TECHNICAL FIELD

The invention relates to a novel thiazolo[3,2-c]pyrimidine based anticancer compounds which may show effect on various cancer types (breast, liver, lung, colon, skin, prostate, kidney, ovarian, etc.) STATE OF THE ART

The cancer is a disease group approximately 110 types of which are diagnosed currently and known during or before the birth of the humankind. Cancer affects many people from different races especially in this age, decreases the life quality and often causes fatal consequences by progressing aggressively. The number of the patients diagnosed with cancer is up to 70 in 2014 only. 50% of the diagnosed patients consist of patients with lung, breast and thyroid cancer.

4 main methods should be followed for cancer treatment as surgery, radiotherapy, chemotherapy and hormone therapy. In chemotherapy which is the most important one among these methods, various combinations of strong anticancer molecules such as 5- flourouracil, cyclophosphamide, methotrexate, doxorubicin, camptothecin, paclitaxel, and docetaxel. Developing various anticancer molecules is important with regard to achieving success in cancer treatments.

Anticancer agents used in cancer treatments globally vary by the cancer types they are used. However, the biggest problem in the cancer treatment is that healthy cells dies in both radiotherapy and chemotherapy together with cancer cells and that side effects occur as well as various complications. For example, doxorubicin which is one of the molecules used in the cancer treatment may cause cardiotoxic side effects when it is used for a long time period. Also, the other widely known anticancer molecules; camptothecin, paclitaxel, and docetaxel are in a disadvantageous position because of their high dose side effects, because they are isolated in very small amounts naturally and because they have a complex structure and they are obtained by multi-stage synthetic ways. Thus, obtaining new molecules which are capable of doing apoptosis in low doses and which have less side effects, with easier synthetic methods has become important with regard to both atom-economic reasons and time.

Anticancer of our work and thiazolopyrimidines which are capable of performing various activities have 3 main structural subgroups. (Figure 1)

It is known in the literature that thiazolo[3,2-a]- and thiazolo[4,5-cf]-pyrimidine derivatives being the first two of the subgroups may be prepared by various multi-stage synthetic methods. However, some of the said preparation methods have low efficiency and difficult product isolation.

Killing effects of these two main thiazolopyrimidine compounds that are commonly seen in the literature on various human cancer cells (HepG2-liver, PC-3-prostate, HCT- 116-colon, A549-lung, A431-skin, T98G-glioblastoma, HL-60-leukemia, SF-268-CNS and MCF-7-breast) are also known.

Furthermore, in the scientific literature there has not been any compound example or drug molecule having different biological activities including anticancer effect of thiazolo[3,2-c]pyrimidines being the last main structural group of thiazolopyrimidines.

Consequently, there is a need for a development in the relevant technical field because of the inefficiency of the present solutions about the subject and disadvantages and deficiencies mentioned above. DESCRIPTION OF THE INVENTION

The present invention relates to the preparation method of thiazolo[3,2-c]pyrimidine based anticancer compounds developed for providing novel advantages in the technical field and to anticancer activities.

The product of the invention is thiazolo[3,2-c]pyrimidine based compounds having the potential to be a new type of anticancer agent and similar to 5-flourouracil (5-FU) from the said anticancer agent molecules. The object of the invention is to provide anticancer effect on various cancer types (breast, liver, lung, colon, skin, prostate, kidney, ovarian, etc.) with said thiazolo[3,2- cjpyrimidine compounds. Thiazolo[3,2-c]pyrimidine based compounds of the invention are biologically active molecules having anticancer (killer) effect and prepared with a multi-component, single- stage and multi-effect method.

Another object of the invention is to present a method having a high efficiency (80- 100%) and an easy isolation and providing fast result by means of a single-stage synthetic preparation method used for thiazolo[3,2-c]pyrimidines.

Target thiazolo[3,2-c]pyrimidines may be easily obtained with high amounts via normal heating method applied for the synthesis in 2-3 hours and via microwave heating method in 3-6 minutes. These effective and easy methods are very valuable with regard to synthetic chemistry and industrial production in extensive productions.

The value of these compounds is increased in both drug industry and medical chemistry field by the fact that an important effect of these obtained thiazolo[3,2- cjpyrimidines like anticancer (especially breast-MCF-7, liver-HEPG2/C3A, etc.) has been proved.

Drawings The applications of the present invention summarized briefly above and addressed in more detail below may be understood by referring to the exemplary applications defined in the appended drawings of the invention. However, it has to be mentioned that the appended drawings illustrates the typical applications of this invention only, thus it can not be assumed that they limit the scope of the invention, because it may allow for equally effective applications.

Figure 1. The view illustrating the 3 main structural subgroups of thiazolopyrimidines Figure 2. The view specifically illustrating the adjacent ring structure of thiazolo[3,2- cjpyrimidines Figure 3. The view illustrating the general thiazolo[3,2-c]pyrimidine adjacent structure and additional group positions

Figure 4. The view illustrating the obtaining of thiazolo[3,2-c]pyrimidine compounds Figure 5. General structural view of enamines

Figure 6. Structural view of formaldehyde

Figure 7. Structural view of amines

Figure 8. The view describing the formation of thiazolo[3,2-c]pyrimidine compound Figure 9. Main structure of 1 ,2,3,4-tetrahydropyrimidine compound For the ease of understanding, identical reference numbers are used in the possible cases in order to define the identical members common in the figures. The figures have not been drawn to scale and they may be simplified for clarity. It has to be considered that the members and properties of this application may be included in the other applications usefully without any need for further explanation.

DETAILED DESCRIPTION OF THE INVENTION

In this detailed description, novel thiazolo[3,2-c]pyrimidine based anticancer compounds of the invention and preferred alternatives of the preparation methods thereof are described for better understanding the subject and without any limiting effect.

Present compounds (approximately 40) have been obtained by derivation with the groups whose 2, 7 and 9 positions are defined on thiazolo[3,2-c]pyrimidine adjacent specific ring structure apparent in Figure 2. Anticancer effects (cytotoxic) of the obtained 40 thiazolo[3,2-c]pyrimidine compounds on the human cancer cells (breast- MCF-7, liver-HEPG2/C3A, lung-A549, etc.) with different doses (1 , 10, 100, 200, 300, 500 mM) and in changing durations (24, 48, 72 hours) have been studied and in variable rates anticancer effects have been ascertained. Anticancer effects of 15 derivatives (on the breast-MCF-7, liver-HEPG2/C3A cells) have been ascertained in considerable levels, at least 5 of these derivatives are strong, 6 of them are in middle strength and 4 of them provide middle-weak level anticancer effect.

In addition to the obtained compounds, novel thiazolo[3,2-c]pyrimidine compounds may be prepared by modifying 2, 3, 6, 7, 8, 9 positions on the general thiazolo[3,2- cjpyrimidine adjacent structure with various functional groups mentioned below and various activities may be performed with these new derivatives

Functional groups where 2, 3, 6, 7, 8, 9 positions may be modified on the main thiazolo[3,2-c]pyrimidine adjacent structure:

R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ : H, halogen, non-metal, saturated and unsaturated alkyl C chain, saturated and unsaturated C ring or heteroring, adjacent rings, ether group, alcohol group, aldehyde group, ketone group, ester group, amide group, nitryl group, carboxilic acid group, anhydride group, acyl halide group, cyanate, isocyanate, thiocyanate groups, imine, oxime, ketene groups, hydrazine, hydrazone, hydrazide, semicarbazide groups.

R ² : H, halogen, non-metal, saturated and unsaturated alkyl C chain, saturated and unsaturated C ring or heteroring, adjacent rings, ether group, aldehyde group, ester group, amide group, nitryl group, carboxilic acid group, anhydride group, acyl halide group, cyanate, isocyanate, thiocyanate groups, imine, oxime, ketene groups, hydrazine, hydrazone, hydrazide, semicarbazide groups. In the compounds which may be prepared by said method, functional groups where 1 ,2, 3, 4, 5, 6 positions may be modified on the main 1 ,2,3,4-tetrahydropyrimidine structure instead of thiazole ring are mentioned below. In this structure, it is especially anticipated that rings comprising various sized (4-8 membered) carbon or heteroatom from C6-N1 positions will be adjacent.

R ² , R ³ , R ⁴ , R ⁵ : H, halogen, non-metal, saturated and unsaturated alkyl C chain, saturated and unsaturated C ring or heteroring, adjacent rings, ether group, alcohol group, aldehyde group, ketone group, ester group, amide group, nitryl group, carboxilic acid group, anhydride group, acyl halide group, cyanate, isocyanate, thiocyanate groups, imine, oxime, ketene groups, hydrazine, hydrazone, hydrazide, semicarbazide groups, and organometallic groups.

R ¹ , R ⁶ : saturated and unsaturated alkyl C chain, saturated and unsaturated C ring or heteroring, adjacent 4-8-carbon rings or heterorings comprising 0,N,S atom. Conventional Heating Method consists of following process steps;

• Dissolving enamine (1 or 2) (1.0 fold) and primer amine (4) (1 fold) in water or asetonitrile (20 mL) and stirring the suspension for 5 minutes,

• Adding formaldehyde (3) (37% v/v aqueous solution, 2.0 fold) to the suspension drop-by-drop via a syringe and boiling the mixture under argon atmosphere for 2-6 hours, · Controlling the reaction process with TLC and upon the completion of the process cooling the reaction mixture to the room temperature,

• Filtering the present precipitate via a sintered funnel and washing the precipitate with water (2 x 10 mL),

• After the drying process, purifying the raw products via flash column chromatography with silica gel by using various ethyl acetate-hexane mixtures and obtaining pure thiazolo[3,2-c]pyrimidine anticancer compunds (5 or 6) Microwave Heating Method consists of following steps;

• Mixing enamine (1 or 2) (1.0 fold) and primer amine (4) (1 fold) and formaldehyde (3) (37% v/v aqueous solution, 2.0 fold) in water or asetonitrile (20 mL) and subjecting the present suspension to pre-stirring in a microwave reactor for 15 seconds,

• Heating the reaction mixture accurately in 90°C and in dynamic mode for 4-6 minutes, · Controlling the reaction process with TLC in every 2 minuets and upon the completion of the process, cooling the reaction mixture to the room temperature by stopping the microwave system,

• Filtering the present precipitate via a sintered funnel and washing the precipitate with water (2 x 10 mL), • After the drying process, purifying the raw products via flash column chromatography with silica gel by using various ethyl acetate-hexane mixtures and obtaining pure thiazolo[3,2-c]pyrimidine anticancer compunds (5 or 6)

Method applied for anticancer activities consists of the following process steps;

• Culturing MCF-7 (human breast) or HEPG2/C3A (human liver) cancer cells into 96-well plates such that there will be 2x10 ⁴ cell/well,

• Applying thiazolo[3,2-c]pyrimidine anticancer compounds (5 or 6) synthesized with different concentrations (1 , 10, 100, 200, 300, 500, 1000 mM) and 5-FU (anticancer agent) on the cells for 24-72 hours,

Dissolving all of the applied substances in dimethylsulfoxide (DMSO) and applying the substances dissolved in DMSO on the cancer cells (MCF-7 or HEPG2/C3A) by diluting them such that their final concentration will be 0.5%,

• After 72 hours, measuring the cell viability by MTT test and calculating their viability by taking their percentages according to the control group

Thiazolo[3,2-c]pyrimidine compounds (5 or 6) are obtained by compounding in a rate of one equivalent from enamine (1 or 2), two equivalent from formaldehyde (3) and one equivalent from amine compounds (4). (figure 4)

Enamines (1,2) have both electrophilic and nucleophilic characters. They allow for the ethanamine formation by reaction from the double-bond carbon of the imine formed by compounding formaldehyde (3) and primer amine (4), in the formation of thiazolo[3,2- c]pyrimidines (5, 6). (figure 5)

Formaldehyde (3) is used in proportion of 2 fold, it allows for the imine formation with primer amine (4) and thiazolo[3,2-c]pyrimidine (5,6) formation with ethanamine which is formed again thereafter (figure 6)

Primer amine (4) allows for imine and ethamine formation by compounding with formaldehyde (3), it is the source of nitrogen on the pyrimidine ring and it enables obtaining various thiazolo[3,2-c]pyrimidines (5,6) with various groups (R ² ) on it. (figure

7)

As its main structure, a thiazolo[3,2-c]pyrimidine compound (5 or 6) is formed by compounding c face of a thiazole (A) ring and (B) ring of pyrimidine. Moreover, thiazolo[3,2-c]pyrimidine compounds also diversify with the changes of R ¹ : N0 ₂ , C0 ₂ Et;

R ³ : H, Methyl; R ⁶ : carbonyl and R ² : alkyl or aryl groups.

Thiazolo[3,2-c]pyrimidine compound (5 or 6) formation occurs with ethanamine formation by reacting enamines from the double bond carbon of imine formed by compounding formaldehyde (3) and primer amine (4) firstly, reacting the present ethanamine with a second formaldehyde (3) and its cyclization. (figure 8)

In the figures describing the invention, numbers from 1 to 6 below the chemical structure are used parenthetically right after the chemical structures in the statements of the detailed description of the invention.