Effects of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (NSC-407347), an alkylating agent derived from 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (NSC-45388).

All experiments were performed in the absence of light. A 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC) concentration of less than 10^?4 M had no effect on cell growth of L cells. At higher concentrations, the cells were inhibited to levels which were similar to those obtained with equimolar doses of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DIC). MIC inhibited the incorporation of ³H-thymidine by DNA more than that of ³H-uridine by RNA. Uptake of ³H from ³H-methyl-MIC by DNA was not influenced by the stage of the cell cycle. The greatest binding took place with DNA of the euchromatin fraction. MIC-treated DNA exhibited impaired template activity in vitro in the RNA polymerase s ystem but not with that of DNA polymerase. Chromatography of DNA hydrolysate from ³H-methyl-MIC-treated cells showed three major radioactive peaks, which corresponded to adenine, guanine and 7-methylguanine. Hydroxyurea markedly reduced the uptake of ³H by adenine and guanine but had relatively little effect on the ³H content of 7-methylguanine. Similarity of cytotoxic reactions of MIC to those of DIC supports the thesis that in the animal system DIC is metabolically converted to MIC, a potential methylating agent. Many of the effects of DIC can be accounted for by the action of MIC.     

Synthesis of 5-amino-1-(5-deoxy-beta-D-ribofuranosyl)imidazole-4-carboxamide and related 5'-deoxyimidazole ribonucleosides.

5-Amino-1-(beta-D-ribofuranosyl)imidazole-4-carboxamide (1, AICA ribonucleoside) was converted in two steps to 5-amino-1-(5-deoxy-5-iodo-2,3-O-isopropylidene-beta-D-ribofuranosyl)imidazole-4-carboxamide (3) which was hydrogenated in the presence of Pd/C to yield 5-amino-1-(5-deoxy-2,3-O-isopropylidene-beta-D-ribofuranosyl)imidazole-4-carboxamide (4). The dehydration of 4 yielded 5-amino-1-(5-deoxy-2,3-O-isopropylidene-beta-D-ribofuranosyl)imidazole-4-carbonitrile (7). The compounds 3, 4, and 7 were deblocked with formic acid to furnish 5-amino-1-(5-deoxy-5-iodo-beta-D-ribofuranosyl)imidazole-4-carboxamide (6). 5-amino-1-(5-deoxy-beta-D-ribofuranosyl)imidazole-4-carboxamide (5), and 5-amino-1-(5-deoxy-beta-D-ribofuranosyl)imidazole-4-carbonitrile (8), respectively. Compound 8 was acetylated and then deaminated to give 1-(2,3-di-O-acetyl-5-deoxy-beta-D-ribofuranosyl)imidazole-4-carbonitrile (11). The compounds 8 and 11 were converted into 5-amino-1-(5-deoxy-beta-D-ribofuranosyl)imidazole-4-thiocarboxamide (9) and 1-(5-deoxy-beta-D-ribofuranosyl)imidazole-4-thiocarboxamide (12), respectively. The synthesis of 1-(5-deoxy-beta-D-ribofuranosyl)imidazole-4-carboxamide (13) was achieved for the first time by the treatment of 11 with hydrogen peroxide in the presence of ammonium hydroxide. The compounds were tested for antibacterial, antifungal, and antiviral activity, with 5 and 6 significantly inhibitory to Staphylococcus aureus.     

4—氨基—1—甲基—3—丙基吡唑—5—甲酰胺的合成

以２－戊酮和草酸二乙酯为起始原料,经缩合,环合、甲基化、水解、硝化、氨解、还原等７步反应合成了新型磷酸二酯酶５型（ＰＤＥ５）抑制剂西地那非的关键中间体－４－氨基－１－３－丙基吡唑－５－甲酰胺。     

Synthesis and antileukemic activities of furanyl, pyranyl, and ribosyl derivatives of 4-(3,3-dimethyl-1-triazeno)imidazole-5-carboxamide and 3-(3,3-dimethyl-1-triazeno)pyrazole-4-carboxamide

From the reaction of silylated 4-(3,3-dimethyl-1-triazeno)imidazole-5-carboxamide (DTIC, 5) and 3-(3,3-dimethyl-1-triazeno)pyrazole-4-carboxamide (DTPC, 9) with 2-chlorotetrahydrofuran, we have isolated in both cases a single tetrahydrofuran-2-yl derivative. However, when silylated DTPC was reacted with 2-chlorotetrahydropyran, two tetrahydropyran-2-yl compounds were obtained, and these were shown to be positional isomers on the basis of 1H NMR and UV data. These furanyl and pyranyl derivatives were tested for antileukemic activity (L-1210, in vivo) and the results were compared with the results obtained for the corresponding ribosyl derivatives of DTIC and DTPC.     

In vivo metabolism and reaction with DNA of the cytostatic agent, 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC)

The cytostatic drug dacarbazine [DTIC, 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide] is strongly carcinogenic in rats. Bioactivation of DTIC yields a methylating intermediate but the extent of interaction with cellular macromolecules has not previously been reported. Following a single i.p. injection of [14C-methyl]DTIC, exhalation of 14CO2 occurred with a t1/2 max of approximately 2 hr (0.95 mg/kg) and 2.5 hr (95 mg/kg). Of the total radioactivity administered, 8.5% was exhaled as 14CO2; 54% was excreted via the urine, predominantly as unchanged DTIC. In liver, kidney and lung, formation of 7-[14C]methylguanine in DNA and RNA was directly proportional with dose. DNA methylation by a single dose of DTIC (9.8 mg/kg; 5 hr survival time) was highest in liver (35 mumoles 7-methylguanine/mole guanine), followed by kidney (25 mumoles) and lung (20 mumoles). The remainder tissues showed 7-methylguanine concentrations approximately 50% of those in liver DNA, with the exception of the brain which had a very low extent of DNA modification (approximately 1 mumole/mole guanine). At the specific radioactivity used (48 mCi/mmole), the promutagenic base O6-methylguanine was only detectable in liver, kidney, lung, and stomach DNA (0.6-0.8 mumoles/mole guanine). Autoradiographic studies revealed a diffuse distribution of reaction products in rat liver. In contrast, N-nitrosodimethylamine and related carcinogens known to be bioactivated by the hepatic cytochrome P-450 system show a predominantly centrilobular distribution. This difference may be due to the greater stability of proximate carcinogens generated by alpha-C hydroxylation at one of the methyl groups of DTIC.     

Molecular interaction of the antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1- (2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide with the CB1 cannabinoid receptor

N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716; 1) is a potent and selective antagonist for the CB1 cannabinoid receptor. Using the AM1 molecular orbital method, conformational analysis of 1 around the pyrazole C3 substituent identified four distinct conformations designated Tg, Ts, Cg, and Cs. The energetic stability of these conformers followed the order Tg > Cg > Ts > Cs for the neutral (unprotonated) form of 1 and Ts > Tg > Cs > Cg for its piperidine N-protonated form. Unified pharmacophore models for the CB1 receptor ligands were developed by incorporating the protonated form of 1 into the superimposition model for the cannabinoid agonists 4-[4-(1,1-dimethylheptyl)-2-hydroxyphenyl]perhydro-2alpha,6beta-dihydroxynaphthalene (CP55244; 2) and the protonated form of (R)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55212-2; 3) reported previously (Shim et al. In Rational Drug Design Symposium Series; Parrill, A. L., Reddy, M. R., Eds.; American Chemical Society: Washington, DC, 1999; pp 165-184). Values of K(i) for 1 and a series of 31 structural analogues were determined from radioligand binding analyses by competitive displacement of [3H]CP55940 from cannabinoid receptors in a rat brain membrane preparation. Comparative molecular field analysis (CoMFA) was employed to construct three-dimensional (3D)-quantitative structure-activity relationship (QSAR) models for this data set as unprotonated species assuming the Tg, Cg, and Ts conformers and for the protonated species assuming the Ts, Tg, and Cs conformers. Values of the conventional r2 and cross-validated r2 (r(cv)2) associated with these CoMFA models exceeded the threshold for statistical robustness (r2 > or = 0.90) and internal predictive ability (r(cv)2 > or = 0.50) in each of these six cases except for the protonated species assuming the Tg conformer (i.e., r2 = 0.97; r(cv)2 = 0.36). Results from conformational analyses, superimposition models, and 3D-QSAR models suggest that the N1 aromatic ring moiety of 1 dominates the steric binding interaction with the receptor in much the same way as does the C3 alkyl side chain of cannabinoid agonists and the C3 aroyl ring of the aminoalkylindole agonists. We also determined that several of the conformers considered in this study possess the proper spatial orientation and distinct electrostatic character to bind to the CB1 receptor. We propose that the unique region in space occupied by the C5 aromatic ring of 1 might contribute to conferring antagonist activity. We further propose that the pyrazole C3 substituent of 1 might contribute to conferring either neutral antagonist or inverse agonist activity, depending upon the interaction with the receptor.     

5-三氟甲基-3-(4-甲基-1H-咪唑-1-基)苯胺的合成

3,5-二硝基三氟甲苯(2)与氟化四甲铵进行氟代反应,所得单氟中间体再与4-甲基-1H-咪唑进行取代反应得到5-三氟甲基-3-(4-甲基-1H-咪唑-1-基)-硝基苯(3),然后在Pd/C催化下氢化还原制得抗肿瘤药尼罗替尼的中间体5-三氟甲基-3-(4-甲基-1H-眯唑-1-基)-苯胺(1),总收率约50％(以2计).     

Inverse agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) for the CB(1) cannabinoid receptor

Two subtypes of cannabinoid receptors are currently recognized, CB(1), found in brain and neuronal cells, and CB(2), found in spleen and immune cells. We have characterized 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) as a novel aryl pyrazole antagonist for the CB(1) receptor. CP-272871 competed for binding of the cannabinoid agonist (3)H-labeled (-)-3-[2-hydroxy-4-(1, 1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol ([(3)H]CP-55940) at the CB(1) receptor in rat brain membranes with a K(d) value 20-fold greater than that of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A). CP-272871 also competed for binding with the aminoalkylindole agonist (3)H-labeled (R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]1, 4-benzoxazin-6-yl](1-naphthyl)methanone ([(3)H]WIN-55212-2), as well as the aryl pyrazole antagonist [(3)H]SR141716A. Inverse agonist as well as antagonist properties were observed for both SR141716A and CP-272871 in signal transduction assays in biological preparations in which the CB(1) receptor is endogenously expressed. SR141716A augmented secretin-stimulated cyclic AMP (cAMP) accumulation in intact N18TG2 neuroblastoma cells, and this response was reversed by the agonist desacetyllevonantradol. CP-272871 antagonized desacetyllevonantradol-mediated inhibition of adenylyl cyclase in N18TG2 membranes, and increased adenylyl cyclase activity in the absence of agonist. SR141716A and CP-272871 antagonized desacetyllevonantradol-stimulated (35)S-labeled guanosine-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) binding to brain membrane G-proteins, and decreased basal [(35)S]GTPgammaS binding to G-proteins. K(+) enhanced CP-272871 and SR141716A inverse agonist activity compared with Na(+) or NMDG(+) in the assay. These results demonstrated that the aryl pyrazoles SR141716A and CP-272871 behave as antagonists and as inverse agonists in G-protein-mediated signal transduction in preparations of endogenously expressed CB(1) receptors.     

Synthesis and structure-activity relationships of amide and hydrazide analogues of the cannabinoid CB(1) receptor antagonist N-(piperidinyl)- 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716)

Analogues of the biaryl pyrazole N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716; 5) were synthesized to investigate the structure-activity relationship (SAR) of the aminopiperidine region. The structural modifications include the substitution of alkyl hydrazines, amines, and hydroxyalkylamines of varying lengths for the aminopiperidinyl moiety. Proximity and steric requirements at the aminopiperidine region were probed by the synthesis of analogues that substitute alkyl hydrazines of increasing chain length and branching. The corresponding amide analogues were compared to the hydrazides to determine the effect of the second nitrogen on receptor binding affinity. The N-cyclohexyl amide 14 represents a direct methine for nitrogen substitution for 5, reducing the potential for heteroatom interaction, while the morpholino analogue 15 adds the potential for an additional heteroatom interaction. The series of hydroxyalkyl amides of increasing chain length was synthesized to investigate the existence of additional receptor hydrogen binding sites. In displacement assays using the cannabinoid agonist [(3)H](1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl) cyclohexan-1-ol (CP 55 940; 2) or the antagonist [(3)H]5, 14 exhibited the highest CB(1) affinity. In general, increasing the length and bulk of the substituent was associated with increased receptor affinity and efficacy (as measured in a guanosine 5'-triphosphate-gamma-[(35)S] assay). However, in most instances, receptor affinity and efficacy increases were no longer observed after a certain chain length was reached. A quantitative SAR study was carried out to characterize the pharmacophoric requirements of the aminopiperidine region. This model indicates that ligands that exceed 3 A in length would have reduced potency and affinity with respect to 5 and that substituents with a positive charge density in the aminopiperidine region would be predicted to possess increased pharmacological activity.     

Conformationally constrained analogues of N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4- dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716): design, synthesis, computational analysis, and biological evaluations

Structure-activity relationships (SARs) of 1 (SR141716) have been extensively documented, however, the conformational properties of this class have received less attention. In an attempt to better understand ligand conformations optimal for receptor recognition, we have designed and synthesized a number of derivatives of 1, including a four-carbon-bridged molecule (11), to constrain rotation of the diaryl rings. Computational analysis of 11 indicates approximately 20 kcal/mol energy barrier for rotation of the two aryl rings. NMR studies have determined the energy barrier to be approximately 18 kcal/mol and suggested atropisomers could exist. Receptor binding and functional studies with these compounds displayed reduced affinity and potency when compared to 1. This indicates that our structural modifications either constrain the ring systems in a suboptimal orientation for receptor interaction or the introduction of steric bulk leads to disfavored steric interactions with the receptor, and/or the relatively modest alterations in the molecular electrostatic potentials results in disfavored Coulombic interactions.