CN111991401A - Application of compound in treatment of SARS-CoV-2 infection - Google Patents

Abstract

The invention provides an application of compounds 69-0 and/or tautomers thereof and/or pharmaceutically acceptable salts thereof and the like in preventing, relieving and/or treating COVID19/SARS-CoV-2, products containing the compounds can effectively inhibit the replication or propagation of SARS-CoV-2 or homologous variant viruses thereof and cytopathic effects generated by the replication or propagation, and the products have good curative effect, good safety and low toxic and side effects. The invention also provides a combination therapy pharmaceutical composition. The combined treatment medicine composition can effectively inhibit the proliferation of SARS-CoV-2 in cells, has good curative effect, low safety and low toxic and side effect, can reduce the drug resistance of viruses, and has obvious synergistic effect.

Description

Use of a compound in the treatment of SARS-CoV-2 infection

technical field

The present invention relates to the field of pharmaceutical technology and virus infection disease technology, in particular to the application of compound 69-0 and/or its tautomer and the like.

Background technique

Severe acute respiratory syndrome type 2 coronavirus (SARS-CoV-2), also known as 2019 novel coronavirus, was discovered during the outbreak of the 2019 novel coronavirus pneumonia (COVID-19). SARS-CoV-2 is a virus with high diffusivity. The disease is mainly transmitted by respiratory droplets and contact, and the population is generally susceptible and highly contagious.

SARS-CoV-2 is an enveloped, single-stranded RNA beta coronavirus. The infection of 2019-nCoV begins with the binding of the spike glycoprotein (Spike, S protein) on the surface of the virion to the angiotensin-converting enzyme (ACE2) receptor on the cell surface. After the coronavirus enters the host cell, it disintegrates and releases the nucleocapsid and viral RNA into the cytoplasm. The open reading frame (ORF1a/b) at the 5' end of the viral RNA encodes polyproteins (pp1a and pp1ab). pp1a and pp1ab can be cleaved by papain-like protease (PLpro) and 3CL protease (3-chymotrypsin-like protease, 3CLpro, also known as Mpro) to generate non-structural proteins (NSPs), Such as RNA-dependent RNA polymerase (RNA-dependent RNA polymerase, RdRp) and helicase (helicase). Viral RNA-dependent RNA polymerases (RNA-dependent RNA polymerases, RdRp), also known as non-structural protein 12 (nsp12), can assemble with other non-structural proteins to form a highly efficient The RNA synthesis "machine" that completes the transcription and replication of the genetic material RNA genome. As the core component of this transcriptional replication machine, RNA polymerase is one of the most important targets of antiviral drugs. Disrupting its function is expected to prevent virus replication and ultimately achieve therapeutic purposes. The drug development of the new coronavirus, especially the verification of the drug effect mechanism of RNA polymerase, is very urgent and urgent.

Remdesivir exhibits potent antiviral activity against many RNA viruses. It can inhibit the replication of several different types of RNA viruses, but after clinical trials, it was found that Remdesivir did not significantly reduce the death effect in humans, and its clinical dosage was close to the safe dosage, with obvious side effects. Therefore, it is imminent to find another drug against SARS-CoV-2.

At present, there is no clear therapeutic drug for severe acute respiratory syndrome type 2 coronavirus infection, and clinical treatment is mainly based on isolation, antiviral, symptomatic support, etc. However, these treatments still cannot meet the clinical needs. On the one hand, commonly used antiviral drugs are prone to cause side effects such as neurotoxicity, diarrhea, arrhythmia, and abnormal liver function. It is especially necessary for the elderly with basic metabolic disorders, low immune function, or cancer and other basic diseases to be used with caution. On the other hand, symptomatic treatment is not effective in reversing the condition of critically ill patients, or the effect is not good, and it is easy to cause co-infection, resulting in multiple and diffuse lesions. Therefore, it will be of great significance to carry out targeted drug research against SARS-CoV-2.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION

Due to the low efficacy of Remdesivir on the SARS-CoV-2 virus and its high toxicity and side effects, researchers have also lost their research interest in related derivatives and precursor compounds of Remdesivir. Compound 69-0, as the precursor compound of Remdesivir, has a similar inhibitory mechanism to that of Remdesivir, and the inhibitory activity of compound 69-0 against various viruses is lower than that of Remdesivir, so the researchers did not. It is believed that compound 69-0 can inhibit the SARS-CoV-2 virus. However, the applicant's research found that compound 69-0 unexpectedly has a strong inhibitory effect on the SARS-CoV-2 virus, and has low toxicity, good safety, and low side effects. It can be used as clinical prevention and mitigation. and/or drugs to treat COVID-19/SARS-CoV-2.

Therefore, the applicant proposes the following summary of the invention.

In the first aspect, the present invention provides a compound 69-0 and/or its tautomer and/or its pharmaceutically acceptable salt and/or its ester and/or its solvate and/or its hydrate and / or its polymorphs and / or its pseudopolymorphs and / or its amorphous forms and / or its prodrugs in the preparation of prevention, alleviation and / or treatment of SARS-CoV-2 or its homologous variant viruses Disease or infection caused by the application of the product. The product can effectively inhibit the replication or reproduction of SARS-CoV-2 or its homologous variant virus and its cytopathic effect, and has good curative effect, good safety and low toxic and side effects.

In a second aspect, the present invention also provides a combination therapy drug combination. The combined therapeutic drug combination can effectively inhibit the proliferation of SARS-CoV-2 in cells, has good curative effect, low safety, low toxic and side effects, can reduce the drug resistance of the virus, and has a significant synergistic effect.

Detailed description of the invention

According to literature reports, the activity data of various viruses in different species show that the antiviral activity of Remdesivir is better than that of compound 69-0. For example, remdesivir inhibits EBOV virus in Hela cells, inhibits EBOV in HMVEC cells, inhibits RSV in Hep-2 cells, inhibits HCV in Huh-7 cells, and inhibits SARS-CoV proliferation in HAE cells. IC50 They were 0.1 μM, 0.053 μM, 0.015 μM, 0.057 μM, and 0.069 μM, respectively. In the same experiment, the anti-virus activity of compound 69-0 was >20 μM, 0.78 μM, 0.53 μM, 4.1 μM, and 0.18 μM, respectively. Compared with remdesivir, the activity of compound 69-0 in inhibiting the virus was reduced by 3->200 times. Ribose monophosphorylation in compound 69-0 is considered to be the rate-limiting step in the activation of compound 69-0, while Remdesivir directly introduces monophosphate into ribonucleosides and connects with polar groups through phosphoramide bonds to form phosphorus. Amide prodrugs reduce molecular polarity and increase membrane permeability by masking polar groups. The prodrug is absorbed into the body and then hydrolyzed by a specific enzyme to release the prototype drug. Bypasses the rate-limiting step of monophosphorylation and thus exhibits better inhibitory activity. Therefore, based on empirical data in other viruses, it is speculated that the anti-SARS-CoV-2 activity of remdesivir is better than compound 69-0, and the therapeutic effect of compound 69-0 in SARS-CoV-2 infection is ignored.

The present invention discloses the inhibitory effect of compound 69-0 on SARS-CoV-2 for the first time. Unexpectedly, the activity of compound 69-0 in Vero-E6 cells is better than that of remdesivir. The ratios showed different trends, revealing the potential application value of compound 69-0 in the treatment of SARS-CoV-2 infection.

Therefore, the present invention proposes the following summary of the invention.

In the first aspect, the present invention provides a compound 69-0 and/or its tautomer and/or its pharmaceutically acceptable salt and/or its ester and/or its solvate and/or its hydrate and Use of/or polymorphs thereof and/or pseudopolymorphs thereof and/or amorphous forms thereof and/or prodrugs thereof.

A compound 69-0 and/or its tautomer and/or its pharmaceutically acceptable salt and/or its ester and/or its solvate and/or its hydrate and/or its polymorph and/or pseudopolymorphs thereof and/or amorphous forms thereof and/or prodrugs thereof in any of the following (1)-(5) applications:

(1) Preparation of medicines for preventing, alleviating and/or treating diseases caused by SARS-CoV-2 or its homologous variant viruses;

(2) Preparation of medicines for preventing, alleviating and/or treating SARS-CoV-2 or its homologous variant virus infection;

(3) Preparation of SARS-CoV-2 inhibitors;

(4) Preparation of drugs for inhibiting the replication or reproduction of SARS-CoV-2 in cells;

(5) Preparation of drugs for inhibiting the cytopathic effect of SARS-CoV-2;

The compound 69-0 and/or its tautomers and/or its pharmaceutically acceptable salts and/or its esters and/or its solvates and/or its hydrates and/or its polymorphs and/or pseudopolymorphs thereof and/or amorphous forms thereof and/or prodrugs thereof are administered in a therapeutically effective amount.

The compounds can be administered by injection, orally, or by inhalation.

The disease symptoms include fever, cough, sore throat, pneumonia, acute respiratory infection, severe acute respiratory infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis or septic shock and other infections.

The disease includes respiratory disease or COVID-19.

The diseases caused by the SARS-CoV-2 or its homologous variant virus include: asymptomatic infection with SARS-CoV-2 or its homologous variant virus, mild infection with SARS-CoV-2 or its homologous variant virus, SARS-CoV-2 - CoV-2 or its homologous variant virus moderate infection or SARS-CoV-2 or its homologous variant virus severe infection.

The symptoms of COVID-19 include: asymptomatic, asymptomatic, mild to moderate respiratory symptoms, severe respiratory syndrome, acute respiratory distress syndrome, sepsis, renal failure, refractory metabolic acid Poisoning or bleeding coagulation disorders. In some embodiments, the severe respiratory syndrome includes symptoms such as fever, dry cough, fatigue, wheezing and/or breathlessness.

The SARS-CoV-2 inhibitor includes a SARS-CoV-2 RNA polymerase inhibitor.

The cells include mammalian cells, fish animal cells, avian animal cells or amphibian cells. In some embodiments, the mammal includes a bovine, equine, ovine, porcine, canine, feline, rodent, primate. In some embodiments, the mammals include cats, dogs, pigs, squirrels, and the like. In some embodiments, the bird animals include animals such as parrots or starlings. In some embodiments, the primate includes humans, monkeys, or chimpanzees, and the like.

The medicament may be a composition comprising the compound 69-0 and/or its tautomer and/or its pharmaceutically acceptable salt and/or its ester and/or its solvate and /or its hydrates and/or its polymorphs and/or its pseudopolymorphs and/or its amorphous forms and/or its prodrugs and pharmaceutically acceptable carriers or excipients.

The drugs include solid preparations, injections, external preparations, sprays, liquid preparations or compound preparations and other preparations.

The medicines include dosage forms such as capsules, tablets, pills, creams, emulsions, ointments, suspensions, freeze-dried preparations, capsules, sustained-release preparations, granules, granules, injections, inhalation preparations or sprays.

In a second aspect, the present invention provides a combined treatment combination for COVID-19 pneumonia or its homologous variant virus pneumonia.

A combined treatment combination for COVID-19 pneumonia or its homologous variant virus pneumonia, comprising the drug described in any one of claims 1-7 and at least one other for preventing, relieving and/or treating COVID-19 pneumonia or its homologous Products of mutated viral pneumonia.

In some embodiments, the product includes a composition containing traditional Chinese medicine ingredients and/or western medicine ingredients.

In some embodiments, the active ingredients of the product include: apilimod, R 82913 (CAS No: 126347-69-1), DS-6930 (CAS No: 1242328-82-0), ONO 5334 (CAS number: 868273-90-9), Remdesivir, Oseltamivir phosphate, HanfangchinA, clofazamine, astemizole, Recombinant human angiotensin-converting enzyme 2 (rhACE2) or Favipiravir and/or their pharmaceutically acceptable salts, etc. can prevent, alleviate and/or treat SARS-CoV-2 or its homologous variants Compounds for diseases caused by viruses.

Compared with the prior art, the present invention has the following technical effects:

(1) It can effectively inhibit the replication and/or reproduction of the coronavirus SARS-CoV-2 in cells, and its activity is much higher than that of remdesivir;

(2) It can effectively inhibit the replication or reproduction of SARS-CoV-2 or its homologous variant virus and its cytopathic effect;

(3) Its toxicity is extremely low and its safety is high;

(4) It can reduce the drug resistance of the virus and reduce the toxic and side effects of drugs;

(5) The structure is simple, which is beneficial to synthesis, production and distribution.

Definition of Terms:

Vero-E6 means African green monkey kidney cells; caco-2 means human colon adenocarcinoma cells; calu-3 means human lung adenocarcinoma cells; μM means micromoles per liter; IC50 means half inhibitory concentration; MOI means multiplicity of infection; CCK8 means ; RT-PCR, reverse transcription-polymerase chain reaction; Hela, HeLa cells; EBOV, Ebola virus; HMVEC, human lung microvascular endothelial cells; Hep-2, human laryngeal epidermoid carcinoma cells; RSV, respiratory syncytial cells Virus; Huh-7, human hepatoma cells; HCV, hepatitis C virus; HAE, hereditary angioedema; DMEM medium, Dulbecco's modified Eagle's medium; min, minutes; h, hours; ℃, degrees Celsius ; nm means nanometer; μl means microliter.

The medicines of the above-mentioned various dosage forms can be prepared according to the conventional methods in the field of pharmacy.

In the present invention, the term "therapeutically effective amount" or "prophylactically effective amount" refers to an amount sufficient to treat or prevent a patient's disease but low enough to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of sound medical judgment. A therapeutically effective amount of a compound will depend on the particular compound selected (eg, taking into account the potency, effectiveness and half-life of the compound), the route of administration selected, the disease being treated, the severity of the disease being treated, the severity of the patient being treated. Factors such as age, size, weight and physical ailment, medical history of the patient being treated, duration of treatment, nature of concurrent therapy, desired therapeutic effect, and the like vary, but can still be routinely determined by those skilled in the art.

It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should also be understood that terms such as those defined in general dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art and, unless defined as herein, are not to be taken in an idealized or overly formal sense. explain.

In addition, it should be pointed out that the compound 69-0 and/or its tautomers and/or its pharmaceutically acceptable salts and/or its esters and/or its solvates and/or its hydrates and/or its The specific dosage and method of use of the polymorph and/or its pseudopolymorph and/or its amorphous form and/or its prodrugs for different patients depends on many factors, including the patient's age, weight, sex, Natural health status, nutritional status, active strength of the drug, time of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. It is preferred here to use doses between 0.001 and 1000 mg/kg body weight/day.

Description of drawings

Figure 1 shows the survival rate of Vero-E6 cells under different concentrations of compound 69-0;

Figure 2 shows the survival rate of Vero-E6 cells under different concentrations of Remdesivir;

Figure 3 shows the survival rate of caco-2 cells under different concentrations of compound 69-0;

Figure 4 shows the survival rate of caco-2 cells under different concentrations of Remdesivir;

Figure 5 shows the survival rate of calu-3 cells under different concentrations of compound 69-0;

Figure 6 shows the survival rate of calu-3 cells under different concentrations of Remdesivir;

Figure 7 shows the inhibitory effect of different concentrations of compound 69-0 on SARS-CoV-2 virus in Vero-E6 cells;

Figure 8 shows the inhibitory effect of different concentrations of Remdesivir on SARS-CoV-2 virus in Vero-E6 cells;

Figure 9 shows the inhibitory effect of different concentrations of compound 69-0 on SARS-CoV-2 virus in caco-2 cells;

Figure 10 shows the inhibitory effect of different concentrations of compound 69-0 on SARS-CoV-2 virus in calu-3 cells.

Figure 11 shows the plasma concentration of remdesivir after SD rats were administered remdesivir

Figure 12 shows the plasma concentration time curve of 69-0 after administration of remdesivir or 69-0 in SD rats

Figure 13 shows the plasma concentration time curve of 69-0 after administration of remdesivir or 69-0 in SD rats

Figure 14 shows the survival curve of mice in each treatment group after virus infection

Figure 15 shows the viral load in lung tissue of mice in each treatment group at different time points after virus infection

Figure 16 shows the viral load in liver tissue of mice in each treatment group at different time points after virus infection

Detailed ways

In order to make those skilled in the art better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

The reagents used in the present invention can be purchased from the market or can be prepared by the method described in the present invention.

Example 1: Toxicity evaluation of compound 69-0 on Vero-E6 cells

Vero-E6 cells were seeded in a 96-well plate, and after the cells adhered, the medium was changed to DMEM medium containing 2% serum, and compound 69- 0 or remdesivir, and do a blank drug control, after culturing for 48 hours, add 1/10 volume of CCK8 reagent, continue to culture at 37 ° C for 2 hours, sample at 450nm to detect the absorbance value, and calculate the cell viability, the results are as follows Figure 1 and Figure 2.

Analysis of results: Compound 69-0 had no cytotoxicity to Vero-E6 cells at final concentrations of 50 μM, 10 μM, 5 μM, 1 μM, 0.1 μM, and 0.01 μM, and compound remdesivir had no cytotoxicity to ERO-E6 cells at a final concentration of 50 μM. E6 cells have certain toxicity, indicating that compound 69-0 has extremely low toxicity to Vero-E6 cells and high safety.

Example 2: Toxicity evaluation of compound 69-0 on caco-2 cells

The caco-2 cells were seeded in 96-well plates, and after the cells adhered, the medium was changed to DMEM medium containing 2% serum, and compound 69- 0 or remdesivir, and do a blank drug control, after culturing for 48 hours, add 1/10 volume of CCK8 reagent, continue to culture at 37 ° C for 2 hours, sample at 450nm to detect the absorbance value, and calculate the cell viability, the results are as follows Figure 3 and Figure 4.

Analysis of results: Compound 69-0 had no cytotoxicity to caco-2 cells at final concentrations of 50 μM, 10 μM, 5 μM, 1 μM, 0.1 μM, and 0.01 μM, and compound remdesivir had no cytotoxicity to caco-2 cells at a final concentration of 50 μM. 2 cells have certain toxicity, indicating that compound 69-0 has extremely low toxicity to caco-2 cells and high safety.

Example 3: Toxicity evaluation of compound 69-0 on calu-3 cells

Calu-3 cells were seeded in a 96-well plate, and after the cells adhered, the medium was changed to DMEM medium containing 2% serum, and compound 69- 0 or remdesivir, and do a blank drug control, after culturing for 48 hours, add 1/10 volume of CCK8 reagent, continue to culture at 37 ° C for 2 hours, sample at 450nm to detect the absorbance value, and calculate the cell viability, the results are as follows Figure 5 and Figure 6.

Analysis of results: Compound 69-0 had no cytotoxicity to caco-2 cells at final concentrations of 50 μM, 10 μM, 5 μM, 1 μM, 0.1 μM, and 0.01 μM, and compound remdesivir was at a final concentration of 50 μM. It has certain toxicity to calu-3 cells, indicating that compound 69-0 has extremely low toxicity to calu-3 cells and high safety.

Example 4: Inhibitory effect of compound 69-0 on SARS-CoV-2 virus on Vero-E6 cells

Vero-E6 cells in a 24-well plate were infected with SARS-CoV-2 virus at a MOI of 0.05. After 1 h of infection, the cell supernatant was discarded and replaced with DMEM medium with 2% serum. 5 μM, 1 μM, 0.1 μM or 0.01 μM of compound 69-0 or remdesivir, and do a blank drug control. After 48h of infection, collect 150ul cell supernatant, inactivate at 56°C for 30min, use OMEGA's viral RNA extraction kit (R6874-02) to extract viral RNA in the supernatant, use the new coronavirus N gene probe and RT- The viral RNA was detected by PCR, and the kit was purchased from Daan Gene Company of Sun Yat-sen University (DA0931). The inhibitory effects of different concentrations of drugs on the virus were calculated, and the IC50 of the drugs was calculated. The results are shown in Figures 7 and 8.

Analysis of results: Compound 69-0 can inhibit the replication of SARS-CoV-2 virus in Vero-E6 cells, and its IC50 against SARS-CoV-2 virus in Vero-E6 cells is 0.7022 μM. In Vero-E6 cells, the activity of compound 69-0 was better than that of remdesivir, indicating that compound 69-0 may effectively inhibit the replication and/or reproduction of SARS-CoV-2 virus in Vero-E6 cells.

Example 5: Inhibitory effect of compound 69-0 on SARS-CoV-2 virus on caco-2 cells

The caco-2 cells in a 24-well plate were infected with SARS-CoV-2 virus at 0.1 MOI. After 1 h of infection, the cell supernatant was discarded and replaced with DMEM medium with 2% serum. 5μM, 1μM, 0.1μM or 0.01μM of compound 69-0, and do a blank drug control. After 48 hours of infection, 150 μl of cell supernatant was collected, inactivated at 56°C for 30 min, and the viral RNA in the supernatant was extracted using OMEGA’s viral RNA extraction kit (R6874-02). The viral RNA was detected by PCR, and the kit was purchased from Daan Gene Company of Sun Yat-sen University (DA0931). The inhibitory effects of different concentrations of drugs on the virus were calculated, and the IC50 of the drugs was calculated. The results are shown in Figure 9.

Analysis of results: Compound 69-0 can inhibit the replication of SARS-CoV-2 virus in caco-2 cells, and its IC50 against SARS-CoV-2 virus in caco-2 cells is 3.603 μM, indicating that compound 69-0 The replication and/or multiplication of SARS-CoV-2 virus may be effectively inhibited in caco-2 cells.

Example 6: Inhibitory effect of compound 69-0 on SARS-CoV-2 virus on calu-3 cells

Calu-3 cells in a 24-well plate were infected with SARS-CoV-2 virus at 0.1 MOI. After 1 h of infection, the cell supernatant was discarded and replaced with DMEM medium with 2% serum. The final concentrations were 50 μM, 10 μM, and 5 μM. , 1 μM, 0.1 μM or 0.01 μM of compound 69-0, and do a blank drug control. After 24 hours of infection, 150 ul of cell supernatant was collected and inactivated at 56°C for 30 minutes. The viral RNA in the supernatant was extracted using OMEGA's viral RNA extraction kit (R6874-02), and the new coronavirus N gene probe and RT- The viral RNA was detected by PCR, and the kit was purchased from Daan Gene Company of Sun Yat-sen University (DA0931). The inhibitory effects of different concentrations of drugs on the virus were calculated, and the IC50 of the drugs was calculated. The results are shown in Figure 10.

Analysis of results: Compound 69-0 can inhibit the replication of SARS-CoV-2 virus in calu-3 cells, and its IC50 against SARS-CoV-2 virus in calu-3 cells is 3.209 μM, indicating that compound 69-0 The replication and/or multiplication of SARS-CoV-2 virus may be effectively inhibited in calu-3 cells.

Example 7: Metabolism and bioavailability research experiment of compound 69-0 in Uncle SD

16 male SD rats weighing 220-250g (4 in each group) were randomly divided into four groups, namely Remdersivir intravenous group (group 1), Remdersivir oral group (group 2), 69-0 intravenous group (group 3) ), 69-0 oral group (4 groups). Animals in groups 1 and 3 were given Remdersivir and 69-0 by tail vein injection, respectively, with an administration volume of 0.1 mL/100 g and an administration concentration of 30 mg/mL; animals in groups 2 and 4 were given a single oral gavage, respectively Remdersivir and 69-0 were administered in a volume of 0.5mL/100g and a concentration of 6mg/mL. All animals were fed on an empty stomach 12 hours before administration and 4 hours after administration, and each experimental group was administered with equal concentrations and different volumes. The jugular vein or orbital venous plexus (after isoflurane gas anesthetized the animal) was used to collect blood, and a disposable syringe was used to pierce the vein or capillary for blood collection, and blood was collected at an appropriate speed. Blood was collected at 0.083h (oral group not collected), 0.16h (oral group not collected), 0.25h, 0.5h, 1h (intravenous group not collected), 2h, 4h, 8h, 24h, 48h after administration, respectively. 0.3 mL was placed in a heparin tube, centrifuged at 4000 r/min for 10 min at 4 °C, and the upper plasma was taken and transferred to a refrigerator (about -20 °C) for temporary storage until the measurement. To the obtained plasma sample (50 μL), add methanol/water mixed solvent (1:1, 10 μL) and internal standard solution. After mixing, vortex for 1 minute, and then centrifuge at 14000G for 3 minutes at 4 degrees Celsius. Take 100 μL of sample 15.1 and use the UNIFI software equipped with Waters UPLC/XEVO TQ-S triple quadrupole liquid mass spectrometer to quantitatively process the sample to be tested. The DAS3.0 pharmacokinetic software was used to process the data at the time of the drug, and the parameters such as AUC, Cmax, Tmax and t1/2 were calculated. time curve. The results are shown in Figures 11-13.

Conclusion: After SD rats were given Remdersivir or 69-0 by oral gavage, 69-0 was rapidly absorbed in the body, and the average Tmax was 0.94±0.77h, indicating that 69-0 was absorbed rapidly. After SD rats were given Remdersivir or 69-0 by intravenous injection and oral gavage, the average t1/2 of 69-0 were 4.77±2.33h and 20.55±16.43h, respectively, indicating that the elimination of 69-0 in rats was better than that of Remdersivir in rats. (The average t1/2 is 1.95±1.16, 3.51±0.16h) slower, and 69-0 has good druggability.

Example 8: In vivo efficacy study of compound 69-0 against mouse coronavirus

75 BABL/c mice were randomly divided into 8 groups according to their body weight, namely MHV virus control (group A), 69-0 intravenous treatment group (B1 group), 69-0 oral treatment group (B2 group), 69- 0 intraperitoneal treatment group (group B3), 69-0 intravenous injection control group (C1 group), 69-0 oral control group (C2 group), 69-0 intraperitoneal control group (C3 group), normal control group (D group) . The animals in the treatment group and 69-0 control group were given 69-0 at a dose of 50 mg/kg by tail vein injection, oral gavage or intraperitoneal injection, respectively, and the administration volume was 0.2 mL/20 g, and the administration concentration was 5 mg/mL, double the first dose. The animals were continuously administered for 14 days, and the death of the animals was observed every day. At 24h and 72h after administration, 4 animals/group in each of groups A, B1, B2 and B3 were collected, about 1 g of lungs and livers were taken into sterile centrifuge tubes, stored at -20°C, and nucleic acid was extracted to measure the concentration. Quantitative analysis of viral load in tissues after virus infection by fluorescent PCR. The results are shown in Figures 14-16.

Conclusion: 5 days after the mice were infected, the virus model control group (A) began to die, and by 14 days, the mortality rate was 87.5% (7/8), while the mortality rate of the treatment groups (B1, B2, B3) was 0-20%. It shows that the drug has obvious positive effect on animal survival. The viral loads in the lungs of mice 24 h after infection, the viral loads in the treatment groups (B1, B2, B3) were lower than those in the virus model control group (A), and the differences in the viral loads in the lungs of each group decreased at 72 h. It shows that the effect of the drug in inhibiting virus replication is obvious in the early stage.

It can be known from the experimental results obtained from the above-mentioned embodiments 1-6:

(1) Compound 69-0 has no toxicity in Vero-E6, caco-2, and calu-3 cells, which proves that it has high safety and less toxic and side effects.

(2) Compound 69-0 can inhibit the replication of SARS-CoV-2 in Vero-E6, caco-2, and calu-3 cells, and its IC50 is between 0.7-3.6 μM. Especially in Vero-E6 cells, the activity of compound 69-0 was twice that of remdesivir, indicating that compound 69-0 may effectively inhibit the replication and/or reproduction of SARS-CoV-2 virus in cells .

The method of the present invention has been described through the preferred embodiments, and it is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to realize and apply the technology of the present invention . Those skilled in the art can learn from the content of this document and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications apparent to those skilled in the art are deemed to be included in the present invention.

Claims (8)

1. A compound 69-0 is used in any one of the following (1)-(5): (1) Preparation of medicines for preventing, alleviating or treating diseases caused by SARS-CoV-2 or its homologous variant viruses; (2) Preparation of medicines for preventing, alleviating or treating SARS-CoV-2 or its homologous variant virus infection; (3) preparation of a drug as a drug as a SARS-CoV-2 inhibitor; (4) Preparation of drugs for inhibiting the replication or reproduction of SARS-CoV-2 in cells; (5) Preparation of drugs for inhibiting the cytopathic effect of SARS-CoV-2;

2. The use according to claim 1, wherein the infection comprises pneumonia, acute respiratory infection, hypoxic respiratory failure and acute respiratory distress syndrome, sepsis or septic shock. 3. The use of claim 1, wherein the disease comprises respiratory disease or COVID-19. 4. purposes according to claim 1, described cell comprises mammalian cell, fish animal cell, bird animal cell or amphibian cell; Described mammal comprises bovine animal, equine animal, ovine animal, Porcine, canine, feline, rodent, or primate; such primates include humans. 5. The use according to claim 1, wherein the medicine is a composition comprising the compound 69-0 and a pharmaceutically acceptable carrier or adjuvant. 6. The use according to claim 1, wherein the medicine comprises solid preparation, injection, semi-solid preparation and liquid preparation. 7. purposes according to claim 1, described medicine comprises capsule, tablet, pill, cream, emulsion, ointment, suspension, freeze-dried agent, capsule, sustained release agent, granule, granule, injection Medication or spray. 8. The use according to claim 1, wherein the medicine further comprises at least one other medicine for preventing, relieving or treating COVID-19 pneumonia or its homologous variant virus pneumonia.

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