NMR spectroscopic studies of intermediary metabolites of cyclophosphamide. A comprehensive kinetic analysis of the interconversion of cis- and trans-4-hydroxycyclophosphamide with aldophosphamide and the concomitant partitioning of aldophosphamide between irreversible fragmentation and reversible conjugation pathways

Abstract

Multinuclear (31P, 13C, 2H, and 1H) Fourier-transform NMR spectroscopy, with and without isotopically enriched materials, was used to identify and quantify, as a function of time, the following intermediary (short-lived) metabolites of the anticancer prodrug cyclophosphamide (1, Scheme I): cis-4-hydroxycyclophosphamide (cis-2), its trans isomer (trans-2), aldophosphamide (3), and its aldehyde-hydrate (5). Under a standard set of reaction conditions (1 M 2,6-dimethylpyridine buffer, pH 7.4, 37 degrees C), the stereospecific deoxygenation of synthetic cis-4-hydroperoxycyclophosphamide (cis-12, 20 mM) with 4 equiv of sodium thiosulfate (Na2S2O3) afforded, after approximately 20 min, a "pseudoequilibrium" distribution of cis-2, 3, 5, and trans-2, i.e., the relative proportions of these reactants (57:4:9:30, respectively) remained constant during their continual disappearance. NMR absorption signals indicative of "iminophosphamide" (8) and enol 6 were not detected (less than 0.5-1% of the synthetic metabolite mixture). A computerized least-squares fitting procedure was applied to the individual 31P NMR derived time courses for conversion of cis-2, 3 plus 5 (i.e., "3"), and trans-2 into acrolein and phosphoramide mustard (4), the latter of which gave an expected array of thiosulfate S-alkylation products (e.g., 16) and other phosphorus-containing materials derived from secondary decomposition reactions. This kinetic analysis gave the individual forward and reverse rate constants for the apparent tautomerization processes, viz., cis-2 in equilibrium "3" in equilibrium trans-2, as well as the rate constant (k3) for the irreversible fragmentation of 3. The values of k3 at pH 6.3, 7.4, and 7.8 were equal to 0.030 +/- 0.004, 0.090 +/- 0.008, and 0.169 +/- 0.006 min-1, respectively. Replacement of the HC(O)CH2 moiety n 3 with HC(O)CD2 led to a primary kinetic isotope effect (kH/kD = 5.6 +/- 0.4) for k3. The apparent half-lives (tau 1/2) for cis-2, "3", and trans-2 under the standard reaction conditions, at "pseudoequilibrium" (constant ratio of cis-2/"3"/trans-2), were each equal to approximately 38 min, which is considerably shorter than the widely cited colorimetrically derived half-lives reported by earlier investigators. The values of tau 1/2 for cis-2, "3", and trans-2 were affected by pH in the same manner as that found for k3 but were relatively insensitive to the presence of either K+, Na+, Ca2+, or Mg2+. The presence of certain primary amines led to marked decreases in tau 1/2 and, in some cases, the formation of acyclic adducts of aldehyde 3.(ABSTRACT TRUNCATED AT 400 WORDS)

Cardiotoxicity of commercial 5-fluorouracil vials stems from the alkaline hydrolysis of this drug

“…A critical impurity was identified in the FU Tris vials, namely the oxazolidine of Facet. It is known that Tris reacts readily with propionaldehyde or aldoifosfamide to produce the corresponding oxazolidines (Borch & Getman, 1984;Zon et al, 1984). We demonstrated by synthesis and isolation of this impurity and with conventional spectroscopic techniques that Tris also reacts with Facet to give the fluorinated oxazolidine 2.…”

Low-dose metronomic cyclophosphamide treatment mediates ischemia-dependent K-ras mutation in colorectal carcinoma xenografts

“…The 4-hydroperoxycyclophosphamide is a preactivated derivative of cyclophosphamide that hydrolyzes spontaneously in aqueous solution to form 4-hydroxycyclophosphamide. Mafosfamide (D-17272, NIOMECH, IIT GmbH, Bielefeld, NRW, Germany) is a cyclophosphamide derivative that rapidly generates 4-hydroperoxycyclophosphamide after aqueous dissolution (Takamizawa et al, 1975;Zon et al, 1984).…”

New approach to the activation of anti-cancer pro-drugs by metalloporphyrin-based cytochrome P450 mimics in all-aqueous biologically relevant system

“…Cyclophosphamide (CP), an oxazaphosphorine-type of cytotoxic pro-drug (including ifosfamide, mafosfamide, and trofosfamide), is the single most commonly utilized component in (i) conventional chemotherapy and (ii) high-dose chemotherapy/bone marrow transplant/stemcell rescue regiments for cancer treatments [1][2][3][4][5]. The activation through the hydroxylation of CP by microsomal cytochrome P450 enzymes in liver, leads to 4-hydroxycyclophosphamide (4-OHCP) (Scheme 1), and ultimately to the formation of cytotoxic phosphoramide mustard which alkylates DNA, thus preventing the proliferation of tumor cells [1][2][3][4][5].…”