Lead Optimization toward Proof-of-Concept Tools for Huntington's

Article pubs.acs.org/jmc

Lead Optimization toward Proof-of-Concept Tools for Huntington’s Disease within a 4‑(1H‑Pyrazol-4-yl)pyrimidine Class of Pan-JNK Inhibitors John Wityak,*,† Kevin F. McGee,‡ Michael P. Conlon,‡ Ren Hua Song,‡ Bryan C. Duffy,‡ Brent Clayton,‡ Michael Lynch,‡ Gwen Wang,‡ Emily Freeman,‡ James Haber,‡ Douglas B. Kitchen,‡ David D. Manning,‡ Jiffry Ismail,‡ Yuri Khmelnitsky,‡ Peter Michels,‡ Jeff Webster,‡ Macarena Irigoyen,‡ Michele Luche,‡ Monica Hultman,‡ Mei Bai,‡ IokTeng D. Kuok,‡ Ryan Newell,‡ Marieke Lamers,§ Philip Leonard,§ Dawn Yates,§ Kim Matthews,§ Lynette Ongeri,§ Steve Clifton,§ Tania Mead,§ Susan Deupree,∥ Pat Wheelan,∥ Kathy Lyons,⊥ Claire Wilson,# Alex Kiselyov,† Leticia Toledo-Sherman,† Maria Beconi,† Ignacio Muñoz-Sanjuan,† Jonathan Bard,† and Celia Dominguez† †

CHDI Foundation, Inc., 6080 Center Drive, Suite 100, Los Angeles, California 90045, United States Albany Molecular Research Inc. (AMRI), 26 Corporate Circle, Albany, New York 12212-5098, United States, § BioFocus Discovery Services, Charles River Laboratories, Chesterford Research Park, Little Chesterford, CB10 1XL, United Kingdom ∥ Tandem Laboratories, 2202 Ellis Road, Durham, North Carolina 27703, United States, ⊥ Pharmacokinetics Consultant to CHDI, P.O. Box 64, Holland, New York 14080, United States, # Evotec, 114 Milton Park, Abingdon, OX14 4SA, United Kingdom ‡

S Supporting Information *

ABSTRACT: Through medicinal chemistry lead optimization studies focused on calculated properties and guided by X-ray crystallography and computational modeling, potent pan-JNK inhibitors were identified that showed submicromolar activity in a cellular assay. Using in vitro ADME profiling data, 9t was identified as possessing favorable permeability and a low potential for efflux, but it was rapidly cleared in liver microsomal incubations. In a mouse pharmacokinetics study, compound 9t was brain-penetrant after oral dosing, but exposure was limited by high plasma clearance. Brain exposure at a level expected to support modulation of a pharmacodynamic marker in mouse was achieved when the compound was coadministered with the pan-cytochrome P450 inhibitor 1-aminobenzotriazole.

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INTRODUCTION The c-Jun N-terminal serine/threonine protein kinases (JNKs) comprise a mitogen-activated protein kinase family that regulates signal transduction events in response to environmental stress. To date, three distinct jnk genes have been identified (jnk1, jnk2, and jnk3), expressing 10 isoforms and splice variants of JNK proteins. Whereas JNK1 and JNK2 are ubiquitously expressed, JNK3 is present primarily in brain, with lower expression found in testis, heart, and pancreatic β cells.1 Activation of JNK has been implicated in chronic neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases.2,3 Genetic ablation of the murine jnk3 gene resulted in mice that were resistant to the excitotoxic glutamate-receptor agonist kainic acid, leading to a reduction in seizure activity.4 Recent reports describe orally bioavailable, ATP-competitive JNK inhibitors that have shown beneficial effects in vitro and in vivo.5,6 In addition, JNK substrate-competitive peptides have shown beneficial effects in models of ischemia and Alzheimer’s disease.7,8 Of particular interest were reports implicating increased JNK expression and activity in cellular9−14 and in vivo models15,16 of © 2015 American Chemical Society

Huntington’s disease (HD), an autosomal dominant, progressive neurodegenerative disease that is characterized clinically by motor, cognitive, and behavioral deficits. In addition, a role for JNK3 in fast axonal transport (FAT) was demonstrated in squid axoplasm perfused with mutant huntingtin protein, where JNK3 was shown to phosphorylate kinesin-1 heavy chain and decrease FAT.17 Due to its reported role in neuronal cell death, apoptosis, and cargo transport as well as its restricted tissue distribution, JNK3 is an attractive target for potential therapeutic intervention in HD. We therefore desired a potent, selective, and brain-penetrant JNK3 inhibitor for proof-ofconcept (POC) studies. There have been many reports of JNK inhibitors from a wide variety of ATP competitive and noncompetitive chemotypes.18,19 Review of this literature revealed several compounds for benchmarking efforts, the results of which are summarized in the Supporting Information. Our minimum requirements for Received: September 3, 2014 Published: March 11, 2015 2967

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Journal of Medicinal Chemistry Table 1. Activity Profile of Pyrazole 1

JNK3 IC50 ± SD (nM) 433 ± 208

JNK1 JNK2 IC50 ± SD (nM) IC50 ± SD (nM) 737 ± 281

161 ± 77

p38α IC50 ± SD (nM)

Caco-2 Papp A−B (nm s−1)

Caco-2 Papp B−A (nm s−1)

mLM Clint (μL min−1 mg−1)

hLM Clint (μL min−1 mg−1)

24 ± 18

467

242

81.8

100-fold selectivity against p38 MAPK (itself a partially validated HD target of interest) and adequate cellular potency and brain exposure to affect a pharmacodynamic (PD) marker. Potential choices considered for a cellular assay were inhibition of phosphorylation of c-Jun or ATF-2 substrates with reduction of phosphorylated c-Jun (p-c-Jun) or ATF-2 as the PD marker. In addition, a POC compound would need to have adequate ADME properties, as determined in solubility, permeability/ efflux, and microsomal stability assays. Attesting to the challenge, none of the benchmark compounds were judged to be suitable for advancement into mouse HD models. Concurrent with these benchmarking studies, and to further facilitate identification of novel starting points, a docking-based virtual screen was conducted based on the X-ray crystal structure of JNK3 in complex with an imidazole-pyrimidine inhibitor (1pmq) from the Protein Data Bank (PDB, www.rcsb. org). A compound library of approximately 200 000 compounds from Asinex’s privileged collections was docked against a protein grid generated from the 1pmq structure. Of the 1100 virtual hits selected for wet screening, a set of approximately 90 was confirmed to be actual hits. Among these, pyrazole 1 was identified as an attractive starting point for medicinal chemistry and was advanced into hit to lead studies based upon favorable JNK3 potency, chemical tractability, and a small base of published SAR including potent activity in a cellular p-c-Jun assay.20 Profiling studies revealed 1 to be a pan-JNK inhibitor 2968

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Journal of Medicinal Chemistry Scheme 1. General Synthesis of Pyrazolesa

Reagents and conditions: (a) NaHMDS, THF, 60 °C, 4 days; (b) DMF·DMA, tol, 110 °C, 6 h, then NH2NHR2, RT, 16 h; (c) mCPBA, CH2Cl2, 0 °C, 16 h; (d) 3,4-dihydro-2H-pyran, p-TsOH, RT, 10 min; (e) NH2R1, dioxane, MW, 160 °C, 1 h; (f) HCl·dioxane, MeOH, RT, 2 h.

a

Scheme 2. Synthesis of 3-Trifluoromethylpyrazole 9oa

the hydrophobic pocket revealed such similarity.23,24 This knowledge base informed our SAR exploration around this chemotype and resulted in the design of compounds that would induce the engagement of the Met146 side chain as observed in the adenosine structure, thus blocking the hydrophobic pocket of hydrophobic region I. Docking grids were prepared for JNK1, JNK2, JNK3, and p38; docking and scoring was used as a tool to guide the medicinal chemistry efforts of the group. Synthesis of Inhibitors. We chose to focus an SAR study around the 4-(pyrazol-4-yl)pyrimidine scaffold. Synthesis of these pyrazoles was readily accomplished using a variation on the Knorr pyrazole synthesis illustrated in Scheme 1. Alkylation of 4-methylpyrimidine 2 with ester 3 in the presence of sodium hexamethyldisilazide gave a mixture of keto and enol tautomers 4 and 4′. Reaction with dimethylformamide dimethylacetal (DMF·DMA) then afforded the intermediate enaminone, which was followed by cyclization in the presence of hydrazine or a monosubstituted hydrazine to give pyrazole 5. Oxidation using mCPBA then provided sulfone 6. For compounds in which R2 is a hydrogen atom, the introduction of a tetrahydropyran (THP) protecting group at this stage, giving 7, avoids the large excess of amine necessary in the subsequent amination step. Protection in this way suppresses a competing dimerization process that is prominent with poorly nucleophilic amines (NH2R1). Microwave-assisted amination to provide 8 followed by THP removal under acidic conditions affords 9. It should be noted that in every instance the regiochemistry of cyclization with a monosubstituted hydrazine was confirmed as the 2,3-regioisomer (as depicted in 7−9) through heteronuclear multiple bond correlation (HMBC) and NOE NMR experiments (see Supporting Information). The synthesis of 3-trifluoromethyl pyrazole 9o required an alternative synthetic route due to the poor reactivity of enol ether 10 (Scheme 2). Enol-ether 11a, prepared by standard methods, was treated with triethyl orthoformate to provide enol ether 12. Cyclization with methyl hydrazine provided pyrazole 13, which was converted to 2-thiomethylpyrimidine 14 following a two-step cyclization procedure. Oxidation to the activated sulfone 15 followed by displacement with excess trans-4aminocyclohexanol gave 9o. The synthesis of 5-chloropyrimidine 9 mm is shown in Scheme 3. Cyclization of ethyl acetoacetate 11b with S-methylisothiouronium sulfate gave pyrimidone 16 in good yield. Chlorination using sulfuryl chloride provided chloride 17,

a Reagents and conditions: (a) HC(OEt)3, Ac2O, reflux, 19 h; (b) NH2NHCH3, THF, −10 °C, 1.25 h; (c) DMF·DMA, tol, 110 °C, 20 h; (d) methyl carbamimidothioate, NaOMe, i-PrOH, 12 h; (e) mCPBA, CH2Cl2, 0 °C, 16 h; (f) NH2R1, dioxane, 160 °C, 1 h.

which was further chlorinated using phosphorus oxychloride to give dichloride 18. Regioselective hydrogenolysis of the 4-chloro-substituent led to 5-chloropyrimidine 19. It should be noted that chloride 19 was quite volatile and that caution must be exercised during solvent removal. Reaction of 19 with N-methoxy-N-methyl-1-(trifluoromethyl)cyclopropanecarboxamide then gave ketone 20. Pyrazole formation using DMF·DMA followed by hydrazine provided pyrazole 21 in poor isolated yield. It was likely that the presence of the chloride on the pyrimidine modulated reactivity of several intermediates. Several subsequent steps required more forcing conditions, resulting in lower yields. Oxidization to methylsulfone 22 using mCPBA, THP protection to give pyrazole 23, and methylsulfone displacement using 4,4-difluorocyclohexylamine under microwave-assisted heating afforded 24. Deprotection under standard conditions completed the synthesis of 9 mm in low overall yield. Preparation of pyrimidine 25 is shown in Scheme 4. Reaction of cyclopropanecarboxylic acid 26 with N-methoxy methylamine 2969

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Journal of Medicinal Chemistry Scheme 3. Synthesis of 5-Chloropyrimidine 9 mma

Reagents and conditions: (a) S-methyl isothiouronium sulfate, Na2CO3, H2O, RT, 16 h, 43%; (b) SO2Cl2, FeCl3, AcOH, Ac2O, 100 °C, 36 h, 69%; (c) POCl3, DMA, 115 °C, 15 h, 75%; (d) H2, Pd−C, NaOH, H2O, RT, 24 h, 75%; (e) N-methoxy-N-methyl-1-(trifluoromethyl)cyclopropanecarboxamide, NaHMDS, THF, 0 °C to RT, 16 h, 79%; (f) DMF·DMA, MeOH, 110 °C, 6 h, then NH2NH2, THF, RT, 16 h, 5%; (g) mCPBA, CH2Cl2, RT, 16 h, 49%; (h) 3,4-dihydro-2H-pyran, p-TsOH, 0 °C, 5 min, 99%; (i) 4,4-difluorocyclohexylamine, DMSO, Et3N, MW, 130 °C, 3 h, 17%; (j) HCl·dioxane, MeOH, RT, 2 days, 85%. a

Scheme 4. Synthesis of 2-Aminopyridine 25a

Table 2. JNK and p38α Potency of (4-Chlorophenyl)pyrazol-3-yl Derivativesa

Reagents and conditions: (a) NH(OCH3)CH3·HCl, EDC, HOBt, Et3N, CH2Cl2, RT, 90%; (b) LHMDS, 2-chloro-4-methylpyridine, THF, 55%; (c) DMF·DMA, MeOH, 110 °C, 6 h, then NH2NH2, THF, RT, 16 h, 68%; (d) 3,4-dihydro-2H-pyran, p-TsOH, 0 °C, 5 min, 95%; (e) 31, (t-Bu3P)2Pd(0), NaOt-Bu, dioxane, 130 °C, 16 h, then HCl, i-PrOH, RT, 2 days, 16%. a

a

Values accompanied by standard deviation were averaged from at least two independent experiments.

afforded Wienreb amide 27, which was subsequently used to acylate 2-chloro-4-methylpyridine to give 28 in good yield. Pyrazole formation to provide 29 and protection as the THP to give 30 was followed by Buchwald−Hartwig reaction with 3,3difluorocyclobutylamine (31), giving aminopyridine 32. Removal of the THP under standard conditions then afforded 25.

a 2-amino moiety on the pyrimidine resulted in a 1 to 2 orders of magnitude improvement in JNK3 activity relative to that of 1, with 4-hydroxycyclohexylamine 9c showing potent activity but little selectivity against JNK1, JNK2, or p38α. The lack of selectivity against JNK1 and JNK2 was expected, as the ATP binding site of the JNK isoforms are highly conserved (98% homology), with the only residue differences being Met115 in JNK1/3 versus Leu77 in JNK2 and Leu144 in JNK2/3 versus Ile106 in JNK1. Several groups have used these differences to gain some degree of selectivity among the JNK isoforms.25−29 The lack of selectivity against p38 was also predicted since the chlorophenyl substituent had been preserved and was expected to occupy the hydrophobic region I, as depicted in Figure 1, in both the JNKs and p38. The next set of analogues kept the trans-4-hydroxycyclohexylamine at R1 constant and probed the R3 position (Table 3).

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RESULTS AND DISCUSSION On the basis of the binding mode of 1, we expected to gain potency through the addition of an amine-containing substituent at the pyrimidine 2-position. This would complete the double hinge hydrogen-bond interaction with Met149 and would introduce the possibility of gaining favorable interactions with residues in the sugar region. Our SAR investigation began with preparation of a series of analogues in which the R3 group was fixed as 4-chlorophenyl with the incorporation of a small set of amines at the 2-position (Table 2). The incorporation of 2970

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Journal of Medicinal Chemistry Table 3. JNK and p38α Potency of trans-4Hydroxycyclohexylamine Derivativesa

Noting the generally favorable selectivity against p38α for this set, we obtained a costructure of JNK3 with pyrazole 9e at 2.3 Å resolution (Figure 2). Inspection of the structure revealed the expected double hinge interaction of the N1 pyrimidine and NH-linker moiety with the carbonyl and NH of Met149 and a water-mediated hydrogen-bonding interaction with the side chain of Lys93. The cyclohexyl ring pointed toward the solvent interphase and occupied the hydrophobic region II, with its hydroxyl substituent forming a hydrogen bond to Gln155. Unexpectedly, the pyrazole had rotated about the pyrazole pyrimidine bond, bringing the tert-butyl group and the cyclohexanol in close proximity due to an apparent favorable intramolecular hydrophobic contact. In this horseshoe conformation, the tert-butyl group occupied the sugar pocket and made hydrophobic interactions with Val78, Ala91, and Leu206. Interestingly, since no portion of compound 9e occupied hydrophobic region I, this allowed the side chain of Met146 to populate its natural adenosine-bound rotamer and thus occupy hydrophobic region I, leading to favorable selectivity against p38. The relatively weak JNK3 activity of 9k, which is devoid of a hydrophobic moiety at R3, highlights the importance of the placement of a hydrophobic moiety in the ribose pocket, aiding intramolecular stabilization of the horseshoe conformation. Compounds 9e and 9j were taken into in vitro ADME assays (Table 4). Both compounds showed low solubility, were rapidly metabolized in mLM, and had good stability in human liver microsomal incubations (hLM). Permeability and P-gp mediated efflux were determined in an MDCK-MDR1 transfected cell line. Tetrahydropyran 9e displayed good permeability but had moderate to high P-gp efflux, whereas 9j showed low permeability accompanied by moderate to high efflux. Our strategy to improve efflux to an acceptable level was to reduce the number of hydrogen-bond donors. Tactics included alkylation of the pyrazole ring and replacement of the cyclohexanol moiety, resulting in the compounds of Table 5. Alkylation of the pyrazole as in compounds 9l−o in all cases resulted in high permeability and a low effective efflux ratio (EER), but, other than for 9m, loss of JNK3 potency was

a

Values accompanied by standard deviation were averaged from at least two independent experiments. bNT indicates not tested.

Most significantly, selectivity against p38α was achieved by replacing the 3-aryl moiety with saturated groups. tert-Butyl derivative 9e was the most potent compound of this set, showing an IC50 value of 13 nM. Compounds 9f−h were also potent JNK3 inhibitors, affording inhibition constants of approximately 20 nM. Cyclopropyl 9i gave up 2-fold potency to this group, with tetrahydropyran 9j being another 2-fold less potent. The importance of a lipophilic substituent at R3 to potency was further established by the order of magnitude loss seen with pyrazole 9k. As expected, little selectivity was observed against the JNK1 or JNK2 isoforms.

Figure 2. 2Fo − Fc electron density map contoured at 1σ showing (A) 9e binding in the JNK3 active site; (B) a three-dimensional schematic showing the binding of 9e (pink) to JNK3 (aqua) overlapped with the binding of 1 (green) in the JNK3 active site (yellow). 2971

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Journal of Medicinal Chemistry Table 4. Results of in Vitro ADME Profiling Cmpd

JNK3 IC50 (nM)

aq. sol. (mg mL−1)

mLM Clint (μL min−1 mg−1)

hLM Clint (μL min−1 mg−1)

MDCK Papp A-B (nm s−1)

MDCK-MDR1 EER

9e 9j

13 73

0.028 0.037

498.8 39.8

10 000 >10 000 >10 000 NT NT

8.0 2.5 2.2 1.6 2.0 0.51 >10 3.0 2.7 >10 2.8 0.1 NT

1600 192 138 67 83 7 NA 28 24 NA 48 200

0.081 0.093 0.45 5.8 0.89 0.15 0.18 0.18 0.35 1.5 0.89 NT 0.0051

16 7 28 242 37 2 2 2 3 12 15 NA

NT indicates not tested. NA indicates not calculated. bValue from ref 33.

membranes and modulate functional activity within a cell at a concentration that might reasonably be achievable in brain. ADME-PK. In preparation for planned in vivo pharmacodynamic evaluation in mice of a p-c-Jun biomarker, several of the compounds from Tables 3, 5, and 6 were profiled for microsomal stability; however, despite the structural diversity queried, all showed rapid rates of metabolism in both mouse and rat liver microsomes (data not shown). From these studies, 9t emerged as an early example of a compound having met most of our criteria for progression into further studies. The JNK3 potency (IC50 = 94 nM), cellular potency (IC50 = 180 nM, LPS−TNF-α assay), cellular permeability (MDCK-WT Papp A−B = 265 nm s−1), and P-gp-mediated efflux (EER = 1.8) of 9t were acceptable. While its stability in mLM was poor/ moderate (Clint = 142 mL min−1 mg−1), its rate of disappearance was slower than that of most compounds tested from this series. When assayed at 10 μM in receptor panels of 144 diverse and 75 kinase targets, 9t showed >50% of control specific binding in 9 out of the 219 assays: adenosine A3 (51%), Na+ channel (83%), norepinephrine transporter (92%), CDC2/CDK1 (79%), CDK2 (77%), CDK5 (72%), GSK3β (82%), HGK (54%), and JNK1 (97%). The full report can be found in the Supporting Information. As a follow-up to the receptor profiling study, IC50 values were determined against CDK5 and GSK3β, since these are targets implicated in HD (Table 8). While activity against these would not affect a p-c-Jun readout, they might impact an efficacy readout in an HD model. The data suggests that selectivity against these kinases, which have relatively close homology to JNK3, may be difficult to achieve with this chemotype, although the selectivity observed for 9m shows that >50-fold is possible. This level of selectivity was possibly driven by favorable hydrophobic interactions of the R2 isopropyl moiety with Leu206 in JNK3 and, assuming a similar binding

results in either the Life Technologies assay or in an internally developed NIH3T3 cell-based c-Jun MSD assay (data not shown), we were unable to reasonably match the published potencies. It is perhaps of interest to note that the potency of a recently reported covalent inhibitor, JNK-IN-11 (35)34 was an exception. In the Life Technologies assay, 35 showed an IC50 of 0.1 μM and had similar translation of biochemical to cellular potency as some of the other inhibitors studied. Unfortunately, this compound potently inhibited a number of other kinases, including p38, when tested at 1 μM concentration in selectivity profiling conducted at Cerep (see Supporting Information). As stated earlier, this activity would be a potential confound in interpretation of efficacy results. In addition, it cannot be ruled out that the potent inhibition of p-c-Jun observed may be the result of off-target activity against a variety of kinase families. Due in part to the cellular c-Jun results, compounds were also tested in a second cellular assay, the LPS-induced TNF-α secretion assay in PBMC conducted at Cerep (Celle l’Evescaul, France). It is known that LPS activates the JNKs to induce TNF-α production35 and that this can be suppressed in macrophages by 34.36 The compounds in Table 7 showed markedly better potency and translation against this readout. Dexamethasone is included as the assay positive standard, and its potency was as expected. Interestingly, the dual JNK/p38 inhibitor 9c was approximately equipotent with JNK inhibitor 9r. The expectation was that significantly improved activity might have resulted from potent inhibition of these two MAPK families. Aminopyridine 25b was also one of the more potent compounds in this assay. These results, while encouraging, do little to advance JNK inhibition as an HD therapeutic strategy, but they may become relevant if a connection between elevated TNF-α and HD disease progression can be established. At present, they demonstrate that the compounds can cross cell 2975

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Journal of Medicinal Chemistry

panel of human CYP450 inhibitors is unknown. The CYP4503A4/5 inhibitor ketoconazole was particularly effective at slowing the rate of clearance when dosed at both 3× and 30× its Ki. In addition, pan-CYP450 inhibition using SKF-525a was more effective than inhibition of any individual isoform; it strongly suppressed the rate of metabolism when coadministered at 3× Ki. These in vitro findings were then extended, demonstrating that in vivo suppression of CYP450-mediated metabolism of 9t could result in enhanced brain exposure after oral dosing in mice. Thus, 9t was dosed orally at 30 mpk to one cohort, a second cohort was coadministered 30 mpk 9t with 50 mpk orally of the CYP450-3A4 inhibitor ketoconazole, and a third group was coadministered 30 mpk 9t and 30 mpk orally of 1-aminobenzotriazole (1-ABT), a potent pan-CYP450 inhibitor (Figure 4). The brain concentration at 8 h was approximately 100-fold improved with 1-ABT coadministration relative to that from administration of 9t alone. The concentration of 9t in brain was 7.3 μM by 0.5 h postdose and remained above this level through 4 h, reaching a Cmax of 11.4 μM at 1 h and then falling to 4.8 μM at 8 h. Working back from this exposure level and assuming that a brain concentration would likely need to be at some multiple of the cellular IC50 value, we felt confident that if elevated levels of TNF-α could be correlated with HD disease progression, then 9t would have the potency necessary to go forward as a PD tool. It is important to note that coadministration with the CYP450 inhibitor did not increase BBB permeability or alter the brain to plasma ratio; it only served to decrease clearance, thus improving exposure to all tissues. The plasma exposure of 9t was also similarly improved (not shown). For a highly metabolized compound such as 9t, suppression of metabolism (1) improved exposure to levels that might allow modulation of a PD marker in brain, (2) ensured that PD activity can be attributed to the parent compound and directly linked to JNK inhibition, and (3) since 1-ABT has been shown to be relatively nontoxic in a rat toxicology study,37 it may permit a highly metabolized compound to enter into a mouse HD efficacy study requiring chronic dosing.

Table 8. CDK5 and GSK3β Inhibition of Selected JNK3 Inhibitors Cmpd

JNK3 IC50 (nM)

CDK5 IC50 (nM)

GSK3β IC50 (nM)

9j 9u 9m 9t

73 16 24 88

1100 87 1400 430

260 280 1800 380

conformation with GSK3β and CDK5, unfavorable, or less favorable, interactions with Cys199 (GSK3β) and Ala143 (CDK5). In a mouse BBB penetration study, 9t was dosed as an i.v. bolus in mouse at 5 mpk. It showed a brain to plasma ratio of 1.8:1, reaching a Cmax in brain of 1967 ng-eq mg−1 at a Tmax of 0.25 h. Clearance of the compound was rapid; the half-life was approximately 15 min. By 2 h, the brain concentration had fallen to 100 ng-eq mg−1. Considering the high rate of microsomal metabolism noted for the series, attempts were made to identify specific site(s) of metabolism. A metabolite identification study of 9t in mLM revealed extensive metabolism involving both the R1 cyclohexyl and R3 cyclopropyl groups. While it was not possible to quantify the relative abundance of metabolites due to unknown ionization efficiencies of the various species, clear evidence was obtained demonstrating R1 and R3 hydroxylation(s), dehydration(s), and dealkylation to the 2-aminopyrimidine (structures of these putative metabolites and their respective extracted single ion mass chromatograms can be found in the Supporting Information). Thus far, the incorporation of hydrophobic groups at both R1 and R3 necessary to achieve a level of biochemical potency that might translate into submicromolar cellular potency was incompatible with metabolic stability in mouse. Anticipating that metabolism issues might dictate the need to proceed into an in vivo pharmacodynamic proof-of-concept study with a high-clearance compound, we undertook an in vitro cytochrome P450-mediated metabolism suppression study of 9t in mLM. Using the cytochrome P450 (CYP450) inhibitors listed in Table 9, the objective was to identify which CYP450

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CONCLUSIONS Despite numerous reports of JNK inhibitors, there remains a need for sharp tools having sufficient cellular potency and pharmacokinetic profile to support in vivo proof-of-concept studies in models of Huntington’s disease. With a focus on identification of compounds having calculated properties aligned with BBB penetration for HD, we have expanded the scope of the SAR of the (1H-pyrazol-4-yl)pyrimidine chemotype through incorporation of new substituents that bind in the sugar pocket while significantly expanding the SAR with respect to binding in hydrophobic region II. Through our studies, a binding model to rationalize the observed selectivity for JNK3 versus p38 was established: one which favors small groups at R3 that allow Met146 of JNK3 to occupy hydrophobic region I. Three different cellular assays were used to stratify compounds for further studies. In two different assays used to assess the activity of compounds against a p-c-Jun readout, only the irreversible inhibitor 35 showed a level of activity consistent with a pharmacodynamic tool. While 35 has the necessary level of cellular potency, its permeability in an MDCK-MDR1 assay and its kinase selectivity were insufficient (see Supporting Information). In an LPS−TNF-α secretion assay, all compounds

Table 9. Human CYP450 Inhibitors for Metabolism Suppression Study human CYP450 isoform

selective inhibitor

Ki (μM)

1A2 2B6 2D6 2C8 2C9 2C19 3A4/5

α-naphthoflavone clopidogrel quinidine montelukast sulfaphenazole ticlopidine ketoconazole

0.01 0.5 0.2 0.07 0.3 1.2 0.09

isoforms were principally responsible for metabolism, as evidenced by reduction in the rate of clearance. Table 9 lists the specific inhibitors of human CYP450 isoforms studied, and the results are summarized in Figure 3. The results from coadministration of each inhibitor with 9t suggest that human CYP450-2B6-like, human CYP450-2C19-like, and human CYP450-3A4-like activities are mainly responsible for its metabolism in mLM. It is important to caution that the CYP450 isoforms in mouse are not well-characterized and that the precise CYP450 isoforms being inhibited in mLM by this 2976

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Figure 3. Suppression of metabolism of 9t in mLM incubations by coadministration with known inhibitors of human CYP450 isoforms.

Figure 4. Brain concentrations of 9t over time in mice dosed orally with (A) 30 mpk 9t (▲), (B) 30 mpk 9t coadministered with 50 mpk ketoconazole (◇), and (C) 30 mpk 9t coadministered with 30 mpk 1-ABT (◆). mass spectrometer (ESI), or a Waters Acquity SQD (ESI and APCI). LC−MS analyses were obtained using a Varian 1200L single quadrapole mass spectrometer (ESI, HP-LCMS) or a Waters Acquity SQD (ESI and APCI, UP-LCMS). HPLC analyses were performed using a Grace Alltima C18 column, 3 μ (7 × 53 mm), with UV detection at 254 nm (unless otherwise noted) using a standard solvent gradient program (Methods 1−5; Tables 10−14). All final compounds

showed a much improved translation of biochemical potency, with most showing submicromolar IC50 values. Our focus on calculated properties resulted in compounds showing low efflux, leading to the identification of 9t as a brainpenetrant pan-JNK inhibitor in mouse. All compounds from this chemotype were highly and rapidly metabolized, showing high in vivo clearance in mouse; however, this liability could be overcome through in vivo pan-CYP450 inhibition. Additional studies will be needed to establish a correlation between TNF-α levels and HD disease progression to determine whether 9t is suitable for oral dosing in pharmacodynamic and efficacy models.

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Table 10 method 1a

EXPERIMENTAL SECTION

Unless otherwise noted, reagents and solvents were used as received from commercial suppliers. All nonaqueous reactions were carried out under an atmosphere of dry nitrogen (unless otherwise noted). Proton nuclear magnetic resonance spectra were obtained on a Bruker AVANCE 300 spectrometer at 300 MHz or Bruker AVANCE 500 spectrometer at 500 MHz. Spectra are given in ppm (δ), and coupling constants, J values, are reported in hertz (Hz). Tetramethylsilane was used as an internal standard for 13C and 1H nuclear magnetic resonance. Mass spectra were obtained on either a PerkinElmer Sciex 100 mass spectrometer (APCI), a Varian 1200L single quadrapole

time (min)

flow (mL/min)

%A

%B

0.0 5.0 6.0

3.0 3.0 3.0

90.0 0.0 0.0

10.0 100.0 100.0

a

A = 95% water/acetonitrile with 0.05% v/v trifluoroacetic acid; B = 95% acetonitrile/water with 0.05% v/v trifluoroacetic acid.

were of ≥95% purity, as assessed by 1H NMR and using one of the analytical HPLC methods noted above. hJNK3α1 and p38α and β in Vitro Kinase Assays for Compound IC50 Determinations. Compounds were prepared as 10 mM stocks in 100% DMSO from fresh powder. The compound 2977

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Journal of Medicinal Chemistry

in 50 mM Tris-HCl, pH 7.5, 2 mM EGTA, 2 mM DTT, and 10 mM MgCl2 and test compounds in a 20 μL final reaction volume. The kinase reaction was initiated with the addition of JNK3α1 kinase and incubated 30 min prior to the addition of the Kinase-Glo Reagent per the manufacture’s recommendation. The plate was incubated for an additional 15 min at room temperature, and luminescence was measured in an Analyst GT reader (Molecular Devices, using the default luminescence settings). The luminescence produced is inversely related to kinase activity. Data were analyzed by calculating the percent of inhibition, and each IC50 was determined using the fourparameter logistic equation (model 205, Excel fit-IDBS curve-fitting software). The γ32P-ATP radioactive assay platform was used to determine compound IC50 for human p38α and β isoforms. The assay was performed at room temperature in 96-well plates (Corning, cat. no.3363). Each well received 10 nM hp38α or 40 nM hp38β (Millipore), 2 or 3 μM ATF-2, respectively (BPS Biosciences), 50 μM ATP (Cell Signaling Technology, cat. no. 9804), and 1.125 μCi γ-32PATP (PerkinElmer, cat. no.BLU502A250UC) in 50 mM Tris-HCl, pH 7.5, 2 mM EGTA, 2 mM DTT, and 10 mM MgCl2 and test compounds in a 20 μL final reaction volume. The kinase reaction was initiated with the addition of the p38α or β kinase and incubated 30 min for the hp38α assay and 40 min for the hp38β. Reactions were terminated with the addition of 150 μL of 150 mM phosphoric acid to each well. The reaction mixture was transferred to a pretreated Immobilon filter plate based on the manufacture’s recommendation (Millipore, cat. no. MAIPNOB50). After vacuum filtration, the filter plate was washed four times with 300 μL of 150 mM phosphoric acid. After the final wash, the membrane was allowed to air dry at room temperature, 50 μL of EcoScint scintillation cocktail (National Diagnostics cat. no. LS-271) was added, and radioactivity was measured using a TriLux reader (PerkinElmer). Data were analyzed by calculating the percent of inhibition, and each IC50 was determined using the four -parameter logistic equation (model 205, Excel fit-IDBS software). Compound Synthesis. 4-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)N-(cyclopropylmethyl)pyrimidin-2-amine (9a). Preparation of 1-(4-Chlorophenyl)-2-(2-(methylthio)pyrimidin-4-yl)ethenol/ ethanone (4 and 4′). To a stirred solution at 0 °C of 2 (2.10 g, 15.0 mmol) and methyl 4-chlorobenzonate (3b, 2.64 g, 15.0 mmol) in THF (30.0 mL) was added lithium hexamethyldisilazide (30 mL of a 1.0 M solution in THF, 30.0 mmol), and after the addition was complete, the reaction mixture was warmed to room temperature. After 16 h, the reaction mixture was quenched with saturated ammonium chloride solution, and the layers were separated. The aqueous layer was extracted using ethyl acetate (2 × 25 mL). The combined organic phase was washed with 1.0 N hydrochloric acid, water, and saturated sodium chloride, dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was triturated using heptane/methylene chloride to provide a mixture of (Z)-1-(4- chlorophenyl)-2-(2(methylthio)pyrimidin-4-yl)ethanol (4) and 1-(4-chlorophenyl)-2-(2(methylthio)pyrimidin-4-yl)ethanone (4′), 2.56:1 ratio, 4.09 g, 98% combined yield, as a bright yellow crystalline solid: 1H NMR (300 MHz, CDCl3) δ 2.51 (s, 1.17H), 2.62 (s, 3H), 4.34 (s, 0.78H), 5.96 (s, 1H), 6.66 (d, J = 5.4 Hz, 1H), 6.97 (d, J = 5.1 Hz, 0.39H), 7.38−7.42 (m, 2H), 7.43−7.47 (m, 0.78H), 7.75−7.79 (m, 2H), 7.98 (d, J = 8.7 Hz, 0.78H), 8.32 (d, J = 5.4 Hz, 1H), 8.46 (d, J = 5.1 Hz, 0.39H), 14.60 (br s, 1H); MS (APCI) m/z 279 [M + H]+. Preparation of 4-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)-2(methylthio)pyrimidine (5). A stirred solution of 4 and 4′ (1.00 g, 3.59 mmol) and DMF·DMA (0.640 g, 5.38 mmol) in toluene (10 mL) was heated at reflux for 18 h. After this time, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. To the resulting residue were added anhydrous hydrazine (0.235 g, 7.18 mmol) and ethanol (9.0 mL), and the mixture was stirred at room temperature for 16 h. After this time, the mixture was concentrated under reduced pressure, and the residue was recrystallized using ethyl acetate/heptane to provide 2-methylthiopyrimidine 5 (0.822 g, 85%) as an off-white solid: 1H NMR (300 MHz, CDCl3) δ 2.43 (s, 3H),

Table 11 method 2a time (min)

flow (mL/min)

%A

%B

0.0 5.0 6.0

3.0 3.0 3.0

70.0 0.0 0.0

30.0 100.0 100.0

a

A = 95% water/acetonitrile with 0.05% v/v trifluoroacetic acid; B = 95% acetonitrile/water with 0.05% v/v trifluoroacetic acid.

Table 12 method 3a time (min)

flow (mL/min)

%A

%B

0.0 10.0 11.0

3.0 3.0 3.0

100.0 0.0 0.0

0.0 100.0 100.0

a

A = 95% water/acetonitrile with 0.05% v/v trifluoroacetic acid; B = 95% acetonitrile/water with 0.05% v/v trifluoroacetic acid.

Table 13 method 4a time (min)

flow (mL/min)

%A

%B

0.0 5.0 6.0

3.0 3.0 3.0

100.0 0.0 0.0

0.0 100.0 100.0

a

A = 95% water/acetonitrile with 0.05% v/v trifluoroacetic acid; B = 95% acetonitrile/water with 0.05% v/v trifluoroacetic acid.

Table 14 method 5a time (min)

flow (mL/min)

%A

%B

0.0 20.0 25.0

0.75 0.75 0.75

90.0 0.0 0.0

10.0 100.0 100.0

a

A = water with 0.01% v/v trifluoroacetic acid; B = acetonitrile with 0.01% v/v trifluoroacetic acid. stock solution was serially diluted 1:3 in DMSO for a 10-point concentration dose response in duplicate and transferred to assay plates with a final DMSO assay concentration of 1%. Control compounds such as JNK inhibitor JNK-40138 (36) and p38 inhibitor SB 23906339 (37) were also included in each test plate to monitor assay performance (Figure 5).

Figure 5. Structures of assay standards 36 and 37. The Kinase Glo assay platform from Promega (cat. no. V6714) was used to determine compound IC50 for hJNK3α1. In this format, ATP in the reaction is measured after the addition of the Kinase-Glo Reagent. The assay was performed at room temperature in 384-well plates (Corning cat. no. 3572). Each well received 8 nM JNK3α1 (Invitrogen, cat. no. PR6983A), 2 μM ATF-2 (BPS Biosciences, cat. no. 40520), and 1 μM ATP (Cell Signaling Technology, cat. no. 9804) 2978

DOI: 10.1021/jm5013598 J. Med. Chem. 2015, 58, 2967−2987

Article

Journal of Medicinal Chemistry

349.0968; found, 349.0976; HPLC: Method 1, tR = 2.99 min (>99% AUC). trans-4-(4-(3-tert-Butyl-1H-pyrazol-4-yl)-pyrimidin-2ylamino)cyclohexanol (9e). The compound was prepared using a method similar to that described for the preparation of 9a, 0.189g, 38% as a white solid: mp 232−234 °C; 1H NMR (500 MHz, CD3OD) δ 1.35−1.39 (m, 4H), 1.46−1.52 (m, 9H), 1.96−1.98 (m, 2H), 2.02− 2.05 (m, 2H), 3.30−3.32 (m, 2H), 3.57−3.58 (m, 1H), 3.89 (br s, 1H), 6.71−6.76 (m, 1H), 7.78−7.94 (m, 1H), 8.12 (d, J = 5.0 Hz, 1H); 13C NMR (125 MHz, CD3OD) δ 29.93, 30.56, 32.05, 33.76, 35.17, 50.30, 70.70, 109.9, 118.3, 142.9, 151.9, 158.3, 162.8, 164.4; HRMS: calcd for C17H25N5O, 316.2137; found, 316.2149; HPLC: Method 1, tR = 2.30 min (98.6% AUC). trans-4-(4-(3-(1-Methylcyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-ylamino)cyclohexanol (9f). The compound was prepared using a method similar to that described for the preparation of 9a, 0.125 g, 40% as an off-white solid: mp, no clear melt observed; 1 H NMR (500 MHz, DMSO-d6) δ 0.73−0.83 (m, 2H), 0.92−0.94 (m, 2H), 1.23−1.30 (m, 2H), 1.38−1.44 (m, 5H), 1.86−1.93 (m, 4H), 3.46 (s, 2H), 3.71 (br s, 1H), 4.11 (br s, 1H), 7.25 (d, J = 6.5 Hz, 1H), 8.29−8.38 (m, 2H), 8.50−8.69 (m, 1H); 13C NMR (125 MHz, DMSO-d6) δ 166.5, 153.4, 151.7, 145.6, 139.9, 116.2, 105.7, 67.5, 48.8, 33.5, 29.8, 24.0, 13.6, 13.4; HRMS: calcd for C17H23N5O, 314.1981; found, 314.1983; HPLC: Method 1, tR = 2.18 min (97.5% AUC). trans-4-(4-(3-(1-(Trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-ylamino)cyclohexanol (9g). The compound was prepared using a method similar to that described for the preparation of 9a, 0.064 g, 19% as a white solid: mp 239−240 °C; 1H NMR (500 MHz, DMSOd6) δ 1.21−1.33 (m, 6H), 1.44−1.53 (m, 2H), 1.80−1.88 (m, 4H), 3.38−3.41 (m, 1H), 3.81 (br s, 1H), 4.52 (d, J = 5.4 Hz, 1H), 6.74 (br s, 1H), 6.81−6.83 (m, 1H), 8.02−8.28 (m, 2H), 13.19−13.56 (m, 1H); 19F{1H} (282 MHz, CDCl3) δ −67.27; 13C NMR (125 MHz, DMSO-d6) δ 161.5, 159.4, 157.6, 143.8, 139.9, 130.9, 120.3, 106.3, 68.3, 48.2, 34.2, 30.5, 11.3; HRMS: calcd for C17H20F3N5O, 368.1698; found, 368.1691; HPLC: Method 3, tR = 3.57 min (>99% AUC). trans-4-(4-(3-Cyclobutyl-1H-pyrazol-4-yl)pyrimidin-2ylamino)cyclohexanol (9h). The compound was prepared using a method similar to that described for the preparation of 9a, 0.167 g, 53% as a light yellow solid: mp, no clear melt observed; 1H NMR (500 MHz, DMSO-d6) δ 1.28−1.46 (m, 4H), 1.92−2.03 (m, 6H), 2.31− 2.36 (m, 4H), 3.48 (br s, 1H), 3.69−3.94 (m, 1H), 4.32 (m, 1H), 5.50 (br s, 1H), 7.20 (br s, 1H), 8.24 (br s, 1H), 8.50 (br s, 2H), 13.18 (br s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 167.1, 152.9, 152.1, 145.0, 138.7, 114.4, 105.4, 67.5, 49.8, 33.6, 32.4, 29.7, 27.8, 18.1; HRMS: calcd for C17H23N5O, 314.1981; found, 314.1981; HPLC: Method 1, tR = 2.30 min (98.4% AUC). 4-(3-Cyclopropyl-1H-pyrazol-4-yl)-N-isopropylpyrimidin-2amine (9i). The compound was prepared using a method similar to that described for the preparation of 9a, 0.116 g, 33% as a white solid: mp 225−226 °C; 1H NMR (500 MHz, DMSO-d6) δ 0.85−1.00 (m, 4H), 1.16−1.30 (m, 4H), 1.81−1.84 (m, 2H), 1.89−1.92 (m, 2H), 2.89 (br s, 1H), 3.35−3.40 (m, 1H), 3.63 (br s, 1H), 4.51 (d, J = 4.5 Hz, 1H), 6.74 (br s, 1H), 6.82 (d, J = 5.5 Hz, 1H), 7.93−8.24 (m, 1H), 8.15 (s, 1H), 12.56−12.68 (m, 1H); 13C NMR (125 MHz, DMSO-d6) δ 161.5, 157.6, 151.8, 144.7, 139.5, 130.0, 117.6, 117.2, 105.4, 68.4, 49.1, 34.2, 30.2, 8.5, 8.0, 7.1; HRMS: calcd for C16H21N5O, 300.1824; found, 300.1833; HPLC: Method 1, tR = 2.09 min (>99% AUC). trans-4-(4-(3-(Tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)pyrimidin-2-ylamino)cyclohexanol (9j). The compound was prepared using a method similar to that described for the preparation of 9a, 0.077 g, 23% as a white powder: mp 229 °C; 1H NMR (300 MHz, DMSO-d6) δ 1.05−1.40 (m, 4H), 1.60−2.00 (m, 8H), 3.55− 4.05 (m, 7H), 4.55−4.61 (m, 1H), 6.78 (d, J = 4.5 Hz, 2H), 7.95−8.30 (m, 2H), 12.84−12.96 (m, 1H); 13C NMR (125 MHz, DMSO-d6) δ 161.7, 160.6, 157.7, 153.9, 146.2, 139.4, 130.3, 115.3, 105.8, 68.3, 67.3, 48.5, 34.3, 33.3, 31.9, 31.3, 30.3; HRMS: calcd for C18H25N5O2, 344.2087; found, 344.2090; HPLC: Method 1, tR = 1.98 min (>99% AUC, integration at 230 nm).

2.99 (br s, 1H), 6.84 (d, J = 5.1 Hz, 1H), 7.42−7.50 (m, 4H), 8.21 (s, 1H), 8.35 (d, J = 5.1 Hz, 1H); MS (ESI) m/z 303 [M + H]+. Preparation of 4-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)-2(methylsulfonyl)pyrimidine (6). To a stirred suspension at 0 °C of 5 (0.773 g, 2.56 mmol) in methylene chloride (12.0 mL) was added m-CPBA (1.18 g, 5.11 mmol) portionwise, and then the reaction mixture was warmed to room temperature. After 16 h, a saturated sodium bicarbonate solution (20 mL) was added to the reaction mixture and then stirred for 5 min. The organic layer was separated. The aqueous layer was extracted with ethyl acetate (2 × 15 mL). The combined organic phase was washed with sodium thiosulfate, saturated sodium chloride, and dried (MgSO4), filtered, and concentrated under reduced pressure to provide 2-(methylsulfonyl)pyrimidine 6 (0.847 g, 99%) as an off-white solid: mp 194−195 °C; 1H NMR (500 MHz, CDCl3) δ 3.23 (s, 3H), 4.95 (br s, 1H), 7.33 (d, J = 5.0 Hz, 1H), 7.45−7.50 (m, 4H), 8.34 (s, 1H), 8.69 (d, J = 5.0 Hz, 1H); MS (APCI) m/z 335 [M + H]+. Preparation of 4-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)-N(cyclopropylmethyl)pyrimidine-2-amine (9a). A microwave tube charged with a stir bar and suspension of 6 (0.300 g, 0.90 mmol) and cyclopropanemethylamine (0.128 g, 1.80 mmol) in dioxane (3.0 mL) was irradiated (400 W, 130 °C) for 30 min. After this time, the reaction mixture was concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel; 40−80% ethyl acetate/heptanes, gradient elution) to provide 9a (0.118 g, 41%) as an off-white solid: mp 221−222 °C; 1H NMR (500 MHz, DMSO-d6) δ 0.12 (br s, 2H), 0.94−0.83 (m, 2H), 0.94−0.83 (m, 1H), 2.86 (br s, 2H), 6.58 (s, 1H), 6.95 (s, 1H), 7.56−7.41 (m, 4H), 8.04−8.32 (m, 2H), 13.30−13.38 (m, 1H); 13C NMR (125 MHz, DMSO-d6) δ 162.1, 157.8, 140.2, 132.1 (low intensity), 131.1, 130.9, 130.6, 128.2, 127.8, 117.9, 117.4, 44.8, 10.8, 3.1; HRMS: calcd for C17H16ClN5, 326.1172; found, 326.1180; HPLC: Method 1, tR = 3.24 min (>99% AUC). 4-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)-N-(cyclohexylmethyl)pyrimidine-2-amine (9b). The compound was prepared using a method similar to that described for the preparation of 9a, 0.164 g, 50% as a white solid: mp 222−223 °C; 1H NMR (500 MHz, DMSOd6) δ 0.73 (br s, 2H), 1.11 (br s, 3H), 1.33−1.60 (m, 6H), 2.80 (br s, 2H), 6.59−6.65 (br s, 1H), 6.87 (s, 1H), 7.41−7.56 (m, 4H), 8.03− 8.31 (m, 2H), 13.29−13.37 (m, 1H); 13C NMR (125 MHz, DMSOd6) δ 162.4, 157.8, 140.2, 133.3 (low intensity), 131.1, 130.8, 130.5, 128.2, 127.7, 117.9, 117.4, 46.6, 37.0, 30.3, 26.1, 25.3; HRMS: calcd for C20H22ClN5, 368.1642; found, 368.1646; HPLC: Method 1, tR = 3.85 min (>99% AUC). trans-4-(4-(3-Chlorophenyl)-1H-pyrazol-4-yl)-pyrimidin-2ylamino)cyclohexanol (9c). The compound was prepared using a method similar to that described for the preparation of 9a, with the addition of an extra preparative HPLC purification step after flash column chromatography (Phenomenex Luna 10 μ C18(2) 100 Å column (21.2 × 250 mm); 10−60% then 60−100% [95:5 CH3CN/ H2O]/[95:5 H2O/CH3CN, 0.05% TFA], gradient elution, integration at 254 nm) and then washed using saturated sodium bicarbonate, 0.114 g, 17% as a white solid: mp 248−250 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.10−1.16 (m, 4H), 1.62−1.73 (m, 4H), 3.25−3.40 (br s, 2H), 4.48 (br s, 1H), 6.66 (br s, 1H), 6.85 (s, 1H), 7.46−7.55 (m, 4H), 8.13 (d, J = 5.5 Hz, 1H), 8.27 (br s, 1H), 13.34 (br s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 161.2, 160.1, 157.4, 148.3, 140.5, 133.2, 132.4, 131.2, 130.6, 128.0, 117.8, 106.6, 68.4, 48.4, 34.0, 30.3; HRMS: calcd for C19H20ClN5O, 370.1435; found, 370.1425; HPLC: Method 1, tR = 2.60 min (>99% AUC). 4-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)-N-(pyridine-2-yl)pyrimidin-2-amine (9d). The compound was prepared using a method similar to that described for the preparation of 9a, 0.032 g, 5% as an off-white solid: mp 246−247 °C; 1H NMR (500 MHz, DMSOd6) δ 6.96−7.01 (m, 2H), 7.47−7.57 (m, 5H), 7.65 (d, J = 8.0 Hz, 1H), 8.18−8.47 (m, 3H), 9.85 (br s, 1H), 13.47−13.60 (m, 1H); 13C NMR (125 MHz, DMSO-d6) δ 160.3, 158.6, 158.5, 158.0, 157.9, 157.7, 152.1, 152.0, 151.9, 145.6, 145.5, 145.2, 139.0, 138.6, 133.0, 130.7, 128.5, 117.1, 112.8, 110.9; HRMS: calcd for C18H13ClN6, 2979

DOI: 10.1021/jm5013598 J. Med. Chem. 2015, 58, 2967−2987

Article

Journal of Medicinal Chemistry trans-4-(4-(1-Cyclopentyl-3-cyclopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)cyclohexanol (9l). The compound was prepared using a method similar to that described for the preparation of 9a, 0.155g, 59% as a white foam: mp, no clear melt observed; 1H NMR (500 MHz, CDCl3) δ 0.60−0.63 (m, 2H), 1.08−1.12 (m, 2H), 1.25−1.33 (m, 2H), 1.40 (br s, 1H), 1.41−1.49 (m, 2H), 1.67−1.71 (m, 2H), 1.81−1.86 (m, 1H), 1.96−2.03 (m, 4H), 2.06−2.10 (m, 4H), 2.17−2.19 (m, 2H), 3.66−3.70 (m, 1H), 3.48−3.91 (m, 1H), 4.88 (d, J = 3.9 Hz, 1H), 5.03−5.09 (m, 1H), 6.79 (d, J = 5.0 Hz, 1H), 7.89 (s, 1H), 8.21 (d, J = 5.0 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 161.6, 159.4, 157.3, 141.9, 137.9, 119.1, 107.1 (low intensity), 68.4, 58.4, 48.7, 34.2, 32.4, 30.3, 24.3, 7.7, 5.4; HRMS: calcd for C21H29N5O, 368.2450; found, 368.2455; HPLC: Method 1, tR = 3.09 min (98.3% AUC). trans-4-(4-(1-Isopropyl-3-(1-methylcyclopropyl)-1H-pyrazol4-yl)pyrimidin-2-ylamino)cyclohexanol (9m). The compound was prepared using a method similar to that described for the preparation of 9a, 0.085 g, 33% as a white solid: mp, no clear melt observed; 1H NMR (500 MHz, DMSO-d6) δ 0.67 (br s, 2H), 0.92 (br s, 2H), 1.17−1.33 (m, 4H), 1.42 (d, J = 6.5 Hz, 6H), 1.45 (s, 3H), 1.82−1.89 (m, 4H), 3.36−3.41 (m, 1H), 3.80 (br s, 1H), 4.51 (d, J = 5.5 Hz, 1H), 4.91−4.96 (m, 1H), 6.73 (br s, 1H), 7.84 (s, 1H), 8.17 (br s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 161.7, 159.6, 157.5, 144.1, 138.3, 117.9, 106.8, 68.4, 49.1, 48.5, 34.3, 30.5, 24.2, 22.5, 15.0, 11.2; HRMS: calcd for C20H29N5O, 356.2450; found, 356.2463; HPLC: Method 1, tR = 3.63 min (>99% AUC). trans-4-(4-(3-Cyclopropyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol4-yl)pyrimidin-2-ylamino)cyclohexanol (9n). The compound was prepared using a method similar to that described for the preparation of 9a, 0.251 g, 57% as a white foam: mp, no clear melt observed; 1H NMR (500 MHz, DMSO-d6) δ 0.58−0.64 (m, 2H), 1.13−1.15 (m, 2H), 1.18−1.33 (m, 4H), 1.82−1.95 (m, 5H), 3.36−3.41 (m, 1H), 3.71 (br s, 1H), 4.50 (d, J = 2.5 Hz, 1H), 5.15 (q, J = 9.0 Hz, 2H), 6.84 (d, J = 5.0 Hz, 1H), 7.93 (s, 1H), 8.23 (s, 1H); 19F{1H} NMR (282 MHz, CDCl3) δ −68.75; 13C NMR (125 MHz, DMSO-d6) δ 161.6, 158.7, 157.6, 143.9, 139.7, 123.7 (q, J = 280.4 Hz), 120.4, 107.5 (low intensity), 68.4, 49.5 (q, J = 34.0 Hz), 48.7, 34.2, 30.3, 7.7, 5.2; HRMS: calcd for C18H22F3N5O, 382.1855; found, 382.1848; HPLC: Method 1, tR = 2.75 min (98.7% AUC). (4-(3-Isopropyl-1H-pyrazol-4-yl)-N-(trans-4-methoxycyclohexyl)-pyrimidin-2-amine (9r). The compound was prepared using a method similar to that described for the preparation of 9a, 0.044 g, 19% as a white solid: mp 190−191 °C; 1H NMR (500 MHz, DMSOd6) δ 1.17−1.31 (m, 10H), 1.94 (d, J = 11.0 Hz, 2H), 2.03 (d, J = 11.0 Hz, 2H), 3.08−3.14 (m, 1H), 3.30 (s, 3H), 3.66−3.72 (m, 1H), 3.87− 4.09 (m, 1H), 6.77−6.79 (m, 2H), 7.93−8.31 (m, 1H), 8.13 (d, J = 3.0 Hz, 1H), 12.73−12.89 (m, 1H); 13C NMR (125 MHz, DMSO-d6) δ 161.6, 160.7, 157.7, 148.7, 139.3, 130.1, 115.9, 114.7, 105.5, 77.9, 55.0, 49.0, 30.3, 30.0, 26.4, 24.8, 22.4, 21.7; HRMS: calcd for C17H25N5O, 316.2137; found, 316.2132; HPLC: Method 1, tR = 2.62 min (98.5% AUC). trans-4-(4-(3-Isopropyl-1H-pyrazol-4-yl)pyrimidin-2ylamino)cyclohexanecarbonitrile (9u). The compound was prepared using a method similar to that described for the preparation of 9a, 0.077 g, 25% as a white solid: mp 219−220 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.29−1.35 (m, 8H), 1.51−1.58 (m, 2H), 1.94− 1.97 (m, 2H), 2.07−2.09 (m, 2H), 2.66−2.71 (m, 1H), 3.71 (br s, 1H), 3.84−4.06 (m, 1H), 6.79 (d, J = 5.0 Hz, 1H), 6.90 (br s, 1H), 7.93−8.27 (m, 1H), 8.14 (d, J = 5.0 Hz, 1H), 12.74−12.89 (m, 1H); 13 C NMR (125 MHz, DMSO-d6) δ 161.5, 160.8, 157.6, 156.1, 148.8, 139.4, 130.2, 123.0, 116.0, 114.8, 105.8, 48.0, 30.6, 28.2, 26.6, 24.8, 22.5, 21.8; HRMS: calcd for C17H22N6, 311.1984; found, 311.1984; HPLC: Method 1, tR = 2.74 min (>99% AUC). N-(Cyclopropylmethyl)-4-(3-(1-methylcyclopropyl)-1H-pyrazol4-yl)pyrimidin-2-amine (9y). The compound was prepared using a method similar to that described for the preparation of 9a, 0.064 g, 25% as a white solid: mp 158−160 °C; 1H NMR (500 MHz, DMSOd6) δ 0.20−0.22 (m, 2H), 0.38−0.44 (m, 2H), 0.70−0.82 (m, 2H), 0.87−0.89 (m, 2H), 1.06−1.11 (m, 1H), 1.34−1.38 (m, 3H), 3.22 (br s, 2H), 6.88−6.94 (m, 2H), 7.92−8.20 (m, 2H), 12.72−12.95

(m, 1H); 13C NMR (125 MHz, DMSO-d6) δ 162.2, 159.6, 157.6, 153.7, 146.3, 139.7, 130.4, 118.1, 117.2, 106.1, 44.8, 24.4, 24.2, 14.6, 13.8, 12.3, 11.1, 3.1; HRMS: calcd for C15H19N5, 270.1719; found, 270.1718; HPLC: Method 1, tR = 3.47 min (>99% AUC). N-(1-Ethylpiperidin-4-yl)-4-(3-(1-(trifluoromethyl)cyclopropyl)1Hpyrazol-4-yl)pyrimidin-2-amine (9dd). The compound was prepared using a method similar to that described for the preparation of 9a, 0.058 g, 19% as a crystalline white solid: mp 220−222 °C (dec); 1 H NMR (500 MHz, DMSO-d6) δ 0.90−1.10 (m, 3H), 1.23−1.40 (m, 2H), 1.40−1.60 (m, 4H), 1.74−1.95 (m, 4H), 2.25−2.39 (m, 2H), 2.80−2.92 (m, 2H), 3.85 (br s, 1H), 6.70−6.85 (m, 2H), 8.10−8.30 (m, 2H), 13.20−13.60 (m, 1H); 19F{1H} (282 MHz, DMSO-d6) δ −67.27 (s); 13C NMR (125 MHz, DMSO-d6) δ 161.7, 159.2, 157.8, 126.2 (q, J = 274.2 Hz), 120.4, 106.5, 52.1, 51.7, 47.4, 31.8, 20.4, 12.3, 11.4; HRMS: calcd for C18H23F3N6, 381.2015; found, 381.2029; HPLC: Method 1, tR = 1.94 min (97.3% AUC). trans-4-(4-(1-Methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrimidin-2-ylamino)cyclohexanol (9o). Preparation of 3(Ethoxymethylene)-1,1,1-trifluoropentane-2,4-dione 13. A stirred solution of 1,1,1-trifluoropentane-2,4-dione 12 (12.7 g, 82.4 mmol), acetic anhydride (8.41 g, 82.4 mmol), and triethyl orthoformate (24.4 g, 164.8 mmol) was heated at reflux for 19 h. After this time, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was vacuum-distilled under reduced pressure to provide 13 (6.01 g, 35%) (55:45 mixture of E/Z isomers by NMR integration) as a red oil: bp 70−85 °C at 2 mmHg; 1 H NMR (300 MHz, CDCl3) δ 1.40−1.49 (m, 3H), 2.32 (s, 1.35H), 2.42 (s, 1.65H), 4.30−4.40 (m, 2H), 7.69 (s, 0.55H), 7.96 (s, 0.45H); 19 1 F{ H} NMR (282 MHz, CDCl3) δ −76.43, −71.92. Preparation of 1-(1-Methyl-3-(trifluoromethyl)-1H-pyrazol4-yl)ethanone 14. To a stirred solution at −10 °C of methyl hydrazine (0.657 g, 14.3 mmol) in THF (500 mL) was added a solution of E- and Z-3-(ethoxymethylene)-1,1,1-trifluoropentane-2,4dione 20 (3.00 g, 14.3 mmol) in THF (250 mL) dropwise over 1 h. After the addition was complete, the resulting reaction mixture was stirred at −10 °C for 1.25 h and then allowed to warm to room temperature over 1 h. After this time, the reaction mixture was concentrated under reduced pressure, and the residue was purified using flash column chromatography (silica gel; 20−85% ethyl acetate/ heptanes, gradient elution) to provide 14 (1.92 g, 70%) as a lightyellow solid: mp 79−80; 1H NMR (300 MHz, CDCl3) δ 2.48 (s, 3H), 3.99 (s, 3H), 7.93 (s, 1H); MS (ESI) m/z 253 [M + H]+. Preparation of 4-(1-Methyl-3-(trifluoromethyl)-1H-pyrazol4-yl)-2-(methylthio)pyrimidine 15. A stirred solution of 1-(1methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)ethanone 14 (0.850 g, 4.42 mmol) and DMF·DMA (1.05 g, 8.85 mmol) in toluene (10 mL) was heated at reflux for 18 h. After this time, additional DMF·DMA (1.05 g, 8.85 mmol) was added, and heating at reflux was continued for an additional 2 h. After this time, the reaction mixture was concentrated under reduced pressure, the residue was treated with methyl carbamimidothioate·1/2H2SO4 (0.923 g, 6.64 mmol) and sodium methoxide (30 wt %, 1.17 mL, 6.64 mmol) in 2-propanol (20 mL), and the resulting reaction mixture was heated at reflux for 12 h. After this time, the reaction mixture was cooled to room temperature, acidified to pH 3 with 1 N hydrochloric acid, and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with aqueous saturated sodium chloride solution (30 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel; 25−85% ethyl acetate/heptanes, gradient elution) to provide 15 (0.097 g, 8%) as a golden oil: 1H NMR (300 MHz, CDCl3) δ 2.59 (s, 3H), 4.00 (s, 3H), 7.17 (d, J = 5.1 Hz, 1H), 8.11 (s, 1H), 8.51 (d, J = 5.1 Hz, 1H); 19F{1H} NMR (282 MHz, CDCl3) δ −60.42; LC-MS (ESI) m/z 275 [M + H]+. Preparation of 4-(1-Methyl-3-(trifluoromethyl)-1H-pyrazol4-yl)-2-(methylsulfonyl)-pyrimidine 16. The title compound was prepared by a similar method described for the preparation of 6, 0.098 g, 92% as a white solid: 1H NMR (300 MHz, CDCl3) δ 3.39 (s, 3H), 4.05 (s, 3H), 7.70 (d, J = 5.1 Hz, 1H), 8.31 (s, 1H), 8.87 2980

DOI: 10.1021/jm5013598 J. Med. Chem. 2015, 58, 2967−2987

Article

Journal of Medicinal Chemistry (d, J = 5.1 Hz, 1H); 19F{1H} NMR (282 MHz, CDCl3) δ −60.69; MS (ESI) m/z 307 [M + H]+. Preparation of trans-4-(4-(1-Methyl-3-(trifluoromethyl)-1Hpyrazol-4-yl)pyrimidin-2-ylamino)cyclohexanol (9o). The compound was prepared using a method similar to that described for the preparation of 9a, 0.061 g, 56% as an off-white powder: mp 191−192 °C; 1H NMR (300 MHz, DMSO-d6) δ 1.10−1.35 (m, 4H), 1.80−1.92 (m, 4H), 3.38 (br s, 1H), 3.73 (br s, 1H), 3.96 (s, 3H), 4.51 (d, J = 4.5 Hz, 1H), 6.74 (br d, J = 4.8 Hz, 1H), 6.92 (br s, 1H), 8.24 (br d, J = 4.8 Hz, 1H), 8.55 (br s, 1H); 19F{1H} NMR (282 MHz, DMSO-d6) δ −59.26; 13C NMR (125 MHz, DMSO-d6) δ 161.6, 158.5, 156.9, 134.4, 122.4, 120.3, 119.5, 105.7, 68.4, 48.5, 34.2, 30.2; HRMS: calcd for C15H18F3N5O, 342.1542; found, 342.1536; HPLC: Method 1, tR = 2.47 min (98.4% AUC). N-(4,4-Difluorocyclohexyl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine (9t). Preparation of 2-Methylsulfonyl)-4-(1-(tetrahydro-2H-pyran-2-yl)-3-(1(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidine (6t). To a stirred solution of 2-(methylsulfonyl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidine (6t) (1.06 g, 3.19 mmol) in 3,4-dihydropyrane (5.0 mL) was added p-toluenesulfonic acid (0.061 g, 0.32 mmol) at room temperature. After 10 min, the reaction mixture was concentrated under reduced pressure. The residue was partitioned between saturated sodium bicarbonate and chloroform and separated. The combined organics were dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel; 5−20% ethyl acetate/methylene chloride, gradient elution) to provide 7t (1.03 g, 78%) as an off-white solid: 1H NMR (500 MHz, CDCl3) δ 1.34−1.41 (m, 2H), 1.28−1.73 (m, 5H), 2.00−2.05 (m, 2H), 2.13−2.16 (m, 1H), 3.36 (s, 3H), 3.69−3.74 (m, 1H), 4.09−4.12 (m, 1H), 5.41 (dd, J = 9.5, 2.5 Hz, 1H), 7.82 (d, J = 5.0 Hz, 1H), 8.44 (s, 1H), 8.82 (d, J = 5.0 Hz, 1H); 19F{1H} (282 MHz, CDCl3) δ −68.45 (s); MS (ESI) m/z 417 [M + H]+. Preparation of N-(4,4-Difluorocyclohexyl)-4-(1-tetrahydro2H-pyran-2-yl)-3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol4-yl)pyrimidin-2-amine (8t). The compound was prepared from 7t using a method similar to that described for the preparation of 9a to provide 8t (0.178 g, 47%) as an off-white solid: 1H NMR (300 MHz, CDCl3) δ 1.20−1.25 (m, 2H), 1.46−1.47 (m, 2H), 1.61−1.71 (m, 6H), 2.01−2.14 (m, 8H), 3.67−3.76 (m, 1H), 4.04−4.11 (m, 2H), 5.12 (br s, 1H), 5.41 (dd, J = 9.3, 3.0 Hz, 1H), 6.93 (d, J = 5.4 Hz, 1H), 8.16 (s, 1H), 8.25 (d, J = 5.4 Hz, 1H); 19F {1H} (282 MHz, CDCl3) δ −68.42 (s), −68.39 (s); MS (ESI) m/z 472 [M + H]+. Preparation of N-(4,4-Difluorocyclohexyl)-4-(3-(1(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2amine (9t). To a stirred solution of 8t (0.178 g, 0.38 mmol) in methanol (3.0 mL) was added hydrochloric acid (0.283 mL of a 4.0 M solution in dioxane, 1.13 mmol) at room temperature. After 1.5 h, the reaction mixture was concentrated under reduced pressure. The residue was basified with sodium bicarbonate solution, and the precipitate was collected by filtration. This solid was purified by trituration with methylene chloride followed by methanol to provide 9t (0.052 g, 13%) as a white solid: 1H NMR (300 MHz, DMSO-d6) δ 1.21−1.26 (m, 2H), 1.43−1.66 (m, 4H), 1.84−2.09 (m, 6H), 3.95− 4.14 (m, 1H), 6.87 (d, J = 5.1 Hz, 1H), 6.99 (br s, 1H), 8.05−8.31 (m, 2H), 13.23−13.59 (m, 1H); 19F{1H} (282 MHz, CDCl3) δ −67.22 (s), −67.56 (s); 13C NMR (125 MHz, DMSO-d6) δ 161.5, 159.3, 158.9, 157.8, 143.7, 140.0, 130.9, 125.6, 123.7, 121.8, 120.3, 106.7, 106.5, 46.3, 31.6 (t, J = 23.9 Hz), 28.0, 27.9, 21.3, 11.5, 11.2; HRMS: calcd for C17H18F5N5, 388.1561; found, 388.1554; HPLC: Method 1, tR = 3.22 min (97.4% AUC). trans-4-((4-(1H-Pyrazol-4-yl)pyrimidin-2-yl)amino)cyclohexanol (9k). The compound was prepared using a method similar to that described for the preparation of 9t, 0.0658 g, 58% as a white solid: mp 217−218 °C; 1H NMR (500 MHz, DMSO-d6) 1.24−1.33 (m, 4H), 1.81−1.89 (m, 4H), 3.33−3.40 (m, 1H), 3.67−3.75 (m, 1H), 4.51 (d, J = 4.5 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.81 (d, J = 5.5 Hz, 1H), 8.02 (br s, 1H), 8.17 (d, J = 4.5 Hz, 1H), 8.32 (br s, 1H), 13.42 (br s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 161.4, 158.9, 157.5, 137.4, 128.0, 120.5, 104.8, 68.1, 48.5, 33.8, 30.0; HRMS: calcd for

C13H17N5O, 260.1511; found, 260.1510; HPLC: Method 4, tR = 2.30 min (98.2% AUC). N-Cyclohexyl-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol4-yl)pyrimidin-2-amine Dihydrochloride (9q). The compound was prepared using a method similar to that described for the preparation of 9t, 0.158 g, 94% as a crystalline yellow-brown solid: 1H NMR (500 MHz, DMSO-d6) δ 1.19−1.89 (m, 15H), 4.10 (s, 1H), 7.19 (s, 1H), 8.25−8.35 (m, 2H), 8.62 (s, 1H); 19F{1H} (282 MHz, DMSO-d6) δ −67.36 (s); 13C NMR (125 MHz, DMSO-d6) δ 166.0, 153.5, 146.3, 143.5, 136.6, 126.0 (q, J = 274.2 Hz), 118.4, 106.1, 48.9, 32.0, 24.8, 24.1, 20.6, 11.4; HRMS: calcd for C17H20F3N5, 352.1749; found, 352.1757; HPLC: Method 1, tR = 3.36 min (98.7% AUC). N-Cyclobutyl-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol4-yl)pyrimidin-2-amine (9v). The compound was prepared using a method similar to that described for the preparation of 9t, 0.096 g, 87% as a white solid: mp, no clear melt observed; 1H NMR (300 MHz, DMSO-d6) δ 1.27 (br s, 2H), 1.53 (br s, 2H), 1.60−1.74 (m, 2H), 2.00−2.10 (m, 2H), 2.27−2.30 (m, 2H), 4.64 (br s, 1H), 7.15 (d, J = 6.3 Hz, 1H), 8.28 (d, J = 6.3 Hz, 1H), 8.52 (br s, 2H), 13.74 (br s, 1H); 19F{1H} (282 MHz, DMSO-d6) δ −67.37 (s); 13C NMR (125 MHz, DMSO-d6) δ 165.6, 153.2, 146.8, 143.5, 136.3, 126.0 (q, J = 273.8 Hz), 118.5, 106.3, 45.4, 30.0, 20.8, 14.5, 11.4; HRMS: calcd for C15H16F3N5, 324.1436; found, 324.1446; HPLC: Method 1, tR = 3.00 min (98.5% AUC). N-Cyclopentyl-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine (9w). The compound was prepared using a method similar to that described for the preparation of 9t, 0.143 g, 90% as a white solid: mp, no clear melt observed; 1H NMR (300 MHz, DMSO-d6) δ 1.28 (br s, 2H), 1.50−1.61 (m, 6H), 1.71− 1.73 (m, 2H), 1.94−1.96 (m, 2H), 4.48 (br s, 1H), 7.19 (d, J = 6.3 Hz, 1H), 8.31 (d, J = 6.6 Hz, 1H), 8.45−8.59 (m, 2H), 13.92 (br s, 1H); 19 1 F{ H} (282 MHz, DMSO-d6) δ −67.39 (s); 13C NMR (125 MHz, DMSO-d6) δ 165.8, 153.7, 146.4, 143.2, 136.5, 126.0 (q, J = 272.9 Hz), 118.5, 106.0, 52.3, 32.0, 23.0, 20.7, 11.4; HRMS: calcd for C16H18F3N5, 338.1593; found, 338.1595; HPLC: Method 1, tR = 3.19 min (>99% AUC). N-Cycloheptyl-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol4-yl)pyrimidin-2-amine (9x). The compound was prepared using a method similar to that described for the preparation of 9t, 0.079 g, 50% as an off-white solid: mp, no clear melt; 1H NMR (300 MHz, DMSO-d6) δ 1.27 (br s, 2H), 1.42−1.70 (m, 12H), 1.88−1.95 (m, 2H), 4.27 (br s, 1H), 7.16 (d, J = 6.0 Hz, 1H), 8.30 (d, J = 6.3 Hz, 1H), 8.35 (br s, 1H), 8.52−8.57 (m, 1H), 13.49 (br s, 1H); 19F{1H} (282 MHz, DMSO-d6) δ −67.35 (s); 13C NMR (125 MHz, DMSOd6) δ 165.9, 153.3, 146.5, 136.4, 126.0 (q, J = 274.2 Hz), 118.6, 106.0, 51.2, 34.1, 27.5, 23.0, 20.6, 11.4; HRMS: calcd for C18H22F3N5, 366.1906; found, 366.1901; HPLC: Method 1, tR = 3.55 min (>99% AUC). N-(Tetrahydro-2H-pyran-4-yl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine Hydrochloride (9z). The compound was prepared using a method similar to that described for the preparation of 9t, 0.057 g, 28% as a white solid: mp, no clear melt observed; 1H NMR (500 MHz, DMSO-d6) δ 1.26 (br s, 2H), 1.50 (br s, 2H), 1.54−1.62 (m, 2H), 1.83 (d, J = 11.0 Hz, 2H), 3.35−3.39 (m, 2H), 3.90 (d, J = 11.0 Hz, 2H), 4.23 (br s, 3H), 7.11 (br s, 1H), 8.00 (br s, 1H), 8.29 (d, J = 6.0 Hz, 1H), 8.50 (br s, 1H); 19 1 F{ H} (282 MHz, CDCl3) δ −67.55 (s); 13C NMR (125 MHz, DMSO-d6) δ 165.7, 153.7, 146.9, 143.5, 136.6, 126.1 (q, J = 274.2 Hz), 118.5, 106.4, 65.6, 46.5, 32.0, 20.6, 11.5; HRMS: calcd for C16H18F3N5O, 354.1542; found, 354.1544; HPLC: Method 1, tR = 2.48 min (97.6% AUC). N-(Tetrahydro-2H-pyran-3-yl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine (9aa). The compound was prepared using a method similar to that described for the preparation of 9t, 0.089 g, 80% as a white solid: mp 228−230 °C; 1 H NMR (300 MHz, DMSOd6) δ 1.29 (br s, 2H), 1.50 (br s, 2H), 1.54−1.74 (m, 3H), 1.93−1.95 (m, 1H), 3.30 (br s, 1H), 3.30 (br s, 1H), 3.34 (br s, 1H), 3.70−3.73 (m, 1H), 3.81−3.86 (m, 1H), 4.22 (br s, 1H), 7.24 (br s, 1H), 8.32−8.64 (m, 3H); 19F{1H} (282 MHz, CDCl3) δ −67.46 (s); 13C NMR (125 MHz, DMSO-d6) δ 165.8, 2981

DOI: 10.1021/jm5013598 J. Med. Chem. 2015, 58, 2967−2987

Article

Journal of Medicinal Chemistry

(s, 4H); 19F{1H} (282 MHz, CDCl3) δ −69.27 (s); MS (ESI) m/z 226 [M + H]+. Preparation of 1-(2,2,2-Trifluoroethyl)piperidin-4-one Oxime (40). A stirred solution of 39 (7.43 g, 32.9 mmol) in 1.0 M hydrochloric acid (125 mL) was heated at reflux for 6 h. After this time, the reaction mixture was cooled in an ice/water bath, and the solution was made basic (pH 9) with saturated sodium carbonate and extracted with MTBE (3 × 150 mL). The combined organics were washed with saturated sodium chloride, dried (Na2SO4), filtered, and concentrated under reduced pressure to prove the crude ketone (4.71 g, 79%). The crude ketone was taken up in absolute ethanol (130 mL) and treated with sodium acetate (3.20 g, 39.0 mmol) and hydroxylamine hydrochloride (2.17 g. 31.2 mmol). The resulting reaction mixture was stirred at room temperature for 18 h. After this time, the reaction mixture was concentrated under reduced pressure, and the residue was partitioned between ethyl acetate (200 mL) and water (200 mL) and separated. The organic layer was washed with saturated sodium chloride, dried (Na2SO4), filtered, and concentrated under reduced pressure to provide 40 (4.59 g, 90%) as a clear, colorless oil: 1H NMR (300 MHz, CDCl3) δ 2.37 (t, J = 6.3 Hz, 2H), 2.67 (t, J = 6.3 Hz, 2H), 2.70−2.84 (m, 4H), 3.05 (q, J = 9.6 Hz, 2H), 7.86 (s, 1H); 19F{1H} (282 MHz, CDCl3) δ −69.26 (s); Note: could not obtain adequate ionization for a mass spectrum. Preparation of 1-(2,2,2-Trifluoroethyl)piperidin-4-amine (36). A Parr bottle charged with a solution of 40 (3.51 g, 17.9 mmol) in absolute ethanol (45.0 mL) was purged with nitrogen, and Raney nickel catalyst (Grade 2800 50 wt % slurry in water) (1.8 mL) was added to the solution. The reactor bottle was purged with hydrogen (3×), the pressure was maintained at 30−40 psi, and the bottle was shaken for 72 h at room temperature. After this time, the reaction bottle was purged with nitrogen, the catalyst was carefully removed over a pad of Celite, and the filtrate was concentrated under reduced pressure to afford 36 (2.28 g, 70%) as a light-yellow oil: 1H NMR (300 MHz, CDCl3) δ 1.42−1.50 (m, 2H), 1.70−1.86 (m, 2H), 1.90 (br s, 2H), 2.30−2.47 (m, 2H), 2.59−2.72 (m, 1H), 2.80−3.05 (m, 4H); 19F{1H} (282 MHz, CDCl3) δ −69.05 (s); Note: could not obtain adequate ionization for a mass spectrum. Preparation of N-(1-(2,2,2-Trifluoroethyl)piperidin-4-yl)-4(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin2-amine Dihydrochloride (9ff). The compound was prepared using a method similar to that described for the preparation of 9t using 36 as the amine, 0.113 g, 30% as an off-white powder: mp 84−87 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.25 (s, 2H), 1.51 (s, 2H), 1.65−2.10 (m, 6H), 2.80−3.00 (m, 2H), 3.10−4.90 (m, 6H), 7.26 (d, J = 5.5 Hz, 1H), 8.38 (d, J = 5.5 Hz, 1H), 8.66 (br s, 1H), 8.88 (br s, 1H); 19 1 F{ H} NMR (282 MHz, DMSO-d6) δ −62.0 to −63.1 (m), −67.3 (s); 13C NMR (125 MHz, DMSO-d6) δ 165.8, 153.8, 147.0, 143.2, 136.8, 126.0 (q, J = 274.2 Hz), 124.2, 121.9, 118.5, 106.6, 54.6, 54.3, 53.6, 53.3, 51.3, 45.5, 29.9, 28.6, 20.8, 11.4; HRMS: calcd for C18H20F6N6, 435.1732; found, 435.1726; HPLC: Method 1, tR = 2.60 min (98.7% AUC). (S)-N-(Piperidin-3-yl)-4-(3-(1-trifluoromethyl)cyclopropyl)1Hpyrazol-4-yl)pyrimidin-2-amine (9gg). The compound was prepared using a method similar to that described for the preparation of 9t, 0.045 g, 45% as a white solid: mp 204−205 °C; 1H NMR (300 MHz, DMSO-d6) δ 1.24 (s, 2H), 1.38−1.44 (m, 2H), 1.53 (s, 2H), 1.59−1.62 (m, 1H), 1.83−1.88 (m, 1H), 2.32−2.46 (m, 2H), 2.72− 2.78 (m, 1H), 2.99−3.03 (m, 1H), 3.86 (br s, 1H), 6.72 (br s, 1H), 6.85 (d, J = 5.1 Hz, 1H), 8.20 (s, 2H), 13.30 (br s, 1H); 19F{1H} (282 MHz, DMSO-d6) δ −67.37 (s); HRMS: calcd for C16H19F3N6, 353.1702; found, 353.1688; HPLC: Method 1, tR = 2.65 min (>99% AUC). (R)-N-(Piperidin-3-yl)-4-(3-(1-trifluoromethyl)cyclopropyl)1H-pyrazol-4-yl)pyrimidin-2-amine (9hh). The compound was prepared using a method similar to that described for the preparation of 9t, 0.052 g, 38% as a white solid: mp 204−205 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.24 (s, 2H), 1.37−1.43 (m, 2H), 1.53 (s, 2H), 1.60−1.62 (m, 1H), 1.83−1.85 (m, 1H), 2.34−2.44 (m, 2H), 2.77 (d, J = 12.0 Hz, 1H), 3.00−3.02 (m, 1H), 3.88 (br s, 1H), 6.71 (br s, 1H), 6.85 (d, J = 4.5 Hz, 1H), 8.19 (s, 2H), 13.30 (br s, 1H); 19F{1H} (282 MHz, DMSO-d6) δ −67.37 (s); 13C NMR (125 MHz, DMSO-d6) δ

154.0, 146.7, 143.3, 136.7, 126.0 (q, J = 272.9 Hz), 118.3, 106.4, 69.7, 66.9, 46.4, 28.3, 23.6, 20.9, 11.3, 11.2; HRMS: calcd for C16H18F3N5O, 354.1542; found, 354.1536; HPLC: Method 1, tR = 2.69 min (98.7% AUC). Preparation of N-(Tetrahydrofuran-3-yl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine Hydrochloride (9bb). The compound was prepared using a method similar to that described for the preparation of 9t, 129.4 mg, 52% as a white solid: 1H NMR (300 MHz, DMSO-d6): δ 1.29 (m, 2H), 1.52 (m, 2H), 1.88− 1.98 (m, 1H), 2.19−2.31 (m, 1H), 3.65−3.68 (m, 1H), 3.69−3.72 (m, 1H), 3.80−3.94 (m, 2H), 4.68 (m, 1H), 7.23 (d, J = 6.3 Hz, 1H), 8.35 (d, J = 6.3 Hz, 1H), 8.60 (br s, 2H); 19F{1H} (282 MHz, DMSO-d6) δ −67.33 (s); 13C NMR (125 MHz, DMSO-d6) δ 164.8, 153.9, 147.0, 142.6, 136.1, 125.6 (q, J = 274.2 Hz), 118.2, 106.1, 71.7, 65.8, 51.1, 31.6, 20.1, 11.0; HRMS: calcd for C15H16F3N5O, 340.1385; found, 340.1385; HPLC: Method 1, tR = 2.52 min (>99% AUC). N-(Oxetan-3-yl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1Hpyrazol-4-yl)pyridin-2-amine (9cc). The compound was prepared using a method similar to that described for the preparation of 9t, 0.024 g, 46% as a yellow solid: mp, no clear melt; 1H NMR (500 MHz, DMSO-d6) δ 1.23 (s, 2H), 1.51−1.53 (m, 2H), 3.51 (br s, 2H), 4.16− 4.20 (m, 1H), 4.22−4.27 (m, 1H), 4.27−4.47 (m, 1H), 5.08 (br s, 1H), 6.77 (br s, 1H), 6.92 (s, 1H), 7.89 (br s, 1H), 8.29 (br s, 1H); 19 1 F{ H} (282 MHz, CDCl3) δ −67.16; MS (ESI) m/z 325 [M + H]+; HRMS: calcd for C15H15F3N4O, 325.1276; found, 325.1286; HPLC: Method 2, tR = 2.65 min (>99% AUC). 4-(3-(1-Methylcyclopropyl)-1H-pyrazol-4-yl)-N-(piperidin-4-yl)pyrimidin-2-amine Dihydrochloride (9ee). The compound was prepared using a method similar to that described for the preparation of 9t, 0.033 g, 39% as an off-white solid: mp, no clear melt; 1H NMR (500 MHz, DMSO-d6) δ 0.85−0.95 (m, 4H), 1.38 (s, 3H), 1.75−1.83 (m, 2H), 2.05−2.10 (m, 2H), 3.00−3.10 (m, 2H), 3.30−3.36 (m, 2H), 7.20 (d, J = 5.5 Hz, 1H), 8.20 (br s, 1H), 8.33 (d, J = 5.5 Hz, 1H), 8.81 (br s, 1H), 8.97 (br s, 1H); HRMS: calcd for C16H22N6, 299.1984; found, 299.1992; HPLC: Method 1, tR = 2.34 min (>99% AUC). N-(1-(2,2,2-Trifluoroethyl)piperidin-4-yl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine Dihydrochloride (9ff). Preparation of 2,2,2-Trifluoro-1-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethanone (37). To a stirred solution at 0 °C of 1,4-dioxa-8-azaspiro[4.5]decane (38) (20.0 g, 0.140 mol), triethylamine (28.3 g, 0.279 mol), and DMAP (0.86 g, 0.007 mol) in dichloromethane (490 mL) was added trifluoroacetic anhydride (32.3 g, 0.154 mol) dropwise over 20 min. After the addition was complete, the reaction mixture was stirred at 0 °C for 1 h and then warmed to room temperature over 4 h. After this time, the reaction was quenched with saturated aqueous sodium bicarbonate (100 mL) and separated. The organic layer was washed with water (100 mL) and saturated sodium chloride (100 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure. The yellow oil was dissolved in hot ethyl acetate (200 mL), and the precipitate was filtered. The solution was cooled to room temperature and passed through a plug of silica gel, and the filtrate was concentrated under reduced pressure to afford 37 (32.4 g, 97%) as a yellow oil, which crystallized to a waxy solid upon standing: 1H NMR (300 MHz, CDCl3) δ 1.76 (t, J = 6.0 Hz, 4H), 3.67 (t, J = 5.7 Hz, 2H), 3.77 (t, J = 6.0 Hz, 2H), 4.00 (s, 4H); 19F{1H} (282 MHz, CDCl3) δ −68.91 (s); MS (ESI) m/z 240 [M + H]+. Preparation of 8-(2,2,2-Trifluoroethyl)-1,4-dioxa-8-azaspiro[4.5]decane (39). To a stirred solution of BH3·THF complex in THF (1.0 M; 69.0 mL, 69.0 mmol) was added a solution of 37 (15.0 g, 62.7 mmol) in THF (69.0 mL) dropwise over 10 min. The resulting reaction solution was heated at reflux for 30 min and cooled to room temperature, an additional portion of BH3·THF complex in THF (1.0 M; 69.0 mL, 69.0 mmol) was added, and the reaction mixture was heated at reflux for 24 h. After this time, the reaction mixture was cooled to room temperature and carefully quenched with methanol (100 mL), and the resulting reaction mixture was heated to reflux for 30 min. After this time, the reaction mixture was cooled to room temperature and concentrated under reduced pressure to provide 39 (7.50 g, 53%) as a light yellow oil: 1H NMR (300 MHz, CDCl3) δ 1.73−1.77 (m, 4H), 2.74−2.79 (m, 4H), 3.01 (q, J = 9.6 Hz, 2H), 3.96 2982

DOI: 10.1021/jm5013598 J. Med. Chem. 2015, 58, 2967−2987

Article

Journal of Medicinal Chemistry 161.7, 159.3, 157.9, 140.7, 134.3, 126.2 (q, J = 274.2 Hz), 120.3, 106.5, 51.5, 47.1, 45.7, 30.7, 25.0, 20.4 (q, J = 35.2 Hz), 11.4; HRMS: calcd for C16H19F3N6, 353.1702; found, 353.1705; HPLC: Method 1, tR = 2.02 min (>99% AUC). N-(4-(3-(6-Methylpyridin-3-yl)-1H-1,2,4-triazol-1-yl)phenyl)4-(3-(1-trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine Hydrochloride (9ii). The compound was prepared using a method similar to that described for the preparation of 9t, 0.106 g, 70% as a yellow solid: mp, no clear melt; 1H NMR (500 MHz, DMSO-d6) δ 1.27−1.31 (m, 2H), 1.54−1.56 (m, 2H), 2.71 (s, 3H), 7.21 (d, J = 5.0 Hz, 1H), 7.82 (br s, 1H), 7.87 (d, J = 9.0 Hz, 2H), 8.02 (d, J = 9.0 Hz, 2H), 8.31 (s, 1H), 8.52 (d, J = 5.0 Hz, 1H), 8.75 (br s, 1H), 9.24 (s, 1H), 9.39 (s, 1H), 9.75 (s, 1H); 19F{1H} (282 MHz, CDCl3) δ −67.27 (s, 3F); 13C NMR (125 MHz, DMSO-d6) δ 160.1, 158. 5, 157.0, 156.5, 154.4, 143.9, 141.1, 140.1, 138.3, 134.7, 130.4, 128.0, 127.2 (q, J = 274.2 Hz), 126.9, 120.1, 119.9, 119.8, 109.3, 33.9, 20.4, 20.2, 19.4, 11.3; HRMS: calcd for C25H20F3N9, 504.1872; found, 504.1882; HPLC: Method 1, tR = 3.09 min (>99% AUC). trans-1-Methyl-4-((4-(3-(1-(trifluoromethyl)cyclopropyl)-1Hpyrazol-4-yl)pyrimidin-2-yl)amino)cyclohexanol (9jj). Preparation of (cis)-4-Amino-1-methylcyclohexanol (41). A mixture of (cis)-4-(dibenzylamino)-1-methylcyclohexanol (42) (2.60 g, 8.40 mmol) and palladium hydroxide on carbon (50% water, 20 wt %, 1.17 g, 1.10 mmol) in ethanol (90 mL) was stirred at ambient temperature under hydrogen (balloon pressure) for 15 h. After this time, the reaction was filtered through diatomaceous earth and washed with ethanol. The filtrate was concentrated under reduced pressure to afford 41 as a white solid: 1H NMR (500 MHz, CDCl3) δ 1.22 (s, 3H), 1.40−1.48 (m, 7H), 1.64−1.69 (m, 4H), 2.60−2.63 (m, 1H); MS (ESI) m/z 130 [M + H]+. Preparation of trans-1-Methyl-4-((4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)cyclohexanol (9jj). The compound was prepared using a method similar to that described for the preparation of 9t using amine 41, 0.0620 g, 54% as a white solid: mp 220−222 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.14 (s, 3H), 1.23−1.27 (m, 2H), 1.38−1.45 (m, 5H), 1.51−1.59 (m, 3H), 1.78−1.81 (m, 2H), 3.91 (br s, 1H), 4.23 (s, 1H), 6.72 (br s, 1H), 6.83 (d, J = 5.0 Hz, 1H), 8.03−8.28 (m, 2H), 13.20 (br s, 0.65H, tautomer), 13.56 (br s, 0.35H, tautomer); 13C NMR (125 MHz, DMSO-d6) δ 160.7, 158.3, 156.8, 142.8, 139.0, 129.9, 119.3, 105.4, 67.1, 46.8, 36.8, 27.9, 25.5, 10.3; HRMS: calcd for C18H22F3N5O, 382.1855; found, 382.1853; HPLC: Method 4, tR = 2.90 min (>99% AUC). (cis)-1-Methyl-4-((4-(3-(1-(trifluoromethyl)cyclopropyl)-1Hpyrazol-4-yl)pyrimidin-2-yl)amino)cyclohexanol (9kk). The compound was prepared using a method similar to that described for the preparation of 9t, 0.0820 g, 55% as a white solid: mp 230−232 °C; 1H NMR (500 MHz, CDCl3) δ 1.11 (s, 3H), 1.20−1.26 (m, 2H), 1.31−1.36 (m, 2H), 1.44 (br s, 1H), 1.51−1.70 (m, 7H), 3.78 (br s, 1H), 3.98 (s, 1H), 6.74−6.82 (m, 2H), 8.18 (s, 1H), 8.27 (s, 1H), 13.19 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 161.6, 159.3, 158.9, 157.7, 143.7, 139.9, 134.9, 130.8, 120.4, 106.3, 66.4, 48.5, 37.4, 31.1, 27.8, 11.5, 11.2; HRMS: calcd for C18H22F3N5O, 382.1855; found, 382.1853; HPLC: Method 5, tR = 15.47 min (>99% AUC). N-(3,3-Difluorocyclobutyl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine (9ll). The compound was prepared using a method similar to that described for the preparation of 9t, 0.035 g, 16% over two steps, as a light brown solid: 1 H NMR (500 MHz, DMSO-d6) δ 1.26 (m, 2H), 1.51 (br s, 2H), 2.68−2.78 (m, 2H), 2.93−3.01 (m, 2H), 4.44 (br s, 1H), 7.10 (d, J = 5.5 Hz, 1H), 8.13−8.42 (m, 3H); 13C NMR (125 MHz, DMSO-d6) δ 127.2, 125.0, 121.6, 119.5, 119.4, 119.2, 117.2, 42.3, 42.1, 41.9, 35.6, 35.5; HRMS: calcd for C15H14F5N5, 360.1248; found, 360.1242; HPLC: Method 4, tR = 3.47 min (97.9% AUC). N-(3,3-Difluorocyclobutyl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyridin-2-amine Hydrochloride (25). Preparation of tert-Butyl (3,3-difluorocyclobutyl)carbamate. To an ice-cold, stirred solution of tert-butyl (3-oxocyclobutyl)carbamate in CH2 Cl 2 (18 mL) was added diethylaminosulfur trifluoride (1.8 mL, 13.5 mmol), and the mixture was allowed to slowly warm to room temperature over 4 h. The

reaction was quenched using saturated NaHCO3. The layers were separated, and the organic layer was washed with water, dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel; 50% ethyl acetate/heptanes) to provide tert-butyl (3,3-difluorocyclobutyl)carbamate (770 mg, 70%) as an off-white solid: 1H NMR (300 MHz, CDCl3): δ 1.45 (s, 9H), 2.41−2.53 (m, 2H), 2.91−3.00 (m, 2H), 3.94 (m, 1H), 4.62 (m, 1H). Preparation of 3,3-Difluorocyclobutanamine Hydrochloride (31). To a stirred solution of tert-butyl (3,3-difluorocyclobutyl)carbamate (770 mg, 3.71 mmol) in ethyl acetate (2 mL) was added hydrochloric acid (18.6 mL of a 4 N solution in dioxane, 74.4 mmol). After 20 h, the solvent was removed under reduced pressure to provide 31 (406 mg, 76%) as a light brown solid: 1H NMR (300 MHz, DMSO-d6): δ 2.77−2.99 (m, 4H), 3.57−3.66 (m, 1H), 8.52 (br s, 3H). Preparation of 2-(2-Chloropyridin-4-yl)-1-(1-(trifluoromethyl)cyclopropyl)ethanone (28). To a cold (−15 to −10 °C), stirred solution of 2-chloro-4-methylpyridine (3.24 g, 25.4 mmol) in THF (90 mL) was added lithium hexamethyldisilazide (52.1 mL of a 1.0 M solution in THF, 52.1 mmol) dropwise to maintain an internal reaction temperature between −10 and −5 °C. After 30 min, a solution of N-methoxy-N-methyl-1-(trifluoromethyl)cyclopropanecarboxamide (27, 5.00 g, 25.4 mmol) in THF (40 mL) was added dropwise. The reaction mixture was then allowed to slowly warm to room temperature overnight. The reaction was quenched with saturated ammonium chloride solution (100 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (3 × 50 mL). The combined organic extracts were washed with saturated sodium chloride, dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel; 0−100% ethyl acetate/heptanes, gradient elution) to provide 2-(2-chloropyridin-4-yl)-1-(1-(trifluoromethyl)cyclopropyl)ethanone 28 (3.69 g, 55%) as a light yellow oil: 1H NMR (300 MHz, CDCl3): δ 1.40−1.60 (m, 4H), 4.07 (s, 2H), 7.04 (d, J = 5.1 Hz, 1H), 7.17 (s, 1H), 8.34 (d, J = 5.1 Hz, 1H); MS (ESI) m/z 264 [M + H]+. Preparation of 2-Chloro-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyridine (29). A stirred solution of 2-(2-chloropyridin-4-yl)-1-(1-(trifluoromethyl)cyclopropyl)ethanone 28 (2.52 g, 9.58 mmol) in DMF·DMA (10 mL) was heated at 80 °C for 1 h. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure. The resulting residue was dissolved in EtOH (50 mL). Hydrazine (0.36 mL, 11.5 mmol) and diisopropylamine (2.5 mL, 14.4 mmol) were then added, and the resulting mixture was stirred at room temperature for 2 days. Afterward, the resulting solids were isolated by filtration, washed with cold EtOH (30 mL), and dried under vacuum to provide the desired product as an off-white solid (0.56 g). The filtrate was concentrated under reduced pressure and the residue triturated with CH2Cl2. A pale yellow solid was isolated by filtration and confirmed to be desired product. The solids were combined to provide 2-chloro-4(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyridine 29 (1.84 g, 67%). 1H NMR (500 MHz, DMSO-d6): δ 1.22 (s, 2H), 1.50 (s, 2H), 7.62 (d, J = 5.5 Hz, 1H), 7.68 (s, 1H), 8.33 (br s, 1H), 8.39 (d, J = 5.5 Hz, 1H), 13.45 (s, 1H). Preparation of 2-Chloro-4-(1-(tetrahydro-2H-pyran-2-yl)-3(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyridine (30). A mixture of 2-chloro-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol4-yl)pyridine 29 (1.78 g, 6.20 mmol) and p-toluene sulfonic acid monohydrate in 3,4-dihydro-2H-pyran (10 mL) was stirred at room temperature for 1 h. The reaction mixture was then concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel; 0−75% ethyl acetate/heptanes, gradient elution) to provide 30 (2.32 g, quantitative) as a mixture of regioisomers (85:15) and as a viscous yellow oil: 1H NMR (500 MHz, CDCl3): δ 1.06−1.13 (m, 2H), 1.41−1.76 (m, 5H), 1.95−2.17 (m, 3H), 3.63−3.75 (m, 1H), 4.08−4.15 (m, 1H), 5.40−5.43 (m, 0.85H), 5.66−5.68 (m, 0.15H), 7.37−7.42 (m, 1H), 7.46−7.47 (m, 1H), 7.79−7.85 (m, 1H), 8.35−8.40 (m, 1H). 2983

DOI: 10.1021/jm5013598 J. Med. Chem. 2015, 58, 2967−2987

Article

Journal of Medicinal Chemistry Preparation of N-(3,3-Difluorocyclobutyl)-4-(3-(1(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyridin-2-amine Hydrochloride (25). To a reaction vial containing 2-chloro-4-(1(tetrahydro-2H-pyran-2-yl)-5-(1-(trifluoromethyl)-cyclopropyl)-1H-pyrazol-4-yl)pyridine (30) (0.519 g, 1.40 mmol), 3,3-difluorocyclobutanamine hydrochloride (31) (0.220 g, 1.54 mmol), bis(tri-tert-butylphosphine)palladium (0) (0.072 g, 0.140 mmol), and sodium tert-butoxide (0.336 g, 3.50 mmol) was added degassed dioxane (4 mL) at room temperature under argon. The reaction mixture was sparged with argon for 5 min; the vial was sealed and then heated at 130 °C overnight. After this time, the reaction mixture was cooled to room temperature, diluted with ethyl acetate (50 mL), washed with water (2 × 25 mL) and saturated sodium chloride (25 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel; 0−75% ethyl acetate/heptanes, gradient elution) to provide a pale yellow semisolid (0.107 g). A mixture of the semisolid (0.100 g, 0.220 mmol) in a 5−6 M solution of hydrogen chloride in 2-propanol (10 mL) was stirred at room temperature for 2 days. After this time, the solvent was removed under reduced pressure. The residue was lyophilized from acetonitrile/ water to provide 25 (0.088 g, 16%, for 2 steps) as a light yellow-brown solid: 1H NMR (500 MHz, DMSO-d6) δ 1.25 (m, 2H), 1.58−1.61 (m, 2H), 2.74−2.84 (m, 2H), 3.17−2.25 (m, 2H), 4.27 (m, 1H), 7.19− 7.21 (m, 2H), 7.95 (d, J = 7.5 Hz, 1H), 8.42 (s, 1H), 9.51 (br s, 1H), 13.65 (br s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 150.0, 134.1, 125.4, 119.4, 117.2, 117.1, 115.4, 115.0, 109.8, 105.9, 40.3, 40.1, 39.9, 34.6, 34.5, 34.4; HRMS: calcd for C16H15F5N4, 359.1295; found, 359.1297; HPLC: Method 4, tR = 3.34 min (98.9% AUC). Preparation of 4-(3-Isopropyl-1H-pyrazol-4-yl)pyrimidine (33). A solution of 2-(methylsulfonyl)-4-(3-(isopropyl)-1H-pyrazol4-yl)pyrimidine (6p) (0.265 g, 0.996 mmol) in 1:1 ethanol/chloroform (12.0 mL) was treated with sodium borohydride (0.171 g, 4.52 mmol) at room temperature. After 2 h the reaction was carefully quenched with deionized water (2 mL, caution: hydrogen gas evolution!), and the resulting reaction mixture was stirred for an additional 30 min at room temperature. After this time, the reaction mixture was diluted with ethyl acetate (150 mL), washed with saturated aqueous sodium chloride, dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel; 0−10% methanol/methylene chloride, gradient elution) to provide 33 (0.105 g, 56%) as a white solid: mp 152−154 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.23−1.29 (m, 6H), 3.79−4.04 (m, 1H), 7.71 (d, J = 5.5 Hz, 1H), 8.10−8.45 (m, 1H), 8.65 (d, J = 5.5 Hz, 1H), 9.06 (d, J = 1.0 Hz, 1H), 12.90−13.04 (m, 1H); 13C NMR (125 MHz, DMSO-d6) δ 160.2, 158.4, 156.6, 149.7, 139.6, 117.3, 114.5, 25.2, 21.8; HRMS: calcd for C10H12N4, 189.1140; found, 189.1135; HPLC: Method 1, tR = 2.81 min (>99% AUC). 5-Chloro-N-(4,4-difluorocyclohexyl)4-(3-(1-(trifluoromethyl)cyclopropyl)-1Hpyrazol-4-yl)pyrimidin-2-amine (9 mm). Preparation of 6-Methyl-2-(methylthio)pyrimidin-4(3H)-one (16). To a stirred solution of 2-methyl-2-thiourea sulfate (24.5 g, 176 mmol) in water (420 mL) were added sodium carbonate (34.0 g, 320 mmol) and ethyl acetoacetate (11b, 20.8 g, 160 mmol) at room temperature. After 16 h, the solid was collected by filtration, washed with ice water (5 mL), and dried under reduced pressure at 40 °C to provide 16 (10.7 g, 43%) as a white solid: 1H NMR (300 MHz, DMSO-d6) δ 2.16 (s, 3H), 2.46 (s, 3H), 5.94 (s, 1H), 12.44 (br s, 1H). Preparation of 5-Chloro-6-methyl-2-(methylthio)pyrimidin4(3H)-one (17). To a stirred suspension of 16 (10.7 g, 68.7 mmol) and acetic anhydride (10.9 g, 103 mmol) in acetic acid (220 mL) were added ferric chloride (10.9 g, 103 mmol) and sulfuryl chloride (14.1 g, 104.4 mmol) dropwise at room temperature. After the addition was complete, the resulting reaction mixture was heated at 100 °C for 18 h. After this time, the reaction mixture was cooled to room temperature, another portion of sulfuryl chloride (3.3 g, 25.0 mmol) was added, and the reaction mixture was heated at 100 °C for an additional 18 h. After this time, the reaction mixture was cooled to room temperature and concentrated under reduced pressure, and the residue was triturated with acetone to provide 17 (9.06 g, 69%) as an off-white solid: 1H

NMR (300 MHz, DMSO-d6) δ 2.32 (s, 3H), 2.48 (s, 3H), 12.10 (br s, 1H); MS (ESI) m/z 194 [M + H]+. Preparation of 4,5-Dichloro-6-methyl-2-(methylthio)pyrimidine (18). A stirred suspension of 17 (6.67 g, 35.0 mmol), phosphorus oxychloride (83.8 g, 55.0 mmol), and N,N-dimethylaniline (6.21 g, 5.3 mmol) was heated at 115 °C for 15 h. After this time, the reaction mixture was cooled to room temperature and concentrated under reduced pressure, and the residue was poured into an ice water. After stirring for several minutes, the aqueous mixture was neutralized with a saturated sodium bicarbonate and extracted with methylene chloride (3 × 50 mL). The combined organics were dried (Na2SO4), filtered, and concentrated under reduced pressure. A major portion of the N,N-dimethylaniline was removed by vacuum distillation (10 mmHg/80 °C oil bath), and the residue was purified using flash column chromatography (silica gel; 0−50% methylene chloride/ heptane, gradient elution) to provide 18 (5.46 g, 75%) as an off-white solid: 1H NMR (300 MHz, CDCl3) δ 2.55 (s, 3H), 2.58 (s, 3H). Preparation of 5-Chloro-4-methyl-2-(methylthio)pyrimidine (19). A Parr bottle charged with a suspension of prepared 18 (5.30 g, 25.3 mmol) and palladium on carbon (50% wet, 10% Pd, 1.06 g) in a 0.68 M sodium hydroxide solution (100 mL) was degassed and backfilled with hydrogen twice, and then the resulting mixture was shaken under a hydrogen atmosphere (25−30 psi) at room temperature for 24 h. After this time, the reaction mixture was filtered through a pad of Celite, and the filter cake was washed with methylene chloride. The combined organics was dried (MgSO4), filtered, and distilled (760 mmHg, oil bath 60 to 75 °C) to provide 19 (3.33 g, 75%) as a clear colorless oil: 1H NMR (300 MHz, CDCl3) δ 2.53 (s, 3H), 2.54 (s, 3H), 8.36 (s, 1H); MS (ESI) m/z 175 [M + H]+. Preparation of 2-(5-Chloro-2-(methylthio)pyrimidin-4-yl)-1(1-(trifluoromethyl)-cyclopropyl)ethenol (20). To a stirred solution at 0 °C of 19 (1.51 g, 8.6 mmol) in THF (18.0 mL) was added sodium hexamethyldisilazide (18 mL of a 1.0 M solution in THF, 17.6 mmol), and the resulting reaction mixture was held at 0 °C for 2 h. After this time, a solution of N-methoxy-N-methyl-1-(trifluoromethyl)cyclopropanecarboxamide (1.70 g, 8.6 mmol) in THF (5 mL) was added dropwise, and then the resulting reaction mixture was warmed to room temperature. After 16 h, the reaction mixture was quenched with saturated ammonium chloride, the layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 20 mL). The combined organics were washed with saturated sodium chloride (15 mL), dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel; 2−5% ethyl acetate/heptanes, gradient elution) to provide 20 (2.10 g, 79%) as a light yellow solid: 1H NMR (300 MHz, CDCl3) δ 1.31−1.35 (m, 2H), 1.42−1.45 (m, 2H), 2.55 (s, 3H), 6.10 (d, J = 0.9 Hz, 1H), 8.27 (s, 1H), 14.76 (s, 1H); 19F{1H} (282 MHz, CDCl3) δ −66.80; MS (ESI) m/z 311 [M + H]+. Preparation of 5-Chloro-2-(methylthio)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1Hpyrazol-4-yl)pyrimidine (21). A stirred solution of 20 (2.10 g, 6.8 mmol) in DMF·DMA (11 mL) and methanol (2.5 mL) was refluxed at 110 °C for 6 h. After this time, the reaction mixture was cooled to room temperature, hydrazine hydrate (4.0 mL, 50−60%) was added, and the resulting reaction mixture was stirred at room temperature for 16 h. After this time, the reaction mixture was concentrated under reduced pressure, and the residue was purified using flash column chromatography (silica gel; 2−5%, 40% ethyl acetate/heptanes, gradient elution) to provide 21 (0.104 g, 5%) as an orange solid: 1H NMR (300 MHz, CDCl3) δ 1.26−1.30 (m, 2H), 1.44−1.48 (m, 2H), 2.57 (s, 3H), 8.17 (s, 1H), 8.52 (s, 1H); 19F{1H} (282 MHz, CDCl3) δ −68.90; MS (ESI) m/z 333 [M − H]−. Preparation of 5-Chloro-2-(methylsulfonyl)-4-(3-(1(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidine (22). To a stirred suspension at 0 °C of 21 (0.178 g, 0.53 mmol) in methylene chloride (2.5 mL) was added m-CPBA (75%, 0.193 g, 1.12 mmol) portionwise, and then the reaction mixture was warmed to room temperature. After 16 h, the reaction mixture was diluted with methylene chloride, saturated sodium bicarbonate (20 mL) and saturated sodium thiosulfate were added, and the resulting reaction mixture was stirred for 15 min. After this time, the organic layer was 2984

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Journal of Medicinal Chemistry separated, and the aqueous layer was extracted with 3:1 chloroform/ IPA (2 × 15 mL). The combined organics were washed with saturated sodium thiosulfate and saturated sodium chloride, dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel; 50−100% ethyl acetate/heptanes, gradient elution) to provide 22 (0.096 g, 49%) as a light yellow solid: 1H NMR (300 MHz, CDCl3) δ 1.23−1.34 (m, 2H), 1.39−1.50 (m, 2H), 3.40 (s, 3H), 8.30 (s, 1H), 8.63 (s, 1H), 9.19 (s, 1H); 19F{1H} (282 MHz, CDCl3) δ −63.11, −62.88; MS (ESI) m/z 367 [M + H]+. Preparation of 5-Chloro-2-(methylsulfonyl)-4-(1-(tetrahydro-2H-pyran-2-yl)-3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidine (23). To an ice-cooled stirred solution of 22 (0.126 g, 0.34 mmol) in 3,4-dihydropyrane (2.0 mL) was added p-toluenesulfonic acid (0.032 g, 0.02 mmol). After 5 min, the reaction mixture was concentrated under reduced pressure, and the residue was partitioned between saturated sodium bicarbonate and chloroform and separated. The organics were dried (MgSO 4 ), filtered, and concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel; 50−80% ethyl acetate/ heptane, gradient elution) to provide 23 (0.125 g, > 99%) as a light yellow oil: 1H NMR (300 MHz, CDCl3) δ 1.59−1.85 (m, 5H), 2.04− 2.06 (m, 1H), 2.16−2.19 (m, 1H), 3.31 (s, 3H), 3.50−3.58 (m, 1H), 3.63−3.77 (m, 1H), 3.98−4.05 (m, 1H), 4.08−4.19 (m, 1H), 4.88− 4.92 (m, 1H), 5.43−5.79 (m, 1H), 8.37−8.46 (m, 1H), 8.84 (d, J = 4.5 Hz, 1H); 19F{1H} (282 MHz, CDCl3) δ −68.65, −68.13; MS (APCI) m/z 451 [M + H]+. Preparation of 5-Chloro-N-(4,4-difluorocyclohexyl)-4-(1-tetrahydro-2H-pyran-2-yl)-3-(1-(trifluoromethyl)cyclopropyl)-1Hpyrazol-4-yl)pyrimidin-2-amine (24). A sealed microwave tube charged with a stirred suspension of 23 (0.150 g, 0.33 mmol), 4,4difluorocyclohexanamine hydrochloride (0.118 g, 0.66 mmol), and triethylamine (0.088 g, 0.83 mmol) in DMSO (2.0 mL) was heated at 130 °C for 3 h with microwave irradiation. After this time, the reaction mixture was cooled to room temperature, poured into water (10 mL), and extracted with methylene chloride (3 × 15 mL). The combined organics were washed with saturated sodium chloride, dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel; 30−50% ethyl acetate/heptanes, gradient elution) to provide 24 (0.029 g, 17%) as a yellow solid: 1H NMR (500 MHz, CDCl3) δ 1.21−1.32 (m, 6H), 1.57−1.93 (m, 6H), 2.03−2.09 (m, 6H), 3.63−3.73 (m, 1H), 4.01 (br s, 1H), 4.07−4.12 (m, 1H), 4.90−4.92 (m, 1H), 5.40−5.70 (m, 1H), 8.01−8.12 (m, 1H), 8.24 (d, J = 5.5 Hz, 1H); 19F{1H} (282 MHz, CDCl3) δ −68.45 (s), −68.59 (s); MS (ESI) m/z 506 [M + H]+. Preparation of 5-Chloro-N-(4,4-difluorocyclohexyl)4-(3-(1(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2amine (9 mm). To a stirred solution of 24 (0.028 g, 0.06 mmol) in methanol (2.0 mL) was added hydrochloric acid (0.500 mL of a 5−6 M solution in IPA, ∼0.25 mmol) at room temperature. After 2 days, the reaction mixture was concentrated under reduced pressure. The residue was basified with sodium bicarbonate solution, and the precipitate was collected by filtration to provide 9 mm (0.020 g, 85%) as an off-white solid: 1 H NMR (500 MHz, DMSO-d6) δ 1.28−1.42 (m, 4H), 1.56−1.62 (m, 2H), 1.84−1.89 (m, 4H), 2.05−2.07 (m, 2H), 3.95 (br s, 1H), 7.87−8.15 (m, 1H), 8.32 (s, 1H), 13.23−13.61 (m, 1H); 19F{1H} (282 MHz, CDCl3) δ −67.82 (s), −67.56 (s); HRMS: calcd for C17H17ClF5N5, 422.1171; found, 422.1159; HPLC: Method 2, tR = 19.65 min (97.3% AUC). Molecular Modeling. Virtual Screening. Docking-based virtual screening was performed according to methods described for Glide.40,41 The structure of Jnk3 in complex with an imidazolepyrimidine inhibitor (1pmq) was downloaded from Protein Data Bank (PDB) and prepared using protein preparation wizard in Maestro.42 Ligands were prepared using LigPrep43 using the OPLS-2005 force field and ionization states at physiological pH (± 2) derived with Epic.44 Molecular docking was performed with Glide45 with HTVS precision scoring function. Virtual hit selection was done using a combination of ligand ranking based on Glide gscore, Glide emodel,

and careful visual inspection for the fulfillment of key hydrogenbonding and hydrophobic interactions between the ligand and the protein. Glide Ligand Docking. Having obtained JNK3 complexes with imidazole 1 and analogue 9e, which represented a better representation of the JNK3 binding states with structurally related compounds, grids were prepared for these two structures for use in Glide docking. All ligands were energy-minimized using the OPLS2005 force field in Macromodel46 prior to docking. Molecular docking was performed with Glide45 with the standard precision scoring function. Assessment of docking pose quality and likelihood was done by visual inspection based on a knowledge-base of Glide gscores and Glide emodel rankings generated for a set of JNK3 inhibitor benchmarks from the literature as well as pyrazole compounds from the medicinal chemistry efforts. X-ray Crystallography Studies. JNK3−Adenosine Cocrystallization. The hanging drop vapor diffusion method was used for JNK3 cocrystallization with adenosine. JNK3 protein (1 μL, ∼8.5 mg/mL) containing 25 mM HEPES, pH 6.9, 5% v/v glycerol, 5 mM DTT, 125 mM NaCl, and 1 mM adenosine was mixed with a precipitant solution containing 20−25% PEG 3350 and 0.18−0.24 M potassium fluoride. JNK3 crystals in complex with adenosine were obtained through microseeding using JNK3/AMP-PNP seeds, and crystals appeared within 1 week at 20 °C. X-ray diffraction data were collected in-house using a Rigaku Micromax 007HF rotating anode X-ray generator and a Rigaku R-Axis IV+2 image plate detector. Prior to data collection, the crystals were flash-cooled to 100 K in the precipitant solution. Data were indexed and reduced with MOSFLM and SCALA (CCP4) in the orthorhombic P212121 space group. The structure was determined by the molecular replacement method using PDB entry 3G9L with MOLREP (CCP4). Refinement and model building were performed using REFMAC5 and COOT, respectively (CCP4). Clear electron density for the adenosine molecule was observed after one round of refinement. After alternating cycles of model building, water addition and refinement the JNK3−adenosine complex had a final Rcryst = 25.6% and Rfree = 31.1%. JNK3-Adenosine Crystal Soaking Experiment with 1. A 50 mM stock solution of each CHDI inhibitor compound was prepared in DMSO. JNK3−adenosine cocrystals were transferred from their growth drop to a drop containing 23% PEG 3350, 0.2 M potassium fluoride, 10% v/v DMSO, and 5 mM CHDI ligand. Crystals were extremely sensitive to drying out and were transferred using a large 0.5 mm diameter, 20 μm loop that holds a relatively large volume of precipitant solution and prevents crystal damage. Soaks were performed with pyrazole 1 for 7 days, 24 h, 3 days, and 3 h, respectively. Prior to data collection, the crystals were flash-cooled to 100 K in the soak solution. All ligand soak data sets were collected in-house as described above, and structures were also solved as described above. The topology files for each inhibitor were generated using JLIGAND (CCP4). The JNK3−1 complex had a final Rcryst = 24.7% and Rfree = 31.9%. Soaking with 9e was conducted in a similar manner. The JNK3−9e complex had a final Rcryst = 26.3% and Rfree = 32.4%. Kinetic Solubility Assay. The assay was run using the following materials and reagents: tamoxifen, Sigma (cat. no. T-5648); verapamil, Sigma (cat. no. V-4629); DMSO, Sigma (cat. no. 472301); phosphate buffered saline (PBS), Sigma (cat. no. D8537); 0.45 μm PVDF filters, Titan Sun Sri (cat. no. 44504-PV); 2 mL 96-well plate, Whatman (cat. no. 7701-5200); 96-well plates (UV compatible, nonpolystyrene, clear bottom), Costar (cat. no. 3635); Spectramax PLUS UV/vis spectrophotometer, Molecular Devices. Stock solutions (10 mM) of each compound were prepared in DMSO, and 10 μL of a 10 mM stock solution was transferred into a well in a 2 mL 96-well plate containing 990 μL of PBS (pH 7.4) in triplicate (theoretical concentration of 100 μM). The plate was sealed and incubated in an incubator shaker for 1 h at 25 °C while shaking at 200 rpm. Samples were filtered through a 0.45 μm hydrophilic PVDF 96-well filter plate mounted on a 2 mL 96-well plate by centrifugation at 3600 rpm for 10 min. Filtered samples were then measured in a 96-well UV plate. Standards were prepared by initially preparing a 200 μM solution from the 10 mM stock (e.g., 20 μL of 10 mM stock 2985

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Journal of Medicinal Chemistry solution in 960 μL DMSO) and then performing a 1:1 serial dilution in a 96-well UV plate. PBS with 1% v/v DMSO was used as a blank. The UV plate was scanned using a UV/vis spectrophotometer at a wavelength of 280 nm. Compound solubility was determined by comparing the absorbance value of the filtrate to the standard calibration curve of the compound and measured in micromolar of compound dissolved in PBS with 1% v/v DMSO.

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E. F.; Quinn, K. P.; John-Michael Sauer, J.-M.; Powell, K.; Ruslim, L.; Ren, Z.; Bard, F.; Yednock, T. A.; Griswold-Prenner, I. Design and synthesis of a novel, orally active, brain penetrant, tri-substituted thiophene based JNK inhibitor. Bioorg. Med. Chem. Lett. 2011, 21, 1838−1843. (7) Borsello, T.; Clarke, P. G. H.; Hirt, L.; Vercelli, A.; Repici, M.; Schorderet, D. F.; Bogousslavsky, J.; Bonny, C. A peptide inhibitor of c-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia. Nat. Med. 2003, 9, 1180−1186. (8) Sclip, A.; Antoniou, X.; Colombo, A.; Camici, G. G.; Pozzi, L.; Cardinetti, D.; Feligioni, M.; Veglianese, P.; Bahlmann, F. H.; Cervo, L.; Balducci, C.; Costa, C.; Tozzi, A.; Calabresi, P.; Forloni, G.; Borsello, T. c-Jun N-terminal kinase regulates soluble Ab oligomers and cognitive impairment in AD mouse model. J. Biol. Chem. 2011, 286, 43871−43880. (9) Apostol, B. L.; Illes, K.; Pallos, J.; Bodai, L.; Wu, J.; Strand, A.; Schweitzer, E. S.; Olson, J. M.; Kazantsev, A.; Marsh, J. L.; Thompson, L. M. Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity. Hum. Mol. Genet. 2006, 15, 273−285. (10) Garcia, M.; Charvin, D.; Caboche, J. Expanded huntingtin activates the c-Jun terminal kinase/c-Jun pathway prior to aggregate formation in striatal neurons in culture. Neuroscience 2004, 127, 859− 870. (11) Merienne, K.; Helmlinger, D.; Perkin, G. R.; Devys, D.; Trottier, Y. Polyglutamine expansion induces a protein-damaging stress connecting heat shock protein 70 to the JNK pathway. J. Biol. Chem. 2003, 278, 16957−16967. (12) Lui, Y. F. Expression of polyglutamine-expanded huntingtin activates the SEK1−JNK pathway and induces apoptosis in a hippocampal neuronal cell line. J. Biol. Chem. 1998, 273, 28873− 28877. (13) Garcia, M.; Vanhoutte, P.; Pages, C.; Besson, M. J.; Brouillet, E.; Caboche, J. The mitochondrial toxin 3-nitropropionic acid induces striatal neurodegeneration via a c-Jun N-terminal kinase/c-Jun module. J. Neurosci. 2002, 22, 2174−2184. (14) Ham, J.; Babij, C.; Whitfield, J.; Pfarr, C. M.; Lallemand, D.; Yaniv, M.; Rubin, L. L. A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death. Neuron 1995, 14, 927−939. (15) Apostol, B. L.; Simmons, D. A.; Zuccato, C.; Illes, K.; Pallos, J.; Casale, M.; Conforti, P.; Ramos, C.; Roarke, M.; Kathuria, S.; Cattaneo, E.; Marsh, J. L.; Thompson, L. M. CEP-1347 reduces mutant huntingtin-associated neurotoxicity and restores BDNF levels in R6/2 mice. Mol. Cell. Neurosci. 2008, 39, 8−20. (16) Perrin, V.; Dufour, N.; Raoul, C.; Hassig, R.; Brouillet, E.; Aebischer, P.; Luthi-Carter, R.; Déglon, N. Implication of the JNK pathway in a rat model of Huntington’s disease. Exp. Neurol. 2009, 15, 191−200. (17) Morfini, G. A.; You, Y.-M.; Pollema, S. L.; Kaminska, A.; Liu, K.; Yoshioka, K.; Björkblom, B.; Coffey, E. T.; Bagnato, C.; Han, D.; Huang, C.-F.; Banker, G.; Pigino, G.; Brady, S. T. Pathogenic huntingtin inhibits fast axonal transport by activating JNK3 and phosphorylating kinesin. Nat. Neurosci. 2009, 12, 864−871. (18) LoGrasso, P.; Kamenecka, T. Inhibitors of c-jun-N-terminal kinase (JNK). Mini-Rev. Med. Chem. 2008, 8, 755−766. (19) Siddiqui, M. A.; Reddy, P. A. Small molecule JNK (c-Jun Nterminal kinase) inhibitors. J. Med. Chem. 2010, 53, 3005−3012. (20) Humphries, P. S.; Lafontaine, J. A.; Agree, C. S.; Alexander, D.; Chen, P.; Do, Q.-Q. T.; Li, L. Y.; Lunney, E. A.; Rajapakse, R. J.; Siegel, K.; Timofeevski, S. L.; Wang, T.; Wilhite, D. M. Synthesis and SAR of 4-substituted-2-aminopyrimidines as novel c-Jun N-terminal kinase (JNK) inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 2099− 2102. (21) Kamenecka, T.; Jiang, R.; Song, X.; Duckett, D.; Chen, W.; Ling, Y. Y.; Habel, J.; Laughlin, J. D.; Chambers, J.; Figuera-Losada, M.; Cameron, M. D.; Lin, L.; Ruiz, C. H.; LoGrasso, P. V. Synthesis, biological evaluation, X-ray structure, and pharmacokinetics of

ASSOCIATED CONTENT

S Supporting Information *

Experimental descriptions of in vitro liver microsomal stability assays and permeability/efflux assays in MDCK cells; description of the metabolite identification in mouse liver microsomes and a mouse pharmacokinetic study of 9t coadministered with CYP450 inhibitors; Cerep selectivity panels showing the activity of 9t at 10 μM concentration against diverse enzyme, kinase, and receptor targets; and Cerep kinase panel results of 34 at 10 μM and a summary of JNK inhibitor benchmark profiling. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 310-342-5518; E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank John Mangette for obtaining 13 C NMR and HRMS data on all new compounds. ABBREVIATIONS USED JNK, c-Jun N-terminal kinase; HD, Huntington’s disease; FAT, fast axonal transport; POC, proof of concept; p38 MAPK, p38 mitogen activated protein kinase; ATF-2, activating transcription factor 2; PD, pharmacodynamic; PK, pharmacokinetics; ADME, adsorption distribution metabolism excretion; mLM, mouse liver microsomes; hLM, human liver microsomes; MDCK-MDR1, Madin−Darby canine kidney cells expressing the MDR1 gene; PDB, Protein Data Bank; HMBC, heteronuclear multiple bond correlation; NOE, nuclear Overhauser effect; P-gp, P-glycoprotein

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REFERENCES

(1) Waetzig, V.; Herdegen, T. Context-specific inhibition of JNKs: overcoming the dilemma of protection and damage. Trends Pharmacol. Sci. 2005, 26, 455−461. (2) Bozyczko-Coyne, D.; Saporito, M. S.; Hudkins, R. L. Targeting the JNK pathway for therapeutic benefit in CNS disease. Curr. Drug Targets: CNS Neurol. Disord. 2002, 1, 31−49. (3) Resnick, L.; Fennell, M. Targeting JNK3 for the treatment of neurodegenerative disorders. Drug Discovery Today 2004, 9, 932−939. (4) Yang, D. D.; Kuan, C. Y.; Whitmarsh, A. J.; Rincon, M.; Zheng, T. S.; Davis, R. J.; Rakic, P.; Flavell, R. A. Absence of excitotoxicityinduced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature 1997, 389, 865−870. (5) Chambers, J. W.; Pachori, A.; Howard, S.; Ganno, M.; Hansen, D.; Kamenecka, T.; Song, X.; Duckett, D.; Chen, W.; Ling, Y.-Y.; Cherry, L.; Cameron, M. D.; Lin, L.; Ruiz, C. H.; LoGrasso, P. Small molecule c-Jun-N-terminal kinase inhibitors protect dopaminergic neurons in a model of Parkinson’s disease. ACS Chem. Neurosci. 2011, 2, 198−206. (6) Bowers, S.; Anh, P.; Truong, A. P.; Neitz, R. J.; Neitzel, M.; Probst, G. D.; Hom, R. K.; Peterson, B.; Galemmo, R. A., Jr.; Konradi, A. W.; Sham, H. L.; Tóth, G.; Pan, H.; Yao, N.; Artis, D. R.; Brigham, 2986

DOI: 10.1021/jm5013598 J. Med. Chem. 2015, 58, 2967−2987

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DOI: 10.1021/jm5013598 J. Med. Chem. 2015, 58, 2967−2987