Galardy R E, Grobelny D
J Med Chem. 1985 Oct;28(10):1422-7. doi: 10.1021/jm00148a008.
The rate of loss of phenol or 4-nitrophenol from N alpha-(diphenoxyphosphoryl)-L-alanyl-L-proline (2), N alpha-[bis(4-nitrophenoxy)phosphoryl]-L-alanyl-L-proline (5), and N alpha-[(2-phenylethyl)phenoxyphosphoryl]-L-alanyl-L-proline (12) was determined spectrophotometrically at pH 7.5 and 37 degrees C in both Tris and phosphate buffers. These moderately potent inhibitors of angiotensin converting enzyme (Ki greater than 0.8 microM) all hydrolyze, losing 1 mol of phenol to yield highly potent inhibitors (Ki = 0.5-18 nM). The half-times for loss of 1 mol of phenol in Tris buffer are 22 days (2), 3.4 h (5), and 21 days (12). The half-times in phosphate buffer were not significantly different. The mono(4-nitrophenoxy) ester 6 (Ki = 18 nM) loses its 1 mol of nitrophenol with a half-time of 35 h to yield N alpha-phosphoryl-L-alanyl-L-proline 16 (Ki = 1.4 nM), which hydrolyzes at the P-N bond with a half-time of 2.2 h. Hydrolysis of the P-N bond in 2 and 12 was not observed during the time course of the kinetic experiments. The two phosphoramidate diesters 2 and 5 and the phosphonamidate monoester 12 thus release powerful inhibitors of angiotensin converting enzyme with a known time course at physiological pH and temperature in vitro. A time-dependent increase in inhibitory potency against converting enzyme that paralleled the kinetics of phenyl ester hydrolysis was confirmed in vitro.
在pH 7.5和37℃条件下，于Tris缓冲液和磷酸盐缓冲液中，通过分光光度法测定了Nα-（二苯氧基磷酰基）-L-丙氨酰-L-脯氨酸（2）、Nα-[双（4-硝基苯氧基）磷酰基]-L-丙氨酰-L-脯氨酸（5）和Nα-[(2-苯乙基）苯氧基磷酰基]-L-丙氨酰-L-脯氨酸（12）中苯酚或4-硝基苯酚的损失速率。这些血管紧张素转换酶的中度有效抑制剂（Ki大于0.8μM）均发生水解，损失1摩尔苯酚，生成高效抑制剂（Ki = 0.5 - 18 nM）。在Tris缓冲液中损失1摩尔苯酚的半衰期分别为22天（2）、3.4小时（5）和21天（12）。在磷酸盐缓冲液中的半衰期无显著差异。单（4-硝基苯氧基）酯6（Ki = 18 nM）以35小时的半衰期损失其1摩尔硝基苯酚，生成Nα-磷酰基-L-丙氨酰-L-脯氨酸16（Ki = 1.4 nM），后者以2.2小时的半衰期在P-N键处水解。在动力学实验过程中未观察到2和12中P-N键的水解。因此，这两种磷酰胺酸二酯2和5以及磷酰胺酸单酯12在生理pH和温度条件下的体外实验中，能以已知的时间进程释放血管紧张素转换酶的强力抑制剂。体外实验证实，对转换酶抑制效力的时间依赖性增加与苯基酯水解动力学平行。
