Furthermore, only a single point determination was conducted for the full process in order to demonstrate the suggested conversion route. When N-hydroxymethyl aripiprazole ( Fig. 2) was added to phosphate Selleck Everolimus buffer, a rapid conversion was observed, i.e., a conversion that should not be rate limiting in vivo. The shift of the proton on C8 was followed, see Fig. 1 (C8 marked with *). When the hydroxymethyl group was attached a shift at 6.75 ppm was observed, whereas the shift changed to 6.40 ppm when the group was removed. At 25 °C the apparent first-order rate constant was 0.0044 min−1 and the
half-life was approximately 35 min. At 37 °C, however, the conversion
was so fast that the rate constant could not be measured with sufficient precision. More than half of the N-hydroxymethyl aripiprazole was converted within the first 15 min at 37 °C. Estimated pKa for 3,4-dihydro-2(1H)-quinolinone is 14.6 [ 41]. RG7204 datasheet If this value is assumed similar for the NH-acidic group in aripiprazole a half-life of 12.7 h should be anticipated based upon the prediction suggested by Bundgaard and Johansen [ 28]. This variation may be a reflection of a different chemical space used to make the correlation or that the estimated pKa value for 3,4-dihydro-2(1H)-quinolinone may not be similar to the pKa value for aripiprazole. The predicting defined by Bundgaard and Johansen [ 28] is very sensitive to the Dipeptidyl peptidase pKa value, for compounds with a pKa on 12.4 a half-life of 15 min would be estimated. When aripiprazole lauroxil was added to rat plasma, concentrations of the expected N-hydroxymethyl intermediate in the two-step degradation described in Fig. 1 could be observed when analysed after both 0.5 and 1 h at 37 °C (see Fig. 3). The in
vitro bioconversion from the N-acyloxyalkyl derivate to the parent compound observed in the present study is in accordance with previous in vitro bioconversion studies investigating N-acyloxyalkyl derivates [ 31, , ,  and ]. Moreira and coworkers [ 39] have described the in vitro conversion of O-amidomethyl penicilloate, through an intermediate, with the very slow formation of penicillin G, whereas Buur et al. reported the conversion of N-acyloxymethyl derivates of 5-fluorouracil to occur within a similar timeframe as in the present study [ 25]. This study demonstrates that during the conversion of aripiprazole lauroxil to aripiprazole, N-hydroxymethyl aripiprazole is present in significant amounts, despite being a very short-lived intermediate compound as revealed from the experiment when N-hydroxymethyl aripiprazole was added to a buffer.