Pharmacology of L-744,453, a novel nonpeptidyl endothelin antagonist.

L-744,453 ((+/-)3-[4-(1-carboxy-1-(3,4-methylenedioxyphenyl)methoxy)-3,5-diprop ylphenyl methyl]-3H-imidazo[4,5-c]pyridine) is an endothelin (ET) receptor antagonist from a new structural class, the dipropyl-alpha-phenoxyphenylacetic acid derivatives. L-744,453 competitively and reversibly inhibits [125I]-ET-1 binding to Chinese Hamster Ovary cells expressing cloned human ET receptors (K(i)s: hET(A)=4.3 nM; hET(B)=232 nM), and is selective for endothelin receptors compared to other peptide receptors. It is an antagonist of ET-1 stimulated phosphatidyl inositol hydrolysis in rat uterine slices (IC50=220 nM) and exhibits no agonist activity. This compound also inhibits ET-1 stimulated contraction of rat aortic rings with a K(b) value of 50 nM. L-744,453 protects against ET-1 induced lethality in mice after i.v. (AD50=13 mg/kg i.v.) or oral administration. This compound also antagonizes ET-1 induced increases in diastolic blood pressure in conscious normotensive rats (AD50=0.67 mg/kg i.v.) and anesthetized ferrets (AD50=1.6 mg/kg i.v.). L-744,453 is a potent, selective, orally active endothelin antagonist which may be useful in elucidating the role of endothelin in normal and pathophysiological states.

Conformationally constrained o-tolylpiperazine camphorsulfonamide oxytocin antagonists. Structural modifications that provide high receptor affinity and suggest a bioactive conformation.

A series of new o-tolylpiperazine camphorsulfonamide OT antagonists is described. Analogs containing conformationally constrained 1-acylamino-2-propyl substituents at the camphor C2 endo position exhibit high affinity for OT and AVP-V1a receptors or high affinity and selectivity for OT receptors, depending on functionalities present in the acyl group. Determination of the preferred conformation of potency-enhancing 1-acylamino-2-propyl substituents using molecular mechanics energy calculations and X-ray crystallography, along with topological similarities to a conformationally constrained cyclic hexapeptide OT antagonist, suggests a receptor-bound conformation for this series of non-peptide OT antagonists.

1-((7,7-Dimethyl-2(S)-(2(S)-amino-4-(methylsulfonyl)butyramido)bicyclo [2.2.1]-heptan-1(S)-yl)methyl)sulfonyl)-4-(2-methylphenyl)piperaz ine (L-368,899): an orally bioavailable, non-peptide oxytocin antagonist with potential utility for managing preterm labor.

Modifications to the previously reported spiroindenylpiperidine camphor-sulfonamide oxytocin (OT) antagonist L-366,509 have produced a new series of o-tolylpiperazine (TP) camphor-sulfonamides. A number of analogues in the TP series that incorporate a modified or unmodified L-methionine sulfone amide at the C2 endo position on the camphor ring exhibit high affinity for OT receptors (IC50 = 1.3-15 nM) and good selectivity for binding to OT versus arginine vasopressin V1a and V2 receptors. Several of these analogues were additionally characterized as potent antagonists of OT-stimulated contractions of the isolated and/or in situ rat uterus. Compound 7 (L-368,899) exhibited the best overall profile of OT receptor affinity (IC50 = 8.9 nM, rat uterus; 26 nM, human uterus), potency for inhibition of OT-stimulated contractions of the isolated rat uterus (pA2 = 8.9) and in situ rat uterus (AD50 = 0.35 mg/kg after intravenous (i.v.) administration and 7.0 mg/kg after intraduodenal administration), aqueous solubility (3.7 mg/mL at pH 5.0), and oral bioavailability in several species (35% (rat), 25% (dog), and 21% (chimpanzee) as estimated from radioreceptor determination of drug levels in plasma after oral and i.v. dosing). On the basis of these favorable properties, 7 has begun clinical testing for use as an oral and i.v. tocolytic agent. Molecular modeling alignment studies have provided support for the hypothesis that the TP camphor-sulfonamide portion of the non-peptide structures may serve as a mimetic of the important D-AA2-Ile3 dipeptide (AA = aromatic amino acid) found in many potent OT antagonists from the cyclic hexapeptide and OT analogue structural classes.

Identification of an orally active, nonpeptidyl oxytocin antagonist.

L-366,509, a member of a novel class of nonpeptidyl compounds, has been characterized as an orally active oxytocin (OT) antagonist. L-366,509 exhibits a moderate binding affinity (K(i) values, 370-780 nM) for the rat, rhesus and human uterine OT receptor. L-366,509 also binds to vasopressin receptor subtypes (arginine vasopressin-V1 and V2) with measurable affinity in rat (K(i) values, 25-30 microM) and primate (K(i) values, 2-6 microM) tissues. In rat uterine slices, L-366,509 inhibits (IC50 = 1.6 microM) the stimulation of phosphatidylinositol turnover induced by OT but not bradykinin. In the rat isolated uterus, L-366,509 is a competitive and reversible OT antagonist (pA2 = 7.32). In vivo, L-366,509 given i.v. (10 mg/kg) or intraduodenally (10-50 mg/kg) to rats causes a marked and long-lasting inhibition of OT-stimulated uterine activity. OT antagonist activity in a pregnant rhesus macaque (approximately day 135 gestation) is also observed with L-366,509 after i.v. or p.o. dosing. L-366,509 represents a prototype for a new chemical class of OT antagonists with significant p.o. bioavailability.

In vitro pharmacology of L-158,809, a new highly potent and selective angiotensin II receptor antagonist.

L-158,809 interacted in a competitive manner with rabbit aortic angiotensin II (AII) receptors as determined by Scatchard analysis of the specific binding of [125I]Sar1Ile8-AII. The affinity of L-158,809 (IC50 = 0.3 nM) for AII receptors in this tissue was appreciably greater than that of other reported nonpeptide AII antagonists such as DuP-753 (IC50 = 54 nM) and EXP3174 (IC50 = 6 nM) and similar to the natural ligand, AII. L-158,809 also exhibited a high potency at AII receptors in several other tissues from different animal species (IC50 = 0.2-0.8 nM). In vitro functional assays utilizing AII-induced aldosterone release in rat adrenal cortical cells demonstrated further that L-158,809 acts as a competitive, high affinity antagonist of AII (pA2 = 10.5) and lacks agonist activity. L-158,809 also potently inhibited AII-induced inositol phosphate accumulation in vascular smooth muscle cells and contractile responses to AII in isolated blood vessels. The specificity of L-158,809 for AII receptors was demonstrated by its lack of activity (IC50 greater than 1 microM) in several other receptor binding assays and its inability to affect in vitro functional responses produced by other agonists. L-158,809 demonstrated a very high selectivity for the AT1 compared to the AT2 receptor subtype (AT2 IC50 greater than or equal to 10 microM). The high affinity and selectivity makes L-158,809 a valuable new tool for investigating the physiological and pharmacological actions of AII.

Radioligand binding studies reveal marked species differences in the vasopressin V1 receptor of rat, rhesus and human tissues.

The [3H]arginine-vasopressin ([3H]AVP) binding site in rat, rhesus and human liver and nonpregnant human uterus was characterized and contrasted. [3H]AVP bound with high affinity (Ki values, 0.2-0.6 nM) to preparations of all tissues studied. Competition binding studies using a series of compounds from three structural classes indicate a marked species difference between the rat and primate liver AVP-V1 site. This site in rhesus and human liver however, is essentially identical, indicating that the rhesus liver is an appropriate surrogate for human tissue. These studies also indicate that the AVP-V1 site of nonpregnant human uterus and human liver is equivalent.

Bradykinin agonist activity of a novel, potent oxytocin antagonist.

From a series of potent cyclic hexapeptide oxytocin (OT) antagonists, a compound that exhibited significant bradykinin (BK) agonist activity was identified. L-366,811 (cyclo[L-proline-D-tryptophan-L-isoleucine-D-pipecolic acid-L-piperazine-2-carboxylic acid-N-Me-D-phenylalanine]) stimulated phosphatidylinositol (PI) turnover in rat uterine slices in vitro (approximately EC50, 2 microM) with a maximal effect (15-fold increase over basal) greater than that obtained for either BK or OT. L-366,811 also elicited dose-related contractions of the isolated rat uterus, producing measurable effects at 100 nM. Several other equally potent OT antagonists from the cyclic hexapeptide structural class were either less potent or inactive as activators of uterine PI turnover or contractility. The stimulatory effects of L-366,811 on uterine PI turnover and contractions were blocked by BK antagonists but not by an arginine vasopressin (AVP)/OT antagonist. In radioligand binding studies, L-366,811 exhibited moderate affinity (IC50, 360 nM) for the [3H]BK binding site in rat uterus, consistent with its potency in the functional models. These results indicate that L-366,811 exhibits BK agonist activity in rat uterus in vitro.

Sarafotoxin S6c: an agonist which distinguishes between endothelin receptor subtypes.

In contrast to endothelin-1 (ET-1) and several of its analogues, sarafotoxin S6c (S6c) was a much more potent inhibitor of [125I]-ET-1 binding in rat hippocampus and cerebellum (Ki approximately 20 pM) than in rat atria and aorta (Ki approximately 4500 nM), suggesting the existence of ET-1 receptor subtypes (aorta/atria, ETA; hippocampus/cerebellum, ETB). S6c was a potent activator of PI turnover in hippocampus (EC50 approximately 10 nM) but not atria (EC50 greater than 1 microM), unlike ET-1 which was active in both tissues. S6c, therefore, is a highly selective ETB agonist. Furthermore, S6c was a potent pressor agent in the pithed rat (ED25 mm Hg approximately 0.1 nmoles/kg, i.v.), suggesting that the ETB receptor subtype may be important in cardiovascular function.

Antagonism of oxytocin in rats and pregnant rhesus monkeys by the novel cyclic hexapeptides, L-366,682 and L-366,948.

Two cyclic hexapeptides unrelated in chemical structure to oxytocin (OT) were shown in vivo to be antagonists of the contractile action of OT on the uterus. In anesthetized rats challenged with OT (1 micrograms/kg) administered as an i.v. bolus, L-366,682 [cyclo-(L-Pro-D-Trp-L-Ile-D-pipecolic acid-L-pipecolic acid-D-His)] and L-366,948 (D-2-naphthyl-alanine in place of D-Trp) were equipotent with AD50 values of about 100 micrograms/kg i.v. At doses of L-366,682 or L-366,948 causing approximately 90 to 95% block (approximately the AD95 dose) of OT, the duration of action of the antagonists exceeded 145 min. Both compounds exhibited selectivity in the rat, as a dose of either at 300 micrograms/kg i.v. shifted the dose-response for OT-induced uterine contraction to the right by approximately 5-fold but did not affect the dose-response to prostaglandin F2 alpha. Furthermore, neither compound, at a dose of 3 mg/kg i.v., antagonized the action of arginine vasopressin acting at V-1 (pressor effect in pithed rats) or V-2 (antidiuretic) receptors. In conscious, freely moving, pregnant rhesus monkeys, L-366,948 or L-366,682 given i.v. or s.c. were effective antagonists of uterine contractions elicited by an infusion of OT. OT- or arginine vasopressin-like agonist activity was not observed in any of the in vivo models. It is concluded that L-366,682 and L-366,948 act in vivo as reasonably potent, long-acting and selective antagonists at OT receptors in the rat and rhesus uterus.

In vitro pharmacological profile of a novel structural class of oxytocin antagonists.

A number of structurally novel cyclic hexapeptides have been characterized as potent and selective oxytocin (OT) antagonists in vitro. As a representative of this class of compounds, L-366,948 [[cyclo(L-prolyl-D-2-naphthylalanyl-L-isoleucyl-D-pipecolyl- L-pipecolyl-D- histidyl)]] exhibited a high binding affinity (Ki, low nanomolar) for OT receptors in rat (uterus and mammary) and primate (pregnant rhesus and human myometrium) tissue with a several hundred-fold binding selectivity vs. rat arginine vasopressin (AVP)-V1 (liver) and AVP-V2 (kidney medulla) receptors. In functional assays, L-366,948 was a pure OT antagonist, blocking both OT-stimulated contraction of the isolated rat uterus (pA2, 8.5) and phosphatidylinositol turnover in uterine slices (IC50, 40 vs. 3 nM OT), with no evidence of partial agonist activity. L-366,948 was comparatively weak as an antagonist of AVP-induced contraction of the isolated rat tail artery (AVP-V1 receptor) and AVP-stimulated adenylate cyclase (AVP-V2 receptor) activity in rat kidney medulla and did not influence prostaglandin F2 alpha- or bradykinin-induced contractions of the isolated rat uterus. L-366,948 and related compounds described in this report represent new experimental tools for the study of the pharmacology and physiology of OT.

A structurally unique, potent, and selective oxytocin antagonist derived from Streptomyces silvensis.

The in vitro and in vivo oxytocin/arginine vasopressin (OT/AVP) antagonist properties of two cyclic hexapeptides derived from a newly discovered natural product (L-156,373) of Streptomyces silvensis are described. In radioligand binding assays, L-156,373 [cyclo(L-Pro-D-Phe-N-OH-L-Ile-D-piperazyl-L-piperazyl-N-Me-D -Phe)] exhibited moderate affinity for rat uterine OT receptors (Ki, 150 nM), with some selectivity (approximately 20-fold) vs. liver AVP-V1 and kidney AVP-V2 receptors. Dehydroxylation of N-hydroxyisoleucine and oxidation of the piperazic acid residues of L-156-373 produced an interesting derivative, L-365,209. These structural modifications increased OT receptor affinity and selectivity by 20- and 2.5-5-fold, respectively. In the isolated rat uterus, L-365,209 was a potent (apparent dissociation constant, 1.7 nM) and competitive OT antagonist. L-365,209 also blocked the effects of AVP at both AVP-V1 (phosphatidylinositol turnover in rat hepatocytes) and AVP-V2 (adenylate cyclase in rat kidney medulla) receptors, but only at low micromolar concentrations. L-365,209, given iv to anesthetized rats, antagonized the action of exogenous OT on the uterus (ID50, 460 micrograms/kg) with a relatively long duration of action. L-365,209 represents a unique class of compounds that provides an entirely new approach for the design of antagonists for these neurohypophyseal hormones.

[3H]L-657,743 (MK-912): a new, high affinity, selective radioligand for brain alpha 2-adrenoceptors.

L-657,743 (MK-912), a highly potent and selective alpha 2-adrenoceptor antagonist was tritiated to a high specific activity and its binding characteristics to brain tissue were determined. The specific binding of [3H]L-657,743 to rat cerebrocortex was saturable, reversible, and dependent on tissue concentration. In saturation studies, [3H]L-657,743 binding was resolved into two high affinity components exhibiting Kd values of 86 pM and 830 pM with densities of 82 fmol/mg protein and 660 fmol/mg protein, respectively. Based on the binding potencies of a variety of compounds with differing receptor selectivities, the sites labeled by [3H]L-657,743 were characteristic of alpha 2-adrenoceptors. In contrast to alpha 2-antagonists, alpha 2-agonists displayed shallow competition curves. In the presence of 100 microM GTP, Gpp(NH)p or 150 mM NaCl, the competition curve for epinephrine was shifted to the right, whereas that for yohimbine was unaffected. In studies utilizing human cerebrocortical tissue, [3H]L-657,743 also bound with high affinity to sites characteristic of alpha 2-adrenoceptors.

A peripherally acting alpha-2 adrenoceptor antagonist: L-659,066.

L-659,066 has been characterized as a potent and selective alpha-2 adrenoceptor antagonist. Both in vitro and in vivo, L-659,066 exhibited specificity (comparable to rauwolscine) for alpha-2 over alpha-1 adrenoceptors. Studies comparing L-659,066 with a previously described antagonist, L-657,743, demonstrate that the new compound penetrates the blood-brain barrier only poorly after systemic administration. With a pA2 of 8.44 at alpha-2 adrenoceptors in the isolated rat vas deferens and an IC50 of 3.0 nM against the binding of [3H]rauwolscine to rat cerebrocortical membranes, L-659,066 possessed, respectively, about one-eighth and one-third of the potency of L-657,743. Similar relative potencies were obtained in vivo in pithed rats with regard to blocking peripherally located postjunctional and prejunctional alpha-2 adrenoceptors (L-659,066 = one-seventh and one-fourth of L-657,743, respectively). In tests carried out in vivo with rats for ascertaining alpha-2 adrenoceptor antagonism in the central nervous system--namely, accumulation of cortical dopa and antagonism of mydriasis induced by the alpha-2 agonist, clonidine--L-659,066 had, respectively, less than 1/345th and about 1/5000th of the potency of L-657,743. In mice, L-659,066 had, respectively, approximately 1/29th and 1/1400th of the potency of L-657,743 as an antagonist in vivo of the predominately peripherally mediated inhibition of colonic propulsion caused by clonidine as compared with the mainly centrally mediated antinocisponsive action elicited by the alpha-2 agonist UK 14,304. The foregoing findings are consistent with poor penetration of the blood-brain barrier by L-659,066.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological profile of a new potent and specific alpha 2-adrenoceptor antagonist, L-657,743.

L-657,743,(2S,12bS)1',3'-dimethylspiro(1,3,4,5',6,6',7,12 b-octahydro-2H- benzo[b]furo[2,3-a]quinolizine)-2,4'-pyrimidin-2'-one, was tested in several in vitro and in vivo models for alpha 2-adrenoceptor antagonism. L-657,743 exhibited a high affinity (less than or equal to 1 nM) for alpha 2-adrenoceptors labelled by [3H] rauwolscine or [3H]clonidine with a 240-fold selectivity versus alpha 1-adrenoceptors labelled by [3H]prazosin. L-657,743 was a potent, selective, and competitive alpha 2-adrenoceptor antagonist in the rat isolated vas deferens (pA2 = 9.3 vs. clonidine; pA2 = 7.1 vs methoxamine). In vivo, L-657,743 potently blocked clonidine-induced mydriasis in the rat and stimulated cerebrocortical norepinephrine synthesis, two indices of central alpha 2-adrenoceptor antagonism. L-657,743 exhibited a comparatively low affinity for several monoamine receptor subtypes (D1, D2, 5-HT1, 5-HT2) in radioligand binding assays in vitro and a comparatively low potency to alter the synthesis of brain DA and 5-HT in vivo indicating a marked alpha 2-specificity versus other monoamine receptor mechanisms. Compared to yohimbine, L-657,743 had considerably higher alpha 2-antagonist potency and alpha 2/alpha 1 selectivity and was significantly more alpha 2-specific (i.e., vs. DA, 5-HT receptors).

In vivo pharmacology of L-364,718, a new potent nonpeptide peripheral cholecystokinin antagonist.

The in vivo pharmacological activity of L-364,718, a new, potent peripheral cholecystokinin (CCK) antagonist, was characterized in several species using assay systems that measure various well known actions of CCK upon the gastrointestinal system. Administered p.o., L-364,178 was highly potent in antagonizing cholecystokinin octapeptide (CCK-8)-induced inhibition of gastric emptying in mice (ED50 = 38 micrograms/kg), rats (ED50 = 140 micrograms/kg) and dogs (ED50 = 91 micrograms/kg) as well as CCK-8-induced reduction in food consumption in rats (ED50 = 321 micrograms/kg). Administered i.v., L-364,718 effectively antagonized the contractile effects of CCK on the colon in rabbits (ED50 = 34 micrograms/kg) and the gallbladder in cats (ED50 = 210 micrograms/kg). Secretion of pancreatic protein and amylase elicited by CCK in cats was also antagonized by L-364,718 (ED50 less than 1.0 mg/kg i.v.). The CCK antagonism produced by L-364,718 in all species persisted for at least 2 to 5 hr. In the absence of exogenously administered CCK-8, L-364,718 per se had no effect in any of the assay systems studied, indicating a lack of CCK-like agonist properties. Specificity for CCK was demonstrated by the inability of L-364,718 (1.0-5.0 mg/kg) to antagonize either amino acid- or atropine-induced inhibition of gastric emptying in rats and dogs, respectively. L-364,718 also did not antagonize motilin-induced gallbladder contractions or secretin-induced pancreatic secretion in cats.(ABSTRACT TRUNCATED AT 250 WORDS)

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.