The invention relates to the production of thymoquinone, a bioactive material proven to be effective in cancer studies using Ay-Yildiz heterogeneous catalyst obtained by the hydrothermal method, one of the environmentally friendly methods. PRIOR ART

Today, many studies in the fields of human and veterinary medicine and food and the environment promote the use of herbal products both in the treatment of diseases and in preventive medicine due to the risk posed by drugs and chemicals used for the protection of human and animal health. Preparations obtained from black cumin and black cumin are widely used in the alternative treatment of many diseases such as various rheumatism and inflammatory diseases, diuretic, asthma, degasser and jaundice, especially cancer, in the Middle East and some Asian countries as in our country.

It is the seed of Nigella sativa plants, commonly known as black cumin, grown in countries belonging to the Ranunculaceae family and mostly bordering the Mediterranean Sea. Antioxidant activity, antitumor activity, anti-inflammatory activity, antibacterial activity and stimulant effect of this seed oil on the immune system have been reported. Nigella sativa seeds contain significant amounts of mineral elements. The most abundant element in the black cumin seed is potassium (K), followed by phosphorus (P) and calcium (Ca). According to the decrease in quantities, the other elements present in the seed are Mg, Na, Fe, Zn, Mn and Cu. Nigella seeds contain high amounts of minerals (Mn, Zn, Cu and Fe). Thymoquinone is the main ingredient in black cumin. This compound is rarely found in black cumin. It is a very expensive compound because it is found in small quantities and can be obtained in very small amounts as a result of the extract.

Thymoquinone (C10H12O2; 2-isopropyl-5-methyl-1,4-benzoquinone), the basic bioactive component of black cumin essential oil, has been used as an antidiabetic, antioxidant, anti-inflammatory and antineoplastic drug for more than 2000 years. Studies have shown that thymoquinone has an inhibitory effect on cell proliferation in many cancer species. Types of cancer that thymoquinone is effective include breast cancer, ovarian cancer, intestinal cancer, pancreatic cancer, uterine cancer, neoplastic keratinocytes, human osteosarcoma, soft tissue cancer, lung cancer. Thymoquinone has also been reported to inhibit hormone-refractory prostate cancer by targeting the androgen receptor and transcription factor E2F-1.

Thymoquinone has been shown to act as an anti-tumor by three main mechanisms. The first and most famous is that it forces tumor cells to 'apoptosis', in other words, 'cell suicide'. In pathology, this is called 'programmed cell death' and it is the most important natural defence mechanism of the body against cancer. The second is' inhibition of angiogenesis', in other words, preventing the formation of new vessels that nourish the tumor. In order for the tumor to grow, it must feed and it must become bloodshot, and it does so by creating new vessels, which is what thymoquinone prevents. The third is' cell cycle arrest', in other words, 'cell growth stopping'. This means: As the tumor grows, each cell enters a special cycle of growth, and comes out of that cycle as two cells, which increases the whole tumor mass. Thymoquinone blocks this cycle. This is also the mechanism of action of many chemotherapy agents. The most worrying aspect of chemotherapy drugs is the side effects they cause to normal cells due to their lack of selectivity.

FIGURES LITS

Figure 1: General Process Flow Diagram of the Target Compound Produced Using Catalytic Conditions Optimized for Thymoquinone Output from Thymol Subject to the Present Invention.

Figure 2: General Synthesis Diagram of the Catalyst, Which Is Produced with Organic and Inorganic Compounds to Be Used to Produce the Catalyst Which Will Enable the Transformation of Thymol to Thymoquinone, As Well As Parameters to Be Used in Subcritical Conditions

Figure 3: Molecular Demonstration of the Unit Cell of the Polymeric Catalyst Synthesized under Flydrothermal Conditions

Figure 4. Demonstration of Molecules of the Carboxylic Acid Organic Compound of Pyrazine that Will Create Pyrazine Under Flydrothermal Conditions Figure 4a. Demonstration of Molecules of 2-Pyrazine Acetic Acid Organic Compound that will Form Pyrazine under Flydrothermal Conditions Figure 4b. Demonstration of Molecules of the Pyrazine -2 Carboxylic Acid Organic Compound of Pyrazine that Will form Pyrazine Under Hydrothermal Conditions Figure 4c. Demonstration of Molecules of the Pyrazine-2 Thio-Carboxamide Carboxylic Acid Organic Compound of Pyrazine that Will Create Pyrazine Under Hydrothermal Conditions

Figure 4d. Demonstration of Molecules of Pyrazine-2 Amide Oxime Organic Compound that will Form Pyrazine under Hydrothermal Conditions Figure 4e. Demonstration of Molecules of 2-(Trifluoromethyl) Pyrazine Organic Compound that will Form Pyrazine under Hydrothermal Conditions

The Number given in the figures correspond to:

1. Catalysis

2. Solvent

3. Oxidant 4. Reaction Temperature

5. Reaction Time

6. Metal Salts

7. Mineralizers

8. Hydrothermal Solvent 9. Groups separated under hydrothermal conditions

10. Hydrothermal Synthesis: Subcritical water conditions

11. Bridge Compound M: Metal: Cu, Co, Ni, Zn

X: NOs-, CHsCOO-, Ch, Br, h

BRIEF DESCRIPTION OF THE INVENTION

The object of the present invention is to manufacture the high added value thymoquinone compound based on the cheap compound thymol. It is the development of an experimental environment with high-efficiency catalyst synthesis and optimum catalytic conditions for this production. The study, which incorporates various innovations (High transformation, 100% selectivity, green chemistry), is tried to be developed in different ways in the scientific literature. Homogeneous, heterogeneous and biocatalysts are also included in these studies. However, this study draws attention in terms of high product selectivity and transformation as well as different methods.

The most important prospective goal of the study is to produce thymoquinone, which is used in cancer treatments, in economically high purity. For this, a heterogeneous catalyst synthesized under hydrothermal conditions, which is an environmentally-friendly method, had been used.

Reducing the negative side effects of chemotherapy drugs and increasing the number of studies with thymoquinone in cancer research are our secondary objectives. It is one of our future plans to create the conditions under which the conversion of thymoquinone with different heterogeneous catalysts will be achieved and to contribute to the spread of thymoquinone in the food, cosmetics and packaging sectors, which are other uses of thymoquinone. The selectivity of this catalyst, and the absence of any side-product formation, does not create any purification problems. For this reason, it can be used effectively if it is industrialized.

DETAILED DESCRIPTION OF THE INVENTION

The invention is the production of thymoquinone using Ay-Yildiz heterogeneous catalyst (1) synthesized by the hydrothermal method, one of the environmentally friendly methods. Thymoquinone, a very expensive compound, could be produced purely from the cheap compound, thymol, which has been chosen as the starting material. It has been observed that the heterogeneous catalyst we synthesized is selective and allowed high added value thymoquinone to form 94% without any by-products.

Green Chemistry is the design, development and application of chemical products and processes to reduce or eliminate the use and production of substances harmful to human health and the environment. In contrast to regulatory requirements to prevent pollution, Green Chemistry is an innovative, non-regulatory and economically driven approach to sustainability. One of the most important methods used within the scope of Green Chemistry is the hydrothermal method we use in catalyst synthesis. In this method, catalyst (1) has been produced using only water without using any organic solvent. One of the greatest advantages of water use is that it is environmentally beneficial and cheaper than other solvents. It is non flammable, carcinogenic, mutagenic, non-toxic and thermodynamically stable. The method is quite advantageous in this respect compared to other conventional methods.

In our study, Ay-Yildiz catalyst has been synthesized under hydrothermal conditions in accordance with the rules of Green Chemistry and have not left contaminated waste to the environment since organic solvents have been not used after synthesis. The greatest advantage of the method used compared to other technological systems is that the catalyst (1) obtained in water is insoluble in many organic solvents, especially water, and acts as a heterogeneous catalyst (1). This feature means that catalyst (1) can be used multiple times in catalytic studies and more thymoquinone can be obtained with less catalyst (1).

Another important situation is that this heterogeneous catalyst (1) synthesized under hydrothermal conditions is selective in thymoquinone output due to its high purity and only the target product thymoquinone has been obtained without any by products. It will also provide a great advantage because the catalyst (1) is heterogeneous and can be easily removed from the environment and reused after the procedure.

The present invention process consists primarily of three steps:

- Production of catalyst (1 ),

- Production of thymoquinone from thymol, - Purification of thymoquinone from thymol.

In order to achieve the object of the invention, the experimental oxidation reaction (Catalytic Condition), the synthesis of catalyst (1) under hydrothermal conditions and the general process flow diagram of the studies of the catalyst (1) obtained are shown in the attached picture. The basic steps covering the present invention are accompanied by many intermediate steps. All steps are shown in the diagrams given in Figures 1 and 2. When all process steps are examined, the process is basically the production of catalyst (1) and thymoquinone from thymol under optimum catalytic conditions determined using the catalyst (1) produced. When the treatment processes are examined, the first step is the formation of bridge compounds (11) to be used under subcritical conditions and the production of catalyst (1) by binding metal and/or metals to these compounds. In the second step, thymoquinone has been produced by applying the solvent (2), oxidant (3) to be used in a catalytic environment together with the catalyst (1) obtained purely and the reaction temperature (4) and duration (5) to be applied together.

Production of catalyst (1) started with the use of organic ligands that will undergo a change in the subcritical water environment (Figure 4). When selecting these ligands, at least one of 2,3-pyrazine carboxylic acid, pyrazine carboxylic acid, 2-pyrazine acetic acid, 6-(Methylamino) pyrazine-2 -carboxylic acid, pyrazine-2- thiocarboxamide, pyrazine-2-amide oxide and 2-(Trifluoromethyl) pyrazine compounds that can form bridge ligands have been used in the subcritical environment. The reason for the use of these compounds is that they can lose substrate groups bound under hydrothermal conditions and form as bridge ligands.

Organic compounds to be used in subcritical water conditions must be thermally stable under high temperature and pressure. Therefore, the 2,3-pyrazine carboxylic acid compound has been used. Since the melting point of this compound is 187-189 °C (centigrade), the mold compound does not undergo any degradation under pressure and at 180 °C. Due to this feature, it shows the ability to bind the metal to itself at different pH values. Another important feature of selecting this compound is that it can bind the metal to itself from multiple points due to its numerous functional groups. Even if the bound -COOH groups break, they exhibit the ability to bind to metal through the nitrogen atoms in the main structure. Finally, the main structure obtained from the use of this compound is the pyrazine compound, which acts as a bridge between many metal atoms and allows the formation of the polymeric structure. Organic compounds other than 2,3-pyrazine dicarboxylic acid are compounds with similar characteristics as mentioned above, and differ in operating temperature, operating time and pH values from each other. Therefore, these organic compounds are compounds that can form a similar pyrazine structure under different experimental conditions from the catalyst (1) obtained using 2,3- pyrazine carboxylic acid ligand and show properties to bind the metal to itself.

Then, mineralizer (7) and metal salt (6) have been added to provide complexity to the bridge ligands obtained and their coordination to the metal has been ensured through nitrogen groups. At least one of H2C2O4, NaOH, KOH, NH3 and piperazine has been used as mineralizer (7). These chemicals have been selected to determine the pH level of the solution medium rather than interacting with reagents. One cause of group separation under hydrothermal conditions (e.g. decarboxylation) is the type of mineralizer (7) used. There is no change in organic compounds when such compounds are not used. These compounds are used to determine the degree of acidity or basicity of the solution medium. Under these conditions, several changes occur in organic compounds when the optimum pH value is reached. As a result of the use of these mineralizers (7) in subcritical conditions, chemical reactions occur at the optimized reaction temperature and changes occur in organic compounds. No compound replacement or catalyst synthesis has been performed when chemicals such as CH3COOH (Acetic acid), tritanol amine or HF (Hydrofluoric acid) have been used to determine solution acidity or baseline other than specified mineralizers (7) (H2C2O4, NaOH, KOH, NH3 and piperazine). This indicates that only the mineralizers used (7) allow the catalyst to be synthesized under optimized conditions.

Copper, cobalt, nickel and zinc-containing nitrate, acetate or halogenate salts have been selected as metal salts (6). These metal salts (6) have been used because they are very active in coordination with nitrogen atoms. When examining the main structure in organic compounds used, it is known that the remaining groups of nitrogen have more interest in the transition metals. This has been observed by using lanthanum salts such as lanthanum, serium and samarium as metal salt (6). Under similar experimental conditions, when lanthanide salts have been used instead of the transition metals above, powdered products whose structure could not be illuminated in the environment have been obtained. For this reason, transition metals have been preferred as metals. Copper, cobalt, nickel and zinc salts preferred from transition metals are the most preferred metals in both industry and catalyst production. In addition, they are catalysts obtained by binding to organic structure together with mineralizers (7) used under determined hydrothermal conditions (1). Stoichiometry and pH values, which are the most important parameter in the coordination, are provided by the mineralizers (7) as well as by the molar proportions of organic compounds and metals. Metal: Ligand: Mineralizer: Water ratios have been used as 1.00: 2.00: 1.00: 1858 / 2.00: 1.00: 1.00: 1858 / 2.00: 2.00: 1.00: 1858 / 2.00: 2.00: 2.00: 1858. With optimization studies conducted under these rates, the most effective pH value has been reached and high purity catalyst (1) production has been provided under hydrothermal conditions.

Another step is the production of catalyst (1) in a closed system reactor under hydrothermal conditions with hydrothermal solvent (8), groups separated under hydrothermal conditions (9) and hydrothermal synthesis: subcritical water conditions (10) by optimizing the reaction temperature (4) and duration (5) required for the formation of catalyst (1). For this, studies have been carried out with reaction temperature (4) of 150, 160, 170 and 180 °C respectively and reaction time (5) of 24, 36, 48, 72, 96 and 120 hours respectively. When the reactions have been completed, cooling has been carried out gradually and the reactor has been opened until it reaches room temperature and the catalyst (1) has been separated from the solution phase and dried.

After obtaining catalyst (1), thymoquinone production step from thymol has been performed using the catalyst (1) obtained in the second part (Figure 1). Catalytic studies have been carried out in flask with conventional methods. The thymol compound, which is the input substance, has been taken and dissolved with the help of solvent (2). Then, by adding oxidant (3) to it and mixing process with the help of a magnetic mixer, reaction temperature (4) for catalytic transformation has been adjusted by adding a catalyst (1) and mixing process (5) has been carried out during the reaction period determined for the realization of catalytic transformation. For this purpose, thymol oxidation has been carried out in an oil bath with a temperature-controlled magnetic mixer in a two-necked flask at room temperature, 60 and 80 °C, respectively. Thymol and solvent are added to the flask.

As is known, there are many organic or inorganic solvents. The solvent used in the catalyst studies should have important properties such as fully dissolving the input material, not reacting with the input material and removing it from the medium at the end of the reaction. The reasons for selecting the selected solvent or solvent mixtures are that they have all the aforementioned properties. At least one of methanol, ethanol, chloroform, dichloromethane, acetonitrile and methanol/acetonitrile/water mixtures has been used separately as solvent (2). These solvents (2) have been used because the best distinction in catalytic transformations and analyses is achieved with these solvents (2). The reaction is then continued by adding at least one of the hydrogen peroxide and tert-butyl hydroperoxide chemicals to the reaction media. For thymoquinone release from thymol, an oxidizing agent is absolutely needed in the media. Therefore, hydrogen peroxide and tert-butyl hydroperoxide oxides have been used. These chemicals are active chemicals that meet the oxygen needs in the media. No efficacy is observed when working with hydrogen peroxide at or above 80 °C. Because it starts to fragment above this temperature. Flowever, the most important advantage of hydrogen peroxide is that it can easily degrade at 80-90 °C at the end of the reaction and transform into water. In this sense, it can be easily removed from the media without leaving waste to the media. Tert-butyl hydroperoxide has higher thermal stability compared to other peroxides. Therefore, it is more appropriate to use this oxidant at high temperatures. At least one of the oxidants has been added to the reaction mixture. Then, the catalysts (1) produced in the flasks have been added at different rates and the reaction has been initiated. Since the amount of thymoquinone produced by the increase in the amount of catalyst (1) has been determined to increase, the amount of catalyst (1) has been used differently. After obtaining thymoquinone using a catalyst (1), thymol and thymoquinone have been separated from each other and the thymoquinone compound has been obtained purely. For this purpose, the mixture has been filtered and the catalyst (1) has been removed from the media. The solvent (2) has been separated from the remaining mixture by being evaporated under vacuum. The thymoquinone compound has been then sublimated from the remaining mixture and obtained under vacuum.

In this way, by using Ay-Yildiz heterogeneous catalyst (1) obtained by the hydrothermal method, one of the environmentally friendly methods, which is the main purpose of the study, thymoquinone, a bioactive material proven to be effective in cancer studies, has been produced. In developed countries, one of the three advanced sectors is the chemical sector. There are around 60 million known chemicals in the world, approximately 80 thousand of which are widely used in the industry. In Turkey, only 2600 of these chemicals have become producible. Chemical trade in the world is approximately $4 trillion and our country's share is less than 1%. In order to close the current account deficit of our country, many chemicals with high added value are required to be made more producible in the domestic market.

The thymoquinone compound produced is rarely found in black cumin and obtaining its pure output is hard. With this present invention, based on the cheap compound thymol, the conversion of thymoquinone in the catalytic medium has been carried out at high rates and vacuum and low-temperature thymoquinone crystals have been obtained with the help of the vacuum sublimation device. In this regard, this compound is produced which is not produced in Turkey and is widely used in many types of research. In the following stages, it will be possible to yield more production in industrial quantities. As a result of thymoquinone production in Turkey, it will be available in cancer studies and will make an important contribution to scientific studies.

There is no institution or organization that can produce thymoquinone chemical in Turkey and even it has been seen that it has a limited number of producers in the world. Due to the high cost of the compound, very few groups can carry out scientific studies. Considering that there are many scientists researching cancer in Turkey as well as in the world, a large market may emerge as a result of this study. Although the thymoquinone compound planned to be obtained is considered to be used in different cancer studies, thymoquinone has been reported to be used extensively in various rheumatism and inflammatory diseases, as diuretic, as anti-inflammatory effect in asthma, in the treatment of jaundice disease, as stimulant effect on the immune system, as anti-oxidant, as strengthener of the hypoglycemic immune system, in the treatment of metabolic disorders, anti-histamine treatments and as antimicrobial. With the present invention, the use of thymoquinone compound will become widespread and will be used in many scientific studies for the treatment of various diseases, especially cancer. Drugs that can be used for the treatment of diseases that are proven to be effective may be used as an active ingredient. In this sense, Turkey will be able to make positive contributions to world science.