Mechanisms of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (Dacarbazine) cytotoxicity toward Chinese hamster ovary cells in vitro are dictated by incubation conditions

Decomposition of the antitumor agent 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC, Dacarbazine) produces several potentially toxic compounds, the concentration of which depend on incubation parameters such as pH, temperature and illumination. The action of DTIC on chinese hamster ovary (CHO) cell clone formation in the dark (7-8-day incubation) reflects the slow formation of 2-azahypoxanthine. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8)-deficient cells are resistant to DTIC under these conditions, reflecting their inability to utilize 2-azahypoxanthine. The toxicity of DTIC in conventional survival experiments (1-2-h exposure to drug) is dependent upon illumination and is highly influenced by the pH of the medium. Toxicity of DTIC in these experiments appears to reflect rapid accumulation of the immediate photodecomposition product of the drug, 4-diazoimidazole-5-carboxamide (DZC), since HGPRT-deficient cells are not resistant to DTIC under these conditions. The biologically initiated pathway of DTIC action (enzymatic hydroxylation) has little, if any, role in the action of this agent toward cultured CHO cells.     

The mechanism of alkylation of DNA by 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC), a metabolite of DIC (NSC-45388). Non-involvement of diazomethane

Comparison of the nuclear magnetic resonance spectra of chemically synthesized methyl-d₁-methanol with the methanol produced in the solvolytic decompostion of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC) in D₂O under acidic, basic or neutral conditions indicated that no deuterium was exchanged for the hydrogens on the methyl group. Diazomethane can therefore be ruled out as an intermediate in this reaction.The methyl-d₃-guanine isolated after incubation of methyl-d₃-MIC with calfthymus DNA in vitro displayed, on chemical ionization mass spectrometry, a quasimolecular ion (MH⁺) at m/e 169, which was 3 mass units higher than the quasimolecular ion for an undeuterated 7-methylguanine standard. The major fragment ions for 7-methyl-d₃-guanine on electron impact mass spectrometry likewise were situated at positions 3 mass units higher than the fragment ions for 7-methylguanine itself.These data indicate that the methylation of biological macromolecules by MIC must involve the transfer of an intact methyl group.     

Effects of 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide [NSC 45388, DTIC] on neuroblastoma cells in culture.

The effects of 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboximide (DTIC) on morphological and biochemical parameters of differentiation were studied in mouse neuroblastoma cells in culture. DTIC (10 μg/ml) did not induce formation of neurites in the cells but inhibited cell division, and produced a marked increase in cell size and in activity of three enzymes (tyrosine hydroxylase, choline acetyltransferase and acetylcholinesterase) involved in neurotransmitter metabolism. These effects were apparently not related to an increase in the intracellular level of cyclic AMP.     

Cytogenetic effects of 1-p-(3-methyltriazeno)benzoic acid potassium salt on human lymphocytes in vitro

The triazene derivative 1-p-(3-methyltriazeno)benzoic acid potassium salt (MTBA) shows pharmacological properties similar to those of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC, trade name dacarbazine), which is known to induce antigenic modulation in tumor cells (xenogenization) and is currently used in cancer therapy. Mutagenic, teratogenic and cancerogenic properties of triazene derivatives have been demonstrated but there is no report on their possible clastogenicity. We describe here the in vitro cytogenetic effects of MTBA on human peripheral blood lymphocytes. The drug was tested at different culture times in a range of concentrations from 2 to 500 micrograms/ml. MTBA caused a dose-dependent increase in the frequency of chromosomal breaks. Different blood donors showed different sensitivity to the treatment. Cell proliferation, as evaluated by [3H]thymidine incorporation, was inhibited at the highest concentrations of the drug. These data might be relevant for comparison with in vivo effects of the drug in clinical trials and to investigate the possible relations between xenogenization induced by MTBA and its genetic and cytogenetic effects in human lymphocytes.     

Synthesis and enzymatic polymerisation of 5-amino-1-(2''-deoxy-beta-D-ribofuranosyl)imidazole-4-carboxamide-5''- triphosphate.

The chemical synthesis of 5-amino-1-(2'-deoxy-beta-D-ribofuranosyl)imidazole-4-carboxamide, referred to as dZ, and of its 5'-triphosphate derivative (dZTP), from 2'-deoxyinosine is described. The polymerisation of dZTP using terminal deoxynucleotidyltransferase to give a homopolymer is also presented.     

The synthesis and stability of oligodeoxyribonucleotides containing the deoxyadenosine mimic 1-(2'-deoxy-beta-D-ribofuranosyl)imidazole-4-carboxamide.

Oligodeoxyribonucleotides containing the nucleoside analog 1-(2'-deoxy-beta-D-ribofuranosyl) imidazole-4-carboxamide (1) were synthesized by solid phase phosphoramidite technology. Nucleoside 1, which contains a reactive exocyclic amide moiety, was incorporated into synthetic oligodeoxyribonucleotides with the use of a benzoyl protecting group. The corresponding oligodeoxyribonucleotides with dI or dA in the same position in the sequence were synthesized for UV comparison of helix-coil transitions. The thermal melting studies indicate that 1, which could conceptually adopt either a dA- or a dI-like hydrogen bond configuration, pairs with significantly higher affinity to T than to dC. Nucleoside 1 further resembles dA in the relative order of its base pairing preferences (T >dG >dA >dC), but may be less discriminating than dA in its bias for base pairing with T over dG.     

Simultaneous measurement of nicotinamide and its catabolites,nicotinamide N-oxide,N1-methyl-2-pyridone-5-carboxamide,and N1-methyl-4-pyridone-3-carboxamide,in mice urine

Nicotinamide N-oxide is a major nicotinamide catabolite in mice but not in humans and rats. A high-performance liquid chromatographic method for the simultaneous measurement of nicotinamide, nicotinamide N-oxide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone-3-carboxamide in mice urine was developed by modifying the mobile phase of a reported method for measurement of nicotinamide N-oxide.     

Riboside and Ribotide of 5(or 3)-Aminopyrazole-4-carboxamide

During the investigation for dephosphorylation of 4-hydroxy-1-β-D-ribofuranosylpyrazolo-[3,4-d] pyrimidine 5′-phosphate, it was found that the compound was converted to an unknown substance by alkaline hydrolysis for 3 hr at 140°C. The structure of the substance was assigned to be 5-amino-1-β-D-ribofuranosylpyrazole-4-carboxamide 5′-phosphate. 5(or3)-Amino- pyrazole-4-carboxamide and its riboside were also obtained from 4-hydroxypyrazolo [3,4-d] pyrimidine and its riboside, respectively, under the similar conditions.

5-Amino-1-β-D-ribofuranosyipyrazole-4-carboxamide and 5-amino-1-β-D-ribofuranosyl- pyrazole-4-carboxamide 5′-phosphate are new compounds.     

Adoptive immunity in mice challenged with L1210/DTIC clones

Summary New antigenic specificities, not detectable on parental cells, have been induced by many investigators in mouse lymphomas by treatment with the antitumor agent 5(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC). The antigens are transmissible, after withdrawal of the drug treatment, as an inheritable character. The mechanism of induction, the molecular nature, and the number of the new antigenic specificities have not been completely elucidated. Four clones from murine leukemia L1210 isolated and expanded in vitro were treated in vivo with DTIC and the new sublines were studied in detail. The four drug-treated sublines studied exhibited strong immunogenicity since they were rejected by syngeneic animals. Immunosuppressed animals challenged with 10⁷ A/DTIC or P/DTIC cells were reciprocally protected by the adoptive transfer of spleen cells from donors that had rejected a lethal challenge of A/DTIC or P/DTIC clones. In a similar fashion, the adoptive transfer of spleen cells obtained from animals that had rejected the Q/DTIC or the R/DTIC clones protected immunosuppressed mice challenged with Q/DTIC or R/DTIC cells. No antitumor activity was observed in cross-protective schedules other than those indicated. It was been concluded that (a) the L1210 leukemia line does not have antigenic cells, (b) four DTIC-treated clone sublines were rejected by compatible hosts, and (c) two mutually exclusive sets of antigens were expressed in four antigenic clone sublines.Research supported in part by P.F.O. Contract Grant from C. N. R., Rome, Italy     

Synthesis of long-chain amide analogs of the cannabinoid CB1 receptor antagonist N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716) with unique binding selectivities and pharmacological activities

An extended series of alkyl carboxamide analogs of N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl- 1H-pyrazole-3-carboxamide (SR141716; 5) was synthesized. Each compound was tested for its ability to displace the prototypical cannabinoid ligands ([3H]CP-55,940, [3H]2; [3H]SR141716, [3H]5; and [3H]WIN55212-2, [3H]3), and selected compounds were further characterized by determining their ability to affect guanosine 5'-triphosphate (GTP)-gamma-[35S] binding and their effects in the mouse vas deferens assay. This systematic evaluation has resulted in the discovery of novel compounds with unique binding properties at the central cannabinoid receptor (CB1) and distinctive pharmacological activities in CB1 receptor tissue preparations. Specifically, compounds with nanomolar affinity which are able to fully displace [3H]5 and [3H]2, but unable to displace [3H]3 at similar concentrations, have been synthesized. This selectivity in ligand displacement is unprecedented, in that previously, compounds in every structural class of cannabinoid ligands had always been shown to displace each of these radioligands in a competitive fashion. Furthermore, the selectivity of these compounds appears to impart unique pharmacological properties when tested in a mouse vas deferens assay for CB1 receptor antagonism.     

Chemical xenogenization of murine lymphoma cells with triazene derivatives: Immunotoxicological studies

Summary 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.This work was supported by Progetto Finalizzato Controllo della Crescita Neoplastica contracts no. 83.00815.96 and no. 83.00838.96 (CNR, Rome, Italy)     

Oligodeoxynucleotides Embodying the Ambiguous Base Z, 5-Amino-imidazole-4-carboxamide

Abstract

Short oligomers containing 5-amino-1-(2-deoxy-β-D-ribofuranosyl)imidazole-4-carboxamide (dZ) were synthesized in solution using the phosphotriester methodology. Usual acyl groups were used for the canonical bases. For the exocyclic amino function of Z residue, the hydrogenolyzable benzyloxycarbonyl group was introduced.     

Nucleosides and Nucleotides. 98. Synthesis and Antitumor Activities of 5-Ethynylimidazole-4-carboxamide and -carbonitrile Derivatives

Abstract

5-Ethynyl-1-(2-deoxy-β-D-ribofuranosyl)imidazole-4-carbonitrile (4) and -carboxamide (5) and 5-ethynyl-1-(5-deoxy-β-D-ribofuranosyl)imidazole-4-carbonitrile (11) and -carboxamide (12) have been synthesized from the corresponding 5-iodo derivatives 2 and 7 by a palladium-catalyzed cross-coupling reaction with (tri-methylsilyl)acetylene. The aglycons, 5-ethynylimidazole derivatives 14 and 15 were synthesized by the hydrolytic cleavage of the corresponding nucleosides. The antileukemic activity of these nucleosides and base analogues are also described.     

Analysis and stability study of temozolomide using capillary electrophoresis

The applicability of micellar electrokinetic capillary chromatography (MEKC) for the analysis of temozolomide (TMZ) and its degradants, 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) and 5-amino-imidazole-4-carboxamide (AIC) has been studied. Using short-end injection, the analysis of TMZ and its degradants could be performed within 1.2 min. The obtained precision of migration times was better than 1.6 RSD%, and the limit of quantitation (LOQ) was 0.31–0.93 μg/mL. The therapeutic concentration of TMZ in blood samples can be determined after direct sample injection and conventional on-capillary UV detection. The proposed MEKC method was applied to study the stability of TMZ in water and serum at different pH values. It was established that the half-life of the TMZ in vitro serum at room temperature was 33 min, close to the half-life (28 min) obtained in water at pH 7.9.     

Immunologic cross-reactivity of antigen(s) induced by drug treatment in two leukemic sublines.

Previous studies have demonstrated that new antigenic specificities, not detectable on parental cells, can be induced by in vivo treatment of murine leukemic cells with anti-neoplastic agents. The immunologic properties of leukemic cells altered by treatment with 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide (DIC) were investigated further. Immunologic cross-reactivity between two DIC-treated leukemic sublines has been demonstrated by cell-mediated immunity in vitro and by active or adoptive immunity in vivo. Rabbit antiserum to DIC-treated sublines absorbed with the parental cells showed residual activity against the DIC-sublines that was specifically inhibited by further absorption with DIC-cells.     

Imidazole-4-Carboxamide and 1,2,4-Triazole-3-Carboxamide Deoxynucleotides as Simplified DNA Building Blocks with Ambiguous Pairing Capacity

Abstract

2′-Deoxynucleosides of imidazole-4 (or 1,2,4-triazole-3)-carboxamide, ethyl imidazole-4 (or 1,2,4-triazole-3)-carboxylate were synthesized by enzymatic glycosylation using N-deoxyribosyltransferase from a lactobacterium. The base pairing properties of Y and V when placed opposite the natural DNA bases as well as their self were evaluated by thermal denaturation experiments. DNA templates containing imidazole-4-carboxamide base were used in elongation reaction catalysed by Klenow fragment.     

Synthesis and antimicrobial activity of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles

A number of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles 6a–g (7b–f) were synthesized and tested for antibacterial and antifungal activity. Some of these compounds displayed antifungal activity at non-cytotoxic concentrations. Derivative 6c was 9 times more potent in vitro than miconazole and 20 times more selective against C. neoformans. 6c was also 8- and 125-fold more potent than amphotericin B and fluconazole, respectively. None of the compounds was active against bacteria. Preliminary structure–activity relationship (SAR) studies showed that the NO group at position 4 of the pyrazole ring is essential for the activity. Lipophilicity of the pyrazole moiety, N-alkyl chain length and planarity of the two heterocyclic rings appear to play a decisive role in modulating cytotoxicity and antifungal activity.     

Synthesis and anti-inflammatory activity of some novel 3-phenyl-N-[3-(4-phenylpiperazin-1yl)propyl]-1H-pyrazole-5-carboxamide derivatives

A new series of 3-phenyl-N-[3-(4-phenylpiperazin-1yl)propyl]-1H-pyrazole-5-carboxamide derivatives were synthesized and investigated their anti-inflammatory activities using carrageenan-induced rat paw edema model in vivo. All the synthesized compounds were found to be potent anti-inflammatory agents.     

Synthesis and antiviral activity of 3-(beta-D-ribofuranosyl)-1,2,4-oxadiazole-5-carboxamide

3-(beta-D-Ribofuranosyl)-1,2,4-oxadiazole-5-carboxamide (5) was prepared by condensation reaction of amidoxime 6 with monoethyl oxaloyl chloride followed by reaction with ammonia. The compound 5, however, did not exhibit any significant activity against herpes simplex virus type-I (HSV-I) and semliki forest virus (SFV).     

Cytotoxic and antivascular 1-methyl-4-(3-fluoro-4-methoxyphenyl)-5-(halophenyl)-imidazoles

A series of 1-methyl-4,5-diphenylimidazoles 6 with various patterns of m-halogen substitution at the 5-phenyl ring were tested for cytotoxicity in cancer and nonmalignant cell lines and for their capacity to prevent tube formation in HUVEC cultures. Unlike the monofluoro and difluoro derivatives 6a and 6e, the monobromo and diiodo analogs 6c and 6h were strongly cytotoxic and inhibited the polymerization of tubulin and the tube formation by HUVEC. The dibromo derivative 6g displayed a unique selectivity for KB-3-1 cervix and PC-3 prostate cancer cells. It also inhibited the tube formation by HUVEC and the polymerization of tubulin which is indicative of its potential antiangiogenic activity in solid tumors.