Alanine derivatives, processes for their preparation and medical use

Abstract

The application discloses alanine derivatives, a preparation method and medical uses thereof, and an alanine derivative as shown in a general formula (I) and enantiomers, diastereoisomers, tautomers, N-oxides, solvates, physiologically hydrolysable esters, formulations and pharmaceutically acceptable salts of the alanine derivative. 14 The application discloses alanine derivatives and pharmaceutically acceptable salts thereof, which have inhibitory effects on P2Y 14 receptors. The compounds are confirmed by pharmacological experiments to have significant inhibitory effects on P2Y receptors, and can be used as drugs for treating inflammatory diseases including gouty arthritis and peritonitis.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and relates to compounds, their preparation methods and uses, specifically to a class of novel alanine derivatives and pharmaceutical compositions containing such compounds, their preparation methods, and their pharmaceutical uses as therapeutic agents, particularly P2Y14 receptor antagonists. Background Technology

[0002] When the body is stressed or injured, various important intracellular molecules, such as adenosine-5′-triphosphate (ATP) and uridine-5′-triphosphate (UTP), are released into the extracellular fluid by specific tissues or organs. These nucleotides have the ability to regulate innate and adaptive immune responses by binding to cell surface receptors. These receptors are identified as purinergic receptors, mainly divided into adenosine (P1) and nucleotide (P2) receptors. The P2 family is further divided into two subfamilies: P2X receptors and P2Y receptors (P2YRs). P2YRs belong to the G protein-coupled receptor (GPCR) family and are composed of P2Y1-like receptors (P2Y1-like receptors). 1，2，4，6，11 ) and P2Y12 (P2Y) 12，13，14 The P2Y receptor family of G protein-coupled receptors, which has been reported to contain 8 subtypes (P2Y...), is composed of... 1、2、4、6、11、12、13、14 P2Y is widely distributed in various cells and tissues, and the homology between different subtypes is relatively low, thus different subtypes exhibit high selectivity for ligands. 1、2、4、6 receptor binding G q And activate the PLC path; P2Y 12、13、14 receptor binding G i Inhibits adenylate cyclase activity; P2Y4 receptor coupled with G q / G i Two G proteins; P2Y 11 Coupled G q / G s Two G proteins. The P2Y receptor mediates a series of biological effects, including immune regulation, platelet aggregation, and smooth muscle cell proliferation.

[0003] P2Y 14 The receptor is activated by at least four naturally occurring UDP sugars, particularly UDP glucose (UDPG). P2Y 14 The receptor has been found to be widely expressed in a range of human tissues, including the brain, heart, adipose tissue, placenta, gut, and hematopoietic stem cells. P2Y 14 The receptor activates its coupled G receptors. i The protein inhibits adenylate cyclase (AC), reducing the production of intracellular 3′,5′ cyclic adenosine monophosphate (cAMP) and its corresponding biological effects.

[0004] P2Y14 Receptors have been shown to be potential targets for innate immune inflammatory diseases such as diabetes, cystic fibrosis, aseptic renal inflammation, acute gouty arthritis, and allergic diseases. P2Y 14 The receptor promotes the recruitment and chemotaxis of neutrophils and macrophages, releasing pro-inflammatory cytokines, chemokines, and mast cell mediators. In severe cases of COVID-19, a significant portion of the severe course is caused by systemic inflammation, known as a "cytokine storm." (Based on P2Y) 14 The effect of receptors on neutrophils and the high levels of UDPG found in severely ill COVID-19 patients, a receptor that interacts with P2Y. 14 The mechanism by which receptor antagonists prevent neutrophil chemotaxis dysregulation has been proposed to avoid cytokine storms and systemic inflammation, thereby reducing patient mortality. Furthermore, P2Y... 14 Knocking out the receptor gene suppressed macrophage recruitment and tissue inflammation, and alleviated insulin resistance induced by a high-fat diet. Another study showed that glycogen metabolism also increased UDPG and P2Y levels in macrophages. 14 The number of receptors, blocking this glycogen metabolism pathway can effectively inhibit lipopolysaccharide (LPS)-induced acute peritonitis. In summary, P2Y... 14 Receptors may be potential therapeutic targets for innate immune system diseases.

[0005] Currently regarding P2Y 14 Research on receptor antagonists has only reported compounds of four structural types (pyrimidine-piperidine, 2-naphthyl acid, 3-substituted benzoic acid, and pyrazole-3-carboxylic acid), all of which are still in the preclinical research stage. Among them, 2-naphthyl acid has the highest activity and selectivity; however, currently reported antagonists with 2-naphthyl acid structures suffer from poor solubility, low oral bioavailability, and difficulties in synthesis and purification, posing significant challenges to further discussion of structure-activity relationships and biological evaluation. Therefore, the search for new structural types of P2Y is crucial. 14 Receptor antagonists, which improve upon the poor drug-likeness of 2-naphthyl acid antagonists, have become a promising candidate for discovering highly active and selective P2Y receptor antagonists. 14 A novel strategy using receptor antagonists. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a novel type of structure that possesses P2Y 14 Alanine derivatives with receptor antagonistic effects and their pharmaceutically acceptable salts. Another object of the present invention is to provide a method for preparing the above-mentioned alanine derivatives. A further object of the present invention is to provide the application of the above-mentioned alanine derivatives in the treatment of inflammatory diseases, including gouty arthritis and peritonitis.

[0007] Technical solution: The alanine derivatives of general formula (I) and their enantiomers, diastereomers, tautomers, N-oxides, solvates, physiologically hydrolyzable esters, formulations, and pharmaceutically acceptable salts as described in this invention:

[0008]

[0009] R 1 Selected from: H, C 1-4 alkyl,

[0010] R 2 Selected from:

[0011]

[0012] R 3 Selected from:

[0013] Phenyl, C 3-6 Cycloalkyl, pyridinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyrroloyl, thiophenyl, furanyl, thiazolyl, isothiazole, imidazolyl, pyrazolyl; the above R3 groups may be independently and optionally substituted by 1 to 5 of the following substituents: C 1-6 Alkyl, halogen, cyano, carboxyl, acetyl, methanesulfonyl, halogenated C 1-4 Alkyl, Halogenated C 1-4 Alkyl group, OH, NO2, aldehyde group, C 1-6 Alkoxy, amino, dimethylamino;

[0014] R 4 Selected from: H, C 3-6 Cycloalkyl, phenyl, methylamino, dimethylamino, -OCOC 1-16 Alkyl, C 1-16 Alkyl group, (OCH2CH2) 1-4 OCH3;

[0015] R 5 Selected from:

[0016] Phenyl, pyridinyl, pyrimidinyl, pyrazinyl, pyrroloyl, thiophenyl, furanyl, thiazolyl, isothiazole, imidazolyl, pyrazolyl; the above R 5 The group may be independently and optionally substituted by 1 to 5 of the following substituents: C 1-4 Alkyl, halogen, cyano, carboxyl, acetyl, methanesulfonyl, halogenated C 1-4 Alkyl, Halogenated C 1-4 Alkyl group, OH, NO2, aldehyde group, C 1-4 Alkoxy, amino, dimethylamino;

[0017] The alanine derivatives and their enantiomers, diastereomers, tautomers, N-oxides, solvates, physiologically hydrolyzable esters, formulations, and pharmaceutically acceptable salts are described.

[0018] R 3 Selected from:

[0019] Phenyl, C 5-6 Cycloalkyl, pyridinyl, pyridinyl, pyrazinyl, pyrroloyl, thiophenyl, furanyl, thiazolyl, isothiazole, imidazolyl, pyrazolyl; the above R 3 The group may be independently and optionally substituted by one or two of the following substituents: halogen, cyano, carboxyl, acetyl, methanesulfonyl, nitro, aldehyde, hydroxyl, amino, trifluoromethyl, trifluoromethoxy, methyl, ethyl, n-propyl, isopropyl, tert-butyl;

[0020] R 4 Selected from: H, C 5-6 Cycloalkyl, phenyl, dimethylamino, -OCOC 1-16 Alkyl, C 1-16 Alkyl group, (OCH2CH2) 1-4 OCH3;

[0021] R 5 Selected from:

[0022] Phenyl, pyridinyl, pyrimidinyl, pyrazinyl, pyrroloyl, thiophenyl, furanyl, thiazolyl, isothiazole, imidazolyl, pyrazolyl; the above R 5 The group may be independently and optionally substituted by one or two of the following substituents: halogen, cyano, carboxyl, acetyl, methanesulfonyl, nitro, aldehyde, hydroxyl, amino, trifluoromethyl, trifluoromethoxy, methyl, ethyl, n-propyl, isopropyl, tert-butyl;

[0023] The alanine derivatives and their enantiomers, diastereomers, tautomers, N-oxides, solvates, physiologically hydrolyzable esters, formulations, and pharmaceutically acceptable salts are described.

[0024] The chiral center is a D-type amino acid backbone;

[0025] R 3 Selected from:

[0026] Phenyl, cyclohexane, pyridyl, thiophenyl, furanyl; the above R 3 The group may be independently and optionally substituted by one of the following substituents: halogen, cyano, carboxyl, acetyl, methanesulfonyl, nitro, aldehyde, hydroxyl, amino, trifluoromethyl, trifluoromethoxy, methyl, ethyl, n-propyl, isopropyl, tert-butyl;

[0027] R 4Selected from: H, cyclohexane, phenyl, dimethylamino, methyl, n-hexadecyl, -OCOCH3, (OCH2CH2)4OCH3;

[0028] R 5 Selected from:

[0029] Phenyl, pyridyl, thiophenyl, furanyl; the above R 5 The group may be independently and optionally substituted by one of the following substituents: halogen, cyano, carboxyl, acetyl, methanesulfonyl, nitro, aldehyde, hydroxyl, amino, trifluoromethyl, trifluoromethoxy, methyl, ethyl, n-propyl, isopropyl, tert-butyl;

[0030] The alanine derivatives and their enantiomers, diastereomers, tautomers, N-oxides, solvates, physiologically hydrolyzable esters, formulations, and pharmaceutically acceptable salts thereof, wherein compounds of general formula (I) are selected from the following compounds:

[0031]

[0032]

[0033]

[0034] The method for preparing the alanine derivative and its enantiomers, diastereomers, tautomers, N-oxides, solvates, physiologically hydrolyzable esters, formulations, and pharmaceutically acceptable salts includes the following steps:

[0035] (1) Compound A of general formula A is prepared into compound B of general formula B by esterification reaction;

[0036] (2) Compound B of general formula is condensed with different carboxylic acid derivatives to obtain compound C of general formula;

[0037] (3) Compound C of general formula can be prepared into compound D of general formula via a deprotection reaction;

[0038] (4) Compound D of general formula can be prepared into compound E of general formula via a substitution reaction.

[0039]

[0040] Compound E is the compound of general formula (I), R 1 R 2 R 3 As stated above.

[0041] A pharmaceutical composition comprising a compound of general formula (I) as claimed in claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0042] The compound of general formula (I) or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof, in the preparation of P2Y 14 Uses of receptor antagonist drugs.

[0043] The use of the compound of general formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition thereof in the preparation of a medicament for treating inflammatory diseases.

[0044] The inflammatory diseases mentioned above include gouty arthritis and peritonitis.

[0045] The intended use is that the drug is formulated into different dosage forms by adding pharmaceutically acceptable excipients.

[0046] Those skilled in the art will understand that when a list of alternative substituents includes members that cannot be used to substitute a particular group member due to their valence requirements or other reasons, it is intended to refer to the list to the knowledge of those skilled in the art to include only those members suitable for substituting that particular group. The same applies to the number of possible substituents on a group.

[0047] The compounds used in this invention may be in the form of pharmaceutically acceptable salts or solvates. The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic base or acid (including inorganic bases or acids and organic bases or acids). If the compounds of this invention contain one or more acidic or basic groups, then this invention also includes their corresponding pharmaceutically or toxicologically acceptable salts, specifically their pharmaceutically usable salts. Therefore, according to this invention, compounds of this invention containing acidic groups may be used, for example, in the form of alkali metal salts, alkaline earth metal salts, or ammonium salts. More specific examples of these salts include sodium salts, potassium salts, calcium salts, magnesium salts, or salts formed with ammonia or organic amines (e.g., ethylamine, ethanolamine, triethylamine, or amino acids). According to this invention, compounds of this invention containing one or more basic groups (i.e., protonable groups) may be used as addition salts of their inorganic or organic acids. Examples of suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, neopentanoic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and acids known to those skilled in the art. If the compounds of the present invention contain both acidic and basic groups in their molecule, the present invention also includes internal salts or betaine (zwitterions) in addition to the salt forms mentioned. Various salts can be obtained by conventional methods known to those skilled in the art, for example, by reacting these substances with organic or inorganic acids or bases in a solvent or dispersant, or by anion or cation exchange with other salts. The present invention also includes salts of all compounds of the present invention that, due to low physiological compatibility, cannot be directly applied to pharmaceuticals but can be used, for example, as intermediates in chemical reactions or for the preparation of pharmaceutically acceptable salts.

[0048] The pharmaceutically acceptable excipients described in this invention refer to various conventional excipients required for the preparation of different dosage forms, such as diluents, binders, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, adsorbents, etc., which are prepared into any commonly used oral dosage form using conventional formulation methods, such as granules, powders, tablets, capsules, pills, oral liquids, decoctions, drop pills, etc.

[0049] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention discloses a class of P2Y... 14 Alanine derivatives and their pharmaceutically usable salts that have inhibitory effects on the P2Y receptor have been demonstrated through pharmacological experiments. 14 The receptor has a significant inhibitory effect, and can be used as a drug to treat inflammatory diseases. Attached Figure Description

[0050] Figure 1It is a THP-I cell UDPG / P2Y 14 Expression of downstream proteins in the receptor signaling pathway;

[0051] Figure 2 It is a THP-I cell UDPG / P2Y 14 The relative expression of NLRP3, a downstream protein of the receptor signaling pathway, was analyzed using one-way ANOVA. The data are mean ± standard deviation (n = 4). #### The comparison with the normal group showed P < 0.0001. * The representative was compared with the model control group (P < 0.05). # The representative was compared with the model control group, and the p-value was less than 0.01. *** The representative was compared with the model control group, and the p-value was less than 0.001. **** (Compared with the model control group, P < 0.0001);

[0052] Figure 3 It is a THP-I cell UDPG / P2Y 14 The relative expression of ASC, a downstream protein in the receptor signaling pathway, was analyzed. The data are the mean ± standard deviation (n=4), and analysis of variance was performed using one-way ANOVA. #### The comparison with the normal group showed P < 0.0001. * The representative was compared with the model control group (P < 0.05). # The representative was compared with the model control group, and the p-value was less than 0.01. *** The representative was compared with the model control group, and the p-value was less than 0.001. **** (Compared with the model control group, P < 0.0001);

[0053] Figure 4 It is a THP-I cell UDPG / P2Y 14 The relative expression of Caspase-1 p20, a downstream protein in the receptor signaling pathway, was analyzed using one-way ANOVA. The data are mean ± standard deviation (n = 4). #### The comparison with the normal group showed P < 0.0001. * The representative was compared with the model control group (P < 0.05). # The representative was compared with the model control group, and the p-value was less than 0.01. *** The representative was compared with the model control group, and the p-value was less than 0.001. **** (Compared with the model control group, P < 0.0001);

[0054] Figure 5 It is a THP-I cell UDPG / P2Y 14The relative expression of IL-1β, a downstream protein in the receptor signaling pathway, was analyzed using one-way ANOVA. The data are mean ± standard deviation (n = 4). #### The comparison with the normal group showed P < 0.0001. * The representative was compared with the model control group (P < 0.05). # The representative was compared with the model control group, and the p-value was less than 0.01. *** The representative was compared with the model control group, and the p-value was less than 0.001. **** (Compared with the model control group, P < 0.0001);

[0055] Figure 6 This represents the IL-1β level in the supernatant of THP-I cell culture medium. The data are the mean ± standard deviation, and analysis of variance was performed using one-way ANOVA. #### The comparison with the normal group showed P < 0.0001. * The representative was compared with the model control group (P < 0.05). # The representative was compared with the model control group, and the p-value was less than 0.01. *** The representative was compared with the model control group, and the p-value was less than 0.001. **** (Compared with the model control group, P < 0.0001);

[0056] Figure 7 These are the levels of various serum indicators in a mouse model of LSP-induced peritonitis. The data are mean ± standard deviation, and analysis of variance was performed using one-way ANOVA. #### The comparison with the normal group showed P < 0.0001. * The representative was compared with the model control group (P < 0.05). # The representative was compared with the model control group, and the p-value was less than 0.01. *** The representative was compared with the model control group, and the p-value was less than 0.001. **** (Compared with the model control group, P < 0.0001);

[0057] Figure 8 This represents the joint circumference in a mouse model of acute gouty arthritis induced by MSU. The data are mean ± standard deviation, analyzed using one-way ANOVA. #### The comparison with the normal group showed P < 0.0001. * The representative was compared with the model control group (P < 0.05). # The representative was compared with the model control group, and the p-value was less than 0.01. *** The representative was compared with the model control group, and the p-value was less than 0.001. **** The results showed that the mouse synovial tissue pathological changes were compared with the model control group (P < 0.0001). Detailed Implementation

[0058] The present invention will be specifically illustrated below through embodiments. In this invention, the embodiments described below are for better explanation and are not intended to limit the scope of the invention.

[0059] Example 1

[0060]

[0061] Step 1: Methyl (R)-2-amino-3-(4-fluorophenyl)propionate (1a)

[0062] (R)-2-amino-3-(4-fluorophenyl)propionic acid (5.0 g) was suspended in 100 mL of methanol, and trimethylchlorosilane (13 mL) was slowly added dropwise with stirring. During the addition, the suspension gradually became clear. After the addition was complete, the mixture was reacted at room temperature for 12 h. TLC monitoring showed that after the reaction was complete, the solution was concentrated under reduced pressure to a dry state, yielding 4.67 g of a white solid, with a yield of 87%. ¹H NMR (300 MHz, DMSO-d6) δ (ppm) 8.69 (s, 3H), 7.29 (dd, J = 8.6, 5.7 Hz, 2H), 7.16 (t, J = 8.9 Hz, 2H), 4.26 (t, J = 6.6 Hz, 1H), 3.67 (s, 3H), 3.24–3.05 (m, 2H).

[0063] Step 2: (R)-3-(4-fluorophenyl)-2-(3-(p-tolyl)propamido)methyl propionate (1b)

[0064] Methyl (R)-2-amino-3-(4-fluorophenyl)propionate (1.0 g) was dissolved in 10 mL of acetonitrile, followed by the addition of 3-(4-toluene)propionic acid (0.91 g), N-methylimidazolium (1.32 g), and N,N,N′,N′-tetramethylchloroformamidin hexafluorophosphate (1.55 g). The mixture was stirred at room temperature for 12 h. After complete reaction by TLC, water was added, and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous Na₂SO₄. Purification was achieved by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to give 1.32 g of a pale yellow solid, in 77.51% yield. 1H NMR (300MHz, Chloroform-d) δ (ppm) 8.36 (d, J=7.8Hz, 1H), 7.26-7.17 (m, 2H), 7.15-6.98 (m, 7H), 4.52-4.41 (m, 1H), 3.61 (s, 3H ), 3.01 (dd, J=13.8, 5.5Hz, 1H), 2.86 (dd, J=13.8, 9.4Hz, 1H), 2.69 (t, J=7.6Hz, 2H), 2.35 (dd, J=8.6, 6.8Hz, 2H), 2.27 (s, 3H).

[0065] Step 3: (R)-3-(4-fluorophenyl)-2-(3-(p-tolyl)propamido)propionic acid (1)

[0066] Methyl (R)-3-(4-fluorophenyl)-2-(3-(p-tolyl)propamido)propionate (500 mg) was dissolved in a methanol / tetrahydrofuran (1 / 1) (10 mL) mixed solvent, and 5 mL of 4 mol / L LiOH solution was added. After the addition was complete, the mixture was stirred at room temperature for 5 h. After the reaction was complete, 1 N HCl was added to adjust the pH of the solution to 2, and the mixture was filtered to give 450.6 mg of a white solid, with a yield of 90.8%. 1 HNMR (300MHz, DMSO-d6) δ (ppm) 8.24 (d, J = 8.1Hz, 1H), 7.25-7.14 (m, 2H), 7.12-6.95 (m, 6H), 4.46 -4.37 (m, 1H), 3.07-2.75 (m, 2H), 2.66 (t, J=7.7Hz, 2H), 2.33 (dd, J=8.7, 6.7Hz, 2H), 2.23 (s, 3H).

[0067]

[0068] The following compounds were prepared in a similar manner to that in Example 1:

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] Example 50

[0082]

[0083] Step 1: (R)-3-(4-fluorophenyl)-2-(3-(p-tolyl)propamido)ethyl propionate

[0084] (R)-3-(4-fluorophenyl)-2-(3-(p-tolyl)propamido)propionic acid (0.5 g) was dissolved in 5 mL of LMF. Chloroethane (0.18 g) and potassium carbonate (0.39 g) were slowly added with stirring. The mixture was heated to 50 °C and reacted for 12 h. The reaction was monitored by TLC. After completion, the mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous Na₂SO₄. The anhydrous Na₂SO₄ was then filtered off, and the filtrate was evaporated under reduced pressure to give 0.45 g of an off-white solid, with a yield of 83.45%.

[0085]

[0086] The following compounds were prepared in a similar manner to that used in Example 50:

[0087]

[0088]

[0089]

[0090] The pharmacological experiments and results of the compounds of general formula I in this invention are as follows:

[0091] Experimental methods:

[0092] Stable transition to P2Y 14 The HEK293 cell line containing the receptor was cultured in DMEM medium (containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin). Before the experiment, the cells were seeded onto culture plates and then transferred to serum-free medium at a seeding density of 1 × 10⁻⁶ cells / mL. 5Cells per well were cultured at 37°C, 95% O2, and 5% CO2 humidity. IBMX was added to inhibit PDEs activity to ensure a high cAMP level. Cell cAMP production was stimulated with the AC agonist Forskolin (30 μM), and different concentrations of the test compound (0.01, 0.1, 1, 10, 100 nm) were pre-added, with PPTN used as a positive control. Subsequently, 1 μM of P2Y was added. 14 The receptor agonist UDPG, cAMP Glo TM The Assay kit (PROMEGA Co., Ltd., USA) was used to detect intracellular cAMP levels. The inhibition rate was calculated based on the cAMP levels.

[0093] Table 1. Effects of all compounds on P2Y at the cellular level. 14 Receptor inhibition rate value:

[0094]

[0095]

[0096] Pharmacological study methods for the test compound to inhibit LPS-induced macrophage inflammatory response

[0097] (1) THP-1 cells were prepared at a rate of 1×10 6 The cells were seeded at a density of 100 cells / mL into 6-well plates. 2 mL of RPMI 1640 complete medium containing 100 μg / mL LPMA and 10% fetal bovine serum was added to each well. The plates were then incubated at 37°C in a constant temperature incubator with 5% CO2 and saturated humidity for 72 h. Under a microscope, the cells were observed to grow in a spindle shape and adhere to the plate. Most cells extended pseudopodia, indicating that the cells had differentiated into macrophages.

[0098] (2) After cell differentiation, the cells were divided into 6 groups: Control group, LPS group, LPS+PPTN (100 nM), LPS+test compound 14 (1000 nM), LPS+test compound 14 (100 nM), and LPS+test compound 14 (10 nM), with three replicates for each group. The culture medium was discarded, and 2 mL of RPMI 1640 incomplete culture medium was added to each well and the drug was administered. After 1 h, except for the Control group, LPS was added to each well at 100 ng / mL for stimulation. The cells were then incubated in a constant temperature incubator at 37 °C with 5% CO2 and saturated humidity for 24 h.

[0099] Western blot analysis of UDPG / P2Y in cells 14 The expression of downstream proteins in the receptor signaling pathway, the results are as follows: Figure 1-5As shown, LPS significantly increased the expression of downstream proteins of the P2Y14 receptor in THP-1 cells, indicating successful model establishment; different doses of the test compound could downregulate the expression of downstream proteins of the P2Y14 receptor to varying degrees, and all showed significant differences compared with the model control group; PPTN also showed the expected effect, indicating that the experimental results are true and reliable.

[0100] The level of IL-1β in the cell culture supernatant was detected using an ELISA kit (Shenzhen Xinbosheng). The results are as follows: Figure 6 As shown, LPS significantly increased the IL-1β level in the supernatant of THP-1 cell culture medium, indicating successful model establishment; different doses of the test compound could downregulate the IL-1β level in the cell culture medium supernatant to varying degrees, and all showed significant differences compared with the model control group; PPTN also showed the expected effect, indicating that the experimental results are true and reliable.

[0101] Pharmacological experimental methods for studying the therapeutic effects of test compounds on acute peritonitis at the whole animal level.

[0102] Eight-week-old male C57 mice were injected abdominally with LPS (10 mg / kg) in phosphate-buffered saline (PBS, pH 7.4) to stimulate inflammation, simulating acute peritonitis. One hour before LPS induction, the test compound was injected intraperitoneally (5 and 10 mg / kg), while control mice were injected with an equal volume of PBS (pH 7.4). Blood was collected 24 hours post-injection, centrifuged at 3000 × g for 15 min, and serum was collected and stored at -80℃ for later use. The following indicators were measured:

[0103] (1) The levels of ALT and ASR in serum were detected according to the method of the kit (Nanjing Jiancheng).

[0104] (2) The serum levels of TNF-α, IL-6 and IL-1β were detected according to the ELISA kit (Shenzhen Xinbosheng).

[0105] The results are as follows Figure 7 As shown, LPS significantly increased the levels of various indicators in mouse serum, indicating successful model establishment; different doses of the test compound could downregulate the levels of various indicators in serum to varying degrees, showing significant differences compared with the model control group; dexamethasone also showed the expected effect, indicating that the experimental results are true and reliable.

[0106] Pharmacological experimental methods for studying the therapeutic effects of test compounds on acute gouty arthritis at the whole animal level.

[0107] Male clean-grade SD rats, weighing 200±20g, were provided with free access to water and food, 12 hours of lighting per day, and an ambient temperature of 25±2℃. Animals were divided into several groups: a normal control group, a model control group, and drug-treated groups (dexamethasone and test compound 14). An acute gouty arthritis model was induced by a single intra-articular injection of MSU, while the normal control group and the drug-treated groups received an equal volume of physiological saline injected into the joint cavity. All drug-treated groups received test compound 14 (50μM) via intra-articular injection, with dexamethasone serving as a positive control. The circumference of the rat joints was measured using the suture method at 0h, 2h, 4h, 8h, 12h, and 24h. At 24h, the animals were euthanized by cervical dislocation, and synovial tissue was rapidly collected on an ice platform for pathological examination. Results are as follows: Figure 8 As shown, MSU significantly increased the joint circumference of mice, indicating successful modeling; the tested compound reduced the joint circumference of mice, showing a significant difference compared with the model control group; dexamethasone also showed the expected effect, indicating that the experimental results are true and reliable.

Claims

1. Alanine derivatives of general formula (I) or pharmaceutically acceptable salts thereof: Its characteristics are, The compound of general formula (I) is selected from the following compounds: 。 2. A method for preparing the alanine derivative of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: (1) Compound A of general formula is prepared into compound B of general formula by esterification reaction; (2) Compound B of general formula is condensed with different carboxylic acid derivatives to obtain compound C of general formula; (3) Compound C of general formula can be prepared into compound D of general formula via a deprotection reaction; (4) Compound D of general formula can be prepared into compound E of general formula by substitution reaction; ； Compound E is the compound of general formula (I), R 1 R 2 R 3 As described in claim 1.

3. A pharmaceutical composition, characterized in that, It contains a compound of general formula (I) as described in claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

4. The compound of general formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 3, in the preparation of P2Y 14 Uses of receptor antagonist drugs.

5. Use of the compound of general formula (I) of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 3 in the preparation of a medicament for treating inflammatory diseases selected from gouty arthritis and peritonitis.

6. The use according to claim 4 or 5, characterized in that, The drug is formulated into different dosage forms by adding pharmaceutically acceptable excipients.

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