In vitro studies on L-771,688 (SNAP 6383), a new potent and selective alpha1A-adrenoceptor antagonist.

L-771,688 (SNAP 6383, methyl(4S)-4-(3, 4-difluorophenyl)-6-[(methyloxy)methyl]-2-oxo-3-[(¿3-[4-(2-pyridin yl)-1-piperidinyl]propyl¿amino)carbonyl]-1,2,3, 4-tetrahydro-5-pyrimidine carboxylate) had high affinity (Ki less than or = 1 nM) for [3H]prazosin binding to cloned human, rat and dog alpha1A-adrenoceptors and high selectivity (>500-fold) over alpha1B and alpha1D-adrenoceptors. [3H]Prazosin / (+/-)-beta-[125I]-4-hydroxy-phenyl)-ethyl-aminomethylteralone ([125I]HEAT) binding studies in human and animal tissues known to contain alpha1A and non-alpha1A-adrenoceptors further demonstrated the potency and alpha1A-subtype selectivity of L-771,688. [3H]L-771,688 binding studies at the cloned human alpha1A-adrenoceptors and in rat tissues indicated that specific [3H]L-771,688 binding was saturable and of high affinity (Kd=43-90 pM) and represented binding to the pharmacologically relevant alpha1A-adrenoceptors. L-771,688 antagonized norepinephrine-induced inositol-phosphate responses in cloned human alpha1A-adrenoceptors, as well as phenylephrine or A-61603 (N-[5-4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7, 8-terahydro-naphthlen-1-yl] methanesulfonamide hydrobromide) induced contraction in isolated rat, dog and human prostate, human and monkey bladder neck and rat caudal artery with apparent Kb values of 0.02-0.28 nM. In contrast, the contraction of rat aorta induced by norepinephrine was resistant to L-771,688. These data indicate that L-771,688 is a highly selective alpha1A-adrenoceptor antagonist.

Phenylacetamides as selective alpha-1A adrenergic receptor antagonists.

A novel class of potent and selective alpha-1a receptor antagonists has been identified. The structures of these antagonists were derived from truncating the 4-aryl dihydropyridine subunit present in known alpha-1a antagonists. The design principles which led to the discovery of substituted phenylacetamides, the synthesis and SAR of key analogues, and the results of select in vitro and in vivo studies are described.

Design, synthesis and SAR of a series of 2-substituted 4-amino-quinazoline neuropeptide Y Y5 receptor antagonists.

The design of a novel series of NPY-Y5 receptor antagonists is described. Key elements for the design were the identification of weak Y5 hits from a Y1 program, results from a combinatorial approach and database mining. This led to the discovery of the quinazoline 4 and the aryl-sulphonamide moiety as major components of the pharmacophore for Y5 affinity. The synthesis and SAR towards CGP71683A is described.

2-amino-2-oxazolines as subtype selective alpha(2) adrenoceptor agonists.

Cyclohexylamino oxazoline 1 (AGN 190837), an analogue of 2 (Bay a6781), is a potent alpha(2) adrenoceptor agonist. On the basis of a design generated by receptor-ligand modeling, a number of cyclohexyl and norbornyl analogues were synthesized wherein the propyl group of 1 was replaced by phenylalkyl subsituents. This resulted in compound 6 being an alpha(2c) selective agonist, as well as 7 and 9 being alpha(2a)/alpha(2c) selective.

Synthesis and evaluation of furo[3,4-d]pyrimidinones as selective alpha1a-adrenergic receptor antagonists.

Furo[3,4-d]pyrimidinones were found to be metabolites of dihydropyrimidinones such as 1a-b that are subtype-selective antagonists of the alpha1a-adrenergic receptor. A versatile synthesis that provides access to furo[3,4-d]pyrimidinones in high yield and in enantiomerically pure forms is described along with structure-activity relationships in the series.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 3. Approaches to eliminate opioid agonist metabolites by using substituted phenylpiperazine side chains.

Dihydropyrimidinones, such as 1, represent a novel class of alpha(1a) adrenoceptor antagonists with potential for the treatment of benign prostatic hyperplasia (BPH) (see part 1 of this series). Analysis of the metabolites of 1 revealed that 4-methoxycarbonyl-4-phenylpiperidine is formed as the major metabolite and is an agonist at the mu-opioid receptor. To circumvent any potential liability resulting from the metabolite, we decided to identify alternate templates devoid of agonist activity at the mu-opioid receptor to replace the 4-methoxycarbonyl-4-phenylpiperidine moiety. The present study describes the synthesis and SAR of dihydropyrimidinones linked to substituted 4-phenylpiperazine containing side chains. Compound (+)-38 was identified as a lead compound with a binding and functional profile comparable to that of 1. The putative metabolite 2-carboxamidophenylpiperazine has negligible affinity for the mu-opioid receptor.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 4. Structure-activity relationship in the dihydropyrimidine series.

We have previously disclosed dihydropyridines such as 1a,b as selective alpha(1a) antagonists as a potential treatment for benign prostatic hyperplasia (BPH). The propensity of dihydropyridines toward an oxidation led us to find suitable replacements of the core unit. The accompanying papers describe the structure-activity relationship (SAR) of dihydropyrimidinones 2a,b as selective alpha(1a) antagonists. We report herein the SAR of dihydropyrimidines such as 4 and highlight the similarities and differences between the dihydropyrimidine and dihydropyrimidinone series of compounds.

Design and synthesis of novel dihydropyridine alpha-1a antagonists.

A series of analogs of SNAP 5150 containing heteroatoms at C2 or C6 positions is described. Herein, we report that the presence of alkyl substituted heteroatoms at the C2(6)-positions of the dihydropyridine are well tolerated. In addition, 15 inhibited the phenylephrine induced contraction of dog prostate tissue with a Kb of 1.5 nM and showed a Kb (DBP, dogs, microg/kg)/Kb (IUP, dogs, microg/kg) ratio of 14.8/2.5.

Design and synthesis of novel alpha1a adrenoceptor-selective dihydropyridine antagonists for the treatment of benign prostatic hyperplasia.

We report the synthesis and evaluation of novel alpha1a adrenoceptor subtype-selective antagonists. Systematic modification of the lipophilic 4,4-diphenylpiperidinyl moiety of the dihydropyridine derivatives 1 and 2 provided several highly selective and potent alpha1a antagonists. From this series, we identified the 4-(methoxycarbonyl)-4-phenylpiperidine analogue SNAP 5540 (-) [(-)-63] for further characterization. When examined in an isolated human prostate tissue assay, this compound was found to have a Ki of 2.8 nM, in agreement with the cloned human receptor binding data (Ki = 2.42 nM). Further evaluation of the compound in isolated dog prostate tissue showed a Ki of 3.6 nM and confirmed it to be a potent antagonist (Kb = 1.6 nM). In vivo, this compound effectively blocked the phenylephrine-stimulated increase in intraurethral pressure (IUP) in mongrel dogs, at doses which did not significantly affect the arterial pressure (diastolic blood pressure, DBP), with a DBP Kb/IUP Kb ratio of 16. In addition, (-)-63 also showed greater than 40 000-fold selectivity over the rat L-type calcium channel and 200-fold selectivity over several G protein-coupled receptors, including histamine and serotonin subtypes. These findings prove that alpha1a adrenoceptor-subtype selective antagonists such as (-)-63 may be developed as uroselective agents for an improved treatment of BPH over nonselective alpha1 antagonists such as prazosin and terazosin, with fewer side effects.

Identification of a dihydropyridine as a potent alpha1a adrenoceptor-selective antagonist that inhibits phenylephrine-induced contraction of the human prostate.

A number of novel dihydropyridine derivatives based upon 1, 4-dihydro-3-(methoxycarbonyl)-2, 6-dimethyl-4-(4-nitrophenyl)-5-((3-(4, 4-diphenylpiperidin-1-yl)propyl)aminocarbonyl)pyridine (4) have been synthesized and tested at cloned human alpha adrenoceptors as well as the rat L-type calcium channel. Within this compound series, 5-(aminocarbonyl)-1,4-dihydro-2, 6-dimethyl-4-(4-nitrophenyl)-3-((3-(4, 4-diphenylpiperidin-1-yl)propyl)aminocarbonyl)pyridine (19) displayed good binding affinity and selectivity for the alpha1a adrenoceptor (pKi = 8.73) and potently inhibited (pA2 = 9.23) phenylephrine-induced contraction of the human prostate.

Characterization of specific binding of [125I]L-762,459, a selective alpha1A-adrenoceptor radioligand to rat and human tissues.

L-762,459 ((+/-)1-(3-¿[5-carbamoyl-2-2-[(4-hydroxy-3-iodobenzimidoyl)-amino] -ethoxy-methy¿-6-methyl-4-(4-nitropheny)-1,4-dihydropyridine -3-carbonyl]-amino¿-propyl)-4-phenyl-1-piperidine-4-carboxylic acid methyl ester), an analog of a series of dihydropyridines previously reported to be selective alpha1A-adrenoceptor subtype antagonists was found to have alpha1A-adrenoceptor subtype selectivity (Ki (nM), la = 1.3, lb = 240, Id = 280). Specific [125I]L-762,459 binding was detected in rat cerebral cortex, hippocampus, vas deferens, kidney, heart and prostate tissues known to contain the alpha1A-adrenoceptor subtype, but not in tissues known to contain alpha1B-adrenoceptor (spleen, liver) and alpha1D-adrenoceptor (aorta). Scatchard analysis of [125I]L-762,459 binding in rat cerebral cortex and prostate indicated a single binding site with a Kd of 0.7 nM and Bmax of 11 (cerebral cortex) and 1 (prostate) pmole/g tissue. Specific and saturable [125I]L-762,459 binding was also found in human cerebral cortex, liver, prostate and vas deferens (Kd = 0.2-0.4 nM, Bmax = 0.4-4 pmole/g tissue). The specific binding in rat and human tissues was competed by non-selective alpha1-adrenoceptor compounds (Ki values in nM: prazosin (0.14-1.2), terazosin (1.8-5.9) and phentolamine (2.4-11)) and selective alpha1A-adrenoceptor compounds [Ki values in nM: (+) niguldipine (0.04-1.2) and SNAP 5399 ((+/-)-2-((2-aminoethyl)oxy)methyl-5-carboxamido-6-ethyl-4-(4-nitropheny l)-3-N-(3-(4,4-diphenylpiperidin-1-yl)propyl)carboxamido-1,4-dihyd ropyridine hydrate (0.5-4.8)]. The results were consistent with the selective binding of [125I]L-762,459 to the alpha1A-adrenoceptor. The specific labeling of the alpha1A-adrenoceptor subtype by [125I]L-762,459 may make it a useful tool to localize the distribution of the alpha1A-adrenoceptor.

Localization of mRNA and receptor binding sites for the alpha 1a-adrenoceptor subtype in the rat, monkey and human urinary bladder and prostate.

PURPOSE: To localize the mRNAs and receptor binding sites for the alpha 1a/A, alpha 1b/B and alpha 1d/D- adrenoceptor (AR) subtypes in the rat, monkey and human urinary bladder and prostate. MATERIALS AND METHODS: alpha 1-AR mRNAs were localized on slide mounted tissue sections by in situ hybridization using [35S]-labeled subtype specific oligonucleotide probes. alpha 1-AR receptor binding sites were localized on slide mounted tissue sections by competitive displacement of [3H]-prazosin using subtype selective ligands. RESULTS: Only the alpha 1a-AR subtype mRNA was discernible by in situ hybridization. The alpha 1a-AR mRNA was localized in all smooth muscle areas of the rat, monkey and human urinary bladder and prostate. High levels of alpha 1a mRNA were detected in bladder dome and bladder base urothelium. Competitive displacement studies using the alpha 1A-AR selective ligand SNAP 5272 revealed that the alpha 1A-AR represented over 80% of the total alpha 1-AR in monkey bladder and prostate. In general, localization of the alpha 1A-AR corresponded to the alpha 1a-AR mRNA localization, that is, receptor protein was localized to smooth muscle areas of the bladder dome, trigone and base and prostate. One notable exception was the bladder urothelium, which contained high levels of alpha 1a-AR mRNA, but undetectable levels of alpha 1A-AR protein. The alpha 1a-AR mRNA appeared to be transcribed but not translated in bladder urothelium. CONCLUSIONS: The alpha 1A-AR represents the major subtype in the smooth muscle of rat, monkey and human urinary systems. Selective alpha 1A-AR agents are therefore potentially useful in the treatment of multiple urinary smooth muscle related disorders.

Modeling the G-protein-coupled neuropeptide Y Y1 receptor agonist and antagonist binding sites.

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor (GPCR) superfamily and mediate several physiological responses, such as blood pressure, food intake, sedation and memory retention. To understand the interactions between the NPY Y1 receptor subtype and its ligands, computer modeling was applied to the natural peptide agonist, NPY and a small molecule antagonist, BIBP3226. An agonist and antagonist binding domain was elucidated using mutagenesis data for the Y1 receptor as well as for other GPCR families. The agonist and antagonist ligands which were investigated appear to share common residues for their interaction within the transmembrane regions of the Y1 receptor structure, including Gln120, Asn283 and His306. This is in contrast to findings with tachykinin receptors where the binding domains of the non-peptide antagonists have very little in common with the binding domains of the agonist, substance-P. In addition, a hydrogen bond between the hydroxyl group of Tyr36 of NPY and the side chain of Gln219, an interaction that is absent in the model complex between Y1 and the antagonist BIBP3226, is proposed as one of the potential interactions necessary for receptor activation.

Rilmenidine-induced ocular hypotension: role of imidazoline1 and alpha 2 receptors.

PURPOSE: To examine ocular actions by rilmenidine, an imidazoline1 and alpha 2 adrenoceptor agonist. METHODS: Intraocular pressure was measured in normal and sympathetically denervated rabbits by pneumatonometry. Electrically stimulated 3H-norepinephrine release from sympathetic nerves was determined in isolated, perfused rabbit iris-ciliary bodies. cAMP levels were evaluated in rabbit iris-ciliary bodies by radioimmunoassay. Ca2+ concentrations were measured in rabbit transformed nonpigmented ciliary epithelial cells by fluorescence ratio microscopy. RESULTS: Topical, unilateral administration of rilmenidine produced hypotensive responses in normal rabbits which were antagonized by either bilaterally administered efaroxan, an imidazoline receptor antagonist or rauwolscine, an alpha 2 receptor antagonist. Sympathectomy also eliminated the ocular hypotensive response. Rilmenidine (0.001, 0.01, 0.1, 1 microM) caused 5 +/- 1%, 18 +/- 5%, 35 +/- 10%, and 48 +/- 9% inhibition, respectively, of 3H-norepinephrine overflow whereas 10 microM efaroxan or rauwolscine caused enhancement of norepinephrine release by 102 +/- 23% or 86 +/- 25%, respectively. Furthermore, pretreatment with efaroxan or rauwolscine partially antagonized the inhibition of norepinephrine release induced by rilmenidine. In other experiments, rilmenidine (1 microM) inhibited isoproterenol-stimulated cAMP accumulation in rabbit iris-ciliary bodies by 43 +/- 9% which was antagonized by 10 microM efaroxan or rauwolscine. Rilmenidine induced large increases in [Ca2+]i in rabbit nonpigmented ciliary epithelial cells which were effectively antagonized by efaroxan or rauwolscine. CONCLUSIONS: These in vivo and in vitro data suggest that the ocular hypotensive activity induced by rilmenidine is due, in part, to suppression of sympathetic neuroeffector function in the rabbit ciliary body and that alpha 2 adrenergic receptors and/or imidazoline1 receptors are involved.

Synthesis and evaluation of 2-[(5-methylbenz-1-ox-4-azin-6-yl)imino]imidazoline, a potent, peripherally acting alpha 2 adrenoceptor agonist.

We have synthesized 2-[(5-methylbenz-1-ox-4-azin-6-yl)imidazoline, 3, a potent, peripherally acting alpha 2 adrenoceptor agonist. The agent is conveniently prepared in five steps from 2-amino-m-cresol. The agent has demonstrated good selectivity for alpha 2 adrenoceptors in binding and functional studies. When applied topically to eyes, the agent is efficacious for the reduction of intraocular pressure. The agent does not penetrate the blood-brain barrier and, as a consequence, does not lower blood pressure or induce sedation when administered topically or intravenously. We have determined the pKa and log P in water versus both octanol and dodecane of 3 and a set of related agents. The best physical parameter to explain its lack of central nervous system penetration appears to be log P measured in octanol versus water.

Modeling and mutagenesis of the human alpha 1a-adrenoceptor: orientation and function of transmembrane helix V sidechains.

A 3-dimensional model of the seven transmembrane helical segments (TMs) of the human alpha 1a-adrenoceptor was initially built by analogy to the known structure of bacteriorhodopsin. However, the rotational orientation of TM V about its helical axis, and the roles of several TM V residues in ligand binding and receptor activation remained in question. Accordingly, we determined the effects of six site-specific mutations in TM V on binding affinity and functional potency of a structurally diverse series of agonists and antagonists. Mutation of Ser 192 and Phe 193 disrupted the binding of many of the tested ligands, as measured by displacement of [3H]prazosin. In addition, mutation of Ser 188, Ser 192, and Phe 193 disrupted receptor activation, as measured by [3H]inositol phosphate formation. On the basis of these results, a specific rotational orientation of TM V is proposed as part of a revised receptor model, which also takes into account more recently reported information about the structure of rhodopsin. This revised alpha 1a-adrenoceptor model accounts for direct interactions which are proposed between Ser 188 and Ser 192 and the meta and para hydroxyl groups of norepinephrine, respectively, in the G-protein coupled receptor state.

Localization of the alpha 1A-adrenoceptor in the human prostate.

PURPOSE: We determined the tissue localization of the alpha 1a-adrenoceptor in the human prostate. MATERIALS AND METHODS: Autoradiographic localization of the alpha 1a-adrenoceptor in the human prostate was determined by performing competitive displacement experiments on slide mounted tissue sections using the ligand 125iodine-2-(-[4-hydroxyphenyl]-ethyl-aminomethyl)tetralone (125I-Heat), and the alpha 1-antagonists WB-4101 (4 x 10(-8) M.) and 5-carboxamido-2,6-diethyl-1,4-dihydro-3-[N-(3-[4-hydroxy-4-phenylpipe ridin- yl]propyl)]carboxamido-4-(4-nitrophenyl) (SNAP 5272, 3 x 10(-7) M.). Under these experimental conditions, WB-4101 and SNAP 5272 are selective alpha 1a/alpha 1d-adrenoceptor and alpha 1a-adrenoceptor antagonists, respectively. The autoradiographs were quantitatively analyzed using a computer image analysis system. RESULTS: Specific 125I-Heat binding associated with the epithelium and stroma were independently analyzed. WB-4101 and SNAP 5272 inhibited 100% of the specific 125I-Heat binding in the stroma, suggesting that all of the stromal alpha 1-adrenoceptors are of the alpha 1a subtype. WB-4101 inhibited none of the specific 125I-Heat binding in the epithelium, suggesting that the alpha 1-adrenoceptor in the epithelium is of the alpha 1b subtype. SNAP 5272 displaced only 25% of the specific 125I-Heat binding in the epithelium, suggesting that a relatively small percentage of the epithelial alpha 1-adrenoceptor is of the alpha 1a subtype. CONCLUSIONS: To our knowledge, our study represents the first cellular localization of the alpha 1-adrenoceptor subtypes in the human prostate using highly selective alpha 1-adrenoceptor antagonists and is consistent with the physiological observation that the activity of prostatic smooth muscle is mediated by the alpha 1a-adrenoceptor.

Tiagabine, SK&F 89976-A, CI-966, and NNC-711 are selective for the cloned GABA transporter GAT-1.

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The synaptic action of GABA is terminated by rapid uptake into presynaptic terminals and surrounding glial cells. Molecular cloning has revealed the existence of four distinct GABA transporters termed GAT-1, GAT-2, GAT-3, and BGT-1. Pharmacological inhibition of transport provides a mechanism for increasing GABA-ergic transmission, which may be useful in the treatment of various neuropsychiatric disorders. Recently, a number of lipophilic GABA transport inhibitors have been designed and synthesized, which are capable of crossing the blood brain barrier, and which display anticonvulsive activity. We have now determined the potency of four of these compounds, SK&F 89976-A (N-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid), tiagabine ((R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3- piperidencarboxylic acid), CI-966 ([1-[2-[bis 4-(trifluoromethyl)phenyl]methoxy]ethyl]-1,2,5,6-tetrahydro-3- pyridinecarboxylic acid), and NNC-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,4,6-tetrahydro-3- pyridinecarboxylic acid hydrochloride), at each of the four cloned GABA transporters, and find them to be highly selective for GAT-1. These data suggest that the anticonvulsant activity of these compounds is mediated via inhibition of uptake by GAT-1.