoxazole (12a). Prepared from 1-(3-fluoro-5-(3-methylisoxazol-5-yl)pyridin-2-yl)hydrazine (prepared via Stille coupling route as described for 18a) and 2,2-difluoro-2-(3-methoxyquinolin-6-yl)acetic acid in 20% yield (over 2 steps) according to the general procedure described for 4. Data for 12a: 1H NMR (400 MHz, CDCl3) δ ppm 8.75−8.84 (m, 2 H), 8.21 (d, J = 8.71 Hz, 1 H), 8.17 (d, J = 1.08 Hz, 1 H), 7.83 (dd, J = 8.80, 2.05 Hz, 1 H), 7.47 (d, J = 2.84 Hz, 1 H), 7.39 (dd, J = 9.78, 1.17 Hz, 1 H), 6.54 (s, 1 H), 3.97 (s, 3 H), 2.41 (s, 3 H). LCMS (ESI): m/z (M + H) = 426.0. J

DOI: 10.1021/jm501913a J. Med. Chem. XXXX, XXX, XXX−XXX

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Journal of Medicinal Chemistry 1-(3-Fluoro-5-(3-methylisoxazol-5-yl)pyridin-2-yl)hydrazine (18a, R = methylisoxazole). To a sealable pressure vessel charged with 5-(5,6-difluoropyridin-3-yl)-3-methylisoxazole (26) (5.32 g, 27.1 mmol) and isopropanol (30 mL) was added anhydrous hydrazine (3.0 mL, 94.9 mmol). The vessel was sealed and heated at 60 °C for 18 h until a white precipitate formed. The mixture was cooled to rt and concentrated to dryness. The resulting solids were suspended in saturated aqueous NaHCO3, then collected by vacuum filtration and dried under reduced pressure to yield 1-(3-fluoro-5-(3-methylisoxazol5-yl)pyridin-2-yl)hydrazine (18a) (1.47 g, 77.8% yield) as a tan amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.42 (br s, 1H), 8.35−8.38 (m, 1H), 7.77 (d, J = 1.86 Hz, 1H), 7.74 (s, 1H), 6.70 (s, 1H), 4.33 (br s, 2H), 2.25 (s, 3H). N′-(3-Fluoro-5-(3-methylisoxazol-5-yl)pyridin-2-yl)-2-(3-methoxyquinolin-6-yl)propanehydrazide (19, R = Methylisoxazole, R1 = OMe, R2 = H). To a flask charged with 1-(3-fluoro-5-(3methylisoxazol-5-yl)pyridin-2-yl)hydrazine (18a) (2.63 g, 12.6 mmol), 2-(3-methoxyquinolin-6-yl)propanoic acid hydrochloride (17) (3.25 g, 12.1 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) (7.20 g, 18.9 mmol), and diisopropylethylamine (6.6 mL, 37.9 mmol) was added DMF (45 mL). The resulting solution was maintained at rt for 2 h, then concentrated to 50% volume, which resulted in the formation of a precipitate. The solid was collected by vacuum filtration and washed with EtOAc to give N′-(3-fluoro-5-(3-methylisoxazol-5-yl)pyridin-2yl)-2-(3-methoxyquinolin-6-yl)propanehydrazide (19) (4.71 g, 89% yield) as a gray-white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.17 (d, J = 1.66 Hz, 1H), 9.16 (s, 1H), 8.60 (d, J = 2.93 Hz, 1H), 8.35 (d, J = 1.56 Hz, 1H), 7.89−7.95 (m, 2H), 7.86 (d, J = 1.86 Hz, 1H), 7.74 (d, J = 2.35 Hz, 1H), 7.65 (dd, J = 2.05, 8.71 Hz, 1H), 6.78 (s, 1H), 3.94−4.01 (m, 1H), 3.93 (s, 3H), 2.26 (s, 3H), 1.50 (d, J = 7.14 Hz, 3H). LCMS (ESI): m/z (M + H) = 422.0. 5-(8-Fluoro-3-(1-fluoro-1-(quinolin-6-yl)ethyl)[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisothiazole (20a). Prepared from 1(3-fluoro-5-(3-methylisothiazol-5-yl)pyridin-2-yl)hydrazine (prepared by Stille method as described for 18a) and 2-fluoro-2-(quinolin-6yl)propanoic acid hydrochloride in 59% yield (over 2 steps) according to the general procedure described for 21e. Separation of enantiomers provided 15% yield of (R)-20a (>99% ee) and 11% yield of (S)-20a (>99%ee). Data for (R)-20a: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.95 (dd, J = 1.76, 4.21 Hz, 1H), 8.45 (td, J = 0.90, 7.58 Hz, 1H), 8.05−8.16 (m, 2H), 8.02 (d, J = 0.88 Hz, 1H), 7.71−7.85 (m, 2H), 7.52−7.65 (m, 2H), 2.41−2.48 (m, 3H), 2.40 (s, 3H). LCMS (ESI): m/z (M + H) = 408.0. As anticipated, the 1H NMR and LCMS data for (S)-20a were identical to those obtained for (R)-20a. 5-(8-Fluoro-3-(1-fluoro-1-(3-methoxyquinolin-6-yl)ethyl)[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisothiazole (20b). Prepared from 1-(3-fluoro-5-(3-methylisothiazol-5-yl)pyridin-2-yl)hydrazine (prepared by Stille method as described for 18a) and 2fluoro-2-(3-methoxyquinolin-6-yl)propanoic acid hydrochloride in 70% yield (over 2 steps) according to the general procedure described for 21e. Separation of enantiomers provided 32% yield of (R)-20b (>99% ee) and 39% yield of (S)-20b (>99%ee). Data for (R)-20b: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.67 (d, J = 2.93 Hz, 1H), 8.03 (d, J = 8.80 Hz, 1H), 7.98 (s, 1H), 7.91 (d, J = 1.96 Hz, 1H), 7.83 (d, J = 2.64 Hz, 1H), 7.80 (dd, J = 1.17, 11.44 Hz, 1H), 7.60 (dd, J = 2.15, 8.80 Hz, 1H), 7.56 (s, 1H), 3.89 (s, 3H), 2.37−2.47 (m, 3H), 2.39 (s, 3H). LCMS (ESI): m/z (M + H) = 438.3. 5-(8-Fluoro-3-(1-fluoro-1-(3-methoxyquinolin-6-yl)ethyl)[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisoxazole (20c). Prepared from 1-(3-fluoro-5-(3-methylisoxazol-5-yl)pyridin-2-yl)hydrazine (18a) and 2-fluoro-2-(3-methoxyquinolin-6-yl)propanoic acid hydrochloride in 25% yield (over 2 steps) according to the general procedure described for 21e. Separation of enantiomers provided 10% yield of (R)-20c (>99% ee) and 8% yield of (S)-20c (>99%ee). Data for (R)-20c: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.67 (d, J = 2.93 Hz, 1H), 8.12 (s, 1H), 8.04 (d, J = 8.80 Hz, 1H), 7.92 (d, J = 11.44 Hz, 1H), 7.85 (d, J = 1.96 Hz, 1H), 7.82 (d, J = 2.84 Hz, 1H), 7.61 (dd, J = 2.10, 8.85 Hz, 1H), 7.02 (s, 1H), 3.88 (s, 3H), 2.35−2.46 (m, 3H), 2.23 (s, 3H). LCMS (ESI): m/z (M + H) = 422.2.

6-(1-Fluoro-1-(8-fluoro-6-(1-methyl-1H-pyrazol-4-yl)[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-methoxyquinoline (20d). Prepared from 1-(3-fluoro-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)hydrazine (prepared as described for 6) and 2-fluoro-2-(3-methoxyquinolin-6-yl)propanoic acid hydrochloride in 57% yield (over 2 steps) according to the general procedure described for 21e. Separation of enantiomers provided 22% yield of (R)-20d (>99% ee) and 26% yield of (S)-20d (>99%ee). Data for (R)-20d: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.66 (d, J = 2.93 Hz, 1H), 8.16 (s, 1H), 8.02 (d, J = 8.80 Hz, 1H), 7.91 (s, 1H), 7.88 (d, J = 1.86 Hz, 1H), 7.82 (d, J = 2.84 Hz, 1H), 7.78 (d, J = 0.68 Hz, 1H), 7.72 (d, J = 0.88 Hz, 1H), 7.62 (dd, J = 2.10, 8.85 Hz, 1H), 3.88 (s, 3H), 3.80 (s, 3H), 2.35−2.45 (m, 3H). LCMS (ESI): m/z (M + H) = 421.2. 5-(8-Fluoro-3-(1-(quinolin-6-yl)ethyl)[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisothiazole (21a). Prepared from 1-(3fluoro-5-(3-methylisothiazol-5-yl)pyridin-2-yl)hydrazine (prepared by Stille method as described for 18a) and 2-(quinolin-6-yl)propanoic acid hydrochloride in 52% yield (over 2 steps) according to the general procedure described for 21e. Separation of enantiomers provided 19% yield of (S)-21a (>99% ee) and 19% yield of (R)-21a (>99% ee). Data for (S)-21a: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.86 (dd, J = 1.71, 4.16 Hz, 1H), 8.64 (d, J = 1.17 Hz, 1H), 8.32 (d, J = 7.43 Hz, 1H), 8.00 (d, J = 8.61 Hz, 1H), 7.94 (d, J = 1.96 Hz, 1H), 7.81 (dd, J = 2.01, 8.75 Hz, 1H), 7.69 (dd, J = 1.17, 11.74 Hz, 1H), 7.67 (s, 1H), 7.51 (dd, J = 4.16, 8.26 Hz, 1H), 5.24 (d, J = 7.14 Hz, 1H), 2.44 (s, 3H), 1.90 (d, J = 7.04 Hz, 3H). LCMS (ESI): m/z (M + H) = 390.0. 5-(8-Fluoro-3-(1-(3-methoxyquinolin-6-yl)ethyl)[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisothiazole (21b). Prepared from 1-(3-fluoro-5-(3-methylisothiazol-5-yl)pyridin-2-yl)hydrazine (prepared by Stille method as described for 18a) and 2-(3methoxyquinolin-6-yl)propanoic acid hydrochloride in 35% yield (over 2 steps) according to the general procedure described for 21e. Separation of enantiomers provided 10% yield of (S)-21b (>99% ee) and 8% yield of (R)-21b (>99%ee). Data for (S)-21b: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.55−8.61 (m, 2H), 7.93 (d, J = 8.61 Hz, 1H), 7.75 (d, J = 1.76 Hz, 1H), 7.66−7.70 (m, 2H), 7.62−7.66 (m, 2H), 5.21 (d, J = 7.04 Hz, 1H), 3.89 (s, 3H), 2.44 (s, 3H), 1.90 (d, J = 7.14 Hz, 3H). LCMS (ESI): m/z (M + H) = 420.2. 6-(1-(8-Fluoro-6-(1-methyl-1H-imidazol-4-yl)[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-methoxyquinoline (21c). Prepared from 1-(3-fluoro-5-(1-methyl-1H-imidazol-4-yl)pyridin-2-yl)hydrazine and 2-(3-methoxyquinolin-6-yl)propanoic acid hydrochloride in 34% yield (over 2 steps) according to the general procedure described for 21e. Separation of enantiomers provided 10% yield of (S)-21c (>99% ee) and 8% yield of (R)-21c (>99%ee). Data for (S)-21c: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.57 (d, J = 2.93 Hz, 1H), 8.23 (d, J = 0.98 Hz, 1H), 7.93 (d, J = 8.61 Hz, 1H), 7.67−7.70 (m, 2H), 7.61− 7.66 (m, 3H), 7.58 (dd, J = 2.05, 8.61 Hz, 1H), 5.09 (d, J = 7.14 Hz, 1H), 3.87 (s, 3H), 3.65 (s, 3H), 1.89 (d, J = 7.04 Hz, 3H). LCMS (ESI): m/z (M + H) = 403.2. 6-(1-(8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-methoxyquinoline (21d). Prepared from 1-(3-fluoro-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)hydrazine (prepared as described for 6) and 2-(3-methoxyquinolin-6-yl)propanoic acid hydrochloride in 40% yield (over 2 steps) according to the general procedure described for 21e. Separation of enantiomers provided 18% yield of (S)-21d (>99% ee) and 21% yield of (R)-21d (>99% ee). Data for (S)-21d: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.57 (d, J = 2.84 Hz, 1H), 8.39 (d, J = 1.08 Hz, 1H), 8.20 (s, 1H), 7.88−7.97 (m, 2H), 7.74 (d, J = 1.76 Hz, 1H), 7.69 (d, J = 2.74 Hz, 1H), 7.65 (dd, J = 2.05, 8.71 Hz, 1H), 7.61 (dd, J = 1.03, 12.28 Hz, 1H), 5.00−5.18 (m, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 1.90 (d, J = 7.04 Hz, 3H). LCMS (ESI): m/z (M + H) = 403.2. 5-(8-Fluoro-3-(1-(3-methoxyquinolin-6-yl)ethyl)[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisoxazole (21e). To a THF solution (120 mL) of N′-(3-fluoro-5-(3-methylisoxazol-5-yl)pyridin-2yl)-2-(3-methoxyquinolin-6-yl)propanehydrazide (19) (4.71 g, 11.2 mmol), and triphenylphosphine (4.40 g, 16.8 mmol) was added trimethylsilylazide (2.2 mL, 16.8 mmol) via syringe followed by dropwise addition of diethyl azodicarboxylate (DEAD) (2.6 mL, 16.8 K

DOI: 10.1021/jm501913a J. Med. Chem. XXXX, XXX, XXX−XXX

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Journal of Medicinal Chemistry mmol). The solution was maintained at rt for 30 min. The solution was concentrated and the residue was purified by MPLC, eluting with 50−80% EtOAc in CH2Cl2 to afford 5-(8-fluoro-3-(1-(3-methoxyquinolin-6-yl)ethyl)[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisoxazole (3.21 g, 7.95 mmol, 71% yield) as a racemic white solid. The racemic product 21e was further purified by superfluid chromatography (SFC) by repeated 1 mL injections of a 25 mg/mL solution of 21e onto a Chiralpak AS-H, 2 cm × 25 cm (i.d. × length) column, eluting with 20% methanol/0.2% diethylamine/80% CO2 to provide enantioenriched products (R)-21e (1.31, 29% yield, 97% ee) and (S)-21e (1.39 g, 31% yield, >99% ee). Data for (R)-21e: As anticipated, the 1H NMR and LCMS data for (R)-21e were identical to those obtained for (S)21e. Data for (S)-21e: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.65 (d, J = 1.08 Hz, 1H), 8.58 (d, J = 2.93 Hz, 1H), 7.93 (d, J = 8.61 Hz, 1H), 7.77 (dd, J = 1.12, 11.59 Hz, 1H), 7.72 (d, J = 1.86 Hz, 1H), 7.69 (d, J = 2.35 Hz, 1H), 7.63 (dd, J = 2.05, 8.71 Hz, 1H), 7.00 (s, 1H), 5.23 (d, J = 7.04 Hz, 1H), 3.88 (s, 3H), 2.27 (s, 3H), 1.89 (d, J = 7.04 Hz, 3H). LCMS (ESI): m/z (M + H) = 404.2. 5-(8-Fluoro-3-(1-(3-(2-methoxyethoxy)quinolin-6-yl)ethyl)[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisoxazole (21f). Prepared from 1-(3-fluoro-5-(3-methylisoxazol-5-yl)pyridin-2-yl)hydrazine (18a) and 2-(3-(2-methoxyethoxy)quinolin-6-yl)propanoic acid hydrochloride in 65% yield (over 2 steps) according to the general procedure described for 21e. Separation of enantiomers provided 25% yield of (S)-21f (>99% ee) and 35% yield of (R)-21f (>99%ee). Data for (S)-21f: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.65 (d, J = 0.98 Hz, 1H), 8.58 (d, J = 2.84 Hz, 1H), 7.92 (d, J = 8.61 Hz, 1H), 7.75 (dd, J = 0.93, 11.49 Hz, 1H), 7.71 (d, J = 1.76 Hz, 1H), 7.70 (d, J = 2.84 Hz, 1H), 7.63 (dd, J = 2.01, 8.66 Hz, 1H), 6.98 (s, 1H), 5.22 (q, J = 7.08 Hz, 1H), 4.12−4.35 (m, 2H), 3.64−3.82 (m, 2H), 3.31 (s, 3H), 2.27 (s, 3H), 1.89 (d, J = 7.04 Hz, 3H). LCMS (ESI): m/z (M + H) = 448.2. 6-(2-(8-Fluoro-6-(3-methylisoxazol-5-yl)[1,2,4]triazolo[4,3a]pyridin-3-yl)propan-2-yl)quinolin-3-ol (22). To a flask charged with 25 (51 mg, 0.10 mmol) was added trifluoroacetic acid (1 mL). The solution was heated at 65 °C for 18 h. The solution was cooled to rt and concentrated. The crude residue was purified by MPLC using a gradient of 0−10% MeOH in CH2Cl2 to afford 30 mg (75% yield) of 22. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.38 (s, 1H), 8.55 (d, J = 2.74 Hz, 1H), 7.87 (d, J = 2.05 Hz, 1H), 7.82 (d, J = 8.80 Hz, 1H), 7.72 (dd, J = 0.88, 11.44 Hz, 1H), 7.58 (d, J = 1.08 Hz, 1H), 7.55 (d, J = 2.35 Hz, 1H), 7.20 (dd, J = 2.15, 8.80 Hz, 1H), 6.83 (s, 1H), 2.17 (s, 3H), 1.98 (s, 6H). LCMS (ESI): m/z (M + H) = 404.4. 5-(8-Fluoro-3-(1-(3-methoxyquinolin-6-yl)cyclopropyl)[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisoxazole (23). Prepared from 1-(3-fluoro-5-(3-methylisoxazol-5-yl)pyridin-2-yl)hydrazine (18a) and 1-(3-methoxyquinolin-6-yl)cyclopropanecarboxylic acid hydrochloride in 22% yield (over 2 steps) according to the general procedure described for 21e. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.56 (d, J = 2.93 Hz, 1H), 8.31 (d, J = 1.17 Hz, 1H), 7.89 (d, J = 8.80 Hz, 1H), 7.84 (dd, J = 1.12, 11.59 Hz, 1H), 7.63 (d, J = 2.64 Hz, 1H), 7.50 (d, J = 2.15 Hz, 1H), 7.33 (dd, J = 2.20, 8.85 Hz, 1H), 7.05 (s, 1H), 3.84 (s, 3H), 2.25 (s, 3H), 1.69−1.85 (m, 4H). LCMS (ESI): m/z (M + H) = 416.4. 5-(8-Fluoro-3-(1-(3-methoxyquinolin-6-yl)propyl)[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisoxazole (24). Prepared from 1-(3-fluoro-5-(3-methylisoxazol-5-yl)pyridin-2-yl)hydrazine (18a) and 2-(3-methoxyquinolin-6-yl)butanoic acid hydrochloride in 47% yield (over 2 steps) according to the general procedure described for (21e). Separation of enantiomers provided 17% yield of 24 (>99% ee, S-enantiomer). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.65 (d, J = 1.08 Hz, 1H), 8.58 (d, J = 2.93 Hz, 1H), 7.93 (d, J = 8.61 Hz, 1H), 7.77 (dd, J = 1.12, 11.59 Hz, 1H), 7.72 (d, J = 1.86 Hz, 1H), 7.69 (d, J = 2.35 Hz, 1H), 7.63 (dd, J = 2.05, 8.71 Hz, 1H), 7.00 (s, 1H), 5.23 (q, J = 7.07 Hz, 1H), 3.88 (s, 3H), 2.52 (d, J = 1.96 Hz, 2H), 2.27 (s, 3H), 1.89 (d, J = 7.04 Hz, 3H). LCMS (ESI): m/z (M + H) = 418.2. 5-(3-(2-(3-(Benzyloxy)quinolin-6-yl)propan-2-yl)-8-fluoro[1,2,4]triazolo[4,3-a]pyridin-6-yl)-3-methylisoxazole (25). Prepared from 1-(3-fluoro-5-(3-methylisoxazol-5-yl)pyridin-2-yl)hydrazine (18a) and 2-(3-(benzyloxy)quinolin-6-yl)-2-methylpropa-

noic acid hydrochloride in 25% yield (over 2 steps) according to the general procedure described for (21e). 5-(5,6-Difluoropyridin-3-yl)-3-methylisoxazole (26). A sealable vessel was charged with 5-chloro-2,3-difluoropyridine (5) (0.35 mL, 3.34 mmol), X-Phos (0.22 g, 0.47 mmol), and palladium(II) acetate (53 mg, 0.23 mmol). Dioxane (16 mL) was added followed by 3-methyl-5-(tributylstannyl)isoxazole (27) (1.87 g, 5.02 mmol). The mixture was purged with Ar, sealed and heated at 100 °C for 3 h. The solution was concentrated in vacuo and the residue purified by MPLC, eluting with 25% EtOAc in hexanes to provide the desired product, which was still contaminated with some residual tin salts. The product was triturated with hexanes (3 × 20 mL) to provide 5-(5,6difluoropyridin-3-yl)-3-methylisoxazole (26) (0.348 g, 53.1% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm d 8.42−8.69 (m, 2H), 7.08 (s, 1H), 2.31 (s, 3H). 3-Methyl-5-(tributylstannyl)isoxazole (27). To a solution of tributyl(ethynyl)tin (55.0 g, 174.6 mmol) in benzene (200 mL) was added nitroethane (13.8 mL, 192.0 mmol), phenyl isocyanate (38.0 mL, 349.1 mmol), and triethylamine (1.4 mL, 9.78 mmol). A reflux condenser was attached, and the solution was heated at 50 °C for 18 h. This resulted in a yellow solution containing a white precipitate. The mixture was diluted with hexanes (200 mL) and then filtered to remove the solid. The filter cake was washed with additional hexanes (500 mL), and these rinses were combined with the mother liquor. The combined solution was washed with saturated aqueous K2CO3 (3 × 100 mL) and brine (1 × 100 mL), then dried (Na2SO4) and concentrated. The residue was dissolved in hexanes and passed through a plug of silica, eluting with hexanes (500 mL), then 5% EtOAc in hexanes (∼400 mL). The resulting solution was then concentrated and subjected to high vacuum for 18 h to provide 3methyl-5-(tributylstannyl)isoxazole (27) (64.32 g, 99.02% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ ppm 6.20 (s, 1H), 2.32 (s, 3H), 1.45−1.70 (m, 6H), 1.34 (t, J = 7.53 Hz, 6H), 1.15 (d, J = 8.22 Hz, 6H), 0.89 (t, J = 7.29 Hz, 9H). Biology. Kinase Assay. IC50 measurements of inhibitor activity against the recombinant c-Met kinase domain were determined using homogeneous time-resolved fluorescence using a gastrin peptide as substrate. Each reaction consists of 10 μL of an 8 nM phosphorylated c-Met kinase domain (WT or mutant), increasing concentrations of inhibitor in a volume of 1.6 and 48 μL of buffer (60 mM HEPES, pH 7.4, 50 mM NaCl, 20 mM MgCl2, 5 mM MnCl2, 2 mM DTT, 0.1 mM Na3VO4, 0.05% BSA) for 30 min at room temperature. An amount of 20 μL of ATP and gastrin (final concentrations are 4 μM for ATP (2/3 of Km) and 1 μM for the biotinylated gastrin) in the same buffer is then added to the reaction in a final volume of 80 μL and incubated at room temperature for 60 min. An amount of 5 μL of the above reaction is then added to a reaction mixture containing 11 nM streptavidin−allophycocyanin (S-APC) and 0.1 nM europium-labeled anti-phosphotyrosine antibody (Eu-PT66) in a final volume of 85 μL for 30 min at room temperature before data capture using a fluorescence plate reader. Cell-Based Assay. IC50 measurements of inhibitor activity on HGF-mediated c-Met autophosphorylation were determined in serumstarved PC-3 cells using a quantitative electrochemiluminescence immunoassay. PC-3 cells were plated in high glucose DMEM with 10% FBS at a density of 20 000 cells/well in 96-well plates. The next day, cells were starved in low glucose DMEM containing 0.1% BSA for 16 h. Cells in the starvation media were then treated with a 10-point serial dilution of the inhibitor for 1 h at 37 °C followed by stimulation with 200 ng/mL of recombinant human HGF for 10 min at 37 °C. Cells were washed once with PBS and lysed (1% Triton X-100, 50 mM Tris, pH 8.0, 100 mM NaCl, Na3VO4, and protease inhibitors). Cell lysates were then used to measure the levels of c-Met phosphorylation using a quantitative assay as follows: A biotinylated antibody against cMet (R&D Systems no. BAF358) was preincubated with streptavidin beads (IGEN no. 110029) for 30 min at rt with rotation. Cell lysates (25 μL) were then added to 25 μL of biotin labeled anti-c-Met antibodies for 1 h at room temperature with shaking. An antiphosphotyrosine antibody 4G10 (Upstate no. 05-321) (12.5 μL) was then added and allowed to incubate for 1 h at rt followed by the L

DOI: 10.1021/jm501913a J. Med. Chem. XXXX, XXX, XXX−XXX

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Journal of Medicinal Chemistry addition of 12.5 μL of an ORI-Tag-labeled antimouse IgG (IGEN no. 110087) for 30 min at rt. PBS (175 μL) was then added to each reaction, and levels of c-Met phosphorylation were measured using an IGEN instrument (Biomek FX). The IC50 values are calculated using Xlfit4-parameter equation. Microsomal Incubations. Rat and mouse liver microsomes were incubated at 0.25 mg/mL protein, 1.0 μM substrate, and 1 mM NADPH in pH 7.4 phosphate buffer at 37 °C. Aliquots were removed from the incubation at 0, 10, 20, 30, and 40 min and added to an equal volume of acetonitrile. The samples were centrifuged and analyzed by LC/MS/MS. CLint was calculated from the in vitro half-life (t1/2) of the disappearance of substrate according to the following equation: CLint = 0.693/(t1/2 × 0.25 mg protein/mL).30

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(3) Comoglio, P. M.; Giordano, S.; Trusolino, L. Drug development of MET inhibitors: targeting oncogene addition and expedience. Nat. Rev. Drug Discovery 2008, 7, 504−516. (4) (a) Cui, J. J. Targeting receptor tyrosine kinase MET in cancer: small molecule inhibitors and clinical progress. J. Med. Chem. 2014, 57, 4427−4453. (b) Porter, J. Small molecule c-Met kinase inhibitors: a review of recent patents. Expert Opin. Ther. Pat. 2010, 20, 159−177. (5) Albrecht, B. A.; Harmange, J.-C.; Bauer, D.; Berry, L.; Bode, C.; Boezio, A. A.; Chen, A.; Choquette, D.; Dussault, I.; Fridrich, C.; Hirai, S.; Hoffman, D.; Larrow, J. F.; Kaplan-Lefko, P.; Lin, J.; Lohman, J.; Long, A. M.; Moriguchi, J.; O’Connor, A.; Potashman, M. H.; Reese, M.; Rex, K. R.; Siegmund, A.; Shah, K.; Shimanovich, R.; Springer, S. K.; Teffera, Y.; Yang, Y.; Zhang, Y.; Bellon, S. F. Discovery and optimization of triazolopyridazines as potent and selective inhibitors of the c-Met kinase. J. Med. Chem. 2008, 51, 2879−2882. (6) All potency values listed as “kinase” or “HTRF” refer to the IC50 measurements of inhibitor activity against the recombinant c-Met kinase domain as described in the Experimental Section under “Kinase Assay.” All potency values listed as “cell” are IC50 measurements of inhibitor activity on HGF-mediated c-Met autophosphorylation and were determined in serum-starved PC-3 cells using a quantitative electrochemiluminescence immunoassay, also described in the Experimental Section under the description of “Cell-Based Assay”. For both assays, n ≥ 2. (7) Boezio, A. A.; Berry, L.; Albrecht, B. K.; Bauer, D.; Bellon, S. F.; Bode, C.; Chen, A.; Choquette, D.; Dussault, I.; Hirai, S.; KaplanLefko, P.; Larrow, J. F.; Lin, M.-H. J.; Lohman, J.; Potashman, M. H.; Rex, K.; Santostefano, M.; Shah, K.; Shimanovich, R.; Springer, S. K.; Teffera, Y.; Yang, Y.; Zhang, Y.; Harmange, J.-C. Discovery and optimization of potent and selective triazolopyridazine series of c-Met inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 6307−6312. (8) Silverman, R. B. Drug Metabolism. The Organic Chemistry of Drug Design and Drug Action; Academic Press: New York, 1992; pp 300− 301. (9) For a review of kinase inhibitor design, see the following: Zuccotto, F.; Ardini, E.; Casale, E.; Angiolini, M. Through the “gatekeeper door”: exploiting the active kinase conformation. J. Med. Chem. 2010, 53, 2681−2694. The hinge binder residue for c-Met is Met1160. (10) General Stille coupling conditions described for the synthesis of intermediate 26 are in the Experimental Section. (11) Wang, Y.; Sarris, K.; Sauer, D. R.; Djuric, S. W. A simple and efficient automatable one step synthesis of triazolopyridines from carboxylic acids. Tetrahedron Lett. 2007, 48, 2237−2240. (12) (a) Laurence, C.; Brameld, K. A.; Graton, J.; Le Questel, J.-Y.; Renault, E. The pKBHX database: toward a better understanding of hydrogen-bond basicity for medicinal chemists. J. Med. Chem. 2009, 52, 4703−4086. (b) Laurence, C.; Gal, J.-F. Thermodynamic and Spectroscopic Scales of Hydrogen-Bond Basicity and Affinity. In Lewis Basicity and Affinity Scales: Data and Measurement; Wiley: New York, 2010; pp 119−135. (13) Incubation of inhibitors with RLM and glutathione led to displacement of the 8-fluorine even in the absence of NADPH. For a review on GSH conjugation see the following: Hayes, J. D.; Pulford, D. J. The glutathione S-transferase supergene family: regulation of GST* and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 1995, 30, 445−600. (14) Hinson, J. A.; Roberts, D. W.; James, L. P. Adverse Drug Reactions. In Handbook of Experimental Pharmacology; Uetrecht, J., Ed.; Springer: New York, 2010; Vol. 196, Part 3, pp 369−405. (15) Roberge, J. Y.; Yu, G.; Mikkilineni, A.; Wu, X.; Zhu, Y.; Lawrence, R. M.; Ewing, W. R. Synthesis of triazolopyridines and triazolopyrimidines using a modified Mitsunobu reaction. ARKIVOC 2007, 12, 132−147. (16) The more potent inhibitor when R2 = F (20) is the (R) enantiomer, and when R2 = H (21), it is the (S) enantiomer. In both cases the methyl group is oriented as drawn in Scheme 2; the stereochemical assignment changes due to the priority assignment rules for fluorine vs hydrogen.

ASSOCIATED CONTENT

S Supporting Information *

Crystallographic data collection and refinement statistics for (R)-20c (PDB code 4XMO) and the cocrystal structure of (S)21f (PDB code 4XYF) with c-Met, the kinase selectivity panel assay data for (S)-21f, and the rat bile-duct cannulation ion chromatograms (10b and (S)-21f). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 617-444-5027. Fax: 617-621-3907. E-mail: emily. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Michael Zhang and Yajing Yang for enzyme and cell assay support, Loren Berry for in vitro PKDM analysis, and Larry Miller and Matt Potter for chiral separation of products bearing stereogenic centers.

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ABBREVIATIONS USED HGF, hepatocyte growth factor; RTK, receptor tyrosine kinase; TDI, time-dependent inhibition; CYP3A4, cytochrome P450 3A4; SAR, structure−activity relationship; CLint, intrinsic clearance; GST, glutathione S-transferase; GSH, glutathione; CL, clearance; Vss, volume of distribution; RLM, rat liver microsome; HLM, human liver microsome; BDC, bile duct cannulation; MsLM, mouse liver microsome; DLM, dog liver microsome; MLM, monkey liver microsome; X-Phos, dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl

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REFERENCES

(1) Gherardi, E.; Birchmeier, W.; Birchmeier, C.; Vande Woude, G. Targeting MET in cancer: rationale and progress. Nat. Rev. Cancer 2012, 12, 89−103. (2) (a) Schmidt, L.; Duh, F. M.; Chen, F.; Kishida, T.; Glenn, G.; Choyke, P.; Scherer, S. W.; Zhuang, Z.; Lubensky, I.; Dean, M.; Allikmets, R.; Chidambaram, A.; Bergerheim, U. R.; Feltis, J. T.; Casadevall, C.; Zamarron, A.; Bernues, M.; Richard, S.; Lips, C. J.; Walther, M. M.; Tsui, L. C.; Geil, L.; Orcutt, M. L.; Stackhouse, T.; Lipan, J.; Slife, L.; Brauch, H.; Decker, J.; Niehans, G.; Hughson, M. D.; Moch, H.; Storkel, S.; Lerman, M. I.; Linehan, W. M.; Zbar, B. Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nat. Genet. 1997, 16, 68−73. (b) Liu, X.; Yao, W.; Newton, R. C.; Scherle, P. A. Targeting the c-Met signaling pathway for cancer therapy. Expert Opin. Invest. Drugs 2008, 17, 997−1011. M

DOI: 10.1021/jm501913a J. Med. Chem. XXXX, XXX, XXX−XXX

Article

Journal of Medicinal Chemistry (17) Atomic coordinates and structure factors for the cocrystal structures of (R)-20c and (S)-21f (pictured in graphical abstract and in Supporting Information) bound to c-Met have been deposited in the PDB with accession codes 4XMO and 4XYF, respectively. (18) Müller, K.; Faeh, C.; Diederich, F. Fluorine in pharmaceuticals: looking beyond intuition. Science 2007, 317, 1881−1886. (19) Note the change of (R) vs (S) assignment changes from compound 20 to compound 21 as the presence of the fluorine atom alters the priority of substituents for stereochemical assignment. (20) For a more detailed description of the methods used in these experiments see the following: Teffera, Y.; Colletti, A. E.; ChristopheHarmange, J.-C.; Hollis, L. S.; Albrecht, B. K.; Boezio, A. A.; Liu, J.; Zhao, Z. Chemical reactivity of methoxy 4-O-aryl quinolines: identification of glutathione displacement products in vitro and in vivo. Chem. Res. Toxicol. 2008, 21, 2216−2222. (21) For an analysis of electronic properties of various fivemembered heterocycles, see the following: Katritzky, A. R.; Pozharskii, A. F. Handbook of Heterocyclic Chemistry, 2nd ed.; Pergamon: Amsterdam, 2000; p 378. (22) See Supporting Information. (23) Similar to compounds 10 and 12, the in vitro predicted clearance (CLint) for compounds 20 and 21 agreed with the observed in vivo clearance. The unbound clearance (CLu) values for (S)-21e and (S)-21f were 14 and 25 L h−1 kg−1, respectively. (24) The parent compound (S)-21e was also observed in all species (peak not shown). (25) The parent compound (S)-21f was also observed in all species (peak not shown). (26) Kinase counterscreen performed at Ambit; see Supporting Information for heat map. (27) Plasma protein binding (PPB) values for (S)-21f: mouse = 85.4%, human = 94.7%, dog = 85.1%, rat = 97.8%, and monkey = 92.9%. (28) For a discussion of the relationship between c-Met target coverage and tumor growth inhibition, see the following: Yamazaki, S.; Skaptason, J.; Romero, D.; Lee, J. H.; Zou, H. Y.; Christensen, J. G.; Koup, J. R.; Smith, B. J.; Koudriakova, T. Pharmacokinetic− pharmacodynamic modeling of biomarker response and tumor growth inhibition to an orally available cMet kinase inhibitor in human tumor xenograft mouse models. Drug Metab. Dispos. 2008, 36, 1267−1274. (29) Procedure adapted from the following: Lee, S.; Beare, N. A.; Hartwig, J. F. Palladium-catalyzed α-arylation of esters and protected amino acids. J. Am. Chem. Soc. 2001, 123, 8410−8411. (30) Obach, R. S. Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: an examination of in vitro half-life approach and nonspecific binding to microsomes. Drug Metab. Dispos. 1999, 27, 1350−1359.

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DOI: 10.1021/jm501913a J. Med. Chem. XXXX, XXX, XXX−XXX