CN112920176B

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

Info

Publication number: CN112920176B
Application number: CN202010451831.6A
Authority: CN
Inventors: 余洛汀; 刘志昊
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-05-25
Filing date: 2020-05-25
Publication date: 2022-11-04
Anticipated expiration: 2040-05-25
Also published as: WO2021238007A1; CN112920176A

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 compound capable of inducing PRC2 protein complex core subunit degradation, pharmaceutical composition and application

Technical Field

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

Background

With the intensive research on the pathogenesis of tumors, human beings have become aware that the occurrence and development of tumors are associated not only with changes in DNA sequences caused by mutations or deletions of genes in cells, etc., but also with genetic disorders caused by changes in other heritable substances, so-called epigenetic phenomena. The epigenetic modification can regulate and control the processes of protooncogene activation, cancer suppressor gene inactivation, DNA damage repair, tumor stem cell differentiation and the like in an organism in a mode of DNA methylation, histone modification, chromatin remodeling or non-coding RNA interference, thereby regulating and controlling the growth, development and pathological change of the organism.

Polycomb group (PcG) proteins were first discovered to be a class of proteins that regulate the establishment of drosophila organisms by participating in the regulation of Hox gene expression during drosophila development and cell differentiation, and were later identified as epigenetic regulators critical to a variety of cellular functions. In mammals, PRC1 and PRC2 are the two major polythobic inhibitory complexes in PcG proteins that regulate transcription of genes by histone post-translational modifications. The PRC1 with different functions can be formed by different combinations of a plurality of subunits, wherein the classical PRC1 consists of CBX (chromabox), PCGF (polycomb group factor), HPH (human polymorphotic homolog) and RING1A/B (reactive intervening new gene) core subunits, mainly mediates H2AK119ub1 through the catalytic subunit RING1A/B, and plays an important role in the processes of embryonic development, stem cell characteristic maintenance, tumorigenesis and the like; PRC2 includes EZH2, SUZ12, EED and RbAp46/48 (retinoblastoma-associated proteins 46/48, also called RBBP 4/7) several core subunits, which mediate H3K27me2 and H3K27me3, mainly through the catalytic subunit EZH2, regulating the transcription of genes associated with cell cycle, senescence, differentiation and tumorigenesis.

Wherein, EZH2 as a catalytic subunit of PRC2 can catalyze H3K27me3 under the co-participation of two other core subunits SUZ12 and EED of the complex, and forms a complex which blocks transcriptional extension on nucleosomes together with PRC 1-mediated H2AK119ub1, thereby maintaining transcriptional silencing of downstream genes. EZH2 alone cannot exert catalytic activity, and it must rely on at least two subunits, SUZ12 and EED, of PRC2 to exert HMTase activity to inhibit transcription of multiple downstream genes. SUZ12 is a core subunit essential for maintaining the integrity of PRC2, the stability of EZH2 and the activity of HMTase, and the C-terminal VEFS (Vrn 2-Emf2-Fis2-SUZ 12) domain of the SUZ can be stably combined with EZH2 on one hand, maintain the assembly of PRC2 complex, and can assist the HMTase activity of PRC2 through allosteric action on the other hand. Interaction and binding of the repeating WD domain of the EED subunit with EZH2 are essential conditions to ensure that EZH2 has HMTase activity, and specific binding of the WD domain of the C-terminal of the EED to H3K36me3 is essential for HMTase allosteric activation, and thus the EED is also a core subunit of PRC2 that exerts HMTase activity. The retinoblastoma-associated proteins 46 and 48 (RbAp 46/48) subunits are highly homologous histone chaperones, which play an important role in the maintenance of chromatin structure. In PRC2, although direct participation of RbAp46 and RbAp48 is not required for EZH2 to exert HMTase activity, the introduction of PRC2 into nucleosomes requires that RbAp48 bind to the histone H3-H4 heterodimer, so RbAp48 is also a core subunit ensuring that PRC2 normally exerts HMTase activity.

However, each core subunit of the PRC2 complex can play its own non-PRC 2 catalytic function, in addition to participating in the formation of a cancer suppressor gene that catalyzes H3K27me3 and up-regulates transcriptional silencing by the PRC2 complex. For example, EZH2, a multifunctional protein, not only mediates gene silencing by catalyzing H3K27me3, but also mediates transcriptional activation of genes in certain tumors in a manner independent of HMTase activity. Multiple studies indicate that EZH2 can also serve as a transcription activator to methylate non-histone proteins or directly interact with other proteins to form a transcription complex, and the transcription of genes is activated. After the 21-position serine (Ser) of EZH2 is phosphorylated by protein kinase B (AKT), pEZH2 can be used as a co-activator of Androgen Receptor (AR) and related compounds to promote the growth of castration-resistant prostate cancer (CRPC) cells, i.e., AKT mediates the phosphorylation of Ser21 of EZH2, promotes EZH2 to bind 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, which is found to provide a new combined treatment concept for treating metastatic and hormone-refractory prostate cancer. It has also been found that phosphorylated EZH2 can also directly bind to and methylate Lys180 of STAT3, mediating tyrosine (Tyr) phosphorylation at position 705 of STAT3 to enhance STAT3 activity, thereby contributing to the tumorigenicity of glioblastoma and glioblastoma stem cells. These results are in stark contrast to the results of EZH2 catalyzing the non-histones GATA4 and ROR α to inhibit gene transcription.

Furthermore, EZH2 has also been found to activate transcription in colorectal cancer cells in a manner independent of methyltransferase activity. Human proliferating cell nuclear antigen related factor PAF recruits EZH2, promotes EHZ2 to combine and interact with TCF/beta-catenin, forms transcription initiation complex with transcription factor TCF/LEF at the promoter of c-myc, cyclinD1, axin2 and other genes, induces the transcription activation of tumor formation related target genes in Wnt pathway, and promotes the progress of colorectal cancer. In addition, in the AR positive prostate cancer, the EED directly interacts with an androgen receptor without depending on the function of PRC2, regulates the expression level of AR and the downstream target of AR, and promotes the development of the prostate cancer. EEDs may also affect the catalytic activity of HDAC independently of the direct interaction of PRC2 function with histone deacetylase HDAC.

Therefore, the current EZH2 inhibitors or EED inhibitors are only used to inhibit the activity of PRC2, but cannot effectively inhibit the oncogenic activity of EZH2, EED, etc. independent of the PRC2 catalytic function, and even cannot degrade the various core subunits of PRC2 simultaneously to completely block the oncogenic activity of the PRC2 complex subunits.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a bifunctional compound capable of inducing the core subunit of a PRC2 protein complex to degrade, a pharmaceutical composition and an application thereof, so as to improve the problems.

The invention is realized in the following way:

in a first aspect, the embodiments of the present invention provide a bifunctional compound capable of inducing degradation of a core subunit of a PRC2 protein complex, which includes a compound represented by any one of formulas I to 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 isotopically substituted compound thereof;

wherein N in the formula I-III is an integer of 1-10, X is O, N or S, Y is O, H ₂ Or S.

In a second aspect, the embodiments of the present invention further provide a method for preparing a bifunctional compound, where in formulas I to III, when X is N, S, and Y is O, the synthetic route of the compound shown in formulas I to III is as follows:

when X and Y are both O in the formulas I-III, the synthetic route of the compound of the formula I is as follows:

the synthetic route for the compounds of formula II is:

the synthetic route for the compound of formula III is:

in a third aspect, the embodiments of the present invention further provide a pharmaceutical composition, which includes pharmaceutically acceptable auxiliary components and the bifunctional compound of the foregoing embodiments.

In a fourth aspect, the embodiments of the present invention further provide an application of the bifunctional compound or the pharmaceutical composition of the foregoing embodiments in preparing a kinase inhibitor.

In a fifth aspect, the embodiments of the present invention further provide an application of the bifunctional compounds or pharmaceutical compositions of the foregoing embodiments in preparing a medicament for treating tumors; optionally, the tumor comprises breast cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, or gastric cancer.

In a sixth aspect, the present invention further provides an application of the bifunctional compound or the pharmaceutical composition of the foregoing embodiments in preparing a degradation agent for degrading a core subunit of a PRC2 protein complex, optionally, degrading the core subunit of the PRC2 protein complex into EZH1, EZH2, EED, SUZ12, and RbAp46/48 subunits for simultaneously degrading the PRC2 protein complex.

In a seventh aspect, the present invention also provides a use of the bifunctional compound or the pharmaceutical composition of the foregoing embodiments in preparing an oral or intravenous injection preparation, where the oral or intravenous injection preparation at least includes the bifunctional compound or the pharmaceutical composition, and optionally further includes an excipient and/or an adjuvant.

One of the schemes of the above embodiments of the present invention has at least the following beneficial effects: the bifunctional compound can effectively induce the core subunit of the PRC2 protein complex to degrade, further achieve the purposes of treating cancers mediated by the PRC2 complex and subunits thereof including EZH2, EED, SUZ12, rbAp46 and RbAp48, completely blocking the carcinogenic activity of the PRC2 complex subunit, has better anticancer activity compared with the effect of simply inhibiting the PRC2 complex activity, such as an EZH2 inhibitor and an EED inhibitor, and has the purposes of treating various solid tumors such as breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, ovarian cancer and the like and various tumor diseases such as hematological tumors and the like. The bifunctional compound or the pharmaceutical composition is used as a kinase inhibitor for treating various tumors of human, and has better antitumor activity and lower toxicity.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIGS. 1 and 2 show Western blotting to detect protein levels of EZH2, SUZ12, EED, rbAp48 and histone H3K27me3 in WSU-DLCL-2 cells;

FIG. 3 shows that the synthetic molecule degrades each core subunit of PRC2 and reduces the level of H3K27me 3;

fig. 4 shows that E7 effectively degrades PRC2 and inhibits H3K27me3.A. Western blotting was performed to detect the inhibition of E7 at different time points on each subunit of PRC2 and H3K27me3 in WSU-DLCL-2 cells. B. Western blotting was performed to detect the inhibition of PRC2 subunits and H3K27me3 by E7 with different concentrations acting on WSU-DLCL-2 cells for 48H. RT-qPCR detects mRNA levels of each core subunit of PRC2 in WSU-DLCL-2 cells. Statistical results are expressed as the mean of triplicates ± SD. D. Detecting the degradation effect of E7 on each subunit of PRC2 in DLBCL, PCa and ovarian cancer cell strains through western blotting;

figure 5 shows that E7 acts in conjunction with EZH2 subunit of PRC2. CETSA detects the thermal stability of EZH2, SUZ12, EED and RbAp48 proteins in the WSU-DLCL-2 cells treated by E7 and corresponding immunoblotting grey statistics results. The statistics are all expressed as the mean ± Standard Deviation (SD) of three times. B. Western immunoblotting was performed to detect the competitive effect of EZH2 inhibitor and EED inhibitor with E7 in WSU-DLCL-2 cells. C. Detecting various methylation modification products of E7 on histone H3 in WSU-DLCL-2 cells by Western blotting;

fig. 6 shows that E7 degrades PRC2 via ubiquitin proteasome pathway. Immunoprecipitation experiments examined the effect of 1 μ M E7 on EZH2 (A), SUZ12 (B) and EED (C) ubiquitination modification for 48 h. WSU-DLCL-2 cells are pretreated for 4 hours by 1 mu M lenalidomide or 5 mu M MLN4924/MG-132, then the cells are treated for 48 hours by 1 mu M E7, and cell lysates are collected for immunoblotting analysis;

FIG. 7 is a graph showing that E7 activates the catalytic function of EZH2 in WSU-DLCL-2 (A), pfeiffer (B) and A549 (C) cells to mediate the transcription of silenced genes. Cells were treated with indicated compounds for 48h and then assayed for mRNA levels using RT-qPCR. Statistical results are expressed as the triple mean ± SD, { P ≦ 0.05, { P ≦ 0.01, };

FIG. 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. Cells were treated with indicated compounds for 48h and then assayed for mRNA levels using RT-qPCR. Statistical results are expressed as the triple mean ± SD, { P ≦ 0.05, { P ≦ 0.01, };

FIG. 9 shows that E7 effectively inhibited the proliferation of WSU-DLCL-2 (A), A549 (B) and NCI-H1299 (C) cells. Proliferation curves of cells treated with 10 μ M E7, EPZ6438 or GSK126 (left) and light microscopy images at different time points (right), statistical results are expressed as the cubic mean ± SD;

FIG. 10 shows MTT assay to detect the inhibition of WSU-DLCL-2 (A), pfeiffer (B), A549 (C) and NCI-H1299 (D) by E7 action 3D, 5D, 7D. Survival curves and IC50 values were fitted by GraphPad prism5.0 software. Statistical results are expressed as mean ± SD of two times.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

The following provides specific descriptions of the bifunctional compound capable of inducing core subunit degradation of PRC2 protein complex, the pharmaceutical composition and the application thereof.

Some embodiments of the present invention provide a bifunctional compound that can induce degradation of a core subunit of a PRC2 protein complex, comprising a compound of any one of formulas I-III, a pharmaceutically acceptable salt or prodrug thereof, a solvate thereof, a hydrate thereof, a polymorph thereof, a tautomer, a stereoisomer, or an isotopically substituted compound thereof;

The term "pharmaceutically acceptable" as used herein means, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response, and the like, which upon administration to a recipient, provides, directly or indirectly, the bifunctional compounds of the above-described embodiments of the present invention. The term "hydrate" refers to a compound that further binds stoichiometric or non-stoichiometric water by non-covalent intermolecular forces. The term "polymorph" denotes a solid crystalline form of a compound or a complex thereof, which can be characterized by physical means, such as x-ray powder diffraction patterns or infrared spectroscopy.

The present inventors have conducted extensive studies and practices in the case where the conventional EZH2 inhibitors, EED inhibitors, and the like, only inhibit the activity of the PRC2 complex, but do not effectively inhibit the oncogenic activity of the PRC2 complex, such as EZH2, EED, and the like, which are independent of the PRC2 catalytic function, and further do not inhibit other core subunits of the PRC2 complex, such as EZH1, SUZ12, and RbAp46/48, and the like, and have creatively found that the bifunctional compounds of the above three formulae can effectively induce the degradation of the PRC2 protein complex core subunit, thereby achieving a better anticancer activity for treating cancers mediated by the PRC2 complex and its subunits, including EZH2, EED, SUZ12, rbAp46, and RbAp48, and completely blocking the oncogenic activity of the PRC2 complex subunit, compared to simply inhibiting the activity of the PRC2 complex, such as EZH2 inhibitors and EED inhibitors, and have a better anticancer activity for treating various solid tumors, such as breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, ovarian cancer, and blood cancer.

It is to be noted that the bifunctional compound capable of inducing the degradation of the core subunit of the PRC2 protein complex may also be a tautomer, stereoisomer or mixture of all proportions of any one of the compounds I to III.

In order to further optimize the degradability and the combination of properties of the bifunctional compounds, in some embodiments, n in the above formulas I-III is an integer of 2 to 6.

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

When X is N, S and Y is O in the formulas I-III, the synthetic routes of the compounds shown in the formulas I-III are as follows:

when X and Y in formulas I-III are both O, the three classes of compounds can be synthesized through some synthetic routes, and specifically, the synthetic route of the compound in formula I is as follows:

the synthetic route for the compound of formula II is:

the synthetic route for the compound of formula III is:

further, some embodiments of the present invention also provide a pharmaceutical composition comprising a pharmaceutically acceptable auxiliary ingredient and the bifunctional compound of the previous embodiments. Wherein the auxiliary components are general components which are added in the process.

Further, the pharmaceutical composition may be in the form of a liquid or a solid. That is, the pharmaceutical composition includes, but is not limited to, an aqueous solution, powder, granule, tablet or lyophilized powder, and in order to make the pharmaceutical composition sufficiently act on the body, in some embodiments, when the pharmaceutical composition is an aqueous solution, the pharmaceutical composition further contains water for injection, saline solution, aqueous glucose solution, saline for injection or infusion, glucose for injection or infusion, geline solution or geline solution containing lactate.

Further, some embodiments of the present invention also provide the use of the aforementioned bifunctional compound or pharmaceutical composition for preparing a kinase inhibitor. Particularly has better inhibition performance on EZH2 enzyme.

Further, some embodiments of the present invention also provide an application of the bifunctional compound or the pharmaceutical composition in preparing a medicament for treating tumors; alternatively, the tumor comprises a solid tumor such as breast cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer or gastric cancer.

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

Further, some embodiments of the present invention also provide the use of the bifunctional compound or the pharmaceutical composition described above for preparing an oral or intravenous formulation. The oral or intravenous injection comprises at least the bifunctional compound or the pharmaceutical composition, optionally together with excipients and/or adjuvants.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

This example provides the synthesis of 9 bifunctional compounds G4-G12 and their associated chemical data. The synthetic route of G4-G12 is as follows:

the preparation process comprises the following steps:

the first step is as follows: preparation 1b, ammonolysis.

1a (3.28g, 20mmol, 1.0eq) and 3-aminopiperidine-2, 6-diketone (3.1g, 24mmol, 1.2eq) are dissolved in 50mL of acetic anhydride, heated and refluxed for 6 hours at 140 ℃, reduced pressure distillation is carried out after the reaction is finished, the reaction solvent is removed, the residue is poured into water, light gray precipitate is separated out, suction filtration is carried out, and a filter cake is washed by water and dried to obtain a product 1b (3.84g, 71%). HRMS M/z calculated for C13H10N2O5[ M + H ] +:275.0662, found.

The second step: 1c-1k general synthetic procedure.

1b (1mmol, 1.0 eq), DIPEA (3mmol, 3.0 eq) and dibromoalkane (1.2mmol, 1.2eq) were dissolved in DMF (10 mL) and reacted at 85-100 ℃ for 3-6h. After the reaction was complete, ethyl acetate (50 mL) and water were added for extraction, na ₂ SO ₄ Drying, concentrating to obtain residue, and purifying by column chromatography to obtain corresponding product。

Compound 1c: white solid (195.8 mg, 48%). 1H NMR (400mhz, dmso-d 6) δ 11.08 (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 (dd, J =12.5,8.5, 8.8hz, 3h), 1.dt (J, 8.8, 6.1tmh), hrc/z/C, for 1.90 (J =8, 6.1tmh, 12 hz/1H) ₁₇ H ₁₇ BrN ₂ O ₅ [M+Na]+:431.0213,found:431.0219。

Compound 1d: white solid (236.3mg, 56%). 1H NMR (400mhz, dmso-d 6) δ 11.08 (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, 6.59, 1.59, 6.7hz, 6.6H), 1.9, 6.7hz, 6H, 6C, 12 z, hrc, 2C, z/C, z ₁₈ H ₁₉ BrN ₂ O ₅ [M+Na] ⁺ :445.0370,found:445.0367。

Compound 1e: white solid (161.3mg, 37%). 1H NMR (400MHz, DMSO-d 6) delta 11.09 (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/z calcaled 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-d 6) δ 11.09 (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 calulated for C ₂₀ H ₂₃ BrN ₂ O ₅ [M+H] ⁺ :451.0863,found:451.0852。

Compound 1g: white solid (232.4mg, 50%). 1H NMR (400mhz, dmso-d 6) δ 11.08 (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 (ms, 8H), telcel/C, for C, for 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-d 6) delta 11.09 (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 (mJ =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 =7.6Hz, 2H), 1.37 (d, J =7.2Hz, 4H), 1.33-1.25 (m, 4H), HRMS m/z calcaulded for C ₂₂ H ₂₇ BrN ₂ O ₅ [M+H] ⁺ :479.1176,found:479.1164。

Compound 1i: white solid (201.7mg, 41%). 1H NMR (400mhz, dmso-d 6) δ 11.09 (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 calcaulded for C ₂₃ H ₂₉ BrN ₂ O ₅ [M+Na] ⁺ :515.1152,found:515.1147。

Compound 1j: white solid (293.5mg, 58%). 1H NMR (400mhz, dmso-d 6) δ 11.08 (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 calcualted for C ₂₄ H ₃₁ BrN ₂ O ₅ [M+Na] ⁺ :529.1308,found:529.1298。

Compound 1k: white solid (176.8mg, 34%). 1H NMR (400mhz, dmso-d 6) δ 11.09 (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, 2h), 2.15-1.96 (m, 1H), 1.77 (q, J =8.6,8.2hz, 4h), 1.53-1.41 (m, 2H), 1.35 (s, 4H), 1.26 (s, 10H), hrtems/z for C, C ₂₅ H ₃₃ BrN ₂ O ₅ [M+Na] ⁺ :543.1465,found:543.1472。

The third step: 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 of DMF and reacted at 85 ℃ for 5 hours. After the reaction is finished, extracting by ethyl acetate and Na ₂ SO ₄ Drying, distilling under reduced pressure to remove the solvent, and separating by silica gel column chromatography to obtain the corresponding product G4-G12.

G4:1- [ (2S) -butan-2-yl ] -N- [ (4, 6-dimethyl-2-oxa-1, 2-dihydropyridin-3-yl) methyl ] -6- {6- [4- (4- { [2- (2, 6-dicarballyl-3-yl) -1, 3-dioxa-2, 3-hydro-1H-isoindol-4-yl ] oxy } butyl) piperazin-1-yl ] pyridin-3-yl } -3-methyl-1H-indole-4-carboxamide.

White solid (72.1mg, 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＝12.9,5.4Hz,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 )δ173.23,170.41,169.18,167.33,165.82,163.57,160.55,158.47,158.47,156.49,149.77,146.00,143.12,138.19,137.49,136.37,133.72,131.17,130.22,126.48,124.86,123.42,122.24,120.28,116.73,116.50,115.60,110.18,108.15,107.82,107.36,69.20,57.79,52.98,52.09,49.24,45.40,35.54,31.44,29.97,26.85,22.93,22.50,21.31,19.44,18.66,12.15,11.20.HRMS m/z calculated for C ₄₈ H ₅₄ N ₈ O ₇ [M+H] ⁺ :855.4188,found:855.4185。

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

White solid (45.6mg, 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,J＝19.9,7.1Hz,4H),1.41(d,J＝6.6Hz,3H),1.24(d,J＝5.1Hz,2H),0.73(t,J＝7.3Hz,3H). 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) -butan-2-yl ] -N- [ (4, 6-dimethyl-2-oxa-1, 2-dihydropyridin-3-yl) methyl ] -6- {6- [4- (6- { [2- (2, 6-dicarballyl-3-yl) -1, 3-dioxa-2, 3-hydro-1H-isoindol-4-yl ] oxy } hexyl) piperazin-1-yl ] pyridin-3-yl } -3-methyl-1H-indole-4-carboxamide.

Light yellow solid (63.3mg, 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 C NMR(101MHz,DMSO-d ₆ )δ173.23,170.40,169.17,167.31,165.78,163.57,158.47,156.51,149.77,145.99,143.12,138.20,137.49,136.36,133.73,131.17,130.22,126.48,124.86,123.42,122.24,120.29,116.72,116.49,115.60,110.18,108.14,107.82,107.36,69.26,58.33,53.07,52.08,49.22,45.36,35.54,31.44,29.97,28.86,27.04,26.66,25.68,22.50,21.31,19.44,18.66,12.15,11.20.HRMS m/z calculated for C ₅₀ H ₅₈ N ₈ O ₇ [M+H] ⁺ :883.4501,found:882.4502。

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

Grey solid (73.6mg, 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.HRMS m/z calculated for C ₅₁ H ₆₀ N ₈ O ₇ [M+H] ⁺ :897.4658,found:897.4656。

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

White solid (41.6mg, 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 )δ173.22,170.39,169.17,167.31,165.77,163.57,158.47,156.50,149.77,145.99,143.12,138.20,137.48,136.36,133.72,131.17,130.22,126.47,124.86,123.41,122.24,120.27,116.70,116.50,115.58,108.14,107.82,107.35,69.30,58.45,53.07,52.08,49.22,45.36,35.53,31.44,29.97,29.36,29.10,28.87,27.36,26.71,25.72,22.49,21.30,19.44,18.66,12.14,11.20.HRMS m/z calculated for C ₅₂ H ₆₂ N ₈ O ₇ [M+H] ⁺ :911.4814,found:911.4819。

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

White solid (85.6mg, 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) -butan-2-yl ] -N- [ (4, 6-dimethyl-2-oxa-1, 2-dihydropyridin-3-yl) methyl ] -6- {6- [4- (10- { [2- (2, 6-dicarbonylpiperidin-3-yl) -1, 3-dioxa-2, 3-hydro-1H-isoindol-4-yl ] oxy } sunflower) piperazin-1-yl ] pyridin-3-yl } -3-methyl-1H-indole-4-carboxamide.

White solid (54.1mg, 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 )δ173.32,170.41,169.32,167.35,165.79,163.67,158.45,156.49,150.14,145.91,143.24,138.17,137.54,136.41,133.63,131.03,130.20,126.45,124.95,123.37,122.12,120.22,116.60,116.43,115.60,110.12,108.15,107.45,69.28,58.43,52.98,52.14,49.20,45.26,35.54,31.38,29.94,29.36,29.06,28.83,27.41,26.61,25.71,22.47,21.27,19.44,18.62,12.08,11.16.HRMS m/z calculated for C ₅₄ H ₆₆ N ₈ O ₇ [M+H] ⁺ :939.5127,found:939.5124。

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

Pale yellow solid (97.6mg, 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 )δ173.21,170.38,169.17,167.31,165.76,163.57,158.46,156.50,149.76,145.99,143.12,138.19,137.47,136.36,133.72,131.17,130.21,126.49,124.87,123.42,122.24,120.26,116.70,116.50,115.58,110.18,108.14,107.82,107.37,69.28,58.43,53.04,52.08,49.22,45.33,35.54,31.44,29.97,29.42,29.12,28.89,27.44,26.71,25.74,22.49,21.30,19.44,18.66,12.14,11.19.HRMS m/z calculated for C ₅₅ H ₆₈ N ₈ O ₇ [M+H] ⁺ :953.5283,found:953.5284。

G12:1- [ (2S) -butan-2-yl ] -N- [ (4, 6-dimethyl-2-oxa-1, 2-dihydropyridin-3-yl) methyl ] -6- {6- [4- (12- { [2- (2, 6-dicarballyl-3-yl) -1, 3-dioxa-2, 3-hydro-1H-isoindol-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.HRMS m/z calculated for C ₅₆ H ₇₀ N ₈ O ₇ [M+H] ⁺ :967.5440,found:967.5438。

Example 2

This example provides the synthesis of 9 bifunctional compounds E4-E12 and their associated chemical data. The synthetic route of E4-E12 is as follows:

the preparation process comprises the following steps:

the first step is as follows: methyl 2-methyl-3-bromo-5-nitrobenzoate 2a (5.5g, 20mmol, 1.0eq), ammonium chloride (5.6 g)100mmol,5.0 eq) was dissolved in aqueous ethanol (60mL, H ₂ EtOH = 1), iron powder (11.2g, 200mmol,10.0 eq) was added in three portions after heating to 80 ℃. After reacting for 1h, detecting by Thin Layer Chromatography (TLC) that the reaction is finished, filtering with diatomaceous earth, distilling the filtrate under reduced pressure to remove solvent, extracting the residue with DCM, and extracting with Na ₂ SO ₄ Concentration after drying afforded product 2b (4.41g, 92%) without further purification. 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 is that: 2b (4 g,17.4mmol, 1.0eq) and tetrahydropyranone (4.4 g,52.2mmol, 3.0eq) were dissolved in chloroform (50 mL), acetic acid (2.1g, 34.8mmol, 2.0eq) was added, and after stirring at room temperature for 3 hours, sodium triacetoxyborohydride (2.7g, 43.6mmol, 2.5eq) was added, and stirring was continued overnight. After completion of the reaction, the solvent was removed by distillation under the reduced pressure, and the residue was purified by silica gel column chromatography to obtain product 2c (3.3g, 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。

The third step: under nitrogen protection, 30mL of 1, 2-dichloroethane was added to 2c (3 g,9.1mmol, 1.0eq) in a 50mL round-bottomed flask, anhydrous acetaldehyde (2.3g, 27.3mmol, 3.0eq) was carefully injected under the liquid level of the solution with slow stirring, and acetic acid (1.1g, 18.2mmol, 2.0eq) was further added over 30min, and the reaction solution was orange-yellow. After the reaction mixture was allowed to spontaneously warm to room temperature, it was stirred for 1h. The mixture was then cooled to 0 ℃ and sodium triacetoxyborohydride (1.48g, 23mmol) was added slowly in portions, the rate of addition was controlled to maintain the reaction temperature below 5 ℃ and after 2h was allowed to invert to room temperature and stir overnight. After TLC reaction is finished, cooling the reaction system to 0 ℃, adding 100mL of ice water, slowly adding excessive saturated aqueous solution of sodium bicarbonate while stirring, stirring for 30min, standing, separating, extracting the aqueous phase with dichloromethane,the organic layers were combined and washed twice with water and the layers were separated. The organic phase was separated and concentrated under reduced pressure to constant weight to give a yellow to pale 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。

The fourth step: under the protection of nitrogen, 2d (2g, 5.6 mmol) is added into 25mL of methanol at one time, after the temperature is raised to 60 ℃,20 mL of sodium hydroxide aqueous solution (2M) is slowly dropped while keeping the temperature, and the color of the reaction solution gradually changes from light green clear liquid to emulsion and finally to light green clear liquid. After incubation for 1h, the reaction was monitored by TLC for completion. The reaction solution was transferred to a rotary evaporator, most of methanol was removed under reduced pressure, 100mL of water was added to the residue, and stirring was carried out for 10min, whereby the solid was completely dissolved. Heating to 65 ℃, adding hydrochloric acid (2M) to adjust the pH to be 2-3, precipitating, stopping the hot bath, cooling to room temperature, and stirring for 0.5h. Suction filtration, the filter cake is fully washed by ice water, the filter cake is dried in a vacuum drying oven by taking phosphorus pentoxide as a drying agent, and the drying is carried out for 10h at the temperature of 60 ℃, thus obtaining 2e (1.75g, 91.2%) 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-dimethylpyridin-2 (1H) -one (0.61g, 4mmol) and 2e (1.5g, 4.4mmol) were dissolved in DMSO (10 mL), HOAT (0.55g, 1.5mmol) and EDCI (0.84g, 2.2mmol) were added and the reaction was stirred at 45 ℃ for 20H. After completion of the reaction monitored by TLC, the reaction solution was poured into ice water (100 mL), stirred for 30min to precipitate, filtered, washed with water, dried, dissolved in a mixture of methanol and chloroform (10),68％)。 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。

And a sixth step: to a mixed solution of 1, 4-dioxane and water (4, 1, 30mL) was dissolved 2f (1.2g, 2.5mmol, 1.0eq), tert-butyl4- (4, 5-tetramethylol-1, 3, 2-dioxaborolan-2-yl) benzyl) piperazine-1-carboxylate (1.21g, 3mmol, 1.2eq), and K ₂ CO ₃ (3.75mmol,0.52g)，Pd(dppf)Cl ₂ (0.2mmol, 146mg), under nitrogen protection, brought to 100 ℃ for reaction for 8h, and then cooled to room temperature. The reaction solution was distilled under reduced pressure to remove the solvent, and then dissolved in ethyl acetate, and filtered with celite. Extracting the filtrate, drying with anhydrous sodium sulfate, concentrating, mixing with sample, and purifying by silica gel column chromatography to obtain the product. Without further purification, the reaction mixture was added directly to 25mL of a trifluoroacetic acid/dichloromethane solution (10%) and reacted at room temperature for 1h, then the solvent was distilled off under reduced pressure, and the residue was neutralized to pH =8 with a saturated sodium carbonate solution (2M), and a precipitate was precipitated, suction filtered, washed with water, and dried to obtain 2g of a product (1.04g, 74%). 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: E4-E12 is synthesized and nucleophilic substitution reaction is carried out.

2g(0.25mmol,1eq),NaHCO ₃ (0.5mmol, 2.0eq) and 1c-1k (0.3mmol, 1.2eq) were dissolved in DMF (5 mL) at 85 deg.CReacting for 3-8h, extracting with ethyl acetate, and adding Na ₂ SO ₄ Drying, distilling under reduced pressure to remove solvent, and separating by silica gel column chromatography to obtain corresponding product E4-E12.

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

Yellow solid (61.7mg, 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＝7.2Hz,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-dioxopiperidin-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 (46.4mg, 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.HRMS m/z calculated for C ₅₃ H ₆₆ N ₇ O ₈ [M+H] ⁺ :928.4967,found:928.4982。

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

Yellow solid (101.3mg, 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 )δ173.31,170.41,169.65,167.34,165.80,163.55,156.49,150.28,149.38,143.31,139.95,138.96,137.62,137.55,137.50,133.65,133.08,129.98,126.82,123.41,121.97,121.20,120.24,116.61,115.61,108.09,69.26,66.78,62.02,58.28,58.07,52.99,52.64,49.20,41.69,35.37,31.39,30.75,29.42,28.92,28.78,27.20,26.23,25.67,22.47,19.41,18.63,15.01,13.14.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-dioxopiperidin-3-yl) -1, 3-dioxa-2, 3-dihydro-1H-isoindol-4-yl ] oxy } octyl) piperazin-1-yl ] methyl } -5- [ ethyl (oxa-4-yl) amino ] -4-methyl- [1,1' -diphenyl ] -3-carboxamide.

Yellow solid (69.3mg, 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 )δ173.20,170.38,169.51,167.31,165.75,163.47,156.49,149.95,149.33,143.19,140.07,138.96,137.49,133.71,133.07,129.85,126.83,123.36,122.09,121.28,120.26,116.70,115.58,107.82,69.28,66.79,62.17,58.34,53.22,49.21,41.68,35.37,31.43,30.79,29.33,29.08,28.86,27.30,26.64,25.70,22.49,19.41,18.66,15.02,13.20.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) piperazin-1-yl ] methyl } -5- [ ethyl (oxa-4-yl) amino ] -4-methyl- [1,1' -diphenyl ] -3-carboxamide.

Yellow solid (97.1mg, 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 )δ173.29,170.40,169.63,167.33,165.78,163.53,156.49,150.22,149.37,143.29,139.97,138.97,137.54,133.66,133.08,129.97,126.83,123.40,121.98,121.21,120.25,116.62,115.61,108.04,69.27,66.78,62.05,58.29,52.99,52.64,49.20,41.68,35.38,31.40,30.75,29.33,29.24,29.02,28.83,27.26,25.68,22.47,19.41,18.64,15.01,13.15.HRMS m/z calculated 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) piperazin-1-yl ] methyl } -5- [ ethyl (oxa-4-yl) amino ] -4-methyl- [1,1' -diphenyl ] -3-carboxamide.

Yellow solid (81.2mg, 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-dioxopiperidin-3-yl) -1, 3-dioxa-2, 3-dihydro-1H-isoindol-4-yl ] oxy } undecyl) piperazin-1-yl ] methyl } -5- [ ethyl (oxa-4-yl) amino ] -4-methyl- [1,1' -diphenyl ] -3-carboxamide.

Yellow solid (119.8mg, 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 )δ171.64,170.14,168.71,167.12,165.66,165.16,158.71,156.73,150.71,149.55,145.96,142.63,139.36,136.40,136.14,135.81,135.64,133.85,132.78,125.51,123.20,122.08,120.05,118.89,117.16,115.62,109.90,106.77,77.26,69.47,67.31,58.71,58.41,52.89,49.14,45.18,41.63,36.15,31.51,30.52,29.47,29.41,29.31,29.15,28.89,27.49,26.65,25.73,22.67,19.70,18.65,14.68,12.76.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-dioxopiperidin-3-yl) -1, 3-dioxa-2, 3-dihydro-1H-isoindol-4-yl ] oxy } dodecyl) piperazin-1-yl ] methyl } -5- [ ethyl (oxa-4-yl) amino ] -4-methyl- [1,1' -diphenyl ] -3-carboxamide.

Yellow solid (48.1mg, 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＝7.7Hz,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] ⁺ :1012.5906,found:1012.5910。

Example 3

This example provides the synthesis of 9 bifunctional compounds S4-S12 and their associated chemical data. The synthetic route of S4-S12 is as follows:

the preparation process comprises the following steps:

the first step is as follows: s1 (4 mmol) and S2 (4.4 mmol) were dissolved in DMSO (10 mL), HOAT (0.55g, 1.5 mmol) and EDCI (0.84g, 2.2mmol) were added, and the reaction mixture was stirred at 45 ℃ for 20h. After completion of the reaction monitored by TLC, the reaction solution was poured into ice water (100 mL), stirred for 30min to precipitate, filtered, washed with water, dried, dissolved in a mixture of methanol and chloroform (10. 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 )δ168.51,163.56,151.17,150.82,140.70,140.36,133.52,128.40,125.01,120.94,118.74,114.96,67.28,58.57,41.39,35.99,30.35,27.42,24.97,22.31,22.22,16.72,14.78,12.79。

The second step is that: s3 (2.5mmol, 1.0eq) and borate (3mmol, 1.2eq) were dissolved in a mixed solution of 1, 4-dioxane and water (4 ₂ CO ₃ (3.75mmol,0.52g)，Pd(dppf)Cl ₂ (0.2mmol, 146mg), reacting at 100 ℃ for 8h under the protection of nitrogen, and cooling to room temperature. The reaction solution was distilled under reduced pressure to remove the solvent, and then dissolved in ethyl acetate, and filtered with celite. Extracting the filtrate, drying with anhydrous sodium sulfate, concentrating, mixing with sample, and purifying by silica gel column chromatography to obtain the product. Without further purification, the reaction mixture was added directly to 25mL of a trifluoroacetic acid/dichloromethane solution (10%) and reacted at room temperature for 1h, then the solvent was distilled off under reduced pressure, and the residue was neutralized to pH =8 with a saturated sodium carbonate solution (2M), and a precipitate was precipitated, suction filtered, washed with water, and dried to obtain 2g of S4 (1.04g, 74%) as a product. 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, 2.0 eq) and 1c-1k (0.3 mmol,1.2 eq) were dissolved in DMF (5 mL) and reacted at 85 ℃ for 5h. After the reaction was completed, after ethyl acetate extraction, na ₂ SO ₄ Drying, distilling under reduced pressure to remove the solvent, and separating by silica gel column chromatography to obtain the corresponding product S6-S11.

S6:5- (6- (4- (6- ((2- (2, 6-pyridodin-3-yl) -1, 3-dioxaisoindol-4-yl) oxa) cyclohexyl) piperazin-1-yl) pyridin-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 (48.1mg, 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-pyridinedion-3-yl) -1, 3-dioxaisoindol-4-yl) oxa) cyclohexyl) piperazin-1-yl) pyridin-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 (48.1mg, 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-pyridodin-3-yl) -1, 3-dioxaisoindol-4-yl) oxa) cyclohexyl) piperazin-1-yl) pyridin-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 (83mg, 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-pyridinedion-3-yl) -1, 3-dioxaisoindol-4-yl) oxa) cyclohexyl) piperazin-1-yl) pyridin-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 (48.1mg, 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-pyridinedion-3-yl) -1, 3-dioxaisoindol-4-yl) oxa) cyclohexyl) piperazin-1-yl) pyridin-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 (58.1mg, 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-pyridodin-3-yl) -1, 3-dioxaisoindol-4-yl) oxa) cyclohexyl) piperazin-1-yl) pyridin-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 (91mg, 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) piperazin-1-yl ] methyl } -5- [ ethyl (oxa-4-yl) amino ] -4-methyl- [1,1' -diphenyl ] -3-carboxamide.

Tan solid (42.3mg, 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＝11.1Hz,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,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) -butan-2-yl ] -N- [ (4, 6-dimethyl-2-oxa-1, 2-dihydropyridin-3-yl) methyl ] -6- {6- [4- (5- { [2- (2, 6-dicarbonyl piperidin-3-yl) -1, 3-dioxa-2, 3-hydro-1H-isoindol-4-yl ] thioether } 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-dioxaisoindolin-4-yl) amino) butanoyl) piperazin-1-yl) pyridin-3-yl) -3-methyl-1H-indole-4-carboxamide.

Light 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 assay

1. Laboratory instruments and materials

The instrument used in the biological experiment in the embodiment of the invention is as follows, a super clean bench BHC-1000IIA/B3: sujingtai Biotechnology Inc.; thermostated water bath Poly science 9505: polyscience, inc.; and (3) sterilizing pan MLS-3780: SANYO corporation; oven: binder corporation; ultrapure water Milli-Q Integral 10: millipore Corp; microplate reader Multiscan MK3, cell incubator, low speed centrifuge Sorvall ST1: thermofisiher corporation; flow cytometry: BD company; pH meter ORION STAR a211: thermoscientific, inc.; ultrasonication Instrument Sonic Materials Inc: danbury corporation; a constant-temperature shaking table Thermolyne at 37 ℃, a small vertical electrophoresis tank Mini-PROTEAN 3, a membrane transferring tank Mini Trans-blot: bio-Rad company; radiographic cassette AX-II: guangdong Yuehua medical instruments Co., ltd; full-automatic X-ray film washing machine HQ-320XT: huqiu image Co., ltd; slice baking machine Leica RM 2125: leica corporation; ordinary optical microscope and inverted microscope: olympus corporation; fluorescence inverted phase contrast microscope: carl Zeiss Corp; fluorescence positive phase contrast microscope: leica DM2500 (lycra) and Carl Zeiss (Carl Zeiss); vernier caliper (0-150 mm): shanghai Shenhan measuring tools, inc.

The cell line used in the present embodiment was purchased from ATCC (American Type Culture Collection). 6, 24, 96-well plates for cell culture, 15mL, 50mL centrifuge tubes, 25cm ² Culture square bottle and 75cm ² Culture flasks were purchased from Doudidin. 10mL dishes were purchased from WHB. Matrigel (Matrigel) was purchased from BD company. Dimethyl sulfoxide (DMSO), MTT, SDS, polysorbate Tween-20, sodium dodecyl sulfate SDS, glycine, tris Tris hydroxymethyl aminomethane, PEG-400 were purchased from Sigma. Ammonium persulfate APS, sodium hydroxide, ammonium persulfate, concentrated hydrochloric acid, isopropanol, methanol and other common analytical purification reagents are available from Corlon chemical company. Physiological saline was purchased from Koran pharmaceuticals, inc. Annexin V-FITC/PI kit, PI reagent, crystal violet dye, RIPA cell lysis buffer (strong) and acrylamide buffer, hematoxylin dye solution are purchased from Biyuntian biotechnology company. N, N, N ', N' -tetramethylethylenediamine TEMED, G250 protein quantification solution was purchased from Bio-Rad. PVDF membrane and chromogenic luminescent substrates were purchased from Millipore. Skimmed milk powder was purchased from illite dairy; self-developed films were purchased from Kodak corporation. 10% common reagents such as APS, 1mol/L Tris-HCl (pH: 6.8), 1.5mol/L Tris-HCl (pH: 8.8), and electrophoresis buffer, TBS buffer, TBST buffer, etc. were prepared in this experiment. All antibodies were purchased from Cell signaling Technology (Beverly, MA). The blocking goat serum, rabbit serum and DAB color development kit were purchased from Kyoto China fir Jinqiao, beijing. The TUNEL detection kit was purchased from Promega corporation (Roche Applied Science).

First, the product compounds of examples 1 to 5 and their positive controls GSK126 and EPZ6438 were tested for EZH2 enzyme inhibitory activity using AlphaScreen technology. The results are shown in Table 1.

TABLE 1

Note: each compound was tested 2 times and the numerical values in the table represent the mean.

As can be seen from the results in Table 1, the inhibitory activities of G4-G12 and E4-E12 on the enzyme EZH2 are all at the nanomolar level, wherein the inhibitory activities of G4-G8 and E4-E8 on EZH2 with n being 2-6 are all equivalent to the inhibitory activities of the respective positive controls GSK126 and EPZ6438, and when n is 7-10, the inhibitory activities of G9-G12 and E9-E12 on EZH2 are respectively reduced by about 6-60 times and 4-14 times compared with the positive controls. In general, most molecules can keep the EZH2 enzyme inhibition activity of the EZH2 inhibitor, and the E series keeps better activity than the G series; of all the molecules synthesized, E7 had the best inhibitory activity (IC) against the EZH2 enzyme ₅₀ =2.7 nM), E7 activity for EZH2 enzyme inhibition is better than positive control.

2. PRC2 protein complex subunit degrading ability

Protein levels of EZH2, SUZ12, EED, rbAp48 and histone H3K27me3 in WSU-DLCL-2 cells were first detected by Western blotting. WSU-DLCL-2 cells were incubated with 1. Mu.M test compound for 48h, respectively, with equal amounts of EPZ6438, GSK126, DMSO as controls, and the results are shown in FIG. 1.

The degradation effect of the synthesized two types of PRC2 degradation agents on EZH2 and other core subunits SUZ12, EED and RbAp48 of PRC2 in the DLBCL cell strain WSU-DLCL-2 is detected through an immunoblotting experiment. The results are shown in FIG. 1, and quantification of the levels of EZH2, SUZ12, EED and RbAp48 protein in WSU-DLCL-2 cells is performed 48h after 1 μ M treatment of the test compound. Quantification of H3K27me3 protein levels in WSU-DLCL-2 cells after 48H of C.1. Mu.M degradant treatment. The protein expression was quantified using Image J software and the statistics were expressed as the three mean values. + -. Standard Deviation (SD). Times.P < 0.05,. Times.P < 0.01,. Times.P < 0.001. In the G series, G4 only shows weak degradation effect on EZH2, SUZ12, EED and RbAp48 subunits, G5-G7 only shows weak degradation effect on SUZ12 and EED subunits, and G8-G12 shows obvious degradation effect on EZH2, SUZ12, EED and RbAp48 subunits; in the E series, E4 with the shortest alkyl chain has very obvious degradation effect on all core subunits EZH2, SUZ12, EED and RbAp48 of PRC2, E5 and E6 obviously degrade each core subunit of PRC2 less than E4, although the middle alkyl chain is extended to 7 carbon atoms (E7) and shows strong degradation effect on each subunit of PRC2, the degradation capability of E8-E12 on each core subunit of PRC2 is rather gradually weakened along with the continuous extension of the middle alkyl chain; while the same dose of EZH2 enzyme inhibitors GSK126 and EPZ6438 did not change the protein level of each core subunit of PRC2 in the cell almost completely under the same conditions.

In addition, good degradation effects for PRC2 subunits were exhibited for both types of degradation molecules represented by M5, K7, which are linked to an alkyl chain via a nitrogen atom as well as a sulfur atom, but no degradation effects were exhibited for PRC2 subunits for degradation molecules via a carbon alkyl chain, which are otherwise represented by N4. In the case where the linker of the carbon alkyl chain type represented by N4 or the ligands at both ends are different, the effect is inferior to that of the compounds of examples 4 and 5.

The inhibitory effect of the synthesized PRC2 degrading agents on the activity of EZH2 enzyme in WSU-DLCL-2 cells is evaluated by detecting the level of H3K27me3 in the cells. As shown in FIG. 3, H3K27me3 can be almost inhibited to different degrees after 1 μ M of test molecules acts on WSU-DLCL-2 cells for 48H, and the trend of the reduction of the H3K27me3 protein level is generally consistent with the trend of PRC2 subunit degradation, wherein the inhibition rate of G8-G12, E4 and E7-E11 on H3K27me3 is more than 60%, and the test molecules have relatively strong inhibition activity. In general, almost all of the two types of PRC2 degradation molecules targeting EZH2 can degrade each core subunit of PRC2 and inhibit H3K27me3, but their PRC2 degradation ability and H3K27me3 inhibition ability are different due to the difference in Linker alkyl chain length. The PROTAC E7 not only shows the best in vitro EZH2 enzyme inhibition activity, but also shows the best activities of degrading PRC2 core subunit (degradation rate: EZH2 72%, SUZ 12%, EED 81%, rbAp 48%) and inhibiting H3K27me3 (inhibition rate 86%), therefore, the compound E7 is selected to continue the subsequent chemical and biological research.

3. E7 efficient degradation PCR2

The time-and dose-effect relationship of E7-induced PRC2 degradation was evaluated in WSU-DLCL-2 cells. In the aspect of aging, 1 mu M of E7 is used for observing the degradation of each core subunit of PRC2 and the inhibition of H3K27me3 in different time periods, and the result shows that the degradation of E7 to EZH2 is gradually enhanced along with the prolonging of the action time within 0-1H, but the E7 has little influence on other subunits of PRC2 and H3K27me 3; in 2-12h, the degradation of each subunit of PRC2 by E7 disappears, and the inhibition of H3K27me3 is weak and unstable. It is therefore speculated that E7 mainly degrades free EZH2 not involved in the formation of the PRC2 complex during the initial phase of action (0-12 h), whereas it takes longer to induce the degradation of EZH2 and other subunits of the PRC2 complex to form a complete ternary complex. After 24H of E7 action, protein levels of PRC2 core subunits EZH2, SUZ12, EED and RbAp48 and its catalytic product H3K27me3 began to decrease significantly, and after that, as the action time was prolonged, the degradation of PRC2 subunits by E7 and the inhibition of H3K27me3 were gradually enhanced until almost complete degradation of PRC2 was achieved by action for 96H. In the aspect of dose effect, the degradation of each core subunit of PRC2 and the inhibition of H3K27me3 by the action of E7 with different concentrations for 48H are examined. As shown in B in FIG. 4, low concentrations of E7 (0-0.5. Mu.M) had only a weak degradation effect on each subunit of PRC 2; when the concentration reaches 1 mu M, E7 can remarkably degrade each subunit of PRC2 and inhibit H3K27me3, and the remarkable effect can be continuously maintained when the action concentration is increased to 5 mu M; however, when the concentration of E7 is too high and reaches 10 μ M, a "hook effect" (hook effect) is generated, namely, the excessive E7 causes the E7 to form binary complexes with EZH2 and E3 ubiquitin ligase respectively, and the effective action concentration of the E7 participating in the formation of EZH2-E7-E3 ubiquitin ligase ternary complexes is reduced, which is shown as the reduction of the degradation of each core subunit of PRC2 and the inhibition of H3K27me3. From the above results it follows: the 1 mu M E7 acting on WSU-DLCL-2 cells 48H can obviously and stably degrade the EZH2, SUZ12, EED and RbAp48 subunits of PRC2 and effectively inhibit H3K27me3.

In addition, mRNA levels of EZH2, SUZ12, EED and RbAp48 in WSU-DLCL-2 cells treated with 1. Mu.M E7 for 48h were examined using a fluorescent real-time quantitative PCR (RT-qPCR) experiment to determine that the decrease in expression was a result of E7 functioning at the protein level rather than the gene level. As shown by C in fig. 4, E7 hardly changed the mRNA levels of EZH2, SUZ12, EED and RbAp48 subunits of PRC2, as did the sole EZH2 methyltransferase inhibitors EPZ6438 and GSK126, indicating that E7 did not affect transcription of genes expressing these proteins, but rather acted at the translational or post-translational stages of these proteins.

The ability of E7 to degrade PRC2 in several other tumor cell lines driven by the dysfunction of EZH2 was further examined to exclude the specific effect of E7 on DLBCL cell line WSU-DLCL-2. Results as shown in D in fig. 4,1 μ M of E7 acting 48H in DLBCL (WSU-DLCL-2, pfeiffer), PCa (LNCaP, DU 145) and ovarian cancer (a 2780, SKOV 3) cells all significantly degraded the core subunits EZH2, SUZ12, EED and RbAp48 of PRC2 and effectively reduced the levels of the catalytic product H3K27me3, indicating that the effects of E7 in degrading various subunits of PRC2 and inhibiting H3K27me3 can be exerted in a variety of tumor types driven by EZH2 dysfunction, not just in DLBCL. The current EZH2 enzyme inhibitors exhibit good inhibitory activity in clinical studies mainly against a few types of tumors such as lymphoma and sarcoma, thus also suggesting that E7 may be able to play a role in more types of tumor cells than most EZH2 enzyme inhibitors.

4. E7 degradation of PRC2 by binding to EZH2

The cell thermal drift experiment (CETSA) can judge the binding condition of a drug and a protein by detecting the change of protein thermal stability caused by the drug in cells, and the principle is as follows: binding of a drug to the corresponding protein within the cell increases the structural stability of the protein, allowing the protein to withstand higher temperatures without degradation. The binding of E7 to each core subunit of PRC2 was therefore investigated by CETSA. Incubating E7 and WSU-DLCL-2 cells pretreated by MG-132 for a certain time, extracting cell lysate after the E7 is combined with corresponding proteins in the cells, and detecting the degradation condition of the cells of a control group and EZH2, SUZ12, EED and RbAp48 proteins in the cells treated by E7 incubated for 6min at different temperatures (45, 48, 51, 54, 57 and 60 ℃). The result is shown in a in fig. 5, the EZH2 protein in the control group is obviously degraded already when incubated at 51 ℃ for 6min, while the EZH2 protein treated by E7 is degraded to a considerable extent when heated to 57 ℃, so that E7 obviously improves the thermal stability of the EZH2 protein, which indicates that E7 is combined with EZH2 in cells; while the other proteins SUZ12, EED and RbAp48, whether treated with E7 or not, are degraded to almost the same extent at the same temperature, i.e. E7 does not change the thermostability of these proteins, indicating that E7 is not bound to these proteins. It was thus demonstrated that selective binding of E7 to the EZH2 subunit of PRC2 leads to its degradation.

The binding of E7 to EZH2 subunits was further validated by competitive binding experiments of E7 to EZH2 inhibitors and EED inhibitors. As can be seen from B in fig. 5, E7 significantly decreased the protein levels of EZH2, SUZ12, EED, rbAp48 and H3K27me3 in WSU-DLCL-2 cells, and when cells were treated with EZH2 inhibitors EPZ6438 or GSK126 simultaneously with E7, the degradation of PRC2 core subunit by E7 was attenuated, but H3K27me3 was still strongly inhibited; meanwhile, the cell treated by EED inhibitors EED226 and E7 does not affect the degradation effect of E7 on each subunit of PRC2 at all, and does not affect the inhibition of E7 on H3K27me3. EPZ6438 and GSK126 interfere with the degradation of PRC2 subunits by E7 because they both compete with E7 for binding to the SAM binding pocket of EZH2, blocking the binding of E7 to EZH2, resulting in a reduced concentration of the actual effect of E7; EPZ6438 and GSK126 do not influence the inhibition effect on H3K27me3 because they have the activity of inhibiting H3K27me3 as an EZH2 methyltransferase inhibitor, so that they occupy the binding site of E7 and also play the role of inhibiting H3K27me3, and can still maintain strong inhibition on H3K27me3. The binding site of EED226 is the H3K27me3 binding pocket of EED, and its binding with EED does not occupy the binding site of E7, so it does not affect the degradation of PRC2 subunit by E7 and the inhibition of H3K27me3. This indirectly demonstrates that E7 binds to the EZH2 subunit of PRC2.

The three-dimensional structure of the PRC2 complex is now being resolved, and thus the method that most directly and unambiguously demonstrates the binding between the E7 and each subunit of PRC2 is characterized by the eutectic structure of E7 and PRC2. We will therefore follow to further confirm the binding of E7 to the EZH2 subunit of PRC2 by way of E7 forming a co-crystal with PRC2.

Detecting the methylation modification level of other Lys sites of histone H3 in WSU-DLCL-2 cells,to investigate the selective inhibition effect of E7 on EZH2 HMTase activity, C in fig. 5 shows that E7 only selectively inhibits H3K27me3 and H3K27me2 catalyzed by EZH2, while it has almost no effect on other HMTase catalytic products H3K27me1, H3K9me3 and H3K4me3, indicating that E7 selectively inhibits EZH2 HMTase activity; furthermore, the in vitro enzyme inhibitory activity of E7 against EZH1, which is highly homologous to EZH2, was examined to show that E7 has inhibitory activity against EZH2 (IC) ₅₀ =2.7 nM) inhibitory activity (IC) against EZH1 ₅₀ =180 nM) was 66 times stronger, it was found that E7 had a highly selective inhibitory effect on EZH2 HMTase activity. This also provides additional proof that E7 functions by targeting EZH 2.

5. E7 degradation of PRC2 by ubiquitin proteasome pathway

According to the action principle of PROTACs, E7 induces degradation of other subunits of EZH2 and PRC2 on the premise that ubiquitin molecules are recruited to target proteins, and the degradation of polyubiquitinated proteins can be realized only by using the identification and degradation effect of UPS. The ubiquitination modification of EZH2, SUZ12 and EED subunits in E7-treated WSU-DLCL-2 cells was thus examined by immunoprecipitation experiments (IP). As shown in FIG. 6, the ubiquitination levels of the E7-treated EZH2 (A in FIG. 6), SUZ12 (B in FIG. 6) and EED (C in FIG. 6) subunits were all significantly higher than those of the protein subunits in the control group, indicating that the E7 action leads to undifferentiated ubiquitination modification of the EZH2, SUZ12 and EED subunits in the cells.

Furthermore, by interfering with the function of UPS members, it was reverse demonstrated that E7 degrades various subunits of PRC2 via the ubiquitin proteasome pathway. Lenalidomide, MLN4924 and MG-132 were used in this experiment to individually verify whether inhibition of ubiquitination modification of target proteins or inhibition of proteasome activity could destroy the degradation of E7, to further determine the pathway of E7 degradation of PRC2. Lenalidomide is a CRBN ligand with a structure very similar to that of thalidomide, and can compete with E7 for binding to E3 ubiquitin ligase to hinder the formation of an EZH2-E7-E3 ubiquitin ligase ternary complex, as shown in D of fig. 6, WSU-DLCL-2 cells are pretreated with lenalidomide before the cells are treated with E7, which results in weakening the degradation of EZH2, SUZ12, EED and RbAp48 subunits of PRC2 by E7; MLN4924 can inhibit ubiquitination modification of target protein by specifically targeting NEDD Activating Enzyme (NAE), so pretreatment of WSU-DLCL-2 cells with MLN4924 can also obviously inhibit degradation of each subunit of PRC2 by E7. MG-132 is a proteasome inhibitor, and D in FIG. 6 shows that pre-inhibition of the activity of the proteasome in WSU-DLCL-2 cells by MG-132 also stabilizes the PRC2 subunits against E7 degradation. These results indicate that inhibition of the ubiquitination modification process in UPS or the activity of proteasome can effectively interfere with the degradation of E7 to each protein subunit of PRC2, indicating that E7 can mediate indiscriminate ubiquitination of each subunit of PRC2, and achieve the degradation of each subunit of PRC2.

6. E7 Regulation of transcription of EZH2 downstream genes

After determining that E7 can effectively degrade EZH2 and other core subunits of PRC2 through ubiquitin proteasome pathway by binding to EZH2, the influence of E7 on the oncogenic function of EZH2 is further examined. Current research has found that EZH2 plays two major roles in driving tumorigenesis and progression: the transcription factor is used as a transcription inhibitor to mediate the transcription silencing of downstream genes in a mode depending on the catalytic activity of methyltransferase; the other is as a transcription co-activator, which mediates the transcriptional activation of downstream target genes in a manner independent of the activity of methyltransferase. In view of the fact that EZH2 regulates the transcription of different genes in different tumor types in completely different roles to drive the progress of tumors, the embodiment of the invention respectively considers the influence of E7 on the regulation of the transcription of multiple genes by EZH2 in different types of tumor cell lines.

7. Catalytic function mediated transcriptional silencing of E7 activated EZH2

In some DLBCL, EZH2 mutations cause abnormally high levels of methylation modification of histone H3K27, leading to high chromatin structure condensation, leading to transcriptional silencing of downstream cancer suppressor genes that trigger tumorigenesis. Therefore, we first performed WSU-DLCL-2 (EZH 2) on two strains of EZH 2-mutated DLBCL cells ^Y641F ) And Pfeiffer (EZH 2) ^A677G ) The regulation of E7 on several genes enriched on H3K27me3, ADRB2, CDKN2A, TXINP and TNFRSF21, was examined. The RT-qPCR results for A and B in FIG. 7 show that WSU-DLCL-2 and Pfeiffer cells were treated with E7 for 48hmRNA levels of all genes tested were almost significantly upregulated, and E7 upregulated the ADRB2 and TNFRSF21 genes more strongly than the EZH2 enzyme inhibitors EPZ6438 and GSK126, respectively, but slightly less strongly than EPZ6438 and GSK126 for CDKN2A and TXINP. In conclusion, in the DLBCL cell strains WSU-DLCL-2 and Pfeiffer with the EZH2 mutation, E7 can effectively activate the transcriptional silencing mediated by the catalytic function of the EZH2 and inhibit the catalytic activity of the EZH 2. In some SWI/SNF mutated tumors, EZH2 has obvious promotion effect on tumor cell proliferation, and the promotion effect depends on the catalytic function of EZH2 in PRC2, so we also tested the regulation of E7 on the transcription of the above genes in NSCLC cell strain A549 cells with SWI/SNF mutations. Results as 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 expression of ADRB2 and TNFRSF 21. The reason why the E7 up-regulates these genes in A549 is not as obvious as in WSU-DLCL-2 and Pfeiffer is that the catalytic activity of EZH2 does not play a dominant role in the proliferation of SWI/SNF mutated tumor cells such as A549, and the non-catalytic activity is the main cause of the proliferation of such tumor cells, so that in A549 cells, the transcription of genes such as ADRB2, CDKN2A and the like is not inhibited by the catalytic activity of EZH2, so that the expression of these genes is not affected even if E7 inhibits the catalytic activity of EZH 2. The results can prove that E7 effectively inhibits the catalytic activity of EZH2 and the transcriptional silencing mediated by the EZH2 by degrading EZH2 and other core subunits of PRC2.

8. E7 inhibition of non-catalytic function-mediated transcriptional activation of EZH2

The effect of E7 on the transcription of several downstream genes ARL6IP, BRIC5, CENPK, CEP76, CHEK1 and TACC3 activated by the non-catalytic activity of EZH2 was examined in tumor cells A549, NCI-H1299 and MDA-MB-468, driven by the non-catalytic function of EZH 2. As shown in fig. 8, in these cells, E7 treatment for 48h resulted in a more significant down-regulation of the EZH 2-activated genes, whereas EZH2 methyltransferase inhibitors EPZ6438 and GSK126 had little effect on the expression of these genes. The above results demonstrate that E7 effectively inhibits the non-catalytic activity of EZH2 and its abnormal activation of transcription of downstream genes by degrading EZH 2.

The results show that, no matter how EZH2 drives tumorigenesis and development, procac E7 can finally effectively inhibit EZH2 driving action in tumorigenesis and development by degrading EZH2 and other core subunits of PRC2, or inhibiting gene silencing mediated by EZH2 as a transcription inhibitor, or inhibiting abnormal transcriptional activation mediated by EZH2 as a transcription coactivator. That is, E7 can destroy not only the catalytic function of EZH2 dependent on PRC2 but also the non-catalytic function thereof independent of PRC2, so that the carcinogenic activity of EZH2 can be completely suppressed in a comprehensive manner. Therefore, for EZH 2-driven tumors, especially tumors driven by the non-catalytic function, this strategy of inducing EZH2 degradation should have better inhibitory effect than simply inhibiting EZH2 methyltransferase activity, so we next preliminarily evaluated and compared the activity difference of E7 and EZH2 enzyme inhibitors EPZ6438 and GSK126 in inhibiting EZH 2-driven tumor cell proliferation.

9. E7 inhibition of proliferation of EZH 2-aberrant tumor cells

The growth inhibition of E7 and EPZ6438, GSK126 on several EZH 2-driven tumor cells was monitored by cell counting and observation. As shown in A in FIG. 9, E7 almost completely inhibited the growth of WSU-DLCL-2 cells, exhibiting good proliferation inhibitory activity; although EPZ6438 and GSK126 both show certain proliferation inhibition activity on WSU-DLCL-2 cells, the inhibition degree of the EPZ6438 and the GSK126 is much lower than that of E7, and therefore the E7 can effectively inhibit the growth of tumor cells driven by the catalytic function of EZH 2. The proliferation activity of SWI/SNF mutated A549 and NCI-H1299 cells is mainly dependent on the non-catalytic function of EZH2 in PRC2, so that the proliferation inhibition of E7, EPZ6438 and GSK126 on the two cell strains is also examined. The results are shown as B and C in FIG. 9, and similar to those observed in WSU-DLCL-2 cells, E7 has very significant proliferation inhibitory activity against both A549 and NCI-H1299 cell lines; although GSK126 can also obviously inhibit the proliferation of two cell strains, the inhibition activity is not as good as that of E7; EPZ6438 showed only very limited proliferation-inhibiting activity on both 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 EZH 2.

The MTT detection result in FIG. 10 also shows that E7 shows good time-dependent inhibition effect on the cell viability of WSU-DLCL-2, pfeiffer and A549 and NCI-H1299 tumor cells driven by the catalytic function of EZH2, and IC is IC ₅₀ IC values were all at low micromolar levels, especially for DLBCL cell line Pfeiffer, E7 action for 7 days ₅₀ It was only 0.17. Mu.M. The above results further demonstrate that E7 has a good inhibitory effect on the viability of tumor cells driven by the catalytic and non-catalytic functions of EZH 2.

In summary, the above biological experimental data show that the compounds of the present invention have a degradation effect on each subunit of the PRC2 complex, and exhibit a broader and stronger anti-tumor effect than the inhibitor.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A bifunctional compound capable of inducing PRC2 protein complex core subunit degradation, which is characterized by comprising a compound shown as any one of formulas I-III and pharmaceutically acceptable salts thereof;

wherein n in the formula I is an integer of 2 and 5-10, X is O, and Y is O;

in the formula II, n is an integer of 4 and 6-9, X is O, and Y is O;

in the formula III, n is an integer of 2 and 6-10, X is O or S, and Y is O.

2. The bifunctional compound of claim 1, wherein X and Y in formulas I-III are both O.

3. The bifunctional compound of claim 1, wherein the bifunctional compound has the formula

4. A process for the preparation of a bifunctional compound as claimed in claim 1 wherein, in formula III, where X is S and Y is O, the synthesis route of the compound of formula III is:

the synthetic route for the compounds of formula II is:

the synthetic route for the compound of formula III is:

5. a pharmaceutical composition comprising a pharmaceutically acceptable auxiliary ingredient and a bifunctional compound as defined in any one of claims 1 to 3.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is an aqueous solution, a powder, a granule, a tablet, or a lyophilized powder.

7. The pharmaceutical composition of claim 6, wherein when the pharmaceutical composition is an aqueous solution, the pharmaceutical composition further comprises water for injection, a saline solution, an aqueous glucose solution, or a Grignard solution;

the saline solution comprises saline for injection or infusion, the aqueous glucose solution comprises glucose for injection or infusion, and the Grignard solution comprises a Grignard solution comprising lactate.

8. Use of a bifunctional compound according to any one of claims 1 to 3 or a pharmaceutical composition according to any one of claims 5 to 7 for the preparation of a kinase inhibitor.

9. Use of a bifunctional compound according to any one of claims 1 to 3 or a pharmaceutical composition according to any one of claims 5 to 7 for the preparation of a medicament for the treatment of a tumor.

10. The use of claim 9, wherein the tumor comprises breast cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, or gastric cancer.

11. Use of a bifunctional compound according to any one of claims 1 to 3 or a pharmaceutical composition according to any one of claims 5 to 7 for the preparation of a degradation agent for degrading a core subunit of a PRC2 protein complex.

12. The use of claim 11, wherein the core subunit of the PRC2 protein complex is a subunit of EZH1, EZH2, EED and SUZ12 that degrades the PRC2 protein complex simultaneously.

13. Use of a bifunctional compound according to any one of claims 1 to 3 or a pharmaceutical composition according to any one of claims 5 to 7 for the preparation of an oral or intravenous formulation comprising at least said bifunctional compound or pharmaceutical composition.

14. The use according to claim 13, further comprising an excipient and/or an adjuvant.