The chemical reduction of diaziquone: products and free radical intermediates.

Sodium borohydride reduced diaziquone (AZQ) can cause cross-links between DNA molecules, between DNA and proteins and cause single- and double-strand DNA breaks. In order to understand these effects better, we investigated the reduction of diaziquone by borohydride, and looked at reaction products. We found that a major product was formed during the oxidation of the colorless 2-electron reduced AZQ, and that this product was a monoaziridinyl quinone. We interpret this result to mean that both the leaving aziridine as well as the remaining one can alkylate. This mode of alkylation does not explain cross-links which may occur by a different mechanism requiring simultaneous opening of the aziridine rings. Most of the antitumor activity of borohydride reduced AZQ is probably exerted during the oxidation of the 2-electron reduced AZQ (AZQH2).

Identification of metabolites of the cell-differentiating agent hexamethylene bisacetamide in humans.

Hexamethylene bisacetamide, a compound which in vitro induces differentiation in a wide variety of human and animal cancer cell lines, is being investigated in phase I clinical trials. After i.v. administration of hexamethylene bisacetamide to humans, urine contained the parent compound and at least five metabolites formed by deacetylation and oxidation pathways. Identification of urinary metabolites was accomplished by gas chromatography-mass spectrometric analysis after isolation by ion exchange chromatography or extraction with ethyl acetate. Metabolites with amino or alcohol groups were trifluoroacetylated and acidic functional groups were esterified with 2,2,2-trifluoroethanol or methanol. The structure of each metabolite was confirmed by comparison with authentic standards. Metabolites identified included the major metabolite, 6-acetamidohexanoic acid; the monodeacetylated product, N-acetyl-1,6-diaminohexane; the bis-deacetylated diamine, 1,6-diaminohexane; and the amino acid, 6-aminohexanoic acid and its lactam, caprolactam.

Diaziquone given as a continuous infusion is an active agent for relapsed adult acute nonlymphocytic leukemia.

Diaziquone given as a bolus has not been effective in patients with relapsed or refractory leukemia. Because of in vitro data suggesting enhancement of diaziquone-induced cytotoxicity for human and murine leukemia cells with increased duration of drug exposure and the relatively short terminal plasma half-life of diaziquone, 49 patients (34 acute nonlymphocytic leukemia [ANLL], six chronic myelogenous leukemia in blast crisis [CML-B], five acute lymphocytic leukemia [ALL], four 2 degrees ANLL) with leukemia were given diaziquone as a continuous infusion for seven days. The maximum tolerated dose was 28 mg/m2/d for seven days. The dose-limiting toxicity was the duration of bone marrow aplasia (median, 49 days to greater than 500 PMNs in responders; range, 28 to 101 days). Nonhematologic toxicity was minimal. Responses occurred only in patients with relapsed ANLL, of whom 26 were treated at effective doses. There were six complete responses (CR) (23%) and two partial responses (PR) (8%), although five of eight responders never achieved platelet counts greater than 100,000/microL. Thrombocytopenia in these patients was felt to be a manifestation of diaziquone effect, not persistence of leukemia. The median duration of CR was 195 days (range, 88 to 860+). One patient had active CNS leukemia at the start of treatment and has had a durable (28+ month) CR in both sites of disease. Diaziquone produced prolonged aplasia in patients with secondary ANLL and CML-B (five of ten patients died aplastic), whereas patients with ALL all had regrowth of leukemia and two failed to become aplastic. The lack of significant nonhematologic toxicity and the activity in patients with relapsed ANLL render diaziquone of interest as second-line therapy or consolidation therapy in first remission for patients with ANLL.

Effects of pH and temperature on the stability and decomposition of N,N'N"-triethylenethiophosphoramide in urine and buffer.

N,N',N"-Triethylenethiophosphoramide (thiotepa) was dissolved at 100 micrograms/ml in urine or in 0.1 M sodium acetate buffer and incubated at 37 degrees or 22 degrees. After 0, 15, 30, 60, 90, and 120 min of incubation, 0.1-ml samples were extracted into ethyl acetate and analyzed by gas-liquid chromatography (1.8-m X 2-mm column packed with 3% OV225 on 100/120 Supelcoport; oven at 180 degrees; injection port and nitrogen-phosphorus detector at 230 degrees). Thiotepa was more stable at 22 degrees than at 37 degrees and at pH 6 to 7 than at pH 4 to 5.5. After 2 hr of incubation at 37 degrees, thiotepa concentrations decreased by 40% at pH 5.0 but only 10% at pH 6 or 7. Although thiotepa concentrations declined as described above, alkylating activity, as assessed by p-nitrobenzyl pyridine reactivity, was stable at all temperatures and pHs tested. Partition coefficients of thiotepa degradation products into toluene, ethyl acetate, diethyl ether, and hexane were determined after 0 and 120 min of incubation in urine at pH 4.0. The extractability of alkylating activity into these organic solvents decreased dramatically after 120 min. Thiotepa degradation products were extracted from urine at pH 4.0 after 0, 30, 60, and 120 min incubation at 37 degrees and were separated by thin-layer chromatography. In addition to thiotepa (Rf 0.15), 3 degradation products possessing p-nitrobenzyl pyridine alkylating activity (Rf 0.35, 0.52, and 0.60) were observed during the course of incubation. The structures of the materials with Rf 0.35 and 0.52 were identified by mass spectrometry and indicated that thiotepa degradation occurs by successive addition of HCl molecules with opening of the aziridine rings and conversion to 2-chloroethyl moieties.

Putrescine metabolism: enzymatic formation and non-enzymatic isotope exchange of delta1-pyrroline.

The deamination of putrescine catalysed by diamine oxidase was carried out in deuterium oxide and deuterated buffers. Enamine and alpha, beta-unsaturated intermediates were excluded, based on the observation that deuterium was not incorporated into delta 1-pyrroline during its enzymatic formation in deuterium oxide. When the reaction mixture was buffered with phosphate, isolated delta 1-pyrroline contained two deuterium atoms at C-3, indicating that a phosphate-promoted, non-enzymatic isotope exchange had occurred. Using 5,5-dimethyl-delta 1-pyrroline as a model compound, the nature of the non-enzymatic deuterium exchange was studied and a bifunctional catalysis mechanism proposed. The results suggest that the choice of buffer could alter the conclusions drawn from enzyme mechanism studies involving imine-enamine tautomerism .

Intermolecular and intramolecular isotope effects in the deamination of putrescine catalyzed by diamine oxidase.

The enzymatic deamination of 1,4-diaminobutane (putrescine) catalyzed by hog kidney diamine oxidase was studied with the aid of deuterium labeled substrates and mass spectrometry. An intermolecular deuterium isotope effect for the deamination of putrescine labeled with deuterium in all 4 alpha positions was observed to be 1.26. 1,4-Diaminobutane-1,1-d2 was synthesized and intramolecular isotope effects determined. The preference of diamine oxidase for the unlabeled alpha position was about 4 times greater than for the deuterated methylene. This work shows that intramolecular deuterium isotope effects are observable in enzyme systems other than cytochrome P-450.

Pyrrolines as prodrugs of gamma-aminobutyric acid analogues.

delta 1-Pyrroline, 5-methyl-delta 1-pyrroline, and 5,5-dimethyl-delta 1-pyrroline have been identified as substances metabolized to gamma-aminobutyric acid (GABA), 4-aminopentanoic acid (methylGABA), and 4-amino-4-methylpentanoic acid (dimethylGABA), respectively. An enzyme system residing in the soluble fraction of rabbit liver catalyzes the conversion of delta 1-pyrroline to GABA and its lactam, 2-pyrrolidinone. Acetaldehyde, allopurinol, and cyanide inhibited the reaction. Incubation of deuterium-labeled delta 1-pyrroline with mouse brain homogenates produced deuterated GABA. Mouse liver 10,000 g supernatant and mouse brain homogenates converted 5-methyl-delta 1-pyrroline to methylGABA, and 5,5-dimethyl-delta 1-pyrroline to dimethylGABA. Four hours after intraperitoneal injection of 5-methyl-delta 1-pyrroline (200 mg/kg), methylGABA was detected in mouse brain (0.27 mumol/g). DimethylGABA (1.21 mumol/g) was determined in mouse brain 30 min after intraperitoneal administration of 5,5-dimethyl-delta 1-pyrroline (200 mg/kg). Neither methylGABA nor dimethylGABA penetrated into the central nervous system when administered in the periphery. The present studies suggest that pyrrolines may represent a chemical class of brain-penetrating precursors of pharmacologically active analogues of GABA.

Applications of deuterium labeling in the study of the in vitro conversion of delta 1-pyrroline to 4-aminobutanoic acid and 2-pyrrolidinone.

delta 1-Pyrroline is a putrescine metabolite that is biotransformed by rabbit liver preparations to 4-aminobutanoic acid and its lactam, 2-pyrrolidinone. Analysis of dilute aqueous solutions of delta 1-pyrroline by proton nuclear magnetic resonance indicated the the predominating species in the liver incubation preparations was delta 1-pyrroline monomer, although other species, such as 4-aminobutyraldehyde an delta 1-pyrroline timer, may exist in equilibrium with the monomer. [2H12]-delta 1-Pyrroline trimer was synthesized from [2H5]pyrrolidine by conversion to the N-chloro derivative followed by dehydrohalogenation. 4-Aminobutanoic acid was measured by a gas chromatographic mass spectrometric assay after derivatization with dimethylformamide dimethyl acetal. The 4-aminobutanoic acid homologue, 5-aminovaleric acid, served as internal standard. 2-Pyrrolidinone was hydrolyzed and measured as 4-aminobutanoic acid. A comparison of the amounts of product formed following incubation of labeled and unlabeled delta 1-pyrroline indicated a significant isotope effect in the formation of 2-pyrrolidinone. The influence of the label was much less on 4-aminobutanoic acid production. The results suggest that there are two separate pathways involved in the reaction.

Metabolism of methotrexate in man after high and conventional doses.

Methotrexate has been found to be extensively metabolized to 7-hydroxymethotrexate in patients receiving conventional doses (less than 10 mg/kg) and high doses (greater than 10 mg/kg). Twelve hours after administration, plasma levels of this metabolite in several patients treated with low doses exceeded those of methotrexate. No 7-hydroxymethotrexate was found in CSF after CNS administration of methotrexate; however, small amounts of the metabolite was found in the CSF after intravenous high dose infusion. We conclude that methotrexate is significantly metabolized in man at all doses used clinically.

The use of N-succinyl derivatives in the study of amino acids and peptides by mass spectrometry.

The terminal amino group of amino acids and peptides is blocked as the N-succinyl derivative by reaction with succinic anhydride. The product is then converted to the N,O-permethyl derivative in order to increase its volatility for use in mass spectrometry. The permethylated N-succinyl derivative retains the advantages of the permethylated N-acetyl derivative in regard to ease of preparation on a small scale, volatility and the presence of characteristic fragmentation patterns in their mass spectra. However, peaks in the high mass region are more abundant due to loss of CH3O-from the N-succinyl carbomethoxyl group as well as from the C-terminal carbomethoxyl group. Ions characteristic of the sequence and of individual amino acids are observed, and molecular weight can be determined from the relatively abundant ion at [M--CH3O]+ and from the weak molecular ion.