Development and Use of an Acetylation Writer Inhibitor

Abstract

Bifunctional compounds (degradation inducers) that target HAT EP300 for degradation are disclosed. Pharmaceutical compositions containing the degrader inducers and methods of using the compounds to treat disease are also disclosed.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority under 35 U.S.C.§ 119(e) to U.S. Provisional Patent Application No. 62 / 754,934, filed on November 2, 2018, and incorporates by reference the entire disclosure thereof herein.

Background Art

[0002] High - risk neuroblastoma (NB) is a pediatric tumor of the peripheral sympathetic nervous system, derived from primitive neural crest cells, with a low survival rate. These neuroendocrine tumors are characterized by high expression of oncogenic MYC family members. (Matthay et al., Nat. Rev. Dis. Primers 2:16078 (2016); Zimmerman et al., Cancer Discov. 8(3):320 - 35(2018)). MYCN is an essential member of a positive feed - forward autoregulatory loop of transcription factors (TFs) that establish cell fate in MYCN - amplified NB. This group of TFs is called the core regulatory circuit (CRC), and each member is regulated by super - enhancer (SE) genes that are critically required for NB survival. One mechanism by which MYC family oncogenes promote tumor growth is by invading gene enhancers and mobilizing the transcriptional and epigenetic machinery. (Zeid et al., Nat. Genet. 50(4):515 - 23(2018)). A combination of pharmacological inhibition of transcription initiation and elongation via SEs has been shown to rapidly disrupt NB CRC in vitro and in vivo, leading to transcriptional collapse and apoptosis. (Durbin et al., Nat. Genet. 50(9):1240 - 60(2018)). Since transcriptional inhibition is insufficient to promote tumor regression in vivo (Morton et al., Mol. Oncol. 7(2):248 - 58(2013)), another approach is needed.

[0003] EP300, also known as histone acetyltransferase (HAT) p300, has recently been identified as an essential component for the survival of NB cells (Durbin et al., Nat. Genet. 50(9):1240 - 60(2018)). Similar to its paralog, cAMP response element (CREB) - binding protein (CBP, CREBBP), EP300 catalyzes the H3K27ac mark typical of SE elements (Dancy et al.., Chem. Rev. 115(6):2419 - 52(2015)). Many tumor types show dependence on EP300 rather than CBP, suggesting that this finding may be a generalizable property of different subsets of human cancers. Other EP300 - dependent and MYC - family - dependent cancers include acute myeloid leukemia (AML), multiple myeloma (MM), melanoma, rhabdomyosarcoma, and diffuse large B - cell lymphoma. Recently reported EP300 inhibitors have shown highly selective inhibition of EP300 / CBP in vitro and in vivo (Lasko et al., Nature 550:128 - 132(2017); Michaelides, et al., ACS Med. Chem. Lett. 9:28 - 33(2018)). However, the molecules did not show selectivity between EP300 and CBP.

Prior Art Documents

Non - Patent Documents

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Summary of the Invention

[0005] The first aspect of the present invention relates to a bifunctional compound having a structure represented by formula I:

Chemical Formula

[0006] In some embodiments, the EP300 targeting ligand is A-485 or an analog thereof as defined herein.

[0007] Another aspect of the present invention relates to a bifunctional compound having a structure represented by formula II:

Chemical Formula

[0008] Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a bispecific compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof and a pharmaceutically acceptable carrier.

[0009] A further aspect of the present invention relates to a method for preparing a bispecific compound of formula (I) or (II) or a pharmaceutically acceptable salt or stereoisomer thereof.

[0010] A further aspect of the present invention relates to a method for treating a disease or disorder associated with dysfunctional or dysregulated EP300 activity, which involves administering to a subject in need thereof a therapeutically effective amount of a bispecific compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof.

[0011] In some embodiments, the disease or disorder is high-risk neuroblastoma (NB).

[0012] In some embodiments, the disease or disorder is acute myeloid leukemia (AML), multiple myeloma (MM) or diffuse large B-cell lymphoma. In other embodiments, the disease or disorder is a solid tumor. In other embodiments, the disease or disorder is melanoma, rhabdomyosarcoma, colon cancer, rectal cancer, gastric cancer, breast cancer or pancreatic cancer.

[0013] Although not intended to be bound by any particular theory of operation, the bifunctional compounds of formula (I) of the present invention are thought to cause the degradation of EP300 by mobilizing the ubiquitin / proteasome system of cells, and the function of the ubiquitin / proteasome system of cells is to constantly identify and remove damaged proteins in proximity to p300 as a result of the binding of p300 and the targeting ligand. The degradation inducer is released after the EP300 molecule is destroyed and remains active. Thus, by involving and utilizing the body's own natural protein disposal system, the bifunctional compounds of the present invention may represent a potential improvement over current small molecule inhibitors of EP300. Thus, the effective intracellular concentration of the degradation inducer may be significantly lower than that of small molecule EP300 inhibitors. In summary, the bifunctional compounds of the present invention may represent an advance over known EP300 inhibitors, may overcome one or more limitations regarding their use, and may be selective in targeting EP300 rather than CBP.

[0014] A further aspect of the present invention is a method of using a bifunctional compound of formula (II) as a probe for an EP300 protein, the method comprising contacting a lysed cell suspected of containing EP300 with a compound of formula (II) and streptavidin immobilized on a support (e.g., beads), isolating a complex formed by molecular and protein binding via biotin-streptavidin binding, and confirming the presence of EP300 in the isolated complex. Confirmation can be achieved by standard techniques in the art, such as immunoblotting. BRIEF DESCRIPTION OF THE DRAWINGS

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DETAILED DESCRIPTION OF THE INVENTION

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the subject matter of this specification belongs. As used in this specification and the appended claims, unless the contrary is specified, the following terms have the meanings indicated for the purpose of facilitating the understanding of the present invention.

[0064] As used in the description and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, and reference to "an inhibitor" includes mixtures of two or more such inhibitors and the like.

[0065] Unless otherwise noted, the term "about" means within 10% of a particular value modified by the term "about" (e.g., within 5%, 2%, or 1% of).

[0066] The transitional term "comprising", which is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0067] For the compounds of the present invention and to the extent that the following terms are used herein to further describe them, the following definitions apply.

[0068] As used herein, the term "aliphatic" refers to acyclic hydrocarbon groups and includes branched and unbranched, alkyl, alkenyl, or alkynyl groups.

[0069] As used herein, the term "alkyl" refers to saturated straight-chain or branched-chain monovalent hydrocarbon radicals. In one embodiment, the alkyl radical is C1-C 18is a radical. In other embodiments, the alkyl radical is a C0-C6 radical, a C0-C5 radical, a C0-C3 radical, a C1-C 12 radical, a C1-C8 radical, a C1-C6 radical, a C1-C5 radical, a C1-C4 radical or a C1-C3 radical (wherein C0 alkyl refers to a bond). Examples of alkyl groups include methyl, ethyl, 1-propyl, 2-propyl, i-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl. In some embodiments, the alkyl group is a C1-C3 alkyl group. In some embodiments, the alkyl group is a C1-C2 alkyl group.

[0070] As used herein, the term "alkylene" consists of only carbon and hydrogen, contains no unsaturation, and refers to a linear or branched divalent hydrocarbon chain having 1 to 12 carbon atoms, such as methylene, ethylene, propylene, n-butylene, etc., which links the remainder of the molecule to a radical group. The alkylene chain can be bonded to the remainder of the molecule via a single bond and to the radical group via a single bond. In some embodiments, the alkylene group contains 1 to 8 carbon atoms (C1-C8 alkylene). In other embodiments, the alkylene group contains 1 to 5 carbon atoms (C1-C5 alkylene). In other embodiments, the alkylene group contains 1 to 4 carbon atoms (C1-C4 alkylene). In other embodiments, the alkylene contains 1 to 3 carbon atoms (C1-C3 alkylene). In other embodiments, the alkylene group contains 1 to 2 carbon atoms (C1-C2 alkylene). In other embodiments, the alkylene group contains 1 carbon atom (C1 alkylene).

[0071] As used herein, the term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more (e.g., 1, 2, 3, or 4) halo groups.

[0072] As used herein, the term "alkenyl" refers to a straight or branched chain monovalent hydrocarbon radical having at least one carbon-carbon double bond. Alkenyl radicals include radicals having "cis" and "trans" orientations, or "E" and "Z" orientations. In one example, the alkenyl radical is a C2-C 18 group. In other embodiments, the alkenyl radical is a C2-C 12 group, a C2-C 10 group, a C2-C8 group, a C2-C6 group or a C2-C3 group. Examples include ethenyl or vinyl, prop-1-enyl, prop-2-enyl, 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-diene, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl and hexa-1,3-dienyl.

[0073] As used herein, the term "alkynyl" refers to a straight or branched chain monovalent hydrocarbon radical having at least one carbon-carbon triple bond. In one example, the alkynyl radical is a C2-C 18 group. In other examples, the alkynyl radical is a C2-C 12 , a C2-C 10 , a C2-C8, a C2-C6 or a C2-C3. Examples include ethynylprop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl and but-3-ynyl.

[0074] As used herein, the term "aldehyde" is represented by the formula -C(O)H. The terms "C(O)" and C=O are used interchangeably herein.

[0075] As used herein, the term "alkoxyl" or "alkoxy" refers to an alkyl group as defined above having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, and the like. "Ether" is two hydrocarbons covalently bonded by oxygen. Thus, the alkyl substituent that makes the alkyl into an ether is an alkoxyl that can be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl, or is similar thereto.

[0076] As used herein, the term "halogen" (or "halo" or "halide") refers to fluorine, chlorine, bromine, or iodine.

[0077] As used herein, the term "carboxylic acid" is represented by the formula -C(O)OH, and "carboxylate" is represented by the formula -C(O)O-.

[0078] As used herein, the term "ester" is represented by the formula -OC(O)Z 1 or -C(O)OZ 1 wherein Z 1 is each independently an alkyl group, haloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, cycloalkyl group, cycloalkenyl group, heterocycloalkyl group, or heterocycloalkenyl group as described herein.

[0079] As used herein, the term "ether" is represented by the formula Z 1 OZ 2 wherein Z 1 and Z 2 are each independently an alkyl group, haloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, cycloalkyl group, cycloalkenyl group, heterocycloalkyl group, or heterocycloalkenyl group as described herein.

[0080] As used herein, the term "ketone" has the formula Z 1 C(O)Z 2 wherein A 1 and A 2 are each independently any of the alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl groups described herein.

[0081] As used herein, the term "sulfonyl" refers to a sulfo-oxo group having the formula --S(O)2Z 1 wherein Z 1 is hydrogen or any of the alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl groups described herein.

[0082] As used herein, the term "sulfonylamino" (or "sulfonamide") has the formula --S(O)2NH2.

[0083] As used herein, the term "thiol" has the formula --SH.

[0084] As used herein, the term "cyclic group" broadly refers to any group used alone or as part of a larger moiety, and includes saturated ring systems, partially saturated ring systems or aromatic ring systems, such as carbocyclic groups (cycloalkyl, cycloalkenyl), heterocyclic groups (heterocycloalkyl, heterocycloalkenyl), aryl groups and heteroaryl groups. A cyclic group can have one or more (e.g., fused) ring systems. Thus, for example, a cyclic group can contain one or more carbocyclic groups, heterocyclic groups, aryl groups or heteroaryl groups.

[0085] As used herein, the term "carbocyclic" (also "carbocyclyl") refers to a group used alone or as part of a larger moiety, and includes a saturated, partially unsaturated or aromatic ring system having 3 to 20 carbon atoms, whether alone or as part of a larger moiety (e.g., an alkcarbocyclic group). The term carbocyclyl includes mono-cyclic, bi-cyclic, tri-cyclic, fused ring systems, bridged ring systems and spiro-ring systems and combinations thereof. In one embodiment, the carbocyclyl contains 3 to 15 carbon atoms (C3-C 15 ). In one embodiment, the carbocyclyl contains 3 to 12 carbon atoms (C3-C 12 ). In another embodiment, the carbocyclyl is C3-C8, C3-C 10 or C5-C 10 . In another embodiment, the carbocyclyl as a monocyclic ring contains C3-C8, C3-C6 or C5-C6. In some embodiments, the carbocyclyl as a bicyclic ring contains C7-C 12 . In another embodiment, the carbocyclyl as a spiro system contains C5-C 12It includes. Representative examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, perdeuteriocyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, phenyl and cyclododecyl. Bicyclic carbocycles having 7 to 12 ring atoms include [4,3], [4,4], [4,5], [5,5], [5,6] or [6,6] ring systems, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, naphthalene and bicyclo[3.2.2]nonane. Representative examples of spirocarbocycles include spiro[2.2]pentane, spiro[2.3]hexane, spiro[2.4]heptane, spiro[2.5]octane and spiro[4.5]decane. The term carbocycle includes aryl ring systems as defined herein. The term carbocycle also includes cycloalkyl rings (e.g., saturated or partially unsaturated mono-carbon rings, bi-carbon rings or spiro-carbon rings). The term carbocyclic group also includes carbocyclic rings fused to one or more (e.g., 1, 2 or 3) different cyclic groups (e.g., aryl rings or heterocyclic rings), where the radical or point of attachment is on the carbocyclic ring.

[0086] Accordingly, the term carbocyclic, as used herein, refers to R c wherein is an alkylene chain, the formula --R c -carbocyclic groups also include carbocyclic alkyl groups. The term carbocyclic, as used herein, also refers to R c wherein is an alkylene chain, the formula --O--R c -carbocyclic groups bonded through an oxygen atom also include carbocyclic alkoxy groups.

[0087] As used herein, the term "heterocyclyl" refers to "carbocyclyl" used alone or as part of a larger moiety, and includes a saturated, partially unsaturated, or aromatic ring system in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., O, N, N(O), S, S(O), or S(O)2). The term heterocyclyl includes mono-cyclic, bi-cyclic, tri-cyclic, fused ring systems, bridged ring systems, and spiro-ring systems and combinations thereof. In some embodiments, heterocyclyl refers to a 3- to 15-membered heterocyclyl ring system. In some embodiments, heterocyclyl refers to a 3- to 12-membered heterocyclyl ring system. In some embodiments, heterocyclyl refers to a saturated ring system such as a 3- to 12-membered saturated heterocyclyl ring system. In some embodiments, heterocyclyl refers to a heteroaryl ring system such as a 5- to 14-membered heteroaryl ring system. The term heterocyclyl also includes C3-C8 heterocycloalkyl, which is a saturated or partially unsaturated mono-cyclic, bi-cyclic, or spiro-ring system containing 3 to 8 carbons and one or more (1, 2, 3, or 4) heteroatoms.

[0088] In some embodiments, the heterocyclyl group contains 3 to 12 ring atoms, including monocyclic, bicyclic, tricyclic, and spiro ring systems, where the ring atoms are carbon and 1 to 5 of the ring atoms are heteroatoms such as nitrogen, sulfur, or oxygen. In some embodiments, the heterocyclyl contains a 3- to 7-membered monocyclic ring having one or more heteroatoms selected from nitrogen, sulfur, or oxygen. In some embodiments, the heterocyclyl contains a 4- to 6-membered monocyclic ring having one or more heteroatoms selected from nitrogen, sulfur, or oxygen. In some embodiments, the heterocyclyl contains a 3-membered monocyclic ring. In some embodiments, the heterocyclyl contains a 4-membered monocyclic ring. In some embodiments, the heterocyclyl contains a 5- to 6-membered monocyclic ring. In some embodiments, the heterocyclyl group contains 0 to 3 double bonds. In any of the foregoing embodiments, the heterocyclyl contains 1, 2, 3, or 4 heteroatoms. Any nitrogen heteroatom or sulfur heteroatom may optionally be oxidized (e.g., NO, SO, SO2), and any nitrogen heteroatom may optionally be quaternized (e.g., [NR4] + Cl - , [NR4] + OH -). Representative examples of heterocyclyl include oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 1,2-dithietanyl, 1,3-dithietanyl, pyrrolidinyl, dihydro-1H-pyrrolyl, dihydrofuranyl, tetrahydropyranyl, dihydrothienyl, tetrahydrothienyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl, oxazinanyl, thiazinanyl, thioxanyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl, oxazepinyl, oxazepanyl, diazepanyl, 1,4-diazepanyl, diazepinyl, thiazepinyl, thiazepanyl, tetrahydrothiopyranyl, oxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,1-dioxoisothiazolidinonyl, oxazolidinonyl, imidazolidinonyl, 4,5,6,7-tetrahydro[2H]indazolyl, tetrahydrobenzimidazolyl, 4,5,6,7-tetrahydrobenzo[d]imidazolyl, 1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridinyl, thiazinyl, thiophenyl, oxazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, thiapyranyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrimidinonyl, pyrimidinedionyl, pyrimidine-2,4-dionyl, piperazinonyl, piperazinedionyl, pyrazolidinylimidazolinyl, 3-azabicyclo[3.1.0]hexanyl, 3,6-diazabicyclo[3.1.1]heptanyl, 6-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 2-azabicyclo[3.2.1]octanyl, 8-azabicyclo[3.2.1] Octanyl, 2-azabicyclo[2.2.2]octanyl, 8-azabicyclo[2.2.2]octanyl, 7-oxabicyclo[2.2.1]heptane, azaspiro[3.5]nonanyl, azaspiro[2.5]octanyl, azaspiro[4.5]decanyl, 1-azaspiro[4.5]decan-2-onyl, azaspiro[5.5] Examples include undecanyl, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindazolyl, and 1,1-dioxohexahydrothiopyranyl. Examples of 5-membered heterocyclyl containing a sulfur atom or an oxygen atom and 1 to 3 nitrogen atoms include thiazolyl containing thiazol-2-yl and thiazol-2-yl N-oxide, thiadiazolyl containing 1,3,4-thiadiazol-5-yl and 1,2,4-thiadiazol-5-yl, oxazolyl such as oxazol-2-yl, and oxadiazolyl such as 1,3,4-oxadiazol-5-yl and 1,2,4-oxadiazol-5-yl. Exemplary 5-membered ring heterocyclyl containing 2 to 4 nitrogen atoms include imidazolyl such as imidazol-2-yl; triazolyl such as 1,3,4-triazol-5-yl; 1,2,3-triazol-5-yl, 1,2,4-triazol-5-yl, and tetrazolyl such as 1H-tetrazol-5-yl. Representative examples of benzo-fused 5-membered heterocyclyl include benzoxazol-2-yl, benzothiazol-2-yl, and benzimidazol-2-yl. Exemplary 6-membered heterocyclyl includes 1 to 3 nitrogen atoms and optionally a sulfur atom or an oxygen atom, for example, pyridyl such as pyrid-2-yl, pyrid-3-yl, and pyrid-4-yl; pyrimidyl such as pyrimid-2-yl and pyrimid-4-yl; triazinyl such as 1,3,4-triazin-2-yl and 1,3,5-triazin-4-yl; pyridazinyl, particularly pyridazin-3-yl, and pyrazinyl. Pyridine N-oxide and pyridazine N-oxide, as well as pyridyl groups, pyrimid-2-yl groups, pyrimid-4-yl groups, pyridazinyl groups, and 1,3,4-triazin-2-yl groups are further examples of heterocyclyl groups. In some embodiments, the heterocyclic group includes a heterocycle fused to one or more (e.g., 1, 2, or 3) different cyclic groups (e.g., carbocyclic or heterocyclic), where the radical or point of attachment is on the heterocycle, and in some embodiments, the point of attachment is a heteroatom contained in the heterocycle.

[0089] Accordingly, as used herein, the term heterocyclic includes N - heterocyclyl groups that refer to heterocyclyl groups containing at least one nitrogen, where the point of attachment of the heterocyclyl group to the remainder of the molecule is through a nitrogen atom of the heterocyclyl group. Representative examples of N - heterocyclyl groups include 1 - morpholinyl, 1 - piperidinyl, 1 - piperazinyl, 1 - pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl. The term heterocyclic also includes, as used herein, C - heterocyclyl groups that refer to heterocyclyl groups containing at least one heteroatom, where the point of attachment of the heterocyclyl group to the remainder of the molecule is through a carbon atom of the heterocyclyl group. Representative examples of C - heterocyclyl radicals include 2 - morpholinyl, 2 - or 3 - or 4 - piperidinyl, 2 - piperazinyl, and 2 - or 3 - pyrrolidinyl. The term heterocyclic also includes, as disclosed above, R c is an alkylene chain of the formula --R c - heterocyclyl, including heterocyclylalkyl groups. The term heterocyclic also includes, as used herein, R c is an alkylene chain of the formula --O--R c - heterocyclyl, including heterocyclylalkoxy groups that refer to radicals attached through an oxygen atom of the heterocyclyl group.

[0090] As used herein, the term "aryl," used alone or as part of a larger moiety (e.g., "aralkyl" where a terminal carbon atom on an alkyl group is the point of attachment, e.g., a benzyl group), "aralkoxy" where an oxygen atom is the point of attachment, or "aroxyalkyl" where the point of attachment is on an aryl group), refers to a group that includes a monocyclic, bicyclic, or tricyclic carbon ring system, including fused rings, where at least one ring in the system is aromatic. In some embodiments, an aralkoxy group is a benzoxy group. The term "aryl" may be used interchangeably with the term "aryl ring." In one embodiment, aryl includes groups having 6 to 18 carbon atoms. In another embodiment, aryl includes groups having 6 to 10 carbon atoms. Examples of aryl groups include phenyl, naphthyl, anthracyl, biphenyl, phenanthrenyl, naphthacenyl, 1,2,3,4-tetrahydronaphthalenyl, 1H-indenyl, 2,3-dihydro-1H-indenyl, naphthyridinyl, and the like, which may be substituted or independently substituted by one or more substituents described herein. A particular aryl is phenyl. In some embodiments, an aryl group comprises an aryl ring fused to one or more (e.g., 1, 2, or 3) different cyclic groups (e.g., carbocyclic or heterocyclic), where the radical or point of attachment is on the aryl ring.

[0091] Thus, the term aryl, as disclosed above, refers to R c is an alkylene chain such as methylene or ethylene; c -aryl groups (e.g., benzyl). In some embodiments, the aralkyl group is an optionally substituted benzyl group. The term aryl, as used herein, also refers to an R c is an alkylene chain such as methylene or ethylene; c - includes aralkoxy, which refers to a group bonded through the oxygen atom of an aryl.

[0092] As used herein, the term "heteroaryl," alone or as part of a larger moiety (e.g., "heteroarylalkyl" (also "heteroaralkyl") or "heteroarylalkoxy" (also "heteroaralkoxy")), refers to a monocyclic, bicyclic or tricyclic ring system having 5 to 14 ring atoms, wherein at least one ring is aromatic and contains at least one heteroatom. In one embodiment, heteroaryl includes a 5- to 6-membered monocyclic aromatic group in which one or more of the ring atoms are independently optionally substituted nitrogen, sulfur or oxygen. In another embodiment, heteroaryl includes a 5- to 6-membered monocyclic aromatic group in which one or more of the ring atoms are nitrogen, sulfur or oxygen. Representative examples of heteroaryl groups include thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, imidazopyridyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, tetrazolo[1,5-b]pyridazinyl, purinyl, deazapurinyl, benzoxazolyl, benzofuryl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzimidazolyl, indolyl, 1,3-thiazol-2-yl, 1,3,4-triazol-5-yl, 1,3-oxazol-2-yl, 1,3,4-oxadiazol-5-yl, 1,2,4-oxadiazol-5-yl, 1,3,4-thiadiazol-5-yl, 1H-tetrazol-5-yl, 1,2,3-triazol-5-yl and pyrid-2-yl N-oxide. The term "heteroaryl" also includes groups in which the heteroaryl is fused to one or more cyclic (e.g., carbocyclic or heterocyclic) rings, where the radical or point of attachment is on the heteroaryl ring.Non-limiting examples include indolyl, indolizinyl, isoindolyl, benzothienyl, benzothiophenyl, methylenedioxyphenyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzodioxazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic, bicyclic, or tricyclic. In some embodiments, a heteroaryl group comprises a heteroaryl ring fused to one or more (e.g., 1, 2, or 3) different cyclic groups (e.g., carbocycles or heterocycles), where the radical or point of attachment is on the heteroaryl ring, and in some embodiments, the point of attachment is a heteroatom contained in the heterocycle.

[0093] Thus, the term heteroaryl, as used herein, encompasses N-heteroaryl groups, which refer to heteroaryl groups as defined above that contain at least one nitrogen, where the point of attachment of the heteroaryl group to the remainder of the molecule is through a nitrogen atom of the heteroaryl group. The term heteroaryl, as used herein, also encompasses C-heteroaryl groups, which refer to heteroaryl groups as defined above, where the point of attachment of the heteroaryl group to the remainder of the molecule is through a carbon atom of the heteroaryl group. The term heteroaryl also encompasses R, as disclosed above. c is an alkylene chain as defined above c The term heteroaryl, as used herein, also includes heteroarylalkyl groups, which refer to the group R c is an alkylene group as defined above c -includes heteroaralkoxy (or heteroarylalkoxy) groups, which refer to groups attached through an oxygen atom of a heteroaryl.

[0094] Any of the groups described herein may be substituted or unsubstituted. As used herein, the term "substituted" means that such substitution is in accordance with the valences of the atoms being substituted and the substituents, and the substitution is accompanied by the implicit condition that it results in a stable compound, i.e., a compound that does not undergo spontaneous transformation, for example, by rearrangement, cyclization, elimination, etc. and broadly refers to any acceptable substituent. Representative substituents include halogen, hydroxyl group, and any number of carbon atoms, for example, from 1 to 14 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) heteroatoms, for example, any other organic grouping that may contain oxygen, sulfur, and nitrogen grouped in a linear, branched, or cyclic structural form.

[0095] Thus, representative examples of substituents include an alkyl group, a substituted alkyl group (e.g., C1-C6, C1-5, C1-4, C1-3, C1-2, C1), an alkoxy group (e.g., C1-C6, C1-5, C1-4, C1-3, C1-2, C1), a substituted alkoxy group (e.g., C1-C6, C1-5, C1-4, C1-3, C1-2, C1), a haloalkyl group (e.g., CF3), an alkenyl group (e.g., C2-C6, C2-5, C2-4, C2-3, C2), a substituted alkenyl group (e.g., C2-C6, C2-5, C2-4, C2-3, C2), an alkynyl group (e.g., C2-C6, C2-5, C2-4, C2-3, C2), a substituted alkynyl group (e.g., C2-C6, C2-5, C2-4, C2-3, C2), a cyclic group (e.g., C3-C12, C5-C6), a substituted cyclic group (e.g., C3-C12, C5-C6), a carbocyclic group (e.g., C3-C12, C5-C6), a substituted carbocyclic group (e.g., C3-C12, C5-C6), a heterocyclic group (e.g., C3-C12, C5-C6), a substituted heterocyclic group (e.g., C3-C12, C5-C6), an aryl group (e.g., benzyl and phenyl), a substituted aryl group (e.g., substituted benzyl or substituted phenyl), a heteroaryl group (e.g., pyridyl or pyrimidyl), a substituted heteroaryl group (e.g., substituted pyridyl or substituted pyrimidyl), an aralkyl group (e.g., benzyl), a substituted aralkyl group (e.g., substituted benzyl), a halo group, a hydroxyl group, an aryloxy group (e.g., C6-C12, C6), a substituted aryloxy group (e.g., C6-C12, C6), an alkylthio group (e.g., C1-C6), a substituted alkylthio group (e.g., C1-C6), an arylthio group (e.g., C6-C12, C6), a substituted arylthio group (e.g., C6-C12, C6), a cyano group, a carbonyl group, a substituted carbonyl group, a carboxyl group, a substituted carboxyl group, an amino group, a substituted amino group, an amide group, a substituted amide group, a thio group, a substituted thio group, a sulfinyl group, a substituted sulfinyl group, a sulfonyl group, a substituted sulfonyl group, a sulfinamido group, a substituted sulfinamido group, a sulfonamido group, a substituted sulfonamido group, a urea group, a substituted urea group, a carbamate group, a substituted carbamate group, an amino acid group, and a peptide group may be mentioned.

[0096] The term "binding" related to the interaction between a targeting ligand and a target protein, which in the present invention is EP300 and its variants (collectively referred to as "EP300"), typically refers to an intermolecular interaction that can be preferential or substantially specific (also referred to herein as "selective") in that the binding of the targeting ligand to other proteinaceous entities present in the cell, such as CBP, is not functionally important. The bifunctional compounds of the present invention can preferentially bind to EP300, including its variants, and recruit them for target degradation.

[0097] The term "binding" related to the interaction between a degron and an E3 ubiquitin ligase typically refers to an intermolecular interaction that may or may not exhibit an affinity level equal to or exceeding that between a targeting ligand and a target protein, yet the affinity is sufficient to achieve target degradation and the recruitment of the ligase for the selective degradation of the target protein.

[0098] Generally, the bifunctional compounds of one aspect of the present invention have a structure represented by Formula I:

Chemical formula

[0099] Targeting ligand In some embodiments, the EP300 targeting ligand is A-485 or an analog thereof. A-485 is represented by Structure TL-1:

Chemical formula

[0100] A-485, also known as N-[(4-fluorophenyl)methyl]-2-{(1R)-5-[(methylcarbamoyl)amino]-2’,4’-dioxo-2,3-dihydro-3’H-spiro[indene-1,5’-[1,3]oxazolidine]-3’-yl}-N-[(2S)-1,1,1-trifluoropropan-2-yl]acetamide, and its spirocyclic analogs are described in U.S. Patent Application Publication No. 2016 / 0235716 and Michaelides, et al., ACS Med. Chem. Lett. 9:28-33 (2018).

[0101] Accordingly, in some embodiments, the bifunctional compound of formula (I) is of structure I-1:

Chem.

[0102] In some embodiments, the EP300 targeting ligand is an A-485 analog and is represented by structure TL-1a:

Chem.

[0103] Accordingly, in some embodiments, the bifunctional compound of formula (I) is of structure I-1a:

Chem.

[0104] In some embodiments, the EP300 targeting ligand is an A-485 analog and is represented by structure TL-1b: [Chemical formula] wherein A is CH2, NH or O; B is CH2 or CO; R is H, halo (e.g., Cl or F), CN, CF3, alkyl or alkoxy; R1 is a C3-C5 carbocyclic group or alk carbocyclic group, or a 3- to 5-membered N-heterocyclic group or alk N-heterocyclic group (where N is attached to an alkyl group), and the alkyl group is a C1-C10 alkyl (e.g., C1-C3) alkyl group.

[0105] In some embodiments, R1 is [Chemical formula] as follows.

[0106] Accordingly, in some embodiments, the bifunctional compound of formula (I) is of structure I-1b: [Chemical formula] (wherein A is CH2, NH or O; B is CH2 or CO; R is halo (e.g., Cl or F), CN, CF3, alkyl or alkoxy; R1 is a C3-C5 carbocyclic group or alk carbocyclic group, or a 3- to 5-membered N-heterocyclic group or alk N-heterocyclic group, and the alkyl group is a C1-C10 alkyl (e.g., C1-C3) alkyl group), or a pharmaceutically acceptable salt or stereoisomer thereof.

[0107] In some embodiments, the EP300 targeting ligand is of structure TL-1c: [Chemical formula] represented by, where Q is CH2, O, N, CO, C(O)O, C(O)N, CH2N, CH2C(O), CH2C(O)O, CH2C(O)N or CH2CH2N; R2 is [Chemical formula] a C3-C5 carbocyclic group or alk carbocyclic group, or a 3-5 membered N-heterocyclic group or alk N-heterocyclic group, and the alkyl group is a C1-C10 alkyl (e.g., C1-C3) alkyl group.

[0108] Thus, in some embodiments, the bifunctional compound of formula (I) is of structure I-c: [Chemical formula] or represented by its pharmaceutically acceptable salt or stereoisomer.

[0109] In some embodiments, the targeting ligand is represented by structure TL-1d: [Chemical formula]

[0110] Thus, in some embodiments, the bifunctional compound of formula (I) is of structure I-1d: [Chemical formula] or represented by its pharmaceutically acceptable salt or stereoisomer.

[0111] In some embodiments, the EP300 targeting ligand is represented by structure TL-1e: [Chemical formula]

[0112] Thus, in some embodiments, the bifunctional compound of formula (I) is of structure I-1e:

Chem.

[0113] In some embodiments, the EP300 targeting ligand is represented by structure TL-1f:

Chem.

[0114] Thus, in some embodiments, the bifunctional compound of formula (I) is of structure I-1f:

Chem.

[0115] The linker (「L」) provides a covalent bond between the targeting ligand and the degron. The structure of the linker may not be important as long as it does not substantially interfere with the activity of the targeting ligand or the degron. In some embodiments, the linker is an alkylene chain (e.g., having 2 to 20 alkylene units). In other embodiments, the linker can be an alkylene chain or a divalent alkylene chain, either of which can be interrupted by and / or terminated with (at one or both ends) at least one of a C3-C carbocyclene, a 3- to 12-membered heterocyclene, a 5- to 12-membered heteroarylene, or any combination thereof, where R’ is H or C1-C6 alkyl, and the interrupting group and one or both of the terminating groups may be the same or different. 12

[0116] ​In other embodiments, the linker can be a polyethylene glycol chain. In other embodiments, the linker can be a polyethylene glycol chain or a divalent alkylene chain, either of which can be interrupted by and / or terminated with (at one or both ends) at least one of a carbocyclene, a 3- to 12-membered heterocycle, a 5- to 12-membered heteroarylene, or any combination thereof, where R’ is H or C1-C6 alkyl, and the ending group(s) at one or both ends can be the same or different. 3~12

[0117] In some embodiments, the linker can be a C1-C alkylene chain terminated with an NH group in which nitrogen is also bonded to the degron. 10

[0118] In certain embodiments, the linker has 1 to 10 alkylene units and is

Chemical formula

Chemical formula

[0119] ​​ In other embodiments, the linker has 1 to 8 PEG units and

Chemical formula

[0120] "Carbocyclene" refers to a divalent carbocyclic radical which may be substituted.

[0121] "Heterocyclene" refers to a divalent heterocyclyl radical which may optionally be substituted.

[0122] "Heteroarylene" refers to a divalent heteroaryl radical which may optionally be substituted.

[0123] Representative examples of linkers that may be suitable for use in the present invention include alkylene chains, such as the following:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0124] In some embodiments, the linker is a polyethylene glycol chain, and examples thereof include the following: [Chemical formula] In the formula, n is an integer from 1 to 10, and examples thereof include the following: [Chemical formula] In some embodiments, the polyethylene glycol chain may be terminated with a functional group, and examples thereof are as follows: [Chemical formula] [Chemical formula]

[0125] In some embodiments, the bifunctional compound of formula (I) contains a linker represented by structure L10: [Chemical formula] In the formula, X is CH2, NH, NMe, NEt, or O, and n is an integer from 0 to 11.

[0126] In some embodiments, the linker is represented by any one of structures L11-L26: [Chemical formula] [Chemical formula]

[0127] Thus, in some embodiments, the bifunctional compound of the present invention is any one of structures I-2 to I-12: [Chemical formula] [Chemical formula] Targeting ligand Degron or a pharmaceutically acceptable salt or stereoisomer thereof.

[0128] In some embodiments, the bifunctional compound of the present invention is a structure selected from the group consisting of: [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemistry] [Chemistry] [Chemistry] [Chemistry] [Chemistry] [Chemistry] [Chemistry] [Chemistry] or represented by its pharmaceutically acceptable salt or stereoisomer. Degron

[0129] The degron (「D」) is a functional moiety that binds to the E3 ubiquitin ligase. In some embodiments, the degron binds to cereblon (CRBRN). In some embodiments, the degron binds to the von Hippel-Lindau (VHL) tumor suppressor.

[0130] In some embodiments, the bifunctional compound of formula (I) comprises a degron that binds to cereblon. Representative examples of degrons that bind to cereblon are described in U.S. Patent Application Publication No. 2018 / 0015085 (e.g., indolinones such as isoindolinone and isoindoline-1,3-dione included in formulas IA and IA' therein, and cross-linked cycloalkyl compounds included in formulas IB and IB' therein).

[0131] In some embodiments, the compound of formula (I) binds to cereblon and comprises a degron represented by structure D1: [Chemistry] In the formula, Y is CH2 or CO; Z is NH, O or OCH2CO, and the wavy line ( [Chemical formula] ) represents a linker and a binding point to the EP300 targeting moiety.

[0132] In some embodiments, the degron is represented by Structure D1-a: [Chemical formula]

[0133] In some embodiments, the degron binds to VHL. Representative examples of degrons that bind to VHL are as follows: [Chemical formula] [Chemical formula] (wherein Y' is a bond, N, O or C); [Chemical formula] (wherein Z is a cyclic group, for example, a C5-C6 carbocyclic group or a C5-C6 heterocyclic group); and [Chemical formula]

[0134] Other degrons that bind to VHL and may be suitable for use in the present invention are disclosed in U.S. Patent Application Publication No. 2017 / 0121321 A1.

[0135] Thus, in some embodiments, the bifunctional compound of formula (I) is represented by any structure generated by the combination of the structures of TL-1, TL-1a, TL-1b, TL-1c and L1-L11 described herein with the structure of the degron containing D1, D1a and D2-a to D2-e, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0136] In some embodiments, the compounds of the invention are structures selected from the group consisting of:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0137] Biotinylated compound Another aspect of the invention relates to a bifunctional compound having a structure represented by formula II:

Chemical formula

[0138] Thus, in some embodiments, the bifunctional compound of formula (II) has a structure selected from the group consisting of:

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0139] The bifunctional compounds of formula (I) and formula (II) can be in the form of the free acid or free base or a pharmaceutically acceptable salt. As used herein, in the context of salts, the term "pharmaceutically acceptable" refers to a compound that does not inactivate the biological activity or properties of the compound and is relatively non-toxic, i.e., the compound in salt form can be administered to a subject without causing undesirable biological effects (such as dizziness or stomach upset) or interacting in a harmful manner with any of the other components of the composition in which it is contained. The term "pharmaceutically acceptable salt" refers to the product obtained by the reaction of a compound of the present invention with a suitable acid or base. Examples of pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic bases, such as Li salts, Na salts, K salts, Ca salts, Mg salts, Fe salts, Cu salts, Al salts, Zn salts, and Mn salts. Examples of pharmaceutically acceptable non-toxic acid addition salts include salts of amino groups formed by inorganic acids, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, 4-methylbenzenesulfonate, or p-toluenesulfonate, etc. Certain compounds of the present invention can form pharmaceutically acceptable salts with various organic bases such as lysine, arginine, guanidine, diethanolamine, or metformin.

[0140] The bifunctional compounds of the present invention may have at least one chiral center and can therefore be in the form of stereoisomers, including all isomers of the individual compounds that differ only in the orientation of their atoms in space as used herein. The term stereoisomers includes enantiomers (compounds containing the (R-) or (S-) configuration of the compound), mixtures of enantiomers of the compound (physical mixtures of enantiomers, and racemates or racemic mixtures), geometric (cis / trans or E / Z, R / S) isomers of the compound, and isomers of compounds having multiple chiral centers that are not mirror images of each other (diastereoisomers). The chiral centers of the compounds can undergo epimerization in vivo. Therefore, for these compounds, administration of the compound in its (R-) form is considered equivalent to administration of the compound in its (S-) form. Thus, the compounds of the present invention can be made and used in the form of the individual isomers, substantially free of other isomers, or in the form of mixtures of various isomers, such as racemic mixtures of stereoisomers.

[0141] In some embodiments, the bifunctional compounds of formula (I) or formula (II) are isotope derivatives in that they have at least one desired isotope substitution of an atom in an amount exceeding the natural abundance of the isotope, i.e., an enriched amount. In one embodiment, the compound contains deuterium or multiple deuterium atoms. Substitution with a relatively heavy isotope such as deuterium, i.e., 2 H, can be advantageous in some situations because it can result in certain therapeutic advantages arising from relatively greater metabolic stability, e.g., an increase in in vivo half-life or a decrease in the required dose.

[0142] Furthermore, the compounds of formula (I) include N-oxides, crystalline forms (also known as polymorphs), active metabolites of compounds having the same type of activity, tautomers, and the use of the compounds in non-solvated and solvated forms with pharmaceutically acceptable solvents such as water, ethanol. Solvated forms of the conjugates presented herein are also considered to be disclosed herein.

[0143] Synthesis methods In another aspect, the present invention relates to a method for preparing a bifunctional compound of the present invention or a pharmaceutically acceptable salt or stereoisomer thereof. Generally, the compounds of the present invention or pharmaceutically acceptable salts or stereoisomers thereof can be prepared by any process known to be applicable to the preparation of chemically related compounds. The compounds of the present invention will be better understood in connection with the synthetic schemes described in various examples and showing non-limiting methods by which the compounds of the present invention can be prepared.

[0144] Pharmaceutical composition Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a bifunctional compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as known in the art refers to a pharmaceutically acceptable material, composition or vehicle suitable for administering the compounds of the present invention to a mammal. Suitable carriers can include, for example, liquids (both aqueous and non-aqueous analogs, and combinations thereof), solids, encapsulating materials, gases, and combinations thereof (e.g., semi-solids), as well as gases that function to transport or convey the compound from one organ, or part of the body, to another organ, or part of the body. A carrier is "acceptable" in the sense that it is physiologically inert to the other components of the formulation, compatible with them, and not harmful to the subject or patient. The composition may also contain one or more pharmaceutically acceptable excipients, depending on the type of formulation.

[0145] Generally, the bifunctional compounds of formula (I) and their pharmaceutically acceptable salts and stereoisomers can be formulated into a given type of composition according to conventional pharmaceutical practice, for example, conventional mixing, dissolving, granulating, tablet coating, levigation, emulsifying, encapsulating, entrapping and compression processes (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A.R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.C. Boylan, 1988 - 1999, Marcel Dekker, New York). The type of formulation depends on the mode of administration which may include enteral (e.g., oral, buccal, sublingual and rectal), parenteral (e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) and intrasternal injection or infusion techniques, intraocular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, intradermal, intravaginal, intraperitoneal, mucosal, nasal, intratracheal instillation, bronchial instillation, and inhalation) and topical (e.g., transdermal). Generally, the most appropriate route of administration depends on various factors including, for example, the nature of the drug (e.g., its stability in the gastrointestinal environment) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration). For example, parenteral (e.g., intravenous) administration can also be advantageous in that the compound can be administered relatively rapidly, as in the case of single dose treatment and / or acute conditions.

[0146] In some embodiments, the bifunctional compounds of formula (I) are formulated for oral administration or intravenous administration (e.g., systemic intravenous injection).

[0147] Therefore, the bifunctional compound of formula (I) can be formulated into solid compositions (e.g., powders, tablets, dispersible granules, capsules, cachets, and suppositories), liquid compositions (e.g., solutions in which the compound is dissolved, suspensions in which solid particles of the compound are dispersed, emulsions, and solutions containing liposomes, micelles, or nanoparticles, syrups, and elixirs); semi-solid compositions (e.g., gels, suspensions, and creams); and gases (e.g., propellants for aerosol compositions). The compound can also be formulated for immediate release, intermediate release, or sustained release.

[0148] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with carriers such as sodium citrate or dicalcium phosphate, and additional carriers or excipients such as a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, etc., c) humectants such as glycerol, d) disintegrants such as cross-linked polymers (e.g., cross-linked polyvinylpyrrolidone (crospovidone), cross-linked sodium carboxymethylcellulose (croscarmellose sodium), sodium starch glycolate, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate), e) solution retarding agents such as paraffin, f) absorption promoters such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, etc., h) absorbents such as kaolin and bentonite clays, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents. Solid compositions of the same type may also be used as fillers in soft and hard gelatin capsule fillings using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol. Solid dosage forms of tablets, dragees, capsules, pills, and granules may be prepared using coatings and shells such as enteric coatings and other coatings. They may further contain opacifying agents.

[0149] In some embodiments, the bifunctional compound of formula (I) can be formulated into soft and hard gelatin capsules. Representative excipients that can be used include pregelatinized starch, magnesium stearate, mannitol, sodium stearyl fumarate, anhydrous lactose, microcrystalline cellulose, and croscarmellose sodium. The gelatin shell can contain gelatin, titanium dioxide, iron oxide, and coloring agents.

[0150] Liquid dosage forms for oral administration include solutions, suspensions, emulsions, microemulsions, syrups, and elixirs. In addition to the compound, the liquid dosage form can contain aqueous or non-aqueous carriers commonly used in the art (depending on the solubility of the compound), such as water or other solvents, solubilizing agents, and emulsifying agents, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. Oral compositions can also contain excipients, such as wetting agents, suspending agents, coloring agents, sweetening agents, flavoring agents, and fragrances.

[0151] Injectable preparations can include sterile aqueous solutions or oily suspensions. They can be formulated according to standard techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can also be, for example, sterile injectable solutions, suspensions or emulsions in a non-toxic parenterally acceptable diluent or solvent, such as a solution of 1,3 - butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, U.S.P. and isotonic saline solution. Further, sterile fixed oils have conventionally been used as a solvent or suspending medium. For this purpose, any mildly stimulating fixed oil containing synthetic monoglycerides or synthetic diglycerides can be used. Further, fatty acids such as oleic acid are used in the preparation of injectables. Injectable formulations can be sterilized, for example, by filtration through a bacteria - retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use. The effect of the compound can be prolonged by delaying its absorption, which can be achieved by using a liquid suspension of low water - solubility or a crystalline or amorphous material. Prolonged absorption of the compound from parenterally administered formulations can also be achieved by suspending the compound in an oily vehicle.

[0152] In certain embodiments, the compounds of formula (I) of the present invention can be administered locally rather than systemically, for example, in many cases in depot or sustained release formulations, via direct injection of the conjugate into an organ. In certain embodiments, the long-acting formulations are administered by implantation (e.g., subcutaneously or intramuscularly) or intramuscular injection. Injectable depot forms are prepared by forming a microcapsule matrix of the compound in a biodegradable polymer such as polylactide-polyglycolide, poly(orthoester) and poly(anhydride). The release rate of the compound can be controlled by varying the ratio of the compound to the polymer and the nature of the particular polymer used. Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions compatible with body tissues. Further, in other embodiments, the compound is delivered in a targeted drug delivery system, such as liposomes coated with an organ-specific antibody. In such embodiments, the liposomes target the organ and are selectively taken up by the organ.

[0153] The bifunctional compounds of formula (I) can be formulated for buccal or sublingual administration and examples thereof include tablets, troches and gels.

[0154] The bifunctional compounds can be formulated for administration by inhalation. Various forms suitable for administration by inhalation include aerosols, mists or powders. The pharmaceutical composition can be delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In some embodiments, the dosage unit of the pressurized aerosol can be determined by providing a valve for delivering a metered amount. In some embodiments, for example, capsules and cartridges containing gelatin and a powder mixture of the compound with a suitable powder base such as lactose or starch for use in an inhaler or insufflator can be formulated.

[0155] The difunctional compound of formula (I), as used herein, can be formulated for topical administration, which refers to intradermal administration by applying the formulation to the epidermis. Compositions of these types are typically in the form of ointments, pastes, creams, lotions, gels, solutions, and sprays.

[0156] Representative examples of carriers useful for formulating compositions for topical application include solvents (e.g., alcohols, polyhydric alcohols, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffer solutions (e.g., hypotonic saline or buffered saline). For example, creams can be formulated using saturated or unsaturated fatty acids such as stearic acid, palmitic acid, oleic acid, palmito-oleic acid, cetyl alcohol, or oleyl alcohol. Creams can also contain nonionic surfactants such as polyoxy-40-stearate.

[0157] In some embodiments, the topical formulation may also include excipients, examples of which are penetration enhancers. These agents can transport pharmacologically active compounds through the stratum corneum to the epidermis or dermis, preferably with little or no systemic absorption. A wide variety of compounds have been evaluated with respect to their effect in enhancing the rate of drug penetration through the skin. See, for example, Percutaneous Penetration Enhancers, Maibach H.I. and Smith H.E. (eds.), CRC Press, Inc., Boca Raton, Fla. (1995), and Buyuktimkin et al., Chemical Means of Transdermal Drug Permeation Enhancement in Transdermal and Topical Drug Delivery Systems, Gosh T.K., Pfister W.R., Yum S.I. (Eds.), Interpharm Press Inc., Buffalo Grove, Ill. (1997). Representative examples of penetration enhancers include triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe vera gel), ethyl alcohol, isopropyl alcohol, octolyphenylpolyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decylmethyl sulfoxide, fatty acid esters (e.g., isopropyl myristate, methyl laurate, glycerol monooleate and propylene glycol monooleate) and N-methylpyrrolidone.

[0158] Representative examples of other excipients that can be included in topical and other types of formulations (to the extent they are compatible) include preservatives, antioxidants, humectants, emollients, buffers, solubilizers, skin protectants, and surfactants. Suitable preservatives include alcohols, quaternary amines, organic acids, parabens, and phenols. Suitable antioxidants include ascorbic acid and its esters, sodium bisulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherol, and chelating agents such as EDTA and citric acid. Suitable humectants include glycerin, sorbitol, polyethylene glycol, urea, and propylene glycol. Suitable buffers include citrate buffer, hydrochloride buffer, and lactate buffer. Suitable solubilizers include quaternary ammonium chloride, cyclodextrin, benzyl benzoate, lecithin, and polysorbate. Suitable skin protectants include vitamin E oil, allantoin, dimethicone, glycerin, petrolatum, and zinc oxide.

[0159] Transdermal formulations typically use a transdermal delivery device and a transdermal patch in which the compound is formulated into a lipophilic emulsion or buffered aqueous solution and dissolved and / or dispersed in a polymer or adhesive. The patch can be constructed for continuous, pulsatile, or on-demand delivery of the pharmaceutical. Transdermal delivery of the compound can be achieved by an iontophoresis patch. The transdermal patch can provide controlled delivery of the compound by using a rate-controlling membrane or by trapping the compound in a polymer matrix or gel to slow the absorption rate. Absorption enhancers may be used to increase absorption and examples include absorbable pharmaceutically acceptable solvents that aid passage through the skin.

[0160] Ophthalmic formulations include eye drops.

[0161] Formulations for rectal administration include enemas, rectal gels, rectal foams, rectal aerosols, and retention enemas, which may contain conventional suppository bases such as cocoa butter or other glycerides, and synthetic polymers such as polyvinylpyrrolidone, PEG, etc. Also, compositions for rectal or vaginal administration are formulated as suppositories that can be prepared by mixing a compound with suitable non-irritating carriers and excipients that are all solid at ambient temperature but liquid at body temperature, and thus melt in the rectal or vaginal cavity to release the compound, such as cocoa butter, mixtures of fatty acid glycerides, polyethylene glycol, suppository wax, and combinations thereof.

[0162] Dosage As used herein, the term "therapeutically effective amount" refers to an amount of a bifunctional compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof; or a composition comprising a bifunctional compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof that is effective to produce a desired therapeutic response in a particular patient afflicted with a disease or disorder due to abnormal EP300 activity. Thus, the term "therapeutically effective amount" includes an amount of a compound of the present invention or a pharmaceutically acceptable salt or stereoisomer thereof that, upon administration, induces a positive modification in the disease or disorder being treated (e.g., to selectively inhibit / degrade EP300), is sufficient to prevent the onset or progression of the disease or disorder, alleviates to some extent one or more of the symptoms of the disease or disorder being treated in the subject, simply kills or inhibits disease (e.g., neuroblastoma) cells, or reduces the amount of EP300 in the disease cells.

[0163] The total daily dosage of the bifunctional compound of formula (I) and its method of use can be determined according to standard medical practice, for example, by the attending physician using sound medical judgment. The specific therapeutically effective dosage for any particular subject depends on various factors including the disease or disorder being treated and its severity (e.g., its current state); the age, weight, general health, sex and diet of the subject; the time of administration, route of administration and rate of excretion of the specific compound being used; the duration of treatment; drugs used in combination with or simultaneously with the specific compound being used; and similar factors well known in the medical arts (see, e.g., Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th Edition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001).

[0164] The bifunctional compounds of formula (I) and their pharmaceutically acceptable salts and stereoisomers can be effective over a wide dosage range. In some embodiments, the total daily dosage (e.g., in the case of an adult) can range from about 0.001 to about 1600 mg, 0.01 to about 1600 mg, 0.01 to about 500 mg, about 0.01 to about 100 mg, about 0.5 to about 100 mg, 1 to about 100 - 400 mg / day, about 1 to about 50 mg / day, and about 5 to about 40 mg / day, and in yet other embodiments can range from about 10 to about 30 mg / day. The individual dosages can be formulated to contain the desired dosage, depending on the number of times the compound is administered per day. By way of example, capsules can be formulated to contain from about 1 to about 200 mg of the compound (e.g., 1, 2, 2.5, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150 and 200 mg).

[0165] In some embodiments, the amount of the bifunctional compound of formula (I) administered depends on the patient being treated, the severity of the disorder, the rate of administration, the nature of the compound, and the discretion of the prescribing physician. The dosage is in the range of about 0.001 to about 100 mg / kg body weight / day, preferably about 1 to about 35 mg / kg / day, for single or divided doses. For a 70 kg human, this corresponds to about 0.05 to about 7 g / day, preferably about 0.1 to about 2.5 g / day. In some cases, dosage levels below the lower limit of the foregoing range may be sufficient, and in other cases, higher dosages may be used without causing harmful side effects.

[0166] Method of Use In some aspects, the invention relates to a method of treating a disease or disorder associated with abnormal (e.g., dysfunctional or dysregulated) EP300 activity, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof.

[0167] A disease or disorder can be said to be characterized or mediated by abnormal EP300 activity or MYC activity (e.g., an increase in the level of EP300 or otherwise functionally abnormal EP300 compared to a non-diseased state). A "disease" is generally considered to be the health condition of a subject in which the subject cannot maintain homeostasis and the subject's health continues to deteriorate if the disease is not improved. In contrast, a "disorder" of a subject is a health condition in which the subject can maintain homeostasis, but the subject's health condition is less favorable than it would be in the absence of the disorder. A disorder does not necessarily further degrade the animal's health condition if left untreated.

[0168] In some embodiments, the compounds of the present application may be useful for the treatment of cell proliferative diseases and disorders (e.g., cancer or benign neoplasms). As used herein, the term "cell proliferative disease or disorder" refers to a condition characterized by deregulated or abnormal cell proliferation or both, including non-cancerous conditions such as neoplasms, pre-cancerous states, benign tumors, and cancer.

[0169] As used herein, the term "subject" (or "patient") includes any member of the animal kingdom that is susceptible to, or has, the indicated disease or disorder. In some embodiments, the subject is a mammal, such as a human or non-human mammal. The methods are applicable to companion animals, such as dogs and cats, and to livestock, such as cows, horses, sheep, goats, pigs, and other domesticated and wild animals. A subject "in need of" treatment according to the invention may have, or be suspected of having, a particular disease or disorder that can be definitely diagnosed, or alternatively, may exhibit a sufficient number of risk factors, or a sufficient number or combination of signs or symptoms, such that a medical professional could suspect that the subject has the disease or disorder. Thus, subjects having, and suspected of having, a particular disease or disorder are not necessarily two separate groups.

[0170] Exemplary types of non-cancerous (e.g., cell proliferative) diseases or disorders that may be suitable for treatment with the compounds of the invention include inflammatory diseases and conditions, autoimmune diseases, neurodegenerative diseases, heart diseases, viral diseases, chronic and acute renal diseases or kidney injuries, metabolic diseases, and allergic and genetic diseases.

[0171] Representative examples of certain non-cancerous diseases and disorders include rheumatoid arthritis, alopecia areata, lymphoproliferative conditions, autoimmune blood disorders (e.g., hemolytic anemia, aplastic anemia, anhidrotic ectodermal dysplasia, polycythemia vera, and idiopathic thrombocytopenia), cholecystitis, rheumatoid spondylitis, osteoarthritis, gout, scleroderma, sepsis, septic shock, dacryoadenitis, cryopyrin-associated periodic syndrome (CAPS), endotoxin shock, endometritis, gram-negative sepsis, keratoconjunctivitis sicca, toxic shock syndrome, asthma, adult respiratory distress syndrome, chronic obstructive pulmonary disease, chronic lung inflammation, chronic transplant rejection, hidradenitis suppurativa, inflammatory bowel disease, Crohn's disease, Behçet's syndrome, systemic lupus erythematosus, glomerulonephritis, multiple sclerosis, juvenile diabetes, autoimmune retinitis uveitis, autoimmune vasculitis, thyroiditis, Addison's disease, lichen planus, appendicitis, bullous pemphigus, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, myasthenia gravis, immunoglobulin A nephropathy, Hashimoto's disease, Sjögren's syndrome, vitiligo, Wegener's granulomatosis, testicular sarcoidosis, autoimmune oophoritis, sarcoidosis, rheumatic carditis, ankylosing spondylitis, Graves' disease, autoimmune thrombocytopenic purpura, psoriasis, psoriatic arthritis, eczema, herpesiform dermatitis, ulcerative colitis, pancreatic fibrosis, hepatitis, hepatic fibrosis, CD14-mediated sepsis, non-CD14-mediated sepsis, acute kidney disease and chronic kidney disease, irritable bowel syndrome, pyresis, restenosis, cervicitis, stroke and ischemic injury, nerve trauma, acute pain and chronic pain, allergic rhinitis, allergic conjunctivitis, chronic heart failure, congestive heart failure, acute coronary syndrome, cachexia, malaria, leprosy, leishmaniasis, Lyme disease, Reiter's syndrome, acute synovitis, muscle degeneration, bursitis, tendinitis, tenosynovitis, herniated, ruptured, or dislocated intervertebral disc syndrome (herniated, ruptured,or prolapsed intervertebral disk syndrome), osteoporosis, rhinitis, thrombosis, silicosis, pulmonary sarcosis, bone resorption diseases, for example, osteoporosis, fibromyalgia, AIDS and other viral diseases, for example, herpes zoster, herpes simplex type I or herpes simplex type II, influenza virus and cytomegalovirus, type I diabetes and type II diabetes, obesity, insulin-resistant retinopathy and diabetic retinopathy, 22q11.2 deletion syndrome, Angelman syndrome, Canavan disease, celiac disease, Charcot-Marie-Tooth disease, color blindness, cat cry syndrome, Down syndrome, cystic fibrosis, Duchenne muscular dystrophy, hemophilia, Klinefelter syndrome, neurofibromatosis, phenylketonuria, Prader-Willi syndrome, sickle cell disease, Tay-Sachs disease, Turner syndrome, urea cycle disorders, thalassemia, otitis, pancreatitis, mumps, pericarditis, pleurisy, pharyngitis, pleurisy, phlebitis, pneumonia, uveitis, polymyositis, proctitis, interstitial pulmonary fibrosis, dermatomyositis, atherosclerosis, arteriosclerosis, amyotrophic lateral sclerosis, asociality, aneurysm, vaginitis, depression and sudden infant death syndrome are mentioned.,

[0172] In other embodiments, the method relates to treating a subject having cancer. Generally, the compounds of the present invention can be effective in treating carcinomas (solid tumors including both primary and metastatic tumors), sarcomas, melanomas, and blood cancers (cancers affecting the blood including lymphocytes, bone marrow and / or lymph nodes), for example, leukemia, lymphoma and multiple myeloma. Adult tumors / cancers and pediatric tumors / cancers are included. The cancer can be a tumor that is angiogenic, not yet substantially angiogenic or non-angiogenic.,

[0173] Typical examples of cancer include adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi's sarcoma, and AIDS-related lymphomas), appendiceal cancer, childhood cancers (e.g., pediatric cerebellar astrocytoma, pediatric cerebral astrocytoma), basal cell carcinoma, cutaneous cancer (non-melanoma), biliary tract cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, brain cancer (e.g., glioma and glioblastoma, e.g., brainstem glioma, gestational trophoblastic tumor glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodeimal tumor, visual pathway and hypothalamic glioma), breast cancer, bronchial adenoma / carcinoid, carcinoid tumor, nervous system cancer (e.g., central nervous system cancer, central nervous system lymphoma), cervical cancer, chronic myeloproliferative disorders, colorectal cancer (e.g., colon cancer, rectal cancer), lymphoid neoplasms, mycosis fungoides, Sézary syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastrointestinal cancer (e.g., gastric cancer, small intestine cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST)), cholangiocarcinoma, germ cell tumor, ovarian germ cell tumor, head and neck cancer, neuroendocrine tumor, Hodgkin lymphoma, Ann Arbor stage III and stage IV pediatric non-Hodgkin lymphoma, ROS1-positive refractory non-Hodgkin lymphoma, leukemia, lymphoma, multiple myeloma, hypopharyngeal cancer, intraocular melanoma, ocular cancer, pancreatic islet cell tumor (pancreatic endocrine part), kidney cancer (e.g., Wilms tumor, renal cell carcinoma), liver cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), ALK-positive anaplastic large cell lymphoma, ALK-positive progressive malignant solid neoplasm, Waldenström's macroglobulinemia, melanoma, intraocular (ocular) melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, multiple endocrine neoplasia (MEN), myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer (e.g., oral cancer (mouthcancer), lip cancer, oral cavity cancer, tongue cancer, hypopharyngeal cancer, throat cancer, laryngeal cancer), ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, low malignant potential ovarian tumor), pancreatic cancer, islet cell pancreatic cancer, paranasal sinus cancer and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma, metastatic anaplastic thyroid cancer, anaplastic thyroid cancer, papillary thyroid cancer, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, uterine cancer (e.g., endometrial uterine cancer, uterine sarcoma, corpus cancer), squamous cell carcinoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, juvenile xanthogranuloma, transitional cell carcinoma of the renal pelvis and ureter and other urinary organs, urethral cancer, gestational trophoblastic tumor, vaginal cancer, vulvar cancer, hepatoblastoma, rhabdoid tumor, and Wilms tumor.

[0174] Sarcomas that may be treatable with the bifunctional compounds of the present invention similarly include both soft tissue cancers and bone cancers, representative examples of which include osteosarcoma or osteogenic sarcoma (bone) (e.g., Ewing sarcoma), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelioma or mesothelioma (intracoelomic membrane), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelioma (blood vessels), liposarcoma (adipose tissue), glioma or astrocytoma (neural connective tissue found in the brain), myxosarcoma (primitive embryonic connective tissue), mesenchymal or mixed mesodermal tumor (mixed connective tissue type), and histiocytic sarcoma (immunocarcinoma).

[0175] In some embodiments, the methods of the present invention involve treating a subject having a proliferative disorder or disorder of cells of the hematopoietic system, liver, brain, lung, colon, pancreas, prostate, ovary, breast, skin, and endometrium.

[0176] As used herein, "hematopoietic cell proliferative diseases or disorders" include lymphoma, leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplastic syndromes, benign monoclonal gammopathy, lymphomatoid papulosis, polycythemia vera, chronic myelogenous leukemia, essential thrombocythemia, and primary myelofibrosis. Thus, representative examples of blood cancers include multiple myeloma, lymphoma (T cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma (diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and ALK+ anaplastic large cell lymphoma (e.g., B cell non-Hodgkin lymphoma selected from diffuse large B cell lymphoma (e.g., germinal center B cell-like diffuse large B cell lymphoma or activated B cell-like diffuse large B cell lymphoma), Burkitt lymphoma / leukemia, mantle cell lymphoma, mediastinal (thymic) large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma / Waldenström macroglobulinemia, metastatic pancreatic adenocarcinoma, refractory B cell non-Hodgkin lymphoma, and relapsed B cell non-Hodgkin lymphoma, pediatric lymphoma, and lymphoma of lymphocytic and cutaneous origin, such as small lymphocytic lymphoma, leukemia including pediatric leukemia, hairy cell leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, acute myeloid leukemia (e.g., acute monocytic leukemia), chronic lymphocytic leukemia, small lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, as well as mast cell leukemia, myeloid neoplasms, and mast cell neoplasms.

[0177] As used herein, "proliferative disease or disorder of the liver" includes any form of proliferative disorder that affects the liver. Proliferative disorders of the liver can include liver cancer (e.g., hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and hepatoblastoma), pre-cancer or pre-cancerous conditions of the liver, benign growths or lesions of the liver, and malignant growths or lesions of the liver, as well as metastatic foci in tissues and organs within the body outside of the liver. Proliferative disorders of the liver can include hyperplasia, dysplasia, and abnormal formation of the liver.

[0178] As used herein, "proliferative disease or disorder of the brain" includes any form of proliferative disorder that affects the brain. Proliferative disorders of the brain can include brain tumors (e.g., glioma, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, and primitive neuroectodermal tumors (medulloblastoma)), pre-cancer or pre-cancerous conditions of the brain, benign growths or lesions of the brain, and malignant growths or lesions of the brain, as well as metastatic foci in tissues and organs within the body outside of the brain. Proliferative disorders of the brain can include hyperplasia, dysplasia, and abnormal formation of the brain.

[0179] As used herein, "proliferative disease or disorder of the lung" includes any form of proliferative disorder that affects lung cells. Proliferative disorders of the lung can include lung cancer, pre-cancer and pre-cancerous conditions of the lung, benign growths or lesions of the lung, hyperplasia, dysplasia, and abnormal formation of the lung, as well as metastatic foci in tissues and organs within the body outside of the lung. Lung cancer includes any form of cancer of the lung, e.g., malignant lung neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors. Lung cancer includes small cell lung cancer ("SLCL"), non-small cell lung cancer ("NSCLC"), adenocarcinoma, small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and mesothelioma. Lung cancer can include "scar carcinoma", bronchioloalveolar carcinoma, giant cell carcinoma, spindle cell carcinoma, and large cell neuroendocrine carcinoma. Lung cancer also includes lung neoplasms having histological and ultrastructural heterogeneity (e.g., mixed cell type). In some embodiments, the compounds of the invention can be used to treat non-metastatic or metastatic lung cancer (e.g., NSCLC, ALK-positive NSCLC, NSCLC having ROS1 rearrangement, lung adenocarcinoma, and lung squamous cell carcinoma).

[0180] As used herein, "colonic cell proliferative disease or disorder" includes any form of cell proliferative disorder affecting colonic cells, including colon cancer, pre-cancer or pre-cancerous conditions of the colon, adenomatous polyps of the colon, and metachronous lesions of the colon. Colon cancer includes sporadic and hereditary colon cancers, malignant colonic neoplasms, carcinoma in situ, typical and atypical carcinoid tumors, adenocarcinoma, squamous cell carcinoma, and squamous cell carcinoma. Colon cancer may be associated with hereditary syndromes, such as hereditary non-polyposis colorectal cancer, familial adenomatous polyposis, MYH-related polyposis, Gardner syndrome, Peutz-Jeghers syndrome, Turcot syndrome, and juvenile polyposis. Colonic cell proliferative disorders may also be characterized by hyperplasia, dysplasia, or abnormal formation of the colon.

[0181] As used herein, "pancreatic cell proliferative disease or disorder" includes any form of cell proliferative disorder affecting pancreatic cells. Pancreatic cell proliferative disorders may include pancreatic cancer, pre-cancer or pre-cancerous conditions of the pancreas, hyperplasia of the pancreas, abnormal formation of the pancreas, benign proliferation or lesions of the pancreas, and malignant proliferation or lesions of the pancreas, as well as metastatic foci in tissues and organs within the body outside the pancreas. Pancreatic cancer includes all forms of cancer of the pancreas, including ductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cell carcinoma, mucinous adenocarcinoma, osteoclast-like giant cell carcinoma, mucinous cystadenocarcinoma, acinar cell carcinoma, unclassified large cell carcinoma, small cell carcinoma, pancreatoblastoma, papillary neoplasms, mucinous cystadenomas, papillary cystic neoplasms, and serous cystadenomas, as well as pancreatic neoplasms having histological and ultrastructural heterogeneity (e.g., mixed cells).

[0182] As used herein, "prostatic cell proliferative disease or disorder" includes any form of cell proliferative disorder affecting the prostate. Prostatic cell proliferative disorders may include prostate cancer, pre-cancer or pre-cancerous conditions of the prostate, benign proliferation or lesions of the prostate, and malignant proliferation or lesions of the prostate, as well as metastatic foci in tissues and organs within the body outside the prostate. Prostatic cell proliferative disorders may include hyperplasia, dysplasia, and abnormal formation of the prostate.

[0183] As used herein, "ovarian cell proliferative disease or disorder" includes any form of cell proliferative disorder that affects ovarian cells. Ovarian cell proliferative disorders can include pre-cancer or pre-cancerous conditions of the ovary, benign proliferations or lesions of the ovary, ovarian cancer, as well as metastatic foci in tissues and organs within the body outside the ovary. Ovarian cell proliferative disorders can include hyperplasia, dysplasia, and abnormal formation of the ovary.

[0184] As used herein, "breast cell proliferative disease or disorder" includes any form of cell proliferative disorder that affects breast cells. Breast cell proliferative disorders can include breast cancer, pre-cancer or pre-cancerous conditions of the breast, benign proliferations or lesions of the breast, as well as metastatic foci in tissues and organs within the body outside the breast. Breast cell proliferative disorders can include hyperplasia, dysplasia, and abnormal formation of the breast.

[0185] As used herein, "skin cell proliferative disease or disorder" includes any form of cell proliferative disorder that affects skin cells. Skin cell proliferative disorders can include pre-cancer or pre-cancerous conditions of the skin, benign proliferations or lesions of the skin, melanoma, malignant melanoma, or other malignant proliferations or lesions of the skin, as well as metastatic foci in tissues and organs within the body outside the skin. Skin cell proliferative disorders can include hyperplasia, dysplasia, and abnormal formation of the skin.

[0186] As used herein, "endometrial cell proliferative disease or disorder" includes any form of cell proliferative disorder that affects endometrial cells. Endometrial cell proliferative disorders can include pre-cancer or pre-cancerous conditions of the endometrium, benign proliferations or lesions of the endometrium, endometrial cancer, as well as metastatic foci in tissues and organs within the body outside the endometrium. Endometrial cell proliferative disorders can include hyperplasia, dysplasia, and abnormal formation of the endometrium.

[0187] In some embodiments, the compound or pharmaceutically acceptable salt or stereoisomer of the invention is a disease or the disorder is high-risk neuroblastoma (NB).

[0188] In some embodiments, the disease or disorder is acute myeloid leukemia (AML), multiple myeloma (MM), melanoma, rhabdomyosarcoma or diffuse large B-cell lymphoma. In other embodiments, the disease or disorder is a small solid tumor. In other embodiments, the disease or disorder is colon cancer, rectal cancer, gastric cancer, breast cancer or pancreatic cancer.

[0189] The bifunctional compound of formula (I) can be administered to a patient, e.g., a cancer patient, as a monotherapy or in combination therapy. The treatment can be, i.e., alone or in combination with other treatments, "frontline / first choice" as initial treatment for patients who have not previously received an anticancer treatment regimen, alone or in combination with other treatments, "second choice" as treatment for patients who have previously received an anticancer treatment regimen, alone or in combination with other treatments, "third choice", "fourth choice", etc. as treatment. Treatment can also be given to patients who have received a partially successful previous treatment but have become intolerant to a particular treatment. The treatment can also be given as adjuvant treatment, i.e., to patients who currently have no detectable disease or after surgical resection of a tumor to prevent cancer recurrence. Thus, in some embodiments, the bifunctional compound can be administered to patients who have received another therapy, e.g., chemotherapy, radioimmunotherapy, surgery, immunotherapy, radiotherapy, targeted therapy or any combination thereof.

[0190] The method of the present invention may involve administering to a patient a bifunctional compound of formula (I) or a pharmaceutical composition thereof in a single dose or multiple doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 or more doses). For example, the frequency of administration can range from once a day to about once every 8 weeks. In some embodiments, the frequency of administration ranges from about once a day over 1, 2, 3, 4, 5 or 6 weeks, and in other embodiments, it involves a 28-day cycle including daily administration for 3 weeks (21 days) and a 7-day "off" period. In other embodiments, the bifunctional compound can be administered twice a day (BID) for two and a half days (total of 5 doses), or once a day (QD) for 2 days (total of 2 doses). In other embodiments, the bifunctional compound can be administered once a day (QD) for 5 days.

[0191] In some aspects, the present invention relates to a method of using a bifunctional compound of formula (II) as a probe for EP300 or CPB. For example, the bifunctional compound of formula II can be used with streptavidin for the identification, isolation, handling and pull-down of target proteins, as well as with interacting proteins (IP) for target identification, or with Chem-seq (Anders et al., Nat. Biotechnol. 32(1):92-6 (2014)). For example, Dynabeads® M-270 streptavidin (Thermo Fisher Scientific; catalog number 65305) may be used to utilize the avidin-biotin interaction for directly isolating any biotinylated molecule. This probe molecule can be used for assay development (see the AlphaScreen® assay described in Example 26).

[0192] Combination therapy The difunctional compound of formula (I) can be used in combination with or simultaneously with at least one other active agent, such as an anti-cancer agent or anti-cancer regimen, when treating diseases and disorders. In this context, the terms "in combination with" and "simultaneously" mean that the agents are co-administered, which includes substantially simultaneous administration by the same or separate dosage forms and by the same or different modes of administration, or sequentially, for example, as part of the same treatment regimen or as sequential treatment regimens. Thus, when administered sequentially, at the start of administration of the second compound, the first of the two compounds may still be detectably present at an effective concentration at the treatment site, in some cases. The order and time intervals can be determined such that they can act together (e.g., synergistically to provide an increased benefit compared to when they are administered in other ways). For example, the therapeutic agents can be administered simultaneously at different times or sequentially in any order. However, if not administered simultaneously, they can be administered close enough in time to provide the desired therapeutic effect, which can be in a synergistic manner. Thus, these terms are not limited to exactly simultaneous administration of the active agents.

[0193] In some embodiments, the treatment regimen may include administering a compound of formula (I) of the invention in combination with one or more additional therapeutic agents known to be used in the treatment of a disease or condition (e.g., cancer). The dosage of the additional anti-cancer therapeutic agent may be the same as or lower than the known or recommended dosage. See Hardman et al., eds., Goodman & Gilman’s The Pharmacological Basis Of Basis Of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; Physician’s Desk Reference, 60th ed., 2006. For example, anti-cancer agents that may be used in combination with the compounds of the invention are known in the art. See, e.g., U.S. Patent No. 9,101,622 (section 5.2) and U.S. Patent No. 9,345,705 B2 (paragraphs 12-18). Representative examples of additional active agents and treatment regimens include radiation therapy, chemotherapeutic agents (e.g., mitotic inhibitors, angiogenesis inhibitors, antihormonal agents, autophagy inhibitors, alkylating agents, intercalating antibiotics, growth factor inhibitors, antiandrogens, signal transduction pathway inhibitors, microtubule inhibitors, platinum coordination complexes, HDAC inhibitors, proteasome inhibitors and topoisomerase inhibitors), immunomodulators, therapeutic antibodies (e.g., monospecific and bispecific antibodies) and CAR-T therapy.

[0194] In some embodiments, the compound of formula (I) of the present invention, and an additional anti-cancer therapeutic agent, may be administered at intervals of less than 5 minutes, less than 30 minutes, less than 1 hour, about 1 hour intervals, about 1 to about 2 hour intervals, about 2 hours to about 3 hour intervals, about 3 hours to about 4 hour intervals, about 4 hours to about 5 hour intervals, about 5 hours to about 6 hour intervals, about 6 hours to about 7 hour intervals, about 7 hours to about 8 hour intervals, about 8 hours to about 9 hour intervals, about 9 hours to about 10 hour intervals, about 10 hours to about 11 hour intervals, about 11 hours to about 12 hour intervals, about 12 hours to 18 hour intervals, 18 hours to 24 hour intervals, 24 hours to 36 hour intervals, 36 hours to 48 hour intervals, 48 hours to 52 hour intervals, 52 hours to 60 hour intervals, 60 hours to 72 hour intervals, 72 hours to 84 hour intervals, 84 hours to 96 hour intervals or 96 hours to 120 hour intervals. Two or more anti-cancer therapeutic agents may be administered during the same patient visit.

[0195] In some embodiments involving cancer treatment, the bifunctional compound of formula (I), and an additional anti-cancer agent or therapeutic agent are administered cyclically. Cycling therapy involves administering one anti-cancer therapeutic agent over a period of time, followed by administering a second anti-cancer therapeutic agent over a period of time, and repeating this sequential administration, i.e., the cycle, to reduce the development of resistance to one or both of the anti-cancer therapeutic agents, avoid or reduce side effects of one or both of the anti-cancer therapeutic agents, and / or improve the effectiveness of the therapy. In one example, cycling therapy involves administering a first anti-cancer therapeutic agent over a period of time, followed by administering a second anti-cancer therapeutic agent over a period of time, and optionally, followed by administering a third anti-cancer therapeutic agent over a period of time, etc., and repeating this sequential administration, i.e., the cycle, to reduce the development of resistance to one or both of the anti-cancer therapeutic agents, avoid or reduce side effects of one of the anti-cancer therapeutic agents, and / or improve the effectiveness of the anti-cancer therapeutic agent.

[0196] In some embodiments, the bifunctional compound of formula (I) can be used in combination with other anti-NB agents or anti-cancer agents, examples of which include dinutuximab (e.g., in the case of NB), cyclophosphamide (e.g., in the case of neuroblastoma), busulfan + melphalan hydrochloride, carboplatin + etoposide phosphate and melphalan hydrochloride (doxorubicin hydrochloride, unituximab (dinutuximab), vincristine sulfate, (entrectinib (e.g., in the case of brain cancer, central nervous system (CNS) cancer), Hu3F8 + provided natural killer cells (e.g., in the case of persistent or recurrent neuroblastoma), Hu3F8 + granulocyte macrophage colony-stimulating factor (GM-CSF) (e.g., in the case of relapsed / refractory neuroblastoma), Hu3F8 / GM-CSF immunotherapy + isotretinoin (e.g., in the case of strengthening the first remission of NB patients), venetoclax (e.g., in the case of persistent or recurrent cancers including leukemia and non-Hodgkin lymphoma), a bivalent vaccine with the immunological adjuvant OPT-821 combined with oral β-glucan (e.g., in the case of NB), trametinib (e.g., in the case of germ cell tumor, liver cancer, kidney cancer, neuroblastoma, pediatric brain tumor, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, soft tissue sarcoma, Wilms tumor), cobimetinib (e.g., in the case of melanoma, pediatric brain tumor and soft tissue sarcoma), and intrathecal radioimmunotherapy using 131I-8H9 (e.g., in the case of brain tumor, primary, brain cancer, and CNS cancer).

[0197] Pharmaceutical kit The composition can be assembled into a kit or a pharmaceutical system. A kit or a pharmaceutical system according to this aspect of the invention includes a carrier or package such as a box, carton, tube, etc. that tightly encloses one or more containers, such as vials, tubes, ampoules or bottles, containing the bifunctional compound or pharmaceutical composition of formula (I) of the invention. The kit or pharmaceutical system of the invention may also include printed instructions for using the compound and composition.

[0198] In some embodiments, a method of using a bifunctional compound of formula (II) as a probe for EP300 involves contacting a lysed cell suspected of containing EP300 with a compound of formula (II) and streptavidin immobilized on a carrier (e.g., beads such as magnetic beads), isolating the complex formed by molecular and protein binding via biotin-streptavidin binding, and confirming the presence of EP300 in the complex via techniques standard in the art (e.g., immunoblotting).

[0199] These and other aspects of the invention, while intended to illustrate particular embodiments of the invention, are not intended to limit its scope as defined by the claims and will be further understood in view of the following examples.

Examples

[0200] Example 1: Synthesis of 12 - ((2 - (2,6 - dioxopiperidin - 3 - yl) - 1,3 - dioxoisoindolin - 5 - yl)amino) - N - ((R) - 3’ - (2 - ((4 - fluorobenzyl)((S) - 1,1,1 - trifluoropropan - 2 - yl)amino) - 2 - oxoethyl) - 2’,4’ - dioxo - 2,3 - dihydrospiro[indene - 1,5’ - oxazolidine] - 5 - yl)dodecanamide (Compound (CPD) 1).

Chemical formula

[0201] A solution of compound A (18.5 g, 163.5 mmol) and 1-(bromomethyl)-4-fluorobenzene (37.2 g, 196.5 mmol) in DMF (200 mL) was added with K2CO3 (67.5 g, 490 mmol). The reaction mixture was stirred overnight at room temperature (rt), and then quenched with water. The mixture was extracted with EtOAc and washed with water and brine. The organic extract was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (methanol:CH2Cl2, 1:20) to obtain compound A-1 (15 g, 41.5%).

Chemical formula

[0202] To a stirred solution of compound A-1 (15 g, 67.8 mmol) in dry CH2Cl2 (300 mL) was added 2-bromoacetyl bromide (27.4 g, 135.6 mmol). The resulting mixture was stirred at room temperature for 2 hours and then quenched with NaHCO3. The reaction was extracted with CH2Cl2. The organic extract was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether:ethyl acetate, 1:1) to obtain intermediate SM-2 (15.3 g, 66%) as an oil.

Chemical formula

[0203] To a stirred solution of SM-1 (31 g, 164 mmol) in a 1:1 mixture of toluene and MeCN (800 mL) were added ZnI2 (5.2 g, 16.4 mmol) and trimethylsilyl cyanide (TMSCN) (33 g, 328 mmol). The mixture was heated under reflux for 1 hour. The reaction was cooled and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate, gradient) to obtain intermediate int-1 (28 g, 60%).

[0204] Int-1 (28 g, 97.2 mmol) was taken in ethanol (400 ml) and cooled to 4 °C. Acetyl chloride (280 ml, 3.9 mol) was added dropwise via an addition funnel at a rate such that the temperature was maintained below 20 °C. The reaction mixture was stirred for 48 h, at which point the reaction mixture was concentrated under reduced pressure to give a pale yellow solid. The pale yellow solid was partitioned between ethyl acetate and aqueous NaHCO3 solution. The organic extract was dried, concentrated, and triturated with a 1:1 mixture of petroleum ether and ethyl acetate. The crude product int-2 (15 g) was used directly in the subsequent step.

[0205] A solution of the crude product int-2 (15 g) in THF (300 mL) and triethylamine (12 g, 116 mmol) was cooled to 5 °C and carefully treated with a 40 ml THF solution of triphosgene (8.6 g, 29 mmol) such that the temperature was maintained below 10 °C. The mixture was stirred for 1 h, treated with 6N HCl until pH 2 was reached, and the resulting mixture was stirred at 5 °C for an additional 30 min. Approximately half of the volume of the solvent from the reaction mixture was removed under reduced pressure and the remaining mixture was partitioned between water and ethyl acetate. The organic extract was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate, gradient) to give the intermediate int-4 (8 g) as a yellow solid.

Chemical formula

[0206] To a stirred suspension of int-4 (8 g, 30.6 mmol) in methanol (80 mL) was added aqueous concentrated HCl (20 mL, 12 M). The mixture was heated to reflux for 3 h. The resulting precipitate was filtered and dried to give the intermediate int-5 (4 g, 60%) as a yellow solid.

[0207] To a solution of int-5 (4g, 18 mmol) and SM-2 (6.2g, 18 mmol) in DMF (40 mL) was added K2CO3 (7.4g, 54 mmol). The reaction mixture was then stirred at room temperature for 2 hours, quenched with water, extracted with EtOAc, washed with water and brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (methanol:CH2Cl2, 1:20) to obtain the intermediate int-6 (6g, 69.5%) as a white solid.

Chemical formula

[0208] Int-7 was synthesized according to Michaelides et al., ACS Med. Chem. Lett. 2018, 9:28-33 (2018).

Chemical formula

[0209] Int-8 was synthesized according to Michaelides et al., ACS Med. Chem. Lett. 2018, 9:28-33 (2018).

Chemical formula

[0210] To a stirred solution of int-6 (6 mg, 0.01 mmol) and compound SM-3 (6 mg, 0.01 mmol) in DMF (1 mL), DIPEA (35 μL, 0.1 mmol) was added followed by 1-[(bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (12 mg, 0.02 mmol) at room temperature all at once. The reaction mixture was stirred at room temperature for 30 minutes. After consumption of the starting material monitored by thin layer chromatography (TLC), the reaction mixture was diluted with water and EtOAc. The combined organic extracts were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography using 1:1 EtOAc / petroleum ether to afford CPD1 (6.2 mg, 45%) as a yellow solid.

[0211] Example 2: Synthesis of 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-N-((R)-3'-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidine]-5-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide (CPD2).

[0212] To a stirred solution of int-6 (6 mg, 0.01 mmol) and compound SM-4 (6 mg, 0.01 mmol) in DMF (1 mL), DIPEA (17 μL, 0.05 mmol) was added followed by HATU (11.4 mg, 0.025) at room temperature all at once (exothermic reaction). The reaction mixture was stirred at room temperature for 30 minutes. After consumption of the starting material (by TLC), the reaction mixture was diluted with water and EtOAc. The combined organic extracts were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography using 1:1 EtOAc / petroleum ether to afford CPD2 (6 mg, 40%) as a yellow solid. [Chemical formula]

[0213] Example 3: Synthesis of 8 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1,3 - dioxoisoindolin - 4 - yl)amino)-N - ((R)-3’-(2 - ((4 - fluorobenzyl)((S)-1,1,1 - trifluoropropan - 2 - yl)amino)-2 - oxoethyl)-2’,4’ - dioxo - 2,3 - dihydrospiro[indene - 1,5’ - oxazolidine]-5 - yl)octanamide (CPD3).

Chemical Structure

[0214] CPD3 was prepared from int - 6 and the appropriate IMiD intermediate in the same manner as CPD2 in Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to obtain CPD3 as a yellow powder (53% yield).

[0215] Example 4: Synthesis of 10 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1,3 - dioxoisoindolin - 5 - yl)amino)-N - ((R)-3’-(2 - ((4 - fluorobenzyl)((S)-1,1,1 - trifluoropropan - 2 - yl)amino)-2 - oxoethyl)-2’,4’ - dioxo - 2,3 - dihydrospiro[indene - 1,5’ - oxazolidine]-5 - yl)decanamide (CPD4).

Chemical Structure

[0216] CPD4 was prepared from int - 6 and the appropriate IMiD intermediate in the same manner as CPD2 in Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to obtain CPD4 as a yellow powder (15 mg, 40% yield).

[0217] C 46 H 48Calculated MS (ESI) value of F4N6O9: 904.34; measured values: [M+1] 905.54, 906.53.

[0218] Example 5: Synthesis of 2-((1R)-5-(3-(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)octyl)ureido)-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidine]-3’-yl)-N-(4-fluorobenzyl)-N-((S)-1,1,1-trifluoropropan-2-yl)acetamide (CPD5).

Chem.

[0219] CPD5 was prepared from int-6 and the appropriate IMiD intermediate in the same manner as CPD2 in Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to obtain CPD5 as a yellow powder (2 mg, 12% yield).

[0220] C 45 H 47 Calculated MS (ESI) value of F4N7O9: 905.34; measured values: [M+1] 906.60, 907.29.

[0221] Example 6: Synthesis of 12-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-N-((R)-3’-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidine]-5- yl)dodecanamide (CPD6).

Chem.

[0222] CPD6 was prepared from int-6 and the appropriate IMiD intermediate in the same manner as CPD2 of Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to afford CPD6 as a yellow powder (7 mg, 72% yield).

[0223] 1 H NMR (500 MHz, acetone-d6) δ 9.90 (d, J = 4.0 Hz, 1H), 9.26 (d, J = 5.1 Hz, 1H), 7.90 (d, J = 7.7 Hz, 1H), 7.59 (td, J = 7.8, 2.7 Hz, 1H), 7.49 (d, J = 9.7 Hz, 3H), 7.36 - 7.31 (m, 1H), 7.22 (t, J = 8.6 Hz, 2H), 7.13 - 7.00 (m, 3H), 6.42 (d, J = 5.9 Hz, 1H), 5.51 (p, J = 7.8 Hz, 1H), 5.07 (ddd, J = 12.1, 7.6, 4.2 Hz, 2H), 4.97 - 4.81 (m, 1H), 4.67 (dd, J = 71.1, 16.7 Hz, 1H), 4.43 (dd, J = 90.6, 16.6 Hz, 1H), 3.38 (q, J = 6.3 Hz, 2H), 3.28 - 3.04 (m, 2H), 3.03 - 2.85 (m, 3H), 2.85 - 2.70 (m, 4H), 2.56 (dddd, J = 14.5, 12.1, 8.6, 4.2 Hz, 1H), 2.39 (t, J = 7.2 Hz, 2H), 2.27 - 2.17 (m, 1H), 2.07 (p, J = 2.2 Hz, 2H), 1.73 - 1.67 (m, 4H), 1.39 (dd, J = 38.4, 5.3 Hz, 12H).

[0224] C 48 H 52 MS (ESI) calcd for C46H61F4N6O9: 932.37; found: [M + 1] 933.36, 934.38.

[0225] Example 7: Synthesis of 12 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1,3 - dioxoisoindolin - 4 - yl)amino)-N - ((R)-3’-(2 - ((4 - fluorobenzyl)((S)-1,1,1 - trifluoropropan - 2 - yl)amino)-2 - oxoethyl)-2’,4’ - dioxo - 2,3 - dihydrospiro[indene - 1,5’ - oxazolidine]-5 - yl)dodecanamide (CPD7).

Chemical Structure

[0226] CPD7 was prepared from int - 6 and the appropriate IMiD intermediate in a similar manner to CPD2 of Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to obtain CPD7 as a yellow powder (14 mg, 72% yield).

[0227] 11H NMR (500 MHz, acetone-d6) δ 9.91 (s, 1H), 9.31 (s, 1H), 7.85 (s, 1H), 7.60 - 7.56 (m, 1H), 7.52 - 7.47 (m, 3H), 7.34 (dd, J = 8.3, 5.3 Hz, 1H), 7.22 (t, J = 8.8 Hz, 2H), 7.12 - 7.00 (m, 3H), 6.60 (t, J = 5.9 Hz, 1H), 5.51 (p, J = 7.7 Hz, 1H), 5.12 - 5.04 (m, 2H), 4.96 - 4.84 (m, 1H), 4.67 (dd, J = 69.9, 16.7 Hz, 1H), 4.43 (dd, J = 91.2, 16.6 Hz, 1H), 3.80 (t, J = 5.9 Hz, 2H), 3.71 (t, J = 5.1 Hz, 2H), 3.51 - 3.47 (m, 2H), 3.22 (dt, J = 15.3, 7.3 Hz, 1H), 3.09 (ddd, J = 15.2, 8.8, 4.0 Hz, 1H), 3.00 - 2.95 (m, 2H), 2.84 (d, J = 16.7 Hz, 4H), 2.78 - 2.75 (m, 2H), 2.63 - 2.50 (m, 4H), 2.22 (ddt, J = 12.8, 5.4, 2.5 Hz, 1H), 2.07 (t, J = 2.2 Hz, 2H), 1.50 (d, J = 6.8 Hz, 1H), 1.43 (d, J = 7.2 Hz, 2H).

[0228] C 45 H 46 F4N6O 12 The calculated value of MS (ESI) for [Compound Name]: 938.31; Measured value: [M + 1] 939.40, 940.50.

[0229] Example 8: Synthesis of 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)-N-((R)-3’-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidine]-5-yl)propanamide (CPD8).

Chemical Structure

[0230] CPD8 was prepared from int-6 and the appropriate IMiD intermediate in the same manner as CPD2 of Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to give CPD8 as a yellow powder (10.8 mg, 66% yield).

[0231] 1 H NMR (500 MHz, acetone-d6) δ 9.88 (s, 1H), 9.65 (d, J = 7.3 Hz, 1H), 7.87 (s, 1H), 7.59 - 7.49 (m, 4H), 7.32 (dt, J = 22.6, 5.7 Hz, 2H), 7.21 (d, J = 8.8 Hz, 1H), 7.01 (d, J = 2.1 Hz, 1H), 6.93 (dd, J = 8.3, 2.2 Hz, 1H), 6.32 (t, J = 5.5 Hz, 1H), 5.51 (p, J = 7.7 Hz, 1H), 5.06 (dd, J = 12.6, 5.4 Hz, 2H), 4.96 - 4.84 (m, 1H), 4.67 (dd, J = 70.8, 16.7 Hz, 1H), 4.43 (dd, J = 88.2, 16.6 Hz, 1H), 3.99 (h, J = 6.6 Hz, 2H), 3.48 (q, J = 7.4 Hz, 2H), 3.25 (dq, J = 36.5, 7.5, 6.9 Hz, 6H), 3.09 (ddd, J = 16.3, 8.8, 3.9 Hz, 2H), 3.01 - 2.91 (m, 2H), 2.82 - 2.73 (m, 4H), 2.55 (ddd, J = 14.4, 8.2, 3.9 Hz, 1H), 2.40 (t, J = 7.0 Hz, 2H), 2.22 - 2.14 (m, 4H), 2.07 (p, J = 2.2 Hz, 2H), 1.89 - 1.83 (m, 2H), 1.68 (p, J = 7.2 Hz, 2H), 1.62 - 1.57 (m, 2H), 1.48 - 1.41 (m, 7H).

[0232] C 52 H 59 F4N7O 10 MS (ESI) calcd for: 1017.43; found: [M+1] 1018.67, 1019.62.

[0233] Example 9: Synthesis of 12-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)-N-((R)-3’-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidine]-5-yl)dodecanamide (CPD9).

Chemical Structure

[0234] CPD9 was prepared from int-6 and the appropriate IMiD intermediate in a similar manner to CPD2 in Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to obtain CPD9 as a white powder (2 mg, 20% yield).

[0235] 1 H NMR (500 MHz, acetone-d6) δ 9.96 (s, 1H), 9.28 (s, 1H), 7.98 (s, 1H), 7.90 - 7.85 (m, 3H), 7.53 (d, J = 2.0 Hz, 2H), 7.49 (d, J = 3.0 Hz, 2H), 7.22 (t, J = 8.7 Hz, 2H), 5.51 (p, J = 7.8 Hz, 1H), 5.20 - 5.13 (m, 2H), 5.10 - 5.00 (m, 2H), 4.96 - 4.83 (m, 2H), 4.56 (dd, J = 42.2, 16.6 Hz, 2H), 4.33 (d, J = 16.8 Hz, 1H), 3.37 - 3.19 (m, 6H), 3.14 - 2.97 (m, 4H), 2.78 - 2.71 (m, 2H), 2.54 (ddd, J = 14.5, 8.3, 3.8 Hz, 2H), 2.38 (t, J = 7.4 Hz, 2H), 2.29 - 2.24 (m, 2H), 1.69 (t, J = 7.3 Hz, 3H), 1.59 - 1.47 (m, 7H), 1.43 (d, J = 7.3 Hz, 4H).

[0236] C 50 H 54 F4N6O 11MS(ESI) calculated value: 990.38; measured values: [M+1] 991.57, 992.37.

[0237] Example 10: Synthesis of 6 - ((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)-N-(4-(((R)-3’-(2-((4-fluorobenzyl)((R)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidine]-5-yl)amino)-4-oxobutyl)hexanamide (CPD10).

Chemical Structure

[0238] CPD10 was prepared from int-6 and the appropriate IMiD intermediate in the same manner as CPD2 in Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to obtain CPD10 as a yellow powder (12 mg, 71% yield).

[0239] 11H NMR (500 MHz, acetone-d6) δ 9.87 (s, 1H), 9.62 (d, J = 5.5 Hz, 1H), 7.87 (d, J = 5.6 Hz, 1H), 7.58 - 7.43 (m, 6H), 7.22 (t, J = 8.7 Hz, 2H), 7.01 (d, J = 2.2 Hz, 1H), 6.92 (dd, J = 8.4, 2.2 Hz, 1H), 6.35 (t, J = 5.5 Hz, 1H), 5.51 (p, J = 7.8 Hz, 1H), 5.06 (dt, J = 10.7, 3.3 Hz, 2H), 4.96 - 4.84 (m, 1H), 4.67 (dd, J = 70.1, 16.7 Hz, 1H), 4.43 (dd, J = 90.3, 16.6 Hz, 1H), 3.25 (dq, J = 39.8, 7.6, 7.0 Hz, 6H), 3.10 (ddt, J = 16.1, 8.7, 3.9 Hz, 2H), 3.01 - 2.92 (m, 2H), 2.80 - 2.71 (m, 4H), 2.54 (ddd, J = 14.4, 8.3, 3.9 Hz, 1H), 2.40 (t, J = 7.0 Hz, 2H), 2.25 - 2.13 (m, 4H), 1.84 (q, J = 6.9 Hz, 2H), 1.70 - 1.64 (m, 4H).

[0240] Example 11: Synthesis of 4-(3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanamide)-N-((R)-3’-(2-((4-fluorobenzyl)((R)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidine]-5-yl)butanamide (CPD11).

Chemical Structure

[0241] CPD11 was prepared from int-6 and the appropriate IMiD intermediate in the same manner as CPD2 in Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to obtain CPD11 as a yellow powder (10 mg, 56% yield).

[0242] 1 1H NMR (500 MHz, acetone-d6) δ 9.98 (s, 1H), 9.57 (s, 1H), 7.87 (s, 1H), 7.62 - 7.57 (m, 1H), 7.51 - 7.45 (m, 3H), 7.25 (dt, J = 31.7, 7.3 Hz, 3H), 7.13 - 7.01 (m, 3H), 6.62 (q, J = 5.9 Hz, 1H), 5.51 (p, J = 7.8 Hz, 1H), 5.13 - 4.86 (m, 4H), 4.67 (dd, J = 69.4, 16.7 Hz, 1H), 4.43 (dd, J = 92.2, 16.6 Hz, 1H), 3.73 (tt, J = 13.1, 6.0 Hz, 6H), 3.60 - 3.48 (m, 6H), 3.33 - 3.18 (m, 4H), 3.10 (ddd, J = 20.1, 9.9, 5.5 Hz, 2H), 3.05 - 2.92 (m, 2H), 2.75 (ddd, J = 16.4, 7.2, 4.7 Hz, 4H), 2.59 - 2.51 (m, 1H), 2.40 (q, J = 7.4, 6.8 Hz, 4H), 2.23 (dtd, J = 10.4, 5.5, 2.8 Hz, 1H), 1.84 (t, J = 6.8 Hz, 2H).

[0243] C 49 H 53 F4N7O 13 Calculated value of MS (ESI) for it: 1023.36; Measured value: [M + 1] 1024.67, 1025.62.

[0244] Example 12: Synthesis of 6 - ((2 - (2,6 - dioxopiperidin - 3 - yl) - 1,3 - dioxoisoindolin - 5 - yl)amino) - N - (4 - (3 - (((R) - 3’ - (2 - ((4 - fluorobenzyl)((S) - 1,1,1 - trifluoropropan - 2 - yl)amino) - 2 - oxoethyl) - 2’,4’ - dioxo - 2,3 - dihydrospiro[indene - 1,5’ - oxazolidine] - 5 - yl)amino) - 3 - oxopropyl)phenyl)hexanamide (CPD12).

Chemical Structure

[0245] CPD12 was prepared from int-6 and the appropriate IMiD intermediate in the same manner as CPD2 of Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to give CPD12 as a yellow powder (12.5 mg, 79% yield).

[0246] 1 H NMR (500 MHz, acetone-d6) δ 9.86 (s, 1H), 9.30 (d, J = 4.8 Hz, 1H), 9.03 (s, 1H), 7.98 (s, 1H), 7.86 (s, 1H), 7.56 (d, J = 8.1 Hz, 4H), 7.47 (s, 2H), 7.21 (d, J = 10.9 Hz, 3H), 7.06 - 6.91 (m, 3H), 6.35 (t, J = 5.6 Hz, 1H), 5.51 (p, J = 7.9 Hz, 1H), 5.11 - 5.01 (m, 3H), 4.88 (t, J = 13.7 Hz, 1H), 4.67 (dd, J = 70.4, 16.8 Hz, 1H), 4.53 - 4.31 (m, 1H), 3.32 - 3.18 (m, 4H), 3.14 - 3.04 (m, 2H), 2.54 (ddd, J = 13.3, 8.1, 3.7 Hz, 2H), 2.38 (t, J = 7.4 Hz, 2H), 2.18 (dt, J = 11.2, 5.4 Hz, 2H), 1.73 (q, J = 8.0 Hz, 6H), 1.51 (d, J = 6.8 Hz, 6H).

[0247] C 51 H 49 F4N7O 10 MS (ESI) calculated for: 995.35; found: [M+1] 996.70, 997.73.

[0248] Example 13: Synthesis of 12 - ((2-(2,6 - dioxopiperidin - 3 - yl)-1,3 - dioxoisoindolin - 5 - yl)amino)-N-(4-(3 - (((R)-3’-(2 - ((4 - fluorobenzyl)((S)-1,1,1 - trifluoropropan - 2 - yl)amino)-2 - oxoethyl)-2’,4’ - dioxo - 2,3 - dihydrospiro[indene - 1,5’ - oxazolidine]-5 - yl)amino)-3 - oxopropyl)phenyl)dodecanamide (CPD13). [Chem.]

[0249] CPD13 was prepared from int-6 and the appropriate IMiD intermediate in the same manner as CPD2 of Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to give CPD13 as a yellow powder (15 mg, 88% yield).

[0250] 1 H NMR (500 MHz, acetone-d6) δ 9.86 (s, 1H), 9.30 (d, J = 4.8 Hz, 1H), 9.01 (s, 1H), 8.73 (d, J = 4.4 Hz, 1H), 8.42 (d, J = 8.3 Hz, 1H), 7.98 (s, 1H), 7.86 (s, 1H), 7.57 (d, J = 8.2 Hz, 2H), 7.52 - 7.48 (m, 2H), 7.22 - 7.17 (m, 3H), 7.09 - 6.90 (m, 3H), 6.32 (t, J = 5.6 Hz, 1H), 5.51 (p, J = 7.7 Hz, 1H), 5.11 - 5.01 (m, 3H), 4.88 (t, J = 13.9 Hz, 1H), 4.67 (dd, J = 70.0, 16.7 Hz, 1H), 4.42 (dd, J = 91.7, 16.6 Hz, 1H), 3.25 (dq, J = 43.4, 7.4, 6.9 Hz, 4H), 3.09 (ddd, J = 16.3, 8.6, 3.8 Hz, 2H), 2.96 (s, 4H), 2.79 (s, 4H), 2.68 (t, J = 7.4 Hz, 3H), 2.54 (ddd, J = 14.1, 8.1, 3.7 Hz, 2H), 2.34 (t, J = 7.4 Hz, 3H), 2.18 (t, J = 3.7 Hz, 1H), 1.68 (q, J = 7.5 Hz, 5H), 1.50 - 1.41 (m, 9H).

[0251] MS (ESI) calculated for C57H61F4N7O10: 1079.44, found: [M+1] 1080.65, 1081.48.

[0252] Example 14: Synthesis of 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)-N-(4-(3-(((R)-3’-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidine]-5-yl)amino)-3-oxopropyl)phenyl)propenamide (CPD14).

Chemical formula

[0253] CPD14 was prepared from int-6 and the appropriate IMiD intermediate in the same manner as CPD2 in Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to obtain CPD14 as a yellow powder (12 mg, 70% yield).

[0254] 11H NMR (500 MHz, acetone-d6) δ 9.93 (s, 1H), 9.28 (d, J = 4.7 Hz, 1H), 9.06 (s, 1H), 7.86 (s, 1H), 7.57 (dd, J = 7.7, 3.7 Hz, 3H), 7.49 (ddd, J = 8.6, 4.6, 1.7 Hz, 3H), 7.24 - 7.15 (m, 4H), 7.07 (dd, J = 29.3, 7.8 Hz, 3H), 6.60 (t, J = 5.7 Hz, 1H), 5.51 (p, J = 7.8 Hz, 1H), 5.13 - 5.01 (m, 3H), 4.96 - 4.86 (m, 1H), 4.67 (dd, J = 70.3, 16.7 Hz, 1H), 4.42 (dd, J = 91.8, 16.6 Hz, 1H), 3.78 (t, J = 6.0 Hz, 2H), 3.70 (t, J = 5.3 Hz, 2H), 3.61 - 3.53 (m, 2H), 3.49 (q, J = 5.0 Hz, 3H), 3.24 - 3.19 (m, 1H), 3.12 - 3.06 (m, 1H), 2.97 - 2.92 (m, 4H), 2.79 (p, J = 1.9 Hz, 2H), 2.76 (dd, J = 6.5, 4.1 Hz, 2H), 2.67 (t, J = 7.5 Hz, 2H), 2.56 (t, J = 6.0 Hz, 3H), 2.22 (ddt, J = 9.5, 5.2, 2.6 Hz, 1H), 1.50 (d, J = 6.6 Hz, 4H), 1.43 (d, J = 7.3 Hz, 2H).

[0255] Example 15: Synthesis of N-((R)-2-(2-((R)-5-acetamido-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidin]-3’-yl)-N-(4-fluorobenzyl)acetamido)propyl)-12-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)dodecanamide (CPD15).

Chemical Structure

[0256] CPD15 was prepared from the (R,R)-isomer of int-7 and the appropriate IMiD intermediate in the same manner as CPD2 of Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0-10%) to give CPD15 as a yellow powder (10 mg, 69% yield).

[0257] 1 H NMR (500 MHz, acetone-d6) δ 9.86 (s, 1H), 9.32 (s, 1H), 7.86 (s, 1H), 7.57 (dd, J = 8.3, 1.4 Hz, 1H), 7.52 - 7.40 (m, 4H), 7.36 - 7.31 (m, 2H), 7.25 - 7.16 (m, 2H), 7.08 - 7.01 (m, 3H), 6.93 (ddd, J = 8.3, 3.3, 2.1 Hz, 2H), 6.32 (q, J = 6.1 Hz, 1H), 5.06 (dd, J = 12.6, 5.4 Hz, 1H), 4.84 (d, J = 16.0 Hz, 1H), 4.77 (d, J = 16.5 Hz, 1H), 4.72 (d, J = 7.3 Hz, 1H), 4.65 (d, J = 16.5 Hz, 1H), 4.52 (d, J = 16.5 Hz, 1H), 4.44 (d, J = 16.0 Hz, 1H), 4.37 (dt, J = 8.4, 6.3 Hz, 1H), 4.27 (d, J = 16.4 Hz, 1H), 4.00 (p, J = 6.6 Hz, 1H), 3.52 - 3.39 (m, 3H), 3.28 (dt, J = 10.2, 4.1 Hz, 5H), 3.25 - 3.18 (m, 2H), 3.10 (ddd, J = 16.4, 8.7, 4.1 Hz, 2H), 3.01 - 2.92 (m, 2H), 2.82 - 2.74 (m, 6H), 2.57 (dddd, J = 17.6, 14.2, 8.2, 3.7 Hz, 2H), 2.28 - 2.14 (m, 4H), 1.68 (td, J = 7.4, 4.2 Hz, 3H), 1.61 - 1.53 (m, 3H).

[0258] C 50 H 58 FN7O 10 The calculated value of MS (ESI) for C

[0259] Example 16: Synthesis of N-((S)-2-(2-((R)-5-acetamido-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidin]-3’-yl)-N-(4-fluorobenzyl)acetamido)propyl)-12-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)dodecanamide (CPD16). [Chemical formula]

[0260] CPD16 was prepared from int-7 and the appropriate IMiD intermediate in the same manner as CPD2 in Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to give CPD16 as a yellow powder (9.6 mg, 68% yield).

[0261] 11H NMR (500 MHz, acetone-d6) δ 9.86 (s, 1H), 9.33 (d, J = 4.0 Hz, 1H), 7.87 (s, 1H), 7.55 (dd, J = 18.0, 8.4 Hz, 3H), 7.51 - 7.45 (m, 2H), 7.42 (dd, J = 8.4, 2.0 Hz, 1H), 7.38 - 7.32 (m, 2H), 7.28 (s, 1H), 7.19 (t, J = 8.8 Hz, 1H), 7.11 - 7.03 (m, 2H), 7.02 (d, J = 2.2 Hz, 2H), 6.93 (dt, J = 8.4, 1.6 Hz, 1H), 6.33 (d, J = 5.7 Hz, 1H), 5.06 (dd, J = 12.6, 5.4 Hz, 1H), 4.88 (d, J = 16.1 Hz, 1H), 4.79 (d, J = 16.6 Hz, 1H), 4.72 (d, J = 6.3 Hz, 1H), 4.61 (d, J = 16.6 Hz, 1H), 4.48 (d, J = 16.5 Hz, 1H), 4.40 (d, J = 16.0 Hz, 1H), 4.30 (d, J = 16.5 Hz, 1H), 4.01 (p, J = 6.6 Hz, 1H), 3.53 - 3.39 (m, 2H), 3.32 - 3.19 (m, 6H), 3.10 (ddt, J = 17.0, 8.7, 4.3 Hz, 2H), 3.01 - 2.93 (m, 2H), 2.83 - 2.72 (m, 6H), 2.57 (dddd, J = 18.5, 14.4, 8.2, 3.7 Hz, 2H), 2.29 - 2.13 (m, 4H), 1.68 (p, J = 7.2 Hz, 3H), 1.58 (p, J = 7.3 Hz, 3H).

[0262] C 50 H 58 FN7O 10 The calculated value of MS (ESI) for [C 50 H 58 FN7O 10 ] is 935.42; the measured values are [M + 1] 936.74, 937.65.

[0263] Example 17: Synthesis of N-((S)-2-(2-((R)-5-acetamido-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidin]-3’-yl)-N-(4-fluorobenzyl)acetamido)propyl)-3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline)-5-yl)amino)ethoxy)ethoxy)ethoxy)propanamide (CPD17).

Chemical formula

[0264] CPD17 was prepared from int-7 and the appropriate IMiD intermediate in the same manner as CPD2 in Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to give CPD17 as a yellow powder (8.4 mg, 86% yield).

[0265] 11H NMR (500 MHz, acetone-d6) δ 9.96 (s, 1H), 9.31 (s, 1H), 7.85 (d, J = 15.6 Hz, 1H), 7.61 - 7.57 (m, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.47 (dt, J = 8.5, 4.2 Hz, 1H), 7.43 - 7.38 (m, 1H), 7.36 - 7.30 (m, 1H), 7.21 - 6.99 (m, 5H), 6.62 (t, J = 5.8 Hz, 1H), 5.09 (ddd, J = 12.8, 5.5, 1.7 Hz, 1H), 4.89 (d, J = 16.0 Hz, 1H), 4.80 (d, J = 16.5 Hz, 1H), 4.70 (d, J = 9.4 Hz, 1H), 4.61 (d, J = 16.5 Hz, 1H), 4.42 - 4.35 (m, 1H), 3.76 - 3.73 (m, 2H), 3.70 (td, J = 6.2, 1.4 Hz, 3H), 3.65 - 3.62 (m, 4H), 3.59 - 3.57 (m, 2H), 3.56 - 3.51 (m, 4H), 3.48 - 3.37 (m, 2H), 3.25 (dtt, J = 18.2, 9.8, 4.1 Hz, 2H), 3.10 (ddt, J = 12.6, 8.8, 3.9 Hz, 1H), 3.02 - 2.91 (m, 2H), 2.80 - 2.75 (m, 4H), 2.61 - 2.37 (m, 4H), 2.22 (ddt, J = 13.0, 5.7, 2.1 Hz, 2H).

[0266] C 47 H 52 FN7O 13 The calculated value of MS (ESI) for C H FN7O: 941.36; measured value: [M + 1] 942.67, 943.65.

[0267] Example 18: Synthesis of N - ((S) - 2 - (2 - ((R) - 5 - acetamido - 2’,4’ - dioxo - 2,3 - dihydrospiro[indene - 1,5’ - oxazolidine] - 3’ - yl) - N - (4 - fluorobenzyl)acetamido)propyl) - 8 - ((2 - (2,6 - dioxopiperidin - 3 - yl) - 1,3 - dioxoisoindolin - 5 - yl)amino)octanamide (CPD18).

Chemical Structure

[0268] CPD18 was prepared from int-7 and the appropriate IMiD intermediate in the same manner as CPD1 of Example 1. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 10%) to give CPD18 as a yellow powder (7.2 mg, 82% yield).

[0269] 1 H NMR (500 MHz, acetone-d6) δ 9.87 (s, 1H), 9.28 (d, J = 4.3 Hz, 1H), 7.81 (d, J = 2.9 Hz, 1H), 7.53 - 7.48 (m, 3H), 7.35 - 7.32 (m, 2H), 7.18 (t, J = 8.8 Hz, 1H), 7.07 (t, J = 8.8 Hz, 2H), 6.95 (t, J = 2.6 Hz, 1H), 6.85 (dt, J = 8.3, 1.8 Hz, 1H), 6.31 - 6.17 (m, 1H), 5.06 (dd, J = 12.6, 5.4 Hz, 1H), 4.92 (d, J = 16.1 Hz, 1H), 4.80 (d, J = 16.5 Hz, 1H), 4.72 (d, J = 4.1 Hz, 1H), 4.62 (d, J = 16.6 Hz, 1H), 4.49 (d, J = 16.5 Hz, 1H), 4.43 - 4.34 (m, 2H), 4.28 (dd, J = 16.5, 1.4 Hz, 1H), 3.51 (ddq, J = 9.3, 4.6, 2.2 Hz, 1H), 3.28 - 3.21 (m, 4H), 3.10 (dt, J = 8.3, 4.0 Hz, 1H), 2.98 - 2.92 (m, 1H), 2.80 - 2.71 (m, 4H), 2.56 (ddt, J = 9.9, 7.6, 4.2 Hz, 1H), 2.28 - 2.16 (m, 4H), 1.67 - 1.62 (m, 4H), 1.48 - 1.37 (m, 4H).

[0270] C 44 H 46 FN7O 10 MS (ESI) calculated for: 851.33; found: [M + 1] 852.53, 853.44.

[0271] Example 19: Synthesis of N1-((R)-3’-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidine]-5-yl)-N4-(6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexyl)succinimide (CPD19).

Chemical Structure

[0272] CPD19 was prepared from int-8 and the appropriate VHL-N2 intermediate in the same manner as CPD2 in Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 15%) to give CPD19 as a white powder (11 mg, 58% yield).

[0273] C 55 H 64 F4N8O 10 Calculated MS (ESI) for C49H54F4N8O11S: 1104.44; Found: [M+1] 1105.73, 1106.72.

[0274] Example 20: Synthesis of N1-((S)-3’-(2-((4-fluorobenzyl)((R)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidine]-5-yl)-N4-((S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl)succinamide (CPD20).

Chemical Structure

[0275] CPD20 was prepared from int-8 and the appropriate VHL-N2 intermediate in the same manner as CPD2 of Example 2. The crude product was purified by ISCO chromatography (MeOH / DCM, 0 - 15%) to give CPD20 as a yellow powder (12 mg, 59% yield).

[0276] C 57 H 68 F4N8O 13 Calculated MS (ESI) for S: 1180.46; Found: [M+1] 1181.77, 1182.68.

[0277] Example 21: Synthesis of N1-((S)-3’-(2-((4-fluorobenzyl)((R)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2’,4’-dioxo-2,3-dihydrospiro[indene-1,5’-oxazolidine]-5-yl)-N4-((S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl)succinamide (CPD21).

Chemical Structure

[0278] CPD21 was prepared from int-8 and the appropriate VHL-N2 intermediate in the same manner as CPD2 of Example 2.

[0279] Example 22: Synthesis of N1-((R)-3'-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidine]-5-yl)-N4-(4-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamoyl)benzyl)succinamide (CPD22).

Chem.

[0280] CPD22 was prepared from int-8 and the appropriate VHL-N2 intermediate in a manner similar to that of CPD2 in Example 2.

[0281] Example 23: Synthesis of N1-((R)-3'-(2-((4-fluorobenzyl)((S)-1,1,1-trifluoropropan-2-yl)amino)-2-oxoethyl)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidine]-5-yl)-N5-(15-oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramide (CPD23).

Chem.

[0282] Compound 3 (30 mg, >95% yield by LCMS) was obtained from int-6 (30 mg, 0.06 mmol) and biotin-PEG3-20-atom-acid (47.5 mg, 0.08 mmol) in a manner similar to that of Compounds 1 and 2.

[0283] Example 24: Cellular degradation of p300 / CBP in Kelly high-risk neuroblastoma (NB) cells.

[0284] Both EP300 and CBP are multi-domain proteins containing a kinase-inducible domain (KID) interaction domain (KIX), bromo-, zinc finger, and acetyltransferase (HAT) domains. The encoded epigenetic modification domains enable both EP300 and CBP to bind transcription factor recognition to chromatin remodeling and critically mediate gene expression. Studies on the development of small molecules targeting EP300 / CBP have focused on the bromodomain, KIX domain, and HAT domain (Figure 2A).

[0285] The effects of several EP300 / CBP inhibitors, C646, CBP30, and A485, were tested on Kelly NB cells (Figure 2B). The results showed that the pan-HAT inhibitor A485 was superior to other known EP300 / CBP inhibitors in promoting growth changes in NB cell models at sub-μM levels (Figures 2C and 2D). Kelly NB cells treated with increasing doses of A485 in the CellTiter-Glo® proliferation assay showed growth inhibition (Figure 2E) and induction of apoptosis (Figure 2F).

[0286] Cell lysis assay Kelly neuroblastoma cells were seeded in 6-well plates at 1,000,000 cells / well and treated in a dose-dependent manner for 24 - 72 hours. Whole cell lysates were recovered using ice-cold lysis buffer [300 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.5% Triton X-100, 1% SDS, 1 mM dithiothreitol (DTT), Roche® protease inhibitor cocktail (1:1000), 25 units / mL benzonase] and blotted at a protein concentration of 20 μg. Histones were extracted using the EpiQuik™ Total Histone Extraction Kit (OP-0006-100, Epigentek) and blotted at a protein concentration of 4 μg.

[0287] Using the standard protocol, compounds CPD1 to CPD22 were tested via the ATPlite (trademark) assay (ATPlite (trademark) 1000 assay kit, PerkinElmer (registered trademark), USA). The Kelly cell line was treated with the compounds for 72 hours, and cell growth inhibition was measured using the ATPlite (trademark) assay kit. The signals were normalized against DMSO-treated cells.

[0288] The results shown in FIGS. 15A to 15G indicate that the compounds of the present invention caused inhibition of Kelly NB cells. CPD1 (FIG. 15A), CPD8 (FIG. 15C) and CPD16 (FIG. 15E) exceeded the positive control A485. The non-cell-permeable CPD2 (negative control) did not inhibit cell growth (FIG. 15A).

[0289] Blotting Whole cell lysates were separated on NuPAGE® 3–8% Tris-acetate polyacrylamide gels (EA03785BOX, Invitrogen®), and histone extraction lysates were separated on Bolt 4–12% Bis-Tris polyacrylamide gels (NW04125BOX, Invitrogen®). Gels were then transferred to nitrocellulose membranes (LC2001, Invitrogen®). Primary and secondary antibodies used included anti-p300 (ab10485, Abcam®) at 1:500 dilution, anti-CBP (ab2832, Abcam®) at 1:1000 dilution, anti-actin (3700S, Cell Signaling Technology) at 1:5000 dilution, anti-H3 (4499S, Cell Signaling Technology®) at 1:1000 dilution, anti-H3K27ac (ab4729, Abcam®) at 1:1000 dilution, IRDye® 800 goat anti-rabbit (926-32211, LiCor® Biosciences) at 1:5000 dilution, and IRDye® 680 goat anti-mouse (926-68070, LiCor®) at 1:5000 dilution. Visualization was performed using an Odyssey infrared imaging system (LiCor® Biosciences) (Figure 3B, Figure 4F, and Figure 4G).

[0290] Example 25: Dependence on EP300 in NB cells.

[0291] Screening and low-throughput data suggest that EP300, rather than CBP, is selectively required for NB cell proliferation. The experimental method for the colony formation assay is as described in Durbin et al., Nat Genet. 50(9):1240-60 (2018). The results are shown in Figure 3A.

[0292] Example 26: AlphaLISA® and AlphaScreen™ assays.

[0293] We developed an AlphaLISA® assay for the EP300 catalytic function (Figure 3B) and an AlphaScreen™ assay for the EP300 bromodomain and cereblon (CRBN) using biotinylated tag - attached small molecules as probes for the EP300 bromodomain and CRBN, respectively (Figure 3C).

[0294] The AlphaScreen™ assay was performed in a 384 - well plate format using a white AlphaPlate (PerkinElmer®, USA), and the transfer of pre - diluted compounds (100 nL) was carried out using a Janus Workstation (PerkinElmer®, USA). All subsequent steps were performed in an assay buffer (50 mM 4 - (2 - hydroxyethyl)-1 - piperazineethanesulfonic acid (HEPES), pH 7.5, 0.1% (wt / vol) bovine serum albumin (BSA) and 0.01% (vol / vol) Tween - 20). Briefly, 10 μL of the assay buffer containing the enzyme (2 nM final) was pre - incubated with the compound dilutions for 15 minutes. The enzyme reaction was initiated by adding a substrate (5 μL) consisting of acetyl - Co - A 2 - OG (5 μM final).

[0295] The final concentration of FAS was 10 μM. The final concentration of histone tail-GGK biotin was 100 nM. The enzymatic reaction was allowed to proceed for 30 minutes and stopped by adding 5 μL of assay buffer containing ethylenediaminetetraacetic acid (EDTA) (40 mM) and NaCl (1200 mM). The final concentration of dimethyl sulfoxide (DMSO) was 1%. Streptavidin donor beads (0.08 mg / mL) and Protein A conjugate acceptor beads (0.08 mg / mL) were pre-incubated with an antibody against methyl mark (final 300 ng / mL) for 1 hour, and the presence of histone H3 product acetylation mark was detected using the pre-incubated AlphaScreen™ beads (5 μL). Detection was allowed to proceed for 2 hours at room temperature, and the assay plate was read using an Envision™ 2104 plate reader. Data were normalized against the (enzyme-free) control, and the IC 50 values were determined by non-linear regression curve fitting using GraphPad Prism (Figures 4E - 4H).

[0296] The IC 50 values of CDP1 - CPD22 generated from the assay are shown in Table 1. Table 1. IC 50 values in Kelly cells. [Table 1]

[0297] Cell growth inhibition was observed with the degradation-inducing agents CPD1, CPD8, CPD10, CPD13, CPD15, and CPD16 of the present invention. No cell growth inhibition was detected or determined with the remaining compounds.

[0298] EP300 is important for the survival of NB cancer cells (Figure 3A). The compound CPD1 of the present invention selectively dimerized the target protein (EP300) to the E3 ligase (CRBN) without affecting CBP. CPD1 induced the selective degradation of EP300 in NB cancer cells and killed NB cells with much higher potency compared to the inhibitor alone.

[0299] Example 27: Evaluation of the adapter function of a putative EP300-degrading inducer.

[0300] To experimentally evaluate the adapter function of a putative EP300-degrading inducer, a luminescence proximity assay (AlphaScreen™, PerkinElmer®) for human recombinant EP300 and CRBN-DDB1 proteins was used as previously reported for BRD4 / CRBN-DDB1 (Figure 5E) (Winter, et al. Science 348(6241):1376-81 (2015)).

[0301] Example 28: Inhibition of cell proliferation by CPD1, CPD2, and A-485 using a 4-day ATPlite™ assay.

[0302] To prepare dilution series with water, aliquots of an ATP standard solution (ATPlite™ 1000 assay kit, PerkinElmer®, USA) were used. 100 μL of a series of complete medium without cells was pipetted into the wells of the plate. 50 μL of mammalian cell lysis solution was added to each well, and the plate was shaken on an orbital shaker at 700 rpm for 5 minutes. 10 μL of the ATP dilution series was added to the wells, and the plate was shaken on an orbital shaker at 700 rpm for 5 minutes. 50 μL of substrate solution was added, and the plate was shaken on an orbital shaker at 700 rpm for 5 minutes. The plate was dark adapted for 10 minutes before measuring luminescence to create a standard curve. The results are summarized in Figures 7A - 7C. In MM.1S and U266 cells, the compound CPD1 of the present invention outperformed the inhibitor A485.

[0303] Example 29: Inhibition of cell proliferation by CPD1, CPD2, and A-485 using a 6-day 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

[0304] The medium from cell culture was discarded. For adherent cells, the medium was carefully aspirated. For floating cells, the 96-well plate was centrifuged at 1,000 x g for 5 minutes at 4°C using a microplate-compatible centrifuge, and the medium was carefully aspirated. The amount of the existing medium needs to be the same for each sample. 50 μL of serum-free medium and 50 μL of MTT solution were added to each well. The plate was incubated at 37°C for 3 hours.

[0305] After incubation, 150 μL of MTT solvent was added to each well. The plate was wrapped with foil and shaken on an orbital shaker for 15 minutes. The absorbance was read at OD = 590 nm. The reading was performed within 1 hour. The results are summarized in FIGS. 8A - 8C. In MM.1S and U266 cells, the compound CPD1 of the present invention exceeded inhibitor A485.

[0306] Example 30: Selective degradation of EP300 by CPD1.

[0307] The experimental protocol was the same as in Example 24. Protein levels were determined by immunoblotting using an EP300 antibody, a CBP antibody, or an H3K27ac antibody. Total H3 was used as a loading control. Protein levels were measured either by a dose-dependent method or a time-dependent method.

[0308] The results shown in FIGS. 9A - 9D showed a clear degradation of EP300 without affecting CBP levels in the first 36 hours, thereby confirming the selectivity of CPD1 for EP300 over CBP. Degradation of CBP was observed after 48 hours (FIG. 9B).

[0309] In the proteomics test, it was shown that EP300 was selectively and significantly degraded by the degradation-inducing agent CPD1 of the present invention (FIG. 9C). No change in CBP levels was observed. The results in FIG. 9D showed that CPD1 inhibited Kelly cell growth in a dose-dependent manner over 10 days using a colony formation assay.

[0310] Example 31: Biological activity evaluation of CPD1 and the isomer (S,S)-CPD1.

[0311] The results shown in FIGS. 10A-10D indicate that the chiral center of the targeting ligand of CPD1(R,S) plays an important role in the potency of the compound. No cell degradation of EP300 was observed with the (S,S) isomer.

[0312] Example 32: Define the substrate specificity for the compounds of the present invention.

[0313] The AlphaScreen™ assay was used to determine the binding of the degrader compounds of the present invention to both the CRBN domain and the HAT domain. The experimental protocol was the same as in Example 26.

[0314] The results shown in FIGS. 11A-11C indicate that the degrader CPD1 of the present invention bound to both the CRBN (FIG. 11C) and the HAT domain (FIG. 11B).

[0315] Example 33: CPD1 is a CRBN-dependent degrader molecule.

[0316] CPD1 was tested against the CRBN knockout (k / o) Kelly cell line. The experimental protocol was the same as in Example 24.

[0317] The results shown in FIGS. 12A-12D indicate that CPD1 did not degrade EP300 or CBP in the CRBN k / o strains (CRBN-1 and CRBN-3), thereby confirming that EP300 degradation by the compounds of the present invention is CRBN-dependent. The cell growth inhibition curves in FIGS. 12C and 12D indicate that CPD1 had no effect on the CRBN k / o cell lines.

[0318] Example 34: In vivo test of CPD1 on CD1 mice.

[0319] For the maximum tolerated dose (MTD), CPD1 was tested in mice (Figure 6A). Four mice per group were treated with 10 mg / kg (mpk), 20 mpk, and 40 mpk. This compound was also tested in human CRBN knock-in mice, and blood samples were collected after 21 days of treatment. All the blood samples tested were normal.

[0320] The results shown in Figures 13A - 13C indicated that no weight loss was observed in the mice, demonstrating that CPD1 showed good tolerance in the in vivo test. Also, humanized CRBN I391V No toxicity was observed in the mice (Figure 13C).

[0321] Blood samples were collected 14 days later from mice treated daily by intraperitoneal injection (IP) at 40 mpk. The results are summarized in Table 2. Blood sample measurements indicated that CPD1 was not toxic to the mice. Table 2: Blood analysis of in vivo test [Table 2]

[0322] To examine the liver from the MTD test, immunohistochemistry (IHC) staining was used. The staining showed that in normal mice using 40 mpk, liver tissue had EP300 knockout after 14 days of treatment (Figure 13C). A decrease in P300 level was observed in vivo. No changes in CBP level and H3K27Ac level were observed.

[0323] Example 35: CPD1 has anti-tumor activity in vivo.

[0324] The in vivo effect was evaluated using a xenograft model of the Kelly cell line. Kelly cells were transplanted into animals at 2M. Treatment was started on the 10th day by IP at 40 mpk.

[0325] The results shown in FIGS. 14A-14B indicate that CPD1 reduced tumor progression and extended the survival period of the animals.

[0326] Example 36: Evaluation of the cellular efficacy and off-target effects of EP300-degrading agents.

[0327] To report on-target proteolysis, intracellular EP300 degradation is evaluated using a degradation assay based on high-throughput flow cytometry. EP300 is cloned into an EGFP reporter system. Using FRT / Flp recombination, EP300 is expressed as an EGFP fusion, followed by expression of the P2A site, and mCherry or mCardinal as a normalization marker to express the EP300-EGFP fusion protein and the RFP reporter at equivalent and stable levels.

[0328] Using a 96-well flow cytometry setup, target proteolysis is measured at single-cell resolution as the loss of signal from EGFP to RFP (Lu et al., Chem. Biol. 22(6):755-63(2015); Winter et al., Science 348(6241):1376-81(2015); Zengerle et al., ACS Chem. Biol. 10(8):1770-7(2015); Winter et al., Mol. Cell. 67(1):5-18(2017)). This assay shows increased sensitivity and robustness compared to other methods, and the 96-well flow cytometry setup enables complete IC 50 profiling for a large number of molecules. In parallel, a similar IKZF1-reporting cell line is developed to monitor IKZF1 degradation to evaluate the specificity of putative EP300-degrading agents (Kronke et al., Science 343(6168):301-5(2014); Lu et al., Science 343(6168):305-9(2014).

[0329] To demonstrate the requirement of CRBN for EP300 degradation in cells, isogenic NB cell line, Kelly cells with gene-modified knockout of CRBN by CRISPR / Cas9 technology were generated (Winter et al., Science 348(6241):1376-81(2015); Winter et al., Mol. Cell. 67(1):5-18(2017)). WT Kelly cells and Kelly CRBN- / - cells were incubated with increasing concentrations of EP300 degradation inducer, and EP300 proteolysis was visualized by immunoblotting. In this cell line, proliferation and viability were also scored to determine if there was off-target toxicity that might interfere with cell growth. Along with immunoblotting of EP300, CBP and IKZF in the treated cell lines, this cell line was used to identify additional potent and selective EP300 degradation inducers.

[0330] Example 37: Evaluation of EP300 degradation inducer function in NB cell models.

[0331] A panel of NB cell lines was profiled for additional EP300 degradation inducers ( "potent and selective compounds / molecules") with the most promising biochemical and on-target cell activities. Degradation of EP300 in NB cell line (Kelly) was tested in dose-range and time-range studies by immunoblot analysis compared to appropriate inactive controls (A-485, CBP30 and lenalidomide). Again, compared to A-485, the effect on viability was evaluated for a panel of MLL-r leukemia cell lines (CellTiter-Glo®; Promega™). The effect on global acetylation of H3K27 was evaluated by immunoblotting. Lead molecules were used in the cell property evaluation methods described below.

[0332] To rigorously evaluate the mechanism of EP300 degradation induced by the degraders, only A-485 and phthalimide are used as negative controls to first test for CP300 degradation in NB cells. The CRBN levels in NB are also evaluated. Next, as described, competition compounds and gene editing strategies are used to address the requirements for proteasome function, EP300 binding, and CRBN binding (Winter et al., Science 348(6241):1376-81(2015)). To support the cellular requirements for CRBN binding, E3 complex activation, and proteasome function for effective EP300 degradation, carfilzomib (a proteasome inhibitor), MLN4924 (a Nedd8-activating enzyme inhibitor), or lenalidomide (a CRBN binder) are used to pretreat Kelly cells and BEC2 cells separately prior to treatment with the putative EP300 degrading agent.

[0333] To evaluate the cellular results of lead EP300 degrader treatment on protein stability in NB cell lines, a unbiased, whole proteome approach is used. Proteomics based on multiplexed quantitative mass spectrometry is used to evaluate the specificity and results of EP300 degrader treatment in NB cells (McAlister et al., Anal. Chem. 84(17):7469-78(2012); McAlister et al., Anal. Chem. 86(14)7150-8(2014)). Kelly cells and BEC2 cells are treated with the putative EP300 degrader, or DMSO, for 24 hours. A 24-hour incubation was selected to capture the major immediate results of small molecule action and to mitigate the predicted confounding effects on the suppressed transactivation of EP300 target genes. The specificity of EP300 degradation is prioritized over potency in combination with off-target effects (e.g., degradation of CRBN neo-substrate IKZF1 Kronke et al., Science 343(6168):301-5(2014); Lu et al., Science 343(6168):305-9(2014)). All of these evaluations are used to identify candidates for in vivo testing.

[0334] Example 38: Optimization of PK properties of EP300 degrader for in vivo testing.

[0335] To accurately evaluate the translational potential of EP300 degrader in vivo, the PK properties of the lead EP300 degrader are optimized. For example, CPD1 was evaluated for its PK properties such as half-life and maximum tolerated dose (MTD) (see Figure 6A, Example 34). Using the A-485 scaffold, novel EP300 degrading agents with excellent potency and selectivity are generated. The lead compounds are produced in large quantities for in vivo PK and efficacy testing.

[0336] Example 39: Characterization of the effects of EP300 degradation on gene expression, chromatin occupancy, and histone modification compared to EP300 inhibitors.

[0337] Using transcriptional profiling by RNA-seq and chromatin evaluation by ChIP-seq and ATAC-seq, achieve a comprehensive molecular evaluation of the impact of EP300 degradation on NB cells. Conduct a comprehensive molecular evaluation of the impact of these compounds on chromatin using transcriptional profiling by RNA-seq and epigenomic profiling by ChIP-seq. Specifically, perform a kinetic study of the transcriptional response at 0, 6, and 24 hours in cells treated with A485 or a lead EP300 degradation inducer on resistant strains. Examine the impact on the localization of EP300 / CBP proteins, enhancer activity (H3K27ac), and promoter integrity (H3K4me3) at 0, 6, and 24 hours after treatment by ChIP-seq and Chem-seq. Determine the accessible chromatin and potential cis-regulator elements before and after drug reaction by ATAC-seq. Integrate these datasets to test the hypothesis that EP300 degradation not only inhibits its enzymatic activity but also abrogates its non-enzymatic function leading to the collapse of the EP300-driven gene expression program in cells not previously exposed to EP300 inhibitors.

[0338] Example 40: Evaluation of the antitumor effect of an EP300 degradation inducer compared to an EP300 inhibitor in vitro.

[0339] Use the newly developed EP300 degradation inducer to examine EP300 degradation in detail in NB in vitro in contrast to enzyme inhibition. A panel of human NB cells (CellTiter-Glo®) in a dose-response format with A-485 and the EP300 degradation inducer in a direct comparison. Furthermore, evaluate cell proliferation, EP300 proteolysis, and H3K79 methylation levels over time by immunoblotting and cell cycle / cell death by flow cytometry analysis.

[0340] Example 41: Evaluation of EP300 degradation in combination with a standard therapeutic agent or other inhibitor as a combined approach in NB.

[0341] Determine the in vitro synergistic effect on NB cells through evaluation of cell proliferation by a standard approach (CellTiter-Glo®). Use a robotic pinning system that allows titration of two different compounds at multiple concentrations on plates of cultured cells. After 48 - 72 hours of incubation, determine cell viability by ATP content (CellTiter-Glo®) using a multi-label plate reader. Plot the results in the CompuSyn package according to the method described in Chou et al., Adv. Enzyme Regul. 22:27 - 55 (198) to determine whether there is an additive or synergistic effect of the drugs. At the combined doses that appear to have a synergistic effect, determine cell cycle arrest as compared to apoptosis through Annexin V staining and evaluation of DNA content. In any experiment, evaluate EP300 protein levels by immunoblotting to ensure proteolysis.

[0342] Example 42: Evaluation of the toxicity of EP300 degradation-inducing agents and inhibitors in zebrafish and mice.

[0343] The maximum tolerated dose (MTD) of each candidate was determined using zebrafish embryos. Three-day-old zebrafish embryos were exposed to various drug concentrations and the adverse effects on health were monitored. The therapeutic dose was then selected by determining the highest concentration that did not result in morphological and behavioral adverse effects. The experiment was first conducted using compound CPD1 as an additional design guideline for the MTD evaluation of the lead EP300 degrader (Figure 6B). In a similar manner, the MTD of the degrader for mice was also determined. Briefly, the test compound was administered to mice, blood and plasma were separated at specific intervals (0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours) after administration, and the presence of the test substance was quantified by LC / MS / MS (n = 5 mice / compound). In vivo mouse PK experiments were used to determine these parameters and inform iterative rounds of medicinal chemistry for the optimization of the EP300 degrader (Figure 6A). Since the degrader has a relatively large molecular weight, appropriate formulations were considered to enhance the solubility and stability of the degrader for in vivo testing. Additionally, these parameters, including the established MTD, were used to guide the in vivo test design, such as the administration method, dose, and schedule for the following experiments. The information obtained was also used to guide the optimization of the degrader performed in Example 36.

[0344] Example 43: Evaluation of compounds using zebrafish model and mouse PDX model.

[0345] In the NB zebrafish xenograft model, the in vivo effects of A-485 and the degradative inducer CPD1 are compared. First, for visualization, human NB cells are stably transfected with EGFP. These cells are then transplanted into 2-day-old zebrafish embryos using yolk and intravenous injection as described previously. (Haldi et al., Angiogenesis 9(3):139-51(2016); He et al., J. Pathology 227(4):431-45(2012); Tulotta et al., Methods Mol. Biol. 1451:155-69(2016)). One day later, zebrafish embryos transplanted with fluorescent human NB cells are arrayed in 24-well plates and treated with the test drugs. Twelve recipient fish are exposed to the individual degradative inducer and inhibitor added to the fish water at the MTD, as well as the DMSO control. Five days after treatment, fluorescence is used to analyze in vivo cell proliferation, cell proliferation and invasion (Tulotta et al., Methods Mol. Biol. 1451:155-69(2016)) and compare between groups. Welch's t-test is used to address heterogeneity of variance. Drugs showing significant tumor suppression are further analyzed at various concentrations. To elucidate the cellular mechanisms underlying tumor suppression by the active drug or drug combination, treated NB cells are analyzed for cell proliferation (by immunohistochemistry for PCNA and phosphorylated histone H3), cell viability and apoptosis (by TUNEL and anti-caspase 3 staining), senescence (by β-galactosidase staining), and autophagy (by lysosome staining). After 0, 1, 2, 3, 4, and 5 days of treatment, the PD effects on EP300 protein level, H3K27 acetylation, MYCN level, and gene expression are performed by qRT-PCR on NB cells isolated from dissociated zebrafish embryos.

[0346] Next, in the NB (Kelly) xenograft model, the in vivo effects of A-485 and the lead EP300 degrader are compared by injecting 1x106 cells each into 30 six- to eight-week-old NOD-SCID-IL2Rnull (NSG) mice. These mice begin to exhibit clinical symptoms two to three weeks after transplantation. After confirming engraftment, the animals are divided into either an efficacy cohort or a pharmacodynamics (PD) cohort (3 animals / group for the PD study and 5 animals / group for the efficacy study). In both cohorts, A485 or the degrader is administered daily by intraperitoneal (IP) injection at the MTD / day. The control group is injected intraperitoneally with the vehicle determined in the formulation study of Example 10. The body weights of all animals are measured to evaluate toxicity, and tumor sizes are measured every three days. After 21 days of drug treatment followed by a seven-day drug-free period, efficacy (survival rate) is determined. Animals evaluated for PD are dosed for 14 days and euthanized after the infusion is completed. At the end of the study, the animals are euthanized and tissues are harvested. For histological examination, the femur, a portion of the spleen, a portion of the liver, and any enlarged lymph nodes are fixed in 10% formalin. qRT-PCR is performed to assess the PD effects on EP300 protein levels, H3K27 acetylation, and gene expression. Using an EP300 degrader that functions well in the xenograft study, a similar experiment is conducted in the NB PDX model. Thirty six- to eight-week-old NSG mice are injected with 1x106 human PDX cells (secondary transplantation). As described above, the mice are monitored for tumor engraftment. Also, the transplanted mice are divided into treatment cohorts (3 animals / group for the PD study and 5 animals / group for the efficacy study) treated with vehicle, the EP300 degrader, or A-485 (administered as described above). All analyses are performed as described above. These studies are conducted on a total of three to five different PDXs.

[0347] Example 44: Evaluation of a combination of EP300 degrader for NB treatment against the PDX model.

[0348] Using the PDX model, evaluate the combined effect of an EP300 inhibitor with a specified EP300 degrader inducer. These experiments are conducted as described in Example 42, except that the animals are randomized into four treatment cohorts of eight mice each (3 / group in the PD test, 5 / group in the efficacy test): vehicle, EP300 degrader inducer, second inhibitor, or degrader inducer / inhibitor.

[0349] All patent publications and non-patent publications are indicative of the level of skill of those of ordinary skill in the art to which the present invention pertains. All of these publications are hereby incorporated by reference into this specification to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.

[0350] Although the invention herein has been described with reference to particular embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the invention. Accordingly, it should be understood that numerous modifications may be made to the exemplary embodiments, and other configurations may be devised without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A bifunctional compound having a structure represented by Formula I, or a pharmaceutically acceptable salt or stereoisomer thereof. : 【Chemical 1】 (In Formula I, the histone acetyltransferase (HAT) targeting ligand has a structure represented by any one of Structures TL-1 and TL-1a to TL-1f: 【Chemical 2】 【Chemical Formula 4】 In formula TL-1a, A is CH ２ , NH or O; B is CH ２ or CO; R is H, halo, CN, CF ３ , alkyl or alkoxy, and 【Chemical Formula 6】 In Formula TL-1b, A, B, and R are the same as those described above, R １ is a C3-C5 carbocyclic group or an alk carbocyclic group, or a 3- to 5-membered N-heterocyclic group or an alk N-heterocyclic group, and the alkyl group is a C1-C10 alkyl group, 【Chemical Formula 9】 In Formula TL-1c, Q is CH ２ , O, N, CO, C(O)O, C(O)N, CH ２ N, CH ２ C(O), CH ２ C(O)O, CH ２ C(O)N or CH ２ CH ２ N; R ２ is 【Chemical Formula 10】 , a C3-C5 carbocyclic group or alkcarbocyclic group, or a 3- to 5-membered N-heterocyclic group or alkN-heterocyclic group, wherein the alkyl group is a C1-C10 alkyl group, 【Chemical Formula 12】 【Chemical Formula 14】 and, 【Chemical 16】 the linker is represented by any one of Structures L10 to L26: 【Chemical Formula 18】 In formula L10, X is CH ２ , NH, NMe or O, and n is an integer from 0 to 11, 【Chemical 19】 the degron binds to cereblon (CRBN) and is represented by Structure D1: 【Chemical Formula 40】 In formula D1, Y is CH ２ or CO; Z is NH, O or OCH ２ CO, and the wavy line ( 【Chemical Formula 41】 ) represents a binding point for the linker and the EP300 targeting moiety, or the degron binds to von Hippel-Lindau tumor suppressor factor (VHL) and is represented by a structure selected from the group consisting of: 【Chemical 43】 【Chemical 44】 (In Formula D2-c, Y' is a bond, N, O, or C)); 【Chemical 45】 (In formula D2-d, Z is a C ５ ~C ６ carbocyclic group or a C ５ ~C ６ heterocyclic group); and 【Chemical 46】 ）。

2. R １ is [Chemical Formula 7] The bifunctional compound according to Claim 1, which is

3. The bifunctional compound according to Claim 1, wherein the linker is represented by any one of Structures L11, L12, L14, L15, and L17: 【Chemical 19】 。

4. The bifunctional compound according to Claim 1, which is represented by any one of Structures I-2 to I-12: 【Chemical formula 21】 【Chemical 22】 (In each formula, X and n are the same as those described above) or a pharmaceutically acceptable salt or stereoisomer thereof.

5. The bifunctional compound according to Claim 1, which is represented by a structure selected from the group consisting of: 【Chemical 23】 【Chemical 24】 ​ 【Chemical 26】 【Chemical 27】 【Chemical 28】 【Chemical 29】 【Chemical Formula 30】 【Chemical 31】 【Chemical 32】 【Chemical 33】 【Chemical 34】 【Chemical 35】 【Chemical 36】 【Chemical 37】 【Chemical 38】 【Chemical 39】 (In each formula, A, B, X, n, Q, R, R １ , R ２ are the same as those described above) or a pharmaceutically acceptable salt or stereoisomer thereof.

6. The bifunctional compound according to Claim 1, wherein the degron is represented by Structure D1: 【Chemical Formula 40】 wherein Y is CH ２ or CO; Z is NH, O or OCH ２ CO, and the wavy line ( 【Chemical 41】 ) represents a binding point for the linker and the EP300 targeting moiety, the bifunctional compound according to any one of Claims 1 to 5.

7. The bifunctional compound according to Claim 6, wherein the degron is represented by Structure D1-a: 【Chemical Formula 42】 。

8. The bifunctional compound according to any one of Claims 1 to 5, wherein the degron is represented by a structure selected from the group consisting of: 【Chemical 43】 【Chemical 44】 (In the formula, Y' is a bond, N, O, or C)); 【Chemical 45】 (wherein Z is a C ５ to C ６ carbocyclic group or a C ５ to C ６ heterocyclic group); and 【Chemical 46】 。

9. 【Fig. 47】 【Chemical Formula 48】 【Chemical 49】 【Chemical Formula 50】 【Chemical 51】 【Chemical 52】 The bifunctional compound according to Claim 1, which is as well as pharmaceutically acceptable salts and stereoisomers thereof.

10. A bifunctional compound having a structure represented by Structure 24: 【Chemical 71】 or a stereoisomer thereof.

11. A pharmaceutical composition comprising a therapeutically effective amount of a bifunctional compound or a pharmaceutically acceptable salt or stereoisomer according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, wherein the pharmaceutical composition is in the form of a tablet or a capsule.

13. A pharmaceutical composition for treating a disease or disorder associated with dysregulated EP300 activity, comprising a therapeutically effective amount of a bifunctional compound or a pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1 to 9.

14. wherein the disease or disorder is an EP300-dependent and MYC family-dependent cancer, or wherein the disease or disorder is high-risk neuroblastoma (NB), and optionally the pharmaceutical composition is for administration in combination with an additional therapeutically effective amount of an anti-NB agent, or wherein the disease or disorder is acute myeloid leukemia (AML), multiple myeloma (MM) or diffuse large B-cell lymphoma, or wherein the disease or disorder is a solid tumor, and optionally the solid tumor is melanoma, rhabdomyosarcoma, colon cancer, rectal cancer, gastric cancer, breast cancer or pancreatic cancer, the pharmaceutical composition according to claim 13.

15. The pharmaceutical composition according to claim 13 or 14, wherein the therapeutically effective amount of the bifunctional compound or a pharmaceutically acceptable salt or stereoisomer thereof is orally administered to the subject in the form of a tablet, a capsule, or a liquid.

16. The pharmaceutical composition according to any one of claims 13 to 15, wherein the bifunctional compound is administered to the subject in the form of a salt, and optionally the subject is human.

17. A method for isolating or detecting an EP300 bromodomain using the bifunctional compound according to claim 10, wherein the method comprises contacting lysed cells suspected of containing EP300 with the bifunctional compound and streptavidin immobilized on a carrier, isolating a complex formed by molecular and protein binding via biotin-streptavidin binding, and confirming the presence of EP300 in the complex, a method for isolating or detecting an EP300 bromodomain, optionally the carrier comprises beads, the method.

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