CN107033100A - A kind of benzothiazole derivant, preparation method and its medical usage - Google Patents

Abstract

The present invention relates to the field of medicinal chemistry, in particular to a benzothiazole derivative (formula I) with anti-inflammatory activity. Preliminary activity tests prove that the compound of the present invention has good Keap1-Nrf2 protein-protein interaction inhibitory activity, which can interfere with Keap1-Nrf2 The combination of Nrf2, thereby activating Nrf2, has potential anti-inflammatory activity and can be used to treat many diseases related to inflammation, such as tumor, Parkinson's disease, Alzheimer's disease, chronic obstructive pulmonary disease, atherosclerosis, chronic kidney disease, diabetes or rheumatoid arthritis.

Description

A kind of benzothiazole derivative, preparation method and medical use thereof

technical field

The invention relates to the field of medicinal chemistry, in particular to a benzothiazole derivative, including the preparation method of these derivatives and the application in the field of anti-inflammation.

Background technique

With aging population, continuous expansion of urbanization and continuous changes in living environment, many environmental and age-related diseases are increasingly becoming a serious health problem worldwide. These diseases include cancer, inflammation, Alzheimer's disease, pulmonary disease, heart disease, diabetes and Parkinson's disease, etc. How to effectively prevent and treat these diseases is a topic of concern all over the world.

Oxidative stress directly or indirectly damages the physiological functions of intracellular proteins, lipids and other macromolecules, and is considered to be related to tumors, inflammation, Parkinson's disease and Alzheimer's disease. Nrf2 (nuclear factor E2-related factor 2) is a key factor in cellular oxidative stress response and is regulated by Keap1 (Keleh-like epichlorohydrin-related protein-1) protein (Free Radic.Biol.Med.2004,36,1208 ), regulate the expression of antioxidant proteins and phase II detoxification enzymes through the interaction of antioxidant response elements ARE (antioxidant response elements), such as NAD(P)H:quinine oxidoreductase1, NQO1), heme plus oxygen 1 (hemoxygenase-1, HO-1) and so on. A large number of studies have shown that under normal conditions, Nrf2 is combined with its inhibitory protein Keap1 and exists in cells. When oxidative stress occurs, the cysteine residue of Keap1 is modified, and the conformational change leads to the release of Nrf2 protein, which enters the nucleus and binds to ARE to promote the expression of target genes, thereby exerting a cytoprotective effect. Under pathological conditions, the body cannot dissociate Keap1-Nrf2. However, Nrf2 protein can be degraded by ubiquitination, and cannot enter the nucleus to induce the expression of antioxidant genes, leading to the occurrence of diseases (Med.Res.Rev.2012, 32, 687).

In the past 30 years, many laboratories have carried out a lot of work, screening small molecule agonists of Nrf2 from natural compounds or synthetic compound libraries, and simulating the biological process of modifying the cysteine residue of Keap1 by chemical small molecules to release Nrf2 makes it enter the nucleus and play a protective role in cells. Nrf2 activators have been used to treat inflammatory-related diseases. For example, dimethyl fumarate has been approved by the FDA for the treatment of multiple sclerosis, and CDDO-Me is currently in Phase II clinical trials for the treatment of pulmonary arterial hypertension. In addition, some natural products and quasi-natural products (curcumin, resveratrol, chalcone, etc.) with anti-inflammatory activity have also been confirmed to activate Nrf2 (patent publication number: CN 105566241A). But unfortunately, long-term use of covalent binding to modify Keap1 protein and continuous activation of Nrf2 will cause greater cytotoxicity and easily lead to carcinogenesis.

In recent years, designing small molecules that directly inhibit Keap1-Nrf2 protein interaction has become a new strategy to discover the regulation of Keap1-Nrf2-ARE pathway (Curr. Top. Med. Chem. 2007, 7, 972). Currently, the cell penetration of peptide Keap1-Nrf2 interaction inhibitors limits their application (Org. Biomol. Chem., 2013, 11, 3553; Bioorg. Med. Chem. Lett. 2013, 23, 3029); Small molecule Keap1-Nrf2 binding inhibitors have become a research hotspot in this field. At present, many pharmaceutical companies and universities are conducting research in this area, and a series of small molecule direct inhibitors of Keap1-Nrf2 interaction have been reported (Medchemcomm., 2014, 5, 93; ChemMedChem., 2014, 9, 699; J.Med.Chem., 2014, 57, 1121; J.Med.Chem., 2016, 59, 3991), showing strong potential in the research and development of anti-Parkinson's disease and anti-Alzheimer's drugs in the treatment of tumors, inflammation Application prospects.

Contents of the invention

The invention discloses a benzothiazole derivative represented by formula I, its preparation method and its medical application. Preliminary activity tests prove that the compounds of the present invention have good Keap1-Nrf2 protein-protein interaction inhibitory activity, can interfere with the combination of Keap1-Nrf2, thereby activate Nrf2, have potential anti-inflammatory activity, and can be used to treat many diseases related to inflammation, such as Cancer, Parkinson's disease, Alzheimer's disease, chronic obstructive pulmonary disease, atherosclerosis, chronic kidney disease, diabetes, or rheumatoid arthritis.

Compound of the present invention, (E)-allyl-(2-(2-(benzo[d]thiazole-2)-2-nitrile vinyl)-5-(diethylamino)phenyl)carbonate (Formula I), structural formula is as follows:

The present invention also discloses the preparation method of the compound of formula I, as follows:

1) In the synthesis step of the compound of formula 1, the preferred base used is triethylamine, diethylamine, sodium carbonate, sodium bicarbonate, potassium bicarbonate, potassium carbonate, cesium carbonate, dimethylaminopyridine, pyridine or 1, 8-diazabicycloundec-7-ene; the solvent is preferably one of methanol, ethanol, N,N-dimethylformamide, dimethyl sulfoxide, 1,4-dioxane and acetonitrile or several kinds; the reaction temperature is 0°C to 80°C, and the reaction time is 1 to 24 hours.

2) In the synthesis steps from the compound of formula 1 to the compound of formula I, the base is preferably triethylamine, diethylamine, dimethylaminopyridine, pyridine or 1,8-diazabicycloundec-7-ene; solvent Preferably one or more of dichloromethane, acetone, N,N-dimethylformamide, 1,4-dioxane, dimethyl sulfoxide and acetonitrile; the reaction temperature is -10°C to 80°C , the reaction time is 1 to 24 hours.

Based on the protein-protein interaction of Nrf2-Keap1, the present invention discovers a benzothiazole derivative with good pharmacological activity. In the fluorescence polarization-based Keap1-Nrf2 protein-protein interaction inhibition experiment, the compound showed better activity than the positive control S47 (J.Med.Chem.2014, 57, 1121). In cell experiments, the compound can make rat cardiomyocytes Nrf2 enter the nucleus, and up-regulate the levels of antioxidant factors HO-1 and NQO1, while inhibiting the levels of inflammatory factors TNF-α, IL-1β, and IL-6 induced by LPS up. Therefore, the compound is expected to be used as a lead compound to be further developed as an anti-inflammatory drug, and has potential application prospects in the preparation of drugs for the prevention and/or treatment of tumors, inflammation, Parkinson's disease and Alzheimer's disease.

Description of drawings

Fig. 1 surface plasmon resonance experiment determines the affinity diagram of the compound to Keap1 protein;

Figure 2 Gel electrophoresis;

Figure 3 The localization map of Nrf2 observed in each group of cells under a confocal microscope;

Figure 4 compound promotes the mRNA level up-regulation diagram of HO-1 and NQ01;

Fig. 5 is a diagram showing that compounds inhibit lipopolysaccharide (LPS)-induced up-regulation of inflammatory factors TNF-a, IL-1β, and IL-6 mRNA levels.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments.

Embodiment 1: the preparation of formula 1 compound

3.5 g of benzothiazole-2-acetonitrile and 3.9 g of 4-(diethylamino) salicylaldehyde were dissolved in 120 mL of methanol solution, protected by nitrogen, followed by 1 mL of triethylamine, and stirred at room temperature for 12 hours. Afterwards, the reaction solution was concentrated, and the residue was purified by column chromatography to obtain 5.5 g of compound formula 1 as a yellow solid, with a yield of 79%. ¹ H NMR (600MHz, CDCl ₃ ) δ8.01 (1H, d, J = 6Hz), 7.90 (1H, d, J = 6Hz), 7.47 (1H, dd, J = 6, 6Hz), 7.35 (1H, dd,J=6,6Hz),7.29(1H,d,J=6Hz),6.48(1H,d,J=6Hz),6.40(1H,s),3.42(4H,t,J=6Hz),1.22 (6H,t,J=6Hz). ¹³ C NMR(150MHz,CDCl ₃ )δ156.1,151.6,142.2,137.2,130.9,130.2,126.2,124.8,122.6,122.4,121.7,121.4,111.0,108.2,97.1,96.9 ,45.3,12.7.ESI-MS m/z:350.1[M+H] ⁺ .

Embodiment 2: the preparation of formula I compound

Dissolve 700mg of the compound of formula 1 in 20mL of dichloromethane, ice-bath to 0°C, add 424mg of triphosgene and 1mL of triethylamine. After the reaction was carried out for 1 hour, 166 mg of allyl alcohol was added and stirred for 12 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 220 mg of compound formula I as a yellow solid, with a yield of 25%. ¹ H NMR (400MHz, CDCl ₃ ) δ8.82 (1H, s), 8.02 (1H, d, J = 8Hz), 7.92 (1H, d, J = 8Hz), 7.51-7.35 (3H, m), 6.63 (1H,d,J=12Hz),6.14-6.07(1H,m),6.48(1H,s),5.48(1H,d,J=8Hz),5.31(1H,d,J=8Hz),4.86( 2H, s Hz), 3.45 (4H, q, J = 8.0Hz), 1.24 (6H, t, J = 7.0Hz). ¹³ C NMR (100MHz, CDCl ₃ ) δ161.5, 159.6, 155.8, 153.1, 152.4, 151.9 , ^{139.8,137.0,132.7,130.8,126.1,124.5,122.3,121.6,118.2,114.2,109.9,108.6,97.0,67.0,45.1,12.7} .

Example 3: Determination of Compound Binding Inhibition Activity to Keap1-Nrf2 Protein by Fluorescence Polarization Method

Fluorescence polarization method was used to measure the binding inhibitory activity of Keap1-Nrf2 protein. 10nM, added to the serially diluted Keap1 protein, incubated at room temperature in the dark for 30 minutes, and the fluorescence anisotropy value was read with a SpectraMax M5e microplate reader (ex: 485nm, em: 535nm). Protein binding constants were fitted using Mathematica 7 (Wolfram Research Inc.) from the fluorescence anisotropy values. The specific formula is as follows:

The experiment was carried out at a single concentration (100 μM) of the test compound, and the compound S47 (J.Med.Chem.2014, 57, 1121) was used as the control compound, and the inhibition rate was

Then the compound was dissolved in DMSO and diluted to the required concentration (1% DMSO) with buffer (10mM HEPES, pH 7.4, 3.4mM EDTA, 150mMNaCl, 0.005% Tween–20), and 20μL compound was added to 60μL containing 10nM fluorescent probe (FITC -βAla-DEETGEF-OH), 400nM Keap1 protein solution, incubated at room temperature for 1 hour in the dark. The fluorescence anisotropy value was read with Bioteck Synergy 2 microplate reader (ex: 485nm, em: 535nm). Protein binding constants were fitted using Mathematica 7 (Wolfram Research Inc.) from the fluorescence anisotropy values. The specific formula is as follows:

where a=K _D1 +K _D2 +L _ST +L _T -R _T ; b=(L _T -R _T )K _D1 +(L _ST -R _T )K _D2 +K _D1 K _D2 ; c=-K _D1 K _D2 R _T ;

In the formula, the Q value is the ratio of the fluorescence intensity of the fluorescent probe after binding with the highest protein concentration to the fluorescence intensity of the free state, F _SB is the binding part of the fluorescent probe, A _B and A _F are the binding and free state respectively The anisotropy value of the probe, K _D1 is the protein binding constant of the probe, L _ST is the probe concentration, _RT is the protein concentration, K _D2 is the protein binding inhibition constant of the compound.

Test results The inhibitory rate of the compound of formula I to the Keap1-Nrf2 protein protein interaction at a concentration of 100 μM was 81.25%, and its protein binding inhibition constant K _D2 value was 5.1 μM, which was slightly better than that of the positive control S47 (K _D2 value was 7.6 μM) .

Embodiment 4: Surface plasmon resonance experiment determines the affinity of the compound to the Keap1 protein

Calculate the ligand coupling level: 1.5×RL=10440RU; pre-enrichment (PH Scouting), select the appropriate coupling pH and ligand concentration: dilute the ligand in 10mM NaAC with different pH (the final concentration of the ligand is about 25-50μg/mL), flowing through the surface of the blank chip to test the effect of electrostatic adsorption; coupling ligand (Immobilization): first use the blank chip electrostatic adsorption to test the pre-enrichment ability, and then treat with EDC and NHS to activate the chip surface, Then the ligand was injected, coupled for 600s, and finally the surface of the chip was blocked with Ethanolamine.

A series of calibration solutions with DMSO concentrations ranging from 4.5% to 5.8% were prepared, and the calibration solutions were injected before all analytes, after all analytes, and after every 30 analytes to correct the concentration error of DMSO in the analytes.

The stock solution of the analyte was diluted step by step with the working buffer to a series of concentrations (0, 250, 500, 1000, 2000, 4000, 8000, 16000, 32000, 64000nM. The Kinetics/Affinity program was used to inject samples at a flow rate of 30μL/min, injection 120s, dissociation 120s.

The obtained data were analyzed using Biacore T200Evaluation Software. Calculate the solvent DMSO concentration calibration curve, select the appropriate concentration range and binding curve, subtract the reference channel and zero concentration, use the steady-state model for fitting analysis, and obtain the affinity value and parameters. The experimental results are shown in Figure 1, the affinity constant between the compound and Keap1 protein is 48.1.μM.

report

Parameters

Example 5: Compounds promote the transfer of Nrf2 to the nucleus

Rat cardiomyocyte H9C2 cells were planted in a small dish, and the cells were treated with 4 μM, 8 μM and 16 μM compounds for 6 h, and the treated cells in the control group and the experimental group were discarded, washed once with 1×PBS, and added 1ml pre-cooled 1×PBS, scrape the cells with a cell scraper, add the cells to a pre-cooled centrifuge tube with a pipette gun, centrifuge at 4°C, 1000rpm for 5min, suck up the supernatant, and leave the cell pellet for later use; use the kit to extract the nuclear protein separately and non-nuclear proteins; take 10 μl of extracted cytoplasmic protein samples and 5 μl of nuclear protein samples into eppendorf tubes, add ddH ₂ O to make up 10 μl, and use BCA to measure protein concentration; make 12% separating gel and 5% stacking gel; protein samples at 100°C Denaturation for 5 minutes, loading 12 μg of each sample; 60V gel injection, 120v constant pressure run gel; transfer tank 80V/160mA constant current transfer membrane, 2.5h; 5% skimmed milk prepared by TBST at room temperature for 1.5h; discard milk powder and use TBST After rinsing, dilute the primary antibody with 5% BSA solution prepared by TBST at a ratio of 1:1000, and incubate the PVDF membrane overnight at 4°C; wash the membrane with TBST, 3 times, 5 min each time; use 5% skimmed milk prepared by TBST Solution The secondary antibody was diluted at a ratio of 1:5000, and incubated at room temperature for 1 h; the membrane was washed with TBST, 3 times, 5 min each time; the membrane was developed with Milipore substrate using a chemiluminescent instrument, and the gel electrophoresis pattern was analyzed and quantified by Image J. The results showed that 8 μM and 16 μM compounds significantly increased the level of Nrf2 in the nucleus (shown in Figure 2).

On the other hand, H9C2 cells were treated with 8 μM compound for 6 hours, the culture medium was discarded, washed with PBS once, the cells were fixed and blocked, the primary antibody of Nrf2 was added overnight for 4 hours, the secondary antibody was incubated at 37 degrees for 1 hour, DAPI counterstained nuclei, The localization of Nrf2 in the cells of each group was observed under the confocal microscope. In the control group, Nrf2 was mainly located in the cytoplasm, and the 8 μ M compound treated the cells for 6 hours, and the Nrf2 in the nucleus increased significantly (as shown in Figure 3).

Example 6: Compounds up-regulate HO-1 and NQ01 mRNA levels

Nrf2 nuclear entry can activate downstream antioxidant elements, therefore, we further analyzed whether the compound can activate downstream antioxidant elements HO-1 and NQ01 by RT-PCR. Plant H9C2 cells in a small dish, treat H9C2 cells with 8 μM compound for 2 hours, 4 hours and 6 hours respectively, lyse the cells with TRIZOL, extract intracellular RNA, reverse to DNA, and use real-time quantitative PCR to analyze the levels of HO-1 and NQ01. The results are shown in Figure 4, the compound treated cells for 2h, 4h and 6h significantly up-regulated the mRNA levels of HO-1 and NQ01.

Example 7: The compound inhibits lipopolysaccharide (LPS)-induced up-regulation of inflammatory factors TNF-a, IL-1β, and IL-6 mRNA levels.

Plant H9C2 cells in a small dish, stimulate H9C2 cells with 1 μg/ml LPS to establish an inflammation model, co-treat H9C2 cells with 8 μM compound and lipopolysaccharide for 6 hours, lyse the cells with TRIZOL, extract intracellular RNA, reverse to DNA, and use real-time quantitative PCR instrument analysis. The results are shown in Figure 5, 8μM compound treatment of cells for 6h can significantly inhibit LPS-induced up-regulation of inflammatory factors TNF-a, IL-1β, IL-6mRNA levels.

Claims (6)

1. a kind of benzothiazole derivant, it is characterised in that it is (E)-pi-allyl-(2- (2- (benzo [d] thiazole -2) -2- nitriles Vinyl) -5- (diethylamino) phenyl) carbonic ester (Formulas I), its structural formula is：

2. the preparation method of derivative described in a kind of claim 1, it is characterised in that comprise the following steps：

(1) benzothiazole -2- acetonitriles and the compound of 4- (lignocaine) salicylides formula 1 are used；

(2) compound of formula 1 is reacted with triphosgene, allyl alcohol, obtains compound of formula I.

3. a kind of derivative of claim 1 is as Keap1-Nrf2 protein protein interaction inhibitor in medicine is prepared Purposes.

4. a kind of pharmaceutical composition, the derivative containing claim 1 or its pharmaceutically acceptable salt and pharmaceutically receive Carrier.

5. the derivative and its pharmaceutically acceptable salt described in a kind of claim 1 are used to prepare treatment and/or prevention and scorching Purposes in the medicine of the related disease of disease.

6. the purposes described in a kind of claim 5, it is characterised in that the disease related to inflammation is tumour, Parkinson's, old age Dementia, COPD, the hardening of artery congee, chronic kidney disease disease, diabetes or rheumatoid arthritis.