A bifunctional mdm2 protein degrader, preparation method, pharmaceutical composition and application thereof

Abstract

The present application provides protein degradation targeting chimeras, methods of making, pharmaceutical compositions, and uses thereof. In particular, the protein degradation targeting chimeras comprise the structure: MDM2 target protein inhibitor-C(O)NH-L 2 -Y 1 -B 1 wherein the groups are as defined in the specification. The compounds are useful for treating conditions or disorders (e.g., cancer) that are responsive to degradation of MDM2 protein.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of small molecule drugs, and in particular, the present application provides an MDM2 protein degrading agent, a preparation method, a pharmaceutical composition and an application thereof. BACKGROUND

[0002] The p53 gene is the highest related anticancer gene to human tumors, and has the functions of maintaining genome stability, inhibiting or preventing cell transformation, thereby inhibiting the occurrence of tumors. The research on new anti-tumor drugs targeting p53 has become a hot spot in this field, and the research results show that MDM2 (murine double minute 2) is a key negative regulator of p53, p53 activates MDM2 transcription, and MDM2 in turn inhibits p53 activity, and the two form an automatic regulation feedback loop to keep p53 and MDM2 at low levels under normal circumstances. The abnormal expression of MDM2 in tumor cells leads to rapid degradation of p53 and inactivation of the p53 pathway, thereby affecting the inhibition level of tumor, and thus releasing p53 from the control of MDM2, activating the P53 pathway, is expected to achieve the effect of inhibiting the growth of tumor cells and inducing apoptosis. Unlike the activation of P53 in normal cells due to rare causes, tumor cells are under continuous cellular stress including hypoxia and activation of pro-apoptotic oncogenes. Thus, there is a strong selective advantage for the inactivation of the p53 pathway in tumors, and researchers have proposed that the elimination of p53 function may be a prerequisite for tumor survival. In order to support this point of view, three research groups have used mouse models to prove that the loss of p53 function is a continuous requirement for tumor maintenance. When researchers restore the p53 function of p53-inactivated tumors, the tumors will regress.

[0003] In 50% of solid tumors and 10% of liquid tumors, p53 is inactivated by mutation and / or deletion. In cancer, other major members of the p53 pathway are also genetically or epigenetically altered. MDM2 is a cancer protein that inhibits p53 function, and it has been reported that MDM2 is genetically amplified and activated with an incidence as high as 10%. MDM2 in turn is inhibited by another tumor suppressor, p14ARF. Alterations downstream of p53 are thought to be responsible for at least partially inactivating the p53 pathway in p53 wild-type. To support this concept, some p53 wild-type tumors appear to show a decrease in apoptotic function, but their ability to undergo cell cycle arrest is still intact. One cancer treatment strategy involves the use of small molecules that bind MDM2 and counteract its interaction with p53. MDM2 inhibits p53 activity through three mechanisms: 1) acting as an E3 ubiquitin ligase to promote p53 degradation; 2) binding to the p53 transcriptional activation domain and blocking p53 transcriptional activation domain; and 3) exporting p53 from the nucleus to the cytoplasm. All three mechanisms would be blocked by counteracting the MDM2-p53 interaction. This treatment strategy can be applied specifically to p53 wild-type tumors, and studies have shown that using small molecule MDM2 protein degraders holds promise for reducing tumor growth in vitro and in vivo. Further, in patients with p53-inactivated tumors, normal tissues with wild-type p53 can be selectively protected from damage due to inhibition of MDM2.

[0004] Proteolysis targeting chimera (PROTAC) is a Nobel Prize-winning technology. The principle of PROTAC technology is to link target proteins and E3 ligases in cells with bifunctional small molecules, so that target proteins are ubiquitinated and recognized by proteasomes, resulting in degradation of target proteins. Eukaryotic cells are always trying to maintain appropriate protein levels, and they are generating and degrading thousands of proteins at every moment. The key factor for maintaining protein balance is a small protein molecule called ubiquitin. When it is linked to a protein, it will cause these proteins to be transported to the proteasome for degradation. Bifunctional protein degraders contain target protein inhibitors, linker groups, and ligands for E3 ubiquitin ligase proteins.

[0005] In summary, there is a need in the art to develop new bifunctional MDM2 protein degraders. SUMMARY

[0006] An object of the present application is to provide a new MDM2 protein degrader.

[0007] In a first aspect of the present application, a proteolysis targeting chimera is provided, said proteolysis targeting chimera comprising:

[0008] MDM2 target protein inhibitor -C(O)NH-L 2 -Y 1 -B 1

[0009] wherein,

[0010] -L 2 -Y 1 - is a linker group, wherein L 2 is -(Y 2 ) r -, Y 2 is selected from the group consisting of -CH2-, -O-, -N(R 2b )-;

[0011] and r is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0012] Y 1 is selected from the group consisting of -C≡C-, -CH=CH-, -CH2-, -O-, -N(R 2b )-, -C(=O)N(R 2c )-, -N(R 2d )C(=O)CH2O- and -N(R 2e )C(=O)CH2N(R 2f )-; or Y 1 is absent;

[0013] wherein the carboxamide nitrogen atom of -N(R 2d )C(=O)CH2O- and -N(R 2e )C(=O)CH2N(R 2f )- and the carbon atom of -C(=O)N(R 2e )- is attached to L 2 ;

[0014] R 2b , R 2c , R 2d , R 2e and R 2f are each independently selected from the group consisting of hydrogen and C1-4alkyl;

[0015] B 1 is selected from the group consisting of:

[0016]

[0017] said MDM2 target protein inhibitor is a structural fragment of a compound of formula I

[0018]

[0019] wherein,

[0020] is a 5-, 6-, or 7-membered heterocyclyl; wherein said heterocyclyl comprises 1-3 N atoms, and 0-2 heteroatoms selected from the group consisting of S and O;

[0021] X is C=O or S=(O)2;

[0022] n is 1, 2, 3, or 4;

[0023] each R is independently selected from the group consisting of H, cyano, halogen, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted C6-C 10 aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S, and O;

[0024] Z1is selected from the group consisting of H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C3-C8cycloalkyl (including monocyclic, fused, or bridged cyclic forms), substituted or unsubstituted C6-C 10 aryl;

[0025] Q is selected from the group consisting of:

[0026] m, p are each independently 1, 2, 3, or 4;

[0027] each Z2or Z3is each independently selected from the group consisting of none, substituted or unsubstituted C1-C7alkylene, NR1, O, S, C=O, S=(O)2;

[0028] Z4is selected from wherein said R1is selected from the group consisting of H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C6-C 10 aryl, cyano, -C(=O)-NRdRe, -C(=O)-substituted or unsubstituted C1-C6alkoxy, -C(=O)-substituted or unsubstituted C1-C6alkyl, -C(=O)-substituted or unsubstituted C3-C10cycloalkyl, -C(=O)-substituted or unsubstituted C2-C6alkenyl, -C(=O)-substituted or unsubstituted C2-C6alkynyl;

[0029] Rd, Reare each independently selected from the group consisting of H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C10cycloalkyl, substituted or unsubstituted C6-C 10Aryl; or the Rd and Re form a 4-10 membered heterocycle with adjacent N atoms, the heterocycle containing 1-2 nitrogen atoms and 0-2 S or O atoms;

[0030] R2 is selected from the following group: substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C3-C8 cycloalkyl groups, substituted or unsubstituted C6-C6 cycloalkyl groups. 10 Aryl, or substituted or unsubstituted, 5-10 membered heteroaryl groups having 1-3 heteroatoms selected from the group consisting of N, S and O;

[0031] Unless otherwise specified, “substitution” means being substituted by one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, oxo, -CN, hydroxyl, amino, carboxyl, unsubstituted or substituted by one or more substituents selected from the group consisting of: C6-C10 aryl, halogenated C6-C10 aryl, 5-10-membered heteroaryl having 1-3 heteroatoms selected from N, S, and O, halogenated 5-10-membered heteroaryl having 1-3 heteroatoms selected from N, S, and O; the substituents are selected from the group consisting of: halogen, C1-C6 alkoxy.

[0032] The additional condition is that when Z4 is When Q is

[0033] In another preferred embodiment, the described Selected from the following group:

[0034] In another preferred embodiment, the compound of formula I has the structure described in formula II:

[0035]

[0036] In another preferred example, n is 3.

[0037] In another preferred embodiment, R is selected from the group consisting of: substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C6-C6 alkyl groups. 10 Aryl.

[0038] In another preferred embodiment, the compound of formula I has the structure described in formula III:

[0039]

[0040] Ra and Rb are each independently substituted or unsubstituted C6-C. 10aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S, and O;

[0041] Rcand Rdare each independently selected from the group consisting of H, cyano, halogen, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy;

[0042] each group is defined as described above.

[0043] In another preferred embodiment, Raand Rbare each independently substituted or unsubstituted phenyl.

[0044] In another preferred embodiment, the compound of formula I has the structure according to formula IV:

[0045]

[0046] In another preferred embodiment, Z 1 substituted or unsubstituted C6-C10aryl (including monocyclic, fused, or bridged ring forms), substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S, and O (including monocyclic, fused, or bridged ring forms), and 10 aryl.

[0047] In another preferred embodiment, the protein degradation targeting chimera has the structure according to formula V:

[0048]

[0049] wherein Z5is a group consisting of L 2 -Y 1 -B 1 .

[0050] In another preferred embodiment, -L 2 -Y 1 - is a linker group, wherein L 2 is -(Y 2 ) r -, Y 2 is selected from the group consisting of -CH2-, -O-;

[0051] and r is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0052] Y 1 is selected from the group consisting of -C≡C-, -CH=CH-, -CH2-, -O-, -N(R 2b )-, -C(=O)N(R 2c )-.

[0053] In a second aspect of the present application, a pharmaceutical composition is provided, comprising (1) the protein degradation targeting chimera according to the first aspect of the present application, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof; and (2) a pharmaceutically acceptable carrier.

[0054] In a third aspect of the present application, the use of the protein degradation targeting chimera according to the first aspect of the present application, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or the pharmaceutical composition according to the second aspect of the present application, for the manufacture of a pharmaceutical composition for preventing and / or treating a disease associated with the activity or expression amount of MDM2 is provided.

[0055] In a fourth aspect of the present application, an MDM2 protein degradation agent is provided, comprising the protein degradation targeting chimera according to the first aspect of the present application, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0056] In another preferred embodiment, the pharmaceutical composition is used to treat a disease selected from the group consisting of bladder cancer, breast cancer, colon cancer, rectal cancer, kidney cancer, liver cancer, lung cancer (small cell lung cancer and non-small cell lung cancer), esophageal cancer, gall bladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, and skin cancer (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage (including leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, and Burkett's lymphoma); hematopoietic tumors of myeloid lineage (including acute and chronic myelogenous leukemias, myelodysplastic syndrome, and promyelocytic leukemia); tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma and other sarcomas such as soft tissue sarcoma and bone sarcoma); tumors of the central and peripheral nervous system (including astrocytoma, neuroblastoma, glioma, and schwannoma); and other tumors (including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer, and Kaposi's sarcoma), endometrial cancer, head and neck cancer, glioblastoma, malignant ascites, hematopoietic cancers, thyroid hyperplasia (especially Grave's disease), cysts, asthma, chronic obstructive pulmonary disease (COPD), emphysema, psoriasis, contact dermatitis, conjunctivitis, allergic rhinitis, systemic lupus erythematosus (SLE), ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, Alzheimer's disease, atherosclerosis, Huntington's disease, inflammatory diseases, hypoxia, ulcers, viral infections, bacterial infections, and bacterial sepsis.

[0057] In another preferred embodiment, the disease is a p53 wild-type cancer.

[0058] In another preferred embodiment, the cancer is a p53 wild-type and CDKN2A mutant cancer.

[0059] In another aspect, the present application provides a diagnostic for determining which patients should be administered a compound of the present application.

[0060] In another preferred embodiment, there is provided a method of inhibiting MDM2 activity or expression in vitro, the method comprising the step of contacting a protein degradation targeting chimera according to the first aspect of the present application, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, with an MDM2 protein.

[0061] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features described in detail below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. DETAILED DESCRIPTION

[0062] The present inventors have found a class of MDM2 protein degrading agents with excellent inhibitory effects through extensive and in-depth research. On this basis, the present inventors have completed the present application.

[0063] DEFINITIONS

[0064] As used herein, the term "alkyl" includes straight-chain or branched-chain alkyl groups. For example, C1-C8 alkyl refers to straight-chain or branched-chain alkyl groups having 1-8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like.

[0065] As used herein, the term "alkenyl" includes straight-chain or branched-chain alkenyl groups. For example, C2-C6 alkenyl refers to straight-chain or branched-chain alkenyl groups having 2-6 carbon atoms, such as ethenyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

[0066] As used herein, the term "alkynyl" includes straight-chain or branched-chain alkynyl groups. For example, C2-C6 alkynyl refers to straight-chain or branched-chain alkynyl groups having 2-6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

[0067] As used herein, the term "C3-C10 cycloalkyl" refers to cycloalkyl groups having 3-10 carbon atoms. It can be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It can also be a bicyclic form, such as a bridged ring or a spiro ring form. 10 "Cycloalkyl" refers to cycloalkyl groups having 3-10 carbon atoms. It can be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It can also be a bicyclic form, such as a bridged ring or a spiro ring form.

[0068] As used herein, the term "C1-C8 alkylamino" refers to an amine group substituted with a C1-C8 alkyl group, which can be mono-substituted or di-substituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di-tert-butylamino, and the like.

[0069] As used herein, the term "C1-C8 alkoxy" refers to straight-chain or branched-chain alkoxy groups having 1-8 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, and the like.

[0070] As used herein, the term "3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S and O" means a saturated or partially saturated cyclic group having 3-10 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It can be monocyclic or bicyclic, e.g. bridged or spirocyclic. Specific examples can be oxetanyl, azetidinyl, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl and pyrrolidinyl, etc.

[0071] As used herein, the term "C6-C 10 "Aryl" means an aromatic group having 6-10 carbon atoms, e.g. phenyl or naphthyl and the like.

[0072] As used herein, the term "5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O" means a cyclic aromatic group having 5-10 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It can be monocyclic or fused. Specific examples can be pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)-triazolyl and (1,2,4)-triazolyl, tetrazolyl, furanyl, thiophenyl, isoxazolyl, thiazolyl, oxazolyl, etc.

[0073] Unless specifically indicated otherwise, the groups described herein can be "substituted or unsubstituted". The groups of the present application can be substituted with a substituent selected from the group consisting of halogen, nitrile, nitro, hydroxy, amino, C1-C6alkyl-amine, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, haloC1-C6alkyl, haloC2-C6alkenyl, haloC2-C6alkynyl, haloC1-C6alkoxy, allyl, benzyl, C6-C10aryl, C1-C6alkoxy-C1-C6alkyl, C1-C6alkoxy-carbonyl, phenoxycarbonyl, C2-C6alkynyl-carbonyl, C2-C6alkenyl-carbonyl, C3-C6cycloalkyl-carbonyl, C1-C6alkyl-sulfonyl, etc. 12 "Aryl" means an aromatic group having 6-10 carbon atoms, e.g. phenyl or naphthyl and the like.

[0074] As used herein, "halogen" or "halo" means F, Cl, Br, and I. More preferably, the halogen or halo is selected from F, Cl and Br. "Halo" means substituted with an atom selected from F, Cl, Br, and I.

[0075] Unless otherwise stated, the structural formulae described herein are intended to include all isomeric forms (e.g., enantiomeric, diastereomeric, and geometric (or conformational) isomers) of the structure: for example, the R, S configurations for asymmetric centers, the (Z), (E) isomers for double bonds, and the like. Thus, individual stereochemically isomeric or enantiomeric, diastereomeric, or geometric (or conformational) isomers of the compounds of the present application, as well as mixtures of those isomers, are within the scope of the present application.

[0076] As used herein, the term "tautomer" means structural isomers that differ in energy by a low barrier, and thus interconvert. For example, prototropic tautomers (i.e., proton shift) include interconversion by proton migration, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion by reorganization of some of the bonding electrons.

[0077] As used herein, the term "solvate" means a complex of a compound of the present application with solvent molecules in a specific stoichiometric ratio.

[0078] As used herein, the term "hydrate" means a complex of a compound of the present application with water in a specific stoichiometric ratio.

[0079] MDM2 protein degraders

[0080] As used herein, "a compound of the present application" refers to a compound of Formula I, and also includes various crystalline forms, pharmaceutically acceptable salts, hydrates, or solvates of the compounds of Formula I:

[0081]

[0082] wherein,

[0083] is a 5-, 6-, or 7-membered heterocyclyl; wherein said heterocyclyl comprises 1-3 N atoms, and 0-2 heteroatoms selected from the group consisting of S and O;

[0084] X is C=O or S=(O)2;

[0085] n is 1, 2, 3, or 4;

[0086] each R is independently selected from the group consisting of H, cyano, halogen, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted C6-C10aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S, and O; 10 aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S, and O;

[0087] Z1is selected from the group consisting of H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C3-C8cycloalkyl (including monocyclic, fused or bridged cyclic forms), substituted or unsubstituted C6-C 10 aryl;

[0088] Q is selected from the group consisting of:

[0089] each m, p is independently 1, 2, 3 or 4;

[0090] each Z2or Z3is independently selected from the group consisting of substituted or unsubstituted C1-C7alkylene, NR1, O, S, C=O, S=(O)2;

[0091] Z4is selected from the group consisting of wherein said R1is selected from the group consisting of H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C6-C 10 aryl, cyano, -C(=O)-NRdRe, -C(=O)-substituted or unsubstituted C1-C6alkoxy, -C(=O)-substituted or unsubstituted C1-C6alkyl, -C(=O)-substituted or unsubstituted C3-C10cycloalkyl, -C(=O)-substituted or unsubstituted C2-C6alkenyl, -C(=O)-substituted or unsubstituted C2-C6alkynyl;

[0092] each R1is independently selected from the group consisting of H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C10cycloalkyl, substituted or unsubstituted C6-C 10 aryl; or said Rdand Re, together with the adjacent N atom form a 4-8 membered heterocyclic ring containing 1-2 nitrogen atoms and 0-1 S or O atoms;

[0093] R2is selected from the group consisting of substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted C6-C 10 aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O;

[0094] Unless otherwise indicated, the term "substituted" means substituted with one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halo, C1-C6alkyl, halo-substituted C1-C6alkyl, C1-C6alkoxy, halo-substituted C1-C6alkoxy, C3-C8cycloalkyl, halo-substituted C3-C8cycloalkyl, oxo, -CN, hydroxy, amino, carboxy, a group selected from the group consisting of C6-C10aryl, halo-substituted C6-C10aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S, and O, halo-substituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S, and O; said substituents being selected from the group consisting of halo, C1-C6alkoxy;

[0095] with the proviso that when Z4is then Q is

[0096] As used herein, "pharmaceutically acceptable salt" refers to those salts of the compounds of the present application which are suitable for use as a medicament. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that of the compounds of the present application with acids. Suitable acids for salt formation include, but are not limited to, hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric, phosphoric, and the like inorganic acids, formic, acetic, propionic, oxalic, malonic, succinic, fumaric, maleic, lactic, malic, tartaric, citric, picric, methanesulfonic, benzenesulfonic, benzenesulfonic, and the like organic acids; and acidic amino acids such as aspartic acid, glutamic acid, and the like. Suitable cations for salt formation include those of the alkali and alkaline earth metals, such as sodium ion, lithium ion, potassium ion, calcium ion, magnesium ion, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0097] In another preferred embodiment, the m, n, p, Z1, Z2, Z3, Z4, Q, R are each independently the group corresponding to each of the compounds of Table 1.

[0098] A preferred MDM2 protein degrader is a compound of the following formula:

[0099]

[0100] Bifunctional protein degrader targeting chimer as an MDM2 protein degrader

[0101] In the present application, there is also provided a bifunctional protein degrader targeting chimer as an MDM2 protein degrader, said targeting chimer having the structure of the following formula:

[0102] MDM2 target protein inhibitor -C(O)NH-L 2 -Y 1 -B 1

[0103] wherein,

[0104] -L 2 -Y 1 - is a linker group, wherein L 2 is -(Y 2 ) r -, Y 2 is selected from the group consisting of -CH2-, -O-, -N(R 2b )-;

[0105] and r is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0106] Y 1 is selected from the group consisting of -C≡C-, -CH=CH-, -CH2-, -O-, -N(R 2b )-, -C(=O)N(R 2c )-, -N(R 2d )C(=O)CH2O- and -N(R 2e )C(=O)CH2N(R 2f )-; or Y 1 is absent;

[0107] wherein the carboxamide nitrogen atom of -N(R 2d )C(=O)CH2O- and -N(R 2e )C(=O)CH2N(R 2f )- and the carbon atom of -C(=O)N(R 2e )- is attached to L 2 ;

[0108] R 2b , R 2c , R 2d , R 2e and R 2f are each independently selected from the group consisting of hydrogen and C1-4alkyl;

[0109] B 1 is selected from the group consisting of:

[0110]

[0111] The MDM2 target protein inhibitor is a compound of formula I as described above.

[0112] Pharmaceutical compositions and methods of administration

[0113] Because the compounds of the present application have excellent inhibitory activity against MDM2, the compounds of the present application and various crystal forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compounds of the present application as an active ingredient can be used for the treatment (stabilization, alleviation or cure) of cancer. The cancers that can be treated with the compounds of the present application include, but are not limited to, carcinomas such as bladder cancer, breast cancer, colon cancer, rectal cancer, kidney cancer, liver cancer, lung cancer (small cell lung cancer and non-small cell lung cancer), esophageal cancer, gall bladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer and skin cancer (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage (including leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic tumors of myeloid lineage (including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia); tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma, and other sarcomas such as soft tissue sarcoma and osteosarcoma); tumors of the central and peripheral nervous system (including astrocytoma, neuroblastoma, glioma and schwannoma); and other tumors (including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi's sarcoma). Other cancers that can be treated with the compounds of the present application include endometrial cancer, head and neck cancer, glioblastoma, malignant ascites, and hematopoietic cancers.

[0114] Specific cancers that can be treated with the compounds of the present application include soft tissue sarcoma, bone cancer (e.g., osteosarcoma), breast tumor, bladder cancer, Li-Fraumeni syndrome, brain tumor, rhabdomyosarcoma, adrenal cortex cancer, colorectal cancer, non-small cell lung cancer, and acute myelogenous leukemia (AML).

[0115] The pharmaceutical composition of the present application comprises a safe and effective amount of the compound of the present application and a pharmaceutically acceptable excipient or carrier. The "safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1-2000 mg of the compound of the present application per dose, more preferably, 10-200 mg of the compound of the present application per dose. Preferably, the "one dose" is one capsule or tablet.

[0116] "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler substances or gel materials, which are suitable for human use and which are of sufficient purity and sufficiently low toxicity. By "compatible" it is meant that the components of the composition are capable of being commingled with the compounds of the application, and with each other, in the dosage form with no interaction that significantly affects the efficacy of the compounds. Examples of suitable pharmaceutically acceptable carriers are celluloses and their derivatives (e.g., sodium carboxymethylcellulose, ethylcellulose sodium, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., lecithin), wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

[0117] The mode of administration of the compounds or pharmaceutical compositions of the present application is not narrowly critical and representative modes of administration include, but are not limited to, oral, parenteral (intravenous, intramuscular or subcutaneous).

[0118] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or with such other ingredients as binders, (a) fillers or extenders, e.g., starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, e.g., hydroxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, e.g., glycerol; (d) disintegrating agents, e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solution retarders, e.g., paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, e.g., cetyl alcohol and glycerol monostearate; (h) absorbents, e.g., kaolin and bentonite clay; and (i) lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage form can also comprise buffering agents.

[0119] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They can optionally contain opacifying agents, and can also be of a composition that they release the active compound or compounds in a certain part of the digestive tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0120] ​Liquid dosage forms for oral administration include pharmaceutically- acceptable emulsions, solutions, suspensions, syrups, or elixirs. In addition to the active compounds, the liquid dosage forms can include inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, and the like, or mixtures thereof.

[0121] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0122] Suspensions, in addition to the active compounds, can contain suspending agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, agar-agar, or mixtures thereof, and the like.

[0123] Compositions for parenteral injection can contain physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

[0124] The compounds of the present application can be administered alone or in combination with other pharmaceutically acceptable compounds (e.g., chemical anticancer drugs).

[0125] In combination therapy, the pharmaceutical composition further comprises one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds (e.g., chemical anticancer drugs). One or more (2, 3, 4, or more) of the other pharmaceutically acceptable compounds (e.g., chemical anticancer drugs) can be used simultaneously, separately or sequentially with the compounds of the present application in the treatment of cancer or related diseases.

[0126] The pharmaceutical composition is used in a safe and effective amount of the compounds of the present application for a mammal (e.g., human) in need of treatment, wherein the dosage is pharmaceutically effective amount, and for a 60 kg body weight human, the daily dosage is usually 1-2000 mg, preferably 20-500 mg. Of course, the specific dosage will take into account a variety of factors, such as the means of administration, the health of the patient, and the nature of the pharamaceutical.

[0127] The main advantages of the present application include:

[0128] (1) The compound of the present application has a novel structure and excellent MDM2 inhibitory effect. In the present application, the existing sulfone compound is modified into a sulfenamide compound, which can maintain the activity or expression amount of inhibiting MDM2 while reducing the plasma protein binding rate, increasing the free drug, and easily transporting to organs and tissues to play a role.

[0129] (2) The compound of the present application has very low toxicity to normal cells, and thus can be applied to a treatment subject in a large dose range.

[0130] (3) The compound of the present application has good drugability. Compared with the existing compound, the compound of the present application has better solubility and shows good bioavailability in in vivo experiments. In addition, compared with the existing compound, the compound of the present application is easy to form a pharmaceutically acceptable salt, thus being helpful for further forming a preparation.

[0131] (4) The compound of the present application and the pharmaceutical composition containing the compound of the present application as the main active ingredient can be used for treating cancer-related diseases.

[0132] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples, for which no specific conditions are noted, are generally carried out according to the conventional conditions or the conditions suggested by the manufacturers. Unless otherwise specified, percentages and parts are calculated by weight.

[0133] Each compound appearing in the examples is prepared by the following route:

[0134] Example 1

[0135] Synthesis of 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-1-((2S)-3-methyl-1-(propyl-2-sulfonamidyl)butyl-2-)-2-oxopiperidin-3-yl)acetic acid

[0136]

[0137] Step 1: Synthesis of methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-5-hydroxy-2-methylpentanoate

[0138]

[0139] Methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-5-hydroxy-2-methylpentanoate (37 g) was dissolved in ethanol (300 mL) and cooled to 0-5 °C. NaBH4(2.85 g) was added portionwise and the reaction was stirred at 0-10 °C for 2 h. TLC indicated that the reaction was essentially complete. Acetic acid (-8 mL) was added dropwise until hydrogen evolution ceased. The solvent was concentrated and the residue was dissolved in ethyl acetate (300 mL). The solution was washed sequentially with water, saturated NaHCO3, and dried over anhydrous Na2SO4. The solvent was concentrated to give 37 g of methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-5-hydroxy-2-methylpentanoate.

[0140] Step 2: Methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-5-hydroxy-2-methylpentanoate

[0141]

[0142] Methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-5-hydroxy-2-methylpentanoate (37 g) was dissolved in ethanol (300 mL) and cooled to 0-5 °C. NaBH4(2.85 g) was added portionwise and the reaction was stirred at 0-10 °C for 2 h. TLC indicated that the reaction was essentially complete. Acetic acid (-8 mL) was added dropwise until hydrogen evolution ceased. The solvent was concentrated and the residue was dissolved in ethyl acetate (300 mL). The solution was washed sequentially with water, saturated NaHCO3, and dried over anhydrous Na2SO4. The solvent was concentrated to give 37 g of methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-5-hydroxy-2-methylpentanoate.

[0143] Step 3: 5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one

[0144]

[0145] Methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-5-hydroxy-2-methylpentanoate (37 g) was dissolved in ethanol (300 mL) and cooled to 0-5 °C. NaBH4(2.85 g) was added portionwise and the reaction was stirred at 0-10 °C for 2 h. TLC indicated that the reaction was essentially complete. Acetic acid (-8 mL) was added dropwise until hydrogen evolution ceased. The solvent was concentrated and the residue was dissolved in ethyl acetate (300 mL). The solution was washed sequentially with water, saturated NaHCO3, and dried over anhydrous Na2SO4. The solvent was concentrated to give 37 g of methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-5-hydroxy-2-methylpentanoate.

[0146] Step 4: (±)(3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one

[0147]

[0148] Dissolve 5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one (6.7 g) and bromoacrylate (7.26 g) in THF (25 ml), cool to -50 °C, add dropwise a solution of LiHMDS (26 ml of 1 M in THF), allow to warm to 0 °C and stir for 1 h, TLC track disappearance of starting material, add saturated ammonium chloride solution, extract with ethyl acetate, column chromatography to isolate 6.0 g of product, the isomer is more difficult to purify by column, dissolve 6.0 g of product in 50 ml of n-heptane / toluene (10:1), heat to reflux to dissolve, slowly cool to room temperature, 2.8 g of solid (±)(3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one precipitates.

[0149] 1 HNMR (CDCI3, 400 MHz): 7.22-7.11 (m, 4H), 6.873 (d, 1H, J = 1.9 Hz), 6.745 (d, 1H, J = 7.8 Hz), 6.58-6.54 (m, 2H), 5.804 (m, 1H), 5.677 (d, 1H, J = 5.1 Hz), 5.157 (d, 1H, J = 10.2 Hz), 5.125 (dd, 1H, J = 1.6, 15.3 Hz), 3.787 (dt, 1H, J = 4.5, 12.2 Hz), 2.598 (dd, 1H, J = 7.9, 14.1 Hz), 2.488 (dd, 1H, J = 7.1, 13.7 Hz), 1.954 (t, 1H, J = 14.0 Hz), 1.897 (dd, 1H, J = 4.5, 14.0 Hz), 1.389 (s, 3H).

[0150] Step 5: 2-((2R,3R)-2-(3-chlorophenyl)-3-(4-chlorophenyl)-3-hydroxypropyl)-N-((S)-1- hydroxy-3-methylbutyl-2)-2-methylpent-4-enamide

[0151]

[0152] (3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro- 2H-pyran-2-one (1.0 g) was dissolved in toluene (1 ml), L-valinol (0.825 g) was added and heated to 100°C for 5 hours, TLC tracking showed the reaction was almost complete, after cooling, ethyl acetate was added, washed with IN hydrochloric acid, saturated sodium bicarbonate, dried over anhydrous magnesium sulfate and concentrated to give 2-((2R,3R)-2-(3-chlorophenyl)-3-(4-chlorophenyl)-3- hydroxypropyl)-N-((S)-1-hydroxy-3-methylbutyl-2)-2-methylpent-4-enamide 1.48 g.

[0153] Step 6: trifluoromethanesulfonic acid (3S,5S,6R,8S)-8-allyl-6-(3-chlorophenyl)-5-(4- chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridine salt

[0154]

[0155] 2-((2R,3R)-2-(3-chlorophenyl)-3-(4-chlorophenyl)-3-hydroxypropyl)-N-((S)-1- hydroxy-3-methylbutyl-2)-2-methylpent-4-enamide (63.6 g) was dissolved in DCM (640 ml), 2,6-lutidine (57 g) was added, cooled to -78°C, Tf20 (97.6 g) was added dropwise, after the addition was complete, the temperature was allowed to rise to room temperature and the reaction was allowed to proceed overnight, washed with 0.5 M TfOH solution (200 ml), extracted with ethyl acetate (500 ml x 2), the organic phase was concentrated and dissolved in DCM (400 ml), column chromatography was used for separation, the conditions were:

[0156] Silica gel: 120 g

[0157] Amount loaded each time: 10 g

[0158] Mobile phase: A is n-heptane, B is acetone

[0159] Time (min) Mobile phase ratio 0-5 25％ 5-15 25％-35％ 15-30 35％ 30-35 25％

[0160] The less polar isomer (27.4 g, 34.8%) was obtained as trifluoromethanesulfonic acid (3S,5S,6R,8S)-8-allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8- methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridine salt:

[0161] 1HNMR (d6-DMSO, 400 MHz): 8.15 ~ 7.10 (m, 8H), 5.812 (m, 1H), 5.346 (dd, 1H, J = 1.2, 16.8 Hz), 5.238 (dd, 1H, J = 1.5, 10.1 Hz), 5.173 (d, 1H, J = 11.3 Hz), 5.003 (dd, 1H, J = 5.5, 10.2 Hz), 4.870 (t, 1H, J = 10.2 Hz), 4.323 (m, 1H), 4.057 (ddd, 1H, 3.1, 13.7, 10.6 Hz), 2.812 (dd, 1H, J = 7.1, 13.7 Hz), 2.717 (dd, 1H, J = 7.4, 13.7 Hz), 2.316 (t, 1H, 13.7 Hz), 1.993 (dd, 1H, J = 13.7, 3.5 Hz), 1.303 (s, 3H), 0.579 (d, 3H, J = 6.7 Hz), 0.524 (d, 3H, J = 7.0 Hz), 0.428 (m, 1H).

[0162] and the more polar isomer (22.8 g, 28.9%) as trifluoromethanesulfonic acid (3S,5R,6S,8R)-8-allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridine salt:

[0163] 1 HNMR (d6-DMSO, 400 MHz): 7.50 ~ 7.05 (m, 8H), 5.902 (m, 1H), 5.290 (dd, 1H, J = 1.6, 17.2 Hz), 5.230 (dd, 1H, J = 2.4, 10.2 Hz), 5.140 (d, 1H, J = 10.2 Hz), 5.084 (dd, 1H, J = 3.9, 10.2 Hz), 4.927 (t, 1H, J = 10.2 Hz), 3.878 (m, 1H), 3.423 (m, 1H), 2.733 (dd, 1H, J = 8.3, 14.1 Hz), 2.657 (dd, 1H, J = 6.7, 13.7 Hz), 2.334 (t, 1H, 13.7 Hz), 2.005 (dd, 1H, J = 13.7, 2.7 Hz), 1.334 (s, 3H), 0.884 (d, 3H, J = 6.6 Hz), 0.662 (d, 3H, J = 6.6 Hz).

[0164] Step 7: (3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylthio)-3-methylbutyl-2)-3-methylpiperidin-2-one

[0165]

[0166] To a solution of potassium tert-butoxide (82.2 ml of 1 M in THF) was added dropwise isopropyl mercaptan (10.4 g) under nitrogen protection, the temperature was controlled below 30 °C. After the addition was completed, the mixture was stirred for 10 min. Then a solution of (3S,5S,6R,8S)-8-allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridine trifluoromethanesulfonate (17.8 g) in DMF (80 ml) was added. The mixture was heated to 50 °C and stirred for 3 h. The reaction was monitored by TLC. The mixture was poured into 600 ml of water and extracted with ethyl acetate (200 ml x 3). The organic phase was washed with water and concentrated. The residue was purified by column chromatography to give 13.5 g of (3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylthio)-3-methylbutyl-2)-3-methylpiperidin-2-one as an oil.

[0167] Step 8: (3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfoxide)-3-methylbutyl-2)-3-methylpiperidin-2-one

[0168]

[0169] To a solution of (3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylthio)-3-methylbutyl-2)-3-methylpiperidin-2-one (2.03 g) in methanol (25 ml) was added 0.60 ml of 30% hydrogen peroxide. The mixture was stirred at 20-25 °C for 2 days. The reaction was quenched by the addition of sodium thiosulfate solution. The mixture was extracted with ethyl acetate and dried over anhydrous magnesium sulfate. The filtrate was concentrated and purified by column chromatography to give 1.99 g of (3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfoxide)-3-methylbutyl-2)-3-methylpiperidin-2-one as a mixture of two isomers in 95% yield.

[0170] 1HNMR (CDC13, 400 MHz): 7.27-7.00 (m, 7H), 6.978 (d, IH, J = 6.8 Hz), 5.898 (m, IH), 5.260 (d, IH, J = 16.8 Hz), 5.180 (dd, IH, J = 1.2, 10.0 Hz), 4.860 (d, IH, J = 8.4 Hz), 3.620 (t, IH, J = 10.5 Hz), 3.541 (ddd, IH, J = 2.3, 11.0, 13.3 Hz), 3.017 (m, 2H), 2.860 (dd, IH, J = 2.7, 12.9 Hz), 2.741 (dd, IH, J = 8.2, 14.1 Hz), 2.716 (dd, IH, J = 2.8, 13.3 Hz), 2.585 (dd, IH, J = 6.7, 13.7 Hz), 2.188 (m, IH), 2.101 (t, IH, J = 13.7 Hz), 1.922 (dd, IH, J = 2.8, 13.7 Hz), 1.343 (d, 3H, J = 7.0 Hz), 1.277 (d, 3H, J = 7.0 Hz), 1.199 (s, 3H), 0.863 (t, IH, J = 6.6 Hz), 0.675 (d, 3H, J = 6.7 Hz), 0.481 (d, 3H, J = 7.0 Hz).

[0171] Step 9: 2,2,2-Trifluoroacetyl N-(((S)-2-((3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4- chlorophenyl)-3-methyl-2-oxopiperidin-l-yl)-3-methylbutyl)(isopropyl)(oxy)-l6-sulfamidate

[0172]

[0173] (3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfinyl)-3- methylbutyl-2)-3-methylpiperidin-2-one (336 mg) and trifluoroacetamide (142.4 mg) were dissolved in DCM (4 ml), then MgO (126.5 mg) and Rh2(OAc)4(6.9 mg) were added, and finally PhI(OAc)2(303.7 mg) was added. The mixture was stirred at room temperature overnight, filtered, and the solid was washed with DCM. The solution was concentrated and then passed through a column, eluting with n-heptane / ethyl acetate = 2:1, to give 58 mg of the product 2,2,2-trifluoroacetamidoN-(((S)-2-((3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-1-yl)-3-methylbutyl)(isopropyl)(oxido)-S- sulfamidamide) as a mixture of two isomers in 14% yield.

[0174] 1 HNMR (CDCI3, 400 MHz): 7.3-7.1 (m, 6H), 6.932 (s, 1H), 6.860 (t, 1H, J = 3.2 Hz), 5.904 (m, 1H), 5.239 (m, 2H), 4.959 (d, 1H, J = 10.8 Hz), 4.450 (dd, 1H, J = 7.6, 15.2 Hz), 4.135 (m, 1H), 3.368 (m, 2H), 3.186 (ddd, 1H, J = 1.6, 7.6, 9.2 Hz), 2.658 (m, 2H), 2.390 (m, 1H), 2.229 (t, 1H, J = 13.6 Hz), 1.898 (dd, 1H, J = 3.2, 14.0 Hz), 1.522 (d, 3H, J = 7.0 Hz), 1.484 (d, 3H, J = 7.0 Hz), 1.282 (t, 1H, J = 7.2 Hz), 1.256 (s, 3H), 0.684 (d, 3H, J = 6.8 Hz), 0.620 (d, 3H, J = 7.2 Hz).

[0175] Step 10: 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-1-((2S)-3-methyl-1-(N-(2,2,2- trifluoroacetyl)-2-propylsulfamidamide)butyl-2-)-2-oxopiperidin-3-yl)acetic acid

[0176]

[0177] Step 1: 2,2,2-trifluoroacetyl N-(((S)-2-((3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4- chlorophenyl)-3-methyl-2-oxopiperidin-l-yl)-3-methylbutyl)(isopropyl)(oxy)-l6-sulfamidyl amine) (150 mg) and ruthenium trichloride (7.3 mg) were dissolved in DCM / water (5 ml / 5 ml), then tetrabutylammonium hydrogen sulfate (15.8 mg) and sodium periodate (300 mg) were added, stirred at room temperature overnight, filtered, extracted with DCM, the solution was dried and concentrated, then columned, elution with n-heptane / ethyl acetate = 1:1, 120 mg of product 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-l-((2S)-3-methyl-l-(N-(2,2,2- trifluoroacetyl)-2-propylsulfamidyl)butyl-2-)-2-oxopiperidin-3-yl)acetic acid as a mixture of two isomers, yield 77.6%.

[0178] 1 H NMR (400 MHz, Methanol-d4) δ 7.27 (br, 4H), 7.19 - 6.89 (m, 4H), 5.00 (dd, 1H, J = 20.4, 11.0 Hz), 4.44 (ddd, 1H, J = 28.6, 15.1, 9.2 Hz), 4.08 (m, 1H), 3.71 - 3.48 (m, 2H), 3.39 - 3.17 (m, 2H), 3.05 - 2.92 (m, 1H), 2.67 - 2.55 (m, 1H), 2.34 - 1.93 (m, 4H), 1.51 (d, 3H, J = 7.1 Hz), 1.47 (d, 3H, J = 7.1 Hz), 1.46 (s, 3H), 1.35 (d, 3H, J = 3.2 Hz), 0.47 (d, 3H, J = 7.0 Hz).

[0179] Step 1: 2,2,2-trifluoroacetyl N-(((S)-2-((3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4- chlorophenyl)-3-methyl-2-oxopiperidin-l-yl)-3-methylbutyl)(isopropyl)(oxy)-l6-sulfamidyl amine) (150 mg) and ruthenium trichloride (7.3 mg) were dissolved in DCM / water (5 ml / 5 ml), then tetrabutylammonium hydrogen sulfate (15.8 mg) and sodium periodate (300 mg) were added, stirred at room temperature overnight, filtered, extracted with DCM, the solution was dried and concentrated, then columned, elution with n-heptane / ethyl acetate = 1:1, 120 mg of product 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-l-((2S)-3-methyl-l-(N-(2,2,2- trifluoroacetyl)-2-propylsulfamidyl)butyl-2-)-2-oxopiperidin-3-yl)acetic acid as a mixture of two isomers, yield 77.6%.

[0180]

[0181] (2S)-3-methyl-l-(N-(2,2,2-trifluoroacetyl)-2-propylsulfonamidyl)butyl-2-)-2-oxopiperidin-3-yl)acetic acid (100 mg) was dissolved in methanol (2.0 ml), then potassium carbonate (417 mg) was added, stirred at room temperature for 2 hours, water and ethyl acetate were added, pH was adjusted to 6 with 3N HC1, separated, the solution was dried and concentrated, then columned, the eluent was n-heptane / ethyl acetate = 1:1 to 0:1, two isomer products were obtained, 31 mg of the more polar one and 18 mg of the less polar one, total yield 57%. The less polar compound was cultured into single crystal, and the configuration of the compound was obtained by single crystal X-ray diffraction experiment

[0182] Rs more polar compound 12: 1 H NMR (400 MHz, Chloroform-d) δ 7.22 (br, 4H), 7.10 - 6.91 (m, 3H), 6.84 (dd, 1H, J = 5.4, 3.3 Hz), 5.38 (d, 1H, J = 11.0 Hz), 4.12 - 3.97 (m, 1H), 3.40 - 3.20 (m, 2H), 3.13 (m, 1H), 2.94 - 2.72 (m, 2H), 2.37 (t, 1H, J = 13.7 Hz), 2.19 (m, 1H), 1.91 (dd, 1H, J = 13.8, 3.0 Hz), 1.57 (d, 1H, J = 6.8 Hz), 1.41 (s, 3H), 1.37 (d, 3H, J = 7.1 Hz), 1.35 (d, 3H, J = 7.1 Hz), 1.30 - 1.13 (m, 3H), 0.61 (d, 2H, J = 6.6 Hz), 0.44 (d, 2H, J = 6.8 Hz).

[0183] Ss less polar compound 13: 1H NMR (400 MHz, Chloroform-d) δ 7.24 (br, 4H), 7.09 - 6.95 (m, 3H), 6.86 (dd, 1H, J = 5.4, 3.1 Hz), 5.30 (d, 1H, J = 11.0 Hz), 3.99 (dd, 1H, J = 13.6, 10.4 Hz), 3.46 (t, 1H, J = 8.6 Hz), 3.30 (ddd, 1H, J = 14.0, 10.9, 3.1 Hz), 3.13 (m, 1H), 2.99 - 2.76 (m, 3H), 2.37 (t, 1H, J = 13.7 Hz), 2.12 (dt, 1H, J = 14.9, 6.9 Hz), 1.90 (dd, 1H, J = 13.8, 3.0 Hz), 1.43 - 1.34 (m, 6H), 0.61 (d, 3H, J = 6.6 Hz), 0.42 (d, 3H, J = 6.9 Hz).

[0184] Example 2

[0185] Compound 4-(2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-1-((S)-3-methyl-1-((S)-2-propylsulfonamidinyl)butan-2-yl)-2-oxopiperidin-3-yl)acetamido)benzoic acid

[0186]

[0187] Step 1: Synthesis of methyl 4-(2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-1-((2S)-3-methyl-1-(N-(2,2,2-trifluoroacetyl)-2-propylsulfonamidinyl)butyl-2-)-2-oxopiperidin-3-yl)acetamido)benzoate

[0188]

[0189] To a solution of 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-1-((2S)-3-methyl-1-(N-(2,2,2-trifluoroacetyl)-2-propylsulfonamidyl)butyl-2-)-2-oxopiperidin-3-yl)acetic acid (800 mg), methyl p-aminobenzoate (219 mg), DMAP (324 mg) and EDC.HC1 (508.4 mg) were added successively to DCM (16 ml) at 0 °C under nitrogen. The reaction was stirred at room temperature overnight. TLC showed the reaction was complete. The reaction was quenched by adding water dropwise under ice-bath. The pH was adjusted to 2 by cold 1 N HC1 solution. The organic phase was washed by 1 N HC1 once more, water, saturated brine, dried over anhydrous MgS04, concentrated and the residue was separated by column chromatography to give 600 mg of white foamy solid with 62.6% yield. 1 H NMR (400 MHz, CDC13) δ 8.84 (s, 1H), 8.07 (d, J = 8.7 Hz, 2H), 7.71 (d, J = 8.7 Hz, 2H), 7.27 - 7.06 (m, 6H), 6.97 (s, 1H), 6.91 (t, J = 3.6 Hz, 1H), 4.97 (d, J = 10.9 Hz, 1H), 4.56 (dd, J = 13.9, 10.6 Hz, 1H), 4.14 (q, J = 7.2 Hz, 1H), 3.94 (s, 3H), 3.39 (dd, J = 16.4, 11.7 Hz, 3H), 2.89 (q, J = 14.4 Hz, 2H), 2.38 (t, J = 13.8 Hz, 1H), 2.15 - 1.92 (m, 2H), 1.58 (d, J = 6.8 Hz, 3H), 1.53 (d, J = 6.9 Hz, 3H), 1.46 (s, 3H), 0.75 (d, J = 6.7 Hz, 3H), 0.36 (d, J = 7.0 Hz, 3H).

[0190] Step 2: Synthesis of 4-(2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-1-((S)-3-methyl-1-((S)-2-propylsulfonamidyl)butan-2-yl)-2-oxopiperidin-3-yl)acetamido)benzoic acid

[0191]

[0192] Methyl 4-(2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-1-((2S)-3-methyl-1-(N-(2,2,2- trifluoroacetyl)-2-propylsulfamidyl)butan-2-yl)-2-oxopiperidin-3-yl)acetamido)benzoate (600 mg) and LiOH.H2O (127 mg) were added to a solution of MeOH / H2O / THF (2.4 ml / 1.2 ml / 1.2 ml) at room temperature and stirred at room temperature. TLC was used to monitor the reaction completion. The solvent was concentrated, the residue was dissolved in 10 ml water, 20 ml ethyl acetate, 1 N HCl solution was added to adjust pH = 2, the mixture was partitioned, the aqueous layer was extracted with ethyl acetate again, the combined organic phase was washed with water, saturated brine, dried over anhydrous magnesium sulfate, concentrated, and the residue was lyophilized to give a white solid 250 mg, 48.4% yield.1H NMR (400 MHz, Methanol-d4) δ 8.05 (d, J = 7.5 Hz, 2H), 7.81 (d, J = 8.7 Hz, 2H), 7.20 (m, 6H), 6.86 (d, J = 19.2 Hz, 2H), 5.13 (s, 1H), 5.01 (s, 1H), 4.43 (s, 1H), 3.57 (m, 3H), 3.08 (d, J = 13.6 Hz, 1H), 2.67 (d, J = 13.9 Hz, 1H), 2.48 - 2.06 (m, 3H), 1.47 (d, J = 38.1 Hz, 9H), 0.77 (s, 3H), 0.59 (s, 3H). LC-MS: M+1 = 686.2

[0193] Following the same experimental procedure, methyl 4-aminobenzoate was replaced by methyl 2-methoxy-4-aminobenzoate to give methyl 4-(2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-1-((2S)-3-methyl-1-(propan-2-ylsulfamidyl)butan-2-yl)-2-oxopiperidin-3-yl)acetamido)-2-methoxybenzoate.

[0194] Example 3

[0195] Synthesis of 4-(2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-1-((2S)-3-methyl-1-(propan-2-ylsulfamidyl)butan-2-yl)-2-oxopiperidin-3-yl)acetamido)-N-(5-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pent-4-yn-1-yl)-2-methoxybenzamide

[0196]

[0197] Step 1 :

[0198]

[0199] To a solution of 3-bromo-2-bromomethyl-benzoic acid methyl ester (5.00 g, 16.2 mmol) and 3-amino-piperidine-2,6-dione hydrochloride (2.94 g, 17.9 mmol) in acetonitrile (50 mL) was added triethylamine (8.21 g, 81.1 mmol) and heated to reflux overnight. The reaction mixture turned a deep purple color. After cooling to room temperature, the mixture was filtered and the solid was washed with water (20 mL) and MTBE (20 mL). The solid was dried under vacuum to give 2.70 g of a solid in 52% yield. 1 H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 7.5 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 5.16 (dd, J = 13.3, 5.1 Hz, 1H), 4.43 (d, J = 17.6 Hz, 1H), 4.27 (d, J = 17.6 Hz, 1H), 2.92 (ddd, J = 18.1, 13.6, 5.4 Hz, 1H), 2.65 - 2.51 (m, 1H), 2.45 (dd, J = 13.3, 4.3 Hz, 1H), 2.02 (ddd, J = 13.0, 6.1, 3.3 Hz, 1H).

[0200] Step 2:

[0201]

[0202] To a solution of compound 3-(4-bromo-l-oxoisoindolin-2-yl)piperidine-2,6-dione (1.00 g, 3.09 mmol) and compound tert-butyl pent-4-yn-l-ylcarbamate (680 mg, 3.71 mmol) in dry DMF (10 mL) was added Cul (59 mg, 0.31 mmol) and Pd(PPh3)2Cl2(217 mg, 0.31 mmol) after purging with nitrogen. The reaction mixture was heated to 80 °C under nitrogen overnight. After cooling to room temperature, ethyl acetate and water were added and the mixture was extracted. The organic phase was dried, concentrated and purified by column chromatography to give 1.12 g of compound tert-butyl (5-(2-(2,6-dioxopiperidin-3-yl)-l-oxoiso-4-yl)pent-4-yn-l-yl)carbamate in 85% yield. LC-MS, positive ion [M+l] = 426.

[0203] Step 3:

[0204]

[0205] Compound tert-butyl 5-(2-(2,6-dioxopiperidin-3-yl)-l-oxoiso-4-yl)pent-4-yn-l- yl)carbamate (1.10 g, 2.59 mmol) was added to 4 M HC1 / dioxane solution (15 ml), stirred at room temperature overnight, concentrated to give 650 mg of compound 5-(2-(2,6-dioxopiperidin-3-yl)-l-oxoiso-4-yl)pent-4-yn-l-yl)amine HC1 salt, yield 69%. LC-MS, positive ion [M+l] = 326.

[0206] Step 4:

[0207]

[0208] Compound 4-(2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-l-((2S)-3- methyl-l-(propan-2-ylsulfamidyl)butan-2-yl)-2-oxopiperidin-3-yl)acetamido)-2- methoxybenzoic acid (71.6 mg, 0.1 mmol) and compound 5-(2-(2,6-dioxopiperidin-3-yl)-l- oxoiso-4-yl)pent-4-yn-l-yl)amine HC1 salt (43.4 mg, 0.12 mmol) were added to anhydrous DMF (2.0 ml), stirred at room temperature for 5 min, then HATU (45.6 mg, 0.12 mmol) was added, followed by DIPEA (51.7 mg, 0.4 mmol), stirred at room temperature overnight, preparative HPLC separation gave 20 mg of solid. 1H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 10.43 (s, 1H), 8.17 (t, J = 5.8 Hz, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 7.3 Hz, 1H), 7.63 (dd, J = 7.7, 1.1 Hz, 1H), 7.58 - 7.47 (m, 2H), 7.36 (s, 3H), 7.32 - 7.11 (m, 3H), 6.98 (d, J = 7.7 Hz, 1H), 6.94 (s, 1H), 5.29 (d, J = 10.9 Hz, 1H), 5.15 (dd, J = 13.3, 5.0 Hz, 1H), 4.55 - 4.45 (m, 1H), 4.35 (d, J = 17.9 Hz, 1H), 3.95 (s, 1H), 3.85 (s, 3H), 3.45 (q, J = 6.7 Hz, 2H), 3.18 (s, 1H), 3.08 (d, J = 13.5 Hz, 1H), 2.93 (t, J = 15.2 Hz, 1H), 2.68 - 2.52 (m, 5H), 2.18 - 2.06 (m, 3H), 2.01 (d, J = 11.7 Hz, 1H), 1.84 (q, J = 6.9 Hz, 2H), 1.35 (d, J = 3.7 Hz, 3H), 1.33 (d, J = 3.7 Hz, 3H), 1.28 (s, 3H), 0.59 (d, J = 6.6 Hz, 3H), 0.41 (d, J = 6.9 Hz, 3H).

[0209] Example 4

[0210] 4-(2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-1-((2S)-3-methyl-1- (propan-2-ylsulfamidyl)butan-2-yl)-2-oxopiperidin-3-yl)acetamido)-N-(5-(2-(2,6- dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pentyl)-2-methoxybenzamide

[0211]

[0212] Step 1:

[0213]

[0214] Compound 5-(2-(2,6-dioxopiperidin-3-yl)-l-oxoiso-4-yl)pent-4-yn-l-yl)amine hydrochloride (79 mg) was added to methanol (10 ml), after nitrogen purging, 10% Pd / C (75 mg) was added, then purged with hydrogen, after 3 days of reaction at room temperature under normal pressure, LC-MS showed the reaction was complete, after nitrogen purging, filtered, the filtrate was concentrated to get 68 mg solid.

[0215] Step 2:

[0216]

[0217] Compound 4-(2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-l-((2S)-3-methyl-l-(propan-2-ylsulfamidyl)butan-2-yl)-2-oxopiperidin-3-yl)acetamido)-2-methoxybenzoic acid (98 mg) and compound 5-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindol-4-yl)pentan-l -amine hydrochloride (60 mg) were added to anhydrous DMF (2.0 ml), stirred at room temperature for 5 min, after the solid was dissolved, HATU (62.4 mg) was added, then DIPEA (70.8 mg,) was added, stirred at room temperature overnight, preparative HPLC separation to get 20 mg solid.

[0218] Example 5

[0219] 4-(2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-l-((2S)-3-methyl-l-(propan-2-ylsulfamidyl)butan-2-yl)-2-oxopiperidin-3-yl)acetamido)-N-(4-((2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindol-4-yl)amino)butyl)-2-methoxybenzamide

[0220]

[0221] Step 1:

[0222]

[0223] To a solution of tert-butyl (4-oxobutyl)carbamate (774 mg, 4.14 mmol) and 3-(4-amino-l-oxoisoindolin-2-yl)piperidine-2,6-dione (1.18 g, 4.55 mmol) in DMF (8.0 mL) was added sodium triacetoxyborohydride (1.75 g, 8.28 mmol) at room temperature. The reaction mixture was stirred overnight. The reaction mixture was diluted with ethyl acetate and washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated to give a crude product. The crude product was purified by column chromatography to give 530 mg of a solid.

[0224] Step 2

[0225]

[0226] Step 3 in Reference Example 3

[0227] Step 3

[0228]

[0229] Step 4 in Reference Example 3.

[0230] Example 6

[0231]

[0232] Step 1:

[0233]

[0234] Step 2 in Reference Example 3.

[0235]

[0236] Step 3 in Reference Example 3

[0237] Step 3:

[0238]

[0239] Step 4 in Reference Example 3.

[0240] The characterization data of each compound are shown in the following table.

[0241]

[0242]

[0243]

[0244] Bioassay Example 1 Homogeneous Time-Resolved Fluorescence Assay (HTRF Assay)

[0245] Standard assay conditions for the in vitro HTRF assay consist of a total reaction volume of 50 ul in black 384-well Costar polypropylene plates in 1X PBS buffer pH 7.4 with 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, MA), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, MA), and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed in E. coli as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2). Residues 1-83 of human p53 were expressed in E. coli as an amino-terminal AviTag <tm>- TrxA-6xHis fusion protein (biotinylated p53). Each protein was isolated from cell homogenate by affinity chromatography.

[0246] Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound in 10% DMSO (various concentrations, serial dilutions) for 20 min at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2 + compound mixture, followed by incubation for 60 min at room temperature. 10 uL of detection buffer consisting of SA-XLent, anti-GST caveolin antibody and KF was added to the GST-hMDM2, biotinylated-p53 and compound reaction and placed at room temperature to reach equilibrium and remain equilibrated for > 4 hr. The final concentration of DMSO in this reaction was 2%. Time resolved fluorescence readout was measured on a microplate multi-label reader. Percent inhibition was calculated relative to nutlin-3.

[0247] An improved HTRF assay (HTRF2 assay) was performed when the potency of the MDM2 protein degrader was increased. All assay conditions were identical to the above conditions except for the following reagent concentration changes: 0.2 nM GST-hMDM2 (1-188), 0.5 nM biotinylated-p53 (1-83), 0.18 nM SA-XLent, and 100 mM KF.

[0248] Results are given in the table below. + indicates < 10 nm, ++ indicates 10-100 nm, +++ indicates > 100 nm

[0249] Table 1 HTRF assay

[0250] No. IC 50 ]]> 055 + 056 + 057 + 058 + 059 + 060 + 061 + 062 + 063 +

[0251] Bioassay Example 2 p21 assay

[0252] Inhibition of the interaction between hMDM2 and p53 leads to activation of the p53 pathway via stabilization and accumulation of p53. p53 activates the transcription of many genes, one of which is p21 <waf1 cip1>To assess the potency of the hMDM2 protein degraders, the p21 transcript levels in compound-treated cells relative to dimethyl sulfoxide (DMSO)-treated control cells were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR).

[0253] On day 1, SJSA-1 cells were seeded at a density of 3 x 105cells / well in 100 ul of growth media (RPMI 1640; 10 mM HEPES; 1 mM sodium pyruvate; IX penicillin-streptomycin-glutamine (PSQ); and 10% fetal bovine serum (all reagents from Invitrogen; Carlsbad, CA)) in 96-well cell culture plates. The cells were incubated overnight at 37 °C and 5% CO2. 4

[0254] On day 2, the hMDM2 protein degraders were serially diluted in DMSO (Sigma-Aldrich; St. Louis, MO). Five ul of each compound dilution was added to 245 ul of filtered assay media (RPMI 1640, 10 mM HEPES, 1 mM sodium pyruvate, and IX PSQ) containing 10% FBS. Alternatively, the assay was also performed in the presence of 10% human serum or 10% mouse serum, or in the absence of any serum. The growth media was removed from the seeded SJSA-1 cells and replaced with 100 ul / well of assay media. One hundred ul of media containing the diluted inhibitor was then added to each well to a final volume of 200 ul. The dose titration of the compounds produced final concentrations ranging from 0.049 uM - 50 uM, plus a DMSO control. The cells were incubated in the presence of the inhibitors for 7 hours at 37 °C and 5% CO2. At the end of the incubation, the media was removed from the cells, and the plates were stored at -80 °C.

[0255] On day 3, total RNA was purified from the inhibitor- and DMSO-treated SJSA-1 cells using a Qiagen BioRobot Universal workstation following the RNeasy 96 BioRobot 8000 kit protocol from the manufacturer (Qiagen; Valencia, CA).

[0256] ​To measure the levels of p21 transcripts present, qRT-PCR was used. Levels of p21 and the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were determined from total RNA from each inhibitor- or DMSO-treated well in technical replicates. qRT-PCR reactions were determined on an Applied Biosystems Prism 7900HT instrument using the following cycling conditions: 48°C for 30 minutes, followed by 95°C for 10 minutes, then 40 cycles consisting of 95°C for 15 seconds and 60°C for 1 minute, using the relative quantification (ΔΔCt) method. Data were analyzed using the Applied Biosystems SDS 2.2 software with GAPDH as the endogenous control and DMSO-treated samples as calibrators. The SDS 2.2 software calculated the relative quantification (RQ) or fold increase in p21 levels relative to the DMSO control for each treated sample. The maximum (100%) p21 induction fold was defined by the maximum value of the fitted curve for the reference compound. The p21 induction fold at each inhibitor dose tested was converted to a value representing a percentage of the maximum. Dose response curves were made using XLFit software (ID Business Solutions, Alameda, CA) to calculate the IC50values for each inhibitor tested. 50 Transit values: + indicates < 1 μM, ++ indicates 1-10 μM, +++ indicates > 10 μM

[0257] Table 2 Cell assay (SJSA-1 cells)

[0258] No. 1 2 3 4 5 6 7 IC 50 ]]> 055 + 056 + 057 ++ 058 ++ 059 ++ 060 ++ 061 ++ 062 ++ 063 ++

[0259] All documents referred to in this disclosure are incorporated by reference as if each were individually incorporated. In addition, it is to be understood that various alterations and modifications will become apparent to the skilled artisan after reviewing the above teachings of the present application and that the same are intended to be encompassed by the present disclosure. < / tm>

Claims

1. A protein degradation targeting chimera, characterized in that, The protein degradation targeting chimera has a structure selected from the group consisting of: 。 2. A pharmaceutical composition, characterized by, A pharmaceutical composition for preventing and / or treating a disease associated with the activity or expression amount of MDM2.

3. The protein degradation targeting chimera of claim 1, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or the use of the pharmaceutical composition of claim 2, wherein A pharmaceutical composition for preventing and / or treating a disease associated with the activity or expression amount of MDM2.

4. An MDM2 protein degrader, characterized by, The inhibitor comprises the protein degradation targeting chimera of claim 1, or a tautomer thereof, or a pharmaceutically acceptable salt thereof.

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