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.

In vitro 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) inhibited specific 125I-[Sar1, Ile8]angiotensin II binding to angiotensin AT1 receptor (Ki = 0.11-0.20 nM) in rabbit aorta, rat adrenal and human angiotensin AT1 receptor in CHO (Chinese hamster ovary transformed) cells and to AT2 receptor (Ki = 0.14-0.23 nM) in rat adrenal and brain receptors. L-163,017 also had a high affinity in the presence of bovine serum albumin (2 mg/ml), for angiotensin AT1 and AT2 receptors on human adrenal (Ki 3.9 and 4.3 nM), aorta (Ki 0.45 and 0.96 nM) and kidney (Ki 3.6 and 2.3 nM). The much higher Ki values in human tissues were likely due to the presence of bovine serum albumin in the binding assay buffer since L-163,017 had Ki values of 0.13 +/- 0.04 and 2.0 +/- 0.04 nM in the absence and presence of bovine serum albumin, respectively, in inhibiting 125I-[Sar1,Ile8]angiotensin II binding to angiotensin AT1 receptor in rat adrenal membranes. Scatchard analysis of 125I-[Sar1,Ile8]angiotensin II binding in the presence of bovine serum albumin (2 mg/ml) in rabbit aorta and bovine cerebellum indicated a competitive interaction of L-163,017 with angiotensin AT1 and AT2 receptors (Ki values 2.5 and 2.1 nM respectively). L-163,017 inhibited angiotensin II-induced aldosterone release in rat adrenal demonstrating that L-163,017 acted as a competitive antagonist (pA2 = 9.9) and lacked agonist activity. L-163,017 also inhibited angiotensin II responses in rat vascular tissues. The specificity of L-163,017 was shown by its lack of activity on the above functional responses produced by other agonists and in several binding assays.

Roles of endogenous cholecystokinin in biliary, pancreatic and gastric function: studies with L-364,718, a specific cholecystokinin receptor antagonist.

A new, highly potent antagonist of gut cholecystokinin (CCK) receptors has been examined for effects upon postprandial biliary and exocrine pancreatic secretion in conscious dogs with chronic duodenal pouches. This drug (L-364,718) markedly inhibited the postprandial increases in biliary volume, bile acid and bilirubin secretion. However, even at high p.o. doses relative to its ability to antagonize the effects of exogenous CCK, no effects were observed upon the pancreatic secretion of either fluid volume or amylase and lipase under similar conditions. In additional studies, pretreatment with L-364,718 did not significantly reverse the inhibitory effects of a fatty acid salt (sodium oleate) upon gastric emptying in gastric fistula dogs. Moreover, pretreatment with L-364,718 had no significant effects upon postprandial acid and pepsin secretion in lesser curvature (vagally innervated) pouch dogs or did it affect basal (interdigestive) gastric secretion in rats. These results suggest that endogenous CCK plays a critical physiological role in regulating postprandial biliary outflow, but not pancreatic enzyme secretion or the gastric emptying of at least liquid fatty substances. The latter two findings stand in contrast with classical views regarding the physiological function(s) of this hormone.

Effects of atropine upon various components mediating postprandial gastric acid secretion in dogs.

Dose-response curves in chronic gastric fistula dogs were first obtained to chemical stimulants of the three accepted physiological excitatory components regulating postprandial gastric acid secretion. These were: 2-deoxy-D-glucose, a central vagal stimulant; gastrin, a hormone; and histamine, a paracrine factor. Using equiactive doses of each, a dose of atropine just maximal for suppressing all of the anatomical phases of food-induced acid secretion in vagally innervated pouch dogs was found to inhibit substantially the responses to all three of the above stimulants. The above results argue in favor of an interdependent model among the above factors for regulating postprandial gastric acid secretion in the dog.

L-646,462, a cyproheptadine-related antagonist of dopamine and serotonin with selectivity for peripheral systems.

The selectivity, peripheral vs. central actions, of the antidopaminergic agent L-646,462 was assessed in two ways. First, elevation of prolactin in serum (peripheral) and homovanillic acid in the striatum were measured in rats. L-646,462 was found to have a central/peripheral activity ratio of 143, whereas comparable values derived for haloperidol, metoclopramide and domperidone were 1.4, 9.4 and 1305, respectively. Second, the ID50 values required to block apomorphine-induced emesis in beagles (peripheral receptor-mediated response) were compared with those required to block apomorphine-induced stereotypy (central receptor-mediated response) in rats. Central/peripheral ID50 ratios of 234, 9.2, 129 and 7040 were obtained, respectively, for L-646,462, haloperidol, metoclopramide and domperidone. The selectivity of L-646,462 for peripheral serotonin (5-HT) receptors in rats was determined by measuring its effectiveness in blocking 5-HT-induced paw edema (peripheral response) and 5-hydroxytryptophan-induced head twitch (central response); a ratio of 114 was obtained. This value agrees nicely with the ratio of 143 derived in the rat ( vide supra) for peripheral selectivity for dopamine receptors. L-646,462 is, therefore, selective in vivo, preferentially blocking dopamine and 5-HT receptors located outside the blood-brain barrier. With regard to dopamine-receptors, L-646,462 was about equipotent and more selective than metoclopramide, while being less potent and less selective than domperidone. Unlike metoclopramide or domperidone, L-646,462 also possessed a reasonably potent 5-HT receptor antagonist effect in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse locomotor activity: an in vivo test for dopamine autoreceptor activation.

A pharmacologic test is described for assessing selective dopamine (DA) autoreceptor activation using locomotor activity (LMA) of the mouse as the dependent variable. In this test, three criteria must be satisfied to indicate selectivity for the DA autoreceptor. First, a dose-related fall in LMA, taken as a measure of DA autoreceptor activation, should be produced by the putative autoreceptor agonist. Second, to demonstrate a DA system is involved, the reduction in LMA should be blocked by a DA receptor antagonist. Finally, the test compound should produce no LMA-stimulating (i.e., postsynaptic DA receptor agonist) effects over prolonged periods of observation. Using these criteria, 15 DA agonists were evaluated for DA autoreceptor selectivity. Four agents satisfied all criteria as selective DA autoreceptor agonists: CF 25-397, N-n-3-propyl-3- hydroxyphenylpiperidine , 6,7-dihydroxy-2- dimethylaminotetralin (TL-99) and 2-amino-6,7- dibenzoyloxy -1,2,3,4- tetrahydronapthalene . Seven DA agonists produced U-shaped dose-response curves indicative of activity at both the autoreceptor and postsynaptic DA receptor. These agents were: apomorphine, n-propylnorapomorphine, pergolide, RU 24213, RU 24926, (-)-6-ethyl-9-oxaergoline and lisuride. SKF 38393 failed to exert any significant effect on the LMA of the mouse. Both lergotrile and bromocriptine produced dose-related falls in LMA, but both caused a rebound increase in LMA before their durations of action were terminated. Although 3,4-dihydroxyphenylamino-2-imidazoline did produce a dose-related fall in LMA, the inhibition produced by 3,4-dihydroxyphenylamino-2-imidazoline was not reduced by sulpiride, suggesting a nondopaminergic action for 3,4-dihydroxyphenylamino-2-imidazoline.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological, biochemical and behavioral assessment of dopamine autoreceptor activation by a series of dopamine agonists.

The rank order of potency to activate central dopamine autoreceptors of seven compounds known to possess central nervous system dopamine agonist activity were assessed with the following techniques: (1) inhibition of dopaminergic neuronal firing in anesthetized rats, (2) inhibition of dopamine synthesis in rats pretreated with gamma-butyrolactone, and (3) inhibition of mouse locomotor activity. The compounds were also examined for their ability to induce stereotypic behaviors in rats as an index of postsynaptic dopamine receptor activation. The compounds under investigation were apomorphine, N-n-propyl-norapomorphine, lergotrile, bromocriptine, RU 24926 and 6-ethyl-9-oxaergoline (EOE). There was a high degree of correlation between the rank order of potency of the compounds in all three of the presumptive autoreceptor tests and with minor variations the following rank order of potency was found: N-n-propylnorapomorphine greater than or equal to EOE greater than apomorphine greater than lergotrile greater than or equal to RU 24926 greater than bromocriptine. However, in the induction of stereotypies, the rank order of potency was considerably different: N-n-propylnorapomorphine greater than apomorphine greater than EOE greater than RU 24926 greater than lergotrile greater than bromocriptine. There was a poor and statistically significant degree of correlation between the rank order of potency of the test compounds to induce stereotyped behaviors and any of the other three test procedures. Altogether, these data confirm and extend the suspected dopaminergic agonist properties of the compounds under investigation and additionally lend credence to the assumption that the three putative autoreceptor assays employed do in fact reflect dopaminergic autoreceptor activation.

Different actions of TL-99 and 3-PPP in producing contraversive turning in the 6-OHDA-lesioned rat.

The putatively selective dopamine autoreceptor agonists TL-99 and 3-PPP were compared with apomorphine for the production of contraversive turning in the 6-hydroxydopamine-lesioned rat. Although less potent than apomorphine, 3-PPP produced dose-related contralateral turning. The contralateral turning produced by TL-99 plateaued at the 3 mg/kg i.p. dose level. Yohimbine significantly enhanced the TL-99-induced turning, whereas it failed to modify the 3-PPP turning. The results suggest that the alpha 2-adrenergic properties of TL-99 at doses of greater than 3.0 mg/kg masked its dopaminergic effects. Hence, 3-PPP is clearly the more selective agent for DA receptors.

Further evidence for selective antagonism of taurine by 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide.

The putative taurine antagonist 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide (TAG), was examined for activity in blocking contraversive turning evoked by the injection of taurine into the substantia nigra of the rat. Microinjected intranigrally as pretreatment to taurine, TAG caused a 71% reduction in taurine elicited turning. On the other hand, TAG pretreatment did not diminish the number of turns evoked by the intranigral injection of the GABA agonist, muscimol. These results and the lack of effect of TAG in a number of central radioligand binding assays provide further indications that TAG may be a selective taurine antagonist.

Further evidence for a GABA-like action on kojic amine.

Data are reported to support the hypothesis that kojic amine is a GABA agonist in an in vivo model of GABAergic activity. The unilateral intranigral microinjection of kojic amine elicited dose-related turning contraversive from the injection site. The direction of the turning is identical to that observed after the intranigral administration of muscimol or other GABA agonists. Furthermore, the numbers of turns elicited by kojic amine was significantly reduced following pretreatment with the GABA antagonist, bicuculline. The results suggest that kojic amine is a GABA agonist.