Oxazepam 3-acetate

S. K. Yang, K. Liu, F. P. Guengerich, Enantioselective Hydrolysis of Oxazepam 3-Acetate by Esterases in Human and Rat Liver Microsomes and Rat Brain S9 Fraction , Chirality 1990, 2, 150-155. 

Hgure 2 Separation of the enantiomers of oxazepam acetate and oxazepam on a chiral stationary phase. Column 4.6 mm X 25 cm stationary phase (S)-dinitrobenzoylleucine covalently bonded to 5 pm silica mobile phase, hexane-ethanol-acetonitrile (91.5 5.7 2.8, v/v/v), 2mlmin detector, UV 231 nm. Racemic oxazepam 3-acetate was hydrolyzed by human liver microsomes, giving racemic oxazepam. The micro-somes strongly preferred the (R) enantiomer after incomplete hydrolysis the remaining oxazepam acetate had a composition of 91.8% (S) and 8.2% (R) enantiomers. (Reproduced from Yang SK, Liu K, and Guengerich FP (1990) Enantioseleclive hydrolysis of oxazepam 3-acetate by esterases in human and rat liver microsomes and rat brain S9 fraction. Chirality 2 150.)... 

The intracellular localization of carboxylesterases is predominantly microsomal, the esterases being localized in the endoplasmic reticulum [73] [79] [93], They are either free in the lumen or loosely bound to the inner aspect of the membrane. The carboxylesterases in liver mitochondria are essentially identical to those of the microsomal fraction. In contrast, carboxylesterases of liver lysosomes are different, their isoelectric point being in the acidic range. Carboxylesterase activity is also found in the cytosolic fraction of liver and kidney. It has been suggested that cytosolic carboxylesterases are mere contaminants of the microsomal enzymes, but there is evidence that soluble esterases do not necessarily originate from the endoplasmic reticulum [94], In guinea pig liver, a specific cytosolic esterase has been identified that is capable of hydrolyzing acetylsalicylate and that differs from the microsomal enzyme. Also, microsomal and cytosolic enzymes have different electrophoretic properties [77]. Cytosolic and microsomal esterases in rat small intestinal mucosa are clearly different enzymes, since they hydrolyze rac-oxazepam acetate with opposite enantioselectivity [95], Consequently, studies of hydrolysis in hepatocytes reflect more closely the in vivo hepatic hydrolysis than subcellular fractions, since cytosolic and microsomal esterases can act in parallel. 

Brush-type CSPs are robust and allow high sample loads. As an example, Figure 2 shows the separation of oxazepam acetate and its hydrolysis product on dinitrobenzoylleucine. 

It will perhaps be recalled from the earlier volume that such N-oxides are prone to undergo the Polonovski rearrangement when treated with acetic anhydride, and that this was illustrated by the formation of oxazepam. It is not surprising that the N-methyl analogue (4) also undergoes this process, and hydrolysis of the resulting acetate gives... 

Figure 3. Inhibition of the reaction between p-nitrophenyl acetate and HSA by drugs. Data for oxazepam (0), fiufenamic acid (D), and naproxen (O) obtained at pH 7.4 in 0.03M triethanolamine/HCl at 25°C with 3.5 X 10 5M HSA. Dissociation constants are given in Table II.

Benzodiazepine derivatives. A wide variety of chiral benzodiazepines including oxazepam, lorazepam, and temazepam as well as their hemisuccinate and acetate derivatives have been resolved (85,92). 

TLC methods for the separation of various benzodiazepine tranquilizers have been reported. Thus, Okumura and Nagaoka (1979) separated various benzodiazepines in methanol-water (2 1 or 3 1), and spots were detected by fluorescence quenching. Tranquilizers and their metabolites have been chromatographed on Whatman HP-KF plates (Whatman Technical Series, Volume 2). Samples prepared in methanol were separated in the mobile phase ethyl acetate-methanol (95 5) spots were detected by fluorescence quenching under 254-nm ultraviolet (UV) light the compounds along with their values were n-desmethycheordiazepoxide, 0.11 chlordiazepoxide, 0.21 demoxepam, 0.38 oxazepam, 0.45 and diazepam, 0.60. 

---