Harmon Paul A, Biffar Stephen, Pitzenberger Steven M, Reed Robert A
Department of Pharmaceutical Analysis and Control, Merck Research Laboratories, P.O. Box 4, West Point, Pennsylvania, 19486, USA.
Pharm Res. 2005 Oct;22(10):1716-26. doi: 10.1007/s11095-005-6947-z. Epub 2005 Sep 22.
The rapid oxidation of rofecoxib under alkaline conditions has been previously reported. The oxidation was reported to involve gamma-lactone ring opening to an alcohol, which further oxidized to a dicarboxyclic acid. The oxidation was suspected to be mediated by peroxy radicals. This work further investigates the mechanism of oxidation under the alkaline solution conditions.
The pH dependence of the oxidation reaction was determined in 50% acetonitrile/50% aqueous phosphate buffer (pH 9-12). The oxidation reaction products were also examined at early timepoints (from 40 s to several minutes) with only 5% water content. The evolution of hydrogen peroxide by the oxidation reaction was quantitatively followed by reaction with triphenylphosphine (TPP) and high-pressure liquid chromatography determination of the resultant triphenylphosphine oxideformed. Rofecoxib was exposed to the alkaline pH conditions in the presence of formaldehyde, and the primary reaction product was isolated and characterized by liquid chromatography-mass spectrometry and proton 1D, heteronuclear multiple quantum coherence (HMQC), gradient heteronuclear multiple bond correlation (gHMBC), and carbon 1D nuclear magnetic resonance techniques. Transient reaction products were examined for hydroperoxide groups by reaction with TPP.
The oxidation reaction occurs only near pH 11 and above. In the presence of excess formaldehyde, oxidation products are no longer observed but a new product is observed in which two formaldehyde molecules have added to the methylene carbon atom of the gamma-lactone ring. The evolution of hydrogen peroxide corresponds quantitatively to the molar amount of the (minor) aldehyde oxidation product formed. It is demonstrated that the rofecoxib anhydride species is actually the primary product of the oxidation reaction. The existence of a transient hydroperoxide species is shown by reaction with TPP and concomitant conversion to a previously identified alcohol.
The oxidation of rofecoxib under these high pH conditions is mediated by rofecoxib enolate ion formation. The enolate ion reacts with either formaldehyde or dissolved oxygen at the C5 position. In the case of oxygen, a transient hydroperoxide species is formed. The major and minor products of the oxidation derive from competitive routes of decomposition of this hydroperoxide. The major route involves a second enolate ion formation, which decomposes with heterolytic cleavage of the RO-OH bond to give the rofeocoxib anhydride and hydroxide ion. The anhydride is rapidly hydrolyzed under the alkaline conditions to give the observed rofecoxib dicarboxylate product. The minor hydroxy-furanone product is formed from hydroxide ion attack on the hydroperoxide intermediate.
先前已报道罗非昔布在碱性条件下会快速氧化。据报道，该氧化反应涉及γ - 内酯环开环生成醇，醇进一步氧化为二羧酸。据推测，该氧化反应由过氧自由基介导。本研究进一步探究碱性溶液条件下的氧化机制。
在50%乙腈/50%磷酸盐水溶液缓冲液（pH 9 - 12）中测定氧化反应的pH依赖性。还在水含量仅为5%的早期时间点（40秒至几分钟）检测氧化反应产物。通过与三苯基膦（TPP）反应并采用高压液相色谱法测定生成的三苯基氧化膦，定量跟踪氧化反应中过氧化氢的生成。使罗非昔布在甲醛存在下暴露于碱性pH条件，分离出主要反应产物，并通过液相色谱 - 质谱联用以及质子1D、异核多量子相干（HMQC）、梯度异核多键相关（gHMBC）和碳1D核磁共振技术对其进行表征。通过与TPP反应检测瞬态反应产物中的氢过氧化物基团。
氧化反应仅在pH 11及以上时发生。在过量甲醛存在下，不再观察到氧化产物，但观察到一种新产物，其中两个甲醛分子加成到γ - 内酯环的亚甲基碳原子上。过氧化氢的生成量与形成的（少量）醛氧化产物的摩尔量定量对应。结果表明，罗非昔布酸酐实际上是氧化反应的主要产物。通过与TPP反应并伴随转化为先前鉴定的醇，表明存在瞬态氢过氧化物。
在这些高pH条件下，罗非昔布的氧化由罗非昔布烯醇负离子的形成介导。烯醇负离子在C5位置与甲醛或溶解氧反应。在与氧反应的情况下，形成瞬态氢过氧化物。氧化反应的主要和次要产物源自该氢过氧化物分解的竞争途径。主要途径涉及再次形成烯醇负离子，其通过RO - OH键的异裂分解生成罗非昔布酸酐和氢氧根离子。酸酐在碱性条件下迅速水解，生成观察到的罗非昔布二羧酸盐产物。次要的羟基呋喃酮产物由氢氧根离子进攻氢过氧化物中间体形成。
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