AhR agonist as well as preparation method and application thereof

Abstract

The invention provides an AhR (Aryl Hydrocarbon Receptor) agonist, the structural general formula of the AhR agonist is as shown in a formula (1): # imgabs0 #, and the invention also provides a preparation method and application of the chlorochalcone AhR agonist. The halogenated chalcone AhR agonist can effectively activate AhR in various cells and up-regulate the expression of AhR downstream protein CYP1A1, so that the anti-inflammatory pharmacological activity is exerted. The AhR agonist can also be used for relieving various inflammatory diseases, such as dextran sodium sulfate induced mouse ulcerative colitis and lipopolysaccharide induced mouse acute lung injury models. The AhR agonist also has the advantages of simple preparation process, high yield, oral effectiveness and the like, can be prepared into various forms such as enteric solvents, tablets, powder, suspensions, injections, capsules, dripping pills and the like, and has a good development prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a novel AhR (Aryl hydrocarbon receptor) agonist and a preparation method and application thereof. Background Art

[0002] Chronic inflammation can cause a variety of intractable diseases, such as inflammatory bowel disease, cancer, psoriasis, atopic dermatitis, hypertension, diabetes, chronic kidney disease, stroke, etc., and in severe cases, it can lead to death. Recent studies have shown that the aryl hydrocarbon receptor (AhR) plays an important role in the occurrence and development of inflammatory diseases. AhR is a typical ligand-activated transcription factor that is widely expressed in immune, epithelial, endothelial and stromal cells in mammalian barrier tissues. AhR plays a vital role in various physiological processes, such as xenobiotic metabolism, cell development and differentiation, and stem cell maintenance. In addition, AhR is associated with a variety of diseases, including inflammation, metabolic disorders, cancer, and autoimmune diseases. As a typical ligand-activated transcription factor, AhR can be activated by a variety of chemical substances with diverse structures, including endogenous substances (such as arachidonic acid metabolites), plant components (such as flavonoids), drugs, and environmental toxicants. AhR undergoes conformational changes after binding to the ligand, exposing its N-terminal nuclear localization sequence and promoting the translocation of the AhR chaperone complex to the nucleus. Once in the nucleus, AhR dimerizes with the aryl hydrocarbon receptor nuclear transporter (ARNT). The complex then binds to the AhR response enhancer element (AHRE) in the 5' regulatory region of the AhR driver gene, activating the transcription of various target genes, including genes for metabolic enzymes such as CYP1A1 and CYP1B1. Increasing evidence shows that the AhR signaling pathway plays a key role in maintaining the balance of the body's immune system, and activating the AhR-CYP1A signaling axis in a specific cellular environment has become a new therapeutic strategy for restoring barrier systems (such as intestinal, lung, and skin barriers) and alleviating inflammatory damage to organs.

[0003] In recent years, many biopharmaceutical companies at home and abroad have actively laid out the research and development pipeline of AhR agonists, but currently only one AhR agonist has been approved for marketing. As the first AhR agonist to be marketed, benzodiazepine (retinoic acid cream, approved for marketing by CFDA in 2019 and approved for marketing by the US FDA in 2022) was approved for the treatment of psoriasis, breaking the 30-year situation of no new topical drugs being marketed in this field. However, benzodiazepine can cause symptoms such as bleeding, redness and erosion of the skin. Compared with topical AhR agonist drugs, oral AhR agonist drugs have higher requirements on the drugability and safety of candidate drugs, resulting in no oral AhR agonist drugs being approved for marketing. Therefore, the industry is in urgent need of developing oral, effective and safe AhR agonists for the treatment of inflammatory diseases. Summary of the invention

[0004] The present invention provides a novel AhR agonist and a preparation method and application thereof, in order to solve the bottleneck problem that there is no oral AhR agonist anti-inflammatory drug in clinic at present.

[0005] The novel chalcone aromatic hydrocarbon receptor (AhR) agonist developed by the present invention has a general structural formula as shown in formula (1): Wherein R1 is any one of hydroxyl, methyl, trifluoromethyl and methoxy; R2 is any one of hydrogen, hydroxyl and methoxy. Specifically, the aryl hydrocarbon receptor (AhR) agonist includes molecules 1-6:

[0006]

[0007] The present invention also provides a method for preparing the above-mentioned chalcone aromatic hydrocarbon receptor (AhR) agonist, using acetophenone derivatives and 4-hydroxy-6-methoxy-3-chlorobenzaldehyde as raw materials, reacting under alkaline conditions for 3 hours and then obtaining the above-mentioned pure product by column chromatography.

[0008] The present invention also provides experimental results of the above compounds activating AhR transcriptional activity at the living cell level, as well as the anti-inflammatory effects of the above compounds at the cellular level and their ability to repair barriers. The present invention also examined the anti-inflammatory effects of the above compounds in animals as a whole, and found that such agonists can effectively reduce the levels of pro-inflammatory cytokines in intestinal tissue and bronchoalveolar lavage fluid, increase the expression level of intestinal tight junction proteins, and restore the integrity of the intestinal mucosal barrier; at the same time, reduce the infiltration of inflammatory cells in lung tissue and tissue structure damage, thereby alleviating a variety of inflammatory diseases.

[0009] Furthermore, the present invention also provides the use of the above-mentioned compound in the preparation of drugs for stimulating AhR and its gene product CYP1A1, as well as the preparation type and administration method thereof.

[0010] Compared with the prior art, the present invention has significant technical progress. The use of the AhR agonist of the present invention for the treatment of inflammatory diseases has the following advantages:

[0011] (1) Easy to synthesize: The AhR agonist provided by the present invention can be efficiently obtained by chemical synthesis, and the synthesis process is simple and easy.

[0012] (2) Significant agonistic effect: The AhR agonist provided by the present invention can significantly increase the transcriptional activity and expression level of AhR at both the cellular and animal levels.

[0013] (3) Good safety: No obvious toxicity was observed after continuous oral administration of the AhR agonist provided by the present invention for 14 days.

[0014] (4) Oral efficacy: The AhR agonist provided by the present invention can be taken orally and exhibits significant anti-inflammatory activity in a variety of inflammatory disease models. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The general structural formula of the novel chlorochalcone.

[0016] Figure 2 Synthetic route of novel chlorochalcone AhR agonists.

[0017] Figure 3 AhR agonist activity of chlorochalcone AhR agonists.

[0018] Figure 4 Anti-inflammatory effects of chlorochalcone AhR agonists in macrophages.

[0019] Figure 5 Chlorochalcone AhR agonists increase the levels of tight junction proteins.

[0020] Figure 6 Chlorochalcone AhR agonists alleviate DSS-induced ulcerative colitis.

[0021] Figure 7 Chlorochalcone AhR agonists alleviate lipopolysaccharide-induced acute lung injury in mice. DETAILED DESCRIPTION

[0022] The present invention will be further described in the following examples, but the present invention is not limited thereto.

[0023] Preparation Example 1 Synthesis route of novel chlorochalcone AhR agonists (such as Figure 2 (shown)

[0024] [Experimental Materials] Acetophenone derivatives, 4-hydroxy-6-methoxy-3-chlorobenzaldehyde, alkaline reagents, thin layer plates

[0025] [Experimental procedures] The organic solvent includes but is not limited to ethanol, dichloromethane, methanol, and ethyl acetate. The alkaline reagent includes but is not limited to sodium hydroxide.

[0026] (1) Dissolve acetophenone derivative (100 mg) and 1 equivalent of 4-hydroxy-6-methoxy-3-chlorobenzaldehyde in 5 mL of a mixed solvent (ethanol: water = 2:1), then slowly add 0.5 mL of a sodium hydroxide solution (1 M). The mixed solution is stirred at room temperature for 48 hours. The reaction is monitored by thin layer chromatography (TLC), and after the reaction is complete, the mixture is extracted with ethyl acetate. The organic phase is washed with brine, dried over anhydrous sodium sulfate, and the solvent is dried by spin drying. The obtained crude product is purified by column chromatography.

[0027] (2) The compound protected by methoxymethyl ether is dissolved in methanol and concentrated hydrochloric acid is added dropwise under stirring. The reaction solution is stirred at room temperature for 2 hours. The reaction is monitored by thin layer chromatography (TLC). After the reaction is complete, the mixture is diluted with water and extracted with ethyl acetate. The organic layer is washed with brine, dried over anhydrous sodium sulfate, the solvent is dried by spin drying, and purified by column chromatography.

[0028]

[0029] Yellow solid powder, (E)-4'-hydroxy-4-hydroxy-6-methoxy-3-chlorochalcone (1, yield 44.8%). 1 HNMR(400MHz,DMSO-d6)δ10.86(s,1H),10.36(s,1H),8.06(dd,J=6.8,2.0Hz,3H),7.88(d ,J=15.6Hz,1H),7.78(d,J=15.7Hz,1H),6.88(d,J=8.7Hz,2H),6.66(s,1H),3.85(s,3H). 13 C NMR (101MHz, DMSO-d6) δ187.47,162.40,158.65,156.73,136.57,131.51,129.88,129.47,119.86,116.21,115.72,112.68,100.59,56.32.

[0030]

[0031] Yellow solid powder, (E)-4'-methyl-4-hydroxy-6-methoxy-3-chlorochalcone (2, yield 37.6%). 1 HNMR(600MHz,DMSO-d6)δ10.90(s,1H),8.10-8.01(m,3H),7.92(d,J=15.7Hz,1H),7 .80(d,J=15.7Hz,1H),7.37(d,J=7.9Hz,2H),6.67(s,1H),3.85(s,3H),2.41(s,3H). 13C NMR (151MHz, DMSO-d6) δ188.85,158.83,143.71,137.50,135.85,129.74,129.62,129.03,119.76,116.05,112.80,100.61,56.36,21.64.

[0032]

[0033] Yellow solid powder, (E)-4'-trifluoromethyl-4-hydroxy-6-methoxy-3-chlorochalcone (3, yield 36.8%) 1 HNMR(DMSO-d6,500MHz): δ11.02(s,1H,4OH),8.32(d,2H,J=8.5Hz,H3'H5'),8.11(s,1H,H6),7.99(d,1H,J= 16.2Hz,Hα),7.93(d,2H,J=8.5Hz,H2'H6'),7.84(d,1H,J=16.2Hz,Hβ),6.68(s,1H,H3),3.87(s,3H,4MeO); 13 C NMR (DMSO-d6, 125MHz): δ188.7, 159.2, 157.5, 141.7, 139.0, 130.0, 129.6×2, 126.2(q)×2, 119.4, 115.8, 112.9, 100.6, 56.4.

[0034]

[0035] Yellow solid powder, (E)-4'-hydroxy-5'-hydroxy-4-hydroxy-6-methoxy-3-chlorochalcone (4, yield 40.3%). 1 H NMR (400MHz, DMSO-d6) δ10.84(s,1H),9.88(s,1H),9.33(s,1H),8.02(s,1H),7.84(d,J=15.6Hz,1H),7.72(d,J= 15.7Hz,1H),7.62(dd,J=8.3,2.1Hz,1H),7.50(d,J=2.1Hz,1H),6.84(d,J=8.3Hz,1H),6.66(s,1H),3.85(s,3H). 13C NMR(101MHz,DMSO-d6)δ187.56,158.63,156.64,151.09,145.86,136.36,130 .36,129.54,122.41,120.02,116.26,115.80,115.47,112.65,100.61,56.31.

[0036]

[0037] Yellow solid powder, (E)-4'-methoxy-5'-hydroxy-4-hydroxy-6-methoxy-3-chlorochalcone (5, yield 42.2%). 1 H NMR (400MHz, DMSO-d6) δ10.87(s,1H),9.37(s,1H),8.05(s,1H),7.87(d,J=15.6Hz,1H),7.81-7.69( m,2H),7.52(d,J=2.1Hz,1H),7.04(d,J=8.5Hz,1H),6.67(s,1H),5.77(s,1H),3.86(d,J=9.8Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ187.82,158.70,156.75,152.48,146.97,136.76,131.49 ,129.61,122.04,119.87,116.20,115.28,112.68,111.58,100.60,56.25,55.39.

[0038]

[0039] Yellow solid powder, (E)-4'-hydroxy-5'-methoxy-4-hydroxy-6-methoxy-3-chlorochalcone (6, yield 36.2%). 1 H NMR(600MHz,DMSO-d6)δ10.85(s,1H),9.99(s,1H),8.05(s,1H),7.89(d,J=15.6Hz,1H),7.81-7 .75(m,2H),7.60(d,J=2.0Hz,1H),6.90(d,J=8.3Hz,1H),6.66(s,1H),3.87(s,3H),3.85(s,3H). 13C NMR(151MHz,DMSO-d6)δ187.45,158.67,152.13,148.18,136.55,130.25,129 .44,124.02,119.79,116.19,115.38,112.72,112.08,100.62,56.33,56.19.

[0040] Conclusion: The synthesis process of this type of compound is simple and easy to operate with high yield.

[0041] Example 2 Evaluation of AhR agonist activity of the above compounds

[0042] (1) 293T-AhR-luc cells were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO 2 . After trypsinization, the cells were seeded in a 96-well plate at a density of 8,000 cells per well.

[0043] (2) After 24 hours, remove the culture medium. Dissolve the compound in dimethyl sulfoxide, add the above compound (5, 10, 20 and 30 μM) and positive agonist (benzenemod, 10 μM) to each well in a volume of 100 μL, and incubate for 24 hours before use. Reagent (BioTianmei Biotechnology Co., Ltd.) to detect luciferase activity (AhR transcription activity). AhR luciferase activity was measured by microplate reader ( Figure 3 ).

[0044]

[0045] Conclusion: The above compounds can stimulate AhR at concentrations of 5, 10, 20 and 30 μM.

[0046] Example 3 Anti-inflammatory effects of novel AhR agonists in macrophages

[0047] (1) Bone marrow was collected from the tibia and femur of mice, and bone marrow cells were flushed out. The cells were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 40 ng / mL macrophage colony stimulating factor for 6 days to differentiate them into bone marrow-derived macrophages.

[0048] (2) Bone marrow-derived macrophages were stimulated with lipopolysaccharide for 24 hours, and the above compounds were respectively tested for their anti-inflammatory effects. The levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β were measured by enzyme-linked immunosorbent assay ( Figure 4 ).

[0049] Conclusion: The above compounds can reduce the secretion of proinflammatory cytokines such as IL-6, TNF-α, and IL-1β, showing good anti-inflammatory activity.

[0050] Example 4 Effect of novel AhR agonists on upregulating intestinal tight junction proteins

[0051] (1) 200,000 normal human intestinal epithelial cells were seeded in a 6-well plate. After 24 hours, the culture medium was removed and 3% DSS, AhR agonist (5 μM, 3% DSS), AhR agonist (10 μM, 3% DSS), AhR agonist (20 μM, 3% DSS), and AhR agonist (30 μM, 3% DSS) were added. The cells were incubated for another 24 hours and the cell proteins were extracted.

[0052] (2) The cell protein concentration was determined using a BCA protein assay kit. The proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred to a PVDF membrane. The membrane was blocked with 5% bovine serum albumin and then incubated with primary antibodies such as anti-occludin and anti-GAPDH at 4°C overnight. Subsequently, the membrane was incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The protein bands were visualized using Enhanced Chemiluminescence ( Figure 5 ).

[0053] Conclusion: Compared with the control group, the level of tight junction protein Occludin in the DSS group was significantly downregulated. On the contrary, AhR agonist significantly upregulated the level of Occludin protein, indicating that the above-mentioned AhR agonist repaired the intestinal barrier in the cell DSS model.

[0054] Conclusion: The above compounds can upregulate tight junction proteins and repair the intestinal barrier by stimulating AhR and its downstream gene products.

[0055] Example 5 Novel AhR agonist alleviates DSS-induced ulcerative colitis

[0056] (1) Prepare male C57BL / 6J mice, and keep them under the following conditions: 12h / 12h light-dark cycle, 22±2℃, relative humidity 55%±5%

[0057] (2) The mice were randomly divided into 5 groups, with 6 mice in each group. During the experiment, 3% (w / v) DSS was added to the drinking water to induce colitis, and the drug (AhR agonist) was administered by gavage. The control group drank normal water for 8 days, followed by normal drinking water for 2 days. The mice were then killed and samples were collected for testing.

[0058] (3) Hematoxylin-eosin staining was used to evaluate the pathological condition of intestinal tissue. The expression levels of proinflammatory cytokines IL-1β, TNF-α, IL-6, and IFN-γ in intestinal tissue were quantified by enzyme-linked immunosorbent assay. Briefly, tissue supernatants were collected and analyzed according to the manufacturer's instructions. In addition, the expression levels of tight junction proteins occludin and ZO-1 in the mouse intestine were quantified by Western blotting and immunofluorescence staining ( Figure 6 ).

[0059] Conclusion: AhR agonist can significantly restore the normal structure of colon tissue in mice, alleviate the elevated levels of inflammatory factors induced by DSS, promote the expression of tight junction proteins in intestinal tissue, and restore the integrity of the intestinal mucosal barrier. It is a potential anti-inflammatory drug for the treatment of ulcerative colitis.

[0060] Example 6 Novel AhR agonist alleviates lipopolysaccharide-induced acute lung injury in mice

[0061] (1) Prepare male C57BL / 6J mice, and keep them under the following conditions: 12h / 12h light-dark cycle, 22±2℃, relative humidity 55%±5%

[0062] (2) The mice were randomly divided into 5 groups, with 6 mice in each group. During the experiment, the above AhR agonists were first administered by gavage for 3 days, and lipopolysaccharide was instilled into the trachea to establish an acute lung injury model in mice. The mice were killed and samples were collected for testing.

[0063] (3) Hematoxylin-eosin staining was used to observe inflammatory cell infiltration and lung tissue structure, bronchoalveolar lavage fluid test was used to evaluate the inflammatory response of lung disease, and enzyme-linked immunosorbent assay was used to measure the levels of proinflammatory cytokines TNF-α, IL-6, and IL-1β. Figure 7 ).

[0064] Conclusion: AhR agonists can alleviate LPS-induced acute lung injury in mice in vivo and effectively inhibit the secretion of proinflammatory factors in bronchoalveolar lavage fluid, indicating that the above AhR agonists can be used as anti-inflammatory drugs to treat acute lung injury.

Claims

1. An agonist of an aromatic hydrocarbon receptor AhR, characterized in that: Its general structural formula is shown in formula (1): Wherein R1 is any one of hydroxyl, methyl, trifluoromethyl and methoxy; R2 is any one of hydrogen, hydroxyl and methoxy.

2. The agonist of aryl hydrocarbon receptor AhR according to claim 1, characterized in that: Any of the following structural formulas, 3. The method for preparing the aryl hydrocarbon receptor AhR agonist according to claim 1, characterized in that: With acetophenone derivatives and 4-hydroxy-6-methoxy-3-chlorobenzaldehyde as raw materials, the reaction is carried out under alkaline conditions for 3 hours and then column chromatography is performed to obtain chalcone compounds.

4. Use of the aryl hydrocarbon receptor AhR agonist according to claim 1 or 2 in the preparation of AhR agonist drugs.

5. The use according to claim 4, characterized in that: The invention can be used to prepare drugs for treating various inflammatory diseases including drugs for treating ulcerative colitis, drugs for treating acute pneumonia, and drugs for treating skin inflammation.

6. The use according to claim 4, characterized in that: The dosage form is any one of enteric solvent, tablet, powder, suspension, injection, enema, emulsion, film, suppository, capsule, granule, pill or injection.

7. The use according to claim 4, characterized in that: The administration route includes oral administration, topical administration or injection.

8. An AhR agonist drug, characterized in that: Contains an aromatic hydrocarbon receptor AhR agonist as claimed in claim 1 or 2.

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