Hydroxyfluorene compound and application thereof

Abstract

The invention discloses a hydroxyfluorene compound (formula I) and application thereof, the compound can effectively inhibit a PD-1 / PD-L1 signal channel, an anti-tumor effect is achieved through an immunosuppression effect, the biological activity on the molecular level and the cellular level is remarkably superior to that of an existing medicine, and the hydroxyfluorene compound has a clinical application prospect. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a hydroxyfluorene compound and application thereof, in particular to a hydroxyfluorene compound with anti-tumor activity and application thereof. Background Art

[0002] PD-1 (programmed death protein 1) is an important receptor in the immune checkpoint. It mainly inhibits the immune response of T cells by binding to its ligand PD-L1, preventing the immune system from attacking normal tissues. In the tumor immune escape mechanism, tumor cells suppress the immune activity of T cells by overexpressing PD-L1, thereby promoting tumor growth and metastasis. Abnormal activation of the PD-1 / PD-L1 signaling pathway has been shown to be closely related to the occurrence and development of various cancers. Therefore, PD-1 / PD-L1 is considered a key target in cancer immunotherapy. In recent years, PD-1 / PD-L1 inhibitors, as breakthrough drugs for immunotherapy, have achieved significant therapeutic effects in various tumor types. However, existing PD-1 / PD-L1 inhibitors still have certain limitations, such as poor inhibitory effects and immune-related side effects. There is an urgent need to develop new and more effective inhibitors. Summary of the Invention

[0003] Purpose of the invention: The present invention aims to provide a hydroxyfluorene compound with PD-1 / PD-L1 inhibitory activity and its application.

[0004] Technical solution: The hydroxyfluorene compound of the present invention has a structure of formula I,

[0005]

[0006] wherein n is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0007] R is selected from hydrogen, hydroxy, carboxyl, halogen, amino, mercapto, cyano, C 1-4 Alkyl-OC(O)-, 6-10 membered aryl, 4-10 membered aromatic heterocyclic group containing 1-4 N, O, S ring heteroatoms, 4-7 membered cycloalkyl, 4-7 membered heterocyclic group containing 1-2 N, O, S ring heteroatoms, the 6-10 membered aryl, 4-10 membered aromatic heterocyclic group, 4-7 membered cycloalkyl, 4-7 membered heterocyclic group is replaced by hydrogen, -C(O)-, hydroxyl, carboxyl, halogen, amino, mercapto, cyano, nitro, C 1-4 Alkyl-OC(O)-, carboxyl C 1-4 Alkyl, hydroxyl C 1-4 Alkyl substitution.

[0008] In order to further enhance the inhibitory activity against PD-1 / PD-L1, and considering that the solubility of the compound may lead to cell penetration problems, the present invention designed and synthesized a series of cyclized terphenyl compounds. By introducing hydroxyl groups to promote hydrogen bonding interactions with the target, the solubility of the compound is enhanced, membrane permeability is improved, and physicochemical properties are improved. At the same time, it facilitates binding to the PD-1 / PD-L1 protein pocket. The introduction of hydrophilic groups of different sizes at the tail is beneficial to enhance the distribution of the drug in the target tissue, thereby enhancing immune activity and improving the anti-tumor effect of the compound.

[0009] Preferably, in the structure, n is selected from 0, 1, 2, 3, and 4.

[0010] Preferably, in the structure, R is selected from hydrogen, hydroxyl, carboxyl, halogen, amino, cyano, CH3-OC(O)-, phenyl, pyridyl, tetrahydropyrrolyl, and the phenyl, pyridyl, tetrahydropyrrolyl are substituted by hydrogen, -C(O)-, hydroxyl, carboxyl, halogen, amino, cyano, nitro, CH3-OC(O)-.

[0011] Preferably, in the structure, R is selected from any one of the following structures:

[0012]

[0013] More preferably, in the structure,

[0014] When n is 0, R is selected from

[0015]

[0016] When n is 1, R is selected from

[0017] When n is 2, 3, 4, 5, 6, 7, or 8, R is selected from -OH-COOH-COOCH3.

[0018] Preferably, the hydroxyfluorene compound is selected from any one of the following compounds:

[0019]

[0020]

[0021] The preparation method of the hydroxyfluorene compound of the present invention comprises the following steps:

[0022]

[0023] (1) Compound 1 is substituted to obtain compound 2;

[0024] (2) Compound 2 is coupled and cyclized to obtain compound 4;

[0025] (3) Compound 4 was demethylated and coupled to obtain compound 6;

[0026] (4) Compound 6 is substituted and reduced to obtain compound I;

[0027] Wherein, n and R are defined as above.

[0028] The specific method is as follows:

[0029] (1) Preparation of Compound 2

[0030] The reaction solvent is N,N-dimethylformamide. During the preparation, compound 1 and N-iodosuccinimide are dissolved in an organic solvent, palladium acetate is added, and nitrogen is protected. The reaction temperature is 100°C and the reaction time is 12 hours.

[0031] (2) Preparation of Compound 3

[0032] Deionized water was selected as the reaction solvent. During the preparation, compound 2 was dispersed in water, heated under reflux for 1 hour, and then 4-methoxyphenylboronic acid, potassium hydroxide, and palladium acetate were added. Under nitrogen protection, the reaction temperature was 100°C and condensed under reflux for 16 hours.

[0033] (3) Preparation of Compound 4

[0034] The reaction solvent is dichloromethane. During the preparation, compound 3 is dissolved in an organic solvent, and methanesulfonic acid is added. The reaction temperature is 40° C. and the reaction time is 16 hours.

[0035] (4) Preparation of Compound 5

[0036] The reaction solvent is glacial acetic acid. During the preparation, compound 4 is dissolved in an organic solvent, and a hydrobromic acid aqueous solution is added. The mixture is protected from light and refluxed at 120° C. for 12 hours.

[0037] (5) Preparation of Compound 6

[0038] The reaction solvent is tetrahydrofuran. During the preparation, compound 5 and benzo-1,4-dioxane-6-boronic acid are dissolved in an organic solvent, and potassium carbonate aqueous solution and tetrakistriphenylphosphine palladium are added. Under nitrogen protection, the mixture is condensed and refluxed at 80°C for 12 hours.

[0039] (6) Preparation of Compound 7

[0040] The reaction solvent is N,N-dimethylformamide, dimethyl sulfoxide or acetone, and the reaction is carried out under alkaline conditions. During preparation, compound 6 is dissolved in an organic solvent, a base is added, and after stirring at room temperature for 5 minutes, a halogenated hydrocarbon is added. The reaction temperature is 60-80°C and the reaction time is 12 hours.

[0041] (7) Preparation of Compound I

[0042] The reaction solvents are methanol and tetrahydrofuran. During the preparation, compound 7 is dissolved in an organic solvent, protected by nitrogen, sodium borohydride is added, and stirred at room temperature for 5 minutes.

[0043] The pharmaceutically acceptable salts of the hydroxyfluorene compounds of the present invention are salts formed by reacting the compounds with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid, an alkali metal ion base, an alkaline earth metal ion base, an aluminum ion base, a zinc ion base, choline, ammonia water, ammonia gas, ethylenediamine, triethylamine, triethanolamine, piperazine, or meglumine.

[0044] Preferably, "pharmaceutically acceptable salts" refer to salts of compounds prepared by reacting the compounds with the specified substituents with relatively nontoxic acids or bases. When the compound contains relatively acidic functional groups, base addition salts can be obtained by contacting the free form of the compound with a sufficient amount of base in neat solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When the compound contains relatively basic functional groups, acid addition salts can be obtained by contacting the free form of the compound with a sufficient amount of acid in neat solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonate or bicarbonate), phosphoric acid (forming phosphate, monohydrogen phosphate, dihydrogen phosphate), sulfuric acid (forming sulfate or bisulfate), hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid, etc.; organic acid salts also include salts of organic acids such as amino acids (such as arginine, etc.) and glucuronic acid. When certain specific compounds contain basic and acidic functional groups, they can be converted into either base or acid addition salts. Preferably, the salt is contacted with a base or acid in a conventional manner, and the parent compound is isolated to thereby regenerate the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.

[0045] "Pharmaceutically acceptable salts" can be synthesized from parent compounds containing acid or basic groups by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0046] The compounds of the present invention also include stereoisomers, tautomers, prodrugs, solvates, isotopic compounds, and crystals thereof.

[0047] Preferably, the stereoisomer is a configurational isomer produced by a chiral atom in the structure, such as an R configuration or an S configuration produced by a chiral carbon atom; when there are multiple chiral atoms in the structure, each chiral atom can produce a different configuration, thereby producing configurational isomers formed by different chiral centers.

[0048] Preferably, the tautomers are isomers formed by conjugated interconversion of double bonds in unsaturated heterocycles, including carbon-carbon double bond interconversion, carbon-heteroatom, and heteroatom-heteroatom double bond interconversion, such as tautomers formed by double bond interconversion in imidazole ring systems and pyrazole rings.

[0049] Preferably, the prodrug is an ester or amide prodrug with a carboxyl group, a hydroxyl group or an amino group introduced therein, more preferably a C1-C4 alkyl ester, a C1-C4 carboxylate or a C1-C4 alkyl amide.

[0050] Preferably, the solvate is a small molecule binding state formed by the compound and solvent molecules, more preferably a hydrate or alcoholate; the solvate can further form a salt with a corresponding acid to obtain a salt of the solvate.

[0051] Preferably, the isotope compound is a compound in which hydrogen is replaced by deuterium.

[0052] Preferably, the crystal is a specific crystal structure formed by the compound during the crystallization process, including different crystal forms of the compound itself, as well as different crystal forms of its salts, solvates, and salts of solvates.

[0053] The pharmaceutical composition of the present invention comprises the hydroxyfluorene compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0054] Preferably, the pharmaceutically acceptable carrier can be an excipient widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, so that the active ingredient dissolves at a desired rate after administration to the subject, or promotes effective absorption of the active ingredient after administration of the composition to the subject. The pharmaceutical excipient can be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipient can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners.

[0055] The pharmaceutical composition of the present invention can be prepared by any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, and lyophilizing.

[0056] The pharmaceutical compositions of the present invention can be administered in any form, including transmucosal, oral (solid and liquid formulations), inhalation, ophthalmic, rectal, topical, and parenteral (infusion, injection, implant, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical compositions of the present invention can also be in a controlled-release or sustained-release dosage form (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, hard capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or transmucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, and drops. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other formulation forms include, but are not limited to, eye drops, other ophthalmic formulations; aerosols, such as nasal sprays, inhalers; and suppositories and lozenges suitable for parenteral administration.

[0057] The hydroxyfluorene compound or pharmaceutically acceptable salt thereof and the pharmaceutical composition of the present invention are used in the preparation of drugs for PD-1 / PD-L1 inhibitors.

[0058] Preferably, the drug is an anti-tumor drug.

[0059] More preferably, the drug is a drug for immunotherapy of tumors.

[0060] More preferably, the drug is a drug for treating multiple myeloma, lung cancer, melanoma, liver cancer, kidney cancer, leukemia, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, rectal cancer, colon cancer, ovarian cancer, testicular cancer, breast cancer, bladder cancer, gallbladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastric cancer, astrocytoma, neuroblastoma, glioma, neurilemmoma or mesothelioma.

[0061] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0062] The compounds designed in the present invention can effectively inhibit the PD-1 / PD-L1 signaling pathway and achieve anti-tumor effects through immunosuppression. The compounds' inhibitory effect on the PD-1 / PD-L1 complex protein reaches the nanomolar concentration level, and their biological activity at the molecular and cellular levels is significantly better than that of existing drugs, showing promising clinical application prospects. DETAILED DESCRIPTION

[0063] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0064] Example 1: 2-Bromo-6-iodobenzoic acid (Compound 2)

[0065]

[0066] Add o-bromobenzoic acid (6.03 g, 30 mmol, 1 eq), N-iodosuccinimide (8.10 g, 36 mmol, 1.2 eq), N,N-dimethylformamide (50 mL) and palladium acetate (674 mg, 3.0 mmol, 0.1 eq) to a round-bottom flask. Heat and stir at 100°C under a nitrogen atmosphere for more than 12 hours. After the reaction is completed, cool to room temperature and dilute the reaction solution with ethyl acetate. The diluted reaction solution is washed with 0.5 M hydrochloric acid solution and water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product is purified by column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain compound 2. 6.63 g of white solid is obtained with a yield of 72.0%.

[0067] 1 H NMR (600MHz, CDCl3) δ7.72 (d, J = 7.9 Hz, 1H), 7.51 (d, J = 8.1 Hz, 1H), 6.91 (t, J = 8.0 Hz, 1H).

[0068] Example 2: 3-Bromo-4'-methoxy-(1,1'-biphenyl)-2-carboxylic acid (Compound 3)

[0069]

[0070] Compound 2 (2.0 g, 6.1 mmol, 1 eq) was dispersed in water (130 mL) and heated under reflux for 1 h. 4-Methoxyphenylboronic acid (1.2 g, 7.9 mmol, 1.3 eq), potassium hydroxide (1.02 g, 18.3 mmol, 3.0 eq), and palladium acetate (137 mg, 0.61 mmol, 0.1 eq) were then added. The mixture was heated under reflux for more than 16 hours under a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to room temperature and the pH of the solution was adjusted to 1 with 1 M hydrochloric acid solution. The solution was extracted with ethyl acetate, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was relatively pure and could be used directly in the next reaction. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 5:1) to obtain compound 3. 1.33 g of white solid was obtained with a yield of 70.6%.

[0071] 1 H NMR (600MHz, CDCl3) δ7.55-7.46 (m, 1H), 7.32-7.22 (m, 4H), 6.87 (d, J = 8.6Hz, 2H), 3.77 (s, 1H).

[0072] Example 3: 1-Bromo-7-methoxy-9H-fluoren-9-one (Compound 4)

[0073]

[0074] Compound 3 (1.7 g, 5.6 mmol, 1 eq) was added to a round-bottom flask and dissolved in dichloromethane (15 mL). Methanesulfonic acid (18 mL) was added, heated to 40 ° C, and the reaction was stirred for more than 16 hours. After the reaction was completed, it was cooled to room temperature and water was added dropwise in an ice-water bath to quench the reaction. It was then extracted with dichloromethane and dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (petroleum ether: dichloromethane = 5:4) to give compound 4. 0.592 g of yellow solid was obtained with a yield of 36.6%.

[0075] 1 H NMR (600MHz, DMSO) δ = 7.76 (d, J = 8.1, 1H), 7.73 (d, J = 7.0, 1H), 7.45 (m, 2H), 7.18 (m, 2H), 3.85 (s, 3H).

[0076] Example 4: 1-Bromo-7-hydroxy-9H-fluoren-9-one (Compound 5)

[0077]

[0078] Compound 4 (650 mg, 2.2 mmol, 1 eq) was placed in a round-bottom flask, and glacial acetic acid (4 mL) and a 48% aqueous solution of hydrobromic acid (12 mL) were added. The mixture was heated to 120°C in the dark and stirred under reflux for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, diluted with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 20:3) to obtain compound 5. 0.322 g of an orange solid was obtained, with a yield of 53.2%.

[0079] 1 H NMR (600MHz, DMSO) δ10.19(s,1H),7.66-7.60(m,2H),7.42(dd,J=10.8,4.4Hz,1H),7.38(d,J=8.0Hz,1H),7.00-6.95(m,2H).

[0080] Example 5: 1-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-7-hydroxy-9H-fluoren-9-one (Compound 6)

[0081]

[0082] Compound 5 (1.44 g, 5.2 mmol, 1 eq) was placed in a round-bottom flask, and benzo-1,4-dioxane-6-boronic acid (1.13 g, 6.3 mmol, 1.2 eq) was added and dissolved in tetrahydrofuran (18 mL). A solution of potassium carbonate (2.17 g, 15.7 mmol, 3 eq) in water (6.7 mL) and palladium tetratriphenylphosphine (302 mg, 0.26 mmol, 0.05 eq) were added, and the mixture was heated to 80 ° C and stirred at reflux for 12 hours under a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. Drying with anhydrous magnesium sulfate, filtering, rotary evaporation, and purification by silica gel column chromatography gave compound 6. 1.294 g of orange-red solid was obtained with a yield of 75.2%.

[0083] The detection data of compound 6 are as follows:

[0084] 1 H NMR (600MHz, DMSO) δ10.01(s,1H),7.58(dd,J=17.0,7.7Hz,2H),7.54-7.47(m,1H),7.07(d,J=7.7Hz,1H),7.02(t,J= 4.9Hz, 1H), 6.97 (dd, J=8.3, 2.0Hz, 1H), 6.94 (dd, J=8.0, 2.3Hz, 1H), 6.91-6.88 (m, 2H), 4.29 (dd, J=8.9, 5.0Hz, 4H).13 C NMR (151MHz, DMSO) δ = 192.31, 158.94, 145.92, 143.54, 142.62, 140.86, 135.35, 134.85, 133.79, 13 0.24,129.84,128.72,122.19,122.06,120.86,118.52,117.86,116.36,110.58,64.21,64.04.HRMS m / z calculated for C 25 H 22 O6[M+Na] + :441.13086,found441.13007

[0085] Example 6: 2-((8-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)-isonicotinonitrile (Compound 7a)

[0086]

[0087] Compound 6 (331 mg, 1 mmol, 1 eq) and potassium carbonate (278 mg, 2 mmol, 2 eq) were added to a round-bottom flask and dissolved in N,N-dimethylformamide (4 mL). The mixture was stirred at room temperature for 5 minutes. 2-Bromo-4-cyanopyridine (260 mg, 1.1 mmol, 1.1 eq) was added, and the mixture was heated to 70°C and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7a as a yellow solid (0.480 g) with a yield of 87.4%.

[0088] The detection data of compound 7a are as follows:

[0089] 1 H NMR (600MHz, CDCl3) δ=8.30(d,J=5.0,1H),7.59(d,J=8.0,1H),7.48(d,J=3.2,2H),7.36(s,1H),7.26(s,1H ),7.23(s,2H),7.18(dd,J=5.1,2.8,1H),7.06(s,1H),7.03(d,J=8.4,1H),6.93(d,J=8.3,1H),4.31(s,4H). 13C NMR (151MHz, DMSO)δ=191.19,162.81,153.94,148.92,144.66,143.63,142.65,141.08,139.59,135.11,134.91,131.10,12 9.97,128.95,127.35,122.74,122.24,122.05,120.67,119.56,117.90,116.98,116.38,116.33,114.70,64.19,64.02.HRMS m / z calculated for C 27 H 16 N2O4[M+H] + :433.11828,found 433.11813

[0090] Example 7: 3-(((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)methyl)-benzonitrile (Compound 7b)

[0091]

[0092] Compound 6 (83 mg, 0.25 mmol, 1 eq) and sodium hydroxide (20 mg, 0.5 mmol, 2 eq) were added to a round-bottom flask and dissolved in N,N-dimethylformamide (1 mL). The mixture was stirred at room temperature for 5 minutes. 3-Cyanobenzyl bromide (74 mg, 0.375 mmol, 1.5 eq) was added, and the mixture was heated to 70°C and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7b as a yellow solid (0.099 g) with a yield of 78.2%.

[0093] The detection data of compound 7b are as follows:

[0094] 1 H NMR (600MHz, DMSO) δ = 7.95 (s, 1H), 7.82 (s, 2H), 7.75 (d, J = 8.3, 1H), 7.65 (t, J = 7.7, 2H), 7.54 (t, J = 7.3, 1H), 7.24 ( d,J=8.2,1H),7.14(m,2H),7.03(s,1H),6.99(d,J=8.9,1H),6.90(d,J=8.3,1H),5.26(s,2H),4.30(d,J=4.9,4H). 13C NMR (151MHz, DMSO) δ = 191.88, 159.35, 145.36, 143.60, 142.67, 140.97, 138.43, 135.82, 135.23, 134.94, 132.44, 131.75, 131.05, 13 0.38,130.13,129.77,128.82,122.23,122.10,120.98,119.02,118.65,117.85,116.39,111.48,109.92,68.50,64.21,64.04.HRMS m / z calculated forC 29 H 19 NO4[M+H] + :446.13868,found 446.14703

[0095] Example 8: (R)-5-(((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)methyl)-pyrrolidin-2-one (Compound 7c)

[0096]

[0097] Compound 6 (83 mg, 0.25 mmol, 1 eq) and potassium carbonate (207 mg, 1.5 mmol, 6 eq) were added to a round-bottom flask and dissolved in acetone (5 mL). (R)-5-bromomethyl-2-pyrrolidone (96 mg, 0.54 mmol, 2.16 eq) was then added, and the mixture was heated to 60°C and refluxed under a nitrogen atmosphere for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7c as a yellow solid (0.111 g) with a yield of 51.9%.

[0098] The detection data of compound 7c are as follows:

[0099] 1 H NMR (600MHz, DMSO) δ=7.81(d,J=27.9,1H),7.57(d,J=7.9,1H),7.48(m,2H),7.07(m,3H),7.00(d,J=2.0,1H),6.95(dd,J=8.3,2 .0,1H),6.87(d,J=8.3,1H),4.30(dd,J=8.4,4.9,4H),4.03(m,1H),3.93(d,J=8.2,2H),2.31(m,2H),2.21(dd,J=15.0,4.7,2H).13 C NMR (151MHz, DMSO) δ = 191.94, 176.95, 159.85, 145.45, 143.61, 142.67, 140.97, 135.57, 134.94, 130.16, 128.82, 127. 51,122.89,122.24,122.02,120.60,118.97,117.86,116.41,109.79,71.32,64.22,64.06,52.44,29.53,22.85.HRMS m / z calculatedfor C 26 H 21 NO5[M+Na] + :450.13086,found450.13119

[0100] Example 9: (S)-5-(((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)methyl)-pyrrolidin-2-one (Compound 7d)

[0101]

[0102] Compound 6 (165 mg, 0.5 mmol, 1 eq) and potassium carbonate (414 mg, 3 mmol, 6 eq) were added to a round-bottom flask and dissolved in acetone (10 mL). (S)-5-bromomethyl-2-pyrrolidone (192 mg, 1.08 mmol, 2.16 eq) was then added, and the mixture was heated to 60°C and refluxed under a nitrogen atmosphere for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7d. A yellow solid (0.120 g) was obtained with a yield of 56.1%.

[0103] The detection data of compound 7d are as follows:

[0104] 1 H NMR (600MHz, DMSO) δ=7.81(d,J=27.9,1H),7.57(d,J=7.9,1H),7.48(m,2H),7.07(m,3H),7.00(d,J=2.0,1H),6.95(dd,J=8.3,2 .0,1H),6.87(d,J=8.3,1H),4.30(dd,J=8.4,4.9,4H),4.03(m,1H),3.93(d,J=8.2,2H),2.31(m,2H),2.21(dd,J=15.0,4.7,2H). 13C NMR (151MHz, DMSO) δ = 191.95, 176.96, 159.84, 145.45, 143.61, 142.67, 140.97, 135.57, 135.21, 134.93, 130.33, 130. 16,128.82,122.24,122.01,120.59,118.96,117.86,116.41,109.79,71.32,64.22,64.06,52.45,29.53,22.85.HRMS m / z calculatedfor C 26 H 21 NO5[M+Na] + :450.13095,found450.13119

[0105] Example 10: 3-((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)-pyridine-2-carbonitrile (Compound 7e)

[0106]

[0107] Compound 6 (166 mg, 0.5 mmol, 1 eq) and potassium carbonate (139 mg, 1 mmol, 2 eq) were added to a round-bottom flask and dissolved in N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 5 minutes. 3-Bromo-2-cyanopyridine (101 mg, 0.55 mmol, 1.1 eq) was added, and the mixture was heated to 70°C and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7e as a yellow solid (0.152 g) with a yield of 70.2%.

[0108] The detection data of compound 7e are as follows:

[0109] 1 HNMR (600MHz, DMSO) δ = 8.52 (d, J = 4.2, 1H), 7.94 ( d, J = 8.1, 1H), 7.80 ( d, J = 7.3, 1H), 7.73 ( dd, J = 8.5, 4.4, 1H), 7.62 ( dd, J = 12.2, 8.2, 2H),7.50(d,J=8.0,1H),7.37(s,1H),7.24(d,J=7.7,1H),7.05(s,1H),7.00(d,J=8.3,1H),6.90(d,J=8.3,1H),4.29(d,J=5.1,4H). 13C NMR (151MHz, DMSO) δ = 190.98, 156.55, 155.28, 145.78, 144.52, 143.69, 142.70, 141.18, 139.93, 135.74, 135.12, 131.29, 12 9.91,129.39,128.95,125.91,125.82,123.78,122.85,122.24,119.80,117.89,116.44,115.26,115.14,64.21,64.04.HRMS m / z calculated for C 27 H 16 N2O4[M+Na] + :455.09982,found 455.10023

[0110] Example 11: 5-((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)-pyridine-3-carbonitrile (Compound 7f)

[0111]

[0112] Compound 6 (83 mg, 0.25 mmol, 1 eq) and potassium hydroxide (28 mg, 0.5 mmol, 2 eq) were added to a round-bottom flask and dissolved in dimethyl sulfoxide (1 mL). The mixture was stirred at room temperature for 5 minutes. 2-Cyano-5-bromopyridine (69 mg, 0.375 mmol, 1.5 eq) was added, and the mixture was heated to 80°C and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7f as a yellow solid (0.045 g) with a yield of 41.8%.

[0113] The detection data of compound 7f are as follows:

[0114] 1 H NMR (600MHz, DMSO) δ = 8.59 (s, 1H), 8.07 (d, J = 8.6, 1H), 7.93 (d, J = 8.0, 1H), 7.79 (d, J = 7.3, 1H), 7.62 (dd, J = 10.1, 4.3, 2H), 7. 45(d,J=8.0,1H),7.32(s,1H),7.23(d,J=7.7,1H),7.05(s,1H),7.00(d,J=8.4,1H),6.90(d,J=8.3,1H),4.29(d,J=5.0,4H). 13C NMR (151MHz, DMSO) δ = 191.20, 164.80, 153.60, 152.21, 144.63, 143.68, 143.57, 142.70, 141.14, 139.95, 135.06, 131.25, 12 9.99,129.04,128.18,127.77,122.28,122.22,119.75,117.93,117.38,117.00,116.45,112.29,103.94,64.22,64.05.HRMS m / zcalculated for C 27 H 16 N2O4[M+Na] + :455.09995,found455.10023

[0115] Example 12: 1-(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)-7-(2-hydroxyethoxy)-9H-fluoren-9-one (Compound 7g)

[0116]

[0117] Compound 6 (83 mg, 0.25 mmol, 1 eq) and sodium hydroxide (20 mg, 0.5 mmol, 2 eq) were added to a round-bottom flask and dissolved in N,N-dimethylformamide (1 mL). The mixture was stirred at room temperature for 5 minutes. 2-Bromoethanol (47 mg, 0.375 mmol, 1.5 eq) was added, and the mixture was heated to 70°C and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7 g as a yellow solid (0.032 g) with a yield of 34.2%.

[0118] The test data of compound 7g are as follows:

[0119] 1 H NMR (600MHz, DMSO) δ = 7.71 (d, J = 8.2, 1H), 7.64 (d, J = 7.4, 1H), 7.53 (t, J = 7.5, 1H), 7.14 (m, 2H), 7.04 (dd, J = 9.9, 1.9, 2H), 6. 98(d,J=8.3,1H), 6.90(d,J=8.3,1H), 4.92(t,J=5.5,1H), 4.29(d,J=4.8,4H), 4.06(t,J=4.7,2H), 3.72(dd,J=9.9,4.9,2H). 13C NMR(151MHz,DMSO)δ=191.97,160.10,145.48,143.56,142.64,140.93,135.25,135.20,134.88,130.20,1 30.14,128.80,122.20,121.99,120.53,118.87,117.83,116.35,109.56,70.14,64.19,64.03,59.46.HRMS m / z calculated for C 23 H 18 O5[M+Na] + :397.10443,found 397.10464

[0120] Example 13: 1-(2,3-Dihydrobenzo[b][1,4]dioxane-6-yl)-7-(3-hydroxypropoxy)-9H-fluoren-9-one (Compound 7h)

[0121]

[0122] Compound 6 (166 mg, 0.5 mmol, 1 eq) and potassium carbonate (138 mg, 1 mmol, 2 eq) were added to a round-bottom flask and dissolved in N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 5 minutes. 3-Bromo-1-propanol (127 mg, 0.55 mmol, 1.1 eq) was added, and the mixture was heated to 70°C and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7h as a yellow solid (0.120 g) with a yield of 61.7%.

[0123] The detection data of compound 7h are as follows:

[0124] 1 H NMR (600MHz, DMSO) δ = 7.70 (d, J = 8.2, 1H), 7.63 (d, J = 7.4, 1H), 7.53 (t, J = 7.0, 1H), 7.12 (t, J = 8.2, 2H), 7.03 (s, 2H), 6.98 (d, J = 8. 3,1H),6.90(d,J=7.2,1H),4.58(t,J=4.6,1H),4.30(d,J=5.1,4H),4.11(t,J=6.1,2H),3.57(q,J=5.3,2H),1.87(p,J=6.1,2H). 13C NMR(151MHz, CDCl3)δ=191.80,161.63,159.10,144.89,142.75,141.94,140.78,135.16,134.98,133.29,129.74,129.35,12 8.64,121.52,120.02,119.44,117.17,117.07,115.67,108.67,66.86,64.93,63.48,63.33,59.07,35.56,30.87,30.47.HRMS m / zcalculated for C 24 H 20 O5[M+Na] + :411.11972,found411.12029

[0125] Example 14: 3-(((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)methyl)-benzoic acid methyl ester (Compound 7i)

[0126]

[0127] Compound 6 (83 mg, 0.25 mmol, 1 eq) and sodium hydroxide (20 mg, 0.5 mmol, 2 eq) were added to a round-bottom flask and dissolved in N,N-dimethylformamide (1 mL). The mixture was stirred at room temperature for 5 minutes. Methyl 3-bromomethylbenzoate (115 mg, 0.5 mmol, 2 eq) was added, and the mixture was heated to 70°C and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7i as a yellow solid (0.074 g) with a yield of 61.8%.

[0128] The detection data of compound 7i are as follows:

[0129] 1 HNMR (600MHz, DMSO) δ = 8.00 (d, J = 7.6, 2H), 7.74 (d, J = 8.1, 1H), 7.65 (d, J = 7.4, 1H), 7.61 (d, J = 7.8, 2H), 7.54 (t, J = 7.5, 1H), 7.24 (d, J=8.2,1H),7.13(d,J=10.6,2H),7.03(s,1H),6.98(d,J=9.0,1H),6.90(d,J=8.2,1H),5.31(s,2H),4.29(d,J=5.2,4H),3.86(s,3H). 13C NMR (151MHz, DMSO) δ = 191.87, 166.01, 159.49, 145.36, 143.57, 142.65, 140.94, 137.49, 135.70, 135.21, 134.88, 132.36, 131.48, 131. 42,129.84,129.00,128.76,128.68,128.64,128.12,122.20,122.04,117.82,116.35,109.84,69.03,64.19,64.02,62.28,52.18.HRMS m / z calculated for C 30 H 22 O6[M+Na] + :501.12949,found501.13086

[0130] Example 15: 4-(((8-(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)methyl)-benzoic acid methyl ester (Compound 7j)

[0131]

[0132] Compound 6 (83 mg, 0.25 mmol, 1 eq) and sodium hydroxide (20 mg, 0.5 mmol, 2 eq) were added to a round-bottom flask and dissolved in N,N-dimethylformamide (1 mL). The mixture was stirred at room temperature for 5 minutes. Methyl 4-bromomethylbenzoate (115 mg, 0.5 mmol, 2 eq) was added, and the mixture was heated to 70°C and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7j as a yellow solid (0.065 g, 54.3% yield).

[0133] The detection data of compound 7j are as follows:

[0134] 1 H NMR (600MHz, DMSO) δ = 8.07 (s, 1H), 7.94 (d, J = 7.9, 1H), 7.75 (dd, J = 13.0, 8.0, 2H), 7.64 (d, J = 7.3, 1H), 7.53 (m, 3H), 7.24 (d, J=8.2,1H),7.13(m,2H),7.03(s,1H),6.98(d,J=9.9,1H),6.90(d,J=8.2,1H),5.29(s,2H),4.30(d,J=4.8,4H),3.87(s,3H). 13C NMR (151MHz, DMSO) δ = 191.87, 165.97, 159.46, 143.59, 142.66, 134.92, 133.07, 132.39, 132.01, 131.99, 131.49, 131.43, 129.34, 129. 01,128.77,128.69,127.50,126.29,122.86,122.21,121.02,118.98,117.83,116.37,109.89,69.02,64.20,64.03,62.36,52.11.HRMS m / z calculated for C 30 H 22 O6[M+Na] + :501.12817,found 501.13086

[0135] Example 16: 5-(((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)-pentanoic acid methyl ester (Compound 7k)

[0136]

[0137] Compound 6 (83 mg, 0.25 mmol, 1 eq) and sodium hydroxide (20 mg, 0.5 mmol, 2 eq) were added to a round-bottom flask and dissolved in N,N-dimethylformamide (1 mL). The mixture was stirred at room temperature for 5 minutes. Methyl 5-bromovalerate (98 mg, 0.5 mmol, 2 eq) was added, and the mixture was heated to 70°C and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7k as a yellow solid (0.079 g, 71.2% yield).

[0138] The detection data of compound 7k are as follows:

[0139] 1 H NMR (600MHz, DMSO) δ = 7.69 (d, J = 8.1, 1H), 7.63 (d, J = 7.4, 1H), 7.52 (t, J = 7.5, 1H), 7.11 (d, J = 7.7, 2H), 7.02 (m, 2H), 6.98 ( d,J=8.3,1H),6.90(d,J=8.3,1H),4.29(d,J=5.0,4H),4.04(t,J=5.9,2H),3.59(s,3H),2.39(t,J=7.1,2H),1.71(m,5H). 13CNMR(151MHz,DMSO)δ=191.97,173.20,159.96,145.49,143.57,142.65,140.92,135.25,135.20,134.87,130.21,130.15 ,128.79,122.20,121.99,120.49,118.87,117.82,116.36,109.43,67.66,64.19,64.03,51.18,32.84,27.89,21.08.HRMS m / z calculated forC 27 H 24 O6[M+H] + :445.16456,found445.16202

[0140] Example 17: 4-(((8-(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)-butyric acid methyl ester (Compound 71)

[0141]

[0142] Compound 6 (83 mg, 0.25 mmol, 1 eq) and sodium hydroxide (20 mg, 0.5 mmol, 2 eq) were added to a round-bottom flask and dissolved in N,N-dimethylformamide (1 mL). The mixture was stirred at room temperature for 5 minutes. Methyl 4-bromobutyrate (136 mg, 0.75 mmol, 3 eq) was added, and the mixture was heated to 70°C and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 71 as a yellow solid (0.072 g) with a yield of 66.7%.

[0143] The detection data of compound 71 are as follows:

[0144] 1 H NMR (600MHz, DMSO) δ = 7.69 (d, J = 8.2, 1H), 7.62 ( d, J = 7.3, 1H), 7.52 ( t, J = 7.5, 1H), 7.11 ( d, J = 7.9, 2H), 7.02 ( m, 2H), 6.98 ( d,J=8.3,1H),6.90(d,J=8.3,1H),4.30(d,J=4.8,4H),4.06(t,J=6.2,2H),3.61(s,2H),2.49(t,J=7.3,2H),1.98(m,2H). 13CNMR(151MHz, CDCl3)δ=192.97,173.71,160.29,146.11,143.96,143.15,141.97,136.30,136.18,134.47,130.94,130.53,129.8 4,122.73,121.21,120.58,118.35,118.28,116.86,109.87,77.44,77.23,77.02,67.35,64.68,64.53,51.88,30.65,24.69.HRMS m / zcalculated for C 26 H 22 O6[M+Na] + :453.12738,found 453.13086

[0145] Example 18: 3-(((8-(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)methyl)-benzoic acid (Compound 7m)

[0146]

[0147] Compound 7i (114 mg, 0.24 mmol, 1 eq) was dissolved in tetrahydrofuran (6.7 mL) and methanol (6.7 mL). A solution of potassium hydroxide (166 mg, 1.2 mmol, 5 eq) in water (1.66 mL) was added. The reaction solution was heated to 80°C and refluxed for 12 hours. After the reaction was complete, the solution was cooled to room temperature and the pH was adjusted to 1-4 with 1 M hydrochloric acid solution. The solution was extracted with ethyl acetate. The product was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7m as a yellow solid (0.084 g, 75.2% yield).

[0148] The detection data of compound 7m are as follows:

[0149] 1 HNMR (600MHz, DMSO) δ=8.05(s,1H),7.92(d,J=8.0,1H),7.73(t,J=8.9,2H),7.65(d,J=7.3,1H),7.54(dd,J=11.9,7.3,2H ),7.24(d,J=8.2,1H),7.13(m,2H),7.03(s,1H),6.99(d,J=8.3,1H),6.90(d,J=8.2,1H),5.28(s,2H),4.30(d,J=4.7,4H). 13CNMR(151MHz,DMSO)δ=191.89,167.10,159.54,143.58,142.66,137.27,134.89,131.96,131.50,131.43,131.01,130.76,130.14,1 28.81,128.77,128.69,128.32,128.28,127.60,127.18,122.22,118.95,117.83,116.37,109.85,69.14,64.20,64.03,62.41.HRMS m / zcalculated for C 29 H 20 O6[M+Na] + :487.11441,found 487.11521

[0150] Example 19: 4-(((8-(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)methyl)-benzoic acid (Compound 7n)

[0151]

[0152] Compound 7j (67 mg, 0.14 mmol, 1 eq) was dissolved in tetrahydrofuran (3.9 mL) and methanol (3.9 mL). A solution of sodium hydroxide (56 mg, 1.4 mmol, 5 eq) dissolved in water (0.56 mL) was added. The reaction solution was heated to 80°C and refluxed for 12 hours. After the reaction was complete, the solution was cooled to room temperature and the pH was adjusted to 1-4 with 1M hydrochloric acid. The solution was extracted with ethyl acetate. The product was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7n as a yellow solid (0.045 g, 69.8% yield).

[0153] The detection data of compound 7n are as follows:

[0154] 1 HNMR (600MHz, DMSO) δ = 7.97 (d, J = 8.1, 2H), 7.74 ( d, J = 8.2, 1H), 7.65 ( d, J = 7.3, 1H), 7.59 ( d, J = 8.1, 2H), 7.54 ( t, J = 7.6, 1H), 7 .24(dd,J=8.2,2.1,1H),7.13(m,2H),7.03(s,1H),6.98(d,J=10.0,1H),6.89(d,J=8.3,1H),5.30(s,2H),4.29(d,J=4.7,4H). 13C NMR(151MHz, DMSO)δ=191.87,167.04,159.48,145.37,143.58,142.66,141.70,140.96,135.72,135.22,134.90,132.01,131.50,131 .43,129.49,128.77,128.69,127.37,126.15,122.22,121.00,118.96,117.83,116.37,109.89,69.08,64.20,64.03,62.42.HRMSm / z calculated for C 29 H 20 O6[M+Na] + :487.11407,found 487.11521

[0155] Example 20: 5-(((8-(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)-pentanoic acid (Compound 7o)

[0156]

[0157] Compound 7k (100 mg, 0.22 mmol, 1 eq) was dissolved in tetrahydrofuran (6.1 mL) and methanol (6.1 mL). Potassium hydroxide (152 mg, 1.1 mmol, 5 eq) dissolved in water (1.52 mL) was added. The reaction solution was heated to 80°C and refluxed for 12 hours. After the reaction was complete, the solution was cooled to room temperature and the pH was adjusted to 1-4 with 1 M hydrochloric acid solution. The solution was extracted with ethyl acetate. The product was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7o as a yellow solid (0.069 g, 72.3% yield).

[0158] The detection data of compound 7o are as follows:

[0159] 1 H NMR (600MHz, DMSO) δ=12.06(s,1H),7.71(d,J=8.2,1H),7.63(s,1H),7.54(d,J=7.6,1H),7.12(t,J=7.6,2H),7.03(s,2H),6.98(d,J=8.4,1 H), 6.90 (d, J = 9.3, 1H), 4.30 (d, J = 5.1, 4H), 4.05 (t, J = 6.1, 2H), 2.29 (t, J = 7.4, 2H), 1.74 (dd, J = 14.0, 6.4, 2H), 1.66 (dt, J = 14.3, 7.2, 2H). 13C NMR (151MHz, DMSO) δ = 192.00, 174.38, 160.01, 145.53, 143.59, 142.67, 140.94, 135.25, 135.22, 134.90, 130.23, 130. 17,128.81,122.23,122.01,120.51,118.89,117.86,116.39,109.44,67.76,64.22,64.05,33.26,28.02,21.15.HRMS m / z calculated for C 26 H 22 O6[M+Na] + :453.13007,found 453.13086

[0160] Example 21: 4-(((8-(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)-9-oxo-9H-fluoren-2-yl)oxy)-butanoic acid (Compound 7p)

[0161]

[0162] Compound 7l (100 mg, 0.23 mmol, 1 eq) was dissolved in tetrahydrofuran (6.4 mL) and methanol (6.4 mL). Potassium hydroxide (159 mg, 1.15 mmol, 5 eq) dissolved in water (1.59 mL) was added. The reaction solution was heated to 80°C and refluxed for 12 hours. After the reaction was complete, the solution was cooled to room temperature and the pH was adjusted to 1-4 with 1 M hydrochloric acid solution. The solution was extracted with ethyl acetate. The product was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound 7p as a yellow solid (0.068 g, 70.5% yield).

[0163] The detection data of compound 7p are as follows:

[0164] 1 H NMR (600MHz, DMSO) δ = 12.16 (s, 1H), 7.71 (d, J = 8.1, 1H), 7.63 (s, 1H), 7.53 (d, J = 7.7, 1H), 7.12 (t, J = 8.1, 2H), 7.03 (s, 2H), 6.98 (d, J = 8.3, 1H), 6.89 (d, J = 8.2, 1H), 4.29 (d, J = 4.9, 4H), 4.06 (t, J = 6.3, 2H), 2.39 (t, J = 7.2, 2H), 1.95 (m, 2H). 13C NMR(151MHz, CDCl3)δ=191.80,176.33,159.06,144.91,142.75,141.94,140.78,133.27,131.09,129.34,127.50,12 1.53,120.02,119.42,117.15,117.08,115.66,108.67,66.02,63.48,63.32,30.41,29.25,28.68,23.28,13.11.HRMS m / z calculated for C 26 H 22 O6[M+Na] + :439.11447,found 439.11521

[0165] Example 22: 2-((8-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)-isonicotinonitrile (Compound I-1)

[0166]

[0167] Compound 7a (181 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound I-1 as a white solid (0.151 g, 82.5% yield).

[0168] The test data of compound I-1 are as follows:

[0169] 1 HNMR (600MHz, DMSO) δ=8.39(d,J=5.0,1H),7.84(d,J=8.1,1H),7.75(d,J=7.5,1H),7.65(s,1H),7.58(d,J=5.1,1H),7.44(s,1H),7.31(s, 1H),7.28(s,1H),7.22(d,J=7.7,1H),7.17(dd,J=7.9,6.6,2H),6.91(d,J=8.3,1H),5.81(d,J=8.2,1H),5.51(d,J=8.1,1H),4.28(s,4H). 13C NMR (151MHz, DMSO) δ = 170.34, 163.37, 152.85, 149.13, 142.97, 142.96, 142.75, 140.06, 139.26, 136.23, 133.24, 129.21, 1 28.29,122.66,121.59,120.95,120.39,118.60,118.13,117.29,116.75,114.52,72.88,64.13,59.76,20.76,14.08.HRMS m / z calculated for C 27 H 18 N2O4[M+Na] + :457.11588,found 457.11557

[0170] Example 23: 3-(((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)methyl)-benzonitrile (Compound I-2)

[0171]

[0172] Compound 7b (187 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to yield compound I-2 as a white solid (0.141 g, 75.2% yield).

[0173] The detection data of compound I-2 are as follows:

[0174] 1 H NMR (600MHz, DMSO) δ = 7.83 (m, 2H), 7.71 (d, J = 8.1, 1H), 7.64 (dt, J = 11.0, 6.3, 2H), 7.55 (t, J = 7.7, 1H), 7.39 (t, J = 7.6, 1H), 7.28 (s, 1H), 7.21 (s, 1H),7.16(m,2H),7.04(dd,J=8.2,1.7,1H),6.91(d,J=8.3,1H),5.73(d, J=8.3,1H),5.43(dd,J=12.3,7.0,2H),4.56(d,J=5.7,1H),4.29(s,4H). 13C NMR (151MHz, DMSO) δ = 158.23, 149.25, 144.27, 142.95, 142.69, 139.13, 138.86, 133.39, 132.48, 132.33, 131.67, 131.19, 131.08, 1 30.47,129.75,129.68,129.33,129.08,121.58,120.81,117.29,116.71,111.70,111.45,72.93,68.27,64.13,64.07,61.85.HRMS m / z calculated for C 29 H 21 NO4[M+Na] + :470.13627,found470.13577

[0175] Example 24: (R)-5-(((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)methyl)-pyrrolidin-2-one (Compound I-3)

[0176]

[0177] Compound 7c (179 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound I-3 as a white solid (0.169 g, 89.2% yield).

[0178] The detection data of compound I-3 are as follows:

[0179] 1H NMR (600MHz, DMSO) δ = 7.90 (s, 1H), 7.84 (s, 1H), 7.70 (d, J = 8.2, 1H), 7.64 (d, J = 7 .5,1H),7.39(t,J=7.6,1H),7.28(s,1H),7.15(s,1H),7.14(d,J=5.3,1H),6.96( d,J=8.3,1H),6.92(d,J=8.3,1H),5.72(d,J=8.2,1H),5.42(d,J=8.3,1H),4.29( s,4H),4.00(d,J=4.3,1H),3.53(d,J=5.1,2H),2.26(m,2H),2.15(d,J=9.7,2H). 13 C NMR (151MHz, DMSO) δ = 176.96, 158.72, 149.18, 142.96, 142.70, 142.51, 140.74, 139.15, 133.43, 132.09, 129.09, 127 .42,121.60,120.75,117.90,117.30,116.73,114.52,111.54,72.93,70.98,64.15,53.73,52.58,29.53,24.84.HRMS m / z calculated for C 26 H 23 NO5[M+Na] + :452.14684,found452.14630

[0180] Example 25: (S)-5-(((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)methyl)-pyrrolidin-2-one (Compound I-4)

[0181]

[0182] Compound 7d (179 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound I-4 as a white solid (0.162 g, 85.5% yield).

[0183] The detection data of compound I-4 are as follows:

[0184] 1 H NMR (600MHz, DMSO) δ = 7.90 (s, 1H), 7.70 (d, J = 8.2, 1H), 7.64 (d, J = 7.5, 1H), 7. 39(t,J=7.6,1H),7.28(s,1H),7.15(dd,J=14.4,6.8,3H),6.96(d,J=8.3,1H), 6.92(d,J=8.3,1H),5.72(d,J=8.2,1H),5.42(d,J=8.3,1H),4.29(s,4H),3.9 9(m,1H),3.53(d,J=5.0,2H),2.18(d,J=13.2,2H),1.88(td,J=10.2,4.7,2H). 13 C NMR (151MHz, DMSO) δ = 176.95, 158.72, 149.17, 142.96, 142.70, 142.51, 140.74, 139.15, 133.43, 132.09, 129.09, 127 .42,121.59,120.75,117.89,117.30,116.72,114.52,111.53,72.94,70.98,64.08,53.73,52.58,29.53,24.84.HRMS m / z calculated for C 26 H 23 NO5[M+Na] + :452.14684,found452.14667

[0185] Example 26: 3-((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)-pyridine-2-carbonitrile (Compound I-5)

[0186]

[0187] Compound 7e (182 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the product was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound I-5 as a white solid (0.175 g, 91.2% yield).

[0188] The test data of compound I-5 are as follows:

[0189] 1HNMR (600MHz, DMSO) δ=8.52(d,J=4.0,1H),7.92(d,J=8.0,1H),7.79(d,J=7.3,1H),7.75(dd,J=8.3,4.4,1H),7.53(d,J=8.6,1H),7.47(t,J=7.6, 1H),7.40(s,1H),7.31(s,1H),7.27(t,J=6.3,2H),7.19(d,J=8.0,1H),6 .93(t,J=11.1,1H),5.84(d,J=7.9,1H),5.57(d,J=8.0,1H),4.30(s,4H). 13 C NMR (151MHz, DMSO) δ = 157.02, 153.91, 149.95, 145.52, 143.00, 142.95, 142.80, 139.84, 139.33, 136.65, 133.16, 129.39, 12 9.31,128.49,125.51,123.64,121.67,121.58,119.91,118.76,117.29,116.79,116.62,115.23,72.86,64.15,64.08.HRMS m / z calculated for C 27 H 18 N2O4[M+Na] + :457.11587,found457.11539

[0190] Example 27: 5-((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)-pyridine-3-carbonitrile (Compound I-6)

[0191]

[0192] Compound 7f (182 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was extracted with a dichloromethane / water system, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound I-6 as a white solid (0.171 g, 88.9% yield).

[0193] The test data of compound I-6 are as follows:

[0194] 1H NMR (600MHz, DMSO) δ=8.68(d,J=1.7,1H),8.33(dd,J=8.7,2.1,1H),7.86(d,J=8.1,1H),7.76(d,J=7.4,1H),7.45(t,J=7.6,1H),7.36(s,1 H),7.30(d,J=1.4,1H),7.26(dd,J=14.6,8.2,2H),7.20(m,2H),6.93(d,J=8.3,1H),5.83(d,J=8.2,1H),5.54(d,J=8.2,1H),4.29(s,4H). 13 C NMR (151MHz, DMSO) δ = 165.31, 152.43, 152.32, 149.29, 143.42, 143.00, 142.78, 140.02, 139.30, 136.57, 133.26, 129.2 5,128.41,121.82,121.62,120.99,118.67,118.39,117.33,117.10,116.79,112.00,103.62,72.93,64.16,64.09.HRMS m / z calculated for C 27 H 18 N2O4[M+Na] + :457.11588,found 457.11572

[0195] Example 28: 1-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-7-(2-hydroxyethoxy)-9H-fluoren-9-ol (Compound I-7)

[0196]

[0197] Compound 7g (157 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with a dichloromethane / water system, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound I-7 as a white solid (0.121 g, 72.1% yield).

[0198] The detection data of compound I-7 are as follows:

[0199] 1H NMR (600MHz, DMSO) δ = 7.67 (d, J = 8.3, 1H), 7.62 (d, J = 7.4, 1H), 7.39 (t, J = 7.5, 1H), 7.28 (d, J = 1.5, 1H), 7.15 (m, 3H), 6.93 (m, 2H), 5.72 (d, J = 8.1, 1H), 5.43 (d, J = 8.2, 1H), 4.95 (t, J = 5.5, 1H), 4.28 (s, 4H), 4.03 (t, J = 4.8, 2H), 3.75 (dd, J = 9.9, 5.1, 2H). 13 C NMR(151MHz,DMSO)δ=159.05,149.22,143.04,142.77,142.52,140.91,139.19,133.53,131.86,129.20,1 27.41,121.69,120.83,117.92,117.37,116.83,114.54,111.45,73.00,69.83,64.24,64.17,59.71.HRMS m / z calculated for C 23 H 20 O5[M+Na] + :399.12029,found 399.11972

[0200] Example 29: 1-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-7-(3-hydroxypropoxy)-9H-fluoren-9-ol (Compound I-8)

[0201]

[0202] Compound 7h (163 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound I-8 as a white solid (0.131 g, 75.6% yield).

[0203] The test data of compound I-8 are as follows:

[0204] 1H NMR (600MHz, DMSO) δ = 7.67 (d, J = 8.3, 1H), 7.62 (d, J = 7.4, 1H), 7.38 (t, J = 7.5, 1H), 7.27 (s, 1H), 7.13 (m, 3H), 6.92 (t, J = 9.6, 2H) ,5.70(d,J=8.2,1H),5.39(d,J=8.2,1H),4.58(t,J=5.0,1H),4.28(s,4H),4.07(t,J=6.3,2H),3.58(q,J=5.8,2H),1.88(m,2H). 13 C NMR (151MHz, DMSO) δ = 158.94, 149.13, 142.93, 142.67, 142.45, 140.81, 139.10, 133.43, 131.67, 129.03, 127. 27,121.57,120.70,117.78,117.28,116.68,114.42,111.23,72.91,64.78,64.12,64.05,57.29,32.11.HRMS m / z calculated for C 24 H 22 O5[M+Na] + :413.13594,found413.13483

[0205] Example 30: 3-(((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)methyl)-benzoic acid methyl ester (Compound I-9)

[0206]

[0207] Compound 7i (201 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound I-9 as a white solid (0.158 g, 78.5% yield).

[0208] The detection data of compound I-9 are as follows:

[0209] 1H NMR (600MHz, DMSO) δ = 8.08 (s, 1H), 7.94 (d, J = 7.9, 1H), 7.77 (d, J = 7.7, 1H), 7.70 (d, J=8.3,1H),7.64(d,J=7.4,1H),7.58(t,J=7.7,1H),7.39(t,J=7.6,1H),7.26(d,J=1 .9,1H),7.21(d,J=1.8,1H),7.15(m,2H),7.04(dd,J=8.3,2.2,1H),6.92(d,J=8.3, 1H),5.72(d,J=8.1,1H),5.46(d,J=8.2,1H),5.24(s,2H),4.29(s,4H),3.87(s,3H). 13 C NMR (151MHz, DMSO) δ = 166.07, 158.39, 149.20, 142.93, 142.67, 142.51, 140.66, 139.11, 137.96, 133.39, 132.35, 132.22, 129.83, 129. 05,128.98,128.55,128.08,127.41,121.56,120.76,117.88,117.27,116.68,114.75,111.76,72.91,68.81,64.12,64.05,52.19.HRMS m / z calculated for C 30 H 24 O6[M+Na] + :503.14651,found 503.14624

[0210] Example 31: 4-(((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)methyl)-benzoic acid methyl ester (Compound I-10)

[0211]

[0212] Compound 7j (201 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound I-10 as a white solid (0.162 g, 80.2% yield).

[0213] The test data of compound I-10 are as follows:

[0214] 1 H NMR (600MHz, DMSO) δ=8.00(d,J=8.0,2H),7.70(d,J=8.3,1H),7.63(m,3H),7.39(t,J=7.5,1H),7.27(s,1H),7.21(s,1H),7.16(t,J =6.1,2H),7.03(d,J=8.2,1H),6.91(d,J=8.3,1H),5.72(d,J=8.3,1H),5.42(d,J=8.3,1H),5.26(s,2H),4.29(s,4H),3.86(s,3H). 13 C NMR (151MHz, DMSO) δ = 166.04, 158.37, 149.22, 142.96, 142.70, 142.53, 140.67, 139.13, 133.41, 132.29, 129.40, 129.35, 129.09, 129. 05,128.99,127.53,127.45,126.32,121.59,120.79,117.92,117.30,116.72,114.78,111.79,72.94,68.82,64.15,64.08,52.15.HRMS m / z calculated for C 30 H 24 O6[M+Na] + :503.14651,found 503.14572

[0215] Example 32: 4-(((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)methyl)-benzoic acid methyl ester (Compound I-11)

[0216]

[0217] Compound 7k (187 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound I-11 as a white solid (0.162 g, 86.2% yield).

[0218] The test data of compound I-11 are as follows:

[0219] 1 H NMR (600MHz, DMSO) δ=7.67(d,J=8.3,1H),7.63(d,J=7.5,1H),7.38(t,J=7.5,1H),7.27(s,1H),7.14(m,2H),7.09(s,1H),6.91(t,J =9.0,2H),5.70(d,J=8.2,1H),5.38(d,J=8.2,1H),4.28(s,4H),4.01(t,J=5.9,2H),3.59(s,3H),2.40(t,J=7.1,2H),1.72(m,4H). 13 C NMR (151MHz, DMSO) δ = 173.26, 158.84, 149.14, 142.94, 142.68, 142.46, 140.80, 139.10, 133.43, 131.75, 129.05, 127.30 ,121.58,120.73,117.81,117.29,116.70,114.40,111.28,72.91,67.27,64.13,64.06,51.23,32.92,28.08,21.21.HRMS m / zcalculated for C 27 H 22 O6[M+Na] + :469.16216,found469.16153

[0220] Example 33: 4-(((8-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)-butyric acid methyl ester (Compound I-12)

[0221]

[0222] Compound 71 (181 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound I-12 as a white solid (0.139 g, 76.5% yield).

[0223] The test data of compound I-12 are as follows:

[0224] 1H NMR (600MHz, DMSO) δ=7.68(d,J=8.3,1H),7.63(d,J=7.5,1H),7.39(t,J=7.5,1H),7.28(s,1H),7.16(t,J=9.1,2H),7.11(s,1H),6.92(dd,J=10. 9,3.8,2H),5.71(d,J=8.2,1H),5.40(dd,J=8.2,1.1,1H),4.29(s,4H),4 .03(s,2H),3.63(d,J=1.0,3H),2.51(s,2H),2.00(dd,J=13.4,6.5,2H). 13 C NMR (151MHz, DMSO) δ = 173.05, 158.68, 149.16, 142.94, 142.68, 142.47, 140.77, 139.15, 133.42, 131.85, 129.06, 127 .33,121.58,120.74,117.83,117.28,116.70,114.43,111.26,72.90,66.69,64.13,64.06,51.35,29.96,24.27.HRMS m / z calculated for C 26 H 24 O6[M+Na] + :455.14651,found455.14563

[0225] Example 34: 3-(((8-(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)methyl)-benzoic acid (Compound I-13)

[0226]

[0227] Compound 7m (195 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford compound I-13 as a white solid (0.176 g, 90.1% yield).

[0228] The test data of compound I-13 are as follows:

[0229] 1H NMR (600MHz, DMSO) δ=12.75(s,1H),8.08(s,1H),7.94(d,J=7.6,1H),7.74(d,J=7 .5,1H),7.70(d,J=8.3,1H),7.64(d,J=7.4,1H),7.55(t,J=7.6,1H),7.39(t,J=7 .5,1H),7.29(s,1H),7.23(s,1H),7.16(t,J=7.4,2H),7.04(d,J=6.4,1H),6.92( d,J=8.3,1H),5.73(d,J=7.7,1H),5.43(d,J=8.1,1H),5.24(s,2H),4.29(s,4H). 13 C NMR (151MHz, DMSO) δ = 167.21, 158.49, 149.24, 142.99, 142.72, 142.56, 140.72, 139.16, 137.75, 133.45, 132.24, 131.97, 131.04, 1 29.10,128.81,128.32,127.45,121.62,120.79,117.91,117.33,116.74,114.79,111.81,72.98,68.95,64.17,64.10,59.78.HRMS m / zcalculated for C 29 H 22 O6[M+Na] + :489.130860,found 489.13055

[0230] Example 35: 4-(((8-(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)methyl)-benzoic acid (Compound I-14)

[0231]

[0232] Compound 7n (195 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound I-14 as a white solid (0.173 g, 88.5% yield).

[0233] The test data of compound I-14 are as follows:

[0234] 1 H NMR (600MHz, DMSO) δ=12.97(s,1H),8.06(s,1H),7.92(d,J=7.7,1H),7.72(dd,J= 18.1,7.9,2H),7.63(t,J=7.3,1H),7.54(t,J=7.6,2H),7.39(t,J=7.6,1H),7.27 (s,1H),7.21(s,1H),7.15(dd,J=12.4,4.5,2H),7.04(dd,J=8.3,1.7,1H),6.91( d,J=8.3,1H),5.72(d,J=8.2,1H),5.42(d,J=8.3,1H),5.23(s,2H),4.29(s,4H). 13 C NMR (151MHz, DMSO) δ = 158.42, 149.22, 142.96, 142.69, 142.53, 140.68, 139.13, 133.41, 132.23, 132.05, 132.04, 131.52, 131.46, 1 29.08,128.80,128.72,127.43,127.34,121.59,120.78,117.90,117.30,116.71,114.77,111.77,72.94,68.93,64.14,64.07.HRMS m / zcalculated for C 29 H 22 O6[M+Na] + :489.13086,found 489.13062

[0235] Example 36: 5-(((8-(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)-pentanoic acid (Compound I-15)

[0236]

[0237] Compound 7o (181 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound I-15 as a white solid (0.144 g, 79.2% yield).

[0238] The test data of compound I-15 are as follows:

[0239] 1 H NMR (600MHz, DMSO) δ = 12.05 (s, 1H), 7.67 (d, J = 8.3, 1H), 7.63 (d, J = 7.5, 1H), 7 .38(t,J=7.6,1H),7.27(s,1H),7.15(dd,J=11.9,8.1,2H),7.11(s,1H),6.92( dd,J=11.3,8.5,2H),5.71(d,J=7.4,1H),5.39(d,J=8.1,1H),4.29(s,4H),4. 01(t,J=6.2,2H),2.30(t,J=7.3,2H),1.76(m,2H),1.68(dt,J=13.8,7.0,2H). 13 C NMR (151MHz, DMSO) δ = 158.90, 149.15, 142.96, 142.69, 142.48, 140.83, 139.11, 133.45, 131.75, 129.07, 127.31, 121 .59,120.74,117.82,117.30,116.72,114.43,111.29,72.93,67.37,64.15,64.08,60.74,33.37,29.21,28.21.HRMS m / z calculated for C 26 H 24 O6[M+Na] + :455.14651,found445.14651

[0240] Example 37: 5-(((8-(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)-9-hydroxy-9H-fluoren-2-yl)oxy)-butanoic acid (Compound I-16)

[0241]

[0242] Compound 7p (175 mg, 0.42 mmol, 1 eq) was dissolved in methanol (55 mL) and tetrahydrofuran (55 mL). Sodium borohydride (189 mg, 2.856 mmol, 6.8 eq) was added under a nitrogen atmosphere and stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction, the mixture was extracted with dichloromethane / water, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain compound I-16 as a white solid (0.133 g, 75.8% yield).

[0243] The test data of compound I-16 are as follows:

[0244] 1 H NMR (600MHz, DMSO) δ=12.17(s,1H),7.68(d,J=8.3,1H),7.63(d,J=7.4,1H),7.39(t,J=7.6,1H),7.28(s,1H),7.16(dd,J=15.3,4.8,2H ),7.11(s,1H),6.93(m,2H),5.71(d,J=8.2,1H),5.40(d,J=8.2,1H),4.29(s,4H),4.03(t,J=6.3,2H),2.42(t,J=7.3,2H),1.97(m,2H). 13 C NMR (151MHz, DMSO)δ=174.14,158.77,149.17,142.95,142.69,142.49,140.80,139.11,133.44,131.83,129.07,127 .33,121.59,120.75,117.84,117.30,116.71,114.48,111.27,72.93,66.81,64.14,64.08,60.74,29.20,24.32.HRMS m / z calculated for C 23 H 18 O5[M+Na] + :353.07843,found 353.07782

[0245] Example 38: Evaluation of the Inhibition of Tumor Proliferation by Compounds in Vitro

[0246] (1) Experimental methods

[0247] 1) Plating: Take tumor cells (4T1, MDA-MB-231, HepG2 and Jurkat) in the logarithmic growth phase and plate them at a cell count of 5×10 3 Cells were seeded into 96-well plates; 100 μL of cell suspension was added to each well; the cells were placed in a cell culture incubator overnight to allow attachment.

[0248] 2) Administration: Discard the old culture medium, wash twice with PBS (skip this step for Jurkat cells), and add 100 μL of target compound solution in gradient concentrations to each well; add complete culture medium containing 0.1% DMSO to the control group; place the 96-well plate in a cell culture incubator and continue incubation for 48 hours;

[0249] 3) Testing: After preparing complete culture medium containing 10% CCK-8 reagent, discard the old culture medium and add 110 μL of complete culture medium containing CCK-8 reagent to each well. Incubate in an incubator for 2 hours. Directly add 10 μL of CCK-8 reagent to each well of Jurkat cells. Measure absorbance using a microplate reader at 450 nm.

[0250] (2) Experimental results

[0251] Table 1 Experimental results of the target compounds inhibiting tumor cell proliferation

[0252]

[0253] As shown in Table 1, the cytotoxicity of the compounds designed in the present invention to the three tumor cells (4T1, Jurkat and B16-F10) is basically the same as that of the positive control drug BMS-1166 and is lower, and cannot directly inhibit the growth of these three tumor cells.

[0254] Example 39: Evaluation of the inhibitory activity of compounds on HepG2 / Jurkat T cells

[0255] (1) Experimental methods

[0256] 1) Activation: Jurkat cells in the logarithmic growth phase were centrifuged, resuspended in complete medium containing PHA (2 μg / mL), transferred to T25 culture flasks, and cultured in a cell culture incubator for 48 hours. HepG2 cells in the logarithmic growth phase were taken and the cell count was 5×10 3 100 μL of cell suspension per well was inoculated into a 96-well plate and cultured overnight in a cell culture medium to allow the cells to adhere. The old culture medium was aspirated, and 100 μL of IFN-γ (10 ng / mL) stimulation solution was added to each well, and the plate was cultured in a cell culture incubator for 24 hours.

[0257] 2) Co-culture: Aspirate the old culture medium from the HepG2 cell 96-well plate, seed the activated Jurkat cells into the HepG2 cell 96-well plate at a target-to-drug ratio of 5:1, add the drug solution at a gradient concentration, and culture the 96-well plate in a cell culture incubator for 48 hours;

[0258] 3) After preparing complete culture medium containing 10% CCK-8 reagent, aspirate the mixture containing Jurkat cells and drug, wash twice with PBS, add 110 μL of complete culture medium containing CCK-8 reagent to each well, and incubate in an incubator for 2 hours; measure the absorbance (OD value) using a microplate reader, selecting 450 nm as the detection wavelength.

[0259] (2) Experimental results

[0260] Table 2 Inhibitory activity of target compounds on HepG2 / Jurkat cells

[0261] Compound Inhibition rate (compound concentration was 50 μM) BMS-1166 72.0％ I-1 80.2％ I-2 85.9％ I-3 90.2％ I-4 91.5％ I-5 90.9％ I-6 82.5％ I-7 77.9％ I-8 80.6％ I-9 81.9％ I-10 79.7％ I-11 78.3％ I-12 77.5％ I-13 83.6％ I-14 87.4％ I-15 85.2％ I-16 75.0％

[0262] As shown in Table 2, in the HepG2 / Jurkat cell co-culture model, the inhibitory rate of the compounds designed in the present invention on HepG2 cells at a concentration of 50 μM was higher than that of the positive control drug BMS-1166 (72.0%), indicating that the strategy of optimizing and modifying the compounds in the present invention is feasible.

[0263] Example 40: HTRF experimental evaluation of target compounds

[0264] (1) Experimental methods

[0265] 1) Preparation of test solution: Dilute the original Tag1-PD-L1 (40X) and Tag2-PD1 (40X) working solutions to 1X with PPI Europium Detection Buffer and set aside. Dilute the original anti-Tag1-PD-L1 (50X) and anti-Tag2-PD1 (50X) working solutions to 1X with Detection Buffer. Mix the diluted anti-Tag1-PD-L1 (1X) and anti-Tag2-PD1 (1X) working solutions in a 1:1 ratio and set aside.

[0266] 2) Positive control, negative control, and buffer control groups: Add 4μL Tag1-PD-L1, 4μL Tag2-PD1, 5μL anti-Tag1-PD-L1, 5μL anti-Tag2-PD1, and 2μL Detection Buffer to each well of the positive control group. Add 4μL Tag1-PD-L1, 5μL anti-Tag1-PD-L1, 5μL anti-Tag2-PD1, and 6μL Detection Buffer to each well of the negative control group. Add 20μL Detection Buffer to each well of the buffer control group. Centrifuge the 384-well plate at 1000 rpm for 1 minute after adding each reagent to prevent the reagent from sticking to the well walls.

[0267] 3) Drug panel setup: Add 2 μL of compound working solution, 4 μL of Tag1-PD-L1, 4 μL of Tag2-PD1, 5 μL of anti-Tag1-PD-L1, and 5 μL of anti-Tag2-PD1 to each well of the drug panel. Centrifuge the 384-well plate at 1000 rpm for 1 minute after each reagent addition to prevent the reagent from sticking to the well walls.

[0268] 4) Incubation: Seal the plate and incubate at room temperature for 1 hour.

[0269] 5) Testing: Fluorescence values were read using a multifunctional microplate reader (excitation wavelength 320 nm, emission wavelengths 620 nm and 665 nm).

[0270] (2) Experimental results

[0271] Table 3 Inhibitory activity of target compounds against PD-1 / PD-L1 protein complex

[0272]

[0273]

[0274] As can be seen from Table 3, in the HTRF experiment of PD-1 / PD-L1 protein binding inhibition, the compounds designed in the present invention had a higher inhibition rate on the PD-1 / PD-L1 protein complex at a concentration of 5 μM than the positive control drug BMS-1166 (65.0%), indicating that the strategy of optimizing and modifying them in the present invention is feasible.

Claims

1. A hydroxyfluorene compound, characterized in that: Having the structure of Formula I, wherein n is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8; R is selected from hydrogen, hydroxy, carboxyl, halogen, amino, mercapto, cyano, C 1-4 Alkyl-OC(O)-, 6-10 membered aryl, 4-10 membered aromatic heterocyclic group containing 1-4 N, O, S ring heteroatoms, 4-7 membered cycloalkyl, 4-7 membered heterocyclic group containing 1-2 N, O, S ring heteroatoms, the 6-10 membered aryl, 4-10 membered aromatic heterocyclic group, 4-7 membered cycloalkyl, 4-7 membered heterocyclic group is replaced by hydrogen, -C(O)-, hydroxyl, carboxyl, halogen, amino, mercapto, cyano, nitro, C 1-4 Alkyl-OC(O)-, carboxyl C 1-4 Alkyl, hydroxyl C 1-4 Alkyl substitution.

2. The hydroxyfluorene compound according to claim 1, characterized in that In the structure, n is selected from 0, 1, 2, 3, and 4.

3. The hydroxyfluorene compound according to claim 1, characterized in that In the structure, R is selected from hydrogen, hydroxyl, carboxyl, halogen, amino, cyano, CH3-OC(O)-, phenyl, pyridyl, tetrahydropyrrolyl, and the phenyl, pyridyl, tetrahydropyrrolyl are substituted by hydrogen, -C(O)-, hydroxyl, carboxyl, halogen, amino, cyano, nitro, CH3-OC(O)-.

4. The hydroxyfluorene compound according to claim 1, characterized in that In the structure, R is selected from any one of the following structures:

5. The hydroxyfluorene compound according to claim 4, characterized in that In the structure described, When n is 0, R is selected from When n is 1, R is selected from When n is 2, 3, 4, 5, 6, 7, or 8, R is selected from -OH-COOH-COOCH3.

6. The hydroxyfluorene compound according to claim 1, characterized in that It is selected from any one of the following compounds:

7. A pharmaceutically acceptable salt of the hydroxyfluorene compound according to claim 1, characterized in that: Salts formed by the compound and hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid, alkali metal ion base, alkaline earth metal ion base, aluminum ion base, zinc ion base, choline, ammonia water, ammonia gas, ethylenediamine, triethylamine, triethanolamine, piperazine, or meglumine.

8. A pharmaceutical composition, characterized in that The invention comprises the hydroxyfluorene compound according to claim 1 or the pharmaceutically acceptable salt according to claim 7 and a pharmaceutically acceptable carrier.

9. Use of the hydroxyfluorene compound according to claim 1, the pharmaceutically acceptable salt according to claim 7, or the pharmaceutical composition according to claim 8 in the preparation of a PD-1 / PD-L1 inhibitor.

10. The use according to claim 9, characterized in that The medicine is an anti-tumor medicine.

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