Dopamine- and tyramine-based derivatives of triazenes: activation by tyrosinase and implications for prodrug design.

A range of triazene derivatives were synthesized and investigated as prodrug candidates for melanocyte-directed enzyme prodrug therapy (MDEPT). The prodrugs contained a tyramine or dopamine promoiety required for tyrosinase activation and this was joined via a urea functional group to the cytotoxic triazene. The stability of each of the prodrugs in phosphate buffer, human plasma and in mushroom tyrosinase is discussed. The identification of the main peak formed after the tyrosinase reaction was attempted by LC-MS and the conversion of prodrug to the quinone was confirmed.

Towards an efficient prodrug of the alkylating metabolite monomethyltriazene: synthesis and stability of N-acylamino acid derivatives of triazenes.

A series of 3-[alpha-(acylamino)acyl]-1-aryl-3-methyltriazenes 6a-l, potential cytotoxic triazene prodrugs, were synthesised by coupling 1-aryl-3-methyltriazenes to N-acylamino acids. Their hydrolysis was studied in isotonic pH 7.4 phosphate buffer and in human plasma, while hydrolysis of the derivative 6a was studied in more depth across a range of pH values. Prodrugs 6a-l hydrolyse by cleavage of the triazene acyl group to afford the corresponding monomethyltriazenes. Studies in human plasma demonstrate that acylation of the alpha-amino group of the amino acid carrier is an effective means of reducing the chemical reactivity of the alpha-aminoacyl derivatives while retaining a rapid rate of enzymatic hydrolysis. These derivatives displayed logP values that suggest they should be well absorbed through biological membranes.

Determination and pharmacokinetic study of 1-p-(3.3-dimethyl-1-triazeno) benzoic acid in cancer patients by capillary gas chromatography.

A capillary gas chromatographic method was developed for determining 1-p-(3.3-dimethyl-1-triazeno) benzoic acid in the plasma and urine of cancer patients under pharmacokinetic study. The drug was extracted with ethyl acetate and methylated with diazomethane. Octadelane (10 microg/ml) was added as internal standard. The separation was carried out on an OV-1 quartz capillary column, 15 m x 0.32 mm (0.52 microm), with high-purity nitrogen as carrier gas and flame ionization detector (FID) as detector. The column temperature was held at 130 degrees C for 9 min and then programmed to 240 degrees C, at a rate of 35 degrees C/min. The temperature of both injector and detector was 260 degrees C. The standard curve was linear from 0.4 to 40 microg/mL in plasma, and from 0.8 to 20 microg/mL in urine, with correlation coefficients of 0.9979 and 0.9932. The relative standard deviations (RSD) were less than 9.7%. The minimum recovery of this method was 81.8%. This method was applied to the pharmacokinetic studies of 1-p-(3.3-dimethyl-1-triazeno) benzoic acid in cancer patients after a single dose (i.v.) of 160, 420 or 760 mg/m(2) was administered. They all conformed to the two-compartment open model and showed linear pharmacokinetics. The excretion of this drug in the urine was minimal.

Triazene drug metabolites: part 15. Synthesis and plasma hydrolysis of anticancer triazenes containing amino acid carriers.

PURPOSE: The synthesis of chemically stable triazene prodrugs capable of hydrolysing under physiological conditions to liberate cytotoxic monomethyltriazene alkylating agents. METHODS: A series of 3-aminoacyl-1-aryl-3-methyltriazenes was synthesised through reaction of 1-aryl-3-methyltriazenes with N-BOC protected amino acids using the DCC method of activation, followed by deprotection of the amino function using HCl in nitromethane. Half-lives for the hydrolysis of these compounds to the corresponding monomethyltriazenes at 37 degrees C in isotonic phosphate buffer and in 80% human plasma containing 20% phosphate buffer were determined by HPLC. RESULTS: The aminoacyltriazene prodrugs hydrolyse in isotonic phosphate buffer with t1/2 values ranging from 26 to 619 minutes. In human plasma, several decompose at the same rate as in phosphate buffer whereas those containing more lipophilic groups decompose more slowly. A beta-alanyl derivative was found to be more stable in phosphate buffer (t1/2 = 180 minutes) than in plasma (t1/2 = 53 minutes). An N-acetylated alpha-alanyl derivative was found to be chemically stable in phosphate buffer (t1/2 = 10 hours) but liberated the cytotoxic drug in t1/2 = 41 minutes in plasma, demonstrating its ability to act as a substrate for plasma enzymes. CONCLUSIONS: Aminoacyltriazenes are prodrugs of the antitumour monomethyltriazenes hydrolysing in human plasma with a range of reactivities. The acylation of the alpha-amino group seems to be an effective and simple means of reducing the chemical reactivity of the alpha-aminoacyl derivatives while retaining a rapid rate of enzymatic hydrolysis.

Metabolism of 1,3-di-(4-[N-(4,6-dimethyl-2-pyrimidinyl)sulphamoyl] phenyl)triazene (DDPSPT) in the rat.

1. Six hours after rats were orally dosed with 1,3-di-(4-[N-(4,6-dimethyl-2-pyrimidinyl)sulphamoyl][U-14C]phenyl) triazene (14C-DDPSPT), approx. 81% of the 14C remained in the gastrointestinal tract (gut) and less than 3% was excreted in the urine. 2. Six hours after dosing, more than half of the 14C in the gut was present as DDPSPT. 14C-Labelled metabolites in the gut included 4-amino-N-(4,6-dimethyl-2-pyrimidinyl)-benzenesulphonamide (Sulmet), N4-glucosyl-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulphonamide (N4-gluc-Sulmet), 4-acetamido-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulphonamide (N4-acetyl-Sulmet), and [N-4,6-dimethyl-2-pyrimidinyl) benzenesulphonamide] (desamino-Sulmet). 3. 14C-Labelled compounds in the blood, liver and skeletal muscle included DDPSPT, Sulmet, N4-gluc-Sulmet, N4-acetyl-Sulmet and desamino-Sulmet. 4. There was little or no reaction of DDPSPT with cysteine, bovine serum albumin, AMP, GMP, or calf thymus deoxyribonucleic acid in vitro (pH 3, 5, 7 or 8).

High-performance liquid chromatographic assay for the determination of p-(3,3-dimethyl-1-triazeno)benzoic acid in mouse plasma.

A reproducible and sensitive method is described for assaying p-(3,3-dimethyl-1-triazeno)-benzoic acid (pCOOH-DMT) and for identifying the N-desmethyl metabolite, p-(3-methyl-1-triazeno)benzoic acid (pCOOH-MMT) using high-performance liquid chromatography. The method measures concentrations as low as 1.25 nmol/ml of plasma. Extraction efficiency of internal standard or of added triazenes averages 88% and the coefficient of variation of the method is less than 10%. pCOOH-DMT is stable at room temperature at pH 7.4, whereas pCOOH-MMT undergoes rapid decomposition (half-life 6 min). pCOOH-MMT is more stable in an albumin-containing solution or in plasma, but not in boiled 9000 g mouse liver. After 80 min incubation with a 9000 g mouse liver fraction and reduced nicotinamideadenine dinucleotide phosphate, only 24% pCOOH-DMT was metabolized. Plasma pharmacokinetic studies in mice treated with 200 mg/kg intraperitoneally showed that the potassium salt of pCOOH-DMT has a half-life of 67 min.