Preliminary communication

Observations on the differential metabolism and biological activity of the optical isomers of cyclophosphamide

‘Nitro­gen mustard’ bis­(2-chloro­eth­yl)amine derivatives (2R,4S,5R)- and (2S,4S,5R)-2-[bis­(2-chloro­eth­yl)amino]-3,4-dimethyl-5-phenyl-1,3,2-oxaza­phos­pho­lidin-2-one (2a and 2b, respectively), C₁₄H₂₁Cl₂N₂O₂P, and (2R,4R)- and (2S,4R)-2-[bis­(2-chloro­eth­yl)amino]-4-isobutyl-1,3,2-oxaza­phospho­lidin-2-one (3a and 3b, respectively), C₁₀H₂₁Cl₂N₂O₂P, were synthesized as a mixture of diastereomers through a 1:1 reaction of enanti­omerically pure chiral amino alcohols with bis­(2-chloro­eth­yl)phospho­ramidic dichloride. Flash column chromatography yielded diastereomerically pure products, as supported by ³¹P NMR. The crystal structures of 2b and 3b were obtained to determine their absolute configuration at phospho­rus, and ³¹P NMR chemical shift trends are proposed based on the spatial relationship of the bis­(2-chloro­eth­yl)amine moiety and the chiral substituent of the amino alcohol. Oxaza­phospho­lidinones were observed to have a more downfield ³¹P NMR chemical shift when the aforementioned substituents are in a syn configuration and vice versa for when they are anti.

This chapter presents the synthesis of heterocycles with a stereogenic phosphorus or sulfur atom derived from aminoalcohols or aminonaphthols, which has been reported recently (usually after 2002). It also contains selected references to the earlier papers and is divided into three sections, describing methods of synthesis of the three particular classes of heterocyclic derivatives. The first two are devoted to heterocycles with a stereogenic phosphorus atom and discuss the protocols for the preparation of 1,3,2-oxazaphospholanes (1,3,2-oxazaphospholidines) with a tri- and tetracoordinated phosphorus atom, 1,3,2-oxazaphosphorinanes and larger rings containing a stereogenic phosphorus atom forming part of the nitrogen-phosphorus-oxygen (NPO) grouping. The third section concerning heterocycles with a stereogenic sulfur atom describes the synthesis of all kinds of 1,2,3-oxathiazolidine 2-oxides and tetrahydro-1,2,3-oxathiazine-2-oxides.

This article presents an overview of the methods of bioanalysis of oxazaphosphorines, in particular, cyclophosphamide, ifosfamide, and trofosfamide as well as their metabolites. The metabolism of oxazaphosphorines is complex and leads to a large variety of metabolites and therefore the spectrum of methods used is relatively broad. The various methods used are shown in a table and the particularly important assays are described.

The enantiomers of the cytostatic drug ifosfamide and the two metabolites 2- and 3-dechloroethylifosfamide were isolated from plasma and urine by liquid-liquid extraction with ethyl acetate, resolved on a Chirasil-l-val gas chromatographic column and detected by a nitrogen-phosphorus-selective flame ionisation detector. Resolution of the racemic compounds for identification purposes was also accomplished with high-performance liquid chromatography on a chiral column. The validated gas chromatographic method was suitable to determine the total concentrations and the enantiomeric composition of ifosfamide and its dechloroethylated metabolites in plasma and urine samples from treated patients. Some metabolic preferences in the metabolism of ifosfamide were found.

A reversed-phase liquid chromatographic method to determine the concentration of 4-hydroxycyclophosphamide, a labile cytotoxic metabolite of cyclophosphamide, in plasma is described. In order to stabilize 4-hydroxycyclophosphamide, as well as to increase the selectivity and the sensitivity, a 2,4-dinitrophenylhydrazone derivative was formed. Plasma proteins were precipitated with acetonitrile prior to the derivatization with 2,4-dinitrophenylhydrazine at pH 2. The derivatization was performed at 50°C for 5 min. The chromatographic system consisted of an octadecyl silica column and a mobile phase containing phosphate buffer and acetonitrile. Quantitation was performed using an UV detector operating at 357 nm. Optimal derivatization was obtained by adding 0.2 ml 2,4-dinitrophenylhydrazine (3.8 mg/ml) to 0.5 ml of the deproteinized plasma supernatant. The relative recovery of 4-hydroxycyclophosphamide from plasma is ⩾97%. Concentration levels down to 22 ng/ml of 4-hydroxycyclophosphamide in plasma could be determined with a R.S.D. of about 13%. No degradation of the derivative was observed after 24 h at room temperature. The t_{$\text{1}\text{2}$} for 4-hydroxycyclophosphamide in blood is ca. 4 min at 37°C, whereas 4-hydroxycyclophosphamide is stable for at least 1 h at 4°C. Application of the method for the pharmacokinetic monitoring of 4-hydroxycyclophosphamide is described.

The synthesis and biological evaluation of chiral, diastereomeric phosphorus mustards derived from natural and unnatural serine are reported herein.