TR201413242A2 - Novel cyano-bridged hetero-nuclear coordination complexes and the uses thereof as pharmaceutical agent - Google Patents

Abstract

The present invention relates to novel cyano-bridged coordination polymers, coordination salts and the uses thereof as pharmaceutical agent. Particularly, the present invention relates to coordination complexes formed by dicyanoargentate (I), [Ag(CN)21-] and a metal selected from transition metals like Nickel (II), Copper (II), Cadmium (II) or Zinc (II) and the N-(2-hydroxyethyl)-ethylenediamine ligand. The uses of this complex as potential pharmaceutical agent and preferably as anti-proliferative, anti-bacterial, anti-fungal or antiviral agent are described.

Description

DESCRIPTION NEW CYANO-BRIDGE HETERONUCLEAR COORDINATION COMPLEX AND THEIR PHARMACEUTICAL AGENT USES AS Field of Invention The present invention includes novel cyano-bridged coordination polymers, coordination salts and relates to their use as pharmaceutical agents. More specifically, the present invention dicyanoargentate (I), transition from [Ag(CN)21'] to Nickel (Il), Copper (Il), Cadmium (Il) or Zinc (II) a metal selected from among the metals and N-(Z-hydroxyethyl)-ethylenediamine ligandinin It is related to the coordination complexes it forms.

History of the Invention Metals and their coordination complexes are widely used in pharmaceutical chemistry. is used. One of the most important of these was Rosenberg et al. by cis-platinum, a platinum-based anti-cancer drug discovered (Prior art).

Ci/Il, Pt"`\ NH3 Platinum-based therapeutics represent an important cornerstone in the treatment of malignant tumors. is doing. Metal complexes allow the formation of different geometries around the metal. different coordination numbers and how pharmaceuticals interact with targets in different ways It has different oxidation steps that allow it to pass through. Also, cationic metals interact with charged targets of the biological system, for example the anionic backbone of DNA For example, cisplatin binds covalently to DNA and is transcribed. causing cell death. Although cisplatin; testicular, AfumuFtalikrbrenkejenHehfîtnümaîservîkalîarSWHem om, melanoma, urinary tract Although cisplatin is one of the agents, it is a dose-limiting agent including nephrotoxicity and neurotoxicity. development of new antitumor agents as well as safer and more effective therapies. the need continues.

Besides cisplatin, there are other platinum coordination complexes used therapeutically. are available. For example, terpyridine-platinum (II) complexes are associated with mammalian topoisomerases and It is an effective inhibitor of human thioredoxin reductases (Figure 2).

Figure 2. Terpyridine-Platinum Complex In addition to platinum, the effect of ruthenium, palladium, gold and titanium transition metals can cause various cancers. cells were studied. For example, a rheumatoid arthritis medication - a gold(I) phosphine complex, namely llauronafin”, via inhibition of the enzyme thioredoxin glutathione reductase It is effective in the treatment of parasitic disease. Similarly, for example a Ruthenium-ethacrynic acid Anticancer activities of various ruthenium complexes such as coordination complex known. In vitro HIV-1 reverse transcriptase of vanadium-porphyrin complexes Its medicinal effect on inhibition is known.

In addition, new potential therapeutic metals are still under investigation and investigation. have complexes. Ligands having electron donor atom or atoms It is known that it shows increased cytotoxicity with the increase of coordination capacity. This In studies, the design of ligands used for metal-based pharmaceuticals is important.

With the increase of the coordination capacity of ligands with electron donor atom or atoms, MWWHWüWWWW/ogwm Inorganic Chem., and transport has important effects on various mechanisms such as metal ion conservation or cleavage. known to play a role.

Coordination polymers are also therapeutic therapeutics that have been extensively studied in the past. among agents. From organic chemistry to inorganic chemistry, from biology to materials science and coordination due to its interdisciplinary nature from electrochemistry to pharmacology Polymers are known to be useful in many applications.

Polynuclear cyano complexes among coordination polymers, chemistry, biology of particular interest because of its novel properties that find potential applications in it awakens.

N-(2-hydroxyethyl)-ethylenediamine - hereinafter referred to as "h/deten"- N- and/or O It is a multithreaded selant ligand that can bind with its ends (Figure 3).

Figure 3. N-(2-hydroxyethyl-ethylenediamine (hydetene) In this invention, metal coordination having hideten ligand with silver(l) metal antiproliferative, antibacterial, antifungal, antiviral, anti-inflammatory and found to exhibit immunomodulatory activities. With HeLa, HT-29, C6 and Vero cell In vitro studies have shown that these new coordination complexes are selective and effective. It has proven its antiproliferative effect. At the same time, nine different showed promising antibacterial activity in experiments with bacterial strains. has been proven. The observed highly significant antiproliferative effect of metal coordination compounds its source suggests that it belongs to the complex [Ag(CN)2]' group.

In addition, the inventors of the present application surprisingly found the metal subject of the invention. has much lower cytotoxic activity than the [Ag(CN)2] ion of the coordination complex found that it was. This result indicates that the new metal coordination compound, which is the subject of the invention, is anticancer. Although it reveals the meaning of the activity of the [Ag(CN)2] ion, it is not individual. Biological structure in the new structure created by designing with complex groups containing other metals. shows that it contributes significantly to the emergence of activities.

Brief Description of the Invention One aspect of the present invention is a metal (M) and N-(2-hydroxyethyl)-ethylenediamine ligandin. with the structure represented by the following Formula I, formed by dicyanidoargentate (I) relates to a coordination complex: WQMMgggcwhmcma Formula I In a preferred embodiment of the invention, said metal (M) is Nickel (II), Copper (II), It is selected from transition metals such as Cadmium (Il) or Zinc (Il).

In a more preferred embodiment of the invention, said metal (M) is Nickel (II).

Another aspect of the present invention is the new coordination with the structure represented by Formula I. It relates to a method for synthesizing the complex.

WWWmWmkompßkW with use in the manufacture of a medicament for the treatment of inflammatory and immunomodulatory conditions Another aspect of the present invention is that proliferative, bacterial, fungal, viral, For the treatment of inflammatory and immunomodulatory conditions, the novelty of the present invention coordination complex or a pharmaceutically acceptable salt thereof by a method comprising administering to a patient a therapeutically effective amount Here we also describe the new coordination complex of the present invention or its pharmaceutical containing a pharmaceutically acceptable salt and pharmaceutically acceptable excipients. A pharmaceutical composition is also disclosed.

Another aspect of the present invention is preferably a tablet, capsule, intravenous formulation, intranasal formulation, transdermal formulation, formulation for muscle injection, syrup, is a pharmaceutical composition in suppository or aerosol form.

Brief Description of the Figure Figure 1. Antiproliferative effects of ANG, [Ag(CN)2]' and 5-FU against HeLa, HT-29, C6 and Vero cells Figure 2. Cytotoxicity of AN5, [Ag(CN)2]1" and 5-FU on HT-29, HeLa and C6 cells activities Figure 3. Effect of ANß on HeLa, HT-29, C6 and Vero cell morphologies Figure 4. Effect of AN6 on DNA fragmentation (AN5; 1: DNA standard; 2: HeLa Control; 3: Detailed Description of the Invention New bimetallic dicyanoargentate, referred to as M(hydeten)2Ag(CN)2][Ag(CN)2].H2O relates to polymers; mghiwuwç Mwcmu Formula I where M; Among the transition metals such as Nickel (Il), Copper (II), Cadmium (Il) or Zinc (Il) In one embodiment of the invention, the compound - hereinafter AN5 (also What will be referred to as Ni(hydeten)2Ag(CN)2][Ag(CN)2].H2O) - has the following structure: m“îtîwuwcwiiqßcmu ANG A given compound is one or more of the special geometric, optical, enantiomeric, diasteriomeric, It can be found in stereoisomeric and conformational form. Compounds of the present invention are also may be in the form of a pharmaceutically acceptable salt because preparing the corresponding salt It may be convenient to purify and use. The compounds may also be in the form of a hydrate because the related hydrate form, eg mono-hydrate, di-hydrate etc. convenient to prepare, purify and use it could be. Compounds may also be in a "chemically preserved" form because the corresponding protected It may be appropriate to prepare, purify and use the form. “Chemically preserved "form" means one or more of which are protected from chemical reactions by means of a protecting group. means a compound with a chemically active functional group.

Another aspect of the present invention is that the Formula I compound or its pharmaceutically acceptable relates to an encompassing method.

In one embodiment of the invention, compound AN5 or its pharmaceutically acceptable a therapeutically effective amount of a salt of a proliferative, bacterial, fungal, viral, administration to a patient for the treatment of inflammatory and immunomodulatory conditions A comprehensive method is provided.

Another aspect of the invention is the Formula I compound or a pharmaceutically acceptable solution thereof. salt with a pharmaceutical composition containing pharmaceutically acceptable excipients. In one embodiment of the invention, compound AN5 or a pharmaceutically acceptable one thereof. provide a pharmaceutical composition comprising its salt and pharmaceutically acceptable excipients. is being done.

Another aspect of the present invention is preferably a tablet, capsule, intravenous formulation, intranasal formulation, transdermal formulation, formulation for muscle injection, syrup, relates to a pharmaceutical composition in suppository or aerosol form.

General Synthesis Method The coordination complexes of the invention consist of a stoichiometric amount of metal salt, N-(2- hydroxyethyl)-ethylenediamine (hydetene) ligandine and dicyanoargentate (I), [Ag(CN)z]' aqueous solution It is synthesized by reacting Also, for solubilization and crystallization solvents such as ethanol, methanol, chloroform, isopropyl alcohol and acetonitrile as solvents used. Reaction schemes are shown below. edebiüFbiFRwmnHerapöthçnngPtkirbiîîiktmîoi erati , a terial, fungal, **7 General characterization methods Elemental Analysis and IR Spectorometry were used for characterization.

Use of Compounds The present invention has in vitro antiproliferative, antibacterial, antiviral, antifungal, anti-inflammatory and provides active compounds with immunomodulatory activity.

The term "active" for antiproliferative activity refers to the ability to regulate cell proliferation. relates to compounds and includes compounds with intrinsic activity.

The present invention provides antiproliferative agents. "antiproliferative agent" used here The term refers to a compound that treats a proliferative condition. In vitro or not, unwanted excess or abnormal cells are undesirable or controllable. It can be used in the sense of untreated cellular proliferation.

Examples of proliferative conditions include - but are not limited to - cancer, tumor, including – but not limited to, leukemia, bone disease, neoplasm and atherosclerosis as - malignant cellular proliferation can be given.

Antiproliferative compounds of the present invention are used in the treatment of cancer.

The term "active" used for antibacterial activity means that which kills or kills bacteria. It relates to compounds and substances that slow its growth.

The term "active" for antifungal activity means that it kills the fungus or kills the fungus. Slowly, it's about biology and substances.

The term "active" for antiviral activity refers to those that kill the virus or slow its growth. relates to compounds and substances.

The term "active" for "inflammatory" activity, or a substance that reduces inflammation. relates to the nature of the substance or treatment. Anti-inflammatory drugs inflammatory they heal the pain by reducing it. relates to items.

The invention is also active for use in a method of treating the human or animal body. Provides compounds. Such a method provides the subject with a therapeutically effective amount of active ingredient. administration of the compound, preferably in the form of a pharmaceutical composition.

In the context of treating a condition as used herein, the term "treatment" is often used to refer to some desired relates to treatment and therapy in which therapeutic effects are achieved.

As used herein, the term "therapeutically effective amount" refers to the active compound or desired containing the active compound active to produce part of the therapeutic effect. relates to the amount of the compound or dosage form.

The invention also includes, for example, an active ingredient for the manufacture of a medicament for the treatment of a proliferative condition. ensures the use of the compound.

The invention further provides a method of treating the human or animal body, the method; A therapeutically effective amount of a pharmaceutical composition is administered to a subject in need of treatment. It includes the administration of active compounds in the form of a pulmonary, rectal, vaginal, or parenteral - but not limited to - site applicable.

Formulations such as carrier, filler, buffer, adjuvant, stabilizer, or other material. at least one active ingredient in combination with one or more pharmaceutically acceptable excipients It is preferred that it be formulated as a pharmaceutical composition containing the component.

The term 'pharmaceutically acceptable' means excessive toxicity, irritation, allergic response or other suitable for use in contact with a subject's tissues without causing problems relates to compounds, materials or compositions.

The formulations may be prepared by any method known in the pharmaceutical literature and the unit may be presented in dosage form. Formulations - but not limited to - tablet, capsule, syrup, lozenge, pill, capsule, sachet, ointment, gel, cream, spray, paste, aerosol or in suppository form.

Activity Antiproliferative activity tests Chemicals and cell lines used in this experiment Trypsin-EDTA (25%) (Vendor - Sigma), penicillin streptomycin solution (Vendor - Sigma), Fetal Bovine Serum (Heat-inactivated)) (Vendor - Sigma), Dulbecco's Modified Eagle's Medium - High glucose (Vendor - Sigma), Cell proliferation ELISA, BrdU kit (Vendor - Roche); HeLa (Human Uterine Cancer Cell); HT-29 (Colon Cancer Cell); C6 (Rat Brain Tumor Cell); Vero (African Green Monkey Kidney Epithelial Cell Line) n devices ELISA (Rayto RT-; Vacuum pump; incubator with COZ water jacket (Nuaire US Autoflow); biological safety cabinets (Esco class II type A2); centrifuge (Hettich EBA20); Microscope (Olympus CX21) Closet; HT-29; C6; Vero Cell Cultures (Passaging Process) The experiment was carried out in a vertical Iaminar-flow biological safety cabinet (Class ll). Cell lines fed Cells are confluent in sterile cell culture flasks containing cell culture medium (fed DMEM). cultured until The culture medium was decanted and the cells adhered to the flask were treated with 10 ml of trypsin. It was incubated with EDTA solution for 2-5 minutes (or incubated at 37°C in a CO2 incubator).

The flask was gently shaken to separate the cells from the flask surface.

The flask was then filled with 10 ml of cell culture medium to neutralize trypsin and The cell suspension was transferred to a sterile 50 ml Falcon tube. The cell suspension was then A cell pellet was formed at the bottom of the tube by centrifugation (600 x g) for 10 min.

The cell medium was decanted under sterile conditions and the remaining cell pellet was 1-3 ml of cell culture. was suspended with On the other hand, sterile 250 ml cell culture flasks are fed with 20 ml. filled with cell culture medium, 1 ml of suspended cells was added to these flasks. transferred and the flasks were placed in the CO2 incubator. This splitting is done every 4 days. was repeated. Cells were checked for growth and contamination every 4 days under an inverted microscope. followed.

Freezing and storage of cells 1 ml of cell suspension in DMEM + 10% DMSO solution (cell freezing solution) (approximately 1x106 cell lines/ml) were transferred to 2 ml Cryovial tubes and the tubes were either directly flushed with liquid. placed in nitrogen or kept at -80°C for 1 day then liquid nitrogen placed inside.

Counting Cells mixed and 10 µl cell suspension 10 µl cell suspension covered with a coverslip pipetted into the hemocytometer (counting chamber) well. In the 5 big squares in the counting area cells were counted under the microscope. The total number of cells is calculated with the help of the following equation calculated: Number of cells = number of cells in 5 wells x dilution factor Preparation of DMEM (Dulbecco's Modified Eagle's Medium-High Glucose) Solution solved with magnetic stirrer. 2.2 g of NaHCOs were added and the pH was adjusted to 7.2 with HCl solution.

The total volume set to 1000 ml. This solution was put in the biological safety cabinet, vacuum Fetal Bovine Serum (10% final) was filtered into sterile bottles with a 0.22 um sterile filter under concentration ) and Penicillin-Streptomycin solution (%Z final concentration) and stored at + 4°C. The final solution was called nutrient DMEM or nutrient culture medium.

Preparation of stock solutions of samples mg samples were dissolved in 100 µl of sterile DMSO. Later in DMSO test samples dissolved in 2 ml of two to prepare 5mg/ml master stocks with 5% DMSO diluted once with distilled water (ddHzO). Stock solutions were placed in sterile falcon tubes (15) with 0.22 µm filters. ml) was filtered and kept at -20°C. Final stock solutions of samples (by dilution of 1 mg/ml and 100% pl test samples was prepared.

CONTROL DMSO SAMPLE SAMPLE SAMPLE SAMPLE SAMPLE SAMPLE SAMPLE Sema 1. Experimental Setup The experimental setup was arranged as shown in Scheme 1. Whole procedure biological safety performed in their cabins. 96-well sterile cell culture plates were used. 100 pl cells suspension (30,000 cells) was added to the wells. Except for the control, 8 different The volume was brought to 200 µl with cell culture medium. as negative and positive controls, respectively. [Ag(CN)2]" and 5-fluorouracil were added to the corresponding wells. Cells were incubated for 24 hours (37°C and 5% CO2). At the end of this period, cell proliferation was determined by BrdU Cell ELISA method. determined. All samples were tested three times and the tests were repeated three times.

BrdU Cell ELISA Assay Experiment setup on a 96-well microplate as shown in Scheme 1. arranged and left for incubation at 37°C, 5% CO2 for 24 hours. After 24 hours, 20 µl of BrdU staining solution was added to the wells and 5% at 37°C. It was left for incubation at CO2 for 4 hours. The solution was decanted from the stained wells and 200 µl of FixDenat was added and left for incubation at temperature for 30 minutes.

After removing the FixDenat solution, 200 µl of anti-BrdU-POD solution was added and the room was left for incubation at temperature for 90 minutes.

After removal of the anti-BrdU-POD solution, all wells were rinsed with 200 µl of wash solution. Washed 3 times. Then, 100 µl of substrate solution was added to each well and the room was left for incubation at temperature for 30 minutes. 450 nm and 450 nm with ELISA reader before and after addition of µl 1M HzSO4 Absorbances at 659 nm were measured. is half of the ryori and a compound's biological or a measure of its effectiveness in inhibiting its biochemical function. Here The IC50 concentration used is the concentration at which 50% of proliferation is inhibited. 90, 100 µl) was prepared. These test samples were applied to cells with the BrDU or SRB methods. tested. Standard curves were created with these absorbance values and the IC50 values in Excel. or calculated using SigmaPlot programs.

Cytotoxic activity tests HeLa, HeLa, Cytotoxic activity on HT-29 and C6 and Vero cells Lactate Dihydrogenase Enzyme (LDH) tested by comparison with cytotoxicity assay. Incubation with test material The increase in cell death during the period of time caused an increase in the LDH enzyme. try this its basis is the calculation of cytoplasmic enzyme availability caused by cell death. is based on. LDH is a stable cytoplasmic enzyme that occurs in almost all enzymes.

For this purpose, the LDH cell cytotoxicity kit (from Roche) was used.

Cells in cell culture (prepared similarly to the BrdU method) transferred to microplates. Growing cells IC50, AN“ concentrations 37% overnight was also incubated. After the incubation period, the cell-free cell culture supernatant gathered. 100 ml of this supernatant was transferred to another microplate and the entire reaction The mixture was added to all supernatants to make a final volume of 100 µl per well and minutes incubated. during incubation, matching enzymatic reaction LDH transferred to the supernatant with the tetrazolium salt was reduced to form formazan. This Therefore, LDH transferred to the supernatant is directly related to formazan formation. observed. The amount of formazan dye in the pools is approximately 500 nm absorbance. Detected by ELISA plate reader.

The results were reported as % cytotoxicity and the optical properties of solvent-treated cells were reported. density was accepted as 100%. % cytotoxicities in accordance with the following equation Determination of Effect on Cell Morphologies HeLa, HT-29, C6 and Vero cancer cells overnight at 37°C with ICSO concentration of AN5 were incubated throughout and viewed with a microscope (Leica lL) attached to a digital camera.

Determination of AN6's Anticancer Mechanisms of Action Exponentially growing HeLa, C6 and HT-29 cells were incubated at 37°C overnight, DNA Isolation and DNA fragmentation were visualized by agarose gel electrophoresis.

Antibacterial activity tests Bacterial species used in these experiments: S. Aureus (HPB-1), Bacillus Subtilis (HPB-3), E. Coli (HPB-8), Bacillus Cereus (HPB-10), Enterobacter Aerogenes (HPB-15), Salmonella Gallinarum (HPB-28), Pseudomonas Aureginosa (HPB-37), Salmonella Enteritidis (HPB-40), Steptococcus Pyogeresis (HPB-41); Agars: Nutritive Agar (BA) and Brain Heart Agar (BKA); Chemicals: methanol, distilled water, azithromycin (AZM), cefoperazone-sulbactam (SCF). First of all, Nutrient Agar (BA) media were prepared for bacteria and only one was used to transfer BA. disposable petri dishes were used. Powder BA was dissolved in distilled water and mixed and sterilized in an autoclave at 121°C for 15 minutes. BA in gelled form 20 ml of the solution was transferred to a sterilized petri dish at 40-50°C aseptic environment and overnight. left at room temperature. Petri dishes were sealed and stored at 4°C for later use.

Preparation of pure microorganism cultures: 133 pl 1/10 diluted bacteria suspensions were suspended and bacteria were added with a swab.

McF setup: Bacterial strains used in microbiological studies were added to agar media. passaged and incubated in the oven for 18 hours and colonies inoculated into liquid medium.

After grafting, the liquid medium became cloudy due to bacterial growth. Detecting blur McF (McFarland) standards were used for The tonality of turbidity depending on the number of bacteria. The 0.5 McF standard was used in this experiment. your blurriness After observation, 9 ml of broth medium was prepared and 1 ml of this turbid solution was prepared. added to the environment. As a result, the dilution of the bacteria was achieved.

Disk diffusion method: Bacteria prepared according to 0.5 McF standard and petri dish with glass rods. were cultured in BA medium in their containers. The BA medium was dried for some time and the test samples and transferred to sterilized discs to be used for soaking the negative control samples.

Chemical solutions were soaked into the discs using a 20 µl micropipette. Petri dishes overnight It was incubated at 37°C and the activity zone diameters were observed. EXAMPLES The following examples are provided to illustrate the present invention and not to cover the scope of the invention. it is not intended to offend.

AN5 synthesis In the present invention, compound AN6 consists of a Ni metal atom coordinated with the hideten ligand. obtained by mixing. 1 mmol AgNOa salt and 2 mmol KCN were dissolved in distilled water-alcohol mixture to produce silver. cyanide anion, [Ag(CN)z]' was obtained. Because cyano groups exhibit polar character, 7 was a soluble and therefore most of the synthesis was in aqueous medium. carried out. A turbid solution was obtained by adding 1 mmol NiCl2.6H20 to this solution. the solution was stirred and heated on a magnetic stirrer. To this solution N-(2-hydroxyethyl)- Added ethylenediamine (hydetene) Iigandi. Then ethanol, methanol, chloroform, isopropyl Iigandin was easily dissolved by the addition of solvents such as alcohol and acetonitrile. this is my wife It was stirred at 60°C for 1 hour. The resulting mixture was filtered and the remaining solution left at room temperature for crystal formation.

Characterization of ANG by Elemental Analysis: Calculated percentages for C12H26N303Ag2Ni Characterization of ANG via the IR Spectrum: Infrared of the coordination polymer ANG The spectrum shows all characteristic peaks of the hideten ligand with shifts.

H) peaks and other characteristic peaks observed in the spectrum at 1602-400 cm'l are hidden in the structure. indicates the presence of ligand. The cleavage in the NH stretch vibrations is the result of the hidten ligand It is explained by the coordination of nitrogen atoms to the metal. at the OH stress peak It is possible to say that the shift is due to the weak H-bond formation. v(C-H) CH2 groups are formed as a result of the coordination of ligand to Ni (Il) cleavage of the bands. explained by the differentiation of their chemical environments.

When the characteristic triple bond region in the spectrum of AN5 is examined, it is seen that v(CEN) titer It is seen that it splits and appears as two separate peaks at the frequencies of 2161 and 2142 cm"1.

As expected, the cyanide stretch vibration is increased to higher frequencies by complex formation. appears to have slipped. Higher than the two peaks resulting from the cleavage of the cyanido peak bridging cyanido ligands at a lower frequency (2142 cm'l) which can be said to belong to the terminal cyanido ligands. In addition, neutral Iigandin bending and stretching vibrations of ö(NH2), 6(CH2), u(C-N) and u(C-O) caused by the presence of indicates the presence of hideten ligand in the structure of the complex. Also, in the complex Ni"-C, Ni”-N and carbon dioxide formed as a result of the coordination of ea'bea and cyano ligands to metals. is coming out.

Antiproliferative effects of AN6 on HeLa, HT-29, C6 and Vero cells with different dosages. was studied and the antiproliferative effects were compared with 5-fluorouracil (5-FU) and [AgCN)2]'.

The results are shown in Figure 2. Experiment results showed that ANG was determined by HeLa (p<0.05), HT-29 (p<0.05) and It inhibited proliferation of C6 (P<0.05) cells much more than the control compound 5-FU has shown that it does.

Growing cells ICSO, ANG concentrations (1.43 ug/ml for HeLa, 1.45 ug/ml for C6 and 0.96 µg/ml for HT-29) was incubated at 37°C overnight. AN6 and [Ag(CN)2]` in its structure ion and the cancer drug 5-FU on HeLa, HT-29 and C6 and Vero cells. activity was compared with the LDH cytotoxicity assay (Figure 3).

The results obtained indicate that [Ag(CN)2]', which has slightly more antiproliferative activity than ANE. showed that it has much lower cytotoxic activity than the ion. This result While revealing the meaning of ANG showing anticancer activity, [Ag(CN)2]' formed by designing the ion not individually but with other metal-containing complex groups. contributes significantly to the emergence of biological activities in the new structure. shows.

This complex has high antiproliferative activity but low cytotoxicity. It is an important feature sought in drugs used in the treatment of This complex is antiproliferative activity and low cytotoxicity, both prevent proliferation of cancer cells. effectively inhibit and have little toxic effect on normal cells. means. This compound, which has low cytotoxicity, is effective against cancer cells. It has a cytostatic effect, that is, it inhibits the cell cycle and prevents the proliferation of cells. is thought to have stopped. Also, some cell cycle related cytostatic effects of this compound. Although it is estimated to occur by stopping the expression of genes, these It is not yet known whether there are fanci genes. In vivo studies related to this needs to be done.

In conclusion, in vitro tests showed that AN6 was found in all tested cancer cell lines, showed that it has activity.

Effect of ANö on HeLa, HT-29, C6 and Vero Cell Morphologies Cells were incubated at 37°C with a 1C50 concentration of ANö overnight and digitally viewed with a microscope (Leica ll) attached to the camera (Figure 4).

In this study, HeLa, HT-29, C6 and Vero cancer cells were treated with AN5 and The effect on cell morphology was observed and photographed. This substance depends on the cell type. changed and generally caused their cell number and separation from the plastic surface. rounding, ballooning, granular structure or It was seen that it caused aggregation (Figure 4). Low doses of these test substances The changes in the morphology of the cells using the antiproliferative effects of these compounds parallels its effects.

Determination of AN5's Anticancer Mechanisms of Action In this study, the presence of apoptosis mechanism on the basis of AN6 antiproliferative activity was determined by DNA. banding (DNA laddering) experiment. For this purpose, the IC50 of the relevant compound DNA isolate from HeLa, HAT-29, C6 and Vero cells treated with concentrations of It has been determined that the apoptotic effects of this compound in this cell are due to the banding in the cell DNA. determined by observation.

Exponentially growing HeLa, C6 and HT-29 cells were incubated at 37°C overnight, DNA Isolation and DNA fragmentation were visualized by agarose gel electrophoresis. AN6 DNA (AN5; 1: DNA standard; 2: HeLa Control; 3: HeLa+AN6; 4: HT- As seen in Figure 5, isolated from control cells untreated with the complex. DNA isolated from substance-treated cells compared to DNA isolated from A clear banding was observed in the samples. AN5 with DNA banding test results The antiproliferative effect of the coordination compound is very likely to result in apoptosis. It was interpreted as that it performed by stimulating the mechanism. These results show that this compound It clearly supports the results of antiproliferative activity and morphological effect.

Antibacterial tests were carried out on 9 different bacterial cells and the antibacterial agent Cefafrezone-sulbactam (SCF) was used as control agent. Formed disc diffusion ages listed in Table 1 below: Effect zones of bacteria test materials (mm) Table 1. Disk diffusion diameters of ANG on bacteria (mm) . I ' .inn the effect obtained AN6 is more effective than SCF, which is used as an antibiotic, when compared to its zones. showed antibacterial activity (Table 1).

MIC (Minimum Inhibition Concentration) values of ANG were also carried out with different dilutions. determined through experiments. The results are listed in Table 2.

Bacteria MIC Values AN5 Positive Control Table 2. MIC (Minimum Inhibition Concentration) values of SCF antibiotic of ANG When Table 2 above, where MIC values are given, is examined, it is seen that AN5 is low. It is seen that it still maintains its effectiveness against the tested bacteria even at high concentrations. Especially when looking at the effect of ANs on HPB-8 bacteria at 1/64 dilution It is effective on bacteria even in its condition, that is, it does not allow bacterial growth. is seen. 1/64 for AN5 as bacteria grow in subsequent dilutions. the diluted state is the workable limit, therefore at very low concentrations. The result of these studies is that the active substance can also be found at very low concentrations. in terms of its effects on bacteria, therefore its use in this field. will have many advantages.

Claims (6)

1. A coordination complex having the structure represented by the below mentioned Formula l and formed by the dicyanidoargentate (I) and the N-(2-hydr0xyethyl)- ethylenediamine and a metal (M):

2. A coordination complex according to Claim 1, characterized in that said metal (M) is selected from the transition metals like Nickel (Il), Copper (Il), Cadmium (ll) or Zinc

3. A coordination complex according to Claim 1 or 2, characterized in that said metal (M) is Nickel (II) and has the following Formula ll structure. Formula II

4. A method for synthesizing a coordination complex according to Claim 1, characterized in that a metal salt, N-(2-hydr0xyethyl)-ethylenediamine (hydeten)

5. A method according to Claim 4, characterized in that said metal is a transition metal.

6. A method according to Claim 4 or 5, characterized in that said metal (M) is selected from the transition metals like Nickel (ll), Copper (II), Cadmium (II) or Zinc (II). A method according to any one of the preceding claims between Claim 4 and 6, characterized in that said metal is Nickel (II). Use of the coordination complex, which is according to any one of the preceding claims between Claim 1 and 3 or a pharmaceutically acceptable salt thereof, for the treatment of a disease selected from proliferative diseases, microbial diseases or inflammatory diseases. A pharmaceutical composition comprising a coordination complex which is according to any one of the preceding claims between Claim 1 and 3 or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable excipients. A pharmaceutical composition according to Claim 9, characterized in that the composition is preferably in syrup, suppository or aerosol form or formulation for muscle injection, transdermal formulation, intranasal formulation, intravenous formulation, tablet or capsule form.