Discovery of Dual Leucine Zipper Kinase (DLK ... - ACS Publications

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Discovery of Dual Leucine Zipper Kinase (DLK, MAP3K12) Inhibitors with Activity in Neurodegeneration Models Snahel Patel,† Frederick Cohen,†,# Brian J. Dean,§ Kelly De La Torre,† Gauri Deshmukh,§ Anthony A. Estrada,† Arundhati Sengupta Ghosh,‡ Paul Gibbons,† Amy Gustafson,∥ Malcolm P. Huestis,† Claire E. Le Pichon,‡,∞ Han Lin,‡ Wendy Liu,† Xingrong Liu,§ Yichin Liu,∥ Cuong Q. Ly,† Joseph P. Lyssikatos,†,× Changyou Ma,⊥ Kimberly Scearce-Levie,‡ Young G. Shin,§,○ Hilda Solanoy,‡ Kimberly L. Stark,‡ Jian Wang,⊥ Bei Wang,‡,△ Xianrui Zhao,† Joseph W. Lewcock,*,‡ and Michael Siu*,† Departments of †Discovery Chemistry, ‡Neurosciences, §Drug Metabolism and Pharmacokinetics, and ∥Biochemical and Cellular Pharmacology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States ⊥ Department of Chemistry, WuXi AppTec Co., Ltd., 288 Fute Zhonglu, Wai Gao Qiao Free Trade Zone, Shanghai, 200131, P. R. China ABSTRACT: Dual leucine zipper kinase (DLK, MAP3K12) was recently identified as an essential regulator of neuronal degeneration in multiple contexts. Here we describe the generation of potent and selective DLK inhibitors starting from a high-throughput screening hit. Using proposed hingebinding interactions to infer a binding mode and specific design parameters to optimize for CNS druglike molecules, we came to focus on the di(pyridin-2-yl)amines because of their combination of desirable potency and good brain penetration following oral dosing. Our lead inhibitor GNE-3511 (26) displayed concentration-dependent protection of neurons from degeneration in vitro and demonstrated dose-dependent activity in two different animal models of disease. These results suggest that specific pharmacological inhibition of DLK may have therapeutic potential in multiple indications.

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required for stress-induced JNK signaling in neurons.13,14 Recent work from our group and others have identified the importance of DLK as a central regulator of neuronal degeneration in multiple contexts including models of Parkinson’s disease, glaucoma/optic neuropathy, and excitotoxic neurodegeneration, suggesting that this kinase may represent an appealing target for treatment of these and related indications.15−21 To date, however, only one study has examined the consequence of DLK inhibition using previously identified compounds which also target other kinases.18 In this contribution, we describe the development of the first potent and selective DLK inhibitors derived from a high-throughput screening effort.

INTRODUCTION Neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, represent significant unmet medical needs, yet no therapies exist that are able to slow the course of neuronal loss. Consequently, considerable interest exists for molecules that target the mechanisms underlying degeneration in these and related contexts, as they represent an attractive approach for treatment.1,2 For example, the c-Jun N-terminal kinase (JNK) signaling pathway has received significant attention in this regard because of its proposed role in both acute and chronic neurodegenerative paradigms. Genetic deletion or inhibition of JNKs in neurons has been shown to be potently neuroprotective in a number of settings,3−7 and evidence of pathway activity exists in multiple indications ranging from traumatic brain injury (TBI) to Alzheimer’s disease.8,9 On the basis of these data, significant effort has been reported toward the development of brain-penetrant JNK inhibitors for the treatment of these indications,10 but these endeavors have not yet resulted in progression of compounds into the clinic, highlighting the challenges associated with this approach.11,12 We hypothesized that upstream regulators of JNK may exist and targeting these regulators may provide an alternative strategy to inhibiting JNK activity directly. The MAP3K DLK (dual leucine zipper kinase) appears to be an ideal candidate for this role, as it displays neuronal-specific expression and is © 2014 American Chemical Society

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RESULTS AND DISCUSSION In order to identify novel small molecule DLK inhibitors, our internal compound collection was screened against the kinase domain of DLK.22 For the primary screen, percent inhibition was measured at a compound concentration of 5 μM. For compounds that showed >50% inhibition, half-maximal Special Issue: New Frontiers in Kinases Received: September 10, 2014 Published: October 23, 2014 401

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Schrodinger’s Jaguar at the B3LYP/6-31G** level of theory confirmed that the proposed planar conformation was the lowest energy conformation by over 1.7 kcal/mol.35,36 As a result of this low energy conformation, the piperidine benzamide and methyl groups are oriented as depicted in Figure 2 toward solvent and the P-loop regions, respectively. On the basis of this proposed binding mode, we evaluated 2aminopyridines as 2-aminothiazole bioisosteric replacements. Either a 2-amino-4-methylpyridine or 2-amino-5-methylpyridine could potentially overlay with the 2-amino-5-methylthiazole of 1. Upon experimental evaluation, the matched pairs 2-amino-5-methylpyridine 2 (Ki = 0.401 μM)/2-amino-4methylpyridine 3 (Ki = 0.085 μM) and 2-amino-5-trifluoromethylpyridine 4 (Ki = 0.691 μM)/2-amino-4-trifluoromethylpyridine 5 (Ki = 0.024 μM) proved the C4 position to be the most optimal for potency (Table 1). Despite the decrease in lipophilic ligand efficiency (LipE) on going from an aminothiazole to an aminopyridine, the removal of a potential toxicophore was realized.37 The consistent LipE (∼3) of the C4-alkylpyridine analogs in Table 1 suggested that the

inhibitory concentrations were determined. This assay was also used for characterization of subsequent hit optimization efforts followed by a cell-based assay that measured levels of JNK phosphorylation (p-JNK) induced by DLK overexpression. On the basis of these activity measurements and physicochemical properties, N-(pyrimidin-4-yl)thiazol-2-amine 1 (Figure 1) was

Figure 1. HTS hit 1.

selected for optimization. Inhibitor 1 had acceptable potency (DLK Ki = 0.035 μM, p-JNK IC50 = 0.641 μM), permeability (MDR1-MDCK BA/AB = 2.2, AB = 4.3 × 10−6 cm/s), ligand efficiency23 (LE = 0.37), and lipophilic ligand efficiency24,25 (LipE = 3.6). The property space of inhibitor 1 (ClogP and ClogD = 3.9, tPSA = 71 Å2, cpKa = 6.8, HBD = 1, MW = 398, CNS MPO = 4.2) was acceptable for optimization of a kinase inhibitor for brain penetration.26,27 In addition to improvement in potency, selectivity against the JNK pathway kinases (DLK → MKK4/7 → JNK2/3 → cJun) and MLK 1/2/3 (kinases most related to DLK)28 is also necessary before in vivo evaluation. For initial SAR development, alternatives to the literature prevalent kinase hinge-binding 2-amino-5-substituted thiazole motif29,30 were examined because of concerns about selectivity and metabolic liabilities of this group (reactive metabolite formation).31 With no crystal structures of DLK in the public domain to guide initial SAR, we proposed a binding mode for compound 1, as outlined in Figure 2, where the 2-aminothiazole binds to

Table 1. Heteroarylamine SARa

Figure 2. Proposed binding mode of HTS hit 1 in DLK.

the kinase domain hinge residues via two hydrogen-bonding interactions and a nonclassical thiazole C4−H interaction.32 The favorable electrostatic pyrimidine N3 to thiazole S1 interaction was proposed to orient these rings coplanar.33,34 Generation of reasonable low energy conformations using OpenEye’s OMEGA tool of the N-(2,6-dimethylpyrimidin-5yl)-5-methylthiazol-2-amine substructure of compound 1 and quantum mechanical minimization of resultant structures using

a

Compounds were tested as racemates. All assay results represent the geometric mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the reported mean. bLipE = −log Ki − ClogP. 402

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Table 2. 2- and 6-Pyrimidine Substitutiona

a

Compounds 12−14 were tested as racemates. All assay results represent the geometric mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the reported mean. bLiver microsome-predicted hepatic clearance. cH/R/M = human/rat/mouse. dLipE = −log Ki − ClogP.

mouse). Further potency exploration and an attempt to increase microsomal stability of pyrrolidine 13 (due to known soft spots)41 led us to synthesize 3,3-difluoropyrrolidine at the C2 position of the pyrimidine. Selection of the gemfluorinated moiety was based on its demonstrated ability to improve pharmacokinetic profile relative to mono- and tetrafluorinated counterparts.42,43 3,3-Difluoropyrrolidine 14 maintained biochemical and cellular potency (Ki = 0.007 μM and p-JNK IC50 = 0.408 μM) with good to moderate liver microsome stability. Transposition of the 3-piperidine to the 4piperidine 15 eliminated the stereogenic center while maintaining potency (Ki = 0.002 μM, p-JNK IC50 = 0.410 μM). These basic amines with two hydrogen bond donors were efflux substrates in an in vitro MDR1−MDCK assay (14, efflux ratio of 68). To reduce the basicity of the analogs and the number of hydrogen bond donors, we examined the 4tetrahydropyran 16 and N-acetyl-4-piperidine 17, both of which maintained reasonable potency; however, this potency came at the expense of in vitro metabolic stability. Reducing lipophilicity at the C2 position (by either removing a carbon or introduction of a polar atom) resulted in the loss of biochemical potency (3,3-difluoroazetidine 18 Ki = 0.166 μM; morpholine 19, Ki = 0.431 μM) with no apparent improvement in metabolic stability.

improvement in observed potency was due to lipophilicity instead of specific interactions between this position of the ligand with DLK (i.e., methylpyridine 3 LipE = 2.9 and trifluoromethylpyridine 5 LipE = 2.9). Further evaluation of C4 substituents showed isopropyl 7, cyclopropyl 8, cyano 10, and methoxy 11 were tolerated with the cyano being the most lipophilic efficient group (albeit with high tPSA). An approximate 10- to 20-fold shift between the biochemical and cellular assay was observed with these inhibitors, which is in line with the ATP Km reported for DLK.38 Benzamide replacements with tPSA < 80 Å2 (for CNS penetration) proved to be unfruitful (not reported). Therefore, we adopted a strategy to further optimize trifluoromethylpyridine 5 by redistribution of the polarity and atoms of the benzamide to examine the C2 position of the pyrimidine core. For comparison, removal of the benzamide of 5 (compound 12, Table 2) lost binding affinity for DLK (∼30×). We surmised that further hit optimization could be developed by growing the vector presumably under the P-loop region.39,40 A cyclic secondary amine was initially chosen at the C2 position of the pyrimidine to avoid additional hydrogen bond donors on the inhibitors that may diminish brain penetration properties. Pyrrolidine 13 showed improved biochemical and cellular potency (DLK Ki = 0.007 μM, p-JNK IC50 = 0.109 μM) with moderate stability in liver microsomes (human, rat, and 403

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examining the functional consequence of endogenous DLK inhibition in neurons.14 As expected, the IC50 for compound 20 in the axon degeneration assay was higher than the p-JNK IC50 because near complete inhibition of DLK activity is likely required to achieve neuroprotection in this context. In order to further reduce lipophilicity of 20 and related analogs, the previously identified polar and lipophilic efficient 4cyano-2-aminopyridine hinge binder (Table 1) was used for further exploration on the pyridine core. Cyanopyridine 21 (direct analog of 20) maintained lipophilic ligand efficiency (LipE = 3.5) but introduced efflux. As in the case with the pyrimidine scaffold, 4-tetrahydropyran 22 was tolerated and gave rise to a potent inhibitor of DLK. The 4-cyano-4tetrahydropyran 23 improved metabolic stability, yet the molecule possessed a high tPSA value. Truncated analogs of 23 that maintain a quaternary carbon center (24 and 25) were potent and avoided efflux in an in vitro MDR1−MDCK assay despite relatively high tPSA. The steric hindrance around the tertiary nitrile 24 and tertiary alcohol 25 potentially reduces the “effective” polar surface area of these compounds.44 An oxetane45−47 was utilized to reduce basicity of the piperidine in order to avoid efflux, and the resultant N-oxetanylpiperidine GNE-3511 (26)22 proved to be one of the most cell potent

In an effort to modify the central core to reduce tPSA, a pyridine scaffold was explored. Our initial efforts focused on changing the 2,4-diaminopyrimidine to a 2,6-diaminopyridine scaffold in order to maintain an overall planar conformation (Figure 3). The initial analog in this endeavor (20, Table 3)

Figure 3. Core change to reduce tPSA while maintaining planar conformation of inhibitors.

gave rise to an improvement in potency (Ki = 0.001 μM, p-JNK IC50 = 0.069 μM) and lipophilic efficiency compared to pyrimidine 17. Compound 20 also reached sufficient cellular potency to elicit protection of primary rat dorsal root ganglion (DRG) neurons in an in vitro axon degeneration assay with an IC50 of 0.171 μM. This assay provides a method to further characterize the activity of selected compounds through Table 3. Pyridine Core C4-Substituent SARa

a

All assay results represent the geometric mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the reported mean. bMDCK−MDR1 human P-gp transfected cell line. Basolateral-to-apical/apical-to-basolateral. Units = ×10−6 cm s−1. cLiver microsome-predicted hepatic clearance. dH/R/M = human/rat/mouse. eLipE = −log Ki − ClogP. 404

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Table 4. Pyridine Core C6-Substituent SARa

a

All assay results represent the geometric mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the reported mean. bMDCK−MDR1 human P-gp transfected cell line. Basolateral-to-apical/apical-to-basolateral. Units = ×10−6 cm s−1. cLiver microsome-predicted hepatic clearance. dH/R/M = human/rat/mouse. eLipE = −log Ki − ClogP.

Table 5. Kinase Selectivity of Compound 26a IC50 (nM)

a

DLK Ki (nM)

MKK4

MKK7

JNK1

JNK2

JNK3

MLK1

MLK2

MLK3

5000

>5000

129

514

364

67.8

767

602

IC50 values were determined at Invitrogen.

inhibitors. The N-oxetanylazetidine 27 was equally potent against DLK but was labile in microsomes despite a lower ClogP. On the basis of these results, the 2-(6-(3,3difluoropyrrolidin-1-yl)pyridin-2-ylamino)isonicotinonitrile subscaffold clearly tolerated a variety of substituents on the pyridine C4 position. Examination of the C6 position of the pyridine was continued with the 2-(4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-ylamino)isonicotinonitrile subscaffold (Table 4). 3Methoxyazetidine 28 and azetidine 29 lost potency with no benefit in metabolic stability or efflux properties (in the case of 28). In addition to amines, ethers such as the cyclobutyl ether analog 30 maintained significant potency (Ki = 0.0002 μM). Larger alkyl groups provided increased binding affinity for DLK at this position (methyl 31 Ki = 0.093 μM, ethyl 32 Ki = 0.018 μM, and cyclopropyl 33 Ki = 0.001 μM). Of the examples in Tables 3 and 4, compound 26 provided the best balance of potency, in vitro metabolic stability, and efflux properties. Inhibitor 26 had very good selectivity over the other JNK pathway kinases and homologues of DLK (Table 5). Compound 26 also exhibited very good overall kinase selectivity in an Invitrogen panel of 298 kinases at 0.1 μM (>200 × DLK Ki) (Figure 4). Lastly, inhibition of DLK by 26 translated to potent protection of primary neurons in an in vitro axon degeneration assay with IC50 = 0.107 μM.

Pharmacokinetic evaluation revealed 26 exhibited moderate (mouse, rat, and cynomolgus) to high (dog) in vivo plasma clearances, moderate volumes of distribution, short half-lives, and brain penetration sufficient to enable examination in animal models of neurodegeneration (Table 6). DLK inhibitor 26 was then tested in the mouse optic nerve crush model of axonal injury, which mimics the degeneration that occurs in glaucoma or optic neuropathy.48,49 Our previous studies demonstrated that loss of DLK expression resulted in protection of retinal ganglion cell neurons from degeneration as well as an attenuation of downstream signaling following injury.17,18 In this and other neuronal injury models, phosphorylation of c-Jun (p-c-Jun) is strongly induced by injury in a DLK/JNK dependent fashion and could thus be used as a pharmacodynamic readout of DLK inhibition in vivo.3,5,17 Animals were dosed orally with either inhibitor 26 at two dose levels or vehicle control 30 min prior to nerve crush injury. Six hours after insult, levels of p-c-Jun in retina were measured using a MSD assay. Treatment with inhibitor 26 resulted in a dosedependent reduction of p-c-Jun present in retina (Figure 5). To examine the activity of inhibitor 26 in brain and further confirm its ability to inhibit DLK activity in vivo, the MPTP (1methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson’s disease was used.50 In this model, MPTP treatment results in dramatic increases in p-c-Jun within the dopaminergic neurons of the substantia nigra by 24 h which leads to the 405

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Figure 4. Kinase selectivity of compound 26. Invitrogen panel of 298 kinases. Kinases with >80% inhibition at 0.1 μM are EphA1, Lck, Src, STK16, Syk, Flt3 (D835Y), Src_N1.

Table 6. PK Properties of DLK Inhibitor 26a species

CLp (mL min−1 kg−1)

Vdss (L kg−1)

t1/2 (h)

F (%)

Bu/Pu b

mouse rat dog cynomolgous

56 30 41 16

2.5 3.7 6.5 3.1

0.6 1.8 4 2.4

45 63 32 19

0.24 at 6 h 0.7

CSF/Pu c 0.4 0.4 0.6

Compounds were dosed iv (1 mg kg−1) as a 60% PEG solution and po (5 mg kg−1) as an aqueous suspension with 1% methylcellulose. bunbound brain/unbound plasma AUC ratio (unless noted otherwise). cCSF/unbound plasma AUC ratio.

a

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degeneration of these neurons at later time points.5 To test the ability of 26 to reduce the induction of p-c-Jun, animals were dosed with 37.5 or 75 mg/kg or vehicle 30 min prior to the initiation of MPTP treatment and again at 12 and 24 h after the first dose in order to maintain necessary concentrations of compound in brain. Brains were dissected at 1 h after the final dose of 26, and the number of p-c-Jun positive neurons was measured. The high dose of inhibitor 26 resulted in a complete suppression of c-Jun phosphorylation, while the low dose reduced the number of p-c-Jun positive cells to an intermediate level. Taken together, these data demonstrated that compound 26 is an orally bioavailable and brain-penetrant DLK inhibitor with activity in multiple animal models of neurodegenerative disease. This, along with the potent neuroprotective activity observed with DLK inhibitor 26 in vitro, suggests that DLK inhibition has attractive therapeutic potential.

CHEMISTRY The preparation of compounds 1−12 was performed as shown in Scheme 1 by reacting commercially available (±)-tert-butyl 3-(6-chloro-2-methylpyrimidin-4-yl)piperidine-1-carboxylate (34, CAS no. 1361116-19-9) with the required heteroarylamine via a Buchwald−Hartwig reaction.51−53 Subsequent acidmediated N-Boc deprotection resulted in a crude amine which was directly coupled with benzoic acid. The syntheses of 13−19 were carried out as outlined in Scheme 2. C6Substituted dichloropyrimidines tert-butyl 4-(2,6-dichloropyrimidin-4-yl)piperidine-1-carboxylate (39, CAS no. 1439823-014) and 2,4-dichloro-6-(tetrahydro-2H-pyran-4-yl)pyrimidine (40, CAS no. 1417519-39-1) are commercially available. The synthesis of 3-(2,6-dichloropyrimidin-4-yl)piperidine-1-carboxylic acid tert-butyl ester (38) commenced with the acylation of (±)-1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid (35) with potassium monoethyl malonate to form ketoester 36 (CDI, MgCl2, CH3CN).54 Condensation of 36 with urea under basic conditions produced 2,4-dihydroxypyrimidine 37, which 406

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Figure 5. GNE-3511 (26) displays activity in two animal models of neurodegeneration. (A) Levels of p-c-Jun in retinal lysates were measured by ELISA following nerve crush and treatment with inhibitor 26. Values are presented relative to uncrushed vehicle controls. N = 5 for vehicle and N = 6 for both inhibitor 26 treated groups: (∗∗∗) p < 0.001. (B) Number of p-c-Jun positive cells/mm2 in animals following MPTP treatment with or without inhibitor 26 treatment as compared to controls. N = 8/group from two cohorts: (∗∗) p < 0.01, (∗∗∗) p < 0.01. For both panels, bars represent the mean and error bars represent SEM. Dots represent data points from individual animals in the study. In panel B, circles and triangles represent animals from independent cohorts.

Scheme 1. Preparation of 2-Methylpyrimidine Analogsa

The pyridine analogs (Tables 3 and 4) were synthesized according to the synthetic routes shown in Schemes 3−7. The majority of the pyridine analogs were synthesized from 2,6dichloro-4-iodopyridine (43) using various metal-mediated cross-coupling reactions. Suzuki−Miyaura cross coupling of N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (44) with 2,6-dichloro-4-iodopyridine (43, Scheme 3) provided an intermediate pyridine suitable for additional substitution with 3,3-difluoropyrrolidine via SNAr displacement followed by a Buchwald−Hartwig amination with 2-amino-4-trifluoromethypyridine. The resulting alkene 47 was reduced through hydrogenation. Protecting group removal followed by acylation with acetyl chloride afforded 20. To circumvent an alkene reduction, Negishi cross coupling of 2,6-dichloro-4-iodopyridine (43) with N-Boc 4-iodopiperidine (50, Scheme 4) provided the intermediate 2,6-dichloropyridine 51.55 From this intermediate, SNAr with 3,3-difluoropyrrolidine provided the corresponding 4-substituted 2-amino-6-chloropyridine 52. Ntert-Butoxycarbonyl deprotection and subsequent reductive amination with oxetan-3-one provided the N-oxetanylpiperidine 53.56 Compound 26 was obtained from chloropyridine 53 via Buchwald−Hartwig amination with 2-amino-4-cyanopyridine. Alternatively, initial introduction of the oxetane (or acetyl) followed by 2-amino-4-cyanopyridine provided intermediate 54 which allowed for late-stage palladium catalyzed Buchwald−Hartwig amination49−51 (compounds 21, 28, 29), Beller etherification 57 (ether 30), and B-alkyl Suzuki coupling58,59 (ethyl 32 and cyclopyropyl 33). Tetrahydropyran 22 and N-oxetanylazetidine 27 were obtained using the reaction sequence in Scheme 5 from an initial Negishi coupling of 4-iodotetrahydropyran and N-Boc-3-iodoazetidine, respectively. Introduction of a quaternary nitrile at the C4 of pyridines occurred via regioselective addition of α-lithiated nitriles to 2,4,6-trichloropyridine (61) to provide 4-substituted-2,6dichloropyridines 62 and 63 (Scheme 6).60−63 From these intermediates, sequential substitution with 2-amino-4-cyanopyridine and 3,3-difluoropyrrolidine provided 23 and 24. The tertiary alcohol 25 was synthesized as shown in Scheme 7. Methyl Grignard addition to methyl 2,6-dichloroisonicotinate (66) gave 2-(2,6-dichloropyridin-4-yl)propan-2-ol (67). SNAr displacement of the resultant 2,6-dichloropyridine 67 with 3,3difluoropyrrolidine provided 2-amino-6-chloropyridine 68

a

Reagents and conditions: (a) (i) R-NH2, Pd2(dba)3, Xantphos, tBuONa, 1,4-dioxane, 80 °C, 3 h; (ii) HCl, 1,4-dioxane, MeOH, 23 °C, 16 h; (iii) benzoic acid, HBTU, Et3N, DMF, 40 °C, 16 h; (b) (i) 4(trifluoromethyl)pyridin-2-amine, Pd2(dba)3, Xantphos, t-BuONa, 1,4dioxane, 80 °C, 3 h; (ii) HCl, 1,4-dioxane, MeOH, 23 °C, 16 h, 10% yield.

upon treatment with phosphoryl trichloride resulted in the (±)-tert-butyl 3-(2,6-dichloropyrimidin-4-yl)piperidine-1-carboxylate (38). Buchwald−Hartwig reaction of the requisite C6-substituted-2,4-dichloropyrimidines 38−40 with 2-amino-4trifluoromethylpyridine followed by SNAr with the selected secondary amine and acid-mediated Boc group removal (if appropriate) furnished 13−16, 41, and 42. Acetamide analogs 17−19 were obtained by acylation with acetic anhydride. 407

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Scheme 2. Preparation of 3-Piperidine, 4-Piperidine, and 4-Tetrahydropyran Analogs 13−19a

Reagents and conditions: (a) potassium monoethyl malonate, CDI, MgCl2, CH3CN, 23 °C, 16 h, 86% yield; (b) urea, NaOMe, EtOH, 80 °C, 72 h, 42% yield; (c) (i) POCl3, reflux, 3 h; (ii) (Boc)2O, NaHCO3, H2O, THF, 23 °C, 16 h, 17% yield; (d) (i) 4-(trifluoromethyl)pyridin-2-amine, Pd2(dba)3, Xantphos, t-BuONa, 1,4-dioxane, 80 °C, 3 h; (ii) R-amine, (i-Pr)2NEt, DMF, 85 °C, 16 h; (iii) HCl, 1,4-dioxane, MeOH, 16 h; (e) Ac2O, DMAP, (i-Pr)2NEt, CH3CN, CH2Cl2, 0 °C, 1 h. a

Scheme 3. Preparation of 20a

Reagents and conditions: (a) Pd(dppf)Cl2, K2CO3, 1,4-dioxane, H2O, 90 °C, 56% yield; (b) 3,3-difluoropyrrolidine hydrochloride, (i-Pr)2NEt, 1,4dioxane, 120 °C microwave, 37% yield; (c) 2-amino-4-trifluoromethylpyridine, t-BuONa, RuPhos, RuPhos palladium(II) phenethylamine chloride, 120 °C microwave, 38% yield; (d) H2, MeOH, 10% Pd/C, 40 bar, 84% crude yield. (e) HCl in 1,4-dioxane, CH2Cl2; (f) (i-Pr)2NEt, THF, acetyl chloride, 45% yield.

a

which upon treatment with 2-amino-4-cyanopyridine afforded 25.

low tPSA and hydrogen bond donor count led to compound

CONCLUSIONS Recent data from multiple models have revealed a central role for dual leucine zipper kinase (DLK, MAP3K12) in the regulation of neuronal degeneration. From a high-throughput screen, we identified aminothiazole 1 which served as a practical starting point for the lead optimization of a brain-penetrant kinase inhibitor. Modification of the 2-aminothiazole hinge binder, pyrimidine scaffold, and substituents while maintaining

penetrant inhibitor of DLK reported. DLK inhibitor 26

26, which represents the first potent, selective, and brain-

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protected primary neurons in an in vitro axon degeneration assay as well as reduced phosphorylation of the downstream transcription factor c-Jun, a marker of neuronal injury, in both the optic nerve crush and MPTP mouse models of neurodegeneration following oral dosing. 408

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Scheme 5. Preparation of 22 and 27a

Scheme 4. Preparation of 2-(4-(piperidin-4-yl)pyridin-2ylamino)isonicotinonitrilesa

a

Reagents and conditions: (a) (i) 4-bromotetrahydro-2H-pyran, Zn, TMSCl, 1,2-dibromoethane; (ii) Pd(dppf)Cl2, CuI, DMA, 80 °C, 70% yield; (b) 3,3-difluoropyrrolidine hydrochloride, NMP, 130 °C; (c) 2amino-4-cyanopyridine, t-BuONa, RuPhos, RuPhos palladium(II) phenethylamine chloride, 120 °C microwave, 29% yield; (d) (i) 1Boc-3-iodoazetidine, Zn, TMSCl, 1,2-dibromoethane; (ii) Pd(dppf)Cl2, CuI, DMA, 80 °C, 54% yield; (e) (i) TFA, CH2Cl2, (ii) Et3N, 3oxetanone, Na(OAc)3BH, THF, 50 °C, 80% yield; (f) 2-amino-4cyanopyridine, Pd2dba3, Xantphos, Cs2CO3, 1,4-dioxane, 80 °C, 51% yield; (g) RuPhos palladium(II) phenethylamine chloride, RuPhos, 3,3-difluoropyrrolidine hydrochloride, t-BuONa, 3 Å molecular sieves, THF, 90 °C, 25% yield.

a

Reagents and conditions: (a) (i) Zn, TMSCl, 1,2-dibromoethane; (ii) Pd(dppf)Cl2, CuI, DMA, 80 °C, 62% yield; (b) 3,3-difluoropyrrolidine hydrochloride, NMP, 130 °C, 47% yield; (c) (i) TFA, CH2Cl2, (ii) Et3N, 3-oxetanone, Na(OAc)3BH, THF; 85% yield for 53, 99% crude yield for 54a; (d) 2-amino-4-cyanopyridine, Pd2(dba)3, BINAP, Cs2CO3, 1,4-dioxane, reflux, 77% yield; (e) (i) TFA, CH2Cl2, (ii) Ac2O, Et3N, DMAP, CH2Cl2; quantitative (crude); (f) 2-amino-4cyanopyridine, Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane, 80 °C; 78% yield; (g) RuPhos palladium(II) phenethylamine chloride, RuPhos, Ramine, t-BuONa, 3 Å molecular sieves, THF, 90 °C; (h) R−OH, Pd(OAc)2, Ad-BippyPhos, Cs2CO3, 3 Å molecular sieves, toluene, H2O, 120 °C; (i) Pd(OAc)2, n-BuPAd2, R-BF3K, Cs2CO3, toluene, H2O, 110 °C.

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X-100, blocked for 1 h with SuperBlock before the overnight incubation with the primary antibodies at 4 °C. The secondary antibodies were incubated for 2 h, washed with PBS, and then stained with Hoechst 33342 dye. The cell plates were imaged on Opera Imaging Platform. In Vitro Axon Degeneration Cell Assay. Assay was conducted as previous described14,64 with the following modifications. Dorsal root ganglion (DRG) neurons were freshly dissected from E14.5 rat embryos. The resulting cell suspension was filtered through a 50 μm sieve (Partec) to remove remaining tissue pieces, centrifuged 5 min at 1000 rpm, and resuspended in DRG culture medium (DMEM/F12 containing 1× N3 supplement, 0.18% glucose, 25 ng/mL NGF). Neurons were then plated on a 384-well dish at a density of 1200− 2000 cells per well on top of the astrocyte monolayer. To inhibit cell proliferation, medium was supplied with 200 μM uridine and 100 μM 5-fluorodeoxyuridine the next day. DRGs were cultured for 4 days prior to the assay. In Vitro Transporter Assays. Madin−Darby kidney cells (MDCK) stably transfected with human MDR1 (Pgp) were obtained from the National Institutes of Health, (Bethesda, MD). Cells were

EXPERIMENTAL SECTION

Methods. DLK Biochemical Assay. The biochemical assay was performed as previously described.22 p-JNK Cell Assay. 7500 HEK293 cells stably transfected with Doxinducible human DLK in 40 μL of DMEM with 10% serum were seeded into each well of 384-well poly-D-lysine coated plates. The plates were incubated at 37 °C for 20−24 h prior to the addition of 5 μL of 60 μM doxycycline in DMEM. After incubation with doxycycline at 37 °C for approximately 20 h, 5 μL of DLK inhibitors in DMEM was added, and cells were incubated at 37 °C for an additional 5.5 h. The cells were then washed with PBS, permeabilized with 0.1% Triton 409

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Scheme 6. Preparation of Tertiary Nitriles 23 and 24a

harvested with trypsin and seeded on Millipore Millicell 24-well plates at initial concentrations of 2.0 × 105 cells/mL and allowed to grow for 5 days. Cell monolayers were equilibrated in transport buffer (Hank’s balanced salt solution with 10 mM Hepes, pH 7.4) for 60 min at 37 °C with 5% CO2 and 95% relative humidity prior to the experiment. Dose solutions were prepared in transport buffer and consisted of test compounds (5 μM) and the monolayer integrity marker lucifer yellow (100 μM). The dose solutions were added to the donor chambers, and transport buffer was added to all receiver chambers. The transport was examined in the apical to basolateral (A−B) and basolateral to apical (B−A) directions. The receiver chambers were sampled (50 μL) at 60, 120, and 180 min and were replenished with fresh transport buffer after the 60 and 120 min samplings. Lucifer yellow permeability was used as a marker of monolayer integrity during the experiment. Compound concentrations in the donor and receiving compartments were determined by LC−MS/MS analysis. The apparent permeability (Papp) in the apical to basal A−B and basal to apical B−A directions was calculated as follows: Papp = (dQ/dt)[1/(AC0)], where dQ/dt = rate of compound appearance in the receiver compartment, A = surface area of the inset, and C0 = initial substrate concentration at T = 0. The efflux ratio (ER) was calculated as Papp(B−A)/Papp(A−B). Animal Models. All experiments with mice were performed under animal protocols approved by the Animal Care and Use Committee at Genentech and adhere to ACS Ethical Guidelines for animal studies. For all in vivo studies, C57Bl/6 mice were dosed with compound 26 orally as an MCT suspension. Optic nerve crush studies were conducted as described,17 except p-c-Jun was measured at 6 h by MSD ELISA (Meso Scale Discovery). For MPTP studies, animals were administered four ip doses of 20 mg/kg MPTP (1-methyl-4-phenyl1,2,3,6-tetrahydropyridine), with each dose separated by 4 h. Twentyfive hours after the first MPTP dose, animals were sacrificed and processed essentially as described.11 For each animal, the p-c-Jun positive cell number/mm2 in five sections was averaged to generate a value for that animal. Synthesis. General. All commercially available reagents and solvents were used as received. Reactions using air- or moisturesensitive reagents were performed under an atmosphere of nitrogen using freshly opened EMD DriSolv solvents. Reaction progress was monitored by TLC and/or LCMS. Flash column chromatography was performed with Isco CombiFlash Companion systems using prepacked silica gel columns (40−60 μm particle size RediSep or 20−40 μm spherical silica gel RediSep Gold columns or similar columns from other vendors). Preparative reverse phase HPLC purifications were performed on a Varian Prostar instrument, using a Phenomenex Gemini-NX C-18 (3 cm × 5 cm, 5 μm) stationary phase, with 0.1% aqueous formic acid/acetonitrile or 0.1% aqueous ammonium hydroxide/acetonitrile gradients as the mobile phase (typically 5− 85% acetonitrile over 10 min) with a flow rate of 60 mL/min. NMR spectra were measured on a Bruker 300 or 400 MHz spectrometer, and chemical shifts were reported in ppm downfield from TMS using residual nondeuterated solvent as internal standards (CHCl3, 7.26 ppm; DMSO, 2.50 ppm; MeOH, 3.31 ppm). The following abbreviations are used: br = broad signal, s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet. The purity of final compounds was verified by HPLC to be >95% in all cases using either of the following methods: (1) Agilent 1200 instrument with an Agilent SB C-18 (2.1 mm × 30 mm, 1.8 μm particle size) stationary phase and a gradient of water/acetonitrile (5− 95% over 10 min, 0.05% TFA in both phases) at a flow rate of 0.4 mL/ min. Quantification of target and impurities was done by UV detection at 254 nm. (2) Shimadzu LC-2010A/2020A instrument with an Ultimate C-18 (3.0 mm × 50 mm, 3 μm particle size) stationary phase and a gradient of water/acetonitrile (10−80% over 6 min and then 80% over 2 min, 0.05% TFA in both phases) at a flow rate of 1.2 mL/ min and oven temperature of 40 °C. Quantification of target and impurities was done by UV detection at 254 nm. (±)-(3-(2-Methyl-6-((5-methylthiazol-2-yl)amino)pyrimidin4-yl)piperidin-1-yl)(phenyl)methanone (1). A mixture of (±)-tertbutyl 3-(6-chloro-2-methylpyrimidin-4-yl)piperidine-1-carboxylate (34) (62 mg, 0.20 mmol), 5-methylthiazol-2-amine (32 mg, 0.28

a

Reagents and conditions: (a) tetrahydro-2H-pyran-4-carbonitrile, LHMDS, THF, −78 to 24 °C, 71% yield; (b) (CH3)2CHCN, LHMDS, THF, −78 to 24 °C, 84% yield; (c) 2-amino-4cyanopyridine, Pd2(dba)3, BINAP, Cs2CO3, 1,4-dioxane, 80 °C, 44% yield; (d) 3,3-difluoropyrrolidine hydrochloride, (i-Pr)2NEt, DMSO, 80−90 °C, 50% yield; (e) RuPhos palladium(II) phenethylamine chloride, RuPhos, 2-amino-4-cyanopyridine, t-BuONa, 3 Å molecular sieves, THF, 90 °C, 47% yield; (f) 2-amino-4-cyanopyridine, Pd(P(tBu)3)2, K3PO4, 1,4-dioxane, reflux, 4% yield.

Scheme 7. Preparation of Tertiary Alcohol 25a

Reagents and conditions: (a) CH3MgBr, THF, −78 °C, 70% yield; (b) 3,3-difluoropyrrolidine hydrochloride, (i-Pr)2NEt, DMSO, 80−90 °C, 50% yield; (c) 2-amino-4-cyanopyridine, Pd(P(t-Bu)3)2, K3PO4, 1,4-dioxane, reflux. 62% yield. a

maintained in Dulbecco’s modified Eagle medium supplemented with 10% FBS, 80 ng/mL colchicine, and 5 μg/mL Plasmocin. Cells were 410

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(±)-(3-(6-((4-Cyclopropylpyridin-2-yl)amino)-2-methylpyrimidin-4-yl)piperidin-1-yl)(phenyl)methanone (8). The title compound (28 mg, 34% yield) was prepared in a manner analogous to 1 by substituting 4-cyclopropylpyridin-2-amine for 5-methylthiazol2-amine. LCMS: m/z = 414.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.03−9.80 (m, 1H), 8.10 (d, J = 5.3 Hz, 1H), 7.70−7.19 (m, 7H), 6.79−6.50 (m, 1H), 4.87−4.21 (m, 1H), 3.81−3.47 (m, 1H), 3.21−2.79 (m, 2H), 2.83−2.66 (m, 2H), 2.09−1.43 (m, 7H), 1.13− 0.95 (m, 2H), 0.84−0.44 (m, 2H). (±)-(3-(2-Methyl-6-(pyridin-2-ylamino)pyrimidin-4-yl)piperidin-1-yl)(phenyl)methanone (9). The title compound (16 mg, 21% yield) was prepared in a manner analogous to 1 by substituting pyridin-2-amine for 5-methylthiazol-2-amine. LCMS: m/z = 374.1 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.07 (s, 1H), 8.59−8.38 (m, 1H), 8.26−7.95 (m, 1H), 7.64−7.28 (m, 5H), 7.06− 6.88 (m, 1H), 4.72−4.28 (m, 1H), 3.86−3.42 (m, 1H), 3.21−2.89 (m, 3H), 2.87−2.57 (m, 1H), 2.22−1.93 (m, 1H), 1.95−1.37 (m, 2H). (±)-2-((6-(1-Benzoylpiperidin-3-yl)-2-methylpyrimidin-4-yl)amino)isonicotinonitrile (10). The title compound (37 mg, 46% yield) was prepared in a manner analogous to 1 by substituting 2aminoisonicotinonitrile for 5-methylthiazol-2-amine. LCMS: m/z = 399.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.12 (s, 1H), 7.47−7.27 (m,7H), 4.63−4.38 (m, 1H), 3.76−3.55 (m, 1H), 3.14−2.63 (m, 3H), 2.56−2.39 (m, 1H), 2.10−1.97 (m, 1H), 1.88−1.47 (m, 3H). (±)-(3-(6-((4-Methoxypyridin-2-yl)amino)-2-methylpyrimidin-4-yl)piperidin-1-yl)(phenyl)methanone (11). The title compound (37 mg, 45% yield) was prepared in a manner analogous to 1 by substituting for 4-methoxypyridin-2-amine for 5-methylthiazol-2amine. LCMS: m/z = 404.2 [M + H]+. 1H NMR (400 MHz, DMSOd6) δ 10.01 (s, 1H), 8.11 (d, J = 5.8 Hz, 1H), 7.55−7.27 (m, 7H), 6.65−6.57 (m, 1H), 4.64−4.40 (m, 1H), 3.81 (s, 3H), 3.72−3.55 (m, 1H), 3.15−2.89 (m, 2H), 2.80−2.67 (m, 1H), 2.50−2.31 (m, 2H), 2.04−1.44 (m, 4H). (±)-2-Methyl-6-(piperidin-3-yl)-N-(4-(trifluoromethyl)pyridin-2-yl)pyrimidin-4-amine (12). A mixture of (±)-tert-butyl 3-(6-chloro-2-methylpyrimidin-4-yl)piperidine-1-carboxylate (34) (62 mg, 0.20 mmol), 4-(trifluoromethyl)pyridin-2-amine (45 mg, 0.28 mmol), sodium tert-butoxide (27 mg, 0.28 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (35 mg, 0.060 mmol), and tris(dibenzylideneacetone)dipalladium(0) (55 mg, 0.060 mmol) in 1,4-dioxane (10 mL) was stirred at 90 °C for 16 h. The reaction mixture was filtered and diluted with water (15 mL). The resulting solution was extracted with ethyl acetate (2 × 15 mL). The collected organic was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was dissolved in methanol (2 mL), and 4.0 M solution of hydrogen chloride in 1,4-dioxane (0.5 mL, 2 mmol) was added to the solution. After 16 h, the mixture was concentrated under reduced pressure and purified by preparative reverse phase HPLC to afford the title compound as an off-white solid (7 mg, 10% yield). LCMS: m/z = 388.1 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.55 (d, J = 5.2 Hz, 1H), 8.19 (s, 1H), 7.40 (s, 1H), 7.36−7.22 (m, 1H), 3.17 (s, 2H), 3.05 (d, J = 7.8 Hz, 1H), 2.97−2.88 (m, 1H), 2.76− 2.57 (m, 2H), 1.99−1.87 (m, 1H), 1.80 (s, 3H), 1.73−1.55 (m, 2H), 1.54−1.40 (m, 1H). (±)-tert-Butyl 3-(3-Ethoxy-3-oxopropanoyl)piperidine-1-carboxylate (36). To a solution of piperidine-1,3-dicarboxylic acid 1-tertbutyl ester (35) (160 g, 0.70 mol) in acetonitrile (1.6 L) was added CDI (136 g, 0.839 mol) portionwise at 23 °C. After 1 h, potassium monoethylmalonate (119 g, 0.697 mol) and MgCl2 (66.4 g, 0.697 mol) were added. After 16 h, the reaction mixture was concentrated under reduced pressure. The residue was diluted with cold water, and the resulting solution was neutralized with citric acid. The mixture was extracted with dichloromethane (3 × 600 mL). The collected organic was concentrated under reduced pressure. Purification by flash column chromatography (2:1 heptane/ethyl acetate) afforded the title compound as an off-white solid (180 g, 86%). 1H NMR (400 MHz, DMSO-d6 at 80 °C) δ 4.14−3.91 (m, 2H), 3.87−3.69 (m, 1H), 3.64− 3.00 (m, 3H), 2.90−2.88 (m, 1H), 2.63−2.50 (m, 1H), 2.63−2.50 (m,

mmol), sodium tert-butoxide (27 mg, 0.28 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (35 mg, 0.060 mmol), and tris(dibenzylideneacetone)dipalladium(0) (55 mg, 0.060 mmol) in 1,4-dioxane (10 mL) was stirred at 90 °C for 16 h. The reaction mixture was filtered and diluted with water (15 mL). The resulting solution was extracted with ethyl acetate (2 × 15 mL). The collected organic was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was dissolved in methanol (2 mL), and 4.0 M solution of hydrogen chloride in 1,4-dioxane (0.5 mL, 2 mmol) was added. After 16 h, the mixture was concentrated under reduced pressure. The resulting crude amine, benzoic acid (29 mg, 0.24 mmol), triethylamine (0.139 mL, 0.997 mmol), and N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (114 mg, 0.301 mmol) in N,Ndimethylformamide were stirred at 23 °C for 16 h. The mixture was concentrated under reduced pressure and purified by preparative reverse phase HPLC to afford the title compound as an off-white solid (16 mg, 21% yield). LCMS: m/z = 394.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 7.62−7.27 (m, 5H), 7.09 (s, 1H), 6.73 (s, 1H), 4.86−4.25 (m, 1H), 3.95−3.52 (m, 1H), 3.16−2.64 (m, 3H), 2.35 (s, 3H), 2.11−1.90 (m, 1H), 1.90−1.32 (m, 3H). (±)-(3-(2-Methyl-6-((5-methylpyridin-2-yl)amino)pyrimidin4-yl)piperidin-1-yl)(phenyl)methanone (2). The title compound (49 mg, 63% yield) was prepared in a manner analogous to 1 by substituting 5-methylpyridin-2-amine for 5-methylthiazol-2-amine. LCMS: m/z = 388.3 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H), 8.12 (s, 1H), 7.65−7.32 (m, 8H), 4.68−4.37 (m, 1H), 3.82−3.49 (m, 1H), 3.16−2.84 (m, 1H), 2.75−2.61 (m, 1H), 2.48− 2.12 (m, 6H), 2.12−1.93 (m, 1H), 1.89−1.46 (m, 3H). (±)-(3-(2-Methyl-6-((4-methylpyridin-2-yl)amino)pyrimidin4-yl)piperidin-1-yl)(phenyl)methanone (3). The title compound (25 mg, 32% yield) was prepared in a manner analogous to 1 by substituting 4-methylpyridin-2-amine for 5-methylthiazol-2-amine. LCMS: m/z = 388.1 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.14 (d, J = 5.1 Hz, 1H), 7.74−7.31 (m, 7H), 6.94−6.73 (m, 1H), 4.74−4.35 (m, 1H), 3.86−3.45 (m, 1H), 3.19−2.62 (m, 3H), 2.42−2.21 (m, 4H), 2.13−1.92 (m, 1H), 1.90−1.42 (m, 3H). (±)-(3-(2-Methyl-6-((5-(trifluoromethyl)pyridin-2-yl)amino)pyrimidin-4-yl)piperidin-1-yl)(phenyl)methanone (4). The title compound (60 mg, 68% yield) was prepared in a manner analogous to 1 by substituting 5-(trifluoromethyl)pyridin-2-amine for 5-methylthiazol-2-amine. LCMS: m/z = 442.1 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H), 8.64 (d, J = 2.4 Hz, 1H), 8.25−7.97 (m, 1H), 7.88 (s, 1H), 7.73−7.25 (m, 6H), 4.79−4.28 (m, 1H), 3.91−3.38 (m, 1H), 3.21−2.88 (m, 2H), 2.88−2.63 (m, 1H), 2.25−1.93 (m, 1H), 1.93−1.25 (m, 3H). (±)-(3-(2-Methyl-6-((4-(trifluoromethyl)pyridin-2-yl)amino)pyrimidin-4-yl)piperidin-1-yl)(phenyl)methanone (5). The title compound (18 mg, 20% yield) was prepared in a manner analogous to 1 by substituting 4-(trifluoromethyl)pyridin-2-amine for 5-methylthiazol-2-amine. LCMS: m/z = 442.4 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.87 (s, 1H), 9.21 (s, 1H), 8.15−7.51 (m, 4H), 6.70− 5.79 (m, 4H), 3.69−3.44 (m, 1H), 3.16−2.93 (m, 3H), 1.63−1.43 (m, 1H), 1.37−1.03 (m, 4H), 1.03−0.76 (m, 3H). (±)-(3-(6-((4-Ethylpyridin-2-yl)amino)-2-methylpyrimidin-4yl)piperidin-1-yl)(phenyl)methanone (6). The title compound (36 mg, 41% yield) was prepared in a manner analogous to 1 by substituting 4-ethylpyridin-2-amine for 5-methylthiazol-2-amine. LCMS: m/z = 442.4 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.46−8.24 (m, 1H), 8.26−8.02 (m, 1H), 7.80−7.35 (m, 6H), 6.97−6.78 (m, 1H), 2.83−2.53 (m, 5H), 2.10−1.89 (m, 2H), 1.96−1.48 (m, 4H), 1.33−1.04 (m, 4H). (±)-(3-(6-((4-Isopropylpyridin-2-yl)amino)-2-methylpyrimidin-4-yl)piperidin-1-yl)(phenyl)methanone (7). The title compound (31 mg, 37% yield) was prepared in a manner analogous to 1 by substituting 4-isopropylpyridin-2-amine for 5-methylthiazol-2amine. LCMS: m/z = 416.3 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.32−8.05 (m, 2H), 7.75−7.23 (m, 6H), 6.97−6.76 (m, 1H), 4.79−4.30 (m, 2H), 3.78−3.48 (m, 1H), 3.11− 2.62 (m, 6H), 2.44−2.21 (m, 2H), 2.13−1.46 (m, 7H). 411

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Journal of Medicinal Chemistry

Article

2-(3,3-Difluoropyrrolidin-1-yl)-6-(piperidin-4-yl)-N-(4(trifluoromethyl)pyridin-2-yl)pyrimidin-4-amine (15). The title compound (32 mg, 74% yield) was prepared in a manner analogous to 13 by substituting tert-butyl 4-(2,6-dichloropyrimidin-4-yl)piperidine1-carboxylate (39) and 3,3-difluoropyrrolidine hydrochloride for tertbutyl 3-(2,6-dichloropyrimidin-4-yl)piperidine-1-carboxylate (38) and pyrrolidine, respectively. LCMS: m/z = 429.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 8.71 (s, 1H), 8.52 (d, J = 5.1 Hz, 1H), 7.29 (d, J = 5.1 Hz, 1H), 6.48 (s, 1H), 3.89 (t, J = 13.2 Hz, 2H), 3.73 (t, J = 7.3 Hz, 2H), 3.06 (d, J = 12.1 Hz, 2H), 2.67−2.52 (m, 4H), 1.92−1.68 (m, 2H), 1.58 (m, 2H). 2-(3,3-Difluoropyrrolidin-1-yl)-6-(tetrahydro-2H-pyran-4-yl)N-(4-(trifluoromethyl)pyridin-2-yl)pyrimidin-4-amine (16). The title compound (31 mg, 72% yield) was prepared in a manner analogous to 13 by substituting 2,4-dichloro-6-(tetrahydro-2H-pyran4-yl)pyrimidine (40) and 3,3-difluoropyrrolidine hydrochloride for tert-butyl 3-(2,6-dichloropyrimidin-4-yl)piperidine-1-carboxylate (38) and pyrrolidine, respectively. LCMS: m/z = 430.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H), 8.70 (s, 1H), 8.52 (d, J = 5.2 Hz, 1H), 7.28 (d, J = 4.9 Hz, 1H), 6.50 (s, 1H), 3.97−3.84 (m, 4H), 3.79−3.60 (m, 2H), 3.43 (td, J = 11.1, 3.5 Hz, 2H), 2.72−2.54 (m, 2H), 1.90- 1.59 (m, 5H). 1-(4-(2-(3,3-Difluoropyrrolidin-1-yl)-6-(4-(trifluoromethyl)pyridin-2-ylamino)pyrimidin-4-yl)piperidin-1-yl)ethanone (17). To an ice-cooled solution of 2-(3,3-difluoropyrrolidin-1-yl)-6(piperidin-4-yl)-N-(4-(trifluoromethyl)pyridin-2-yl)pyrimidin-4-amine (15) (42 mg, 0.10 mmol), 4-(dimethylamino)pyridine (1.2 mg, 0.0098 mmol) and N,N-diisopropylethylamine (65 mg, 0.50 mmol) in acetonitrile (5 mL) and dichloromethane (5 mL) was added acetic anhydride (0.020 mL, 0.22 mmol). After 1 h, the reaction mixture was poured into saturated aqueous ammonium chloride solution (10 mL), and the resulting solution was extracted with ethyl acetate (3 × 5 mL). The combined organic was washed saturated aqueous sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by preparative reverse phase HPLC afforded the title compound as an off-white solid (37 mg, 80% yield). LCMS: m/z = 471.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H), 8.70 (s, 1H), 8.52 (d, J = 5.2 Hz, 1H), 7.31−6.90 (m, 1H), 6.49 (s, 1H), 4.63−4.35 (m, 1H), 4.02−3.84 (m, 3H), 3.73 (t, J = 7.3 Hz, 2H), 3.25−3.02 (m, 1H), 2.74−2.55 (m, 4H), 1.83 (t, J = 14.5 Hz, 2H), 1.70−1.37 (m, 2H). 1-(4-(2-(3,3-Difluoroazetidin-1-yl)-6-((4-(trifluoromethyl)pyridin-2-yl)amino)pyrimidin-4-yl)piperidin-1-yl)ethanone (18). The title compound (32 mg, 68% yield) was prepared in a manner analogous to 17 by substituting 3,3-difluoroazetidine hydrochloride for 3,3-difluoropyrrolidine hydrochloride. LCMS: m/z = 457.1 [M + H]+. 1H NMR (400 MHz, CD3OD) δ 8.53 (s, 1H), 8.46 (d, J = 5.6 Hz, 1 H), 7.21 (d, J = 4.8 Hz, 1 H), 6.60 (s, 1H), 4.65−4.61 (m, 1 H), 4.44 (t, J = 12.0 Hz, 4 H), 4.05−4.01 (m, 1 H), 3.29−3.19 (m, 1 H), 2.79−2.69 (m, 2H), 2.12 (s, 3 H), 2.00−1.91 (m, 2H), 1.75−1.66 (m, 2 H). 1-(4-(2-Morpholino-6-((4-(trifluoromethyl)pyridin-2-yl)amino)pyrimidin-4-yl)piperidin-1-yl)ethanone (19). The title compound (34 mg, 75% yield) was prepared in a manner analogous to 17 by substituting morpholine for 3,3-difluoropyrrolidine hydrochloride. LCMS: m/z = 451.0 [M + H]+. 1H NMR (400 MHz, CDCl3), δ ppm: 8.54−8.43 (m, 2 H), 7.31−7.26 (m, 2H), 6.90−6.60 (br s, 1H), 4.90−4.60 (m, 1H), 4.18−3.87 (m, 8H), 3.60−3.40 (m, 1H), 2.80−2.70 (m, 1H), 2.52−2.50 (m, 1H), 2.30−2.26 (m, 4H), 2.10−1.96 (m, 1H), 1.80−1.40 (m, 3H). tert-Butyl 4-(2,6-Dichloropyridin-4-yl)-5,6-dihydropyridine1(2H)-carboxylate (45). A mixture of 2,6-dichloro-4-iodopyridine (43) (1.0 g, 3.7 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-5,6-dihydro-2H-pyridine-1-carboxylate (44) (1.4 g, 4.4 mmol), potassium carbonate (1.0 g, 7.3 mmol), and Pd(dppf)Cl2·CH2Cl2 (0.30 g, 0.37 mmol) in 1,4-dioxane (12 mL) and water (3 mL) was purged with nitrogen and heated at 90 °C for 16 h. The mixture was diluted with water (100 mL), and the resulting mixture was extracted with ethyl acetate (2 × 100 mL). The combined organic was washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated under

1H), 2.50−2.48 (m, 1H), 1.90 (m, 1H), 1.64−1.63 (m, 1H), 1.53− 1.40 (m, 1H), 1.35 (s, 9H), 1.28 (m, 1H), 1.2 (m, 3H). tert-Butyl 3-(2,6-Dihydroxypyrimidin-4-yl)piperidine-1-carboxylate (37). A solution of (±)-tert-butyl 3-(3-ethoxy-3oxopropanoyl)piperidine-1-carboxylate (36) (180 g, 0.60 mol), urea (361 g, 6.02 mol), and sodium methoxide (325 g, 6.02 mol) in ethanol (1.8 L) was refluxed for 72 h. After completion of the reaction, the solvent was concentrated under reduced pressure. The residue was dissolved in water and neutralized with citric acid. The resulting solution was extracted with dichloromethane (3 × 800 mL). The collected organic was sequentially washed with water (2 × 800 mL), saturated aqueous sodium chloride solution (800 mL) and concentrated under reduced pressure. Trituration with petroleum ether afforded the title compound as a white solid (75 g, 42%). LCMS: m/z = 294.6 [M − H]−. 1H NMR (300 MHz, DMSO-d6) δ 10.96 (br s, 1H), 10.81 (br s, 1H), 5.3 (s, 1H), 3.79−3.75 (m, 2H), 2.82−2.75 (m, 2H), 2.34 (m, 1H), 1.89−1.83 (m, 1H), 1.58−1.54 (m, 2H), 1.37 (m, 10H). tert-Butyl 3-(2,6-Dichloropyrimidin-4-yl)piperidine-1-carboxylate (38). tert-Butyl 3-(2,6-dihydroxypyrimidin-4-yl)piperidine1-carboxylate (37) (75 g, 0.25 mol) was dissolved in phosphoryl trichloride (750 mL) and heated to reflux for 3 h. The mixture was concentrated to 10% of the original volume and poured slowly onto ice. The solution was adjusted to pH ≈ 9 with slow addition of solid sodium bicarbonate. Tetrahydrofuran (500 mL) and di-tert-butyl dicarbonate (83 g, 0.38 mol) were added, and the resulting mixture was stirred at 23 °C for 16 h. The mixture was filtered, and the filtrate was extracted with dichloromethane (3 × 400 mL). The collected organic was washed sequentially with water (3 × 400 mL), saturated aqueous sodium chloride solution (400 mL) and concentrated under reduced pressure. Purification by flash column chromatography (1:1 heptane/ethyl acetate) afforded the title compound as a white solid (14 g, 17%). LCMS: m/z = 278 [M − tBu + 2H]+. 1H NMR (400 MHz, DMSO-d6) δ 7.79 (s, 1H), 4.00 (br s, 1H), 3.80 (br s, 1H), 3.20−3.00 (m, 1H), 2.90−2.84 (m, 2H), 1.95 (m, 1H), 1.72−1.60 (m, 2H), 1.38 (m, 10H). 6-(Piperidin-3-yl)-2-(pyrrolidin-1-yl)-N-(4-(trifluoromethyl)pyridin-2-yl)pyrimidin-4-amine (13). A solution of tert-butyl 3(2,6-dichloropyrimidin-4-yl)piperidine-1-carboxylate (38) (33 mg, 0.10 mmol), 4-(trifluoromethyl)pyridin-2-amine (23 mg, 0.14 mmol), sodium tert-butoxide (14 mg, 0.14 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (18 mg, 0.031 mmol), and tris(dibenzylideneacetone)dipalladium(0) (28 mg, 0.030 mmol) in 1,4-dioxane (5 mL) was stirred at 23 °C for 1 h before heating to 60 °C for 13 h. The mixture was filtered and diluted with water (10 mL). The resulting solution was extracted with ethyl acetate (2 × 10 mL). The collected organic was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dissolved in N,N-dimethylformamide (5 mL) before the addition of pyrrolidine (21 mg, 0.30 mmol) and N,N-diisopropylethylamine (64 mg, 0.50 mmol). The reaction mixture was heated to 85 °C. After 16 h, the reaction was concentrated under reduced pressure. The resulting residue was dissolved in methanol (2 mL) before the addition of a 4.0 M solution of hydrogen chloride in 1,4-dioxane (0.5 mL, 2 mmol). After 16 h, the mixture was concentrate under reduced pressure. Purification by preparative reverse phase HPLC afforded the title compound as an off-white solid (14 mg, 37% yield). LCMS: m/z = 393.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.85 (s, 1H), 8.50 (d, J = 5.1 Hz, 1H), 7.26 (d, J = 4.7 Hz, 1H), 6.32 (s, 1H), 3.19 (d, J = 12.4 Hz, 2H), 3.04 (s, 2H), 2.78 (t, J = 11.5 Hz, 1H), 2.64 (dd, J = 15.1, 6.7 Hz, 2H), 1.94 (s, 5H), 1.80−1.46 (m, 4H). 2-(3,3-Difluoropyrrolidin-1-yl)-6-(piperidin-3-yl)-N-(4(trifluoromethyl)pyridin-2-yl)pyrimidin-4-amine (14). The title compound (18 mg, 43% yield) was prepared in a manner analogous to 13 by substituting 3,3-difluoropyrrolidine hydrochloride for pyrrolidine. LCMS: m/z = 429.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.29 (d, J = 17.8 Hz, 1H), 8.69 (s, 1H), 8.53 (d, J = 5.1 Hz, 1H), 7.29 (d, J = 5.1 Hz, 1H), 6.48 (s, 1H), 3.90 (t, J = 13.2 Hz, 3H), 3.73 (t, J = 7.3 Hz, 3H), 3.03 (d, J = 12.6 Hz, 2H), 2.82−2.56 (m, 4H), 1.92 (d, J = 13.7 Hz, 1H), 1.77−1.50 (m, 3H). 412

dx.doi.org/10.1021/jm5013984 | J. Med. Chem. 2015, 58, 401−418

Journal of Medicinal Chemistry

Article

reduced pressure. Purification by flash column chromatography (15% ethyl acetate in heptane) afforded the title compound as a light yellow solid (670 mg, 56% yield). LCMS: m/z = 330 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 7.21 (s, 2H), 6.32 (s, 1H), 4.12 (d, J = 2.7 Hz, 2H), 3.63 (t, J = 5.6 Hz, 2H), 2.45 (s, 2H), 1.49 (s, 9H). tert-Butyl 4-(2-Chloro-6-(3,3-difluoropyrrolidin-1-yl)pyridin4-yl)-5,6-dihydropyridine-1(2H)-carboxylate (46). A mixture of tert-butyl 4-(2,6-dichloro-4-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (45) (240 mg, 0.72 mmol), 3,3-difluoropyrrolidine hydrochloride (126 mg, 0.878 mmol), and N,N-diisopropylethylamine (0.38 mL, 2.2 mmol) in 1,4-dioxane (3.0 mL) was heated in a CEM microwave at 120 °C. After 30 min, additional 3,3-difluoropyrrolidine hydrochloride (251 mg, 1.75 mmol) and N,N-diisopropylethylamine (0.35 mL, 2.01 mmol) in N,N-dimethylformamide (1.0 mL) were added to the reaction mixture. The resulting solution was heated at 130 °C under microwave irradiation for 30 min and then heated in an oil bath at 110 °C for 16 h The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (2 × 50 mL). The collected organic was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by flash column chromatography (9:1 heptane/ethyl acetate) afforded the title compound as a thick oil (106 mg, 37% yield). LCMS: m/z = 400 [M + H]+. te rt -But yl 4-( 2-(3,3 -Di fluoropyrrolidin-1-yl)-6 -(4(trifluoromethyl)pyridin-2-ylamino)pyridin-4-yl)-5,6-dihydropyridine-1(2H)-carboxylate (47). A mixture of tert-butyl 4-[2chloro-6-(3,3-difluoropyrrolidin-1-yl)-4-pyridyl]-3,6-dihydro-2H-pyridine-1-carboxylate (46) (150 mg, 0.37 mmol), 4-(trifluoromethyl)pyridin-2-amine (73 mg, 0.45 mmol), sodium tert-butoxide (54 mg, 0.56 mmol), 2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl (22 mg, 0.047 mmol), and RuPhos palladium(II) phenethylamine chloride (27 mg, 0.033 mmol) in 1,4-dioxane (2.0 mL) was capped in a microwave vial, purged with nitrogen, and heated at 120 °C under microwave irradiation (CEM) for 15 min. The reaction mixture was diluted with ethyl acetate (15 mL), filtered through Celite, and concentrated under reduced pressure. Purification by flash column chromatography (4:1 heptane/ethyl acetate) yielded the title compound as an off-white solid (75 mg, 38% yield). LCMS: m/z = 526 [M + H]+. te rt -But yl 4-( 2-(3,3 -Di fluoropyrrolidin-1-yl)-6 -(4(trifluoromethyl)pyridin-2-ylamino)pyridin-4-yl)piperidine-1carboxylate (48). A solution of tert-butyl 4-[2-(3,3-difluoropyrrolidin-1-yl)-6-[[4-(trifluoromethyl)-2-pyridyl]amino]-4-pyridyl]-3,6-dihydro-2H-pyridine-1-carboxylate (47) (75 mg, 0.14 mmol) in methanol (50 mL) was hydrogenated in a H-Cube (10% Pd/C cartridge, 40 bar of hydrogen, 30 °C, flow rate = 1 mL/min). LCMS indicated incomplete reaction, and the reaction solution was further hydrogenated (10% Pd/C cartridge, 40 bar of hydrogen, 40 °C, flow rate of 1 mL/min). The reaction solution was concentrated to afford the title compound as a light brown solid (63 mg, 84% crude yield). LCMS: m/z = 528 [M + H]+. 6-(3,3-Difluoropyrrolidin-1-yl)-4-(piperidin-4-yl)-N-(4(trifluoromethyl)pyridin-2-yl)pyridin-2-amine (49). To a solution of tert-butyl 4-[2-(3,3-difluoropyrrolidin-1-yl)-6-[[4-(trifluoromethyl)-2-pyridyl]amino]-4-pyridyl]piperidine-1-carboxylate (48) (60 mg, 0.1 mmol) in dichloromethane (1.0 mL) was slowly added 4 M HCl in 1,4-dioxane (2.0 mL) at 23 °C. After 1 h, the mixture was concentrated under reduced pressure and dried under vacuum to afford the title compound as an off-white solid (50 mg, 86% crude yield). LCMS: m/z = 428 [M + H]+. 1-(4-(2-(3,3-Difluoropyrrolidin-1-yl)-6-(4-(trifluoromethyl)pyridin-2-ylamino)pyridin-4-yl)piperidin-1-yl)ethanone (20). To a solution of 6-(3,3-difluoropyrrolidin-1-yl)-4-(4-piperidyl)-N-[4(trifluoromethyl)-2-pyridyl]pyridin-2-amine (49) (0.050 g, 0.11 mmol) and N,N-diisopropylethylamine (0.051 mL, 0.29 mmol) in tetrahydrofuran (2.0 mL) was added acetyl chloride (0.009 mL, 0.1 mmol) at 23 °C. After 1 h, the reaction mixture was diluted with ethyl acetate (10 mL), and the resulting solution was washed with 1 N aqueous sodium bicarbonate solution. The collected organic was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by preparative reverse phase HPLC

afforded the title product as a yellow solid (25 mg, 45% yield). LCMS: m/z = 470 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.82 (s, 1H), 8.67 (s, 1H), 8.43 (d, J = 5.1 Hz, 1H), 7.13 (d, J = 5.2 Hz, 1H), 6.50 (s, 1H), 5.98 (s, 1H), 4.52 (d, J = 13.2 Hz, 1H), 3.91 (d, J = 13.5 Hz, 1H), 3.83 (t, J = 13.3 Hz, 2H), 3.63 (t, J = 7.2 Hz, 2H), 3.12 (t, J = 12.2 Hz, 1H), 2.69−2.53 (m, 3H), 2.03 (s, 3H), 1.77 (t, J = 13.8 Hz, 2H), 1.64−1.51 (m, 1H), 1.50−1.33 (m, 2H). tert-Butyl 4-(2,6-Dichloro-4-pyridyl)piperidine-1-carboxylate (51). To a suspension of zinc dust (4.06 g, 62.1 mmol) in N,N-dimethylacetamide (5 mL) under a nitrogen atmosphere was cautiously added a mixture of trimethylsilyl chloride (0.946 mL, 7.30 mmol) and 1,2-dibromoethane (0.636 mL, 7.30 mmol) over 10 min. After stirring for 15 min, a solution of N-(tert-butoxycarbonyl)-4iodopiperidine (50) (16.74 g, 51.11 mmol) in N,N-dimethylacetamide (20 mL) was added over 30 min and stirring was continued for an additional 30 min. In the open atmosphere, this mixture was filtered through Celite as quickly as possible, rinsing with a small amount of N,N-dimethylacetamide. The resulting yellow solution was slowly added to a separately prepared, nitrogen flushed suspension of [1,1bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.35 g, 1.83 mmol), copper(I) iodide (695 mg, 3.65 mmol), and 2,6-dichloro-4iodopyridine (43) (10.0 g, 36.5 mmol) in N,N-dimethylacetamide (30 mL), and this mixture was stirred at 80 °C for 16.5 h. After cooling to 23 °C, the mixture was partitioned between ethyl acetate and water. Filtration through Celite was necessary to break the emulsion, after which the collected organic was washed with water. The organic was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. Purification by flash column chromatography (7:3 heptane/ethyl acetate) afforded the title compound as a white solid (7.5 g, 62%). 1H NMR (400 MHz, CDCl3) δ 7.11 (s, 2H), 4.19 (m, 2H), 2.78 (m, 2H), 2.71 (m, 1H), 1.81 (m, 2H), 1.65 (m, 2H), 1.48 (s, 9H). tert-Butyl 4-(2-Chloro-6-(3,3-difluoropyrrolidin-1-yl)pyridin4-yl)piperidine-1-carboxylate (52). A solution of 3,3-difluoropyrrolidine hydrochloride (533 mg, 3.72 mmol), tert-butyl 4-(2,6dichloropyridin-4-yl)piperidine-1-carboxylate (51) (1.00 g, 1.17 mmol), and N,N-diisopropylethylamine (1.23 mL, 7.03 mmol) in Nmethylpyrrolidinone (2.3 mL) was heated at 130 °C in the microwave (CEM) for 1.5 h. The reaction mixture was diluted with ethyl acetate (50 mL), and the resulting solution was washed with saturated aqueous ammonium chloride solution (2 × 20 mL). The aqueous washes were extracted with ethyl acetate (25 mL). The combined organic was washed with water (25 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by flash column chromatography (4:1 heptane/ethyl acetate) afforded the title compound as a clear oil (220 mg, 47% yield). 1H NMR (400 MHz, CDCl3), δ 6.51 (s, 1 H), 6.03 (s, 1 H), 4.25 (m, 2 H), 3.83 (t, J = 13.1 Hz, 2 H), 3.67 (t, J = 7.3 Hz, 2 H), 2.77 (m, 2 H), 2.58−2.42 (m, 3 H), 1.78 (m, 2 H), 1.62−1.53 (m, 2 H), 1.48 (s, 9 H). 2-Chloro-6-(3,3-difluoropyrrolidin-1-yl)-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridine (53). To an ice-cooled solution of tert-butyl 4-(2-chloro-6-(3,3-difluoropyrrolidin-1-yl)pyridin-4-yl)piperidine-1carboxylate (52) (0.220 g, 0.547 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (2 mL). After 2 h, the reaction mixture was concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane (10 mL) and washed with saturated aqueous sodium bicarbonate solution (5 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was dissolved in tetrahydrofuran (2 mL). Oxetan-3-one (0.112 mL, 1.10 mmol) and sodium triacetoxyborohydride (366 mg, 1.64 mmol) were sequentially added to the solution at 23 °C. After 35 min, the reaction mixture was partitioned between ethyl acetate (10 mL) and saturated aqueous ammonium chloride solution (10 mL). The organic was separated, and the aqueous layer was further extracted with ethyl acetate (2 × 5 mL). The combined organic was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by flash column chromatography (98:2 dichloromethane/methanol + 0.1% ammonium hydroxide) afforded the title compound as a clear oil (166 mg, 85% yield). 1H NMR (CDCl3, 400 MHz), δ: 6.54 (s, 1 H), 6.07 413

dx.doi.org/10.1021/jm5013984 | J. Med. Chem. 2015, 58, 401−418

Journal of Medicinal Chemistry

Article

(s, 1 H), 4.67 (t, J = 6.3 Hz, 2 H), 4.63 (t, J = 6.3 Hz, 2 H), 3.82 (t, J = 13.2 Hz, 2 H), 3.66 (t, J = 7.2 Hz, 2 H), 3.50 (m, 1 H), 2.86 (m, 2 H), 2.52−2.38 (m, 3 H), 1.94−1.72 (m, 6 H). 2-((6-(3,3-Difluoropyrrolidin-1-yl)-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (26, GNE3511). To a solution of 2-chloro-6-(3,3-difluoropyrrolidin-1-yl)-4-(1(oxetan-3-yl)piperidin-4-yl)pyridine (53) (216 mg, 0.603 mmol), 2amino-4-cyanopyridine (122 mg, 1.03 mmol), cesium carbonate (399 mg, 1.22 mmol), and 2,2′-bis(diphenylphosphino)-1,1′-binaphthalene (44 mg, 0.068 mmol) in 1,4-dioxane was added tris(dibenzylideneacetone)dipalladium(0) (28 mg, 0.031 mmol). The reaction mixture was heated at reflux under nitrogen for 13 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with water (10 mL). The organic was separated, and the aqueous wash was further extracted with ethyl acetate (2 × 10 mL). The combined organics were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by flash column chromatography (97:3 dichloromethane/methanol + 0.1% ammonium hydroxide) provided the title compound as a yellow solid (205 mg, 77% yield). 1H NMR (400 MHz, DMSO-d6), δ: 9.72 (s, 1 H), 8.46 (s, 1 H), 8.41 (d, J = 5.0 Hz, 1 H), 7.20 (dd, J = 5.1, 1.2 Hz, 1 H), 6.60 (s, 1 H), 6.00 (s, 1 H), 4.54 (t, J = 6.4 Hz, 2 H), 4.44 (t, J = 6.0 Hz, 2 H), 3.85 (t, J = 13.2 Hz, 2 H), 3.65 (t, J = 7.2 Hz, 2 H), 3.40 (m, 1 H), 2.80 (m, 2 H), 2.56 (m, 2 H), 2.39 (m, 1 H), 1.84 (m, 2 H), 1.76−1.61 (m, 4 H). 13C NMR (126 MHz, DMSO-d6) δ 157.97, 155.37, 154.60, 152.44, 149.19, 128.44 (JCF = 245.43 Hz), 119.88, 117.38, 115.98, 113.44, 98.62, 96.98, 74.66, 58.55, 53.44 (JCF = 31.39 Hz), 49.63, 44.08, 41.64, 38.88, 33.06 (JCF = 23.66 Hz), 31.64. HRMS (ESI) m/z: [M + H]+ calcd for C23H27F2N6O, 441.2209. Found: 441.2211. 2,6-Dichloro-4-[1-(oxetan-3-yl)-4-piperidyl]pyridine (54a). To an ice-cooled solution of tert-butyl 4-(2,6-dichloro-4-pyridyl)piperidine-1-carboxylate (51) (2.84 g, 8.57 mmol) in dichloromethane (17 mL) was added trifluoroacetic acid (8.4 mL), and the solution was warm to room temperature. After stirring for 1 h, the solution was concentrated under reduced pressure to afford a white solid which was resuspended in anhydrous tetrahydrofuran (34 mL) with triethylamine (6.0 mL, 43 mmol) and 3-oxetanone (0.82 mL, 13 mmol). After stirring for 30 min, sodium triacetoxyborohydride (5.73 g, 25.7 mmol) was added and stirring continued for 2 h. The reaction mixture was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate. The collected organic was dried over magnesium sulfate, filtered, and concentrated under reduced pressure to afford title compound as a beige solid (2.67 g, >99% crude yield over 2 steps). 1H NMR (400 MHz, CDCl3) δ 7.13 (s, 2H), 4.73−4.63 (m, 4H), 3.60− 3.52 (m, 1H), 3.00−2.88 (m, 2H), 2.60−2.48 (m, 1H), 2.05−1.94 (m, 2H), 1.93−1.78 (m, 4H). 2-((6-Chloro-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (55a). To 2,6-dichloro-4-[1-(oxetan-3-yl)4-piperidyl]pyridine (0.720 g, 2.51 mmol), 2-amino-5-cyanopyridine (308 mg, 2.51 mmol), tris(dibenzylideneacetone)dipalladium(0) (59 mg, 0.064 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (0.090 g, 0.16 mmol), and cesium carbonate (1.14 g, 3.51 mmol) was added anhydrous 1,4-dioxane (10 mL) under nitrogen. The reaction mixture was stirred at 80 °C for 20 h before cooling to room temperature and filtering through Celite (rinsing with dichloromethane). After concentration under reduced pressure, flash column chromatography (9:1 dichloromethane/methanol) afforded the title compound as a colorless solid (725 mg, 78% yield). LCMS: m/z = 370 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.38 (br s, 1H), 8.49 (d, J = 5.1 Hz, 1H), 7.94 (s, 1H), 7.63 (s, 1H), 7.30 (d, J = 5.1 Hz, 1H), 6.98 (s, 1H), 4.57−4.51 (m, 2H), 4.47−4.41 (m, 2H), 3.46−3.38 (m, 1H), 2.85−2.77 (m, 2H), 1.90−1.75 (m, 4H), 1.70−1.58 (m, 2H). General Procedure for Buchwald−Hartwig Reaction of Secondary Amines. A vial charged with the 2-((6-chloro-4-(1(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (55) (1 equiv), the amine (usually as the HCl salt, 3 equiv), chloro(2dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)[2-(2aminoethylphenyl)]palladium(II) methyl-tert-butyl ether adduct (10 mol %), 2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl (10

mol %), and sodium tert-butoxide (6 equiv) was purged under nitrogen before the addition of anhydrous tetrahydrofuran (0.1 M). The mixture was stirred at 90 °C for 16 h before filtering through Celite, rinsing with dichloromethane. After concentration under reduced pressure, the reaction residue was purified by preparative reverse phase HPLC to afford the title compound. 2-((6-(3-Methoxyazetidin-1-yl)-4-(1-(oxetan-3-yl)piperidin-4yl)pyridin-2-yl)amino)isonicotinonitrile (28). Reaction of 3methoxyazetidine following the general Buchwald−Hartwig procedure afforded the title compound (47% yield). LCMS: m/z = 421 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.77 (br s, 1H), 8.45 (s, 1H), 8.40 (d, J = 5.1 Hz, 1H), 7.18 (d, J = 5.1 Hz, 1H), 6.64 (s, 1H), 5.86 (s, 1H), 4.58−4.50 (m, 2H), 4.47−4.39 (m, 2H), 4.35 (m, 1H), 4.20− 4.09 (m, 2H), 3.80−3.66 (m, 2H), 3.44−3.35 (m, 1H), 3.26 (s, 3H), 2.82−2.72 (m, 2H), 2.41−2.32 (m, 1H), 1.88−1.76 (m, 2H), 1.76− 1.57 (m, 4H). 2-((6-(Azetidin-1-yl)-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin2-yl)amino)isonicotinonitrile (29). Reaction of azetidine following the general Buchwald−Hartwig procedure afforded the title compound (47% yield). LCMS: m/z = 391 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.76 (br s, 1H), 8.51 (s, 1H), 8.40 (d, J = 5.1 Hz, 1H), 7.18 (d, J = 5.1 Hz, 1H), 6.59 (s, 1H), 5.80 (s, 1H), 4.57−4.49 (m, 2H), 4.49−4.40 (m, 2H), 4.00−3.92 (m, 4H), 3.43−3.35 (m, 1H), 2.82−2.73 (m, 2H), 2.40−2.27 (m, 3H), 1.88−1.78 (m, 2H), 1.77− 1.68 (m, 2H), 1.68−1.51 (m, 2H). 2-((6-Cyclobutoxy-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin2-yl)amino)isonicotinonitrile (30). A vial charged with 6-chloro-N(4-(difluoromethyl)pyridin-2-yl)-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-amine (55) (0.040 g, 0.11 mmol), palladium(II) acetate (2.4 mg, 0.011 mmol), 5-di(1-adamantylphosphino)-1-(1,3,5-triphenyl-1Hpyrazol-4-yl)-1H-pyrazole (13 mg, 0.022 mmol), and cesium carbonate (106 mg, 0.325 mmol) was purged with nitrogen before the addition of 3-cyclobutanol (86 μL, 1.1 mmol) and anhydrous toluene (1.1 mL). The mixture was stirred at 120 °C for 16 h. The mixture was diluted with dichloromethane, filtered through Celite (rinsing with dichloromethane). The organic was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative reverse phase HPLC to afford the title compound as a colorless solid (28 mg, 64% yield). LCMS: m/z = 406.3 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.98 (br s, 1H), 8.50−8.39 (m, 2H), 7.25 (dd, J = 5.1, 1.3 Hz, 1H), 6.83 (s, 1H), 6.20 (s, 1H), 5.11 (m, 1H), 4.57−4.50 (m, 2H), 4.47−4.41 (m, 2H), 3.40 (m, 1H), 2.79 (d, J = 11.3 Hz, 2H), 2.47−2.37 (m, 3H), 2.18−2.02 (m, 2H), 1.88−1.79 (m, 3H), 1.78−1.70 (m, 2H), 1.70−1.54 (m, 3H). General Procedure for Suzuki−Miyaura Reaction Trifluoroborate Potassium Salts. A vial charged with the 2-((6-chloro-4-(1(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (55) (1 equiv), palladium(II) acetate (10 mol %), butyldi-1-adamantylphosphine (15 mol %), the potassium trifluoroborate salt (1.2 equiv), and cesium carbonate (3 equiv) was purged under nitrogen before the addition of degassed toluene (0.2 M) and degassed water (2 M). The mixture was stirred at 110 °C overnight and then diluted with dichloromethane, filtered through Celite (rinsing with dichloromethane). The organics were dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The reaction residue thus obtained was purified by preparative reverse phase HPLC to afford the title compound. 2-((6-Ethyl-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (32). Reaction of ethyl trifluoroborate potassium salt with 6-chloro-N-(4-(difluoromethyl)pyridin-2-yl)-4-(1(oxetan-3-yl)piperidin-4-yl)pyridin-2-amine (55) (50.0 mg, 0.135 mmol) following the general Suzuki−Miyaura procedure afforded the title compound as a colorless solid (25 mg, 50% yield). LCMS: m/ z = 364 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.99 (br s, 1H), 8.44 (d, J = 5.0 Hz, 1H), 8.35 (s, 1H), 7.27 (s, 1H), 7.21 (dd, J = 5.1, 1.4 Hz, 1H), 6.75 (s, 1H), 4.57−4.50 (m, 2H), 4.46−4.42 (m, 2H), 3.47−3.36 (m, 1H), 2.86−2.74 (m, 2H), 2.69 (q, J = 7.5 Hz, 2H), 2.48−2.41 (m, 1H), 1.91−1.81 (m, 2H), 1.81−1.72 (m, 2H), 1.72− 1.56 (m, 2H), 1.26 (t, J = 7.6 Hz, 3H). 414

dx.doi.org/10.1021/jm5013984 | J. Med. Chem. 2015, 58, 401−418

Journal of Medicinal Chemistry

Article

2-((6-Cyclopropyl-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2yl)amino)isonicotinonitrile (33). Reaction of cyclopropyl trifluoroborate potassium salt with 6-chloro-N-(4-(difluoromethyl)pyridin-2yl)-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-amine (55) (50.0 mg, 0.135 mmol) following the general Suzuki−Miyaura procedure afforded the title compound as a colorless solid (8.5 mg, 17% yield). LCMS: m/z = 376 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.42 (d, J = 5.0 Hz, 1H), 8.34 (s, 1H), 7.22 (dd, J = 5.1, 1.3 Hz, 1H), 7.07 (s, 1H), 6.82 (d, J = 0.9 Hz, 1H), 4.58−4.52 (m, 2H), 4.47−4.42 (m, 2H), 3.46−3.36 (m, 1H), 2.84−2.75 (m, 2H), 2.47− 2.38 (m, 1H), 2.07−1.98 (m, 1H), 1.90−1.80 (m, 2H), 1.80−1.72 (m, 2H), 1.72−1.57 (m, 2H), 0.98−0.90 (m, 4H). 2-((6-Methyl-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (31). 31 was made from tert-butyl 4-(2chloro-6-methyl-4-pyridyl)piperidine-1-carboxylate via Buchwald− Hartwig, Boc deprotection, and reductive amination as previously reported (WO 2013174780).22 LCMS: m/z = 350 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.99 (br s, 1H), 8.44 (d, J = 5.1 Hz, 1H), 8.18 (s, 1H), 7.36 (s, 1H), 7.21 (d, J = 5.1 Hz, 1H), 6.75 (s, 1H), 4.54 (dd, J = 6.5, 6.5 Hz, 2H), 4.45 (dd, J = 6.5, 6.5 Hz, 2H), 3.42 (m, 1H), 2.80 (m, 2H), 2.44 (m, 1H), 2.40 (s, 3H), 1.86 (m, 2H), 1.76 (m, 2H), 1.63 (m, 2H). 2-((4-(1-Acetylpiperidin-4-yl)-6-chloropyridin-2-yl)amino)isonicotinonitrile (55b). A solution of tert-butyl 4-(2,6-dichloro-4pyridyl)piperidine-1-carboxylate (51) (1.79 g, 5.41 mmol) in trifluoroacetic acid (5.4 mL) was stirred for 1 h before the solution was concentrated under reduced pressure to afford the trifluoroacetate salt as a white solid. Dissolution of the residue in anhydrous dichloromethane, (11 mL, 0.5 M) was followed by the addition of triethylamine (2.3 mL, 16 mmol), 4-dimethylaminopyridine (33 mg, 0.27 mmol), and acetic anhydride (0.79 mL, 8.1 mmol). After stirring 3.5 h, the reaction mixture was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate solution. The collected organic was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. Three quarters of the crude material was subjected to same procedure as the synthesis of 2-((6chloro-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (55a) employing 2-amino-5-cyanopyridine to afford the title compound as a white solid (1.07 g, 74% over three steps). LCMS: m/z = 356 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 5.0 Hz, 1H), 7.90 (s, 1H), 7.39 (br s, 1H), 7.20 (s, 1H), 7.08 (d, J = 5.0 Hz, 1H), 6.81 (s, 1H), 4.87−4.78 (m, 1H), 4.00−3.92 (m, 1H), 3.22−3.13 (m, 1H), 2.79−2.70 (m, 1H), 2.68−2.57 (m, 1H), 2.14 (s, 3H), 1.99−1.86 (m, 2H), 1.69−1.58 (m, 2H). 2-(4-(1-Acetylpiperidin-4-yl)-6-(3,3-difluoropyrrolidin-1-yl)pyridin-2-ylamino)isonicotinonitrile (21). Reaction of 3,3-difluoropyrrolidine hydrochloride and 2-((4-(1-acetylpiperidin-4-yl)-6-chloropyridin-2-yl)amino)isonicotinonitrile (55b) following the general Buchwald−Hartwig procedure afforded the title compound. 1H NMR (400 MHz, DMSO-d6) δ 9.81 (s, 1H), 8.51−8.43 (m, 1H), 8.41 (d, J = 5.0 Hz, 1H), 7.20 (dd, J = 5.0, 1.4 Hz, 1H), 6.58 (s, 1H), 6.00 (s, 1H), 4.59−4.45 (m, 1H), 4.01−3.77 (m, 3H), 3.71−3.55 (m, 2H), 3.19− 3.01 (m, 1H), 2.71−2.54 (m, 4H), 2.03 (s, 3H), 1.85−1.68 (m, 2H), 1.67−1.34 (m, 2H). 2,6-Dichloro-4-(tetrahydro-2H-pyran-4-yl)pyridine (56). Reaction of 4-bromotetrahydro-2H-pyran (2.88 mL, 25.6 mmol) following the procedure for the preparation of tert-butyl 4-(2,6dichloro-4-pyridyl)piperidine-1-carboxylate (51) afforded the title compound as a white solid (3.0 g, 70% yield). 1H NMR (CDCl3, 400 MHz) δ 7.11 (s, 1H), 4.09 (m, 2H), 3.57−3.42 (m, 2H), 2.85− 2.70 (m, 1H), 1.77 (m, 4H). 2-Chloro-6-(3,3-difluoropyrrolidin-1-yl)-4-(tetrahydro-2Hpyran-4-yl)pyridine (57). Reaction of 2,6-dichloro-4-(tetrahydro2H-pyran-4-yl)pyridine (1.20 g, 5.20 mmol) following the procedure for the preparation of tert-butyl 4-(2-chloro-6-(3,3-difluoropyrrolidin1-yl)pyridin-4-yl)piperidine-1-carboxylate (52) afforded the title compound. LCMS: m/z = 303 [M + H]+. 2-(6-(3,3-Difluoropyrrolidin-1-yl)-4-(tetrahydro-2H-pyran-4yl)pyridin-2-ylamino)isonicotinonitrile (22). Reaction of 2-chloro6-(3,3-difluoropyrrolidin-1-yl)-4-(tetrahydro-2H-pyran-4-yl)pyridine

(57) (100 mg, 0.33 mmol) following the procedure for the preparation of tert-butyl 4-(2-(3,3-difluoropyrrolidin-1-yl)-6-(4-(trifluoromethyl)pyridin-2-ylamino)pyridin-4-yl)-5,6-dihydropyridine-1(2H)-carboxylate (47) afforded the title compound as an off-white solid (37 mg, 29% yield). LCMS: m/z = 386 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.84 (s, 1H), 8.47 (s, 1H), 8.41 (d, J = 5.0 Hz, 1H), 7.21 (d, J = 5.0 Hz, 1H), 6.61 (s, 1H), 6.00 (s, 1H), 3.94 (d, J = 10.9 Hz, 2H), 3.86 (t, J = 13.2 Hz, 2H), 3.65 (t, J = 7.2 Hz, 2H), 3.47−3.38 (m, 2H), 2.70−2.52 (m, 3H), 1.71−1.62 (m, 4H). tert-Butyl 3-(2,6-Dichloropyridin-4-yl)azetidine-1-carboxylate (58). Reaction of 1-Boc-3-(iodo)azetidine (11.3 g, 39.0 mmol) following the procedure for the preparation of tert-butyl 4-(2,6dichloro-4-pyridyl)piperidine-1-carboxylate (51) afforded the title compound (1.73 g, 54% yield). LCMS: m/z = 303 [M + H]+. 1H NMR (400 MHz, CDCl3) δ: 7.23 (s, 2H), 4.35 (t, J = 8.7 Hz, 2H), 3.92 (dd, J = 8.7, 5.6 Hz, 2H), 3.73−3.61 (m, 1H), 1.47 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 157.03, 156.08, 151.09, 121.24, 80.24, 55.31, 32.48, 28.32. 2,6-Dichloro-4-(1-(oxetan-3-yl)azetidin-3-yl)pyridine (59). Reaction of tert-butyl 3-(2,6-dichloropyridin-4-yl)azetidine-1-carboxylate (58) (940 mg, 3.10 mmol) following the procedure for the preparation of 2-chloro-6-(3,3-difluoropyrrolidin-1-yl)-4-(1-(oxetan-3yl)piperidin-4-yl)pyridine (53) except that the reductive amination was performed at 50 °C afforded the title compound as a red oil (640 mg, 80% over two steps). 1H NMR (400 MHz, CDCl3) δ 7.27 (s, 2H), 4.75−4.68 (m, 2H), 4.57−4.52 (m, 2H), 3.82−3.77 (m, 1H), 3.77− 3.71 (m, 2H), 3.67−3.58 (m, 1H), 3.32−3.27 (m, 2H). 2-((6-Chloro-4-(1-(oxetan-3-yl)azetidin-3-yl)pyridin-2-yl)amino)isonicotinonitrile (60). Reaction of 2,6-dichloro-4-(1-(oxetan-3-yl)azetidin-3-yl)pyridine (58) (1.50 g, 5.79 mmol) following the procedure for the preparation of 2-((6-chloro-4-(1-(oxetan-3yl)piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (55) afforded the title compound as a white solid (1.0 g, 51%). 1H NMR (400 MHz, CDCl3) δ 8.39 (dd, J = 5.0, 0.8 Hz, 1H), 7.97 (d, J = 0.8 Hz, 1H), 7.54 (br s, 1H), 7.29 (s, 1H), 7.09 (dd, J = 5.1, 1.3 Hz, 1H), 6.96 (s, 1H), 4.76−4.69 (m, 2H), 4.61−4.53 (m, 2H), 3.85−3.79 (m, 1H), 3.79− 3.72 (m, 2H), 3.71−3.60 (m, 1H), 3.34−3.29 (m, 2H). 2-((6-(3,3-Difluoropyrrolidin-1-yl)-4-(1-(oxetan-3-yl)azetidin3-yl)pyridin-2-yl)amino)isonicotinonitrile (27). Reaction of 2-((6chloro-4-(1-(oxetan-3-yl)azetidin-3-yl)pyridin-2-yl)amino)isonicotinonitrile (50.0 mg, 0.146 mmol) following the general Buchwald−Hartwig procedure afforded the title compound as a white solid (15 mg, 25%). LCMS: m/z = 413 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.83 (br s, 1H), 8.47 (s, 1H), 8.41 (d, J = 5.0 Hz, 1H), 7.21 (d, J = 5.0 Hz, 1H), 6.71 (s, 1H), 6.02 (s, 1H), 4.62− 4.53 (m, 2H), 4.44−4.36 (m, 2H), 3.93−3.80 (m, 2H), 3.80−3.71 (m, 1H), 3.70−3.59 (m, 4H), 3.59−3.48 (m, 1H), 3.24−3.18 (m, 2H), 2.63−2.53 (m, 2H). 4-(2,6-Dichloro-4-pyridyl)tetrahydropyran-4-carbonitrile (62). To a stirring solution of 2,4,6-trichloropyridine (4.65 g, 24.7 mmol) and tetrahydropyran-4-carbonitrile (2.29 g, 20.6 mmol) in tetrahydrofuran (100 mL, 0.2 M) at −78 °C under nitrogen was added lithium bis(trimethylsilyl) amide (29 mL, 29 mmol, 1.0 M in tetrahydrofuran). After 5 min, the cooling bath was removed. After stirring further for 40 min, the reaction was quenched by the addition of saturated aqueous ammonium chloride solution. The organic was removed under reduced pressure. The resulting mixture was extracted with dichloromethane. The collected organic was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. Purification by flash column chromatography (7:3 heptane/ethyl acetate) afforded the title compound as a white solid (3.75 g, 71% yield). 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 0.6 Hz, 2H), 4.17− 4.05 (m, 2H), 3.93−3.84 (m, 2H), 2.15−1.99 (m, 4H). 2-((6-Chloro-4-(4-cyanotetrahydro-2H-pyran-4-yl)pyridin-2yl)amino)isonicotinonitrile (64). To 4-(2,6-dichloro-4-pyridyl)tetrahydropyran-4-carbonitrile (62) (1.50 g, 5.83 mmol), 2-amino-5cyanopyridine (716 mg, 5.83 mmol), tris(dibenzylideneacetone)dipalladium(0) (138 mg, 0.151 mmol), 2,2′-bis(diphenylphosphino)1,1′-binaphthyl (187 mg, 0.300 mmol), and cesium carbonate (2.66 g, 8.17 mmol) was added anhydrous 1,4-dioxane (23 mL, 0.25 M) under 415

dx.doi.org/10.1021/jm5013984 | J. Med. Chem. 2015, 58, 401−418

Journal of Medicinal Chemistry

Article

nitrogen. The reaction mixture was stirred at 80 °C for 18 h before cooling to room temperatue and filtering through Celite. After concentration under reduced pressure, purification by flash column chromatography (1:1 heptane/ethyl acetate) afforded the title compound as a colorless solid (880 mg, 44% yield). LCMS: m/z = 340 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.44 (d, J = 5.1 Hz, 1H), 7.82 (s, 1H), 7.67 (s, 1H), 7.52 (br s, 1H), 7.13 (d, J = 5.1 Hz, 1H), 7.05 (s, 1H), 4.21−4.07 (m, 2H), 3.97−3.79 (m, 2H), 2.26−2.08 (m, 2H), 2.08−1.99 (m, 2H). 2-(4-(4-Cyanotetrahydro-2H-pyran-4-yl)-6-(3,3-difluoropyrrolidin-1-yl)pyridin-2-ylamino)isonicotinonitrile (23). Reaction of 3,3-difluoropyrrolidine hydrochloride with 2-((6-chloro-4-(4cyanotetrahydro-2H-pyran-4-yl)pyridin-2-yl)amino)isonicotinonitrile (64) following the general Buchwald−Hartwig procedure afforded the title compound (47% yield). LCMS: m/z = 411.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.01 (br s, 1H), 8.47−8.41 (m, 2H), 7.29− 7.22 (m, 1H), 6.93 (d, J = 0.9 Hz, 1H), 6.18 (d, J = 1.0 Hz, 1H), 4.08− 3.97 (m, 2H), 3.97−3.84 (m, 2H), 3.76−3.60 (m, 4H), 2.67−2.56 (m, 2H), 2.17−1.98 (m, 4H). 2-(2,6-Dichloropyridin-4-yl)-2-methylpropanenitrile (63). To a solution of 2,4,6-trichloropyridine (6.0 g, 33 mmol) and 2methylpropanenitrile (1.9 g, 28 mmol) in anhydrous tetrahydrofuran (30 mL) was added dropwise lithium bis(trimethylsilyl)amide (38.6 mL, 38.6 mmol, 1 M in tetrahydrofuran) at −78 °C under nitrogen. After 15 min, the reaction mixture was warmed to room temperature for 2 h. The reaction mixture was diluted with saturated aqueous ammonia chloride solution (30 mL), and the resulting solution was extracted with ethyl acetate (3 × 60 mL). The combined organic was concentrated under reduced pressure. Purification by flash column chromatography afforded the title compound as a white solid (6.0 g, 84% yield). LCMS: m/z = 214.7 [M + H]+. 2-(2-Chloro-6-(3,3-difluoropyrrolidin-1-yl)pyridin-4-yl)-2methylpropanenitrile (65). A mixture of 2-(2,6-dichloropyridin-4yl)-2-methylpropanenitrile (63) (0.30 g, 1.4 mmol), 3,3-difluoropyrrolidine hydrochloride (1 g, 7 mmol), and N,N-diisopropylethylamine (1.8 g, 14 mmol) in dimethyl sulfoxide (20 mL) was heated between 80−90 °C for 16 h. After cooling to room temperature, the reaction solution was poured into water (10 mL), and the resulting mixture was extracted with ethyl acetate (3 × 20 mL). The collected organic was concentrated under reduced pressure. Purification by flash column chromatography afforded the title compound as a white solid (0.20 g, 50% yield). LCMS: m/z = 285.8 [M + H]+. 2-(4-(2-Cyanopropan-2-yl)-6-(3,3-difluoropyrrolidin-1-yl)pyridin-2-ylamino)isonicotinonitrile (24). To a solution of 2-(2chloro-6-(3,3-difluoropyrrolidin-1-yl)pyridin-4-yl)-2-methylpropanenitrile (65) (0.20 g, 0.70 mmol), 2-amino-4-cyanopyridine (167 mg, 1.40 mmol), and K3PO4 (0.30 g, 1.4 mmol) in anhydrous 1,4-dioxane (15 mL) was added Pd(t-Bu3P)2 (36 mg, 0.070 mmol) under nitrogen. The resulting solution was heated to reflux for 3 h. After cooling to room temperature, the mixture was filtered and concentrated under reduced pressure. Purification by preparative reverse phase HPLC afforded the title compound as a white solid (11 mg, 4.2% yield). LCMS: m/z 368.9 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.38− 8.35 (m, 2H), 7.40 (s, 1H), 7.03 (d, J = 5.2 Hz, 1H), 6.44 (s, 1H), 6.03 (s, 1H), 3.87 (t, J = 13.2 Hz, 2H), 3.78 (t, J = 7.2 Hz, 2H), 2.59−2.48 (m, 2H), 1.71 (s, 6H). 2-(2,6-Dichloropyridin-4-yl)propan-2-ol (67). To a solution of methyl 2,6-dichloroisonicotinate (61) (1.0 g, 4.9 mmol) in tetrahydrofuran (30 mL) was added CH3MgBr (4.1 mL, 12.3 mmol, 3 M in tetrahydrofuran) dropwise at −78 °C under nitrogen. After 1 h, the mixture was warmed to room temperature for 1 h. The reaction mixture was diluted with saturated aqueous ammonia chloride solution (15 mL), and the resulting mixture was extracted with ethyl acetate (3 × 30 mL). The collected organic was concentrated under reduced pressure. Purification by flash column chromatography afforded the title compound as a white solid (700 mg, 70% yield). LCMS: m/z = 205.7 [M + H]+. 2-(2-Chloro-6-(3,3-difluoropyrrolidin-1-yl)pyridin-4-yl)propan-2-ol (68). A solution of 2-(2,6-dichloropyridin-4-yl)propan2-ol (67) (0.300 g, 1.46 mmol), 3,3-difluoropyrrolidine hydrochloride

(1.0 g, 7.3 mmol), and N,N-diisopropylethylamine (1.88 g, 14.6 mmol) in dimethyl sulfoxide (10 mL) was heated between 80 and 90 °C for 16 h. After cooling to room temperature, the mixture was poured into water (10 mL), and the resulting mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic was concentrated under reduced pressure. Purification by flash column chromatography afforded the title compound as a white solid (0.20 g, 50% yield). LCMS: m/z = 276.9 [M + H]+, 2-(6-(3,3-Difluoropyrrolidin-1-yl)-4-(2-hydroxypropan-2-yl)pyridin-2-ylamino)isonicotinonitrile (25). To a mixture of 2-(2chloro-6-(3,3-difluoropyrrolidin-1-yl)pyridin-4-yl)propan-2-ol (68) (0.10 g, 0.36 mmol), 2-amino-4-cyanopyridine (86.3 mg, 0.725 mmol), and K3PO4 (155 mg, 0.72 mmol) in anhydrous 1,4-dioxane (15 mL) was added Pd(t-Bu3P)2 (18 mg, 0.036 mmol) under nitrogen. The resulting mixture was heated to reflux for 3 h. After cooling to room temperature, the mixture was filtered and concentrated under reduced pressure. Purification by preparative reverse phase HPLC afforded the title compound as a white solid (0.080 g, 62% yield). LCMS: m/z = 359.9 [M + H]+. 1H NMR (400 MHz, CD3OD) δ 8.52 (s, 1H), 8.34−8.33 (m, 1H), 7.06−7.04 (m, 1H), 6.63 (s, 1H), 6.19 (s, 1H), 3.99 (t, J = 13.2 Hz, 2H), 3.73 (t, J = 7.2 Hz, 2H), 2.60−2.49 (m, 2H), 1.51 (s, 6H).

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AUTHOR INFORMATION

Corresponding Authors

*J.W.L.: phone, 650-467-2877; e-mail, lewcock.joseph@gene. com. *M.S.: phone, 650-467-7764; e-mail, [email protected]. Present Addresses

# F.C.: Achaogen, 7000 Shoreline Court, No. 371, South San Francisco, CA 94080, U.S. ∞ C.E.L.P.: National Institute for Neurological Disorders and Stroke, National Institutes of Health, 35A Convent Drive, Bethesda, MD 20892, U.S. × J.P.L.: Biogen Idec, 14 Cambridge Center, Cambridge, MA 02142, U.S. ○ Y.G.S.: College of Pharmacy, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Republic of Korea (South). △ B.W.: Well Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, U.S.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the following individuals for their contributions: Chris Hamman, Mengling Wong, and Michael Hayes for compound purification; Emile Plise and Jonathan Cheong for MDR1-MDCK data; Xiaolin Zhang, Allan Jaochico, and Xiao Ding for bioanalytical data; Amy Sambrone for formulations work; York Rudhard and the Evotec team for biochemical and cell-based potency data; Wuxi for support with analog synthesis; and Genentech compound management for sample handling.

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ABBREVIATIONS USED CDI, carbonyldiimidazole; ClogD, calculated logarithm of distribution coefficient; CNS MPO, central nervous system multiparameter optimization; cpKa, calculated pKa; DMEM, Dulbecco’s modified Eagle medium; DLK, dual leucine zipper kinase; DRG, dorsal root ganglion; FBS, fetal bovine serum; IP, intraperitoneal; JNK, c-Jun N-terminal kinase; LipE, lipophilic ligand efficiency; MAP3K12, mitogen-activated protein kinase kinase kinase 12; MCT, methylcellulose Tween 80; MKK, 416

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program that couples apoptotic and regenerative responses to axonal injury. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 4039−4044. (18) Welsbie, D. S.; Yang, Z.; Ge, Y.; Mitchell, K. L.; Zhou, X.; Martin, S. E.; Berlinicke, C. A.; Hackler, L., Jr.; Fuller, J.; Fu, J.; Cao, L. H.; Han, B.; Auld, D.; Xue, T.; Hirai, S.; Germain, L.; Simard-Bisson, C.; Blouin, R.; Nguyen, J. V.; Davis, C. H.; Enke, R. A.; Boye, S. L.; Merbs, S. L.; Marsh-Armstrong, N.; Hauswirth, W. W.; DiAntonio, A.; Nickells, R. W.; Inglese, J.; Hanes, J.; Yau, K. W.; Quigley, H. A.; Zack, D. J. Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 4045−4050. (19) Pozniak, C. D.; Sengupta Ghosh, A.; Gogineni, A.; Hanson, J. E.; Lee, S. H.; Larson, J. L.; Solanoy, H.; Bustos, D.; Li, H.; Ngu, H.; Jubb, A. M.; Ayalon, G.; Wu, J.; Scearce-Levie, K.; Zhou, Q.; Weimer, R. M.; Kirkpatrick, D. S.; Lewcock, J. W. Dual leucine zipper kinase is required for excitotoxicity-induced neuronal degeneration. J. Exp. Med. 2013, 210, 2553−2567. (20) Miller, B. R.; Press, C.; Daniels, R. W.; Sasaki, Y.; Milbrandt, J.; DiAntonio, A. A dual leucine kinase-dependent axon self-destruction program promotes Wallerian degeneration. Nat. Neurosci. 2009, 12, 387−389. (21) Ferraris, D.; Yang, Z.; Welsbie, D. Dual leucine zipper kinase as a therapeutic target for neurodegenerative conditions. Future Med. Chem. 2013, 5, 1923−1934. (22) Cohen, F.; Huestis, M.; Ly, C.; Patel, S.; Siu, M.; Zhao, X. Preparation of substituted dipyridines as DLK inhibitors for treating neurodegeneration. PCT Int. Appl. WO 2013174780, 2013. (23) Hopkins, A. L.; Groome, C. R.; Alex, A. Ligand efficiency: a useful metric for lead selection. Drug Discovery Today 2004, 9, 430− 431. (24) LipE or LLE = −log Ki − ClogP. Leeson, P. D.; Springthorpe, B. The influence of drug-like concepts on decision-making in medicinal chemistry. Nat. Rev. Drug Discovery 2007, 6, 881−890. (25) Ryckmans, T.; Edwards, M. P.; Horne, V. A.; Correia, A. M.; Owen, D. R.; Thompson, L. R.; Tran, I.; Tutt, M. F.; Young, T. Rapid assessment of a novel series of selective CB2 agonists using parallel synthesis protocols: a lipophilic efficiency (LipE) analysis. Bioorg. Med. Chem. Lett. 2009, 19, 4406−4409. (26) CNS MPO (central nervous system multiparameter optimization) of >4 is desirable. Wager, T. T.; Hou, X.; Verhoest, P. R.; Villalobos, A. Moving beyond rules: the development of a central nervous system multiparameter optimization (CNS MPO) approach to enable alignment of druglike properties. ACS Chem. Neurosci. 2010, 1, 435−449. (27) Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman, M. D.; Verhoest, P. R.; Villalobos, A.; Will, Y. Defining desirable central nervous system drug space through the alignment of molecular properties, in vitro ADME, and safety attributes. ACS Chem. Neurosci. 2010, 1, 420−434. (28) Gallo, K. A.; Johnson, G. L. Mixed-lineage kinase control of JNK and p38 MAPK pathways. Nat. Rev. Mol. Cell Biol. 2002, 3, 663−672. (29) For example: Das, J.; Chen, P.; Norris, D.; Padmanabha, R.; Lin, J.; Moquin, R. V.; Shen, Z.; Cook, L. S.; Doweyko, A. M.; Pitt, S.; Pang, S.; Shen, D. R.; Fang, Q.; de Fex, H. F.; McIntrye, K. W.; Shuster, D. J.; Gillooly, K. M.; Behina, K.; Schieven, G. L.; Wityak, J.; Barrish, J. C. 2-Aminothiazole as a novel kinase inhibitor template. Structure−activity relationship studies toward the discovery of N-(2chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1- piperazinyl)]-2methyl-4-pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (dasatinib, BMS-354825) as a potent pan-Src kinase inhibitor. J. Med. Chem. 2006, 49, 6819−6832. (30) For example: Janetka, J. W.; Ashwell, S. Checkpoint kinase inhibitors: a review of the patent literature. Expert Opin. Ther. Pat. 2009, 19, 165−197. (31) Dalvie, D. K.; Kalgutkar, A. S.; Khojasteh-Bakht, S. C.; Obach, R. S.; O’Donnell, J. P. Biotransformation reactions of five-membered aromatic heterocyclic rings. Chem. Res. Toxicol. 2002, 15, 269−299. (32) Pierce, A. C.; ter Haar, E.; Binch, H. M.; Kay, D. P.; Patel, S. R.; Li, P. CH···O and CH···N hydrogen bonds in ligand design: a novel

mitogen-activated protein kinase kinase; MLK, mixed-lineage kinase; MDR1, multidrug resistance protein 1; MDCK, Madin−Darby canine kidney; MPTP, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine; MSD, Meso Scale Discovery detection; NGF, nerve growth factor; PBS, phosphate buffered saline; P-gp, P-glycoprotein; SNAr, nucleophilic aromatic substitution; TBI, traumatic brain injury

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