Azetidine and Piperidine Carbamates as Efficient, Covalent Inhibitors

Article pubs.acs.org/jmc

Cite This: J. Med. Chem. 2017, 60, 9860−9873

Azetidine and Piperidine Carbamates as Efficient, Covalent Inhibitors of Monoacylglycerol Lipase Christopher R. Butler,*,† Elizabeth M. Beck,† Anthony Harris,‡ Zhen Huang,† Laura A. McAllister,† Christopher W. am Ende,‡ Kimberly Fennell,‡ Timothy L. Foley,‡ Kari Fonseca,† Steven J. Hawrylik,‡ Douglas S. Johnson,† John D. Knafels,‡ Scot Mente,† G. Stephen Noell,‡ Jayvardhan Pandit,‡ Tracy B. Phillips,‡ Justin R. Piro,† Bruce N. Rogers,‡ Tarek A. Samad,† Jane Wang,§ Shuangyi Wan,§ and Michael A. Brodney† †

Pfizer Worldwide Research and Development, 1 Portland Street, Cambridge, Massachusetts 02139, United States Pfizer Worldwide Research and Development, 445 Eastern Point Road, Groton, Connecticut 06340, United States § WuXi AppTec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China ‡

S Supporting Information *

ABSTRACT: Monoacylglycerol lipase (MAGL) is the main enzyme responsible for degradation of the endocannabinoid 2arachidonoylglycerol (2-AG) in the CNS. MAGL catalyzes the conversion of 2-AG to arachidonic acid (AA), a precursor to the proinflammatory eicosannoids such as prostaglandins. Herein we describe highly efficient MAGL inhibitors, identified through a parallel medicinal chemistry approach that highlighted the improved efficiency of azetidine and piperidine-derived carbamates. The discovery and optimization of 3-substituted azetidine carbamate irreversible inhibitors of MAGL were aided by the generation of inhibitor-bound MAGL crystal structures. Compound 6, a highly efficient and selective MAGL inhibitor against recombinant enzyme and in a cellular context, was tested in vivo and shown to elevate central 2-AG levels at a 10 mg/kg dose.

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INTRODUCTION Inflammation serves as an evolutionarily conserved protective mechanism to reduce infections and clear antigens as well as cellular debris to minimize tissue damage and promote subsequent tissue healing following the resolution of the inflammatory cascade. Exaggerated or persistent inflammatory response in the periphery or the central nervous system (CNS) can become pathological, leading to chronic inflammatory conditions. Neuronal homeostasis in the CNS is particularly vulnerable to chronic inflammation, and most neurological disorders are associated with an imbalanced inflammatory response in the brain. Hallmarks of neuroinflammation include activation of resident immune cells (microglia and astrocytes), production of proinflammatory cytokines and chemokines, as well as deterioration of the blood−brain barrier. This cascade leads to heightened risk of neuronal dysfunction and neurodegeneration and in some cases infiltration of peripheral cells and proteins into the CNS.1 We have previously shown that monoacylglycerol lipase (MAGL), an enzyme that terminates the signaling of the endocannabinoid 2-arachidonoylglycerol (2-AG), also controls arachidonic acid (AA) production in the brain.2,3 MAGL © 2017 American Chemical Society

catalyzes the hydrolytic conversion of 2-AG to arachidonic acid and glycerol.2 2-AG accounts for 50% of the production of AA in the brain,4,5 whereas phospholipid hydrolysis by phospholipase A2 (PLA2) serves as the primary source of arachidonic acid production in the periphery. AA plays a major role in the inflammatory response, both as a signaling molecule and also as a precursor to multiple pathways of proinflammatory eicosanoids (Figure 1). As such, MAGL exerts a bidirectional control on brain inflammation by modulating brain endocannabinoid and arachidonate levels in opposing directions. Therefore, MAGL inhibition potently decreases the proinflammatory eicosanoids and cytokines in the brain5 and enhances 2-AG signaling at CB1/2 receptors. Genetic deletion or pharmacological blockade of MAGL attenuates biomarkers of neuroinflammation,6−11 including cytokines and gliosis, in mouse models of Alzheimer’s disease.12 The use of steroids is currently the main therapeutic option to attenuate pronounced brain inflammation; therefore development of potent, selective inhibitors of MAGL represents a research avenue with Received: October 13, 2017 Published: November 17, 2017 9860

DOI: 10.1021/acs.jmedchem.7b01531 J. Med. Chem. 2017, 60, 9860−9873

Journal of Medicinal Chemistry

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alcoholic subunit with a relatively low pKa to serve as a suitable leaving group. As shown in Figure 2, exemplar literature MAGL inhibitors typically employ alcohols such as 4-nitrophenol and hexafluoroisopropyl (HFIP) alcohol.18−21 In addition to the requirement of a suitable leaving group, two key structural elements for the inhibition of MAGL can be elucidated in the comparison of the structural features of 2-AG to known inhibitors.13 As illustrated by both the lipophilic arachidonate and bis-dioxolane tail groups in 1, significant lipophilicity is required distal to the catalytic serine (Figure 3,

Figure 3. Arachidonic acid and related pharmacophore. Figure 1. Role of MAGL in endocannabinoid and arachidonic acid pathways.

pink). This lipophilic tail is attached via a linear linker (yellow) that partially recapitulates the alkyl chain of 2-AG. These structural requirements underpin the challenge in developing orally bioavailable MAGL inhibitors suitable for in vivo evaluation: incorporating the requisite lipophilic tail region and leaving group (blue) without dramatically increasing clearance, which is known to be unfavorably impacted by increased lipophilicity.22 Early optimization of the linker becomes crucial to facilitate subsequent tuning of lipophilicity and molecular weight in the tail region. This communication will describe the identification of efficient core amine systems by monitoring the optimization using lipophilic efficiency (LipE) and fit quality (FQ).23,24

significant therapeutic potential for the treatment of acute and chronic diseases associated with neuroinflammation. MAGL is a 33 kDa serine hydrolase with an active site containing a classical Ser-His-Asp catalytic triad.13 The characteristic reactivity of the active site enables the opportunity for covalent modification of the key serine residue (Ser122). Covalent inhibition is attractive in that it offers the potential for extended duration of pharmacodynamic modulation relative to pharmacokinetic profile of the inhibitor.14 Thus, a sustained elevated pharmacological response can be achieved without the corresponding sustained exposure, as would be required for a reversible, noncovalent inhibitor. In order to take advantage of these potential attributes, covalent inhibitors require two key elements: (a) sufficient reactivity for the residue that is to be modified in the protein and (b) specific target binding affinity. Serine hydrolases as a class have been successfully inhibited by a number of electrophile-containing molecules that range in their inherent reactivity.15,16 More specifically, inhibition of MAGL has been accomplished most often in the literature with a carbamate electrophile that efficiently engages Ser122, as observed in JZL-184 (1, Figure 2)3,17 but is not susceptible to subsequent catalytic turnover, thus inhibiting the enzyme. In order to facilitate inhibitory acyl transfer with Ser122, these carbamates generally contain an

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RESULTS AND DISCUSSION With the goal of identifying a potent, brain-penetrant, and low clearance MAGL inhibitor, initial efforts for linker optimization occurred via two related approaches enabled by the diversity available from Pfizer’s molecule collection (Figure 4). The first approach (A) was to utilize small, fragment-like monofunctional amines lacking the lipophilic tail portion of the molecule to prepare HFIP carbamates. The second approach (B) utilized bifunctional monomers, either by incorporating an equivalent lipophilic tail, deprotecting, and then forming the key HFIP carbamate or alternatively by inverting the sequence to introduce the carbamate and subsequent deprotection and incorporation of lipophilicity. Whereas the combination of these two approaches significantly broadened the availability of linker architecture, it required a normalization of potency to account for the stark differences in lipophilicity and molecular weight between the two approaches and was ultimately accomplished using LipE and FQ. LipE (calculated as the pIC50 − log D) distinguishes potency enhancements attributable strictly to increased lipophilicity from those of target-specific interactions. Fit quality (FQ), calculated as the ligand efficiency (LE) of a compound adjusted for its number of heavy atoms, corrects the potency values for the corresponding molecular weight.24 The utilization of FQ is particularly important when comparing series and compounds of significant differences in size, as LE

Figure 2. Literature MAGL inhibitors. Data shown were obtained from in-house assays. 9861



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Figure 4. Parallel approaches to linker exploration.

Figure 5. Efficiency plots for MAGL inhibitors: (a, left) lipophilic efficiency plot comparing log D (x-axis) vs MAGL pIC50; (b, right) fit quality plot comparing number of heavy atoms (x-axis) vs ligand efficiency (LE).

alone lends bias to smaller compounds in such a comparison. These two metrics are especially useful for optimizing brain availability, as increases in lipophilicity and MW diminish brain penetration via active efflux.25 The LipE and FQ for compounds prepared via these approaches are plotted in Figure 5. This analysis revealed that cyclic, secondary amines such as piperidines (blue dots) and azetidines (green diamonds) were preferred cores for MAGL, whereas alternative ring systems or substitution patterns, especially those that were not linear, were generally less efficient. Whereas azetidines and piperidines exhibited similar LipE values, with a number of exemplars of each registering between 5 and 6, a number of azetidines significantly differentiated when gauging FQ. Highlighted compounds were screened against MAGL and against FAAH as an initial, albeit imperfect, surrogate for serine hydrolase selectivity to efficiently guide design. Specifically, 3substituted azetidines emerged as some of the most efficient

linkers, as exemplified by 3−5 (Table 1). Oxadiazole 3 displayed potent inhibition of MAGL (IC50 = 0.38 nM) nearly equivalent to that of the literature tool KML29 (MAGL IC50 = 0.19 nM), albeit with significantly decreased molecular weight, as reflected in its high FQ (0.97) and LipE. Pyrimidine 4 was slightly less potent; however its decreased lipophilicity translated to an improvement in LipE (5.7) relative to 3 (LipE = 5.2). Moreover, while 3 maintained the previously observed selectivity for MAGL over FAAH (∼1000-fold), 4 exhibited enhanced selectivity to >15 000-fold. Piperazinesubstituted 5 displayed similar potency (MAGL IC50 = 3.2 nM), efficiency (LipE = 5.5, FQ = 0.87), and selectivity over FAAH (781-fold). Pyrazole 6, a slightly less polar variant of oxadiazole 3, displayed modestly improved potency and a subtle improvement in LipE and FQ. Collectively, these lead analogs 3−6 exemplified useful, efficient scaffolds for further optimization. In general, the 9862



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Table 1. In Vitro Data for 3−6

a

IC50 values obtained from MAGL inhibition assay. bkinact/KI ratio. cIC50 values obtained from FAAH inhibition assay.

Figure 6. X-ray crystal structure of 3 bound to the active site serine in MAGL (PDB code 6AX1). The residues making up the catalytic triad (Ser122, His269, and Asp239) have been shown in stick representation. Hydrogen bonds are indicated by dashed lines. A glycerol molecule (gray) was modeled into electron density seen in the exit tunnel. Glycerol was part of the buffer used to purify the protein and was present in the crystallization drop.

proposed binding mode of compound 1.13 The hydrophobicity of the pocket surrounding the oxadiazole and phenyl rings reinforces the need for minimal polarity in the tail region of the molecule. Recognition of the pyrazole as an efficient linker off of the azetidine suggested modulation of the pyrazole substituent to expand the SAR and understand if further improvements in efficiency were available (Table 2). To gauge the efficiency of the pyrazole itself, the phenyl ring was stripped back and replaced with a simple methyl (7), resulting in a significant attenuation of potency and decreased efficiency. This result further reinforced the structural implication that a lipophilic tail

diversity in ring structures presented clear synthetic opportunities to grow these subunits in a wide range of vectors. To guide design of the optimal group for the distal end of the MAGL binding pocket, a crystal structure of 3 bound to the active site of MAGL was obtained (Figure 6). The HFIP leaving group has clearly been displaced by the active site serine (Ser), resulting in a protein−inhibitor carbamate adduct. The azetidine resides in the opening of the arachidonate port, illustrating the narrow tunnel required and empirically suggested from our initial SAR efforts. The limited rotatable bonds of 3 efficiently places the phenyl oxadiazolyl substituent into one of the distal pockets, in a similar fashion to the 9863



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Table 2. In Vitro Data for 7−12

a

MAGL IC50: IC50 values obtained from MAGL inhibition assay. FAAH IC50: IC50 values obtained from FAAH inhibition assay. bkinact/Ki.

Scheme 1

Figure 7. Click chemistry activity-based protein profiling in human brain vascular pericytes. Live cells were treated at various concentrations of 14 for 1 h at 37 °C. (A) In-gel fluorescence. (B) Normalized MAGL intensity was fitted as a function of the concentration of 14.

corresponding potency gain, resulting in decreased efficiency. Difluorinated analog 9 did not impact MAGL potency or efficiency but enhanced selectivity over FAAH. Introduction of

was required. Similarly, the substitution of an additional phenyl ring at the 3-position of the pyrazole (8) maintained potency relative to 6, but the additional lipophilicity outpaced the 9864



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Figure 8. Effect of 6 on brain 2-AG and corresponding exposure following subcutaneous administration in wild-type mice: (a) 2-AG response following 10 mg/kg dose of 6; (b) mean unbound plasma and brain exposure; (c) 2-AG response following 1 mg/kg dose of 6; (d) mean unbound plasma and brain exposure.

of both the alkyne and azetidine was followed by formation of the azetidinyl HFIP carbamate. Human brain vascular pericytes were treated with increasing concentrations of 14, followed by cell lysis and click chemistry with TAMRA-azide. The probelabeled proteins were visualized by in-gel fluorescence to illustrate MAGL engagement and inhibition (Figure 7). Plotting the percentage of MAGL inhibition demonstrated a cellular IC50 of 3 nM. Interesting, at concentrations below the IC50, the compound was selective only for MAGL; however at higher concentrations, an off-target protein was labeled corresponding to a molecular weight of ∼40 kDa. With robust potency against recombinant MAGL as well as in human pericytes, compound 6 was dosed in vivo in an effort to assess the relationship between drug levels and increases in 2-AG levels in the brain. As shown in Figure 8, a subcutaneous dose of 10 mg/kg to mouse elicited significant elevations (∼5fold, Figure 8a) in brain levels of 2-AG out to an 8 h time point. Additionally, compound 6 showed free access to the CNS in mouse, as measured by an AUC-derived Cb,u/Cp,u ratio of 0.6. The corresponding exposure for the 10 mg/kg dose exhibited a Tmax at 0.5 h (2 nM Cb,u) and had maintained free levels above the measured IC50 to 8 h (Figure 8b). Further, a lower dose of 1 mg/kg provided a dampened response, with a maximal 3-fold increase of 2-AG occurring at the 0.5 h time point and no clear elevation beyond that time point (Figure 8c). At this dose, the

a pyrazine in place of the phenyl ring (10) resulted in a minor decrease in MAGL potency, but the significant drop in lipophilicity resulted in a marked improvement in LipE. Homologation to a benzyl-substituted pyrazole loses efficiency at MAGL as well as selectivity over FAAH. An attempt to decrease lipophilicity with the introduction of a 4-substituted tetrahydropyran (12) improved FAAH selectivity but resulted in a 10× loss in MAGL potency. The anticipated decrease in lipophilicity, however, offset the potency loss and resulted in similar LipE as the parent. With an exemplar such as 6 that was well-optimized for in vitro MAGL potency, we next wanted to get a preliminary gauge of the broader selectivity against the other serine hydrolases. To this end, 6 was screened against a panel of 42 serine hydrolases at 1 and 10 μM concentrations of inhibitor (see Table S2, Supporting Information). In this panel, 6 demonstrated significant inhibition (>70%) of ABHD6, CES1, CES2, MAGL, and PLA2G7 at both concentrations tested and FAAH only at the top concentration. To further explore the cellular potency and selectivity profile against other serine hydrolases, click chemistry activity-based protein profiling (CCABPP) was utilized.26 As such, an alkyne-containing clickable probe (14, Scheme 1) was prepared from bromophenyl pyrazole 13. After installation of the alkyne via Sonagashira reaction with ethynyltrimethylsilane, subsequent deprotection 9865



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Figure 9. (a) Structure of compound 15. (b, d) Effect of 15 on brain 2-AG following subcutaneous administration in wild-type mice of (c) 1 mg/kg and (e) 10 mg/kg doses.

at 10 μM; see Table S2, Supporting Information). Compound 15 was then administered in vivo at the same doses as compound 6. Again, good brain penetration was observed, with an AUC-derived Cb,u/Cp,u ratio of 0.5. At both doses a robust, persistent 2-AG elevation was observed. At the lower, 1 mg/kg, dose of 15 the 2-AG response peaked at 2 h (Figure 9b), similar to the high dose of 6 albeit achieving a considerably lower Cmax (0.14 nM, Figure 9c). At the higher, 10 mg/kg, dose, 15 demonstrated a robust 2-AG response out to 24 h (Figure 9d). Interestingly, at neither dose did central, free levels of compound 15 exceed the IC50 (2 nM) measured in vitro, yet 2-AG was significantly elevated in both. In aggregate, the PK/PD data from 6 and 15 illustrated a disconnect in the exposure/2-AG elevation relationship for these compounds. Compound 6 achieved free exposures significantly higher than its in vitro IC50 but showed only a marginal response, whereas 15 never exceeded its in vitro value but exhibited a sustained 2-AG elevation. It cannot be discounted that the lack of exquisite selectivity for MAGL as assessed by the serine hydrolase panel could potentially convolute the interpretation of the data. However, the similar

free brain exposure of compound 6 could only be measured at 0.5 h and was 0.02 nM, 100-fold lower than the 10 mg/kg dose and well below the in vitro IC50 (Figure 8d). Whereas the higher dose confirmed MAGL inhibition with treatment of compound 6, collectively, the data were somewhat confounding. First, the modest response at both doses was surprising in comparison to previously run literature compounds with similar potency. Analysis of the juxtaposed PK/PD alone would suggest that 6 was simply rapidly cleared, and the lack of exposure precluded any opportunity for a 2-AG response. Additionally, however, it was striking that the 1 mg/kg dose showed 100-fold lower free brain exposure than the 10 mg/kg dose. This nonlinear PK suggested that another phenomenon may be occurring as well. In an effort to understand whether this phenomenon was specific to the azetidine core itself or alternatively the phenyl pyrazole, the corresponding piperidine 15 was prepared in similar fashion (Figure 9a). Compound 15 retained similar in vitro activity against MAGL (IC50 = 2 nM) and serine hydrolase selectivity panel (>70% inhibition of ABHD6, CES2, MAGL at both 1 and 10 μM and CES1, FAAH, PLA2G7 only 9866



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10 μL of MAGL enzyme in assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, and 25% glycerin). An equal volume of 7-HCA in assay buffer with 10% DMSO was added either immediately (T = 0 min) or after a 30 min incubation (T = 30 min) to initiate the reaction. The final concentration of MAGL enzyme was 88 pM, and 7-HCA substrate was 5 μM. After these dilutions, the final concentration of the test compound ranged from 3 μM to 0.03 nM. The reaction was allowed to progress for 60 min, after which the plate was read at an Ex/Em of 340/465. Percent inhibitions were calculated based on control wells containing no compound (0% inhibition) and a control compound (e.g., a MAGL inhibitor whose activity is known or was previously reported in the literature, such as one with about 100% inhibition). IC50 values were generated based on a four-parameter fit model using ABASE software from IDBS.28 Assessment of Rate of MAGL Inactivation (MAGL kinact/KI). To measure MAGL inactivation, the same protocol for the (T = 0 min) MAGL inhibition IC50 assay was performed with data collected every minute to acquire enzyme progress curves at decreasing concentrations of compound. Kobs values were calculated from these data, and kinact/KI ratios were determined from a plot of Kobs values vs compound concentrations. Assessment of FAAH Inhibition. Assessment of FAAH inhibition utilizes human recombinant fatty acid amide hydrolase lipase and the fluorogenic substrate arachidonoyl-AMC (Sigma A6855). 400 nL of a test compound at decreasing concentration was spotted into a 384well back plate (PerkinElmer, 6007279) using a Labcyte Echo, followed by addition of 10 μL of FAAH enzyme (Cayman 10010183) in assay buffer (50 mM Tris, pH 9.0, 1 mM EDTA). After a 30 min incubation at room temperature, 10 μL of arachidonyl-AMC was added in assay buffer with 16% DMSO. Final concentration of FAAH enzyme was 0.0125 unit, and the AAMC substrate was used at a concentration of 5 μM. The final concentration of the test compound ranged from 30 μM to 0.3 nM. The reaction was allowed to progress for 60 min, after which the plate was read at an Ex/Em of 355/460. Percent inhibitions were calculated based on control wells containing no compound (0% inhibition) and a control compound at a concentration known to inhibit FAAH to 100%. IC50 values were generated based on a four-parameter fit model using ABASE software from IDBS. Cellular Pharmacology. Human brain vascular pericytes and pericyte growth supplement were purchased from ScienCell Research Laboratories. Media, other supplements for cell culture, and reagents for sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS− PAGE) were purchased from Thermo Fisher Scientific unless otherwise noted. TAMRA azide was purchased from Lumiprobe. DMSO solutions of compound 14 were prepared, and aliquots were stored at −20 °C. Cell Culture and Treatment. Human brain vascular pericytes were cultured in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12), supplemented with 5% heat-inactivated fetal bovine serum (FBS), 1% pericyte growth supplement, and 100 U/mL penicillin−streptomycin in a humidified environment maintained with 5% CO2 at 37 °C. For labeling with probe 14, cells were cultured on six-well plates overnight, and 1000× stock solutions of probe 14 in DMSO were added to the growth media, and cells were maintained for 1 h at 37 °C. At the end of treatment, cells were washed with 1 mL of cold phosphate buffered saline (PBS) and were scrapped in 1 mL of cold PBS. Cells were pelleted by centrifuging at 10 000g at 37 °C for 1 min. The supernatant was removed, and cell pellets were kept at −80 °C until further analysis. Click Chemistry Activity-Based Protein Profiling. To the cell pellets was added PBS containing 0.25% SDS, and the cells were lysed with gentle sonication. The proteome concentration was measured with bicinchoninic acid (BCA) assay. The proteomes were normalized to 1.0 mg/mL and subject to Cu-catalyzed click chemistry with TAMRA azide following a reported protocol.29 The reaction mixtures were diluted with lithium dodecyl sulfate (LDS) sample buffer and NuPAGE reducing agent and were analyzed with 1.0 mm NuPAGE 4−12% Bis-Tris 15-well gels in 2-[N-morpholino]ethanesulfonic acid (MES) running buffer. TAMRA fluorescence signals were collected on

selectivity profile observed for both 6 and 15 minimizes offtarget impacts as cause for the discrepancy, as the same off target hydrolases are engaged to a similar extent. A number of scenarios could potentially help to explain this PK/PD disconnect, including (a) that the compounds were not covalently inhibiting MAGL and instead reverting to a paradigm wherein in vivo inhibition and resulting pharmacodynamics response were driven by AUC coverage of the target or (b) that the inhibitors were indeed covalently modifying MAGL but that the resultant adducts varied in their hydrolytic stability. The combination of the cellular ABPP of 14 and the crystal structure of 3 covalently bound to MAGL reinforced to us that the former scenario was unlikely. That same data, however, reinforce the second scenario in that the azetidine carbamates were indeed forming a carbamate adduct with MAGL but that those complexes were less hydrolytically stable than those derived from the corresponding piperidine. To this end, acylated azetidines have been shown to react differently than acyclic or larger ring size amides due to decreased planarity in the amide moiety itself, conveyed by the ring strain associated with the azetidine itself.27 Moreover, a comparison of the PK data suggested that in brain, the azetidine carbamate had a much faster rate of clearance than the corresponding piperidine, which could potentially be explained by initial reaction with MAGL, followed by a subsequent hydrolysis step. This, in effect, would suggest that the target for inhibition was also its primary clearance pathway. Efforts to further test this hypothesis, further deconvolute the observed PK/PD disconnect, and improve serine hydrolase selectivity are underway and will be reported in due course.

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CONCLUSION In summary, highly efficient MAGL inhibitors were identified through a parallel medicinal chemistry approach that highlighted the improved efficiency of piperidine and azetidinederived carbamates. Through monitoring improvements in FQ and LipE, efforts were focused on the optimization of 3substituted azetidines, including five- and six-membered heterocycles. The incorporation of a pyrazole served as an efficient linker to facilitate further growth into the distal lipophilic pocket. A crystal structure of 3 aided in subsequent design of inhibitors and suggested minimizing the exposed polarity on the inhibitor. Replacement of the oxadiazole with a pyrazole, substituted with either a phenyl (6) or pyrazine (10) ring, maximized potency and LipE, respectively. The cellular potency, an exemplar of these compounds, was confirmed using ABPP. An in vivo experiment monitoring the effect of a key exemplar on 2-AG elevation confirmed in vivo MAGL inhibition, but a modest response suggested that the azetidine-derived analogs were exhibiting dampened PD effects than would be expected for a covalent modification of MAGL, whereas the corresponding piperidine 15 demonstrated a robust 2-AG elevation. Further efforts to understand this phenomenon are ongoing and will be reported in due course.

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EXPERIMENTAL SECTION

Biology. In Vitro Pharmacology. Assessment of MAGL Inhibition (MAGL IC50). Assessment of MAGL inhibition utilized human recombinant monoacylglycerol lipase and the fluorogenic substrate 7-hydroxycoumarinyl arachidonate (7-HCA, Biomol ST502). 400 nL of a test compound at decreasing concentration (ranging from 150 μM down to 1.5 nM) was spotted into a 384-well back plate (PerkinElmer, 6007279) using a Labcyte Echo, followed by addition of 9867



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Analysis of Compound in Plasma and Brain and Pharmacokinetic Parameters. All samples were quantified via HPLC−MS/MS. Cerebellum samples were thawed, diluted 1:4 (w/v) with water, and homogenized using an H-speed dispersator. Standards and controls were prepared in a similar manner using a brain homogenate prepared from untreated animals. Plasma and brain homogenate samples were prepared for analysis by deproteination with methanol/acetonitrile (1:1, v/v) containing an internal standard. The sample volume for analysis was 10 and 50 μL for the plasma and brain matrices, respectively. The samples were precipitated with 200 μL of methanol/ acetonitrile, vortexed, and centrifuged. Following centrifugation, the supernatant was transferred to a clean 96-well injection block and diluted 40× with water containing 0.1% formic acid. A sample volume of 10 μL for the plasma samples and 5 μL for the brain samples was subsequently injected on the LC−MS/MS for analysis. The lower limit of quantification (LLOQ) was 1.0 ng/mL for plasma and 0.5 ng/mL for brain homogenate (conversion to 2.5 ng/g) samples. The upper limit of quantification (ULOQ) was 1000 ng/mL for plasma and 500 ng/mL for brain homogenate (conversion to 2500 ng/g). Pharmacokinetic parameters were determined by noncompartmental analysis using Watson LIMS, version 7.5. X-ray Structure of MAGL Bound to 3. Synthetic DNA encoding full-length human MAGL (residues 1−303), with an N-terminal 6×His tag and a C-terminal Strep tag, was cloned into E. coli (Rosetta strain) and expressed and purified essentially as described previously.30 The protein was purified by a combination of nickel ion and streptactin affinity chromatography and concentrated to 50 mg/mL in a final buffer consisting of 15 mM HEPES, pH 8.2, 2 mM TCEP, and 10% glycerol, with hexaethylene glycol monododecyl ether added to 0.1 mM. Crystallization was carried out in sitting drops with 0.30 μL of protein at 20 mg/mL added to 0.30 μL of reservoir solution (0.07 M sodium cacodylate, pH 5.1−5.9 and 33−51% MPD). Apo crystals obtained from these drops were harvested and transferred to a cryoprotectant soaking solution that consisted of 70 mM sodium cacodylate, pH 5.1; 10% MPD; 30% PEG-MME-2K with the addition of 1 mM compound 3. Crystals were left to soak overnight (12−18 h) and flash-frozen in liquid nitrogen for data collection. X-ray diffraction data to 2.26 Å resolution were collected at 100 K with radiation of wavelength 1 Å at beamline 17-ID of the Advanced Photon Source at Argonne, on a Pilatus-6M detector. Data were processed using autoPROC31 with XDS32 for data integration and the program Scala from the CCP4 Suite33 for merging and scaling. The structure was solved by molecular replacement using PDB entry 3HJU30 as the search model, and refined with autoBUSTER.34 Model building and fitting were done using Coot.35 Data collection and refinement statistics are given in Table S1. Chemistry. General Methods. Solvents and reagents were of reagent grade and were used as supplied by the manufacturer. All reactions were run under a N2 atmosphere. Organic extracts were routinely dried over anhydrous sodium sulfate. Concentration refers to rotary evaporation under reduced pressure. Chromatography refers to flash chromatography using disposable RediSepRf 4 to 120 g silica columns or Biotage disposable columns on a CombiFlash Companion or Biotage Horizon automatic purification system. Microwave reactions were carried out in a SmithCreator microwave reactor from Personal Chemistry. Purification by mass-triggered HPLC was carried out using Waters XTerra PrepMS C18 columns, 5 μm, 30 mm × 100 mm. Compounds were presalted as TFA salts and diluted with 1 mL of dimethylsulfoxide. Samples were purified by mass triggered collection using a mobile phase of 0.1% trifluoroacetic acid in water and acetonitrile with a starting gradient of 100% aqueous to 100% acetonitrile over 10 min at a flow rate of 20 mL/min. Elemental analyses were performed by QTI, Whitehouse, NJ. All target compounds were analyzed using ultrahigh performance liquid chromatography/ultraviolet/evaporative light scattering detection coupled to time-of-flight mass spectrometry (UHPLC/UV/ELSD/ TOFMS). Unless otherwise noted, all tested compounds were found to be >95% pure by this method.

a Typhoon FLA 9500 biomolecular imager (GE Healthcare) with 532 nm laser excitation and ≥575 nm long-pass emission filter. Data Analysis. In-gel fluorescence data were visualized with ImageJ software (version 1.47, NIH). Fluorescence intensity was quantified with background subtraction using ImageStudio Lite (version 4.0.21, LI-COR). The intensities of the two MAGL bands were averaged, and data from two biological replicates were plotted as mean ± standard deviation in GraphPad Prism (version 7.02) and were fit to an inhibitor dose−response curve to obtain the cellular IC50. In Vivo Experiments. All procedures performed on animals in this study were in accordance with established guidelines and regulations and were reviewed and approved by the Pfizer (or other) Institutional Animal Care and Use Committee. Assessment of 2-AG Elevation. C57Bl6 mice (7 weeks old) were housed 4 per cage in an environmentally controlled animal facility (12 h light/dark cycle from 6:00 a.m. to 6:00 p.m.; temperature was maintained between 20.5 and 22.0 °C; relative humidity was maintained between 50 and 65%). Food and water were available ad libitum throughout the study. Mice were subcutaneously administered 6, 14, or vehicle (DMSO/Cremophor/saline at a volume ratio of 5:5:90). Plasma and brain samples of 6 and 14 treated mice were collected at 0.5, 1, 2, 4, 8, 12, and 24 h postdose (3 mice per time point). Plasma and brain samples of vehicle treated mice were collected at 1 h postdose. Blood was collected by first anesthetizing mice with isoflurane followed by cardiac puncture. Whole blood was placed into tubes containing NaF and K2EDTA (0.5 M, 20 μL per 1 mL of blood) to inhibit plasma hydrolase activity and as an anticoagulant, respectively. Blood was centrifuged at 4000 rcf for 5 min to harvest plasma. Immediately following blood sampling mice were euthanized by cervical dislocation and their brains were removed. Brain and cerebellum were immediately frozen in liquid nitrogen and stored at −80 °C until analysis. Pharmacodynamic Measurement. Plasma samples were prepared for 2-AG measurement on wet ice. Briefly, samples were spiked with IS2 (internal standard 2) solution [1 μg/mL d5-2-AG (deuterated 2 arachidonoylglycerol) (Cayman Chemical Ann Arbor, MI) in ACN] followed by precipitation and organic extraction in EtOAC/hexane (v:v 50:50). Samples were then centrifuged at 15 700 rcf for 15 min. Supernatant was removed and dried under nitrogen gas at room temperature. Samples were then reconstituted in ACN and directly injected for LC−MS/MS analysis. Calibration standards and quality control samples were prepared using the same methodology. The 2AG calibration curve consisted of 2 500 ng/mL of 2-AG in water/ PMSF/buffer [100 mM NH4OAc in water (pH 2.0 adjusted with formic acid)] (v:v:v 100:5:20). For 2-AG measurement samples were directly injected onto a C 18 UPLC column held at 65 °C at a flow rate of 0.5 mL/min. Retention time for 2-AG was 2.71 min. Retention time for d5-2-AG was 2.70 min. Mass spectrometry was run in positive electrospray ionization (ESI) mode with selected reaction monitoring (SRM) detection. The 2-AG m/z was 379.4/287.3. The d5-2-AG m/z was 384.4/287.4. Brain samples were prepared on wet ice. Briefly, brain homogenates were prepared by homogenizing brain with 3 volumes (w:v) of homogenization buffer (1% PMSF and 5% 100 mM NH4OAc in water, pH 2.0, adjusted with formic acid). The brain homogenates were then spiked with IS2 [1 μg/mL d5-2-AG, (deuterated 2 arachidonoylglycerol) (Cayman Chemical Ann Arbor, MI) in ACN] solution followed by precipitation and organic extraction in ACN. Samples were then centrifuged at 15 700 rcf for 15 min at 4 °C. Supernatant was removed from each sample directly injected for LC−MS/MS analysis. A d5-2AG internal standard (Cayman Chemical Ann Arbor, MI) was added to each sample. Calibration standards and quality control samples were prepared using the same methodology. The 2-AG calibration curve consisted of 50 50000 ng/mL 2-AG in homogenization buffer. For 2AG measurement samples were directly injected onto a C 18 UPLC column held at 50 °C at a flow rate of 0.5 mL/min. Retention time for 2-AG was 3.30 min. Retention time for d5-2-AG was 3.29 min. Mass spectrometry was run in positive ESI mode with SRM detection. The 2-AG m/z was 379.4/287.3. 9868



Article

1,1,1,3,3,3-Hexafluoropropan-2-yl 3-[4-(Pyrimidin-2-yl)piperazin-1-yl]azetidine-1-carboxylate (5). To a solution of 2[4-(azetidin-3-yl)piperazin-1-yl]pyrimidine36 (100 mg, 0.30 mmol) and DIPEA (196 mg, 1.5 mmol) in CH2Cl2 (6 mL) was added a solution of triphosgene, 1,1,1,3,3,3-hexafluoropropan-2-ol, DIPEA, and DMAP in CH2Cl2 dropwise at 0 °C. The mixture was stirred at 16 °C for 3 h. The mixture was concentrated in vacuo and purified by preparative high performance liquid chromatography to give 21 mg (17%) of 1,1,1,3,3,3-hexafluoropropan-2-yl 3-[4-(pyrimidin-2-yl)piperazin-1-yl]azetidine-1-carboxylate (5) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.31 (d, J = 4.7 Hz, 2 H), 6.51 (t, J = 4.7 Hz, 1 H), 5.65 (sep, J = 6.2 Hz, 1 H), 4.23−3.97 (m, 4 H), 3.93−3.79 (m, 4 H), 3.29−3.16 (m, 1 H), 2.44−2.39 (m, 4 H). 13C NMR (101 MHz, CDCl3) δ 161.5, 157.7, 151.7, 120.6 (q, 1JCF = 283.2 Hz), 110.2, 67.6 (sep, 2JCF = 34.5 Hz), 54.2, 54.0, 53.3, 49.4, 43.2. HRMS calculated for C15H17F6N5O2 [M + H]+ 414.1359, found 414.1361. 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(1-Phenyl-1H-pyrazol3-yl)azetidine-1-carboxylate (6). Step 1: tert-Butyl 3-(1-Phenyl1H-pyrazol-3-yl)azetidine-1-carboxylate. A mixture of tert-butyl 3[(2E)-3-(dimethylamino)prop-2-enoyl]azetidine-1-carboxylate (800 mg, 3.15 mmol) and phenyl hydrazine (340 mg, 3.15 mmol) in EtOH (10 mL) was heated to reflux and stirred for 16 h. The reaction mixture was concentrated in vacuo and purified by silica gel chromatography to give a mixture of tert-butyl 3-(1-phenyl-1Hpyrazol-3-yl)azetidine-1-carboxylate (200 mg, 21.25%) and tert-butyl 3-(1-phenyl-1H-pyrazol-5-yl)azetidine-1-carboxylate (280 mg, 29.8%) as a brown solid. Data for tert-butyl 3-(1-phenyl-1H-pyrazol-3yl)azetidine-1-carboxylate: 1H NMR (400 MHz, CDCl3) δ ppm 7.82 (d, J = 2.4 Hz, 1 H), 7.65−7.57 (m, 2 H), 7.37 (t, J = 7.9 Hz, 2 H), 7.24−7.18 (m, 1 H), 6.37 (d, J = 2.4 Hz, 1 H), 4.35−4.19 (m, 2 H), 4.04 (dd, J = 8.4 and 6.2 Hz, 2 H), 3.83 (s, 1 H), 1.45−1.32 (m, 9 H). Step 2: 3-(Azetidin-3-yl)-1-phenyl-1H-pyrazole. To a solution of tert-butyl 3-(1-phenyl-1H-pyrazol-3-yl)azetidine-1-carboxylate (200 mg, 0.67 mmol) in EtOAc (0.5 mL) was added a solution of HCl in MeOH (1 mL, 4 mmol, 4 M) dropwise at 0 °C. The resulting mixture was stirred at room temperature (25 °C) for 1 h. The reaction was concentrated in vacuo and the residue was triturated with MTBE (20 mL × 3) and evaporated to give the 150 mg (97%) of the crude 3(azetidin-3-yl)-1-phenyl-1H-pyrazole HCl salt as a dark yellow solid, which was used in the next step directly. Step 3: 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(1-phenyl-1H-pyrazol-3-yl)azetidine-1-carboxylate (6). A solution of hexafluoroisopropanol (108 mg, 0.64 mmol), DMAP (8.3 mg, 0.06 mmol), and DIPEA (99 mg, 0.77 mmol) in CH2Cl2 (5 mL) was added dropwise to a solution, precooled (0 °C, under an N2 atm), of triphosgene (63 mg, 0.21 mmol) in CH2Cl2 (3 mL). The resulting mixture was stirred at 25 °C for 16 h. This solution was then added to a stirring solution of 3-(azetidin-3-yl)-1-phenyl-1H-pyrazole hydrochloride (150 mg, 0.64 mmol) and DIPEA (248 mg, 1.92 mmol) in CH2Cl2 (2 mL). The resulting mixture was stirred at 25 °C for 6 h. The reaction mixture was quenched with water (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layer was dried over sodium sulfate, filtered, concentrated in vacuo, and purified by prepHPLC to give 50 mg (20%) of 1,1,1,3,3,3-hexafluoropropan-2-yl 3-(1phenyl-1H-pyrazol-3-yl)azetidine-1-carboxylate (6) as a white solid. 1 H NMR (400 MHz, CDCl3) δ ppm 7.91 (d, J = 2.5 Hz, 1 H), 7.67 (d, J = 8.0 Hz, 2 H), 7.46 (s, 2 H), 7.26 (s, 1 H), 6.42 (d, J = 2.5 Hz, 1 H), 5.70 (dt, J = 12.5 and 6.2 Hz, 1 H), 4.59−4.43 (m, 2 H), 4.42−4.25 (m, 2 H), 4.13−3.99 (m, 1 H). 13C NMR (101 MHz, CDCl3) δ 153.6, 151.8, 139.9, 129.5, 128.1, 126.6, 120.6 (q, 1JCF = 282.4 Hz), 119.1, 105.3, 67.6 (sep, 2JCF = 34.5 Hz), 56.2, 55.5, 28.2. HRMS calculated for C16H13F6N3O2 [M + H]+ 396.0777, found 396.0766. 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(1-Methyl-1H-pyrazol3-yl)azetidine-1-carboxylate (7). To a solution of triphosgene (89 mg, 0.3 mmol) in CH2Cl2 (1 mL) was added a solution of 1,1,1,3,3,3-hexafluoropropan-2-ol (153 mg, 0.9 mmol), DIPEA (118 mg, 0.9 mmol), and DMAP (11 mg, 0.09 mmol) in CH2Cl2 (1 mL) dropwise at −10 °C. To this solution was added a solution of 3(azetidin-3-yl)-1-methyl-1H-pyrazole (125 mg, 0.9 mmol) and DIPEA (353 mg, 2.7 mmol) in CH2Cl2 (1 mL), and the resulting mixture was

UHPLC/MS Analysis. The UHPLC was performed on a Waters ACQUITY UHPLC system (Waters, Milford, MA), which was equipped with a binary solvent delivery manager, column manager, and sample manager coupled to ELSD and UV detectors (Waters, Milford, MA). Detection was performed on a Waters LCT premier XE mass spectrometry (Waters, Milford, MA). The instrument was fitted with an Acquity BEH (Bridged Ethane Hybrid) C18 column (30 mm × 2.1 mm, 1.7 μm particle size, Waters, Milford, MA) operated at 60 °C. 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(3-Phenyl-1,2,4-oxadiazol-5-yl)azetidine-1-carboxylate (3). Step 1. To a solution of Nhydroxybenzenecarboximidamide (6.5 g, 48 mmol), 1-(tertbutoxycarbonyl)azetidine-3-carboxylic acid (11.5 g, 57 mmol), and HATU (21.8 g, 57 mmol) in CH2Cl2 (80 mL) at room temperature was added DIPEA (18.5 g, 143 mmol), and the resulting mixture was stirred for 16 h. The mixture was then portioned with water (40 mL × 3) and then saturated sodium chloride (40 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was dissolved in NMP (80 mL) and heated to 140 °C for 5 h. The mixture was cooled to room temperature, poured onto water, and extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with water and saturated sodium chloride, dried over sodium sulfate, and concentrated in vacuo. The residue was purified on a silica gel column (gradient, petroleum ether to 1:1 petroleum ether/ethyl acetate) to afford 10.6 g (74% yield) of tert-butyl 3-(3-phenyl-1,2,4oxadiazol-5-yl)azetidine-1-carboxylate as a yellow oil Step 2. To a mixture of tert-butyl 3-(3-phenyl-1,2,4-oxadiazol-5yl)azetidine-1-carboxylate (3.5 g, 11.6 mmol) in ethyl acetate (10 mL) was added a solution of HCl in ethyl acetate (4 M, 40 mL), and the reaction was stirred at room temperature for 30 min. The reaction was then concentrated in vacuo to give 2.62 g (95%) of the HCl salt of 5(azetidin-3-yl)-3-phenyl-1,2,4-oxadiazole as a white solid. Step 3. To a solution of bis(trichloromethyl) carbonate (252 mg, 0.84 mmol) in CH2Cl2(40 mL) were added 1,1,1,3,3,3-hexafluoropropan-2-ol (426 mg, 2.52 mmol) and DIPEA (330 mg, 2.52 mmol) at 0 °C under N2. The reaction mixture was stirred at 20 °C for 7 h. A solution of 5-(azetidin-3-yl)-3-phenyl-1,2,4-oxadiazole hydrochloride (600 mg, 2.52 mmol) and DIPEA (1.3 g, 10 mmol) in CH2Cl2 (10 mL) was added to the mixture and then was stirred at 20 °C for 16 h. The reaction mixture was then diluted with CH2Cl2 and washed with water (50 mL × 3) and saturated sodium chloride (50 mL), dried over sodium sulfate, and concentrated in vacuo to give the crude product. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate, 5:1) to give 470 mg (47%) of 1,1,1,3,3,3hexafluoropropan-2-yl 3-(3-phenyl-1,2,4-oxadiazol-5-yl)azetidine-1carboxylate (3) as a clear oil. 1H NMR (400 MHz, CD3OD) δ ppm 8.11- 8.07 (m, 2 H), 7.59−7.49 (m, 3 H), 6.12 (dt, J = 12.5 and 6.3 Hz, 1 H), 4.68−4.53 (m, 2 H), 4.51−4.38 (m, 2 H), 4.38−4.30 (m, 1 H). 13C NMR (101 MHz, CDCl3) δ 177.9, 168.7, 151.4, 131.5, 129.0, 127.5, 126.2, 120.5 (q, 1JCF = 283.2 Hz), 67.8 (sep, 2JCF = 34.5 Hz), 54.1, 53.4, 26.3. LC/MS (ESI, m/z) 396.0 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(2-Morpholinopyrimidin-4-yl)azetidine-1-carboxylate (4). To a solution of triphosgene (38 mg, 0.13 mmol) in CH2Cl2 (5 mL) were added 1,1,1,3,3,3-hexafluoropropan-2-ol (66 mg, 0.39 mmol) and DIPEA (50 mg, 0.39 mmol) at 0 °C under N2. The reaction mixture was stirred at 20 °C for 4 h. To it were added 4-(4-(azetidin-3-yl)pyrimidin-2yl)morpholine (100 mg, 0.39 mmol) and DIEA (202 mg, 1.56 mmol) in CH2Cl2 (5 mL), and the reaction mixture was stirred at 20 °C for 16 h. The reaction mixture was partitioned between CH2Cl2,(20 mL) and water (30 mL × 3) and then washed with saturated sodium chloride (30 mL), dried over sodium sulfate, concentrated in vacuo to give the crude product which was purified by prep TLC on silica gel (petroleum ether/ethyl acetate, 2:1) to afford 57 mg (35%) of 1,1,1,3,3,3-hexafluoropropan-2-yl 3-(2-morpholinopyrimidin-4-yl)azetidine-1-carboxylate (4) as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm 8.26 (d, J = 5.1 Hz, 1 H), 6.54 (d, J = 4.8 Hz, 1 H), 6.09 (spt, J = 6.4 Hz, 1 H), 4.47−4.34 (m, 2 H), 4.34−4.20 (m, 2 H), 3.87−3.82 (m, 1 H), 3.81−3.70 (m, 8 H). LC/MS (ESI, m/z) 415.3 [M + H]+. 9869



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stirred at 15 °C overnight. The reaction mixture was then filtered, concentrated, and purified by prep TLC (30% ethyl acetate in petroleum ether) to give 20 mg (7%) of 1,1,1,3,3,3-hexafluoropropan2-yl 3-(1-methyl-1H-pyrazol-3-yl)azetidine-1-carboxylate (7). 1H NMR (400 MHz, CD3OD) δ ppm 7.82−7.29 (m, 1 H), 6.24 (d, J = 2.2 Hz, 1 H), 6.05 (dquin, J = 12.7 and 6.4 Hz, 1 H), 4.53−4.35 (m, 2 H), 4.24−4.08 (m, 2 H), 3.94 (tt, J = 8.8 and 6.3 Hz, 1 H), 3.85 (s, 3 H). 13C NMR (101 MHz, CD3OD) δ 153.75, 153.34, 133.44, 104.63, 68.88 (sep, 2JCF = 34.06 Hz), 57.69, 57.01, 38.87, 29.25, CF3 carbons not observed. HRMS calculated for C11H11F6N3O2 [M + H]+ 332.0828, found 332.0825. 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(1,5-Diphenyl-1H-pyrazol-3-yl)azetidine-1-carboxylate (8). To a solution of triphosgene (31 mg, 0.11 mmol) in CH2Cl2 (10 mL) were added 1,1,1,3,3,3hexafluoroisopropanol (54 mg, 0.32 mmol) and DIPEA (41 mg, 0.32 mmol) at 0 °C under N2. The reaction mixture was stirred at 27 °C for 7 h. To this solution was added a solution of 3-(azetidin-3-yl)-1,5diphenyl-1H-pyrazole (100 mg, 0.32 mmol) and DIPEA (166 mg, 1.3 mmol) in CH2Cl2 (5 mL), and then the reaction mixture was stirred at 27 °C for 16 h. The reaction mixture was then partitioned between CH2Cl2 (15 mL) and water (50 mL), and the organic layer was washed with water (50 mL × 3) and saturated sodium chloride (50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give the crude product, which was purified by prep TLC (petroleum ether/EtOAc 6:1) to give 71 mg (46%) of 1,1,1,3,3,3-hexafluoropropan-2-yl 3-(1,5-diphenyl-1H-pyrazol-3-yl)azetidine-1-carboxylate (8) as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm 7.64− 7.01 (m, 11 H), 6.71−6.53 (m, 1 H), 6.19−5.97 (m, 1 H), 4.61−4.44 (m, 2 H), 4.38−4.22 (m, 2 H), 4.14−3.98 (m, 1 H). 13C NMR (101 MHz, CDCl3) δ 152.6, 151.7, 144.5, 139.8, 130.2, 129.0, 128.6, 128.5, 128.4, 127.6, 125.2, 120.6 (q, 1JCF = 282.4 Hz), 105.3, 67.6 (sep, 2JCF = 34.5 Hz), 56.2, 55.6, 28.2. LC/MS (ESI, m/z) 470.0 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-[1-(3,4-Difluorophenyl)1H-pyrazol-3-yl]azetidine-1-carboxylate (9). Step 1: tert-Butyl 3[1-(3,4-Difluorophenyl)-1H-pyrazol-3-yl]azetidine-1-carboxylate (A). A mixture of tert-butyl 3-(1H-pyrazol-3-yl)azetidine-1-carboxylate (200 mg, 0.9 mmol), 3,4-difluorophenylboronic acid (170 mg, 1.1 mmol), Cu(OAc)2 (244 mg, 1.3 mmol), and pyridine (213 mg, 2.7 mmol) in CH2Cl2 (30 mL) was stirred at 40 °C for 20 h. The reaction mixture was then filtered, and the filtrate was diluted with water (20 mL) and extracted with CH2Cl2 (20 mL × 3). The combined organic layer was washed with water (20 mL), saturated sodium chloride (20 mL), dried over sodium sulfate, concentrated in vacuo, and purified by silica gel chromatography (ethyl acetate/petroleum ether, gradient 0− 10%,) to give 150 mg (50%) of tert-butyl 3-[1-(3,4-difluorophenyl)1H-pyrazol-3-yl]azetidine-1-carboxylate (A) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.82 (d, J = 2.5 Hz, 1 H), 7.56 (ddd, J = 11.2, 6.9, and 2.5 Hz, 1 H), 7.40- 7.33 (m, 1 H), 7.28−7.11 (m, 1 H), 6.44 (d, J = 2.5 Hz, 1 H), 4.36−4.26 (m, 2 H), 4.13−4.05 (m, 2 H), 3.87 (tt, J = 8.7, 6.1 Hz, 1 H), 1.50−1.41 (m, 9 H). Step 2: 3-(Azetidin-3-yl)-1-(3,4-difluorophenyl)-1H-pyrazole (B). To a solution of A (150 mg, 0.45 mmol) in CH2Cl2 (0.5 mL) was added a solution of HCl in EtOAc (0.56 mL 2.2 mmol, 4 M) dropwise at 0 °C. The resulting mixture was stirred at room temperature for 1 h and then concentrated in vacuo. The residue was triturated with TBME (10 mL × 3) and then concentrated to give 120 mg (99%) of crude 3-(azetidin-3-yl)-1-(3,4-difluorophenyl)-1H-pyrazole (B) as a white solid, which was used in the next step without purification. Step 3: 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-[1-(3,4-difluorophenyl)-1H-pyrazol-3-yl]azetidine-1-carboxylate (9). A solution of hexafluoroisopropanol (66.0 mg, 0.39 mmol), DMAP (4.8 mg, 0.04 mmol), and DIPEA (61 mg, 0.47 mmol) in CH2Cl2 (5 mL) was added dropwise to a solution of triphosgene (38.5 mg, 0.13 mmol) in CH2Cl2 (3 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at 30 °C for 16 h. The solution was then added to a solution of B (120 mg, 0.39 mmol) and DIPEA (152 mg, 1.2 mmol) in CH2Cl2 (2 mL). The resulting mixture was stirred at 30 °C for 6 h. The reaction mixture was quenched with water (20 mL) and extracted with CH2Cl2 (20 mL × 3). The combined organic layer was dried over sodium sulfate, filtered, concentrated in vacuo, and purified by prep-

HPLC to give 24 mg (14%) of 1,1,1,3,3,3-hexafluoropropan-2-yl 3-[1(3,4-difluorophenyl)-1H-pyrazol-3-yl]azetidine-1-carboxylate (9) as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm 8.26−8.14 (m, 1 H), 7.75 (ddd, J = 11.7, 7.1, and 2.6 Hz, 1 H), 7.63−7.52 (m, 1 H), 7.45−7.28 (m, 1 H), 6.51 (d, J = 2.4 Hz, 1 H), 6.16−5.99 (m, 1 H), 4.58−4.40 (m, 2 H), 4.34−4.19 (m, 2 H), 4.05 (tt, J = 8.9 and 6.1 Hz, 1 H). LCMS m/z 430.1 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-[1-(pyrazin-2-yl)-1Hpyrazol-3-yl]azetidine-1-carboxylate (10). Step 1: tert-Butyl 3[1-(Pyrazin-2-yl)-1H-pyrazol-3-yl]azetidine-1-carboxylate. To a mixture of tert-butyl 3-(1H-pyrazol-3-yl)azetidine-1-carboxylate (100 mg, 0.45 mmol), 2-bromopyrazine (92 mg, 0.45 mmol), Pd(OAc)2 (15 mg, 0.067 mmol), and X-Phos (64 mg, 0.13 mmol) in THF (10 mL) was added Cs2CO3 (292 mg, 0.90 mmol). The reaction mixture was stirred under a N2 atmosphere and heated to reflux for 18 h. The reaction mixture was then filtered, and filtrate was diluted with EtOAc (20 mL). The solution was then washed with water (20 mL), saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated in vacuo, and purified by prep TLC (petroleum ether/ EtOAc, 1:1) to give 50 mg (37%) of tert-butyl 3-[1-(pyrazin-2-yl)-1Hpyrazol-3-yl]azetidine-1-carboxylate. 1H NMR (400 MHz, CD3OD) δ ppm 9.39−9.04 (m, 1 H), 8.55 (d, J = 2.7 Hz, 1 H), 8.49−8.46 (m, 1 H), 8.44 (dd, J = 2.7 and 1.5 Hz, 1 H), 6.57 (d, J = 2.7 Hz, 1 H), 4.41− 4.21 (m, 2 H), 4.16−4.04 (m, 2 H), 4.01−3.85 (m, 1 H) 1.45 (s, 9 H) Step 2: 2-[3-(Azetidin-3-yl)-1H-pyrazol-1-yl]pyrazine. A solution of tert-butyl 3-[1-(pyrazin-2-yl)-1H-pyrazol-3-yl]azetidine-1-carboxylate (50 mg, 0.17 mmol) in HCl/EtOAc (5 mL, 4 M) was stirred at rt for 1 h. The reaction was then concentrated in vacuo to afford 33 mg (96%) of 2-[3-(azetidin-3-yl)-1H-pyrazol-1-yl]pyrazine as a light oil which was used in the next step without further purification. Step 3: 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-[1-(Pyrazin-2-yl)-1Hpyrazol-3-yl]azetidine-1-carboxylate (10). To a solution of triphosgene (16.2 mg, 0.055 mmol) in CH2Cl2(1 mL) was added a solution of hexafluoroisopropanol (27.6 mg, 0.16 mmol), DIPEA (21.2 mg, 0.16 mmol), and DMAP (0.2 mg, 1.6 μmol) in CH2Cl2 (1 mL) dropwise at 0 °C. The mixture was stirred at rt for 7 h. A solution of 2[3-(azetidin-3-yl)-1H-pyrazol-1-yl]pyrazine (33 mg, 0.16 mmol) and DIPEA (63.6 mg, 0.49 mmol) in CH2Cl2 (3 mL) was then added to the above solution. The resultant reaction mixture was stirred at rt for an additional 18 h. The reaction mixture was concentrated in vacuo and then dissolved in EtOAc (10 mL), washed with water (10 mL × 2) and saturated sodium chloride, then dried over anhydrous sodium sulfate and filtered. The solution was then concentrated in vacuo and purified by prep-HPLC to afford 18 mg (28%) of 1,1,1,3,3,3hexafluoropropan-2-yl 3-[1-(pyrazin-2-yl)-1H-pyrazol-3-yl]azetidine1-carboxylate as a solid. 1H NMR (400 MHz, CD3OD) δ ppm 9.39−9.04 (m, 1 H), 8.55 (d, J = 2.7 Hz, 1 H), 8.49−8.46 (m, 1 H), 8.44 (dd, J = 2.7 and 1.5 Hz, 1 H), 6.57 (d, J = 2.7 Hz, 1 H), 4.41− 4.21 (m, 2 H), 4.16−4.04 (m, 2 H), 4.01−3.85 (m, 1 H), 1.45 (s, 9 H). LCMS m/z 395.7 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(1-Benzyl-1H-pyrazol3-yl)azetidine-1-carboxylate (11). Step 1: tert-Butyl 3-(1HPyrazol-3-yl)azetidine-1-carboxylate. A mixture of 1-azetidinecarboxylic acid, 3-[(2E)-3-(dimethylamino)-1-oxo-2-propen-1-yl]-, 1,1-dimethylethyl ester37 (1.9 g, 7.5 mmol), and hydrazine hydrate (412 mg, 8.2 mmol) in EtOH (20 mL) was heated to 80 °C and stirred for 16 h. The reaction was concentrated in vacuo to afford 1.8 g (112%) of crude tert-butyl 3-(1H-pyrazol-3-yl)azetidine-1-carboxylate as a brown oil, which was used for next step directly. 1H NMR (400 MHz, CDCl3) δ ppm 7.56 (d, J = 2.3 Hz, 1 H), 6.30 (d, J = 2.3 Hz, 1 H), 4.31 (t, J = 8.66 Hz, 2 H), 4.04 (dd, J = 8.5 and 6.0 Hz, 2 H), 3.90−3.79 (m, 1 H), 1.48−1.41 (m, 9 H). Step 2: tert-Butyl 3-(1-Benzyl-1H-pyrazol-3-yl)azetidine-1-carboxylate. A mixture of tert-butyl 3-(1H-pyrazol-3-yl)azetidine-1carboxylate (300 mg, 1.3 mmol), benzyl bromide (345 mg, 2.0 mmol), cesium carbonate (1.31 g, 4.0 mmol), and potassium iodide (11.2 mg, 0.067 mmol) in CH3CN (10 mL) was stirred at 25 °C for 16 h. The reaction mixture was diluted with water, extracted with EtOAc (30 mL × 3), washed with saturated sodium chloride (30 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The 9870



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1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(1-(4-Ethynylphenyl)1H-pyrazol-3-yl)azetidine-1-carboxylate (14). Step 1: tert-Butyl 3-(1-(4-((Trimethylsilyl)ethynyl)phenyl)-1H-pyrazol-3-yl)azetidine-1carboxylate. Nitrogen was bubbled through a solution of 13 (500 mg, 1.32 mmol) in triethylamine (7 mL) and dimethylformamide (1 mL) for 15 min. To this was added ethynyltrimethylsilane (0.38 mL, 260 mg, 2.6 mmol), PdCl2(PPh3)2 (189 mg, 0.264 mmol), and CuI (25 mg, 0.26 mmol), and the reaction was heated to 70 °C. After 3 h, the reaction was cooled to room temperature and poured over water. The mixture was extracted with dichloromethane (3×), and the combined organic fraction was dried with anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel flash chromatography (EtOAc/heptane) to afford 450 mg (86%) of tert-butyl 3-(1-(4-((trimethylsilyl)ethynyl)phenyl)-1H-pyrazol-3-yl)azetidine-1-carboxylate as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 1.6 Hz, 1H), 7.64−7.58 (m, 2H), 7.57−7.51 (m, 2H), 6.45 (d, J = 1.6 Hz, 1H), 4.32 (app t, J = 8.6 Hz, 2H), 4.12−4.06 (m, 2H), 3.95−3.84 (m, 1H), 1.46 (s, 9H), 0.26 (s, 9H). MS ES+ [2M + H]+ 791.3. Step 2: tert-Butyl 3-(1-(4-Ethynylphenyl)-1H-pyrazol-3-yl)azetidine-1-carboxylate. To a solution of tert-butyl 3-(1-(4((trimethylsilyl)ethynyl)phenyl)-1H-pyrazol-3-yl)azetidine-1-carboxylate (430 mg, 1.1 mmol) in methanol (5 mL) was added potassium carbonate (455 mg, 3.26 mmol). The reaction mixture was stirred at room temperature for 1 h and then poured over 1 N KHSO4. The mixture was extracted with CH2Cl2 (3×), and the combined organic fraction was dried with anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel flash chromatography (EtOAc/heptane) to afford 298 mg (85%) of tert-butyl 3-(1-(4-ethynylphenyl)-1H-pyrazol-3-yl)azetidine-1-carboxylate as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 2.5 Hz, 1H), 7.66−7.62 (m, 2H), 7.59−7.54 (m, 2H), 6.45 (d, J = 2.5 Hz, 1H), 4.32 (app t, J = 8.6 Hz, 2H), 4.10 (dd, J = 6.1, 8.6 Hz, 2H), 3.89 (tt, J = 6.0, 8.8 Hz, 1H), 3.12 (s, 1H), 1.46 (s, 9H). MS ES+ [M + H]+ 324.1. 3-(Azetidin-3-yl)-1-(4-ethynylphenyl)-1H-pyrazole). To a solution of tert-butyl 3-(1-(4-ethynylphenyl)-1H-pyrazol-3-yl)azetidine-1-carboxylate (272 mg, 0.84 mmol) in CH2Cl2 (4 mL) was added trifluoroacetic acid (1 mL). The reaction mixture was stirred at rt for 2 h 30 min and then poured over 1 N NaOH. The mixture was extracted with CH2Cl2 (3×), and the combined organic fraction was dried with anhydrous MgSO4, filtered, and concentrated in vacuo to afford 180 mg (96%) of 3-(azetidin-3-yl)-1-(4-ethynylphenyl)-1H-pyrazole) as a white foam which was used directly in the subsequent step. 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(1-(4-Ethynylphenyl)1H-pyrazol-3-yl)azetidine-1-carboxylate (14). To a solution of 1,1′-carbonyldiimidazole (91 mg, 0.56 mmol) in tetrahydrofuran (4 mL) was added 1,1,1,3,3,3-hexafluoro-2-propanol (0.47 mL, 750 mg, 4.5 mmol). The reaction mixture was stirred at room temperature for 15 min, and then 3-(azetidin-3-yl)-1-(4-ethynylphenyl)-1H-pyrazole) (50 mg, 0.22 mmol) was added in one portion. The reaction mixture was stirred an additional 2 h at room temperature and then concentrated under reduced pressure. The crude pale yellow oil was purified by silica gel flash chromatography (EtOAc/heptane) to afford 14 (36 mg, 39% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 2.3 Hz, 1H), 7.67−7.61 (m, 2H), 7.61−7.54 (m, 2H), 6.42 (d, J = 2.7 Hz, 1H), 5.69 (sep, J = 6.2 Hz, 1H), 4.55−4.44 (m, 2H), 4.38−4.26 (m, 2H), 4.02 (tt, J = 6.2, 9.0 Hz, 1H), 3.13 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 154.02, 151.73, 139.73, 133.36, 128.01, 120.64 (q, 1JCF = 281.70 Hz), 120.13, 118.50, 105.89, 82.79, 77.98, 67.60 (sep, 2JCF = 34.48 Hz), 56.08, 55.43, 28.19. HRMS calculated for C18H14F6N3O2 [M + H]+ 418.0985, found 418.0984. 1,1,1,3,3,3-Hexafluoropropan-2-yl 4-(1-Phenyl-1H-pyrazol3-yl)piperidine-1-carboxylate (15). Step 1: tert-Butyl (E)-4-(3(Dimethylamino)acryloyl)piperidine-1-carboxylate. tert-Butyl 4-acetylpiperidine-1-carboxylate (1 g, 4.4 mmol) was dissolved in N,Ndimethylformamide dimethyl acetal (15 mL), and the mixture was warmed to 110 °C and stirred for 40 h. The reaction mixture was then concentrated in vacuo to give 1.24 g of tert-butyl (E)-4-(3-

residue was purified by prep-HPLC to give 186 mg (44%) of tert-butyl 3-(1-benzyl-1H-pyrazol-3-yl)azetidine-1-carboxylate as a pale yellow gum. Step 3: 3-(Azetidin-3-yl)-1-benzyl-1H-pyrazole. A solution of tertbutyl 3-(1-benzyl-1H-pyrazol-3-yl)azetidine-1-carboxylate (210 mg, 0.67 mmol) and trifluoroacetic acid (1.5 mL) in CH2Cl2 (6.0 mL) was stirred at 20 °C for 2 h. The reaction mixture was concentrated in vacuo to give 305 mg of 3-(azetidin-3-yl)-1-benzyl-1H-pyrazole as yellow oil, which was directly used in the next step. Step 4: 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(1-Benzyl-1H-pyrazol-3-yl)azetidine-1-carboxylate (11). To a solution of 1,1,1,3,3,3hexafluoropropan-2-ol (500 mg, 2.98 mmol) in anhydrous CH3CN (8 mL) was added bis(perfluorophenyl) carbonate (1.1 g, 2.8 mmol) at 10 °C under a nitrogen atmosphere and was then stirred for 30 min at 22 °C. The reaction was cooled to 5 °C, and Et3N (2.27 g, 22 mmol) was added. The reaction was allowed to warm to rt and stirred at 22 °C for 2 h to give a light purple solution. The solution was then cooled to 5 °C and added dropwise to a mixture of 3-(azetidin-3-yl)-1-benzyl1H-pyrazole (305 mg, 0.69 mmol) and Et3N (350 mg, 3.5 mmol) in anhydrous CH3CN (8 mL) at 5 °C. The reaction mixture was allowed to warm to rt and stirred at 22 °C for 16 h, then concentrated in vacuo, and the residue was purified by prep-HPLC to give 102 mg (36%) of 1,1,1,3,3,3-hexafluoropropan-2-yl 3-(1-benzyl-1H-pyrazol-3yl)azetidine-1-carboxylate as clear oil. 1H NMR (400 MHz, CDCl3) δ ppm 7.40−7.29 (m, 4 H), 7.24−7.18 (m, 2 H), 6.23 (d, J = 2.5 Hz, 1 H), 5.69 (spt, J = 6.3 Hz, 1 H), 5.28 (s, 2 H), 4.47 (dt, J = 12.1 and 9.0 Hz, 2 H), 4.30−4.20 (m, 2 H), 3.97 (tt, J = 8.9 and 6.4 Hz, 1 H). HRMS (ESI) m/z calculated for C17H15F6N3O2H+ [M + H+]: 408.1127. Found 408.1141. 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(1-(Tetrahydro-2Hpyran-4-yl)-1H-pyrazol-3-yl)azetidine-1-carboxylate (12). Step 1: tert-Butyl 3-(1-(Tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)azetidine-1-carboxylate. To a solution of tert-butyl 3-(1H-pyrazol3-yl)azetidine-1-carboxylate (400 mg, 1.8 mmol) and tetrahydro-2Hpyran-4-yl methanesulfonate (390 mg, 2.2 mmol) in DMF (10 mL) was added cesium carbonate (1.75 g, 5.4 mmol). The resulting mixture was stirred at 120 °C for 16 h. The mixture was then cooled and diluted with water (50 mL), extracted with EtOAc (20 mL × 3), washed with saturated sodium chloride (30 mL), dried over sodium sulfate, filtered, and concentrated to give the crude product which was purified by prep-HPLC to give 230 mg (42%) of tert-butyl 3-(1(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)azetidine-1-carboxylate. Step 2: 3-(Azetidin-3-yl)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole. A solution of tert-butyl 3-(1-(tetrahydro-2H-pyran-4-yl)-1Hpyrazol-3-yl)azetidine-1-carboxylate (220 mg, 0.72 mmol) and trifluoroacetic acid (2.2 mL) in CH2Cl2 (8.0 mL) was stirred at 25 °C for 3 h. The solution was then concentrated in vacuo to give 312 mg of 3-(azetidin-3-yl)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole as a yellow oil. Step 3: 1,1,1,3,3,3-Hexafluoropropan-2-yl 3-(1-(Tetrahydro-2Hpyran-4-yl)-1H-pyrazol-3-yl)azetidine-1-carboxylate (12). To a solution of 1,1,1,3,3,3-hexafluoropropan-2-ol (540 mg, 3.2 mmol) in anhydrous CH3CN (8 mL) was added bis(perfluorophenyl) carbonate (1.1 g, 2.8 mmol) at 10 °C under a nitrogen atmosphere, and the mixture was then stirred for 30 min at 22 °C. The reaction was then cooled to 5 °C, and Et3N (1.6 g, 15.8 mmol) was added. The reaction was allowed to warm to rt and stirred for 2 h. This solution was then cooled to 5 °C and added dropwise to a solution of 3-(azetidin-3-yl)-1(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (280 mg, 0.64 mmol) and Et3N (325 mg, 3.2 mmol) in anhydrous CH3CN (8 mL). The reaction mixture was allowed to warm to rt and stirred at 22 °C for 15 h, then concentrated, and the crude product was purified by prep-HPLC to give 63 mg (24% over 3 steps) of 1,1,1,3,3,3-hexafluoropropan-2-yl 3(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)azetidine-1-carboxylate as a clear oil. 1H NMR (400 MHz, CDCl3) δ ppm 7.40 (d, J = 3.0 Hz, 1 H), 6.19 (d, J = 3.0 Hz, 1 H), 5.67 (spt, J = 6.3 Hz, 1 H), 4.48−4.37 (m, 2 H), 4.34−4.25 (m, 1 H), 4.25−4.16 (m, 2 H), 4.12−4.03 (m, 2 H), 3.92 (tt, J = 9.0 and 6.0 Hz, 1 H), 3.56−3.46 (m, 2 H), 2.10−1.96 (m, 4 H). HRMS (ESI) m/z calculated for C15H17F6N3O3H+ [M + H+]: 402.1244. Found 402.1247. 9871



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(dimethylamino)acryloyl)piperidine-1-carboxylate as brown oil, which was used directly in the next step. Step 2: tert-Butyl 4-(1H-Pyrazol-3-yl)piperidine-1-carboxylate. Hydrazine monohydrate (659 mg, 6.6 mmol, 50% purity) was added to a solution of crude tert-butyl (E)-4-(3-(dimethylamino)acryloyl)piperidine-1-carboxylate (1240 mg, 4.4 mmol) in ethanol (25 mL) at 15 °C. The reaction mixture was warmed to 85 °C and stirred for 20 h. The mixture was then cooled and concentrated in vacuo, and the resulting residue was purified by silica gel flash chromatography (EtOAc/petroleum ether) to give 610 mg (55%) of tert-butyl 4-(1Hpyrazol-3-yl)piperidine-1-carboxylate as yellow oil. 1H NMR (400 MHz, CDCl3) δ ppm 7.51 (d, J = 2.5 Hz, 1 H), 6.14 (d, J = 2.5 Hz, 1 H), 4.29−4.10 (m, 2 H), 2.97−2.79 (m, 3 H), 2.03−1.89 (m, 2 H), 1.64 (qd, J = 12.4 and 4.3 Hz, 2 H), 1.54−1.54 (m, 9 H). Step 3: tert-Butyl 4-(1-Phenyl-1H-pyrazol-3-yl)piperidine-1-carboxylate. A suspension of phenyl boronic acid (291 mg, 2.4 mmol), copper(II) acetate (21.7 mg, 0.12 mmol), and 4 Å molecular sieves (1.3 g) in CH2Cl2(15 mL) was stirred at 15 °C for 3 min. To this suspension was added tert-butyl 4-(1H-pyrazol-3-yl)piperidine-1carboxylate (300 mg, 1.2 mmol), and the mixture was stirred at 35 °C under O2 (g) . After 20 h the reaction mixture was concentrated in vacuo. Pyridine (15 mL) and copper(II) acetate (21.7 mg) were then added, and the reaction mixture was heated to 100 °C and stirred under O2 (g). After 20 h, the mixture was filtered and the filtrate was diluted with water and extracted with EtOAc (3×). The combined organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (EtOAc/petroleum ether) to afford 180 mg (46%) of tert-butyl 4-(1-phenyl-1H-pyrazol-3-yl)piperidine-1-carboxylate as a yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.61 (d, J = 2.0 Hz, 1 H), 7.55−7.36 (m, 5 H), 6.22 (d, J = 2.4 Hz, 1 H), 4.28−4.04 (m, 2 H), 2.89−2.76 (m, 1 H), 2.65 (t, J = 13.4 Hz, 2 H), 1.79 (d, J = 14.7 Hz, 2 H), 1.55 (d, J = 3.9 Hz, 2 H), 1.50−1.41 (m, 9 H). Step 4: 4-(1-Phenyl-1H-pyrazol-3-yl)piperidine. To a solution of tert-butyl 4-(1-phenyl-1H-pyrazol-3-yl)piperidine-1-carboxylate (180 mg, 0.55 mmol) in CH2Cl2 (4 mL) was added trifluoroacetic acid (1 mL). The reaction mixture was stirred at 20 °C for 2 h. The mixture was concentrated under reduced pressure to afford 188 mg of 4-(1phenyl-1H-pyrazol-3-yl)piperidine as a brown gum, which was used directly in the next step. Step 5: 1,1,1,3,3,3-Hexafluoropropan-2-yl 4-(1-Phenyl-1H-pyrazol-3-yl)piperidine-1-carboxylate (15). To a solution of 1,1,1,3,3,3,hexafluoropropan-2-ol (140 mg, 0.83 mmol) in anhydrous acetonitrile (5 mL) were added bis(pentafluorophenyl)carbonate (328 mg, 0.83 mmol) and triethylamine (337 mg, 3.3 mmol) at 0 °C. The solution was stirred at 20 °C under N2 for 2 h to afford a purple solution. Separately, a solution of 4-(1-phenyl-1H-pyrazol-3-yl)piperidine (188 mg, 0.55 mmol) in anhydrous acetonitrile (5 mL) was treated with triethylamine (279 mg, 2.7 mmol) at 0 °C and stirred for 3 min, and then the above solution was added dropwise. The reaction mixture was stirred at 20 °C for 40 h and then concentrated in vacuo, and the residue was treated with additional 1,1,1,3,3,3-hexafluoropropan-2-yl (perfluorophenyl)carbonate (1.67 mmol in 10 mL of anhydrous acetonitrile), prepared as described above. The reaction mixture was stirred at 20 °C under N2. After 16 h, the reaction mixture was concentrated in vacuo and the residue was purified by reversed-phase HPLC (column, Agela Durashell C18 150 mm × 25 mm, 5 μm; mobile phase A of 0.05% NH4OH in water (v/v); mobile phase B of 0.05% NH4OH in MeCN (v/v); gradient from 58% to 78% B over 10 min, HOLD at 100% B for 2 min; flow rate of 35 mL/min) and then lyophilized to afford 80 mg (34%) of 15 as a white solid. 1H NMR (400 MHz, MeOD) δ ppm 8.09 (d, J = 2.4 Hz, 1 H), 7.73−7.66 (m, 2 H), 7.46 (t, J = 7.8 Hz, 2 H), 7.33−7.26 (m, 1 H), 6.38 (d, J = 2.9 Hz, 1 H), 6.21−6.11 (m, 1 H), 4.20 (td, J = 8.8 and 3.9 Hz, 2 H), 3.25− 3.08 (m, 2 H), 3.01 (tt, J = 11.5 and 3.8 Hz, 1 H), 2.11−2.02 (m, 2 H), 1.82−1.65 (m, 2 H). 13C NMR (101 MHz, MeOD) δ 158.93, 152.71, 141.51, 130.54, 129.56, 127.45, 123.84, 121.07, 120.20, 106.03, 69.16 (sep, 2JCF = 33.75 Hz), 46.13, 45.56, 36.51, 32.97, 32.59. HRMS calculated for C18H17F6N3O2 [M + H]+ 422.1298, found 422.1292.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01531. Data processing and model refinement statistics for MAGL complex with 3, replicates of human brain vascular pericytes treated with 14, and serine hydrolase selectivity panel for compounds 6 and 15 (PDF) Molecular formula strings and some data (CSV) Accession Codes

The PDB code for compound 3 bound to the active site serine in MAGL is 6AX1. Authors will release the atomic coordinates and experimental data upon article publication.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 617-395-0670. E-mail: christopher.r.butler@pfizer. com. ORCID

Christopher R. Butler: 0000-0002-9387-5011 Christopher W. am Ende: 0000-0001-8832-9641 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Steven V. O’Neil for helpful discussions in manuscript preparation. Use of the IMCA-CAT beamline 17ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357.

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ABBREVIATIONS USED AA, arachidonic acid; 2-AG, 2-arachidonoylglycerol; CC-ABPP, click chemistry activity based protein profiling; CNS, central nervous system; FAAH, fatty acid amide hydrolase; HFIP, hexafluoroisopropanol; LipE, lipophilic efficiency; FQ, fit quality; MAGL, monoacylglycerol lipase; PLA2, phospholipase A2

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REFERENCES

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