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The goal of this study was to identify a structurally distinct D(4)-selective agonist with superior oral bioavailability to our first-generation clinical candidate 1a (ABT-724) for the potential treatment of erectile dysfunction. Arylpiperazines such as (heteroarylmethyl)piperazine 1a, benzamide 2, and acetamides such as 3a,b exhibit poor oral bioavailability. Structure-activity relationship (SAR) studies with the arylpiperidine template provided potent partial agonists such as 4d and 5k that demonstrated no improvement in oral bioavailability. Further optimization with the (N-oxy-2-pyridinyl)piperidine template led to the discovery of compound 6b (ABT-670), which exhibited excellent oral bioavailability in rat, dog, and monkey (68%, 85%, and 91%, respectively) with comparable efficacy, safety, and tolerability to 1a. The N-oxy-2-pyridinyl moiety not only provided the structural motif required for agonist function but also reduced metabolism rates. The SAR study leading to the discovery of 6b is described herein.

A new series of dopamine D4 receptor agonists, 1-aryl-3-(4-pyridinepiperazin-1-yl)propanone oximes, was designed through the modification of known dopamine D4 receptor agonist PD 168077. Replacement of the amide group with a methylene-oxime moiety produced compounds with improved stability and efficacy. Structure-activity relationsips (SAR) of the aromatic ring linked to the N-4-piperazine ring confirmed the superiority of 2-pyridine as a core for D4 agonist activity. A two-methylene linker between the oxime group and the N-1-piperazine ring displayed the best profile. New dopamine D4 receptor agonists, exemplified by (E)-1-(4-chlorophenyl)-3-(4-pyridin-2-ylpiperazin-1-yl)propan-1-one O-methyloxime (59a) and (E)-1-(3-chloro-4-fluorophenyl)-3-(4-pyridin-2-ylpiperazin-1-yl)propan-1-one O-methyloxime (64a), exhibited favorable pharmacokinetic profiles and showed oral bioavailability in rat and dog. Subsequent evaluation of 59a in the rat penile erection model revealed in vivo activity, comparable in efficacy to apomorphine. Our results suggest that the oximes provide a novel structural linker for 4-arylpiperazine-based D4 agonists, possessing leadlike quality and with potential to develop a new class of potent and selective dopamine D4 receptor agonists.

2-[4-(3,4-Dimethylphenlyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393) was identified as a potent dopamine D4 receptor antagonist with excellent receptor selectivity. [3H]-spiperone competition binding assays showed that A-381393 potently bound to membrane from cells expressing recombinant human dopamine D4.4 receptor (Ki=1.5 nM), which was 20-fold higher than that of clozapine (Ki=30.4 nM). A-381393 exhibited highly selective binding for the dopamine D4.4 receptor (>2700-fold) when compared to D1, D2, D3 and D5 dopamine receptors. Furthermore, in comparison to clozapine and L-745870, A-381393 exhibits better receptor selectivity, showing no affinity up to 10 microM for a panel of more than 70 receptors and channels, with the exception of moderate affinity for 5-HT2A (Ki=370 nM). A-381393 potently inhibited the functional activity of agonist-induced GTP-gamma-S binding assay and 1 microM dopamine induced-Ca2+ flux in human dopamine D4.4 receptor expressing cells, but not in human dopamine D2L or D3 receptor cells. In contrast to L-745870, A-381393 did not exhibit any significant intrinsic activity in a D4.4 receptor. In vivo, A-381393 has good brain penetration after subcutaneous administration. A-381393 inhibited penile erection induced by the selective D4 agonist PD168077 in conscious rats. Thus, A-381393 is a novel selective D4 antagonist that will enhance the ability to study dopamine D4 receptors both in vitro and in vivo.

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.

Tritiation of the dopamine D(4) receptor selective agonist A-369508 ([2-[4-(2-cyanophenyl)-1-piperazinyl]-N-(3-methylphenyl) acetamide) has provided a radioligand for the characterization of dopamine D(4) receptors. [(3)H] A-369508 binds with high affinity to the major human dopamine D(4) receptor variants D(4.2), D(4.4) and D(4.7) (K(d)=1.7, 4, and 1.2 nM, respectively). It also binds to the rat dopamine D(4) receptor, (K(d)=4.4 nM), implying similar binding affinity across human and rat receptors. A-369508 shows >400-fold selectivity over D(2L), >350-fold selectivity over 5-HT(1A) and >700-1,000-fold selectivity over all other receptors tested. Agonist activity determined by inhibition of forskolin-induced cAMP in Chinese hamster ovary cells transfected with the human dopamine D(4.4) receptor (EC(50)=7.5 nM, intrinsic activity=0.71) indicates that A-369508 is a potent agonist at the human dopamine D(4) receptor. Similar data was observed in other functional assays. [(3)H] A-369508 binds to a single, high affinity site on membranes containing the human dopamine D(4.4) receptor. When compared to the D(2)-like antagonist [(3)H] spiperone, competition binding for agonists like dopamine and apomorphine were 2-10-fold more potent with [(3)H] A-369508, while the antagonists clozapine, haloperidol and L-745870 bind with similar affinity to both ligands. Binding to rat brain regions demonstrated that the most abundant area was cerebral cortex (51.2 fmol/mg protein) followed by hypothalamus, hippocampus, striatum and cerebellum. [(3)H] A-369508 is a useful tool to define the localization and physiological role of dopamine D(4) receptors in central nervous system and can facilitate measuring accurate affinities (K(i)) for structure/activity relationship studies designed to identify dopamine D(4) receptor selective agonists.

The goal of this study was to develop a new approach to study the pharmacology of the dopamine D(4) receptor that could be used in comparative studies with dopamine D(2) and D(3) receptors. Stable HEK-293 cell lines co-expressing recombinant human D(2L), D(3) or D(4) receptors along with Galpha(qo5) cDNA were prepared. Dopamine induced a robust, transient calcium signal in these cell lines with EC(50)s for D(2L), D(3) and D(4) of 18.0, 11.9 and 2.2 nM, respectively. Reported D(4)-selective agonists CP226269 and PD168077 were potent, partial D(4) agonists exhibiting 31-1700-fold selectivity for D(4) over D(3) or D(2). Non-selective D(2)-like agonists apomorphine and quinpirole showed full efficacy but did not discriminate across the three receptors. D(3)-selective agonists 7-hydroxy-DPAT and PD128907 were potent but non-selective D(2)-like agonists. The reported D(3) partial agonist BP-897 exhibited minimal agonist activity at D(3) but was a potent D(3) antagonist and a partial D(4) agonist. Other D(2)-like antagonists, haloperidol, clozapine, and domperidone showed concentration-dependent inhibition of dopamine responses at all three receptors with K(i) ranging from 0.05 to 48.3 nM. The D(3) selective antagonist S33084 and D(4)-selective antagonist L-745870 were highly selective for D(3) and D(4) receptors with K(b) of 0.7 and 0.1 nM, respectively. Stable co-expression of D(2)-like receptors with chimeric Galpha(qo5) proteins in HEK-293 cells is an efficient method to study receptor activation in a common cellular background and an efficient method for direct comparison of ligand affinity and efficacy across human D(2L), D(3) and D(4) receptors.

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.

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.

A series of subtype selective dopamine D(4) receptor ligands from the hetroarylmethylphenylpiperazine class have been discovered that exhibit a remarkable structure-activity relationship (SAR), revealing a substituent effect in which regiosubstitution on the terminal arylpiperazine ring can modulate functional or intrinsic activity. Other structure-dependent efficacy studies in the dopamine D(4) field have suggested a critical interaction of the heteroarylmethyl moiety with specific protein microdomains in controlling intrinsic activity. Our studies indicate that for some binding orientations, the phenylpiperazine moiety also plays a key role in determining efficacy. These data also implicate a kinetic or efficiency term, contained within measured functional affinities for agonists, which support a sequential binding and conformational stabilization model for receptor activation. The structural similarity between partial agonist and antagonist, within this subset of ligands, and lack of bioisosterism for this substituent effect are key phenomena for these hypotheses.

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.

Nitric oxide (NO) activates corpus cavernosum smooth muscle soluble guanylate cyclase (sGC) and increases the synthesis of cGMP that results in smooth muscle relaxation and ultimately, penile erection. To characterize sGC and define the potential synergy between NO and the allosteric activator YC-1 in corpus cavernosum, rat sGC was activated by either sodium nitroprusside (SNP) or YC-1, and YC-1 potentiated the effects of SNP with a 200-fold activation of sGC. Both SNP and YC-1 decreased the Km and increased the Vmax. ODQ significantly inhibited sGC activated by SNP with IC50 of 0.5 nM, but did not affect the sGC activated by YC-1 as well as basal sGC activity. SNP and YC-1 synergistically increased intracellular cGMP levels in rabbit corpus cavernosum smooth muscle cell cultures. YC-1 significantly relaxed rabbit cavernosum tissue strips in organ baths with an EC50 of 8.4 microM. In the presence of L-nitroarginine methyl ester to block endogenous NO production, co-administration of SNP shifted the dose response of YC-1 to the left, showing the synergism of SNP and YC-1 in tissue strips. In view of the clinical efficacy of phosphodiesterase-5 inhibitors, activation of sGC may provide an alternative means for enhancing the activity of neurally derived NO during sexual stimulation in the corpus cavernosum, representing a novel approach for the treatment of erectile dysfunction.