CN113143930B - Application of compound in preparing SARS-Cov-2 E protein inhibitor - Google Patents

Abstract

The invention discloses an application of a compound in preparing SARS-Cov-2E protein inhibitor. In a first aspect of the present application there is provided the use of a compound, or a pharmaceutically acceptable salt thereof, of the formula a-L-B; the product has at least one function of a1 to a 3: a1. inhibiting ion channel activity of SARS-Cov-2E protein; a2. reducing the pathogenicity of SARS-Cov-2; a3. preventing and/or treating SARS-Cov-2 caused coronavirus pneumonia. According to the application of the embodiment of the application, at least the following beneficial effects are achieved: the applicant finds that the compound with the structure or the pharmaceutically acceptable salt thereof has good effect on inhibiting the ion channel activity of SARS-Cov-2E protein in the experimental process, and can be used for preparing products with corresponding functions.

Description

Use of compounds in the preparation of SARS-Cov-2 E protein inhibitors

Technical Field

The present application relates to the technical field of antiviral drugs, and in particular to the use of compounds in the preparation of SARS-Cov-2 E protein inhibitors.

Background Art

COVID-19 is a pneumonia caused by the novel coronavirus SARS-CoV-2. In addition to the development of diagnostic reagents and vaccines, drugs that can directly treat COVID-19 are also needed. Many highly pathogenic viruses encode small molecule membrane proteins with ion channel activity, and the ion channels of viruses are crucial to the pathogenicity of viruses and can be used as ideal drug targets. For example, the anti-influenza A virus drug amantadine is an ion channel antagonist. Existing research results show that all coronaviruses, including SARS-CoV-2, encode a small hydrophobic envelope (E) protein. Taking severe acute respiratory syndrome coronavirus (SARS-CoV) as an example, the ion channel activity of its E protein is crucial to the pathogenicity and pathogenesis of the virus. The E protein sequences of SARS-CoV and SARS-CoV-2 have a high degree of homology. In view of this, the E protein of SARS-CoV-2 is likely to also have ion channel activity and play a crucial role in the pathogenicity of the virus. Therefore, SARS-CoV-2 E protein inhibitors targeting the E protein of SARS-CoV-2 are very likely to play an important role in the treatment of COVID-19. However, no effective inhibitors of the E protein of the new coronavirus SARS-CoV-2 have been found so far.

Summary of the invention

The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes the use of a compound in the preparation of a SARS-Cov-2 E protein inhibitor.

In a first aspect of the present application, a compound or a pharmaceutically acceptable salt thereof is provided for use in preparing a product, wherein the compound has the general formula A-L-B;

Among them, A is

^R1 is selected from any one of hydrogen, substituted or unsubstituted hydrocarbon group, substituted or unsubstituted acyl group;

R ² to R ⁵ are independently selected from any one of a carbonyl group and a substituted or unsubstituted methylene group;

L is selected from at least one of substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted heteroalkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted heteroalkynylene, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, and substituted or unsubstituted heteroarylene;

R⁶选自氢、卤素、氰基、硝基、叠氮基、取代或未取代的烷基、取代或未取代的烯基、取代或未取代的炔基、取代或未取代的芳基、-OR⁷、-SR⁸、-NR⁹R¹⁰中的任一种,R⁷～R¹⁰分别独立选自氢、取代或未取代的烷基、取代或未取代的烯基、取代或未取代的炔基、取代或未取代的芳基中的任一种；B is

^R6 is selected from any one of hydrogen, halogen, cyano, nitro, azido, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, ^-OR7 , ^-SR8 , ^-NR9R10 , ^R7 to ^R10 are independently selected from any one of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl ^;

The product has at least one of the functions a1 to a3:

a1. Inhibit the ion channel activity of SARS-Cov-2 E protein;

a2. Reduce the pathogenicity of SARS-Cov-2;

a3. Prevent and/or treat novel coronavirus pneumonia caused by SARS-Cov-2.

According to the application of the embodiment of the present application, there are at least the following beneficial effects:

The applicant discovered during the experiment that the compound having the above structure or a pharmaceutically acceptable salt thereof has a good effect on inhibiting the ion channel activity of the SARS-Cov-2 E protein, and thus can be used to prepare products with corresponding functions.

Among them, pathogenicity refers to the ability of the virus to cause host infection, and the indicators of the degree of infection of the virus to the host include pathogenicity and virulence. Existing studies have shown that the ion channel protein of the virus has a very important influence on the replication and release of the virus. Therefore, by inhibiting the ion channel activity of the virus-specific protein, it becomes an important way to inhibit the pathogenicity of the virus. Specific to this scheme, the above-mentioned compound can affect the ion channel activity of the SARS-Cov-2 E protein to a great extent, and naturally it can also have an important impact on its pathogenicity, and then can prevent and/or treat the new coronavirus pneumonia caused by SARS-Cov-2 to a certain extent.

In some embodiments of the present application, ^R1 is selected from any one of hydrogen, substituted or unsubstituted hydrocarbon group, substituted or unsubstituted acyl group, wherein the carbon number of the hydrocarbon group or the acyl group is 1 to 20; further, the carbon number is 1 to 10; further, the carbon number is 1 to 5 or 1 to 3; non-limiting examples of substituents of the hydrocarbon group or the acyl group include halogen, cyano, nitro, etc.

In some embodiments of the present application, non-limiting examples of substituents for the methylene group in R ² to R ⁵ include halogen, cyano, nitro, hydrocarbon group, halogenated hydrocarbon group, and the like.

In some embodiments of the present application, R ² to R ⁵ are independently selected from any one of a carbonyl group, a methylene group, and a methylene group substituted with a methyl group.

In some embodiments of the present application, A is selected from any one of the following:

In some embodiments of the present application, A is selected from any one of the following:

In some embodiments of the present application, non-limiting examples of substituents on the group of L include halogen, cyano, nitro, hydrocarbon group, halogenated hydrocarbon group, and the like.

In some embodiments of the present application, the number of carbon atoms in L is 1-40; further, the number of carbon atoms is 1-30; further, the number of carbon atoms is 1-20 or 1-15.

其中，L¹选自亚烷基、卤代亚烷基、亚烷氧基、卤代亚烷氧基、含氮亚杂环基、卤代含氮亚杂环基中的任一种或不存在。In some embodiments of the present application, L is

Wherein, ^L1 is selected from any one of an alkylene group, a halogenated alkylene group, an alkyleneoxy group, a halogenated alkyleneoxy group, a nitrogen-containing heterocyclic group, and a halogenated nitrogen-containing heterocyclic group, or is absent.

In some embodiments of the present application, L is selected from any one of the following:

In some embodiments of the present application, non-limiting examples of substituents for the alkyl, alkenyl, alkynyl, and aryl groups on the R ⁶ group include halogen, cyano, nitro, hydrocarbon, halogenated hydrocarbon, and the like.

In some embodiments of the present application, R ⁶ is selected from any one of alkyl, halogenated alkyl, alkoxy, and halogenated alkoxy.

In some embodiments of the present application, R ⁶ is any one of trifluoromethyl and methoxy.

In some embodiments of the present application, the compound has the following general formula:

In some embodiments of the present application, the compound is selected from any one of the following:

、

,

In some embodiments of the present application, the pharmaceutically acceptable salt is a hydroxy naphthoate. Non-limiting examples of hydroxy naphthoates include 1-hydroxy-2-naphthoate, 3-hydroxy-2-naphthoate, 4-hydroxy-2-naphthoate, 5-hydroxy-2-naphthoate, 6-hydroxy-2-naphthoate, 7-hydroxy-2-naphthoate, 8-hydroxy-2-naphthoate, etc.

Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is the result of the ion channel activity assay of the SARS-CoV-2E protein in Example 1 of the present application.

Figure 2 is the measurement result of the inhibitory effect of the salt of compound AZD5153 in Example 2 of the present application on the ion channel activity of SARS-CoV-2E protein.

Figure 3 is a binding model of the salt of compound AZD5153 and SARS-CoV-2E protein in Example 2 of the present application.

FIG4 is a result of measuring the inhibitory effect of compound ABBV-075 on the ion channel activity of SARS-CoV-2E protein in Example 3 of the present application.

DETAILED DESCRIPTION

The following will clearly and completely describe the concept of the present application and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of them. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present application.

The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

In the description of this application, "several" means more than one, "more" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

Example 1

SARS-CoV-2E protein ion channel activity assay

(1) Expression of SARS-CoV-2E protein

The SARS-CoV-2E protein gene (NCBI reference sequence: NC_045512.2) was synthesized by GENEWIZ and subcloned into the pcDNA3.1 vector with IRES-GFP. The above plasmid was transiently transfected into the Chinese hamster ovary cell line (CHO) using Lipofectamine 3000 reagent to express the SARS-CoV-2E protein.

(2) Testing the ion channel activity of SARS-CoV-2E protein

Using untransfected CHO cells as a control, the ion channel activity of SARS-CoV-2E protein was detected by whole-cell voltage clamp experiment. The specific process is as follows:

The cells were clamped at 0 mV, and a step voltage from -100 mV to 100 mV was applied to the cells, with an increment of 10 mV. The maximum current stimulated by the cells under different voltages was detected, and the results are shown in Figure 1.

At -100mV voltage, the current size of untransfected CHO cells was -88.5±24.4pA (n=13), and the current size of CHO cells expressing SARS-CoV-2E protein was -169.9±73.7pA (n=15); at 100mV voltage, the current size of untransfected CHO cells was 154.9±42.8pA (n=13), and the current size of CHO cells expressing SARS-CoV-2E protein was 300.6±136.5pA (n=15). Compared with CHO cells that do not express SARS-CoV-2E protein, CHO cells expressing SARS-CoV-2E protein stimulated significantly increased inward and outward currents under voltage stimulation. The unpaired t test showed that the current size of the two groups of cells under the same voltage stimulation was significantly different (p<0.05).

The above experimental results indicate that SARS-CoV-2E protein has ion channel activity.

Example 2

Through homology modeling, a pentameric structural model of the SARS-CoV-2E protein was established. Since the E protein sequences of SARS-CoV and SARS-CoV-2 have a high homology (94.7%), the structural model of the SARS-CoV-2E protein was obtained by homology modeling based on the protein structure of SARS-CoV. Using the structural model of the SARS-CoV-2E protein, a total of 5,000 compounds were virtually screened using the listed drug libraries, clinical drug libraries, and natural product libraries. The screening results include the 6-hydroxy-2-naphthoate salt of the compound AZD5153 (CAS No: 1869912-40-2, molecular formula: C ₃₆ H ₄₁ N ₇ O ₆ , molecular weight: 667.7540), the structural formula of which is as follows:

Verify the compound's inhibitory effect on the ion channel activity of SARS-CoV-2E protein

The compound with a final concentration of 40 μM was added to the cell recording external solution, and the SARS-CoV-2E protein was detected to have ion channel activity using the method in Example 1. The results are shown in FIG2 .

Under the action of this compound, the current of CHO cells expressing SARS-CoV-2E protein was -56.8±21.1pA (n=13) at a voltage of -100mV; the current of CHO cells expressing SARS-CoV-2E protein was 102.4±11.6pA (n=13) at a voltage of 100mV. Compared with the case without adding the compound, the cell current was significantly reduced, and the unpaired t-test showed that the current of the two groups of cells under the same voltage stimulation was significantly different (p<0.05).

The above experimental results show that the compound has a strong inhibitory effect on the ion channel activity of SARS-CoV-2E protein.

Figure 3 is a partial schematic diagram of the binding model of the salt of compound AZD5153 and SARS-CoV-2E protein in Example 2 of the present application. Referring to Figure 3, the hydrophobic pocket of SARS-CoV-2E protein is composed of Val25, Phe26, Val29 of one subunit and the interface formed by Phe23 and Phe26 of the adjacent subunit, wherein Phe23 and Val29 are the key sites for the interaction between AZD5153 and E protein, and the piperazine group of AZD5153 (A in the A-L-B general formula) may interact with residues such as Phe23, Phe26, and Val29 of two adjacent subunits, while its triazolopyridazine group (B in the A-L-B general formula) may interact with residues such as Leu18, Leu19, and Asn15 of the other two subunits of the pentamer, thereby inhibiting the conduction of cations therebetween by changing its steric hindrance, thereby affecting the ion channel activity of SARS-CoV-2E protein. Therefore, the compounds of the general formula A-L-B or their salts that meet the requirements of the embodiments of the present application can significantly inhibit the ion channel activity of the SARS-CoV-2E protein, and thus can be used to reduce the pathogenicity of SARS-Cov-2, or prevent and/or treat the new coronavirus pneumonia caused by SARS-Cov-2.

Example 3

Existing studies have shown that the compound AZD5153 can bind to the bromodomain on the BET (bromodomain and extraterminaldomain) protein BRD4 (bromodomain-containing protein 4, i.e., bromodomain protein 4), to a certain extent preventing the protein-protein interaction between the BET protein and the acetylated histones and transcription factors, and targeting the inhibition of the BRD4 protein, thereby delaying the proliferation of tumor cells and even inducing apoptosis of tumor cells, thereby achieving an anti-tumor effect (Expert Opinion on Therapeutic Patents, 2014, 24 (2): 185-199; Nature Structural & Molecular Biology, 2014, 21 (12): 1047-1057). To this end, the inventors further used other known BRD4 inhibitors as examples to study whether they can also achieve the inhibition of the ion channel activity of the SARS-CoV-2E protein. The pyridone compound ABBV-075 was used in the verification process, and its structural formula is as follows:

The method of Example 2 was used for detection, and the results are shown in Figure 4. The experimental results showed that compared with the case where no compound ABBV-075 was added, the cell current did not decrease significantly, and the unpaired t-test showed that there was no significant difference in the current magnitude between the two groups of cells under the same voltage stimulation (p>0.05).

Example 4

1-Hydroxy-2-naphthoate, 3-hydroxy-2-naphthoate and 6-hydroxy-2-naphthoate of the compound with the following structural formula were respectively taken, and their inhibitory effects on the ion channel activity of SARS-CoV-2E protein were verified by the method in Example 2. For the preparation of the compound, see Journal of Medicinal Chemistry, 2016, 59(17): 7801-7817:

、

,

The results showed that there was a significant decrease in cell current after the addition of the corresponding compounds compared with when no corresponding compounds were added, indicating that these compounds also have a significant inhibitory effect on the ion channel activity of the SARS-CoV-2E protein.

It can be seen from the above experiments that the compounds with specific structures provided in the embodiments of the present application can be used as ion channel activity inhibitors of SARS-CoV-2E protein. In addition, studies on various coronaviruses have shown that ion channel proteins have a very important effect on the replication and release of viruses. Therefore, the above compounds and their salts can also have an important impact on the pathogenicity of SARS-CoV-2, and thus can prevent and/or treat the new coronavirus pneumonia caused by SARS-Cov-2 to a certain extent.

The present application is described in detail above in conjunction with the embodiments, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims (2)

1. The purposes of compound or pharmaceutically acceptable salt thereof in the preparation product, it is characterized in that, described compound is selected from following any one:

,

; The product has at least one function in a1~a3: a1. Inhibit the ion channel activity of SARS-Cov-2 E protein; a2. Reduce the pathogenicity of SARS-Cov-2; a3. Prevention and/or treatment of novel coronavirus pneumonia caused by SARS-Cov-2. 2. The use according to claim 1, characterized in that the pharmaceutically acceptable salt is hydroxynaphthoate.

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