METHOD THEREOF

Field of the Invention

The invention relates to a drug for use in anticancer and antiviral therapy comprising a nucleoside analogue that irreversibly stops the synthesis of DNA/RNA and the synthesis method thereof.

State of the Art

Nucleoside analogues are synthetic compounds that are structurally similar to natural nucleosides, used as antiviral or anticancer drugs, modified at one of their specific sites that interfere with DNA or RNA synthesis and limit or stop their replication. These are antimetabolites, a class of drugs that inhibit DNA synthesis directly or through inhibition of DNA precursor synthesis in de novo or salvage pathways.

Cytotoxicnucleoside analogues are the first chemotherapeutic agents to be administered for the medical treatment of cancer. These compounds have been developed to include various purine and pyrimidinenucleoside derivatives in both solid tumors and malignant blood diseases. These agents act as anti-metabolites competing with physiological nucleosides and interact with multiple intracellular targets to induce cytotoxicity.

Nucleoside analogues are administered to cells in forms that will enter the cells with nucleoside carriers before they are phosphorylated to the active triphosphate forms by kinases. These analogues may exhibit cytotoxic activity by being incorporated into DNA and RNA macromolecules and being modified and/or inhibiting various enzymes involved in nucleic acid synthesis such as DNA polymerases and ribonucleotide reductases. These effects result in inhibition of DNA synthesis or induction of apoptotic cell death as shown in figure 1.

In the prior art, there are certain molecules/drugs that bind to the DNA/RNA chain developed for anticancer and antiviral therapy, blocking their synthesis.

The patent document CN103435672A of the known state of the art may be an example. The present invention describes the structure and synthesis of a modified benzyl-containing novel nucleoside phosphate prodrug. The invention describes a phosphate structure containing substituted benzyl, wherein the phosphate structure is shown as formula (I) and a formula (II). Phosphate structure can be used as prodrugs for various nucleoside analogues including non-cyclic nucleoside, carbon ring nucleoside, furan ring nucleoside etc., and can increase the bioactivity of nucleoside compounds. It is therefore disclosed that the structure is applicable to the treatment of viral infections and cancer. The molecule described in said document is a nucleotide and comprises a furan ring. Ribose or deoxyribose is in the form of a furan ring. The furan ring forms a common part of nucleosides and nucleotides found in all living things. The compound described herein is not a nucleoside; it is a nucleotide and comprises a phosphate group. Since the phosphate group contains negative charges, it cannot enter the cell (due to its polar nature). Therefore, researchers have removed polarity by linking benzyl groups to the phosphate group to introduce the molecule of the subject matter into the cell. In this way, it enters the cell and binds to DNA with weak bonds. This binding with DNA is affected by other interactions that occur in the environment and it is always possible to separate weak bonds. This may lead to the elimination of inhibition of DNA.

Another example to the known state of the art is the non-patent document titled “ Furo [2,3- djpyrimidine based derivatives as kinase inhibitors and anticancer agents”. This document describes Furo [2, 3-d] pyrimidine -based derivatives as kinase inhibitors and anticancer agents. Furopyrimidines are fused heterocyclic ring systems. Structurally, they are bioisotere to purines, and implements pharmacological actions in various directions. They play an important role in different disease conditions. Furo [2, 3-d] pyrimidine derivatives have been investigated for their inhibitory activity against different protein kinase enzymes. Said document is considered to be the first study on the synthesis, anticancer activity of furo [2, 3-d] pyrimidine derivatives, inhibition of various protein kinase enzymes and structure-activity relationships reported until today. The molecule described in said document is not a newly synthesized drug. Researchers have made some modifications on thymine and other pyrimidine bases, which prevent the formation of hydrogen (FI) bonds in the DNA double helix. It also serves as kinase inhibitors. In this respect, it acts by blocking the synthesis of nucleotides. Thus, said document does not provide a drug which irreversibly stops DNA/RNA synthesis, taking into account the delivery mechanism and chemical structure of the molecule described.

In order to eliminate the disadvantages of the above-mentioned documents, for anticancer and antiviral treatment, it is intended to develop a drug comprising a nucleoside analogue comprising a furan ring which irreversibly halts DNA/RNA synthesis, and a synthesis method thereof.

Detailed Description of the Invention

The invention discloses a drug comprising a novel nucleoside analogue for use in anticancer and antiviral therapy. The following embodiments of the invention include a furan ring.

The synthesis method of the drug of the invention is also within the scope of the invention. In the synthesis method developed, furan ring is transformed into SP ³ hybridization. The developed structure has the same structure as the furan ring (found in the structure of ribose/deoxyribose monosaccharide) of all natural nucleosides/nucleotides in the RNA/DNA structure. The developed molecule enters the cells with this form, after entering the cell it is phosphatized by kinase enzymes and by competing with natural nucleotides, it enters the RNA/DNA synthesis chain and irreversibly stops RNA DNA synthesis.

Unlike existing drugs in the art, it stops the synthesis of RNA DNA by irreversibly binding to the RNA DNA synthesis chain by phosphodiester bonds.

By taking heat effects into account from a thermodynamic point of view, it can be decided whether or not a chemical reaction occurs. Thermodynamic quantum chemical data are widely used to investigate the reaction mechanisms of organic compounds. The spontaneity of a chemical reaction and the stability of the reaction products can be estimated from thermodynamic properties such as Enthalpy, Gibbs free energy (Cohen, N., Benson, S., W, 1993. "Estimation of heats of formation of organic compounds by additivity methods.", Chemical Reviews, 93, 2419-2438).

Free energy is the main criterion representing the interaction between the molecules making a bond. The size and signal of free energy is important to determine the likelihood of the formation of bimolecular structures. Larger negative values indicate improved thermodynamic properties. From our calculations, negative values were obtained, which means that no additional energy consumption is required to bond, that is the reaction can take place automatically (Garbett, N., C, Chaires, J., B, 2012. "Thermodynamic studies for drug design and screening.", Expert Opinion on Drug Discovery., 7, 299- 314).

As an example, when compared to the 2-deoxythymidine molecule (a nucleoside naturally found in all living things) and the newly synthesized molecule; the free energy value of the 2-deoxythymidine molecule was determined to be -874.918273 Eh. The free energy of the synthesized drug of the invention was determined as -1756.889248 Eh, in other words higher (Table 1).

Table 1. Enthalpy, Free Energies of Compounds

These results show that the newly synthesized drug molecule is more energetic and more preferable in configuration. After entering the cell, it is phosphated with kinase enzymes and competes with natural nucleotides to bind to the active sites of the synthesis enzymes. After the newly synthesized nucleoside shown in Figure 2 is introduced into the cell and phosphatized with kinase enzymes, as shown in Figure 3, the 5 ’end of the furan ring binds to the DNA synthesis chain, and since there is no hydroxyl (OH) group bound to carbons numbered 2’ and 3’, it causes irreversible stopping of DNA synthesis.

Thus, as shown in FIG. 3, the drug of the invention binds to the DNA chain with strong phosphodiester bonds, thereby irreversibly stopping DNA synthesis.

The drug of the invention is bound to the DNA double strand by the natural procedure, thereby causing replication to stop due to the absence of the OH group attached to C3 in the furan ring. In this way, it causes the cancer cell division to stop.

The furan ring of the anticancer drug of the invention can enter HIV and cancer cells and will interfere with natural nucleosides by binding to DNA double strand, because it has SP ³ hybridization, the structure of the natural nucleosides found in the receiver’s body.

The invention will not show side effects of anticancer nucleoside analogues known in the art to the receiver. These causes can be used in the treatment of patients having HIV, and any type of cancer.

The process steps of the synthesis method of one embodiment of the invention are shown in Figure 4. In its most general terms, it includes these process steps: acetylation (a) generation, bonding of nitrogen base (b) , deprotection (c), tosylation (d), cyclization (e), oxidation (f), and formation of fused ring (synthesis of new nucleoside analogue) (g). These steps are explained in more detail below. . Synthesis of (2R, 3S) -2- (acetatoxymethyl) tetrahydrofuran-3-yl acetate Acetylation (a)

To the solution of D-deoxyribose (5.1 mmol) in concentrated acetic acid (10 ml) is added acetic anhydride (5 ml) and sulfuric acid (0.01 ml). The resulting solution is stirred at room temperature for 12 hours, then to the mixture is added water (50 ml) and it is extracted with 40ml chloroform for three times, dried on MgS04 and evaporated under reduced pressure to obtain the compound shown by number 1 in Figure 4. . l-[(2R,4S,5R)-4-hydroxy-5(hydroxymethyl)tetrahydrofuran-2-yl )-5 methylpyrimidin-2,4 (1 H, 3H)] -dione

Bonding of nitrogen base (b)

Acetylated sugar (1.25 mmol) is stirred in xylene over 20 hours by refluxing with thymine mercury salt (3.07 mmol), the reaction is monitored by TLC (thin layer chromatography). Traces of thymine salt are filtered through a suspension of hot xylene and washed with dichloromethane (20 ml). In order to remove the remaining mercury salts, organic layer is washed with 20 ml 20% aqueous potassium iodide twice, then washed with 20 ml water twice, dried over anhydrous MgS04 and the solvent is removed, purified by column chromatography to obtain the compound represented by number 2 in Figure 4. . Synthesis of Thymidine Deprotection (c)

From sodium methoxide (0.1 gr, 1.85 mmol) (0.29 mmol) in methanol (20 ml), it is added to 1- [(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl) -5 methylpyrimidin-2,4 (1H, 3H)]- dione solution and stirred for 1 hour under reflux. The solvent is removed under reduced pressure to obtain the compound shown by number 3 in Figure 4. . Synthesis of l-[(2-Deoxy-3,5-bis (p-toluenesulfonyl)] -b-D-eritro-pentfuranosyl) thymidine Tosylation (d)

A 3 L, three-necked, round bottom flask is prepared as equipped with a Claisen adapter containing a top mixer and racket, a 500 mL dropper funnel, an additional funnel and a thermometer. To the flask, thymidine (0.82 mol) and pyridine (750 mL, 9.65 mol) is added. The mixture is stirred and then cooled in an ice bath to 0-3 °C and charged to the dropping funnel with p-toluenesulfonyl chloride (1.8mol). Then p-toluenesulfonyl chloride is added dropwise. The temperature was raised to 10 °C over 40 minutes. The mixture is cooled to 0°C and stirred for 1 hour and then stored at 5°C for 18 hours. The resulting light brown mixture is then poured into vigorously stirred water (3 L) containing ice. The desired product crystallizes immediately. After stirring for half an hour the product is collected by filtration and washed 3 times with 100 ml water. The solid white part is dried under vacuum overnight. It is recrystallized from hot acetone to obtain the compound shown by number 4 in Figure 4. . 1- [(1R, 3R, 5R) -2,6-dioxabicyclo [3.2.0] heptan-3-yl) -5-methylpyrimidine-2,4 (1 H, 3H)] - dione

Cyclization (e)

To l-[(2-Deoxy-3,5-bis(methylsulfonyl) - ery — ro pentofuranosyl)] thymine (0.62 mol) is added portionwise a solution of potassium hydroxide (1.87 mol) prepared in water (1.6 L), upon which the reaction mixture becomes yellow-orange in color. The stirred solution is then heated with reflux over 45 minutes. After cooling the reaction mixture to room temperature, concentrated hydrochloric acid (54 mL) is added. Approximately 1 L of water is removed to give a white slurry and allowed to cool for 2 hours in an ice bath. The solid is filtered and washed with a small amount of ice water and then dried in vacuo. It is recrystallized from ethanol to obtain the compound shown by number 5 in Figure 4.

6. 1- (2,3-Dideoxy-P-D-glycero-pent-2-enofuranosyl) thymine Oxidation (f)

A three -necked, 1 L, round-bottomed flask equipped with a mechanical stirrer, thermometer and nitrogen influx, is charged with dry dimethyl sulfoxide (DMSO) (400 mL) and oxetan 5 (90.0 g, 0.402 mol). To this solution is added 1.5 g portions of 97% KOtBu (74.0 g, 0.643 mol) over 25 minutes. The temperature is maintained between 18 °C and 22 °C by an external ice bath. After the addition is complete, the reaction is stirred for an additional 1 hour. No increase in temperature is observed and thin layer chromatography (TLC) indicates that the reaction is approximately 90% complete. The reaction is stirred at 21 °C for 16 hours; then TLC indicates the reaction is complete. The viscous solution is poured into a cold (4 °C) toluene (3 L), precipitating a beige solid. The temperature of the mixture rises to 7 °C upon addition of DMSO solution. The mixture is spun occasionally for 20 minutes and then filtered on an 18.5 cm Buchner funnel. The collected yellowish oily solid is washed twice with cold toluene and allowed to dry for 1 hour under suction. The solid is dissolved in 300 mL of water, upon which two layers are formed. The mixture is placed in a separatory funnel and the upper layer (containing residual toluene) is discarded. The aqueous layer is placed in a 1 L beaker equipped with a pH probe, a magnetic stir bar and a thermometer. The solution is cooled to 10 °C using an external ice bath. To the stirred solution, concentrated HC1 is added dropwise at a rate at which the temperature is kept below 15 °C. Upon the addition of HC1 (50.5 mL, 0.61 mol), pH becomes 7±0.1 and a precipitate begins to form. Potassium chloride (70 g) was added to this dense mixture and stirring was continued for 1 hour at 5 °C. The precipitate is collected and left under vacuum for 2 hours and then air-dried for 16 hours. The solid is triturated and slurried in hot acetone (500 mL) and then filtered. The residue on the filter papers is rinsed twice with 200 ml of hot acetone, then slurried again with hot acetone (300 mL), filtrated and washed again with hot acetone (2 x 200 ml). The combined filtrate is concentrated to dryness to obtain the compound shown by number 6 in Figure 4. . Synthesis of 1 - [(IS, 2R, 4S, 5R) -6,6-dichloro-4- (hydroxymethyl) -3-oxabicyclo [3.1.0] hexane-2-yl) -5-methylpyrimidine-2,4 ( 1H, 3H)] -dione Formation of fused ring (Synthesis of new nucleoside analogue) (g)

The mixture of (2S, 5S) -5- (hydroxymethyl) -2-, 5- dihydrofuran -2- ol (3 g, 13.39 micromole), % 50 aq. NaOH and tetrabutyl amonium bromide (4.45 gr, 0.43 micromole) in trichloromethane (CHCL) (37 ml) is stirred vigorously at r.t., and after 24 hours the reaction is complete. Water (50 mL) is slowly added to the reaction mixture and then extracted with diethyl ether (2 x 75 mL). The combined organic layer is washed with water (50 mL), dried over anhydrous MgS0 ₄ , filtrated and concentrated under vacuum. The resulting residue is purified by column chromatography to obtain 1 - ((IS, 2R, 4S, 5R) -6,6-dichloro-4-(hydroxymethyl)-3-oxabicyclo [3.1.0] hexane-2-yl) -5- methylpyrimidine-2,4 (1H, 3H)-dione as shown by number 7 in Figure 4. In one embodiment of the invention, in some steps of the synthesis of the drug, chemicals such as acetyl chloride (at step 1), methanesulfonyl chloride (at step 4) and sodium hydroxide (at step 5) may optionally be used.

Due to its resemblance to natural nucleoside analogues, the invention can be used in the treatment of HIV and cancer, which are important health problems. For patients exposed to acute and late side effects due to chemotherapy, a more comfortable treatment chance can be created and leads to significant improvement in chemotherapy. In addition, the success of radiotherapy to be given together with the developed nucleoside analogue will increase. Successful treatment of cancer patients without interruption of chemotherapy will allow avoiding additional costs such as interruption and resumption of treatment.

Since the synthesis of the developed drug would be done in Turkey beginning from the very first step, and being more cost-effective, it would contribute into national economy of the country.

In summary, the invention is a drug for anticancer and antiviral therapy comprising a nucleoside analogue having the molecular structure shown in Figure 2, wherein the nucleoside analogue comprises a furan ring with SP ³ hybridization that is irreversibly linked to the RNA/DNA synthesis chain by phosphodiester linkages. All natural nucleosides/nucleotides in the RNA/DNA structure have the same structure as the furan ring.

Briefly, the synthesis method of the present invention comprises the following process steps: i. adding 5 ml of acetic anhydride and 0.01 ml of sulfuric acid to a solution of 5.1 mmol of D- deoxyribose in 10 ml of concentrated acetic acid, stirring the resulting solution at room temperature for 12 hours, then adding 50 ml of water to the mixture and extracting the mixture 3 times with 40 ml of chloroform, drying it over MgS04 and evaporating it under reduced pressure to obtain the acetylated sugar, ii. stirring 1.25 mmol acetylated sugar with 3.07 mmol of thymine mercury salt in xylene for 20 hours under reflux, monitoring the reaction by TLC (thin layer chromatography), filtering the traces of thymine salt from the hot xylene suspension and washing with 20 ml of dichloromethane, washing the organic layer twice with 20 ml of 20% aqueous potassium iodide to remove residual mercury salts, then washing twice with 20 ml water, drying over anhydrous MgS04 and removing the solvent, purification with column chromatography to obtain the compound 1-((2R, 4S, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-il) -5 methylpirimidin-2,4 (1 H, 3H)-dione, iii. adding 0.1 gr, 1.85 mmol sodium methoxide 0.29 mmol in 20 ml methanol to the solution of 1 - ((2R, 4S, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -5 methylpirimidin-2,4 (1H, 3H) -dione and stirring for 1 hour under reflux, removing the solvent under reduced pressure to obtain thymidine, iv. adding 0.82 mol thymidine and 750 ml, 9.65 mol pyridine to a 3 L, three-neck, round bottom flask equipped with a distillation adapter with a top mixer and racket, a 500 ml dropper funnel, an additional funnel and a thermometer, stirring the mixture and cooling it to 0-3 °C in an ice bath and loading into the dropping funnel with 1.8 moles of p-toluenesulfonyl chloride followed by dropwise addition of p-toluenesulfonyl chloride, raising the temperature to 10 ° C for 40 minutes, cooling the mixture to 0 °C and stirring for 1 hour and then storing at 5 °C for 18 hours, pouring the resulting mixture into 3 L of stirred water containing ice, collecting the crystallized mixture by filtration after stirring for half an hour and washing thrice with 100 ml water, drying the solid under vacuum, recrystallizing it from acetone to obtain the compound 1- (2-Deoxy-3,5-bis (p-toluenesulfonyl) -b- D-eritro-pentfuranosyl) thymine, v. adding stepwise 1.87 mol potassium hydroxide solution prepared in 1.6 L water to 0.62 mol l-[(2-

Deoxy-3,5-bis(methylsulfonyl) - ery — ro-pentofuranosyl)] thymine, heating the stirred solution with reflux for 45 minutes, after cooling the reaction mixture to room temperature, adding 54 ml of concentrated hydrochloric acid, removing about 1 L of water to give a slurry and allowing to cool in an ice bath for 2 hours, filtering the solid and washing with ice water and then drying in vacuo and recrystallising from ethanol to obtain the compound 1-((1R, 3R, 5R) -2,6-dioxabicyclo [3.2.0] heptan-3-yl) -5-methylpyrimidine-2,4 (1 H, 3H) -dione, vi. charging 400 mL dry dimethyl sulfoxide (DMSO) and 90.0 g, 0.402 mol oxetan 5 to a three-neck, 1 L round bottom flask with a mechanical stirrer, thermometer and nitrogen inlet, adding 1.5 g portions of 97%, 74.0 g, 0.643 mol KOtBu to this solution over 25 minutes, keeping the temperature between 18 °C and 22 °C by means of an external ice bath, after the addition is complete, stirring the reaction for an additional 1 hour, monitoring that the reaction is complete by about 90% by TLC thin layer chromatography without an increase in temperature, stirring the reaction at 21 °C for 16 hours followed by monitoring by TLC that the reaction is complete, pouring the viscous solution into 3 L of toluene at 4 °C, precipitating the solid, raising the temperature of the mixture to 7 °C upon addition of DMSO solution, rotating the mixture for 20 minutes and then filtering in an 18.5 cm funnel, washing the collected solid twice with toluene and drying under suction for 1 hour, dissolving the solid in 300 mL of water, forming two layers, placing the mixture in a separatory funnel and discarding the residual toluene -containing top layer, placing the aqueous layer in a 1 L beaker equipped with a pH probe, magnetic stir bar and thermometer, cooling the solution to 10 °C using an external ice bath, adding concentrated HC1 dropwise to the stirred solution at a rate at which the temperature is kept below 15 °C, after adding 50.5 mL, 0.61 mol HC1, adjusting pH to 7 ± 0.1 and forming a precipitate, adding 70 g of potassium chloride to this mixture and continuing stirring at 5 °C for 1 hour, collecting the precipitate and leaving under vacuum for 2 hours and then air drying for 16 hours, triturating the solid and slurrying in 500 mL of hot acetone, then filtering, rinsing the residue from the filter papers twice with 200 mL of hot acetone, then slurrying again with 300 ml of acetone, filtering and once again rinsing twice with 200 mL of hot acetone, concentrating the combined filtrate to dryness to obtain the compound l-(2,3-Dideoxy-P-D-glycero-pent-2- enofuranosyl) thymine, vii. vigorously stirring the mixture of 3 g, 13.39 micromole (2S, 5S) -5- hydroxymethyl -2-, 5- dihydrofuran -2- ol, aqueous 50 % NaOH and 4.45 gr, 0.43 micromole tetra butyl amonium bromide in 37 mL trichloromethane (CHCL) at room temperature, completing of the reaction after 24 hours, adding 50 mL of water to the reaction mixture and then extracting twice with 75 mL diethyl ether, washing the combined organic layer with 50ml of water, drying over anhydrous MgS0 ₄ , filtrating and concentrating under vacuum, purifying the resulting residue by column chromatography to obtain the compound 1 - ((IS, 2R, 4S, 5R) -6,6-dichloro-4-(hydroxymethyl)-3-oxabicyclo [3.1.0] hexane-2-yl) -5- methylpyrimidine-2,4 (1H, 3H)-dione.

In step i, acetyl chloride; in step iv methanesulfonyl chloride and in step v sodium hydroxide may be used.

A nucleoside analogue obtained by the above-mentioned synthesis method and a drug comprising said nucleoside analogue is within the scope of the invention.

Description of the Figures

Figure 1: A Figure relating to the known state of the art.

Figure 2: A view of the structure of the nucleoside analogue of the present invention.

Figure 3: The illustration of the stopping of the DNA synthesis by the nucleoside analogue of the present invention.

Figure 4: The illustration of the synthesis steps according to an embodiment of the invention.

Descriptions of Reference Numbers in Figures a . Acetylation b . Bonding of nitrogen base c . Deprotection d. Tosylation e . Cyclization f . Oxidation g . Formation of fused ring (Synthesis of new nucleoside analogue)