N-Acyl pyrazoles: Effective and tunable inhibitors of serine hydrolases
Graphical abstract
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.
References (43)
- et al.
2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain
Biochem Biophys Res Commun
(1995) - et al.
Fatty acid amide signaling molecules
Bioorg Med Chem Lett
(2010)et al.Oleamide: an endogenous sleep-inducing lipid and prototypical member of a new class of biological signaling molecules
Curr Pharm Design
(1998) Cannabinoid receptors as therapeutic targets
Annu Rev Pharmacol Toxicol
(2006)et al.The endogenous cannabinoid system and its role in nociceptive behaviour
J Neurobiol
(2004)- et al.
Molecular characterization of an enzyme that degrades neuromodulatory fatty acid amides
Nature
(1996)et al.Molecular characterization of human and mouse fatty acid amide hydrolases
Proc Natl Acad Sci USA
(1997) - et al.
Discovery of a potent, selective, and efficacious class of reversible α-ketoheterocycle inhibitors of fatty acid amide hydrolase as analgesics
J Med Chem
(2005)et al.Reversible inhibitors of fatty acid amide hydrolase that promote analgesia: evidence for an unprecedented combination of potency and selectivity
J Pharmacol Exp Ther
(2004) - et al.
Blockade of endocannabinoid-degrading enzymes attenuates neuropathic pain
J Pharmacol Exp Ther
(2009) - et al.
Full fatty acid amide hydrolase inhibition combined with partial monoacylglycerol lipase inhibition: Augmented and sustained antinociceptive effects with reduced cannabimimetic side effects in mice
J Pharmacol Exp Ther
(2015)et al.Simultaneous inhibition of fatty acid amide hydrolase and monoacylglycerol lipase shares discriminative stimulus effects with delta(9)-tetrahydrocannabinol in mice
J Pharmacol Exp Ther
(2015)et al.Equipotent inhibition of fatty acid amide hydrolase and monoacylglycerol lipase – dual targets of the endocannabinoid system to protect against seizure pathology
Neurotherapeutics
(2012)et al.Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo
Proc Natl Acad Sci USA
(2009) - et al.
Synthesis and structure–activity relationship for a novel class of potent and selective carbamoyl-triazole based inhibitors of hormone sensitive lipase
J Med Chem
(2004)et al.Click-generated triazole ureas as ultrapotent in vivo-active serine hydrolase inhibitors
Nat Chem Biol
(2011)et al.Development and characterization of endocannabinoid hydrolases FAAH and MAGL inhibitors bearing a benzotriazol-1-yl carboxamide scaffold
Bioorg Med Chem
(2012)et al.Piperazine and piperidine triazole ureas as ultrapotent and highly selective inhibitors of monoacylglycerol lipase
Chem Biol
(2013)et al.Optimization of N-benzoylindazole derivatives as inhibitors of human neutrophil elastase
J Med Chem
(2013)et al.Development and optimization of piperidyl-1,2,3-triazole ureas as selective chemical probes of endocannabinoid biosynthesis
J Med Chem
(2013)et al.Discovery and optimization of piperidyl-1,2,3-triazole ureas as potent, selective, and in vivo-active inhibitors of alpha/beta-hydrolase domain containing 6 (ABHD6)
J Med Chem
(2013)et al.Loratadine analogues as MAGL inhibitors
Bioorg Med Chem Lett
(2015)et al.Development and pharmacological characterization of selective blockers of 2-arachidonoyl glycerol degradation with efficacy in rodent models of multiple sclerosis and pain
J Med Chem
(2016) - et al.
Novel mechanistic class of fatty acid amide hydrolase inhibitors with remarkable selectivity
Biochemistry
(2007) - et al.
Structure–activity relationships of α-ketooxazole inhibitors of fatty acid amide hydrolase
J Med Chem
(2007)et al.Potent and selective α -ketoheterocycle-based inhibitors of the anandamide and oleamide catabolizing enzyme, fatty acid amide hydrolase
J Med Chem
(2007)et al.Optimization of α-ketooxazole inhibitors of fatty acid amide hydrolase
J Med Chem
(2008)et al.Optimization of the central heterocycle of α -ketoheterocycle inhibitors of fatty acid amide hydrolase
J Med Chem
(2008)et al.Reversible competitive α-ketoheterocycle inhibitors of fatty acid amide hydrolase containing additional conformational constraints in the acyl side chain: orally active, long acting analgesics
J Med Chem
(2011)