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Twenty new 2-(cyanoalkyl)tetrazoles (15 and 16) and twenty new 2-(hydroxyalkyl)tetrazoles (17 and 18) were synthesized and investigated in vitro for their abilities to inhibit selectively rat brain monoamine oxidase (MAO) B over MAO A. Most of them were MAO B inhibitors and those bearing a substituted 4-(arylmethoxy)phenyl group in the position 5 of the tetrazole ring had IC50 values between 8 microM for 18d and 2 nM for 16a (30 nM for lazabemide) with a selectivity toward MAO B of 37,000 for 16a. The reversibility of their inhibitory activity was demonstrated by in vitro dialysis tests. The 5-[4-(phenylmethoxy)phenyl]-2-(2-cyanoethyl)tetrazole (16a) its derivative 16h and the 5-[4-(phenylmethoxy)phenyl]-2-(2-hydroxyethyl)tetrazole (18a) and its derivative 18h were found to be potent, in vitro selective, and competitive MAO B inhibitors. Tetrazole 16a can be considered one of the most active and selective competitive MAO B inhibitors known up to now. This compound was selected for ex vivo experiments and was shown to be a strong and reversible MAO B inhibitor with a short duration of action after oral administration at 5 mg/kg. The structure-activity approach gives rise to the great importance of lipophilicity over electronic effects of the compounds in these series.

Eighteen new 5-aryl-1,3,4-oxadiazol-2(3H)-one derivatives and sulfur analogues were prepared and evaluated in vitro for their inhibitory properties on monoamine oxidase (MAO) types A and B. The most active compounds in these series acted preferentially against MAO B with IC50 values in the range of 1.8-0.056 μM. The 5-(4-biphenylyl)-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one 23 and its oxadiazolethione analogue 33 were found to act as potent, selective and competitive MAO B inhibitors with a slight slow-binding character. Both compounds inhibited MAO A in a classical competitive manner. According to their Ki (MAO B) values of 2.6 and 4 × 10−8 M, respectively, and their Ki (MAO A)/Ki (MAO B) ratios of 270 and 500, respectively, 23 and 33 can be placed among the most active and selective competitive MAO B inhibitors known up to now. The structure-activity relationships are discussed.

Twenty new 2-(cyanoalkyl)tetrazoles (15 and 16) and twenty new 2-(hydroxyalkyl)tetrazoles (17 and 18) were synthesized and investigated in vitro for their abilities to inhibit selectively rat brain monoamine oxidase (MAO) B over MAO A. Most of them were MAO B inhibitors and those bearing a substituted 4-(arylmethoxy)phenyl group in the position 5 of the tetrazole ring had IC50 values between 8 microM for 18d and 2 nM for 16a (30 nM for lazabemide) with a selectivity toward MAO B of 37,000 for 16a. The reversibility of their inhibitory activity was demonstrated by in vitro dialysis tests. The 5-[4-(phenylmethoxy)phenyl]-2-(2-cyanoethyl)tetrazole (16a) its derivative 16h and the 5-[4-(phenylmethoxy)phenyl]-2-(2-hydroxyethyl)tetrazole (18a) and its derivative 18h were found to be potent, in vitro selective, and competitive MAO B inhibitors. Tetrazole 16a can be considered one of the most active and selective competitive MAO B inhibitors known up to now. This compound was selected for ex vivo experiments and was shown to be a strong and reversible MAO B inhibitor with a short duration of action after oral administration at 5 mg/kg. The structure-activity approach gives rise to the great importance of lipophilicity over electronic effects of the compounds in these series.

Eighteen new 5-aryl-1,3,4-oxadiazol-2(3H)-one derivatives and sulfur analogues were prepared and evaluated in vitro for their inhibitory properties on monoamine oxidase (MAO) types A and B. The most active compounds in these series acted preferentially against MAO B with IC50 values in the range of 1.8-0.056 μM. The 5-(4-biphenylyl)-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one 23 and its oxadiazolethione analogue 33 were found to act as potent, selective and competitive MAO B inhibitors with a slight slow-binding character. Both compounds inhibited MAO A in a classical competitive manner. According to their Ki (MAO B) values of 2.6 and 4 × 10−8 M, respectively, and their Ki (MAO A)/Ki (MAO B) ratios of 270 and 500, respectively, 23 and 33 can be placed among the most active and selective competitive MAO B inhibitors known up to now. The structure-activity relationships are discussed.

A series of new analogues of 15-deoxyspergualin (DSG), an immunosuppressive agent commercialized in Japan, was synthesized and tested in a graft-versus-host disease (GVHD) model in mice. Various substitutions of the spermidine "D" region were made in order to determine its optimum structure in terms of in vivo immunosuppressive activity. Various positions of methylation were first investigated leading to the discovery of the monomethylated malonic derivative 56h in which the pro-R hydrogen of the methylene alpha to the primary amine of the spermidine moiety has been replaced by a methyl group. Synthesis of the similarly methylated analogue of the previously reported glycolic derivative LF 08-0299 afforded 60e which demonstrated a powerful activity at a dose as low as 0.3 mg/kg in the GVHD model and was much more potent than DSG in the demanding heart allotransplantation model in rats. The improvement of in vivo activity was supposed to be related to an increase of the metabolic stability of the methylated analogues compared to the parent molecules. Due to its very low active dose, compatible with a subcutaneous administration in humans, and its favorable pharmacological and toxicological profile, 60e was selected as a candidate for clinical evaluation.

A series of new analogues of 15-deoxyspergualin (DSG), an immunosuppressive agent currently commercialized in Japan, was synthesized and tested in a graft-versus-host disease (GVHD) model in mice. Using the general concept of bioisosteric replacement, variations of the hydroxyglycine central "C" region were made in order to determine its optimum structure in terms of in vivo immunosuppressive activity. By this way, the malonic derivative 13a was discovered as the first example of a new series of potent immunosuppressive agents encompassing a retro-amide bond linked to the hexyl-guanidino moiety. Structure-activity relationships of this series were studied by synthesizing compounds 13g-i and 13k-s. Variation of the "right-amide" of 13a led to the urea 19a and the carbamates 23 and 27a which proved to be equally active as DSG in our GVHD model. Finally 27a was found to be the most potent derivative, being slightly more active than DSG in a heart allotransplantation model in rats. Due to the absence of chiral center in its structure and to its improved chemical stability compared to DSG, 27a was selected as a candidate for clinical evaluation.

PURPOSE To test the efficiency of locally administrated tresperimus in experimental autoimmune uveoretinitis (EAU). METHODS EAU was induced in Lewis rats by S-antigen (S-Ag) immunization. Three intravitreal injections of tresperimus (prevention or prevention/treatment protocols) were performed at different time points after immunization. The pharmacokinetics of tresperimus was evaluated in the ocular tissues and plasma. The in vitro effect of tresperimus was evaluated on macrophages. EAU was graded clinically and histologically. Blood ocular barrier permeability was evaluated by protein concentration in ocular fluids. Immune response to S-Ag was examined by delayed type hypersensitivity, the expression of inflammatory cytokines in lymph nodes, ocular fluids and serum by multiplex ELISA, and in ocular cells by RT-PCR. RESULTS In vitro, tresperimus significantly reduced the production of inflammatory cytokines by lipopolysaccharide-stimulated macrophages. In vivo, in the treatment protocol, efficient tresperimus levels were measured in the eye but not in the plasma up to 8 days after the last injection. Tresperimus efficiently reduced inflammation, retinal damage, and blood ocular barrier permeability breakdown. It inhibited nitric oxide synthase-2 and nuclear factor κBp65 expression in ocular macrophages. IL-2 and IL-17 were decreased in ocular media, while IL-18 was increased. By contrast, IL-2 and IL-17 levels were not modified in inguinal lymph nodes draining the immunization site. Moreover, cytokine levels in serum and delayed type hypersensitivity to S-Ag were not different in control and treated rats. In the prevention/treatment protocol, ocular immunosuppressive effects were also observed. CONCLUSIONS Locally administered tresperimus appears to be a potential immunosuppressive agent in the management of intraocular inflammation.

Monoamine oxidase (MAO) assay specificity based on substrate specificity was investigated by substrate competition experiments. 10 μM serotonin (5-HT) and 5 μM β-phenylethylamine (PEA) were found to ensure total substrate specificity for, respectively, MAO types A and B. Twenty-five 2-aryl-4 H -1,3,4-oxadiazin-5(6 H )-one derivatives were synthesized and tested in vitro for their inhibitory effects on MAO A and B. Most of them inhibited preferentially MAO B. The 2-(4-biphenylyl)-4-(2-cyanoethyl)-4 H -1,3,4-oxadiazin-5(6 H )-one 32 was the most efficient MAO B inhibitor and acted as a competitive inhibitor on the two enzymes. Its K i values for MAO A and B were 11 and 0.15 μM, respectively. Structure-activity relationships suggest that these oxadiazinones should interact with a hydrophobic site and a nucleophilic site on MAO B for binding, while the functional group of the N-4 substituent should compete with the substrate for the active site of the enzyme.

Some 5-aryl-3-carbazoyl-1,3,4-oxadiazol-2(3H)-one derivatives 6 and 9 have been synthesized in two ways. The expected thermal ring transformation into 2,5-disubstituted 1,3,4-oxadiazoles did not occur but, by acid hydrolysis of 5-aryl-3-[3-benzylidene-2-methyl(or phenyl)carbazoyl]-1,3,4-oxadiazol-2(3H)-ones 6, a new ring transformation took place and the corresponding 4-benzamido-1-methyl(or phenyl)-1,2,4-triazolidine-3,5-dione derivatives 11 were formed. The 4-amino-1-phenyl-1,2,4-triazolidine-3,5-dione (19) has been prepared and its structure was confirmed by some reactions.

Monoamine oxidase (MAO) assay specificity based on substrate specificity was investigated by substrate competition experiments. 10 μM serotonin (5-HT) and 5 μM β-phenylethylamine (PEA) were found to ensure total substrate specificity for, respectively, MAO types A and B. Twenty-five 2-aryl-4 H -1,3,4-oxadiazin-5(6 H )-one derivatives were synthesized and tested in vitro for their inhibitory effects on MAO A and B. Most of them inhibited preferentially MAO B. The 2-(4-biphenylyl)-4-(2-cyanoethyl)-4 H -1,3,4-oxadiazin-5(6 H )-one 32 was the most efficient MAO B inhibitor and acted as a competitive inhibitor on the two enzymes. Its K i values for MAO A and B were 11 and 0.15 μM, respectively. Structure-activity relationships suggest that these oxadiazinones should interact with a hydrophobic site and a nucleophilic site on MAO B for binding, while the functional group of the N-4 substituent should compete with the substrate for the active site of the enzyme.