Pharmaceutical salt of sulfonamide compound, crystal form of salt, composition, and use

Abstract

Disclosed are a pharmaceutical salt of a sulfonamide compound, a crystal form of the salt, a composition, and a use. Specifically, disclosed is a pharmaceutical salt of compound A, wherein the pharmaceutical salt of compound A is an alkali metal salt or an alkaline earth metal salt. The pharmaceutical salt of the compound has good solubility and excellent inhibitory activity against PR3 enzyme and neutrophil elastase, and can improve sepsis survival rates.

Description

A pharmaceutical salt of a sulfonamide compound, the crystal form of the salt, its composition and uses. Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a pharmaceutical salt of a sulfonamide compound, the crystal form of the salt, its composition, and its uses. Background Technology

[0002] Compound A, chemically named O-methyl-N-(2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoyl)-D-serine, exhibits excellent neutrophil elastase inhibitory activity (IC50 of 2.47 nM), significantly superior to the marketed cevelex (44 nM). Compound A can be used to treat various acute lung diseases, such as acute lung injury and acute respiratory distress syndrome.

[0003] This invention discovered that compound A is poorly soluble in water. The solubility of a compound is an important physical property in clinical drug applications, as drug efficacy is related to the concentration of drug that can be achieved in the bloodstream. Different salts or crystal forms of compounds typically possess different physical properties; therefore, finding salts or crystal forms with suitable solubility for compound A is of great significance for its clinical application.

[0004] PR3 is a serine protease secreted by neutrophils with a wide range of biological activities, including proteolytic activity, antibacterial activity, and regulation of inflammatory processes. Studies have reported that PR3 enzymes are closely related to the development and progression of various inflammatory diseases, including sepsis; therefore, inhibitors of PR3 enzymes could serve as targets for the prevention and treatment of these diseases. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the deficiency of PR3 enzyme inhibitors in the prior art. This invention provides a pharmaceutical salt of a sulfonamide compound, the crystal form of the salt, its composition, and its uses. The pharmaceutical salt of the compound of this invention has good solubility, excellent inhibitory activity against PR3 enzyme, and improves the survival rate of sepsis patients.

[0006] In a first aspect, the present invention provides a pharmaceutical salt of compound A, wherein the pharmaceutical salt of compound A is an alkali metal salt or an alkaline earth metal salt;

[0007] The alkali metal salt mentioned is a commonly used alkali metal salt of this type of compound in the art.

[0008] The alkaline earth metal salts mentioned are commonly used alkaline earth metal salts of this type of compound in the art.

[0009] In one embodiment of the present invention, the pharmaceutical salt of compound A is a monosodium salt, a monolithium salt, a monopotassium salt, a magnesium salt, or a calcium salt of compound A; preferably, it is a monosodium salt, a monolithium salt, a monopotassium salt, or a magnesium salt of compound A; and even more preferably, it is a monosodium salt of compound A.

[0010] The pharmaceutical salts of compound A described in this invention include amorphous or crystalline forms.

[0011] In one aspect of the present invention, the monosodium salt of compound A includes an amorphous or crystalline form of the monosodium salt of compound A; preferably, it is an amorphous form of the monosodium salt of compound A, crystalline form I of the monosodium salt of compound A, crystalline form II of the monosodium salt of compound A, or crystalline form III of the monosodium salt of compound A.

[0012] The present invention confirms the crystal structure of the pharmaceutical salt of compound A by powder X-ray diffraction (PXRD).

[0013] The monosodium salt of compound A, crystal form I, has X-ray powder diffraction patterns at 5.1±0.2°, 10.4±0.2°, 12.0±0.2° and 17.8±0.2° when expressed in 2θ angles.

[0014] The monosodium salt of compound A, crystal form II, has X-ray powder diffraction patterns at 3.4±0.2°, 5.5±0.2°, 10.3±0.2°, 12.2±0.2° and 16.3±0.2° when expressed in 2θ angles.

[0015] The monosodium salt of compound A, crystal form III, has characteristic peaks at 4.3±0.2°, 8.6±0.2°, 9.5±0.2°, and 18.0±0.2° in its X-ray powder diffraction pattern expressed in 2θ angles.

[0016] In one aspect of the present invention, the X-ray diffraction pattern of the monosodium salt amorphous powder of compound A is basically as shown in Figure 1.

[0017] In one embodiment of the present invention, the monosodium salt of compound A, crystal form I, has an X-ray powder diffraction pattern in 2θ angles that also has diffraction peaks at one or more of the following 2θ angles: 4.6±0.2°, 13.5±0.2°, 16.5±0.2° and 19.5±0.2°.

[0018] In one aspect of the present invention, the monosodium salt of compound A, crystal form I, has an X-ray powder diffraction pattern expressed in 2θ angles, with diffraction peaks at the following 2θ angles: 4.6±0.2°, 5.1±0.2°, 6.2±0.2°, 7.4±0.2°, 10.4±0.2°, 11.3±0.2°, 12.0±0.2°, 13.5±0.2°, 13.9±0.2°, 15.3±0.2°, 16.0±0.2°, 16.5±0.2°, 16.9±0.2°, 17.8±0.2°, and 19.5±0.2°.

[0019] In one embodiment of the present invention, the characteristic peak data of the X-ray powder diffraction pattern of the monosodium salt I of compound A, expressed in terms of 2θ angle, are as follows, wherein the error range of the 2θ value is ±0.2°:

[0020] In one embodiment of the present invention, the crystal form I of the monosodium salt of compound A, and its X-ray powder diffraction pattern expressed in 2θ angle are basically as shown in Figure 2.

[0021] In one embodiment of the present invention, the monosodium salt of compound A, crystal form II, has X-ray powder diffraction patterns expressed in 2θ angles that also have diffraction peaks at one or more of the following 2θ angles: 14.6±0.2°, 17.2±0.2°, 17.8±0.2° and 19.4±0.2°.

[0022] In one embodiment of the present invention, the monosodium salt of compound A, crystal form II, has an X-ray powder diffraction pattern expressed in 2θ angles, with diffraction peaks at the following 2θ angles: 3.4±0.2°, 5.0±0.2°, 5.5±0.2°, 8.9±0.2°, 9.5±0.2°, 10.3±0.2°, 12.2±0.2°, 14.6±0.2°, 16.3±0.2°, 17.2±0.2°, 17.8±0.2°, and 19.4±0.2°.

[0023] In one embodiment of the present invention, the characteristic peak data of the X-ray powder diffraction pattern of the monosodium salt II of compound A, expressed in terms of 2θ angle, are as follows, wherein the error range of the 2θ value is ±0.2°:

[0024] In one aspect of the present invention, the crystal form II of the monosodium salt of compound A has an X-ray powder diffraction pattern represented by a 2θ angle, which is basically as shown in Figure 3.

[0025] In one embodiment of the present invention, the monosodium salt of compound A, crystal form III, has X-ray powder diffraction patterns expressed in 2θ angles that also have diffraction peaks at one or more of the following 2θ angles: 5.5±0.2°, 13.2±0.2°, 16.2±0.2° and 16.9±0.2°.

[0026] In one embodiment of the present invention, the monosodium salt of compound A, crystal form III, has X-ray powder diffraction patterns expressed in 2θ angles, with diffraction peaks at the following 2θ angles: 4.3±0.2°, 5.5±0.2°, 8.6±0.2°, 9.5±0.2°, 11.1±0.2°, 13.2±0.2°, 16.2±0.2°, 16.6±0.2°, 16.9±0.2°, 17.4±0.2°, and 18.0±0.2°.

[0027] In one embodiment of the present invention, the characteristic peak data of the X-ray powder diffraction pattern of the monosodium salt III of compound A, expressed in terms of 2θ angle, are as follows, wherein the error range of the 2θ value is ±0.2°:

[0028] In one embodiment of the present invention, the crystal form III of the monosodium salt of compound A has an X-ray powder diffraction pattern represented by a 2θ angle, which is basically shown in Figure 4.

[0029] The pharmaceutical salt of compound A described in this invention can be prepared by conventional methods for such pharmaceutical salts in the art.

[0030] In a second aspect, the present invention provides a method for preparing a pharmaceutical salt of compound A, comprising: reacting compound A or another pharmaceutical salt of compound A with compound B in a solvent to prepare a pharmaceutical salt of compound A;

[0031] Wherein, compound B is an alkali metal hydroxide, an alkali metal inorganic acid salt, an alkali metal alkoxide, an alkali metal organic acid salt, an alkaline earth metal hydroxide, or an alkaline earth metal inorganic acid salt.

[0032] In one embodiment of the present invention, the other pharmaceutical salts of compound A are alkali metal salts of compound A.

[0033] In one aspect of the present invention, a method for preparing an alkali metal salt of compound A is provided, comprising: reacting compound A and compound B in a solvent to prepare an alkali metal salt of compound A; wherein, compound B is an alkali metal hydroxide, an alkali metal inorganic acid salt, an alkali metal alkoxide, or an alkali metal organic acid salt; preferably an alkali metal hydroxide, an alkali metal alkoxide, or an alkali metal organic acid salt.

[0034] In one aspect of the present invention, a method for preparing an alkaline earth metal salt of compound A is provided, comprising: reacting compound A and compound B in a solvent to prepare an alkaline earth metal salt of compound A; wherein, compound B is an alkaline earth metal hydroxide.

[0035] In one aspect of the present invention, a method for preparing an alkaline earth metal salt of compound A is provided, comprising: reacting an alkali metal salt of compound A with compound B in a solvent to prepare an alkaline earth metal salt of compound A; wherein, compound B is an alkaline earth metal inorganic acid salt.

[0036] The alkali metal hydroxide is a commonly used alkali metal hydroxide in this type of reaction, preferably lithium hydroxide, sodium hydroxide or potassium hydroxide; sodium hydroxide is also preferred.

[0037] The alkali metal inorganic acid salt is a commonly used alkali metal inorganic acid salt in this type of reaction, preferably an alkali metal carbonate or an alkali metal bicarbonate, and even more preferably sodium carbonate, potassium carbonate, sodium bicarbonate or potassium bicarbonate.

[0038] The alkali metal alkoxide is a commonly used alkali metal alkoxide in this type of reaction, preferably a methanol salt, ethanol salt, n-propoxide salt, isopropoxide salt, n-butoxide salt, or tert-butoxide salt of an alkali metal, preferably sodium methoxide, potassium methoxide, lithium methoxide, sodium ethoxide, potassium ethoxide, sodium isopropoxide, potassium isopropoxide, sodium tert-butoxide, or potassium tert-butoxide; and even more preferably sodium methoxide, sodium ethoxide, sodium isopropoxide, sodium tert-butoxide, potassium methoxide, or potassium ethoxide.

[0039] The alkali metal organoacid salt is a commonly used alkali metal organoacid salt in this type of reaction in the art, preferably sodium formate, sodium acetate, sodium propionate, sodium butyrate, sodium valerate, sodium hexanoate, sodium octanoate, sodium isooctanoate, potassium octanoate, or potassium isooctanoate; even more preferably sodium octanoate, sodium isooctanoate, potassium octanoate, or potassium isooctanoate.

[0040] The alkaline earth metal hydroxide is a commonly used alkaline earth metal hydroxide in this type of reaction, preferably magnesium hydroxide or calcium hydroxide.

[0041] The alkaline earth metal inorganic acid salt is a commonly used alkaline earth metal inorganic acid salt in this type of reaction, preferably an alkaline earth metal hydrochloride, alkaline earth metal carbonate or alkaline earth metal bicarbonate; more preferably an alkaline earth metal hydrochloride, preferably magnesium chloride or calcium chloride; and even more preferably magnesium chloride.

[0042] In one aspect of the present invention, a method for preparing a monosodium salt of compound A is provided, comprising: reacting compound A and compound B in a solvent to prepare a monosodium salt of compound A, wherein the compound B is sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium methoxide, sodium ethoxide, sodium isopropoxide, sodium tert-butoxide, sodium octanoate, or sodium isooctanoate.

[0043] In one aspect of the present invention, a method for preparing a magnesium salt of compound A is provided, comprising: reacting a monosodium salt of compound A with compound B in a solvent to prepare a magnesium salt of compound A, wherein the compound B is magnesium chloride.

[0044] The solvent is selected from one or more solvents commonly used in this type of reaction in the art, such as water and organic solvents.

[0045] The organic solvent is a commonly used organic solvent in this type of reaction in the art; preferably, it is one or more of alcohol solvents, ketone solvents, or ether solvents; more preferably, it is one or two of alcohol solvents or ether solvents; preferably, the alcohol solvent is selected from one or more of methanol, ethanol, n-propanol, isopropanol, n-butanol, or tert-butanol, preferably one or two of methanol or ethanol; the ketone solvent is selected from one or two of acetone or butanone; and the ether solvent is selected from tert-butyl methyl ether.

[0046] In one embodiment of the present invention, the solvent is selected from one or more of water, methanol, ethanol or tert-butyl methyl ether.

[0047] When the monosodium salt of compound A described in this invention is in crystalline form, the crystalline form of the monosodium salt of compound A can be prepared by a recrystallization step. The recrystallization can be carried out using conventional methods in the art, including cooling crystallization, solvent evaporation, or solvent / antisolvent crystallization. Different recrystallization methods can be used individually or in combination.

[0048] In a third aspect, the present invention provides a method for preparing the crystal form of the monosodium salt of compound A as described above, comprising a recrystallization step: recrystallizing the monosodium salt of compound A in a solvent.

[0049] Wherein: the crystal form of the monosodium salt of compound A includes any obtainable crystal form of the monosodium salt of compound A, including crystal form I, crystal form II or crystal form III of the monosodium salt of compound A.

[0050] The monosodium salt of compound A in the recrystallization step includes any form of monosodium salt of compound A, including amorphous or crystalline forms of monosodium salt of compound A, including amorphous or crystalline form I, crystalline form II, or crystalline form III of monosodium salt of compound A.

[0051] The recrystallization solvent is selected from solvents commonly used in recrystallization in the art, such as organic solvents or mixtures of water and organic solvents.

[0052] The organic solvent is a commonly used organic solvent for recrystallization in the art; preferably, it is one or more of alcohol solvents, ketone solvents, or ether solvents; more preferably, it is one or two of alcohol solvents or ether solvents; preferably, the alcohol solvent is selected from one or more of methanol, ethanol, n-propanol, isopropanol, n-butanol, or tert-butanol, and more preferably one or two of methanol or ethanol; the ketone solvent is selected from one or two of acetone or butanone; and the ether solvent is selected from tert-butyl methyl ether.

[0053] In one embodiment of the present invention, the recrystallization solvent is selected from organic solvents or a mixture of water and organic solvents, wherein the organic solvent is selected from one or two of methanol, ethanol or tert-butyl methyl ether.

[0054] In one embodiment of the present invention, the recrystallization solvent is selected from a mixed solvent of methanol and tert-butyl methyl ether. Preferably, the volume ratio of methanol to tert-butyl methyl ether is 1:0.1-30, more preferably 1:0.1-10, and even more preferably 1:0.5-5.

[0055] In one embodiment of the present invention, the recrystallization solvent is selected from ethanol or a mixture of ethanol and water. Preferably, the volume ratio of ethanol to water is greater than or equal to 1:10, more preferably greater than or equal to 1:1, and more preferably 1-30:1.

[0056] In one embodiment of the present invention, the recrystallization solvent is selected from a mixed solvent of ethanol and water, wherein the volume ratio of ethanol to water is 1-8:1.

[0057] In one embodiment of the present invention, the recrystallization solvent is selected from a mixed solvent of ethanol and water, wherein the volume ratio of ethanol to water is greater than or equal to 9:1, preferably 9-30:1.

[0058] Seed crystals may be added during the recrystallization step.

[0059] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable salt of compound A and pharmaceutically acceptable excipients;

[0060] Wherein, the pharmaceutical salt of compound A is as described in any embodiment of the present invention; preferably, the pharmaceutical salt of compound A is selected from the monosodium salt of compound A.

[0061] In one aspect of the present invention, the pharmaceutically acceptable excipients include one or more of the following: excipients, surfactants, pH adjusters, osmotic pressure adjusters, lubricants, disintegrants, stabilizers, suspending agents, flavoring agents, solvents, emulsifiers, dispersants, or solubilizers.

[0062] In one embodiment of the present invention, the pharmaceutical composition is selected from solution preparations, aerosols, inhaled dry powder preparations, inhaled solutions, lyophilized powder preparations, injections, tablets, capsules, granules, etc.; preferably, lyophilized powder preparations. The lyophilized powder preparation includes lyophilized powder preparations for injection or lyophilized powder preparations for inhalation, and the lyophilized powder preparation is administered by infusion, injection, or inhalation after reconstitution; preferably, it is administered by injection.

[0063] In one embodiment of the present invention, the pharmaceutically acceptable excipient includes a pH adjuster. Preferably, the pH adjuster is selected from one or more of organic bases and their salts, organic acids and their salts, inorganic acids and their salts, and inorganic bases and their salts.

[0064] In one aspect of the invention, the pharmaceutically acceptable excipient includes an excipient. Preferably, the excipient is selected from one or more of lactose, mannitol, xylitol, sorbitol, and glucose, more preferably lactose or mannitol, and more preferably mannitol.

[0065] In one embodiment of the present invention, the pharmaceutical composition is a lyophilized powder formulation. Preferably, the pH of the solution of the lyophilized powder formulation before lyophilization or after reconstitution is 3-10, more preferably 5-9, even more preferably 6-9, and further preferably 6.5-9, for example 7-9.

[0066] In one aspect of the present invention, a lyophilized powder formulation is provided, wherein the lyophilized powder formulation contains a monosodium salt of compound A and a pH adjuster.

[0067] In one aspect of the present invention, a lyophilized powder formulation is provided, the lyophilized powder formulation containing a monosodium salt of compound A, a pH adjuster, and mannitol.

[0068] In further research on compound A or its pharmaceutical salt, it was discovered that, in addition to exhibiting excellent inhibitory activity against neutrophil elastase, compound A or its pharmaceutical salt also exhibits excellent inhibitory activity against neutrophil serine protease 3 (PR3). Therefore, compound A or its pharmaceutical salt can be used as a dual inhibitor of neutrophil elastase and / or PR3 enzyme for the prevention or treatment of related diseases.

[0069] In a fifth aspect, the present invention provides the use of compound A as described above, or a pharmaceutical salt of compound A as described above, or a pharmaceutical composition as described above, wherein the use is selected from:

[0070] (1) Preparation of PR3 enzyme inhibitors;

[0071] (2) To prepare drugs for the treatment and / or prevention of diseases related to PR3 enzymes;

[0072] Preferably, the PR3 enzyme-related disease is sepsis.

[0073] In a sixth aspect, the present invention provides the use of a pharmaceutical salt of compound A as described above or of a pharmaceutical composition as claimed in claim 7 or 8, wherein the use is selected from:

[0074] (1) Preparation of neutrophil elastase inhibitor

[0075] (2) To prepare drugs for the treatment and / or prevention of diseases associated with neutrophil elastase;

[0076] (3) Prepare medicines for the treatment and / or prevention of the following diseases: acute lung injury or acute respiratory distress syndrome.

[0077] definition

[0078] 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. Furthermore, the following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe this invention.

[0079] The term "amorphous" refers to a substance formed when the particles (molecules, atoms, ions) of a substance are arranged in a non-periodic manner in three-dimensional space. Its characteristic is that it has a diffuse X-ray powder diffraction pattern without sharp peaks.

[0080] The term "crystal form" refers to a solid with a highly regular chemical structure, including but not limited to single-component or multi-component crystals, and / or polymorphs of compounds, solvates, hydrates, inclusion compounds, eutectics, salts, solvates of salts, hydrates of salts, etc.

[0081] The term "pharmaceuticalally acceptable excipient" refers to any formulation or carrier medium capable of delivering an effective amount of the active substance of the present invention without interfering with the biological activity of the active substance and without toxic side effects on the host or patient. Representative excipients include excipients, surfactants, pH adjusters, osmotic pressure adjusters, lubricants, disintegrants, stabilizers, suspending agents, flavoring agents, solvents, emulsifiers, dispersants, or solubilizers.

[0082] The term "pharmaceutical composition" refers to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient and a pharmaceutically acceptable excipient, intended for administration to mammals, such as humans, in need of such treatment.

[0083] The term “treatment” refers to reversing, alleviating, inhibiting, or preventing the progression of an obstacle or condition to which the term applies, or one or more symptoms of such an obstacle or condition. As used herein, the term “treatment” refers to the action of the verb “to treat,” as previously defined.

[0084] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0085] The reagents and raw materials used in this invention are all commercially available.

[0086] High-resolution mass spectrometry: Detection instrument: Ultra-high performance liquid chromatography / supercritical fluid chromatography-quadrupole time-of-flight mass spectrometry (G2-XS) (Acquity 2D-UPLC / Acquity UPC2 / Xevo G2-XS QTOF). Mass spectrometry analysis conditions: Electrospray ionization source, positive ion mode, scan range m / z 50-1200.

[0087] The positive and progressive effects of this invention are as follows: This invention provides a pharmaceutical salt of a sulfonamide compound, the crystal form of the salt, its composition, and its uses. The pharmaceutical salt of the sulfonamide compound of this invention and the crystal form of the salt have one or more of the following effects: good solubility in water, high stability, good inhibitory effect on PR3 enzyme and neutrophil elastase; and can improve the survival rate of sepsis. Attached Figure Description

[0088] Figure 1. PXRD pattern of the amorphous form of the monosodium salt of compound A.

[0089] Figure 2. PXRD pattern of monosodium salt crystal form I of compound A.

[0090] Figure 3. PXRD pattern of monosodium salt form II of compound A.

[0091] Figure 4. PXRD pattern of monosodium salt form III of compound A. Detailed Implementation

[0092] The present invention is further illustrated below by way of embodiments, but these embodiments are not intended to limit the invention to their scope. Experimental methods not specifically described in the following embodiments were performed according to conventional methods and conditions, or as selected according to the product instructions. The raw materials used in the embodiments of the present invention can be purchased or obtained according to conventional methods in the art.

[0093] Example 1: O-methyl-N-(2-((4-(neovaleroxy)phenyl)sulfonamido)benzoyl)-D-serine

[0094] Step 1: N-(tert-butoxycarbonyl)-O-methyl-D-serine benzyl ester

[0095] A mixture of N-(tert-butoxycarbonyl)-O-methyl-D-serine (1 g, 4.6 mmol), CbzCl (1.56 g, 9.2 mmol), TEA (0.9 g, 9.2 mmol), and DMAP (112 mg, 0.92 mmol) in DCM (10 mL) was stirred overnight at 0 °C. The mixture was quenched with H2O (10 mL) and extracted with DCM (30 mL × 3). The organic phases were combined, washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (13% EtOAc / PE) to give N-(tert-butoxycarbonyl)-O-methyl-D-serine benzyl ester (1.3 g, 92% yield) as a colorless oil. MS (ESI): m / z 210.1 [M-99] + .

[0096] Step 2: O-methyl-D-serine benzyl ester

[0097] A mixture of N-(tert-butoxycarbonyl)-O-methyl-D-serine benzyl ester (1.3 g, 4.2 mmol) and TFA (5 mL) in DCM (50 mL) was stirred at room temperature for 2 hours. The mixture was then dried under vacuum to give a white solid product (800 mg, crude product). The crude product was used directly in the next step. MS (ESI): m / z = 210.2 [M+H] + .

[0098] Step 3: N-(2-aminobenzoyl)-O-methyl-D-serine benzyl ester

[0099] The product of step 2 (200 mg, 0.62 mmol), 2H-benzo[d][1,3]oxazine-2,4(1H)-dione (186 mg, 1.14 mmol), and TEA (192 mg, 1.9 mmol) were mixed in THF (5 mL) and stirred overnight at room temperature. The reaction mixture was concentrated and purified by silica gel column chromatography (25% EtOAc / PE) to give N-(2-aminobenzoyl)-O-methyl-D-serine benzyl ester (100 mg, 49% yield) as a colorless oil. MS (ESI): m / z 329.1 [M+H] + .

[0100] Step 4: O-methyl-N-(2-((4-(neovaleroxy)phenyl)sulfonamido)benzoyl)-D-serine benzyl ester

[0101] The product from step 3 (100 mg, 0.3 mmol), 4-(chlorosulfonyl)phenyl pentanoate (93 mg, 0.33 mmol), and Py (1.8 g, 2.4 mmol) were mixed in DCM (5 mL) and stirred overnight at room temperature. The reaction mixture was concentrated and purified by silica gel column chromatography (20% EtOAc / PE) to give a colorless oil of O-methyl-N-(2-((4-(neopentyloxy)phenyl)sulfonamide)benzoyl)-D-serine benzyl ester (150 mg, 86% yield). MS (ESI): m / z 569.2 [M+H] + .

[0102] Step 5: O-methyl-N-(2-((4-(neovaleroxy)phenyl)sulfonamido)benzoyl)-D-serine

[0103] The product from step 4 (150 mg, 0.26 mmol) and a mixture of 10% Pd / C (50 mg) in MeOH (5 mL) were stirred overnight at room temperature under H2 atmosphere. The mixture was filtered. The filtrate was concentrated and purified by PREP-HPLC (using TFA buffer: A: 10 mM TFA aqueous solution; B: acetonitrile; column: Waters XBridge Peptide BEH C18, 19 × 250 mm, 10 μm). The reaction yielded a white solid product, compound A (76 mg, 60% yield). MS (ESI): m / z = 479.1 [M+H] + .

[0104] 1 H NMR (400MHz, DMSO-d6): δ13.0(brs,1H),11.39(s,1H),9.05(d,J=7.3Hz,1H),7.82-7.77(m,3H),7.50-4.48(m,2H ),7.28(dd,J=7.2,2.4Hz,2H),7.21–7.12(m,1H),4.62-4.58(m,1H),3.77-3.66(m,2H),3.29(s,3H),1.27(s,9H).

[0105] Example 2: A single lithium salt of compound A (O-methyl-N-(2-((4-(neoptiacyloxy)phenyl)sulfonamido)benzoyl)-D-serine lithium)

[0106] In a 50 ml reaction flask, 1.0 g of compound A (free acid, prepared in Example 1) and 9.0 ml of methanol were added, and the temperature was raised to 35 °C. 0.6 ml of a 3.3 mol / L lithium hydroxide aqueous solution was added dropwise, and the mixture was stirred for 15 min. 9.0 ml of tert-butyl methyl ether was added, and the mixture was stirred until no solid precipitated. The product was concentrated and dried under vacuum to obtain 1.06 g of solid. The product had poor properties, with some parts being slightly viscous and syrupy. HPLC analysis showed a purity of 93.80%, with a maximum single impurity content of 4.04%, which was difficult to remove.

[0107] Example 3: Monopotassium salt of compound A (O-methyl-N-(2-((4-(neoptiacyloxy)phenyl)sulfonamido)benzoyl)-D-serine potassium)

[0108] In a 50 ml reaction flask, 1.0 g of compound A (free acid, prepared in Example 1) and 9.0 ml of methanol were added, and the temperature was raised to 35 °C. 0.5 ml of a 4.0 mol / L potassium hydroxide aqueous solution was added dropwise, and the mixture was stirred for 15 min. 9.0 ml of tert-butyl methyl ether was added, and the mixture was stirred until no solid precipitated. The product was concentrated and dried under vacuum to obtain 1.1 g of solid. The product had poor properties, with some parts being slightly viscous and syrupy. HPLC analysis showed a purity of 93.67%, with a maximum single impurity content of 4.11%, which was difficult to remove.

[0109] Example 4: Monosodium salt of compound A (O-methyl-N-(2-((4-(neoptiacyloxy)phenyl)sulfonamido)benzoyl)-D-serine sodium)

[0110] In a 100 ml reaction flask, add 6.0 g of compound A (free acid, prepared in Example 1), 12.0 ml of methanol, and 12.0 ml of tert-butyl methyl ether. Add 2.26 ml of sodium methoxide methanol solution (30% wt 1.0 eq), the system remains clear with minimal temperature increase. Then add 18.0 ml of tert-butyl methyl ether and stir overnight at room temperature (25-35°C). Add 24.0 ml of tert-butyl methyl ether, stir in a -5°C cold bath to obtain a solid, filter, wash the filter cake with tert-butyl methyl ether (6.0 ml × 2), dry the wet product to obtain 6.16 g of white solid. HPLC analysis showed a purity of 99.74%.

[0111] High-resolution mass spectrometry: [M+Na] + =501.1305.

[0112] 1H NMR (600MHz, DMSO-d6) δ11.10(s,1H),7.85–7.83(m,1H),7.82(d,J=8.7Hz,2H),7.37(d,J=8.3Hz,1H),7.13(d,J=8.6 Hz,2H)7.12–7.10(m,1H),6.70(t,J=7.4Hz,1H),4.53(t,J=5.0Hz,1H),3.71–3.62(m,2H),3.29(s,3H),1.27(s,9H).

[0113] Example 5: Magnesium salt of compound A (O-methyl-N-(2-((4-(neoptiacyloxy)phenyl)sulfonamido)benzoyl)-D-serine magnesium)

[0114] In a 100ml reaction flask, add 1.7g of the monosodium salt of compound A (prepared in Example 4) and 20.0ml of water, heat to 40-50℃ and stir until dissolved. Take 320mg of magnesium chloride, add 14.0ml of water, stir to dissolve, and add dropwise to the aqueous solution of the monosodium salt of compound A. A solid precipitates during the addition. After the addition is complete, stir at 40-50℃ for 1-2 hours. Then cool to 20-30℃ and stir to form a slurry for 1-2 hours. Filter, dry the filter cake, and obtain 1.4g of white solid. HPLC analysis shows a purity of 99.75%. Test Example 1: Solubility of pharmaceutical salt of compound A.

[0115] Compound A or its different pharmaceutical salts, as well as cevelexat sodium, were tested for their solubility in water. The results are shown in Table 1 below:

[0116] Solubility test conditions: Weigh an appropriate amount of the sample, which has been ground into a fine powder (meaning that all of it can pass through a No. 5 sieve and contains no less than 95% of the powder that can pass through a No. 6 sieve), and place it in an appropriate amount of water at 25℃±2℃. Shake vigorously for 30 seconds every 5 minutes and observe the dissolution within 30 minutes. If no solute particles are visible to the naked eye, it is considered to be completely dissolved.

[0117] Table 1

[0118] Compound A is poorly soluble in water, but its pharmaceutical salts can significantly improve its solubility. Furthermore, the solubility of the monolithium, monopotassium, and monosodium salts of compound A is significantly better than that of the magnesium salt. The solubility of the monosodium salt of compound A is significantly better than that of cevelexat sodium.

[0119] Example 6 Monosodium salt crystal form I of compound A

[0120] In a 100ml reaction flask, add 4.0g of the monosodium salt of compound A (prepared in Example 4), 16.0ml of methanol, and heat to 45-55℃ with stirring to dissolve. Filter, collect the filtrate, add 16ml of tert-butyl methyl ether, and stir at 25-35℃ overnight to allow crystallization. Add 48ml of tert-butyl methyl ether dropwise over 15-30 minutes. After the addition is complete, stir and slurry for 3-5 hours. Filter, wash the filter cake with 8ml of tert-butyl methyl ether, dry under vacuum, and then use an oil pump to remove excess filter cake, yielding monosodium salt of compound A in crystal form I. The PXRD pattern is shown in Figure 2.

[0121] Example 7 Monosodium salt crystal form II of compound A

[0122] In a 100ml reaction flask, add 4.0g of the monosodium salt of compound A (prepared in Example 4), and 16.0ml of an ethanol / water (8:1 v / v) mixture. Heat to 45-55℃ and stir to dissolve. Filter, collect the filtrate, and stir at 20-30℃ overnight to allow crystals to precipitate. Add 48ml of tert-butyl methyl ether dropwise over 15-30 minutes. After the addition is complete, stir and slurry for 3-5 hours. Filter, wash the filter cake with 8ml of tert-butyl methyl ether, dry under vacuum, and then use an oil pump to remove excess water, yielding monosodium salt of compound A, crystal form II. The PXRD pattern is shown in Figure 3.

[0123] Example 8: Monosodium salt crystal form III of compound A

[0124] In a 100ml reaction flask, add 4.0g of the monosodium salt of compound A (prepared in Example 4), and 16.0ml of an ethanol / water (volume ratio 16:1). Heat to 45-55℃ and stir to dissolve. Filter, collect the filtrate, and stir at 35-45℃ overnight to allow crystals to precipitate. Add 48ml of tert-butyl methyl ether dropwise over 15-30 minutes. After the addition is complete, stir and slurry at 20-30℃ for 3-5 hours. Filter, wash the filter cake with 8ml of tert-butyl methyl ether, dry under vacuum, and then use an oil pump to remove excess water, yielding monosodium salt of compound A, crystal form III. The PXRD pattern is shown in Figure 4.

[0125] Following the method of Example 8, the monosodium salt of compound A was recrystallized in an ethanol / water (9:1) mixed solution to obtain monosodium salt crystal form III of compound A.

[0126] Stability of various crystal forms of monosodium salt of compound A in Test Example 2

[0127] A certain amount of monosodium salt crystals I, II, and III of compound A were taken and dried in a forced-air drying environment at 40℃, 50℃, and 60℃ for 24 h, respectively. Samples were taken and PXRD was measured. The results are shown in Table 2 below:

[0128] Table 2

[0129] The monosodium salts of compound A, crystal forms I and II, are easily converted to amorphous forms during the drying process (PXRD spectra are shown in Figure 1). Crystal form III remains stable throughout the drying process, and the stability of crystal form III is significantly better than that of crystal forms I and II.

[0130] Table 3 Note: The above methods for investigating light exposure, high temperature, high humidity, accelerated treatment over 3 months, and long-term treatment over 3 months all refer to the methods in the Chinese Pharmacopoeia.

[0131] The crystal form III of the monosodium salt of compound A remained unchanged after 30 days of influencing factors (light, high humidity, high temperature), 3 months of accelerated treatment, and 3 months of long-term treatment, indicating that crystal form III has good stability.

[0132] Example 9 Pharmaceutical Composition

[0133] Table 4

[0134] Mannitol was added to 60% of the prescribed water for injection and stirred to dissolve. The monosodium salt of compound A was then added and stirred to disperse. 0.1M NaOH solution was added to adjust the pH to 7.5. After the monosodium salt of compound A was completely dissolved, water was added to bring the volume to 5 ml. The solution was filled, freeze-dried, and capped. The lyophilized powder formulation of the monosodium salt of compound A was obtained.

[0135] Activity test 1: Inhibitory effect of monosodium salt of compound A on PR3 enzyme.

[0136] The PR3 protease solution was incubated with the substrates Boc-Ala-Ala-NVa-SBzl and DTNB for 20 min. The sample wells were then filled with the test sample, and the absorbance was measured at 410 nM. The luminescence signal was positively correlated with the protease activity. The absorbance value of each well was measured using a microplate reader, and the IC50 value of the test sample for PR3 enzyme activity was obtained by plotting and calculation.

[0137] This experiment used XLfit software, developed by IDBS and integrated into the Microsoft Excel environment, for test data processing and analysis. First, the average reaction signals of the positive control wells (HPE) and negative control wells (ZPE) were calculated separately. Then, the percentage inhibition rate of each compound well was calculated using the formula: "Inhibition rate per well = (Average signal value of negative control - Signal value per well) / (Average signal value of negative control - Average signal value of positive control) * 100%". Next, the concentration and corresponding inhibition rate data were imported into XLfit software. Using the Dose Response One Site 205 model in the software, a four-parameter method was employed to fit the inhibition rate-concentration curve, and the half-maximum inhibitory concentration (IC50 value) of the compound was calculated.

[0138] Table 5

[0139] In vitro enzyme activity assays showed that the monosodium salt of compound A had a good inhibitory effect on PR3 enzyme, and the inhibitory effect of the monosodium salt of compound A on PR3 enzyme was significantly better than that of cevelexta sodium.

[0140] Animal Experiment 1: Efficacy evaluation of the monosodium salt of compound A in a rat model of acute sepsis induced by cecal ligation and perforation (CLP) surgery.

[0141] Male SD rats, 7-8 weeks old, were selected. After acclimatization, they were grouped. Based on their body weight, the animals were randomly assigned to groups using the BioBook randomization function to achieve approximately equal weight across groups and reduce inter-group bias. The grouping and drug administration details are shown in the table below.

[0142] Table 6

[0143] iv: intravenous injection; a: BID: twice daily, approximately 4 hours apart; b: TID: three times daily, approximately 4 hours apart.

[0144] Drug preparation method: Use 0.9% NaCl and ultrapure water as solvents, add monosodium salt of compound A or cevelexat sodium, then add 0.1M NaOH to bring the pH to 7.5, and continue stirring until dissolved.

[0145] One day before surgery (Day 1), after grouping, on the day of surgery (Day 0), all rats were anesthetized with isoflurane (1-4%). ​​In groups 2-4, after anesthesia, a 1.5-2 cm incision was made along the midline of the abdomen to separate the cecum. The cecum was tightly ligated between its distal and proximal ends using 4-0 silk sutures. A puncture was then performed on the ligated cecum, and a small amount of feces was expelled. The cecum was then returned to its original position in the abdomen, and the abdominal cavity was sutured shut. In group 1, after abdominal incision, the cecum was separated, returned to its original position in the abdomen, and the abdominal cavity was sutured shut without puncture. All surgeries were performed under strict aseptic conditions. Each rat received a subcutaneous injection of 20 mg / kg nefopam for analgesia and 10 mL / kg saline for fluid replacement postoperatively for 3 consecutive days. All animals were observed postoperatively until they regained consciousness.

[0146] The medication is administered intravenously after surgery. It is given twice on the day of surgery (Day 0), with an interval of approximately 4 hours. On Day 1 (postoperative day 1) and thereafter, it is given three times daily, with an interval of approximately 4 hours. See Table 9 for the detailed administration schedule.

[0147] Twenty-four hours after surgery, all animals were anesthetized by inhalation of 1-4% isoflurane. Approximately 200 μL of whole blood was collected through the orbital vein, centrifuged at 2000g at 4°C for 10 minutes, and the serum was separated and stored at 80°C for later detection of cytokines IL-6 and TNF-α.

[0148] Survival rates were observed twice daily, and differences were statistically analyzed. The experiment ended on Day 6 after the last administration of the drug. All remaining animals in all groups were euthanized by CO2 inhalation, and their livers and bilateral kidneys were collected, fixed in 10% neutral formalin solution, and stored for future use.

[0149] Table 7 Note: Differences noted in cells: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001; Compared with the sham surgery group, # P<0.05, ## P<0.01, ### P<0.001.

[0150] In a rat model of acute sepsis induced by cecal ligation and perforation (CLP) surgery, the monosodium salt of compound A significantly improved the survival rate of rats, with better results than cevelexatol sodium.

[0151] Activity test 2: Inhibitory effect of compound A or its sodium salt on neutrophil elastase.

[0152] Compound preparation: All compounds were dissolved in DMSO at a concentration of 10 mM and stored at -20 °C.

[0153] Prepare a 1x assay buffer by serially diluting the positive compound (Sivelestat) and the analyte in DMSO three times, resulting in 10 concentration points. Add 1 μL of the serially diluted compound and 65.67 μL of assay buffer to each well to obtain the 3X working solutions of the positive compound and the analyte (DMSO final concentration 1.5%). Seal the wells with sealing film and shake on a shaker for 15 minutes.

[0154] Prepare a 1x activation buffer and dilute rhELA2 in an activation buffer containing DPPI. Incubate at 37°C for 2 hours to activate rhELA2. Add 5 μl of the 3x diluted working solution to a 384-well plate. Dilute rhELA2 with 1x assay buffer to obtain a 3x rhELA2 working solution. Add 5 μl of the 3x rhELA2 working solution to each well of the 384-well plate. Seal the plate and incubate at room temperature in the dark for 30 minutes.

[0155] Prepare a 3x substrate solution in 1x assay buffer. Add 5 μl of the 3x substrate working solution to a 384-well plate. Centrifuge the 384-well plate at 1000 rpm for 1 minute. Incubate at room temperature in the dark for 1 hour. Read the fluorescence values ​​(FLU) at 360 nm / 450 nm using a plate reader.

[0156] The inhibition rate was calculated as % inhibition rate = (1 - (FLU compound - FLU positive wells) / (FLU reference wells - FLU positive wells)). FLU reference wells were those with only enzyme added and no inhibitor added, while FLU positive wells were those with both enzyme and 3000 nM Sivelestat-2. The IC50 value was then calculated using Graphpad 8.0 software: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)).

[0157] Table 8

[0158] Compound A exhibits excellent inhibitory activity against neutrophil elastase.

Claims

1. A pharmaceutical salt of compound A, characterized in that, The pharmaceutical salt of compound A is an alkali metal salt or an alkaline earth metal salt; 2. The pharmaceutical salt of compound A according to claim 1, characterized in that, The pharmaceutical salt of compound A is a monosodium salt, a monolithium salt, a monopotassium salt, a magnesium salt, or a calcium salt of compound A; preferably, it is a monosodium salt of compound A.

3. The pharmaceutical salt of compound A according to claim 2, characterized in that, The monosodium salt of compound A includes the amorphous or crystalline form of the monosodium salt of compound A; preferably the amorphous form of the monosodium salt of compound A, crystalline form I of the monosodium salt of compound A, crystalline form II of the monosodium salt of compound A, or crystalline form III of the monosodium salt of compound A. The monosodium salt of compound A, crystal form I, has X-ray powder diffraction patterns at 5.1±0.2°, 10.4±0.2°, 12.0±0.2° and 17.8±0.2° when expressed in 2θ angles. The monosodium salt of compound A, crystal form II, has X-ray powder diffraction patterns at 3.4±0.2°, 5.5±0.2°, 10.3±0.2°, 12.2±0.2° and 16.3±0.2° when expressed in 2θ angles. The monosodium salt of compound A, crystal form III, has characteristic peaks at 4.3±0.2°, 8.6±0.2°, 9.5±0.2° and 18.0±0.2° in its X-ray powder diffraction pattern expressed in 2θ angles; Preferably, the pharmaceutical salt of compound A satisfies one or more of the following conditions: (1) The basic X-ray diffraction pattern of the monosodium salt of compound A is shown in Figure 1; (2) The monosodium salt of the compound A, crystal form I, has X-ray powder diffraction patterns in 2θ angles that also have diffraction peaks at one or more of the following 2θ angles: 4.6±0.2°, 13.5±0.2°, 16.5±0.2° and 19.5±0.2°; (3) The monosodium salt of compound A, crystal form II, has an X-ray powder diffraction pattern in 2θ angles that also shows diffraction peaks at one or more of the following 2θ angles: 14.6±0.2°, 17.2±0.2°, 17.8±0.2°, and 19.4±0.2°; and (4) The monosodium salt of the compound A, crystal form III, has X-ray powder diffraction patterns in 2θ angles with diffraction peaks at one or more of the following 2θ angles: 5.5±0.2°, 13.2±0.2°, 16.2±0.2° and 16.9±0.2°.

4. A method for preparing a pharmaceutical salt of compound A, characterized in that, It includes: reacting compound A or other pharmaceutical salts of compound A with compound B in a solvent to prepare a pharmaceutical salt of compound A; Wherein, compound B is an alkali metal hydroxide, an alkali metal inorganic acid salt, an alkali metal alkoxide, an alkali metal organic acid salt, an alkaline earth metal hydroxide, or an alkaline earth metal inorganic acid salt; Other pharmaceutical salts of compound A may be alkali metal salts of compound A.

5. The method for preparing the pharmaceutical salt of compound A according to claim 4, characterized in that, It satisfies one or more of the following conditions: (1) Compound A and compound B react to prepare an alkali metal salt of compound A, wherein the compound B is an alkali metal hydroxide, an alkali metal inorganic acid salt, an alkali metal alkoxide or an alkali metal organic acid salt; (2) The alkali metal salt of compound A reacts with compound B to prepare the alkaline earth metal salt of compound A; wherein, compound B is an alkaline earth metal inorganic acid salt; (3) The alkali metal hydroxide is lithium hydroxide, sodium hydroxide or potassium hydroxide; the alkali metal inorganic acid salt is an alkali metal carbonate or an alkali metal bicarbonate; the alkali metal alkoxide is an alkali metal methanol salt, ethanol salt, n-propoxide salt, isopropoxide salt, n-butoxide salt or tert-butoxide salt; the alkali metal organic acid salt is sodium formate, sodium acetate, sodium propionate, sodium butyrate, sodium valerate, sodium hexanoate, sodium octanoate, sodium isooctanoate, potassium octanoate or potassium isooctanoate; (4) The alkaline earth metal hydroxide is magnesium hydroxide or calcium hydroxide; the alkaline earth metal inorganic acid salt is alkaline earth metal hydrochloride, alkaline earth metal carbonate, or alkaline earth metal bicarbonate; and (5) The solvent is one or more of water and organic solvents.

6. A method for preparing the crystal form of a monosodium salt of compound A, characterized in that, Includes a recrystallization step: recrystallization of the monosodium salt of compound A in a solvent; Preferably, the method for preparing the crystal form of the monosodium salt of compound A satisfies one or more of the following conditions: (1) The crystal form of the monosodium salt of compound A includes crystal form I, crystal form II or crystal form III of the monosodium salt of compound A; (2) The monosodium salt of compound A in the recrystallization step includes the amorphous or crystalline form of the monosodium salt of compound A. (3) The solvent for recrystallization is selected from organic solvents or a mixture of water and organic solvents; and (4) Seed crystals may be added optionally during the recrystallization step.

7. A pharmaceutical composition, characterized in that, It contains pharmaceutical salts of compound A and pharmaceutically acceptable excipients; 8. The pharmaceutical composition according to claim 7, characterized in that, It satisfies one or more of the following conditions: (1) The pharmaceutical salt of compound A as described in any one of claims 1-3; (2) The pharmaceutical composition is a solution preparation, aerosol, inhaled dry powder, inhaled solution, lyophilized powder preparation, injection, tablet, capsule or granule; preferably a lyophilized powder preparation; more preferably, the pH of the solution of the lyophilized powder preparation before lyophilization or after reconstitution is 3-10; (3) The pharmaceutically acceptable excipients include pH adjusters; preferably, the pH adjusters are selected from one or more of organic bases and their salts, organic acids and their salts, inorganic acids and their salts, and inorganic bases and their salts; and (4) The pharmaceutically acceptable excipients include excipients; preferably, the excipients are selected from one or more of lactose, mannitol, xylitol, sorbitol and glucose.

9. Use of a compound A, a pharmaceutical salt of compound A as claimed in any one of claims 1-3, or a pharmaceutical composition as claimed in claim 7 or 8. The uses are selected from: (1) Preparation of PR3 enzyme inhibitors; (2) Prepare a drug for treating and / or preventing diseases related to PR3 enzymes; preferably, the disease is sepsis.

10. Use of a pharmaceutical salt of compound A as described in any one of claims 1-3 or a pharmaceutical composition as described in claim 7 or 8, wherein the use is selected from: (1) Preparation of neutrophil elastase inhibitor (2) To prepare drugs for the treatment and / or prevention of diseases associated with neutrophil elastase; (3) Prepare medicines for the treatment and / or prevention of the following diseases: acute lung injury or acute respiratory distress syndrome.

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