Aminoisoquinoline derivatives and their application in the preparation of antiviral drugs

Abstract

This invention provides an aminoisoquinoline derivative and its application in the preparation of antiviral drugs. The invention reveals that the aminoisoquinoline derivative exhibits significant inhibitory effects against various RNA and DNA viruses, demonstrating broad-spectrum activity. This small-molecule inhibitor has low toxicity and does not affect the normal growth of host cells. It shows good antiviral efficacy even at low concentrations. The results of this invention indicate that the compound has the potential to be used in the preparation of antiviral drugs and has promising clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of aminoisoquinoline derivatives in the preparation of antiviral drugs. Background Technology

[0002] Stearoyl-CoA desaturases (Scd) are primarily involved in fatty acid synthesis in organisms, and their protein family members are highly expressed in multiple species. Of particular interest is Scd1, a delta-9 desaturase within the Scd family, whose catalytic activity plays a crucial role in the replication of various RNA viruses. Scd1's possible mechanism of action involves increasing membrane fluidity in physiologically relevant phospholipid bilayers through its catalytic product oleic acid, or altering the curvature of biological membrane structures. This characteristic of oleic acid molecules facilitates the formation of closed vesicle-like structures within cells, and viruses require lipid droplets and specialized double-membrane vesicles in the host for replication.

[0003] Isoquinoline derivatives are a very important class of nitrogen-containing heterocyclic compounds, widely found in natural products, drug molecules, and organic functional materials. Studies have shown that these compounds possess significant physiological activities, such as antitumor, antianalgesic, antiarrhythmic, and antithrombotic effects. Therefore, isoquinoline derivatives have wide applications in medicine, agricultural chemistry, and materials science. Particularly in biomedicine, isoquinoline derivatives can lower progesterone levels, measure functional activity, and have anti-ulcer properties. With further research, isoquinoline derivatives have also exhibited good pharmacological activities, such as antiviral, anti-inflammatory, and anti-insect activities.

[0004] Currently, with the application of molecular targeted therapy in the treatment of malignant tumors, more and more virological studies have uncovered new specific molecular sites. Targeted therapy against specific molecular sites can also greatly improve the host's antiviral efficacy.

[0005] Therefore, it is necessary to develop a new broad-spectrum antiviral drug. Summary of the Invention

[0006] The purpose of this invention is to provide the application of aminoisoquinoline derivatives in the preparation of antiviral drugs. This application has found that aminoisoquinoline derivatives have significant inhibitory effects on a variety of RNA and DNA viruses, and have good broad-spectrum activity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect of the present invention, an aminobenzoic acid derivative is provided, the aminobenzoic acid derivative having the following structural formula:

[0009]

[0010] Where R is H or an alkali metal atom.

[0011] In a second aspect of the invention, a pharmaceutically acceptable salt of an aminoisoquinoline derivative is provided.

[0012] The preparation method of the aminobenzoic acid derivative includes reacting 7-amino-1,2,3,4-tetrahydroisoquinoline, naphthic acid, and cyclopropionic acid to obtain the derivative. The aminobenzoic acid derivative was successfully prepared by detection.

[0013] Furthermore, the pharmaceutically acceptable salt includes inorganic or organic salts.

[0014] The inorganic salts include one of hydrochloride, hydrobromide, sulfate, nitrate and phosphate; the organic salts include one of methanesulfonate, maleate, tartrate, succinate, acetate, trifluoroacetate, fumarate, citrate, citric acid, benzenesulfonate, benzoate, benzenesulfonate, lactate and malate.

[0015] In a third aspect of the invention, the use of aminoisoquinoline derivatives in the preparation of antiviral drugs is provided.

[0016] Furthermore, the virus includes RNA viruses and DNA viruses, wherein the RNA virus includes one of foot-and-mouth disease virus, parainfluenza virus, rubella virus, and Coxsackie virus, and the DNA virus includes one of mumps virus and adenovirus.

[0017] Furthermore, the concentration of the aminoisoquinoline derivative is 0.01 μM-10 μM.

[0018] Furthermore, the aminoisoquinoline derivatives in the antiviral drug target the host stearoyl-CoA desaturase Scd1 for antiviral activity.

[0019] Furthermore, the antiviral drug also includes pharmaceutically acceptable excipients and carriers.

[0020] Furthermore, the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, or colorants.

[0021] Furthermore, the dosage form of the antiviral drug includes at least one of granules, tablets, pills, capsules, injections, or dispersants.

[0022] Furthermore, the antiviral mechanism of the antiviral drug includes: inhibiting viral nucleic acid replication, viral protein expression, and infection within cells.

[0023] In a fourth aspect of the invention, an antiviral drug is provided, wherein the active ingredient of the antiviral drug comprises the aminoisoquinoline derivatives or pharmaceutically acceptable salts thereof.

[0024] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0025] This invention relates to the application of aminoisoquinoline derivatives in the preparation of antiviral drugs. The application reveals that aminoisoquinoline derivatives exhibit significant inhibitory effects against various RNA and DNA viruses, demonstrating broad-spectrum activity. These small-molecule inhibitors exhibit low toxicity and do not affect the normal growth of host cells. They also demonstrate good antiviral efficacy at low concentrations. The results of this invention indicate that the compounds have the potential to be used in the preparation of antiviral drugs and possess promising clinical application prospects. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The structural formula is that of an aminobenzoic acid derivative;

[0028] Figure 2 The results are for FMDV titer detection.

[0029] Figure 3 HY-Q28157 is used to suppress FMDV replication;

[0030] Figure 4 Effect of HY-Q28157 treatment on Scd1 expression levels in cells;

[0031] Figure 5 The effect of co-treatment of cells with HY-Q28157 and oleic acid on the expression level of FMDV 3D;

[0032] Figure 6 The effect of HY-Q28157 treatment method on its antiviral effect; among which Figure 6 A represents the method of drug administration. Figure 6 B shows that different administration methods all have significant inhibitory effects on FMDV; Figure 6 C is a schematic diagram illustrating the effect of adding HY-Q28157 8 hours or 4 hours before viral infection on viral infection. Figure 6 D represents the qRT-PCR detection result.

[0033] Figure 7HY-Q28157 inhibits the replication of RNA viruses HPIV1, HPIV2, HPIV3, Rubella, and MuV. Detailed Implementation

[0034] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0035] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0036] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0037] The technical solution of this application embodiment is to solve the above-mentioned technical problems, and the general idea is as follows:

[0038] 1. Targeting Scd1 as a specific site, an aminobenzoic acid derivative was designed and synthesized, with the following structural formula: Figure 1 R shown represents H or an alkali metal atom.

[0039] When R is H, it is named aminobenzoic acid derivative HY-Q28157, with the chemical formula C. 24 H 22 N2O2 has a molecular weight of 370.44.

[0040] 2. The toxicity of HY-Q28157 to host cells was detected by CCK-8 assay.

[0041] 3. The qRT-PCR experiment confirmed that the target of the inhibitor HY-Q28157 is Scd1.

[0042] 4. Through TCID 50 The experiment used foot-and-mouth disease virus (FMDV) as a model, and the virus titer was 5 × 10⁻⁶. - 6 TCID 50 / mL.

[0043] 5. The working concentration of the inhibitor HY-Q28157 was determined by qRT-PCR experiments, showing that the small molecule compound HY-Q28157 can significantly inhibit foot-and-mouth disease virus (FMDV) at the RNA level at a concentration of 0.01 μM.

[0044] 6. Different inhibitor treatments were set up to explore the optimal administration method of the inhibitor HY-Q28157. The results are as follows: Figure 6 As shown in Figure B, different administration methods all have significant inhibitory effects on FMDV, but the treatment method of administering the drug before infection is more effective.

[0045] 7. To verify the broad-spectrum antiviral efficacy of the inhibitor HY-Q28157 in parainfluenza virus, rubella virus, coxsackie virus, mumps virus, and adenovirus.

[0046] In summary, the results of this invention indicate that the compound has the potential to be used to prepare a broad-spectrum antiviral drug with good prospects for clinical application.

[0047] The present application will now be described in detail with reference to embodiments and experimental data.

[0048] Example 1: The small molecule compound HY-Q28157 significantly inhibited foot-and-mouth disease virus (FMDV).

[0049] I. Experimental reagents: Syrian hamster kidney cells BHK-21, rhesus monkey kidney cells LLC-MK2, African green monkey kidney cells Vero, and human non-small cell lung cancer cells A549, CCK-8 Kit.

[0050] II. Experimental Procedure:

[0051] 1. BHK-21 cells were cultured at a concentration of 5 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL of cell suspension per well in 96-well plates. When cell confluence reached approximately 70-80%, the original culture medium was aspirated, and fresh MEM medium containing HY-Q28157 at concentrations of 0.05 μM, 0.1 μM, 2 μM, 5 μM, and 10 μM was added to each well, with eight replicates for each concentration. DMSO was used as a blank control. After incubation at 37°C with 5% CO2 for 48 h, the original culture medium was aspirated, and 100 μL of fresh medium containing 10% CCK-8 solution was added, followed by incubation for 2 h. The absorbance of each well was measured at 450 nm using a Labserv K3 microplate reader. Cell viability was calculated based on the absorbance of each well. The viability percentage (%) was calculated as follows: OD 450 Sample / OD 450 Blank control × 100%.

[0052] Table 1. Toxicity of different concentrations of aminoisoquinoline derivative HY-Q28157 on four cell types

[0053]

[0054]

[0055] 2. Use titers of 10 respectively. -4 TCID 50 / mL, 10 -5 TCID 50 / mL, 5×10 -6 TCID 50 / mL and 10 -6 TCID 50 BHK-21 cells were infected with FMDV at a concentration of / mL, and the time to cytopathic effect (CPE) was observed after infection with cells at different viral titers.

[0056] The results are as follows Figure 2 As shown, the final titer determined to be 5 × 10⁻⁶ was used in subsequent experiments. -6 TCID 50 / mL of FMDV was used to detect the antiviral effect of HY-Q28157.

[0057] 3. When the BHK-21 cell density in the T-25 culture flask reaches 80%-90%, digest the cells with trypsin and seed them into 24-well plates. Gently shake the 24-well plate to distribute the cells evenly and incubate at 37°C in a 5% CO2 incubator. After cell attachment, use a 5×10⁻⁶ droplet of [agent name missing] to [cell concentration missing]. -6 TCID 50 Infect cells with FMDV at a concentration of 100 μL / mL, adding 100 μL of virus solution to each well, and incubate at 37°C with 5% CO2 for 1 h. Discard the virus solution in the wells, add MEM medium containing 2% FBS, and add compound HY-Q28157 to final concentrations of 0.01 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, and 10 μM, respectively. Collect the cells when CPE occurs, extract RNA, and perform qRT-PCR detection.

[0058] The results are as follows Figure 3 As shown, the small molecule compound HY-Q28157 can significantly inhibit foot-and-mouth disease virus (FMDV) at the RNA level at a concentration of 0.01 μM.

[0059] 4. To verify that the designed and synthesized small molecule compound HY-Q28157 regulates FMDV replication by inhibiting Scd1, BHK-21 cells were seeded into 24-well plates. After seeding, the plates were gently shaken to ensure even cell distribution, and then incubated at 37°C in a 5% CO2 incubator. After cell adhesion, cells were treated with a titer of 5 × 10⁻⁶. -6FMDV was used to infect cells, with 100 μL of virus solution added to each well. The cells were incubated at 37°C with 5% CO2 for 1 hour, then the medium was replaced with 2% MEM and cultured further. Cells were harvested when CPE appeared and analyzed by qRT-PCR. Results showed that ( Figure 4 Similar to the commercially available Scd1 inhibitor MK-8245, HY-Q28157 does not affect Scd1 RNA levels. Oleic acid (OA) is the end product of Scd1 catalysis. Treatment with HY-Q28157 or the commercially available Scd1 inhibitor MK-8245, followed by the addition of 200 μM OA, revealed by qRT-PCR that both HY-Q28157 and MK-8245 inhibited FMDV 3D RNA levels, but the addition of OA significantly restored 3D expression. Figure 5 ).

[0060] Example 2: The effect of the method of adding small molecule compound HY-Q28157 on its antiviral activity.

[0061] To investigate the effect of the method of adding the small molecule compound HY-Q28157 on its antiviral activity, two methods were selected: adding HY-Q28157 8 hours before viral infection and then replenishing the same concentration of HY-Q28157 during culture change after viral infection, or adding it only 1 hour after viral infection during culture change. A schematic diagram of the drug addition method is shown below. Figure 6 A. After culturing cells for 24 hours, samples were collected and qRT-PCR was performed to investigate the effect of different administration methods on the antiviral efficacy of HY-Q28157.

[0062] The results are as follows Figure 6 As shown in Figure B, different administration methods all showed significant inhibitory effects on FMDV, but the pre-infection administration method was more effective. Furthermore, to determine the optimal timing for pre-infection compound administration, HY-Q28157 was added 8 hours or 4 hours before viral infection to investigate its impact on viral infection. The schematic diagram is shown below. Figure 6 C, and the results were obtained by qRT-PCR detection. Figure 6 D) Adding HY-Q28157 8 hours or 4 hours before infection had no significant difference in the effect on FMDV infection.

[0063] Example 3: The broad-spectrum regulation of viral replication by the small molecule compound HY-Q28157 through inhibition of Scd1.

[0064] To verify the broad-spectrum regulation of viral replication by the small molecule compound HY-Q28157 through inhibition of Scd1, the regulation of viral nucleic acid levels by HY-Q28157 was investigated in RNA viruses: parainfluenza virus type 1 (HPIV1), type 2 (HPIV2), and type 3 (HPIV3), rubella virus, and mumps virus (MuV), and DNA viruses: adenovirus type 3 (ADV3) and adenovirus type 5 (ADV5). LLC-MK2 and A549 cells, the host cells corresponding to the above viruses, were seeded into 24-well plates. After coating, the plates were gently shaken to ensure even distribution of cells, and incubated at 37°C with 5% CO2. After cell adhesion, the corresponding host cells were infected with an appropriate titer of virus, with 100 μL of virus solution added to each well, and incubated at 37°C with 5% CO2 for 1 h. Discard the virus solution in the well plates, add MEM medium containing 2% FBS, and add compound HY-Q28157 to final concentrations of 0.1 μM and 5 μM, respectively. Collect the cells when CPE occurs, extract RNA, and perform qRT-PCR detection. The results are shown in Table 2. Figure 7 As shown.

[0065] Table 2. HY-Q28157 inhibits the replication of DNA viruses ADV3 and ADV5.

[0066]

[0067] From Table 2, Figure 7 It was found that the small molecule compound HY-Q28157 inhibited the nucleic acid levels of HPIV1, HPIV2, HPIV3, Rubella, MuV, ADV3, and ADV5 to varying degrees, indicating that HY-Q28157 has a certain broad-spectrum inhibitory effect on viral replication. It has the potential to be further developed into a clinically effective broad-spectrum antiviral drug.

[0068] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0069] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0070] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A pharmaceutically acceptable salt of an aminoisoquinoline derivative, characterized in that, The aminoisoquinoline derivatives have the following structural formula: Where R stands for H.

2. The pharmaceutically acceptable salt of the aminoisoquinoline derivative according to claim 1, characterized in that, Pharmaceutically acceptable salts include inorganic or organic salts.

3. The use of a pharmaceutically acceptable salt or structural formula of the aminoisoquinoline derivative according to any one of claims 1-2 in the preparation of an antiviral drug, characterized in that, The virus is an RNA virus and a DNA virus. The RNA virus is one of foot-and-mouth disease virus, parainfluenza virus, rubella virus, and Coxsackie virus. The DNA virus is one of mumps virus and adenovirus.

4. The application according to claim 3, characterized in that, The concentration of the aminoisoquinoline derivative is 0.01 μM-10 μM.

5. An antiviral drug, characterized in that, The active ingredient of the antiviral drug comprises a pharmaceutically acceptable salt of an aminoisoquinoline derivative as described in any one of claims 1-2, or an aminoisoquinoline derivative with the structural formula as described in claim 1.

6. The antiviral drug according to claim 5, characterized in that, The antiviral drugs also include pharmaceutically acceptable excipients.

7. The antiviral drug according to claim 6, characterized in that, The excipients include at least one of fillers, disintegrants, binders, lubricants, sweeteners, or colorants.

8. The antiviral drug according to claim 6, characterized in that, The dosage form of the antiviral drug includes at least one of granules, tablets, pills, capsules, injections, or dispersants.

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