Flexible base nucleoside analogue and medical application thereof

Abstract

The invention relates to a compound shown in a formula I or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the compound or the pharmaceutically acceptable salt thereof as an active component, and application of the compound or the pharmaceutically acceptable salt thereof in preparation of antiviral drugs. .

Description

Technical Field

[0001] This invention relates to flexible nucleoside analogs or pharmaceutically acceptable salts thereof for antiviral purposes, pharmaceutical compositions containing such analogs or pharmaceutically acceptable salts thereof as active ingredients, and the use of said analogs or pharmaceutically acceptable salts thereof in the preparation of antiviral drugs. Background Technology

[0002] RNA viruses are highly mutated and dangerous pathogens. Their genetic material generates numerous errors during replication (potentially producing thousands of mutations per replication), allowing them to quickly evade drug attacks and develop drug resistance. These viruses can also infect humans and animals through various routes, including airborne transmission and mosquito bites, posing a severe challenge to global viral outbreaks and infectious disease control.

[0003] Currently, nucleoside analogues for anti-RNA virus treatment have several drawbacks. First, their molecular structures are too rigid, like a fixed-shape key, only able to match specific viral targets. Once the virus mutates (e.g., altering the target structure), the drug quickly becomes ineffective. Second, the effective dose is very close to the toxic dose, easily damaging normal human cells and resulting in poor safety. Furthermore, the antiviral activity of nucleoside analogues needs improvement. Therefore, there is an urgent clinical need for broad-spectrum nucleoside analogues with novel structures to combat RNA viruses.

[0004] Flexible nucleosides are nucleoside molecules that can flexibly adjust their structure like a "transformer key." These molecules "split" a rigid purine ring into an imidazole ring and a pyrimidine ring, connected by a conformationally flexible C-C single bond, resulting in a flexible conformational nucleoside analog. This flexible base structure not only enhances the tunability of the molecular conformation but also allows it to better adapt to the spatial and electronic characteristics of viral enzyme binding sites. Through this design, nucleoside molecules can bind more effectively to the active sites of viral target enzymes, thereby overcoming drug resistance problems caused by viral mutations. Furthermore, this flexibility also improves the interaction between the drug and the target, for example, by forming more hydrogen bonds, hydrophobic interactions, or van der Waals forces, further enhancing its antiviral activity.

[0005] HP083 is an acyclic flexible nucleoside analog derived from the purine bases of the nucleoside antiviral drug acyclovir. Studies have shown that HP083 exhibits potent and broad-spectrum inhibitory effects against a variety of highly pathogenic RNA viruses, such as dengue virus (DENV), Zika virus (ZIKV), coronaviruses (SARS-CoV, MERS-CoV), and Ebola virus (EBOV), and its toxicity to normal cells is reduced by more than 90% compared to traditional nucleoside drugs.

[0006] However, flexible base nucleoside analogs based on the classic five-membered cyclic nucleotide structure can maintain their antiviral activity, which is of great significance for expanding the molecular diversity of nucleoside antiviral drugs and discovering the lead structures of broad-spectrum anti-RNA virus drugs.

[0007] Summary of the Invention

[0008] This invention designs and synthesizes a series of compounds represented by Formula I:

[0009]

[0010] in:

[0011] X represents carbon, nitrogen, oxygen, or sulfur.

[0012] R1 is hydrogen, deuterium, hydroxyl, halogen, amino, alkyl, NH-alkyl, O-alkyl, or alkyl-substituted amino group;

[0013] R2 can be hydrogen, deuterium, hydroxyl, sulfur, halogen, trifluoromethyl, alkyl, cyano, amino, NH-alkyl, N-alkyl, O-alkyl, or NH-OH.

[0014] R3 is hydrogen, a halogen, OR 4、 Cyano, C1–C6 alkyl, C1–C6 alkoxy, C2–C6 alkenyl, C2–C6 alkynyl;

[0015] R4 represents hydrogen, azide, and C. 1-20 Alkyl group, amino group C 1-20 Alkyl group, C 1-6 Alkylamino, C 1-6 Alkyl group, C 1-6 Alkoxy, C 1-6 Alkyl groups, α-amino acids (the carboxyl group of the α-amino acid is linked to the hydroxyl group on the furan ring by an ester bond).

[0016] R5 can be hydrogen, deuterium, monophosphate, diphosphate, triphosphate, or various prodrugs (such as lipids, lipid phosphates, lipid esters, amino acid prodrugs, etc.).

[0017] L can be -CH2-, sulfur, vinyl, ethynyl, or -O-(CH2). n -、-NH-(CH2) n - (n is an integer selected from 0–2, for example 0, 1, 2.)

[0018] W1 can be hydrogen, hydroxyl, sulfur, fluorine, amino, NH-alkyl, or O-alkyl.

[0019] W2 can be hydrogen, hydroxyl, sulfur, fluorine, amino, NH-alkyl, or O-alkyl.

[0020] Y1 can be hydrogen, deuterium, hydroxyl, sulfur, halogen, trifluoromethyl, alkyl, cyano, amino, NH-alkyl, N-alkyl, O-alkyl, or NH-OH.

[0021] Y2 is hydrogen, deuterium, hydroxyl, halogen, amino, alkyl, NH-alkyl, O-alkyl, or alkyl-substituted amino groups;

[0022] Activity evaluation results showed that the compound of formula I exhibited high in vitro anti-H1N1 influenza virus activity, low cytotoxicity, and good safety.

[0023] Based on the above results, this invention is complete.

[0024] This invention provides compounds of formula I or pharmaceutically acceptable salts thereof:

[0025]

[0026] In some embodiments, the compound shown in Formula I has the structure shown in Formula Ia or Formula Ib.

[0027]

[0028] Among them, Y 1、 The definitions of Y2 and R3, R4, and R5 are as described in any embodiment of the present invention.

[0029] In some embodiments, the compound represented by Formula I is selected from:

[0030]

[0031] The present invention also provides pharmaceutical compositions comprising a compound of Formula I or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable excipient or carrier. These pharmaceutical compositions may be solutions, tablets, capsules, or injections. These pharmaceutical compositions may be administered by injection or orally.

[0032] The present invention also provides the use of a compound of Formula I or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing a compound of Formula I or a pharmaceutically acceptable salt thereof as an active ingredient, in the preparation of an antiviral drug.

[0033] The present invention also provides the use of a compound of Formula I or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing a compound of Formula I or a pharmaceutically acceptable salt thereof as an active ingredient, in the preparation of a medicament as an RNA-dependent RNA polymerase.

[0034] As used herein, the term "pharmaceutical composition" refers to a composition containing one or more of the compounds described herein or their pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier or excipient. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity. Carriers described herein include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycerol, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose materials, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, and lanolin. The excipients refer to additives in pharmaceutical preparations other than the active pharmaceutical ingredient (API). They are stable, have no incompatibilities with the API, do not produce side effects, do not affect efficacy, are not easily deformed, cracked, moldy, or infested by insects at room temperature, are harmless to the human body, have no physiological effects, do not react chemically or physically with the API, and do not affect the determination of the API's content. Examples of excipients include binders, fillers, disintegrants, and lubricants in tablets; wine, vinegar, and medicinal juices in traditional Chinese medicine pills; the base portion in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solvents, osmotic pressure regulators, and colorants in liquid preparations.

[0035] The compounds of the present invention or their pharmaceutically acceptable salts or pharmaceutical compositions thereof may be administered via parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intramuscular routes or as inhalers.

[0036] The compounds of the present invention, or their pharmaceutically acceptable salts, or their pharmaceutical compositions, can be formulated into various suitable dosage forms depending on the route of administration.

[0037] When taken orally, the compounds of this invention can be formulated into any orally acceptable dosage form, including but not limited to tablets, capsules, aqueous solutions, or aqueous suspensions. Tablets typically use carriers including lactose and corn starch, and lubricants such as magnesium stearate may also be added. Capsule formulations typically use diluents including lactose and dried corn starch. Aqueous suspension formulations usually involve mixing the active ingredient with suitable emulsifiers and suspending agents. Optionally, sweeteners, flavorings, or colorings may also be added to the above oral dosage forms.

[0038] When applied topically to the skin, the compounds of the present invention can be formulated into suitable ointments, lotions, or creams, wherein the active ingredients are suspended or dissolved in one or more carriers. Carriers that can be used in ointment formulations include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsified waxes, and water; carriers that can be used in lotions or creams include, but are not limited to, mineral oil, sorbitan monostearate, Tween 60, hexadecyl ester wax, hexadecene aromatic alcohol, 2-octyldodecyl alcohol, benzyl alcohol, and water.

[0039] The compounds of this invention can also be administered in sterile injectable formulations, including sterile injectable water or oil suspensions or sterile injectable solutions. The carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspension media.

[0040] Typically, an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof sufficient to achieve a preventive or therapeutic effect is from about 0.001 mg / kg body weight / day to about 10,000 mg / kg body weight / day. Where suitable, the dose is from about 0.01 mg / kg body weight / day to about 1000 mg / kg body weight / day. The dose range can be from about 0.01 to 1000 mg / kg body weight daily, every two days, or every three days, and more typically from 0.1 to 500 mg / kg body weight. Exemplary treatment regimens are administration once every two days, once weekly, or once monthly. The formulation is usually given multiple times, with intervals between single doses that can be daily, weekly, monthly, or annually. Alternatively, the formulation can be given in the form of a sustained-release formulation, in which case a less frequent dosing is required. The dose and frequency vary depending on the half-life of the formulation in the subject. It can also vary depending on whether it is a prophylactic or therapeutic treatment. In prophylactic use, a relatively low dose is given for a long period at relatively low frequency intervals. In therapeutic applications, it is sometimes necessary to administer relatively high doses at relatively short intervals until the progression of the disease is slowed or stopped, and preferably until the individual shows partial or complete improvement in the symptoms of the disease. After this, a preventive treatment can be given to the patient.

[0041] In some embodiments, the synthesis of the compound represented by formula Ia begins with 1-acetoxy-2,3,5-tribenzoyloxy-β-D-ribofuranosyl (1a) as the starting material, which is coupled with 4,5-diiodoimidazole to generate intermediate (2a). 2a is then deprotected to generate intermediate (4,5-diiodoimidazole-1-yl)-1-β-D-ribofuranosyl (3a). The three hydroxyl groups of 3a are then reprotected with benzyl groups to give 2,3-dibenzyloxy-5-benzyloxymethyl-1-(4,5-diiodoimidazole-1-yl)-1-β-D-ribofuranosyl (4a). 4a is reacted with a Grignard reagent to give 2,3-dibenzyloxy-5-benzyloxymethyl-1-(4-iodoimidazole-3-yl)-1-β-D-ribofuranosyl (5a). 5a is then coupled with a 2,4-substituted pyrimidine borate ester to generate intermediate (6a). 6a was deprotected to obtain the target product Ia; the three hydroxyl groups of the sugar ring were modified with acetate, propionate, isobutyrate and other esters respectively to obtain the target compound Ib.

[0042] The synthetic routes for the compounds represented by formulas Ia and Ib are shown below:

[0043]

[0044] In formula Ia or Ib, R1 and R2 are defined as described in any embodiment of the present invention. Detailed Implementation

[0045] The following specific embodiments of the present invention will further illustrate the substantive content of the invention. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the invention. Unless otherwise specified in the following embodiments, conditions are performed according to conventional conditions or the manufacturer's recommendations. Raw materials whose manufacturers are not specified are all commercially available conventional products.

[0046] While many of the materials and methods of operation used in the following embodiments are well known in the art, the present invention is still described in as much detail as possible herein. It will be apparent to those skilled in the art that, unless otherwise stated, the materials and methods of operation used in the following embodiments are well known in the art.

[0047] Example 1: Synthesis of (2R,3R,4S,5R)-2-(4-(2,4-dimethoxypyrimidin-5-yl)1H-imidazol-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Ia-1)

[0048]

[0049] 1.1 Synthesis of (2R,3R,4R,5R)-2,3-dibenzoyloxy-5-benzoyloxymethyl-1-(4,5-diiodoimidazole-1-yl)-β-D-ribofuranosyl (2a)

[0050]

[0051] Take a 1000 mL three-necked round-bottom flask, dissolve 50 g (99.18 mmol) of 1-acetoxy-2,3,5-tribenzoyloxy-β-D-furanose (1a) and 32 g (99.18 mmol) of 4,5-diiodoimidazole in 450 mL of acetonitrile, add 50 mL (198.36 mmol) of N,O-bis(trimethylsilylacetamide), stir at room temperature for 4 h under nitrogen protection, cool the reaction solution to 0 °C, slowly add 20 mL (109.10 mmol) of trimethylsilyl trifluoromethanesulfonate, heat to 60 °C and react for 18 h, perform TLC to detect complete reaction, turn off stirring. Cool the reaction solution to room temperature, rotary evaporate under reduced pressure, add an appropriate amount of DCM to dissolve, wash twice with saturated NaHCO3 solution and saturated NaCl solution, dry with anhydrous sodium sulfate, concentrate under reduced pressure to obtain a brown oily substance. The crude product was separated by column chromatography using 200-300 mesh silica gel with a PE:EA ratio of 7:1 as the eluent, and finally 70.00 g of yellowish-brown solid (2a) was obtained, with a yield of 92.4%.

[0052] 1.2 Synthesis of (2R,3R,4S,5R)-(4,5-diiodoimidazole-1-yl)-1-β-D-furanose (3a)

[0053]

[0054] 50 g (65.45 mmol) of 2,3-dibenzoyloxy-5-benzoyloxymethyl-1-(4,5-diiodoimidazole-1-yl)-β-D-ribofuranosyl (2a) was weighed and added to 500 mL of methanol. Under nitrogen protection, the mixture was stirred until fully dissolved. The reaction temperature was lowered to 0 °C, and 7.07 g of sodium methoxide (130.90 mmol) was added in portions. The mixture was stirred at 0 °C for 1 h. The reaction was stopped after TLC detection to confirm completeness. The mixture was concentrated under reduced pressure, and a small amount of silica gel powder was added for mixing. The mixture was then separated by column chromatography using 200-300 mesh silica gel with DCM:MeOH = 20:1 as the eluent. 25.8 g of a brown oily substance (2R,3R,4S,5R)-(4,5-diiodoimidazole-1-yl)-1-β-D-ribofuranosyl (3a) was obtained, with a yield of 87.2%.

[0055] 1.3 Synthesis of 1-((2R,3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl)-4,5-diiodo-1H-imidazolium (4a)

[0056]

[0057] Take a 500 mL three-necked round-bottom flask and transfer 3 g (12.1 mmol) of 4,5-diiodoimidazole-1-yl-1-β-D-furanose (3a) into the flask. Under argon protection, add an appropriate amount of ultra-dry THF into the flask. Under ice-water bath stirring, slowly add 73 mL (73 mmol) of 1 M boranetetrahydrofuran complex solution. After the addition is complete, transfer to an oil bath and heat under reflux for 18 h. Perform TLC detection to confirm the reaction is complete. Stop heating and allow the reaction solution to cool to room temperature. Under stirring, slowly add an appropriate amount of methanol solution until no bubbles are produced. Concentrate under reduced pressure to dryness to obtain a grayish-white solid. Repeat twice. Add a small amount of methanol solution, heat under reflux and stir until fully dissolved. Slowly add ethyl acetate to the solution until a large amount of white solid is produced. Filter and add excess concentrated hydrochloric acid to the filtrate to form a salt. Concentrate under reduced pressure to dryness, add a small amount of silica gel powder and mix. Then, perform column chromatography separation and purification using 200-300 mesh silica gel. Use DCM:MeOH = 30:1 eluent for column chromatography separation to obtain 1.98 g of white solid (4a), with a yield of 60.43%.

[0058] 1.4 Synthesis of 1-((2R,3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl)-4-iodo-1H-imidazolium (5a)

[0059]

[0060] Under an argon atmosphere, compound 3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl)-4-iodo-1H-imidazolium (4a) (1.00 g, 1.385 mmol, 1.0 eq) was dissolved in anhydrous THF and stirred in a 100 mL three-necked round-bottom flask until completely dissolved. Then, 1 M ethyl magnesium bromide solution (1.66 mL, 1.662 mmol, 1.2 eq) was slowly added dropwise, and stirring continued at room temperature for 1 hour after the addition was complete. After confirming the completion of the reaction by TLC, the reaction was quenched with saturated ammonium chloride solution. After removing the solvent under reduced pressure, column chromatography was performed with 100-200 mesh silica gel and isocratic elution with petroleum ether / ethyl acetate (3:1) to finally obtain 0.46 g of yellow oily product 5a, with a yield of 55.2%.

[0061] 1.5 Synthesis of 5-(1-((2R,3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl)-1H-imidazol-4-yl)-2,4-dimethoxypyrimidine (6a-1)

[0062]

[0063] 3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl)-4-iodo-1H-imidazolium (5a) (1.00 g, 1.680 mmol, 1.0 eq) was transferred to a round-bottom flask, and tetrahydrofuran (80 mL) was added. The mixture was stirred until completely dissolved. Then, 2,4-dimethoxypyrimidin-5-boronic acid pinacol ester (0.49 g, 1.850 mmol, 1.1 eq), potassium carbonate (0.79 g, 5.710 mmol, 3.0 eq), bis(triphenylphosphine)palladium dichloride (0.26 g, 0.370 mmol, 0.2 eq), and deionized water (5 mL) were added sequentially. After purging with nitrogen, the reaction mixture was refluxed in an oil bath at 85 °C for 4 hours, and the reaction progress was monitored by TLC. After the reaction was complete, the mixture was allowed to cool naturally to room temperature, and the solvent was removed under vacuum. The residue was extracted with an ethyl acetate / water system, the organic layer was washed with saturated brine, and dried over anhydrous sodium sulfate. Concentration of the organic phase yielded 0.82 g of a brown oily product 6a-1, with a yield of 80.4%.

[0064] 1.6 Synthesis of (2R,3R,4S,5R)-2-(4-(2,4-dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Ia-1)

[0065]

[0066] Compound 6a-1 (0.70 g, 1.150 mmol, 1.0 eq) was transferred to a round-bottom flask, and ultradry DCM (50 mL) was added. The mixture was purged with argon, and the reaction solution temperature was lowered to -78°C. Boron trichloride (3.85 mL, 4.610 mmol, 4.0 eq) was slowly injected. After injection, the temperature was raised to -30°C and stirred for 1 h. The reaction was then quenched with 2 mL of methanol. The reaction mixture was separated by silica gel column chromatography, using a dichloromethane / methanol (10:1) eluent. The target fraction was collected, concentrated, and dried to give 0.21 g of Ia-1 as a white solid, with a yield of 55.0%. HRMS (ESI) m / z: calcd. for C 14 H 18 N4O6 [M + H] + 339.1304, found 339.1305. 1H NMR (600 MHz, DMSO-d6) δ 8.86 (s, 1H), 7.97 (d, J= 1.3 Hz, 1H), 7.74 (d, J = 1.3 Hz, 1H), 5.61 (d, J = 5.7 Hz, 1H), 5.44 (brs,1H), 5.18 (brs, 1H), 5.09–5.07 (m, 1H), 4.20 (t, J = 5.5 Hz, 1H), 4.09–4.08(m, 1H), 4.05 (s, 3H), 3.97–3.94 (m, 1H), 3.93 (s, 3H), 3.63 (dt, J = 11.7,3.5 Hz, 1H), 3.57 (dt, J = 11.7, 4.0 Hz, 1H).

[0067] Example 2: Synthesis of (2R,3R,4S,5R)-2-(4-(2-amino-4-methylpyrimidin-5-yl)-1H-imidazol-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Ia-2)

[0068]

[0069] 2.1 Synthesis of 5-(1-((2R,3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl)-1H-imidazol-4-yl)-4-methylpyrimidin-2-amine (6a-2)

[0070]

[0071] Compound 5a (1.00 g, 1.680 mmol, 1.0 eq) was added to a round-bottom flask, and 2,4-dimethoxypyrimidine-5-boronic acid pinacol ester was replaced with 2-amino-4-methylpyrimidine-5-boronic acid pinacol ester (0.44 g, 1.850 mmol, 1.1 eq). The remaining steps were the same as those for synthesis 10a, yielding 0.83 g of a brown oily compound 6b-2 in 85.5% yield.

[0072] 2.2 Synthesis of (2R,3R,4S,5R)-2-(4-(2-amino-4-methylpyrimidin-5-yl)-1H-imidazol-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Ia-2)

[0073]

[0074] Compound 6a-2 (0.50 g, 0.866 mmol, 1.0 eq) was transferred to a round-bottom flask, and the remaining procedures were the same as those for the synthesis of compound SW-HX-21, yielding 0.13 g of Ia-2 as a white solid, in 47.0% yield. HRMS (ESI) m / z: calcd. for C 13 H 17 N5O4 [M + H] + 308.1359, found 308.1413. 1 H NMR (600 MHz, DMSO-d6) δ 8.45 (s, 1H), 7.94 (d, J = 0.9 Hz, 1H), 7.52 (d, J = 0.9 Hz, 1H), 6.48 (s, 2H), 5.59 (d, J = 5.8 Hz, 1H), 5.43 (brs, 1H), 5.17 (brs, 1H), 5.02(t, J = 5.4 Hz, 1H), 4.24–4.22 (m, 1H), 4.08 (t, J = 4.2 Hz, 1H), 3.91 (q, J= 3.8 Hz, 1H), 3.64–3.60 (m, 1H), 3.57–3.53 (m, 1H), 2.43 (s, 3H).

[0075] Example 3: Synthesis of (2R,3R,4S,5R)-2-(4-(2-amino-4-(trifluoromethyl)pyrimidin-5-yl)-1H-imidazol-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Ia-3)

[0076]

[0077] 3.1 Synthesis of 5-(1-((2R,3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl)-1H-imidazol-4-yl)-4-(trifluoromethyl)pyrimidine-2-amine (6a-3)

[0078]

[0079] Compound 5a (1.00 g, 1.680 mmol, 1.0 eq) was added to a round-bottom flask, and 2,4-dimethoxypyrimidine-5-boronic acid pinacol ester was replaced with 2-amino-4-trifluoromethylpyrimidine-5-boronic acid pinacol ester (0.54 g, 1.850 mmol, 1.1 eq). The remaining steps were the same as those for the synthesis of 6a-1, yielding 0.80 g of brown oily compound 6a-3 in 75.4% yield.

[0080] 3.2 Synthesis of (2R,3R,4S,5R)-2-(4-(2-amino-4-(trifluoromethyl)pyrimidin-5-yl)-1H-imidazol-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Ia-3)

[0081]

[0082] Compound 6a-3 (0.80 g, 1.267 mmol, 1.0 eq) was transferred to a round-bottom flask, and the remaining procedures were the same as those for the synthesis of compound Ia-1, yielding 0.26 g of a yellow solid, compound Ia-3, in 57.5% yield. HRMS(ESI) m / z: calcd. for C 13 H 14 F3N5O4 [M + H] + 362.1076, found 362.1074. 1 H NMR (600 MHz, DMSO-d6) δ 8.67 (s, 1H), 7.96 (d, J = 1.1 Hz, 1H), 7.27 (s, 2H), 7.52 (s, 1H), 5.59 (d, J = 5.7 Hz, 1H), 5.40 (d, J = 6.2 Hz, 1H), 5.16 (d, J= 4.3 Hz, 1H), 4.99 (t, J = 5.2 Hz, 1H), 4.18 (q, J = 5.5 Hz, 1H), 4.05 (q, J= 3.7 Hz, 1H), 3.90 (q, J = 3.7 Hz, 1H), 3.60 (ddd, J = 11.8, 5.3, 3.8 Hz, 1H), 3.53 (ddd, J = 11.8, 5.1, 3.8 Hz, 1H).

[0083] Example 4: Synthesis of (2R,3R,4S,5R)-2-(4-(2-aminopyrimidin-5-yl)-1H-imidazol-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Ia-4)

[0084]

[0085] 4.1 Synthesis of 5-(1-((2R,3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxymethyl)tetrahydrofuran-2-yl)-1H-imidazol-4-yl)pyrimidine-2-amine (6a-4)

[0086]

[0087] Compound 5a (1.00 g, 1.680 mmol, 1.0 eq) was added to a round-bottom flask, and 2,4-dimethoxypyrimidine-5-boronic acid pinacol ester was replaced with 2-aminopyrimidine-5-boronic acid pinacol ester (0.41 g, 1.850 mmol, 1.1 eq). The remaining steps were the same as those for the synthesis of 6a-1, yielding 0.81 g of compound 6a-4, a brown oil, in 85.7% yield.

[0088] 4.2 Synthesis of (2R,3R,4S,5R)-2-(4-(2-aminopyrimidin-5-yl)-1H-imidazol-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Ia-4)

[0089]

[0090] Compound 6a-4 (0.50 g, 0.888 mmol, 1.0 eq) was transferred to a round-bottom flask, and the remaining procedures were the same as those for the synthesis of compound Ia-1, yielding 0.13 g of a yellow solid, compound Ia-4, in 46.1% yield. HRMS(ESI) m / z: calcd. for C 12 H 15 N5O4 [M + H] + 294.1202, found 294.1196. 1H NMR (600MHz, DMSO-d6) δ 8.59 (s, 1H), 8.20 (s, 1H), 7.90 (d, J = 1.3 Hz, 1H), 7.73(d, J = 1.3 Hz, 1H), 6.80 (s, 1H), 6.59 (s, 2H), 5.55 (d, J = 5.9 Hz, 1H), 5.20 (s, 1H), 5.05 (s, 1H), 4.22 (t, J = 6.6 Hz, 1H), 3.92 (t, J = 3.9 Hz, 1H), 3.89 (q, J = 4.0 Hz, 1H).

[0091] Example 5: Synthesis of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4-(2,4-dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)tetrahydrofuran-3,4-dimethyldiacetate (Ib-1)

[0092]

[0093] 40 mL of ultradry DMF was added to a 100 mL round-bottom flask, followed by compound Ia-1 (0.30 g, 0.887 mmol, 1.0 eq). The mixture was stirred until completely dissolved. Then, 4-dimethylaminopyridine (0.011 g, 0.090 mmol, 0.10 eq) and acetic anhydride (0.36 g, 3.548 mmol, 4.0 eq) were added sequentially. The reaction was stirred at room temperature for 2 hours and confirmed to be complete by TLC. An equal volume of water / ethyl acetate mixture was added to the reaction system, and the mixture was extracted with ethyl acetate. The organic layer was washed with sodium bicarbonate solution and brine, dried, and concentrated. Purification was then performed by silica gel column chromatography (dichloromethane / methanol = 20:1) to give 0.24 g of white solid Ib-1, with a yield of 59.5%. HRMS (ESI) m / z: calcd. for C 20 H 24 N4O9 [M + H] + 465.1621, found 465.1620. 1H NMR (600MHz, CD3OD) δ 8.84 (s, 1H), 7.98 (d, J = 1.3 Hz, 1H), 7.69 (d, J = 1.3 Hz, 1H), 6.03 (d, J = 5.8 Hz, 1H), 5.52 (t, J = 5.7 Hz, 1H), 5.44 (dd, J = 5.5, 4.0 Hz, 1H), 4.48 (q, J = 3.6 Hz, 1H), 4.39 (dd, J = 12.5, 3.2 Hz, 1H), 4.36 (dd, J = 12.5, 3.9 Hz, 1H), 4.16 (s, 3H), 4.03 (s, 3H), 2.14 (s, 3H), 2.14 (s, 3H), 2.08 (s, 3H).

[0094] Example 6: Synthesis of (2R,3R,4R,5R)-2-(4-(2,4-dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)-5-((propionyloxy)methyl)tetrahydrofuran-3,4-dimethyldipropionate (Ib-2)

[0095]

[0096] Compound Ia-1 (0.30 g, 0.887 mmol, 1.0 eq) was added to ultradry DMF, and acetic anhydride was replaced with propionic anhydride (0.46 g, 3.548 mmol, 4.0 eq). The same procedure as for the synthesis of compound Ib-1 was followed to obtain 0.29 g of white solid Ib-2 in 64.6% yield.

[0097] HRMS (ESI) m / z: calcd. for C 23 H 30 N4O9 [M + H] + 507.2091, found 507.2090. 1H NMR (600 MHz, CD3OD) δ 8.85 (s, 1H), 7.66 (d, J = 1.3 Hz, 1H), 7.57 (d, J = 1.3 Hz, 1H), 6.04 (d, J = 8.1 Hz, 1H), 5.56 (t, J = 5.7 Hz, 1H), 5.47 (dd, J = 5.5, 3.9 Hz, 1H), 4.60 (q, J = 3.6 Hz, 1H), 4.50 (dd, J = 12.5, 3.5 Hz, 1H), 4.43 (dd, J = 12.5, 3.9 Hz, 1H), 4.18 (s, 3H), 4.04 (s, 3H),2.48–2.42 (m, 6H), 1.17 (t, J = 3.5 Hz, 3H), 1.14 (t, J = 3.5 Hz, 3H), 1.12(t, J = 3.5 Hz, 3H).

[0098] Example 7: Synthesis of (2R,3R,4R,5R)-2-(4-(2,4-dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)-5-((isobutyryloxy)methyl)tetrahydrofuran-3,4-diylbis(2-methylpropionate) (Ib-3)

[0099]

[0100] Compound Ia-1 (0.30 g, 0.887 mmol, 1.0 eq) was added to ultradry DMF, and acetic anhydride was replaced with isobutyric anhydride (0.56 g, 3.548 mmol, 4.0 eq). The same procedure as for the synthesis of compound Ib-1 was followed to give 0.30 g of compound Ib-3 as a white solid, with a yield of 61.7%. HRMS (ESI) m / z: calcd. for C 26 H 36 N4O9 [M +H] + 549.2540, found 549.2558. 1H NMR (600 MHz, CD3OD) δ8.87 (s, 1H), 8.02 (d,J = 1.3 Hz, 1H), 7.69 (d, J = 1.3 Hz, 1H), 6.12(d, J = 6.1 Hz, 1H), 5.54 (t,J = 5.6 Hz, 1H), 5.47 (dd, J = 5.8, 4.0 Hz, 1H), 4.58–4.51 (m, 1H), 4.50 (dd, J = 11.8, 4.5 Hz, 1H), 4.45 (dd, J = 11.9, 4.4 Hz, 1H), 4.17 (s, 3H), 4.08 (s,3H),2.39–2.34 (m, 3H) 1.26 (d, J = 7.0 Hz, 3H), 1.23 (d, J = 7.0 Hz, 3H), 1.20 (d, J = 7.0 Hz, 3H), 1.17 (d, J = 7.0 Hz, 3H).

[0101] Example 8: Synthesis of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4-(2-amino-4-methylpyrimidin-5-yl)-1H-imidazol-1-yl)tetrahydrofuran-3,4-diacyldiacetate (Ib-4)

[0102]

[0103] 40 mL of ultra-dry DMF was added to a 100 mL round-bottom flask, followed by compound Ia-2 (0.40 g, 1.302 mmol, 1.0 eq). The mixture was stirred until completely dissolved. Then, 4-dimethylaminopyridine (0.020 g, 0.130 mmol, 0.1 eq) and acetic anhydride (0.53 g, 5.208 mmol, 4.0 eq) were added sequentially. The reaction was allowed to proceed at room temperature for 2 hours, and the endpoint was observed by TLC. The post-treatment was the same as for Ib-1, and column chromatography was used to purify 0.39 g of white solid Ib-4, with a yield of 69.2%. HRMS (ESI) m / z: calcd. forC 19 H 23 N5O7 [M + H] + 434.1675, found 434.1692. 1H NMR (600 MHz, CD3OD) δ 8.36 (s,1H), 8.00 (d, J = 1.3 Hz, 1H), 7.41 (d, J = 1.3 Hz, 1H), 6.03 (d, J = 5.7 Hz,1H), 5.53 (t, J = 5.7 Hz, 1H), 5.42 (dd, J = 5.6, 4.2 Hz, 1H), 4.46 (q, J =3.9 Hz, 1H), 4.41 (dd, J = 12.3, 4.1 Hz, 1H), 4.37 (dd, J = 12.3, 3.4 Hz,1H), 2.45 (s, 3H), 2.13 (s, 3H), 2.11 (s, 3H), 2.09 (s, 3H).

[0104] Example 9: Synthesis of (2R,3R,4R,5R)-2-(4-(2-amino-4-methylpyrimidin-5-yl)-1H-imidazol-1-yl)-5-((propionyloxy)methyl)tetrahydrofuran-3,4-dimethyldipropionate (Ib-5)

[0105]

[0106] Compound Ia-2 (0.40 g, 1.302 mmol, 1.0 eq) was added to ultradry DMF, and acetic anhydride was replaced with propionic anhydride (0.68 g, 5.208 mmol, 4.0 eq). The rest of the procedure was the same as for the synthesis of Ib-4, yielding 0.38 g of compound Ib-5 as a white solid, in 61.7% yield. HRMS (ESI) m / z: calcd. for C 22 H 29 N5O7 [M + H] + 476.2145, found 476.2143. 1H NMR (600 MHz, DMSO-d6) δ 8.45 (s, 1H), 8.00 (d, J = 1.2 Hz,1H), 7.55 (d, J = 1.2 Hz, 1H), 6.55 (s, 2H), 6.04 (d, J = 6.1 Hz, 1H), 5.55(t, J = 6.0 Hz, 1H), 5.40 (dd, J = 5.9, 3.7 Hz, 1H), 4.35 (dq, J = 7.1, 3.5Hz, 2H), 4.32–4.29 (m, 1H), 2.41 (s, 3H), 2.40–2.32 (m, 6H), 1.06 (t, J = 7.5Hz, 3H), 1.03 (t, J = 7.5 Hz, 3H), 1.00 (t, J = 7.5 Hz, 3H).

[0107] Example 10: Synthesis of (2R,3R,4R,5R)-2-(4-(2-amino-4-methylpyrimidin-5-yl)-1H-imidazol-1-yl)-5-((isobutyryloxy)methyl)tetrahydrofuran-3,4-diylbis(2-methylpropionic acid) (Ib-6)

[0108]

[0109] Compound Ia-2 (0.40 g, 1.302 mmol, 1.0 eq) was added to ultradry DMF, and acetic anhydride was replaced with isobutyric anhydride (0.82 g, 5.208 mmol, 4.0 eq). The rest of the procedure was the same as for the synthesis of Ib-4, yielding 0.50 g of compound Ib-6 as a white solid, in 74.2% yield. HRMS (ESI) m / z: calcd. for C 25 H 35 N5O7 [M + H] + 518.2614, found 518.2610. 1H NMR (600 MHz, DMSO-d6) δ 8.44 (s, 1H), 7.99 (d, J= 1.3 Hz, 1H), 7.52 (d, J = 1.3 Hz, 1H), 6.54 (s, 2H), 6.05 (d, J = 6.1 Hz, 1H), 5.55–5.40 (m, 2H), 4.36 (t, J = 4.1 Hz, 1H), 4.33 (d, J = 5.0 Hz, 2H), 2.63 –2.54 (m, 3H), 2.41 (s, 3H), 1.14 (d, J = 3.5 Hz, 3H), 1.13 (d, J = 3.5Hz, 3H), 1.11 (d, J = 6.9 Hz, 3H), 1.09 (d, J = 7.0 Hz, 3H), 1.07 (d, J = 7.0Hz, 3H), 1.05 (d, J = 7.0 Hz, 3H).

[0110] Example 11: Synthesis of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4-(2-amino-4-(trifluoromethyl)pyrimidin-5-yl)-1H-imidazol-1-yl)tetrahydrofuran-3,4-diacetate (Ib-7)

[0111]

[0112] 40 mL of ultra-dry DMF was added to a 100 mL round-bottom flask, followed by compound Ia-3 (0.40 g, 1.110 mmol, 1.0 eq). The mixture was stirred until completely dissolved. Then, 4-dimethylaminopyridine (0.013 g, 0.11 mmol, 0.1 eq) and acetic anhydride (0.45 g, 4.43 mmol, 4 eq) were added sequentially. After the reaction was completed at room temperature for 2 h, the same post-treatment and purification process as for Ib-1 was performed (DCM:MeOH = 20:1). The final result was 0.36 g of compound Ib-7 as a white solid, with a yield of 67.0%. HRMS (ESI) m / z: calcd.for C 19 H 20 F3N5O7 [M + H] + 488.1393, found 488.1388. 1H NMR (600 MHz, CD3OD) δ8.58 (s, 1H), 8.00 (d, J = 1.3 Hz, 1H), 7.42, (s, 1H), 6.04 (d, J = 5.8 Hz,1H), 5.48 (t, J = 5.7 Hz, 1H), 5.41 (dd, J = 5.5, 4.0 Hz, 1H), 4.48 (q, J =3.6 Hz, 1H), 4.41 (dd, J = 12.4, 3.7 Hz, 1H), 4.36 (dd, J = 12.4, 3.7 Hz,1H), 2.13 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H).

[0113] Example 12: Synthesis of (2R,3R,4R,5R)-2-(4-(2-amino-4-(trifluoromethyl)pyrimidin-5-yl)-1H-imidazol-1-yl)-5-((propionyloxy)methyl)tetrahydrofuran-3,4-dimethyldipropionate (Ib-8)

[0114]

[0115] Compound Ia-3 (0.40 g, 1.110 mmol, 1.0 eq) was added to ultradry DMF, and acetic anhydride was replaced with propionic anhydride (0.58 g, 4.430 mmol, 4.0 eq). The rest of the procedure was the same as for the synthesis of Ib-7, yielding 0.40 g of compound Ib-8 as a white solid, in 68.1% yield. HRMS (ESI) m / z: calcd. for C 22 H 26 F3N5O7 [M + H] + 530.1862, found 530.1858. 1H NMR (600 MHz, CD3OD) δ 8.57 (s, 1H), 8.00 (d, J =1.4 Hz, 1H), 7.41 (s, 1H), 6.03 (d, J = 5.9 Hz, 1H), 5.50 (t, J = 5.7 Hz,1H), 5.43 (dd, J = 5.5, 3.9 Hz, 1H), 4.48 (q, J = 3.5 Hz, 1H), 4.43 (dd, J =12.4, 3.6 Hz, 1H), 4.37 (dd, J = 12.4, 3.6 Hz, 1H), 2.46–2.37 (m, 6H), 1.16(t, J = 7.6 Hz, 3H), 1.14 (t, J = 7.6 Hz, 3H), 1.11 (t, J = 7.6 Hz, 3H).

[0116] Example 13: Synthesis of (2R,3R,4R,5R)-2-(4-(2-amino-4-9-(trifluoromethyl)pyrimidin-5-yl)-1H-imidazol-1-yl)-5-((isobutyryloxy)methyl)tetrahydrofuran-3,4-diylbis(2-methylpropionate) (Ib-9)

[0117]

[0118] Compound Ia-3 (0.40 g, 1.110 mmol, 1.0 eq) was added to ultradry DMF, and acetic anhydride was replaced with isobutyric anhydride (0.70 g, 4.430 mmol, 4.0 eq). The rest of the procedure was the same as for the synthesis of Ib-7, yielding 0.40 g of compound Ib-9 as a white solid, in 63.7% yield. HRMS (ESI) m / z: calcd. for C 25 H 32 F3N5O7 [M + H] + 571.2254, found 572.2332. 1H NMR (600 MHz, CD3OD) δ 8.56 (s, 2H), 8.01 (d, J =1.3 Hz, 1H), 7.41 (d, J = 1.3 Hz, 1H), 6.05 (d, J = 6.1 Hz, 1H), 5.48 (t, J =5.8 Hz, 1H), 5.42 (dd, J = 5.4, 3.6 Hz, 1H), 4.49 (q, J = 3.4 Hz, 1H), 4.44 (dd, J = 12.4, 3.6 Hz, 1H), 4.35 (dd, J = 12.4, 3.0 Hz, 1H), 2.68–2.57 (m,3H), 1.22 (d, J = 7.0 Hz, 3H), 1.21 (d, J = 7.0 Hz, 3H), 1.20 (d, J = 7.0 Hz,3H), 1.17 (d, J = 7.0 Hz, 3H), 1.16 (d, J = 7.0 Hz, 3H), 1.14 (d, J = 7.0 Hz,3H).

[0119] Example 14: Synthesis of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4-(2-aminopyrimidin-5-yl)-1H-imidazol-1-yl)tetrahydrofuran-3,4-diacyldiacetate (Ib-10)

[0120]

[0121] 40 mL of ultra-dry DMF was added to a 100 mL round-bottom flask, followed by compound Ia-4 (0.30 g, 1.020 mmol, 1.0 eq). The mixture was stirred until fully dissolved. Then, 4-dimethylaminopyridine (0.012 g, 0.100 mmol, 0.1 eq) and acetic anhydride (0.42 mg, 4.090 mmol, 4.0 eq) were added sequentially. After reacting at room temperature for 2 hours and verifying by TLC, the product underwent the same post-treatment and purification steps as Ib-1 (eluent DCM: MeOH = 20:1) to obtain 0.27 g of white solid product Ib-10, with a yield of 62.2%. HRMS (ESI) m / z: calcd. for C 18 H 21 N5O7 [M + H] + 420.1519, found 420.1518. 1H NMR(600 MHz, CD3OD) δ 8.63 (s, 2H), 7.97 (d, J = 1.3 Hz, 1H), 7.66 (d, J = 1.3Hz, 1H), 6.028 (d, J = 5.5 Hz, 1H), 5.50 (t, J = 5.5 Hz, 1H), 5.43 (dd, J =5.6, 4.4 Hz, 1H), 4.50 (s, 1H), 4.47 (q, J = 4.0 Hz, 1H), 4.40–4.39 (m, 2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.09 (s, 3H).

[0122] Example 15: Synthesis of (2R,3R,4R,5R)-2-(4-(2-aminopyrimidin-5-yl)-1H-imidazol-1-yl)-5-((propionyloxy)methyl)tetrahydrofuran-3,4-dimethyldipropionate (Ib-11)

[0123]

[0124] Compound Ia-4 (0.30 g, 1.020 mmol, 1.0 eq) was added to ultradry DMF, and acetic anhydride was replaced with propionic anhydride (0.53 g, 4.080 mmol, 4.0 eq). The rest of the procedure was the same as for the synthesis of Ib-10, yielding 0.28 g of compound Ib-11 as a white solid, in 59.5% yield. HRMS (ESI) m / z: calcd. for C 21 H 27 N5O7 [M + H] + 462.1988, found 462.1985. 1H NMR (600 MHz, CD3OD) δ 8.62 (s, 2H), 7.98 (d, J =1.4 Hz, 1H), 7.65 (d, J = 1.4 Hz, 1H), 6.02 (d, J = 5.5 Hz, 1H), 5.52 (t, J =5.5 Hz, 1H), 1.14 (t, J= 7.6 Hz, 3H), 1.11 (t, J = 7.5 Hz, 3H).

[0125] Example 16: Synthesis of (2R,3R,4R,5R)-2-(4-(2-aminopyrimidin-5-yl(-1H-imidazol-1-yl)-5-((isobutyryloxy)methyl)tetrahydrofuran-3,4-diylbis(2-methylpropionic acid) (Ib-12)

[0126]

[0127] Compound Ia-4 (0.30 g, 1.020 mmol, 1.0 eq) was added to ultradry DMF, and acetic anhydride was replaced with isobutyric anhydride (0.65 g, 4.080 mmol, 4.0 eq). The rest of the procedure was the same as for the synthesis of SW-HX-34, yielding 0.31 g of compound Ib-12 as a white solid, in 61.0% yield. HRMS (ESI) m / z: calcd. for C 24 H 33 N5O7 [M + H] + 504.2458, found 504.2460. 1H NMR (600 MHz, CD3OD) δ 8.68 (s, 2H), 7.97 (d, J =1.3 Hz, 1H), 7.65 (d, J = 1.4 Hz, 1H), 6.03 (s, 1H), 5.51 – 5.50 (m, 1H),5.44 – 5.43 (m, 1H), 4.57 (s, 1H), 4.42 (dd, J = 4.0, 2.3 Hz, 1H), 4.39 (dd,J = 3.3, 1.3 Hz, 1H), 2.64 (m, 3H), 1.21 (d, J = 7.0 Hz, 3H), 1.20 (d, J =7.0 Hz, 3H), 1.17 (d, J = 7.0 Hz, 3H), 1.16 (d, J = 7.0 Hz, 3H), 1.15 (d, J =7.0 Hz, 3H), 1.14 (d, J = 7.0 Hz, 3H).

[0128] Example 17: Evaluation of in vitro anti-H1N1 influenza virus activity

[0129] 17.1 Cytotoxicity assay

[0130] Cytotoxicity assays were performed using the MDCK cell model, and the cytotoxicity of the target compound was assessed using the CCK-8 assay. First, MDCK cells were seeded in 96-well plates at 1 × 10⁶ cells per well. 4 Cells were cultured at 37°C and 5% CO2 for 12 hours until a confluent monolayer was formed, at which point the experiment began. The test compound was diluted with serum-free viral isolation medium at an initial concentration of 400.0 µM, followed by 2-fold serial dilutions, resulting in eight concentration gradients, with a maximum drug concentration of 200 µM. 100 µL of the test compound at different concentrations was added to each well of the cell plate, along with 100 µL of serum-free viral isolation medium. The cells were incubated at 37°C and 5% CO2 for 48 hours. After incubation, the supernatant was discarded, and the cells were washed twice with PBS. 100 µL of serum-free viral isolation medium and 10 µL of CCK-8 reagent were added to each well, and the cells were incubated for another 4 hours. Finally, the absorbance was measured at 450 nm using a microplate reader, cell viability was calculated, and the CC50 value (half-maximal toxicity concentration) was obtained using GraphPad Prism 8 software.

[0131] 17.2 In vitro anti-H1N1 virus experiment

[0132] The in vitro anti-H1N1 influenza virus activity assay used an MDCK cell model, with remdesivir as a positive control, and virus and cell controls were also set up. First, MDCK cells were seeded in 96-well plates at 5 × 10⁶ cells per well. 4 Cells were cultured at 37°C and 5% CO2 for 24 hours. Before the experiment, the preserved H1N1 virus was thawed and diluted to 10 to 100 TCID50. The test compound was diluted twice with virus growth medium to the starting concentration, followed by 3-fold serial dilutions, for a total of 8 concentration gradients, with 3 replicates for each concentration. The diluted drug solution was mixed with the virus solution, and 120 µL of the mixture was added to each well. The mixture was transferred to a pre-prepared MDCK cell plate and cultured at 37°C and 5% CO2 for 96 hours. During the culture period, cytopathic effects were observed. When more than 85% of the virus control wells showed cytopathic effects, the supernatant was discarded, and the cells were washed twice with PBS. 100 µL of neutral red staining solution was added to each well for 3-5 hours. After staining, the staining solution was discarded, and the cells were washed 2-3 times with PBS. 100 µL of acidic ethanol was added to dissolve the dye, and the absorbance was measured at 540 nm using a microplate reader. Experimental data were analyzed using GraphPad Prism 8 software to calculate EC50 values ​​(half-maximal inhibitory concentration) and assess the antiviral activity of the target compounds.

[0133] Table 1. Results of cytotoxicity and anti-H1N1 virus activity of the compounds

[0134] As shown in Table 1, the compounds of the present invention exhibit high activity against H1N1 influenza virus. The activities of Ia-2, Ia-3, Ib-1, Ib-3, Ib-4, Ib-5, Ib-6, Ib-7, and Ib-8 are significantly higher than those of the positive control drug Remdesivir, and they also have low cytotoxicity and good safety.

[0135] Example 18: Acute toxicity test in mice

[0136] Sixty SPF - level ICR healthy adult male mice, weighing 18–22 g, were selected. An appropriate amount of compound Ia - 3 was weighed according to the requirements of each dose group to prepare concentration gradients: 10, 50, 100, 160, 200 mg / mL. The experiment adopted a gradient - dose design and a total of 6 experimental groups were set up, including 1 blank control group and 5 dose groups. The blank control group (n = 10) was given an equal volume of normal saline (0.1 mL / 10 g) as a negative control. The dose groups were as follows: low - dose group (100 mg / kg, n = 10), medium - dose group 1 (500 mg / kg, n = 10), medium - dose group 2 (1000 mg / kg, n = 10), high - dose group 1 (1600 mg / kg, n = 10), and high - dose group 2 (2000 mg / kg, n = 10). Each dose group was administered by oral gavage at a dosing volume of 0.1 mL / 10 g to systematically evaluate the dose - effect relationship of compound Ia - 3.

[0137] Table 2 Survival status of test animals within 14 days

[0138] The results in Table 2 show that the LD of compound Ia - 3 50 is significantly higher than 2000 mg / kg, showing excellent acute safety characteristics.

[0139] Example 19: Metabolism experiment in SD rats

[0140] In this study, healthy male SD rats were selected as the experimental animal model. A total of 3 rats were used, with a body weight range of 210–220 g and a week - age of 6–8 weeks. They were all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Experimental animal production license number: SCXK (Beijing) 2021–0011). The dosing plan was designed for intravenous injection, and the dose was set at 2 mg / kg. The blood - sampling time points included 9 time points: before dosing (0 h) and 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, 24 h after dosing.

[0141] Table 3 Pharmacokinetic parameters of Ia - 3 in plasma of SD rats after single intravenous administration of Ia - 3 (2 mg·kg-1, N = 3)

[0142] Table 3 shows that compound Ia - 3 exhibits good pharmacokinetic characteristics and safety in SD rats. After single intravenous administration (2 mg / kg), the compound shows a moderate elimination half - life (t 1 / 2 = 3.65 ± 0.20 h), indicating a reasonable clearance rate in vivo. All test animals did not show abnormal reactions, indicating good tolerance.

[0143] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. The compound represented by Formula I or a pharmaceutically acceptable salt thereof, in: X represents carbon, nitrogen, oxygen, or sulfur. R1 is hydrogen, deuterium, hydroxyl, halogen, amino, alkyl, NH-alkyl, O-alkyl, or alkyl-substituted amino group; R2 can be hydrogen, deuterium, hydroxyl, sulfur, halogen, trifluoromethyl, alkyl, cyano, amino, NH-alkyl, N-alkyl, O-alkyl, or NH-OH. R3 is hydrogen, a halogen, OR 4、 Cyano, C1–C6 alkyl, C1–C6 alkoxy, C2–C6 alkenyl, C2–C6 alkynyl; R4 represents hydrogen, azide, and C. 1-20 Alkyl group, amino group C 1-20 Alkyl group, C 1-6 Alkylamino, C 1-6 Alkyl group, C 1-6 Alkoxy, C 1-6 Alkyl groups, α-amino acids (the carboxyl group of the α-amino acid is linked to the hydroxyl group on the furan ring by an ester bond). R5 can be hydrogen, deuterium, monophosphate, diphosphate, triphosphate, or various prodrugs (such as lipids, lipid phosphates, lipid esters, amino acid prodrugs, etc.). L can be -CH2-, sulfur, vinyl, ethynyl, or -O-(CH2). n -、-NH-(CH2) n - (n is an integer selected from 0–2, for example 0, 1, 2.) W1 can be hydrogen, hydroxyl, sulfur, fluorine, amino, NH-alkyl, or O-alkyl. W2 can be hydrogen, hydroxyl, sulfur, fluorine, amino, NH-alkyl, or O-alkyl. Y1 can be hydrogen, deuterium, hydroxyl, sulfur, halogen, trifluoromethyl, alkyl, cyano, amino, NH-alkyl, N-alkyl, O-alkyl, or NH-OH. Y2 is hydrogen, deuterium, hydroxyl, halogen, amino, alkyl, NH-alkyl, O-alkyl, or alkyl-substituted amino.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound has the structure shown in formula Ia or formula Ib. in, Y1 can be hydrogen, deuterium, hydroxyl, sulfur, halogen, trifluoromethyl, alkyl, cyano, amino, NH-alkyl, N-alkyl, O-alkyl, or NH-OH. Y2 is hydrogen, deuterium, hydroxyl, halogen, amino, alkyl, NH-alkyl, O-alkyl, or alkyl-substituted amino groups; R3 is hydrogen, a halogen, OR 4、 Cyano, C1–C6 alkyl, C1–C6 alkoxy, C2–C6 alkenyl, C2–C6 alkynyl; R4 represents hydrogen, azide, and C. 1-20 Alkyl group, amino group C 1-20 Alkyl group, C 1-6 Alkylamino, C 1-6 Alkyl group, C 1-6 Alkoxy, C 1-6 Alkyl groups, α-amino acids (the carboxyl group of the α-amino acid is linked to the hydroxyl group on the furan ring by an ester bond). R5 can be hydrogen, deuterium, monophosphate, diphosphate, triphosphate, or various prodrugs (such as lipids, lipid phosphates, lipid esters, amino acid prodrugs, etc.).

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from: 。 4. A pharmaceutical composition comprising the compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable excipient or carrier.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is a solution, tablet, capsule or injection.

6. Use of the compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient, in the preparation of a medicament as an antiviral drug.

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