Preparation and biological effects of pure stereoisomeric novel soft anticholinergics.

A series of pure stereoisomeric soft glycopyrrolate analogues 3, 4 and 5 was synthesized using chiral intermediates and by careful separation of the stereoisomers formed during the last quaternization step of the synthesis. The stereochemistry of the products was elucidated using various 1D and 2D NMR techniques. Anticholinergic activity of the new compounds was determined by receptor binding studies and performing tests on isolated organs and by in vivo tests. Receptor binding revealed that in the higher alkyl ester series the (2R, 1'R, 3'R) and the (2R, 1'S, 3'S) isomers were the compounds showing the highest receptor affinity furthermore it demonstrated the confines of the length of the alkyl chain. In vitro isolated organ experiments correlated well with the receptor binding results, and in vivo investigations indicated the soft character of the compounds.

Binding characteristics of GYKI-46 903, a non-competitive ligand at 5-HT3 receptors.

GYKI-46903 [(+)-(5S,6R)-4-(4-fluorophenyl)-6-propionyloxy-1-aza-bicyclo[3.3.1]-non-3-ene-hydrochloride], a cognition enhancer identified as a non-competitive antagonist of 5-HT3receptors in isolated guinea-pig ileum, was investigated for allosteric action at 5-HT3 receptors in rat cortical membranes by using [3H]granisetron. Equilibrium and kinetic protocols were applied and the competitive antagonist granisetron was included as a negative control. In competition studies, both granisetron and GYKI-46 903 displaced the radioligand with K(i) values of 0.20 +/- 0.02 and 79.84 +/- 0.28 nM, respectively. The inhibition curve for GYKI-46 903 resulted in a Hill slope significantly greater than unity ( 1.37 +/- 0.11), whereas the slope for granisetron was 0.88 +/- 0.08, not different from unity. These results indicate non-competitive and competitive interactions, respectively. Scatchard analysis yielded a linear plot, suggesting a single population of binding sites with a Kd of 0.13 +/- 0.01 nM and a Bmax) of 13.15 +/- 0.34 fmol per mg of protein. Scatchard plots obtained in the absence and presence of granisetron (0.1-3 nM) or GYKI-46 903 (30-1000 nM) revealed a concentration-dependent increase in Kd values by either of these compounds. Granisetron left the Bmax unchanged, but there was a significant increase in the Bmax by GYKI-46 903, which could point to an atypical allosteric interaction. The Schild plot derived from the Kd shifts induced by granisetron was linear with a slope of 1.02, not different from unity, as expected from a competitive interaction. The Schild regression for GYKI-46 903 was linear with a slope of 1.20, deviating significantly from unity, which may also indicate an allosteric interaction. Both the association and dissociation curves of [3H]granisetron were monoexponential. The dissociation rate constant (K(-1)) and the association rate constant (K(+1)) were 0.32 +/- 0.01 min(-1) and 1.15 min(-1) x nM(-1), respectively. The dissociation driven by an excess concentration of ondansetron ( 1 microM) in the absence and presence of granisetron (0.1-3 nM) or GYKI-46 903 (30-10 000 nM) was not influenced by the compounds under study, as compared with the control, indicating the lack of an allosteric effect on the dissociation. Summing up, the binding profile of GYKI-46 903 may reflect a mixed type of action, including a negative allosteric interaction.

Thienocycloheptapyridazines as new muscarinic agents.

A series of thienocycloheptapyridazines (3aa-dd), structurally related to Minaprine, was synthesized and compounds tested for their affinity towards muscarinic receptors. All of them showed Ki values in the micromolar range towards both the antagonist 3H-QNB and the agonist 3H-OXO-M, thus indicating that they act as antagonists at the muscarinic receptors. Moreover a theoretical study was performed on their interaction behaviour with a three dimensional (3-D) model of the human m1 muscarinic receptor.

GYKI-46 903, a non-competitive antagonist for 5-HT3 receptors.

The effects of GYKI-46 903 ((+)endo-4-propionyloxy-6-(4-fluorophenyl)-1-azabicyclo [3.3.1]non-6-ene HCl), on 5-HT3 receptors have been studied and compared with ondansetron in peripheral organs in vitro and in vivo, and in a receptor binding assay in membranes prepared from rat cerebral cortex. GYKI-46 903 was found to be a non-competitive antagonist at 5-HT3 receptors present in non-stimulated longitudinal muscle strip of guinea-pig ileum (pD2' against serotonin = 5.54), and also in 5-methoxytryptamine-pretreated electrically stimulated ileal preparations (pD2' against serotonin = 5.26). On the contrary, ondansetron was found to be a competitive antagonist for 5-HT3 receptors; the pA2 value against serotonin was 7.40 in non-stimulated ileum, and it was 7.08 in electrically stimulated ileal preparation pretreated with 5-methoxytryptamine. In displacement studies, the pIC50 values of GYKI-46 903 and ondansetron against [3H]granisetron binding to rat cerebral cortex membranes were 6.91 and 8.58 respectively. GYKI-46 903, when administered by intravenous infusion, antagonized the decrease in heart rate evoked by serotonin (Bezold-Jarisch reflex) in anaesthetized rats, and the maximal reversal was less than 50%. This was in striking contrast with ondansetron, which, after intravenous injection, completely antagonized the serotonin-induced bradycardia with an ID50 value of 3.28 ug/kg. These data classify GYKI-46 903 as a non-competitive antagonist for 5-HT3 receptors.

Synthesis of thiazole derivatives with positive inotropic effect.

Thiazole derivatives with potential positive inotropic effect were synthesized. The highest activity (medium positive inotropic effect) was exhibited by ethyl 2-(2-hydroxyethylamino)-4-phenylthiazole-5-carboxylate (3b) of which several derivatives have been prepared. Among these derivatives only those which could presumably easily removed in the living organism to give 3b exhibited a medium positive inotropic effect (6, 8b). It was assumed that the effect of 3b had a beta-agonist character.

The effect of a long-term CCl4 action on the DNA content of rat liver cell nuclei. A cytophotometric study.

Inbred CFY male and female rats were given subcutaneous injections of carbon tetrachloride solution in sunflower oil for 12 weeks. Cytophotometric DNA determination was made on liver cell nuclei. Cell nuclei from normal liver showed modal peak of DNA in the diploid range. In the treated animals there were modal peaks of DNA in diploid and tetraploid regions as well as remarkable numbers of over-tetraploid nuclei. The hepatic lesions classified histologically were more severe in males than in females. The ploidy distributions were not related to the histopathological features and sex.