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)

The crystal and molecular structure of enantiomerically homogeneous cyclophosphamide (C7H I 5N202PC12) has been determined by x-ray diffraction with the absolute configuration being established by the anomalous dispersion of the CI and P atoms. It is found that the dextrorotatory enantiomer of cyclophosphamide ([a]&'2.3O (c 3.0, methanol)) has the R configuration a t phosphorus. The compound crystallized in the rhombohedral space group R3 with the three molecules in the cell related by the threefold axis forming a trimeric unit by NH-O=P hydrogen bonding. Cell parameters are a = 10.520 ( 5 ) 8, and a = 108.9 (1)'. The conformation of the enantiomerically homogeneous cyclophosphamide a s compared to the racemate differs mainly in the orientation of one of the chloroethyl chains. Cyclophosphamide(2-[bis(2-chloroethyl)amino]-2H-1,3,2-0xazaphosphorinane 2-oxide, 1) is a widely used anticancer,drug which is prepared synthetically and administered clinically in racemic form (Cytoxan).2 The broad spectrum of activity3 exhibited by 1 has led to considerable interest in its metabolism, and a substantial amount of chemical and biochemical data supports the degradative pathway shown in Scheme I. Fragmentation of enzymatically produced 4-hydroxycyclophosphamide ( 2 ) and/or its putative aldehyde tautomer, aldophosphamide (3), affords acrolein and phosphoramide mustard (4), generally regarded as the ultimate DNA cross-linking agent. Competing enzymatic conversion of 2 and/or 3 into 4-ketocyclophosphamide ( 5 ) and carboxyphosphamide (6) is associated with drug deto~ification.~ Since biological systems normally exhibit a marked enantiomeric selectivity, it was expected5 and recently found6 that the antipodal forms of 1 exhibit significantly different therapeutic indices7 with (-)-I being more effective against PC6 mouse Karle et al. / (+)-Cyclophosphamide Absolute Configuration

Development of myometrium in young female rats was stimulated by administration of diethylstilboestrol. Plasma membrane and sarcoplasmic reticulum from rat myometrium were separated by a new and rapid method using a Percoll gradient. Calcium uptake was inhibited in plasma membrane vesicles isolated from oxytocin-treated myometrium, while no consistent effect of oxytocin was found on the Ca2+ uptake in the sarcoplasmic reticulum. Oxytocin regulated the plasma membrane Ca2+ pump by decreasing its apparent affinity for Ca2+ without affecting its maximal velocity. The K1/2 for Ca2+ in the absence of calmodulin was 0.41 +/- 0.04 microM in normal membranes; this was increased to 0.93 +/- 0.12 microM in oxytocin-treated membranes. Calmodulin decreased the K1/2 for Ca2+ to 0.27 +/- 0.027 microM and oxytocin also increased this, to 0.46 +/- 0.061 microM. The effect of oxytocin on the plasma membrane Ca2+ pump was highly dependent on the hormonal status of the animals. When the diethylstilboestrol was administered together with progesterone, the inhibitory action of oxytocin was totally suppressed, consistent with the expected action of this agent. The results suggest that regulation of the plasma membrane Ca2+ pump may be important in the prolonged elevation of intracellular Ca2+ caused by oxytocin.

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Die Hydrierung des Benzyloxycyclophosphamids (I) ergibt Hydroxy‐ (IIa) und Cyclophosphamid (IIb).