CN114181258B - Nucleoside compounds for antiviral treatment and application thereof - Google Patents

Abstract

The invention discloses a nucleoside compound for antiviral treatment and application thereof, the structure of the nucleoside analogue is shown as a formula I, the compound can inhibit coronavirus, porcine epidemic diarrhea virus and avian influenza virus, and can be used for treating and preventing diseases caused by various viruses of human and animals, such as acute pneumonia caused by new coronavirus and epidemic diarrhea caused by porcine epidemic diarrhea virus, and the compound has remarkable and better effect and better safety.

Description

Nucleoside compounds for antiviral treatment and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and in particular relates to a nucleoside compound for antiviral treatment and application thereof.

Background

Human coronaviruses were isolated by UK scientists the earliest of the 20 th century, and 7 new types of coronaviruses 2019 (2019-nCoV, causing the new coronavirus pneumonia COVID-19) were currently known as 7 th coronaviruses that could infect humans, the remaining 6 types being HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing middle east respiratory syndrome), respectively. Of these, 4 only caused common cold symptoms, the other 3 were highly pathogenic pneumonia-associated coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and novel coronavirus (SARS-CoV-2), respectively. Coronaviruses are enveloped RNA viruses and are classified into the alpha, beta, gamma, delta 4 genera. The genome is a single-stranded positive-strand RNA, is about 30kb long, wherein 20kb is a non-coding region, and 10kb codes for viral proteins, including non-structural proteins (such as some replication-related enzymes) and structural proteins, including spike (S), envelope (E), membrane (M) and nucleocapsid (N).

The COVID-19 infected person mainly shows symptoms such as muscle soreness, general debilitation, fever, chest distress, dyspnea and the like, and can cause acute respiratory distress syndrome, multiple organ failure and the like under serious conditions; mortality in these cases has been about 3.5% of all cases of infection worldwide; although the mortality rate is far lower than severe acute respiratory syndrome and middle east respiratory syndrome, both are 9.14% and 34.4%, respectively; but SARS-CoV-2 is highly infectious and has a long latency period that can be transmitted by asymptomatic infected persons, potentially triggering supertransmission. SARS-CoV-2 belongs to a large class of forward single-stranded RNA viruses of coronaviruses, and the genome has a total length of 26000-32000 bases; according to genomic sequence analysis, SARS-CoV-2 is about 89% identical to bats SARS-like CoVZXC21, 82% similar to human SARS-CoV, has the same major structural and molecular features as other coronaviruses, including spike protein S, envelope protein E, membrane protein M and nucleocapsid protein N of structural proteins responsible for viral envelope formation, stability and interaction with the RNA genome. The virus interacts with host cells under the mediation of spike protein S through the receptor angiotensin converting enzyme 2 (angiotensin converting enzyme 2, ace 2); spike protein S is also dependent on another host factor, transmembrane protease serine 2 (transmembrane serine protease, TMPRSS 2), activation of TMPRSS2 can induce fusion of the virus with the cell membrane. Extensive studies have revealed the origin of the virus, the mechanism of transmission and the clinical manifestation of the infection, however, the pathogenesis of SARS-CoV2 and its interaction with the individual's immune system is poorly understood. Among the infected patients with covd-19, some individuals present asymptomatic, while some present with serious complications, such as interstitial pneumonia and respiratory failure. In order to develop new therapeutic regimens and safe and reliable viral vaccines, it is necessary to understand the pathogenesis of SARS-CoV-2 and the relationship of interactions with the immune system. SARS-CoV-2 is the seventh member of the coronavirus family that infects humans. Four human coronaviruses (HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU 1) are capable of causing a broad range of upper respiratory tract infections, whereas SARS-CoV and MERSCoV cause atypical pneumonia;

SARS-CoV and MERS-CoV enter the cell via endocytic pathways, using surface spike protein S to bind to ciliated bronchial epithelial cell ACE2 receptor and dipeptidyl peptidase 4 (dipeptidyl peptidase, dpp 4) receptor of type ii lung cells, respectively. Spike protein S is the most important surface membrane protein of SARS-CoV-2, consists of two subunits S1 and S2, and is cleaved by the receptor TMPRSS 2. The receptor binding domain in the S1 subunit mediates attachment and cellular tropism of the virus to the host cell, while the S2 subunit contains the essential elements required for membrane fusion, with the help of two tandem domain heptad repeat sequences 1 and 2, the virus fuses with the cell membrane. After the virus enters the host cell, viral RNA is exposed. Viral genomic RNA released in the cytoplasm will translate two different polyproteins, protein phosphatase 1a and protein phosphatase 1b. These polyproteins encode nonstructural proteins that form a replication transcription complex (replication and transcription complex, RTC) in double membrane vesicles. RTC replication proceeds serially and forms a collection of subgenomic RNAs responsible for encoding accessory and structural proteins. The newly formed viral genomic RNA, together with nucleocapsid proteins, envelope glycoproteins, endoplasmic reticulum and Golgi apparatus, aggregate to form viral particle buds, which then attach to the plasma membrane resulting in viral release.

Acute SARS-CoV2 infection is very similar to seasonal influenza, with the most common symptoms being fever, cough, shortness of breath, and muscle soreness, with the later stages possibly developing pneumonia, acute respiratory distress, renal failure, and the like. The lung is the main site of SARS-CoV-2 infection; studies have shown that chest CT in patients with SARS-CoV2 typically shows bilateral, erised, glassy turbidity lesions in the posterior and peripheral lungs, a major feature of COVID-19. Biopsy sample pathology studies of lung, liver and heart in patients dying from covd-19 indicate that lung is the major affected tissue, and its pathological changes include type ii lung cell proliferation, alveolar epithelial cell damage, hyaline membrane and diffuse alveolar damage. ACE2 is the primary receptor for viral spike protein S, providing an entry point for the capture of sarkov 2 and infection of human cells. SARS-CoV2 has been reported to enter cells through receptors such as DC-SIGN (CD 209), CD147 and L-SIGN (CD 209L), and thus drugs that interfere with the interaction of spike protein S with ACE2, CD147, DC-SIGN or L-SIGN, or their gene expression, may inhibit viral invasion.

But both SARS in 2003, MERS in 2012 and C0VID-19 (disease caused by novel coronaviruses) pose serious threats and impacts on people's life health worldwide. Severe acute respiratory syndrome is an acute respiratory infectious disease caused by SARS coronavirus (SARS-CoV), and is mainly clinically manifested as: the incubation period is 1-16 days, usually 3-5 days. The disease is urgent, the infectivity is strong, the fever is the first symptom, the body temperature is often over 38 ℃, irregular heat or relaxation heat, heat retention and the like are carried out, and the heat is Cheng Duowei to 2 weeks; with headache, muscle soreness, systemic debilitation and diarrhea. Dry cough and little phlegm appear after 3-7 days of onset, and even blood streak phlegm is not obvious in lung signs. The illness state reaches the peak in 10-14 days, the infection and poisoning symptoms such as fever, hypodynamia and the like are aggravated, frequent cough, shortness of breath and dyspnea occur, and asthma and palpitation are forced to lie in bed for rest when the patient moves slightly. This period is susceptible to secondary infections of the respiratory tract. After the course of the disease is 2-3 weeks, fever is gradually reduced, and other symptoms and signs are relieved or even disappear. The absorption and recovery of the pulmonary inflammatory changes are slow, and the pulmonary inflammatory changes still can be completely absorbed and recovered to be normal after the normal body temperature is about 2 weeks. The clinical symptoms of the light patients are light. The serious patients are easy to suffer from respiratory distress syndrome. The disease condition of the children patient is lighter than that of the adult. Few patients do not experience fever as a primary symptom, especially those with recent surgical history or underlying disease. In 2003, published data by the world health organization shows that the death rate of human SARS is approximately 11% worldwide due to the number of SARS deaths 919. Middle east respiratory syndrome (Middle East respiratorysyndrome, MERS) is a serious respiratory disease caused by MERS-CoV, and the main symptoms include fever, cough, dyspnea and the like, and digestive system symptoms such as diarrhea, nausea, vomiting and the like can also occur, which can lead to serious complications such as pneumonia and renal failure. From 2012 to date MERS-CoV has resulted in over 2468 worldwide infections, over 851 deaths, with MERS patient mortality around 38% based on World Health Organization (WHO) statistics (431/1139). According to World Health Organization (WHO) statistics, MERS patient mortality was around 38% (431/1139).

The disease caused by the novel coronavirus (C0 VID-19) is caused by infection with the novel coronavirus (SARS-CoV-2). The general symptoms are: fever, hypodynamia, dry cough, and gradual dyspnea; some patients may have slight symptoms and may even have no obvious fever. The serious symptoms are as follows: acute respiratory distress syndrome, septic shock, metabolic acidosis that is difficult to correct, and clotting dysfunction. SARS-CoV-2 virus positivity can be detected by SARS-CoV-2 infected patients on nasopharyngeal swab, sputum, respiratory tract secretion, blood, feces and the like, and chest imaging of the patients shows that multiple small patches and interstitial changes appear at early stage, and the lung external zone is obvious. Further, it develops a double lung multiple abrasion glass shadow and an infiltration shadow, and severe cases can cause lung excessive changes and less pleural effusion.

According to the new coronavirus infection pneumonia diagnosis and treatment scheme (trial fifth edition), no specific antiviral drug exists for treating the new coronavirus infection patients clinically at present, and isolation and support treatment are generally adopted for the infected patients, but interferon aerosol inhalation and lopinavir/ritonavir can be tried out. Ongoing clinical trial drugs against 2019-ncovi include either chinese patent drugs (e.g., bicaluri) or anti-other viral drugs (e.g., adefovir, arbidol, darunavir, etc.), which have inhibitory effects on 2019-ncovi in vitro cell assays. Among them, the Remdesivir (Remdesivir) structure is shown below, which has shown a better therapeutic effect in 1 new patients infected with coronaviruses in the United states. In China formally introduced in month 2 of 2020, a new type of phase III clinical trial of coronavirus infection is currently underway.

Remdesivir (Remdesivir) is a novel adenosine analog monophosphate amide prodrug that is converted into a pharmacologically active form of ribonucleophos triphosphate (NTP) in cells, inhibits viral RNA polymerase, and inhibits viral nucleic acid synthesis. The phase III clinical study of ebola virus infection treatment is currently underway. It also has been found to have broad-spectrum antiviral activity against various RNA viruses such as respiratory syncytial virus, dove's virus, lassa fever virus, MERS-CoV, SARS, nipa virus, etc. in vitro.

The two hydroxyl groups in the active molecule group of the medicine are made into a phosphate structure, so that the metabolic stability and the safety of the medicine can be further improved while the activity of the medicine is ensured, the treatment dosage is reduced, and the pharmacological activity of the medicine is not influenced.

At present, no research report on phosphate compounds of the Ruidexivir compound exists, and the phosphate compounds of the Ruidexivir compound are researched in view of important roles in controlling novel coronavirus epidemic situations, so that the metabolic stability and safety of the Ruidexivir are further improved, and the Ruidexivir compound has important clinical significance.

Porcine epidemic diarrhea is an acute intestinal disease caused by porcine epidemic diarrhea virus PEDV, and mainly causes severe watery diarrhea, vomiting and dehydration of piglets, and the mortality rate of 7-day-old piglets infected with the virus is as high as that of piglets after the infection of the virus

100%. PEDV belongs to a single-stranded positive strand RNA virus, which belongs to a member of the genus alphacoronavirus of the family coronaviridae. Since 2010, PEDV variant strains with higher pathogenicity and mortality are widely popular in China, and the existing prevention and control means are difficult to deal with the threat caused by PEDV, so that huge losses are caused to the aquaculture industry in China.

At present, no specific medicine is available for porcine epidemic diarrhea, and vaccine immunization is the most main prevention and control measure, but for newborn piglets, vaccine immunization has some problems. The absorption of milk from the sow containing PEDV antibodies by newborn piglets affects the immune protection effect induced by the vaccine, in addition, PEDV vaccines mainly play a role through mucosal immunity, but the mucosal immune system of the piglets is not yet developed completely, so that the immune effect is greatly reduced. At present, besides vaccine immunization, antibiotics, hormone and traditional Chinese medicine compounds are used for relieving diarrhea symptoms of piglets, but no nucleotide analogues are reported at present, so that research on corresponding medicines has important clinical significance for treating porcine epidemic diarrhea.

Disclosure of Invention

The invention aims to: the invention provides a nucleoside compound for antiviral treatment and application thereof.

The technical scheme is as follows: the nucleoside compounds for antiviral treatment of the present invention,

wherein:

a is P;

R ¹ and R is ² Independently selected from H, -OR ³ 、-C(＝O)R ³ Or (b)Each R ³ Independently H, (C) ₁ -C ₈ ) An alkyl group.

Further, the nucleoside compound for antiviral treatment, A is P;

R ¹ and R is ² Independently selected from H, -OR ³ 、-C(＝O)R ³ Or (b)

Each R ³ Independently is H, isopropyl.

The nucleoside compounds for antiviral treatment of the present invention are selected from:

the invention also provides a pharmaceutical composition which contains the nucleoside compound for antiviral treatment or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Further, the virus is 2019 novel coronavirus COVID-19, HCoV-229E coronavirus, HCoV-OC43 coronavirus, SARS coronavirus, MERS coronavirus, porcine epidemic diarrhea virus PEDV, and avian influenza virus.

The application of the nucleoside compound for antiviral treatment in preparing a medicament for treating or preventing coronavirus, porcine epidemic diarrhea virus PEDV or avian influenza virus.

The beneficial effects are that: compared with the existing medicines, the compound disclosed by the invention has a novel structure, has the nucleoside compounds for inhibiting coronaviruses, porcine epidemic diarrhea viruses and avian influenza viruses, can be used for treating and preventing diseases caused by various viruses of human beings and animals, such as acute pneumonia caused by new coronaviruses and epidemic diarrhea caused by porcine epidemic diarrhea viruses, and has the advantages of remarkable and better effect and better safety.

Drawings

FIG. 1 shows the results of in vivo toxicity experiments of the compound BALB/C;

FIG. 2 toxicity of drugs to Vero cells;

FIG. 3 fluorescent quantitation of the activity of drugs in inhibiting porcine epidemic diarrhea virus infection on Vero cells.

Detailed Description

1. Synthesis of Compounds

The preparation of the compounds of the present invention may be carried out as follows:

example 1

Step 1

NaOH (985 mg,24.63 mmol) was weighed, 13mL of water was added to make a solution and placed under ice bath and stirred for 5 minutes, then bis (N, N-dimethylamino) phosphonyl chloride (2 g,11.73 mmol) was added over 8 minutes, then stirred to room temperature until the reaction was complete. Then spin-drying the solvent, adding ethanol for dissolution, and concentrating the filtrate after suction filtration to obtain the compound 1.

Step 2

Compound 2 (100 mg,0.34 mmol) and compound 1 (60 mg,0.34 mmol) were weighed, put into a eggplant-shaped bottle, dissolved by adding 3mL of N-methylpyrrolidone, reacted overnight to completion in an oil bath at 80 ℃, and after concentrating the solvent, compound I (96 mg, yield 80%) was obtained by high pressure.

¹ H NMR(400MHz,DMSO-d ₆ )δ8.01(d,J＝52.0Hz,3H),6.93(d,J＝7.6Hz,2H),5.24(s,1H),4.88–4.61(m,2H),4.61–4.43(m,1H),4.26(q,J＝32.0,27.0Hz,2H),3.31(s,1H).

MS:m/z 354.1(M+H).

Example 2

Step 1

NaOH (247 mg,6.16 mmol) was weighed, 3.5mL of water was added to prepare a solution and placed under ice bath stirring and cooling for 5 minutes, then bis (N, N-dimethylamino) phosphonyl chloride (500 mg,2.93 mmol) was added over 8 minutes, then the reaction was stirred to completion at room temperature. Then spin-drying the solvent, adding ethanol for dissolution, and concentrating the filtrate after suction filtration to obtain the compound 1.

Step 2

Compound 3 (200 mg,0.33 mmol) and compound 1 (116 mg,0.66 mmol) were weighed, put into a eggplant-shaped bottle, dissolved by adding 4mL of N-methylpyrrolidone, reacted overnight to completion in an oil bath at 80 ℃, and after concentrating the solvent, compound II (171 mg, yield 78%) was obtained by high pressure.

¹ H NMR(400MHz,DMSO-d ₆ )δ8.19(s,2H),7.97(d,J＝7.1Hz,1H),7.32(t,J＝8.1Hz,2H),7.17(t,J＝7.4Hz,3H),7.05–6.79(m,2H),6.04(dd,J＝23.2,10.7Hz,1H),5.35(q,J＝5.6Hz,1H),4.95–4.84(m,1H),4.54–4.39(m,1H),4.38–4.21(m,2H),4.20–

4.08(m,1H),3.95(dd,J＝11.3,5.9Hz,1H),3.89–3.76(m,2H),1.48–1.33(m,1H),1.33–1.10(m,7H),0.79(t,J＝7.4Hz,6H).

MS:m/z 665.2(M+H).

Example 3

Step 1

Compound I (2057 mg,5.81 mmol) was weighed into a eggplant-shaped bottle, then 24mL of triethylamine was added to dissolve the substrate completely, and then 4-dimethylaminopyridine (36 mg,0.29 mmol) was added and the reaction system was stirred under ice for 5-10 minutes. Isobutyric anhydride (1011 mg,6.39 mmol) was then added slowly. The reaction mixture was stirred until the reaction was complete, at room temperature. The solvent was concentrated by evaporation under reduced pressure, the residue was added to water, extracted with ethyl acetate and saturated sodium hydrogencarbonate, the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (dichloromethane-methanol) to give compound iii (1536 mg, yield 62.5%).

¹ H NMR(400MHz,DMSO-d ₆ )δ8.01(d,J＝52.0Hz,3H),6.93(d,J＝7.6Hz,2H),5.24(s,1H),4.88–4.61(m,2H),4.61–4.43(m,1H),4.26(q,J＝32.0,27.0Hz,2H),2.58–2.52(m,1H),3.31(s,1H),1.07(d,J＝7.0Hz,6H).

MS:m/z 424.1(M+H).

Example 4

Step 1

Compound II (500 mg,0.75 mmol) was weighed into a eggplant-shaped bottle, 3mL of acetonitrile was added to dissolve completely, phenol (71 mg,0.75 mmol), CDI (122 mg,0.75 mmol) and N, N-dimethylaniline (182 mg,1.50 mmol) were added in this order, and after nitrogen protection, the mixture was stirred at room temperature for 12 hours. After completion of the reaction, the solvent was removed under reduced pressure to give a crude product, and the obtained crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate) to give compound IV (495 mg, yield 89%).

¹ H NMR(400MHz,DMSO-d ₆ )δ8.38(s,1H),8.00(s,2H),7.31(t,J＝8.1Hz,2H),7.28–7.21(m,2H),7.17(d,J＝7.9Hz,3H),7.00(s,1H),6.92(d,J＝5.1Hz,2H),6.13–6.08(m,1H),5.37(s,1H),4.93(d,J＝9.5Hz,1H),4.48(s,1H),4.28(s,1H),4.16(s,2H),3.94(t,J＝7.7Hz,2H),3.86–3.79(m,2H),1.48–1.33(m,1H),1.33–1.10(m,7H),0.78(d,J＝8.3Hz,6H).

MS:m/z 741.1(M+H).

Specific compounds synthesized and their names are shown in the following table.

2. Anti-coronavirus Activity test

Test materials and methods:

1. virus strain: coronaviruses HCoV-OC43, HCoV-229E were stored at-80 ℃.

2. Sample treatment: samples were made up with DMSO as stock solution and then 3-fold diluted with culture medium, 8 dilutions each.

3. The testing method comprises the following steps: h460 cells were inoculated into 96-well plates and placed in 5% CO ₂ Culturing at 37 ℃. After 24 hours, coronavirus infected with about 100TCID50 was added with a maintenance solution containing samples of different dilutions and positive control drugs, and cell control wells and virus control wells were simultaneously set with 5% CO ₂ Culturing at 37 ℃. Observing cytopathic degrees (CPE) of each group when the virus control group has a pathological degree (CPE) of 4+, and calculating half-Toxic Concentration (TC) of the sample on cells by using a Reed-Muench method ₅₀ ) And half-maximal Inhibitory Concentration (IC) against virus ₅₀ )。

The cell results of the compounds are shown in the following table:

note that:

(1) In the table "-" indicates that the sample has no antiviral activity at the maximum non-toxic dose.

(2)TC ₅₀ : half of the toxic concentration of the drug; IC (integrated circuit) ₅₀ : half inhibitory concentration of drug against virus; SI: selection index, si=tc ₅₀ /IC ₅₀ 。

The cell activity result shows that the compound II is basically equivalent to Remdesivir, but has better safety.

3. Toxicity and activity test for resisting porcine epidemic diarrhea virus

Test materials and methods:

1. virus strain: porcine epidemic diarrhea virus PEDV-AH2012, stored at-80 ℃.

2. Sample treatment: the samples were made up into mother liquor with DMSO and diluted to the corresponding concentrations with DMEM broth.

3. The testing method comprises the following steps:

(1) Test for detecting toxicity of medicine to Vero cell

Vero cells were seeded in 96-well cell plates at 37℃with 5% CO ₂ Culturing under the condition until the cells grow to form a monolayer. Discarding culture medium, washing with PBS for 3 times, adding medicine diluted with DMEM at different concentrations, repeating each dilution at 37deg.C and 5% CO ₂ Culturing under the condition for 48h, discarding culture solution, adding DMEM culture solution (100 ul/well) containing 10% CCK-8, and culturing at 37deg.C with 5% CO ₂ Incubation was continued for 2h in the incubator, absorbance (450 nm) was measured with an enzyme-labeled instrument, and cell activities were calculated for 48h of treatment with different concentrations of drug.

The toxicity of the drug on Vero cells is shown in figure 2. The cytotoxicity test results show that the medicine has no effect on the cell activity at the high concentration of 50 mu M.

(2) Fluorescent quantitative determination of the Activity of the drug for inhibiting porcine epidemic diarrhea Virus infection on Vero cells were inoculated into 24-well plates and placed in 5% CO ₂ Culturing at 37deg.C, inoculating porcine epidemic diarrhea virus AH2012 strain (MOI=0.1) into Vero cells after cells are adhered to form a monolayer, and culturing at 37deg.C with 5% CO ₂ After incubation in incubator for 1 hour, the incubation was performed three times with PBS, and then a maintenance solution containing samples of different concentrations was added, and the incubation was performed at 37℃with 5% CO ₂ After 24h incubation in the incubator, cell samples were collected, RNA was extracted and reverse transcribed into cDNA, and the activity of the samples in inhibiting porcine epidemic diarrhea virus infection was determined by fluorescent quantitative detection on Vero (fig. 3). The fluorescent quantitative results show that the drug significantly inhibits the proliferation of PEDV in Vero cells and exhibits dose dependency.

4. Results of drug stability in liver microsomes of different species

Taking out liver microsomes (20 mg protein/mL) from a refrigerator at-80 ℃, placing the liver microsomes on a water bath constant temperature oscillator at 37 ℃ for pre-incubation for 3min, and melting for later use. According to the ratio of "constitution of the experimental incubation system", a mixed solution of the incubation system (without beta-NADPH) was prepared. And (3) preparing a proper amount of the compound into a 10mM stock solution by using DMSO, and diluting the stock solution into a 100 mu M working solution of the test compound by using 50% acetonitrile-water for later use. Control group (without β -NADPH): respectively taking 25 mu L of PB solution into 75 mu L of the mixed solution of the incubation system, swirling for 30s, uniformly mixing, and carrying out reaction to obtain 100 mu L of total volume. Incubation was performed in a 37℃water bath thermostatted shaker and timing was started with sampling time points of 0min and 60min. Sample group: 25. Mu.L of beta-NADPH solution (4 mM) was added to 75. Mu.L of the reaction system, vortexed for 30s, mixed well, and the total volume of the reaction was 100. Mu.L, and the samples were repeated. Incubation was performed in a 37℃water bath thermostated shaker and timing was started with sampling times of 0min,5min,15min,30min,60min. At each time point, the sample tube was removed and 300. Mu.L of cold stop reagent (containing internal standard) was added to stop the reaction. After vortexing for 5min, centrifugation was carried out for 10min (5500 Xg). 150 μl of the supernatant was added with 150 μl of water, vortexed and mixed well, and analyzed by LC-MS/MS.

Constitution of the experimental incubation system:

the results are shown below:

NA indicates that no corresponding compound was detected.

Microsome stability results show that the metabolic stability of compound II is far better than that of adefovir in human, monkey and SD rats, and the compound II has better advantages for oral administration.

5. In vivo toxicity test of candidate Compound BALB/C

25 BALB/C mice were randomly divided into 5 groups: solvent Control (Control), positive Control Remdeivir (50 mg/kg, 2 times a day), remdeivir (100 mg/kg, 2 times a day), compound II (50 mg/kg, 2 times a day), compound II (100 mg/kg, 2 times a day), each of which was 5, subjects were subcutaneously administered at respective concentrations in 5mL/kg, 100mg/kg groups at a dose of 10mL/kg, and Remdeivir and compound II were continuously administered daily for 21 days.

The preparation method of the compound solution comprises the following steps: 10% sulfobutyl-beta-cyclodextrin formulation:

5.0g of sulfobutyl-beta-cyclodextrin powder was weighed into a beaker, 50mL of citric acid buffer was pipetted into the beaker, dissolved and transferred into the container.

Preparation of Remdesivir:

25mg of Remdeivir is weighed, 2mL of 20% polyethylene glycol aqueous solution is added, 3mL of 10% sulfobutyl-beta-cyclodextrin aqueous solution is added after the solution is cleared, and 5mg/mL of Remdeivir test solution is obtained.

50mg of Remdeivir is weighed, 2mL of 20% polyethylene glycol aqueous solution is added, 3mL of 10% sulfobutyl-beta-cyclodextrin aqueous solution is added after the solution is cleared, and 10mg/mL of Remdeivir test solution is obtained.

Preparation of compound II:

25mg of compound II is weighed, 2mL of 20% polyethylene glycol aqueous solution is added, 3mL of 10% sulfobutyl-beta-cyclodextrin aqueous solution is added after the solution is cleared, and 5mg/mL of compound II test solution is obtained.

50mg of compound II is weighed, 2mL of 20% polyethylene glycol aqueous solution is added, 3mL of 10% sulfobutyl-beta-cyclodextrin aqueous solution is added after the solution is cleared, and 10mg/mL of compound II test solution is obtained.

The results of body weight changes in mice after continuous administration are shown in FIG. 1.

Conclusion: as shown in fig. 1, a significant decrease in 100mg/kg body weight occurred in remdesired, the mice were in the worst state, but no death occurred. The 50mg/kg group of Remdesivir showed comparable changes in body weight as the 100mg/kg group of Compound II, and the body weight tended to decrease compared to the blank group. While the 50mg/kg group of compound II still had a body weight comparable to the blank after 21 days. Thus, compound II showed better safety than Remdesivir.

Claims (6)

1. A nucleoside compound for antiviral treatment has a structure shown in formula (I),

wherein:

a is P;

R ¹ and R is ² Independently selected from H, -OR ³ 、-C(＝O)R ³ Or (b)

Each R ³ Independently H, (C) ₁ -C ₈ ) Alkyl and aryl.

2. The nucleoside compound for antiviral treatment according to claim 1, wherein

A is P;

R ¹ and R is ² Independently selected from H, -OR ³ 、-C(＝O)R ³ Or (b)

Each R ³ Independently is H, isopropyl.

3. The nucleoside compound for antiviral treatment according to claim 1, which is any one of the following:

4. a pharmaceutical composition comprising a nucleoside compound of any one of claims 1 to 3 for antiviral treatment and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition of claim 4, wherein the virus is 2019 novel coronavirus covd-19, HCoV-229E coronavirus, HCoV-OC43 coronavirus, SARS coronavirus, MERS coronavirus, porcine epidemic diarrhea virus PEDV, or avian influenza virus.

6. Use of a nucleoside compound for antiviral treatment according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment or prophylaxis of coronavirus, porcine epidemic diarrhea virus PEDV or avian influenza virus.