40277-05-2Relevant academic research and scientific papers
Oxime derivatives of the intermediary oncostatic metabolites of cyclophosphamide and ifosfamide: Synthesis and deuterium labeling for applications to metabolite quantification
Ludeman,Shulman-Roskes,Wong,Han,Anderson,Strong,Colvin
, p. 393 - 398 (1995)
There is ongoing interest in the selective, quantitative analysis of the cyclophosphamide metabolites 4-hydroxycyclophosphamide (2a) and aldophosphamide (3a) because these tautomers are generally believed to play a key role in oncostatic selectivity and metabolite transport. O-(2,3,4,5,6- Pentafluorobenzyl)hydroxylamine (C6F5CH2ONH2, 1 equiv) provided for the complete conversion (by 31P NMR, 60% reaction within 15 min at 20 °C) of 2a/3a (17 mM in H2O/CH3OH) to E/Z-aldophosphamide O-(2,3,4,5,6- pentafluorobenzyl)oxime [C6F5CH2ON=CHCH2CH2OP-(O)(NH2)N(CH2CH2Cl)2; E:Z = 54:46 (±3% average deviation)]. Under these conditions, the oxime exhibited little (6%) decomposition over 3 weeks. Parallel studies showed that 4-hydroxyifosfamide/aldoifosfamide reacted completely to give the analogous aldoifosfamide oxime [C6F5- CH2ON=CHCH2CH2OP(O)(NHCH2CH2Cl)2; E:Z = 52:48 (±1% average deviation)] with 50% reaction within 15 min at 20 °C with no product decomposition over 3 weeks. In aqueous methanol and with 2 equiv C6F5CH2ONH2, clinically useful 4-hydroperoxycyclophosphamide (10 mM; τ = 10 min, 37 °C) and its isomer 4-hydroperoxyifosfamide (10 mM; τ = 25 min, 20 °C) underwent complete conversion to the corresponding aldehyde oximes. Each oxime was synthesized with deuterium in the chloroethyl moieties for use as internal standards in GC/MS applications.
Synthesis of cyclophosphamide metabolites by a peroxygenase from Marasmius rotula for toxicological studies on human cancer cells
K?nig, Rosalie,Küpper, Jan-Heiner,Kammerer, Sarah,Kiebist, Jan,Scheibner, Katrin,Schmidtke, Kai-Uwe,Steinbrecht, Susanne,Thiessen, Markus
, (2020)
Cyclophosphamide (CPA) represents a widely used anti-cancer prodrug that is converted by liver cytochrome P450 (CYP) enzymes into the primary metabolite 4-hydroxycyclophosphamide (4-OH-CPA), followed by non-enzymatic generation of the bioactive metabolites phosphoramide mustard and acrolein. The use of human drug metabolites as authentic standards to evaluate their toxicity is essential for drug development. However, the chemical synthesis of 4-OH-CPA is complex and leads to only low yields and undesired side products. In past years, fungal unspecific peroxygenases (UPOs) have raised to powerful biocatalysts. They can exert the identical selective oxyfunctionalization of organic compounds and drugs as known for CYP enzymes with hydrogen peroxide being used as sole cosubstrate. Herein, we report the efficient enzymatic hydroxylation of CPA using the unspecific peroxygenase from Marasmius rotula (MroUPO) in a simple reaction design. Depending on the conditions used the primary liver metabolite 4-OH-CPA, its tautomer aldophosphamide (APA) and the overoxidized product 4-ketocyclophosphamide (4-keto-CPA) could be obtained. Using a kinetically controlled approach 4-OH-CPA was isolated with a yield of 32% (purity > 97.6%). Two human cancer cell lines (HepG2 and MCF-7) were treated with purified 4-OH-CPA produced by MroUPO (4-OH-CPAUPO). 4-OH-CPAUPO–induced cytotoxicity as measured by a luminescent cell viability assay and its genotoxicity as measured by γH2AX foci formation was not significantly different to the commercially available standard. The high yield of 4-OH-CPAUPO and its biological activity demonstrate that UPOs can be efficiently used to produce CYP-specific drug metabolites for pharmacological assessment.
4-Peroxycyclophosphamide use thereof
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Page/Page column 2, (2010/09/18)
4-Peroxycyclophosphamide is provided along with its method of preparation. The compound is useful for treating human cancer, particularly human primary and metastatic malignant brain tumors.
Effects of N-substitution of the activation mechanisms of 4-hydroxycyclophosphamide analogues
Kwon,Borch
, p. 1491 - 1496 (2007/10/02)
The activation mechanisms of the N-substituted 4-hydroxycyclophosphamide analogues 4-hydroxyifosfamide (2b), 4-hydroxytrofosfamide (2c), and 3-methyl-4-hydroxycyclophosphamide (2d) were compared with that of the unsubstituted parent compound 2a. The reaction kinetics of cis-2b, -2c, and -2d are qualitatively similar to those of 2a in that they undergo ring opening to the respective aldophosphamide intermediates 3, which can reclose to the cis- or trans-4-hydroxy isomers or undergo base-catalyzed β-elimination to generate the corresponding phosphoramide mustard products 4. In contrast to the general acid catalysis observed for ring opening of 2a and 2d, the N-(chloroethyl)-substituted analogues 2b and 2c undergo specific base-catalyzed ring opening. This mechanistic difference was also illustrated by the rapid action of 2a and 2d with sodium 2-mercaptoethanesulfonate (Mesna) under acidic conditions to give the 4-(alkylthio)-substituted cyclophosphamide derivatives 5a and 5d. Compounds 2b and 2c did not react with Mesna to generate 5b and 5c under these conditions. Both the fraction of aldehyde/hydrate present at equilibrium and the cytotoxicity against L1210 cells in vitro decreased in the order 2c > 2b > 2a > 2d. The plasma-catalyzed acceleration of phosphoramide mustard generation previously reported for 2a was also observed for these analogues.
The Mechanism of Activation of 4-Hydroxycyclophosphamide
Borch, Richard F.,Millard, Jo Ann
, p. 427 - 431 (2007/10/02)
4-Hydroxycyclophosphamide (2/3) of unknown stereochemistry is the initial metabolite formed after administration of cyclophosphamide (1).Ultimate conversion to the cytotoxic metabolite phosphoramide mustard (6) is initiated by ring opennig of 4-hydroxycyclophosphamide to produce aldophosphamide (4).The ring-opennig reaction and subsequent equilibration of 2-4 are subject to general-acid catalysis, and the equilibrium composition is independent of buffer structure and pH.In contrast, formation of 6 from 4 proceeds by general-base-catalyzed β-elimination. trans-4-Hydroxycyclophosphamide undergoes ring opening ca. 4 times faster than the cis isomer, and cyclization of 4 favors the trans isomer by a factor of ca. 3 over the cis isomer.The rapid equilibration of 2-5 and the absence of elimination to give 6 at pH ca. 5 provides a convenient method to prepare a stable equilibrium mixture of activated cyclophosphamide metabolites suitable for in vitro use.
Activation Mechanisms of Mafosfamide and the Role of Thiols in Cyclophosphamide Metabolism
Kwon, Chul-Hoon,Borch, Richard F.,Engel, Jurgen,Niemeyer, Ulf
, p. 395 - 399 (2007/10/02)
cis-Mafosfamide (cis-5) (ASTA Z7557), a stable analogue of cis-4-hydroxycyclophosphamide (cis-2), undergoes rapid decomposition in aqueous phosphate buffer or plasma at pH 7.4 and 37 deg C.The reaction kinetics of cis-5 are complex, and trans-mafosfamide (trans-5) and cis-2 are produced and subsequently disappear over the course of the reaction.The rates of decomposition of cis-5 as well as cis-2 were much faster in plasma than in buffer.The cis-trans isomerization of cis-5 occured by a specific-base-catalyzed process via iminocyclophosphamide (8) as a transient intermediate.In contrast, formation of cis- and trans-mafosfamide (5) from cis-2 and MESNA (sodium 2-mercaptoethanesulfonate) proceeded by an acid-catalyzed process via the hemithioacetal intermediate (6).The significance of these findings with respect to cyclophosphamide metabolism is discussed.
31P Nuclear Magnetic Resonanse Spectroscopic Observation of the Intracellular Transformations of Oncostatic Cyclophosphamide Metabolites
Boyd, Victoria L.,Robbins, Joan D.,Egan, William,Ludeman, Susan M.
, p. 1206 - 1210 (2007/10/02)
(31)P NMR spectroscopy was used to directly monitor, for the first time, the intracellular chemistry of the ultimate active metabolite of cyclophosphamide, namely, phosphoramide mustard.These NMR studies utilized a human histiocytic lymphoma cell line (U937), embedded in agarose gel threads, and perfused with medium containing synthetically derived metabolites (4-hydroxycyclophosphamide (2), aldophosphamide (3), and phosphoramide mustard (4)).Metabolites 2 or 3 or both readily crossed the cell membrane; in contrast, the membrane was relatively impermeable to 4.Intracellular concentrations of 4 could, therefore, be attributed primarily to the intracellular fragmentation of 3.Signals suggestive of either carboxyphosphamide or 4-ketophosphamide were not detected.Spectral data were used to calculate a rate constant of (5.4 +/- 0.3) * 10-3 min-1 for the intracellular disappearance of 4 at 23 deg C.The intracellular pH was determined to be 7.1 from the chemical shift of the internal inorganic phosphate signal.
In situ preparation and fate of cis-4-hydroxycyclophosphamide and aldophosphamide: 1H and 31P NMR evidence for equilibration of cis- and trans-4-hydroxycyclophosphamide with aldophosphamide and its hydrate in aqueous solution
Borch,Hoye,Swanson
, p. 490 - 494 (2007/10/02)
cis-4-Hydroxycyclophosphamide (2) and aldophosphamide (4) were generated in aqueous phosphate or cacodylate buffer by dimethyl sulfide reduction of cis-4-hydroperoxycyclophosphamide and by sodium periodate cleavage of 3,4-dihydroxybutyl N,N-bis(2-chloroethyl)phosphorodiamate, respectively; the reactions of 2 and 4 were examined by 1H and 31P NMR. Within 30-60 min (pH or pD 7.0, 25 °C) the same pseudoequilibrium mixture was established in both reactions, with cis- and trans-4-hydroxycyclophosphamide (2 and 3), aldophosphamide (4), and its hydrate (5) present in the approximate ratio of 4:2:0.3:1. Structures of the intermediates were assigned unambiguously based upon analysis of the chemical shifts and coupling constants in the proton spectra determined in D2O buffers, and the 31P assignments followed by correlation of component ratios at equilibrium. Free energy differences of 0.4, 0.4, and 0.7 kcal/mol at 25 °C were estimated between 2, 3, 5, and 4, respectively, with 2 being the most stable. The aldehyde 4 reacted most rapidly with water to give hydrate 5; cyclization of 4 to 3 occurred faster than to 2. Compound 5 is formed much faster than 3 from the diol cleavage, but 5 and 3 are produced at comparable rates from 2, suggesting that conversion of 2 to 3 can proceed by a mechanism other than ring opening. The rate of equilibration appears to be independent of buffer structure, indicating that bifunctional catalysis is not important in the ring-opening reaction. β-Elimination from 4 is rate limiting for the production of acrolein, and the rate for phosphate is 2- to 3-fold faster than for cacodylate under identical conditions. These results provide the first definitive evidence for the stability of the elusive aldehyde 4 in aqueous solution and for the existence of a preequilibrium among 2-5 prior to rate-limiting expulsion of phosphoramide mustard from 4.
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 ...
Zon, Gerald,Ludeman, Susan Marie,Brandt, Joan A.,Boyd, Victoria L.,Oezkan, Gunay,et al.
, p. 466 - 485 (2007/10/02)
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 deg C), the stereospecific deoxygenation of synthetic cis-4-hydroperoxycyclophosphamide (cis-12, 20 mM) with 4 equiv of sodium thiosulfate (Na2S2O3) afforded, after ca.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 ( "3" 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 in 3 with HC(O)CD2 led to a primary kinetic isotope effect (kH/kD = 5.6 +/- 0.4) for k3.The apparent half-lives (τ*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 ca.38 min, which is considerably shorter than the widely cited colorimetrically derived half-lives reported by earlier investigators.The values of τ*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(+), Ca(2+), or Mg(2+).The presence of certain primary amines led to marked decreases in τ*1/2 and, in some cases, the formation of acyclic adducts of aldehyde 3.The relatively stable adduct formed from 3 and tris(hydroxymethyl)aminomethane (Tris) at pH 7.4 and 37 deg C gave rise to a 31P NMR signal that other investigators have mistakenly ascribed to 2. 31P NMR spectroscopy was also used to examine, in considerable detail, the manifold effects of N-acetyl-L-cysteine upon the chemistry of 2, "3", and 4, which featured the formation of a mixture of diastereomeric, acyclic ...
Base-catalyzed hydrolysis of 4-hydroperoxycyclophosphamide: Evidence for iminocyclophosphamide as an intermediate
Borch,Getman
, p. 485 - 490 (2007/10/02)
cis-4-Hydroperoxycyclophosphamide (5) undergoes facile reaction with acqueous phosphate or Tris buffers at pH 7-8 and 30 °C. The kinetics of 5 are complex,and the trans-4-hydroperoxy isomer 6 is produced and subsequently disappears over the course of the reaction. Addition of hydrogen peroxide to the reaction mixture retards the disappearance rate of 5 and increases the amount of 6 generated. Rate constants for the reversible disappearance of 5 and appearance of 6 and 4-hydrocyclophosphamide (2) have been determined by nonlinear least-squares methods. The reaction is catalyzed by hydroxide ion, Tris free base, and HPO42-, with catalytic constants of 0.032 min-1 (pH 8.0), 0.052, and 0.115 M-1, respectively. The major product in the presence of Tris is the oxazolidine arising from the addition of Tris to aldophosphamide, not 2 as assumed previously. These results are consistent with a mechanism involving general-base-catalyzed elimination to produce iminocyclophosphamide as a transient intermediate; the imine can react with the hydrogen peroxide evolved in the reaction to give 5 and 6, with water to give 2, or, in general, by addition of a nucleophile to C-4. The significance of these findings with respect to other 4-substituted cyclophosphamides is discussed.
