A novel mitochondria-targeted phosphorescence probe for hypochlorite ions detection in living cells.

Monitoring hypochlorite anion (ClO-) in living cells is particularly meaningful and valuable, because over-exposure of the ClO- may cause a potential health hazard towards animals and humans. Considering the special structure and properties of the gemini surfactant, a novel amphiphilic gemini-iridium complex Ir[(ppy-iso)2(bpy-tma2Br2)] (Ir-iso) with isoniazide as a recognition site for ClO- was designed. The Ir-iso possessed an excellent water-solubility as well as a strong ClO- binding capacity, as revealed from the rapid response of emission signal towards ClO-. It was worth noting that such probe had a highly-specific selectivity with a low detection limit (20.5 nM) and was suitable in physiological environment. The cell viability assay, cell imaging, and co-location studies further proved that the Ir-iso had little cytotoxicity and was specifically localized in the mitochondria of breast cancer cells, being a promising candidate of chemo-sensor to detect the endogenous ClO- in living cells.

Hydrazone, benzohydrazones and isoniazid-acylhydrazones as potential antituberculosis agents.

Aim: To evaluate the potential of three benzohydrazones (1-3), four acylhydrazones derived from isoniazid (INH-acylhydrazones) (4-7) and one hydrazone (8) as antituberculosis agents. Materials & methods: Inhibitory and bactericidal activities were determined for the reference Mycobacterium tuberculosis (Mtb) strain and clinical isolates. Cytotoxicity, drug combinations and ethidium bromide accumulation assays were also performed. Results: The tested compounds (1-8) presented excellent antituberculosis activity with surprisingly inhibitory (0.12-250 µg/ml) and bactericidal values, even against multidrug-resistant Mtb clinical isolates. Compounds showed high selectivity index, with values reaching 1833.33, and a limited spectrum of activity. Some of the compounds (2 & 8) are also great inhibitors of bacillus efflux pumps. Conclusion: Benzohydrazones and INH-acylhydrazones may be considered scaffolds for the development of new anti-Mtb drugs.

New lipophilic isoniazid derivatives and their 1,3,4-oxadiazole analogues: Synthesis, antimycobacterial activity and investigation of their mechanism of action.

The development of novel drugs is essential for the treatment of tuberculosis and other mycobacterial infections in future. A series of N-alkyl-2-isonicotinoylhydrazine-1-carboxamides was synthesized from isoniazid (INH) and then cyclized to N-alkyl-5-(pyridin-4-yl)-1,3,4-oxadiazole-2-amines. All derivatives were characterised spectroscopically. The compounds were screened for their in vitro antimycobacterial activity against susceptible and multidrug-resistant Mycobacterium tuberculosis (Mtb.) and nontuberculous mycobacteria (NTM; M. avium, M. kansasii). The most active carboxamides were substituted by a short n-alkyl, their activity was comparable to INH with minimum inhibitory concentrations (MICs) against Mtb. of 0.5-2 µM. Moreover, they are non-toxic for HepG2, and some of them are highly active against INH-resistant NTM (MICs ≥4 µM). Their cyclization to 1,3,4-oxadiazoles did not increase the activity. The experimentally proved mechanism of action of 2-isonicotinoylhydrazine-1-carboxamides consists of the inhibition of enoyl-ACP reductase (InhA) in a way similar to INH, which is blocking the biosynthesis of mycolic acids. N-Dodecyl-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine as the most efficacious oxadiazole inhibits growth of both susceptible and drug-resistant Mtb. strains with uniform MIC values of 4-8 µM with no cross-resistance to antitubercular drugs including INH. The mechanism of action is not elucidated but it is different from INH. Obtained results qualify these promising derivatives for further investigation.

New hydrazides derivatives of isoniazid against Mycobacterium tuberculosis: Higher potency and lower hepatocytotoxicity.

Tuberculosis (TB) is one of the leading causes of death worldwide. The emergence of multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) and TB-HIV co-infection are major public health challenges. The anti-TB drugs of first choice were developed more than 4 decades ago and present several adverse effects, making the treatment of TB even more complicated and the development of new chemotherapeutics for this disease imperative. In this work, we synthesized two series of new acylhydrazides and evaluated their activity against different strains of Mtb. Derivatives of isoniazid (INH) showed important anti-Mtb activity, some being more potent than all anti-TB drugs of first choice. Moreover, three compounds proved to be more potent than INH against resistant Mtb. The Ames test showed favorable results for two of these substances compared to INH, one of which presented expressly lower toxicity to HepG2 cells than that of INH. This result shows that this compound has the potential to overcome one of the main adverse effects of this drug.

Synthesis and mechanistic investigation of iron(II) complexes of isoniazid and derivatives as a redox-mediated activation strategy for anti-tuberculosis therapy.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB) represents a major threat to global health. Isoniazid (INH) is a prodrug used in the first-line treatment of tuberculosis. It undergoes oxidation by a catalase-peroxidase KatG, leading to generation of an isonicotinoyl radical that reacts with NAD(H) forming the INH-NADH adduct as the active metabolite. A redox-mediated activation of isoniazid using an iron metal complex was previously proposed as a strategy to overcome isoniazid resistance due to KatG mutations. Here, we have prepared a series of iron metal complexes with isoniazid and analogues, containing alkyl substituents at the hydrazide moiety, and also with pyrazinamide derivatives. These complexes were activated by H2O2 and studied by ESR and LC-MS. For the first time, the formation of the oxidized INH-NAD adduct from the pentacyano(isoniazid)ferrate(II) complex was detected by LC-MS, supporting a redox-mediated activation, for which a mechanistic proposition is reported. ESR data showed all alkylated hydrazides, in contrast to non-substituted hydrazides, only generated alkyl-based radicals. The structural modifications did not improve minimal inhibitory concentration (MIC) against MTB in comparison to isoniazid iron complex, providing support to isonicotinoyl radical formation as a requirement for activity. Nonetheless, the pyrazinoic acid hydrazide iron complex showed redox-mediated activation using H2O2 with generation of a pyrazinoyl radical intermediate and production of pyrazinoic acid, which is in fact the active metabolite of pyrazinamide prodrug. Thereby, this strategy can also unveil new opportunities for activation of this type of drug.

Synthesis, Characterization, Antimicrobial and Antiproliferative Activity Evaluation of Cu(II), Co(II), Zn(II), Ni(II) and Pt(II) Complexes with Isoniazid-Derived Compound.

Hydrazone complexes of Cu(II), Co(II), Zn(II), Ni(II) and Pt(II) with N-isonicotinoyl-N'-(3-metoxy-2 hydroxybenzaldehyde)-hydrazone (HL) were synthesized and characterized by different physico-chemical techniques including elemental and thermal analysis, magnetic susceptibility measurements, molar electric conductivity, as well as IR (infrared), ¹H-NMR and 13C-NMR (hydrogen and carbon nuclear magnetic resonance, UV-Vis (ultraviolet-visible), FAB (fast atom bombardment), EPR (electron paramagnetic resonance), and mass spectroscopies. The crystal structure of ligand was determined by single crystal X-ray diffraction studies. Spectral data showed that hydrazone behaves as an ONO tridentate ligand through the azomethine nitrogen, phenolate and keto oxygen atoms. For the copper(II) complexes, metal-ligand bonding parameters were evaluated from the EPR spectra. These parameters indicate the presence of in-plane π bonding. In addition, the f values of complexes 1-4 indicate small distortion from planarity. The effect of these complexes on proliferation of human breast cancer (MCF-7 and SKBR-3), human melanoma (A375), lung adenocarcinoma cells (NCI-H1573) and their antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Candida albicans strains were studied and compared with those of free ligand. The ligand and complexes 1-3 showed significant antimicrobial activity against the Gram-positive bacteria Staphylococcus aureus and Candida albicans in comparison to the control drugs. The complexes 2-4 could be potential antitumor agents, leading to a significant improvement of the cytotoxic activity when compared with HL.

Synthesis, spectral characterization, molecular structure and pharmacological studies of N'-(1, 4-naphtho-quinone-2yl) isonicotinohydrazide.

A simple and efficient procedure was employed for the synthesis of N'-(1,4-naphtho-quinone-2-yl) isonicotinohydrazide (NIH) by the reaction of 2-hydroxy-1,4-naphthaquinone (lawsone) and isonicotinoyl hydrazine in methanol using ultrasonic irradiation. Lawsone is the principal dye, isolated from the leaves of henna (Lawsonia inermis). Structural modification was done on the molecule aiming to get a more active derivative. The structure of the parent compound and the derivative was characterized by elemental analyses, infrared, electronic, (1)H, (13)C NMR and GC-MS spectra. The fluorescence spectral investigation of the compound was studied in DMSO and ethanol. Single crystal X-ray diffraction studies reveal that NIH crystallizes in monoclinic space group. The DNA cleavage study was monitored by gel electrophoresis method. The synthesized compound was found to have significant antioxidant activity against DPPH radical (IC50=58 µM). The in vitro cytotoxic studies of the derivative against two human cancer cell lines MCF-7 (human breast cancer) and HCT-15 (human colon carcinoma cells) using MTT assay revealed that the compound exhibited higher cytotoxic activity with a lower IC50 value indicating its efficiency in killing the cancer cells even at low concentrations. These results suggest that the structural modifications performed on lawsone could be considered a good strategy to obtain a more active drug.

New strategy for selective and sensitive assay of cathepsin B using a dityrosine-based material.

The increasing number of reports for disease-related proteases has necessitated materials for the fast, sensitive, and specific assessment of protease activities. The purpose of this study was to synthesize and test a dityrosine-based substrate for the selective assay of a specific cysteine cathepsin. DBDY-Gly-INH)2 was synthesized from the conjugation of N,N'-diBoc-dityrosine (DBDY) with two molecules of glycine and isoniazid (INH) for this purpose. The fluorescence of DBDY (λex=284-320nm, λem=400-420nm) disappeared due to the quenching effect of INH. However, the protease-catalyzed hydrolysis resulted in the release of INH and recovered the fluorescence of DBDY. When reacted with 13 proteases, DBDY-Gly-INH)2 was hydrolyzed by the cysteine proteases only. Meeting the growing need to discriminate cysteine cathepsins (e.g., cathepsins B, L, and S found at high levels in various cancers), DBDY-Gly-INH)2 was tested as a substrate for cathepsins B, L, and S. Only cathepsin B catalyzed the hydrolysis reaction among the three cathepsins. The reaction rate followed the Michaelis-Menten kinetics, and the KM and kcat/KM values were 2.88µM and 3.87×10(3)M(-1)s(-1), respectively, which were comparable to those for the materials reported for the selective assay of cathepsin B. Considering the simple preparation of DBDY-(Gly-INH)2, DBDY-(Gly-INH)2 is believed to be valuable for the sensitive and selective assay of cathepsin B activity.

Synthesis of highly potent novel anti-tubercular isoniazid analogues with preliminary pharmacokinetic evaluation.

Thirty two novel isoniazid analogues were prepared by one-pot three component condensations of isoniazid (INH), 3-mercaptopropionic acid and various aryl/heteroaryl aldehydes. The synthesized compounds were evaluated for their anti-TB activity against Mycobacterium tuberculosis H37Rv (MTB) and cytotoxicity. Among the compounds, compound N-(2-(4-(benzyloxy) phenyl)-4-oxo-1,3-thiazinan-3-yl) isonicotinamide (17) inhibited MTB with MIC of 0.12 µM and was three times more potent than INH. The main pharmacokinetic parameters after intravenous administration (10 mg/kg body weight) in male Wistar rats viz. t(½), K(el), mean plasma clearance and mean volume of distribution were found to be 1.14±0.20 h, 0.62±0.10 h(-1), 22.48±0.16 mL/kg/min and 1.99±0.49 L, respectively. The systemic absorption was slow after oral administration (50 mg/kg body weight). The peak plasma concentration was found to be 1.31±0.06 µg/mL attained in 3 h. The bioavailability was found to be 16.7%.

Planejamento racional de novos tuberculostáticos com base na estrutura das enzimas enoil redutase e timidina monofosfato quinase do Mycobacterium tuberculosis / Rational design of new antituberculosis agents based on the structures of Mycobacterium tuberculosis enzymes enoyl reductase and thymidylate kinase

Nos últimos quinze anos, a tuberculose (TB) ressurgiu tanto em países em desenvolvimento quanto naqueles desenvolvidos. Em 1993, a Organização Mundial da Saúde (OMS) declarou a TB como emergência global de saúde pública. Atualmente, um terço da população mundial está infectada pelo Mycobacterium tuberculosis e mais de 10% destes indivíduos desenvolverão a doença ativa. Em 2006, estimaram-se 9 milhões de novos casos de TB em todo o mundo. No Brasil, aproximadamente 95.000 novos casos são registrados anualmente, com incidência de 50 casos em cada 100.000 habitantes. Tendo em vista o quadro alarmante da TB no mundo e, em especial no Brasil, e considerando os índices elevados de resistência do microrganismo aos fármacos convencionalmente utilizados na terapêutica, há necessidade urgente do desenvolvimento de novos tuberculostáticos. Além disso, a busca por novos alvos de ação se faz necessária, já que os antimicobacterianos utilizados na terapia anti-TB têm como alvo apenas pequeno número de enzimas relacionadas a funções essenciais do microrganismo. A biossíntese bacteriana de ácidos graxos tem despertado atenção especial como alvo atraente no desenvolvimento de novos agentes antibacterianos. Diferenças bioquímicas e funcionais fazem com que as enzimas envolvidas em tal processo sejam alvos potencialmente atraentes para o desenvolvimento de novos agentes antibacterianos/antimicobacterianos. As enoil-acp redutases são enzimas determinantes na etapa de alongamento de ácidos graxos, produtos intermediários na biossíntese dos principais constituintes da parede celular micobacteriana, os ácidos micólicos...

Synthesis, characterization, and antifungal studies of transition metal complexes of omega-bromoacetoacetanilide isonicotinylhydrazone.

Isonicotinic acid hydrazide or isonicotinylhydrazide, commonly known as isoniazid, is an antibacterial agent that has been used to treat tuberculosis. It interacts with microbial cell walls. Schiff's bases or anils are the compounds having >C=N-N< linkages, which have immense applications as catalysts, stabilizers, pigments, dyes, and drugs. They have good ability to form chelates with many metal ions. Isoniazid can form Schiff's bases with diketones such as acetoacetanilide. Acetoacetanilide isonicotinylhydrazone and its metal chelates exhibit anticancer activity. Our studies on N-methylacetoacetanilide isonicotinylhydrazone and its metal chelates revealed that they are active against pathogenic fungal strains. Hence, it is worthwhile to synthesize new complexes of ligands having different substituents on the acetoacetanilide moiety. We synthesized five new metal chelates of omega-bromoacetoacetanilide isonicotinylhydrazone. The ligand behaved as a tridentate monoanion or as a tridentate dianion in the complexes. These compounds were characterized mainly by elemental analysis; conductivity measurements; and electronic, infrared, and nuclear magnetic resonance spectral studies. We also carried out antifungal studies of these compounds against four selected pathogenic fungal strains using a cup-plate technique. Both the ligand and its metal chelates were active against all fungal strains investigated. However, the chelates were found to be more active than the ligand.

Development of potential iron chelators for the treatment of Friedreich's ataxia: ligands that mobilize mitochondrial iron.

Friedreich's ataxia (FA) is a crippling neurodegenerative disease that is due to iron (Fe) overload within the mitochondrion. One therapeutic intervention may be the development of a chelator that could remove mitochondrial Fe. We have implemented the only well characterized model of mammalian mitochondrial Fe overload to examine the Fe chelation efficacy of novel chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) class. In this model we utilize reticulocytes treated with the haem synthesis inhibitor succinylacetone which results in mitochondrial Fe-loading. Our experiments demonstrate that in contrast to desferrioxamine, several of the PCIH analogues show very high activity at mobilizing (59)Fe from (59)Fe-loaded reticulocytes. Further studies on these ligands in animals are clearly warranted considering their potential to treat FA.

Development of novel aroylhydrazone ligands for iron chelation therapy: 2-pyridylcarboxaldehyde isonicotinoyl hydrazone analogs.

Previous studies have demonstrated that aroylhydrazone iron (Fe) chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class have high Fe chelation efficacy both in vitro and in vivo. Depending on their design, these drugs may have potential as agents for the treatment of Fe overload disease or cancer. Considering the high potential of this class of ligands, we have synthesized seven novel aroylhydrazones in an attempt to identify Fe chelators more efficient than desferrioxamine (DFO) and more soluble than those of the PIH class. These compounds belong to a new series of tridentate chelators known as the 2-pyridylcarboxaldehyde isonicotinoyl hydrazones (PCIH). In this study we have examined the Fe chelation efficacy and antiproliferative activity of these chelators including their effects on the expression of genes (WAF1 and GADD45) known to be important in mediating cell cycle arrest at G1/S. From seven chelators synthesized, three ligands, namely 2-pyridylcarbox-aldehyde benzoyl hydrazone (PCBH), 2-pyridylcarboxaldehyde m-bromobenzoyl hydrazone (PCBBH), and 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH), showed greater Fe chelation activity than DFO and comparable or greater efficiency than PIH. These ligands were highly effective at both mobilizing 59Fe from cells and preventing 59Fe uptake from 59Fe-transferrin and caused a marked increase in the RNA-binding activity of the iron-regulatory proteins (IRP). Our studies have also demonstrated that compared with the cytotoxic Fe chelator, 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), these ligands have far less effect on cellular growth and 3H-thymidine, 3H-leucine, or 3H-uridine incorporation. In addition, in contrast to 311, which markedly increased WAF1 and GADD45 mRNA expression, PCBH and PCTH did not have any effect, whereas PCBBH increased the expression of GADD45 mRNA. Collectively, these results demonstrate the potential of several of these ligands as agents for the management of Fe overload disease.

Synthesis of 2-[123I and 124I]-iodoisonicotinic acid hydrazide--potential radiotracers for tuberculosis.

The radiochemical syntheses of methyl 2-[123I]-iodoisonicotinate, 2-[123I]-iodoisonicotinic acid hydrazide and 2-[124I]-iodoisonicotinic acid hydrazide was accomplished. Iodine-123 was incorporated in the methyl ester molecule by an exchange reaction in glacial acetic acid. The average efficiency of iodine exchange reaction was (92.6 +/- 4.5)%. This radiotracer was extracted with ether and the solvent was evaporated. The residue was re-dissolved in anhydrous ethanol and treated with hydrazine under anhydrous conditions to obtain 2-[123I]-iodoisonicotinic acid hydrazide. The overall radiochemical yield was 69%. Biodistribution data of both radio-tracers in male Sprague-Dawley rats were collected. This is the first report of SPECT radiopharmaceuticals which may be useful for differential diagnosis of intracranial masses (tuberculoma vs glioma), and CNS tuberculosis in immunosuppressed subjects.

Syntheses of iron bis(pyridoxal isonicotinoylhydrazone)s and the in vivo iron-removal properties of some pyridoxal derivatives.

Pyridoxal isonicotinoylhydrazone (PINH; 1) and its isomeric O-acetates (E and Z) were synthesized and complexed with ferrous ions to afford the hitherto unisolated chelates iron(II) bis(pyridoxal isonicotinoylhydrazone)s (11) and iron(II) bis(O-acetylpyridoxal isonicotinoylhydrazone)s (12). The analytical and spectroscopic data of the new coordination compounds are presented. In addition, a series of imino derivatives of pyridoxal of structures 2-3 and 5-10 have been prepared and tested in vivo as chelators of storage iron, and the cumulative net excretion of radioiron in urine and in feces was estimated. This study reestablishes that PINH is a potent iron chelator in vivo comparable in efficiency with parenteral desferrioxamine (DF) and indicates that it requires further attention.

Determination of hydrazine metabolites of isoniazid in human urine by gas chromatography.

A method is described for the determination of isoniazid, acetylisoniazid, acetylhydrazine, diacetylhydrazine and hydrazine in urine. Isoniazid, acetylhydrazine and hydrazine are reacted in aqueous solution with p-chlorobenzaldehyde to form hydrazones. Following the addition of appropriate internal standards, these hydrazones are then extracted into an organic solvent and determined by gas chromatography using a nitrogen-sensitive detector. Acetylisoniazid and diacetylhydrazine are determined similarly after hydrolysis to isoniazid and acetylhydrazine, respectively.