WO2023217063A1

WO2023217063A1 - Benzo[d]isoxazole compound and use thereof

Info

Publication number: WO2023217063A1
Application number: PCT/CN2023/092667
Authority: WO
Inventors: 许�永; 沈慧; 徐鸿瑞; 吴天帮; 张�成; 于昊楠; 胡建康; 罗国龙; 李俊骅; 卢吉布; 吴锡山; 张岩
Original assignee: 广州智药生物科技有限公司
Priority date: 2022-05-13
Filing date: 2023-05-08
Publication date: 2023-11-16
Also published as: CN117088864A

Abstract

The present application provides a benzo[d]isoxazole compound and use thereof. The compound has a structure represented by the following formula I. The benzo[d]isoxazole compound of the present application can induce the degradation of BET protein and/or GSPT1 protein, and has an anti-proliferation effect on cancer cells. Therefore, the compound provided by the present application and a composition comprising same can be used for preparing a drug for treating or preventing a disease such as oncogenesis, an inflammation, a virus infection, a cell proliferative disorder, an autoimmune disease, and septicemia.

Description

A kind of benzo[d]isoxazole compound and its application

Technical field

This application belongs to the field of chemical medicine technology, and specifically relates to a benzo[d]isoxazole compound and its application.

Background technique

Epigenetics is the study of heritable changes in gene expression without changes in DNA sequence. Epigenetics mainly controls gene expression through DNA methylation, histone modification, chromatin remodeling and non-coding RNA regulation. Among them, histones are the core of chromatin and participate in post-transcriptional modifications, including acetylation, methylation, phosphorylation, and ubiquitination.

Bromodomain and extra terminal domain (BET) proteins of the bromodomain (BRD) superfamily of proteins are important epigenetic "readers". BET family proteins include four members, BRD2, BRD3, BRD4 and BRDT, and the protein contains two tandem N-terminal bromodomains (BD1 and BD2). The N-terminal bromodomain consists of four α-helices (αZ, αA, αB and αC), ZA loop and BC loop. This class of proteins relies on the hydrophobic pocket of the bromodomain to recognize acetylated histone lysine residues to function. BET protein affects cell growth, proliferation and differentiation, apoptosis and necrosis and other processes. Multiple studies have shown that BET dysfunction is related to the occurrence and development of cancer, cell proliferative disorders, inflammation, autoimmune diseases, sepsis, or viral infections (such as the new coronavirus).

GSPT1 is a translation termination factor that mediates stop codon recognition and promotes the release of nascent peptides from ribosomes by binding to eRF1. GSPT1 is also involved in several other key cellular processes, such as cell cycle regulation, cytoskeletal organization, apoptosis, and transcription. Therefore, downregulation of GSPT1 levels impairs the control of cell proliferation and promotes cell migration and scarring. GSPT1 has been implicated as an oncogenic driver in several different cancer types, including breast, hepatocellular, gastric, and prostate cancer.

Abnormal expression of BET protein and GSPT1 protein is related to diseases. Studies have found that targeted protein degraders can not only inhibit the activity of target proteins, but also use the ubiquitin-proteasome system in the body to clear target proteins to achieve therapeutic purposes. Several BET or GSPT1 degraders have been developed.

CN109311890A discloses a BET protein degrading agent. The compound disclosed by the invention degrades the BET bromodomain protein and can be used for the treatment of various diseases and conditions. In particular, the disclosed compound can be used for the treatment of which BET bromodomain protein degradation provides Methods of benefiting diseases or conditions, such as cancer and proliferative diseases.

CN110062759A discloses a condensed 1,4-oxynitrogen heterocyclic compound as a BET protein degradation agent. The compounds disclosed in this invention are BET bromodomain protein degraders, and therefore can be used to treat or prevent the degradation of BET bromodomains (such as BRD2, BRD3, BRD4, BRD-t or isoforms or mutants thereof) providing beneficial effects. disease or condition.

CN112543764A discloses a compound that is excellent in its cytotoxic effect on cancer cells, its effect in inducing the degradation of BET protein in cancer cells, and its inhibitory effect on the binding of BET protein and acetylated histones, and can be used as an anti- Cancer agent, BET protein degradation inducer or BET protein inhibitor.

CN111032043A discloses a 2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl) Compositions and methods of use of methyl)-2,2-difluoroacetamide on bone marrow mononuclear cells from patients with myelodysplastic syndrome when tested as a single agent or in combination with everolimus Role of caspase-3 activation and GSPT1 degradation.

Currently, multiple published BET or GSPT1 degraders have good preclinical anti-tumor activity in vivo or in vitro, but their research is still in the early stages. Therefore, there is an urgent need to develop a new and efficient compound that can induce the degradation of BET family proteins and/or GSPT1 proteins.

Taken together, degradation agents targeting BET proteins and/or GSPT1 proteins may be beneficial for the development of therapeutic strategies targeting diseases including cancer, inflammatory diseases, cell proliferative disorders, autoimmune diseases, sepsis, and viral infections. of.

Contents of the invention

The present application provides a class of benzo[d]isoxazole compounds, which can regulate BET protein levels and/or functions, and/or regulate GSPT1 protein levels and/or functions. , has a cytotoxic effect on cancer cells. This will provide a new class of promising drugs for the treatment of diseases including cancer, inflammatory diseases, cell proliferative disorders, autoimmune diseases, sepsis and viral infections.

In the first aspect, this application provides a benzo[d]isoxazole compound, which has the structure shown in Formula I below:

Among them, X is selected from O or S;

R ₁ is selected from H, hydroxyl, substituted or unsubstituted C1-C5 (such as C1, C2, C3, C4, C5) alkyl, substituted or unsubstituted C1-C5 (such as C1, C2, C3, C4, C5 )alkoxy;

R ₂ and R ₃ are each independently selected from any group in the following groups (i) or (ii):

(i)H, halogen, amino, nitro, carboxyl, cyano, hydroxyl, substituted or unsubstituted C1-C5 (such as C1, C2, C3, C4, C5) alkyl, substituted or unsubstituted C1-C6 (e.g. C1, C2, C3, C4, C5, C6) alkoxy, substituted or unsubstituted C3-C10 (e.g. C3, C4, C5, C6, C7, C8, C9, C10) cycloalkyl, substituted or Unsubstituted C3-C10 (such as C3, C4, C5, C6, C7, C8, C9, C10) heterocycloalkyl, substituted or unsubstituted C6-C20 (such as C6, C10, C12, C14, C16, C18 , C20, etc.) aryl, substituted or unsubstituted C4-C20 (such as C4, C5, C6, C10, C12, C14, C16, C18, C20, etc.) heteroaryl; or

(ii)-N(R _a )SO ₂ R _b , -SO ₂ N(R _a )R _b , -N(R _a )COR _b , -CON(R _a )R _b -, -N(R _a ) CH ₂ R _b , -NHCH(R _a )R _b , -N(R _a )R _b , -CH(R _a )R _b , -COR _b , -COOR _b , -OCOR _b , -SR _b or -OR _b ;

R _a is selected from H, substituted or unsubstituted C1-C6 (such as C1, C2, C3, C4, C5, C6) alkyl, substituted or unsubstituted C3-C10 (such as C3, C4, C5, C6, C7 , C8, C9, C10) cycloalkyl, substituted or unsubstituted C3-C10 (such as C3, C4, C5, C6, C7, C8, C9, C10) cycloalkenyl, substituted or unsubstituted C1-C10 ( For example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10) heterocycloalkyl;

R _b is selected from any one of the following groups (v), (vi), (vii) or (viii):

(v) Substituted or unsubstituted C6-C20 (such as C6, C10, C12, C14, C16, C18, C20, etc.) aryl, substituted or unsubstituted C4-C20 (such as C4, C5, C6, C10, C12 , C14, C16, C18, C20, etc.) heteroaryl;

(vi) Substituted or unsubstituted C3-C10 (such as C3, C4, C5, C6, C7, C8, C9, C10) cycloalkyl, substituted or unsubstituted C3-C10 (such as C3, C4, C5, C6 or

(vii) Substituted or unsubstituted C1-C5 (such as C1, C2, C3, C4, C5) alkyl, substituted or unsubstituted C2-C10 (such as C2, C3, C4, C5, C6, C7, C8, C9, C10) alkenyl, substituted or unsubstituted C2-C10 (such as C2, C3, C4, C5, C6, C7, C8, C9, C10) alkynyl;

Y ₁ is selected from -NH(CH ₂ ) _n CON(R _c ) -, -O(CH ₂ ) _n CON(R _c ) -, -S(CH ₂ ) _n CON(R _c ) -, -(CH ₂ ) _n CON(R _c )-or single bond, n is 1-8 (such as 1, 2, 3, 4, 5, 6, 7, 8);

R _c is selected from H, substituted or unsubstituted C3-C10 (such as C3, C4, C5, C6, C7, C8, C9, C10) cycloalkanes group, substituted or unsubstituted C3-C10 (such as C3, C4, C5, C6, C7, C8, C9, C10) cycloalkenyl, substituted or unsubstituted C3-C10 (such as C3, C4, C5, C6, C7, C8, C9, C10) heterocycloalkyl, substituted or unsubstituted C6-C20 (such as C6, C10, C12, C14, C16, C18, C20, etc.) aryl, substituted or unsubstituted C4-C20 ( For example, C4, C6, C10, C12, C14, C16, C18, C20, etc.) heteroaryl, substituted or unsubstituted C2-C10 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10) Alkenyl;

Y ₂ is selected from -O-, -NH-, -CH ₂ - or single bond;

L is selected from the group consisting of single bond, alkylene group, alkenylene group, alkynylene group, ether group, thioether group, ester group, amine group, amide group, urethane group, ureido group, sulfone group, aryl group, hetero group Any one or a combination of at least two of aryl, carbonyl, cycloalkyl and heteroaryl;

E has the structure shown in formula II, where It is the group connection position;

Among them, Y ₃ is selected from -O-, -S-, -CHR _d -, -C(=O)-, -SO ₂ -, -NR _e -;

R _d and R _e are each independently selected from H, substituted or unsubstituted C1-C10 (such as C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkyl, substituted or unsubstituted C3-C8 (such as C3, C4, C5, C6, C7, C8, etc.) cycloalkyl, substituted or unsubstituted C3-C8 (such as C3, C4, C5, C6, C7, C8, etc.) heterocyclyl;

Y ₄ , Y ₅ , Y ₆ , Y ₇ are each independently selected from CH or N;

T ₁ , T ₂ , and T ₃ are each independently selected from O or S; and

R ₄ and R ₅ are each independently selected from -H, hydroxyl, substituted or unsubstituted C1-C10 (such as C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkyl, substituted or Unsubstituted C3-C10 (such as C3, C4, C5, C6, C7, C8, C9, C10, etc.) cycloalkyl, substituted or unsubstituted C1-C10 (such as C1, C2, C3, C4, C5, C6 , C7, C8, C9, C10, etc.) O-containing heterocycloalkyl, substituted or unsubstituted C1-C10 (such as C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) containing N Heterocycloalkyl, substituted or unsubstituted C1-C10 (such as C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) contains S heterocycloalkyl.

Preferably, the Y4, Y5, Y6 and Y7 are independently selected from CH.

Preferably, any one of the Y4, Y5, Y6, and Y7 groups is CH, and at least one of the other three groups is N.

Preferably, any one of the groups Y4, Y5, Y6, and Y7 is CH, and the connecting position of the group is located on the CH.

Preferably, the L is selected from any one of the following groups:

Wherein, m, n and o are independently selected from positive integers between 1-8; Y ₈ is selected from substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted Substituted C1-C10 contains O heterocycloalkyl, substituted or unsubstituted C1-C10 contains N heterocycloalkyl, substituted or unsubstituted C1-C10 contains S heterocycloalkyl;

Preferably, the Y ₃ is selected from -CH- or -C(=O)-;

Preferably, the T ₁ , T ₂ and T ₃ are selected from O.

Preferably, the benzo[d]isoxazole compound has the structure shown in the following formula III:

Among them, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , X, Y ₁ , Y ₂ , L, Y ₃ , Y 4 , Y ₅ , Y ₆ , Y ₇ , T ₁ , T ₂ , _T ₃ The definition of groups is the same as above.

Preferably, the benzo[d]isoxazole compound has the structure shown in the following formula IV:

Among them, the definitions of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , X, Y ₁ , Y ₂ , L, and Y ₃ are the same as above.

Preferably, the benzo[d]isoxazole compound has the structure shown in the following formula V or VI:

Among them, the definitions of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , X, Y ₁ , Y ₂ and L groups are the same as above.

Preferably, the R ₁ is selected from substituted or unsubstituted C1-C5 alkyl, more preferably unsubstituted C1-C5 alkyl, further preferably unsubstituted C1-C3 alkyl;

Preferably, any one of R ₂ and R ₃ is selected from any group in group (ii), and the other group is selected from any group in group (i), preferably the Either group of R ₂ and R ₃ is selected from any group of -N(R _a )SO ₂ R _b or -SO ₂ N(R _a )R _b , and the other group is selected from H;

Preferably, the R _a is selected from H, substituted or unsubstituted C1-C6 alkyl, more preferably H;

Preferably, the R _b is selected from any group in group (v), more preferably a substituted or unsubstituted C6-C20 aryl group, further preferably a substituted C6-C10 aryl group;

Preferably, the R _b is selected from substituted phenyl, and the substituent of the phenyl is C1-C3 alkoxy and/or halogen;

Preferably, the Y ₁ is selected from -O(CH ₂ ) _n CON(R _c )- or a single bond;

Preferably, the n is 1-3, and R _c is selected from H;

Preferably, the Y ₂ is selected from -O-, -NH-, -CH ₂ - or a single bond;

Preferably, the L is selected from any one of the following groups:

Wherein, m, n and o are independently selected from positive integers between 1-6, Y ₈ is selected from unsubstituted C1-C6 alkyl;

Preferably, the benzo[d]isoxazole compound has the following structure represented by formula VII:

Wherein, Y ₁ is selected from -O(CH ₂ ) _n CON(R _c )- or a single bond, n is 1-3, and R _c is selected from H; and

L is selected from any one of the following groups:

Wherein, m, n and _o are independently selected from positive integers between 1-6, Y ₈ is selected from unsubstituted C1-C6 alkyl;

Y ₂ is selected from -O-, -NH-, -CH ₂ - or a single bond; and

Y ₃ is selected from -CH- or -C(=O)-.

Preferably, the aryl group is selected from phenyl or naphthyl.

Preferably, the heteroaryl group has an aromatic 5-8 (eg, 5, 6, 7, 8)-membered monocyclic ring, an 8-12-membered (eg, 8, 9, 10, 11, 12)-membered bicyclic ring, or an 11-14-membered bicyclic ring. (e.g. 11, 12, 13, 14) membered tricyclic ring system; the single ring has 1-4 (e.g. 1, 2, 3, 4) heteroatoms, and the bicyclic ring has 1-6 (e.g. 1, 2, 4) heteroatoms. 3, 4, 5, 6) heteroatoms, the tricyclic ring has 1-9 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9) heteroatoms; the heteroatoms are selected from O, N or S.

Preferably, the alkyl group, alkoxy group, cycloalkyl group, heterocycloalkyl group, aryl group, heteroaryl group, alkenyl group, cycloalkenyl group, and alkynyl group have a substituent, and the substituent group is selected from halogen, hydroxyl , any one of methoxy, ethoxy, amino, nitro, ether, thioether, ester, amide, urethane, urea or sulfone groups.

Preferably, the heteroaryl group is selected from pyridazinyl, indolyl, quinazolinyl, pyrrolyl, thienyl, indazolyl, pyrazolyl, quinolyl, pyridyl, furyl, imidazolyl, Any one of pyrazinyl, pyrimidinyl, thiazolyl, isoquinolinyl, benzothiazolyl or naphthyridyl.

Preferably, the benzo[d]isoxazole compounds include any one of the following compounds 1-19:

In a second aspect, the present application provides a pharmaceutically acceptable salt, stereoisomer, N-oxide, prodrug molecule, solvate, and deuterated compound of the compound described in the first aspect.

In a third aspect, this application provides a pharmaceutical composition, which includes active ingredients and pharmaceutically acceptable excipients;

Wherein, the active ingredient includes at least one compound described in the first aspect and/or at least one pharmaceutically acceptable salt, stereoisomer, N-oxide, prodrug molecule, solvent as described in the second aspect compounds, deuterated compounds.

In the fourth aspect, the application provides a compound as described in the first aspect, a pharmaceutically acceptable salt, a stereoisomer, an N-oxide, a prodrug molecule, a solvate, a deuterated compound as described in the second aspect, The use of the pharmaceutical composition described in the third aspect in preparing a preparation for degrading BET and/or GSPT1 protein.

In the fifth aspect, the application provides a compound as described in the first aspect, a pharmaceutically acceptable salt, a stereoisomer, an N-oxide, a prodrug molecule, a solvate, a deuterated compound as described in the second aspect, The use of the pharmaceutical composition according to the third aspect in the preparation of medicaments for preventing or treating cancer, cell proliferative disorders, inflammation, autoimmune diseases, sepsis or viral infections.

Preferably, the cancer is selected from the group consisting of acute monocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia mixed lineage leukemia, NUT-midline carcinoma, multiple myeloma, glioma, lung cancer, neurological blastoma, Burkitt lymphoma, cervical cancer, esophageal cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer cancer or breast cancer. The inflammatory and autoimmune diseases are selected from the group consisting of inflammatory pelvic disease, urethritis, pneumonia, meningitis, myocarditis, ulcerative colitis, organ transplant rejection, asthma, allergic rhinitis, chronic obstructive pulmonary disease, and autoimmune diseases. Autoimmune alopecia, anemia, autoimmune hemolytic and systemic lupus erythematosus, rheumatoid arthritis, Hashimoto's thyroiditis or atopic dermatitis. The viral infection is selected from the group consisting of poliovirus, hepatitis A virus, rubella virus, Japanese encephalitis virus, hepatitis C virus, human papilloma virus, rabies virus, herpes virus, Barr virus or human immunodeficiency virus, novel Coronavirus or infection.

In this application, exemplary cancers that may be treated with compounds of this application include, but are not limited to, leukemias (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, Acute promyeloid leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, mixed leukemia, chronic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), multiple myeloma , polycythemia vera, cutaneous T lymphocytoma, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenström's macroglobulinemia, heavy chain disease, and solid tumors; said entities Tumors are sarcomas and cancers, for example, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovialoma, mesothelioma, Ewing leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, Papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, adrenal tumor, acoustic neuroma, limb Melanoma, acrohidromas, adenoid cystic carcinoma, adipose tissue tumors, adrenocortical carcinoma, adult T-cell leukemia/lymphoma, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft sarcoma, ameloblastoma fibroma , anaplastic large cell lymphoma, anaplastic thyroid cancer, angiomyolipoma, astrocytoma, atypical rod-shaped tumors, B-cell chronic lymphocytic leukemia, B-cell prelymphocytic leukemia, B-cell lymphoma, Biliary tract cancer, bladder cancer, blastoma, bone tumors, brown tumors, Burkitt lymphoma, brain cancer, carcinoma in situ, chondroma, cementoma, myeloid sarcoma, choroid plexus papilloma, renal clear cell Sarcoma, craniopharyngioma, cervical cancer, small round cell tumor, diffuse B-cell lymphoma, neuroepithelial tumors, dysgerminoma, endocrine gland tumors, endodermal sinus tumors, esophageal cancer, fibroma, follicular lymphoma tumour, follicular astrocytoma, thyroid cancer, gastrointestinal cancer, germ cell tumor, choriocarcinoma of pregnancy, giant cell fibroblastoma, giant cell tumor of bone, glioblastoma, pleomorphic Glioblastoma, glioma, granulosa cell tumor, androcytoma, gallbladder cancer, gastric cancer, hemangioblastoma, head and neck cancer, hemangiopericytoma malignancy, hepatoblastoma, cytolymphoid Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, fatal midline cancer, testicular Leydig cell tumor, liposarcoma, lung cancer, lymphangioma, Lymphoepithelioma, acute lymphangiosarcoma, lymphocytic leukemia, chronic lymphocytic leukemia, small cell lung cancer, non-small cell lung cancer, malt lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, marginal zone B-cell lymphoma , mast cell leukemia, mediastinal germ cell tumors, medullary breast carcinoma, medullary thyroid carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, metastatic cell carcinoma, mixed Mueller tumor, mucinous Neoplasms, muscle tissue tumors, mycoid mycoid liposarcoma, nasopharyngeal carcinoma, neuroblastoma, neurofibroma, neuroma, eye cancer, optic nerve sheath meningioma, visual pathway glioma, oral cancer, thyroid papilla neoplastic paraganglioma, pineoblastoma, pituitary cell tumor, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, peritoneal carcinoma, pharyngeal carcinoma, renal medullary carcinoma, adult Retinocytoma, rhabdomyomas, rectal cancer, trophoblastic tumors, skin cancer, small cell carcinoma, soft tissue sarcoma, somatostatinoma, spinal cord tumors, splenic marginal zone lymphoma, synovial sarcoma, small intestinal cancer, T-cell lymphoma, Testicular cancer, transitional cell cancer, urachal cancer, urogenital cancer, uterine cancer, verrucous cancer, vulvar cancer, or vaginal cancer.

In this application, the cell proliferative disorders include: benign soft tissue tumors, brain and spinal cord tumors, eyelid and orbital tumors, granulomas, lipomas, meningiomas, multiple endocrine tumors, nasal polyps, pituitary tumors, prolactinoma tumor, fat Epileptic keratosis, gastric polyps, thyroid nodules, hepatic hemangiomas, vocal cord nodules, polyps, cysts, pilonidal disease, dermatofibromas, Pilar cysts or pyogenic granulomas.

In this application, the inflammatory diseases include: inflammatory pelvic disease, urethritis, sunburned skin, sinusitis, pneumonia, encephalitis, meningitis, myocarditis, nephritis, osteomyelitis, myositis, hepatitis, gastritis, enteritis, dermatitis , Gingivitis, Pancreatitis, Psoriasis, Allergies, Crohn's Disease, Bowel Syndrome, Ulcerative Colitis, Tissue Transplant Rejection, Organ Transplant Rejection, Asthma, Allergic Rhinitis, Chronic Obstructive Pulmonary Disease, Autoimmune Diseases, autoimmune alopecia, anemia, glomerulonephritis, dermatomyositis, multiple sclerosis, scleroderma, vasculitis, autoimmune hemolytic and thrombocytopenia, pulmonary hemorrhage nephritic syndrome, atherosclerosis, Addison's disease, Parkinson's disease, Alzheimer's disease, diabetes, septic shock, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, chronic idiopathic platelet Purpura purpura, myasthenia gravis, Hashimoto's thyroiditis, atopic dermatitis, degenerative joint disease, Guillain-Barre syndrome, mycosis fungoides, or acute inflammatory reaction.

In this application, the viral infection includes: poliovirus, hepatitis A virus, rubella virus, Japanese encephalitis virus, hepatitis C virus, human papillomavirus, rabies virus, herpes virus, Barr virus or human immunodeficiency virus, novel coronavirus, or infection.

[Explanation of terms]

Various aspects and features of the present application are further described below.

Various terms and phrases used in this application have general meanings known to those skilled in the art. Even so, this application still hopes to provide a more detailed description and explanation of these terms and phrases. If the mentioned terms and phrases have the same meaning as If the publicly known meanings are inconsistent, the meaning expressed in this application shall prevail. The following are definitions of various terms used in this application. These definitions apply to the terms used throughout the specification of this application unless otherwise stated in a specific case. The definitions of various groups of the compounds of the present application are provided below. Unless otherwise defined, they are used uniformly in the description and claims.

As mentioned in this application, the terms "halo", "halogen", "halogen atom", "halo" and the like mean fluorine, chlorine, bromine or iodine, in particular fluorine, chlorine or bromine.

As mentioned in this application, the term "alkyl" refers to an alkyl group having a specified number of carbon atoms, which may be a straight-chain alkyl group or a branched-chain alkyl group, such as the "C1-C20 alkyl group" When, it refers to a linear or branched chain alkyl group with 1 to 20 carbon atoms. Specific groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, Tert-butyl, etc., and similar groups.

As mentioned in this application, the term "cycloalkyl" refers to a cyclic alkyl group with a specified number of ring carbon atoms. For example, when "C3-C10 cycloalkyl" is mentioned, it refers to a cyclic alkyl group with a specified number of ring carbon atoms. -10 cycloalkyl, specific groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc., and similar groups.

As referred to in this application, the term "alkenyl" refers to an alkenyl group (a hydrocarbyl group with one or more C=C double bonds) having the specified number of carbon atoms, which may be a straight-chain alkyl or Branched alkenyl, such as the "C2-C20 alkenyl" refers to a linear alkyl or branched alkenyl group with 2-20 carbon atoms. Specific groups such as vinyl, propenyl, Allyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 1,3-pentadienyl, 1-hexenyl group, 2-hexenyl, etc., and similar groups.

As used herein, the term "cycloalkenyl" refers to a cyclic alkenyl group (a hydrocarbyl group with one or more C=C double bonds) having a specified number of carbon atoms, such as the "C3- C10 cycloalkenyl" refers to a cyclic alkenyl group with 3-10 carbon atoms. Specific groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, etc. and similar groups.

As referred to in this application, the term "alkynyl" refers to an alkynyl group (a hydrocarbyl group with one or more C≡C triple bonds) having a specified number of carbon atoms, which may be a straight-chain alkyl or Branched-chain alkynyl, such as the "C2-C20 alkynyl", refers to a straight-chain alkyl or branched alkynyl group with 2-20 carbon atoms. Specific groups such as ethynyl and propynyl ,1-butynyl, 2-butynyl, 1-pentynyl, 2-hexynyl, etc., and similar groups.

As referred to herein, the term "heterocycloalkyl" refers to a non-aromatic heterocyclic ring in which one or more of the atoms forming the ring are heteroatoms such as O, N, or S. Heterocyclyl groups may include monocyclic or polycyclic (eg, having 2, 3, or 4 fused rings) ring systems as well as spirocyclic rings. Examples of preferred "heterocycloalkyl" groups include, but are not limited to: aziridinyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, oxazolidinyl, thiazolidinyl , imidazolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, morpholinyl, thiomorpholinyl, piperazinyl, piperidinyl, and similar groups. Also included within the definition of heterocycloalkyl are those moieties having one or more aromatic rings fused to a non-aromatic heterocycloalkyl ring (eg with a shared bond), such as 2,3-dihydrobenzene Furyl, 1,3-benzodioxolyl, benzo-1,4-dioxanyl, phthalimide, naphthalimide, and the like group. Heterocycloalkyl groups having one or more fused aromatic rings can be attached through an aromatic moiety or a non-aromatic moiety.

As referred to in this application, the term "aryl" refers to a monocyclic or polycyclic (eg having 2, 3 or 4 fused rings) aromatic hydrocarbon, such as phenyl, naphthyl, anthracenyl, phenanthrenyl, indene groups, and similar groups.

As referred to herein, the term "heteroaryl" refers to an aromatic heterocyclic ring having at least one heteroatom ring member such as O, N, or S. Heteroaryl groups include monocyclic or polycyclic (eg, having 2, 3, or 4 fused rings) ring systems. Any N atoms forming a ring in the heterocyclic group can also be oxidized to form N-oxides. Examples of preferred "heteroaryl" groups include, but are not limited to: pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, thienyl, imidazolyl, triazolyl, tetrazolyl , Thiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, pyrrolyl, pyrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, benzofuryl, benzothienyl, Benzothiazolyl, indolyl, indazolyl, quinolyl, isoquinolinyl, purinyl, carbazolyl, benzimidazolyl, pyrrolopyridyl, pyrrolopyrimidinyl, pyrazopyridinyl, Pyrazolopyrimidinyl, and similar groups.

As mentioned in this application, the term "single bond" refers to the direct connection of the two groups connected to this position; for example, when Y ₁ is a single bond, L is directly connected to the benzene ring, and the formula I is The structure can be expressed as: When Y ₂ is a single bond, the group Y ₁ is directly connected to E, and the structure shown in formula I can be expressed as: A more specific example is the case where Y ₂ is a single bond, that is, compound 18 provided in this application.

As referred to in this application, the term "compound", as used herein, is meant to include all stereoisomers, geometric isomers, tautomers, isotopes.

Compounds of the present application may be asymmetric, such as having one or more stereocenters. Unless otherwise limited, all stereoisomers can be enantiomers and diastereomers. Compounds of the present application containing asymmetrically substituted carbon atoms can be isolated into optically pure or racemic forms. Optically pure forms can be prepared by resolution of racemates, or by the use of chiral synthons or chiral reagents.

Compounds of the present application may also include tautomeric forms. New tautomer forms result from the interchange of a single bond and an adjacent double bond together with the migration of a proton.

Compounds of the present application may also include all isotopic forms of atoms present in intermediates or final compounds. Isotopes include those atoms that have the same atomic number but different mass numbers. For example, isotopes of hydrogen include deuterium and tritium.

As used herein, the term "pharmaceutical composition", which may also refer to a "composition", may be used to effect treatment and/or prevention of the diseases or conditions described herein in a subject, particularly a mammal. .

As used herein, the term "disease and/or disorder" refers to a physical state of the subject that is associated with the disease and/or disorder described herein. For example, the diseases and/or conditions described herein may refer to both a physical state and a disease state. In this article no distinction is made between physical states and disease states, or they may refer to each other.

As mentioned in this application, the term "pharmaceutically acceptable salt" means that the salt is not only physiologically acceptable to the subject, but also refers to a synthetic substance that has pharmaceutical use, such as in the preparation of chiral The salt formed as an intermediate during the resolution, although the salt of this intermediate cannot be directly administered to the subject, the salt can play a role in obtaining the final product of the present application.

There are two forms of pharmaceutically acceptable salts of the compound represented by formula I: one is the salt formed with acid; the other is the salt formed with alkali or alkali metal. Acids that form pharmaceutically acceptable salts with the compounds of Formula I include inorganic acids and organic acids. Suitable inorganic acids include: hydrochloric acid, sulfuric acid and phosphoric acid. Suitable organic acids include aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic acids and sulfonic acid organic acids, examples of which include but are not limited to: formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, Lactic acid, malic acid, tartaric acid, glycine, arginine, citric acid, fumaric acid, alkyl sulfonic acid, aromatic sulfonic acid, etc. Alkali metals that form pharmaceutically acceptable salts with the compounds represented by Formula I include lithium, sodium, potassium, magnesium, calcium, aluminum, zinc, etc.; bases that form pharmaceutically acceptable salts with the compounds represented by Formula I include choline. , diethanolamine, morpholine, etc.

As mentioned in this application, the term "prodrug" refers to some derivatives of the compound represented by Formula I that are converted in vivo (for example: hydrolysis, reduction or oxidation) into Formula I by means of metabolism in the body. Compounds, these derivatives are called prodrugs. For example, the compound represented by Formula I and containing a hydroxyl group can be reacted with an acid to prepare the corresponding ester. The prepared ester is a prodrug and can hydrolyze the parent drug in vivo. Acids suitable for preparing "prodrugs" include, but are not limited to: acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, oxalic acid, salicylic acid, succinic acid, fumaric acid, maleic acid, methylene Bis-β-hydroxynaphthoic acid, gentisic acid, isethionic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.

Compared with the existing technology, this application has the following beneficial effects:

(1) This application provides a class of structurally novel compounds and their pharmaceutically acceptable salts, stereoisomers, tautomers, N-oxides or prodrug molecules thereof, which can induce the degradation of BET proteins ( Preferably, induce the degradation of BRD2), or/and effectively induce the degradation of GSPT1;

(2) The compounds and compositions provided in this application can be used to prepare drugs for treating or preventing tumor formation, inflammation, viral infection, cell proliferation disorders, autoimmune diseases, sepsis and other diseases.

Description of the drawings

Figure 1 shows the down-regulation of BET protein levels in 22Rv1 cells by compounds 7, 8 and 9 at different concentrations.

Figure 2 is a graph showing the down-regulation of BET protein levels in 22Rv1 cells by compound 9 at different concentrations.

Figure 3 shows the BET protein levels in MV4;11 cells downregulated by compound 9 at different concentrations.

Figure 4 shows the down-regulation of BET protein levels in 22Rv1 cells by compound 9 at different times.

Figure 5 shows the BET protein levels in MV4;11 cells downregulated by compound 9 at different times.

Figure 6 is a graph showing the down-regulation of GSPT1 protein levels in 22Rv1 cells by compound 9 at different concentrations.

Figure 7 is a graph showing the down-regulation of GSPT1 protein levels in MV4;11 cells by compound 9 at different concentrations.

Figure 8 is a graph showing compound 9 down-regulating GSPT1 protein levels in 22Rv1 cells at different times.

Figure 9 is a graph showing compound 9 down-regulating GSPT1 protein levels in MV4;11 cells at different times.

Detailed ways

The technical solutions of the present application will be further described below through specific implementations. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

Preparation example

This preparation example provides the T-1/T-2 synthesis steps of raw materials required for the preparation process of benzo[d]isoxazole compounds.

The synthetic route of compound T-1/T-2 is as follows:

(a) N-(2,4-dimethoxyphenyl)acetamide (1-1)

Dissolve 2,4-dimethoxyaniline (25g, 163.21mmol) and triethylamine (36.29mL, 104.4mmol) in 40mL dichloromethane. Under ice-water bath conditions, add acetic anhydride (21.6mL, 228.49mmol) dropwise. After the reaction is completed, 10% citric acid aqueous solution is added to the reaction system. Extract with dichloromethane solution, extract the organic layer with dilute hydrochloric acid, saturated sodium bicarbonate, and saturated brine respectively, and dry over anhydrous sodium sulfate. After concentration under reduced pressure, the target product was obtained as a dark brown solid (30 g, yield 94%).

¹ H NMR (400MHz, DMSO) δ 8.99 (s, 1H), 7.64 (d, J = 8.7Hz, 1H), 6.59 (s, 1H), 6.45 (d, J = 8.8Hz, 1H), 3.79 ( s, 3H), 3.73 (s, 3H), 2.01 (s, 3H). MS (ESI) m/z [M+H] ⁺ theoretical value: 196.09; actual value: 196.1.

(b) N-(5-acetyl-4-hydroxy-2-methoxyphenyl)acetamide (1-2)

Dissolve 1-1 (30g, 153.85mmol) in 50mL dichloromethane, slowly add aluminum trichloride (86.98g, 652.32mmol) in batches under ice bath, and add acetyl chloride (34.6mL, 489.24mmol) under argon protection. , stir the reaction at 43°C. After the reaction, the reaction solution was slowly poured into ice water to quench for 2 hours, and then extracted with dichloromethane. The organic layer was extracted with water and saturated brine respectively, and dried over anhydrous sodium sulfate. After the organic layer was concentrated under reduced pressure, the crude product was subjected to column treatment (PE:EA=1:1) to obtain the target product (26.1g, yield 76%).

¹ H NMR (500MHz, DMSO) δ12.54 (s, 1H), 9.17 (s, 1H), 8.26 (s, 1H), 6.59 (s, 1H), 3.88 (s, 3H), 2.52 (s, 3H) ), 2.05(s, 3H).MS(ESI)m/z[M+H] ⁺ theoretical value: 224.08; actual value: 224.1.

(c) (E)-N-(4-hydroxy-5-(1-(hydroxyimino)ethyl)-2-methoxyphenyl)acetamide (1-3)

Dissolve 1-2 (26.1g, 116.80mmol) in 200mL ethanol aqueous solution (absolute ethanol: water = 3:1, v/v), add hydroxylamine hydrochloride (12.99g, 186.88mmol) and sodium acetate (15.33g, 186.88 mmol), reflux at 80°C for 2 hours. The reaction was monitored by TLC. After the reaction, the solvent was spin-dried under reduced pressure, water was added, filtered under reduced pressure, and the filter cake was washed and dried to obtain the target product (24.5 g, yield 88%).

¹ H NMR (500MHz, DMSO) δ11.74 (s, 1H), 11.33 (s, 1H), 9.03 (s, 1H), 7.84 (s, 1H), 6.54 (s, 1H), 3.80 (s, 3H ), 2.17(s, 3H), 2.02(s, 3H).MS(ESI)m/z[M+H] ⁺ theoretical value: 239.10; actual value: 239.1.

(d)N-(6-methoxy-3-methylbenzisoxazol-5-yl)acetamide (1-4)

Dissolve 1-3 (24.5g) in 200mL of 1,4-dioxane, and add N,N-dimethylformamide dimethyl acetal under vigorous stirring at 40-60°C. After the reaction system is stable, the temperature is raised to 100°C and reacted for 7 minutes. Cool to room temperature, dissolve in dichloromethane, extract with water and saturated brine respectively, and dry over anhydrous sodium sulfate. After the organic layer was concentrated under reduced pressure, the target product (22.2 g, yield 98%) was obtained.

¹ H NMR (500MHz, DMSO) δ9.26 (s, 1H), 8.24 (s, 1H), 7.36 (s, 1H), 3.94 (s, 3H), 2.47 (s, 3H), 2.11 (s, 3H) ).MS(ESI)m/z[M+H] ⁺ theoretical value: 221.08; actual value: 221.1.

(e) 6-methoxy-3-methylbenzo[d]isoxazole-5-amine (1-5)

Dissolve 1-4 (22.2g, 100.64mmol) in 400mL hydrochloric acid (3mol/L) and reflux at 90°C for 3h. The reaction was monitored by TLC. After the reaction is completed, add sodium hydroxide solution (3mol/L) to adjust the pH to neutral (7-9), precipitate out, filter under reduced pressure, wash with a small amount of water, and dry to obtain a tan solid (17.1g, yield 95%) .

¹ H NMR (500MHz, DMSO) δ7.14 (s, 1H), 6.80 (s, 1H), 4.80 (s, 2H), 3.88 (s, 3H), 2.39 (s, 3H).

(f)5-amino-3-methylbenzisoxazole-6-ol (1-6)

Dissolve 1-5 (17.1g, 95.96mmol) in 100mL dichloromethane, dissolve BBr3 in 100mL dichloromethane, slowly add to the reaction system under ice bath, and react overnight at room temperature. After the reaction is completed, methanol and amine chloride solution are slowly added dropwise to quench. Add water, adjust the pH to 7 with sodium hydroxide solution, and extract with ethyl acetate. The organic layer was extracted with water and saturated brine respectively, and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure and dried to obtain the target product (6 g, yield 38%).

(g) 5-bromo-N-(6-hydroxy-3-methylbenzo[d]isoxazol-5-yl)-2-methoxybenzenesulfonamide (1-7)

Dissolve 1-6 (6g, 36.57mmol) and 4-chlorobenzenesulfonyl chloride (10.44g, 36.57mmol) in dichloromethane, add 7mL of pyridine, and react at room temperature for 4 hours. After the reaction is completed, add water for extraction, and the organic layer is extracted with Extract with dilute hydrochloric acid aqueous solution, water, saturated brine, and dry over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure and the crude product was subjected to column treatment (DCM:MeOH=250:1-125:1) to obtain the target product (2.5g, yield 17%).

¹ H NMR (500MHz, DMSO) δ10.70 (s, 1H), 9.09 (s, 1H), 7.74 (dd, J=8.8, 2.5Hz, 1H), 7.69 (d, J=2.5Hz, 1H), 7.54 (s, 1H), 7.18 (d, J=8.9Hz, 1H), 6.88 (s, 1H), 3.82 (s, 3H), 2.44 (s, 3H).

(h) tert-butyl 2-(5-(5-bromo-2-methoxyphenyl)sulfonylamino)-3-methylbenzo[d]isoxazol-6-yl)acetate (1- 8)

Compound 1-7 (500 mg, 1.21 mmol) was dissolved in ultra-dry DMF, and KI (20 mg, 0.12 mmol) and KHCO ₃ (182 mg, 1.82 mmol) were added. Then dissolve tert-butyl bromobutyrate (0.21 mL, 1.21 mmol) in 2 mL of ultra-dry DMF, stir in an ice bath, add to the reaction system in batches under argon protection, and stir for 4 hours at 40°C. After the reaction, dichloromethane and water were added to the reaction system for three extractions. The organic layers were combined and extracted with saturated brine. The organic layers were separated and combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrated crude product was separated and purified through a silica gel column (PE:EA=10:1, v/v) to obtain the target product as a white solid (300 mg, yield 47%).

(i) 2-(5-(5-bromo-2-methoxyphenyl)sulfonylamino)-3-methylbenzo[d]isoxazol-6-yl)acetic acid (T-1)

Compound 1-8 (300 mg, 0.59 mmol) was dissolved in dichloromethane solvent (5 mL), trifluoroacetic acid (1 mL) was added, the reaction was stirred at room temperature, and the reaction was monitored by TLC. After the reaction, the solvent was evaporated under reduced pressure. The product was directly used in the next reaction.

(j) 4-(5-(5-(5-bromo-2-methoxyphenyl)sulfonamido)-3-methylbenzo[d]isoxazol-6-yl)oxybutyric acid Tert-butyl ester (1-9)

Compound 1-7 (500 mg, 1.21 mmol) was dissolved in ultra-dry DMF, and KI (20 mg, 0.12 mmol) and KHCO ₃ (182 mg, 1.82 mmol) were added. Then dissolve tert-butyl bromobutyrate (0.21 mL, 1.21 mmol) in 2 mL of ultra-dry DMF, stir in an ice bath and add to the reaction system in batches under argon protection, stir for 4 hours at 40°C, and monitor the reaction by TLC. . After the reaction, dichloromethane and water were added to the reaction system for three extractions. The organic layers were combined and extracted with saturated brine. The organic layers were separated and combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrated crude product was separated and purified through a silica gel column (PE:EA=10:1, v/v) to obtain the target product as a white solid (134 mg, yield 20%).

(k) 4-(5-(5-bromo-2-methoxyphenyl)sulfonamido)-3-methylbenzisoxazol-6-yl)oxy)butyric acid (T-2)

Compound 1-9 (134 mg, 0.24 mmol) was dissolved in dichloromethane solvent (5 mL), trifluoroacetic acid (1 mL) was added, the reaction was stirred at room temperature, and the reaction was monitored by TLC. After the reaction, the solvent was evaporated under reduced pressure. The product was directly used in the next reaction.

Example 1

2-(5-(5-bromo-2-methoxyphenyl)sulfonylamino)-3-methylbenzo[d]isoxazol-6-yl)-N-(2-(2-( 2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide)ethyl)

The synthetic route of compound 1 is as follows:

(a) 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (2-1)

Dissolve 3-hydroxyphthalic anhydride (5.00g, 30.47mmol) and 3-amino-2,6-piperidine-dione hydrochloride (5.00g, 30.38mmol) in toluene solvent, and add TEA (4.65 mL), heated to reflux at 115°C overnight, and the reaction was monitored by TLC. After the reaction, the solvent was dried under reduced pressure, and the crude product was separated and purified through a silica gel column (DCM:MeOH=50:1, v/v) to obtain the target product 2-1, a dark green solid (8.00g, yield 96 %).

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.18 (brs, 1H), 11.08 (s, 1H), 7.65 (t, J = 7.8Hz, 1H), 7.32 (d, J = 7.1Hz, 1H) , 7.25 (d, J=8.4Hz, 1H), 5.09-5.04 (m, 1H), 2.92-2.85 (m, 1H), 2.64-2.53 (m, 2H), 2.04-2.00 (m, 1H).

(b) tert-butyl 2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetate (2-2)

Compound 2-1 (2.00g, 7.33mmol) was dissolved in ultra-dry DMF, and KI (122mg, 0.733mmol) and KHCO ₃ (1.10g, 11.00mmol) were added. Then tert-butyl bromoacetate (1.28 mL, 8.80 mmol) was dissolved in 5 mL of ultra-dry DMF, and added to the reaction system in batches under argon protection, stirred at 60°C overnight, and monitored the reaction by TLC. After the reaction, dichloromethane and water were added to the reaction system for extraction. The extraction step was repeated three times. The organic layers were combined and extracted with saturated brine. The organic layers were separated and combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated and purified through a silica gel column (DCM:MeOH=100:1, v/v) to obtain the target product 2-2 as a white solid (2.15g, yield 76%).

¹ H NMR (400MHz, DMSO-d ₆ ) δ 11.11 (s, 1H), 7.80 (dd, J=8.3, 7.5Hz, 1H), 7.48 (d, J=7.2Hz, 1H), 7.38 (d, J=8.6Hz, 1H), 5.10 (dd, J=12.8, 5.3Hz, 1H), 4.97 (s, 2H), 2.93-2.85 (m, 1H), 2.61-2.53 (m, 2H), 2.06-2.01 (m, 1H), 1.43 (s, 9H).

(c) 2-(2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetic acid (2-3)

Compound 2-2 (2.05g, 5.20mmol) was dissolved in dichloromethane (100mL), trifluoroacetic acid (5mL) was added, the reaction was stirred at room temperature, and the reaction was monitored by TLC. After the reaction, the solvent was evaporated under reduced pressure. The target product 2-3 was obtained as a white solid (1.40g, yield 81%), and the product was directly used in the next reaction.

¹ H NMR (400MHz, DMSO-d ₆ ) δ 11.11 (s, 1H), 7.79 (dd, J=8.4, 7.4Hz, 1H), 7.48 (d, J=7.2Hz, 1H), 7.39 (d, J=8.6Hz, 1H), 5.10 (dd, J=12.8, 5.3Hz, 1H), 4.99 (s, 2H), 2.94-2.90 (m, 1H), 2.61-2.53 (m, 2H), 2.06-2.01 (m,1H).

(d) (2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido )ethyl) tert-butyl carbamate (2-4)

Dissolve compound 2-3 (300 mg, 0.90 mmol) in ultra-dry DMF, add HATU (411 mg, 1.08 mmol), add DIPEA (0.5 mL, 2.70 mmol) under argon protection, stir and react at room temperature for 0.5 h, and then add N-BOC-ethylenediamine (173 mg, 1.08 mmol), stir the reaction at room temperature, and monitor the reaction by TLC. After the reaction, dichloromethane and water were added to the reaction system for three extractions. The organic layers were combined and extracted once with saturated brine. The organic layers were separated and combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated and purified through a silica gel column (DCM:MeOH=100:1, v/v) to obtain the target product 2-4, a light yellow-green solid (136 mg, yield 23%).

¹ H NMR (400MHz, DMSO-d ₆ ) δ 11.11 (s, 1H), 8.03 (t, J = 5.1Hz, 1H), 7.81 (t, J = 8.0Hz, 1H), 7.50 (d, J = 7.2Hz, 1H), 7.39 (d, J=8.5Hz, 1H), 6.84 (t, J=5.2Hz, 1H), 5.15-5.09 (m, 1H), 4.76 (s, 2H), 3.19-3.15 ( m, 2H), 3.03-2.99 (m, 2H), 2.90-2.85 (m, 1H), 2.62-2.50 (m, 2H), 2.05-2.00 (m, 1H), 1.36 (s, 9H).

(e) N-(2-aminoethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoquinolin-4-yl)oxy )acetamide(2-5)

Compound 2-4 (136 mg, 0.36 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (1 mL) was added, the reaction was stirred at room temperature, and the reaction was monitored by TLC. After the reaction, the solvent was evaporated under reduced pressure. The product is 2-5, which is directly used in the next reaction.

(f)2-(5-(5-bromo-2-methoxyphenyl)sulfonylamino)-3-methylbenzo[d]isoxazol-6-yl)-N-(2-( 2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide)ethyl) (Example 1)

Compound T1 (50 mg, 0.11 mmol) and HATU (46 mg, 0.12 mmol) were dissolved in DMF, DIPEA (0.1 mL, 0.53 mmol) was added, and the reaction was stirred for 0.5 h. Then add 2-5 (44 mg, 0.12 mmol) to the reaction system, stir the reaction at room temperature, and monitor the reaction with TLC. After the reaction is completed, dichloromethane and water are added to the reaction system for extraction. Repeat three times. The organic layers are combined and extracted with saturated brine. The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography column (DCM:MeOH=100:1, v/v), and the target product was obtained as compound 1, a white solid (33 mg, yield 38%).

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.12 (s, 1H), 9.67 (s, 1H), 8.21 (s, 1H), 8.17 (s, 1H), 7.77 (t, J=7.8Hz, 1H), 7.71-7.69 (m, 3H), 7.45 (d, J=7.2Hz, 1H), 7.36 (d, J=8.5Hz, 1H), 7.31 (s, 1H), 7.08 (d, J=8.5 Hz, 1H), 5.13-5.10 (m, 1H), 4.79 (s, 2H), 4.36 (s, 2H), 3.53 (s, 3H), 3.24-3.20 (m, 4H), 2.93-2.86 (m, 1H), 2.62-2.55(m, 2H), 2.47(s, 3H), 2.04-2.03(m, 1H); ¹³ C NMR (126MHz, DMSO-d ₆ ) δ172.71, 169.82, 167.36, 166.67, 166.66 , 165.37, 161.14, 155.92, 154.99, 153.31, 137.13, 136.84, 133.01, 131.36, 129.50, 123.12, 120.32, 118.53, 116.80, 115.99, 115.00, 114.85, 1 10.61, 93.49, 67.67, 67.55, 56.18, 48.79, 38.03, 30.92 , 21.98, 9.43; MS (ESI) m/z [MH] ^- theoretical value: 825.09 and 827.09, actual value: 825.8 and 827.5; HPLC analysis: MeOH-H ₂ O (85: 15), 9.26min, 98.26% purity .

Example 2

2-((5-((5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(3- (2-((2-(2,6))-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)propyl) Acetamide

The synthetic method refers to the synthetic route of Example 1.

The only difference is that the N-BOC-ethylenediamine raw material is replaced by an equal molar amount of N-tert-butoxycarbonyl-1,3-propanediamine raw material.

Compound 2 was a white solid, and the yield was 28%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ11.11 (s, 1H), 9.74 (s, 1H), 8.25-8.23 (m, 1H), 8.12-8.11 (m, 1H), 7.80 (t, J =7.6Hz, 1H), 7.71-7.68 (m, 3H), 7.48 (d, J = 7.0Hz, 1H), 7.40 (d, J = 8.3Hz, 1H), 7.29 (s, 1H), 7.07 (d , J=8.6Hz, 1H), 5.13-5.10(m, 1H), 4.80(s, 2H), 4.38(s, 2H), 3.55(s, 3H), 3.22-3.06(m, 4H), 2.97- 2.81 (m, 1H), 2.67-2.53 (m, 2H), 2.49-2.44 (m, 3H), 2.09-1.98 (m, 1H), 1.65-1.53 (m, 2H); ¹³ C NMR (126MHz, DMSO -d ₆ )δ172.74, 169.84, 166.85, 166.71, 166.42, 165.42, 155.91, 155.06, 154.96, 153.53, 137.03, 136.91, 133.03, 131.28, 120.41, 116.81, 116. 02, 114.99, 114.85, 110.58, 93.55, 67.75, 67.64, 56.17, 48.80, 36.20, 36.10, 30.93, 29.21, 21.99, 9.43; MS (ESI) m/z [MH] ^- theoretical value: 839.11 and 841.11; actual value: 839.5 and 841.8; HPLC analysis: MeOH-H ₂ O(85:15), 11.20min, 97.32% purity.

Example 3

2-((5-((5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(4- (2-((2-(2,6))-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)butyl )acetamide

The synthetic method refers to the synthetic route of Example 1.

The only difference is that the N-BOC-ethylenediamine raw material is replaced by an equal molar amount of N-tert-butoxycarbonyl-1,4-butanediamine raw material.

Compound 3 was a white solid with a yield of 25%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.12 (s, 1H), 9.70 (s, 1H), 8.06 (t, J = 5.4Hz, 1H), 7.96 (t, J = 5.4Hz, 1H) , 7.80 (t, J=7.9Hz, 1H), 7.72-7.70 (m, 3H), 7.49 (d, J=7.2Hz, 1H), 7.38 (d, J=8.5Hz, 1H), 7.30 (s, 1H), 7.08(d, J=9.5Hz, 1H), 5.15-5.10(m, 1H), 4.77(s, 2H), 4.38(s, 2H), 3.54(s, 3H), 3.15-3.10(m ¹³ C NMR (126MHz, DMSO-d ₆ ) δ 172.72, 169.83, 166.69 (2×C), 166.23, 165.48, 161.14, 155.91, 155.04, 154.99, 153.43, 137.14, 136.90, 133.01, 131.26, 12 9.58, 123.19, 120.40 , 118.60, 116.81, 116.03, 115.00, 114.83, 110.58, 93.50, 67.71, 67.65, 56.17, 48.80, 38.01 (2×C), 30.93, 26.54, 26.49, 21.97, 9.42; MS (ESI) m/z [MH] ^- Theoretical values: 853.12 and 855.12; actual values: 853.4 and 855.5; HPLC analysis: MeOH-H ₂ O (85:15), 9.40 min, 95.21% purity.

Example 4

2-(5-(5-bromo-2-methoxyphenyl)sulfonylamino)-3-methylbenzo[d]isoxazol-6-yl)-N-(5-(2-( 2,6-Dioxypiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide)valeramide

The synthetic method refers to the synthetic route of Example 1.

The only difference is that the N-BOC-ethylenediamine raw material is replaced by an equimolar amount of mono-BOC-pentanediamine raw material.

Compound 4 was a white solid with a yield of 29%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.11 (s, 1H), 9.77 (s, 1H), 8.03 (brs, 1H), 7.92 (brs, 1H), 7.80 (t, J=7.8Hz, 1H), 7.72-7.70 (m, 3H), 7.48 (d, J=7.2Hz, 1H), 7.39 (d, J=8.5Hz, 1H), 7.29 (s, 1H), 7.08 (d, J=8.8 Hz, 1H), 5.15-5.12 (m, 1H), 4.78 (s, 2H), 4.39 (s, 2H), 3.55 (s, 3H), 3.13-3.08 (m, 4H), 2.94-2.87 (m, 1H), 2.63-2.56(m, 2H), 2.49(s, 3H), 2.06-2.04(m, 1H), 1.39-1.36(m, 4H), 1.30-1.21(m, 2H). ¹³ C NMR ( 126MHz, DMSO-d ₆ ) δ 172.76, 169.86, 166.73, 166.65, 166.26, 165.51, 161.09, 155.92, 155.07, 154.97, 153.59, 137.03, 136.90, 133.02, 131.26, 129 .85, 120.39, 116.79, 116.01, 114.99, 114.84 , 110.59, 93.49, 67.77, 67.64, 56.18, 48.83, 38.27, 38.26, 30.95, 28.79, 28.66, 23.69, 22.00, 9.44.MS(ESI)m/z[MH] ^-Theoretical value: 867.14 and 869.14; actual value: 867.5 and 869.2. HPLC analysis: MeOH-H ₂ O (85:15), 9.44 min, 98.43% purity.

Example 5

2-(5-(5-bromo-2-methoxyphenyl)sulfonylamino)-3-methylbenzo[d]isoxazol-6-yl)-N-(6-(2-( 2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamido)hexanoamide)

The synthetic method refers to the synthetic route of Example 1.

The only difference is that the N-BOC-ethylenediamine raw material is replaced by an equal molar amount of N-(6-aminohexyl)carbamic acid tert-butyl ester raw material.

Compound 5 was a white solid with a yield of 69%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.12 (s, 1H), 9.70 (s, 1H), 8.03 (s, 1H), 7.92 (s, 1H), 7.81 (t, J=7.7Hz, 1H), 7.71 (d, J=10.0Hz, 3H), 7.49 (d, J=7.0Hz, 1H), 7.40 (d, J=8.4Hz, 1H), 7.29 (s, 1H), 7.09 (d, J=8.6Hz, 1H), 5.21-5.05 (m, 1H), 4.77 (s, 2H), 4.40 (s, 2H), 3.56 (s, 3H), 3.22-3.01 (m, 4H), 2.96-2.82 (m, 1H), 2.58 (m, 2H), 2.37 (s, 3H), 2.03 (m, 1H), 1.45-1.32 (m, 4H), 1.22 (m, 4H). ¹³ C NMR (126MHz, DMSO -d ₆ )δ172.74, 169.85, 166.72, 166.62, 166.19, 165.51, 161.17, 155.92, 155.02, 154.99, 153.47, 137.13, 136.90, 133.02, 131.23, 129.66, 123. 22, 120.39, 118.67, 116.84, 116.05, 115.00, 114.84, 110.61, 93.45, 67.72, 67.68, 56.18, 48.83, 38.25, 30.95, 29.11, 28.94, 26.04, 25.97, 22.00, 9.43.MS(ESI)m/z[MH] ^-Theoretical value: 881.15 and 883.15; actual value : 881.6 and 883.5. HPLC analysis: MeOH-H ₂ O (85:15), 9.86 min, 98.60% purity.

Example 6

2-(5-(5-bromo-2-methoxyphenyl)sulfonylamino)-3-methylbenzo[d]isoxazol-6-yl)-N-(2-(2-( 2,6-Dioxypiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide)acetamide

The synthetic route of compound 6 is as follows:

(a) 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (3-1)

Dissolve 3-fluorophthalanhydride (5.00g, 30.10mmol), 3-amino-2,6-piperidine-dione hydrochloride (4.95g, 30.10mmol) and sodium acetate (2.95g, 36.00mmol) in acetic acid The solution was heated to reflux at 120°C overnight, and the reaction was monitored by TLC. After the reaction is completed, add ice water until the solid is washed out, perform suction filtration, wash the filter cake with water several times, and dry the filter cake in a drying oven at 60°C. The target product was obtained as 3-1, an off-white solid (7.32g, yield 88%).

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.15 (s, 1H), 7.95 (m, 1H), 7.79 (d, J = 7.4Hz, 1H), 7.73 (t, J = 8.9Hz, 1H) , 5.18-5.14(m, 1H), 2.93-2.85(m, 1H), 2.62-2.52(m, 2H), 2.09-2.05(m, 1H).

(b) (2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)tert-butylcarbamate (3- 2)

Compound 3-1 (100 mg, 0.36 mmol) was dissolved in ultra-dry DMF, N-BOC-ethylenediamine (0.06 mL, 0.40 mmol) was added under argon protection, the reaction was stirred at 80°C, and the reaction was monitored by TLC. After the reaction is completed, dichloromethane and water are added to the reaction system for three extractions, then the organic layers are combined and extracted once with saturated brine. The organic layers were separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified and separated through a silica gel column (DCM:MeOH=100:1, v/v) to obtain the target product 3-2, a yellow solid (30 mg, yield 20%).

¹ H NMR (400MHz, DMSO-d ₆ ) δ11.08 (s, 1H), 7.57 (t, J=7.8Hz, 1H), 7.14 (d, J=8.6Hz, 1H), 7.03-7.00 (m, 2H), 6.70 (t, J=5.2Hz, 1H), 5.05 (dd, J=12.7, 5.2Hz, 1H), 3.37-3.32 (m, 2H), 3.12-3.11 (m, 2H), 2.93-2.84 (m, 1H), 2.61-2.54 (m, 2H), 2.03-2.00 (m, 1H), 1.36 (s, 9H).

(c) 4-((2-Aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (3-3)

Compound 3-2 (30 mg, 0.07 mmol) was dissolved in dichloromethane solvent (5 mL), trifluoroacetic acid (0.5 mL) was added, the reaction was stirred at room temperature, and the reaction was monitored by TLC. After the reaction, the solvent was dried under reduced pressure. Product 3-3 was directly used in the next reaction.

(d)2-(5-(5-bromo-2-methoxyphenyl)sulfonylamino)-3-methylbenzo[d]isoxazol-6-yl)-N-(2-( 2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide)acetamide (Example 6)

Compound T-1 (34 mg, 0.07 mmol) and HATU (33 mg, 0.09 mmol) were dissolved in DMF, DIPEA (0.07 mL, 0.37 mmol) was added, and the reaction was stirred for 0.5 h. Then add 2-3 (23 mg, 0.07 mmol) to the reaction system, stir the reaction at room temperature, and monitor the reaction with TLC. After the reaction is completed, dichloromethane and water are added to the reaction system for extraction. Repeat three times. The organic layers are combined and extracted with saturated brine. The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography column (DCM:MeOH=100:1, v/v), and the target product was obtained as compound 6, a yellow solid (20 mg, yield 36%).

¹ H NMR (400MHz, CDCl ₃ ) δ 8.40 (s, 1H), 7.90 (s, 1H), 7.63-7.59 (m, 2H), 7.52-7.42 (m, 3H), 7.05-7.04 (m, 1H) ), 6.91-6.86 (m, 3H), 6.35 (t, J=5.0Hz, 1H), 4.95-4.93 (m, 1H), 4.49 (s, 2H), 3.88 (s, 3H), 3.56-3.55 ( m, 2H), 3.46-3.45 (m, 2H), 2.88-2.73 (m, 3H), 2.47 (s, 3H), 2.22-2.15 (m, 1H), . ¹³ C NMR (126MHz, DMSO-d ₆ )δ172.75, 170.05, 168.66, 167.23, 167.19, 161.09, 155.92, 154.96, 153.32, 146.18, 137.12, 136.18, 132.20, 131.27, 129.48, 123.09, 118.56, 1 16.95, 115.01, 114.86, 110.59, 110.55, 109.32, 93.48 , 67.67, 56.19, 48.53, 41.45, 37.85, 30.96, 22.13, 9.41.MS (ESI) m/z [MH] ^- theoretical value: 767.08 and 769.08; actual value: 767.0 and 769.5. HPLC analysis: MeOH-H ₂ 0 (70:30), 13.75min, 97.84% purity.

Example 7

2-(5-(5-bromo-2-methoxyphenyl)sulfonylamino)-3-methylbenzo[d]isoxazol-6-yl)-N-(3-(2-( 2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamidopropyl)

The synthetic method refers to the synthetic route of Example 6.

Compound 7 was a yellow solid with a yield of 46%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.10 (s, 1H), 9.70 (s, 1H), 8.17 (t, J = 4.9Hz, 1H), 7.71-7.70 (m, 3H), 7.56 ( t, J=5.8Hz, 1H), 7.32 (s, 1H), 7.08 (d, J=8.7Hz, 1H), 7.05-6.98 (m, 2H), 6.66 (t, J=6.0Hz, 1H), 5.06-5.02(m, 1H), 4.41(s, 2H), 3.54(s, 3H), 3.33-3.26(m, 2H), 3.23-3.19(m, 2H), 2.95-2.78(m, 1H), 2.63-2.56 (m, 2H), 2.49 (s, 3H), 2.03-2.01 (m, 1H), 1.71-1.65 (m, 2H). ¹³ C NMR (126MHz, DMSO-d ₆ ) δ 172.74, 170.04 , 168.81, 167.25, 166.54, 161.14, 155.91, 154.98, 153.40, 146.20, 137.11, 136.20, 132.23, 131.20, 129.63, 123.16, 118.61, 117.00, 115.03, 1 14.83, 110.59, 110.44, 109.22, 93.44, 67.68, 56.17, 48.53 , 35.94, 30.95, 28.87, 20.73, 22.13, 9.41.MS (ESI) m/z [MH] ^- theoretical value: 781.10 and 783.10; actual value: 781.1 and 783.0. HPLC analysis: MeOH-H ₂ O (85: 15 ), 9.10min, 95.11% purity.

Example 8

2-((5-((5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(4- ((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)acetamide

The synthetic method refers to the synthetic route of Example 6.

Compound 8 was a yellow solid, and the yield was 54%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ11.09 (s, 1H), 9.69 (s, 1H), 8.08 (s, 1H), 7.71-7.69 (m, 3H), 7.56 (t, J=7.7 Hz, 1H), 7.30 (s, 1H), 7.09-7.06 (m, 2H), 7.02-7.01 (m, 1H), 6.52 (t, J=5.1Hz, 1H), 5.06-5.03 (m, 1H) , 4.39(s, 2H), 3.54(s, 3H), 3.32-3.31(m, 2H), 3.16-3.14(m, 2H), 2.98-2.80(m, 1H), 2.67-2.55(m, 2H) , 2.49-2.38 (s, 3H), 2.09-1.97 (m, 1H), 1.63-1.41 (m, 4H). ¹³ C NMR (126MHz, DMSO-d ₆ ) δ172.75, 170.05, 168.91, 167.26, 166.25 , 161.12, 155.90, 154.97, 153.43, 146.34, 137.11, 136.24, 132.16, 131.23, 129.64, 123.20, 118.61, 117.14, 114.99, 114.82, 110.58, 110.40, 1 09.05, 93.47, 67.70, 56.15, 48.53, 41.47, 38.01, 30.95 , 26.59, 26.16, 22.13, 9.41.MS (ESI) m/z [MH] ^- theoretical value: 795.12 and 797.11; actual value: 795.5 and 797.1. HPLC analysis: MeOH-H ₂ O (70: 30), 18.16min , 98.93% purity.

Example 9

2-((5-((5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(5- ((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentyl)acetamide

The synthetic method refers to the synthetic route of Example 6.

Compound 9 was a yellow solid with a yield of 81%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.08 (s, 1H), 9.70 (s, 1H), 8.04 (t, J = 5.1Hz, 1H), 7.72-7.69 (m, 3H), 7.59- 7.56 (m, 1H), 7.30 (s, 1H), 7.09-7.01 (m, 3H), 6.50 (t, J=5.6Hz, 1H), 5.06-5.03 (m, 1H), 4.39 (s, 2H) , 3.54(s, 3H), 3.32-3.21(m, 2H), 3.12-3.09(m, 2H), 2.91-2.84(m, 1H), 2.63-2.55(m, 2H), 2.49(s, 3H) , 2.04-2.01 (m, 1H), 1.59-1.53 (m, 2H), 1.45-1.39 (m, 2H), 1.34-1.26 (m, 2H). ¹³ C NMR (126MHz, DMSO-d ₆ ) δ172. 74, 170.04, 168.93, 167.26, 166.17, 161.14, 155.90, 154.98, 153.45, 146.36, 137.11, 136.24, 132.16, 131.20, 129.68, 123.22, 118.64, 117.10 ,115.01,114.81,110.58,110.39,109.05,93.46,67.72, 56.15, 48.53, 41.76, 38.17, 30.95, 28.86, 28.34, 23.66, 22.13, 9.41. MS (ESI) m/z [MH] ^- theoretical value: 809.13 and 811.13; actual value: 809.8 and 811.5. HPLC analysis: MeOH- H ₂ O (85:15), 5.46 min, 98.91% purity.

Example 10

2-((5-((5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(6- ((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)acetamide

The synthetic method refers to the synthetic route of Example 6.

Compound 10 was a white solid, and the yield was 71%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.09 (s, 1H), 9.69 (s, 1H), 8.02 (brs, 1H), 7.71-7.69 (m, 3H), 7.57 (t, J=7.7 Hz, 1H), 7.29 (s, 1H), 7.09-7.06 (m, 2H), 7.01 (d, J=7.0Hz, 1H), 6.50 (t, J=5.0Hz, 1H), 5.05 (dd, J =12.6, 5.3Hz, 1H), 4.39(s, 2H), 3.55(s, 3H), 3.32-3.24(m, 2H), 3.11-3.08(m, 2H), 2.91-2.84(m, 1H), 2.61-2.53(m, 2H), 2.49(s, 3H), 2.03-2.01(m, 1H), 1.53-1.50(m, 2H), 1.39-1.23(m, 6H). ¹³ C NMR (126MHz, DMSO -d ₆ )δ172.75, 170.04, 168.94, 167.27, 166.15, 161.15, 155.90, 154.97, 153.47, 146.39, 137.09, 136.23, 132.16, 131.19, 129.69, 123.22, 118. 65, 117.11, 115.00, 114.81, 110.59, 110.36, 109.02，93.45，67.72，56.16，48.53，41.75，38.25，30.96，29.08，28.61，26.09，26.02，22.14，9.40.MS(ESI)m/z[MH] ^-理论值：823.15和825.15；实际值：823.5 and 825.5. HPLC analysis: MeOH-H ₂ O (70:30), 22.45 min, 99.55% purity.

Example 11

2-((5-((5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(2- ((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethyl)acetamide

The synthetic method refers to the synthetic route of Example 6.

The only difference is that the 3-fluorophthalic anhydride raw material is replaced by an equal molar amount of 4-fluorophthalic anhydride raw material.

The target product was obtained as compound 11 as a yellow solid (70 mg, yield 54%).

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.06 (s, 1H), 9.63 (s, 1H), 8.30-8.29 (m, 1H), 7.71-7.70 (m, 3H), 7.56 (d, J =8.5Hz, 1H), 7.32 (s, 1H), 7.15 (t, J = 5.5Hz, 1H), 7.09 (d, J = 9.5Hz, 1H), 6.99 (s, 1H), 6.87 (d, J =8.4Hz, 1H), 5.05-5.01(m, 1H), 4.39(s, 2H), 3.56(s, 3H), 3.27-3.25(m, 2H), 3.17-3.16(m, 1H), 2.87- 2.85 (m, 1H), 2.64-2.50 (m, 2H), 2.47 (s, 3H), 2.36 (brs, 1H), 2.00-1.99 (m, 1H). ¹³ C NMR (126MHz, DMSO-d ₆ ) δ172.68, 170.03, 167.51, 167.01, 166.89, 161.06, 155.87, 154.91, 154.11, 153.27, 137.08, 134.17, 131.22, 129.44, 124.98, 123.05, 118.44, 1 16.40, 114.99, 114.83, 110.52, 93.49, 67.69, 56.15, 48.59, 48.51, 41.68, 37.50, 30.91, 22.14, 9.35. MS (ESI) m/z [M+H] ⁺ theoretical value: 769.08 and 71.08; actual value: 769.11 and 771.17. HPLC analysis: MeOH-H ₂ O ( 80:20), 4.70min, 95.15% purity.

Example 12

2-((5-((5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(3- ((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)propyl)acetamide

The synthetic method refers to the synthetic route of Example 7.

Compound 12 was a yellow solid, and the yield was 38%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.06 (s, 1H), 9.69 (s, 1H), 8.15 (t, J = 5.6Hz, 1H), 7.71-7.70 (m, 3H), 7.55 ( d, J=8.3Hz, 1H), 7.30 (s, 1H), 7.09-7.08 (m, 2H), 6.92 (s, 1H), 6.80 (d, J=8.4Hz, 1H), 5.05-5.01 (m , 1H), 4.42(s, 2H), 3.56(s, 3H), 3.24-3.22(m, 2H), 3.14-3.12(m, 2H), 2.89-2.85(m, 1H), 2.63-2.56(m , 2H), 2.47 (s, 3H), 2.01-1.98 (m, 1H), 1.72-1.69 (m, 2H). ¹³ C NMR (126MHz, DMSO-d ₆ ) δ 172.72, 170.08, 167.62, 167.09, 166.47, 161.10, 155.89, 154.92, 154.26, 153.48, 137.05, 134.13, 131.15, 129.65, 124.98, 123.22, 118.56, 116.00, 115.01, 114.83, 110.57, 93 .48, 67.73, 56.15, 52.99, 48.61, 41.39, 36.24, 30.94, 28.29, 22.20, 9.36. MS (ESI) m/z [M+H] ⁺ theoretical value: 783.10 and 785.10; actual value: 783.08 and 785.13. HPLC analysis: MeOH-H ₂ O (80: 20), 4.70min, 98.83% pure.

Example 13

2-((5-((5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(4- ((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)butyl)acetamide

For the synthesis method, refer to the synthesis route of Example 8.

Compound 13 was a yellow solid with a yield of 49%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.05 (s, 1H), 9.69 (s, 1H), 8.08 (brs, 1H), 7.72-7.69 (m, 3H), 7.55 (d, J=8.3 Hz, 1H), 7.31 (s, 1H), 7.11-7.07 (m, 2H), 6.94 (s, 1H), 6.83 (d, J=8.3Hz, 1H), 5.04-5.01 (m, 1H), 4.39 (s, 2H), 3.54 (s, 3H), 3.31-3.29 (m, 2H), 3.16-3.13 (m, 2H), 2.91-2.87 (m, 1H), 2.64-2.59 (m, 2H), 2.47 (s, 3H), 2.01-1.97 (m, 1H), 1.55-1.47 (m, 4H). ¹³ C NMR (126MHz, DMSO-d ₆ ) δ 172.69, 170.07, 167.63, 167.06, 166.23, 161.09, 155.87 , 154.93, 154.35, 153.41, 137.08, 134.16, 131.21, 129.60, 125.01, 123.18, 118.52, 115.85, 114.98, 114.80, 110.55, 93.47, 67.70, 56.12, 54.8 2, 48.58, 42.08, 37.98, 30.92, 26.74, 25.60, 22.17 , 9.36.MS(ESI)m/z[M+H] ⁺ theoretical value: 797.12 and 799.11; actual value: 797.23 and 799.35.HPLC analysis: MeOH-H ₂ O (80:20), 4.84min, 99.59% purity .

Example 14

2-((5-((5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(5- ((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)pentyl)acetamide

The synthetic method refers to the synthetic route of Example 9.

Compound 14 was a yellow solid, and the yield was 36%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.05 (s, 1H), 9.70 (s, 1H), 8.05 (brs, 1H), 7.72-7.69 (m, 3H), 7.55 (d, J=8.3 Hz, 1H), 7.30 (s, 1H), 7.09-7.06 (m, 2H), 6.92 (s, 1H), 6.82 (d, J=8.4Hz, 1H), 5.04-5.01 (m, 1H), 4.39 (s, 2H), 3.54 (s, 3H), 3.13-3.08 (m, 4H), 2.88-2.84 (m, 1H), 2.63-2.55 (m, 2H), 2.47 (s, 3H), 2.00-1.97 (m, 1H), 1.58-1.53 (m, 2H), 1.45-1.39 (m, 2H), 1.34-1.30 (m, 2H). ¹³ C NMR (126MHz, DMSO-d ₆ ) δ 172.70, 170.07, 167.63, 167.07, 166.16, 161.12, 155.87, 154.93, 154.36, 153.44, 137.07, 134.14, 131.17, 129.63, 125.01, 123.17, 118.59, 115.81, 114.99, 11 4.79, 110.56, 93.45, 67.69, 56.13, 48.58, 42.37, 38.17, 30.92, 28.86, 27.85, 23.83, 22.18, 9.36. MS (ESI) m/z [M+H] ⁺ theoretical value: 811.13 and 813.13; actual value: 811.21 and 813.17. HPLC analysis: MeOH-H ₂ O (80: 20), 5.06 min, 97.47% purity.

Example 15

2-(5-(5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)-N-(2-(2-( 2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)hexyl)acetamide

For the synthesis method, refer to the synthesis route of Example 10.

Compound 15 was a yellow solid with a yield of 50%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.05 (s, 1H), 9.70 (s, 1H), 8.03 (brs, 1H), 7.71-7.69 (m, 3H), 7.55 (d, J=8.4 Hz, 1H), 7.29 (s, 1H), 7.09-7.07 (m, 2H), 6.93 (s, 1H), 6.83 (d, J=8.7Hz, 1H), 5.04-5.01 (m, 1H), 4.39 (s, 2H), 3.54 (s, 3H), 3.14-3.07 (m, 4H), 2.88-2.84 (m, 1H), 2.63-2.55 (m, 2H), 2.49 (s, 3H), 2.00-1.98 (m, 1H), 1.55-1.49 (m, 2H), 1.39-1.31 (m, 4H), 1.27-1.24 (m, 2H). ¹³ C NMR (126MHz, DMSO-d ₆ ) δ 172.70, 170.07, 167.63, 167.07, 166.13, 161.12, 155.87, 154.93, 154.38, 153.44, 137.07, 134.15, 131.17, 129.63, 125.02, 123.17, 118.58, 115.77, 114.98, 11 4.79, 110.56, 99.45, 93.43, 67.70, 56.13, 48.58, 42.35, 38.24, 30.92, 29.08, 28.12, 26.17, 26.09, 22.18, 9.36.MS (ESI) m/z [M+H] ⁺ theoretical value: 825.15 and 827.15; actual value: 825.31 and 827.36. HPLC analysis: MeOH-H ₂ O(80:20), 5.28min, 98.81% purity.

Example 16

2-(5-(5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(3-( 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)propionyl-2-yn-1-yl)acetamide

The synthetic route of compound 16 is as follows:

(a) tert-butyl (3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)propionic acid-2-yne-1- base) carbamate (4-1)

Compound 3-(4-iodo-1-oxoisoindolin-2-yl)piperidine-2,6-dione (3.0g, 9.28mmol), N-Boc-aminopropyne (4.32g, 27.85mmol), Pd(PPh ₃ ) ₂ Cl ₂ (652mg, 0.93mmol) and CuI (177mg, 0.93mmol) were added to a dry two-neck bottle and sealed. After adding 30mL ultra-dry DMF to dissolve and 19.36mL triethylamine, use After argon gas was exchanged, the reaction was carried out at 80°C for 12 hours, and the reaction was monitored by TLC. After the reaction is completed, dichloromethane and water are added to the reaction system for extraction. Repeat three times. The organic layers are combined and extracted with saturated brine. The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified through a silica gel chromatography column (DCM:MeOH=100:1, v/v) to obtain the target product 4-1, a brown solid (2.4 g, yield 65%).

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.02 (s, 1H), 7.74 (d, J = 7.5Hz, 1H), 7.66 (d, J = 7.2Hz, 1H), 7.55 (t, J = 7.6Hz, 1H), 7.40 (br, 1H), 5.17 (dd, J=13.3, 5.1Hz, 1H), 4.45 (d, J=17.7Hz, 1H), 4.30 (d, J=17.7Hz, 1H) , 4.02 (d, J=5.4Hz, 2H), 2.97-2.90 (m, 1H), 2.65-2.57 (m, 1H), 2.43-2.34 (m, 1H), 2.08-2.00 (m, 1H), 1.40 (s,9H).

(b) 3-(4-(3-Aminoprop-1-yn-1-yl)-1-oxoisoquinolin-2-yl)piperidine-2,6-dione (4-2)

Compound 4-1 (1.00g, 2.52mmol) was dissolved in dichloromethane solvent (10mL), trifluoroacetic acid (2mL) was added, and the reaction was stirred at room temperature for 2h, and the reaction was monitored by TLC. After the reaction, dichloromethane and water were added to the reaction system for three extractions. The organic layers were combined and extracted with saturated brine. The organic layers were separated and combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was separated and purified through a silica gel column (DCM:MeOH=100:1, v/v) to obtain the target product 4-2, a brown solid (730 mg, yield 98%).

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.06 (s, 1H), 8.37 (s, 2H), 7.82 (d, J = 7.5Hz, 1H), 7.71 (d, J = 7.2Hz, 1H) , 7.60 (t, J=7.6Hz, 1H), 5.21 (dd, J=13.3, 5.1Hz, 1H), 4.49 (d, J=17.8Hz, 1H), 4.35 (d, J=17.8Hz, 1H) , 4.06 (s, 2H), 3.00-2.90 (m, 1H), 2.67-2.58 (m, 1H), 2.39-2.29 (m, 1H), 2.12-2.02 (m, 1H).

(c)2-(5-(5-bromo-2-methoxyphenyl)sulfonamido)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-( 3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)propionyl-2-yn-1-yl)acetamide (Example 16)

Compound T-1 (80 mg, 0.17 mmol) and HATU (100 mg, 0.30 mmol) were dissolved in DMF, DIPEA (0.10 mL, 0.51 mmol) was added, and the reaction was stirred for 0.5 h. Then add 3-2 (79 mg, 0.20 mmol) to the reaction system, stir the reaction at room temperature, and monitor the reaction with TLC. After the reaction is completed, dichloromethane and water are added to the reaction system for extraction. Repeat three times. The organic layers are combined and extracted with saturated brine. The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography column (DCM:MeOH=100:1, v/v), and the target product was obtained as compound 16, a white solid (70 mg, yield 55%).

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.00 (s, 1H), 9.76 (s, 1H), 8.81 (t, J = 5.5Hz, 1H), 7.76 (d, J = 7.3Hz, 1H) , 7.72-7.71 (m, 2H), 7.70-7.67 (m, 1H), 7.65 (d, J=7.0Hz, 1H), 7.55 (t, J=7.6Hz, 1H), 7.33 (s, 1H), 7.08(d, J=8.9Hz, 1H), 5.16-5.13(m, 1H), 4.48(s, 2H), 4.31-4.29(m, 2H), 3.61(s, 3H), 2.94-2.87(m, 1H), 2.63-2.57(m, 2H), 2.47(s, 3H), 2.44-2.36(m, 2H), 2.02-1.99(m, 1H). ¹³ C NMR (126MHz, DMSO-d ₆ ) δ172. 69.170.88 ,117.64,115.03,114.86,110.56,93.60,91.77,77.63, 67.65, 56.21, 51.61, 46.85, 31.12, 28.46, 22.35, 9.37. HRMS (ESI) m/z [M+H] ⁺ theoretical value: 750.51 and 752.51; actual value: 750.0840 and 752.0827. HPLC analysis: MeOH-H ₂ O(80:20), 4.92min, 96.37% purity.

Example 17

2-((5-((5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(3- (2-(2,6-dioxopiperidin-3-yl)-1-oxoisoquinolin-4-yl)propyl)acetamide

The synthetic route of compound 17 is as follows:

Steps (a) and (b) are the same as in Example 16.

The only difference is that step (d) is carried out: 3-(4-(3-aminopropyl)-1-oxoisoquinolin-2-yl)piperidine-2,6-dione (4-3)

Compound 4-2 (300 mg, 1.01 mmol) was dissolved in 20 mL of methanol. After replacing the gas with argon, 10% palladium on carbon was added, and the system was replaced with hydrogen three times. The reaction was stirred at room temperature for 24 h, and the reaction was monitored by TLC. After the reaction, the palladium carbon was removed by suction filtration and concentrated under reduced pressure. The crude product was purified through a silica gel chromatography column (DCM:MeOH=100:1-50:1, v/v) to obtain the target product 4-3, a white solid. (270 mg, yield 89%), proceed directly to the next reaction.

The synthesis method of step (e) refers to step (c) of Example 16, the only difference is that the raw material compound 4-2 is replaced by the raw material compound 4-3 prepared in this example.

Compound 17 was a white solid with a yield of 39%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.00 (s, 1H), 9.70 (s, 1H), 8.81 (t, J = 5.9Hz, 1H), 7.71-7.68 (m, 3H), 7.58 ( d, J=7.3Hz, 1H), 7.47-7.40 (m, 2H), 7.32 (s, 1H), 7.08 (d, J=8.7Hz, 1H), 5.15-5.11 (m, 1H), 4.46-4.41 (m, 3H), 4.29 (d, J=8.6Hz, 1H), 3.54 (s, 3H), 3.17-3.15 (m, 3H), 2.95-2.90 (m, 1H), 2.64-2.59 (m, 2H ), 2.47 (s, 3H), 2.43-2.36 (m, 2H), 2.02-1.99 (m, 1H), 1.74-1.71 (m, 1H). ¹³ C NMR (126MHz, DMSO-d ₆ ) δ 172.75 , 170.94, 168.25, 166.31, 161.12, 155.87, 154.94, 153.40, 140.46, 137.08, 136.65, 131.57, 131.32, 131.18, 129.60, 128.25, 123.14, 120.72, 1 18.60, 115.00, 114.81, 110.55, 93.43, 67.69, 56.14, 51.51 , 46.14, 37.97, 31.13, 29.13, 28.44, 22.47, 9.37.MS(ESI)m/z[M+H] ⁺ theoretical value: 754.11 and 756.11; actual value: 754.09 and 756.19.HPLC analysis: MeOH-H ₂ O (80:20), 4.88min, 96.98% purity.

Example 18

4-(5-(5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(3-( 2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)propionyl-2-yn-1-yl)butanamide

The synthetic route of compound 18 is as follows:

For the synthesis method, refer to the synthesis route of Example 16.

The only difference is that the T-1 raw material is replaced by an equal molar amount of T-2 raw material.

The compound was a white solid with a yield of 64%.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 11.01 (s, 1H), 9.20 (s, 1H), 8.36 (t, J = 5.3Hz, 1H), 7.74-7.70 (m, 3H), 7.61- 7.59(m, 2H), 7.52-7.49(m, 1H), 7.24(s, 1H), 7.14(d, J＝8.8Hz, 1H), 5.17-5.14(m, 1H), 4.43(d, J＝ 17.7Hz, 1H), 4.30 (d, J=17.7Hz, 1H), 4.19 (d, J=5.4Hz, 2H), 3.90 (t, J=6.4Hz, 2H), 3.72 (s, 3H), 2.95 -2.88(m, 1H), 2.61-2.53(m, 1H), 2.45(s, 3H), 2.43-2.36(m, 1H), 2.22(t, J=7.3Hz, 2H), 2.03-1.99(m , 1H), 1.81-1.74 (m, 2H). ¹³ C NMR (126MHz, DMSO-d ₆ ) δ 172.72, 171.47, 170.88, 167.44, 161.32, 155.90, 154.77, 154.57, 143.86, 136.97, 134.05, 131 .94, 131.01, 129.87, 128.57, 123.14, 123.03, 117.75, 117.69, 115.16, 113.86, 110.56, 92.56, 92.40, 77.33, 68.09, 56.25, 51.56, 46.81, 31.11, 28. 59, 23.97, 22.40, 9.33.HRMS(ESI)m/ z[M+H] ⁺ theoretical value: 778.11 and 780.11; actual value: 778.1144 and 780.1147. HPLC analysis: MeOH-H ₂ O (80:20), 5.03 min, 97.71% purity.

Example 19

4-(5-(5-bromo-2-methoxyphenyl)sulfonamide)-3-methylbenzo[d]isoxazol-6-yl)oxy)-N-(2-( 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)propyl)butanamide

For the synthesis method, refer to the synthesis route of Example 17.

Compound 19 was a white solid with a yield of 37.59%.

¹ H NMR (500MHz, DMSO-d6) δ11.01 (s, 1H), 9.76 (s, 1H), 8.80 (t, J=5.5Hz, 1H), 7.74 (d, J=7.3Hz, 1H), 7.72-7.70 (m, 2H), 7.70-7.68 (m, 1H), 7.65 (d, J=7.0Hz, 1H), 7.55 (t, J=7.6Hz, 1H), 7.33 (s, 1H), 7.08 (d, J=8.9Hz, 1H), 5.16-5.13 (m, 1H), 4.48 (s, 2H), 4.31-4.29 (m, 2H), 4.19 (d, J=5.4Hz, 2H), 3.90 ( t, J=6.4Hz, 2H), 3.61 (s, 3H), 2.94-2.87 (m, 1H), 2.63-2.57 (m, 2H), 2.47 (s, 3H), 2.44-2.36 (m, 2H) , 2.03-1.99 (m, 1H). ¹³ C NMR (126MHz, DMSO-d6) δ172.69, 170.88, 167.44, 166.39, 160.96, 155.90, 154.93, 152.96, 143.88, 137.17, 134.20, 132.02, 1 31.40, 129.10, 128.65, 123.29, 122.99, 117.90, 117.64, 115.03, 114.86, 110.56, 93.60, 91.77, 77.63, 67.65, 56.21, 51.61, 46.85, 31.12, 28.59, 23.97, 22.40 ,9.37.HRMS(ESI)m/z[M+ H] ⁺ theoretical value: 782.14 and 784.14; actual value: 782.1486 and 784.1462. HPLC analysis: MeOH-H ₂ O (80:20), 5.03 min, 98.76% purity.

Test example 1

Cell proliferation inhibition assay (CellTiter-Glo)

Experimental purpose: Use CellTiter-Glo detection reagent to evaluate the anti-tumor cell proliferation activity of the compound of this application.

Experimental method: Inoculate 500-1500 cells/20μL per well in a 384 or 96-well plate, and culture in a constant temperature incubator overnight. The compound was diluted to the specified concentration with the corresponding cell culture medium (+10% FBS), and 10 μL of the diluted compound was added to each well of the well plate, and the culture was continued for 72-120 h. Then add 25 μL of CellTiter-Glo reagent to each well, and measure the fluorescence signal using a multifunctional microplate reader (PerkinElmer).

The experimental results are shown in Table 1 below:

Table 1

Note: For anti-cancer cell proliferation inhibitory activity, less than 1 μM indicates strong activity and is expressed in specific numerical values; 1-10 μM indicates moderate activity; 10-20 μM indicates weak activity; greater than 20 μM indicates weak anti-cell proliferation inhibitory activity.

According to the results in Table 1, it was found that the compounds of Example 7, Example 8, Example 9, Example 16 and Example 18 of the present application have good anti-proliferation inhibitory activity against various tumor cells.

Test example 2

Western blotting experiment

Experimental purpose: Western Blot technology can be used to detect, characterize and semi-quantitate proteins.

Experimental method: Collection of total protein samples: Collect cells after drug administration, fully lyse the cells, and then take the supernatant for SDS-PAGE electrophoresis separation. Then transfer the protein on the SDS-PAGE gel to a membrane. After the transfer is complete, put the membrane into 5% skim milk for blocking. After blocking, cut the membrane according to the protein molecular weight. The corresponding primary antibody was diluted to an appropriate concentration with blocking solution and incubated with PVDF membrane overnight at 4°C. After primary antibody incubation, wash the membrane 3-6 times with 1×TBST, and then incubate with the corresponding secondary antibody dilution for 1-2 hours at room temperature. After the incubation, wash the membrane 3-6 times with 1×TBST, 10 min each time. Finally, chemiluminescence and development were performed.

The experimental results are shown in Figure 1-9 below:

As shown in Figure 1, compound Examples 7, 8, and 9 of the present application downregulate the level of BRD2 in the BET family in cells in a concentration-dependent manner;

As shown in Figure 2-3, the compound Example 9 of the present application down-regulates the intracellular BRD2 protein level in a concentration-dependent manner; among them, as shown in Figure 2, it can be clearly seen that 333nM compound 9 can significantly induce 22Rv1 cells after 9 hours of treatment. Degradation of intracellular BRD2 protein; as shown in Figure 3, 37nM compound 9 can significantly induce the degradation of intracellular BRD2 protein in MV4; 11 cells for 6 hours.

As shown in Figure 4-5, the compound Example 9 of the present application down-regulates the intracellular BRD2 protein level in a time-dependent manner; among them, as shown in Figure 4, when 300 nM Compound 9 treats 22Rv1 cells, it can be clearly seen that the level of intracellular BRD2 protein can be significantly reduced after 12 hours of treatment. Induces the degradation of intracellular BRD2 protein; as shown in Figure 5, 300nM compound 9 treated MV4; 11 cells. It can be clearly seen that after 6 hours of treatment, the degradation of intracellular BRD2 protein can be significantly induced.

As shown in Figures 6-7, the compound Example 9 of the present application can induce the degradation of GSPT1 protein. As shown in Figure 6, under the conditions of 6 hours of action, it can be seen that 37nM begins to significantly induce the degradation of GSPT1 protein in 22Rv1 cells; such as As shown in Figure 7, it can be seen that 12nM can significantly induce the degradation of GSPT1 protein in MV4;11 cells.

As shown in Figures 8-9, the compounds of the present application down-regulate the GSPT1 protein level in a time-dependent manner. As shown in Figure 8, it can be clearly seen that the degradation of GSPT1 protein in 22Rv1 cells can be induced in 4 hours; as shown in Figure 9, The degradation effect of GSPT1 protein in MV4;11 cells can be significantly induced within 4 hours.

The applicant declares that this application uses the above examples to illustrate the benzo[d]isoxazole compounds and their applications described in this application, but this application is not limited to the above process steps, which does not mean that this application must rely on the above. process steps can be implemented. Those skilled in the art should understand that any improvements to the present application, equivalent replacement of the raw materials selected in the present application, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present application.

Claims

A kind of benzo[d]isoxazole compound, which has the structure shown in the following formula I:

Among them, X is selected from O or S;

R 1 is selected from H, hydroxyl, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C5 alkoxy;

R 2 and R 3 are each independently selected from any group in the following groups (i) or (ii):

(i)H, halogen, amino, nitro, carboxyl, cyano, hydroxyl, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C10 Cycloalkyl, substituted or unsubstituted C3-C10 heterocycloalkyl, substituted or unsubstituted C6-C20 aryl, substituted or unsubstituted C4-C20 heteroaryl; or

(ii)-N(R a )SO 2 R b , -SO 2 N(R a )R b , -N(R a )COR b , -CON(R a )R b -, -N(R a ) CH 2 R b , -NHCH(R a )R b , -N(R a )R b , -CH(R a )R b , -COR b , -COOR b , -OCOR b , -SR b or -OR b ;

R a is selected from H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C3-C10 cycloalkenyl, substituted or unsubstituted C1-C10 hetero Cycloalkyl;

R b is selected from any one of the following groups (v), (vi), (vii) or (viii):

(v) Substituted or unsubstituted C6-C20 aryl group, substituted or unsubstituted C4-C20 heteroaryl group;

(vi) Substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C3-C10 cycloalkenyl, substituted or unsubstituted C3-C10 heterocycloalkyl; or

(vii) Substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl;

Y 1 is selected from -NH(CH 2 ) n CON(R c ) -, -O(CH 2 ) n CON(R c ) -, -S(CH 2 ) n CON(R c ) -, -(CH 2 ) n CON(R c )-or single bond, n is 1-8;

R c is selected from H, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C3-C10 cycloalkenyl, substituted or unsubstituted C3-C10 heterocycloalkyl, substituted or unsubstituted C6- C20 aryl, substituted or unsubstituted C4-C20 heteroaryl, substituted or unsubstituted C2-C10 alkenyl;

Y 2 is selected from -O-, -NH-, -CH 2 - or single bond;

L is selected from the group consisting of single bond, alkylene group, alkenylene group, alkynylene group, ether group, thioether group, ester group, amine group, amide group, urethane group, ureido group, sulfone group, aryl group, hetero group Any one or a combination of at least two of aryl, carbonyl, cycloalkyl and heteroaryl;

E has the structure shown in formula II, where It is the connection position of the group;

Among them, Y 3 is selected from -O-, -S-, -CHR d -, -C(=O)-, -SO 2 -, -NR e -;

R d and R e are each independently selected from H, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C8 cycloalkyl base, substituted or unsubstituted C3-C8 heterocyclyl;

Y 4 , Y 5 , Y 6 , Y 7 are each independently selected from CH or N;

T 1 , T 2 , and T 3 are each independently selected from O or S; and

R 4 and R 5 are each independently selected from -H, hydroxyl, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C1-C10 O-containing heterocycloalkyl base, substituted or unsubstituted C1-C10 containing N heterocycloalkyl group, substituted or unsubstituted C1-C10 containing S heterocycloalkyl group.
The benzo[d]isoxazole compound according to claim 1, wherein the Y 4 , Y 5 , Y 6 and Y 7 are independently selected from CH;

Preferably, any one of the groups Y 4 , Y 5 , Y 6 , and Y 7 is CH, and at least one of the other three groups is N;

Preferably, any one of the groups Y 4 , Y 5 , Y 6 , and Y 7 is CH, and the connection position of the group is located on the CH;

Preferably, the L is selected from any one of the following groups:

Wherein, m, n and o are independently selected from positive integers between 1-8; Y 8 is selected from substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted Substituted C1-C10 contains O heterocycloalkyl, substituted or unsubstituted C1-C10 contains N heterocycloalkyl, substituted or unsubstituted C1-C10 contains S heterocycloalkyl;

Preferably, the Y 3 is selected from -CH- or -C(=O)-;

Preferably, the T 1 , T 2 and T 3 are selected from O.
The benzo[d]isoxazole compound according to claim 1 or 2, wherein the benzo[d]isoxazole compound has the structure shown in the following formula III:

Among them, R 1 , R 2 , R 3 , R 4 , R 5 , X, Y 1 , Y 2 , L, Y 3 , Y 4 , Y 5 , Y 6 , Y 7 , T 1 , T 2 , T 3 The definition of a group is the same as in claim 1 or 2;

Preferably, the benzo[d]isoxazole compound has the structure shown in the following formula IV:

Among them, the definitions of R 1 , R 2 , R 3 , R 4 , R 5 , X, Y 1 , Y 2 , L, and Y 3 are the same as those in claim 1 or 2;

Preferably, the benzo[d]isoxazole compound has the structure shown in the following formula V or VI:

Wherein, the definitions of R 1 , R 2 , R 3 , R 4 , R 5 , X, Y 1 , Y 2 and L groups are the same as those in claim 1 or 2;

Preferably, the R 1 is selected from substituted or unsubstituted C1-C5 alkyl, preferably unsubstituted C1-C5 alkyl, further preferably unsubstituted C1-C3 alkyl;

Preferably, any one of R 2 and R 3 is selected from any group in group (ii), and the other group is selected from any group in group (i), preferably the Either group of R 2 and R 3 is selected from any group of -N(R a )SO 2 R b or -SO 2 N(R a )R b , and the other group is selected from H;

Preferably, the R a is selected from H, substituted or unsubstituted C1-C6 alkyl, preferably H;

Preferably, the R b is selected from any group in group (v), preferably a substituted or unsubstituted C6-C20 aryl group, and further preferably a substituted C6-C10 aryl group;

Preferably, the R b is selected from substituted phenyl, and the substituent of the phenyl is C1-C3 alkoxy and/or halogen;

Preferably, the Y 1 is selected from -O(CH 2 ) n CON(R c )- or a single bond;

Preferably, the n is 1-3, and R c is selected from H;

Preferably, the Y 2 is selected from -O-, -NH-, -CH 2 - or a single bond;

Preferably, the L is selected from any one of the following groups:

Wherein, m, n and o are independently selected from positive integers between 1-6, Y 8 is selected from unsubstituted C1-C6 alkyl;

Preferably, the benzo[d]isoxazole compound has the following structure represented by formula VII:

Among them, Y 1 is selected from -O(CH 2 ) n CON(R c )- or a single bond, n is 1-3, and R c is selected from H;

L is selected from any one of the following groups:

Wherein, m, n and o are independently selected from positive integers between 1-6, Y 8 is selected from unsubstituted C1-C6 alkyl;

Y 2 is selected from -O-, -NH-, -CH 2 - or a single bond; and

Y 3 is selected from -CH- or -C(=O)-.
The benzo[d]isoxazole compound according to any one of claims 1-3, wherein the aryl group is selected from phenyl or naphthyl;

Preferably, the heteroaryl group has an aromatic 5-8 membered monocyclic ring, 8-12 membered bicyclic ring or 11-14 membered tricyclic ring system; the single ring has 1-4 heteroatoms, and the bicyclic ring has 1 -6 heteroatoms, the tricyclic ring has 1-9 heteroatoms; the heteroatoms are selected from O, N or S;

Preferably, the heteroaryl group is selected from pyridazinyl, indolyl, quinazolinyl, pyrrolyl, thienyl, indazolyl, pyrazolyl, quinolyl, pyridyl, furyl, imidazolyl, Any one of pyrazinyl, pyrimidinyl, thiazolyl, isoquinolinyl, benzothiazolyl or naphthyridyl;

Preferably, the alkyl group, alkoxy group, cycloalkyl group, heterocycloalkyl group, aryl group, heteroaryl group, alkenyl group, cycloalkenyl group, and alkynyl group have a substituent, and the substituent group is selected from halogen, hydroxyl , any one of amino group, methoxy group, ethoxy group, nitro group, ether group, thioether group, ester group, amide group, urethane group, urea group or sulfone group.
The benzo[d]isoxazole compound according to any one of claims 1-4, wherein the benzo[d]isoxazole compound includes any one of the following compounds 1-19:
A pharmaceutically acceptable salt, stereoisomer, N-oxide, prodrug molecule, solvate, deuterated compound of benzo[d]isoxazole compound according to any one of claims 1-5 compound.
A pharmaceutical composition comprising active ingredients and pharmaceutically acceptable excipients;

Wherein, the active ingredient includes at least one benzo[d]isoxazole compound as described in any one of claims 1-5 and/or at least one pharmaceutically acceptable salt as claimed in claim 6, Stereoisomers, N-oxides, prodrug molecules, solvates, deuterated compounds.
A benzo[d]isoxazole compound as claimed in any one of claims 1 to 5, a pharmaceutically acceptable salt, stereoisomer, N-oxide, prodrug molecule as claimed in claim 6, Use of solvates, deuterated compounds or the pharmaceutical composition of claim 7 in the preparation of preparations that degrade BET and/or GSPT1 proteins.
A benzo[d]isoxazole compound as claimed in any one of claims 1 to 5, a pharmaceutically acceptable salt, stereoisomer, N-oxide, prodrug molecule as claimed in claim 6, Use of solvates, deuterated compounds or the pharmaceutical composition of claim 7 in the preparation of medicaments for preventing or treating cancer, cell proliferative disorders, inflammation, autoimmune diseases, sepsis or viral infections.
Use according to claim 9, wherein the cancer is selected from the group consisting of acute monocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia mixed lineage leukemia, NUT midline carcinoma, multiple myeloma, glaucoma, tumor, lung cancer, neuroblastoma, Burkitt lymphoma, cervical cancer, esophageal cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer or breast cancer;

The inflammatory and autoimmune diseases are selected from the group consisting of inflammatory pelvic disease, urethritis, pneumonia, meningitis, myocarditis, ulcerative colitis, organ transplant rejection, asthma, allergic rhinitis, chronic obstructive pulmonary disease, and autoimmune diseases. Autoimmune alopecia, anemia, autoimmune hemolytic and systemic lupus erythematosus, rheumatoid arthritis, Hashimoto's thyroiditis or atopic dermatitis;

The viral infection is selected from the group consisting of poliovirus, hepatitis A virus, rubella virus, Japanese encephalitis virus, hepatitis C virus, human papilloma virus, rabies virus, herpes virus, Barr virus, human immunodeficiency virus, novel Coronavirus or infection.