FIELD OF THE INVENTION
The present invention relates to synthesis of metal complexes of ligand and studying their activity. More particularly the present invention relates to synthesis of Cu (II) carbohydrazone coordination complexes and studying them for anticancerous activity.
BACKGROUND OF THE INVENTION
To succeed in the therapy, the increased susceptibility of malignant cells to pro-apoptotic action of chemotherapeutic agents exceeding that of normal counterparts would be necessary. However, in tumor cells the apoptotic pathways are frequently misregulated due to inactivation or down-regulation of pro-apoptotic genes [1] or as a consequence of over expression of anti apoptotic factors. Therefore, the application of new agents possessing higher selectivity towards malignant cells is necessary. Here the present invention relates to a novel copper (II) complex [Formula I] of a carbohydrazone, a method of synthesize therefore, and its antineoplastic effect against MCF-7 breast cancer cell line, a representative cell line for Estrogen Receptor (ER) positive breast cancers. The antiproliferative effect of [Formula I] was also checked in SKBrS (p53 mutated and ER negative breast cancer cell line) and HBL-1 00 (model for pre-neoplastic breast cancer, [2].
Carbohydrazones are the next higher homologue of semicarbazones, both are studied for their biological properties [3]. Carbohydrazones are the condensation products of carbohydrazides [4] with aldehydes or ketones. The chelating behaviour of 2- acetyl pyridine carbohydrazones [5] towards Cu(ll) ion has been reported. Several mono-and bis-carbohydrazone ligands have been synthesized and characterized and their coordinating properties towards Cu (II) have been studied along with their antimicrobial and anti-mutagenic activity [6]. Carbohydrazones are structurally similar to thiocarbohydrazones, which on complexation with Cu(II) are proposed as anticancer drug analogues [7,8] like thiosemicarbazones and their Cu(II) complexes [9, 10], except that they have an oxygen atom in place of sulfur. Hence we considered it worthwhile to study the anticancer effect of carbohydrazones and their complexes. We synthesized various Cu (II) complexes of N"- N'"- bis (di-2-pyridylmethylene) carbonic dihydrazide (H2L) [II], a recently reported ligand by us, using different copper salts and is found that the [Cu2(HL)(HS04)- H20]S04- 6 H2O [Formula I] have

more activity against MCF -7 breast cancer cell line compared to standard anticancer chemotherapeutic agents.
The compound specified above attains its LD50 in MCF - 7 cell line at lower concentration (in ^M), which is established through a cell viability assay, MTT assay. We compared this with cisplatin, a standard chemotherapeutic drug that requires a higher LD50 of SO^iM in MCF -7 cells. First stage of apoptosis starts with the loss of membrane potential of mitochondrion. There was a clear evidence for the loss of mitochondrial membrane potential with the treatment of [Formula I] in MCF -7 cells. The p53 gene is mutated in over 50% of human tumors and in some inflammatory disorders like rheumatoid arthritis [12, 13]. MCF-7 cells are known to have functional p53 protein [14]. This tumor suppressor protein p53 plays an important role in mediating response to stress such as that induced by DNA damage and hypoxia resulting in either growth arrest or apoptosis [15]. Role of endogenous p53 in regulating endogenous caspases is well proved, MCF -7 cells when treated with doxorubicin, a standard chemotherapeutic drug, increases the level of p53 protein [16]. There was a profound activation of p53 protein, which clearly suggests that the apoptosis take place through a p53 dependant pathway. When treated with [Formula
11 seven-fold activation for caspase 9 can be seen after 24h. A transition metal complexes of phenanthrenequinone thiosemicarbazone showed maximum antiproliferative activity against, T47D breast cancer ceils, probably due to inhibition of steroid binding to the cognitive receptor or by preventing dimerization of the estrogen receptor [17]. MCF -7 cells are Estrogen Receptor alpha (ERa) over expressing and this suggests us to investigate on the differential expression of ERa in MCF7 in response to this investigational [Formula I]. However, significant variation in the expression of ERa was not observed. PARP (Poly (ADP-ribose) polymerase-!) has been implicated in DNA repair and maintenance of genomic integrity: PARP cleavage is most appropriately viewed as the marker for apoptosis, rather than the executor of the process [18]. Our results also show a profound activation for PARP. DNA break due to investigational [Formula I] was observed with COMET assay for
12 h but DNA break for cisplatin for the same duration of treatment was negligible.

PRIOR ART OF THE INVENTION
The related art of interest will be discussed in the order of perceived relevance to the present invention.
"si I Patent No I Their Claim I Our Claim
_No
1 6,831,061 Semicarbazonc and They are not mentioning
Thiosemicarbazone apicidin Cu2+ complex of
derivatives have anticancer activitv carbohydra/nne
2 6,927.042 Semicarbazide and carbazide for j We arc mentioning
glycoprotein synthesis anticancer activity of
Cu2+ complex of
I carbohydrazone ^
3 6,649,658 Verbenone derivative of Our complex is sulphosalicilate anticancer ,
4 6,743,786 Vanadium cottipounds for treating We claim Cu2-
' cancer complex for treating
' cancer ^ _ _
5 5,490,951 Highly reactive form of copper is We claim for Cu2-i-
j well discussed complex of
i carbohydrazone
6 4,657,928 Organic copper complexes as We claim for the
superoxide scavengers hence anticancer propeny.
. ■ radioproteclants
7 5,440,062 Copper complex ofdialkyi T^'c claim for Cti2^
; subsiitvited aminotroponeitt\ines conaplcx of
used as catalyst carbohydrazone used as
_^ I antitumor agent
~8 I 6,627,176 TCopfKr complex of isonotrile ligand We claim for Cu2+
I ' having anticancer activity complex of
, carbohydrazone
9 6,056,939 i-lcteropolymcttalic agents of We claim for Cu2+
I DTPA.DOTA 35 imaging agents and complex of
! as radiopharamcuticals carbohydrazone used as
i ■ antitumor at;ent
[Tb 6,093,382 Metal complexes bound with folate We claim for Cu2+
us chemolherapcutic agents. complex of
I carbohydrazone used as
I antitumor agent
! 11 6.812,247 ' Copper complex ufheteroaryl We claim for Cu2'-
carbonyl compounds complex of
^ : carbohydrazone _____
12 7,056^.924 Heterocyclic derivatives used for I We claim for Cu2 •
cardiovascular diseases ! complexes as antitumor
LilS^'lL __
~n 6,331.542 Phenoxyphenyl sulphonyl | We claim for Cu2+
carbohydrazide as protease complex of dipyridyl-
inhibitors 2yl-methylene
carbohydrazides as i
anticancer agents J

interested only in research. The clinical utility of cisplatin is limited by the emergence of resistance in many tumor types (Perez, 1998). Therapeutic application against this phenomenon has not yet been reported.
The matriarchal platinum anti-cancer agent, cisplatin, has been known for 150 years, its efficacy in the treatment of a variety of types of cancer was only discovered serendipitously by Rosenberg in the 1960's [1-3]. Since its approval for clinical use in 1978, it has proved very successful in the treatment of cancers. The cancer cells show a primary or acquired resistance to cisplatin. Therefore, extensive effort to develop novel cisplatin analogues with equivalent or greater antitumor activity and a lower toxicity has been made. Among them, carboplatin has reduced renal and gastrointestinal side effects than cisplatin. However, carboplatin has no enhanced therapeutic efficacy over cisplatin and has not circumvented the acquired resistance to cisplatin. Even then cisplatin is used as a standard chemotherapeutic agent. Therefore there is a need for novel compounds that will be more effective and less toxic than the above compounds.
The combination of cisplatin plus gemcitabine is active in relapsed breast cancer patients. The activity observed in drug-resistant patients suggests relative non-cross resistance with other drug combinations. Cisplatin in combination with other agents (eg, etoposide or 5-fluorouracil), it provides response rates equivalent to other front¬line/combinations. Our ongoing biochemical modulation and translational correlative trials should clarify which specific mechanisms are most relevant to clinical cisplatin resistance. Such investigations have the potential to improve the ability to predict likelihood of response and should identify potential targets like compound [Formula I] for pharmacological or molecular intervention. Hence we suggest that this compound [Formula I] may be used instead of cisplatin in combination therapy.
Utility of instant invention
This compound [Formula I] is found to be more potent than cisplatin in breast
cancers. Hence we claim that
a. Antiproliferative effect of [Formula I] more pronounced than conventional
Cisplatin.
b. [Formula I] shows a p53 dependant apoptotic behavior in MCF-7 breast cancer
cell line.

c. [Formula I] acts in a ERa independent pathway in ER positive cancer cells.
d. The present compound [Formula I] is a novel anticancer Cu (II) complex of a
recently reported ligand.
In future [Formula I] can be a potential anticancer target against drug resistant cancers.
Present national and international knowledge on the utility of this invention Carbohydrazones and their complexes are leastly studied compared to thiocarbohydrazones. Synthesis of Cu (II) complexes of some carbohydrazones are reported by Baachi et al., 1996 & 1999 [4, 5] and Chohan et al., 2004 [19]. Nationally, Pandey et al., 1986 [20] have synthesized oxovanadium (IV) complexes of some carbohydrazones. Some La(III) and Pr(III) complexes are reported by Tripathi et al., 2000[21]. Many disubstituted carbazones and their complexes were studied by Siddalingaiah et al., 2004 [22] and Dimmock et al., 1997 [23] have reported cytotoxic evaluation of some carbohydrazones. However, the ligand used here is reported first time by us [11] and any of its metal complexes have not been reported.
Antitumor functions of 1, 2-naphtho-quinone-2-thiosemicarbazone (NQTS) and its metal complexes [Cu (II), Pd (II), and Ni (II)] against the MCF -7 human breast cancer cells and the possible mechanisms of action were determined [24]. Aryloxy-aryl semicarbazones and related compounds were synthesized and evaluated for anticonvulsant activities [25].
National status points that copper (II) and manganese (II) complexes of N' [(2-hydroxy phenyl) carbano thioyl)] pyridine-2-carbohydrazide showed better anti proliferative activity against colon cancer cell line HT29. These complexes are found to be highly potent agent for which act against myristoyilation of c-Src [26]. T.S. Lobana, Department of Chemistry, Guru Nanak Dev University, Amritsar, India has worked in the field of complexes of thiosemicarbazones [27]. P. Yogeeswari, Pharmacy Group, Birla Institute of Technology and Science, Pilani, Rajasthan, India has worked in the synthesis and study of anticancer compounds (metal complexes of Aryl thiosemicarbazones, quinolones, semicarbazones & prodrugs). S. P. Gupta of Birla Institute of Technology and Science, Pilani, India, who has worked in quantitative structure-activity relationship studies on anticancer drugs.

Disruption of p53 only made MCF-7 cells sensitive to cisplatin [28]. In this context our [Formula I] will show its proof of action leading to apoptosis.
This is the first report for the molecular mechanism of [Formula I]'s apoptotic action in human breast MCF -7 cancer cells.
Novelty, non-obviousness, inventive step and utility of this invention, with brief justification
The compound [Formula I] is a novel complex of a known ligand (recently reported by us) and its anticancer property is not identified till date. Our study suggested clear molecular evidence for its anti-neoplastic activity. This compound [Formula I] is more sensitive to cisplatin resistant MCF -7 cell line. From our utility 6b it is clear that p53 deficient cancers may be more resistant to this compound [Formula I]. We justify our study as this investigational [Formula I] strongly inhibited the proliferation of MCF -7 than cisplatin. About two- fold higher cisplatin concentration was necessary than [Formula I] to cause the same effect. This observation remains in concordance with reports of Gadek et ai, 2003 revealing the same resistance of MCF 7 cells to cisplatin [29]. Our results implicate that [Formula I] inhibits more efficaciously the proliferation of human breast carcinoma cells than cisplatin.
Reference:
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4. Kurzer,T.; Wilkinson, 1970, M. Chern. Rev. 70: 111-149.
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Trans. 2699-2705.
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7. B. Maubaraki, K.S. Murray, J.D. Ranford, X.Wang, Y. Xu, 1998, Chern. Commun. 353354.
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13. Tak, P. P., Zvaifler, N. J., Green, D. R., and Firestein, G. S., 2000, Immunol. Today, 21: 78-82

14. Tam,S.W.,Theodoras,A,M.,Shay,J,W.,Draetta,G,F and Pagano,M., 1994, Oncogene 9: 2663-2674.
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Subhash Padhye and Yinfa Ma, 2004, Toxicol, and Applied Pharmacol.
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Patent Reference
30. Lee H W,Jung Y H, Han J W, Lee S Y, Lee Y W, Lee H Y, Zee O P, 2004,United States, Patent No. 6,831,061
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6,331,542
None of the above inventions and patents, taken either singly or in combination, is seen to describe the instant invention as claimed.
OBJECTS OF THE PRESENT INVENTION
The principal object of the present invention is to synthesis a metal complex of ligand.
Another object of the present invention is to synthesis a Cu (II) carbohydrazone
coordination complex.
Yet another object of the present invention is to study the anticancer property of Cu
(II) carbohydrazone coordination complex.
Still yet another object of the present invention is to provide cancer therapy which is
more effective than the conventional chemotherapeutic agents in general and cisplatin
in particular in breast cancer cells.
Still another object of the present invention is to provide a molecular mechanism for
the action of Cu (II) carbohydrazone coordination complex in MCF-7 breast cancer
cells.

Still another object of the present invention is to provide a molecular mechanism for
the action of Cu (II) carbohydrazone coordination complex in other breast cancer
cells.
Still another object of the present invention is to arrive at a pharmaceutical
composition by combining the active moiety of Cu (II) carbohydrazone coordination
complex, investigational compound [Formula I] with standard chemotherapeutics.
STATEMENT OF THE INVENTION
Accordingly, the present invention provides a compound of structural formula-I

BHjO

Formula-I
a composition comprising compound of structural formula-I


o

Formula-I

and pharmaceutically acceptable additive(s); a synergistic composition for management of cancer, said composition comprising compound of structural formula I


o

Formula I
at a concentration ranging from 1 |iM to 10 |iM and cisplatin at a concentration ranging from 5 ^M to 50 \iM, optionally along with pharmaceutically acceptable additives; a process to prepare compound of structural formula-I,


HO

o

Formula-I
wherein said process comprising steps of dissolving ligand and metallic salt in
organic solvent to obtain solution; refluxing and filtering the solution followed by allowing the filtrate to precipitate; and washing and drying the precipitate to obtain compound of structural formula I; an in-vitro method to inhibit proliferation of cancerous cells by allowing compound of structural formula I

6HjO

Formula -1
optionally along with known anticancer drugs to bind to its cell receptors.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure: 1 EPR spectrum of the compound [Formula I]
Figure: 2A and 2B Histogram of Compound [Formula I] showing significant growth
inhibitory effect on MCF-7K, SKBr-3 and HBL-100 breast cancer cell lines
Figure: 3A Compound [Formula I] induces apoptosis through caspase 8 and 9
activation
Figure: 3B DNA damage is significant in compound [Formula I] treated cells
Figure: 4 Growth inhibitory effects due to compound [Formula 1] treatment was
through apoptosis
Figure: 5 Compound [Formula I] showed loss of mitochondrial membrane potential
(Av|/m)
Figure: 6A Compound [Formula I] showed maximum p53 expression
Figure: 6B Apoptosis take place through an ER a independent pathway
Figure: 6C Compound [Formula 1] showed maximum PARP expression
Figure: 7A Cell viability assay of complexes
Figure: 7B Cell viability assay of ligand
Figure: 8 Cell proliferation assay for the combination of Compound [Formula 1] with
Cisplatin and Etoposide

DETAILED DESCRIPTION OF THE PRESENT INVENTION
The present invention is in relation to a compound of structural formula-I

6H2O

Formula-I
In another embodiment of the present invention, said compound is a metal complex
having molecular formula [Cui (HL) (HSO4) • H2O] S04- 6 H2O.
In yet another embodiment of the present invention, said metal is selected from a
group comprising transition metals Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu and Zn,
preferably Cu.
In still another embodiment of the present invention, said metal is coordinately
complexed with a ligand N"- N'"- bis (di-2-pyridylmethylene) carbonic dihydrazide.
The present invention is in relation to a composition comprising compound of
structural formula-I


^S 6H,0
O \\ ^
o

Formula-I

and pharmaceutically acceptable additive(s).
In another embodiment of the present invention, said composition is for management
of cancer.
In yet another embodiment of the present invention, the additives are selected from a
group comprising granulating agents, binding agents, lubricating agents,
disintegrating agents, sweetening agents, coloring agents, flavoring agents, coating
agents, plasticizers, preservatives, suspending agents, emulsifying agents and
spheronization agents.
In still another embodiment of the present invention, said composition is formulated
into various dosage forms selected from a group comprising tablet, troches, lozenges,
aqueous or oily suspensions, dispersible powders or granules, emulsion in hard or soft
gel capsules, syrups and elixirs.
The present invention is in relation to a synergistic composition for management of cancer, said composition comprising compound of structural formula I


o

at a concentration ranging from 1 ^M to 10 ^M and cisplatin at a concentration
ranging from 5 ^M to 50 |J.M, optionally along with pharmaceutically acceptable
additives.
In another embodiment of the present invention, the concentration of compound of
structural formula I is about 5 nM and concentration of cisplatin is about 10 ^iM.
In yet another embodiment of the present invention, said cancer is preferably breast
cancer.
The present invention is in relation to a process to prepare compound of structural
formula-I,

6H20

Formula-I
wherein said process comprising steps of:
(a) dissolving ligand and metallic salt in organic solvent to obtain solution;
(b) refluxing and filtering the solution followed by allowing the filtrate to
precipitate; and
(c) washing and drying the precipitate to obtain compound of structural formula I.
In another embodiment of the present invention, said metal is in salt form and is
selected from a metal group comprising transifion metals Sc, Ti, V, Cr, Mn, Fe, Co,
Ni, Cu and Zn, preferably copper sulphate.
In yet another embodiment of the present invention, the metal is complexed with a
ligand N"- N'"- bis (di-2-pyridylmethylene) carbonic dihydrazide
In still another embodiment of the present invention, the reflux is carried out for a
time period ranging from 15-25 minutes, preferably about 20 minutes.
In still another embodiment of the present invention, said organic solvent is alcohol,
preferably methanol.
In still another embodiment of the present invention, said precipitate is washed with
absolute ethanol and diethyl ether and dried in vacuo.
In still another embodiment of the present invention, the compound yield is ranging
from 80 % to 90 %, preferably about 83 %.
The present invention is in relation to an in-vitro method to inhibit proliferation of
cancerous cells by allowing compound of structural formula I

6HjO

Formula -1
optionally along with known anticancer drugs to bind to its cell receptors.
In another embodiment of the present invention, said cancerous cells are breast cancer
cells.
In yet another embodiment of the present invention, said anticancer drugs are
Cisplatin and Etoposide, preferably Cisplatin.
The technology of the instant Application is further elaborated with the help of following examples. However, the examples should not be construed to limit the scope of the invention.
Example: 1
Chemical synthesis and characterization of compound [Cu2(HL)(HS04)'
H20]S04- 6 H2O [Formula I]
Synthesis of H2L is reported elsewhere [11]. To a solution of H2L (0.4225 g, 1 mmol) in hot methanol (40 ml), CuS04- 5H2O (0.50 g, 2 mmol) in hot methanol were added and refluxed the solution for 20 minutes. The solution was filtered and kept for precipitation. The brown precipitate was filtered off and washed with absolute ethanol and diethyl ether, and dried over P4O10 in vacuo. Yield: 721 mg (83%). Anal. Calcd for C23H,8Cu2N809S2- 7H2O: C, 31.88; H, 3.88; N, 12.90: Found: C, 31.83; H, 3.72; N, 12.91%. Uv/vis [CH3OH; Xmax/nm (s/dm^ mol"' cm"']: 221 (22720), 270 (22750), 304 (17360), 359 (8870), 458 (11590). IR (KBr, cm"'): 3408 (br, vHjO) 3220 (sh, vNH), 3083 (sh, vCH), 1597vs, 1562, 1515, 1473, 1425 (m, vC=C+vC=N), 1366 (m, vC-N), 1310 (s, vC-0), 1235 (s, vN-N), 1110-1145 (vs, V3SO4), 976 (m, v,S04) 790,

748 (m, 8C-H), 616 (vs, V4SO4), 414 (s, vCu-Nazo), 355 (m, vCu-0), 275 (s, vCu-Npy). TGA data show a loss of six water molecules (12.4%) within the temperature range 40-I12°C and then the loss of one molecule of water (2%) in the range 112-210°C. Molar conductivity (AM) of 10"^ M methanol solution of [Formula I] was found to be
1 T 1
81 ohm' cm mole" . Room temperature magnetic moment is found to be 2.0 HB- EPR spectrum of the compound [Formula I] in DMF solution at 77K shows two different noninteracting Cu(Il) species with one species having §11= 2.244, S-L= 2.051,^„ =
560 MHz and A^ = 50 MHz. The other species shows §11 = 2.294, S^ = 2.066, v4„ =
500 MHz and Aj_ = 50 MHz values. However no half-field signals were found in the frozen solution spectrum.
The schematic representation of the chemical reaction to achieve the product of instant invention is as shown below:

V^

N

\/

methanol
+ 2 CUSO4. 5H,0 *■ Compound [I]
reflux

Schematic representation
Figure 1 provides EPR spectrum of the compound [Formula I] in DMF solution at 77K. Blue is the simulated spectrum which indicates two different Cu(ll) species with one species having §11 =2.244, §-L = 2.05 I, 4| = 5 60 M H z and ^^ = 60 M H z. T he other
species shows §11 =2.296, §J-= 2.066, A^^= Ui MHz, and ^j^=40 MHz values,
Howeverno half-field signals were found.
Example: 2
The formation of [Cu2(HL'')Cl3(OH2)]- 1.5 H2O has been exclusively confirmed by single crystal X-ray studies by the reaction between bis (methyl 2-pyridyl ketone) carbonohydrazone (H2L^) and CuCb in absolute ethanol for 2 h refluxion. Here the NNO and NNN coordination is confirmed [5]. The other binuclear compound with

Cu(02CMe)2- 2 H2O of the same ligand has been given a molecular formula of Cu2(HL'*)(02CMe)3. The present tentative structure of compound [Formula I] was assigned based on spectral features as all attempts to crystallize the compound [Formula I] went vain.
Example: 3
Biological activity
We assessed the differential effect of compound [Formula I] in MCF -7, HBL-IOO and SKBr3 cell line. These cells were grown in 96-well plate, and then treated with or without the indicated concentrations of drugs for 48 h. At the end of treatment, cell viability was assessed by MTT assay. Cell viability was calculated as percentage over control and all results are expressed as the mean percentage of control ± S.D. of quadruplicate determinations from three independent experiments (Figure 2 A & B).
Figure: 2A. Compound [Formula I] show significant growth inhibitory effect on MCF-7, SKBr- 3 and HBL-IOO breast cancer cell lines
Cells were seeded into a 96-well mlcrotitre plate (5000 cells/well) in DMEM with 10% FBS. Once the cells had attached, the compound [Formula I] were added at different concentrations in quadruplicate samples and incubated for 48 h at 37 °C in DMEM with 2.5% FBS. After incubation, the compound [Formula I] containing medium was removed and equal volumes of fresh medium were added along with 100^1 of 3-(4-5dimethylthiozol-2-yl) 2-5diphenyl- tetrazolium bromide (MTT, USB Amersham Biosciences, Buckinghamshire, UK) (5 mg/mL) to each well. The yellowish MTT was reduced to dark colored formazan by viable cells only. After incubating for 3 h, the formazan crystals formed were solubilized with MTT lysis buffer (20% SDS in 50% dimethyl formamide). The plates were kept, protected from light, for 4h at 37°C in an incubator. The color developed was quantitated with an ELISA plate reader (Bio RadSystems, Hercules, CA) (measuring wave length: 570 nm, reference wave length: 630 nm). All results are expressed as the mean percentage of control ±S.D. of quadruplicate determinations from three independent experiments. The differences among the mean values were analyzed using 1-way ANOVA followed by Tukey's post hoc t- test analysis. The one-way ANOVA revealed that the

average mean values of cell survival differed significantly as a function of concentration of Compound [Formula I] (P> 0.05).
Figure: 2B: The MTT assay for the compound [Formula I[ at a lower concentration in MCF-7 cells
Cell viability assay, MTT assay was performed with compound [Formula I] in MCF-7 breast cancer cells at a concentration from lyiM to 10 |aM.
To show that the cell death induced by compound [Formula I] in MCF -7 cells were by apoptosis, we performed caspase assay and Comet assay (Figure 3A & 3B).
Figure: 3A. Compound [Formula I] induces apoptosis through Caspase 8 and 9 Activation.
The enzymatic activity of caspase 8 was induced by the Compound [Formula I] was assayed spectrofluorimetrically. The cells were treated with the indicated concentrations of the drugs and incubated them for 6, 12, 18 & 24 h. After the incubation, cells were scrapped and pelleted, the pellet was lysed in radio immunoprecipitation buffer (10 mM phenyl methyl sulfonyl fluoride, 1 mg/mL of aprotinin, 100 mM EGTA, 100 mM sodium orthovanadate, and 100 mM dithiothreitol) and centrifuged at 15,000 rpm for 15 min; supernatant was collected as a whole cell lysate. 50 ng of total protein was incubated with 50 \iM of caspase-8 or 9 fluorimetric substrate in a total volume of 500 (xl of reaction buffer at 37°C for 1 h in dark. The released AMC (7-amino-4-methyl-coumarin) was quantitated using spectrofluorimeter (LS-50B model, Perkin Elmer) with excitation and emission wavelengths 400 and 505 nm respectively. Both caspase 8 & 9 in Compound [Formula I] treated MCF-7 cells show more than 2.5 fold activation at 24 h.
Figure: 3B. DNA damage is significant in Compound [Formula I] treated cells.
The alkaline single cell gel electrophoresis (comet assay) was performed. Briefly, the cells (treated with or without compound[l]) were pelleted and resuspended in 0.5% low melting point agarose at 37°C and layered on a frosted microscope slide previously coated with a thin layer of 0.5% normal melting agarose and kept for 5 min at 4°C. After solidification, the slides were immersed, in lysing solution (2.5 M NaCI, 100 mM EDTA, 10 mM Tris, pH 10.5, 1% TritonX-100, and 10% dimethyl sulfoxide)

for 1 hat48"C.The slides were then eiectrophorosed for 20-30 min at 25 V, The slides after electrophoresis were washed with 0.4 M Tris (pH 7.5) and stained with ethidium bromide (1 mg/ml) and observed under a fluorescent microscope. The control and cisplatin treated MCF-7 cells show no comets indicating the DNA is intact but in compound [Formula I] treated cells show DNA damage as comets with tails of different size.
The morphological analysis for apoptosis was done by Hoechst staining (Figure 4). Figure: 4 Growth inhibitory effects due to Compound [Formula I] treatment was through apoptosis. Morphological analysis of MCF-7 cells using Hoechst 33342 (Molecular Probes, USA) stain, showed bright blue fluorescence after 4 h of Compound [FormulaI] treatment but fluorescence was negligible in control cells.
To identify the mitochondrial involvement during apoptosis, the mitochondrial membrane potential loss was investigated through BD mitochondrial Apoalert ^^ staining kit as per manufactures protocol (Figure 5).
Figure: 5 Compound [Formula I] showed loss of mitochondrial membrane potential (A\|/m). The cells with and without drugs were washed with serum-free medium and then once with IX assay buffer, supplied with the kit. Then the cells were incubated with the MitoSensorTM (ApoAlertTM Mitochondrial Membrane Sensor Kit, Clontech, PaloAlto, CA) (final concentration, 5 mg/mL) at 37°C in an incubator for 15-20 min. After rinsing the cells with serum-free medium, the cells were viewed under fluorescent microscope. MitoSensor dye aggregates in the mitochondria of healthy cells and fluoresces red. In apoptotic cells, mitochondrial potential is altered, and the dye cannot accumulate in mitochondria; it remains in the cytoplasm and fluoresces green. The control cells showed no loss in A\|/m. Treatment with indicated concentrations of compound [Formula I] up to 4 h shows Avj/m as green fluorescence of the unpolymerized dye. Compound [Formula 1] treated cell shows intense loss in A\|/m.
The apoptotic cells were observed in bright green fluorescence in contrary, the non-apoptotic cells showed red fluorescent bodies, which are representative of live mitochondria in control cells. The cells are analyzed with Nikon ^^ fluorescent

microscope and acquired with Image Master ^"^ so ftw are. Im m u n o b lo ttin g for p5j, ERa.PARP and p actin (as control) (Figure 6 A, B & C). Using lysate from treated cells against untreated cells as control were done so as to confirm the apoptotic pathway.
Figure: 6A. Compound [Formula I] showed maximum p53 expression. Cells were treated with the indicated concentrations of Compound [Formula I] diluted in DMEM containing 2.5% FBS for 6, 12, 18 & 24 h. The cells were collected for whole cell lysate preparation as described in Figure 3A. Fifty micrograms of protein was separated on a 10% PAGE, transferred onto a nitrocellulose membrane, and probed with monoclonal anti-p53 antibody (Sigma Aldrich, USA) at 4°C overnight, followed by incubation in alkaline phosphatase-labeled antimouse IgG (1 :3000 dilution). The blot was developed by incubating in the substrate solution (NBT/BCIP) for 5 min. The blot was scanned and photographed with gel documentation system. Maximum p53 expression was observed at 8 h which shows that p53 dependant apoptosis is taking place in these cells in response to compound[Formula I].
Figure: 6B. Apoptosis take place through an ER a independent pathway. Cells were treated with the indicated concentration of compound [Formula I] diluted in D.M.E.M containing 2.5% FBS. Western blot were performed using anti-estrogen receptor a antibody ((Santa Cruz, CA) (1: 500 dilution). There was no significant difference in the expression of ERa in treated samples when compared to control.
Figure: 6C. Compound [Formula I] showed maximum PARP expression. MCF-7 cells were treated with compound [Formula I] for 6, 12, 18 & 24 h. Whole cell lysate were isolated from the samples using radioimmuno precipitation buffer. Western blot were performed using anti-PARP antibody (Sigma Aldrich, USA) (1: 500 dilution). The active cleaved 85 kDa band was observed to be increasing with time, as a result of apoptosis.

Example: 4
The cell viability assay results of the copper complexes with homologous structures and their respective ligands are shown in Figure 7 A and Figure 7 B.
Figure: 7 Cell viability assay of complexes (Figure 7A) and ligand (Figure 7B):
MCF-7 cells were seeded into a 96-well microtitre plate (5000 cells/well) in DMEM with 10% FBS. Once the cells had attached, the complexes and ligands were added at
0
different concentrations in quadruplicate samples and incubated for 48 h at 37 C. After incubation, the complexes and ligands containing medium was removed and equal volumes of fresh medium were added along with 20 |4,1 of 3-(4-5dimethylthiozol-2-yl) 2-5diphenyl-tetrazolium bromide (MTT, USB Amersham Biosciences, Buckinghamshire,UK) (5 mg/mL) to each well. The yellowish MTT was reduced to dark colored formazan by viable cells only. After incubating for 3 h, the formazan crystals formed were solubilized with MTT lysis buffer (20% SDS in 50%
0
dimethyl formamide). The plates were kept, protected from light, overnight at 37 C in an incubator. The color developed was quantitated with an ELISA plate reader (Bio RadSystems, Hercules, CA) (measuring wave length: 570 nm, reference wave length: 630 nm). All results are expressed as the mean percentage of control ± S.D. of quadruplicate determinations from three independent experiments.
Copper complexes such as CSl, CS7, and CSS showed antiproliferative effect in which CSS showed more effect, which can be compared to compound [Formula I]. All other complexes CP5, CS4 and CTl showed a proliferative effect hence cannot be considered as anticancer targets. In the case of ligands used in MTT assay all of them
1 2
such as H L, H L and H L showed proliferative effect.
2 2 2
CSl, CS4, CS7 and CSS - Cu (II) complexes of H^L
1 CP5, CP7 - Cu (II) complex of H^L
2
CTl -Cu (II) complex of H L
H L- is chemically N"- N'"- bis (di-2-pyridylmethylene) carbonic dihydrazide or A^'-
[(lZ)-dipyridin-2-ylmethylene]-A'"'-(dipyridin-2-ylmethylene) carbonohydrazide or bis (di-2-pyridyl ketone) carbohydrazone

1 H L - is chemically (2EJ)-N- [(l£)-quinolin-2-ylmethylene]-2-(quinolin-2-
ylmethylene) hydrazinecarbohydrazonothioic acid
2
H L - is chemically (2£)-iV-[(l£)-quinolin-2-ylmethylene]-2-(quinolin-2-ylmethylene) hydrazinecarbohydrazonic acid.
Cells were seeded into a 96-well microtitre plate (5000 cells/well) in DMEM with 10% FBS. Once the cells had attached, the complexes and ligands were added at
0
different concentrations in quadruplicate samples and incubated for 48 h at 37 C. After incubation, the complexes and ligands containing medium was removed and equal volumes of fresh medium were added along with 20 |il of 3-(4-5dimethylthiozol-2-yl) 2-5diphenyl-tetrazolium bromide (MTT, USB Amersham Biosciences, Buckinghamshire, UK) (5 mg/mL) to each well. The yellowish MTT was reduced to dark colored formazan by viable cells only. After incubating for 3 h, the formazan crystals formed were solubilized with MTT lysis buffer (20% SDS in 50% dimethyl formamide). The plates were kept, protected from light, overnight at
0
37 C in an incubator. The color developed was quantitated with an ELISA plate reader (Bio RadSystems, Hercules, CA) (measuring wave length: 570 nm, reference wave length: 630 nm). All results are expressed as the mean percentage of control ± S.D. of quadruplicate determinations from three independent experiments. The differences among the mean values were analyzed using 1-way ANOVA followed by Tukey's post hoc /- test analysis. The one-way ANOVA revealed that the average mean values of cell survival differed significantly as a fianction of concentration of complexes and ligands (P> 0.05).
Copper complexes such as CSl, CS7, and CSS showed antiproliferative effect in which CSS showed more effect, which can be compared to compound [Formula I]. All other complexes CP5, CS4 and CTl showed a proliferative effect hence cannot be considered as anticancer targets. In the case of ligands used in MTT assay all of them such as H2L' H2L and H2L^ showed proliferative effect.

CSl. CS4. CS7 and CSS - are Cu TIP complexes of H^L
CP5. CP7 - are Cu (ID complexes of H^L'
CTl - are Cu (11) complex of H^L^
H7L - is chemically N"- N'"- bis (di-2-pvridvlmethvlene) carbonic dihydrazide or
A^'-[nZ)-dipyridin-2-ylmethvlene]-iV"-(dipyridin-2-ylmethylene) carbonohydrazide or bis (di-2-pyridyl ketone) carbohydrazone
H^L' - is chemically (2E)-N- [(lF)-quinolin-2-ylmethylene]-2-(quinolin-2-ylmethylene) hydrazinecarbohydrazonothioic acid
H^L^- is chemically (2£")-A^-[('l£^-quinolin-2-ylmethylene]-2-(quinolin-2-ylmethylene) hydrazine carbohydrazonic acid
Example: 5
Cell proliferation assay for the combination of Compound [Formula I] with Cisplatin and Etoposide. The results are provided in figure: 8 Figure: 8 Cell proliferation assay for the combination of Compound [Formula I] with Cisplatin and Etoposide: The differential effect of compound [Formula 1] in MCF-7 cell line in combination with standard chemotherapeutic drugs, etoposide and cisplatin. These cells were grown in 96-well plate, and then treated with or without the indicated concentrations of the compounds for 48 h. In combination therajjy the first drug was given an initial pretreatment of 4 h. At the end of treatment, cell viability was assessed by MTT assay. Cell viability was calculated as percentage over control and all results are expressed as the mean percentage of control ± S.D. of quadruplicate determinations from three independent experiments. LDgo concentrations were used for combination study. The graph clearly suggests that combination of cisplatin and compound [Formula I] was more effective than combination of Etoposide with compound [Formula I].
The results of the sequential studies are provided in figure 8, wherein it is evident that compound [Formula I] when treated initially, followed by treatment with cisplatin helped in reducing the percentage viability of cells over control. Thus, when compound [Formula I] administered in combination with cisplatin helped in achieving better results and hence found to have synergistic effect. The sum of the effect

obtained by this combination is significantly greater than when the drugs treatea independently.
26

We claim:
1) A compound of structural formula-I

2) The compound as claimed in claim 1, wherein said compound is a metal
complex having molecular formula [Cu2 (HL) (HSO4) • H2O] S04- 6 H2O.
3) The compound as claimed in claim 2, wherein said metal is selected from a
group comprising transition metals Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu and Zn,
preferably Cu.
4) The compound as claimed in claim 2, wherein said metal is coordlnately
complexed with a ligand N"- N'"- bis (di-2-pyridylmethylene) carbonic
dihydrazide.
5) A composition comprising compound of structural formula-I


and pharmaceutically acceptable additive(s).
6) The composition as claimed in claim 5, wherein said composition is for
management of cancer.
7) The composition as claimed in claim 5, wherein the additives are selected
from a group comprising granulating agents, binding agents, lubricating
agents, disintegrating agents, sweetening agents, coloring agents, flavoring
agents, coating agents, plasticizers, preservatives, suspending agents,
emulsifying agents and spheronization agents.
8) The composition as claimed in claim 5, wherein said composition is
formulated into various dosage forms selected from a group comprising tablet,
troches, lozenges, aqueous or oily suspensions, dispersible powders or
granules, emulsion in hard or soft gel capsules, syrups and elixirs.
9) A synergistic composition for management of cancer, said composition
comprising compound of structural formula I

Formula I
at a concentration ranging from 1 |JM to 10 (xM and cisplatin at a concentration ranging from 5 |xM to 50 fiM , optionally along with pharmaceutically acceptable additives.
10) The composition as claimed in claim 9, wherein the concentration of
compound of structural formula I is about 5 nM and concentration of cisplatin
is about 10 (xM.
11) The composition as claimed in claim 9, wherein said cancer is preferably
breast cancer.


Formula-I
wherein said process comprising steps of:
(a) dissolving ligand and metallic salt in organic solvent to obtain solution;
(b) refluxing and filtering the solution followed by allowing the filtrate to precipitate; and
(c) washing and drying the precipitate to obtain compound of structural formula
I.
13) The process as claimed in claim 12, wherein said metal is in salt form and is
selected from a metal group comprising transition metals Sc, Ti, V, Cr, Mn,
Fe, Co, Ni, Cu and Zn, preferably copper sulphate.
14) The process as claimed in claim 12, wherein the metal is complexed with a
ligand N"- N'"- bis (di-2-pyridylmethylene) carbonic dihydrazide
15) The process as claimed in claim 12, wherein the reflux is carried out for a time
period ranging from 15-25 minutes, preferably about 20 minutes.
16) The process as claimed in claim 12, wherein said organic solvent is alcohol,
preferably methanol.
17) The process as claimed in claim 12, wherein said precipitate is washed with
absolute ethanol and diethyl ether and dried in vacuo.
18) The process as claimed in claim 12, wherein the compound yield is ranging
from 80 % to 90 %, preferably about 83 %.

19) An in-vitro method to inhibit proHferation of cancerous cells by allowing
compound of structural formula 1

Formula -1
optionally along with known anticancer drugs to bind to its cell receptors.
20) The in-vitro method as claimed in claim 19, wherein said cancerous cells are
breast cancer cells.
21) The in-vitro method as claimed in claim 19, wherein said anticancer drugs are
Cisplatin and Etoposide, preferably Cisplatin.
22) A compound of structural formula-I, a composition, a synergistic composition,
a process and an in-vitro method as herein described along with accompanying
examples and drawings.