Application of compound of 3CL protease inhibitor

Abstract

The present invention provides compounds that are 3CL protease inhibitors and their use in the treatment / or prophylaxis of viral infections, such as coronavirus infections. Experiments prove that the compounds have a relatively strong inhibition effect on 3CL protease, and can block virus replication by inhibiting the activity of the virus main protease 3CL so as to achieve an antiviral treatment effect. The compound has a novel structure prospect for treating and / or preventing coronavirus infection.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a variety of compounds that can be used as 3CL protease inhibitors and uses of the compounds. Background Art

[0002] Although most SARS-CoV-2 patients have a good prognosis, it is highly pathogenic to high-risk groups such as the elderly, and has developed into severe or even fatal acute respiratory distress syndrome. SARS-CoV-2 belongs to the genus Betacoronavirus and is an enveloped positive single-stranded RNA (+ssRNA) virus. It enters the host cell through the spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2), uncoats and releases the viral RNA genome into the cytoplasm, and then the viral RNA genome is translated into two polyprotein precursors ppla and pplab by the cell. The main protease (Mpro), also known as 3CL protease (3C-like protease, 3CLpro), is a conserved cysteine ​​protease that can process the polyprotein precursors ppla and pplab at no less than 11 conserved sites, releasing most of the non-structural functional proteins (NSPS), and participating in the replication and transcription process of the virus. The key role of 3Clpro in the viral life cycle and the lack of homologous proteins in human cells make 3Clpro a very attractive target for anti-SARS-CoV-2. Currently, many inhibitors targeting 3Clpro have been reported, and non-covalent, non-peptide small molecule 3CL protease inhibitors are used as 3CL protease inhibitors to treat coronavirus-related diseases, especially the new coronavirus infection caused by SARS-CoV-2.

[0003] Pfizer's 3CL protease inhibitor PF-07321332 has shown good antiviral effects in preclinical and clinical trials, but its covalent and peptidomimetic structural characteristics result in poor pharmacokinetic properties, and it must be combined with a CYP3A4 inhibitor to obtain good oral bioavailability. Therefore, the drug Paxlovid is a combination of PF-07321332 and the anti-HIV-1 drug ritonavir, which requires twice-a-day administration. Many patients with impaired liver and kidney function and whose drugs are dependent on or induce CYP3A4 metabolic activity cannot benefit from it. In addition, PF-07321332 has a complex structure, up to 6 chiral centers, a long synthetic route, and limited supply of the key intermediate caronanoic anhydride, which also affects scale-up production and clinical accessibility.

[0004] Therefore, the development of new 3CL protease inhibitors is of great significance. Summary of the invention

[0005] The purpose of the present invention is to provide a non-covalent, non-peptide novel structure 3CL protease inhibitory compound, which inhibits the activity of the viral main protease 3CL and blocks the replication of the virus, thereby achieving an antiviral therapeutic effect.

[0006] The present invention provides the following compounds, stereoisomers, tautomers or mixtures thereof and isotopic derivatives of the compounds, and pharmacologically acceptable salts of the compounds:

[0007]

[0008] It should be noted that the preferred embodiments of the present invention can be combined arbitrarily.

[0009] The purpose of the present invention also includes providing stereoisomers, tautomers or mixtures thereof of the compounds shown in the present invention, and the use of pharmaceutically acceptable salts of the compounds in the preparation of drugs for treating and / or preventing coronavirus infection.

[0010] Experiments have confirmed that these compounds have a strong inhibitory effect on 3CL protease, and can achieve antiviral therapeutic effects by inhibiting the activity of the main viral protease 3CL, blocking viral replication, and thus providing novel and promising compounds for the treatment and / or prevention of coronavirus infection.

[0011] Beneficial effects of the present invention:

[0012] In vitro experiments showed that the compounds of the present invention, luteolin-7-O-β-D-glucuronide, 2,5-dihydroxyphenylacetic acid, neoeriocin, licorice chalcone E, oleanolic acid and epitheanogalloside 3-O-gallic acid, have the effect of inhibiting the SARS-CoV-2 target 3CL protease. Therefore, the research results of the present invention can broaden the clinical application scope of such compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 .Structural formulas of luteolin-7-O-β-D-glucuronide, 2,5-dihydroxyphenylacetic acid, neoeriocin, licoricechalcone E, oleanolic acid and epitheanogalloside 3-O-gallate.

[0014] Figure 2 .Inhibition rate (%) of negative control group, positive control group (ebselen), luteolin-7-O-β-D-glucuronide, 2,5-dihydroxyphenylacetic acid, neoeriocin, licorice chalcone E, oleanolic acid and epitheanogalloside 3-O-gallate on 3CL protease activity.

[0015] Figure 3 .(A) The inhibitory activity dose-effect curve of glycyrrhizic chalcone E on 3CL protease; (B) The inhibitory activity dose-effect curve of oleanolic acid on 3CL protease; (C) The inhibitory activity dose-effect curve of epitheagalloin 3-O-gallic acid on 3CL protease. DETAILED DESCRIPTION

[0016] The following is a further description of the substantial content of the invention with reference to the accompanying drawings, but does not limit the invention in any way. The following is a further description of the invention with reference to specific embodiments.

[0017] Unless otherwise specified, the various chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels, but do not limit the scope of the method to be protected by the present invention.

[0018] Luteolin-7-O-β-D-glucuronide, 2,5-dihydroxyphenylacetic acid, neoeriocin, licorice chalcone E, oleanolic acid, and epithea galloline 3-O-gallate were purchased from Chengdu Pusi Biotechnology Co., Ltd. 3CLpro activity detection kit and dimethyl sulfoxide (DMSO) were purchased from Shanghai Biotech Co., Ltd. The detection platform was the Ensight multi-function microplate reader of PE.

[0019] Example 1: 3CL protease inhibitory activity experiment

[0020] The 3CLpro activity detection kit used in this experiment contains Assay Buffer (AB), 3CLpro stock solution and positive molecule Ebselen, as well as a fluorescent substrate (Probe) based on FRET technology. According to the instructions, a 50μL detection system was prepared, and the Assay Reagent (AR) used was composed of 90μL AB and 1μL 3CLpro stock solution.

[0021] (1) Preliminary screening of 3CL protease inhibitory activity

[0022] A negative control group NC (AB: 40 μL; probe: 10 μL), a positive control group PC (AR: 35 μL; probe: 10 μL; ebselen: 5 μL) and a sample group (AR: 35 μL; probe: 10 μL; sample group compounds: luteolin-7-O-β-D-glucuronide, 2,5-dihydroxyphenylacetic acid, neoeriocin, licorice chalcone E, oleanolic acid, or epithea gallate 3-O-gallic acid, the concentration of each sample group compound was 100 μM, the volume was 5 μL, and the solvent was Assaybuffer containing 0.1% DMSO) were set up. A 1.5 mL PBS was added to each group of reagents. In the EP tube, each reagent group or concentration group has 3 replicate wells. After vortex mixing for 15 seconds, the plate reader is immediately used for detection (the detection temperature of the plate reader is set at 37°C, the excitation light wavelength is 325nm, the emission light wavelength is 393nm, and the double track oscillation is 5s before reading the plate) to read the fluorescence signal value.

[0023] The results showed that luteolin-7-O-β-D-glucuronide, 2,5-dihydroxyphenylacetic acid, neoeriocin, licorice chalcone E, oleanolic acid and epithea gallate 3-O-gallic acid could inhibit the activity of 3CL protease. At a compound concentration of 100 μM, the inhibition rate was more than 90% ( Figure 2 and Table 1), indicating that the above compounds are all 3CL protease inhibitors.

[0024] Table 1 Inhibition rate (%) of each sample on 3CL protease activity.

[0025]

[0026] (2) Evaluation of 3CL protease inhibitory activity dose

[0027] A negative control group NC (AB: 40 μL; probe: 10 μL) and a sample group (AR: 35 μL; probe: 10 μL; sample group compounds: licorice chalcone E and oleanolic acid were set at 8 concentrations: 200 μM, 40 μM, 8 μM, 1.6 μM, 0.32 μM, 0.064 μM, 0.013 μM, 0.003 μM, and the solvent was Assay buffer containing 0.1% DMSO; the concentrations of epithea gallate 3-O-gallic acid were: 200 μM, 50 μM, 12.5 μM, 3.125 μM, 0.781 μM, 0.195 μM, 0.049 μM, 0.012 μM, the volume was 5 μL, and the solvent was Assay buffer containing 0.1% DMSO. Each reagent or concentration group was added into a 1.5 mL EP tube, and each reagent or concentration group was divided into 3 replicate wells. After vortex mixing for 15 seconds, the plate was immediately tested using an ELISA reader (the detection temperature of the ELISA reader was set at 37°C, the excitation wavelength was 325 nm, the emission wavelength was 393 nm, and the double-track oscillation was performed for 5 seconds before reading the plate) to read the fluorescence signal value.

[0028] The results showed that licorice chalcone E, oleanolic acid and epithea gallate 3-O-gallate could inhibit the activity of 3CL enzyme in a dose-dependent manner ( Figure 3 ), is a 3CL protease inhibitor, with a specific IC 50 See Table 2 for values.

[0029] Table 2 IC values ​​of compounds with 3CL inhibitory activity 50 value.

[0030]

Claims

1. The use of a compound refers to the use of the compound in the preparation of a 3CL protease inhibitor or in the preparation of a drug for treating and / or preventing viral infection, wherein the compound is one or more of the compounds shown in the following formula, and one or more of the stereoisomers, tautomers or mixtures thereof and isotopic derivatives thereof, or pharmaceutically acceptable salts of the compound, wherein the compound structure is as follows:

2. The use according to claim 1, characterized in that: It comprises the compound according to any one of claims 1, and one or more of the stereoisomers, tautomers or mixtures thereof, or pharmaceutically acceptable salts of the compound as active ingredients.

3. The use according to claim 1 or 2, characterized in that: in, The viral infection is coronavirus infection.

4. The use according to claim 2, characterized in that: The prepared 3CL protease inhibitor or the prepared drug for treating and / or preventing viral infection also includes a pharmaceutically acceptable carrier or excipient.

5. The use according to claim 1 or 2, characterized in that: The compound is one or more of the pharmaceutically acceptable salts of luteolin-7-O-β-D-glucuronide, 2,5-dihydroxyphenylacetic acid, neoeriocin, licorice chalcone E, oleanolic acid and epitheanogalloside 3-O-gallic acid.

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