Compound with NF-kappa B signal inhibition activity, preparation method and application

Abstract

The invention discloses a small molecule compound with NF-kappa B signal inhibition activity, the structure of the small molecule compound is shown in the following formula, and cell experiments prove that the small molecule compound has a remarkable inhibition effect on TNF-alpha induced NF-kappa B signal activation and has no obvious cytotoxicity. The compounds and the pharmaceutical composition can be used for preventing or treating inflammatory diseases, such as anti-inflammatory treatment of sepsis, acute lung injury and chronic obstructive pulmonary disease. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of drug synthesis, and in particular relates to a compound with NF-κB signal inhibitory activity, a preparation method and an application thereof. Background Art

[0002] NF-κB signaling was first identified by Ranjan Sen and David Baltimore through electrophoretic migration analysis of terminally labeled DNA fragments, which identified the binding of nuclear factor in B lymphocytes to the kappa enhancer of the gene encoding immunoglobulin kappa light chain and named it nuclear factor binding factor near kappa light chain. NF-κB signaling is a strictly controlled signal transduction pathway, which is mainly manifested by the activation of the NF-κB transcription factor protein family, including Rel (c-Rel), RELB, RelA / p65, NFκB1p50 / p105 and NFκB2p52 / p100. These proteins dimerize to form active transcription factors, further regulating gene expression and affecting various biological processes, including immune system regulation and inflammatory response. As an early transcription factor, the activation of NF-κB does not require newly translated proteins for regulation. Therefore, it can respond to harmful cell stimulation at the first time. The NF-κB pathway can be activated by cell stress, inflammatory cytokines, growth factors, ultraviolet rays, etc. After being transported from the cytoplasm to the nucleus, activated NF-κB binds to a specific DNA sequence to form a DNA / NF-κB complex, which initiates transcription of downstream DNA sequences and synthesizes proteins, thereby exerting different biological functions and playing an important role in the occurrence and development of various diseases. NF-κB signal activation is closely related to the progression of inflammatory diseases. It has been found that NF-κB signal activation is related to the progression of multiple diseases such as sepsis, acute lung injury and chronic obstructive pulmonary disease (Lancet, 2018 392, 75–87; Mil. Med. Res, 2022, 9, 7; J. Exp. Med, 1995, 182, 1951-1958; Autoimmun. Rev, 2021, 20, 102741). Multiple inflammatory factors (TNF-α, etc.), endotoxin (LPS) or cigarette extracts can activate NF-κB signals, promote the release of inflammatory factors, and induce inflammatory cells to gather in the lungs. At present, many clinical drugs have been found to exert their efficacy by inhibiting NF-κB signaling. For example, glucocorticoids, which are commonly used in clinical practice, inhibit NF-κB signaling activation by inhibiting the binding of NF-κB to DNA, but long-term use of glucocorticoids can lead to various adverse reactions. Given the key role of the NF-κB signaling pathway in the progression of inflammatory diseases, new therapeutic drugs targeting it have also been a research hotspot. Summary of the invention

[0003] The purpose of the embodiments of the present application is to provide a small molecule compound with NF-κB signaling inhibitory activity, targeting the potential effect of NF-κB signaling inhibition in treating various diseases.

[0004] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0005] A compound having the structural formula:

[0006]

[0007] Among them, R 1 -R 6 The substituents may be the same or different.

[0008] Furthermore, R 1 and R 5 Each independently selected from a 4- to 10-membered aromatic ring and a 4- to 10-membered aromatic heterocycle, wherein the aromatic ring and the aromatic heterocycle may be further substituted by one or more Ra;

[0009] Ra is selected from H, halogen, cyano, C1-C3 alkyl;

[0010] R 2 , R 3 , R 4 , R 6 Each is independently selected from H, halogen, cyano, and C1-C3 alkyl.

[0011] Among them, R 1 and R 5 The general structural formula of the substituent is:

[0012]

[0013] Furthermore, X is one of H, Cl, Br and CN.

[0014] Furthermore, R 2 Substituents are H or -CH 3 One of them.

[0015] Furthermore, R 3 Substituents are H or -CH 3 One of them.

[0016] Furthermore, R 4 , R 6 Substituents are H or -CH 3 One of them.

[0017] Furthermore, the R 2 , R 3 Same, the R 4, R 6 Same; said R 1 and R 5 same.

[0018] More specifically, the compound is selected from the following structures:

[0019]

[0020] An active small molecule capable of effectively inhibiting NF-κB signaling includes the above-mentioned compound and its pharmaceutically acceptable salt or cocrystal, deuterated substance, solvate, and enantiomer.

[0021] A pharmaceutical composition comprising, as an active ingredient, a compound according to any one of the above technical solutions or a pharmaceutically acceptable salt or cocrystal, deuterated substance, solvate, or enantiomer thereof.

[0022] A use of the compound described in any of the above technical solutions or its pharmaceutically acceptable salt or cocrystal, deuterated product, solvate, enantiomer in the preparation of drugs for improving or treating various diseases caused by excessive activation of NF-κB signal.

[0023] Preferably, the activation of NF-κB signal is TNF-α-induced NF-κB signal activation.

[0024] A method for preparing the compound described in any of the above technical solutions, comprising subjecting the compounds represented by formula (I) and formula (II) to a cyclization reaction under the action of strontium carbonate to obtain;

[0025]

[0026] R 1 , R 5 , R 2 , R 3 , R 4 , R 6 The definitions are the same as those in formula (III) or (IV).

[0027] Preferably, the compounds represented by formula (I) and formula (II) are selected from the same compound.

[0028] A method for preparing the compound described in any of the above technical solutions comprises: adding the compounds represented by formula (I) and formula (II) and cesium carbonate into a reaction container, dissolving them with acetonitrile and reacting the reaction solution at room temperature for 10 to 30 hours. After the reaction is complete, water is added to the reaction solution, the aqueous phase is extracted with ethyl acetate, the organic phases are combined and the solvent is evaporated under reduced pressure, and finally the product is separated by silica gel column chromatography.

[0029] The molar ratio of the compound represented by formula (I) and formula (II) to cesium carbonate is 1:1-5.

[0030] The beneficial effects of the present invention are:

[0031] The active small molecule compounds of the present invention can effectively inhibit NF-κB signals, and cell experiments have confirmed that they have a significant inhibitory effect on TNF-α-induced NF-κB signal activation, and have no physiological toxicity. The compounds are expected to provide an ideal way to improve the treatment of various diseases caused by excessive activation of NF-κB signals. For example, these compounds and pharmaceutical compositions can be used for the prevention or treatment of inflammatory diseases, such as anti-inflammatory treatment of sepsis, acute lung injury and chronic obstructive pulmonary disease.

[0032] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The effect of the preferred N2 compound on the expression of inflammatory factors in LPS-induced RAW264.7 cells (n=3, ##P<0.01 vs Ctrl group; *P<0.05, **P<0.01 vs LPS group). DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the examples and drawings. The following examples are only intended to illustrate the present invention, but are not intended to limit the present invention in any way.

[0035] Example 1

[0036] 1-(3-(4-chlorobenzoyl)-2-(4-chlorophenyl)indolizine-1-yl)pyridin-2(1H)-one N1

[0037] The preparation method of N1 compound is:

[0038]

[0039] Preparation of intermediate 2: In a 500 mL round-bottom flask, add 1 (1.54 g, 100 mmol, 1.0 eq), CuBr 2 (1.57 g, 110 mmol, 1.1 eq), dissolved in 200 mL of ethyl acetate, the reaction solution was heated to 90°C and reacted for 24 h. After TLC monitoring of the complete reaction of the raw materials, 300 mL of water was added to the reaction solution, the aqueous phase was extracted with ethyl acetate (3×100 mL), the organic phases were combined and the solvent was distilled off under reduced pressure, and finally silica gel column chromatography was performed with petroleum ether: ethyl acetate = 10:1~5:1 system to obtain intermediate 2 (1.86 g, 80% yield, yellow-brown solid).

[0040] Preparation of intermediate 3: Add intermediate 2 (2.32 g, 100 mmol, 1.0 eq) and 2-chloropyridine (3.4 g, 300 mmol, 3.0 eq) to a 500 mL round-bottom flask, dissolve with 200 mL acetonitrile, heat the reaction solution to 120 ° C and react for 12 hours. After TLC monitoring that the raw materials are basically reacted, the reaction solution is filtered, the filter cake is washed with a small amount of ethyl acetate and dried, and the filter cake is retained to obtain the crude intermediate 3. The crude intermediate 3 is slurried with 200 mL of ethyl acetate overnight, then filtered, the filter cake is washed with a small amount of ethyl acetate and dried, and the filter cake is retained to obtain intermediate 3 (2.80 g, 90% yield, brown solid).

[0041] Preparation of product N1: Add 3 (3.11 g, 100 mmol, 1.0 eq) and cesium carbonate (6.5 g, 200 mmol, 2.0 eq) to a 500 mL round-bottom flask, dissolve in 200 mL acetonitrile and react at room temperature for 24 h. After TLC monitoring shows that the raw materials are basically reacted, add 300 mL of water to the reaction solution, extract the aqueous phase with ethyl acetate (3×100 mL), combine the organic phases and evaporate the solvent under reduced pressure, and finally separate by silica gel column chromatography using DCM: MeOH = 10:1~5:1 system to obtain product N1 (1.37 g, 30% yield, yellow solid).

[0042]

[0043] Melting point: 196.6-197.2℃. 1 H NMR (400 MHz, CDCl 3 )δ9.80(d,J=7.2Hz,1H),7.39–7.27(m,5H),7.06–6.96(m,5H),6.90–6.86(m,3H),6.70(d,J=9.6Hz,1H),6.02(td,J=6.8,1.2Hz,1H). 13 C NMR (125 MHz, CDCl 3 )δ185.36,163.62,140.48,139.66,137.74,137.41,133.94,133.56,133.26,131.64,130.70,130.0 5,128.24,128.20,127.78,125.95,121.59,119.00,116.14,115.36,115.07,106.40.HRMS(ESI):m / z calcd for[M+H] + :459.0662,found:459.0658.

[0044] Example 2

[0045] 1-(3-(4-bromobenzoyl)-2-(4-bromophenyl)indolizine-1-yl)pyridin-2(1H)-one N2

[0046]

[0047] The preparation method is the same as that of Example 1, except that 1-(4-chlorophenyl)ethanone is replaced with 1-(4-bromophenyl)ethanone, yield 28%, yellow solid, melting point: 194.5-195.3°C. 1 H NMR (400 MHz, CDCl 3 )δ9.82(d,J=7.6Hz,1H),7.39–7.29(m,3H),7.28–7.23(m,2H),7.15(dd,J=18.0,8.4Hz,4H),7.04(td,J=7.2 ,1.2Hz,1H),6.88(dd,J=6.8,2.0Hz,1H),6.82(d,J=8.4Hz,2H),6.70(d,J=9.6Hz,1H),6.03(t,J=6.8Hz,1H). 13 C NMR (125 MHz, CDCl 3 )δ185.45,163.60,140.48,139.62,138.17,133.60,133.38,131.90,131.16,130.78,130.76,130.52,1 28.29,126.02,125.88,122.18,121.57,118.92,116.12,115.34,115.13,106.41.HRMS(ESI):m / zcalcd for[M+H] + :546.9651,found:546.9658.

[0048] Example 3

[0049] 4-(3-(4-cyanobenzoyl)-1-(2-oxopyridin-1(2H)-yl)indolizine-2-yl)benzonitrile N3

[0050]

[0051] The preparation method is the same as that of Example 1, except that 1-(4-chlorophenyl)ethanone is replaced with 1-(4-cyanophenyl)ethanone, yield 28%, brown solid, melting point: 287.5-288.3°C. 1 H NMR (400 MHz, CDCl 3)δ9.87(d,J=7.2Hz,1H),7.48(d,J=8.4Hz,2H),7.40–7.35(m,3H),7.31(dd,J=20.0,8.4Hz,4H),7 .16–7.09(m,3H),6.87(dd,J=6.8,1.6Hz,1H),6.68(d,J=9.2Hz,1H),6.05(td,J=6.8,1.2Hz,1H). 13 C NMR (100 MHz, CDCl 3 )δ184.11,163.43,143.29,140.65,139.11,136.48,134.15,132.88,131.60,131.41,131.07,129.70,12 8.41,126.94,121.76,118.55,117.86,117.64,116.22,115.98,114.47,111.74,106.58.HRMS(ESI):m / z calcd for[M+H] + :441.1352,found:441.1359.

[0052] Example 4

[0053] 1-(3-(Furan-2-carbonyl)-2-(furan-2-yl)indolizine-1-yl)pyridin-2(1H)-one N4

[0054]

[0055] The preparation method is the same as that of Example 1, except that 1-(4-chlorophenyl)ethanone is replaced with 1-(2-furan)ethanone, yield 37%, brown solid, melting point: 209.3-210.2°C. 1 H NMR (500 MHz, CDCl 3 )δ9.39(d,J=7.0Hz,1H),7.50–7.46(m,1H),7.35–7.33(m,1H),7.29(d,J=9.0Hz,1H),7.19–7.1 4(m,3H),6.93–6.89(m,1H),6.85–6.80(m,2H),6.32(dd,J=3.8,1.8Hz,1H),6.26–6.20(m,3H). 13 C NMR (125 MHz, CDCl 3)δ173.44,163.29,152.72,145.93,145.34,143.42,140.64,140.11,132.91,127.25,124.89,122.0 1,121.55,118.03,117.64,116.13,114.47,113.75,111.87,111.46,110.65,106.55.HRMS(ESI):m / z calcd for[M+H] + :371.1032,found:371.1029.

[0056] Example 5

[0057] 1-(3-Benzoyl-6-methyl-2-phenylindolizine-1-yl)-5-methylpyridin-2(1H)-one N5

[0058]

[0059] The preparation method is the same as that of Example 1, except that 2-chloropyridine is replaced with 2-chloro-5-methylpyridine, yield 26%, brown solid, melting point: 207.1-207.6 ℃. 1 H NMR (500 MHz, CDCl 3 )δ9.65(s,1H),7.42(dd,J=8.0,1.3Hz,2H),7.26–7.21(m,2H),7.14–7.10(m,2H),6.9 9–6.90(m,7H),6.76(d,J=9.0Hz,1H),6.67(s,1H),2.41(d,J=1.0Hz,3H),1.85(s,3H). 13 C NMR (125 MHz, CDCl 3 )δ186.96,163.04,143.39,139.55,137.33,133.90,132.11,131.78,130.79,130.47,129.46,128.46,127. 71,127.34,127.10,126.02,124.55,120.61,118.99,115.72,115.47,115.05,18.72,16.80.HRMS(ESI):m / z calcd for[M+H] + :419.1760,found:419.1767.

[0060] Example 6 (Activity Evaluation) Evaluation of the Cytotoxicity of N1-N5 Series Compounds and Detection of Their Inhibitory Effects on TNF-a-Induced NF-κB Signal Activation

[0061] The specific experiments are as follows:

[0062] Assay 1: Cytotoxicity of N1-N5 compounds on HEK293T cells using CCK-8 assay

[0063] HEK293T cells were plated at 4 × 10 3 The cells / well were seeded into 96-well plates, and each plate was set up with 9 groups, including 1 control group (only the drug solvent dimethyl sulfoxide was added) and 8 drug-added groups (1, 5, 10, 50, 100, 500, 1000, and 5000 nM of N1-N5 series compounds were added, respectively. Each group was set up with 5 replicate wells; the culture medium was complete culture medium (DMEM+10% FBS+1% double antibody), and cultured for 24, 48, and 72 hours, respectively. At the corresponding time point, 10 μl of CCK-8 reagent was added to each well, and the cells were incubated in a cell culture incubator for 1 hour under dark conditions, taken out, fully shaken on a microplate reader, and the absorbance value at 450 nm was measured (the more active cells, the higher the absorbance), and the IC value of each compound on the cytotoxicity of HEK293T cells was measured. 50 As follows (Table 1).

[0064] Assay 2: Detection of the inhibitory effect of N1-N5 series compounds on TNF-a-induced NF-κB signaling activation

[0065] In order to quickly and conveniently detect the NF-κB signaling inhibitory activity of the target compound, HEK293T cells (NF-κB-Luc-293T cells) that stably express NF-κB-Luciferase Reporter were selected as tools for activity detection. NF-κB-Luc-293T cells were cultured at 5×10 5 Cells / well were inoculated in 24-well plates, and after the cells adhered, they were starved for 8 hours using serum-free medium. Gradient concentrations of candidate compounds were added, and the final dose was selected according to assay 1. Six drug doses that did not produce cytotoxicity were selected, such as: 5, 10, 50, 100, 500, and 1000 nM. After 30 minutes of action, 20 ng / ml of TNF-α was added to stimulate for 10 minutes, and the cells were collected. The firefly luciferase activity of each group was determined according to the instructions of the commercially available luciferase kit (such as Promage, TM040 kit).

[0066]

[0067] To determine the IC 50 , a S-shaped curve was fitted to % inhibition vs. Log 10 The compound concentration curve was used to calculate the IC value of the compound for NF-κB signal inhibition. 50 .

[0068] Table 1. IC values ​​of compounds N1-N5 on HEK293T cells 50 IC of TNF-α-induced NF-κB signaling activation 50

[0069] Compound Assay 1: Cytotoxicity (nM) <![CDATA[Measurement 2: NF-κB signaling inhibitory IC 50 (nM)]]> N1 1224.74±12.76 25.18±4.17 N2 918.85±18.16 8.19±1.94 N3 894.78±20.49 18.18±7.98 N4 1021.34±21.83 23.13±6.18 N5 673.51±18.11 36.19±7.88

[0070] Example 7 (Evaluation of anti-inflammatory activity) The above N1-N5 compounds, preferably N2, were tested for their inhibitory effect on the expression of inflammatory factors in the LPS-induced (lipopolysaccharide) macrophage cell line RAW264.7.

[0071] Cell RNA extraction and reverse transcription operation:

[0072] 1. Perform RNA extraction experiments in a clean bench.

[0073] 2. After carefully removing the culture medium, wash the cells three times with PBS, add 1 ml of Trizol to each well, pipette the cells repeatedly, and transfer the lysate into a 1.5 ml RNase-free EP tube and let it stand for 10 min.

[0074] 3. Add 1 / 5 chloroform to each tube and shake vigorously in a vortex apparatus, then let it stand on ice for 5 minutes and centrifuge at 4°C and 12,000 g for 15 minutes.

[0075] 4. After centrifugation, the liquid can be seen to be divided into three layers. Transfer the top layer of liquid to a new RNase-free EP tube, add an equal volume of isopropanol, gently mix the liquid, let it stand at room temperature for 10 minutes, and then centrifuge it at 4°C and 12,000g for 10 minutes.

[0076] 5. Remove the supernatant and add 800 μL of ice-cold 75% ethanol (prepared with 0.1% DEPC water) to wash the precipitate three times, and centrifuge at 4°C and 8000 g for 8 min.

[0077] 6. Remove the supernatant, dry the precipitate at room temperature, add 20 μL RNase-Free water to dissolve it, and take 2 μL for concentration detection and reverse transcription.

[0078] 7. Prepare the reverse transcription reaction mixture on ice in a 200 μL RNase-free centrifuge tube according to the system in the table below. The reverse transcription system is as follows:

[0079]

[0080] The above mixture was mixed and centrifuged and reverse transcription was performed according to the following system:

[0081] 25℃ / 10min, 50℃ / 30min, 85℃ / 5min, 4℃ / Forever.

[0082] Real-time fluorescence quantitative PCR detection of inflammatory factor expression levels:

[0083] Using the reverse transcription product cDNA as a template, the designed quantitative primer sequence was used for real-time fluorescence quantitative PCR detection. The 20μL reaction system is as follows:

[0084]

[0085] Amplification conditions were: 95°C / 30 seconds, 58°C / 30 seconds, 72°C / 30 seconds, 40 cycles; 4°C / forever.

[0086] Table 2. Primers for quantitative PCR detection of inflammatory factors

[0087]

[0088] The experimental results showed that N2 compound 3.3nM and 10nM could significantly inhibit the expression of IL-1α, IL-1β, IL-6 and TNF-α in RAW264.7 cells induced by LPS in a dose-dependent manner ( Figure 1 ), indicating that the compound has a significant improvement effect on the expression of inflammatory factors in bacterial infection.

Claims

1. A compound having NF-κB signaling inhibitory activity, characterized in that: The general structural formula of the compound is: Enantiomers; Among them, R 1 and R 5 Each independently selected from a 4- to 10-membered aromatic ring and a 4- to 10-membered aromatic heterocycle, wherein the aromatic ring and the aromatic heterocycle may be further substituted by one or more Ra; Ra is selected from H, halogen, cyano, C1-C3 alkyl; R 2 , R 3 , R 4 , R 6 Each is independently selected from H, halogen, cyano, and C1-C3 alkyl.

2. The compound according to claim 1, characterized in that The R 1 and R 5 are independently selected from the following groups: wherein X is one of H, Cl, Br and CN; The R 2 , R 3 , R 4 , R 6 Each is independently selected from one of H or -CH3.

3. The compound according to claim 1, characterized in that The R 1 and R 5 Each is independently selected from the following structures:

4. The compound according to claim 1, characterized in that The R 2 , R 3 Same, the R 4 , R 6 Same; said R 1 and R 5 same.

5. The compound according to claim 1, characterized in that The structural formula of the compound is:

6. A pharmaceutical composition, characterized in that A compound according to any one of claims 1 to 5 comprising an active ingredient.

7. Use of the compound according to any one of claims 1 to 5 or the composition according to claim 6 in the preparation of drugs for improving or treating various diseases caused by excessive activation of NF-κB signaling.

8. The use according to claim 7, characterized in that: The activation of NF-κB signal is TNF-α-induced NF-κB signal activation.

9. A method for preparing the compound according to any one of claims 1 to 5, characterized in that: The compound represented by formula (I) and formula (II) is subjected to a ring-forming reaction under the action of strontium carbonate to obtain; R 1 , R 5 , R 2 , R 3 , R 4 , R 6 Same definition as claim 1.

10. The preparation method according to claim 9, characterized in that: The compounds represented by formula (I) and formula (II) are selected from the same compounds.

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