CN114601827A - Application of GN44028 in treatment of SARS-CoV-2 infection - Google Patents

Abstract

The invention relates to application of GN44028 or a pharmaceutically acceptable salt thereof in preparing medicines for preventing and treating diseases related to angiotensin converting enzyme 2(ACE 2). Specifically, the invention relates to application of GN44028 shown in a general formula (I) or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing and treating ACE2 related diseases, in particular to prevention and treatment of novel coronary viral pneumonia (COVID-19).

Description

Application of GN44028 in treatment of SARS-CoV-2 infection

Technical Field

The invention relates to application of GN44028 or a pharmaceutically acceptable salt thereof in preparing medicines for preventing or treating angiotensin converting enzyme 2(ACE2) related diseases. Specifically, the invention relates to application of GN44028 shown in a general formula (I) or a medicinal salt thereof in preparing a medicament for preventing and treating ACE2 related diseases, in particular preventing and treating novel coronary viral pneumonia (COVID-19).

Background

More than 6000 million people have been diagnosed in the world with COVID-19 caused by the novel coronavirus (SARS-CoV-2), more than two million people die, and no medicine for effectively controlling infection, transmission and treatment exists at present. Angiotensin converting enzyme 2(ACE2) is the primary receptor for SARS-CoV-2 entry into host cells; the expression level of ACE2 in the host cell determines the susceptibility of the cell to SARS-CoV-2 infection.

GN44028 was the first report of a hypoxia-inducible factor HIF-1 inhibitor in 2013. IC thereof₅₀At 14nM, it inhibited hypoxia-induced HIF-1. alpha. transcriptional activity, but not HIF-1. alpha. mRNA expression, accumulation of HIF-1. alpha. protein or HIF-1. alpha./HIF-1. beta. heterodimerization (Minegishi H, et al. ACS Med Chem Lett.2013 Jan 27; 4(2): 297-.

ACE2 is a key receptor for the entry of novel coronaviruses into human cells, and by inhibiting the expression of ACE2 protein, the risk of infection with novel coronaviruses can be significantly reduced, disease progression can be alleviated, and the novel coronaviruses can be used for treatment of infected patients. At present, no treatment medicine with a definite curative effect is available for severe acute respiratory syndrome type 2 coronavirus infection, and isolation, antivirus, symptomatic support and other treatments are mainly adopted in clinical treatment, but the treatment can not meet the clinical requirements. Therefore, it is of great significance to develop the targeted drug research aiming at SARS-CoV-2.

Disclosure of Invention

In a screening test, the GN44028 has better inhibitory activity in an ACE2 gene promoter reporter gene screening model and has activity of reducing ACE2 protein expression in cells and lung tissues of experimental animals, so that the GN44028 and structural analogues thereof have wide research and application prospects in the fields of inhibiting ACE2 expression, preventing, controlling and treating novel coronaviruses.

In order to solve the defects in the prior art, the invention provides GN44028 shown as a general formula (I) or a pharmaceutically acceptable salt thereof, which is applied to any one of the following (1) to (3):

wherein the application is as follows:

(1) preparing a medicament for preventing, relieving or treating diseases caused by SARS-CoV-2 or homologous variant virus thereof;

(2) preparing a medicament for preventing, relieving or treating SARS-CoV-2 or its homologous variant virus infection;

(3) for the preparation of a medicament as an ACE2 inhibitor.

Wherein the infection comprises pneumonia, acute respiratory infection, hypoxic respiratory failure and acute respiratory distress syndrome, sepsis or septic shock; wherein the disease comprises a respiratory disease or COVID-19.

The symptoms of the disease comprise fever, cough, pharyngalgia, pneumonia, acute respiratory infection, severe acute respiratory infection, hypoxic respiratory failure and acute respiratory distress syndrome, sepsis or septic shock and other infections.

The disease comprises respiratory disease or COVID-19.

The diseases caused by SARS-CoV-2 or its homologous variant virus include: SARS-CoV-2 or its homologous variant virus asymptomatic infection, SARS-CoV-2 or its homologous variant virus mild infection, SARS-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, mild to moderate respiratory symptoms, severe respiratory syndrome, acute respiratory distress syndrome, sepsis, renal failure, refractory metabolic acidosis or bleeding coagulation disorders; the severe respiratory syndrome comprises symptoms of fever, dry cough, hypodynamia, asthma and/or hypodynamia.

The research of the applicant finds that the compound GN44028 unexpectedly can generate strong inhibitory action on SARS-CoV-2 virus, has low toxicity, good safety and low toxic and side effects, and can be used as a medicament for clinically preventing, relieving and/or treating COVID-19/SARS-CoV-2.

The invention provides a pharmaceutical composition, which comprises GN44028 shown in a general formula (I) and a pharmaceutically acceptable carrier or auxiliary material.

In another aspect, the invention provides formulations of GN44028, including solid, injectable, semi-solid, liquid formulations.

The invention provides a GN44028 preparation, and the pharmaceutical preparation also comprises capsules, tablets, pills, creams, emulsions, ointments, suspensions, freeze-drying agents, capsules, sustained-release agents, granules, injection agents or sprays.

The invention also provides a combination therapeutic drug combination, which comprises GN44028 and at least one other drug, antibody or vaccine for preventing, relieving or treating COVID-19 pneumonia or homologous variant virus pneumonia thereof. (ii) a The other medicines, antibodies or vaccines for preventing, relieving or treating the COVID-19 pneumonia or the homologous variant virus pneumonia thereof comprise a composition containing traditional Chinese medicine components and/or western medicine components; the traditional Chinese medicine components and/or the western medicine components comprise: apilimod (apilimod), R82913 (CAS number: 126347-69-1), DS-6930(CAS number: 1242328-82-0), ONO 5334(CAS number: 868273-90-9), Reidesvir, Oseltamivir phosphate (Oseltamivir phosphate), hanfangchinA (HandfangchinA), clofazamine (clofazamine), astemizole (astemizole), recombinant human angiotensin converting enzyme 2(rhACE2) or Favipiravir (Favipiravir) and/or their pharmaceutically acceptable salts can prevent, alleviate and/or treat diseases caused by SARS-CoV-2 or its homologous mutant virus. The other medicines for preventing, relieving or treating COVID-19 pneumonia or homologous variant virus pneumonia thereof also comprise vaccines, therapeutic or prophylactic antibodies, active peptides or protein medicines aiming at SARS-CoV-2 or other homologous variant viruses and other viruses using ACE2 as a receptor.

The combined treatment medicine composition can effectively inhibit SARS-CoV-2 from entering cells and expanding in the cells, has good curative effect and low toxic and side effect, can reduce the drug resistance of viruses, and has obvious synergistic effect.

The present invention provides a method of modulating ACE2 in vitro, the method comprising a reporter gene model, a lung cancer cell model, or an animal tissue model, wherein the method comprises contacting ACE2 with a compound of claim 1.

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

(1) can effectively inhibit the coronavirus SARS-CoV-2 from entering cells and replicating and/or amplifying in the cells;

(2) can effectively inhibit the replication and/or amplification of SARS-CoV-2 or the homologous variant virus thereof and the cytopathic effect generated by the replication and/or amplification;

(3) the toxicity is extremely low, and the safety is high;

(4) can reduce the drug resistance of the virus, reduce the toxic and side effects of the drug, and the like;

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

Detailed Description

The elemental carbon, hydrogen, oxygen, nitrogen or halogen referred to in the groups and compounds of the invention include isotopes thereof, and the elemental carbon, hydrogen, oxygen or nitrogen referred to in the groups and compounds of the invention are optionally further replaced by one or more of their corresponding isotopes, wherein isotopes of carbon include¹²C、¹³C and¹⁴c, isotopes of hydrogen including protium (H), deuterium (D, also called deuterium), tritium (T, also called deuterium), isotopes of oxygen including¹⁶O、¹⁷O and¹⁸isotopes of O, nitrogen including¹⁴N and¹⁵isotopes of N, F¹⁹Isotopes of F, chlorine including³⁵Cl and³⁷cl, isotopes of bromine including⁷⁹Br and⁸¹Br。

"pharmaceutical composition" means a mixture of one or more compounds described herein or a physiologically/pharmaceutically acceptable salt thereof with other ingredients, wherein the other ingredients comprise physiologically/pharmaceutically acceptable carriers and excipients.

"carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

"excipient" refers to an inert substance added to a pharmaceutical composition to further depend on the administration of the compound. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and different types of starch, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like.

"prodrug" refers to a drug that can be converted to a biologically active drug under physiological conditions or by solvolysis. The prodrugs of the invention are prepared by modifying functional groups in estradiol, which modifications may be removed by conventional procedures or in vivo, to provide estradiol.

An "effective dose" refers to an amount of a compound that elicits a physiological or medical response in a tissue, system, or subject that is sought, including an amount of the compound that, when administered to a subject, is sufficient to prevent the onset of, or alleviate to some extent, one or more symptoms of the condition or disorder being treated.

"pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of non-toxic acids or bases, including salts of inorganic acids and bases, organic acids and bases.

"Relative expression" means a Relative expression level.

"Vehicle" means a Vehicle.

Drawings

FIG. 1 is a graph showing the activity of GN44028 in inhibiting the expression of ACE2 protein in cells in test example 2

FIG. 2 is a graph showing the activity of GN44028 in inhibiting the expression of ACE2 protein in mouse lung tissue in test example 3

Detailed Description

Test example 1 screening of Compound inhibiting ACE2 Gene expression

293T cells for serial passage were plated in 96-well plates at 1X 10 cells per well⁴A cell; test compounds were dissolved in DMSO, diluted in complete medium and added to culture wells at a final concentration of 2. mu.M, duplicate wells were set, DMSO dilutions were used as a blank, and after 1 hour incubation the reporter plasmid pGL3-ACE2(50 ng/well) and Renilla luciferase (Renilla) reporter plasmid pRL-SV40(10 ng/well) were transfected with PEI. The pRL-SV40 plasmid expresses Renilla red luciferase and is used as a reference for standardizing the efficiency of cells transfected by the pGL3-ACE2 plasmid, so that the difference of luciferase activities of fireflies in each space caused by the difference of transient transfection efficiency is eliminated. 72 hours after transfection, the activity of firefly Luciferase and Renilla Luciferase in the cells was detected by Dual-Glo Luciferase Assay System (Promega); the ratio of firefly luciferase signal to renilla luciferase signal was used as a relative reporter signal, and the percentage of the activity of ACE2 promoter inhibited by the compound was obtained as compared to the blank control. Screening compounds for cell toxicity was determined using CellTiter-glo (Promega). The compounds were then tested for inhibitory activity against pGL3-ACE2 reporter after 3-fold dilution of 8 concentration gradients starting with 2. mu.M, and IC was calculated₅₀。

And (4) conclusion: GN44028 has better inhibitory activity in ACE2 gene promoter reporter gene screening model, IC₅₀Was 30 nM.

Test example 2 Activity test for inhibiting expression of ACE2 protein in cells

After continuously subculturing human lung cancer cells A549 and treating the cells for 72 hours by using GN44028 with different concentrations, collecting the cells, cracking the cells in a lysis solution containing SDS and a protease inhibitor, determining the protein concentration by using a BCA method, separating the cells by SDS-PAGE, detecting the ACE2 protein expression amount in the cells by using Western Blotting, and performing semi-quantitative analysis by using GAPDH as an internal reference.

As shown in FIG. 1, GN 440282.5. mu.M and GN 5.0. mu.M inhibited ACE2 protein expression in cells by 35% and 50%, respectively.

Test example 3 Activity test for inhibiting expression of ACE2 protein in mouse lung tissue

GN44028 was dissolved in DMSO + HS-15+ 0.9% NS (5:5:90, v/v/v), administered to adult male ICR mice at 10, 50mg/kg by intragastric administration once a day for 7 days continuously. The animals are sacrificed 4 hours after the last administration, lung tissues are rapidly collected, liquid nitrogen is used for quick freezing, then homogenate is carried out in lysate containing protease inhibitors to extract total protein, the protein concentration is determined by a BCA method, the ACE2 protein expression level in the tissues is detected by Western Blotting, and GAPDH is used as internal standard for semi-quantitative analysis.

According to the results in FIG. 2, it was shown that continuous administration of 50mg/kg GN44028 significantly reduced the expression level of ACE2 protein in mouse lung tissue.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (8)

1. The application of GN44028 shown as a general formula (I) or a pharmaceutically acceptable salt thereof in any one of the following (1) to (3):

(1) preparing a medicament for preventing, relieving or treating diseases caused by SARS-CoV-2 and other viruses taking ACE2 as a receptor or homologous variant viruses thereof;

(2) preparing a medicament for preventing, relieving or treating SARS-CoV-2 and other virus taking ACE2 as a receptor or homologous variant virus infection thereof;

(3) for the preparation of medicaments as ACE2 inhibitors.

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 a respiratory disease or COVID-19.

4. The use according to claim 1, wherein the medicament is a composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier or adjuvant.

5. The use of claim 1, wherein the medicament comprises a solid formulation, an injection, a semi-solid formulation, a liquid formulation.

6. The use of claim 1, wherein the medicament comprises a capsule, a tablet, a pill, a cream, an emulsion, an ointment, a suspension, a lyophilized formulation, a capsule, a sustained release formulation, a granule, an injectable formulation, or a spray.

7. The use of claim 1, wherein the medicament further comprises at least one other medicament, antibody or vaccine for preventing, ameliorating or treating COVID-19 pneumonia and pneumonia of other ACE 2-receptor viruses or their homologous variant viruses.

8. A method of modulating ACE2 in vitro, the method comprising a reporter gene model, a lung cancer cell model, or an animal tissue model, wherein the method comprises contacting ACE2 with the compound of claim 1.

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