Phenylimidazolidin-2-one derivatives as selective 5-HT3 receptor antagonists and refinement of the pharmacophore model for 5-HT3 receptor binding.

A possible bioisosterism between the benzamido and the phenylimidazolidin-2-one moieties has been suggested on the basis of the similarity between the molecular electrostatic potential (MEP) of metoclopramide, a D2 receptor antagonist with weak 5-HT3 receptor antagonist properties, and zetidoline, a D2 receptor antagonist. Starting from this premise, a series of phenylimidazolidin-2-one derivatives bearing a basic azabicycloalkyl or an imidazolylalkyl moiety were synthesized and evaluated for 5-HT3 receptor radioligand binding affinity ([3H]-GR 43,694). In vitro 5-HT3 receptor antagonist activity was tested in the guinea pig ileum assay (GPI). A number of high-affinity ligands were shown to be potent 5-HT3 receptor antagonists in vivo as determined by inhibition of the Bezold--Jarisch reflex in the anesthetized rat. In general, the imidazolylalkyl derivatives were found to be more active than azabicycloalkyls. 1-(3,5-Dichlorophenyl)-3-[(5-methyl-1H-imidazol-4-yl)methyl]imidazoli din-2-one (58), in particular, displayed very high affinity for the 5-HT3 receptor (Ki of 0.038 nM) with a Kb of 5.62 nM in the GPI assay, being more potent than the reference compounds (ondansetron, tropisetron, granisetron, and BRL 46,470) tested. 58 showed an ID50 comparable to that of ondansetron (2.2 micrograms/kg i.v.) in the Bezold--Jarisch reflex. A molecular modeling study based on this structurally novel series of compounds allowed the refinement of previously reported 5-HT3 receptor antagonist pharmacophore models.

D-amino acid scan of endothelin: importance of amino acids adjacent to cysteinyl residues in isomeric selectivity.

A systematic approach to map the functionally important determinants of endothelin-1 (ET-1) by a D-amino acid scan is described. Correct orientation of the amino acid side chains was generally of paramount importance both for binding at the ETA receptor and for contracting activity. This was particularly valid for positions 2, 8, 14, 16-21 (the four Cys residues were kept unaltered). Nevertheless, increment of binding affinity was observed by inversion of configuration at positions 6, 7, 9 and 10. In addition, [D-Lys9]ET was an agonist about four times more potent than the natural compound. Usually both 1,4- and 1,3-isomers (corresponding, respectively, to the correct and misfolded disulfide bridges of ET) were obtained, and usually the isomer formed in larger amount had the higher HPLC retention time and the higher biological activity. However, four out of seventeen single-point D-amino acid analogues could be isolated only in one isomeric form. In three cases (D-Ser2, D-Ser4, D-Val12), the inverted amino acid was adjacent to a Cys residue, and in one case (D-Lys9) it was one amino acid apart, thus suggesting a possible effect of the bridged cysteinyl residues in isomeric selectivity.

Reversal of multidrug resistance by new dihydropyridines with low calcium antagonist activity.

The clinical use of Ca++ antagonist agents as modulators of multidrug resistance is limited by their strong vasodilator activity. This study reports data obtained by testing a series of new 1,4 dihydropyridine derivatives (DHPs) for their in vitro resistance modulating activity and their Ca++ antagonist effect. All the tested DHPs are active to increase doxorubicin activity with dose modifying factor values ranging between 2 and 47 on P388/DX cells and 12 and 36 on LoVo/DX cells. Their resistance modulating action is exerted through an increase of DX intracellular level. The Ca++ antagonist activity of DHPs, evaluated as capacity to inhibit the KCl-induced contractions in isolated Guinea pig ileum strips, is not related to their resistance modulating activity. This finding makes it possible to select, for further in vivo evaluations, compounds IX, X and XI, which have strong ability to overcome multidrug resistance and low Ca++ antagonist effect.

Alanine scan of endothelin: importance of aromatic residues.

A systematic approach to map the functional important determinants of endothelin-1 (ET) by an alanine scan is described. Studies on the in vitro receptor binding affinity and on the agonist contracting activity defined that residues Asp8, Tyr13, Phe14, Leu17, and Trp21 were of major biological significance. A striking observation was that four out of these five sites were hydrophobic amino acids. Ala analogues of the aromatic residues at position 13, 14, and 21 displayed sharply reduced receptor binding affinity (< 2% of ET) and can be considered important for receptor contact. Ala analogues of Asp8 and Leu17 lost most (> 90%) of the agonist activity but retained a receptor affinity nearly equivalent to ET and can be considered to be important for signal transduction. Three other positions, Val12, Asp18, and Ile20 (which are adjacent to the biologically important sites of Tyr13, Leu17, and Trp21), resulted as partially tolerant to Ala substitution, retaining 14-50% of the potency of ET. Ala analogues of the Et isomeric disulfide arrangement (Cys1,11 and Cys3,15) were always less active than the corresponding analogues with the native disulfide pairings (Cys1,15 and Cys3,11).

Imidazol-1-yl and pyridin-3-yl derivatives of 4-phenyl-1,4-dihydropyridines combining Ca2+ antagonism and thromboxane A2 synthase inhibition.

A series of derivatives of 4-phenyl-1,4-dihydropyridine bearing imidazol-1-yl or pyridin-3-yl moieties on the phenyl ring were synthesized with the aim of combining Ca2+ antagonism and thromboxane A2 (TxA2) synthase inhibition in the same molecule. Some of these compounds showed significant combined Ca2+ antagonism and TxA2 synthase inhibition in vitro, while others showed only one single activity. Structural requirements for significant single or combined activities are discussed. Theoretical conformational analysis, by molecular mechanics and semiempirical AM1 calculations, was performed for 1,4-dihydro-2,6-dimethyl-4-[3-(1H-imidazol-1-yl)phenyl]- 3,5-pyridinedicarboxylic acid, diethyl ester (FCE 24265) and two close congeners. FCE 24265, which inhibited TxB2 production in rat whole blood with IC50 = 1.7 x 10(-7) M and antagonized K+ induced contraction in guinea pig aorta with IC50 = 6.0 x 10(-8) M, was selected for further pharmacological evaluation. Our results show that this compound is less potent than nifedipine both in vitro and in vivo yet presents a favorable profile in vivo, lowering blood pressure without inducing reflex tachycardia. Moreover, its additional potent and selective TxA2 synthase inhibitory activity makes this compound an interesting pharmacologic tool in pathologies where both enhanced TxA2 synthesis and cellular Ca2+ overload are involved.

Pharmacological characterization of FCE 27262, a combined thromboxane synthase inhibitor and PGH2/TXA2 receptor antagonist.

The literature supports the hypothesis that the association of a thromboxane (TX)A2 synthase inhibitor and a PGH2/TXA2 receptor antagonist has a superior antithrombotic effect when compared to both aspirin and single agent alone; a compound endowed with the dual mechanism of action might therefore be of therapeutic value for the management of thrombotic disorders. FCE 27262, an imidazol-1-yl-ethylideneaminooxypentanoic acid, displaces in vitro the binding of [3H]SQ 29,548 to washed human platelets (IC50 = 6.0 +/- 0.6 x 10(-8) M) and antagonizes human platelet aggregation induced by U 46619 in PRP with an IC50 (95% confidence limits) of 4.5(3.3-5.1) x 10(-7) M. It also selectively antagonizes the isolated vessel contraction induced by U 46619. In the rat aorta the Kb (95% confidence limits) was 1.6(0.6-4.3) x 10(-7) M. Additionally it inhibits in vitro TXB2 production in rat and human whole blood, the IC50 being, respectively, 5.9(3.3-9.6) x 10(-8) M and 3.8(2.9-5.0) x 10(-8) M. When administered orally to fed rats it also inhibits ex vivo TXB2 production in whole blood during clotting, the ID50 being 0.62(0.4-0.8) mg/kg. Both in vitro and ex vivo the effect of FCE 27262 on TXA2 synthase was selective, the production of PGE2, the product of a different isomerase from the common precursors, PG-endoperoxides, being concomitantly enhanced. In a canine model of electrically-induced coronary thrombosis, FCE 27262 (1 mg/kg i.v.) inhibits ex vivo TXB2 synthesis (> 95%), antagonizes U 46619-induced platelet aggregation and prolongs occlusion time (controls: 72 +/- 8 min, FCE 27262: 215 +/- 38 min; p < 0.01). In the same model both aspirin (5 mg/kg i.v.) and a pure PGH2/TXA2 receptor antagonist (L 670596), at a dose giving a similar degree of TXA2 synthase inhibition and receptor blockade, respectively, are significantly less effective. Thus, FCE 27262 combines thromboxane synthase inhibition and PGH2/TXA2 receptor antagonism in one molecule, resulting in enhanced antithrombotic activity. FCE 27262 thus may be an appropriate pharmacological tool to test the therapeutic potential of the dual mechanism of action.

Human preproendothelin-1 is converted into active endothelin-1 by baculovirus-infected insect cells.

To investigate biochemical and biological parameters involved in preproendothelin-1 (preproET-1) maturation we infected Spodoptera frugiperda (Sf21) cells with a suitable engineered baculovirus vector carrying the cDNA encoding the entire human 212 amino acids precursor. Culture supernatants were tested by RIA using an anti-ET-1 serum, ET-1-like immunoreactive material (IRM) was detected in the infected Sf21 cells but not in control, wild-type or mock-infected cells. Fractionation of the culture supernatant by RP-HPLC coupled to an ET-1 specific RIA yielded two main peaks corresponding to the retention times of human bigET-1 and ET-1. Furthermore, culture supernatant of preproET-1 expressing Sf21 cells elicited a characteristic dose-response vasoconstrictive activity on rabbit vena cava, consistent with the amount of ET-1 as estimated by RP-HPLC coupled to RIA. These results suggest that insect cells possess the enzymatic activities necessary for human preproET-1 full maturation even though no such peptide has ever been found in insect cells.

Thrombomodulin is a cofactor for thrombin degradation of recombinant single-chain urokinase plasminogen activator "in vitro" and in a perfused rabbit heart model.

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) to a two-chain derivative (tcu-PA) fibrinolytically inactive. This reaction was accelerated in vitro by purified rabbit lung thrombomodulin in equimolar complex with thrombin. Polyclonal antibodies to rabbit thrombomodulin prevented this effect. We also observed that heparin and other sulfated polysaccharides had an accelerating effect on thrombin cleavage of recombinant scu-PA. Their effect was concentration-dependent and then reversed at high levels. The effect of heparin and heparan sulfate was independent and synergic with respect to thrombomodulin. All observations except the effect of heparin, could be confirmed in a Langendorff isolated rabbit heart model. From competition experiments carried out with scu-PA derivatives and mutants, we postulate that the amino-terminal sequence of rscu-PA, containing the epidermal growth factor (EGF)-like and the kringle domains is involved in the cofactor effect of thrombomodulin on scu-PA inactivation by thrombin. We conclude that a regulatory mechanism of scu-PA inactivation is present at the cell surface.

Heterologous in vivo processing of human preproendothelin 1 into bioactive peptides.

Endothelin (ET) is an extremely potent vasoconstrictor peptide of 21 amino acids, originally found in the supernatant of cultured vascular endothelial cells. To gain insights into its biosynthetic pathway, we expressed a synthetic RNA coding for the 212-amino acid precursor of human ET-1 (preproET-1) in Xenopus oocytes. Cell homogenates and oocyte incubation medium were tested by RIA using an anti-ET-1 serum. ET-1-like immunoreactivity was detected in oocytes injected with preproET-1 synthetic RNA but not in control oocytes and was much higher in medium than in cell homogenates. When preproET-1 was expressed in oocytes treated with monensin, a dramatic decrease in secretion of immunoreactive material was observed, indicating that secretion is mediated by the Golgi complex. ET-1-like immunoreactive material present in oocyte incubation medium was fractionated by reverse-phase HPLC into two main peaks, corresponding to the retention times of human big ET-1 and ET-1. Incubation medium of oocytes expressing the synthetic preproET-1 RNA elicited a characteristic vasoconstrictor response on rabbit vena cava, consistent with the biological activity that would be predicted from the amount of ET-1-like immunoreactivity measured. These results suggest that common pathways of ET maturation exist in widely different cells and that Xenopus oocytes may represent a useful tool in studying the cell biology of ET-1 synthesis.

New 2-substituted 2,3,3a,4-tetrahydro-1H-imidazo[5,1-c] [1,4]benzoxazin-1-ones as alpha 2-adrenoceptor antagonists.

The synthesis of new 2-imidazol(in)yl-alkyl derivatives of 2,3,3a,4-tetrahydro-1H-imidazo[5,1-c][1,4]benzoxazin-1-one is reported. Some compounds of the series have shown high affinity for alpha 2 receptors, high alpha 2/alpha 1 selectivity and alpha 2 antagonism in vitro (vas deferens). Owing to their selective alpha 2-antagonism associated to a novel structure, compounds 8 and 20 have been selected for further biological investigation as potential antidepressants.

Mechanism of the antihypertensive effect of FCE 22716, a new ergoline derivative, in the spontaneously hypertensive rat.

Both acute and chronic oral administration (1-20 mg/kg) of FCE 22716, a new ergoline derivative, resulted in a dose-related fall of arterial blood pressure lasting for more than 6 h. Tachycardia was observed only at high dosages. Yohimbine, propranolol and indometacin did not modify its antihypertensive effect; on the other hand pretreatment with prazosin, a selective alpha 1-adrenoceptor antagonist and pithing, almost completely neutralized its antihypertensive effect. Haloperidol, a dopamine antagonist that crosses the blood-brain barrier, also antagonized FCE 22716 activity. The lack of effects of domperidone (DA2-receptor antagonist selectively acting on the periphery) together with the finding that norepinephrine and epinephrine levels were unchanged after treatment with FCE 22716, seem to rule out an involvement of peripheral DA2-receptors. Both in vitro (isolated organs) and in vivo the compound antagonized responses mediated by stimulation of alpha 1-adrenoceptors and S2-receptors. Radioligand binding studies in different cerebral regions are in line with the above reported in vitro and in vivo results. These data suggest that FCE 22716 is endowed with a multitarget mechanism of action, mainly involving blockade of alpha 1-adrenoceptors and S2-receptors.

Byciclic compounds with potential antiulcer and/or antisecretory activity. III--2-substituted tetrahydrothiazolo-[5,4-c]pyridines.

As the ultimate result of a long-term search for new bicyclic molecules potentially endowed with antiulcer and/or antisecretory activity, simple urea derivatives of 2-guanidino-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine [(VIII), Fig. 1; X = 0, R = alkyl], displaying a strong inhibition of stress- and ASA-induced gastric ulcers and of basal gastric secretion, were found. Their potency does compare very favourably with that of cimetidine and ranitidine and approaches that of famotidine. On spontaneously beating guinea pig atria they behaved as inhibitors of the histamine H2-receptor. In contrast with cimetidine and ranitidine but like other recently described inhibitors (famotidine included), the most active compounds (VIII) caused a surmountable antagonism only at low concentrations and an unsurmountable antagonism at higher concentrations. The onset of action was slow, while the inhibitory effect was hardly reversed by washing. The rational development of the research and the synthetic approach, as well as a quantitative assessment of both in vitro and in vivo potencies in comparison with the best known, clinically used drugs, are shown in detail.