O-Methylhydroxylamine as a New Trapping Reagent for Quantitative Studies of 4-Hydroxycyclophosphamide and Aldophosphamide

Zon, Gerald; Ludeman, Susan M.; Sweet, E. M.; Egan, William; Phillips, L.

doi:10.1002/jps.2600710417

Cited by 11 publications

(4 citation statements)

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“…independently prepared by separate reactions of CH3ONH2 with 2/342 and acrolein, respectively; the E and Z stereochemical assignments for the diastereomers of 18 and 19 were based on reported structure-spectrum correlations. 42,82 The relatively rapid conversion of acrolein into aldehyde B and hydrate A accounted for the previously described 2H NMR results concerning the near absence of acrolein-a-d and the loss of its deuterium, e.g., enolization of aldehyde B. The completely reversible nature of this "postfragmentation" chemistry was in accord with the fact that stable derivatives of acrolein are isolable via incubations of 2/3 in the presence of a trapping agent.46,83 Other investigators35 have stated that the acrolein produced from 2/3 may "polymerize", but no details were given.…”

Section: Resultsmentioning

confidence: 99%

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

Zon¹,

Ludeman²,

Brandt³

et al. 1984

J. Med. Chem.

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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)

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Section: Resultsmentioning

confidence: 99%

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

Zon¹,

Ludeman²,

Brandt³

et al. 1984

J. Med. Chem.

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“…Product was desorbed from silica gel using CHCl3-CH3OH (1:1), and 15 was obtained as a white solid in 56% yield (0.25 mmol, 59 mg, 56% E and 44% Z-isomers, mp range 165-195 °C with decomposition over 200 °C). Signal assignments and isomer composition by 1 H NMR were based on literature reports of spectral data for similar oximes (24). 1 H NMR (DMSO-d6) δ 12.40 (br s, 1H, exchanges with D2O, NH), 7.78 (s, 1H, C-8 proton), 7.45 (t, J ) 6 Hz, 0.56 H, ONdCH of E-isomer), 6.85 (t, J ) 5 Hz, 0.44 H, ONdCH of Z-isomer), 6.20 (s, 2H, exchangeable with D2O, NH2), 4.50 (E) and 4.48 (Z) [two t, J ) 7 (E) and 6 (Z) Hz, 2H total, OCH2], 3.75 (Z) and 3.69 (E) [two s, 3H total, OCH3] and 2.69 (Z) and 2.59 (E) [two apparent q, J ) 6 (Z) and 6 (E) Hz, 2H total, OCH2CH2].…”

Section: Methodsmentioning

confidence: 99%

“…Aldophosphamide (3) is readily converted to stable oximes through reactions with hydroxylamines (24,45). In anticipation that this chemistry might have some applications to the ultimate goal of detecting O 6 -alkylated residues in DNA, the stability of the O-methylhydroxyloxime of O 6 -PAG (15) was studied in a limited manner.…”

Section: Methodsmentioning

confidence: 99%

“…Product 17 was obtained as a white solid in 19% yield [0.04 mmol, 10 mg, 35% E-and 65% Z-isomers, Rf 0.65 in CHCl3-CH3OH (7:3), mp 200-206 °C]. Signal assignments and isomer composition by 1 H NMR were based on literature reports of spectral data for similar oximes (24). 1 H NMR (DMSO-d6) δ 12.47 (br s, 1H, exchangeable with D2O, NH), 7.86 (s, 1H, C-8 proton), 7.72 (apparent t, J ) 6 Hz, 0.35 H, ONdCH of E-isomer), 7.11 (apparent t, J ) 4 Hz, 0.65 H, ONdCH of Z-isomer), 6.31 (br s, 2H, exchangeable with D2O, NH2), 5.16 (Z) and 4.98 (E) [two d, J ) 4 (Z) and 6 (E) Hz, 2H total, OCH2], and 3.86 (Z) and 3.80 (E) (two s, 3H total, OCH3).…”

Section: E/z-o 6 -(N-ethanalyl)guanine O-methyloxime (E/z-17; E/z-o 6...mentioning

confidence: 99%

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Modified Guanines Representing O⁶-Alkylation by the Cyclophosphamide Metabolites Acrolein and Chloroacetaldehyde: Synthesis, Stability, and ab Initio Studies

Balu

Gamcsik

Colvin

et al. 2002

Chem. Res. Toxicol.

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Alkylation of DNA by acrolein and/or chloroacetaldehyde may result in the mutations that lead to the therapy-induced leukemia associated with cyclophosphamide (and ifosfamide) treatment. O(6)-(n-Propanalyl)guanine (O(6)-PAG) and O(6)-(ethanalyl)guanine (O(6)-EAG) were synthesized for use as authentic standards in investigations of DNA alkylation by acrolein and chloroacetaldehyde, respectively. Preparation of the O-methyl oximes of these aldehydes aided in confirming the structural assignments of O(6)-PAG and O(6)-EAG. HPLC was used to study the stability of O(6)-PAG under a variety of conditions. The decomposition of O(6)-PAG was attributed to an alpha,beta-elimination reaction resulting in the formation of guanine and acrolein. In 0.1 M phosphate-DMSO (9:1), O(6)-PAG (1-10 mM) had a half-life of approximately 1 h (pH 7.4, 37 degrees C). In 0.05 M Tris-DMSO (9:1), the apparent half-life of O(6)-PAG (1-10 mM) was approximately 16 h (pH 7.4, 37 degrees C). The increased lifetime under the latter conditions was attributed to a reversible reaction between Tris and the aldehydic functionality of O(6)-PAG to give a more stable oxazolidine. Under conditions similar to those that would be used for hydrolysis of DNA [0.1 M HCl-DMSO (98:2), pH 1.3, 70 degrees C, 30 min], there was an estimated 10-35% loss of O(6)-PAG. Under the same conditions, O(6)-EAG had apparent half-lives of 6.6 h (phosphate-DMSO) and 2.5 days (Tris-DMSO) and the estimated loss at pH 1.3 over 30 min (70 degrees C) was 15-20%. Ab initio quantum chemical calculations were used to understand the energy factors that underlie the occurrence of O- versus N-alkylations as well as possible, subsequent intramolecular cyclizations. Simulations of the free energies of reactions between acrolein and guanine indicated that N-alkylation was favored over O(6)()-alkylation and that cyclizations to tautomers were most favorable if they involved the N-1 or NH(2) positions.

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Photolabile Carbonyl Protecting Group: A New Tool for Light‐Controlled Release of Anticancer Agents

2009

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O-Methylhydroxylamine as a New Trapping Reagent for Quantitative Studies of 4-Hydroxycyclophosphamide and Aldophosphamide

Cited by 11 publications

References 12 publications

Modified Guanines Representing O⁶-Alkylation by the Cyclophosphamide Metabolites Acrolein and Chloroacetaldehyde: Synthesis, Stability, and ab Initio Studies

Photolabile Carbonyl Protecting Group: A New Tool for Light‐Controlled Release of Anticancer Agents

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O-Methylhydroxylamine as a New Trapping Reagent for Quantitative Studies of 4-Hydroxycyclophosphamide and Aldophosphamide

Cited by 11 publications

References 12 publications

Modified Guanines Representing O6-Alkylation by the Cyclophosphamide Metabolites Acrolein and Chloroacetaldehyde: Synthesis, Stability, and ab Initio Studies

Photolabile Carbonyl Protecting Group: A New Tool for Light‐Controlled Release of Anticancer Agents

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Product

Resources

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Modified Guanines Representing O⁶-Alkylation by the Cyclophosphamide Metabolites Acrolein and Chloroacetaldehyde: Synthesis, Stability, and ab Initio Studies