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A monomeric complex of ZnII with ornidazole [Zn(Onz)2Cl2] decreases formation of the nitro-radical anion (R–NO2˙−), and this is realized by recording it in an enzyme assay using xanthine oxidase, which is a model nitro-reductase. Although the formation of R–NO2˙− is essential for drug action, as it is also associated with neurotoxic side effects, it is imperative to control its generation in order to avoid excess presence. With a decrease in R–NO2˙−, while the neurotoxic side effects should decrease, it can be expected that a compromise with regard to therapeutic efficacy will be seen since the complex will be less active in the free radical pathway. Since R–NO2˙− is crucial for the functioning of 5-nitroimidazoles, we attempted to find out if its biological activity is affected in any way in our effort to control its formation. For this purpose, Entamoeba histolytica (HM1:IMS Strain) was chosen as a biological target to realize the performance of the complex with respect to ornidazole (R–NO2). The experiments revealed that the complex not only compares well with ornidazole, but in fact, under longer exposure times, it also performs better than it. This efficacy of the complex was seen despite a decrease in R–NO2˙−, as identified by an enzyme assay, and this was probably due to certain attributes of the complex formation that are not known for ornidazole. These attributes outweigh any loss in efficacy in the free radical pathway following complex formation. This is certainly an advantage of complex formation and helps to improve the therapeutic index. This study has attempted to look at some of the possible reasons why the complex performs better than ornidazole. One reason is its ability to bind to DNA better than ornidazole does, and this can be understood by following the interaction of ornidazole and its Zn(II) complex with calf-thymus DNA using cyclic voltammetry. Therefore, this study showed that despite a decrease in R–NO2˙−, the complex does not compromise its efficacy, and this was examined using a biological target. In addition, the complex is likely to have less toxic side effects on the host of the disease-causing microbes.

A monomeric complex of Zn

Emodin, a hydroxy-9,10-anthraquinone, resembles anthracycline anticancer drugs at the core and possesses anticancer activities. A CuII complex of emodin [CuII(emod)2]2− was synthesized and its crystal structure was determined by Rietveld refinement of the PXRD data by using an appropriate structural model based on spectroscopy. This is the third report on the crystal structure of a hydroxy-9,10-anthraquinone with a 3d-transition metal ion. Since the formation of reactive oxygen species (ROS) by anthracycline-based anticancer drugs is important for antitumor activity and given the fact that the generation of ROS is responsible for cardiotoxic side effects, it is essential to be able to control their formation. Complex formation decreases ROS generation and could thereby lead to a decrease in cardiotoxic side effects. However, in an attempt to decrease complications, there is also the possibility of compromising the therapeutic efficacy. For this reason, the activities of emodin and its modified form [Cu(II) complex] were studied on the carcinoma cell lines HeLa and Hep G2 to see how they compared with each other in terms of performance. Studies were also performed on WI 38 lung fibroblast normal cells. The studies revealed that, in spite of the decreased ROS formation, followed by the DCFDA assay, the Cu(II) complex showed better activity on carcinoma cell lines. This suggests that the complex has other attributes that enable it to perform better than emodin. Consequently, one such attribute, namely DNA binding, was thoroughly investigated by varying the ionic strength and the temperature of the medium. It was found that the complex was able to bind DNA better than emodin, and, more importantly, since both generate a good amount of anionic species in solution under increased ionic strength of the medium, both bind DNA better; the increase in binding with increase in ionic strength being higher for the complex. The study suggests that with a substantial decrease in ROS generation by the complex, there are likely to be less toxic side effects, which is a key advantage of the complex, leading to an improvement in the therapeutic index. The complex showed almost no activity on WI 38 normal cells.

The formation of reactive oxygen species (ROS) by anthracycline anticancer drugs is essential for their antitumor activity but they also make these drugs cardiotoxic. When complexed with metal ions there is a decrease in ROS formation and therefore in cardiotoxicity. Interestingly, in spite of producing fewer ROS, some of the complexes are effective antitumor agents, often better than the parent anthracycline. Purpurin (LH3), a hydroxy-9,10-anthraquinone, resembles doxorubicin at the core. An MnII complex of LH3 [MnII(LH2)2] was synthesized to see the extent to which the complex resembles metal–anthracyclines with regard to structure and function. The crystal structure was determined by Rietveld refinement of PXRD data using an appropriate structural model developed on the basis of spectroscopic information. This is only the second report on the crystal structure of a hydroxy-9,10-anthraquinone with a 3d-transition metal ion. Bond lengths and bond angles were obtained by structural refinement. The structure is supported by DFT calculations. DNA binding of the complex is slightly better than for purpurin but more importantly unlike purpurin, the binding constant values remained constant even with an increase in the pH of the medium. The NADH dehydrogenase assay and the DCFDA-ROS generation assay showed that generation of superoxide in the former and ROS in general in the latter were significantly less for the complex than for purpurin. Even with decreased ROS formation, the complex is able to maintain the biological activity of purpurin.

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.

THA, a structural analogue of the family of anthracycline anticancer drugs, was used to understand how environmental changes affect its biophysical interactions with DNA. A variation of the ionic strength of the medium was attempted at a constant pH. Under such varying conditions, the binding constant and the site size of interaction were evaluated. Owing to the close structural similarity with anthracyclines and the fact that the quinone moiety in these drugs control the chemical reactions, the effect of the ionic strength on the physicochemical and biophysical attributes of hydroxy-9,10-anthraquinones is important. With an increase in ionic strength, the dissociation of the first proton of THA is affected to a greater extent compared to the second proton. Since the pKa1 is connected to the generation of the mono-anion of THA, an increase in its value implies that there would be less anion in the medium leading to improved DNA binding. Increased NaCl concentration neutralizes the negative charges on the DNA backbone manifesting in an overall increase in the binding constant for THA. This fact might be exploited for the use of such molecules in cancer patients. However, in the case of a CuII complex of THA and the formation of anions being almost negligible, there is a marked improvement in DNA binding. The cytotoxic action of THA (LH3) and its CuII complex [CuII(LH2)2] was also studied on three breast carcinoma cell lines and a primary human dermal fibroblast cell line. The complex was seen to perform better than THA. The results could be explained with the help of the comet assay, the γH2AX foci assay, and DAPI staining followed by western blotting with an apoptotic protein marker. The findings of THA and its CuII complex were compared to anthracycline doxorubicin.

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.