1-methyl-2-carboxy-3-acetanilide indole compounds and use thereof

Abstract

1-methyl-2-carboxyl-3-acetanilide indole compounds and applications thereof. The compounds can block the interaction between NLRP3 and NEK7, effectively inhibit the activation and assembly of NLRP3 inflammasome, and have good prevention and treatment effect on NLRP3 inflammasome related inflammatory diseases such as Alzheimer's disease and Parkinson's disease. Thus, the compounds can be used for preparing drugs for treating and / or preventing NLRP3 inflammasome related inflammatory diseases, and provide a new choice for the development of NLRP3 inflammasome small molecule inhibitors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a class of 1-methyl-2-carboxyl-3-acetanilide indole compounds and applications thereof. BACKGROUND

[0002] NLRP3 inflammasome is a kind of multi-protein complex, which is composed of pattern recognition receptor NLRP3, linker protein ASC and caspase-1 precursor. NLRP3 inflammasome can be activated by a variety of stimulants, including viruses, bacteria, uric acid, ATP and beta-amyloid protein, etc. After the activation and assembly of NLRP3 inflammasome, the self-cleavage of caspase-1 precursor occurs, forming caspase-1 with cleavage activity, which in turn promotes the cleavage of IL-1 beta precursor and IL-18 precursor and forms mature IL-1 beta and IL-18, promotes immune response and inflammatory reaction, and thus participates in the occurrence and development of various diseases, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, type II diabetes, depression, gout, rheumatoid arthritis and atherosclerosis, etc. Therefore, targeting NLRP3 inflammasome has become a front hot spot for the development of therapeutic drugs for related inflammatory diseases. Although some inhibitors targeting NLRP3 inflammasome have been reported for the improvement of related diseases, there is no drug in clinic that can inhibit the activation of NLRP3 inflammasome. Therefore, it is of great significance to continue to find new and effective NLRP3 inflammasome inhibitors for the treatment of NLRP3 inflammasome related inflammatory diseases. SUMMARY

[0003] The purpose of the present application is to provide a class of 1-methyl-2-carboxyl-3-acetanilide indole compounds or pharmaceutically acceptable salts thereof and applications thereof, which can inhibit the activation of NLRP3 inflammasome and can be used for the preparation of treating and / or preventing NLRP3 inflammasome related inflammatory diseases.

[0004] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0005] A class of 1-methyl-2-carboxyl-3-acetanilide indole compounds or pharmaceutically acceptable salts thereof, as shown in formula (I);

[0006]

[0007] In the formula, R1 represents H, F or methoxy; R2 represents H, Cl or methoxy; R3 represents H, F, Cl or methoxy; R4 represents H, Cl, methyl or methoxy; and R5 represents H or methoxy.

[0008] A kind of pharmaceutical composition includes the 1-methyl-2-carboxyl-3-acetanilide indole compound or its pharmaceutically acceptable salt described and optionally one or more pharmaceutically acceptable carriers and / or excipients.

[0009] The application of the 1-methyl-2-carboxyl-3-acetanilide indole compound or its pharmaceutically acceptable salt as shown in formula (I) in inhibiting NLRP3 inflammasome activation in vitro.

[0010] The 1-methyl-2-carboxyl-3-acetanilide indole compound or its pharmaceutically acceptable salt as shown in formula (I) has the structure of compound 26 and compound 27:

[0011]

[0012] The 1-methyl-2-carboxyl-3-acetanilide indole compound or its pharmaceutically acceptable salt as described in compound 26 and compound 27 is applied to prepare a drug for treating and / or preventing NLRP3 inflammasome related inflammatory diseases.

[0013] The in vitro NLRP3 activation is induced by lipopolysaccharide (LPS) and ATP combination, and the drug dose is 2-50 μM.

[0014] The administration mode of the drug is intraperitoneal injection.

[0015] The drug for treating and / or preventing NLRP3 inflammasome related inflammatory diseases is used for treating and / or preventing neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, vascular dementia, neuropathic pain or progressive spinal cord lateral sclerosis.

[0016] The drug for treating and / or preventing NLRP3 inflammasome related inflammatory diseases is used for treating and / or preventing Alzheimer's disease and Parkinson's disease.

[0017] The drug dose for Alzheimer's disease and Parkinson's disease is preferably 5-20 mg / kg.

[0018] The beneficial effects of the present application are:

[0019] The application provides a kind of 1-methyl-2-carboxyl-3-acetanilide indole compound, the compound can inhibit the activation of NLRP3 inflammasome in mouse monocyte macrophage (BMDM) in vitro.Further, the compound can be embedded in the binding position of NLRP3 and NEK7 complex, and has good binding with NLRP3 and NEK7, thereby blocking the interaction of NLRP3 and NEK7, and has good prevention and treatment effect on NLRP3 inflammasome related inflammatory diseases such as Alzheimer's disease and Parkinson's disease.Therefore, the application provides a kind of 1-methyl-2-carboxyl-3-acetanilide indole compound in the application of treating and / or preventing NLRP3 inflammasome related inflammatory diseases, which provides a new choice for the development of NLRP3 inflammasome small molecule inhibitors. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The virtual screening process of the compound for blocking NLRP3-NEK7 binding; wherein, Figure 1 A and B in the formula (I) are the interaction schematic diagram and local enlarged view of 50 compounds with NLRP3-NEK7 complex (PDB:6NPY) binding interface pocket; Figure 1 C and D in the formula (I) are respectively the three-dimensional and two-dimensional schematic diagram of the interaction of compound No.26 with NLRP3-NEK7 complex binding interface pocket; Figure 1 E and F in the formula (I) are respectively the three-dimensional and two-dimensional schematic diagram of the interaction of compound No.27 with NLRP3-NEK7 complex binding interface pocket;

[0021] Figure 2 The compound inhibits NLRP3 inflammasome activation and NEK7 protein expression in vitro; wherein, Figure 2 A in the formula (I) is the inhibition rate of 50 screened compounds for inhibiting IL-1β in vitro; Figure 2 B and C in the formula (I) are respectively the influence of compound No.26 and No.27 at different concentrations on Caspase-1, IL-1β protein in cell supernatant and NLRP3, ASC, pro-Caspase-1, pro-IL-1β and NEK7 protein expression in lysis solution.

[0022] Figure 3 Protein purification of NLRP3 and NEK7; wherein, Figure 3 A in the formula (I) is NLRP3 protein expressed and purified on Sf9 insect cells; Figure 3 B in the formula (I) is NEK7 protein expressed and purified on E. coli system.

[0023] Figure 4 Affinity detection of the compound and NLRP3; wherein, Figure 4A in FIG. 1 is a reaction curve diagram of SPR detecting the affinity of the compound to NLRP3; Figure 4 B in FIG. 1 is the affinity value of the compound to NLRP3.

[0024] Figure 5 is the affinity detection of the compound to NEK7; wherein, Figure 5 A in FIG. 2 is a reaction curve diagram of SPR detecting the affinity of the compound to NEK7; Figure 5 B in FIG. 2 is the affinity value of the compound to NEK7.

[0025] Figure 6 is the AD behavior result diagram of the compound on APP / PS-1 double transgenic Alzheimer's disease mice, wherein A, B are the open field test display diagram; C is the new object recognition test display diagram; D-F are the water maze test display diagram;

[0026] Figure 7 is the AD behavior result diagram of the compound on MPTP-induced Parkinson's model mice, wherein A, B are the open field test display diagram; C is the pole climbing test display diagram. DETAILED DESCRIPTION

[0027] The present application is further described below in conjunction with the accompanying drawings, but the present application is not limited to the following examples.

[0028] Example 1: Virtual screening process of compounds blocking NLRP3-NEK7 binding:

[0029] According to the protein crystal structure (PDB: 6NPY) of NLRP3-NEK7 complex reported in the literature (Sharif, H. et al. Nature. 2019, 570(7761): 338-343), a grid file was generated for virtual screening using the key amino acid residues (E800, D748, E743, Y916, E862, V705 in NLRP3; Q129, R121, R131, R136 in NEK7) that bind in the LRR region of the two proteins. The grid file was generated using the Receptor Grid Generation module of Schrodinger software 2018 version. The Chemdiv (Version 2020) database was selected as the compound library for virtual screening. The Chemdiv library contains 1,507,829 small molecule compounds, and the database has skeletal diversity. The LigPrep module of Maestro was used to prepare the compound library under the OPLS3 force field.

[0030] Protonation was performed at pH 7.0±2.0 using the Epik method, adding metal ion binding states, desalting, generating tautomers, and maintaining the original atomic chirality. To ensure the global conformation of small molecules during virtual screening, conformation generation was performed on small molecules, with a maximum of 32 conformations per small molecule. The prepared compound library was saved, and NLRP3-NEK7 blocker virtual screening was performed using the Virtual Screening Workflow module in Maestro. A three-step screening mode with step-wise strategy (i.e. HTVS [high-throughput virtual screening mode]→SP [standard precision mode]→XP [ultra-precision mode]) was used, with the accuracy gradually increasing. Small molecule flexible docking was used in each step, and energy optimization was performed after docking. The top 10% of small molecules before docking scoring were retained for the next round of screening.

[0031] Then, the top 1500 molecules with higher scoring ranking were retained for solubility, binding mode analysis, and structural diversity analysis, and finally 50 small molecule compounds were screened for subsequent cell activity screening, as shown in Figure 1 A and B.

[0032] Example 2: Inhibition of NLRP3 inflammasome activation and NEK7 protein expression by compounds in vitro:

[0033] Mouse monocyte macrophage J774A.1 was cultured in DMEM medium containing 10% fetal bovine serum. Cells were seeded in a 96-well plate at a density of 5×105 cells / well and cultured at 37°C with 5% CO2 for 24 h. According to the previous screening conditions, the cells were treated with 500 nM LPS for 4.5 h, then 50 compounds were added (concentration of 10 μM), and 5 mM ATP was added 1 h later for 1 h, and then the cells and supernatant were collected. The inhibition rate of IL-1β after drug treatment was detected by ELISA kit, and the results showed that Figure 2 A), compounds 21, 23, 26, 27, 28, 30, 31, 39, 40, 46, 47, 49 and 50 had significant inhibitory activity on IL-1β, and compounds 26 and 27 had the best inhibitory effect; molecular docking results also showed that Figure 1 C-F), compounds 26 and 27 could bind to the NLRP3 and NEK7 binding interface pocket with good affinity; in addition, as represented by these two compounds, the effect of the compounds on the expression of Caspase-1, IL-1β protein in the supernatant and NLRP3, ASC, pro-Caspase-1, pro-IL-1β and NEK7 protein in the lysis solution at concentrations of 2, 10 and 50 μM was further detected, and the results showed that Figure 2Compounds 26 and 27 can effectively inhibit NLRP3 inflammasome activation and NEK7 protein expression, thereby blocking the interaction between NLRP3 inflammasome and NEK7. The structures of the compounds that significantly inhibit IL-1β activity in vitro are as follows:

[0034]

[0035] Among the compounds 21, 23, 26, 27, 28, 30, 31, 39, 40, 46, 47, 49 and 50, R1 represents H, F or methoxy; R2 represents H, Cl or methoxy; R3 represents H, F, Cl or methoxy; R4 represents H, Cl, methyl or methoxy; and R5 represents H or methoxy, as shown in the following table:

[0036]

[0037]

[0038] Among the compounds 26 and 27, the structures are as follows:

[0039]

[0040] Example 3: Protein purification of NLRP3 and NEK7

[0041] 1. Protein purification of NLRP3

[0042] NLRP3 protein expression and purification were performed on the Sf9 insect cell platform. The recombinant protein sequence: full-length protein plus Tag has 1284 amino acids, the theoretical molecular weight is 144.71 kDa, and the A280 concentration conversion coefficient is 0.92; after removing the MBP-His-Flag tag, it has 901 amino acids, the theoretical molecular weight is 102.3 kDa, and the A280 concentration conversion coefficient is 1.12. The virus prepared by the DPR1077 plasmid without fusing the MBP tag was used to infect Sf9 insect cells in parallel to optimize the purification, Figure 3 Figure A in the above shows that the NLRP3 protein has good expression and high protein purity.

[0043] 2. Protein purification of NEK7

[0044] The NEK7 protein was expressed and purified in an E. coli expression system. The plasmid was constructed by full gene synthesis and subcloned into the pSmart-I (His-SUMO tag) expression vector, which was further transformed into Rosetta (DE3) E. coli competent cells. The recombinant NEK7 protein with a purity of > 90% was obtained by the processes of activation, induction, collection of bacterial cells, expression detection, and protein purification, and was verified by SDS-PAGE and Western Blot (B) in Example 1. Figure 3

[0045] Example 4: Detection of the affinity of the compounds to NLRP3 and NEK7

[0046] Surface plasmon resonance (SPR) was used to detect the affinity of the 13 compounds with significant in vitro inhibition of IL-1β activity to NLRP3 and NEK7. The protein concentration used was 20 μg / mL, and 10 mM sodium acetate with different pH values was used to explore the appropriate coupling pH value of the protein and the chip. Finally, pH 4.0 was selected for immobilization, and the final protein immobilization amount was: NLRP3 387 RU, NEK7 445 RU. The elution buffer was PBSP + 2% DMSO; the injection time was 1 minute; the dissociation time was 2 minutes; and the flow rate was 30 μL / min. The 21, 23, 26, 27, 28, 30, 31, 39, 40, 46, 47, and 49 compounds were used as the mobile phase for sampling detection, and the data were analyzed and processed by biacore software. Figure 4 and Figure 5 It was shown that all the small molecules had certain binding to the two proteins, and the binding force of compounds 26 and 27 was the best as a whole, which was consistent with the in vitro activity results, while the 47th compound had poor solubility, resulting in non-standard graph.

[0047] Example 5: Improvement of the compounds on the related behavior disorders of APP / PS-1 double transgenic Alzheimer's disease mice

[0048] 1. Experimental animals and treatment methods

[0049] ​The experimental animals were APP / PS-1 double transgenic AD mice and wild-type normal control mice (WT). They were raised in an animal room with constant temperature (24±2℃), constant humidity (50-60%), natural lighting and good ventilation, and free access to water and food. All experimental procedures were in accordance with the regulations of the Animal Use and Management Committee of the Air Force Medical University. The mice were randomly divided into WT group, model group (APP / PS-1), compound 26 group (10, 50 mg / kg) and compound 27 group (10, 50 mg / kg), 6 in each group. Compound 26 and compound 27 were dissolved in 0.9% saline containing 1% DMSO, and the WT group and model group (APP / PS-1) were injected with the same volume of 0.9% saline containing 1% DMSO. They were given intragastrically once a day for 4 consecutive weeks.

[0050] 2. Behavioral detection method

[0051] (1) Open field test: The open field reaction box is a soundproof box equipped with lighting and air exchange devices, with a size of 30×30×30 cm 3 . The four sides and the bottom are square boxes made of transparent resin glass, and there are four digital cameras in the box that can cover the entire inside of the open field. Before the experiment, the relevant parameters are set, and during the experiment, the mouse is placed in the central area of the box, and the camera is turned on to record the mouse's activity. Each box is placed with one mouse at a time. Let the mouse freely move for 15 min, identify the track, and export the relevant indicators. The recorded indicators include the total activity distance of the mouse in the experimental box and the central area activity time. We define the 15×15 cm 2 area in the middle of the bottom of the experimental box as the central area. After each experiment, the inner wall of the box is cleaned with alcohol to prevent the smell of the animals from affecting subsequent experiments.

[0052] (2) Novel object recognition test: Two identical objects are placed in the open field with a distance of 25 cm between them. First, the mouse is placed in the open field for 10 min to adapt to the environment, and then it is placed back in the cage for 2 h. When the test starts, one of the objects in the open field is replaced with another new object of the same material but different shape and color. Then the mouse is allowed to freely explore the open field for 5 min. The ability of the mouse to recognize and remember is evaluated by comparing the time spent exploring the new object and the old object. The analysis index is discrimination index (DI), which is (exploration time of new object-exploration time of old object) / (exploration time of new object+exploration time of old object)×100%.

[0053] (3) Morris water maze test: The pool was divided into four areas: east, south, west, and north. Lytron 621, an opaque agent, was poured into each area, raising the water level 1-2 cm above the hidden platforms. During training, mice were randomly placed into the pool from one of the areas, facing the pool wall, and allowed to swim until they found the hidden platforms. Each training session lasted 90 seconds. After finding a platform, the animal was allowed to stay on it for 30 seconds. If the animal failed to find the platform within the allotted time, it was assisted in finding it. The interval between training sessions was 30 seconds, and training was conducted twice daily for a total of 7 days. The animals' behavior was recorded video, and software analysis was used to determine the time spent finding each platform, the number of times the animal traversed the platform, and the duration of each stay.

[0054] 3. Experimental Results

[0055] Open field experiments show that ( Figure 6 Compounds A and B), 26, and 27 significantly increased the total distance traveled and central activity time in AD model mice at doses of 10 mg / kg and 50 mg / kg, indicating improved motor and exploratory abilities; the new object recognition experiment showed ( Figure 6 Compounds 26 and 27, at doses of 10 mg / kg and 50 mg / kg, significantly increased the ability of AD model mice to recognize new objects, indicating that the mice's exploratory desire and learning recognition ability were enhanced; the Morris water maze test showed that ( Figure 6 In the study of AD model mice (DF), with increasing training sessions, mice administered different doses of compounds 26 and 27 via gavage were able to quickly locate platforms hidden on the water surface, and the number of times they crossed the platforms and the duration of their stay on the platforms also significantly increased, indicating improved spatial memory. These behavioral results suggest that long-term administration of compounds 26 and 27 can significantly reduce activity reduction in AD mice and improve their learning and cognitive functions.

[0056] Example 6: The effect of the compound on improving MPTP-induced behavioral disorders in MPTP-induced Parkinson's disease model mice

[0057] 1. Laboratory animals and treatment methods

[0058] The experimental animals were SPF male C57BL / 6 mice, 6-8 weeks old, purchased from the Experimental Animal Center of Air Force Military Medical University. One week before the experiment, the mice were acclimated to the environment and housed in an animal room with constant temperature (24±2℃), constant humidity (50-60%), natural lighting and good ventilation, with free access to water and food. All experimental procedures were in accordance with the regulations of the Animal Use and Management Committee of Air Force Military Medical University. The mice were intraperitoneally injected with 20mg / kg of tetrahydro-pyridine (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP) for 7 consecutive days to establish a mouse model of Parkinson's disease. Subsequently, the mice were randomly divided into control group, MPTP group, compound 26 group (10, 50mg / kg) and compound 27 group (10, 50mg / kg), 6 mice in each group. Compound 26 and compound 27 were dissolved in 0.9% saline containing 1% DMSO. The control group and model group were injected with the same volume of 0.9% saline containing 1% DMSO, and gavaged once a day for 4 consecutive weeks.

[0059] 2. Behavioral testing methods

[0060] (1) Open field test: The open field reaction box is a soundproof box equipped with lighting and air exchange devices, with dimensions of 30×30×30cm 3 . The four sides and the bottom are square boxes made of transparent resin glass. There is a digital camera above the inside of the box that can cover the entire inside of the open field. Before the experiment, the relevant parameters are set. During the experiment, the mouse is placed in the central area of the box, and the camera is turned on to record the mouse's activity. Each box is placed with one mouse at a time. The mouse is allowed to move freely for 15 minutes, and the trajectory is recognized to derive relevant indicators. The recorded indicators include the total activity distance of the mouse in the experimental box and the central area activity time. We define the 15×15cm 2 area in the middle of the bottom of the experimental box as the central area. After each experiment, the inner wall of the box is cleaned with alcohol to prevent the residual odor of the animals from affecting subsequent experiments.

[0061] (2) Pole test: This test is a commonly used method to evaluate the motor coordination of PD mice. A metal rod with a diameter of 1cm and a length of 50cm is wrapped with bandage gauze to prevent the mouse from falling. The metal rod is placed in the cage at the bottom. The mouse is placed head down at the top of the rod. The time for the mouse to return to the cage freely is recorded until the mouse's hind legs reach the bottom of the cage. The mice are trained for 2 days before the formal test. Each mouse is tested three times, with an interval of 15 minutes between each test. The average of the three tests is taken as the final value for analysis.

[0062] 3. Experimental results

[0063] The open field test showed thatFigure 7 Compound No. 26 and Compound No. 27 can significantly increase the total distance and central activity time of the MPTP-induced PD model mice at the doses of 10 mg / kg and 50 mg / kg, indicating that the movement and exploration ability of the mice are improved; the pole test shows that Compound No. 26 and Compound No. 27 can significantly reduce the time of the PD model mice returning to the cage at the doses of 10 mg / kg and 50 mg / kg, indicating that the movement dysfunction of the mice is improved. Figure 7 Compound No. 26 and Compound No. 27 can significantly reduce the time of the PD model mice returning to the cage at the doses of 10 mg / kg and 50 mg / kg, indicating that the movement dysfunction of the mice is improved.

Claims

1. Use of a compound of formula (I) in the preparation of a medicament for treating and / or preventing NLRP3 inflammasome-related inflammatory diseases, characterized in that, The compound of formula (I) is: ； or a pharmaceutically acceptable salt thereof.

2. Use according to claim 1, characterized in that: The disease is Alzheimer's disease and Parkinson's disease.

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