Benzofuran [2, 3-b] quinoline derivative as well as preparation method and anti-renal fibrosis application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a benzofuran [2, 3-b] quinoline derivative as well as a preparation method and application thereof in resisting renal fibrosis. The structure of the benzofuran [2, 3-b] quinoline derivative, the optical isomer thereof or the pharmaceutical salt thereof is shown as a formula (I): # imgabs0 #, wherein R1 is selected from C1-C8 alkoxy, halogen, a 3-8 membered aliphatic heterocyclic ring, a substituted 3-8 membered aliphatic heterocyclic ring and-NR3R4; r2 is selected from = O, hydroxyl and hydrogen; the substituent group of the substituted 3-8-membered aliphatic heterocyclic ring is selected from C1-C8 alkyl, C1-C8 alkyl ester group, = O and 3-8-membered aliphatic ring. A plurality of derivatives with novel structures are synthesized, the derivatives have the potential of becoming anti-renal fibrosis drugs, renal fibrosis is relieved by dual regulation and control of TGF-beta1 / Smad2 / Smad3 and PI3K / AKT signal pathways, and a new strategy and thought are provided for treatment of CKD.

Description

Technical Field

[0001] The present invention belongs to the fields of medicine and chemical industry, and particularly relates to a benzofuran[2,3-b]]quinoline derivative, a preparation method thereof, and an application thereof in resisting renal fibrosis. Background Art

[0002] Renal fibrosis (RF) is a hallmark pathological feature of end-stage chronic kidney disease (CKD). External stimulation of the kidneys triggers damage and phenotypic changes in certain renal cells. Its core mechanisms involve complex processes such as tubular epithelial cell transdifferentiation, excessive extracellular matrix (ECM) deposition, and inflammatory responses. Epidemiological data show that the prevalence of RF is as high as 8%-16% and is increasing annually.

[0003] Meflunisal, as a new anti-renal fibrosis drug, exerts its anti-fibrotic effects by reducing inflammation and oxidative stress levels. However, the research on meflunisal (ZHC116) in anti-renal fibrosis is still in the exploratory stage.

[0004] Prior art CN116284018A discloses that the furanoquinoline alkaloid fennel isolated from Rutaceae plants has significant anti-renal fibrosis activity. However, fennel suffers from problems such as rapid metabolism, poor water solubility, and potential hepatotoxicity, and its activity is still insufficient. Summary of the Invention

[0005] The present invention aims to provide a benzofuran[2,3-b]quinoline derivative, a preparation method thereof and an application thereof in preparing a medicine for treating chronic kidney disease.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] A benzofuran[2,3-b]quinoline derivative, an optical isomer thereof or a pharmaceutically acceptable salt thereof, the structure of which is shown in formula (I):

[0008]

[0009] wherein R1 is selected from the group consisting of: C1-C8 alkoxy, halogen, 3-8 membered aliphatic heterocycle, substituted 3-8 membered aliphatic heterocycle, -NR3R4;

[0010] R2 is selected from: =O, hydroxy, hydrogen;

[0011] The substituents for the 3-8 membered aliphatic heterocyclic ring are selected from the group consisting of: C1-C8 alkyl, C1-C8 alkyl ester, =O, and 3-8 membered aliphatic ring;

[0012] R3 and R4 are each independently selected from: hydrogen, C1-C8 alkyl, C6H5-CH2-, 3-8 membered aliphatic ring,

[0013] When R2 is a hydroxyl group, R1 is not

[0014] Preferably, the 3-8 membered aliphatic heterocycle includes: oxirane, aziridine, thiirane, oxetane, azetidine, thietane, tetrahydrofuran, tetrahydropyrrole, tetrahydrothiophene, tetrahydropyran, piperidine, tetrahydrothiopyran, dioxane, piperazine, hexahydropyrazine, morpholine, and thiophene.

[0015] Preferably, the substituted 3-8 membered aliphatic heterocycle is a substituted piperazine.

[0016] Preferably, the substituent for the 3-8 membered aliphatic heterocyclic ring is selected from the group consisting of: C1-C5 alkyl, C1-C4 alkyl ester, =O, and 3-5 membered aliphatic ring.

[0017] Preferably, the substituents for the 3-8 membered aliphatic heterocycle are selected from:

[0018] Preferably, the substitution position of the substituted piperazine is the 3-position or the 4-position.

[0019] Preferably, R1 is selected from C1-C3 alkoxy, fluorine, chlorine, bromine, and iodine.

[0020] Preferably, R1 is selected from:

[0021]

[0022] Preferably, the benzofuran[2,3-b]quinoline derivative, its optical isomer or its pharmaceutically acceptable salt has the following structural formula:

[0023]

[0024]

[0025]

[0026] Based on the same inventive concept, the present invention also claims a method for preparing the benzofuran[2,3-b]quinoline derivatives, their optical isomers or pharmaceutically acceptable salts thereof, which, when R2 is =O, comprises the following steps:

[0027] S1, using 3,4-dimethoxyaniline as the starting material, and obtaining intermediate 1 through acetal substitution reaction;

[0028] S2, intermediate 1 undergoes cyclization reaction in a high temperature solvent to obtain intermediate 2;

[0029] S3 and intermediate 2 undergo chlorination reaction to generate intermediate 3;

[0030] S4. Intermediate 3 undergoes a nucleophilic substitution reaction to obtain the benzofuran[2,3-b]quinoline derivative, its optical isomer or a pharmaceutically acceptable salt thereof.

[0031] Preferably, when R2 is a hydroxyl group, the method for preparing the benzofuran[2,3-b]quinoline derivative, its optical isomer or its pharmaceutically acceptable salt comprises the following steps:

[0032] S1, using 3,4-dimethoxyaniline as the starting material, and obtaining intermediate 1 through acetal substitution reaction;

[0033] S2, intermediate 1 undergoes cyclization reaction in a high temperature solvent to obtain intermediate 2;

[0034] S3 and intermediate 2 undergo chlorination reaction to generate intermediate 3;

[0035] S5, intermediate 3 is reduced to obtain intermediate 4;

[0036] S6. Intermediate 4 undergoes a nucleophilic substitution reaction to obtain the benzofuran[2,3-b]quinoline derivative, its optical isomer or a pharmaceutically acceptable salt thereof.

[0037] Preferably, when R2 is hydrogen, the method for preparing the benzofuran[2,3-b]quinoline derivative, its optical isomer or its pharmaceutically acceptable salt comprises the following steps:

[0038] S1, using 3,4-dimethoxyaniline as the starting material, and obtaining intermediate 1 through acetal substitution reaction;

[0039] S2, intermediate 1 undergoes cyclization reaction in a high temperature solvent to obtain intermediate 2;

[0040] S3 and intermediate 2 undergo chlorination reaction to generate intermediate 3;

[0041] S7. The intermediate 3 is subjected to a reduction reaction to obtain the benzofuran[2,3-b]quinoline derivative, its optical isomer or a pharmaceutically acceptable salt thereof.

[0042] In the preparation method, the structure of intermediate 1 is: The structure of intermediate 2 is: The structure of intermediate 3 is: The structure of intermediate 4 is: The structure of intermediate 5 is:

[0043] Preferably, in step S1, NaH and a catalyst are added to the acetal substitution reaction.

[0044] Preferably, in step S1, the amount of NaH added in the acetal substitution reaction is 2.0-3.0 equiv, preferably 2.5 equiv.

[0045] Preferably, in step S1, the catalyst in the acetal substitution reaction is one or more of TEA or DIPEA, preferably TEA.

[0046] Preferably, in step S1, the temperature of the acetal substitution reaction is 20-90°C, preferably 20-30°C.

[0047] Preferably, in step S2, the temperature of the cyclization reaction is 250-280°C.

[0048] Preferably, in step S2, during the cyclization reaction, the concentration of intermediate 1 is 0.01-0.02 mol·L -1 .

[0049] Preferably, in step S3, a solvent and a catalyst are added to the chlorination reaction.

[0050] Preferably, in step S3, the catalyst for the chlorination reaction is one or more of SOCl2, (COCl)2 or POCl3, preferably POCl3.

[0051] Preferably, in step S3, the solvent for the chlorination reaction is CH2Cl2.

[0052] Preferably, in step S5, a reducing agent is added to the reduction reaction.

[0053] Preferably, in step S5, the reducing agent for the reduction reaction is one or more of NaBH3CN, NaBH(OAc)3, NaBH4 or LiAlH4, preferably NaBH4.

[0054] A benzofuran[2,3-b]quinoline derivative, an optical isomer thereof or a pharmaceutically acceptable salt thereof, the structure of which is shown in formula (II):

[0055]

[0056] Wherein R5 is selected from: C3-C6 alkoxy,

[0057] Preferably, R5 is selected from

[0058] Based on the same inventive concept, the present invention also claims a method for preparing the benzofuran[2,3-b]quinoline derivatives, their optical isomers or pharmaceutically acceptable salts thereof, comprising the following steps:

[0059] S1, using 3,4-dimethoxyaniline as the starting material, synthesizing furanone through condensation reaction; then synthesizing intermediate 1 through CN condensation reaction under alkaline conditions;

[0060] S2, intermediate 1 undergoes cyclization reaction in a high temperature solvent to obtain intermediate 2;

[0061] S3 and intermediate 2 undergo chlorination reaction to generate intermediate 3;

[0062] S4, intermediate 3 is reduced to obtain intermediate 4;

[0063] S5, intermediate 4 undergoes dehydration elimination reaction to obtain intermediate 5;

[0064] S6. The intermediate 5 reacts to obtain the benzofuran[2,3-b]quinoline derivative, its optical isomer or a pharmaceutically acceptable salt thereof.

[0065] Based on the same inventive concept, the present invention also claims protection for the use of the benzofuran[2,3-b]quinoline derivatives, their optical isomers or pharmaceutically acceptable salts thereof in the preparation of drugs for chronic kidney disease.

[0066] Preferably, the drug further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.

[0067] The pharmaceutically acceptable "carriers, adjuvants or excipients" mentioned above refer to conventional drug carriers in the pharmaceutical field, such as: diluents, excipients such as water, fillers such as starch and sucrose, adhesives such as cellulose derivatives, gelatin, etc.; in addition, other excipients such as flavoring agents, sweeteners, etc. can also be added to the composition.

[0068] The compounds of the present invention may be in the form of a preferred compound or a crystalline solvate. Methods of solvation are well known in the art, and suitable solvates are pharmaceutically acceptable solvates. In a specific embodiment, the solvate is a hydrate.

[0069] Preferably, the chronic kidney disease is renal fibrosis.

[0070] The present invention will be further explained below:

[0071] The present invention evaluated the effects of all derivatives on the proliferation of rat renal fibrosis cells NRK-49F after 24 hours of treatment. The results showed that the inhibitory activity of the derivatives of the present invention on NRK-49F cells was generally better than that of the lead compound vanillin (IC50 = 645.8 μM) and the positive control drug meflunisal (IC50 = 408.2 μM), indicating that benzofurano[2,3-b]quinoline derivatives have anti-renal fibrosis potential. Among them, the IC50 values of compounds 7a, 10a-b, 10e-g, 10o, 11a-d and 11h were all less than 100 μM (IC50 = 13.8-99.63 μM). These compounds demonstrated potential anti-renal fibrosis activity.

[0072] The beneficial effects of the present invention are:

[0073] The present invention synthesized multiple novel derivatives; (1) CCK8 assays preliminarily confirmed that the compounds of the present invention exhibited strong inhibitory activity against NRK-49F cells (IC50 = 13.8-80.84 μM). (2) In vivo acute toxicity experiments confirmed that the compounds of the present invention exhibited no significant toxicity at a dose of 2000 mg / kg, exhibiting good safety and optimal anti-renal fibrosis activity. Second, in two animal models of renal fibrosis, the compounds of the present invention showed significant therapeutic effects: in the adenine model, the compounds of the present invention significantly reduced the blood creatinine level, effectively improved the degree of renal damage and fibrosis, and the efficacy was comparable to that of the control drug alloprolol; in the UUO model, the compounds of the present invention significantly inhibited collagen deposition, and the efficacy was comparable to that of positive losartan; Third, mechanism studies showed that the compounds of the present invention inhibited the excessive accumulation of extracellular matrix proteins (α-SMA, Collagen I, Vimentin and Fibronectin) by dually regulating the TGF-β1 / Smad2 / Smad3 and PI3K / AKT signaling pathways, thereby exerting an anti-fibrotic effect; (3) Pharmacokinetic studies showed that the compounds of the present invention have excellent drug metabolism characteristics, with an oral bioavailability of 67% and a half-life of 2h, and good drugability.

[0074] Therefore, the compounds of the present invention have the potential to become anti-renal fibrosis drugs, and can alleviate renal fibrosis by dually regulating the TGF-β1 / Smad2 / Smad3 and PI3K / AKT signaling pathways, providing new strategies and ideas for the treatment of CKD. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Results of acute toxicity experiments of the compounds in vivo; A is the change in body weight; B is the change in average food intake; (n=6, compared with the control group, *P<0.05, **P<0.01, ***P<0.001).

[0076] Figure 2 Effects of the compound on the adenine diet-induced renal fibrosis model in mice; A represents the change in creatinine level, and B represents the change in urea nitrogen level; (n=6, compared with the control group, *P<0.05, **P<0.01, ***P<0.001).

[0077] Figure 3 HE staining and Masson staining results show the effects of the compound on the adenine diet-induced mouse renal fibrosis model.

[0078] Figure 4 The staining scores of the effects of the compounds on the adenine diet-induced mouse renal fibrosis model; A is the HE score; B is the Masson score.

[0079] Figure 5 is the expression level of renal fibrosis-related proteins in the kidney tissue of mice in each group.

[0080] Figure 6 Statistical graphs of the relative gray values of renal fibrosis-related proteins in each group of mice; A is the relative gray value of vimentin; B is the relative gray value of type I collagen FN; C is the relative gray value of fibronectin; D is the relative gray value of α-smooth muscle actin (n=3, compared with the control group, *P<0.05, **P<0.01, ***P<0.001).

[0081] Figure 7 The results of kidney pathological staining of each group of mice in the UUO model were shown in Figure 5; A is HE staining, and B is Masson staining.

[0082] Figure 8 The scores of the compound on kidney pathological staining in each group of mice in the UUO model; A is the HE score; B is the Masson score.

[0083] Figure 9 The expression levels of renal fibrosis-related proteins in the renal tissue of mice in each group of UUO model.

[0084] Figure 10 Statistical graphs of the relative gray values of renal fibrosis-related proteins in the renal tissues of mice in each group of the UUO model; A is the relative gray value of vimentin; B is the relative gray value of type I collagen FN; C is the relative gray value of fibronectin; D is the relative gray value of α-smooth muscle actin (n=3, compared with the control group, *P<0.05, **P<0.01, ***P<0.001). Specific implementation methods

[0085] The following examples may help those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0086] Example 1

[0087] Synthesis of benzofuran[2,3-b]quinoline derivatives, their optical isomers or pharmaceutically acceptable salts thereof

[0088] The synthetic routes of benzofuran[2,3-b]quinoline derivatives, their optical isomers or pharmaceutically acceptable salts thereof are as follows:

[0089] The basic structural unit of vanillaminin is 4,6,7-trimethoxyfuran[2,3-b]quinoline. The present invention uses 3,4-dimethoxyaniline as the starting material and simultaneously synthesizes furanone via a self-condensation reaction of diethyl malonate. Subsequently, intermediate 1 is synthesized through a CN condensation reaction under alkaline conditions. After obtaining intermediate 1, intermediate 1 is heated in a high-temperature solvent to achieve an intramolecular cyclization reaction, thereby constructing the basic skeleton 2 of vanillaminin. Next, a chlorination reaction is carried out under the synergistic action of the surfactants methyltrioctylammonium chloride and phosphorus oxychloride to produce the key intermediate 3. At this point, the following two synthetic pathways can be selected: first, a reduction and elimination reaction is performed on the ring structure of furanone to obtain the parent nucleus structure of the furan ring, followed by a nucleophilic substitution reaction to synthesize the target product, with an overall yield of 25%. Second, a methoxy substituent is introduced on the pyridine ring through a nucleophilic substitution reaction, and the furanone is reduced and further reduced in the presence of a Lewis acid to obtain the final product, with an overall yield of 22%.

[0090]

[0091] For the first step CN condensation reaction, that is:

[0092]

[0093] The present invention screened the NaH equivalent, organic base type, and reaction temperature. The results, shown in Table 1, show that the highest yield was achieved when the NaH equivalent was 2.5 equiv. Increasing or decreasing the NaH equivalent significantly decreased the yield. The highest yield was achieved when TEA was used as the organic base and the reaction was carried out at room temperature. The overall yield was low due to the milligram-level raw materials selected for the reaction during the screening process, which precluded recrystallization. For large-scale production, the present invention prioritized recrystallization and employed the conditions described in Entry 6, significantly improving the yield to 71%.

[0094] Table 1 Optimization of conditions for acetal substitution reaction of intermediate 1

[0095]

[0096] In the synthesis process of heating cyclization (2), that is:

[0097]

[0098] The present invention mainly studies the reaction temperature and reaction concentration, and finds that when the temperature is lower than 250 ° C, compound 1 will remove the ester to generate the compound shown in 2-1 (MS: 235.9). When the reaction concentration is too high (0.05 mol·L -1 Above), 1 will undergo intermolecular reaction, mainly generating compound 2-2 as shown above (MS: 464.9). When the reaction concentration drops to 0.01-0.02 mol·L -1 When , the main generation is 2 (MS: 261.9).

[0099] In the synthesis process of chlorination reaction (3), namely:

[0100]

[0101] The present invention mainly screens the chlorination reagent and whether a surfactant and a solvent are added. The results are shown in Table 2. When other chlorination reagents such as SOCl2 and (COCl)2 are used, the yield is quite low. When POCl3 is directly used as the solvent, the yield increases but is relatively low. When the chlorination reagent is POCl3, and the surfactant methyl trioctyl ammonium chloride is added and CH2Cl2 is used as the solvent, the yield is greatly improved.

[0102] Table 2 Optimization of conditions for the chlorination reaction of intermediate 3

[0103]

[0104] In the synthesis process of hydrogenation reduction (4), that is:

[0105]

[0106] This study mainly screened the reducing agents. As shown in Table 3, it was found that the yield was the highest when NaBH4 was used for reduction.

[0107] Table 3 Optimization of reduction reaction conditions for intermediate 4

[0108]

[0109] The synthesis of the specific compound is as follows:

[0110] 1. Synthesis of intermediates:

[0111] (1) Synthesis of Compound 1: In a 500 mL round-bottom flask, 22 g of NaH (60%) was added to 75 mL of dry THF for dispersion. The mixture was stirred in an ice bath and ClAcCl (69 mL) was slowly added dropwise over 1 h. After the addition was complete, the mixture was set aside. In another 500 mL reaction flask, ClAcCl (19.2 mL) was dissolved in 102 mL of dry THF. The solution prepared in the previous step was slowly added dropwise over 1 h in an ice bath, with the reaction temperature controlled below 30°C. After the addition was complete, the mixture was stirred at room temperature for 1 h, and then TEA (60.6 mL) was added and stirred for 2 h. Subsequently, a solution of 3,4-dimethoxyaniline (35.1 g) in THF (150 mL) was slowly added dropwise over 1 h in an ice bath, with the temperature controlled above 30°C. After the addition was complete, the mixture was stirred at room temperature overnight. After the reaction of the starting material was complete, as monitored by thin-layer chromatography (PE:EA = 1:1), the mixture was concentrated under reduced pressure to remove THF, extracted with 100 mL of EA and 50 mL of water, and the organic phase was filtered to yield 40 g of pure compound 1. The aqueous phase was extracted multiple times with EA, and the organic phase of the filtrate was dried, concentrated under reduced pressure, and then dispersed with diethyl ether by stirring. Filtering afforded the crude product, which was recrystallized from 50 mL of ethanol to yield 9.3 g of compound 1 as an off-white solid, in a 71% yield.

[0112] (2) Synthesis of compound 2: In a 1000 mL reaction flask, compound 1 (5 g) was added with 30 mL (0.5 mol·L -1 )Ph2O was ultrasonically dispersed into a suspension and slowly added to 600mL (0.02mol·L -1 ) in boiling Ph2O and refluxed for 10 min. Thin layer chromatography (PE:EA=1:1) indicated complete reaction of the starting material. The product was monitored by DCM:MeOH=10:1. The mixture was cooled to room temperature. The reaction solution was poured into 1000 mL of PE, the resulting solid was filtered, and the filter cake was rinsed with PE to obtain 4.3 g of crude brown solid Compound 2, with a yield of 81%.

[0113] (3) Synthesis of Compound 3: 6 g of Compound 2 from the previous step and 1 g of methyltrioctylammonium chloride were added to a 100 mL reaction flask. Under N2 protection, 45 mL of CH2Cl2 and 9 mL of POCl3 were added. The mixture was refluxed under N2 protection for 5 h. Thin-layer chromatography (DCM:MeOH=10:1 or PE:EA=1:1) indicated that the reaction was complete. The reaction solution was poured into 100 mL of crushed ice and quenched completely. Na2CO3 / NaHCO3 was added to neutralize the mixture. The mixture was extracted with DCM and counter-extracted with water. The organic phases were combined, concentrated, and recrystallized from DCM / PE. Filtering yielded 5.5 g of crude Compound 3. The yield was 85%.

[0114] (4) Synthesis of compound 4: Compound 3 from the previous step was treated with 200 mL (0.1 mol·L -1) was dissolved in a 1:1 CH2Cl2:MeOH mixture. 3.72 g NaBH4 (5.0 equiv) was added under ice-cooling and the reaction was stirred at room temperature for 1 h. After the reaction was complete, thin-layer chromatography (PE:EA = 1:1) analysis confirmed that the starting material had reacted completely. 10 mL of water was added to quench the NaBH4. The CH2Cl2 and MeOH were removed under reduced pressure at 40°C, and the mixture was extracted with DCM (3 x 100 mL). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated to remove the DCM, yielding 5.88 g of crude compound 4. The yield was 90%. Alternatively, compound 3 was reduced with LiAlH4 in the presence of the Lewis acid AlCl3 in a two-step process to afford 8 and 9.

[0115] (5) Synthesis of compound 5: Compound 4 from the previous step was added with dry 1,4-dioxane (200 mL, 0.1 mol·L -1 ) was stirred, 14 g (5.0 equiv) of KHSO₄ was added, and the temperature was raised to 110°C and refluxed with stirring overnight. Thin-layer chromatography (PE:EA=2:1) analysis indicated that the starting material had reacted completely. Dioxane was removed under reduced pressure, and the mixture was extracted with 100 mL of DCM. The mixture was washed with saturated aqueous Na₂CO₃ (3 x 50 mL) until neutral. The aqueous phase was extracted with DCM (3 x 100 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated to afford 5.1 g of crude compound 5 in a 93% yield.

[0116] (6) Synthesis of vanilla linalool: The crude compound 5 in the previous step was added with MeOH (135 mL, 0.15 mol·L -1 ) was stirred at room temperature, and sodium methoxide methanol solution (5.4 mol·L -1 ) 7.9 mL (7.6 equiv) was added and heated under reflux at 40-50°C for 3 days. Thin-layer chromatography (petroleum ether: ethyl acetate 1:1) indicated that the starting material had reacted completely. MeOH was removed under reduced pressure at 40°C, and the mixture was extracted with 100 mL of DCM and 30 mL of water. The aqueous phase was extracted with DCM (3 x 100 mL), combined, dried, filtered, and concentrated to yield the crude product. This crude product was separated by normal phase chromatography on a silica gel column (PE:EA = 3:1 to PE:EA = 1:1) to afford 3.0 g of vanillin, with a yield of 60% and an overall yield of 25% over six steps. In the synthesis of step 6, increasing the reaction temperature to 90°C resulted in the formation of byproducts 13 and 14, but the reaction time was shortened to half.

[0117] (7) Synthesis of vanilla glutinin: Under N2 protection, 5 g of compound 3 was added to the reaction flask, 90 mL of MeOH was added as solvent, 5 mL of sodium methoxide methanol solution was added and stirred evenly, and then refluxed overnight. After the reaction was completed as monitored by thin-layer chromatography, MeOH was removed under reduced pressure at 40°C, and the mixture was extracted with DCM / H2O. After aqueous back extraction, the organic phases were combined, dried over anhydrous Na2SO4, filtered and concentrated to remove DCM, and 3.95 g of crude vanilla glutinin was obtained, with a yield of 80%.

[0118] 2. Synthesis of compounds:

[0119] (1) Synthesis of fennel (Route 2): After adding 1 g of compound 6 and 1.7 g of AlCl3 to a reaction flask, 72 mL of THF was added under N2 protection. 0.24 g of LiAlH4 was added in batches under ice bath conditions. The mixture was stirred at room temperature overnight. After completion of the reaction, the mixture was quenched with H2O-NaOH-H2O and extracted with EA / saturated brine. After aqueous counter extraction, the organic phase was concentrated to obtain a crude product. The crude product was separated by silica gel normal phase chromatography (PE:EA=3:1 to PE:EA=1:1) to obtain 0.52 g of fennel with a yield of 55% and a total yield of 22% over the five steps.

[0120] (2-1) Synthesis of compound 6a: To a reaction flask were added 100 mg of intermediate 5, 5.0 equiv of NaH, and n-butanol (56.3 mg, 2.0 equiv). Under nitrogen protection, DMF was added as the reaction solvent. The reaction was allowed to proceed at room temperature for 12 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 20.6 mg of a white amorphous powder in 18% yield.

[0121] (2-2) Synthesis of compound 6b: To a reaction flask were added 100 mg of intermediate 5, 5.0 equiv of NaH, and 71.5 mg of phenol (2.0 equiv). Under nitrogen protection, DMF was added as the reaction solvent. The reaction was allowed to proceed at room temperature for 12 h. After completion of the reaction, the mixture was monitored by TLC and separated by silica gel column chromatography to obtain 29.3 mg of a white amorphous powder in a 24% yield.

[0122] (2-3) Synthesis of compound 6c: To a reaction flask were added 100 mg of intermediate 5, 5.0 equiv of NaH, and 77.6 mg of 3-methylpentan-1-ol (2.0 equiv). Under nitrogen protection, DMF was added as the reaction solvent. The reaction was allowed to proceed at room temperature for 12 h. After completion of the reaction, the mixture was separated by silica gel column chromatography to obtain 60 mg of a white amorphous powder in a 48% yield.

[0123] (3-1) Synthesis of compound 7a: To a reaction flask, 100 mg of vanillin, 0.2 equiv of Pd(OH)2 and 0.2 equiv of Pd, 10.0 equiv of HCOOH, and 10.0 equiv of TEA were added. MeOH was used as the reaction solvent. Under hydrogen protection, the reaction was allowed to proceed for 12 h. After completion of the reaction, the product was separated by silica gel column chromatography to obtain 80.6 mg of a white amorphous powder in 80% yield.

[0124] (3-2) Synthesis of compound 7b: To a reaction flask was added 100 mg of intermediate 5, 0.2 equiv of Pd(OH)2 and 0.2 equiv of Pd, 10.0 equiv of HCOOH, and 10.0 equiv of TEA. MeOH was used as the reaction solvent. Under hydrogen protection, the reaction was allowed to proceed for 12 h. After completion of the reaction as monitored by TLC, the product was separated by silica gel column chromatography to obtain 82.6 mg of a white amorphous powder in 82% yield.

[0125] (4) Synthesis of compound 10a: To a reaction flask was added 100 mg of intermediate 3 and N,N-dimethyl-2-(methylamino)acetamide (207.9 mg, 5.0 equiv). 5.0 equiv of DIPEA was used as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 121 mg of a white amorphous powder in a yield of 94%.

[0126] (4) Synthesis of compound 10b: To a reaction flask, 100 mg of intermediate 3 and 1-isopropylpiperazine (229.4 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst, and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 129 mg of a white amorphous powder in a yield of 97%.

[0127] (4) Synthesis of compound 10c: To a reaction flask, 100 mg of intermediate 3 and tert-butyl piperazine-1-carboxylate (333.4 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 144.5 mg of a white amorphous powder in a yield of 94%.

[0128] (4) Synthesis of compound 10d: To a reaction flask was added 100 mg of intermediate 3 and n-propylamine (105.7 mg, 5.0 equiv). 5.0 equiv of DIPEA was added as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 101.7 mg of a white amorphous powder in a yield of 94%.

[0129] (4) Synthesis of compound 10e: To a reaction flask, 100 mg of intermediate 3 and N1,N1-dimethylethane-1,2-diamine (157.7 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst, and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 45 mg of a white amorphous powder in a yield of 38%.

[0130] (4) Synthesis of compound 10f: To a reaction flask was added 100 mg of intermediate 3 and isopropylamine (105.7 mg, 5.0 equiv). 5.0 equiv of DIPEA was used as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 28 mg of a white amorphous powder in a yield of 26%.

[0131] (4) Synthesis of compound 10g: To a reaction flask, 100 mg of intermediate 3 and cyclopropylamine (102.2 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 74.2 mg of a white amorphous powder in a yield of 69%.

[0132] (4) Synthesis of compound 10h: To a reaction flask, 100 mg of intermediate 3 and N-methyl-1-benzylamine (216.8 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst, and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 67.8 mg of a white amorphous powder in a yield of 52%.

[0133] (4) Synthesis of compound 10i: To a reaction flask, 100 mg of intermediate 3 and 1-methylpiperazin-2-one (204.3 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 89.6 mg of a white amorphous powder in a 70% yield.

[0134] (4) Synthesis of compound 10j: To a reaction flask, 100 mg of intermediate 3 and 177.4 mg of cyclohexylamine (5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 28.2 mg of a white amorphous powder in a yield of 23%.

[0135] (4) Synthesis of compound 10k: To a reaction flask, 100 mg of intermediate 3 and propargylamine (98.6 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 50 mg of a white amorphous powder in a yield of 47%.

[0136] (4) Synthesis of compound 101: To a reaction flask, 100 mg of intermediate 3 and 1-cyclopropylpiperazine (225.8 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 96.5 mg of a white amorphous powder in a yield of 73%.

[0137] (4) Synthesis of compound 10m: To a reaction flask, 100 mg of intermediate 3 and 1-(tert-butyl)piperazine (254.5 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 107.6 mg of a white amorphous powder in a yield of 78%.

[0138] (4) Synthesis of compound 10n: To a reaction flask, 100 mg of intermediate 3 and dimethylamine (80.6 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst, and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 100.2 mg of a white amorphous powder in a yield of 97%.

[0139] (4) Synthesis of compound 10o: To a reaction flask, 100 mg of intermediate 3 and 1-methylpiperazine (179 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 94.7 mg of a white amorphous powder in a yield of 77%.

[0140] (4) Synthesis of compound 10p: To a reaction flask, 100 mg of intermediate 3 and (R)-tert-butyl 3-aminopyrrolidine-1-carboxylate (333.4 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 124.5 mg of a white amorphous powder in an 81% yield.

[0141] (4) Synthesis of compound 10q: To a reaction flask, 100 mg of intermediate 3 and (S)-tert-butyl 3-aminopyrrolidine-1-carboxylate (207.9 mg, 5.0 equiv) were added. 5.0 equiv of DIPEA was used as a catalyst and CH2Cl2 (0.1 M) was used as the reaction solvent. The mixture was refluxed at 50°C for 6 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain 124.5 mg of a white amorphous powder in an 81% yield.

[0142] (5) Synthesis of compound 11a: 10a obtained above was added to a reaction flask, and 3.0 equiv of NaBH4 was added. MeOH was used as the reaction solvent and the mixture was reduced at room temperature for 3 h. After completion of the reaction as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain a white amorphous powder in a yield of 65%.

[0143] (5) Synthesis of compound 11b: 10b obtained above was added to a reaction flask, and 3.0 equiv of NaBH4 was added. MeOH was used as the reaction solvent and the mixture was reduced at room temperature for 3 h. After completion of the reaction monitored by TLC, the mixture was separated by silica gel column chromatography to obtain a white amorphous powder with a yield of 73%.

[0144] (5) Synthesis of compound 11c: 10c obtained above was added to a reaction flask, and 3.0 equiv of NaBH4 was added. MeOH was used as the reaction solvent and the mixture was reduced at room temperature for 3 h. After the reaction was completed as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain a white amorphous powder in a yield of 56%.

[0145] (5) Synthesis of compound 11d: The above-obtained 10d was added to a reaction flask, and 3.0 equiv of NaBH4 was added. MeOH was used as the reaction solvent and the mixture was reduced at room temperature for 3 h. After the reaction was completed as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain a white amorphous powder in a yield of 60%.

[0146] (5) Synthesis of compound 11e: The above-obtained 10e was added to a reaction flask, and 3.0 equiv of NaBH4 was added. MeOH was used as the reaction solvent and the mixture was reduced at room temperature for 3 h. After the reaction was completed as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain a white amorphous powder in a yield of 38%.

[0147] (5) Synthesis of compound 11f: The above-obtained 10f was added to a reaction flask, and 3.0 equiv of NaBH4 was added. MeOH was used as the reaction solvent and the mixture was reduced at room temperature for 3 h. After the reaction was completed as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain a white amorphous powder in an 88% yield.

[0148] (5) Synthesis of compound 11g: 10 g of the above-obtained product was added to a reaction flask, 3.0 equiv of NaBH4 was added, and MeOH was used as the reaction solvent. The product was reduced at room temperature for 3 h. After the reaction was completed as monitored by TLC, it was separated by silica gel column chromatography to obtain a white amorphous powder with a yield of 86%.

[0149] (5) Synthesis of compound 11h: The above-obtained 10h was added to a reaction flask, and 3.0 equiv of NaBH4 was added. MeOH was used as the reaction solvent and the mixture was reduced at room temperature for 3 h. After the reaction was completed as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain a white amorphous powder with a yield of 73%.

[0150] (5) Synthesis of compound 11i: The above-obtained 10i was added to a reaction flask, and 3.0 equiv of NaBH4 was added. MeOH was used as the reaction solvent and the mixture was reduced at room temperature for 3 h. After the reaction was completed as monitored by TLC, the mixture was separated by silica gel column chromatography to obtain a white amorphous powder in a yield of 59%.

[0151] 2.5.3 Structural characterization of compounds

[0152] Compound 1, White amorphous powder, 71% yield; 1H NMR (400MHz, DMSO) δ9.26 (s, 1H), 6.23 (d, J = 2.1Hz, 1H), 6.16–6.08 (m, 2H), 3.79 (s, 2H) ,3.34(q,J=7.1Hz,2H),2.89(d,J=1.5Hz,6H),1.65–1.63(m,3H).HR-MS(ESI)m / z:calcd for C 15 H 17 NO6[M+H] + :308.1129,found:308.1148.

[0153] Compound 2, White amorphous powder, 81% yield; 1 HNMR (400 MHz, DMSO) δ

[0154] 7.22(s,1H),6.92(s,1H),4.94(s,2H),3.88(s,3H),3.84(s,3H).HR-MS(ESI)m / z:calcd for C 13 H 11 NO5[M+H] + :262.0710,found:262.0724.

[0155] Compound 3, White amorphous powder, 84% yield; 1 HNMR (500 MHz, DMSO) δ

[0156] 7.46(s,1H),7.41-7.31(m,1H),5.33(t,J=5.0Hz,1H),4.89(s,2H),4.00(s,3H),3.97(s,3H).HR-MS(ESI)m / z:calcd for C 13 H 10 ClNO4[M+H] + :280.0371,found:280.0388.

[0157] Compound 4, White amorphous powder, 80% yield; 1H NMR (500MHz, CDCl3) δ7.21 (s, 1H), 7.19 (s, 1H), 5.64 (s, 1H), 4.73 (dd, J = 10.5, 6.6Hz, 1H) ,4.63(dd,J=10.5,2.3Hz,1H),4.01(d,J=11.1Hz,6H),3.05(s,1H).HR-MS(ESI)m / z:calcd forC 13 H 12 ClNO4[M+H] + :282.0528,found:282.0553.

[0158] Compound 5, White amorphous powder, 93% yield; 1 H NMR (500MHz, CDCl3) δ7.74(d,J=2.6Hz,1H),7.48(s,1H),7.43(s,1H),6.94(d,J=2.6Hz,1H),4.07(d,J=11.7Hz,7H).HR-MS(ESI)m / z:calcd for C 13 H 10 ClNO3[M+H] + :264.0422,found:264.0446.

[0159] Compound 6a, White amorphous powder, 18% yield; 1 H NMR (400MHz, CDCl3) δ7.56(d,J=2.7Hz,1H),7.50(s,1H),7.34(s,1H),6.97(d,J=2.7Hz,1H),4.66(t,J=6.4H z,2H),4.03(s,3H),4.02(s,3H),1.97(dt,J=14.4,6.5Hz,2H),1.64(d,J=1.7Hz,2H),1.05(t,J=7.4Hz,3H). 13 C NMR (101MHz, CDCl3) δ163.13,155.10,152.61,147.75,142.69,142.40,113.29,106.79,1 04.79,102.61,100.36,71.28,56.05,55.95,31.80,19.28,13.88.HR-MS(ESI)m / z:calcd for C 17 H 19 NO4[M+H] +:302.1387,found:302.1399.

[0160] Compound 6b, White amorphous powder, 24% yield; 1 H NMR (400MHz, CDCl3) δ7.56 (d, J=2.7Hz, 1H), 7.51 (dd, J=5.5, 2.8Hz, 3H), 7.47 –7.39(m,3H),7.35(s,1H),6.95(d,J=2.7Hz,1H),4.03(s,3H),3.97(s,,3H). 13 C NMR (101MHz, CDCl3) δ163.03,154.83,152.71,147.94,142.77,136.29,128.87,128.53,127 .29,113.49,106.81,104.57,103.15,100.39,73.27,56.07,56.00..HR-MS(ESI)m / z:calcd for C 19 H 15 NO4[M+H] + :322.1074,found:365.1087.

[0161] Compound 6c, White amorphous powder, 48% yield; 1 H NMR (400MHz, CDCl3) δ8.32(d,J=12.2Hz,1H),7.19(s,1H),6.74(s,1H),6.21(d,J=12.2Hz,1H),3.94(s,3H),3.92 –3.84(m,5H),1.85–1.67(m,2H),1.60–1.48(m,2H),1.44–1.36(m,1H),1.26–1.16(m,3H),0.93(d,J=2.2Hz,3H). 13 C NMR (101MHz, CDCl3) δ161.67,155.28,151.74,146.08,137.25,130.63,122.53,112.88,105.09, 100.68,97.46,69.01,56.07,55.83,35.98,31.39,29.50,19.10,,11.28.HR-MS(ESI)m / z:calcd for C 19 H 23 NO4[M+H] +:330.1700,found:330.1723.

[0162] Compound 7a, White amorphous powder, 80% yield; 1 H NMR (500MHz, CDCl3) δ7.27(s,1H),7.14(s,1H),4.56(t,J=8.2Hz,2H),4.16(s,3H),3.96(d,J=4.7Hz,6H),3.57(t,J=8.2Hz,2H). 13 C NMR (101MHz, CDCl3) δ168.50,158.11,151.99,146.89,143.87,113.36,106.87,101.07,98.92,68.31,57.96,55.94,55.92,28.35.HR-MS(ESI)m / z:calcd for C 14 H 15 NO4[M+H] + :262.1074,found:262.1088.

[0163] Compound 7b, White amorphous powder, 82% yield; 1 H NMR (400MHz, CDCl3) δ7.21 (s, 1H), 6.95 (s, 1H), 4.65 (t, J = 8.3Hz, 2H), 3.97 (s, 3H), 3.95 (s, 3H), 3.32 (td, J = 8.4, 1.3Hz, 2H). 13 C NMR (126MHz, CDCl3) δ166.80,151.74,147.50,143.16,131.50,119.78,118.85,107.33,105.94,69.04,55.99,55.97,27.84.HR-MS(ESI)m / z:calcd forC 13 H 12 ClNO3[M+H] + :266.0578,found:266.0584.

[0164] Compound 10a, White amorphous powder, 94% yield; 1H NMR (400MHz, CDCl3) δ7.59 (s, 1H), 7.13 (s, 1H), 4.64 (d, J = 2.2Hz, 4H), 3.99 (d ,J=7.8Hz,3H),3.97(d,J=9.0Hz,3H),3.48(s,3H),3.02(s,3H),2.99(s,,3H). 13 C NMR (101MHz, CDCl3) δ194.47,174.09,168.82,157.72,155.25,150.62,146.94,114.85,107. 70,107.20,101.72,73.51,58.46,56.38,56.23,45.00,36.35,35.89.HR-MS(ESI)m / z:calcd for C 18 H 21 N3O5[M+H] + :360.1554,found:360.1576.

[0165] Compound 10b, White amorphous powder, 97% yield; 1 H NMR (500MHz, CDCl3) δ7.25(s,1H),7.15(s,1H),4.63(s,2H),4.01(s,3H),3.95(s,3H),3.86(s,4H),2.82(s,5H),1.14(s,3H),1.13(s,3H). 13 C NMR (126MHz, CDCl3) δ193.57,174.06,155.73,154.53,149.99,146.44,113.73,107.88, 106.21,100.97,73.24,56.15,55.97,54.69,53.76,49.70,18.48.HR-MS(ESI)m / z:calcd for C 20 H 25 N3O4[M+H] + :372.1918,found:372.1938.

[0166] Compound 10c, White amorphous powder, 24% yield; 1 H NMR (600MHz, CDCl3) δ7.18(s,1H),7.16(s,1H),4.64(s,2H),4.01(s,3H),3.95(s,3H),3.72(s,8H),1.51(s,9H).13 C NMR (101MHz, CDCl3) δ194.15,174.07,156.06,155.16,154.81,150.39,147.24,114..29,108 .16,105.53,102.16,80.53,73.52,56.42,56.18,53.15,29.82,28.55.HR-MS(ESI)m / z:calcd for C 22 H 27 N3O6[M+H] + :430.1973,found:430.1987.

[0167] Compound 10d, White amorphous powder, 35% yield; 1 H NMR(400MHz, CDCl3)δ7.36(s,1H),7.14–7.03(m,1H),4.58(s,2H),3.92(s,3H),3. 87(s,3H),3.81(dd,J=12.6,6.8Hz,2H),1.88–1.73(m,2H),1.05(t,J=7.4Hz,3H). 13 CNMR(101MHz,CDCl3)δ196.39,173.66,154.55,153.63,150.55,146.08,109.48, 108.66,105.96,94.92,73.57,56.12,48.55,23.80,11.21.HR-MS(ESI)m / z:calcd for C 16 H 18 N2O4[M+H] + :303.1339,found:303.1356.

[0168] Compound 10e, White amorphous powder, 38% yield; 1 H NMR(500MHz, CDCl3)δ7.20(dd,J=13.1,4.0Hz,1H),7.03(s,1H),4.53(d,J=10.0Hz,2H),3.95( dd,J=10.9,5.4Hz,2H),3.90(d,J=9.4Hz,3H),3.88(s,3H),2.64(t,J=5.8Hz,2H),2.31(s,6H). 13C NMR (101MHz, CDCl3) δ195.42,173.97,154.45,152.41,149.30,146.46,109.76,108.5 5,104.38,94.61,73.35,58.11,56.14,56.11,45.09,44.11.HR-MS(ESI)m / z:calcdfor C 17 H 21 N3O4[M+H] + :332.1605,found:332.1627.

[0169] Compound 10f, White amorphous powder, 26% yield; 1 H NMR (400MHz, CDCl3) δ7.73(d,J=5.9Hz,1H),7.34(s,1H),7.15(s,1H),4.65(s,2H),3.99(s,3H),3.94(s,3H),1.74(s,1H),1.47(d,J=6.3Hz,6H). 13 C NMR (126MHz, CDCl3) δ196.34,173.72,154.50,152.62,150.81,146.28,109.07,1 08.74,105.62,94.92,73.54,56.14,56.06,47.73,24.17.HR-MS(ESI)m / z:calcd for C 16 H 18 N2O4[M+H] + :303.1339,found:303.1367.

[0170] Compound 10g, White amorphous powder, 69% yield; 1 H NMR (400MHz, CDCl3) δ8.27(s,1H),7.16(s,1H),4.64(s,2H),4.00(s,3H),3.95(s,3H),3.49(s,1H),1.17–1.07(m,2H),1.03–0.88(m,2H). 13C NMR (126MHz, CDCl3) δ196.82,196.74,173.42,154.60,153.96,150.92,145.73,109. 48,108.40,106.85,94.80,73.60,56.12,55.92,28.09,10.90.HR-MS(ESI)m / z:calcd for C 16 H 16 N2O4[M+H] + :301.1183,found:301.1208.

[0171] Compound 10h, White amorphous powder, 52% yield; 1 H NMR (400MHz, CDCl3) δ7.39–7.37(m,1H),7.36(d,J=6.7Hz,3H),7.33(s,1H),7.23(s,1H) ,7.18(s,1H),4.88(s,2H),4.67(s,2H),4.01(d,J=4.0Hz,3H),3.65(s,3H),3.31(s,3H). 13 C NMR (101MHz, CDCl3) δ193.97,174.12,157.43,154.90,150.16,147.06,137.61,130.17,129.88,129.02,1 27.90,127.69,114.69,108.18,106.02,101.94,73.55,61.38,56.39,55.85,43.02.HR-MS(ESI)m / z:calcd for C 21 H 20 N2O4[M+H] + :365.1496,found:365.1551.

[0172] Compound 10i, White amorphous powder, 70% yield; 1 H NMR (400MHz, CDCl3) δ7.20(s,1H),7.11(d,J=4.5Hz,1H),4.68(s,2H),4.37(s,2H),4.08–3.99(m,5H),3.97(s,3H),3.64–3.51(m,2H),3.11(s,3H). 13C NMR (101MHz, CDCl3) δ194.52,173.80,165.80,155.62,154.02,150.66,147.85,114.53,108. 17,104.65,102.88,73.61,56.50,56.34,56.01,50.17,49.59,34.61.HR-MS(ESI)m / z:calcd for C 18 H 19 N3O5[M+H] + :358.1397,found:358.1401.

[0173] Compound 10j, White amorphous powder, 23% yield; 1 H NMR (400MHz, CDCl3) δ7.35(d,J=4.8Hz,1H),7.15(d,J=8.4Hz,1H),4.66(s,2H),4.33–4.16(m,1H),4.00(s,2H),3.95(s,2H),2.29–2 .18(m,2H),2.03(dt,J=11.5,7.1Hz,1H),1.90(dd,J=9.3,3.8Hz,2H),1.68(dt,J=33.0,12.9Hz,3H),1.52(dd,J=23.7,11.7Hz,3H). 13 C NMR (101MHz, CDCl3) δ196.64,173.95,154.55,152.81,150.91,146.31,109.27,108.97,1 05.70,95.15,73.67,56.28,56.04,54.74,34.40,25.41,24.65.HR-MS(ESI)m / z:calcdfor C 19 H 22 N2O4[M+H] + :343.1652,found:343.1686.

[0174] Compound 10k, White amorphous powder, 47% yield; 1 H NMR (400MHz, CDCl3) δ7.77(s,1H),7.53(d,J=7.3Hz,1H),7.17(s,1H),4.69–4.64(m,4H),4.00(d,J=6.4Hz,6H),2.49(t,J=2.5Hz,1H). 13C NMR (101MHz, CDCl3) δ196.58,173.34,154.91,152.75,150.62,146.68,109.21,1 08.64,105.00,95.70,79.38,74.05,73.56,56.22,36.34.HR-MS(ESI)m / z:calcd for C 16 H 14 N2O4[M+H] + :299.1026,found:299.1047.

[0175] Compound 101, White amorphous powder, 73% yield; 1 H NMR (400MHz, CDCl3) δ7.16 (s, 1H), 7.06 (s, 1H), 4.55 (s, 2H), 3.91 (d, J = 18.5Hz ,6H),3.71(t,J=4.5Hz,4H),2.84(s,4H),1.77–1.62(m,1H),0.50–0.42(m,4H). 13 CNMR(101MHz,CDCl3)δ193.72,174.09,156.01,154.69,150.05,146.69,113.98,107.98 ,106.01,101.37,73.32,56.22,56.03,54.15,53.36,38.51,5.98.HR-MS(ESI)m / z:calcd for C 20 H 23 N3O4[M+H] + :370.1761,found:370.1773.

[0176] Compound 10m, White amorphous powder, 78% yield; 1 H NMR (400MHz, CDCl3) δ7.18(d,J=8.6Hz,1H),7.07–6.98(m,1H),4.60–4.51(m,2H),4.01–3.88(m,6H),3.82(s,4H),2.83(s,4H),1.15–1.07(m,9H). 13C NMR (101MHz, CDCl3) δ193.57,174.15,155.65,154.55,150.06,146.41,113.65,107. 90,106.39,100.79,73.26,56.16,56.01,54.13,47.01,25.84.HR-MS(ESI)m / z:calcd for C 21 H 27 N3O4[M+H] + :386.2074,found:386.2079.

[0177] Compound 10n, White amorphous powder, 97% yield; 1 H NMR (400MHz, CDCl3) δ7.22(s,1H),7.09(s,1H),4.56(s,2H),3.94(s,3H),3.90(s,3H),3.38(s,6H). 13 CNMR(126MHz, CDCl3)δ193.46,174.19,156.55,154.43,150.04,146.63,113.62,108.16,106.61,100.38,73.31,56.11,45.69.HR-MS(ESI)m / z:calcd for C 15 H 16 N2O4[M+H] + :289.1183,found:289.1204.

[0178] Compound 10o, White amorphous powder, 77% yield; 1 H NMR (500MHz, CDCl3) δ7.29(s,1H),7.18(s,1H),4.66(s,2H),4.04(s,3H),3.98(s,3H),3.87(dd,J=11.7,6.9Hz,4H),2.73(s,4H),2.45(s,3H). 13 C NMR (101MHz, CDCl3) δ193.72,174.02,155.88,154.69,150.04,146.67,113.93,107. 94,105.97,101.38,73.29,56.20,56.01,55.91,53.23,46.27.HR-MS(ESI)m / z:calcd for C 18 H 21N3O4[M+H] + :344.1605,found:344.1623.

[0179] Compound 10p, White amorphous powder, 81% yield; 1 H NMR (400MHz, CDCl3) δ7.22(s,1H),7.18(s,1H),4.68(s,2H),4.01(s,3H),3.95(s,3H),1.61(s,6H),1.47(s,9H). 13 C NMR (101MHz, CDCl3) δ196.44,173.57,154.87,154.37,152.15,146.75,109.09,108.9 4,104.51,95.37,80.12,73.55,56.22,56.18,29.70,28.45.HR-MS(ESI)m / z:calcdfor C 22 H 27 N3O6[M+H] + :430.1973,found:430.1987.

[0180] Compound 10q, White amorphous powder, 81% yield; 1 H NMR (400MHz, CDCl3) δ7.22(s,1H),7.17(s,1H),4.67(s,2H),4.01(s,3H),3.95(s,3H),1.62(s,5H),1.47(s,10H). 13 C NMR (101MHz, CDCl3) δ196.44,173.57,154.87,154.37,152.15,146.75,109.09,108. 94,104.51,95.37,80.12,73.55,56.22,56.18,29.70,28.45.HR-MS(ESI)m / z:calcd for C 22 H 27 N3O6[M+H] + :430.1973,found:430.1987.

[0181] Compound 11a, White amorphous powder, 65% yield; 1H NMR(500MHz, CDCl3)δ7.23(s,1H),7.11(s,1H),6.33(s,1H),5.60(d,J=5.3Hz,1H),5.30(s,1H),4.70–4.58(m,2H ),4.50(dd,J=9.9,5.8Hz,,1H),4.21–4.11(m,1H),3.97(d,J=3.0Hz,6H),3.32(s,3H),2.94(s,3H),2.88(s,3H). 13 C NMR (101MHz, CDCl3) δ169.88,167.41,153.03,152.10,146.76,146.02,114.25,111.29, 108.03,103.86,69.37,56.20,56.02,55.93,40.36,36.19,35.85.HR-MS(ESI)m / z:calcd for C 18 H 23 N3O5[M+H] + :362.1710,found:362.1715.

[0182] Compound 11b, White amorphous powder, 73% yield; 1 H NMR (400MHz, CDCl3) δ7.10(s,1H),7.05–6.98(m,1H),5.41(dt,J=11.8,5.9Hz,1H),4.40(t,J=7.3Hz,2H),3.89(s,3H),3.86 (s,3H),3.54(dd,J=18.1,13.6Hz,2H),3.22–3.07(m,2H),2.73(dt,J=13.0,6.5Hz,4H),1.18(s,1H),1.06(d,J=6.5Hz,6H). 13 C NMR (101MHz, CDCl3) δ166.31,154.13,151.24,145.72,144.37,114.60,109.43,106. 30,102.63,68.83,55.01,54.86,53.82,48.04,17.46,17.39.HR-MS(ESI)m / z:calcd for C 20 H 27 N3O4[M+H] + :374.2074,found:374.2086.

[0183] Compound 11c, White amorphous powder, 56% yield; 1 H NMR (400MHz, CDCl3) δ7.14(s,1H),7.05–6.94(m,1H),5.38(d,J=5.5Hz,1H),4.49–4.35(m,1H),4.29(dd,J=10.4, 1.2Hz,1H),3.88(s,3H),3.87(s,3H),3.51–3.32(m,5H),2.35–2.24(m,2H),2.20(s,1H),1.38(d,J=4..3Hz,9H). 13 C NMR (101MHz, CDCl3) δ166.05,154.23,153.75,151.44,146.02,144.29,114.72,109.98, 106.16,102.19,79.18,78.76,68.77,55.05,54.78,53.66,27.37.HR-MS(ESI)m / z:calcd for C 22 H 29 N3O6[M+H] + :432.2129,found:432.2134.

[0184] Compound 11d, White amorphous powder, 60% yield; 1 H NMR (500MHz, CDCl3) δ6.89(s,1H),6..34(s,1H),5.30(d,J=5.5Hz,1H),4.54–4.44(m,3H),3.88( s,3H),3.81(s,3H),3..62(dd,J=13.4,6.6Hz,2H),1.66(d,J=7.3Hz,2H),0.98(t,J=7.4Hz,3H). 13 C NMR(126MHz, CDCl3)δ167.58,151.53,147.89,146.00,144.21,110.04,107.85,99.60,97.11,70.70,56.01,45.63,23.70,11.30.HR-MS(ESI)m / z:calcd for C 16 H 20 N2O4[M+H] + :305.1496,found:305.1503.

[0185] Compound 11e, White amorphous powder, 38% yield; 1 H NMR (500MHz, CDCl3) δ7.03 (s, 1H), 6..85 (s, 1H), 5.47 (t, J = 3.7Hz, 1H), 4.51 (t, J = 5. 1Hz,2H),4.25–4.18(m,1H),3.94(d,J=14.7Hz,6H),2.81–2.70(m,4H),2.34(s,6H). 13 C NMR (126MHz, CDCl3) δ167.50,151.82,148.46,146.25,144.42,110.64,107.67, 100.55,98.09,70.30,58.11,55.97,55.90,44.97,40.91.HR-MS(ESI)m / z:calcd for C 17 H 23 N3O4[M+H] + :334.1761,found:334.1766.

[0186] Compound 11f, White amorphous powder, 88% yield; 1 H NMR(400MHz, CDCl3) δ6.83(d,J=9.6Hz,1H),6.35(s,1H),5.17(dd,J=5.8,2.0Hz,1H),4.52–4.40(m,3 H),4.31(s,1H),3.87(d,J=9.1Hz,3H),3.77(d,J=16.4Hz,3H),1.38–1.29(m,3H),1.15–1.06(m,3H). 13 C NMR (101MHz, CDCl3) δ167.18,151.38,147.25,145.89,143.67,110.29,107.46, 99.74,97.34,70.83,56.06,55.91,44.26,23.87,23.73.HR-MS(ESI)m / z:calcd for C 16 H 20 N2O4[M+H] + :305.1496,found:305.1497.

[0187] Compound 11g, White amorphous powder, 86% yield; 1H NMR (400MHz, CDCl3) δ6.87(s,1H),6.72–6.65(m,1H),5.78–5.71(m,1H),,4.61–4.52(m,2H),3.97(d,J=14.7Hz,3H),3.8 6(s,3H),3.66(d,J=11..2Hz,2H),3.43–3.29(m,1H),1.06–0.95(m,1H),0.88(td,J=10.1,5.8Hz,1H),0.79–0.60(m,2H). 13 CNMR (101MHz, CDCl3) δ167.41,151.35,149.37,145.66,143.46,110.11,106.47,100.6 9,97.94,97.90,70.28,55.80,55.70,25.69,10.66,7.86,7.81.HR-MS(ESI)m / z:calcd for C 16 H 18 N2O4[M+H] + :303.1339,found:303.1359.

[0188] Compound 11h, White amorphous powder, 73% yield; 1 H NMR(400MHz, CDCl3)δ7.15(s,1H),7.03(d,J=8.6Hz,1H),5.39(d,J=5.5Hz,1H),4.48–4 .38(m,1H),4.37–4.29(m,1H),3.87(t,J=15.8Hz,10H),3.51(s,2H),3.15–3.03(m,2H). 13 C NMR (101MHz, CDCl3) δ167.09,155.11,152.53,147.04,145.50,115.59,110.51,107.32,103.40,69.98,67.23,56.09,55.85.HR-MS(ESI)m / z:calcd for C 17 H 18 N2O5[M+H] + :333.1445,found:333.1459.

[0189] Compound 11i, White amorphous powder, 59% yield; 1H NMR (500MHz, CDCl3) δ6.91(d,J=8.5Hz,1H),6.79(t,J=6.8Hz,1H),5.43–5.33(m,1H),4.45–4.38(m,2H),3.99(dd,J=12.5,9. 0Hz,1H),3.91–3.81(m,6H),3.65(dd,J=18.4,5.3Hz,1H),1.28(dd,J=20.0,1.7Hz,1H),1.25–1.14(m,4H),0.96–0.79(m,6H). 13 C NMR (101MHz, CDCl3) δ167.45,152.14,148.95,146.42,143.57,110.33,106.65,100.86,96.42, 76.46,69.99,62.78,59.23,56.14,55.78,37.82,25.75,14.95,11.80.HR-MS(ESI)m / z::calcd forC 19 H 26 N2O5[M+H] + :363.1914,found:363.1929.

[0190] Example 2

[0191] Evaluation of the anti-renal fibrosis activity of benzofurano[2,3-b]quinoline derivatives in vitro

[0192] The NRK-49F cell line, a fibroblast-like cell line, is widely used to study myofibroblast differentiation and extracellular matrix (ECM) deposition. Compared to primary fibroblasts, NRK-49F cells exhibit greater proliferation stability and experimental reproducibility, making them particularly suitable for high-throughput drug screening and signaling pathway research.

[0193] NRK-49F cells were cultured in DMEM complete medium (containing 10% FBS and 1% double antibody), and HK-2 cells were cultured in DMEM / F12 complete medium (containing 10% FBS and 1% double antibody).

[0194] (1) Cell recovery

[0195] Preparation: Wipe the clean bench with 75% ethanol and sterilize all consumables and reagents with 75% ethanol before placing them inside the clean bench. Irradiate with UV light for 30 minutes, then wipe the bench again with 75% ethanol. Thawing: Remove the frozen cells from the liquid nitrogen tank and quickly thaw them in a 37°C water bath. Centrifugation: Prepare complete culture medium and transfer 3 mL to a 15 mL centrifuge tube. Disinfect the surface of the thawed cryovial and place it in the clean bench. Transfer the cryovial to a centrifuge tube containing complete culture medium and centrifuge at 800 rpm for 3 minutes. Cell Culture: Discard the supernatant and resuspend the cell pellet in 1 mL of complete culture medium. Transfer the cell suspension to a 100 mm cell culture dish and add 9 mL of complete culture medium. Label the cells with the person, date, cell name, and passage number. Gently cross-mix the cells and culture them in a 37°C, 5% CO2 incubator.

[0196] (2) Cell passage

[0197] Preparation: Passage is performed when the cell density reaches 80%-90%. Disinfect the required consumables and reagents with 75% ethanol and place them in a clean bench, and wipe the table with a 75% ethanol cotton ball. Cell digestion: Discard the original culture medium and wash the cells three times with 3mL of sterile PBS. Add 3mL of 0.05% trypsin to digest the adherent cells. After observing under a microscope until the intercellular connections disappear and the cell morphology becomes round, discard the trypsin and add 3mL of complete culture medium to stop digestion. Cell packaging and culture: Gently blow the cells until they are suspended, take 1mL of cell suspension and add it to three new 100mm culture dishes, and add 9mL of complete culture medium to each dish. After gently cross-mixing, place in a 37°C, 5% CO2 incubator for continued culture.

[0198] (3) CCK-8 experiment

[0199] Cell plating: After counting, NRK-49F and HK-2 cells were seeded at a density of 4,000 cells / well (100 μL / well) in 96-well plates and incubated in a 37°C, 5% CO2 incubator. Drug treatment: When the cell density reached 50%-60%, the synthetic compound dissolved in DMSO was diluted with complete culture medium to prepare drug-containing culture medium at concentrations of 10, 20, 40, 80, 160, and 320 μmol / L. The drug-containing culture medium was filtered through a 0.22 μm filter and set aside. The supernatant in the 96-well plate was discarded, and 100 μL of drug-containing culture medium was added to each well and incubated in an incubator for 24 hours. CCK8 assay: The supernatant in the cell plate was discarded and the cells were washed twice with sterile PBS. Under dark conditions, a working solution of culture medium containing 10% CCK-8 reagent (without FBS) was prepared and 100 μL was added to each well and incubated in an incubator for 2 hours. Absorbance was measured at 450 nm using a microplate reader. Calculate cell viability as follows: Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100. Where As is the absorbance of the experimental well, Ab is the absorbance of the blank well, and Ac is the absorbance of the control well. The results are shown in the table below.

[0200] Table 4 IC50 values of compounds against NRK-49F cells

[0201]

[0202]

[0203] Note: - indicates no activity, and IC50 values are the average of three repeated experiments (n=3).

[0204] The in vitro antiproliferative activity of benzofurano[2,3-b]quinoline derivatives against rat renal fibroblast NRK-49F cells was determined using the CCK-8 assay. As shown in Table 4, the benzofurano[2,3-b]quinoline derivatives of the present invention exhibited a moderate inhibitory effect on NRK-49F cell proliferation, significantly outperforming fennel and meflunisal. Compounds 7a, 10a-b, 10e-g, 10o, 11a-d, and 11h all exhibited IC50 values below 100 μM. Compounds 10b (IC50 = 20.35 μM), 10e (IC50 = 29.14 μM), 10g (IC50 = 20.74 μM), 11a (IC50 = 13.8 μM), and 11b (IC50 = 21.66 μM) exhibited particularly significant activity.

[0205] Example 3

[0206] In vivo anti-renal fibrosis activity and mechanism of benzofurano[2,3-b]quinoline derivatives

[0207] 1. Animal acute toxicity test method

[0208] All animal experiments complied with international ethical standards and were approved by the Animal Ethics Committee of Central South University.

[0209] (1) Experimental animal grouping: Four-week-old KM mice were randomly divided into five groups, with six mice in each group, half male and half female: control group (0.5% CMC-Na + saline), 10e group (2000 mg / kg 10e + 0.5% CMC-Na + saline), 10o group (2000 mg / kg 10o + 0.5% CMC-Na + saline), 11a group (2000 mg / kg 11a + 0.5% CMC-Na + saline) and 11d group (2000 mg / kg 11d + 0.5% CMC-Na + saline).

[0210] (2) Drug administration process, data, and specimen collection: 4-week-old KM mice were fed adaptively for 5 days and housed in an experimental environment with a 12-hour light-dark cycle, a temperature of 25±2°C, and a relative humidity of 30% to 60% (24th floor, Science and Education Building, Xiangya Hospital, Central South University). A maximum dose single administration method was used. After 5 days of pre-adaptation, the mice were fasted overnight and given the drug suspension by gavage in the morning of the 6th day at a dose of 10 mL / kg. After administration, the mice were closely observed for 8 hours, and then observed once a day in the morning and evening for 14 days. The observation time was adjusted according to the toxic reaction. The time of appearance and disappearance of various toxic reactions of each animal was recorded in detail. The observation content included skin, mucous membranes, coat color, eyes, respiration, circulation, autonomic activity, and central nervous system behavioral manifestations, and the time of death of the animals was accurately recorded. The animals were weighed before and after administration, 1 week after the animal died, and at the end of the experiment. All animals (including dead or sacrificed animals) were autopsied and abnormal organs were recorded. Heart, liver, lung, kidney, and spleen were dissected and weighed, and the relative organ mass was calculated and histopathological examination was performed. All organs were rinsed with ice-cold saline, fixed with 10% formalin, and stained with HE for pathological examination (reference Zheng B, Yuan M, Wang S, et al. Fraxinellone alleviates kidney fibrosis by inhibiting CUG-binding protein 1-mediated fibroblast activation [J]. Toxicol Appl Pharmacol, 2021, 420: 115530.).

[0211] The results are as follows Figure 1 A. Figure 1As shown in Figure B, after oral administration of 2000 mg / kg to healthy KM mice for 14 days at 10e, 10o, 11a, and 11d, the average daily food intake of each mouse remained essentially unchanged, while body weight increased daily. Notably, a statistically significant difference was observed between the 10o and 11d administration groups (P < 0.05).

[0212] It can be seen that the compounds of the present invention have basically no acute toxicity. Subsequently, compound 11d (with better activity, but not the best) was selected for subsequent animal model studies.

[0213] 2 Animal experiments on adenine-induced renal fibrosis

[0214] (1) Grouping: 7-week-old male C57BL / 6J mice were randomly divided into six experimental groups, with 8 mice in each group.

[0215] Control group: fed with normal feed and given 0.5% CMC-Na + saline; model group: fed with adenine feed and given 0.5% CMC-Na + saline; positive drug group (allopurinol: 20 mg / kd / d): fed with adenine feed and given 20 mg / kg allopurinol + 0.5% CMC-Na + saline.

[0216] Based on previous studies on vanilla, the dosages adopted in the present invention are: low-dose group (10 mg / kg / d): fed with adenine feed, and given 10 mg / kg for 11 days + 0.5% CMC-Na + saline; medium-dose group (20 mg / kg / d): fed with adenine feed, and given 20 mg / kg for 11 days + 0.5% CMC-Na + saline; high-dose group (40 mg / kg / d): fed with adenine feed, and given 40 mg / kg for 11 days + 0.5% CMC-Na + saline;

[0217] (2) Administration and sampling: After 7 days of adaptive feeding, 7-week-old male C57BL / 6J mice were fed adenine feed and gavage at the same time, with a volume of 10 mL / kg, for 21 days, with administration at a fixed time every day. Daily food intake was recorded, and body weight changes were monitored every 3 days. The day after the last administration, the mice were weighed and anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). After the skin pinch reflex disappeared, blood was collected from the eyeballs and blood samples were collected. The mice were then fixed, the skin was cut open along the midline of the abdomen to expose the abdominal cavity, and the diaphragm was cut open to expose the chest cavity. 10 mL of normal saline was perfused from the right ventricle until the liver and kidney turned white, the kidneys were separated, and the capsule was removed. The left kidney was cut in half longitudinally, one half was fixed in 4% paraformaldehyde, and the other half and the cortex of the intact right kidney were separated and frozen in liquid nitrogen for subsequent testing. The blood samples were allowed to stand at 37°C for 30 minutes, centrifuged at 4°C and 3000 rpm for 20 minutes, and the supernatant was collected for testing of renal function indicators.

[0218] The experimental methods involved are as follows:

[0219] (1) Tissue protein extraction: All experimental procedures were performed on ice.

[0220] Tissue processing: Cut kidney tissue into small fragments (approximately 20 mg). Lysis: Add 150-250 μL of lysis buffer (protein lysis buffer (strong): phosphatase inhibitor A (PA): PB: protease inhibitor = 100:1:1:1) to every 20 mg of tissue. Disruption and centrifugation: Disrupt the tissue using a tissue disruptor, then centrifuge at 4°C, 12,000 rpm for 20 minutes. Aspirate the supernatant. Protein dilution: Dilute the supernatant to an appropriate concentration (usually 10- or 20-fold) for subsequent protein concentration determination.

[0221] (2) Protein concentration determination:

[0222] Dilute the 2 mg / mL protein standard from the Thermo BCA kit with PBS to 0, 0.5, 0.75, 1, 1.5, and 2 mg / mL. Prepare BCA working solution (Solution A:Solution B) at a 50:1 ratio. Add 200 μL of working solution to each well of a 96-well microtiter plate, add 10 μL of protein standard dilution solution to each of 6 wells, and add 10 μL of the protein sample to be tested to the remaining wells. Incubate at 37°C for 30 min. Measure absorbance at 562 nm using a microplate reader. Draw a standard curve (Y = KX + B) and calculate the concentration of the protein to be tested.

[0223] (3) Sample loading system configuration: Calculate the required protein volume based on the protein concentration and sample loading amount, and prepare a sample loading system of the same concentration (1 / 5 loading buffer (5×SDS) plus 4 / 5 protein lysis buffer). Centrifuge for 1 min, heat in a 100°C dry heat apparatus for 5 min, and store at -20°C until use.

[0224] (4) Western Blot Experiment Process:

[0225] Gel Preparation: Wash the glass plates, rinse with distilled water, and air dry. Align the bottoms of the two glass plates and mount them on a gel preparation rack. Prepare a lower layer of gel according to the 1.5mm formula. After adding it, lightly cover it with 1-2mL of ethanol and let it sit for about 15 minutes until the lower layer of gel solidifies. Invert the plate, absorb the ethanol, add the upper layer of gel, and immediately insert the comb. Electrophoresis: After the gel solidifies, pour in fresh electrophoresis buffer, remove the comb, and add the marker and protein sample. Connect the electrophoresis tank according to the positive and negative poles. Set the initial voltage to 80V. After the marker is clearly separated, adjust it to 120V until the target protein band reaches the bottom of the separation gel. Transfer: Prepare 1× transfer buffer and pre-cool it to -20°C. Cut a PVDF membrane (8.5cm × 5.5cm) and activate it with methanol. Place the following on the transfer electrode plate in the following order: transparent electrode plate - sponge - filter paper - membrane - gel - filter paper - sponge - black electrode plate, avoiding bubbles. Pour pre-chilled transfer buffer into the membrane, confirm the positive and negative electrodes are connected, place the membrane in an ice box, and transfer at a constant current of 260mA for 90 minutes. Antibody Incubation: After transfer, place the PVDF membrane in an incubation box and add blocking solution (1g nonfat dry milk dissolved in 20mL TBST). Block the membrane with slow shaking at room temperature for 1 hour. Wash the membrane three times with TBST at room temperature, each for 10 minutes with rapid shaking. Cut the target protein band according to the marker, add the diluted primary antibody solution, and incubate at 4°C with slow shaking overnight. Recover the primary antibody, wash three times with TBST, add the diluted secondary antibody solution, and slowly shake at room temperature for 1 hour. Wash three times with TBST. Development: Mix equal volumes of ECL luminescent solution A and solution B, preparing the membrane immediately before use. Apply the developer solution evenly to the PVDF membrane, develop the membrane in a developer, and save the image. Analyze the relative grayscale values of the protein bands using Image J software. Antibody Removal and Reincubation: After development, rinse with distilled water for 5 minutes. Add sufficient Western primary and secondary antibody removal buffer (strongly alkaline) and rinse on a shaker for 5 minutes. Discard the removal buffer, rinse with distilled water, and then re-block and proceed with subsequent operations.

[0226] The results showed that after oral administration of 10, 20 and 40 mg / kg of adenine for 11 days and 21 days, the serum creatinine and urea nitrogen levels of mice in the 0.2% adenine diet model were compared. Figure 2 As shown in A and 2B, compared with the control group, the serum creatinine and urea nitrogen levels of the model group mice were significantly increased (P < 0.001). After 11 days of treatment with 20 mg / kg, the serum creatinine and urea nitrogen of the drug-treated group were significantly decreased (P < 0.05).

[0227] The HE-stained sections of the left kidney of mice were observed under an optical microscope to evaluate the degree of tubulointerstitial damage and tubulointerstitial fibrosis in response to the adenine diet. Figure 3As shown: There was no obvious pathological change in the renal tissue of the mice in the normal group, while the renal tissue of the mice in the model group showed obvious tubular damage, including obvious sclerosis and reduction of the kidney, uneven surface, and granular changes; diffuse infiltration of inflammatory cells in the renal interstitium, proliferation of interstitial fibrous tissue, and fibrosis formation; most of the glomeruli underwent sclerosis and hyaline changes, capillary destruction, and mesangial matrix proliferation; due to the obstruction of glomerular blood flow, the renal tubules atrophied, and in mild cases, the residual kidneys increased compensatorily. This also shows that the model was successful. After 11 days of treatment, the renal lesions in mice were alleviated. The tubular damage score was performed on the HE-stained sections, as shown below: Figure 4 As shown in Figure A, compared with the normal group, the renal injury score in the adenine model group was significantly increased (P<0.001). After 11 days of treatment with allopurinol in the adenine model, allopurinol at 10 mg / kg, 20 mg / kg, 40 mg / kg, and 20 mg / kg for 11 days all reduced the renal tubular injury score (P<0.05). Among them, the 20 mg / kg dose for 11 days had the best effect, which was comparable to allopurinol.

[0228] Masson-stained sections of the left kidney of mice were observed under an optical microscope to assess the degree of tubulointerstitial fibrosis induced by adenine diet. Figure 3 As shown. No obvious collagen deposition was observed in the renal tubular spaces of mice in the normal group, while collagen deposition in the renal tubular spaces of mice in the model group was severe. After 11 days of treatment, the renal tubular collagen deposition in mice was partially alleviated. The degree of renal fibrosis was scored on Masson-stained sections. Figure 4 As shown in Figure B: Compared with the normal group, the renal fibrosis score in the model group was significantly increased (P<0.001). In the adenine model, after 11 days of treatment with allopurinol, 10, 20, and 40 mg / kg for 11 days and allopurinol all reduced the renal fibrosis score (P<0.05), with the 20 mg / kg dose for 11 days having the best effect, comparable to allopurinol.

[0229] The expression levels of FN, CollagenⅠ, Vn and α-SMA proteins in renal tissue were detected by Western blot. Figure 5 and Figure 6 As shown: In the adenine model, compared with the control group, the expression of FN, CollagenⅠ, Vn and α-SMA in the kidneys of the model group mice was significantly upregulated (P<0.001). Compared with the model group, the expression of FN, CollagenⅠ, Vn and α-SMA proteins was significantly downregulated after 11 days of treatment (FN: P<0.05; CollagenⅠ: P<0.01; Vn: P<0.001; α-SMA: P<0.01), among which the 20 mg / kg concentration had the best effect (P<0.001) ( Figure 6 A. Figure 6 B. Figure 6 C. Figure 6 D).

[0230] 3UUO surgery-induced renal fibrosis animal experiment

[0231] (1) Experimental animal grouping: 7-week-old male C57BL / 6J mice were randomly divided into 6 groups, with 8 mice in each group:

[0232] Control group: Only the kidney was exposed without ureteral ligation, and 0.5% CMC-Na+ saline was administered. Model group: The left ureter was ligated and 0.5% CMC-Na+ saline was administered. Positive drug group (losartan: 20 mg / kg / day): The left ureter was ligated and 20 mg / kg of losartan plus 0.5% CMC-Na+ saline was administered. Low-dose group (10 mg / kg / day): The left ureter was ligated and 10 mg / kg was administered for 11 days plus 0.5% CMC-Na+ saline. Medium-dose group (20 mg / kg / day): The left ureter was ligated and 20 mg / kg was administered for 11 days plus 0.5% CMC-Na+ saline. High-dose group (40 mg / kg / day): The left ureter was ligated and 40 mg / kg was administered for 11 days plus 0.5% CMC-Na+ saline.

[0233] (2) Model construction: All instruments were sterilized by high pressure before the experiment. The mice were weighed after one week of adaptive feeding and anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). After the skin reflex disappeared, the skin was prepared and the left back was disinfected. The mice were fixed in a prone position, and a longitudinal incision of about 1.5 cm was made on the left side of the midline of the abdomen. The skin and muscles were cut in sequence to enter the abdominal cavity. The ureters were separated and double-ligated with 4.0 sutures at the proximal and distal ends of the renal pelvis, and the ureters were cut in the middle of the two ligatures. The kidneys were repositioned, the abdominal cavity was flushed with 1 mL of gentamicin, the muscles and skin were sutured layer by layer, and finally disinfected with iodine tincture for 3 consecutive days. Pay attention to protecting the kidneys during the operation. In the sham operation group, only the kidneys were exposed, and no ureteral ligation or disconnection was performed.

[0234] (3) Dosing and specimen collection: On the day after surgery, administer the drug by gavage as described above, at a dose of 10 mL / kg, once a day for 14 days, at a fixed time each day. The day after the last dose, weigh the rats and administer anesthesia with an intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). After the skin reflex disappears, blood is collected from the eyeballs. Subsequently, the abdominal and thoracic cavities are opened, and 10 mL of normal saline is perfused from the right ventricle until the liver and kidneys become pale. Separate the left kidney, remove the capsule, cut it in half along the midline, fix one half in 4% paraformaldehyde, and separate the cortex of the other half and freeze it in liquid nitrogen for subsequent testing. The blood sample processing method is the same as 2.

[0235] After left ureteral ligation, UUO mice were orally administered 10, 20, and 40 mg / kg for 11 days and 14 days later. The results showed that the degree of tubulointerstitial damage and tubulointerstitial fibrosis in UUO-induced renal fibrosis mice were evaluated by HE and Masson staining sections of the left kidney under an optical microscope. Figure 7 As shown in the figure: There was no obvious pathological change in the renal tissue of the mice in the sham operation group, while the renal tissue of the mice in the model group showed obvious tubular damage, including severe dilation and atrophy of the cortical tubules, swelling and vacuolar degeneration of the renal tubular epithelial cells, and increased infiltration of interstitial inflammatory cells. This also shows that the model was successful. After 11 days of treatment, the renal lesions of the mice were partially alleviated. The renal tubular damage score was performed on the HE-stained sections. Figure 8 As shown in Figure A, compared with the sham operation group, the renal injury score in the UUO model group was significantly increased (P<0.001). After 11 days of treatment with losartan in the UUO model, 10, 20, 40 mg / kg for 11 days and 20 mg / kg of losartan all reduced the renal tubular injury score (P<0.001), with 20 mg / kg for 11 days having the best effect, comparable to losartan.

[0236] Masson-stained sections of the left kidney of mice were observed under an optical microscope to assess the degree of tubulointerstitial fibrosis induced by adenine diet. Figure 7 As shown in the figure, no obvious collagen deposition was observed in the renal tubular spaces of mice in the normal group, while collagen deposition in the renal tubular spaces of mice in the model group was severe. 11 days of treatment partially alleviated the renal tubular collagen deposition in mice. The degree of renal fibrosis was scored on Masson-stained sections. Figure 8 As shown in Figure B, compared with the sham operation group, the renal fibrosis score in the UUO model group was significantly increased (P < 0.001). After treatment with 11 days of losartan and 10, 20, and 40 mg / kg of 11 days of losartan in the UUO model, the renal fibrosis score was reduced (P < 0.001), with 20 mg / kg of 11 days of treatment having the best effect, comparable to losartan.

[0237] The expression levels of FN, CollagenⅠ, Vn and α-SMA proteins in renal tissue were detected by Western blot. Figure 9 and Figure 10As shown: In the UUO model, compared with the control group, the expression of FN, CollagenⅠ, Vn and α-SMA in the kidneys of the model group mice was significantly upregulated (P<0.001). Compared with the model group, the expression of FN, CollagenⅠ, Vn and α-SMA proteins was significantly downregulated after 11 days of treatment (FN: P<0.05; CollagenⅠ: P<0.01; Vn: P<0.01; α-SMA: P<0.05), among which the 20 mg / kg concentration had the best effect (P<0.001) ( Figure 10 A. Figure 10 B. Figure 10 C. Figure 10 D).

[0238] Experimental data show that the compounds of the present invention are essentially non-toxic. In-depth studies of the active mechanism of action have shown that the compounds exert their anti-renal fibrosis effects through a dual regulatory pathway: on the one hand, they inhibit TGF-β / Smad2 / Smad3 signal transduction, and on the other hand, they block PI3K / AKT pathway activation. This synergistic effect has been verified in two animal models of renal fibrosis, adenine-induced and unilateral ureteral ligation (UUO). Notably, the degree of fibrosis improvement observed in the UUO model was significantly better than that in the adenine model (P < 0.05), suggesting that the compounds of the present invention have potential application value in the treatment of obstructive nephropathy.

[0239] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. A benzofurano[2,3-b]quinoline derivative, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, wherein the structure is shown in formula (I): wherein R1 is selected from the group consisting of: C1-C8 alkoxy, halogen, 3-8 membered aliphatic heterocycle, substituted 3-8 membered aliphatic heterocycle, -NR3R4; R2 is selected from: =O, hydroxy, hydrogen; The substituents for the 3-8 membered aliphatic heterocyclic ring are selected from the group consisting of: C1-C8 alkyl, C1-C8 alkyl ester, =O, and 3-8 membered aliphatic ring; R3 and R4 are each independently selected from: hydrogen, C1-C8 alkyl, 3-8 membered aliphatic ring, When R2 is a hydroxyl group, R1 is not 2. The derivative according to claim 1, characterized in that 3-8 membered aliphatic heterocycles include: oxirane, aziridine, thiirane, oxetane, azetidine, thietane, tetrahydrofuran, tetrahydropyrrole, tetrahydrothiophene, tetrahydropyran, piperidine, tetrahydrothiopyran, dioxane, piperazine, hexahydropyrazine, morpholine, and thiophene.

3. The derivative according to claim 1, characterized in that The substituted 3-8 membered aliphatic heterocycle is substituted piperazine.

4. The derivative according to claim 1, characterized in that R1 is selected from C1-C3 alkoxy, fluorine, chlorine, bromine, iodine, 5. The derivative according to claim 1, characterized in that The benzofuran[2,3-b]quinoline derivatives, their optical isomers or their pharmaceutically acceptable salts have the following structural formula:

6. The method for preparing the derivative according to any one of claims 1 to 5, characterized in that: When R2 is =0, the process comprises the following steps: S1, using 3,4-dimethoxyaniline as the starting material, and obtaining intermediate 1 through acetal substitution reaction; S2, intermediate 1 undergoes cyclization reaction in a high temperature solvent to obtain intermediate 2; S3 and intermediate 2 undergo chlorination reaction to generate intermediate 3; S4, intermediate 3 is substituted to obtain the benzofuran[2,3-b]quinoline derivative, its optical isomer or pharmaceutically acceptable salt thereof; The structure of intermediate 1 is: The structure of intermediate 2 is: The structure of intermediate 3 is:

7. The method for preparing the derivative according to any one of claims 1 to 5, characterized in that: When R2 is a hydroxyl group, the method comprises the following steps: S1, using 3,4-dimethoxyaniline as the starting material, and obtaining intermediate 1 through acetal substitution reaction; S2, intermediate 1 undergoes cyclization reaction in a high temperature solvent to obtain intermediate 2; S3 and intermediate 2 undergo chlorination reaction to generate intermediate 3; S5, intermediate 3 is reduced to obtain intermediate 4; S6, reacting intermediate 4 to obtain the benzofuran[2,3-b]quinoline derivative, its optical isomer or its pharmaceutically acceptable salt; The structure of intermediate 1 is: The structure of intermediate 2 is: The structure of intermediate 3 is: The structure of intermediate 4 is:

8. The method for preparing the derivative according to any one of claims 1 to 5, characterized in that: When R2 is hydrogen, the method comprises the following steps: S1, using 3,4-dimethoxyaniline as the starting material, and obtaining intermediate 1 through acetal substitution reaction; S2, intermediate 1 undergoes cyclization reaction in a high temperature solvent to obtain intermediate 2; S3 and intermediate 2 undergo chlorination reaction to generate intermediate 3; S7, performing a reduction reaction on the intermediate 3 to obtain the benzofuran[2,3-b]quinoline derivative, its optical isomer or a pharmaceutically acceptable salt thereof; The structure of intermediate 1 is: The structure of intermediate 2 is: The structure of intermediate 3 is:

9. A benzofurano[2,3-b]quinoline derivative, an optical isomer thereof or a pharmaceutically acceptable salt thereof, characterized in that: Its structure is shown in formula (II): Wherein R5 is selected from: C3-C6 alkoxy, 10. Use of the derivative according to any one of claims 1 to 5 or claim 9, or an optical isomer thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating chronic kidney disease, preferably, the chronic kidney disease is renal fibrosis.

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