A cyclic nucleoside analogue and its preparation method and application

Abstract

The present invention relates to a cyclic nucleoside analogue and a preparation method and application thereof. The cyclic nucleoside analogue has a structure shown in the following formula (I): wherein R 1 H, C1-C 15 Alkyl chain, C3-C 15 Cycloalkyl or C2-C 15 Olefins; where R 2 is halogen, alkyl, alkoxy, cyano or nitro; wherein R 3 is H or acyl; wherein R 4 is H, acyl or alkyl; wherein n is 0, 1, 2 or 3.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a cyclic nucleoside analogue and a preparation method and application thereof. Background Art

[0002] So far, many pathogenic viruses have been found, such as influenza virus, respiratory syncytial virus (RSV), severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) virus, Ebola virus, and new coronavirus (SARS-CoV-2), which cause great harm to human health.

[0003] However, so far, only a few drugs such as Remdesivir, Molnupiravir and Paxlovid are available for clinical use, among which Molnupiravir has relatively poor efficacy. Remdesivir is approved by the FDA for the treatment of patients over 12 years old and weighing at least 40 kg. Due to its strong first-pass effect in the liver and low bioavailability, Remdesivir can only be administered by injection, which limits its clinical application.

[0004] In addition, the study found that 102 viruses in 13 virus families can also infect mammals. For example, coronaviruses can also infect cats, pigs, dogs and other mammals. Cats carry feline coronavirus (FCoV), which can easily develop into infectious peritonitis (FIP) after infection. There is still no approved treatment for FIP, and the mortality rate caused by the disease is as high as over 95%. Another example is porcine epidemic diarrhea virus (PEDV), which is also a coronavirus. Pigs of all ages can be infected and fall ill, especially lactating pigs. The incidence of sows varies greatly, about 15-90%, and there is currently no effective treatment.

[0005] Therefore, developing new, efficient and safe antiviral drugs for the treatment of the above diseases has important clinical value and significance. Brief description of the invention

[0006] The present invention fills the gap in the prior art, provides a cyclic nucleoside analog that can effectively treat related diseases caused by viral infection, and provides a preparation method thereof and its application in the preparation of drugs against RNA viruses (such as including but not limited to the new coronavirus SARS-CoV-2, feline infectious peritonitis virus FCoV, porcine epidemic diarrhea virus, respiratory syncytial virus, influenza virus, Ebola virus, dengue virus, etc.).

[0007] Therefore, one object of the present invention is to provide a cyclic nucleoside analog.

[0008] Another object of the present invention is to provide a method for preparing cyclic nucleoside analogs.

[0009] Another object of the present invention is to provide a use of a cyclic nucleoside analog.

[0010] Based on the above objectives, the present invention provides the following technical solutions.

[0011] In one aspect, the present invention provides a cyclic nucleoside analog (i.e., a nucleoside-3',5'-cyclic phosphate compound) or a pharmaceutically acceptable salt thereof, wherein the cyclic nucleoside analog has a structure shown in the following formula (I):

[0012]

[0013] Among them, R 1 H, C1-C 15 Alkyl chain, C3-C 15 Cycloalkyl or C2-C 15 Olefins;

[0014] Where R 2 is halogen, alkyl, alkoxy, cyano or nitro;

[0015] Where R 3 is H or acyl;

[0016] Where R 4 is H, acyl or alkyl;

[0017] Where n is 0, 1, 2 or 3.

[0018] Preferably, R 1 is H, C1-C6 chain alkyl or C3-C7 cycloalkyl;

[0019] More preferably, R 1 is H, C1-C3 chain alkyl or C3-C7 cycloalkyl;

[0020] Most preferably, R 1 It is a C1-C3 chain alkyl group, for example, methyl, ethyl, propyl, isopropyl.

[0021] Preferably, R 2 is halogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 alkoxy, cyano or nitro;

[0022] More preferably, R 2 is halogen, C1-C6 chain alkyl or C3-C7 cycloalkyl;

[0023] Preferably, R 3 is H or a C1-C6 straight chain or branched chain alkanoyl;

[0024] More preferably, R 3is H or a C1-C3 straight chain or branched chain alkanoyl;

[0025] Preferably, R 4 is H, C1-C6 straight or branched alkanoyl, C1-C6 chain alkyl or C3-C7 cycloalkyl;

[0026] More preferably, R 4 is H, C1-C3 straight or branched alkanoyl, C1-C3 chain alkyl or C3-C7 cycloalkyl;

[0027] Most preferably, R 4 is H;

[0028] Preferably, n is 0 or 1.

[0029] In a most preferred technical solution, the cyclic nucleoside analog is selected from the following compounds:

[0030]

[0031] In another aspect, the present invention provides a method for preparing the above-mentioned cyclic nucleoside analog, the method comprising the following steps:

[0032] The first step: in an organic solvent, the compound of formula (II) reacts with a silicon reagent under the action of an alkaline reagent to generate a compound of formula (III);

[0033] Step 2: reacting the compound (III) prepared in the first step with an acylating agent to obtain compound (IV);

[0034] Step 3: removing silicon protection from the compound of formula (IV) prepared in step 2 in the presence of a desilication agent to obtain a compound of formula (V);

[0035] Step 4: reacting the compound of formula (V) prepared in step 3 with the compound of formula (VI) to generate the compound of formula (VII);

[0036] Step 5: The compound of formula (VII) prepared in step 4 is reacted with a halide and a base to generate the target compound (I);

[0037]

[0038] Preferably, in the first step, the organic solvent is selected from one or more of DMF, CH2Cl2 and THF; preferably DMF;

[0039] Preferably, in the first step, the alkaline agent is selected from one or more of imidazole, triethylamine and NaH; preferably imidazole;

[0040] Preferably, in the first step, the silicon reagent is selected from one or more of tert-butyldiphenylsilyl chloride, tert-butyldimethylsilyl chloride and triisopropylsilyl chloride; preferably triisopropylsilyl chloride;

[0041] Preferably, in the second step, the acylating agent is C2-C 20 Alkyl chlorides, for example, selected from acetyl chloride, propionyl chloride, isopropyl chloride, n-butyl chloride and C5-C 20 One or more of alkyl acyl chlorides; preferably acetyl chloride;

[0042] Preferably, in the third step, the desilication agent is selected from one or more of triethylamine trihydrofluoride and TBAF, preferably triethylamine trihydrofluoride.

[0043] Preferably, in the fifth step, the halogenated compound is selected from one or more of N-chlorodiisopropylamine, NBS and NCS, preferably N-chlorodiisopropylamine.

[0044] Preferably, in the fifth step, the base is selected from one or more of imidazole, 1-(cyanomethyl)imidazole, DMAP, DBU, DIPEA and potassium tert-butoxide, preferably potassium tert-butoxide.

[0045] Preferably, in the fifth step, the reaction temperature is -40°C to room temperature, preferably -40°C to -10°C.

[0046] Preferably, in the fifth step, the reaction solvent is selected from acetonitrile and THF, preferably THF.

[0047] Those skilled in the art know that 5'(3')-phosphate modification of nucleosides usually adopts the phosphoric acid di / triester method or the phosphoramidite method. The former uses phosphoryl chloride reagents such as phosphorus oxychloride, phosphorus trichloride, etc., and the reaction conditions are fierce and the yield is low, so it is now less used; the latter generally uses phosphoramidite and nucleosides under the action of tetrazole to prepare trivalent nucleotide phosphoramidite intermediates, and various oxidants such as hydrogen peroxide, iodine, tert-butyl peroxide or meta-chloroperbenzoic acid are used to oxidize to become a stable pentavalent phosphate. The synthesis yield is significantly better than the former, which is a method commonly used in the art. In the course of research, the inventor tried to use the phosphoramidite method, but only a very small amount of product was obtained in the reaction solution, which did not have industrial value. To this end, the inventor tried the following method for stereoselective preparation of 3',5'-nucleoside cyclic phosphates, and successfully prepared the target compound of the present application. As shown in the above synthetic route, the hydrogen phosphate compound (VII) is synthesized by four-step reaction. The key is that in the fifth step, N-chlorodiisopropylamine, NBS or NCS are selected, and imidazole, 1-(cyanomethyl)imidazole, DMAP, DBU, DIPEA and potassium tert-butoxide are selected as bases. The optimal solution is N-chlorodiisopropylamine and potassium tert-butoxide. The purity of the target compound in the reaction system is increased to 81%, and the yield is about 76%. In particular, by optimizing the reaction conditions of this step, the stereoselectivity of the phosphorus atom when constructing -3',5'-cyclic phosphate is greatly improved (Sp-product / Rp-product ratio is about 6 to 1) and the generation of alkylation by-products is reduced. In addition, most intermediates in the reaction route of the present invention can be directly crystallized, and the post-treatment is simple and more suitable for industrial production.

[0048] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned cyclic nucleoside analog or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0049] In another aspect, the present invention provides a use of the above-mentioned cyclic nucleoside analog or a pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition in the preparation of a drug for treating a disease caused by a viral infection.

[0050] Preferably, the virus is selected from the group consisting of: Filoviridae, Arenaviridae, Coronaviridae, Flaviviridae and Paramyxoviridae. Specifically, the virus includes but is not limited to the novel coronavirus SARS-CoV-2, feline infectious peritonitis virus FCoV, porcine epidemic diarrhea virus, respiratory syncytial virus, influenza virus, Ebola virus, dengue virus and the like.

[0051] The nucleoside compound or pharmaceutical composition of the present invention can be used alone or in combination with one or more other antiviral drugs, including but not limited to: RNA polymerase inhibitors Favipiravir, Galidesivir, molnupiravir, EIDD-1931; 3CL protease inhibitors Nirmatrelvir, Ensitrelvir (S-217622), GC-376, Lopinavir, Nelfinavir; and others such as interferon, hydroxychloroquine, cyclosporine, ivermectin, ribavirin, penciclovir, azithromycin, paxlovidin, etc. or their combinations. The combined use can provide "synergistic effect" and "synergistic effect", which can be co-formulated and used or delivered simultaneously in the form of a combined preparation, or can be used alternately in the form of a separate preparation, such as a separate tablet, pill or capsule, or injected separately through a separate syringe, to obtain a synergistic effect.

[0052] The cyclic nucleoside analogs of the present application have a significant inhibitory effect on the replication of the new coronavirus, low cytotoxicity, high therapeutic index, high oral exposure and high oral bioavailability in animals, and the cyclic nucleoside analogs of the present application are superior to other similar compounds in terms of solubility and stability. The preparation method of the present application optimizes the reaction route, especially the reaction conditions, and the purity and yield of the prepared product are significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 is the hydrogen spectrum of compound 1 in formula (I).

[0054] Figure 2 It is the carbon spectrum of compound 1 in formula (I).

[0055] Figure 3 is the phosphorus spectrum of compound 1 in formula (I).

[0056] Figure 4 It is the high-resolution mass spectrum of compound 1 in formula (I).

[0057] Figure 5 It is the crystal structure analysis spectrum of compound 1 in formula (I). DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0059] Example 1

[0060] Preparation of ethyl 2-((((2S,4aR,6R,7R,7aR)-7-acetoxy-6-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-6-cyano-2-oxytetrahydro-4H-furo[3,2-d][1,3,2]diphosphin-2-yl)oxy)methyl)benzoate (Compound 1)

[0061] The synthetic route is as follows:

[0062]

[0063] 1) Synthesis of intermediate 1

[0064]

[0065] Dissolve 29.8 g of sodium hydroxide in 500 ml of purified water, stir to dissolve, add 100 g of phthalide, and react at 70° C. for 5 h. After the reaction, concentrate to obtain a white solid, add 500 ml of ethyl acetate to slurry, filter, wash the filter cake twice with ethyl acetate, and dry at 60° C. for 6 hours to obtain 104 g of intermediate 1 with a yield of 80%.

[0066] 2) Synthesis of Intermediate 2

[0067]

[0068] Prepare iodoethane / DMSO mixed solution: Mix 50 ml DMSO and 49 g iodoethane evenly for later use.

[0069] Add 50g of intermediate 1 and 150mL of DMSO to a reaction bottle and stir to dissolve. Slowly add the prepared iodoethane / DMSO mixed solution at 20-30°C, control the dripping to be complete in 10-20 minutes, and react for 5 hours. After the reaction, add 400ml of ethyl acetate and 400ml of purified water, separate the liquids, wash the organic phase twice with purified water, and then wash it twice with saturated brine, combine the organic phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 42g of intermediate 2, with a yield of 81%. Ms: [M+Na] + 203.1

[0070] 1H NMR (400MHz, DMSO) δ1.282-1.317(t,3H),4.241-4.294(dd,J1=7.2Hz,J2=14Hz,2H),4.795-4.809(d,J=4Hz,2H),5 .196-5.224(t,J=5.6Hz,1H),7.328-7.369(m,1H),7.560-7.601(m,1H),7.680-7.713(m,1H),7.753-7.772(m,1H)

[0071] 13 C NMR (400MHz, DMSO) δ14.07,60.56,60.97,61.02,126.46,126.82,127.69,129.68,132.11,144.13,166.61.

[0072] 3) Synthesis of intermediate 4

[0073]

[0074] Add 10g (2R, 3R, 4R, 5R) -2- (4-aminopyrrolo [2, 1-f] [1, 2, 4] triazine-7-yl) -3-fluoro-4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-carbonitrile (GS-441524), 5.84g imidazole and 57mL N, N-dimethylformamide to the reaction bottle, stir to dissolve, and slowly add 13g TIPDSCl at 5°C. After the addition, react at 5°C for 1 hour, add 90g ethyl acetate and 120ml purified water for extraction, separate the liquids, and collect the organic phase. Add 45ml ethyl acetate to the aqueous phase for extraction, separate the liquids, combine the organic phases, wash twice with purified water, and concentrate. Add 55ml n-hexane, heat to reflux, stir for 0.5 hours, cool to 10-20°C for crystallization, and stir for crystallization for 0.5 hours. The filter cake was filtered and washed with n-hexane and dried at 60°C for 4 hours to obtain 14.6 g of intermediate 4 with a yield of 80%. Ms: [M+H] + 534.3,[M+Na] + 556.2

[0075] 1H NMR (400MHz, DMSO) δ0.814-1.030(m,28H),3.879-3.918(m,1H),4.099-4.199(m,3H),4.532-4.557(t,1H),6. 441-6.455(d,J=6.0Hz,1H),6.775-6.786(d,J=4.8Hz,1H),6.849-6.876(d,J=4.8Hz,1H),7.886-7.911(m,3H)

[0076] 13 C NMR (400MHz, DMSO) δ12.04,12.16,12.28,12.42,12.55,12.75,16.69,16.76,16.790,17.01,17.08,17.14,17. 30,22.04,30.94,59.76,69.17,73.96,79.46,81.09,100.72,109.86,116.32,116.51,123.55,148.01,155.52

[0077] 4) Synthesis of Intermediate 6

[0078]

[0079] 14.6 g of intermediate 4, 88 mL of acetonitrile, 2.8 g of acetic anhydride, and 0.67 g of dimethylaminopyridine were added to the reaction bottle, stirred, and when the temperature dropped to 0°C, 8.8 g of triethylamine hydrofluoride was slowly added dropwise at a uniform rate. After the addition was completed, the temperature was controlled at 0°C for 1 hour. After the reaction was completed, the mixture was concentrated to obtain a crude intermediate 5.

[0080] Add 88 mL of tetrahydrofuran to the crude intermediate 5, stir, and add triethylamine hydrofluoride dropwise at 20°C. After the addition, react for 8 hours. After the reaction, concentrate, add 97 ml of ethyl acetate and 73 ml of purified water for extraction, and separate the liquids. The aqueous phase is extracted twice with 97 ml of ethyl acetate, combined with three ethyl acetates, and concentrated. Add 27 ml of ethyl acetate to the crude product, heat to reflux, beat for 0.5 hours, cool to 5-10°C, and stir to crystallize for 0.5 hours. Filter by suction, wash the filter cake once with 8 ml of ethyl acetate, and dry it at 60°C for 4 hours to obtain 7.3 g of intermediate 6, with a yield of 80%, Ms: [M+H] + 334.1,[M+Na] + 356.1.

[0081] 1H NMR(400MHz,DMSO)δ2.084(s,3H),3.518-3.642(m,2H),4.252-4.281(m,1H),4.936-4.966(m,1H),5.0 27-5.056(m,1H),5.122-5.146(m,1H),6.455-6.471(m,1H),6.875-6.920(m,2H),7.910-7.949(m,3H)

[0082] 13 C NMR (400MHz, DMSO) δ20.74,60.53,72.18,72.57,78.01,83.49,100.84,111.06,116.80,116.87,122.80,148.00,155.63,169.70

[0083] 5) Synthesis of intermediate 7

[0084]

[0085] 32.18g DPP and 300ml acetonitrile were added to the reaction bottle, stirred, 30ml ultra-dry pyridine was added, 21.9g intermediate 2 was slowly added dropwise, and the reaction was continued for 1h. After the reaction, the reaction solution of intermediate 3 was obtained. Without treatment, 30g intermediate 6 was directly added to the reaction solution, and the temperature was raised to 50℃ for reaction for 2h. After the reaction, the solution was concentrated and purified by column chromatography to obtain 12.6g intermediate 7 with a yield of 25%. Ms: [M+H] + 558.1,[M+Na] + 580.1

[0086] 1 H NMR (400MHz, DMSO) δ1.267-1.308(m,3H),2.082(s,3H),4.243-4.296(m,4H),4.471-4. 481(d,J=4Hz,1H),5.035-5.065(t,J1=6Hz,J2=12Hz,1H),5.124-5.149(m,1H),5.333- 5.367(m,2H),6.050-6.076(d,J=10.4H,1H),6.597-6.617(dd,J1=1.6Hz,J2=6.4Hz,2H ),6.844-6.903(m,2H),7.450-7.471(m,1H),7.488-7.602(m,2Hz),7.839-7.918(m,4H)

[0087] 13C NMR (400MHz, DMSO) δ13.97,20.63,20.68,26.32,60.95,63.93,63.98,64.06,64.50,64.54,71.49,71.52,72.12,72.16,76.50,78.52,80.61 ,80.67,100.85,110.96,116.45,119.95,122.14,127.93,128.11,128 .21,130.28,132.62,137.15,137.22,147.98,155.57,165.98,169.57

[0088] 31 P NMR(400MHz,DMSO)δ9.80-10.02

[0089] 6) Preparation of Compound 1

[0090]

[0091] Weigh 56.23g of intermediate 7, add 560mL of tetrahydrofuran to the reaction flask, stir to dissolve, cool to -40°C, add 34.3g of N-chlorodiisopropylamine dropwise, and complete the dripping. Dissolve 16.87g of potassium tert-butoxide in 280mL of tetrahydrofuran to obtain a turbid liquid, and add a tetrahydrofuran solution of potassium tert-butoxide to the above reaction liquid. After dripping, react at -30 to -40°C for 0.5 hour. After the reaction, adjust the pH to 7 with about 50ml of 15% citric acid, separate the layers, wash the organic phase once with saturated brine, concentrate, and purify by column chromatography to obtain 42.2g of compound 1 and compound 2, with a yield of 76% for compound 1. (5 mg / ml, THF-acetonitrile (1:1)), compound 2: (5 mg / ml, THF-acetonitrile (1:1)).

[0092] Compound 1: 1H NMR (400MHz, DMSO) δ1.349-1.385 (t, J1=7.2Hz, J2=14.4Hz, 3H), 2.295 (s, 3H), 4.297-4.388 ( m,3H),4.459-4.521(m,1H),4.608-4.698(m,1H),5.162-5.205(m,1H),5.605-5.622(m,2H), 5.940(brs,2H),6.028-6.042(d,J=5.6Hz,1H),6.676-6.688(d,J=4.8Hz,1H),6.972-6.984( d,J=4.8Hz,1H),7.404-7.445(m,1H),7.551-7.632(m,2H),7.786(s,1H),8.008-8.027(m,1H)

[0093] 13 C NMR (400MHz, DMSO) δ14.19,20.85,61.35,67.70,67.75,69.18,69.27,71.60,71.68,71.89,71.94,81.99,100.65,113. 14,114.22,116.95,121.59,128.54,128.55,128.77,130.97,132.64,136.67,136.75,147.32,155.10,166.42,169.10

[0094] 31 P NMR (400MHz, DMSO) δ-6.88. Ms:[M+H] + 558.1424

[0095] Compound 2:

[0096] 1H NMR (400MHz, DMSO) δ1.321-1.356(t,3H),2.160(s,3H),4.302-4.356(dd,J1=7.2Hz,J 2=14.4Hz,2H),4.508-4.628(m,2H),4.769-4.835(m,1H),5.023-5.060(dd,J1=5.2Hz ,J2=9.6Hz,1H),5.507-5.526(d,J=7.6Hz,2H),6.049-6.062(d,J=5.2Hz,1H),6.932- 6.959(m,2H),7.505-7.543(t,J=7.6Hz,1H),7.623-7.717(m,2H),7.943-8.076(m,4H)

[0097] 13 C NMR (400MHz, DMSO) δ14.01,14.06,20.28,20.74,61.06,67.85,67.90,68.77,68.86,71.21,71.29,71.64,71.71,75.64,75.67,80.61 ,101.25,111.04,114.52,117.21,120.08,127.79,128.19,128.47,130.37,132.77,136.66,136.74,148.43,155.55,166.07,168.43

[0098] 31 P NMR (400MHz, DMSO) δ-5.78

[0099] Ms:[M+H] + 558.1409,[M+Na] + 580.1232

[0100] The hydrogen spectrum, carbon spectrum, phosphorus spectrum, high-resolution mass spectrum, and crystal structure analysis spectrum of the prepared compound 1 are as follows: Figures 1 to 5 shown.

[0101] Example 2

[0102] This experiment was conducted to investigate the effects of various conditions in the preparation process on the final product. The experiment was conducted with reference to the synthesis of compound 1 in Example 1, and the conditions are shown in Table 1.

[0103] Table 1: Results of optimization screening of synthesis conditions for compound 1

[0104]

[0105]

[0106] Note: a: All reaction temperatures in Table 1 are room temperature. b: The purity of the main product was determined by liquid chromatography and calculated by area normalization method.

[0107] In order to study the antiviral activity, solubility and stability of compounds 1 and 2 (compounds 1 and 2 were prepared from Example 1), the following experiments were performed.

[0108] Experimental Example 1: Inhibitory effect and cytotoxicity of compound 1 on the replication of novel coronavirus

[0109] 1) Preparation method of the sample to be tested:

[0110] Compound 1 was dissolved in DMSO to a 100 mM working solution, and then serially diluted with DMEM medium (containing penicillin and streptomycin, wherein the ratio of penicillin and streptomycin in the medium was 2%, v / v, the final concentration of penicillin was 100 U / ml, and the final concentration of streptomycin was 0.1 mg / ml), with a total of 8 dilutions. In the cytotoxicity test, the test concentrations of compound 1 were 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, 0.78 μM, and 0.39 μM. In the antiviral activity test, the test concentrations of compound 1 were 500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM, 15.6 nM, 7.8 nM, and 3.9 nM.

[0111] 2) Inhibition experiment of the sample to be tested (sample containing compound 1) on the replication of SARS-CoV-2 in Vero E6 cells (IC 50 )

[0112] 1×10 5Vero E6 cells were inoculated into 48-well plates at 200 μL / well, and cultured overnight at 37°C and 5% CO2. The cells were used when the monolayer grew to about 90%. In the P3 laboratory, 100 μL of the sample to be tested and 100 μL of virus dilution (containing 100 TCID50 virus) were added to each well at the same time, and incubated for 1 hour at 37°C and 5% CO2. The sample to be tested was set up with 8 concentration gradients, and 4 replicates were set up for each gradient; at the same time, a solvent control group of 1‰ DMSO (1‰ DMSO + virus), a cell control (no sample to be tested and virus), and a virus control (only virus) were set up. After 1 hour, the virus-sample mixed culture medium was aspirated and discarded, and washed twice with 1×PBS and replaced with a culture medium containing only the sample to be tested for continued culture, 300 μL / well, 8 gradients, and 4 replicates were set up for each gradient. At the same time, a solvent control group of 1‰ DMSO (1‰ DMSO + virus), a cell control (no sample to be tested and virus), and a virus control (only virus) were set up. After culturing at 37°C and 5% CO2 for 48 h, the cell supernatant was collected and viral RNA was extracted for real-time PCR virus quantification to calculate the inhibition rate of virus replication (IC 50 ).

[0113] 3) Toxicity test of the sample to be tested (sample containing compound 1) on Vero-E6 cells (CC 50 )

[0114] The CCK8 kit was used to detect the cell viability of the samples with different concentrations (three replicates for each concentration) (measured 24 hours after adding the samples to be tested), and the CC was calculated. 50 The value (50% Cytotoxic Concentration) is the concentration of the sample to be tested that produces toxicity to 50% of VeroE6 cells.

[0115] 4) According to cell activity CC 50 and cytotoxic IC 50 , calculate the therapeutic index (SI) of the sample to be tested: SI = CC 50 / IC 50 .

[0116] Table 2: Inhibitory activity results of compound 1 against the new coronavirus

[0117]

[0118] Experimental Example 2: Solubility and stability of compounds 1 and 2 in different solvents

[0119] The solubility test method is:

[0120] 1. Prepare PBS buffer (pH 7.4), simulated pre-meal small intestinal fluid FaSSIF (pH 6.5), and simulated pre-meal gastric fluid FaSSGF (pH 1.6).

[0121] 2. Dissolve the standard in acetonitrile as reference solution.

[0122] 3. Compound 1 and compound 2 were prepared into saturated solutions using PBS buffer (pH 7.4), small intestinal fluid FaSSIF (pH 6.5), and gastric fluid FaSSGF (pH 1.6) prepared in 1, respectively. The solutions were shaken at a constant temperature of 37°C, and samples were taken after 24 hours and filtered.

[0123] 4. Use the external standard method and acetonitrile water as the mobile phase for high performance liquid chromatography detection.

[0124] 5. Record the peak area and calculate the solubility of each sample.

[0125] The test method for solution stability is:

[0126] 1. Prepare PBS buffer (pH 7.4), simulated pre-meal small intestinal fluid FaSSIF (pH 6.5), and simulated pre-meal gastric fluid FaSSGF (pH 1.6).

[0127] 2. Take appropriate amounts of compound 1 and compound 2, and add equal volumes of PBS buffer (pH 7.4), simulated pre-meal small intestinal fluid FaSSIF (pH 6.5), and simulated pre-meal gastric fluid FaSSGF (pH 1.6) prepared in 1.

[0128] 3. Take samples at 1, 4, 8 and 24 hours respectively and dissolve them in acetonitrile to make up the volume.

[0129] 4. Use the external standard method and acetonitrile water as the mobile phase for high performance liquid chromatography detection.

[0130] 5. Record the peak area and calculate the residual content % of each sample.

[0131] The test results are shown in Table 3-4.

[0132] Table 3 Saturation solubility test results of compounds 1 and 2

[0133]

[0134] Table 4 Solution stability test results of compounds 1 and 2

[0135]

[0136] Solid stability test method:

[0137] 1. Preparation of test solution

[0138] A volumetric flask (5 mL) containing compound 1 (2.5 mg) was placed in an environment of 40°C ± 2°C / 75% ± 5% RH (Relative Humidity) for 4 hours and 24 hours, respectively, and then cooled naturally to room temperature. Acetonitrile was then added to dissolve and diluted to the scale and shaken to obtain the test solutions of compound 1 after being placed for 4 hours and 24 hours, respectively.

[0139] The preparation method of the test solution of compound 2 after being placed for 4 hours and 24 hours is the same as that of compound 1.

[0140] 2. Determination

[0141] The test solutions of compound 1 and compound 2 which were left for 4 hours and 24 hours were respectively measured, and the content of compound 1 or compound 2 in each test solution was calculated by external standard method using liquid chromatograph as the test instrument.

[0142] 3. Solid stability test results

[0143] The results are shown in Table 5.

[0144] Table 5 Solid stability test results

[0145]

[0146] As shown in Table 3, the solubility of Compound 1 and Compound 2 in different biological media is quite different, especially in the simulated gastric fluid of the fasting state, the solubility of Compound 1 is much higher than that of Compound 2, which is increased by about 200 times.

[0147] From the solution stability results in Table 4, it can be seen that the stability of compound 2 in the above-mentioned media is worse than that of compound 1. When placed at a constant temperature of 37°C for 24 hours in PBS, FaSSIF and FeSSIF media, the content of compound 1 is greater than 90%, while the content of compound 2 is less than 60%. When placed at a constant temperature of 37°C for 24 hours in FaSSGF medium, the content of compound 1 is 58.06%, and the content of compound 2 is 7.97%. The stability of compound 1 is significantly better than that of compound 2.

[0148] Table 5 shows the solid stability results. Compound 1 was placed at 40°C / 75%RH for 24 hours, and the content did not change significantly. Compound 2 was placed at the same conditions for 4 hours and 24 hours, and the content decreased by about 5% and 30%, respectively, and the content decreased significantly. It can be seen that the solid stability of compound 1 is significantly better than that of compound 2.

[0149] Based on the above results, it can be seen that compound 1 has greatly improved solubility, liquid and solid stability compared to compound 2, and has greater drugability and potential for development as a clinical candidate compound.

[0150] Experimental Example 3: Pharmacokinetic study of compound 1 in different animal species

[0151] 1. Pharmacokinetic evaluation in rats

[0152] Pharmacokinetic process: 24 SD rats (weight 271.5-348.3g) were taken, 12 in each group, half male and half female. The oral administration group fasted (>12h) before the experiment, had free access to water, and was given food 2h after administration. The animals in the intravenous administration group were free to eat. Compound 1 was dissolved in an appropriate solvent. The intravenous administration group preparation was prepared on the day of administration, and the oral administration group preparation was prepared during the stable period. The solvent for the intravenous administration group was 5% DMSO + 5% ethanol + 40% PEG400 + 50% saline, and the solvent for the oral administration group was 0.5% MC (w / v) aqueous solution. Compound 1 was administered to rats by oral gavage (10 mg / kg) / tail vein injection (3 mg / kg), and the intravenous administration group was administered before, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration; the oral administration group was administered before, 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 24 h after administration, respectively. About 0.5 ml of orbital blood was collected with a micro blood collection pipette. The fresh whole blood sample was placed in a K2EDTA anticoagulant tube and gently inverted to mix at a ratio of whole blood: stabilizer (0.4 M DDV dissolved in isopropanol) = 10:0.25 (v / v) (the blood collection tube was placed in wet ice throughout the process). Plasma was obtained after centrifugation for 10 min (4°C, 2000g), and frozen in a -80°C refrigerator for testing. The organic solvent precipitation method was used to quantitatively take plasma samples, add acetonitrile precipitant, vortex thoroughly to mix, and centrifuge for 10 min (4000 rpm, 4° C.). The supernatant was taken out and the concentration of raw material 1 in plasma was quantitatively tested by LC-MS to calculate the pharmacokinetic parameters.

[0153] Standard curve: Take 8 1.5ml EP tubes, add 0.1, 0.2, 0.5, 1, 2, 5, 9, 10μl of raw material 1 stock solution respectively, make up to 450μl of working solvent, mix well, add 50μl of rat blank plasma to each tube, vortex for 5min, centrifuge at 12000rpm / min for 5min, take the supernatant, centrifuge again at 12000rpm / min for 5min, take the supernatant and put it into 1.5ml EP tube to obtain 10, 20, 50, 100, 200, 500, 900, 1000ng / ml raw material 1 standard curve samples (including internal standard dexamethasone, 5.00ng / ml).

[0154] Sample processing: Take 10 μl of drug-containing plasma obtained from the pharmacokinetic process, place it in a 1.1 ml strip tube with 200 μl of internal standard working solution (5.00 ng / ml Dexamethasone in 100% acetonitrile), vortex for 10 min, 4000 rpm, 4°C, centrifuge for 10 min, take 20 μl of supernatant into a 1.1 ml strip tube, add 180 μl of diluent II (acetonitrile: deionized water = 1:5), and use a vortex to mix the sample. Transfer 180 μl of the mixed sample solution to a 96-well plate, and inject 5 μl of the sample into the LC-MS system to detect the sample.

[0155] LC-MS detection: chromatographic column, YMC-Triart C18 (50×2.1mm, S-3μm, 12nm, Shimadzu Corporation, Japan), column temperature was ambient temperature, injection volume was 5μL, mobile phase was acetonitrile-deionized water, flow rate was 0.6ml / min, gradient elution; ion source, TRIPLE QUAD 5500 mass spectrometer electrospray ionization source ESI; detection method, multiple reaction monitoring (MRM); scanning mode, positive ion mode; ESI spray voltage 5500V, ion source temperature 500℃, spray gas 50psi, auxiliary heating gas 50psi, curtain gas 30psi.

[0156] 2. Pharmacokinetic evaluation in dogs

[0157] Pharmacokinetic process: 6 beagle dogs (weight 6.8-8.7kg), half male and half female, were selected, and a total of 6 animals were used for oral and intravenous administration groups, and the metabolic clearance time was no less than 7 days. The oral administration group fasted (>12h) before the experiment, had free access to water, and was given food 2h after administration. Animals in the intravenous administration group were free to eat. Compound 1 was dissolved in an appropriate solvent, the intravenous administration group preparation was prepared on the day of administration, and the oral administration group preparation was prepared during the stable period. The solvent for the intravenous administration group was 5% DMSO + 5% ethanol

[0158] +40% PEG400+50% saline, the solvent for the oral administration group was 0.5% MC (w / v) aqueous solution. Compound 1 was administered orally (3 mg / kg) / intravenously (1 mg / kg) to dogs. The intravenous administration group was administered before administration, 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 24h after administration; the oral administration group was administered before administration, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24h after administration. About 1.0 ml of blood was collected by peripheral vein puncture. The fresh whole blood sample was placed in a K2EDTA anticoagulant tube, and the whole blood: stabilizer (0.4M DDV dissolved in isopropanol) = 10: 0.25 (v / v) ratio was gently inverted and mixed (the blood collection tube was placed in wet ice throughout the process), and the plasma was obtained after centrifugation for 10 min (4°C, 2000g), and frozen in a -80°C refrigerator for testing. The organic solvent precipitation method was used to quantitatively take plasma samples and add acetonitrile precipitant. After thorough vortex mixing, the samples were centrifuged for 10 min (4000 rpm, 4°C). The supernatant was taken out for quantitative analysis by LC-MS to calculate the pharmacokinetic parameters.

[0159] Standard curve: Take 8 1.5ml EP tubes, add 0.1, 0.2, 0.5, 1, 2, 5, 9, 10μl of raw material 1 stock solution respectively, make up to 450μl of working solvent, mix well, add 50μl of canine blank plasma to each tube, vortex for 5min, centrifuge at 12000rpm / min for 5min, take the supernatant, centrifuge again at 12000rpm / min for 5min, take the supernatant and put it into 1.5ml EP tube to obtain 10, 20, 50, 100, 200, 500, 900, 1000ng / ml raw material 1 standard curve samples (including internal standard dexamethasone, 5.00ng / ml).

[0160] Sample processing: Take 10 μl of drug-containing plasma obtained from the pharmacokinetic process, place it in a 1.1 ml strip tube with 200 μl of internal standard working solution (5.00 ng / ml dexamethasone in 100% acetonitrile), vortex for 10 min, 4000 rpm, 4°C, centrifuge for 10 min, take 20 μl of supernatant into a 1.1 ml strip tube, add 180 μl of diluent II (acetonitrile: deionized water = 1:5), and use a vortex to mix the sample. Transfer 180 μl of the mixed sample solution to a 96-well plate, and inject 5 μl of the sample into the LC-MS system to detect the sample.

[0161] LC-MS detection: chromatographic column, YMC-Triart C18 (50×2.1mm, S-3μm, 12nm, Shimadzu Corporation, Japan), column temperature was ambient temperature, injection volume was 5μL, mobile phase was acetonitrile-deionized water, flow rate was 0.6ml / min, gradient elution; ion source, TRIPLE QUAD 5500 mass spectrometer electrospray ionization source ESI; detection method, multiple reaction monitoring (MRM); scanning mode, positive ion mode; ESI spray voltage 5500V, ion source temperature 500℃, spray gas 50psi, auxiliary heating gas 50psi, curtain gas 30psi.

[0162] 3. Pharmacokinetic evaluation in monkeys

[0163] Pharmacokinetic process: 6 male cynomolgus monkeys (weight 3.80±0.62kg) were taken, with 3 animals in each group. The oral administration group fasted for 16-17h before the experiment, had free access to water, and was given food 2h after administration. Animals in the intravenous administration group were free to eat. Compound 1 was dissolved in an appropriate solvent. The preparation for the intravenous administration group was prepared on the day of administration, and the preparation for the oral administration group was prepared during the stable period. The solvent for the intravenous administration group was 5% DMSO + 5% ethanol + 40% PEG400 + 50% saline, and the solvent for the oral administration group was 0.5% MC (w / v) aqueous solution. For the oral (30 mg / kg) / intravenous (4 mg / kg) groups of cynomolgus monkeys, about 1.0 ml of blood was collected from the forelimb vein at 0.033 (only intravenous administration group), 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration. The fresh whole blood samples were placed in K2EDTA anticoagulant tubes, and gently inverted and mixed according to the ratio of whole blood: stabilizer (0.4 MDDV dissolved in isopropanol) = 10: 0.25 (v / v) (the blood collection tube was placed in wet ice throughout the process), and the plasma was obtained after centrifugation for 10 min (4°C, 3000 rpm), and frozen in a -80°C refrigerator for testing. Using the organic solvent precipitation method, the plasma samples were quantitatively taken and added with the precipitant (100 ng / ml toluenesulfonamide in acetonitrile solution), and after sufficient vortex mixing, centrifuged for 15 min (4000 rpm, 4°C), and the supernatant was taken out for quantitative analysis by LC-MS method to calculate the pharmacokinetic parameters.

[0164] Standard curve: Dissolve raw material 1 in DMSO to prepare a 1.00 mg / ml stock solution. Dilute the stock solution with methanol to prepare a series of standard curve working solutions. Take 50 μl of monkey blank plasma and add 5 μl of the standard curve working solution of each concentration of the analyte, vortex for 1.0 min, and prepare standard curve samples of raw material 1 with final concentrations of 1.00, 2.00, 5.00, 20.0, 100, 500, 1000 and 2000 ng / ml.

[0165] Sample processing: Take 50 μl of drug-containing plasma obtained in the pharmacokinetic process, add 200 μl of precipitant (100 ng / ml toluenesulfonamide in acetonitrile), vortex at room temperature for 2 min to mix thoroughly, centrifuge at 4000 rpm, 4°C for 15 min, and take the supernatant to the LC-MS system to detect the sample.

[0166] LC-MS detection: chromatographic column, Kinetex C18 (30×2.1 mm, 2.6 μm, Phenomenex, USA), column temperature was room temperature, injection volume was 3 μL, mobile phase was 0.05% FA and 5 mM NH4Ac aqueous solution-80% CAN aqueous solution, flow rate was 0.6 ml / min, gradient elution; ion source, TRIPLE QUADTM 6500+ (SCIEX) mass spectrometer electrospray ionization source ESI; detection method, multiple reaction monitoring (MRM); scanning mode, positive ion mode; ESI spray voltage was 5500 V, ion source temperature was 500°C, spray gas was 50 psi, auxiliary heating gas was 50 psi, and curtain gas was 35 psi.

[0167] The pharmacokinetic results are shown in Table 6:

[0168] Table 6 Pharmacokinetic parameters of metabolites of compound 1 in different species

[0169]

[0170] F(%) = (AUC of raw material 1 after oral administration of compound 1) 最终 / oral dose level) / (AUC of raw material 1 after intravenous injection of compound 1 最终 / intravenous dose level)×100%.

Claims

1. A method for preparing a cyclic nucleoside analog represented by formula (I): in, R 1 H, C1-C 15 Alkyl chain, C3-C 15 Cycloalkyl or C2-C 15 Olefins; Where R 2 is halogen, alkyl, alkoxy, cyano or nitro; Where R 3 is H or acyl; Where R 4 is H, acyl or alkyl; Where n is 0, 1, 2, or 3, The method comprises the following steps: The first step: in an organic solvent, the compound of formula (II) reacts with a silicon reagent under the action of an alkaline reagent to generate a compound of formula (III); Step 2: reacting the compound (III) prepared in the first step with an acylating agent to obtain compound (IV); Step 3: removing silicon protection from the compound of formula (IV) prepared in step 2 in the presence of a desilication agent to obtain a compound of formula (V); Step 4: reacting the compound of formula (V) prepared in step 3 with the compound of formula (VI) to generate the compound of formula (VII); Step 5: The compound of formula (VII) prepared in step 4 is converted into target compound (I) under the condition of halide and base; In the fifth step, the halide is N-chlorodiisopropylamine, the base is potassium tert-butoxide, and the reaction solvent is THF.

2. The preparation method according to claim 1, wherein In the first step, the organic solvent is selected from one or more of DMF, CH2Cl2 and THF.

3. The preparation method according to claim 1, wherein In the first step, the organic solvent is selected from DMF.

4. The preparation method according to any one of claims 1 to 3, wherein In the first step, the alkaline agent is selected from one or more of imidazole, triethylamine or NaH.

5. The preparation method according to any one of claims 1 to 3, wherein In the first step, the alkaline agent is selected from imidazole.

6. The preparation method according to any one of claims 1 to 3, wherein In the first step, the silicon reagent is selected from one or more of tert-butyldiphenylsilyl chloride, tert-butyldimethylsilyl chloride and triisopropylsilyl chloride.

7. The preparation method according to any one of claims 1 to 3, wherein In the first step, the silicon reagent is selected from triisopropylsilyl chloride.

8. The preparation method according to any one of claims 1 to 3, wherein In the second step, the acylating agent is C2-C 20 Alkyl acid chlorides.

9. The preparation method according to any one of claims 1 to 3, wherein In the second step, the acylating agent is selected from acetyl chloride, propionyl chloride, isopropyl chloride, n-butyl chloride and C5-C 20 One or more of alkyl acid chlorides.

10. The preparation method according to any one of claims 1 to 3, wherein In the second step, the acylating agent is acetyl chloride.

11. The preparation method according to any one of claims 1 to 3, wherein In the third step, the desiliconizing agent is selected from one or more of triethylamine trihydrofluoride and TBAF.

12. The preparation method according to any one of claims 1 to 3, wherein In the third step, the desiliconizing agent is triethylamine trihydrofluoride.

13. The preparation method according to any one of claims 1 to 3, wherein In the fifth step, the reaction temperature is -40°C to room temperature.

14. The preparation method according to any one of claims 1 to 3, wherein In the fifth step, the reaction temperature is -40°C to -10°C.

15. The preparation method according to any one of claims 1 to 3, wherein R 1 It is H, C1-C6 chain alkyl or C3-C7 cycloalkyl.

16. The preparation method according to any one of claims 1 to 3, wherein R 1 It is H, C1-C3 chain alkyl or C3-C7 cycloalkyl.

17. The preparation method according to any one of claims 1 to 3, wherein R 1 It is methyl, ethyl, propyl and isopropyl.

18. The preparation method according to any one of claims 1 to 3, wherein R 2 It is halogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 alkoxy, cyano or nitro.

19. The preparation method according to any one of claims 1 to 3, wherein R 2 It is halogen, C1-C6 chain alkyl or C3-C7 cycloalkyl.

20. The preparation method according to any one of claims 1 to 3, wherein R 3 It is H or a C1-C6 straight chain or branched chain alkanoyl group.

21. The preparation method according to any one of claims 1 to 3, wherein R 3 It is H or a C1-C3 straight chain or branched chain alkanoyl group.

22. The preparation method according to any one of claims 1 to 3, wherein R 4 It is H, C1-C6 straight or branched alkanoyl, C1-C6 chain alkyl or C3-C7 cycloalkyl.

23. The preparation method according to any one of claims 1 to 3, wherein R 4 It is H, C1-C3 straight or branched alkanoyl, C1-C3 chain alkyl or C3-C7 cycloalkyl.

24. The preparation method according to any one of claims 1 to 3, wherein R 4 For H.

25. The preparation method according to any one of claims 1 to 3, wherein n is 0 or 1.

26. The preparation method according to any one of claims 1 to 3, wherein The cyclic nucleoside analogs are selected from the following compounds:

Images