Application of tetrahydroisoquinoline compounds in the preparation of drugs for preventing and / or treating coronavirus infection

Abstract

The present invention belongs to the technical field of antiviral drugs, and relates to the use of a tetrahydroisoquinoline compound in the preparation of a drug for preventing and / or treating coronavirus infection. Specifically, the tetrahydroisoquinoline compound has a structure as shown in Formula I, and has little toxicity to coronavirus-susceptible human cells, but can significantly inhibit the infection of coronavirus to susceptible human cells, has a significant inhibitory effect on coronavirus infection at the cellular level, and is dose-dependent, so it can be used as a coronavirus inhibitor.

Description

Technical Field

[0001] The present invention belongs to the technical field of antiviral drugs and relates to the medical use of tetrahydroisoquinoline compounds, in particular to the use of the compounds in the preparation of drugs for preventing and / or treating coronavirus infection. Background Art

[0002] Coronavirus is a large class of viruses that are widely present in nature and has the largest genome among the known RNA viruses. Coronavirus only infects vertebrates, such as primates, rodents, carnivores and artiodactyls in the class Mammalia, and Galliformes in the class Aves. SARS-CoV-2 is the seventh known coronavirus that can infect humans. The other six are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV. The first four have low pathogenicity and generally cause respiratory symptoms similar to the common cold. SARS-CoV can cause severe acute respiratory syndrome (SARS), and MERS-CoV can cause Middle East respiratory syndrome (MERS), which broke out in 2002 and 2012 respectively (Zaki AM, van Boheemen S., Bestebroer TM, et al., Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia[J], N.Engl.J.Med., 2012, 367(19):1814-1820; Cui J., Li F., Shi ZL, Origin and evolution of pathogeniccoronaviruses[J], Nat. Rev. Microbiol., 2019, 17(3): 181-192).

[0003] Corona Virus Disease 2019 (COVID-19) is a contact infectious disease caused by SARS-CoV-2 (new coronavirus), characterized by fever, dry cough, fatigue and a certain mortality rate. Since the outbreak of the novel coronavirus in December 2019, the number of confirmed cases has exceeded that of SARS and is still increasing, causing serious social harm and economic losses. The World Health Organization has announced that the novel coronavirus epidemic has constituted an "international public health emergency". Since the outbreak, related research has mainly focused on epidemiological surveys, sequence comparison and analysis of clinically isolated strains, and virus detection methods. However, research on the invasion, replication, and immune regulation mechanisms of the novel coronavirus is still in the primary exploratory stage, and no vaccine can provide adequate protection. Therefore, research and development of antiviral drugs against coronaviruses (especially SARS-CoV-2) has important social significance and economic value.

[0004] Alkaloids are a class of nitrogen-containing alkaline organic compounds widely found in nature. Most of them have complex ring structures, and the nitrogen atom is usually present in the ring. They have significant biological activity and are one of the important active ingredients in Chinese herbal medicines. According to the basic nucleus structure of alkaloids, they can be divided into about 60 categories, such as organic amines, pyrrolidines, pyridines, and isoquinolines. Among them, isoquinolines are the largest class of alkaloids, with isoquinoline or tetrahydroisoquinoline as the nucleus, and are derived from phenylalanine or tyrosine. They can be roughly divided into simple isoquinolines (such as garveline), benzyl isoquinolines (such as papaverine), dibenzyl isoquinolines (such as tetrandrine), aporphine (such as magnoline), protoberberine (such as berberine), protropine (such as scopolamine), morphine (such as morphine), etc.

[0005] Chinese invention patent application CN109419804A discloses that alkaloid components derived from Cephalotaxus plants can effectively inhibit 9 viruses (including coronavirus) from 7 families from infecting host cells and animals, significantly reduce or even eliminate viruses in infected animals, wherein the alkaloid components include homoharringtonine (HHT), cephalotaxine, cephalotaxine, and its parent nucleus structure is benzazepine One more methylene group than tetrahydroisoquinoline. Chinese invention patent application CN110960532A discloses that a composition of Macleaya cordata benzylisoquinoline alkaloids and trans-resveratrol has a significant anti-coronavirus effect, particularly involving the inhibitory effect on the COVID-19 coronavirus, wherein the parent nucleus structure of protopine and allocryptine is an open-ring structure containing a ketocarbonyl group derived from tetrahydroisoquinoline. Chinese invention patent application CN111481546A discloses that a cypermethrin alkaloid or its salt has an excellent coronavirus inhibitory effect, especially showing high inhibitory activity in the inhibition test results of the new coronavirus 2019-nCoV, wherein the parent nucleus structure of the cypermethrin alkaloid is hexahydropyrroloisoquinoline, which has one more pyrrolidine than tetrahydroisoquinoline.

[0006] Li Zhen et al. found that matrine may directly inactivate SARS-CoV-2 and inhibit its replication and proliferation in the body, while sophoracarpine may play an antiviral role by inhibiting the attachment and penetration of SARS-CoV-2 to host cells. The parent nucleus structures of matrine and sophoracarpine are both tetrahydroisoquinoline-containing tetracyclic ring structures (Li Zhen, Yu Kexian, Study on the mechanism of multi-path anti-coronavirus of matrine [J], Chinese Herbal Medicine, 2020, 51(4): 888-894). Zeng Binglin et al.'s review revealed that lycorine has great potential in antiviral research, especially in SARS-CoV-2 research. The parent nucleus structure of lycorine is a pentacyclic ring structure containing tetrahydroisoquinoline (Zeng Binglin et al., Research Progress on Pharmacological Activity and Structure-Activity Relationship of Lycorine [J], Natural Product Research and Development, 2020).

[0007] In summary, there are few reports on the research of tetrahydroisoquinoline alkaloids in anti-coronavirus infection. Summary of the invention

[0008] Problem that the invention aims to solve

[0009] In order to solve the problem that there is no tetrahydroisoquinoline compound capable of combating coronavirus infection, the present invention provides the use of the corresponding tetrahydroisoquinoline compound in combating coronavirus (especially SARS-CoV-2) infection.

[0010] Solutions for solving problems

[0011] In a first aspect, the present invention provides the use of a compound of formula I or a pharmaceutically acceptable form thereof in the preparation of a medicament for preventing and / or treating coronavirus infection, wherein

[0012]

[0013] Each R1 is independently selected from hydrogen, C 1-6 Alkyl, C2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl and one of the following groups:

[0014]

[0015] and

[0016] At least one R1 is one of the following groups:

[0017]

[0018] Each R2 is independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl and C 6-10 Aryl;

[0019] Each R3 is independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, C 6-10 Aryl and 5-10 membered heteroaryl;

[0020] The pharmaceutically acceptable form is selected from pharmaceutically acceptable salts.

[0021] In a second aspect, the present invention provides the use of a compound of formula II or a pharmaceutically acceptable form thereof in the preparation of a medicament for preventing and / or treating coronavirus infection, wherein

[0022]

[0023] Each R1 is independently selected from hydrogen, and the two R1 are not hydrogen at the same time;

[0024] Each R2 is independently C 1-4 Alkyl, preferably methyl, ethyl and isopropyl, more preferably methyl;

[0025] Each R3 is independently selected from hydrogen and C 1-4 Alkyl, preferably hydrogen, methyl, ethyl and isopropyl, more preferably hydrogen and methyl;

[0026] The pharmaceutically acceptable form is selected from pharmaceutically acceptable salts.

[0027] In a third aspect, the present invention provides the use of the following compounds or pharmaceutically acceptable forms thereof in the preparation of a medicament for preventing and / or treating coronavirus infection:

[0028]

[0029] The pharmaceutically acceptable form is selected from pharmaceutically acceptable salts.

[0030] In a fourth aspect, the present invention provides the use of a pharmaceutical composition comprising a compound of Formula I or Formula II or a pharmaceutically acceptable form thereof in the preparation of a medicament for preventing and / or treating coronavirus infection.

[0031] In a fifth aspect, the present invention provides a compound of formula I or formula II or a pharmaceutically acceptable form thereof, or a pharmaceutical composition comprising a compound of formula I or formula II or a pharmaceutically acceptable form thereof, which is used as an anti-coronavirus drug.

[0032] In a sixth aspect, the present invention provides a method for preventing and / or treating a disease caused by coronavirus infection, comprising the step of administering a therapeutically effective amount of a compound of Formula I or Formula II or a pharmaceutically acceptable form thereof, or a therapeutically effective amount of the above-mentioned pharmaceutical composition, to an individual in need thereof.

[0033] Effects of the Invention

[0034] The results of in vitro cell tests show that the tetrahydroisoquinoline compounds of the present invention (especially dauricine, dauricine, liesinine, isoliensinine and methyl neferine) have little toxic effect on coronavirus-susceptible human cells, but can significantly inhibit the infection of SARS-CoV-2 and SARS-CoV pseudovirus-susceptible human cells, and have a significant inhibitory effect on SARS-CoV-2 and SARS-CoV pseudovirus infection at the cellular level, and are dose-dependent, so they can be used as coronavirus inhibitors, especially as SARS-CoV-2 inhibitors, to treat COVID-19 caused by human infection with the virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The CC of dauricine and dauricine on HEK293T-ACE2 cells is shown. 50 Measurement results.

[0036] Figure 2 IC values ​​of dauricine and dauricine for inhibiting SARS-CoV-2 pseudovirus are shown 50 Measurement results.

[0037] Figure 3 IC values ​​of dauricine and dauricine for inhibiting SARS-CoV pseudovirus are shown 50 Measurement results.

[0038] Figure 4The CC of liensinine, isoliensinine and methyl neferine on HEK293T-ACE2 cells is shown. 50 Measurement results.

[0039] Figure 5 The IC values ​​of neferine, isoliensinine and neferine for inhibiting SARS-CoV-2 pseudovirus are shown. 50 Measurement results.

[0040] Figure 6 The IC values ​​of neferine, isoliensinine and neferine for inhibiting SARS-CoV pseudovirus are shown. 50 Measurement results. DETAILED DESCRIPTION

[0041] Bis-tetrahydroisoquinoline compounds

[0042] The present invention provides the use of a compound of formula I or a pharmaceutically acceptable form thereof in the preparation of a medicament for preventing and / or treating coronavirus infection, wherein

[0043]

[0044] Each R1 is independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, C 6-10 Aryl, 5-10 membered heteroaryl and one of the following groups:

[0045]

[0046] and

[0047] At least one R1 is one of the following groups:

[0048]

[0049] Each R2 is independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl and C 6-10 Aryl;

[0050] Each R3 is independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, C 6-10 Aryl and 5-10 membered heteroaryl;

[0051] The pharmaceutically acceptable form is selected from pharmaceutically acceptable salts.

[0052] In one embodiment of the present invention, in the compound of formula I above,

[0053] Each R1 is independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 6-10 Aryl and one of the following:

[0054]

[0055] and

[0056] At least one R1 is one of the following groups:

[0057]

[0058] In a preferred embodiment of the present invention, in the compound of formula I above,

[0059] Each R1 is independently selected from hydrogen and one of the following groups:

[0060]

[0061] and

[0062] The two R1's are not hydrogen at the same time.

[0063] In a more preferred embodiment of the present invention, in the compound of formula I above,

[0064] Each R1 is independently selected from hydrogen, And the two R1 are not hydrogen at the same time.

[0065] In one embodiment of the present invention, each R2 in the above-mentioned compound of formula I is independently selected from hydrogen, C 1-6 Alkyl and C 3-6 Cycloalkyl.

[0066] In a preferred embodiment of the present invention, each R2 in the above-mentioned compound of formula I is independently selected from hydrogen and C 1-6 alkyl.

[0067] In a more preferred embodiment of the present invention, each R2 in the above-mentioned compound of formula I is independently C 1-6 alkyl.

[0068] In one embodiment of the present invention, each R3 in the above compounds of formula I is independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl and C 6-10 Aryl.

[0069] In a preferred embodiment of the present invention, each R3 in the above-mentioned compound of formula I is independently selected from hydrogen, C 1-6 Alkyl and C 3-6 Cycloalkyl.

[0070] In a more preferred embodiment of the present invention, each R3 in the above-mentioned compound of formula I is independently selected from hydrogen and C 1-6 alkyl.

[0071] In one embodiment of the present invention, in the compound of formula I above,

[0072] Each R1 is independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 6-10 Aryl and one of the following:

[0073]

[0074] and

[0075] At least one R1 is one of the following groups:

[0076]

[0077] Each R2 is independently selected from hydrogen, C 1-6 Alkyl and C 3-6 Cycloalkyl;

[0078] Each R3 is independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl and C 6-10 Aryl.

[0079] In a preferred embodiment of the present invention, in the compound of formula I above,

[0080] Each R1 is independently selected from hydrogen and one of the following groups:

[0081]

[0082] and

[0083] The two R1 are not hydrogen at the same time;

[0084] Each R2 is independently selected from hydrogen and C 1-6 alkyl;

[0085] Each R3 is independently selected from hydrogen, C 1-6 Alkyl and C 3-6 Cycloalkyl.

[0086] In a more preferred embodiment of the present invention, in the compound of formula I above,

[0087] Each R1 is independently selected from hydrogen, and the two R1 are not hydrogen at the same time;

[0088] Each R2 is independently C 1-6 Alkyl, preferably C 1-4 Alkyl, more preferably methyl, ethyl and isopropyl, most preferably methyl;

[0089] Each R3 is independently selected from hydrogen and C 1-6 Alkyl, preferably hydrogen and C 1-4 Alkyl, more preferably hydrogen, methyl, ethyl and isopropyl, most preferably hydrogen and methyl.

[0090] In a more preferred embodiment of the present invention, the compound of formula I is a compound of formula II, wherein

[0091]

[0092] Each R1 is independently selected from hydrogen, and the two R1 are not hydrogen at the same time;

[0093] Each R2 is independently C 1-4 Alkyl, preferably methyl, ethyl and isopropyl, more preferably methyl;

[0094] Each R3 is independently selected from hydrogen and C 1-4 As the alkyl group, hydrogen, methyl, ethyl and isopropyl are preferred, and hydrogen and methyl are more preferred.

[0095] Specifically, the compounds of formula I or formula II include the following specific bis-tetrahydroisoquinoline compounds.

[0096]

[0097]

[0098] Pharmaceutically acceptable form

[0099] Unless otherwise specified, the term "pharmaceutically acceptable form" referred to in the context of this specification refers to a pharmaceutical form derived from the original form (usually the free form) of the active pharmaceutical ingredient (API) that can be administered to an individual in need thereof, including salts (e.g., addition salts formed between a free compound and an acid or base).

[0100] Pharmaceutical composition

[0101] Unless otherwise specified, the term "pharmaceutical composition" mentioned in the context of this specification refers to a composition that can be used as a medicament, which comprises a pharmaceutically active ingredient (API) and optionally one or more pharmaceutically acceptable carriers.

[0102] Unless otherwise specified, the term "pharmaceutically acceptable carrier" mentioned in the context of this specification refers to pharmaceutical excipients that are compatible with the active ingredients of the drug and harmless to the individual, including (but not limited to) diluents (or fillers), binders, disintegrants, lubricants, wetting agents, thickeners, glidants, flavoring agents, olfactory agents, preservatives, antioxidants, pH adjusters, solvents, cosolvents, surfactants, etc.

[0103] The present invention provides the use of a pharmaceutical composition comprising the above-mentioned compound of formula I or formula II (or its specific compound) or a pharmaceutically acceptable form thereof in the preparation of a medicament for preventing and / or treating coronavirus infection.

[0104] In one embodiment of the present invention, the above pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0105] Prevention and / or treatment

[0106] Unless otherwise indicated, the term "prevention" mentioned in the context of this specification refers to the preventive administration of an active ingredient (e.g., a compound of Formula I or Formula II of the present invention (or its specific compound) or a pharmaceutically acceptable form thereof) or a pharmaceutical composition (e.g., a pharmaceutical composition of the present invention) to a healthy individual in need thereof to prevent the occurrence of a disease.

[0107] Unless otherwise indicated, the term "treatment" mentioned in the context of this specification refers to the therapeutic administration of (e.g., a compound of Formula I or Formula II of the present invention (or its specific compound) or a pharmaceutically acceptable form thereof) or a pharmaceutical composition (e.g., a pharmaceutical composition of the present invention) to a sick individual in need thereof to slow down, interrupt or terminate the progression of the disease, but not to completely eliminate all symptoms.

[0108] Coronavirus infection

[0109] Unless otherwise specified, the term "coronavirus" mentioned in the context of this specification refers to a virus belonging to the genus Coronavirus in the family Coronaviridae of the order Nidovirales in terms of systematic classification.

[0110] In one embodiment of the invention, the coronavirus is selected from HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2.

[0111] In a preferred embodiment of the present invention, the coronavirus is selected from SARS-CoV, MERS-CoV and SARS-CoV-2.

[0112] In a more preferred embodiment of the present invention, the coronavirus is selected from SARS-CoV and SARS-CoV-2.

[0113] Diseases caused by coronavirus infection

[0114] The present invention provides a method for preventing and / or treating a disease caused by coronavirus infection, comprising the step of administering a therapeutically effective amount of the above-mentioned compound of Formula I or Formula II (or its specific compound) or a pharmaceutically acceptable form thereof, or a therapeutically effective amount of the above-mentioned pharmaceutical composition to an individual in need thereof.

[0115] In one embodiment of the present invention, the disease caused by coronavirus infection is selected from severe acute respiratory syndrome (SARS) and novel coronavirus infection (COVID-19).

[0116] Severe Acute Respiratory Syndrome (SARS)

[0117] Unless otherwise specified, the term "severe acute respiratory syndrome" mentioned in the context of this specification refers to the acute respiratory infectious disease caused by SARS-CoV, which the World Health Organization (WHO) named severe acute respiratory syndrome.

[0118] Middle East Respiratory Syndrome (MERS)

[0119] Unless otherwise specified, the term "Middle East Respiratory Syndrome" mentioned in the context of this specification refers to the acute respiratory infectious disease caused by MERS-CoV, which was named Middle East Respiratory Syndrome by the World Health Organization (WHO).

[0120] Novel Coronavirus Infection (COVID-19)

[0121] Unless otherwise specified, the term "new coronavirus infection" referred to in the context of this specification refers to the acute respiratory infectious disease caused by SARS-CoV-2, which the World Health Organization (WHO) named Middle East Respiratory Syndrome.

[0122] Therapeutically effective amount

[0123] Unless otherwise specified, the term "therapeutically effective amount" mentioned in the context of this specification refers to the amount of a drug (active ingredient or combination thereof or pharmaceutical composition comprising active ingredient or combination thereof) sufficient to affect a disease or symptom when administered to an individual with the intent to prevent and / or treat a disease or at least one clinical symptom thereof. It will be understood by those skilled in the art that the "therapeutically effective amount" may vary depending on factors such as the nature of the active ingredient, the extent of the disease and / or symptom, and the individual's condition.

[0124] Application

[0125] Unless otherwise indicated, the term "administration" mentioned in the context of this specification refers to systemic and / or local administration. The term "systemic administration" refers to non-local administration, so that the administered substance may affect several organs or tissues of the individual, or so that the administered substance may pass through several organs or tissues of the individual to reach the target of action. It will be understood by those skilled in the art that "systemic administration" covers various forms of administration, including (but not limited to) parenteral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, oral administration, etc. The term "local administration" refers to administration at or around a specific site. It will be understood by those skilled in the art that "local administration" covers various forms of administration, such as direct injection into a specific site or injection into its surroundings (e.g., administration within a tumor).

[0126] individual

[0127] Unless otherwise indicated, the term "subject" referred to in the context of this specification includes (but is not limited to) humans (males or females of any age group, such as infants, toddlers or adolescents in pediatric subjects, and young adults, middle-aged or elderly in adult subjects) and non-human animals (such as monkeys, cows, pigs, horses, sheep, mice, rabbits, cats or dogs in mammals).

[0128] In one embodiment of the invention, the subject in the method is a human.

[0129] The technical solutions of the present invention will be further explained or illustrated in conjunction with specific embodiments below. Unless otherwise specified, the instruments, materials or reagents used in the following embodiments can be obtained by conventional commercial means.

[0130] Test materials

[0131] Human embryonic kidney cells (HEK293T-ACE2) overexpressing ACE2, as well as SARS-CoV-2 and SARS-CoV retrovirus (MLV)-based pseudoviruses were prepared and stored in the laboratory of Suzhou Institute of Systems Medicine; the cell growth medium was DMEM containing 10% FBS.

[0132] Example 1: Toxicity test of dauricine and dauricine on cells.

[0133] 1. Test method:

[0134] 1) HEK293T-ACE2 cells with good growth status were taken for digestion and subculture, and the cell density was adjusted to 1×10 4 / mL, inoculate 96-well plates, 100 μL / well, and culture in a 37°C, 5% CO2 incubator for 16 h;

[0135] 2) After 16 hours, the culture medium in the wells was discarded, and then the cell maintenance solution was added, and dauricine and dauricine were added at the same time to make the final concentrations of 128μM, 64μM, 32μM, 16μM, 8μM, 4μM, 2μM, 1μM, 0.5μM, 0.25μM and 0.125μM. At the same time, a cell control group was set up and placed in a 37°C, 5% CO2 incubator for culture;

[0136] 3) After 48 hours, use reagents to detect cell viability.

[0137] 2. Test results:

[0138] Determination of cell survival rate can reflect the toxic effects of dauricine and dauricine on HEK293T-ACE2 cells. The results are shown in Figure 1 .from Figure 1 As can be seen, the half cytotoxic concentration (CC 50 ) were 23.6 μM and 20.8 μM, respectively, indicating that dauricine and dauricine had less toxic effects on HEK293T-ACE2 cells.

[0139] Example 2: Anti-SARS-CoV-2 and SARS-CoV pseudovirus activity test of dauricine and dauricine.

[0140] 1. Test method:

[0141] 1) HEK293T-ACE2 cells with good growth status were taken for digestion and subculture, and the cell density was adjusted to 1×10 4 / mL, inoculate 96-well plates, 100 μL / well, and culture in a 37°C, 5% CO2 incubator for 16 h;

[0142] 2) After 16 h, dauricine and dauricine were diluted with cell maintenance medium to a concentration of 16 μM, 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM and 0.25 μM, respectively;

[0143] 3) Mark the lid of the 96-well plate, discard the culture medium in the wells, add 100 μL of diluted dauricine and dauricine to each well in the marked order, add 100 μL of SARS-CoV-2 or SARS-CoV pseudovirus to each well, so that the final concentrations of dauricine and dauricine are 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM and 0.125 μM, set up a virus control group, and culture in a 37°C, 5% CO2 incubator;

[0144] 4) After 48 h, pseudovirus infection detection was performed using luciferase substrate reagent.

[0145] 2. Test results:

[0146] from Figure 2 and Figure 3 It can be seen that the inhibitory effects of dauricine and dauricine on SARS-CoV-2 and SARS-CoV pseudovirus are dose-dependent. Among them, the half-maximal inhibitory dose concentration (IC 50 ) were 0.22 μM and 0.23 μM, respectively, and the half maximal inhibitory dose concentration (IC 50 ) were 0.16 μM and 0.13 μM, respectively.

[0147] Example 3: Toxicity test of liesinine, isoliensinine and neferine on cells.

[0148] 1. Test method:

[0149] 1) HEK293T-ACE2 cells with good growth status were taken for digestion and subculture, and the cell density was adjusted to 1×10 4 / mL, inoculate 96-well plates, 100 μL / well, and culture in a 37°C, 5% CO2 incubator for 16 h;

[0150] 2) After 16 hours, the culture medium in the wells was discarded, and then the cell maintenance solution was added, and neferine, isoliensinine and neferine were added at the same time to make the final concentrations of 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM and 0.125 μM. At the same time, a cell control group was set up and placed in a 37°C, 5% CO2 incubator for culture;

[0151] 3) After 48 hours, use reagents to detect cell viability.

[0152] 2. Test results:

[0153] Determination of cell survival rate can reflect the toxic effects of liesinine, isoliensinine and methyl neferine on HEK293T-ACE2 cells. The results are shown in Figure 4 .from Figure 4 As can be seen, the half cytotoxic concentration (CC 50 ) were 29.8 μM, 23.5 μM and 39.9 μM, respectively, indicating that liensinine, isoliensinine and methyl liensinine had less toxic effects on HEK293T-ACE2 cells.

[0154] Example 4: Anti-SARS-CoV-2 and SARS-CoV pseudovirus activity test of liesinine, isoliensinine and methyl neferine.

[0155] 1. Test method:

[0156] 1) HEK293T-ACE2 cells with good growth status were taken for digestion and subculture, and the cell density was adjusted to 1×10 4 / mL, inoculate 96-well plates, 100 μL / well, and culture in a 37°C, 5% CO2 incubator for 16 h;

[0157] 2) After 16 h, the cells were maintained in a cell maintenance medium to dilute liesinine, isoliensinine and neferine to concentrations of 16 μM, 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM and 0.25 μM, respectively;

[0158] 3) Mark the lid of the 96-well plate, discard the culture medium in the wells, add 100 μL of diluted liesinine, isoliensinine and neferine to each well in the marked order, add 100 μL of SARS-CoV-2 or SARS-CoV pseudovirus to each well, so that the final concentrations of liesinine, isoliensinine and neferine are 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM and 0.125 μM, set up a virus control group, and culture in a 37°C, 5% CO2 incubator;

[0159] 4) After 48 h, pseudovirus infection detection was performed using luciferase substrate reagent.

[0160] 2. Test results:

[0161] from Figure 5 and Figure 6 It can be seen that the inhibitory effects of liesinine, isoliensinine and methyl neferine on SARS-CoV-2 and SARS-CoV pseudovirus are dose-dependent. Among them, the half maximum inhibitory dose concentration (IC 50) were 0.28μM, 0.27μM and 0.29μM, respectively, and the half maximal inhibitory dose concentration (IC 50 ) were 0.13 μM, 0.14 μM and 0.15 μM respectively.

Claims

1. Use of the following compounds or their pharmaceutically acceptable forms in the preparation of drugs for preventing and / or treating coronavirus infection: The pharmaceutically acceptable form is selected from pharmaceutically acceptable salts.

2. A pharmaceutical composition comprising a compound used in the application according to claim 1 or a pharmaceutically acceptable form thereof in the preparation of a medicament for preventing and / or treating coronavirus infection, wherein the pharmaceutically acceptable form is selected from a pharmaceutically acceptable salt.

3. The use according to claim 1 or 2, characterized in that: The coronavirus is selected from HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2.

4. The use according to claim 3, characterized in that: The coronavirus is selected from SARS-CoV, MERS-CoV and SARS-CoV-2.

5. The use according to claim 3, characterized in that: The coronavirus is selected from SARS-CoV and SARS-CoV-2.

6. The use according to claim 3, characterized in that: The disease caused by the coronavirus infection is selected from severe acute respiratory syndrome and new coronavirus infection.

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