Inhibition of glutathion-S-transferase and amidase by impurities in technical grade benfuracarb

Objectives of this study were to determine if impurities in technical grade benfuracarb inhibit glutathione-S-transferase and amidase and to identify structures of impurities in technical grade benfuracarb. Technical grade benfuracarb, active ingredient, and impurity inhibited glutathione-S-transferase, and their $I_{50}$ were $9.7{\times}10^{-4}M,\;>1.0{\times}10^{-3}M,\;1.8{\times}10^{-4}M$, respectively. Such inhibition, however, was not higher than that by ethacrynic acid, a selective inhibitor to GST. Technical grade benfuracarb, active ingredient, and impurity also inhibited amidase, and their $I_{50}$ were $6.0{\times}10^{-5}M,\;4.3{\times}10^{-4}M,\;7.6{\times}10^{-5}M$, respectively. Our results show that the inhibition of both detoxifying enzymes by impurities in benfuracarb was 10-fold lower than that by active ingredient, suggesting that both active ingredient and impurities are involved in the inhibition of both detoxifying enzymes. Of four impurities (IM $1{\sim}4$) that were separated from technical grade benfuracarb, IM 2 and IM 3 inhibited GST and amidase. Based on data from IR, $^1H$-NMR, $^{13}C$-NMR and MS, it was determined that IM 2 is ethyl-N-isopropylamino propionate and IM 3 is ethyl-N-isopropyl-N(chlorosulfenyl)aminopropionate.

Benfuracarb 원제에 함유된 불순물들의 glutathione-S-transferase와 amidase 저해 특성

본 논문의 목적은 benfuracarb 원제(90.2%)에 함유된 불순물의 glutathione-S-transferase와 amidase에 대한 저해 특성과 해당 불순물의 구조를 밝히는데 있다. Benfuracarb 원제, 유효성분 및 불순물은 glutathione-S-transferase(GST)를 효과적으로 저해하였으나, 그 저해력은 GST 효소 저해제인 ethacrynic acid의 저해력보다는 낮았다. 즉, GST에 대한 benfuracarb 원제, 유효성분 및 불순물의 $I_{50}$은 각각 $9.7{\times}10^{-4}M,\;>1.0{\times}10^{-3}M,\;1.8{\times}10^{-4}M$이었으나, ethacrynic acid의 $I_{50}$은 $1.7{\times}10^{-5}M$이었다. Benfuracarb 원제, 유효성분 및 불순물은 amidase를 저해하였는데, 이들의 효소저해력은 iprobenfos의 저해력($I_{50},\;8.2{\times}10^{-7}M$)보다는 낮은 $6.0{\times}10^{-5}M,\;4.3{\times}10^{-4}M,\;7.6{\times}10^{-5}M$이었다. Benfuracarb 원제에는 4종의 불순물(IM $1{\sim}4$)이 검출되었는데, 이들 중 IM 2와 3은 GST와 amidase의 활성을 저해하였던 반면, IM 4는 효소활성을 저해하지 않았다. 이들 불순물 중 효소활성 저해특성을 갖는 IM 2와 3을 IR, $^1H$-NMR, $^{13}C$-NMR, LC-MS를 이용하여 구조를 분석한 결과, IM 2는 ethyl-N-isopropylamino propionate로, 그리고 IM 3은 ethyl-N-isopropyl-N-(chlorosulfenyl) aminopropionate로 확인되었다.