Synthesis of N2-(substituted benzyl)-3-(4-methylphenyl)indazoles as novel anti-angiogenic agents

To search for novel compounds with potent anti-angiogenic activity, a series of N(1)-(substituted benzyl)-3-(4-methylphenyl)-1H-indazoles (16, 18, 20, 22, 24, 26, 28, 30, 32) and N(2)-(substituted benzyl)-3-(4-methylphenyl)-2H-indazoles (17, 19, 21, 23, 25, 27, 29, 31, and 33) were synthesized. The structures of these regioisomers were established by IR, UV, and NMR spectral data. 3-(4-Methylphenyl)-1H-indazole (6) and the N(2)-substituted derivatives (17, 19, 21, 23, 25, 29, 31, 33) were evaluated for their anti-angiogenic activity. Most of them showed more prominent activity than ethyl 4-(1-benzyl-1H-indazol-3-yl)benzoate (YD-3). Among these tested compounds, 2-(4-chlorobenzyl)-3-(4-methylphenyl)-2H-indazole (19), 2-(4-methylbenzyl)-3-(4-methylphenyl)-2H-indazole (25), and 2-(4-methoxybenzyl)-3-(4-methylphenyl)-2H-indazole (31) showed significant anti-angiogenic activity and are worthy of further investigation.     

Synthesis and biological properties of new 5-nitroindazole derivatives

A series of new 3-alkoxy- or 3-hydroxy-1-[omega-(dialkylamino)alkyl]-5-nitroindazoles have been synthesized and their trichomonacidal, antichagasic and antineoplastic properties studied. Five derivatives (5, 6, 8, 9 and 17) showed remarkable trichomonacidal activity against Trichomonas vaginalis at 10 microg/mL concentration. Three compounds (8, 10, 11) exhibited interesting antichagasic activity and these same compounds moderate antineoplastic activity against TK-10 and HT-29 cell lines. Unspecific cytotoxicity against macrophages has also been evaluated and only compounds 9, 10 and 11 resulted cytotoxic at the higher dose evaluated (100 microg/mL), loosing cytotoxicity at lower doses. QSAR studies have been carried out. X-ray crystallographic study of compound 8 has been performed.     

Regioselective synthesis of indazole N1- and N2-(beta-D-ribonucleosides)

The regioselective synthesis of 4-nitroindazole N1- and N2-(beta-D-ribonucleosides) (8, 9, 1b and 2b) is described. The N1-regioisomers are formed under thermodynamic control of the glycosylation reaction [fusion reaction or Silyl Hilbert-Johnson glycosylation for 48 h (66%)], while the kinetic control (Silyl Hilbert-Johnson glycosylation for 5 h) afforded only the N2-isomer (64%). The structures of the nucleosides 1b and 2b were assigned by single crystal X-ray analyses. The 4-amino-N1-(beta-D-ribofuranosyl)-1H-indazole (3b) was obtained from the nitro nucleoside 1b by catalytic hydrogenation. Compound 3b shows fluorescence while the 4-nitroindazole nucleosides 1b and 2b do not possess this property.     

The N2-guanine adduct but not the C8-guanine or N6-adenine adducts formed by 4-nitroquinoline 1-oxide blocks the 3'-5' exonuclease action of T4 DNA polymerase

When O-acetyl-4-(hydroxyamino)quinoline 1-oxide (Ac-4HAQO) reacts with double-stranded DNA at 37 degrees C the major products, N2-guanine, C8-guanine, and N6-adenine adducts, are formed in the proportions of 5:3:2, respectively. When the reaction is carried out with single-stranded DNA at 0 degree C, the products are found in the ratio 1:7:2. Unique 174-bp DNA fragments were modified in these ways and used as substrates for the 3'-5' exonuclease activity of T4 DNA polymerase. The results obtained showed that the exonuclease is blocked by the N2-guanine adduct but not the other two adducts. Interpretation of the cleavage patterns suggested that the enzyme stopped 2 nucleotides before the N2-guanine adduct. The N2-guanine adduct lies in the minor groove of the DNA double helix, while the other two adducts are found in the major groove. Apparently, only the former hinders progression of the enzyme.     

Identification of AMP N1-oxide in royal jelly as a component neurotrophic toward cultured rat pheochromocytoma PC12 cells

An extract of royal jelly (RJ) induced processes from cultured rat pheochromocytoma PC12 cells. Active components were isolated, and identified as adenosine monophosphate (AMP) and AMP N1-oxide. AMP N1-oxide was more than 20 times as active as AMP, judging from the minimal concentration to elicit activity. AMP N1-oxide was thought to be responsible for about half of the process-forming activity of whole RJ. Chemically-synthesized AMP N1-oxide was active similarly to the molecule purified from RJ, confirming AMP N1-oxide as the active entity. AMP N1-oxide also suppressed proliferation of PC12 cells and stimulated expression of neurofilament M, a specific protein of mature neurons, demonstrating the stimulatory activity of AMP N1-oxide to induce neuronal differentiation of PC12 cells. Pharmacological experiments suggested that AMP N1-oxide actions are mediated by adenyl cyclase-coupled adenosine receptors, including A2A. Thus AMP N1-oxide is a key molecule that characterizes RJ, and is not found in natural products other than RJ.     

Synthesis of 3-(1-alkyl/aminoalkyl-3-vinyl-piperidin-4-yl)-1-(quinolin-4-yl)-propan-1-ones and their 2-methylene derivatives as potential spermicidal and microbicidal agents

A series of twenty two derivatives of 3-(1-alkyl/aminoalkyl-3-vinyl-piperidin-4-yl)-1-(quinolin-4-yl)-propan-1-one and their 2-methylene derivatives were synthesized from naturally abundant cinchonine (I). Tartarate salts of these compounds were prepared and evaluated for spermicidal activity. The most active compounds (24, 27, 34, 36, and 38) showing potent spermicidal activity were further evaluated against different strains of Trichomonas vaginalis, for antimicrobial activity, in HeLa cell lines for cytotoxicity and against Lactobacillus jensenii for eco-safety. The tartarate of 3-(1-pentyl-3-vinyl-piperidin-4-yl)-1-(quinolin-4-yl)-propan-1-one (27) was found to be more active than N-9 in spermicidal activity.     

Fates of N′-Methylnicotinamide and Nicotinamide N-Oxide in Mice

This experiment was performed to investigate the possibility that N′ -methylnicotinamide (N′-methyl-3-pyridinecarboxamide) and nicotinamide N-oxide have niacin activity or not in animals. When 20 mg N′-methylnicotinamide per mouse was administered, urinary excretion of nicotinamide, N¹-methylnicotinamide (MNA), N¹-methyl-2-pyridone-5-carboxamide (2-Py), and N¹-methyl-4-pyridone-3-carboxamide (4-Py) increased 24-, 3-, 3-, and 3-fold, respectively, compared with the control values. The increased ratios of MNA, 2-Py, and 4-Py were almost the same as those when 20 mg nicotinamide was administered. Therefore, the relative activity of N′-methylnicotinamide to nicotinamide as niacin was considered to be about 1. When 20 mg nicotinamide N-oxide per mouse was administered, urinary excretion of nicotinamide, MNA, 2-Py, and 4-Py increased 6.4-, 1.8-, 1.6-, and 1.7-fold, respectively, compared with the control values. The increased ratios of MNA, 2-Py, and 4-Py were about 1/2 of those when 20 mg nicotinamide was administered, so the relative activity of nicotinamide N-oxide to nicotinamide as niacin is considered to be about 1/2. In conclusion, it was found the possibility that the reactions N′-methylnicotinamide → nicotinamide and nicotinamide N-oxide → nicotinamide occur, at least in mice, and that therefore N′-methylnicotinamide and nicotinamide N-oxide have niacin activity.     

反义寡核苷酸体外抗流感病毒活性

为了获得具有抗流感病毒活性的反义寡核苷酸,针对A型流感病毒基因组3′和5′端保守序列,设计并合成了多条硫代寡核苷酸(ODN)：3′端反义ODN(IV3#)与3′端正义ODN(IV3S);5′端反义ODN(IV4#)与5′端正义ODN(IV4S)以及由5′和3′端正义/反义保守序列组成的复合序列ODN(IV6#和IV7#)。测定了PSODN的体外细胞毒性和在MDCK细胞中对流感病毒复制的影响。结果表明：(1)PSODN浓度高达50μmol/L时对MDCK细胞末表现有毒性作用;(2)与流感病毒基因组5′端互补的ODN IV4#以及由5′和3′端保守序列构成的IV6#ODN和IV7#ODN均具有较高的抗病毒活性;如IV4#ODN浓度为1μmol/L时对流感病毒A/京防/861(H1N1)抑制率近50%,浓度为10μmol/L或更高时抑制率超过70%,且IV4#抑制病毒活性呈现明显的序列和剂量依赖性;(3)IV4#ODN不仅对A型流感病毒H1N1亚型有抑制作用,对H3N2亚型也表现较高的抑制活性;(4)病毒感染复数(MOI)对IV4#ODN抗病毒活性有一定影响,当MOI较低时,IV4#ODN表现的剂量效应关系更加明显。抗流感病毒反义寡核苷核IV4#ODN的发现为进一步研究流感新型药物奠定了实验基础。〖HTH〗关键词〖HTSS〗：流感病毒, 反义寡核苷酸, 体外细胞毒性, 抗病毒活性, 感染复数     

Chemistry of the N′-Oxides of Nicotine and Myosmine

Nicotine-N′-oxide (II) was purified to give a crystalline form which has m.p. 170~171°C, and . The reaction of nicotine-N′-oxide with acetic anhydride afforded a good yield of 1′-(3-pyridyl)-4′-(N′-acetylmethylamino)-l′-propanone (V) which was hydrolyzed to give pseudoöxynicotine (III). Nicotine-N′-oxide (II) either with acetyl chloride or with benzoyl chloride, under similar conditions, furnished pseudoöxynicotine (III) without giving the corresponding acyl compound as an intermediate. 2′-Methyl-6′-(3-pyridyl)-tetrahydro-l′,2′-oxazine (XII) rearranged from nicotine-N′-oxide reacted neither with acetic anhydride nor acetyl chloride. Reduction of the oxime (IV) of pseudoöxynicotine dihydrochloride gave dl-l′-amino-l′-(3-pyridyl)-4′-methyl-aminobutane (VII) as a main product. The hydrazone (VIII) of pseudoöxynicotine dihydrochloride subjected to a modification of the Wolf-Kischner reaction, was reduced to yield dihydrometanicotine (IX). The pyrolysis of N′-methylmyosmine (IV) gave N′-methylnicotinamide (XI) and nicotyrine (X) in low yields. The presence of N′-methylmyosmine in the autoxidation mixture of nicotine was also established. Oxidation of nornicotine (XIV) with hydrogen peroxide furnished myosmine-N′-oxide (XV) whose identity was established by its chemical and physical properties. This oxide, on pyrolysis, gave nornicotyrine (XVII) and myosmine (XVI).     

Novel thrombin inhibitors incorporating weakly basic heterobicyclic P1-arginine mimetics: optimization via modification of P1 and P3 moieties

Optimization of lead compounds 1 and 2 resulted in novel, selective, and potent thrombin inhibitors incorporating weakly basic heterobicyclic P(1)-arginine mimetics. The design, synthesis, and biological activity of racemic thrombin inhibitors 17-29 and enantiomerically pure thrombin inhibitors 30-33 are described. The arginine side-chain mimetics used in this study are 4,5,6,7-tetrahydro-1,3-benzothiazol-2-amine, 4,5,6,7-tetrahydro-2H-indazole, and 2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-3(2H)-ylamine.     

4-Nitroquinoline-1-oxide: factors determining its mutagenicity in bacteria

In bacteria, 4-nitroquinoline-1-oxide (NQO) causes primarily mutations of the base-substitution type although frameshift mutations are also induced. The adducts formed are presumably recognized by error-prone DNA repair enzymes as evidenced by the much greater activity in plasmid pKM101-bearing tester strains. Although reduction of the nitro group appears to be required for mutagenic activity, this reduction is not catalyzed by the nitroreductase required for the demonstration of the mutagenicity in bacteria of other nitro-containing mutagens (nitrofurans, 2-nitronaphthalene, nitrofluorenes). The reduction of the nitro group appears to be catalyzed by a different nitroreductase. The mutagenicity of the non-carcinogenic 3-methyl-4-nitroquinoline-1-oxide (meNQO) may be related to this newly recognized nitroreductase. It is proposed, further, that the ultimate mutagenic intermediates derived from NQO and MeNQO differ.     

Ring-substituted quinolines. Part 2: Synthesis and antimycobacterial activities of ring-substituted quinolinecarbohydrazide and ring-substituted quinolinecarboxamide analogues

Additional structural modifications of the new chemical entity, 2,8-dicyclopentyl-4-methylquinoline (DCMQ; MIC = 6.25 μg/mL, M. tuberculosis H37Rv) resulted in the synthesis of four new series of the ring-substituted quinolinecarbohydrazides (series 1–4) constituting 22 analogues. All new derivatives were evaluated for in vitro antimycobacterial activities against drug-sensitive M. tuberculosis H37Rv strain. Certain ring-substituted-2-quinolinecarbohydrazide analogues described herein showed good inhibitory activity. In particular, analogues 4-(1-adamantyl)-2-quinolinecarbohydrazide (2d), 4,5-dicyclopentyl-2-quinolinecarbohydrazide (2e), 4,8-dicyclopentyl-2-quinolinecarbohydrazide (2f), and 4,5-dicyclohexyl-2-quinolinecarbohydrazide (2g) have exhibited the MIC value of 6.25 μg/mL. Further investigation of the most suitable lead prototype, 4-(1-adamantyl)-2-quinolinecarbohydrazide (2d, series 1) led to the synthesis of N2-alkyl/N2,N2-dialkyl/N2-aryl-4-(1-adamantyl)-2-quinolinecarboxamides (series 5) consisting of 13 analogues. Some of the synthesized carboxamides 7a, 7h, and 7m reported herein have exhibited excellent antimycobacterial activities in the range of 6.25–3.125 μg/mL against drug-sensitive and drug-resistant M. tuberculosis H37Rv strains.     

Some substrates and inhibitors of cytosolic epoxide hydrolase induce sister-chromatid exchanges in mammalian cells, but do not induce gene mutations in Salmonella typhimurium and V79 cells

Trans-stilbene oxide, trans-β-methylstyrene, 7,8-oxide, trans-β-ethylstyrene, 7,8-oxide, trans-β-propylstyrene 7,8-oxide and 4-fluorochalcone oxide were investigated for genotoxic activity in bacterial and mammalian cells, in the absence of external xenobiotic-metabolising systems. All compounds strongly enhanced the frequency of sister-chromatid exchanges (SCE) in cultured human lymphocytes. None of them was mutagenic in Salmonella typhimurium (reversion of the his⁻ strains TA98, TA100 and TA104). The limit of detection was 1/20,000 to 1/10⁶ of the activity of the positive control, benzo[a]pyrene 4,5-oxide, depending on the compound and the bacterial strain. Trans-β-methylstyrene 7,8-oxide and 4-fluorochalcone oxide were additionally tested for induction of SCE and gene mutations in the same target cells, namely Chinese hamster V79 cells. Their influence on the level of SCE was similar to that observed in human lymphocytes, whilst gene mutations (at the hprt locus) were not induced. The four investigated styrene oxide derivatives are known to be excellent substrates for a mammalian enzyme, cytosolic epoxide hydrolase (cEH). 4-Fluorochalcone oxide is a potent selective inhibitor of this enzyme and is structurally similar to the investigated styrene oxide derivatives. These properties of the test compounds however cannot explain the observed discrepancies in the results, since the genetic end point (SCE versus gene mutations) was decisive, and SCE were induced in cEH-proficient human lymphocytes as well as in cEH-deficient V79 cells.     

Indole alkaloids from the leaves of Philippine Alstonia scholaris

The first seco-uleine alkaloids, manilamine (1) (18-hydroxy-19,20-dehydro-7,21-seco-uleine) and N4-methyl angustilobine B (2), were isolated from the (pH 5) alkaloid extract of Philippine Alstonia scholaris leaves together with the known indole alkaloids 19,20-(E)-vallesamine (3), angustilobine B N4-oxide (4), 20(S)-tubotaiwine (5), and 6,7-seco-angustilobine B (6). The structure of the alkaloids was established from MS and NMR experiments.     

The role of cellular catalase on the radiosensitization of bacterial vegetative cells by N2O

The effect of N2O on eight strains of bacteria was measured in dilute suspensions. The dose-modifying factors (DMF) of N2O on M. radiodurans R1, P. radiora 0-1, M. lysodeikticus and B. pumilus E601 (vegetative cells) were 3 . 4, 2 . 9, 2 . 4 and 1 . 7, respectively. But P. radiora RP-C, P. fluorescens B3-1, E. coli B/r and E. coli K-12 were hardly sensitized by N2O. From measurements of catalase activity of each bacterium, it was found that the DMF increases with increased catalase activity, suggesting that cellular catalase promotes the sensitizing action of N2O.     

Antibacterial and cytotoxic activity of prenylated bicyclic acylphloroglucinol derivatives from Hypericum amblycalyx

Two new bicyclic acylphloroglucinol derivatives, hypercalyxone A (1-[5,7-dihydroxy-2-methyl-3-(3-methyl-but-2-enyl)-2-(4-methyl-pent-3-enyl)-chroman-8-yl]-2-methyl-propan-1-one, 1) and B (1-[5,7-dihydroxy-2-methyl-3-(3-methyl-but-2-enyl)-2-(4-methyl-pent-3-enyl)-chroman-8-yl]-2-methyl-butan-1-one, 2), have been isolated from the petroleum ether extract of the aerial parts of Hypericum amblycalyx, together with two further compounds (1-[5,7-dihydroxy-2-methyl-2-(4-methyl-pent-3-enyl)-chroman-8-yl]-2-methyl-propan-1-one, 3 and 1-[5,7-dihydroxy-2-methyl-2-(4-methyl-pent-3-enyl)-chroman-8-yl]-2-methyl-butan-1-one, 4), which have been described only as semi-synthetic products. In addition, the known triterpene lup-20(29)-en-3-one was obtained. Structure elucidation was based on 1D and 2D NMR studies, as well as on data derived from mass spectrometry. The four acylphloroglucinol derivatives were evaluated for their cytotoxic and antibacterial activity. All compounds showed moderate cytotoxic activity against KB and Jurkat T cancer cells. Especially compounds 3 and 4 exhibited a strong antibacterial activity against different Gram-positive strains.     

Oxidation pattern of the anticancer drug ellipticine by hepatic microsomes - similarity between human and rat systems

Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochrome P450 (CYP). We investigated the ability of CYP enzymes in rat, rabbit and human hepatic microsomes to oxidize ellipticine and evaluated suitable animal models mimicking its oxidation in humans. Ellipticine is oxidized by microsomes of all species to 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N(2)-oxide. However, only rat microsomes generated the pattern of ellipticine metabolites reproducing that formed by human microsomes. While rabbit microsomes favored the production of ellipticine N(2)-oxide, human and rat microsomes predominantly formed 13-hydroxyellipticine. The species difference in expression and catalytic activities of individual CYPs in livers are the cause of these metabolic differences. Formation of 7-hydroxy- and 9-hydroxyellipticine was attributable to CYP1A in microsomes of all species. However, production of 13-hydroxy-, 12-hydroxyellipticine and ellipticine N(2)-oxide, the metabolites generating DNA adducts, was attributable to the orthologous CYPs only in rats and humans. CYP3A predominantly generates these metabolites in rat and human microsomes, while CYP2C3 activity prevails in microsomes of rabbits. The results underline the suitability of rat species as a model to evaluate human susceptibility to ellipticine.     

X-ray refinement study on the binding of cytidylic acid (2'-CMP) to ribonuclease A

The X-ray structure of the inhibitor complex of bovine ribonuclease A with cytidylic acid (2'-CMP) has been determined at 2.3 A (1 A = 0.1 nm) resolution and refined by restrained least-squares refinement to R = 0.132 for 5650 reflections. Incorporation of the inhibitor molecule has occurred with little disturbance of the protein main-chain atoms, although significant displacement of some side-chain atoms has occurred, particularly in the region of the active site. The binding of 2'-CMP to ribonuclease A is different from that of the related cytidine-N(3)-oxide 2'-phosphate, which has an extra oxygen on N(3) of the cytidine base. The PO4(2-) group is held by hydrogen bond interactions to the side-groups of His 12, Glu 11 and His119. Thr45 is involved in stabilizing the enzyme-ligand complex by forming hydrogen bond interactions between O(gamma) and the pyrimidine base N(3) atom and between the main-chain N(45) and O(2) of the base. Phe120 is much closer to the inhibitor than in the cytidine N(3)-oxide 2'-phosphate structure.     

In Vitro antifungal activity of 2-(4-substituted phenyl)-3(2H)-isothiazolones

The in vitro antifungal activity of several N2-phenyl-3(2H)-isothiazolones substituted at C4 of the phenyl moiety with heterocyclic nucleus or groups of different physico-chemical properties against four human pathogenic fungi was determined by broth macrodilution method; results were compared with those obtained with itraconazole and ketoconazole. These isothiazolones showed moderate to high activity against some or all tested strains and in comparison with the reference drugs, 5-chloro-2-(4-nitrophenyl)isothiazol-3-one (1g), 5-chloro-2-phenylisothiazol-3-one (1c), 4-[4-(5-chloro-3-oxo-3H-isothiazol-2-yl)phenyl]-1,4-dihydrotriazol-5-one (1s) and 2-(4-nitrophenyl)isothiazol-3-one (2g) against Aspergillus niger, 5-chloro-2-(4-nitrophenyl)isothiazol-3-one (1g) and 4-[4-(5-chloro-3-oxo-3H-isothiazol-2-yl)phenyl]piperazine-1-carboxamide (1q) against Trichophyton mentagrophytes had comparable activity, compounds 1g and 2g showing higher activity against Microsporum canis. Antifungal activity was favored by the presence of chlorine at C5 of the isothiazolone and/or the presence of nitro group or heterocyclic nucleus at C4 of the phenyl ring and proper hydrophilicity of the molecule.     

Sialidase activity of influenza A virus in an endocytic pathway enhances viral replication

N2 neuraminidase (NA) genes of the 1957 and 1968 pandemic influenza virus strains possessed avian-like low-pH stability of sialidase activity, unlike most epidemic strains. We generated four reverse-genetics viruses from a genetic background of A/WSN/33 (H1N1) that included parental N2 NAs of 1968 pandemic (H3N2) and epidemic (H2N2) strains or their counterpart N2 NAs in which the low-pH stability of the sialidase activity was changed by substitutions of one or two amino acid residues. We found that the transfectant viruses bearing low-pH-stable sialidase (WSN/Stable-NAs) showed 25- to 80-times-greater ability to replicate in Madin-Darby canine kidney (MDCK) cells than did the transfectant viruses bearing low-pH-unstable sialidase (WSN/Unstable-NAs). Enzymatic activities of WSN/Stable-NAs were detected in endosomes of MDCK cells after 90 min of virus internalization by in situ fluorescent detection with 5-bromo-4-chloro-indole-3-yl-alpha-N-acetylneuraminic acid and Fast Red Violet LB. Inhibition of sialidase activity of WSN/Stable-NAs on the endocytic pathway by pretreatment with 4-guanidino-2,4-dideoxy-N-acetylneuraminic acid (zanamivir) resulted in a significant decrease in progeny viruses. In contrast, the enzymatic activities of WSN/Unstable-NAs, the replication of which had no effect on pretreatment with zanamivir, were undetectable in cells under the same conditions. Hemadsorption assays of transfectant-virus-infected cells revealed that the low-pH stability of the sialidase had no effect on the process of removal of sialic acid from hemagglutinin in the Golgi regions. Moreover, high titers of viruses were recovered from the lungs of mice infected with WSN/Stable-NAs on day 3 after intranasal inoculation, but WSN/Unstable-NAs were cleared from the lungs of the mice. These results indicate that sialidase activity in late endosome/lysosome traffic enhances influenza A virus replication.