Optimization of alpha-ketooxazole inhibitors of fatty acid amide hydrolase

A series of alpha-ketooxazoles containing conformational constraints in the flexible C2 acyl side chain of 2 (OL-135) and representative oxazole C5 substituents were prepared and examined as inhibitors of fatty acid amide hydrolase (FAAH). Exceptionally potent and selective FAAH inhibitors emerged from the series (e.g., 6, Ki = 200 and 260 pM for rat and rhFAAH). With simple and small C5 oxazole substituents, each series bearing a biphenylethyl, phenoxyphenethyl, or (phenoxymethyl)phenethyl C2 side chain was found to follow a well-defined linear relationship between -log Ki and Hammett sigmap of a magnitude (rho = 2.7-3.0) that indicates that the substituent electronic effect dominates, confirming its fundamental importance to the series and further establishing its predictive value. Just as significantly, the nature of the C5 oxazole substituent substantially impacts the selectivity of the inhibitors whereas the effect of the C2 acyl chain was more subtle but still significant even in the small series examined. Combination of these independent features, which display generalized trends across a range of inhibitor series, simultaneously improves FAAH potency and selectivity and can provide exquisitely selective and potent FAAH inhibitors.     

Structure-activity relationships of alpha-ketooxazole inhibitors of fatty acid amide hydrolase

A systematic study of the structure-activity relationships of 2b (OL-135), a potent inhibitor of fatty acid amide hydrolase (FAAH), is detailed targeting the C2 acyl side chain. A series of aryl replacements or substituents for the terminal phenyl group provided effective inhibitors (e.g., 5c, aryl = 1-napthyl, Ki = 2.6 nM), with 5hh (aryl = 3-ClPh, Ki = 900 pM) being 5-fold more potent than 2b. Conformationally restricted C2 side chains were examined, and many provided exceptionally potent inhibitors, of which 11j (ethylbiphenyl side chain) was established to be a 750 pM inhibitor. A systematic series of heteroatoms (O, NMe, S), electron-withdrawing groups (SO, SO2), and amides positioned within and hydroxyl substitutions on the linking side chain were investigated, which typically led to a loss in potency. The most tolerant positions provided effective inhibitors (12p, 6-position S, Ki = 3 nM, or 13d, 2-position OH, Ki = 8 nM) comparable in potency to 2b. Proteome-wide screening of selected inhibitors from the systematic series of >100 candidates prepared revealed that they are selective for FAAH over all other mammalian serine proteases.     

Latest advances in the discovery of fatty acid amide hydrolase inhibitors

Introduction: Fatty acid amide hydrolase (FAAH) is the major catabolic enzyme of the endocannabinoid N-arachidonoylethanolamine (anandamide) that, with different degrees of efficiency, also hydrolyzes other endogenous fatty acid ethanolamides. FAAH is increasingly being considered a relevant therapeutic target, especially in models of inflammatory pain. The opportunity to selectively increase the endocannabinoid tone only in those tissues where such an enhancement can be beneficial might result in a therapeutic benefit with more limited side effects, compared to the use of direct agonists of anandamide-binding receptors. Thus the research for selective FAAH inhibitors has become a hot topic in current drug discovery.

Areas covered: This review highlights the advances in the development of different compounds belonging to different chemical families that have been proposed as FAAH inhibitors. Several classes of inhibitors have been reported so far, and they may be classified into two major classes: reversible and irreversible compounds. These inhibitors are reviewed herein with an emphasis on their potency and selectivity.

Expert opinion: In recent years, tremendous efforts have been made to develop the FAAH inhibitors, and consequently many novel chemical templates have been discovered. It is still a major challenge to identify the first inhibitor of FAAH suitable for clinical exploitation that satisfies the requirements of potency, selectivity versus proteins related to anandamide activity as well as other potential off-targets, reversibility versus irreversibility, and efficacy toward rat versus human FAAH.     

The endogenous cannabinoid anandamide has effects on motivation and anxiety that are revealed by fatty acid amide hydrolase (FAAH) inhibition

Converging evidence suggests that the endocannabinoid system is an important constituent of neuronal substrates involved in brain reward processes and emotional responses to stress. Here, we evaluated motivational effects of intravenously administered anandamide, an endogenous ligand for cannabinoid CB1-receptors, in Sprague-Dawley rats, using a place-conditioning procedure in which drugs abused by humans generally produce conditioned place preferences (reward). Anandamide (0.03-3 mg/kg intravenous) produced neither conditioned place preferences nor aversions. However, when rats were pre-treated with the fatty acid amide hydrolase (FAAH) inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester; 0.3 mg/kg intraperitoneal), which blocks anandamide's metabolic degradation, anandamide produced dose-related conditioned place aversions. In contrast, URB597 alone showed no motivational effects. Like URB597 plus anandamide, the synthetic CB1-receptor ligand WIN 55,212-2 (50-300 microg/kg, intravenous) produced dose-related conditioned place aversions. When anxiety-related effects of anandamide and URB597 were evaluated in a light/dark box, both a low anandamide dose (0.3 mg/kg) and URB597 (0.1 and 0.3 mg/kg) produced anxiolytic effects when given alone, but produced anxiogenic effects when combined. A higher dose of anandamide (3 mg/kg) produced anxiogenic effects and depressed locomotor activity when given alone and these effects were potentiated after URB597 treatment. Finally, anxiogenic effects of anandamide plus URB597 and development of place aversions with URB597 plus anandamide were prevented by the CB1-receptor antagonist AM251 (3 mg/kg intraperitoneal). Thus, additive interactions between the effects of anandamide on brain reward processes and on anxiety may account for its aversive effects when intravenously administered during FAAH inhibition with URB597.     

The fatty acid amide hydrolase (FAAH) inhibitor PF-3845 acts in the nervous system to reverse LPS-induced tactile allodynia in mice

BACKGROUND AND PURPOSE

Inflammatory pain presents a problem of clinical relevance and often elicits allodynia, a condition in which non-noxious stimuli are perceived as painful. One potential target to treat inflammatory pain is the endogenous cannabinoid (endocannabinoid) system, which is comprised of CB₁ and CB₂ cannabinoid receptors and several endogenous ligands, including anandamide (AEA). Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without the psychomimetic side effects associated with Δ⁹-tetrahydrocannabinol (THC).

EXPERIMENTAL APPROACH

Allodynia was induced by intraplantar injection of LPS. Complementary genetic and pharmacological approaches were used to determine the strategy of blocking FAAH to reverse LPS-induced allodynia. Endocannabinoid levels were quantified using mass spectroscopy analyses.

KEY RESULTS

FAAH (−/−) mice or wild-type mice treated with FAAH inhibitors (URB597, OL-135 and PF-3845) displayed an anti-allodynic phenotype. Furthermore, i.p. PF-3845 increased AEA levels in the brain and spinal cord. Additionally, intraplantar PF-3845 produced a partial reduction in allodynia. However, the anti-allodynic phenotype was absent in mice expressing FAAH exclusively in the nervous system under a neural specific enolase promoter, implicating the involvement of neuronal fatty acid amides (FAAs). The anti-allodynic effects of FAAH-compromised mice required activation of both CB₁ and CB₂ receptors, but other potential targets of FAA substrates (i.e. µ-opioid, TRPV1 and PPARα receptors) had no apparent role.

CONCLUSIONS AND IMPLICATIONS

AEA is the primary FAAH substrate reducing LPS-induced tactile allodynia. Blockade of neuronal FAAH reverses allodynia through the activation of both cannabinoid receptors and represents a promising target to treat inflammatory pain.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

Recent advances in the discovery and evaluation of fatty acid amide hydrolase inhibitors

Importance of the field: Cannabis has been used for both medicinal and recreational purposes since ancient times. Although cannabinoid-based medicines hold great promise in several challenging therapeutic areas such as pain management and mode control, their development has been hampered by psychoactive and other CNS-related side effects. The identification of fatty acid amide hydrolase (FAAH), a key enzyme responsible for the degradation of endocannabinoids, has brought in tremendous opportunities in that inhibition of FAAH leads to local elevation of endocannabinoids under certain stimuli, thus, avoiding the side effects from global activation of cannabinoid receptors by exogenous cannabimimetic compounds. The search for selective FAAH inhibitors has thus become a strong focus in current drug discovery. Areas covered in this review: This review summarizes our current understanding of FAAH including its structure, catalytic mechanism and biological functions with emphases on its role in the regulation of endocannabinoids and other signaling lipids. The review then highlights the most recent discovery and biological activities of different classes of FAAH inhibitors. Last, the review discusses challenges and potential drawbacks in the development of FAAH inhibitor-based therapy. What the reader will gain: Readers will have an overview of FAAH and obtain a rationale on FAAH as an attractive therapeutic target for the development of medicines for treating pain, inflammation, anxiety and other diseases. More importantly, readers will gain knowledge on various newly established FAAH inhibitor scaffolds and their development potentials, and such information will hopefully stimulate ideas for the designing of new inhibitors with superior activity profiles. The discussions on the potential challenges in developing FAAH inhibitors will impose more caution in the decision-making process, thus, lowering the possibility of late stage failure. Take home message: FAAH is an attractive target for modulating the endocannabinoid system, thus, treating many disease conditions including pain and mode control without the CNS side effects associated with cannabis usage. In recent years, tremendous effort has been focused in the FAAH inhibitor research field, and consequently many novel chemical templates have been discovered. FAAH hydrolyzes several important signaling lipids, but the long-term effects of FAAH inhibition in humans remain to be seen. While it is challenging to identify the right molecule with the right level of intervention of the FAAH function for treating a disease condition, it is possible to avoid mechanism-related undesired effects. With the entry of several compounds into clinical trials, FAAH inhibitor-based medicines are on the horizon.     

Recent advances in the discovery and evaluation of fatty acid amide hydrolase inhibitors

Importance of the field: Cannabis has been used for both medicinal and recreational purposes since ancient times. Although cannabinoid-based medicines hold great promise in several challenging therapeutic areas such as pain management and mode control, their development has been hampered by psychoactive and other CNS-related side effects. The identification of fatty acid amide hydrolase (FAAH), a key enzyme responsible for the degradation of endocannabinoids, has brought in tremendous opportunities in that inhibition of FAAH leads to local elevation of endocannabinoids under certain stimuli, thus, avoiding the side effects from global activation of cannabinoid receptors by exogenous cannabimimetic compounds. The search for selective FAAH inhibitors has thus become a strong focus in current drug discovery.

Areas covered in this review: This review summarizes our current understanding of FAAH including its structure, catalytic mechanism and biological functions with emphases on its role in the regulation of endocannabinoids and other signaling lipids. The review then highlights the most recent discovery and biological activities of different classes of FAAH inhibitors. Last, the review discusses challenges and potential drawbacks in the development of FAAH inhibitor-based therapy.

What the reader will gain: Readers will have an overview of FAAH and obtain a rationale on FAAH as an attractive therapeutic target for the development of medicines for treating pain, inflammation, anxiety and other diseases. More importantly, readers will gain knowledge on various newly established FAAH inhibitor scaffolds and their development potentials, and such information will hopefully stimulate ideas for the designing of new inhibitors with superior activity profiles. The discussions on the potential challenges in developing FAAH inhibitors will impose more caution in the decision-making process, thus, lowering the possibility of late stage failure.

Take home message: FAAH is an attractive target for modulating the endocannabinoid system, thus, treating many disease conditions including pain and mode control without the CNS side effects associated with cannabis usage. In recent years, tremendous effort has been focused in the FAAH inhibitor research field, and consequently many novel chemical templates have been discovered. FAAH hydrolyzes several important signaling lipids, but the long-term effects of FAAH inhibition in humans remain to be seen. While it is challenging to identify the right molecule with the right level of intervention of the FAAH function for treating a disease condition, it is possible to avoid mechanism-related undesired effects. With the entry of several compounds into clinical trials, FAAH inhibitor-based medicines are on the horizon.     

Inhibition of fatty acid amide hydrolase modulates anxiety-like behavior in PCP-treated rats

Sub-chronic administration of PCP produces a social interaction deficit that is reversed by URB597, an inhibitor of the catabolic enzyme of the endocannabinoid anandamide. Since increased anxiety may contribute to social withdrawal and URB597 has been shown to have an anxiolytic action, we studied whether this drug affected saline- and PCP-treated rats' performance in the Elevated-Plus-Maze task, which has been used to assess anxiety-like effects. Sub-chronic PCP produced a CB₁-dependent decrease in anxiety-like behavior that was reversed by URB597 in a CB₁-independent fashion, as it was not blocked by the CB₁ antagonist AM251. These findings suggest that PCP-treated rats have altered endocannabinoid transmission and that anxiety does not contribute to the PCP-induced social withdrawal.