O-hydroxyacetamide carbamates as a highly potent and selective class of endocannabinoid hydrolase inhibitors

Abstract

The two major endocannabinoid transmitters, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are degraded by distinct enzymes in the nervous system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. FAAH and MAGL inhibitors cause elevations in brain AEA and 2-AG levels, respectively, and reduce pain, anxiety, and depression in rodents without causing the full spectrum of psychotropic behavioral effects observed with direct cannabinoid receptor-1 (CB1) agonists. These findings have inspired the development of several classes of endocannabinoid hydrolase inhibitors, most of which have been optimized to show specificity for either FAAH or MAGL or, in certain cases, equipotent activity for both enzymes. Here, we investigate an unusual class of O-hydroxyacetamide carbamate inhibitors and find that individual compounds from this class can serve as selective FAAH or dual FAAH/MAGL inhibitors in vivo across a dose range (0.125-12.5 mg kg(-1)) suitable for behavioral studies. Competitive and click chemistry activity-based protein profiling confirmed that the O-hydroxyacetamide carbamate SA-57 is remarkably selective for FAAH and MAGL in vivo, targeting only one other enzyme in brain, the additional 2-AG hydrolase ABHD6. These data designate O-hydroxyacetamide carbamates as a versatile chemotype for creating endocannabinoid hydrolase inhibitors that display excellent in vivo activity and tunable selectivity for FAAH-anandamide versus MAGL (and ABHD6)-2-AG pathways.

Keywords: 2-arachidonoylglycerol; Activity-based protein profiling; anandamide; carbamate; endocannabinoid; hydrolase.

Figure 1

Representative structures of reversible and irreversible FAAH inhibitors.

Scheme 1. Synthesis of SA-57 and SA-57yne

(a) DIEA, DMSO, 60 °C; (b) 2 M MeNH₂ in THF, DMSO; (c) triethylsilylacetylene, PdCl₂(CH₃CN)₂, X-Phos, CH₃CN, 85 °C; (d) TBAF, THF, 0 °C.

Figure 2

Concentration dependent, in vitro competitive ABPP analysis of PF-7845 and SA-57 in the mouse brain membrane proteome using the serine hydrolase-directed probe FP-Rh. PF-7845 selectively blocks FP-Rh labeling of FAAH across the full inhibitor concentration range tested, whereas SA-57 selectively blocks labeling of FAAH at lower concentrations (10 nM to 1 μM), but also inhibits MAGL and ABHD6 at higher concentrations (10 μM).

Figure 3

Concentration dependent, in vitro competitive ABPP analysis of PF-7845 and SA-57 against recombinant human and mouse FAAH, MAGL, and ABHD6 enzymes expressed by transient transfection in HEK293 cells. Inhibitory activity against (a) mFAAH, (b) hFAAH, and (c) mMAGL, hMAGL, and mABHD6.

Figure 4

Time-dependent inhibition of mouse FAAH (a) and MAGL (b) enzymes by PF-7845, SA-57, and JZL184 as determined by competitive ABPP.

Figure 5

Competitive ABPP of brain serine hydrolase activities from mice treated with PF-7845 (a) and SA-57 (b) at the indicated doses (0.01–12.5 mg kg^–1, i.p.) for 2 h.

Figure 6

Structures (a) and in vitro competitive ABPP analysis (b) of PF-7845yne and SA-57yne in a mouse brain membrane proteome using a FP-Rh probe.

Figure 7

Competitive ABPP (a and b) and CC-ABPP (c and d) analysis of brain proteomes from mice treated with clickable probes PF-7845yne (a and c) and SA-57yne (b and d) at the indicated doses (0.01–12.5 mg kg^–1, i.p.) for 2 h.

Figure 8

Brain lipid profile for mice treated with PF-7845, SA-57, or vehicle at indicated doses (0.125–12.5 mg kg^–1, i.p.) for 2 h. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for vehicle-treated versus inhibitor-treated mice. Data are presented as means ± SEM, n = three mice per group.