CN118005694A

CN118005694A - Cyclic nucleoside analogue and preparation method and application thereof

Info

Publication number: CN118005694A
Application number: CN202410012205.5A
Authority: CN
Inventors: 周宜遂; 苏亚宁; 董英杰; 王守亮; 荆慧敏; 李梦琳; 吴虓飞; 王天照
Original assignee: Beijing Shuanghe Runchuang Technology Co ltd
Current assignee: Beijing Shuanghe Runchuang Technology Co ltd
Priority date: 2023-01-06
Filing date: 2024-01-04
Publication date: 2024-05-10

Abstract

The invention relates to a cyclic nucleoside analogue, a preparation method and application thereof. The cyclic nucleoside analog has a structure represented by the following formula (I): Wherein R ¹ is H, C ₁-C₁₅'s alkanyl, C ₃-C₁₅ cycloalkyl or C ₂-C₁₅ alkene; wherein 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

Cyclic nucleoside analogue and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a cyclic nucleoside analogue, and a preparation method and application thereof.

Background

Up to now, a number of pathogenic viruses have been found to pose a great hazard to human health, such as influenza virus, respiratory Syncytial Virus (RSV), atypical pneumonia (SARS) virus, middle East Respiratory Syndrome (MERS) virus, ebola virus, novel coronavirus (SARS-CoV-2), and the like.

However, only a few drugs such as adefovir, molnupiravir (Mo Nupi) and Paxlovid are available in clinic so far, and the Mo Nupi has relatively poor therapeutic effect. The adefovir is approved by the FDA for treatment of the age of 12 years and has a weight of at least 40 kg, and the adefovir has strong liver first pass effect and low bioavailability, so that the adefovir can only be injected for administration, and the clinical application of the adefovir is limited.

In addition, it was found that 102 viruses in 13 virus families can infect mammals, for example, coronaviruses can also infect multiple mammals such as cats, pigs and dogs. Worldwide estimated to haveCat (FCoV) carries feline coronavirus, which easily develops into infectious peritonitis (FIP) after infection, and no therapeutic drug against FIP is currently approved, which causes mortality of up to 95% or more. In addition, porcine Epidemic Diarrhea Virus (PEDV) is also a coronavirus, pigs of various ages can be infected and ill, especially suckling pigs are most seriously ill, the incidence rate of sows is greatly changed by about 15-90%, and no effective treatment method exists at present.

Therefore, the development of novel high-efficiency safe antiviral drugs for the treatment of the diseases has important clinical value and significance.

Brief description of the invention

The invention fills the blank of the prior art, provides a cyclic nucleoside analogue capable of effectively treating related diseases caused by virus infection, and provides a preparation method and application thereof in preparing medicaments for resisting RNA viruses (such as 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).

It is therefore an object of the present invention to provide a cyclic nucleoside analogue.

Another object of the present invention is to provide a process for producing a cyclic nucleoside analog.

It is a further object of the present invention to provide the use of a cyclic nucleoside analogue.

Based on the above purpose, the present invention provides the following technical solutions.

In one aspect, the present invention provides a cyclic nucleoside analog (i.e., nucleoside-3 ',5' -cyclic phosphate compound) having a structure represented by the following formula (I):

Wherein R ¹ is H, C ₁-C₁₅'s alkanyl, C ₃-C₁₅ cycloalkyl or C ₂-C₁₅ alkene;

Wherein 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.

Preferably, R ¹ is H, C ₁-C₆'s alkanyl or C ₃-C₇ cycloalkyl;

More preferably, R ¹ is H, C ₁-C₃'s alkanyl or C ₃-C₇ cycloalkyl;

Most preferably, R ¹ is C ₁-C₃ alkanyl, such as methyl, ethyl, propyl, isopropyl.

Preferably, R ² is halogen, C ₁-C₆ alkanyl, C ₃-C₇ cycloalkyl, C ₁-C₆ alkoxy, cyano or nitro;

more preferably, R ² is halogen, C ₁-C₆'s alkanyl or C ₃-C₇ cycloalkyl;

Preferably, R ³ is H or a linear or branched alkanoyl of C ₁-C₆;

more preferably, R ³ is H or a linear or branched alkanoyl of C ₁-C₃;

Preferably, R ⁴ is a linear or branched alkanoyl of H, C ₁-C₆, an alkanyl of C ₁-C₆ or a cycloalkyl of C ₃-C₇;

More preferably, R ⁴ is a linear or branched alkanoyl of H, C ₁-C₃, an alkanyl of C ₁-C₃ or a cycloalkyl of C ₃-C₇;

most preferably, R ⁴ is H;

Preferably, n is 0 or 1.

In a most preferred embodiment, the cyclic nucleoside analog is selected from the following compounds:

in another aspect, the present invention provides a method for preparing the above cyclic nucleoside analog, comprising the steps of:

The first step: reacting a compound of formula (II) with a silicon reagent in an organic solvent under the action of an alkaline reagent to produce a compound of formula (III);

and a second step of: the compound of the formula (III) prepared in the first step reacts with an acylating reagent to obtain a compound of the formula (IV);

And a third step of: removing silicon protection of the compound of the formula (IV) prepared in the second step in the presence of a desilication reagent to obtain a compound of the formula (V);

Fourth step: the compound of the formula (V) prepared in the third step reacts with the compound of the formula (VI) to generate the compound of the formula (VII);

Fifth step: the compound of the formula (VII) prepared in the fourth step generates a target compound (I) under the conditions of a halide and alkali;

Preferably, in the first step, the organic solvent is selected from one or more of DMF, CH ₂Cl₂ and THF; DMF is preferred;

Preferably, in the first step, the basic reagent is selected from one or more of imidazole, triethylamine and NaH; imidazole is preferred;

preferably, in the first step, the silicon reagent is selected from one or more of t-butyldiphenylchlorosilane, t-butyldimethylchlorosilane, and triisopropylchlorosilane; preferably triisopropylchlorosilane;

Preferably, in the second step, the acylating agent is a C ₂-C₂₀ alkyl acyl chloride, for example one or more selected from acetyl chloride, propionyl chloride, isopropyl acyl chloride, n-butyl acyl chloride and C ₅-C₂₀ alkyl acyl chloride; preferably acetyl chloride;

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

Preferably, in the fifth step, the halide is selected from one or more of N-chlorodiisopropylamine, NBS and NCS, preferably N-chlorodiisopropylamine.

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

Preferably, in the fifth step, the reaction temperature is from-40℃to room temperature, preferably from-40℃to-10 ℃.

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

As known to those skilled in the art, the modification of nucleosides with 5 '(3') -phosphates is usually carried out by the phosphodi/triester method or the phosphoramidite method. The former adopts phosphoryl chloride reagent such as phosphorus oxychloride, phosphorus trichloride and the like, has strong reaction condition and low yield, and is used less at present; the latter generally adopts phosphoramidite and nucleoside to prepare trivalent nucleotide phosphoramidite intermediate under the action of tetrazole and the like, adopts various oxidants such as hydrogen peroxide, iodine, peroxy-tert-butyl alcohol or m-chloroperoxybenzoic acid and the like to oxidize and become stable pentavalent phosphate, and the synthesis yield is obviously superior to the former, and is a method commonly adopted in the field. In the research process, the inventor tries to adopt a phosphoramidite method, but a reaction solution only obtains a very small amount of products and has no industrial value. For this reason, the inventors have tried the following methods for stereoselectively preparing 3',5' -nucleoside cyclic phosphates, and succeeded in preparing the objective compounds of the present application. The key point of the four-step reaction shown in the above synthetic route is that in the fifth step, N-chlorodiisopropylamine, NBS or NCS is selected, imidazole, 1- (cyanomethyl) imidazole, DMAP, DBU, DIPEA, potassium tert-butoxide and the like are selected as alkali, the optimal scheme is adopted to improve the purity of the target compound in the reaction system to 81%, and the yield is about 76%. In particular, by optimizing the reaction conditions in this step, the phosphorus atom stereoselectivity (Sp-product/Rp-product ratio of about 6 to 1) in the construction of the-3 ',5' -cyclic phosphate is greatly improved and the formation of alkylation by-products is reduced. In addition, most intermediates in the reaction route can be directly crystallized to obtain the intermediate, and the post-treatment is simple and convenient, so that the method is more suitable for industrial production.

In yet 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 adjuvant.

In yet another aspect, the present invention provides the use of a cyclic nucleoside analogue as described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, in the manufacture of a medicament for the treatment of a disease of viral infection.

Preferably, the virus is selected from: filoviridae, arenaviridae, coronaviridae, flaviviridae and paramyxoviridae. Specific examples include, but are 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.

The nucleoside compounds or pharmaceutical compositions of the present invention may be used alone or in combination with one or more other antiviral agents including, but not limited to: RNA polymerase inhibitors fampicin, GALIDESIVIR, MOLNUPIRAVIR, EIDD-1931;3CL protease inhibitor NIRMATRELVIR, ENSITRELVIR (S-217622), GC-376, lopinavir, nelfinavir; and others such as interferon, hydroxychloroquine, cyclosporine, ivermectin, ribavirin, penciclovir, alzvudine, praecox, and the like, or combinations thereof. The combination may provide "synergy" and "synergy" for co-formulation and simultaneous use or delivery in a combined preparation, or alternatively may be used in the form of separate preparations, such as separate tablets, pills or capsules, or separately injected via separate syringes, to achieve synergy.

The cyclic nucleoside analogues of the present application have significant inhibition of novel coronavirus replication, low cytotoxicity, high therapeutic index, high oral exposure and high oral bioavailability in animals, and are superior to other similar compounds in solubility and stability. The preparation method optimizes the reaction route, particularly the reaction condition, and obviously improves the purity and the yield of the prepared product.

Drawings

FIG. 1 is a hydrogen spectrum of compound 1 in formula (I).

FIG. 2 is a carbon spectrum of compound 1 in formula (I).

FIG. 3 is a phosphorus spectrum of compound 1 in formula (I).

FIG. 4 is a high resolution mass spectrum of compound 1 of formula (I).

FIG. 5 is a diagram showing the analysis of the crystal structure of Compound 1 in formula (I).

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the detailed description is presented herein for purposes of illustration only and is not intended to limit the invention.

Example 1

Preparation of ethyl 2- ((((2S, 4aR,6R,7 aR) -7-acetoxy-6- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -6-cyano-2-oxo-tetrahydro-4H-furo [3,2-d ] [1,3,2] dioxan-2-yl) oxy) methyl) benzoate (Compound 1)

The synthetic route is as follows:

1) Synthesis of intermediate 1

29.8G of sodium hydroxide was dissolved in 500ml of purified water, and 100g of phthalide was added thereto with stirring to react at 70℃for 5 hours. After the reaction, the mixture was concentrated to give a white solid, which was slurried with 500ml of ethyl acetate, filtered, and the cake was washed twice with ethyl acetate and air-dried at 60℃for 6 hours to give 104g of intermediate 1 in 80% yield.

2) Synthesis of intermediate 2

Preparing an iodoethane/DMSO mixed solution: 50ml of DMSO and 49g of ethyl iodide were mixed well for use.

50G of intermediate 1 and 150mL of DMSO are added into a reaction bottle, stirred and dissolved, the prepared iodoethane/DMSO mixed solution is slowly added dropwise at 20-30 ℃, the dripping is controlled to be completed in 10-20 minutes, and the reaction is carried out for 5 hours. After the reaction, 400ml of ethyl acetate and 400ml of purified water were added, the mixture was separated, and the organic phase was washed twice with purified water and twice with saturated brine, and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 42g of intermediate 2 in a yield of 81%. Ms, [ M+Na ] ⁺ 203.1

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)

¹³C 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.

3) Synthesis of intermediate 4

10G of (2R, 3R,4R, 5R) -2- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -3-fluoro-4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-carbonitrile (GS-441524), 5.84g of imidazole and 57mL of N, N-dimethylformamide were added to a reaction flask, stirred and dissolved, and 13g TIPDSCl was slowly added dropwise at 5 ℃. After the addition, the mixture was reacted at 5℃for 1 hour, extracted with 90g of ethyl acetate and 120ml of purified water, and the organic phase was collected. The aqueous phase was extracted with 45ml of ethyl acetate, the organic phases were combined, washed twice with purified water and concentrated. 55ml of normal hexane is added, the mixture is heated to reflux, stirred for 0.5 hour, cooled to 10-20 ℃ for crystallization, and stirred for 0.5 hour. Suction filtration, washing of the filter cake with n-hexane, forced air drying at 60℃for 4 hours gave 14.6g of intermediate 4 in 80% yield. Ms [ M+H ] ⁺534.3,[M+Na]⁺ 556.2

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)

¹³C 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

4) Synthesis of intermediate 6

14.6G of intermediate 4, 88mL of acetonitrile, 2.8g of acetic anhydride and 0.67g of dimethylaminopyridine are added into a reaction bottle, stirred, cooled to 0 ℃, 8.8g of triethylamine hydrofluoric acid salt is slowly added dropwise at a constant speed, and the mixture is reacted for 1 hour at the temperature of 0 ℃. After the reaction, concentrating to obtain a crude product of the intermediate 5.

88ML of tetrahydrofuran was added to the crude intermediate 5, followed by stirring, and triethylamine hydrofluoric acid salt was added dropwise at 20 ℃. After the completion of the dropping, the reaction was carried out for 8 hours. After the reaction, the mixture was concentrated, 97ml of ethyl acetate and 73ml of purified water were added to extract the mixture, and the mixture was separated. The aqueous phases were extracted twice with 97ml of ethyl acetate, respectively, and the three ethyl acetates were combined and concentrated. Adding 27ml of ethyl acetate into the crude product, heating to reflux, pulping for 0.5 hour, cooling to 5-10 ℃, stirring and crystallizing for 0.5 hour. Suction filtration, washing the filter cake once with 8ml of ethyl acetate, and forced air drying at 60℃for 4 hours gave 7.3g of intermediate 6 in 80% yield, ms: [ M+H ] ⁺334.1,[M+Na]⁺ 356.1.1.

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),5.027-5.056(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)

¹³C 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

5) Synthesis of intermediate 7

32.18G DPP and 300ml acetonitrile are added into a reaction bottle, stirred, 30ml ultra-dry pyridine is added, 21.9g intermediate 2 is slowly added dropwise, and the reaction is carried out for 1h after the dropwise addition. After the reaction, obtaining a reaction solution of the intermediate 3, carrying out untreated reaction, directly adding 30g of the intermediate 6 into the reaction solution, and heating to 50 ℃ for reaction for 2 hours. After the reaction, the mixture was concentrated and purified by column chromatography to obtain 12.6g of intermediate 7 in 25% yield. Ms [ M+H ] ⁺558.1,[M+Na]⁺ 580.1

¹H 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)

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

³¹P NMR(400MHz,DMSO)δ9.80-10.02

6) Preparation of Compound 1

56.23G of intermediate 7 and 560mL of tetrahydrofuran are weighed and added into a reaction bottle, stirring and dissolving are carried out, 56.23g of intermediate 7 and 560mL of tetrahydrofuran are weighed and added into the reaction bottle, stirring and dissolving are carried out, cooling is carried out to-40 ℃, and 34.3-g N-diisopropylamine chloride is dropwise added, and the dripping is completed. 16.87g of potassium tert-butoxide was dissolved in 280mL of tetrahydrofuran to obtain a turbid solution, and a tetrahydrofuran solution of potassium tert-butoxide was added dropwise to the reaction solution. After the dripping, the mixture is reacted for 0.5 hour at the temperature of between minus 30 and minus 40 ℃. After the reaction, the pH was adjusted to 7 with about 50ml of 15% citric acid, the layers were separated, and the organic phase was washed once with saturated brine, concentrated and purified by column chromatography to give 42.2g of Compound 1 and Compound 2, compound 1: the yield thereof was found to be 76%.(5 Mg/ml, THF-acetonitrile (1:1)), compound 2: /(I)(5 Mg/ml, THF-acetonitrile (1:1)).

Compounds of formula (I) 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)

¹³C 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

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

Compound 2:

¹H NMR(400MHz,DMSO)δ1.321-1.356(t,3H),2.160(s,3H),4.302-4.356(dd,J₁＝7.2Hz,J₂＝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)

¹³C 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

³¹P NMR(400MHz,DMSO)δ-5.78

Ms:[M+H]⁺558.1409,[M+Na]⁺580.1232

the prepared compound 1 has hydrogen spectrum, carbon spectrum, phosphorus spectrum, high resolution mass spectrum and crystal structure analysis spectrum shown in figures 1-5.

Example 2

In order to examine the effect of each condition on the final product during the preparation, the experiment was performed with reference to the synthesis of compound 1 in example 1, the conditions being shown in table 1.

Table 1: compound 1 synthesis condition optimizing screening result

Note that: all reaction temperatures in Table 1 were at room temperature. And b, detecting the purity of the main product by liquid chromatography and calculating by an area normalization method.

In order to investigate the antiviral activity, solubility and stability of the compounds 1 and 2 (the compounds 1 and 2 were prepared from example 1), the following experiments were performed.

Experimental example 1: inhibition and cytotoxicity of Compound 1 against novel coronavirus replication

1) The preparation method of the sample to be tested comprises the following steps:

Compound 1 was dissolved in DMSO to 100mM working solution, and serial dilutions were then made in DMEM medium (containing penicillin and streptomycin at a ratio of 2% in the medium, v/v, penicillin final concentration of 100U/ml, streptomycin final concentration of 0.1 mg/ml) for a total of 8 dilutions. Cytotoxicity assay compound 1 was tested at concentrations of 50. Mu.M, 25. Mu.M, 12.5. Mu.M, 6.25. Mu.M, 3.13. Mu.M, 1.56. Mu.M, 0.78. Mu.M, 0.39. Mu.M. Antiviral Activity assay, compound 1 was tested at 500nM, 250nM, 125nM, 62.5nM, 31.3nM, 15.6nM, 7.8nM, 3.9nM.

2) Test for inhibition of SARS-CoV-2 replication in Vero E6 cells by test sample (sample containing Compound 1) (IC ₅₀)

Vero E6 cells were seeded at 1X 10 ⁵ cells/well and 200. Mu.L/well in 48 well plates and incubated overnight at 37℃with 5% CO ₂ until the monolayer had grown to about 90% for use. In the P3 laboratory, 100. Mu.L of the sample to be tested and 100. Mu.L of a virus diluent (containing 100TCID50 virus) were simultaneously added to each well, and incubated at 37℃for 1h with 5% CO ₂. Setting 8 concentration gradients of the sample to be tested, and repeating 4 holes of each gradient; vehicle control groups 1%DMSO (1%DMSO+virus), cell control (without test sample and virus), and virus control (with virus only) were also established. After 1h, the virus-sample mixed medium was aspirated and washed twice with 1 XPBS and then replaced with medium containing only the sample to be tested for further culture, 300. Mu.L/well, 8 gradients were set, 4 duplicate wells per gradient, and vehicle control groups of 1%DMSO (1%DMSO+virus), cell control (no sample to be tested and virus added), and virus control (virus only) were established. After culturing at 37℃for 48h with 5% CO ₂, the cell supernatant was collected and viral RNA was extracted for Real-time PCR virus quantification, and the inhibition ratio (IC ₅₀) of the test sample to viral replication was calculated.

3) Toxicity test of the test sample (sample containing Compound 1) on Vero-E6 cells (CC ₅₀)

And (3) detecting the survival rate (24 h after adding the to-be-detected sample) of the to-be-detected sample added with different concentrations (3 compound wells per concentration) by adopting a CCK8 kit, and calculating a CC ₅₀ value (50%Cytotoxic Concentration), namely the concentration of the to-be-detected sample when toxicity is generated on 50% of Vero E6 cells.

4) Based on the cellular activity CC ₅₀ and the cytotoxicity IC ₅₀, the therapeutic index (SI) of the test sample is calculated: si=cc ₅₀/IC₅₀.

Table 2: results of inhibitory Activity of Compound 1 against novel coronaviruses

Experimental example 2: solubility and stability of Compounds 1 and 2 in different vehicles

The method for testing the solubility comprises the following steps:

1. PBS buffer (pH 7.4), simulated preprandial intestinal fluid FaSSIF (pH 6.5), simulated preprandial gastric fluid FaSSGF (pH 1.6) were prepared.

2. The standard was dissolved in acetonitrile as a control solution.

3. Compound 1 and compound 2 were each prepared as saturated solutions in PBS buffer (ph 7.4), small intestine solution FaSSIF (ph 6.5), gastric juice FaSSGF (ph 1.6) prepared as in 1, and were shaken at constant temperature of 37 ℃ for 24 hours, sampled, and filtered.

4. And adopting an external standard method, taking acetonitrile water as a mobile phase, and carrying out high performance liquid chromatography detection.

5. Peak areas were recorded and the solubility of each sample was calculated.

The test method of the solution stability comprises the following steps:

2. Appropriate amounts of Compound 1 and Compound 2 were taken and added to equal volumes of PBS buffer (pH 7.4) prepared in 1, simulated preprandial intestinal fluid FaSSIF (pH 6.5), simulated preprandial gastric fluid FaSSGF (pH 1.6).

3. Samples were taken at 1,4, 8, 24 hours, respectively, and dissolved with acetonitrile to volume.

5. Peak areas were recorded and the% residual content of each sample was calculated.

The test results are shown in tables 3-4.

Table 3 results of saturated solubility measurements for compounds 1 and 2

Table 4 results of solution stability determination of compounds 1 and 2

Solid stability test method:

1. preparation of the solution to be tested

The measuring flask (5 mL) containing Compound 1 (2.5 mg) was placed in an atmosphere of 40 ℃ + -2 ℃/75% + -5% RH (Relative Humidity), left for 4h and 24h, respectively, and then cooled naturally to room temperature. Then acetonitrile is added for dissolution and diluted to the scale, and then shaking is carried out, thus obtaining the solution to be detected of the compound 1 after being placed for 4 hours and 24 hours respectively.

The preparation method of the compound 2 in the solution to be tested which is placed for 4 hours and 24 hours is the same as that of the compound 1.

2. Measurement

Measuring the solution to be measured of the compound 1 and the compound 2 respectively, standing for 4 hours and 24 hours, taking a liquid chromatograph as a testing instrument, and calculating the content of the compound 1 or the compound 2 in each solution to be measured by adopting an external standard method.

3. Results of solid stability test

The results are shown in Table 5.

TABLE 5 results of solid stability measurements

Table 3 the solubility results show that compound 1 and compound 2 have a large difference in solubility in different biological media, especially compound 1 has a much higher solubility in simulated gastric fluid than compound 2 in fasted state, by a factor of about 200.

Table 4 solution stability results show that compound 2 is less stable than compound 1 in each of the above media, and the results of compound 1 content are greater than 90% and compound 2 content is less than 60% when the solution is kept at 37℃for 24 hours in PBS, faSSIF and FeSSIF media; the mixture is placed in a FaSSGF medium for 24 hours at a constant temperature of 37 ℃, the content of the compound 1 is 58.06 percent, the content of the compound 2 is 7.97 percent, and the stability of the compound 1 is obviously better than that of the compound 2.

Table 5 the solid stability results show that compound 1 was left for 24 hours at 40 ℃/75% rh without significant changes in content. The content of the compound 2 is reduced by about 5 percent and 30 percent respectively after the compound is placed for 4 hours and 24 hours under the same condition, and the content is obviously reduced. It can be seen that the solid stability of compound 1 is significantly better than compound 2.

From the above results, it can be seen that compound 1 has a greater improvement in solubility, liquid and solid stability than compound 2, and has the potential to become a drug and develop into a clinical candidate compound.

Experimental example 3: in vivo pharmacokinetic study of Compound 1 in different animal species

1. Pharmacokinetic evaluation in rats

Pharmacokinetic process: 24 SD rats (weight 271.5-348.3 g) were taken, each group of 12 rats, each half of which was fasted (> 12 h) before oral administration, free drinking water, and food was given 2h after administration. Animals in the intravenous group had free feeding. Compound 1 was dissolved in an appropriate solvent, and the intravenous administration formulation was prepared on the day of administration, and the oral administration formulation was prepared during the stationary phase. The vehicle for intravenous administration group was 5% DMSO+5% ethanol+40% PEG400+50% physiological saline, and the vehicle for oral administration group was 0.5% MC (w/v) aqueous solution. Compound 1 was administered by intragastric (10 mg/kg)/tail vein injection (3 mg/kg), and the intravenous administration groups were administered before, 0.083, 0.25, 0.5, 1,2,4,6, 8, and 24 hours after administration, respectively; the oral administration groups were respectively before administration, and 0.25, 0.5, 1,2,3,4, 6,8 and 24 hours after administration, about 0.5ml of orbital blood was taken by a micropipette, and fresh whole blood samples were placed in K2EDTA anticoagulation tubes according to whole blood: stabilizer (0.4M DDV in isopropanol) =10: 0.25 (v/v) mixing the materials in a gentle reverse manner (blood sampling tube is placed in wet ice in the whole process), centrifuging for 10min (4 ℃ C., 2000 g) to obtain plasma, and cooling in a refrigerator at-80 ℃ to be tested. And (3) quantitatively taking a plasma sample by adopting an organic solvent precipitation method, adding an acetonitrile precipitant into the plasma sample, fully and uniformly vortex, centrifuging for 10min (4000 rpm,4 ℃), taking out supernatant, quantitatively testing the concentration of the raw material 1 in the plasma by adopting an LC-MS method, and calculating pharmacokinetic parameters.

Standard curve: taking 8 EP tubes of 1.5ml, adding 0.1, 0.2, 0.5, 1, 2, 5, 9 and 10 mu l of stock solution of raw material 1 respectively, supplementing the working solvent to 450 mu l, uniformly mixing, adding 50 mu l of rat blank plasma respectively, swirling for 5min, centrifuging for 5min at 12000rpm/min, taking supernatant, centrifuging for 5min again at 12000rpm/min, taking supernatant in the EP tube of 1.5ml, and obtaining 10, 20, 50, 100, 200, 500, 900, 1000ng/ml of raw material 1 standard curve sample (containing internal standard dexamethasone, 5.00 ng/ml).

Sample detection treatment: the pharmacokinetic process resulted in 10. Mu.l of the drug-containing plasma was placed in a 1.1ml of a row of tubes with 200. Mu.l of an internal standard working solution (5.00 ng/ml Dexamethasone in 100% acetonitrile), vortexed for 10min,4000rpm,4℃and centrifuged for 10min, 20. Mu.l of the supernatant was taken up in 1.1ml of row of tubes, 180. Mu.l of diluent II (acetonitrile: deionized water=1:5) was added and the samples were mixed using a vortexing apparatus. 180. Mu.l of the mixed sample solution was transferred to a 96-well plate, and 5. Mu.l of the sample was injected to the LC-MS system for detection.

LC-MS detection: chromatographic column, YMC-Triart C (50×2.1mm, S-3 μm,12nm, shimadzu corporation), column temperature at ambient temperature, sample injection amount 5 μl, mobile phase acetonitrile-deionized water, flow rate 0.6ml/min, gradient elution; an ion source, TRIPLE QUAD 5500 mass spectrometer electrospray ion source ESI; detection means, multiplex Reaction Monitoring (MRM); scanning mode, positive ion mode; ESI spray voltage 5500V, ion source temperature 500 ℃, spray gas 50psi, auxiliary heating gas 50psi, air curtain gas 30psi.

2. Pharmacokinetic evaluation in dogs

Pharmacokinetic process: 6 beagle dogs (weight 6.8-8.7 kg) are taken, the male and female animals are half, 6 animals are shared by oral administration and intravenous administration groups, and the metabolic clearance time is not less than 7 days. The oral dosing group had fasted (> 12 h) prior to the experiment, was free to drink water and was given food 2h after dosing. Animals in the intravenous group had free feeding. Compound 1 was dissolved in an appropriate solvent, and the intravenous administration formulation was prepared on the day of administration, and the oral administration formulation was prepared during the stationary phase. The solvent of intravenous administration group is 5% DMSO+5% ethanol

The vehicle for the orally administered group was 0.5% MC (w/v) in water, +40% PEG400+50% physiological saline. Compound 1 was administered orally (3 mg/kg)/intravenously (1 mg/kg) and the intravenous groups were administered for 0.033, 0.083, 0.25, 0.5, 1,2, 4, 8, 24 hours before, after, respectively; the oral administration groups were orally administered before administration, and about 1.0ml of blood was collected by peripheral venipuncture at 0.083, 0.25, 0.5, 1,2, 4,6, 8, and 24 hours after administration, and fresh whole blood samples were placed in K2EDTA anticoagulation tubes according to whole blood: stabilizer (0.4M DDV in isopropanol) =10: 0.25 (v/v) mixing the materials in a gentle reverse manner (blood sampling tube is placed in wet ice in the whole process), centrifuging for 10min (4 ℃ C., 2000 g) to obtain plasma, and cooling in a refrigerator at-80 ℃ to be tested. Adopting an organic solvent precipitation method, quantitatively taking a plasma sample, adding an acetonitrile precipitant, fully and uniformly vortex, centrifuging for 10min (4000 rpm,4 ℃), taking out supernatant, quantitatively analyzing by adopting an LC-MS method, and calculating pharmacokinetic parameters.

Standard curve: taking 8 EP tubes of 1.5ml, adding 0.1, 0.2, 0.5, 1,2, 5, 9 and 10 mu l of stock solution of raw material 1 respectively, supplementing the working solvent to 450 mu l, uniformly mixing, adding 50 mu l of canine blank plasma respectively, swirling 5min, centrifuging at 12000rpm/min for 5min, taking supernatant, centrifuging at 12000rpm/min for 5min again, taking supernatant in the EP tube of 1.5ml, and obtaining 10, 20, 50, 100, 200, 500, 900, 1000ng/ml of standard curve sample of raw material 1 (containing internal standard dexamethasone, 5.00 ng/ml).

Sample detection treatment: the pharmacokinetic process resulted in 10. Mu.l of the drug-containing plasma was placed in a 1.1ml of a serial tube with 200. Mu.l of an internal standard working solution (5.00 ng/ml dexamethasone in 100% acetonitrile), vortexed for 10min,4000rpm, and centrifuged for 10min at 4℃to give 20. Mu.l of the supernatant to 1.1ml of serial tube, and 180. Mu.l of diluent II (acetonitrile: deionized water=1:5) was added to mix the samples using a vortexing apparatus. 180. Mu.l of the mixed sample solution was transferred to a 96-well plate, and 5. Mu.l of the sample was injected to the LC-MS system for detection.

3. Pharmacokinetic evaluation in vivo in monkeys

Pharmacokinetic process: 6 male cynomolgus monkeys (body weight: 3.80.+ -. 0.62 kg) were taken, 3 animals per group. The oral administration group had a fast of 16-17 hours before the experiment, was free to drink water, and was given food 2 hours after the administration. Animals in the intravenous group had free feeding. Compound 1 was dissolved in an appropriate solvent, and the intravenous administration formulation was prepared on the day of administration, and the oral administration formulation was prepared during the stationary phase. The vehicle for intravenous administration group was 5% DMSO+5% ethanol+40% PEG400+50% physiological saline, and the vehicle for oral administration group was 0.5% MC (w/v) aqueous solution. The cynomolgus monkey oral (30 mg/kg)/intravenous administration (4 mg/kg) groups were subjected to vein blood sampling of about 1.0ml for forelimb at 0.033 (intravenous administration only) group, 0.083, 0.25, 0.5, 1,2, 4, 6, 8, 24 hours after administration, respectively, fresh whole blood samples were placed in K2EDTA anticoagulation tubes according to whole blood: stabilizer (0.4 MDDV in isopropanol) =10: 0.25 (v/v) mixing gently upside down (blood sampling tube is placed in wet ice in the whole course), centrifuging for 10min (4 ℃ C., 3000 rpm) to obtain plasma, and cooling in a refrigerator at-80 ℃ to be tested. Adopting an organic solvent precipitation method, quantitatively taking a plasma sample, adding a precipitator (100 ng/ml acetonitrile solution of toluene sulfobutyramide), fully and uniformly vortex, centrifuging for 15min (4000 rpm,4 ℃), taking out supernatant, quantitatively analyzing by adopting an LC-MS method, and calculating pharmacokinetic parameters.

Standard curve: stock 1 was dissolved in DMSO to prepare a stock solution of 1.00 mg/ml. Stock solutions were diluted with methanol to a series of standard curve working solutions. Mu.l of working solution of each concentration standard curve of the analyte is respectively added into 50 mu.l of monkey blank plasma, and the working solution is vortexed for 1.0min to prepare 1 standard curve samples of raw materials with final concentrations of 1.00, 2.00, 5.00, 20.0, 100, 500, 1000 and 2000 ng/ml.

Sample detection treatment: 50 μl of the medicated plasma obtained in the pharmacokinetic process was taken, 200 μl of a precipitant (100 ng/ml acetonitrile solution of toluene sulfobutyramide) was added, and the mixture was vortexed at room temperature for 2min to thoroughly mix, 4000rpm,4℃and centrifuged for 15min, and the supernatant was taken to obtain a sample for detection by an LC-MS system.

LC-MS detection: a chromatographic column Kinetex C18 (30X 2.1mm,2.6 μm, phenomenex, U.S.A.), column temperature at room temperature, sample injection amount 3. Mu.L, mobile phase 0.05% aqueous solution of FA and 5mM NH4 Ac-80% CAN aqueous solution, flow rate 0.6ml/min, gradient elution; ion source TRIPLE QUADTM + (SCIEX) mass spectrometer electrospray ion source ESI; detection means, multiplex Reaction Monitoring (MRM); scanning mode, positive ion mode; ESI spray voltage 5500V, ion source temperature 500 ℃, spray gas 50psi, auxiliary heating gas 50psi, air curtain gas 35psi.

The pharmacokinetic results are detailed in table 6:

TABLE 6 results of pharmacokinetic parameters of Compound 1 metabolites at different species

F (%) = (AUC _{Final result}/oral dose level of starting material 1 after oral administration of compound 1)/(AUC _{Final result}/intravenous dose level of starting material 1 after intravenous administration of compound 1) ×100%.

Claims

1. A cyclic nucleoside analog or a pharmaceutically acceptable salt thereof, wherein the cyclic nucleoside analog has a structure represented by the following formula (I):

Wherein 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.

2. The cyclic nucleoside analog or pharmaceutically acceptable salt thereof according to claim 1, wherein R ¹ is H, C ₁-C₆'s alkanyl or C ₃-C₇ cycloalkyl; more preferably, R ¹ is H, C ₁-C₃'s alkanyl or C ₃-C₇ cycloalkyl; most preferably, R ¹ is C ₁-C₃ alkanyl, such as methyl, ethyl, propyl, isopropyl.

3. The cyclic nucleoside analog or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein R ² is halogen, alkanyl of C ₁-C₆, C ₃-C₇ cycloalkyl, alkoxy of C ₁-C₆, cyano or nitro; more preferably, R ² is halogen, C ₁-C₆'s alkanyl or C ₃-C₇ cycloalkyl.

4. The cyclic nucleoside analog or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein R ³ is H or C ₁-C₆ linear or branched alkanoyl; more preferably, R ³ is H or a linear or branched alkanoyl of C ₁-C₃.

5. The cyclic nucleoside analog or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein R ⁴ is a linear or branched alkanoyl of H, C ₁-C₆, an alkanyl of C ₁-C₆ or a C ₃-C₇ cycloalkyl;

most preferably, R ⁴ is H;

Preferably, n is 0 or 1.

6. The cyclic nucleoside analogue or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the cyclic nucleoside analogue is selected from the following compounds:

7. a process for the preparation of a cyclic nucleoside analogue as claimed in any one of claims 1 to 6, which comprises the steps of:

Fifth step: the compound of the formula (VII) prepared in the fourth step is formed into a target compound (I) under the conditions of a halide and a base.

8. The preparation method according to claim 7, wherein, in the first step, the organic solvent is selected from one or more of DMF, CH ₂Cl₂, and THF; DMF is preferred;

preferably, in the first step, the alkaline reagent is selected from one or more of imidazole, triethylamine or NaH; imidazole is preferred;

Preferably, in the second step, the acylating agent is a C ₂-C₂₀ alkyl acyl chloride, preferably one or more selected from acetyl chloride, propionyl chloride, isopropyl acyl chloride, n-butyl acyl chloride and C ₅-C₂₀ alkyl acyl chloride; preferably acetyl chloride;

preferably, in the third step, the desilication agent is selected from one or more of triethylamine trihydrofluoride and TBAF, preferably triethylamine trihydrofluoride;

Preferably, in the fifth step, the halide is selected from one or more of N-chlorodiisopropylamine, NBS or NCS, preferably N-chlorodiisopropylamine;

preferably, in the fifth step, the base is selected from one or more of imidazole, 1- (cyanomethyl) imidazole, DMAP, DBU, DIPEA and potassium t-butoxide, preferably potassium t-butoxide;

Preferably, in the fifth step, the reaction solvent is selected from acetonitrile and THF, preferably THF;

9. A pharmaceutical composition comprising the cyclic nucleoside analogue of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

10. Use of a cyclic nucleoside analogue or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 9 in the manufacture of a medicament for the treatment of a viral infection;

Preferably, the virus is selected from: filoviridae, arenaviridae, coronaviridae, flaviviridae, and paramyxoviridae; specific examples include, but are 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.