Buynak J D, Wu K, Bachmann B, Khasnis D, Hua L, Nguyen H K, Carver C L
Department of Chemistry, Southern Methodist University, Dallas, Texas 75275.
J Med Chem. 1995 Mar 17;38(6):1022-34. doi: 10.1021/jm00006a022.
Several 7-alkylidenecephalosporins were synthesized and biologically evaluated as beta-lactamase inhibitors. The three beta-lactamase enzymes used in this study included two type C beta-lactamases, derived from Enterobacter cloacae P99 and E. cloacae SC12368, and one type A beta-lactamase, derived from Escherichia coli WC3310. Of the cephalosporins prepared, compound 7e, the sodium salt of 7-[(Z)-(2'-pyridyl)methylene]cephalosporanic acid sulfone, was found to have excellent inhibitory properties against both type C enzymes. Also, compound 7f, the sodium salt of 7-[(Z)-(tert-butoxycarbonyl)methylene]cephalosporanic acid sulfone showed high activity as an inhibitor of the type A enzyme. The inhibition kinetics of 7e were further explored. The IC50 value of 7e indicated that this compound was approximately 20-fold more active than tazobactam against the enzyme derived from E. cloacae P99 and 167-fold more active than tazobactam against the enzyme derived from E. cloacae SC12368. A plot of enzymatic activity vs incubation time with stoichiometric amounts of inhibitor reveals a rapid deactivation of the enzyme followed by an extremely slow reactivation. 7e exhibited a second-order rate constant of k3' = 5.3 x 10(6) L/mol.min, and a partition ratio of approximately 20:1 inhibitor:enzyme was determined for this inhibitor. After separation of excess inhibitor with Sephadex filtration, a rate constant of enzyme reactivation was measured at kreactiv = 1.0 x 10(-3) s-1. Following 24 h of incubation of enzyme with a large excess of inhibitor and sephadex filtration to remove excess inhibitor, the enzyme was able to recover only 43% of its original activity, indicating an irreversible component to the inhibition. Potential mechanisms of inhibition for both 7e and 7f are suggested.
合成了几种7-亚烷基头孢菌素，并对其作为β-内酰胺酶抑制剂进行了生物学评价。本研究中使用的三种β-内酰胺酶包括两种C型β-内酰胺酶，分别来源于阴沟肠杆菌P99和阴沟肠杆菌SC12368，以及一种A型β-内酰胺酶，来源于大肠杆菌WC3310。在所制备的头孢菌素中，化合物7e，即7-[(Z)-(2'-吡啶基)亚甲基]头孢烷酸砜的钠盐，被发现对两种C型酶均具有优异的抑制特性。此外，化合物7f，即7-[(Z)-(叔丁氧羰基)亚甲基]头孢烷酸砜的钠盐，作为A型酶的抑制剂表现出高活性。进一步研究了7e的抑制动力学。7e的IC50值表明，该化合物对来源于阴沟肠杆菌P99的酶的活性比他唑巴坦高约20倍，对来源于阴沟肠杆菌SC12368的酶的活性比他唑巴坦高167倍。用化学计量的抑制剂绘制酶活性与孵育时间的关系图，显示酶迅速失活，随后是极其缓慢的再活化。7e表现出二级速率常数k3' = 5.3 x 10(6) L/mol.min，并且确定该抑制剂的分配比约为20:1（抑制剂:酶）。用葡聚糖凝胶过滤分离过量抑制剂后，测得酶再活化的速率常数为kreactiv = 1.0 x 10(-3) s-1。将酶与大量过量的抑制剂孵育24小时并通过葡聚糖凝胶过滤去除过量抑制剂后，酶仅能恢复其原始活性的43%，表明抑制作用存在不可逆成分。提出了7e和7f的潜在抑制机制。
