Regio- and stereoselective cycloadditions of cyclic nitrones to maleic diamide forced in a peptide: synthesis of potent ligands of human NK-2 receptor.

The regioselectivity and the stereoselectivity induced by relatively small peptidomimetic maleic diamide 1 in cycloaddition reactions with cyclic nitrones 2-5 was studied. The high regio- and stereoselectivity observed, sensibly increased by nonpolar solvents, was the effect of a double-asymmetric induction produced by the nitrone substituent on the pseudopeptidic tether. A new class of potent human tachykinin NK-2 receptor ligands was synthesized.

Molecular determinants of peptide and nonpeptide NK-2 receptor antagonists binding sites of the human tachykinin NK-2 receptor by site-directed mutagenesis.

A series of 14 mutants on nine selected residues of the human tachykinin NK(2) receptor was produced and stably transfected into CHO cells to investigate the binding of the peptide MEN 11420 and the nonpeptide SR 48968 antagonists. The main interactions found for MEN 11420 were with Thr171, Tyr206, Tyr266 and Phe270. In the case of SR 48968 crucial residues were Tyr266 and Tyr289. While some overlapping of the binding sites exists, the binding modes suggested by this study appear not to allow structural correlation, and therefore general SAR, between these two antagonists.

Antimuscarinic, calcium channel blocker and tachykinin NK2 receptor antagonist actions of otilonium bromide in the circular muscle of guinea-pig colon.

We have analyzed, by the sucrose gap method, the action of otilonium bromide, a quaternary ammonium derivative in use for the symptomatic therapy of irritable bowel syndrome, on the electrical and mechanical responses initiated by different stimuli in the circular muscle of the guinea-pig proximal colon. Otilonium bromide produced a concentration-dependent inhibition of membrane depolarization (IC50 4.1 microM), action potentials (APs) and contraction (IC50 3.7 microM) produced by the muscarinic receptor agonist, methacholine. It also produced a concentration-dependent inhibition of APs and accompanying contraction (IC50 31 microM) produced by KCl (30 mM), and had a biphasic effect on the cholinergic excitatory junction potential (e.j.p.) produced by single pulse electrical field stimulation: at low concentrations (0.1-0.3 microM) otilonium bromide enhanced the e.j.p. and, at higher concentrations (IC50 22 microM and 16 microM toward depolarization and contraction), produced a concentration-dependent inhibition. Otilonium bromide eliminated the APs superimposed on the depolarization induced by the tachykinin NK1 receptor agonist, [Sar9]substance P-sulphone and suppressed the corresponding contraction (IC50 43 microM) but had little effect on the sustained membrane depolarization induced by this agonist. On the other hand, otilonium bromide produced a similar inhibitory effect on both membrane depolarization and contraction (IC50 38 microM and 45 microM, respectively) induced by the tachykinin NK2 receptor agonist [betaAla8]neurokinin A (4-10). When tested in the presence of nifedipine (1 microM), otilonium bromide had no effect on the membrane depolarization induced by [Sar9]substance P-sulphone but inhibited in a concentration-dependent manner the depolarization induced by [betaAla8]neurokinin A (4-10) (IC50 41 microM). In contrast, the blocker of receptor-operated cation channels, SKF 96365, inhibited with similar potency the depolarization induced by both [Sar9]substance P-sulphone and [betaAla8]neurokinin A (4-10) (IC50 60 microM and 54 microM, respectively). In radioligand binding experiments otilonium bromide produced a concentration-dependent inhibition of the binding of both an agonist ([125I]neurokinin A, Ki 7.2 microM) and an antagonist ([3H]SR 48968, Ki 2.2 microM) to membranes of Chinese hamster ovary cells transfected with the human tachykinin NK2 receptor. In conclusion, the present findings demonstrate that, in the microM range of concentrations, otilonium bromide acts as a muscarinic and tachykinin NK2 receptor antagonist and as a calcium channel blocker. The latter property is likely to account for its ability to suppress contraction initiated by the tachykinin NK1 receptor agonist. Therefore multiple mechanisms of action account for the ability of otilonium bromide to reduce stimulated motility of intestinal smooth muscle.

Relevance of aromatic residues in transmembrane segments V to VII for binding of peptide and nonpeptide antagonists to the human tachykinin NK(2) receptor.

We used membranes from Chinese hamster ovary cells stably transfected with the human tachykinin NK(2) receptor, either wild-type or mutated, at four aromatic residues (His(198), Tyr(266), Phe(270), Tyr(289)) located in transmembrane segments V to VII, to assess the role of these residues in the binding of natural tachykinins and peptide and nonpeptide antagonists. Three radioligands, the agonist [(125)I]neurokinin A (NKA), the peptide antagonist [(3)H]MEN 11420, and the nonpeptide antagonist [(3)H]SR 48968 bound to the wild-type receptor with high affinity (K(d) = 2.4 nM, 0.3 nM, and 4.0 nM, respectively). Four of the six mutant receptors tested retained high affinity for at least one of the radioligands. H(198)A mutation abrogated the binding of NKA but not that of MEN 11420 or SR 48968 (K(d) = 4.8 and 11.5 nM, respectively); Y(266)F mutation abrogated the binding of MEN 11420 but not that of NKA or SR 48968 (K(d) = 2.8 nM and 1.2 nM, respectively); F(270)A mutation abrogated the binding of both NKA and MEN 11420 but not that of SR 48968 (K(d) = 1.6 nM); Y(289)F mutation abrogated the binding of SR 48968 but not that of NKA and MEN 11420 (K(d) = 2.0 and 2.9 nM, respectively). Y(266)A and Y(289)A mutations abrogated the binding of all radioligands. Among the unlabeled antagonists, the affinity of the nonpeptide GR 159897, at variance with SR 48968, resulted heavily compromised by H(198)A and Y(266)F mutations; the peptide antagonists R396 and MEN 10376 essentially followed the binding profile of NKA, but R396 showed markedly increased affinity for the Y(289)F mutant receptor. Taken together, these results indicate that different, partially overlapping sets of sites may be involved in the binding of agonists and diverse antagonists to the human tachykinin NK(2) receptor.

Synthesis of biphenylyltetrazole derivatives of 1-aminopyrroles as angiotensin II antagonists.

Based on preliminary molecular modelling study, the synthesis of two different classes of biphenylyltetrazole derivatives of 1-aminopyrroles, as potentially active non-peptide angiotensin II (AII) antagonists, is reported. Some NH-Boc protected l-aminopyrroles were deprotected, N-acylated, N-alkylated with 5-[4'-bromomethyl-1,1'-biphenyl-2-yl]-1-triphenylmethyl-1H-tetrazo le, and then detritylated to give the first class of title compounds. Other 1-NH-Boc protected 1,2-diaminopyrroles were regioselectively subjected to the 1-alkylation with 5-[4'-bromomethyl-1,1'-biphenyl-2-yl]-1-triphenylmethyl-1H-tetrazo le, to the acylation of the amino group at 2-position of the pyrrole ring, and then to the detritylation process to yield the second class of title compounds.

Defects of tyrosine289phenylalanine mutation on binding and functional properties of the human tachykinin NK2 receptor stably expressed in chinese hamster ovary cells.

A point mutation was made at position 289 in the transmembrane segment 7 of the human tachykinin NK2 receptor to yield a tyrosine/phenylalanine (Tyr/Phe) substitution. Chinese hamster ovary cells stably transfected with the wild-type or Tyr289Phe mutant NK2 receptor both bound neurokinin A (NKA) and the synthetic NK2 receptor-selective agonists, GR 64349 and [betaAla8]NKA(4-10), with high and even affinities. Neurokinin B (NKB) and substance P (SP) also displayed sizeable binding affinities, albeit with lower affinity as compared to NKA. In a functional assay (production of inositol-1,4,5-trisphosphate, IP3), NKA, GR 64349, and [betaAla8]INKA(4-10) stimulated IP3 accumulation via the wild-type and mutant receptors with similar potencies. On the other hand, NKB and SP exhibited a dramatic reduction in their agonist efficacies at the mutant receptor, NKB acting as a partial agonist (maximum effect = 50% of the response to NKA) and SP being totally inactive. The results obtained with phenoxybenzamine inactivation experiments indicated that a large and similar receptor reserve existed for both the wild-type and the mutant receptor. SP, which displayed sizeable binding affinity for the mutant receptor but did not stimulate IP3 accumulation, antagonized the agonist effect of NKA. The antagonist action of SP at the mutant NK2 receptor cannot be ascribed to receptor internalization. The Tyr/Phe replacement at position 289 markedly reduced the binding affinity and antagonist potency of the non-peptide ligand, SR 48968, without affecting the binding affinity and antagonist potency of the bicyclic peptide antagonist MEN 11420. The results indicate that the hydroxyl radical function of Tyr289 in transmembrane segment 7 of the human NK2 receptor is, directly or indirectly, involved in stimulus transduction when the NK2 receptor is occupied by NKB or SP, but not when using NKA or NK2 receptor-selective agonists.

Independent coupling of the human tachykinin NK2 receptor to phospholipases C and A2 in transfected Chinese hamster ovary cells.

The human tachykinin NK2 receptor stably expressed in Chinese hamster ovary cells (CHO-hNK2R cells) was characterized by studying the effect of neurokinin A (NKA), the preferred natural ligand, and that of other agonists and antagonists in both binding experiments and functional assays. Competition experiments using [125I]NKA showed that CHO-hNK2R cells express binding sites which have high affinity for NKA (Ki=3.4+/-0.9 nM), GR 64349 (Ki=12+/-3 nM) and [betaAla8]NKA(4-10) (Ki=21+/-8 nM) and for the antagonists MEN 10627 (Ki=0.55+/-0.2 nM), and MEN 11420 (Ki=2.4+/-0.8 nM). In contrast, the tachykinin NK1 and NK3 receptor agonists [Sar9,Met(O2)11]SP and senktide, respectively, were recognized with low affinity (Ki>10 microM). NKA (EC50=68+/-18 nM) induced a rapid and concentration-dependent increase in the intracellular level of inositoltrisphosphate (IP3). The concentration-response curve to GR 64349 (EC50=155+/-14 nM) was close to that of NKA, whereas [betaAla8]NKA(4-10) (EC50=445+/-78 nM) and SP (EC50=3197+/-669 nM) were 7- and 50-fold less potent, respectively. In addition, NKA stimulated the release of arachidonic acid and the production of prostaglandin E2 (PGE2) in a concentration-dependent manner. Also in this assay, NKA was found to be more potent than the other agonists tested (the EC50 values were 3+/-0.3, 9+/-3, 7.8+/-0.9 and 217+/-37 nM for NKA, GR 64349, [betaAla8]NKA(4-10) and SP, respectively). MEN 10627 and MEN 11420 were potent and competitive antagonists in blocking NKA-induced IP3 formation and PGE2 release: MEN 10627 and MEN 11420 displayed comparable potencies in blocking the two functional responses initiated by occupancy of the NK2 receptor by NKA. Pretreatment of the cells with pertussis toxin (500 ng/ml for 18 h) did not significantly modify the basal or stimulated phosphatidylinositol turnover but reduced the basal and NKA-induced PGE2 release by about 35%. The phospholipase C inhibitor U-73122 (10 microM) prevented the NKA-induced formation of IP3 but did not affect PGE2 release. Conversely, the phospholipase A2 inhibitor quinacrine (100 microM) blocked the release of arachidonic acid and PGE2 without affecting the NKA-stimulated formation of IP3. Chelation of extracellular calcium with 3 mM EGTA inhibited the NKA-induced PGE2 release by 81% but was without effect on basal and NKA-stimulated IP3 production. The calcium channel blockers verapamil (10 microM) and omega-conotoxin GVIA (0.1 microM) did not modify the basal PGE2 production and had no significant effect on the response to tachykinins while the blocker of non-selective cation channels, SKF-96365 (10 microM), inhibited the response to NKA by about 74%. SKF-96365 did not affect the basal or the NKA-induced IP3 formation. In conclusion, our data demonstrate that the human tachykinin NK2 receptor expressed in CHO cells displays binding affinity and functional properties which are those of a native NK2 receptor. No pharmacological evidence for heterogeneity of the human NK2 receptor was obtained in this study. Our findings indicate that the human tachykinin NK2 receptor is independently coupled to both PLC and PLA2 signaling pathways. Activation of the PLA2 pathway may be linked to the opening of a voltage-independent cation channel which activates a Ca2+-dependent PLA2.

Characterization of [3H]MEN 11420, a novel glycosylated peptide antagonist radioligand of the tachykinin NK2 receptor.

[3H]MEN 11420, a radiolabeled glycosylated peptide antagonist of the tachykinin NK2 receptor, has been investigated in ligand-receptor binding assays using membranes of CHO cells transfected with the human tachykinin NK2 receptor. [3H]MEN 11420 bound to a single class of high affinity binding sites: its binding was inhibited by natural tachykinins (potency ranking: NKA >> SP > or = NKB), as well as by peptide (MEN 11420 > MEN 10376 >> R 396) and nonpeptide (SR 48968 > GR 159897) selective NK2 receptor antagonists. These data indicate that [3H]MEN 11420 is a potent radioligand for the human tachykinin NK2 receptor that may represent a useful tool for studying ligand-receptor interactions at the molecular level.

Antihistaminic and antiallergic properties of dextro-mequitamium iodide in upper and lower guinea pig airways: comparison with azelastine.

1. The ability of dextro-mequitamium iodide (d-Meq) to antagonize bronchomotor and inflammatory effects mediated by histamine and antigen challenge in the upper or lower guinea pig airways or both and its potential activity against the recruitment and activation of eosinophils in the bronchial wall have been evaluated in comparison with azelastine. 2. In receptor-binding studies, d-Meq displayed a nanomolar affinity for H1 and muscarinic receptors, and it was endowed with potent bronchodilating properties in the nanomolar range toward tonic contractions induced by histamine and carbachol. 3. d-Meq (100-1,000 nmol/guinea pig) and azelastine (100-5,000 nmol/guinea pig) administered by aerosol significantly inhibited histamine- and antigen-induced increases in insufflation pressure in sensitized animals. 4. d-Meq (1,000-6,000 nmol/kg i.v.) dose dependently inhibited the histamine- or antigen-induced increase in vascular permeability in the upper airways. 5. d-Meq was more effective against histamine than antigen challenge, and its potency was similar or greater than that of azelastine. 6. Aerosolized d-Meq (1,000 nmol/animal) reduced antigen-induced eosinophil accumulation in the bronchoalveolar lavage (BAL) fluid from sensitized guinea pigs. 7. Eosinophils recovered from the BAL fluid of antigen-challenged animals showed an increased chemotaxis in response to LTB4 or platelet-activating factor. Both d-Meq and azelastine (300 nmol/animal) reduced this increase without affecting direct chemotaxis induced by leukotriene B4 (LTB4). 8. These findings provide evidence that local administration of d-Meq might be useful in the treatment of allergic disorders, such as rhinitis and asthma.

MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist.

1. The pharmacological profile was studied of MEN 11420, or cyclo[[Asn(beta-D-GlcNAc)-Asp-Trp-Phe-Dap-Leu]cyclo(2beta-5beta )], a glycosylated derivative of the potent, selective, conformationally-constrained tachykinin NK2 receptor antagonist MEN 10627 (cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2beta-5beta)). 2. MEN 11420 competitively bound with high affinity to the human NK2 receptor stably transfected in CHO cells, displacing radiolabelled [125I]-neurokinin A and [3H]-SR 48968 with Ki values of 2.5+/-0.7 nM (n = 6) and 2.6+/-0.4 nM (n = 3), respectively. 3. MEN 11420 showed negligible binding affinity (pIC50 < 6) at 50 different receptors (including tachykinin NK1 and NK3 receptors) and ion channels. 4. In the rabbit isolated pulmonary artery and rat urinary bladder MEN 11420 potently and competitively antagonized tachykinin NK2 receptor-mediated contractions (pK(B) = 8.6+/-0.07, n = 10, and 9.0+/-0.04, n = 12; Schild plot slope = -1.06 (95% c.l. = -1.3; -0.8) and -1.17 (95% c.l. = -1.3; -1.0), respectively). MEN 11420 produced an insurmountable antagonism at NK2 receptors in the hamster trachea and mouse urinary bladder. However, in both preparations, the effect of MEN 11420 was reverted by washout and an apparent pK(B) of 10.2+/-0.14, n = 9, and 9.8+/-0.15, n = 9, was calculated in the hamster trachea and mouse urinary bladder, respectively. 5. MEN 11420 showed low affinity (pK(B) < 6) at guinea-pig and rat tachykinin NK1 (guinea-pig ileum and rat urinary bladder) and NK3 (guinea-pig ileum and rat portal vein) receptors. On the whole, the affinities (potency and selectivity) showed by MEN 11420 for different tachykinin receptors, measured either in binding or in functional bioassays, were similar to those shown by the parent compound, MEN 10627. 6. The in vivo antagonism of the contractions produced by [betaAla8]neurokinin A(4-10) (1 nmol kg(-1)) was observed after intravenous (dose range: 1-10 nmol kg(-1)), intranasal (3-10 nmol kg(-1)), intrarectal (30-100 nmol kg(-1)) and intraduodenal (100-300 nmol kg(-1)) administration of MEN 11420. MEN 11420 was more potent (about 10 fold) and longer lasting than its parent compound MEN 10627, possibly due to a greater metabolic stability. 7. A dose of MEN 11420 (100 nmol kg(-1), i.v.), that produced potent and long lasting inhibition of the contraction of the rat urinary bladder induced by challenge with the NK2 selective receptor agonist [betaAla8]neurokinin A(4-10) (10-300 nmol kg(-1)), was without effect on the responses produced by the NK1 receptor selective agonist [Sar9]substance P sulphone (1-10 nmol kg(-1)). 8. These findings indicate that MEN 11420 is a potent and selective tachykinin NK2 receptor antagonist. The introduction of a sugar moiety did not produce major changes in the affinity profile of this antagonist as compared to MEN 10627, but markedly improved its in vivo potency and duration of action. With these characteristics, MEN 11420 is a suitable candidate for studying the pathophysiological significance of tachykinin NK2 receptors in humans.

Tachykinin NK2 receptors: characterization in the rat small intestine by the use of a peptide agonist and a nonpeptide antagonist as radioligands.

The tachykinin NK2 receptors present in membranes of rat small intestine were characterized by means of tachykinin receptor agonists and antagonists, by using the natural agonist, NKA, or the nonpeptide antagonist SR 48968, as radioligands. The affinity of the antagonists was independent of the radioligand used, whereas the NK2 receptor selective agonists showed a different binding profile according to the radioligand. In particular, when using [125I]NKA, NKA and NKA(4-10) bound to a high affinity site, whilst [beta-Ala8]NKA(4-10) and GR 64349 bound to a high and a low affinity site; the high affinity site was still detected in the presence of 100 microM GppNHp which produced a strong inhibition of the specific binding of [125I]NKA. On the other hand, when using [3H]SR 48968, NKA bound to a high affinity site, [beta-Ala8]NKA(4-10) bound to a low affinity site and NKA(4-10) and GR 64349 bound to a high and a low affinity site; in this case, the high affinity site was no longer detected in the presence of 1 microM GppNHp, which did not reduce the specific binding of [3H]SR 48968 to NK2 receptors. We interpreted these data as an indication that in the rat small intestine membranes [125I]NKA labels multiple conformations of G protein-coupled NK2 receptor which are distinguished by the use of selective receptor agonists, but not by peptide or nonpeptide antagonists.

Tachykinin receptors and intestinal motility.

Substance P (SP) and neurokinin A (NKA) are synthesized by enteric cholinergic motorneurons that project to the longitudinal and circular muscle of the mammalian intestine. Thus, acetylcholine, SP, and NKA are the excitatory neuromuscular transmitters in the intestine. Tachykinin NK1 and NK2 receptors are expressed by smooth muscle cells in most regions of the intestine: the corelease of SP and NKA from nerves thus realizes paradigms of tachykininergic cotransmission. Examples have been found in which a cooperative model can be applied to account for the action of SP-NKA acting at NK1 and NK2 receptors (e.g., circular muscle of guinea-pig duodenum), as well as examples in which the message produced by activation of the two receptors diverges sharply in producing responses that have a markedly different time course and use different effector systems (e.g., circular muscle of guinea-pig colon). NK3 receptors are expressed on both excitatory and inhibitory motor neurons: indirect contractions (via release of acetylcholine and tachykinins) and relaxations (via release of nitric oxide) can be evoked in the gut by selective stimulation of NK3 receptors. Although a role of NK3 receptors in certain enteric reflexes has been evidenced, the importance of this system in mediating hexamethonium-resistant enteric transmission appears less important than previously speculated.

Human umbilical vein smooth muscle cells as a model to study thrombin generation and function: effect of thrombin inhibitors.

The aim of the present work was to study how human umbilical vein smooth muscle cells (HUVSMC) can initiate the coagulation process and to investigate the responses of these cells to thrombin. Exposure of HUVSMC to recalcified human plasma led to a time-dependent production of thrombin, measured both as amidolytic activity and as release of fibrinopeptide A. Thrombin activity was dose-dependently reduced by an anti-human tissue factor antibody (76 +/- 3% at 10 micrograms/ml) and by inhibitors like heparin, rec-hirudin, hirulog 1, Napap and hirunorm, a novel hirudin-like thrombin inhibitor (IC50 = 2 +/- 0.4, 8 +/- 1, 130 +/- 22, 199 +/- 29 and 68 +/- 8 nM, respectively). The release of fibrinopeptide A was similarly prevented (IC50 = 14 +/- 1, 132 +/- 25 and 50 +/- 8 nM for rec-hirudin, Napap and hirunorm, respectively). Exogenously added thrombin increased thymidine incorporation into HUVSMC to 240 +/- 30% of basal (EC50 = 0.49 +/- 0.09 nM) and thrombin inhibitors blocked this effect (IC50 = 10 +/- 3, 37 +/- 17, 343 +/- 165 and 1402 +/- 758 nM for rec-hirudin, hirunorm, Napap and hirulog-1, respectively). Also recalcified human plasma was mitogenic for HUVSMC and its effect was mainly due to endogenously generated thrombin, as shown by the use of thrombin inhibitors. In conclusion, HUVSMC are capable of initiating the extrinsic coagulation cascade, leading to the formation of thrombin which promotes clotting and stimulates DNA synthesis. Thrombin inhibitors prevent both coagulative and cellular effects of thrombin.

Down-regulation of substance P receptors during colitis induced by trinitrobenzene sulfonic acid in rats.

Neurochemical and functional studies were performed to investigate the role of substance P (SP) during trinitrobenzene sulfonic acid (TNB)-induced colitis. Time course studies showed that tissutal SP-like immunoreactivity levels decreased in acute or chronic phases of the experimental colitis. The affinity of SP was not significantly reduced up to 1 week after TNB-induced colitis but a decreased density of SP binding sites was observed at all times. The subcutaneous administration of neurokinin (NK)1 receptor antagonist RP 67580 (0.1-1 mumol/kg daily x 1 week) did not affect the injury induced by the hapten. These findings suggest that changes in SP seem to be the effect rather than the cause of colitis and differ from those observed in human inflammatory bowel diseases.

A review of the design, synthesis and biological activity of the bicyclic hexapeptide tachykinin NK2 antagonist MEN 10627.

We review the reported data on the design, the conformational features and the pharmacological properties of the bicyclic peptide tachykinin NK2 receptor antagonist MEN 10,627 or cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2 beta-5 beta). MEN 10,627 possesses a highly constrained structure characterized by two consecutive beta-turns, as confirmed by the almost coincident results of NMR and X-ray analyses. The compound has been efficiently synthesized by solid-phase methodology using either Boc or Fmoc strategies. It is quite stable to metabolic degradation and is endowed with high affinity and selectivity for NK2 receptor expressed in various species. At the hamster NK2 receptor MEN 10,627 is about 30-fold more potent than the nonpeptide NK2 receptor antagonist, SR 48,968, while the converse is true for the rabbit NK2 receptor. MEN 10,627 and SR 48,968 show comparable affinities for the human NK2 receptor. MEN 10,627 produces a long lasting inhibition of the response to the selective NK2 receptor agonist [beta Ala8]NKA(4-10) in the rat urinary bladder in vivo after intravenous, intranasal and intraduodenal administration. Therefore different administration routes are possible for this compound that overcomes the usual drawbacks for the application of peptides as drugs.

Role of calcium in angiotensin II-induced prostaglandin release and DNA synthesis in rat vascular smooth muscle cells.

Cellular calcium modulates enzyme activity, cell proliferation, and differentiation. In vascular smooth muscle cells (VSMC), calcium may contribute to increased vascular contractility and structural alterations in both hypertension and atherosclerosis. We investigated the role of calcium in angiotensin II (AII)-induced prostaglandin release and DNA synthesis in VSMC. Prostaglandin levels were determined by radioimmunoassay, and DNA synthesis was determined by the incorporation of [3H]thymidine. AII dose-dependently stimulated the release of prostaglandin E2 and prostaglandin I2, and this effect was synergistically enhanced by the Ca2+ ionophore A23187. Conversely, the AII response was inhibited by EGTA, a chelator of Ca2+ ions and by verapamil and nifedipine, two Ca2+ channel blockers or by incubation of the cells without exogenous Ca2+. TMB-8, an inhibitor of calcium mobilization, also strongly reduced angiotensin response. Similar results were obtained for angiotensin III (AIII) and vasopressin, two other agonists of prostaglandin production. AII- or serum-stimulated DNA synthesis was almost abolished by EGTA, whereas TMB-8, verapamil, and nifedipine had little or no effect. The production of prostaglandins triggered by angiotensins and vasopressin in VSMC is dependent on both intracellular and extracellular calcium, with calcium entering through L-type Ca2+ channels. Extracellular calcium is important for AII and serum mitogenic activity, but L-type Ca2+ channels do not appear to be implicated.

N-3-substituted pyrimidinones as potent, orally active, AT1 selective angiotensin II receptor antagonists.

A novel series of nonpeptide angiotensin II (A II) antagonists containing a pyrimidinone ring which carries a C-linked biphenyltetrazole moiety and a carboxyheteroaryl group on the 3-position have been prepared. Their affinity for the AT1 receptor was determined in a binding assay on rat adrenal cortical membranes. The in vivo antihypertensive properties were tested by evaluating the inhibition of the pressor response to A II followed by iv and id administration. Extensive molecular modeling studies, including comparison of molecular electrostatic potential distributions, conformational analysis, and overlays on a computational pharmacophore model of A II, were used to evaluate structural parameters of the new compounds, in comparison to other known A II antagonists (e.g., DUP-753 and SK&F 108566). According to the modeling studies, the introduction of a (carboxyheteroaryl)methyl moiety at the 3-position of the pyrimidinone ring led to derivatives with increased potency. Methyl 2-[[4-butyl-2-methyl-6-oxo-5-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl ]- 4-yl]methyl]-1-(6H)-pyrimidinyl]methyl]-3-thiophenecarboxylate (3k, LR-B/081), one of the most potent compounds in the series (Ki = 1.4 nM), exhibited a marked antihypertensive activity on oral administration to conscious renal hypertensive rats, with long duration of action. It was selected for clinical evaluation in the treatment of hypertension in man.

4-Diazinyl- and 4-pyridinylimidazoles: potent angiotensin II antagonists. A study of their activity and computational characterization.

A series of N-[biphenylyl(tetrazolyl)methyl]-2-butylimidazoles containing variously substituted diazine or pyridine moieties either as their free bases or N-oxide derivatives attached to the 4-position of the imidazole ring was synthesized and tested for interaction with the AT1 receptors of rat adrenal cortex membranes (receptor binding assay). Some compounds were then chosen for further evaluation in vivo in the A II-induced pressor response in conscious normotensive rats. The most potent in the AT1 binding assay were found to be compounds in which the diazine or pyridine ring nitrogen is adjacent to the point of attachment between the two heteroaromatic rings such as 2-butyl-4-(3,6-dimethylpyrazin-2-yl)-1-[[2'-(1H-tetrazol-5-y l)-biphenyl-4- yl]methyl]-1H-imidazole (3b) or 2-butyl-4-[5-(methoxycarbonyl)pyrid-2-yl]-1-[[2'-(1H-tetrazol++ +-5- yl)biphenyl-4-yl]methyl]-1H-imidazole (6c). The binding affinities and oral activities of the pyridine N-oxide imidazoles in which a stabilizing group ortho to the pyridine ring nitrogen is present were markedly improved as in 2-butyl-4-[(3-methoxycarbonyl)-6-methyl-N-oxopyridin-2-yl]-1-[[2'- (1H- tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-imidazole 31b. Molecular modeling studies were carried out to determine the molecular electrostatic potential values of related model systems and to correlate their receptor interaction energies with the observed activities of our compounds.

Stereoselective inhibition of muscarinic receptor subtypes by the eight stereoisomers related to rociverine.

The chemical structure corresponding to 1-hydroxy[1,1'-bicyclohexyl]-2-carboxylic acid 2-(diethylamino)-1-methylethyl ester has the classical profile of ester-type antimuscarinic drugs. The presence of three chiral carbons leads to eight stereoisomers and the substitutions on the cyclohexyl ring generate cis-isomers (1, named rociverine) and trans-isomers (2). The aim of this study was to determine the binding pattern of the eight stereoisomers and two derived compounds, (1S,2S)-1-hydroxy[1,1'-bicyclohexyl]-2-carboxylic acid 2-(dimethylamino)-1-ethyl ester (3) (1S,2S)-1-hydroxy[1,1'-bicyclohexyl]-2-carboxylic acid (S)-2-(diethylamino)-1-methylethyl ester methyl iodide (4), at the five cloned muscarinic receptors stably expressed in chinese hamster ovary cells, in order to define how stereochemical modifications could affect the affinity. Our data showed that cis-stereoisomers exhibited higher variations in affinity than trans-stereoisomers. Among the cis-stereoisomers, those with the (1R,2R) configuration showed considerably higher affinities (up to 240-fold) than those with the (1S,2S) configuration. The (1S,2S) configuration was important for binding selectivity; this was confirmed also by the use of the two additional compounds.