Fatty acid amide hydrolase inhibitors display broad selectivity and inhibit multiple carboxylesterases as off-targets.

Fatty acid amide hydrolase (FAAH) is the primary regulator of several bioactive lipid amides including anandamide. Inhibitors of FAAH are potentially useful for the treatment of pain, anxiety, depression, and other nervous system disorders. However, FAAH inhibitors must display selectivity for this enzyme relative to the numerous other serine hydrolases present in the human proteome in order to be therapeutically acceptable. Here we employed activity-based protein profiling (ABPP) to assess the selectivity of FAAH inhibitors in multiple rat and human tissues. We discovered that some inhibitors, including carbamate compounds SA-47 and SA-72, and AM404 are exceptionally selective while others, like URB597, BMS-1, OL-135, and LY2077855 are less selective, displaying multiple off-targets. Since proteins around 60kDa constitute the major off-targets for URB597 and several other FAAH inhibitors with different chemical structures, we employed the multi-dimensional protein identification technology (MudPIT) approach to analyze their identities. We identified multiple carboxylesterase isozymes as bona fide off-targets of FAAH inhibitors. Consistently, enzymatic assay confirmed inhibition of carboxylesterase activities in rat liver by FAAH inhibitors. Since carboxylesterases hydrolyze a variety of ester-containing drugs and prodrugs, we speculate that certain FAAH inhibitors, by inhibiting carboxylesterases, might have drug-drug interactions with other medicines if developed as therapeutic agents.

Discovery of 2-(4-pyridin-2-ylpiperazin-1-ylmethyl)-1H-benzimidazole (ABT-724), a dopaminergic agent with a novel mode of action for the potential treatment of erectile dysfunction.

A new class of agents with potential utility for the treatment of erectile dysfunction has been discovered, guided by the hypothesis that selective D4 agonists are erectogenic but devoid of the side effects typically associated with dopaminergic agents. The lead agent 2-(4-pyridin-2-ylpiperazin-1-ylmethyl)-1H-benzimidazole (1, ABT-724) was discovered by optimization of a series of benzimidazole arylpiperazines. This highly selective D4 agonist was found to be very potent and efficacious in vivo, eliciting penile erections in rats at a dose of 0.03 micromol/kg, with a positive response rate of 77% erectile incidence. Even at high doses, it was devoid of side effects in animal models of central nervous system behaviors, emesis, or nausea. The structure-activity relationship of the parent benzimidazole series leading to 1 is described, with the detailed in vitro and in vivo profiles described. Distinctive structural features were discovered that are associated with D4 selective agonism in this series of analogues.

A-350619: a novel activator of soluble guanylyl cyclase.

Nitric oxide (NO) is a key mediator in many physiological processes and one of the major receptors through which NO exerts its effects is soluble guanylyl cyclase. Guanylyl cyclase converts GTP to cyclic GMP as part of the cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, inhibition of platelet aggregation and immune response. The properties of A-350619, a novel soluble guanylyl cyclase activator, were examined to determine the modulatory effect on the catalytic properties of soluble guanylyl cyclase. A-350619 increased V(max) from 0.1 to 14.5 micromol/min/mg (145 fold increase), and lowered K(m) from 300 to 50 microM (6 fold decrease). When YC-1 (another sGC activator) and A-350619 were combined, a 156 fold increase in V(max) and a 5 fold decrease in Km were observed, indicating that the modulation of the enzyme brought about by YC-1 and A-350619 are not additive, suggesting a common binding site. Activation of soluble guanylyl cyclase by A-350619 was partially inhibited by ODQ, a specific inhibitor of soluble guanylyl cyclase by oxidation of the enzyme heme. YC-1 and A-350619 after pre-treatment with N-omega-nitro-L-arginine, an NO-synthase inhibitor, relaxed cavernosum tissue strips in a dose-dependent manner with EC(50) of 50 microM and 80 microM, respectively. Addition of SNP potentiated the relaxation effect of YC-1 and A-350619, shifting the dose-response curve to the left to 3 microM and 10 microM, respectively. Consistent with its biochemical activity, A-350619 (1 micromol/kg) alone induced penile erection in a conscious rat model. Activation of soluble guanylyl cyclase in cavernosum tissue as an alternate method of enhancing the effect of NO may provide a novel treatment of sexual dysfunction.

Synthesis and evaluation of benzothiazole-based analogues as novel, potent, and selective fatty acid amide hydrolase inhibitors.

High-throughput screening (HTS) identified benzothiazole analogue 3 as a potent fatty acid amide hydrolase (FAAH) inhibitor. Structure-activity relationship (SAR) studies indicated that the sulfonyl group, the piperidine ring and benzothiazole were the key components to their activity, with 16j being the most potent analogue in this series. Time-dependent preincubation study of compound 3 was consistent with it being a reversible inhibitor. Activity-based protein-profiling (ABPP) evaluation of 3 in rat tissues revealed that it had exceptional selectivity and no off-target activity with respect to other serine hydrolases. Molecular shape overlay of 3 with a known FAAH inhibitor indicated that these compounds might act as transition-state analogues, forming putative hydrogen bonds with catalytic residues and mimicking the charge distribution of the tetrahedral transition state. The modeling study also indicated that hydrophobic interactions of the benzothiazole ring with the enzyme contributed to its extraordinary potency. These compounds may provide useful tools for the study of FAAH and the endocannabinoid system.

Rapid hit to lead evaluation of pyrazolo[3,4-d]pyrimidin-4-one as selective and orally bioavailable mGluR1 antagonists.

Our HTS effort yielded a preferential mGluR1 pyrimidinone antagonist 1 with lead-like characteristics. Rapid hit to lead (HTL) study identified compounds with improved functional activity and selectivity such as 1b with little improvements in ADME properties. Addition of an aminosulfonyl group on the N-1 aromatic ring led to 2f, a compound with similar in vitro biochemical profiles as those of 1b but drastically improved in vitro ADME properties. These improvements were paralleled by rat PK study characterized by low clearance and quantitative bioavailability. Compound 2f represented a true lead-like molecule that is amenable for further lead optimization (LO) evaluation.

Acrylamide analog as a novel nitric oxide-independent soluble guanylyl cyclase activator.

Soluble guanylyl cyclase (sGC) is a target enzyme for endogenous nitric oxide (NO), and it converts GTP to cyclic GMP (guanosine 3',5'-cyclic monophosphate) as part of a cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, and inhibition of platelet aggregation. Here we examine a representative of the novel class sCG activators, A-778935 ((+/-)-cis-3-[2-(2,2-dimethyl-propylsulfanyl)-pyridin-3-yl]-N-(3-hydroxy-cyclohexyl)-acrylamide). A-778935 activated sGC synergistically with sodium nitroprusside (SNP) over a wide range of concentration, inducing up to 420-fold activation. A specific inhibitor of sGC, ODQ (1H-[1,2,4]-oxadiazolo[4,3-alpha]quinoxalin-1-one), did not block basal sGC activity, but competitively inhibited the activation by A-778935. A-778935, with or without SNP, did not activate heme-deficient sGC, indicating that the activation of sGC by A-778935 is fully heme-dependent. A-778935 increased intracellular cGMP level dose-dependently in smooth muscle cells. In the presence of 1 microM SNP, a lower concentration of A-778935 increased cGMP than A-778935 alone, and the cGMP concentration reached the same level at 100 microM of A-778935. A-778935 relaxed cavernosum tissue strips in a dose-dependent manner; and in the presence of 1 microM SNP, A-778935 relaxed the strips more potently, shifting the dose-response curve to the left. This novel activator of sGC may have potential efficacy for the treatment of a variety of disorders associated with reduced NO signaling.

Synthesis and evaluation of 3-aryl piperidine analogs as potent and efficacious dopamine D4 receptor agonists.

A series of 3-aryl piperidine analogs with 2-piperidinoalkylamino or 2-piperidinoalkyloxy fused bicyclic rings were prepared and found to be potent and efficacious human dopamine D4 agonists. The synthesis and structure-activity relationship (SAR) studies that led to the identification of these compounds are discussed.

Activation of dopamine D4 receptors by ABT-724 induces penile erection in rats.

Apomorphine, a nonselective dopamine receptor agonist, facilitates penile erection and is effective in patients suffering from erectile dysfunction. The specific dopamine receptor subtype(s) responsible for its erectogenic effect is not known. Here we report that the dopamine D(4) receptor plays a role in the regulation of penile function. ABT-724 is a selective dopamine D(4) receptor agonist that activates human dopamine D(4) receptors with an EC(50) of 12.4 nM and 61% efficacy, with no effect on dopamine D(1), D(2), D(3), or D(5) receptors. ABT-724 dose-dependently facilitates penile erection when given s.c. to conscious rats, an effect that is blocked by haloperidol and clozapine but not by domperidone. A proerectile effect is observed after intracerebroventricular but not intrathecal administration, suggesting a supraspinal site of action. s.c. injections of ABT-724 increase intracavernosal pressure in awake freely moving rats. In the presence of sildenafil, a potentiation of the proerectile effect of ABT-724 is observed in conscious rats. The ability of ABT-724 to facilitate penile erection together with the favorable side-effect profile indicates that ABT-724 could be useful for the treatment of erectile dysfunction.

Synthesis and activity of 2-[4-(4-[3H]-2-cyanophenyl)piperazinyl]-N-(2,4,6-[3H]3-3-methylphenyl)acetamide: a selective dopamine D4 receptor agonist and radioligand.

The first selective dopamine D4 agonist radioligand is described. The synthesis of these piperazine radioligands relied on the transformation of brominated precursors 4a and 4b with tritium gas in the presence of a sensitive cyano functional group. The specific activity of these two radioligands was measured and [3H]6b found to be suitable for use in D4 saturation and competition binding studies. The synthesis, biological, and radioactivity of this new agonist radioligand as well as preliminary SAR will be discussed.

Central mechanisms regulating penile erection in conscious rats: the dopaminergic systems related to the proerectile effect of apomorphine.

Apomorphine has been used as a pharmacological probe of dopaminergic receptors in a variety of central nervous system disorders. The utility of apomorphine as an agent for the treatment of erectile dysfunction has also been demonstrated clinically. Apomorphine is a nonselective dopaminergic receptor agonist with potent binding affinity (Ki) of 101, 32, 26, 2.6, and 10 nM for D1, D2, D3, D4, and D5, respectively. When administered either subcutaneously (s.c.) or intracerebroventricularly (i.c.v.), apomorphine fully evoked penile erections in conscious rats with maximum effect at 0.1 micromol/kg s.c. and 3 nmol/rat i.c.v., respectively. Apomorphine was less efficacious when injected intrathecally (i.t.) to L4-L6 spinal levels (50% at 30-100 nmol/rat i.t.). Penile erection facilitated by apomorphine was blocked by haloperidol and clozapine (i.p. and i.c.v.) but not by domperidone (a peripherally acting dopaminergic receptor antagonist). In this model using conscious rats, penile erection was significantly induced by quinpirole (D2-D3-D4 receptor agonist), but not by R(+)-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol (SKF38393) and R(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzapine (SKF81297) (D1 receptor agonists), or a D2 receptor agonist R-5,6-dihydro-N,N-dimethyl-4H-imidazo[4,5,1-ij]quinolin-5-amine (PNU-95666E). The role of D4 receptors in penile erection was demonstrated using selective D4 receptor agonists [(4-phenylpiperazinyl)-methyl]benzamide (PD168077) and 5-fluoro-2-[[4-(2-pyridinyl)-1-piperazinyl]methyl]-1H-indole (CP226269), whether administered systemically (s.c.) or locally in the brain (i.c.v.). The ability of apomorphine to activate D3 receptors in relation to its proerectile activity remains to be elucidated by use of selective subtype agonists. These results suggest that the proerectile action of apomorphine in rats is mediated at supraspinal levels and that this effect is not mimicked by a D2 receptor agonist but associated with activation of D4 receptors.

Synthesis and functional activity of (2-aryl-1-piperazinyl)-N-(3-methylphenyl)acetamides: selective dopamine D4 receptor agonists.

Diaryl piperazine acetamides were identified as potent and selective dopamine D(4) receptor agonists. Our strategy is based on an amide bond reversal of an acid sensitive, dopamine D(4) receptor partial agonist, PD 168077. This reversal provided compounds with excellent potency and improved stability. Systematic evaluation of the substitution on the aryl piperazine portion revealed a significant effect on functional activity. The synthesis and biological activity of these new dopamine D(4) agonists is discussed.