HDAC degraders

Abstract

The present disclosure provides compounds of formula (I). The compounds can be used to degrade the histone deacetylase (HDAC) family of enzymes, particularly HDAC1, HDAC2, and HDAC3, which are present in corepressor complexes. Thus, the compounds can be used to treat cancer. The present invention extends to pharmaceutical compositions containing these compounds and to the use of these compounds in therapy.

Description

[Technical field]

[0001] The present invention relates to compounds that can degrade the histone deacetylase (HDAC) family of enzymes, particularly HDAC1, HDAC2 and HDAC3, which are present in the corepressor complex.The present invention extends to pharmaceutical compositions that contain these compounds, and to the use of these compounds in therapy.The present invention describes how these compounds can be used to treat cancer. [Background technology]

[0002] The histone deacetylase (HDAC) family of enzymes, often referred to as epigenetic erasers, function by removing acetyl moieties from histone tails, thereby altering chromatin structure and gene expression. Currently, five HDAC inhibitors have been approved for clinical use in clinical trials to treat T-cell lymphoma and multiple myeloma along with other compounds. Humans contain 18 HDAC enzymes, 11 of which have a divalent zinc cation at the catalytic site, and seven sirtuins are dependent on NAD+ for their activity. Zn inhibitors such as valproic acid or SAHA (Zolinza) 2+ Inhibitors of the dependency enzymes are currently in clinical use to treat CTCL and bipolar disorder, however, these agents exhibit limited selectivity, and this lack of selectivity has been implicated as the cause of the debilitating side effects exhibited by patients using HDAC inhibitors.

[0003] HDAC1, HDAC2, and HDAC3 are localized in the nucleus, constitute approximately 50% of total cellular deacetylase activity, and are the most prominent HDACs in gene expression. They do not function as single entities, but exist in vivo as catalytic subunits of much larger multiprotein corepressor complexes such as Sin3, NuRD, CoREST, MiDAC, and NCoR. These complexes play a key role in integrating HDAC enzymes into specific regions of the genome and display functions in different cell types.

[0004] The present invention arose from the inventors' research to develop other compounds that target the HDAC family of enzymes. Summary of the Invention

[0005] According to a first aspect, there is provided a compound of formula (I), or a pharma- ceutically acceptable salt, solvate, tautomeric or polymorphic form thereof:

[0006] [ka]

[0007] During the ceremony, L is a linker having a backbone containing at least one group, the or each group being independently an optionally substituted C1-C 30 Alkylene, optionally substituted C2-C 30 Alkenylene, optionally substituted C2-C 30 Alkynylene, NR 3 , O, S, SO, SO2, optionally substituted C6-C 12 arylene, and optionally substituted 5-10 membered heteroarylene, wherein the backbone length of the linker is 7-50 atoms; R 1 is an E3 ligand; Each R 2 are independently halogen, OR 3 , N.R. 3 R 4 , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C 12 an aryl group, or a 5-10 membered heteroaryl group; R 3 and R 4 are independently H, C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; p is 0 or an integer from 1 to 4.

[0008] Said compound of formula (I) is a proteolytic targeting chimera (PROTAC). PROTAC is a heterobifunctional molecule that combines the ligand of protein of interest (POI) with the ligand of E3 ligand, and recruits E3 ligase to cause polyubiquitination and degradation of POI. Advantageously, compound of formula (I) can induce protein-protein interaction with HDAC enzyme and successful degradation. In particular, the inventors have found that compound of formula (I) can increase histone acetylation level and reduce cell viability in HCT116 colon cancer cell line, which is comparable to clinical candidate CI-994.

[0009] To the best of our knowledge, this is the first time that a selective benzamide HDAC inhibitor such as CI-994 has been taken, converted into a PROTAC, and shown to degrade HDAC1, HDAC2, and HDAC3. Given the significance and importance of HDAC drugs in diseases, and the advantages of PROTAC technology, the medical significance and potential utility may be very high.

[0010] The term "alkylene" as used herein refers to a divalent saturated straight chain hydrocarbon, unless otherwise specified. The terms "alkenylene" and "alkynylene" as used herein refer to a divalent olefinically unsaturated straight chain hydrocarbon or a divalent acetylenically unsaturated straight chain hydrocarbon, respectively, unless otherwise specified. The term "arylene" refers to a divalent aromatic 6-12 membered hydrocarbon group. The term "heteroarylene" refers to a divalent aromatic 5-10 membered ring system in which at least one ring atom is a heteroatom.

[0011] The or each of the optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, and optionally substituted heteroaryl is unsubstituted or is selected from the group consisting of halogen, oxo, OH, NH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3-10 membered heterocycle, C6-C 12The or each optionally substituted aryl may be unsubstituted or substituted with one or more of halogen, OH, NH, C1-C6 alkyl, C2-C6 alkenyl and / or C2-C6 alkynyl, C3-C6 cycloalkyl, 3-10 membered heterocycle, C6-C7 alkyl, C8-C9 alkyl, C9-C10 alkyl, C10-C10 alkyl, C10-C10 alkyl, C2-C10 alkyl, C2-C10 alkyl, C3 ...3-C10 alkyl, C3-C10 alkyl, C3-C10 alkyl, C3-C10 alkyl, C3-C 12 It may be substituted with one or more of aryl and / or 5-10 membered heterocycles.

[0012] The term "backbone of the linker" means

[0013] [ka]

[0014] and R 1 It will be understood that the bond refers to the shortest continuous chain of bond atoms between

[0015] In some embodiments, p is 1. Thus, the compound of formula (I) can be a compound of formula (Iai) or (Iaii):

[0016] [ka]

[0017] R 2 R can be a halogen or a 5- or 10-membered heteroaryl group. 2 In embodiments where R is a 5- or 10-membered heteroaryl group, it can be a 5- or 6-membered heteroaryl group. 2 R can be 1H-pyrrolyl, furanyl or thiophenyl. 2 In embodiments where R is a halogen, it may be fluorine, chlorine, iodine or bromine. 2 is fluorine or chlorine, most preferably fluorine.

[0018] Thus, the compound of formula (I) may be of formula (Iaiii) or of formula (Iaiv):

[0019] [ka]

[0020] In other embodiments, p is 0. Thus, the compound of formula (I) may be a compound of formula (Ib):

[0021] [ka]

[0022] It will be understood that an E3 ligand is a ligand for an E3 ligase. The E3 ligand may be for von Hippel-Lindau (VHL) E3 ubiquitin ligase, cereblon E3 ubiquitin ligase, cIAP1 E3 ubiquitin ligase or MDM2 E3 ubiquitin ligase, or a biologically active isoform or analog thereof. It will be understood that there are multiple possible binding positions for these E3 ligands, and the linker may bind the E3 ligand at any of the possible binding positions.

[0023] For example, R 1 may have the following structure:

[0024] [ka]

[0025] During the ceremony, X 1 ~X 7 and X 13 are NH or O, R 7is H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted 3-6 membered heterocycle, optionally substituted phenyl, or optionally substituted 5- or 6-membered heterocycle. The alkyl, alkenyl, alkynyl, cycloalkyl, heterocycle, phenyl, or heteroaryl may be unsubstituted or substituted with one or more of halogen, oxo, OH, or NH2. The halogen may be fluorine, chlorine, bromine, or iodine, preferably fluorine or chlorine, more preferably fluorine.

[0026] More preferably, R 1 is the following:

[0027] [ka]

[0028] Preferably, X 1 and X 2 is NH. Preferably, X 3 is O. Preferably, X 6 is O. Preferably, X 7 is NH. Preferably, X 13 is O.

[0029] Preferably, R 7 is optionally substituted C1-C3 alkyl, optionally substituted C2-C3 alkenyl, optionally substituted C2-C3 alkynyl, optionally substituted cyclopropyl, or optionally substituted 3-membered heterocycle. More preferably, R 7 is methyl or:

[0030] [ka]

[0031] It is.

[0032] Most preferably, R 1 teeth,

[0033] [ka]

[0034] It is.

[0035] The backbone of the linker may be 7-40 atoms in length, more preferably 8-30 atoms, 9-25 atoms, 10-20 atoms, or 11-15 atoms in length, and most preferably 13 atoms in length.

[0036] The backbone of the linker may be 7 to 40 atoms in length, more preferably 8 to 30 atoms, 9 to 25 atoms, 10 to 20 atoms, or 11 to 18 atoms in length, and most preferably 13 to 16 atoms in length.

[0037] As described in the examples, the inventors have obtained particularly promising results in vivo for compounds with longer linker lengths. This is surprising, since the in vivo results showed that molecules with shorter linkers are more active. Furthermore, the inventors would have expected that compounds with longer linkers would have difficulty passing through cell membranes. The fact that compounds with longer linker lengths are more active could not have been predicted.

[0038] L is -L 1 -L 2 where L 1 does not exist or -L 3 -L 4 -L 5 -L 6 - and L 2 -L 7 -L 8 -L 9 -L 10 -where: L 3is an optionally substituted C1 to C6 alkylene, an optionally substituted C2 to C6 alkenylene, or an optionally substituted C2 to C6 alkynylene; L 4 is NR 5 ,O.S.

[0039] [ka]

[0040] where the asterisk represents L 5 , or L 5 If there is no L 6 Indicates the attachment point to; L 5 is absent, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C6-C 12 arylene, or optionally substituted 5- to 10-membered heteroarylene; L 6 is an optionally substituted C6 to C 12 arylene, or optionally substituted 5- to 10-membered heteroarylene; L 7 is absent, optionally substituted C1-C6 alkylene, optionally substituted C2-C6 alkenylene, or optionally substituted C2-C6 alkynylene; L 8 does not exist or

[0041] [ka]

[0042] where the asterisk represents L 9 Indicates the attachment point to; L 9 is an optionally substituted C1 to C 20 Alkylene, optionally substituted C2-C 20Alkenylene or optionally substituted C2-C 20 alkynylene, and the skeleton of the alkylene, alkenylene or alkynylene group is O or NR 6 or may be interrupted by one or more heteroatoms selected from L 9 teeth,

[0043] [ka]

[0044] where the asterisk represents L 10 , or L 10 If not, R 1 Indicates the attachment point to; L 10 is absent, C(O) or:

[0045] [ka]

[0046] where the asterisk represents R 1 Indicates the attachment point to; L 11 is absent, optionally substituted C1 to C5 alkylene, optionally substituted C2 to C5 alkenylene, or optionally substituted C2 to C5 alkynylene; L 12 and L 13 are independently an optionally substituted C1 to C5 alkylene, an optionally substituted C2 to C5 alkenylene, or an optionally substituted C2 to C5 alkynylene; X 8 ~X 12 are independently O or NR 6 and; R 5 and R 6 are independently H, C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; m is an integer of 0, 1 to 10; and n is an integer from 1 to 10.

[0047] In some embodiments, L 1 exists.

[0048] L 3 can be an optionally substituted C1-C3 alkylene, an optionally substituted C2-C3 alkenylene, or an optionally substituted C2-C3 alkynylene. 3 can be -CH2-.

[0049] L 4 is NR 5 or

[0050] [ka]

[0051] Preferably, X 8 is NR 5 Preferably, X 9 is O. Preferably, R 5 is H.

[0052] L 5 can be an optionally substituted C1-C3 alkylene, an optionally substituted C2-C3 alkenylene, or an optionally substituted C2-C3 alkynylene. 5 can be -CH2-.

[0053] Or, L. 5 may be an optionally substituted phenyl, or an optionally substituted 5- or 6-membered heteroaryl. The 5- or 6-membered heteroaryl may be an optionally substituted pyridinyl, an optionally substituted pyridazinyl, an optionally substituted pyrimidinyl, or an optionally substituted triazinyl. The phenyl or heteroaryl may be unsubstituted.

[0054] Or, L. 5 may not be present.

[0055] L 6 may be an optionally substituted phenyl or an optionally substituted 5- or 6-membered heteroaryl. The 5- or 6-membered heteroaryl may be an optionally substituted pyrrolyl, an optionally substituted imidazolyl, an optionally substituted pyrazolyl, an optionally substituted triazolyl, an optionally substituted pyridinyl, an optionally substituted pyridazinyl, an optionally substituted pyrimidinyl or an optionally substituted triazinyl. The phenyl or heteroaryl may be unsubstituted or substituted with phenyl or a 5- or 6-membered heteroaryl.

[0056] Thus, in some embodiments, L 1 teeth:

[0057] [ka]

[0058] and the asterisk may be L 2 The attachment points to the

[0059] Or, L. 1 may not be present.

[0060] L 7 can be an optionally substituted C1-C3 alkylene or an optionally substituted C2-C3 alkylene. 7 can be -CH2-.

[0061] In some embodiments, L 7 does not exist.

[0062] L 8 teeth:

[0063] [ka]

[0064] Preferably, X 10 is NR 6 Preferably, R 6 is H.

[0065] In some embodiments, L 8 does not exist.

[0066] In some embodiments, L 9 is an optionally substituted C5 to C 15 Alkylene, optionally substituted C5-C 15 Alkenylene or optionally substituted C5-C 15 The backbone of the alkylene, alkenylene or alkynylene group must not be interrupted by any heteroatoms. More preferably, L 9 is an optionally substituted C8 to C 13 Alkylene, optionally substituted C8-C 13 Alkenylene or optionally substituted C8-C 13 Alkynylene, most preferably C 10 ~C 12 Thus, in some embodiments, L 9 Ha-(CH2) 11 --It could be.

[0067] Or, L. 9 is an optionally substituted C3 to C 10 Alkylene, optionally substituted C3-C 10 Alkenylene or optionally substituted C3-C 10 The backbone of the alkylene, alkenylene or alkynylene group must not be interrupted by any heteroatoms. More preferably, L 9is optionally substituted C5-C9 alkylene, optionally substituted C5-C9 alkenylene, or optionally substituted C5-C9 alkynylene, and most preferably C6-C8 alkylene. Thus, in some embodiments, L 9 can be -(CH2)7-.

[0068] Or, L. 9 is an optionally substituted C3 to C 15 Alkylene, optionally substituted C3-C 15 Alkenylene or optionally substituted C3-C 15 It may be alkynylene, and the backbone of the alkylene, alkenylene or alkynylene group is interrupted by one or more heteroatoms. More preferably, L 9 is an optionally substituted C5 to C 12 Alkylene, optionally substituted C5-C 12 Alkenylene or optionally substituted C5-C 12 Alkynylene, most preferably C7-C 10 Alkylene alkynylene, wherein the backbone of the alkylene, alkenylene or alkynylene group is interrupted by one or more heteroatoms. Preferably, the backbone of the alkylene, alkenylene or alkynylene group is interrupted by 1 to 4, more preferably 1 to 3, most preferably 1 to 2 heteroatoms. Preferably, the or each heteroatom is O.

[0069] In some embodiments, L 9 teeth:

[0070] [ka]

[0071] where v is an integer of at least 1, w is an integer of at least 2, and the asterisk represents L 10 , or L 10 If does not exist, R 1 The attachment points to the

[0072] In another embodiment, L 9 teeth:

[0073] [ka]

[0074] It can be. 11 may be independently absent, optionally substituted C1-C3 alkylene, optionally substituted C2-C3 alkenylene, or optionally substituted C2-C3 alkynylene. More preferably, L 11 is CH2. L 12 can be an optionally substituted C2-C3 alkylene, an optionally substituted C2-C3 alkenylene, or an optionally substituted C2-C3 alkynylene. In some embodiments, L 12 is -CH2CH2-. n is an integer from 1 to 5. In some embodiments, n can be 1, 2, or 3. L 13 may be independently an optionally substituted C1-C3 alkylene, an optionally substituted C2-C3 alkenylene, or an optionally substituted C2-C3 alkynylene. More preferably, L 13 is CH2. In some embodiments, m is 0. In other embodiments, m is 1.

[0075] In some embodiments, L 10 is C(O).

[0076] In another embodiment, L 10 does not exist.

[0077] Therefore, L 2 teeth:

[0078] [ka]

[0079] and the asterisk represents R 1 The attachment points to the

[0080] In a preferred embodiment, L 2 teeth:

[0081] [ka]

[0082] wherein The asterisk stands for R. 1 Indicates the attachment point to; q is an integer of at least 5; r is an integer of at least 4; s is an integer of at least 2; t is an integer of at least 1; u is an integer of at least 3; v is an integer of at least 1; and w is an integer of at least two.

[0083] In a preferred embodiment, L is

[0084] [ka]

[0085] where q, r, s, t, u, v and w are as defined above.

[0086] Preferably, q is an integer from 5 to 20, more preferably from 7 to 15, and most preferably from 10 to 12. In some embodiments, q is 5, 8, or 11. In a preferred embodiment, q is 11.

[0087] Preferably, r is an integer from 4 to 20, more preferably from 7 to 15, and most preferably from 9 to 11. In some embodiments, r is 4, 7, 10, or 12. In a preferred embodiment, r is 10. In other embodiments, r is 12.

[0088] Preferably, s is an integer of 2 to 10, more preferably an integer of 2 to 5. In a preferred embodiment, s is 2 or 3.

[0089] Preferably, t is an integer of 1 to 10, more preferably an integer of 1 to 5. In a preferred embodiment, t is 1 or 2.

[0090] Preferably, u is an integer from 2 to 15, from 3 to 12, from 4 to 10, or from 6 to 8. In some embodiments, u is 7.

[0091] Preferably, v is an integer from 1 to 13, 3 to 12, 4 to 10, or 6 to 9. In some embodiments, v is 6. In other embodiments, v is 9.

[0092] Preferably, w is an integer from 2 to 14, from 3 to 12, from 5 to 10, or from 7 to 9. In some embodiments, w is 8.

[0093] The compound of formula (I) may be a compound of formula (Ic), (Id), (Ie) or (If):

[0094] [ka]

[0095] where q, r, s and t are as defined above.

[0096] The compound of formula (I) may be a compound of formula (101) to (116):

[0097] [ka]

[0098] [ka]

[0099] [ka]

[0100] [ka]

[0101] The term "pharmaceutically acceptable salt" may be understood to refer to any salt of a compound provided herein that retains its biological properties and is not toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counterions known in the art. Such salts include, but are not limited to: (1) Hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, adepic acid, aspartic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, glutaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, picric acid, cinnamic acid, mandelic acid, phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanoic acid, acid addition salts formed with organic or inorganic acids, such as benzenesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphoric acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptone, 3-phenylpropionate, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, benzoic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, cyclohexylsulfamic acid, quinic acid, muconic acid, and such acids; or (2) A base addition salt formed when an acidic proton is present in the parent compound, which is one of the following: (a) replaced by a metal ion, for example an alkali metal ion, an alkaline earth ion or an aluminum ion, or an alkali metal or alkaline earth metal hydroxide, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, aluminum hydroxide, lithium hydroxide, zinc hydroxide, barium hydroxide, or ammonia; or (b) Interacts with organic bases such as aliphatic, alicyclic, or aromatic organic amines, such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, etc.

[0102] Pharmaceutically acceptable salts include sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like. When the compound contains a basic functional group, for example, hydrohalides such as hydrochlorides, hydrobromides, and hydroiodides, carbonates or hydrogen carbonates, sulfates or hydrogen sulfates, borates, phosphates, hydrogen phosphates, dihydrogen phosphates, pyroglutamates, sugar salts, stearates, and sulfamates. , Nitrate, Orotate, Oxalate, Palmitic Acid, Pamoic Acid, Acetic Acid, Trifluoroacetic Acid, Trichloroacetic Acid, Propionic Acid, Hexanoic Acid, Cyclopentylpropionic Acid, Glycolic Acid, Glutaric Acid, Pyruvic Acid, Lactic Acid, Malonic Acid, Succinic Acid, Tannic Acid, Tartaric Acid, Tosylic Acid, Sorbic Acid, Ascorbic Acid, Malic Acid, Maleic Acid, Fumaric Acid, Tartaric Acid, Camsylic Acid, Citric Acid, Cyclamic Acid, Benzoic Acid, Isethionic Acid, Essylic Acid, Formic Acid, 3-(4-Hydroxyethyl)-2-(2-methyl-1,2-propanediol, 1 ... (oxybenzoyl)benzoic acid, picric acid, cinnamic acid, mandelic acid, phthalic acid, lauric acid, methanesulfonic acid (mesylic acid), methylsulfuric acid, naphthylic acid, 2-napsylic acid, nicotinic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid (besylic acid), 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphor acid salt, camphorsulfonate, 4-methylbicyclo[ 2.2.2]-Oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethyl acetate, tert-butyl acetate, lauryl sulfate, gluceptic acid, gluconic acid, glucuronic acid, hexafluorophosphoric acid, hybenzic acid, benzoic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, cyclohexylsulfamic acid, quinic acid, muconic acid, xinofoic acid, and other non-toxic organic or inorganic acid salts.

[0103] Hemisalts of acids and bases can also be formed, such as hemisulfate salts.

[0104] The term "solvate" may be understood to refer to a compound provided herein or a salt thereof that further comprises a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0105] In a second aspect, there is provided a compound of formula (I) as defined in the first aspect, or a pharma- ceutically acceptable complex, salt, solvate, tautomer or polymorph thereof, for use in therapy.

[0106] In a third aspect, there is provided a compound of formula (I) or a pharma-ceutically acceptable complex, salt, solvate, tautomeric or polymorphic form thereof as defined in the first aspect for use in the treatment of cancer.

[0107] In a fourth aspect, there is provided a method of treating, preventing or ameliorating cancer in a subject, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of formula (I) as defined in the first aspect, or a pharma- ceutically acceptable complex, salt, solvate, tautomer or polymorph thereof.

[0108] The cancer may be a solid tumor or solid cancer. The cancer may be blood cancer, intestinal cancer, brain cancer, breast cancer, cervical cancer, endometrial cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. The blood cancer may be myeloma, leukemia, or lymphoma. The lymphoma may be cutaneous T-cell lymphoma (CTCL). The intestinal cancer may be colon cancer or rectal cancer. The brain tumor may be glioma or glioblastoma. The breast cancer may be BRCA-positive breast cancer. The breast cancer may be HER2-positive breast cancer or HER2-negative breast cancer. The breast cancer may be triple-negative breast cancer. The liver cancer may be hepatocellular carcinoma. The lung cancer may be non-small cell lung cancer or small cell lung cancer. The skin cancer may be melanoma.

[0109] It will be understood that the compounds of formula (I) described herein, or their pharmaceutically acceptable salts or solvates, can be used in pharmaceutical preparations that can be used in monotherapy (i.e., use of the inhibitor alone) to treat, ameliorate, or prevent cancer. Alternatively, the inhibitor or its pharmaceutically acceptable salts or solvates can be used as an adjunct or in combination with known therapies to treat, ameliorate, or prevent cancer.

[0110] The compound of formula (I) can be combined into a composition having many different forms, depending on the method in which the composition is used.Thus, for example, the composition can be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle solution, transdermal patch, liposomal suspension, or any other suitable form that can be administered to the person or animal in need of treatment.It will be understood that the vehicle of the pharmaceutical agent according to the present invention should be well tolerated by the subject to which it is administered.

[0111] The medicament containing the compound of formula (I) can be used in many ways. The composition containing the compound of formula (I) can be administered by inhalation (e.g., intranasal). The composition can also be formulated for topical use. For example, a cream or ointment can be applied to the skin.

[0112] The compound of formula (I) can also be incorporated into a sustained or delayed release device. Such a device can be inserted, for example, on or under the skin, and the drug can be released over a period of weeks or months. The device can be placed at least adjacent to the treatment site. Such a device can be particularly advantageous when long-term treatment with the inhibitor used according to the present invention is required, which usually requires frequent administration (e.g., at least daily injections).

[0113] The compounds of formula (I) and compositions containing the compounds can be administered to a subject by injection into the bloodstream or by direct injection into the bloodstream at the site in need of treatment, such as a cancerous tumor or adjacent thereto. Injection can be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), intradermal (bolus or infusion) or intramuscular (bolus or infusion).

[0114] In a preferred embodiment, the compound of formula (I) is administered orally. Thus, the compound of formula (I) may be contained within a composition that can be taken orally, for example, in the form of a tablet, capsule or liquid.

[0115] It will be understood that the amount of the compound of formula (I) required is determined by its biological activity and bioavailability, which depends on the mode of administration, the physicochemical properties of the compound of formula (I), and whether it is used as a monotherapy or in a combination therapy. The frequency of administration is also influenced by the half-life of the compound of formula (I) in the subject being treated. The optimal dose to be administered can be determined by those skilled in the art and varies depending on the specific inhibitor being used, the strength of the pharmaceutical composition, the mode of administration, and the progression of the cancer. Additional factors that depend on the specific subject being treated require adjustment of the dosage, such as the subject's age, weight, sex, diet, and time of administration.

[0116] The compound of formula (I) can be administered before, during, or after the onset of the cancer to be treated. The daily dose can be given in a single dose. Alternatively, the compound of formula (I) can be given two or more times a day, and given twice a day.

[0117] Generally, a daily dose of 0.01 μg / kg body weight to 500 mg / kg body weight of the compound of formula (I) can be used for the treatment, amelioration or prevention of cancer. More preferably, the daily dose is 0.01 mg / kg body weight to 400 mg / kg body weight, more preferably, 0.1 mg / kg body weight to 200 mg / kg body weight, and most preferably, about 1 mg / kg body weight to 100 mg / kg body weight.

[0118] The patient undergoing treatment takes one dose upon waking, followed by a second dose in the evening (if a two dose regimen) or doses at 3 or 4 hour intervals thereafter. Alternatively, a sustained release device may be used to provide the patient with an optimal dose of the inhibitor according to the invention without the need for repeated doses.

[0119] Known procedures, such as those conventionally used in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), may be used to form a specific formulation containing the inhibitor according to the present invention and a precise treatment regimen (such as daily dose and frequency of administration of the inhibitor).The inventors believe that they are the first to describe a pharmaceutical composition for treating cancer based on the use of the compound of formula (I) above.

[0120] Thus, in a fifth aspect, there is provided a pharmaceutical composition for treating cancer in a subject, said composition comprising a compound of formula (I) as defined in the first aspect, or a pharma- ceutically acceptable complex, salt, solvate, tautomeric or polymorphic form thereof, and a pharma- ceutically acceptable vehicle.

[0121] The pharmaceutical composition may be used for the therapeutic amelioration, prevention or treatment in a subject with cancer.

[0122] The pharmaceutical composition may further comprise a known therapeutic for treating, ameliorating, or preventing cancer.

[0123] The present invention also provides, in a sixth aspect, a method for preparing a composition according to the fifth aspect, comprising contacting a therapeutically effective amount of a compound of formula (I), or a pharma- ceutically acceptable complex, salt, solvate, tautomer or polymorph thereof, and a pharma- ceutically acceptable vehicle.

[0124] A "subject" may be a vertebrate, a mammal, or a livestock animal. Thus, the compounds, compositions and medicaments of formula (I) according to the present invention may be used to treat any mammal, such as livestock (e.g. horses), pets, or for other veterinary purposes. However, most preferably, the subject is a human.

[0125] A "therapeutically effective amount" of a compound of formula (I) is any amount that, when administered to a subject, is the amount of drug necessary to treat said cancer.

[0126] For example, the therapeutically effective amount of the compound of formula (I) used may be about 0.01 mg to about 800 mg, preferably about 0.01 mg to about 500 mg. The amount of the compound of formula (I) is preferably about 0.1 mg to about 250 mg, and most preferably about 0.1 mg to about 20 mg.

[0127] A "pharmaceutically acceptable vehicle" as referred to herein is any known compound, or combination of known compounds, known to those skilled in the art to be useful in formulating a pharmaceutical composition.

[0128] In one embodiment, the pharma- ceutically acceptable vehicle may be solid, and the composition may be in the form of a powder or tablet. A solid pharma- ceutically acceptable vehicle may contain one or more substances that may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet disintegrants. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid mixed with the finely divided active agent (i.e., inhibitor) according to the present invention. In tablets, the inhibitor may be mixed with a vehicle having the necessary compression properties in the appropriate proportions and compressed into the desired shape and size. The powders and tablets preferably contain up to 99% of the inhibitor. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes, and ion exchange resins. In other embodiments, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

[0129] However, the pharmaceutical vehicle may be liquid and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used to prepare solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The compound of formula (I) may be dissolved or suspended in a pharma- ceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture of both, or a pharma- ceutically acceptable oil or fat. The liquid vehicle may contain other suitable pharmaceutical excipients, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity regulators, stabilizers, or osmolality regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing the above-mentioned excipients, such as cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric alcohols, such as glycols, and polyhydric alcohols) and their derivatives, and oils (such as fractionated coconut oil and peanut oil). For parenteral administration, the vehicle can also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be halogenated hydrocarbon or other pharma- ceutically acceptable propellants.

[0130] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized, for example, by intramuscular, intrathecal, epidural, intraperitoneal, intravenous, and especially subcutaneous injection.The compound of formula (I) can be prepared as a sterile solid composition that can be dissolved or suspended at the time of administration using sterile water, physiological saline, or other suitable sterile injection medium.

[0131] The compounds of formula (I) and compositions of the present invention may be administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (an oleic acid ester of sorbitol and its anhydride copolymerized with ethylene oxide), and the like. The compounds of formula (I) used according to the present invention may also be administered orally in the form of a liquid or solid composition. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, and liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0132] Every feature described in this specification (including the accompanying claims, abstract and drawings), and / or every step of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0133] For a better understanding of the present invention, and to show how an embodiment of the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: [Brief description of the drawings]

[0134] [Figure 1] Figure 1 shows benzamide-based HDAC inhibitors. CI-994 shows selective inhibition of HDAC1, HDAC2, and HDAC3, inhibition of cell proliferation, induction of apoptosis, and antitumor activity. MS-275 is a selective inhibitor of HDAC1 and HDAC3 with antitumor activity, currently in Phase III clinical trials for breast cancer. MGCD0103 is an HDAC1, HDAC2, HDAC3, and HDAC11 inhibitor in clinical trials for solid tumors and non-small cell lung cancer. [Figure 2A]FIG. 2A is a schematic diagram showing that when CI-994 is located in the binding pocket of HDAC2, the acetyl group of CI-994 is exposed on the surface from the HDAC2 catalytic active site (PDB: 4LY1). [Figure 2B] 2B and 2C show the structures of compounds synthesized by the present inventors. [Figure 2C] 2B and 2C show the structures of compounds synthesized by the present inventors. [Figure 3A] FIG. 3A is a graph showing AMC-fluorescent histone deacetylase inhibition assay with LSD1-CoREST-HDAC complex. [Figure 3B] FIG. 3B shows histone 3 lysine 56 acetylation (H3K56Ac) levels in E14 mouse embryonic stem cells after 24 hours. CI-994=40 μM, panobinostat=30 nM [Figure 4A] Figure 4A shows immunoblots with HDAC1, HDAC2, and HDAC3 antibodies after 24 hours of treatment of HCT116 colon cancer cell lines with the indicated reagents. Values ​​represent percentages relative to DMSO control = 100%. [Figure 4B] FIG. 4B shows histone H3 lysine 9 acetylation levels 24 hours after the indicated treatments in HCT116 colon cancer cell lines. [Figure 4C] FIG. 4C shows that NC-PROTAC4 containing the inactive VHL diastereoisomer does not induce degradation of HCT116 cells. CI-994=40 μM, panobinostat=30 nM [Diagram 5] FIG. 5 shows fluorescence-activated cell sorting (FACS) data for compound 4 and CI-994 in the HCT116 colon cancer cell line. [Figure 6A] FIG. 6 shows the structures of further compounds synthesized by the inventors. [Figure 6B] FIG. 6 shows the structures of further compounds synthesized by the inventors. [Figure 7]FIG. 7 shows the effect of Compound 2 on HDAC1 / HDAC2 levels in HCT116 cells after 24 hours of treatment. [Figure 8] FIG. 8 shows the effect of other representative compounds on HDAC1 / HDAC2 in HCT116 cells after 24 hours of treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0135] Example 1: Synthesis of four preliminary proteolytic targeting chimeras (PROTACs) For PROTAC design, we selected benzamide-based HDAC inhibitors that show selectivity for HDAC1, HDAC2, and HDAC3. CI-994 (see Figure 1) has reported Ki values ​​of 0.41 μM for HDAC1, 0.75 μM for HDAC3, and approximately 100 μM for HDAC8. This inhibitor exhibits HDAC1, HDAC2, and HDAC3-related phenotypes, inhibiting cell proliferation, inducing apoptosis, and exhibiting broad antitumor activity in vitro and in vivo. CI-994 is in clinical trials for its antitumor properties, and the similar benzamides MS-275 and MGCD0103 (see Figure 1) are currently in Phase III clinical trials for breast cancer and non-small cell lung cancer. More recently, CI-994 has also been reported for its neuroprotective effects in mice after spinal cord injury, treating cognitive impairment, and reducing atrial fibrillation.

[0136] We functionalized CI-994 from the acetyl group of the phenyl moiety (Figure 2A) because the acetyl group of the similar benzamide inhibitor protrudes from the catalytic active site and is surface exposed in HDAC2 (see PROTAC1-PROTAC4 shown in Figure 2B and Figure 2C). Therefore, we hypothesized that functionalization at this position may maintain HDAC binding. As previous PROTAC studies have shown that the linker length plays an important role in inducing degradation, short alkyl linker lengths were prepared (n=6) and long linker lengths were prepared (n=12). The alkyl linker was selected to speed up the synthesis. Two different E3 ligands were also selected; von Hippel-Lindau (VHL) ligand and cereblon ligand as the choice of E3 ligand, as well as the length of the linker may also affect the success of protein-protein binding with E3 ligase and thus degradation.

[0137] Example 2: Preliminary PROTAC Evaluation In Vivo We evaluated their preliminary PROTACs in vitro using an established fluorescent deacetylase assay with the LSD1-CoREST-HDAC1 complex. We used this complex as a prototypical HDAC multiprotein complex to determine whether such heterobifunctional molecules, PROTAC1-4, could still engage HDAC enzymes in the context of an intact multiprotein complex. The IC of PROTAC1-PROTAC4 50 values ​​were determined alongside CI-994 as a positive control; as a negative control, we also synthesized a Boc-protected CI-994, compound 5 (see FIG. 3). Compound 5 is unable to chelate zinc at the HDAC active site and should not exhibit HDAC inhibition.

[0138] Consideration We found that CI-994 has an IC of approximately 0.5 μM, consistent with the literature. 50 We observed that the interim PROTACs, which have short linker lengths, all had IC values ​​directly comparable to CI-994 and did not exhibit HDAC inhibition (see Figure 3A). 50The CoREST complex engaged HDAC1 at 100% inhibition, whereas the longer linker lengths PROTAC2 and PROTAC4 still maintained inhibition but had a 10- to 15-fold reduction in inhibition compared to PROTAC1 and PROTAC3.

[0139] Example 3: Preliminary PROTAC Evaluation in Cells We proceeded to evaluate the effects of these compounds on HDAC activity in cells. In a previous study, we demonstrated that acetylated histone H3 Lys56 (H3K56ac) is a direct substrate of HDAC1 in embryonic stem (ES) cells.

[0140] Consideration To investigate the ability of PROTAC1-PROTAC4 to reduce the activity of HDAC1, HDAC2, and HDAC3 in cells, they began by measuring H3K56ac using quantitative Western blotting (see Figure 3B). CI-994 and the pan-HDAC inhibitor panobinostat were used as positive controls. H3K56 acetylation levels were increased with panobinostat and CI-994 as expected, and interestingly, only PROTACs with longer linker lengths, namely PROTAC2 and PROTAC4, increased acetylation to similar levels (see Figure 3B).

[0141] The inventors speculate that PROTAC1 and PROTAC3 are active in vitro but may not be able to reach cellular targets for HDAC inhibition or degradation.

[0142] The conformation that PROTAC2 and PROTAC4 reduce histone deacetylase activity in vitro and in cells prompted us to quantify HDAC1, HDAC2, and HDAC3 protein abundance. Although partial degradation was observed in ES cells (data not shown), degradation was more pronounced in the human colon cancer cell line HCT116. After 24 hours, degradation was observed in a dose-dependent manner (see Figure 4A). VHL-based PROTAC4 was a more effective degrader than Cereblon PROTAC2 (see Figure 7). HDAC1 and HDAC2 were almost completely degraded at 10 μM with PROTAC4, but HDAC3 levels were also reduced. At 1 μM, approximately 50% degradation was observed for HDAC1, HDAC2, and HDAC3. Interestingly, even 10 nM PROTAC4 did not restore full HDAC1 and HDAC2 levels.

[0143] Acetylation levels of histone H3 Lys9 (H3K9ac), another established residue for HDAC activity, were also measured in HCT116 cells after 24 h treatment with PROTAC4. PROTAC4 acetylation levels at 10 μM were highly elevated compared to DMSO control (Figure 4B), consistent with the reduction in HDAC1 and HDAC2 levels at 10 μM (Figure 4A). However, at 1 μM, including HDAC1, HDAC2, and HDAC3, H3K9 acetylation levels were similar to DMSO control, at levels of approximately 40–48%. This indicates that acetylation is greatly increased, at least in H3K9ac, indicating that near-complete HDAC degradation is necessary, and partial degradation is not sufficient. To confirm that HDAC degradation is occurring via the VHL-mediated proteasomal degradation pathway, we synthesized NC-PROTAC4 with an inactive diastereoisomer of VHL (Figure 4C). When compared side-by-side with PROTAC4, no degradation of HDAC1 and HDAC2 was observed, confirming that degradation occurs via the VHL-mediated E3 ligase pathway.

[0144] Example 4: Evaluation of the effect of PROTAC4 on the cell cycle of colon cancer HCT116 cell line using fluorescence-activated cell sorting (FAC) Next, we investigated the effect of PROTAC4 on the cell cycle in the colon cancer HCT116 cell line.

[0145] After 48 hours, significant cell death was observed with 10 μM PROTAC4 (Figure 5). At 40 μM, cell death after 48 hours was at a similar level to CI-994. The effect of PROTAC4 on cell viability was comparable to CI-994, but the IC of PROTAC4 on the LSD1-CoREST-HDAC1 complex in vitro was significantly lower. 50 It is important to note that near complete HDAC1 and HDAC2 degradation is observed with PROTAC4 at 10 μM, compared to 16.8 μM with CI-994 at 0.5 μM. Thus, the phenotype observed with PROTAC4 is most likely due to degradation rather than inhibition of the CoREST complex as well as other HDAC1- and HDAC2-containing complexes in cells.

[0146] Example 5: Synthesis of additional PROTACs The inventors further synthesized PROTAC5 to PROTAC10 (see Figures 2B, 2C, 6A, and 6B).

[0147] Example 6: Synthesis and Evaluation of Additional PROTACs The inventors further synthesized the following PROTACs:

[0148] [ka]

[0149] [ka]

[0150] [ka]

[0151] The compounds were screened at 0.1 μM, 1 μM and 10 μM in the HCT116 cell line. The maximum degradation observed was recorded, regardless of concentration. Screening of new compounds was performed in 6-well plates with 400,000 cells per well, 5 mL medium to increase throughput. Representative compounds are shown in Figure 8.

[0152] The activity of the compounds was analyzed and compared to PROTAC4, and the results are shown in the table below. CI-994 and BocCI-994 serve as positive and negative controls for the CoREST, NuRD, MiDAC, and SMRT assays, respectively.

[0153] IC of PROTAC4 50 Note that values ​​differ from those in Figure 3 because the assays were performed under different conditions.

[0154] [Table 1]

[0155] [Table 2]

[0156] Consideration We found that CoREST, NuRD, MiDAC and SMRT complexes had comparable IC to CI-994. 50 In addition, when tested in cells, the compounds were found to also be HDAC degraders.

[0157] Without wishing to be bound by theory, the inventors note that if low degradation is observed, this may indicate that the PROTAC targets a specific complex. For example, the thiophene group in the head group of PROTAC22 is believed to make the PROTAC selective for CoREST. Therefore, the relatively low % HDAC degradation observed with this compound may be due to this selectivity.

[0158] conclusion Although the PROTAC approach has been applied to many protein targets, importantly, not all proteins are prone to degradation. This has been observed with PROTACs based on nonselective pan-kinase inhibitors. Uncomplexed, cytoplasmically localized HDAC6 has also recently been identified as a preferential target for degradation by hydroxamic acid-based PROTACs. We identified degraders of nuclear-localized HDAC1, HDAC2, and HDAC3, which are components of a multiprotein corepressor complex. Near-complete HDAC1, HDAC2, and HDAC3 degradation is required to significantly affect the acetylation levels of specific HDAC markers in cells. However, potential complex selective degraders predict overall acetylation levels, and specific HDAC acetylation markers, which may not be increased compared to pan-HDAC inhibitors, but may show higher efficacy or reduced side effects for certain disease-associated HDAC corepressor complexes. Unexpectedly, we found that the length of the alkyl linker is important for cell permeability. Compounds 1 and 3, despite having lower molecular weights than compounds 2 and 4 and being better in vitro HDAC inhibitors, failed to alter histone acetylation levels in cells. The length and chemical nature of the PROTAC linker likely have a major impact on activity in cells. HDAC degradation as described above may provide an alternative strategy to chemical probes and therapeutics of key HDAC corepressor complexes.

[0159] material and method General method Starting materials and solvents used were purchased from commercially available sources and used without further purification. Anhydrous THF and anhydrous DCM were dried using an Innovative Technology inc. PureSolv solvent purification system. All chemical names have been generated using ChemDraw Professional. All reactions were stirred and carried out under nitrogen unless otherwise stated. All reactions were monitored using TLC run on aluminum supported silica. Preparative column chromatography and flash column chromatography using a Biotage Isolera purification system were carried out using silica gel 60 (230-400 mesh).

[0160] Analytical and semi-preparative HPLC were performed on a ThermoFisher Ultimate 3000 system equipped with Chromeleon software on a Phenomenex Luna C18 column. Method 1, A = H2O, B = CH3CN, 5-100% B, 10 mL / min flow rate, 45 min gradient. Method 2, A = 0.1% TFA in H2O, B = 0.1% TFA in CH3CN, 5-100% B, 10 mL / min flow rate, 45 min gradient.

[0161] Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker 500 ( 1 H, 500MHz; 13 C 125MHz) or Bruker400( 1 H, 400MHz; 13 C 100MHz instrument was used, and deuterated solvent -CDCl3 (δ H =7.26ppm,δ C =77.00 ppm), DMSO-d6(δ H =2.50ppm,δ C =39.51 ppm), CD3OD(δ H =3.31ppm,δ C = 49.15 ppm), or CD3CN(δ H =1.94ppm,δ C= 1.39, 118.69 ppm) at ambient temperature. Peak selection, integration and calculation of coupling constants were performed using ACDLabs software (Chemsketch and Spectrus Processor). High-resolution mass spectra (HRMS) were recorded on a Water Aquity XEVO Q ToF machine and measured at m / z.

[0162] General Method A. DIPEA (1.5 equiv.) was added to a solution of the substituted aniline (1 equiv.) in dry DCM (2.5 mL / mmol) at 0° C., followed by 4-nitrobenzoyl chloride (1.1 equiv.) as a solution in dry DCM. The mixture was stirred at 0° C. for 30 min and then at room temperature overnight. The reaction mixture was diluted with DCM and then washed with saturated NaHCO4, 1M HCl and saturated NaCl. The organic layer was then dried over Na2SO4 and concentrated in vacuo before being purified as appropriate to give the desired compound.

[0163] General Method B. Triethylamine (3 equiv.) was added to a solution of 4-aminobenzamide starting material (1 equiv.) in dry THF (10 mL / mmol) at 0° C., followed by dropwise addition of acetyl chloride (1.2 equiv.). The mixture was stirred at 0° C. for 30 min and then at room temperature for 2 h. The reaction mixture was concentrated in vacuo to give the corresponding crude product, which was purified by chromatography to give the desired compound.

[0164] General Method C. TFA (20 equiv.) was added to a stirred solution of Boc-protected HDAC inhibitor / inhibitor-linker (1 equiv.) in DCM (10 mL / mmol) and the resulting reaction mixture was stirred at room temperature for 6 h. The reaction mixture was concentrated in vacuo, dissolved in MeOH (0.1 M), stirred in MP-carbonate resin (3.02 mmol / g loading capacity) for 2-3 h, and then filtered. The filtrate was concentrated in vacuo to give the desired HDAC inhibitor / inhibitor-linker.

[0165] General Method D. To a solution of dicarboxylic acid (1 equiv.) in 1,4-dioxane / DMF (1:1, 4 mL / mmol) was added benzyl bromide (1 equiv.) followed by NaHCO3 (1 equiv.). The resulting suspension was heated at 90° C. overnight. The reaction mixture was allowed to cool to room temperature and then concentrated under reduced pressure. The crude residue was then suspended in EtOAc and washed with saturated NaCl and water. The organic phase was dried over MgSO4, filtered and concentrated in vacuo to give the corresponding crude product, which was purified by chromatography to give the desired compound.

[0166] General Method E. To a solution of the acid linker (1.1-1.3 equiv.) in anhydrous DMF (10 mL / mmol) at 0°C, DIPEA (3 equiv.) and HATU (1.3-1.5 equiv.) were added. The reaction mixture was stirred for 15 min, after which a solution of the amine (1 equiv.) in DMF was slowly added and the resulting solution was stirred at room temperature overnight. The reaction mixture was diluted with EtOAc and then washed with saturated NaHCO3 and saturated NaCl. The organic layer was dried over MgSO4, filtered and concentrated in vacuo to give the corresponding crude product, which was purified by chromatography to give the desired compound.

[0167] General Method F. To a solution of benzyl ester protected HDACi-linker conjugate (1 equiv.) in THF was added 10% Pd / C (10% wt). The reaction flask was filled with nitrogen and evacuated three times using a Schlenk line, after which a balloon of hydrogen was added and the resulting mixture was stirred vigorously for 4-18 h. The hydrogen balloon was removed and the flask was flushed with nitrogen. The reaction mixture was filtered through a glass microfiber filter paper and the filtrate was concentrated in vacuo to give the desired compound.

[0168] General Method G. To a solution of HDACi-linker acid (1.2 equiv.) in dry DMF (1 mL) at 0°C, DIPEA (3 equiv.) and HATU (1.3 equiv.) were added. After the reaction mixture was stirred for 15 min, a solution of (4R)-3-methyl-L-valyl-4-hydroxy-N-[[4-(4-methyl-5-thiazolyl)phenyl]methyl]-L-prolinamide hydrochloride (VH_032 amine, 0.08-0.10 mmol) in DMF (1 mL) was slowly added and the resulting solution was stirred at room temperature for 16 h. The reaction mixture was diluted with EtOAc (10 mL) and then washed with saturated NaHCO3 (2 x 5 mL) and saturated NaCl (2 x 5 mL). The organic layer was dried over MgSO4, filtered and concentrated in vacuo to give the corresponding crude product, which was purified by chromatography to give the desired compound.

[0169] General Method H. TFA (0.4 mL or 20 equiv.) was added to a stirred solution of Boc-protected PROTAC (1 equiv.) in DCM (2 mL) and the resulting reaction mixture was stirred at room temperature for 4-6 h. The reaction mixture was concentrated in vacuo, dissolved in MeOH (2 mL), stirred in MP-carbonate resin (3.02 mmol / g loading capacity) for 2-3 h, and then filtered. The filtrate was concentrated in vacuo and the resulting solid was dissolved in MeCN:H2O (1:1) and lyophilized to remove residual TFA impurities to give the final PROTAC.

[0170] General Method I. A mixture of E3 ligand phenol (1 equiv.), alkyl bromide (1 equiv.) and K2CO3 (3 equiv.) in dry DMF (0.8 mL) was stirred overnight at 70° C. The reaction was concentrated in vacuo to give the corresponding crude product, which was purified by chromatography to give the desired compound.

[0171] General Method J. To a solution of 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic acid (CRBN acid, 1 eq.) in anhydrous DMF (2 mL) was added DIPEA (3 eq.) and HATU (1.2 eq.) at 0° C. The reaction mixture was stirred for 15 min, after which a solution of HDACi-linker amine (1 eq.) in DMF (1 mL) was slowly added and the resulting solution was stirred at room temperature for 18 h. The reaction mixture was diluted with EtOAc (10 mL) and then washed with saturated NaHCO3 (2×10 mL) and saturated NaCl (2×10 mL). The organic layer was dried over MgSO4, filtered and concentrated in vacuo to give the corresponding crude product, which was purified by chromatography to give the final PROTAC.

[0172] General Method K. To a solution of HDAC i-linker acid (1.2 equiv.) in anhydrous DMF (1 mL) at 0° C. was added DIPEA (3 equiv.) and HATU (1.3 equiv.). The reaction mixture was stirred for 15 min, after which tert-butyl ((S)-1-(((S)-2-((2S,4S)-4-amino-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate hydrochloride (A 410099.1 amine, 0.04 mmol) in DMF (1 mL) was added slowly and the resulting solution was stirred at room temperature for 16 h. The reaction mixture was diluted with EtOAc (10 mL) and then washed with saturated NaHCO3 (2 x 5 mL) and saturated NaCl (2 x 5 mL). The organic layer was dried over MgSO4, filtered and concentrated in vacuo to give the corresponding crude product, which was purified by chromatography to give the desired compound.

[0173] Synthesis of HDAC inhibitors (HDACi)

[0174] [ka]

[0175] tert-Butyl (2-aminophenyl)carbamate (2): A solution of Boc2O (6.05 g, 27.7 mmol) in THF (50 mL) was added dropwise over 3 h to a solution of 7 (3.00 g, 27.7 mmol) and triethylamine (4.64 mL, 33.3 mmol) in THF (25 mL), after which the mixture was stirred at room temperature for 15 h. The reaction mixture was concentrated in vacuo to give a grey crystalline solid, which was then redissolved in EtOAc (50 mL). This solution was washed with water (2 x 30 mL) and saturated brine (2 x 30 mL), filtered over Na2SO4, and then concentrated in vacuo to give a yellow / grey solid. The crude solid was purified by column chromatography (solid load, 10-25% EtOAc in hexanes) to give 8 (4.72 g, 22.5 mmol, 82% yield) as a yellow / grey solid. HRMS (ESI) m / z: [M+Na] + calculated for C 11 H 16 N2O2Na: 231.1109, found 231.1112. tert-Butyl (5-fluoro-2-nitrophenyl)carbamate (4): A solution of 2 (4.61 g, 19.2 mmol) in THF (5 mL) was added dropwise to a solution of 3 (3.00 g, 19.2 mmol), DMAP (59 mg, 0.48 mmol) and triethylamine (1.54 mL, 11.06 mmol) in THF (35 mL) at 0° C., then the mixture was stirred at 80° C. for 15 h. The reaction mixture was concentrated in vacuo, then redissolved in EtOAc (100 mL) and 2M HCl (100 mL) was added. The EtOAc layer was collected, then the aqueous layer was extracted with additional EtOAc (2×100 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give an orange oil (5.10 g). The crude product was purified by column chromatography (10-20% EtOAc in hexanes) to give 4 (2.77 g, 10.7 mmol, 56% yield) as a yellow crystalline solid. HRMS (ESI, direct infusion) m / z: [MH] - calculated for C 11 H 12N2O4F: 255.0781, found 255.0780. tert-Butyl (2-amino-5-fluorophenyl)carbamate (5): To a solution of 4 (1.68 g, 6.56 mmol) in MeOH (50 mL) was added 10% Pd / C (170 mg). The reaction flask was filled with nitrogen and evacuated three times using a Schlenk line, after which a balloon of hydrogen was added and the resulting mixture was stirred vigorously for 4.5 h. The hydrogen balloon was removed and the flask was flushed with nitrogen. The reaction mixture was filtered through a glass microfiber filter, then the filtrate was concentrated in vacuo to give 5 (1.43 g, 6.26 mmol, 96% yield) as a beige solid. MS (ESI) m / z: 227 [M+H] + . tert-Butyl (4-bromo-2-nitrophenyl)carbamate (7): A solution of Boc2O (2.21 g, 10.14 mmol) in THF (5 mL) was added dropwise to a solution of 6 (2.00 g, 9.22 mmol), DMAP (0.113 g, 0.92 mmol) and triethylamine (1.54 mL, 11.06 mmol) in THF (35 mL) at 0 °C, then the mixture was stirred at 60 °C for 16 h. The reaction mixture was concentrated in vacuo and then purified by column chromatography (0-50% EtOAc in hexanes) to give 7 (1.524 g, 4.76 mmol, 47% yield) as a yellow crystalline solid. HRMS (ESI) m / z: [(M-Boc)+H] + calculated for C6H6BrN2O4( 81 Br): 218.9592, found 218.9594. tert-Butyl (2-nitro-4-(thiophen-2-yl)phenyl)carbamate (8): To a solution of DME / water (2:1, 36 mL) was added thiophen-2-ylboronic acid (0.77 g, 6.05 mmol), 7 (1.60 g, 5.05 mmol), Na2CO3 (0.80 g, 7.57 mmol), and Pd(PPh3)4 (0.38 g, 0.33 mmol). The resulting mixture was vigorously stirred at 110 °C for 16 h. The reaction mixture was further diluted with water (40 mL) and then the product was extracted with EtOAc (3 x 50 mL). The organic layers were combined, washed with water (2 x 70 mL), dried over MgSO4, filtered, and concentrated in vacuo to give a brown solid (1.920 g). The crude product was purified by column chromatography (dry load, 0-50% EtOAc in hexanes) to give 8 (1.21 g, 3.74 mmol, 74% yield) as an orange crystalline solid. HRMS (ESI) m / z: [MH] - calculated for C 15 H 15 N2O4S: 319.0753, found 319.0753. tert-Butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate (9): To a solution of 8 (1.181 g, 3.69 mmol) in MeOH (15 mL) was added 10% Pd / C (0.120 g). The reaction flask was filled with nitrogen and evacuated three times using a Schlenk line, after which a balloon of hydrogen was added and the resulting mixture was stirred vigorously for 4 h. The hydrogen balloon was removed and the flask was flushed with nitrogen. The reaction mixture was filtered through a glass microfiber filter and the filtrate was then concentrated in vacuo to give 9 (1.057 g, 3.60 mmol, 98% yield) as a brown solid. HRMS (ESI) m / z: [M+H] + calculated for C 15 H 19 N2O2S: 291.1167, found 291.1167. tert-Butyl (2-(4-nitrobenzamido)phenyl)carbamate (10a): Following general procedure A, 10a was obtained from 2 (4.17 g, 20.0 mmol) and 4-nitrobenzoyl chloride (4.09 g, 22.0 mmol). The crude product was triturated in EtOH and then filtered to give 10a (5.52 g, 15.3 mmol, 76% yield) as a pale yellow solid. HRMS (ESI) m / z: [M+Na] + calculated for C 18 H 19 N3O5Na: 380.1222, found 380.1223. tert-Butyl (5-fluoro-2-(4-nitrobenzamido)phenyl)carbamate (10b): Following general procedure A, 10b was obtained from 5 (1.27 g, 5.60 mmol) and 4-nitrobenzoyl chloride (1.15 g, 6.17 mmol). The crude product was purified by column chromatography (0-50% EtOAc in hexanes) to give 10b (1.87 g, 4.93 mmol, 88% yield) as a fluffy light brown solid. HRMS (ESI) m / z: [M+Na] + calculated for C 18 H 18 N3O5FNa: 398.1128, found 398.1125. tert-Butyl (2-(4-nitrobenzamido)-4-(thiophen-2-yl)phenyl)carbamate (10c): Following general procedure A, 10c was obtained from 9 (400 mg, 1.38 mmol) and 4-nitrobenzoyl chloride (284 mg, 1.53 mmol). The crude product was triturated in EtOH and then filtered to give 10c (459 mg, 1.04 mmol, 76% yield) as a pale green solid. HRMS (ESI) m / z: [M+H] + calculated for C 22 H 22 N3O5S: 440.1280, found 440.1280. tert-Butyl (2-(4-aminobenzamido)phenyl)carbamate (11a): To a solution of 10a (5.52 g, 15.3 mmol) in MeOH / THF (1:1, 100 mL) was added 10% Pd / C (0.55 g). The reaction flask was filled with nitrogen and evacuated three times using a Schlenk line, after which a balloon of hydrogen was added and the resulting mixture was stirred vigorously for 18 h. The hydrogen balloon was removed and the flask was flushed with nitrogen. The reaction mixture was filtered through Celite, which was then washed with additional MeOH (3×50 mL) and the filtrate was concentrated in vacuo to give 11a (5.23 g, 15.3 mmol, 100% yield) as a fluffy white crystalline solid. HRMS (ESI) m / z: [M+Na] + calculated for C 18 H 21 N3O3Na: 350.1481, found 350.1486. tert-Butyl (2-(4-aminobenzamido)-5-fluorophenyl)carbamate (11b): To a solution of 10b (1.525 g, 4.063 mmol) in MeOH (100 mL) was added 10% Pd / C (0.155 g). The reaction flask was filled with nitrogen and evacuated three times using a Schlenk line, after which a balloon of hydrogen was added and the resulting mixture was stirred vigorously for 7 h. The hydrogen balloon was removed and the flask was flushed with nitrogen. The reaction mixture was filtered through a glass microfiber filter and the filtrate was then concentrated in vacuo to give 11b (1.335 g, 3.827 mmol, 94% yield) as a light grey crystalline solid. HRMS (ESI) m / z: [M+H] + calculated for C 18 H 21 N3O3F: 346.1567, found 346.1563. tert-Butyl (2-(4-aminobenzamido)-4-(thiophen-2-yl)phenyl)carbamate (11c): To a solution of 10c (0.333 g, 0.76 mmol) in MeOH / DCM (1:1, 120 mL) was added SnCl2 (0.863 g, 4.55 mmol) and the resulting mixture was stirred at room temperature for 1 week. The reaction mixture was cooled to 0 °C and then saturated Na2CO3 (40 mL) was added slowly. The product was extracted with DCM (4 x 40 mL) and then the organic layers were combined and washed with brine (2 x 80 mL). The organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give 11c (0.283 g, 0.62 mmol, 82% yield) as a yellow crystalline solid. HRMS (ESI) m / z: [M+H] + calculated for C 22 H 24 N3O3S: 410.1538, found 410.1530. tert-Butyl (2-(4-acetamidobenzamido)phenyl)carbamate (12a): Following general procedure B, 12a was obtained from 11a (200 mg, 0.611 mmol) and acetyl chloride (0.052 mL, 0.733 mmol). The crude product was purified by column chromatography (dry load, 100% EtOAc) to give 12a (193 mg, 0.517 mmol, 85% yield) as a white solid. HRMS (ESI) m / z: [M+Na] + calculated for C 20 H 23 N3O4Na: 392.1586, found 392.1586. tert-Butyl (2-(4-acetamidobenzamido)-5-fluorophenyl)carbamate (12b): Following general procedure B, 12b was obtained from 11b (95.7 mg, 0.277 mmol) and acetyl chloride (0.024 mL, 0.333 mmol). The crude product was purified by column chromatography (dry load, 50-100% EtOAc) to give 12b (101.0 mg, 0.258 mmol, 93% yield) as a pale yellow / brown solid. HRMS (ESI) m / z: [M+H] +calculated for C 20 H 23 FN3O4: 388.1673, found 388.1672. tert-Butyl (2-(4-acetamidobenzamido)-4-(thiophen-2-yl)phenyl)carbamate (12c): Following general procedure B, 12c was obtained from 11c (96.6 mg, 0.236 mmol) and acetyl chloride (0.020 mL, 0.283 mmol). The crude product was purified by column chromatography (0-100% EtOAc) to give 12c (72.8 mg, 0.160 mmol, 68% yield) as a pale yellow / green solid. HRMS (ESI) m / z: [M+H] + calculated for C 24 H 26 N3O4S: 452.1644, found 452.1643. 4-Acetamido-N-(2-aminophenyl)benzamide (CI-994): Boc deprotection of 12a (47.5 mg, 0.129 mmol) was carried out following general procedure C to give CI-994 (35.6 mg, 0.126 mmol, 97% yield) as a yellow / white solid. HRMS (ESI) m / z: [M+H] + calculated for C 15 H 16 N3O2: 270.1243, found 270.1247. 4-Acetamido-N-(2-amino-4-fluorophenyl)benzamide (BRD3308): Boc deprotection of 12b (87.0 mg, 0.225 mmol) was carried out following general procedure C to give BRD3308 (59.3 mg, 0.202 mmol, 90% yield) as a yellow / brown solid. HRMS (ESI) m / z: [M+H] + calculated for C 15 H 15 FN3O2: 288.1148, found 288.1139. 4-Acetamido-N-(2-amino-5-(thiophen-2-yl)phenyl)benzamide (Cpd-60): Boc deprotection of 12c (48.2 mg, 0.107 mmol) was carried out according to general procedure C to give Cpd-60 (35.5 mg, 0.100 mmol, 93% yield) as a pale yellow / green solid. HRMS (ESI) m / z: [M+H] + calculated for C 19 H 18 N3O2S: 352.1120, found 352.1120. Synthesis of the linker

[0176] [ka]

[0177] 9-(Benzyloxy)-9-oxononanoic acid (14a): Following general procedure D, 14a was obtained from 13a (2.00 g, 10.63 mmol) and benzyl bromide (1.26 mL, 10.63 mmol). The crude product was purified by column chromatography (50% EtOAc in hexanes) to give 14a (1.083 g, 3.85 mmol, 36% yield) as a clear, colorless oil. HRMS (ESI) m / z: [M+Na] + calculated for C 16 H 22 O4Na: 301.1416, found 301.1415. 12-(Benzyloxy)-12-oxododecanoic acid (14b): Following general procedure D, 14b was obtained from 13b (2.00 g, 8.68 mmol) and benzyl bromide (1.03 mL, 8.68 mmol). The crude product was purified by column chromatography (50% EtOAc in hexanes) to give 14b (0.998 g, 2.99 mmol, 34% yield) as a clear, colorless oil. HRMS (ESI) m / z: [M+Na] + calculated for C 19 H 28 O4Na: 343.1885, found 343.1887. 14-(Benzyloxy)-14-oxotetradecanoic acid (14c): Following general procedure D, 14c was obtained from 13c (1.50 g, 5.81 mmol) and benzyl bromide (0.69 mL, 5.81 mmol). The crude product was purified by column chromatography (50% EtOAc in hexanes) to give 14c (0.628 g, 1.78 mmol, 31% yield) as a clear, colorless oil. HRMS (ESI) m / z: [M+H] + calculated for C 21 H 33 O4: 349.2379, found 349.2374. 3-Oxo-1-phenyl-2,5,8,11-tetraoxatridecan-13-oic acid (16): 16 was obtained from 15 (1.00 g, 3.15 mmol) and benzyl bromide (0.37 mL, 3.15 mmol) following general procedure D. The crude product was purified by column chromatography (0-10% MeOH in DCM) to give 16 (0.484 g, 1.53 mmol, 49% yield) as a clear yellow oil. HRMS (ESI) m / z: [M+H] + calculated for C 15 H 21 O 7: 313.1287, found 313.1290. Bi-tert-butyl-2,2'-(hexane-1,6-diylbis(oxy))diacetate (17): Hexane-1,6-diol (1.50 g, 12.69 mmol) was dissolved in DCM (52 ml) and then tert-butyl-2-bromoacetate (15.0 ml, 102.28 mmol) was added dropwise followed by tetra-n-butylammonium bromide (4.50 g, 13.96 mmol). The reaction mixture was then cooled to 0°C before NaOH (37% w / w, 52 ml) was added. The reaction mixture was then stirred vigorously at room temperature for 16 hours. The crude reaction mixture was biphasic with the organic layer (upper layer) being yellow. The organic layer was separated and then the aqueous layer was washed with DCM (3 x 15 ml). The organic layers were combined and dried over MgSO4 before being filtered and concentrated in vacuo to give a clear pale yellow oil (6.87 g) which slowly crystallized. The crude product was purified by column chromatography (1% MeOH in DCM) to give 17 (1.015 g, 2.90 mmol, 23% yield) as a clear, colorless oil. HRMS (ESI) m / z: [M+Na] + calculated for C 18 H 34 O6Na: 369.2253, found 369.2255. Di-tert-butyl-2,2'-(nonane-1,9-diylbis(oxy))diacetate (18) and tert-butyl-2-((9-hydroxynonyl)oxy)acetate (19): Nonane-1,9-diol (2.00 g, 12.48 mmol) was dissolved in DCM (50 ml) followed by dropwise addition of tert-butyl-2-bromoacetate (5.53 ml, 37.44 mmol) followed by addition of tetra-n-butylammonium bromide (4.43 g, 13.73 mmol). The reaction mixture was then cooled to 0°C before adding NaOH (37% w / w, 50 ml). The reaction mixture was then stirred vigorously at room temperature for 16 hours. The crude reaction mixture was biphasic with the organic layer (upper layer) being pale yellow in color. The organic layer was separated and the aqueous layer was then washed with DCM (3 x 15 ml). The organic layers were combined and dried over MgSO4, then filtered and concentrated in vacuo to give a clear pale yellow oil. The crude product was purified by column chromatography (10-30% EtOAc in hexanes) to give 18 (2.80 g, 7.14 mmol, 57% yield) and 19 (1.387 g, 5.01 mmol, 40% yield) as a clear, colorless oil. HRMS (ESI) m / z: [M+Na] + calculated for C 15 H 30 O4Na: 297.2042, found 297.2046. 2,2'-(Hexane-1,6-diylbis(oxy))diacetic acid (20a): To a stirred solution of 17 (0.460 g, 1.33 mmol) in DCM (2 mL) was added TFA (0.4 mL) and the resulting reaction mixture was stirred at room temperature for 4 h. The reaction mixture was concentrated in vacuo to give 20a (319.0 mg, 1.36 mmol, 99% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 10 H 19 O6: 235.1182, found 235.1182. 2,2'-(nonane-1,9-diylbis(oxy))diacetic acid (20b): To a stirred solution of 18 (1.55 g, 3.99 mmol) in DCM (3 mL) was added TFA (3 mL) and the resulting reaction mixture was stirred at room temperature for 4.5 h. The reaction mixture was concentrated in vacuo to give 20b (1.09 g, mmol, 3.95 mmol, 99% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 13 H 25 O6: 277.1651, found 277.1656. 2-((6-(2-(benzyloxy)-2-oxoethoxy)hexyl)oxy)acetic acid (21a): Following general procedure D, 21a was obtained from 20a (0.304 g, 1.30 mmol) and benzyl bromide (0.15 mL, 1.30 mmol). The crude product was purified by column chromatography (0-100% EtOAc in hexanes) to give 21a (0.110 g, 0.34 mmol, 26% yield) as a yellow oil. HRMS (ESI) m / z: [M+H] + calculated for C 17 H 25 O6: 325.1651, found 325.1659. 2-((9-(2-(benzyloxy)-2-oxoethoxy)nonyl)oxy)acetic acid (21b): Following general procedure D, 21b was obtained from 20b (1.047 g, 3.79 mmol) and benzyl bromide (0.45 mL, 3.79 mmol). The crude product was purified by column chromatography (0-50% EtOAc in hexanes) to give 21b (0.463 g, 1.24 mmol, 33% yield) as a clear pale yellow oil. HRMS (ESI) m / z: [M+H] + calculated for C 20 H 31 O6: 367.2121, found 367.2121. 9-(2-(tert-butoxy)-2-oxoethoxy)nonanoic acid (22): Pyridinium dichromate (685.5 mg, 1.822 mmol) was added portionwise to a solution of 19 (100.0 mg, 0.364 mmol) in dry DMF (2 mL) and stirred at room temperature for 6 h. The reaction mixture was diluted with 10% citric acid solution (30 mL) and extracted with EtOAc (4×20 mL). The combined organic layers were washed with saturated NaCl (3×30 mL), dried over Na2SO4, filtered, and concentrated in vacuo to give 22 (109.8 mg, 0.361 mmol, 99% yield) as a pale yellow oil. HRMS (ESI -ve, Direct Infusion) m / z: [MH] - calculated for C 15 H 27 O5: 287.1858, found 287.1862. Benzyl-9-(2-(tert-butoxy)-2-oxoethoxy)nonanoate (23): To a solution of 22 (215.3 mg, 0.747 mmol) in 1,4-dioxane / DMF (1:1, 8 mL) was added benzyl bromide (0.10 mL, 0.821 mmol) followed by NEt3 (0.12 mL, 0.885 mmol) and the resulting mixture was stirred at room temperature for 5 h. The reaction mixture was concentrated in vacuo to give a yellow oil and the crude product was then purified by column chromatography (0-40% EtOAc in hexanes) to give 23 (159.9 mg, 0.418 mmol, 56% yield) as a clear pale yellow oil. HRMS (ESI) m / z: [M+Na] + calculated for C 22 H 34 O5Na: 401.2304, found 401.2303. 2-((9-(benzyloxy)-9-oxononyl)oxy)acetic acid (24): To a stirred solution of 23 (159.9 mg, 0.422) in DCM (2 mL) was added TFA (1 mL) and the resulting reaction mixture was stirred at room temperature for 4 h. The reaction mixture was concentrated in vacuo to give 24 (132.5 mg, 0.407 mmol, 98% yield) as a clear, colorless oil. HRMS (ESI) m / z: [M+H] +calculated for C 18 H 27 O5: 323.1858, found 323.1862. Synthesis of HDACi-linker conjugates with VHL ligands

[0178] [ka]

[0179] Benzyl-12-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-12-oxododecanoate (25a): Following general procedure E, 25a was obtained from 14a (257 mg, 0.802 mmol) and 11a (200 mg, 0.611 mmol). The crude product was purified by column chromatography (50% EtOAc in hexanes) to give 25a (274 mg, 0.430 mmol, 70% yield) as a pale yellow solid. HRMS (ESI) m / z: [M+Na] + calculated for C 37 H 47 N3O6Na: 652.3363, found 652.3364. Benzyl-9-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-9-oxononanoate (25b): Following general procedure E, 25b was obtained from 14b (0.475 g, 1.71 mmol) and 11a (0.430 g, 1.31 mmol). The crude product was purified by column chromatography (0-50% EtOAc in hexanes) to give 25b (0.540 g, 0.94 mmol, 53% yield) as a yellow tar. HRMS (ESI) m / z: [M+H] + calculated for C 34 H 42 N3O6: 588.3074, found 588.3067. Benzyl-14-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-14-oxotetradecanoic acid (25c): Following general procedure E, 25c was obtained from 14c (421.4 mg, 1.21 mmol) and 11a (300.0 mg, 0.92 mmol). The crude product was purified by column chromatography (0-50% EtOAc in hexanes) to give 25c (372.4 mg, 0.56 mmol, 61% yield) as a yellow solid. HRMS (ESI) m / z: [M+H] + calculated for C 39 H 52 N3O6: 658.3856, found 658.3880. Benzyl-2-((6-(2-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)hexyl)oxy)acetate (25d): Following general procedure E, 25d was obtained from 21a (78.2 mg, 0.241 mmol) and 11a (71.8 mg, 0.219 mmol). The crude product was purified by column chromatography (0-100% EtOAc in hexanes) to give 25d (76.5 mg, 0.120 mmol, 55% yield) as a pale yellow oil. HRMS (ESI) m / z: [M+H] + calculated for C 35 H 44 N3O8: 634.3128, found 634.3125. tert-Butyl-2-((9-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-9-oxononyl)oxy)acetate (25e): Following general procedure E, 25e was obtained from 22 (85.0 mg, 0.295 mmol) and 11a (88.4 mg, 0.270 mmol). The crude product was purified by column chromatography (0-100% EtOAc in hexanes) to give 25e (66.3 mg, 0.110 mmol, 41% yield) as a yellow tar. HRMS (ESI) m / z: [M+H] + calculated for C33 H 48 N3O7: 598.3492, found 598.3484. Benzyl-9-(2-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)nonanoate (25f): Following general procedure E, 25f was obtained from 24 (130.0 mg, 0.403 mmol) and 11a (110.0 mg, 0.336 mmol). The crude product was purified by column chromatography (0-100% EtOAc in hexanes) to give 25f (157.4 mg, 0.247 mmol, 73% yield) as a clear, colorless tar. HRMS (ESI) m / z: [M+H] + calculated for C 36 H 46 N3O7: 632.3336, found 632.3335. Benzyl-2-((9-(2-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)nonyl)oxy)acetate (25g): Following general procedure E, 25g was obtained from 21b (145.0 mg, 0.396 mmol) and 11a (99.6 mg, 0.304 mmol). The crude product was purified by column chromatography (0-70% EtOAc in hexanes) to give 25g (157.8 mg, 0.229 mmol, 75% yield) as a clear, colorless tar. HRMS (ESI) m / z: [M+H] + calculated for C 38 H 50 N3O8: 676.3598, found 676.3605. Benzyl-12-((4-((2-((tert-butoxycarbonyl)amino)-4-fluorophenyl)carbamoyl)phenyl)amino)-12-oxododecanoic acid (25h): Following general procedure E, 25h was obtained from 14a (246.4 mg, 0.767 mmol) and 11b (201.8 mg, 0.584 mmol). The crude product was purified by column chromatography (10-80% EtOAc in hexanes) to give 25h (332.4 mg, 0.508 mmol, 87% yield) as a dark orange tar. HRMS (ESI) m / z: [M+H] + calculated for C 37 H 47 FN3O6: 648.3449, found 648.3452. Benzyl-2-((9-(2-((4-((2-((tert-butoxycarbonyl)amino)-4-fluorophenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)nonyl)oxy)acetate (25i): Following general procedure E, 25i was obtained from 21b (167.7 mg, 0.458 mmol) and 11b (120.0 mg, 0.347 mmol). The crude product was purified by column chromatography (25-30% EtOAc in hexanes) to give 25i (114.8 mg, 0.164 mmol, 42% yield) as an orange tar. HRMS (ESI) m / z: [M+H] + calculated for C 38 H 49 FN3O8: 694.3504, found 694.3517. Benzyl-2-(2-(2-(2-((4-((2-((tert-butoxycarbonyl)amino)-5-(thiophen-2-yl)phenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)ethoxy)ethoxy)acetate (25j): Following general procedure E, 25j was obtained from 16 (114.0 mg, 0.362 mmol) and 11c (114.0 mg, 0.278 mmol). The crude product was purified by column chromatography (0-100% EtOAc in hexanes, product eluted with 80-90% EtOAc) to give 25i (117.5 mg, 0.162 mmol, 58% yield) as a dark yellow tar. HRMS (ESI) m / z: [M+H] + calculated for C 37 H 42 N3O9S: 704.2642, found 704.2637. 12-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-12-oxododecanoic acid (26a): Benzyl ester hydrogenolysis of 25a (171 mg, 0.271 mmol) was carried out following general procedure F to give 26a (146 mg, 0.266 mmol, 98% yield) as an off-white solid. HRMS (ESI) m / z: [M+Na] + calculated for C 30 H 41 N3O6Na: 562.2893, found 562.2886. 9-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-9-oxononanoic acid (26b): Benzyl ester hydrogenolysis of 25b (370.8 mg, 0.63 mmol) was carried out following general procedure F to give 26b (295.1 mg, 0.59 mmol, 93% yield) as a white crystalline solid. HRMS (ESI) m / z: [M+H] + calculated for C 27 H 36 N3O6: 498.2604, found 498.2597. 14-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-14-oxotetradecanoic acid (26c): Benzyl ester hydrogenolysis of 25c (348.6 mg, 0.530 mmol) was carried out according to general procedure F to give 26c (302.0 mg, 0.527 mmol, 99% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 32 H 46 N3O6: 568.3387, found 568.3391. 2-((6-(2-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)hexyl)oxy)acetic acid (26d): Benzyl ester hydrogenolysis of 25d (76.5 mg, 0.121 mmol) was carried out according to general procedure F to give 26d (65.8 mg, 0.120 mmol, 99% yield) as a clear, colorless oil. HRMS (ESI) m / z: [M+H] + calculated for C 28 H 38 N3O8: 544.2659, found 544.2645. 2-((9-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-9-oxononyl)oxy)acetic acid (26e): To a solution of 25e (62.7 mg, 0.111 mmol) in DCM (2.7 mL) was added a solution of NaOH in MeOH (4 M, 0.3 mL). The reaction was stirred at room temperature for 16 h. The reaction mixture was concentrated in vacuo, then redissolved in water (10 mL) and acidified to pH 2 with HCl (3 M, ca. 1 mL). The product was then extracted with EtOAc (2×20 mL), dried over Na2SO4, filtered and concentrated in vacuo to give 26e (55.4 mg, 0.101 mmol, 91% yield) as a yellow solid. HRMS (ESI) m / z: [M+H] + calculated for C 29 H 40 N3O7: 542.2866, found 542.2861. 9-(2-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)nonanoic acid (26f): Benzyl ester hydrogenolysis of 25f (120.2 mg, 0.190 mmol) was carried out according to general procedure F to give 26f (105.6 mg, 0.189 mmol, 99% yield) as a clear, colorless tar. HRMS (ESI) m / z: [M+H] + calculated for C 29 H 40 N3O7: 542.2866, found 542.2863. 2-((9-(2-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)nonyl)oxy)acetic acid (26g): Benzyl ester hydrogenolysis of 25g (134.8 mg, 0.199 mmol) following general procedure F afforded 26g (118.7 mg, 0.199 mmol, 100% yield) as a clear, colorless tar. HRMS (ESI) m / z: [M+H] + calculated for C 31 H 44 N3O8: 586.3128, found 586.3128. 12-((4-((2-((tert-butoxycarbonyl)amino)-4-fluorophenyl)carbamoyl)phenyl)amino)-12-oxododecanoic acid (26h): Benzyl ester hydrogenolysis of 25h (219.6 mg, 0.339 mmol) was carried out following general procedure F to give 26h (192.9 mg, 0.266 mmol, 98% yield) as a pale purple / brown solid. HRMS (ESI) m / z: [M+H] + calculated for C 30 H 41 FN3O6: 558.2979, found 558.2974. 2-((9-(2-((4-((2-((tert-butoxycarbonyl)amino)-4-fluorophenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)nonyl)oxy)acetic acid (26i): Benzyl ester hydrogenolysis of 25i (114.0 mg, 0.164 mmol) was carried out according to general procedure F to give 26i (99.8 mg, 0.164 mmol, 100% yield) as a yellow / brown tar. HRMS (ESI) m / z: [M+H] + calculated for C 31 H 43 FN3O8: 604.3034, found 604.3046. 2-(2-(2-(2-((4-((2-((tert-butoxycarbonyl)amino)-5-(thiophen-2-yl)phenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)ethoxy)ethoxy)acetic acid (26j): To a solution of 25j (111.6 mg, 0.159 mmol) in MeOH (10 mL) was added NaOH (160 mg) until the solution reached pH 10 (0.4 M NaOH in MeOH), and the resulting solution was then stirred at room temperature for 4 h. The reaction mixture was concentrated in vacuo, then dissolved in water (10 mL) and washed with EtOAC (20 mL). The aqueous layer was acidified to pH 2 with HCl (1M) and the product was extracted with EtOAc (2×20 mL), then the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give 26j (96.7 mg, 0.154 mmol, 87% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 30 H 36 N3O9S: 614.2172, found 614.2161. tert-Butyl (2-(4-(12-(((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)-12-oxododecaneamido)benzamido)phenyl)carbamate (27a): Following general procedure G, 27a was obtained from 26a (65.5 mg, 0.121 mmol) and VH_032 amine (50.0 mg, 0.099 mmol). The crude product was purified by column chromatography (0-5% MeOH in DCM) to give 27a (61.9 mg, 0.064 mmol, 64% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 52 H 70 N7O8S: 952.5007, found 952.5009. tert-Butyl (2-(4-(9-(((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)-9-oxononanamido)benzamido)phenyl)carbamate (27b): Following general method G, 27b was obtained from 26b (59.9 mg, 0.120 mmol) and VH_032 amine (50.0 mg, 0.099 mmol). The crude product was purified by column chromatography (0-5% MeOH in DCM) to give 27b (76.1 mg, 0.083 mmol, 84% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 49 H 64 N7O8S: 910.4537, found 910.4529. tert-Butyl (2-(4-(14-(((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)-14-oxotetradecanamido)benzamido)phenyl)carbamate (27c): Following general method G, 27c was obtained from 26c (53.5 mg, 0.094 mmol) and VH_032 amine (40.0 mg, 0.080 mmol). The crude product was purified by column chromatography (0-5% MeOH in DCM) to give 27c (62.5 mg, 0.063 mmol, 79% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 54 H 74 N7O8S: 980.5320, found 980.5303. tert-Butyl (2-(4-(2-((6-(2-(((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)-2-oxoethoxy)hexyl)oxy)acetamido)benzamido)phenyl)carbamate (27d): Following general method G, 27d was obtained from 26d (67.2 mg, 0.124 mmol) and VH_032 amine (50.0 mg, 0.099 mmol). The crude product was purified by column chromatography (0-10% MeOH in DCM) to give 27d (92.4 mg, 0.093 mmol, 94% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 50 H 66 N7O 10 S: 956.4592, found 956.4590. tert-Butyl (2-(4-(9-(2-(((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)-2-oxoethoxy)nonanamido)benzamido)phenyl)carbamate (27e): Following general method G, 27e was obtained from 26e (52.3 mg, 0.099 mmol) and VH_032 amine (40.0 mg, 0.080 mmol). The crude product was purified by column chromatography (0-10% MeOH in DCM) to give 27e (68.4 mg, 0.071 mmol, 89% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 51 H 68 N7O9S: 954.4799, found 954.4798. tert-Butyl (2-(4-(2-((9-(((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)-9-oxononyl)oxy)acetamido)benzamido)phenyl)carbamate (27f): Following general method G, 27f was obtained from 26f (52.8 mg, 0.097 mmol) and VH_032 amine (40.0 mg, 0.080 mmol). The crude product was purified by column chromatography (0-10% MeOH in DCM) to give 27f (72.9, 0.076 mmol, 95% yield) as a pale yellow / white solid. HRMS (ESI) m / z: [M+H] + calculated for C 51 H 68 N7O9S: 954.4799, found 954.4792. tert-Butyl (2-(4-(2-((9-(2-(((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)-2-oxoethoxy)nonyl)oxy)acetamido)benzamido)phenyl)carbamate (27g): Following general method G, 27g was obtained from 26g (56.2 mg, 0.096 mmol) and VH_032 amine (40.0 mg, 0.080 mmol). The crude product was purified by column chromatography (0-8% MeOH in DCM) to give 27g (68.4 mg, 0.068 mmol, 85% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 53 H 72 N7O 10 S: 998.5061, found 998.5048. tert-Butyl (5-fluoro-2-(4-(12-(((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)-12-oxododecanamido)benzamido)phenyl)carbamate (27h): Following general method G, 27h was obtained from 26h (53.2 mg, 0.095 mmol) and VH_032 amine (40.0 mg, 0.080 mmol). The crude product was purified by column chromatography (0-10% MeOH in DCM) to give 27h (58.7 mg, 0.060 mmol, 75% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 52 H 69 FN7O8S: 970.4912, found 970.4913. tert-Butyl (5-fluoro-2-(4-(2-((9-(2-(((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)-2-oxoethoxy)nonyl)oxy)acetamido)benzamido)phenyl)carbamate (27i): Following general method G, 27i was obtained from 26i (57.6 mg, 0.096 mmol) and VH_032 amine (40.0 mg, 0.080 mmol). The crude product was purified by column chromatography (0-8% MeOH in DCM) to give 27i (75.7, 0.072 mmol, 90% yield) as a pale yellow / white solid. HRMS (ESI) m / z: [M+H] + calculated for C 53 H 71 FN7O 10 S: 1016.4967, found 1016.4928. tert-Butyl (2-(4-((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-azapentadecanamido)benzamido)-4-(thiophen-2-yl)phenyl)carbamate (27j): Following general method G, 27j was obtained from 26j (35.1 mg, 0.057 mmol) and VH_032 amine (24.0 mg, 0.048 mmol). The crude product was purified by column chromatography (0–10% MeOH in DCM) to give 27j (30.3 mg, 0.029 mmol, 61% yield) as a yellow tar. HRMS (ESI) m / z: [M+H] + calculated for C 52 H 64 N7O 11 S2: 1026.4105, found 1026.4066.

[0180] [ka]

[0181] N1-(4-((2-aminophenyl)carbamoyl)phenyl)-N12-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)dodecanediamide (PROTAC4): Boc deprotection of 27a (37.6 mg, 0.0395 mmol) was carried out according to general method H to afford PROTAC4 (25.8 mg, 0.0288 mmol, 73% yield) as a pale yellow solid. Prior to biological evaluation, the PROTAC was further purified by semi-preparative HPLC (5–95% MeCN in HO, 260 nm, 45 min gradient). 1 H NMR (400 MHz, Methanol-d4) δ Hppm 8.86 (s, 1 H, 39-CH), 7.95 (d, J = 8.8 Hz, 2 H, 15-CH), 7.72 (d, J = 8.8 Hz, 2 H, 14-CH), 7.43 - 7.48 (m, 2 H, 36-CH), 7.38 - 7.42 (m, 2 H, 35-CH), 7.18 (dd, J = 7.8, 1.3 Hz, 1 H, 23-CH), 7.07 (app. td, J = 7.8, 1.3 Hz, 1 H, 21-CH), 6.90 (dd, J = 7.8, 1.3 Hz, 1 H, 20-CH), 6.76 (app. td, J = 7.8, 1.3 Hz, 1 H, 22-CH), 4.60 - 4.66 (m, 1 H, 24-CH), 4.55 - 4.60 (m, 1 H, 31-CH), 4.50 - 4.55 (m, 1 H, 33-CH), 4.47 - 4.50 (m, 1 H, 29-CH), 4.31 - 4.39 (m, 1 H, 33-CH), 3.86 - 3.93 (m, 1 H, 28-CH), 3.76 - 3.83 (m, 1 H, 28-CH), 2.47 (s, 3 H, 41-CH3), 2.40 (t, J = 7.5 Hz, 2 H, 11-CH2), 2.18 - 2.33 (m, 3 H, 2-CH2, 30-CH), 2.03 - 2.12 (m, 1 H, 30-CH), 1.70 (quin, J = 7.5 Hz, 2 H, 10-CH2), 1.53 - 1.64 (m, 2 H, 3-CH2), 1.28 - 1.41 (m, 12 H, (4 - 9)-CH2), 1.03 (s, 9 H, 26-CH3). HRMS (ESI) m / z: [M + H] + calculated for C 47 H 62 N7O6S: 852.4476, found 852.4482.

[0182]

Chem.

[0183] N1-(4-((2-aminophenyl)carbamoyl)phenyl)-N9-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)nonanediamide (PROTAC6): Boc deprotection of 27b (37.6 mg, 0.0395 mmol) was carried out according to general method H to afford PROTAC6 (37.0 mg, 0.044 mmol, 99% yield) as a pale white solid. Prior to biological evaluation, the PROTAC was further purified by semi-preparative HPLC (5–95% MeCN in HO, 260 nm, 45 min gradient). 1 H NMR (400 MHz, Methanol-d4) δ Hppm 8.86 (singlet, 1 H, 36-CH), 7.95 (doublet, J = 8.7 Hz, 2 H, 12-CH), 7.71 (doublet, J = 8.7 Hz, 2 H, 11-CH), 7.45 (doublet, J = 8.4 Hz, 2 H, 33-CH), 7.40 (doublet, J = 8.4 Hz, 2 H, 32-CH), 7.18 (apparent doublet of doublets, J = 7.8, 1.3 Hz, 1 H, 20-CH), 7.07 (apparent triplet of doublets, J = 7.8, 1.3 Hz, 1 H, 18-CH), 6.90 (apparent doublet of doublets, J = 7.8, 1.3 Hz, 1 H, 17-CH), 6.76 (apparent triplet of doublets, J = 7.8, 1.3 Hz, 1 H, 19-CH), 4.63 (singlet, 1 H, 21-CH), 4.55 - 4.61 (multiplet, 1 H, 28-CH), 4.50 - 4.55 (multiplet, 1 H, 30-CH), 4.47 - 4.50 (multiplet, 1 H, 29-CH), 4.31 - 4.38 (multiplet, 1 H, 30-CH), 3.87 - 3.94 (multiplet, 1 H, 25-CH), 3.76 - 3.83 (multiplet, 1 H, 25-CH), 2.46 (singlet, 3 H, 38-CH3), 2.39 (triplet, J = 7.5 Hz, 2 H, 8-CH3), 2.17 - 2.33 (multiplet, 3 H, 2-CH2, 27-CH), 2.03 - 2.11 (multiplet, 1 H, 27-CH), 1.70 (quintet, J = 7.5 Hz, 2 H, 7-CH2), 1.61 (quintet, J = 6.9 Hz, 2 H, 3-CH2), 1.27 - 1.44 (multiplet, 6 H, (4 - 6)-CH2), 1.03 (singlet, 9 H, 23-CH3). HRMS (ESI) m / z: [M + H] + calculated for C 44 H 56 N7O6S: 810.4013, found 810.4005.

[0184]

Chem.

[0185] N1-(4-((2-aminophenyl)carbamoyl)phenyl)-N14-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)tetradecanediamide (PROTAC34): Boc deprotection of 27c (62.5 mg, 0.064 mmol) was carried out according to general method H to give PROTAC34 (51.6 mg, 0.058 mmol, 91% yield) as a white solid. 1 H NMR (400 MHz, Methanol-d4) δ Hppm 8.86 (singlet, 1 H, 41, CH), 7.95 (doublet, J = 8.7 Hz, 2 H, 17-CH), 7.72 (doublet, J = 8.7 Hz, 2 H, 16-CH), 7.43 - 7.47 (multiplet, 2 H, 38-CH), 7.38 - 7.43 (multiplet, 2 H, 37-CH), 7.18 (doublet of doublets, J = 7.8, 1.3 Hz, 1 H, 25-CH), 7.06 (apparent triplet of doublets, J = 7.8, 1.3 Hz, 1 H, 23-CH), 6.90 (doublet of doublets, J = 7.8, 1.3 Hz, 1 H, 22-CH), 6.76 (apparent triplet of doublets, J = 7.8, 1.3 Hz, 1 H, 24-CH), 4.63 (singlet, 1 H, 26-CH), 4.55 - 4.60 (multiplet, 1 H, 33-CH), 4.52 (doublet, J = 15.5 Hz, 1 H, 35-CH), 4.46 - 4.50 (multiplet, 1 H, 31-CH), 4.34 (doublet, J = 15.5 Hz, 1 H, 35-CH), 3.86 - 3.93 (multiplet, 1 H, 30-CH), 3.75 - 3.82 (multiplet, 1 H, 30-CH), 2.46 (singlet, 3 H, 43-CH3), 2.39 (triplet, J = 7.4 Hz, 2 H, 13-CH2), 2.17 - 2.32 (multiplet, 3 H, 2-CH2,32-CH), 2.03 - 2.11 (multiplet, 1 H, 32-CH), 1.70 (quintet, J = 7.4 Hz, 2 H, 12-CH2), 1.52 - 1.65 (multiplet, 2 H, 3-CH2), 1.27 - 1.39 (multiplet, 16 H, (4 - 11)-CH2), 1.03 (singlet, 9 H, 28-CH3). HRMS (ESI) m / z: [M+H] + calculated for C 49 H 66 N7O6S: 880.4795, found 880.4762.

[0186]

Chem.

[0187] (2S,4R)-1-((S)-2-(2-((6-(2-((4-((2-aminophenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)hexyl)oxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (PROTAC12): Boc deprotection of 27d (86.3 mg, 0.090 mmol) was carried out according to general method H to afford PROTAC12 (71.7 mg, 0.083 mmol, 90% yield) as a pale yellow solid. Prior to biological evaluation, the PROTAC was further purified by semi-preparative HPLC (5–95% MeCN in HO, 260 nm, 45 min gradient). 1 H NMR (400 MHz, Methanol-d4) δ Hppm 8.85 (s, 1 H, 37-CH), 7.96 (d, J=8.6 Hz, 2 H, 13-CH), 7.75 (d, J=8.6 Hz, 2 H, 12-CH), 7.42 - 7.45 (m, 2 H, 34-CH), 7.37 - 7.41 (m, 2 H, 33-CH), 7.18 (dd, J=7.7, 1.1 Hz, 1 H, 21-CH), 7.07 (td, J=7.7, 1.1 Hz, 1 H, 19-CH), 6.89 (dd, J=7.7, 1.1 Hz, 1 H, 18-CH), 6.76 (td, J=7.7, 1.1 Hz, 1 H, 20-CH), 4.69 (s, 1 H, 22-CH), 4.55 - 4.62 (m, 1 H, 29-CH), 4.45 - 4.55 (m, 2 H, 27,31-CH), 4.31 - 4.38 (m, 1 H, 31-CH), 4.05 - 4.11 (m, 2 H, 9-CH2), 3.95 - 4.00 (m, 1 H, 2-CH), 3.89 - 3.94 (m, 1 H, 2-CH), 3.83 - 3.89 (m, 1 H, 26-CH), 3.75 - 3.82 (m, 1 H, 26-CH), 3.53 - 3.62 (m, 4 H, 3,8-CH2), 2.46 (s, 3 H, 39-CH3), 2.18 - 2.27 (m, 1 H, 28-CH), 2.03 - 2.13 (m, 1 H, 28-CH), 1.62 - 1.73 (m, 4 H, 4,7-CH2), 1.42 - 1.52 (m, 4 H, 5,6-CH2), 1.03 (s, 9 H, 24-CH3). HRMS (ESI) m / z: [M+H] + calculated for C 45 H 58 N7O8S: 856.4068, found 856.4064.

[0188]

Chem.

[0189] (2S,4R)-1-((S)-2-(2-((9-((4-((2-aminophenyl)carbamoyl)phenyl)amino)-9-oxononyl)oxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (PROTAC13): Boc deprotection of 27e (66.9 mg, 0.070 mmol) was carried out according to general method H to afford PROTAC13 (52.5 mg, 0.060 mmol, 86% yield) as a pale yellow solid. Prior to biological evaluation, the PROTAC was further purified by semi-preparative HPLC (5–95% MeCN in HO, 260 nm, 45 min gradient). 1 H NMR (400 MHz, Methanol-d4) δ Hppm 8.85 (s, 1 H, 38-CH), 7.94 (d, J=8.7 Hz, 2 H, 14-CH), 7.72 (d, J=8.7 Hz, 2 H, 13-CH), 7.43 - 7.47 (m, 2 H, 35-CH), 7.37 - 7.42 (m, 2 H, 34-CH), 7.17 (dd, J=7.7, 1.1 Hz, 1 H, 22-CH), 7.07 (app. td, J=7.7, 1.1 Hz, 1 H, 20-CH), 6.89 (dd, J=7.7, 1.1 Hz, 1 H, 19-CH), 6.76 (app. td, J=7.7, 1.1 Hz, 1 H, 21-CH), 4.69 (s, 1 H, 23-CH), 4.56 - 4.63 (m, 1 H, 30-CH), 4.47 - 4.55 (m, 2 H, 28,32-CH), 4.35 (d, J=15.5 Hz, 1 H, 32-CH), 3.94 - 3.99 (m, 1 H, 2-CH), 3.89 - 3.94 (m, 1 H, 2-CH), 3.84 - 3.89 (m, 1 H, 27-CH), 3.76 - 3.82 (m, 1 H, 27-CH), 3.53 (t, J=6.3 Hz, 2 H, 3-CH2), 2.46 (s, 3 H, 40-CH3), 2.37 (t, J=7.5 Hz, 2 H, 10-CH2), 2.19 - 2.27 (m, 1 H, 29-CH), 2.04 - 2.12 (m, 1 H, 29-CH), 1.59 - 1.71 (m, 4 H, 4,9-CH2), 1.33 - 1.43 (m, 8 H, (5-8)-CH2), 1.03 (s, 9 H, 25-CH3). HRMS (ESI) m / z: [M+H] + calculated for C 46 H 60 N7O7S: 854.4275, found 854.4277.

[0190]

Chem.

[0191] (2S,4R)-1-((S)-2-(9-(2-((4-((2-aminophenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)nonanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (PROTAC14): Boc deprotection of 27f (60.2 mg, 0.063 mmol) was carried out according to general method H to afford PROTAC14 (50.6 mg, 0.059 mmol, 93% yield) as a pale yellow solid. Prior to biological evaluation, the PROTAC was further purified by semi-preparative HPLC (5–95% MeCN in HO, 260 nm, 45 min gradient). 1 H NMR (400 MHz, Methanol-d4) δ Hppm 8.86 (s, 1 H, 38-CH), 7.98 (d, J=8.7 Hz, 2 H, 14-CH), 7.77 (d, J=8.7 Hz, 2 H, 13-CH), 7.45 (d, J=8.3 Hz, 2 H, 35-CH), 7.41 (d, J=8.3 Hz, 2 H, 34-CH), 7.18 (dd, J=7.6, 1.3 Hz, 1 H, 22-CH), 7.07 (app. td, J=7.6, 1.3 Hz, 1 H, 20-CH), 6.90 (dd, J=7.6, 1.3 Hz, 1 H, 19-CH), 6.77 (app. td, J=7.6, 1.3 Hz, 1 H, 21-CH), 4.63 (s, 1 H, 23-CH), 4.56 - 4.59 (m, 1 H, 30-CH), 4.52 (d, J=15.5 Hz, 1 H, 32-CH), 4.46 - 4.50 (m, 1 H, 28-CH), 4.35 (d, J=15.5 Hz, 1 H, 32-CH), 4.09 (s, 2 H, 10-CH2), 3.86 - 3.92 (m, 1 H, 27-CH), 3.76 - 3.82 (m, 1 H, 27-CH), 3.60 (t, J=6.6 Hz, 2 H, 9-CH2), 2.47 (s, 3 H, 40-CH3), 2.18 - 2.32 (m, 3 H, 2-CH2,29-CH), 2.03 - 2.12 (m, 1 H, 29-CH), 1.68 (quin, J=6.6 Hz, 2 H, 8-CH2), 1.56 - 1.64 (m, 2 H, 3-CH2), 1.32 - 1.45 (m, 8 H, (4-7)-CH2), 1.03 (s, 9 H, 25-CH3). HRMS (ESI) m / z: [M+H] + calculated for C 46 H 60 N7O7S: 854.4275, found 854.4268.

[0192]

Chem.

[0193] (2S,4R)-1-((S)-2-(2-((9-(2-((4-((2-aminophenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)nonyl)oxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (PROTAC16): Boc deprotection of 27g (66.7 mg, 0.067 mmol) was carried out according to general method H to afford PROTAC16 (58.2 mg, 0.064 mmol, 96% yield) as a pale yellow solid. Prior to biological evaluation, the PROTAC was further purified by semi-preparative HPLC (5–95% MeCN in HO, 260 nm, 45 min gradient). 1 H NMR (400 MHz, Methanol-d4) δ Hppm 8.86 (s, 1 H, 40-CH), 7.97 (d, J=8.6 Hz, 2 H, 16-CH), 7.76 (d, J=8.6 Hz, 2 H, 15-CH), 7.45 (d, J=8.3 Hz, 2 H, 37-CH), 7.39 (d, J=8.3 Hz, 2 H, 36-CH), 7.18 (dd, J=7.7, 1.1 Hz, 1 H, 24-CH), 7.07 (app. td, J=7.7, 1.1 Hz, 1 H, 22-CH), 6.90 (dd, J=7.7, 1.1 Hz, 1 H, 21-CH), 6.76 (app. td, J=7.7, 1.1 Hz, 1 H, 23-CH), 4.69 (s, 1 H, 25-CH), 4.56 - 4.61 (m, 1 H, 32-CH), 4.53 (d, J=15.5 Hz, 1 H, 34-CH), 4.46 - 4.50 (m, 1 H, 30-CH), 4.34 (d, J=15.5 Hz, 1 H, 34-CH), 4.07 (s, 2 H, 12-CH2), 3.97 (d, J=15.4 Hz, 1 H, 2-CH2), 3.92 (d, J=15.4 Hz, 1 H, 2-CH2), 3.83 - 3.90 (m, 1 H, 29-CH), 3.75 - 3.82 (m, 1 H, 29-CH), 3.50 - 3.59 (m, 4 H, 3,11-CH2), 2.46 (s, 3 H, 42-CH3), 2.17 - 2.26 (m, 1 H, 31-CH), 2.03 - 2.11 (m, 1 H, 31-CH), 1.59 - 1.69 (m, 4 H, 4,10-CH2), 1.32 - 1.44 (m, 10 H, (5-9)-CH2), 1.03 (s, 9 H, 27-CH3). HRMS (ESI) m / z: [M+H] + calculated for C 48 H 64 N7O8S: 898.4537, found 898.4531.

[0194]

Chem.

[0195] N1-(4-((2-amino-4-fluorophenyl)carbamoyl)phenyl)-N12-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)dodecanediamide (PROTAC17): Boc deprotection of 27h (58.7 mg, 0.061 mmol) was carried out according to general method H to afford PROTAC17 (49.2 mg, 0.056 mmol, 92% yield) as a pale yellow solid. 1 H NMR (400 MHz, Methanol-d4) δ H ppm 8.86 (s, 1 H, 39-CH), 7.94 (d, J=8.7 Hz, 2 H, 15-CH), 7.72 (d, J=8.7 Hz, 2 H, 14-CH), 7.43 - 7.47 (m, 2 H, 36-CH), 7.37 - 7.42 (m, 2 H, 35-CH), 7.11 (dd, J HH =8.6, J HF =6.0 Hz, 1 H, 23-CH), 6.58 (dd, J HF =10.7, J HH =2.8 Hz, 1 H, 20-CH), 6.41 (app. td, J HF =8.6, J HH=8.6, 2.8 Hz, 1 H, 22-CH), 4.63 (s, 1 H, 24-CH), 4.55 - 4.60 (m, 1 H, 31-CH), 4.52 (d, J=15.5 Hz, 1 H, 33-CH), 4.47 - 4.50 (m, 1 H, 29-CH), 4.35 (d, J=15.5 Hz, 1 H, 33-CH), 3.86 - 3.93 (m, 1 H, 28-CH), 3.76 - 3.82 (m, 1 H, 28-CH), 2.46 (s, 3 H, 41-CH3), 2.39 (t, J=7.5 Hz, 2H, 11-CH2), 2.17 - 2.31 (m, 3 H, 2-CH2,30-CH), 2.03 - 2.12 (m, 1 H, 30-CH), 1.69 (quin, J=7.5 Hz, 2 H, 10-CH2), 1.53 - 1.63 (m, 2 H, 3-CH2), 1.29 - 1.37 (m, 12 H, (4-9)-CH2), 1.03 (s, 9 H, 26-CH3). HRMS (ESI) m / z: [M+H] + calculated for C 47 H 61 FN7O6S: 870.4388, found 870.4376.

[0196] [ka]

[0197] (2S,4R)-1-((S)-2-(2-((9-(2-((4-((2-amino-4-fluorophenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)nonyl)oxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (PROTAC20): Boc deprotection of 27i (70.8 mg, 0.070 mmol) was carried out according to general method H to afford PROTAC20 (63.3 mg, 0.068, 98% yield) as a light brown solid. 1H NMR (400 MHz, Methanol-d4) δ H ppm 8.86 (s, 1 H, 40-CH), 7.96 (d, J=8.7 Hz, 2 H, 16-CH), 7.75 (d, J=8.7 Hz, 2 H, 15-CH), 7.42 - 7.47 (m, 2 H, 37-CH), 7.37 - 7.42 (m, 2 H, 36-CH), 7.11 (dd, J HH =8.6, J HF =6.1 Hz, 1 H, 24-CH), 6.58 (dd, J HF =10.7, J HH =2.8 Hz, 1 H, 20-CH), 6.41 (td, J HF =8.6, J HH =8.6, 2.8 Hz, 1 H, 23-CH), 4.68 (s, 1 H, 25-CH), 4.59 (dd, J=9.0, 7.8 Hz, 1 H, 32-CH), 4.52 (d, J=15.6 Hz, 1 H, 34-CH), 4.47 - 4.50 (m, 1 H, 30-CH), 4.34 (d, J=15.6 Hz, 1 H, 34-CH), 4.07 (s, 2 H, 12-CH2), 3.95 (d, J=15.4 Hz, 1 H, 2-CH2), 3.94 (d, J=15.4 Hz, 1 H, 2-CH2), 3.84 - 3.89 (m, 1 H, 29-CH), 3.74 - 3.82 (m, 1 H, 29-CH), 3.50 - 3.59 (m, 4 H, (3,11)-CH2), 2.46 (s, 3 H, 42-CH3), 2.18 - 2.27 (m, 1 H, 31-CH), 2.02 - 2.12 (m, 1 H, 31-CH), 1.58 - 1.68 (m, 4 H, (4,10)-CH2), 1.32 - 1.43 (m, 12 H, (5-9)-CH2), 1.03 (s, 9 H, 27-CH3). HRMS (ESI) m / z: [M+H] + calculated for C 48 H 63FN7O8S: 916.4443, found 916.4426.

[0198] [ka]

[0199] (2S,4R)-1-((S)-14-((4-((2-amino-5-(thiophen-2-yl)phenyl)carbamoyl)phenyl)amino)-2-(tert-butyl)-4,14-dioxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (PROTAC22): Boc deprotection of 27j (30.3 mg, 0.029 mmol) was carried out according to general method H to afford PROTAC22 (26.8 mg, 0.029 mmol, 99% yield) as a pale yellow solid. 1 H NMR (400 MHz, Methanol-d4) δ Hppm 8.84 (s, 1 H, 39-CH), 7.98 (d, J=8.7 Hz, 2 H, 11-CH), 7.75 (d, J=8.7 Hz, 2 H, 10-CH), 7.49 (d, J=2.1 Hz, 1 H, 19-CH), 7.40 - 7.44 (m, 2 H, 36-CH), 7.36 - 7.39 (m, 2 H, 35-CH), 7.34 (dd, J=8.3, 2.1 Hz, 1 H, 17-CH), 7.22 (d, J=5.0 Hz, 1 H, 23-CH), 7.19 (d, J=3.7 Hz, 1 H, 21-CH), 7.01 (dd, J=5.0, 3.7 Hz, 1 H, 22-CH), 6.89 (d, J=8.3 Hz, 1 H, 16-CH), 4.68 (s, 1 H, 24-CH), 4.54 - 4.60 (m, 1 H, 31-CH), 4.51 (d, J=15.5 Hz, 1 H, 33-CH), 4.46 - 4.49 (m, 1 H, 29-CH), 4.31 (d, J=15.5 Hz, 1 H, 33-CH), 4.15 (d, J=15.8 Hz, 1 H, 7-CH), 4.09 (d, J=15.8 Hz, 1 H, 7-CH), 4.02 (d, J=15.6 Hz, 1 H, 2-CH), 3.91 (d, J=15.6 Hz, 1 H, 2-CH), 3.82 - 3.87 (m, 1 H, 28-CH), 3.69 - 3.81 (m, 9 H, 28-CH,(3-6)-CH2), 2.45 (s, 3 H, 41-CH3), 2.16 - 2.24 (m, 1 H, 30-CH), 2.03 - 2.11 (m, 1 H, 30-CH), 1.02 (s, 9 H, 26-CH3). HRMS (ESI) m / z: [M+H] + calculated for C 47 H 56 N7O9S2: 926.3581, found 926.3556. Synthesis of HDAC PROTAC, which replaces VHL

[0200] [ka]

[0201] tert-Butyl (2-(4-(12-bromodecanamido)benzamido)phenyl)carbamate (29): Following general procedure E, 29 was obtained from 28 (332.6 mg, 1.19 mmol) and 11a (300.0 mg, 0.92 mmol). The crude product was purified by column chromatography (0-50% EtOAc in hexanes) to give 29 (207.0 mg, 0.35 mmol, 38% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 30 H 43 81 BrN3O4: 590.2416, found 590.2411. tert-Butyl (2-(4-(12-(2-(((2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)dodecanamido)benzamido)phenyl)carbamate (30a): Following general procedure I, 30a was obtained from (2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VH 032 phenol, 17.2 mg, 0.034 mmol) and 29 (20.0 mg, 0.034 mmol). The crude product was purified by column chromatography (0-10% MeOH in DCM) to give 30a (17.8 mg, 0.018 mmol, 52% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 54 H 74 N7O9S: 996.5269 , found 996.5239. tert-Butyl (2-(4-(12-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)dodecanamido)benzamido)phenyl)carbamate (30b): According to general method I, (2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VH 101 phenol, 18.6 mg, 0.034 mmol) and 29 (20.0 mg, 0.034 mmol) gave 30b. The crude product was purified by column chromatography (1-10% MeOH in DCM) to give 30b (24.2 mg, 0.022 mmol, 64% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 56 H 75 FN7O9S: 1040.5331, found 1040.5304.

[0202] [ka]

[0203] (2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-N-(2-((12-((4-((2-aminophenyl)carbamoyl)phenyl)amino)-12-oxododecyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-2-carboxamide (PROTAC35): Boc deprotection of 30a (17.8 mg, 0.018 mmol) was carried out according to general method H to give PROTAC35 (16.1 mg, 0.018 mmol, 99% yield) as a white solid. 1 H NMR (400 MHz, Methanol-d4) δ Hppm 8.86 (s, 1 H, 28-CH), 7.95 (d, J=8.7 Hz, 2 H, 15-CH), 7.72 (d, J=8.7 Hz, 2 H, 14-CH), 7.47 (d, J=8.2 Hz, 1 H, 32-CH), 7.17 (dd, J=7.7, 1.3 Hz, 1 H, 23-CH), 7.07 (app. td, J=7.7, 1.3 Hz, 1 H, 21-CH), 6.94 - 7.00 (m, 2 H, 25,31-CH), 6.90 (dd, J=7.7, 1.3 Hz, 1 H, 20-CH), 6.76 (app. td, J=7.7, 1.3 Hz, 1 H, 22-CH), 4.57 - 4.65 (m, 2 H, 36,41-CH), 4.48 - 4.52 (m, 1 H, 38-CH), 4.46 (d, J=16.1 Hz, 1 H, 34-CH), 4.39 (d, J=16.1 Hz, 1 H, 34-CH), 4.05 (t, J=6.3 Hz, 2 H, 1-CH2), 3.86 - 3.92 (m, 1 H, 39-CH2), 3.75 - 3.81 (m, 1 H, 39-CH2), 2.48 (s, 3 H, 30-CH3), 2.40 (t, J=7.5 Hz, 2 H, 11-CH2), 2.17 - 2.25 (m, 1 H, 37-CH), 2.07 - 2.15 (m, 1 H, 37-CH), 1.99 (s, 3 H, 45-CH3), 1.78 - 1.87 (m, 2 H, 2-CH2), 1.71 (quin, J=7.3 Hz, 2 H, 10-CH2), 1.46 - 1.56 (m, 2 H, 3-CH2), 1.32 - 1.42 (m, 12 H, (4-9)-CH2), 1.02 (s, 9 H, 43-CH3). HRMS (ESI) m / z: [M+H] + calculated for C 49 H 66 N7O7S: 896.4744, found 896.4744.

[0204]

Chem.

[0205] (2S,4R)-N-(2-((12-((4-((2-aminophenyl)carbamoyl)phenyl)amino)-12-oxododecyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamide)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (PROTAC36): Boc deprotection of 30b (24.2 mg, 0.022 mmol) following general procedure H, followed by purification by column chromatography (2-5% MeOH in DCM) afforded PROTAC36 (11.7 mg, 0.012 mmol, 57% yield) as a white solid. 1 H NMR (400 MHz, Methanol-d4) δ H ppm 8.86 (s, 1 H, 28-CH), 7.95 (d, J=8.7 Hz, 2 H, 15-CH), 7.72 (d, J=8.7 Hz, 2 H, 14-CH), 7.47 (d, J=7.7 Hz, 1 H, 32-CH), 7.17 (dd, J=7.8, 1.3 Hz, 1 H, 23-CH), 7.06 (app. td, J=7.8, 1.3 Hz, 1 H, 21-CH), 6.96 - 7.02 (m, 2 H, 25,31-CH), 6.90 (dd, J=7.8, 1.3 Hz, 1 H, 20-CH), 6.76 (app. td, J=7.8, 1.3 Hz, 1H, 22-CH), 4.74 (d, J HF= 0.8 Hz, 1 H, 41-CH), 4.60 - 4.67 (m, 1 H, 36-CH), 4.49 - 4.53 (m, 1 H, 38-CH), 4.47 (d, J = 16.0 Hz, 1 H, 34-CH), 4.39 (d, J = 16.0 Hz, 1 H, 34-CH), 4.06 (t, J = 6.3 Hz, 2 H, 1-CH2), 3.82 - 3.88 (m, 1 H, 39-CH), 3.76 - 3.81 (m, 1 H, 39-CH), 2.48 (s, 3 H, 30-CH3), 2.40 (t, J = 7.5 Hz, 2 H, 11-CH2), 2.19 - 2.27 (m, 1 H, 37-CH), 2.09 - 2.16 (m, 1 H, 37-CH), 1.78 - 1.89 (m, 2 H, 2-CH2), 1.64 - 1.76 (m, 2 H, 10-CH2), 1.48 - 1.57 (m, 2 H, 3-CH2), 1.28 - 1.40 (m, 16 H, (4-9)-CH2,46-CH2), 1.04 (s, 9 H, 43-CH3). HRMS (ESI) m / z: [M+H] + calculated for C 51 H 67 FN7O7S: 940.4807, found 940.4781. Synthesis of HDAC PROTAC Containing IAP Ligand

[0206]

Chemical Structure

[0207] tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(12-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-12-oxododecanamido)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (32a): Following general procedure K, 32a was obtained from 26a (24.3 mg, 0.045 mmol) and A 410099.1 amine (24.5 mg, 0.038 mmol). The crude product was purified by column chromatography (0-10% MeOH in DCM) to give 32a (26.1 mg, 0.023 mmol, 60% yield) as a pale yellow solid. HRMS (ESI) m / z: [M+H] + calculated for C 62 H 89 N8O 10 : 1105.6702, found 1105.6704. tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(2-(2-(2-(2-((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)ethoxy)ethoxy)acetamido)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (32b): Following general procedure K, 32b was obtained from 31 (24.0 mg, 0.045 mmol) and A 410099.1 amine (24.5 mg, 0.038 mmol). The crude product was purified by column chromatography (0-10% MeOH in DCM) to give 32b (34.0 mg, 0.031 mmol, 81% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 58 H 81 N8O 13: 1097.5923, found 1097.5881.

[0208] [ka]

[0209] N 1 -(4-((2-aminophenyl)carbamoyl)phenyl)-N 12 -((3S,5S)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-3-yl)dodecanediamide (PROTAC26): Two Boc deprotections of 32a (26.1 mg, 0.0227 mmol) were carried out according to general method H to give PROTAC26 (21.0 mg, 0.0223 mmol, 98% yield) as a pale yellow solid. 1 H NMR (400 MHz, Methanol-d4) δ Hppm 7.95 (d, J = 8.7 Hz, 2 H, 15-CH), 7.73 (d, J = 8.7 Hz, 2 H, 14-CH), 7.35 - 7.42 (m, 1 H, 35-CH), 7.18 (dd, J = 7.8, 1.2 Hz, 1 H, 23-CH), 7.11 - 7.15 (m, 2 H, 36,37-CH), 7.05 - 7.10 (m, 2 H, 21,38-CH), 6.90 (dd, J = 7.8, 1.2 Hz, 1 H, 20-CH), 6.77 (app. td, J = 7.8, 1.2 Hz, 1 H, 22-CH), 5.02 - 5.09 (m, 1 H, 29-CH), 4.41 - 4.53 (m, 3 H, 24,26,40-CH), 4.16 (dd, J = 10.3, 6.2 Hz, 1 H, 27-CH), 3.57 (dd, J = 10.3, 5.4 Hz, 1 H, 27-CH), 3.13 (q, J = 6.8 Hz, 1 H, 48-CH), 2.72 - 2.84 (m, 2 H, 32-CH2), 2.46 - 2.55 (m, 1 H, 25-CH), 2.40 (t, J = 7.4 Hz, 2 H, 11-CH2), 2.29 (s, 3 H, 50-CH3), 2.19 (t, J = 7.5 Hz, 2 H, 2-CH2), 1.67 - 1.96 (m, 13 H, 10,30,31-CH2,25,41,(42 - 46)-CH), 1.57 - 1.64 (m, 2 H, 3-CH2), 1.32 - 1.42 (m, 12 H, (4 - 9)-CH2), 1.23 - 1.29 (m, 3 H, (43 - 45)-CH), 1.20 (d, J = 6.8 Hz, 3 H, 49-CH3), 1.03 - 1.05 (m, 2 H, 42,46-CH). HRMS (ESI) m / z: [M + H] + calculated for C 52 H 73 N8O6: 905.5653, found 905.5648.

[0210]

Chem.

[0211] (2S,4S)-4-(2-(2-(2-(2-((4-((2-aminophenyl)carbamoyl)phenyl)amino)-2-oxoethoxy)ethoxy)ethoxy)acetamido)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC28): Double Boc deprotection of 32b (34.0 mg, 0.031 mmol) was carried out according to general method H to afford PROTAC28 (26.8 mg, 0.029 mmol, 95% yield) as a white solid. 1 H NMR (400 MHz, Methanol-d4) δ Hppm 7.98 (d, J=8.7 Hz, 2 H, 11-CH), 7.80 (d, J=8.7 Hz, 2 H, 10-CH), 7.33 - 7.39 (m, 1 H, 31-CH), 7.19 (dd, J=7.8, 1.3 Hz, 1 H, 19-CH), 7.03 - 7.15 (m, 4 H, 17,32,33,34-CH), 6.90 (dd, J=7.8, 1.3 Hz, 1 H, 16-CH), 6.75 (app. td, J=7.8, 1.3 Hz, 1 H, 18-CH), 5.00 - 5.08 (m, 1 H, 25-CH), 4.58 - 4.65 (m, 1 H, 20-CH), 4.46 (dd, J=8.9, 4.7 Hz, 1 H, 22-CH), 4.43 (d, J=8.4 Hz, 1 H, 36-CH), 4.08 - 4.18 (m, 3 H, 7-CH2,23-CH), 4.01 (s, 2 H, 2-CH2), 3.79 - 3.87 (m, 2 H, PEG-CH2), 3.70 - 3.79 (m, 6 H, PEG-CH2x 3), 3.62 - 3.68 (m, 1 H, 23-CH), 3.09 (q, J=6.8 Hz, 1 H, 44-CH), 2.68 - 2.85 (m, 2 H, 28-CH2), 2.44 - 2.53 (m, 1 H, 21-CH2), 2.26 (s, 3 H, 46-CH3), 1.62 - 1.98 (m, 11 H, 26,27-CH2,21,37,(38-42)-CH), 1.17 - 1.27 (m, 3 H, (39-41)-CH), 1.16 (d, J=6.9 Hz, 3 H, 45-CH3), 0.98 - 1.13 (m, 2 H, 38,42-CH). HRMS (ESI) m / z: [M+H] + calculated for C 48 H 65 N8O9: 897.4875, found 897.4860. tert-Butyl ((S)-1-(((S)-2-((S)-2-(4-(3-((12-((4-((2-((tert-butoxycarbonyl))amino)phenyl)carbamoyl)phenyl)amino)-12-oxododecyl)oxy)benzoyl)thiazol-2-yl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (33) 33 was obtained from tert-butyl ((S)-1-(((S)-1-cyclohexyl-2-((S)-2-(4-(3-hydroxybenzoyl)thiazol-2-yl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (LCL161 phenol, 20.5 mg, 0.034 mmol) and 29 (20.0 mg, 0.034 mmol) according to general method I. The crude product was purified by column chromatography (1-8% MeOH in DCM) to give 33 (14.7 mg, 0.013 mmol, 37% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 61 H 84 N7O 10 S: 1106.6000, found 1106.5963.

[0212] [ka]

[0213] N-(2-aminophenyl)-4-(12-(3-(2-((S)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidin-2-yl)thiazole-4-carbonyl)phenoxy)dodecanamido)benzamide (PROTAC 37): Two Boc deprotections of 33 (14.7 mg, 0.013 mmol) were performed according to general method H, followed by purification by column chromatography (0–10% MeOH in DCM) to afford PROTAC 37 (9.4 mg, 0.0103 mmol, 82% yield) as a white solid. 11H NMR (400 MHz, Methanol-d4) δ H ppm 8.32 (s, 1 H, 32-CH), 7.95 (d, J=8.7 Hz, 2 H, 15-CH), 7.65 - 7.76 (m, 4 H, 14,27,29-CH), 7.42 (app. t, J=8.0 Hz, 1 H, 26-CH), 7.15 - 7.22 (m, 2 H, 23,25-CH), 7.07 (app. td, J=7.8, 1.3 Hz, 1 H, 21-CH), 6.90 (dd, J=7.8, 1.3 Hz, 1 H, 20-CH), 6.77 (app. td, J=7.8, 1.3 Hz, 1 H, 22-CH), 5.48 (dd, J=7.8, 3.1 Hz, 1 H, 34-CH), 4.52 - 4.60 (m, 1 H, 39-CH), 4.04 (t, J=6.4 Hz, 2 H, 1-CH2), 3.95 - 4.01 (m, 1 H, 37-CH), 3.86 - 3.93 (m, 1 H, 37-CH), 3.20 (q, J=6.8 Hz, 1 H, 47-CH), 2.40 (t, J=7.5 Hz, 2 H, 11-CH2), 2.29 - 2.37 (m, 4 H, 35-CH,49-CH3), 2.18 - 2.28 (m, 2 H, 35,36-CH), 2.08 - 2.17 (m, 1 H, 36-CH), 1.77 - 1.85 (m, 2 H, 2-CH2), 1.54 - 1.77 (m, 8 H, 10-CH2,40-CH,(41-45)-CH),1.46 - 1.53 (m, 2 H, 3-CH2), 1.32 - 1.43 (m, 12 H, (4-9)-CH2), 1.24 (d, J=6.8 Hz, 3 H, 48-CH3), 1.00 - 1.18 (m, 5 H, (41-45)-CH). HRMS (ESI) m / z: [M+H] + calculated for C 51 H 68 N7O6S: 906.4952, found 906.4925. Synthesis of HDAC PROTACs Containing CRBN Ligands

[0214] [ka]

[0215] 9-((tert-Butoxycarbonyl)amino)nonanoic acid (35a): A solution of di-tert-butyl dicarbonate (0.305 g, 1.40 mmol) in 1,4-dioxane / water (2:1, 2 mL) was slowly added to a solution of 34a (0.220 g, 1.27 mmol) and NaOH (0.051 g, 1.27 mmol) in 1,4-dioxane / water (2:1, 7 mL) at 0° C., after which the mixture was stirred at room temperature for 64 h. The reaction mixture was concentrated in vacuo to give a pale yellow solid (0.391 g), then the basic residue was redissolved in water (20 mL) and washed with EtOAc (2×20 mL). The aqueous phase was then acidified to pH 1 with 1 M HCl (ca. 2 mL) and extracted with EtOAc (3×20 mL). The organic phases were combined, dried over Na2SO4, filtered, and concentrated in vacuo to give 35a (0.260 g, 0.94 mmol, 74% yield) as a clear yellow tar (slowly crystallizing). HRMS (ESI) m / z: [M+H] + calculated for C 14 H 28 NO4: 274.2018, found 274.2017. 12-((tert-Butoxycarbonyl)amino)dodecanoic acid (35b): A solution of Boc2O (2.23 g, 10.22 mmol) in 1,4-dioxane / water (2:1, 10 mL) was slowly added to a solution of 34b (2.00 g, 9.29 mmol) and NaOH (0.37 g, 9.29 mmol) in 1,4-dioxane / water (2:1, 50 mL) at 0° C. and stirred at room temperature for 18 h. The reaction mixture was concentrated in vacuo, then the basic residue was redissolved in water (100 mL) and washed with EtOAc (2×50 mL). The aqueous phase was then acidified to pH 1 with 1M HCl (ca. 15 mL) and extracted with EtOAc (3×100 mL). The organic phases were combined, dried over Na2SO4, filtered, and concentrated in vacuo to give 35b (2.41 g, 7.56 mmol, 81% yield) as a fluffy white solid. HRMS (ESI) m / z: [M+Na] + calculated for C 17 H 33 NO4Na: 338.2307, found 338.2307. tert-Butyl (2-(4-(9-((tert-butoxycarbonyl)amino)nonamido)benzamido)phenyl)carbamate (36a): Following general procedure E, 36a was obtained from 35a (247.5 mg, 0.91 mmol) and 11a (250.0 mg, 0.76 mmol). The crude product was purified by column chromatography (0-80% EtOAc in hexanes) to give 36a (255.8 mg, 0.43 mmol, 57% yield). HRMS (ESI) m / z: [M+H] + calculated for C 32 H 47 N4O6: 583.3496, found 583.3492. tert-Butyl (2-(4-(12-((tert-butoxycarbonyl)amino)dodecanamido)benzamido)phenyl)carbamate (36b): Following general procedure E, 36b was obtained from 35b (318 mg, 1.01 mmol) and 11a (300 mg, 0.92 mmol). The crude product was purified by column chromatography (50% EtOAc in hexanes) to give 36b (374 mg, 0.59 mmol, 65% yield) as a pale yellow crystalline solid. HRMS (ESI) m / z: [M+Na] + calculated for C 35 H 52 N4O6Na: 647.3785, found 647.3792. 4-(9-aminononanamido)-N-(2-aminophenyl)benzamide (37a): Boc deprotection of 36a (96.0 mg, 0.165 mmol) was carried out following general procedure C to give 37a (54.0 mg, 0.140 mmol, 85% yield) as a pale yellow solid. HRMS (ESI) m / z: [M+H] + calculated for C 22 H 31 N4O2: 383.2447, found 383.2440. 4-(12-aminododecanamido)-N-(2-aminophenyl)benzamide (37b): Boc deprotection of 36b (159 mg, 0.255 mmol) was carried out following general procedure C to give 37b (104 mg, 0.242 mmol, 95% yield) as a pale yellow solid. HRMS (ESI) m / z: [M+H] + calculated for C 25 H 37 N4O2: 425.2917, found 425.2918.

[0216] [ka]

[0217] N-(2-aminophenyl)-4-(9-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)nonanamido)benzamide (PROTAC5): PROTAC5 was obtained from CRBN acid (33.9 mg, 0.102 mmol) and 37a (39.0 mg, 0.102 mmol) following general method J. The crude product was purified by column chromatography (5% MeOH in DCM) to give PROTAC5 (22.5 mg, 0.032 mmol, 31% yield) as a pale yellow solid. 1 H NMR (400 MHz, Methanol-d4) δ Hppm 7.94 (d, J = 8.7 Hz, 2 H, 12-CH), 7.79 (dd, J = 8.4, 7.3 Hz, 1 H, 25-CH), 7.71 (d, J = 8.7 Hz, 2 H, 11-CH), 7.52 (dd, J = 7.3, 0.5 Hz, 1 H, 26-CH), 7.41 (dd, J = 8.4, 0.5 Hz, 1 H, 24-CH), 7.18 (app. dd, J = 7.7, 1.4 Hz, 1 H, 20-CH), 7.08 (app. td, J = 7.7, 1.4 Hz, 1 H, 18-CH), 6.90 (app. dd, J = 7.7, 1.4 Hz, 1 H, 17-CH), 6.77 (app. td, J = 7.7, 1.4 Hz, 1 H, 19-CH), 5.13 (dd, J = 12.5, 5.5 Hz, 1 H, 31-CH), 4.75 (s, 2 H, 22-CH2), 3.29 - 3.33 (m, 2 H, 1-CH2), 2.82 - 2.93 (m, 1 H, 34-CH), 2.66 - 2.79 (m, 2 H, 34-CH, 35-CH), 2.39 (t, J = 7.5 Hz, 2 H, 8-CH2), 2.10 - 2.18 (m, 1 H, 35-CH), 1.70 (quin, J = 7.1 Hz, 2 H, 7-CH2), 1.58 (quin, J = 7.1 Hz, 2 H, 2-CH2), 1.35 - 1.40 (m, 8 H, (3 - 6)-CH2). HRMS (ESI) m / z: [M+H] + calculated for C 37 H 41 N6O8; 697.2986, found 697.2981.

[0218]

Chem.

[0219] N-(2-aminophenyl)-4-(12-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)dodecanamido)benzamide (PROTAC2): PROTAC2 was obtained from CRBN acid (31.6 mg, 0.095 mmol) and 37b (40.4 mg, 0.095 mmol) following general method J. The crude product was purified by column chromatography (5% MeOH in DCM) to give PROTAC2 (25.4 mg, 0.033 mmol, 34% yield) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ Hppm 11.11 (br s, 1 H, NH), 10.11 (s, 1 H, NH), 9.54 (s, 1 H, NH), 7.93 (d, J=8.8 Hz, 2 H, 15-CH), 7.91 (s, 1H, NH), 7.81 (dd, J=8.4, 7.2 Hz, 1 H, 28-CH), 7.70 (d, J=8.8 Hz, 2 H, 14-CH), 7.50 (d, J=7.2 Hz, 1 H, 29-CH), 7.39 (d, J=8.4 Hz, 1 H, 27-CH), 7.15 (dd, J=7.7, 1.4 Hz, 1 H, 23-CH), 6.96 (app. td, J=7.7, 1.4 Hz, 1 H, 21-CH), 6.78 (dd, J=7.7, 1.4 Hz, 1 H, 20-CH), 6.59 (app. td, J=7.7, 1.4 Hz, 1 H, 22-CH), 5.12 (dd, J=12.9, 5.4 Hz, 1 H, 34-CH), 4.87 (s, 2 H, NH2), 4.76 (s, 2 H, 25-CH2), 3.13 (q, J=6.5 Hz, 2 H, 1-CH2), 2.85 - 2.95 (m, 1 H, 37-CH), 2.53 - 2.63 (m, 2 H, 37-CH,38-CH), 2.33 (t, J=7.4 Hz, 2 H, 11-CH2), 2.00 - 2.07 (m, 1 H, 38-CH), 1.59 (quin, J=7.0 Hz, 2 H, 10-CH2), 1.39 - 1.47 (m, 2 H, 2-CH2), 1.23 - 1.31 (m, 14 H, (3-9)-CH2). HRMS (ESI) m / z: [M+H] + calculated for C 40 H 47 N6O8: 739.3455, found 739.3455. Using click chemistry to synthesize HDAC PROTACs containing VHL ligands

[0220] [ka]

[0221] 4-({[(Prop-2-yn-1-yloxy)carbonyl]amino}methyl)benzoic acid (38): Propargyl alcohol (0.12 mL, 1.98 mmol) and carbonyldiimidazole (0.32 g, 1.98 mmol) were dissolved in THF (5 mL) and stirred at 10 °C for 1 h. 4-(Aminomethyl)benzoic acid (0.30 g, 1.98 mmol), DBU (0.29 mL, 1.98 mmol) and triethylamine (0.27 mL, 1.98 mmol) were suspended in THF (5 mL), the CDI intermediate solution was combined and the reaction was stirred at room temperature for 16 h. The solvent was removed under reduced pressure, suspended in HO (10 mL) and the solution was acidified to pH 5 with 1 M HCl solution. The resulting precipitate was collected by gravity filtration and air-dried to give 38 (0.35 g, 75% yield) as a white powder. HRMS (ESI) m / z: [M+H] + calculated for C 12 H 12 NO4: 234.0766, found 234.0764. tert-Butyl-N-{2-[4-({[(prop-2-yn-1-yloxy)carbonyl]amino}methyl)benzamido]phenyl}carbamate (39): 38 and HATU (0.49 g, 1.28 mmol) were dissolved in dry DMF (5 mL) and stirred at room temperature for 1 h. tert-Butyl-N-(2-aminophenyl)carbamate (0.18 g, 0.85 mmol) and diisopropylethylamine (0.30 mL, 1.71 mmol) were added and the reaction was stirred at room temperature for 16 h. The solvent was removed under reduced pressure and suspended in EtOAc (20 mL). The organic solution was washed with saturated NaHCO3 (2 x 20 mL), brine (20 mL), the organic layers were combined, dried over MgSO4, filtered and the solvent was removed under reduced pressure. The crude oil was purified by flash chromatography on silica (hexane:EtOAc 30:70 to 50:50 gradient) to give 39 (0.28 g, 76% yield) as a pale pink oil. HRMS (ESI) m / z: [M+H] + calculated for C 23H 26 N3O5:424.1872, found 424.1871. 8-(4-(((4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)benzyl)carbamoyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)octanoic acid (40): 39 (0.13 g, 0.29 mmol), 8-azido-octanoic acid (0.05 g, 0.29 mmol) and CuI (0.01 g, 0.10 mmol) were suspended in degassed DMF:MeCN (5 mL, 2:1) under inert atmosphere. 2,6-Lutidine (0.06 g, 0.52 mmol) was added and the reaction was stirred at room temperature for 16 h. The solvent was removed under reduced pressure, suspended in EtOAc (10 mL) and washed with saturated NaHCO3. NaHCO3 (2 x 10 mL), brine (2 x 10 mL), the organic layers were combined, dried over MgSO4, filtered and the solvent removed under reduced pressure. The crude oil was purified by flash chromatography on silica (3-5% MeOH:DCM solvent gradient) to give 40 (0.08 g, 46%) as an off-white solid. HRMS (ESI) m / z: [M+Na] + calculated for C 31 H 40 N6O7Na: 631.2856, found 631.2856. (1-(8-(((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)-8-oxooctyl)-1H-1,2,3-triazol-4-yl)methyl (4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)benzyl)carbamate (41): Following general procedure G, 41 was obtained from 40 (0.075 g, 0.12 mmol) and VH_032 amine (0.05 g, 0.10 mmol). The crude product was purified by flash chromatography on silica (2-7% MeOH:DCM solvent gradient) to give 41 (0.08 g, 78%) as an off-white crystalline solid. HRMS (ESI) m / z: [M+Na] +calculated for C 53 H 68 N 10 O9SNa: 1043.4789, found 1043.4758.

[0222] [ka]

[0223] (1-(8-(((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)-8-oxooctyl)-1H-1,2,3-triazol-4-yl)methyl (4-((2-aminophenyl)carbamoyl)benzyl)carbamate (PROTAC29): Boc deprotection of 41 (0.08 g, 0.08 mmol) was carried out according to general procedure H to give PROTAC29 (0.06 g, 84%) as an off-white solid. 1H NMR (400 MHz, Methanol-d4) δH ppm 8.75 (s, 1H, 45-CH), 7.87 (s, 1H, 1-CH), 7.82 (m, 2H, 9-CH), 7.35-7.33 (m, 2H, 41-CH), 7.31-7.27 (m, 4H, 8-CH, 42-CH), 7.08-7.06 (m, 1H, 14-CH), 6.98-6.94 (m, 1H, 16-CH), 6.80-6.77 (m, 1H, 17-CH), 6.67-6.63 (m, 1H, 15-CH), 5.05 (s, 2H, 3-CH2), 4.51 (s, 1H, 29-CH), 4.47-4.39 (m, 2H, 36-CH, 39-CH), 4.38-4.36 (m, 1H, 34-CH), 4.28-4.19 (m, 3H, 20-CH2, 39-CH), 4.25 (s, 2H, 6-CH2), 3.79-3.76 (m, 1H, 33-CH), 3.69-3.65 (m, 1H, 33-CH), 2.35 (s, 3H, 47-CH3), 2.17-2.06 (m, 3H, 26-CH2, 35-CH), 1.99-1.92 (m, 1H, 35-CH), 1.80-1.73 (m, 2H, 0.91 (s, 9H, 31-CH3). HRMS (ESI) m / z: [M+Na] + calculated for C 48 H 60 N 10 O7SNa: 943.4265, found 943.4246. Synthesis of PROTAC4 negative controls using inactive isomers of VHL ligands

[0224] [ka]

[0225] tert-Butyl (2-(4-(12-(((S)-1-((2S,4S)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)))pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-12-oxododecaneamido)benzamido)phenyl)carbamate (27a*): To a solution of 26a (51.4 mg, 0.095 mmol) in anhydrous DMF (1 mL) at 0 °C was added DIPEA (0.04 mL, 0.238 mmol) and HATU (39.3 mg, 0.103 mmol). The reaction mixture was stirred for 15 min, then a solution of (2S,4S)-1-[(2S)-2-amino-3,3-dimethyl-butanoyl]-4-hydroxy-N-[[4-(4-methylthiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide dihydrochloride (VH_032*amine, 40.0 mg, 0.079 mmol) in DMF (1 mL) was added slowly, and the resulting solution was stirred at room temperature for 16 h. The reaction mixture was diluted with EtOAc (10 mL) and then washed with saturated NaHCO3 (2 x 5 mL) and saturated NaCl (2 x 5 mL). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo to give a dark yellow tar (104 mg). The crude product was purified by column chromatography (0-5% MeOH in DCM) to give 27a* (50.8 mg, 0.053 mmol, 67% yield) as a white solid. HRMS (ESI) m / z: [M+H] + calculated for C 52 H 70 N7O8S: 952.5007, found 952.4999.

[0226] [ka]

[0227] N1-(4-((2-aminophenyl)carbamoyl)phenyl)-N12-((S)-1-((2S,4S)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)dodecanediamide (NC-PROTAC4): Boc deprotection of 27a* (29.0 mg, 0.030 mmol) was carried out according to general method H to afford NC-PROTAC4 (25.6 mg, 0.029 mmol, 98% yield) as a pale yellow solid. Prior to biological evaluation, the product was further purified by semi-preparative HPLC (5–95% MeCN in HO, 260 nm, 45 min gradient). 1 H NMR (400 MHz, Methanol-d4) δ Hppm 8.86 (s, 1 H, 39-CH) 7.95 (d, J=8.7 Hz, 2 H, 15-CH) 7.72 (d, J=8.7 Hz, 2 H, 14-CH) 7.44 (d, J=8.5 Hz, 2 H, 36-CH) 7.40 (d, J=8.5 Hz, 2 H, 35-CH) 7.18 (dd, J=7.8, 1.3 Hz, 1 H, 23-CH) 7.07 (app. td, J=7.8, 1.3 Hz, 1 H, 21-CH) 6.90 (dd, J=7.8, 1.3 Hz, 1 H, 20-CH) 6.76 (app. td, J=7.8, 1.3 Hz, 1 H, 22-CH), 4.47 - 4.58 (m, 3 H, 24-CH,31-CH,33-CH) 4.32 - 4.40 (m, 2 H, 29-CH,33-CH) 4.03 (dd, J=10.5, 5.1 Hz, 1 H, 28-CH) 3.69 (dd, J=10.5, 3.5 Hz, 1 H, 28-CH) 2.47 (s, 3 H, 41-CH3) 2.42 - 2.45 (m, 1 H, 30-CH) 2.40 (t, J=7.4 Hz, 2 H, 11-CH2) 2.18 - 2.32 (m, 2 H, 2-CH2) 1.97 (dt, J=13.3, 4.3 Hz, 1 H, 30-CH) 1.70 (quin, J=7.4 Hz, 2 H, 10-CH2) 1.53 - 1.63 (m, 2 H, 3-CH2) 1.33 - 1.39 (m, 4 H, 4-CH2,9-CH2) 1.29 - 1.33 (m, 8 H, (5-8)-CH2) 1.03 (s, 9 H, 26-CH3). HRMS (ESI) m / z: [M+H] + calculated for C 47 H 62 N7O6S: 852.4482, found 852.4483. In vitro HDAC assay using CoREST complex Inhibition studies against the LSD1-HDAC-CoREST complex were performed using a two-enzyme fluorescence-based HDAC assay. Inhibitors / PROTACs were dissolved in DMSO at 50 mM and serially diluted 1:2 using HDAC assay buffer (50 mM Tris pH 7.5, 150 mM NaCl) to obtain a concentration range. 10 μL of these solutions were added to individual wells, followed by the addition of 20 μL of HDAC complex dissolved in HDAC assay buffer (18 nM) and 20 μL of the substrate Boc-(Ac)Lys-AMC dissolved in HDAC assay buffer.

[0228] The assay was performed in black 96-well plates with a reaction volume of 50 μL per well. All determinations were performed in triplicate. Control wells without inhibitor were used to determine when inhibition ceased. After 20 min of incubation at 37°C and 150 rpm, deacetylation was stopped by adding 50 μL of developer solution containing trypsin (50 mM Tris pH 7.5, 100 mM NaCl, 10 mg / mL trypsin). Fluorescence intensity was measured using a plate reader (PerkinElmer, 2030 Multilabel Reader, VICTOR X5, λ ex 335nm, λ em Fluorescence was measured at 460 nm. HDAC activity was calculated by subtracting the mean blank fluorescence from the well fluorescence. IC50 values ​​were determined using Graphpad Prism software.

[0229] Cell lines and materials E14 wild-type (WT) mouse embryonic stem (mES) cells were maintained on gelatinized plates in standard mES medium consisting of knockout Dulbecco's modified Eagle's medium (KO DMEM) (GIBCO, 10829-018) supplemented with 15% fetal bovine serum (FBS) (Sigma, F9665), 1X glutamine / penicillin / streptomycin (GIBCO, 10378-016), 100 μM β-mercaptoethanol (Sigma) and leukemia inhibitory factor (synthesized in-house). HCT116 human colon carcinoma cells were grown in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, 41965-039) supplemented with 10% fetal bovine serum (FBS) (Sigma) and 1X glutamine / penicillin / streptomycin (GIBCO, 10378-016). Both cell lines were incubated at 37 °C with 5% CO. Cells were treated with PROTACs (0.01-40 μM) along with HDAC inhibitors CI-994 (10 / 40 μM) and panobinostat (30 nM) as controls.

[0230] Western blotting HCT116 cells or mES cells were treated 24 hours after seeding. After 24 hours of treatment, cells were harvested and lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% NP-40, 0.5% Triton X-100) containing protease inhibitors (Sigma, P8340), incubated on ice for 30 minutes, and then centrifuged (18,000rcf, 15 minutes, 4°C). The supernatant was collected and protein concentration was quantified by Bradford assay using Protein Assay Dye Reagent Concentrate (BIO-RAD). For histone extraction, an equal volume of 0.4N H2SO4 was added to the pellet and the extract was left at 4°C overnight. After overnight incubation, the tubes were centrifuged (18,000rcf, 15 minutes, 4°C) and the supernatant (histone extract) was collected.

[0231] Western blots were run on NuPAGETM 4-12% Bis-Tris gels loaded with 20-30 μg protein or 10 μL acid-extracted histones per lane using NuPAGETM LDS Sample Buffer (4X). PageRuler Plus Prestained Ladder was used for size standards. After gel electrophoresis at 140V for 75-90 min, separated proteins were transferred to nitrocellulose membranes at 30V for 60 min. Membranes were probed with primary antibodies (listed below) for 60-90 min. Blots were developed with complementary IRDye-conjugated secondary antibodies and bands were visualized using an Odyssey Infrared Imaging System. Image processing and quantification of band intensity were performed using Image Studio Lite.

[0232] Antibody information Primary antibody; α-Tubulin - Sigma, t5168 (1:10,000 dilution) HDAC1-Abcam, 109411 (1:2,000 dilution) HDAC2-Merck Millipore, 05-814 (1:2,000 dilution) HDAC3-Abcam, 32369 (1:2,000 dilution) H3-Merck Millipore, 05-499 (1:1,000 dilution) H3K9Ac-Upstate, 06-942 (1:1,000 dilution) H3K27Ac-Merck Millipore, 07-360 (1:1,000 dilution) H3K56Ac-active motif, 39281 (1:1,000 dilution) secondary antibody; IRDye® 680LT-LI-COR Biosciences, 926-68023 (1:10,000 dilution) IRDye® 800CW-LI-COR Biosciences, 926-32210 (1:10,000 dilution) Cell viability assay To analyze cell death, cells were treated with DMSO, CI-994 (40 μM), or PROTAC4 (1–40 μM) 24 h after seeding. After 24 h of treatment, cells were harvested and fixed with 70% (vol / vol) ethanol overnight at -20 °C. Cells were washed with PBS and then incubated with 50 μg of propidium iodide and RNase A (10 μg / mL) for 30 min at room temperature in the dark. Samples were analyzed using a BD FACSCanto II flow cytometer (BD Biosciences) in the PE_A channel with BD FACSDiva software.

Claims

1. A compound of formula (I) or a pharma- ceutically acceptable salt, solvate, or tautomeric form thereof: 【Chemistry 1】 During the ceremony, L is a linker that is -L 1 -L 2 -, the backbone length of said linker being 7 to 50 atoms, wherein L 1 is absent or -L 3 -L 4 -L 5 -L 6 -; L 2 is -L 7 -L 8 -L 9 -L 10 -, where L 3 is an optionally substituted C 1 -C 6 alkylene, an optionally substituted C 2 -C 6 alkenylene, or an optionally substituted C 2 -C 6 alkynylene; L4 is NR5, O, S, 【Chemistry 2】 wherein the asterisk indicates the point of attachment to L 5 , or to L 6 if L 5 is absent; and L 5 is absent, optionally substituted C 1 -C 6 alkylene, optionally substituted C 2 -C 6 alkenylene, or optionally substituted C 2 -C 6 alkynylene, optionally substituted C 6 -C 12 arylene, or optionally substituted 5-10 membered heteroarylene; L 6 is an optionally substituted C 6 -C 12 arylene or an optionally substituted 5-10 membered heteroarylene; L 7 is absent, optionally substituted C 1 -C 6 alkylene, optionally substituted C 2 -C 6 alkenylene, or optionally substituted C 2 -C 6 alkynylene; L 8 is absent or 【Chemistry 3】 wherein the asterisk indicates the point of attachment to L 9 ; and L 9 is an optionally substituted C 1 -C 20 alkylene, an optionally substituted C 2 -C 20 alkenylene, or an optionally substituted C 2 -C 20 alkynylene, the backbone of said alkylene, alkenylene, or alkynylene group being optionally interrupted by one or more heteroatoms selected from O or NR 6 , or L9 is 【Chemistry 4】 wherein the asterisk indicates the point of attachment to L 10 , or to R 1 if L 10 is absent; and L 10 is absent, C(O) or: 【Chemistry 5】 where the asterisk indicates the point of attachment to R 1 . and L 11 is independently absent, optionally substituted C 1 -C 5 alkylene, optionally substituted C 2 -C 5 alkenylene, or optionally substituted C 2 -C 5 alkynylene; L 12 and L 13 are independently an optionally substituted C 1 -C 5 alkylene, an optionally substituted C 2 -C 5 alkenylene, or an optionally substituted C 2 -C 5 alkynylene; X 8 -X 12 are independently O or NR 6 ; R 5 and R 6 are independently H, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl; m is an integer from 0, 1 to 10; and n is an integer from 1 to 10, R １ is an E3 ligand for E3 ligase, and R 1 is any one of the following: 【Chemistry 6】 During the ceremony, X 1 to X 7 and X 13 are each NH or O; R 7 is H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted 3-6 membered heterocycle, optionally substituted phenyl, or optionally substituted 5- or 6-membered heteroaryl, where when R 7 is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocycle, optionally substituted phenyl, or optionally substituted heteroaryl, said alkyl, said alkenyl, said alkynyl, said cycloalkyl, said heterocycle, said phenyl, or said heteroaryl is unsubstituted or substituted with one or more of halogen, oxo, OH, or NH 2 ; Each R ２ are independently halogen, OR ３ , N.R. ３ R ４ , C １ ~C ６ Alkyl, C ２ ~C ６ Alkenyl, C ２ ~C ６ Alkynyl, C ６ ~C １２ an aryl group, or a 5-10 membered heteroaryl group; R ３ and R ４ are independently H, C １ ~C ６ Alkyl, C ２ ~C ６ Alkenyl, or C ２ ~C ６ alkynyl; Unless otherwise specified, the or each optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, and optionally substituted heteroarylene is unsubstituted or substituted with one or more of halogen, oxo, OH, NH 2 , C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 6 cycloalkyl, 3-10 membered heterocycle, C 6 -C 12 aryl and / or 5-10 membered heteroaryl, and the or each optionally substituted arylene is unsubstituted or substituted with one or more of halogen, OH, NH 2 , C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 6 cycloalkyl, 3-10 membered heterocycle, C 6 -C 12 substituted with one or more of aryl and / or 5-10 membered heteroaryl; p is 0 or an integer from 1 to 4;

2. The compound of claim 1, wherein p is 0 or p is 1 and the compound of formula (I) is a compound of formula (Iaiii) or (Iaiv): 【Chemistry 7】

3. R １ but: 【Chemistry 8】 3. The compound according to claim 1 or 2,

4. X １ , X ２ and X ７ is NH, and X ３ and X １３ is O, and R ７ is methyl or: 【Chemistry 9】 The compound according to any one of claims 1 to 3,

5. The compound according to any one of claims 1 to 4, wherein the backbone of the linker is 7 to 40 atoms in length.

6. L １ but: 【Chemistry 10】 where the asterisk represents L ２ indicates the point of attachment to or L １ The compound according to any one of claims 1 to 5, wherein is absent.

7. L ２ but: 【Chemistry 11】 where the asterisk represents R １ indicates the point of attachment to The compound according to any one of claims 1 to 6,

8. L ２ but: 【Chemistry 12】 During the ceremony, The asterisk indicates R. １ indicates the point of attachment to q is an integer of at least 5; r is an integer of at least 4; s is an integer of at least 2; t is an integer of at least 1; u is an integer of at least 3; v is an integer of at least 1; and w is an integer of at least 2; 8. The compound of claim 7,

9. q is an integer from 5 to 20; r is an integer from 4 to 20; s is an integer from 2 to 10; t is an integer from 1 to 10; u is an integer from 3 to 15; and The compound according to claim 8, wherein w is an integer from 2 to 14.

10. L is: 【Chemistry 13】 During the ceremony, The asterisk indicates R. １ indicates the point of attachment to q is an integer of at least 5; r is an integer of at least 4; s is an integer of at least 2; t is an integer of at least 1; u is an integer of at least 3; v is an integer of at least 1; and w is an integer of at least 2; The compound according to any one of claims 1 to 9,

11. q is an integer from 5 to 20; r is an integer from 4 to 20; s is an integer from 2 to 10; t is an integer from 1 to 10; u is an integer from 3 to 15; and The compound according to claim 9 or claim 10, wherein w is an integer from 2 to 14.

12. The compound of claim 1, wherein the compound of formula (I) is a compound of formula (Ic), (Id), (Ie) or (If): 【Chemistry 14】

13. 13. The compound of claim 12, which is a compound of formula (Ic) and q is 11 or a compound of formula (Id) and r is 10.

14. The compound according to claim 1, which is a compound represented by any one of formulas (101) to (116): 【Chemistry 15-1】 【Chemistry 15-2】 【Chemistry 15-3】 【Chemistry 15-4】

15. A pharmaceutical composition comprising a compound of formula (I) as claimed in any one of claims 1 to 14, or a pharma- ceutically acceptable complex, salt, solvate, or tautomeric form thereof, and a pharma- ceutically acceptable vehicle.

16. 15. A pharmaceutical composition for treating cancer in a subject comprising a compound of formula (I) as defined in any one of claims 1 to 14, or a pharma- ceutically acceptable complex, salt, solvate, or tautomeric form thereof, and a pharma- ceutically acceptable vehicle.

17. 17. The pharmaceutical composition of claim 16, wherein the cancer is blood cancer, intestinal cancer, brain cancer, breast cancer, cervical cancer, endometrial cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.

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