Analysis of Metabolites Produced after Mixing OPC-67683 and M. bovis BCG
(A) 15 μl of 14C OPC-67683 (0.5mg/ml: 0.056 μCi/μl) was added to 585 μl of 7H9/TN-ADC broth or bacterial culture and incubated for 48 h. After incubation, a 2-fold volume of acetonitrile was added and mixed well. The lysate was centrifuged for 5 min at 15,000 rpm. The supernatant was analyzed using HPLC with flow scintillation analyzer to determine the metabolite pattern.
(B) The identified metabolite (desnitro-imidazooxazole) was synthesized at Otsuka Pharmaceutical, and the fragment pattern of the metabolite by electrospray ionization mass spectroscopy was then compared with that of another compound newly synthesized based on the predicted structure.