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  • In conclusion we have shown that

    2021-11-25

    In conclusion, we have shown that the brain penetrating ability of heteroaryl piperazine and piperadine ureas can be modulated through small structural changes to the heteroarenes, the piperazine or piperidine core, and the biaryl ether tail. Compounds exhibiting / ratios ranging as high as 4.2:1 to as low as 0.02:1 have been preparable. The degree of brain penetration was not predictable from PK experiments as large volumes of distribution did not correlate with a high /. As the described compounds are mechanism-based inhibitors of the FAAH enzyme, low / ratios did not preclude central target engagement, but did slow the onset of inhibition.
    Fatty eph receptor amide hydrolase is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including anandamide (-arachidonylethanolamine, AEA). AEA was identified as an endogenous agonist for the cannabinoid receptors CB1 and CB2 in 1992. The anti-nociceptive benefit of stimulating the CB1/CB2 receptors has long been recognized and for some time it has been postulated that inhibiting FAAH would show similar therapeutic benefits through increasing lifetime of AEA in various tissues., Evidence supporting this hypothesis includes the fact that FAAH knock-out mice show increased levels of AEA in the brain accompanied with decreased susceptibility to neuropathic and inflammatory pain. Likewise, inhibition of FAAH with small molecule inhibitors (e.g., OL-135 and URB-597) in vivo leads to similar increases in AEA levels and decreased sensitivities in pain models. Except for the recent report by Wang, most FAAH inhibitors reported to date appear to rely on covalent modification of the active site serine 241. Herein we describe the identification of a novel, potent and noncovalent series of FAAH inhibitors with in vitro potencies comparable to covalent FAAH inhibitors. Compounds and were identified during the course of a high-throughput screen (HTS) of the Amgen compound collection. As indicated in , and inhibited rat and human FAAH with potencies comparable to that of OL-135 and URB-597 in our assays. Subsequent studies revealed these ureas to be suicide substrates., Incubation of or with FAAH followed by crystallization resulted in complexes where the urea carbonyl acylated S241 with expulsion of the aniline fragment as indicated equation 1. The complete loss of FAAH inhibitory activity seen with amide is consistent with mechanism-based inactivation suggested by co-crystallization studies. Interestingly, simple N-methylation of also resulted in a complete loss of FAAH activity (cf. , and , ). We were interested in developing reversible (and preferably noncovalent) FAAH inhibitors with the expectation that they might have a different selectivity/safety profile compared to suicide substrates like or . We imagined that it might be possible to create reversible FAAH inhibitors based on spirolactams or cyclic ureas related to . Starting from a urea exemplified by (), we reasoned that by installing steric hindrance adjacent to the carbonyl group would reduce its susceptibility to nucleophilic attack. In addition, tethering the aniline to the opposite side of the scissile bond would prevent it from leaving the active site after initial attack by S241 thus creating the possibility of reversible inhibition through re-cyclization of the postulated enzyme-acyl intermediate. Our strategy (summarized in for a spirolactam) called for designing a serine electrophile that carried a substituent to target the acyl-chain binding pocket (ABP) of FAAH (i.e., R in ). Once suitable scaffolds were identified we intended to optimize inhibitor affinity for the ABP and eventually remove the electrophile. Toward this end, we designed a number of bicyclic and spirocyclic ureas and lactams as potential FAAH inhibitor electrophiles. The designs were prioritized through their theoretical binding to the active site of FAAH using the program Glide and in-house X-ray crystallographic data. Selected designs were synthesized and used to produce libraries. Early on in this effort, we identified 4-[2-benzofuran]-2-yl-pyrimidine as a highly potent piperidine nitrogen substituent. For example, spirolactam showed inhibition of rFAAH with an IC of 1.2μM. Interestingly, the -methyl spirolactam showed slightly improved FAAH inhibition (IC=0.50μM) in contrast to the complete loss of activity seen upon N-methylating , which suggested two distinct binding modes for these two series of compounds. Encouraged by initial results we used 4-[2-benzofuran]2-yl-pyrimidine as a key fragment to evaluate additional serine-electrophile designs, that were selected from our molecular modeling efforts.