PROTACs compounds targeting degradation of bcl6 and applications thereof

Abstract

The present application relates to the technical field of antitumor drugs, and particularly relates to a kind of PROTACs compound targeted to degrade BCL6 and application thereof, and the compound is the compound shown in formula (I) or its stereoisomer, geometric isomer, tautomer, nitroxide, solvate, pharmaceutically acceptable salt, metabolite or prodrug.The PROTACs compound targeted to degrade BCL6 provided in the present application connects BCL6 protein ligand and E3 ubiquitin ligase ligand through chemical groups;The compound can be used to inhibit the proliferation, growth, migration, infiltration, clonal formation and metastasis of tumor cells, promote the apoptosis of cancer cells, promote the autophagy of tumor cells, overcome the drug resistance of chemotherapy or targeted therapy of tumor, inhibit the growth of tumor stem cells, and prolong the survival period of tumor patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antitumor drugs, in particular to a kind of PROTACs compound targeted to degrade BCL6 and application thereof. BACKGROUND

[0002] BCL6 (B-cell lymphoma 6, BCL6) is a transcriptional repressor, belonging to the BTB / POZ zinc finger transcription factor family. BCL6 was first discovered in diffuse large B-cell lymphoma and is a 95 kDa multi-domain protein. Its BTB domain is the active site region of BCL6 to exert transcriptional inhibition, responsible for recruiting BCL6 co-repressors (SMRT, B-COR and N-COR) and forming a dimeric complex, and then inhibiting the expression of genes related to cell cycle, apoptosis, differentiation and DNA damage response. Competitive binding BCL6 modulators can be designed for the BTB domain to effectively inhibit its transcriptional activity. BCL6 is highly expressed in various lymphomas (such as follicular lymphoma, diffuse large B-cell lymphoma) and various solid tumors (such as lung cancer, breast cancer and glioblastoma), and is positively correlated with the occurrence, development and poor prognosis of these tumors. Therefore, targeting BCL6 has therapeutic potential against the development of breast cancer and other malignant tumors.

[0003] Current BCL6 inhibitors competitively target the interaction between BCL6-BTB domain and its co-repressor complex to inhibit its function, which can be mainly divided into polypeptide inhibitors and small molecule inhibitors. BCL6 degraders work by competitively binding to the BCL6 BTB domain and then inducing BCL6 protein degradation, which can be divided into molecular glue degraders and PROTAC degraders. The first BCL6 polypeptide inhibitor BBD-BPI can kill various BCL6 high-expression DLBCL cell lines and Burkitt lymphoma cell lines in vitro. However, the anti-cancer activity of BBD-BPI in vitro only exists at 10.2 μM and above, and it shows no activity in vivo. The small molecule inhibitor 79-6 also inhibits the activity of BCL6 at the micromolar level. 79-6 can effectively inhibit the growth of multiple breast cancer cell lines in vitro at a high concentration of hundreds of micromoles. TMX-2164 is a new tyrosine-directed reversible covalent inhibitor, with an IC 50 at the level of single-digit micromoles per liter, but no in vivo anti-tumor activity has been reported. CCT374705 has weak ability to inhibit tumor growth in a DLBCL xenograft tumor model when administered orally. In summary, neither polypeptide nor small molecule BCL6 inhibitors have been approved for clinical treatment of tumors.

[0004] The BCL6 molecular glue degraders reported in the literature are of various types, but none of these degraders directly targets BCL6 protein in solid tumors.

[0005] Protein degradation targeting chimera (PROTAC) is a kind of compound that can induce the polyubiquitination of target protein and lead to the degradation of target protein. PROTAC is a new direction in the field of drug research and development, and this strategy greatly expands the range of potential drug targets. By mobilizing the intrinsic protein degradation system ubiquitin-proteasome system (UPS) in cells to down-regulate pathogenic target proteins, PROTAC is expected to overcome the limitations of traditional inhibitors, challenge “difficult drug” targets, and provide new therapeutic means for drug development. PROTAC degrades target proteins rather than occupies the active site of target proteins to prevent them from functioning, so there is no problem of target protein mutation drug resistance, and PROTAC has more advantages than traditional inhibitors. And PROTAC can be designed to use the specific expression of E3 in tumor cells to selectively degrade target proteins in tumors, thereby improving the selectivity and safety of drugs. At present, more than 10 PROTACs have entered the clinical trial stage worldwide, among which ARV-471 targeting ER is used for the treatment of ER+ breast cancer and has entered the III phase clinical trial (NCT05654623).

[0006] In summary, various solid tumors such as breast cancer are of great concern due to their high incidence and mortality, and the development of new targeted drugs can provide new strategies for the treatment of various malignancies. BCL6, as a carcinogenic factor in various tumors, is closely related to the occurrence and development of cancer. The present application aims to provide a class of small molecule compounds and degrading agents that can target transcription factor BCL6. This class of small molecule organic compounds and degrading agents has important potential therapeutic value and market development prospects for patients with various solid tumors. SUMMARY

[0007] The purpose of the present application is to provide a class of PROTACs compounds targeting and degrading BCL6, and the use of the compounds or pharmaceutical compositions containing the compounds in the preparation of antitumor drugs.

[0008] The technical scheme of the present application is as follows: a PROTACs compound targeting and degrading BCL6, the compound is shown as formula (I):

[0009]

[0010] BL represents the BCL6 protein ligand, EL represents the E3 ubiquitin ligase ligand, and L represents the connecting group connecting BL and EL;

[0011] The BL is a structure shown as formula (II):

[0012]

[0013] X is CH or N;

[0014] R 1 selected from any one or more of the following groups: hydrogen, halogen, hydroxyl, amino, C1-C6 alkyl or C1-C6 alkoxy;

[0015] R 2 selected from any one of the following groups: hydrogen, halogen or C1-C3 alkoxy;

[0016] R 3 and R 4 are each independently selected from any one of the following groups: hydrogen, C1-C3 alkyl or C1-C3 alkoxy;

[0017] said EL is represented by any one of the following structures:

[0018]

[0019] wherein: A is selected from any one of CH2, C(O) or SO2;

[0020] B, C, D are each independently selected from any one of CH or CF;

[0021] Ar is a 6-membered aromatic or heteroaromatic ring, and is selected from any one of the following aromatic rings: benzene, pyridine or pyrimidine;

[0022] R 5 selected from any one of the following groups: hydrogen, methyl, halogen or methoxy;

[0023] R 6 selected from any one of the following groups: hydrogen, alkyl or alkoxy;

[0024] said L is selected from any one of the following groups:

[0025] .

[0026] Preferably, said EL is selected from any one of the following structures:

[0027] .

[0028] Preferably, said L is selected from any one of the following structures:

[0029] .

[0030] wherein: m is an integer between 0 and 10;

[0031] n is an integer between 1 and 10;

[0032] a and b are each independently selected from an integer between 0 and 5.

[0033] As a preference, the PROTACs class of compounds comprises one or more of the following compounds:

[0034] 2-(2,6-dioxopiperidin-3-yl)-4-((2-(3-(4-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3- oxopropoxy)ethyl)amino)isoindoline-1,3-dione;

[0035] 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-((2-(3-(4-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3- oxopropoxy)ethyl)amino)isoindoline-1,3-dione;

[0036] 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3- oxopropoxy)ethyl)amino)isoindoline-1,3-dione;

[0037] 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3- oxopropoxy)ethyl)amino)isoindoline-1,3-dione;

[0038] 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3- oxopropoxy)ethyl)amino)isoindoline-1,3-dione;

[0039] 2-(2,6-dioxopiperidin-3-yl)-4-(4-(3-(4-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3- oxopropoxy)ethyl)amino)isoindoline-1,3-dione;

[0040] 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(4-(5-fluoro-4-((2H -1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)-4-oxobutyl)piperidin- 1 -yl)isoindoline- 1,3 -dione;

[0041] 2-(2,6-dioxopiperidin-3-yl)-5-(4-(4-(4-(5-fluoro-4-((2 H -1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)-4-oxobutyl)piperidin- 1 -yl)isoindoline- 1,3 -dione;

[0042] 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2 H -1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)-2-oxoethyl)piperazin- 1 -yl)isoindoline- 1,3 -dione;

[0043] 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(5-fluoro-4-((2 H -1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)-2-oxoethyl)piperazin- 1 -yl)isoindoline- 1,3 -dione;

[0044] 2-(1 -(2-(2,6-dioxopiperidin-3-yl)- 1,3 -dioxoisoindolin-4-yl)piperidin-4-yl)- N -(1 -(5-fluoro-4-((2 H -1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)- N -methylacetamide;

[0045] 2-(2,6-dioxopiperidin-3-yl)-4-(4-((4-(5-fluoro-4-((2 H -1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)methyl)piperidin- 1 -yl)isoindoline- 1,3 -dione;

[0046] 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(5-fluoro-4-((2 H -1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)piperidin- 1 -yl)isoindoline- 1,3 -dione;

[0047] 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2 H- 1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)ethyl)piperidin- 1 -yl)isoindoline- 1,3 -dione;

[0048] 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(5-fluoro-4-((2 H - 1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)ethyl)piperidin- 1 -yl)isoindoline- 1,3 -dione;

[0049] 2-(2,6-dioxopiperidin-3-yl)-4-(4-(3-(4-(5-fluoro-4-((2 H - 1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)propyl)piperidin- 1 -yl)isoindoline- 1,3 -dione;

[0050] 2-(2,6-dioxopiperidin-3-yl)-4-(2-(4-(5-fluoro-4-((2 H - 1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)-7-azaspiro[3.5]nonan-7-yl)isoindoline- 1,3 -dione;

[0051] 2-(2,6-dioxopiperidin-3-yl)-4-(3-((4-(5-fluoro-4-((2 H - 1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)methyl)piperidin- 1 -yl)isoindoline- 1,3 -dione;

[0052] 2-(2,6-dioxopiperidin-3-yl)-4-(3-(2-(4-(5-fluoro-4-((2 H - 1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)ethyl)piperidin- 1 -yl)isoindoline- 1,3 -dione;

[0053] 2-(2,6-dioxopiperidin-3-yl)-4-(3-((4-(5-fluoro-4-((2 H - 1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)methyl)pyrrolidin- 1 -yl)isoindoline- 1,3 -dione;

[0054] 2-(2,6-dioxopiperidin-3-yl)-4-(3-(2-(4-(5-fluoro-4-((2 H - 1 -benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin- 1 -yl)ethyl)pyrrolidin- 1 -yl)isoindoline- 1,3 -dione;

[0055] 2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-((1-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)(methyl)amino)ethyl)piperidin-1-yl)isoindoline-1,3-dione;

[0056] 2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-((1-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)(methyl)amino)ethyl)piperidin-1-yl)isoindoline-1,3-dione;

[0057] 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-((1-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)(methyl)amino)ethyl)piperidin-1-yl)isoindoline-1,3-dione;

[0058] 4-(4-(2-(4-(5-chloro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)(methyl)amino)ethyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione;

[0059] 2-(2,6-dioxopiperidin-3-yl)-4-[4-((4-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)(methyl)amino)ethyl)piperidin-1-yl]isoindoline-1,3-dione;

[0060] 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)-[1,4'-bipiperidin]-1'-yl)isoindoline-1,3-dione;

[0061] 3-(4-(2-(4-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)(methyl)amino)ethyl)piperidin-1-yl)-2-oxoethoxy)phenyl)piperidine-2,6-dione;

[0062] 3-(4-(2-(3-(4-(5-fluoro-4-((2 H-1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethoxy)phenyl)piperidine-2,6-dione;

[0063] 3-(4-(2-(3-(4-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethoxy)phenyl)piperidine-2,6-dione;

[0064] 3-(4-(4-(2-(4-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)piperidin-1-yl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione;

[0065] 3-(4-(5-(4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)pent-1-yn-1-yl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione;

[0066] (2 S 4 R )-1-(( S )-2-(7-(4-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-7-oxohepanoylamino)-3,3-dimethylbutyryl)-4-hydroxy- N -(( S )-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide;

[0067] (2 S 4 R )-1-(( S )-2-(3-(3-(4-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)propionylamino)-3,3-dimethylbutyryl)-4-hydroxy- N -(( S )-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide;

[0068] (2 S 4 R )-1-(( S)-2-(tert-butyl)-16-(4-(5-fluoro-4-((2 H -1-phenyl-6-(pyrrolidin-1-yl)-1H-1,3-benzodiazol-2-yl)pyrimidin-2-yl)piperazin-1-yl)-4,16-dioxo-7,10,13-trioxa-3-azahexadecanoyl)-4-hydroxy- N -(( S )-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0069] The present application also provides a pharmaceutical composition containing a therapeutic dose of the aforementioned compound. “Pharmaceutical composition” means a mixture of one or more compounds described in the present application or a physiologically acceptable salt or prodrug thereof with other chemical components. The purpose of the pharmaceutical composition is to facilitate administration to the organism, facilitate absorption of the active ingredient and thereby exert biological activity.

[0070] As preferred, the pharmaceutical composition comprises the PROTACs compound targeting degradation of BCL6 or a pharmaceutically acceptable salt thereof;

[0071] and a pharmaceutically acceptable carrier;

[0072] and / or the pharmaceutical composition further comprises a second agent for preventing and / or treating cancer;

[0073] and / or the pharmaceutical composition further comprises an excipient, diluent, adjuvant, vehicle or a combination thereof;

[0074] and / or the pharmaceutical composition is formulated into an injectable fluid, aerosol, cream, gel, pill, capsule, syrup or transdermal patch.

[0075] As preferred, the second agent comprises a chemotherapeutic anticancer drug or a targeted anticancer drug;

[0076] The chemotherapeutic drug is selected from paclitaxel, cisplatin, doxorubicin, etoposide or gemcitabine;

[0077] The targeted anticancer drug is selected from BCR-ABL inhibitor imatinib, BTK inhibitor ibrutinib, BET inhibitor JQ1 or KRAS G12C inhibitor AMG510;

[0078] The present application also provides use of the compound or the pharmaceutical composition in the preparation of a BCL6 modulator.

[0079] The present application also provides use of the compound or the pharmaceutical composition in the preparation of an antitumor drug targeting degradation of BCL6.

[0080] As a further description of the above-mentioned solution: the tumor includes one or more of diffuse large B-cell lymphoma, follicular lymphoma, Burkitt's lymphoma, breast cancer, prostate cancer, glioma, mantle cell lymphoma, myeloma, ovarian cancer, pancreatic cancer, osteosarcoma, renal cancer, cervical cancer, colorectal cancer, liver cancer or lung cancer.

[0081] An effective amount of the PROTACs compound targeting degradation of BCL6 and its related analogues or pharmaceutically acceptable salts, metabolites or prodrugs thereof or the pharmaceutical composition is administered to an individual in need thereof for inhibiting proliferation, growth, migration, infiltration, clonogenicity and metastasis of tumor cells, promoting apoptosis of cancer cells, promoting autophagy of tumor cells, overcoming drug resistance of chemotherapy or targeted therapy of tumors, inhibiting growth of tumor stem cells, and / or prolonging survival of tumor patients.

[0082] Compared with the prior art, the beneficial effects of the present application include:

[0083] 1. Unlike the existing BCL6 degradation agent which mainly targets the treatment of hematological tumors represented by DLBCL, most of the compounds of the present application can effectively degrade BCL6 protein in various liquid tumor and solid tumor cells, such as the degradation activity of representative compound LY-14 (LY-708B) on BCL6 in breast cancer cells below 100 nM.

[0084] 2. The compounds of the present application can significantly inhibit the proliferation of cells including DLBCL cells and various solid tumor cells, such as the IC 50 <100 nM.

[0085] 3. The representative compound LY-14 (LY-708B) of the present application can effectively inhibit the migration process of breast cancer cells.

[0086] 4. The representative compound LY-14 (LY-708B) of the present application has obvious inhibitory effect on the growth of breast cancer cell HCC1806 xenograft model, and does not cause weight loss in mice, showing good tolerance. BRIEF DESCRIPTION OF DRAWINGS

[0087] Figure 1 The degradation activity of some compounds of the present application on BCL6 in breast cancer cell HCC1806 (A) and breast cancer cell T47D (B);

[0088] Figure 2A. The degradation of BCL6 in various tumor cells by some compounds of the present application; A is the degradation of BCL6 protein in colorectal cancer cell HT29, skin cancer cell A375, lymphoma cell SU-DHL-4 by compounds LY-3, LY-4, LY-13, LY-15 and LY-17, and the degradation of BCL6 protein in breast cancer cell HCC1806, breast cancer cell MDA-MB-468, lymphoma cell SU-DHL-4 by LY-14 (LY-708B) and negative control; B is the DC50 of LY-14 (LY-708B) in HCC1806 and MDA-MB-468 cells;

[0089] Figure 3 The effect of some compounds of the present application on the proliferation of breast cancer cell HCC1806 (A) and breast cancer cell T47D (B);

[0090] Figure 4 The effect of representative compound LY-14 (LY-708B) in the examples of the present application on the proliferation of breast cancer cells; A represents the growth inhibition curves of LY-708B on breast cancer cells HCC1806 and MDA-MB-468, and LY-708B on lymphoma cell SU-DHL4, the horizontal coordinate represents the concentration of the compound treatment, and the vertical coordinate represents the cell survival rate; B represents the growth inhibition curves of LY-708B and LY-722D on breast cancer cells HCC1806 and MDA-MB-468, C represents the growth inhibition curves and IC 50 Data;

[0091] Figure 5 Wound healing experiments (A) were performed on breast cancer cells HCC1806 and MDA-MB-468 with representative compound LY-14 (LY-708B) in the examples of the present application, and the effect on cell migration (B) was detected;

[0092] Figure 6 The effect of representative compound LY-14 (LY-708B) in the examples of the present application on the colony formation of breast cancer cells HCC1806 and MDA-MB-468, A is the colony formation experiment to evaluate the effect of compound LY-708B on the survival of breast cancer cells HCC1806 and MDA-MB-468; B is the data statistics of breast cancer cell HCC1806 and MDA-MB-468 colony formation experiment;

[0093] Figure 7Effect of representative compound LY-14 (LY-708B) in the embodiment of the present application on apoptosis of breast cancer cells HCC1806 and MDA-MB-468; A is the effect of 0 nM, 50 nM, 100 nM and 200 nM LY-708B on cell apoptosis detected by flow cytometry after treating cells for 48 hours; B is the data statistics of A; C is the effect of LY-708B on apoptosis-related proteins detected by Western Blot;

[0094] Figure 8 Effect of representative compound LY-14 (LY-708B) in the embodiment of the present application on cell cycle of breast cancer cells HCC1806 and MDA-MB-468; A is the effect of 0-200 nM LY-708B on cell cycle detected by flow cytometry after treating cells for 24 hours; B is the data statistics of A; C is the effect of LY-708B on cycle-related proteins detected by Western Blot;

[0095] Figure 9 In vivo treatment effect of representative compound LY-14 (LY-708B) in the embodiment of the present application on breast cancer cell HCC1806 xenograft tumor model; A is the administration time schedule of the tumor in mice; B is the tumor removed at the end of the tumor formation experiment in nude mice; C is the growth curve of the tumor during the tumor formation process; D is the volume of the tumor removed in the tumor formation experiment; E is the weight of the tumor removed in the tumor formation experiment; F is the level of creatinine (CRE), a liver toxicity evaluation index of mice; G is glutamic-oxalacetic transaminase (AST) of mice, a liver toxicity evaluation index; H is the level of glutathione-alanine transaminase (ALT) of mice, a liver toxicity evaluation index; I is the body weight data record of mice; J is the expression level of BCL6 protein in the transplanted tumor detected by Western Blot;

[0096] Figure 10 Binding activity evaluation of representative compound LY-14 (LY-708B) in the embodiment of the present application and BCL6 protein; A is the binding capacity of compound LY-708B and BCL6 protein detected by surface plasmon resonance (SPR); B is the binding constant of compound LY-708B and BCL6 protein detected by SPR. DETAILED DESCRIPTION

[0097] In the present application, the term "C1-C6 alkyl" refers to a straight-chain or branched alkyl group having 1 to 6 carbon atoms, which includes, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl, etc.

[0098] The term "C1-C6alkoxy" refers to a straight or branched chain alkoxy group having from 1 to 6 carbon atoms and at least one oxygen atom, including but not limited to methoxy, ethoxy, methoxymethyl, propoxy, ethoxymethyl, methoxyethyl, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, 2-methyl-2-butoxy, 3-methyl-2-butoxy, 3-methyl-1-butoxy, 2-methyl-1-butoxy, n-hexoxy, and the like.

[0099] The term "C1-C3alkoxy" refers to a straight or branched chain alkoxy group having from 1 to 3 carbon atoms and at least one oxygen atom, including but not limited to methoxy, ethoxy, methoxymethyl, propoxy, ethoxymethyl, methoxyethyl, or isopropoxy.

[0100] The term "C1-C3alkyl" refers to a straight or branched chain alkyl group having from 1 to 3 carbon atoms, including but not limited to methyl, ethyl, propyl, or isopropyl. The term "halogen" refers to fluorine, chlorine, bromine, or iodine. Preferably, the "C1-C3alkyl" is methyl and the "halogen" is fluorine or chlorine.

[0101] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present application, prepared from a compound of the present application having a particular substituent with a relatively non-toxic acid or base. When a compound of the present application contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the base in a pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, amine, organic amine, or magnesium salts or similar salts. When a compound of the present application contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the acid in a pure solution or in a suitable inert solvent. The practice of the present application includes the use of pharmaceutically acceptable acid addition salts, including mineral salts such as hydrochlorides, hydrobromides, nitrates, carbonates, bicarbonates, phosphates, monohydrogenphosphates, dihydrogenphosphates, sulfates, bisulfates, hydroiodides, hydrosulfates, and the like; and organic salts such as acetates, propionates, isobutyrates, maleates, malonates, benzoates, succinates, suberates, fumarates, lactates, mandelates, phthalates, benzenesulfonates, p-toluenesulfonates, citrates, tartrates, methanesulfonates, and the like; also salts with amino acids such as arginate, asparginate, glutamine, and the like; and salts with organic acids such as gluconate, glucoseaminoate, and the like. Certain specific compounds of the present application contain both basic and acidic functionalities and, as such, are capable of forming either base or acid addition salts. The term "pharmaceutically acceptable" means that the compound, material, composition or dosage form, or the salt thereof, is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and / or desirable, and includes a relatively broad utility.

[0102] In addition to salt forms, the compounds of the present application can exist in prodrug form. Prodrugs of the compounds of the present application are readily converted by chemical or physiological processes, in an organism or cell, into the compounds of the present application. Any compound that will be converted by a subject into a biologically active substance (i.e., a compound of Formula (I)-(V)) in vivo is a prodrug within the scope and spirit of the present application. For example, a compound containing a carboxylic acid group can form a physiologically hydrolysable ester which serves as a prodrug by being hydrolyzed in vivo to yield the compound of Formula (I)-(V) itself.

[0103] Certain compounds of the present application can possess asymmetric carbon atoms (optical centers) or double bonds. The racemates, diastereomers, geometric isomers and individual isomers are all intended to be within the scope of the present application, including cis- and trans-forms, (-)- and (+)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, as well as racemic mixtures and other mixtures thereof, all enantiomers and mixtures thereof, are included within the scope of the present application.

[0104] The term "excipient" refers generally to a carrier, diluent and / or vehicle with which an effective pharmaceutical composition is formulated.

[0105] The term "effective amount" or "therapeutically effective amount" with respect to a pharmaceutical or pharmacological agent refers to a sufficient amount of the agent to achieve the intended effect without being toxic to the recipient. For oral dosage forms of the present application, an "effective amount" of one active agent in a composition refers to the amount of the other active agent in the composition that is required to achieve the intended effect. The determination of an effective amount is dependent on the age and general condition of the recipient, as well as the particular active agent, and an effective amount for a given case can be determined by one of ordinary skill in the art with routine experimentation.

[0106] The terms "active ingredient", "therapeutic agent", "active agent" or "active substance" of the present application refer to a chemical entity that is effective in treating a target disorder, disease or condition.

[0107] When any variable (e.g., R) occurs more than one time in a compound, its definition in each occurrence is independent of the other. Thus, for example, if a group is substituted with 0-2 R groups, said group can be optionally substituted with up to two R groups, and at each occurrence R is selected independently of the other. Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0108] The application provides a kind of PROTACs compound targeted degradation BCL6 and its derivative or pharmaceutically acceptable salt, and pharmaceutical composition containing such compound.The PROTACs compound of the application can selectively induce BCL6 protein degradation.The PROTACs compound targeted degradation BCL6 is successfully prepared by using different kinds and different length of linker to connect BCL6 small molecule ligand and E3 ubiquitin ligase ligand.

[0109] The low-temperature reaction device used in the application is EYELA (PSL-1810) magnetic stirring low-temperature constant-temperature water tank; EYELA (N-1100) rotary evaporator; the purity of the obtained compound is analyzed by Agilent 1200 series LC system high-performance liquid chromatograph, and the ultraviolet detection wavelength is 254 nm and 365 nm; the structure of the compound is identified by nuclear magnetic resonance spectrometer, the nuclear magnetic resonance spectrometer is Bruker 500 type, and the solvent used is DMSO- d 6; the purification of reaction intermediates and final products is carried out by using chromatographic column (silica gel 200-300 mesh), and the silica gel is purchased from Qingdao Marine Chemical Plant. Unless otherwise specified, all reactions are tracked by TLC.

[0110] In the drawings of the application, one-way analysis of variance is used to determine p the value, * represents p <0.05, ** represents p <0.01, *** represents p <0.001, **** represents p <0.0001, ns represents no significant difference, and the data is shown as mean ± standard deviation.

[0111] Preparation of the compound of the application in example 1

[0112] Preparation of key intermediate BCL6 ligand 6-((5-fluoro-2-(piperazin-1-yl)pyrimidin-4-yl)amino)-2 H -1-benzopyran-2-one (BL1):

[0113]

[0114] 6-amino-2 H -benzopyran-2-one (3.0 g, 18.62 mmol) and 2,4-dichloro-5-fluoropyrimidine (6.2 g, 37.23 mmol) were dissolved in ethanol (EtOH, 30 mL), and N , N-Diisopropylethylamine (DIPEA, 7.2 g, 55.86 mmol) was reacted overnight at 60 °C. The solvent was then removed by vacuum distillation, followed by extraction with ethyl acetate and water. The organic phase was retained, dried over anhydrous sodium sulfate, and then distilled under reduced pressure. Column chromatography purification yielded 6-((2-chloro-5-fluoropyrimidin-4-yl)amino)-2 H -Benzopyran-2-one (5.2 g, yield 96%).

[0115] 6-((2-chloro-5-fluoropyrimidin-4-yl)amino)-2 H 1-Benzopyran-2-one (5.2 g, 17.83 mmol) and tert-butylpiperazine-1-carboxylic acid ester (3.3 g, 17.83 mmol) were dissolved in n-butanol ( n Add 5 mL of BuOH to the reaction mixture, then add DIPEA (6.9 g, 53.49 mmol) and react overnight at 120 °C. Vacuum concentration is used to remove most of the n-butanol, followed by extraction with DCM (dichloromethane) and water. The organic phases are combined and distilled under reduced pressure. The crude product obtained is purified by column chromatography to give 4-(5-fluoro-4-((2-oxo-2-)- H- Benzopyran-6-yl)amino)pyrimidin-2-yl)piperazine-1-carboxylic acid tert-butyl ester (6.0 g, yield 76.2%).

[0116] 4-(5-fluoro-4-((2-oxo-2) H- 6.0 g (13.59 mmol) of benzopyran-6-yl)amino)pyrimidin-2-yl)piperazine-1-carboxylic acid tert-butyl ester (DCM) was dissolved in 50 mL of dimethyl methacrylate (DCM), and TFA (trifluoroacetic acid, 5 mL) was added. The mixture was stirred at room temperature for 6 hours. The DCM and TFA were evaporated to dryness under reduced pressure, and the crude product was extracted with saturated sodium bicarbonate solution and DCM. The crude product was purified by column chromatography to give the key intermediate 6-((5-fluoro-2-(piperazine-1-yl)pyrimidin-4-yl)amino)-2-yl)piperazine-1-carboxylic acid tert-butyl ester. H -1-Benzopyran-2-one (BL1) (5.0 g, yield 75.4%).

[0117] The key intermediate, BCL6 ligand 4-(5-fluoro-4-((2) H Preparation of tert-butyl 1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazine-1-carboxylate (BL2):

[0118] Similar to the synthesis of the key intermediate BL1, tert-butylpiperazine-1-carboxylate was replaced with tert-butylmethyl(4-piperidinyl)carbamate, and the crude product obtained was purified by column chromatography to give compound BL2.

[0119] Key intermediate BCL6 ligand 6-((5-chloro-2-(piperazin-1-yl)pyrimidin-4-yl)amino)-2 H Preparation of 1-benzopyran-2-one (BL3):

[0120] Similar to the synthetic scheme of key intermediate BL1, 2,4-dichloro-5-fluoropyrimidine was replaced by 2,4,5-trichloropyrimidine, and the crude product thus obtained was purified by column chromatography to obtain compound BL3.

[0121] Example 1-1 2-(2,6-dioxopiperidin-3-yl)-4-((2-(3-(4-(5-fluoro-4-((2 H Preparation of 1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)amino)isoindoline-1,3-dione (LY-1):

[0122]

[0123] Dissolve 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (200 mg, 0.7241 mmol) and tert-butyl 3-(2-aminoethoxy)propanoate (177 mg, 0.8689 mmol) in DMSO (2 mL), and then add DIPEA (187 mg, 1.448 mmol) to the reaction solution. React at 90 ℃ under nitrogen protection overnight. After the reaction is completed, extract with EA and water, wash the organic phase with saturated sodium chloride solution, combine the organic phases, dry over anhydrous sodium sulfate, and distill under reduced pressure. Purify by column chromatography to obtain tert-butyl 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoate (150 mg, yield 46.5%).

[0124] Dissolve tert-butyl 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoate (54 mg, 0.1210 mmol) in DCM (3 mL), and then add TFA (1 mL). React at room temperature for 5 hours. After the reaction is completed, distill off the solvent under reduced pressure to obtain 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoic acid, which is directly used in the next step without further purification.

[0125] 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)propionic acid (0.1210 mmol) and intermediate BL1 (41 mg, 0.1210 mmol) were dissolved in DMSO (2 mL), and then HATU (60 mg, 0.1573 mmol) and DIPEA (47 mg, 0.3630 mmol) were added to the reaction solution. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was extracted with EA and water, the organic phase was collected, washed with saturated sodium chloride solution, the organic phases were combined, dried over anhydrous sodium sulfate, and distilled under reduced pressure. The crude product obtained was purified by column chromatography to obtain the target product 2-(2,6-dioxopiperidin-3-yl)-4-((2-(3-(4-(5-fluoro-4-((2 H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)amino)isoindoline-1,3-dione (LY-1). 1 H NMR (500 MHz, DMSO- d 6) δ 11.07 (s,1H), 9.52 (s,1H), 8.13 (d, J =2.6 Hz, 1H), 8.08-8.03 (m, 2H), 7.84 (dd, J =9.0, 2.6 Hz, 1H), 7.55 (dd, J =8.5, 7.1 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.12 (d, J =8.6 Hz, 1H), 7.01 (d, J =7.0 Hz, 1H), 6.56 (t, J =5.8 Hz, 1H), 6.49 (d, J =9.5 Hz, 1H), 5.02(dd, J =12.9, 5.4 Hz, 1H), 3.71 (t, J =6.5 Hz, 2H), 3.65-3.51 (m, 9H), 3.46 (q, J =5.5 Hz, 2H), 3.29-3.26 (m, 1H), 2.90-2.79 (m, 1H), 2.63 (t, J =6.5 Hz, 2H), 2.59-2.51 (m, 2H), 2.03-1.96 (m, 1H).

[0126] Example 1-2 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-((2-(3-(4-(5-fluoro-4-((2 H Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-((2-(3-(4-(5-fluoro-4-((2

[0127]

[0128] In a similar manner to the synthetic scheme of Example 1-1, 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione was replaced by 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisoindoline-1,3-dione, and the crude product thus obtained was purified by column chromatography to obtain compound LY-2. 1 H NMR (500 MHz, DMSO- d 6) δ 11.06 (s, 1H), 9.52 (d, J = 1.7 Hz, 1H), 8.12 (d, J = 2.6 Hz, 1H), 8.08-8.02 (m, 2H), 7.83 (dd, J =9.0, 2.6 Hz, 1H), 7.50 (d, J = 10.2 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H),7.17(d, J = 7.2 Hz, 1H), 6.77 (s, 1H), 6.50 (d, J = 9.5 Hz, 1H), 5.03 (dd, J =12.9, 5.4 Hz, 1H), 3.69 (t, J = 6.4 Hz, 2H), 3.60 (dd, J = 11.9, 6.4 Hz, 6H),3.52 (t, J = 5.2 Hz, 4H), 3.47-3.41 (m, 2H), 2.91-2.80 (m, 1H), 2.65-2.52 (m,4H), 2.01-1.94 (m, 1H).

[0129] Example 1-3 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(5-fluoro-4-((2 HPreparation of 1-(2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2

[0130]

[0131] Preparation of 1-(2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2 1 H NMR (500 MHz, DMSO- d 6) δ11.10 (s, 1H), 9.57 (s, 1H), 8.15-8.05 (m, 3H), 7.86 (d, J = 9.1 Hz, 1H),7.72-7.64 (m, 1H), 7.44-7.28 (m, 3H), 6.51 (d, J = 9.8 Hz, 1H), 5.09 (dd, J =13.1, 5.4 Hz, 1H), 3.85-3.50 (m, 10H), 3.06-2.81 (m, 4H), 2.66-2.54 (m, 2H),2.11-1.96 (m, 1H), 1.89-1.67 (m, 4H).

[0132] Preparation of 1-(2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2 H Preparation of 1-(2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2

[0133]

[0134] Preparation of 1-(2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2 1 H NMR (500 MHz, DMSO-d6) δ 11.08 (s, 1H), 9.54 (d, J = 1.6 Hz, 1H),8.14 (d, J = 2.6 Hz,1H),8.10-8.04 (m,2H),7.85 (dd, J= 9.1, 2.6 Hz, 1H), 7.67 (dd, J = 8.5, 7.1 Hz,1H), 7.40(d, J = 9.0 Hz, 1H), 7.32 (t, J = 8.1 Hz, 2H),6.50 (d, J = 9.5 Hz, 1H), 5.08 (dd, J =12.7, 5.5 Hz, 1H), 3.72-3.54 (m, 10H), 2.92-2.83 (m, 3H),2.63-2.52 (m, 2H),2.37 (d, J = 6.8 Hz,2H), 2.05-1.99 (m, 1H),1.97-1.90 (m, 1H),1.80 (d, J =12.5 Hz,2H), 1.48-1.37 (m,2H); HRMS (ESI): calcd for C 37 H 35 FN8NaO7[M+Na] + : 745.2505, found 745.2500.

[0135] Example 1-5 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(5-fluoro-4-((2 H Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(5-fluoro-4-((2

[0136]

[0137] Similarly to the synthetic scheme of Example 1-1, 2-(2,6-dioxopiperidin-3-yl)-4- fluoroisoindoline-1,3-dione was replaced by 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline- 1,3-dione, and tert-butyl 3-(2-aminoethoxy)propanoate was replaced by tert-butyl 2- (piperidin-4-yl)acetate. The crude product thus obtained was purified by column chromatography to obtain compound LY-5. 1 H NMR (500 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.57 (s, 1H),8.13 (d, J =2.6 Hz, 1H), 8.11-8.04 (m, 2H), 7.85 (dd, J= 9.0, 2.7 Hz, 1H), 7.64 (d, J =8.5 Hz, 1H), 7.40 (d, J = 8.9 Hz, 1H), 7.33-7.29 (m, 1H), 7.23 (dd, J = 8.7, 2.4 Hz, 1H), 6.50 (d, J = 9.5 Hz, 1H), 5.06 (dd, J = 12.8, 5.5 Hz, 1H), 4.10-4.00 (m, 2H), 3.68-3.51 (m, 8H), 2.98 (t, J = 12.6 Hz, 2H), 2.93-2.83 (m,1H), 2.65-2.55 (m, 3H), 2.33 (d, J = 6.8 Hz, 2H), 2.09-1.97 (m, 3H), 1.77 (d, J = 12.7 Hz, 2H).

[0138] Examples 1-6 2-(2,6-dioxopiperidin-3-yl)-4-(4-(3-(4-(5-fluoro-4-((2) H Preparation of LY-6 (-1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropyl)piperidin-1-yl)isoindoline-1,3-dione):

[0139]

[0140] Similar to the synthesis scheme in Example 1-1, tert-butyl 3-(2-aminoethoxy)propionate was replaced with tert-butyl 3-(piperidin-4-yl)propionate, and the crude product obtained was purified by column chromatography to give compound LY-6. 1 H NMR (500 MHz, DMSO- d 6) δ 11.08 (s, 1H), 9.54 (s, 1H), 8.14 (d, J = 2.6 Hz, 1H), 8.11-8.05(m, 2H), 7.86 (dd, J = 9.0, 2.6 Hz, 1H), 7.67 (dd, J = 8.5, 7.1 Hz, 1H), 7.40(d, J = 8.9 Hz, 1H), 7.32 (t,J = 8.1 Hz, 2H), 6.50 (d, J = 9.6 Hz, 1H), 5.08 (dd, J = 12.7, 5.5 Hz, 1H), 3.75-3.51 (m, 10H), 2.96-2.79 (m, 3H), 2.63-2.52(m, 3H), 2.42 (t, J = 7.5 Hz, 2H), 2.06-1.97 (m, 1H), 1.80 (d, J = 12.2 Hz,2H), 1.55-1.50 (m, 2H), 1.40-1.32 (m, 2H).

[0141] Example 1-7 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(4-(5-fluoro-4-((2 H -1-phenoxazin-2-ylamino)pyrimidin-2-yl)piperazin-1-yl)-4-oxobutyl)piperidin-1-yl)isoindoline-1,3-dione (LY-7) was prepared according to the similar synthetic scheme of Example 1-1, replacing 3-(2-aminoethoxy)propionic acid tert-butyl ester with 4-(piperidin-4-yl)butyric acid tert-butyl ester. The crude product thus obtained was purified by column chromatography to obtain the compound LY-7.

[0142]

[0143] Example 1-7 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(4-(5-fluoro-4-((2 1 H NMR (500 MHz, DMSO- d 6) δ 11.07 (s, 1H), 9.55 (s, 1H), 8.14 (d, J = 2.6 Hz, 1H), 8.10-8.05 (m, 2H), 7.85 (dd, J = 9.0, 2.6 Hz, 1H), 7.67 (dd, J = 8.5, 7.1 Hz, 1H), 7.40 (d, J = 8.9 Hz, 1H), 7.35-7.28 (m, 2H), 6.50 (d, J = 9.6 Hz, 1H), 5.08 (dd, J= 12.7, 5.4 Hz, 1H), 3.72-3.51 (m, 10H), 2.91-2.81 (m, 3H), 2.65-2.56 (m, 2H), 2.36 (t, J = 7.5 Hz, 2H), 2.05-1.98 (m, 1H), 1.77 (d, J = 12.4 Hz, 2H), 1.63-1.53 ​​(m, 2H), 1.44 (s, 1H), 1.38-1.26 (m, 4H).

[0144] Examples 1-8 2-(2,6-dioxopiperidin-3-yl)-5-(4-(4-(4-(5-fluoro-4-((2) H Preparation of -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-4-oxobutyl)piperidin-1-yl)isoindoline-1,3-dione (LY-8):

[0145]

[0146] Similar to the synthesis scheme in Example 1-1, 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisodihydroindole-1,3-dione was replaced with 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione, and tert-butyl 3-(2-aminoethoxy)propionate was replaced with tert-butyl 4-(piperidin-4-yl)butyrate. The crude product obtained was purified by column chromatography to give compound LY-8. 1 H NMR (500 MHz, DMSO- d 6) δ 11.07 (s, 1H), 9.54 (s, 1H), 8.14 (d, J =2.6 Hz, 1H), 8.10-8.04 (m, 2H), 7.85 (dd, J = 9.0, 2.6 Hz, 1H), 7.64 (d, J =8.5 Hz, 1H), 7.40 (d, J = 9.0 Hz, 1H), 7.30 (d, J = 2.3 Hz, 1H), 7.22 (dd, J = 8.7, 2.4 Hz, 1H), 6.50 (d, J = 9.5 Hz, 1H), 5.06 (dd, J= 12.8, 5.4 Hz,1H), 4.04 (d, J = 13.1 Hz, 2H), 3.63 (d, J = 25.9 Hz, 4H), 3.53 (d, J = 5.3Hz, 4H), 3.01-2.83 (m, 3H), 2.65-2.53 (m, 2H), 2.35 (t, J = 7.5 Hz, 2H),2.06-1.98 (m, 1H),1.75 (d, J = 12.9 Hz, 2H),1.63-1.46 (m,4H), 1.22-1.10 (m,3H)。

[0147] Example 1-9 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2 H -1-phenylpyrazol-2-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-2-oxoethyl)piperazin-1-yl)isoindoline-1,3-dione (LY-9) was prepared according to the similar synthetic scheme of Example 1-1 by replacing 3-(2-aminoethoxy)propionic acid tert-butyl ester with 2-(piperazin-1-yl)acetic acid tert-butyl ester. The crude product thus obtained was purified by column chromatography to give compound LY-9.

[0148]

[0149] Example 1-9 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2 1 H NMR (500 MHz,DMSO- d 6) δ 11.08 (s, 1H), 9.54 (s, 1H), 8.12 (d, J = 2.5 Hz, 1H), 8.10-8.03(m, 2H), 7.86 (dd, J = 9.2, 2.6 Hz, 1H), 7.73-7.65 (m, 1H), 7.40 (d, J = 9.0Hz, 1H), 7.37-7.29 (m, 2H), 6.48 (d, J = 9.6 Hz, 1H), 5.09 (dd, J= 12.9, 5.4Hz, 1H), 3.74-3.52 (m, 10H), 3.29-3.27 (m, 4H), 2.94-2.82 (m, 1H),2.71-2.55(m, 6H),2.10-1.95 (m, 1H).

[0150] Example 1-10 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(5-fluoro-4-((2 H Preparation of (2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(5-fluoro-4-((2

[0151]

[0152] In a similar manner to the synthetic scheme of Example 1-1, 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione was replaced with 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione, and tert-butyl 3-(2-aminoethoxy)propanoate was replaced with tert-butyl 2-(piperazin-1-yl)acetate, and the thus obtained crude product was purified by column chromatography to obtain Compound LY-10. 1 H NMR (500 MHz, DMSO- d 6) δ 11.07 (s, 1H),9.54 (d, J = 1.7 Hz, 1H),8.12 (d, J = 2.6 Hz, 1H),8.08 (d, J = 3.6 Hz, 1H),8.04 (d, J = 9.6 Hz, 1H),7.85 (dd, J = 9.0, 2.6 Hz, 1H),7.67 (d, J = 8.5 Hz, 1H), 7.39 (d, J = 9.0 Hz,1H),7.35-7.31 (m,1H), 7.24 (dd, J = 8.8, 2.3 Hz, 1H), 6.48 (d, J = 9.6 Hz,1H), 5.07 (dd, J = 12.8, 5.5 Hz, 1H), 3.71-3.61 (m, 6H), 3.56 (d, J= 5.7 Hz, 2H), 3.45 (s, 4H), 3.27 (s, 2H), 2.94-2.84 (m, 1H), 2.65-2.52 (m, 6H), 2.06-1.98 (m, 1H).

[0153] Examples 1-11 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)piperidin-4-yl)- N -(1-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)- N Preparation of methylacetamide (LY-11):

[0154]

[0155] Similar to the synthesis scheme in Example 1-1, tert-butyl 3-(2-aminoethoxy)propionate was replaced with tert-butyl 2-(piperidin-4-yl)acetate, and the BCL6 ligand was replaced with BL2 instead of BL1. The crude product obtained was purified by column chromatography to obtain compound LY-11. 1 H NMR (500 MHz, DMSO- d 6) δ11.08 (s,1H), 9.50 (s,1H), 8.13 (dd, J =9.4, 2.5 Hz, 1H), 8.05 (d, J = 3.6 Hz, 1H), 8.01 (d, J =9.6 Hz, 1H), 7.85 (dd, J =9.1, 2.5 Hz, 1H), 7.67 (dd, J =8.6, 7.0 Hz, 1H), 7.40 (d, J =9.0 Hz, 1H), 7.33 (dd, J =11.0, 6.1 Hz, 2H), 6.49 (dd, J =9.6, 2.1 Hz, 1H), 5.08 (dd, J =12.7, 5.5 Hz, 1H), 4.60 (d, J= 8.7 Hz, 2H), 3.68 (s, 2H), 3.00-2.92 (m, 1H),2.92-2.83 (m, 4H), 2.67-2.56 (m, 3H), 2.44-2.39 (m, 1H), 2.38-2.34 (m, 1H),2.32-2.27 (m, 1H), 2.06-1.90 (m, 3H), 1.83-1.75 (m, 2H), 1.68-1.56 (m, 2H),1.52 (d, J =11.4 Hz, 2H),1.47-1.37 (m, 2H), 1.36-1.32 (m,1H); HRMS (ESI):calcd for C 39 H 39 FN8NaO7 [M+Na] + :773.2818, found 773.2826.

[0156] Example 1-12 2-(2,6-dioxopiperidin-3-yl)-4-(4-((4-(5-fluoro-4-((2 H Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-(4-((4-(5-fluoro-4-((2

[0157]

[0158] Dissolve 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (500 mg, 1.810 mmol) and 4-hydroxymethylpiperidine (312 mg, 2.715 mmol) in 2 mL of solvent DMSO, and add DIPEA (468 mg, 3.620 mmol) to the mixed solution. Protect under nitrogen, and react at 90°C overnight. After the reaction is completed, quench with water, extract with EA and water, collect the organic phase, extract again with saturated sodium chloride solution, reserve the organic phase, dry with anhydrous sodium sulfate, distill under reduced pressure to obtain a crude product. Purify the crude product by column chromatography to obtain 2-(2,6-dioxopiperidin-3-yl)-4-(4-(hydroxymethyl)piperidin-1-yl)isoindoline-1,3-dione (677 mg).

[0159] Dissolve 2-(2,6-dioxopiperidin-3-yl)-4-(4-(hydroxymethyl)piperidin-1-yl)isoindoline-1,3-dione (677 mg, 1.823 mmol) and 4-methylbenzenesulfonyl chloride ( p- TsCl, 1043 mg, 5.469 mmol) was dissolved in DCM (3 mL), followed by the addition of triethylamine (TEA, 597 mg, 5.469 mmol) and a catalytic amount of DMAP (4-dimethylaminopyridine), and the reaction was allowed to proceed at room temperature for 5 hours. After the reaction was completed, the DCM and water were extracted, the organic phase was collected, dried over anhydrous sodium sulfate, and distilled under reduced pressure. The crude product was purified by column chromatography to obtain (1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperidin-4-yl)methyl 4-methylbenzenesulfonate (477 mg, yield 49.8%).

[0160] (1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperidin-4-yl)methyl 4-methylbenzenesulfonate (50 mg, 0.09532 mmol) and intermediate BL1 (22 mg, 0.06354 mmol) were dissolved in anhydrous N , N dimethylformamide (DMF, 2 mL), followed by the addition of DIPEA (16 mg, 0.1271 mmol). The reaction was allowed to proceed at 80°C overnight under nitrogen protection. After the reaction was completed, EA and water were extracted, and the crude product thus obtained was purified by column chromatography on silica gel to obtain the target product 2-(2,6-dioxopiperidin-3-yl)-4-(4-((4-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (LY-12). 1 H NMR (500 MHz, DMSO- d 6) δ 11.04 (d, J = 1.9 Hz, 1H), 9.50 (s, 1H), 8.13 (t, J =2.6 Hz, 1H), 8.07-8.00 (m, 2H), 7.84 (dd, J = 9.0, 2.6 Hz, 1H), 7.56 (dd, J =8.6, 6.9 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.14-7.05 (m, 2H), 6.49 (d, J= 9.6 Hz, 1H), 5.08-5.00 (m, 1H), 3.70-3.56 (m, 6H), 3.53-3.45 (m, 2H), 2.91-2.80 (m, 1H), 2.61-2.52 (m, 1H), 2.47-2.19 (m, 8H), 2.15-2.09 (m, 1H), 2.03-1.94 (m, 1H), 1.70-1.54 (m, 3H).

[0161] Example 1-13 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(5-fluoro-4-((2 H -1-phenylpyrrol-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)piperidin-1-yl)isoindoline-1,3-dione (LY-13) was prepared according to the following scheme:

[0162]

[0163] The crude product thus obtained was purified by column chromatography to obtain compound LY-13. 1 H NMR (500 MHz, DMSO- d 6) δ 11.10 (s, 1H),9.52 (s, 1H), 8.16 (d, J = 2.5 Hz, 1H), 8.08-8.02 (m, 2H), 7.85 (dd, J = 9.1,2.6 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.40 (d, J = 8.9 Hz, 1H), 7.34 (d, J =7.7 Hz, 2H), 6.49 (d, J = 9.5 Hz, 1H), 5.09 (dd, J = 12.8, 5.5 Hz, 1H), 3.75(d, J = 11.4 Hz, 2H), 3.62 (s, 4H),2.94-2.84 (m, 3H), 2.59 (s, 5H),2.07-1.96(m, 2H), 1.95-1.82 (m, 2H),1.65 (d, J = 12.5 Hz, 3H)。

[0164] Example 1-14 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2 H Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2

[0165]

[0166] In a similar manner to the synthetic scheme of Example 1-12, 4-hydroxymethylpiperidine was replaced by 4-piperidineethanol, and the crude product thus obtained was purified by column chromatography to give compound LY-14 (LY-708B). 1 H NMR (500 MHz, DMSO- d 6) δ 11.08 (s,1H),9.49 (s,1H),8.13 (d, J = 2.6 Hz,1H),8.07-8.00 (m,2H),7.85 (dd, J =9.0,2.6Hz, 1H),7.67 (dd, J =8.5,7.0 Hz, 1H),7.39 (d, J =9.0 Hz, 1H), 7.32 (d, J =7.8,5.3Hz,2H), 6.49 (d, J = 9.5 Hz, 1H), 5.08 (dd, J =12.7, 5.5 Hz,1H),3.72-3.58 (m,6H),2.94-2.81 (m, 3H),2.65-2.52 (m,2H),2.46-2.33 (m,6H), 2.07-1.96 (m, 1H),1.79 (d, J =12.3 Hz, 2H), 1.55-1.31(m, 5H);HRMS (ESI):calcd for C 37 H 38 FN8O6 [M+H] + :709.2893, found 709.2897.

[0167] Example 1-15 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(5-fluoro-4-((2 HPreparation of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(3-(4-(5-fluoro-4-(2

[0168]

[0169] In a similar manner to the synthetic scheme of Example 1-12, 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione was replaced by 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione, and 4-hydroxymethylpiperidine was replaced by 4-piperidinethanol, and the crude product thus obtained was purified by column chromatography to obtain the compound LY-15. 1 H NMR (500 MHz, DMSO- d 6) δ 11.07 (s, 1H), 9.50 (s, 1H), 8.18-8.00 (m, 3H), 7.84 (dd, J = 8.9,2.6 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.40 (dd, J = 9.0, 4.7 Hz, 1H), 7.31(s, 1H), 7.23 (d, J = 8.7 Hz, 1H), 6.49 (d, J = 9.5 Hz, 1H), 5.06 (dd, J =12.7, 5.4 Hz, 1H), 4.04 (d, J = 13.0 Hz, 2H), 3.72-3.55 (m, 4H), 3.45 (s,1H), 3.06-2.83 (m, 4H), 2.65-2.54 (m, 3H), 2.46-2.28 (m, 4H), 2.07-1.96 (m,1H), 1.82-1.72 (m, 2H), 1.62 (s, 2H), 1.43 (s, 2H)。

[0170] Example 1-16 2-(2,6-dioxopiperidin-3-yl)-4-(4-(3-(4-(5-fluoro-4-(2 H Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(3-(4-(5-fluoro-4-(2

[0171]

[0172] In a similar manner as described in example 1-12, 4-hydroxymethylpiperidine was replaced by 4-piperidinopropanol. The crude product thus obtained was purified by column chromatography to give compound LY-16. 1 H NMR (500 MHz, DMSO- d 6) δ 11.08 (s, 1H),9.49 (s, 1H), 8.13 (d, J = 2.6 Hz, 1H), 8.08-8.01 (m, 2H), 7.85 (dd, J = 9.0,2.6 Hz, 1H), 7.67 (dd, J = 8.5, 7.1 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.36-7.29 (m, 2H), 6.49 (d, J = 9.5 Hz, 1H), 5.08 (dd, J = 12.7, 5.5 Hz, 1H),3.72-3.57 (m, 6H), 2.95-2.80 (m, 3H), 2.65-2.56 (m, 2H), 2.45-2.25 (m, 6H),2.07-1.98 (m, 1H), 1.78 (d, J = 12.3 Hz, 2H), 1.57-1.27 (m, 7H)。

[0173] Example 1-17 2-(2,6-dioxopiperidin-3-yl)-4-(2-(4-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-7-azaspiro[3.5]nonan-7-yl)isoindoline-1,3-dione (LY-17):

[0174]

[0175] In a similar manner as described in example 1-12, 4-hydroxymethylpiperidine was replaced by 7-azaspiro[3.5]nonan-2-ol. The crude product thus obtained was purified by column chromatography to give compound LY-17. 1 H NMR (500 MHz, DMSO- d 6) δ 11.08(s, 1H), 9.51 (s, 1H), 8.13 (d, J= 2.6 Hz, 1H), 8.08-8.02 (m, 2H), 7.85 (dd, J = 9.0, 2.6 Hz, 1H), 7.67 (dd, J = 8.5, 7.1 Hz, 1H), 7.40 (d, J = 8.9 Hz, 1H), 7.33 (t, J = 8.2 Hz, 2H), 6.49 (d, J = 9.5 Hz, 1H), 5.08 (dd, J = 12.7,5.5 Hz, 1H), 3.62 (s, 3H), 3.21 (d, J = 38.3 Hz, 5H), 2.93-2.82 (m, 1H), 2.65-2.56 (m, 2H), 2.33 (d, J = 30.4 Hz, 4H), 2.02 (d, J = 8.8 Hz, 5H), 1.79-1.56 (m, 3H), 1.53-1.40 (m, 2H); HRMS (ESI): calcd for C 38 H 37 FN8NaO6 [M+Na] + :743.2712, found 743.2727.

[0176] Examples 1-18 2-(2,6-dioxopiperidin-3-yl)-4-(3-((4-(5-fluoro-4-((2) H Preparation of 1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (LY-18):

[0177]

[0178] Similar to the synthesis schemes in Examples 1-12, 4-hydroxymethylpiperidine was replaced with 3-hydroxymethylpiperidine, and the crude product obtained was purified by column chromatography to give compound LY-18. 1 H NMR (500 MHz, DMSO- d 6) δ 11.08 (s, 1H),9.48 (s, 1H), 8.12 (d, J = 2.6 Hz, 1H), 8.07-7.98 (m, 2H), 7.84 (dd,J = 9.0, 2.6 Hz, 1H), 7.73-7.63 (m, 1H), 7.43-7.29 (m, 3H), 6.48 (d, J = 9.6 Hz, 1H), 5.08 (dd, J = 12.5, 5.5 Hz, 1H), 3.76 (d, J = 11.9 Hz, 1H), 3.59 (s, 4H), 2.95-2.77 (m, 2H), 2.69-2.58 (m, 3H), 2.39-2.14 (m, 6H), 2.11-1.97 (m, 2H), 1.87-1.66 (m, 4H), 1.47 (s, 1H).

[0179] Example 1-19 2-(2,6-dioxopiperidin-3-yl)-4-(3-(2-(4-(5-fluoro-4-((2 H -1-phenylpyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)piperidin-1-yl)isoindoline-1,3-dione (LY-19) was prepared according to the following scheme:

[0180]

[0181] According to the similar synthetic procedure of Example 1-12, 4-hydroxymethylpiperidine was replaced by 3-piperidineethanol. The crude product thus obtained was purified by column chromatography to give compound LY-19. 1 H NMR (500 MHz, DMSO- d 6) δ 11.08 (s, 1H), 9.50 (d, J = 8.9 Hz, 1H), 8.21-8.02 (m, 3H), 7.84 (dd, J = 9.0, 2.6 Hz, 1H), 7.74-7.62 (m, 1H), 7.45-7.34 (m, 2H), 7.30 (d, J = 7.1 Hz, 1H), 6.49 (d, J = 9.6 Hz, 1H), 5.09 (dd, J= 12.8, 5.5 Hz, 1H), 3.76-3.54 (m, 6H), 2.93-2.82(m, 2H), 2.64-2.55 (m, 5H), 2.46-2.33 (m, 5H), 2.09-1.98 (m, 1H), 1.88-1.62(m, 4H), 1.43 (s, 2H); HRMS (ESI): calcd for C 37 H 38 FN8O6 [M+H] + :709.2893, found709.2909.

[0182] Example 1-20 2-(2,6-dioxopiperidin-3-yl)-4-(3-((4-(5-fluoro-4-((2 H Preparation of (S)-2-(2,6-dioxopiperidin-3-yl)-4-(3-((4-(5-fluoro-4-((2

[0183]

[0184] Similarly to the synthetic scheme of Example 1-12, 4-hydroxymethylpiperidine was replaced by 3-hydroxymethylpyrrolidine, and the crude product thus obtained was purified by column chromatography to obtain compound LY-20. 1 H NMR (500 MHz, DMSO- d 6) δ 11.05 (s, 1H),9.50 (s, 1H), 8.14 (d, J = 2.5 Hz, 1H), 8.08-7.99 (m, 2H), 7.84 (dd, J = 9.0,2.6 Hz, 1H), 7.56 (dd, J = 8.6, 7.0 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.18-7.06 (m, 2H), 6.48 (d, J = 9.5 Hz, 1H), 5.06 (dd, J = 12.9, 5.4 Hz, 1H),3.69-3.51 (m, 6H), 3.47-3.34 (m, 2H), 2.93-2.81 (m, 1H), 2.65-2.55 (m, 3H),2.48-2.42 (m, 3H), 2.37 (d, J= 8.1 Hz, 2H), 2.14-1.95 (m, 3H), 1.69 (d, J = 9.2 Hz, 1H); HRMS (ESI): calcd for C 35 H 34 FN8O6 [M+H] + :681.2580, found 681.2587.

[0185] Example 1-21 2-(2,6-dioxopiperidin-3-yl)-4-(3-(2-(4-(5-fluoro-4-((2 H -1-phenylpyrrol-2-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)pyrrolidin-1-yl)isoindoline-1,3-dione (LY-21) was prepared according to the following scheme:

[0186]

[0187] According to the similar synthetic procedure of Example 1-12, 4-hydroxymethylpiperidine was replaced by pyrrolidin-3-ethanol, the crude product thus obtained was purified by column chromatography to obtain compound LY-21. 1 H NMR (500 MHz, DMSO- d 6) δ 11.04 (d, J = 2.1 Hz, 1H), 9.49 (s, 1H), 8.13 (t, J = 2.5 Hz, 1H), 8.07-8.00 (m, 2H), 7.84(dd, J = 9.0, 2.6 Hz, 1H), 7.55 (dd, J = 8.7, 6.9 Hz, 1H), 7.39 (d, J = 9.0Hz, 1H), 7.15-7.06 (m, 2H), 6.49 (d, J = 9.6 Hz, 1H), 5.05 (ddd, J = 12.6,5.4, 2.7 Hz, 1H), 3.68-3.55 (m, 6H), 3.53-3.43 (m, 1H), 3.38-3.33 (m, 1H),2.91-2.79 (m, 1H), 2.56 (dd, J= 19.0, 4.0 Hz, 2H), 2.46-2.32 (m, 6H), 2.29-2.19 (m, 1H), 2.17-2.06 (m, 1H), 2.05-1.93 (m, 1H), 1.69-1.57 (m, 3H); HRMS (ESI): calcd for C 36 H 36 FN8O6 [M+H] + :695.2736, found 695.2743.

[0188] Example 1-22 2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(4-(5-fluoro-4-((2 H Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(4-(5-fluoro-4-((2

[0189]

[0190] In a similar manner as described in the synthesis of Example 1-12, 4- hydroxymethylpiperidine was replaced by 2-(2-aminoethoxy)-1-ethanol. The crude product thus obtained was purified by column chromatography to give compound LY-22. 1 H NMR (500 MHz,DMSO- d 6) δ 11.07(s, 1H),9.49 (s, 1H), 8.11 (d, J = 2.6 Hz, 1H),8.06-7.98 (m,2H),7.84 (dd, J =9.0, 2.6 Hz, 1H),7.58 (dd, J = 8.6, 7.0 Hz, 1H),7.38 (d, J = 9.0 Hz, 1H),7.16(d, J = 8.6 Hz, 1H),7.03 (d, J = 7.0 Hz, 1H), 6.61 (t, J = 5.8 Hz, 1H),6.47(d, J = 9.5 Hz, 1H),5.03 (dd, J= 12.9, 5.4 Hz, 1H), 3.65-3.54 (m, 8H),3.52-3.45 (m, 2H),2.91-2.79 (m, 1H), 2.58-2.50 (m, 3H), 2.48-2.42 (m, 5H), 2.04-1.95 (m, 1H).

[0191] Example 1-23 2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-((1-(5-fluoro-4-((2 H -1-phenylpyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)(methyl)amino)ethoxy)ethyl)amino)isoindoline-1,3-dione (LY-23):

[0192]

[0193] In a similar manner to the synthetic scheme of Example 1-12, 4-hydroxymethylpiperidine was replaced by 2-(2-aminoethoxy)-1-ethanol, and the BCL6 ligand was replaced by BL2 from BL1, and the crude product thus obtained was purified by column chromatography to obtain the compound LY-23. 1 HNMR (500 MHz, DMSO- d 6) δ 11.08 (s, 1H), 9.46 (s, 1H), 8.11 (d, J = 2.6 Hz,1H), 8.02 (d, J = 3.6 Hz, 1H), 7.99 (d, J = 9.6 Hz, 1H), 7.85 (dd, J = 9.0,2.5 Hz, 1H), 7.56 (dd, J = 8.6, 7.1 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.14(d, J = 8.6 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.60 (t, J = 5.9 Hz, 1H), 6.48(d, J = 9.5 Hz, 1H), 5.04 (dd, J = 12.7, 5.4 Hz, 1H), 4.50 (d, J = 13.0 Hz,2H), 3.60 (t,J = 5.4 Hz, 2H), 3.52-3.43 (m, 4H), 2.93-2.72 (m, 4H), 2.58 (d, J = 3.6 Hz, 4H), 2.19 (d, J = 9.5 Hz, 3H), 2.06-1.94 (m, 2H), 1.71 (d, J = 12.1 Hz, 2H), 1.38-1.27 (m, 1H).

[0194] Example 1-242-(2,6-dioxopiperidin-3-yl)-4-(4-(2-((1-(5-fluoro-4-((2H-1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)(methyl)amino)ethyl)piperidin-1-yl)isoindoline-1,3-dione 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-((1-(5-fluoro-4-((2-oxo-2H-chromen-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)(methyl)amino)ethyl)piperidin-1-yl)isoindoline-1,3-dione (LY-24) Preparation:

[0195]

[0196] In a similar manner as described in the synthesis of Example 1-12, 4-hydroxymethylpiperidine was replaced by 4-piperidineethanol, and the BCL6 ligand was replaced by BL2 from BL1, and the crude product thus obtained was purified by column chromatography to give the compound LY-24. 1 H NMR (500 MHz, DMSO- d 6) δ 11.08 (s, 1H), 9.47 (s, 1H),8.13 (d, J = 2.6 Hz, 1H),8.04 (d, J = 3.6 Hz, 1H), 8.01 (d, J = 9.6 Hz, 1H), 7.85 (dd, J = 9.0, 2.6 Hz, 1H), 7.66(dd, J = 8.5, 7.1 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.31 (dd, J= 7.9, 2.3Hz, 2H), 6.49 (d, J = 9.5 Hz, 1H), 5.08 (dd, J = 12.8, 5.5 Hz, 1H), 4.54 (d, J = 12.8 Hz, 2H), 3.67 (d, J = 11.6 Hz, 2H), 2.91-2.75 (m, 5H), 2.65-2.53 (m,3H), 2.21-2.14 (m, 3H), 2.05-1.97 (m, 1H), 1.77 (d, J = 12.4 Hz, 4H), 1.54-1.30 (m, 9H); HRMS (ESI): calcd for C 39 H 42 FN8O6 [M+H] + :737.3206, found737.3202.

[0197] Example 1-25 4-(4-(2-(4-(5-chloro-4-((2) H Preparation of -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (LY-25):

[0198]

[0199] Similar to the synthesis schemes in Examples 1-12, 4-hydroxymethylpiperidine was replaced with 4-piperidineethanol, and the BCL6 ligand was replaced with BL3 instead of BL1. The crude product obtained was purified by column chromatography to obtain compound LY-25. 1 H NMR (500 MHz, DMSO- d 6) δ 11.08 (s, 1H), 8.96 (s, 1H), 8.11-7.98 (m, 3H), 7.82 (dd, J = 9.0,2.6 Hz, 1H), 7.71-7.63 (m, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.32 (t, J = 7.1Hz, 2H), 6.50 (d, J = 9.5 Hz, 1H), 5.08 (dd, J= 12.8, 5.5 Hz, 1H), 3.72-3.56(m, 6H), 2.92-2.81 (m, 3H), 2.65-2.55 (m, 2H), 2.43-2.32 (m, 6H), 2.08-1.97(m, 1H), 1.78 (d, J = 12.3 Hz, 2H), 1.54-1.32 (m, 5H); HRMS (ESI): calcd forC 37 H 37 ClN8NaO6 [M+Na] + :747.2417, found 747.2417.

[0200] Examples 1-26 2-(2,6-dioxopiperidin-3-yl)-4-(4-((4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione 2-(2,6-dioxopiperidin-3-yl)-4-(4-((4-(4-(5-fluoro-4-((2-oxo-2) H Preparation of -chromen-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (LY-26):

[0201]

[0202] 6-((5-fluoro-2-(piperazin-1-yl)pyrimidin-4-yl)amino)-2 H 1-Benzopyran-2-one (200 mg, 0.5865 mmol) and tert-butyl 4-oxopyridine-1-carboxylate (584 mg, 2.933 mmol) were dissolved in methanol (MeOH, 5 mL), followed by the addition of sodium cyanoborohydride (NaBH3CN, 110 mg, 1.760 mmol) and acetic acid (AcOH, 35 mg, 0.5865 mmol). The reaction was carried out overnight at room temperature, and the solvent was evaporated under reduced pressure to obtain the crude product. Column chromatography was used to purify the crude product to obtain 4-(4-(5-fluoro-4-((2-)-) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)piperidine-1-carboxylic acid tert-butyl ester (370 mg, yield 99%).

[0203] tert-Butyl 4-(4-(5-fluoro-4-((2 H tert-Butyl 4-(4-(5-fluoro-4-((2 H chromen-2-one (270 mg, 90% yield).

[0204] tert-Butyl 4-(4-(5-fluoro-4-((2 H chromen-2-one (31 mg, 0.08578 mmol) and intermediate synthesized in example 1-12 (1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperidin-4-yl)methyl 4-methylbenzenesulfonate (30 mg, 0.05719 mmol) were dissolved in DMF (1 mL) followed by addition of DIPEA (15 mg, 0.1144 mmol) and 80 o C and stirred overnight. After completion of the reaction, EA and water were extracted and distilled under reduced pressure. The crude product thus obtained was purified by column chromatography on silica gel to obtain the target product 2-(2,6-dioxopiperidin-3-yl)-4-(4-((4-(5-fluoro-4-((2 H tert-Butyl 4-(4-(5-fluoro-4-((2 1 H NMR (500 MHz, DMSO- d 6) δ 11.06 (d, J = 4.2 Hz, 1H), 9.49 (s, 1H), 8.13 (d, J = 2.6 Hz, 1H), 8.07-8.01 (m, 2H), 7.85 (dd, J = 9.0, 2.6 Hz, 1H), 7.59-7.53 (m, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.11 (dd, J= 15.8, 7.4 Hz, 2H), 6.49 (d, J = 9.5 Hz, 1H), 5.05 (dd, J =12.8, 5.5 Hz, 1H), 3.55 (d, J = 47.1 Hz, 8H), 2.95-2.82 (m, 2H), 2.66-2.50(m, 4H), 2.39-2.33 (m, 1H), 2.26-2.14 (m, 2H), 2.09 (d, J = 5.6 Hz, 2H),2.04-1.96 (m, 2H), 1.78 (s, 3H), 1.59 (s, 3H), 1.46 (s, 3H), 1.38-1.28 (m,2H); HRMS (ESI): calcd for C 41 H 45 FN9O6 [M+H] + :778.3471, found 778.3486.

[0205] Examples 1-27 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(5-fluoro-4-((2 H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-[1,4'-bipiperidine]-1'-yl)isoindoline-1,3-dione 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(5-fluoro-4-((2-oxo-2) H Preparation of -chromen-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-[1,4'-bipiperidin]-1'-yl)isoindoline-1,3-dione (LY-27):

[0206]

[0207] Similar to the synthesis schemes in Examples 1-26, 4-hydroxymethylpiperidine was replaced with 4-hydroxypiperidine, and the crude product obtained was purified by column chromatography to give compound LY-27. 1 H NMR (500 MHz, DMSO- d 6) δ 11.09 (s, 1H),9.49 (s, 1H), 8.14 (d, J= 2.6 Hz, 1H), 8.07-8.01 (m, 2H), 7.85 (dd, J = 9.0,2.6 Hz, 1H), 7.71-7.64 (m, 1H), 7.39 (d, J = 8.9 Hz, 1H), 7.36-7.30 (m, 2H),6.49 (d, J = 9.5 Hz, 1H), 5.09 (dd, J = 12.7, 5.5 Hz, 1H), 3.74 (d, J = 11.5Hz, 2H), 3.60 (s, 4H), 3.00-2.81 (m, 5H), 2.65-2.52 (m, 6H), 2.26-2.12 (m,3H),2.05-1.99 (m, 1H),1.89-1.72 (m, 4H), 1.71-1.58 (m, 2H), 1.48-1.27 (m,3H)。

[0208] Example 1-28 3-(4-(2-(4-(5-Fluoro-4-((2 H Preparation of 3-(4-(2-(4-(5-fluoro-4-((2

[0209]

[0210] To a solution of 2,6-bis(benzyloxy)-3-bromopyridine (1.0 g, 2.701 mmol), 4- hydroxybenzeneboronic acid pinacol ester (709 mg, 3.511 mmol) and potassium carbonate (K2CO3, 1.12 g, 8.103 mmol) in 3 mL of water and 10 mL of 1,4-dioxane was added a catalytic amount of 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (Pd(dppf)Cl2). The reaction vessel was purged with nitrogen and heated to 100 °C overnight. After the reaction was completed, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography to give the intermediate 4-(2,6-bis(benzyloxy)pyridin-3-yl)phenol (about 1.3 g).

[0211] Dissolve 4-(2,6-bis(benzyloxy)pyridin-3-yl)phenol (1 g, 2.608 mmol) and tert-butyl 2-bromoacetate (510 mg, 2.608 mmol) in DMF, add potassium carbonate (720 mg, 5.216 mmol), and stir at room temperature for 6 hours. Extract with EA and water, combine the organic phases, dry and concentrate, and purify by column chromatography to obtain tert-butyl 2-(4-(2,6-bis(benzyloxy)pyridin-3-yl)phenoxy)acetate (930 mg, yield 71.7%).

[0212] Dissolve tert-butyl 2-(4-(2,6-bis(benzyloxy)pyridin-3-yl)phenoxy)acetate (930 mg, 1.869 mmol) in 20 mL of methanol, protect with nitrogen, add an appropriate amount of palladium-carbon, and protect again with nitrogen. After removing the nitrogen from the reaction bottle, introduce hydrogen. Stir at room temperature overnight, and after the reaction is complete, remove the methanol under reduced pressure to obtain crude product, which is purified by column chromatography to obtain tert-butyl 2-(4-(2,6-dioxopiperidin-3-yl)phenoxy)acetate (about 780 mg).

[0213] Dissolve tert-butyl 2-(4-(2,6-dioxopiperidin-3-yl)phenoxy)acetate (61 mg, 0.1901 mmol) in DCM (3 mL), add 1 mL of trifluoroacetic acid, stir at room temperature for 7 hours, and after the reaction is complete, evaporate the DCM and trifluoroacetic acid to obtain intermediate 2-(4-(2,6-dioxopiperidin-3-yl)phenoxy)acetic acid, which is used directly in the next step without further purification.

[0214] Dissolve 2-(4-(2,6-dioxopiperidin-3-yl)phenoxy)acetic acid (0.1901 mmol) and BCL6 ligand BL1 (65 mg, 0.1901 mmol) in DMSO (1 mL), add HATU (93 mg, 0.2471 mmol) and DIPEA (73 mg, 0.5703 mmol) in sequence, and react at room temperature for 6 hours. After the reaction is complete, extract with EA and water, wash with saturated brine, dry and concentrate to obtain crude product, which is purified by column chromatography to obtain the target product 3-(4-(2-(4-(5-fluoro-4-((2 H -1-phenalene-2-keto-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-2-oxoethoxy)phenyl)piperidine-2,6-dione (LY-28, 64 mg, yield 59.2%). 1 H NMR (500 MHz, DMSO- d 6) δ 10.79 (s, 1H), 9.55 (d, J = 1.7 Hz, 1H), 8.14(d,J = 2.6 Hz, 1H), 8.11-8.04 (m, 2H), 7.85 (dd, J = 9.0, 2.6 Hz, 1H), 7.40(d, J = 8.9 Hz, 1H), 7.16-7.09 (m, 2H), 6.93-6.86 (m, 2H), 6.50 (d, J = 9.5Hz, 1H), 4.85 (s, 2H), 3.78 (dd, J = 11.6, 4.9 Hz, 1H), 3.71 (s, 2H), 3.65(s, 2H), 3.56 (s, 4H), 2.70-2.60 (m, 1H), 2.48-2.44 (m, 1H), 2.21-2.11 (m,1H), 2.05-1.97 (m, 1H);HRMS (ESI): calcd for C 30 H 27 FN6NaO6 [M+Na] + : 609.1868, found 609.1878.

[0215] Example 1-29 3-(4-(2-(3-(4-(5-Fluoro-4-((2 H Example 1-29 3-(4-(2-(3-(4-(5-Fluoro-4-((2

[0216]

[0217] Example 1-29 3-(4-(2-(3-(4-(5-Fluoro-4-((2 1 H NMR (500 MHz, DMSO- d 6) δ10.78 (s, 1H),9.55 (s, 1H),8.13 (d, J = 2.6 Hz, 1H), 8.11-8.04 (m, 2H), 7.85 (dd, J = 9.0, 2.6 Hz, 1H), 7.39 (d, J= 9.0 Hz, 1H), 7.12-7.06 (m, 2H), 6.90-6.83 (m, 2H), 6.49 (d, J = 9.5 Hz, 1H), 4.10-4.04 (m, 2H), 3.78-3.69 (m, 5H),3.67-3.62 (m, 2H), 3.62-3.58 (m, 2H), 3.57-3.51 (m, 4H), 2.68-2.58 (m, 3H),2.48-2.43 (m, 1H), 2.19-2.06 (m, 1H), 2.03-1.93 (m, 1H).

[0218] Example 1-30 3-(4-((4-(4-(5-Fluoro-4-((2 H Preparation of (3-(4-((4-(4-(5-fluoro-4-((2

[0219]

[0220] Dissolve 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione (300 mg, 1.157 mmol) and 4-bromobutanoic acid tert-butyl ester (387 mg, 1.736 mmol) in N - methylpyrrolidone (NMP, 1 mL), then add DIPEA (2.893 mmol, 374 mg) to the reaction solution, and heat and stir at 90°C overnight. After the reaction is complete, extract with EA and water, collect the organic phase, and distill under reduced pressure, to obtain 4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butanoic acid tert-butyl ester (246 mg, yield 53.0%) by column chromatography separation and purification.

[0221] tert-Butyl 4-((2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-4-yl)amino)butanoate (52 mg, 0.1295 mmol) was dissolved in 2 mL of DCM, TFA (0.5 mL) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction was completed, the solvent was evaporated, and the residue was dissolved in DMSO (2 mL). Then, BCL6 ligand BL1 (44 mg, 0.1295 mmol), HATU (64 mg, 0.1684 mmol), and DIPEA (50 mg, 0.3885 mmol) were added to the reaction solution, and the mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was extracted with EA and water, and dried and concentrated. The obtained crude product was separated and purified by column chromatography to obtain the target product 3-(4-(2-(3-(4-(5-fluoro-4-((2 H -1-phenylpyrrol-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethoxy)phenyl)piperidine-2,6-dione (LY-30, 19 mg, yield 22.0%). 1 H NMR (500MHz, DMSO- d 6) δ 11.00 (s, 1H), 9.54 (d, J = 1.6 Hz, 1H), 8.13 (d, J = 2.6 Hz,1H), 8.10-8.04 (m, 2H),7.85 (dd, J = 9.0, 2.6 Hz,1H), 7.40 (d, J = 9.0 Hz,1H), 7.28 (t, J = 7.7 Hz, 1H), 6.92 (dd, J = 7.5, 0.8 Hz, 1H), 6.76 (d, J =8.1 Hz, 1H), 6.50 (d, J = 9.5 Hz, 1H), 5.59 (t, J = 5.6 Hz, 1H), 5.10 (dd, J = 13.3, 5.2 Hz, 1H),4.22 (d, J = 17.1 Hz, 1H),4.12 (d, J = 17.1 Hz, 1H), 3.65(s,2H), 3.59 (d, J= 6.2 Hz, 2H), 3.56-3.49 (m, 4H), 3.14 (d, J = 5.7 Hz,2H), 2.97-2.86 (m, 1H), 2.40 (d, J = 6.9 Hz, 2H), 2.32-2.23 (m, 1H), 1.66-1.58 (m, 4H).

[0222] Example 1-31 3-(4-(4-(2-(4-(5-fluoro-4-((2) H Preparation of 1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)piperidin-1-yl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione (LY-31):

[0223]

[0224] 3-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione (200 mg, 0.6189 mmol), 4-piperidineethanol (240 mg, 1.858 mmol), and cesium carbonate (Cs₂CO₃, 610 mg, 1.858 mmol) were dissolved in dioxane (6 mL). The air in the reaction flask was replaced with nitrogen, and the catalyst Pd-PEPPSI-IHept was rapidly added. Cl (30 mg), the air in the reaction flask was replaced with nitrogen again, and the mixture was heated to 100°C and reacted overnight. After the reaction was completed, the mixture was filtered and the filtrate was collected. The crude product was obtained by vacuum distillation and purified by column chromatography to obtain the intermediate 3-(4-(4-(2-hydroxyethyl)piperidin-1-yl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione (40 mg, yield 17.3%).

[0225] 3-(4-(4-(2-hydroxyethyl)piperidin-1-yl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione (40 mg, 0.1036 mmol) and p -TsCl (30 mg, 0.1554 mmol) was dissolved in DCM (3 mL), and then TEA (32 mg, 0.3109 mmol) and a catalytic amount of DMAP were added. The reaction was carried out overnight at room temperature. After the reaction was completed, DCM and water were extracted, the organic phase was collected, dried over anhydrous sodium sulfate, and distilled under reduced pressure. The crude product was purified by column chromatography to obtain the intermediate 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)piperidin-4-yl)ethyl 4-methylbenzenesulfonate (18 mg, yield 33.1%).

[0226] 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)piperidin-4-yl)ethyl 4-methylbenzenesulfonate (18 mg, 0.0342 mmol) and intermediate BL1 (18 mg, 0.0514 mmol) were dissolved in anhydrous DMF (1 mL), followed by the addition of DIPEA (13 mg, 0.1020 mmol); the reaction was carried out overnight at 80 °C. After the reaction was complete, the product was extracted with EA and water, and then distilled under reduced pressure. The crude product obtained was purified by column chromatography to give the target product 3-(4-(4-(2-(4-(5-fluoro-4-((2- ... H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)piperidin-1-yl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione (LY-31). 1 H NMR (500 MHz, DMSO- d 6) δ 10.98 (s, 1H), 9.50 (s, 1H), 8.13 (d, J = 2.6 Hz, 1H), 8.08-8.01 (m, 2H), 7.85 (dd, J = 9.0, 2.6 Hz,1H), 7.46-7.36 (m, 2H), 7.29 (d, J = 7.4 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 6.49 (d, J = 9.5 Hz, 1H), 5.11 (dd, J = 13.3, 5.1 Hz, 1H), 4.42 (d, J = 17.3Hz, 1H), 4.29 (d, J = 17.3 Hz, 1H), 4.12-4.05 (m, 1H), 3.62 (s, 4H), 3.17 (d, J = 5.0 Hz, 2H), 2.98-2.86 (m, 1H), 2.77-2.66 (m, 2H), 2.65-2.56 (m, 2H), 2.39(d, J = 27.7 Hz, 6H), 2.04-1.95 (m, 1H), 1.79 (d, J= 12.5 Hz, 2H), 1.47 (s, 4H); HRMS (ESI): calcd for C 37 H 40 FN8O5 [M+H] + :6695.3100, found 695.3109.

[0227] Example 1-32 3-(4-(5-(4-(5-Fluoro-4-((2 H Preparation of 3-(4-(5-(4-(5-fluoro-4-((2

[0228]

[0229] To a solution of 3-(4-bromo-l-oxoisoindolin-2-yl)piperidine-2,6-dione (150 mg, 0.4642 mmol), 4-pentyn-l-ol (47 mg, 0.5570 mmol), anhydrous TEA (188 mg, 1.857 mmol) and Cul (18 mg, 0.0928 mmol) in anhydrous DMF (2 mL) was added Pd(PPh3)2Cl2(95 mg, 0.0928 mmol) after the system was purged with nitrogen. The reaction was carried out under nitrogen at 90 °C overnight. After the reaction was completed, EA and water were used for extraction, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained crude product was separated and purified by column chromatography to obtain the intermediate 3-(4-(5-hydroxypent-l-yn-l-yl)-l-oxoisoindolin-2-yl)piperidine-2,6-dione (85 mg, yield 56.1%).

[0230] To a solution of 3-(4-(5-hydroxypent-l-yn-l-yl)-l-oxoisoindolin-2-yl)piperidine-2,6-dione (85 mg, 0.2605 mmol) and p -TsCl (75 mg, 0.3908 mmol) in DCM (3 mL) was added TEA (79 mg, 0.7815 mmol) and a catalytic amount of DMAP, and the reaction was carried out at room temperature overnight. After the reaction was completed, DCM and water were used for extraction, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained crude product was separated and purified by column chromatography to obtain the intermediate 5-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-4-yl)pent-4-yn-l-yl 4-methylbenzenesulfonate (68 mg, yield 50.8%).

[0231] 5-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)pent-4-yn-1-yl 4-methylbenzenesulfonate (55 mg, 0.1071 mmol) and intermediate BL1 (55 mg, 0.1606 mmol) were dissolved in anhydrous DMF (2 mL), followed by the addition of DIPEA (28 mg, 0.2142 mmol). The reaction was carried out overnight at 80 °C. After the reaction was complete, the product was extracted with EA and water, and then distilled under reduced pressure. The crude product obtained was purified by column chromatography to obtain the target product 3-(4-(5-(4-(5-fluoro-4-((2-))) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)pent-1-yn-1-yl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione (LY-32, 26 mg, yield 36.6%). 1 H NMR (500 MHz, DMSO- d 6) δ 11.00 (s,1H), 9.50 (s, 1H), 8.13 (d, J = 2.6 Hz, 1H), 8.07-8.00 (m, 2H), 7.85 (dd, J =9.0, 2.6 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.64 (d, J = 7.5 Hz, 1H), 7.52(t, J = 7.6 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 6.48 (d, J = 9.5 Hz, 1H), 5.14 (dd, J = 13.3, 5.1 Hz, 1H), 4.46 (d, J = 17.6 Hz, 1H), 4.32 (d, J = 17.7 Hz,1H), 3.68-3.56 (m, 4H), 2.96-2.85 (m, 1H), 2.62-2.50 (m, 3H), 2.48-2.35 (m,7H), 2.06-1.97 (m, 1H), 1.83-1.74 (m, 2H) ; HRMS (ESI): calcd for C 35 H 33 FN7O5[M+H] +:650.2522, found 650.2540.

[0232] Example 1-33(2S,4R)-1-((S)-2-(7-(4-(5-fluoro-4-((2H-1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-7-oxoheptanoylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide(2S,4R)-1-((S)-2-(7-(4-(5-fluoro-4-((2-oxo-2) Preparation of H-chromen-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (LY-33):

[0233]

[0234] (2) S 4 R )-1-(( S )-2-amino-3,3-dimethylbutyryl)-4-hydroxy- N -(( S 1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (80 mg, 0.1663 mmol) and 7-(tert-butoxy)-7-oxoheptanoic acid (36 mg, 0.1663 mmol) were dissolved in 1 mL of DMF. Then, HATU (82 mg, 0.2162 mmol) and DIPEA (65 mg, 0.4989 mmol) were added to the reaction mixture, and the mixture was stirred overnight at room temperature. After the reaction was complete, EA and water were extracted, the organic phase was collected, and the crude product was purified by column chromatography to obtain the intermediate 7-((( S )-1-((2 S 4 R )-4-hydroxy-2-((( S 1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-7-oxoheptanate tert-butyl ester (76 mg, yield 71.1%).

[0235] 7-(((S )-1-((2 S 4 R )-4-hydroxy-2-((( S 1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)carbamoyl)pyrrolidone-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-7-oxoheptanoate tert-butyl ester (76 mg, 0.1182 mmol) was dissolved in 3 mL of DCM, followed by the addition of 1 mL of TFA. The mixture was stirred at room temperature for 6 hours. After the reaction was complete, the solvent and most of the TFA were removed by vacuum distillation, and the resulting solution was directly dissolved in 1 mL of DMF. Subsequently, BCL6 ligand BL1 (40 mg, 0.1182 mmol), HATU (58 mg, 0.1537 mmol), and DIPEA (46 mg, 0.3546 mmol) were added sequentially to the reaction solution, and the mixture was reacted overnight at room temperature. After the reaction was complete, the mixture was extracted with EA and water, and the organic phase was collected and distilled under reduced pressure. The crude product obtained was purified by column chromatography to obtain the target product (2). S 4 R )-1-(( S )-2-(7-(4-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-7-oxohepanoylamino)-3,3-dimethylbutyryl)-4-hydroxy- N -(( S 1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (LY-33, 58 mg, yield 54.0%). 1 H NMR (500 MHz, DMSO- d 6) δ 9.53 (d, J = 1.7 Hz, 1H),8.98 (s, 1H), 8.36 (d, J = 7.8 Hz, 1H), 8.14 (d, J = 2.6 Hz, 1H), 8.10-8.04(m, 2H), 7.85 (dd, J = 9.0, 2.6 Hz, 1H), 7.79 (d, J = 9.3 Hz, 1H), 7.44-7.36(m, 5H), 6.50 (d, J = 9.6 Hz, 1H), 5.09 (d, J = 3.5 Hz, 1H), 4.91 (p, J= 7.0Hz, 1H), 4.51 (d, J = 9.3 Hz, 1H), 4.42 (t, J = 8.1 Hz, 1H), 4.27 (s, 1H), 3.70-3.48 (m, 11H), 2.45 (s, 3H), 2.36-2.29 (m, 2H), 2.28-2.19 (m, 1H), 2.17-2.06 (m, 1H), 2.05-1.96 (m, 1H), 1.83-1.74 (m, 1H), 1.57-1.44 (m, 5H), 1.37(d, J = 7.0 Hz, 3H), 0.92 (s, 9H).

[0236] Examples 1-34 (2) S 4 R )-1-(( S )-2-(3-(3-(4-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)propionylamino)-3,3-dimethylbutyryl)-4-hydroxy- N -(( S )-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (2 S 4 R )-1-(( S )-2-(3-(3-(4-(5-fluoro-4-((2-oxo-2 H -chromen-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy- N -(( S Preparation of )-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (LY-34):

[0237]

[0238] In a similar manner as the synthetic scheme of Example 1-33, 7-(tert-butoxy)-7- oxoheptanoic acid was replaced by 3-(3-(tert-butoxy)-3-oxopropoxy)propanoic acid, the crude product thus obtained was purified by column chromatography to obtain Compound LY-34. 1 H NMR (500MHz, DMSO- d 6) δ 9.54 (d, J = 1.6 Hz, 1H), 8.98 (s, 1H), 8.36 (d, J = 7.8 Hz,1H), 8.15 (d, J = 2.6 Hz, 1H), 8.10-8.03 (m, 2H), 7.89-7.82 (m, 2H), 7.45-7.32 (m, 5H), 6.50 (d, J = 9.5 Hz, 1H), 5.10 (d, J = 3.6 Hz, 1H), 4.90 (t, J = 7.3 Hz, 1H), 4.52 (d, J = 9.3 Hz, 1H), 4.42 (t, J = 8.1 Hz, 1H), 4.27 (s,1H), 3.69-3.49 (m, 15H), 2.63-2.58 (m, 2H), 2.45 (s, 3H), 2.40-2.31 (m, 1H),2.01 (t, J = 10.2 Hz, 1H), 1.84-1.73 (m, 1H), 1.36 (d, J = 6.9 Hz, 3H), 0.92(s, 9H)。

[0239] Example 1-35 (2 S ,4 R )-1-(( S )-2-(tert-butyl)-16-(4-(5-fluoro-4-((2 H -1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-4,16-dioxo-7,10,13- trioxa-3-azahexadecanoyl)-4-hydroxy- N -(( S )-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (2 S ,4 R )-1-(( S)-2-(tert-butyl)-16-(4-(5-fluoro-4-((2-oxo-2 H -chromen-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-4,16-dioxo-7,10,13-trioxa-3-azahexadecanoyl)-4-hydroxy- N -(( S Preparation of )-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (LY-35):

[0240]

[0241] Similar to the synthesis schemes in Examples 1-33, 7-(tert-butoxy)-7-oxoheptanoic acid was replaced with 2,2-dimethyl-4-oxo-3,7,10,13-tetraoxahexadecane-16-acid, and the crude product obtained was purified by column chromatography to give compound LY-35. 1 H NMR (500 MHz, DMSO- d 6) δ 9.56-9.51 (m, 1H), 8.98 (s, 1H), 8.37 (d, J =7.8 Hz, 1H), 8.15 (d, J = 2.6 Hz, 1H), 8.10-8.04 (m, 2H), 7.85 (dd, J = 9.0,2.2 Hz, 2H), 7.46-7.34 (m, 5H), 6.50 (d, J = 9.5 Hz, 1H), 5.09 (d, J = 3.6Hz, 1H), 4.98-4.85 (m, 1H), 4.52 (d, J = 9.3 Hz, 1H), 4.42 (t, J = 8.0 Hz,1H), 4.27 (s, 1H), 3.71-3.39 (m, 23H), 2.62 (t, J = 6.7 Hz, 2H), 2.45 (s,3H), 2.37-2.30 (m, 1H), 2.05-1.97 (m, 1H), 1.83-1.76 (m, 1H), 1.37 (d, J= 7.0 Hz, 3H), 0.92 (s, 9H).

[0242] Evaluation of the effect of the compounds of the present application on the inhibition of BCL6 expression in tumor cells

[0243] Example 2-1 Cell culture

[0244] The cancer cells used in the present application were obtained from Kunming Medical University. The culture medium was RPMI 1640 medium + 5% FBS and DMEM-Basic medium + 5% FBS, and the cells were incubated in a constant temperature cell incubator at 37°C and 5% CO2.

[0245] Example 2-2 Evaluation of the BCL6 degradation activity induced by the compounds of the present application

[0246] A series of compounds of the present application synthesized in Example 1, which target the degradation of BCL6, were dissolved in DMSO to a final concentration of 0.1 micromole / liter and 1 micromole / liter, respectively, and the control group was added with an equal amount of DMSO. Breast cancer cells T47D and HCC1806 were treated for 24 h. The cells were collected by centrifugation and denatured lysis to collect total protein. Then, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the inhibition and degradation of BCL6 by the compounds.

[0247] The Western Blot test scheme is as follows: after completing the cell culture under experimental conditions, the cells were collected after washing with cold phosphate buffered saline (PBS). The cell pellet was dissolved with 2x protein sample buffer (1M Tris-HCl pH=6.8, 50% glycerol, 10% SDS, 2-mercaptoethanol and 1% bromophenol blue), and boiled at 100°C for 10 min. The prepared protein sample was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to a PVDF membrane (Millipore). Blocking was performed in 5% skim milk solution for specific coupling reactions between proteins on the PVDF membrane and antibodies. Then, the skim milk solution was discarded, and the membrane was washed with PBST (0.5% Tween 20 added to PBS). The coupling reaction between the proteins on the membrane and the antibodies was carried out at 4°C overnight, and then the membrane was washed again with PBST. Finally, the secondary antibody (Cell signaling Technology) conjugated with horseradish peroxidase (HRP) was diluted in PBST and reacted at room temperature for 2 hours. The protein content on the membrane was determined using Luminata Forte HRP substrate (Millipore).

[0248] The gray values of BCL6 and Tubulin in different channels were analyzed by using Image J software, the relative gray ratio of BCL6 / Tubulin corresponding to each compound and the relative gray ratio of BCL6 / Tubulin in the control group were calculated in Excel, and the degradation rate (%) of BCL6 protein was calculated according to the following formula. Tubulin is the internal reference protein.

[0249]

[0250] The degradation ability of the compounds of the present application to BCL6 in tumor cells is represented by the degradation rate (%) of BCL6 protein of the compounds at a concentration of 0.1 micromole / liter and 1 micromole / liter. Among them, "a", "b", "c", "d" respectively represent that the degradation rate of the compounds provided by the present application to BCL6 in tumor cells at this concentration is less than 25%, between 25% and 50%, between 50% and 75%, and greater than 75%, and "NT" represents that the degradation ability of the compound to BCL6 at the specified concentration is not evaluated. The evaluation results of the degradation efficiency of the compounds of the present application to BCL6 in breast cancer cells T47D and HCC1806 are shown in Table 1.

[0251] Table 1 Evaluation results of the compounds of the present application in inducing BCL6 degradation in different tumor cells

[0252]

[0253] The results in Table 1 show that most of the compounds of the present application can at least effectively induce the degradation of BCL6 protein in one strain of breast cancer cells. Among them, multiple compounds such as LY-3, LY-14 (LY-708B), LY-16, LY-18, LY-19, LY-20, LY-21, LY-25 and LY-31 can induce more than 50% of BCL6 protein degradation in breast cancer T47D or HCC1806 cells at a concentration of 1 micromole / liter; LY-14 (LY-708B) and LY-25 can induce more than 75% of BCL6 protein degradation in breast cancer HCC1806 and T47D cells at a concentration of 1 micromole / liter.

[0254] In order to further illustrate the ability of the compounds of the present application to induce the degradation of BCL6 protein in breast cancer cells, Figure 1 The Western Blot results of some compounds of the present application in inducing the degradation of BCL6 in breast cancer cells HCC1806 and T47D are shown, and the results show that the compounds of the present application represented by LY-3 and LY-14 (LY-708B) have obvious BCL6 degradation ability. Figure 1BI-3802 is a compound that has been reported to have only BCL6 degradation activity without anticancer activity, and is used as a control in the present example.

[0255] Example 2-3 DC of representative compound LY-14 (LY-708B) in the present example in breast cancer cells HCC1806 and MDA-MB-468 cells 50 and Dmax

[0256] To evaluate the ability of the compounds of the present application to degrade BCL6 in different breast cancer cells, the representative compound LY-14 (LY-708B) in the present example was selected to treat HCC1806 and MDA-MB-468 cells for 24 h, and then the cells were collected and lysed with an appropriate amount of SDS protein lysis buffer. The cells and lysis buffer were mixed well by blowing. After mixing, the protein was denatured at 98°C for 10 minutes using a metal bath. The denatured protein was separated by 10% SDS-PAGE electrophoresis (concentration gel 80 V, 40 min; separation gel 120 V, 90 min), and then transferred to a PVDF membrane using a wet transfer method (260 mA, 95 min). The membrane was blocked with 5% skim milk for 1 hour, and then incubated with primary antibody (BCL6 (Abcam, ab172610), Tubulin (MCE, HY-P80372)) at 4°C overnight. The membrane was washed with TBST buffer three times (10 min each time), and then incubated with secondary antibody (Millipore, 401315) at room temperature for 1 hour. The membrane was washed with TBST buffer three times (10 min each time), and then developed after incubation with enhanced chemiluminescence horseradish peroxidase substrate (ThermoFisher Scientific, 32106).

[0257] The gray values of BCL6 and Tubulin in different channels were analyzed using Image J software, and the relative gray ratio of BCL6 / Tubulin corresponding to each compound and the relative gray ratio of BCL6 / Tubulin in the control group were calculated in Excel. The BCL6 protein degradation rate (%) was calculated according to the following formula.

[0258]

[0259] DC was calculated using Graphpad Prism software 50 and D max , and the BCL6 protein degradation curve of the compound concentration was plotted, as shown in Figure 2 B. Figure 2In this embodiment, A represents the assay results of the degradation abilities of multiple compounds LY-3, LY-4, LY-13, LY-15, and LY-17 on BCL6 protein in colorectal cancer cells HT29, A375 skin cancer cells, and SU-DHL-4 lymphoma cells, and the assay result of the degradation ability of LY-14 (LY-708B) on BCL6 protein in HCC1806, MDA-MB-468, and SU-DHL-4 cells. Low concentrations of LY-14 (LY-708B) significantly degraded BCL6 protein in HCC1806, MDA-MB-468, and diffuse large B-cell lymphoma SU-DHL-4 cells without a hook effect, demonstrating a stronger effect than the other five BCL6 proteins. Figure 2 As shown in Figure B, LY-14 (LY-708B) in DCs of HCC1806 and MDA-MB-468 cells 50 The concentrations were 0.065 μmol / L and 0.074 μmol / L, respectively. max The percentages were 87% and 91%, respectively. The negative control LY-14 (LY-708B) (LY-722D, with glutarimide methylation on the CRBN ligand) failed to induce BCL6 degradation due to the loss of CRBN recruitment ability. The results show that the representative compound LY-14 (LY-708B) and others in the embodiments of this invention can significantly degrade BCL6 protein in breast cancer cell lines, further demonstrating the effectiveness of the compounds of this invention in degrading BCL6 protein.

[0260] Compound degradation activity such as Figure 2 As shown, LY-14 (LY-708B) stably degrades BCL6 protein in a concentration-dependent manner, and its effect is stronger than that of compound LY-722D.

[0261] LY-722D is 4-(4-(2-(4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)piperidin-1-yl)-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, with the following structural formula:

[0262]

[0263] Example 3 Evaluation of the antiproliferative activity of the compounds of the present invention in tumor cells

[0264] CCK8 assay was used to determine the inhibitory activity of the compound on breast cancer cell proliferation.

[0265] The CCK8 method is used to detect cell growth inhibition. The compound to be tested is dissolved in DMSO (dimethyl sulfoxide), and then the compound concentration of 10 μM, 1 μM and 0.1 μM of 0.1% DMSO solution is prepared in the cell culture medium.

[0266] The CCK8 cell proliferation kit is used for detection: cells in the logarithmic growth phase are digested to prepare a single cell suspension, and are inoculated into a 96-well plate at a certain density. The experimental groups and control groups are set, and are placed in an incubator for a certain period of time to allow the cells to adhere and grow. According to the experimental requirements, 1 μL of different concentrations of compounds dissolved in DMSO is added to each well of the cell culture, so that the final concentration of each compound is one hundredth of the previous concentration, and the control group is only added with the same volume of DMSO. Continue to culture for an appropriate period of time. After 72 h, 10 μL of fresh medium containing CCK8 is added to each well, and incubated for 1 hour to allow the reagent to fully react with the living cells. The absorbance value of each well is detected by a microplate reader at a specific wavelength (around 450 nm). By comparing the absorbance data of different groups, the cell proliferation is analyzed. The higher the absorbance, the more living cells and the better the proliferation. The percentage of the absorbance value of the experimental group relative to the absorbance value of the control group represents the survival rate of the cells or the proliferation level of the cells, that is, the control group is set as 100%.

[0267] The effects of other different concentrations of the compounds of the present application on the proliferation of breast cancer cells are continuously analyzed, and the GraphPad Prism software is used to display the inhibition rate data of different compounds on cell proliferation. The test results are shown in Table 2.

[0268] The proliferation inhibition ability of the compounds of the present application on tumor cells is represented by the cell survival rate. Among them, “+”, “++”, “+++” and “++++” respectively represent that the half maximal inhibitory concentration (IC 50 ) of the compound is greater than 10 micromoles / liter, between 1.0 micromole / liter and 10 micromole / liter, between 0.1 micromole / liter and 1.0 micromole / liter, and less than 0.1 micromole / liter. The IC 50 of the compounds of the present application on breast cancer cells HCC1937 and T47D is shown in Table 2.

[0269] Table 2 Anti-proliferative activity evaluation results of the compounds of the present application in different breast cancer tumor cells

[0270]

[0271] The results of Table 2 show that the compounds of the present application can effectively inhibit the proliferation of breast cancer cells HCC1806 and T47D, and most of the compounds such as LY-4, LY-9, LY-11, LY-14 (LY-708B), LY-15, LY-18, LY-19, LY-21, LY-24, LY-25 and LY-31 have certain inhibitory effect on the breast cancer cells HCC1806 and T47D, and the IC50 of the compounds is less than 10 micromole / liter. 50 The IC50 of the compounds LY-4, LY-19 and LY-25 on the two breast cancer cells HCC1806 and T47D is less than 1.0 micromole / liter, and the cell survival rate of LY-14 (LY-708B) is less than 50% at 0.1 μM. 50 As shown in Table 3, some of the compounds have good inhibitory activity on the proliferation of breast cancer cells HCC1806 (A) and breast cancer cells T47D (B). Figure 3 Figure 3 Figure 3

[0272] In order to further test the inhibitory activity of the compounds of the present application on the proliferation of breast cancer, the representative compound LY-14 (LY-708B) in the examples of the present application was selected to continue to determine the inhibitory activity of the compound on the proliferation of various breast cancer cells and lymphoma cells SU-DHL-4 by using the CCK8 kit. Figure 4 The effect of the compound LY-14 (LY-708B) on the proliferation of various breast cancer cells is shown in Table 4. Figure 4 A shows the growth inhibition curves of LY-708B on breast cancer cells HCC1806 and MDA-MB-468, and the growth inhibition curves of LY-708B on lymphoma cells SU-DHL4. Figure 4 B shows the growth inhibition curves of LY-708B and LY-722D on breast cancer cells HCC1806 and MDA-MB-468, Figure 4 C shows the growth inhibition curves and IC50 data of LY-708B on 9 types of breast cancer cell lines. Figure 4 As shown in Table 5, LY-14 (LY-708B) has strong inhibitory activity on the proliferation of breast cancer cells and lymphoma cells.

[0273] ​​​To further evaluate the effect of the compounds of the present application on the proliferation of other tumor cells, the present application selected a representative compound LY-14 (LY-708B) to test its anti-proliferative activity in a variety of other cancer cells, and the results are shown in Table 3. The test results show that the compound LY-14 (LY-708B) has strong anti-proliferative activity on lung cancer cells, liver cancer cells, leukemia cells, colorectal cancer cells, skin cancer cells, human glioma cells and human pancreatic cancer cell lines, indicating that the compounds of the present application have good therapeutic potential for a variety of tumors. Table 3 Evaluation results of the anti-proliferative activity of the compound LY-14 (LY-708B) of the present application in different tumor cells

[0274]

[0275] Example 4 Cell scratch test to test the migration inhibitory effect of the compounds of the present application on tumor cells

[0276] The method for detecting the migration of cells by using the scratch healing test of the compounds with good activity: the cells were seeded in a 6-well plate, and after the cells grew into a dense layer, a scratch was made in the middle of the cell layer with a 200 μL gun head. The cell debris was washed away with 1xPBS, and then complete medium containing low concentration of serum and different concentrations of compounds were added and placed in the incubator for continuous culture. Immediately, 3 scratches were randomly selected for image acquisition, and the signal was collected at the same position every 4 hours, and the experiment was ended when the scratches in one group were close to healing. The migration ability of the cells was analyzed by analyzing the healing degree of the scratch area. Cell migration rate = (initial scratch width - final scratch width) / initial scratch width x 100%.

[0277] The test results are shown in

[0278] Figure A, a scratch test was performed in MDA-MB-468 and HCC1806 cell lines Figure 5 , LY-14 (LY-708B) was used for treatment at different concentrations (0.05 μM, 0.1 μM and 0.2 μM), and photographs were taken every 12 h. It can be found that with the increase of the concentration, LY-14 (LY-708B) can significantly inhibit the migration rate of cancer cells Figure 5 (B). Figure 5

[0279] Example 5 Spheroid formation test to test the proliferation inhibitory effect of the compounds of the present application on tumor cells

[0280] ​After digesting and counting the cultured cells, seed an appropriate amount of cell suspension into 6-well plates (800 cells per well), add different concentrations of the drug, and culture with an equal volume of DMSO as a control. Culture for 7-14 days until clonal formation is visible to the naked eye. Once clonal formation is complete, remove the culture medium and wash the cells 1-2 times with PBS. Fix the cells with an appropriate amount of paraformaldehyde solution for 10-15 minutes to fix them at the bottom of the culture plate. Remove the fixative and wash the cells again with PBS. Add crystal violet staining solution and stain for 10-30 minutes to stain the clones purple or blue for easy observation and counting. Wash away excess staining solution with PBS and air dry the culture plate or dish. Observe and count the clones under a microscope. The size and number of clones can be defined according to experimental needs; generally, a cell cluster containing more than 50 cells is considered a clone. Use automated cell counting software or count the number of clones manually.

[0281] Experimental results: Figure 6 The effect of compound LY-14 (LY-708B) on colony formation of breast cancer cells HCC1806 and MDA-MB-468 was investigated. Figure 6 In Figure A, a clonogenic assay was performed to evaluate the effect of compound LY-708B on the survival of breast cancer cells HCC1806 and MDA-MB-468. Figure 6 B represents statistical data from colony formation experiments of breast cancer cells HCC1806 and MDA-MB-468; for example... Figure 6 As shown, colony formation experiments were performed in MDA-MB-468 and HCC1806 cell lines. After culturing for 14 days with different concentrations of LY-14 (LY-708B), staining and photography were performed, and the results were quantified. It was found that with increasing concentration, LY-14 (LY-708B) could inhibit the number of cancer cell colonies formed.

[0282] Example 6: Apoptosis Experiment to Test the Apoptosis-Promoting Effect of the Compounds of the Present Invention on Tumor Cells

[0283] Apoptosis detection was performed by staining cells with FITC Annexin V and PI dye, following the instructions of the kit, and the percentage of apoptotic cells was analyzed by flow cytometry.

[0284] Experimental results: Figure 7 The effect of compound LY-14 (LY-708B) on apoptosis in breast cancer cells HCC1806 and MDA-MB-468; Figure 7 In Figure A, the effect of LY-708B treatment at 0 nM, 50 nM, 100 nM, and 200 nM on cell apoptosis was detected by flow cytometry. Figure 7 B in the text represents the statistical data of A. Figure 7C represents the effect of LY-708B on apoptosis-related proteins detected by Western blotting; for example... Figure 7 As shown, MDA-MB-468 cells were treated with LY-14 (LY-708B) for 48 h, and the apoptosis rate was analyzed by staining. Quantitative results showed that the number of apoptotic cells significantly increased with increasing LY-14 (LY-708B) concentration. Data were analyzed using a t-test. ***, p<0.001. DMSO served as the control group.

[0285] For apoptosis detection, cells were stained with FITC Annexin V and PI dye according to the kit instructions, and the cell cycle distribution ratio was analyzed by flow cytometry.

[0286] Experimental results: Figure 8 The effect of the representative compound LY-14 (LY-708B) on the cell cycle of breast cancer cells HCC1806 and MDA-MB-468; Figure 8 In Figure A, the effect of LY-708B treatment at 0-200 nM on the cell cycle was detected by flow cytometry. Figure 8 B in the text represents the statistical data of A. Figure 8 C represents the effect of LY-708B on cycle-related proteins detected by Western blotting; for example... Figure 8 As shown, MDA-MB-468 cells were treated with LY-14 (LY-708B) for 24 h, and their cell cycle distribution was analyzed by staining. Quantitative results showed that with increasing LY-14 (LY-708B) concentration, the number of cells arrested in the G1 / S phase significantly decreased, while the number of cells arrested in the G2 / M phase significantly increased. Data were analyzed using a t-test. ***, p < 0.001. DMSO served as the control group.

[0287] Example 7 Evaluation of the inhibitory activity of the representative compound LY-14 (LY-708B) of this invention on the growth of breast cancer xenografts in nude mice.

[0288] To evaluate the potential therapeutic effect of the compounds of this invention on tumors in vivo, HCC1806 cells in logarithmic growth phase were harvested, digested, collected, and counted. Cells were counted at 1 × 10⁻⁶ cells per cell point. 6 After centrifuging to remove the supernatant, the cells were resuspended in pre-chilled 1×PBS, then Matrigel was added and thoroughly mixed with a pre-chilled pipette tip. Using an insulin injection needle, 75 μL was injected into the fourth pair of mammary fat pads of mice at each site. One week after inoculation, the in situ tumor formation in mice was observed. When the tumor volume reached 50-60 mm... 3When the mice were randomly divided into groups of 5, different drug treatment concentration groups were set. According to the volume of 100 μL per mouse, the drug injection solution was prepared according to the formula of 10% (DMSO or drug) drug mixture + 30% PEG300 + 5% Towen80 + 30% normal saline. Intraperitoneal administration was continued every 3 days, with one day off, and the body weight of the mice was recorded, and the tumor of the mice was measured with a vernier caliper, and the tumor volume was calculated by the formula V = 0.5 × length × width². After the drug treatment was completed, the mice were sacrificed by cervical dislocation. The tumor tissue was separated using surgical instruments, weighed, photographed, and the mouse serum was taken for subsequent liver and kidney function detection.

[0289] Figure 9 Inhibition of human breast cancer xenograft tumor in mice in vivo by compound LY-14 (LY-708B); Figure 9 A is the construction of a mouse orthotopic breast cancer model and the dosing time and frequency diagram; after 9 consecutive doses, the mice were euthanized, the tumor was removed and photographed; Figure 9 C is the tumor volume measured every 2 days after the start of drug treatment, represented by a broken line graph, and Figure 9 As can be seen from C, compared with the control group, after administration of compound LY-14 (LY-708B), the tumor volume of the mice was lower than that of the control group; the tumor weight of the mice in the control group (DMSO) and the experimental group (5 mg / kg and 10 mg / kg) was lower than that in the control group; Figure 9 I is the change of the body weight of the mice after administration of compound LY-14 (LY-708B); there is no difference in the body weight of the mice in the experimental and control groups; Figure 9 F-H is after the completion of the administration, the mice were anesthetized, the orbital blood was taken, the level of glutamic oxalacetic transaminase in the serum was detected to evaluate the hepatotoxicity of compound LY-14 (LY-708B); the level of glutamic-pyruvic transaminase in the serum was detected to evaluate the hepatotoxicity of compound LY-14 (LY-708B) and the level of creatinine in the serum was detected to evaluate the nephrotoxicity of compound LY-14 (LY-708B); P <0.01, P <0.001, ns represents no difference; from Figure 9 As can be seen, compound LY-14 (LY-708B) significantly inhibited the growth of tumor tissue in mice and did not cause liver and kidney toxicity in mice. From Figure 9 As can be seen from J, in the tumor tissue of the mice in the control group (DMSO) and the experimental group (5 mg / kg and 10 mg / kg), the level of BCL6 in the tumor tissue of the experimental group was significantly reduced, proving that LY-14 (LY-708B) has the effect of degrading BCL6 in vivo.

[0290] The use and welfare of the experimental animals in this experiment were performed in compliance with the regulations of the International Assessment and Recognition Committee of Experimental Animals (AAALAC). The health status and death of the animals were monitored every day, and routine examinations included observation of the effects of the test substances and drugs on the daily behavior of the animals, such as behavioral activity, body weight change, and appearance signs.

[0291] The results show that the representative compound LY-14 (LY-708B) in the embodiments of the present application has good in vivo anticancer effect, can effectively inhibit the growth of breast cancer cells in mice, and does not significantly affect the body weight and liver and kidney functions of the mice.

[0292] Example 8 Evaluation of the representative compound LY-14 (LY-708B) in the embodiments of the present application on the binding activity of BCL6 protein

[0293] The binding ability of LY-14 (LY-708B) and BCL6 was determined by SPR (surface plasmon resonance detection) experiment: using a Biacore S200 instrument, the BCL6 protein was coupled to a CM5 chip, the coupling buffer was sodium acetate buffer pH = 4.5; the regeneration liquid was Glyline pH = 2.5; the running buffer was PBSP (PBS + 0.005% P20 + 5% DMSO); the binding test was performed using LY-14 (LY-708B) concentrations of 0 µM, 0.125 µM, 0.25 µM, 1 µM, 2 µM, 4 µM, 8 µM, 16 µM and 32 µM, respectively, and the coupled BCL6 protein.

[0294] Test results: Figure 10 The binding activity of the representative compound LY-14 (LY-708B) in the embodiments of the present application and BCL6 protein was evaluated. Figure 10 In the embodiment A, the binding ability of the compound LY-708B and BCL6 protein was detected by surface plasmon resonance (SPR). Figure 10 In the embodiment B, the binding constant of the compound LY-708B and BCL6 protein was detected by SPR.

[0295] As Figure 10As shown, the compound LY-14 (LY-708B) at 0.125 μM started to have the ability to bind to the BCL6 protein (RU>0, the vertical coordinate in the figure represents the ability of the compound LY-14 (LY-708B) to bind to the BCL6 protein, the greater the value, the stronger the binding ability, and the horizontal coordinate represents the reaction time), and the binding signal increased with the increase of the molar concentration of the compound LY-14 (LY-708B), and according to the results, the affinity constant of the compound LY-14 (LY-708B) to the BCL6 protein can reach 10-7. The results show that the compound LY-14 (LY-708B) can directly bind to the BCL6 protein.

[0296] In summary, the compound of the present application not only shows the activity of inducing the degradation of the BCL6 protein, but also shows the obvious anti-proliferation effect, and shows the obvious dose-dependent inhibition of the growth of tumors in vivo, and is a potential effective compound for treating tumors.

[0297] The protection scope of the present application is not limited to the above examples. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.

Claims

1. A PROTACs-like compound that targets and degrades BCL6, characterized in that, The PROTACs class of compounds includes one or more of the following compounds: 2-(2,6-dioxopiperidin-3-yl)-4-((2-(3-(4-(5-fluoro-4-((2))) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)amino)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-((2-(3-(4-(5-fluoro-4-((2))) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)amino)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazine-1-formyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2)) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-2-oxoethyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(5-fluoro-4-((2)) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-2-oxoethyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(4-(3-(4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-4-oxobutyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-5-(4-(4-(4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-4-oxobutyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2)) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-2-oxoethyl)piperazin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(5-fluoro-4-((2)) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-2-oxoethyl)piperazin-1-yl)isoindoline-1,3-dione; 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)piperidin-4-yl)- N -(1-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)- N -Methylacetamide; 2-(2,6-dioxopiperidin-3-yl)-4-(4-((4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(5-fluoro-4-((2)) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(5-fluoro-4-((2)) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(4-(3-(4-(5-fluoro-4-(2H-1-benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)propyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(2-(4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-7-azaspiro[3.5]nonane-7-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(3-((4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(3-(2-(4-(5-fluoro-4-((2)) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(3-((4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)methyl)pyrrolidine-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(3-(2-(4-(5-fluoro-4-((2)) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)pyrrolidine-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(4-(5-fluoro-4-((2))) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethyl)amino)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-((1-(5-fluoro-4-((2))) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)methyl)amino)ethoxy)ethyl)amino)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-((1-(5-fluoro-4-((2))) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperidin-4-yl)(methyl)amino)ethyl)piperidin-1-yl)isoindoline-1,3-dione; 4-(4-(2-(4-(5-chloro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(4-((4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)piperidin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione; 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-[1,4'-bipiperidine]-1'-yl)isoindoline-1,3-dione; 3-(4-(2-(4-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-2-oxoethoxy)phenyl)piperidin-2,6-dione; 3-(4-(2-(3-(4-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethoxy)phenyl)piperidine-2,6-dione; 3-(4-((4-(4-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-4-oxobutyl)amino)-1-oxoisoindoline-2-yl)piperidine-2,6-dione; 3-(4-(4-(2-(4-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethyl)piperidin-1-yl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione; 3-(4-(5-(4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)pent-1-yn-1-yl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione; (2 S 4 R )-1-(( S )-2-(7-(4-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-7-oxohepanoylamino)-3,3-dimethylbutyryl)-4-hydroxy- N -(( S )-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide; (2 S 4 R )-1-(( S )-2-(3-(3-(4-(5-Fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-3-oxopropoxy)propionylamino)-3,3-dimethylbutyryl)-4-hydroxy- N -(( S )-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide; (2 S 4 R )-1-(( S )-2-(tert-butyl)-16-(4-(5-fluoro-4-((2) H -1-Benzopyran-2-one-6-yl)amino)pyrimidin-2-yl)piperazin-1-yl)-4,16-dioxo-7,10,13-trioxa-3-azahexadecanoyl)-4-hydroxy- N -(( S )-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

2. A pharmaceutical composition, characterized in that, Includes PROTACs-like compounds and excipients for targeted degradation of BCL6 as described in claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that, The composition further includes a second pharmaceutical agent selected from at least one of paclitaxel, cisplatin, doxorubicin, etoposide, gemcitabine, imatinib, ibrutinib, JQ1, or AMG510.

4. The use of a compound of claim 1 or a pharmaceutical composition of any one of claims 2-3 in the preparation of an antitumor drug for targeting the degradation of BCL6, wherein the tumor is at least one of diffuse large B-cell lymphoma, breast cancer, colorectal cancer, lung cancer, liver cancer, kidney cancer, osteosarcoma, cervical cancer, skin cancer, leukemia, ovarian cancer, glioma, or pancreatic cancer.

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