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Introduction

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Materials and Methods

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Result and Discussion

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Conclusion

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References

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INTRODUCTION Transition metal complexes play a crucial role in antitumor therapy. Complexes of platinum, ruthenium as well as lanthanum and gallium have been investigated in preclinical as well as in clinical studies. The best known platinum (II) agents approved worldwide, Cisplatin or carbplatin, are used in nearly 50% of all cancer therapies. Due to their severe toxicity, their high activity is not always satisfactory and side effects are frequently encountered. Therefore, ongoing efforts word wide is aiming to identify novel metal-based substances with less toxicity and new mechanisms of action. Cancer The medical term for cancer is neoplasm, which PHDQV´DUHODWLYHO\DXWRQRPRXVJURZWK RIWLVVXHV´ [1]. It is a collective term for a group of diseases characterized by the loss of control of the growth, division and spread of a group of cells. It can form an encapsulated benign tumor, leading to invasion and destruction of adjacent tissues. On the other hand, non encapsulated malignant tumors grow rapidly, and can spread to various regions of the body and metastabise. Metastasis, being the cause of 90% of cancer deaths, is a secondary growth though originating from the original primary tumor. Cancer is caused by abnormalities in the genetic material of the affected cells. On the way to tumorigenesis there occurs an accumulation of successive mutations in proto-oncogenes and suppressor genes that deregulates the cell cycle. The events key to tumorigenesis are for instance point mutations in DNA sequences, chromosomal aberrations such as translocations or deletions and changes that affect the chromatin structure such as methylation of DNA or acetylation of histones. Cancer is still one of the more difficult diseases to treat and was responsible for ca. 13% (7.4 million) of all deaths worldwide in 2004 [2]. An increase in cancer death rates to 12 million is estimated by 2030. Cancer therapy is mostly based largely on surgery, radiotherapy, hormone and chemotherapy. However, the clinical results are often only a short prolongation in patient survival [3].

Cancer Chemotherapy The word 'chemotherapy' was first introduced by the German chemist and immunologist Paul Ehrlich, which means treatment of the diseases with chemicals. Moreover, Ehrlich documented the effectiveness of animal screenings to test agents for their potential biological activities [4]. Chemotherapeutical drugs aim at killing malignant tumor cells more or less selectively.Throughout the years, there were many key advances in the development of cancer chemotherapy, beginning in the first half of the 20th century (Fig 1.1. and Fig. 1.2.) [4]. Over ten years, a series of antimetabolites was discovered such as antifoliates, thiopurines, 5- fluorouracil or methotrexate. The aim of these drugs is to inhibit the enzymatic reactions required to insert the metabolite into the replicating DNA. For instance, mercaptopurine, the main representative of the group of purine antagonists is used in the treatment of acute leukemias. Another chemotherapeutic, 5-fluorouracil, a pirymidine analog, noncompetitively inhibits thymidylate synthase. It is used in colorectal cancer and pancreatic cancer.A serious problem in treatment with chemotherapeutic agents is their narrow margin of safety (i.e., therapeutic index). The non-selective biodistribution throughout the body is the cause of their toxicity. The administration requires a large total dose resulting only in high local concentrations of the drug in a tumor cell.

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Figure 1. Key advances in the cancer chemotherapy in the years 1900-1960 [4]. A more effective way to deliver a drug is by targeted therapy. This aims at a selective accumulation of the drug in the target cells independent of the method and route of drug administration. Administration of prodrugs is one of the ways to improve the selectivity of the cancer drugs. This approach takes advantage of some unique properties of the tumor such as hypoxia, selective enzyme expression and low extracellular pH. The drug resistance remains still one of the main problems of chemotherapy. Cells can develop a cross-resistance to structurally similar drugs, which is known as multidrug resistance (MDR) . The mechanisms that cause MDR are for instance drug modification or inactivation (like in the cause of cisplatin by glutathione), decreased permeability or increased efflux of the drug.

Figure 2. Key advances in the cancer chemotherapy in the years 1963-2007 .

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In 1965, Barnett Rosenberg discovered unexpectedly that the platinum complex, cisdiamminedichloridoplatinum(II) resulted in inhibition of the division processes but not the cell growth of the bacteria Escherichia coli. This discovery gave rise to search for the anticancer agents among the metal complexes.Since the discovery of the anti-cancer activity of Cisplatin some 40 years ago there has been a growing interest in the field of metal complex-based chemotherapy. In addition to platinum, medicinal chemists have investigated complexes with other transition metals such as ruthenium, copper, cobalt, palladium and gold as a way of killing cancer cells. This has given rise to the field of metal-based anticancer agents, a discipline that has very rapidly expanded and brought about the discovery of many interesting and effective anticancer agents.

Metal ion complexes as anticancer agents Before the great success of cisplatin, the idea of using inorganic chemicals to treat cancer was rather uncommon. Heavy metals, causing cases of poisoning, were not assumed to act as potential anticancer drugs because of their toxicity. The earliest attempt to introduce metals in the drug development was so called Fowler´s solution, giving rise of arsenotherapy [5]. Some of similar compounds are still been used today, such as As2O3 [6].Work with metal complexes regarding their structure-efficacy-relationships started in 1931 when two German scientists, Collier and Krauss, introduced a rational for the activity of metal FRPSOH[HVWKDWVWDWHV´7KHHIIHFWRIDKHDY\PHWDORQ experimental murine cancer is not only due to the metal alone but also to the structure of the FRPSRXQGV DQG WKH W\SH RI FRPSRXQG´>@$V %DUQHWW 5RVHQEHUJ GLVFRYHUHG cisplatin in 1965, he found that the agent can successfully inhibit the cell division without any influencing cell growth. Cisplatin was first V\QWKHVL]HGLQDQGNQRZQDV3H\URQH¶VFKORULGH5RVHQEHUJ9DQ&DPS  Co. tried to synthesize the compound by using chemical techniques; however the attempts met with failure, as they obtained complex was deprived of biological activity. It turned out that the Pt compound exists in two isomeric forms, cis and trans. The second, transplatin is more stable and was inactive. The biological studies that followed after the discovery confirmed the anticancer activity of cisplatin against Sarcoma-180 in vitro and in vivo. Publication of these results in _ature in 1969 gave rise to expansive research of metal ion complexes as anticancer agents [8, 9].Metal ion complexes very quickly turned out to be interesting and attractive compounds in the development of anticancer drugs because of their chemical reactivity. In addition, the possibility of many different coordination geometries enables the synthesis of compounds with stereochemistry that are quite unique and not obtainable in the group of pure organic compounds [10]. The critical factors in the structure of metal ion complexes are the type of ligands and the oxidation state of the metal, which regulate the biological activity of the metal-based drugs. Moreover, oxidation state often dictates a particular coordination geometry [11].In the recent years much effort has been made to increase the number of therapeutic metal complexes. Historically, metals and metal complexes have played a key role in the development of pharmacy and modern chemotherapy. However, they still remain a tiny minority of all therapeutics on the market today [12].

Development of a new anticancer agent The development of a new anticancer agent is a multi-stage process and includes such steps as synthesis, characterization, proof of biological activity, pre-clinical and clinical screenings [13,14]. Testing for the biological activity requires the measurement of the biological effect in in vitro (in the cancer cell lines) and in vivo (in animals) screens. Almost all of the successful metal based pharmaceuticals have originated from university research groups or small companies. If larger pharmaceutical companies do not take over extended work in the area of metal complexes, many developments will be lost. There are three methods to develop new tumor-inhibiting complexes [15]: ϱ 

1. through the synthesis of classical and non classical derivatives of cisplatin, 2. Synthesizing tumor-inhibiting non-platinum complexes, 3. Synthesizing the platinum complexes linked to carrier systems that have the ability to accumulate the drug in organs and tissues. The first possibility seems to be less promising because it will lead to drugs that will not much differ from cisplatin. However, attempts can be made to reduce the toxic side effects in comparison to the parent compound, as was the case in the development of carbplatin, or change the tumor selectivity, as in the case of oxaliplatin.The second approach aims at compounds with a central heavy metal ion other than platinum. Here, the range of activity can be changed owing to different chemical properties. This is a more difficult and risky approach of scientific research compared to the first strategy but the opportunities for a breakthrough are greater. Finally, the linkage of platinum complex to carrier molecules is a concept known as drug targeting. The aim of this approach is to synthesize a platinum drug that possesses high selectivity towards malignant cells. It can be done for tumors containing biochemical targets different in structure or quantity from the normal tissues. The major problem in the development of anticancer drugs is the large leap from the preclinical in vitro and in vivo studies to clinical trials. The cause for this is the great difference between the experimental animal models and the individual patient tumors, making the therapeutic situation of the cancer patients much more complex [3]

Classes of metal based pharmaceuticals There are various classes of metal based pharmaceuticals that can be divided into seven groups depending on which part of the structure is responsible for the biological activity of the compound [14]: 1. Entire inert complex is active. Here the complexes are synthesized from smaller and simpler components. An example is the group of ruthenium complexes that are highly potent inhibitors of protein kinases [16]. 2. Entire reactive complex is active. The copper complexes of NSAIDs that can mimic the actions of some enzymes, for instance superoxide dismutase, and have an analgesic effect. Very active complexes are the macrocyclic [17] and porphyrin [18] Mn(II) complexes. A five-coordinate intermediate is probably involved in the mechanism of action of the macrocylic complex.

Figure 3. Structures of cisplatin (A) and BBR3464 (B). 3. The metal ion or one of its biotransformation products is active. The complexes that deliver an active metal are for instance insulin potentiating vanadium complexes. Orthovanadate mimics phosphate and inhibits the protein tyrosine phosphatases causing an increased cellular uptake of insulin [19.20,21]. However, the problem is with the bioavailability from the gastric system. One of the maltolato complexes of vanadium(IV), bis(ethylmaltolato)oxovanadium (IV), appeared to be effective and entered phase I of the human trials with no adverse effects [22]. One of the least toxic transition metals it is unfortunately taken up and stored in bones, resulting in side effects. ϲ 

4. The metal is a radiation enhancer. While radiation causes much damage to the tissues that are close to irradiated tumor, it is worth introducing metal ion complexes that have the potential to enhance the effectiveness of the radiation in the tumor cells.Motexafin gadolinium, a dagolinium complex, showed high activity while the metal was localized to a high degree in tumor tissue [23], enhancing the treatment of brain tumours. 5. The metal ion is radioactive. Many d-block and f-block metals for instance 67Cu and 90Y have radioactive isotopes that emit high energy beta particles, which are suitable for tumor treatment. It is essential to protect the non-target radiosensitive organs such as kidneys or bone marrow. Radiolabelling of small particles radionuclide carriers that target proteins overexpressed in tumours has a great potential. 6. The metal is a radiation enhancer. While radiation causes much damage to the tissues that are close to irradiated tumor, it is worth introducing metal ion complexes that have the potential to enhance the effectiveness of the radiation in the tumor cells.Motexafin gadolinium, a dagolinium complex, showed high activity while the metal was localized to a high degree in tumor tissue [24], enhancing the treatment of brain tumours.

Antiinfective metal ion complexes Another therapeutical challenge in medicine apart from pharmaceuticals active against cancer is antiinfective therapy. There are many substances active against bacteria, fungi or viruses, however, even the best antibiotics can be ineffective at treating diseases because of ever increasing drug resistance. Therefore, much effort is directed at treating bacterialinfections with compounds of various chemical structures that are more potent than the already existing drugs on the market. One of the first antibacterial agents used were inorganic mercury salts. However, the compounds had only a bacteriostatic activity. The agents bound to the sulfhydryl groups of bacterial enzymes and inhibited their growth. No longer in use is mercury chloride. Moreover, silver has good antibacterial activity and is used not only in the form of silver nitrate but also in the salts of sulfonamides, such as sulfadiazine and sulphathiazole for the treatment of burns. Other metal salts of zinc, copper and gold also show antibacterial avtivity [25]. Antibacterial therapy aims among others at interfering in the biosynthesis of bacterial cell wall or the synthesis of the bacterial DNA or proteins. Apart from zinc(II) pyrithione, which is used in the anti-dandruff schampoos, so far there are no copper(II), cobalt(II) or platinum(II) complexes used therapeutically. Nevertheless, there is much research going into finding a good agent that could be of better activity than known antibiotics.The derivatives of benzimidazole, such as Mebendazole and Albendazole, act asanthelminthic agents. The agents bind ZLWKKLJKDIILQLW\WRȕ-tubulin of nematodes and block the development of microtubules. While such compounds possess good biological activity, it might be worth expanding the structures by formation of metal complexes that can also be potent antiinfective agents. Other antiinfective agents are metalo-organic compounds. The only two that are still in use are arsen compound Melarsoprol (Fig. 1.4. a) and antimony compound Stibogluconate [37]. The agents inactivate the SH-groups in enzymes through a reaction with the organic bound metal. Although Stibophen also contains antimony, it is no longer used due to the toxic side effects. Stibogluconat-Sodium is used in the treatment of Leishmaniasis.

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Figure 4. The structures of Melarsoprol (a) and Stibogluconate (b).

Biological properties of Copper Metal There has been much attention paid to copper because it is an essential element for life. It is associated with a number of copper-dependent enzymes that are key in biological processes.[26,27] Elevated copper levels in plasma can be important for the etiology of some illness [40]. For example, copper ions are closely involved in neurodegenerative disorders [41-43], especially in Parkinson's disease [28-29]. Moreover, there has been interest in the medical uses of copper, in particular as a complexing ion of known biologically active ligands and drugs. Throughout the years of scientific research copper(II) complexes have been found to possess various activities such as antiulcer [30], antiamoebic [31], antidiabetic [32], anticonvulsant [33], antiinflamatory [34-36], antimicrobial [37] and antitumor [38]. In particular, anti-inflammatory, antimicrobial and anti-cancer activity of copper complexes has been studied. The three sub-chapters below briefly review copper complexes with such pharmaceutical properties. Copper complexes with anti-inflammatory activity Copper has been used since the Middle ages for the treatment of arthritis. It has been shown that the copper complexes of the non steroid anti-inflammatory drugs are more active compared to the free ligands and the inorganic copper salts. Carboxylic acids of derivatives of salicylic acid or penicillamine can serve as the ligands. It was noted that the anti-inflammatory activity of these complexes is independent of the amount of copper in the tested compound [39]. This form of administration is also safer due to the protection of the gastrointestinal tract compared to the free drugs. The mechanism may be due to the transport of copper in the form of copper complexes into the cell. An induction of the copper-dependent enzymes such as superoxide dismutase (Cu,Zn-SOD) results in protection of the cells from the accumulation and harmful activity of superoxide ions by dismutation into dioxygen and hydrogen peroxide [40]. The binuclear copper complex of the well known NSAIDS drug, diclofenac, was found to inhibit the activity of lipooxygenase [41]. Another interesting compound is the copper(II) complex of 3,5-diisopropylsalicylic acid, [Cu(II)(3,5-Dips)2]2 that shows not only anti-inflammatory but also antiulcer, anticarcinogenic, anticonvulsant, antidiabetic and analgetic properties [42-46]. There has been some interest in copper(II) complexes with anti-inflammatory activity. Many of the Cu(II)-complexes have been tested but the number of drugs on the market is low. Two drugs containing Cu(II) salicylate were approved in Australia in the late 1970s and early 1980s; i.e., Alcusal and Dermusal, as the gels for the pain treatment. The former ϴ 

is an alcohol adduct and the later a DMSO adduct of salicylic acid. The parenterally administered Cu(II) complex, Permalon, has been reported but is not comercially available. Moreover, an indometacin complex Cu-Algesic, [Cu2(Indo)4(DMF)2] is in veterinary use. It is claimed that the application to dogs is deprived of GI side affects in comparison to free indometacin [27].

Copper complexes with antimicrobial and antifungal activity It is well known that the derivatives of thiosemicarbazones possess the antibacterial activity. In addition, there are many studies that show the antimicrobial activity of copper(II) complexes with these ligands [47-49].Common anti-bacterial agents have been also used as ligands to complex with copper ions. It was noticed that the antimicrobial activity against M. Smegmatis of a metal ion complex in comparison to free ciprofloxacin, a bacterial gyrase inhibitor, increased three times [50]. It may result from facilitated diffusion of the drug through the cell membranes, presumably by an increase in the lipophilicity of the drug [51]. The activity against Streptococcus can also be influenced by the slow release of the ligands inside the bacterial cell [52]. This was noticed with the copper complex of isoniazide and ethambutol. It seems that intercellular reduction of Cu(II) into Cu(I) can acitivate the oxygen which is toxic for bacteria [53]. Moreover, a copper(II) complex of sulfacetamide, (N-[4-(amino-fenil)sulfonil]acetamide), has been intensively used in treatment of ophthalmic and dermatologic infections [54, 55]. Further studies of the copper(II) complexes of sulfacetamide and sulfanilamide [56] and sulfisoxazole [57] have showed promising results. In general, sulfonamide copper(II) complexes show antimicrobial activity against both types, Gramm(+) (Staphylococcus aureus, Bacillus subtilis) and Gramm(-) (Escherichia coli, Pseudomonas euruginosa) [58]. More often though a slightly higher activity is found in Gram(-) bacteria [59]. The copper(II) complexes of benzimidazoles have not only shown antibacterial activity against S. epidermidis [60] but also some strong activity was found against fungi [61]. In addition, complexes of thiabendazole appeared to be active [62]. Moreover, antifungal activity against Aspergillus sp. and Penicillium sp. was also found for a copper(II) complex of p-amino acetophenone benzoylhydrazone [63]. There are also copper(II)complexes that possess antimalarial activity. A complex of the derivative of naphthoquinone has been reported to act against the parasite Plasmodium falciparum [64]. Furthermore, a copper(II) complex of pyridine-2-carboxamidrazone appeared to have potent antimalarial activity. The presence of a metal ion in the structure may be a new strategy to develop effective metal-based antimalarials. The enhancement of antimalarial activity by copper coordination to ligands may also be due to reduction of Cu(II) to Cu(I) [65]. Copper complexes with antitumor activity In 1946 it was noticed that certain inorganic copper salts act against hepatic tumors in animals [66]. Scientists observed that the copper complexes were more active than the inorganic copper salts. Thiosemicarbazones were some of the first compounds that showed antineoplastic activity. After the discovery of Damagk et al. [67] that certain monothiosemicarbazones show antitubercular action, new copper mono- and bis-compounds of this group were synthesized in search of antitumor activity.

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Figure 5. Structures of copper(II) monothiosemicarbazones (a) and copper(II) bisthiosemicarbazones (b). 3-Ethoxy-2-oxybutyraldehyde bisthiosemicarbazone was one of the first compounds tested for in vivo antitumor activity [68]. Its activity is likely to be due to inhibition of the synthesis of DNA and the changes in the oxidation and reduction stages of copper.

Figure 6. Cu(II)(KTS) ± one of the first copper(II) complexes tested for antitumor activity Another interesting compound was a macrocyclic copper chelate (Figure 1.7.), which was active in vitro and in vivo against P 388 leukemia and B 16 melanoma cells [69].

Figure 7. A square-planar chelate of a macrocyclic ligand with antitumor activity In a paper coauthored by Linus Pauling, it was reported that a copper(II) complex of glycylglycylhistidine associated with vitamin C gave complete regressions of osteosarcoma in a human patients [70]. Casiopeinas represent a series of mixed chelate copper complexes that are being evaluated as anticancer agents. Their structures in figure 8 are [Cu(N-N)(O-O)]NO3 or [Cu(NN)(ON)] NO3. The mechanism of action includes oxidative damage and disruption of mitochondrial ϭϬ 

function. Recent studies suggest that Casiopeina IIgly initiates the overproduction of reactive oxygen species (ROS), leading to mitochondrial dysfunction and cell death [71]. Casiopeina IIgly is hepatotoxic in rats after i.v. administration [72] but it turned out to be the most promising compound, showing strong inhibition of a glioma C6 cancer cell line in vivo and in vitro. Other Casiopeinas appear to have various grades of genotoxicity [73].

Figure 8. The structures of Casiopeina IIgly (a) and Casiopeina IIIi There has also been much interest in phenanthroline copper(II) complexes. Unsubstituted (Figure 1.9. a) or substituted with methyl groups, the phenanthroline complexes turned out to possess in vitro (Figure 1.9. b, c) and in vivo antitumor activity in rodents (Figure 1.9. c) [74- 75, respectively].

Figure 9. The structures of differently substituted ring of phenanthroline A copper complex of o-phenanthroline and _-(9H-purin-6-yl)benzenesulfonamide turned out to be active in human Caco-2 cells and Jurkat T lymphocytes [76]. Polymeric copper(II) phenanthroline ϭϭ 

complexes with different contents of copper were prepared and the one with a high content of copper showed antitumor activity [77]. After it became apparent that some tumor cells have reduced superoxide dismutase (SOD) activity compared to normal cells, copper complexes with SOD mimetic activity where considered for their chemotherapeutic activity [78]. Compounds that displayed SOD-like activity were copper(II) salicylate [79] and copper(II) 3,5- diisopropylsalicylate . The later showed activity against sarcoma 180 and Ehrlich ascites carcinoma [80, 81]. However, whether the antitumor activity is a direct result of the SOD mimetic activity is not known.

Figure 10. Structures of copper(II) salicylate (a) and a derivative (b). Copper coordination compounds of derivatives of imidazole compounds have been one of the most often synthesized compounds in search for biologically active compounds. The strategy to obtain an antitumor drug was to synthesize a square-planar compound with zero net charge and bearing various moderately good leaving ligand [82]. Recently, Saczewski and coworkers showed that copper complexes of benzimidazole have in vitro antitumor activity. This research group synthesized a VHULHV RI QRYHO Į-diimine containing bidentate benzimidazole ligands in search for antitumor and SOD-mimicking activities. Some copper complexes possessed antitumor activity. Moreover, ϭϮ 

complexes C and D were selective towards lung cancer cell lines LCLC-103H and A-427, respectively. The IC50 values of SOD activity were low (in the range 0.09-ȝ0 KRZHYHU and the results showed no correlation between IC50 values for SOD activity and cancer cell growth inhibition [83]. The known antineoplastic drug, bleomycin, which causes DNA strand scission, was originally isolated in the form of copper complex from the culture medium Streptomyces verticullus [83]. However, it was found to be an excellent ligand for several other metal ions, in particular iron [84]. Copper(II) bleomycin is a stable complex, but can be converted to instable Cu(I) complex and as such can react rapidly with thiols [85]. Cisplatin (cis-diamminedichloroplatinum(II)) is a widely used chemotherapeutic agent for the treatment of testicular cancer and it is used in combination regimens for a variety of other tumors, including ovarian, cervical, bladder, lung and those of the head and neck. Despite the success of cisplatin, problems regarding intrinsic or acquired resistance and side effects have encouraged the development of new platinum drugs.[86]Even though platinum-based complexes had been in primary focus of research on chemotherapy agents, the interests in this field have shifted to non-platinumbased agents, in order to find different metal complexes with less side effects and similar, or better, cytotoxicity. The choice of metal ion is the most important factor in the design of metal-based chemotherapeutic agent. Varieties of metal complexes have been used as drugs and are well known to increase their activity or when administered as metal complexes show higher activity towards specific targets.[87] Copper is a bioessential and bio-relevant element.[88] Cu(II) is an essential element in human normal metabolism because of its functions as cofactor of several metalloenzymes. Copper is widely distributed in the biological system and copper complexes are known to have a broad spectrum of biological action. It has been demonstrated that Cu accumulates in tumors due to selective permeability of the cancer cell membranes. Because of this, a number of copper complexes have been screened for anticancer activity and some of them were found active both in vivo and in vitro.[89] Furthermore, copper(II)-based complexes appear to be very promising candidates for anticancer therapy; an idea supported by a considerable number of research articles describing the synthesis and cytotoxic activities of numerous copper(II) complexes. [90] COPPER(II) COMPLEXES AS ANTICANCER AGENTS Gokhale et al.[91] investigated the crystal structure of the neutral, distorted square planar copper(II) complex, cis-[dichloro (N1-(2-benzyloxybenzylidene)pyridine-2-carboxamidrazone) copper(II)] (92). Antitumor activity of the ligand and its copper complex were investigated against the human breast cancer cell line MCF-7 by using MTT method. At low concentration the complex (1) showed cell death. The IC50 YDOXHIRUWKHFRSSHUFRPSOH[LVȝ0ZKLFKLVIRXUWLPHVOHVVWKDQWKDWRIWKH ligand. This reveals that copper on complexation with the ligand shows potent antiproliferative activity. Chaviara et al.[93] have synthesized and characterized a new series of copper compounds with the starting materials [Cu(dienX2Y2)] and their adducts [Cu(dienXXY2)(2a-5mt)] (where dien=diethylenetriamine, dienXX=Schiff bases of diethylenetriamine with 2-furaldehyde or 2thiophene-carboxaldehyde, X=O, S, Y=Cl, Br, NO3 and 2a-5mt=2-amino-5-methylthiazole). Antibacterial activity of the compounds against E. coli (XL1), B. subtilis (ATCC 6633), and B. cereus (ATCC 11778) shows higher activity of the copper compounds. Antiproliferation activity of the compounds were evaluated against MRC5 (normal human lung fibroblasts), MCF7 (human breast cancer), T47D (human breast cancer), HT-29 (human colon cancer), HeLa (human cervical cancer) and OAW42 (human ovarian cancer) cell lines. The compounds with the ligand show higher ϭϯ 

cytotoxicity than the starting compounds. Among the copper compounds synthesized, CudienOOBr2. was found to be highly active against the MCF-7 cell line with an IC50 YDOXH ȝ0 7KXV WKH antiproliferative studies showed that the copper compounds exhibit higher bioactivity compared to the starting materials and the compounds itself. Jayendra et al.[94] have reported the synthesis of copper complexes of Salicylaldehyde semi/thiosemicarbazones and their structural characterization by X-ray analysis. The cytotoxicity activity of the ligands and its copper complexes were examined on human breast cancer cell line MCF-7. The ligands were inactive against the cell line, whereas the copper complexes were found to be more potent. Among the copper complexes, complexes have more anti proliferative activity. The enhanced antiproliferative activity of the copper complexes can be explained on the basis of their facile Cu(II)/Cu(I) redox couple which can generate considerable intracellular oxygen by the reduced cuprous centres. MCF-7 cells were found to be more sensitive to such additional oxidative stress due to their down-regulated antioxidant defense enzymes leading generally to apoptotic death. Zhao et al.[95] synthesized and characterized new complexes of Pt(II), Pd(II), Cu(II), and Hg(II) with the Schiff base ligand MeCONHCH2CH2N=CHPy (L) (Py = pyridine). The anticancer potential of the complexes was evaluated in two human cancer cell lines MOLT-4 and MCF-7. Among the compounds studied, the copper complex was found to be more active against cancer cell lines, due to its high solubility in water. The IC50 value obtained for copper complex against MCF-7 cell line is  ȝ0 ZKLFK LV ORZHU WKDQ WKH FLVSODWLQ ,& 50  ȝ0  7KH UHVXOWV VXJJHVW WKDW WKH FRSSHU complex exhibits promising potentials as an anticancer compound, having comparable cytotoxic activity with that of cisplatin. Lin-Yun et al. [96] have reported the synthesis and characterization of three new Cu(II) complexes. DNA binding studies by spectrophotometric titration spectra indicates that complexes interact with CT-DNA through intercalation mode, whereas theinteraction of the complex (1) was week. The cytotoxicity assay have been evaluated against four cancerous cell lines (human breast carcinoma cells MCF-7, human esophageal cancer cells Eca-109, human cervical cancer Hela cells, and human lung adenocarcinoma A549 cells) by MTT method. The complexes inhibit the proliferation of the MCF-7 cell with IC50 in the range of 37.12±ȝ0ZKLFKLVVPDOOHUWKDQWKHUHSRUWHGFRPSOH[ [Cu(dpa)[email protected] ȝ0 GXHWRWKHVROXELOLW\DQGPROHFXODUFRQIRUPDWLRQRIFRPSOH[HV. Design and chemistry of macrocyclic complexes has received much attention in recent years [97] due to their potential applications and importance in the area of medicinal chemistry [9899]. Thus, the study of macrocyclic complexes is becoming a growing class of research[100]. Macrocycles are best prepared by the aid of metal ions as templates to direct the condensation reaction towards ring closure [101]. The field of macrocyclic chemistry of metals is developing very fast because of its variety of application[102] and importance in the area of inorganic medicinal chemistry [103]. The rational design and construction of inorganic and organometallic metallomacrocycles by transition metal-directed multi-component self assembly has a major impact on pharmacological activity.[104-105] The incorporation of metal centers into supramolecular system gives rise to novel electronic and/or magnetic properties as well as fascinating structural features. Copper (II) is the most ϭϰ 

studied metal ion among all the transition metal ions [106-107] Cu(II) complexes are known to play a significant role either in naturally occurring biological systems or as pharmacological agents [108-109]. In recent years, several families of copper complexes have been studied as potential antitumor agents. Therefore, molecular docking studies of this copper complex was done on Estrogen Receptor Į Estrogen Receptor ȕ and compared with Tamoxifen and Afimoxifene. Results of copper complex was highly significantly, thus led to synthesis, characterization and pharmacological evaluation of compounds. Kovala-Demertzi et al.[110] have synthesized the complexes of mefenamic acid [Mn(mef)2(H2O)2] , [Co(mef)2(H2O)2] , [Ni(mef)2(H2O)2], [Cu(mef)2(H2O)]2 and [Zn(mef)2] . DPPH radical scavenging activity of the complexes shows their high antioxidant activity, which are time and concentration dependent. In vitro inhibitory effect of the complexes against soybean lipoxygenase, bglucuronidase and trypsin- induced proteolysis shows the highest inhibitory effect of the complexes. In vitro antiproliferative activity of the complexes against human cancer cell line MCF-7 (human breast cancer cell line), T24 (bladder cancer cell line), A-549 (non-small cell lung carcinoma) and a mouse L-929 (a fibroblast-like cell line cloned from strain L) was determined by using SRB and MTT assay. [Cu(mef)2(H2O)]2 complex with IC50 YDOXH “ ȝ0 DJDLQVW MCF-7 cell line was considered to be the potent anticancer drug. This result indicates that coupling of mefenamic acid to Cu(II) metal center result in metallic complexes with important biological properties. Zhao et al.[111] have synthesized and characterized novel platinum and copper metallodendrimers [G0-Py4-[PtCl2]4] and [G0-Py4- [CuCl2]7] . Cytotoxicity studies of the complexes were evaluated against MOLT-4, MCF-7 and Chang Liver cells by MTT assay. The cytotoxicity of the multinuclear complexes were compared to their mono-nuclear analogues, [(MeCONHCH2CH2N=CHPy)(PtCl2)] and [(MeCONHCH2CH2N=CHPy)(CuCl2)]. Both the platinum coordinated complexes and exhibits low cytotoxic activity compared to that of the copper complexes . The copper complexes also showed high cytotoxic activities on MCF-7 cell line, with IC50 values of 73.1 ± 4.9 and 10.2 ± 1.5 lM respectively, whereas cisplatin has been found to have no significant activity against MCF-7 within the tested range of concentrations. The higher activity of the copper complexes may be due to their higher solubility than the platinum complexes. Thus low solubility reduces the possibility of the complexes entering into the cells to react with the DNA to cause an increase in cytotoxicity.Therefore, it is concluded that, metal-based complexes with higher solubility would result in higher Cytotoxicity Chakraborty et al.[112] synthesized a series of copper complexes. Among the copper complexes, [Cu(Pyimpy)Cl2], represented as CuP1 was found to be the best compound.

ϭϱ 

Fig1.11 Structure of mononuclear and multinuclear platinum and copper complexes Chakraborty et al.[113] synthesized a series of copper complexes . Among the copper complexes, [Cu(Pyimpy)Cl2], represented as CuP1 was found to be the best compound. CT-DNA binding by fluorescence studies indicates that the EB and calf thymus DNA complex fluorescence quenches with LQFUHDVLQJFRQFHQWUDWLRQVRI&X3IURPWRȝ0GXHWRWKHGLVSODFHPHQWRI'1$ERXQG(%WR free state. Such quenching effects were not observed with the ligand (pyimpy) or the copper salt (CuCl2). Antiproliferative effect of copper complexes was evaluated against the MCF-7, HEK 293 and PC3 cancer cells by MTT assay. Among the copper complexes CuP1 shows high cytotoxicity with low IC50 value against the cell lines due to its high solubility comparing to the rest of the complexes. Due to this reason CuP1 was selected for further analysis of anticancer activities. CuP1 causes intensified fragmentation of DNA in MCF-7 cells. CuP1 was further tested on rat breast tumor models and was found to inhibit tumor growth. As a result, CuP1 possesses prominent anticancer properties in vitro as well as in vivo.

Fig1.12. Basic structure of the family of copper complexes ϭϲ 

Puszko et al.[114] have synthesized and characterized three mononuclear copper(II) complexes with three ligands 2-Methyl-4-nitropyridine N-oxide ; 2,6-dimethyl-4-nitropyridine N-oxide; 2,3,6trimethyl-4-nitropyridine N-oxide . The free ligands and the copper complexes were tested for their in vitro anti-proliferative activity against MCF-7 (breast cancer) and SW-707 (colon adenocarcinoma). The ligands and the complexes were found to be active against the MCF-7 cell line. From the ID50 values it was found that, the increasing number of methyl groups in the ligand causes a decrease of the activity for both free ligands and the complexes. The results show that the complexes without or with one methyl group exhibit remarkable cytotoxic activity. Manan et al.[115] have synthesized Schiff base ligand SMISA and cobalt(II), nickel(II), copper(II), zinc(II) and cadmium(II) metal complexes of S-methyldithiocarbazate with isatin. The copper(II) compound was crystallized in an orthorhombic crystal system with a space group C 2cb. All the complexes were screened for their cytotoxic activities against MCF-7. The ligand was found to be inactive against the cell line and the other complexes have lesser activity, whereas the Cu(II) complex was highly active with an IC50 value 0. ȝJP/ Ghosh et al.[116] have synthesized five copper complexes and the complexes were characterized by spectroscopic techniques. The anti-cancer activities of have been investigated against various Cancer cell lines, in comparison with the widely used anti-cancer drug cisplatin under identical conditions by using MTT assay. The complexes exhibited more cytotoxicity for MCF-7 cell than the starting material CuCl2.2H2O and the ligand. Thus, all the copper complexes shows high activity against the MCF-7 cell line. Puszko et al.[117] have carried out the synthesis and characterization of nitrato copper(II) complexes of dimethyl substituted 4-nitropyridine N-oxide. In vitro cytotoxicity of the complexes were tested against human cancer cell lines: MCF-7 (breast), SW-707 (colon) and P-388 (murine leukemia). The complexes have been found to be less active against the MCF-7 cell line due to the increasing number of methyl groups in the ligand, which causes a decrease of the activity for both free ligands and their complexes. These results may suggest that for this complex the activity is most likely due to all of the individual components in solution and/or some synergistic effects. Amit Kumar et al.[18] have reported the synthesis, characterization, structure determination of a copper complex of anthracenyl terpyridine and Zn 2+ complex. Binding studies carried out by Absorption method with CT DNA indicates that the complex 1 binds with CTDNA through partial intercalation mode. Cleavage studies by agarose gel electrophoresis method shows that the complex cleaves the plasmid DNA in the absence of any chemical stimulant. Antiproliferative activity of the complexes against Cervical carcinoma cells (HeLa, SiHa, CaSki); breast cancer cells (MCF-7); hepatocellular carcinoma cells (HepG2) and human lung carcinoma cells (H1299) was carried out by using MTT assay. From the IC 50 values (IC50, 0.8 ± 0.2 to 5.4 ± 0.1 mM) it was observed that copper complex shows higher activity compared to that of the cisplatin or carboplatin. Thus the studies reveal that complex can be used as a effective chemo-therapeutics against various cancerous cells. Osowole et al.[119] have reported the synthesis and characterization of the Schiff base, 3-{[(4,6-dimethoxypyrimidin-2yl)imino]methyl}naphthalen-2-ol and its VO(IV), Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Pd(II) complexes. The anticancer activities of the compounds were analysed against the MCF-7 cell line. From the IC50 values it is concluded that Pd(II) and Cu(II) had best activity with IC50 YDOXHVDQGȝ0UHVSHFWLYHO\ZKLFKZHUHZLWKLQ the same order as Cis-SODWLQ  ȝ0  ZKHUHDV WKH OLJDQG DQG RWKHU PHWDO FRPSOH[HV KDYH EHHQ found to be inactive. The high activity of the Pd (II) and Cu(II) complexes may be atttibuted to their planar structure which has been documented to avoid possible steric hindrance during physiological ϭϳ 

actions. Thus the coordination enhances the activity of the complexes. Vyas et al.[120] have reported the synthesis of aryl/heterocyclic analogs of retinoid trans-2-octenal with and without copper complexation. Cytotoxic activity of the copper complexes against hormone-dependent (MCF-7), hormone-independent (MDA-MB-231and BT-20) breast cancer and androgen-independent (PC3) prostate cancer cell lines have been investigated by using MTT assay. Copper complexes shows significant inhibition against hormone-dependent breast cancer cell line MCF-7 and no activity against hormone-independent cell lines. Significant activity of the copper complexes shows that the activity of the complexes may be due to the presence of copper which helps in enhancing the biological activity. Jaividhya et al.[121] have synthesized and characterized mononuclear copper(II) complexes with general formula [Cu(L)(H2O)2] and [Cu(L)(diimine)]. The interaction of the complexes with calf thymus DNA has been investigated using UV-visible and fluorescence spectroscopy, and viscosity measurements. The complexes and interact with calf thymus DNA more strongly than the other complexes through partial intercalation mode. The complexes cleave the double strand DNA in the absence of a reductant. since DNA cleavage is considered [122,123] as essential for a drug to act as an anticancer agent, the cytotoxicity of the complexes against both human breast cancer cell lines (MCF-7) and human cervical epidermoid carcinoma cell lines (ME 180) were investigated by using MTT assay. All the copper complexes have been found to exhibit cytotoxicity higher than that of cisplatin. From the studies it has been reported that the copper complexes can be used as an anticancer drug for treating human breast cancer and possibly other forms of cancer. Zhong-Ying et al.[124] have synthesized a Schiff base copper(II) complex, with a general formula [CuII(5-Cl-pap)(OAc)(H2O)]·2H2O.Cell viability of the complexes was examined by MTT assay against the Human breast cancer cell line MCF-7. The results indicates that the complex inhibit the growth of MCF-7 cells in a dose- and time-dependent manner. The IC50 values at  DQG  K ZHUH  ȝ0 DQG  ȝ0 IRU WKH FRSSHU FRPSOH[ ZKLOH  ȝ0 DQG  ȝ0 IRU cisplatin. This suggests that the copper complex potentially inhibits the MCF-7 cells. The antiproliferative effects of complex on MCF-7 cells were further determined using clonogenic assay, which suggests that the copper complex inhibits clonogenic growth of MCF-7 cells in a dosedependent manner. Thus, these studies suggest that the copper complex may be a potent antitumor drug toward MCF-7 breast cancer cells by inducing apoptosis via amitochondrial pathway. Kaushik et al.[125] have synthesized a copper complex [Cu(tBuPhimp)(Cl)] derived from tridentate ligand tBuPhimpH. The nuclease activity of the complex has been studied using supercoiled pBR322 DNA, and the extent of DNA cleavage was measured by gel electrophoresis. The nuclease activity of the copper complex was happened in the absence of any external agent. The anticancer activity of the copper complex was studied against MCF-7 cell line. IC50 value for the copper complex was found WR EH  “  ȝ0 ZKLFK ZDV IRXQG WR EH EHWWHU WKDQ WKH ,& 50 value obtained for cis cisplatin (17.98 ± 1.18). Thus the copper complex was found to have high anticancer activity. Rajalakshmi et al.[126] have synthesized and characterized two copper(II) complexes [Cu(meotpy)(dmp)](NO3)2 and [Cu(bitpy)(dmp)](NO3)2. DNA binding studies of the complexes shows the potent binding nature of the complexes through intercalative mode. Cleavage of DNA by the agarose gel electrophoresis technique indicates that, the copper (II) complexes are able to hydrolytically cleave DNA without any coreagents. The cytotoxicity of the complexes were studied against a human cancerous breast adenocarcinoma cell line (MCF-7) and a noncancerous monkey kidney fibroblast cell line (VERO) using the MTT assay. The results showed that the complexes exhibited a significant inhibitory ϭϴ 

potency towards proliferation of the MCF-7 cell line. The % cell viability was observed to be 87.77 for complexes. From the studies it is concluded that the copper complex shows higher cytotoxicity towards a cancerous cell line than complex , which can be used as a potent antiproliferative agent against a cancer cell line. Kellett et al.[126] synthesized two copper complexes [Cu(ph)(1,10phen)].2H22DQG>&X SK ¶-bipy)].2H2O . The in-vitro Cytotoxic activity of the copper complexes was assessed against breast (MCF-7), prostate(DU145), colon(HT29), and intrinsically cisplatinresistantovarian(SK-OV-3) human cancer cell lines by using MTT method. The inhibitory activity of the copper complex was almost identical in all cell lines with IC50 YDOXHVEHWZHHQWRȝ07KH ¶-bipy-containing complex , was active against all cell line after 96h with IC50 value ranges EHWZHHQWRȝ0,WLVHYLGHQWIURPWKHVWXGLHVWKDWWKH-phen copper complex , was more DFWLYHWKDQWKH¶-bipy copper complex . DNA interaction studies, using fluorescence and viscosity measurements indicates intercalation mode of binding of complex and minor groove binding of complex .Complexes cleave super helical DNA in the presence of the added reductant(L-ascorbic acid) Complex 1 induce single and double-stranded damage at cRQFHQWUDWLRQV ȝ0 DQG •ȝ0 respectively and complex 2 DW FRQFHQWUDWLRQV ȝ0 DQG ȝ0 UHVSHFWLYHO\ %RWK FRPSOH[HV demonstrated potent superoxide dismutase mimetic(SODm ) activity in the presence hydrogen peroxide, and catalase mimetic(CATm) activity was detected only in the presence of the cocatalyst, imidazole. The ability of complexes to induce DSBs within cancer cells was confirmed in SK-OV-3 cancer cells using immune detection of ࣥ±H2AX foci by confocal microscopy and flow cytometry. Murtaza et al.[127] have synthesized and characterized A series of homoleptic copper(II) complexes ZLWK 11¶1¶¶-trisubstituted guanidines, [Cu(II) {PhCONHC(NHR)NPh}2] (where R ¼ phenyl , nbutyl , sec-butyl , cyclohexyl , 1-naphthyl , 2,4-dichlorophenyl , 3,4-dichlorophenyl , and 3,5dichlorophenyl . In vitro antimicrobial screening was carried out against four bacterial strains, two Gram positive Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 10240) and two Gram negative, Escherichia coli (ATCC 15224), Salmonellasetubal (ATCC 19196), and four fungal strains i.e. Mucor species, Aspergillus niger, Aspergillu flavus, and Fusarium solani. In-vitro cytotoxicity of the complexes were investigated against seven human cancer cell lines A498 (renal cancer), MCF-7 (estrogen receptor (ER)+/progesterone receptor (PgR)+ breast cancer), EVSA-T (estrogen receptor (ER)-/progesterone receptor (PgR) - breast cancer), H226 (lung cancer), IGROV (ovarian cancer), M19 MEL (melanoma) and WIDR (colon cancer). ID50 values of the copper complexes were in the high range of those of cisplatin. All the copper complexes show similar results against the human cancer cell lines. The in vitro cytotoxic activity of the copper complexes may be due to the size or electronic properties of the substituent present in the complexes. Abdi et al.[128] have synthesized and characterized a water soluble 1,3,5-triazine-based copper(II) complex, [Cu(tptz)2]2+, where tptz is 2,4,6-tris(2-pyridyl)-1,3,5-triazine. The solid state structure of the complex has been determined by single-crystal X-ray crystallography. The interaction of the complex with DNA investigated by electronic absorption, competitive fluorescence titration, voltammetric techniques and a gel electrophoresis mobility shift assay reveal that the complex binds to DNA via intercalation mode with the partial insertion of a planar polypyridyl ligand between the base stacks of double-stranded DNA. In vitro cytotoxic activity of the copper complex on the human breast adenocarcinoma MCF-7 cell line has been evaluated in comparison with cisplatin by using MTT assay. The IC50 YDOXHIRUWKHFRSSHUFRPSOH[ZDVȝ0ZKLFKZDVIRXQGWREHWLPHV ϭϵ 

better than cisplatin(IC50=ȝ0 7KHVHUHVXOWVLQGLFDWHWKDWWKHCu (II) complex has a pronounced inhibitory effect on the proliferation of MCF-7 cancer cells than cisplatin and other reportedCu(II) complexes. The potent antiproliferation activity of this Cu(II) complex may be due to its effective binding to DNA and its water-solubility. Altogether, these results suggest that the [Cu(tptz) 2](NO3)2 complex may function as a highly effective metal-based anticancer chemotherapeutic agent. El-Aziz et al.[28] have synthesized Cu(II), Fe(III), Co(II), Ni(II) and Zn(II) complexes from N-(pyridine-2ylmethylene)benzo[d]thiazol-2-amine Schiff base(L) and characterized by elemental analysis, electrical conductance, mass spectra, magnetic susceptibility and spectral techniques (IR, UV±Vis, 1H NMR). In-vitro potential cytotoxicity of the Schiff base and its Cu(II), Ni(II) and Zn(II) complexes was tested against human breast carcinoma (MCF 7). The studies reveal that the Cu(II) complex exhibits highly cytotoxic activity towards human breast cancer (MCF 7) cell lines. From these studies, it is clearly observed that complexation with metals has a synergistic effect on the antiproliferative activity of these compounds especially against hormone responsive cancers.Bao-Li et al.[129] have synthesized two novel copper-¶-bipyridine Copper complexes are characterized by X-ray single-crystal diffraction, elemental analysis and IR spectra. The complexes were also screened for their in-vitro cytotoxic ability against various cancer cell lines HepG-2, HeLa, NCIH460, MCF-7 and HL-60 by MTT assay. Among the complexes, complex 2 exhibits higher cytotoxicity than complex 1 against each of the selected cell lines. The better cytotoxic activity of complex is due to its stronger DNA binding ability, which consequently leads to cell death. El-Seidy et al.[130] reported the synthesis and characterization of a series of novel metal complexes with the Schiff base ligands 4-((((1H-benzo[d]imidazol-2-yl)methyl)imino)methyl) benzene-1,3-diol, H3L1, and (((1H-benzo[d]imidazol-2-yl)methyl) thio)propanenitrile, HL2. The cytotoxic activity of the Schiff base ligands H3L1, HL2 and its metal complexes was measured in vitro using the Sulfo Rhodamine-B stain (SRB) assay against MCF-7 cell line, using the method of Skehan at The National Cancer Institute, Cairo University, Cairo, Egypt. The IC50 values obtained with human breast cancer cell line (MCF-7) revealed that the H3L1 ligand and HL2-silver complexes were between 2.53 and 2.8 and HL2-copper complexes were 3.35 times more cytotoxic than Tamxifen . Copper complexes inhibits the growth in tumor cells due to the intracellular generation of hydroxyl radicals from H2O2 produced during normal cellular activities by the reduction of Cu(II) to Cu(I).Yin et al.[131] have synthesized and characterized a new polymeric demethylcantharidato (DCA) bridged copper(II) complex. X-ray single crystal diffraction analysis revealed that the copper(II) complex crystalizes in the orthorhombic Pbca space group with an asymmetric unit containing one Cu(II) ion, one tetradentate DCA ligand, one phen ligand, and one lattice water molecule. The copper(II) complex was analyzed for its anticancer activity against six human cancerous cell lines like HCT-8, A549, HeLa, HepG2, BGC-823 and MCF-7. The cytotoxic studies reveals the potent cytotoxic nature of the Copper(II) complex. The copper(II) complex, [Cu(DCA)(phen)]n·nH2O has been found to be an excellent antitumor agent due to the presence of an extended planar aromatic ring.Jevtovic et al.[132] synthesized three new copper complexes [Cu(PLSC)Cl 2](1), [Cu(PLSC)(H2O)(SO4)]2.3H2O(2), [Cu2(PLSC)2(NCS)2](NCS)2(3) with pyridoxal semicarbazone (PLSC). The compounds were evaluated for their in vitro cytotoxicity towards two human breast adenocarcinoma cell lines (MCF7 and MDA-MB-231, respectively). The results suggest that compound 1 exhibit no antiproliferative effect. Compound 2 and 3 exhibits cytotoxic effects on both cell lines, but only after 72h treatment by the highest tested concentrations. Thus the copper ϮϬ 

complexes exhibited cytotoxic effects against human breast cancer cell line with reported IC 50 values ranging from 5- ȝ0*ZDUDP HW DO[133] have synthesized a series of copper(II), manganese(II), nickel(II) and zinc(II) complexes. The metal complexes were found to inhibit the growth of MCF-7 cells in a dose-dependent manner. The free ligand MTT assay showed no significant inhibition activities at a concentration even higher than the complex. The cytotoxicity of the metal complexes against human breast cancer MCF-7 cells was obtained by the MTT assay.The metal complexes were found to inhibit the growth of MCF-7 cells in a dose-dependent manner. The free ligand MTT assay showed no significant inhibition activities at a concentration even higher than the complex which confirmed that chelation of ligand with metal ions was significance for the activity of this novel compounds.Aazam et al.[134] have reported the synthesis of a new Schiff base N2O2 ligand and its Ni/Cu complexes. The invitro cytotoxicity activity were analyzed by means of cell viability cures and expressed with IC50 values. The IC50 value of the Cu complex against MCF-7 cell was found to be 19.25 mM , which indicated that the Cu complex has the higher cytotoxicity activity than free ligand; while Ni complex did not show any significant activity on the tested cancer cell line.Pravin et al.[135] synthesized a series of carboplatin type Cu(II) and Zn(II) metalloinsertors and characterized by spectral and analytical methods. In vitro cytotoxic activity of the complexes against MCF-7 human cancer cell line has been carried out by the MTT assay method. Cisplatin and carboplatin are used as positive controls to assess the cytotoxicity of the test compounds. The IC 50 values show that Copper complexes exhibit significant activity against MCF-7 cell lines, which are almost equal to the activity of the well-known anticancer drugs, cisplatin and carboplatin. The studies clearly indicate that the free CuCl2 does not show any significant activity on all the cancer cells (IC50 = 465-642 mM), which confirms that the chelation of the ligand (nature of p-substituents) with the Cu(II) ion is the responsible factor for the observed varying cytotoxic properties of the complexes . It is also stated that the better cytotoxic properties of the complexes may be attributed to the extended planar structure induced by the ʌĺʌ conjugation resulting from the chelation of the Cu(II) ion with the ligands. Raman et al.[36] have reported the in-vivo and in- vitro anti-tumor functions of mixed-ligand metal complexes of Cu(II), Ni(II) and Zn(II). All the complexes have been found to exhibit cytotoxic effects against cancerous cell lines with potency more than that of the widely used drug cisplatin and hence they have the potential to act as promising anticancer agents Comparing to other complexes, [CuL(Met)Cl2]complex have been found to possess the most potent inhibitory effect against the human cancer cell lines. Its IC50 value is very low to that of cisplatin indicating high cytotoxic effects against human cancer cell line MCF-7.Imran et al.[37] synthesized a new oxopyrrolidine-based ligand (L) from pyroglutamic acid and its Cu(II) and Ni(II) metal complexes. The in-vitro anti-cancer effects of L, CuL and NiL were studied against MCF-7 cell line using MTT assay method. The studies show that all the reported compounds were viable in the range of 92±DWȝJP/í1. Viability % of all the compounds at different concentration with respect to the standard drug doxorubicin was depicted in the bar diagram. From the results it was observed that CuL complex shows best. Ganeshpandian et al.[137] have synthesized mixed ligand copper(II) complexes of the type [Cu(L)(2,9-dmp)]2+. DNA binding affinity of the Copper (II) complexes were evaluated by Absorption, Fluorescence spectral techniques and Viscosity measurements. These studies reveal that copper complexes bind to DNA through Intercalative mode. All the complexes cleave pUC19 Ϯϭ 

supercoiled DNA in the absence of an activating agent. The cytotoxicity of the complexes against MCF 7 breast cancer cell lines has been investigated by using MTT assay. IC 50 values of the complexes at 24h and 48h were lower than that of cisplatin, which indicates the high potent nature of the Copper(II) complexes.Lu et al.[138] have synthesized two water-soluble ternary copper(II) complexes, [Cu(L)Cl](ClO4) and[Cu(L)Br2] . The interaction of two complexes with CT-DNA (calf thymus-DNA) and BSA (bovine serumalbumin) studied by means of various spectroscopy methods, reveals that the complexes binds with CT-DNA through intercalation mode, and could quench the intrinsic fluorescence of BSA in a static quenching process. The DNA cleavage activity of the complexes by agarose gel electrophoresis method shows that the complexes cleave supercoiled pBR322 plasmid DNA without any external agents. The in-vitro cytotoxicity of the complexes have been evaluated against three human tumor cell lines HeLa (human cervical carcinoma), MCF-7 (human breast adenocarcinoma), and A549 (lung adenocarcinoma carcinoma) by MTT assay. IC 50 YDOXHVRIWKHFRSSHUFRPSOH[HVKDYHEHHQIRXQGWREHȝ0DJDLQVWMCF-7 cell line, which was much less than cisplatin (IC50  ȝ0  DQG FDUERSODWLQ ,&50   ȝ0 7KLV VKRZV WKH SRWent nature of the copper complexes against MCF-7 cell line than other cell line. The quinoline ring system present in the copper(II) complexes makes them to act as potent anticancer agents Iglesias et al.[139] have reported the synthesis and characterization of six copper complexes with general formula [Cu(L-dipeptide)(phen)]·nH2O (where, phen= 1,10-phenanthroline). DNA binding studies of the complexes by absorption spectra indicates that the complexes may bind through groove or intercalation binding. From WKH Q ELQGLQJ FRQVWDQW µ.E¶ YDOXH LW LV FRQFOXGHG WKDW WKH FRPSOH[HV binds trough groove binding and partial intercalation. Cytotoxic activity of the complexes was studied against theuman cancerous cell lines like: HeLa (human cervical adenocarcinoma),MCF-7 (human metastatic breast adenocarcinoma) and A549 (human lung epithelial carcinoma). All the complexes shows Cytotoxic activity. Among the six copper complexes, [Cu(Ala-Phe)(phen)] complex(3) induces MCF-7 cell death at an IC50   ȝ0 ZKLFK LV PRUH Dctive than Cisplatin(IC50 ȝ0  )URP WKLV UHVXOW LW LV FRQFOXGHG WKDW WKH FRSSHU FRPSOH[ 3) may be a good candidate to act as a anticancer agent. Aazam et al.[41] have synthesized 2,3-bis-[(3-ethoxy-2hydroxybenzylidene)amino]but-2 enedinitrile Schiff base ligand and its corresponding copper/nickel complexes. Free ligand, its metal complexes and metals nanoparticles have been characterized by various spectral studies. The in vitro cytotoxic activity of free ligand and its complexes were assessed against two cancer cell lines (HeLa and MCF-7 cells)and one healthy cell line (HEK293 cell) by using MTT assay. From the studies it has been evaluated that the copper complex is more active against the cancer cell line, while the nickel complex is inactive. The IC 50 values forCu complex were 14.04 and 19.25 mM for HeLa and MCF-7 cell, respectively, which indicated that the Cu complex exhibits significant activity against HeLa and MCF-7 cell lines. Wen-Ji Song et al.[42] synthesized and characterized two novel copper(II) complexes and from the Schiff bases HL1 and HL2. DNA binding studies by electronic spectra, fluorescence spectra and viscosity measurements indicates the partial intercalation mode of binding of the copper complexes. DNA cleavage studies by agarose gel electrophoresis studies reveals that the complex cleaves supercoiled DNA through the oxidative mechanism. In-vitro antiproliferative activities of complex and the Schiff bases was evaluated against human breast cancer cells (MCF-7) and human colorectal cancer cells (COLO205) by using MTT assay. The IC50 value of the complex against MCF-7 cell line was found to be 16.9 ± ȝPRO/-1, which was more than that of the ligands. Form the studies it is observed that the ϮϮ 

chemical structure of compounds is essential to express the biological activity, and it can be important in designing and synthesizing novel anti-cancer drugs.Lu et al.[43] synthesized and characterized two new complexes, [CuLCl]ClO4 and [Zn2L2SO4(H2O)2](ClO4)2 . The DNA binding studies of two complexes with CT-DNA was investigated by UV absorption, fluorescence spectroscopy and viscosity measurements reveal that the complexes bind through moderate intercalative mode. Cleavage studies by gel electrophoresis method shows efficient DNA cleavage activity of the complexes. Antiproliferative activity of the complexes was evaluated against four cell line panels consisting of 7404, HeLa, MCF-7 and HepG-2 cancer cells by MTT assay. Thus the studies reveal that the copper complex has the potential to act as an effective metal-based anticancer drug. Ozdemir et al.[44] V\QWKHVL]HGDQGFKDUDFWHUL]HGDQHZ1¶-acetyl butane sulfonic acid hydrazide, C4H9-SO2-NH-NH-COCH3 (Absh) and its Cu(II) complex [Cu(Absh)2(CH3COO)2]. The antibacterial activities of synthesized compounds were studied against Gram positive bacteria, Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Bacillus cereus (NRRL-B-3711), Enterococcus faecalis (ATCC 29212) and Gram negative bacteria, Escherichia coli (ATCC 11230), Pseudomonas aeruginosa (ATCC 15442), Klebsiella pneumonia (ATCC 70063) by using the disc diffusion and micro dilutionmethods shows the higher antibacterial activity of the copper(II) complex than its legend. In-vitro antitumor activity of the compounds was evaluated against breast cancer cell lines MCF-7 by using MTT assay. IC50 YDOXH RI WKH &RSSHU ,,  FRPSOH[ ZDV IRXQG WR EH ȝPRO/-1, which was less than that of the ligand Absh(IC50  ȝPRO/-1) and nearer to the standard compound Docetaxel(IC50 ȝPRO/-1). The high potency of the copper(II) complex may be due to the coordination of the ligand Absh with Cu(II) ion, as Cu(II) is known to be an essential element in human normal metabolism Anbu et al.[140] have synthesized a new type of copper(II) complex, [CuL(phen)2](NO3) (CuIP), where L = ((E)-1¶-(2-oxoindolin-3-ylidene)benzohydrazide). The interaction of the ligand L and CuIP with calf thymus DNA (CT DNA) has been investigated by absorption, fluorescence and viscosity titration methods, which suggests groove binding and/or a partial intercalative mode of binding. CuIP shows a good binding propensity with the bovine serum albumin (BSA) protein, with KBSA value of 1.25(±0.24) x106 M-1. Molecular docking studies on DNA and human serum albumin (HSA) reveal that the interaction between CuIP with HSA is dominated by hydrophobic forces. The in vitro anti-proliferative activity of the CuIP against the human cervical (HeLa) and breast (MCF7) cancer cells; noncancer breast epithelial (MCF10a) cells have been investigated by MTT assay. IC50 values of CuIP indicate higher anticancer potency of the CuIP against the human cervical (HeLa) and breast (MCF7) cancer cells, than against the noncancer breast epithelial cells. Chandra et al.[46] KDYHUHSRUWHGWKHV\QWKHVLVDQGFKDUDFWHUL]DWLRQRI6FKLII¶VEDVHOLJDQG / K\GUD]LQHFDUER[DPLGH[3-methyl-2-thienyl methylene] and its Ni and Cu metal complexes with general composition [M(L)2X2], where M = Ni(II) and Cu(II), X = Cl-, NO3- , CH3COO- and ½SO42-. Cytotoxicity of hydrazine carboxamide, 2-[3-methyl-2-thienylmethylene] and its Ni(II) and Cu(II) complexes were evaluated using SRB fluorometric assay and driamycin (ADR) was applied as positive control and DMSO as negative control. The results are reported in GI50 which means concentration of drug causing 50% inhibition of cell growth. From the GI50 values it was concluded that the Cu(II) complexes has high cytotoxicity activity than the Ni(II) complexes. These studies indicate that the incorporation of metal ion increases the inhibition of the cell growth.Mathan kumar et al.[141] have Ϯϯ 

synthesized four new mononuclear mixed ligand copper(II) complexes of the type [Cu(L)(diimine)] (1±4). In vitro cytotoxicity of the new complexes 1±4 was evaluated using MTT assay against the MCF-7 cancer cell line. All the complexes showscytotoxicity in dose dependent manner. The potency of the complexes to kill the cancer cells follows the order 4 > 3 > 1 > 2. Thus the complex 4 is more effective with DQ ,&   ȝ0 WKDQ L and the complexes 1-3 , due to the presence of bulky groups at position N(4) of the thiosemicarbazone moiety. Inci et al.[142] reported the synthesis and characterization of two new water-soluble copper(II) complexes, [Cu(dmphen)2(NO3)]NO3 (1), [Cu(dmphen)(tyr)(H2O)] NO3.H2O (2) and the diquarternary salt of dmphen. The CT-DNA binding of these compounds investigated by UV±vis spectroscopy, fluorescence spectroscopy and thermal denaturation studies indicate that the compounds bind to DNA via an intercalation binding mode. The cleavage studies of the compounds with pBR322 DNA indicate that these compounds exhibit efficient DNA cleavage. The cytotoxicity of the compounds was investigated against different cancer cell lines (A549, Caco-2 and MCF-7) and a healthy cell line (BEAS-2B). Both the copper complexes exhibit higher cytotoxicity activity with low IC 50 YDOXHV  ȝ0  DQG VKRZ VHOHFWLYH cytotoxicity.Among the copper complexes, complex 1 shows higher cytotoxicity against the Caco-2 cell line than cisplatin; complex 2 shows higher cytotoxicity than cisplatin against the MCF-7 cell line. The differences in the activity of the copper complexes may be due to the difference between the coordinated modes of complexes 1 and 2. Thus the copper complexes exhibited higher cytotoxic effects on the cancer cell lines with lower IC 50 values indicating their efficiency in killing cancer cells even at low concentrations compared with cisplatin. Lakshmipraba et al.[143] have synthesized and characterized water soluble polyethyleneimine±copper(II) complexes, [Cu(phen)(Ltyr)BPEI]ClO4 with various degree of copper(II) complex units in the polymer chain. Binding of polymer±copper(II) complexes to DNA was studied by electronic absorption, CD spectroscopy, fluorescence and cyclic voltammetry studies. The studies suggests that the polymer-complexes can bind to DNA by electrostatic interaction. In vitro cytotoxicity of polymer±copper(II) complexes was evaluated by MTT assay on MCF-7 cells. The polymer±copper(II) complex inhibited the growth of the cancer cells significantly, in a dose- and duration-dependent manner. The IC50 values of the FRPSOH[HVDUH“DQG“ȝJPO-1 after 24 h and 48 h respectively. The polymer± copper (II) complex showed highly effective cytotoxic activity against MCF-7 cancer cells which was lesser than the cisplatin (IC50  “  DQG  “  ȝJPO-1 for24 h and 48 h). However, cisplatin shows toxic side effects, which is not expected with the polymer±copper(II) complex. Hence it can be used as a potent anticancer agent. Drug resistance has become a growing problem in the treatment of infectious diseases caused by bacteria [114].Theserious medical problem of bacterial and fungal resistance and the rapid rate at which it develops has led toincreasing levels of resistance to classical antibiotics, [115-116], and the discovery and development of effective antibacterial drugs with novel mechanisms of action have, thus, become urgent tasks for research programs oninfectious diseases [117].The importance of metal ions in biological systems is well established. One of the most interesting features of metal-coordinated systems is the concerted spatial arrangement of the ligands around the metal ion. Among metal ions of biological importance, the Cu (II) ion is involved in a large number of distorted complexes [118]. Over the past two decades, considerable attention has been paid to metal complexes of Schiff bases containing nitrogen and other donor atoms [119-120]. Bio- organo metallic chemistry is Ϯϰ 

dedicated to the study of metallic complexes and their biological applications [121], including the design of new drugs that are more effective than those already known. Thiosemicarbazones are well established as an important class of sulfur-donor Schiff baseligands and their metalcomplexing ability is responsible for the remarkable biological activities observed for these compounds. As part of our work involving the preparation of free imino-macrocyclic compounds and their metal complexes, we were interested in obtaining the free [2+2] condensation product from carbohydrazone and thiosemicarbazide. The aim of this work was to compare and assess the reactivity of different macroligands containing the same functionalgroup, but with variable number of substituents and different structures, with a copper metal ion. In addition, we determined the optimal conditions to control the nature of the copper complexes, since the different structures can improve their potential applications. Therefore, in the present book, we report the synthesis, structural characterization and biological evaluation of mononuclear Cu (II) macrocyclic complexes derived from malonanilic carbohydrazone and thiosemicarbazide. All complexes exhibited potent anticancer, antioxidant and antibacterial activities.

Ϯϱ 

MATERIALS AND METHODS Accession of Target Protein: The three-GLPHQVLRQDO VWUXFWXUHV RI (VWURJHQ 5HFHSWRU Į (5Į 3'% ,' '7  DQG (VWURJHQ 5HFHSWRU ȕ (5ȕ 3'% ,' 478  ZHUH GRZQORDGHG IURP the RCSB protein Data Bank in pdb format. [122,123] Selection of Ligands: Chemical structure of Copper complex was prepared by Marvin sketch. Chemical structures of Tamoxifen and Afimoxifene were prepared by using Chem BioDraw Ultra 12.0 and Chem 3D Ultra 8.0 and saved in pdb format. Optimization of Target Protein and ligands: Two proteins (ER Į DQG (5 ȕ) have bound ligands attached, which were deleted by using Discovery Studio 4.5. PDB PDB Coordinates of target protein were optimized by using Discovery Studio 4.5 ad UCSF Chimera 1.10.2. and saved as a pdb file. Copper complex was optimized by Marvin Sketch. Tamoxifen and Afimoxine were optimized by Chem 3D Ultra 8.0 using energy minimization process. Docking Analysis A computational approach of ligand-protein docking was done to analyze the binding scores and interactions. Docking was done by PyRx 0.8 which uses autodock vina based scoring function.14 ³*ULGSRLQW´KDVEHHQDVVLJQHGIRUHDFKZLWKUHVSHFWWR(5ĮDQG(5ȕHDFK$IWHUJHWWLQJELQGLQJ scores, respective files were uploaded to Pymol 1.1 and Discovery Studio 4.5 for knowing the different protein-ligand interactions and amino acids involved.

Ϯϲ 

RESULTS AND DISCUSSION Docking results of copper complex, tamoxifen and afimoxifene oQ ERWK (VWURJHQ 5HFHSWRU Į (5Į DQG(VWURJHQ5HFHSWRUȕ (5ȕ DUHJLYHQLQ7DEOHDQG7DEOH The respective docking images are given in Figure 1-17[124] Table 1: Docking results of copper complex, tamoxifen and afimoxifene on ERĮ Sl. No.

Compound

1

Copper complex Tamoxifen Afimoxifene

4. 5.

BindingAffinity RMSD RMSD (kcal/mol) Upper bound Lower bound -9.9

0.0

0.0

-9.4 -9.6

0.0 0.0

0.0 0.0

Amino acids involved in Hbonding Thr 278 Asp 282 Arg 82

Table 2: Docking results of copper complex, tamoxifen and afimoxifene on ERȕ Sl. No.

Compound

1.

Copper complex

2. 3.

Tamoxifen Afimoxifene

BindingAffinity RMSD RMSD (kcal/mol) Lower bound Upper bound -9.4 0.0 0.0

-6.4 -6.5

0.0 0.0

Ϯϳ 

0.0 0.0

Amino acids involved in Hbonding Glu 109 Glu 286 Ser 287 Ser 363 Ser 287 Ser 287

Figure 1

Figure 2

Figure 3

Figure 1-&RSSHUFRPSOH[ZLWKLQ(VWURJHQ5HFHSWRUĮ

Figure 4

Figure 5

Figure 6

Figure 4-6: Copper complex within Estrogen 5HFHSWRUȕ

Figure 7

Figure 8

Figure 7-7DPR[LIHQLQ(VWURJHQ5HFHSWRUĮ

Figure 9

Figure 10 Ϯϴ



Figure 11

Figure 9-7DPR[LIHQZLWKLQ(VWURJHQ5HFHSWRUȕ

Figure 12

Figure 13

Figure 14

Figure 12-$ILPR[LIHQHZLWKLQ(VWURJHQ5HFHSWRUĮ

Figure 15

Figure 16

Figure 17

Figure 15-17: AfimR[LIHQHZLWK(VWURJHQ5HFHSWRUȕ Synthesis of Complexes: All the materials, chemicals and solvent used in this study were of analytical grade. Thiosemicarbazide, copper salts were purchased from S.D. fine, Merck, and Ranbaxy and were used as received.All the complexes were synthesized by the template method i.e. by condensation of bis-hydrazone and thiosemicarbazide in the presence of the divalent copper salts. To a stirred hot methanolic solution §FP3) of thiosemicarbazide (2 mmol) and bis-hydrazone were added to a divalent copper salts (1 mmol) dissolved in the minimum quantity of methanol. The resulting solution was refluxed for 8 ± 10 hrs. The mixture was concentrated to half its volume and kept in desiccators overnight. On overnight cooling, a dark colored precipitate formed which was filtered, washed with methanol and dried in vaccuo. The obtained yield was 40-60%. The complexes were soluble in DMF and DMSO. They were found to be thermally stable upto 260-280° C, after which decomposition occurred. Analytical and Physical measurements: Microanalyses for C, H and N were performed using an elemental analyzer (perkin Elmer 2400) at the SAIF, Punjab University, Chandigarh. The magnetic susceptibility measurements were performed at SAIF, IIT Roorkee, using a vibrating sample magnetometer (model PAR 155). The copper contents in the complexes were determined Ϯϵ 

by a literature method [125]. The IR spectra were recorded on a FT-IR spectrophotometer (Perkin Elmer) in the range 4000-200 cm-1 using nujol mull. The H1NMR spectra (at room temperature, DMSO ± d6) were recorded on a Bruker AVANCE II 400 NMR spectrometer (400 MHz) at the SAIF, Punjab University, Chandigarh. The electronic spectra (in DMSO) were recorded on a cary 14 spectrophotometer at room temperature. The FAB (Fast atom bombardment) mass spectra (at room temperature) were recorded on a TOF MS ES+ mass spectrometer. The conductivity was measured using a digital conductivity meter (HPG system G- 3001). The melting points were determined in capillaries using an electrical melting point apparatus. In-vitro antibacterial activity: All synthesized macrocyclic complexes were tested for invitro antibacterial activity against some bacterial strains using Muller ± Hinton agar [126]. Four test pathogenic bacterial strains, viz. E. Coli, Bacillus subtilis, S. Aureus and P. aeruginosa were considered for the determination of the MICs (minimum inhibitory concentration) of macrocyclic copper (II) complexes. The sterilized (autoclaved at 121 ° C for 15 min) medium (40-50 °C) was poured into the petri dishes to give a depth of 3-4 mm and allowed to solidify. The suspension of the microorganism streaked on plates. The paper discs were placed on the solidified medium. The plates were incubated for 1 hrs at room temperature and incubated at 37°C for 24 hrs [127]. In-vitro anti tumor activity: For Cytotoxicity assays, the cells were plated at 1x104 cells/well in 96-well plate, and grown in DMEM supplemented with 10% FBS for 48 hrs before treatment. The cells were treated with various doses of compounds (1-ȝ0  IRU  KUV IROORZHG E\ Sulforhodamine±B (SRB) performed as reported earlier [128-129]. Briefly, at the end of the experiment, the cells were fixed with 10% TCA for 1 h at 4 oC. The supernatant was aspirated; plates were washed with deionized water 3 times and air-GULHGȝORI ZY 65%LQ acetic acid was added to each well and incubated for 30 min at room temperature. Unbound SRB was removed by 3 washes with 1% acetic acid and the plates air-GULHG  ȝO RI XQEXIIHUHG 10mM Tris base, pH 10.5 was added for extracting the bound stain. The absorbance was read at 560nm in a SpectraMax Me2 Elisa Microplate Reader (Molecular Devices Inc.). Suitable untreated controls were also concomitantly employed. For the morphological analysis, 0.2x106 cells/well were plated into 6-well plate in DMEM medium supplemented with 10% FBS for 48 hrs before treatment. The cells were treated with various doses of compounds (0.1-ȝ0 IRU the next 48 hrs and at the end of experiment cells were observed under phase contrast microscope & photographed (Nikon Eclipse Ti, Japan). Antioxidant activity: The ability of synthetic compounds to scavenge hydrogen peroxide was determined as reported previously. A solution of hydrogen peroxide (40 mM) was prepared in SKRVSKDWH EXIIHU S+   ([WUDFWV  ȝJP/  LQ GLVWLOOHG ZDWHU ZHUH DGGHG WR D K\GURJHQ peroxide solution (0.6 mL, 40mM). Absorbance of hydrogen peroxide at 230 nm was determined 10 minutes later against a blank solution containing the phosphate buffer without hydrogen ϯϬ 

peroxide. The percentages of hydrogen peroxide scavenging of H2O2 by compounds were calculated as follows: % Scavenged [H2O2] = [(AC ± AS)/AC] x 100 Where AC is the absorbance of the control and AS is the absorbance in the presence of the compounds [128-129]. All the complexes are stable to the atmosphere. The complexes are soluble in DMF and DMSO and partial soluble in methanol. The elemental analyses are consistent with the proposed structure of the complexes. Conductivity measurements in DMSO indicated them to be nonelectrolyte. The tests of the anions were negative only after decomposition of the complexes, indicating their presence outside the coordination sphere. The analytical data of the reported complexes are given in table 3.

Table 3: Analytical and Physical data of the copper complexes S Molecular N formula

Color &

Yiel d

M.P. (°C)

molecular weight 1

[Cu(C36H36N12O2 S2)]Cl2

2

[Cu(C36H36N12O2 S2)](NO3)2

3

[Cu(C36H36N12O2 S2)]SO4

Greenis h Yellow Greenis h Light Green

Molar Conduc -tance ȍ-1cm2 mol-1)

%M

%C

%H

%N

52 % 230

57.5

7.26/ 49.56/ (7.32) (49.80)

3.98/ 14.1/19. (4.15) 36

39 % 155160

127.2

6.80/ 46.86/ (6.90) (46.93)

3.81/

59 % 220

10.7

7.25/ 48.75/ (7.28) (48.90)

4.10/

20.9/21.

(3.91) 29 21.5/(21

(4.13) .8)

IR Spectra: The presence of a single medium band in the region 400 - 4000 cm-1 in all the complexes may be assigned to N-H stretching vibrations and relevant IR spectra of complexes are given in table 4.

ϯϭ 

Table 4: Relevant IR spectra of the copper complexes (cm-1) S.No.

Copper Complex

Ȟ 1-H)

Ȟ $U-H)

Ȟ(C=N) Ȟ(C=S)

Ȟ 0-N)

1

[Cu(C36H36N12O2S2)]Cl2

3310

3061

1620

1170

435

2

[Cu(C36H36N12O2S2)](NO3)2

3305

3065

1625

1176

445

3

[Cu(C36H36N12O2S2)]SO4

3300

3071

1628

1189

455

It was noted that a pairs of bands FRUUHVSRQGLQJ WR Ȟ 1+2) stretching vibrations appeared at 3200-3210 cm-1 in the IR spectrum of thiosemicarbazide but was absent in the IR spectra of all the copper complexes. Furthermore, no strong absorption band was observed in the spectra of the complexes near 1700 cm-1 indicating, the absence of the (C=O) group of carbohydrazone and thus combining the condensation of the carbonyl group of carbohydrazone and the amino group of thiosemicarbazide. A strong absorption band in the region 1595-1625 cm-1 may be assigned to C=N stretching vibrations [131-132]. These results provide strong evidence for the formation of the macrocyclic frame133. The lower values of (C = N) may be explained based on a drift of the lone pair electron density of the azomethine nitrogen towards the central metal atom[134-135]. Another set of medium intensity bands in the region 1500-1585 cm-1 ZHUHDWWULEXWHGWRȞ F F aromatic stretching vibrations of the phenyl groups and the bands around 845-875 cm-1 may be assigned C-H out of plane bending vibrations of the phenyl groups. The C-N stretching vibration may occur in the range 1015-1355 cm-1.The far IR spectra of the complexes showed bands in the region 422-435 cm-1 FRUUHVSRQGLQJ WR Ȟ 0-N) stretching vibrations[136-137] which give insight into the coordination of the azomethine nitrogen to the central copper atom [138]. 1

H-NMR Spectra: The 1HNMR spectra of all complexes were obtained in the CDCl3 at room

WHPSHUDWXUHXVLQJ706DVDQLQWHUQDOVWDQGDUG7KHDURPDWLFUHJLRQVKRZVDVKDUSVLQJOHWDWį SSPDVVLJQHGWRWKHSKHQ\OSURWRQVDQGDVLQJOHWDWįSSPGXHWRPHWK\OSURWRQV7KH O-+SURWRQRIDSKHQROLFJURXSVKRZVDVKDUSVLQJOHWDWį.47 ppm. The multiplets observed in the region 6.81-7.93 ppm may be assigned to the aromatic ring protons of carbohydrazone and the thiosemicarbazide moiety. The H1NMR spectra of copper complexes shows signals corresponding to ±CH3, -NH2, NH (hydrazone) and -OH protons at 2.28 (5, 3H), 7.40-7.48 (M, 3H), 8.059-8.38 (2H), 10.09 (s, 1H) and 11.83 (s, 1H) respectively. The NMR spectrum of copper chelates confirms the participation of ±NH2 group and imino ±NH group in the coordination with metal ions. (Figure 5) ϯϮ 

Table 5: Relevant H1NMR peaks of copper complexes (ppm) S.No.

Complex

į &+2)

į 1+

į(HC=N) į(CH3) į $U&H)

1

[Cu(C36H36N12O2S2)]Cl2

2.45

9.89

8.35

2.2

6.7

2

[Cu(C36H36N12O2S2)](NO3)2

2.4

9.99

8.2

2.1

6.8

3

[Cu(C36H36N12O2S2)]SO4

2.5

9.96

8.06

2.1

6.5

6RPHK\GURJHQDWRPYDOXHVRIįZHUHQRWREVHUYHGSUHFLVHO\GXHWRoverlapping with the signals of the aromatic hydrogen atoms of carbohydrazone ligand. 1HNMR integrations and signal multiplicity are in agreement with the proposed structures. In the 1HNMR spectra of the complexes a high frequency shift of Ca 0.13 ppm, for the methyl hydrogen atoms (C-CH3), compared to the spectra of the thiosemicarbazones, and evidences the coordinator through the azomethine nitrogen atom. Electronic spectra of Cu (II) complexes exhibit bands in the range 15270 ± 16680 cm-1 and 18,200 ± 19200 cm-1 respectively. Magnetic measurements and electronic spectra: The magnetic moments of copper complexes were found in the range 1.75-1.83 µB corresponding to one unpaired e- in the copper (II) ion. The absorption spectra of the copper complexes exhibited bands in the region 17700-19680 cm-1 which showed that these complexes were distorted octahedral in nature

[139-140]

. Assuming

tetragonal distortion in the molecule, the d ± orbital energy level sequence for these complexes may be represented as x2 ± Y2 > Z2 > XY >XZ > YZ and the shoulder may be assigned to Z2ĺ x2 ± Y2 (2B1g ĺ 2B2g DQGWKHEURDGEDQGFRQWDLQVERWKWKH; [Cu(C36H36N12O2S2)]Cl2 complexes. The good values of ϯϯ 

activity found for these complexes, around 10 µ mol/L, show that the complexation of thiosemicarbazone to Cu (II) may be a good strategy to obtain antitumor agents. (Figure 18-20). The similarity of the values of LD50 found for the Cu(II) complexes is an evidence in favour of the same biochemical action mechanism, but different from those of the cisplatin, inactive in this case. Three macrocyclic copper (II) complexes were screened for their potential anticancer/cytotoxic activity in table no. 6. Table 6: In-vitro cytotoxicity activity of copper complexes Complex

R

LD50 value

1

[Cu(C36H36N12O2S2)]Cl2

R1 = CH3, R2 = Cl, and R3 = OH

15 µM

2

[Cu(C36H36N12O2S2)](NO3)2

R1 = CH3, R2 = NO3, and R3 = OH

10 µM

3

[Cu(C36H36N12O2S2)]SO4

R1 = CH3, R2 = SO4 and R3 = OH

10 µM

йŽĨŽŶƚƌŽů

S. No.

ϮϬϬ ϭϬϬ Ϭ ͲϭϬϬ ŽŶĐĞŶƚƌĂƚŝŽŶ;ђDͿ

(a)

(b)

(c)

Figure 18: (a) Control showing cancer cell line MCF-7 (b) Cytotoxic activity of compound 1 at 15µM (c) Graph showing the Dose dependent effect of compound 1 on MCF-7 cell line

ϯϰ 

ϭϬϬ ϱϬ Ϭ ͲϱϬ

ŽŶƚƌŽů Ϭ͘ϱђD ϱђD ϭϱђD

йŽĨŽŶƚƌŽů

ϭϱϬ

ŽŶĐĞŶƚƌĂƚŝŽŶ;

(a)

(b)

(c)

Figure 19: (a) Control showing cancer cell line MCF-7 (b) Cytotoxic activity of compound 2

(a)

(b)

ϭϱђ͙

ͲϮϬϬ

ϱђD

Ϭ

Ϭ͘ϱ͙

ϮϬϬ ŽŶ͙

йŽĨŽŶƚƌŽů

at 15µM (c) Graph showing the Dose dependent effect of compound 2 on MCF-7 cell line

ŽŶĐĞŶƚƌĂƚŝŽŶ;ђD

(c)

Figure 20: (a) Control showing cancer cell line MCF-7 (b) Cytotoxic activity of compound 3 at 15µM (c) Graph showing the Dose dependent effect of compound 3 on MCF-7 cell line In-vitro antibacterial activity: The three macrocyclic Cu(II) complexes were also evaluated for their potential antibacterial activity against B. Subtilis, S. aureus , E. Coli and P. aereoginasa. Table 5-7 highlight the antibacterial activity against B. Subtilis, S. aureus , E. Coli as observed by dise diffusion method. None of the compounds were found to be active against P. aereoginasa at any concentration. Table 7: In-vitro antibacterial activity of compounds 1-3 against B. subtilis Ring Diameter (mm) Compound

Tetracyclin DMSO (1mg/ml)

(1mg/ml)

4

6

8

10

1.

20

ND

ND

ND

ND

6

ND

2.

20

ND

ND

ND

ND

ND

ND

3.

15

ND

ND

ND

ND

ND

ND

ϯϱ 

Compound Dose (mg) 2

Table 8 In-vitro antibacterial activity of compounds 1-3 against S. aureus Ring Diameter (mm) Compound Tetracyclin

DMSO

Compound Dose (mg)

(1mg/ml)

(1mg/ml)

2

4

6

8

10

1.

15

ND

17

14

ND

ND

ND

2.

18

ND

10

11

8

7

16

3.

15

ND

12

6

6

12

16

Table 9 In-vitro antibacterial activity of compounds 1-3 against E. coli Ring Diameter (mm) Compound

Tetracyclin

DMSO

Compound Dose (mg)

(1mg/ml)

(1mg/ml)

2

4

6

8

10

1.

ND

ND

6

6

6

8

ND

2.

15

ND

6

6

6

16

12

3.

ND

ND

8

8

8

10

11

The minimum inhibitory concentrations of complexes were determined by disc diffusion method. The minimum inhibitory concentration at which no growth observed was taken as MIC values. None of the compounds active against P. aerogenosa at any concentration. The higher antibacterial activity of the copper (II) complexes may be due to coordination and chelation tends to make copper complexes act as more powerful and potent bacteriostatic agents thus inhibiting the growth of the bacteria. In a complex, the positive charge of the copper is partially shared with the donor atoms present in the complexes DQGWKHVHPD\EHȆ electrons delocalization over the whole. The increase activity of the copper chelate can be explained on the basis of chelation theory. On chelation, the polarity of the copper ion if reduced largely due to the overlap of the ligand orbital and the partial sharing of the positive charge of the metal ion with the donor groups. This increases the lipophilic character of these complexes seems to be the reason of their enhanced potent antibacterial activity. There are some other factors which also increase the ϯϲ 

activity such as solubility, conductivity and bond length between the metal and the ligand. All three copper complexes were active due to the presence of thio group in the coordinating ligand. In-vitro antioxidant activity: Macrocyclic and their metal complexes have been suggested as promising agents for the diagnosis and treatment of different disease

[142-144]

. All three

compounds showed significant free radical scavenging action against hydrogen peroxide (H2O2) induced release of free radicals at different concentration 200, 400, 800 and 1000 µg/ml. Ascorbic acid used as reference standard.

Table 10: In-vitro antioxidant activity of Copper complexes S.No.

% Scavenging (Mean ± SEM) of triplication Compound

400 µg/ml

800 µg/ml

1000 µg/ml

1

[Cu(C36H36N12O2S2)]Cl2

26.22 ± 0.082

31.26 ±

33.62 ±

40.64 ±

0.176

0.210

0.094

2

[Cu(C36H36N12O2S2)](NO3)2

33.18 ± 0.034

36.94 ±

37.60 ±

42.62 ±

0.022

0.091

0.090

35.94 ±

36.17 ±

35.92 ±

0.026

0.042

0.021

39.69 ±

42.26 ±

40.19 ±

0.024

0.014

0.012

3

4

[Cu(C36H36N12O2S2)]SO4

Ascorbic acid

200 µg/ml

36.16 ± 0.042

40.16 ± 0.022

(Standard)

All compounds were found to possess potent antioxidant activity in the range of 80-90 %. When screened for their radical scavenging activity against H2O2.

ϯϳ 

CONCLUSION The present work highlights the potent in-vitro anticancer activity of the Copper(II) complexes against the MCF-7 cell line. To overcome drug resistance and reduce the undesirable side effects, it becomes essential to synthesize drugs with high potential. Among transition metal complexes copper complexes were found to have high activity. The wide range of applications of copper complexes may provide a target oriented information to the chemist to develop variable copper complexes with potent anti proliferative activity. Based on molecular docking studies and other various studies like elemental analysis, conductance measurements and magnetic susceptibilities, as well as IR, H1NMR, electronic and mass spectral studies, a distorted octahedral geometry may be proposed for all these complexes. The proposed structures are shown in figure 21-23.

H N

H3C

H3C

Cl

NH

S

N 2+

NH N

N

2Cl

Cu

N

N

N NH

S

Cl CH3

NH

N H

Figure 21: Structure of copper complex 1

ϯϴ 

CH3

-

H N

H3C

H3C

Cl

NH

S

NH N

N

N

2+

2NO 3

Cu

N

-

N

N NH

S

NH

Cl

CH3

N H

CH3

Figure 22: Structure of copper complex 2 H N

H3C

H3C

Cl

NH

S

N 2+

NH N

N

SO 4

Cu

N

N

N NH

S

NH

Cl CH3

--

CH3

N H

Figure 23: Structure of copper complex 3

These copper complexes have shown excellent molecular docking studies by giving significant binding energies i.e. -9.9 kcal/mol and - NFDOPRO RQ (VWURJHQ 5HFHSWRU Į DQG (VWURJHQ 5HFHSWRU ȕ UHVSHFWLYHO\ DQG FRPSDUHG ZLWK WZR DQWLFDQFHU GUXJV XVHG LQ EUHDVW FDQFHU LH Tamoxifen and Afimoxifene.These three Cu(II) complexes show modest in-vitro cytotoxic properties against breast cancer cell line MCF- 7. LD 50 values are compared with cisplatin and the results revealed that complex possess better activity. More detailed studies are needed to understand the mechanism of action at the cellular level and the role of the metal. Investigations of antibacterial screening data revealed that the compounds 1, 2 and 3 exhibited maximum zone of inhibition against the bacterial strains E. coli, S. aurous, B. Subtilis. It has been suggested that chelation reduces the polarity of the metal ion, mainly because of the partial sharing of its ϯϵ 

positive charge with a donor group within the whole chelate ring system. This process of chelation increases the lipophilic nature of the central metal atom, which in turn, favour its permeation through the lipid layer of the membrane, thus causing the metal complexes to cross the bacterial membrane more effectively thereby increasing the activity of the complexes. In addition to this many other factors, such as solubility, dipole moment and conductivity as well as the influence of the metal ion, may be possible reasons for the remarkable antibacterial activities of these complexes. Analysis of result revealed that the all three macrocyclic Cu(II) complexes exhibited good radical scavenging activity as compared to the standard ascorbic acids. Apparently, potency of all three complexes were found to be relatively low to cis-platin compounds which are capable of inducing cell death via apoptosis are regarded as potent anticancer drugs. Cell shrinkage and rounding, membrane bubbling, chromatin condensation and nuclear fragmentation are important characteristics of apoptosis. In our study, prominent morphological changes, which are associated with apoptosis, live cell rounding and shrinkage and nuclear fragmentation were observed when MCF ± 7 breast cancer cell line were treated with the macrocyclic Cu(II) complexes(10 hrs) for more potent 24 hrs. The data reported in this book may be helpful guide for the medicinal chemist who is working in this area.

ϰϬ 

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