Proteolytic targeting chimera (PROTAC) compositions using ubiquitin conjugating enzyme ligands

Abstract

Provided herein are E2 binding PROTACs and uses thereof.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 332,305, filed on April 19, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] Proteolysis targeting chimeras (PROTACs) have become an attractive technology for regulating proteins of interest (POIs). PROTACs are heterobifunctional small molecules with the following three chemical elements: a ligand that binds to a target protein, a ligand that binds to an E3 ubiquitin ligase, and a linker for conjugating the two ligands. See, for example, Sun et al., SigTransduct Target Ther 4, 64 (2019). Unlike the competitive and occupancy-driven processes of traditional inhibitors, PROTACs have a catalytic mode of action and have the potential to degrade target pathogenic proteins and regulate related signal transduction pathways. Several PROTACs targeting about 50 proteins (many of which are clinically validated drug targets) have been developed in clinical trials for the treatment of cancer and other diseases.

[0004] However, the problem with current PROTACs is that they are limited to engaging E3 ubiquitin ligases, which are prone to high mutation. Recent studies have shown that acquired resistance to PROTACs is associated with mutations in core components of E3 ligases. (Zhang et al., Mol Cancer Ther. 2019 July; 18(7): 1302-1311. doi: 10.1158 / 1535-7163. MCT-18-1129. Epub 2019 May 7). Therefore, alternative approaches to traditional PROTAC technology are needed. Summary of the invention

[0005] It has now been discovered that ubiquitin-binding enzymes (E2) (e.g., UBE2K) can be used as effective targets in the design and use of PROTACs. Unlike E3 ligases, E2s (such as UBE2K) have very low mutation frequencies, thus reducing the risk of PROTAC acquired resistance in clinical practice.

[0006] Therefore, compounds and compositions for recruiting target proteins to E2 enzymes (e.g., UBE2K) for ubiquitination and subsequent degradation, and methods of using the same, are provided herein. In one aspect, the disclosure provides PROTAC compounds with E2 enzyme binding moieties, target protein binding moieties, and linkers. In one aspect, the polyubiquitination activity and / or increased continuous synthesis capacity of the E2 enzyme are independent of the E3 ligase. In one aspect, the E2 enzyme binding moiety is used to stabilize E2-E2 dimers (e.g., homodimers or heterodimers).

[0007] Also provided are uses of the disclosed PROTACs for degrading a target protein of interest.

[0008] Further provided are uses of the disclosed PROTACs for treating a disease, such as cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The E2 binding ability of PROTACs based on the present disclosure is shown.

[0010] Figure 2 Dose-dependent degradation of BRD4 in HeLa cells using E2-binding PROTACs based on the present disclosure is shown. DETAILED DESCRIPTION

[0011] Provided herein are PROTAC compounds comprising an E2 binding portion, a target protein binding portion, and a linker that covalently links the E2 binding portion to the target protein binding portion.

[0012] As used herein, "E2 binding portion", "E2 binder" or "E2 ligand" refers to a chemical portion that binds an E2 enzyme. E2 binding portions include, but are not limited to, E2 binding portions that bind an E2 enzyme selected from the group consisting of UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2G1, UBE2G2, UBE2H, UBE2J1, UBE2J2, UBE2K, UBE2L3, UBE2N, UBE2NL, UBE2O, UBE2Q1, UBE2Q2, UBE2QL, UBE2R1, UBE2R2, UBE2S, UBE2T, UBE2U, UBE2V1, UBE2V2, UBE2W, and BIRC6. In one aspect, the E2 enzyme to which the E2 ligand binds is UBE2K.

[0013] In one aspect, as part of the first chemical embodiment, the E2 binding moiety is a compound of formula I:

[0014]

[0015] in

[0016] Z 1 and Z 2 are each independently N or CH;

[0017] R 1 is (C1-C6)alkyl, halogenated (C1-C6)alkyl, (C1-C6)alkoxy, halogenated (C1-C6)alkoxy or –NR c R d wherein the two available hydrogen atoms on the halo(C1-C6)alkyl and halo(C1-C6)alkoxy groups may be taken together with the carbon atoms to which they are attached to form a 3- to 6-membered cycloalkyl group optionally substituted with 1 to 3 groups selected from the group consisting of halo, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy and halo(C1-C6)alkoxy;

[0018] R 2 is CN, halo, OH, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy or halo(C1-C6)alkoxy; or R 1 and R 2 When on adjacent carbon atoms, together with the carbon atoms to which they are attached, form a 5- or 6-membered oxygen-containing heterocyclic group optionally substituted with 1 to 3 groups selected from halo, (C1-C6)alkyl and halo(C1-C6)alkyl;

[0019] R 3 is hydrogen, (C1-C6)alkyl or halogenated (C1-C6)alkyl;

[0020] Y is CH2, -CHR a , -CR a R b , S or SO;

[0021] p is 0 or 1;

[0022] R a and R b are each independently halo, (C1-C6)alkyl or halo(C1-C6)alkyl; or R a and R b Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl or a 3- to 6-membered heterocyclyl, each of which is optionally substituted with 1 to 3 groups selected from halo, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, (C1-C6)alkylOH, (C1-C6)alkylO(C1-C6)alkyl and OH;

[0023] R c and R d each independently represents hydrogen, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkylO(C1-C6)alkyl, halo(C1-C6)alkylO(C1-C6)alkyl, (C1-C6)alkyl-O-halo(C1-C6)alkyl, halo(C1-C6)alkyl-O-halo(C1-C6)alkyl or (C1-C6)alkylOH; or R c and R d Together with the nitrogen atom to which they are attached, they form a 4- to 7-membered heterocyclyl group optionally substituted with 1 to 3 groups selected from the group consisting of halo, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy and oxo;

[0024] Z is a tricyclic fused ring having the formula:

[0025]

[0026] Ring A is aromatic;

[0027] The wavy bond on ring A indicates the point of attachment to Y;

[0028] The wavy bond next to the W indicates the connection point to the connector (L);

[0029] When the valence permits, X, X 1 and X 2 Each independently selected from -CR 7 , N, O and S;

[0030] The dashed lines in ring B represent single or double bonds;

[0031] W is NH, -N(C1-C6) alkyl), O or S;

[0032] R 5 is selected from hydrogen, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, -S(C1-C6)alkyl, -SH, OH, (C3-C6)cycloalkyl, (C4-C7)heterocyclyl, and -NR e R f wherein the (C3-C6)cycloalkyl and (C4-C7)heterocyclyl are each optionally substituted with 1 to 3 groups selected from the group consisting of halo, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy and oxo;

[0033] When the dashed line in ring B is a single bond, R6 is hydrogen or (C1-C6) alkyl, or when the dotted line in ring B is a double bond, R 6 does not exist;

[0034] d, d1, d2 and d3 are each independently selected from CR 8 and N;

[0035] R e and R f Each is independently hydrogen, (C1-C4)alkyl, (C1-C4)alkylNH(C1-C4)alkyl, (C1-C4)alkylN[(C1-C4)alkyl]2, (C1-C4)alkylO(C1-C4)alkyl;

[0036] R 7 is hydrogen or (C1-C6)alkyl; and

[0037] R 8 is halogen, hydrogen, (C1-C6)alkyl, halo(C1-C6)alkyl, CN or OH, wherein the linker (L) and the target protein binding portion are as defined herein.

[0038] When used in conjunction to describe a chemical group that may have multiple points of attachment, the hyphen (-) indicates the point of attachment of the group to the variable defining it. For example, -NH(C1-C6)alkyl means that the point of attachment of the group is at the nitrogen atom.

[0039] The terms "halo" and "halogen" refer to an atom selected from fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br) and iodine (iodo, -I).

[0040] The term "alkyl" when used alone or as part of a larger moiety (such as "haloalkyl"), means a saturated straight or branched monovalent hydrocarbon radical. Unless otherwise specified, an alkyl group typically has 1 to 4 carbon atoms, i.e., (C1-C4) alkyl.

[0041] "Alkoxy" means an alkyl group represented by -O-alkyl attached through an oxygen linking atom. For example, "(C1-C4)alkoxy" includes methoxy, ethoxy, propoxy, and butoxy.

[0042] The term "haloalkyl" embraces monohaloalkyl groups, polyhaloalkyl groups, and perhaloalkyl groups, wherein the halogen is independently selected from fluorine, chlorine, bromine, and iodine.

[0043] "Haloalkoxy" is a haloalkyl group attached to another moiety through an oxygen atom such as, for example but not limited to, -OCHCF2 or -OCF3.

[0044] "Oxo" refers to a divalent functional group =0, ie, an oxygen atom attached to another atom, usually carbon or sulfur, by a double bond.

[0045] The term "heteroaryl" refers to an aromatic ring of a specified size (e.g., 5-, 6-, 7-, 8- or 9-membered ring) containing 1 to 4 heteroatoms independently selected from N, O and S. The heteroaryl group can be monocyclic or bicyclic. Monocyclic heteroaryl includes, for example, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, etc. Bicyclic heteroaryl includes a group in which a monocyclic heteroaromatic ring is fused to one or more aryl or heteroaromatic rings. Non-limiting examples include indolyl, imidazopyridyl, benzoxazolyl, benzooxydiazolyl, indazolyl, benzimidazolyl, benzothiazolyl, pyrazolopyridyl, thienopyridyl, thienopyrimidinyl, indolizinyl, etc. When specified, optional substituents on a heteroaryl group may be present at any substitutable position.

[0046] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic ring containing 1 to 4 heteroatoms of a specified size (e.g., 3, 4, 5, 6 or 7-membered ring) independently selected from N, O and S. The heterocyclyl ring can be connected to its side group at any heteroatom or carbon atom forming a stable structure. Examples of such saturated or partially unsaturated heterocyclyls include but are not limited to oxirane, thiirane, aziridine, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, pyrrolidinyl, pyridone, pyrrolidinone, piperidinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, morpholinyl, dihydrofuranyl, dihydropyranyl, dihydropyridyl, tetrahydropyridyl, dihydropyrimidinyl, oxetanyl, azetidinyl and tetrahydropyrimidinyl. When specified, optional substituents on a heterocyclyl group may be present at any substitutable position and include, for example, the position at which the heterocyclyl is attached.

[0047] The term "cycloalkyl" refers to a monocyclic hydrocarbon of a specified size (e.g., 3-, 4-, 5-, 6-, or 7-ring). Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, and cyclooctyl. When specified, the optional substituents on the cycloalkyl group may be present in any substitutable position, and include, for example, positions that connect the cycloalkyl.

[0048] Unless otherwise stated, structures depicted herein are also intended to encompass all isomeric (e.g., enantiomers, diastereomers, and geometric (or conformational)) forms of the structure; for example, R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the present invention. Unless otherwise stated, all tautomeric forms of the present compounds are within the scope of the present invention.

[0049] The compounds described herein may exist in the form of pharmaceutically acceptable salts. For use in medicine, the salts of the compounds described herein refer to non-toxic "pharmaceutically acceptable salts". Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic / anionic or alkaline / cationic salts. Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include, for example, salts of inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid and sulfuric acid) and organic acids (such as acetic acid, benzenesulfonic acid, benzoic acid, methanesulfonic acid and p-toluenesulfonic acid). The compounds taught by the present invention having acidic groups such as carboxylic acids can form pharmaceutically acceptable salts with pharmaceutically acceptable bases. Suitable pharmaceutically acceptable alkaline salts include, for example, ammonium salts, alkali metal salts (such as sodium and potassium salts) and alkaline earth metal salts (such as magnesium and calcium salts). Compounds with quaternary ammonium groups also contain counter anions, such as chloride, bromide, iodide, acetate, perchlorate, etc. Other examples of such salts include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, benzoates, and salts containing amino acids such as glutamic acid.

[0050] In one aspect, as part of the second chemical embodiment, the E2 binding moiety is a compound of formula II:

[0051]

[0052] wherein the variables are as described above for Formula I, and wherein the linker (L) and the target protein binding moiety are as defined herein.

[0053] In one aspect, as part of the third chemical embodiment, W in the compound of Formula I or Formula II is S, wherein the variables are as described above for Formula I, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0054] In one aspect, as part of the fourth chemical embodiment, R in the compound of formula I or formula II 3 is hydrogen, wherein the variables are as described above for Formula I or the third chemical embodiment, and wherein the linker (L) and the target protein binding moiety are as defined herein.

[0055] In one aspect, as part of the fifth chemical embodiment, Z in the compound of formula I or formula II 1 and Z 2 Each is CH, wherein the variables are as described above for Formula I or the third chemical embodiment or the fourth chemical embodiment, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0056] In one aspect, as part of the sixth chemical embodiment, Y in the compound of Formula I or Formula II is CH2, wherein the variables are as described above for Formula I or any one of the third to fifth chemical embodiments, wherein the linker (L) and the target protein binding portion are as defined herein.

[0057] In one aspect, as part of the seventh chemical embodiment, p in the compound of Formula I or Formula II is 0, wherein the variables are as described above for Formula I or any one of the third to sixth chemical embodiments, wherein the linker (L) and the target protein binding portion are as defined herein.

[0058] In one aspect, as part of the eighth chemical embodiment, R in the compound of formula I or formula II 1 is (C1-C6)alkyl, halo(C1-C6)alkyl, halo(C1-C6)alkoxy, (C1-C6)alkoxy or –NR c R d ; and R c and R d Together with the nitrogen atom to which they are attached, they form a 5- to 6-membered heterocyclyl optionally substituted with 1 to 3 halogens, wherein the variables are as described above for Formula I or any one of the third to seventh chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein. Alternatively, as part of the eighth chemical embodiment, R in the compound of Formula I or Formula II is 1 is (C1-C3)alkyl, halogenated (C1-C3)alkyl, halogenated (C1-C6)alkoxy, (C1-C3)alkoxy or –NR c R d ; and R c and R d Together with the nitrogen atom to which they are attached, they form a 5- to 6-membered nitrogen-containing heterocyclic group optionally substituted with 1 to 3 halogens, wherein the variables are as described above for Formula I or any one of the third to seventh chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein. In another alternative, as part of the eighth chemical embodiment, R in the compound of Formula I or Formula II is 1is OCF3, OCHF2, OCH3, CH3, pyrrolidinyl or piperidinyl, wherein the pyrrolidinyl or piperidinyl is optionally substituted with 1 to 3 halogenations, wherein the variables are as described above for Formula I or any one of the third to seventh chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0059] In one aspect, as part of the ninth chemical embodiment, X in the compound of Formula I or Formula II is N, wherein the variables are as described above for Formula I or any one of the third to eighth chemical embodiments, wherein the linker (L) and the target protein binding portion are as defined herein.

[0060] In one aspect, as part of the tenth chemical embodiment, X in the compound of formula I or formula II is 1 is CH or N, wherein the variables are as described above for Formula I or any one of the third to ninth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0061] In one aspect, as part of the eleventh chemical embodiment, X in the compound of formula I or formula II is 2 is CH, N, S or O, wherein the variables are as described above for Formula I or any one of the third to tenth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0062] In one aspect, as part of the twelfth chemical embodiment, in the compound of Formula I or Formula II, X is N, X1 is N, and X2 is CH; X is N, X 1 is N, and X 2 is N; X is N, X 1 is CH, and X 2 is S; or X is N, X 1 is CH, and X 2 is O, wherein the variables are as described above for Formula I or any one of the third to eleventh chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0063] In one aspect, as part of the thirteenth chemical embodiment, d in the compound of formula I or formula II is N or -CR 8 , wherein the variables are as described above for Formula I or any one of the third to twelfth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0064] In one aspect, as part of the fourteenth chemical embodiment, d 2 N or -CR 8, wherein the variables are as described above for Formula I or any one of the third to thirteenth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0065] In one aspect, as part of the fifteenth chemical embodiment, the compound of formula I or formula II wherein d 1 For-CR 8 , wherein the variables are as described above for Formula I or any one of the third to fourteenth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0066] In one aspect, as part of the sixteenth chemical embodiment, d 3 For-CR 8 , wherein the variables are as described above for Formula I or any one of the third to fifteenth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0067] In one aspect, as part of the seventeenth chemical embodiment, Z in the compound of formula I or formula II is selected from

[0068] wherein the variables are as described above for Formula I or any one of the third to sixteenth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein. Alternatively, as part of the seventeenth chemical embodiment, Z in the compound of Formula I or Formula II is selected from

[0069]

[0070] wherein the variables are as described above for Formula I or any one of the third to sixteenth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0071] In one aspect, as part of the eighteenth chemical embodiment, R in the compound of formula I or formula II 8 is hydrogen or halogen, wherein the variables are as described above for Formula I or any one of the third to seventeenth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein. Alternatively, as part of the eighteenth chemical embodiment, R in the compound of Formula I or Formula II is 8 is hydrogen, wherein the variables are as described above for Formula I or any one of the third to seventeenth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0072] In one aspect, as part of the nineteenth chemical embodiment, R in the compound of formula I or formula II5 is hydrogen, wherein the variables are as described above for Formula I or any one of the third to eighteenth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0073] In one aspect, as part of the twentieth chemical embodiment, R in the compound of formula I or formula II 6 is selected from hydrogen and (C1-C4)alkyl, wherein the variables are as described above for Formula I or any one of the third to nineteenth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein. Alternatively, as part of the twentieth chemical embodiment, R in the compound of Formula I or Formula II is 6 is selected from hydrogen and methyl, wherein the variables are as described above for Formula I or any one of the third to nineteenth chemical embodiments, wherein the linker (L) and the target protein binding moiety are as defined herein.

[0074] In one aspect, as part of the twenty-first chemical embodiment, the E2 binding moiety is a compound of formula III:

[0075]

[0076] Where d, d1, d2 and d3 are CR 8 , and wherein the variables are as described above for Formula I, and wherein the linker (L) and the target protein binding portion are as defined herein.

[0077] In one aspect, as part of the twenty-second chemical embodiment, p in the compound of formula III is 0, wherein the variables are as described above for formula I, and wherein the linker (L) and the target protein binding moiety are as defined herein.

[0078] In one aspect, as part of the twenty-third chemical embodiment, the E2 binding moiety has the structure:

[0079]

[0080] In one aspect, as part of the twenty-third chemical embodiment, Z in the compound of formula III is S, wherein the variables are as described above for formula I or the twenty-second embodiment, and wherein the linker (L) and the target protein binding moiety are as defined herein.

[0081] In one aspect, as part of the twenty-fourth chemical embodiment, the E2 binding moiety is selected from the moieties disclosed in US Provisional 62 / 223,626.

[0082] As used herein, "target protein binding moiety" refers to a chemical moiety that binds a protein of interest (POI). POIs of the disclosed compounds include, but are not limited to, BRD4, STAT3, BCL2, and WRN. In one aspect, the POI is BRD4.

[0083] In one aspect, the target protein binding portion disclosed herein (e.g., in any one of the first to twenty-fourth embodiments above) has the following structure:

[0084]

[0085] The wavy bond indicates the point of connection to the linker (L).

[0086] The term "linker" refers to a chemical moiety that covalently links the E2 binding portion to the target protein binding portion. In one aspect, the linker is not cleavable in vivo. In one aspect, the linker includes optimal spatial and chemical properties to achieve optimal therapeutic activity. In one aspect, the linker does not interfere with the ability of the E2 binding portion and / or the target protein binding portion to bind to its corresponding target. In one aspect, the linker includes an optionally substituted straight or branched chain alkyl group, which is optionally interspersed with one or more heteroatoms selected from O, N and S. In one aspect, the linker is a straight or branched chain alkyl group substituted with one or more oxo groups (=O) and is interspersed with one or more heteroatoms selected from O and N.

[0087] In one aspect, a linker (L) as disclosed herein (eg, in any one of the first to twenty-fourth embodiments described above) has the following structure:

[0088] wherein the asterisk (*) indicates the point of connection to the E2 binding moiety; e is an integer from 0 to 4 (e.g., 0, 1 or 2); and j is an integer from 0 to 6 (e.g., 1). Alternatively, the linker (L) as disclosed herein (e.g., in any one of the first to twenty-fourth embodiments described above) is selected from one of the following structures:

[0089]

[0090] Additional compounds are disclosed in the examples and are included in the present disclosure, including pharmaceutically acceptable salts and neutral forms thereof.

[0091] Use, preparation and application

[0092] One or more compounds described herein may be present as part of a pharmaceutical composition. Thus, in one aspect, a pharmaceutical composition is provided, comprising a disclosed compound or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

[0093] The term "pharmaceutically acceptable" refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound formulated with it. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin).

[0094] In certain aspects, pharmaceutical compositions as described herein are formulated to be used for patients who need such compositions. Pharmaceutical compositions as described herein can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implantable reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In certain embodiments, the composition is administered orally, intraperitoneally or intravenously. The sterile injectable form of pharmaceutical compositions as described herein can be aqueous or oily suspensions. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to technology known in the art.

[0095] The compounds and compositions described herein can generally be used to modulate the activity of a target protein (e.g., STAT3, BCL2, WRN, or BRD4). In some aspects, the compounds, pharmaceutically acceptable salts, and pharmaceutical compositions described herein degrade target proteins (e.g., STAT3, BCL2, WRN, or BRD4).

[0096] Thus, provided herein is a method for treating a disease in a subject that is responsive to degradation of a target protein (e.g., STAT3, BCL2, WRN, or BRD4), the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a disclosed compound or a pharmaceutically acceptable salt thereof.

[0097] Also provided are uses of a compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a disclosed compound or a pharmaceutically acceptable salt thereof, for treating a disease that responds to degradation of a target protein (e.g., STAT3, BCL2, WRN, or BRD4).

[0098] Also provided herein is a method for degrading a target protein (e.g., STAT3, BCL2, WRN or BRD4) in a cell, the method comprising introducing into the cell a compound comprising an E2 binding portion, a target protein binding portion (i.e., a binding portion that binds to the target protein to be degraded), and a linker that covalently links the E2 binding portion to the target protein binding portion, thereby degrading the target protein in the cell.

[0099] Also provided herein is a method for degrading a target protein (e.g., STAT3, BCL2, WRN or BRD4) in a subject, the method comprising administering to the subject an effective amount of a compound comprising an E2 binding portion, a target protein binding portion (i.e., a binding portion that binds to the target protein to be degraded) and a linker that covalently links the E2 binding portion to the target protein binding portion, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof, thereby degrading the target protein in the subject.

[0100] In addition, provided herein is a method for treating a disease in a subject that is responsive to degradation of a target protein (e.g., STAT3, BCL2, WRN, or BRD4), the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound comprising an E2 binding portion, a target protein binding portion (i.e., a binding portion that binds to the target protein when degraded to treat the disease), and a linker that covalently links the E2 binding portion to the target protein binding portion, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof, thereby treating the disease.

[0101] Also provided is the use of a compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the disclosed compound or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for treating a disease that responds to degradation of a target protein (e.g., STAT3, BCL2, WRN, or BRD4).

[0102] The terms "subject" and "patient" are used interchangeably and refer to mammals in need of treatment, e.g., companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). Typically, the subject is a human in need of treatment.

[0103] As used herein, the terms "treatment", "treat" and "treating" refer to reversing, alleviating, delaying the onset of a disease or disorder as described herein or one or more of its symptoms, or inhibiting its progression. In some aspects, treatment can be administered after one or more symptoms have occurred, i.e., therapeutic treatment. In other aspects, treatment can be administered in the absence of symptoms. For example, treatment can be administered to susceptible individuals before the onset of symptoms (e.g., according to symptom history and / or according to exposure to a specific organism or other susceptibility factors), i.e., preventive treatment. Treatment can also be continued after symptoms have subsided, for example, to delay their recurrence.

[0104] The term "effective amount" or "therapeutically effective amount" refers to the amount of a compound described herein that will elicit a biological or medical response in a subject, for example, a dosage between 0.01 mg / kg to 100 mg / kg body weight per day.

[0105] The specific dosage and treatment regimen for any particular patient will depend on various factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination and the judgment of the treating physician and the severity of the specific disease being treated. The amount of the compound described herein in the composition will also depend on the specific compound in the composition.

[0106] In some aspects, diseases responsive to degradation of a target protein include, but are not limited to, cancer and other proliferative disorders, inflammatory diseases, sepsis, autoimmune diseases, and viral infections.

[0107] In some aspects, the disease to be treated by the methods of the invention is cancer. Examples of cancers treated using the compounds and methods described herein include, but are not limited to, adrenal cancer, acinar cell carcinoma, acoustic neuroma, acromelanoma, acrohidrosis, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphocytic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatous odontogenic tumor, adenosquamous carcinoma, adipose tissue tumor, adrenocortical carcinoma, adult T-cell leukemia / lymphoma, invasive NK cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft tissue sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, germ cell tumor, bone cancer, Brenner tumor tumor), Brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, chondroma, cementoma, medullary sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, renal clear cell sarcoma, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease disease), desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelioma, dysgerminoma, embryonal carcinoma, endocrine adenoma, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, fetus in fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of bone, glioma, glioblastoma multiforme, glioma, glioma cerebri, glucagonoma, Gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma, hematological malignancies, hepatoblastoma, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, fatal midline cancer, leukemia, leydig cell tumortumor), liposarcoma, lung cancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non-small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary carcinoma of the thyroid, medulloblastoma, melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor tumor), myxoma, multiple myeloma, muscle tissue tumor, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, schwannoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, eye cancer, oligodendroglioma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic neuroma, oral cancer, osteosarcoma, ovarian cancer, superior pulmonary sulcus tumor, papillary thyroid cancer, paraganglioma, pinealoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell tumor, multiblastoma, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma peritonei peritonei), renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, schwannomatosis, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, soft tissue sarcoma, somatostatinoma, sootwart, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease, small intestine cancer, squamous cell carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroid cancer, transitional cell carcinoma, laryngeal cancer, urachal cancer, genitourinary cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, optic pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Watson's tumortumor) and Wilms' tumor.

[0108] In some aspects, the disease to be treated by the methods of the invention is inflammatory pelvic disease, urethritis, sunburn of the skin, sinusitis, pneumonia, encephalitis, meningitis, myocarditis, nephritis, osteomyelitis, myositis, hepatitis, gastritis, enteritis, dermatitis, gingivitis, appendicitis, pancreatitis, cholecystitis, agammaglobulinemia, psoriasis, allergies, Crohn's disease, irritable bowel syndrome, ulcerative colitis, Sjogren's disease, tissue graft rejection, hyperacute rejection of transplanted organs, asthma, allergic rhinitis, chronic obstructive pulmonary disease (COPD), autoimmune polyglandular disease (also known as autoimmune polyglandular syndrome), autoimmune alopecia, pernicious anemia, glomerulonephritis, dermatomyositis, multiple sclerosis, scleroderma, vasculitis, autoimmune hemolytic and thrombocytopenic states, Goodpasture's syndrome (Goodpasture's syndrome), atherosclerosis, Addison's disease, Parkinson's disease, Alzheimer's disease, type I diabetes, septic shock, systemic lupus erythematosus (SLE), rheumatoid arthritis, psoriatic arthritis, juvenile arthritis, osteoarthritis, chronic idiopathic thrombocytopenic purpura, Waldenstrom macroglobulinemia, myasthenia gravis, Hashimoto's thyroiditis, atopic dermatitis, degenerative joint disease, vitiligo, autoimmune hypopituitarism, Guillain-Barre syndrome, Behcet's disease, scleritis, mycosis fungoides, acute inflammatory responses (such as acute respiratory distress syndrome and ischemia / reperfusion injury), and Graves' disease.

[0109] Examples

[0110] The following representative examples are intended to help illustrate the present disclosure and are not intended to, and should not be construed to, limit the scope of the invention. Unless otherwise noted, general starting materials used were obtained from commercial sources or prepared in other examples.

[0111] abbreviation:

[0112]

[0113] Synthesis of N-((5-thiolidene-5,6-dihydropyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (Compound 1):

[0114]

[0115] Step 1: Synthesis of N-((1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide.

[0116] To a stirred solution of 2-(trifluoromethoxy)benzoic acid (1) (25 g, 0.121 mmol) in DMF (250 mL) was added HATU (46.1 g, 0.121) at 0 ° C, followed by (2H-pyrazol-3-yl)methylamine (11.7 g, 0.1213) and DIPEA (39.1 g, 0.303 mmol). The reaction was then stirred at room temperature for 12 h. The mixture was then diluted with water (2.5 L) and extracted with EtOAc (2 x 500 mL). The combined organic layers were washed once with H2O (250 mL), saturated NaHCO3 solution (250 mL), and finally saturated NaCl solution (250 mL). The organic layer was dried over Na2SO4 and concentrated to obtain a crude product. It was then purified by flash column chromatography (eluent: 70% EtOAc / Pet ether) to give N-((lH-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (18.3 g, 53.0%) as an off-white solid. 1 H NMR (400MHz, DMSO) δ12.69-12.58(m,1H),8.92-8.82(m,1H),7.76–7.50(m,3H),7.49–7.27(m,2H),6.15(d,J=12.1Hz,1H),4.42(t,J=11.1Hz,2H). LC-MS m / z(M+H): 286.1.

[0117] Step 2: Synthesis of N-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide.

[0118] To a stirred solution of N-((1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (18.3 g, 64.15 mmol) in toluene (400 mL) was added 3,4-dihydro-2H-pyran (5.39 g, 64.1 mmol) at room temperature. The mixture was then heated at 80 °C for 4 h and concentrated. The residue was diluted with EtOAc (250 mL), washed once with saturated NaHCO3 solution (100 mL) and H2O (100 mL). The organic layer was dried over Na2SO4 and concentrated. The crude product obtained was triturated with petroleum ether (200 mL) and stirred for 12 h, whereupon the precipitated solid was filtered and dried under vacuum to afford N-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (12.57 g, 51.5%) as an off-white solid. 1 H NMR (400MHz, DMSO) δ8.87 (s, 1H), 7.80 (d, J = 2.2Hz, 1H), 7.57 (t, J = 7.0Hz, 2 H),7.46–7.38(m,2H),6.20(d,J=2.2Hz,1H),5.32(d,J=10.3Hz,1H),4.38( d,J=5.9Hz,2H),3.90(d,J=11.0Hz,1H),3.67–3.52(m,1H),2.07(dd,J=24. 7,11.0Hz,1H),1.98–1.80(m,2H),1.65(s,1H),1.51(d,J=3.5Hz,2H); LC-MS m / z(M+H):370.1.

[0119] Step 3: Synthesis of N-((5-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide.

[0120] To a stirred solution of N-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (18.2 g, 49.30 mmol) in anhydrous THF (200 mL) was added 1.6 M n-butyl lithium in hexane (6.31 g, 98.61 mmol) at -78 °C over a period of 10 min. The mixture was then stirred at the same temperature for 1 h, followed by anhydrous THF (200 mL) containing iodine (13.76 g, 54.2 mmol) over a period of 15 min. After the iodine addition was complete, the reaction mixture was slowly warmed to -20 °C over a period of 45 min. The reaction was then quenched with saturated NaHSO solution (200 mL) and extracted with EtOAc (2 x 150 mL). The combined organic layers were dried over NaSO and concentrated on a rotary evaporator to give the crude compound. The crude compound was purified by flash column chromatography (eluent: 20% EtOAc + Pet ether) to give N-((5-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (12.57 g, 51.5%) as an off-white solid. 1 H NMR(400MHz,DMSO)δ8.91(t,J=5.6Hz,1H),7.63–7.53(m,2H),7.46-7.41(m ,2H),6.43(s,1H),5.33(d,J=9.8Hz,1H),4.36(d,J=5.6Hz,2H),3.90(d,J= 10.9Hz,1H),3.61-3.58(m,1H),3.56-3.53(m,1H),2.31-2.22(m,1H),1.97 (d,J=12.3Hz,1H),1.83(d,J=12.1Hz,1H),1.67(s,1H),1.50(s,2H); LC-MS m / z(M+H):396.0.

[0121] Step 4: Synthesis of N-((5-(2-aminophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide.

[0122] To a stirred solution of N-((5-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (6.64 g, 13.40 mmol) and (2-aminophenyl)boronic acid (2.20 g, 16.08 mmol) in 1,4-dioxane-water (61 mL+19 mL) was added Na2CO3 (3.55 g, 33.49 mmol). The mixture was then degassed with argon for 10 min and tetrakis(triphenylphosphine)palladium(0) (1.54 g, 1.33 mmol) was added thereto. The resulting mixture was heated at 100 °C for 12 h, then diluted with H2O (15 mL) and extracted with EtOAc (2 x 50 mL). The combined organic layers were dried over Na2SO4 and concentrated. The obtained crude product was purified by flash column chromatography (eluent: 30% EtOAc + hexanes) to give N-((5-(2-aminophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (4.6 g, 74.5%) as a yellow gummy solid. LC-MS m / z (MH): 461.11.

[0123] Step 5: Synthesis of N-((5-(2-aminophenyl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide.

[0124] To a stirred solution of N-((5-(2-aminophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (3.70 g, 8.03 mmol) in DCM (20 mL) was added 1,4-dioxane (4 M, 30 mL) containing HCl at 0°C. The mixture was then warmed and stirred at room temperature for 12 h. It was then concentrated and further co-distilled with DCM (2 x 25 mL) to obtain the crude compound. The crude compound was diluted with H2O (25 mL), basified with saturated NaHCO3 solution (25 mL) and extracted with EtOAc (2 x 25 mL). The combined organic layers were dried over Na 2 SO 4 and concentrated to give N-((5-(2-aminophenyl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (4.75 g) as a yellow gummy solid; LC-MS m / z (MH): 377.19.

[0125] Step 6: Synthesis of N-((5-thiolidene-5,6-dihydropyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (Compound 1):

[0126] To a stirred solution of N-((5-(2-aminophenyl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (4.75g, 12.62mmol) in pyridine (110mL) and H2O (30mL) was added carbon disulfide (110mL) at room temperature. The reaction mixture was then heated at 100 °C for 12h. It was then diluted with cold H2O (500mL), the precipitated solid was filtered and air-dried to obtain a crude compound. It was then purified by preparative HPLC to give N-((5-sulfylidene-5,6-dihydropyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (1.6g, 30.30%) as an off-white solid. 1 H NMR (400MHz, DMSO) δ13.52(s,1H),9.17(t,J=5.8Hz,1H),8.07(d,J=7.8Hz,1H),7.76–7.6 5(m,1H),7.62-7.56(m,3H),7.51–7.36(m,3H),7.23(s,1H),4.64(d,J=5.9Hz,2H); LC-MS m / z(M+H):419.26.

[0127] Synthesis of (S)-N-((5-((2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (Compound 2):

[0128]

[0129] Step 1: Synthesis of tert-butyl (2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)ethyl)carbamate.

[0130] To a stirred suspension of N-((5-mercaptopyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (Compound 1, 100 mg, 0.239 mmol) in acetone (5 mL) was added KCO (33 mg, 0.239 mmol) followed by tert-butyl (2-bromoethyl)carbamate (53 mg, 0.239 mmol) at room temperature. The resulting reaction mixture was stirred at 70 °C for 5 h. The reaction mixture was concentrated under reduced pressure and the resulting residue was subjected to column chromatography using 2-3% MeOH in dichloromethane to afford tert-butyl (2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)ethyl)carbamate (12 mg, 89.5%) as an off-white solid. 1 H NMR (400MHz, DMSO) δ9.17(t,J=6.0Hz,1H),8.19(d,J=6.7Hz,1H),7.83(d,J=8.0Hz,1H),7.73–7.65(m,2H),7.63–7.54(m, 2H),7.52–7.42(m,2H),7.23(s,1H),7.16(s,1H),4.66(d,J=5.9Hz,2H),3.40(dd,J=12.9,5.0Hz,4H),1.34(s,9H); LC-MS m / z(M+H):561.93.

[0131] Step 2: Synthesis of N-((5-((2-aminoethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide 2,2,2-trifluoroacetate.

[0132] To a stirred solution of tert-butyl (2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)ethyl)carbamate (120 mg, 0.213 mmol) in DCM (5 mL) was added TFA (0.3 mL) at 0° C. The reaction mixture was warmed and stirred at room temperature for 4 h and concentrated to give N-((5-((2-aminoethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide 2,2,2-trifluoroacetate (100 mg, 82%) as an off-white solid. 1H NMR (400MHz, DMSO) δ9.19(t,J=6.0Hz,1H),8.21(d,J=6.6Hz,1H),7.97(d,J=16.3Hz,2H),7.88(d,J=8.2Hz,1H),7.76–7.66(m,2H),7.61( dd,J=12.7,4.7Hz,2H),7.52–7.44(m,2H),7.26(s,1H),4.67(d,J=6.0Hz,2H),3.61(t,J=6.6Hz,2H),3.32(dd,J=11.8,6.0Hz,2H); LC-MS m / z(M+H):462.21.

[0133] Step 3: Synthesis of (S)-N-((5-((2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (Compound 2):

[0134] To a stirred solution of N-((5-((2-aminoethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide:TFA (100 mg, 0.173 mmol) in DMF (3 mL) was added DIPEA (112 mg, 0.869 mmol), HATU (99 mg, 0.260 mmol) followed by (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (Compound 4, 83 mg, 0.208 mmol). The resulting reaction mixture was stirred at room temperature for 12 h, then diluted with cold water (20 mL) and stirred for 10 min. The resulting solid was collected by filtration, washed with water (20 mL) and dried under vacuum to give (S)-N-((5-((2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (50 mg, 34%) as a white solid. 1H NMR (400MHz, DMSO) δ9.17(s,1H),8.59(s,1H),8.20(d,J=7.9Hz,1H),7.82(d,J=7.7Hz,1H),7.69(s,2H),7.59(s,2H),7.54–7.35(m,6H) ,7.24(s,1H),4.66(d,J=5.9Hz,2H),4.51(d,J=7.9Hz,1H),3.52(s,4H),3.27–3.18(m,2H),2.58(s,3H),2.40(s,4H),1.60(s,3H); LC-MS m / z(M+H):844.39.

[0135] Synthesis of 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)acetic acid (Compound 3):

[0136]

[0137] Step 1: Synthesis of 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)ethyl ester:

[0138] To a stirred suspension of N-((5-thiolidene-5,6-dihydropyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (300 mg, 0.717 mmol) in acetone (15 mL) was added KCO (99 mg, 0.717 mmol) followed by ethyl 2-bromoacetate (87 mg, 0.717 mmol) at room temperature. The reaction mixture was stirred at 70 °C for 8 h and concentrated under reduced pressure. The residue was subjected to flash chromatography using 2-3% MeOH in DCM to give ethyl 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)acetate (280 mg, 77%) as a brown solid. 1 H NMR(400MHz, DMSO)δ9.17(t,J=5.9Hz,1H),8.20(d,J=7.7Hz,1H),7.74–7.66(m,3H),7.64–7.57(m,2H),7.5 2–7.42(m,2H),4.68(d,J=5.9Hz,2H),4.25(s,2H),4.17(q,J=7.1Hz,2H),1.21(dd,J=9.3,4.8Hz,3H); LC-MS m / z(M+H):505.1.

[0139] Step 2: Synthesis of 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)acetic acid (Compound 3):

[0140] To a stirred solution of ethyl 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)acetic acid (280 mg, 0.55 mmol) in THF-H2O (7 mL + 3 mL) at 0 ° C. LiOH.H2O (28 mg, 0.66 mmol) was added, and the reaction mixture was warmed and stirred at room temperature for 4 h. It was concentrated and diluted with H2O (10 mL) and acidified with 1N HCl (5 mL). The solid formed was collected by filtration and dried under vacuum to give 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)acetic acid (200 mg, 75%) as a white solid. 1 H NMR (400MHz, DMSO) δ9.18(t,J=5.9Hz,1H),8.19(d,J=7.7Hz,1H),7.75(d,J=8.1Hz,1H),7.69(dd,J=10.7,4.4Hz,2H) ,7.64–7.55(m,2H),7.50(d,J=7.6Hz,1H),7.47–7.42(m,1H),7.25(s,1H),4.68(d,J=5.9Hz,2H),4.19(s,2H); LC-MS m / z(M+H):477.1.

[0141] Synthesis of (S)-N-(2-aminoethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide TFA salt (Compound 5):

[0142]

[0143] Step 1: Synthesis of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (Compound 4):

[0144] To a stirred solution of (S)-tert-butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (500 mg, 1.094 mmol) in DCM (20 mL) at 0°C was added TFA (1 mL). The reaction mixture was warmed and stirred at room temperature for 12 h. The resulting reaction mixture was concentrated and co-distilled twice with DCM (10 mL each) to give (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid:TFA salt (400 mg, 91%) as a light yellow gummy solid. LC-MS: m / z=401.20

[0145] Step 2: Synthesis of tert-butyl (S)-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)carbamate:

[0146] To a stirred solution of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid:TFA (250 mg, 0.623 mmol) and tert-butyl (2-aminoethyl)carbamate (150 mg, 0.935 mmol) in DMF (5 mL) at 0°C was added HATU (355 mg, 0.935 mmol) followed by DIPEA (0.16 mL, 0.935 mmol). The resulting reaction mixture was stirred at room temperature for 12 h, then quenched with ice-cold water (20 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The residue was purified by flash chromatography using 3% MeOH in DCM to give (S)-tert-butyl(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)carbamate (110 mg, 32.4%) as a light yellow gummy solid. LC-MS: m / z=543.1.

[0147] Step 3: Synthesis of (S)-N-(2-aminoethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide:TFA salt (Compound 5):

[0148] To a stirred solution of (S)-tert-butyl(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)carbamate (100 mg, 0.181 mmol) in DCM (5 mL) was added TFA (0.2 mL) at 0° C. The reaction mixture was warmed and stirred at room temperature for 12 h. It was then concentrated, and the residue was co-distilled twice with DCM (10 mL) (to remove residual TFA) to give (S)-N-(2-aminoethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide (78 mg, 97.5%) as a light yellow gummy solid. LC-MS: m / z=443.2.

[0149] Synthesis of (S)-N-((5-((2-((2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)amino)-2-oxoethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (Compound 6):

[0150]

[0151] To a stirred solution of 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)acetic acid (100 mg, 0.209 mmol) in DMF (3 mL) was added DIPEA (40.6 mg, 0.314 mmol) and HATU (119.6 mg, 0.314 mmol) followed by (S)-N-(2-aminoethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide:TFA salt (139 mg, 0.314 mmol). The reaction mixture was stirred at room temperature for 12 h then diluted with water (20 mL) and extracted with EtOAc (2×20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative HPLC to give (S)-N-((5-((2-((2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)amino)-2-oxoethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (35 mg, 18.5%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ9.17(t,J=5.6Hz,1H),8.34(bs,1H),8.25(bs,1H),8 .16(d,J=7.6Hz,1H),7.78(d,J=8.0Hz,1H),7.69-7.54(m,4H),7.50-7.38(m, 6H),7.22(s,1H),4.67(d,J=6.0Hz,2H),4.44(t,J=7.2Hz,1H),4.11(s,2H),3 .20-3.16(m,4H),2.57(s,3H),2.39(s,3H),1.60(s,3H); LC-MS: m / z=901.53.

[0152] Synthesis of (S)-N-((5-((2-((2-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)amino)-2-oxoethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (Compound 7):

[0153]

[0154] Step 1: Synthesis of tert-butyl (S)-(2-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)carbamate:

[0155] A solution of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (400 mg, 1.0 mmol), DIPEA (0.52 mL, 3.0 mmol), HATU (570 mg, 1.5 mmol) and tert-butyl (2-(2-aminoethoxy)ethyl)carbamate (245 mg, 1.2 mmol) was stirred at room temperature for 16 h. The mixture was diluted with ice-cold water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with ice water (2 x 50 mL) and brine (50 mL), dried over Na2SO4 and concentrated under vacuum. The crude compound was purified by 0-5% MeOH in dichloromethane to give (S)-tert-butyl(2-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)carbamate (290 mg, 49%) as an off-white solid. LC-MS (ESI): m / z 587.1 (M+H)

[0156] Step 2: Synthesis of (S)-N-(2-(2-aminoethoxy)ethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide:TFA.

[0157] To a stirred solution of (S)-tert-butyl(2-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)carbamate (290 mg, 0.49 mmol) in DCM (3 mL) at 0°C was added TFA (0.5 mL) and the mixture was stirred at room temperature for 4 h. After completion of the reaction, the reaction mixture was concentrated under vacuum. The crude compound was purified by preparative HPLC (water:ACN) to afford (S)-N-(2-(2-aminoethoxy)ethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide. TFA was obtained as a light yellow solid (200 mg, 83%). 1 H NMR (400MHz, DMSO) δ8.25(d,J=5.3Hz,1H),7.79(s,3H),7.56–7.47(m,2H),7.42(d,J=8.4Hz,2H),4.52(t,J=7.1Hz,1H),3.61(d,J=5.2Hz,2 H),3.49(t,J=5.5Hz,2H),3.38–3.24(m,4H),3.08–2.97(m,2H),2.61(d,J=5.3Hz,3H),2.42(d,J=4.5Hz,3H),1.62(s,3H); LC-MS(ESI):m / z 487.4(M+H) + .

[0158] Step 3: Synthesis of (S)-N-((5-((2-((2-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thienyl[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)amino)-2-oxoethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide:

[0159] To a stirred solution of 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)acetic acid (100 mg, 0.209 mmol) in DMF (1.5 mL) was added DIPEA (40.6 mg, 0.314 mmol) and HATU (119.6 mg, 0.314 mmol) followed by (S)-N-(2-(2-aminoethoxy)ethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide:TFA salt (153 mg, 0.314 mmol). The reaction mixture was stirred at room temperature for 12 h then diluted with water (30 mL) and extracted with EtOAc (2 x 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative HPLC to give (S)-N-((5-((2-((2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)amino)-2-oxoethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (39 mg, 19.6%) as an off-white solid. 1 H NMR(400MHz, DMSO-d6)δ9.18(t,J=5.6Hz,1H),8.41-8.38(m,2H),8.17(d,J=7.2H z,1H),7.67(t,J=7.2Hz,2H),7.62-7.60(m,3H),7.58-7.54(m,4H),7.38(d,J=8. 4Hz,2H),7.23(s,1H),4.67(d,J=6.0Hz,2H),4.57(t,J=8Hz,1H),4.16-4.07(m,2 H),3.49-3.44(m,4H),3.15-3.31(m,6H),2.45(s,3H),2.26(s,3H),1.53(s,3H). LC-MS: m / z=945.41 (M+H) + .

[0160] Synthesis of (S)-N-((5-((14-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2,13-dioxo-6,9-dioxa-3,12-diazatetradecyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (Compound 9):

[0161]

[0162] Step 1: Synthesis of (S)-tert-butyl (2-(2-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethoxy)ethyl)carbamate:

[0163] To a stirred solution of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (860 mg, 2.145 mmol) and tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (799 mg, 3.217 mmol) in DMF (10 mL) at 0°C was added HATU (1.2 g, 3.217 mmol) followed by DIPEA (0.53 mL, 3.217 mmol). The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was quenched with ice-cold water (30 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography using 3% MeOH in DCM to give (S)-tert-butyl(2-(2-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethoxy)ethyl)carbamate (590 mg, 43.7%) as a light yellow gummy solid. LC-MS: m / z=631.26 (M+H) + .

[0164] Step 2: Synthesis of (S)-N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide: TFA salt.

[0165] To a stirred solution of (S)-tert-butyl(2-(2-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethoxy)ethyl)carbamate (590 mg, 0.934 mmol) in DCM (10 mL) was added TFA (2 mL) at 0° C. The reaction mixture was warmed and stirred at room temperature for 12 h. The reaction solvent was evaporated and co-distilled twice with DCM (10 mL) to give (S)—N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide TFA salt (423 mg, 85%) as a light yellow gummy solid. 1 HNMR (400MHz, DMSO) δ8.30(t,J=5.6Hz,1H),7.81(bs,3H),7.56-7.48(m,2H),7.47-7.38(m,2H),4.55–4.50(m,1H),3.61(d,J=5.6,8.7 Hz, 6H), 3.47 (t, J = 5.6Hz, 2H), 3.31-3.25 (m, 4H), 3.02-2.98 (m, 2H), 2.31 (s, 3H), 2.42 (s, 3H), 1.64 (s, 3H); LC-MS: m / z = 531.3 (530+H) + .

[0166] Step 3: Synthesis of (S)-N-((5-((14-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2,13-dioxo-6,9-dioxa-3,12-diazatetradecyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide:

[0167] To a stirred solution of 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)acetic acid (100 mg, 0.210 mmol) in DMF (1.5 mL) was added DIPEA (40.6 mg, 0.315 mmol) and HATU (119.7 mg, 0.315 mmol) followed by (S)—N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide:TFA salt (167.2 mg, 0.315 mmol). The resulting reaction mixture was stirred at room temperature for 12 h, then diluted with water (30 mL) and extracted with EtOAc (2 x 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative HPLC to give (S)-N-((5-((14-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2,13-dioxo-6,9-dioxa-3,12-diazatetradecyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (88.9 mg, 42.78%) as an off-white solid. 1 H NMR (400MHz, DMSO-d6) δ9.17(t,J=6.0Hz,1H),8.37(t,J=5.6Hz,1H),8.27(t,J=5.6Hz,1H),8.1 9(d,J=8.0Hz,1H),7.77(d,J=8.0Hz,1H),7.69-7.65(m,2H),7.62-7.55(m,2H),7.50-7.38(m,6 H),7.24(s,1H),4.68(d,J=5.6Hz,2H),4.50(t,J=7.2Hz,1H),4.10(s,2H),3.48(s,4H),3.42-3 .40(m,4H),3.29-3.21(m,6H),2.55(s,3H),2.36(s,3H),1.58(s,3H); LC-MS: m / z=989.45(M+H) + .

[0168] Synthesis of (R)-N-((5-(2-((14-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2,13-dioxo-6,9-dioxa-3,12-diazatetradecyl)oxy)phenyl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (Compound 10):

[0169]

[0170] Step 1: Synthesis of N-((5-(2-aminophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide:

[0171] To a stirred solution of N-((5-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (250 mg, 0.505 mmol), (2-hydroxyphenyl)boronic acid (83.5 mg, 0.606 mmol) in 1,4-dioxane (16 mL) and water (4 mL) was added Na2CO3 (133.7 mg, 1.262 mmol) and the resulting mixture was degassed with argon for 5 min followed by the addition of Pd(PPh3)4 (58.3 mg, 0.05 mmol). The resulting reaction mixture was stirred at 100°C for 12 h and concentrated. The obtained residue was diluted with water (30 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product obtained was purified by flash chromatography using 30% EtOAc in hexanes to give N-((5-(2-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (160 mg, 68.6%) as an off-white solid. LC-MS: m / z=460.43 (MH) + .

[0172] Step 2: Synthesis of ethyl 2-(2-(1-(tetrahydro-2H-pyran-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetate:

[0173] To a stirred solution of N-((5-(2-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (160 mg, 0.346 mmol) in acetone (7 mL) was added KCO (47.8 mg, 0.346 mmol) followed by ethyl 2-chloroacetate (42.4 mg, 0.346 mmol). The reaction mixture was stirred at 80 °C for 12 h and concentrated. The residue was then diluted with water (20 mL) and extracted with EtOAc (2 x 20 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The crude product was purified by flash chromatography using 30% EtOAc in hexanes to give ethyl 2-(2-(1-(tetrahydro-2H-pyran-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetate (105 mg, 55.3%) as a yellow gummy solid. LC-MS: m / z=546.35 (MH) + .

[0174] Step 3: Synthesis of 2-(2-(1-(tetrahydro-2H-pyran-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetic acid.

[0175] To a stirred solution of ethyl 2-(2-(1-(tetrahydro-2H-pyran-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetate (105 mg, 0.191 mmol) in THF (2.5 mL) and water (2.5 mL) was added LiOH.HO (30 mg, 0.767 mmol). The reaction mixture was stirred at room temperature for 12 h and extracted with EtOAc (20 mL). The aqueous layer was acidified with 1N HCl and extracted with EtOAc (3×20 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The crude compound was triturated with diethyl ether (10 mL) to give 2-(2-(1-(tetrahydro-2H-pyran-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetic acid (95 mg, 95.3%) as an off-white solid. LC-MS: m / z=518.4 (MH) + .

[0176] Step 4: Synthesis of 2-(2-(3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetic acid:

[0177] To a stirred solution of 2-(2-(1-(tetrahydro-2H-pyran-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetic acid (140 mg, 0.282 mmol) in DCM (5 mL) at 0 ° C. 1,4-dioxane (5 mL) containing 4M HCl was added. The reaction mixture was stirred at room temperature for 12 h. After the reaction was complete, the solvent was distilled off, and the residue was neutralized with saturated NaHCO3 solution and stirred for 10 min. The obtained solid was collected by filtration and dried under vacuum to give 2-(2-(3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetic acid (95 mg, 73%) (95 mg, 73%) as an off-white solid. 1 H NMR (400MHz, DMSO-d6) δ15.26(s,1H),8.87(t,J=5.6Hz,1H),7.68-7.54(m,3H),7.46(q,J=15.2Hz,2H),7.25(t,J=7.2Hz,1 H),7.09(d,J=8.4Hz,1H),7.00(t,J=7.6Hz,1H),6.55(s,1H),4.44(d,J=5.6Hz,2H),4.34(s,2H); LC-MS: m / z=436.2(435+H) + .

[0178] Step 5: Synthesis of (R)-N-((5-(2-((14-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2,13-dioxo-6,9-dioxa-3,12-diazatetradecyl)oxy)phenyl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide:

[0179] To a stirred solution of 2-(2-(3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetic acid (70 mg, 0.160 mmol) and (S)-N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide (155.5 mg, 0.241 mmol) in DMF (3 mL) at 0°C was added HATU (91.6 mg, 0.241 mmol) followed by DIPEA (62 mg, 0.482 mmol). The reaction mixture was stirred at room temperature for 12 h then quenched with ice-cold water (30 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative HPLC to give (R)-N-((5-(2-((14-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2,13-dioxo-6,9-dioxa-3,12-diazatetradecyl)oxy)phenyl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (25 mg, 16%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ13.0(s,1H),8.97-8.76(m,1H),8.34-8.20(m,2H),7.7 1-7.55(m,3H),7.47-7.39(m,6H),7.28(t,J=7.6Hz,1H),7.06-6.97(m,2H),6. 64(s,1H),4.68(s,1H),4.55-4.31(m,4H),3.50(s,4H),3.45-3.41(m,4H),3.2 4-3.22(m,4H),2.57(s,3H),2.38(s,3H),1.59(s,3H); LC-MS: m / z=948.5(M+H) + .

[0180] Synthesis of (S)-N-((1-(1-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2,13-dioxo-6,9-dioxa-3,12-diazapentadecan-15-yl)-5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (Compound 11):

[0181]

[0182] Step 1: Synthesis of N-((1-(tetrahydro-2H-pyran-2-yl)-5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide.

[0183] To a sealed tube containing 1,4-dioxane (16 mL) and water (4 mL) containing N-((5-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (1.4 g, 2.826 mmol) under argon atmosphere was added thiophene-2-ylboronic acid (0.398 g, 3.106 mmol), Na2CO3 (0.749 g, 7.065 mmol) and Pd(PPh3)4 (0.326 g, 0.282 mmol). The reaction mixture was then stirred at 100°C in a preheated oil bath for 12 h. The mixture was cooled, then diluted with water and extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, eluent: 0-15% EtOAc in petroleum ether to afford N-((1-(tetrahydro-2H-pyran-2-yl)-5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (0.434 g, 34.1%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ8.94(t,J=6.0Hz,1H),7.71(dd,J=5.2,0.8Hz,1H),7.64-7.54(m, 2H),7.50-7.39(m,2H),7.58(dd,J=3.6,0.8Hz,1H),7.25-7.17(m,1H),6.40(s,1H),5.33 (dd,J=10.0,2.0Hz,1H),4.48-4.34(m,2H),3.96(d,J=12.0Hz,1H),3.70-3.55(m,1H),2. 43-2.38(m,1H),2.02-1.90(m,1H),1.89-78(m,1H),1.72-1.57(m,1H),1.56-1.47(m,2H).

[0184] Step 2: Synthesis of N-((5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide.

[0185] To a stirred solution of N-((1-(tetrahydro-2H-pyran-2-yl)-5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (570 mg, 1.262 mmol) in DCM (10 mL) and 1,4-dioxane (10 mL) was added 4M HCl in dioxane (15 mL) at 0°C. The reaction mixture was then stirred at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure, the residue was basified with saturated NaHCO3 solution, the precipitated solid was collected by filtration and washed with Et2O. The solid was then dried to give (N-((5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (434 mg, 93.5%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ12.70(brs,1H),8.95(s,1H),7.70-7.55(m,2H),7.53-7.3 9(m,3H),7.32(s,1H),7.07(s,1H),6.43(s,1H),4.44(d,J=5.2Hz,2H); LC-MS:m / z 386.14(M+H) + .

[0186] Step 3: Synthesis of ethyl 2-(5-(thiophen-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-1-yl)acetate.

[0187] To a stirred solution of N-((5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (433 mg, 1.178 mmol) in acetonitrile (15 mL) was added KCO (244 mg, 1.767 mmol) and ethyl 2-bromoacetate (0.15 mL, 1.414 mmol) at 0°C. The reaction mixture was then heated and stirred at 80°C for 12 h before being cooled to room temperature, diluted with water and extracted three times with EtOAc. The combined organic layers were dried over NaSO, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 0-20% EtOAc in hexanes) to give ethyl 2-(5-(thiophen-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-1-yl)acetate (80 mg, 14.9%) as an off-white solid. LC-MS: m / z 454.1 (M+H) + .

[0188] Step 4: Synthesis of 2-(5-(thiophen-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-1-yl)acetic acid:

[0189] To a stirred solution of ethyl 2-(5-(thiophen-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-1-yl)acetic acid (90 mg, 0.198 mmol) in THF (2 mL) and water (1 mL) was added LiOH.HO (21 mg, 0.496 mmol) at 0°C. The reaction mixture was then warmed and stirred at room temperature for 12 h. It was concentrated, the residue was acidified with citric acid (PH~2) and extracted with 10% MeOH in DCM. The combined organic layers were dried over NaSO, filtered and concentrated under reduced pressure to give 2-(5-(thiophen-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-1-yl)acetic acid (40 mg, 45.8%) as an off-white solid. 1 H NMR(400MHz, DMSO-d6)δ13.20(s,1H),8.94(t,J=5.6Hz,1H),7.68(d,J=9.2Hz,1H),7.64-7.53(m,2H),7.5 0-7.39(m,2H),7.22(d,J=7.6Hz,1H),7.20-7.12(m,1H),6.39(s,1H),4.96(s,2H),4.40(t,J=5.6Hz,2H). LC-MS:426.1,m / z=(M+H) + .

[0190] Step 5: Synthesis of (S)-N-((1-(1-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2,13-dioxo-6,9-dioxa-3,12-diazapentadecan-15-yl)-5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide.

[0191] To a stirred solution of (S)-N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide (44 mg, 0.077 mmol) in DMF (2.5 mL) at 0°C was added DIPEA (23 mg, 0.176 mmol), HATU (30 mg, 0.077 mmol) and 2-(5-(thiophen-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-1-yl)acetic acid (40 mg, 0.070 mmol). The reaction mixture was then warmed and stirred at room temperature for 16 h. The reaction mixture was diluted with ice-cold water and extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: 0-4% methanol in DCM) to give (S)-N-((1-(1-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2,13-dioxo-6,9-dioxa-3,12-diazapentadecan-15-yl)-5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (33 mg, 37.8% yield) as a light yellow gummy solid. 1 H NMR(400MHz,DMSO-d6)δ8.93(t,J=5.6Hz,1H),8.32-8.20(m,2H),7.6(d,J=4 .8Hz,1H),7.63-7.53(m,2H),7.50-7.35(m,6H),7.29-7.24(m,1H),7.18-7.1 0(m,1H),6.35(s,1H),4.79(s,2H),4.53-4.45(m,1H),4.38(t,J=6.0Hz,2H), 3.53(s,4H),3.49-3.39(m,5H),3.30-3.20(m,8H),2.58(s,3H),2.39(s,3H). LC-MS: m / z 938.49 (M+H) + .

[0192] Synthesis of (S)-N-(5-amino-1-(thiazolo[4,5-c]quinolin-2-yl)pentyl)-2-(difluoromethoxy)benzamide (Compound 12):

[0193]

[0194] Step 1: Synthesis of 3-aminoquinolin-4-ol.

[0195] 10% Pd / C (5g) was added to a stirred solution of 3-nitroquinoline-4-ol (10g, 3.125mmol) in MeOH (10mL) at room temperature under a nitrogen atmosphere. The reaction flask was then pressurized with hydrogen (50psi), and the mixture was stirred for 12h. The resulting reaction mixture was filtered through diatomite, and the filtrate was concentrated under reduced pressure. The crude product obtained was purified by column chromatography (eluent: hexane containing 50% EtOAc) to obtain 3-aminoquinoline-4-ol (7.70g, 90.53%) as an off-white solid. LC-MS C9H8N2O m / z(M+H):161.1.

[0196] Step 2: Synthesis of (R)-benzyl (6-((4-hydroxyquinolin-3-yl)amino)-6-oxohexane-1,5-diyl)tert-butyl dicarbamate.

[0197] To a stirred solution of 3-amino 4-hydroxyquinolone (2.50 g, 3.125 mmol) and N6-((benzyloxy)carbonyl)-N2-(tert-butoxycarbonyl)-D-lysine (7.12 g, 3.75 mmol) in DCM was added HATU (7.12 g, 3.75 mmol) at 0 ° C., followed by DIPEA (8 mL, 9.375 mmol). The reaction mixture was then stirred at room temperature for 12 h and concentrated. The residue was diluted with H2O (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic layers were washed once with brine solution (20 mL), the organic layers were separated, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The obtained crude product was purified by column chromatography (eluent: 40% EtOAc in hexanes) to give (R)-benzyl (6-((4-hydroxyquinolin-3-yl)amino)-6-oxohexane-1,5-diyl) dicarbamate tert-butyl ester (3.0 g, 88.23%) as an off-white solid. LC-MS C 28 H 34 N4O6 m / z(M+H):523.1.

[0198] Step 3: Synthesis of (R)-benzyl(1-(thiazolo[4,5-c]quinolin-2-yl)pentane-1,5-diyl)tert-butyl dicarbamate.

[0199] To a stirred solution of (R)-benzyl diaminoformate (1-(thiazolo[4,5-c]quinolin-2-yl)pentane-1,5-diyl) tert-butyl ester (3.0 g, 5.747 mmol) in THF was added Lawesson's reagent (1.85 g, 4.59 mmol) at room temperature. The reaction mixture was then stirred at 70 ° C for 8 h, and the reaction was quenched with saturated NH4Cl solution. It was further diluted with H2O (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic layer was washed once with brine (20 mL), the organic layer was separated, dried over anhydrous Na2SO4 and concentrated. The obtained crude compound was purified by column chromatography (eluent: 60% EtOAc in hexane) to give (R)-benzyl(1-(thiazolo[4,5-c]quinolin-2-yl)pentane-1,5-diyl)tert-butyl dicarbamate (2.2 g, 73.48%) as a light yellow solid. LC-MS: m / z 521.1 (M+H) + .

[0200] Step 4: Synthesis of (R)-(5-amino-5-(thiazolo[4,5-c]quinolin-2-yl)pentyl)carbamic acid benzyl ester:

[0201] To a stirred solution of (R)-benzyl (1-(thiazolo[4,5-c]quinolin-2-yl)pentane-1,5-diyl) tert-butyl dicarbamate (1.0 g, 1.919 mmol) in DCM (25 mL) was added TFA (5 mL) at 0°C and the reaction was stirred at room temperature for 4 h. After the reaction was complete, the solvent was distilled off and then diluted with H2O (20 mL), basified with saturated aqueous NaHCO3 solution (10 mL) and extracted with EtOAc (2 x 30 mL). The combined organic layers were washed once with brine (20 mL), the organic layers were separated, dried over anhydrous Na2SO4 and concentrated. The crude product was used in the next step without further purification (620 mg, 76.78%). LC-MS: m / z 421.1 (M+H) + .

[0202] Step 5: Synthesis of benzyl (S)-(5-(2-(difluoromethoxy)benzamido)-5-(thiazolo[4,5-c]quinolin-2-yl)pentyl)carbamate.

[0203] To a stirred solution of (R)-(5-amino-5-(thiazolo[4,5-c]quinolin-2-yl)pentyl)benzylcarbamate (620 mg, 1.4726 mmol) and 2-(difluoromethoxy)benzoic acid (309 mg, 1.6456 mmol) in DCM (20 mL) at 0 ° C. EDC.HCl (428 mg, 2.244 mmol), HOBt (302 mg, 2.244 mmol) and TEA (1.4 mL, 7.48 mmol) were added. The reaction mixture was then stirred at room temperature for 12 h and concentrated. The residue was treated with NH4Cl aqueous solution and water (20 mL) and extracted with EtOAc (2 x 20 mL). The combined organic layer was washed once with brine (20 mL), the organic layer was separated, dried over anhydrous Na2SO4 and concentrated. The crude product obtained was purified by column chromatography (eluent: 5% MeOH in DCM) to afford benzyl (S)-(5-(2-(difluoromethoxy)benzamido)-5-(thiazolo[4,5-c]quinolin-2-yl)pentyl)carbamate (350 mg, 40.32%) as an off-white solid. 1 H NMR (400MHz, DMSO) δ9.43 (s, 1H), 8.45 (d, J = 7.2Hz, 1H), 8.27 (t, J = 5.6, Hz 1H),8.20(d,J=8.4Hz,2H),7.83(t,J=7.6Hz,1H),7.75(t,J=7.6Hz,1H),7.50-7.44(m,2H),7.39-7.30(m,3H) ,7.26-7.20(m,5H),5.10-5.04(m,3H),3.25-3.21(m,2H),2015-2.11(m,1H),1.96-1.94(m,1H),1.549(m,4H). LC-MS:m / z591.0(M+H) + .

[0204] Step 6: Synthesis of (S)-N-(5-amino-1-(thiazolo[4,5-c]quinolin-2-yl)pentyl)-2-(difluoromethoxy)benzamide:

[0205] To a stirred solution (5mL) of (S)-(5-(2-(difluoromethoxy)benzamido)-5-(thiazolo[4,5-c]quinolin-2-yl)pentyl)benzylcarbamate (50mg, 0.084mmol) in DCM was added AcOH containing 40% HBr at 0°C, and the reaction mixture was stirred at room temperature for 4h. It was then concentrated, and the obtained crude product was quenched with saturated NaHCO3 solution (10mL) and extracted with EtOAc (2x 10mL). The combined organic layers were washed with brine (10mL), the organic layers were dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product obtained was purified by preparative HPLC (buffered solution formic acid) to give (S)-N-(5-amino-1-(thiazolo[4,5-c]quinolin-2-yl)pentyl)-2-(difluoromethoxy)benzamide as a green viscous compound (5 mg, 38.64%). 1 H NMR(400MHz,DMSO)δ9.39(s,1H),8.26-8.17(m,3H),7.83-7.72(m,2H),7.52-7.42(m,2H),7 .30-6.93(m,3H),4.31(m,1H),3.3-3.23(m,3H),2.16-1.766(m,2H),1.54(m,5H); LC-MS:m / z 457.29(M+H) + .

[0206] Synthesis of N-(5-amino-1-(5-thiolidene-5,6-dihydro-[1,2,4]triazolo[1,5-c]quinazolin-2-yl)pentyl)-2-(difluoromethoxy)benzamide (Compound 13):

[0207]

[0208] Step 1: Synthesis of N6-(tert-butoxycarbonyl)-N2-(2-(difluoromethoxy)benzoyl)lysine methyl ester.

[0209] To a stirred solution of N6-(tert-butoxycarbonyl)lysine methyl ester (1.70 g, 5.851 mmol) and 2-(difluoromethoxy)benzoic acid (1.20 g, 6.43 mmol) in DCM (15 mL) at 0°C was added EDC.HCl (1.84 gm, 9.64 mmol), HOBt (1.30 gm, 9.64 mmol), followed by TEA (4 mL, 29.250 mmol). The reaction mixture was then stirred at room temperature for 12 h and concentrated. The residue was diluted with H2O (20 mL) and extracted with EtOAc (2x20 mL). The combined organic layers were washed once with saturated brine (20 mL), the organic layers were separated, dried over anhydrous Na2SO4 and concentrated. The obtained crude product was purified by column chromatography (eluent: 5% MeOH in DCM) to give N6-(tert-butoxycarbonyl)-N2-(2-(difluoromethoxy)benzoyl)lysine methyl ester (1.10 g, 40.32%) as an off-white solid. LC-MS: m / z (M+H): 331.1.

[0210] Step 2: Synthesis of tert-butyl (5-(2-(difluoromethoxy)benzamido)-6-hydrazino-6-oxohexyl)carbamate:

[0211] To a stirred solution of N6-(tert-butoxycarbonyl)-N2-(2-(difluoromethoxy)benzoyl)lysine methyl ester (500 mg, 1.62 mmol) in EtOH (5 mL) was added an aqueous solution of hydrazine (1 mL), and the reaction mixture was stirred at room temperature for 15 min and at 100 ° C for 3 h. The reaction mixture was then concentrated, and the residue was treated with H2O (10 mL) and extracted with EtOAc (2 x 20 mL). The combined organic layer was washed once with brine (20 mL), the organic layer was separated, dried over anhydrous Na2SO4 and concentrated. The obtained crude product was purified by column chromatography (hexane containing 20% ​​EtOAc) to give tert-butyl (5-(2-(difluoromethoxy)benzamido)-6-hydrazine-6-oxohexyl)carbamate (200 mg, 40%) as a dark brown liquid. LC-MS: m / z 431.10 (M+H).

[0212] Step 3: Synthesis of tert-butyl (5-(2-(difluoromethoxy)benzamido)-5-(5-thiolidene-5,6-dihydro-[1,2,4]triazolo[1,5-c]quinazolin-2-yl)pentyl)carbamate.

[0213] To a stirred solution of tert-butyl (5-(2-(difluoromethoxy)benzamido)-6-hydrazine-6-oxohexyl)carbamate (120 mg, 0.27 mmol) in EtOH (10 mL), 2-isothiocyanate benzonitrile (49 mg, 0.30 mmol) was added, and the reaction mixture was stirred at room temperature for 15 min and then at 100 ° C for 2 h. The reaction mixture was then concentrated, diluted with H2O (10 mL) and extracted with EtOAc (2 x 20 mL). The combined organic layer was washed once with brine (20 mL), the organic layer was separated, dried over anhydrous Na2SO4 and concentrated. The crude product obtained was purified by preparative HPLC (buffered solution NH4OAc, MeOH) to afford tert-butyl (5-(2-(difluoromethoxy)benzamido)-5-(5-thiolide-5,6-dihydro-[1,2,4]triazolo[1,5-c]quinazolin-2-yl)pentyl)carbamate as a white solid (10 mg, 6.28%). 1 H NMR (400MHz, DMSO) δ14.01(s,1H),8.91(d,J=8.4,1H),8.20(d,J=7.6,1H),7.81-7.77(m,1H),7.67-7.50(m,4H), 7.40-6.79(m,4H),5.99-5.71(m,1H),2.92-2.91(m,2H),2.00-1.91(m,2H),1.44(m,4H),1.34(s,9H); LC-MS:m / z 571.10(MH).

[0214] Step 4: N-(5-amino-1-(5-sulfanylidene-5,6-dihydro-[1,2,4]triazolo[1,5-c]quinazolin-2-yl)pentyl)-2-(difluoromethoxy)benzamide.

[0215] To a stirred solution of tert-butyl (5-(2-(difluoromethoxy)benzamido)-5-(5-sulfanylidene-5,6-dihydro-[1,2,4]triazolo[1,5-c]quinazolin-2-yl)pentyl)carbamate (50 mg, 0.087 mmol) in methanol (5 mL) at 0°C was added acetyl chloride (0.1 mL) and the reaction was stirred at room temperature for 16 h. The reaction mixture was then concentrated and the crude product was purified by preparative HPLC (buffered solution NH4OAc, MeOH) to give N-(5-amino-1-(5-sulfanylidene-5,6-dihydro-[1,2,4]triazolo[1,5-c]quinazolin-2-yl)pentyl)-2-(difluoromethoxy)benzamide (20 mg, 48.48%) as a light yellow solid. 1H NMR (400MHz, DMSO) δ14.05(s,1H),8.95(d,J=8.4Hz,1H),8.19(d,J=7.6Hz,1H),7.80(t,J=8.4Hz,1H),7.74(s,2H),7.68(d,J=8.0Hz, 1H),7.59-7.52(m,3H),7.39-7.02(m,3H),5.33(q,J=8.8Hz,1H),2.82-2.75(m,2H),2.10-1.92(m,2H),1.63-1.47(m,4H); LC-MS: m / z 473.38(M+H).

[0216] Synthesis of (R)-N-(5-amino-1-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)-2-(difluoromethoxy)benzamide (Compound 14):

[0217]

[0218] Step 1: Synthesis of (R)-benzyl (6-(methoxy(methyl)amino)-6-oxohexane-1,5-diyl)tert-butyl dicarbamate.

[0219] N, O-dimethylhydroxylamine hydrochloride (260 mg, 26.29 mmol) and DIPEA (1 g, 78.86 mmol) were added to a stirred solution of (R)-N-benzyloxycarbonyl-N6-(tert-butoxycarbonyl)-L-lysine (1 g, 26.29 mmol) and HATU (1.5 g, 39.43 mmol) in DMF (10 mL). The reaction mixture was then stirred at room temperature for 12 h. After the reaction was complete, it was diluted with water (10 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layer was concentrated. The crude compound was purified by flash column chromatography (eluent: hexane containing 20% ​​ethyl acetate) to obtain (R)-diaminobenzyl (6-(methoxy(methyl)amino)-6-oxohexane-1,5-diyl) tert-butyl ester (600 mg, 54.6%) as a colorless viscous liquid. LC-MS m / z(M+H-Boc):324.1.

[0220] Step 2: Synthesis of (S)-tert-butyl benzyl diaminocarbamate (8-(2-methoxyphenyl)-6-oxooct-7-yn-1,5-diyl) ester.

[0221] To a stirred solution of 1-ethynyl-2-methoxybenzene (100 mg, 0.2361 mmol) in anhydrous THF (2 mL) was added THF (0.6 mL, 0.9445 mmol) containing n-BuLi at -78 ° C in 10 minutes. The reaction mixture was stirred at -78 ° C for 1 h, followed by dropwise addition of THF containing (R)-diaminobenzyl (6-(methoxy(methyl)amino)-6-oxohexane-1,5-diyl) tert-butyl ester (from step 1) in 10 min. The reaction mixture was then warmed and stirred at 0 ° C for 2 h. It was then quenched by adding aqueous ammonium chloride (10 mL) and extracted with EtOAc (2 × 50 mL). The combined organic layers were dried over Na2SO4 and concentrated. The crude compound was purified by flash column chromatography (eluent: 20% EtOAc in hexanes) to afford (S)-benzyl (8-(2-methoxyphenyl)-6-oxooct-7-yn-1,5-diyl) dicarbamate tert-butyl ester (60 mg, 51.4%) as a colorless viscous liquid. 1 H NMR (400MHz, DMSO) δ7.51-7.43(m,2H),7.37-7.29(m,5H),6.97-6.89(m,2H),5.66(d,J=7.2Hz,1H),5.13(s,2H),4.59-4.57(m,2H),3 .86(s,3H),3.12(q,J=6.4Hz,2H),2.12-2.07(m,1H),1.87-1.81(m,1H),1.54-1.51(m,2H),1.41(s,9H); LC-MSm / z(M+H-Boc): 395.1.

[0222] Step 3: Synthesis of (R)-benzyl (1-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentane-1,5-diyl)tert-butyl dicarbamate.

[0223] To a stirred solution of (S)-benzyl diaminoformate (8-(2-methoxyphenyl)-6-oxooct-7-yn-1,5-diyl) tert-butyl ester (60 mg, 0.1213 mmol) in ethanol (1 mL) was added hydrazine hydrate (15.5 mg, 0.4852 mmol). The resulting solution was heated at 100 ° C for 12 h and concentrated. Water (10 mL) was added to the residue, and the mixture was extracted with EtOAc (2x50 mL). The combined organic layers were dried over Na2SO4 and concentrated. The crude compound was purified by flash column chromatography (eluent: hexane containing 70-80% EtOAc) to obtain (R)-benzyl diaminoformate (1-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentane-1,5-diyl) tert-butyl ester (50 mg, yield 86.6%) as a white viscous liquid. 1 H NMR (400MHz, DMSO) δ12.60(bs,1H),7.61(bs,2H),7.36-7.30(m,6H),7.11(d,J=7.6Hz,1H),6.99(t,J=7.2Hz,1H),6.77(t,J=5.6Hz,1H) ,6.56(s,1H),5.03(s,2H),4.62(d,J=8.0Hz,1H),3.86(s,3H),2.88(q,J=6.4Hz,2H),1.73-1.68(m,2H),1.35(s,9H),1.23-1.15(m,4H). LC-MS m / z(M+H):509.11.

[0224] Step 4: Synthesis of (R)-tert-butyl(5-amino-5-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)carbamate.

[0225] To a stirred solution of (R)-benzyl diaminoformate (1-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentane-1,5-diyl) tert-butyl ester (100 mg, 0.2103 mmol) in methanol (3 mL) was added 10% Pd / C at room temperature. The reaction was then stirred at room temperature for 12 h in the presence of a hydrogen atmosphere. After the reaction was complete, the reaction mixture was filtered through a celite pad and washed with methanol (2 x 20 mL). The combined filtrate was concentrated on a rotary evaporator to obtain the desired compound (R)-(5-amino-5-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)tert-butyl carbamate (52 mg, 66.12% yield) as a white solid. 1H NMR (400MHz, DMSO) δ12.59(bs,1H),7.74(bs,1H),7.30(t,J=7.2Hz,1H),7.11(d,J=8.4Hz,1H),6.99(t,J=8Hz,1H),6.76(t,J=5.2Hz, 1H),6.61(s,1H),3.91(t,J=6.4Hz,1H),3.87(s,3H),2.88(q,J=6.4Hz,2H),1.68-1.66(m,2H),1.35(s,9H),1.35-1.31(m,4H); LC-MS m / z(M+H):375.1.

[0226] Step 5: Synthesis of tert-butyl (R)-(5-(2-(difluoromethoxy)benzamido)-5-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)carbamate.

[0227] To a stirred solution of (R)-(5-amino-5-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)carbamic acid tert-butyl ester (50 mg, 0.1337 mmol), 2-(difluoromethoxy)benzoic acid (25 mg, 0.1337 mmol), EDC.HCl (38.5 mg, 0.2006 mmol) and HOBt (27 mg, 0.2006 mmol) in DCM (2 mL) was added TEA (41 mg, 0.4012 mmol) at room temperature. The reaction mixture was then stirred at room temperature for 12 h and concentrated. The residue was then diluted with water (10 mL) and extracted with EtOAc (2 x 50 mL). The combined EtOAc layer was dried over sodium sulfate and concentrated. The crude compound was purified by flash column chromatography (eluent: 2% MeOH + DCM) to afford tert-butyl (R)-(5-(2-(difluoromethoxy)benzamido)-5-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)carbamate (45 mg, 61.88%) as an off-white solid. LC-MS m / z (M+H): 545.1.

[0228] Step 6: Synthesis of (R)-N-(5-amino-1-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)-2-(difluoromethoxy)benzamide.

[0229] To a stirred solution of (R)-(5-(2-(difluoromethoxy)benzamido)-5-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)carbamic acid tert-butyl ester (40 mg, 0.0735 mmol) in MeOH (2 mL) was added acetyl chloride (2 mL) dropwise at 0 ° C. The reaction mixture was then stirred at room temperature for 5 h and concentrated. The obtained crude compound was washed with diethyl ether (2x10 mL) and dried on a rotary evaporator. The obtained crude compound was further purified by preparative HPLC to obtain (R)-N-(5-amino-1-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)-2-(difluoromethoxy)benzamide (14 mg, 42.88%) as an off-white solid. 1 H NMR (400MHz, DMSO) δ12.69(bs,1H),8.58(d,J=7.6Hz,1H),7.71(bs,1H),7.55-7.50(m,2H),7.37-6.99(m,6H) ,6.65(s,1H),5.13(t,J=8Hz,1H),3.87(s,3H),2.63-2.60(m,2H),1.88-1.74(m,2H),1.41-1.39(m,4H),LC-MS m / z(M+H):445.1.

[0230] The following degrader molecules can be prepared using amide coupling between targeting molecules and E2 binders.

[0231]

[0232] Bioassay

[0233] UBE2K dissociation constant (Kd) of E2-binder compound 1

[0234] UBE2K ligand binding to single Ub UBE2K and UBE2K was measured by surface plasmon resonance using a Biacore SPR S200 instrument. Proteins and ligands were prepared for experimental measurements as described below.

[0235] Avi-tagged proteins were biotinylated and immobilized on S-Series / Sensor Chip SA. Proteins were immobilized based on the molar weight of the following proteins: biotinylated Avi peptide (1000 μM), biotinylated UBE2K-Ub complex (12 μM), and biotinylated UBE2K wild type (3 μM). Proteins were immobilized using a buffer consisting of 1X HBS-N buffer + 0.05% Tween 20 (250 mL). A stock solution of compound 1 was prepared in an affinity buffer consisting of 1% DMSO, 1X PBS-P+ buffer. Further diluted stock solutions were prepared in 0% DMSO, 1X PBS-P+ buffer or 3% DMSO, 1X PBS-P+ buffer and used as running buffer. Compound 1 was prepared as a 2mM or 750 μM stock solution in the above buffer. Titration was performed as follows using a liquid handler / TECAN EVO (WALL-E). Titration was performed using 1 or 2 96 deep well plates (1.2 mL) - aliquots of 3% DMSO, 1X PBS-P + buffer. The remaining titrations were diluted 1:2 until 3.12 μM by WALL-E (250 μL + 250 μL). The titrations of compound 1 were then transferred to 384 deep well plates side by side (125 μL) in duplicate. The plate was then covered with a 384-well S200 compatible foil and used for affinity methods. A solvent calibration buffer with DMSO concentration ± 0.5% DMSO running buffer was run in each experimental run.

[0236] Data were collected and processed according to the methods described in the Biacore S200 evaluation software. As described in Dahl, G et al., SLAS Discovery, 2017, Vol. 22(2) 203–209, the data were fitted to a steady state using the software and the data represented by the sensorgram to deconvolve the binding and dissociation constants. The result was a Kd (μM) of 2.11 for compound 1. The data indicate that compound 1 is a useful UBE2K binder for subsequent PROTAC generation.

[0237] Dose-dependent degradation of BRD4 in HeLa cells

[0238] UBE2K binder compound 1 was used as a precursor to generate PROTAC compounds 2, 6, 7, and 9. See Experimental section above. Each of these PROTACs was first tested for its ability to bind to the E2 enzyme UBE2K.

[0239] All compounds except compound 2 bind to UBE2K-Ub. Figure 1 .

[0240] The ability of compounds 6, 7, and 9 to degrade BRD4 was then investigated. Figure 2As shown, compounds 7 and 9 showed concentration-dependent loss of BRD4. This data shows that targeting E2 (particularly UBE2K) is a viable strategy to generate novel PROTACs with therapeutic relevance.

[0241] Although we have described multiple embodiments, it is apparent that our basic examples can be altered to provide other embodiments utilizing the compounds and methods of the present invention. It should therefore be understood that the scope of the invention is defined by the appended claims rather than by the specific embodiments that have been shown by way of example.

[0242] The contents of all references cited throughout this application (including literature references, granted patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly known to those of ordinary skill in the art.

Claims

1. A compound comprising an E2 binding portion, a target protein binding portion and a linker (L) covalently linking the E2 binding portion to the target protein binding portion.

2. The compound of claim 1, wherein the E2 binding portion is selected from an E2 binding portion that binds an E2 enzyme selected from the group consisting of UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2G1, UBE2G2, UBE2H, UBE2J1, UBE2J2, UBE2K, UBE2L3, UBE2N, UBE2NL, UBE2O, UBE2Q1, UBE2Q2, UBE2QL, UBE2R1, UBE2R2, UBE2S, UBE2T, UBE2U, UBE2V1, UBE2V2, UBE2W, and BIRC6.

3. The compound of claim 1 or 2, wherein the E2 binding moiety is an E2 binding moiety that binds UBE2K.

4. The compound according to any one of claims 1 to 3, wherein the E2 binding moiety is a compound of formula I: in Z 1 and Z 2 are each independently N or CH; R 1 is (C1-C6)alkyl, halogenated (C1-C6)alkyl, (C1-C6)alkoxy, halogenated (C1-C6)alkoxy or –NR c R d wherein the two available hydrogen atoms on the halo(C1-C6)alkyl and halo(C1-C6)alkoxy groups may be taken together with the carbon atoms to which they are attached to form a 3- to 6-membered cycloalkyl group optionally substituted with 1 to 3 groups selected from the group consisting of halo, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy and halo(C1-C6)alkoxy; R 2 is CN, halo, OH, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy or halo(C1-C6)alkoxy; or R 1 and R 2 When on adjacent carbon atoms, together with the carbon atoms to which they are attached, form a 5- or 6-membered oxygen-containing heterocyclic group optionally substituted with 1 to 3 groups selected from halo, (C1-C6)alkyl and halo(C1-C6)alkyl; R 3 is hydrogen, (C1-C6)alkyl or halogenated (C1-C6)alkyl; Y is CH2, -CHR a , -CR a R b , S or SO; p is 0 or 1; R a and R b are each independently halo, (C1-C6)alkyl or halo(C1-C6)alkyl; or R a and R b Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl or a 3- to 6-membered heterocyclyl, each of which is optionally substituted with 1 to 3 groups selected from halo, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, (C1-C6)alkylOH, (C1-C6)alkylO(C1-C6)alkyl and OH; R c and R d each independently represents hydrogen, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkylO(C1-C6)alkyl, halo(C1-C6)alkylO(C1-C6)alkyl, (C1-C6)alkyl-O-halo(C1-C6)alkyl, halo(C1-C6)alkyl-O-halo(C1-C6)alkyl or (C1-C6)alkylOH; or R c and R d Together with the nitrogen atom to which they are attached, they form a 4- to 7-membered heterocyclyl group optionally substituted with 1 to 3 groups selected from the group consisting of halo, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy and oxo; Z is a tricyclic fused ring having the formula: Ring A is aromatic; The wavy bond on ring A indicates the point of attachment to Y; The wavy bond next to the W indicates the connection point to the connector (L); When the valence permits, X, X 1 and X 2 Each independently selected from -CR 7 , N, O and S; The dashed lines in ring B represent single or double bonds; W is NH, -N(C1-C6) alkyl), O or S; R 5 is selected from hydrogen, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, -S(C1-C6)alkyl, -SH, OH, (C3-C6)cycloalkyl, (C4-C7)heterocyclyl, and -NR e R f wherein the (C3-C6)cycloalkyl and (C4-C7)heterocyclyl are each optionally substituted with 1 to 3 groups selected from the group consisting of halo, (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy and oxo; When the dashed line in ring B is a single bond, R 6 is hydrogen or (C1-C6) alkyl, or when the dotted line in ring B is a double bond, R 6 does not exist; d, d1, d2 and d3 are each independently selected from CR 8 and N; R e and R f Each is independently hydrogen, (C1-C4)alkyl, (C1-C4)alkylNH(C1-C4)alkyl, (C1-C4)alkylN[(C1-C4)alkyl]2, (C1-C4)alkylO(C1-C4)alkyl; R 7 is hydrogen or (C1-C6)alkyl; and R 8 is halogen, hydrogen, (C1-C6)alkyl, halo(C1-C6)alkyl, CN or OH.

5. The compound according to claim 4, wherein the E2 binding moiety is a compound of formula II:

6. The compound according to claim 4 or 5, wherein W is S.

7. A compound according to any one of claims 4 to 6, wherein R 3 For hydrogen.

8. A compound according to any one of claims 4 to 7, wherein Z 1 and Z 2 Each is CH.

9. A compound according to any one of claims 4 to 8, wherein Y is CH2.

10. The compound according to any one of claims 4 to 9, wherein p is 0.

11. A compound according to any one of claims 4 to 10, wherein R 1 is (C1-C6)alkyl, halo(C1-C6)alkyl, halo(C1-C6)alkoxy, (C1-C6)alkoxy or –NR c R d ; and R c and R d Together with the nitrogen atom to which they are bound, they form a 5- to 6-membered heterocyclic group which is optionally substituted by 1 to 3 halogen groups.

12. A compound according to any one of claims 4 to 11, wherein R 1 is (C1-C3)alkyl, halogenated (C1-C3)alkyl, halogenated (C1-C6)alkoxy, (C1-C3)alkoxy or –NR c R d ; and R c and R d Together with the nitrogen atom to which they are bound, they form a 5- to 6-membered nitrogen-containing heterocyclic group which is optionally substituted by 1 to 3 halogen groups.

13. A compound according to any one of claims 4 to 12, wherein R 1 is OCF3, OCHF2, OCH3, CH3, pyrrolidinyl or piperidinyl, wherein the pyrrolidinyl or piperidinyl is optionally substituted with 1 to 3 halo groups.

14. A compound according to any one of claims 4 to 13, wherein X is N.

15. A compound according to any one of claims 4 to 14, wherein X 1 It is CH or N.

16. A compound according to any one of claims 4 to 15, wherein X 2 is CH, N, S or O.

17. A compound according to any one of claims 4 to 16, wherein X is N, X 1 is N, and X 2 is CH; X is N, X 1 is N, and X 2 is N; X is N, X 1 is CH, and X 2 is S; or X is N, X 1 is CH, and X 2 is O.

18. A compound according to any one of claims 4 to 17, wherein d is N or -CR 8 .

19. A compound according to any one of claims 4 to 18, wherein d 2 N or -CR 8 .

20. A compound according to any one of claims 4 to 19, wherein d 1 For-CR 8 .

21. A compound according to any one of claims 4 to 20, wherein d 3 For-CR 8 .

22. A compound according to any one of claims 4 to 21, wherein Z is selected from 23. A compound according to any one of claims 4 to 22, wherein Z is selected from 24. A compound according to any one of claims 4 to 23, wherein R 8 It is hydrogen or halogenated.

25. A compound according to any one of claims 4 to 24, wherein R 8 For hydrogen.

26. A compound according to any one of claims 4 to 25, wherein R 5 For hydrogen.

27. A compound according to any one of claims 4 to 26, wherein R 6 is selected from hydrogen and (C1-C4)alkyl.

28. A compound according to any one of claims 4 to 27, wherein R 6 is selected from hydrogen and methyl.

29. The compound according to any one of claims 1 to 4, wherein the E2 binding moiety is a compound of formula III: Where d, d1, d2 and d3 are CR 8 .

30. The compound of claim 29, wherein p is 0.

31. The compound according to claim 29 or 30, wherein Z is S.

32. A compound according to any one of claims 1 to 4, wherein the E2 binding portion has the following structure:

33. The compound of any one of claims 1 to 32, wherein the target protein binding moiety is one that binds a protein selected from the group consisting of STAT3, BCL2, WRN, and BRD4.

34. The compound of any one of claims 1 to 33, wherein the target protein binding moiety is a target protein binding moiety that binds BRD4.

35. A compound according to any one of claims 1 to 33, wherein the target protein binding portion has the following structure: The wavy bond indicates the point of connection to the linker (L).

36. according to the compound described in any one of claims 1 to 35, wherein joint (L) comprises optionally substituted straight chain or branched alkyl group, and described alkyl group is optionally interspersed by one or more heteroatoms selected from O, N and S.

37. The compound according to any one of claims 1 to 36, wherein the linker (L) is a straight or branched alkyl group substituted with one or more oxo groups (=O) and interrupted by one or more heteroatoms selected from O and N.

38. A compound according to any one of claims 1 to 37, wherein the linker (L) has the following structure: wherein the asterisk (*) indicates the point of attachment to the E2 binding moiety; e is an integer from 0 to 4; and j is an integer from 0 to 6.

39. The compound according to claim 38, wherein e is 0, 1 or 2.

40. The compound of claim 38 or 39, wherein j is 1.

41. The compound of claim 38, wherein the linker (L) is selected from one of the following structures:

42. A compound according to any one of claims 1 to 3, wherein the compound has the following structural formula: or a pharmaceutically acceptable salt thereof.

43. A pharmaceutical composition comprising a compound according to any one of claims 1 to 42 or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

44. A method of treating a disease responsive to degradation of a target protein, the method comprising administering to a subject an effective amount of a compound according to any one of claims 1 to 42 or a pharmaceutically acceptable composition according to claim 43.

45. The method of claim 44, wherein the target protein is selected from the group consisting of STAT3, BCL2, WRN and BRD4.

46. ​​The method of claim 44 or 45, wherein the disease is selected from cancer, inflammatory disease, sepsis, autoimmune disease and viral infection.

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