Aminobenzoic acid derivatives, pharmaceutically acceptable salts, antiviral drugs and uses

Abstract

The present application relates to an aminobenzoic acid derivative, a pharmaceutically acceptable salt, an antiviral drug and its application, which has the following general formula structure: wherein R1 is H or an alkali metal atom, R2 is H or an alkali metal atom, and R3 is a halogen atom. The present application designs an aminobenzoic acid derivative with the host Scd1 as a specific target. Through antiviral detection, it is found that the small molecule compound has a significant inhibitory effect on foot-and-mouth disease virus, parainfluenza virus, rubella virus, coxsackie virus, mumps virus and adenovirus, and has no obvious toxic effect on the host cells BHK‑21, LLC‑MK2 and A549 corresponding to foot-and-mouth disease virus, parainfluenza virus, rubella virus, coxsackie virus, mumps virus and adenovirus. The present application proves that the Scd1-specific aminobenzoic acid derivative has good antiviral activity and can be developed and applied as an antiviral drug, which opens up new ideas for the exploration of antiviral drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an aminobenzoic acid derivative, a pharmaceutically acceptable salt, an antiviral drug and application. BACKGROUND

[0002] Stearoyl-CoA desaturase (Scd) is mainly involved in the synthesis of fatty acids in organisms, and the Scd family is contained in many species. Scd1 is a delta-9 desaturase, which is a rate-limiting enzyme for catalyzing the biosynthesis of saturated fatty acid precursors into monounsaturated fatty acids. The catalytic product of the enzyme, monounsaturated fatty acids (such as oleic acid, diacylglycerol, phospholipid, triglyceride (TG), palmitoleic acid and cholesteryl ester), plays a key role in the formation of the viral replication complex.

[0003] Aminobenzoic acid and its derivatives have been widely used in the field of biological medicine. For example, in the research related to liver cancer, it is found that a derivative of aminobenzoic acid can inhibit the growth of liver cancer tumors and reduce the drug resistance to sorafenib. In addition, aminobenzoic acid derivatives are used as preservatives in a large number of skin care products commonly used by people. Aminobenzoic acid ethyl ester is also used for local anesthesia in surgery due to its analgesic effect. Aminobenzoic acid and its derivatives have been widely used in antiviral reactions. For example, aminobenzoic acid derivatives designed for HIV-1 host restriction factor A3G protein have good inhibitory effect on HIV virus. The main mechanisms of their antiviral effects include protecting host cells, inhibiting viral replication, and changing the stability of viral capsid proteins.

[0004] At the cellular and molecular level, specific target protein inhibitors designed for known antiviral proteins can achieve the effect of specifically killing viruses in the host body.

[0005] It is still difficult to find suitable aminobenzoic acid derivatives as inhibitors targeting Scd1 for antiviral activity. SUMMARY

[0006] The present application provides an aminobenzoic acid derivative, a pharmaceutically acceptable salt, an antiviral drug and application. The present application proves that the Scd1-specific aminobenzoic acid derivative has good antiviral activity and can be used as an antiviral drug for research and development, which opens up a new way for exploring antiviral drugs.

[0007] In a first aspect, an aminobenzoic acid derivative is provided, which has the following general structure:

[0008]

[0009] wherein R1 is H or an alkali metal atom, R2 is H or an alkali metal atom, and R3 is a halogen atom.

[0010] In some embodiments, R1 is H, R2 is H, and R3 is Cl.

[0011] In a second aspect, there is provided a pharmaceutically acceptable salt of the aminobenzoic acid derivative as described above.

[0012] In some embodiments, the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt.

[0013] In some embodiments, the inorganic acid salt is selected from at least one of a hydrochloride, a hydrobromide, a hydrofluoride, a sulfate, a nitrate, and a phosphate.

[0014] In some embodiments, the organic acid salt is selected from at least one of a formate, an acetate, a propionate, a citrate, a methanesulfonate, an ethanesulfonate, a malonate, a succinate, a fumarate, a maleate, a lactate, a malate, an oxalate, and a tartrate.

[0015] In a third aspect, there is provided an antiviral medicament comprising an active substance, the active substance comprising the aminobenzoic acid derivative as described above, and one or more of the pharmaceutically acceptable salts as described in any one of the above.

[0016] In some embodiments, the virus comprises one or more of a foot-and-mouth disease virus, a parainfluenza virus, a rubella virus, a coxsackie virus, a mumps virus, and an adenovirus.

[0017] In a fourth aspect, there is provided use of the aminobenzoic acid derivative as described above, or the pharmaceutically acceptable salt as described in any one of the above, in the manufacture of an antiviral medicament.

[0018] In some embodiments, the virus comprises one or more of a foot-and-mouth disease virus, a parainfluenza virus, a rubella virus, a coxsackie virus, a mumps virus, and an adenovirus.

[0019] The technical solutions provided in the present application have the following beneficial effects:

[0020] The present application designs an amino benzoic acid derivative with host Scd1 as a specific target. Through antiviral detection, it is found that the small molecule compound has obvious inhibitory effect on foot-and-mouth disease virus, parainfluenza virus, rubella virus, coxsackie virus, mumps virus and adenovirus, and has no obvious toxic effect on the corresponding host cells BHK-21, LLC-MK2 and A549 of foot-and-mouth disease virus, parainfluenza virus, rubella virus, coxsackie virus, mumps virus and adenovirus. The present application proves that the amino benzoic acid derivative specific to Scd1 has good antiviral activity, and can be used as an antiviral drug for research and application, which opens up a new way for exploring antiviral drugs. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0022] Figure 1 FMDV titer detection schematic diagram provided for the embodiments of the present application;

[0023] Figure 2 HY-Q05978 inhibits FMDV 3D replication results provided for the embodiments of the present application;

[0024] Figure 3 The results of the influence of HY-Q05978 on the expression level of Scd1 after treating cells are provided for the embodiments of the present application;

[0025] Figure 4 The results of the influence of HY-Q05978 and oleic acid on the expression level of FMDV 3D after treating cells are provided for the embodiments of the present application;

[0026] Figure 5 The results of the influence of HY-Q05978 on the antiviral effect of different adding methods are provided for the embodiments of the present application, wherein (A) is the different adding methods of HY-Q05978, (B) is the antiviral effect influence results corresponding to different adding methods, (C) is the different adding time of HY-Q05978, and (D) is the antiviral effect influence results corresponding to different adding time;

[0027] Figure 6 The results of HY-Q05978 inhibiting RNA viruses HPIV1, HPIV2, HPIV3, Rube l l a and MuV replication provided by the embodiments of the present application. DETAILED DESCRIPTION

[0028] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0029] The embodiments of the present application provide an aminobenzoic acid derivative, which has the following general structure:

[0030]

[0031] wherein R1 is H or an alkali metal atom, R2 is H or an alkali metal atom, and R3 is a halogen atom. As an example, the alkali metal can be selected from Li, Na and K, and the halogen atom can be selected from Br, Cl and I.

[0032] As a preferred example, R1 is H, R2 is H, and R3 is Cl, and the structure is as follows:

[0033]

[0034] The chemical formula of the aminobenzoic acid derivative is C 21 H 16 Cl NO4, and the molecular weight is 381.81. The aminobenzoic acid derivative is referred to as “HY-Q05978”.

[0035] The present application also provides a pharmaceutically acceptable salt of the aminobenzoic acid derivative.

[0036] The pharmaceutically acceptable salt includes, but is not limited to, at least one inorganic acid salt of the compound of the general formula, such as hydrochloride, hydrobromide, hydrofluoride, sulfate, nitrate and phosphate, and / or at least one organic acid salt of the compound of the general formula, such as formate, acetate, propionate, citrate, methanesulfonate, ethanesulfonate, malonate, succinate, fumarate, maleate, lactate, malate, oxalate and tartrate.

[0037] In a specific embodiment, the pharmaceutically acceptable salt is selected from at least one inorganic acid salt of hydrochloride, hydrobromide, hydrofluoride, sulfate, nitrate and phosphate, and / or the pharmaceutically acceptable salt is selected from at least one organic acid salt of formate, acetate, propionate, citrate, methanesulfonate, ethanesulfonate, malonate, succinate, fumarate, maleate, lactate, malate, oxalate and tartrate.

[0038] The present application also provides an antiviral drug comprising an active substance, which comprises the aminobenzoic acid derivative provided in the above embodiments, and one or more of the pharmaceutically acceptable salts provided in the above embodiments.

[0039] The above antiviral drug can further comprise a pharmaceutical carrier, which refers to the pharmaceutical carriers commonly used in the pharmaceutical field, such as pharmaceutically acceptable solid or liquid excipients and / or adjuvants, etc.

[0040] That is, the aminobenzoic acid derivative provided in the above embodiments and / or the pharmaceutically acceptable salt provided in the above embodiments can be combined with pharmaceutically acceptable solid or liquid excipients and / or adjuvants to form any dosage form suitable for human or animal use.

[0041] The aminobenzoic acid derivative and the pharmaceutically acceptable salt thereof provided in the present application can be prepared into ordinary preparations, or into sustained-release preparations, controlled-release preparations, targeted preparations, and various microparticle drug delivery systems.

[0042] In order to prepare the aminobenzoic acid derivative and the pharmaceutically acceptable salt thereof provided in the present application into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropyl alcohol, etc.; the binders can be starch paste, dextrin, sugar syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; the lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0043] The tablets can be further prepared into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0044] To make the administration unit into a capsule, the effective component, i.e. the aminobenzoic acid derivative and pharmaceutically acceptable salt thereof provided in the present application, can be mixed with a diluent and a glidant, and the mixture can be directly placed into a hard capsule or a soft capsule. Alternatively, the effective component, i.e. the aminobenzoic acid derivative and pharmaceutically acceptable salt thereof provided in the present application, can be first mixed with a diluent, a binder and a disintegrant to form granules or pellets, and then placed into a hard capsule or a soft capsule. The diluent, binder, wetting agent, disintegrant and glidant used for preparing the tablet of the aminobenzoic acid derivative and pharmaceutically acceptable salt thereof provided in the present application can also be used for preparing the capsule of the aminobenzoic acid derivative and pharmaceutically acceptable salt thereof provided in the present application.

[0045] To make the aminobenzoic acid derivative and pharmaceutically acceptable salt thereof provided in the present application into an injection, water, ethanol, isopropyl alcohol, propylene glycol or a mixture thereof can be used as a solvent, and an appropriate amount of a solubilizer, a co-solvent, a pH regulator and an osmotic pressure regulator commonly used in the art can be added. The solubilizer or co-solvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. If a freeze-dried powder injection is prepared, mannitol, glucose, etc. can also be added as a supporting agent. In addition, if necessary, a coloring agent, a preservative, a flavoring agent or other additives can also be added to the pharmaceutical preparation. To achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present application can be administered by any known administration method.

[0046] The present application also provides a use of the above-mentioned aminobenzoic acid derivative or pharmaceutically acceptable salt thereof in the preparation of an antiviral drug.

[0047] Specifically, the virus includes one or more of foot-and-mouth disease virus, parainfluenza virus, rubella virus, coxsackie virus, mumps virus and adenovirus.

[0048] The following examples are used to further illustrate the present application, but should not be construed as limiting the present application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art.

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

[0050] Experimental steps:

[0051] 1. BHK-21 cells were seeded at 5 x 10 4The density of one cell per well was inoculated in a 96-well plate, 100 μL of cell suspension was inoculated in each well, and when the cell confluence reached about 70-80%, the original culture medium in the well was removed, and fresh MEM culture medium containing small molecule compound HY-Q05978 at a concentration of 0.05 μM, 0.1 μM, 2 μM, 5 μM, 10 μM was added, 8 replicates were set for each concentration, and DMSO was used as a blank control, and the HY-Q05978 concentration was 0 μM. After incubation at 37°C in a 5% CO2 incubator for 48 h, the original culture medium in the well was removed, and 100 μL of fresh culture medium containing 10% CCK-8 solution was added and incubated for 2 h. Using Labserv K3 type enzyme labeler, the absorbance of each well was measured at 450 nm. The survival rate of cells was calculated according to the absorbance of each well, and the calculation method of survival rate (%) was: OD 450 Sample / OD 450 Blank control) x 100%.

[0052] Table 1 Toxicity of different concentrations of small molecule compound HY-Q05978 to three kinds of cells

[0053]

[0054]

[0055] From the data in Table 1, it can be seen that the above-mentioned small molecule compound HY-Q05978 has no obvious toxicity to host cells.

[0056] 2, respectively using titers of 10 -4 TCID 50 / mL, 10 -5 TCID 50 / mL, 5 x 10 -6 TCID 50 / mL and 10 -6 TCID 50 / mL of foot-and-mouth disease virus FMDV to infect BHK-21 cells, and observe the time of cytopathic effect (CPE) after the cells are infected with viruses at different titers. The results are shown in Figure 1 5 x 10 -6 TCID 50 / mL titers, the time of cytopathic effect is moderate, and finally the titer of 5 x 10 -6 TCID 50 / mL of FMDV is determined for detecting the antiviral effect of small molecule compound HY-Q05978 in subsequent experiments.

[0057] 3. When the density of BHK-21 cells in T-25 culture flasks reaches 80%-90%, digest the cells with trypsin and inoculate them into 24-well plates. Gently shake the 24-well plates to evenly distribute the cells and incubate them in a 37°C incubator containing 5% CO2. After the cells adhere to the wall, use a titer of 5×10 -6 TCID 50 Infect the cells with FMDV at a concentration of 100 μL / mL. Add 100 μL of virus solution to each well and incubate in a 37°C incubator with 5% CO2 for 1 hour. Discard the virus solution in the well plate, add MEM medium containing 2% FBS, and add the small molecule compound HY-Q05978 to final concentrations of 0.01 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, and 10 μM, respectively. DMSO was used as a blank control with a HY-Q05978 concentration of 0 μM. When CPE appeared in the cells, samples were collected, RNA was extracted, and qRT-PCR was performed. Figure 2 As shown, the results showed that the small molecule compound HY-Q05978 could significantly inhibit FMDV at the RNA level at a concentration of 0.01 μM, where mock was a negative control group without DMSO added.

[0058] 4. To verify that the small molecule compound HY-Q05978 regulates FMDV replication by inhibiting Scd1, BHK-21 cells were seeded into 24-well plates. After plating, the plates were gently shaken to evenly distribute the cells and incubated in a 37°C incubator containing 5% CO2. After the cells adhered, a titer of 5×10 -6 Infect cells with FMDV. Add 100 μL of virus solution to each well and incubate at 37°C in a 5% CO2 incubator for 1 hour. Then switch to MEM medium containing 2% FBS and continue culturing. Add the small molecule compound HY-Q05978 to a final concentration of 0.01 μM. When CPE appears in the cells, collect samples and perform qRT-PCR detection. Figure 3 As shown, the mock control group was negative and no DMSO was added, and the DMSO control group was blank. The HY-Q05978 concentration was 0 μM. The results showed that, similar to the commercial Scd1 inhibitor MK-8245, HY-Q05978 did not affect the RNA level of Scd1. Oleic acid (OA) is the end product of Scd1 catalysis. Cells were treated with HY-Q05978 or the commercial Scd1 inhibitor MK-8245 and then incubated with 200 μM oleic acid. qRT-PCR detection revealed that both HY-Q05978 and MK-8245 could inhibit the RNA level of FMDV 3D, but the addition of oleic acid significantly restored the expression of 3D, as shown in Figure 2. Figure 4 As shown, the data of the inhibitor+DMSO group are approximately zero.

[0059] 5、To study the influence of the way of adding small molecule compound HY-Q05978 on its antiviral effect. HY-Q05978 was added 8h before virus infection and the same concentration of HY-Q05978 was supplemented when the liquid was changed after virus infection, or only added when the liquid was changed 1h after virus infection. The drug administration mode is shown in Figure 5 A. The cells were cultured for 24h and then sampled for qRT-PCR detection to explore the influence of different drug administration methods on the antiviral effect of HY-Q05978. As shown in Figure 5 B, DMSO was the blank control group, and the HY-Q05978 concentration was 0μM. The results showed that different drug administration methods had significant inhibitory effect on FMDV, but the effect of pre-infection administration was better. In addition, to determine the time of adding HY-Q05978 before infection, HY-Q05978 was added 8h or 4h before virus infection to explore its influence on virus infection. The mode is shown in Figure 5 C, and the qRT-PCR detection results are shown in Figure 5 D, which showed that there was no obvious difference between adding HY-Q05978 8h or 4h before infection on the infection of FMDV.

[0060] 6、To verify the broad-spectrum of small molecule compound HY-Q05978 in regulating virus replication by inhibiting Scd1. The regulation of small molecule compound HY-Q05978 on the nucleic acid level of RNA viruses: parainfluenza virus type 1 (HP IV1), type 2 (HP IV2), type 3 (HP IV3), rube l l a (Rube l l a), mumps virus (MuV), and DNA viruses: adenovirus type 3 (ADV3), type 5 (ADV5) was verified. The above-mentioned virus corresponding host cells LLC-MK2 and A549 cells were inoculated into 24-well plates, and after plating, the cells were gently shaken to distribute evenly, and incubated in a 37℃ incubator with 5% CO2. After the cells adhered, the corresponding host cells were infected with appropriate titer of virus, 100μL of virus solution was added to each well, and incubated in a 37℃ incubator with 5% CO2 for 1h. The virus solution in the well plate was discarded, and MEM medium containing 2% FBS was added, and compound HY-Q05978 was added to a final concentration of 0.1μM and 5μM, respectively. When CPE appeared, the sample was collected, RNA was extracted, and qRT-PCR detection was performed. See Figure 6 and Table 2 below, DMSO was the blank control group, and the HY-Q05978 concentration was 0μM. The results showed that small molecule compound HY-Q05978 inhibited the nucleic acid levels of HP IV1, HP IV2, HP IV3, Rube l l a, MuV, ADV3, and ADV5 to varying degrees, indicating that HY-Q05978 had a certain broad-spectrum in inhibiting virus replication.

[0061] Table 2 HY-Q05978 inhibits DNA viruses ADV3 and ADV5 replication

[0062]

[0063] It can be seen that, for the host stearoyl-CoA desaturase Scdl, the amino benzoic acid derivative HY-Q05978 provided by the embodiments of the present application has the following advantages in antiviral:

[0064] 1. The small molecule inhibitor has small toxicity and does not affect the normal growth of host cells.

[0065] 2. The small molecule inhibitor has good antiviral effect at low concentration.

[0066] 3. The small molecule inhibitor has significant inhibitory effect on various RNA viruses and DNA viruses, and has good broad-spectrum.

[0067] The above is only a specific embodiment of the present application, which enables those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features applied herein.

Claims

1. A pharmaceutically acceptable salt of an aminobenzoic acid derivative, characterized in that: Aminobenzoic acid derivatives have the following general structure: Among them, R1 is H, R2 is H, and R3 is Cl.

2. The pharmaceutically acceptable salt according to claim 1, wherein: The pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt.

3. The pharmaceutically acceptable salt according to claim 2, wherein: The inorganic acid salt is selected from at least one of hydrochloride, hydrobromide, hydrofluoride, sulfate, nitrate and phosphate.

4. The pharmaceutically acceptable salt according to claim 2, wherein: The organic acid salt is selected from at least one of formate, acetate, propionate, citrate, methanesulfonate, ethanesulfonate, malonate, succinate, fumarate, maleate, lactate, malate, oxalate and tartrate.

5. An antiviral drug, characterized in that: It comprises an active substance, and the active substance comprises one or more of the pharmaceutically acceptable salts according to any one of claims 1 to 4.

6. The antiviral drug according to claim 5, characterized in that: The virus is one or more of foot-and-mouth disease virus, parainfluenza virus, rubella virus, coxsackie virus, mumps virus and adenovirus.

7. Use of the pharmaceutically acceptable salt according to any one of claims 1 to 4 in the preparation of an antiviral drug, wherein the virus is one or more of foot-and-mouth disease virus, parainfluenza virus, rubella virus, coxsackie virus, mumps virus and adenovirus.

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