CN112516143B

CN112516143B - Application of dibenzyl tetrahydroisoquinoline derivative in preparation of anti-coronavirus medicines

Info

Publication number: CN112516143B
Application number: CN202011375253.9A
Authority: CN
Inventors: 黄露义; 唐霓; 汪凯; 黄爱龙; 何长龙
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2023-03-21
Anticipated expiration: 2040-11-30
Also published as: CN112516143A

Abstract

The invention discloses application of a dibenzyltetrahydroisoquinoline derivative in preparation of an anti-coronavirus medicament. The structure of the dibenzyl tetrahydroisoquinoline derivative is shown as a formula I, and the dibenzyl tetrahydroisoquinoline derivative can effectively inhibit the infection capability of various coronaviruses on cells, and particularly has excellent inhibitory activity on SARS-CoV-2 (S-D614), SARS-CoV-2 (S-G614), SARS-CoV and MERS-CoV coronavirus infection. The bisbenzylic tetrahydroisoquinoline derivative has good application prospect in preparing anti-coronavirus medicines and medicines for preventing and/or treating diseases caused by coronavirus infection.

Description

Application of dibenzyl tetrahydroisoquinoline derivative in preparation of anti-coronavirus medicines

Technical Field

The invention relates to the field of biological medicines, in particular to application of a dibenzyl tetrahydroisoquinoline derivative in preparing an anti-coronavirus medicine and a medicine for treating diseases caused by coronavirus infection.

Background

The coronaviruses which can infect human and are discovered at present are 7 types: hcoV-229E, hcoV-NL63, hcoV-HKU1, hcoV-OC43, severe acute respiratory syndrome coronavirus (SARS-CoV), zhongdong respiratory syndrome coronavirus (MERS-CoV), and 2019 novel coronavirus (2019-nCoV, SARS-CoV-2). Of these, the first 4 (HcoV-229E, hcoV-NL63, hcoV-HKU1, hcoV-OC 43) coronaviruses are seasonal epidemics in the human population worldwide, causing mild respiratory disease in most patients; the mortality rate caused by SARS-CoV and MERS-CoV infection is very high; SARS-CoV-2 is a new type of beta genus coronavirus, belonging to single-stranded RNA virus, with extremely strong infectivity and general susceptibility to the crowd.

The Receptor Binding Domain (RBD) of the coronavirus surface spike S protein is combined with a receptor on a cell, the S protein forms a trimeric compound, the S protein can be cut into two subunits of S1 and S2 by cell surface protease, the S1 subunit comprises the receptor binding domain RBD and an N-terminal domain NTD, a receptor binding motif is arranged in the middle of the RBD and is called as RBM, and the amino acid sequence of the RBM is changed greatly in the S protein of each coronavirus. The S2 subunit has three functional domains, including Fusion Peptide (FP), HR1, and HR2, which mediate the fusion of the virus with the cell into which the viral genetic material can enter for replication. If the cell surface does not have protease for cutting S protein, the virus can enter the cell in an endocytic mode, and a virus membrane can be fused with an endocytic vacuole under the action of an acid environment in the endocytic vacuole and cathepsin CatB/L to release gene substances of the virus into the cell. The modes of virus entering cells are surface membrane fusion and endocytic membrane fusion, and the surface fusion is the main mode of entry. After entry of the coronavirus into the cell, under the action of coronavirus enzymes, e.g. M ^pro (3 CL protease), rdRp (RNA-dependent RNA polymerase), replicate new viral particles and release cells. Therefore, blocking virus entry into cells is a very important anti-coronavirus strategy.

At present, although there is a certain understanding on the epidemiology, clinical characteristics, diagnosis, treatment and prevention outcome of the novel coronavirus pneumonia caused by SARS-CoV-2, the treatment still can not meet the clinical requirements, and the treatment is lack of specific drugs, and the form is still very severe. Existing antiviral drugs are mostly ineffective against SARS-CoV-2. For example, the anti-HIV drug kresoxim (lopinavir and ritonavir) has poor effect and great side effect in clinical trials for treating novel coronavirus pneumonia. Although antibodies, small proteins and lipopeptides targeting the spike S protein can effectively block the invasion of coronavirus into cells, the price is high, and effective small molecule drugs are still very deficient. Therefore, there is a need to develop novel anti-coronavirus small molecule drugs that can effectively block the invasion of coronavirus into cells.

Disclosure of Invention

The invention aims to provide application of a dibenzyltetrahydroisoquinoline derivative, or pharmaceutically acceptable salt, or crystal form, or solvate thereof in preparing an anti-coronavirus medicament and a medicament for treating diseases caused by coronavirus infection.

The invention provides an application of the following compounds, or pharmaceutically acceptable salts, or crystal forms, or solvates, or stereoisomers, or isotopic substitution compounds thereof in preparing anti-coronavirus medicines:

in the formula I, the compound is shown in the specification,

is composed of

R ₁ 、R ₂ 、R ₃ 、R ₄ Each independently selected from hydrogen, hydroxy, C _1～3 Alkyl radical, C _1～3 An alkoxy group;

R ₀ selected from hydrogen, hydroxy, C _1～3 Alkyl radical, C _1～3 Alkoxy radical, R ₅ Selected from hydrogen, hydroxy, C _1～3 Alkyl radical, C _1～3 An alkoxy group; or R ₀ And R ₅ Connection formation

R ₆ 、R ₇ 、R ₈ Each independently selected from hydrogen, hydroxy, C _1～3 Alkyl radical, C _1～3 An alkoxy group;

the compound of formula I is not

(tetrandrine),

(isoliensinine ),

(liensinine ),

(berbamine).

Further, the compound is one of the following compounds:

further, the coronavirus is a coronavirus or a variant thereof: SARS-CoV-2, SARS-CoV, MERS-CoV, hcoV-229E, hcoV-NL63, hcoV-HKU1, hcoV-OC43;

the coronavirus variant is a wild-type coronavirus or a mutant coronavirus.

Further, the variant of SARS-CoV-2 is wild-type coronavirus S-D614 or mutant coronavirus S-G614.

Further, the compounds are capable of inhibiting the entry of coronaviruses into cells.

Further, the compounds are capable of inhibiting cell fusion by coronaviruses.

The invention also provides the application of the compound, or the pharmaceutically acceptable salt, the crystal form, the solvate, the stereoisomer or the isotopic substitution compound thereof in preparing the medicine for preventing and/or treating the diseases caused by the coronavirus infection or the complications thereof; coronaviruses are as described above.

Further, the disease is a respiratory tract infection disease.

Further, the disease is a novel coronavirus pneumonia.

The invention also provides an anti-coronavirus medicine, which is a preparation prepared by taking the compound, or pharmaceutically acceptable salt, crystal form, solvate, stereoisomer or isotopic substitution compound thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

In the present invention, thalictrum wilsonii alkali is compound S1, fangchinoline alkali is compound S20, isofangchinoline alkali is compound S21, dauricinine is compound S24, neferine is compound S25, and daucosterol is compound S27.

Different variants of coronaviruses include wild-type coronavirus strains and various mutated mutant coronavirus strains.

SARS-CoV-2 (S-D614), the wild type SARS-CoV-2 virus strain, is a circulating virus strain at the initial stage of 2019 new coronavirus epidemic situation; SARS-CoV-2 (S-G614), the SARS-CoV-2 strain in which the S protein 614 position is mutated into glycine, is the most prevalent strain at present.

The dibenzyl tetrahydroisoquinoline derivative disclosed by the invention can effectively inhibit the infection capability of various coronaviruses on cells, and particularly has excellent inhibitory activity on SARS-CoV-2 (S-D614), SARS-CoV-2 (S-G614), SARS-CoV and MERS-CoV coronavirus infection. The dibenzyl tetrahydroisoquinoline derivative has good application prospect in preparing anti-coronavirus medicines and medicines for preventing and/or treating diseases caused by coronavirus infection.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1: inhibitory activity, specificity and cytotoxicity results of S1, S20, S21, S24, S25, S27, cepharanthine (Cepharanthine) and Tetrandrine (Tetrandrine) on HEK293T cells against SARS-CoV-2 (S-G614) pseudovirus and VSVG virus infection.

FIG. 2: s1, S25, cepharanthine (Cepharanthine) and Tetrandrine (Tetrandrine) were used to determine the inhibition curves of the ability of different coronaviruses (SARS-CoV-2 (S-D614), SARS-CoV-2 (S-G614), SARS-CoV and MERS-CoV) to invade cells.

FIG. 3: s1, S25, cepharanthine (CEP), tetrandrine (TET) inhibition of cell membrane fusion.

FIG. 4 is a schematic view of: and S1 inhibits the activity test result of SARS-CoV-2 pseudovirus invading cells on three cells of HEK293T, calu-3 and A549.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

The thalidomide (S1), the fangchinoline (S20), the isofangchinoline (S21), the dauricinine (S24), the neferine (S25), the dauricinine (S27), the cepharanthine and the tetrandrine are all purchased from commercial products.

Example 1: inhibitory Activity, specificity and cytotoxicity of Compounds on HEK293T cells against SARS-CoV-2 pseudovirus infection

1. Pseudoviral packaging

2x10 to ⁷ HEK293T cells were seeded in 10cm dishes (80-90% density),

after 12h, transfection was carried out (see Lipo8000 instructions) at a ratio of Opti-MEM 500. Mu.l + Lipo 8000. Mu.l + the following plasmids:

pNL4-3-Luc-R-E：VSVG＝5:1(10μg，2μg)；

pNL4-3-Luc-R-E：pCMV3-SARS-CoV-2.Spike(D614)＝1:1(6μg，6μg)；

pNL4-3-Luc-R-E：pCMV3-SARS-CoV-2.Spike(G614)＝1:1(6μg，6μg)；

pNL4-3-Luc-R-E：pCMV3-SARS-CoV.Spike＝1:1(6μg，6μg)；

pNL4-3-Luc-R-E：pCMV3-MERS-CoV.Spike＝1:1(6μg，6μg)

and (3) replacing the transfection solution after 12h, normally culturing for 48h and 72h, collecting the supernatant, centrifuging for 5min (4 ℃,4000 rpm), and collecting the virus supernatant to obtain the pseudovirus for later use.

2. Pseudovirus infection and titer determination

The experiment was performed using the Lipofectamine 3000 transfection kit (Invitrogen) and according to the manufacturer's instructions. After 48 hours of transfection, pseudoviruses expressing the spike proteins S-SARS, S-MERS, S-D614 and S-G614 were collected in the supernatant. Centrifuged and filtered through a 0.45 μm filter and stored at-80 ℃. The pNL4-3.Luc. R-E-plasmid was co-transfected with pMD2.G to collect VSVG pseudovirus. RT-qPCR was used in conjunction with primers and probes targeting LTRs to quantify copies of pseudoviruses by determining the number of viral RNA genomes per ml of viral stock. An upstream primer: 5 'TGTGTGCCCGTCTGTGT-3', a downstream primer: 5 'GAGTCCTGCGAGAGAGC-3', probe: 5'-FAM-CAGTGGCGCCCGAACAGGGA-BHQ1-3'. Viral RNA was extracted using TRIzol reagent (Invitrogen, rockville, MD). Total RNA was then amplified using TaqMan one-step RT-PCR premix reagents (Applied Biosystems, thermo Fisher). A standard curve was generated using a pNL4-3.Luc. R-E-vector with known copy number. All pseudoviruses we harvested were titrated to a uniform titer (copy number/mL) for subsequent studies.

3. Test Compounds for inhibitory Activity, specificity and cytotoxicity against pseudoviral infection

(1) Half the Effective Concentration (EC) ₅₀ ) Testing

HEK293T cells 3X 10 ⁶ 4 mug of pReceiver-M02-ACE2 plasmid is transfected in a 6cm culture dish (80% -90% density), and stable ACE2 overexpression cell strains are selected by G418. At a rate of 1X 10 per hole ⁴ Each cell was inoculated with ACE 2-overexpressed HEK293T cell line in a 96-well plate, and after 12h, 60. Mu.l of pseudovirus supernatant, which was diluted in a cell culture medium gradient, was added, together with Polybrene (5. Mu.g/ml) to increase the virus infection efficiency, in 3 duplicate wells per group. Replacing with fresh culture medium after 8h of pseudovirus infection, measuring luciferase activity after 72h, and selecting luciferase with activity up to 10 ⁴ The virus titer corresponding to RLU was followed for compound detection. In the experiment toCepharanthine and tetrandrine were used as positive controls.

(2) Specificity Index (SI) test

Specificity Index (SI) = VSVG EC ₅₀ (μM)/SARS-CoV-2EC ₅₀ (μM)

(3) Half the Cytotoxic Concentration (CC) ₅₀ ) Testing

Use of

The AQueous One Solution cell proliferation assay (G3582, promega) assesses cell viability. Briefly, HEK293T cells were transferred to 96-well plates (2 × 10) ⁴ Cells/well) in medium containing a gradient of compounds at 37 ℃,5% CO ₂ The culture was carried out in an atmosphere for 72 hours. The medium was then removed and the cells were incubated with 100. Mu.l of fresh medium. Transfer 20. Mu.l

AQueous One reagent was applied to each sample well at 5% CO ₂ Incubate for 1-4 hours at 37 ℃ in a humid atmosphere. The absorbance was recorded at 490nm using a microplate reader (Synergy H1, bioTek). From the readings, the CC of each compound was calculated ₅₀ 。

4. Results of the experiment

TABLE 1 inhibitory Activity, specificity and cytotoxicity results of test Compounds on HEK293T cells against SARS-CoV-2 (S-G614) pseudovirus and VSVG Virus infection

The results of the experiment are shown in FIG. 1 and Table 1. The experimental result shows that the compounds S1, S20, S21, S24, S25 and S27 have obvious inhibition effect on SARS-CoV-2 pseudovirus invading cells, but have no inhibition effect on VSVG virus infection basically. It shows that the compound of the present invention does not act on the HIV virus backbone, and has good specificity to SARS-CoV-2 (S-G614) virus.

Meanwhile, compounds S1, S20, S21, S24 and S25. S27 has a larger CC on HEK293T cells ₅₀ Thus, the safety was good.

Example 2: inhibitory Activity of Compounds on different cells against SARS-CoV-2 pseudovirus infection

1. Test for inhibitory Activity of test Compound against infection with pseudoVirus

The target compound was tested for its activity against viral infection on different cells by luciferase activity, with cepharanthine and tetrandrine as positive controls. HEK293T, calu-3 or A549 cells (2X 10) cultured in 96-well plates ⁴ Cells/well) were incubated with graded concentrations of each compound for 1 hour and the same amount of pseudovirus SARS-CoV-2 (S-G614) (50. Mu.L, 3.8X 10 ⁴ Copy) infection. Fresh DMEM medium was replaced 8 hours after infection. 72 hours after infection, cells were harvested and lysed with 30. Mu.l lysis buffer (Promega), RLU values were measured using luciferase assay reagent (Promega) according to product description, and the half-Effective Concentration (EC) of test compound against SARS-CoV-2 (S-G614) pseudovirus infection was calculated ₅₀ ). All data were tested at least 3 times and expressed as mean ± Standard Deviation (SD)

2. Results of the experiment

TABLE 2 inhibitory Activity of Compounds on different cells against SARS-CoV-2 (S-G614) pseudoviral infection

The results of the experiment are shown in table 2 and fig. 4. Experimental results show that S1, S24, S25 and S27 have strong inhibition effect on SARS-CoV-2 pseudovirus infection on three cells of HEK293T, calu-3 and A549. In particular S1, which blocks the EC of SARS-CoV-2 pseudovirus invading cells on three cells of HEK293T, calu-3 and A549 ₅₀ The inhibitory activity of the compounds is respectively 0.111 mu M,0.28 mu M and 2.46 mu M, and the inhibitory activity on HEK293T and Calu-3 cells is even better than that of positive control cepharanthine and tetrandrine.

Example 3: inhibitory Activity of Compounds against different coronavirus infections

1. Experimental methods

HEK293T cells (1X 10) ⁴ Per well) density into a 96-well plate, diluting a compound to be tested (50 mu M to 32.7 nM) with 2.5-fold gradient concentration after 12h, incubating the diluted compound with the equal volume of virus supernatant at 37 ℃ for 30min, adding the incubated compound into cells, setting a negative virus-free control group and a solvent blank control group as well as stephanine and tetrandrine as positive controls, culturing the cells at 37 ℃ for 48h in 3 duplicate wells. Then 30 mul of 1 Xcell lysate is added into each hole to crack cells, the cells are cracked for 15min at room temperature, 15 mul of supernatant is centrifugally taken and added with 15 mul of detection substrate, and luciferase activity is immediately detected by a microplate reader after the mixture is fully mixed. Calculating half Effective Concentration (EC) of each group according to the inhibition rate of drugs with different concentrations on coronavirus ₅₀ )。

2. Results of the experiment

TABLE 3 inhibitory Activity of S1 and S25 against different coronavirus infections

As a result, as shown in Table 3 and FIG. 2, both S1 and S25 efficiently blocked the invasion of SARS-CoV-2 (S-D614), SARS-CoV-2 (S-G614), SARS-CoV and MERS-CoV pseudovirus into cells.

Example 4: effect of Compounds on cell fusion

1. Experimental method

HEK293T was transfected as effector cells with plasmid pAdTrack-TO4-GFP encoding GFP or plasmid pS-G614 encoding the corresponding SARS-CoV-2S protein. 293T-ACE2 cells were used as target cells. 8 hours after transfection, effector cells were washed twice with PBS and pre-treated with compounds or DMSO. 24 hours after transfection, effector cells with S protein expression were overlaid on target cells at a ratio of 1. After 4 hours of co-incubation, images of syncytia were captured using an inverted fluorescence microscope. Three regions were randomly selected in each well to account for confluent and unfused cells.

2. Results of the experiment

The HEK293T cell expressing SARS-CoV-2S protein and human ACE2 protein by transfection respectively can generate cell membrane fusion phenomenon. The experimental results show (FIG. 3) that the addition of S1 (5. Mu.M) and S25 (5. Mu.M) significantly inhibited the cell membrane fusion phenomenon, compared to the blank control group.

Example 5: pharmacokinetic Property testing of Compounds

1. Experimental methods

Pharmacokinetic analysis of S1 and S25 was performed in male ICR mice. The vehicle was 5% DMSO,15% Solutol,80% physiological saline, pH7.0, administered by submandibular intravenous injection at a dose of 2mg/kg and intragastric oral administration at a dose of 10mg/kg to ICR mice. Blood was collected at the indicated times and immediately centrifuged to separate plasma, and plasma concentrations were determined using LC-MS/MS analysis.

2. Results of the experiment

TABLE 4 pharmacokinetic test results of intravenous injection and intragastric administration of S1 and S25 in mice

The pharmacokinetic properties of S1 and S25 in mice are shown in table 4, and it can be seen that both S1 and S25 have good pharmacokinetic properties, especially the compound S1, which is better.

In conclusion, the invention provides the application of the dibenzyltetrahydroisoquinoline derivative shown in the formula I in preparing anti-coronavirus medicines. The bisbenzylidenetetrahydroisoquinoline derivative can effectively inhibit the infection capability of various coronaviruses on cells, and particularly has excellent inhibitory activity on SARS-CoV-2 (S-D614), SARS-CoV-2 (S-G614), SARS-CoV and MERS-CoV coronavirus infection. The bisbenzylic tetrahydroisoquinoline derivative has good application prospect in preparing anti-coronavirus medicines and medicines for preventing and/or treating diseases caused by coronavirus infection.

Claims

1. Use of a compound or a pharmaceutically acceptable salt thereof for the manufacture of a medicament against coronavirus, characterised in that: the coronavirus is a coronavirus SARS-CoV-2 variant, the variant of SARS-CoV-2 is a mutant coronavirus S-G614;

the compound is:

2. use according to claim 1, characterized in that: the compounds are capable of inhibiting the entry of coronavirus into a cell.

3. Use according to claim 1, characterized in that: the compounds are capable of inhibiting cell fusion by coronaviruses.

4. Use of a compound or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention and/or treatment of a disease caused by a coronavirus infection or a complication thereof, characterized in that: the coronavirus is a coronavirus SARS-CoV-2 variant, the variant of SARS-CoV-2 is a mutant coronavirus S-G614; the compound is:

5. use according to claim 4, characterized in that: the disease is respiratory tract infection disease.

6. Use according to claim 5, characterized in that: the disease is a novel coronavirus pneumonia.