Bioactivation of 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843) to reactive intermediates that bind covalently to macromolecules and produce genotoxicity.

U-89843 is a novel pyrrolo[2,3-d]pyrimidine antioxidant with prophylactic activity in animal models of lung inflammation. During preclinical safety evaluation, U-89843 was found to give a positive response in the in vitro unscheduled DNA synthesis (UDS) assay, an assay which measures DNA repair following chemically-induced DNA damage in metabolically competent rat hepatocytes. Incubation of [14C]U-89843 with liver microsomes resulted in covalent binding of radioactive material to macromolecules by a process that was NADPH-dependent. U-89843 has been shown to undergo C-6 methylhydroxylation to give U-97924, in rat both in vivo and in vitro, in a reaction catalyzed by cytochrome P450 2C11. Synthetical U-97924 is chemically reactive and undergoes dimerization in aqueous solution. The dimerization of U-97924 was significantly inhibited by addition of nucleophiles such as methanol, glutathione, and N-acetylcysteine. Characterization of the corresponding methanol, glutathione, and N-acetylcysteine adducts of U-97924 supported the hypothesis of a reaction pathway involving reactive iminium species formed via dehydration of U-97924. The metabolism-dependent irreversible covalent binding of radioactive material to liver microsomal protein and DNA also is dramatically reduced in the presence of reduced glutathione (GSH). A trifluoromethyl analog of U-89843 was prepared in an effort to block the corresponding metabolic hydroxylation pathway. This new compound (U-107634) was found to be negative in the in vitro UDS assay, and its metabolic susceptibility toward hydroxylation at the C-6 methyl group was eliminated. These observations suggest that the positive in vitro UDS results of U-89843 are mediated by the bioactivation of U-89843, leading to reactive electrophilic intermediates derived from the (hydroxymethyl)pyrrole metabolite U-97924.

Characterization of the serotonin1A receptor antagonist activity of WAY-100135 and spiperone.

The effects of the putative serotonin (5-HT)1A receptor antagonists WAY-100135 (WAY) and spiperone on the neuronal activity recorded from medullary and dorsal raphe 5-HT neurons and the inferior cardiac sympathetic nerve were investigated in chloralose anesthetized cats. We also determined the effectiveness of WAY and spiperone to antagonize the sympathoinhibitory effects of the 5-HT1A agonist 8-hydroxy-(2-di-n-propylamino)tetralin (8-OH DPAT). Intravenous administration of both WAY and spiperone produced a dose-related inhibition of the firing of medullary 5-HT neurons. WAY also inhibited firing of serotonergic neurons in the dorsal raphe nucleus. WAY treatment had no significant effect on inferior cardiac sympathetic nerve discharge (SND), whereas spiperone treatment caused a small, but significant, increase in SND. WAY treatment did not significantly affect 8-OH DPAT-induced inhibition of unit firing. Spiperone, however, did display antagonist activity at the presynaptic autoreceptor site. WAY and spiperone pretreatments resulted in significant rightward shifts in the 8-OH DPAT inhibition of SND dose-response curves and reversed the depressant effects of 8-OH DPAT. These results suggest that WAY and spiperone act as 5-HT1A antagonists postsynaptically, but WAY appears to have more potent agonist efficacy at the 5-HT1A presynaptic autoreceptor site in the cat. However, because all drugs were administered intravenously, conclusions regarding direct effects of WAY and spiperone on 5-HT1A receptors must be made cautiously.

Synthesis of acylguanidine analogues: inhibitors of ADP-induced platelet aggregation.

Routine screening of compounds for inhibition of ADP-induced platelet aggregation in vitro revealed that 1,1'-hexamethylenebis[3-cyclohexyl-3-[(cyclohexylimino) (4-morpholinyl) methyl]urea] (1) was active and represented the first example of a bis(acylguanidine) with possible antithrombotic activity. In order to develop a structure-activity relationship for this class of compounds, we synthesized a number of new bis(acylguanidines). These were tested in vitro, and several analogues were also active. Ex vivo testing revealed that compounds 22, 41, 58, and 70-73 were orally active in rats or guinea pigs.

Synthesis and platelet aggregation inhibitory activity of 4,5-bis(substituted)-1,2,3-thiadiazoles.

Routine screening of compounds for inhibition of collagen-induced platelet aggregation in vitro revealed 4,5-bis-(4-methoxyphenyl)-1,2,3-thiadiazole (2) was active and it represents the first example of a 1,2,3-thiadiazole with possible antithrombotic activity. In order to develop a structure-activity relationship for this heterocycle, a number of new 4(5)-mono- and -disubstituted 1,2,3-thiadiazoles were synthesized. These were tested in our screen and a number of additional active compounds were found. The most active compounds (2, 5a, 5b, and 6c) were those in which the heterocycle was substituted with benzene rings possessing para electron-donating groups.

Distribution of a Fatty Acid cyclase enzyme system in plants.

Extracts from tissues of 24 plant species were tested for the enzyme that catalyzes the conversion of 13-l-hydroperoxy-cis-9,15-trans-11-octadecatrienoic acid to the cyclic fatty acid 12-oxo-cis-10,15-phytodienoic acid. The enzyme was detected in 15 of the 24 tissues examined, and was demonstrated in seedlings, leaves, and fruits.