CN113214203A

CN113214203A - Small molecule compound based on EZH2 protein degradation and application thereof

Info

Publication number: CN113214203A
Application number: CN202011479303.8A
Authority: CN
Inventors: 文石军; 黄蓬; 涂亚林; 孙亚猛; 胡寓旻
Original assignee: Sun Yat Sen University Cancer Center
Current assignee: Sun Yat Sen University Cancer Center
Priority date: 2020-12-16
Filing date: 2020-12-16
Publication date: 2021-08-06

Abstract

The invention discloses a series of micromolecule compounds capable of selectively degrading EZH2 protein and application thereof, and the compounds can be prepared into proper pharmaceutical dosage forms for treating related tumors mediated by EZH 2.

Description

Small molecule compound based on EZH2 protein degradation and application thereof

Technical Field

The invention belongs to the field of medicines, and relates to a series of micromolecule compounds capable of selectively degrading EZH2 protein and application thereof, wherein the compounds can be prepared into a proper pharmaceutical dosage form for treating related tumors mediated by EZH 2.

Background

Histone lysine methyltransferase EZH2(enhancer of zeste homolog 2, EZH2) is a core component of Polycomb repressive complex 2 (PRC 2) and is responsible for the enzymatic catalytic function of the complex. PRC2 is responsible for trimethylation of histone H3K27 (H3K27me3) in cells and thereby regulates gene expression (primarily silencing of the gene of interest). EZH2 was found to show different forms of abnormalities in various tumors such as non-hodgkin's lymphoma, T-cell acute lymphocytic leukemia, breast cancer, prostate cancer, ovarian cancer, melanoma, etc., including EZH2 acquired mutations, chromatin remodeling complex SWI-SNF mutations, EZH2 high expression, EZH2 abnormal phosphorylation, etc. In tumor cells, the EZH2 abnormality is closely related to malignant phenotypes such as tumor proliferation and invasion and metastasis, and the enzyme activity of knockout of EZH2 or inhibition of EZH2 can obviously inhibit the malignant phenotypes of certain tumors, so EZH2 becomes an important drug target.

At present, a series of small molecule inhibitors interfering with the enzymatic activity of EZH2 have been reported, and show certain antitumor effects in preclinical and clinical tests. These drugs act on the enzyme-catalytic active domain of EZH2, and competitively inhibit the enzyme activity of EZH2 with the EZH2 substrate S-adenosyl-L-methionine, but do not affect the protein stability and expression degree of EZH 2. In recent years, the EZH2 inhibitor only with enzyme catalytic activity inhibition has the defects of high dosage, drug resistance and insensitivity to most solid tumors except a few blood tumors (such as diffuse large B-cell lymphoma of EZH2 mutation). On the other hand, EZH2 inhibitors with only inhibition of enzyme catalytic activity are not sensitive to many tumors including SWI-SNF mutations and EZH2 mutations. This may be associated with oncogene function of EZH2 not being completely dependent on the enzymatic activity of EZH2, as in castration-resistant prostate cancer, breast cancer and in T-cell acute lymphoblastic leukemia and neuroblastoma, oncogene function of EZH2 is also independent of the enzymatic activity of EZH 2. The overall knock-down of EZH2 can obviously inhibit the proliferation of tumor cells or promote the apoptosis of the tumor cells, and the high expression of EZH2 in clinical samples, but not the methylation level of H3K27, is the index of poor prognosis of the tumors. Therefore, the oncogene function of EZH2 is not completely dependent on the enzymatic activity of EZH2, and the EZH2 protein is taken as a whole drug target, so that inhibiting the enzymatic activity of EZH2 and reducing the protein level of EZH2 may be better strategies for developing EZH2 inhibitors.

Traditional drug development, including existing EZH2 inhibitors, has focused on directly modulating protein or enzyme activity to treat disease, while protein-targeted degradation agents are an alternative to targeted therapy by removing specific oncogenic proteins from cells using their own protein destruction mechanisms, the proteasome degradation system. The cell needs ubiquitin-proteasome degradation system to maintain normal protein level, and the most critical process is ubiquitination of protein, which is transported to proteasome for degradation when target protein is linked to ubiquitin, a small protein. Protein degradation targeting chimeras (PROTACs) are compounds obtained by linking two active small molecule fragments via a linker chain, one active end of which can bind to the targeted protein and the other active end of which can bind to E3 ligase including VHL and CRBN. Such bifunctional PROTAC molecules are capable of targeting proteins to human ubiquitin, which are then degraded by the proteasome, and they can continue to target the remaining proteins after they are degraded, thereby rapidly reducing the levels of unwanted proteins. Compared with the traditional medicine, the protein degradation medicine has the advantages of low dosage, better selectivity and action effect, overcoming drug-resistant tumors and the like, becomes a new high place for international medicine enterprises, and part of the medicine enters a clinical experiment stage.

Some literatures report that knockout of EZH2 by RNA interference technology is more effective and more effective in killing tumors, however, small molecule compounds that specifically and effectively degrade EZH2 are lacking at present. The invention utilizes PROTAC technology to develop specific compounds which can simultaneously degrade EZH2 and inhibit the enzymatic activity thereof. The compounds are mainly designed based on E3 ligase VHL and CRBN, and have good treatment effect on cancer cells such as lymph cancer.

Disclosure of Invention

Based on the research background and the related theory, by utilizing the protein targeting degradation technology, the invention synthesizes a series of micromolecule EZH2 degradation agents capable of specifically degrading EZH2 protein, and finds that the micromolecule compounds have stronger anti-tumor effect compared with an EZH2 inhibitor.

The invention relates to a micromolecular compound based on EZH2 protein degradation, which is a molecule with a structural characteristic I, wherein X is a linker comprising acyclic or cyclic saturated or unsaturated carbon, glycol, amide, amino, ether or carbonyl-containing groups; y may be a group of an affinity E3 ligase VHL or cereblon (crbn);

preferably, Y in said formula I is a fragment of the affinity E3 ligase VHL shown in formula II and having the structural feature III:

preferably, the structural formula III, X may be, but is not limited to, the following connecting chains X1, X2 and X3:

preferably, n shown by X1 can be, but is not limited to, 1,2, 3; n as indicated by X2 may be, but is not limited to, 2, 3, 4.

Preferably, the structural formula I, Y may be a compound represented in structure IV, having the structural feature V, and being a fragment of the affinity E3 ligase CRBN:

preferably, the structural formulae V, X may be, but are not limited to, the following connecting chains X4, X5, X6 and X7:

preferably, n of X6 can be, but is not limited to, 3, 4; or n in X7 can be, but is not limited to, 3, 4.

Preferably, the compounds of structural feature III may each be one of the following:

preferably, the compounds having the structural feature V may each be one of the following:

the present invention also provides a pharmaceutical composition comprising ED 1-ED 13 of any one of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

It is noted that the compounds of formula III and formula V are useful in EZH2 mediated tumors, particularly in lymphomas.

Further, the compounds and compositions of the present invention may be used with other drugs to provide combination therapy. The other drugs may form part of the same composition or may be provided as separate compositions for simultaneous or non-simultaneous administration.

Drawings

FIG. 1 is a graph of the effect of compounds of the invention on the level of EZH2 protein;

FIG. 2 is a graph of the effect of varying concentrations of compounds ED2 and ED3 on the protein levels of EZH2 in accordance with the present invention;

FIG. 3 is a graph showing the effect of inventive compounds ED2, ED3 and control compound EPZ6438 on lymphoma cell viability.

Detailed Description

The present invention will be further described with reference to the accompanying drawings, and it should be noted that the following examples are provided to illustrate the detailed embodiments and specific operations based on the technical solutions of the present invention, but the scope of the present invention is not limited to the examples.

Example 1: synthesis of Compound ED2

Synthesis of intermediate a 9:

reagents and reaction conditions: a) fe, NH₄Cl,MeOH,90℃；b)tetrahydro-4H-pyran-4-one,AcOH,Na(AcO)₃BH,DCE；e)acetaldehyde,AcOH,Na(AcO)₃BH,DCE；d)NaOH,EtOH,60℃；e)Boc₂O,H₂,Raney-Ni,MeOH；f)AcCl,MeOH；g)A5，HOBt,EDCI,NMM,DMSO。

As shown in the above reaction procedure, the synthesis was carried out starting from commercially available A1, and 5 equivalents of iron powder and 2 equivalents of ammonium chloride were added to the mixture, followed by reflux overnight in a methanol solution at 90 ℃. After TLC detection reaction is completed, filtering the reaction system, collecting filtrate, carrying out reduced pressure distillation, and carrying out silica gel column chromatography separation and purification on the concentrated product by using an ethyl acetate/petroleum ether system to obtain a light yellow liquid product A2 with the yield of about 90%.

1 equivalent of A2, 1.5 equivalents of tetrahydropyranone and 6 equivalents of acetic acid were added to a round bottom flask containing dry, anhydrous 1, 2-Dichloroethane (DCE) and stirred at room temperature for 30 minutes. Then, 3 equivalents of sodium triacetoxyborohydride was added under ice bath, and the ice bath was removed to react at room temperature for 24 hours. After TLC detection reaction is completed, the solvent is removed by reduced pressure distillation, the concentrated crude product is extracted by using an ethyl acetate/water system, an organic phase is collected and dried by using anhydrous sodium sulfate, the solvent is removed by reduced pressure distillation, and the concentrated product is separated and purified by using an ethyl acetate/petroleum ether system and silica gel column chromatography to obtain a white solid A3 with the yield of about 91%.

Dissolving A3 in an appropriate amount of anhydrous DCE, adding 6 equivalents of acetic acid and injecting 1.5 equivalents of acetaldehyde into the reaction system, stirring and reacting for 30 minutes at room temperature, adding 6 equivalents of sodium triacetoxyborohydride in an ice bath, and reacting for 24 hours at room temperature. The same A3 was post-treated to give A4 as a pale yellow liquid with a yield of about 95%.

A4 is dissolved in ethanol solution and added with 1.5 equivalents of sodium hydroxide solution to react for 3h under reflux at 60 ℃. And after TLC detection reaction is completed, extracting the system, extracting with dichloromethane/water, collecting an aqueous phase, adjusting the pH of the aqueous phase to 3-4 by using a 1M hydrochloric acid solution, extracting with ethyl acetate, collecting an organic phase, and distilling under reduced pressure to obtain a hydrochloride of the product A5.

A commercially available A6 solution in methanol (1 eq) was reacted overnight under hydrogen protection with addition of Raney nickel in appropriate amount and 1.2 eq Boc anhydride three times with hydrogen displacement gas. After TLC detection reaction is completed, filtering the reaction system, collecting filtrate, carrying out reduced pressure distillation, and carrying out silica gel column chromatography separation and purification on the concentrated product by using an ethyl acetate/petroleum ether system to obtain a white solid A7, wherein the yield is about 99%.

4 equivalents of acetyl chloride were slowly added to the methanol solution under ice bath conditions, and after 30 minutes A7 was added to the reaction system and reacted at room temperature for 3 hours. After the TLC detects that the reaction is complete, the extraction operation is carried out. The product A8 hydrochloride is obtained by first performing extraction with dichloromethane/water, collecting the aqueous phase, adjusting the pH of the aqueous phase to 3-4 with 1M hydrochloric acid solution, performing extraction with ethyl acetate, collecting the organic phase, and performing reduced pressure distillation.

1 equivalent of A5, 1.2 equivalents of A8, 1.5 equivalents of HOBT and 1.5 equivalents of EDCI were added to the DMSO (dry) solution and N-methylmorpholine was injected into the reaction and stirred at room temperature overnight. After TLC detection reaction is completed, ethyl acetate/water system is used for extraction, organic phase is collected and dried by anhydrous sodium sulfate, the solvent is removed by reduced pressure distillation, and the concentrated product is separated and purified by silica gel column chromatography using methanol/dichloromethane system to obtain light yellow solid A9 with the yield of about 95%.¹H NMR(400MHz,CDCl₃)δ7.22(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(d,J＝5.3Hz,1H),2.39(s,3H),2.24(d,J＝2.3Hz,6H),1.66(dd,J＝7.8,3.3Hz,4H),0.85(t,J＝7.0Hz,3H)。

Synthesis of intermediate B10:

reagents and reaction conditions: a) NaOH, (Boc)₂O,MeOH；b)B3,Pd(AcO)₂,KOAc,DMA,150℃；c)MeOH,AcCl；d)B6,DMAP,EDCI,TEA,CH₂Cl₂；e)MeOH,AcCl；f)B9,HATU,DIPEA,THF。

To a methanol solution containing one equivalent of B1, 1.5 equivalents of Boc anhydride and 2 equivalents of sodium hydroxide were sequentially added at 0 ℃ and then reacted at room temperature for 5 hours. After TLC detection reaction is completed, methanol is removed by reduced pressure distillation, 1M hydrochloric acid solution is used for adjusting pH to 5, then dichloromethane is used for extraction for three times, organic phases are combined and washed by saturated saline water for multiple times, anhydrous sodium sulfate is used for drying out water, and the B2 is obtained by reduced pressure distillation, wherein the yield is about 94%.

1 equivalent of B4 was dissolved in anhydrous N, N-Dimethylacetamide (DMA) and commercially available 1 equivalent of B3 was added under argon as well as 0.02 equivalents of palladium acetate and 2 equivalents of potassium acetate. The reaction was stirred at 150 ℃ and refluxed overnight. After TLC detection reaction is finished, dichloromethane is used for extraction, a small amount of dilute hydrochloric acid is used for washing a reaction system, water is used for washing for multiple times, DMA is removed as far as possible, an organic phase is collected, and after anhydrous sodium sulfate is dried, the yellow liquid B4 is obtained by concentration. Under ice bath conditions, 5 equivalents of acetyl chloride was added to methanol, and after 1 hour of reaction, one equivalent of B4 was added to the reaction system and reacted for 1 hour. After the TLC detection reaction is finished, the reaction system is dried by spinning to obtain yellow solid B5 with the yield of about 100 percent.

One equivalent of B5 was dissolved in an appropriate amount of anhydrous dichloromethane, followed by the sequential addition of 1 equivalent of commercially available B6, 2 equivalents of TEA, and a catalytic amount of DMAP. 2 equivalents of EDCI were added at 0 ℃ and the reaction was allowed to proceed for 3h at room temperature. After TLC detection reaction is finished, dichloromethane/water system is used for extraction, organic phase is collected, anhydrous sodium sulfate is dried, and concentrated crude product is subjected to silica gel column chromatography and purification by using methanol/dichloromethane system to obtain yellow liquid B7, wherein the yield is about 70%.

Using anhydrous tetrahydrofuran as a solvent, 1 equivalent of B8 (obtained by treating B7 with HCl in methanol) and 1 equivalent of commercially availableB9, 3 equivalents of DIPEA and 1.5 equivalents of HATU. After TLC detection reaction, ethyl acetate/water system is used for extraction, then organic phase is collected, anhydrous sodium sulfate is dried and then vacuum distillation is carried out to obtain crude product, and then methanol/dichloromethane system is used for silica gel column chromatography separation and purification to obtain yellow solid B10, wherein the yield is about 70%. B10 was used after removal of the Boc protecting group.¹H NMR(400MHz,CDCl₃)δ8.68(s,1H),7.45(t,J＝5.5Hz,1H),7.36–7.29(m,4H),5.20(d,J＝9.1Hz,1H),4.74(t,J＝7.9Hz,1H),4.54(dd,J＝15.2,6.7Hz,2H),4.30(dd,J＝15.0,5.1Hz,1H),4.16(d,J＝9.2Hz,1H),4.03(d,J＝11.3Hz,1H),3.59(dd,J＝11.3,3.4Hz,1H),2.50(s,3H),2.10(dd,J＝13.1,8.3Hz,1H),1.40(s,9H),0.91(s,9H)。

Synthesis of ED2

Reagents and reaction conditions: a) BnBr, NaH, THF; b) tert-butyl Bromoacetate, TBACl, CH₂Cl₂,NaOH；c)Pd/C,H₂,EtOH；d)TsCl,DMAP,TEA,CH₂Cl₂；e)4-hydroxyphenylboronic acid pinacol ester,K₂CO_3,DMF,70℃；f)A9,K₃PO₄,Pd(PPh)_4,DMF,90℃；g)20％TFA/CH₂Cl₂；h)B10,HATU,DIPEA,DMF。

The synthesis was carried out starting from commercially available C1 by adding 1.5 equivalents of NaH in portions to a solution of 3 equivalents of C1 in THF at 0 deg.C, stirring at room temperature for one hour, then adding 1 equivalent of benzyl bromide at 0 deg.C and reacting at room temperature overnight. After TLC detection reaction is completed, adding saturated ammonium chloride aqueous solution to quench the reaction, extracting by using ethyl acetate/water system, combining organic phases, drying by using anhydrous sodium sulfate, distilling under reduced pressure to obtain a crude product, and separating and purifying the crude product by using silica gel column chromatography and chromatography by using the ethyl acetate/water system to obtain colorless liquid C2, wherein the yield is about 56%.

Using dichloromethane/35% sodium hydroxide aqueous solution as a solvent (volume ratio is 1: 1), 1 equivalent of C2, 1 equivalent of tetrabutylammonium chloride and 4 equivalents of tert-butyl bromoacetate are added in sequence, and the mixture is reacted at room temperature overnight. And after TLC detection reaction is completed, extracting by using a dichloromethane/water system, combining organic phases, drying by using anhydrous sodium sulfate, distilling under reduced pressure to obtain a crude product, and separating and purifying the crude product by using ethyl acetate/water system through silica gel column chromatography to obtain colorless liquid C3 with the yield of about 84%. Ethanol was used as a solvent, C3 and palladium on charcoal (10%) were added in this order, and the mixture was purged with hydrogen balloon three times and reacted at room temperature under hydrogen for 5 hours. After the TLC detection reaction is completed, the reaction solution is filtered, the filtrate is collected and distilled under reduced pressure to obtain C4, and the yield is about 100%.

With anhydrous dichloromethane as a solvent, 1 equivalent of C4, 1.5 equivalents of TEA, 1.2 equivalents of TsCl, and a catalytic amount of DMAP were added in that order. And after TLC detection reaction is finished, removing dichloromethane in a reaction system by reduced pressure distillation, extracting by using an ethyl acetate/water system, combining organic phases, drying by using anhydrous sodium sulfate, then carrying out reduced pressure distillation to obtain a crude product, and carrying out silica gel column chromatography and purification on the crude product by using an ethyl acetate/petroleum ether system to obtain colorless liquid C5 with the yield of about 60%.

Dissolving 1 equivalent of C5 in an appropriate amount of DMF solution, sequentially adding 1.2 equivalents of 4-hydroxyphenylboronic acid pinacol ester and 1.5 equivalents of potassium carbonate, and reacting overnight in a 70 ℃ oil bath. After TLC detection reaction is finished, ethyl acetate/water system is used for extraction, organic phase anhydrous sodium sulfate is combined, reduced pressure distillation is carried out to obtain a crude product, and the crude product is subjected to silica gel column chromatography separation and purification by using ethyl acetate/petroleum ether system to obtain light yellow liquid C6 with the yield of about 65%.

1 equivalent of A9 was dissolved in DMF solution, and 1.2 equivalents of C6, 2.5 equivalents of potassium phosphate and a catalytic amount of tetrakis (triphenylphosphine) palladium were added in that order, and the mixture was purged three times with argon gas, and stirred under reflux at 90 ℃ overnight under argon protection. After TLC detection reaction is completed, ethyl acetate/water system is used for extraction, organic phase anhydrous sodium sulfate is combined, reduced pressure distillation is carried out to obtain a crude product, and the crude product is subjected to silica gel column chromatography and purification by using ethyl acetate/petroleum ether system to obtain yellow gel solid C7 with the yield of about 75%.

Adding C7 to 20%Trifluoroacetic acid in dichloromethane, and reacting for three hours. After the TLC detection reaction is completed, the organic phase is directly dried by spinning, trifluoroacetic acid is removed as far as possible, and the organic phase is dried for standby. The t-butoxy-removed C7 was dissolved in an appropriate amount of DMF, and 6 equivalents of DIPEA, 1.2 equivalents of HATU, and 1.2 equivalents of B10 (Boc protecting group removed) were added in this order to react at room temperature for 4 hours. After TLC detection reaction is completed, ethyl acetate/water system is used for extraction, organic phase anhydrous sodium sulfate is combined, reduced pressure distillation is carried out to obtain a crude product, and the crude product is separated and purified by silica gel column chromatography using dichloromethane/methanol system to obtain light yellow solid ED2 with the yield of about 55%. ED2:¹H NMR(500MHz,d6-DMSO)δ11.47(s,1H),8.95(s,1H),8.60(s,1H),8.18(s,1H),7.51(d,J＝7.8Hz,2H),7.44(d,J＝8.8Hz,1H),7.38(t,J＝9.7Hz,4H),7.32(s,1H),7.16(s,1H),7.01(d,J＝7.4Hz,2H),5.85(s,1H),5.17(s,1H),4.57(d,J＝9.3Hz,1H),4.46(t,J＝8.1Hz,1H),4.38(d,J＝15.9Hz,2H),4.33–4.21(m,3H),4.10(s,2H),3.97(s,2H),3.81(d,J＝9.9Hz,2H),3.71–3.58(m,4H),3.23(t,J＝11.2Hz,2H),3.06(d,J＝6.3Hz,2H),2.99(s,1H),2.41(s,3H),2.23(s,3H),2.20(s,3H),2.10(s,3H),2.07–1.98(m,3H),1.91(s,1H),1.64(d,J＝11.2Hz,2H),1.50(d,J＝10.6Hz,2H),0.94(s,9H),0.81(t,J＝5.9Hz,3H).¹³C NMR(125MHz,d6-DMSO)δ171.74,169.19,169.15,168.44,163.02,158.07,151.41,149.52,148.80,147.72,142.73,139.55,139.41,136.89,132.27,131.93,131.13,129.70,128.88,128.67,128.16,127.68,127.47,122.50,121.64,120.47,114.88,107.38,69.52,68.90,67.67,66.33,64.59,58.78,57.87,56.62,55.71,41.68,41.18,40.11,40.02,39.94,39.85,39.78,39.69,39.61,39.52,39.35,39.19,39.02,37.91,36.16,35.84,34.89,30.30,29.01,26.28,26.21,18.95,18.87,18.19,15.95,15.89,14.50,12.72.HRMS(ESI,m/z)calcd for C₅₆H₇₂N₇O₉S[M+H]⁺:1018.5107,found:1018.5121。

example 2: synthesis of Compound ED1

Reagents and reaction conditions: a) BnBr, NaH, THF; b) tert-butyl Bromoacetate, TBACl, CH2Cl2, NaOH; c) Pd/C, H2, EtOH; d) TsCl, DMAP, TEA, CH2Cl 2; (e)4-hydroxyphenylboronic acid pinacol ester, K2CO3, DMF,70 ℃; f) a9, K3PO4, Pd (PPh)4, DMF,90 ℃; g) 20% TFA/CH2Cl 2; h) b10, HATU, DIPEA, DMF.

As shown in the above reaction procedure, the synthesis of ED1 is described in the ED2 synthesis procedure. ED1:¹H NMR(400MHz,d6-DMSO)δ11.47(s,1H),8.92(s,1H),8.63(t,J＝5.9Hz,1H),8.17(t,J＝4.8Hz,1H),7.53–7.46(m,3H),7.42(d,J＝8.3Hz,2H),7.36(d,J＝8.2Hz,2H),7.29(s,1H),7.15(s,1H),7.09(d,J＝8.7Hz,2H),5.85(s,1H),5.18(d,J＝3.4Hz,1H),4.61(d,J＝9.6Hz,1H),4.51–4.34(m,4H),4.30(d,J＝4.8Hz,2H),4.26(d,J＝5.5Hz,1H),4.24–4.12(m,3H),4.06(s,2H),3.86(t,J＝4.3Hz,2H),3.80(d,J＝11.7Hz,2H),3.69(dd,J＝10.5,3.7Hz,1H),3.62(d,J＝10.8Hz,1H),3.22(t,J＝11.0Hz,2H),3.08–3.01(m,2H),3.01–2.92(m,2H),2.38(s,3H),2.22(s,3H),2.21(s,3H),2.10(s,3H),2.09–2.02(m,1H),1.96–1.88(m,1H),1.63(d,J＝11.4Hz,2H),1.55–1.45(m,2H),0.99–0.91(m,9H),0.79(t,J＝7.0Hz,3H).¹³C NMR(101MHz,d6-DMSO)δ171.77,169.12,168.47,162.99,158.03,151.33,149.47,148.75,147.63,142.71,139.51,139.39,136.83,132.38,131.94,131.09,129.63,128.84,128.62,128.10,127.62,127.39,122.51,121.61,120.46,114.96,107.36,69.60,69.54,68.87,66.98,66.29,58.76,57.83,56.57,55.71,41.71,41.20,40.15,39.94,39.73,39.52,39.31,39.10,38.89,37.89,35.79,34.89,30.29,26.22,18.93,18.85,18.17,15.86,14.47,12.67.HRMS(ESI,m/z)calcd for C₅₅H₇₀N₇O₉S[M+H]⁺:1004.4805,found:1004.4805。

example 3: synthesis of compound ED3:

reagents and reaction conditions: a) tert-butyl Bromoacetate, NaH, DMF; b) TsCl, DMAP, TEA, CH₂Cl₂；c)4-hydroxyphenylboronic acid pinacol ester,K₂CO₃,DMF,70℃；d)A9,K₃PO₄,Pd(PPh)₄,DMF,90℃；e)20％TFA/CH₂Cl₂；(f)B10,HATU,DIPEA,DMF。

As shown in the above reaction procedure, the synthesis of ED3 is described in the ED2 synthesis procedure. ED3:¹H NMR(500MHz,d6-DMSO)δ11.46(s,1H),8.95(s,1H),8.59(t,J＝5.6Hz,1H),8.17(s,1H),7.51(d,J＝8.1Hz,2H),7.45(d,J＝9.5Hz,1H),7.42–7.36(m,4H),7.33(s,1H),7.17(s,1H),6.98(d,J＝8.0Hz,2H),5.85(s,1H),5.16(s,1H),4.57(d,J＝9.5Hz,1H),4.45(t,J＝8.0Hz,1H),4.38(dd,J＝16.4,6.1Hz,2H),4.29(d,J＝4.2Hz,2H),4.25(dd,J＝16.0,5.3Hz,1H),4.13(s,2H),3.97(d,J＝16.9Hz,2H),3.80(t,J＝14.1Hz,4H),3.70–3.59(m,6H),3.23(t,J＝11.4Hz,2H),3.06(d,J＝6.6Hz,2H),3.01(d,J＝10.2Hz,1H),2.42(s,3H),2.23(s,3H),2.20(s,3H),2.10(s,3H),2.05(d,J＝7.3Hz,1H),1.98(dd,J＝17.1,10.8Hz,1H),1.91(dd,J＝14.8,6.5Hz,1H),1.65(d,J＝11.4Hz,2H),1.57–1.44(m,3H),1.23(s,5H),0.94(s,9H),0.82(t,J＝6.7Hz,3H).¹³C NMR(101MHz,d6-DMSO)δ171.81,169.22,168.71,163.08,158.06,151.44,149.64,147.75,142.80,139.54,139.43,136.92,132.29,131.99,131.18,129.73,128.71,127.67,127.49,122.57,121.64,120.49,114.92,107.51,70.53,69.79,69.68,69.06,68.93,67.21,66.36,58.81,57.90,56.61,55.79,41.74,40.15,39.94,39.73,39.52,39.31,39.10,38.89,37.94,35.75,34.95,31.31,30.36,29.04,28.85,28.75,28.71,28.59,26.23,25.15,22.12,21.41,18.99,18.23,15.91,14.53,13.98,12.73.HRMS(ESI,m/z)calcd for C₅₇H₇₄N₇O₁₀S[M+H]⁺:1048.5212,found:1048.5216。

example 4: synthesis of Compound ED4

Synthesis of intermediate A11

Reagents and reaction conditions: a) 4-methoxyarylphenylboronic acid, K₂CO₃,Pd(PPh₃)₄,DMF,100℃；b)NaOH,EtOH,60℃。

For the synthesis of a10 see the synthesis of C7. And after A10 is obtained, dissolving the A10 in ethanol solution, adding 1.5 equivalent of sodium hydroxide solution dissolved in water, stirring and refluxing for 3h at 60 ℃, adjusting the pH of the system to 3-4 by using dilute hydrochloric acid after TLC detection reaction is completed, extracting by using a dichloromethane/water system, combining organic phases, and carrying out reduced pressure distillation to obtain A11.

Synthesis of ED4

Reagents and reaction conditions: a) diethyl amine, CH₂Cl₂；b)Boc₂O,NaOH,THF/H₂O；c)B10,HATU,DIPEA；d)20％AcCl/MeOH；e)A11,HATU,DIPEA,DMF。

As shown in the above reaction procedure, the synthesis of ED4 is described in the synthesis of ED 2.ED 4:¹H NMR(400MHz,d6-DMSO)δ11.44(s,1H),8.95(s,1H),8.60–8.50(m,2H),8.20(t,J＝5.0Hz,1H),7.91(d,J＝8.4Hz,2H),7.70(d,J＝8.4Hz,2H),7.44(d,J＝9.9Hz,2H),7.38(s,4H),7.29(s,1H),5.86(s,1H),5.16(d,J＝3.5Hz,1H),4.57(d,J＝9.6Hz,1H),4.47(d,J＝8.1Hz,1H),4.35(d,J＝6.0Hz,2H),4.30(d,J＝4.8Hz,2H),4.25(dd,J＝15.8,5.7Hz,1H),3.97(s,2H),3.82(d,J＝10.3Hz,3H),3.70–3.55(m,9H),3.46(d,J＝5.8Hz,4H),3.25(t,J＝11.2Hz,4H),3.15–2.97(m,5H),2.43(s,3H),2.25(s,3H),2.21(s,3H),2.10(s,3H),2.06(s,1H),1.90(s,1H),1.66(d,J＝11.7Hz,3H),1.52(d,J＝8.8Hz,3H),1.23(s,2H),0.93(s,9H),0.83(t,J＝7.0Hz,3H).¹³C NMR(101MHz,d6-DMSO)δ172.16,169.69,169.42,169.09,166.42,163.48,151.88,150.00,149.52,148.21,142.78,140.23,139.87,136.73,133.60,131.59,130.20,129.16,128.30,127.93,126.84,123.54,121.50,100.00,70.88,70.06,69.86,69.53,69.33,66.80,59.21,58.38,57.06,56.20,42.16,41.66,40.64,40.43,40.23,40.02,39.81,39.60,39.39,38.39,36.20,35.38,30.78,26.67,19.43,18.66,16.37,15.10,13.26.HRMS(ESI,m/z)calcd for C₅₈H₇₃N₈O₁₀S[M-H]^-:1073.5176,found:1073.5195。

example 5: synthesis of compound ED5:

reagents and reaction conditions: a) tert-butyl Bromoacetate, NaH, DMF; b) TsCl, DMAP, TEA; c)4-hydroxyphenylboronic acid pinacol ester, K₂CO₃,DMF,70℃；d)K₃PO₄,Pd(PPh)₄,DMF,A9,90℃；e)20％TFA/CH₂Cl₂；f)B10,HATU,DIPEA,DMF。

As shown in the above reaction procedure, the synthesis of ED5 is described in the synthesis of ED 2.ED 5:¹H NMR(400MHz,d6-DMSO)δ11.47(s,1H),8.97(d,J＝3.9Hz,1H),8.60(t,J＝5.9Hz,1H),8.17(t,J＝4.8Hz,1H),7.53(d,J＝8.6Hz,2H),7.43(d,J＝4.6Hz,1H),7.41(s,1H),7.38(d,J＝9.1Hz,4H),7.35(s,1H),7.18(s,1H),6.99(d,J＝8.7Hz,2H),5.85(s,1H),5.17(s,1H),4.57(d,J＝9.6Hz,1H),4.50–4.39(m,2H),4.37(d,J＝6.1Hz,2H),4.33–4.20(m,4H),4.13–4.06(m,3H),3.97(s,2H),3.82(d,J＝10.2Hz,3H),3.76–3.71(m,2H),3.66(d,J＝3.6Hz,2H),3.60(d,J＝3.5Hz,10H),3.24(t,J＝11.2Hz,3H),3.17(s,3H),3.07(d,J＝6.9Hz,2H),3.03(d,J＝14.5Hz,1H),2.43(s,3H),2.23(s,3H),2.21(s,3H),2.10(s,3H),2.08–2.03(m,1H),1.95–1.86(m,1H),1.65(d,J＝10.9Hz,2H),1.57–1.45(m,2H),1.23(s,2H),0.94(s,9H),0.82(t,J＝6.9Hz,3H).¹³C NMR(101MHz,d6-DMSO)δ171.76,169.14,168.59,163.02,158.04,151.42,149.51,148.81,147.73,142.73,139.52,139.43,136.85,132.27,131.97,131.14,129.69,128.67,127.64,127.46,122.54,121.63,120.46,114.87,107.38,70.48,69.94,69.88,69.62,68.94,68.87,67.19,66.32,58.75,57.85,56.55,55.70,48.59,41.68,41.24,40.15,39.94,39.73,39.52,39.31,39.10,38.89,37.91,35.70,34.89,30.32,26.17,18.94,18.18,15.89,14.48,12.70.HRMS(ESI,m/z)calcd for C₅₉H₇₆N₇O₁₁S[M-H]^-:1090.5329,found:1090.5319。

example 6: synthesis of Compound ED6

Reagents and reaction conditions:a)diethylamine,CH₂Cl₂；b)tert-butyl bromoacetate,NaOH,CH₂Cl₂；(c)B10,HATU,DIPEA；d)20％AcCl/MeOH；e)A11,HATU,DIPEA,DMF。

as shown in the above reaction procedure, the synthesis of ED6 is described in the synthesis of ED 2.ED 6:¹H NMR(400MHz,d6-DMSO)δ11.45(s,1H),8.97(s,1H),8.55(t,J＝5.7Hz,2H),8.21(t,J＝4.9Hz,2H),7.91(dd,J＝12.4,9.0Hz,4H),7.72(d,J＝8.3Hz,3H),7.46(s,2H),7.40(q,J＝8.3Hz,5H),7.30(s,1H),5.87(s,1H),5.14(d,J＝3.5Hz,1H),4.55(d,J＝9.4Hz,2H),4.42(dd,J＝15.3,7.1Hz,3H),4.35(s,2H),4.30(d,J＝4.9Hz,3H),4.22(dd,J＝15.8,5.4Hz,2H),4.07–4.01(m,1H),4.01–3.95(m,1H),3.83(d,J＝10.8Hz,4H),3.70–3.56(m,9H),3.56–3.47(m,14H),3.47–3.41(m,8H),3.29–3.20(m,7H),3.15–2.99(m,6H),2.44(s,3H),2.26(s,3H),2.22(s,3H),2.11(s,3H),2.03(d,J＝8.3Hz,2H),1.97–1.86(m,1H),1.67(d,J＝11.3Hz,2H),1.54(dd,J＝17.5,10.3Hz,3H),1.44(s,1H),1.40–1.28(m,2H),1.25(d,J＝12.1Hz,2H),0.93(s,9H),0.83(t,J＝7.0Hz,3H).¹³C NMR(101MHz,d6-DMSO)δ172.39,170.46,170.02,169.44,166.43,163.49,151.89,150.04,149.54,148.19,143.30,143.25,142.80,140.21,139.96,136.71,133.93,133.58,131.63,130.12,129.11,128.30,127.91,126.84,124.50,123.56,122.06,121.50,107.88,70.19,70.09,69.95,69.34,67.40,66.80,59.19,58.38,56.80,42.14,41.79,41.70,40.62,40.41,40.20,39.99,39.78,39.57,39.37,38.39,36.15,35.81,35.39,30.80,26.79,19.42,19.02,18.65,16.38,15.10,13.26.HRMS(ESI,m/z)calcd for C₆₁H₇₉N₈O₁₁S[M-H]^-:1131.5594,found:1131.5584。

example 7: synthesis of Compound ED7

Reagents and reaction conditions: a) diethyl amine, CH₂Cl₂；b)tert-butyl bromoacetate,NaOH,CH₂Cl₂；c)B10,HATU,DIPEA；d)20％AcCl/MeOH；e)A11,HATU,DIPEA,DMF。

As shown in the above reaction procedure, the synthesis of ED7 is described in the synthesis of ED 2.ED 7:¹H NMR(400MHz,d6-DMSO)δ11.47(s,1H),8.98(s,1H),8.58(s,2H),8.23(s,1H),7.93(d,J＝8.2Hz,3H),7.72(d,J＝8.0Hz,2H),7.46(s,1H),7.39(dd,J＝16.2,7.9Hz,4H),7.29(s,1H),5.86(s,1H),5.15(s,1H),4.55(d,J＝9.3Hz,1H),4.42(d,J＝4.8Hz,2H),4.35(s,1H),4.30(d,J＝4.0Hz,2H),4.26–4.16(m,1H),4.08–4.01(m,1H),3.98(t,J＝6.5Hz,1H),3.83(d,J＝10.2Hz,2H),3.70–3.59(m,4H),3.55(d,J＝15.6Hz,7H),3.45(dd,J＝18.5,4.6Hz,11H),3.25(t,J＝11.2Hz,3H),3.17–2.96(m,4H),2.86(d,J＝15.4Hz,1H),2.72(d,J＝15.1Hz,1H),2.44(s,3H),2.39–2.29(m,2H),2.25(s,3H),2.21(s,3H),2.10(s,3H),2.07–2.00(m,1H),1.99(s,1H),1.90(s,1H),1.66(d,J＝10.9Hz,2H),1.52(d,J＝7.4Hz,4H),1.31(dd,J＝14.5,7.2Hz,3H),1.23(s,1H),1.17(t,J＝7.1Hz,1H),0.93(s,9H),0.83(t,J＝7.0Hz,3H).¹³C NMR(101MHz,d6-DMSO)δ172.40,170.48,170.01,169.70,169.45,166.43,163.49,151.89,150.10,149.53,148.16,143.26,142.78,140.17,139.93,136.70,133.91,133.56,131.63,130.10,129.09,128.29,127.90,126.83,123.55,122.02,121.47,107.92,73.34,70.23,70.18,70.15,70.09,69.93,69.36,69.33,67.37,66.79,64.24,59.19,58.36,56.81,49.06,43.51,42.13,41.68,40.57,40.36,40.15,39.94,39.73,39.52,39.31,38.37,36.14,35.81,35.39,30.78,30.53,30.43,26.78,19.42,19.01,18.65,16.37,15.10,13.97,13.24.HRMS(ESI,m/z)calcd for C₆₃H₈₃N₈O₁₂S[M-H]^-:1175.5857,found:1175.5874。

example 8: synthesis of Compound ED8

Reagents and reaction conditions: a) NaOAc, AcOH, reflux; b) DIPEA, DMF,90 ℃; c) 20% TFA/CH₂Cl₂；d)HATU,DIPEA。

Commercially available 1 equivalent of D1, 1.1 equivalent of D2, and 1.2 equivalents of sodium acetate were added to a round bottom flask containing the appropriate amount of acetic acid and the reaction was stirred at room temperature overnight. After completion of the TLC detection reaction, ethyl acetate was usedExtraction was performed by eluting with ester/water, the organic phase was collected, the solvent was removed by distillation under reduced pressure, and the crude product was purified by silica gel column chromatography using a methanol/dichloromethane system to give D3 as a yellow solid in about 80% yield.¹H NMR(400MHz,DMSO)δ11.15(s,1H),8.00–7.91(m,1H),7.79(d,J＝7.3Hz,1H),7.74(t,J＝8.9Hz,1H),5.16(dd,J＝12.9,5.4Hz,1H),2.89(ddd,J＝17.2,14.0,5.5Hz,1H),2.69–2.55(m,2H),2.06(ddd,J＝10.7,5.5,3.1Hz,1H)。

1 equivalent of D3 was dissolved in the appropriate amount of DMF and 1.2 equivalents of D3 and 2 equivalents of DIPEA were added. The reaction system is reacted for 12 hours at 90 ℃. After the completion of the TLC detection reaction, extraction was performed using an ethyl acetate/petroleum ether system, the organic phase was collected, the solvent was distilled off under reduced pressure, and the crude product was subjected to silica gel column chromatography using a methanol/dichloromethane system to isolate purified yellow gel-like solid D5 in a yield of about 39%. Then, the Boc protecting group was removed with trifluoroacetic acid to obtain yellow liquid D6.

Referring to the synthesis method of ED2, compound ED8 was obtained in 65% yield by a one-step amide formation reaction starting from D6 and a 11. ED 8:¹H NMR(400MHz,d6-DMSO)δ11.44(s,1H),11.07(s,1H),8.52(s,1H),8.20(s,1H),7.90(d,J＝8.4Hz,2H),7.71(d,J＝8.4Hz,2H),7.60–7.53(m,1H),7.46(s,1H),7.29(s,1H),7.12(d,J＝8.6Hz,1H),7.01(d,J＝7.0Hz,1H),6.57(s,1H),5.86(s,1H),5.04(dd,J＝12.8,5.4Hz,1H),4.30(d,J＝4.9Hz,2H),3.83(d,J＝10.4Hz,3H),3.09(dd,J＝22.8,15.6Hz,7H),2.89(dd,J＝22.5,8.5Hz,2H),2.58(d,J＝16.9Hz,2H),2.25(s,3H),2.21(d,J＝5.4Hz,3H),2.11(s,3H),2.08–1.99(m,2H),1.65(d,J＝20.5Hz,6H),1.53(d,J＝8.2Hz,3H),1.23(s,1H),0.84(t,J＝6.9Hz,3H).¹³C NMR(101MHz,d6-DMSO)δ173.28,170.56,169.44,167.77,166.28,163.49,150.04,149.53,146.90,142.73,140.22,136.74,133.88,133.80,132.68,128.25,126.84,123.54,122.06,121.48,117.72,110.87,109.52,107.85,66.80,58.38,49.02,42.05,41.66,40.61,40.41,40.20,39.99,39.78,39.57,39.36,35.38,31.44,30.79,27.04,26.74,22.63,19.42,18.66,15.10,13.26.HRMS(ESI,m/z)calcd for C₄₇H₅₁N₆O₉[M-H]^-:843.3723,found:843.3684。

example 9: synthesis of Compound ED9

Reagents and reaction conditions: a) DIPEA, DMF,90 ℃; b) 20% TFA/CH₂Cl₂；c)HATU,DIPEA。

Reference to the synthetic procedure for ED8 gave compound ED 9. ED9:¹H NMR(400MHz,d6-DMSO)δ11.47(s,1H),11.07(s,1H),8.53(t,J＝5.5Hz,1H),8.21(t,J＝4.7Hz,1H),7.89(d,J＝8.3Hz,2H),7.69(d,J＝8.4Hz,2H),7.58–7.52(m,2H),7.46(s,1H),7.29(s,1H),7.14(d,J＝8.6Hz,1H),7.01(d,J＝7.0Hz,1H),6.62(t,J＝5.6Hz,1H),5.86(s,1H),5.03(dd,J＝12.9,5.3Hz,1H),4.30(d,J＝4.9Hz,2H),3.83(d,J＝9.6Hz,3H),3.66(t,J＝5.3Hz,3H),3.61(d,J＝6.0Hz,3H),3.47(dd,J＝10.3,5.3Hz,8H),3.27(d,J＝11.1Hz,4H),3.10(dd,J＝13.9,6.8Hz,2H),3.03(s,1H),2.92–2.80(m,1H),2.56(d,J＝16.1Hz,2H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.04–1.94(m,1H),1.67(d,J＝10.2Hz,2H),1.60–1.46(m,3H),1.23(s,2H),0.83(t,J＝6.9Hz,3H).¹³C NMR(101MHz,d6-DMSO)δ191.60,173.23,170.52,169.41,167.75,166.46,163.49,158.59,158.28,150.09,149.54,146.91,143.28,142.78,140.18,136.71,133.93,133.55,132.54,128.28,126.80,123.57,122.05,121.50,117.93,111.13,109.72,107.92,69.26,66.80,58.38,49.03,42.17,41.70,40.60,40.39,40.18,39.97,39.76,39.55,39.34,35.38,31.43,30.81,22.61,19.42,18.65,15.10,13.25.HRMS(ESI,m/z)calcd for C₄₇H₅₁N₆O₁₀[M-H]^-:859.3672,found:859.3682。

example 10: synthesis of Compound ED10

Synthesis of intermediate D14

Reagents and reaction conditions a) pyridine,110 ℃; b) tert-butyl Bromoacetate, K₂CO₃,DMF；c)20％TFA/CH₂Cl₂。

Commercially available 1 equivalent of D10 and 1.1 equivalent of D11 were added to a round bottom flask containing pyridine as a solvent, and the reaction was stirred at 110 ℃ under reflux for 14 h. After TLC detection reaction, ethyl acetate/water system is used for extraction, organic phase is collected, after reduced pressure distillation, the obtained crude product is subjected to silica gel column chromatography separation and purification by using methanol/dichloromethane system, so that brown solid D12 is obtained, and the yield is about 80%.¹H NMR(500MHz,DMSO)δ11.18(s,1H),11.09(s,1H),7.66(t,J＝7.7Hz,1H),7.31(t,J＝7.4Hz,1H),7.25(d,J＝8.4Hz,1H),5.07(dd,J＝12.8,5.2Hz,1H),2.95–2.82(m,1H),2.66–2.52(m,2H),2.01(dd,J＝13.6,7.8Hz,1H)。

1 equivalent of D12 was dissolved in an appropriate amount of DMF, and then 1.2 equivalents of t-butyl bromoacetate and 1.5 equivalents of potassium carbonate were added to react at room temperature for two hours. After TLC detection reaction is completed, ethyl acetate/water system is used for extraction, organic phase is collected, after reduced pressure distillation, the obtained crude product is subjected to silica gel column chromatography and purification by using methanol/dichloromethane system, and white solid D13 is obtained, and the yield is about 90%. D13 was added to a 20% trifluoroacetic acid in dichloromethane and reacted for three hours. After TLC detection is finished, directly carrying out reduced pressure distillation to remove dichloromethane, then adding ethyl acetate and methanol for many times to carry out reduced pressure distillation to remove residual trifluoroacetic acid as far as possible to obtain D14, wherein the yield is about 100%.

Synthesis of ED10

Reagents and reaction conditions: a) boc2O, EtOH; b) TsCl, TEA, DMAP, CH₂Cl₂；c)Na₂CO₃,DMF,80℃；d)20％TFA/CH₂Cl₂；e)HATU,DIPEA。

1 equivalent of D15 and 1.2 equivalents of Boc₂Adding O into a round-bottom flask containing a proper amount of ethanol, and reacting for 2 hours at room temperature. After completion of the TLC detection reaction (ninhydrin development), the solvent was distilled off under reduced pressure to obtain colorless liquid D16 with a yield of about 100%. At 0 deg.C, 1.5 equivalents of trisEthylamine, 1.2 equivalents of p-toluenesulfonyl chloride, and a catalytic amount of DMAP were added to a solution of 1 equivalent of D16 in dichloromethane, and the reaction was allowed to slowly return to room temperature for 16 hours. After TLC detection reaction is completed, the solvent is removed by reduced pressure distillation, ethyl acetate/water system is used for extraction, organic phases are combined and washed by half-saturated saline for a plurality of times, and colorless liquid D17 is obtained, wherein the yield is about 67%.

1 equivalent of D12, 1.2 equivalents of D17 and 1.5 equivalents of sodium carbonate were added to a round bottom flask containing DMF and reacted at 80 ℃ for 42 h. After the completion of the TLC detection reaction, extraction was performed using a dichloromethane/water system, the organic phase was collected, washed with half-saturated brine several times to remove DMF as much as possible, and after distillation under reduced pressure, the obtained crude product was subjected to silica gel column chromatography purification using a methanol/dichloromethane system to obtain D18 as a white solid with a yield of about 100%. Then, the Boc protecting group was removed with trifluoroacetic acid to obtain yellow liquid D19.

Referring to the synthesis method of ED8, compound ED10 was obtained in about 60% yield by amide-forming reaction starting from D19 and a 11. ED 10:¹H NMR(400MHz,d6-DMSO)δ11.46(s,1H),11.09(s,1H),8.52(d,J＝5.7Hz,1H),8.21(s,1H),7.91(d,J＝8.3Hz,2H),7.82–7.75(m,1H),7.71(d,J＝8.2Hz,2H),7.52–7.40(m,3H),7.29(s,1H),5.86(s,1H),5.08(dd,J＝12.8,5.3Hz,1H),4.31(s,4H),3.82(d,J＝13.5Hz,4H),3.70–3.63(m,3H),3.57(dd,J＝11.1,5.5Hz,5H),3.43(d,J＝5.4Hz,3H),3.29–3.18(m,4H),3.09(d,J＝6.8Hz,2H),3.01(d,J＝11.0Hz,1H),2.94–2.79(m,2H),2.63–2.53(m,2H),2.26(s,2H),2.21(d,J＝5.2Hz,3H),2.11(s,3H),2.07(d,J＝3.3Hz,1H),2.06–1.98(m,2H),1.90(d,J＝11.1Hz,1H),1.66(d,J＝10.2Hz,2H),1.52(d,J＝8.2Hz,2H),1.24(t,J＝15.7Hz,2H),0.83(t,J＝6.8Hz,3H).¹³C NMR(101MHz,d6-DMSO)δ172.75,169.91,168.96,166.79,165.91,165.27,163.02,155.82,149.54,149.05,142.77,142.30,139.74,136.94,136.22,133.45,133.23,133.10,127.80,126.35,123.07,121.59,121.02,119.99,116.33,115.38,107.38,70.10,69.70,68.94,68.88,68.70,66.32,57.91,48.77,41.22,40.15,39.94,39.73,39.52,39.31,39.10,38.89,34.92,30.94,30.32,21.99,18.94,18.17,14.63,12.79.HRMS(ESI,m/z)calcd for C₄₉H₅₇N₆O₁₁[M+H]⁺905.4080,found:905.4096。

example 11: synthesis of Compound ED11

Reagents and reaction conditions: a) boc₂O,CH₂Cl₂；b)D14,HATU,DIPEA,DMF；c)TFA,CH₂Cl₂,50℃；d)HATU,DIPEA。

Dissolving 1 equivalent of Boc anhydride in an appropriate amount of DCM, slowly dropwise adding into a4 equivalent of D20 solution in an appropriate amount of DCM under the ice bath condition, and reacting for 3h when a white solid appears during dropwise adding. After TLC detection reaction is completed (ninhydrin is developed), adding half saturated common salt solution into the system, then extracting with DCM for three times, combining organic phases, washing the organic phases with half saturated common salt solution for multiple times, collecting the organic phases, and carrying out vacuum distillation to obtain a white solid D21 with the yield of about 64%.

1.2 equivalents of D21, 1 equivalent of D14, 3 equivalents of DIPEA and 1 equivalent of HATU were added sequentially to a round bottom flask containing DMF and the reaction was stirred at room temperature for 19 h. After the completion of the TLC detection reaction, the system was extracted, the organic phase was collected and the solvent was distilled off under reduced pressure. And (3) separating and purifying the crude product by using a methanol/dichloromethane system through silica gel column chromatography to obtain a light yellow solid, and then removing a protecting group by using trifluoroacetic acid to obtain a white solid D22.

Referring to the synthesis method of ED8, compound ED11 was obtained in about 60% yield by amide-forming reaction starting from D22 and a 11. ED 11:¹H NMR(500MHz,d6-DMSO)δ11.47(s,1H),11.12(s,1H),8.47(t,J＝5.3Hz,1H),8.22(t,J＝4.7Hz,1H),7.95(t,J＝5.4Hz,1H),7.90(d,J＝8.2Hz,2H),7.81(t,J＝7.9Hz,1H),7.71(d,J＝8.2Hz,2H),7.51–7.43(m,2H),7.39(d,J＝8.5Hz,1H),7.29(s,1H),5.86(s,1H),5.12(dd,J＝12.7,5.4Hz,1H),4.77(s,2H),4.30(d,J＝4.7Hz,2H),3.83(d,J＝11.6Hz,2H),3.25(t,J＝9.3Hz,5H),3.15(dd,J＝12.5,6.2Hz,3H),3.10(d,J＝6.9Hz,3H),3.02(t,J＝10.8Hz,1H),2.90(dd,J＝23.2,7.3Hz,3H),2.56(dd,J＝24.7,11.7Hz,2H),2.25(s,3H),2.21(s,3H),2.10(s,3H),2.08–2.00(m,2H),1.66(d,J＝11.1Hz,2H),1.53(d,J＝7.4Hz,5H),1.45(s,3H),1.29(d,J＝18.1Hz,5H),1.27–1.20(m,2H),0.83(t,J＝6.9Hz,3H).¹³C NMR(126MHz,d6-DMSO)δ172.82,169.92,169.01,166.77,166.69,165.76,165.55,163.05,155.07,149.63,149.08,142.82,142.24,139.77,136.97,136.31,133.42,133.06,127.81,126.39,123.06,121.61,121.04,120.40,116.85,116.08,107.44,67.66,66.37,57.93,48.84,41.16,40.02,39.94,39.85,39.78,39.69,39.61,39.52,39.44,39.35,39.19,39.02,38.31,34.93,30.97,30.32,29.15,29.01,26.21,26.06,22.03,18.99,18.22,14.68,12.81.HRMS(ESI,m/z)calcd for C₅₁H₆₀N₇O₁₀[M+H]⁺:930.4396,found:930.4413。

example 12: synthesis of Compound ED12

Reagents and reaction conditions: a) boc₂O,CH₂Cl₂；b)D14,HATU,DIPEA,DMF；c)TFA,CH₂Cl₂,50℃；d)HATU,DIPEA,DMF。

Synthesis of ed12.ed12 with reference to the synthesis of ED 8:¹H NMR(400MHz,d6-DMSO)δ11.45(s,1H),11.10(s,1H),8.45(t,J＝5.5Hz,1H),8.21(t,J＝4.7Hz,1H),7.91(d,J＝8.5Hz,3H),7.84–7.78(m,1H),7.71(d,J＝8.3Hz,2H),7.49(d,J＝7.2Hz,1H),7.46(s,1H),7.40(d,J＝8.5Hz,1H),7.29(s,1H),5.86(s,1H),5.12(dd,J＝12.8,5.4Hz,1H),4.76(s,2H),4.30(d,J＝4.7Hz,2H),3.83(d,J＝10.8Hz,2H),3.29–3.20(m,6H),3.18–3.06(m,6H),2.96–2.84(m,3H),2.62(s,1H),2.60–2.53(m,1H),2.26(s,3H),2.21(s,3H),2.11(s,3H),2.05(dd,J＝12.5,4.2Hz,2H),1.67(d,J＝11.1Hz,2H),1.52(s,4H),1.44(s,3H),1.28(s,13H),0.83(t,J＝6.8Hz,3H).¹³C NMR(101MHz,d6-DMSO)δ173.22,170.32,169.44,167.20,167.09,166.18,165.99,163.49,155.52,150.02,149.52,143.25,142.67,140.22,137.39,136.76,133.88,133.51,128.24,126.82,123.55,122.06,121.49,120.90,117.32,116.53,107.86,68.17,66.80,58.38,49.31,41.68,40.62,40.41,40.20,40.00,39.79,39.58,39.37,38.81,35.39,31.42,30.80,29.60,29.45,29.21,29.15,26.93,26.74,22.47,19.42,18.65,15.10,13.26.HRMS(ESI,m/z)calcd for C₅₃H₆₄N₇O₁₀[M+H]⁺:958.4709,found:958.4677。

example 13: synthesis of Compound ED13

Reagents and reaction conditions: a) boc2O, EtOH; b) TsCl, TEA, DMAP, CH₂Cl₂；c)Na₂CO₃,DMF；d)20％TFA/CH₂Cl₂；e)HATU,DIPEA,DMF。

Synthesis of ed13.ed13 with reference to the synthesis of ED 8:¹H NMR(400MHz,d6-DMSO)δ11.45(s,1H),11.09(s,1H),8.52(dd,J＝12.1,6.3Hz,1H),8.20(t,J＝4.9Hz,1H),7.90(t,J＝9.3Hz,2H),7.81–7.76(m,1H),7.74–7.67(m,2H),7.50(d,J＝8.5Hz,1H),7.44(d,J＝7.3Hz,2H),7.30(s,1H),5.86(s,1H),5.08(dd,J＝12.8,5.4Hz,1H),4.30(dd,J＝11.9,6.1Hz,4H),3.85(d,J＝5.9Hz,2H),3.82–3.75(m,3H),3.66–3.60(m,3H),3.54(d,J＝5.2Hz,10H),3.46–3.39(m,4H),3.28–3.19(m,3H),3.09(d,J＝7.0Hz,1H),3.03(s,1H),2.94–2.80(m,1H),2.66–2.53(m,2H),2.25(s,1H),2.21(d,J＝5.4Hz,3H),2.10(s,3H),2.09–1.97(m,3H),1.90(d,J＝11.7Hz,1H),1.66(d,J＝10.6Hz,1H),1.59–1.47(m,2H),1.30–1.17(m,3H),0.83(t,J＝7.0Hz,2H).¹³C NMR(101MHz,d6-DMSO)δ172.74,169.89,166.79,165.24,163.00,155.82,136.95,133.22,127.80,126.35,120.02,116.32,115.37,107.37,70.15,69.83,69.73,69.62,69.23,68.90,68.85,68.68,66.31,48.76,40.15,39.94,39.73,39.52,39.31,39.10,38.89,30.94,30.32,22.00,18.94,18.91,18.18,14.63,12.79.HRMS(ESI,m/z)calcd for C₅₁H₆₁N₆O₁₂[M+H]⁺:949.4342,Found949.4323。

example 14: western blotting (Westren Blot) to detect the degradation of EZH2 by compounds

(1) Extraction of total protein: the cells that were not treated with the drug or treatment group were transferred to a centrifuge tube, harvested by centrifugation and washed twice with PBS and transferred to a 1.5ml EP tube. 100-200ul lysis buffer lysate was added to each EP tube based on the number of cells, and the mixture was left to stand at room temperature for 10 minutes. The EP is then placed on a thermostatted metal bath and cooked for 15-30 minutes at 95 ℃. After cooling to room temperature, the mixture was placed in a centrifuge and centrifuged at 12000rpm at 4 ℃ for about 15 minutes. The supernatant was carefully transferred to a new clean EP tube. (2) Protein content determination: protein content determination was performed with reference to Pierce BCA protein quantification kit instructions. (3) And (3) carrying out SDS-PAGE gel electrophoresis gel preparation: and (3) placing and airing the cleaned glass plate, and preparing 10% of separation glue and 5% of concentrated glue according to the formula in sequence. And (3) adding 5xSDS loading buffer to the protein sample in proportion to make the final concentration of SDS be 1 x. After mixing uniformly, the sample is put into a constant temperature metal bath and boiled for 10 minutes at the temperature of 97 ℃. After calculation, 30ug of protein sample was added in equal amount to each loading well. Electrophoresis: the voltage of the gel concentration part is set to be 80V, and after the band enters the separation gel, the constant voltage is adjusted to be 120V until the bromophenol blue band runs to the bottom end. (4) Preparing a PVDF membrane soaked by methanol, filter paper and black sponge soaked by the membrane transferring solution, separation glue, a membrane transferring clamp and the filter paper, and placing the membrane transferring clamp and the filter paper in a tray filled with the membrane transferring solution. The rotating film clamp is black, the sponge, the filter paper, the separating gel PVDF film, the other piece of filter paper and the sponge are sequentially placed on the rotating film clamp, and a glass rod is used for carefully removing bubbles in the process. Clamping and placing into a film rotating groove for fixing. The wet transfer method is adopted, the constant current is 0.25A, and the film is transferred for 1-3h according to different molecular weights. (5) Immunoblot blocking: the PVDF membrane after membrane conversion is put into a plastic box containing 5% skimmed milk powder, sealed at room temperature for 1 hour, and washed with TBST for 10 minutes. Incubating primary antibody: diluting the primary antibody with a dilution solution of Biyuntian corporation according to the instruction, putting the transfer printing film into the diluted primary antibody, and incubating overnight in a shaking table at 4 ℃. Hatching a secondary antibody: the next day the membranes were washed with TBST for 10 min X3 times. The transfer film was then transferred to a diluted secondary antibody, incubated at room temperature for 1 hour, and then eluted again with TBST for 10 minutes × 3 times. ECL development: and uniformly delivering the prepared ECL luminous liquid to a strip to be detected, placing the strip for one minute in a dark place, developing by using a chemiluminescence imaging system, and taking a picture.

Example 15: killing effect of MTS detection compound on tumor cells

Taking the lymph cancer cell strains Su-DHL-2, Su-DHL-4 and Su-DHL-6 as examples, taking the cells in the logarithmic growth phase, centrifuging, discarding the supernatant, washing with PBS twice, then counting by resuspension, and evenly inoculating the cells in a 96-well plate at 3000 cells/100 ul per well. Duplicate wells and blanks were set up. The drug was diluted with fresh medium at the desired concentration, 100. mu.l per well was added to a 96-well plate in which cells were plated, and incubated in an incubator containing CO2 at 37 ℃ for 5 days. The reaction was continued for 4 hours by adding 20. mu.l of MTS solution to the 96-well plate. The absorbance (OD) of the different wells was measured at 490nm wavelength using a microplate reader.

To further demonstrate the effect of the compounds of the invention on the level of EZH2 protein. In the invention, after 22Rv1 cells are treated with compound E1-E13 or DMSO at a specified concentration for 48 hours, the levels of EZH2 and H3K27me3 are determined by using a protein immunoblot (Wesren blot) experiment, and Tubulin is used as a control. As shown in figure 1, the results show that compounds ED2, ED3 most significantly reduced EZH2 protein levels, and also reduced H3K27me3 levels.

Further, as shown in fig. 2, the effect of different concentrations of compounds ED2 and ED3 on EZH2 protein levels in the present invention. The present invention measured EZH2 and H3K27me3 levels using a western blot (Wesrern blot) assay with histone H3 and Tubulin as controls 24 hours after treatment of 22Rv1 cells with the indicated concentrations of compounds. The results show that ED2 and ED3 can degrade EZH2 protein levels in a concentration-dependent manner.

As shown in figure 3, the effect of compounds ED2, ED3 and the existing EZH2 inhibitor EPZ6438 of the present invention on lymphoma cell viability is demonstrated. The cell survival rate is determined by using MTS after the compound under the specified concentration is respectively treated for five days on three lymphoma cell strains Su-DHL-2, Su-DHL-6 and Su-DHL-6. The results show that the compounds ED2 and ED3 of the invention have a stronger killing effect than EPZ 6438.

Various corresponding changes and modifications can be made by those skilled in the art based on the above technical solutions and concepts, and all such changes and modifications should be included in the protection scope of the present invention.

Claims

1. A small molecule compound based on the degradation of EZH2 protein, characterized by a molecule of structural feature I, wherein X is a linker comprising an acyclic or cyclic, saturated or unsaturated carbon, glycol, amide, amino, ether, or carbonyl-containing group; y may be a group of an affinity E3 ligase VHL or cereblon (crbn);

2. the EZH2 protein degradation-based small molecule compound of claim 1, wherein Y in structural formula I is a compound of structural formula II, a fragment of affinity E3 ligase VHL, and structural feature III:

3. the EZH2 protein degradation-based small molecule compound of claim 2, wherein the structural formula III, X can be, but is not limited to, the following connecting chains X1, X2 and X3:

4. the EZH2 protein degradation-based small molecule compound of claim 3, wherein n in X1 can be, but is not limited to, 1,2, 3; n as indicated by X2 may be, but is not limited to, 2, 3, 4.

5. The EZH2 protein degradation-based small molecule compound of claim 1, wherein the structural formula I, Y can be a fragment of the affinity E3 ligase CRBN shown in structure IV and the structural feature is V:

6. the EZH2 protein degradation-based small molecule compound of claim 5, wherein the structural formula V, X can be, but is not limited to, the following connecting chains X4, X5, X6 and X7:

7. the EZH2 protein degradation-based small molecule compound of claim 6, wherein n of X6 can be, but is not limited to, 3, 4; or n in X7 can be, but is not limited to, 3, 4.

8. The EZH2 protein degradation-based small molecule compound of claim 2, wherein the compounds with structural feature III can be one of the following:

9. the EZH2 protein degradation-based small molecule compound of claim 5, wherein the compounds with structural feature V can be one of the following:

10. a pharmaceutical composition comprising a compound of any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

11. Use of a pharmaceutical composition according to claim 10 for the preparation of a medicament for the treatment of a tumor.