Labeled oxazaphosphorines for applications in mass spectrometry studies. 2. Synthesis of deuterium-labeled 2-dechloroethylcyclo-phosphamides and 2- and 3-dechloroethylifosfamides

The prodrugs cyclophosphamide (CP) and ifosfamide (IF) each metabolize to an active alkylating agent through a cytochrome P450-mediated oxidation at the C-4 position. Competing with this activation pathway are enzymatic oxidations at the exocyclic αand α’ carbons of CP

and IF. Each reaction at a side chain produces a hemiaminal; rearrangement results in a fragmentation of the parent drug to give a dechloroethyl oxazaphosphorine and chloroacetaldehyde. No toxicities have been linked unambiguously to the dechloroethyl metabolites; however, chloroacetaldehyde is associated with neurotoxicity, and this side effect, especially during IF treatment, can be dose-limiting.

The incidence of oxidation at one position relative to another is believed to be at least one factor underlying the high degree of interpatient variability in both CP and IF pharmacokinetics.

As a result, there is much interest in determining the influence of different variants, such as race, gender, age, and polymorphisms, on the extent of C-4 and side chain oxidations in CP and IF. There are multiple reports of applications of mass spectrometry (MS) to such studies, particularly those relating to the quantification of C-4 oxidation. Typically, deuterated analogs of the C-4 oxidized analytes are used as internal standards. The availability of labeled dechloroethyl metabolites for use as internal standards would facilitate extensions of such work to the reliable quantification of each dechloroethylation reaction.

[2,2,3,3-2H4]-3-Amino-1-propanol ([2,2,3,3-2H4]-6).

As reported, 3-hydroxypropionitrile was reacted with NaOD/D2O(≥98 atom %D) to give [2,2-2H2]-3-deuterioxypropionitrile (DOCH2CD2CN) in 65% yield [Rf 0.52, EtOAc-hexanes (3:1)]. This nitrile was then reduced with LiAlD4(98 atom %D) according to the literature to give the fina product [96% yield].

[4,4,5,5-2H4]-2-Dechloroethylcyclophosphamide ([4,4,5,5-2H4]-3).Equivalent to [4,4,5,5-2H4]-3-dechloroethylifosfamide ([4,4,5,5-2H4]-5).

A solution of [2,2,3,3-2H4]-6 (461mg, 5.8mmol) in CH2Cl2 (6mL) wasnadded dropwise to a cooled (ice bath) solution of freshly distilled POCl3 (0.55mL, 5.9mmol) in CH2Cl2 (17mL). Et3N (1.66mL, 11.9mmol) was then added, and the mixture was stirred for 5 h at ice bath temperature. 2-Chloroethylamine hydrochloride (770mg, 6.6mmol) was added as a solid, in one portion, followed by Et3N (1.66mL, 11.9mmol). The reaction mixture was stirred at room temperature overnight and was then filtered through a pad (2’’) of Celite 545 (Thermo Fisher Scientific, Hampton, NH). The filtrate was concentrated, and the residue was flash-chromatographed using CH3OH-CH2Cl2 (5:95). The product was still contaminated, and therefore, the material was flash-chromatographed again using EtOH-EtOAc (1:9). Pure product was obtained [527mg, 45%, Rf 0.19 in EtOH-EtOAc (1:9)].


With minor modification to a literature preparation of unlabeled material, AlD3 [from LiAlD4 (98 atom %D, 1.0 g, 22.6mmol)] was reacted with [2,2,3,3-2H4]-7 (1.5 g, 6.4mmol) for ~12 h. The flask was then cooled (ice bath), and the reaction was quenched with the dropwise addition of 1M sodium potassium tartrate (5.0mL). The mixture was diluted with water (100mL) and CH2Cl2 (50mL); 40% NaOH (~1.0–1.5mL) was added to insure a basic pH. The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3 × 50mL). All organic layers were combined

dried (Na2SO4), filtered, and evaporated to afford the product (86%) as a pure, colorless oil [Rf 0.50 in NH3-saturated CH3OH : CH2Cl2 (1:9)].

There is ongoing interest in determining the influence of C-4 versus side chain oxidations on the interpatient variability in response to treatment with the anticancer agents CP and IF. MS has been applied to both clinical and in vitro studies, particularly those relating to the quantification of C-4 oxidation. The syntheses of deuterium-labeled internal standards have been reported for use in C-4 oxidation studies; deuterated standards for companion investigations of dechloroethylation reactions were the synthetic goals of this work. Oxidative dechloroethylation of CP gives one possible product, whereas the same reaction of IF leads to two possible products. Tetra- and/or hexadeuterated analogs of each of these metabolites were synthesized by multistep but otherwise generally straightforward pathways.



James B. Springer, J. Label Compd. Radiopharm 2014, 57, 110-114