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Triazene metabolism. VI. 3-Azidomethyl-3-alkyl-1-aryltriazenes, a new class of anti-tumour triazene with potential pro-drug applications.

The synthesis of a new series of 3-azidomethyl-3-methyl-1-aryltriazenes is described. 3-Acetoxymethyl-3-methyl-1-aryltriazenes react with a large molar excess of sodium azide in aqueous acetone to afford the 3-azidomethyltriazenes in high yield. The rate of formation of the azidomethyltriazene increasing azide concentration, suggesting either an SN2 mechanism or a significant ionic strength effect on an SN1 reaction. In the absence of azide ion, the acetoxymethyltriazene undergoes a slow hydrolysis to give a bis-anilinomethane, which presumably arises via hydrolysis of the triazene to the aniline followed by condensation with formaldehyde released during the hydrolysis. The azidomethyltriazenes undergo facile hydrolysis in aqueous buffer solution with identical kinetic parameters to those of the hydrolysis of hydroxymethyltriazenes, suggesting that the azides may be good pro-drugs for the cytotoxic monomethyltriazene, the hydrolysis product derived from the hydroxymethyltriazene. Indeed, the azidomethyltriazenes have comparable anti-tumour activity against the P388 and PC6 tumours to other triazenes in this series. Furthermore, the azidomethyltriazenes display selective toxicity towards a human tumour cell line (the BE cell line) which is deficient in the repair of O6-methylguanine lesions, suggesting that these triazenes are capable of generating the monomethyltriazene without the need for metabolic activation.

Antineoplastic Agents

X-ray studies on anti-tumour triazenes. Structures of 1-(4-carbamoylphenyl)-3,3-dimethyltriazene 1-oxide and 3,3-dimethyl-1-(4-nitrophenyl)triazene.

The molecular structures of the title compounds have been determined by X-ray crystallographic methods. The analyses revealed differences in the geometry, and by inference the bond delocalization in these two triazenes owing to the presence in the 1-oxide structure of an N-O bond. The geometries are compared to the crystal structures of the isomeric 3-oxides [Kuroda & Wilman (1985). Acta Cryst. C41, 1543-1545; Neidle, Webster, Kuroda & Wilman (1987). Acta Cryst. C43, 674-676] which shows the dominance of an alternative tautomeric equilibrium for the triazene groups.

Computer Graphics

Triazene metabolism. IV. Derivatives of hydroxymethyltriazenes: potential prodrugs for the active metabolites of the anti-tumour triazene, DTIC.

A series of derivatives of the anti-tumour hydroxymethyltriazenes have been investigated for activity in vivo and in vitro. Acetoxymethyltriazenes are active in vivo against the TLX5, P388 and PC6 tumours in mice, and inhibit the growth of TLX5, Np and Li cells in vitro without metabolic activation. The acetoxymethyltriazenes are comparable with the hydroxymethyltriazenes and monomethyltriazenes in their spectrum of activity and thus appear to be prodrugs for these species. On the other hand, a methoxymethyltriazene was found to be active on the TLX5 tumour in vivo, but did not inhibit the growth of Np cells in vitro. This latter observation is consistent with the anticipated chemical stability of the methoxymethyltriazene and the requirement for metabolic O-demethylation to generate an active species. Acetoxymethyltriazenes do not require metabolic intervention and break down chemically in phosphate buffer to the hydroxymethyltriazene, which in turn loses formaldehyde to give the incipient methylating agent, the monomethyltriazene.

Animals

Formation of triazene from sulphonamide and nitrite in acidic solution.

Formation of triazene derivatives from sodium nitrite and sulphanilamide, sulphamethizole or sulphamethoxazole in acidic solution containing saliva or canned vegetable juice was studied using reverse-phase high-performance liquid chromatography with ultraviolet light detection at 365 nm. At the optimal pH of 3.2-3.7, the mean yield of the triazene formed by reaction of sulphanilamide and nitrite in saliva was 28.8%, calculated on the nitrite content, and there was a high positive correlation (r = 0.993) between the nitrite concentration in the saliva and the triazene yield. In contrast, at the optimal pH values for triazene formation from sulphamethizole and sulphamethoxazole (3.0-3.5 and 2.5-2.9 respectively), triazene yields were much lower, accounting for only 0.9 and 1.0%, respectively, of the nitrite present. Triazene formation was partly inhibited in the presence of vegetable juice.

Chromatography, High Pressure Liquid

DNA methylating activity in murine lymphoma cells xenogenized by triazene derivatives.

We investigated whether epigenetic rather than mutational events may be involved in the induction of novel immunogenicity ("xenogenization") in murine lymphoma treated with triazene derivatives. To this end, we assessed the DNA methylation pattern of tumor cells during the course of in vivo or in vitro xenogenization by 2 triazenes, and compared it with the changes induced by the DNA hypomethylating agent 5-azacytidine (5-aza). While all of the tested agents were able to increase tumor cell immunogenicity, their effects on DNA methylating activity were opposite. In particular, the novel immunogenicity conferred by 5-aza treatment correlated well with the extent of hypomethylation induced, whereas xenogenization by triazenes was accompanied by a limited increase in DNA methylating activity. Interestingly, the antimutagenic compound quinacrine, which is known to block the xenogenizing activity of triazenes, was incapable of hypermethylating effects, whereas the hypermethylating agent cytosine arabinoside (ara-C) was apparently unable to interfere with the induction of novel immunogenicity by triazenes.

Animals

Mutagenicity of dimethyl heteroaromatic triazenes in the Ames test: the role of hydrophobicity and electronic effects.

The mutagenicities of five heterocyclic 3,3-dimethyltriazenes have been evaluated in the Ames test. The octanol-water partition coefficients (P) for these triazenes have been measured, and their electron distributions and molecular orbital energies were calculated using the MNDO semiempirical molecular orbital method. Molecular structures of three triazenes have been determined using X-ray crystallography. The mutagenicities of these five triazenes, which range from nearly inactive to very highly mutagenic, are well predicted by quantitative structure-activity relationships that had been derived previously for the mutagenicity of aryltriazenes. The form of these equations indicates that more hydrophobic and more electron-rich triazenes are more active in the Ames test. This supports the hypothesis that the ease of initial triazene activation by cytochrome P-450 governs the mutagenicity of these compounds.

Biotransformation

Functional-group modifications of dextran for linkage to a diazonium group. A potential vehicle for tumour targeting of antineoplastic triazenes.

Several derivatives of dextran, containing a triazene side-chain, which are analogues of the antitumour agent DTIC (Dacarbazine), were prepared. The extent of triazene incorporation was measured by determination of the nitrogen content, and the presence of triazene groups was corroborated by u.v.- and n.m.r.-spectroscopic measurements. Some dextran-triazenes exhibited cytotoxic activity against M21 tumour cells in vitro.

Animals

Immunogenic changes of murine lymphoma cells following in vitro treatment with aryl-triazene derivatives.

A series of dimethyl aryl-triazene derivatives and related monomethyl compounds were studied for their efficacy in mediating a strong increase in immunogenicity (i.e., chemical xenogenization, CX) of murine leukemic cells following in vitro treatment. It was found that all compounds under investigation were able to induce CX. The dimethyl derivatives were able to induce CX only after metabolic activation, whereas related monomethyl compounds were active per se. The antigenicity acquired by triazene-treated leukemic cells was very marked; intact hosts histocompatible with the parental line were able to reject up to 10(7) cells. Antigenic tumor cells retained their immunogenic properties even after a large number of transplant generations in the absence of the drug. This means that marked immunogenicity of triazene-treated cells is a stable and heritable characteristic.

Animals

Aryl-monoalkyl and cyclic triazenes: direct-acting mutagens.

An aryl-monoalkyl triazene, methyl-p-tolyl triazene (MTT) and a cyclic triazene (delta2-triazoline) are direct-acting mutagens for Salmonella typhimurium bacteria and for cell-free Hemophilus influenzae DNA. MTT causes reversion of the hisG46 base-substitution mutation, but no reversion of the hisD3052 frameshift mutation. Induced mutation frequency is not strongly influenced by modifications in the genetic background of the S. typhimurium Ames tester strains, but is mildly enhanced by the addition of a pool of amino acids to the plating medium and is strongly enhanced by liquid preincubation before plating.

Base Sequence

DNA sequence specificity of guanine N7-alkylations for a series of structurally related triazenes.

The base sequence selectivity for reaction at the guanine-N7 position was examined for a series of structurally related triazenes by a modification of a standard DNA sequencing method. The monomethyl and monochloroethyl triazenes alkylate guanines extensively at the N7 position with a general preference for runs of contiguous guanines, similar to, but not as striking as that observed previously for the chloroethylnitrosoureas. In contrast to the nitrosoureas, the triazenes had patterns of base sequence selectivity that differed somewhat from agent to agent, with the monochloro-ethylphenyltriazene having the pattern most different from the others in the series. Thus, the nature of the nonalkylating portion of the molecule can influence the ultimate alkylation preference. The monoethylating analogues alkylated weakly with little sequence preference, and the dimethyl analogues were essentially unreactive in this system.

Alkylation

Carcinogenicity of cytostatic triazenes.

Introduction of the anticancer drug dacarbazine is the result of an attempt to design antagonists of purine biosynthesis. The mechanism of action of dacarbazine depends mainly on enzymatic transformation into as yet unknown reactive (electrophilic) intermediates. Recent studies have led to the identification and synthesis of 5-(3-hydroxymethyl-3-methyl)imidazole-4-carboxamide (HMTIC), a carbinolamine metabolite with methylating capacity. Although dacarbazine and related cytostatic triazenes are effective in the treatment of malignant melanoma and other human malignancies, dacarbazine has been demonstrated to be a carcinogen in laboratory rodents. Chronic administration of dacarbazine to rats of each sex induced predominantly thymic lymphosarcomas and mammary adenocarcinomas that were transplantable. Intraperitoneally injected 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC), a metabolite of dacarbazine, induced a high incidence of mammary adenofibromas and a low incidence of uterine leiomyosarcomas. Animals treated with 5-diazoimidazole-4-carboxamide developed a low incidence of thymic, stomach, bladder or mammary tumours. Animals receiving 5-aminoimidazole-4-carboxamide developed a variety of tumours. No secondary malignancy has been reported in humans after treatment with dacarbazine alone. Despite their adverse effects, dacarbazine and related cytostatic triazene derivatives are useful clinically since their haematological toxicity is relatively moderate. As a rule, they are not cross-resistant with nitrogen mustard alkylating agents. It is hoped that research and development of second-generation N-(1-hydroxyalkyl)triazene compounds will lead to improvements in their clinical efficacy.

Animals

Chemical xenogenization of murine lymphoma cells with triazene derivatives: immunotoxicological studies.

Equitoxic doses of 5-(3-3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) and aryl-triazene derivatives (compounds all capable of inducing a marked increase in murine tumor cell immunogenicity) were studied for their effects on the host immune system. At different times after drug exposure the animals were tested for allograft responses, competence in producing lymphocytes active in lethal graft-versus-host disease, delayed-type hypersensitivity, humoral antibody production, and mitogen responsiveness. While some of the aryl-triazenes tested (DM-COOK DM-NO2) showed a pattern of immunodepression similar to that of DTIC, others were less (MIC, MM-COOK, MM-Cl) or far less (DM-Cl, MM-NO2) active than DTIC in impairing host immunocompetence, although all retained or even augmented their ability to induce chemical xenogenization.

Animals

Antimetastatic action of some triazene derivatives against the Lewis lung carcinoma in mice.

Two dimethyltriazenoimidazoles, DTIC and BRL 51308, and a benzenoid dimethyltriazene, CB 10286, have been examined for their effects in mice bearing Lewis lung carcinoma. A slight reduction of primary tumor growth was found after treatment with DTIC and BRL 51308, whereas CB 10286 caused no significant effect. On the contrary, all the tested compounds sharply reduced the number of lung metastases and also resulted in a high proportion of animals free of metastases at death. No significant cytotoxic effect of the triazenes was observed in small established pulmonary tumors, as determined by evaluating the effects of treatment on the fractional incorporation of 3H-TdR into DNA of the lung colonies. These results are in contrast to those obtained with a purely cytotoxic agent, cyclophosphamide, and indicate that all three triazene derivatives tested have selective antimetastatic properties.

Animals

Comparison of the cytotoxicity in vitro of temozolomide and dacarbazine, prodrugs of 3-methyl-(triazen-1-yl)imidazole-4-carboxamide.

The present study tested the hypothesis that the experimental antineoplastic imidazotetrazinone temozolomide degrades in the biophase to 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) and exerts its cytotoxicity via this species. MTIC is a metabolite of the antimelanoma agent dacarbazine and is thought to be responsible for the antineoplastic activity of the latter. Cytotoxicity in vitro was investigated in TLX5 murine lymphoma cells. MTIC and temozolomide were cytotoxic in the absence of mouse-liver microsomes, whereas dacarbazine required metabolic activation. The generation of MTIC from either dacarbazine, its primary metabolite 5-[3-(hydroxymethyl)-3-methyl-triazen-1-yl]-imidazole-4-carboxamid e (HMMTIC) or temozolomide was studied by reversed-phase high-performance liquid chromatography in incubation mixtures under the conditions of the cytotoxicity assay. MTIC was found in incubations of temozolomide with or without microsomes. Dacarbazine yielded MTIC (and HMMTIC) only when microsomes were included in the incubation mixture. Although the mode of action of temozolomide seems to be similar to that of dacarbazine, the results obtained in this study show that these agents differ markedly in their ability to generate the active species MTIC.

Animals

In vitro formation of a triazene compound by reaction of sulphadimidine and nitrite.

The interaction between the veterinary drug sodium sulphadimidine and nitrite has been studied under acid conditions and the formation of 1,3-di-(4-[N-(4,6-dimethyl-2-pyrimidinyl)sulphamoylphenyl)triazene (DDPSPT) was demonstrated. This compound was not mutagenic when tested on Salmonella typhimurium and Drosophila melanogaster. In addition to the formation of DDPSPT, desaminosulphadimidine was identified as a minor reaction product.

Animals

Activity of the anorectic agent 1,3-bis[2-cyano-5-(trifluoromethyl)-phenyl]triazene in in vitro and in vivo liver promoting assays.

A substituted 1,3-diaryltriazene, 1,3-bis[2-cyano-5-(trifluoromethyl)phenyl]triazene (BPT), was studied for promoting activity in vitro and in vivo. BPT inhibited intercellular molecular exchange between cultured hepatocytes and rat liver epithelial cells, although the effect was not consistent. For the in vivo assay, male F344 rats were first exposed to N-2-fluorenylacetamide (FAA) for 8 weeks to induce liver altered foci, after which those maintained on control diet for an additional 12 weeks developed a 33% incidence of liver neoplasms. In rats given 0.02% BPT in the diet as a second exposure, the final incidence of liver neoplasms was 92%, which was comparable to the enhancement by phenobarbital (PB), a known liver neoplasm promoter. In the rats given BPT after FAA, the area occupied by gamma-glutamyltranspeptidase (GGT)-positive preneoplastic and neoplastic lesions was significantly higher than in the rats exposed to FAA only. Feeding of BPT alone for 12 weeks did not induce either liver altered foci or neoplasms and it was non-genotoxic in the hepatocyte DNA repair test. Therefore, although additional studies are needed to firmly establish the basis for the enhancement of liver carcinogenesis, BPT is suggested to be a new type of liver neoplasm promoter.

2-Acetylaminofluorene

Use of cluster analysis in the development of structure-activity relations for antitumor triazenes.

A series of antitumor triazenes in which the members of the series are physicochemically distinct was designed using the cluster analysis approach as proposed by Hansch and his co-workers. The series that resulted was tested against Sarcoma 180 in the mouse and the antitumor activities were analyzed using regression techniques. The structure-activity relations that resulted are discussed in terms of proposed mechanisms of action.

Animals

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).

Administration, Oral