Use of nucleoside phosphoramidate derivative CL-13 in the preparation of antiviral drugs

Abstract

This invention discloses the application of a nucleoside phosphoramide ester derivative, CL-13, in the preparation of antiviral drugs. The virus is selected from flaviviruses or enteroviruses, and the structure of CL-13 is shown below. This invention discovers a novel use of compound CL-13 in antiviral applications. Experimental results show that CL-13 has significant inhibitory effects on Zika virus (ZIKV), dengue virus type 2 (DENV2), Japanese encephalitis virus (JEV), enterovirus 71 (EV71), and tick-borne encephalitis virus (TBEV), with the inhibitory effect being concentration-dependent and exhibiting a broad antiviral spectrum. Simultaneously, CL-13 possesses strong antiviral activity and shows no significant cytotoxicity within the effective antiviral concentration range, exhibiting a high therapeutic index, good safety profile, and excellent drug development potential. Therefore, CL-13 combines broad-spectrum, high-efficiency, and low-toxicity characteristics, making it suitable for preparing drugs to prevent or treat diseases caused by the aforementioned viral infections, and has significant application prospects.

Description

Technical Field

[0001] This invention belongs to the field of antiviral drug technology, specifically relating to the application of a nucleoside phosphoramide ester derivative CL-13 in the preparation of antiviral drugs. Background Technology

[0002] Zika virus (ZIKV), dengue virus type 2 (DENV2), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) all belong to the Flaviviridae family. They are mainly transmitted through insect vectors and can cause a series of clinical symptoms such as fever, rash, joint pain, and central nervous system damage, seriously endangering public health. Enterovirus 71 (EV71) is one of the main pathogens causing hand-foot-mouth disease, often leading to severe infections in infants and young children, and currently there is still no specific treatment.

[0003] In recent years, these viruses have experienced multiple outbreaks and epidemics globally, posing a continuous challenge to the prevention and control of infectious diseases. Although some vaccines or investigational drugs have entered clinical trials, existing antiviral drugs generally suffer from narrow antiviral spectrum, limited efficacy, susceptibility to drug resistance, or high cytotoxicity. Small molecule drugs that can simultaneously and effectively inhibit multiple important RNA viruses while maintaining excellent safety are still relatively scarce. Therefore, developing novel antiviral small molecule compounds with broad-spectrum, high efficacy, low toxicity, and clearly defined mechanisms of action is of significant practical importance and clinical application value in combating infections caused by viruses such as ZIKV, DENV2, JEV, EV71, and TBEV. Summary of the Invention

[0004] Objective: To address the lack of highly effective and safe clinical treatments for important RNA viruses such as Zika virus (ZIKV), dengue virus type 2 (DENV2), Japanese encephalitis virus (JEV), and enterovirus 71 (EV71), and the limitations of existing antiviral drugs such as narrow antiviral spectrum, limited therapeutic efficacy, and poor safety profiles, this invention provides the application of the nucleoside phosphoramide ester derivative CL-13 in the preparation of antiviral drugs. The results of this invention overcome the shortcomings of existing technologies and provide new drug options and technical solutions for the prevention and treatment of related viral diseases.

[0005] Technical Solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0006] In a first aspect, the present invention provides the use of a nucleoside phosphoramide ester derivative CL-13 or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug, wherein the virus is selected from flaviviruses or enteroviruses, and the structure of CL-13 is shown below:

[0007] .

[0008] CL-13 is a novel nucleoside phosphoramide ester derivative with an N-type nucleus. 6 -Methyladenine, with the ribose portion modified by 2'-fluoro-2'-deoxy-2'-fluoromethyl, and a phosphoramide ester prodrug group attached at the 5'-position, exhibits good cell permeability and metabolic stability.

[0009] Preferably, the flavivirus is selected from at least one of Zika virus, dengue virus type 2, Japanese encephalitis virus, and tick-borne encephalitis virus.

[0010] Preferably, the enterovirus is selected from enterovirus 71.

[0011] As a specific implementation scheme, in the antiviral drug, the nucleoside phosphoramide ester derivative CL-13 or its pharmaceutically acceptable salt is the sole active ingredient, or one of the active ingredients.

[0012] In a second aspect, the present invention provides the use of compositions comprising nucleoside phosphoramide derivative CL-13 or a pharmaceutically acceptable salt thereof in the preparation of antiviral drugs, wherein the virus is selected from flaviviruses or enteroviruses, and the structure of CL-13 is shown below:

[0013] .

[0014] Preferably, the flavivirus is selected from at least one of Zika virus, dengue virus type 2, Japanese encephalitis virus, and tick-borne encephalitis virus.

[0015] Preferably, the enterovirus is selected from enterovirus 71.

[0016] As a specific implementation scheme, in the antiviral drug, the nucleoside phosphoramide ester derivative CL-13 or its pharmaceutically acceptable salt is the sole active ingredient, or one of the active ingredients.

[0017] The antiviral drug of the present invention contains CL-13 as the sole or main active ingredient, as well as pharmaceutically acceptable carriers, diluents, excipients, disintegrants, lubricants, sweeteners, solubilizers, preservatives, etc.; it can also be used in combination with other antiviral drugs, immunomodulators, and anti-inflammatory drugs to synergistically enhance the antiviral efficacy.

[0018] The compound CL-13 of this invention has been disclosed in patents CN115721661A and CN 103980332A, and is mainly used for anti-coronavirus CoV and hepatitis C virus HCV. However, in subsequent research, the applicant discovered that compound CL-13 has broad-spectrum antiviral activity against flaviviruses such as Japanese encephalitis, dengue fever, and Zika. Compound CL-13 mainly exerts its antiviral effect by inhibiting viral polymerase in its triphosphate form. Although these viruses are all positive-sense RNA viruses, and their RNA replication-dependent RNA polymerases (RdRp) have certain conserved catalytic modules, there are still some differences, which are important factors determining the different mechanisms of action of compound CL-13.

[0019] The RdRp catalytic center is mainly composed of several modules, including motif AG. The composition of the basic active unit and the substrate binding site of the catalytic center of coronavirus RdRp are most different from those of flaviviruses such as HCV, JEV, ZIKA, DEN, and TBEV. The motif B of coronavirus RdRp is closer to the motif C than that of other RdRp, resulting in a relatively narrow substrate binding space. When compound CL-13 is recognized, the 2-methylamino group on the base causes compound CL-13 to pair with uridine on the template chain in a special cis conformation.

[0020] In the anti-HCV activity of 2'-fluoromethyl compounds, the fluoromethyl group and fluorine substitution at the 2' position play a crucial role, primarily by inhibiting the formation of the hydrogen bond required for catalysis between the conserved serine (S282) on RdRp motif B and the nucleoside, leading to chain elongation termination. Compared to HCV, other flaviviruses such as JEV, ZIKA, TBEV, and DEN exhibit higher similarity in the catalytic module arrangement of RdRp, with a relatively larger distance between motif C and B. This results in a greater distance between the fluoromethyl group at the 2' position and the conserved serine during substrate recognition, thus weakening the activity. However, compared to other 2'-fluoromethyl nucleoside compounds, compound CL-13 possesses a unique 2-methylamino base modification, which can synergistically work with the fluoromethyl group at the 2' position, enhancing the intervention effect on the conserved serine of motif B. Therefore, it achieves broad-spectrum antiviral activity against flaviviruses such as JEV, ZIKA, TBEV, and DEN.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0022] CL-13 has significant inhibitory effects on Zika virus (ZIKV), dengue virus type 2 (DENV2), Japanese encephalitis virus (JEV), enterovirus type 71 (EV71), and tick-borne encephalitis virus (TBEV). The inhibitory effect is concentration-dependent and has a broad antiviral spectrum.

[0023] CL-13 exhibits strong antiviral activity, showing low micromolar levels of IC50 against ZIKV, DENV2, JEV, and TBEV. 50 At high concentrations, its inhibitory effect is close to or even better than that of the positive control drug. Meanwhile, CL-13 shows no significant toxicity to cells within the effective antiviral concentration range, has a high therapeutic index, good safety profile, and excellent potential for drug development.

[0024] In summary, CL-13 possesses the characteristics of being broad-spectrum, highly effective, and low in toxicity, making it suitable for the preparation of drugs to prevent or treat diseases caused by the aforementioned viral infections, and thus has significant application prospects. Attached Figure Description

[0025] Figure 1 This describes the cytotoxic effects of compounds CL-13 and MCH1623 on RD cells in Example 1.

[0026] Figure 2 This describes the cytotoxic effects of compounds CL-13 and MCH1623 on huH7 cells in Example 1.

[0027] Figure 3 This is a preliminary evaluation diagram of the anti-ZIKV activity of compounds CL-13 and MCH1623 in Example 2.

[0028] Figure 4 This is a preliminary evaluation diagram of the anti-DENV2 activity of compounds CL-13 and MCH1623 in Example 2.

[0029] Figure 5 This is a preliminary evaluation diagram of the anti-JEV activity of compounds CL-13 and MCH1623 in Example 2.

[0030] Figure 6 This is a preliminary evaluation diagram of the anti-EV71 activity of compounds CL-13 and MCH1623 in Example 2.

[0031] Figure 7 The inhibitory activity and IC50 of compounds CL-13 and MCH1623 in Example 3 against ZIKV-infected huH7 cells were investigated. 50 Comparative analysis.

[0032] Figure 8 The inhibitory activity and IC50 of compounds CL-13 and MCH1623 in Example 3 against DENV2-infected huH7 cells were investigated. 50 Comparative analysis.

[0033] Figure 9 The inhibitory activity and IC50 of compounds CL-13 and MCH1623 in Example 3 against JEV-infected huH7 cells were investigated. 50 Comparative analysis. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] In the following examples, the structural formula of MCH1623 is shown below, and it was prepared according to the literature Bioorganic Chemistry, 2026, 170, 109472.

[0036]

[0037] The structural formula of NITD008 is shown below, and it was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0038]

[0039] The preparation methods for compounds 13 and 14 are as follows:

[0040]

[0041] Preparation of compound 2

[0042] Compound 1 (500 g, 3.85 mol) was dissolved in ethyl acrylate (1200 mL) and placed at 0 °C. DABCO (215 g, 1.93 mol) was added in portions. After the addition was complete, the mixture was stirred at this temperature for 12 h, and then stirred for another 6 days at room temperature. The reaction of the starting material was monitored by TLC until complete. Ethyl acrylate was removed by concentration under reduced pressure. The resulting organic phase was dissolved in ethyl acetate (2000 mL) and washed with 0.5 mol / L hydrochloric acid solution until the aqueous layer was colorless. The organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to give a yellow oily crude compound 2 (620 g, 70% yield).

[0043] Preparation of compound 3

[0044] NBS (124 g, 0.70 mol) was dissolved in dichloromethane (1200 mL) and placed at 0 °C. A solution of DMS (47 g, 0.76 mol) in dichloromethane (200 mL) was slowly added using a constant-pressure dropping funnel. After the addition was complete, the mixture was stirred at this temperature for 30 min. Then, compound 2 (146 g, 0.64 mol) in dichloromethane (200 mL) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 15 h. The reaction mixture was monitored by TLC until the reaction was complete. The reaction solution was concentrated at low temperature. After concentration, the organic matter was extracted three times with ether and 500 mL of saturated brine. The organic phases were combined and washed with 0.5 mol / L sodium hydroxide solution until the organic layer was colorless. The mixture was then washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a yellow oily crude compound 3 (141 g, yield 75%).

[0045] Preparation of compound 4

[0046] Compound 3 (118 g, 0.4 mol) was dissolved in ethanol (1000 mL), and anhydrous CH3COONa (100 g, 1.2 mol) was added. The mixture was heated to reflux. The reaction was monitored by TLC until the reactants were completely reacted. The insoluble solids were removed by filtration, and the filtrate was cooled to 0 °C. o C. Add K2CO3 (107.4 g, 0.8 mol) and heat to 80°C. o C. Continue stirring until the reaction is complete as monitored by TLC. After the reaction solution is cooled to room temperature, filter under vacuum, retain the filtrate, concentrate under reduced pressure to remove ethanol, and concentrate again. The crude product is purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:10 - 1:3) to give a pale yellow oily compound 4 (66.2 g, yield 62%).

[0047] Preparation of compound 5

[0048] Compound 4 (200 g, 0.87 mol) was dissolved in anhydrous tetrahydrofuran (1700 mL), followed by the sequential addition of n-C4F9SO2F (525 g, 1.74 mol) and Et3N·3HF (210 g, 1.30 mol), and then placed at 0 °C. o At temperature C, slowly add Et3N (440 g, 4.35 mol) dropwise using a constant-pressure dropping funnel. After the addition is complete, maintain a temperature of 20-25°C. oUnder C conditions, stirring continued, and the reaction was monitored by TLC until complete. Petroleum ether (2000 mL) was added to the system and stirred for 5 min. The upper organic phase was separated. The lower aqueous phase was extracted multiple times with ethyl acetate / petroleum ether (volume ratio 1:10). All organic phases were combined and concentrated. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:80) to give a light yellow oily compound 5 (90.8 g, yield 45%).

[0049] Preparation of compound 6

[0050] Compound 5 (81 g, 0.35 mol) was dissolved in acetone (1500 mL), and NaHCO3 (88 g, 1.05 mol) and ethylene glycol (87 g, 1.40 mol) were added sequentially. KMnO4 (66 g, 0.42 mol) was slowly added in portions at 0 °C, and the mixture was stirred at 0 °C for 30 min after the addition was complete. The reaction was monitored by TLC until complete. The reaction was quenched by slowly adding saturated sodium bisulfite solution (300 mL) at low temperature, and stirred at room temperature for 2 h. The solid was removed by filtration, and the filter cake was washed with acetone. The filtrate was distilled under reduced pressure to remove acetone, and the concentrated aqueous phase was extracted three times with ethyl acetate. The combined organic phases were washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated. The organic compound was azeotropically removed by adding toluene (150 mL), and finally recrystallized from a toluene (100 mL) / n-heptane (500 mL) mixture to give compound 6 (32.5 g, 35% yield) as a white solid.

[0051] Preparation of compound 8

[0052] Compound 6 (8 g, 30 mmol) was dissolved in a mixed solvent of acetonitrile (15 mL) and isopropyl acetate (60 mL), and Et3N (7.28 g, 72 mmol) was added. The reaction solution was placed in an ice bath, and SOCl2 (4.12 g, 34.6 mmol) was slowly added dropwise, keeping the temperature below 20°C. The mixture was stirred at this temperature for 30 min. The reaction was monitored by TLC until complete. The reaction was quenched with water (30 mL), extracted, and the organic phase was washed successively with water (30 mL) and saturated sodium bicarbonate solution (30 mL). Acetonitrile (15 mL) and sodium bicarbonate (5.04 g, 60 mmol) were then added to the organic phase. A sodium hypochlorite aqueous solution (60 mL) with an available chlorine content of 8%–10% was slowly added dropwise using a constant pressure dropping funnel, keeping the temperature of the reaction system below 10°C. The mixture was stirred at room temperature for 2 h after the addition was complete. The organic phase was separated and washed with saturated sodium sulfite solution and dried over anhydrous sodium sulfate. 0.5 mL of triethylamine was added to stabilize the product, which was then concentrated and purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:6) to give a colorless oily compound 7 (7.4 g, yield 75%).

[0053] Preparation of compound 9

[0054] Compound 8 (8 g, 24.4 mmol), Et3N·3HF (1.96 g, 12.2 mmol), and Et3N (2.96 g, 29.3 mmol) were mixed at 90°C. o Stir at C for 4 h; after cooling to room temperature, add concentrated hydrochloric acid (4 mL, 48 mmol), 90 o Stir for 30 minutes under C conditions, then add 30 mL of saturated barium chloride solution. o Stirred at C for 4 h; add n-propanol (200 mL) to concentrate, and azeotropically remove water from the residue with toluene (150 mL), repeating twice. Dissolve the solid residue in acetonitrile (60 mL), and add BzCl (10.29 g, 73.2 mmol), DMAP (298 mg, 2.44 mmol), and Et3N (7.41 g, 73.2 mmol) sequentially. After the reaction was monitored by TLC, the reaction was quenched with water, extracted, and the organic phase was washed successively with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate and concentrated; purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:5) to give a white solid compound 9 (3.61 g, yield 38%).

[0055] Preparation of compound 11

[0056] Compound 9 (10 g, 25.6 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL) and heated at -20°C. o(t-BuO)3AlLiH (1 mol / L THF, 31 mL, 31 mmol) was added under C conditions. After stirring at this temperature for 4 h, (t-BuO)3AlLiH (1 mol / L THF, 7.4 mL, 7.4 mmol) was added again. Stirring was continued for 2 h, followed by quenching with water to extinguish the reaction. The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated. The resulting oil was heated to 45°C. o Incubate at -20°C for 8 hours to obtain a white solid crude compound 10, which can be directly added to the next reaction without further purification. Dissolve the above crude compound 10 in anhydrous dichloromethane (75 mL) at -20°C. o PPh3 (8.0 g, 30.7 mmol) was added under C conditions, and the mixture was stirred for 10 min before adding CBr4 (11.0 g, 33.18 mmol). The reaction solution was then heated to 0 °C. o C and stirred for 2 h. After the reaction was completed by TLC monitoring, water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:5) to give a white solid compound 11 (6.16 g, yield 53%).

[0057]

[0058] Preparation of compound 12

[0059] 2-Amino-6-chloropurine (4.5 g, 26.43 mmol) was dissolved in tert-butanol (90 mL), and t-BuOK (3.0 g, 26.43 mmol) was added. The mixture was stirred at room temperature for 1 h. The resulting reaction solution was added to a solution of compound 11 (4 g, 8.81 mmol) in acetonitrile (10 mL), and the mixture was stirred at 70 °C for 16 h. After cooling to room temperature, the reaction was quenched with saturated ammonium chloride aqueous solution, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography (eluent gradient: ethyl acetate / petroleum ether = 1:4 - 1:1) to give a white solid compound 12 (1.91 g, 40% yield). 1HNMR(CDCl3, 400 MHz) δ 7.96 − 7.93 (m, 2H), 7.89 − 7.87 (m, 2H), 7.84 (s, 1H),7.58 − 7.54 (m, 1H), 7.47 − 7.43 (m, 1H), 7.40 − 7.36 (m, 2H), 7.28 − 7.24(m, 2H, overlapped with the peak of chloroform), 6.67 (dd, J=22.0, 8.8 Hz,1H), 6.26 (d, J = 18.0 Hz, 1H), 5.60 (s, 2H), 5.21 − 5.16 (m, 1H), 4.86 (dt,J = 10.8, 5.6 Hz, 1H), 4.78 − 4.44 (m, 3H); 19 F NMR (376 MHz, CDCl3) δ -174.20, -234.72; 13 CNMR (CDCl3, 100 MHz) δ 166.3, 165.6, 159.1, 152.4, 152.3,141.7, 133.9, 133.3, 129.9, 129.6, 129.0, 128.5, 128.3, 127.9, 125.9, 100.6(dd, J C−F =190.3, 17.4 Hz), 89.4 (dd, J C−F = 39.6, 1.8 Hz), 81.8 (dd, J C−F =174.6, 24.8 Hz), 78.1, 71.3 (dd, J C−F = 15.6, 5.2 Hz), 62.8; HRMS (ESI) m / z:[M + Na] + Theoretical calculated value C 25 H 20 ClF2N5O5Na 566.1013, actual measured value 566.1016.

[0060]

[0061] Preparation of compound 13

[0062] Compound 12 (0.5 g, 0.91 mmol) was dissolved in methanol (2 mL) and placed in a thick-walled, pressure-resistant sealed tube. Freshly prepared 33% dimethylamine methanol solution (1 mL) was added, and the mixture was stirred at 85 °C for 12 h. The reaction solution was concentrated and purified by silica gel column chromatography (eluent gradient: methanol / dichloromethane = 1:30 - 1:10) to give a white solid compound 13 (0.24 g, yield 75%). 1 HNMR (400 MHz, MeOD) δ 7.93 (s, 1H), 6.27 (d, J = 18.6 Hz, 1H), 4.76 (dd, J =23.7, 9.2 Hz, 1H), 4.61 (dt, J = 47.6, 11.4 Hz, 1H), 4.36 (ddd, J = 45.8,25.6, 11.2 Hz, 1H), 4.10 (dt, J = 9.2, 2.7 Hz, 1H), 4.05 (dd, J = 12.5, 2.2Hz, 1H),, 3.88 (dd, J = 12.6, 3.1 Hz, 1H), 3.38 (s, 6H); 19 F NMR (376 MHz, MeOD) δ -179.45, -238.68; 13 C NMR (101 MHz, MeOD) δ 161.7, 154.5, 152.0,142.9, 124.9, 101.2 (dd, J C−F = 185.7, 17.6 Hz), 88.9 (d, J C−F = 40.2 Hz), 83.7, 81.5 (dd, J C−F = 174.3, 30.0 Hz), 69.1 (d, J C−F = 16.5 Hz), 60.8, 54.3;HRMS (ESI) m / z: [M + H] + Theoretical calculated value C 13 H 19 F2N6O3345.1481, actual measured value 345.1487.

[0063]

[0064] Preparation of compound 14

[0065] Compound 12 (0.5 g, 0.91 mmol) was dissolved in methanol (2 mL) and placed in a thick-walled, pressure-resistant sealed tube. Freshly prepared 33% cyclopropylamine methanol solution (1 mL) was added, and the mixture was stirred at 85 °C for 12 h. The reaction solution was concentrated and purified by silica gel column chromatography (eluent gradient: methanol / dichloromethane = 1:30 - 1:10) to give a white solid compound 14 (0.24 g, yield 72%). 1 HNMR (400 MHz, MeOD) δ 7.97 (s, 1H), 6.26 (d, J = 18.5 Hz, 1H), 4.77 (dd, J =23.8, 9.3 Hz, 1H), 4.63 (dt, J = 47.7, 11.3 Hz, 1H), 4.37 (ddd, J = 45.8,26.2, 11.2 Hz, 1H), 4.09 (dd, J = 9.3, 3.0 Hz, 1H), 4.04 (dd, J = 12.6, 2.2Hz, 1H), 3.89 (dd, J = 12.5, 3.3 Hz, 1H), 2.92 – 2.87 (m, 1H), 0.85 – 0.80(m, 2H), 0.66 – 0.56(m, 2H); 19 F NMR (376 MHz, MeOD) δ -179.62, -238.83; 13 CNMR (101 MHz, MeOD) δ 161.9, 157.6, 151.4, 138.1, 114.8, 101.5 (dd, J C−F =185.8, 17.3 Hz), 89.3 (d, J C−F = 40.0 Hz), 83.6, 81.9 (dd, J C−F = 174.7, 28.1Hz), 69.3 (dd, J C−F = 17.1, 4.6 Hz), 61.0, 39.9, 7.6. HRMS (ESI) m / z: [M + H] + Theoretical calculated value C 14 H 19 F2N6O3357.1481, actual measured value 357.1487.

[0066] Example 1: Cytotoxicity of CL-13 and MCH1623 (CC) 50 ) Measurement

[0067] 1.1 Experimental Materials

[0068] 1.1.1 Test compounds: CL-13 (purity ≥98%, prepared according to the synthesis method of compound III in patent CN115721661A) and MCH1623 (purity ≥98%) were dissolved in DMSO to prepare a 100 mM stock solution and stored at 4℃ in the dark. During the experiment, the compounds were serially diluted with complete culture medium, and the concentration gradients were set as follows: 0.01 μM, 0.05 μM, 0.15 μM, 0.4 μM, 1.25 μM, 3.7 μM, 11.1 μM, 33.3 μM, and 100 μM.

[0069] 1.1.2 Negative control: 0.1% DMSO solution.

[0070] 1.1.3 Test cells: RD cells (human rhabdomyosarcoma cell line) and huH7 cells (human liver cancer cell line) were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator.

[0071] 1.1.4 Reagents and Instruments: CCK-8 cell proliferation assay kit (Beyotime Biotechnology); 96-well cell culture plate; Thermo Fisher Multiskan FC microplate reader; GraphPad Prism 8.0 data fitting software.

[0072] 1.2 Experimental Methods

[0073] 1.2.1 Cell Plating: RD cells and huH7 cells in logarithmic growth phase were plated at 1×10⁻⁶ cells per cell line. 4 Cells were seeded at a density of 100 μL per well in 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator for 24 h until the cell confluence reached 70%-80%.

[0074] 1.2.2 Drug treatment: Remove the original culture medium from each well, add 100 μL of complete culture medium containing different concentrations of CL-13 and MCH1623 to each well, and set up a 0.1% DMSO negative control group. Each treatment is set up in 6 replicates, and the wells are placed in an incubator at 37℃ and 5% CO2 for 48 h.

[0075] 1.2.3 CCK-8 assay: After incubation, add 10 μL of CCK-8 solution to each well, gently shake to mix, and incubate in a 37℃, 5% CO2 incubator in the dark for 2 h; use a microplate reader to measure the absorbance (OD) of each well at 450 nm. 450 ), record the detection data.

[0076] 1.2.4 Cell Viability Calculation and CC 50 Fitting:

[0077] Cell viability (%) = (Experimental group OD) 450 Value / Negative control group OD 450 Value) × 100%

[0078] Plotting compound concentration (μM) on the x-axis and cell viability (%) on the y-axis, a nonlinear dose-response curve was fitted using GraphPad Prism 8.0 software to calculate the half-maximal cytotoxicity concentration (CC). 50 (That is, the concentration of the compound that causes 50% cell death).

[0079] 1.3 Experimental Results

[0080] 1.3.1 RD cytotoxicity results:

[0081] CL-13: Within the concentration range of 0.01–100 μM, RD cell viability remained above 90%, with no significant dose-dependent decrease. The fitted curve did not show a typical cell death trend, and the measured CC... 50 =NA (No valid calculated value, indicating that 50% cytotoxicity was not achieved within the tested concentration range);

[0082] MCH1623: As the compound concentration increases, the viability of RD cells decreases in a gradient, and the fitted curve shows that CC 50 =13.75μM, meaning that a concentration of 13.75 μM can cause 50% of RD cells to die.

[0083] 1.3.2 huH7 cytotoxicity results:

[0084] CL-13: Within the concentration range of 0.01~100μM, huH7 cell viability did not show a significant decrease in lethality, and the fitted curve CC 50 =NA, with no significant cytotoxicity;

[0085] MCH1623: Increased concentration significantly reduced huH7 cell viability; fitted curve showed CC 50 =14.17 μM, with clear dose-dependent toxicity to huH7 cells.

[0086] 1.3.3 Conclusion: CL-13 showed no significant cytotoxicity to RD and huH7 cells within the tested concentration range, indicating extremely high safety; MCH1623 exhibited dose-dependent toxicity to both cell types, limiting its potential as a drug.

[0087] Example 2: Initial screening of in vitro inhibitory activity of CL-13 and MCH1623 against ZIKV / DENV2 / JEV / EV71 / TBEV

[0088] 2.1 Experimental Materials

[0089] 2.1.1 Test compounds: CL-13, MCH1623, and similar compounds 13 and 14, were stored in 100 mM DMSO stock solution at 4°C protected from light; during the experiment, they were diluted with complete culture medium to two working concentrations of 50 μM and 10 μM.

[0090] 2.1.2 Positive control: NITD008 (Sigma, purity ≥98%), dissolved and diluted in DMSO to a working concentration of 5 μM.

[0091] 2.1.3 Negative control: 0.1% DMSO solution.

[0092] 2.1.4 Virus strains: Zika virus (ZIKV), dengue virus type 2 (DENV2), Japanese encephalitis virus (JEV), enterovirus 71 (EV71), and tick-borne encephalitis virus (TBEV) were all standard strains isolated and identified in the laboratory, and were tested using TCID. 50 Viral titers were determined using a method with a uniform multiplicity of infection (MOI) of 0.1.

[0093] 2.1.5 Cells and reagents: Vero cells (Chinese Academy of Sciences Cell Bank), DMEM complete medium containing 10% fetal bovine serum; 4% paraformaldehyde fixative, 0.25% Triton X-100 permeabilization solution; ZIKV / DENV2 / JEV / EV71 / TBEV specific rabbit primary antibody, Alexa Fluor 488 labeled goat anti-rabbit secondary antibody; DAPI staining solution (Beyotime Biotechnology); ImageJ image analysis software.

[0094] 2.2 Experimental Methods

[0095] 2.2.1 Cell Plating: Vero cells in logarithmic growth phase were plated at a density of 1×10⁻⁶ cells / cells. 5 The cells were seeded at a density of 1 cell per well in 96-well plates and cultured at 37°C with 5% CO2 for 24 h until the cell confluence reached 70%-80%.

[0096] 2.2.2 Grouping: Remove the culture medium and add 100 μL of complete culture medium containing the corresponding test substance / control to each well, as follows:

[0097] Experimental group 1: 50 μM CL-13; Experimental group 2: 10 μM CL-13;

[0098] Experimental group 3: 50 μM MCH1623; Experimental group 4: 10 μM MCH1623;

[0099] Positive control group: 5 μM NITD008; Negative control group: 0.1% DMSO.

[0100] Each group has 3 duplicate wells, and is pretreated at 37℃ for 1 hour.

[0101] 2.2.3 Viral infection: Wash cells twice with PBS, add viral infection solution containing the corresponding concentration of compound / control (ZIKV / DENV2 / JEV / EV71 / TBEV, MOI=0.1), and adsorb at 37℃ for 1 h; remove the viral solution, wash twice with PBS, replace with fresh maintenance medium (2% FBS DMEM) containing the corresponding concentration of compound, culture ZIKV / DENV2 / JEV / TBEV for 48 h, and EV71 for 36 h.

[0102] 2.2.4 Fixation and staining: Remove the culture medium, fix with 4% paraformaldehyde at room temperature for 30 min; wash with PBS, permeate with 0.25% Triton X-100 for 15 min; add virus-specific primary antibody (1:500 dilution) and incubate at 37℃ for 1 h; incubate with Alexa Fluor488-labeled secondary antibody (1:1000 dilution) at room temperature in the dark for 1 h; stain the nucleus with DAPI for 5 min, and acquire images using a fluorescence microscope.

[0103] 2.2.5 Quantitative analysis: The viral fluorescence intensity was quantified using ImageJ software, and the cell number was normalized to the DAPI signal. The inhibition rate was calculated as (1 - fluorescence value of experimental group / fluorescence value of negative control group) × 100%.

[0104] 2.3 Experimental Results

[0105] 2.3.1 Cytotoxicity verification: There were no significant differences in cell morphology and number between the treatment groups and the negative control group, and no obvious cytopathic effects were observed, indicating that the experimental system was reliable.

[0106] 2.3.2 Inhibitory effects on five viruses:

[0107]

[0108] - indicates not detected, ND indicates not detected.

[0109] 2.3.3 Conclusion: CL-13 and MCH1623 both exhibited concentration-dependent inhibitory activity against four RNA viruses: ZIKV, DENV2, JEV, and EV71. CL-13 showed significantly better overall antiviral activity than MCH1623 and exhibited no cytotoxicity within the effective antiviral concentration range. At a concentration of 50 μM, CL-13 showed significantly higher inhibitory activity against ZIKV, DENV2, JEV, and EV71 than similar compounds 13 and 14. At a concentration of 50 μM, CL-13's inhibitory activity against tick-borne encephalitis virus (TBEV) was comparable to remdesivir and significantly higher than the control group DMSO.

[0110] Example 3: Half-maximal inhibitory concentration (IC50) of CL-13 and MCH1623 against ZIKV / DENV2 / JEV 50 ) Measurement

[0111] 3.1 Experimental Materials

[0112] 3.1.1 Test compounds: CL-13, MCH1623, 100 mM DMSO stock solution stored at 4℃ protected from light; serial dilutions were performed using complete culture medium during experiments.

[0113] CL-13 concentration gradient: 50 μM, 16.7 μM, 5.56 μM, 1.85 μM, 0.617 μM, 0.206 μM, 0.068 μM, 0.022 μM, 0.0076 μM;

[0114] MCH1623 concentration gradient: 9 μM, 3 μM, 1 μM, 0.33 μM, 0.11 μM, 0.037 μM, 0.012 μM, 0.004 μM, 0.001 μM.

[0115] 3.1.2 Negative control: 0.1% DMSO solution.

[0116] 3.1.3 Virus strains and cells: ZIKV, DENV2, and JEV were all laboratory standard strains with a uniform MOI of 0.1; huH7 cells (human liver cancer cell line) were cultured in DMEM complete medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0117] 3.1.4 Reagents and instruments: 4% paraformaldehyde, 0.25% Triton X-100; ZIKV / DENV2 / JEV specific rabbit primary antibody, Alexa Fluor 488 labeled secondary antibody, DAPI staining solution; Olympus IX83 fluorescence microscope; ImageJ and GraphPad Prism 8.0 software.

[0118] 3.2 Experimental Methods

[0119] 3.2.1 Cell plating: huH7 cells in logarithmic growth phase were seeded at a rate of 1×10⁻⁶ cells / cells. 5 Inoculate 1 cell per well into a 96-well plate and incubate at 37°C and 5% CO2 for 24 h until confluence reaches 70%-80%.

[0120] 3.2.2 Drug treatment: Remove the culture medium, add 100 μL of complete culture medium containing different concentrations of CL-13 / MCH1623 to each well, set up a 0.1% DMSO negative control group, and make 3 replicates per group. Pre-treat at 37℃ for 1 h.

[0121] 3.2.3 Virus infection and culture: After washing with PBS, add the corresponding virus (ZIKV / DENV2 / JEV) infection solution (MOI=0.1) and adsorb at 37℃ for 1 h; replace with maintenance medium containing the corresponding concentration of compound and culture at 37℃ and 5% CO2 for 48 h.

[0122] 3.2.4 Staining and Imaging: Same as in Example 2.2.4, acquire images of DAPI (cell nucleus) and viral antigen channels.

[0123] 3.2.5 IC 50 Calculations: ImageJ was used to quantify viral fluorescence intensity, normalized to the DAPI signal, and the inhibition rate at each concentration was calculated; GraphPad Prism 8.0 was used to fit a nonlinear dose-response curve and calculate the half-maximal inhibitory concentration (IC50). 50 .

[0124] 3.3 Experimental Results

[0125] 3.3.1 ZIKV-resistant IC 50 result:

[0126] CL-13: With increasing concentration, the ZIKV fluorescence signal gradient decreases, and the fitted curve shows IC50. 50 =0.91 μM, the inhibition rate is close to 100% at a concentration of 50 μM, showing a typical S-type dose-response relationship;

[0127] MCH1623: Within the concentration range of 9 μM to 0.001 μM, the ZIKV fluorescence signal was not significantly different from that of the negative control group, indicating no obvious inhibitory activity and failing to fit the effective IC50. 50 .

[0128] 3.3.2 DENV2-resistant IC 50 result:

[0129] CL-13: The fluorescence signal of DENV2 decreases with increasing concentration, and the fitted curve shows that IC50... 50 =0.89 μM, the inhibition rate is close to 100% at high concentrations, and the dose dependence is significant;

[0130] MCH1623: Within the tested concentration range, the DENV2 fluorescence signal showed no significant gradient change, indicating no effective inhibitory activity and failing to fit the IC50. 50 .

[0131] 3.3.3 Anti-JEV IC 50 result:

[0132] CL-13: JEV fluorescence signal decreases gradually with increasing concentration, and the fitted curve shows IC50. 50 =0.12 μM, with extremely strong inhibitory activity, achieving efficient inhibition even at low concentrations;

[0133] MCH1623: Within the tested concentration range, the JEV fluorescence signal showed no significant change, indicating no effective inhibitory activity and failing to fit the IC50 value. 50 .

[0134] 3.3.4 Cytotoxicity verification: There was no difference in morphology and number of huH7 cells in each concentration group compared with the negative control group, and no obvious cytotoxicity was observed at the experimental concentrations.

[0135] 3.3.5 Conclusion: CL-13 exhibits potent, concentration-dependent in vitro inhibitory activity against ZIKV, DENV2, and JEV, with an IC50 value of [missing value]. 50 The concentrations were 0.91 μM, 0.89 μM, and 0.12 μM, respectively. MCH1623 showed no effective inhibitory effect on the three viruses within the tested concentration range, clarifying the activity difference between the two compounds.

[0136] This invention systematically verified the antiviral activity and safety of CL-13 and MCH1623 through three embodiments:

[0137] 1. Safety Verification (Example 1): CL-13 showed no significant cytotoxicity to RD and huH7 cells within the tested concentration range. CC 50 =NA, with extremely high safety; MCH1623 has significant dose-dependent cytotoxicity, limiting its potential as a drug.

[0138] 2. Broad-spectrum activity screening (Example 2): CL-13 has concentration-dependent inhibitory activity against five viruses: ZIKV, DENV2, JEV, EV71 and TBEV. At high concentrations, the inhibitory effect is better than that of the positive control NITD008, and the activity is significantly better than that of MCH1623.

[0139] 3. Precise efficacy determination (Example 3): IC50 of CL-13 against ZIKV, DENV2, and JEV 50 The concentrations of 0.91 μM, 0.89 μM, and 0.12 μM, respectively, confirmed its potent antiviral activity; MCH1623 showed no effective inhibitory activity.

[0140] Based on the experimental data above, CL-13 is a candidate compound that combines broad-spectrum and potent antiviral activity with high safety. It can be used to prepare drugs or formulations against RNA viruses such as ZIKV, DENV2, JEV, EV71, and (TBEV), providing new candidate small molecule compounds for antiviral drug development.

[0141] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. The use of a nucleoside phosphoramide ester derivative CL-13 or a pharmaceutically acceptable salt thereof in the preparation of antiviral drugs, characterized in that, The virus is selected from flaviviruses or enteroviruses, and the structure of CL-13 is shown below: 。 2. The application according to claim 1, characterized in that, The flavivirus is selected from at least one of Zika virus, dengue virus type 2, Japanese encephalitis virus, and tick-borne encephalitis virus.

3. The application according to claim 1, characterized in that, The enterovirus was selected from enterovirus 71.

4. The application according to claim 1, characterized in that, In the antiviral drug, the nucleoside phosphoramide ester derivative CL-13 or its pharmaceutically acceptable salt is either the sole active ingredient or one of the active ingredients.

5. The use of a composition comprising a nucleoside phosphoramide ester derivative CL-13 or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug, characterized in that, The virus is selected from flaviviruses or enteroviruses, and the structure of CL-13 is shown below: 。 6. The application according to claim 5, characterized in that, The flavivirus is selected from at least one of Zika virus, dengue virus type 2, Japanese encephalitis virus, and tick-borne encephalitis virus.

7. The application according to claim 5, characterized in that, The enterovirus was selected from enterovirus 71.

8. The application according to claim 5, characterized in that, In the antiviral drug, the nucleoside phosphoramide ester derivative CL-13 or its pharmaceutically acceptable salt is either the sole active ingredient or one of the active ingredients.

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