Identification of ABX-1431, a Selective Inhibitor of Monoacylglycerol

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Cite This: J. Med. Chem. 2018, 61, 9062−9084

Identification of ABX-1431, a Selective Inhibitor of Monoacylglycerol Lipase and Clinical Candidate for Treatment of Neurological Disorders Justin S. Cisar,† Olivia D. Weber,† Jason R. Clapper,† Jacqueline L. Blankman,† Cassandra L. Henry,† Gabriel M. Simon,‡ Jessica P. Alexander,† Todd K. Jones,† R. Alan B. Ezekowitz,† Gary P. O’Neill,† and Cheryl A. Grice*,† †

Abide Therapeutics, 10835 Road to the Cure, Suite 250, San Diego, California 92121, United States Vividion Therapeutics, 3565 General Atomics Court, Suite 100, San Diego, California 92121, United States

J. Med. Chem. 2018.61:9062-9084. Downloaded from pubs.acs.org by 95.181.176.235 on 10/25/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: The serine hydrolase monoacylglycerol lipase (MGLL) converts the endogenous cannabinoid receptor agonist 2-arachidonoylglycerol (2-AG) and other monoacylglycerols into fatty acids and glycerol. Genetic or pharmacological inactivation of MGLL leads to elevation in 2-AG in the central nervous system and corresponding reductions in arachidonic acid and eicosanoids, producing antinociceptive, anxiolytic, and antineuroinflammatory effects without inducing the full spectrum of psychoactive effects of direct cannabinoid receptor agonists. Here, we report the optimization of hexafluoroisopropyl carbamate-based irreversible inhibitors of MGLL, culminating in a highly potent, selective, and orally available, CNS-penetrant MGLL inhibitor, 28 (ABX-1431). Activity-based protein profiling experiments verify the exquisite selectivity of 28 for MGLL versus other members of the serine hydrolase class. In vivo, 28 inhibits MGLL activity in rodent brain (ED50 = 0.5−1.4 mg/kg), increases brain 2-AG concentrations, and suppresses pain behavior in the rat formalin pain model. ABX-1431 (28) is currently under evaluation in human clinical trials.

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with Δ9-tetrahydrocannabinol (THC) and other exocannabinoid CB1 agonists.5,15 Thus, MGLL inhibitors have potential to produce therapeutic effects through both enhanced CB1/2 signaling and reduced proinflammatory prostanoid action and may demonstrate significantly reduced side effects relative to direct CB1 receptor agonists.16,17 Several academic and industrial groups have reported MGLL inhibitors that act via reversible18−22 or irreversible mechanisms.23−30 Irreversible MGLL inhibitors (Figure 1B) such as JZL184 (1) carbamoylate the catalytic serine nucleophile, resulting in persistent enzyme inactivation.5 A distinct advantage of irreversible over reversible inhibitors is that pharmacodynamic effects may be maintained for extended periods with a low dose of administered drug because enzyme activity only returns in vivo following the synthesis of new protein (vs recovery shortly after compound clearance).31,32 Additionally, MGLL and other mechanistically related serine hydrolase enzymes can be assayed using activity-based protein profiling (ABPP) methods,33−35 which allow rapid assessment of compound selectivity in vitro and in living systems.36 As will be described below, we have leveraged ABPP as a core

INTRODUCTION Monoacylglycerol lipase (MGLL, also known as MAG lipase, MAGL, and MGL) is a serine hydrolase enzyme responsible for controlling the content and signaling of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system (Figure 1A).1−4 MGLL also regulates 2-AG and other MAG lipids in peripheral tissues.5 In select tissues, such as the brain, MGLL-mediated metabolism of 2-AG serves as an important source of arachidonic acid and contributes to subsequent biosynthesis of proinflammatory prostanoids.6 Thus, MGLL is a critical point of regulation of both the endocannabinoid and eicosanoid signaling pathways in the CNS and select peripheral tissues. Pharmacological inactivation of MGLL in animals increases 2-AG content of brain and peripheral tissues and has been found to produce antinociceptive, anxiolytic, and antiinflammatory effects that are dependent on CB1 and/or CB2 cannabinoid receptors.3,7−9 MGLL inhibitors also promote CB1/2-independent effects on neuroinflammation that may reflect reductions in proinflammatory prostanoid signaling in models of traumatic brain injury,10,11 neurodegeneration,6,12,13 and status epilepticus.14 Importantly, MGLL-mediated modulation of endocannabinoid signaling in animals does not produce the full spectrum of neurobehavioral effects observed © 2018 American Chemical Society

Received: June 14, 2018 Published: August 1, 2018 9062

DOI: 10.1021/acs.jmedchem.8b00951 J. Med. Chem. 2018, 61, 9062−9084

Journal of Medicinal Chemistry

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Figure 1. (A) Enzymatic reaction catalyzed by MGLL and (B) representative covalent MGLL inhibitors.

ery of 28 (ABX-1431),43 a highly potent, selective, and CNSpenetrant MGLL inhibitor with excellent drug-like properties suitable for once-per-day oral administration. Our lead optimization strategy was implemented using ABPP to measure the potency and selectivity of candidate MGLL inhibitors directly in native brain tissue proteomes. ABPP was also used to measure brain target engagement and selectivity in animals, which served to rapidly identify compounds with desirable CNS activity. Finally, we show that the optimized product of these efforts, 28, has low nanomolar potency for MGLL and is active at low doses in an animal model of pain.

screening technology to identify MGLL inhibitors with high potency and selectivity that would provide robust pharmacological responses at low compound exposures. The carbamate substructure is present in a number of selective covalent MGLL inhibitors (2−7, Figure 1B) that are active both in vitro and in vivo.36−38 At the outset of this program, both N-hydroxysuccinimidyl (NHS) carbamates39 and O-hexafluoroisopropyl (HFIP) carbamates24 were reported to inhibit MGLL in vitro and in vivo and demonstrated efficacy in preclinical models.40,41 While the NHS carbamates such as 4 have good proteome-wide selectivity,39 we observed aqueous instability due to hydrolysis of the imide carbonyl, thus posing a potential challenge for clinical development. As previously noted,24 the HFIP carbamate constitutes a reactive group of tempered electrophilicity (pKa value of hexafluoroisopropanol is ∼9.3) and the hexafluoroisopropanol component structurally resembles glycerol in the natural MAG substrates of MGLL. In our experience, the HFIP carbamate has excellent aqueous chemical stability over a broad pH range (typically >95% stable after 24 h at pH 2−8). Finally, the safety of hexafluoroisopropanol, which is presumably liberated upon MGLL carbamoylation, is supported by the clinical experience of the widely used anesthetic sevofluorane, of which hexafluoroisopropanol is a circulating metabolite.42 Thus, we focused our efforts on elaboration of the HFIP carbamate substructure to optimize both MGLL potency and drug-like properties.43−45 At the outset of these studies, the reported covalent MGLL inhibitors were limited by their selectivity and/or drug-like properties. More recent reports from Pfizer29 have demonstrated a series of MGLL inhibitors with high potency and selectivity and good physicochemical properties suitable for intravenous administration.30 Herein, we describe the discov-

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RESULTS AND DISCUSSION

Our goal was to develop a MGLL inhibitor that was highly potent and selective, orally active, CNS penetrant, and suitable for clinical use. While previously reported tool compounds and inhibitors lacked at least one of these critical properties, several HFIP carbamates such as KML29 (2)24 exhibited high potency and selectivity for MGLL. In general, the HFIP carbamate has high serine hydrolase selectivity; however, common off-targets include the serine hydrolases fatty acid amide hydrolase (FAAH),46 α/β hydrolase domain containing 6 (ABHD6),47 phospholipase A2 group VII (PLA2G7),48 and the carboxyesterases (CES).29,49 To optimize both MGLL potency and serine hydrolase selectivity simultaneously, our initial screening analysis included competitive gel-based ABPP methods50 in multiple human proteomes. Potency (IC50) measurements in native lysates derived from the human PC3 cell line using the HFIP carbamate ABPP probe JW91249 provided initial information about potency for MGLL and selectivity assessments for the off-targets ABHD6 and PLA2G7. Additional in vitro selectivity analysis was carried out in human post-mortem 9063



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prefrontal cortex proteome using a fluorophosphonate activity probe (FP-Rh),51 which enabled detection of many additional serine hydrolases enzymes, including FAAH. MGLL potency and off-target selectivity was first established for symmetrical biaryl analogues of 9, a previously reported24 MGLL inhibitor (Table 1). The symmetrical biaryl com-

Table 2. Pyrazole Analogues and in Vitro Potency for MGLL and the Off-Targets ABHD6 and PLA2G7

Table 1. Symmetrical Biaryl Analogues 9−13 and in Vitro Potency for MGLL and the Off-Targets ABHD6 and PLA2G7

a

IC50s determined using ABPP with a 30 min inhibitor preincubation in PC3 lysate. bcLogP values calculated using XLogP52 (Dotmatics).

improved selectivity profile. In analogues 21−23, the position of the chlorine substituent of 20 was varied. Chlorine was preferred at the R4 or R3 positions, with both analogues 20 and 23 displaying high MGLL potency and off-target selectivity. Analysis of in vitro metabolic stability in human liver microsomes indicated that the overall profile of 20 was superior to that of 23. On the basis of the properties of 20, we investigated additional R2/R4-substituted analogues to optimize selectivity and drug-like properties further, as shown in Table 4. Morpholine analogues 20 (R4 = Cl) and 24 (R4 = CF3) afforded similar MGLL and selectivity profiles; however, the trifluoromethyl analogue 24 possessed improved human liver microsome stability. Analogues containing morpholine (20, 24) and piperidine (25, 26) at the R4 position were all potent against human MGLL (PC3 lysates); however, 25 and 26 (R4 = piperidine) displayed significantly lower rodent MGLL potency (mouse brain homogenates) as compared to 20 and 24 (R4 = morpholine). Pyrrolidine analogue 28 (R4 = CF3) displayed high potency and selectivity for MGLL in human PC3 lysates and rodent MGLL brain homogenates. Furthermore, 28 demonstrated improved microsome stability as compared to 20 and 24. Interestingly, other R4 substituents such as chlorine (27) and bromine (29) gave similar MGLL potency and selectivity; however, the polar-aprotic sulfone moiety (30) significantly reduced selectivity against ABHD6 and PLA2G7. Introduction of an alkyne (31) or proton (32) at the R4 position resulted in a reduction of MGLL potency.

a

IC50s determined using ABPP with a 30 min inhibitor preincubation in PC3 lysate. bcLogP values calculated using XLogP52 (Dotmatics).

pounds in Table 1 included both phenyl (10) as well as heterocyclic (11−13) analogues. Analogue 10 (Table 1) maintained potency and selectivity; however, the compound was not further pursued due to its high lipophilicity. The more hydrophilic biaryl analogues 11−13 generally maintained MGLL potency but displayed low selectivity against ABHD6 and PLA2G7. Thus, replacements for the biaryl motif were explored to optimize selectivity. Given their reduced lipophilicity, 1,3,4-substituted pyrazoles (14−18) linked to the piperazine HFIP carbamate were examined as replacements for the biaryl substituent (Table 2). Phenyl substituted pyrazoles were tolerated with respect to MGLL potency (14, 15, 17, 18). Replacing phenyl with an Nacetylpiperidinyl moiety (16), however, resulted in reduction in MGLL potency. While the phenyl substituted pyrazole motif was effective in potently inhibiting MGLL and reducing lipophilicity, it offered little advantage over the biaryl analogues with respect to ABHD6 and PLA2G7 off-target selectivity. The impact of removing one of the aryl groups of the biaryl analogues (Table 3) was evaluated, resulting in analogues exemplified by compound 19, which maintained MGLL potency but was less selective against ABHD6. Importantly, introduction of a morpholine ring at R2 (20) resulted in an 9064



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Table 3. Impact of R2−R4 Variation on in Vitro Potency for MGLL, the Off-Targets ABHD6 and PLA2G7, and the Intrinsic Clearance Values in Human Liver Microsomes

a

IC50s determined using ABPP with a 30 min inhibitor preincubation in PC3 lysate. bClint (μL/min/mg protein) in human liver microsomes.

Table 4. Impact of R2 and R4 Variation on Potency for MGLL, Off-Targets, in Vivo Target Engagement, and Microsome Stability

a

Determined using ABPP with a 30 min inhibitor preincubation in PC3 lysate. bBrain target engagement measured 4 h following 5 mg/kg dose po using ABPP. cClint (μL/min/mg protein).

In addition to assessing in vitro potency and selectivity, the in vivo target engagement of inhibitors following oral dosing in mice was routinely measured using ABPP methods36 (Table 4). Mice were administered test compounds (5 mg/kg, po), and whole brains were collected from the euthanized animals 4 h postdose for target engagement analysis by gel-based ABPP using the FP-Rh activity probe. Assessment of target engagement under these conditions reflects the cumulative impact of the compound across multiple parameters including oral absorption, brain penetrance, pharmacokinetics, and the molecular interaction of the compound and enzyme in the target tissue. Analogues 24 and 28 showed high potency in vivo, with both molecules demonstrating complete MGLL inhibition in the mouse brain 4 h following a single 5 mg/kg oral gavage (po) dose. A broader survey of substituents at R2 while preserving either a trifluoromethyl or chlorine group at R4 (Table 5) was conducted. MGLL potency was maintained over a broad range

of R2 substitutions including elaborated morpholine (33−36) and pyrrolidine (37−41) functionalities. Among the allowed substituents were larger ring systems including bridged morpholine analogues 33−35 and fused pyrrolopyrimidine analogue 36. Elaborated pyrrolidines 37−41 were also tolerated at the R2 position, including highly potent fused ring (R2 = hexahydro-1H-furo[3,4-c]pyrrole, 39) and spirocyclic (R2 = 2-methyl-8-oxa-2-azaspiro[4.5]decane, 41) analogues. Together, in vitro human potency, human in vitro selectivity, and in vivo target engagement data provided a means to prioritize compounds. Analogues such as 36 and 39 had favorable in vitro MGLL potency (MGLL IC50 = 11, 2, 4 nM, respectively) but submaximal in vivo MGLL engagement in the brain and were thus deprioritized. Introduction of a 18F fluorine atom would provide PET tracer candidates that could be used to image MGLL activity distribution in the CNS and enable drug target occupancy studies. 18F-based radiotracers should have longer half-lives 9065



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Table 5. Impact of R2 Variation on Potency for MGLL, Off-Targets, and in Vivo MGLL Target Engagement

a Determined using ABPP with a 30 min inhibitor preincubation in PC3 lysate. bBrain target engagement measured 4 h following 5 mg/kg dose po using ABPP.

MGLL activity with an IC50 value of 0.0022 μM (Supporting Information, Table S1), which is ∼6-fold more potent than that observed in vitro. In the cell-based assay, >100-fold selectivity for MGLL over ABHD6 (IC50 = 0.253 μM) and PLA2G7 (IC50 = 494 μM) was maintained (Supporting Information, Table S1). In mouse, rat, and dog brain proteomes, 28 was found to be selective and potent for MGLL as judged by gel-based ABPP (Figure 2A−C, Supporting Information, Table S1). Additional analysis of 28 selectivity was carried out using MS-based ABPP methods in human post-mortem prefrontal cortex homogenates and shown to be highly selective against a panel of 37 serine hydrolases (Figure 2E). The kinetics for inactivation of human MGLL by 28 were determined by measuring the kinact/Ki, a time independent measure of inhibition. The average kinact/Ki value of 28 was 20,200 M−1 s−1 against purified human MGLL using a fluorescent substrate assay. Carbamate electrophiles are expected to inhibit MGLL via covalent labeling of the active-site serine nucleophile (Ser122) and thus afford a durable carbamoylated inhibitor−enzyme adduct that is catalytically inactive (Figure 3A).5 To confirm the covalent mechanism of action, the interaction of 28 with MGLL was characterized via a mass spectrometry-based assay. Following incubation of recombinant human MGLL with 28 and subsequent tryptic digestion, analysis by high resolution nanoLC-MS/MS revealed an adduct corresponding to the peptide mass of the predicted carbamate adduct of 28 with Ser122 (Figure 3B). This adduct was only observed in the

and sensitivity over the previously reported MGLL radiotracers, all of which contain the 11C radioisotope.53−55 The R2 position was selected for fluorine incorporation, as the SAR demonstrated a broad range of tolerated substitution patterns. Out of several candidates, 42 was prepared and found to be potent and selective against MGLL and active in the in vivo target engagement assay in rodents. Additional results involving the successful preclinical and clinical application of 42 as a MGLL PET tracer will be described elsewhere. Together, these medicinal chemistry efforts identified 28 (ABX-1431)43 and 34 (ABD-1970)44 as inhibitors warranting further consideration. ABD-1970 (34) was extensively characterized and used as a tool to probe MGLL pharmacology, and its activity in rodents and human biological systems will be described in a separate publication. ABX-1431 (28) was selected as the lead compound for clinical development and is described further herein. ABPP was used to profile 28 in multiple human and animal proteomes using gel-based and mass spectrometric based technologies. Using gel-based ABPP, 28 was assayed in human cell line lysates (Table 4, Supporting Information, Figure S1) and human post-mortem prefrontal cortex homogenates (Figure 2D) following a 30 min inhibitor incubation time. Overall, 28 was determined to be a potent human MGLL inhibitor (average IC50 = 0.014 μM) with >100-fold selectivity against ABHD6 and >200-fold selectivity against PLA2G7 (Table 4, Supporting Information, Table S1). Treatment of intact human PC3 cells with 28 following a 30 min inhibitor incubation time caused concentration dependent inhibition of 9066



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Figure 2. In vitro potency and selectivity for 28. (A−D) Gel-based ABPP in mouse, rat, dog, and human brain membrane homogenates. Brain membrane homogenates were treated with 28 (0.001−10 μM, 30 min, 37 °C) and subsequently labeled with FP-Rh (1 μM, 30 min, room temperature), quenched in SDS-PAGE loading buffer, and analyzed by in-gel fluorescence scanning. Human ABHD6 was not visible by in-gel fluorescence scanning. (E) MS-based ABPP: Human prefrontal cortex total homogenates were treated with 10 μM 28, labeled with FP-biotin, and enriched with avidin beads. Trypsinized peptides were analyzed by LC MS/MS.

presence of inhibitor and not in DMSO-treated samples. Furthermore, DMSO-treated samples contained greater levels of the unmodified Ser122-containing peptide than those treated with 28. These data indicate that 28 inhibits MGLL via carbamoylation of the catalytic nucleophile Ser122. A biochemical assay was employed to measure the in vitro stability of the 28−MGLL adduct. Accordingly, the MGLL− 28 covalent adduct was prepared by incubation of recombinant human MGLL and 28 (1 μM). Exogenous compound was then removed via repeated washing, and recovery of enzyme activity was monitored over 24 h (Supporting Information, Table S2). Over this time frame, MGLL activity regained less than three percent activity, suggesting that the carbamoylated inhibitor−MGLL adduct is stable and nonhydrolyzable for at least 24 h. Pharmacokinetic analysis in rats and dogs of 28 (Figure 4, Table 6) indicated low to moderate systemic clearance, moderate volume of distribution, and high oral bioavailability (64% in rat, 57% in dog). While 28 was stable in human and dog plasma, it was not stable in rat plasma (Supporting Information, Table S2). This result is thought to be a consequence of increased expression of the CES enzymes,56,57

which are known to metabolize ester and carbamate xenobiotics and are more abundant in rodent plasma than other higher mammal species including dogs, primates, and humans.57 Thus, in vivo studies that required measurement of 28 in rat blood, where degradation could occur ex vivo, were conducted using a modified procedure that stabilizes 28 during sample preparation (see Experimental Section). Additional selectivity analysis revealed that 28 has low propensity to inhibit human recombinant CYP enzymes (IC50 > 50 μM for CYP1A2, CYP2C9, CYP2C19, CYP3A4/5; IC50 = 6.5 μM for CYP2D6) and the hERG channel (IC20 ∼ 7 μM). Analysis in various transporter assays indicated that 28 is not an inhibitor or a substrate for P-gp, BCRP, and OCT2 at 10 μM. Finally, 28 (10 μM) did not display significant binding or activity (defined as >50% of control ligand binding or activity) against a panel of 95 targets including enzymes, receptors, transporters, and ion channels. To determine the in vivo MGLL potency of 28, the compound was administered by oral gavage to mice (Figure 5A−C). Four hours after a single oral dose, 28 dosedependently inhibited mouse brain MGLL activity with ED50 = 1.4 mg/kg (Figure 5B). At a dose ∼20-fold higher (32 mg/ 9067



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Figure 3. Mechanism of 28 inhibition of MGLL. (A) Proposed reaction of 28 and MGLL and the resulting carbamoylated active-site serine nucleophile (Ser 122). (B) Extracted ion chromatograms (EICs) showing the unmodified (top) and 28-modified (bottom) active-site tryptic peptide in DMSO-treated (red) and 28-treated (blue) samples. The 28-modified adduct is calculated to increase the parent peptide mass by 339.15585 amu. The mass of the base-peak was used for each EIC trace: m/z 1050.967 and 1118.799, respectively.

Figure 4. Concentration of 28 vs time profiles following a single iv or po administration in rat (A) and dog (B).

at the R4 position of the benzyl ring, was used. The alkyne group serves as a latent affinity handle suitable for conjugation to reporter tags by copper-catalyzed azide−alkyne cycloaddition (CuAAC or click chemistry).58 In vitro, compound 31 inhibited mouse MGLL with slightly reduced potency (mouse MGLL IC50 = 0.159 μM, Table 4) relative to 28 (mouse MGLL IC50 = 0.027 μM, Table 4). Following oral administration of compound 31 to mice (25 mg/kg, po), the only probe-labeled proteins, as determined by gel-based ABPP, were MGLL and ABHD6, demonstrating the high proteomewide selectivity of the 28 chemotype (Figure 5D). Further characterization of 28 revealed similar in vivo activity when the compound was administered orally to rats (Figure 6). The ED50 of 28 for inhibition of brain MGLL activity was 0.5 mg/kg (Figure 6A.B), whereas a 6-fold higher dose was required to produce substantial inhibition of ABHD6 (ED50 = 2.9 mg/kg). Similar to the mouse, the rodent-specific

Table 6. Pharmacokinetic Parameters of 28 in Rat and Dog species

oral Cmax (μM)

tmax (h)

CL (mL/min/kg)

Vdss (L/kg)

iv t1/2 (h)

%F

rat dog

0.688 0.777

8.0 1.0

14.7 6.3

3.2 1.2

3.6 6.1

64 57

kg), 28 demonstrated high selectivity, with the only other serine hydrolases inhibited being ABHD6 (ED50 ∼ 32 mg/kg) (Figure 5A) and the carboxylesterease CES1c (ED50 ∼ 32 mg/ kg, Supporting Information, Figure S2), a rodent-specific CES isoform with no direct human homologue. On the basis of comparison of the ED50 values, 28 displays ∼23-fold selectivity for MGLL versus ABHD6 and CES1c. As expected, inhibition of brain MGLL activity resulted in dose-dependent increases in brain 2-AG concentrations (Figure 5C). To enable in vivo proteome-wide selectivity assessment of the 28 chemotype, an analogue of 28, 31, containing an alkyne 9068



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Figure 5. ABX-1431 (28) is a potent and selective inhibitor of MGLL in mouse brain in vivo. (A−C) Mice were administered vehicle or 28 (0.5− 32 mg/kg, po, n = 3), and after 4 h, sacrificed brains were collected and prepared for (A,B) gel-based ABPP with the serine hydrolase directed probe FP-Rh or (C) 2-AG quantification by LCMS/MS. (D) Click probe (31) analysis in mouse brain 4 h following a single oral 25 mg/kg dose. *** p < 0.001 vs vehicle.

antinociceptive effects in mice following either single or multiple day dosing. Characterization of the relationship between MGLL inhibition resulting from treatment with 28, and antinociceptive effects will be described elsewhere.

CES1c isoform was inhibited by 28 (ED50 = 1.2 mg/kg, Supporting Information, Figure S3). Inhibition of MGLL activity in the brain in rats by 28 corresponded to a dosedependent elevation of brain 2-AG tissue concentrations (Figure 6C). To evaluate the potential antinociceptive effects of 28, the compound was evaluated in the intraplantar formalin paw test in rats. The formalin paw test is a model of pain induced by local injection of formalin (2.5%, 50 μL) into one hind paw, producing an early response (0−5 min) due to direct stimulation of nociceptors and a delayed response (10−30 min) driven by inflammation and central sensitization.59 Pain behavior in these studies was quantified by total time the rats spent licking the affected paw. A single 3 mg/kg oral dose of 28 administered to rats 4 h prior to formalin injection significantly reduced formalin-evoked paw licking duration in both phases (Figure 6D), demonstrating the antinociceptive properties of 28. The formalin model evaluates effects of a compound on spontaneous pain, thus effects on paw licking can be due to antinociceptive mechanisms or sedation. To differentiate between these possibilities, the effects of 28 on spontaneous locomotor activity was evaluated in rats that were not administered formalin. In support of an antinociceptive effect, single 10 mg/kg oral administration of 28 did not alter locomotor activity in rat (vehicle locomotor counts = 53.0 ± 3.7; 28 locomotor counts = 54.9 ± 6.6; n = 10 animals per group). The doses of 28 used in these studies produced near complete MGLL inhibition and maximal elevation of brain 2AG content; however, previously published studies of MGLL inhibitors suggest that partial MGLL inhibition and submaximal elevation of brain 2-AG is sufficient to produce

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CHEMISTRY The synthesis of 28 was carried out in four steps with 38% overall yield (Scheme 1). The sequence began with SNAr reaction between aldehyde 28a and pyrrolidine 28b to give 2pyrroldinebenzaldehyde 28c. Reductive amination of 28c with Boc-piperazine provided 28d. Subsequent Boc deprotection and carbamoylation with hexafluoroisopropanol and triphosgene and diisopropylethylamine yielded 28. Additional analogues (20−29) were prepared using this approach, and a general scheme is shown in (Scheme 2). Preparation of the biaryl analogues 10−13 (Scheme 3) began with synthesis of biarylmethanol intermediates 10b− 13b. Intermediates 10b and 11b were formed via nucleophilic addition of two equivalents of aryl magnesium or lithium reagents to ethyl formate. Reduction of commercially available ketone 12a yielded 12b. Biaryl methanol 13b was produced by treatment of 13a with n-butyl lithium and subsequent reaction with oxazole-4-carbaldehyde. The biarylmethanol intermediates 10b−13b were subsequently activated using methanesulfonyl chloride or thionyl chloride and reacted with Bocpiperazine to yield intermediates 10c−13c. Protecting group removal and carbamoylation with hexafluoroisopropanol gave final products 10−13. Preparation of pyrazole analogues 14−18 are shown in Scheme 4. For the preparation of 14, 15, 17, and 18, pyrazole carbaldehydes 14c−18c were combined with Boc-piperazine 9069



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Figure 6. ABX-1431 (28) is a potent and selective inhibitor of MGLL in rat brain in vivo and produces antinociceptive effects in rats. (A−C) Rats were administered vehicle or 28 and after 4 h were sacrificed and brains collected and prepared for (A,B) gel-based ABPP with the serine hydrolase directed probe FP-Rh or (C) 2-AG quantification by LCMS/MS. (D) Single doses of 28 (3 mg/kg, PO) were administered 4 h prior to formalin (2.5%, 50 μL, intraplantar), and reduced pain behavior was measured as total paw licking duration (n = 10 rats per group, *p < 0.05, **p < 0.01 vs respective vehicle).

Scheme 1. Preparation of 28 (ABX-1431)

Scheme 2. General Strategy for Preparation of Aldehyde Intermediates and Benzyl Substituted HFIP carbamates

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Scheme 3. Preparation of 10−13

Scheme 4. Preparation of 14−18

under reductive amination conditions and subsequently deprotected and carbamoylated to give the desired HFIP carbamates. The synthesis of 16 required an alternative route and began with reaction of the methyl hydrazine with 16a and subsequent cyclization with N-(chloromethylene)-N-methylmethanaminium chloride to give pyrazole carbaldehyde 16c. Reductive amination of 16c with Boc-piperazine resulted in 16d. The distal pyridine moiety was alkylated, reduced, and

acylated to give 16f. Finally, Boc-deprotection of 16f and carbamoylation with hexafluoroisopropanol gave 16. The preparation of analogues 30 and 31 (Scheme 5) required slight deviations from the general routes described above. 30d was subjected to sulfonation conditions to produce 30e. Subsequent Boc-deprotection of 30e and carbamoylation with hexafluoroisopropanol afforded 30. Analogue 31 was produced by incorporation of a Sonogashira coupling to unite 9071



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Scheme 5. Preparation of 30 and 31

Scheme 6. Preparation of 32, 40, and 41

of Boc-piperazine and bis-HFIP carbonate and followed by subsequent TFA-mediated Boc-deprotection. 32b could be reacted with benzaldehydes 32c, 40c, and 42c to give either a final product (32) or an advanced intermediate such as 40d or 42d. The compounds 40d and 42d could be treated with TFA to remove the distal Boc protecting group and subjected to alkylation conditions to give final products 40 and 42.

29c and TMS-acetylene. The resulting product, 31f, was fully deprotected to yield 31g. The penultimate intermediate 31g was carbamoylated with hexafluoroisopropanol to give final compound 31. An alternative approach to HFIP carbamate analogues was used to prepare analogues 32, 40, and 42 (Scheme 6). In these cases, key common intermediate 32b was prepared by reaction 9072



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DCM (5 mL), and NMM (90 μL, 0.819 mmol). The reaction was cooled to 0 °C, and TMSI (70 μL, 0.491 mmol) was added dropwise. After 15 min at 4 °C, the reaction was quenched with saturated sodium carbonate and extracted with DCM (3 × 50 mL). The organics were dried over sodium sulfate, filtered, and concentrated. The residue was chromatographed on a silica gel column (100% DCM to 6% 2 M NH3 in MeOH) and 10d (143 mg, 70%). 1H NMR (400 MHz, CDCl3) δ 7.47 (dd, J = 8.4, 1.1 Hz, 2H), 7.16−7.11 (m, 1H), 7.11−7.06 (m, 2H), 4.64 (s, 1H), 2.88−2.80 (m, 4H), 2.44− 2.33 (m, 5H), 2.28 (s, 7H). LCMS (ESI, m/z): 349 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl 4-(Bis(4-chloro-2methylphenyl)methyl)-piperazine-1-carboxylate (10). The title compound was prepared from 10d as described for the preparation of 28. (18 mg, 59%). 1H NMR (400 MHz, CDCl3) δ 7.47 (dd, J = 8.4, 2.1 Hz, 2H), 7.22−7.16 (m, 2H), 7.16−7.10 (m, 2H), 5.83−5.70 (m, 1H), 4.70 (s, 1H), 3.58−3.50 (m, 4H), 2.51−2.40 (m, 4H), 2.30 (s, 6H). LCMS (ESI, m/z): 263 [bis(4-chloro-2-methylphenyl)methane cation]+. Bis(1-methyl-1H-indazol-5-yl)methanol (11b). A round-bottom flask was charged with 5-bromo-1-methyl-1H-indazole (300 mg, 1.42 mmol) and THF (45 mL). The solution was cooled to −78 °C, and an n-butyllithium solution (2.3 M in THF, 680 μL, 1.56 mmol) was added dropwise. After 30 min, a solution of ethyl formate (57 μL, 0.697 mmol, in 10 mL THF) was added dropwise, and the reaction was stirred at −78 °C for 10 min and rt for 3 h. The reaction was quenched with saturated NH4Cl and extracted with ethyl acetate (3 × 50 mL). The organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (100% DCM to 10% MeOH in DCM) to yield 11b (134 mg, 32%) as a brown oil. 1H NMR (400 MHz, CDCl3) δ 7.90 (s, 2H), 7.77 (s, 2H), 7.39 (dd, J = 8.7, 1.2 Hz, 2H), 7.31 (d, J = 8.7 Hz, 2H), 6.07 (s, 1H), 4.02 (s, 7H). LCMS (ESI, m/ z): 293 [M + H]+. tert-Butyl 4-(Bis(1-methyl-1H-indazol-5-yl)methyl)piperazine-1carboxylate (11c). A round-bottom flask was charged with 11b (50 mg, 0.17 mmol) and DCM (5 mL). Thionyl chloride (25 μL, 342 mmol) was added, resulting in a cloudy mixture. After 15 min, the mixture became a clear-pink solution and was stirred at rt for 48 h. The solution was concentrated under reduced pressure. Acetonitrile was added, and the solution was concentrated two times. Acetonitrile (7 mL) and tert-butyl piperazine-1-carboxylate (60 mg, 0.32 mmol) were added, and the solution was stirred at rt overnight. The reaction was concentrated under reduced pressure and purified by silica chromatography (100% DCM to 3% MeOH in DCM) to yield 11c (53 mg, 56%) as a light-brown oil. 1H NMR (400 MHz, CDCl3) δ 7.97−7.89 (m, 2H), 7.79 (s, 2H), 7.54 (dd, J = 8.8, 1.5 Hz, 2H), 7.32 (d, J = 8.7 Hz, 2H), 4.49 (s, 1H), 4.03 (s, 6H), 3.47 (s, 4H), 2.41 (s, 4H), 1.45 (s, 9H). LCMS (ESI, m/z): 483 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(bis(1-methyl-1H-indazol-5yl)methyl)-piperazine-1-carboxylate (11). The title compound was prepared from 11c as described for compound 10d and compound 28 (12 mg, 35%). 1H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 7.5 Hz, 2H), 7.77 (d, J = 6.7 Hz, 2H), 7.55−7.45 (m, 2H), 7.36−7.28 (m, 2H), 5.80−5.67 (m, 1H), 4.51 (s, 1H), 4.02 (s, 6H), 3.58 (s, 4H), 2.47 (d, J = 5.5 Hz, 4H). LCMS (ESI, m/z): 555 [M + H]+. Di(pyridin-3-yl)methanol (12b). A round-bottom flask was charged with di(pyridin-3-yl)methanone (500 mg, 2.72 mmol), MeOH (30 mL), and DCM (15 mL) and cooled to 0 °C. NaBH4 (51 mg, 1.35 mmol) was added in one portion. The solution was stirred for 1 h at 0 °C and quenched with 1N NaOH, and the reaction was extracted with DCM (3 × 50 mL). The organic layers were combined, dried over sodium sulfate, and concentrated under reduced pressure. Crude 12b (505 mg, 100%) was used in the next step without further purification. 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 2H), 8.24 (d, J = 4.8 Hz, 2H), 7.47 (d, J = 7.9 Hz, 2H), 7.09−7.01 (m, 2H), 5.67 (s, 1H). tert-Butyl 4-(Di(pyridin-3-yl)methyl)piperazine-1-carboxylate (12c). A round-bottom flask was charged with 12b (600 mg, 3.22 mmol) and DCM (50 mL). Thionyl chloride (353 μL, 4.83 mmol) was added, and the reaction was stirred for 18 h at rt. The solution

CONCLUSION While the beneficial pharmacology of direct CB1 and CB2 agonists is well recognized, these agents are hindered by numerous side effects. Significant preclinical evidence suggests that MGLL inhibitors that elevate the endogenous CB receptors’ agonist 2-AG have the potential to produce the beneficial effects of direct CB agonists while achieving a superior side effect profile. Our detailed optimization of the HFIP carbamate motif, achieved by integrating ABPP screening methods that efficiently determine MGLL potency and selectivity both in vitro and in vivo, led to discovery of 28. ABX-1431 (28) is a potent and selective MGLL inhibitor with favorable ADME properties and is currently being evaluated in clinical trials.

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Chemistry General Methods. All commercially available chemicals were obtained from Aldrich, Acros, Fisher, Fluka, Maybridge, or the like and were used without further purification, except where noted. Anhydrous solvents and oven-dried glassware were used for synthetic transformations sensitive to moisture and/or oxygen. All reactions are typically carried out under an inert nitrogen atmosphere using oven-baked glassware unless otherwise noted. Flash chromatography is performed using 230−400 mesh silica gel 60 using Isco Combiflash instruments. NMR spectra were generated on either Bruker 300 or Bruker 400 instruments. Chemical shifts are typically recorded in ppm relative to tetramethylsilane (TMS) with multiplicities given as s (singlet), bs (broad singlet), d (doublet), t (triplet), dt (double of triplets), q (quadruplet), qd (quadruplet of doublets), hept (heptuplet), and m (multiplet). Chemical purities were >95% for all final compounds as assessed by LC/MS analysis using a Shimadzu 2020 or an Agilent 1200 LCMS with detection at 210 and 254 nm. The column used was an xBridge 50 mm × 3 mm using a 0−100% acetonitrile in water (with 0.05% TFA additive) gradient over 1 min at 1 mL/min at 40 °C. Alternatively, an ACE 2.1mm × 100 mm C18 column was used with a 0−100% acetonitrile in water (with 0.1% formic acid additive) gradient over 1 min at 1 mL/min at 25 °C. Bis(4-chloro-2-methylphenyl)methanol (10b). A round-bottom flask was charged with a 4-chloro-2-methylphenyl magnesium bromide solution (10 mL of a 0.5 M THF solution, 5 mmol) and THF (50 mL). The solution was cooled to −78 °C, and an ethyl formate solution (200 μL, 2.50 mmol, in 10 mL THF) was added dropwise. The reaction was allowed to stir at −78 °C for 15 min and allowed to warm to rt slowly and stirred for 18 h. The reaction was diluted in ethyl acetate and washed with brine (3 × 100 mL). The organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (100% hexanes to 15% ethyl acetate in hexanes) to yield 10b (643 mg, 46%) as a clear crystalline solid. 1H NMR (400 MHz, CDCl3) δ 7.25−7.14 (m, 6H), 6.07 (s, 1H), 2.26 (s, 6H), 1.57 (s, 2H). LCMS (ESI, m/z): 263 [M + H]+. tert-Butyl 4-(Bis(oxazol-4-yl)methyl)piperazine-1-carboxylate (10c). A round-bottom flask was charged with 10b (200 mg, 0.711 mmol) and DCM (7 mL). Thionyl chloride (100 μL, 1.37 mmol) was added, and the reaction was stirred at rt for 24 h. The reaction was concentrated. Acetonitrile was added, and the reaction was concentrated two times. Acetonitrile (6 mL), tert-butyl piperazine-1carboxylate (200 mg, 1.07 mmol), and K2CO3 (200 mg, 1.42 mmol) were added, and the reaction was heated to 80 °C for 4 h followed by 120 °C for 18 h. The reaction was poured into brine and extracted with ethyl acetate (2 × 100 mL). The residue was chromatographed on a silica gel column (100% hexanes to 20% ethyl acetate) and yielded 10c (184 mg, 57%) as a clear oil. 1H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 7.11 (s, 2H), 4.65 (s, 1H), 3.44−3.37 (m, 4H), 2.38 (s, 4H), 2.29 (s, 6H), 1.46 (s, 9H). LCMS (ESI, m/z): 471 [M + H]+. 1-(Bis(4-chloro-2-methylphenyl)methyl)piperazine (10d). A round-bottom flask was charged with 10c (184 mg, 409 mmol), 9073



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was concentrated under reduced pressure. The residue was chromatographed on a silica gel column (100% DCM to 5% 2 M NH3 in MeOH) and yielded 3,3′-(chloromethylene)-dipyridine (415 mg, 64%). 1H NMR (400 MHz, CDCl3) δ 8.66 (d, J = 2.4 Hz, 2H), 8.59 (dd, J = 4.7, 1.6 Hz, 2H), 7.76 (m, 2H), 7.34 (m, 2H), 6.17 (s, 1H). A round-bottom flask was charged with 3,3′-(chloromethylene)dipyridine (415 mg, 2.03 mmol), tert-butyl piperazine-1-carboxylate (1.20 g, 6.45 mmol), and acetonitrile (50 mL). The reaction was heated to 80 °C for 2 h, concentrated under reduced pressure, and purified by silica chromatography (100% DCM to 5% 2 M NH3 in MeOH) to yield 12c (220 mg, 31%). 1H NMR (400 MHz, CDCl3) δ 8.64 (d, J = 2.0 Hz, 3H), 8.48 (dd, J = 4.8, 1.7 Hz, 3H), 7.70 (dt, J = 7.9, 1.9 Hz, 3H), 7.24 (ddd, J = 7.9, 4.8, 0.7 Hz, 3H), 5.28 (s, 1H), 4.36 (s, 1H), 3.47−3.39 (m, 6H), 2.34 (s, 6H), 1.42 (s, 9H). LCMS (ESI, m/z): 355 [M + H]+ 1,1,1,3,3,3-Hexafluoropropan-2-yl4-(di(pyridin-3-yl)methyl)piperazine-1-carboxylate (12). The title compound was prepared from 12c as described for compound 10d and compound 28 (23 mg, 33%). 1H NMR (400 MHz, CDCl3) δ 8.66 (s, 3H), 8.51 (d, J = 4.7 Hz, 3H), 7.75−7.67 (m, 3H), 7.27 (t, J = 6.3 Hz, 3H), 5.72 (hept, J = 6.2 Hz, 1H), 4.41 (s, 1H), 3.58 (d, J = 4.4 Hz, 7H), 2.43 (dt, J = 10.4, 4.4 Hz, 8H). LCMS (ESI, m/z): 449 [M + H]+. Bis(oxazol-4-yl)methanol (13b). A round-bottom flask was charged with oxazole (476 μL, 7.2 mmol) and THF (100 mL). The solution was cooled to −78 °C. A solution of n-butyllithium (2.3 M in hexanes, 3.5 mL, 8.05 mmol) was added dropwise. After stirring at −78 °C for 40 min, a solution of oxazole-4-carbaldehyde (773 mg, 7.96 mmol, in 8 mL of THF) was added dropwise. The reaction was allowed to warm to rt. After 30 min, the reaction was quenched with saturated NH4Cl and extracted with DCM (3 × 100 mL). The organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (100% DCM to 10% MeOH in DCM) to yield 13b (354 mg, 29%) as a light-brown solid. 1H NMR (400 MHz, CDCl3) δ 7.93 (s, 2H), 7.73 (s, 2H), 5.87 (s, 1H), 3.52 (s, 1H). LCMS (ESI, m/z): 167 [M + H]+. tert-Butyl 4-(Bis(oxazol-4-yl)methyl)piperazine-1-carboxylate (13c). A round-bottom flask was charged with 13b (200 mg, 1.20 mmol), DCM (3 mL), and DIPEA (412 μL, 2.41 mmol). The solution was cooled to 0 °C, and methanesulfonyl chloride (112 μL, 1.45 mmol) was added dropwise. After stirring 0 °C for 30 min, more methanesulfonyl chloride (100 μL, 1.29 mmol) was added. After an additional 30 min at 0 °C, the reaction was quenched with brine and extracted with DCM (3 × 50 m1L). The organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure to yield the crude alkyl chloride. The crude intermediate was dissolved in DCM (3 mL) and treated with tert-butyl piperazine-1-carboxylate (224 mg, 1.20 mmol) and stirred at rt. After 48 h at rt, the reaction was quenched with brine and extracted with DCM (3 × 50 mL). The organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (100% DCM to 4% MeOH in DCM) to yield 13c (114 mg, 28%) as a light-brown oil. 1H NMR (400 MHz, CDCl3) δ 7.87 (s, 2H), 7.69 (s, 2H), 4.78 (s, 1H), 3.45−3.37 (m, 4H), 2.53−2.40 (m, 4H), 1.39 (s, 9H). LCMS (ESI, m/z): 335 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl 4-(bis(oxazol-4-yl)methyl)piperazine-1-carboxylate (13). The title compound was prepared from 13c as described for compound 10d and compound 28 (26 mg, 65%). 1H NMR (400 MHz, CDCl3) δ 7.92 (s, 2H), 7.74 (s, 2H), 5.72 (hept, J = 6.3 Hz, 1H), 4.86 (s, 1H), 3.65−3.55 (m, 4H), 2.64−2.54 (m, 4H). LCMS (ESI, m/z): 429 [M + H]+. tert-Butyl 4-(3-Chloro-2-morpholinobenzyl)piperazine-1-carboxylate (14d). The title compound was prepared from commercially available 1-methyl-3-phenyl-1H-pyrazole-4-carbaldehyde and tertbutyl-1-piperazine-1-carboxylate as described for 28d. 1H NMR (400 MHz, chloroform-d) δ 7.79 (d, J = 8.3 Hz, 2H), 7.39 (t, J = 8.2 Hz, 2H), 7.34−7.28 (m, 2H), 3.91 (s, 3H), 3.45−3.38 (m, 6H), 2.40 (bs, 4H), 1.45 (s, 9H). 1-((1-Methyl-3-phenyl-1H-pyrazol-4-yl)methyl)piperazine (14e). A 100 mL round-bottom flask, equipped with a magnetic stir bar, was

charged with 14d (0.920 g, 2.58 mmol) and dichloromethane (14 mL). The resulting solution was cooled to 0 °C. Hydrochloric acid (3.87 mL, 4 N in dioxane) was added via syringe. The ice bath was removed, and the resulting cloudy suspension was allowed to stir at room temperature overnight. After 20 h, saturated aqueous sodium carbonate (15 mL), water (10 mL), and dichloromethane (15 mL) were added and the resulting solution was stirred for 30 min. The layers were separated, and the aqueous layer was extracted twice with dicholomethane (30 mL). The organic layers were washed with saturated aqueous sodium bicarbonate (30 mL), combined, dried over sodium sulfate, and concentrated. The resulting yellow oil was chromatographed on a 24 g silica column with a gradient (100% dichloromethane to 90% dichloromethane/10% methanol containing 2 M ammonia) to yield 14e as a yellow oil (410 mg, 62%). 1H NMR (400 MHz, chloroform-d) δ 7.79 (d, J = 7.2 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.35−7.28 (m, 2H), 3.92 (s, 3H), 3.43 (s, 2H), 2.90 (t, J = 4.8 Hz, 4H), 2.57−2.43 (m, 5H). 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-((1-methyl-3-phenyl-1Hpyrazol-4-yl)methyl)-piperazine-1-carboxylate (14). A screw cap vial equipped with a magnetic stir bar was charged with triphosgene (13.6 mg, 0.046 mmol, 0.50 equiv) and DCM (1 mL) under nitrogen and cooled to 0 °C. 1,1,1,3,3,3-Hexafluoroisopropanol (19 uL, 0.15 mmol) was added via syringe over 1 min, followed by addition of 2,6lutidine (33 μL, 3.1 equiv) over 1 min. N,N- Dimethylaminopyridine (1 mg, 0.1 equiv) was then added to the reaction mixture and allowed to stir at rt for 1 h. A separate vial, equipped with a magnetic stir bar, was charged with 14e (24.0 mg, 0.092 mmol) and DCM (1 mL). The chloroformate solution was recooled to 0 °C, and the amine solution was added to the chloroformate solution over 1 min via syringe. The ice bath was removed, and the clear, colorless reaction was stirred at rt overnight. Saturated aqueous sodium bicarbonate (1 mL) was added, and the layers were separated. The aqueous layer was washed with DCM (1 mL). The organic layers were combined, concentrated, and applied to a 40 g silica gel column and yielded 14 (72 mg, 78%). 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 7.6 Hz, 2H), 7.40 (t, J = 7.6 Hz, 2H), 7.37−7.28 (m, 2H), 5.75 (hep, J = 6.2 Hz), 3.93 (s, 3H), 3.58−3.53 (m, 4H), 3.47 (s, 2H), 2.53−2.42 (m, 4H). LCMS (ESI, m/z): 451.1 [M + H]+. (Z)-1-[1-(2-Chlorophenyl)ethylidene]-2-methylhydrazine (15b). A round-bottom flask was purged and maintained with an inert atmosphere of nitrogen and charged with 1-(2-chlorophenyl)ethan-1one (3.80 g, 24.6 mmol, 1.20 equiv), methylhydrazine sulfate (3.00 g, 20.8 mmol, 1.00 equiv), and ethanol (30 mL). The resulting solution was heated to reflux overnight. The reaction progress was monitored by LCMS. The resulting mixture was concentrated under reduced pressure to yield 7.0 g of 15b as yellow oil which was used without further purification. LCMS (ES, m/z): 183 [M + H]+. 3-(2-Chlorophenyl)-1-methyl-1H-pyrazole-4-carbaldehyde (15c). A round-bottom flask was purged and maintained with an inert atmosphere of nitrogen and charged with 15b (2.00 g, 10.9 mmol, 1.00 equiv), (chloromethylidene)dimethylazanium chloride (12.7 g, 99.2 mmol, 9.06 equiv), and N,N-dimethylformamide (40 mL). The resulting solution was stirred overnight at 50 °C. The reaction progress was monitored by LCMS. The reaction was then quenched by the addition of saturated sodium carbonate solution (100 mL). The resulting solution was extracted with ethyl acetate (3 × 100 mL), and the organic layers were combined, washed with brine (2 × 100 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (1/1) to yield 1.00 g (41% yield) of 15c as a yellow solid. LCMS (ES, m/z): 221 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-((3-(2-chlorophenyl)-1methyl-1H-pyrazol-4-yl)methyl)piperazine-1-carboxylate (15). The title compound was prepared from 15c as described for 28d−28. 1H NMR (300 MHz, CDC13) δ: 7.26−7.46 (m, 5H), 5.67−5.75 (m, 1H), 3.94 (s, 3H), 3.40 (br, 6H), 2.31 (br, 4H). LCMS (ES, m/z): 485 [M + H]+. 4-[(1Z)-1-(2-Methylhydrazin-1-ylidene)ethyl]pyridine (16b). A round-bottom flask was charged with 1-(pyridin-4-yl)ethan-1-one (12.1 g, 99.9 mmol, 1.00 equiv) in ethanol (100 mL) and 9074



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flask was charged with tert-butyl-4-[[1-methyl-3-(piperidin-4-yl)-1Hpyrazol-4-yl]methyl]piperazine-1-carboxylate (400 mg, 1.10 mmol, 1.00 equiv) in DCM (10 mL) and triethylamine (223 mg, 2.20 mmol, 2.00 equiv) under nitrogen. Acetic anhydride (169 mg, 1.66 mmol, 1.50 equiv) was added dropwise at 0 °C. The resulting solution was stirred for 4 h at rt. The reaction was then quenched with water (10 mL). The mixture was extracted with DCM (3 × 20 mL), and the organic layers were combined, washed with brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with DCM/methanol (10/1) to yield 350 mg (78% yield) of 16f as yellow oil. LCMS (ESI, m/z): 406 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-((3-(1-acetylpiperidin-4-yl)1-methyl-1H-pyrazol-4-yl)methyl)piperazine-1-carboxylate (16). The title compound was prepared from 16f as described for 28d− 28. 1H NMR (300 MHz, chloroform-d) δ 7.19 (s, 1H), 5.70−5.79 (m, 1H), 4.65−4.70 (m, 1H), 3.89−3.93 (m, 1H), 3.81 (s, 3H), 3.54 (br, 4H), 3.37 (br, 2H), 3.10−3.19 (m, 1H), 2.83−2.93 (m, 1H), 2.62−2.71 (m, 1H), 2.43 (br, 4H), 2.11 (s, 3H), 1.83−1.90 (m, 4H). LCMS (ESI, m/z): 500 [M + H]+. tert-Butyl 4-((1-Methyl-3-phenyl-1H-pyrazol-5-yl)methyl)piperazine-1-carboxylate (17d). The title compound was prepared directly from commercially available 1-methyl-3-phenyl-1H-pyrazole5-carbaldehyde and tert-butyl piperazine-1-carboxylate as described for 28d. 1H NMR (400 MHz, CDCl3) 7.78 (d, J = 7.8, 2H), 7.40 (t, J = 7.4, 2H), 7.29 (t, J = 7.7, 1H), 6.45 (s, 1H), 3.95 (s, 3H), 3.54 (s, 2H), 3.43−3.40 (m, 4H), 2.45−2.40 (m, 4H), 1.47 (s, 9H). 1-((1-Methyl-3-phenyl-1H-pyrazol-5-yl)methyl)piperazine (17e). The title compound was prepared from 17d as described for 14e. 1H NMR (400 MHz, chloroform-d) δ 7.78 (d, J = 7.2 Hz, 2H), 7.36 (t, J = 7.2 Hz, 2H), 7.28 (d, 1H), 6.45 (s, 1H), 3.95 (s, 3H), 3.51 (s, 2H), 2.89−2.86 (m, 4H), 2.50−2.40 (m, 4H), 1.63 (bs, 1H). 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-((1-methyl-3-phenyl-1Hpyrazol-5-yl)methyl)piperazine-1-carboxylate (17). The title compound was prepared directly from 17e as described for 28d−28. 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 7.4, 2H), 7.31 (d, J = 7.4, 2H), 7.21 (t, J = 7.4, 1H), 6.33 (s, 1H), 5.68 (hep, J = 6.2, 1H), 3.86 (s, 3H), 3.55−3.45 (bs, 6H), 2.45−2.35 (m, 4H). LCMS (ESI, m/z): 451.1 [M + H]+. tert-Butyl 4-((3-Methyl-1-phenyl-1H-pyrazol-4-yl)methyl)piperazine-1-carboxylate (18d). The title compound was prepared using commercially available 3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (18c) and tert-butyl piperazine-1-carboxylate as described for 28d. 1H NMR (400 MHz, CDCl3) 7.79 (s, 1H), 7.65 (d, J = 8.4, 2H), 7.43 (t, J = 8.4, 2H), 7.23 (t, J = 8.4, 1H), 3.45−3.40 (m, 6H), 2.42− 2.38 (m, 4H), 2.34 (s, 3H), 1.47 (s, 9H). 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-((3-methyl-1-phenyl-1Hpyrazol-4-yl)methyl)piperazine-1-carboxylate (18). The title compound was prepared from 18d as described for 28. 1H NMR (400 MHz, CDCl3) δ 7.71 (s, 1H), 7.56 (d, J = 7.5, 2H), 7.35 (t, J = 7.4, 2H), 7.19 (t, J = 7.4, 1H), 5.68 (hep, J = 6.2, 1H), 3.53−3.48 (m, 4H), 3.37(s, 2H), 2.45−2.35 (m, 4H), 2.26 (s, 3H). LCMS (ESI, m/ z): 451.1 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl 4-(4-chlorobenzyl)piperazine1-carboxylate (19). The title compound was prepared from commercially available 1-(4-chlorobenzyl)piperazine as described for 28 (24 mg, 24%). 1H NMR (400 MHz, chloroform-d) δ 7.26−7.11 (m, 5H), 5.68 (hept, J = 6.3 Hz, 1H), 3.52−3.38 (m, 6H), 2.36 (m, 4H). LCMS (ESI, m/z): 405 [M + H]+. 4-Chloro-2-morpholinobenzaldehyde (20c). The title compound was prepared directly from commercially available 5-chloro-2fluorobenzaldehyde and morpholine as described for 28c. 1H NMR (400 MHz, chloroform-d) δ 10.25 (s, 1H), 7.80−7.73 (m, 1H), 7.17− 7.07 (m, 2H), 3.95−3.88 (m, 4H), 3.14−3.07 (m, 4H). LCMS (ESI, m/z): 226 [M + H]+. tert-Butyl 4-(4-Chloro-2-morpholinobenzyl)piperazine-1-carboxylate (20d). The title compound was prepared from 20c and tert-butyl piperazine-1-carboxylate as described for 28d. 1H NMR (400 MHz, chloroform-d) δ 7.38 (d, J = 8.4 Hz, 1H), 7.11−7.04 (m, 2H), 3.88− 3.81 (m, 4H), 3.53 (s, 2H), 3.41 (t, J = 5.0 Hz, 4H), 3.02−2.95 (m,

methylhydrazine sulfate (28.8 g, 200.00 mmol, 2.00 equiv) under nitrogen. The resulting solution was heated to reflux overnight in an oil bath. The reaction progress was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was chromatographed on a silica gel column with DCM/ methanol (4/1) to yield 25.0 g (crude) of 16b as a yellow solid, which was used without further purification. LCMS (ESI, m/z): 150 [M + H]+. 1-Methyl-3-(pyridin-4-yl)-1H-pyrazole-4-carbaldehyde (16c). A round-bottom flask was charged with N,N-dimethylformamide (250 mL) and phosphoryl trichloride (230 g, 1.50 mol, 8.95 equiv) was added dropwise at 0 °C. The resulting solution was stirred for 1 h at rt. 16b (25.0 g, 168 mmol, 1.00 equiv) was added. The resulting solution was stirred overnight at 50 °C (3 × 50 mL). The reaction progress was monitored by LCMS. The reaction was then quenched with water (500 mL). The pH value of the solution was adjusted to 8 with sodium carbonate. The resulting solution was extracted with ethyl acetate (4 × 500 mL), and the organic layers were combined, washed with brine (2 × 500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with DCM/ methanol (100/1) to yield 20.0 g (64% yield) of 16c as a yellow solid. LCMS (ESI, m/z): 188 [M + H]+. tert-Butyl-4-[[1-methyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl]methyl]piperazine-1-carboxylate (16d). A round-bottom flask was charged with 16c (5.00 g, 26.7 mmol, 1.00 equiv) in dichloroethane (100 mL), tert-butyl piperazine-1-carboxylate (5.50 g, 29.5 mmol, 1.10 equiv), and triethylamine (4.05 g, 40.0 mmol, 1.50 equiv), and the resulting mixture was stirred for 1h at rt. Sodium triacetoxyborohydride (17.0 g, 80.2 mmol, 3.00 equiv) was added. The reaction was stirred overnight and monitored by LCMS. The reaction was then quenched with water (100 mL). The resulting solution was extracted with DCM (3 × 100 mL), and the organic layers were combined, washed with brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with DCM/ methanol (10/1) to yield (9.50, 100% yield) of 16d as yellow oil. LCMS (ESI, m/z): 358 [M + H]+. 1-Benzyl-4-[4-([4-[(tert-butoxy)carbonyl]piperazin-1-yl]methyl)1-methyl-1H-pyrazol-3-yl]pyridin-1-ium bromide (16e). A roundbottom flask was charged with 16d (9.50 g, 26.6 mmol, 1.00 equiv in 100 mL acetone) and (bromomethyl)benzene (4.80 g, 28.1 mmol, 1.06 equiv) under nitrogen. The resulting solution was stirred overnight at 50 °C. The reaction progress was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The crude product was triturated with acetone/ether (1/10) to yield 14.0 g (100% yield) of 1-benzyl-4-[4-([4-[(tert-butoxy)carbonyl]piperazin1-yl]methyl)-1-methyl-1H-pyrazol-3-yl]pyridin-1-ium bromide as a yellow solid which was used without further purification. LCMS (ESI, m/z): 448 [M-Br]+. A round-bottom flask was charged with 1-benzyl4-[4-([4-[(tert-butoxy)carbonyl]piperazin-1-yl]methyl)-1-methyl-1Hpyrazol-3-yl]pyridin-1-ium bromide (14.0 g, 26.5 mmol, 1.00 equiv) in ethanol (150 mL) under nitrogen and sodium borohydride (1.50 g, 39.7 mmol, 1.50 equiv) was added in several batches at 0 °C. The resulting solution was stirred for 2 h at rt. The reaction progress was monitored by LCMS. The reaction was then quenched with water (10 mL). The resulting mixture was concentrated under reduced pressure. The residue was chromatographed on a silica gel column with DCM/ methanol (10/1) to yield 9.00 g (75% yield) of 16e as yellow oil. LCMS (ESI, m/z): 452 [M + H]+. tert-Butyl-4-[[1-methyl-3-(piperidin-4-yl)-1H-pyrazol-4-yl]methyl]piperazine-1-carboxylate (16f). A round-bottom flask was charged with 16e (9.00 g, 19.9 mmol, 1.00 equiv) in ethyl acetate (100 mL) and palladium carbon (2.0 g). The resulting mixture was stirred overnight at rt under an atmosphere of hydrogen (1 atm). The solids were filtered out. The resulting mixture was concentrated under reduced pressure to provide 900 mg (12% yield) of tert-butyl 4-[[1methyl-3-(piperidin-4-yl)-1H-pyrazol-4-yl]methyl]piperazine-1-carboxylate as light-yellow oil which was used without further purification. LCMS (ESI, m/z): 364 [M + H]+. A round-bottom 9075



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8.2, 1.7 Hz, 1H), 7.15−6.97 (m, 2H), 3.08 (t, J = 5.0 Hz, 4H), 1.79 (p, J = 5.4 Hz, 4H), 1.64 (t, J = 5.7 Hz, 4H). LCMS (ES, m/z): 224 [M + H]+. tert-Butyl 4-(4-Chloro-2-(piperidin-1-yl)benzyl)piperazine-1-carboxylate (25d). The title compound was prepared from 25c as described for 28d. 1H NMR (400 MHz, chloroform-d) δ 7.39 (d, J = 7.8 Hz, 1H), 7.11−6.93 (m, 2H), 3.62−3.49 (m, 2H), 3.48−3.32 (m, 4H), 2.86 (t, J = 5.2 Hz, 4H), 2.43 (s, 4H), 1.71 (p, J = 5.6 Hz, 4H), 1.66−1.53 (m, 2H), 1.48 (s, 9H). LCMS (ES, m/z): 394 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(4-chloro-2-(piperidin-1-yl)benzyl)piperazine-1-carboxylate (25). The title compound was prepared from 25d as described in 10d and 10. 1H NMR (400 MHz, chloroform-d) δ 7.38 (d, J = 8.0 Hz, 1H), 7.11−6.92 (m, 2H), 5.79 (hept, J = 6.2 Hz, 1H), 3.56 (d, J = 6.1 Hz, 6H), 2.85 (t, J = 5.1 Hz, 4H), 2.60−2.40 (m, 4H), 1.71 (t, J = 5.5 Hz, 4H), 1.66−1.47 (m, 2H). LCMS (ES, m/z): 488 [M + H]+. 2-(Piperidin-1-yl)-4-(trifluoromethyl)benzaldehyde (26c). The title compound was prepared directly from commercially available 4- trifluoromethyl −2-fluorobenzaldehyde and piperidine as described for 28c (63%, yellow oil). LCMS (ESI, m/z): 258 [M + H]+ tert-Butyl 4-(2-(Piperidin-1-yl)-4-(trifluoromethyl)benzyl)piperazine-1-carboxylate (26d). The title compound was prepared from 26c and tert-butyl piperazine-1-carboxylate as described for 28d (71%, yellow oil). LCMS (ESI, m/z): 428 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl 4-(2-(piperidin-1-yl)-4(trifluoromethyl)benzyl)piperazine-1-carboxylate (26). The title compound was prepared from 26d as described in 28. 1H NMR (300 MHz, chloroform-d) δ 7.58 (d, J = 7.8 Hz, 1H), 7.28 (br, 2H), 5.69−5.81 (m, 1H), 3.60 (s, 2H), 3.55 (br, 4H), 2.85 (t, J = 5.0 Hz, 4H), 2.49 (br, 4H), 1.69−1.73 (m, 4H), 1.58−1.60 (m, 2H). LCMS (ESI, m/z): 522 [M + H]+ 4-Chloro-2-(pyrrolidin-1-yl)benzaldehyde (27c). The title compound was prepared from 4-chloro-2-fluorobenzaldehyde and pyrrolidine as described for 28c. 1H NMR (400 MHz, CDCl3) δ 10.04 (s, 1H), 7.64 (d, J = 8.1 Hz, 1H), 6.82 (s, 1H), 6.77 (d, J = 8.4 Hz, 1H), 3.41−3.33 (m, 4H), 2.06−1.96 (m, 4H). LCMS (ESI, m/z): 210 [M + H]+. tert-Butyl 4-(4-Chloro-2-(pyrrolidin-1-yl)benzyl)piperazine-1-carboxylate (27d). The title compound was prepared from 27c and tertbutyl piperazine-1-carboxylate as described for 28d. 1H NMR (400 MHz, CDCl3) δ 7.32 (d, J = 8.2 Hz, 1H), 6.88−6.79 (m, 2H), 3.49 (s, 2H), 3.43 (t, J = 4.9 Hz, 4H), 3.24 (ddd, J = 6.5, 4.2, 2.1 Hz, 4H), 2.39 (d, J = 6.0 Hz, 4H), 1.94 (td, J = 5.5, 4.8, 2.9 Hz, 4H), 1.47 (d, J = 1.4 Hz, 9H). LCMS (ESI, m/z): 380 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(4-chloro-2-(pyrrolidin-1yl)benzyl)piperazine-1-carboxylate (27). The title compound was prepared from 27d by deprotection as described for 10d and carbamoylation as described for 28. 1H NMR (400 MHz, CDCl3) δ 7.34−7.26 (m, 1H), 6.90−6.80 (m, 2H), 5.78 (hept, J = 6.2 Hz, 1H), 3.61−3.54 (m, 4H), 3.52 (s, 2H), 3.27−3.19 (m, 4H), 2.47 (m, 4H), 2.00−1.87 (m, 4H). LCMS (ESI, m/z): 474 [M + H]+. 2-(Pyrrolidin-1-yl)-4-(trifluoromethyl)benzaldehyde (28c). A flask was charged with 2-fluoro-4-(trifluoromethyl)benzaldehyde (2.00 g, 10.4 mmol, 1.00 equiv), pyrrolidine (1.11 g, 15.6 mmol, 1.50 equiv), potassium carbonate (3.59 g, 26.0 mmol, 2.50 equiv), and dimethyl sulfoxide (20 mL). The reaction was stirred overnight at 85 °C in an oil bath. The resulting mixture was diluted with H2O (20 mL) and extracted with DCM (2 × 20 mL). The organic layers were combined, washed with H2O (3 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (1/9) to provide 1.60 g (63% yield) of 28c as yellow oil. 1H NMR (300 MHz, CDC13) δ 10.14 (s, 1H), 7.78 (d, J = 8.1 Hz, 1H), 6.99−7.05 (m, 2H), 3.37−3.42 (m, 4H), 1.99−2.08 (m, 4H). LCMS (ES, m/z): 244 [M + H]+. tert-Butyl 4-[[2-(Pyrrolidin-1-yl)-4-(trifluoromethyl)phenyl]methyl]piperazine-1-carboxylate (28d). A flask was charged with 28c (1.60 g, 6.58 mmol, 1.10 equiv), tert-butyl piperazine-1carboxylate (1.11 g, 5.96 mmol, 1.00 equiv), and 1,2-dichloroethane (20 mL). The mixture was stirred at rt for 0.5 h. Sodium

4H), 2.43 (t, J = 5.1 Hz, 4H), 1.48 (s, 9H). LCMS (ESI, m/z): 302 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(4-chloro-2morpholinobenzyl)piperazine-1-carboxylate (20). The title compound was prepared from 20d as described in 10d. 1H NMR (400 MHz, chloroform-d) δ 7.40−7.33 (m, 1H), 7.10−7.03 (m, 2H), 5.74 (h, J = 6.3 Hz, 1H), 3.86−3.79 (m, 4H), 3.53 (d, J = 7.0 Hz, 5H), 2.98−2.90 (m, 4H), 2.47 (dt, J = 9.8, 5.1 Hz, 4H). LCMS (ESI, m/z): 490 [M + H]+. 5-Chloro-2-morpholinobenzaldehyde (21c). The title compound was prepared directly from commercially available 5-chloro-2fluorobenzaldehyde and morpholine as described for 28c. LCMS (ESI, m/z): 226 [M + H]+ tert-Butyl 4-(5-Chloro-2-morpholinobenzyl)piperazine-1-carboxylate (21d). The title compound was prepared from 21c as described for 28d. LCMS (ESI, m/z): 396 [M + H]+ 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(5-chloro-2morpholinobenzyl)piperazine-1-carboxylate (21). The title compound was prepared from tert-butyl 4-(5-chloro-2morpholinobenzyl)piperazine-1-carboxylate as described for 28. 1H NMR (300 MHz, chloroform-d) δ 7.45 (s, 1H), 7.23 (d, J = 9.0 Hz, 1H), 7.04 (d, J = 9.0 Hz, 1H), 5.81−5.69 (m, 1H), 3.84−3.81 (m, 4H), 3.56 (br, 6H), 2.91−2.88 (m, 4H), 2.50 (br, 4H). LCMS (ESI, m/z): 490 [M + H]+. 2-Chloro-6-morpholinobenzaldehyde (22c). The title compound was prepared directly from commercially available 2-chloro-6fluorobenzaldehyde and morpholine as described for 28c. LCMS (ESI, m/z): 226 [M + H]+. tert-Butyl 4-(2-Chloro-6-morpholinobenzyl)piperazine-1-carboxylate (22d). The title compound was prepared from 22c and tertbutyl-1-piperazine-1-carboxylate as described for compound 28d. LCMS (ESI, m/z): 396 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(2-chloro-6morpholinobenzyl)piperazine-1-carboxylate (22). The title compound was prepared from 22d as described for 28. 1H NMR (300 MHz, chloroform-d) δ 7.25−7.17 (m, 2H), 7.12−7.09 (m, 1H), 5.86−5.73 (m, 1H), 3.84−3.78 (m, 6H), 3.47 (d, J = 6.0 Hz, 4H), 3.00 (br, 4H), 2.60 (br, 4H). LCMS (ESI, m/z): 490 [M + H]+. 3-Chloro-2-morpholinobenzaldehyde (23c). The title compound was prepared directly from commercially available 3-chloro-2fluorobenzaldehyde and morpholine as described for 28c. LCMS (ESI, m/z): 226 [M + H]+. tert-Butyl 4-(3-Chloro-2-morpholinobenzyl)piperazine-1-carboxylate (23d). The title compound was prepared from 23c and tertbutyl-1-piperazine-1-carboxylate as described for 28d. LCMS (ESI, m/z): 396 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(3-chloro-2morpholinobenzyl)piperazine-1-carboxylate (23). The title compound was prepared from 23d as described for 28. 1H NMR (300 MHz, chloroform-d) δ 7.27 (d, J = 3.0 Hz, 2H), 7.11−7.06 (m, 1H), 5.84−5.71 (m, 1H), 3.90−3.87 (m, 2H), 3.78−3.62 (m, 6H), 3.60− 3.53 (m, 4H), 2.78 (d, J = 10.8 Hz, 2H), 2.49−2.48 (m, 4H). LCMS (ESI, m/z): 490 [M + H]+. 2-Morpholino-4-(trifluoromethyl)benzaldehyde (24c). The title compound was prepared directly from commercially available 2fluoro-4-(trifluoromethyl)benzaldehyde and tert-butyl piperazine-1carboxylate as described for 28c. LCMS (ES, m/z): 260 [M + H]+. tert-Butyl 4-(2-Morpholino-4-(trifluoromethyl)benzyl)piperazine1-carboxylate (24d). The title compound was prepared from 23c as described for 28d. LCMS (ES, m/z): 430 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl 4-(2-morpholino-4 (trifluoromethyl)benzyl)piperazine-1-carboxylate (24). The title compound was prepared from 24d as described for 28. 1H NMR (300 MHz, CDC13) δ: 7.54−7.623 (m, 1H), 7.33−7.42 (m, 2H), 5.72−5.85 (m, 1H), 3.84−3.87 (m, 4H), 3.64 (s, 2H), 3.56−3.57 (m, 4H), 2.96−3.00 (m, 4H), 2.51−2.52 (m, 4H). LCMS (ES, m/z): 524 [M + H]+. 4-Chloro-2-(piperidin-1-yl)benzaldehyde (25c). The title compound was prepared directly from commercially available 5-chloro-2fluorobenzaldehyde and morpholine as described for 28c. 1H NMR (400 MHz, chloroform-d) δ 10.21 (d, J = 1.6 Hz, 1H), 7.74 (dd, J = 9076



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triacetoxyborohydride (3.80 g, 17.9 mmol, 3.00 equiv) was added. The resulting solution was stirred overnight at rt and washed with H2O (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (1/ 3) to provide 2.10 g (77% yield) of 28d as yellow oil. LCMS (ES, m/ z): 414 [M + H]+. 1-[[2-(Pyrrolidin-1-yl)-4-(trifluoromethyl)phenyl]methyl]piperazine (28e). A round-bottom flask was charged with 28d (500 mg, 1.21 mmol, 1.00 equiv) and DCM (10 mL). The mixture was cooled to 0 °C. Trifluoroacetic acid (1 mL) was added dropwise. The resulting solution was stirred overnight at rt. The resulting mixture was concentrated under reduced pressure to provide 421 mg of 28e as a brown solid, which was used without further purification. LCMS (ES, m/z): 314 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-[[2-(pyrrolidin-1-yl)-4(trifluoromethyl)phenyl]methyl]piperazine-1-carboxylate (28). A round-bottom flask was charged with 1,1,1,3,3,3-hexafluoropropan2-ol (84.0 mg, 0.500 mmol, 1.00 equiv), triphosgene (50.0 mg, 0.170 mmol, 0.33 equiv), DCM (10 mL), and N,N-diisopropylethylamine (194 mg, 1.50 mmol, 2.94 equiv). The mixture was stirred at rt for 2 h. 28e (160 mg, 0.510 mmol, 1.00 equiv) was added. The resulting mixture was stirred for 2 h at rt and diluted with DCM (20 mL). The mixture was washed with H2O (2 × 5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (1/3). The product (276 mg) was purified by preparative HPLC using the following gradient conditions: 20% CH3CN/80% phase A increasing to 80% CH3CN over 10 min, then to 100% CH3CN over 0.1 min, holding at 100% CH3CN for 1.9 min, then reducing to 20% CH3CN over 0.1 min and holding at 20% CH3CN for 1.9 min, on a Waters 2767-5 Chromatograph. Column: Xbridge Prep C18, 19 mm × 150 mm 5 μm; mobile phase. Phase A: aqueous of NH4HCO3 (0.05%). Phase B: CH3CN; detector 220 and 254 nm. Purification resulted in 204.9 mg (79% yield) of 28 as lightyellow oil. 1H NMR (300 MHz, CDC13) δ 7.52 (d, J = 8.1 Hz, 1H), 7.08−7.11 (m, 2H), 5.74−5.84 (m, 1H), 3.58 (br, 6H), 3.22−3.26 (m, 4H), 2.46−2.49 (m, 4H), 1.90−1.98 (m, 4H)). LCMS (ES, m/ z): 508 [M + H]+. HRMS (ESI): m/z calculated for C20H22F9N3O2 + H+ [M + H+] 508.1647, found 508.1639. 4-Bromo-2-(pyrrolidin-1-yl)benzaldehyde (29c). The title compound was prepared from 4-bromo-2-fluorobenzaldehyde and pyrrolidine as described for 28c. 1H NMR (400 MHz, CDCl3) δ 10.04 (d, J = 1.2 Hz, 1H), 7.56 (dd, J = 8.4, 1.5 Hz, 1H), 7.00 (d, J = 1.4 Hz, 1H), 6.93 (dd, J = 8.4, 1.5 Hz, 1H), 3.41−3.33 (m, 4H), 2.02 (tt, J = 5.1, 2.1 Hz, 4H). LCMS (ESI, m/z): 254 [M + H]+. 4-(4-Bromo-2-(pyrrolidin-1-yl)benzyl)piperazine-1-carboxylate (29d). The title compound was prepared from 29c and tert-butyl piperazine-1-carboxylate as described for 28d. 1H NMR (400 MHz, CDCl3) δ 7.31−7.23 (m, 1H), 7.02−6.93 (m, 2H), 3.50−3.37 (m, 6H), 3.23 (t, J = 6.5 Hz, 4H), 2.39 (m, 4H), 1.99−1.88 (m, 4H), 1.47 (s, 9H). LCMS (ESI, m/z): 424 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(4-bromo-2-(pyrrolidin-1yl)benzyl)piperazine-1-carboxylate (29). The title compound was prepared from 29d by deprotection as described for 10d and carbamoylation as described for 28. 1H NMR (400 MHz, CDCl3) δ 7.26 (dd, J = 8.1, 3.7 Hz, 1H), 7.05−6.95 (m, 2H), 5.79 (hept, J = 6.0 Hz, 1H), 3.62−3.44 (m, 6H), 3.27−3.18 (m, 4H), 2.53−2.41 (m, 4H), 2.01−1.88 (m, 4H). LCMS (ESI, m/z): 518 [M + H]+. 4-Iodo-2-(pyrrolidin-1-yl)benzaldehyde (30c). The title compound was prepared from commercially available 2-fluoro-4iodobenzaldehyde and pyrrolidine as described for 28c. LCMS (ESI, m/z): 302 [M + H]+. tert-Butyl 4-(4-Iodo-2-(pyrrolidin-1-yl)benzyl)piperazine-1-carboxylate (30d). The title compound was prepared from 30c and tert-butyl piperazine-1-carboxylate as described for 28d. LCMS (ESI, m/z): 472 [M + H]+. tert-Butyl 4-(4-(Methylsulfonyl)-2-(pyrrolidin-1-yl)benzyl)piperazine-1-carboxylate (30e). A round-bottom flask was charged with 30d (380 mg, 0.807 mmol, 1.00 equiv), sodium methanesulfinate

(99.0 mg, 0.968 mmol, 1.20 equiv), copper(I) iodide (31.0 mg, 0.161 mmol, 0.20 equiv), L-proline (37.0 mg, 0.323 mmol, 0.40 equiv), and dimethyl sulfoxide (10 mL). The resulting mixture was stirred overnight at 120 °C and quenched with water (10 mL). The mixture was extracted with ethyl acetate (3 × 20 mL), and the organic layers were combined, washed with brine (2 × 15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with DCM/methanol (98/2) to yield 237 mg (69% yield) of 30e as yellow oil. LCMS (ESI, m/z): 424 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(4-(methylsulfonyl)-2-(pyrrolidin-1-yl)benzyl)piperazine-1-carboxylate (30). The title compound was prepared from 30e as described for 28. 1H NMR (300 MHz, chloroform-d) δ 7.64−7.62 (m, 1H), 7.39 (s, 2H), 5.79−5.71 (m, 1H), 3.59 (br, 6H), 3.28−3.26 (m, 4H), 3.05 (s, 3H), 2.47 (br, 4H), 1.97 (br, 4H). LCMS (ESI, m/z): 518 [M + H]+. 2-(Pyrrolidin-1-yl)-4-[2-(trimethylsilyl)ethynyl]benzaldehyde (31e). A mixture of 29c (1 g, 3.93 mmol, 1 equiv), ethynyltrimethylsilane (1.93 g, 19.6 mmol, 5 equiv), Pd(PPh3)4 (450 mg, 0.39 mmol, 0.1 equiv), copper(I) iodide (149 mg, 0.78 mmol, 0.2 equiv), and Et3N (1100 mg, 12.0 mmol, 3 equiv) in DMF (20 mL) was stirred overnight at 90 °C under nitrogen atmosphere. The resulting mixture was diluted with ethyl acetate (100 mL). The organic layers were washed with brine (3 × 100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE/ethyl acetate (10:1) to afford 31e (1.0 g, 93%) as a yellow oil. LCMS (ESI, m/z): 272 [M + H]+. tert-Butyl 4-[[2-(Pyrrolidin-1-yl)-4-[2-(trimethylsilyl)ethynyl]phenyl]methyl]piperazine-1-carboxylate (31f). A mixture of 31e (900 mg, 3.3 mmol, 1 equiv) and tert-butyl piperazine-1-carboxylate (679 mg, 3.6 mmol, 1.1 equiv) in DCM (20 mL) was stirred for 1 h at rt under nitrogen atmosphere. To the above mixture was added NaBH(OAc)3 (1.75 g, 8.28 mmol, 2.5 equiv) . The resulting mixture was stirred for additional overnight at rt. The reaction was quenched with H2O (20 mL), extracted with DCM (2 × 50 mL), and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE/ethyl acetate (5:1) to afford 31f (900 mg, 61%) as a yellow oil. LCMS (ESI, m/z): 442 [M + H]+. 1-[[4-Ethynyl-2-(pyrrolidin-1-yl)phenyl]methyl]piperazine (31g). A mixture of 31f (900 mg, 2.038 mmol, 1 equiv) and TFA (3 mL) in DCM (15 mL) was stirred for 4 h at rt. The mixture was concentrated under reduced pressure to yield 1-[[2-(pyrrolidin-1-yl)-4-[2(trimethylsilyl)ethynyl]phenyl]methyl]piperazine (1g) as a yellow crude oil. LCMS (ESI, m/z): 342 [M + H]+. Crude 1-[[2-(pyrrolidin1-yl)-4-[2-(trimethylsilyl)ethynyl]phenyl]methyl]piperazine (950 mg, 2.78 mmol, 1 equiv) and K2CO3 (1100 mg, 8.34 mmol, 3 equiv) in MeOH (15 mL) were stirred for 3 h at rt. The reaction was quenched with H2O. The resulting mixture was extracted with ethyl acetate (2 × 100 mL). The combined organic layers dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE/ethyl acetate (1:1) to afford 31g (480 mg, 64%) as a yellow solid. LCMS (ESI, m/z): 270 [M + H]+. 1-(4-[[4-Ethynyl-2-(pyrrolidin-1-yl)phenyl]methyl]piperazin-1yl)-3,3,3-trifluoro-2-(trifluoromethyl)propan-1-one (31). The title compound was prepared from 31g as described for 28. 1H NMR (400 MHz, DMSO-d6) δ 7.34 (d, J = 7.5 Hz, 1H), 6.96−6.88 (m, 2H), 6.61−6.57 (m, 1H), 4.07 (s, 1H), 3.51 (s, 2H), 3.47 (s, 4H), 3.17 (s, 4H), 2.38 (s, 4H), 1.86 (s, 4H). LCMS (ESI, m/z): 464 [M + H]+. Bis(1,1,1,3,3,3-hexafluoropropan-2-yl) carbonate. A flask was charged with sodium hydride (60% dispersion, 8.16 g, 204 mmol). Ether (100 mL) was added, and the reaction was cooled to 0 °C. A solution of 1,1,1,3,3,3-hexafluoro-2-propanol (21 mL, 204 mmol) in 40 mL of ether was added via addition funnel over 10 min. The solution became clear by the end of the addition. The reaction was stirred at 0 °C for 20 min and then allowed to warm to rt and stirred for 20 min. The solution was transferred via cannula (∼10 mL/min) 9077



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to a 0 °C solution of triphosgene (10 g, 33.7 mmol) in ether (40 mL), resulting in an exothermic reaction and precipitation forming. The solution was stirred at rt for 2 h. The reaction was filtered, and solids were washed with 50 mL of ether. The filtrate was carefully concentrated (bath 36 °C, 500 Torr) via rotary evaporation and yielded a biphasic solution, which partitions into a top (organic) and bottom (fluorous) layer. The bottom layer was retained and found to be bis[2,2,2-trifluoro-1-(trifluoromethyl)ethyl] carbonate (8.00 g, 78% by weight solution in ether, 51% yield) solution and stored and used as a solution without further purification. 1H NMR (400 MHz, chloroform-d) δ 5.50 (hept, J = 5.7 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 151.56, 123.85, 121.02, 118.24, 115.41, 72.51, 72.16, 71.80, 71.45, 71.09. 1,1,1,3,3,3-Hexafluoropropan-2-yl piperazine-1-carboxylate (32b). A flask was charged with tert-butyl-1-piperazinecarboxylate (6.0 g, 32.2 mmol) and dissolved in DCM (50 mL). The solution was cooled to 0 °C, and bis[2,2,2-trifluoro-1-(trifluoromethyl)ethyl] carbonate (12.2 g, 33.8 mmol) was added dropwise, and the reaction was stirred at 0 °C for 1 h. The ice bath was removed, and when the reaction reached rt, the reaction was concentrated. MeOH (100 mL) was added and the reaction was concentrated, resulting in a white powder. The crude 1-(tert-butyl) 4-(1,1,1,3,3,3-hexafluoropropan-2yl) piperazine-1,4-dicarboxylate was dissolved in DCM (40 mL), and triflouoroacetic acid (10 mL) was added and the reaction was stirred at room temp for 5 h. The reaction was concentrated and suspended in cold DCM (200 mL) and 1 N NaOH (50 mL). The aqueous layer was extracted with DCM (3 × 100 mL). The organics were dried over anhydrous sodium sulfate, filtered, and carefully concentrated to yield 32b (6.4 g, 70% yield) as a light-orange oil which was moderately volatile. 1H NMR (400 MHz, chloroform-d) δ 5.84−5.71 (m, 1H), 3.60−3.51 (m, 4H), 2.96−2.80 (m, 4H). LCMS (ESI, m/z): 281.2 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(2-(pyrrolidin-1-yl)benzyl)piperazine-1-carboxylate (32). A round-bottom flask was charged with commercially available 2-(pyrrolidin-1-yl)benzaldehyde (32c, 91.0 mg, 0.520 mmol, 1.00 equiv) and DCM (10 mL), 1,1,1,3,3,3hexafluoropropan-2-yl piperazine-1-carboxylate (147 mg, 0.520 mmol, 1.00 equiv). The mixture was stirred for 1 h at rt. Sodium triacetoxyborohydride (331 mg, 1.56 mmol, 3.00 equiv) was added. The resulting solution was stirred overnight at rt and quenched with water (10 mL). The mixture was extracted with DCM (3 × 30 mL), and the organic layers were combined, washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product (300 mg) was purified by preparative HPLC using the following gradient conditions: 35% CH3CN/65% phase A increasing to 65% CH3CN over 10 min, then to 100% CH3CN over 0.1 min, holding at 100% CH3CN for 1.9 min, then reducing to 35% CH3CN over 0.1 min, and holding at 35% for 1.9 min, on a Waters 2767−5 Chromatograph. Column: Xbridge Prep C18, (19 mm × 150)mm 5 μm. Mobile phase: phase A, aqueous NH4HCO3 (0.05%); phase B, CH3CN; detector 220 and 254 nm. Purification resulted in 150.8 mg (66% yield) of 32 as orange oil. 1H NMR (300 MHz, chloroform-d) δ 7.39 (d, J = 7.5 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H), 6.96−6.87 (m, 2H), 5.81−5.69 (m, 1H), 3.56 (br, 6H), 3.18 (t, J = 6.4 Hz, 4H), 2.48 (br, 4H), 1.94−1.90 (m, 4H). LCMS (ESI, m/z): 440 [M + H]+. 2-((1R,5S)-8-Oxa-3-azabicyclo[3.2.1]octan-3-yl)-4(trifluoromethyl)benzaldehyde (33c). The title compound was prepared from commercially available 2-fluoro-4(trifluoromethyl)benzaldehyde and (1R,5S)-8-oxa-3-azabicyclo[3.2.1]octane as described for 28c. LCMS (ESI, m/z): 286 [M + H]+. tert-Butyl-4-(2-((1R,5S)-8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-4(trifluoromethyl)benzyl)piperazine-1-carboxylate (33d). The title compound was prepared from 33c as described for 28d. LCMS (ESI, m/z): 456 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(2-((1R,5S)-8-oxa-3azabicyclo[3.2.1]octan-3-yl)-4-(trifluoromethyl)benzyl)piperazine1-carboxylate (33). The title compound was prepared from 33d as described for 28. 1H NMR (300 MHz, chloroform-d) δ 7.59 (d, J = 7.8 Hz, 1H), 7.33−7.36 (m, 2H), 5.69−5.82 (m, 1H), 4.42 (s, 2H),

3.64 (s, 2H), 3.56−3.57 (m, 4H), 3.09 (d, J = 10.5 Hz, 2H), 2.80 (d, J = 11.1 Hz, 2H), 2.47−2.48 (m, 4H), 1.93−2.12 (m, 4H). LCMS (ESI, m/z): 550 [M + H]+. 2-(8-Oxa-3-azabicyclo[3.2.1]octan-3-yl)-4-chlorobenzaldehyde (34c). The title compound was prepared from commercially available 4-chloro-2-fluorobenzaldehyde and 8-oxa-3-azabicyclo[3.2.1]octane as described for 28c. LCMS (ESI, m/z): 252 [M + H]+. tert-Butyl-4-(2-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-4chlorobenzyl)piperazine-1-carboxylate (34d). The title compound was prepared from 34c and tert-butyl piperazine-1-carboxylate as described for 28d. LCMS (ESI, m/z): 422 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(2-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-4-chlorobenzyl)piperazine-1-carboxylate (34). The title compound was prepared from 34d as described for 28. 1H NMR (300 MHz, chloroform-d) δ 7.36 (d, J = 8.1 Hz, 1H), 7.05− 7.10 (m, 2H), 5.69−5.81 (m, 1H), 4.39−4.40 (m, 2H), 3.47−3.56 (m, 6H), 3.02−3.06 (m, 2H), 2.78−2.82 (m, 2H), 2.34−2.38 (m, 4H), 1.97−2.11 (m, 4H). LCMS (ESI, m/z): 516 [M + H]+. 2-((1R,5S)-3-Oxa-8-azabicyclo[3.2.1]octan-8-yl)-4(trifluoromethyl)benzaldehyde (35c). The title compound was prepared from commercially available 2-fluoro-4-(trifluoromethyl)benzaldehyde and (1R,5S)-3-oxa-8-azabicyclo[3.2.1]octane as described for compound 28c. LCMS (ESI, m/z): 286 [M + H]+. tert-Butyl-4-(2-((1R,5S)-3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-4(trifluoromethyl)benzyl)piperazine-1-carboxylate (35d). The title compound was prepared from 35c and tert-butyl piperazine-1carboxylate as described for 28d. LCMS (ESI, m/z): 456 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(2-((1R,5S)-3-oxa-8azabicyclo[3.2.1]octan-8-yl)-4-(trifluoromethyl)benzyl)piperazine1-carboxylate (35). The title compound was prepared from 35d as described for 28. 1H NMR (300 MHz, chloroform-d) δ 7.57 (d, J = 7.8 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 7.06 (s, 1H), 5.71−5.79 (m, 1H), 3.91 (d, J = 10.2 Hz, 2H), 3.64−3.72 (m, 6H), 3.56 (br, 4H), 2.52 (br, 4H), 1.94−2.07 (m, 4H). LCMS (ESI, m/z): 550 [M + H]+. (R)-2-(Hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl)-4(trifluoromethyl)benzaldehyde (36c). The title compound was prepared from commercially available 4-chloro-2-fluorobenzaldehyde and (R)-octahydropyrrolo[1,2-a]pyrazine as described for 28c. 1H NMR (300 MHz, CDCl3) δ 10.21 (s, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.02−7.09 (m, 2H), 3.34−3.38 (m, 1H), 3.13−3.26 (m, 4H), 2.77− 2.84 (m, 1H), 2.52 (s, 1H), 2.18−2.29 (m, 2H), 1.70−1.97 (m, 3H), 1.46−1.50 (m, 1H). (ESI, m/z): 265 [M + H] +. tert-Butyl-(R)-4-(2-(8a-methylhexahydropyrrolo[1,2-a]pyrazin2(1H)-yl)-4-(trifluoromethyl)benzyl)piperazine-1-carboxylate (36d). The title compound was prepared from 36c as described for 28d. 1H NMR (300 MHz, CDCl3) δ 7.30−7.33 (m, 1H), 7.03−7.09 (m, 2H), 3.73 (br, 2H), 3.49 (br, 2H), 3.17−3.40 (m, 7H), 2.98−3.05 (m, 1H), 2.39−2.82 (m, 7H), 2.08 (br, 2H), 1.90−2.04 (m, 2H), 1.44 (s, 9H). (ESI, m/z): 435 [M + H] +. 1,1,1,3,3,3-Hexafluoropropan-2-yl-(R)-4-(2-(hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl)-4-(trifluoromethyl)benzyl)piperazine-1carboxylate (36). The title compound was prepared from 36d as described for 28. 1H NMR (300 MHz, CDCl3) δ 7.32−7.35 (m, 1H), 7.02−7.08 (m, 2H), 5.71−5.80 (m, 1H), 3.52 (br, 6H), 3.27 (d, J = 10.8 Hz, 1H), 3.08−3.17 (m, 3H), 2.90−2.95 (m, 1H), 2.38−2.62 (m, 6H), 2.23−2.26 (m, 2H), 1.76−1.93 (m, 3H), 1.45−1.52(m, 1H). (ESI, m/z): 529 [M + H] +. ( S) - 2- ( 3- H y d ro x y py r r o l i di n - 1 - y l ) -4 - ( tri flu o r o m e t h y l ) benzaldehyde (37c). The title compound was prepared from commercially available 2-fluoro-4-(trifluoromethyl)benzaldehyde and (S)-pyrrolidin-3-ol as described for 28c. 1H NMR (400 MHz, chloroform-d) δ 10.10 (d, J = 3.6 Hz, 1H), 7.80 (dd, J = 7.9, 2.9 Hz, 1H), 7.10−7.03 (m, 2H), 4.66−4.60 (m, 1H), 3.83−3.71 (m, 2H), 3.44−3.34 (m, 1H), 3.13−3.04 (m, 1H), 2.27−2.03 (m, 2H), 1.32− 1.22 (m, 1H). (ESI, m/z): 260 [M + H] +. tert-Butyl (S)-4-(2-(3-Hydroxypyrrolidin-1-yl)-4-(trifluoromethyl)benzyl)piperazine-1-carboxylate (37d). The title compound was prepared from 37c and tert-butyl piperazine-1-carboxylate as described for 28d. (ESI, m/z): 430 [M + H] +. 1,1,1,3,3,3-Hexafluoropropan-2-yl-(S)-4-(2-(3-hydroxypyrrolidin1-yl)-4-(trifluoromethyl)benzyl)piperazine-1-carboxylate (37). The 9078



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1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(2-(5methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-4-(trifluoromethyl)benzyl)piperazine-1-carboxylate (40). A 40 mL round-bottom flask was charged with 40e (110 mg, 0.201 mmol, 1.00 equiv), 1,2dichloroethane (10 mL), paraformaldehyde (36.0 mg, 0.401 mmol, 2.00 equiv), and sodium triacetoxyborohydride (85.0 mg, 0.401 mmol, 2.00 equiv). The resulting solution was stirred overnight at rt, washed with water (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC using the following gradient conditions: 20% CH3CN/80% phase A increasing to 80% CH3CN over 10 min, then to 100% CH3CN over 0.1 min, holding at 100% CH3CN for 1.9 min, then reducing to 20% CH3CN over 0.1 min and holding at 20% for 1.9 min, on a Waters 2767−5 Chromatograph. Column: Xbridge Prep C18, 19 mm × 150 mm 5 μm. Mobile phase: phase A, aqueous NH4HCO3 (0.05%); phase B, CH3CN; detector 220 and 254 nm. Purification resulted in 53.4 mg (47% yield) of 40 as an off-white solid. 1H NMR (300 MHz, chloroform-d) δ 7.60 (d, J = 7.89 Hz, 1H), 7.30 (s, 1H), 7.29 (s, 1H), 5.70−5.80 (m, 1H), 3.55−3.64 (m, 6H), 3.05−3.15 (m, 2H), 2.88− 3.02 (m, 6H), 3.44−3.52 (m, 4H), 2.41 (s, 5H). LCMS (ESI, m/z): 563 [M + H]+. 2-(8-Oxa-2-azaspiro[4.5]decan-2-yl)-4-(trifluoromethyl)benzaldehyde (41c). The title compound was prepared from commercially available 2-fluoro-4-(trifluoromethyl)benzaldehyde and 8-oxa-2-azaspiro[4.5]decane as described for 28c. LCMS (ESI, m/z): 314 [M + H]+. t e r t - B u t y l 4 - ( 2 - ( 8 - O x a - 2 - a z a s p i r o [ 4 . 5 ] d e c a n - 2 - y l ) -4 (trifluoromethyl)benzyl)piperazine-1-carboxylate (41d). The title compound was prepared from 41c and tert-butyl piperazine-1carboxylate as described for 28d. LCMS (ESI, m/z): 484 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(2-(8-oxa-2-azaspiro[4.5]decan-2-yl)-4-(trifluoromethyl)benzyl)piperazine-1-carboxylate (41). The title compound was prepared from 41d as described for 28. 1 H NMR (300 MHz, chloroform-d) δ 7.47 (d, J = 7.5 Hz, 1H), 7.12− 7.07 (m, 2H), 5.80−5.68 (m, 1H), 4.05−3.56 (m, 10H), 3.33 (t, J = 6.9 Hz, 2H), 3.18 (s, 2H), 2.45 (br, 4H), 1.86 (t, J = 6.9 Hz, 2H), 1.67 (t, J = 5.3 Hz, 4H). LCMS (ESI, m/z): 578 [M + H]+. tert-Butyl 4-(5-Chloro-2-formylphenyl)piperazine-1-carboxylate (42c). The title compound was prepared from commercially available 2-fluoro-4-chloro-benzaldehyde and tert-butyl-1-piperazinecarboxylate as described for 28c (73% yield). 1H NMR (400 MHz, chloroform-d) δ 10.25 (s, 1H), 7.77 (d, J = 8.3 Hz, 1H), 7.18−7.05 (m, 2H), 3.69− 3.61 (m, 4H), 3.09−3.02 (m, 4H), 1.51 (s, 9H). LCMS (ESI, m/z): 325.1 [M + H]+. tert-Butyl 4-(5-Chloro-2-((4-(((1,1,1,3,3,3-hexafluoropropan-2yl)oxy)carbonyl)piperazin-1-yl)methyl)phenyl)piperazine-1-carboxylate (42d). The title compound was prepared from 42c and 1,1,1,3,3,3-hexafluoropropan-2-yl piperazine-1-carboxylate (32b) as described for 32. 1H NMR (400 MHz, chloroform-d) δ 7.17 (d, J = 8.2 Hz, 1H), 6.91−6.83 (m, 2H), 5.57 (hept, J = 6.1 Hz, 1H), 3.36 (s, 10H), 2.78−2.64 (m, 4H), 2.30 (dt, J = 9.6, 4.9 Hz, 4H), 1.31 (s, 9H). LCMS (ESI, m/z): 590.2 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(4-chloro-2-(piperazin-1yl)benzyl)piperazine-1-carboxylate (42e). A flask was charged with 42c (8.0 g, 13.6 mmol) and dissolved in DCM (40 mL). Trifluoroacetic acid (10 mL) was added, and the reaction was stirred at rt for 4 h. The reaction was concentrated, and DCM (100 mL) was added. The solution was poured into a cold 1N NaOH solution (70 mL). The aqueous phase was extracted with DCM (3 × 100 mL). The resulting clear oil 42e (6.7 g, 99% yield) was used without further purification. 1H NMR (400 MHz, chloroform-d) δ 7.38 (d, J = 8.7 Hz, 1H), 7.10−7.02 (m, 2H), 5.77 (hept, J = 6.3 Hz, 1H), 3.55 (d, J = 4.7 Hz, 6H), 3.02 (t, J = 4.7 Hz, 4H), 2.94−2.86 (m, 4H), 2.49 (dt, J = 9.9, 5.0 Hz, 4H). LCMS (ESI, m/z): 489.2 [M + H]+. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(4-chloro-2-(4-(2fluoroethyl)piperazin-1-yl)benzyl)piperazine-1-carboxylate (42). A thick walled tube was charged with 42e (200 mg, 0.409 mmol), potassium carbonate (113 mg, 0.818 mmol), and 2-fluoroethyl 4methylbenzenesulfonate (89 mg, 0.409 mmol) and dissolved in MeCN (11 mL). The reaction was heated to 80 °C for 8 h. The

title compound was prepared from 37d as described for 28. 1H NMR (400 MHz, chloroform-d) δ 7.50 (d, J = 7.9 Hz, 1H), 7.20−7.12 (m, 2H), 5.77 (hept, J = 6.2 Hz, 1H), 4.54 (s, 1H), 3.66 (d, J = 14.1 Hz, 1H), 3.61−3.48 (m, 6H), 3.41 (dd, J = 10.1, 4.4 Hz, 1H), 3.30 (d, J = 10.2 Hz, 1H), 3.18 (td, J = 8.8, 4.7 Hz, 1H), 2.52−2.43 (m, 4H), 2.31−2.17 (m, 2H), 2.05−1.97 (m, 1H). (ESI, m/z): 524 [M + H] +. ( R ) -2 - ( 3 - H y d r o x y p y r r o l i d i n - 1 - y l ) - 4 - ( t r i fl uorom et h yl)benzaldehyde (38c). The title compound was prepared from commercially available 2-fluoro-4-(trifluoromethyl)benzaldehyde and (R)-pyrrolidin-3-ol as described for 28c. 1H NMR (400 MHz, chloroform-d) δ 10.09 (s, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.14−7.02 (m, 2H), 4.63 (s, 1H), 3.83−3.71 (m, 2H), 3.43−3.33 (m, 1H), 3.08 (d, J = 11.4 Hz, 1H), 2.26−2.10 (m, 3H). (ESI, m/z): 260 [M + H] +. tert-Butyl-(R)-4-(2-(3-hydroxypyrrolidin-1-yl)-4-(trifluoromethyl)benzyl)piperazine-1-carboxylate (38d). The title compound was prepared from 38c and tert-butyl piperazine-1-carboxylate as described for 28d. 1H NMR (400 MHz, chloroform-d) δ 7.49 (d, J = 7.8 Hz, 1H), 7.18−7.10 (m, 2H), 4.52 (s, 1H), 3.64 (d, J = 13.9 Hz, 1H), 3.59−3.48 (m, 2H), 3.46−3.35 (m, 5H), 3.31 (d, J = 10.1 Hz, 1H), 3.18 (td, J = 8.8, 4.7 Hz, 1H), 2.40 (s, 4H), 2.29−2.16 (m, 2H), 2.06−1.96 (m, 1H), 1.47 (s, 9H). (ESI, m/z): 430 [M + H] +. 1,1,1,3,3,3-Hexafluoropropan-2-yl-(R)-4-(2-(3-hydroxypyrrolidin1-yl)-4-(trifluoromethyl)benzyl)piperazine-1-carboxylate (38). The title compound was prepared from 38d as described for 28. 1H NMR (400 MHz, chloroform-d) δ 7.45 (d, J = 7.8 Hz, 1H), 7.15−7.07 (m, 2H), 5.72 (hept, J = 6.2 Hz, 1H), 4.49 (s, 1H), 3.62 (d, J = 14.0 Hz, 1H), 3.57−3.43 (m, 6H), 3.36 (dd, J = 10.1, 4.4 Hz, 1H), 3.25 (d, J = 10.1 Hz, 1H), 3.13 (td, J = 8.8, 4.7 Hz, 1H), 2.47−2.38 (m, 4H), 2.26−2.12 (m, 2H), 2.05−1.92 (m, 1H). (ESI, m/z): 524 [M + H] +. 2-(Tetrahydro-1H-furo[3,4-c]pyrrol-5(3H)-yl)-4-(trifluoromethyl)benzaldehyde (39c). The title compound was prepared from commercially available 2-fluoro-4-(trifluoromethyl)benzaldehyde and hexahydro-1H-furo[3,4-c]pyrrole as described for 28c. 1H NMR (400 MHz, chloroform-d) δ 10.24 (s, 1H), 7.93−7.64 (m, 1H), 7.25−7.19 (m, 1H), 7.17 (s, 1H), 4.04−3.93 (m, 2H), 3.79−3.71 (m, 2H), 3.57−3.49 (m, 2H), 3.26−3.18 (m, 2H), 3.13−3.02 (m, 2H). (ESI, m/z): 524 [M + H] +. tert-Butyl-4-(2-(tetrahydro-1H-furo[3,4-c]pyrrol-5(3H)-yl)-4(trifluoromethyl)benzyl)piperazine-1-carboxylate (39d). The title compound was prepared from 39c and tert-butyl piperazine-1carboxylate as described for 28d. 1H NMR (400 MHz, chloroformd) δ 7.62−7.57 (m, 1H), 7.28−7.23 (m, 2H), 4.08−4.02 (m, 2H), 3.66−3.60 (m, 2H), 3.56 (s, 2H), 3.47−3.41 (m, 4H), 3.18−3.11 (m, 2H), 3.08−3.02 (m, 2H), 3.00−2.91 (m, 2H), 2.46−2.37 (m, 4H), 1.48 (s, 9H). 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(2-(tetrahydro-1H-furo[3,4c]pyrrol-5(3H)-yl)-4-(trifluoromethyl)benzyl)piperazine-1-carboxylate (39). The title compound was prepared from 39d as described for 28. 1H NMR (400 MHz, chloroform-d) δ 7.42−7.34 (m, 1H), 7.08− 7.06 (m, 1H), 7.05−7.04 (m, 1H), 7.04−7.02 (m, 1H), 5.59−5.49 (m, 1H), 3.84−3.77 (m, 2H), 3.46−3.28 (m, 8H), 2.97−2.89 (m, 2H), 2.83−2.70 (m, 4H), 2.32−2.22 (m, 4H). (ESI, m/z): 550 [M + H] +. tert-Butyl 5-(2-Formyl-5-(trifluoromethyl)phenyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (40c). The title compound was prepared from commercially available 2-fluoro4(trifluoromethyl)benzaldehyde and tert-butyl hexahydropyrrolo[3,4c]-2(1H)-carboxylate as described for 28c. (ESI, m/z): 385 [M + H] + . tert-Butyl 5-(2-((4-(((1,1,1,3,3,3-Hexafluoropropan-2-yl)oxy)carbonyl)piperazin-1-yl)methyl)-5-(trifluoromethyl)phenyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (40d). The title compound was prepared from 40c and 32b as described for 32. (ESI, m/z): 649 [M + H] +. 1,1,1,3,3,3-Hexafluoropropan-2-yl-4-(2-(hexahydropyrrolo[3,4c]pyrrol-2(1H)-yl)-4-(trifluoromethyl)benzyl)piperazine-1-carboxylate (40e). A 100 mL round-bottom flask was charged with 40d (300 mg, 0.461 mmol, 1.00 equiv), DCM (5 mL), and trifluoroacetic acid (2 mL). The resulting solution was stirred for 1 h at rt and concentrated under reduced pressure to provide 300 mg (crude) of 40e as a yellow oil. LCMS (ESI, m/z): 549 [M + H]+. 9079



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monitored at 25 °C in kinetic mode with excitation at 355 nm and emission at 460 nm. Relative fluorescence units (RFU, normalized to no enzyme controls) were converted to micromolar product using a standard curve for umbelliferone. The reaction progress curves at each concentration of 28 were fit in Prism (Graphpad) using an equation for first-order association, defined by eq 1.

reaction was poured into brine (30 mL) and extracted with DCM (3 × 50 mL). The organics were dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting oil was chromatographed on a silica column with a gradient (0−30% ethyl acetate in hexane) and yielded 42 (100 mg, 43%) as a clear oil. 1H NMR (400 MHz, chloroform-d) δ 7.37 (d, J = 8.7 Hz, 1H), 7.13−7.02 (m, 2H), 5.77 (hept, J = 6.3 Hz, 1H), 4.71 (t, J = 4.8 Hz, 1H), 4.59 (t, J = 4.8 Hz, 1H), 3.62−3.47 (m, 6H), 3.01 (t, J = 4.3 Hz, 4H), 2.84 (t, J = 4.7 Hz, 1H), 2.81−2.64 (m, 5H), 2.50 (dt, J = 9.3, 5.0 Hz, 4H). LCMS (ESI, m/z): 535.2 [M + H]+. Activity-Based Protein Profiling (ABPP). Gel-Based ABPP. The potency (IC50 values following a 30 min preincubation) and selectivity of test compounds were assessed by ABPP coupled to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE)60 and were carried out essentially as previously described.4 The FP-Rh,51 HT-01,61 and JW91249 activity-based probes were synthesized and implemented in a similar manner as previously described. Brain protein homogenates (1 mg/mL, 50 μL) or PC3 membrane fractions (2 mg/mL, 50 μL) were treated with 0.001−10 μM compound or DMSO for 30 min at 37 °C. ABPP analysis was performed by treating the reactions with 1 μM FP-Rh or HT-01 (brain homogenates) or JW912 (PC3 membranes) for 30 min at 25 °C. Reactions were quenched with 4× SDS-PAGE loading buffer. Samples were separated by SDS-PAGE (10% acrylamide), and fluorescence was visualized in-gel with a Bio-Rad ChemiDox XRS fluorescence imager. Rhodamine (FP-Rh) or BODIPY (JW912) fluorescence is shown in gray scale. Fluorescent bands corresponding to MGLL activity (labeled with FP-Rh or JW912) or ABHD6 activity (labeled with HT-01 or JW912) were quantitated using image J (NIH). Intensities were normalized to produce 0−100% enzyme activity based on DMSO controls (100% activity). IC50 values were calculated by curve fitting log-transformed data by nonlinear regression with a four-parameter, sigmoidal dose response function (variable slope) in Prism software (GraphPad). Curves were constrained to 0% (bottom) and 100% (top). IC50 assays were performed in duplicate unless otherwise noted. Cell-Based ABPP of 28 in PC3 Cells. Human prostate cancer PC3 cells (ATCC, CRL-1435) were grown in F-12K medium supplemented with 10% fetal bovine serum at 37 °C with 5% CO2 to ∼80% confluency in 100 mm dishes. 28 was added to cells to give final concentration of 0.1−10 μM compound in serum free media. Cells were incubated with compound for 30 min at 37°C with 5% CO2. Cells were washed, harvested, and lysed by probe sonication. Cell lysates (2 mg/mL) were treated with JW912 (1 μM) and analyzed by SDS-PAGE and in-gel fluorescence scanning. Full details can be found in the Supporting Information. MS-Based ABPP of 28. Homogenate from human prefrontal cortex treated with FP-biotin, enriched with avidin, and trypsinized essentially as previously described.3 Tryptic digests of the inhibitorand DMSO-treated samples were isotopically labeled by reductive dimethylation of primary amines with natural or isotopically heavy formaldehyde62 and subsequently analyzed using MudPIT on a ThermoFisher Velos Pro Orbitrap mass spectrometer. Peptide spectral matching was performed with the complete human UniProt database (ver 11/5/2012) using the ProLuCID algorithm (version 1.3.3), and the resulting matches filtered using DTASelect (version 2.0.47). Quantification of light/heavy ratios was performed using the Cimage algorithm. Data were plotted as percent activity ± SEM. Full MS-based ABPP experimental details can be found in the Supporting Information. Kinetic Characterization and Determination of kinact/KI for 28. In vitro inhibition of recombinant, purified human N-terminal hexahistidine tagged MGLL47 by a dose response (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 μM) of 28 at 25 °C was assessed by a fluorescent plate-based assay monitoring the hydrolysis of the substrate 7hydroxycoumarin arachidonate (7-HCA). The reaction volume was 100 μL and included, in order of addition, 89 μL of 7-HCA substrate (60 μM in assay) in assay buffer (PBS, pH 8, with 0.05% BSA and 0.1% Triton X-100), 1 μL of inhibitor (100×) in DMSO, and 10 μL of 10× hMGLL (0.0005 mg/mL in assay). The progress curves were

Y = Y0 + (plateau − Y0) × (1 − exp( − Kx))

(1)

K from the fit was defined as the kobs, the rate of inactivation. Each experiment was performed with triplicate assay points, and the entire experiment was conducted four separate times. The observed firstorder rate constant for MGLL inactivation, kobs, at multiple concentrations was replotted versus 28 concentration in pro Fit software (Quantum Soft, Switzerland). The data was fit using the Levenberg−Marquadt algorithm to eq 2 that simplifies to eq 3 under conditions of [S] = 2 × Km.63,64 The fit provides kinact and KI from which the ratio kinact/KI was calculated.

k inact[l]

kobs =

(

[l ] + K i 1 + k inact[l] [l] + 3(K i)

[S] Km

)

(2)

(3)

Samples were run with triplicate enzyme samples and corresponding triplicate no enzyme (assay buffer) controls. Site of MGLL Labeling by 28. Detection of the 28−MGLL adduct was carried out in a similar manner as previously described.3,24 Briefly, hMGLL purified wild-type (WT) human MGLL protein was incubated in the presence of 200 μM compound or DMSO for 30 min at room temperature. The samples were treated with urea, tris(2carboxyethyl)phosphine (TCEP), and iodoacetamide. Reactions were then diluted, trypsinized, and analyzed by LC-MS/MS on a Thermo Velos Pro Orbitrap hybrid mass spectrometer. Peptide spectral matching was performed against an Eschericha coli database from UniProt appended with the sequences of hexa-histidine-tagged WT MGLL using the ProLuCID algorithm (version 1.3.3). ProLuCID parameters specified static modification of cysteine residues (+57.02146 for carboxamidomethylation) and variable modifications for methionine oxidation (+15.99491) and serine modification with masses appropriate for the specific inhibitor (28: +339.15585). Full site of MGLL labeling details can be found in the Supporting Information. In Vivo Pharmacokinetic Studies of 28. Rat PK. Six male Sprague−Dawley rats were obtained from Vital River Laboratory Animal Technology Co. Ltd., Beijing, China. All animals were 6−8 weeks old at the time of study and weighed between 197 and 217 g. Animals (n = 3 per dosing route) were dosed with 28 at 1 mg/kg iv (in 70% polyethylene glycol (PEG) 400 in hydroxypropyl-βcylcodextran (HPBCD) in saline) or 5 mg/kg po (in 70% PEG 400 in 0.5% carboxymethylcellulose (CMC, w/v) in saline). Animals were fasted overnight and food withheld until 4 h post dose. Approximately 100 μL of blood were collected via a jugular vein catheter at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after intravenous and 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after oral administration. All blood samples were collected into tubes containing 400 μL of acetonitrile to immediately inactivate blood esterase activity and frozen at −80 °C. Samples were thawed and centrifuged (14000 rpm for 5 min at 4 °C) and the supernatant transferred for LCMS analysis. Dog PK. Six non-naive male beagle dogs were obtained from Marshall Biotechnology, Co. Ltd., Beijing, China. All animals were 10−12 months old at the time of study and weighed between 8.5 and 11.1 kg. Animals (n = 3 per dosing route) were dosed with 28 at 1 mg/kg iv and po (in PEG 400/ethanol/PBS, 7/2/1, v/v/v). Animals in the iv and po groups were fasted overnight and food withheld until 2 h postdose. Approximately 1 mL of blood was collected from a peripheral vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after iv administration and 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after the oral administration. All blood samples were collected into tubes containing 9080



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K2EDTA and processed for plasma. Samples were centrifuged (2000g for 10 min at 4 °C) within 0.5 h of collection, and plasma samples were kept at −80 °C until analysis. Bioanalytical Method of PK Samples. All blood (rat) or plasma (dog) samples were quantified via HPLC-MS/MS using nonvalidated methods. Standard curves were prepared separately for each species. Pharmacokinetic parameters were determined by noncompartmental analysis using Phoenix WinNonlin (version 6.1). In Vivo Pharmacokinetic and Pharmacodynamic Studies. Male ICR mice and male Sprague−Dawley rats were administered 28 by oral gavage. 28 was formulated on the day of dosing, and maximal dispersal of the compound was achieved by vortexing and sonication. Four hours after oral administration of 28, animals were euthanized by isoflurane overdose followed by decapitation and brains removed and immediately frozen in liquid nitrogen. MGLL target engagement in brain tissue was determined by ABPP and 2-AG concentrations were measured in brain as detailed in the subsequent methods sections. Dose Response of 28 in Rodents. Male ICR mice (Harlan Laboratories) or male Sprague−Dawley rats (Charles River Laboratories) 6−12 weeks old at the time of dosing were maintained under a 12 h light/dark cycle and allowed free access to food and water. 28 was prepared fresh on the day of dosing in a PEG400:ethanol:PBS (7/2/1 v/v/v) vehicle (in mice) or 0.5% methylcellulose (in rats). Maximal dispersal of the compound was achieved by bath and probe sonication until a fine white suspension was formed. Homogeneity of the suspension during dosing was achieved by continual inversion of the dosing solution until immediately before dosing each animal. Compounds were administered in a volume of 5 mL/kg. Animals were administered single oral doses of 28 (0.5−32 mg/kg for mice and 0.03−10 mg/kg for rats). Four hours after 28 administration, animals were anesthetized with isoflurane and exsanguinated by cardiac puncture. Following blood collection, animals were killed by decapitation, and brains were removed, hemisected, and frozen in liquid nitrogen. In mice, one hemisphere each was used for analysis of MGLL inhibition by ABPP and 2-AG levels by LC-MS/MS. In rats, adjacent forebrain sections (∼100−200 mg) were excised from a single hemisphere for analysis of MGLL inhibition by ABPP and 2-AG levels by LC-MS/MS. Brain 2-AG Analysis and Quantitation. LC-MS quantitation of 2AG in rodent brains was carried out essentially as previously described3,24,39 and detailed in the Supporting Information. In Vivo Dosing and “Click” Analysis of 31. In vivo administration of the alkynylated click probe 31 in mice was performed to evaluate proteome-wide reactivity of the 28 chemotype in a similar manner as previously described.49 Male C57BL/6 mice (23−25 g at the time of dosing) were administered the alkyne click-probe 31 (25 mg/kg) by oral gavage. Four hours after 31 administration, animals were euthanized by isoflurane overdose followed by decapitation. Brains were immediately removed, hemisected, frozen in liquid nitrogen, and stored at −80 °C until processed. Brain proteomes from a single hemisphere were prepared as described for FP-Rh ABPP analysis and diluted to 1 mg/mL. Rh−N3 was conjugated to the alkyne probe, 31, for in-gel analysis by copper catalyzed azide−alkyne cycloaddition chemistry.49 Effects of 28 in the Rat Formalin Pain Model. Male Sprague− Dawley rats (170−200 g at the time of dosing, Harlan Laboratories, UK) were administered an intraplantar injection of formalin (50 μL, 2.5%) into the dorsal surface of the right hindpaw to induce acute pain (Saretius Ltd., UK). Immediately after, paw licking behavior, a correlate of spontaneous pain,65 was tracked using the Laboras (Laboratorium Animal Behavior Observation, Registration and Analysis System, Metris BV Hoofddorp, The Netherlands) automated rodent behavioral tracking system that can differentiate behaviors and locomotor activity of individually housed rats. The Laboras system consists of sensor platforms which rest on two orthogonally placed force transducers and a third fixed point attached to a base plate. Each force transducer transforms the mechanical vibrations caused by animal movement into electrical signals, which are amplified, filtered, and digitized by the Laboras system. Characteristic movements have

their own unique vibrational patterns which the Laboras software can distinguish. The Laboras cages (22.5 cm (width) × 42.5 cm (length) × 19.0 cm (height)) consist of two halves to sensitize detection of vibrations. Thus the base is separate from the sides, and the lid and sits directly on the sensor platform, while the sides and the lid hang from supports on the base plate. Duration of paw licking behavior (in seconds) was collected from two distinctive phases, the early nociceptive phase occurring 0−5 min after formalin injection and the late phase thought to be associated with central sensitization, which peaks 10−30 min after formalin injection. Animals were orally administered vehicle (0.5% methylcellulose) or 28 (3 mg/kg) 240 min before formalin injection. Paw licking behavior was monitored for 30 min using the Laboras behavior tracking system. Treatment effects were compared to vehicle by One-Way ANOVA and subsequent Dunnett’s post-test. Effects of 28 on Spontaneous Locomotor Behavior in the Rat. Male Sprague−Dawley rats (175−200 g at the time of dosing, Charles River, UK) were orally administered vehicle (0.5% methylcellulose) or 28 (10 mg/kg). Four hours after administration, animals were placed into the Laboras behavior tracking system and spontaneous locomotor frequency was monitored over a 10 min period. Treatment effects were compared to vehicle by One-Way ANOVA and subsequent Dunnett’s post-test. Ethics Statement. All animal experiments at Abide Therapeutics were performed in accordance with the guidelines outlined by the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act and approved by the Explora Biolabs Institutional Animal Care and Use Committee. Animal experiments performed at external institutions were performed in accordance with the individual institutions and their respective country’s policies governing the ethical and human treatment of laboratory animals.

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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.8b00951. Full experimental methods for MGLL−28 recovery assay, tissue and cell preparation, MS-based ABPP, site of MGLL labeling, grain 2-AG quantitation, and tables (PDF) Molecular formula string (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Phone: (858) 427-2590. E-mail: [email protected]. ORCID

Justin S. Cisar: 0000-0002-8693-9670 Notes

The authors declare the following competing financial interest(s): All authors were employed at Abide Therapeutics at the time this work was done.

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ACKNOWLEDGMENTS We thank Ben Cravatt, Micah Niphakis, Sandy Mills, and Jake Weiner for helpful discussions and critical reading of this manuscript. We thank Micah Niphakis for assistance with figure preparation.

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ABBREVIATIONS USED Cmax, maximum concentration of the drug achieved in the plasma following dose administration; tmax, time at which Cmax is attained; Cl, clearance; Vdss, volume of distribution at steady state; HLM, human liver microsomes 9081



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