WO2014003541A1 - Copper(ii) -mixed ligand complexes with anticancer properties - Google Patents

Abstract

The present invention discloses pharmaceutical composition having at least one of the series of ternary copper(II) complexes, [Cu(phen)(aa)(H₂O)]ΝO₃·xΗ₂O from 1,10-phenanthroline and amino acid ligands. The said amino acid ligands are glycine (gly), sarcosine (sar), DL-alanine (DL-ala), or 2, 2-dimethylglycine (C-dMg). The series of ternary copper(II) complexes with anticancer properties are further characterized by selectivity towards cancer cells. The anticancer properties are due to increase reactive oxygen species (ROS) generation, selective induction of nuclear double strand DNA breaks (DSBs), cell cycle arrested at G₀/G₁, proteasome inhibition, oxidative DNA damage and topoisomerase I (topo I) inhibition.

Description

Copper (II) -Mixed Ligand Complexes with Anticancer Properties FIELD OF INVENTION

The -present invention relates to copper ( II ) -mixed ligand complexes with anticancer properties, more particularly to copper ( II ) -polypyridyl complexes with anticancer properties by generation of reactive oxygen species (ROS) , proteasome targeting, topoisomerase inhibition and DNA damage. BACKGROUND OF INVENTION

Cancer is one of the major mortality factors globally. A WHO study has predicted that global could increase by 50% to 15 million by 2020. high mortality

rate, improvement in early detection and treatment leads to better survival rates for people with cancer.

Breast cancer is the most prevalent cause of cancer deaths among women. Although the technology and scientific advancement has led to lower mortality, rate in breast cancer, metastatic breast cancer remains incurable with low survival rates. In addition, nasopharyngeal cancer (NPC) is the most common head and neck cancer in Malaysia and some countries in Asia. Chemotherapy with cisplatin and 5- fluorouracil , radiotherapy or both in combination have been used as treatment for NPC. However, the current treatment regimens shows low efficacy in advanced and metastatic NPC. A new treatment which uses monoclonal antibody drug targeting epidermal growth factor receptor (EGFR) has elicited only positive response from 13% of patients although the treatment was well tolerated with minimum adverse effect. In view of the shortcoming of the current treatment therapies and drugs available, there is a need to explore new drugs which are more effective and safe.

Anticancer drug development has been focused on platinum- based compounds such as cisplatin. In recent years, researchers use copper compounds as alternatives that are less toxic than platinum-based anticancer drugs yet as equally effective in killing cancer cells. However, some copper compounds are not selective.

By understanding the interaction of metal complexes with DNA and proteins, it has been discovered that metal complexes may selectively bind DNA to induce death of cancer cells. For example, cisplatin selectively and covalently binds to nucleobases with the availability of two cis-labile ligands leading to inter- and intra-strand linkages involving only G-bases. Inhibition of restriction enzymes that binds to short, specific nucleotide sequences and cleaves DNA strands at specific site(s), suggests that the metal complexes bind at these specific sequences and have similar selective binding .

Another new approach is by targeting topoisomerases with metal complexes. Topoisomerases are produced when needed for certain biological processes involving DNA, and are degraded by proteasome when not needed. They are important enzymes that modify the topological state of DNA by introducing transient breaks in a number of DNA strands. Mitosis, particularly DNA transcription and replication, require topoisomerases. Topoisomerase I (topo I) inhibitors are established as one of the most widely used anticancer drugs clinically for wide range of tumour cells. Topo I inhibitors are also found to be DNA sequence-selective as evidenced by stabilization of topoisomerase-DNA cleavage complexes at specific sites.

Another molecular target in anticancer drugs development is G-quadruplex, which is a non-B form DNA structure. G- quadruplex DNA motifs, such as TTAGGG-repeats, occur at DNA telomeric ends throughout the human genome. Extensive research into, interaction of small molecules with G- quadruplex have been reported, however similar reports involving metal complexes are less numerous. Another approach employed in killing of cancer cells is by targeting the redox processes and modulating the level of reactive oxygen species (ROS) . The characterized high copper levels and high oxidative stress in cancer cells have been proposed to achieve selective cancer treatment. When an ROS threshold is reached, it initiates cell apoptosis. Since cancer cells are more susceptible in ROS increase, this allows a selective targeting of cancer cell.

Proteasome inhibition is also another means to kill cancer cells. Proteasome is an abundant multi-catalytic protein complex in eukaryotic cells that degrades intracellular proteins that are no longer needed or are mis-folded or damaged. Proteasome controls levels of proteins that are biologically essential for activities such as cell growth, cell-cycle progression and apoptosis. Studies show that cancer cells give higher sensitivity to proteasome inhibition compared to normal cells therefore it seems feasible to target the proteasome for selective cancer therapy. Despite various compounds exhibiting proteasome inhibition, bortezomid is the only drug that has been approved for clinical treatment and mainly for relapsed/refractory multiple myeloma. However, this cancer drug is not selective and has numerous adverse side effects including haematological and neurological complications. In order to increase the reliability of copper compounds in anticancer drug development, there remains a need to develop copper compounds that are therapeutically effective, safe and highly selective.

SUMMARY OF THE INVENTION

The present invention discloses a series of ternary copper(II) complexes, [Cu(phen) (aa) (H₂0) ] Ν0₃·χΗ₂0, (abbreviated as Cu (phen) (aa)N0₃) from 1 , 10-phenanthroline and amino acid ligands. The amino acid ligands are glycine (gly) , sarcosine (sar) , DL-alanine (DL-ala), or 2,2- dimethylglycine (C-dMg) · The series of ternary copper (II) complexes with anticancer properties are further characterized by selectivity towards cancer cells. The anticancer properties are due to increase reactive oxygen species (ROS) levels, selective induction of nuclear double strand DNA breaks (DSBs) , induction of cell cycle arrested at Go/Gl, proteasome inhibition, oxidative DNA damage and topoisomerase I (topo I) inhibition.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates structure of [Cu (phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 (gly: Rl=R2=R3=H; DL-ala: Rl= CH₃; R2=R3=H, ; C-dMg: Rl= R2= CH₃, R3= H; sar: R1=R2=H; R3= CH₃) according to an embodiment herein;

Figure 2a illustrates the morphological changes in breast cancer MDA-MB-231 cells (A) and breast epithelial MCFIOA (B) treated with Cu(phen) (DL-ala) 0₃ at different concentrations as compared to untreated cells;

Figure 2b illustrates the morphological changes in nasopharygeal carcinoma HK1 and nasopharyngeal epithelial NP69 treated with Cu (phen) (DL-ala) N0₃ at different concentrations as compared to untreated cells;

Figure 3a illustrates the dose response curve of the antiproliferative activity (% cell viability) of (a) Cu(phen) (DL-ala) N0₃, (b) Cu(phen) (sar)N0₃, (c)

Cu(phen) (gly)N0₃, (d) Cu (phen) (C-dMg) N0₃, (e) Cu(80HQ)₂ in nasopharyngeal carcinoma HKl and nasopharyngeal epithelial NP69 cells at 24 h;

Figure 3b illustrates the dose response curve for different concentrations of Cu(phen) (C-dMg) N0₃ against various cell lines ;

Figure 4 illustrates the mean IC₅o of different cell lines treated with Cu(phen) (c-dMg)N0₃;

Figure 5a illustrates the percentage of apoptotic cells after treatment with 5 μΜ ternary copper (II) complexes and Cu(80HQ)₂ at 24h in breast cancer MDA-MB-231 and breast epithelial MCFIOA cell lines; Figure 5b illustrates the percentage of apoptosis induced by [Cu(phen) (aa) (H₂0) ]Ν0₃·χΗ₂0 (1-4) (1: aa=gly; 2: aa=DL-ala; 3: aa=C-dMg; 4: aa=sar) and Cu(80HQ)₂ in nasopharyngeal carcinoma HK1 cancer cells and nasopharyngeal epithelial NP69 normal cells at 24 h incubation;

Figure 6a illustrates the radical oxygen species (ROS) production induced by ternary copper (II) complexes treatment for 6 h;

Figure 6b illustrates the radical oxygen species (ROS) production induced by ternary copper (II) complexes treatment for 24 h;

Figure 7a illustrates flow cytometry data for radical oxygen species (ROS) in MDA-MB-231 cells untreated or treated with (A) Cu(phen) (DL-ala)N0₃, (B) Cu(phen) (sar)N0₃, (C) Cu(phen) (gly)N0₃, (D) Cu(phen) (C-dMg)N0₃ for 6 h;

Figure 7b illustrates flow cytometry data for radical oxygen species (ROS) in MCF10A cells untreated or treated 6h with (A) Cu(phen) (DL-ala)N0₃, (B) Cu(phen) (sar)N0₃, (C) Cu(phen) (gly)N0₃, (D) Cu(phen) (C-dMg)N0₃ for 6 h;

Figure 7c illustrates flow cytometry data for radical oxygen species (ROS) of MDA-MB-231 cells untreated or treated with (A) Cu(phen) ( DL-ala) N0₃, (B) . Cu (phen) (sar) 0₃, (C)

Cu(phen) (gly)N0₃, (D) Cu(phen) (C-dMg)N0₃ for 24 h; Figure 7d illustrates flow cytometry data for radical oxygen species (ROS) of MCFIOA cells untreated or treated with (A) Cu(phen) (DL-ala)N0₃, (B) Cu(phen) (sar)N0₃, (C)

Cu(phen) (gly)N0₃, (D) Cu(phen) (C-dMg)N0₃ for 24 h;

Figure 8a illustrates the cell cycle distribution of MDA-MB- 231 cells in the absence or presence of 5 μΜ Cu(phen) (aa)N0₃ complexes at 24 h;

Figure 8b illustrates the cell cycle distribution of MCFIOA cells in the absence or presence of 5 μΜ Cu(phen) (aa)N0₃ complexes at 24 h;

Figure 9 illustrates the fluorescent intensity of γΗ2ΑΧ production induced by Cu(phen) (aa)N0₃ complexes in MDA-MB-231 cells ;

Figure 10a illustrates the immunofluorescence staining for γ-Η2ΑΧ in MCFIOA ,· cells treated with Cu(phen) (aa)N0₃ complexes compared to control cell;

Figure 10b illustrates the immunofluorescence staining for Y-H2AX in MDA-MB-231 cells treated with Cu(phen) (aa)N0₃ complexes compared to control cell;

Figure 11 illustrates the inhibition of chymotrypsin-like activity of 20S proteosome by various Cu(phen) (aa)N0₃ compounds, Cu(80HQ)2 and epoxomicin as control;

Figure 12a _t illustrates Western blot analysis for ubiquitinated protein and ΙκΒ expression (20 μg of total protein lysate/lane) obtained from human breast cancer MDA- MB-231 cells, treated with 1 μΜ and 10 μΜ of Cu (phen) (aa)N0₃ complexes for 24 h;

Figure 12b illustrates the proteasome inhibition by Cu(phen) (c-dMg) N03 on a stably transfected HK1 expressing Ub-G76V-GFP at 9h treatment;

Figure 13 illustrates the gel electrophoresis results of incubating human topoisomerase I with pBR322 in the absence or presence of [Cu(phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 complexes 1-4 (a: gly, 1; b: DL-ala, 2; c: C-dMg, 3; d: sar, 4) ;

Figure 14 illustrates the sets of CD spectra of G-quadruplex 5 ^X-AG3 (T2AG3) 3-3' in the absence and presence of different concentrations of [Cu(phen) (aa) (H₂0)]⁺ (a: gly, 1; b: DL-ala, 2; c: C-dMg, 3; d: sar, 4);

Figure 15a illustrates the positive-ion ESI mass spectrum showing experimental isotopic distribution of

[Cu(phen) (gly)]⁺ species and their theoretical calculated m/z;

Figure 15b illustrates the positive-ion ESI mass spectrum showing experimental isotopic distribution of [Cu(phen) (DL- ala)]⁺ species and their theoretical calculated m/z;

Figure 15c illustrates the positive-ion ESI mass spectrum

i

showing experimental isotopic distribution of [Cu(phen) (C- dMg) ]⁺ species and their theoretical calculated m/z; Figure 15d illustrates the positive-ion ESI mass spectrum showing experimental isotopic distribution of

[Cu(phen) (sar) ]⁺ species and their theoretical calculated m/z;

Figure 16a illustrates the effects of Cu (phen) (DL-ala)N0₃ on weight of male and female mice after administration;

Figure 16b illustrates the inhibition of tumour growth in mice treated with Cu(phen) (DL-ala)N0₃ ; and

Figure 17 illustrates result from the NCI-60 single dose (10 μΜ) screening of 60 cancer cell lines for Cu(phen) (C-d g)N0₃ as mean growth percent.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding; of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

The present invention discloses a series of ternary copper (II) complexes, [Cu(phen) (aa) (H₂0) ] N0₃*xH₂0 from 1,10- phenanthroline and amino acids namely glycine (gly) , sarcosine (sar) , DL-alanine (DL-ala),. or 2 , 2-dimethylglycine (C-dMg) . The structure of [Cu (phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 is disclosed in Figure 1. The choice of Rl, R2 and R3 depends on the choice of amino acid ligand. Rl, R2 and R3 may be selected from H or alkyl groups. The x is a natural number. In one embodiment, the amino acid ligand is Glycine (gly) which has R1=R2=R3=H. In another preferred embodiment, amino acid ligand is Alanine (DL-ala) which has Rl= CH₃; R2=R3=H.In yet another embodiment, amino acid ligand is dimethyl glycine (C-dMg) which has Rl= R2= CH₃, R3= H. In yet another preferred embodiment, amino acid ligand is sarcosine (sar) which has R1=R2=H; R3= CH₃. The effect of the position and number of methyl substituent ( s ) in the auxiliary ligand and methylated glycine derivative, of the mixed-ligand copper (II) complexes with 1 , 10-phenanthroline in their crystal structure has been illustrated by their, binding with G-quadruplex and DNA duplexes, their inhibition of restriction enzymes and topoisomerase I (topo I), and their differential cytotoxicity towards nasopharyngeal cancer (HK1), metastatic breast cancer (MDA-MB-231 ) , and their respective normal, i.e. non-cancer cells (NP69 and MCF10A) .

The series of ternary copper (II) complexes have been characterized by elemental analysis, X-ray crystallography, UV-visible spectroscopy, molar conductivity, and positive- ion electrospray ionization-mass spectra (ESI-MS) . Cu(80HQ)₂, a known proteasome inhibitor, was used for comparison. All complexes may bind selectively to DNA by intercalation and electrostatic forces, and inhibit topo I. Gel electrophoresis, _. fluorescence quenching, and restriction enzyme inhibition assays were conducted to study the binding interaction, binding affinity and selectivity of these complexes for various types of B-form DNA duplexes and G- quadruplex.

Morphological study, apoptosis assay, 3- (4, 5-

Dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay or ( 3- ( 4 , 5-dimethylthiazol-2-yl ) -5- ( 3- carboxymethoxyphenyl ) -2- ( 4-sulfophenyl ) -2H-tetrazolium)

(MTS) assay and γΗ2ΑΧ (intracellular DNA damage) assays showed that the copper complexes of the present invention are cytoselectijve and inhibit cell proliferation in a dose- dependent manrier. The reactive oxygen species (ROS) i

generated in dancer cells by the copper complexes of the

I present invention was distinctly higher than those in normal cells. Treated cancer cells are further characterized by cell cycle arrest at G₀/Gi by copper (II) complexes, with double strand DNA breaks (DSBs) detected in cancer cells. Detection of DNA damage-induced phosphorylation of H2AX at Serl39 in MDA-MB-231 cells suggested that the copper compounds induced double-strand breaks and may activate signaling pathways leading to apoptosis. These findings indicate that ternary copper (II) complexes kill cancer cells by inducing ROS production, DNA damage and cell cycle arrest at Go/Gi phase. In the assay using 2 ' , 7 ' -dichlorofluorescein diacetate (DCFH-DA) , it was found that the compounds of present invention induced significant increase in ROS production in cancer cells compared to non-cancer and untreated cells.

Accumulation of ubiquitinated proteins and ΙκΒ (NF-κΒ inhibitor) in MDA-MB-231 cells via Western blotting suggest proteasome inhibition. All complexes showed slight inhibition of chymotrypsin-like activity of purified 20S proteasome. The compounds could inhibit proteasome in the cancer cells. Thus, the anticancer properties of these compounds involve multiple pathways and are found to exhibit significant selectivity for cancer over non-cancerous cells. Chemistry Each of the tested copper complexes,

[Cu(phen) (aa) (H₂0) ] Ν0₃·χΗ₂0, have a square pyramidal [Cu(phen) (aa) (H₂0)]⁺ cation as shown in Figure 1 and an uncoordinated nitrate anion with or without lattice water molecule. The [Cu(phen) (aa) (H₂0) ] NO3 ·χΗ₂0 where amino acids (aa) are glycine (gly; 1) and methylated glycine derivatives like 2-methylglycine (DL-ala, 2), 2 , 2-dimethylglycine (c-dMg; 3), sarcosine or N-methylglcyine (sar; 4); and x = 0, 1½, 2½ were similarly prepared. In one embodiment, [Cu (phen) (DL-ala) (H₂0) ] Ν0₃·2½Η₂0 are prepared by adding L-alanine to a water-ethanol mixture of Cu (NO3) 2* 3H₂0 and 1 , 10-phenanthroline . The resulting dark blue solution pH was raised to pH 8.1 with NaOH solution before heating in water-bath at 45 °C overnight to obtain the final product as blue needle crystals. As shown in figures 14a-14d, whereby 14a comprises amino acid of gly; 14b comprises amino acid of DL-ala; 14c comprises amino acid of C-dMg; and 14d comprises amino acid of sar. The positive- ion ESI-MS spectra showed only m/z peaks due to [Cu(phen) (aa) ]⁺ with the correct isotopic ratio, thus confirming the presence of the copper (II) complex cations in solution. No copper (II) species arising from the dissociation of the coordinated amino acid was detected in each ESI-MS spectrum. In other words, the solid [Cu(phen) (aa) (H₂0) ] N0₃. xH₂0 dissolved in aqueous solution to yield the N0₃ ⁺, and [Cu(phen) (aa) (H₂0)]⁺. The latter is stable for a long period of time. Repeated attempts yielded suitable crystal for X-ray crystal structure analysis. ax

(Molar absorbtivity )

Compound O h 1 h 24 h

X_max / nm Xmax/ nm _max/ nm

(s/M-'cm^"1) (s/M^{' 1}) (e/M^'W).

Cu(N0₃)₂ 799.50 (12.0) 803.03 (12.1) 839.14 (14.4)

1 615.99 (48.4) 618.89 (52.1) 620.92 (47.8) 2 615.56 (44.4) 611.98 (52.4) 615.73 (63.2)

3 612.03 (59.9) 608.92 (84.1) 610.50 (79.0)

4 619.96 (60.7) 620.00 (62.5) 624.04 (61.0)

Table 1 Table 1 above shows the visible spectral data of l x l CT³ M Cu(N0₃ ) 2 and complexes 1-4 in water-methanol (1:1 v/v) . Whereas in Table 2 shows the molar conductivity (Ω-l cm² mol^"1) of complexes 1-4 and other compounds at 1^χ10^~3 M.

Compounds Time

O h 1 h 24 h

1 53.10 53.37 55.92

2 51.77 52.13 54.61

3 56.13 56.53 58.05

4 56.65 56.78 58.05

Cu(N0₃)₂ 143.35 143.18 143.72

Phen 1.21 1.33 1.61

L-ala 0.85 0.92 1.12

Sar 1.12 1.28 1.59

Gly 1.03 1.11 1.37

C-dmg 1.42 1.51 1.87

KCf 1413 1412 1402

Table 2 Anticancer activity / selectivity - evidence from cell morphology

Breast cancer MDA-MB-231, breast epithelial MCF10A, nasopharyngeal cancer HKl and nasopharyngeal epithelial NP69 cells were treated with compounds at different concentrations and then were examined and imaged for morphological evidence of induced apoptosis. Figures 2a and 2b show the morphological changes in MDA-MB-231 cells (A) , MCF10A (B) , HKl and NP69 treated for 24 h with Cu(phen) (DL- ala)N0₃ at different concentrations as compared to untreated cells as observed under microscopic magnification of 400^χ. All pictures are typical of three independent experiments each performed under identical conditions. Arrow 1 indicates condensation of chromatin and Arrow 2 indicates membrane bleb. Untreated MDA-MB-231 cells maintained their morphology as robust, elongated, adherent, and showed cellular crowding which is suggestive of normal proliferation. In contrast to cells treated with ternary copper (II) complexes, it appeared slow growing, shrinking, characteristic apoptotic blebbing and forming islets of more rounded cells with dependence on dose and incubation period of ternary copper (II) complexes. The results show that all the [Cu (phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 are cancer-targeting at low concentration. However, the known anticancer Cu(80HQ)₂ induced apoptotic morphological changes in both MDA-MB-231 and MCF10A cells at low concentration, suggesting its indiscriminate nature and severe toxicity to both cell lines.

Anticancer activity / selectivity - evidence from MTT/MTS assay Both DA-MB-231 and MCF10A cells were harvested and seeded from exponential growth phase cultures at density 1 ^χ 10⁵ cells/mL in 100 pL medium per well in a 96-well plate, followed by incubation at 37 °C in a 5 % C0₂ overnight for cell attachment. The media incubated in 96-well plate was replaced with fresh media with or without test compounds before incubation for drug to take effect. Cells were treated at different drug concentrations of

[Cu(phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 complexes. After drug exposure, 20 pL MTT solution is added into each well then followed by incubation for 4 h (37 °C, 5 % C0₂) to allow metabolism of MTT. Then, 100 μΤ dimethyl sulfoxide (DMSO) per well is added to dissolve the formazan produced by the metabolism of MTT by viable cells and the optical density (OD) is then measured at . a wavelength of 570 nm with background subtraction at 630 nm. The MTS solution works the same way as MTT solution. Thus either of both solutions may be used in conducting cell viability test. Every experiment includes a set of negative controls (untreated cultures), blank wells -without cells -and a set of positive control [Cu(80HQ)2j . Cell viability of treated samples is calculated in reference to the untreated control that is defined as 100% viable. The degree of cell proliferative inhibition is expressed as the percentage of the untreated cell control using the following formula:

Cell viability (%) =F [100 x (Mean optical density of sample) / (Mean optical density of control) ] .

IC₅₀ values (the concentration of the complex causing 50 % growth inhibition) are derived from dose response curves. The same experiment is conducted for nasopharyngeal cancer cell line HK1 and non-cancer nasopharyngeal epithelial cell line NP69 where all experiments were performed in triplicates and repeated three times. As shown in figure 4 and table 3, the mean IC50 of different cell lines treated with [Cu(phen) (c-dMg) (H₂0)]N0₃ is shown, indicating the efficiency of [Cu(phen) (aa) (H₂0) ] N0₃ ·χΗ₂0 complexes in inhibiting cell growth.

Panel Cell Line IC50 Std. Dev. Std. Error

Liver Cancer HepG2 1.151E-05 6.793E-07 3.922E-07

HCT116 6.997E-06 2.286E-06 1.320E-06

SW48 1.912E-05 2.842E-06 1.641E-06

Colorectal

Cancer SW480 1.100E-05 4.111E-06 2.374E-06

HL60 2.552E-06 4.080E-07 2.356E-07

Leukaemia Jurkat 4.288E-06 4.737E-07 2.735E-07 K562 1.292E-05 3.508E-06 2.025E-06

Lymphoma Namalwa 2.248E-06 1.154E-06 6.661E-07

MCF7 1.727E-06 4.005E-07 2.312E-07

MDA-MB-231 4.865E-06 1.720E-06 9.932E-07

Breast Cancer T47D 2.480E-06 1.731E-07 9.996E-08

C-666-1 3.601E-06 7.876E-07 4.547E-07

HK1 4.971E-06 7.256E-07 4.189E-07

Nasopharyngeal

Cancer Honel 4.276E-06 1.016E-06 5.868E-07

PC9 4.435E-06 8.245E-07 4.761E-07

Lung Cancer A549 1.119E-05 2.218E-06 1.280E-06

Ovarian Cancer SKOV-3 1.484E-05 5.470E-06 3.158E-06

Cervical Cancer HeLa 1.968E-06 3.134E-07 1.810E-07

HK2 1.842E-05 1.290E-06 7.448E-07

>2.500E-

HFK Clone 22 05

>2.500E-

HFK Clone 398 05

Non-malignant NP69 1.261E-05 1.644E-06 9.491E-07

Table 3

MTT/MTS assay showed cellular proliferation inhibition at different concentrations of ternary copper (II) complexes, and decrease in formazan (MTT metabolite) percentage is directly related amount of viable cells. Figure 3a depicts a dose response curve of the anti-proliferative activity (% cell viability) of (a) Cu(phen) (DL-ala)N0₃, (b) Cu(phen) (sar)N0₃, (c) Cu (phen) (gly) N0₃, (d) Cu(phen) (C- dMg)N0₃, (e) Cu(80HQ)₂ in HK1 and NP69 cells at 24 h. Cell viability' is expressed as relative activity of control cells (100%). Results are the mean of at least three independent experiments and error bars show the standard error of the mean. MTT/MTS assay results shown in Figure 3a indicates that DA-MB-231 and MCFIOA treated with copper (II) complexes is dose-dependently decline in viability as concentration increases. Figure 3b shows the dose response curve for different concentrations of Cu(phen) (C-dMg) N0₃ against various cell lines such as colorectal, leukaemia, lymphoma, breast, nasopharyngeal (NPC) , lung, ovarian, cervical and liver cancer. The dose response curve for Cu(phen) (C-dMg) 03 against Adriamycin resistant leukaemic K562/ADM cells was similar to the parental leukaemic K562 cells showing that the compound was effective against resistant cancer cells. Fig 3b and Fig 4 showed that in contrast to its action on cancer cells, the compound Cu(phen) (C-dMg) N03 was less toxic to non-cancer cells/"non-malignant cells" (kidney epithelial HK2, keratinocytes HFK 22 and HFK 398 and nasopharyngeal epithelial NP69 cells) .

The [Cu(phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 complexes are distinctly more antiproliferative in MDA-MB-231 cancer cells compared MCFIOA normal cells, which shows high selectivity on cancer cells. In addition, table 4 shows the IC₅₀ values of 1-4 for HK1 were in the range 2.2 - 5.2 μΜ while the corresponding values for NP69 were greater than 13.0 μΜ indicating that [Cu(phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 complexes are distinctly antiproliferative against HKl than NP69.

Compound ΗΚ1/μΜ ΝΡ69/μ Ratio

NP69 : HKl

Cu(80HQ)₂ 2.0 3.2 1.6

1 5.2 > 25 >4.8

2 3.9 13.8 3.5

3 2.2 > 25 >11.4

4 2.2 > 25 >11.4

Table 4

The MTT/MTS assay result of as a comparison set, Cu(80HQ)₂ shows similar inhibition of cell viability and decreased proliferation for both cell lines in a dose-dependent manner This result further strengthens that Cu(80HQ)₂ is not selective to cancer cells as compared to present invention.

Anticancer activity / selectivity - evidence from apoptosis studies

The four [Cu (phen) (aa) (H₂0) ] N0₃ ·χΗ₂0 complexes of present invention were tested with flow cytometric analysis of apoptosis using annexin V-FITC/PI double staining. Cells were treated with [Cu(phen) (aa) (H₂0) ] N0₃ ·χΗ₂0 complexes and Cu(80HQ)₂ for 24 h where cells without treatment are prepared as control. Apoptosis assay was performed as according to protocol in Annexin V-FITC Apoptosis Detection Kit II and analyzed by fluorescent activated cell sorter. Unstained cells, cells stained with Annexin V-FITC alone and cells stained with PI alone were used as control to set up compensation and quadrants.

Figure 5a shows percentage of apoptotic cells after treatment with 5 μΜ ternary copper (II) complexes and Cu(80HQ)₂ at 24h in MDA-MB-231 and MCF10A cell lines. Results are the mean of three independent experiments and error bars show the standard error of the mean, where * = (p < 0.05), ** = (p < 0.01), *** = (p < 0.005) indicates significant difference in comparison to untreated. Flow cytometric data further confirmed the efficacy and selectivity of [Cu(phen) (aa) (H₂0) ] Ν0₃ ·χΗ₂0 complexes by inducing higher apoptotic cell death in MDA-MB-231 cells, than in MCFlOA cells (which remained at similar levels to untreated MCFlOA) . Figure 5b illustrates percentage of apoptosis induced by 5 μΜ of [Cu(phen) (aa) (H₂0)^" ] Ν0₃·^χΗ₂Ό (1-4) and Cu(80HQ)₂ in HK1 cancer cells and NP69 normal cells at^' 24 h incubation. Where * indicates p < 0.05 for comparing treated cells with untreated cells for the same cell line; and # indicates p < 0.05 for comparing treated cancer cells with treated normal cells with the same compound. The [Cu(phen) (aa) ] Ν0₃·χΗ₂0 complexes are efficient in killing the HK1 cancer cells but less cytotoxic than Cu(80HQ)₂. It is found that normal NP69 cells gives apoptotic percentage that is comparable to that of untreated cells, indicating non-toxicity towards NP69. Cu(80HQ)₂ as control however shows approximately 90 % apoptotic cell death in both cell lines, which indicates indiscriminate high cytotoxicity towards all cell lines (ie cancer and non-cancer cells) . This further proves that copper (II) complexes of the present invention are capable of selectively targeting cancer cells without causing any harm to normal cells.

Anticancer activity / selectivity - evidence from ROS levels studies

Copper complexes are well known for their redox properties and generation of ROS. MDA-MB-231 and MCF10A were treated with various concentrations of [Cu(phen) (aa) (H₂0) ] 0₃ · xH₂0 complexes to determine generation of ROS. The intracellular ROS levels were detected by flow cytometric analysis using the fluorophore, dichlorofluorescin diacetate (DCFH-DA) which is a well-established compound to detect and quantify intracellular H2O2. The DCF fluorescence was measured immediately at 488 nm excitation and 525 nm emission (fluorescein isothiocyanate filter) with 20,000 events recorded. Increase in dichloroflourescein (DCF) fluorescence is directly proportional to oxidation of non-fluorescent dichloroflourescin (DCFH) to DCF, indicating higher ROS level. The amount of ROS was quantified as the mean fluorescence intensity.

Figure 6a and 6b illustrates ROS production induced by ternary copper (II) complexes treatment with different concentrations for 6 h (6a) and 24h (6b) . The average of data obtained in three independent experiments. Results are mean ± S.D. (n=3) . The exposure of MDA-MB-231 to test compounds resulted in significant increase in ROS production compared to untreated cells in dosage and time-dependent manner. Dosage that affects MDA-MB-231 has no statistically significant effect on ROS levels in MCF10A treated cells, suggesting that there is a safe minimum dosage of copper (II) complexes to induce significant ROS increase in cancer cells without sensitizing normal cells in altering its ROS levels. This result therefore supports the use of copper (II) complexes to selectively kill cancer cells, and their further development for selective anticancer therapy.

Figure 7a-7d shows MDA-MB-231 cells were untreated or treated for 6 h (7a) , MCF10A cells were untreated or treated for 6h (7b), MDA-MB-231 cells were untreated or treated for 24h (7c) and MCFIOA cells were untreated or treated for 24h (7d) with (A) Cu(phen) (DL-ala)N0₃, (B) Cu(phen) (sar)N0₃, (C) Cu(phen) (gly)N0₃, (D) Cu(phen) (C-dMg)N0₃. The cells were then stained with DCFH-DA and fluorescence intensity was measured by flow cytometry. An experiment representative of three is shown. Low percentage of apoptotic cells were detected in both untreated MDA-MB-231 and MCF10A cells, demonstrating normal cell viability. MDA-MB-231 cells treated with [Cu(phen) (aa) (H₂0) ] 0₃·χΗ₂0 complexes resulted in increase percentage of apoptotic cells, however the similar pattern is not shown in MCF10A cells indicating the anticancer selectivity of said complexes in cancer cells.

Anticancer activity / selectivity - evidence from cell cycle studies

To determine the effects of [Cu (phen) (aa) (H20) ] 0₃ ·χΗ₂0 complexes on cell cycle distribution, MDA-MB-231 and MCF10A cells were treated with copper (II) complexes and analyzed by flow cytometry. The percentage of cells in each phase of the cell cycle was determined by using Modfit LT software. Figure 8a illustrates the cell cycle distribution of MDA-MB- 231 cells in the absence or presence of 5 μΜ [Cu(phen) (aa) ] Ν0₃·χΗ₂0 complexes at 24 h and Figure 8b illustrates the cell cycle distribution of MCF10A cells in the absence or presence of 5 μΜ [Cu(phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 complexes at 24 h.

The distribution of cells in three major phases of the cycle (Go/Gi, S, G₂/M) in DNA histogram of MDA-MB-231 cells and MCF10A cells after treatment with copper (II) complexes depicted in Figure 8a and 8b. The average percentage of cells in different phases for MDA-MB-231 cells treated with [Cu(phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 complexes were compared with those of untreated cells. Figure 8a and 8b shows the cell .cycle distribution of MDA-MB-231 cells (8a) and MCFIOA (8b) in the absence or presence of 5 μΜ

[Cu (phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 complexes at 24 h. Data are presented as means of the percentage of cells in G₀/Gi, S or G₂/M phase from three independent experiments with S.D. Statistical analysis (p<0.05) shows that the increases in G_o/Gi phase for Cu(phen) (DL-ala)N0₃ and Cu(phen) (C-dMg)N0₃ are significant. This indicates that these two complexes induced cell cycle arrest at Go/Gi and that the methyl substituent at the a-carbon of the amino acid may be important. Treatment with other [Cu(phen) (aa) (H₂0) ] N0₃*xH₂0 complexes resulted in a rise in the percentage of cells in G₀/Gi phase with a concomitant decrease in the percentage S phase and G₂/M phase in MDA-MB-231 but the increases are not statistically significant . The percentage of cells in Go/Gi, S and G₂/M phases for MCFIOA cells treated with [Cu (phen) (aa) (H₂0) ] 0₃·χΗ₂0 complexes were comparable to those for untreated cells. As shown in Figure 8b, the ternary copper (II) complexes did not affect the cell cycle of MCFIOA cells, indicating selectivity on cancer cells. Anticancer activity / selectivity - evidence from intracellular DNA damage studies

Copper (II) complexes of the present invention could generate ROS which causes DNA damage and leads to decrease cell viability, cell cycle arrest and apoptotic cell death. Therefore it is important to identify DNA as one of the targets for [Cu(phen) (aa) (H₂Q) ] Ν0₃·χΗ₂0 complexes.

Intracellular DNA damage that lead to formation of DNA double strand breaks (DSBs) will induce phosphorylated H2AX on Serl39 (denoted as γ-Η2ΑΧ) . Therefore, H2AX phosphorylation on Serl39 was used to investigate DNA DSBs induced by copper (II) complexes in MDA-MB-231 and MCF10A.

Isotype control used in the study was to act as a negative control, to exclude non-specific binding/background fluorescence. Adriamycin, as positive control, could induce Y-H2AX which can be detected in both MDA-MB-231 and MCFIOA cells incubated with tagged specific antibodies against γ- H2AX by immunofluorescence staining and fluorescence microscopy. Three images were taken for analysis with DAPI (4', 6-diamidino-2-phenylindole) channel, FITC channel and phase contrast. Figure 10a and 10b shows the immunofluorescence staining for γ-Η2ΑΧ in MCFIOA and MDA- MB-231 cells treated with 5 μΜ [Cu (phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 complexes compared to control cell. DNA counterstaining is done with DAPI. This indicates that

[Cu(phen) (aa) (H₂0) ] N0₃ ·χΗ₂0 induced selective DNA damage in MDA-MB-231 cells without harming DNA of the MCFIOA cells.

Figure 9 shows the flourescent intensity of γΗ2ΑΧ production induced, by copper (II) complexes in MDA-MB-231 cells. Results are mean ± S.E.M. The γ-Η2ΑΧ fluorescence (green) was . significantly elevated in each individual [Cu(phen) (aa) (H₂0) ] N0₃ · xH₂0-treated MDA-MB-231 cells.

Accordingly, the results suggest that the treatment with copper (II) complexes induced oxidative DNA damage and formation of γ-Η2ΑΧ leading to apoptosis.

Anticancer activity / selectivity - evidence from Proteasome inhibition studies

26S proteasome is a protease complex with chymotrypsin-like activity and its inhibition is a strong apoptosis stimulus. Figure 11 shows direct inhibition of chymotrypsin-like activity of purified 26S proteasome by various copper (II) compounds, as well as Cu(80HQ)₂ and epoxomicin (as positive controls) . To' evaluate inhibition of 26S proteasome, breast cancer MDA-MB-231 cells were treated with

[Cu (phen) (aaj (H20) ]Ν0₃·χΗ₂0 complexes, Cu(80HQ)₂ (as positive control) at / different concentrations for 24 h, followed by Western blotting with specific antibodies. Figure 12a shows Western blot analysis for ubiquitinated protein and ΙκΒ expression obtained from MDA-MB-231 cells treated with 1 μΜ and 10 μΜ of copper (II) ternary complexes for 24 h. β-actin was used as the loading control. The experiment was repeated three times with similar results. Lane 1, untreated; Lane 2, ΙμΜ Cu (phen) (DL-ala) N0₃; Lane 3, ΙΟμΜ Cu (phen) (DL-ala) N0₃; Lane 4, ΙμΜ Cu (phen) (sar) N0₃; Lane 5, ΙΟμΜ Cu (phen) (sar) N0₃; Lane 6: ΙμΜ Cu (phen) (gly) 0₃; Lane 7, ΙΟμΜ Cu (phen) (gly ) N0₃; Lane 8, ΙμΜ Cu(phen) (C-dMg) N0₃; Lane 9, ΙΟμΜ Cu(phen) (C- dMg)N0₃; Lane 10, ΙμΜ Cu(80HQ)₂; Lane 11, 10μΜ Cu(80HQ)₂. It was found that higher intensity and greater accumulation of ubiquitinated proteins occurred when cells were treated with higher concentration (10 μΜ) for all

[Cu(phen) (aa) (H₂0) ] N0₃ ·χΗ₂0 complexes.

Functional proteasome will cause degradation of the ubiquitinated ΙκΒ-α) . Proteasome inhibitor functions to block the degradation of ubiquitinated ΙκΒ-α and this results in the accumulation of ubiquitinated ΙκΒ- . In the experiment, treatment with 10 μΜ [Cu(phen) (aa) (H₂0) ] N0₃ · xH₂0 resulted in increased levels of ubiquitinated Ι Β- (56 kDa) and corresponding reduction in non-ubiquitinated ΙκΒ-α (37 kDa) . This indicates that [Cu (phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 complexes can inhibit proteasome and this contributes to their anticancer property. Proteasome inhibition was also tested in Nasopharyngeal carcinoma HK1 cells using Ub-G76V-GFP as a reporter for proteasome inhibition. Figure 12b shows that on treatment of stably transfected NPC HK1 cells expressing Ub-G76V-GFP at 9h treatment , increased fluorescence was detected indicating the proteasome inhibition by Cu(phen) ( c-d g) 0₃. Anticancer activity / selectivity - evidence from Topoisomerase I assay

Topoisomerases are essential proteins required for biological processes such as DNA replication, transcription and repair, and chromatin assembly by introducing temporary DNA single-strand or double-strand break. Topo I unwind duplex DNA through transient single-strand break, resulting in a more relaxed DNA. To test if copper (II) complexes inhibit of DNA-topo I complex which lead to permanent strand break and lead to apoptotic cell death, Topo I assay was performed.

Supercoiled plasmid DNA pBR322 was used as substrate to study the inhibition of Topo I as the speed of migration of the topoisomers (relaxed DNA) on the electrophoresis gel decreases with the degree of relaxation. Figure 13 illustrates gel electrophoresis results of incubating human topoisomerase I with pBR322 in the absence or presence of 5- 40 μΜ of [Cu (phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 complexes 1-4 (aa for a: gly, 1; b: DL-ala, 2; c: C-dMg, 3; d: sar, 4) where Lane 1 & 5, gene ruler 1 Kb DNA ladder; Lane 2, DNA alone; Lane 3, DNA + 40 μΜ complex (control); Lane 4, DNA + lunit Topo I (control); Lane 6, DNA + 5 μΜ complex + lunit Topo I; Lane 7, DNA + 10 μΜ complex + lunit Topo I; Lane 8, DNA + 20 μΜ complex + lunit Topo I; Lane 9, DNA + 40 μΜ complex + lunit Topo I. It was observed that slower moving bands of topoisomers were formed with increasing concentration of the [Cu(phen) (aa) (H₂0) ] N0₃ ·χ¾0 complexes, indicating they could inhibit the topo I in a concentration-dependent manner. The effect of buffer ionic concentration (NaCl) on the binding affinity was also studied. The binding constant of each copper complex decreased with increasing NaCl concentration which provided evidence that complexes exist as cationic [Cu(phen) (aa) (H₂0)]⁺ and anionic N0₃ ^~ ions in aqueous solution, and they also bind to DNA by electrostatic attraction with the negatively charged phosphate of DNA. At 20 and 40 mM NaCl, the binding constant of 4 decreased more significantly compared to the other copper (II) complexes, suggesting the greater weakening effect of methyl substituent at amino nitrogen than that of the methyl substituent at the -carbon of the coordinated glycine moiety on the DNA binding affinity.

Binding with G-quadruplex The CD spectrum of the G-quadruplex (G-4), annealed from a human telomeric 22-base oligonucleotide 5' -AG3 (T2AG3) 3-3' , showed two maxima at 295 nm and 246 nm, and a minimum at 267 nm. The G-4 was titrated with increasing concentration of the [Cu(phen) (aa) (H₂0) ] NO3 ·χ¾0 complexes as shown in Figure 14. Figure 14 shows the sets of CD spectra of 20 μΜ of G- quadruplex 5 -AG3 (T2AG3) 3-3' in the absence and presence of different concentrations of [Cu(phen) (aa) (H20)]⁺ (aa for a: gly, 1; b: DL-ala, 2; c: C-dMg, 3; d: sar, 4), and each of set of spectra (a - d) contains CD of (I) G-quadruplex alone, (II) G-quadruplex with 60 μΜ complex, (III) G- quadruplex with 120 μΜ complex in TN buffer (5 mM Tris, 50 mM NaCl) at pH 7.5 The three CD spectral peaks of the G-4 did not change with the addition of each copper (II) complex, suggesting retention of the anti-parallel structure.

[Cu (phen) (gly) (H₂0) ] Ν0₃·χΗ₂0 with no methyl substituent, showed the lowest binding affinity on G-quadruplex DNA. The result revealed that the G-quadruplex DNA binding affinity of [Cu(phen) (gly) (H₂0) ] Ν0₃·χΗ₂0 can be improved by substituting methyl group (s) into the coordinated glycine and that the methyl group at the amino nitrogen plays a greater role than at the -carbon of the amino acid.

Therefore, the position and number of methyl substituent in the coordinated glycine moiety modulate the G-4 binding selectivity. The above higher binding affinity for G-4 over duplex DNA is in line with the distinguishing characteristic of a molecule as potential G-4 targeting anticancer drug.

Experiments were conducted to ascertain positive-ion mass spectrum of the complexes of the embodiment herein. Figure 15a illustrates the positive-ion ESI mass spectrum showing experimental isotopic distribution of complex having glycine amino acid as ligand i.e. [Cu(phen) (gly)]⁺ species and their theoretical calculated m/z.

Figure 15b illustrates the positive-ion ESI mass spectrum showing experimental isotopic distribution of complex having alanine _. amino acid as ligand i.e. [Cu(phen) (DL-ala)]⁺ species and their theoretical calculated m/z;

Figure 15c illustrates the positive-ion ESI mass spectrum showing experimental isotopic distribution of complex having dimethyl glycine amino acid as ligand i.e. [Cu(phen) (C-dMg)]⁺ species and their theoretical calculated m/z.

Figure 15d illustrates the positive-ion ESI mass spectrum showing experimental isotopic distribution of complex having sarcosine amino acid as ligand i.e. [Cu(phen) (s.ar)]+ species and their theoretical calculated m/z.

Anticancer activity / selectivity - evidence from In-vivo studies The in-vitro studies above were complemented with in-vivo studies in lab animals such as mice. Figure 16a illustrates the effects of Cu(phen) (DL-ala)NC>3 on weight of male and female mice after administration. NOD SCID gamma (NSG) mice were injected ( intraperitoneally) with normal saline, 2, 4 arid 6mg/kg of Cu(phen) (DL-ala)N0₃ daily for 14 days. No signs of toxicity or significant weight loss were observed as may be observed in the Figure 16a. Figure 16b illustrates the inhibition of tumour growth in mice treated with 4 mg/kg Cu(phen) (DL-ala)N0₃. 1 million HK1- RFP NPC cancer cells were inoculated at the lower right flank of each mice. The treatment was started after 24 hours post inoculation with intraperitoneal injection every 3 days. Tumor growth was monitored manually and by IVIS optical imaging system. As may be seen for the untreated mice, the total radiant efficiency for the untreated cells remained the highest whereas those treated with the compound Cu(phen) (DL-ala)N0₃ showed decrease in the radiant efficiency indicating effective anticancer activity of the compound. Mice treated with the same regime of 4 mg/kg Cu(phen) (DL- ala)N0₃ had normal appearance of the heart, liver and kidneys on histopathological examination indicating the lack of toxicity of the regime to the normal organs indicating selectivity of the compound. NCI-60 Single dose data

Figure 17 illustrates the results from the NCI-60 single dose screen as mean growth percent for Cu(phen) (C-dMg)N0₃. The results from the NCI-60 single dose screen was reported as a mean growth percent. The number reported fot the One- dose assay is growth relative to the no-drug control, and relative to the number of cells at time zero. This _. allowed detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0) . For example, a value of 100 means no growth inhibition. A value of 40 would mean 60% growth inhibition. A value of 0 means no net growth over the course of the experiment. A value of -40 would mean 40% lethality. A value of -100 means all cells are dead. As may be observed, Cu(phen) (C-dMg)NC>3 exhibits significant growth inhibition and lethality against the 60 cell panel at a single dose of 10 uM.

Conclusion

All [Cu(phen) (aa) (H₂0) ] N0₃ ·χΗ₂0 complexes with glycine or methylated glycine derivatives crystallize with the same square pyramidal geometry for the cationic copper (II). Among them, [Cu(phen) (sar) (H₂0)]N0₃ with methyl substituent at the amino nitrogen binds most weakly to CT-DNA (factor of ½) . [Cu(phen) (sar) (H₂0)]N0₃ has the greatest binding selectivity for G-quadruplex over the corresponding duplex DNA. Based on restriction enzyme inhibition, complexes 1 - 4 have DNA binding selectivity, i.e. can only bind to certain sites on the DNA. However, [Cu (phen) (c-dMg) (H₂0) ] N0₃ with two methyl substituents at the -carbon of the glycine moiety, possesses the greatest binding selectivity.

All the [Cu (phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 complexes are considerably more efficient than cisplatin as anticancer drugs against, breast and nasopharyngeal cancer cells. The compounds also showed anticancer activity against a wide range of other cancer cells. Replacing coordinated glycine with methylated glycine can affect the biological property of the ternary complexes. Their antiproliferative and apoptosis-inducing properties are selective towards DA-MB- 231 and HK1 cancer cells over MCFlOA and NP69 normal cells. Two of these complexes with methylated glycine (at a-carbon) appear to also selectively induce cell cycle arrest at G₀/Gi. This selectivity may be attributed to (i) generation of higher ROS levels in cancer cells over normal cells which are responsible for induction of apoptosis, (ii) selective induction of nuclear DSBs which may result from sufficiently high ROS elevation in cancer cells, and (iii) proteasome inhibition which is related to the greater sensitivity of cancer cells towards proteasome inhibition. Although the present invention has been described with reference to the preferred embodiments and examples thereof, it is apparent to those skilled in the art that a variety of modifications and changes may be made without departing from the scope of the present invention which is intended to be defined by the appended claims.

Claims

1. A pharmaceutical composition comprising at least one of the series of ternary copper (II) complexes having the structure of [Cu(phen) (aa) (H₂0) ] Ν0₃·χΗ₂0 wherein x is a fraction or natural number; aa is an amino acid ligand; and phen is 1 , 10-phenathroline or derivatives thereof.

2. The pharmaceutical composition of claim 1 having structure:

Wherein, Rl, R2 and R3 are selected from either H or alkyl groups .

3. The pharmaceutical composition of claim 1, wherein said compounds give following cation in aqueous solution:

4. The pharmaceutical composition of claim 1 or claim 2, wherein a salt of said complexes include nitrate salts, chloride salts or halide salts.

5. The pharmaceutical composition of claim 1, wherein said amino acid ligands are glycine (gly) or DL-alanine (DL-ala)

6. The pharmaceutical composition of claim 1, wherein said amino acid ligands are sarcosine (sar) or 2,2- dimethylglycine (C-dMg) .

7. The pharmaceutical composition of claim 2, wherein said amino acid ligand are selected from group consisting of gly with R1=R2=R3=H; DL ala with R1=CH₃; R2=R3=H, ; C-dMg with R1=R2=CH₃, R3=H; and sar with R1=R2=H; R3=CH₃ and derivatives thereof.

8. The pharmaceutical composition of claim 1 or 2, wherein said complexes show selective activity against nasopharyngeal cancer, breast cancer, colorectal cancer, leukaemia, lymphoma, lung cancer, ovarian cancer, cervical cancer, liver cancer, central nervous system cancer, renal caner, prostate cancer and melanoma.

9. The pharmaceutical composition of claim 1 or claim 2, wherein said complexes are less toxic to normal or non- cancer cells as compared to cancer cells.

10. The pharmaceutical composition of claim 1 or claim 2, wherein said complexes do not show systemic toxicity in animal testing.

11. The pharmaceutical composition of claim 8, wherein said nasopharyngeal cancer further comprises recurrent nasopharyngeal cancer.

12. The pharmaceutical composition of claim 8, wherein said breast cancer further comprises metastatic breast cancer.

13. The pharmaceutical composition of claim 1, wherein said ternary copper (II) complexes increase reactive oxygen species (ROS) generation, induce nuclear double strand DNA breaks (DSBs, inhibit proteasome, arrest of- cell cycle, damage to DNA, induce formation of γ-Η2ΑΧ foci, and inhibit topoisomerase I (topo I) activity or combination thereof in order to kill cancerous cells.

14. The pharmaceutical composition of claim 2, wherein said amino acid of said complexes with methyl substituent ( s ) in the alpha carbon further comprising DL-ala and C-dMg.