Selective Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12

Article pubs.acs.org/jmc

Selective Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12) with Activity in a Model of Alzheimer’s Disease Snahel Patel,† William J. Meilandt,‡ Rebecca I. Erickson,∥ Jinhua Chen,# Gauri Deshmukh,⊥ Anthony A. Estrada,† Reina N. Fuji,∥ Paul Gibbons,† Amy Gustafson,∇ Seth F. Harris,§ Jose Imperio,‡ Wendy Liu,† Xingrong Liu,⊥ Yichin Liu,∇ Joseph P. Lyssikatos,† Changyou Ma,# Jianping Yin,§ Joseph W. Lewcock,*,‡ and Michael Siu*,† †

Department of Discovery Chemistry, ∥Department of Safety Assessment, §Department of Structural Biology, ‡Department of Neurosciences, ⊥Department of Drug Metabolism and Pharmacokinetics, and ∇Department of Biochemical and Cellular Pharmacology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States # Department of Chemistry, WuXi AppTec Co., Ltd., 288 Fute Zhonglu, Wai Gao Qiao Free Trade Zone, Shanghai 200131, P. R. China S Supporting Information *

ABSTRACT: Significant data exists to suggest that dual leucine zipper kinase (DLK, MAP3K12) is a conserved regulator of neuronal degeneration following neuronal injury and in chronic neurodegenerative disease. Consequently, there is considerable interest in the identification of DLK inhibitors with a profile compatible with development for these indications. Herein, we use structure-based drug design combined with a focus on CNS drug-like properties to generate compounds with superior kinase selectivity and metabolic stability as compared to previously disclosed DLK inhibitors. These compounds, exemplified by inhibitor 14, retain excellent CNS penetration and are well tolerated following multiple days of dosing at concentrations that exceed those required for DLK inhibition in the brain.

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INTRODUCTION Dual leucine zipper kinase (DLK, MAP3K12) dependent activation of the JNK/c-Jun pathway in neurons is essential for induction of the neuronal stress response following insult.1−5 Abrogation of this stress response in DLK null animals results in potent protection of neurons from degeneration in multiple neuronal injury models.1,3,6−8 Although DLK also appears to be required for axon regeneration following peripheral nerve injury,1,2,5,9 recent work has demonstrated that genetic deletion or pharmacological inhibition of DLK results in attenuation of synapse loss, neuronal degeneration, and functional decline in models of both Alzheimer’s Disease and Amyotrophic Lateral Sclerosis (ALS).10 Based on these findings, the net effect of DLK inhibition would be expected to provide functional protection in the context of chronic neuronal degeneration, making DLK an attractive therapeutic target for the treatment of neurodegenerative disease.11,12 Previously, we have disclosed two series of small molecule DLK inhibitors that effectively reduce c-Jun phosphorylation in nerve crush and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) based acute injury mouse models.13,14 These compounds have served as valuable tools to enable an improved understanding of the consequences of DLK inhibition in vivo, yet challenges remain in the development of DLK inhibitors for treatment of chronic neurodegenerative disease. For these © 2017 American Chemical Society

indications, a good safety profile and large therapeutic window following chronic dosing are required, which are challenging attributes to achieve with CNS-penetrant kinase inhibitors.15 Therefore, we used structure-based design to further evolve our pyrazole scaffold (represented by compound 1) to address these challenges. Here we describe the discovery of a series of inhibitors with an improved potency, PK, kinase selectivity, and tolerability profile with potential for prolonged administration. These compounds appear more suitable than previously reported DLK inhibitors for use in chronic neurodegenerative indications.

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RESULTS AND DISCUSSION In prior studies, compound 1 (DLK Ki = 0.042 μM, p-JNK cellular IC50 = 0.536 μM, ClogP 3.7, tPSA 78 Å2, HBD 1, LipE 3.7) exhibited encouraging free drug exposure and DLK inhibition in an optic nerve crush model.14,16,17 Although promising, further optimization was necessary to improve potency, kinase selectivity, and drug-like properties befitting a brain-penetrant therapeutic (Figure 1). Examination of the crystal structure of 1 bound to DLK14 led us to explore alternative hydrogen bond Received: June 12, 2017 Published: September 20, 2017 8083

DOI: 10.1021/acs.jmedchem.7b00843 J. Med. Chem. 2017, 60, 8083−8102

Journal of Medicinal Chemistry

Article

MDR1 BA/AB = 1.6) showed better efflux ratios and in vitro metabolic stability.20 Interestingly, 3-methyl-2-aminopyridine 8 (Ki > 1.6 μM, MDR1 BA/AB = 8.2) and 3-chloro-2-aminopyridine 9 (Ki = 1 μM, MDR1 BA/AB = 3.6) were efflux substrates and highlighted the importance of C3-substitutents to effectively mask the 2-amino group. With the new pyridine hinge binder, pyrazole N1 substitution (binding under the P-loop) afforded flat SAR (data not shown); thus, we targeted the N-oxetanylpiperidine fragment for modification. Initially, we looked to reduce lipophilicity without increasing tPSA by bridging the piperidine ring to an exo-3azabicyclo[3.1.0]hexane to assess if productive interactions could be achieved in the region of the solvent-exposed opening. Gratifyingly, this subtle change provided significant improvements in binding affinity and lipophilic efficiencies while preserving tPSA (Table 2). The matched pairs 2-amino-3trifluoromethylpyridines 7 (Ki = 0.154 μM, LipE = 3.5, pKa = 6.55)/10 (Ki = 0.007 μM, LipE = 5.6, pKa = 5.9) and 2-amino3-methoxypyridines 6 (Ki = 0.142 μM, LipE = 4.6)/12 (Ki = 0.007 μM, LipE = 6.5) showed approximately 20-fold improvement in binding affinity through minimal alteration of the scaffold. Crystal structures of piperidine 7 and 3-azabicyclo[3.1.0]hexane 10 bound to the DLK kinase domain were determined at 2.96 and 2.45 Å resolutions, respectively. We observed a consistent disposition for the core scaffold (Figure 3a) to form hydrogen bonds to the kinase hinge element and engaged with the flexible glycine-rich P-loop, suggesting that the potency difference is not due to fundamental changes in the protein conformation or coarse adjustments of the core of the small molecule. However, the azabicyclo[3.1.0]hexane portion did show a shifted position relative to the piperidine moiety it replaced, assuming a more orthogonal orientation relative to the core ring systems and a slight shift toward the hinge side of the protein cavity en route to the more solvent exposed region. This approach to backbone carbonyl groups of Ala194 and Gln195 (Figure 3b) and the conformational rigidity of the 3-azabicyclo[3.1.0]hexane may account for some of the improved potency of compound 10. Finally, the terminal oxetane also differs significantly in position between the two structures. In brief, a combination of subtle effects is believed to contribute to the notable improvement in DLK binding affinity. A variety of 3-substituents on the 2-aminopyridine hinge binder maintained favorable interactions with the Met-190 gatekeeper side chain. The trifluoromethylpyridine 10 (MDR1 BA/AB = 0.97, AB = 16 × 10−6 cm/s) and difluoromethylpyridine 11 (MDR1 BA/AB = 1.9, AB = 2.2 × 10−6 cm/s) retained desirable permeability properties and metabolic stability in liver microsomes (Table 2). In addition to fluoroalkyls, C3 alkoxy analogs, such as methoxy analog 12 (Ki = 0.007 μM, p-JNK IC50 = 0.873 μM, MDR1 BA/AB = 2.1), difluoromethoxy analog 13 (Ki = 0.008 μM, p-JNK IC50 = 0.328 μM, MDR1 BA/AB = 6.6), and trifluoromethoxy analog 14 (Ki = 0.003 μM, p-JNK IC50 = 0.195 μM, MDR1 BA/AB = 1.3) have comparable biochemical potencies. The difluoromethoxy analog 13 in particular suffered from an efflux liability, presumably due to the polar nature of this group.21 We then investigated 3-substituents of the 3-azabicyclo[3.1.0]hexane toward solvent as shown in Table 3. Substitution toward the solvent exposed region was more tolerant of changes with minimal effect on binding affinity as the oxetanylmethyl 15, difluoroethyl 17, 2-methoxyethyl 18, and 2-hydroxypropyl 19 had similar potency, permeability, and microsomal stability as oxetane 10. The N-acetyl 16 (MDR1 BA/AB = 3.8, AB = 5.7 × 10−6 cm s−1)

Figure 1.

donor/acceptor interactions of the 2-aminopyridine hinge binder, and we hypothesized that altering the hinge contacts could provide new leads for optimization. Initially, we targeted replacement of the 2-aminopyridine with a 7-azaindole18 (pyrrolo[2,3-b]pyridine) where the NH and C6−H could maintain polarized hydrogen bond interactions to the carbonyl moieties of Glu191 and Cys193, respectively (Figure 2). The 7-azaindole portion would also shift the central pyrazole core,

Figure 2. Two-dimensional representation of the hydrogen bonds of compound 1 with the hinge residues of DLK (PDB code: 5CEQ). Two-dimensional representation of proposed hydrogen bonds of 3-substituted-1H-pyrrolo[2,3-b]pyridine.

changing the interactions of pyrazole N1 substituents with the P-loop.14 Additionally, 7-azaindole C3-substituents would match the positioning of the C4-nitrile of compound 1 and provide interactions with both the P-loop and Met190 gatekeeper side chain.14 Using the 4-(1-isopropyl-1H-pyrazol-5-yl)-1-(oxetan-3-yl)piperidine framework of compound 1 and targeting properties for brain penetration (HBD ≤ 1, tPSA ≤ 80 Å2), we initially evaluated the C3-substituted fluoro-, chloro-, methyl-, and trifluoromethyl-7-azaindoles (Table 1). These compounds showed moderate biochemical and cellular potencies as measured by reduction of MKK4 phosphorylation and JNK phosphorylation (p-JNK) in HEK293 cells stably overexpressing DLK, respectively.13,14 The cellular assay was previously found to correlate well with potency against endogenous DLK.13,14 Furthermore, these 7-azaindoles displayed increased lipophilic efficiencies when compared to 1 (LipE = 3.7). Unfortunately, the 7-azaindole analogs showed large efflux ratios in an MDR1 assay despite their low tPSA (3-fluoro-7-azaindole 2, BA/AB = 3.9, tPSA = 58; 3-trifluoromethyl-7-azaindole 5, BA/AB = 4.5, tPSA = 58); thus, balancing brain penetration with other properties proved to be difficult for this hinge binding motif.19 Accordingly, we examined a 3-substituted 2-aminopyridine (with C5 connectivity to the pyrazole scaffold) as an isostere of the 7-azaindole. Specific pyridine C3-substitutents were selected to mask the additional HBD and mimic the planar azaindole motif. Although less potent compared to the 7-azaindoles, 2-amino-3-methoxypyridine 6 (Ki = 0.142 μM, MDR1 BA/AB = 2.2) and 2-amino-3-(trifluoromethyl)pyridine 7 (Ki = 0.154 μM, 8084



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Table 1a

a

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

Table 2a

compd

R

Ki (μM)

pJNK IC50 (μM)

MDR1-MDCK BA/AB (AB)b

LM CLhepc (mL min−1 kg−1) H/R/Md

ClogP

tPSA

LipEe

10 11 12 13 14

CF3 CF2H OCH3 OCF2H OCF3

0.007 0.035 0.007 0.008 0.003

0.25 1.2 0.873 0.328 0.195

0.97 (16) 1.9 (2.2) 2.1 (6.8) 6.6 (5.4) 1.3 (10.1)

5/13/41 2/10/23 4/12/18 1/10/32 4/14/30

2.6 1.1 1.6 1.0 2.8

69 69 78 78 78

5.6 6.4 6.5 6.2 5.8

a

All assay results represent the geometric mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the reported mean. bMDCK-MDR1 human P-gp transfected cell line. (BA/AB) = Basolateral-to-apical/apical-to-basolateral ratio. AB = Apical-tobasolateral flux, Units = ×10−6 cm s−1. cLiver microsome-predicted hepatic clearance. dH/R/M = human/rat/mouse. eLipE = −log Ki − ClogP. 8085



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Figure 3. Overlay of two crystallographic structures of piperidine 7 (protein/ligand/waters in dark green/bright green/pale green, PDB 5VO2) and azabicyclo[3.1.0]hexane 10 (ivory/brown/ivory, PDB 5VO1) bound to the DLK kinase domain. (a) Viewed down into the ATP-binding pocket with the glycine-rich P loop removed from the foreground. The kinase “hinge” is at the upper left, providing a canonical kinase binding motif of hydrogen bonds with the small molecule ligand. The core 5-(pyrazol-3-yl)pyridin-2-amines maintain a consistent position, while the solvent-oriented oxetane differs between piperidine (7) or the azabicyclo[3.1.0]hexane (10). (b) A rotated view of the overlay with the hinge at the rear, highlighting the proximity of the azabicyclo[3.1.0]hexane moiety to carbonyl groups of DLK residues 194 and 195 and showing the slight shift of the isopropyl moiety of the ligand at the center of the image.

Table 3a

a

Compound 19 was tested as racemate. All assay results represent the geometric mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the reported mean. bbMDCK-MDR1 human P-gp transfected cell line. (BA/AB) = Basolateral-to-apical/ apical-to-basolateral ratio. AB = Apical-to-basolateral flux, Units = ×10−6 cm s−1. cLiver microsome-predicted hepatic clearance. dH/R/M = human/ rat/mouse. eLipE = −log Ki − ClogP.

maintained potency at the expense of an efflux liability. From this analysis, the oxetane group provided the best balance of potency and properties and became the preferred substitution toward solvent.

The P-loop region of DLK was explored via the pyrazole N1 substitution with the 2-amino-3-trifluoromethylpyridine and 2-amino-3-trifluoromethoxypyridine hinge binders. Increasing lipophilicity in this area improved cellular potency and afforded 8086



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Table 4a

a

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

kinases, serine/threonine kinases, and lipid kinases. Single point testing at 1 μM revealed few kinases with ≥50% inhibition (Figure 4 Compound 14, Flt3, PAK4, STK33, TrkA; Compound 24, Axl, CSF1R, DMPK, EphA7, Flt3, TrkA). No kinases were inhibited to a degree of >70% by either compound, constituting a significant improvement compared to previous DLK inhibitors.13,14 Binding affinity (Kd) of compounds 14 and 24 were tested for the homologous mixed lineage kinase LZK as no kinase activities assays are available and were determined to be 0.460 μM (42 × DLK Kd) and 0.120 μM (24 × DLK Kd), respectively (Table 5). The specific rearrangement of the hinge binder along with an exo-3-azabicyclo[3.1.0]hexane group is believed to favorably position these inhibitors in DLK with specific interactions to hinge residue Phe192 and gatekeeper residue Met190,

good permeability (methyl 20 and 21 < ethyl 22 < trifluoroethyl 23 ∼ cyclopropylmethyl 24 and 25 < isobutyl 26) (Table 4). Polar atoms were less tolerated at this position (2-methylbutan2-ol 27 Ki = 0.023 μM, p-JNK IC50 = 0.829 μM) while cycloalkyls such as cyclobutyl 28 and cyclopentyl 29 deteriorated lipophilic efficiency. The in vitro data from a number of compounds displayed an encouraging combination of properties and were suitable for further evaluation. Based on their optimal combination of properties including cellular potency, microsomal stability, and lipophilicity, compounds 14 and 24 were progressed to a range of in vitro screens to examine potential safety liabilities (Table 5). Kinase selectivity was assessed using a panel of 220 kinases (Life Technologies) that included cytoplasmic and receptor tyrosine 8087



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peridin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (GNE3511).13 Inhibitors 14 and 24 were also evaluated in a broad panel of biochemical radioligand-binding and enzyme assays, including 42 targets of major classes of biogenic amine receptors, neuropeptide receptors, ion channels, and neurotransmitter transporters. The results of these assays did not reveal any significant off-target binding for either compound. hERG ion channel inhibition from a patch clamp assay was minimal for 14 (14% at 10 μM) and moderate for 24 (42% at 10 μM). Taken together, these results demonstrated that 14 and 24 were highly selective and had selectivity profiles suitable for chronic dosing. To confirm that the selective DLK inhibition elicited by 14 and 24 was sufficient to result in neuroprotection, we examined these azabicyclo[3.1.0]hexane pyrazoles in a high-content in vitro axon degeneration assay.22 A dose dependent protection of axons was observed with both 14 and 24 (Figure 5. Compound 14 EC50 = 0.574 μM, Compound 24 EC50 = 0.457 μM), which correlated well with the IC50 in the p-JNK assay, though the neuroprotection EC50 was shifted as near complete DLK/JNK inhibition is required to elicit protection in this acute setup.13,14,22 In addition, these compounds were significantly (∼5 fold) more potent in this functional assay as compared to the potency we previously reported for Compound 1 (EC50 = 2.05 μM).14 For all compounds, the maximum protection of axons achieved was roughly 80% of a positive control in which degeneration was not induced. These results demonstrate that selective inhibition of DLK is sufficient to elicit potent neuroprotection, though LZK or other pathways

Table 5. In Vitro Screening Assays 24 hERG ion channela (inhibition @ 1, 10 μM) receptor panelb (binding hits >50% of control @ 10 uM) JNK pathway and related kinases IC50c

LZK and DLK binding affinityd

14

11%, 42%

6%, 14%

none

none

JNK1 > 10 μM JNK2 > 10 μM JNK3 > 10 μM MLK1 = 3.5 μM MLK2 = 5.15 μM MLK3 > 10 μM LZK Kd = 0.120 μM DLK Kd = 0.0051 μM

JNK1 > 10 μM JNK2 > 10 μM JNK3 > 10 μM MLK1 = 5.92 μM MLK2 = 7.88 μM MLK3 > 10 μM LZK Kd = 0.460 μM DLK Kd = 0.011 μM

a ChanTest FASTPatch hERG Assay. bSee Supporting Information; CEREP: A1, A2A, α1, α2, β1, β2, BZD, D1, D2S, ETA, GABAA1 (α1,β2,γ2), GABAB(1b), Kainate, H1, H2, M1, M2, M3, NK1, N neuronal α4β2, N muscle-type, Opiod, PPARα, PPARδ, Rolipram, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT3, 5-HT4e, Ca2+ channel (L, dihydropyridine site), Ca2+ channel (L, verapamil site) (phenylalkylamine), KATP channel, hERG (membrane preparation), KV channel, Na+ channel (site 2), Cl− channel (GABA-gated), Norepinephrine transporter, Dopamine transporter, 5-HT transporter, Acetylcholinesterase. c Biochemical IC50 values were determined at Life Technologies. dKd values were determined at DiscoverX.

which are typically different in kinases inhibited by Compound 1 and 2-((6-(3,3-difluoropyrrolidin-1-yl)-4-(1-(oxetan-3-yl)-pi-

Figure 4. Kinase selectivity of compounds 14 and 24 @ 1 μM (333 × and 250 × DLK Ki, respectively). Values are reported as percent inhibition and determined in a 220 kinase panel at Life Technologies. EGFR* = EGFR(T790M, L858R), RAF1* = RAF1(Y340D, Y341D), MYLK* = MYLK(smMLCK), MYLK3* = MYLK3(caMLCK), and p38_alpha* = p38_alpha(direct). Kinases with ≥50% inhibition for compound 14 are Flt3 (IC50 = 0.709 μM), PAK4 (IC50 = 0.673 μM), STK16 (IC50 = 1.52 μM), and TrkA (IC50 = 1.55 μM). Kinases with ≥50% inhibition for compound 24 are Axl (IC50 = 0.448 μM), CSF1R (IC50 = 1.22 μM), DMPK (IC50 = 1.38 μM), EphA7 (IC50 = 0.665 μM), Flt3 (IC50 = 0.238 μM), and TrkA (IC50 = 0.383 μM). Biochemical IC50 values were determined at Life Technologies. 8088



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Kpuu (brain). The slightly more lipophilic and more potent N-cyclopropylmethyl analog 24 also maintained good bioavailability and brain penetration but with moderate CLp and half-life in rat. Both compounds also displayed good unbound clearance in C57BL/6 mice, with 24 exhibiting a longer half-life. However, based on the superior rat exposure profile and better stability in higher species liver microsomes/hepatocytes, 14 was determined to be the best compound to advance to further studies. Compound 14 was then dosed in cynomolgus monkeys and displayed low plasma clearance (total and unbound), moderate volume of distribution, long half-life, good bioavailability, and Kpuu (CSF/unbound plasma). The cell-based potency, protein binding, and in vivo exposure observed for 14 suggested that sustained DLK inhibition in brain could be achieved at reasonable dose levels. To test this directly, 9−10 month old PS2APP mice24 were dosed orally with either 15 mg kg−1 or 50 mg kg−1 of compound 14. This mouse model of Alzheimer’s Disease displays amyloid plaque pathology as well as aberrant DLK/JNK pathway activity in neurons that reflects what is observed in Alzheimer’s patients and thus represents an excellent model for testing of DLK inhibitors in the context of chronic neurodegeneration.7,24 A single dose of 14 resulted in significantly decreased phosphorylation of the downstream transcription factor c-Jun at 6 h that correlated well with levels of compound in brain (Figure 6A,B), with the 50 mg kg−1 dose resulting in a near complete inhibition of p-c-Jun. Although phosphorylation of c-Jun is multiple steps downstream of DLK, p-c-Jun was selected as the primary marker for in vivo DLK inhibition as c-Jun phosphorylation in the adult brain is highly specific to stressed neurons while the upstream signaling components MKK4/7 and JNK display high levels of basal phosphorylation unrelated to the PS2APP phenotype, which could confound calculations of in vivo potency.3,6,25 Consistent with this, a smaller reduction in p-MKK4 and a trend toward reduced p-JNK was also observed following dosing of 14 (Figure 6C,D). P-c-Jun was reduced by 42% at the 15 mg/kg dose where levels of 14 were 2.09 μM in plasma and 2.79 μM in brain (Figure 6B,E). When corrected for protein binding (plasma = 86.8% and brain = 93.2%), free concentration of 14 was found to be 0.277 μM in plasma and 0.190 μM in brain. Furthermore, when the relationship between free brain concentrations of 14 was based on data from individual animals rather than dose groups (Figure 6D), a strong PK/PD relationship could be observed with an approximate IC50 of 0.190 μM. These results are consistent with the observations of PK/PD studies with two other DLK inhibitors developed at Genentech when tested in a nerve injury model.13,14 In all of these studies, the free drug levels required for 50% inhibition of p-c-Jun were consistent with the cell-based p-JNK IC50 assay when corrected for serum binding. The safety profile of 14 was then evaluated in a 7-day repeatdose study in Sprague−Dawley rats, and the compound was shown to be tolerated at doses up to 75 mg kg−1 (Table 7). Females had higher exposure (∼2-fold by AUC24h and Cmax) than males. DLK inhibitor 14 maintained good brain penetration based on the unbound brain/unbound plasma and CSF/unbound plasma ratios (Kpuu) assessed 24 h after the last dose. Findings related to dosing with inhibitor 14 included dose-dependent effects on body weight (reduced gain at 25 mg kg−1 or loss at 75 mg kg−1). Hematology findings associated with 14 comprised of a dose-dependent decrease in reticulocyte count and immature reticulocyte fraction with no effect on mature red cell mass. At 75 mg kg−1 in males only, decreases in monocyte and eosinophil counts were also observed. The hematological effects

Figure 5. Evaluation of compounds using an in vitro axon degeneration assay. Representative curves for compound 14 (red) and compound 24 (blue) show EC50 values of 0.574 μM and 0.457 μM, respectively. Max activity reflects the extent of axonal protection relative to a positive control where degeneration was not induced as measured by high content imaging of axons followed by automated quantification. Each data point represents averages from three independent assays ± SD.

may also contribute to axonal degeneration in this setup as been observed in other experimental paradigms.13,23 In Vivo Compound Characterization. Based on these promising in vitro results, 14 and 24 were progressed to in vivo studies. Pharmacokinetic (PK) data from these compounds in various preclinical species are shown in Table 6. In Sprague−Dawley rats, Table 6. Pharmacokinetic Properties of DLK inhibitors 24 and 14a 24 CLp (mL min−1 kg−1) CLu (mL min−1 kg−1)b Vd (L kg−1) t1/2 (h) F

Kpuu (AUC) CLp (mL min−1 kg−1) CLu (mL min−1 kg−1)c Vd (L kg−1) t1/2 (h) F

Kpuu (6 h) CLp (mL min−1 kg−1) CLu (mL min−1 kg−1)d Vd (L kg−1) t1/2 (h) F CSF Kpuu (AUC)

Sprague−Dawley Rat PK 31 342 7.3 3.4 63% (1 mg kg−1)

0.908 C57BL/6 Mouse PK 9.3 113 2.7 3.7 93% (15 mg kg−1) 99% (30 mg kg−1) 73% (100 mg kg−1) 0.81 (PO 5 mg kg−1) Cyno PK

14 8.0 45 5.4 8.5 57% (5 mg kg−1) 56% (25 mg kg−1) 49% (75 mg kg−1) 55% (150 mg kg−1) 0.78 24.6 186 3.60 2.05 60% (50 mg kg−1)

0.540 (PO 50 mg kg−1) 10 53 5.1 7.2 103% (1 mg kg−1) 0.710

a

Oral doses were formulated as aqueous suspensions in 1% methylcellulose; intravenous doses were formulated in 10% DMSO, 10% Cremophor EL in saline. bRat PPB: inhibitor 24 = 90.9%; inhibitor 14 = 82.3%. cMouse PPB: inhibitor 24 = 91.8%; inhibitor 14 = 86.8%. dCyno PPB: inhibitor 14 = 80.9%.

N-isopropyl pyrazole 14 showed low plasma clearance (total and unbound), long half-life, good bioavailability, and high 8089



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Figure 6. Single dose of compound 14 reduces p-c-Jun in the PS2APP model of Alzheimer’s disease. (A) Representative Western blot images of p-MKK4, p-JNK, p-c-Jun, and a GAPDH control from cortical lysates of PS2APP mice treated with vehicle (MCT) or compound 14 at 15 or 50 mg/kg PO (n = 7−8 animals per group). (B−D) Quantification of p-MKK4, p-JNK, and p-c-Jun following compound 14 dosing. A significant treatment effect was observed for p-c-Jun at both dose levels while p-MKK4 levels were significantly reduced only at the 50 mg/kg dose as compared to vehicle (****p < 0.0001, **p < 0.01 versus vehicle by Dunnett’s post hoc test, #p < 0.05 by Student’s t test) (C) Total concentration of 14 in brain 6 h postdose. (D) In vivo PK/PD relationship for brain exposure versus p-c-Jun reduction in individual animals from both dose groups determined an IC50 = 0.190 μM.

Table 7. Inhibitor 14 Rat in Vivo Toxicity Study dose (mg kg−1 d−1)

sex

day 7 free plasma AUC0−24h (multiplea)

day 7 free plasma Cmax (multiplea)

day 7 Kpuu at 24 h

25

M (3)

12.2 μM·h (3.9×)

0.81 μM (6.3×)

0.75

F (3)

28.0 μM·h (9.0×)

1.45 μM (11×)

0.59

M (3) F (3)

34.3 μM·h (11×) 67.1 μM·h (22×)

1.88 μM (15×) 3.19 μM (25×)

0.84 0.72

75

treatment-related findingsb reduced body weight, gain decreased RET, IRF increased GLDH, ALT, T.Bili (females only) no histopathological findings body weight loss, decreased RET, IRF, and increased MCHC decreased MON and EOS (males only); increased GLDH, ALT, T.Bili (females only) bone marrow hypocellularity; spleen, thymus, lymph nodes (lymphoid hypocellularity/apoptosis)

a Multiple = Exposure multiple above targeted free AUC24h of 3.096 μM·h or free concentration of 0.129 μM bALT, Alanine aminotransferase; EOS, Eosinophil; GLU, Glucose; GLDH, glutamate dehydrogenase; IRF, Immature reticulocyte fraction; MCHC, Mean corpuscular hemoglobin concentration; MON, Monocyte; RET, Reticulocyte; T.Bili, Total bilirubin.

correlated with hypocellularity of the bone marrow that was observed at 75 mg kg−1. Other findings included lymphocyte hypocellularity/apoptosis in the spleen, thymus, and lymph nodes, and increases in liver transaminases and total bilirubin with no corresponding histopathology. Based on the tight

correlation between IC50 for DLK inhibition in vitro and in vivo as well as the observation that a ∼ 50% reduction in DLK is sufficient to elicit neuroprotection (Figure 5),26 exposure multiples for this study were calculated based on the p-JNK cell IC50 for inhibitor 14 corrected for protein binding in the cell media 8090



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(IC50 = 0.195 μM, 0.129 μM when corrected for bovine serum binding).27 This equated to free AUC24h levels of 3.096 μM·h. On day 7, inhibitor 14 achieved exposure multiples of 11 × AUC and 15 × Cmax in males, and 22 × AUC and 25 × Cmax in females. In summary, DLK inhibitor 14 was tolerated in rats at concentrations well exceeding those required for DLK inhibition. Chemistry. The preparation of compounds 2−9 is shown in Scheme 1. Sandmeyer reaction of tert-butyl 4-(3-amino-1isopropyl-1H-pyrazol-5-yl)piperidine-1-carboxylate (30) resulted in iodopyrazole 31. Subsequent N-Boc deprotection

of intermediates 45−56 with the appropriately substituted 2-aminopyridyl boronates afforded analogs 10−13, 15−21, 23, and 26−29. An alternative route was also developed and is exemplified by the preparation of compounds 14, 22, 24, and 25 in Scheme 3. Amide coupling of (exo)-3-(tert-butoxycarbonyl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid 57 with N,O-dimethylhydroxylamine hydrochloride afforded Weinreb amide 58. Subsequent addition of methylmagnesium bromide obtained methyl ketone 59. Acylation of the lithium enolate of ketone 59 with ethyl oxalate followed by direct treatment of the ethyl 2,3dioxobutanoate with isopropyl hydrazine, ethyl hydrazine, or methylcyclopropyl hydrazine yielded pyrazolyl esters 60−62, regioselectively. The synthesis of aminopyrazoles 66−68 was accomplished by the following sequence: saponification of esters 60−62; Curtius rearrangement of the resulting acids 63−65; and N-Cbz removal by hydrogenolysis afforded 3-aminopyrazoles 69−71. Sandmeyer reaction of amino pyrazoles 69−71 provided 3-iodopyrazoles 72−74. Acid mediated N-Boc deprotection and reductive amination with 3-oxetanone yielded oxetanes 75−77. Final Suzuki−Miyaura cross coupling of iodides 75−77 with the required amino pyridyl boronate furnished compounds 14, 22, 24, and 25.

Scheme 1a

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CONCLUSIONS Building upon the broad research interest in DLK as a potential new therapeutic target for treatment of neurodegenerative conditions, we have further optimized recently described pyrazole inhibitor 1 using a property and structural-based approach. Important to this effort were structure guided variation of key polar contacts with the hinge residues of DLK and introduction of an exo-3-azabicyclo[3.1.0]hexane group that improved potency, kinase selectivity, and CNS pharmacokinetic properties. This effort led to the identification of 14, which has favorable in vitro safety properties and in vivo tolerability, enabling interrogation of DLK inhibition in models of chronic neurodegeneration. The notable in vivo properties of these azabicyclo[3.1.0]hexane pyrazoles have advanced our knowledge of the mechanisms of DLK inhibition and our efforts to progress DLK inhibitors toward clinical study.

a Reagents and conditions: (a) NaI, NaNO2, p-toluenesulfonic acid, CH3CN, H2O, 0 °C, 0.5 h, 60% yield; (b) (i) HCl, 1,4-dioxane, CH3OH, 0−23 °C, 2 h; (ii) 3-oxetanone, NaBH3CN, CH3OH, 50 °C, 3 h, 95% yield; (c) R−(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl), Pd(dppf)Cl2, 1,4-dioxane, H2O, Cs2CO3, 110 °C microwave, 0.5 h.

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followed by reductive amination with 3-oxetanone provided oxetane 32. Suzuki−Miyaura cross coupling with the requisite azaindole or aminopyridine boronate furnished compounds 2−9. The syntheses of 10−13, 15−21, 23, and 26−29 are outlined in Scheme 2. Addition of deprotonated acetonitrile to 3-(tert-butyl) 6-ethyl exo-3-azabicyclo[3.1.0]hexane-3,6-dicarboxylate (33) produced α-cyanoketone 34. Hydrazine addition to nitrile 34 with in situ cyclodehydration afforded 3-aminopyrazole 35, which was converted to the corresponding 3-iodopyrazole 36 via a Sandmeyer reaction. N1-Pyrazole alkylation with 2-bromopropane afforded N-isopropyl 37, which upon deprotection provided amine 38 for further derivatization. Additional pyrazole N1 groups were obtained by alkylation of iodopyrazole 36 with iodomethane, trifluoroethyl triflate, isobutyl bromide, 2,2-dimethyloxirane, cyclobutyl bromide, and cyclopentyl bromide to provide N-methyl 39, trifluoroethyl 40, isobutyl 41, 2-methyl-2-hydroxypropyl 42, cyclobutyl 43, and cyclopentyl 44, respectively. Functionalization of the exo-3-azabicycl[3.1.0]hexane 38 by reductive amination, acylation, and alkylation provided differentially substituted intermediates 45−50. Similarly, acid mediated N-Boc deprotection of 39−44 followed by reductive amination with 3-oxetanone furnished 3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexanes 51−56. Suzuki−Miyaura coupling

EXPERIMENTAL SECTION

Methods. ClogP. Values were calculated using MoKa v2.6.6, available from Molecular Discovery Ltd. pKa. Values were determined by Analiza Inc. using capillary electrophoresis. DLK Biochemical Assay. The biochemical assay was performed as previously described.28 p-JNK Cell Assay. The p-JNK cell assay for generation of IC50 values was performed as previously described.13 In Vitro Axon Degeneration Cell Assay. The assay was conducted as previously described.29 MKK4 and MKK7 Biochemical Assays. The assay was conducted as previously described.14 In Vitro Transporter Assays. The in vitro transporter assays were performed as previously described.13 Life Technologies Kinase Assays. Compounds were tested in Life Technologies’ SelectScreen (Madison, WI) against 220 representative kinases at a concentration of 1 μM, which is 333- and 250-fold greater than the Ki for compounds 14 and 24, respectively, against human recombinant DLK in the DLK enzyme assay. The kinase assays were carried out using Z′-LYTE Technology (Life Technologies, Madison WI), which measured labeled peptide phosphorylation via fluorescence resonance energy transfer (FRET) following protocols developed and performed by Life Technologies.30 8091



Article

Scheme 2a

Reagents and conditions: (a) CH3CN, t-BuOK, THF, 23 °C, 1 h, 94% crude yield; (b) hydrazine monohydrate, i-PrOH, 80 °C, 16 h, 80% yield (2-steps); (c) NaI, NaNO2, p-toluenesulfonic acid monohydrate, CH3CN, H2O, 23 °C, 1 h, 26% yield; (d) 2-bromopropane, Cs2CO3, DMF, 23 °C, 10−16 h, 23% yield; (e) R-I, R-Br, or R-OSO2CF3, Cs2CO3 or K2CO3, DMF or CH3CN, 23 °C, 10−16 h; (f) 2,2-dimethyloxirane, Cs2CO3, DMF, 90 °C, 16 h, 47% yield; (g) (TFA, CH2Cl2) or (HCl, EtOAc) or (HCl, 1,4-dioxane, CH3OH), 23 °C, 1−3 h; (h) 3-oxetanone, NaBH3CN, CH3OH, 23−60 °C, 1−3 h; (i) R-Br or R-OSO2Me, (i-Pr)2NEt, THF or DMF, 40−50 °C, 16 h; (j) Ac2O, (i-Pr)2NEt, 25 °C, 1 h, 75% yield; (k) 2-methyloxirane, CH3OH, 23 °C, 16 h, 55% yield; (l) R-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl), Pd(dppf)Cl2, Cs2CO3, 1,4-dioxane, H2O, 100−120 °C microwave, 0.5 h. a

DiscoverX KINOMEscan Profiling Service. Binding affinities were determined at DiscoverX (Fremont, CA) for LZK and DLK.31 Structure Determination. Protein crystallography was performed as previously described,14 and refinement statistics are included with the PDB deposition. Animal Models. All experiments were performed under animal protocols approved by the Animal Care and Use Committee at Genentech and adhere to ACS Ethical Guidelines for animal studies. For all in vivo studies, PS2APP mice,24 Sprague−Dawley rats, and cynomolgus monkeys were dosed with compound 14 orally as a MCT suspension. Homozygous PS2APP mice (9−10 mo) were given a single oral dose of vehicle or 15 mg kg−1 or 50 mg kg−1 of compound 14, and

terminal plasma was collected from deeply anaesthetized animals via cardiac puncture. The animals were then perfused with PBS, and the brains were extracted. The left cortex and cerebellum were collected and frozen at −80 °C until further processing. The cortex was homogenized in 10V of RIPA buffer for Western blots as described previously.7 The cerebellum was used for brain PK measurements. General. All commercially available reagents and solvents were used as received. Reactions using air or moisture sensitive reagents were performed under an atmosphere of nitrogen using freshly opened EMD DriSolv solvents. Reaction progress was monitored by TLC or LCMS. Flash chromatography was performed with Isco CombiFlash Companion systems using prepacked silica gel columns (40−60 μm particle 8092



Article

Scheme 3a

Reagents and conditions: (a) N,O-dimethylhydroxylamine hydrochloride, HATU, Et3N, DMF, 23 °C, 2 h, 84% yield; (b) MeMgBr, THF, Et2O, 23 °C, 2 h, 89% yield; (c) (i) (CO2Et)2, LiHMDS, 25 °C, 3 h; (ii) isopropylhydrazine or ethylhydrazine or cyclopropylmethylhydrazine, EtOH, 50 °C, 0.3 h; (d) aq. NaOH, EtOH, 23 °C, 2 h; (e) diphenylphosphoryl azide, BnOH, (i-Pr)2NEt, toluene, 90 °C, 4 h; (f) H2 (1 atm), CH3OH, 10% Pd/C, 23 °C, 1.5 h; (g) NaI, NaNO2, p-toluenesulfonic acid, CH3CN, H2O, 0−23 °C, 3 h; (h) (i) (TFA, CH2Cl2) or (HCl, EtOAc), 23 °C, 1 h; (ii) 3-oxetanone, acetic acid, NaBH3CN, CH3OH, 40−60 °C, 2 h; (i) R-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) Pd(dppf)Cl2, K2CO3 or Cs2CO3, CH3CN or 1,4-dioxane, H2O, 105 °C microwave or reflux, 0.25−3.5 h. a

size RediSep or 20−40 μm spherical silica gel RediSep Gold columns, or similar columns from other vendors). Preparative reverse phase HPLC purifications were performed on a Varian Prostar instrument, using a Phenomenex Gemini-NX C-18 (3 cm × 5 cm; 5 μM) stationary phase, with 0.1% aqueous formic acid/acetonitrile or 0.1% aqueous ammonium hydroxide/acetonitrile gradients as the mobile phase (typically 5−85% acetonitrile over 10 min) with a flow rate of 60 mL/min. NMR spectra were measured on Bruker 300, 400, or 500 MHz spectrometers, and chemical shifts were reported in ppm downfield from TMS using residual nondeuterated solvent as internal standards (CHCl3, 7.26 ppm; DMSO, 2.50 ppm; MeOH, 3.31 ppm). The following abbreviations are used: br = broad signal, s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet. The purity of final compounds was verified by HPLC to be >95% in all cases using either of the following methods: (1) Agilent 1200 instrument with an Agilent SB C-18 (2.1 mm × 30 mm; 1.8 μm particle size) stationary phase, and a gradient of water/ acetonitrile (5−95% over 10 min; 0.05% TFA in both phases) at a flow rate of 0.4 mL/min; quantification of target and impurities was done by UV detection at 254 nm; (2) Shimadzu LC-2010A/2020A instrument with an Ultimate C-18 (3.0 mm × 50 mm; 3 μm particle size) stationary phase, and a gradient of water/acetonitrile (10−80% over 6 min then 80% over 2 min; 0.05% TFA in both phases) at a flow rate of 1.2 mL/min and oven temperature of 40 °C; quantification of target and impurities was done by UV detection at 254 nm.

Experimental Details. 3-Fluoro-5-(1-isopropyl-5-(1-(oxetan-3yl)piperidin-4-yl)-1H-pyrazol-3-yl)-1H-pyrrolo[2,3-b]pyridine (2). To a solution of 4-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-1-(oxetan-3-yl)piperidine (32) (80 mg, 0.2 mmol), 3-fluoro-5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (104 mg, 0.40 mmol), and cesium carbonate (139 mg, 0.40 mmol) in 10:1 1,4-dioxane/water (2 mL) was added 1,1′-bis(diphenylphosphino)ferrocene-palladium(II) dichloride (15 mg, 0.02 mmol). The mixture was heated at 110 °C using microwave irradiation. After 30 min, the reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by preparative reverse phase HPLC to afford a white solid (30.5 mg, 40% yield). LCMS: m/z [M + H]+ calcd for C21H27FN5O 384.2, found 384.0; 1 H NMR (400 MHz, DMSO-d6) δ 11.45 (s, 1 H), 8.71−8.70 (m, 1 H), 8.25−8.23 (m, 1 H), 7.43−7.42 (m, 1 H), 6.59 (s, 1 H), 4.57−4.51 (m, 2 H), 4.44−4.41 (m, 1 H), 3.41−3.36 (m, 1 H), 3.07−3.04 (m, 1 H), 2.78−2.69 (m, 2 H), 2.52−2.50 (m, 1 H), 2.48−2.36 (m, 2 H), 1.92−1.81 (m, 3 H), 1.78−1.71 (m, 2 H), 1.42 (d, J = 6.4 Hz, 6 H). 3-Chloro-5-(1-isopropyl-5-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrazol-3-yl)-1H-pyrrolo[2,3-b]pyridine (3). The title compound (2 mg, 6% yield) was prepared in a manner analogous to 2 by substituting 3-chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine for 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)-1H-pyrrolo[2,3-b]pyridine. LCMS: m/z [M + H]+ calcd for C21H27ClN5O 400.2, found 399.9; 1H NMR (400 MHz, CD3OD) δ 8.72 (s, 1 H), 8.32 (s, 1 H), 7.45 (s, 1 H), 6.55 (s, 1 H), 4.76−4.73 (m, 2 H), 8093



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4.68−4.61 (m, 3 H), 3.60−3.57 (m, 1 H), 2.97−2.94 (m, 2 H), 2.88−2.82 (m, 1 H), 2.11−1.99 (m, 4 H), 1.913−1.82 (m, 2 H), 1.55 (d, J = 6.4 Hz, 6 H). 5-(1-Isopropyl-5-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrazol-3-yl)3-methyl-1H-pyrrolo[2,3-b]pyridine (4). The title compound (21.7 mg, 12% yield) was prepared in a manner analogous to 2 by substituting 3methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine for 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)-1H-pyrrolo[2,3-b]pyridine. LCMS: m/z [M + H]+ calcd for C22H30N5O 380.2, found 380.0; 1H NMR (400 MHz, CD3OD) δ 9.03 (s, 1 H), 8.81 (s, 1 H), 7.50 (s, 1 H), 6.88−6.85 (m, 1 H), 4.97−4.88 (m, 2 H), 4.79−4.76 (m, 1 H), 4.18−4.09 (m, 2 H), 3.90−3.82 (m, 1 H), 3.69−3.56 (m, 3 H), 3.25−3.13 (m, 1 H), 3.14 (s, 1 H), 2.46 (s, 3 H), 2.35−2.18 (m, 4 H), 1.57 (d, J = 6.0 Hz, 6 H). 5-(1-Isopropyl-5-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrazol-3-yl)3-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine (5). The title compound (12.8 mg, 6% yield) was prepared in a manner analogous to 2 by substituting 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine for 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine. LCMS: m/z [M + H]+ calcd for C22H27F3N5O 434.2, found 433.9; 1H NMR (400 MHz, CD3OD) δ 8.78 (s, 1 H), 8.40 (s, 1 H), 7.88 (s, 1 H), 6.55 (s, 1 H), 4.79−4.75 (m, 2 H), 4.69−4.64 (m, 3 H), 3.74−3.71 (m, 1 H), 3.07−3.02 (m, 2 H), 2.93−2.87 (m, 1 H), 2.35−2.15 (m, 2 H), 2.06−2.03 (m, 2 H), 1.89−1.86 (m, 2 H), 1.54 (d, J = 6.4 Hz, 6 H). 5-(1-Isopropyl-5-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrazol-3-yl)3-methoxypyridin-2-amine (6). The title compound (5.8 mg, 6% yield) was prepared in a manner analogous to 2 by substituting 3-methoxy-5(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine for 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine. LCMS: m/z [M + H]+ calcd for C20H30N5O2 372.2, found 372.3; 1H NMR (400 MHz, DMSO-d6) δ 7.92 (s, 1H), 7.53 (s, 1H), 7.01 (s, 2H), 6.51 (s, 1H), 4.99−4.47 (m, 5H), 3.94 (s, 3H), 3.06 (s, 1H), 2.49−2.36 (m,4H), 2.01 (s, 5H), 1.42 (d, J = 6.5 Hz, 6H). 5-(1-Isopropyl-5-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrazol-3-yl)3-(trifluoromethyl)-pyridin-2-amine (7). The title compound (24.5 mg, 31% yield) was prepared in a manner analogous to 2 by substituting 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridin-2-amine for 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)-1H-pyrrolo[2,3-b]pyridine. LCMS: m/z [M + H]+ calcd for C20H27F3N5O 410.2, found 410.0; 1H NMR (400 MHz, CD3OD) δ 8.53 (s, 1 H), 8.13 (s, 1 H), 6.42 (s, 1 H), 4.79−4.76 (m, 2 H), 4.72−4.69 (m, 2 H), 4.62−4.55 (m, 1 H), 3.92−3.86 (m, 1 H), 3.18−3.15 (m, 2 H), 2.97−2.87 (m, 1 H), 2.46−2.39 (m, 2 H), 2.06−2.03 (m, 2 H), 1.92−1.82 (m, 2 H), 1.48 (d, J = 6.8 Hz, 6 H). 5-(1-Isopropyl-5-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrazol-3-yl)3-methylpyridin-2-amine (8). The title compound (21.5 mg, 24% yield) was prepared in a manner analogous to 2 by substituting 3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2amine for 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1Hpyrrolo[2,3-b]pyridine. LCMS: m/z [M + H]+ calcd for C20H30N5O 356.2, found 356.3; 1H NMR (400 MHz, DMSO-d6) δ 8.19 (s, 1H), 7.93 (s, 1H), 6.88 (s, 2H), 6.45 (s, 1H), 4.82−4.39 (m, 3H), 2.53−2.44 (m, 9H), 2.17 (s, 3H), 1.97 (d, J = 13.0 Hz, 2H), 1.41 (d, J = 6.5 Hz, 6H). 3-Chloro-5-(1-isopropyl-5-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrazol-3-yl)pyridin-2-amine (9). The title compound (21.9 mg, 30% yield) was prepared in a manner analogous to 2 by substituting 3-chloro5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine for 3fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b] pyridine. LCMS: m/z [M + H]+ calcd for C19H27ClN5O 376.2, found 376.0; 1H NMR (400 MHz, CD3OD) δ 8.33 (s, 1 H), 7.96 (s, 1 H), 6.19 (s, 1 H), 4.84 (s, 2 H), 4.69−4.62 (m, 4 H), 4.42−4.39 (m, 1 H), 3.52−3.49 (m, 1 H), 2.88−2.85 (m, 2 H), 2.64−2.58 (m, 1 H), 1.98−1.90 (m, 4 H), 1.83−1.79 (m, 2 H), 1.51 (d, J = 6.8 Hz, 6 H). 5-(1-Isopropyl-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan6-yl)-1H-pyrazol-3-yl)-3-(trifluoromethyl)pyridin-2-amine (10). To a solution of (exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3yl)-3-azabicyclo[3.1.0]hexane (45) (0.50 g, 1.34 mmol), 5-(4,4,5,5tetramethyl-1,3-dioxolan-2-yl)-3-(trifluoromethyl)pyridin-2-amine (463 mg, 1.61 mmol), and cesium carbonate (655 mg, 2.01 mmol) in

10:1 1,4-dioxane/water (5 mL) was added 1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride (196 mg, 0.27 mmol) under nitrogen. The mixture was heated at 100 °C under microwave irradiation. After 30 min, the reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by flash column chromatography (6% ethyl acetate in hexanes) to afford a white solid (297 mg, 54% yield). LCMS: m/z [M + H]+ calcd for C20H25F3N5O 408.2, found 408.0; 1H NMR (CDCl3, 400 MHz): δ 8.54 (d, J = 2.1 Hz, 1 H), 7.98 (d, J = 2.1 Hz, 1 H), 6.50 (br s, 2 H), 6.39 (s, 1 H), 4.67 (m, 1 H), 4.56 (t, J = 6.6 Hz, 2 H), 4.48 (t, J = 6.0 Hz, 2 H), 3.75 (m, 1 H), 3.12 (d, J = 8.7 Hz, 2 H), 2.42 (m, 2 H), 2.15 (m, 1 H), 1.81 (m, 2 H), 1.42 (d, J = 6.5 Hz, 6 H). 3-(Difluoromethyl)-5-(1-isopropyl-5-((exo)-3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexan-6-yl)-1H-pyrazol-3-yl)pyridin-2-amine formate (11). To a solution of (exo)-6-(3-iodo-1-isopropyl-1H-pyrazol5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane (45) (80 mg, 0.2 mmol), 3-(difluoromethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (85 mg, 0.3 mmol), and cesium carbonate (163 mg, 0.50 mmol) in 5:1 1,4-dioxane/water (4 mL) was added 1,1′-bis (diphenylphosphino)ferrocene-palladium(II)dichloride (15 mg, 0.020 mmol), and the mixture was purged with nitrogen. The mixture was heated at 110 °C under microwave irradiation for 30 min. The reaction mixture was partitioned between ethyl acetate (20 mL) and saturated aqueous sodium chloride solution (15 mL). The organic layer was concentrated. Purification by preparative reverse phase HPLC afforded a white solid (8 mg, 10% yield). LCMS: m/z [M + H]+ calcd for C20H26F2N5O 390.2, found 390.2; 1H NMR (400 MHz, CD3OD) δ 8.40 (s, 1H), 8.16 (s, 1H), 8.04−8.02 (m, 1H), 6.83 (t, J = 55.2 Hz, 1 H), 6.21 (s, 1H), 4.76−4.72 (m, 2H), 4.67−4.65 (m, 2H), 3.94−3.90 (m, 1H), 3.40−3.20 (m, 2H), 2.69−2.66 (m, 2H), 2.32−2.30 (s, 1H), 1.94−1.93 (m, 2H), 1.53−1.51(m, 6H). 5-(1-Isopropyl-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan6-yl)-1H-pyrazol-3-yl)-3-methoxypyridin-2-amine (12). The title compound (19 mg, 19% yield) was prepared in a manner analogous to 11 by substituting 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3(methoxy)pyridin-2-amine for 3-(difluoromethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine. LCMS: m/z [M + H]+ calcd for C20H28N5O2 370.2, found 370.2; 1H NMR (400 MHz, DMSO-d6) δ 7.87 (s, 1H), 7.32 (s, 1H), 6.29 (s, 1H), 5.89 (s, 2H), 4.72−4.46 (m, 5H), 3.84 (s, 3H), 3.18 (s, 2H), 2.48−2.42 (m, 3H), 2.18 (s, 1H), 1.84 (s, 2H), 1.43 (d, J = 6.6 Hz, 6H). 3-(Difluoromethoxy)-5-(1-isopropyl-5-((exo)-3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexan-6-yl)-1H-pyrazol-3-yl)pyridin-2-amine (13). The title compound (13.6 mg, 26% yield) was prepared in a manner analogous to 11 by substituting 3-(difluoromethoxy)-5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine for 3-(difluoromethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2amine. LCMS: m/z [M + H]+ calcd for C20H26F2N5O2 406.2, found 406.0; 1H NMR (400 MHz, CD3OD): δ 8.16 (s, 1 H), 7.74 (s, 1 H), 6.88 (t, J = 73.2 Hz, 1 H), 6.19 (s, 1 H), 4.80−4.72 (m, 3 H), 4.66−4.63 (m, 2 H), 3.85−3.80 (m, 1 H), 3.23 (d, J = 9.2 Hz, 2 H), 2.54−2.52 (m, 2 H), 2.32− 2.30 (m, 1 H), 1.88−1.87 (m, 2 H), 1.54 (d, J = 6.4 Hz, 6 H). 5-(1-Isopropyl-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan6-yl)-1H-pyrazol-3-yl)-3-(trifluoromethoxy)pyridin-2-amine (14). To a suspension of (exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane (75) (45 g, 120 mmol), 5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethoxy)pyridin-2amine (44 g, 144 mmol), and cesium carbonate (59 g, 180 mmol) in 10:1 1,4-dioxane/water (990 mL) was added Pd(dppf)Cl2 (8.82 g, 12 mmol). The mixture was purged with nitrogen for 30 min and heated to reflux for 3.5 h. The reaction was cooled to room temperature and filtered. The filtrate was concentrated in vacuo, and the resulting residue was purified by flash column chromatography (1% methanol in dichloromethane) to afford product (20 g, 39% yield). 1H NMR (400 MHz, CD3OD) δ 8.25 (s, 1 H), 7.83 (s, 1 H), 6.19 (s, 1 H), 4.80−4.70 (m, 3 H), 4.64−4.61 (m, 2 H), 3.83−3.76 (m, 1 H), 3.21 (d, J = 8.8 Hz, 2 H), 2.50 (d, J = 8.8 Hz, 2 H), 2.30−2.28 (m, 1 H), 1.87−1.85 (m, 2 H), 1.52 (d, J = 6.4 Hz, 6 H). 13C NMR (126 MHz, DMSO-d6) δ 151.93, 145.33, 143.50, 143.21, 130.10, 125.11, 121.62, 119.57, 119.12, 99.08, 74.58, 55.99, 50.44, 49.29, 24.38, 22.51, 13.98. HRMS (ESI) m/z: [M + H]+ 8094



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Calcd for C20H25N5O2F3 424.1955; found 424.1944. Anal. Calcd for C20H24N5O2F3: C, 56.73; H, 5.71; N, 16.54. Found C, 56.58; H, 5.60; N, 16.50; Melting point 198.4−199.1 °C. 5-(1-Isopropyl-5-((exo)-3-(oxetan-3-ylmethyl)-3-azabicyclo[3.1.0]hexan-6-yl)-1H-pyrazol-3-yl)-3-(trifluoromethyl)pyridin-2amine (15). The title compound (26 mg, 24% yield) was prepared in a manner analogous to 11 by substituting (exo)-6-(3-iodo-1-isopropyl1H-pyrazol-5-yl)-3-(oxetan-3-ylmethyl)-3-azabicyclo[3.1.0]hexane for (exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexane and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)-3-(trifluoromethyl)pyridin-2-amine for 3-(difluoromethyl)-5(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine. LCMS: m/z [M + H]+ calcd for C21H26F3N5O 422.2, found 422.2; 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 7.97 (s, 1H), 6.50 (s, 2H), 6.37 (s, 1H), 4.74−4.51 (m, 3H), 4.33−4.16 (m, 2H), 3.16−2.95 (m, 3H), 2.74 (d, J = 7.4 Hz, 2H), 2.43−2.30 (m, 2H), 2.15−1.93 (m, 1H), 1.76 (d, J = 2.8 Hz, 2H), 1.41 (d, J = 6.6 Hz, 6H). 1-((Exo)-6-(3-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexan-3-yl)ethanone (16). To a solution of 1-((exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexan-3-yl)ethanone (47) (90.0 mg, 0.25 mmol), 5(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridin-2-amine (134 mg, 0.330 mmol), and cesium carbonate (163 mg, 0.5 mmol) in 6:1 1,4-dioxane/water (3 mL) was added 1,1′bis(diphenylphosphino)ferrocene-palladium(II)dichloride (18 mg, 0.025 mmol). The mixture was heated at 120 °C under microwave irradiation for 30 min. The reaction mixture was concentrated under reduced pressure and purified by preparative reverse phase HPLC to afford a white solid (65 mg, 66% yield). LCMS: m/z [M + H]+ calcd for C19H23F3N5O 394.2, found 394.1; 1H NMR (400 MHz, CD3OD) δ 8.51 (s, 1 H), 8.12 (s, 1 H), 6.28 (s, 1 H), 4.80−4.74 (m, 1 H), 3.93−3.87 (m, 2 H), 3.79−3.75 (m, 1 H), 3.54−3.50 (m, 1 H), 2.10−2.07 (m, 4 H), 2.04−2.00 (m, 1 H), 1.76−1.74 (m, 1 H), 1.51 (d, J = 6.8 Hz, 6 H). 5-(5-((Exo)-3-(2,2-difluoroethyl)-3-azabicyclo[3.1.0]hexan-6-yl)-1isopropyl-1H-pyrazol-3-yl)-3-(trifluoromethyl)pyridin-2-amine (17). To a solution of (exo)-3-(2,2-difluoroethyl)-6-(3-iodo-1-isopropyl-1Hpyrazol-5-yl)-3-azabicyclo[3.1.0]hexane (48) (73 mg, 0.19 mmol), 5(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridin-2-amine (102 mg, 0.38 mmol), and cesium carbonate (125 mg, 0.380 mmol) in 6:1 1,4-dioxane/water (2 mL) was added 1,1′bis(diphenylphosphino)ferrocene-palladium(II)dichloride (14 mg, 0.019 mmol). The mixture was heated at 120 °C under microwave irradiation for 30 min. The reaction mixture was concentrated under reduced pressure. Purification by preparative reverse phase HPLC afforded an off-white solid (38 mg, 48% yield). LCMS: m/z [M + H]+ calcd for C19H23F5N5 416.2, found 416.1; 1H NMR (400 MHz, CD3OD): δ 8.50 (s, 1 H), 8.11 (s, 1 H), 6.22 (s, 1 H), 6.06−5.76 (m, 1 H), 4.78−4.71 (m, 1 H), 3.28 (d, J = 8.8 Hz, 2 H), 2.93−2.84 (m, 2 H), 2.65−2.63 (m, 2 H), 2.22−2.21 (m, 1 H), 1.84−1.82 (m, 2 H), 1.51 (d, J = 6.8 Hz, 6 H). 5-(1-Isopropyl-5-((exo)-3-(2-methoxyethyl)-3-azabicyclo[3.1.0]hexan-6-yl)-1H-pyrazol-3-yl)-3-(trifluoromethyl)pyridin-2-amine (18). The title compound (54 mg, yield 20%).) was prepared in a manner analogous to 11 by using (exo)-6-(3-iodo-1-isopropyl-1Hpyrazol-5-yl)-3-(2-methoxyethyl)-3-azabicyclo[3.1.0]hexane for (exo)6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3(trifluoromethyl)pyridin-2-amine for 3-(difluoromethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine. LCMS: m/z [M + H]+ calcd for C20H27F3N5O 410.2, found 410.0; 1H NMR (400 MHz, CD3OD): δ 8.50 (s, 1 H), 8.10 (s, 1 H), 6.24 (s, 1 H), 4.78−4.75 (m, 1 H), 3.60−3.56 (m, 2 H), 3.49−3.46 (m, 2 H), 3.39 (s, 3 H), 3.01−2.94 (m, 2 H), 2.72−2.68 (m, 2 H), 2.36−1.34 (m, 1 H), 2.02−1.98 (m, 2 H), 1.52 (d, J = 6.4 Hz, 6 H). 1-((Exo)-6-(3-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexan-3-yl)propan-2-ol (19). To a solution of 1-((exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexan-3-yl)propan-2-ol (50) (70 mg, 0.2 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridin-2-amine (75 mg, 0.26 mmol), and cesium carbonate (130 mg,

0.4 mmol) in 6:1 1,4-dioxane/water (2 mL) was added 1,1′-bis (diphenylphosphino)ferrocene-palladium(II)dichloride (14.6 mg, 0.02 mmol). The mixture was heat at 110 °C under microwave irradiation for 30 min. The reaction mixture was concentrated under reduced pressure. Purification by preparative reverse phase HPLC afforded a white solid (23 mg, 27% yield). LCMS: m/z [M + H]+ calcd for C20H27F3N5O 410.2, found 410.1; 1H NMR (400 MHz, CD3OD) δ 8.48 (s, 1H), 8.10 (s, 1H), 6.18 (s, 1H), 4.81−4.76 (m, 1H), 3.86−3.82 (m, 1H), 3.23 (d, J = 9.2 Hz, 2H), 2.57−2.47 (m, 2H), 2.42−2.40 (m, 2H), 2.34−2.32 (m, 1H), 1.79 (s, 2H), 1.50 (d, J = 6.4 Hz, 6H), 1.13 (d, J = 6.4 Hz, 3H). 5-(1-Methyl-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan-6yl)-1H-pyrazol-3-yl)-3-(trifluoromethyl)pyridin-2-amine (20). The title compound (34 mg, 30% yield) was prepared in a manner analogous to 11 by using (exo)-6-(3-iodo-1-methyl-1H-pyrazol-5-yl)-3-(oxetan-3yl)-3-azabicyclo[3.1.0]hexane for (exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridin-2-amine for 3-(difluoromethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine. 1H NMR (400 MHz, CD3OD): δ 8.49 (s, 1 H), 8.09 (s, 1 H), 6.26 (s, 1 H), 4.73−4.70 (m, 2 H), 4.63−4.60 (m, 2 H), 3.89 (s, 3 H), 3.79−3.75 (m, 1 H), 3.23 (d, J = 8.8 Hz, 2 H), 2.50 (d, J = 8.8 Hz, 2 H), 2.29 (s, 1 H), 1.86 (s, 2 H). 5-(1-Methyl-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan-6yl)-1H-pyrazol-3-yl)-3-(trifluoromethoxy)pyridin-2-amine (21). The title compound (17.4 mg, 15% yield) was prepared in a manner analogous to 11 by using (exo)-6-(3-iodo-1-methyl-1H-pyrazol-5-yl)-3(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane for (exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethoxy)pyridin-2-amine for 3-(difluoromethyl)-5-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)pyridin-2-amine. 1H NMR (400 MHz, CD3OD) δ 8.24 (s, 1 H), 7.85 (s, 1 H), 6.23 (s, 1 H), 4.73−4.60 (m, 4 H), 3.88 (s, 3 H), 3.79 (s, 1 H), 3.23 (d, J = 8.8 Hz, 2 H), 2.50 (d, J = 8.8 Hz, 2 H), 2.29 (s, 1 H), 1.86 (s, 2 H). 5-(1-Ethyl-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan-6yl)-1H-pyrazol-3-yl)-3-(trifluoromethyl)pyridin-2-amine (22). A solution of (exo)-6-(1-ethyl-3-iodo-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexane (76) (25 mg, 0.07 mmol), 5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridin-2amine (25 mg, 0.088 mmol), and 1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride (10 mg, 0.013 mmol) in acetonitrile (2 mL) and 1 M aqueous potassium carbonate (1 mL) was heated at 105 °C under microwave irradiation for 15 min. The reaction mixture was concentrated, and the resulting residue was purified by preparativeHPLC to afford a white solid (9.5 mg, 35% yield). LCMS: m/z [M + H]+ calcd for C19H23F3N5O 394.2, found 394.1; 1H NMR (400 MHz, CD3OD): δ 8.50 (s, 1H), 8.10 (s, 1H), 6.24 (s, 1H), 4.74−4.70 (m, 2H), 4.65−4.61 (m, 2H), 4.29−4.22 (m, 2H), 3.85−3.81 (m, 1H), 3.31−3.23 (m, 2H), 2.57−2.54 (m, 2H), 2.30 (s, 1H), 1.89 (s, 2H), 1.47−1.43 (m, 3H). 5-(5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan-6-yl)-1(2,2,2-trifluoroethyl)-1H-pyrazol-3-yl)-3-(trifluoromethyl)pyridin-2amine (23). The title compound (7.6 mg, 8% yield) was prepared in a manner analogous to 11 by using (exo)-6-(3-iodo-1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane for (exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexane and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)-3-(trifluoromethyl)pyridin-2-amine for 3-(difluoromethyl)-5(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine. LCMS: m/z [M + H]+ calcd for C19H20F6N5O 448.2, found 448.1; 1H NMR (400 MHz, CDCl3) δ: 8.56 (dd, J = 2.2, 0.8 Hz, 1H), 8.09 (dd, J = 2.2, 0.7 Hz, 1H), 6.10 (d, J = 0.7 Hz, 1H), 5.03 (s, 2H), 4.78 (q, J = 8.3 Hz, 2H), 4.70 (t, J = 6.7 Hz, 2H), 4.64−4.53 (m, 2H), 3.81 (tt, J = 6.8, 5.8 Hz, 1H), 3.16 (d, J = 8.8 Hz, 2H), 2.50 (d, J = 8.8, 2.0, 0.8 Hz, 2H), 2.25 (t, J = 3.3 Hz, 1H), 1.85−1.76 (m, 2H). 5-(1-(Cyclopropylmethyl)-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan-6-yl)-1H-pyrazol-3-yl)-3-(trifluoromethyl)pyridin-2amine (24). To a suspension of (exo)-6-(1-(cyclopropylmethyl)-3iodo-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane (77) 8095



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C21H25F3N5O 420.2, found 420.2; 1H NMR (400 MHz, CD3OD): δ 8.53 (s, 1 H), 8.14 (s, 1 H), 6.25 (s, 1 H), 5.03−4.99 (m, 1 H), 4.76−4.73 (m, 2 H), 4.66−4.63 (m, 2 H), 3.91−3.87 (m, 1 H), 3.32−3.30 (m, 2 H), 2.78−2.62 (m, 4 H), 2.50−2.43 (m, 2 H), 2.28−2.26 (m, 1 H), 1.95− 1.90 (m, 4 H). 5-(1-Cyclopentyl-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan-6-yl)-1H-pyrazol-3-yl)-3-(trifluoromethyl)pyridin-2-amine (29). The title compound (52 mg, 44% yield) was prepared in a manner analogous to 11 by using (exo)-6-(1-cyclopentyl-3-iodo-1H-pyrazol-5yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane for (exo)-6-(3-iodo-1isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridin-2-amine for 3-(difluoromethyl)-5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)pyridin-2-amine. LCMS: m/z [M + H]+ calcd for C22H27F3N5O 434.2, found 433.9; 1H NMR (400 MHz, CD3OD): δ 8.52 (s, 1 H), 8.12 (s, 1 H), 6.24 (s, 1 H), 4.94−4.90 (m, 1 H), 4.76−4.73 (m, 2 H), 4.67−4.64 (m, 2 H), 3.88−3.82 (m, 1 H), 3.26 (d, J = 9.2 Hz, 2 H), 2.59−2.56 (m, 2 H), 2.34−2.32 (m, 1 H), 2.18− 2.09 (m, 4 H), 2.04−1.98 (m, 2 H), 1.90 (s, 2 H), 1.89−1.74 (m, 2 H). tert-Butyl 4-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)piperidine-1-carboxylate (31). To an ice-cooled solution of tert-butyl 4-(3-amino-1isopropyl-1H-pyrazol-5-yl)piperidine-1-carboxylate (30) (0.60 g, 2.0 mmol) in 8:1 acetonitrile/water (15 mL) were added p-toluenesulfonic acid (1.14 g, 6.00 mmol) and sodium nitrite (0.28 g, 4.0 mmol). After 30 min, sodium iodide (0.60 mg, 4.0 mmol) was slowly added, and the reaction was warmed to 23 °C for 3 h. The reaction mixture was poured into water (50 mL), and the resulting solution was extracted with ethyl acetate (3 × 50 mL). The collected organic phase was washed with saturated aqueous sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification of the resulting residue by flash column chromatography (15% ethyl acetate in petroleum ether) afforded a yellow solid (0.5 g, 60% yield). LCMS: m/z [M + H]+ calcd for C16H27IN3O2 420.1, found 420.1. 4-(3-Iodo-1-isopropyl-1H-pyrazol-5-yl)-1-(oxetan-3-yl)piperidine (32). To an ice-cooled solution of tert-butyl 4-(3-iodo-1-isopropyl-1Hpyrazol-5-yl)piperidine-1-carboxylate (31) (1.5 g, 3.6 mmol) in methanol (20 mL) was added 4 M HCl/1,4-dioxane solution (2.5 mL) in methanol (2.5 mL). The reaction was warmed to 23 °C. After 2 h, the mixture was concentrated, and the resulting residue (1.20 g) was dissolved in methanol (30 mL). Oxetan-3-one (1.35 g, 18.8 mmol) was then added, and the mixture was maintained at 23 °C. After 2 h, sodium cyanoborohydride (0.730 g, 11.3 mmol) was added to the reaction, and the mixture was heated at 50 °C. After 3 h, the reaction mixture was diluted with water (100 mL), and the resulting mixture was extracted with ethyl acetate (3 × 100 mL). The collected organic was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by flash column chromatography (5:1 → 2:1 petroleum ether/ethyl acetate) afforded a white solid (1.34 g, 95% yield). LCMS: m/z [M + H]+ calcd for C14H23IN3O 376.3, found 376.1; 1 H NMR (400 MHz, CDCl3) δ 6.13 (s, 1 H), 4.68−4.60 (m, 4 H), 4.39−4.33 (m, 1 H), 3.52−3.49 (m, 1 H), 2.83 (d, J = 11.6 Hz, 2 H), 2.61−2.53 (m, 1 H), 1.95−1.83 (m, 4 H), 1.78−1.65 (m, 2 H), 1.46 (d, J = 6.8 Hz, 6 H). tert-Butyl (exo)-6-(2-cyanoacetyl)-3-azabicyclo[3.1.0]hexane-3carboxylate (34). To an ice-cooled solution of (exo)-3-tert-butyl 6-ethyl 3-azabicyclo[3.1.0]hexane-3,6-dicarboxylate (33) (27.0 g, 0.106 mol) in tetrahydrofuran (500 mL) and acetonitrile (21.7 g, 0.529 mol) was added potassium tert-butoxide (21.1 g, 0.188 mmol) over 30 min. The resulting mixture was warmed to 23 °C. After 1 h, the reaction mixture was partitioned between 0.5 M aqueous HCl (200 mL) and ethyl acetate (400 mL). The organic phase was separated, and the aqueous layer was extracted with ethyl acetate (2 × 400 mL). The combined organic phase was washed with saturated aqueous sodium chloride solution (150 mL), dried over anhydrous sodium sulfate, and concentrated to afford crude product (25 g, 94% crude yield) which was used without further purification. tert-Butyl (exo)-6-(3-amino-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (35). A solution of crude (exo)-tert-butyl 6-(2-cyanoacetyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (34) (25 g,

(40 g, 0.1 mol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3(trifluoromethyl)pyridin-2-amine (36 g, 0.12 mol), and cesium carbonate (51 g, 0.15 mol) in 10:1 dioxane/water (880 mL) was added 1,1′-bis(diphenylphosphino)ferrocene palladium dichloride (7.3 g, 8.9 mmol). The mixture was purged with nitrogen for 15 min and heated to reflux for 3.5 h. The reaction was cooled to room temperature and filtered. The filtrate was concentrated in vacuo, and the resulting residue was purified by flash column chromatography (1% methanol in dichloromethane) to afford product (23 g, 53%). 1H NMR (CDCl3 400 MHz), δ 8.56 (s, 1H), 8.10 (s, 1H), 6.04 (s, 1H), 5.02 (m, 2H), 4.71 (m, 2H), 4.63 (m, 2H), 4.08 (d, J = 6.8 Hz, 2H), 3.81 (m, 1H), 3.16 (d, J = 8.8 Hz, 2H), 2.50 (m, 2H), 2.27 (t, J = 3.2 Hz, 1H), 1.79 (m, 2H), 1.35 (m, 1H), 0.61 (m, 2H), 0.44 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 154.52, 148.83, 145.32, 144.25, 131.05 (q, J = 4.30 Hz), 125.21 (q, z = 270.47 Hz), 117.85, 105.54 (q, J = 31.0 Hz), 98.57, 74.40, 55.80, 52.71, 50.30, 24.97, 14.19, 11.40, 3.50. HRMS (ESI) m/z: [M + H]+ Calcd for C21H25N5OF3 420.2006; found: 420.2001. Anal. Calcd for C21H24N5OF3·0.5H2O: C, 58.87; H, 5.88; N, 16.35. Found C, 58.85; H, 5.65; N, 16.33; Melting Point 198.6−200 °C. 5-(1-(Cyclopropylmethyl)-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan-6-yl)-1H-pyrazol-3-yl)-3-(trifluoromethoxy)pyridin-2amine (25). The title compound (29.4 mg, 96% yield) was prepared in a manner analogous to 22 by substituting (exo)-6-(1-(cyclopropylmethyl)-3-iodo-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane for (exo)-6-(1-ethyl-3-iodo-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)-3-(trifluoromethoxy)pyridin-2-amine for 5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridin-2-amine. LCMS: m/z [M + H]+ calcd for C21H25F3N5O2 436.2, found 436.1; 1H NMR (400 MHz, CD3OD): δ 8.26 (s, 1 H), 7.83−7.82 (m, 1 H), 6.21 (s, 1 H), 4.72 (t, J = 6.8 Hz, 2 H), 4.62 (t, J = 6.0 Hz, 2 H), 4.09 (d, J = 7.2 Hz, 2 H), 3.83−3.77 (m, 1 H), 3.20 (d, J = 9.2 Hz, 2 H), 2.51 (d, J = 8.0 Hz, 2 H), 2.33−2.31 (m, 1 H), 1.88 (s, 2 H), 1.37−1.31 (m, 1 H), 0.62−0.58 (m, 2 H), 0.47−0.45 (m, 2 H). 5-(1-Isobutyl-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan6-yl)-1H-pyrazol-3-yl)-3-(trifluoromethyl)pyridin-2-amine (26). The title compound (11.9 mg, 11% yield) was prepared in a manner analogous to 11 by using (exo)-6-(3-iodo-1-isobutyl-1H-pyrazol-5-yl)3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane for (exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridin-2-amine for 3-(difluoromethyl)-5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)pyridin-2-amine. 1H NMR (400 MHz, CDCl3): δ 8.56 (s, 1 H), 8.10 (s, 1 H), 6.02 (s, 1 H), 4.96 (s, 2 H), 4.73−4.63 (m, 4 H), 3.98 (d, J = 7.2 Hz, 2 H), 3.84−3.82 (m, 1 H), 3.17 (d, J = 8.8 Hz, 2 H), 2.51−2.24 (m, 4 H), 1.78 (s, 2 H), 0.98 (d, J = 6.8 Hz, 6 H). 1-(3-(6-Amino-5-(trifluoromethyl)pyridin-3-yl)-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan-6-yl)-1H-pyrazol-1-yl)-2-methylpropan-2-ol (27). The title compound (7.3 mg, 8% yield) was prepared in a manner analogous to 11 by using 1-(3-iodo-5-((exo)-3-(oxetan-3yl)-3-azabicyclo[3.1.0]hexan-6-yl)-1H-pyrazol-1-yl)-2-methylpropan2-ol for (exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)3-azabicyclo[3.1.0]hexane and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridin-2-amine for 3-(difluoromethyl)-5(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine. LCMS: m/z [M + H]+ calcd for C21H27F3N5O2 438.2, found 438.1; 1H NMR (400 MHz, CD3OD): δ 8.52 (s, 1 H), 8.12 (s, 1 H), 6.26 (s, 1 H), 4.74−4.67 (m, 2 H), 4.63−4.60 (m, 2 H), 4.18 (s, 2 H), 3.82−3.76 (m, 1 H), 3.20 (d, J = 9.2 Hz, 2 H), 2.50 (d, J = 8.4 Hz, 2 H), 2.41−2.40 (m, 1 H), 1.88 (s, 2 H), 1.27 (s, 6 H). 5-(1-Cyclobutyl-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan-6-yl)-1H-pyrazol-3-yl)-3-(trifluoromethyl)pyridin-2-amine (28). The title compound (5.4 mg, 13% yield) was prepared in a manner analogous to 11 by using (exo)-6-(1-cyclobutyl-3-iodo-1H-pyrazol-5yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane for (exo)-6-(3-iodo-1isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridin-2-amine for 3-(difluoromethyl)-5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)pyridin-2-amine. LCMS: m/z [M + H]+ calcd for 8096



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∼0.1 mol) and hydrazine monohydrate (20 mL) in isopropanol (500 mL) was heated at 80 °C. After 16 h, the reaction was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in dichloromethane (300 mL) and washed sequentially with water (300 mL) and brine (300 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated. Purification by flash column chromatography (ethyl acetate) afforded a white solid (21 g, 80% 2-steps). LCMS: m/z [M + H]+ calcd for C13H21N4O2 265.2, found 265.1. tert-Butyl (exo)-6-(3-iodo-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (36). To an ice-cooled solution of (exo)-tertbutyl 6-(3-amino-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (35) (10.5 g, 39.77 mmol) and p-toluenesulfonic acid hydrate (20.3 g, 119 mmol) in 5:1 acetonitrile/water (120 mL) was added dropwise a solution of sodium nitrite (8.20 g, 119 mmol) and sodium iodide (17.9 g, 119 mmol) in water (10 mL). The mixture was warmed to 23 °C. After 1 h, the reaction mixture was partitioned between water (50 mL) and ethyl acetate (30 mL). The organic was separated and the aqueous was extracted with ethyl acetate (2 × 30 mL). The combined organic was washed with saturated aqueous sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by flash column chromatography (ethyl acetate) afforded a yellow solid (3.8 g, 26% yield). 1H NMR (400 MHz, CDCl3): δ 6.05 (s, 1 H), 3.80−3.66 (m, 2 H), 3.49−3.41 (m, 2 H), 1.96−1.94 (m, 1 H), 1.82−1.80 (m, 1 H), 1.78−1.70 (m, 1 H), 1.46−1.44 (m, 9 H). tert-Butyl (exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexane-3-carboxylate (37). To a solution of (exo)tert-butyl 6-(3-iodo-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (36) (7.00 g, 18.7 mmol) and cesium carbonate (12.2 g, 37.3 mmol) in N,N-dimethylformamide (80 mL) was added 2-bromopropane (4.60 g, 37.3 mmol) at 23 °C. After 10 h, the reaction mixture was diluted with ethyl acetate (60 mL). The solids were filtered, and the filtrate was concentrated under reduced pressure. Purification by flash column chromatography (10% ethyl acetate in petroleum ether) afforded a tan solid (1.8 g, 23% yield). Rf = 0.4 in 20% ethyl acetate in petroleum ether; 1H NMR (400 MHz, CDCl3): δ 5.95 (s, 1 H), 4.60−4.53 (m, 1 H), 3.77−3.74 (m, 1 H), 3.67−3.64 (m, 1 H), 3.47−3.44 (m, 2 H), 1.80−1.75 (m, 2 H), 1.59−1.55 (m, 1 H), 1.48−1.46 (m, 15 H). (Exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane (38). To an ice-cooled solution of (exo)-tert-butyl 6-(3-iodo-1isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (37) (1.0 g, 2.4 mmol) in dichloromethane (20 mL) was added trifluoroacetic acid (3 mL). The resulting mixture was stirred warmed to 23 °C. After 3 h, the mixture was concentrated under reduced pressure. The residue was redissolved in dichloromethane and concentrated under reduced pressure to afford a yellow oil (0.63 g, 81% crude yield), which was used further without purification. tert-Butyl (exo)-6-(3-iodo-1-methyl-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexane-3-carboxylate (39). To a suspension of (exo)-tert-butyl 6-(3-iodo-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane3-carboxylate (36) (1.13 g, 3.00 mmol) and potassium carbonate (459 mg, 3.33 mmol) in acetonitrile (20 mL) was added iodomethane (3.00 g, 21.1 mmol) at 23 °C. After 16 h, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. Purification by flash column chromatography (10% ethyl acetate in petroleum ether) afforded a white solid (200 mg, 39% yield). LCMS: m/z [M + H]+ calcd for C14H21IN3O2 390.1, found 390.1. tert-Butyl (exo)-6-(3-iodo-1-(2,2,2-trifluoroethyl)-1H-pyrazol-5yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (40). A suspension of tert-butyl (exo)-6-(3-iodo-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane3-carboxylate (36) (0.600 g, 1.60 mmol), 2,2,2-trifluoroethyl trifluoromethanesulfonate (742 mg, 3.20 mmol), and cesium carbonate (1.04 g, 3.20 mmol) in N,N-dimethylformamide (6 mL) was heated at 60 °C for 2 h. The reaction mixture was diluted with ethyl acetate (60 mL), and the resulting mixture was sequentially washed with saturated aqueous sodium bicarbonate solution (40 mL), water (20 mL), and saturated aqueous sodium chloride solution (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by flash column chromatography (0 → 15% ethyl acetate in heptane) afforded the a yellow solid (684 mg, 94% yield).

tert-Butyl (exo)-6-(3-iodo-1-isobutyl-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexane-3-carboxylate (41). The title compound (400 mg, 31%) was prepared in a manner analogous to 37 by substituting 1-bromo-2-methylpropane for 2-bromopropane. LCMS: m/z [M + H]+ calcd for C17H27IN3O2 432.1, found 432.2. tert-Butyl (exo)-6-(1-(2-hydroxy-2-methylpropyl)-3-iodo-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (42). A suspension of tert-butyl (exo)-6-(3-iodo-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (36) (200 mg, 0.533 mmol), 2,2-dimethyloxirane (154 mg, 2.14 mmol), and cesium carbonate (347 mg, 1.07 mmol) in N,N-dimethylformamide (5 mL) was heated 90 °C for 16 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure. Purification by flash column chromatography (40% ethyl acetate in petroleum ether) afforded a white solid (111 mg, 47% yield). LCMS: m/z [M + H]+ calcd for C17H27IN3O3 448.1, found 447.9. tert-Butyl (exo)-6-(1-cyclobutyl-3-iodo-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexane-3-carboxylate (43). The title compound (350 mg, 16% yield) was prepared in a manner analogous to 37 by substituting bromocyclobutane for 2-bromopropane. Rf = 0.3 in 12:1 petroleum ether/ethyl acetate; 1H NMR (400 MHz, CDCl3): δ 5.97 (s, 1 H), 4.81−4.76 (m, 1 H), 3.79−3.65 (m, 2 H), 3.47−3.44(m, 2 H), 2.74−2.70 (m, 2 H), 2.42−2.34 (m, 2 H), 1.90−1.54 (m, 5 H), 1.47 (s, 9 H). tert-Butyl (exo)-6-(1-cyclopentyl-3-iodo-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexane-3-carboxylate (44). The title compound (810 mg, 30% yield) was prepared in a manner analogous to 37 by substituting bromocyclopentane for 2-bromopropane. Rf = 0.4 in 6:1 petroleum ether/ethyl acetate; 1H NMR (400 MHz, CDCl3): δ 5.95 (s, 1 H), 4.60−4.53 (m, 1 H), 3.77−3.74 (m, 1 H), 3.67−3.64 (m, 1 H), 3.47−3.44 (m, 2 H), 1.80−1.75 (m, 2 H), 1.59−1.55 (m, 1 H), 1.48−1.46 (m, 15 H). (Exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexane (45). To a solution of crude (exo)-6-(3-iodo1-isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane (38) (0.78 g, ∼2.46 mmol) and triethylamine (0.2 mL) in methanol (10 mL) was added oxetan-3-one (0.880 g, 12.3 mmol) at 23 °C for 1 h before the addition of sodium cyanoborohydride (774 mg, 12.3 mmol). After 3 h, the mixture was partitioned between water (15 mL) and ethyl acetate (20 mL). The aqueous layer was separated and extracted with ethyl acetate (2 × 20 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by flash column chromatography (10% ethyl acetate in petroleum ether) afforded a white solid (0.62 g, 68% 2-steps). LCMS: m/z [M + H]+ calcd for C14H21IN3O 374.1, found 373.8. (Exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-ylmethyl)-3-azabicyclo[3.1.0]hexane (46). A solution of (exo)-6-(3-iodo-1isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane (38) (0.500 g, 1.58 mmol), 3-(bromomethyl)oxetane (476 mg, 3.15 mmol), and N,N-diisopropylethylamine (2.75 mL, 15.8 mmol) in N,N-dimethylformamide (5 mL) was heated at 50 °C for 16 h. The reaction mixture was concentrated under reduced pressure to afford a yellow oil (611 mg, 99% crude yield), which was used without further purification. LCMS: m/z [M + H]+ calcd for C15H23IN3O 388.1, found 388.3. 1-((Exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexan-3-yl)ethanone (47). To a solution of (exo)-6-(3-iodo-1isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane (38) (106 mg, 0.33 mmol) and N,N-diisopropylethylamine (0.12 mL, 0.67 mmol) in dichloromethane (2 mL) was added acetic anhydride (68 mg, 0.67 mmol) at 23 °C. After 1 h, the mixture was concentrated, and the resulting residue was purified by preparative TLC (70% ethyl acetate in petroleum ether) to afford an off-white solid (90 mg, 75% yield). LCMS: m/z [M + H]+ calcd for C13H19IN3O 360.1, found 360.1. (Exo)-3-(2,2-difluoroethyl)-6-(3-iodo-1-isopropyl-1H-pyrazol-5yl)-3-azabicyclo[3.1.0]hexane (48). A solution of (exo)-6-(3-iodo-1isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane (38) (83 mg, 0.26 mmol), 2,2-difluoroethyl trifluoromethanesulfonate (280 mg, 1.31 mmol), and N,N-diisopropylethylamine (0.10 mL, 0.52 mmol) in tetrahydrofuran (5 mL) was heated at 40 °C. After 14 h, the reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by preparative TLC (30% ethyl acetate in 8097



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petroleum ether) to afford a white solid (73 mg, 73% yield). LCMS: m/z [M + H]+ calcd for C13H19F2IN3 382.1, found 381.9. (Exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(2-methoxyethyl)3-azabicyclo[3.1.0]hexane (49). The title compound (145 mg, 89% crude yield) was prepared in a manner analogous to (exo)-6-(3-iodo-1isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-ylmethyl)-3-azabicyclo[3.1.0]hexane (46) by substituting 1-bromo-2-methoxyethane for 3-(bromomethyl)oxetane. LCMS: m/z [M + H]+ calcd for C14H23IN3O 376.1, found 376.1. 1-((Exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexan-3-yl)propan-2-ol (50). To a solution of (exo)-6-(3-iodo1-isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane (38) (342 mg, 1.08 mmol) in methanol (5 mL) was added 2-methyloxirane (189 mg, 3.24 mmol) at 23 °C. After 16 h, the reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by flash column chromatography (30% ethyl acetate in petroleum ether) to afford a white solid (220 mg, 55% yield). LCMS: m/z [M + H]+ calcd for C14H23IN3O 376.1, found 375.8. (Exo)-6-(3-iodo-1-methyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexane (51). To an ice-cooled solution of tert-butyl (exo)-6-(3-iodo-1-methyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane3-carboxylate (39) (550 mg, 1.41 mmol) in ethyl acetate (10 mL) was added 4 M hydrogen chloride in ethyl acetate (5 mL) at 23 °C. After 1 h, the reaction mixture was concentrated to afford crude amine (408 mg), which was used without further purification. LCMS (ESI): m/z = 290.1 [M + H]+. To a solution of crude (exo)-6-(3-iodo-1-methyl-1H-pyrazol5-yl)-3-azabicyclo[3.1.0]hexane hydrochloride (408 mg) in methanol (20 mL) were added triethylamine (0.5 mL), oxetan-3-one (508 mg, 7.06 mmol), and acetic acid (0.5 mL). The reaction was heated at 50 °C for 1 h. After cooling to 23 °C, sodium cyanoborohydride (133 mg, 2.10 mmol) was added, and the mixture was heated at 60 °C for 1 h. The reaction mixture was concentrated, and the resulting residue was dissolved in ethyl acetate (30 mL). The organic solution was washed with saturated aqueous sodium chloride solution (15 mL) and concentrated. Purification by preparative reverse phase HPLC afforded an off-white solid (320 mg, 66% yield). LCMS: m/z 346.1 [M + H]+. (Exo)-6-(3-iodo-1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane (52). The title compound (88 mg, 44% yield) was prepared in a manner analogous to 51 by substituting tert-butyl (exo)-6-(3-iodo-1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexane-3-carboxylate for tert-butyl (exo)-6-(3-iodo-1methyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate. LCMS: m/z [M + H]+ calcd for C13H16F3IN3O 414.0, found 414.2. (Exo)-6-(3-iodo-1-isobutyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexane (53). The title compound (330 mg, 28% yield) was prepared in a manner analogous to 51 by substituting tertbutyl (exo)-6-(3-iodo-1-isobutyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate for tert-butyl (exo)-6-(3-iodo-1-methyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate. LCMS: m/z [M + H]+ calcd for C15H23IN3O 388.1, found 388.2. 1-(3-iodo-5-((exo)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexan-6yl)-1H-pyrazol-1-yl)-2-methylpropan-2-ol (54). To a round-bottomed flask charged with tert-butyl (exo)-6-(1-(2-hydroxy-2-methylpropyl)-3iodo-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (42) (111 mg, 0.248 mmol) was added 4 M hydrogen chloride in 1,4-dioxane (4 mL) at 23 °C. After 1 h, the reaction mixture was concentrated to afford an off-white solid (86 mg), which was used without further purification. LCMS: m/z = 347.8 [M + H]+. To a solution of 1-(5((exo)-3-azabicyclo[3.1.0]hexan-6-yl)-3-iodo-1H-pyrazol-1-yl)-2methylpropan-2-ol hydrochloride (86 mg) in methanol (5 mL) were added oxetan-3-one (54 mg, 0.74 mmol) and acetic acid (0.1 mL). The mixture was heated at 60 °C for 30 min. After cooling to 23 °C, sodium cyanoborohydride (47 mg, 0.74 mmol) was added, and the mixture was heated at 60 °C for 1.5 h. The reaction mixture was concentrated, and the resulting residue was purified by preparative-TLC (70% ethyl acetate in petroleum ether) to afford a white solid (88 mg, 89% yield). LCMS: m/z [M + H]+ calcd for C15H23IN3O2 404.1, found 403.8. (Exo)-6-(1-cyclobutyl-3-iodo-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexane (55). The title compound (250 mg, 80% yield) was prepared in a manner analogous to 51 by substituting

tert-butyl (exo)-6-(1-cyclobutyl-3-iodo-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate for tert-butyl (exo)-6-(3-iodo-1-methyl1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate. LCMS: m/z [M + H]+ calcd for C15H21IN3O 386.1, found 385.9. (Exo)-6-(1-cyclopentyl-3-iodo-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexane (56). The title compound (138 mg, 97% yield) was prepared in a manner analogous to 51 by substituting tertbutyl (exo)-6-(1-cyclopentyl-3-iodo-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate for tert-butyl (exo)-6-(3-iodo-1-methyl1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate. LCMS: m/z [M + H]+ calcd for C16H23IN3O 400.1, found 400.0. tert-Butyl (exo)-6-(methoxy(methyl)carbamoyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (58). To a solution of 3-(tert-butoxycarbonyl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid (57) (27 g, 0.12 mol), N,O-dimethylhydroxylamine hydrochloride (11.7 g, 0.12 mol), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (45 g, 0.12 mol) in N,N-dimethylformamide (300 mL) was added triethylamine (36 g, 0.36 mol) at 23 °C. After 2 h, the reaction mixture was evaporated under reduced pressure, and the resulting residue was diluted with ethyl acetate (300 mL). The organic phase was sequentially washed with aqueous 1 N sodium hydroxide solution (2 × 100 mL), water, and saturated aqueous sodium chloride solution. The collected organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by flash column chromatography (20% ethyl acetate in petroleum ether) afforded a white solid (27 g, 84% yield). 1H NMR (400 MHz, CDCl3) δ: 3.74 (s, 3H), 3.68−3.65 (m, 1H), 3.58−3.55 (m, 1H), 3.48−3.44 (m, 2H), 3.20 (s, 3H), 2.09−2.04 (m, 2H), 1.97 (s, 1H), 1.45 (s, 9H). tert-Butyl (exo)-6-acetyl-3-azabicyclo[3.1.0]hexane-3-carboxylate (59). To an ice-cooled solution of tert-butyl (exo)-6-(methoxy(methyl)carbamoyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (58) (27 g, 0.1 mol) in tetrahydrofuran (500 mL) was added dropwise a solution of 3 M methylmagnesium bromide in diethyl ether (167 mL, 0.5 mol). The mixture was warmed to 23 °C. After 2 h, the reaction mixture was diluted with saturated aqueous ammonium chloride solution (250 mL), and the resulting mixture was extracted with ethyl acetate (2 × 300 mL). The combined organic phase was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by flash column chromatography (10% ethyl acetate in petroleum ether) afforded an offwhite solid (20 g, 89% yield). 1H NMR (400 MHz, CDCl3) δ: 3.68−3.66 (m, 1H), 3.60−3.56 (m, 1H), 3.43−3.40 (m, 2H), 2.26 (s, 3H), 2.09−2.07 (m, 2H), 1.83−1.81 (m, 1H), 1.45 (s, 9H). tert-Butyl (exo)-6-(3-(ethoxycarbonyl)-1-isopropyl-1H-pyrazol-5yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (60). To an ice-cooled solution of tert-butyl (exo)-6-acetyl-3-azabicyclo[3.1.0]hexane-3-carboxylate (59) (137 g, 610 mmol) and diethyl oxalate (134 g, 916 mmol) in tetrahydrofuran (1.26 L) was added dropwise lithium bis(trimethylsilyl)amide (1.2 L, 1.2 mol, 1.0 M in tetrahydrofuran). The mixture was warmed to 23 °C. After 1 h, the reaction mixture was poured into 0.5 M hydrochloric acid (20 L). The resulting mixture was extracted with ethyl acetate (3 × 2 L). The combined organic was washed with saturated aqueous sodium chloride solution (2 L), dried over anhydrous sodium sulfate, filtered, and concentrated. To a solution of the resulting residue (250 g) in ethanol (3.05 L) was added isopropylhydrazine hydrochloride (216 g, 1.95 mol) at 23 °C. After 13 h, the reaction was concentrated in vacuo. This sequence was repeated four additional times on the same scale. The combined material was purified by flash column chromatography (6 → 12% ethyl acetate in petroleum ether) to afford a yellow solid (770 g, 69% yield). 1H NMR (400 MHz, CDCl3): δ 6.35 (s, 1H), 4.71−4.61 (m, 1H), 4.34 (q, J = 7.2 Hz, 2H), 3.78−3.63 (m, 2H), 3.45 (br. d., J = 11.2 Hz, 2H), 1.88−1.74 (m, 3H), 1.59 (t, J = 3.2 Hz, 1H), 1.51 (d, J = 6.8 Hz, 7H), 1.44 (s, 10H), 1.34 (t, J = 7.2 Hz, 3H). tert-Butyl (exo)-6-(3-(ethoxycarbonyl)-1-ethyl-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexane-3-carboxylate (61). To a solution of tertbutyl (exo)-6-acetyl-3-azabicyclo[3.1.0]hexane-3-carboxylate (59) (2.5 g, 11 mmol) and diethyl oxalate (2.4 g, 0.020 mol) in tetrahydrofuran (50 mL) was added lithium bis(trimethylsilyl)amide (22 mL, 66.6 mmol, 3.0 M in tetrahydrofuran) at −70 °C. The mixture was warmed to 23 °C. 8098



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tert-Butyl (exo)-6-(3-(((benzyloxy)carbonyl)amino)-1-ethyl-1Hpyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (67). To a solution of (exo)-5-(3-(tert-butoxycarbonyl)-3-azabicyclo[3.1.0]hexan6-yl) −1-ethyl-1H-pyrazole-3-carboxylic acid (64) (1.5 g, 4.8 mmol), N,N-diisopropylethylamine (1.1 mL, 6.3 mmol), and benzyl alcohol (2.6 g, 24 mmol) in toluene (30 mL) at 90 °C was added dropwise a solution of diphenylphosphorazidate (1.98 g, 7.2 mmol) in toluene (5 mL). Upon complete addition of DPPA, the reaction mixture was maintained at 90 °C for 4 h. The reaction mixture was concentrated in vacuo, and the resulting residue was purified by flash column chromatography (20% ethyl acetate in petroleum ether) to afford a white solid (1.4 g, 70% yield). 1H NMR (400 MHz, CDCl3): δ 7.52 (s, 1H), 7.39−7.34 (m, 5H), 6.11 (s, 1H), 5.18 (s, 2H), 4.06−4.00 (m, 2H), 3.78−3.64 (m, 2H), 3.49−3.45 (m, 2H), 1.88−1.79 (m, 2H), 1.59−1.56 (m, 1H), 1.48 (s, 9H), 1.34−1.32 (m, 6H). tert-Butyl (exo)-6-(3-(benzyloxycarbonylamino)-1-(cyclopropylmethyl)-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (68). The title compound (50 g, 99% crude yield) was prepared in a manner analogous to 67 substituting 5-((exo)-3-(tert-butoxycarbonyl)3-azabicyclo[3.1.0]hexan-6-yl)-1-(cyclopropylmethyl)-1H-pyrazole-3carboxylic acid for (exo)-5-(3-(tert-butoxycarbonyl)-3-azabicyclo[3.1.0]hexan-6-yl)-1-ethyl-1H-pyrazole-3-carboxylic acid. The material was used without purification. 1H NMR (400 MHz, CDCl3): δ 7.76 (s, 1H), 7.32−7.37 (m, 5H), 6.12 (s, 1H), 5.18 (m, 2H), 3.85−3.86 (m, 2H), 3.63−3.75 (m, 2H), 3.45−3.48 (m, 2H), 1.80−1.88 (m, 2H), 1.53 (m, 1H), 1.47 (s, 9H), 1.13−1.19 (m, 1H), 0.51−0.55 (m, 2H), 0.28−0.30 (m, 2H). tert-Butyl (exo)-6-(3-amino-1-isopropyl-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexane-3-carboxylate (69). A solution of tert-butyl (exo)-6-(3-(((benzyloxy)carbonyl)amino)-1-isopropyl-1H-pyrazol-5yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (66) (77 g, 1.7 mol) and 10% palladium on carbon (10 g) in methanol (1.2 L) was stirred under hydrogen (50 psi) at room temperature. After 1 h, the reaction was filtered, and the filtrate was concentrated in vacuo. This reaction was performed 10× to provide crude product as a colorless oil (510 g), which was without further purification. LCMS: m/z [M + H]+ calcd for C16H27N4O2 307.2, found 307.2. tert-Butyl (exo)-6-(3-amino-1-ethyl-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexane-3-carboxylate (70). To a solution of tertbutyl (exo)-6-(3-(((benzyloxy)carbonyl)amino)-1-ethyl-1H-pyrazol-5yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (67) (1.3 g, 3.0 mmol) in methanol (15 mL) was added Pd/C (200 mg, 10% on carbon). The mixture was stirred under hydrogen (1 atm) at 23 °C for 1.5 h. The reaction mixture was filtered, and the filtrate was concentrated in vacuo to afford a colorless oil (850 mg, 80% crude yield), which was used without further purification. LCMS: m/z [M + H]+ calcd for C15H25N4O2 293.2, found 293.2. tert-Butyl (exo)-6-(3-amino-1-(cyclopropylmethyl)-1H-pyrazol-5yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (71). The title compound (50 g, 87% yield) was prepared in a manner analogous to 70 and substituting tert-butyl (exo)-6-(3-(benzyloxycarbonylamino)-1(cyclopropylmethyl)-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3carboxylate for tert-butyl (exo)-6-(3-(((benzyloxy)carbonyl)amino)-1ethyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate. 1H NMR (400 MHz, DMSO-d6) δ 5.05−5.07 (s, 1H), 4.37 (m, 2H), 3.72−3.74 (m, 2H), 3.52−3.54 (m, 2H), 3.36−3.39 (m, 2H), 1.75 (m, 2H), 1.51−1.53 (m, 1H), 1.39 (s, 9H), 1.11−1.16 (m, 1H), 0.42−0.47 (m, 2H), 0.25−0.28 (m, 2H). tert-Butyl (exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexane-3-carboxylate (72). To a solution of tertbutyl (exo)-6-(3-amino-1-isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (69) (125 g, 408 mmol) and p-toluenesulfonic acid mono hydrate (233 g, 1.22 mol) in acetonitrile (2.36 L) and water (602 mL) at 10 °C was added dropwise a solution of sodium nitrite (70.4. 1.02 mmol) and sodium iodide (153 g, 1.02 mol) in water (602 mL). Upon complete addition of reagents, the reaction was warmed to 23 °C. After 1 h, the reaction was cooled to 0 °C and diluted with sodium sulfite aqueous solution (500 mL). The resulting mixture was extracted with ethyl acetate (3 × 2 L). The combined organic was washed with saturated aqueous sodium chloride solution (1 L),

After 3 h, the reaction mixture was poured into 0.5 M hydrochloric acid (100 mL), and the resulting mixture was extracted with ethyl acetate (2 × 50 mL). The combined organic phase was washed with saturated aqueous sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. A solution of the resulting residue and ethylhydrazine (1.5 g, 25 mmol) in ethanol (40 mL) was heated at 50 °C. After 20 min, the reaction was concentrated in vacuo. Purification by flash column chromatography (20% ethyl acetate in petroleum ether) afforded a yellow solid (2.6 g, 69% yield). 1H NMR (400 MHz, CDCl3): δ 6.39 (s, 1H), 4.40−4.25 (m, 4H), 3.78−3.68 (m, 2H), 3.51−3.47 (m, 2H), 1.85−1.60 (m, 2H), 1.49−1.47 (m, 1H), 1.46−1.36 (m, 15H). tert-Butyl (exo)-6-(1-(cyclopropylmethyl)-3-(ethoxycarbonyl)-1Hpyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (62). The title compound (260 g, 52% yield) was prepared in a manner analogous to 61 by substituting (cyclopropylmethyl)hydrazine for ethylhydrazine. 1 H NMR (400 MHz, CDCl3): δ 6.40 (s, 1H), 4.36−4.41 (m, 2H), 4.11−4.12 (m, 2H), 3.66−3.77 (m, 2H), 3.47−3.51 (m, 2H), 1.83−1.88 (m, 2H), 1.63−1.65 (m, 1H), 1.48 (m, 9H), 1.34−1.40 (m, 3H), 1.32 (m, 1H), 0.58−0.63 (m, 2H), 0.41−0.43 (m, 2H). 5-((Exo)-3-(tert-butoxycarbonyl)-3-azabicyclo[3.1.0]hexan-6-yl)1-isopropyl-1H-pyrazole-3-carboxylic acid (63). To a solution tertbutyl (exo)-6-(3-(ethoxycarbonyl)-1-isopropyl-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexane-3-carboxylate (60) (385 g, 1.06 mol) in ethanol (2.54 L) was added 6 M aqueous sodium hydroxide solution (794 mL) at room temperature. After 30 min, ethanol was removed from the reaction in vacuo, and the remaining solution was diluted with water (4 L). The aqueous solution was acidified to pH = 2 with 2 M hydrochloric acid. The resulting solids were filtered, rinsed with water (2 L), and dried. This reaction was performed twice to afford crude product (710 g, 99% yield), which was used without further purification. LCMS: m/z [M + H]+ calcd for C17H26N3O4 336.2, found 336.2. 5-((Exo)-3-(tert-butoxycarbonyl)-3-azabicyclo[3.1.0]hexan-6-yl)1-ethyl-1H-pyrazole-3-carboxylic acid (64). To a solution of tert-butyl (exo)-6-(3-(ethoxycarbonyl)-1-ethyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (61) (2.64 g, 7.5 mmol) in ethanol (30 mL) was added 7.6 M aqueous sodium hydroxide (5 mL) at 23 °C. After 2 h, the organic was removed under reduced pressure, and the remaining aqueous solution was diluted with water (10 mL). The aqueous solution was acidified to pH = 2 with 2 M hydrochloric acid. The mixture was extracted with ethyl acetate (2 × 30 mL). The combined organic was washed with brine, dried over sodium sulfate, filtered, and concentrated to afford a brown solid (1.5 g, 63% crude yield), which was used without further purification. 1H NMR (400 MHz, DMSO-d6): δ 12.5 (br, 1H), 6.38 (s, 1H), 4.2−4.19 (m, 2H), 3.61−3.56 (m, 2H), 3.43−3.38 (m, 2H), 1.93−1.90 (m, 2H), 1.74−1.71 (m, 1H), 1.40 (s, 9H), 1.36−1.32 (m, 3H). 5-((Exo)-3-(tert-butoxycarbonyl)-3-azabicyclo[3.1.0]hexan-6-yl)1-(cyclopropylmethyl)-1H-pyrazole-3-carboxylic acid (65). The title compound (420 g, 87% yield) was prepared in a manner analogous to 64 using (exo)-tert-butyl 6-(1-(cyclopropylmethyl)-3-(ethoxycarbonyl)1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate for (exo)tert-butyl 6-(3-(ethoxycarbonyl)-1-ethyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate. 1H NMR (400 MHz, CDCl3): δ 6.37 (s, 1H), 4.07−4.09 (m, 2H), 3.57 (m, 2H), 3.43 (m, 2H), 1.93 (m, 2H), 1.74 (m, 1H), 1.39 (s, 9H), 1.24 (m, 1H), 0.50−0.52 (m, 2H), 0.35−0.36 (m, 2H). tert-Butyl (exo)-6-(3-(((benzyloxy)carbonyl)amino)-1-isopropyl1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (66). To a solution of 5-((exo)-3-(tert-butoxycarbonyl)-3-azabicyclo[3.1.0]hexan-6-yl)-1-isopropyl-1H-pyrazole-3-carboxylic acid (63) (355 g, 1.06 mol), N,N-diisopropylethylamine (277 mL, 1.59 mmol), and benzyl alcohol (343 g, 3.18 mol) in toluene (3 L) at 100 °C was added dropwise diphenyl phosphorylazide (437 g, 1.59 mmol) in toluene (500 mL). The resulting reaction mixture was stirred at 100 °C. After 3 h, the reaction was concentrated in vacuo. The residues from two reactions performed on the same scale were combined and purified by flash column chromatography (9 → 25% ethyl acetate in petroleum ether) to afford a yellow solid (770 g, 82% yield). LCMS: m/z [M + H]+ calcd for C24H33N4O4 441.3, found 441.1. 8099



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dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. Purification by flash column chromatography (6 → 9% ethyl acetate in petroleum ether) afforded a yellow oil (400 g, 58% yield). 1H NMR (400 MHz, CDCl3): δ 5.94 (s, 1H), 4.55 (m, 1H), 3.73 (br. d., J = 11.2 Hz, 1H), 3.64 (br. d., J = 11.2 Hz, 1H), 3.44 (br. d., J = 11.2 Hz, 2H), 1.76 (br. d., J = 19.2 Hz, 2H), 1.54 (t, J = 3.2 Hz, 1H), 1.47−1.40 (m, 15H). tert-Butyl (exo)-6-(1-ethyl-3-iodo-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (73). To a solution of tert-butyl 6-(3amino-1-ethyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (70) (0.600 g, 2.05 mmol) and p-toluenesulfonic acid (1.16 g, 6.10 mmol) in acetonitrile (20 mL) and water (2 mL) at 23 °C was added dropwise a solution of sodium nitrite (350 mg. 5.1 mmol) and sodium iodide (770 mg, 5.1 mmol) in water (5 mL). After 3 h, the reaction was diluted with ethyl acetate (50 mL). The aqueous layer was separated and extracted with ethyl acetate (2 × 20 mL). The combined organic phase was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. Purification by flash column chromatography (10% ethyl acetate in petroleum ether) afforded a brown oil (0.50 g, 75% yield). 1 H NMR (400 MHz, CDCl3): δ 5.97 (s, 1H), 4.22−4.16 (m, 2H), 3.79− 3.66 (m, 2H), 3.51−3.45 (m, 2H), 1.82−1.78 (m, 2H), 1.52−1.42 (m, 13H). tert-Butyl (exo)-6-(1-(cyclopropylmethyl)-3-iodo-1H-pyrazol-5yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (74). The title compound (70 g, 41% yield) was prepared in a manner analogous to 73 substituting tert-butyl (exo)-6-(3-amino-1-(cyclopropylmethyl)-1Hpyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate for tert-butyl (exo)-6-(3-amino-1-ethyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane3-carboxylate. Purification by flash chromatography (6 → 9% ethyl acetate in petroleum ether) provided product. 1H NMR (400 MHz, CDCl3) δ 5.96 (s, 1H), 3.99−4.06 (m, 2H), 3.64−3.76 (m, 2H), 3.45−3.51 (m, 2H), 1.78−1.81 (m, 2H), 1.56−1.58 (m, 1H), 1.45 (s, 9H), 1.23−1.26 (m, 1H), 0.55−0.58 (m, 2H), 0.34−0.37 (m, 2H). (Exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexane (75). To an ice-cooled solution of (tert-butyl (exo)-6-(3-iodo-1-isopropyl-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (72) (0.20 kg, 480 mmol) in ethyl acetate (1 L) was added hydrogen chloride in ethyl acetate (0.50 L, 1.2 mol). The mixture was warmed to room temperature. After 2 h, the reaction was concentrated. To a solution of the crude material in methanol (2.1 L) were added triethylamine (334 mL, 2.41 mol) and oxetan-3-one (94.7 g, 1.31 mol) at 10 °C. After 30 min, sodium cyanoborohydride (82.6 g, 1.31 mol) was added portionwise, and the resulting mixture was warmed to room temperature for 3 h. The reaction mixture was diluted with water (700 mL) and extracted with ethyl acetate (3 × 2 L). The collected organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. Purification by flash column chromatography (9 → 20% ethyl acetate in petroleum ether) afforded a white solid (55 g, 31% yield). 1H NMR (400 MHz, CDCl3) δ 5.87 (s, 1H), 4.79−4.73 (m, 1H), 4.68−4.50 (m, 7H), 3.71 (m, 1H), 3.06 (d, J = 8.8 Hz, 2H), 2.39 (m, 2H), 2.14 (t, J = 3.2 Hz, 1H), 1.42 (d, J = 6.8 Hz, 5H), 1.46−1.38 (m, 1H). (Exo)-6-(1-Ethyl-3-iodo-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3azabicyclo[3.1.0]hexane (76). The title compound (0.20 g, 64% yield) was prepared in a manner analogous to 77 substituting tert-butyl (exo)6-(1-ethyl-3-iodo-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate for tert-butyl (exo)-6-(1-(cyclopropylmethyl)-3-iodo-1H-pyrazol-5-yl)-3-azabicyclo[3.1.0]hexane-3-carboxylate. LCMS: m/z [M + H]+ calcd for C13H19IN3O 361.1, found 361.7. (Exo)-6-(1-(cyclopropylmethyl)-3-iodo-1H-pyrazol-5-yl)-3-(oxetan-3-yl)-3-azabicyclo[3.1.0]hexane (77). To an ice-cooled solution of tert-butyl (exo)-6-(1-(cyclopropylmethyl)-3-iodo-1H-pyrazol-5-yl)-3azabicyclo[3.1.0]hexane-3-carboxylate (110 g, 0.26 mmol) in dichloromethane (1.3 L) was added trifluoroacetic acid (260 mL). The reaction mixture was warmed to 40 °C for 2 h and concentrated in vacuo. The resulting residue was dissolved in methanol (1.3 L), and the solution was cooled to 0 °C. Triethylamine (142 g, 1.41 mol) and oxetan-3-one (55.4 g, 0.768 mol) were added, and the mixture was warmed to room temperature for 20 min and cooled to 10 °C. Sodium cyanoborohydride (48.4 g, 0.768 mol) was added, and the resulting suspension was warmed to 40 °C. After 3 h, the reaction was diluted with water (400 mL), and

the resulting mixture was extracted with ethyl acetate (3 × 1.5 L). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by flash column chromatography (9 → 20% ethyl acetate in petroleum ether) provided product (81 g, 82% yield). 1H NMR: (CDCl3 400 MHz), δ 5.94 (s, 1H), 4.68 (m, 2H), 4.60 (m, 2H), 4.03 (d, J = 7.2 Hz, 2H), 3.78 (m, 1H), 3.12 (d, J = 8.8 Hz, 2H), 2.46 (m, 2H), 2.21 (t, J = 3.2 Hz, 1H), 1.71 (m, 2H), 1.27 (m, 1H), 0.57 (m, 2H), 0.39 (m, 2H).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00843. CEREP Receptor panel data (PDF) Molecular formula strings (CSV) Accession Codes

Coordinates and structure factors for DLK kinase domain complexes are available in the PDB with accession codes 5VO1 (7) and 5VO2 (10).

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AUTHOR INFORMATION

Corresponding Authors

*M.S. Phone: 650-467-7764. E-mail: [email protected]. *J.W.L. Phone: 650-745-5247. E-mail: [email protected]. ORCID

Michael Siu: 0000-0002-2822-6584 Present Addresses

R.I.E.: Denali Therapeutics, South San Francisco, CA 94080. A.A.E.: Denali Therapeutics, South San Francisco, CA 94080. J.P.L.: Stemcentrx, South San Francisco, CA 94080. J.W.L.: Denali Therapeutics, South San Francisco, CA 94080. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the following individuals for their contributions: Chris Hamman, Mengling Wong, and Michael Hayes for compound purification; Genentech Biomolecular Resources group for construct generation; Emile Plise and Jonathan Cheong for MDR1-MDCK data; Xiaolin Zhang, Allan Jaochico, and Xiao Ding for bioanalytical data; Amy Sambrone for formulations work; Tim Earr for tissue collection; York Rudhard and the Evotec team for biochemical and cell-based potency data; Wuxi for support with analog synthesis; and Genentech compound management for sample handling. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02- 05CH11231. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393) and the National Center for Research Resources (P41RR001209). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS, NCRR or NIH. 8100



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ABBREVIATIONS USED ClogP calculated logarithm of partition coefficient; CNS MPO central nervous system multiparameter optimization; DMEM Dulbecco’s modified Eagle medium; DLK dual leucine zipper kinase; DRG dorsal root ganglion; DAST diethylaminosulfur trifluoride; ELISA enzyme-linked immunosorbent assay; HBD hydrogen bond donor; JNK c-Jun N-terminal kinase; Kpuu unbound partition coefficient; LipE lipophilic ligand efficiency; MAP3K12 mitogen-activated protein kinase kinase kinase 12; MCT methylcellulose Tween 80; MKK mitogen-activated protein kinase kinase; MLK mixed-lineage kinase; MDR1 multidrug resistance protein 1; MDCK Madin-Darby canine kidney; MSD Meso Scale Discovery detection; NGF nerve growth factor; P-gp P-glycoprotein

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REFERENCES

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Selective Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12

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