Double-target inhibitor targeting LSD1 and EZH2 as well as preparation method and application of double-target inhibitor

Abstract

The invention discloses an LSD1 and EZH2 targeted double-target inhibitor and a preparation method and application thereof, the LSD1 and EZH2 targeted double-target inhibitor comprises a structural fragment containing at least one benzene ring and inhibiting LSD1 protein and a structural fragment containing at least one benzene ring and inhibiting EZH2 protein; the structural fragment for inhibiting the LSD1 protein and the structural fragment for inhibiting the EZH2 protein share a benzene ring. The fused double-target inhibitor is formed by sharing benzene rings on the LSD1 target inhibitor and the EZH2 target inhibitor, the fused double-target inhibitor is small in molecular weight and good in druggability, the problems of high molecular weight, poor metabolism and the like can be avoided, and the anti-tumor effect of the fused double-target inhibitor is superior to that of the single LSD1 inhibitor or the single EZH2 inhibitor and is also superior to that of combination of the LSD1 inhibitor and the EZH2 inhibitor under the same condition.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a dual-target inhibitor targeting LSD1 and EZH2, and a preparation method and application thereof. Background Art

[0002] Epigenetic polypharmacology strategies that combine drugs acting on different biological pathways have gradually become a new approach for modern cancer treatment. Drug combinations or epigenetic drugs that simultaneously inhibit two or more targets can improve treatment efficacy and overcome drug resistance in the clinic. They can activate multiple anti-cancer pathways, such as extrinsic or intrinsic apoptosis, inflammation / immune response, growth arrest, mitotic and autophagic cell death, senescence, etc. Therefore, epigenetic inhibitors have become a common tool in cancer research and pharmacology.

[0003] Lysine-specific demethylase 1 (LSD1), also known as lysine-specific demethylase 1A (KDM1A), is a flavin-dependent monoamine oxidase that removes monomethylation and dimethylation of histone 3 lysine 4 (H3K4) residues, thereby inhibiting gene transcription; it can also remove monomethylation and dimethylation of histone 3 lysine 4 (H3K9) residues, thereby activating gene transcription. LSD1 is closely related to tumorigenesis. Overexpression of LSD1 has been observed to promote tumor activity in different types of malignant tumors, such as colorectal cancer, breast cancer, prostate cancer, small cell lung cancer, and acute myeloid leukemia. Therefore, LSD1 has become a promising anticancer drug target. At present, many LSD1 inhibitors with different structures have been widely developed, including TCP, ORY-1001, and GSK-2879552. Enhancer of zeste homolog 2 (EZH2) is a catalytic unit of polycomb repressive complex 2 (PRC2) that promotes mono-, di-, and tri-methylation of histone 3 lysine 27 (H3K27). EZH2 can silence many genes involved in cell proliferation, cell cycle, cell differentiation, and self-renewal. Among them, trimethylation of H3K27 (H3K27me3) contributes to the modification of chromatin structure, leading to the inhibition of gene transcription, thereby promoting differentiation and inhibiting the proliferation of normal cells. EZH2 is overexpressed in a variety of solid tumors and malignancies, including prostate cancer, breast cancer, kidney cancer, lung cancer, myeloma, and leukemia, and its expression level is usually associated with disease progression and poor prognosis. Therefore, EZH2 has become an anti-cancer target that has attracted much attention from researchers, and many EZH2 inhibitors have been reported, including GSK126, EPZ6438, PF-06821497, and CPI-1205. Among them, EZP6438 has been approved by the FDA for the treatment of locally advanced or metastatic epithelioid sarcoma and follicular lymphoma in 2020. LSD1 and EZH2 inhibitors have limited sensitivity to tumors, and compounds with greater anti-tumor activity are urgently needed. Summary of the invention

[0004] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the object of the present invention is to provide a dual-target inhibitor targeting LSD1 and EZH2 and a preparation method and application thereof.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] In the first aspect of the present invention, a dual-target inhibitor of LSD1 and EZH2 is proposed, comprising a structural fragment containing at least one benzene ring for inhibiting LSD1 protein and a structural fragment containing at least one benzene ring for inhibiting EZH2 protein; the structural fragment inhibiting LSD1 protein and the structural fragment inhibiting EZH2 protein share a benzene ring.

[0007] Compared with dual-target inhibitors formed by connecting with linkers, the present invention uses the benzene rings on the shared LSD1 target inhibitor and the EZH2 target inhibitor to form a fusion dual-target inhibitor, which has a small molecular weight and good drugability, and can avoid problems such as high molecular weight and poor metabolism; the main modified sites can also be concentrated on the N-terminus of the structural fragment that inhibits the LSD1 protein and the connection position of the shared benzene ring.

[0008] In some embodiments of the present invention, the structural fragment that inhibits LSD1 protein and the structural fragment that inhibits EZH2 protein share a benzene ring.

[0009] In some embodiments of the present invention, the structural fragment inhibiting LSD1 protein and the structural fragment inhibiting EZH2 protein are located at the para position or meta position on the common benzene ring.

[0010] In some embodiments of the present invention, the structural fragment that inhibits EZH2 protein includes the structural fragment of EPZ6438 that has the EZH2 protein inhibitory effect.

[0011] In some embodiments of the present invention, the structural fragment that inhibits LSD1 protein includes a structural fragment of trans-phenylcyclopropylamine (TCP) that has an inhibitory effect on LSD1 protein.

[0012] In some embodiments of the present invention, the N-terminus of the structural fragment having LSD1 protein inhibitory effect in the TCP is further connected to any two selected from hydrogen, tert-butyloxycarbonyl, 2-morpholin-2-oxyethyl, 2-oxo-2-(piperazin-1-yl)ethyl, N-(tert-butyloxycarbonyl)-2-oxo-2-(piperazin-1-yl)ethyl, 2-fluorobenzyl, 2-trifluoromethylbenzyl, 2-methoxybenzyl, benzyl, cyclopropylmethyl, cyclohexyl, tetrahydro-2H-pyran-4-yl, tetrahydro-2H-pyran-4-yl)methyl, tetrahydro-2H-pyran-4-yl)ethyl, and pivaloyl.

[0013] In a second aspect of the present invention, a compound of formula I, or a pharmaceutically acceptable salt, enantiomer, diastereomer, tautomer, solvate, crystal form or prodrug thereof is provided:

[0014]

[0015] Among them, R 1 , R2 Each of the following is independently selected from hydrogen, tert-butoxycarbonyl, 2-morpholin-2-oxyethyl, 2-oxo-2-(piperazin-1-yl)ethyl, N-(tert-butoxycarbonyl)-2-oxo-2-(piperazin-1-yl)ethyl, 2-fluorobenzyl, 2-trifluoromethylbenzyl, 2-methoxybenzyl, benzyl, cyclopropylmethyl, cyclohexyl, tetrahydro-2H-pyran-4-yl, tetrahydro-2H-pyran-4-yl)methyl, tetrahydro-2H-pyran-4-yl)ethyl, and pivaloyl.

[0016] In some embodiments of the present invention, the compound of formula I is selected from the compound of formula II Compound of formula III Where R 1 , R 2 The definition of is as described above.

[0017] In some embodiments of the present invention, the compound of formula I is selected from the compound of formula II-A Compound of formula III-A Where R 1 , R 2 The definition of is as described above.

[0018] In some embodiments of the present invention, R 1 Selected from H.

[0019] In some embodiments of the present invention, R 2 Selected from H, Boc.

[0020] In some embodiments of the present invention, the compound of formula I is selected from the following compounds and / or isomers thereof:

[0021]

[0022]

[0023]

[0024] In some embodiments of the present invention, the isomers include enantiomers. In the present invention, the above compounds only show one of the isomers, and the compound of formula I may actually include the enantiomers of the above compounds to form a racemic compound (±). To avoid redundancy, the above compounds do not list their enantiomers.

[0025] In some embodiments of the present invention, the pharmaceutically acceptable salt is selected from hydrochloride, hydrobromide, sulfate, bisulfate, phosphate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, benzenesulfonate, naphthalene disulfonate, acetate, propionate, lactate, trifluoroacetate, maleate, citrate, fumarate, oxalate, tartrate, benzoate, succinate, camphorsulfonate, diethanolamine salt, glycine salt, lysine salt, magnesium salt, meglumine salt, ethanolamine salt, sodium salt, potassium salt, ammonium salt, tromethamine salt, zinc salt, arginine salt, hexafluorophosphate, hydroiodide, trifluoroacetate, xinafoate, isethionate, formate, and maleate.

[0026] The third aspect of the present invention provides a method for preparing the compound of formula I, comprising the following steps:

[0027] The compound of formula IA With the compound of formula IB The reaction produces a compound of formula I; wherein X is a halogen atom (such as F, Cl, Br, I, etc.), R 1 , R 2 The definition of is as described above.

[0028] In some embodiments of the present invention, the reaction is carried out in an alkaline environment using NaOH, KOH, Na 2 CO 3 , K 2 CO 3 The reaction environment is adjusted to be alkaline.

[0029] In some embodiments of the present invention, the reaction is carried out in the presence of a catalyst, wherein the catalyst comprises Pd(PPh 3 ) 4 、Pd(dppf) 2 Cl 2 .

[0030] In some embodiments of the present invention, the compound of formula I-A1 is subjected to a condensation reaction with the compound of formula I-A2 to obtain the compound of formula IA; preferably, the condensation reagent used in the condensation reaction includes HOBT and EDCI.

[0031]

[0032] In some embodiments of the present invention, the compound of formula I-B1 is subjected to a Curtius rearrangement reaction to obtain a compound of formula I-B2, and the compound of formula I-B2 is subjected to a Miyaura borylation reaction. Preferably, the reagent used in the Curtius rearrangement reaction is DPPA, Et 3N, t-Butanol; the reagent used in the Miyaura borylation reaction is Bis(pinacolato)diboron, Pd(dppf)Cl 2 ,AcOK,DMF.

[0033]

[0034] The fourth aspect of the present invention provides a pharmaceutical composition comprising the LSD1 and EZH2 dual-target inhibitor or the compound of formula I.

[0035] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier.

[0036] "Pharmaceutically acceptable excipients or carriers" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0037] The administration method of the compound or pharmaceutical composition of the present invention is not particularly limited. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and topical administration.

[0038] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; (c) humectants, such as glycerol; (d) disintegrators, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates and sodium carbonate; (e) solubilizers, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0039] Solid dosage forms, such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microcapsules with one or more of the above-mentioned excipients.

[0040] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0041] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0042] Suspensions, in addition to the active compounds, may contain suspending agents, for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0043] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0044] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0045] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0046] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage. For a person weighing 60 kg, the daily dosage is usually 1 to 5000 mg, preferably 5 to 2000 mg, such as 10 mg, 20 mg, 30 mg, 50 mg, 100 mg, 200 mg, etc. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill range of skilled physicians.

[0047] The fifth aspect of the present invention provides the use of the LSD1 and EZH2 dual-target inhibitor or the compound of formula I in the preparation of anti-tumor drugs.

[0048] In some embodiments of the present invention, the tumor includes solid tumors and hematological tumors.

[0049] In some embodiments of the present invention, the solid tumor is selected from lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, kidney cancer, uterine cancer, prostate cancer or melanoma; the blood tumor is selected from leukemia or lymphoma.

[0050] The beneficial effects of the present invention are:

[0051] LSD1 and EZH2 are two different histone methyltransferases, EZH2 can promote H3K27 trimethylation and LSD1 can remove H3K4 / K9 monomethyl and dimethyl. Although LSD1 and EZH2 perform different methyl transfer functions, they have the same regulation on gene transcription and can inhibit gene transcription. The inventors found that the LSD1 and EZH2 dual-target inhibitor obtained by sharing a benzene ring with the structural fragment that inhibits the EZH2 protein and the structural fragment that inhibits the LSD1 protein has an unexpected anti-tumor effect, which is better than the single EZH2 inhibitor or BRD4 inhibitor, and is also better than the combination of the two under the same conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1The effect of the compounds of the present invention on the protein levels of H3K27me3 downstream of EZH2 and H3K4me2 and H3K9me2 downstream of LSD1; wherein the upper figure is the detection result, and the lower figure is the quantification result;

[0053] Figure 2 The effects of the compound 18j of the present invention and the existing EZH2 inhibitor EPZ6438 and LSD1 inhibitor TCP on the viability of different prostate cancer cells;

[0054] Figure 3 The anti-tumor effect of the compound 18j of the present invention in combination with EPZ6438, TCP or both in a 22RV1 nude mouse subcutaneous transplanted tumor model;

[0055] Figure 4 These are the anti-tumor results of the compound 18j of the present invention used in combination with EPZ6438, TCP or both in different tumor cell lines. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0057] Example 1: Synthesis of Compound 18j / 19j

[0058] Synthesis of intermediate 6:

[0059]

[0060] Reagents and reaction conditions: (a) Fe, NH 4 Cl,MeOH,90℃; (b)tetrahydro-4H-pyran-4-one,AcOH,Na(AcO) 3 BH,1,2-dichloroethane; (c)acetaldehyde,AcOH,Na(AcO) 3 BH,1,2-dichloroethane; (d)Boc 2 O,H 2 , Raney-Ni, MeOH; (e) HCl, MeOH; (f) NaOH, EtOH, 60℃; (g) HOBt, EDCI, NMM, DMSO.

[0061] As shown in the above reaction process, the commercially purchased compound 1 (900 mg, 3.28 mmol) was used as the starting material for synthesis, iron powder and ammonium chloride were added, and the mixture was refluxed at 90°C in a methanol solution for overnight reaction. After TLC detection of the reaction was complete, the reaction system was filtered, the filtrate was collected and distilled under reduced pressure, and the concentrated product was separated and purified by silica gel column chromatography using an ethyl acetate / petroleum ether system to obtain a light yellow liquid product intermediate 2 (723 mg, 2.96 mmol) with a yield of about 90%.

[0062] Intermediate 2 (700 mg, 2.87 mmol), tetrahydropyrone and acetic acid were added to a round-bottom flask containing dry anhydrous 1,2-dichloroethane (DCE) and stirred at room temperature for 30 min. Then, sodium triacetoxyborohydride was added to an ice bath, and the ice bath was removed to react at room temperature for 24 h. After TLC detection, the solvent was removed by vacuum distillation, and the crude product after concentration was extracted with ethyl acetate / water system. The organic phase was collected and dried with anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The white solid obtained by product purification after concentration was dissolved in an appropriate amount of anhydrous DCE, acetic acid was added and acetaldehyde was injected into the reaction system. After stirring and reacting at room temperature for 30 min, sodium triacetoxyborohydride was added to an ice bath and reacted at room temperature for 24 h. The post-treatment was the same as the above white solid to obtain a light yellow liquid intermediate 3 (890 mg, 2.5 mmol) with a yield of about 87%.

[0063] The compound 4 purchased from business was used as raw material (500mg, 3.37mmol) in methanol solution, and appropriate amount of Raney nickel and Boc anhydride were added, hydrogen was replaced three times, and the reaction was continued overnight under hydrogen protection. After TLC detection reaction was complete, the reaction system was filtered, the filtrate was collected and distilled under reduced pressure, and the product purification after concentration obtained a white solid, which was added to the reaction system of acetyl chloride and methanol solution under ice bath conditions, and room temperature reaction was 3h. After TLC detected that the reaction was complete, extraction operation was performed. First, dichloromethane / water was used to extract, and aqueous phase was collected. With 1M hydrochloric acid solution, it was adjusted that aqueous phase pH was 3-4, and ethyl acetate was used to extract, and organic phase was collected. The hydrochloride of product intermediate 5 was obtained by distillation under reduced pressure.

[0064] Intermediate 3 (787 mg, 210 mmol), intermediate 5 (403.5 mg, 265 mmol), HOBt and EDCI were added to the DMSO solution, and N-methylmorpholine was injected into the reaction system, and stirred at room temperature overnight. After TLC detection, the reaction was complete, and then extracted with ethyl acetate / water system, the organic phase was collected, and dried with anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The concentrated product was separated and purified by silica gel column chromatography using methanol / dichloromethane system to obtain light yellow solid intermediate 6 (1 g, 210 mmol), with a yield of about 95%.1 H NMR (400MHz, CDCl3) δ11.12(s,1H),7.22(d,J=1.8Hz,1H),7.18(d,J=1.8Hz,1H),7.12(t,J=5.6Hz,1H),5.95(s,1H),4.52(d,J=5.9Hz,2H),3.95 (d,J=11.4Hz,2H),3.35-3.27(m,2H),3.02(q,J=6.9Hz,2H),2.93(m,1H) ,2.39(s,3H),2.24(d,J=2.3Hz,6H),1.66(m,4H),0.85(t,J=7.0Hz,3H).

[0065] Synthesis of intermediate 11:

[0066]

[0067] Reagents and reaction conditions: (a) OSMe 3 I,t-BuOK,DMSO,50-60℃,rt,47.3%; (b)NaOH,MeOH,H 2 O, 60℃, 100%; (c) DPPA, Et 3 N,t-Butanol,80℃,61.8%;(d)Bis(pinacolato)diboron,Pd(dppf)Cl 2 ,AcOK,DMF,90.2%.

[0068] Trimethylsulfoxide iodide (2.2 g, 10.14 mmol) and potassium tert-butoxide (1.0 g, 10.36 mmol) were dissolved in anhydrous dimethylsulfoxide (20 mL) and heated to 55 ° C under argon and stirred for 1.5 h. After completion, the reaction mixture was cooled to room temperature and a commercially available solution of trans-4-bromocinnamic acid ethyl ester (Compound 7) (2.0 g, 7.80 mmol) in anhydrous DMSO was slowly added. After stirring at room temperature for 5 h, the mixture was heated with precooled NH 4Cl aqueous solution was slowly dripped into the reaction mixture. The water layer was extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The product after the residue was concentrated was separated and purified by silica gel column chromatography using ethyl acetate / petroleum ether system to obtain intermediate 8 (1.0 g) as a colorless oil with a yield of about 47.3%. 1M NaOH aqueous solution was added to a solution of the colorless oil (1.0 g, 3.72 mmol) in MeOH (5 mL). The reaction mixture was stirred at room temperature for 12 h to obtain intermediate 9 (1.0 g) as a white solid with a yield of about 100%. Intermediate 9 (1.0 g, 4.10 mmol) was dissolved in anhydrous tert-butyl alcohol solution, and diphenylphosphoryl azide (1.1 mL, 5.10 mmol) and triethylamine (850 μL, 4.90 mmol) were added dropwise to the above system at 0 ° C. The reaction mixture was stirred at 80 ° C under argon for 48 h and extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The product after the residue was concentrated was separated and purified by silica gel column chromatography using ethyl acetate / petroleum ether system to obtain intermediate 10 (800 mg) with a yield of about 61.8%. To a solution of intermediate 10 (800 g, 2.6 mmol) in N,N-dimethylformamide (DMF) (10 mL) were added diboric acid pinacol ester (787 mg, 3.10 mmol), potassium acetate (765 mg, 7.80 mmol) and Pd(dppf)Cl 2 (98 mg, 0.10 mmol). The reaction mixture was stirred at 90 ° C under argon for 12 h. It was then extracted with ethyl acetate / water system, the organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The product after the residue was concentrated was separated and purified by silica gel column chromatography using ethyl acetate / petroleum ether system to obtain intermediate 11 (830 mg) as a white solid with a yield of about 90.2%. 1 H NMR (400 MHz, CDCl 3 )δ7.72(d,J=8.0Hz,2H),7.12(d,J=8.0Hz,2H),2.84–2.73(m,1H),.10–2.01(m,1H),1.47(d,J=4.2Hz,9H),1.35(s,12H),1.24–1.17(m,2H).

[0069] Synthesis of intermediate 16:

[0070]

[0071] Reagents and reaction conditions: (a) OSMe 3I,t-BuOK,DMSO,50-60℃,rt,47.3%; (b)NaOH,MeOH,H 2 O, 60℃, 100%; (c) DPPA, Et 3 N,t-Butanol,80℃,61.8%;(d)Bis(pinacolato)diboron,Pd(dppf)Cl 2 ,AcOK,DMF,90.2%.

[0072] As shown in the above reaction process, commercially purchased trans-3-bromocinnamic acid ethyl ester (Compound 12) was used as the raw material, and the reaction process was referred to the synthesis process of Intermediate 11. 1H NMR (400MHz, CDCl3) δ7.64 (d, J = 7.0 Hz, 1H), 7.57 (s, 1H), 7.30 (d, J = 7.7 Hz, 1H), 7.26 (dd, J = 9.8, 3.4 Hz, 1H), 2.86-2.73 (m, 1H), 2.09-2.01 (m, 1H), 1.48 (s, 9H), 1.36 (s, 12H), 1.20-1.10 (m, 2H).

[0073] Synthesis of compound 18j / 19j:

[0074]

[0075] Reagents and reaction conditions: (a) 4-Bromomethyltetrahydropyran, NaH, DMF, 0℃ to rt, 70%; (b) intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H 2 O, 90°C, 88.9%; (c) HCl / MeOH, rt, 100%.

[0076] At 0°C, a solution of sodium hydride (21.6 mg, 0.90 mmol) in anhydrous DMF (2 mL) was slowly added dropwise to a solution of intermediate 11 (100 mg, 0.30 mmol) in anhydrous DMF (2 mL). After stirring at room temperature for 30 min, 4-Bromomethyltetrahydropyran was added, and the reaction mixture was stirred at room temperature for 16 h, then quenched with cold water and extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The product after the residue was concentrated was separated and purified by silica gel column chromatography using ethyl acetate / petroleum ether system to obtain intermediate 17j (80 mg) as a yellow solid with a yield of about 62.8%.

[0077] Toluene / EtOH / H 2 O=3 / 1 / 2 (6 mL) solution was added with intermediate 17j (80 mg, 0.20 mmol), K 2 CO 3 (82.9 mg, 0.60 mmol) and Pd(PPh 3 ) 4 (23.1 mg, 0.06 mmol). Stir at 90 ° C under argon for 12 h. Then extract with ethyl acetate / water system, collect the organic phase, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure. The residue is separated and purified by silica gel column chromatography using methanol / dichloromethane system to obtain compound 18j (113 mg) as a white solid with a yield of about 88.9%. 1 H NMR (400MHz, DMSO) δ11.46(s,1H),8.19(t,J=4.9Hz,1H),7.52(d,J=8.3Hz,2H),7.37(s,1H),7.21(d,J=8.3Hz,2H),7.20(s ,1H),5.86(s,1H),4.29(d,J=4.9Hz,2H),3.83(d,J=10.6Hz,4H),3.31–3.23(m,4H),3.23(d,J=4.1Hz,1H),3.08(dd,J=14.5 ,7.5Hz,2H),3.05–2.97(m,2H),2.71–2.64(m,1H),2.24(s,3H),2.21(s,3H),2.16–2.12(m,1H),2.11(s,3H),1.92–1.80(m ,1H),1.66(d,J=11.0Hz,2H),1.58–1.46(m,4H),1.35(s,9H),1.32–1.25(m,2H).,1.20–1.13(m,2H),0.83(t,J=7.0Hz,3H). 13 C NMR(126MHz,MeOD)δ171.7,164.2,152.2,149.2,143.5,140.4,139.0,138.3,137.8,132.7,126.2,123.6,121.3,120.6, 109.7,79.8,67.3,66.8,58.3,41.7,38.9,35.2,34.0,30.5,30.4,27.3,18.3,17.2,13.7,11.7.HRMS(ESI-TOF)m / z[M+H] + Calculate for C43 H 59 N 4 O 6 727.44291, found727.44220.

[0078] Acetyl chloride (0.5 mL) was added to MeOH (5 mL) to prepare a solution of HCl in MeOH in advance. Compound 18j (50 mg, 0.07 mmol) was added to the freshly prepared HCl solution. After stirring at room temperature for 2 h, the reaction mixture was concentrated under reduced pressure to give compound 19j (43.1 mg) as a white solid without further purification, with a yield of about 100%. 1 HNMR (400MHz, DMSO) δ11.46(s,1H),8.19(t,J=4.9Hz,1H),7.52(d,J=8.3Hz,2H),7.37(s,1H),7.21(d,J=8.3Hz,1H),7.20( s,1H),5.86(s,1H),4.29(d,J=4.9Hz,2H),3.83(d,J=10.6Hz,4H),3.31–3.23(m,2H),3.23(d,J=4.1Hz,1H),3.08(dd,J=14. 5,7.5Hz,2H),3.05–2.97(m,2H),2.71–2.64(m,1H),2.24(s,3H),2.21(s,3H),2.16–2.12(m,1H),2.11(s,3H),1.92–1.80( m,1H),1.66(d,J=11.0Hz,2H),1.58–1.46(m,4H),1.35(s,9H),1.27–1.20(m,1H),1.20–1.13(m,2H),0.83(t,J=7.0Hz,3H). 13 C NMR(126MHz,MeOD)δ171.7,164.2,152.2,149.2,143.5,140.4,139.0,138.3,137.8,132.7,126.2,123.6,121.3,120.6, 109.7,79.8,67.3,66.8,58.3,41.7,38.9,35.2,34.0,30.5,30.4,27.3,18.3,17.2,13.7,11.7.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 43 H 59 N 4 O 6 727.44291, found 727.44220.

[0079] Example 2: Synthesis of Compound 18a / 19a

[0080]

[0081] Reagents and reaction conditions: (a) Intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H 2 O, 90°C, 88%; (b) HCl / MeOH, rt, 100%.

[0082] As shown in the above reaction process, the synthesis of compound 18a / 19a refers to the synthesis process of compound 18j / 19j. Compound 18a: 1 HNMR(500MHz,MeOD)δ7.43(d,J=8.1Hz,2H),7.39(s,1H),7.25(s,1H),7.13(d,J=8.1Hz,2H),6. 07(s,1H),4.44(s,2H),3.87(d,J=10.7Hz,2H),3.31(t,J=11.2Hz,2H),3.15–3.06(m,2H),3.07– 2.99(m,1H),2.67–2.58(m,1H),2.35(s,3H),2.28(s,3H),2.20(s,3H),2.01–1.92(m,1H).,1.7 1(d,J=11.9Hz,2H),1.65–1.53(m,2H),1.40(s,9H),1.12t,J=6.6Hz,2H),0.85(t,J=6.9Hz,3H). 13 C NMR(126MHz,MeOD)δ173.2,165.6,165.2,159.0,153.6,150.6,144.8,142.1,140.4,139.8,139.0,134.0,127.7,127.6,125.0,1 22.7,122.0,111.1,68.2,59.7,43.1,36.7,36.6,33.9,31.8,28.7,25.0,19.7,18.5,16.5,15.0,13.0.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 37 H 49 N 4 O 5 629.36975, found 629.36914.

[0083] Compound 19a: 1 H NMR(500MHz,MeOD)δ7.48(d,J=8.0Hz,2H),7.42(s,1H),7.28(s,1H),7.15(d,J=8.0Hz,2H),6.10(s ,1H),4.48(s,2H),3.90(d,J=10.3Hz,2H),3.34(t,J=11.4Hz,2H),3.13(dd,J=14.0,7.0Hz,2H),3. 10–3.04(m,1H),2.70–2.63(m,1H),2.38(s,3H),2.31(s,3H),2.24(s,3H),2.17–2.11(m,1H),1.74 (d,J=11.6Hz,2H),1.68–1.58(m,2H),1.23–1.21(m,1H),1.20–1.14(m,1H),0.88(t,J=6.8Hz,3H). 13 C NMR(126MHz,MeOD)δ173.0,165.,153.58,150.6,144.8,141.3,140.4,139.6,139.3,134.1,127.8,127.5,125.0,12 2.6,121.9,111.0,68.2,59.7,43.1,36.5,34.2,31.8,24.3,19.7,18.6,16.3,15.0,13.0.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 32 H 41 N 4 O 3 529.31732, found 529.31714.

[0084] Example 3: Synthesis of Compound 18b / 19b

[0085]

[0086] Reagents and reaction conditions: (a) 2-chloro-1-morpholinoethan-1-one, NaH, DMF, 0℃ to rt, 77%; (b) intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H 2 O, 90°C, 88%; (c) HCl / MeOH, rt, 92%.

[0087] As shown in the above reaction process, the synthesis of compound 18b / 19b refers to the synthesis process of compound 18j / 19j. Compound 18b:. 1 HNMR(400MHz,MeOD)δ7.51(d,J=8.2Hz,2H),7.46(s,1H),7.32(s,1H),7.21(d,J=5.1Hz,2H),6.14(s,1H),4.52(s ,2H),4.20(q,J=17.1Hz,2H),3.95(d,J=11.3Hz,2H),3.74–3.67(m,4H),3.62–3.54(m,4H),3.39(t,J=11.6Hz,2H ),3.18(dd,J=14.1,7.0Hz,2H),3.14–3.07(m,1H),3.04–2.93(m,1H),2.42(s,3H),2.35(s,3H),2.31–2.28(m,1H ),2.27(s,3H),1.79(d,J=10.9Hz,2H),1.73–1.60(m,2H),1.44(s,9H),1.39–1.32(m,2H),0.93(t,J=7.0Hz,3H). 13 CNMR(101MHz,MeOD)δ173.,169.74,165.6,153.5,150.6,144.8,141.9,140.4,139.8,139.2 ,134.1,127.6,125.0,122.7,122.0,111.1,81.6,68.2,67.7,67.6,59.8,54.8,46.3,43.57 43.1,41.3,36.6,31.9,28.7,19.7,18.6,15.1,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 43 H 58 N 5 O 7 756.43308, found 756.433308.

[0088] Compound 19b: 1H NMR (400MHz, MeOD) δ7.47(d,J=8.2Hz,2H),7.43(s,1H),7.30(s,1H),7.13(d,J=8.2Hz,2H),6.12(s,1H),4.50(s ,2H),3.93(d,J=11.1Hz,2H),3.68–3.63(m,4H),3.62–3.57(m,4H),3.51–3.46(m,2H),3.41–3.35(m,2H),3.16( dd,J=14.2,7.1Hz,2H),3.12–3.05(m,1H),2.54–2.47(m,1H),2.40(s,3H),2.33(s,3H),2.25(s,3H),2.03–1.99 (m,1H),1.76(d,J=11.1Hz,2H),1.68–1.62(m,2H),1.18–1.13(m,1H),1.07–1.03(m,1H),0.90(t,J=5.2Hz,3H). 13 C NMR(126MHz,MeOD)δ173.0,165.5,153.5,150.5,144.8,142.0,140.4,139.7,139.1,134.0,127.7,127.4,125.0,122.6,121.9,111.0,101.3,68 .2,67.5(d,J=14.3Hz),59.7,46.2,43.3,43.0,41.7,36.5,31.8,30.7, 24.9,24.6,23.6,19.6,18.5,16.6,15.0,14.4.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 38 H 50 N 5 O 5 656.38065, found 656.38068.

[0089] Example 4: Synthesis of Compound 18c / 19c

[0090]

[0091] Reagents and reaction conditions: (a) tert-butyl 4-(2-chloroacetyl)piperazine-1-carboxylate, NaH, DMF, 0℃ to rt, 77%; (b) intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO3 ,Toluene,EtOH,H 2 O, 90°C, 87%; (c) HCl / MeOH, rt, 94%.

[0092] As shown in the above reaction process, the synthesis of compound 18c / 19c refers to the synthesis process of compound 18j / 19j. Compound 18c: 1 HNMR(400MHz,MeOD)δ7.47(d,J=8.2Hz,2H),7.43(s,1H),7.29(s,1H),7.18(d,J=5.4Hz,2H),6.11(s,1H),4.48(s,2H) ,4.18(q,J=17.1Hz,2H),3.91(d,J=11.4Hz,2H),3.56(d,J=4.5Hz,2H),3.47(d,J=25.7Hz,6H),3.36(t,J=10.9Hz,2H), 3.14(dd,J=14.1,7.1Hz,2H),3.11–3.05(m,1H),3.00–2.89(m,1H),2.39(s,3H),2.31(s,3H),2.29–2.25(m,1H),2.24 (s,3H),1.75(d,J=11.2Hz,2H),1.69–1.60(m,2H),1.47(s,9H),1.41(s,9H),1.34–1.30(m,2H),0.89(t,J=7.0Hz,3H). 13 C NMR (126MHz, MeOD) δ172.9,165.6,156.1,153.5,150.6,144.8,141.9,140.3,139.7,139.1,134.1,127.5,124.9,122.7,121.9,111 .0,81.62,68.2,59.7,54.8,45.5,43.1,42.9,41.2,36.5,31.8,30.7,28.7,28.6,19.7,18.6,15.1,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 48 H 67 N 6 O 8 855.50073, found 855.50149.

[0093] Compound 19c: 1H NMR(400MHz,MeOD)δ7.46(d,J=8.2Hz,2H),7.41(s,1H),7.28(s,1H),7.13(d,J=8.2Hz,2H),6.11(s,1H),4 .48(s,2H),3.92(d,J=11.1Hz,2H),3.76(d,J=4.0Hz,2H),3.73–3.67(m,4H),3.37(d,J=10.5Hz,2H),3.19– 3.10(m,6H),3.10–3.05(m,1H),2.57–2.52(m,1H),2.39(s,3H),2.31(s,3H),2.24(s,3H),2.09–2.03(m,1 H),1.75(d,J=12.0Hz,2H),1.68–1.60(m,2H),1.21–1.17(m,1H),1.11–1.04(m,1H),0.89(t,J=4.8Hz,3H). 13 CNMR(126MHz,MeOD)δ173.1,165.6,153.6,150.6,144.8,142.7,140.4,139.8,138.9,134.0,130.8,127.6,127.3,125.0,122.7,121.9,111. 0,68.2,59.7,54.8,46.3,46.1,43.1,36.6,31.8,30.7,30.4,28.1,25.09,23.7,19.7,18.5,17.2,15.0,14.4,13.0.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 38 H 51 N 6 O 4 655.39663, found 655.39716.

[0094] Example 5: Synthesis of Compound 18d / 19d

[0095]

[0096] Reagents and reaction conditions: (a) 1-(bromomethyl)-2-fluorobenzene, NaH, DMF, 0℃ to rt, 66%; (b) intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H 2O, 90°C, 85%; (c) HCl / MeOH, rt, 92%.

[0097] As shown in the above reaction process, the synthesis of compound 18d / 19d refers to the synthesis process of compound 18j / 19j. Compound 18d: 1 HNMR(500MHz,MeOD)δ7.35(d,J=8.2Hz,2H),7.32(s,1H),7.18(d,J=1.5Hz,2H),7.16(s,1H),7.03(t,J=7.1Hz,3H),7.0 0–6.94(m,1H),6.01(s,1H),4.55(d,J=16.1Hz,1H),4.44(d,J=15.8Hz,1H),4.38(s,2H),3.81(d,J=10.7Hz,2H),3.25(t ,J=11.2Hz,2H),3.04(dd,J=13.9,6.9Hz,2H),3.00–2.93(m,1H),2.62–2.57(m,1H),2.29(s,3H),2.21(s,3H),2.14(s,3 H),2.13–2.07(m,1H),1.64(d,J=12.0Hz,2H),1.57–1.49(m,2H),1.32(s,9H),1.28–1.24(m,2H),0.78(t,J=6.9Hz,3H). 13 C NMR(126MHz,MeOD)δ172.9,165.6,162.0(d,J C-F =245.1Hz),153.5,150.6,144.8,141.6,140.3,139.7,139.1,134.1,130. 5(d,J=4.2Hz),130.1(d,J=8.1Hz),127.6,127.5,125.3(d,J=3.3Hz),124 .9,122.7,122.0,116.3,116.2,111.0,81.6,75.8,68.2,59.7,43.1,40.3 ,36.6,31.8,28.7,25.0,19.7,18.6,15.1,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 44 H 54 FN 4 O 5 737.40728, found737.40686.

[0098] Compound 19d: 1H NMR(500MHz,MeOD)δ7.46(s,1H),7.43(d,J=2.8Hz,2H),7.38(d,J=5.8Hz,1H),7.31(s,1H),7.29(d,J=4.0Hz,1H),7.17–7. 12(m,1H),7.10(s,1H),7.06(d,J=2.1Hz,2H),6.11(s,1H),4.49(s,2H),4.00(d,J=3.3Hz,2H),3.91(d,J=10.8Hz,2H),3.38 –3.32(m,2H),3.13(dd,J=12.5,6.5Hz,2H),3.10–3.04(m,1H),2.45(s,1H),2.39(d,J=5.4Hz,3H),2.32(s,3H),2.24(s,3H) ,2.01–1.95(m,1H),1.74(d,J=12.2Hz,2H),1.67–1.62(m,2H),1.20–1.16(m,1H),1.10–1.03(m,1H),0.89(t,J=7.0Hz,3H). 13 C NMR(126MHz,MeOD)δ173.0,165.6,162.6(d,J C-F =245.1Hz),153.5,150.6,144.8,142.1,140.4,139.7,139.0,134.1,13 2.3(d,J=3.8Hz),130.7(d,J=8.0Hz),127.6,127.4,125.4,125.0,122.7 ,121.9,116.4,116.2,111.0,68.2,59.7,47.0,43.1,41.61,36.6,31.8, 28.8,25.5,24.7,19.7,18.5,16.4,15.0,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 39 H 46 FN 4 O 3 637.35485, found 637.35541.

[0099] Example 6: Synthesis of Compound 18e / 19e

[0100]

[0101] Reagents and reaction conditions: (a) 1-(Bromomethyl)-2-methoxybenzene, NaH, DMF, 0℃ to rt, 78%; (b) intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H 2 O, 90°C, 85%; (c) HCl / MeOH, rt, 91%.

[0102] As shown in the above reaction process, the synthesis of compound 18e / 19e refers to the synthesis process of compound 18j / 19j. Compound 18e: 1 HNMR(400MHz,MeOD)δ7.46(d,J=8.2Hz,2H),7.44(s,1H),7.29(s,1H),7.28–7.22(m,1H),7.15(d,J=7.5Hz,1H),7.12(d,J=8 .0Hz,2H),6.96(d,J=8.1Hz,1H),6.91(t,J=7.5Hz,1H),6.13(s,1H),4.63–4.53(m,2H),4.50(s,2H),3.94(d,J=10.3Hz,2H) ,3.79(s,3H),3.39(d,J=11.7Hz,2H),3.15(t,J=7.0Hz,2H),3.11(s,1H),2.74–2.68(m,1H),2.41(s,3H),2.33(s,3H),2.26 (s,3H),2.22–2.17(m,1H),1.77(d,J=12.2Hz,2H),1.69–1.63(m,2H),1.45(s,9H),1.24–1.14(m,2H),0.91(t,J=5.6Hz,3H). 13 C NMR (126MHz, MeOD) δ173.0,165.6,158.5,153.5,150.6,144.8,141.9,140. 3,139.7,139.0,134.1,129.4,129.1,127.6,127.5,127.4,124.9,122.7,12 2.0,121.3,111.3,111.0,81.3,68.2,61.5,59.7,55.7,43.1,40.5,36.6,31 .8,30.7,28.7,23.7,19.7,18.5,15.1,14.4,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 45H 57 N 4 O 6 749.42726, found749.42645.

[0103] Compound 19e: 1 H NMR(500MHz,MeOD)δ7.45(d,J=8.1Hz,2H),7.42(s,1H),7.28(s,1H),7.11(d,J=8.2Hz,2H),6.11(s,1H), 4.48(s,2H),3.91(d,J=10.6Hz,2H),3.58(s,4H),3.46(s,2H),3.36(t,J=9.1Hz,2H),3.16–3.12(m,2H),3 .10–3.05(m,1H),2.80(s,4H),2.50–2.44(m,1H),2.39(s,3H),2.31(s,3H),2.24(s,3H),2.01–1.97(m,1H ),1.75(d,J=11.6Hz,2H),1.66–1.61(m,2H),1.15–1.10(m,1H),1.05–1.00(m,1H),0.89(t,J=7.0Hz,3H). 13 C NMR(126MHz,MeOD)δ173.1,165.6,159.2,153.6,150.6,144.8,142.7,140.4 ,139.8,138.8,134.0,131.4,130.8,129.9,127.6,127.3,125.0,122.7,121 .9,121.4,111.4,111.1,101.4,68.2,59.7,55.7,43.1,41.7,36.6,31.8,30 .7,30.3,28.1,24.9,19.7,18.5,16.7,15.0,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 40 H 49 N 4 O 4 649.37483, found 649.37469.

[0104] Example 7: Synthesis of Compound 18f / 19f

[0105]

[0106] Reagents and reaction conditions: (a) Benzyl bromide, NaH, DMF, 0℃ to rt, 68%; (b) Intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H 2 O, 90°C, 83%; (c) HCl / MeOH, rt, 93%.

[0107] As shown in the above reaction process, the synthesis of compound 18f / 19f refers to the synthesis process of compound 18j / 19j. Compound 18f: 1 HNMR(400MHz,MeOD)δ7.45(d,J=8.2Hz,2H),7.41(s,1H),7.34–7.29(m,2H),7.28(d,J=1.7Hz,1H),7.26(s,2H),7.24–7.21(m,1 H),7.11(d,J=8.1Hz,2H),6.10(s,1H),4.59(d,J=15.5Hz,1H),4.48(s,2H),4.45(d,J=7.7Hz,1H),3.91(d,J=11.0Hz,2H),3.36 (d,J=10.1Hz,2H),3.14(q,J=7.1Hz,2H),3.10–3.03(m,1H),2.70–2.64(m,1H),2.39(s,3H),2.31(s,3H),2.24(s,3H),2.22–2. 14(m,1H),1.74(d,J=11.2Hz,2H),1.68–1.56(m,2H),1.43(s,9H),1.39–1.32(m,1H),1.24–1.16(m,1H),0.88(t,J=7.0Hz,3H). 13 C NMR(101MHz,MeOD)δ173.1,165.6,153.6,150.6,144.8,141.7,140.4,139.9,139.7,139.2,134.1,129.5,128.4,128.2,127.6,12 7.5,125.0,122.7,122.0,111.0,81.5,68.2,59.7,43.1,40.4,36.6,31.8,28.7,19.7,18.5,15.0,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 44 H 55 N 4 O 5719.41670, found 719.41626.

[0108] Compound 19f: 1 H NMR(500MHz,MeOD)δ8.07(s,1H),7.83(s,1H),7.72(d,J=3.6Hz,2H),7.52–7.47(m,2H),7.42(d,J=1.5Hz,2H ),7.40(d,J=9.6Hz,1H),7.24(d,J=6.6Hz,2H),6.67(s,1H),4.57(s,2H),4.37(s,2H),4.25–4.11(m,1H),4.1 0–3.84(m,4H),3.38(dd,J=54.0,12.2Hz,2H),3.07–2.99(m,1H),2.51(s,3H),2.50(s,3H),2.40(s,3H),2.1 6–1.96(m,1H),1.69(d,J=32.6Hz,2H),1.59–1.54(m,1H),1.45–1.36(m,1H),1.32–1.24(m,2H),1.05(s,3H). 13 C NMR (126MHz, DMSO) δ172.3,158.5,147.7,142.7,140.4,137.5,132.2,131.2,130.7,130.3,128.6,128.5,128.2,122.4,1 22.3,116.8,69.4,67.1,52.9,51.4,39.1,36.5,30.8,30.0,22.3,20.2,18.7,15.4,13.9,10.5.HRMS(ESI-TOF)m / z[M+H] + calcdfor C 39 H 47 N 4 O 3 619.36427, found 619.36359.

[0109] Example 8: Synthesis of Compound 18g / 19g

[0110]

[0111] Reagents and reaction conditions: (a) (Bromomethyl)cyclopropane, NaH, DMF, 0℃ to rt, 67%; (b) intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO 3,Toluene,EtOH,H 2 O, 90°C, 84%; (c) HCl / MeOH, rt, 93%.

[0112] As shown in the above reaction process, the synthesis of compound 18g / 19g refers to the synthesis process of compound 18j / 19j. Compound 18g:. 1 HNMR(400MHz,MeOD)δ7.49(d,J=8.2Hz,2H),7.44(s,1H),7.30(s,1H),7.19(d,J=8.2Hz,2H),6.11(s,1H),4.49(s ,2H),3.92(d,J=10.6Hz,2H),3.40–3.35(m,2H),3.15(dd,J=14.0,6.9Hz,2H),3.11–3.05(m,1H),2.84(dd,J=6.9 ,3.8Hz,1H),2.39(s,3H),2.32(s,3H),2.24(s,3H),2.17–2.12(m,1H),1.76(d,J=11.2Hz,2H),1.68–1.62(m,2H) ,1.43(s,9H),1.35–1.32(m,2H),0.90(t,J=7.0Hz,3H),0.53–0.42(m,2H),0.30–0.24(m,1H),0.21–0.13(m,1H). 13 CNMR(126MHz,MeOD)δ173.1,165.6,153.5,150.6,144.8,141.9,140.3,139.7,139.1,134.1,127.6,127.4,125.0,122.7,121. 9,111.0,81.1,68.2,59.7,43.0,40.1,36.5,31.8,28.7,24.9,19.6,18.5,15.0,13.0,10.8,3.9,3.7.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 41 H 55 N 4 O 5 683.41670, found 683.41693.

[0113] Compound 19g: 1 H NMR (400 MHz, CDCl 3)δ7.37(d,J=8.2Hz,2H),7.29(s,1H),7.13(s,1H),7.04(d,J=8.2Hz,2H),5.90(s,1H),4.55(d,J=5.9Hz, 2H),3.94(d,J=11.2Hz,2H),3.31(td,J=11.1,2.7Hz,2H),3.08(q,J=6.9Hz,2H),3.03–2.94(m,1H),2.64 –2.53(m,2H),2.40(s,3H),2.38–2.35(m,1H),2.33(s,3H),2.12(s,3H),1.95–1.91(m,1H),1.70–1.64(m ,4H),1.14–1.10(m,1H),1.00–0.94(m,2H),0.88(t,J=7.0Hz,3H),0.51–0.44(m,2H),0.14–0.08(m,2H). 13 C NMR(126MHz,MeOD)δ173.0,165.6,153.6,150.7,144.9,140.5,140.1,139.4,139.1,134.3,128.0,127.8,125.0,122.6,122.0 ,111.0,68.25,59.7,54.3,43.0,39.2,36.6,31.8,22.5,19.7,18.5,15.1,14.0,13.1,8.4,4.1,4.6.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 36 H 47 N 4 O 3 583.36427, found 583.36414.

[0114] Example 9: Synthesis of Compound 18h / 19h

[0115]

[0116] Reagents and reaction conditions: (a) Cyclohexylmethyl bromide, NaH, DMF, 0℃ to rt, 71%; (b) Intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H 2 O, 90°C, 81%; (c) HCl / MeOH, rt, 93%.

[0117] As shown in the above reaction process, the synthesis of compound 18h / 19h refers to the synthesis process of compound 18j / 19j. Compound 18h: 1 HNMR(500MHz,MeOD)δ7.48(d,J=8.1Hz,2H),7.43(s,1H),7.29(s,1H),7.16(d,J=8.1Hz,2H),6.10(s,1 H),4.47(s,2H),3.90(d,J=10.7Hz,2H),3.65–3.56(m,1H),3.39-3.35(m,2H),3.18–3.11(m,2H),3.10– 3.04(m,1H),2.63–2.58(m,1H),2.38(s,3H),2.30(s,3H),2.23(s,3H),2.17–2.10(m,1H),1.87–1.78(m ,4H),1.74(d,J=12.5Hz,4H),1.71–1.55(m,6H),1.41(s,9H),1.35–1.27(m,2H),0.88(t,J=6.8Hz,3H). 13 C NMR (126MHz, MeOD) δ173.1,165.6,158.3,153.6,150.6,144.8,142.1,140.4,139.8,139.2,134.1,127.7,127.4,125.0,122.7,122.0,111.1,8 1.0,68.2,60.5,59.7,43.1,38.6,36.6,32.9,32.4,31.8,30.7,28.8,2 7.5,27.4,27.3,26.8,19.7,18.5,15.0,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 43 H 59 N 4 O 5 711.44800, found711.44733.

[0118] Compound 19h: 1H NMR (500MHz, MeOD) δ7.45(d,J=8.1Hz,2H),7.41(s,1H),7.28(s,1H),7.10(d,J=8.0Hz,2H),6.09(s,1H),4.47(s ,2H),3.90(d,J=10.8Hz,2H),3.37–3.32(m,2H),3.13(dd,J=13.5,6.6Hz,2H),3.09–3.01(m,1H),2.68–2.60(m, 1H),2.38(s,3H),2.37–2.33(m,1H),2.31(s,3H),2.23(s,3H),2.01–1.96(m,1H),1.97–1.91(m,2H),1.73(d,J= 12.5Hz,4H),1.67–1.56(m,4H),1.33–1.24(m,2H),1.19–1.13(m,2H),1.12–1.05(m,2H),0.88(t,J=6.8Hz,3H). 13 C NMR(126MHz,MeOD)δ173.0,165.3,153.5,150.6,144.8,142.7,140.4,139.8,138.9,134.0,130.0,127.7,127.2,125.0,122.7,121.9,111.0,6 8.2,59.7,58.6,43.0,40.6,36.6,33.8,33.0,31.8,30.8,27.1,26.1,2 6.1,25.1,23.7,19.7,18.6,16.5,15.0,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 38 H 51 N 4 O 3 611.39557, found 611.39508.

[0119] Example 10: Synthesis of Compound 18i / 19i

[0120]

[0121] Reagents and reaction conditions: (a) 4-Bromotetrahydro-2H-pyran, NaH, DMF, 0℃ to rt, 75%; (b) intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H2 O, 90°C, 82.0%; (c) HCl / MeOH, rt, 91%.

[0122] As shown in the above reaction process, the synthesis of compound 18i / 19i refers to the synthesis process of compound 18j / 19j. Compound 18i: 1 HNMR (400MHz, DMSO) δ11.46(s,1H),8.18(t,J=4.9Hz,1H),7.53(d,J=8.3Hz,2H),7.38(s,1H),7.21(s,1H),7.20(d,J= 8.1Hz,2H),5.86(s,1H),4.29(d,J=4.9Hz,2H),3.92–3.81(m,4H),3.79–3.72(m,1H),3.30–3.22(m,4H),3.12–3.04(m, 2H),3.04–2.97(m,1H),2.63–2.58(m,1H),2.24(s,3H),2.21(s,3H),2.16–2.13(m,1H),2.11(s,3H),2.05–1.98(m,1H ),1.92–1.83(m,1H),1.66(d,J=12.1Hz,4H),1.57–1.49(m,2H),1.37(s,9H),1.30–1.25(m,2H),0.83(t,J=7.0Hz,3H). 13 C NMR(101MHz,MeOD)δ173.0,165.6,158.1,153.6,150.6,144.8,141.9,140. 4,139.7,139.2,134.1,127.7,127.4,125.0,122.7,121.9,111.1,81.3,68 .8(d,J=4.4Hz),68.2,59.7,57.6,43.0,38.6,36.6,32.9,32.4,31.8,30.7 ,28.8,27.41,19.7,19.4,18.6,15.1,14.4,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 42 H 57 N 4 O 6 713.42726, found 713.42670.

[0123] Compound 19i: 1H NMR(500MHz,MeOD)δ7.47(d,J=8.1Hz,2H),7.42(s,1H),7.29(s,1H),7.12(d,J=8.1Hz,2H),6.11(s,1H) ,4.49(s,2H),3.98–3.86(m,2H),3.44–3.33(m,4H),3.14(q,J=6.9Hz,2H),3.11–3.05(m,1H),2.92–2.83 (m,1H),2.40(s,3H),2.38–2.34(m,1H),2.32(s,3H),2.25(s,3H),1.96–1.92(m,1H),1.91–1.84(m,2H) ,1.75(d,J=11.9Hz,2H),1.70–1.59(m,2H),1.50–1.41(m,2H),1.14–1.06(m,2H),0.90(t,J=6.9Hz,3H). 13 C NMR(126MHz,MeOD)δ173.0,165.6,153.5,150.6,144.8,142.7,140.4,139.8,138.9,134.0,127.7,127.1,125.0,122.7,121.9,111.0,68 .25,67.8,61.5,59.7,55.5,43.1,40.3,36.6,34.3,34.1,31.8,25.2,20.8,19.7,18.5,16.7,15.0,14.4,13.0.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 37 H 49 N 4 O 4 613.37483, found 613.37427.

[0124] Example 11: Synthesis of Compound 21a / 22a

[0125]

[0126] Reagents and reaction conditions: (a) Intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H 2 O, 90°C, 80.0-90.0%; (b) HCl / MeOH, rt, 90-100%.

[0127] As shown in the above reaction process, the synthesis of compound 21a / 22a refers to the synthesis process of compound 18a / 19a.

[0128] Compound 21a: 1 H NMR (400MHz, MeOD) δ7.46(s,1H),7.37(d,J=2.2Hz,2H),7.33(t,J=7.7Hz,2H),7.08(d,J=7.3Hz,1H ),6.13(s,1H),4.51(s,2H),3.94(d,J=11.1Hz,2H),3.38(t,J=11.3Hz,2H),3.17(dd,J=13.8,6.8Hz ,2H),3.13–3.08(m,1H),2.74–2.65(m,1H),2.41(s,3H),2.35(s,3H),2.26(s,3H),2.11–2.00(m,1H ),1.78(d,J=12.0Hz,2H),1.73–1.60(m,2H),1.46(s,9H),1.20–1.13(m,2H),0.92(t,J=6.9Hz,3H). 13 C NMR (101MHz, MeOD) δ173.0,165.6,159.0,153.,150.6,144.8,143.3,141.6,140.3,140.1,134.3,129.8,126.1,125.9,125.4,125.3,122 .7,122.3,111.0,75.8,68.2,59.7,43.1,36.7,36.6,33.7,31.8,28.7,25.6,25.0,19.7,18.6,16.4,15.1,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 37 H 49 N 4 O 5 629.36975, found 629.36914.

[0129] Compound 22a: 1H NMR(500MHz,MeOD)δ7.41(d,J=1.7Hz,1H),7.33–7.31(m,1H),7.31–7.27(m,2H),7.26(s,1H),6.9 8(d,J=7.4Hz,1H),6.12(s,1H),4.49(s,2H),3.92(d,J=10.9Hz,2H),3.36(t,J=10.8Hz,2H),3.15( q,J=7.0Hz,2H),3.12–3.05(m,1H),2.54–2.49(m,1H),2.39(s,3H),2.32(s,3H),2.24(s,3H),1.98 –1.92(m,1H),1.75(d,J=12.0Hz,2H),1.68–1.62(m,2H),1.08–1.03(m,2H),0.90(t,J=7.0Hz,3H). 13 C NMR(126MHz,MeOD)δ173.1,165.6,153.6,150.6,144.9,144.1,141.6,140.4,140.2,134.3,130.8,129.9,125.5,125.3,125.2,1 22.7,122.2,111.1,68.2,59.7,43.1,36.6,35.7,31.8,30.7,26.2,25.0,19.7,18.5,17.9,15.0,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 32 H 41 N 4 O 3 529.31732, found 529.31714.

[0130] Example 12: Synthesis of Compound 21b / 22b

[0131]

[0132] Reagents and reaction conditions: (a) 4-Bromotetrahydro-2H-pyran, NaH, DMF, 0℃ to rt, 60-80%; (b) intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H 2 O, 90°C, 80.0-90.0%; (c) HCl / MeOH, rt, 90-100%.

[0133] As shown in the above reaction process, the synthesis of compound 21b / 22b refers to the synthesis process of compound 18j / 19j.

[0134] Compound 21b: 1 H NMR (400MHz, MeOD) δ7.34(s,1H),7.27(d,J=2.3Hz,2H),7.24(t,J=6.1Hz,1H),7.20(d,J=8.0Hz,1H),6.95(d,J=7.4Hz, 1H),6.01(s,1H),4.40(s,2H),3.89(dd,J=9.3,5.8Hz,2H),3.86–3.79(m,2H),3.80–3.71(m,1H),3.37–3.24(m,4H),3.0 6(dd,J=13.8,6.7Hz,2H),3.03–2.96(m,1H),2.60–2.54(m,1H),2.30(s,3H),2.23(s,3H),2.14(s,3H),2.13–2.05(m,2 H),1.99–1.91(m,1H),1.66(d,J=11.1Hz,2H),1.63–1.47(m,4H),1.32(s,9H),1.25–1.20(m,2H),0.80(t,J=7.0Hz,3H). 13 C NMR(126MHz,MeOD)δ173.0,165.6,158.1,153.5,150.6,144.8,143.1,141.6 ,140.4,140.0,134.4,129.9,126.1,125.6,125.5,125.3,122.7,122.2,111 .0,81.3,68.9,68.8,68.2,59.7,57.6,43.1,38.6,36.6,32.9,32.5,31.8,3 0.7,28.8,27.7,23.7,19.7,19.1,18.6,15.1,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 42 H 57 N 4 O 6 713.42726, found 713.42719.

[0135] Compound 22b: 1H NMR(500MHz,MeOD)δ7.42(s,1H),7.36–7.31(m,2H),7.31–7.26(m,2H),7.02(s,1H),6.11(s,1H ),4.49(s,2H),3.93(s,4H),3.44–3.34(m,4H),3.17–3.14(m,2H),3.10–3.04(m,1H),3.01–2.9 2(m,1H),2.49–2.43(m,1H),2.39(s,3H),2.32(s,3H),2.24(s,3H),2.08–2.01(m,1H),1.75(d, J=12.0Hz,2H),1.70–1.56(m,4H),1.52–1.43(m,2H),1.18–1.12(m,2H),0.91(t,J=7.0Hz,3H). 13 C NMR (126MHz, MeOD) δ173.0,165.6,153.6,150.6,144.9,143.5,141.8,140.4,140.1,134.3,130.0,125.5,125.4,125.3,122.7,122 .2,111.0,68.2,67.7,59.8,55.6,43.1,39.8,36.6,33.9,33.8,31.8,25.1,19.7,18.5,16.2,15.1,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 37 H 49 N 4 O 4 613.37483, found 613.37561.

[0136] Example 13: Synthesis of Compound 21c / 22c

[0137]

[0138] Reagents and reaction conditions: (a) 4-Bromomethyltetrahydropyran, NaH, DMF, 0℃ to rt, 60-80%; (b) intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H 2 O, 90°C, 80.0-90.0%; (c) HCl / MeOH, rt, 90-100%.

[0139] As shown in the above reaction process, the synthesis of compound 21c / 22c refers to the synthesis process of compound 18j / 19j.

[0140] Compound 21c: 1 H NMR(500MHz,MeOD)δ7.45(s,1H),7.39(s,1H),7.35(t,2H),7.32(d,J=1.5Hz,1H),7.06(d,J=6.6Hz,1H),6.1 2(s,1H),4.50(s,2H),3.92(d,J=9.4Hz,4H),3.41–3.32(m,6H),3.16(dd,J=14.0,7.0Hz,2H),3.13–3.08(m, 1H),2.78–2.71(m,1H),2.40(s,3H),2.33(s,3H),2.25(s,3H),2.22–2.16(m,1H),2.03–1.92(m,1H),1.76(d ,J=11.6Hz,2H),1.70–1.55(m,4H),1.50–1.38(m,2H),1.40(s,9H),1.36–1.30(m,2H),0.91(t,J=7.0Hz,3H). 13 CNMR(126MHz,MeOD)δ173.0,165.6,158.5,153.5,150.6,144.8,143.0,141.6,1 40.4,140.1,134.3,129.9,126.1(d,J=6.8Hz),125.8–125.6(m),125.4(d,J=34 .1Hz),122.7,122.2,111.1,81.2,68.7,68.6,68.2,59.7,43.1,40.3,36.6,35. 4,33.0,30.7,30.7,28.7,25.0,19.7,18.6,15.1,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 43 H 59 N 4 O 6 727.44291, found 727.44214.

[0141] Compound 22c: 1H NMR (500MHz, MeOD) δ7.43(d,J=1.1Hz,1H),7.39(d,J=7.8Hz,1H),7.34(t,J=7.6Hz,2H),7.31(d,J=1.3Hz,1H),7.07(d ,J=7.6Hz,1H),6.12(s,1H),4.49(s,2H),3.95(dd,J=22.8,10.8Hz,4H),3.45–3.35(m,4H),3.15(q,J=7.0Hz,2H),3.12 –3.05(m,1H),2.86(d,J=6.9Hz,2H),2.74–2.65(m,1H),2.40(s,3H),2.32(s,3H),2.30–2.26(m,1H),2.24(s,3H),1.9 4–1.86(m,1H),1.75(d,J=11.8Hz,2H),1.73–1.69(m,2H),1.69–1.56(m,4H),1.28–1.25(m,2H),0.90(t,J=6.9Hz,3H). 13 C NMR (126MHz, MeOD) δ173.0,165.6,153.6,150.6,144.9,141.9,140.5,139.9,134.4,130.1,125.9,125.9,125.3,122.7,122.2,111.1,68.4 8,68.2,61.5,59.8,55.6,54.8,43.1,36.6,34.7,33.0,31.8,30.7,30.4,28.1,23.7,19.7,18.5,15.1,14.,13.1.HRMS(ESI-TOF)m / z[M+H] + calcdfor C 38 H 51 N 4 O 4 627.39048, found 627.39038.

[0142] Example 14: Synthesis of Compound 21d / 22d

[0143]

[0144] Reagents and reaction conditions: (a) 4-(2-Bromoethyl)tetrahydro-2H-pyran, NaH, DMF, 0℃ tort, 60-80%; (b) intermediate 6, Pd(PPh 3 ) 4 ,K 2 CO3 ,Toluene,EtOH,H 2 O, 90°C, 80.0-90.0%; (c) HCl / MeOH, rt, 90-100%.

[0145] As shown in the above reaction process, the synthesis of compound 21d / 22d refers to the synthesis process of compound 18j / 19j.

[0146] Compound 21d: 1 H NMR (400MHz, Cδ7.44(s,1H),7.38(s,1H),7.37–7.32(m,2H),7.31(d,J=1.7Hz,1H),7.05(d,J=7.3Hz,1H),6.10(s,1 H),4.49(s,2H),3.95–3.84(m,4H),3.48–3.40(m,1H),3.39–3.30(m,6H).,3.15(dd,J=14.1,7.1Hz,2H),3.11–3.04 (m,1H),2.75–2.69(m,1H),2.39(s,3H),2.32(s,3H),2.24(s,3H),2.21–2.15(m,1H),1.75(d,J=11.0Hz,2H),1.69– 1.60(m,4H),1.51(dd,J=11.7,7.5Hz,4H),1.40(s,9H),1.36–1.33(m,2H),1.31–1.29(m,2H),0.90(t,J=7.0Hz,5H). 13 C NMR (126MHz, MeOD) δ153.6,150.6,141.6,140.4,140.1,134.3,129.9,127.0,126.1,125.6,125.3,122.7,122.2,111.1,81.1,69.0,68.2,59 .7,43.1,39.9,36.6,34.2,34.0,33.9,33.0,31.8,30.7,30.4,30.3,28.8,23.7,19.7,18.6,17.9,15.1,14.4,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 44 H 61 N 4 O 6 741.45856, found 741.45801.

[0147] Compound 22d: 1H NMR (500MHz, MeOD) δ7.42(d,J=1.6Hz,1H),7.34(d,J=7.7Hz,1H),7.32(d,J=7.5Hz,1H),7.29(d,J=1.8Hz,2H),7.02(d,J= 7.4Hz,1H),6.11(s,1H),4.49(s,2H),3.92(d,J=11.8Hz,2H),3.88(d,J=3.9Hz,2H),3.40–3.33(m,4H),3.15(q,J=6.9Hz, 2H),3.12–3.06(m,1H),2.84–2.79(m,2H),2.47–2.42(m,1H),2.39(s,3H),2.32(s,3H),2.24(s,3H),2.07–2.04(m,1H),2 .04–2.00(m,1H),1.75(d,J=11.2Hz,2H),1.68–1.58(m,6H),1.53–1.47(m,2H),1.16–1.10(m,2H),0.90(t,J=7.0Hz,3H). 13 C NMR (126MHz, MeOD) δ173.0,165.6,153.6,150.6,144.8,143.6,141.7,140. 4,140.1,134.3,130.8,130.0,125.6,125.4,125.3,122.7,122.2,111.0,6 8.9,68.2,59.7,47.3,43.1,42.1,37.1,36.6,34.2,33.0,31.8,30.7,28.1 ,24.8,23.7,19.7,18.5,16.30,15.1,14.4,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 39 H 53 N 4 O 4 641.40613, found 641.40594.

[0148] Example 15: Synthesis of Compound 27

[0149]

[0150] Reagents and reaction conditions: (a) Intermediate 3, Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H 2O, 90℃, 90.1%; (b) NaOH, EtOH, 60℃, 90.4%; (c) Boc 2 O,H 2 , Raney Ni, MeOH, 99.0%; (d) HCl / MeOH, 100%; (e) HOBt, EDCI, NMM, DMSO, 63.4%.

[0151] Compound 27: 1 H NMR(400MHz,MeOD)δ7.46(d,J=8.2Hz,2H),7.42(s,1H),7.28(s,1H),7.17(d,J=8.2Hz,2H),4.5 5(s,2H),3.91(d,J=10.9Hz,2H),3.34(dd,J=20.1,5.3Hz,2H),3.13(dd,J=14.1,7.1Hz,2H),3. 10–3.04(m,1H),2.70–2.65(m,1H),2.52(s,3H),2.40(s,3H),2.31(s,3H),2.02–1.96(m,1H),1 .74(d,J=11.2Hz,2H),1.66–1.58(m,2H),1.44(s,9H),1.17–1.12(m,2H),0.88(t,J=7.0Hz,3H). 13 C NMR(101MHz,MeOD)δ173.1,164.0,159.0,152.5,150.6,144.0,142.0,140.2,139.8,139.0,134.1,127.7,127.6,125.0,1 24.6,122.0,68.2,59.7,43.1,40.4,37.7,33.8,31.8,30.7,28.7,20.9,20.5,16.5,15.1,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 37 H 48 Bn 4 O 5 707.28026, found707.27997.

[0152] Example 16: Synthesis of Compound 31

[0153]

[0154] Reagents and reaction conditions (a) HCl / MeOH, 100%; (b) Pivaloyl chloride, Et 3 N,CH2 Cl 2 ,0℃,90.1%;(c)Bis(pinacolato)diboron,Pd(dppf)Cl 2 ,AcOK,DMF,80.2%; (d)7,Pd(PPh 3 ) 4 ,K 2 CO 3 ,Toluene,EtOH,H 2 O,90℃,89.8%.

[0155] Compound 31: 1 H NMR(400MHz,MeOD)δ7.47(d,J=8.3Hz,2H),7.42(s,1H),7.29(s,1H),7.20(d,J=8.3Hz,2H),6.1 0(s,1H),4.48(s,2H),3.90(d,J=11.0Hz,2H),3.39–3.32(m,2H),3.13(dd,J=14.1,7.1Hz,2H),3 .09–3.04(m,1H),2.87–2.82(m,1H),2.38(s,3H),2.31(s,3H),2.23(s,3H),2.08–2.02(m,1H),1 .74(d,J=11.0Hz,2H),1.70–1.56(m,2H),1.24–1.22(m,2H),1.18(s,9H),0.89(t,J=7.0Hz,3H). 13 C NMR(126MHz,MeOD)δ182.7,173.0,165.6,153.5,150.6,144.8,142.0,140.4,139.8,139.1,134.1,127.8,127.6,125.0,122.7,122 .0,111.0,75.8,68.2,59.7,54.8,43.1,39.5,36.6,33.8,31.8,25.0,24.9,19.7,18.6,16.3,15.0,13.1.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 37 H 49 N 4 O 4 613.37427, found 613.37483.

[0156] Example 17: Western Blot Detection of the Inhibitory Effects of Compounds on LSD1 and EZH2

[0157] (1) Total protein extraction: The cells treated with DMSO or drugs were washed twice with PBS, then lysed in a lysis buffer containing a protease inhibitor cocktail (Selleck) and transferred to a 1.5 mL EP tube. The EP tube was then placed in a thermostatic metal bath and boiled at 95°C for 15 min.

[0158] (2) Protein content determination: Protein content determination was performed according to the instruction manual of Pierce BCA protein quantification kit.

[0159] (3) SDS-PAGE gel electrophoresis gel preparation: Assemble the glass plate, and prepare 12% separation gel and 5% concentration gel according to the formula. Loading: Add 4xSDS loading buffer to the protein sample in proportion to make the final SDS concentration 1x. After mixing evenly, place the sample in a constant temperature metal bath at 95℃ and cook for 15 minutes. After calculation, add 8μg of protein sample to each loading well. Electrophoresis: Set the voltage to 80V at the beginning of the concentration gel part. After the band enters the separation gel, adjust the constant voltage to 120V until the bromophenol blue band runs to the bottom.

[0160] (4) Transfer: Prepare the PVDF membrane soaked in methanol, the filter paper soaked in the transfer solution, the black sponge, the separation gel, the transfer clip and the filter paper and place them in a tray filled with the transfer solution. The black transfer clip is at the bottom. Place the sponge, filter paper, separation gel PVDF membrane, another filter paper and sponge on the transfer clip in sequence. Be careful to use a glass rod to drive away bubbles during the process. Clamp it and put it into the transfer tank to fix it. Use the wet transfer method, constant voltage 90V, and transfer for 1-3 hours according to different molecular weights.

[0161] (5) Blocking of immunoblotting: Place the transferred PVDF membrane in a plastic box containing 5% skimmed milk powder, block at room temperature for 1 hour, and wash 3 times with TBST, 10 minutes each time. Incubate with primary antibody: Dilute the primary antibody with the primary antibody diluent of Bio-Tech according to the instructions, place the transfer membrane in the diluted primary antibody, and incubate overnight on a shaker at 4°C. Incubate with secondary antibody: Wash 3 times with TBST on the next day, 10 minutes each time. Then transfer the transfer membrane to the diluted secondary antibody, incubate at room temperature for 1 hour, and then wash 3 times with TBST again, 10 minutes each time. ECL development: Evenly apply the prepared ECL luminescent solution to the strip to be detected, place it away from light for 1 minute, and then use the chemiluminescence imaging system to develop and take pictures.

[0162] Example 18: MTS detection of the killing effect of compounds on tumor cells

[0163] MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] can be reduced by dehydrogenases in metabolically active cells to a colored formazan product that is soluble in the culture medium and directly detected by visible light absorption at 490 nm using a 96-well plate. Prostate cancer cells were seeded in 96-well plates (1000-1500 cells / well in 100 μL of culture medium) for 24 h. The drug was then diluted with fresh culture medium at the desired concentration and 100 μL per well was added to the plated 96-well plate and placed in a 37°C, CO2-containing chamber. 2 Incubate in an incubator for 6-7 days. After the specified incubation time, add 20 μL of MTS to each well, and incubate the cells for another 4 h at 37°C with 5% carbon dioxide. Then, the absorbance values ​​(OD values) of different wells are measured at a wavelength of 490 nm using an ELISA reader.

[0164] Example 19: Human prostate subcutaneous transplant tumor model experiment in nude mice to test the anti-tumor effect of compound 18j

[0165] (1) Prostate cancer 22RV1 cells in the logarithmic growth phase were collected, washed twice with PBS solution, and resuspended in serum-free RPMI-1640 medium. The number of cells required for inoculation was calculated. 22RV1 model cells: 4.0×10 6 / 100μL;

[0166] (2) 100 μL of cell suspension was injected subcutaneously in the right axilla of 8-week-old male nude mice;

[0167] (3) When the tumor volume reaches 50-100 mm 3 The mice were randomly divided into 5 groups, namely, solvent control group and 5 drug treatment groups;

[0168] (4) Administration: drug treatment groups: EPZ6438 (65 mg / kg), TCP (20 mg / kg), EPZ+TCP (EPZ6438 (65 mg / kg,) + TCP (20 mg / kg)) and 18j (80 mg / kg). Solvent control group (DMSO: castor oil: PBS buffer = 1:1:8) was intraperitoneally injected with drug or solvent 200 μL / mouse per day;

[0169] (5) When subcutaneous tumors were visible to the naked eye, tumor growth measurements were performed every two days. The length (L) and width (W) of the tumor were measured and recorded using a vernier caliper. The tumor volume was calculated as V (mm 3 )=(L×W 2 ) / 2, draw the tumor growth curve according to the tumor volume;

[0170] (6) When the tumor size in the control group reached 1000-2000mm 3 The experiment was terminated at 4 pm, and the nude mice were killed by dislocation. The tumor tissues were collected, photographed, and the tumor weight was weighed.

[0171] In order to further verify the effects of the compounds of the present invention on LSD1 and EZH2 targets, the present invention treated prostate cancer cells with different concentrations of compound 18j or DMSO for 24 hours, and then used Western blot to measure the levels of H3K27me3, H3K9me2 and H3K4me2, with tubulin as a control.

[0172] like Figure 1 The results showed that compound 18j significantly reduced the level of H3K27me3 protein, while significantly increasing the levels of H3K9me2 and H3K4me2, indicating that compound 18j can inhibit the enzymatic function of LSD1 and EZH2

[0173] Figure 2 The effects of the compound 18j of the present invention and the existing EZH2 inhibitor EPZ6438 and LSD1 inhibitor TCP on the viability of different prostate cancer cells were demonstrated. The compounds of the present invention were used to treat the prostate cancer cell lines LNCAP, PC3 and 22RV1 for six or seven days, and the cell survival rate was measured using MTS. The results showed that compared with EPZ6438 (GI 50 19.5μM, >30μM, >30μM), TCP(GI 50 >30 μM) compared with compound 18j (GI 50 1.63μM, 1.04μM, and 3.96μM respectively) have a stronger killing effect.

[0174] like Figure 3 As shown, in the 22RV1 nude mouse subcutaneous transplant tumor model, the compound 18j of the present invention (% TGI = 52.2%) has a more effective anti-tumor effect compared with EPZ6438, TCP or the combination of the two (% TGI = 28.8%).

[0175] Figure 4 The antitumor results of compound 18j and EPZ6438, TCP or their combination in different tumor cell lines show that compared with EPZ6438, TCP or their combination (mostly >30 μM), compound 18j of the present invention (mostly <10 μM) has a more effective antitumor effect.

[0176] Table 1 shows the GI of different compounds 18a-j, 19a-j, 21a-d, 22a-d, EPZ5438, TCP and their combination in prostate cancer cell lines LNCAP, PC3 and 22RV1 50 (μM) and IC of extracellular LSD1 and EZH2 enzyme inhibitory activities 50 (μM).

[0177] Table 1

[0178]

[0179]

[0180]

[0181] a:LSD1 inhibition rate at 3μM; b:EZH2 inhibition rate at 1μM;

[0182] From the data in Table 1, we can see that:

[0183] 1) When connected with different substituents, the dual-target inhibitors have good enzyme inhibition activities against LSD1 and EZH2;

[0184] 2) When the substituent is 4-bromomethyltetrahydropyran, the dual-target inhibitor has the best activity in inhibiting the proliferation of prostate cancer cells;

[0185] 3) The connection position of the benzene ring has little effect on the anti-tumor activity of the compound. Specifically, when the connection position of the benzene ring is at the 3' position, the activity of the dual-target inhibitor is weaker than when it is at the 4' position.

[0186] 4) When the substituent is 4-bromomethyltetrahydropyran and the connection position of the benzene ring is 4', the dual-target inhibitor has the best anti-prostate cancer cell proliferation activity.

[0187] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A dual-target inhibitor of LSD1 and EZH2, characterized in that: The invention comprises a structural fragment containing at least one benzene ring for inhibiting LSD1 protein and a structural fragment containing at least one benzene ring for inhibiting EZH2 protein; the structural fragment containing at least one benzene ring for inhibiting LSD1 protein and the structural fragment containing at least one benzene ring for inhibiting EZH2 protein share a benzene ring.

2. The LSD1 and EZH2 dual-target inhibitor according to claim 1, characterized in that: The structural fragment inhibiting LSD1 protein includes a structural fragment having LSD1 protein inhibitory effect in trans-phenylcyclopropylamine (TCP); preferably, the N-terminus of the structural fragment having LSD1 protein inhibitory effect in TCP is also connected to any two selected from hydrogen, tert-butyloxycarbonyl, 2-morpholine-2-oxyethyl, 2-oxo-2-(piperazin-1-yl)ethyl, N-(tert-butyloxycarbonyl)-2-oxo-2-(piperazin-1-yl)ethyl, 2-fluorobenzyl, 2-trifluoromethylbenzyl, 2-methoxybenzyl, benzyl, cyclopropylmethyl, cyclohexyl, tetrahydro-2H-pyran-4-yl, tetrahydro-2H-pyran-4-yl)methyl, tetrahydro-2H-pyran-4-yl)ethyl, and pivaloyl.

3. A compound of formula I, or a pharmaceutically acceptable salt, enantiomer, diastereomer, tautomer, solvate, crystalline form or prodrug thereof: in, R1 and R2 are independently selected from hydrogen, tert-butoxycarbonyl, 2-morpholin-2-oxyethyl, 2-oxo-2-(piperazin-1-yl)ethyl, N-(tert-butoxycarbonyl)-2-oxo-2-(piperazin-1-yl)ethyl, 2-fluorobenzyl, 2-trifluoromethylbenzyl, 2-methoxybenzyl, benzyl, cyclopropylmethyl, cyclohexyl, tetrahydro-2H-pyran-4-yl, tetrahydro-2H-pyran-4-yl)methyl, tetrahydro-2H-pyran-4-yl)ethyl, and pivaloyl.

4. The compound of formula (I) according to claim 3, characterized in that: The compound of formula I is selected from the compound of formula II Compound of formula III Wherein R1 and R2 are defined as in claim 3.

5. The compound of formula (I) according to claim 3, characterized in that: The compound of formula I is selected from the compounds of formula II-A Compound of formula III-A Wherein R1 and R2 are defined as in claim 3.

6. The compound of formula (I) according to claim 3, characterized in that: The compound of formula I is selected from the following compounds and / or their isomers:

7. A method for preparing a compound of formula I according to any one of claims 3 to 6, characterized in that: The following steps are involved: The compound of formula IA With the compound of formula IB The reaction produces a compound of formula I, wherein X is a halogen atom, and the definitions of R1 and R2 are as described in any one of claims 3 to 6.

8. A pharmaceutical composition, characterized in that: It includes the LSD1 and EZH2 dual-target inhibitor according to claim 1 or 2 or the compound of formula I according to any one of claims 3 to 6.

9. Use of the LSD1 and EZH2 dual-target inhibitor according to claim 1 or 2 or the compound of formula I according to any one of claims 3 to 6 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor includes solid tumors and blood tumors; preferably, the solid tumor is selected from lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, kidney cancer, uterine cancer, prostate cancer or melanoma; the blood tumor is selected from leukemia or lymphoma.

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