Synthesis, structure-activity relationships, and in vivo evaluations of substituted di-tert-butylphenols as a novel class of potent, selective, and orally active cyclooxygenase-2 inhibitors. 2. 1,3,4- and 1,2,4-thiadiazole series.

Two isoforms of the cyclooxygenase (COX) enzyme have been identified: COX-1, which is expressed constitutively, and COX-2, which is induced in inflammation. Recently, it has been shown that selective COX-2 inhibitors have antiinflammatory activity and lack the GI side effects typically associated with NSAIDs. Initial mass screening and subsequent SAR studies have identified 6b (PD164387) as a potent, selective, and orally active COX-2 inhibitor. It had IC50 values of 0.14 and 100 microM against recombinant human COX-2 and purified ovine COX-1, respectively. It inhibited COX-2 activity in the J774A.1 cell line with an IC50 of 0.18 microM and inhibited COX-1 activity in platelets with an IC50 of 3.1 microM. The choline salt of compound 6b was also orally active in vivo with an ED40 of 7. 1 mg/kg in the carrageenan footpad edema (CFE) assay. In vivo studies in rats at a dose of 100 mg/kg showed that this compound inhibited gastric prostaglandin E2 (PGE2) production in gastric mucosa by 77% but caused minimal GI damage. SAR studies of this chemical series revealed that the potency and selectivity are very sensitive to minor structural changes.

Synthetic approaches to the guanosine and xanthosine analogues 5-amino-3-beta-D-ribofuranosylpyrazolo[3,4-e][1,3]oxazin-7-one and 3-beta-D-ribofuranosylpyrazolo[3,4-e][1,3]oxazine-5,7-dione and studies of their antitumor potential.

5-Amino-3-beta-D-ribofuranosylpyrazolo[3,4-e][1,3]oxazin-7-o ne has been synthesized via cyclization of the appropriately protected pyrazofurin derivatives and subsequent transformations of the heterocyclic moiety. This guanosine analogue was marginally cytotoxic to L1210 cells in vitro. The xanthosine analogue 3-beta-D-ribofuranosylpyrazolo[3,4-e][1,3]oxazine-5,7-dione was also synthesized, and was found to be highly cytotoxic. It appeared to act as a prodrug of pyrazofurin.

Detoxication of aliphatic epoxides by diol formation and glutathione conjugation.

The Ames procedure with Salmonella typhimurium strain TA100 was used to follow the detoxication by rat liver fractions of two series of aliphatic epoxides. The epoxides employed were 3-chloro-, 3,3-dichloro- and 3,3,3-trichloropropylene oxides and also p-methoxyphenyl-, phenyl- and p-nitrophenylglycidyl ethers. In our procedure with preincubation of the epoxides with rat liver fractions prior to the Ames tests, there was more detoxication of both systems by glutathione conjugation (non-enzymatic and transferase promoted) than by the hydrolase pathways. Non-enzymatic reaction with glutathione was more pronounced for the chloro series than for the glycidyl ethers. An HPLC system was developed which was capable of quantitative measurements of the phenylglycidyl ethers together with their diol and glutathione conjugate products. A comparison of the HPLC and Ames test results indicates that the glutathione transferase reported to be present in Salmonella could be playing a role in detoxication by the Ames test. Diols were measured more readily by HPLC than by use of the Ames test in the microsomal fraction and were detected in the cytosol with the glycidyl ethers while they were not by the Ames procedure. However, all three epoxides were converted to a greater extent to their glutathione conjugates than to their diols. Thus, while literature references question the availability of the glutathione detoxication system for epoxides produced by membrane-bound enzymes, such detoxication would be of primary importance where direct-acting environmental epoxides come into contact with the cytosolic enzymes prior to possible reaction with bionucleophiles.

Mutagenicity of para-substituted alpha-methylstyrene oxide derivatives with Salmonella.

A series of 5 para-substituted alpha-methylstyrene oxide derivatives have been synthesized and together with alpha-methylstyrene oxide as well as styrene oxide have been studied as to their mutagenicity with the TA100 and TA1535 strains of Salmonella typhimurium. A multiple regression analysis model has been developed which describes the mutagenicity of the alpha-methylstyrene oxides in TA100. An increase in van der Waals volume was the most important variable in the model with greater improvement occurring with inclusion of the Hammett values for the para substituents on the compounds. The alpha-methylstyrene oxides were less active alkylating agents with 4-(p-nitrobenzyl)pyridine than styrene oxide and with pyridine all reactivity was at the beta-epoxide carbon. However all the alpha-methylstyrene oxide derivatives, except for the bromo compound where toxicity was evident, showed mutagenicity values either greater or comparable to that of styrene oxide. These studies would indicate that reactivity at the beta-carbon should also be a factor in describing the mutagenicity of the parent styrene oxide series.