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.     

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.     

Validated high-performance liquid chromatographic assay for simultaneous determination of dacarbazine and the plasma metabolites 5-(3-hydroxymethyl-3-methyl-1-triazeno)imidazole-4-carboxamide and 5-(3-methyl-1-triazeno)imidazole-4-carboxamide

Dacarbazine (DTIC) is a prodrug that is clinically effective in the treatment of Hodgkin’s disease, melanoma and soft tissue sarcoma. To better characterize the clinical pharmacology of parent drug and reactive metabolites, a reversed-phase HPLC method with UV detection was developed for simultaneous determination of dacarbazine and the metabolites 5-(3-hydroxymethyl-3-methyl-1-triazeno)imidazole-4-carboxamide (HMMTIC) and 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC). Chromatographic separation was achieved with a Zorbax SB-CN column and with a mobile phase of 80% 50 mM ammonium phosphate, pH 6.5, 20% methanol and 0.1% triethylamine. HMMTIC, MTIC and DTIC were extracted from plasma with methanol precipitation of the proteins. Recovery of DTIC and the metabolites from whole blood was greater than 92%. Rapid processing of whole blood, methanol extraction and storage at −70°C substantially increased the stability of HMMTIC and MTIC from less than 15 min to 3 days. Precision for HMMTIC, MTIC and DTIC ranged from 3.7 to 16.3% relative standard deviation. The accuracy ranged from 101 to 114% for all three analytes. The validated assay was used to determine the pharmacokinetic data for dacarbazine and its active metabolites for human patients with recurrent glioma receiving DTIC intravenously.     

Inhibition of cell-surface neutral protease of Ehrlich ascites tumor cells by potassium p-(3,3-dimethyl-1-triazeno) benzoate

Ehrlich ascites tumour (EAT) cells possess a trypsin-like neutral protease on the cell surface. The antimetastatic triazene drug potassium p-(3,3-dimethyl-1-triazeno) benzoate (DM-COOK) inhibits this neutral protease, and also trypsin. Incubation of EAT cells with human erythrocytes (ratio of 1 to 5) at 37 degrees C for 18 h caused haemolysis of 28.8% of the erythrocytes. Addition of millimolar concentrations of DM-COOK to a fixed quantity (2.5 X 10(8)) of EAT cells caused a dose-related inhibition of haemolysis. DM-COOK was strongly bound to EAT cells and could not be removed by repeated washing.     

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.     

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.     

Design and optimization of novel (2S,4S,5S)-5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxy-2-isopropylhexanamides as renin inhibitors

Introduction of the 2,2-dimethyl-4-phenylpiperazin-5-one scaffold into the P(3)-P(1) portion of the (2S,4S,5S)-5-amino-6-dialkylamino-4-hydroxy-2-isopropylhexanamide backbone dramatically increased the renin inhibitory activity without using the interaction to the S(3)(sp) pocket. Compound 31 exhibited >10,000-fold selectivity over other human proteases, and 18.5% oral bioavailability in monkey.     

Synthesis and acrosin inhibitory activities of substituted ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives

A series of novel ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives were designed and synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds exhibited acrosin inhibitory activities. Among them, three compounds (5l, 5n, and 5v) were more potent than that of the control TLCK. These provide a new structural type for the development of novel contraceptive acrosin inhibitory agents.     

The design of (-)-(S)-2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate: a cardioselective positive inotropic derivative of Bay K 8644.

The title compound, (-)-(S)-9, is a novel cardioselective calcium channel modulator that exhibits a calcium channel agonist effect on heart, a weak calcium channel antagonist effect on smooth muscle, and releases nitric oxide in vitro. (-)-(S)-9 is a useful lead-compound for the design of positive inotropic agents to treat congestive heart failure, and to study the structure-function relationship of calcium channel modulation.     

New hybrid molecules with anticonvulsant and antinociceptive activity derived from 3-methyl- or 3,3-dimethyl-1-[1-oxo-1-(4-phenylpiperazin-1-yl)propan-2-yl]pyrrolidine-2,5-diones

The purpose of this study was to synthetize the focused library of 34 new piperazinamides of 3-methyl- and 3,3-dimethyl-(2,5-dioxopyrrolidin-1-yl)propanoic or butanoic acids as potential new hybrid anticonvulsants. These hybrid molecules join the chemical fragments of well-known antiepileptic drugs (AEDs) such as ethosuximide, levetiracetam, and lacosamide. Compounds 5–38 were prepared in a coupling reaction of the 3-methyl- or 3,3-dimethyl-2-(2,5-dioxopyrrolidin-1-yl)propanoic (1, 2) or butanoic acids (3, 4) with the appropriately substituted secondary amines in the presence of the N,N-carbonyldiimidazole reagent. The initial anticonvulsant screening was performed in mice (ip) using the ‘classical’ maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests as well as in the six-Hertz (6 Hz) model of pharmacoresistant limbic seizures. The acute neurological toxicity was determined applying the chimney test. The broad spectra of activity across the preclinical seizure models in mice ip displayed compounds 7, 15, and 36. The most favorable anticonvulsant properties demonstrated 15 (ED₅₀ MES = 74.8 mg/kg, ED₅₀ scPTZ = 51.6 mg/kg, ED₅₀ 6 Hz = 16.8 mg/kg) which showed TD₅₀ = 213.3 mg/kg in the chimney test that yielded satisfying protective indexes (PI MES = 2.85, PI scPTZ = 4.13, PI 6 Hz = 12.70) at time point of 0.5 h. As a result, compound 15 displayed comparable or better safety profile than clinically relevant AEDs: ethosuximide, lacosamide or valproic acid. In the in vitro assays compound 15 was observed as relatively effective binder to the neuronal voltage-sensitive sodium and L-type calcium channels. Beyond the anticonvulsant properties, 6 compounds diminished the pain responses in the formalin model of tonic pain in mice.     

Synthesis and optimization of novel (3S,5R)-5-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides as orally active renin inhibitors

We report synthesis and optimization of a series of (3S,5R)-5-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides as renin inhibitors. Chemical modification of ${{\text{P}}_1}^{\prime }$, ${{\text{P}}_2}^{\prime }$ and P₃ portions led to a promising 3,5-disubstituted piperidine 32o showing high renin inhibitory activity and favorable oral exposure in both rats and cynomolgus monkeys with acceptable CYP and hERG current inhibition. Compound 32o exhibited a significant blood pressure lowering effect by oral administration in two hypertensive animal models, double transgenic rats and furosemide pretreated cynomolgus monkeys.     

Metabolism of the anticancer agent 1-(4-acetylphenyl)-3,3-dimethyltriazene

High pressure liquid chromatography was used in combination with mass spectrometry to confirm that the main products of in vitro metabolism of 1-(4-acetylphenyl)-3,3-dimethyltriazene are 1-(4-acetylphenyl-3-methyltriazene and 4-aminoacetophenone. In addition a novel metabolite, 1-[4-(1-hydroxyethyl)-phenyl]-3,3-dimethyltriazene, possessing antitumour activity similar to the parent drug, was identified.     

Design and discovery of new (3S,5R)-5-[4-(2-chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]piperidine-3-carboxamides as potent renin inhibitors

Utilizing X-ray crystal structure analysis, (3S,5R)-5-[4-(2-chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]piperidine-3-carboxamides were designed and identified as renin inhibitors. The most potent compound 15 demonstrated favorable pharmacokinetic and pharmacodynamic profiles in rat.     

1-(4-Methane(amino)sulfonylphenyl)-3-(4-substituted-phenyl)-5-(4-trifluoromethylphenyl)-1H-2-pyrazolines/pyrazoles as potential anti-inflammatory agents

2-Pyrazolins 14a–l and pyrazoles 15a–l were designed as celecoxib analogs for the evaluation of their in vitro COX-1/COX-2 inhibitory activity and the in vivo anti-inflammatory activity. Compounds 14i, 15a, 15d and 15f were the most COX-2 selective derivatives (S.I. = 5.93, 6.08, 5.03 and 5.27 respectively) while the pyrazoline derivatives 14g and 14i exhibited the highest AI activity (ED₅₀ = 190.5 and 160.1 μmol/kg po, respectively).     

The discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1 ,5-a]-pyrimidine: a corticotropin-releasing factor (hCRF1) antagonist

Structure activity relationship studies led to the discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazo lo-[1,5-a]-pyrimidine 11-31 (DMP904), whose pharmacological profile strongly supports the hypothesis that hCRF1 antagonists may be potent anxiolytic drugs. Compound 11-31 (hCRF1 Ki = 1.0+/-0.2 nM (n = 8)) was a potent antagonist of hCRF1-coupled adenylate cyclase activity in HEK293 cells (IC50= 10.0+/-0.01 nM versus 10 nM r/hCRF, n = 8); alpha-helical CRF(9-41) had weaker potency (IC50 = 286+/-63 nM, n = 3). Analogue 11-31 had good oral activity in the rat situational anxiety test; the minimum effective dose for 11-31 was 0.3 mg/kg (po). Maximal efficacy (approximately 57% reduction in latency time in the dark compartment) was observed at this dose. Chlordiazepoxide caused a 72% reduction in latency at 20 mg/kg (po). The literature compound 1 (CP154526-1, 30 mg/kg (po)) was inactive in this test. Compound 11-31 did not inhibit open-field locomotor activity at 10, 30, and 100 mg/kg (po) in rats. In beagle dogs, this compound (5 mg/kg, iv, po) afforded good plasma levels. The key iv pharmacokinetic parameters were t1/2, CL and Vd,ss values equal to 46.4+/-7.6 h. 0.49+/-0.08 L/kg/h and 23.0+/-4.2 L/kg, respectively. After oral dosing, the mean Cmax, Tmax t1/2 and bioavailability values were equal to 1260+/-290 nM, 0.75+/-0.25 h. 45.1+/-10.2 h and 33.1%, respectively. The overall rat behavioral profile of this compound suggests that it may be an anxiolytic drug with a low motor side effect liability.     

Heteroarylamide smoothened inhibitors: Discovery of N-[2,4-dimethyl-5-(1-methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542) and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide (AZD7254)

Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating metastasized basal cell carcinoma. Our lead optimization effort focused on a series of heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline core significantly improved the potency of this series of compounds. These findings predated the availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics calculations to demonstrate the o-Me substitution favors the bioactive conformation by inducing a dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti- tumor effects.     

Induction of mitotic gene conversion by 3,3-dimethyl-I-phenyltriazene, I-(3-hydroxyphenyl-)-3,3-dimethyltriazene and by I-(4-hydroxyphenyl)-3,3-dimethyltriazene in Saccharomyces cerevisiae

The induction of mitotic gene conversion by 3,3-dimethyl-1-phenyltriazene (DMPT), 1-(3-hydroxyphenyl)-3,3-dimethyltriazene (3-HO-PDMT) and by 1-(4-hydroxyphenyl)-3,3-dimethyltriazene (4-HO-PDMT) in the diploid strain D4 of Saccharomyces cerevisiae was investigated. The frequencies of the non-reciprocal intragenic recombinations at two unlinked loci ade2 (adenine) and trp5 (tryptophan) were determined. Although all three triazenes showed marked convertogenic activities, significant differences in their genetic effectiveness have been observed. Thus both phenolic triazenes were found to be much stronger convertogens than the unhydroxylated parent compound, DMPT. An attempt is made to account for the established differences in convertogenicity by chemical reactivity that could be expected from the structural features of the tested alkaryltriazenes.