Reaction #1585224

Reaction equation

N,7-Dimethyl-5-{1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}imidazo[5,1-f][1,2,4]triazin-4-amine

NADPH

→

product

[4-(Methylamino)-5-{1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}imidazo[5,1-f][1,2,4]triazin-7-yl]methanol

Conditions

Workup

1. 1
   ExtractionIncubations were extracted with 4 volumes of acetonitrile
2. 2
   Otherthe supernatant was evaporated at 25° C. in a Turbovap
3. 3
   Othersubjected to preparative reverse-phase HPLC purification (Column: Zorbax Rx-C8 [Agilent], 250×9.6 mm, 5 μm; Mobile phase A: aqueous 5 mM ammonium formate, pH 3; Mobile phase B: acetonitrile; Gradient: 10% to 90% B; UV detection at 254 mm)
4. 4
   workup.ADDITIONThe fractions containing the M+16 metabolite
5. 5
   Otherwere dried down

Procedure

N,7-Dimethyl-5-{1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}imidazo[5,1-f][1,2,4]triazin-4-amine, at a substrate concentration of 50 μM, was incubated for 1 hour with rat liver microsomes and NADPH (1 mM) (21.1 mg/mL protein concentration; 25 mL incubation volume). Incubations were extracted with 4 volumes of acetonitrile. Following centrifugation at 3400 rpm, the supernatant was evaporated at 25° C. in a Turbovap. The residue was reconstituted with 5% aqueous acetonitrile and subjected to preparative reverse-phase HPLC purification (Column: Zorbax Rx-C8 [Agilent], 250×9.6 mm, 5 μm; Mobile phase A: aqueous 5 mM ammonium formate, pH 3; Mobile phase B: acetonitrile; Gradient: 10% to 90% B; UV detection at 254 mm). The fractions containing the M+16 metabolite were dried down to provide the product as a solid. MS m/z 404 (M+H). 1H NMR (600 MHz, DMSO-d6) δ 2.71 (d, J=4.8 Hz, 3H), 3.86 (s, 3H), 4.69 (s, 2H), 6.68 (br q, J=4.7 Hz, 1H), 7.55 (d, J=8.0 Hz, 2H), 7.70 (s, 1H), 7.76 (d, J=8.0 Hz, 2H), 7.85 (s, 1H).

Source