4H-3,1-Benzoxazin-4-ones are alternate substrate inhibitors of the serine proteinase human leukocyte elastase (HL elastase) and form acyl enzyme intermediates during enzyme catalysis. We have synthesized a large variety of benzoxazinones using specific methods that have been adapted to achieve the pattern of ring substitution dictated by theoretical considerations. The results of the inhibition of HL elastase by 175 benzoxazinones are reported herein with reference to hydrophobicity constants D, alkaline hydrolysis rates kOH-, inhibition constants Ki, and their component acylation and deacylation rate constants, kon and koff, respectively. The ranges for the compounds are considerable; alkaline hydrolysis rates and kon span 6, koff covers 5, and ki spans 8 orders of magnitude. Multiple regression on this large data set has been used to isolate the contributions of electronic and steric effects, as well as other factors specific to compound stability and elastase inhibition. Essentially, a simple electronic parameter is sufficient to account for almost all the variance in the alkaline hydrolysis data, indicating that electronic factors are the major determinants of this type of benzoxazinone reactivity. Factors that significantly enhance the potency of benzoxazinones I are R5 alkyl groups and electron withdrawal by R2. Bulk in R7 and R8 and compound hydrophobicity are not significant, but substitution in R6 is highly unfavorable as are substituents linked via carbon to C2. The physiochemical factors that underlie these trends in Ki are further analyzed in terms of equations that describe kon and koff. A conclusion that emerges is that chemically stable, potent benzoxazinone inhibitors of HL elastase with inhibition constants in the nanomolar range can be designed with (1) R5 alkyl groups to inhibit enzyme-catalyzed deacylation, (2) small alkyl substituents linked via heteroatoms to C2 to enhance acylation and limit deacylation rates, and (3) strongly electron-donating groups at C7 to stabilize the oxazinone ring to nucleophilic attack. Thus, 2-(isopropylamino)-5-n-propyl-7-(dimethylamino)benzoxazinone 95 has kOH = 0.01 M-1 s-1, which extrapolates to a half-life at pH 7.4 of over 8.5 years, and 2-ethoxy-5-ethylbenzoxazinone 38 has Ki = 42 pM.

中文翻译：

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设计和合成4H-3,1-苯并恶嗪-4-酮类化合物作为人类白细胞弹性蛋白酶的有效替代底物抑制剂。

4H-3,1-Benzoxazin-4-ones是丝氨酸蛋白酶人类白细胞弹性蛋白酶（HL弹性蛋白酶）的替代底物抑制剂，在酶催化过程中形成酰基酶中间体。我们已经使用特定方法合成了多种苯并恶嗪酮，这些特定方法已被调整以实现理论上的考虑而决定的环取代方式。本文参考疏水常数D，碱水解速率kOH-，抑制常数Ki及其组分酰化和脱酰化速率常数kon和koff分别报道了175种苯并恶嗪酮对HL弹性蛋白酶的抑制结果。化合物的范围是可观的。碱水解速率和kon跨度为6，koff覆盖5，ki跨度为8个数量级。在这个大数据集上的多元回归已被用于隔离电子和空间效应以及化合物稳定性和弹性蛋白酶抑制特异的其他因素的作用。本质上，简单的电子参数足以说明碱性水解数据中几乎所有的变化，表明电子因素是此类苯并嗪酮反应性的主要决定因素。显着增强苯并恶嗪酮I效力的因素是R5烷基和R2撤离电子。R 7和R 8中的体积大和化合物疏水性不显着，但是R 6中的取代是非常不利的，经由碳连接至C 2的取代基也是非常不利的。根据描述kon和koff的方程式，进一步分析了Ki形成这些趋势的物理化学因素。得出的结论是，可以设计具有化学稳定性的高效HL弹性蛋白酶苯并恶嗪酮抑制剂，其抑制常数在纳摩尔范围内，其中（1）R5烷基可抑制酶催化的脱酰作用，（2）通过杂原子与C2连接的小的烷基取代基增强酰化作用并限制脱酰率，以及（3）C7上的强供电子基团可稳定恶嗪酮环对亲核分子的攻击。因此，2-（异丙基氨基）-5-n-丙基-7-（二甲基氨基）苯并恶嗪酮95的kOH = 0.01 M-1 s-1，其外推至pH 7.4的半衰期超过8.5年，而2-乙氧基-5-乙基苯并恶嗪酮38的Ki = 42 pM。可以设计（1）R5烷基以抑制酶催化的脱酰作用，（2）通过杂原子与C2连接的小的烷基取代基来增强HL弹性蛋白酶的强苯甲并嗪酮抑制剂，其抑制常数在纳摩尔范围内，以及（3）C7的强电子给体基团，用于稳定恶嗪酮环对亲核的攻击。因此，2-（异丙基氨基）-5-n-丙基-7-（二甲基氨基）苯并恶嗪酮95的kOH = 0.01 M-1 s-1，其外推至pH 7.4的半衰期超过8.5年，而2-乙氧基-5-乙基苯并恶嗪酮38的Ki = 42 pM。可以设计（1）R5烷基以抑制酶催化的脱酰作用，（2）通过杂原子与C2连接的小的烷基取代基来增强HL弹性蛋白酶的强苯甲并嗪酮抑制剂，其抑制常数在纳摩尔范围内，以及（3）C7的强电子给体基团，用于稳定恶嗪酮环对亲核的攻击。因此，2-（异丙基氨基）-5-n-丙基-7-（二甲基氨基）苯并恶嗪酮95的kOH = 0.01 M-1 s-1，其外推至pH 7.4的半衰期超过8.5年，而2-乙氧基-5-乙基苯并恶嗪酮38的Ki = 42 pM。