Application of naphthyl skeleton compounds in the preparation of coronavirus 3CL protease inhibitors

Abstract

The present invention provides the use of naphthyl skeleton compounds in the preparation of coronavirus 3CL protease inhibitors. The compounds with naphthalene as the skeleton can effectively inhibit 3CL pro It has high activity, a high therapeutic index and a low half effective concentration, which can inhibit the activity of coronavirus and can potentially be used to develop anti-coronavirus drugs.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to the application of a class of compounds with naphthalene as a skeleton in the preparation of coronavirus 3CL protease inhibitors. Background Art

[0002] Research on the new coronavirus has entered a white-hot stage. Coronaviruses mainly include the new coronavirus pneumonia (SARS-CoV-2), severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East syndrome-related coronavirus (MERS-CoV). They are mainly positive-stranded single-stranded RNA viruses with an envelope and a diameter of about 80 to 120 nm. An attractive drug target in coronaviruses is the main protease (M pro , 3CL pro ), as it plays a crucial role in processing the polyprotein translated from viral RNA. pro It has been proven to be a drug discovery target for SARS, MERS and SARS-CoV-2 coronaviruses. Most of the functional proteins (non-structural proteins) of coronaviruses are encoded by the ORF1ab gene, which is first translated into a polyprotein (7096aa) and then converted to 3CL pro It is cut into multiple active proteins such as the viral replication protein RdRp. As an important non-structural protein in coronavirus, it has a cleavage site specificity similar to the 3CL protease of microRNA viruses. pro It is a cysteine ​​protease of about 33kDa (much smaller than S protein) composed of 306 amino acids. It can specifically recognize and cut the 11 cleavage sites of non-structural proteins NSP4-NSP16, thereby releasing other non-structural proteins of coronavirus. Therefore, 3CL pro In the current research on the development of new coronavirus drugs, it is a very popular target. However, there is no effective anti-new coronavirus drug yet. Summary of the invention

[0003] The present invention provides an application of a naphthyl skeleton compound in the preparation of a coronavirus 3CL protease inhibitor, which is expected to solve the current situation that there is no anti-coronavirus drug. The class of compounds with naphthalene as the skeleton can effectively inhibit 3CL pro activity, thereby inhibiting the activity of coronavirus, and can potentially be used to develop anti-coronavirus drugs.

[0004] The present invention is achieved through the following technical solutions:

[0005] The present invention provides an application of a naphthyl skeleton compound in the preparation of a coronavirus 3CL protease inhibitor.

[0006] The present invention also provides an application of a naphthyl skeleton compound in the preparation of a drug for preventing and / or treating diseases caused by coronavirus.

[0007] Preferably, the naphthyl skeleton compound is a pharmaceutically acceptable salt, solvent compound, enantiomer, diastereomer, tautomer or a mixture thereof in any proportion.

[0008] Preferably, the naphthyl skeleton compound is one of the following compounds:

[0009]

[0010] Preferably, the naphthyl skeleton compound is 2,3-dimethoxy-1-naphthaldehyde, 2-hydroxy-7-methoxy-1-naphthaldehyde or 1,4-dimethoxy-2-naphthaldehyde.

[0011] Preferably, the coronavirus is MERS-CoV, SARS-CoV or SARS-CoV-2.

[0012] Preferably, the disease caused by the coronavirus is an infectious disease or its complications caused by MERS-CoV, SARS-CoV, or SARS-CoV-2.

[0013] Preferably, the inhibitor or drug is in the form of tablets, capsules, granules, syrups, premixes or pellets for oral administration, or in the form of ointments or injections for non-oral administration.

[0014] Preferably, the inhibitor is a naphthyl skeleton compound used alone or a combination of a naphthyl skeleton compound and other coronavirus 3CL protease inhibitors.

[0015] Preferably, the drug is a naphthyl skeleton compound used alone or a combination of a naphthyl skeleton compound and other drugs for preventing and / or treating diseases caused by coronavirus.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention first confirmed the efficacy of naphthyl skeleton compounds on coronavirus 3CL pro The inhibitory effect of the drug is high, with a high therapeutic index and a low half effective concentration. Moreover, the use of a class of compounds with naphthalene as the skeleton before infection can effectively increase the antiviral effect. Naphthyl skeleton compounds can be used as effective drugs for the treatment of coronavirus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The inhibitory effect of 2,3-dimethoxy-1-naphthaldehyde on SARS-CoV 3CL protease in the present invention;

[0019] Figure 2 The inhibitory effect of 2-hydroxy-7-methoxy-1-naphthaldehyde on SARS-CoV 3CL protease in the present invention;

[0020] Figure 3 The inhibitory effect of 1,4-dimethoxy-2-naphthaldehyde on SARS-CoV 3CL protease in the present invention;

[0021] Figure 4 It is the inhibitory effect of 2,3-dimethoxy-1-naphthaldehyde on SARS-CoV-2 3CL protease in the present invention;

[0022] Figure 5 The inhibitory effect of 2-hydroxy-7-methoxy-1-naphthaldehyde on SARS-CoV-2 3CL protease in the present invention;

[0023] Figure 6 The inhibitory effect of 1,4-dimethoxy-2-naphthaldehyde on SARS-CoV-2 3CL protease in the present invention. DETAILED DESCRIPTION

[0024] In order to further understand the present invention, the present invention is described below in conjunction with embodiments. These descriptions are only to further explain the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0025] The following raw material sources are for illustrative purposes only.

[0026] 2,3-Dimethoxy-1-naphthaldehyde (CAS: 56252-09-6), 2-hydroxy-7-methoxy-1-naphthaldehyde (CAS: 75965-66-1), 1,4-dimethoxy-2-naphthaldehyde (CAS: 75965-83-2).

[0027] Example 1

[0028] In vitro inhibition test of SARS-CoV 3CL protease by 2,3-dimethoxy-1-naphthaldehyde, 2-hydroxy-7-methoxy-1-naphthaldehyde and 1,4-dimethoxy-2-naphthaldehyde.

[0029] Fluorescence resonance energy transfer was used to evaluate the inhibitory effects of the three compounds on the activity of SARS-CoV 3CL protease (each compound was tested in triplicate).

[0030] Weigh appropriate amounts of the above compounds and prepare solutions with appropriate concentration gradients using DMSO. Mix 5 μL of the prepared solution with 91 μL of Assay Reagent (Assay Buffer: SARS-CoV M pro / 3CLpro =90:1, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) were added to a black 96-well plate in sequence, mixed, and incubated at 37°C in the dark for 10 minutes. Then, 4 μL of Substrate (100 μM Dabcyl-KTSAVLQSGFRKME-Edans, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) was quickly added to each well and mixed. After incubation at 37°C in the dark for 5 minutes, the signal became stable. Within 5-30 minutes, a multifunctional microplate reader (Thermo Fisher Scientific, Varioskan Flash) was used to measure the fluorescence and calculate the inhibition percentage of the sample. The excitation wavelength was 340 nm and the emission wavelength was 490 nm. The Assay Reagent without the compound was used as the 100% enzyme activity control, and the Assay Reagent without SARS-CoV M pro The Assay Buffer of / 3CL protease was used as blank control, and the rest of the treatment methods were the same. Nonlinear regression analysis was performed using GraphPadPrism software to calculate the IC 50 value.

[0031]

[0032] The experimental results are shown in Table 1. In Example 2, 3-dimethoxy-1-naphthaldehyde, 2-hydroxy-7-methoxy-1-naphthaldehyde, and 1,4-dimethoxy-2-naphthaldehyde all have inhibitory activity against SARS-CoV 3CL protease, among which 2,3-dimethoxy-1-naphthaldehyde has a stronger inhibitory effect on SARS-CoV 3CL protease, IC 50 The values ​​were all around 20 μM. Figure 1-3 The inhibitory effects of 2,3-dimethoxy-1-naphthaldehyde, 2-hydroxy-7-methoxy-1-naphthaldehyde, and 1,4-dimethoxy-2-naphthaldehyde on SARS-CoV 3CL protease were shown respectively.

[0033] Table 1 Inhibition rate of SARS-CoV 3CL protease (%)

[0034]

[0035]

[0036] Example 2

[0037] In vitro inhibition test of SARS-CoV-2 3CL protease by 2,3-dimethoxy-1-naphthaldehyde, 2-hydroxy-7-methoxy-1-naphthaldehyde and 1,4-dimethoxy-2-naphthaldehyde.

[0038] Fluorescence resonance energy transfer was used to evaluate the inhibitory effects of the three compounds on SARS-CoV-2 3CL protease activity (each compound was tested three times in parallel).

[0039] Weigh an appropriate amount of the above compounds and prepare a solution with appropriate concentration gradient using DMSO. Mix 5 μL of the prepared solution with 91 μL Assay Reagent (Assay Buffer: SARS-CoV-2M pro / 3CL pro =90:1, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) were added to a black 96-well plate in sequence, mixed, and incubated at 37°C in the dark for 10 minutes. Then, 4 μL of Substrate (100 μM Dabcyl-KTSAVLQSGFRKME-Edans, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) was quickly added to each well and mixed. After incubation at 37°C in the dark for 5 minutes, the signal became stable. Within 5-30 minutes, a multifunctional microplate reader (Thermo Fisher Scientific, Varioskan Flash) was used to measure the fluorescence and calculate the inhibition percentage of the sample. The excitation wavelength was 340 nm and the emission wavelength was 490 nm. The Assay Reagent without the compound was used as the 100% enzyme activity control, and the SARS-CoV-2M was not included. pro The Assay Buffer of / 3CL protease was used as blank control, and the rest of the treatment methods were the same. GraphPadPrism software was used for nonlinear regression analysis to calculate the IC values ​​of samples 2,3-dimethoxy-1-naphthaldehyde, 2-hydroxy-7-methoxy-1-naphthaldehyde, and 1,4-dimethoxy-2-naphthaldehyde. 50 value.

[0040]

[0041] The experimental results are shown in Table 2. In Example 2, 3-dimethoxy-1-naphthaldehyde, 2-hydroxy-7-methoxy-1-naphthaldehyde, and 1,4-dimethoxy-2-naphthaldehyde all have inhibitory activity against SARS-CoV-2 3CL protease, and 2,3-dimethoxy-1-naphthaldehyde has a stronger inhibitory effect on SARS-CoV-2 3CL protease, IC 50 The values ​​are all around 20μM. Figure 4-6 The inhibitory effects of 2,3-dimethoxy-1-naphthaldehyde, 2-hydroxy-7-methoxy-1-naphthaldehyde, and 1,4-dimethoxy-2-naphthaldehyde on SARS-CoV-2 3CL protease were shown respectively.

[0042] Table 2 Inhibition rate of SARS-CoV-2 3CL protease (%)

[0043]

[0044] In summary, 2,3-dimethoxy-1-naphthaldehyde, 2-hydroxy-7-methoxy-1-naphthaldehyde, and 1,4-dimethoxy-2-naphthaldehyde showed excellent inhibitory activity against the 3CL proteases of both SARS-CoV and SARS-CoV-2 coronaviruses, and can be developed and studied as anti-coronavirus drugs.

Claims

1. Use of a naphthyl skeleton compound in the preparation of a drug for preventing and / or treating a disease caused by a coronavirus, characterized in that: The naphthyl skeleton compound is 2,3-dimethoxy-1-naphthaldehyde, 2-hydroxy-7-methoxy-1-naphthaldehyde or 1,4-dimethoxy-2-naphthaldehyde; the disease caused by the coronavirus is an infectious disease caused by SARS-CoV or SARS-CoV-2.

2. The use according to claim 1, characterized in that: The medicine is in the form of tablets, capsules, granules, syrups or pellets for oral administration, or in the form of injections for non-oral administration.

3. The use according to claim 1, characterized in that: The drug is a naphthyl skeleton compound used alone or a combination of a naphthyl skeleton compound and other drugs for preventing and / or treating diseases caused by coronavirus.

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