Simple high-performance liquid chromatographic method to analyze serum creatinine has several advantages over the Jaffé picric acid reaction as demonstrated with a cimetidine dose response in rhesus monkeys.

A simple method for creatinine determination was developed using high-performance liquid chromatography (HPLC) to more accurately monitor serum creatinine levels in experimental animal models when compared to the Jaffé method. The new HPLC procedure will replace the traditional Jaffé method for rhesus monkey kidney function studies. We developed an isocratic method using a polymeric, hydrophilic, silica-based strong cation-exchange bed with a 5.0 mmol/l lithium acetate matrix, pH 4.9, which isolates creatinine with no detectable impurities as determined by three-dimensional ultraviolet-visible spectral analysis. Sample preparation includes deproteination with acetonitrile, evaporation, and resolubilization in mobile phase followed by quantitation with UV detection at 234 nm. Extraction efficiency across the measured range was 96 +/- 2%. From numerous extracted rhesus monkey creatinine curves (n=38) a slope of 251,100 +/- 756 (95% CI) and an intercept of 675.6 +/- 712.7 (95% CI) was calculated. Extraction efficiency and peak purity tests with human plasma were cross-compared with rhesus monkey serum producing equivalent results. An average of 120 samples can be run daily.

Cyclic imides as potent and selective alpha-1A adrenergic receptor antagonists.

We disclose a new compound class of potent and selective alpha-1A adrenergic receptor antagonists exemplified by the geminally, disubstituted cyclic imide 7. The optimization of lead compounds resulting in the cyclic imide motif is highlighted. The results of in vitro and in vivo studies of selected compounds are presented.

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.

Selective alpha1a adrenergic receptor antagonists based on 4-aryl-3,4-dihydropyridine-2-ones.

A series of alpha1a receptor antagonists derived from a 4-aryl-3,4-dihydropyridine-2-one heterocycle is disclosed. Potency in the low nanomolar to picomolar range along with high selectivity was obtained. In vivo efficacy in a prostate contraction model in rats was observed with a few derivatives.

In vitro and in vivo evaluation of dihydropyrimidinone C-5 amides as potent and selective alpha(1A) receptor antagonists for the treatment of benign prostatic hyperplasia.

alpha(1) Adrenergic receptors mediate both vascular and lower urinary tract tone, and alpha(1) receptor antagonists such as terazosin (1b) are used to treat both hypertension and benign prostatic hyperplasia (BPH). Recently, three different subtypes of this receptor have been identified, with the alpha(1A) receptor being most prevalent in lower urinary tract tissue. This paper explores 4-aryldihydropyrimidinones attached to an aminopropyl-4-arylpiperidine via a C-5 amide as selective alpha(1A) receptor subtype antagonists. In receptor binding assays, these types of compounds generally display K(i) values for the alpha(1a) receptor subtype <1 nM while being greater than 100-fold selective versus the alpha(1b) and alpha(1d) receptor subtypes. Many of these compounds were also evaluated in vivo and found to be more potent than terazosin in both a rat model of prostate tone and a dog model of intra-urethral pressure without significantly affecting blood pressure. While many of the compounds tested displayed poor pharmacokinetics, compound 48 was found to have adequate bioavailability (>20%) and half-life (>6 h) in both rats and dogs. Due to its selectivity for the alpha(1a) over the alpha(1b) and alpha(1d) receptors as well as its favorable pharmacokinetic profile, 48 has the potential to relieve the symptoms of BPH without eliciting effects on the cardiovascular system.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 1. Structure-activity relationship in dihydropyrimidinones.

Dihydropyrimidinones such as compound 12 exhibited high binding affinity and subtype selectivity for the cloned human alpha(1a) receptor. Systematic modifications of 12 led to identification of highly potent and subtype-selective compounds such as (+)-30 and (+)-103, with high binding affinity (K(i) = 0.2 nM) for alpha(1a) receptor and greater than 1500-fold selectivity over alpha(1b) and alpha(1d) adrenoceptors. The compounds were found to be functional antagonists in human, rat, and dog prostate tissues. Compound (+)-103 exhibited excellent selectively to inhibit intraurethral pressure (IUP) as compared to lowering diastolic blood pressure (DBP) in mongrel dogs (K(b)(DBP)/K(b)(IUP) = 40) suggesting uroselectivity for alpha(1a)-selective compounds.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 2. Approaches to eliminate opioid agonist metabolites via modification of linker and 4-methoxycarbonyl-4-phenylpiperidine moiety.

We have previously described compound 1a as a high-affinity subtype selective alpha(1a) antagonist. In vitro and in vivo evaluation of compound 1a showed its major metabolite to be a mu-opioid agonist, 4-methoxycarbonyl-4-phenylpiperidine (3). Several dihydropyrimidinone analogues were synthesized with the goal of either minimizing the formation of 3 by modification of the linker or finding alternative piperidine moieties which when cleaved as a consequence of metabolism would not give rise to mu-opioid activity. Modification of the linker gave several compounds with good alpha(1a) binding affinity (K(i) = < 1 nM) and selectivity (>300-fold over alpha(1b) and alpha(1d)). In vitro analysis in the microsomal assay revealed these modifications did not significantly affect N-dealkylation and the formation of the piperidine 3. The second approach, however, yielded several piperidine replacements for 3, which did not show significant mu-opioid activity. Several of these compounds maintained good affinity at the alpha(1a) adrenoceptor and selectivity over alpha(1b) and alpha(1d). For example, the piperidine fragments of (+)-73 and (+)-83, viz. 4-cyano-4-phenylpiperidine and 4-methyl-4-phenylpiperidine, were essentially inactive at the mu-opioid receptor (IC(50) > 30 microM vs 3 microM for 3). Compounds (+)-73 and (+)-83 were subjected to detailed in vitro and in vivo characterization. Both these compounds, in addition to their excellent selectivity (>880-fold) over alpha(1b) and alpha(1d), also showed good selectivity over several other recombinant human G-protein coupled receptors. Compounds (+)-73 and (+)-83 showed good functional potency in isolated human prostate tissues, with K(b)s comparable to their in vitro alpha(1a) binding data. In addition, compound (+)-73 also exhibited good uroselectivity (DBP K(b)/IUP K(b) > 20-fold) in the in vivo experiments in dogs, similar to 1a.

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.

4-Oxospiro[benzopyran-2,4'-piperidines] as selective alpha 1a-adrenergic receptor antagonists.

The 4-oxospiro[benzopyran-2,4'-piperidine] ring system is contained within potent class III antiarrhythmic agents. We highlight how these agents can be chemically transformed into a new class of potent (< 1 nM) and selective (> 25-fold) alpha 1a-receptor subtype adrenergic antagonists.

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.

3-(1-piperazinyl)-4,5-dihydro-1H-benzo[g]indazoles: high affinity ligands for the human dopamine D4 receptor with improved selectivity over ion channels.

3-(4-Piperidinyl)-5-arylpyrazoles, such as 1, were selective for the cloned human dopamine D4 receptor (hD4), but also showed affinity at voltage sensitive calcium, sodium and potassium ion channels. A combination of substituent changes to reduce the basicity of the piperidine nitrogen and conformational restriction to give 4,5-dihydro-1H-benzo[g]indazoles reduced this ion channel affinity at the expense of selectivity for hD4 over other dopamine receptors. Incorporation of piperazine into the 4,5-dihydro-1H-benzo[g]indazoles in place of piperidine gave a novel series of high affinity, selective, orally bioavailable hD4 ligands, such as 16, with improved selectivity over ion channels.

Design and synthesis of N-alkylated saccharins as selective alpha-1a adrenergic receptor antagonists.

Benign prostatic hyperplasia can be managed pharmacologically with alpha-1 adrenergic receptor antagonists. Agents that demonstrate selectivity for the alpha-1a receptor subtype may offer advantages in clinical applications with respect to hypotensive side effects. The N-alkylated saccharins reported here represent a new class of subtype selective alpha-1a adrenergic receptor antagonists which demonstrate potent effects on prostate function in vivo and are devoid of blood pressure side effects.

In vivo pharmacology of an angiotensin AT1 receptor antagonist with balanced affinity for AT2 receptors.

L-163,017 (6-[benzoylamino]-7-methyl-2-propyl-3-[[2'-(N-(3-methyl-1-butoxy) carbonylaminosulfonyl)[1,1']-biphenyl-4-yl]methyl]-3H-imidazo[4,5- b]pyridine) is a potent, orally active, nonpeptide angiotensin II receptor antagonist. Conscious rats and dogs were dosed p.o. and i.v.; in both species the plasma bioequivalents are similar at the angiotensin AT1 and AT2 receptor sites indicating balanced activity is maintained in vivo. L-163,017 prevents the pressor response to intravenous (i.v.) angiotensin II in the conscious rat, dog, and rhesus monkey. L-163,017 also significantly reduces blood pressure in a renin-dependent model of hypertension, similar to an angiotensin converting enzyme inhibitor (Enalapril) and an angiotensin AT1 receptor-selective antagonist (L-159,282). These studies indicate that neither the angiotensin AT2 receptor nor bradykinin is important in the acute antihypertensive activity of angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists.

Pharmacology of losartan, an angiotensin II receptor antagonist, in animal models of hypertension.

BACKGROUND: Clinical experience with angiotensin converting enzyme (ACE) inhibitors has shown that inhibition of the renin-angiotensin system is effective therapy for hypertension and heart failure. Losartan (DuP753, MK954, cozaar) is the first non-peptidic drug that inhibits the renin-angiotensin system by selectively blocking the interaction of angiotensin II with its receptor. DIFFERENCES BETWEEN LOSARTAN AND ACE INHIBITORS: Pharmacological differences between ACE inhibitors and losartan could affect comparative efficacy and/or safety. In addition to angiotensin I, ACE has other substrates (e.g. kinins). Blocking the metabolism of kinins with ACE inhibitors could be beneficial (e.g. vasodilation) and/or elicit side effects (e.g. cough) which will not be produced by losartan. Non-ACE pathways of angiotensin II formation have been described (e.g. angiotensin I convertase) which may limit the ability of ACE inhibitors to prevent formation of angiotensin II in all tissues. Losartan blocks angiotensin II responses irrespective of the route or site of angiotensin II formation. Two binding sites for angiotensin II are widely accepted, AT1 and AT2. Losartan blocks only AT1 sites while ACE inhibitors functionally block angiotensin II interaction with both sites. Since the physiological role for AT2 sites is unknown, the relevance of this difference between ACE inhibitors and losartan is questionable. HYPERTENSION: In animal models of hypertension, the efficacy of losartan is equivalent to the efficacy of ACE inhibitors. In animal models that reflect complications of hypertension, such as kidney dysfunction, cardiac and vascular hypertrophy and stroke, losartan and ACE inhibitors are also equally effective. From these results, kinin potentiation and lack of inhibition of angiotensin I convertase do not lead to differences in pharmacological efficacy between ACE inhibitors and losartan. Therefore, with respect to therapeutic efficacy, results in animal models indicate that losartan will display the beneficial pharmacology of ACE inhibitors without the detrimental side effects attributed to kinin potentiation.

In vivo pharmacology of a novel AT1 selective angiotensin II receptor antagonist, MK-996.

MK-996, N-(4'-(5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridin-3-yl- methyl)1,1'-biphenyl-2-yl)-sulfonylbenzamide, is a potent, orally active, highly selective, nonpeptide angiotensin II (AII) receptor antagonist. MK-996 prevents the pressor response to intravenous AII in the conscious rat, dog, and rhesus monkey (ED50, mg/kg; oral/intravenous = 0.067/0.014, 0.035/0.017, and 0.1/0.036, respectively). In the anesthetized chimpanzee, MK-996 (1 mg/kg, iv) produces 100% (peak) inhibition of the AII pressor response and is still active (52%) at 24 h. To our knowledge this pharmacologic profile in the rat, dog, rhesus monkey, and chimpanzee presents the least species variability of any AII receptor antagonist yet described. Responses to methoxamine and arginine vasopressin are not affected by MK-996. In aortic coarcted (high renin) rats, MK-996 (3 mg/kg, by mouth) reduces blood pressure to normotensive (< 120 mm Hg) levels without reflex tachycardia. This dose of MK-996 reduces blood pressure to approximately the same level as both losartan (3 mg/kg, by mouth) and enalapril (3 mg/kg, by mouth) in this model. The duration of antihypertensive activity of MK-996 is similar to enalapril and shorter than losartan at the doses tested. Additionally, in the rat MK-996 does not potentiate the vasodepressor response to bradykinin and completely prevents the ability of AII to stimulate an increase in plasma levels of aldosterone. Therefore, MK-996 is a potent, orally active, nonpeptide AII receptor antagonist with a long duration of action, little species variability, and anti-hypertensive activity.

Role of endothelium-derived relaxing factor in parasympathetic coronary vasodilation.

Vasodilation following the infusion of acetylcholine is due to the release of endothelium-derived relaxing factor (EDRF). However, the role of EDRF in neurogenic coronary vasodilation, when acetylcholine is released outside the vessel at the adventitial-medial junction, has not been established. The action of EDRF in parasympathetic coronary vasodilation was tested in the present study using a specific inhibitor of EDRF synthesis, nitro-L-arginine methyl ester (L-NAME). Experiments were conducted on closed-chest, alpha-chloralose-anesthetized dogs with the heart paced at a constant rate. Phentolamine and propranolol were administered to block alpha- and beta-adrenergic receptors, and ibuprofen was given to inhibit prostaglandin synthesis. Intracoronary infusion of L-NAME decreased the coronary vasodilation in response to intracoronary acetylcholine or vagal stimulation. The coronary response to the endothelium-independent vasodilator nitroglycerin was unaffected by L-NAME. These data demonstrate that L-NAME specifically inhibits coronary vasodilation caused by acetylcholine and vagal stimulation, indicating that parasympathetic coronary vasodilation is dependent on EDRF.

Role of myocardial oxygen and carbon dioxide in coronary autoregulation.

Myocardial oxygen (PO2) and carbon dioxide tensions (PCO2) are likely mediators of the local control of coronary blood flow. A previous study demonstrated that myocardial PO2 and PCO2, estimated by coronary venous values, interact synergistically to determine coronary flow. This synergistic relation was used in a prospective study to test the hypothesis that myocardial PO2 and PCO2 mediate changes in coronary vascular conductance during autoregulation. The left main coronary artery was pump perfused at controlled pressures in closed-chest anesthetized dogs. Autoregulation curves were obtained by increasing coronary perfusion pressure from 80 to 160 mmHg in 20-mm increments. Steady-state measurements of coronary venous PO2 and PCO2 and coronary conductance were obtained at each perfusion pressure. The coronary venous PO2 and PCO2 were used in the previously determined synergistic relation to predict the coronary vascular conductance during autoregulation. The predicted changes in coronary vascular conductance were compared with the actual changes in coronary vascular conductance for the pressure range of 80-160 mmHg. The data indicate that the synergistic interaction of oxygen and carbon dioxide accounts for approximately 23% of the change in coronary vascular conductance during autoregulation. These results suggest that other factors are also involved in autoregulation.

Synergistic action of myocardial oxygen and carbon dioxide in controlling coronary blood flow.

A two-part experiment was designed to test the hypothesis that myocardial oxygen and carbon dioxide tensions, as measured by coronary venous oxygen and carbon dioxide tensions, determine coronary blood flow during increases in myocardial oxygen consumption. The left main coronary artery was pump-perfused at constant pressure in closed-chest, anesthetized dogs. Oxygenators in the perfusion circuit permitted control of coronary arterial gas tensions. The steady-state relation between coronary venous oxygen and carbon dioxide tensions and coronary flow at a constant myocardial oxygen consumption was determined by locally altering coronary arterial oxygen and carbon dioxide tensions. Values of coronary venous oxygen and carbon dioxide tensions and coronary flow were also obtained at normal coronary arterial gas tensions during pacing-induced increases in myocardial oxygen consumption. The data yielded a hyperbolic relation among coronary venous oxygen and carbon dioxide tension and coronary flow during constant myocardial metabolism, suggesting a synergistic interaction between myocardial oxygen and carbon dioxide tensions in determining coronary flow. This relation was then used to predict the coronary flow change during pacing-induced increases in myocardial metabolism. Approximately 40% of the flow response during pacing-induced increases in myocardial oxygen consumption was predicted. In conclusion, coronary venous oxygen and carbon dioxide tensions synergistically interact to produce steady-state changes in coronary flow at a constant myocardial oxygen consumption. Changes in myocardial oxygen and carbon dioxide tensions can account for about 40% of the change in coronary flow during moderate changes in myocardial oxygen consumption.