Alkaline metal salt or alkaline earth metal salt of compound

Abstract

The present invention pertains to an alkaline metal salt or an alkaline earth metal salt of a compound represented by general formula (1).　In general formula (1), R1 represents -NH- or -CH2-, R2 represents -NR8- or -CHR8-, R3 represents a halogen atom, each R4 independently represents a hydrogen atom or a methyl group, R5 represents S or O, R5 represents a carboxyl group, R7 represents an amino group or a methyl group, R8 represents an alkyl group having 1-3 carbon atoms, and n represents a natural number of 1-11.

Description

Alkali metal or alkaline earth metal salts of the compounds

[0001] The present invention relates to an alkali metal salt or alkaline earth metal salt of a compound, more specifically, an alkali metal salt or alkaline earth metal salt of a compound represented by general formula (1) (e.g., AS1842856).

[0002] 5-Amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid (AS1842856) is known as a FOXO1 inhibitor and has been reported to have the effect of suppressing the expression of angiotensin converting enzyme 2 (ACE2) and / or TMPRSS2 (transmembrane protease, serine 2) (for example, Patent Document 1).

[0003] Japanese Patent Application Laid-Open No. 2022-117366

[0004] The free forms of AS1842856 and its analogs (compounds represented by general formula (1)) have the problem of being poorly soluble in solvents such as water. This has led to operational problems, such as the inability to perform dust removal filtration during crystallization purification. Furthermore, the free forms of AS1842856 and its analogs have the problem of low absorbability when administered orally.

[0005] In view of the above-mentioned problems, an object of the present invention is to improve the solubility of the compound represented by general formula (1) and to improve its absorbability when orally administered.

[0006] The present inventors have made the novel discovery that AS1842856 can form salts only with alkali metals or alkaline earth metals, and further that alkali metal salts or alkaline earth metal salts of AS1842856 are highly absorbable when orally administered. The present invention is based on this discovery.

[0007] The present invention relates to an alkali metal salt or alkaline earth metal salt of a compound represented by the following general formula (1): [In general formula (1), R １ is -NH- or -CH ２ - indicates R ２ is -NR ８- or -CHR ８ - indicates R ３ represents a halogen atom, and R ４ each independently represents a hydrogen atom or a methyl group, R ５ represents S or O, R ５ represents a carboxyl group, and R ７ represents an amino group or a methyl group, and R ８ represents an alkyl group having 1 to 3 carbon atoms, and n represents a natural number from 1 to 11.

[0008] The alkali metal salt or alkaline earth metal salt according to the present invention has improved solubility in water and improved absorbability when orally administered.

[0009] The compound may be a compound represented by the following general formula (2). [In general formula (2), R ３ , R ４ , R ５ , R ８ and n have the same meanings as in general formula (1).

[0010] The compound may be a compound (AS1842856) represented by the following formula (3).

[0011] The alkali metal salts or alkaline earth metal salts mentioned above may be sodium salts.

[0012] The present invention also relates to a pharmaceutical composition containing the above-mentioned alkali metal salt or alkaline earth metal salt according to the present invention as an active ingredient.

[0013] The pharmaceutical composition according to the present invention contains the alkali metal salt or alkaline earth metal salt according to the present invention, and therefore has excellent absorbability when orally administered. Therefore, the pharmaceutical composition may be for oral administration.

[0014] The pharmaceutical composition according to the present invention contains the alkali metal salt or alkaline earth metal salt according to the present invention, and therefore has excellent absorbability when orally administered, and is capable of increasing the blood concentration of the compound, thereby enabling the compound to exert its medicinal effects based on its pharmacological action. Thus, the pharmaceutical composition may be used, for example, for the prevention or treatment of SARS-CoV2 infection.

[0015] According to the present invention, the solubility of the compound represented by general formula (1) can be improved, and the absorbability upon oral administration can also be improved.

[0016] 1(A) and 1(B) are graphs showing the time course of plasma concentrations of AS1842856 when AS1842856 sodium salt (AS-Na) and AS1842856 (free form) (AS) were orally administered to rats. FIG. 1(A) is a graph showing the time course up to 48 hours after administration. FIG. 1(B) is an excerpt from FIG. 1(A) and is a graph showing the time course up to 4 hours after administration. FIG. 2(A) is a graph showing the results of analyzing the mRNA expression levels of each gene in the lungs of COVID-19 model hamsters when AS1842856 sodium salt (AS-Na) and AS1842856 (free form) (AS) were intraperitoneally administered. FIG. 2(A) is a graph showing the results of quantification of the IL6 gene. FIG. 2(B) is a graph showing the results of quantification of the IFNα9 gene. FIG. 2(C) is a graph showing the results of quantification of the IFNγ gene. Figure 2(D) is a graph showing the results of quantification of the CCL2 gene. Figure 2(E) is a graph showing the results of quantification of the CXCL1 gene. Figure 2(E) is a graph showing the results of weight measurement of COVID-19 model mice when AS1842856 sodium salt (AS-Na), AS1842856 (free form) (AS), and phosphate-buffered saline containing 10 w / v% dimethyl sulfoxide (DMSO) were intraperitoneally administered to COVID-19 model mice. Figure 2(F) is a graph showing the survival curve of mice when AS1842856 sodium salt (AS-Na), AS1842856 (free form) (AS), and phosphate-buffered saline containing 10 w / v% dimethyl sulfoxide (DMSO) were intraperitoneally administered to COVID-19 model mice. 1 is a graph showing the results of measuring the amount of viral RNA in the lungs of COVID-19 model mice euthanized on Day 3 after intraperitoneal administration of AS1842856 sodium salt (AS-Na), AS1842856 (free form) (AS), and phosphate-buffered saline containing 10 w / v% dimethyl sulfoxide (DMSO).

[0017] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following describes in detail the embodiments of the present invention, but the present invention is not limited to the following embodiments.

[0018] The present invention relates to an alkali metal salt or alkaline earth metal salt of a compound represented by general formula (1) (for example, AS1842856). [In general formula (1), R１ is -NH- or -CH ２ - indicates R ２ is -NR ８ - or -CHR ８ - indicates R ３ represents a halogen atom, and R ４ each independently represents a hydrogen atom or a methyl group, R ５ represents S or O, R ５ represents a carboxyl group, and R ７ represents an amino group or a methyl group, and R ８ represents an alkyl group having 1 to 3 carbon atoms, and n represents a natural number from 1 to 11.

[0019] The alkali metal salt or alkaline earth metal salt may be in anhydrous form or in hydrated form (for example, monohydrate, dihydrate, trihydrate).

[0020] The alkali metal salt may be any salt of the compound represented by general formula (1) with an alkali metal, and specific examples thereof include lithium salts, sodium salts, potassium salts, rubidium salts, cesium salts, and francium salts.

[0021] The alkaline earth metal salt may be a salt of the compound represented by general formula (1) with an alkaline earth metal, and specific examples thereof include beryllium salts, magnesium salts, calcium salts, strontium salts, barium salts, and radium salts.

[0022] Among the various salts mentioned above, alkali metal salts are preferred, and sodium salts are more preferred, since they have a more significant effect of improving the solubility of the compound represented by general formula (1) and the effect of improving the absorbability when orally administered.

[0023] The compound represented by general formula (1) is preferably a compound represented by the following general formula (2), and more preferably a compound (AS1842856) represented by the following formula (3), because the compound represented by general formula (1) has a more significant effect of improving solubility and improving absorbability upon oral administration.

[0024] [In general formula (2), R ３ , R ４ , R５ , R ８ and n have the same meanings as in general formula (1).

[0025]

[0026] The compound represented by general formula (1) can be produced, for example, according to the method described in International Publication No. 2020 / 163816. For example, commercially available compounds such as AS1842856 may be used.

[0027] The alkali metal salt or alkaline earth metal salt of the compound represented by general formula (1) can be obtained, for example, by reacting the compound represented by general formula (1) with an alkali metal or alkaline earth metal.

[0028] Specifically, for example, an alkali metal salt or alkaline earth metal salt of a compound represented by general formula (1) can be obtained by a production method including a mixing step of mixing a compound represented by general formula (1), a solvent, and a hydroxide of an alkali metal or alkaline earth metal, a reaction step of heating the solution obtained in the mixing step while stirring, a precipitation step of cooling the solution obtained in the reaction step to precipitate an alkali metal salt or alkaline earth metal salt of the compound represented by general formula (1), a recovery step of recovering the solid (e.g., crystal) precipitated in the precipitation step, and a drying step of drying the recovered solid.

[0029] Examples of the solvent used in the mixing step include water and alcohols such as ethanol, propanol, butanol, isopropanol, etc. The solvent may be used alone or in combination of two or more.

[0030] The order of addition in the mixing step is not particularly limited, and for example, an alkali metal or alkaline earth metal hydroxide may be added to a solution or suspension in which a compound represented by general formula (1) is dissolved or suspended in a solvent, a solution or suspension in which an alkali metal or alkaline earth metal hydroxide is dissolved or suspended in a solvent may be added to a solution or suspension in which a compound represented by general formula (1) is dissolved or suspended in a solvent, or the compound represented by general formula (1) may be added to a solution or suspension in which an alkali metal or alkaline earth metal hydroxide is dissolved or suspended in a solvent.

[0031] The compound represented by the general formula (1) and the hydroxide of an alkali metal or alkaline earth metal may be mixed in such a ratio that the hydroxide of the alkali metal or alkaline earth metal is in excess on a molar basis, for example.

[0032] In the reaction step, the solution obtained in the mixing step is heated while being stirred. The heating temperature may be, for example, in the range of 60° C. to 80° C. The heating time may be, for example, in the range of 10 minutes to 60 minutes.

[0033] In the production method according to this embodiment, a step of performing dust removal filtration may be carried out after the reaction step and before the precipitation step, thereby improving the efficiency of crystal precipitation in the precipitation step.

[0034] In the precipitation step, the solution obtained in the reaction step is cooled to precipitate the alkali metal salt or alkaline earth metal salt of the compound represented by general formula (1). The cooling temperature may be, for example, in the range of 35° C. or higher and 50° C. or lower. The precipitation of the alkali metal salt or alkaline earth metal salt can be confirmed visually.

[0035] In the recovery step, the solid (e.g., crystals) precipitated in the precipitation step is recovered. The recovery method is not particularly limited, and can be appropriately selected, for example, a method of recovering the solid by filtration, a method of recovering the solid by centrifugation, or the like.

[0036] In the drying step, the recovered solid is dried. The drying may be, for example, drying under reduced pressure.

[0037] The alkali metal salt or alkaline earth metal salt of the compound represented by general formula (1) according to the present invention has improved solubility and improved absorbability when orally administered, and therefore can be suitably used as an active ingredient of a pharmaceutical composition.

[0038] Therefore, one aspect of the present invention is a pharmaceutical composition containing an alkali metal salt or alkaline earth metal salt of a compound represented by general formula (1) as an active ingredient.

[0039] The pharmaceutical composition according to this embodiment may contain a pharmaceutically acceptable carrier, such as water, physiological saline, or a buffer solution.

[0040] The pharmaceutical composition according to this embodiment may contain other pharmaceutically acceptable ingredients, such as excipients, disintegrants, glidants / lubricants, flavorings, stabilizers, preservatives, antioxidants, binders, etc., within the scope of not impairing the effects of the present invention.

[0041] Examples of excipients include lactose, corn starch, sucrose, glucose, sorbitol, and crystalline cellulose. Examples of disintegrants include starch, agar, gelatin, calcium carbonate, sodium chloride, sodium bicarbonate, calcium citrate, anhydrous silicic acid, dextrin, pectin, carmellose, carmellose calcium, croscarmellose sodium, crospovidone, low-substituted hydroxypropyl cellulose, and sodium starch glycolate. Examples of fluidizers and lubricants include light anhydrous silicic acid, hydrous silicic acid dioxide, magnesium stearate, and talc. Examples of flavoring agents include peppermint, aromatic powder, peppermint oil, borneol, and cinnamon powder. Examples of stabilizers include ascorbic acid and glycerin. Examples of preservatives include ethyl parahydroxybenzoate and propyl parahydroxybenzoate. Examples of antioxidants include butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, propyl gallate, etc. Examples of binders include sucrose, gelatin, gum arabic, methylcellulose, etc.

[0042] The pharmaceutical composition according to this embodiment may be for oral administration or for parenteral administration. Examples of administration routes include oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intrathecal administration, intraperitoneal administration, sublingual administration, rectal administration, vaginal administration, intraocular administration, nasal administration, inhalation, and transdermal administration. Since the alkali metal salt or alkaline earth metal salt of the compound represented by general formula (1) according to the present invention has improved absorbability upon oral administration, the pharmaceutical composition is preferably for oral administration. Furthermore, from the viewpoint of achieving a more pronounced medicinal effect of the pharmaceutical composition according to this embodiment, intravenous administration, oral administration, nasal administration, and inhalation are preferred, while from the viewpoint of reducing the burden on the subject receiving the pharmaceutical composition according to this embodiment, oral administration and inhalation are preferred.

[0043] The dosage of the pharmaceutical composition according to this embodiment may be appropriately determined depending on the type of disease, the gender, age, and weight of the patient. Non-limiting examples of dosages of the pharmaceutical composition according to this embodiment include dosages in which the amount of active ingredient per kg of body weight per day is 0.2 mg to 70 mg, or 2 mg to 60 mg. Typically, when administered to an adult, the dosage is 0.5 mg to 50 mg, preferably 1 mg to 40 mg, per day. This dosage may be administered multiple times as a single dose, or may be administered in multiple divided doses.

[0044] The pharmaceutical composition according to this embodiment contains an alkali metal salt or alkaline earth metal salt of the compound represented by general formula (1) as an active ingredient, and therefore can be suitably used for applications utilizing the pharmacological action of the compound represented by general formula (1) (e.g., AS1842856) (e.g., for the prevention or treatment of a disease).

[0045] For example, a compound represented by general formula (1) (e.g., AS1842856) can be used for the treatment and / or prevention of diseases involving angiotensin converting enzyme 2 (ACE2) and / or TMPRSS2. Specifically, it can be used for the treatment and / or prevention of infectious diseases involving these. Examples of infectious diseases include respiratory infections, which are infections that occur in organs related to the air passages, such as the nose, throat, bronchi, and lungs. Specific examples include common cold syndrome, group A streptococcal infection, respiratory syncytial virus infection, SARS, adenovirus-induced pharyngoconjunctival fever, influenza, Haemophilus influenzae infection, psittacosis, chlamydial pneumonia, tuberculosis, coccidioidomycosis, Corynebacterium ulcerans infection, diphtheria, anthrax, pneumococcal infection, hantavirus pulmonary syndrome, histoplasmosis, whooping cough, rubella, plague, mycoplasmal pneumonia, measles, Legionnaires' disease, leptospirosis, SARS-CoV, and SARS-CoV2 infection. Among these, SARS-CoV2 infection (COVID-19) is preferred. SARS-CoV2 binds its spike protein to the ACE2 receptor of human cells and uses this as a foothold to enter the cells. Furthermore, after SARS-CoV2 binds to ACE2 on the surface of human cells, its efficiency of entry into the cells is enhanced by enzymatic processing of TMPRSS2 in the human cells. Therefore, by suppressing the expression of ACE2 and / or TMPRSS2, it is possible to inhibit cell entry by such viruses, thereby suppressing infection. For this reason, the pharmaceutical composition according to this embodiment can be suitably used for the prevention or treatment of SARS-CoV2 infection.

[0046] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0047] [Test Example 1: Formation of AS1842856 Sodium Salt] AS1842856 was obtained from Jiangsu Aikon Biopharmaceutical R&D Co., Ltd. To AS1842856 (500 mg), 10% aqueous sodium hydroxide solution (2.5 mL, 5 vol / wt) and isopropanol (2.5 mL, 5 vol / wt) were added, stirred at room temperature, and then heated to 70 ° C. to completely dissolve. After stirring for 30 minutes, the mixture was filtered using filter paper with moderate particle retention while maintaining the heated state, and the external bath was cooled to 45 ° C. to confirm the precipitation of crystals. The mixture was then cooled with ice water (approximately 2 ° C.), and the precipitated crystals were collected by filtration and dried under reduced pressure to obtain AS1842856 sodium salt (420 mg, apparent yield 79%). １ H-NMR (DMSO-D6) δ: 8.24 (s, 1H), 7.10-7.70 (bs, 2H), 5.78 (d, 1H), 5.54 (s, 1H), 4.11 (m, 2H), 3.35 -3.47 (m, 1H), 1.93 (d, 2H), 1.73 (d, 2H), 1.61-1.65 (m, 1H), 1.27-1.44 (m, 7H), 1.10-1.25 (m, 1H) Elemental analysis: C １８ H ２１ FN ３ NaO ３ ・H ２ O Theoretical values: C: 55.81 H: 5.98 N: 10.85 Measured values: C: 55.01 H: 6.21 N: 10.68 Elemental analysis data revealed that the obtained sodium salt formed a monohydrate.

[0048] Furthermore, salt formation was carried out in the same manner as above, except that an aqueous potassium hydroxide solution or an aqueous calcium hydroxide solution was used instead of the aqueous sodium hydroxide solution, and the potassium salt of AS1842856 and the calcium salt of AS1842856 were obtained, respectively.

[0049] Salt formation with sulfuric acid, hydrochloric acid, mesylic acid, hydrobromic acid, phosphoric acid, acetic acid, nitric acid, tartaric acid, maleic acid, and tosylic acid was investigated, but no salt formation was observed with any of them.

[0050] Test Example 2: Evaluation of Solubility The solubility of AS1842856 sodium salt and AS1842856 (free form) in solvents (water, ethanol, or 50 v / v% aqueous ethanol) was measured. For the measurements, six types of samples (n = 3 each) were prepared as follows, and the content of AS1842856 in the solvent was quantified using high-performance liquid chromatography (HPLC), and the average value for each was calculated. (Sample 1: Free form / water) 10 mg of AS1842856 (free form) was weighed out, 20 mL of purified water was added, and the mixture was heated at 37.5°C for 30 minutes (shaking every 5 minutes). (Sample 2: Sodium salt / water) 20 mg of AS1842856 sodium salt was weighed out, 20 mL of purified water was added, and the mixture was heated at 37.5°C for 30 minutes (shaking every 5 minutes). (Sample 3: Free form, 50 v / v% ethanol aqueous solution) 30 mg of AS1842856 (free form) was weighed out, and 20 mL of 50% v / v% ethanol aqueous solution was added, followed by heating at 37.5°C for 30 minutes (shaking every 5 minutes). (Sample 4: Sodium salt, 50 v / v% ethanol aqueous solution) 50 mg of AS1842856 sodium salt was weighed out, and 20 mL of 50% v / v% ethanol aqueous solution was added, followed by heating at 37.5°C for 30 minutes (shaking every 5 minutes). (Sample 5: Free form, ethanol) 30 mg of AS1842856 (free form) was weighed out, and 20 mL of ethanol was added, followed by heating at 37.5°C for 30 minutes (shaking every 5 minutes). (Sample 6: Sodium Salt / Ethanol) 50 mg of AS1842856 sodium salt was weighed out, and 20 mL of ethanol was added, followed by heating at 37.5° C. for 30 minutes (shaking every 5 minutes).

[0051] A total of 18 sample solutions prepared as described above were each centrifuged (3000 rpm, 10 minutes), the supernatant was removed, and 2 mL of the filtrate after filtering through a 0.45 μm filter was made up to 50 mL with the mobile phase, and the filtrate was filtered again through a 0.45 μm filter to obtain a sample specimen.

[0052] (Standard Solution) 10 mg of AS1842856 (free form) was weighed out, and 20 mL of dimethyl sulfoxide (DMSO) was added, followed by heating for 30 minutes at 37.5° C. 2 mL of the solution was diluted to 50 mL with the mobile phase, and the filtrate was filtered through a 0.45 μm filter to obtain a standard solution.

[0053] -Analytical equipment and analytical column- (HPLC conditions) Equipment: 1260 Infinity II LC system (Agilent) Analytical column: Inertsil ODS-3, 5 μm, 150 × 4.6 mm I.D. (GL Sciences) Column bath temperature: 40°C (set value) Mobile phase: 30% 10 mmol / L ammonium formate aqueous solution, 70% acetonitrile Flow rate: 1.0 mL / min Injection volume: 40 μL Autosampler temperature: 4°C (set value) Detection: UV 291 nm

[0054] The results are shown in Table 1.

[0055] It was confirmed that the sodium salt of AS1842856 has excellent solubility in solvents (water, ethanol, or 50 v / v % aqueous ethanol solution).

[0056] Test Example 3: Evaluation of oral absorbability Each of AS1842856 sodium salt and AS1842856 (free form) was suspended in a separately prepared 0.5 w / v % aqueous solution of carboxymethylcellulose sodium (CMC-Na) to prepare a test solution for evaluation with an AS1842856 concentration of 100 mg / mL.

[0057] The test solution for evaluation prepared above was orally administered to 6-week-old male SD (Sprague-Dawley) rats. Oral administration was by single forced administration using a standard flexible gastric sonde. The test groups were divided into two groups: one group (n=5) administered the test solution for evaluation prepared using AS1842856 sodium salt so that the amount of AS1842856 per kg of rat body weight was 1000 mg, and the other group (n=5) administered the test solution for evaluation prepared using AS1842856 (free form) so that the amount of AS1842856 per kg of rat body weight was 1000 mg.

[0058] All animals were fasted overnight (approximately 16 hours) prior to administration and were fed after the completion of general observation 4 hours after administration. Blood samples were collected at seven time points: 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 24 hours, and 48 hours after administration. Approximately 0.2 mL of blood was collected from the jugular vein using a heparin sodium-treated syringe under unanesthetized conditions. The collected blood was transferred to a polypropylene container, cooled on ice, and centrifuged (4°C, 6000 x g, 2 minutes) to obtain plasma. The AS1842856 concentration in the obtained plasma was analyzed using liquid chromatography / tandem mass spectrometry (LC / MS / MS). The specific method is described below.

[0059] - Preparation of measurement sample - 30 μL of 50% acetonitrile aqueous solution and 120 μL of 1 μmol / L diclofenac (internal standard, solvent: acetonitrile) were added to 30 μL of plasma and mixed to obtain a mixture. The mixture was centrifuged (4°C, 1900 × g, 5 minutes), and 20 μL of the resulting supernatant was recovered. 480 μL of 20% acetonitrile aqueous solution was added to the recovered supernatant, and the resulting solution was used as a measurement sample.

[0060] -Analytical equipment and analytical column- (LC conditions) Equipment: LC-30AD system (Shimadzu Corporation) Analytical column: L-column 2 ODS, 5 μm, 2.0 × 50 mm (Metal Free) (Chemicals Evaluation and Research Institute, Japan) Column tank temperature: 40 ° C (set value) Mobile phase: A; 10 mmol / L ammonium formate aqueous solution, B; acetonitrile Gradient conditions: Flow rate: 0.4 mL / min Injection volume: 10 μL Autosampler temperature: 4°C (set value)

[0061] (MS / MS conditions) Instrument: QTRAP 6500 (AB Sciex) Ionization method: ESI Polarity: Positive Resolution (Q1 / Q3): Unit / Unit Measurement time: 5.5 minutes Analyte: AS1842856; Q1 (Da) 348.10 / Q3 (Da) 220.10 Diclofenac; Q1 (Da) 296.00 / Q3 (Da) 214.00 Scan type: Multiple reaction monitoring Curtain gas: 30 psi (Nitrogen) Ion source gas 1: 70 psi (Air) Ion source gas 2: 90 psi (Air) Spray voltage: 2500 V Heater temperature: 600°C (set value)

[0062] The results are shown in Figure 1. In Figure 1, "AS-Na" indicates the results for the group administered with AS1842856 sodium salt, and "AS" indicates the results for the group administered with AS1842856 (free form). Each value in Figure 1 is the mean value ± standard deviation calculated from the results of three independent measurements performed on each measurement sample. Figure 1(A) is a graph showing the results of measurement of the AS1842856 concentration in plasma up to 48 hours after administration, and Figure 1(B) is an excerpt from Figure 1(A) and is a graph showing the results of measurement of the AS1842856 concentration in plasma up to 4 hours after administration.

[0063] From the measurement results of the AS1842856 concentration in plasma, the area under the plasma concentration-time curve (AUC (up to 24 hrs): ng h / mL) and maximum plasma concentration (Cmax: ng / mL) were calculated using GraphPad Prism (GraphPad Software, LLC). The results are shown in Table 3.

[0064]

[0065] As shown in Figure 1 and Table 3, AS1842856 sodium salt showed an approximately 11-fold improvement in AUC (up to 24 hours) and an increased Cmax compared to AS1842856 (free form). This confirmed that AS1842856 sodium salt exhibits higher absorbability when orally administered than AS1842856 (free form).

[0066] Test Example 4: Administration test on COVID-19 model hamsters Six-week-old male Syrian hamsters were obtained from Japan SLC and transferred to an Animal Biosafety Level-3 (ABSL-3) research facility for SARS-CoV-2 challenge studies. Animals were maintained under a light-dark cycle, fed a standard pellet diet, and had free access to water. All experimental protocols, including immunization, booster doses, and animal exposure, were approved by the ethics committee.

[0067] Test solutions for administration with an AS1842856 concentration of 10 mg / mL were prepared by dissolving AS1842856 sodium salt and AS1842856 (free form) in separately prepared phosphate-buffered saline (PBS) containing 10 w / v % dimethyl sulfoxide (DMSO).

[0068] All hamsters were maintained in an ABSL3 facility with 1 × 10 ３ PFU (plaque-forming unit) of SARS-CoV-2 (conventional strain: 2019-nCoV / Japan / TY / WK-521 / 2020) was exposed. For oral administration, the hamsters were anesthetized with pentobarbital (100 mg / kg) and then given a single forced dose using a standard micropipette. Starting in the afternoon of the day after exposure (Day 1), administration of the test solution prepared above began, with half the dose administered in the morning and half in the afternoon, for a total of six doses by the morning of Day 4.

[0069] The test groups were divided into three groups: one group (n=4) that received intraperitoneal administration of a test solution prepared using AS1842856 sodium salt at a dose of 50 mg of AS1842856 per kg of body weight per day, one group (n=4) that received intraperitoneal administration of a test solution prepared using AS1842856 (free form) at a dose of 50 mg of AS1842856 per kg of body weight per day, and one group (n=3) that received intraperitoneal administration of 10 w / v% DMSO-containing PBS alone. Intraperitoneal administration was performed via the abdomen using a syringe equipped with a standard injection needle.

[0070] In the afternoon of Day 4, at ABSL3, all hamsters were euthanized, dissected, and the lungs were removed. RNA was collected from the lungs, and the mRNA expression levels of IL6, IFNα9, IFNγ, CCL2, and CXCL10 genes were measured by quantitative PCR.

[0071] The results are shown in Figure 2. In Figure 2, "AS-Na" indicates the results for the group administered with AS1842856 sodium salt, and "AS" indicates the results for the group administered with AS1842856 (free form). Also in Figure 2, "DMSO" indicates the results for the group administered with 10 w / v% DMSO-containing PBS alone, and "Naive" indicates the results for untreated (unadministered) hamsters. As shown in Figure 2, in the group administered with AS1842856-Na salt, the expression of inflammatory cytokines IL6, IFNα9, and IFNγ was significantly suppressed (Figures 2(A), (B), and (C)). Furthermore, in the group administered with AS1842856-Na salt, the expression of chemokines CCL2 and CXCL10 was significantly suppressed (Figures 2(D) and (E)).

[0072] Test Example 5: Administration test on COVID-19 model mice Sixteen-week-old BALB / c mice were obtained from Japan SLC and transferred to an Animal Biosafety Level-3 (ABSL-3) research facility for SARS-CoV-2 challenge studies. Animals were maintained under a light-dark cycle, fed a standard pellet diet, and had free access to water. All experimental protocols, including immunization, booster doses, and animal exposure, were approved by the ethics committee.

[0073] Test solutions for administration with an AS1842856 concentration of 10 mg / mL were prepared by dissolving AS1842856 sodium salt and AS1842856 (free form) in separately prepared PBS containing 10 w / v % DMSO.

[0074] All mice were immunized in an ABSL3 facility with 1 × 10 ４Mice were exposed to PFU of SARS-CoV-2 (QHmusX strain). For nasal administration, mice were anesthetized with pentobarbital (100 mg / kg) and then given a single forced dose using a standard micropipette. Starting the day after exposure (Day 1), administration of the test solution prepared above was initiated and administered once daily.

[0075] The test groups were: a group (n = 10) in which a test solution prepared using AS1842856 sodium salt was intraperitoneally administered at a dose of 50 mg of AS1842856 per kg of mouse body weight per day; a group (n = 10) in which a test solution prepared using AS1842856 (free form) was intraperitoneally administered at a dose of 50 mg of AS1842856 per kg of mouse body weight per day; and a group (n = 10) in which only 10 w / v% DMSO-containing PBS was intraperitoneally administered. Intraperitoneal administration was performed via the abdomen using a syringe with a standard injection needle. Each group was further divided into six groups: a group euthanized on Day 3 (n = 5) and a group euthanized on Day 7 (n = 5).

[0076] All mice were weighed daily and their lungs were excised and the amount of SARS-CoV-2 viral RNA in the lungs was measured by quantitative PCR and normalized for the amount of GAPDH expression in each sample.

[0077] The results of the body weight measurements are shown in Figure 3. In Figure 3, "DMSO" indicates the results for the group administered with 10 w / v% DMSO-containing PBS alone, "AS" indicates the results for the group administered with AS1842856 (free form), and "AS-Na" indicates the results for the group administered with AS1842856 sodium salt. As shown in Figure 3, weight loss was significantly suppressed in the group administered with AS1842856-Na salt.

[0078] The survival curves are shown in Figure 4. In Figure 4, "DMSO" indicates the results for the group administered with 10 w / v% DMSO-containing PBS alone, "AS" indicates the results for the group administered with AS1842856 (free form), and "AS-Na" indicates the results for the group administered with AS1842856 sodium salt. A weight loss of -20% or more was counted as death. As shown in Figure 4, death was significantly suppressed in the group administered with AS1842856-Na salt.

[0079] The results of measuring the viral RNA levels in the lungs of each group euthanized on Day 3 are shown in Figure 5. In Figure 5, "DMSO" indicates the results for the group administered only 10 w / v% DMSO-containing PBS, "AS" indicates the results for the group administered AS1842856 (free form), and "AS-Na" indicates the results for the group administered AS1842856 sodium salt. As shown in Figure 5, the viral load on Day 3 was significantly suppressed in the group administered AS1842856-Na salt.

Claims

1. An alkali metal salt or alkaline earth metal salt of a compound represented by the following general formula (1): [In general formula (1), R １ is -NH- or -CH ２ - indicates R ２ is -NR ８ - or -CHR ８ - indicates R ３ represents a halogen atom, and R ４ each independently represents a hydrogen atom or a methyl group, R ５ represents S or O, R ５ represents a carboxyl group, and R ７ represents an amino group or a methyl group, and R ８ represents an alkyl group having 1 to 3 carbon atoms, and n represents a natural number from 1 to 11.

2. The alkali metal salt or alkaline earth metal salt according to claim 1, wherein the compound is a compound represented by the following general formula (2): [In general formula (2), R ３ , R ４ , R ５ , R ８ and n have the same meanings as in general formula (1).

3. The alkali metal salt or alkaline earth metal salt according to claim 1, wherein the compound is a compound represented by the following formula (3):

4. The alkali metal or alkaline earth metal salt of claim 1, which is a sodium salt.

5. The alkali metal salt or alkaline earth metal salt according to claim 3, which is a sodium salt.

6. A pharmaceutical composition containing the alkali metal salt or alkaline earth metal salt according to any one of claims 1 to 5 as an active ingredient.

7. The pharmaceutical composition according to claim 6, which is for oral administration.

8. The pharmaceutical composition according to claim 6, which is for the prevention or treatment of SARS-CoV2 infection.

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