Method for synthesizing 5'-phosphoniamidate nucleotide prodrug

Abstract

The present invention relates to a method for synthesizing a phosphoniamidate derivative in a single step or directly through ultrasonic irradiation and portionwise addition of the Hovayda-Grubbs catalyst.

Description

Synthesis method of 5'-phosphonamidate nucleotide prodrug

[0001] The present invention relates to a new reaction method for a phosphonamidate nucleotide precursor drug, a method for synthesizing the substance, and the structure thereof.

[0002] Mimics of natural nucleosides are used in various pharmaceuticals, such as antivirals, anticancer agents, and antibacterial agents; however, they present limitations in therapeutic development because they exhibit biological activity only after being triphosphated by various intracellular kinases (nucleoside kinases, mono- / di-phosphate nucleotide kinases) following administration. Regarding this mechanism of biological activity, biological activity was confirmed only for those capable of monophosphate among the thousands of synthesized nucleoside mimics, and only a select few have proceeded with drug development. Furthermore, there are almost no transport receptors for nucleoside mimics on the cell membrane, and the two negative charges of phosphate (-OPO3) 2- It has a limitation in that its bioavailability is low because it is almost impossible to penetrate the cell membrane, which is a non-polar lipid, due to the polar functional groups (-OH, -H, -CONH-). Recently, to overcome these two limitations of nucleoside mimics, phosphoamidate derivatives (ProTide prodrug derivatives) and phosphoamidate prodrug technologies have been developed in which the negatively charged part of a monophosphate nucleoside is covered by a non-polar molecule.

[0003] ProTide derivatives, which are precursors of monophosphate nucleosides, present stability issues as their PO bonds are cleaved by phosphorylases or hydrolysis. To improve the stability of these precursors, synthesis methods for phosphonamidate precursors in which the PO bond is modified to PC have been reported. In most cases, phosphonic acid derivatives are synthesized from alkyl phosphonates using strong Lewis acids under vigorous reaction conditions, and then amino acid derivatives or phenyloxy and alkoxy derivatives are introduced to finally synthesize phosphonamidate precursors. However, this synthesis process is multi-step, and because it utilizes vigorous reaction conditions, controlling reaction conditions is difficult, resulting in low product yields; in particular, product separation and purification are challenging. Furthermore, since most methods use expensive reagents, large-scale reactions are difficult, posing economic feasibility issues. Therefore, there is a need to develop synthesis methods for phosphonamidate precursors using catalytic reactions under mild conditions.

[0004] Accordingly, the inventors of the present invention identified and optimized new reaction conditions for the cross-double segregation reaction of terminal vinyl-vinyl through ultrasonic irradiation and split addition of the Hovayda-Grubbs catalyst, and developed a single-step or direct method for synthesizing phosphonamidate derivatives, thereby completing the present invention.

[0005] Accordingly, the object of the present invention is to provide a method for preparing a 5'-phosphonamidate nucleotide derivative.

[0006] In addition, the object of the present invention is to provide a method for preventing or treating a viral infection, comprising administering a 5'-phosphonamidate nucleotide derivative or a composition containing the same to an individual.

[0007] In addition, the object of the present invention is to provide a method for preventing or treating cancer, comprising administering a 5'-phosphonamidate nucleotide derivative or a composition containing the same to an individual.

[0008] To achieve the above objectives, the present invention provides a method for preparing a 5'-phosphonamidate nucleotide derivative having a structure represented by Formula 3, comprising reacting a compound represented by Formula 1 and a compound represented by Formula 2 under ultrasonic irradiation:

[0009] [Chemical Formula 1]

[0010] [Correction pursuant to Rule 91 25.02.2026]

[0011] [Chemical Formula 2]

[0012]

[0013] [Chemical Formula 3]

[0014]

[0015] In the above chemical formulas 1 to 3,

[0016] R is TBS (tert-butyldimethylsilyl), and

[0017] B and BASE include those selected from the group consisting of uridine, adenine, cytidine, and guanine, and

[0018] OPG includes those selected from the group consisting of silyl groups, benzyl groups, and acetal groups, and

[0019] R1 and R2 each include those independently selected from the group consisting of amino acid alkyl esters, benzyl, phenol, aryl, and combinations thereof.

[0020] In addition, the present invention provides an antiviral composition comprising a phosphonamidate nucleotide derivative prepared by the method according to the present invention as an active ingredient.

[0021] In addition, the present invention provides an anticancer composition comprising a phosphonamidate nucleotide derivative prepared by the method according to the present invention as an active ingredient.

[0022] In addition, the present invention provides a method for preventing or treating a viral infection, comprising administering a phosphonamidate nucleotide derivative according to the present invention or a composition containing the same to an individual.

[0023] In addition, the present invention provides a method for preventing or treating cancer, comprising administering a phosphonamidate nucleotide derivative according to the present invention or a composition containing the same to an individual.

[0024] The present invention develops a method for synthesizing 5'-nucleotide phosphonamidate by directly introducing phosphonamidate into a nucleoside derivative through a double decomposition reaction, thereby enabling the effective synthesis of various derivatives in the development of new antiviral and anticancer drugs, and thus can be usefully utilized in the field of nucleoside mimic drug development.

[0025] Embodiments of the present invention will be described in detail below with reference to the attached drawings. In the following description, detailed descriptions of technologies well known to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such descriptions would unnecessarily obscure the essence of the present invention. Additionally, the terminology used in this specification is used to appropriately express preferred embodiments of the present invention, and may vary depending on the intentions of the user or operator, or the conventions of the field to which the present invention belongs.

[0026] Therefore, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0027] The present invention relates to a method for preparing a 5'-phosphonamidate nucleotide derivative having a structure represented by Formula 3, comprising reacting a compound represented by Formula 1 and a compound represented by Formula 2 under ultrasonic irradiation:

[0028] [Chemical Formula 1]

[0029] [Correction pursuant to Rule 91 25.02.2026]

[0030] [Chemical Formula 2]

[0031]

[0032] [Chemical Formula 3]

[0033]

[0034] In the above chemical formulas 1 to 3,

[0035] R is TBS (tert-butyldimethylsilyl), B and BASE include those selected from the group consisting of uridine, adenine, cytidine and guanine, and OPG includes those selected from the group consisting of silyl groups, benzyl groups and acetal groups, and R1 and R2 each independently include those selected from the group consisting of amino acid alkyl esters, benzyl, phenol, aryl and combinations thereof, but are not limited thereto.

[0036] In the above method, equal amounts of Hoveyda-Grubbs catalyst may be added in divided portions, for example, Hoveyda-Grubbs first-generation or second-generation catalyst may be added in divided portions from 2 to 8 times, but are not limited thereto.

[0037] In one embodiment of the present invention, the method may be carried out according to a process according to the following reaction scheme, but is not limited thereto:

[0038] [Correction pursuant to Rule 91 25.02.2026]

[0039] Phosponamidate nucleotide derivatives can be synthesized through cross methathesis of the vinyl group at the C5'-position of the compound represented by Chemical Formula 1 and the vinyl group of the compound represented by Chemical Formula 2, but are not limited thereto.

[0040] The compound represented by Chemical Formula 1 above can be synthesized to have a nucleoside derivative having a terminal double bond at the 5'-position using a natural nucleoside selected from the group consisting of adenosine, cytidine, and uridine as a starting material, but is not limited thereto. For example, the compound represented by Chemical Formula 1 above can be synthesized by the following reaction scheme, but is not limited thereto.

[0041] [Correction pursuant to Rule 91 25.02.2026]

[0042] [Correction pursuant to Rule 91 25.02.2026]

[0043] The compound represented by Chemical Formula 1 above can be synthesized by reacting an intermediate having a 1,2,3-acyl-5-double bond, synthesized using D-ribose as a starting material, with guanine, but is not limited thereto. For example, the compound represented by Chemical Formula 1 above can be synthesized by the following reaction scheme, but is not limited thereto.

[0044] The compound represented by Chemical Formula 2 above may be synthesized by reacting vinylphosphonic acid with oxalyl chloride using vinylphosphonic acid as a starting material, for example, by reacting vinylphosphonic acid as a starting material with oxalyl chloride and a catalytic amount of dimethylformamide in a dichloromethane solvent, but is not limited thereto. For example, the compound represented by Chemical Formula 2 above may be synthesized by the following reaction scheme, but is not limited thereto.

[0045]

[0046] In addition, the present invention provides an antiviral composition and an anticancer composition comprising a phosphonamidate nucleotide derivative prepared by the method according to the present invention as an active ingredient.

[0047] The above virus may be an RNA virus, but is not limited thereto. The above virus may be SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), dengue virus, or influenza virus, but is not limited thereto. The above influenza virus may be influenza A or influenza B virus, but is not limited thereto.

[0048] The above cancer may be colorectal cancer, stomach cancer, colon cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, uterine cancer, blood cancer, or prostate cancer, but is not limited thereto.

[0049] In the pharmaceutical composition of the present invention, the compound according to the present invention may be administered in a suitable formulation together with a carrier and a diluent known in the art, and may have a formulation such as an oral or parenteral administration, e.g., intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, etc., depending on the desired method.

[0050] The above formulations can be prepared by conventional methods using suitable excipients, fillers, binders, wetting agents, decomposers, lubricants, surfactants, dispersants, buffers, preservatives, solubilizing agents, disinfectants, sweeteners, spices, analgesics, stabilizers, isotonic agents, etc., commonly used in pharmaceutical compositions.

[0051] Each formulation described above may include a pharmaceutically acceptable carrier or additive. Specific examples of said carrier or additive include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidine, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerol, propylene glycol, polyethyl glycol, petroleum jelly, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, and lactic acid. One or more additives may be selected or appropriately combined depending on the formulation form. Furthermore, regarding the method of administering the cell therapy agent, local administration to target cells may be performed in addition to conventional systemic administration such as intravenous or intra-arterial administration, and administration methods combined with catheter technology and surgical procedures may be used.

[0052] The composition of the present invention may contain the compound according to the present invention in a pharmaceutically effective amount together with a pharmaceutically acceptable carrier.

[0053] In the present invention, "pharmaceuticalally effective amount" refers to an amount of an active ingredient that exhibits alleviating, suppressing, improving, and / or curative effects on an immune rejection disease to be treated. The dosage of the compound according to the present invention varies depending on the patient's weight, age, gender, health status, diet, time of administration, method of administration, and severity of the disease. For example, a therapeutically effective dosage can initially be determined using in vitro analysis via cell culture. It is possible to determine a therapeutically effective amount without undergoing excessive experimentation in the field, and this information can be used to determine a useful dosage for humans more accurately. For example, the compound according to the present invention may be administered as an active ingredient in an amount of 0.1 to 100 mg / kg / day.

[0054] In addition, the present invention provides a method for preventing or treating a viral infection, comprising administering a phosphonamidate nucleotide derivative according to the present invention or a composition containing the same to an individual.

[0055] In addition, the present invention provides a method for preventing or treating cancer, comprising administering a phosphonamidate nucleotide derivative according to the present invention or a composition containing the same to an individual.

[0056] The above-mentioned individual may be a mammal, for example, a human, but is not limited thereto.

[0057] The present invention will be described in detail below through examples. However, these examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0058] [Correction pursuant to Rule 91 25.02.2026]

[0059] <Example 1> Synthesis of 5'-Double Bond Nucleoside

[0060] 1-((2R,3R,4R,5R)-3,4-Bis((tert-Butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione(2a)

[0061] TBSCl (12.34 g, 81.9 mmol) and imidazole (8.36 g, 122.85 mmol) were added to a solution of uridine (5.0 g, 20.47 mmol) dissolved in 100 mL of anhydrous dimethylformamide at 0°C. After stirring at room temperature for 24 hours, the reaction mixture was poured into ice water and extracted with diethyl ether (150 mL x 3). The combined organic layer was dried with Na2SO4, filtered, and the filtrate was evaporated under reduced pressure. The residue was dissolved in 160 mL of tetrahydrofuran and treated with 60 mL of trifluoroacetic acid / water (1:2 v / v) solution at 0°C. After stirring at the same temperature for 6 hours, the reaction mixture was neutralized with a saturated aqueous NaHCO3 solution until the pH reached 6 and diluted with dichloromethane (300 mL). After separation, the aqueous layer was extracted with dichloromethane (100 mL x 3). The combined organic layer was washed with brine and dried with Na2SO4. After filtering the solid, the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methyl alcohol = 200:1 → 50:1 v / v) to obtain compound 2a (7.74 g, 16.37 mmol) in 80% yield.

[0062]

[0063] N-1-((2R,3R,4R,5R)-3,4-Bis((tert-Butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropymidin-4-yl)benzamide (2b)

[0064] Compound 2b was prepared with a 75% yield through the same process as compound 2a.

[0065] N-9-((2R,3R,4R,5R)-3,4-Bis((tert-Butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (2c)

[0066] Compound 2c was prepared with an 80% yield through the same process as compound 2a.

[0067]

[0068] 1-((2R,3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-vinyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione(3a)

[0069] IBX (5.90 g, 21.15 mmol) was added to a solution of compound 2a (5.0 g, 10.57 mmol) dissolved in 100 mL of anhydrous dimethylformamide, and the mixture was stirred in an oil bath at 80°C for 12 hours, after which the reaction mixture was cooled to 0°C. After dilution with 200 mL of ethyl acetate, the precipitate was filtered, and the filtrate was concentrated under reduced pressure. The residue was used in the next step without further purification. 25.2 mL of n-BuLi (40.19 mmol, 1.6 M hexane solution) was added to a suspension of Ph3PCH3Br (15.11 g, 42.30 mmol) dissolved in 80 mL of tetrahydrofuran under a nitrogen stream at 0°C. After 30 minutes, a solution of the aldehyde derivative dissolved in 150 mL of anhydrous tetrahydrofuran was added to the reaction mixture at -78°C and stirred at room temperature for 4 hours. The resulting solution was treated with 20 mL of methyl alcohol, and an ether-water solution (360 mL, 3:1 v / v) was added. The organic layer was separated, and the aqueous layer was extracted with diethyl ether (100 mL x 2). The combined organic layer was washed with brine (50 mL x 2) and dried with Na2SO4. After filtering the solid, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 10:1 → 1:1 v / v) to obtain compound 3a (3.96 g, 8.46 mmol) in 80% yield.

[0070]

[0071] N-(1-((2R,3R,4R,5R)-3,4-bis((tert-Butyldimethylsilyl)oxy)-5-vinyltetrahydrofuran-2-yl)-2-oxo-1,2-dihydropymidin-4-yl)benzamide (3b)

[0072] Compound 3b was prepared with a 75% yield through the same process as compound 3a.

[0073]

[0074] N-(9-((2R,3R,4R,5R)-3,4-bis((tert-Butyldimethylsilyl)oxy)-5-vinyltetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (3c)

[0075] Compound 3c was prepared with an 80% yield through the same process as compound 3a.

[0076]

[0077] tert-Butyl-3-((2R,3R,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-vinyltetrahydrofuran-2-yl)-2,6-dioxo-3,6-dihydropyrimidine-1(2H)-carboxylate (4a)

[0078] Boc2O (0.61 g, 2.81 mmol) and DMAP (0.03 g, 0.23 mmol) were added to a solution of compound 3a (1.10 g, 2.34 mmol) dissolved in 50 mL of tetrahydrofuran at 0°C. After stirring at room temperature for 12 hours, the reaction mixture was adsorbed onto silica gel and purified by silica gel column chromatography (hexane:ethyl acetate = 10:1 → 5:1 v / v) to obtain compound 4a (1.28 g, 2.25 mmol) in 80% yield.

[0079]

[0080] 5'-Vinyl-N 4 ,N 4 -bisBoc-cytidinederivative (4b)

[0081] 0.1 mL of NaOMe (0.54 g, 5.4 M methyl alcohol solution) was added to a solution of compound 3a (3.12 g, 5.45 mmol) dissolved in 40 mL of methyl alcohol at 0°C. After stirring at room temperature for 62 hours, the reaction mixture was adsorbed onto silica gel and purified by silica gel column chromatography (dichloromethane:methyl alcohol = 20:1 v / v) to obtain a 2,3-O-diTBS-5'-vinyl cytidine derivative (2.39 g, 5.14 mmol) in a yield of 94%. Boc2O (3.34 g, 15.32 mmol) and DMAP (0.31 g, 2.55 mmol) were added to a solution of a 5'-vinyl cytidine derivative (2.39 g, 5.14 mmol) dissolved in 50 mL of anhydrous tetrahydrofuran, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was adsorbed onto silica gel and purified by silica gel column chromatography (hexane:ethyl acetate = 10:1 v / v) to obtain compound 4b (3.07 g, 4.59 mmol) in 75% yield.

[0082]

[0083] 5'-Vinyl-N 6 ,N 6 -bisBoc-adenosinederivative (4c)

[0084] Compound 4c was prepared with an 80% yield through the same process as compound 4b.

[0085]

[0086] (3R,4R,5R)-5-Vinyltetrahydrofuran-2,3,4-triyl triacetate (6)

[0087] Concentrated sulfuric acid (0.52 g, 5.32 mmol) was added to a solution of D-ribose (8.0 g, 53.28 mmol) dissolved in 200 mL of methyl alcohol at 0°C. After stirring at room temperature for 12 hours, the reaction mixture was neutralized with NaHCO3 at 0°C until the pH reached 6 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methyl alcohol = 10:1 v / v) to obtain 1-0-methyl-D-ribose (8.22 g, 50.08 mmol) in a yield of 94%. TBSCl (30.18 g, 200.29 mmol) and imidazole (23.86 g, 350.51 mmol) were added at 0°C to a solution of 1-0-methyl-D-ribose derivative (8.22 g, 50.08 mmol) dissolved in 150 mL of anhydrous dimethylformamide. After stirring for 12 hours at room temperature, the reaction mixture was diluted with diethyl ether (600 mL) and washed with distilled water (100 mL x 2). The organic layer was dried with Na2SO4, filtered, and concentrated. The residue was dissolved in 200 mL of tetrahydrofuran / distilled water (4:1 v / v) and treated with trifluoroacetic acid (2.85 g, 25.03 mmol) at 0°C. After stirring for 8 hours at the same temperature, the reaction mixture was treated with NaHCO3 at 0°C until the pH reached 6 and extracted with diethyl ether (150 mL x 3). The organic layer was dried with Na2SO4. After filtering the excess solid, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 3:1 v / v at 10:1) to obtain 1-0-methyl2,3,-O-diTBS-D-ribose derivatives in a yield of 85%. IBX (14.26 g, 50.93 mmol) was added to a solution of 1-0-methyl2,3,-O-diTBS-D-ribose derivatives (10 g, 25.46 mmol) dissolved in 150 mL of anhydrous acetonitrile. After stirring at 80°C for 6 hours, the reaction mixture was cooled to 0°C and diluted with 200 mL of ethyl acetate.After filtering the precipitate, the filtrate was concentrated under reduced pressure to obtain the aldehyde derivative. The aldehyde was used in the next step without further purification. 60.4 mL of n-BuLi (96.77 mmol, 1.6 M hexane solution) was added to a suspension of Ph3PCH3Br (36.38 g, 101.86 mmol) dissolved in 200 mL of tetrahydrofuran under a nitrogen stream at 0°C. After 30 minutes, a solution of the aldehyde derivative dissolved in 200 mL of tetrahydrofuran was added to the reaction mixture at -10°C, and the mixture was stirred at room temperature for 4 hours. The resulting solution was treated with 20 mL of methyl alcohol, and an ether-distilled water solution (360 mL, 3:1 v / v) was added. The organic layer was separated, and the aqueous layer was extracted with ether (200 mL x 2). The combined organic layer was washed with brine (80 mL x 2) and dried with MgSO4. After filtering the solid, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 5:1 v / v at 25:1) to obtain a 5-vinyl-D-ribose derivative (7.91 g, 20.37 mmol) in 80% yield. Acetic acid (4.37 g, 77.18 mmol), acetic anhydride (3.93 g, 38.59 mmol), and concentrated sulfuric acid (0.07 g, 0.71 mmol) were added at 0°C to a solution of the 5-vinyl-D-ribose derivative (7.91 g, 20.37 mmol) dissolved in 20 mL of anhydrous dichloromethane. After stirring at room temperature for 12 hours, the reaction mixture was neutralized with a saturated aqueous NaHCO3 solution until pH 7 and extracted with diethyl ether (100 mL x 2). The organic layer was dried with Na2SO4. After filtering the excess solid, the filtrate was concentrated under reduced pressure. The organic layer was dried with Na2SO4 and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane:ethyl acetate = 10:1 → 2:1 v / v) to obtain Compound 6 (1.93 g, 7.) as a mixture of α- / β-isomers.10 mmol) was obtained with a yield of 92%.

[0088]

[0089] (2R,3R,4R,5R)-2-(2-Acetamido-6-((diphenylcarbamoyl)oxy)-9H-purin-9-yl)-5-vinyltetrahydrofuran-3,4-diyl diacetate (8)

[0090] BSA (1.52 g, 7.49 mmol) was added to a suspension of Compound 7 (1.45 g, 3.74 mmol) dissolved in 20 mL of anhydrous acetonitrile. After stirring at 80°C for 30 minutes, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in 10 mL of anhydrous toluene, and a solution of Compound 6 (0.85 g, 3.12 mmol) dissolved in 30 mL of anhydrous toluene was added to the mixture, followed by the addition of TMSOTf (0.83 g, 3.74 mmol) at 0°C. The reaction mixture was stirred at 80°C for 1.5 hours, followed by treatment with 5 mL of saturated aqueous NaHCO3 solution at 0°C. The resulting solution was extracted with ethyl acetate and washed with brine (30 mL). The organic layer was dried with Na2SO4 and filtered. The filtrate was purified by silica gel column chromatography (dichloromethane:methyl alcohol = 30:1 → 20:1 v / v) to obtain compound 8 (1.36 g, 2.24 mmol) in 72% yield.

[0091]

[0092] 2-Acetamido-9-(2R,3R,4R,5R)- 3,4-bis((tert-butyldimethylsilyl)oxy)-5-vinyltetrahydrofuran-2-yl)-9H-purin-6-yl diphenylcarbamate (4d)

[0093] 0.01 mL of NaOMe (0.05 mmol, 5.4 M methyl alcohol solution) was added to a solution of compound 8 (0.54 g, 0.89 mmol) dissolved in 8 mL of methyl alcohol at 0°C. After stirring for 2 hours at the same temperature, the reaction mixture was treated with a 1 NHCl aqueous solution until the pH reached 6 and concentrated under reduced pressure. The residue was dissolved in 4 mL of anhydrous dichloromethane and adsorbed onto pyridine (0.28 g, 3.59 mmol) and silica gel, and then purified by silica gel column chromatography (dichloromethane:methyl alcohol = 20:1 v / v) to obtain a 2,3-O-diTBS-5'-vinyl cytidine derivative (2.39 g, 5.14 mmol) in a yield of 94%. Boc2O (3.34 g, 15.32 mmol) and DMAP (0.31 g, 2.55 mmol) were added to a solution of a 5'-vinyl cytidine derivative (2.39 g, 5.14 mmol) dissolved in 50 mL of anhydrous tetrahydrofuran, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was adsorbed onto silica gel and purified by silica gel column chromatography (hexane:ethyl acetate = 10:1 v / v) to obtain compound 4d (3.07 g, 4.59 mmol) in 75% yield.

[0094]

[0095] <Example 2> Synthesis of Phosponamidate Derivative Having Terminal Double Bonds

[0096]

[0097] Ethyl(phenoxy(vinyl)phosphoryl)-D-alaninate (10) as a mixture of Rp- / Sp-isomers

[0098] Oxalyl chloride (14.68 g, 115.70 mmol) and a catalytic amount of dimethylformamide were added to a solution of vinyl phosphonic acid (9) (5.0 g, 46.28 mmol) dissolved in 80 mL of anhydrous dichloromethane at 0°C. After stirring at room temperature for 3 hours, the turbid solution was cooled to -78°C. Phenol (4.57 g, 48.60 mmol) and triethylamine (5.14 g, 50.92 mmol) were added to the reaction mixture at -78°C for 30 minutes. After stirring at the same temperature for 2 hours, L-alanine-ethyl ester (8.53 g, 55.55 mmol) and triethylamine (11.24 g, 111.10 mmol) were added at -78°C for 30 minutes. After stirring for 2 hours at the same temperature, the mixture was treated with an ether / ice solution and washed with a 2% aqueous HCl solution. The organic layer was dried with Na2SO4 and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate) to obtain compound 10b (9.17 g, 32.40 mmol) in 70% yield.

[0099]

[0100] <Example 3> Synthesis of Phosphonamidate Nucleotide Derivative

[0101] [Correction pursuant to Rule 91 25.02.2026]

[0102] As a mixture of E- / Z- and Rp- / Sp-isomers, tert-Butyl-3-((2R,3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(2-(((S)-1-ethoxy-1-oxopropan-2-yl)amino)(phenoxy)phosphory) vinyl)tetrahydrofuran-2-yl)-2,6-dioxo-3,6-dihydropyrimidine-1(2H)-carboxylate (11a)

[0103] A solution of compound 4a (0.15 g, 0.26 mmol) and compound 10 (0.15 g, 0.52 mmol) dissolved in 5 mL of anhydrous dichloromethane was irradiated at 50°C using a sonicator with 3 mol% Hoveyda-Grubbs second-generation catalyst (495 mg, 0.0079 mmol, 4 times for 2 hours each). After cooling the reaction mixture to room temperature, it was adsorbed onto silica gel and purified by silica gel column chromatography (hexane:ethyl acetate = 20:1 v / v) to obtain a 2,3-O-diTBS-5'-vinyl cytidine derivative (2.39 g, 5.14 mmol) in a yield of 94%. Boc2O (3.34 g, 15.32 mmol) and DMAP (0.31 g, 2.55 mmol) were added to a solution of a 5'-vinyl cytidine derivative (2.39 g, 5.14 mmol) dissolved in 50 mL of anhydrous tetrahydrofuran, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was adsorbed onto silica gel and purified by silica gel column chromatography (hexane:ethyl acetate = 3:1 → 1:1 v / v) to obtain compound 11a (0.18 g, 0.21 mmol) in an 82% yield (it is a mixture of E- / Z-isomers, and the ratio of E- / Z-isomers is 1 It was confirmed to be ~1:1 by H NMR).

[0104]

[0105] As a mixture of E- / Z- and Rp- / Sp-isomers Ethyl-(2-((2R,3R,4R,5R)-5-(4,4-bis((tert-butoxycarbonyl)amino)2-oxopyrimidin-1(2H)-yl)-3,4-bis((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)vinyl)(phenoxy)phosphoryl)-L-alaninate (11b)

[0106] Compound 11b was prepared with a 77% yield through the same process as compound 11a.

[0107]

[0108] As a mixture of E- / Z- and Rp- / Sp-isomers Ethyl-(2-((2R,3R,4R,5R)-5-(6,6-bis((tert-butoxycarbonyl)amino)-9H-purin-9-yl)-3,4-bis((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)vinyl)(phenoxy)phosphoryl)-L-alaninate (11c)

[0109] Compound 11c was prepared with a 68% yield through the same process as compound 11a.

[0110]

[0111] As a mixture of E- / Z- and Rp- / Sp-isomers Ethyl(2-((2R,3R,4R,5R)-5-(2-acetamido-6-(diphenylcarbamoyl)oxy)-9H-purin-9-yl)-3,4-bis((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)vinyl)(phenoxy)phosphoryl)-L-alaninate (11d)

[0112] Compound 11d was prepared with a 61% yield through the same process as compound 11a.

[0113]

[0114] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

1. A method for preparing a phosphonamidate nucleotide derivative having a structure represented by Formula 3, comprising reacting a compound represented by Formula 1 and a compound represented by Formula 2 under ultrasonic irradiation: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] In the above chemical formulas 1 to 3, R is TBS (tert-butyldimethylsilyl), and B and BASE include those selected from the group consisting of uridine, adenine, cytidine, and guanine, and OPG includes those selected from the group consisting of silyl groups, benzyl groups, and acetal groups, and R1 and R2 each include those independently selected from the group consisting of amino acid alkyl esters, benzyl, phenol, aryl, and combinations thereof.

2. In Paragraph 1, A method in which an equal amount of Hoveyda-Grubbs catalyst is added in a split manner in the above method.

3. In Paragraph 1, A method in which a phosphonamidate nucleotide derivative is synthesized through a cross ethathesis reaction between the vinyl group at the C5'-position of the compound represented by Chemical Formula 1 and the vinyl group of the compound represented by Chemical Formula 2.

4. In Paragraph 1, A method in which the compound represented by the above chemical formula 1 is synthesized to have a nucleoside derivative having a terminal double bond at the 5'-position using a natural nucleoside selected from the group consisting of adenosine, cytidine, and uridine as a starting material.

5. In Paragraph 1, A method in which the compound represented by the above chemical formula 1 is synthesized by reacting an intermediate having a 1,2,3-acyl-5-double bond, synthesized using D-ribose as a starting material, with guanine.

6. In Paragraph 1, A method in which the compound represented by the above chemical formula 2 is synthesized by reacting vinylphosphonic acid with oxalyl chloride using vinylphosphonic acid as a starting material.

7. An antiviral composition comprising a phosphonamidate nucleotide derivative prepared by the method of any one of claims 1 to 6 as an active ingredient.

8. An anticancer composition comprising a phosphonamidate nucleotide derivative prepared by the method of any one of claims 1 to 6 as an active ingredient.

9. A method for preventing or treating a viral infection, comprising administering the antiviral composition of claim 7 to an individual.

10. In Paragraph 9, A method for preventing or treating viral infection, wherein the above-mentioned individual is a mammal.

11. A method for preventing or treating cancer, comprising administering the anticancer composition of claim 8 to an individual.

12. In Paragraph 11, A method for preventing or treating cancer, wherein the above-mentioned individual is a mammal.

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