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Purpose To explain the observed radio-protection properties of an azo compound, 2-(2-hydroxyphenylazo)-indole-3∕-acetic acid (HPIA). Materials and methods Mechanism of radioprotection by HPIA was attempted using the stable free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) using UV-Vis and electron paramagnetic resonance (EPR) spectroscopy. The radical destroying ability of HPIA was studied by depletion of reactive oxygen species (ROS) in WI 38 lung fibroblast cells. Results & Discussion Studies indicate HPIA interacts with radical intermediates formed in solution following irradiation by 60Co γ-rays. As a result, reactive radical intermediates do not cause any damage on chosen substrates like thymine or calf thymus DNA when irradiated in presence of HPIA. The study showed that reactive intermediates not only react with HPIA but that the kinetics of their reaction is definitely faster than their interaction either with thymine or with DNA. Had this not been the case, much more damage would have been observed on chosen substrates following irradiation with 60Co γ-rays, in the presence of HPIA than actually observed in experiments, particularly those that were performed in a relatively high dose. Experiments reveal radiation induced-damage caused to thymine in presence of HPIA was ~ 136 to ~ 132times that caused in its absence under different conditions indicating the radio-protection properties of HPIA. In case of calf thymus DNA, damage in presence of HPIA was much lower than in its absence. A fluorometric microplate assay for depletion of ROS by detecting the oxidation of 2′,7′-dichlorofluorescin-diacetate (DCF-DA) into the highly fluorescent compound 2′,7′ dichlorofluorescein (DCF) indicated HPIA brought about a considerable check on ROS-mediated damage to cells by scavenging them right away. Conclusion The study indicates HPIA may be an antioxidant supplement during radiotherapy., Pharmaceutical chemistry, 2-(2-hydroxyphenylazo)-indole-3ï,¤-acetic acid (HPIA), Thymine, Calf thymus DNA, DPPH, DCFDA, Radioprotection

Curcumin is an important molecule with a plethora of pharmacological activities and therapeutic potentials. Despite its efficacy, it remained a potential drug candidate owing to hydrolytic instability and poor aqueous solubility. To overcome the limitations related to low solubility, low bioavailability, and the fact that curcumin is never present in solution as a “single unit”, its complex was prepared with MnII with the idea that binding to a metal ion might help to resolve these issues. The complex was characterized by elemental and spectral analysis. The structure of the complex was determined by density functional theory calculations. The complex was stable at physiological buffer conditions, unlike curcumin. It did not have any detrimental effect on mammalian cells. There was a significant enhancement in the antibacterial activity of the complex compared to curcumin against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. It showed a strong affinity for deoxyribonucleic acid (DNA) evident from a high binding constant value with calf thymus DNA and also from the retarded electrophoretic mobility of bacterial plasmid DNA. The complex showed “superoxide dismutase-like” activity leading to the generation of reactive oxygen species (ROS). The complex caused bacterial membrane perturbation evident from calcein leakage assay, which was further corroborated by scanning and transmission electron microscopic experiments. Overall, the present study shows improved stability and antibacterial potency of a nontoxic complex over curcumin. Its multitargeting mode of action such as ROS-production, effective binding with DNA, and permeabilization of bacterial membrane together allows it to be an effective antibacterial agent that could be taken further for therapeutic use against bacterial infections.

The antibacterial efficacy of a Cu(II) complex of $$\hbox {2-hydroxyphenyl-azo-2}^{\prime }\hbox {-naphthol}$$ (HPAN) was studied on gram-positive Bacillus subtilis and gram-negative Escherichia coli. In vitro antimicrobial activity was determined by an agar-well diffusion assay and minimum inhibitory concentration. $$\hbox {Cu}^{\mathrm{II}}(\hbox {HPAN})_{2}$$ was found to be better than HPAN in antibacterial activity. Although both bacterial strains succumbed to high concentrations of each compound, at low concentrations only $$\hbox {Cu}^{\mathrm{II}}(\hbox {HPAN})_{2}$$ was active on B. subtilis. To explain the observations, reductive cleavage of the azo-bond to aromatic amines was followed by an in vitro enzyme assay using cell extracts of E. coli containing azo-reductase. Interaction of $$\hbox {Cu}^{\mathrm{II}}(\hbox {HPAN})_{2}$$ with calf thymus DNA was compared with HPAN for correlation with antibacterial activity. Enzyme-assay on the reductive degradation of azo bond and DNA interaction do not individually explain trends observed in antibacterial activity. Comparable binding of $$\hbox {Cu}^{\mathrm{II}}(\hbox {HPAN})_{2}$$ and HPAN with calf thymus DNA was attributed to the presence of anionic species of the complex in solution. Significant activity of the complex was probably due to effective cellular uptake of it by bacterial cells as shown by a fluorescence study. The activity of the complex resembled some established antimicrobial agents. Since the complex has a moiety, not common to most antibacterial agents, resistance towards it should be significantly less, hence an advantage. Synopsis: $$\hbox {2-Hydroxyphenyl-azo-2}^{\prime }\hbox {-naphthol}$$ (HPAN) and its $$\hbox {Cu}^{\mathrm{II}}$$ complex were screened for antibacterial activity by agar-well diffusion assay and minimum inhibitory concentration. Studies on azo bond cleavage by enzyme assay and DNA binding of the compounds were done to explain the antibacterial activity. The pronounced activity of $$\hbox {Cu}^{\mathrm{(II)}}$$ -HPAN is attributed to an effective cellular uptake by bacterial cells.

Reductive cleavage of azo bond in a CoIIcomplex of HPIA decreased significantly implying less toxicity. On human colon carcinoma HCT116, ALL MOLT-4 & MCF-7 breast cancer cells complex was more effective than HPIA but less toxic towards normal cells.

A major drawback of azo compounds is their associated toxicity, often carcinogenic, which is related to the reduction of the azo bond. This study intends to re-investigate this behavior by studying 5-amino-2-((3-hydroxy-4-((3-hydroxyphenyl) phenyl) diazenyl) phenol (AHPD), a compound containing two azo bonds. Interaction of AHPD and its dimeric Cu(II) complex with bacterial strains Escherichia coli and Staphylococcus aureus revealed the complex was less toxic. Reductive cleavage of the azo bond in AHPD and the complex followed using cytochrome c reductase (a model azo-reductase) as well as azo-reductase enzymes obtained from bacterial cell extracts. Degradation of the azo bond was less in the complex allowing us to correlate the observed cytotoxicity. Cyclic voltammetry on AHPD and the complex support observations of enzyme assay experiments. These were particularly useful in realizing the formation of amines as an outcome of the reductive cleavage of azo bonds in AHPD that could not be identified through an enzyme assay. Results suggest that complex formation of azo compounds could be a means to control the formation of amines responsible for cytotoxicity. Studies carried out on bacterial cells for mere simplicity bear significance for multicellular organisms and could be important for human beings involved with the preparation and utilization of azo dyes.

Variation of pyridyl based receptors around a Cu(II) center results in structural diversity allowing redox triggered Cu(II)/Cu(I) fluorescence switch for intracellular Cu(I) imaging. Single crystal X-ray structures of 2-hydroxy-6-methylnicotinic acid (L1), its mono nuclear Cu(II) complex, viz. [Cu(L1)(2)(H2O)(SO4)]center dot 2H(2)O (C1a), polymeric Cu(II) complex (C1b) and a dinuclear Cu(II) complex of 2-(naphthalen-2-ylmethylsulfanyl)nicotinic acid (L4), viz. [Cu-2(L4)(4)(H2O)(2)]center dot 1.12H(2)O (C4) with a Cu-Cu distance of 2.6028(7) angstrom are reported. Moreover, C1a interacts with calf thymus DNA, which is useful for disruption of normal DNA activity with plausible applications in biology (binding constant, K' = 1.56 x 10(4)). Chemical or electrochemical reduction of C1a generates a fluorescent Cu(I) complex. The lowest detection limit of L1 for Cu(I) is 50 mu M. The association constant of L1 for Cu+ is 7.09 x 10(2). Quenching constants of Cu(II) for L1, L2, L3 and L4 are 4 x 10(5), 4.2 x 10(6), 8.9 x 10(5) and 4.4 x 10(6), respectively. The magnetic behavior of C1b indicates a ferromagnetic one-dimensional solid.

A major intention of this study was to use the modified toxicity of the azo moiety in [2-(2-hydroxyphenylazo)-1H-indol-3-yl]-acetic acid (HPIA), achieved through complex formation with CoII on some cancer cell lines. This is important because the azo functional group has not been tried in cancer chemotherapy. Keeping in mind aspects of drug resistance of some of the common anticancer drugs, a serious problem and a major clinical challenge in cancer chemotherapy, it is essential that new molecules are identified with anticancer activity. Although cytotoxicity of azo compounds is established there are not many reports that utilize them in cancer treatment. Another important aspect is to prepare compounds having preferential activity on cancer cells over normal cells so that toxic side effects are a minimum. Enzyme assay on the reductive cleavage of the azo bond showed complex formation with CoII almost completely checked the generation of cytotoxic amines implying that the complex could be less cytotoxic which was actually observed in case of normal healthy cells. Even though the complex formed less cytotoxic amines and possessed an almost similar binding ability with DNA like that of HPIA surprisingly its activity on three cancer cell lines namely human colon carcinoma HCT116 cells, acute lymphoblastic leukemia MOLT-4 cells and MCF-7 breast cancer cells was much greater than HPIA. The difference in activity between HPIA and its CoII complex on cancer cells showed no correlation with DNA binding or amine formation like that observed for normal cells. The complex probably possesses multiple modes of action whereby it is able to inhibit one or more cellular processes or functioning of different enzymes involved in the cell cycle of cancer cells for which it was found more effective.

A Cu(II) complex of 2-hydroxyphenyl-azo-2′-naphthol (HPAN) having the formula CuII(HPAN)2 was characterized by different techniques. When HPAN and CuII(HPAN)2 were incubated for 24 hours with human T-acute lymphoblastic leukemia (MOLT-4) cells, almost no activity was observed for HPAN while the complex was active. When incubated for 48 hours, HPAN showed cell death of ∼35% at a concentration of 40 μM while CuII(HPAN)2 was only slightly better than when incubated for 24 hours. Therefore, irrespective of incubation time, the anti-proliferative activity due to CuII(HPAN)2 was similar. However, increase in incubation time did show increased activity for HPAN. Anti-leukemic potential was confirmed by microscopic analysis of cell viability by trypan blue stain and MTT assay. The BrdU assay further confirmed proliferative effects of aqueous Cu(II)/HPAN and anti-proliferative effects of Cu(II)(HPAN)2. Propidium iodide staining of Cu(II)(HPAN)2-treated MOLT-4 cells confirmed apoptosis. Since amines formed as a consequence of reduction of the azo bond are reported to be cytotoxic, we performed an enzyme assay to understand the relative reduction of the azo bond in both compounds. Results suggest reduction of the azo bond was slightly higher for HPAN. DNA binding of CuII(HPAN)2 using fluorescence spectroscopy was compared with that of HPAN to determine the propensity of biological activity. The results being similar, binding of the compounds with DNA and the ease of reduction of the azo bond were not able to explain why CuII(HPAN)2 was better in preventing cell proliferation. The high anti-proliferative activity of CuII(HPAN)2 was attributed to increased cellular uptake. We designed experiments to support this hypothesis using independent approaches. In one, Cu(II) was identified in cell lysates using ferrocyanide, while, in another, CuII(HPAN)2 was detected using flow cytometry. We chose Cu(II) as the metal ion for this work because of its recognized involvement in cancer. Being essential for angiogenesis, it is found in increased levels in cancer cells. Interaction of Cu(II)(aq) with MOLT-4 cells confirmed this as a part of this study also. Hence, our objective was to see if molecules like HPAN that bind Cu(II) could lead to its role reversal, i.e. from supporting the growth of cancer cells to be able to destroy them as CuII(HPAN)2.