Oxidative ipso substitution of 2,4-difluoro-benzylphthalazines -Identification of a rare stable quinone methide and subsequent glutathione conjugate DMD #46268 2 Running Title: Oxidative ipso substitution2,4-difluoro-benzylphthalazines

Abstract In vitro metabolite identification and glutathione trapping studies in human liver microsomes were conducted in order to understand the bioactivation potential of compound 1, 2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol), an inhibitor of the Hedgehog pathway. The results revealed the formation of a unique, stable quinone methide metabolite (M1) via ipso substitution of a fluorine atom and subsequent formation of a glutathione adduct (M2). The stability of this metabolite arises from extensive resonance stabilized conjugation of the substituted benzylphthalazine moiety. CYP450 phenotyping studies revealed that the formation of M1 and M2 were NADPH dependent and primarily catalyzed by CYP3A4 among the studied CYP450 isoforms. In summary, an unusual and stable quinone methide metabolite of compound 1 was identified and a mechanism was proposed for its formation via an oxidative ipso substitution. DMD #46268

Small-molecule factor B inhibitor for the treatment of complement-mediated diseases

Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development

The effect of polymorphic metabolism enzymes on serum phenytoin level

Phenytoin has a widespread use in epilepsy treatment and is mainly metabolized by hepatic cytochrome P450 enzymes (CYP). We have investigated CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*3 allelic variants in a Turkish population of patients on phenytoin therapy. Patients on phenytoin therapy (n = 102) for the prevention of epileptic seizures were included. Polymorphic alleles were analyzed by restriction fragment length polymorphism method. Serum concentrations of phenytoin were measured by fluorescence polarization immune assay method. The most frequent genotype was detected for CYP2C9 wild-type alleles (78.43 %), whereas CYP2C19*2/*2 (5.88 %) was the least frequent genotype group. According to the classification made with both enzyme polymorphisms, CYP2C9*1/*1-CYP2C19*1/*1 (G1: 41.17 %) genotype group was the most frequent whereas CYP2C9*1/*2-CYP2C19*1/*3 (G7: 0.98 %) was the least frequent one. The highest mean phenytoin level (27.95 +/- A 1.85 A mu g/ml) was detected in the G8 genotype group (CYP2C9*1/*3-CYP2C19*2/*3) and the G1 genotype group showed the lowest mean phenytoin level (7.43 +/- A 0.73 A mu g/ml). The mean serum concentration of phenytoin of the polymorphic patients with epilepsy was higher than that for the wild-type alleles both in the monotherapy and polytherapy patients. These results show the importance of the genetic polymorphism analysis of the main metabolizing enzyme groups of phenytoin for the dose adjustment