Liquid Chromatographic Resolution of Racemic 1,4-Benzodiazepin-2-ones on Pirkle-type Chiral Stationary Phases Based on (S)-Leucine

Abstract

It has been well known that two enantiomers of chiral drugs often show different pharmacological activity. In this instance, exact determination of the enantiomeric composition of chiral drugs is essential for the development of chiral drugs and for the better insight into the in vivo distribution and metabolism of the two enantiomers. Among various methods, liquid chromatographic separation of enantiomers on HPLC chiral stationary phases (CSPs) has been demonstrated as very attractive means for the analysis of enantiomeric composition of chiral compounds including chiral drugs. A series of 3-substituted 1,4-benzodiazepin-2-ones such as camazepam, lorazepam, lormetazepam and oxazepam belongs to a class of widely used anxiolytics and/or tranquilizer. The pharmacological activity of a series of 3substituted 1,4-benzodiazepin-2-ones has been shown to be enantiodependent. Consequently some efforts have been devoted to the development of the liquid chromatographic separation of the two enantiomers of 3-substituted 1,4benzodiazepin-2-ones. Among others, Pirkle-type CSPs have been found to be quite useful in the resolution of racemic 3-substituted 1,4-benzodiazepin-2-ones. Especially, CSP 1 (Figure 1) derived from (S)-N-(3,5-dinitrobenzoyl)leucine has been most successfully utilized in the resolution 3-substituted 1,4-benzodiazepin-2-ones. As an improved CSP, we recently developed CSP 2 (Figure 1) by simply replacing the superfluous adsorption site, the N-H hydrogen of the connecting tether of CSP 1, with a phenyl group. CSP 2 has shown greater enantioselectivities than CSP 1 for the enantiomers of π-acidic or π-basic derivatives of a-amino acids. However, CSP 2 has not been applied in the resolution of racemic 3-substituted 1,4-benzodiazepin-2ones. In this study, as an effort to extend the use of CSP 2, we wish to elucidate that CSP 2 is also useful in the resolution of 3-substituted 1,4-benzodiazepin-2-ones and wish to compare the chiral recognition efficiency of CSP 2 with that of CSP 1. 3-Substituted 1,4-benzodiazepin-2-ones (3 and 4) used in this study (Figure 1) were prepared via the reported procedure. For the purpose of comparison, 3-substituted 1,4-benzodiazepin-2-ones (3 and 4) prepared in our laboratory were resolved on CSP 1 and CSP 2 under an identical chromatographic condition and the resolution results were summarized in Table 1. The representative chromatograms on CSP 2 are shown in Figure 2. As shown in Table 1 and Figure 2, the resolutions of 3substituted 1,4-benzodiazepin-2-ones (3 and 4) are excellent on both CSP 1 and CSP 2. Interestingly, the retention factors (k) on CSP 2 are always smaller than those on CSP 1. Even when the content of isopropyl alcohol in mobile phase was varied within the range of 3% to 20%, the retention factors (k) on CSP 2 were still always smaller than those on CSP 1. This means that CSP 2 is better than CSP 1 in terms of reducing analytical time and saving solvent. The smaller retention factors (k) on CSP 2 might be rationalized by considering the chiral recognition mechanism. A chiral recognition mechanism for the resolution of 3substituted 1,4-benzodiazepin-2-ones on CSP 1 was