Technical Field of the Invention

The invention relates to a thiosemicarbazone molecule with anti-inflammatory and wound healing properties that inhibits C30 endopeptidase or in other words 3- chymotrypsin-like protease (3CL major protease) enzyme of SARS-CoV2 virus, lnterleukin-8 (IL-8) cytokine and inducible nitric oxide synthase (NOS2) enzyme of human bronchial epithelial cells and the synthesis method of this molecule.

State of the Art

COVID-19 emerged in China in late 2019 and has been recognised as a pandemic by the World Health Organization due to the rapid increase in the number of cases. COVID-19 has caused more than 5 million cases and more than 300,000 deaths to date. The spike proteins encoded in the RNA of this virus associate with the cellular receptors of target cells to mediate viral infection, and then viral replication begins in the cytoplasm. The main targets of SARS-CoV-2 virus are lungs, immune system organs (organs with lymphoid tissue etc.) and systemic small vessels and this virus causes systemic vasculitis.

Non-structural proteins (nsp) in the COVID-19 genome have a wide range of functions, from transcription of RNA to protein synthesis and modification. Among them, C30 endopeptidase, or 3-chymotrypsin-like protease (3CL main protease) enzyme is an important target for the development of small inhibitor molecules due to its functional structure and enzyme active sites, and therefore, since 3CL main protease enzyme is an important enzyme related to the replication system of the virus, it is used in inhibition trials in drug studiesand has an important place in the fight against the epidemic. In conclusion, 3CL main protease enzyme, also known as 3CL hydrolase enzyme, is an essential enzyme for the maturation of the virus, and therefore studies on the inhibition of 3CL main protease enzyme are carried out in the treatment of COVID-19 [1 ],

1

SUBSTITUTE SHEET (RULE 26) In the state of the art, another enzyme that should be focused on in enzyme inhibition studies on the treatment of COVID-19 is the NOS2 enzyme. NOS2 provides cell protection by synthesizing NO in response to bacterial or proinflammatory stimulation. It is a key enzyme involved in all aspects of the pathophysiological process of pulmonary infections of various aetiologies, including Sars-Cov-2. However, excessive NO production mediates harmful proinflammatory effects. Overexpression of NOS2 is associated with the release of proinflammatory cytokines such as IL-1 (3, IL-6, IL-8 and TNFa.

In the state of the art, another element that is important in COVID-19 disease is lnterleukin-8 (IL-8) cytokine, which is in the category of inflammatory proteins. IL-8 is a proinflammatory cytokine that has a role in neutrophil activation and has been identified in the pathogenesis and progression of this disease. It causes severe respiratory failure and/or ARDS in patients with severe COVID-19. In fact, activation of the immune system and production of inflammatory cytokines are essential for natural antiviral immune responses. However, hyperactivation of the immune system causes an acute increase in circulating pro-inflammatory cytokine levels, leading to a "cytokine storm". Cytokine storm clinically results in systemic inflammation, hyperferritinemia, hemodynamic instability, and multi-organ failure.

In the state of the art, studies on the inhibition of the 3CL main protease enzyme from the enzymes mentioned above have been carried out. In the article of Arslan et al. on tetradentate thiosemicarbazones and iron(lll) and nickel(ll) complexes, new inhibitors that can be used in the treatment of viral diseases, especially COVID-19, innovative compounds with increased efficiency that inhibit the enzyme and receptor mechanisms are mentioned. The inhibitory effect of the synthesized Fe1 compound against the 3CL protease enzyme specific to SARS-CoV-2 virus has been experimentally proven. However, due to their redox activities, iron ions interfere with the vital processes that progress with redox, making a disruptive effect and harming vital processes. In addition, since iron compounds are not easily soluble in water, they accumulate in tissues and cause chronic toxicity in the body [1], Thiosemicarbazone mentioned in the study described is an organosulphur compound with the formula H ₂ NCNHN=CR1. Thiosemicarbazones come in many variations, including where some or all of the NH centres are substituted by organic groups, and are usually obtained by condensation with an aldehyde or ketone. Thiosemicarbazone (TSC) derivatives are of great interest in pharmacology due to their antiviral, antineoplastic, antimalarial, antifungal and antibacterial properties. It is stated that the biological activities of these compounds may be due to the ability of the molecule to form a three-dentate chelate formed between two nitrogen atoms and a sulphur atom and essential heavy metal ions. Some researchers report that chelates are more active than ligands and that TSCs and metal complexes substituted with aromatic aldehydes have a strong pharmacological effect. It is stated that the reduction potentials of copper TSC-Copper complexes are directly related to their inhibitory cardiac mitochondrial oxidative phosphorylation and in vitro cytotoxic effects against tumour cells. In a study comparing the spectrophotometric behaviour of various TSC derivatives and copper complexes with superoxide dismutase (SOD), it is emphasized that these compounds show SOD-like activity as well as their broad-spectrum biological activities. Thiosemicarbazones and Schiff bases inhibit DNA and RNA synthesis. This effect is due to their being the strongest known inhibitor of ribonucleotide diphosphate reductase, which reduces ribonucleotides to deoxyribonucleotides[2].

Transition metals such as iron, copper, zinc, manganese and cobalt are essential metals for human biology as they are integral parts of many metallo-proteins involved in important biological processes. They also function as components of cofactors in enzyme activity. Cobalt is used for the formation of vitamin B12 (cobalamin) in humans. Vitamin B12 is the only metal-containing vitamin and the only known cobalt-containing compound in the human body. Cobalamin and related enzymes catalyse important reactions in the body, such as methionine synthesis, purine and folate metabolism, and the formation of methylmalonic acid, which is important for the tricarboxylic acid cycle. Vitamin B12 provides the synthesis and regulation of DNA and is also used in fatty acid metabolism and amino acid metabolism [3, 4], Cobalt-containing coordination complexes have remarkable applications in biology and medicine due to their interesting redox and magnetic properties [5], Cobalt shows a range of possible oxidation steps from -1 to 4+. In biological systems, cobalt exists only as Co (II) or Co(lll)[2], These two dominant oxidation states exhibit different properties. The biological activity of cobalt complexes was first reported by Dwyer et al. [6], Later, Dwyer identified cobalt phenanthroline complexes with potent antibacterial activity [7], as well as cobalt complexes that can inhibit neuromuscular activity in animals [8], Although Cobalt-Schiff base complexes are used as antimicrobial agents, significant clinical progress has been made in these complexes as an antiviral [9-10], For example, the [Co(acacen)(l_2)]+ complex, (L=2-methylimidazole) is the only cobalt- Schiff base complex to enter clinical trials. A derivative of this complex has demonstrated selectivity against Sp1 zinc finger transcription factors (ZFTF) for potential application in the treatment of human immunodeficiency virus (HIV) [11 ], The pharmaceutical properties of [Co(acacen)(l_2)]+ complexes have triggered many mechanic investigations [12-13],

Although studies on the inhibition of the 3CL main protease enzyme, which supports the maturation of the virus, are carried out in the state of the art, it is obvious that a molecule that only provides the inhibition of the 3CL main protease enzyme will not be sufficient to completely eliminate the deadly effects of COVID-19. Apart from the 3CL main protease enzyme, there are inducible nitric oxide synthase (NOS2) enzyme and IL-8 cytokine that should be inhibited. If NOS enzyme is not inhibited, in other words, if it is not produced in a controlled manner, it causes immunodeficiency in hosts [14], Immunodeficiency is the biggest trigger of COVID-19 disease, and when the immunity declines, inhibiting the 3CL main protease enzyme will not make much sense. The same is true for IL-8 cytokine. If the increase in IL-8 cytokine, which is one of the inflammatory indicators and associated with systemic inflammation, hyperferritinemia, hemodynamic instability and multi-organ failure, is not suppressed in patients hospitalized in the intensive care unit with the diagnosis of severe COVID-19, inhibition of the 3CL main protease enzyme alone will not be sufficient in the treatment of COVID- 19. Apart from all these, almost all drugs developed and put on the market against COVID-19 cause diseases such as hypercholesterolemia and hypertriglyceridemia that seriously disrupt the body balance. In addition, compositions used in the state of the art for healing wounds in BEAS-2B cells, that is the bronchial epithelial cell line, in the later stages of Covid19 disease have synthetic content and said compositions cannot provide protection satisfactorily, are insufficient in cleaning wounds from harmful substances, do not reduce pain, do not support the healing process and do not provide the necessary moistening. In addition, said synthetic compositions adversely affect body functions.

Due reasons such as limitations and inadequacies of molecules developed against COVID-19 and drugs released in the state of the art, Inhibition of the 3CL main protease enzyme alone not being sufficient in the treatment of COVID-19, the inability of drugs in the current technique to fully treat COVID-19 and the fact that the drugs in the state of the art cannot fully treat and cure COVID-19 and bring many problems with it, the development of a new non-synthetic molecule with wound healing properties that inhibits IL-8 cytokine and inducible nitric oxide synthase NOS2 enzyme as well as 3CL main protease enzyme has become necessary.

Brief Description and Aims of the Invention

In the invention, a thiosemicarbazone molecule with anti-inflammatory and wound healing properties that inhibits C30 endopeptidase or in other words 3-chymotrypsin- like protease (3CL major protease) enzyme of SARS-CoV2 virus, lnterleukin-8 (IL-8) cytokine and inducible nitric oxide synthase (NOS2) enzyme of human bronchial epithelial cells and the synthesis method of this molecule is described. The general formula of the molecule, which is also the subject of the invention, which is synthesized together with the method of the invention, is shown with Formula 1 below.

Formula 1

The aim of the invention is to reveal a highly effective molecule in the treatment of COVID-19 with anti-inflammatory and wound healing properties that inhibits the 3CL main protease enzyme, IL-8 cytokine and NOS2 enzyme of human bronchial epithelial cells, for use in the treatment of COVID-19. Introducing a thiosemicarbazone molecule with high therapeutic effects in the treatment of COVID-19 is provided by the synthesis method of said molecule.

Another aim of the invention is to provide a molecule that inhibits the overexpression of NOS2 causing an acute increase in proinflam matory cytokine levels. Inhibition of NOS enzyme, which causes immunodeficiency, which is almost the biggest enemy of COVID-19 disease, is provided by the thiosemicarbazone molecule and the method of obtaining this molecule, which is the subject of the invention. NOS2 provides cell protection by synthesizing NO in response to bacterial or proinflammatory stimulation and is a key enzyme involved in all aspects of the pathophysiological process of pulmonary infections of various aetiologies, including Sars-Cov-2. However, sustained overproduction of NO mediates deleterious proinflammatory effects. NOS2 overexpression is associated with the release of proinflammatory cytokines such as IL- 1 [3, IL-6, IL-8 and TNFa.

In the invention, inhibition of IL-8 cytokine, which is indeed necessary for natural antiviral immune responses, but causes an acute increase in circulating pro-inflammatory cytokine levels with hyperactivation of the immune system, resulting in problems such as systemic inflammation, hyperferritinemia, hemodynamic instability and multi-organ failure, is aimed to be used in the treatment of COVID-19. Inhibition of IL-8 cytokine is provided by the thiosemicarbazone molecule of the invention and the method of obtaining this molecule.

With the invention, fully protecting and healing the wounds in the BEAS-2B cells, which are the bronchial epithelial cell line, in the later stages of the COVID-19 disease, moisturizing the wounds sufficiently, reducing the pain in the wounds and cleaning the wounds from harmful substances are aimed. Said effects are provided by a thiosemicarbazone molecule with wound healing properties and the synthesis method of this molecule. Contrary to the active wound healing agents in the state of the art, no synthetic material is needed to provide said wound healing effect. Therefore, the negative effects of bodily functions arising from said synthetic substances are prevented.

Description of Drawings

Figure 1. MS spectrum data of the compound N ¹ -1 ,1 ,1 -Trifl uoroacety lacetone-N ⁴ -4- methoxysalicylidene-S-methylthiosemicarbazidato-cobalt(ll) Figure 2. ORTEP diagram obtained from X-ray analysis showing the molecular structure of the thiosemicarbazone-cobalt complex that is the subject of the invention.

Figure 3. Bar graph showing the percent inhibition of 3CL main protease enzyme at 1 , 10, 25, 50, 100, 200, 300 pM concentrations of compound MS spectrum data of the compound N ¹ -1 ,1 ,1-Trifluoroacetylacetone-N ⁴ -4-methoxysalicylidene-S- methylthiosemicarbazidato-cobalt(ll)

Figure 4. Bar graph showing that compound N ¹ -1 ,1 ,1-Trifluoroacetylacetone-N ⁴ -4- methoxysalicylidene-S-methylthiosemicarbazidato-cobalt(ll)ha s a proliferative effect at low doses and maintains very high viability even at 100 pM concentration

Figure 5. Bar graph showing that compound N ¹ -1 ,1 ,1-Trifluoroacetylacetone-N ⁴ -4- methoxysalicylidene-S-methylthiosemicarbazidato-cobalt(ll) highly suppresses IL-8 gene expression

Figure 6. Bar graph showing that compound N ¹ -1 ,1 ,1 -Trifluoroacetylacetone-N ⁴ -4- methoxysalicylidene-S-methylthiosemicarbazidato-cobalt(ll) highly suppressesNOS2 gene expression

Figure 7. Microscope images showing high wound healing effect of compound N ¹ - 1 ,1 ,1-Trifluoroacetylacetone-N ⁴ -4-methoxysalicylidene-S-methylthiosemicarbazidato- cobalt(ll)

Figure 8. Column chart covering a 24-hour period showing the high wound-healing effect of compound N ¹ -1 ,1 ,1-Trifluoroacetylacetone-N ⁴ -4-methoxysalicylidene-S- methylthiosemicarbazidato-cobalt(ll)

Figure 9. Column chart covering 48 hours showing high wound healing effect of compoundN ¹ -1 ,1 ,1 -Trifluoroacetylacetone-N ⁴ -4-methoxysalicylidene-S- methylthiosemicarbazidato-cobalt(ll)

Detailed Description of the Invention

The invention relates to a thiosemicarbazone molecule with anti-inflammatory and wound healing properties that inhibits C30 endopeptidase or in other words 3- chymotrypsin-like protease (3CL major protease) enzyme of SARS-COV-2 virus, lnterleukin-8 (IL-8) cytokine and inducible nitric oxide synthase (NOS2) enzyme of human bronchial epithelial cells and the synthesis method of this molecule.

The synthesis reaction of an anti-inflammatory and wound-healing thiosemicarbazone molecule, in other words, the thiosemicarbazone-cobalt complex compound, which is the subject of the invention, that inhibits the IL-8 cytokine and NOS2 enzyme, is shown in Formula 2 below.

Reaction 1

The chemical name of the thiosemicarbazone-cobalt complex compound of the invention is N ¹ -1 , 1 , 1 -T rifluoroacetylacetone-N ⁴ -4-methoxysalicylidene-S- methylthiosemicarbazidato-cobalt(ll). Synthesis method of this thiosemicarbazone- cobalt complex compound comprises the process steps of: i. obtaining solution 1 by dissolving 0.37 g, 1.0 mmol of 1 ,1 ,1 - Trifluoroacetylacetone-S-methylthiosemicarbazone hydroiodide and 0.15 g, 1.0 mmol of 4-methoxysalicylaldehyde in 10 mL of methyl alcohol in a Schlenk tube, ii. obtaining solution 2 by dissolving 0.24 g, 1.0 mmol of cobalt(ll) chloride hexahydrate (COCI2.6H2O) in 5 mL of methyl alcohol, iii. obtaining solution 3 is by adding solution 2 dropwise to solution 1 , iv. adding 0.2 mL of triethylamine (EtsN) to solution 3 and then stirring at 50°C for 1 hour, v. cooling the resulting reaction mixture (solution 3) to room temperature, vi. filtration of the burgundy coloured solid formed 24 hours after the solution 3 was left to cool, vii. recrystallization of the burgundy solid from a solvent mixture of dichloromethane-methyl alcohol (3:1 , v/v), and viii. drying the filtered crystals over P2O5 in vacuo.

Figure 1 shows the MS spectrum of the thiosemicarbazone-cobalt complex compound of the invention.

The mass of compound N ¹ -1 ,1 ,1 -Trifluoroacetylacetone-N ⁴ -4-methoxysalicylidene-S- methylthiosemicarbazidato-cobalt(ll) (Formula 1 ) with the closed formula C15H14COF3N3O3S is 432.28 g/mol. The values found for the ratios of the elements in this compound, % by mass: C is 41 .44; H, 3.02; N, 9.58; S, 7.01 .

IR (crrr ¹ ): v(C=N ¹ ) 1600, v(N ² =C) 1569, v(N ⁴ =C) 1559, v(C-O) 1174, 1161.

+ c ESI MS (m/z, Relative Abundance ⁰ /,.): [M] 432.1 (100%)

Figure 2 shows the ORTEP diagram obtained from X-ray analysis showing the molecular structure of the thiosemicarbazone-cobalt complex that is the subject of the invention. The independent reflections to be used in the solution of the crystalline structure of the complex were collected using the MoKa beam with the BRLIKER D8- QLIEST diffractometer in the X-ray laboratory. The SHELXS-2013 software in the WinGX package program was used during the structure resolution of the crystals, and the SHELXL-2013 software was used for the purification of the dissolved structures. Mercury software was used to visualize the results found. The details obtained as a result of the treatment are given in Table 1 and the bond lengths and angles are given in Table 2.

Table 1. Crystalline data and purification details of the thiosemicarbazone-cobalt complex compound of the invention The expression FOOO in the table is a structure factor expression of the zero ^th order (h = k = I = 0), evaluated in the case of F <ooo). For X-rays, F<ooo) is a positive number. It refers to the number of active electrons in the unit cell.

The linear absorption coefficient (mm ^-1 ) in the table depends on the mass absorption coefficient of the atoms in the molecule, the crystal density and the mass ratio of each atom in the molecule. If the linear absorption coefficient of the material is known, it can be understood which thickness of the material should be used with the least loss of intensity. In order to obtain a maximum intensity reflection, it is necessary to select the appropriate crystal thickness.

Again, the value indicated by S(F ² ) in the table, which is another criterion in determining the degree of accuracy of the structures at the purification stage, is the "Settlement factor" (Goodness of Fit). The value of this factor, indicated by S, should be at the end of the purification. Deviation of S value from 1 indicates inconsistency in the structure. It is unitless. Its value should be in the range of 0.8-1 .2.

The small residual charge values (Apmax, Apmin) in the electron density maps are another criterion that reveals the successful outcome of the purification process. In Table 1 above, which shows the crystalline data and purification details of the thiosemicarbazone-cobalt complex compound that is the subject of the invention, the charge values of the said compound are shown as Apmax, Apmin. Electron density maps consist of peaks corresponding to atomic positions. The intensities of the peaks, the distances and angles between the peaks give us an outline of a molecule that we can construct. Due to the scattering of X-rays from electrons, high-intensity peaks correspond to atoms with a greater number of electrons, and therefore to atoms with a larger atomic number. Its unit is electron/angstrom ³ . Its value should be less than 0.1/- 0.1.

Rint shows the Merge (measure of precision I repeatability) error. With a high Rint value, possible sources of error are identified. It is unitless. Its value must be less than 0.1.

The R in Table 1 is the "confidence factor", which is known as the R index in crystallography, which shows the agreement between the experimental data and the calculated data. The smaller the difference between the experimental and calculated structure factors, the more accurate the crystal structure solution. If the purification was successful, the R value should be quite small. It is unitless. Its value is expected to be 0.1 or less.

Another criterion that reveals the accuracy of the structure after the purification process is the "weighted reliability factor" indicated in Table 1. With the weighted reliability factor, it is aimed to use less reflections with high error in the refinement and thus to provide a better convergence to the real structure, w is the weight function. The weighted confidence factor Rw can take values slightly larger than the confidence factor R. It is unitless. Its value is expected be less than 0.25.

The a, b, c vectors in Table 1 form the reference axes of the crystal, and these vectors can be perpendicular to each other or at different angles between the said vectors. The lengths of these vectors and the angles between them reveal the properties of a particular crystal. Its unit is angstrom.

Table 2. Crystalline data and purification details of the thiosemicarbazone-cobalt complex compound of the invention

It has been experimentally proven that the thiosemicarbazone-cobalt complex of the invention highly inhibits the 3CL main protease enzyme. In addition, it has also been experimentally proven that it has anti-inflammatory and wound-healing effects on BEAS-2B cells, the bronchial epithelial cell line, which are the cells mainly infected by the virus.

The inhibitory activity of the synthesised substance against the SARS-CoV-2 3CL major protease enzyme was evaluated with a commercially available assay (BPS Bioscience 3CL Protease - Catalog #79955). In the analysis performed according to the kit protocol, it was measured how much percent the thiosemicarbazone-cobalt complex, which is the subject of the invention, inhibited the 3CL main protease enzyme at 1 , 10, 25, 50, 100, 200, 300 pMconcentrations. The inhibitory effect of the thiosemicarbazone-cobalt complex, which is the subject of the invention, was analysed by comparing it with an inhibitory standard (GC376) included in the test kit. According to the results obtained, it has been proven that the thiosemicarbazone-cobalt complex, which is the subject of the invention, has a very high inhibitory effect. In Figure 3, there is a bar graph showing the inhibition level of 3CL main protease enzyme at 1 , 10, 25, 50, 100, 200, 300 pM concentrations of compound N ¹ -1 ,1 ,1 -Trifluoroacetylacetone-N ⁴ - 4-methoxysalicylidene-S-methylthiosemicarbazidato-cobalt(ll) in percentages.

In Figure 4, there is a bar graph showing that compound N ¹ -1 ,1 ,1- Trifluoroacetylacetone-N ⁴ -4-methoxysalicylidene-S-methylthiosemicarbazidato- cobalt(ll), a thiosemicarbazone-cobalt complex, which is the subject of the invention, has a proliferative effect at low doses and maintains viability at a very high rate even at 100 pM concentration. The effect of thiosemicarbazone-cobalt complex synthesized in the invention on cell viability at concentrations of 1 , 10, 50, 100, 200, 300 pM was analysed by the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) (Sigma, M-5655) method. 10 pl of the synthesised complexes were added to the 96- well plates at the concentrations of 1 , 10, 50, 100, 200, and 300 pM, respectively. Afterwards, 90 pl of BEAS-2B cells prepared as 10 ⁵ cells/ml were added to 96-well plates and incubated at 37°C to determine the cell viability effects of the thiosemicarbazone-cobalt complex, which is the subject of the invention, after 48 hours. After 48-hours incubation, 10 pl of freshly prepared MTT (5 mg/ml in PBS) was added to the wells and incubated for 3 hours. DMSO was added to dissolve the formazan crystals formed by reduction of MTT in living cells. After being kept in the dark overnight, it was measured in the spectrometer at 570 nm the next day. The absorbance value of the DMSO control was subtracted from the absorbance of the other wells. As a result, Cell viability was calculated by comparison with control cells according to the following formula:

% Cell Viability = (1- Absorbance of wells incubated with substance) I (Absorbance of controls) x 100

As a result of MTT experiments, it has been proven that the thiosemicarbazone-cobalt complex, which is the subject of the invention, has a proliferative effect at low doses and maintains viability at a very high level even at 100 pM concentration. In order to understand the anti-inflammatory effects of the thiosemicarbazone-cobalt complex, which is the subject of the invention, an inflammation model was created in BEAS-2B cells with tumour necrosis factor (TNFa) cytokine. TNFa is a cell signalling cytokine involved in system icinflammation. BEAS-2B cells were inoculated into 6-well plates as 3x10 ⁶ /3ml and incubated for 24 hours with 10, 50, 100 pM concentrations of thiosemicarbazone-cobalt complex. After incubation, 5 ng/ml TNF-a was added according to the experimental groups and incubated for 24 hours. After incubation, RNA isolation of BEAS-2B cells was performed using the kit (RNeasy Mini Kit (Qiagen)). Complementary DNA (cDNA) synthesis was performed from 500 ng RNA with the kit (All-In-One 5X RT MasterMix) by measuring the concentrations of the isolated RNAs. IL-8 mRNA gene expression levels in BEAS-2B cells incubated with agents and TNF-a was analysed by Real Time Polymerase Chain Reaction (qRT-PCR) method using kit (BrightGreen 2X qPCR MasterMix-ROX). IL-8 and NOS2 mRNA gene expression levels were normalised with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequences are given in the sequence list as IL-8 forward primer SEQ ID NO.1 and IL-8 reverse primer as SEQ ID NO.2. Furthermore, NOS2 forward primer is given as SEQ ID NO.3 and NOS2 reverse primer as SEQ ID NO.4.

Figure 5 shows the bar graph showing that the thiosemicarbazone-cobalt complex, which is the subject of the invention, highly suppresses IL-8 gene expression. In addition, as can be seen in detail in Figure 5, the thiosemicarbazone-cobalt complex of the invention suppresses IL-8 gene expression at very high rates even when an inflammatory response is generated in BEAS-2B cells.

In order to understand the anti-inflammatory effect of the thiosemicarbazone-cobalt complex compound, which is the subject of the invention, the activity of said complex compound on NOS2 gene expression was also examined. In the analysis of efficacy, it has been shown that the thiosemicarbazone-cobalt complex compound has an inhibitory effect against the NOS2 gene expression increase due to TNFa cytokine. As a result, it has been proven that the thiosemicarbazone-cobalt complex compound of the invention has a very high suppressive effect against NOS2 gene expression in both controls and TNFa cytokine-induced inflammation model in BEAS-2B cells at a concentration of 10 pM. In Figure 6, there is a bar graph showing that the thiosemicarbazone-cobalt complex compound, which is the subject of the invention, suppresses NOS2 gene expression at a very high rate.

Wound healing test was performed to investigate the wound healing effect of the thiosemicarbazone-cobalt complex compound, which is the subject of the invention, in BEAS-2B cells. BEAS-2B cells were inoculated into a 12-well cell culture dish with 10 ⁶ cells in each well. The seeded BEAS-2B cells were incubated for 24 hours with 10, 50, 100 pM concentrations of the thiosemicarbazone-cobalt complex of the invention after 24 hours. Afterwards, 5 ng/ml TNF-a was added for inflammation and incubated for another 24 hours. After approximately 24 hours, a wound was created with a 10 pL micropipette tip on the surface, which filled the surface of the cell culture container to which they were attached, almost 100%. While creating the wound, the pipette tip was drawn at a constant speed from one end to the other on the cell culture dish in one go. Thiosemicarbazone-cobalt complex and BEAS-2B cells that were not incubated with TNF-a were used as the control group. Wound line was visualized by phase contrast microscope at 0, 24 and 48 hours and wound healing activity was determined by comparing with the control group. At all concentrations, the healing rate was found to be very high compared to the control at 24 and 48 hours after wound formation. In addition, it has been proven that the thiosemicarbazone-cobalt complex, which is the subject of the invention, has a higher rate of wound healing effect compared to the control in the inflammation model induced by TNFa cytokine. In Figure 7, there are microscope images showing that the thiosemicarbazone-cobalt complex, which is the subject of the invention, has a high wound healing effect. In Figure 8 and Figure 9, respectively, there are column charts showing the wound healing rates (%) at 24 and 48 hours.

In an embodiment of the invention, the synthesis method of N ¹ -1 ,1 ,1 - Trifluoroacetylacetone-N ⁴ -4-methoxysalicylidene-S-methylthiosemicarbazidato- cobalt(ll) compound, which is a thiosemicarbazone-cobalt complex compound, comprises the process step of: i. obtaining solution 1 by dissolving 0.37 g, 1.0 mmol of 1 ,1 ,1 - Trifluoroacetylacetone-S-methylthiosemicarbazone hydroiodide and 0.15 g, 1.0 mmol of 4-methoxysalicylaldehyde in 10 mL of methyl alcohol in a Schlenk tube, ii. obtaining solution 2 by dissolving 0.24 g, 1.0 mmol of cobalt(ll) chloride hexahydrate (COCI2.6H2O) in 5 mL of methyl alcohol, iii. obtaining solution 3 is by adding solution 2 dropwise to solution 1 iv. adding 0.2 mL of triethylamine (Et3N) to solution 3 and then stirring at 50°C for 1 hour, v. cooling the resulting reaction mixture (solution 3) to room temperature, vi. filtration of the burgundy coloured solid formed 24 hours after the solution 3 was left to cool, vii. recrystallization of the burgundy solid with a solvent mixture of dichloromethanemethyl alcohol (3:1 , v/v), and viii. drying the filtered crystals over P2O5 in vacuo.

The N ¹ -1 ,1 ,1-Trifluoroacetylacetone-N ⁴ -4-methoxysalicylidene-S- methylthiosemicarbazidato-cobalt(ll) compound shown with Formula 1 , which is the subject of the invention, is suitable for use as an inhibitor of 3CL main protease enzyme of SARS-CoV-2 virus and IL-8 cytokine and NOS2 enzyme of human bronchial epithelial cells in the treatment of COVID-19 disease caused by SARS- CoV-2 virus. Therefore, a drug containing the N ¹ -1 ,1 ,1 -Trifluoroacetylacetone-N ⁴ -4- methoxysalicylidene-S-methylthiosemicarbazidato-cobalt(ll) compound represented by Formula 1 of the invention is indicated for the treatment of COVID-19 disease caused by the SARS-CoV-2 virus.

The cobalt (ll)ion with the central atom in the N ¹ -1 ,1 ,1 -Trifluoroacetylacetone-N ⁴ -4- methoxysalicylidene-S-methylthiosemicarbazidato-cobalt(ll) compound shown with Formula 1 , which is the subject of the invention, is a component of vitamin B12 and prevents iodine uptake by the thyroid. In addition, cobalt(ll) ion controls the transfer of enzymes such as homocysteine methyltransferase and plays a role in methionine metabolism.

The triethylorthoformate metal atom used in the synthesis of the N ¹ -1 ,1 ,1- Trifluoroacetylacetone-N ⁴ -4-methoxysalicylidene-S-methylthiosemicarbazidato- cobalt(ll) compound, which is the subject of the invention, removes water and similar solvent molecules, allowing the cobalt ion to combine with the thiosemicarbazone derivative and the aldehyde molecule faster and to bond to the donor atom system (N2O2) more easily. Triethylamine added in the last step is a base and it facilitates the binding of the cobalt ion to the oxygen atoms by removing the H atom from the -OH groups in the ligand system. The use of Schlenk tube in the reaction cuts off the contact of the mixture with air as much as possible and protects the reaction from the oxidizing effects of air oxygen.

References

[1 ] F.A. Cotton, D.M.L. Goodgame, M. Goodgame, J. Am. Chem. Soc. 83 (1961 ) 4690- 4699.

[2] S. Okamoto, L.D. Eltis, Metallomics 3 (2011 ) 963-970.

[3] Oh, R.; Brown, D. L. Am. Fam. Physician 2003, 67, 979.

[4] Barceloux, D. G. J. Toxicol. Clin. Toxicol. 1999, 37, 201.

[5] A.K. Renfrew et al. I Coordination Chemistry Reviews 375 (2018) 221-233.

[6] F.P. Dwyer, E.C. Gyarfas, W.P. Rogers, J.H. Koch, Nature 170 (1952) 190-191.

[7] F.P. Dwyer, I.K. Reid, A. Shulman, G.M. Laycock, S. Dixson, Aust. J. Exp. Biol. Med. Sci. 47 (1969) 203-218.

[8] F.P. Dwyer, E.C. Gyarfas, R.D. Wright, A. Shulman, Nature 179 (1957) 425- 426.

[9] A. Bottcher, T. Takeuchi, K. I. Hardcastle, T. J. Meade, H. B. Gray, D. Cwikel, M. Kapon, Z. Dori, Inorg. Chem. 1997, 36, 2498-2504.

[10] J. A. Schwartz, E. K. Lium, S. J. Silverstein, J. Virol. 2001 , 75, 4117-4128.

[11 ] A. Y. Louie, T. J. Meade, Proc. Natl. Acad. Sci. USA 1998, 95, 6663-6668.

[12] O. Blum, A. Haiek, D. Cwikel, Z. Dori, T. J. Meade, H. B. Gray, Proc. Natl. Acad. Sci. USA 1998, 95, 6659-6662.

[13] M. C. Heffern, V. Reichova, J. L. Coomes, A. S. Harney, E. A. Bajema, T. J. Meade, Inorg. Chem. 2015, 54, 9066-9074.

[14] 1. Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: A physiologic messenger. Ann Intern Med 1994; 120: 227-37.