CN112920176B - Bifunctional compound capable of inducing PRC2 protein complex core subunit degradation, pharmaceutical composition and application - Google Patents

Abstract

The invention discloses a bifunctional compound capable of inducing PRC2 protein complex core subunit degradation, a pharmaceutical composition and an application, wherein the bifunctional compound comprises a compound shown as any one of formulas I-III, a pharmaceutically acceptable salt or prodrug thereof, a solvate thereof, a hydrate thereof, a polymorph thereof, a tautomer thereof, a stereoisomer thereof or an isotopic substitution compound;

wherein N in the formula I-III is an integer of 1-10, X is O, N or S, Y is O or H ₂ Or S. The bifunctional compound can effectively induce the core subunit of the PRC2 protein complex to degrade, so as to treat cancers mediated by the PRC2 complex and subunits thereof including EZH2, EED, SUZ12, rbAp46 and RbAp48, and completely block the oncogenic activity of the PRC2 complex subunit.

Description

Bifunctional compounds and drugs that induce degradation of the core subunit of the PRC2 protein complex Composition and application

technical field

The invention relates to the technical field of chemistry and medicine, in particular to a bifunctional compound capable of inducing the degradation of the core subunit of PRC2 protein complex, a pharmaceutical composition and its application.

Background technique

With the in-depth research on the pathogenesis of tumors, humans have deeply realized that the occurrence and development of tumors are not only related to DNA sequence changes caused by intracellular gene mutations or deletions, but also related to gene disorders caused by changes in other heritable substances , the so-called epigenetic phenomenon. Epigenetic modification can regulate processes such as proto-oncogene activation, tumor suppressor gene inactivation, DNA damage repair, and tumor stem cell differentiation through DNA methylation, histone modification, chromatin remodeling, or non-coding RNA interference. Thereby regulating the growth, development and pathological changes of the body.

The polycomb group (PcG) protein was originally discovered as a kind of protein that regulates the establishment of the Drosophila body by participating in the regulation of Hox gene expression during the development and cell differentiation of Drosophila, and was later identified as a Functionally critical epigenetic regulators. In mammals, PRC1 and PRC2 are the two major polysphobic repressive complexes among PcG proteins that regulate gene transcription through histone post-translational modifications. Different combinations of multiple subunits can form PRC1 with different functions, among which the classic PRC1 consists of several core subunits: CBX (chrombox), PCGF (polycomb groupfactor), HPH (human polyhomeotic homolog) and RING1A/B (really interesting newgene) It mainly mediates H2AK119ub1 through the catalytic subunit RING1A/B, and plays an important role in embryonic development, maintenance of stem cell characteristics and tumorigenesis; PRC2 includes EZH2, SUZ12, EED and RbAp46/48 (retinoblastoma-associated proteins 46/48 , also known as RBBP4/7) several core subunits, mainly mediate H3K27me2 and H3K27me3 through the catalytic subunit EZH2, and regulate the transcription of genes related to cell cycle, aging, differentiation and tumorigenesis.

Among them, EZH2, as the catalytic subunit of PRC2, can catalyze H3K27me3 with the participation of the other two core subunits of the complex, SUZ12 and EED, and form a complex with PRC1-mediated H2AK119ub1 on the nucleosome to hinder transcription elongation body, thereby maintaining the transcriptional silencing of downstream genes. EZH2 itself cannot exert catalytic activity alone, it must at least rely on the SUZ12 and EED subunits of PRC2 to exert HMTase activity and inhibit the transcription of various downstream genes. SUZ12 is a core subunit that is critical to maintain PRC2 integrity, EZH2 stability and HMTase activity. Its C-terminal VEFS (Vrn2-Emf2-Fis2-SUZ12) domain can stably bind to EZH2 and maintain the PRC2 complex. Assembly, on the other hand, can assist the HMTase activity of PRC2 through allosteric effects. The interaction and binding of the repeated WD domain of the EED subunit with EZH2 is a necessary condition for ensuring that EZH2 has HMTase activity, and the specific binding of the C-terminal WD domain of EED to H3K36me3 is a necessary condition for the allosteric activation of HMTase, thus EED It is also a core subunit of PRC2 to exert HMTase activity. Retinoblastoma-associated protein 46 and 48 (RbAp46/48) subunits are highly homologous histone chaperones that play an important role in the maintenance of chromatin structure. In PRC2, although EZH2 does not require the direct participation of RbAp46 and RbAp48 to exert HMTase activity, the process of introducing PRC2 into nucleosomes requires the combination of RbAp48 and histone H3-H4 heterodimers, so RbAp48 also ensures the normal operation of PRC2 The core subunit that exerts HMTase activity.

However, in addition to participating in the formation of the PRC2 complex to catalyze H3K27me3 and upregulate transcriptionally silenced tumor suppressor genes, each core subunit of the PRC2 complex can also exert its own non-PRC2 catalytic functions. For example, as a multifunctional protein, EZH2 can not only mediate gene silencing by catalyzing H3K27me3, but also mediate gene transcriptional activation in some tumors in a manner independent of HMTase activity. Many studies have shown that EZH2 can also act as a transcriptional activator to methylate non-histone proteins or directly interact with other proteins to form transcriptional complexes to activate gene transcription. After Ser 21 of EZH2 is phosphorylated by protein kinase B (AKT), pEZH2 can act as a co-activator of androgen receptor (AR) and its related complexes to promote castration-resistant prostate cancer (castration). -resistant prostate cancer (CRPC) cell growth, that is, AKT mediates Ser21 phosphorylation of EZH2, promotes EZH2 binding to AR and catalyzes the methylation of AR or AR-related proteins, thereby activating the transcription of a series of target genes downstream of AR. It provides a new combination therapy idea for the treatment of metastatic, hormone-refractory prostate cancer. Other studies have found that phosphorylated EZH2 can also directly bind and methylate Lys180 of STAT3, mediate the phosphorylation of 705 tyrosine (Tyr) of STAT3 and enhance the activity of STAT3, thereby promoting glioblastoma and glial Tumorigenicity of blastoma stem cells. These results are in complete contrast to the results that EZH2 catalyzes non-histone GATA4 and RORα to repress gene transcription.

In addition, EZH2 was also found to activate transcription in a manner independent of methyltransferase activity in colorectal cancer cells. Human proliferating cell nuclear antigen-related factor PAF recruits EZH2, promotes the binding and interaction between EHZ2 and TCF/β-catenin, and forms transcription initiation together with transcription factor TCF/LEF at the promoters of c-myc, cyclinD1, Axin2 and other genes complex, induces transcriptional activation of tumorigenesis-related target genes in the Wnt pathway, and promotes colorectal cancer progression. In addition, in AR-positive prostate cancer, EED directly interacts with the androgen receptor independently of PRC2 function, regulates the expression level of AR and the downstream targets of AR, and promotes the development of prostate cancer. EED can also directly interact with the histone deacetyltransferase HDAC independent of PRC2 function, affecting the catalytic activity of HDAC.

Therefore, the current EZH2 inhibitors or EED inhibitors are only used to inhibit the activity of PRC2, but they cannot effectively inhibit the oncogenic activities of EZH2, EED, etc. degradation, to completely block the oncogenic activity of PRC2 complex subunits.

In view of this, the present invention is proposed.

Contents of the invention

The object of the present invention is to provide a bifunctional compound capable of inducing the degradation of the core subunit of the PRC2 protein complex, a pharmaceutical composition and its application, so as to improve the above problems.

The present invention is achieved like this:

In the first aspect, an embodiment of the present invention provides a bifunctional compound capable of inducing the degradation of the core subunit of the PRC2 protein complex, which includes a compound shown in any one of formulas I-III, a pharmaceutically acceptable salt thereof, or Prodrugs, their solvates, their hydrates, their polymorphs, their tautomers, stereoisomers or isotopically substituted compounds;

Wherein, n in formula I-III is an integer of 1 to 10, X is O, N or S, and Y is O, _H2 or S.

In the second aspect, the embodiment of the present invention also provides a method for preparing a bifunctional compound. In formula I-III, when X is N, S, and Y are O, the synthetic route of the compound shown in formula I-III is:

When X and Y are both O in formula I-III, the synthetic route of formula I compound is:

The synthetic route of formula II compound is:

The synthetic route of formula III compound is:

In the third aspect, the embodiment of the present invention also provides a pharmaceutical composition, which includes a pharmaceutically acceptable auxiliary component and the bifunctional compound of the foregoing embodiment.

In the fourth aspect, the embodiments of the present invention also provide the application of the bifunctional compound or the pharmaceutical composition of the aforementioned embodiment in the preparation of a kinase inhibitor.

In the fifth aspect, the examples of the present invention also provide the application of the bifunctional compound or pharmaceutical composition of the foregoing embodiment in the preparation of a drug for treating tumors; optionally, the tumors include breast cancer, colorectal cancer, prostate cancer, Cancer of the ovary, pancreas, or stomach.

In the sixth aspect, the examples of the present invention also provide the application of the bifunctional compound or pharmaceutical composition of the aforementioned embodiment in the preparation of a degrading agent for degrading the core subunit of the PRC2 protein complex, optionally, degrading the core subunit of the PRC2 protein complex The bases are EZH1, EZH2, EED, SUZ12 and RbAp46/48 subunits that degrade the PRC2 protein complex simultaneously.

In the seventh aspect, the examples of the present invention also provide the application of the bifunctional compound or pharmaceutical composition of the aforementioned embodiment in the preparation of oral or intravenous injection preparations, the oral or intravenous injection at least includes the bifunctional compound or pharmaceutical composition, which can be Optionally, excipients and/or adjuvants are also included.

One of the schemes of the above-mentioned embodiments of the present invention has at least the following beneficial effects: the bifunctional compound can effectively induce the degradation of the core subunit of the PRC2 protein complex, thereby achieving the therapeutic effect of the PRC2 complex and its subunits including EZH2, EED, SUZ12, Cancers mediated by RbAp46 and RbAp48 completely block the oncogenic activity of PRC2 complex subunits, compared to simply inhibiting PRC2 complex activity, such as EZH2 inhibitors, EED inhibitors, have better anticancer activity, It has the application of being able to treat various solid tumors such as breast cancer, colorectal cancer, prostate cancer, pancreatic cancer and ovarian cancer, and various tumor diseases such as blood tumors. The bifunctional compound or pharmaceutical composition is used as a kinase inhibitor to treat various human tumors, and has better antitumor activity and lower toxicity.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

Figure 1 and Figure 2 are the protein levels of EZH2, SUZ12, EED, RbAp48 and histone H3K27me3 detected in WSU-DLCL-2 cells by Western blotting;

Figure 3 shows that the synthetic molecule degrades each core subunit of PRC2 and reduces the level of H3K27me3;

Figure 4 shows that E7 efficiently degrades PRC2 and inhibits H3K27me3. A. Western blotting to detect the inhibition of E7 on PRC2 subunits and H3K27me3 in WSU-DLCL-2 cells at different time points. B. Western blotting to detect the inhibition of PRC2 subunits and H3K27me3 by different concentrations of E7 acting on WSU-DLCL-2 cells for 48 hours. C. RT-qPCR detection of the mRNA levels of PRC2 core subunits in WSU-DLCL-2 cells. Statistical results are expressed in the form of three mean ± SD. D. Western blotting detection of the degradation effect of E7 on PRC2 subunits in DLBCL, PCa and ovarian cancer cell lines;

Figure 5 shows that E7 binds to the EZH2 subunit of PRC2 to play a role. A. CETSA detection of thermal stability of EZH2, SUZ12, EED and RbAp48 proteins in E7-treated WSU-DLCL-2 cells and corresponding Western blot grayscale statistical results. Statistical results are presented in the form of three mean ± standard deviation (SD). B. Western blot detection of the competitive effects of EZH2 inhibitors and EED inhibitors with E7 in WSU-DLCL-2 cells. C. Western blot detection of E7 for various methylation products of histone H3 in WSU-DLCL-2 cells;

Figure 6 shows that E7 degrades PRC2 through the ubiquitin-proteasome pathway. Immunoprecipitation assay was used to detect the effect of 1μM E7 on the ubiquitination modification of EZH2(A), SUZ12(B) and EED(C) for 48h. D. After WSU-DLCL-2 cells were pretreated with 1 μM lenalidomide or 5 μM MLN4924/MG-132 for 4 hours, the cells were treated with 1 μM E7 for 48 hours, and the cell lysate was collected for Western blot analysis;

Figure 7 shows that E7 activates the transcription of genes silenced by the catalytic function of EZH2 in WSU-DLCL-2 (A), Pfeiffer (B) and A549 (C) cells. After the cells were treated with the specified compounds for 48h, the mRNA levels were detected by RT-qPCR. Statistical results are expressed in the form of three mean ± SD, *P<0.05, **p<0.01;

Figure 8 shows that E7 inhibits the transcription of genes activated by the non-catalytic function of EZH2 in A549 (A), NCI-H1299 (B) and MDA-MB-468 (C) cells. After the cells were treated with the specified compounds for 48h, the mRNA levels were detected by RT-qPCR. Statistical results are expressed in the form of three mean ± SD, *P<0.05, **p<0.01;

Figure 9 shows that E7 effectively inhibits the proliferation of WSU-DLCL-2 (A), A549 (B) and NCI-H1299 (C) cells. Proliferation curves (left) and optical microscope images (right) of cells treated with 10μME7, EPZ6438 or GSK126 at different time points, the statistical results are expressed in the form of three mean ± SD;

Figure 10 shows the inhibitory effect of E7 on the viability of WSU-DLCL-2 (A), Pfeiffer (B), A549 (C) and NCI-H1299 (D) cells detected by MTT assay for 3d, 5d, and 7d. Survival curves and IC50 values were fitted by GraphPad Prism5.0 software. Statistical results are expressed in the form of mean ± SD of two times.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

A bifunctional compound capable of inducing the degradation of the core subunit of the PRC2 protein complex, a pharmaceutical composition and applications provided by the present invention are specifically described below.

Some embodiments of the present invention provide a bifunctional compound capable of inducing degradation of the core subunit of the PRC2 protein complex, which includes a compound as shown in any one of formulas I-III, a pharmaceutically acceptable salt or a prodrug thereof , its solvates, its hydrates, its polymorphs, its tautomers, stereoisomers or isotopically substituted compounds;

Wherein, n in formula I-III is an integer of 1 to 10, X is O, N or S, and Y is O, _H2 or S.

The above-mentioned term "pharmaceutically acceptable" means that within the scope of reasonable medical judgment, it is suitable for use in contact with tissues of humans and other mammals without undue toxicity, irritation, allergic reactions, etc. The administration can directly or indirectly provide the bifunctional compound of the above-mentioned embodiment of the present invention. The term "hydrate" means a compound that further incorporates stoichiometric or non-stoichiometric amounts of water through non-covalent intermolecular forces. The term "polymorph" means a solid crystalline form of a compound or complex thereof which can be characterized by physical methods such as X. ray powder diffraction patterns or infrared spectroscopy.

The inventors have found that the existing EZH2 inhibitors and EED inhibitors only inhibit the activity of the PRC2 complex, but cannot effectively inhibit EZH2, EED and other oncogenic activities that do not depend on the catalytic function of PRC2, let alone other core subunits of the PRC2 complex. In the case of simultaneous inhibition by genes such as EZH1, SUZ12 and RbAp46/48, a lot of research and practice have been carried out, and a bifunctional compound described in the above three structural formulas has been creatively found, which can effectively induce the core subunit of the PRC2 protein complex. Gene degradation, and then achieve the treatment of cancers mediated by the PRC2 complex and its subunits, including EZH2, EED, SUZ12, RbAp46, RbAp48, completely blocking the oncogenic activity of PRC2 complex subunits, compared to simply inhibiting PRC2 Complex activities, such as EZH2 inhibitors, EED inhibitors, have better anticancer activity, which can treat breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, ovarian cancer and many other solid tumors and blood tumors. The purposes of a kind of tumor disease.

It should be noted that the bifunctional compound that can induce the degradation of the core subunit of the PRC2 protein complex can also be a tautomer, a stereoisomer, and a mixture of all ratios of the compound shown in any one of I-III .

In order to further optimize the degradation ability and overall performance of the bifunctional compound, in some embodiments, n in the above formulas I-III are all integers of 2-6.

In some preferred embodiments, n in formula I-III is an integer of 2 to 10, X and Y are both O; preferably, n in formula I-III is an integer of 2 to 6, X and Y is O. For example, in some embodiments, the chemical formula of the bifunctional compound can be

When X is N in the formula I-III, S, Y is O, the synthetic route of the compound shown in the formula I-III is:

When X and Y are both O in formula I-III, its three types of compounds can be synthesized through some synthetic routes, specifically, the synthetic route of the compound of formula I is:

The synthetic route of formula II compound is:

The synthetic route of formula III compound is:

Further, some embodiments of the present invention also provide a pharmaceutical composition, which includes a pharmaceutically acceptable auxiliary component and the bifunctional compound of the aforementioned embodiment. Wherein the auxiliary ingredients are conventional ingredients that are generally added in the process.

Furthermore, the above pharmaceutical composition can be in liquid form or in solid form. That is, the pharmaceutical composition includes but is not limited to aqueous solution, powder, granule, tablet or freeze-dried powder. In order to enable the pharmaceutical composition to fully act on the body, in some embodiments, when the pharmaceutical composition is an aqueous solution, the pharmaceutical composition also contains injection Water, saline solution, dextrose in water, saline for injection or infusion, dextrose for injection or infusion, Grüner's solution, or Grüner's solution with lactate.

Furthermore, some embodiments of the present invention also provide the application of the aforementioned bifunctional compound or pharmaceutical composition in the preparation of kinase inhibitors. In particular, it has better inhibitory performance on EZH2 enzyme.

Further, some embodiments of the present invention also provide the application of the aforementioned bifunctional compound or pharmaceutical composition in the preparation of drugs for treating tumors; optionally, tumors include breast cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, Solid tumors such as carcinoma or gastric cancer.

Further, some embodiments of the present invention also provide the application of the aforementioned bifunctional compound or pharmaceutical composition in the preparation of a degradation agent for degrading the core subunit of the PRC2 protein complex. Optionally, the core subunit degrading the PRC2 protein complex is simultaneously degrading EZH1, EZH2, EED, SUZ12 and RbAp46/48 subunits of the PRC2 protein complex.

Further, some embodiments of the present invention also provide the application of the aforementioned bifunctional compound or pharmaceutical composition in the preparation of oral or intravenous injection preparations. The oral or intravenous injection at least includes the bifunctional compound or the pharmaceutical composition, and optionally also includes excipients and/or adjuvants.

The characteristics and performance of the present invention will be described in further detail below in conjunction with the examples.

Example 1

This example provides the synthesis and related chemical data of nine bifunctional compounds G4-G12. The synthetic route of G4-G12 is as follows:

The specific preparation process is:

The first step: preparation 1b, ammonolysis reaction.

1a (3.28g, 20mmol, 1.0eq), 3-aminopiperidine-2,6-dione (3.1g, 24mmol, 1.2eq) was dissolved in 50mL of acetic anhydride, heated to reflux at 140°C for 6h, and decompressed after the reaction was completed The reaction solvent was removed by distillation, and the residue was poured into water, and a light gray precipitate was precipitated, filtered with suction, the filter cake was washed with water, and dried to obtain product 1b (3.84g, 71%). HRMS m/z calculated for C13H10N2O5[M+H]+: 275.0662, found: 275.0686.

The second step: 1c-1k general synthesis steps.

1b (1mmol, 1.0eq), DIPEA (3mmol, 3.0eq) and dibromoalkane (1.2mmol, 1.2eq) were dissolved in DMF (10mL), and reacted at 85-100°C for 3-6h. After the reaction was completed, ethyl acetate (50 mL) and water were added for extraction, dried over Na ₂ SO ₄ and concentrated to obtain a residue, which was purified by column chromatography to obtain the corresponding product.

Compound 1c: white solid (195.8 mg, 48%). 1H NMR (400MHz, DMSO-d6) δ11.08(s, 1H), 7.82(dd, J=8.5, 7.2Hz, 1H), 7.52(d, J=8.5Hz, 1H), 7.45(d, J= 7.2Hz, 1H), 5.08(dd, J=12.8, 5.4Hz, 1H), 4.26(t, J=6.1Hz, 2H), 3.66(t, J=6.7Hz, 2H), 2.88(m, J= 17.1, 13.9, 5.4Hz, 1H), 2.57(dd, J=16.4, 12.1Hz, 2H), 2.03(ddd, J=12.5, 8.5, 5.8Hz, 3H), 1.90(dt, J=8.8, 6.1Hz ,2H). HRMS m/z calculated for C ₁₇ H ₁₇ BrN ₂ O ₅ [M+Na]+: 431.0213, found: 431.0219.

Compound 1d: white solid (236.3 mg, 56%). 1H NMR (400MHz, DMSO-d6) δ11.08(s, 1H), 7.81(dd, J=8.5, 7.2Hz, 1H), 7.52(d, J=8.5Hz, 1H), 7.44(d, J= 7.2Hz, 1H), 5.08(dd, J＝12.8, 5.4Hz, 1H), 4.22(t, J＝6.3Hz, 2H), 3.56(t, J＝6.7Hz, 2H), 2.94–2.82(m, 1H), 2.67–2.53(m, 2H), 2.03(m, J＝13.5, 6.0, 3.4, 2.9Hz, 1H), 1.90(m, J＝6.8Hz, 2H), 1.80(m, J＝6.7Hz , 2H), 1.59 (m, J=9.6, 6.2Hz, 2H). HRMS m/z calculated for C ₁₈ H ₁₉ BrN ₂ O ₅ [M+Na] ⁺ : 445.0370, found: 445.0367.

Compound 1e: white solid (161.3 mg, 37%). 1H NMR (400MHz, DMSO-d6) δ11.09(s, 1H), 7.81(dd, J=8.5, 7.3Hz, 1H), 7.51(d, J=8.5Hz, 1H), 7.44(d, J= 7.2Hz, 1H), 5.08(dd, J＝12.8, 5.4Hz, 1H), 4.21(t, J＝6.3Hz, 2H), 3.54(t, J＝6.7Hz, 2H), 2.96–2.82(m, 1H), 2.67–2.53(m, 2H), 2.03(m, J＝13.5, 6.5, 6.0, 3.4Hz, 1H), 1.84(q, J＝6.8Hz, 2H), 1.76(q, J＝6.7Hz , 2H), 1.47 (dq, J=7.8, 4.5, 4.1Hz, 4H). HRMS m/z calculated for C ₁₉ H ₂₁ BrN ₂ O ₅ [M+Na] ⁺ : 459.0526, found: 459.0523.

Compound 1f: white solid (284.0 mg, 63%). 1H NMR (400MHz, DMSO-d6) δ11.09(s, 1H), 7.81(t, J=7.9Hz, 1H), 7.51(d, J=8.5Hz, 1H), 7.44(d, J=7.2Hz ,1H),5.08(dd,J=12.9,5.4Hz,1H),4.21(t,J=6.4Hz,2H),3.53(t,J=6.7Hz,2H),2.89(m,J=19.1, 14.4,5.4Hz,1H),2.59(d,J＝17.3Hz,2H),2.11–1.96(m,1H),1.79(dp,J＝20.7,6.6Hz,4H),1.54–1.30(m,6H ). HRMS m/z calculated for C ₂₀ H ₂₃ BrN ₂ O ₅ [M+H] ⁺ : 451.0863, found: 451.0852.

Compound 1g: white solid (232.4 mg, 50%). 1H NMR (400MHz, DMSO-d6) δ11.08(s, 1H), 7.80(dd, J=8.5, 7.2Hz, 1H), 7.51(d, J=8.5Hz, 1H), 7.44(d, J= 7.2Hz, 1H), 5.07(dd, J＝12.9, 5.4Hz, 1H), 4.20(t, J＝6.4Hz, 2H), 3.52(t, J＝6.7Hz, 2H), 2.94–2.81(m, 1H),2.69–2.52(m,2H),2.03(m,J＝14.1,6.7,3.3,2.9Hz,1H),1.77(tq,J＝12.4,6.7Hz,4H),1.52–1.28(m, 8H). HRMS m/z calculated for C ₂₁ H ₂₅ BrN ₂ O ₅ [M+H] ⁺ : 465.1020, found: 465.0994.

Compound 1h: white solid (292.0 mg, 61%). 1H NMR (400MHz, DMSO-d6) δ11.09(s, 1H), 7.81(dd, J=8.5, 7.3Hz, 1H), 7.51(d, J=8.5Hz, 1H), 7.44(d, J= 7.2Hz, 1H), 5.08(dd, J＝12.8, 5.4Hz, 1H), 4.20(t, J＝6.4Hz, 2H), 3.52(m, J＝6.7, 2.0Hz, 2H), 2.89(m J =16.8,13.9,5.3Hz,1H),2.58(dd,J=16.9,12.4Hz,2H),2.09–1.99(m,1H),1.84–1.72(m,4H),1.46(t,J=7.6 Hz,2H),1.37(d,J＝7.2Hz,4H),1.33–1.25(m,4H).HRMS m/z calculated for C ₂₂ H ₂₇ BrN ₂ O ₅ [M+H] ⁺ :479.1176,found: 479.1164.

Compound 1i: white solid (201.7 mg, 41%). 1H NMR (400MHz, DMSO-d6) δ11.09(s, 1H), 7.84–7.75(m, 1H), 7.51(d, J=8.5Hz, 1H), 7.44(d, J=7.2Hz, 1H) ,5.07(dd,J=12.8,5.4Hz,1H),4.20(t,J=6.4Hz,2H),3.52(td,J=6.7,2.2Hz,2H),2.88(m,J=17.0,13.9 ,5.4Hz,1H),2.58(dd,J=16.3,12.2Hz,2H),2.03(tt,J=8.0,4.4Hz,1H),1.76(m,J=14.1,11.4,6.7Hz,4H) ,1.45(q,J=7.4Hz,2H),1.36(q,J=6.3Hz,4H),1.30(d,J=18.4Hz,6H).HRMS m/z calculated for C ₂₃ H ₂₉ BrN ₂ O ₅ [M+Na] ⁺ :515.1152, found: 515.1147.

Compound 1j: white solid (293.5 mg, 58%). 1H NMR (400MHz, DMSO-d6) δ11.08(s, 1H), 7.80(t, J=7.9Hz, 1H), 7.51(d, J=8.6Hz, 1H), 7.44(d, J=7.3Hz ,1H),5.07(dd,J=13.0,5.4Hz,1H),4.20(t,J=6.4Hz,2H),3.51(t,J=6.7Hz,2H),2.88(s,1H),2.61 (s,2H),2.03(d,J=13.5Hz,1H),1.76(t,J=9.2Hz,4H),1.45(t,J=7.8Hz,2H),1.41–1.33(m,4H) ,1.27(s,8H).HRMS m/z calculated for C ₂₄ H ₃₁ BrN ₂ O ₅ [M+Na] ⁺ :529.1308,found:529.1298.

Compound 1k: white solid (176.8 mg, 34%). 1H NMR (400MHz, DMSO-d6) δ11.09(s, 1H), 7.81(t, J=7.8Hz, 1H), 7.47(dd, J=27.8, 7.8Hz, 2H), 5.08(dd, J= 13.0,5.5Hz,1H),4.20(t,J=6.5Hz,2H),3.51(t,J=6.7Hz,2H),2.98–2.80(m,1H),2.60(d,J=17.2Hz, HRMS m/z calculated for C ₂₅ H ₃₃ BrN ₂ O ₅ [M+Na] ⁺ : 543.1465, found: 543.1472.

Step 3: Preparation of G4-G12

GSK126 (0.25mmol, 1eq), NaHCO ₃ (0.5mmol, 2.0eq) and 1c-1k (0.3mmol, 1.2eq) were dissolved in 5mL DMF and reacted at 85°C for 5h. After the reaction was completed, it was extracted with ethyl acetate, dried over Na ₂ SO ₄ , evaporated under reduced pressure to remove the solvent, and then separated by silica gel column chromatography to obtain the corresponding products G4-G12.

G4: 1-[(2S)-butane-2-yl]-N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6-{6-[4-(4-{[2-(2,6-Dicarbylpiperidin-3-yl)-1,3-dioxa-2,3-hydrogen-1H-isoindole -4-yl]oxy}butyl)piperazin-1-yl]pyridin-3-yl}-3-methyl-1H-indole-4-carboxamide.

White solid (72.1 mg, 34%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.10(s,1H),8.50(d,J=2.6Hz,1H),8.12(t,J=5.2Hz,1H) ,7.91(dd,J=8.8,2.7Hz,1H),7.81(t,J=7.9Hz,1H),7.72(s,1H),7.53(d,J=8.5Hz,1H),7.44(d, J=7.2Hz,1H),7.25(s,1H),7.17(s,1H),6.89(d,J=8.9Hz,1H),5.86(s,1H),5.09(dd,J=12.9,5.4 Hz, 1H), 4.59(p, J=7.0Hz, 1H), 4.35(d, J=5.1Hz, 2H), 4.26(t, J=6.3Hz, 2H), 3.51(d, J=4.9Hz, 4H), 2.90(td, J＝17.3, 15.5, 5.3Hz, 1H), 2.70–2.53(m, 2H), 2.47(s, 2H), 2.41(t, J＝6.8Hz, 2H), 2.24(s ,3H),2.16(s,3H),2.11(s,3H),2.08–1.96(m,1H),1.82(q,J=6.9Hz,4H),1.69(q,J=7.4Hz,2H) ,1.40(d,J=6.6Hz,3H),1.23(s,2H),0.73(t,J=7.3Hz,3H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ173.23,170.41,169.18,167.33 ,165.82,163.57,160.55,158.47,158.47,156.49,149.77,146.00,143.12,138.19,137.49,136.37,133.72,131.17,130.22,126.48,124.86,123.42,122.24,120.28,116.73,116.50,115.60,110.18,108.15 _{, 107.82,107.36,69.20,57.79,52.98,52.09,49.24,45.40,35.54,31.44,29.97,26.85,22.93,22.50,21.31,19.44,18.66,12.15,11.50 8} O ₇ [M+H] ⁺ :855.4188,found:855.4185.

G5: 1-[(2S)-butane-2-yl]-N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6-{6-[4-(5-{[2-(2,6-Dicarbylpiperidin-3-yl)-1,3-dioxa-2,3-hydrogen-1H-isoindole -4-yl]oxy}pentyl)piperazin-1-yl]pyridin-3-yl}-3-methyl-1H-indole-4-carboxamide.

White solid (45.6 mg, 21%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.10(s,1H),8.50(d,J=2.4Hz,1H),8.12(t,J=5.1Hz,1H) ,7.91(dd,J=8.8,2.6Hz,1H),7.81(t,J=7.9Hz,1H),7.72(s,1H),7.52(d,J=8.5Hz,1H),7.44(d, J＝7.2Hz, 1H), 7.25(s, 1H), 7.18(d, J＝1.4Hz, 1H), 6.89(d, J＝8.9Hz, 1H), 5.86(s, 1H), 5.08(dd, J=12.9, 5.4Hz, 1H), 4.59(p, J=6.9Hz, 1H), 4.36(d, J=5.1Hz, 2H), 4.23(t, J=6.3Hz, 2H), 3.51(t, J＝4.7Hz, 4H), 2.89(m, J＝17.3, 14.0, 5.4Hz, 1H), 2.66–2.53(m, 2H), 2.48(s, 2H), 2.34(d, J＝7.4Hz, 2H ),2.24(s,3H),2.17(s,3H),2.11(s,3H),2.07–1.99(m,1H),1.80(m,J＝8.4,4.8Hz,4H),1.53(m, ¹³ C NMR ( 101MHz,DMSO-d ₆ )δ173.22,170.40,169.18,167.32,165.78,163.57,158.48,156.50,149.77,145.99,143.12,138.20,137.49,136.37,133.72,131.17,130.22,126.48,124.87,123.42,122.24,120.29 ,116.71,116.50,115.60,110.18,108.15,107.83,107.37,69.28,58.29,53.04,52.09,49.23,45.36,35.54,31.44,29.97,28.81,26.37,23.80,22.50,21.31,19.44,18.65,12.15,11.20 .HRMS m/z calculated for C ₄₉ H ₅₆ N ₈ O ₇ [M+H] ⁺ :869.4345,found:869.4343.

G6: 1-[(2S)-butane-2-yl]-N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6-{6-[4-(6-{[2-(2,6-Dicarbonylpiperidin-3-yl)-1,3-dioxa-2,3-hydrogen-1H-isoindole -4-yl]oxy}hexyl)piperazin-1-yl]pyridin-3-yl}-3-methyl-1H-indole-4-carboxamide.

Pale yellow solid (63.3 mg, 29%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.10(s,1H),8.49(d,J=2.5Hz,1H),8.12(t,J=5.1Hz,1H) ,7.90(dd,J=8.8,2.6Hz,1H),7.81(t,J=7.9Hz,1H),7.72(d,J=1.5Hz,1H),7.52(d,J=8.6Hz,1H) ,7.44(d,J=7.2Hz,1H),7.25(s,1H),7.17(d,J=1.4Hz,1H),6.89(d,J=8.9Hz,1H),5.86(s,1H) ,5.08(dd,J=12.9,5.4Hz,1H),4.60(m,J=6.9Hz,1H),4.35(d,J=5.1Hz,2H),4.21(t,J=6.4Hz,2H) ,3.50(t,J=4.8Hz,4H),2.89(m,J=17.5,14.1,5.4Hz,1H),2.66–2.54(m,2H),2.46(s,4H),2.32(d,J ＝7.2Hz,2H),2.24(s,3H),2.16(s,3H),2.11(s,3H),2.07–2.00(m,1H),1.86–1.72(m,4H),1.50(m, J＝7.5Hz, 4H), 1.40(t, J＝7.3Hz, 3H), 1.23(s, 2H), 0.73(t, J＝7.3Hz, 3H). ¹³ CNMR(101MHz, DMSO-d ₆ )δ173 .23,170.40,169.17,167.31,165.78,163.57,158.47,156.51,149.77,145.99,143.12,138.20,137.49,136.36,133.73,131.17,130.22,126.48,124.86,123.42,122.24,120.29,116.72,116.49,115.60,110.18 ,108.14,107.82,107.36,69.26,58.33,53.07,52.08,49.22,45.36,35.54,31.44,29.97,28.86,27.04,26.66,25.68,22.50,21.31 for C ₅₀ H ₅₈ N ₈ O ₇ [M+H] ⁺ :883.4501,found:882.4502.

G7: 1-[(2S)-butane-2-yl]-N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6-{6-[4-(7-{[2-(2,6-Dicarbylpiperidin-3-yl)-1,3-dioxa-2,3-hydrogen-1H-isoindole -4-yl]oxy}heptyl)piperazin-1-yl]pyridin-3-yl}-3-methyl-1H-indole-4-carboxamide.

Gray solid (73.6 mg, 33%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.09(s,1H),8.49(d,J=2.5Hz,1H),8.12(t,J=5.1Hz,1H) ,7.90(dd,J=8.8,2.6Hz,1H),7.81(dd,J=8.5,7.3Hz,1H),7.72(d,J=1.5Hz,1H),7.52(d,J=8.6Hz, 1H), 7.44(d, J=7.2Hz, 1H), 7.25(s, 1H), 7.17(d, J=1.5Hz, 1H), 6.89(d, J=8.9Hz, 1H), 5.86(s, 1H), 5.08(dd, J=12.9, 5.4Hz, 1H), 4.60(q, J=6.9Hz, 1H), 4.35(d, J=5.1Hz, 2H), 4.21(t, J=6.4Hz, 2H), 3.50(s, 4H), 2.88(m, J＝17.3, 14.0, 5.4Hz, 1H), 2.69–2.54(m, 2H), 2.49–2.40(m, 4H), 2.31(d, J＝ 7.6Hz, 2H), 2.24(s, 3H), 2.16(s, 3H), 2.11(s, 3H), 2.03(m, J＝11.9, 6.1, 3.5Hz, 1H), 1.78(m, J＝19.7 ,6.9Hz,4H),1.47(q,J=7.6Hz,4H),1.40(d,J=6.6Hz,3H),1.34(dt,J=9.1,4.3Hz,2H),1.24(d,J =4.8Hz, 2H), 0.73(t, J=7.3Hz, 3H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ173.22, 170.39, 169.17, 167.31, 165.77, 163.56, 158.47, 156.51, 149.77, 145.99, 143.11,138.19,137.48,136.37,133.73,131.17,127.92,126.49,123.41,122.24,120.27,116.71,116.50,115.59,110.18,108.14,107.81,107.37,69.28,58.40,53.07,52.08,49.23,45.36,35.53, 31.44, 29.97, _29.04 , 28.86, _27.36 , _25.74 , 22.49, _21.31 , 19.44, 18.66, 12.14, 11.20 ^. 897.4658,found: 897.4656.

G8: 1-[(2S)-butane-2-yl]-N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6-{6-[4-(8-{[2-(2,6-Dicarbylpiperidin-3-yl)-1,3-dioxa-2,3-hydrogen-1H-isoindole -4-yl]oxy}octyl)piperazin-1-yl]pyridin-3-yl}-3-methyl-1H-indole-4-carboxamide.

White solid (41.6 mg, 18%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.10(s,1H),8.49(d,J=2.6Hz,1H),8.13(t,J=5.1Hz,1H) ,7.90(dd,J=8.8,2.6Hz,1H),7.80(dd,J=8.5,7.2Hz,1H),7.72(d,J=1.5Hz,1H),7.51(d,J=8.6Hz, 1H), 7.44(d, J=7.2Hz, 1H), 7.25(s, 1H), 7.17(d, J=1.4Hz, 1H), 6.89(d, J=8.9Hz, 1H), 5.86(s, 1H), 5.08(dd, J=12.9, 5.4Hz, 1H), 4.60(q, J=6.9Hz, 1H), 4.35(d, J=5.0Hz, 2H), 4.21(t, J=6.3Hz, 2H), 3.50(t, J=5.7Hz, 4H), 2.88(m, J=17.3, 14.1, 5.4Hz, 1H), 2.58(dd, J=20.8, 6.8Hz, 2H), 2.45(s, 4H ),2.31(d,J=7.4Hz,2H),2.24(s,3H),2.16(s,3H),2.11(s,3H),2.07–1.98(m,1H),1.78(m,J= 13.8, 6.7Hz, 4H), 1.48(d, J=7.1Hz, 4H), 1.40(d, J=6.7Hz, 3H), 1.31(d, J=10.9Hz, 4H), 1.23(s, 2H) ,0.73(t,J＝7.3Hz,3H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ173.22,170.39,169.17,167.31,165.77,163.57,158.47,156.50,149.77,145.99,143.12,138.48,1 136.36,133.72,131.17,130.22,126.47,124.86,123.41,122.24,120.27,116.70,116.50,115.58,108.14,107.82,107.35,69.30,58.45,53.07,52.08,49.22,45.36,35.53,31.44,29.97,29.36, 29.10,28.87,27.36,26.71,25.72,22.49,21.30,19.44,18.66,12.14,11.20. HRMS m/z calculated for C ₅₂ H ₆₂ N ₈ O ₇ [M+H] ⁺ : 911.4814,found: 911.4819.

G9: 1-[(2S)-butane-2-yl]-N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6-{6-[4-(9-{[2-(2,6-Dicarbonylpiperidin-3-yl)-1,3-dioxa-2,3-hydrogen-1H-isoindole -4-yl]oxy}nonyl)piperazin-1-yl]pyridin-3-yl}-3-methyl-1H-indole-4-carboxamide.

White solid (85.6 mg, 37%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.11(s,1H),8.49(d,J=2.5Hz,1H),8.13(t,J=5.1Hz,1H) ,7.90(dd,J=8.8,2.6Hz,1H),7.80(t,J=7.9Hz,1H),7.72(s,1H),7.51(d,J=8.5Hz,1H),7.44(d, J＝7.2Hz, 1H), 7.25(s, 1H), 7.22–7.13(m, 1H), 6.89(d, J＝8.9Hz, 1H), 5.86(s, 1H), 5.08(dd, J＝12.9 ,5.4Hz,1H),4.60(q,J=6.9Hz,1H),4.35(d,J=5.1Hz,2H),4.20(t,J=6.3Hz,2H),3.50(s,4H), 2.88(m,J＝17.8,14.4,5.3Hz,1H),2.68–2.54(m,2H),2.45(s,4H),2.30(s,2H),2.24(s,3H),2.16(s, 3H), 2.11(s, 3H), 2.03(dd, J＝11.6, 5.8Hz, 1H), 1.86–1.69(m, 4H), 1.45(d, J＝7.2Hz, 4H), 1.40(d, J ＝6.6Hz, 3H), 1.38–1.27(m, 6H), 1.23(s, 2H), 0.73(t, J＝7.3Hz, 3H). ¹³ C NMR(101MHz, DMSO-d ₆ )δ173.22, 170.39, 169.18,167.31,165.76,163.58,158.47,156.50,149.78,145.99,143.12,138.20,137.47,136.35,133.72,131.17,130.23,126.47,124.86,123.42,122.24,120.25,116.70,116.50,115.58,110.19,108.14, 107.84,107.35,69.28,58.47,56.50,53.07,52.09,49.23,45.35,35.54,31.44,29.97,29.43,29.37,29.10,28.89,27.44,26.75,25.73,22.50,21.30,19.44,18.65,12.15,11.19. HRMS m/z calculated for C ₅₃ H ₆₄ N ₈ O ₇ [M+H] ⁺ : 925.4971, found: 925.4975.

G10: 1-[(2S)-butane-2-yl]-N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6-{6-[4-(10-{[2-(2,6-Dicarbylpiperidin-3-yl)-1,3-dioxa-2,3-hydrogen-1H-isoindole -4-yl]oxy}decyl)piperazin-1-yl]pyridin-3-yl}-3-methyl-1H-indole-4-carboxamide.

White solid (54.1 mg, 23%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.46(s,1H),11.12(s,1H),8.49(d,J=2.5Hz,1H),8.13(t,J=5.2Hz,1H) ,7.90(dd,J=8.8,2.6Hz,1H),7.84–7.76(m,1H),7.72(d,J=1.5Hz,1H),7.50(d,J=8.6Hz,1H),7.43( d,J=7.2Hz,1H),7.25(s,1H),7.18(d,J=1.4Hz,1H),6.89(d,J=8.9Hz,1H),5.86(s,1H),5.08( dd, J=12.8, 5.4Hz, 1H), 4.59(m, J=6.8Hz, 1H), 4.36(d, J=5.1Hz, 2H), 4.20(t, J=6.4Hz, 2H), 3.50( t, J＝4.9Hz, 4H), 2.89(m, J＝17.3, 14.0, 5.3Hz, 1H), 2.65–2.53(m, 2H), 2.45(t, J＝5.0Hz, 4H), 2.29(t ,J=7.4Hz,2H),2.24(s,3H),2.17(s,3H),2.11(s,3H),2.03(m,J=13.3,6.3,5.8,3.2Hz,1H),1.86– 1.71(m,4H),1.45(d,J=7.7Hz,4H),1.40(d,J=6.7Hz,3H),1.29(s,8H),1.23(s,2H),0.73(t,J ＝7.3Hz, 3H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ173.32, 170.41, 169.32, 167.35, 165.79, 163.67, 158.45, 156.49, 150.14, 145.91, 143.24, 138.17, 137.51.3, 133.6 130.20,126.45,124.95,123.37,122.12,120.22,116.60,116.43,115.60,110.12,108.15,107.45,69.28,58.43,52.98,52.14,49.20,45.26,35.54,31.38,29.94,29.36,29.06,28.83,27.41, 26.61,25.71,22.47,21.27,19.44,18.62,12.08,11.16. HRMSm/z calculated for C ₅₄ H ₆₆ N ₈ O ₇ [M+H] ⁺ :939.5127, fo und: 939.5124.

G11: 1-[(2S)-butane-2-yl]-N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6-{6-[4-(11-{[2-(2,6-Dicarbylpiperidin-3-yl)-1,3-dioxa-2,3-hydro-1H-isoindole -4-yl]oxy}undecyl)piperazin-1-yl]pyridin-3-yl}-3-methyl-1H-indole-4-carboxamide.

Pale yellow solid (97.6 mg, 41%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.10(s,1H),8.49(d,J=2.5Hz,1H),8.12(t,J=5.1Hz,1H) ,7.90(dd,J=8.8,2.6Hz,1H),7.80(t,J=7.9Hz,1H),7.72(s,1H),7.50(d,J=8.5Hz,1H),7.43(d, J＝7.2Hz, 1H), 7.25(s, 1H), 7.20–7.11(m, 1H), 6.89(d, J＝8.9Hz, 1H), 5.86(s, 1H), 5.08(dd, J＝12.9 ,5.4Hz,1H),4.60(p,J=6.7Hz,1H),4.35(d,J=5.1Hz,2H),4.20(t,J=6.4Hz,2H),3.51(s,4H), 2.96–2.83(m,1H),2.58(dd,J=15.9,11.8Hz,2H),2.47(s,4H),2.31(d,J=7.8Hz,2H),2.24(s,3H),2.16 (s,3H),2.11(s,3H),2.03(dd,J＝9.3,4.6Hz,1H),1.86–1.70(m,4H),1.46(t,J＝7.5Hz,4H),1.40( d, J=6.7Hz, 3H), 1.28(s, 12H), 0.73(t, J=7.3Hz, 3H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ173.21, 170.38, 169.17, 167.31, 165.76, 163.57,158.46,156.50,149.76,145.99,143.12,138.19,137.47,136.36,133.72,131.17,130.21,126.49,124.87,123.42,122.24,120.26,116.70,116.50,115.58,110.18,108.14,107.82,107.37,69.28, _HRcMS H for C m/ _z 6 N ₈ O ₇ [M+H] ⁺ :953.5283,found:953.5284.

G12: 1-[(2S)-butane-2-yl]-N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6-{6-[4-(12-{[2-(2,6-Dicarbylpiperidin-3-yl)-1,3-dioxa-2,3-hydrogen-1H-isoindole -4-yl]oxy}dodecyl)piperazin-1-yl]pyridin-3-yl}-3-methyl-1H-indole-4-carboxamide.

Yellow solid (33.8 mg, 14%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.10(s,1H),8.49(d,J=2.5Hz,1H),8.12(t,J=5.1Hz,1H) ,7.90(dd,J=8.8,2.6Hz,1H),7.80(dd,J=8.5,7.3Hz,1H),7.72(d,J=1.5Hz,1H),7.50(d,J=8.6Hz, 1H), 7.43(d, J=7.3Hz, 1H), 7.25(s, 1H), 7.17(d, J=1.4Hz, 1H), 6.89(d, J=8.9Hz, 1H), 5.86(s, 1H), 5.08(dd, J=12.9, 5.4Hz, 1H), 4.59(q, J=6.9Hz, 1H), 4.34(t, J=5.7Hz, 2H), 4.19(t, J=6.4Hz, 2H), 3.50(t, J=5.0Hz, 4H), 2.88(m, J=17.4, 14.1, 5.4Hz, 1H), 2.69–2.52(m, 2H), 2.46(d, J=4.8Hz, 4H ),2.30(t,J=7.5Hz,2H),2.24(s,3H),2.16(s,3H),2.11(s,3H),2.03(m,J=15.1,7.9,4.2Hz,1H) ,1.85–1.70(m,4H),1.51–1.43(m,4H),1.40(d,J=6.7Hz,3H),1.27(s,14H),0.73(t,J=7.3Hz,3H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ173.21,170.38,169.17,167.31,165.76,163.57,158.48,156.50,149.76,145.99,138.19,137.47,136.36,133.72,131.17,130.21,126.48,123.42,122.24,120.26 ,116.71,116.50,115.58,110.18,108.14,107.81,107.36,69.28,58.45,53.07,52.08,49.22,45.36,35.54,31.44,29.97,29.50,29.45,29.12,28.88,27.44,26.75,25.73,22.49,21.30 ,19.44,18.66,12.14,11.19.HRMSm/z calculated for C ₅₆ H ₇₀ N ₈ O ₇ [M+H] ⁺ :967.5440,found:967.54 38.

Example 2

This example provides the synthesis and related chemical data of nine bifunctional compounds E4-E12. The synthetic route of E4-E12 is as follows:

The specific preparation process is:

The first step: 2-methyl-3-bromo-5-nitrobenzoic acid methyl ester 2a (5.5g, 20mmol, 1.0eq), ammonium chloride (5.6g, 100mmol, 5.0eq) was dissolved in aqueous ethanol ( 60mL, H ₂ O:EtOH=1:3), after heating up to 80°C, iron powder (11.2g, 200mmol, 10.0eq) was added in three portions. Thin-layer chromatography (TLC) detected the completion of the reaction after 1 h of reaction, diatomaceous earth filter aided, the filtrate was distilled off under reduced pressure to remove the solvent, the residue was extracted with DCM, dried _{over Na2SO4} and concentrated to obtain product 2b (4.41g, 92%). No further purification was required. ¹ H NMR (400MHz, CDCl ₃ ) δ7.31(d, J=2.6Hz, 1H), 6.92(d, J=2.6Hz, 1H), 3.87(s, 3H), 3.82(s, 2H), 2.26 (s,3H).HRMS m/z calculated for C ₉ H ₁₀ BrNO ₂ [M+H] ⁺ :243.9967,found:243.9958.

The second step: 2b (4g, 17.4mmol, 1.0eq) and tetrahydropyrone (4.4g, 52.2mmol, 3.0eq) were dissolved in chloroform (50mL), and acetic acid (2.1g, 34.8mmol, 2.0eq) was added at room temperature After stirring for 3h, sodium triacetylborohydride (2.7g, 43.6mmol, 2.5eq) was added and stirring was continued overnight. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography to obtain product 2c (3.3 g, 57.8%). ¹ H NMR (400MHz, CDCl ₃ ) δ7.23(d, J=2.0Hz, 1H), 6.84(d, J=2.0Hz, 1H), 4.01(dt, J=11.9, 3.6Hz, 2H), 3.87 (s,3H),3.66(d,J=7.4Hz,1H),3.54(td,J=11.6,2.3Hz,2H),3.48(m,1H),2.23(s,3H),2.10–1.99( m, 2H), 1.51 (m, J=13.3, 10.6, 4.3Hz, 2H). HRMS m/z calculated for C ₁₄ H ₁₈ BrNO ₃ [M+H] ⁺ : 328.0542, found: 328.0547.

Step 3: Under nitrogen protection, take 2c (3g, 9.1mmol, 1.0eq) and add 30mL of 1,2-dichloroethane to a 50mL round bottom flask, and carefully inject anhydrous acetaldehyde (2.3g, 27.3mmol, 3.0eq) was placed under the liquid surface of the solution, and acetic acid (1.1g, 18.2mmol, 2.0eq) was added within 30min, and the reaction solution was orange. The reaction mixture was allowed to rise to room temperature naturally, and stirred for 1 h. Then the mixture was cooled to 0°C, sodium triacetoxyborohydride (1.48g, 23mmol) was slowly added in batches, and the addition rate was controlled to keep the temperature of the reaction system below 5°C. After 2h, it was turned back to room temperature and stirred overnight. After the TLC reaction was completed, the reaction system was cooled to 0°C, 100 mL of ice water was added, and an excess of saturated sodium bicarbonate aqueous solution was slowly added while stirring. The organic layers were combined, washed twice with water and separated. The organic phase was separated and concentrated under reduced pressure to constant weight to obtain a yellow to light red oily liquid. ¹ H NMR (400MHz, CDCl ₃ ) δ7.71(d, J=2.0Hz, 1H), 7.37(d, J=2.0Hz, 1H), 3.96(d, J=12.1Hz, 2H), 3.89(s ,3H),3.32(td,J＝11.3,2.9Hz,2H),3.05(q,J＝7.1Hz,2H),2.99–2.85(m,1H),2.45(s,3H),1.80–1.54( m, 4H), 0.87 (t, J=7.0Hz, 3H). HRMS m/z calculated for C ₁₆ H ₂₃ BrNO ₃ [M+H] ⁺ : 356.0861, found: 356.0854.

Step 4: Under the protection of nitrogen, add 2d (2g, 5.6mmol) in 25mL of methanol at one time, after heating up to 60°C, slowly add 20mL of sodium hydroxide aqueous solution (2M) dropwise under heat preservation, the color of the reaction solution changes from light green to The clear liquid gradually turns into an emulsion, and finally turns into a light green clear liquid. After incubation for 1 h, TLC monitored the completion of the reaction. Transfer the reaction solution to a rotary evaporator, remove most of the methanol under reduced pressure, add 100 mL of water to the residue, stir for 10 min, and the solid is completely dissolved. Raise the temperature to 65°C, add hydrochloric acid (2M) to adjust the pH=2-3, precipitate out, stop the heating bath, cool down to room temperature and stir for 0.5h. After suction filtration, the filter cake was fully washed with ice water. After the filter cake was drained, it was dried in a vacuum oven with phosphorus pentoxide as a desiccant at 60° C. for 10 h to obtain 2e (1.75 g, 91.2%) as a white target compound. ¹ H NMR (400MHz, DMSO-d ₆ ) δ13.15(s, 1H), 7.62(d, J=2.1Hz, 1H), 7.49(d, J=2.1Hz, 1H), 3.83(dt, J= 9.5,2.3Hz,2H),3.26(td,J＝11.6,2.1Hz,2H),3.04(q,J＝7.1Hz,2H),3.01–2.92(m,1H),2.40(s,3H), 1.66–1.58(m,2H),1.50(m,J＝11.7,4.3Hz,2H),0.80(t,J＝7.0Hz,3H).HRMS m/z calculated for C ₁₅ H ₂₀ BrNO ₃ [M+ H] ⁺ :342.0699, found: 342.0706.

The fifth step: 3-(aminomethyl)-4,6-lutidine-2(1H)-one (0.61g, 4mmol) and 2e (1.5g, 4.4mmol) were dissolved in DMSO (10mL), added HOAT (0.55g, 1.5mmol) and EDCI (0.84g, 2.2mmol), the reaction solution was stirred at 45°C for 20h. After the completion of the reaction monitored by TLC, the reaction solution was poured into ice water (100mL), stirred for 30min, precipitated out, filtered, washed with water, dried, dissolved in a mixture of methanol and chloroform (10:1), mixed, passed through a silica gel column layer Purification by chromatography afforded 2f (1.42 g, 68%) as a yellow solid. ¹ H NMR (400MHz, DMSO-d ₆ )δ11.47(s, 1H), 8.23(t, J=5.0Hz, 1H), 7.31(d, J=2.0Hz, 1H), 7.09(d, J= 2.0Hz, 1H), 5.86(s, 1H), 4.25(d, J＝4.9Hz, 2H), 3.83(dd, J＝10.8, 3.5Hz, 2H), 3.28–3.18(m, 2H), 3.01( q,J＝7.0Hz,2H),2.97–2.89(m,1H),2.54(s,1H),2.19(s,3H),2.15(s,3H),2.11(s,3H),1.60(d , J=12.4Hz, 2H), 1.49 (m, J=11.7, 4.2Hz, 2H). HRMS m/z calculated for C ₂₃ H ₃₀ BrN ₃ O ₃ [M+H] ⁺ : 476.1543, found: 476.1552.

Step 6: Take 2f (1.2g, 2.5mmol, 1.0eq), tert-butyl4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)piperazine -1-carboxylate (1.21g, 3mmol, 1.2eq) was dissolved in a mixed solution of 1,4-dioxane and water (4:1, 30mL), and K ₂ CO ₃ (3.75mmol, 0.52g) was added, Pd(dppf)Cl ₂ (0.2mmol, 146mg), under nitrogen protection, transferred to 100°C for 8h reaction, and then cooled to room temperature. The reaction solution was distilled off the solvent under reduced pressure, dissolved in ethyl acetate, and filtered with diatomaceous earth. Extract the filtrate, dry it with anhydrous sodium sulfate, concentrate, mix the sample, and purify by silica gel column chromatography to obtain the product. Without further purification, it was directly added to 25 mL of trifluoroacetic acid/dichloromethane solution (10%) and reacted at room temperature for 1 h, then the solvent was distilled off under reduced pressure, and the residue was neutralized to pH=8 by saturated sodium carbonate solution (2M) , precipitated, suction filtered, washed with water, and dried to obtain 2g (1.04g, 74%) of the product. ¹ H NMR (400MHz,DMSO-d ₆ )δ11.47(s,1H),8.63(s,1H),8.20(t,J=5.0Hz,1H),7.60(d,J=7.9Hz,2H) ,7.38(d,J＝8.0Hz,3H),7.28–7.16(m,1H),5.86(s,1H),4.30(d,J＝4.9Hz,2H),3.89–3.77(m,2H), 3.57(s, 2H), 3.25(t, J=11.4Hz, 2H), 3.09(d, J=6.2Hz, 6H), 3.04–2.98(m, 1H), 2.57(t, J=4.8Hz, 4H ),2.25(s,3H),2.21(s,3H),2.11(s,3H),1.66(d,J=12.3Hz,2H),1.53(m,J=12.4,12.0,4.1Hz,2H) , 0.84 (t, J=6.9Hz, 3H). HRMS m/z calculated for C ₃₄ H ₄₅ N ₅ O ₃ [M+H] ⁺ : 572.3595, found: 572.3601.

The seventh step: synthesis of E4-E12, nucleophilic substitution reaction.

2g (0.25mmol, 1eq), NaHCO ₃ (0.5mmol, 2.0eq) and 1c-1k (0.3mmol, 1.2eq) were dissolved in DMF (5mL), and reacted at 85°C for 3-8h. After the reaction was completed, ethyl acetate After ester extraction, Na ₂ SO ₄ was dried, the solvent was distilled off under reduced pressure, and the corresponding products E4-E12 were obtained by silica gel column chromatography.

E4: N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'-{[4-(4-{[2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl]oxy}butyl)piperazine-1- base]methyl}-5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.

Yellow solid (36.0 mg, 15%). ¹ H NMR (400MHz, CDCl ₃ ) δ12.00(s, 1H), 9.66(s, 1H), 8.35(d, J=2.5Hz, 1H), 7.71–7.57(m, 2H), 7.44(d, J＝7.2Hz, 1H), 7.27–7.15(m, 4H), 5.91(s, 1H), 4.93(dd, J＝11.9, 5.4Hz, 1H), 4.54(d, J＝5.9Hz, 2H), 4.19(q, J=6.2Hz, 2H), 3.94(dt, J=11.6, 3.3Hz, 2H), 3.65–3.52(m, 4H), 3.41(s, 1H), 3.31(td, J=11.3, 2.9Hz, 2H), 3.08(q, J＝7.0Hz, 2H), 3.04–2.95(m, 1H), 2.88–2.68(m, 3H), 2.58(t, J＝5.0Hz, 4H), 2.45( t, J＝7.1Hz, 2H), 2.39(s, 3H), 2.33(s, 3H), 2.14(s, 3H), 2.13–2.05(m, 1H), 1.92(t, J＝6.8Hz, 2H ),1.81–1.50(m,9H),0.88(t,J＝7.0Hz,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ171.65,168.71,167.05,165.09,158.69,156.61,150.67,149.55,145.91, 142.60,139.33,136.45,135.87,135.58,133.86,132.83,125.55,123.17,122.11,120.12,118.81,115.69,109.86,106.90,69.25,67.31,58.40,58.31,52.83,49.11,45.25,41.64,36.13,31.43, 30.52,28.76,26.33,23.96,22.71,19.69,18.65,14.68,12.77. HRMS m/z calculated for C ₅₁ H ₆₂ N ₇ O ₈ [M+H] ⁺ :900.4654,found:900.4653.

E5: N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'-{[4-(5-{[2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl]oxy}pentyl)piperazine-1- base]methyl}-5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.

Yellow solid (61.7 mg, 27%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.08(s,1H),8.17(t,J=5.0Hz,1H),7.80(t,J=7.9Hz,1H) ,7.57(d,J=7.9Hz,2H),7.51(d,J=8.5Hz,1H),7.43(d,J=7.2Hz,1H),7.42–7.38(m,1H),7.35(d, J=7.8Hz, 2H), 7.22(d, J=1.7Hz, 1H), 5.86(s, 1H), 5.07(dd, J=12.9, 5.4Hz, 1H), 4.30(d, J=5.0Hz, 2H), 4.23(t, J=6.3Hz, 2H), 3.88–3.78(m, 2H), 3.46(s, 2H), 3.25(t, J=11.4Hz, 2H), 3.09(q, J=7.2 Hz, 2H), 3.01(d, J=10.6Hz, 1H), 2.94–2.82(m, 1H), 2.57(dd, J=15.3, 11.4Hz, 2H), 2.45–2.29(m, 8H), 2.25 (s,3H),2.21(s,3H),2.11(s,3H),2.07–1.97(m,1H),1.83–1.71(m,2H),1.71–1.59(m,4H),1.59–1.48 (m,2H),1.25(d,J=13.0Hz,4H),0.84(t,J=6.9Hz,3H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ173.21,170.38,169.51,167.31,165.79 ,163.46,156.47,149.94,149.34,143.19,140.08,138.94,137.89,137.49,137.47,133.71,133.07,129.86,126.83,123.35,122.09,121.28,120.27,116.71,115.57,107.80,69.16,66.79,62.22,58.34 ,57.66,53.18,49.21,41.67,35.36,31.42,30.79,29.45,26.79,22.92,22.48,19.42,18.66,15.02,13.21. HRMS m/z calculated for C ₅₂ H ₆₄ N ₇ O ₈ [M+H] ⁺ :914.4811,found:914.4812.

E6: N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'-{[4-(6-{[2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl]oxy}hexyl)piperazin-1-yl ]methyl}-5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.

Yellow solid (46.4 mg, 20%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.09(s,1H),8.17(t,J=5.0Hz,1H),7.80(t,J=7.9Hz,1H) ,7.57(d,J=7.8Hz,2H),7.51(d,J=8.5Hz,1H),7.44(d,J=7.2Hz,1H),7.40(s,1H),7.35(d,J= 7.9Hz, 2H), 7.22(s, 1H), 5.86(s, 1H), 5.07(dd, J=12.9, 5.4Hz, 1H), 4.29(d, J=5.0Hz, 2H), 4.20(t, J＝6.4Hz, 2H), 3.83(d, J＝11.2Hz, 2H), 3.47(s, 2H), 3.24(d, J＝11.3Hz, 2H), 3.06(dd, J＝18.9, 11.1Hz, 3H), 2.88(q, J=12.5Hz, 1H), 2.58(d, J=17.5Hz, 2H), 2.38(s, 6H), 2.25(s, 3H), 2.21(s, 3H), 2.11( s,3H),2.03–1.99(m,1H),1.75(m,J=7.0Hz,2H),1.67(d,J=12.0Hz,2H),1.49(dt,J=24.8,6.0Hz,4H ),1.38–1.31(m,2H),1.24(d,J＝6.6Hz,6H),0.84(q,J＝6.9Hz,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ173.31,170.41,169.65, 167.34,165.81,163.54,156.49,150.27,149.38,143.31,139.97,137.57,133.65,133.08,130.00,126.84,123.41,121.97,121.20,120.95,120.27,116.62,115.64,108.07,69.21,66.78,58.28,52.92, _{49.20,41.69,35.37,31.37,30.75,30.73,29.42,29.10,28.74,25.56,22.47,19.41,18.63,15.01,13.14} ^. _{_ _ _} 928.4967,found: 928.4982.

E7: N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'-{[4-(7-{[2-( 2,6-dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl]oxy}heptyl)piperazine-1- base]methyl}-5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.

Yellow solid (101.3 mg, 43%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.10(s,1H),8.17(t,J=5.0Hz,1H),7.80(t,J=7.9Hz,1H) ,7.56(d,J=7.8Hz,2H),7.50(d,J=8.6Hz,1H),7.43(d,J=7.3Hz,1H),7.40(s,1H),7.35(d,J= 7.8Hz, 2H), 7.22(s, 1H), 5.86(s, 1H), 5.08(dd, J=12.9, 5.4Hz, 1H), 4.30(d, J=4.9Hz, 2H), 4.20(t, J＝6.4Hz, 2H), 3.83(d, J＝11.1Hz, 2H), 3.47(s, 2H), 3.28–3.20(m, 2H), 3.09(q, J＝7.6, 7.1Hz, 2H), 3.01(d, J=10.7Hz, 1H), 2.89(m, J=13.6, 12.5, 6.9Hz, 1H), 2.59(d, J=17.0Hz, 2H), 2.37(s, 6H), 2.25(s ,3H),2.21(s,3H),2.11(s,3H),2.03(d,J=12.5Hz,1H),1.75(t,J=7.2Hz,2H),1.67(d,J=12.0Hz ,2H),1.53(dt,J=12.5,7.6Hz,2H),1.43(p,J=7.4Hz,4H),1.33(d,J=6.3Hz,2H),1.28(d,J=7.8Hz ,2H),1.23(s,4H),0.83(t,J=7.0Hz,3H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ173.31,170.41,169.65,167.34,165.80,163.55,156.49,150.28, 149.38,143.31,139.95,138.96,137.62,137.55,137.50,133.65,133.08,129.98,126.82,123.41,121.97,121.20,120.24,116.61,115.61,108.09,69.26,66.78,62.02,58.28,58.07,52.99,52.64, HRMS m/z calculated for C ₅₄ H ₆₈ N ₇ O ₈ [M+H] ⁺ :942.5124,found:942.5135.

E8: N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'-{[4-(8-{[2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl]oxy}octyl)piperazine-1- base]methyl}-5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.

Yellow solid (69.3 mg, 29%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.09(s,1H),8.17(t,J=5.0Hz,1H),7.80(t,J=7.9Hz,1H) ,7.56(d,J=7.8Hz,2H),7.50(d,J=8.6Hz,1H),7.43(d,J=7.2Hz,1H),7.40(d,J=2.1Hz,1H),7.35 (d,J=7.9Hz,2H),7.22(d,J=1.9Hz,1H),5.86(s,1H),5.07(dd,J=12.9,5.4Hz,1H),4.30(d,J= 5.0Hz, 2H), 4.19(t, J=6.4Hz, 2H), 3.88–3.77(m, 2H), 3.47(s, 2H), 3.25(t, J=11.8Hz, 2H), 3.09(q, J＝7.1Hz, 2H), 3.02(q, J＝6.9, 5.4Hz, 1H), 2.88(m, J＝17.5, 14.2, 5.3Hz, 1H), 2.63–2.54(m, 2H), 2.46–2.30 (m,6H),2.25(s,5H),2.21(s,3H),2.11(s,3H),2.06–2.00(m,1H),1.75(t,J＝7.3Hz,2H),1.66( d,J=12.0Hz,2H),1.54(td,J=11.8,4.0Hz,2H),1.43(dt,J=11.6,6.5Hz,4H),1.36–1.20(m,8H),0.83(t , J＝6.9Hz, 3H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ173.20, 170.38, 169.51, 167.31, 165.75, 163.47, 156.49, 149.95, 149.33, 143.19, 140.07, 138.96, 1333.479, 1 129.85,126.83,123.36,122.09,121.28,120.26,116.70,115.58,107.82,69.28,66.79,62.17,58.34,53.22,49.21,41.68,35.37,31.43,30.79,29.33,29.08,28.86,27.30,26.64,25.70, 22.49, 19.41, 18.66, 15.02, 13.20. HRMS m/z calculated for C ₅₅ H ₇₀ N ₇ O ₈ [M+H] ⁺ : 956.5280, found: 956.5281.

E9: N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'-{[4-(9-{[2-( 2,6-dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl]oxy}nonyl)piperazine-1- base]methyl}-5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.

Yellow solid (97.1 mg, 40%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.45(s,1H),11.10(s,1H),8.18(q,J=5.6,5.0Hz,1H),7.80(t,J=7.9Hz, 1H), 7.56(d, J=7.8Hz, 2H), 7.51(d, J=8.5Hz, 1H), 7.43(d, J=7.3Hz, 1H), 7.40(d, J=1.9Hz, 1H) ,7.35(d,J=7.9Hz,2H),7.22(d,J=1.8Hz,1H),5.86(s,1H),5.07(dd,J=12.9,5.4Hz,1H),4.29(d, J=4.9Hz, 2H), 4.19(t, J=6.4Hz, 2H), 3.83(d, J=11.1Hz, 2H), 3.47(s, 2H), 3.24(d, J=11.3Hz, 2H) ,3.09(q,J=7.1Hz,2H),3.01(q,J=7.2,5.6Hz,1H),2.89(td,J=13.4,12.1,6.9Hz,1H),2.57(dd,J=15.9 ,12.0Hz,2H),2.37(s,6H),2.25(s,5H),2.21(s,3H),2.11(s,3H),2.05–1.99(m,1H),1.75(p,J= 6.6Hz, 2H), 1.66(d, J=12.1Hz, 2H), 1.54(td, J=11.9, 4.1Hz, 2H), 1.45(t, J=7.7Hz, 2H), 1.40(d, J= 8.9Hz, 2H), 1.33(d, J=5.8Hz, 2H), 1.25(d, J=11.4Hz, 8H), 0.84(q, J=7.0Hz, 3H). ¹³ C NMR (101MHz, DMSO- d ₆ )δ173.29,170.40,169.63,167.33,165.78,163.53,156.49,150.22,149.37,143.29,139.97,138.97,137.54,133.66,133.08,129.97,126.83,123.40,121.98,121.21,120.25,116.62,115.61,108.04 . for C ₅₆ H ₇₂ N ₇ O ₈ [M+H] ⁺ : 970.5437, found: 970.5474.

E10: N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'-{[4-(10-{[2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl]oxy}decyl)piperazine-1- base]methyl}-5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.

Yellow solid (81.2 mg, 33%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.44(s,1H),11.10(s,1H),8.17(t,J=5.0Hz,1H),7.80(t,J=7.9Hz,1H) ,7.56(d,J=7.9Hz,2H),7.50(d,J=8.5Hz,1H),7.43(d,J=7.3Hz,1H),7.39(d,J=1.8Hz,1H),7.35 (d,J=7.9Hz,2H),7.22(d,J=1.8Hz,1H),5.85(s,1H),5.07(dd,J=12.9,5.3Hz,1H),4.29(d,J= 5.0Hz, 2H), 4.19(d, J=6.5Hz, 2H), 3.86–3.78(m, 2H), 3.47(s, 2H), 3.24(d, J=11.3Hz, 2H), 3.09(q, J＝7.2Hz, 2H), 3.01(d, J＝11.0Hz, 1H), 2.88(m, J＝18.0, 14.1, 5.3Hz, 1H), 2.68–2.54(m, 2H), 2.35(d, J =16.6Hz,6H),2.25(s,5H),2.21(s,3H),2.10(s,3H),2.06–1.98(m,1H),1.75(m,J=6.6Hz,2H),1.66 (d, J=11.4Hz, 2H), 1.53(dt, J=12.2, 5.9Hz, 2H), 1.45(t, J=7.8Hz, 2H), 1.42–1.31(m, 4H), 1.25(d, J＝8.1Hz, 10H), 0.83(t, J＝6.9Hz, 3H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ173.21, 170.38, 167.31, 166.11, 165.75, 163.99, 161.20, 156.49, 149.94, 149.33 ,137.49,133.07,129.85,126.83,121.28,120.27,116.70,115.59,106.37,76.61,69.30,67.68,64.13,58.35,53.14,49.20,35.36,34.70,31.43,29.39,29.09,28.88,26.26,25.51,22.48 , 22.23, 19.00, 18.43, 17.95, 16.80, 15.02, 14.56, 11.44. HRMS m/z calculated for C ₅₇ H ₇₄ N ₇ O ₈ [M+H] ⁺ : 984.5593, found: 984.5593.

E11: N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'-{[4-(11-{[2-( 2,6-dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl]oxy}undecyl)piperazine- 1-yl]methyl}-5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.

Yellow solid (119.8 mg, 47%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.44(s,1H),11.09(s,1H),8.17(t,J=5.0Hz,1H),7.80(t,J=7.9Hz,1H) ,7.56(d,J=7.8Hz,2H),7.51(d,J=8.6Hz,1H),7.43(d,J=7.3Hz,1H),7.39(d,J=1.8Hz,1H),7.35 (d,J=8.0Hz,2H),7.22(d,J=1.8Hz,1H),5.86(s,1H),5.07(dd,J=12.8,5.4Hz,1H),4.29(d,J= 4.9Hz, 2H), 4.19(t, J=6.4Hz, 2H), 3.83(d, J=11.2Hz, 2H), 3.47(s, 2H), 3.24(d, J=11.3Hz, 2H), 3.09 (q, J=7.1Hz, 2H), 3.01(d, J=10.7Hz, 1H), 2.88(m, J=18.6, 14.5, 5.3Hz, 1H), 2.58(d, J=17.4Hz, 2H) ,2.37(s,6H),2.24(s,4H),2.21(s,3H),2.10(s,3H),2.06–1.96(m,2H),1.75(t,J＝7.3Hz,2H), 1.66(d, J=12.4Hz, 2H), 1.52(dd, J=12.1, 3.9Hz, 2H), 1.45(s, 2H), 1.34(s, 4H), 1.24(d, J=4.5Hz, 12H ),0.83(t,J=7.5Hz,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ171.64,170.14,168.71,167.12,165.66,165.16,158.71,156.73,150.71,149.55,145.96,142.636,139 ,136.14,135.81,135.64,133.85,132.78,125.51,123.20,122.08,120.05,118.89,117.16,115.62,109.90,106.77,77.26,69.47,67.31,58.71,58.41,52.89,49.14,45.18,41.63,36.15,31.51 ,30.52,29.47,29.41,29.31,29.15,28.89,27.49,26.65,25.73,22.67,19.70,18.65,14.68,12.76. HRMS m/z calculated for C ₅₈ H ₇₆ N ₇ O ₈ [M+H] ⁺ :998.5750,found:998.5775.

E12: N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'-{[4-(12-{[2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl]oxy}dodecyl)piperazine- 1-yl]methyl}-5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.

Yellow solid (48.1 mg, 19%). ¹ H NMR (400MHz,DMSO-d ₆ )δ11.44(s,1H),11.09(s,1H),8.17(t,J=4.9Hz,1H),7.82–7.75(m,1H),7.60– 7.53(m,2H),7.50(dd,J=8.6,1.9Hz,1H),7.43(dd,J=7.3,1.6Hz,1H),7.39(d,J=2.0Hz,1H),7.38–7.31 (m,2H),7.22(d,J=1.8Hz,1H),5.85(s,1H),5.07(dd,J=12.9,5.3Hz,1H),4.29(d,J=4.9Hz,2H) ,4.22–4.14(m,2H),3.83(d,J=11.1Hz,2H),3.46(s,2H),3.23(d,J=11.3Hz,2H),3.08(q,J=7.1Hz, 2H), 3.00(d, J=11.1Hz, 1H), 2.88(m, J=18.9, 14.2, 5.4Hz, 1H), 2.57(dd, J=15.5, 11.9Hz, 2H), 2.37(s, 6H ),2.24(s,5H),2.21(s,3H),2.10(s,3H),2.06–1.97(m,1H),1.74(t,J＝7.2Hz,2H),1.66(d,J＝ 12.3Hz, 2H), 1.53(m, J=11.9, 4.0Hz, 2H), 1.49–1.41(m, 2H), 1.35(dd, J=16.7, 5.5Hz, 4H), 1.25(d, J=7.7 Hz,14H),0.83(t,J=6.9Hz,3H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ173.20,170.37,169.51,167.31,165.76,163.46,156.50,149.94,149.33,143.19,140.08, 138.96,137.50,133.72,129.85,126.83,122.09,121.28,120.26,116.70,115.58,107.81,69.28,66.80,62.20,58.34,53.26,53.11,49.21,41.68,35.36,31.44,30.79,29.48,29.42,29.40, 29.11,28.88,27.39,26.72,25.72,22.49,19.41,18.65,15.02,13.20. HRMS m/z calculated for C ₅₉ H ₇₈ N ₇ O ₈ [M+H] ⁺ :1 012.5906,found: 1012.5910.

Example 3

This example provides the synthesis and related chemical data of nine bifunctional compounds S4-S12. The synthetic route of S4-S12 is as follows:

The specific preparation process is:

The first step: S1 (4mmol) and S2 (4.4mmol) were dissolved in DMSO (10mL), HOAT (0.55g, 1.5mmol) and EDCI (0.84g, 2.2mmol) were added, and the reaction solution was stirred at 45°C for 20h. After the completion of the reaction monitored by TLC, the reaction solution was poured into ice water (100mL), stirred for 30min, precipitated out, filtered, washed with water, dried, dissolved in a mixture of methanol and chloroform (10:1), mixed, passed through a silica gel column layer Analysis and purification afforded yellow solid S3 (1.57 g). ¹ H NMR (400MHz, Chloroform-d) δ12.78(s, 1H), 7.41(t, J=5.7Hz, 1H), 7.22(d, J=2.0Hz, 1H), 7.18(d, 2.0Hz, 1H), 4.54(d, J=5.7Hz, 2H), 3.94(dt, J=11.6, 3.3Hz, 2H), 3.36–3.25(m, 2H), 3.02(q, J=7.0Hz, 2H), 2.95(m,1H),2.93(dd,J=7.4,4.8Hz,2H),2.44(t,J=6.0Hz,2H),2.27(s,3H),2.19(s,3H),1.76(dd , J＝7.5, 4.3Hz, 4H), 1.68–1.58(m, 4H), 0.85(t, J＝7.0Hz, 3H). ¹³ C NMR (101MHz, CDCl ₃ ) δ168.51, 163.56, 151.17, 150.82, 140.70 .

Step 2: Dissolve S3 (2.5mmol, 1.0eq), borate (3mmol, 1.2eq) in a mixed solution of 1,4-dioxane and water (4:1, 30mL), add K ₂ CO ₃ (3.75mmol, 0.52g), Pd(dppf)Cl ₂ (0.2mmol, 146mg), under nitrogen protection, transferred to 100°C for 8h, and then cooled to room temperature. The reaction solution was distilled off the solvent under reduced pressure, dissolved in ethyl acetate, and filtered with diatomaceous earth. Extract the filtrate, dry it with anhydrous sodium sulfate, concentrate, mix the sample, and purify by silica gel column chromatography to obtain the product. Without further purification, it was directly added to 25 mL of trifluoroacetic acid/dichloromethane solution (10%) and reacted at room temperature for 1 h, then the solvent was distilled off under reduced pressure, and the residue was neutralized to pH=8 by saturated sodium carbonate solution (2M) , precipitated, suction filtered, washed with water, and dried to obtain the product 2g S4 (1.04g, 74%). HRMS m/z calculated for C ₃₅ H ₄₆ N ₆ O ₃ [M+H] ⁺ : 599.3704, found: 599.3711.

The third step: synthesis of S6-S11.

S4 (0.25mmol, 1eq), NaHCO ₃ (0.5mmol, 2.0eq) and 1c-1k (0.3mmol, 1.2eq) were dissolved in DMF (5mL), and reacted at 85°C for 5h. After the reaction was completed, extracted with ethyl acetate, dried over Na ₂ SO ₄ , evaporated under reduced pressure to remove the solvent, and separated by silica gel column chromatography to obtain the corresponding products S6-S11.

S6: 5-(6-(4-(6-((2-(2,6-pyridinedione-3-yl)-1,3-dioxaisindol-4-yl)oxa)ring Hexyl)piperazin-1-yl)pyridine-3-yl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-N-((1-methyl -3-oxa-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)benzamide.

Yellow solid (48.1 mg, 13%). 12.48(s,1H),8.33(d,J=2.4Hz,1H),7.60(dd,J=8.8,2.6Hz,1H),7.41(t,J=5.8Hz,1H),7.25(d,J =1.9Hz,1H),7.20(d,J=1.8Hz,1H),6.62(d,J=8.8Hz,1H),5.07(dd,J=12.9,5.4Hz,1H),4.57(d,J =5.7Hz, 2H), 4.30(d, J=5.0Hz, 2H), 4.23(t, J=6.3Hz, 2H), 3.94(dt, J=11.5, 3.2Hz, 2H), 3.88–3.78(m ,2H),3.57(t,J=5.0Hz,4H),3.46(s,2H),3.31(td,J=11.2,3.1Hz,2H),3.25(t,J=11.4Hz,2H),3.09 (q, J=7.2Hz, 2H), 3.01(d, J=10.6Hz, 1H), 2.94–2.82(m, 1H), 2.57(dd, J=11.4Hz, 2H), 2.45–2.29(m, 8H), 2.21(s,3H), 2.11(s,3H), 2.07–1.97(m,1H), 1.83–1.71(m,2H), 1.59–1.48(m,2H), 1.25(d,J= 13.0Hz, 3H), 0.84 (t, J=6.9Hz, 3H). HRMS m/z calculated for C ₅₄ H ₆₆ N ₈ O ₈ [M+H] ⁺ : 955.5076, found: 955.5071.

S7: 5-(6-(4-(7-((2-(2,6-pyridinedione-3-yl)-1,3-dioxaisindol-4-yl)oxa)ring Hexyl)piperazin-1-yl)pyridine-3-yl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-N-((1-methyl -3-oxa-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)benzamide.

Yellow solid (48.1 mg, 13%). HRMS m/z calculated for C ₅₅ H ₆₈ N ₈ O ₈ [M+H] ⁺ : 969.5233, found: 969.5243.

S8: 5-(6-(4-(8-((2-(2,6-pyridinedione-3-yl)-1,3-dioxaisindol-4-yl)oxa)ring Hexyl)piperazin-1-yl)pyridine-3-yl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-N-((1-methyl -3-oxa-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)benzamide.

Yellow solid (83 mg, 24%). HRMS m/z calculated for C ₅₆ H ₇₀ N ₈ O ₈ [M+H] ⁺ : 1012.5906, found: 1012.5910.

S9: 5-(6-(4-(7-((2-(2,6-pyridinedione-3-yl)-1,3-dioxaisindol-4-yl)oxa)ring Hexyl)piperazin-1-yl)pyridine-3-yl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-N-((1-methyl -3-oxa-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)benzamide.

Yellow solid (48.1 mg, 13%). HRMS m/z calculated for C ₅₇ H ₇₂ N ₈ O ₈ [M+H] ⁺ : 997.5946, found: 997.5938.

S10: 5-(6-(4-(10-((2-(2,6-pyridinedione-3-yl)-1,3-dioxaisindol-4-yl)oxa) ring Hexyl)piperazin-1-yl)pyridine-3-yl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-N-((1-methyl -3-oxa-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)benzamide.

Yellow solid (58.1 mg, 17%). HRMS m/z calculated for C ₅₈ H ₇₄ N ₈ O ₈ [M+H] ⁺ : 1011.5702, found: 1011.5710.

S11: 5-(6-(4-(11-((2-(2,6-pyridinedione-3-yl)-1,3-dioxaisindol-4-yl)oxa) ring Hexyl)piperazin-1-yl)pyridine-3-yl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-N-((1-methyl -3-oxa-2,3,5,6,7,8-hexahydroisoquinolin-4-yl)methyl)benzamide.

Yellow solid (91 mg, 27%). HRMS m/z calculated for C ₅₉ H ₇₆ N ₈ O ₈ [M+H] ⁺ : 1025.5859, found: 1025.5863.

Example 4

K7: N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]-4'-{[4-(7-{[2-( 2,6-Dioxapiperidin-3-yl)-1,3-dioxa-2,3-dihydro-1H-isoindol-4-yl]thio}heptyl)piperazine-1- base]methyl}-5-[ethyl(oxa-4-yl)amino]-4-methyl-[1,1'-diphenyl]-3-carboxamide.

Tan solid (42.3 mg, 13%). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.43(s,1H),11.12(s,1H),8.16(t,1H),7.81(t,J=7.9Hz,1H),7.52(d, J＝7.9Hz, 2H), 7.51(d, 1H), 7.44(d, J＝7.3Hz, 1H), 7.41(s, 1H), 7.36(d, 2H), 7.23(s, 1H), 5.86( s,1H),5.08(dd,J=12.9,5.4Hz,1H),4.30(d,J=4.9Hz,2H),4.20(t,J=6.4Hz,2H),3.83(d,J=11.1 Hz, 2H), 3.47(s, 2H), 3.28–3.20(m, 2H), 3.09(q, J=7.6, 7.1Hz, 2H), 3.01(d, J=10.7Hz, 1H), 2.89(m ,J=13.6,12.5,6.9Hz,1H),2.59(d,J=17.0Hz,2H),2.37(s,6H),2.25(s,3H),2.21(s,3H),2.11(s, 3H), 2.03(d, J=12.5Hz, 1H), 1.75(t, J=7.2Hz, 2H), 1.67(d, J=12.0Hz, 2H), 1.53(dt, 2H), 1.43(p, 6H), 1.27(d,2H), 1.24(s,4H), 0.79(t,3H). HRMS m/z calculated for C ₅₄ H ₆₈ N ₇ O ₈ [M+H] ⁺ : 958.4895, found: 958.4893.

Example 5

M5: 1-[(2S)-butane-2-yl]-N-[(4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl]- 6-{6-[4-(5-{[2-(2,6-Dicarbylpiperidin-3-yl)-1,3-dioxa-2,3-hydrogen-1H-isoindole -4-yl]thioetheryl}pentyl)piperazin-1-yl]pyridin-3-yl}-3-methyl-1H-indole-4-carboxamide.

Light brown solid. HRMS m/z calculated for C ₄₉ H ₅₆ N ₈ O ₇ [M+H] ⁺ :871.3960,found:871.3962.

Example 6

N4: 1-((S)-sec-butyl)-N-((4,6-dimethyl-2-oxa-1,2-dihydropyridin-3-yl)methyl)-6-( 6-(4-(4-((2-(2,6-Dioxapiperidin-3-yl)-1,3-dioxaisoindoline-4-yl)amino)butyryl)piper (oxazin-1-yl)pyridin-3-yl)-3-methyl-1H-indole-4-carboxamide.

Pale yellow solid. ¹ H NMR (400MHz,DMSO-d ₆ )δ11.46(s,1H),11.08(s,1H),8.52(d,J=2.6Hz,1H),8.12(t,J=5.2Hz,1H) ,7.91(m,1H),7.80(t,1H),7.74(s,1H),7.51(d,J=8.5Hz,1H),7.41(d1H),7.28(s,1H),7.13(s, 1H), 6.92(d, 1H), 5.86(s, 1H), 5.12(m, 1H), 4.52(m, 1H), 4.33(d, 2H), 4.26(t, J=6.3Hz, 2H), 3.48(d,4H),2.92(m,,1H),2.66(m,2H),2.47(s,2H),2.41(t,J=6.8Hz,2H),2.27(s,3H),2.18( s,3H),2.09(s,3H),2.03(m,1H),1.82(m,4H),1.67(q,J＝7.4Hz,2H),1.39(d,3H),0.76(t,J =7.3Hz, 3H). HRMS m/z calculated for C ₄₉ H ₅₆ N ₈ O ₇ [M+H] ⁺ : 871.3960, found: 871.3962.

biological test

1. Experimental instruments and materials

The instruments used in the biological experiment in the embodiment of the present invention are as follows, ultra-clean workbench BHC-1000IIA/B3: Sujing Antai Biotechnology Company; constant temperature water bath PolyScience 9505: PolyScience Company; sterilizer MLS-3780: SANYO Company; oven : Binder Company; ultrapure water meter Milli-Q Integral 10: Millipore Company; microplate reader Multiscan MK3, cell culture incubator, low-speed centrifuge Sorvall ST1: Thermofisher Company; flow cytometer: BD Company; pH meter ORION STAR A211: ThermoScientific Company; ultrasonic breaker Sonic Materials Inc: Danbury Company; 37 ℃ constant temperature shaker Thermolyne, small vertical electrophoresis tank Mini-PROTEAN 3, transfer membrane tank MiniTrans-blot: Bio-Rad Company; X-ray photography cassette AX-Ⅱ: Guangdong Yuehua Medical Equipment Factory Co., Ltd.; fully automatic X-ray film processor HQ-320XT: Huqiu Imaging Co., Ltd.; slicer Leica RM 2125: Leica Company; ordinary optical microscope and inverted microscope: Olympus Company; fluorescence inversion Phase-contrast microscope: Carl Zeiss Company; fluorescence upright phase-contrast microscope: Leica DM2500 (Leica) Company and Carl Zeiss (Carl Zeiss) Company; vernier caliper (0-150mm): Shanghai Shenhan Measuring Tools Co., Ltd.

The cell lines used in the embodiments of the present invention are purchased from American Type Culture Collection (ATCC). 6, 24, and 96-well plates for cell culture, 15mL, 50mL centrifuge tubes, 25cm ² culture flasks and 75cm ² culture flasks were purchased from Chengdu Abiding Company. 10mL Petri dishes were purchased from WHB Company. Matrigel was purchased from BD Company. Dimethylsulfoxide (DMSO), MTT, SDS, polysorbate Tween-20, sodium dodecylsulfonate SDS, glycine, Tris, PEG-400 were purchased from Sigma. Ammonium persulfate APS, sodium hydroxide, ammonium persulfate, concentrated hydrochloric acid, isopropanol, methanol and other common analytical pure chemical reagents are from Kelon Chemical Company. Normal saline was purchased from Kelun Pharmaceutical Co., Ltd. Annexin V-FITC/PI kit, PI reagent, crystal violet dye, RIPA cell lysis buffer (strong), acrylamide buffer, and hematoxylin staining solution were purchased from Biyuntian Biotechnology Company. N,N,N',N'-tetramethylethylenediamine TEMED, G250 protein quantification solution was purchased from Bio-Rad. PVDF membrane and developing luminescence substrate were purchased from Millipore Company. Skimmed milk powder was purchased from Yili Dairy Company; self-developing film was purchased from Kodak Company. 10% APS, 1mol/L Tris-HCl (pH: 6.8), 1.5mol/L Tris-HCl (pH: 8.8), electrophoresis buffer, transfer buffer, TBS buffer, TBST buffer and other common reagents are prepared by this experiment . All antibodies were purchased from Cell signaling Technology (Beverly, MA). Goat serum, rabbit serum and DAB chromogenic kit for blocking were purchased from Beijing Zhongshan Jinqiao Company. TUNEL detection kit was purchased from promega company (Roche Applied Science).

First, AlphaScreen technology was used to test the inhibitory activity of the product compounds in Examples 1-5 and their positive controls GSK126 and EPZ6438 on EZH2 enzyme. The test results are shown in Table 1.

Table 1

Note: Each compound was tested twice, and the values in the table represent the average value.

From the results in Table 1, it can be seen that the inhibitory activities of G4-G12 and E4-E12 on EZH2 enzyme are at the nanomolar level, and the inhibitory activities of G4-G8 and E4-E8 with n being 2 to 6 on EZH2 are all consistent with their respective positive The control GSK126 is comparable to EPZ6438. When n is 7-10, the inhibitory activity of G9-G12 and E9-E12 on EZH2 is about 6-60 and 4-14 times weaker than their positive controls, respectively. Overall, most of the molecules can maintain the EZH2 enzyme inhibitory activity of the EZH2 inhibitor itself, and the E series maintains better activity than the G series; among all the synthesized molecules, E7 has the best inhibitory effect on the EZH2 enzyme Activity (IC ₅₀ =2.7nM), E7 was superior to the positive control in terms of EZH2 enzyme inhibitory activity.

2. Subunit degradation ability of PRC2 protein complex

Firstly, the protein levels of EZH2, SUZ12, EED, RbAp48 and histone H3K27me3 in WSU-DLCL-2 cells were detected by Western blotting. WSU-DLCL-2 cells were incubated with 1 μM test compound for 48 hours, and the same amount of EPZ6438, GSK126, and DMSO were used as controls. The results are shown in Figure 1.

The degradation effects of the two synthetic PRC2 degrading agents on EZH2 and other PRC2 core subunits SUZ12, EED, and RbAp48 in the DLBCL cell line WSU-DLCL-2 were detected by immunoblotting. The results are shown in Figure 1, the quantification of EZH2, SUZ12, EED and RbAp48 protein levels in WSU-DLCL-2 cells after 1 μM test compound treatment for 48 hours. C. Quantification of H3K27me3 protein level in WSU-DLCL-2 cells after 1 μM degradation agent treatment for 48 h. Quantitative analysis of protein expression was performed using Image J software, and the statistical results were expressed in the form of three mean ± standard deviation (SD), *P<0.05, **P<0.01, ***P<0.001. In the G series, G4 only showed weak degradation to EZH2, SUZ12, EED and RbAp48 subunits, G5-G7 only showed weak degradation to SUZ12 and EED subunits, while G8-G12 showed only weak degradation to EZH2, SUZ12, EED and RbAp48 subunits showed obvious degradative effects; in the E series, E4 with the shortest alkyl chain had a very obvious degradative effect on all the core subunits EZH2, SUZ12, EED and RbAp48 of PRC2, and E5 and E6 had very obvious degradative effects on the PRC2 core subunits. The degradation of the core subunit was significantly weaker than that of E4. Although the middle alkyl chain extended to 7 carbon atoms (E7), it showed a strong degradation effect on the subunits of PRC2, but with the continued extension of the middle alkyl chain , the ability of E8-E12 to degrade PRC2 core subunits gradually weakened; while the same dose of EZH2 enzyme inhibitors GSK126 and EPZ6438 almost did not change the protein levels of PRC2 core subunits in cells under the same conditions.

In addition, for the two types of degradation molecules represented by M5 and K7 that are linked to the alkyl chain through nitrogen atoms and sulfur atoms, they all show good degradation effects on PRC2 subunits, but for the other represented by N4 The degrading molecule of the chain did not show the degrading effect on the PRC2 subunit. For the example of the carbon alkyl chain type linker represented by N4 or the ligands at both ends are different, its effect is worse than that of the compounds of Example 4 and Example 5.

The inhibitory effect of the two synthetic PRC2 degradation agents on EZH2 activity in WSU-DLCL-2 cells was evaluated by detecting the level of intracellular H3K27me3. As shown in Figure 3, 1 μM test molecules can almost inhibit H3K27me3 to varying degrees after acting on WSU-DLCL-2 cells for 48 hours, and the trend of H3K27me3 protein level reduction is generally consistent with the trend of PRC2 subunit degradation, in which G8-G12, The inhibitory rate of E4 and E7-E11 on H3K27me3 is more than 60%, and has relatively strong inhibitory activity. Overall, the two types of PRC2-degrading molecules targeting EZH2 can almost degrade the core subunits of PRC2 and inhibit H3K27me3, but their ability to degrade PRC2 and inhibit H3K27me3 varies with the length of the Linker alkyl chain difference. Among them, PROTAC E7 not only showed the best in vitro EZH2 enzyme inhibitory activity, but also showed the best degradation of PRC2 core subunit (degradation rate: EZH2 72%, SUZ12 75%, EED 81%, RbAp48 74%) and inhibited H3K27me3 (inhibition rate 86%), therefore, the compound E7 was selected to continue the follow-up chemical biology research.

3. E7 effectively degrades PCR2

The time- and dose-response relationship of E7-induced PRC2 degradation was evaluated in WSU-DLCL-2 cells. In terms of timeliness, the degradation of the core subunits of PRC2 and the inhibition of H3K27me3 by 1μM E7 at different times were investigated. The results showed that as shown in A in Figure 4, within 0-1h, the degradation of E7 to EZH2 increased with the action time. The elongation gradually strengthened, but had almost no effect on other subunits of PRC2 and H3K27me3; at 2-12h, the degradation effect of E7 on PRC2 subunits disappeared, and the inhibitory effect on H3K27me3 was weak and unstable. It is speculated that E7 mainly degrades free EZH2 that is not involved in the formation of the PRC2 complex at the early stage of its action (0-12h), while it induces the degradation of EZH2 and other subunits of the PRC2 complex and takes longer to form a complete ternary complex . After being treated with E7 for 24 hours, the protein levels of PRC2 core subunits EZH2, SUZ12, EED, and RbAp48 and their catalytic product H3K27me3 began to decrease significantly, and after that, with the prolongation of the action time, the degradation of each subunit of PRC2 and the effect of E7 on The inhibition of H3K27me3 was gradually enhanced until the complete degradation of PRC2 was almost achieved at 96h. In terms of dose-effect, the degradation of PRC2 core subunits and the inhibition of H3K27me3 by different concentrations of E7 were investigated for 48 hours. The results are shown in B in Figure 4, low concentrations of E7 (0-0.5 μM) have only a weak degradation effect on PRC2 subunits; when the concentration reaches 1 μM, E7 can significantly degrade PRC2 subunits and inhibit H3K27me3, and This remarkable effect can be maintained continuously when the concentration is increased to 5μM; but when the concentration of E7 is too high, reaching 10μM, a "hook effect" (hook effect) occurs, that is, excessive E7 causes it to interact with EZH2 and E3. Ubiquitin ligases formed binary complexes respectively, but reduced the effective concentration of them participating in the formation of EZH2-E7-E3 ubiquitin ligase ternary complexes, which manifested as the degradation of the core subunits of PRC2 and the inhibition of H3K27me3 weakened. From the above results, it can be concluded that 1 μM of E7 acting on WSU-DLCL-2 cells for 48 hours can significantly and stably degrade the EZH2, SUZ12, EED and RbAp48 subunits of PRC2 and effectively inhibit H3K27me3.

In addition, the mRNA levels of EZH2, SUZ12, EED, and RbAp48 in WSU-DLCL-2 cells treated with 1 μM E7 for 48 h were detected by fluorescence real-time quantitative PCR (RT-qPCR) to determine that the reduction of their expression is due to the protein level of E7. It is not the result of the function at the genetic level. As shown in Figure 4C, E7, like the simple EZH2 methyltransferase inhibitors EPZ6438 and GSK126, hardly changed the mRNA levels of EZH2, SUZ12, EED and RbAp48 subunits of PRC2, indicating that E7 did not affect the expression of these proteins transcription of the genes involved, but rather in the translational or post-translational stages of these proteins.

The ability of E7 to degrade PRC2 in several other tumor cell lines driven by EZH2 dysfunction was further investigated to exclude the specific effect of E7 on the DLBCL cell line WSU-DLCL-2. The results are shown in D in Figure 4, 1μM E7 can significantly degrade the core of PRC2 in DLBCL (WSU-DLCL-2, Pfeiffer), PCa (LNCaP, DU 145) and ovarian cancer (A2780, SKOV3) cells for 48h Subunits EZH2, SUZ12, EED and RbAp48, and effectively reduce the level of the catalytic product H3K27me3, indicating that the role of E7 in degrading PRC2 subunits and inhibiting H3K27me3 can be exerted in a variety of tumor types driven by EZH2 dysfunction, not just Restricted to DLBCL. The current EZH2 enzyme inhibitors have shown good inhibitory activity on a few types of tumors such as lymphoma and sarcoma in clinical research, so this also suggests that, compared with most EZH2 enzyme inhibitors, E7 may be able to function in different types of tumor cells.

4. E7 degrades PRC2 by binding to EZH2

The cell thermal drift test (CETSA) can judge the combination of the drug and the protein by detecting the change of the protein thermal stability caused by the drug in the cell. The principle is: the combination of the drug and the corresponding protein in the cell will improve the structural stability of the protein , so that the protein can withstand higher temperatures without being degraded. Therefore, the combination of E7 and each core subunit of PRC2 was investigated by CETSA. Incubate E7 with WSU-DLCL-2 cells pretreated with MG-132 for a certain period of time. After E7 binds to the corresponding protein in the cells, extract the cell lysate, and detect EZH2 and SUZ12 in the cells of the control group and E7-treated cells. , EED and RbAp48 proteins were incubated at different temperatures (45, 48, 51, 54, 57, 60°C) for 6 minutes of degradation. The results are shown in A in Figure 5. The EZH2 protein in the control group was significantly degraded when it was incubated at 51°C for 6 minutes, while the EZH2 protein treated with E7 did not degrade to a certain level when it was heated to 57°C. It can be seen that E7 significantly improved The thermostability of EZH2 protein indicates that E7 binds to EZH2 in the cell; while several other proteins SUZ12, EED and RbAp48, regardless of whether E7 is treated or not, almost all undergo the same degree of degradation under the same temperature conditions, That is, E7 does not change the thermal stability of these proteins, indicating that E7 does not bind to these proteins. This demonstrates that E7 selectively binds to the EZH2 subunit of PRC2 leading to its degradation.

The binding of E7 to EZH2 subunits was further verified by competitive binding experiments of E7 with EZH2 inhibitors and EED inhibitors. As can be seen from B in Figure 5, in WSU-DLCL-2 cells, E7 can significantly reduce the protein levels of EZH2, SUZ12, EED, RbAp48 and H3K27me3, when the cells were treated with EZH2 inhibitor EPZ6438 or GSK126 simultaneously with E7 , the degradation effect of E7 on PRC2 core subunits was weakened, but H3K27me3 was still strongly inhibited; while the simultaneous treatment of cells with EED inhibitor EED226 and E7 did not affect the degradation effect of E7 on PRC2 subunits, nor affected E7 Inhibition of H3K27me3. The reason why EPZ6438 and GSK126 can interfere with E7’s degradation of PRC2 subunits is because they all compete with E7 for binding to the SAM binding pocket of EZH2, hindering the combination of E7 and EZH2, resulting in a decrease in the actual concentration of E7; while EPZ6438 and GSK126 do not The reason for affecting the inhibition of H3K27me3 is that as EZH2 methyltransferase inhibitors, they themselves have the activity of inhibiting H3K27me3, so they occupy the binding site of E7 and also inhibit H3K27me3, and can still maintain a strong inhibition of H3K27me3. The binding site of EED226 is the H3K27me3 binding pocket of EED, and its combination with EED does not occupy the binding site of E7, so it does not affect the degradation of PRC2 subunits and the inhibition of H3K27me3 by E7. This indirectly proves that E7 binds to the EZH2 subunit of PRC2.

At present, the three-dimensional structure of the PRC2 complex has been resolved, so the most direct and clear way to prove the binding between E7 and PRC2 subunits is to characterize the co-crystal structure of E7 and PRC2. Therefore, we will further confirm the combination of E7 and the EZH2 subunit of PRC2 by forming a co-crystal between E7 and PRC2.

In WSU-DLCL-2 cells, the methylation level of other Lys sites of histone H3 was detected to investigate the selective inhibitory effect of E7 on the activity of EZH2 HMTase. C in Figure 5 shows that E7 only selectively inhibits the activity catalyzed by EZH2 H3K27me3 and H3K27me2, but has little effect on other HMTase catalytic products H3K27me1, H3K9me3 and H3K4me3, indicating that E7 selectively inhibits EZH2 HMTase activity; and detection of E7's in vitro enzyme inhibitory activity on EZH1, which is highly homologous to EZH2, shows that E7 The inhibitory activity on EZH2 (IC ₅₀ =2.7nM) is 66 times stronger than that on EZH1 (IC ₅₀ =180nM), showing that E7 has a highly selective inhibitory effect on EZH2 HMTase activity. This also provides ancillary evidence that E7 functions by targeting EZH2.

5. E7 degrades PRC2 through the ubiquitin-proteasome pathway

According to the mechanism of action of PROTACs, E7 induces the degradation of EZH2 and other subunits of PRC2 on the premise of first recruiting ubiquitin molecules to target proteins, and this can only be achieved by the recognition and degradation of polyubiquitinated proteins by UPS. Therefore, the ubiquitination of EZH2, SUZ12 and EED subunits in E7-treated WSU-DLCL-2 cells was detected by immunoprecipitation (IP). The results are shown in Figure 6. In cells treated with E7, the ubiquitination levels of EZH2 (A in Figure 6), SUZ12 (B in Figure 6) and EED (C in Figure 6) subunits were significantly higher than The ubiquitination levels of each protein subunit in the control group indicated that the effect of E7 led to indifferent ubiquitination modification of EZH2, SUZ12 and EED subunits in cells.

In addition, by interfering with the function of UPS members, it was reversely verified that E7 degrades PRC2 subunits through the ubiquitin-proteasome pathway. In this experiment, lenalidomide, MLN4924 and MG-132 were used to verify whether blocking the ubiquitination modification of the target protein or inhibiting the activity of proteasome can destroy the degradation of E7, so as to further determine the pathway of E7 to degrade PRC2. Lenalidomide is a CRBN ligand with a structure very similar to thalidomide, which can compete with E7 for binding to E3 ubiquitin ligase and hinder the formation of EZH2-E7-E3 ubiquitin ligase ternary complex, as shown in the figure As shown in D of 6, pretreatment of WSU-DLCL-2 cells with lenalidomide before treatment of cells with E7 will weaken the degradation of EZH2, SUZ12, EED and RbAp48 subunits of PRC2 by E7; MLN4924 The ubiquitination modification of the target protein can be inhibited by specifically targeting NEDD-activating enzyme (NAE), so pretreatment of WSU-DLCL-2 cells with MLN4924 will also significantly inhibit the degradation of each subunit of PRC2 by E7. MG-132 is a proteasome inhibitor. D in Figure 6 shows that pre-inhibiting proteasome activity in WSU-DLCL-2 cells with MG-132 can also make PRC2 subunits stable without being degraded by E7. These results indicate that inhibiting the ubiquitination modification process in UPS or the activity of proteasome can effectively interfere with the degradation of PRC2 protein subunits by E7, indicating that E7 does indeed mediate the indifferent ubiquitination of PRC2 subunits and achieve the goal of degrading PRC2 subunits. Degradation of each subunit.

6. Regulation of E7 on EZH2 Downstream Gene Transcription

After confirming that E7 can effectively degrade EZH2 and other core subunits of PRC2 through the ubiquitin-proteasome pathway by binding to EZH2, the effect of E7 on the oncogenic function of EZH2 was further investigated. Current research has found that EZH2 mainly plays two roles in the process of driving tumor development: one is as a transcriptional repressor, which mediates transcriptional silencing of downstream genes in a manner dependent on the catalytic activity of its methyltransferase; Is a transcriptional coactivator that mediates transcriptional activation of downstream target genes in a manner independent of methyltransferase activity. In view of the fact that EZH2 plays different roles in different tumor types to regulate the transcription of different genes to drive tumor progression, in the embodiments of the present invention, the effects of E7 on EZH2 regulation of multiple gene transcriptions were investigated in different types of tumor cell lines.

7. E7 activates transcriptional silencing mediated by the catalytic function of EZH2

In some DLBCL, mutations in EZH2 lead to abnormally high levels of methylation of histone H3K27, resulting in highly condensed chromatin structure, leading to transcriptional silencing of downstream tumor suppressor genes and triggering tumor formation. Thus, we first examined the regulation of E7 on several H3K27me3-enriched genes ADRB2, CDKN2A, TXINP, and TNFRSF21 in two EZH2-mutant DLBCL cell lines, WSU-DLCL-2 (EZH2 ^Y641F ) and Pfeiffer (EZH2 ^A677G ). . The RT-qPCR results of A and B in Figure 7 showed that after WSU-DLCL-2 and Pfeiffer cells were treated with E7 for 48h, the mRNA levels of several detected genes were almost all significantly up-regulated, and E7 had a significant effect on the expression of ADRB2 and TNFRSF21. The upregulation was stronger than that of EZH2 enzyme inhibitors EPZ6438 and GSK126, but slightly weaker than that of EPZ6438 and GSK126 for CDKN2A and TXINP. In conclusion, in the EZH2 mutant DLBCL cell lines WSU-DLCL-2 and Pfeiffer, E7 can effectively activate the transcriptional silencing mediated by the catalytic function of EZH2 and inhibit the catalytic activity of EZH2. In some tumors with SWI/SNF mutations, EZH2 can significantly promote the proliferation of tumor cells, and this proliferation-promoting effect is partly dependent on the catalytic function of EZH2 in PRC2, so we also have NSCLC with SWI/SNF mutations The regulation of E7 on the transcription of the above several genes was detected in the cell line A549. The results are shown in C in Fig. 7, unlike the results observed in DLBCL, E7 only increased the mRNA levels of CDKN2A and TXINP2 genes, but failed to up-regulate the expressions of ADRB2 and TNFRSF21. The extent to which E7 upregulates these genes in A549 is less pronounced than in WSU-DLCL-2 and Pfeiffer is that the catalytic activity of EZH2 does not play a dominant role in the proliferation of tumor cells with SWI/SNF mutations such as A549, and its non-catalytic Activity is the main reason for the proliferation of these tumor cells. Therefore, in A549 cells, the transcription of genes such as ADRB2 and CDKN2A are not inhibited by the catalytic activity of EZH2, so even if E7 inhibits the catalytic activity of EZH2, these genes will not be affected. gene expression. The above results can prove that E7 can effectively inhibit the catalytic activity of EZH2 and its mediated transcriptional silencing by degrading EZH2 and other core subunits of PRC2.

8. E7 inhibits transcriptional activation mediated by the non-catalytic function of EZH2

Detection of several downstream genes ARL6IP, BRIC5, CENPK, CEP76, CHEK1 and TACC3 activated by E7 for the non-catalytic activity of EZH2 in tumor cells A549, NCI-H1299 and MDA-MB-468 driven by the non-catalytic function of EZH2 Transcription effects. As shown in Figure 8, in these types of cells, E7 treatment for 48 hours significantly down-regulated the above-mentioned EZH2-activated genes, while EZH2 methyltransferase inhibitors EPZ6438 and GSK126 had little effect on the expression of these genes. The above results prove that E7 effectively inhibits the non-catalytic activity of EZH2 and its abnormal activation of downstream gene transcription by degrading EZH2.

The above results indicate that no matter how EZH2 drives tumor development, PROTAC E7 can degrade EZH2 and other core subunits of PRC2, or inhibit gene silencing mediated by EZH2 as a transcriptional repressor, or inhibit EZH2 as a transcriptional regulator. Abnormal activation of transcription mediated by coactivators can ultimately effectively inhibit the driving role of EZH2 in tumorigenesis and development. In other words, E7 can not only destroy the catalytic function of EZH2 dependent on PRC2, but also destroy its non-catalytic function independent of PRC2, so it can completely and completely inhibit the oncogenic activity of EZH2. Therefore, for EZH2-driven tumors, especially those driven by its non-catalytic function, this strategy of inducing EZH2 degradation should have a better inhibitory effect than simply inhibiting EZH2 methyltransferase activity, so we initially evaluated and compared The differences in activity of E7 and EZH2 enzyme inhibitors EPZ6438 and GSK126 in inhibiting EZH2-driven tumor cell proliferation were investigated.

9. E7 inhibits the proliferation of EZH2 abnormal tumor cells

The growth inhibition of several EZH2-driven tumor cells by E7, EPZ6438, and GSK126 was monitored by cell counting and observation. As shown in A in Figure 9, E7 can almost completely inhibit the growth of WSU-DLCL-2 cells and exhibit good proliferation inhibitory activity; although both EPZ6438 and GSK126 also exhibit certain proliferation inhibitory activity on WSU-DLCL-2 cells , but its inhibitory degree is significantly lower than that of E7, which shows that E7 can effectively inhibit the growth of tumor cells driven by the catalytic function of EZH2. The proliferation activity of A549 and NCI-H1299 cells with SWI/SNF mutation mainly depends on the non-catalytic function of EZH2 in PRC2, so the proliferation inhibition of E7, EPZ6438 and GSK126 on these two cell lines was also investigated. The results are shown in Figure 9 B and C, similar to the results observed in WSU-DLCL-2 cells, E7 has very significant inhibitory activity on both A549 and NCI-H1299 cell lines; although GSK126 can also significantly EPZ6438 showed very limited proliferation inhibitory activity on these two cell lines. These results fully demonstrate that E7 has a good inhibitory effect on the growth of tumor cells driven by the catalytic and non-catalytic functions of EZH2.

The MTT test results in Figure 10 also show that E7 has a good time for the cell viability of the tumor cells WSU-DLCL-2, Pfeiffer, and the tumor cells A549 and NCI-H1299 driven by the catalytic function of EZH2. The IC ₅₀ of DLBCL cell line Pfeiffer is only 0.17μM for ₇ days of E7-dependent inhibition. The above results further prove that E7 has a good inhibitory effect on the viability of tumor cells driven by the catalytic and non-catalytic functions of EZH2.

In summary, the above biological experiment data show that the compounds of the embodiments of the present invention have degradative effects on each subunit of the PRC2 complex, and exhibit broader and stronger anti-tumor effects than inhibitors.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (14)

1. A bifunctional compound capable of inducing the degradation of the core subunit of the PRC2 protein complex, characterized in that it comprises a compound as shown in any one of formulas I-III and a pharmaceutically acceptable salt thereof;

Wherein, n in formula I is an integer of 2, 5-10, X is O, and Y is O; n in formula II is an integer of 4, 6 to 9, X is O, and Y is O; n in formula III is an integer of 2, 6-10, X is O or S, and Y is O. 2. The bifunctional compound according to claim 1, wherein X and Y in formula I-III are both O. 3. bifunctional compound according to claim 1, is characterized in that, the chemical formula of described bifunctional compound is

4. the preparation method of bifunctional compound as claimed in claim 1 is characterized in that, in formula III, X is S, and when Y is O, the synthetic route of the compound shown in formula III is:

When X and Y are both O in formula I-III, the synthetic route of formula I compound is:

The synthetic route of formula II compound is:

The synthetic route of formula III compound is:

5. A pharmaceutical composition, characterized in that it comprises pharmaceutically acceptable auxiliary components and the bifunctional compound according to any one of claims 1-3. 6. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is an aqueous solution, powder, granule, tablet or freeze-dried powder. 7. The pharmaceutical composition according to claim 6, wherein, when the pharmaceutical composition is an aqueous solution, the pharmaceutical composition also contains water for injection, saline solution, aqueous glucose solution or Grüner's solution; Said saline solution includes saline for injection or infusion, said aqueous dextrose solution includes glucose for injection or infusion, and said Guernsey's solution includes Guernsey's solution containing lactate. 8. Use of the bifunctional compound according to any one of claims 1-3 or the pharmaceutical composition according to any one of claims 5-7 in the preparation of kinase inhibitors. 9. The use of the bifunctional compound according to any one of claims 1-3 or the pharmaceutical composition according to any one of claims 5-7 in the preparation of drugs for treating tumors. 10. The use according to claim 9, wherein the tumor comprises breast cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer or gastric cancer. 11. The use of the bifunctional compound according to any one of claims 1-3 or the pharmaceutical composition according to any one of claims 5-7 in the preparation of a degradation agent for degrading the core subunit of the PRC2 protein complex. 12 . The application according to claim 11 , wherein the core subunit of the PRC2 protein complex degrades the subunits of EZH1, EZH2, EED and SUZ12 of the PRC2 protein complex. 13. The application of the bifunctional compound according to any one of claims 1 to 3 or the pharmaceutical composition according to any one of claims 5 to 7 in the preparation of oral or intravenous injection preparations, the oral or intravenous injection preparations are at least Including said bifunctional compound or pharmaceutical composition. 14. The use according to claim 13, further comprising excipients and/or adjuvants.

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