ALI OMEED AKBAR ALI (TR)
AL-GBURI FIRAS SHAWQI ABDULRAZZAQ (TR)
| WO1993018776A1 | 1993-09-30 |
ZANDER J; ET AL: "Synergistic antimicrobial activities of folic acid antagonists and nucleoside analogs", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 54, no. 3, 2010, pages 1226 - 1231, XP055402508
| CLAIMS 1.A drug for anticancer and antiviral treatment, characterized by comprising: a nucleoside analogue having the above molecular structure, - said nucleoside analogue comprising a furan ring irreversibly bound to the RNA/DNA synthesis chain by phosphodiester bonds and having SP3 hybridization, and folic acid (A) bound to said nucleoside analogue. 2 . The drug according to claim 1, wherein it comprises the same structure as the furan ring which is the structural element of all natural nucleosides/nucleotides in RNA/DNA structure. 3. A synthesis method of a nucleoside analogue for use in anticancer and antiviral treatment, characterized by comprising the steps of; i. performing acetylation, ii . bonding of nitrogen base, iii .deprotection, iv. performing tosylation, v. performing cyclization, vi . performing oxidation, vii .formation of fused ring (synthesis of nucleoside analogue) and viii . synthesising by binding folic acid (A) to the resulting nucleoside analogue. 4 . The synthesis method according to claim 3, comprising the steps of; 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, to the mixture adding 50 ml of water and extracting 3 times with 40 ml of chloroform, drying over MgS04 and obtaining the acetylated sugar by evaporating it under reduced pressure, 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 with TLC (thin layer chromatography), filtration of thymine salt traces from the hot xylene suspension and washing with 20 ml 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 MgSCb and removing the solvent, obtaining 1 ((2R, 4S, 5R) -4-hydroxY-5- (hydroxymethyl) tetrahydrofuran-2-yl) -5 methylpyrimidine-2,4 (1 H, 3H) - dione compound by purifying by column chromatography, iii.Adding 0.1 gr, 1.85 mmol sodium methoxide in 20 ml methanol to the solution of 0.29 mmol 1 - ((2R, 4S, 5R) - 4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) 5metilpirimidin-2,4 (1 H, 3H) -dione and stirring for 1 hour under reflux, obtaining the thymidine by removing the solvent under reduced pressure, iv.Adding 0.82 mol thymidine and 750 ml, 9.65 mol pyridine to a 3 L flask with a three neck, round bottom and 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 the mixture 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 pouring the resulting mixture, obtained after storing at 5 °C for 18 hours, into 3 L of mixed water containing ice, collecting of the crystallized mixture after stirring for half an hour by filtration and washing 3 times with 100 ml water, drying the solid under vacuum, obtaining 1- (2-Deoxy-3,5-bis (p-toluenesulfonyl) -b-D-eritro-pentfuranosyl) thymine compound by recrystallizing from acetone, v. Adding stepwise 1.87 mol potassium hydroxide solution prepared in 1.6 L water, to 0.62 mol 1-[(2-Deoxy-3,5- bis(methylsulfonyl) - ery~~ro-pentofuranosyl)] 1- (2- Deoxy-3,5-bis (p-toluenesulfonyl) -b-D-eritro- pentfuranosyl) thymine, heating the stirred solution under 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 vacuum and obtaining 1-((1R, 3R, 5R) -2,6- dioxabicyclo [3.2.0] heptan-3-yl) -5-methylpyrimidine-2,4 (1 H, 3H) -dione compound by recrystallizing from ethanol, 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, to this solution, adding 1.5 g portions of 97%, 74.0 g, 0.643 mol KOtBu 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 that the reaction is complete by TLC, 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 on it, placing the mixture in a separatory funnel and discarding top layer containing the residual toluene, 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, collect the precipitate and leaving under vacuum for 2 hours and then air-dry for 16 hours, triturating the solid and slurrying in 500 mL of hot acetone, then filtering, rinsing the residue on 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, obtaining 1-(2,3-Dideoxy-p-D-glycero-pent-2-enofuranosyl) thymine compound by concentrating the combined filtrate to dryness, vii. Stirring vigorously the mixture of 3 g, 13.39 micromoles (mmole) (2S, 5S)-5-hydroxymethyl-2-, 5- dihydrofuran-2-ol, %50 aqueous NaOH and 4.45 g, 0.43 micromoles of tetra butyl ammonium bromide in 37 mL trichloromethane (CHCI3) at room temperature, completing of the reaction after 24 hours, adding 50 mL water to the reaction mixture and then extracting twice with 75 mL diethyl ether, washing the combined organic layer with 50ml water, drying over anhydrous MgSCb, filtrating and concentrating under vacuum, purifying the resulting residue by column chromatography, obtaining 1-((1S, 2R, 4S, 5R)-6,6-dichloro-4-hydroxymethyl)-3- oxabicyclo[3.1.0]hexane-2-yl)-5-methylpyrimidine-2,4 (1H, 3H)-dione compound. 5.The synthesis method according to claim 3 or 4, comprising the steps of; - adding of redistilled 4.5g, 6.67mL of thionyl chloride to 4.212g, 19.5 mmol dry powder of folic acid (A) in the claisen bottle, and refluxing the mixture for 1 hour or until the formation of hydrogen chloride ceases, - allowing the reaction mixture to cool, - removing the condenser and stirring the flask with heating to give the folic acid chloride (B) compound for 3 minutes at 60 °C, - adding dropwise of 19.5 mmol folic acid chloride (B) compound to the solution of nucleoside analogue (7) containing furan ring cooled with ice at -12 °C in 5.989 g, 19.5 mmol pyridine, - keeping the resulting reaction mixture at room temperature for 24 hour, and then adding 300 mL of cold-distilled water and 400 mL of chloroform, - separating of the organic layer and washing 3 times with 400 mL of distilled water, - drying on anhydrous MgSCh, filtering and removing the solvent by evaporating with 50 mL toluene 3 times under reduced pressure, - purifying the remaining syrup by a silica gel column to obtain the primary folic acid-bound nucleoside analogue (C). 6.A synthesis method according to claim 3 or 4, comprising the following process steps: - dissolving 2 g, 6.511 mmol 1,2: 5,6-di-O- isopropylidene- -D-glucosofuranose(69), in a mixture of 5 mL dimethyl sulfoxide and 2.5 mL acetic anhydride, - after the reaction mixture was left for 24 hours at room temperature by TLC (thin layer chromatography) and after only a slight trace of the nucleoside analogue (7) containing the furan ring remained, adding ice-water to the reaction mixture and stirring for 15 minutes, - isolating the result by pouring the water layer and washing the syrup 3 times with 30 mL of cooled water, then extracting 3 times with 30 mL of chloroform, - drying the combined chloroform extracts on anhydrous magnesium sulphate (MgSCb) and removing the solvent under low pressure, - purifying the remaining syrup by silica gel column to give the 3-keto derivative which is the modified nucleoside (D) compound, - adding of 2.096 g, 6.555 mmol of folic acid (A) solution and 2 drops of icy acetic acid, in 30 mL of absolute ethanol, to 3-keto derivative which is the modified nucleoside (D) compound and leaving for reflux for 24 hours and monitoring the reaction by TLC, filtering, concentrating and then adding 40 mL of water and extracting 3 times with 40 mL chloroform, drying over MgSCb and removing the solvent under vacuum to obtain the secondary folic acid-bound nucleoside analogue (E). 7. The synthesis method according to claim 4, wherein acetyl chloride is used in step i. 8. The synthesis method according to claim 4, wherein methanesulfonyl chloride is used in step iv. 9. The synthesis method according to claim 4, wherein sodium hydroxide is used in step v. 10. A nucleoside analogue obtained by the synthesis method according to any one of claims 3 to 9. 11. A drug comprising a nucleoside analogue according to claim 10. |
DESCRIPTION
Field of the Invention
The invention relates to a drug for use in anticancer and antiviral therapy comprising a folic acid-bonded 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 and/or RNA synthesis and limit and/or stop 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.
Cytotoxic nucleoside 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 pyrimidine nucleoside derivatives in both solid tumours 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 incorporating and modifying DNA and RNA macromolecules 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 known the state of the art Patent document No.
EP2415486A2 may be reviewed. Said document describes a composition and a conjugate to facilitate delivery to the cancer cell. Conjugates provided by the present invention may confer therapeutic activity by transferring therapeutic compounds across the cellular membranes. The present invention encompasses the development and synthesis of new agents for the provision of molecules, including small molecules, lipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers, and other polymers such as proteins, peptides, hormones, carbohydrates, or polyamines. The agent used in the said document is a nucleotide. In one embodiment of the invention, the nucleoside (R2) is described to exhibit anticancer and antiviral effects. In said document, a serine amino acid residue (2-amino-3-hydroxypropanoic acid) was used as a bridge. This makes the molecule mentioned in the document more bulky, which is a disadvantage.
Another example of the state of the art is the Patent document No. US5608046A. The invention related to this document discloses tetrahydrofuranyl compounds, functionalized to include pendant conjugate groups and useful in diagnostic experiments and as research reagents. New intermediates for the synthesis of compounds are also described. An object of the said invention is to provide new nucleosides for use in combining suspended conjugate groups into oligomeric compounds. It is defined in the said document that oligomers refer to the nucleoside and nucleoside analogues that come together with phosphodiester bonds or other bonds. The said document mentions that local triplex formation of oligomeric compounds is used to inhibit gene transcription. In said document, a normal oligo nucleotide was used without any change in position 3.
In order to eliminate the disadvantages of the aforementioned documents, a drug comprising a nucleoside analogue containing a furan ring and folic acid-bonded to said nucleoside analogue that irreversibly stops DNA/RNA synthesis for antiviral and anticancer therapy and the synthesis method thereof has been developed.
Detailed Explanation of the Invention
The invention relates to a drug comprising an improved nucleoside analogue for use in anticancer and antiviral therapy. The embodiments of the invention described hereinbelow comprise a furan ring and the nucleoside analogue is synthesized as folic acid (A) is bonded thereto.
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 invention is introduced into cancer cells containing a large number of folic acid receptors more quickly and easily, since it contains folic acid (A) bound to the nucleoside analogue. Unlike the known state of the art in the synthesized nucleoside analogue, there is no bridge group used between folic acid (A) and nucleic acid.
As a result of the synthesis, the furan ring was transformed into SP 3 hybridization. Due to this newly occured structure, SP 3 hybridization has the same structure as the furan ring (found in the ribose/deoxyribose monosaccharide structure), the structural element of all natural nucleoside/nucleotides found in the RNA/DNA structure. The molecule enters the cells in this form, after entering the cell, it is phosphatized by kinase enzymes and by competing with natural nucleotides, it irreversibly stops RNA/DNA synthesis by entering the RNA/DNA synthesis chain.
The nucleoside analogue (modified in position 3) expected to complete the DNA chain was used in the invention, however once the newly synthesized nucleoside analogue is bound to DNA due to a change in position 3 in the furan ring, it will stop DNA production and replication.
An embodiment of the present invention enters the cell, it is phosphatized by kinase enzymes and competes with natural nucleotides to bind to the active sites of the synthesis enzymes. After the enhanced nucleoside (C, E) shown in Figure 2 is introduced into the cell and phosphatized by kinase enzymes, it binds to the DNA synthesis chain from the 5' end of the furan ring and since there is no hydroxyl (OH) group bound to carbons numbered 2 and 3', it causes irreversible stopping of DNA synthesis.
Figure 3 shows that the drug of the present invention irreversibly stops DNA synthesis by binding to the DNA chain by strong phosphodiester bonds. 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 developed structure has the same structure as the furan ring which is component element (found in the structure of ribose/deoxyribose monosaccharide) of all natural nucleosides/nucleotides in the RNA / DNA structure.
Since the furan ring of the anticancer drug of the invention has SP3 hybridization, which is the structure of natural nucleosides found in the body of the user, it can enter HIV and cancer cells and stop its synthesis by binding to the DNA double helix competitively with natural nucleosides.
Metabolism in cancer cells is fast and most cancer cells multiply very quickly. Therefore, cancer cells have more folic acid receptors than intact cells. The new agent developed by the invention is provided to be introduced to cancer cells more quickly and more easily. Since pH in cancer cells is lower than physiological pH, folic acid (A) is separated from nucleoside analogue in low pH medium. The nucleoside analogue can be attached to the DNA double helix by natural procedure by being phosphatized by the nucleoside kinases in the cell but it will cause the replication to stop due to the absence of the OH group bound to C3 in the furan ring and thus cause the cancer cell to stop dividing.
The invention will not show side effects of anticancer nucleoside analogues known in the art to the user. Therefore, it can be used in the treatment of HIV and all cancer patients. The invention can be used in the treatment of HIV and cancer patients with significant health problems, due to its similarity to natural nucleoside analogues and its inclusion in cells by folic acid receptors, which are abundant in cancer cells since folic acid (A) is bound to the nucleoside. 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 additional costs such as interruption and resumption of treatment. Since the synthesis of the developed drug from the first step would be done in Turkey, and being more cost-effective, it would contribute into national economy of the country.
The process steps of the synthesis method of one embodiment of the invention are shown in Figure 4. In its most general form, this method comprises the steps of; performing acetylation, binding of nitrogen base, removal of protection, tosylation, cyclization, oxidation, formation of fused ring (synthesis of new nucleoside analogue) and synthesis of the obtained nucleoside analogue by binding folic acid (A) thereto. These steps are explained in more detail below.
1. Synthesis of (2R,3S)-2-(acetatoxymethyl) tetrahydrofuran- 3-yl acetate Acetylation (i)
Acetic anhydride (5 ml) and sulfuric acid (0.01 ml) are added to the solution of D-deoxyribose (5.1 mmol) in concentrated acetic acid (10 ml). The resulting solution is stirred at room temperature for 12 hours, then water (50 ml) is added to the mixture and extracted with 40ml chloroform for three times, dried on MgS0 4 and evaporated under reduced pressure to obtain the compound shown by number 1 in Figure
4.
2. 1-[(2R,4S,5R)-4-hydroxy-5(hydroxymethyl)tetrahydrofuran-2- yl)-5 methylpyrimidin-2,4 (1 H, 3H)]-dione Connecting the nitrogen base (ii)
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). The organic layer is washed twice with 20 ml of 20% en
3. Synthesis of Thymidine Deprotection (iii)
From sodium methoxide (0.1 gr, 1.85 mmol) (0.29 mmol) in methanol (20 ml) is added to 1-[(2R, 4S, 5R)-4-hydroxy-5-
(hydroxymethyl) tetrahidrofuran-2-yl )-5 methylpyrimidine- 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.
4. Synthesis of 1-[(2-Deoxy-3,5-bis (p-toluenesulfonyl)]-b-D- eritro-pentfuranosyl) thymidine Tosylation (iv)
A 3 L, three-necked, round bottom flask, equipped with a Claisen adapter containing a top mixer and racket, a 500 mL dropper funnel, an additional funnel and a thermometer is prepared. 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 crystalizes immediately. After stirring for half an hour the product is collected by filtration and washed 3 times with water 100 ml. 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.
5. 1-[(1R, 3R, 5R)-2,6-dioxabicyclo [3.2.0] heptan-3-yl) -5- methylpyrimidine-2 ,4 (1 H, 3H)] -dione
Cyclization (v)
A solution potassium hydroxide solution (1.87 mol) prepared in 1.6 L water is added stepwise to 1-[(2-Deoxy-3,5- bis (methylsulfonyl) - ery~~ro-pentofuranosyl )] thymine
(0.62 mol), upon which the reaction mixture becomes yellow- orange in colour. The stirred solution is then heated under 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 (vi)
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, 1.5 g portions of 97% KOtBu (74.0 g, 0.643 mol) is added 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 a 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, magnetic stir bar and 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. 7. Synthesis of 1-[(1S, 2R, 4S, 5R)-6,6-dichloro-4-
(hydroxymethyl) -3-oxabicyclo[3.1.0]heksan-2-yl)-5- methylpyrimidine-2 ,4 (1H, 3H)]-dione Formation of fused ring (Synthesis of new nucleoside analogue) (vii)
The mixture of (2S, 5S) -5- (hydroxymethyl) -2-, 5- dihydrofuran -2- ol (3 g, 13.39 micromoles(13.38 mmole)), % 50 aq. NaOH 50mL and tetrabutyl ammonium bromide (4.45 gr 1.161 gr, 0.43 micromoles 3.6 mmole), in trichloromethane (CHCI3) (37 ml), is stirred vigorously at room temperature, after 24 hours the reaction is complete. Water (50 mL) is slowly added to the reaction mixture and then extracted with diethyl ether chloroform (2 x 75 mL). The combined organic layer is washed with water (50 mL), dried over anhydrous MgS04, 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] heksan-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 optionally, chemicals such as acetyl chloride (at step i), methanesulfonyl chloride (at step iv) and sodium hydroxide (at step v) may be used.
In an embodiment of the invention, the nucleoside analogue (7) comprising a furan ring obtained after step vii is synthesized by binding folic acid (A) by two different methods. The nucleoside analogue obtained by the first method was named as the primary folic acid-bound nucleoside analogue (C), and the one obtained by the second (alternative) method, was named as the secondary folic acid (A) bound nucleoside analogue (E). The methods mentioned are described in detail below. A method for synthesis of the primary folic acid-bound nucleoside analogue (C):
Obtaining the folic acid chloride (B) compound given in Figure 5 (Preparation of acid chloride):
To dry powder of folic acid (A) (4.212g, 19.5 mmol) in a
Claisen flask, redistilled thionyl chloride (4.5g, 6.67mL) is added and the mixture is refluxed for 1 hour or until the formation of hydrogen chloride ceases. The reaction mixture is allowed to cool, the condenser is removed, and the flask is stirred with heating for 3 minutes at 60°C to give the folic acid chloride (B) compound.
Obtaining the primary folic acid-bound nucleoside analogue (C) compound given in Figure 5 (Preparation of the ester): Folic acid chloride (B) compound (19.5 mmol) is added dropwise carefully to a solution of nucleoside analogue (7) containing the furan ring(19.5 mmol) in pyridine which was cooled with ice (-12 °C), given in figure 4. The resulting reaction mixture is kept stirred at room temperature for 24 hours and then cold distilled water (300 mL) and chloroform (400 mL) are added. The organic layer is separated and washed with distilled water (3 x 400 mL), dried on anhydrous (MgSCh), filtered and the solvent is removed under reduced pressure [(evaporated with toluene to remove traces of pyridine under reduced pressure) (3x50 mL)]. The remaining syrup is purified by silica gel column to give the primary folic acid-bound nucleoside analogue (C).
An alternative method for synthesis of the secondary folic acid-bonded nucleoside analogue (E):
Obtaining the modified nucleoside (D) compound given in Figure 6:
Nucleoside analogue (7)(6.511 mmol) is dissolved in the mixture of dimethyl sulfoxide (5 mL) and acetic anhydride (2.5 mL). After 24 hours at room temperature,the reaction mixture monitored by TLC (thin-layer chromatography), then ice-water is added to the reaction mixture and stirred for 15 minutes. The result is isolated by spilling the water layer and the syrup is washed with cooled water (3 x 30 mL), then extracted with chloroform (3 x 30 mL). Combined chloroform extracts are dried over anhydrous magnesium sulphate (MgSCh) and the solvent removed under reduced pressure. The remaining syrup is purified by silica gel column to give the 3-keto derivative, which is the modified nucleoside (D) compound.
Obtaining the secondary folic acid-linked nucleoside analogue (E) given in Figure 6:
A solution of folic acid (A) (2.096 g, 6.555 mmol) in absolute ethanol (30 mL) and 2 drops of icy acetic acid are added to the 3-keto derivative which is the modified nucleoside (D) compound, and allowed to reflux for 24 hours, and the reaction is monitored by TLC, filtered, concentrated, and then 40 mL of water is added and extracted with chloroform (3 x 40 mL), dried over anhydrous MgS0 4 , and the solvent removed under vacuum to give the secondary folic acid-bound nucleoside analogue (E).
The primary folic acid-bound nucleoside analogue (C) and the secondary folic acid-bound nucleoside analogue (E) obtained by the aforementioned synthesis methods and a drug comprising these nucleoside analogues are also within the scope of the invention.
In summary, the invention is a drug for anticancer and antiviral treatment, wherein the drug comprises a nucleoside analogue having the molecular structure of the primary folic acid-bound nucleoside analogue (C) or secondary folic acid- bound nucleoside analogue (E), the said nucleoside analogue comprising a furan ring irreversibly bound to the RNA/DNA synthesis chain by phosphodiester bonds and having SP3 hybridization, and folic acid (A) bound to the nucleoside analogue.
In summary, the method of synthesis of the invention comprises the following process steps: i. To a solution of 5.1 mmol of D-deoxyribose in 10 ml of concentrated acetic acid, adding 5 ml of acetic anhydride and 0.01 ml of sulfuric acid, stirring the resulting solution at room temperature for 12 hours, then, to the mixture adding 50 ml of water and extracting 3 times with 40 ml of chloroform, drying over MgS0 4 and obtaining the acetylated sugar by evaporating it under reduced pressure, 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 with TLC (thin layer chromatography) ,filtration of thymine salt traces from the hot xylene suspension and washing with 20 ml 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 MgS0 4 and removing the solvent, by purifying by column chromatography, obtaining 1 - ((2R,
4S, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2- yl) -5 methylpyrimidine-2,4 (1 H, 3H) -dione compound,
Lii. Adding 0.1 gr, 1.85 mmol sodium methoxide in 20 ml methanol to the solution of 0.29 mmol 1 - ((2R, 4S, 5R) -
4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) 5metilpirimidin-2 ,4 (1 H, 3H) -dione and stirring for 1 hour under reflux, obtaining the thymidine by removing the solvent under reduced pressure, iv. To a 3 L flask with a three neck, round bottom and equipped with a distillation adapter with a top mixer and racket, a 500 ml dropper funnel, an additional funnel and a thermometer, adding 0.82 mol thymidine and 750 ml, 9.65 mol pyridine, stirring the mixture and cooling it to 0-3 °C in an ice bath and loading the mixture 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 18 hours, storing at 5 °C, then pouring the resulting mixture into 3 L of mixed water containing ice, after stirring for half an hour collection of the crystallized mixture by filtration and washing 3 times with 100 ml water, drying the solid under vacuum, recrystallizing from acetone, obtaining 1- (2-
Deoxy-3,5-bis (p-toluenesulfonyl) -b-D-eritro- pentfuranosyl) thymine compound, v. Adding stepwise 1.87 mol potassium hydroxide solution prepared in 1.6 L water, to 0.62 mol 1-[(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 recrystallizing from ethanol, obtaining 1-((1R, 3R, 5R) -2,6-dioxabicyclo [3.2.0] heptan-3-yl) -5- methylpyrimidine-2 ,4 (1 H, 3H) -dione compound, i. xiii. 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, to this solution, adding over 25 minutes 1.5 g portions of 97%, 74.0 g, 0.643 mol KOtBu, 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 that the reaction is complete by TLC, 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-dry for 16 hours, triturating(crushing) the solid and slurrying in 500 mL of hot acetone, then filtering, rinsing the residue on 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, obtaining 1- (2,3-Dideoxy-p-D-glycero-pent-2-enofuranosyl) thymine compound by concentrating the combined filtrate to dryness, vi. vigorously stirring the mixture of 3 g, 13.39 micromoles (2S, 5S)-5-hydroxymethyl-2-, 5-dihydrofuran-2-ol, %50 aqueous NaOH and 4.45 g, 0.43 micromoles of tetra butyl ammonium bromide in 37 mL trichloromethane (CHCI3) at room temperature, completing of the reaction after 24 hours, adding 50 mL water to the reaction mixture and then extracting twice with 75 mL diethyl ether, washing the combined organic layer with 50ml water, drying over anhydrous MgSCh, filtrating and concentrating under vacuum, purifying the resulting residue by column chromatography, obtaining 1-((1S, 2R, 4S, 5R)-6,6- dichloro-4-hydroxymethyl) -3-oxabicyclo[3.1.0]hexane-2- yl)-5-methylpyrimidine-2 ,4 (1H, 3H)-dione compound. vii. adding redistilled 4.5g 2.31 g, 6.67mL 1.41 mL thionyl chloride to dry powder of 4.212g 8.6 g, 19.5 mmol folic acid (A) in the claisen flask and refluxing the mixture for 1 hour or until the formation of hydrogen chloride ceases, allowing the reaction mixture to cool, removing the condenser and stirring the flask, at 60 °C for 3 minutes with heating to obtain folic acid chloride (B) compound, to the solution of nucleoside analogue (7) containing furan ring ice-cooled at -12 °C in 5.989 g, 19.5 mmol pyridine is added dropwise of 19.5 mmol folic acid chloride (B) compound, allowing the resulting reaction mixture at room temperature for 24 hours and then 300 mL of cold distilled water and 400 mL chloroform is added, the organic layer is separated and washed with 400mL distilled water 3 times, drying over MgSCb, filtered and the solvent removed by evaporating 3 times with 50 mL toluene under reduced pressure, purifying the remaining syrup by silica gel column to provide the primary folic acid-bound nucleoside analogue (C). or alternatively,
Lii. dissolving 2 g, 6.511 mmol 1,2: 5,6-di-O-isopropylidene-a- D-glucofuranose (69), in a mixture of 5 mL dimethyl sulfoxide and 2.5 mL acetic anhydride, after the reaction mixture remained at room temperature for 24 hours by TLC (thin layer chromatography) and after only a slight trace of the nucleoside analogue (7) containing the furan ring remains, adding ice-water to the reaction mixture and stirring 15 minutes, isolation by spilling the water layer of the resultant and washing the syrup with 30mL of cold water 3 times, then extracting 3 times with 30 mL of chloroform, drying the combined chloroform extracts on anhydrous magnesium sulphate (MgS0 4) and removal of the solvent under reduced pressure, purifying the remaining syrup with a column of silica gel column to provide 3- keto derivative, which is the modified nucleoside (D) compound, 2.096 g, 6.555 mmoles of folic acid (A) solution in 30 mL absolute ethanol and 2 drops of icy acetic acid is added to the 3-keto derivative, which is the modified nucleoside (D) compound and allowed to reflux for 24 hours and monitoring the reaction by TLC, filtering, concentrating and then adding 40 mL water and extracting 3 times with 40 mL chloroform, drying over MgS0 4 and removing the solvent under vacuum to obtain the secondary folic acid bound nucleoside analogue (E).
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 (C, E) 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.
Figure 5: An illustration of the synthesis steps of the folic acid-bound nucleoside analogue of an embodiment of the invention.
Figure 6: Illustration of the folic acid-bound nucleoside analogue synthesis steps of an embodiment of the invention (alternative) .
Descriptions of Reference Numbers in Figures
A. Folic Acid
B. Folic acid chloride
C. Primary folic acid-bound nucleoside analogue
D. Modified nucleoside
E. Secondary folic acid-bound nucleoside analogue 7. Nucleoside analogue comprising furan ring
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