N-Acyl pyrazoles: Effective and tunable inhibitors of serine hydrolases

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Abstract

A series of N-acyl pyrazoles was examined as candidate serine hydrolase inhibitors in which the active site acylating reactivity and the leaving group ability of the pyrazole could be tuned not only through the nature of the acyl group (reactivity: amide > carbamate > urea), but also through pyrazole C4 substitution with electron-withdrawing or electron-donating substituents. Their impact on enzyme inhibitory activity displayed pronounced effects with the activity improving substantially as one alters both the nature of the reacting carbonyl group (urea > carbamate > amide) and the pyrazole C4 substituent (CN > H > Me). It was further demonstrated that the acyl chain of the N-acyl pyrazole ureas can be used to tailor the potency and selectivity of the inhibitor class to a targeted serine hydrolase. Thus, elaboration of the acyl chain of pyrazole-based ureas provided remarkably potent, irreversible inhibitors of fatty acid amide hydrolase (FAAH, apparent Ki = 100–200 pM), dual inhibitors of FAAH and monoacylglycerol hydrolase (MGLL), or selective inhibitors of MGLL (IC50 = 10–20 nM) while simultaneously minimizing off-target activity (e.g., ABHD6 and KIAA1363).

Introduction

The endocannabinoids anandamide1, 2 and 2-arachidonylglycerol (2-AG)3, 4 are endogenous lipid signaling molecules5 that activate cannabinoid (CB1 and CB2) receptors (Fig. 1).2, 6 The characterization and pharmacologic inactivation of the major enzymes that terminate the signaling of anandamide and 2-AG, fatty acid amide hydrolase (FAAH)7 and monoacylglycerol lipase (MGLL), respectively,8, 9, 10, 11 have shown that each elevate endocannabinoid signaling and that each affect many of the same pharmacological processes, including pain and inflammation.12, 12(a), 12(b), 12(c), 12(d), 13, 13(a), 13(b), 14, 15, 15(a), 15(b), 15(c), 15(d), 16, 17, 18 Less has been described about simultaneous inhibition of both enzymes, but indications are that it results in more pronounced pharmacological activity.19 Inhibitors of each enzyme individually or combined could provide therapeutics that replace opioid analgesics for either severe (surgical) or chronic neuropathic pain, avoiding the opioid side effects of dependence, desensitization with chronic dosing, dose-limiting respiratory depression, and constipation that extends hospital stays. Similarly, it is expected such individual or dual enzyme inhibitors could serve as alternatives to medical marijuana or its active constituents in clinical indications where it is presently used, including oncology pain and nausea. Importantly and because they only act on activated signaling pathways, increasing the endogenous concentration of the released signaling molecules only at a site of stimulation, they provide a spatial and temporal pharmacological control not available to direct acting opioid or CB receptor agonists. Herein, we report full details of a study that identified N-acyl pyrazole ureas as candidate serine hydrolase inhibitors and their elaboration to provide selective FAAH inhibitors, dual FAAH/MGLL inhibitors, or selective MGLL inhibitors through modification of distinguishable recognition elements used to target the individual enzyme active sites.

Section snippets

Results and discussion

In the course of efforts targeting serine hydrolases,12, 13 we prepared a series of N-acyl pyrazole amides, carbamates and ureas and examined them as candidate inhibitors of the FAAH in order to define their potential utility (Fig. 2).20 The compounds were first evaluated in a substrate (oleamide) hydrolysis assay with the pure recombinant rat enzyme (rFAAH) and the results are reported as an apparent Ki that was measured after 3 h preincubation at 25 °C with the enzyme (typically 1 nM, but

Conclusions

A series of N-acyl pyrazoles were examined as candidate serine hydrolase inhibitors and it was shown that active site acylating reactivity and the leaving group ability of the pyrazole could be tuned not only through the nature of the acyl group (reactivity: amide > carbamate > urea), but also through pyrazole C4 substitution with electron-withdrawing or electron-donating substituents. Their impact on FAAH inhibitory activity displayed clear and pronounced effects with the activity improving as

Chemistry general methods

All commercial reagents were used without further purification unless otherwise noted. THF was distilled prior to use. All reactions were performed in oven-dried (200 °C) glassware and under an inert atmosphere of anhydrous argon unless otherwise noted. Column chromatography was performed with silica gel 60. TLC was performed on Whatman silica gel (250 μm) F254 glass plates and spots were visualized by UV. PTLC was performed on Whatman silica gel (250 and 500 μm) F254 glass plates. 1H NMR

Acknowledgements

This work was supported by the National Institutes of Health (DA015648, DK114785, DLB; DA037760, BFC). We thank Aleksandar Radakovic and Jelena Momirov for the rFAAH Ki determinations reported in Fig. 14, Fig. 15.

Competing interests

D.L.B. and B.F.C. have financial interests in Abide Therapeutics, which has interests in the development of therapeutics derived from inhibition of serine hydrolases.

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