Proteolysis-targeting chimeric molecule (PROTAC) compositions using ubiquitin-conjugating enzyme ligands

Abstract

Provided herein are E2-linked PROTACs and uses thereof.

Description

[Background technology]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 332,305, filed April 19, 2022, the entire contents of which are incorporated herein by reference.

[0002] Proteolysis-targeting chimeric molecules (PROTACs) have become an attractive technology for modulating proteins of interest (POIs). PROTACs are heterobifunctional small molecules with three chemical elements: a ligand that binds to the target protein, a ligand that binds to the E3 ubiquitin ligase, and a linker to connect these two ligands. See, for example, Sun et al., Sig Transduct Target Ther 4, 64 (2019). Unlike the competitive and occupancy-driven process of traditional inhibitors, PROTACs are catalytic in their mechanism of action, potentially degrading target pathogenic proteins and modulating associated signaling pathways. PROTACs targeting approximately 50 proteins (many of which are clinically validated drug targets) have been developed, and several are in clinical trials for the treatment of cancer and other diseases.

[0003] However, a problem with current PROTACs is that they are limited to binding to E3 ubiquitin ligases, which are highly susceptible to mutations. 1733189996129_0 Recent studies have demonstrated that acquired resistance to PROTACs is associated with mutations in the core components of E3 ligases (Zhang et al., Mol Cancer Ther. 2019 Jul;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

[0004] It has now been discovered that ubiquitin-conjugating enzymes (E2) (e.g., UBE2K) can serve as effective targets in the design and use of PROTACs. Unlike E3 ligands, E2s such as UBE2K have very low mutation frequencies, reducing the risk of PROTAC-acquired resistance in clinical settings.

[0005] Thus, provided herein are compounds and compositions that function to recruit target proteins to E2 enzymes (e.g., UBE2K) for ubiquitination and subsequent degradation, as well as methods of their use. In one aspect, the present disclosure provides PROTAC compounds having an E2 enzyme binding moiety, a target protein binding moiety, and a linker. In one aspect, the polyubiquitination activity and / or increased processivity of the E2 enzyme is independent of E3 ligase. In one aspect, the E2 enzyme binding moiety serves to stabilize the E2-E2 dimer (e.g., homodimer or heterodimer).

[0006] Also provided is the use of the disclosed PROTACs to degrade a target protein of interest.

[0007] Further provided is the use of the disclosed PROTACs for treating diseases, such as cancer. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows the E2 binding ability of PROTACs according to the present disclosure. [Diagram 2] FIG. 1 shows dose-dependent degradation of BRD4 in HeLa cells using E2-conjugated PROTACs according to the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Provided herein is a PROTAC compound that includes an E2-binding moiety, a target protein-binding moiety, and a linker that covalently attaches the E2-binding moiety to the target protein-binding moiety.

[0010] As used herein, "E2 binding moiety," "E2 binder," or "E2 ligand" refers to a chemical moiety that binds to an E2 enzyme. E2 binding moieties include, but are not limited to, moieties that bind to an E2 enzyme selected from 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 embodiment, the E2 enzyme to which the E2 ligand binds is UBE2K.

[0011] In one aspect, as part of the first chemical embodiment, the E2 binding moiety is a compound of formula I: [ka] During the ceremony, Z 1 and Z 2 are each independently N or CH; R 1 is (C1-C6)alkyl, halo(C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, or -NR c R d wherein two available hydrogen atoms on the halo(C1-C6)alkyl and halo(C1-C6)alkoxy can be taken together with the carbon atom to which they are attached to form a 3- to 6-membered cycloalkyl optionally substituted with 1-3 groups selected from 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 2when on adjacent carbon atoms, together with the carbon atoms to which they are attached form a 5- or 6-membered oxygen-containing heterocyclyl 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 halo(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 atom to which they are attached form a 3- to 6-membered cycloalkyl or 3- to 6-membered heterocyclyl, each 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 are each independently 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 form a 4- to 7-membered heterocyclyl optionally substituted with 1 to 3 groups selected from 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: [ka] Ring A is aromatic; the wavy bond on ring A represents the point of attachment to Y; the wavy bond next to W represents the point of attachment to the linker (L); X, X 1 , and X 2 are, as far as valence permits, -CR 7 , N, O, and S; The dotted line in ring B represents a single or double bond; W is NH, -N(C1-C6)alkyl, O, or S; R 5 is 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 each of the (C3-C6)cycloalkyl and (C4-C7)heterocyclyl 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, and oxo; If the dotted 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 CR 8 and N, R e and R f are each 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, R 8is halogen, hydrogen, (C1-C6)alkyl, halo(C1-C6)alkyl, CN, or OH, and the linker (L) and the target protein binding moiety are as defined herein.

[0012] When used in connection with describing a chemical group that may have multiple points of attachment, a hyphen (-) indicates the point of attachment of the group to the variable being defined. For example, -NH(C1-C6)alkyl means that the point of attachment of the group is on the nitrogen atom.

[0013] The terms "halo" and "halogen" refer to an atom selected from fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), and iodine (iodo, -I).

[0014] The term "alkyl", whether used alone or as part of a larger moiety such as "haloalkyl", means a saturated straight-chain or branched monovalent hydrocarbon radical. Unless otherwise specified, an alkyl group typically has 1 to 4 carbon atoms, i.e., (C1-C4) alkyl.

[0015] "Alkoxy" refers to an alkyl radical attached through an oxygen linking atom represented by -O-alkyl. For example, "(C1-C4)alkoxy" includes methoxy, ethoxy, proproxy, and butoxy.

[0016] The term "haloalkyl" includes mono-, poly-, and perhaloalkyl groups, where the halogens are independently selected from fluorine, chlorine, bromine, and iodine.

[0017] A "haloalkoxy" is a haloalkyl group that is attached to another moiety via an oxygen atom, such as, for example, but not limited to, -OCHCF2 or -OCF3.

[0018] "Oxo" refers to the divalent functional group =O, an oxygen atom linked to another atom (typically carbon or sulfur) by a double bond.

[0019] The term "heteroaryl" refers to an aromatic ring of a particular size (e.g., 5-, 6-, 7-, 8-, or 9-membered ring) containing 1-4 heteroatoms independently selected from N, O, and S. Heteroaryl groups can be monocyclic or bicyclic. Monocyclic heteroaryls include, for example, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, and the like. Bicyclic heteroaryls include groups in which a monocyclic heteroaryl ring is fused to one or more aryl or heteroaryl rings. Non-limiting examples include indolyl, imidazopyridinyl, benzoxazolyl, benzoxodiazolyl, indazolyl, benzimidazolyl, benzthiazolyl, pyrazolopyridinyl, thienopyridinyl, thienopyrimidinyl, indolizinyl, etc. When specified, any substituent on a heteroaryl group may be present at any substitutable position.

[0020] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic ring of a particular size (e.g., 3-, 4-, 5-, 6-, or 7-membered ring) containing 1-4 heteroatoms independently selected from N, O, and S. A heterocyclyl ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, oxiranyl, thiiranyl, aziridinyl, tetrahydrofuranyl, tetrahydrothienyl, terahydropyranyl, pyrrolidinyl, pyridinonyl, pyrrolidonyl, piperidinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, morpholinyl, dihydrofuranyl, dihydropyranyl, dihydropyridinyl, tetrahydropyridinyl, dihydropyrimidinyl, oxetanyl, azetidinyl, and tetrahydropyrimidinyl. When specified, optional substituents on a heterocyclyl group may be present at any substitutable position, including, for example, the position at which the heterocyclyl is attached.

[0021] The term "cycloalkyl" refers to a monocyclic hydrocarbon of a particular size (e.g., 3, 4, 5, 6, or 7 membered ring). Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, and cyclooctyl. When specified, any substituent on the cycloalkyl group may be present at any substitutable position, including, for example, the position at which the cycloalkyl is attached.

[0022] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure, such as the R and S configurations of each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the invention are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0023] The compounds described herein may exist in the form of pharmaceutically acceptable salts. When used in medicines, the salts of the compounds described herein refer to non-toxic "pharmaceutically acceptable salts". The pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic / anionic or basic / 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 salts of organic acids (such as acetic acid, benzenesulfonic acid, benzoic acid, methanesulfonic acid, and p-toluenesulfonic acid). Compounds of the present teachings having an acidic group, such as carboxylic acid, can form pharmaceutically acceptable salts with pharmaceutically acceptable base(s). Suitable pharmaceutically acceptable base 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 a quaternary ammonium group also include a counteranion such as chloride, bromide, iodide, acetate, perchlorate, etc. Other examples of such salts include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, benzoates, and salts with amino acids such as glutamic acid.

[0024] In one aspect, as part of a second chemical embodiment, the E2 binding moiety is a compound of formula II: [ka] wherein the variables are as described above for Formula I, and the linker (L) and target protein binding moieties are as defined herein.

[0025] In one aspect, as part of a third chemical embodiment, W in a compound of Formula I or Formula II is S, where the variables are as described above for Formula I, and the linker (L) and the target protein binding moiety are as defined herein.

[0026] In one aspect, as part of a fourth chemical embodiment, R in a compound of formula I or formula II 3 is hydrogen, where the variables are as described above for Formula I or the third chemical embodiment, and the linker (L) and the target protein binding moiety are as defined herein.

[0027] In one aspect, as part of the fifth chemical embodiment, Z in a compound of formula I or formula II 1 and Z 2 each is CH, where the variables are as described above for Formula I, or the third or fourth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0028] In one aspect, as part of the sixth chemical embodiment, Y in a compound of Formula I or Formula II is CH2, where the variables are as described above for Formula I or any one of the third through fifth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0029] In one aspect, as part of the seventh chemical embodiment, p in a compound of Formula I or Formula II is 0, where the variables are as described above for Formula I or any one of the third through sixth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0030] In one aspect, as part of an eighth chemical embodiment, R in a 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 form a 5-6 membered heterocyclyl optionally substituted with 1-3 halo, where the variables are as described above for formula I or any one of the third through seventh chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein. Alternatively, as part of the eighth chemical embodiment, R in a compound of formula I or formula II is 1 is (C1-C3)alkyl, halo(C1-C3)alkyl, halo(C1-C6)alkoxy, (C1-C3)alkoxy, or -NR c R d and R c and R d are taken together with the nitrogen atom to which they are attached to form a 5-6 membered nitrogen-containing heterocyclyl optionally substituted with 1-3 halo, where the variables are as described above for formula I or any one of the third through seventh chemical embodiments, and 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 a compound of formula I or formula II is 1 is OCF3, OCHF2, OCH3, CH3, pyrrolidinyl, or piperidinyl, wherein pyrrolidinyl or piperidinyl is optionally substituted with 1-3 halo, where the variables are as described above for formula I, or any one of the third through seventh chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0031] In one aspect, as part of a ninth chemical embodiment, X in a compound of Formula I or Formula II is N, where the variables are as described above for Formula I or any one of the third through eighth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0032] In one aspect, as part of a tenth chemical embodiment, X in a compound of formula I or formula II 1 is CH or N, where the variables are as described above for formula I or any one of the third through ninth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0033] In one aspect, as part of an eleventh chemical embodiment, X in a compound of formula I or formula II 2 is CH, N, S, or O, where the variables are as described above for formula I, or any one of the third through tenth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0034] In one aspect, as part of the twelfth chemical embodiment, X in the compound of formula I or formula II is N, and X 1 is N and X 2 is CH or X is N, and X 1 is N and X 2 is N or X is N and X 1 is CH and X 2 is S or X is N and X 1 is CH and X 2 is O, where the variables are as described above for formula I, or any one of the third through eleventh chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0035] In one aspect, as part of the thirteenth chemical embodiment, d in the compound of formula I or formula II is N or -CR 8where the variables are as described above for formula I, or any one of the third through twelfth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0036] In one aspect, as part of the fourteenth chemical embodiment, d in a compound of formula I or formula II 2 is N or -CR 8 where the variables are as described above for formula I, or any one of the third through thirteenth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0037] In one aspect, as part of the fifteenth chemical embodiment, d in a compound of formula I or formula II 1 HA-CR 8 where the variables are as described above for formula I, or any one of the third through fourteenth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0038] In one aspect, as part of the sixteenth chemical embodiment, d in a compound of formula I or formula II 3 HA-CR 8 where the variables are as described above for formula I, or any one of the third through fifteenth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0039] In one aspect, as part of the seventeenth chemical embodiment, Z in the compound of formula I or formula II is [ka] [ka] [ka] [ka] wherein the variables are as described above for formula I or any one of the third through sixteenth chemical embodiments, and 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: [ka] [ka] [ka] [ka] [ka] where the variables are as described above for formula I, or any one of the third through sixteenth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0040] In one aspect, as part of the eighteenth chemical embodiment, R in a compound of formula I or formula II 8 is hydrogen or halo, where the variables are as described above for Formula I or any one of the third through seventeenth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein. Alternatively, as part of the eighteenth chemical embodiment, R in a compound of Formula I or Formula II is 8 is hydrogen, where the variables are as described above for formula I, or any one of the third through seventeenth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0041] In one aspect, as part of the nineteenth chemical embodiment, R in a compound of formula I or formula II 5is hydrogen, where the variables are as described above for formula I, or any one of the third through eighteenth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0042] In one aspect, as part of the twentieth chemical embodiment, R in a compound of formula I or formula II 6 is selected from hydrogen and (C1-C4) alkyl, where the variables are as described above for formula I or any one of the third through nineteenth chemical embodiments, and 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, where the variables are as described above for formula I, or any one of the third through nineteenth chemical embodiments, and the linker (L) and the target protein binding moiety are as defined herein.

[0043] In one aspect, as part of a twenty-first chemical embodiment, the E2 binding moiety is a compound of formula III: [ka] In the formula, d, d1, d2, and d3 are each CR 8 where the variables are as described above for Formula I, and the linker (L) and the target protein binding moiety are as defined herein.

[0044] In one aspect, as part of the twenty-second chemical embodiment, p in the compound of formula III is 0, where the variables are as described above for formula I, and the linker (L) and the target protein binding moiety are as defined herein.

[0045] In one aspect, as part of a twenty-third chemical embodiment, the E2 binding moiety has the structure: [ka]

[0046] In one aspect, as part of the twenty-third chemical embodiment, Z in the compound of formula III is S, where the variables are as described above for formula I or the twenty-second embodiment, and the linker (L) and the target protein binding moiety are as defined herein.

[0047] In one aspect, as part of the twenty-fourth chemical embodiment, the E2 binding moiety is selected from those disclosed in US Provisional Application No. 62 / 223,626.

[0048] As used herein, "target protein binding moiety" refers to a chemical moiety that binds to a protein of interest (POI). The POIs of the disclosed compounds include, but are not limited to, BRD4, STAT3, BCL2, and WRN. In one embodiment, the POI is BRD4.

[0049] In one aspect, a target protein binding moiety disclosed herein (e.g., in any one of the first to twenty-fourth embodiments above) has the structure: [ka] where the wavy bond represents the point of attachment to the linker (L).

[0050] The term "linker" refers to a chemical moiety that covalently links the E2 binding moiety to the target protein binding moiety. In one embodiment, the linker is not cleavable in vivo. In one embodiment, the linker comprises optimal spatial and chemical properties to achieve optimal therapeutic activity. In one embodiment, the linker does not interfere with the ability of the E2 binding moiety and / or the target protein binding moiety to bind to their respective targets. In one embodiment, the linker comprises an optionally substituted linear or branched alkyl group, which is optionally interrupted by one or more heteroatoms selected from O, N, and S. In one embodiment, the linker is a linear or branched alkyl group substituted with one or more oxo groups (=O) and also interrupted by one or more heteroatoms selected from O and N.

[0051] In one aspect, the linker (L) disclosed herein (e.g., in any one of the first to twenty-fourth embodiments above) has the structure: [ka] wherein the asterisk (*) represents the point of attachment 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) disclosed herein (e.g., in any one of the first to twenty-fourth embodiments above) is selected from one of the following structures: [ka] [ka] [ka]

[0052] Additional compounds are disclosed in the Examples and are included in the present disclosure, including their pharma- ceutically acceptable salts and neutral forms.

[0053] Uses, Formulation and Administration One or more compounds described herein may be present as part of a pharmaceutical composition.Thus, in one aspect, a pharmaceutical composition is provided that comprises a disclosed compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0054] The term "pharmaceutically acceptable" refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. 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 phosphoric acid, 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 silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0055] In certain aspects, the pharmaceutical compositions described herein are formulated for administration to a patient in need of such compositions. The pharmaceutical compositions described herein can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intraperitoneally, or intravenously. Sterile injectable forms of the pharmaceutical compositions described herein can be aqueous or oily suspensions. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.

[0056] The compounds and compositions described herein are generally useful for modulating the activity of a target protein (e.g., STAT3, BCL2, WRN, or BRD4). In some embodiments, the compounds, pharma- ceutically acceptable salts, and pharmaceutical compositions described herein degrade a target protein (e.g., STAT3, BCL2, WRN, or BRD4).

[0057] Accordingly, provided herein is a method of treating a disease responsive to degradation of a target protein (e.g., STAT3, BCL2, WRN, or BRD4) in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a compound described herein or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising a disclosed compound or a pharma- ceutically acceptable salt thereof.

[0058] Also provided is the use of a compound described herein, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising the disclosed compound or a pharma- ceutically acceptable salt thereof, for treating a disease responsive to degradation of a target protein (e.g., STAT3, BCL2, WRN, or BRD4).

[0059] 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 that includes an E2 binding moiety, a target protein binding moiety (i.e., a binding moiety that binds to a target protein to be degraded), and a linker that covalently attaches the E2 binding moiety to the target protein binding moiety, thereby degrading the target protein in the cell.

[0060] Also provided herein is a method of 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 or a pharma- ceutically acceptable salt thereof, the compound or a pharmaceutical composition comprising the compound or a pharma- ceutically acceptable salt thereof, the compound or a pharmaceutical composition comprising the compound or a pharma- ceutically acceptable salt thereof, thereby degrading the target protein in the subject.

[0061] Further provided herein is a method of treating a disease responsive to degradation of a target protein (e.g., STAT3, BCL2, WRN, or BRD4) in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a compound or a pharma- ceutically acceptable salt thereof, the compound or a pharmaceutical composition comprising the compound or a pharma- ceutically acceptable salt thereof, the compound or a pharmaceutical composition comprising the compound or a pharma- ceutically acceptable salt thereof, thereby treating the disease.

[0062] Also provided is the use of a compound described herein, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising a disclosed compound or a pharma- ceutically acceptable salt thereof, for the manufacture of a medicament for treating a disease responsive to degradation of a target protein (e.g., STAT3, BCL2, WRN, or BRD4).

[0063] The terms "subject" and "patient" may be used interchangeably and refer to a mammal in need of treatment, such as 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.

[0064] As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset, or inhibiting the progression of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment can be administered after one or more symptoms have developed, i.e., a therapeutic treatment. In other embodiments, treatment can be administered in the absence of symptoms. For example, treatment can be administered to susceptible individuals prior to the onset of symptoms (e.g., in light of a history of symptoms and / or exposure to a particular microorganism or other susceptibility factors), i.e., a prophylactic treatment. Treatment can also be continued after symptoms have disappeared, e.g., to delay recurrence.

[0065] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound described herein that elicits a biological or medical response in a subject, e.g., a dosage of 0.01 to 100 mg / kg body weight / day.

[0066] The specific dosage and treatment regimen for any particular patient will vary depending on a variety of factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of a compound described herein in the composition will also depend on the particular compound in the composition.

[0067] In some aspects, diseases responsive to target protein degradation include, but are not limited to, cancer and other proliferative diseases, inflammatory diseases, sepsis, autoimmune diseases, and viral infections.

[0068] In some embodiments, the disease treated by the methods of the invention is cancer. Examples of cancers that may be treated using the compounds and methods described herein include adrenal gland carcinoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrohidroma, acute eosinophilic leukemia, acute erythroleukemia, acute lymphocytic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenoid odontogenic tumor, adenosquamous carcinoma, adipose tissue tumor, adrenal cortical carcinoma, adult T-cell leukemia / lymphoma, aggressive NK cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, anaplastic large cell lymphoma, undifferentiated ... 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, blastoma, bone cancer, Brenner tumor, brown tumor, Burkitt lymphoma, breast cancer, brain tumor, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear cell sarcoma of the kidney, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large intestine large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine tumor, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, fetal in foetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid carcinoma, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of bone, glial tumor, giant cell tumor of bone, glial cell tumor, glioblastoma multiforme, glioma, gliomatosis cerebri, glucagon-producing tumor, gonadoblastoma, granulosa cell tumor, ginandroblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer 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, lethal midline carcinoma, leukemia, Leydig cell tumor, 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 breast carcinoma, medullary thyroid carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic urothelial carcinoma, mixed Müllerian tumor, mucinous tumor, multiple myeloma, muscle tissue tumor, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, schwannoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, eye cancer, oligoastrocytoma, oligodendroglioma Glioma, eosinophilic granular cell tumor, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid carcinoma, paraganglioma, pineoblastoma, pineocytoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryomatosis, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary peritoneal cancer, prostate cancer, pancreatic cancer, These include, but are not limited to, pharyngeal cancer, pseudomyxoma peritonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, schwannoma, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, soft tissue sarcoma, somatostatinoma, sooty warts, spinal cord tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease, small intestine cancer, squamous cell carcinoma, gastric cancer, T-cell lymphoma, testicular cancer, theca cell tumor, thyroid cancer, transitional cell carcinoma, pharyngeal cancer, urachal cancer, genitourinary cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, optic pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor. ,

[0069] In some embodiments, the disease treated by the methods of the present 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, allergy, Crohn's disease, irritable bowel syndrome, ulcerative colitis, Sjogren's syndrome, tissue transplant rejection, hyperacute rejection of transplanted organs, asthma, allergic rhinitis, chronic obstructive pulmonary disease (COPD), autoimmune polyglandular disease (also called autoimmune polyglandular syndrome), autoimmune alopecia, pernicious anemia, glomerulonephritis, dermatomyositis, multiple sclerosis, scleroderma, vasculitis. , autoimmune hemolytic and thrombocytopenic disorders, Goodpasture's syndrome, atherosclerosis, Addison's disease, Parkinson's disease, Alzheimer's disease, type 1 diabetes, septic shock, systemic lupus erythematosus (SLE), rheumatoid arthritis, psoriatic arthritis, juvenile arthritis, osteoarthritis, chronic idiopathic thrombocytopenic purpura, Waldenström's macroglobulinemia, myasthenia gravis, Hashimoto's disease, atopic dermatitis, osteoarthritis, vitiligo, autoimmune hypopituitarism, Guillain-Barre syndrome, Behcet's disease, scleritis, mycosis fungoides, acute inflammatory reactions (such as acute respiratory distress syndrome and ischemia / reperfusion injury), and Graves' disease. EXAMPLES

[0070] The following representative examples are intended to help illustrate the disclosure and are not intended, nor should they be construed, to limit the scope of the invention. Typical starting materials used were obtained from commercial sources or prepared in other examples unless otherwise noted. [Table 1]

[0071] Synthesis of N-((5-thioxo-5,6-dihydropyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (Compound 1). [ka]

[0072] Step 1: Synthesis of N-((1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide. To a stirred solution of 2-(trifluoromethoxy)benzoic acid (1) (25 g, 0.121 mmol) in DMF (250 mL) at 0° C. was added HATU (46.1 g, 0.121), followed by (2H-pyrazol-3-yl)methanamine (11.7 g, 0.1213) and DIPEA (39.1 g, 0.303 mmol). The reaction was then stirred at room temperature for 12 h. After that, the mixture was diluted with water (2.5 L) and extracted with EtOAc (2×500 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 layers were dried over Na2SO4 and concentrated to give the crude product. The crude product was then purified by flash column chromatography (eluent: 70% EtOAc / petroleum ether) to give N-((1H-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,3 LC-MS m / z(M+H):286.1.

[0073] Step 2: Synthesis of N-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide. 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) and washed once with saturated NaHCO3 solution (100 mL) and H2O (100 mL). The organic layer was dried over Na2SO4 and concentrated. The resulting crude product was triturated with petroleum ether (200 mL), stirred for 12 h, and the precipitated solid was filtered and dried under vacuum to give 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.

[0074] Step 3: Synthesis of N-((5-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide. 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 dry THF (200 mL) was added 1.6 M n-butyllithium (6.31 g, 98.61 mmol) in hexanes at -78 °C over 10 min. The mixture was then stirred at the same temperature for 1 h, followed by the addition of iodine (13.76 g, 54.2 mmol) in dry THF (200 mL) over 15 min. After complete addition of iodine, the reaction mixture was allowed to warm slowly to -20 °C over 45 min. The reaction was then quenched with saturated NaHSO3 solution (200 mL) and extracted with EtOAc (2 x 150 mL). The combined organic layers were dried over Na2SO4 and concentrated on a rotary evaporator to give the crude compound. The crude compound was purified by flash column chromatography (eluent: 20% EtOAc + petroleum 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.

[0075] Step 4: Synthesis of N-((5-(2-aminophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide. 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. The resulting mixture was heated at 100° C. for 12 h, then diluted with H2O (15 mL) and extracted with EtOAc (2×50 mL). The combined organic layers were dried over Na2SO4 and concentrated. The resulting crude product was purified by flash column chromatography (eluent: 30% EtOAc + hexane) to give N-((5-(2-aminophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide as a yellow gummy solid (4.6 g, 74.5%). LC-MS m / z(MH): 461.11.

[0076] Step 5: Synthesis of N-((5-(2-aminophenyl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide. 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 HCl in 1,4-dioxane (4 M, 30 mL) 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 (2×25 mL) to give the crude compound. The crude compound was diluted with H2O (25 mL), basified with saturated NaHCO3 solution (25 mL) and extracted with EtOAc (2×25 mL). The combined organic layers were dried over Na2SO4 and concentrated to give N-((5-(2-aminophenyl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide as a yellow gummy solid (4.75 g). LC-MS m / z(M-H):377.19.

[0077] Step 6: Synthesis of N-((5-thioxo-5,6-dihydropyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (Compound 1). To a stirred solution of N-((5-(2-aminophenyl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (4.75 g, 12.62 mmol) in pyridine (110 mL) and HO (30 mL) was added carbon disulfide (110 mL) at room temperature. The reaction mixture was then heated at 100° C. for 12 h. It was then diluted with cold HO (500 mL) and the precipitated solid was filtered and air-dried to give the crude compound. It was then purified by preparative HPLC to give N-((5-thioxo-5,6-dihydropyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide as an off-white solid (1.6 g, 30.30%). 1H 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.

[0078] 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). [ka]

[0079] Step-1: Synthesis of tert-butyl (2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)ethyl)carbamate. 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 K2CO3 (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 give an off-white solid of (2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)ethyl)tert-butylcarbamate (12 mg, 89.5%). 1H 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, LC-MS m / z(M+H):561.93.

[0080] Step-2: Synthesis of N-((5-((2-aminoethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide 2,2,2-trifluoroacetate. 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 as an off-white solid (100 mg, 82%). 1 H 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.

[0081] 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). N-((5-((2-aminoethyl)thio)pyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide: To a stirred solution of TFA (100 mg, 0.173 mmol) in DMF (3 mL), DIPEA (112 mg, 0.869 mmol), HATU (99 mg, 0.260 mmol) were added 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 as a white solid (50 mg, 34%). 1 H 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.

[0082] Synthesis of 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)acetic acid (compound 3). [ka]

[0083] Step-1: Synthesis of ethyl 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)acetate. To a stirred suspension of N-((5-thioxo-5,6-dihydropyrazolo[1,5-c]quinazolin-2-yl)methyl)-2-(trifluoromethoxy)benzamide (300 mg, 0.717 mmol) in acetone (15 mL) was added K2CO3 (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 with 2-3% MeOH in DCM to give ethyl 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)acetate as a brown solid (280 mg, 77%). 1 H NMR(400MHz,DMSO)δ917(t,J=5.9Hz,1H),8.20(d,J=7.7Hz,1H),7.74-7.66(m,3H),7.64-7.57(m,2H),7.52 -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.

[0084] Step 2: Synthesis of 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)acetic acid (compound 3). To a stirred solution of 2-((2-((2-(trifluoromethoxy)benzamido)methyl)pyrazolo[1,5-c]quinazolin-5-yl)thio)ethyl acetate (280 mg, 0.55 mmol) in THF-HO (7 mL+3 mL) was added LiOH.HO (28 mg, 0.66 mmol) at 0° C. and the reaction mixture was allowed to warm and stirred at room temperature for 4 h. The reaction mixture was concentrated, diluted with HO (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 as a white solid (200 mg, 75%). 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.

[0085] 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). [ka]

[0086] 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). 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) tert-butyl acetate (500 mg, 1.094 mmol) in DCM (20 mL) was added TFA (1 mL) at 0° C. The reaction mixture was warmed and stirred at room temperature for 12 h. The resulting reaction mixture was concentrated and coevaporated 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 as a pale yellow gummy solid (400 mg, 91%). LC-MS: m / z=401.20

[0087] Step-2: Synthesis of (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 tert-butyl. (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: To a stirred solution of TFA (250 mg, 0.623 mmol) and tert-butyl (2-aminoethyl)carbamate (150 mg, 0.935 mmol) in DMF (5 mL) was added HATU (355 mg, 0.935 mmol) followed by DIPEA (0.16 mL, 0.935 mmol) at 0° C. The resulting reaction mixture was stirred at room temperature for 12 h, quenched with ice-cold water (20 mL) and extracted with EtOAc (3×20 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)-(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)tert-butylcarbamate as a pale yellow gummy solid (110 mg, 32.4%). LC-MS: m / z=543.1.

[0088] 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). To a stirred solution of (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)tert-butyl carbamate (100 mg, 0.181 mmol) in DCM (5 mL) was added TFA (0.2 mL) at 0° C. The reaction mixture was allowed to warm and stirred at room temperature for 12 h. The reaction mixture was then concentrated and the residue was co-evaporated 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 as a pale yellow gummy solid (78 mg, 97.5%). LC-MS: m / z=443.2.

[0089] 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). [ka] 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 layer was 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 as an off-white solid (35 mg, 18.5%). 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.

[0090] 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). [ka]

[0091] Step 1: Synthesis of (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 tert-butyl. 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 (2×50 mL). The combined organic layers were washed with ice-water (2×50 mL) and brine (50 mL), dried over Na2SO4, and concentrated in vacuo. The crude compound was purified with 0-5% MeOH in dichloromethane to give (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)tert-butylcarbamate as an off-white solid (290 mg, 49%). LC-MS (ESI): m / z 587.1 (M+H)

[0092] 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. To a stirred solution of (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)tert-butylcarbamate (290 mg, 0.49 mmol) in DCM (3 mL) was added TFA (0.5 mL) at 0° C. and the mixture was stirred at room temperature for 4 h. After completion of the reaction, the reaction mixture was concentrated in vacuo. The crude compound was purified by preparative HPLC (water:ACN) to give (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 as a pale 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) + .

[0093] Step-3: 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. 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 (2×20 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-(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 as an off-white solid (39 mg, 19.6%). 1H NMR(400MHz,DMSO-d6)δ9.18(t,J=5.6Hz,1H),8.41-8.38(m,2H),8.17(d,J=7.2Hz,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,2H),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) + .

[0094] Synthesis of (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 (Compound 9). [ka]

[0095] Step-1: Synthesis of (S)-(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 tert-butyl. 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) was added HATU (1.2 g, 3.217 mmol) followed by DIPEA (0.53 mL, 3.217 mmol) at 0° C. 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 (3×30 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)-(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)tert-butylcarbamate as a pale yellow gummy solid (590 mg, 43.7%). LC-MS: m / z=631.26 (M+H). + .

[0096] 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. To a stirred solution of (S)-(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)tert-butylcarbamate (590 mg, 0.934 mmol) in DCM (10 mL) was added TFA (2 mL) at 0° C. The reaction mixture was allowed to warm 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 as a pale yellow gummy solid (423 mg, 85%). 1 H NMR(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) + .

[0097] Step-3: Synthesis of (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. 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]triazole[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 hours, then diluted with water (30 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-((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 as an off-white solid (88.9 mg, 42.78%). 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) + .

[0098] 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). [ka]

[0099] Step-1: Synthesis of N-((5-(2-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide. 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), the mixture was degassed with argon gas for 5 min, followed by the addition of Pd(PPh3)4 (58.3 mg, 0.05 mmol). The reaction mixture was stirred at 100 °C for 12 h and concentrated. The residue was diluted with 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 resulting crude product was purified by flash chromatography using 30% EtOAc in hexane to give N-((5-(2-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide as an off-white solid (160 mg, 68.6%). LC-MS: m / z=460.43 (MH). + .

[0100] Step-2: Synthesis of ethyl 2-(2-(1-(tetrahydro-2H-pyran-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetate. 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 K2CO3 (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 Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography using 30% EtOAc in hexane to give ethyl 2-(2-(1-(tetrahydro-2H-pyran-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetate as a yellow gummy solid (105 mg, 55.3%). LC-MS: m / z=546.35 (MH). + .

[0101] Step-3: Synthesis of 2-(2-(1-(tetrahydro-2H-pyran-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetic acid. 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.H2O (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 x 20 mL). The combined organic layers were dried over Na2SO4 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 as an off-white solid (95 mg, 95.3%). LC-MS: m / z=518.4 (MH). + .

[0102] Step-4: Synthesis of 2-(2-(3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-5-yl)phenoxy)acetic acid. 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) was added 4M HCl in 1,4-dioxane (5 mL) at 0° C. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the solvent was evaporated and the residue was neutralized with saturated NaHCO3 solution and stirred for 10 min. The resulting 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 as an off-white solid (95 mg, 73%). 1H 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) + .

[0103] 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. 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 (3×20 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) + .

[0104] 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). [ka]

[0105] Step 1: Synthesis of N-((1-(tetrahydro-2H-pyran-2-yl)-5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide. To a sealed tube containing N-((5-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide (1.4 g, 2.826 mmol) in 1,4-dioxane (16 mL) and water (4 mL) was added thiophen-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) under argon atmosphere. The reaction mixture was then stirred at 100° C. for 12 h in a preheated oil bath. The mixture was cooled and 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 give N-((1-(tetrahydro-2H-pyran-2-yl)-5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide as an off-white solid (0.434 g, 34.1%). 1H 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).

[0106] Step 2: Synthesis of N-((5-(thiophen-2-yl)-1H-pyrazol-3-yl)methyl)-2-(trifluoromethoxy)benzamide. 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, and 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 as an off-white solid (434 mg, 93.5%). 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) + .

[0107] Step 3: Synthesis of ethyl 2-(5-(thiophen-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-1-yl)acetate. 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 K2CO3 (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, then cooled to room temperature, 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 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 / 454.1 (M+H) + .

[0108] Step 4: Synthesis of 2-(5-(thiophen-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-1-yl)acetic acid. To a stirred solution of 2-(5-(thiophen-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-1-yl)ethyl acetate (90 mg, 0.198 mmol) in THF (2 mL) and water (1 mL) was added LiOH.H2O (21 mg, 0.496 mmol) at 0 °C. The reaction mixture was then warmed and stirred at room temperature for 12 h. The reaction mixture was concentrated and the residue was acidified with citric acid (pH approx. 2) and extracted with 10% MeOH in DCM. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-(5-(thiophen-2-yl)-3-((2-(trifluoromethoxy)benzamido)methyl)-1H-pyrazol-1-yl)acetic acid as an off-white solid (40 mg, 45.8%). 1H 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),7.50-7.39(m,2) H),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) + .

[0109] 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. 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) 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) at 0° C. The reaction mixture was then allowed to warm 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 as a pale yellow gummy solid (33 mg, 37.8% yield). 1 H NMR(400MHz,DMSO-d6)δ8.93(t,J=5.6Hz,1H),8.32-8.20(m,2H),7.6(d,J=4.8H z,1H),7.63-7.53(m,2H),7.50-7.35(m,6H),7.29-7.24(m,1H),7.18-7.10(m,1 H),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) + .

[0110] Synthesis of (S)-N-(5-amino-1-(thiazolo[4,5-c]quinolin-2-yl)pentyl)-2-(difluoromethoxy)benzamide (Compound 12). [ka]

[0111] Step-1: Synthesis of 3-aminoquinolin-4-ol. To a stirred solution of 3-nitroquinolin-4-ol (10 g, 3.125 mmol) in MeOH (10 mL) was added 10% Pd / C (5 g) at room temperature under nitrogen atmosphere. The reaction flask was then pressurized with hydrogen gas (50 psi) and the mixture was stirred for 12 h. The resulting reaction mixture was filtered over Celite and the filtrate was concentrated under reduced pressure. The resulting crude product was purified by column chromatography (eluent: 50% EtOAc in hexanes) to give 3-aminoquinolin-4-ol as an off-white solid (7.70 g, 90.53%). LC-MS C9H8N2O m / z (M+H): 161.1.

[0112] Step-2: Synthesis of (6-((4-hydroxyquinolin-3-yl)amino)-6-oxohexane-1,5-diyl)(R)-dicarbamate benzyl tert-butyl. 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) followed by DIPEA (8 mL, 9.375 mmol) at 0° C. 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 (2×30 mL). The combined organic layers were washed once with brine solution (20 mL), the organic layer was separated, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting crude product was purified by column chromatography (eluent: 40% EtOAc in hexanes) to give (6-((4-hydroxyquinolin-3-yl)amino)-6-oxohexane-1,5-diyl)(R)-benzyl tert-butyl dicarbamate as an off-white solid (3.0 g, 88.23%). LC-MS C 28 H 34 N4O6m / z(M+H):523.1.

[0113] Step-3: Synthesis of (1-(thiazolo[4,5-c]quinolin-2-yl)pentane-1,5-diyl)(R)-dicarbamate benzyl tert-butyl. To a stirred solution of (1-(thiazolo[4,5-c]quinolin-2-yl)pentane-1,5-diyl)(R)-benzyl tert-butyl dicarbamate (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 aqueous NH4Cl. It was further diluted with H2O (30 mL) and extracted with EtOAc (2×30 mL). The combined organic layers were washed once with brine (20 mL) and the organic layer was separated, dried over anhydrous Na2SO4 and concentrated. The crude compound obtained was purified by column chromatography (eluent: 60% EtOAc in hexane) to give (1-(thiazolo[4,5-c]quinolin-2-yl)pentane-1,5-diyl)(R)-benzyl tert-butyl dicarbamate as a pale yellow solid (2.2 g, 73.48%). LC-MS: m / z 521.1 (M+H). + .

[0114] Step-4: Synthesis of (R)-(5-amino-5-(thiazolo[4,5-c]quinolin-2-yl)pentyl)benzylcarbamate. To a stirred solution of (1-(thiazolo[4,5-c]quinolin-2-yl)pentane-1,5-diyl)(R)-benzyl 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 completion of the reaction, the solvent was evaporated, then diluted with H2O (20 mL), basified with saturated aqueous NaHCO3 (10 mL) and extracted with EtOAc (2×30 mL). The combined organic layers were washed once with brine (20 mL), the organic layer was 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) + .

[0115] Step-5: Synthesis of (S)-(5-(2-(difluoromethoxy)benzamido)-5-(thiazolo[4,5-c]quinolin-2-yl)pentyl)benzylcarbamate. 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) was added EDC.HCl (428 mg, 2.244 mmol), HOBt (302 mg, 2.244 mmol) and TEA (1.4 mL, 7.48 mmol) at 0° C. The reaction mixture was then stirred at room temperature for 12 h and concentrated. The residue was treated with aqueous NH4Cl and water (20 mL) and extracted with EtOAc (2×20 mL). The combined organic layers were washed once with brine (20 mL) and the organic layer was separated, dried over anhydrous Na2SO4 and concentrated. The resulting crude product was purified by column chromatography (eluent: 5% MeOH in DCM) to give (S)-(5-(2-(difluoromethoxy)benzamido)-5-(thiazolo[4,5-c]quinolin-2-yl)pentyl)benzylcarbamate as an off-white solid (350 mg, 40.32%). 1 H NMR(400MHz,DMSO)δ9.43(s,1H),8.45(d,J=7.2Hz,1H),8.27(t,J=5.6,Hz1H ),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 / z 591.0(M+H) + .

[0116] Step-6: Synthesis of (S)-N-(5-amino-1-(thiazolo[4,5-c]quinolin-2-yl)pentyl)-2-(difluoromethoxy)benzamide. To a stirred solution of (S)-(5-(2-(difluoromethoxy)benzamido)-5-(thiazolo[4,5-c]quinolin-2-yl)pentyl)benzylcarbamate (50 mg, 0.084 mmol) in DCM (5 mL) was added 40% HBr in AcOH at 0 °C and the reaction was stirred at room temperature for 4 h. The reaction was then concentrated and the crude product obtained was quenched with saturated NaHCO3 solution (10 mL) and extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with brine (10 mL) and the organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product obtained was purified by preparative HPLC (buffer formic acid) to give (S)-N-(5-amino-1-(thiazolo[4,5-c]quinolin-2-yl)pentyl)-2-(difluoromethoxy)benzamide as a green sticky 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) + .

[0117] Synthesis of N-(5-amino-1-(5-thioxo-5,6-dihydro-[1,2,4]triazolo[1,5-c]quinazolin-2-yl)pentyl)-2-(difluoromethoxy)benzamide (Compound 13). [ka]

[0118] Step-1: Synthesis of methyl N6-(tert-butoxycarbonyl)-N2-(2-(difluoromethoxy)benzoyl)lysinate. To a stirred solution of methyl N6-(tert-butoxycarbonyl)lysinate (1.70 g, 5.851 mmol) and 2-(difluoromethoxy)benzoic acid (1.20 g, 6.43 mmol) in DCM (15 mL) 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) at 0° C. The reaction mixture was then stirred at room temperature for 12 hours and concentrated. The residue was diluted with H2O (20 mL) and extracted with EtOAc (2×20 mL). The combined organic layers were washed once with saturated brine (20 mL), the organic layer was separated, dried over anhydrous Na2SO4 and concentrated. The resulting crude product was purified by column chromatography (eluent: 5% MeOH in DCM) to give methyl N6-(tert-butoxycarbonyl)-N2-(2-(difluoromethoxy)benzoyl)lysinate as an off-white solid (1.10 g, 40.32%). LC-MS: m / z(M+H): 331.1.

[0119] Step-2: Synthesis of tert-butyl (5-(2-(difluoromethoxy)benzamido)-6-hydrazinyl-6-oxohexyl)carbamate. To a stirred solution of N6-(tert-butoxycarbonyl)-N2-(2-(difluoromethoxy)benzoyl)methyl lysinate (500 mg, 1.62 mmol) in EtOH (5 mL), aqueous hydrazine (1 mL) was added 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 (2x20 mL). The combined organic layers were washed once with brine (20 mL) and the organic layer was separated, dried over anhydrous Na2SO4 and concentrated. The resulting crude product was purified by column chromatography (20% EtOAc in hexanes) to give tert-butyl (5-(2-(difluoromethoxy)benzamido)-6-hydrazinyl-6-oxohexyl)carbamate as a dark brown liquid (200 mg, 40%). LC-MS: m / z 431.10 (M+H).

[0120] Step-3: Synthesis of tert-butyl (5-(2-(difluoromethoxy)benzamido)-5-(5-thioxo-5,6-dihydro-[1,2,4]triazolo[1,5-c]quinazolin-2-yl)pentyl)carbamate. To a stirred solution of tert-butyl (5-(2-(difluoromethoxy)benzamido)-6-hydrazinyl-6-oxohexyl)carbamate (120 mg, 0.27 mmol) in EtOH (10 mL) was added 2-isothiocyanatobenzonitrile (49 mg, 0.30 mmol) 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 (2×20 mL). The combined organic layers were washed once with brine (20 mL) and the organic layer was separated, dried over anhydrous Na2SO4 and concentrated. The resulting crude product was purified by preparative HPLC (buffer NHOAc, MeOH) to give tert-butyl (5-(2-(difluoromethoxy)benzamido)-5-(5-thioxo-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).

[0121] Step-4: N-(5-amino-1-(5-thioxo-5,6-dihydro-[1,2,4]triazolo[1,5-c]quinazolin-2-yl)pentyl)-2-(difluoromethoxy)benzamide. To a stirred solution of tert-butyl (5-(2-(difluoromethoxy)benzamido)-5-(5-thioxo-5,6-dihydro-[1,2,4]triazolo[1,5-c]quinazolin-2-yl)pentyl)carbamate (50 mg, 0.087 mmol) in methanol (5 mL) was added acetyl chloride (0.1 mL) at 0° C. 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 (buffer NHOAc, MeOH) to give N-(5-amino-1-(5-thioxo-5,6-dihydro-[1,2,4]triazolo[1,5-c]quinazolin-2-yl)pentyl)-2-(difluoromethoxy)benzamide as a pale yellow solid (20 mg, 48.48%). 1 H 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).

[0122] Synthesis of (R)-N-(5-amino-1-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)-2-(difluoromethoxy)benzamide (Compound 14). [ka]

[0123] Step 1: Synthesis of (6-(methoxy(methyl)amino)-6-oxohexane-1,5-diyl)(R)-dicarminic acid benzyl tert-butyl ester. 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), N,O-dimethylhydroxylamine hydrochloride (260 mg, 26.29 mmol) and DIPEA (1 g, 78.86 mmol) were added. The reaction mixture was then stirred at room temperature for 12 h. After completion of the reaction, it was diluted with water (10 mL) and extracted with EtOAc (3×50 mL). The combined organic layers were concentrated. The crude compound was purified by flash column chromatography (eluent: 20% ethyl acetate in hexane) to give (6-(methoxy(methyl)amino)-6-oxohexane-1,5-diyl)(R)-benzyl tert-butyl dicarbamate (600 mg, 54.6%) as a colorless viscous liquid. LC-MS m / z(M+H-Boc):324.1.

[0124] Step 2: Synthesis of (8-(2-methoxyphenyl)-6-oxooct-7-yn-1,5-diyl)(S)-benzyl tert-butyl dicarbamate. To a stirred solution of 1-ethynyl-2-methoxybenzene (100 mg, 0.2361 mmol) in dry THF (2 mL) was added n-BuLi (0.6 mL, 0.9445 mmol) in THF at -78 °C over 10 min. The reaction mixture was stirred at -78 °C for 1 h, followed by the dropwise addition of (6-(methoxy(methyl)amino)-6-oxohexane-1,5-diyl)(R)-benzyl tert-butyl dicarbamate (from step 1) in THF over 10 min. The reaction mixture was then warmed and stirred at 0 °C for 2 h. It was then quenched by the addition of aqueous ammonium chloride (10 mL) and extracted with EtOAc (2 x 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 give (8-(2-methoxyphenyl)-6-oxooct-7-yn-1,5-diyl)(S)-benzyl tert-butyl dicarbamate (60 mg, 51.4%) as a colorless viscous liquid. 1H 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-MS m / z(M+H-Boc):395.1.

[0125] Step 3: Synthesis of (1-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentane-1,5-diyl)(R)-dicarbamate benzyl tert-butyl. To a stirred solution of (8-(2-methoxyphenyl)-6-oxooct-7-yn-1,5-diyl)(S)-benzyl tert-butyl dicarbamate (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 (2 × 50 mL). The combined organic layers were dried over Na2SO4 and concentrated. The crude compound was purified by flash column chromatography (eluent: 70-80% EtOAc in hexanes) to give (1-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentane-1,5-diyl)(R)-benzyl tert-butyl dicarbamate (50 mg, 86.6% yield) as a white sticky 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.

[0126] Step 4: Synthesis of (R)-(5-amino-5-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)carbamate tert-butyl. To a stirred solution of (1-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentane-1,5-diyl)(R)-benzyl tert-butyl dicarbamate (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 hours under hydrogen atmosphere. After completion of the reaction, the reaction mixture was filtered through a Celite pad and washed with methanol (2×20 mL). The combined filtrate was concentrated on a rotary evaporator to give the desired compound (R)-(5-amino-5-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)carbamate tert-butyl (52 mg, 66.12% yield) as a white solid. 1 H 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, LC-MS m / z(M+H):375.1.

[0127] Step 5: Synthesis of (R)-(5-(2-(difluoromethoxy)benzamido)-5-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)carbamate tert-butyl. To a stirred solution of (R)-(5-amino-5-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)tert-butylcarbamate (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 diluted with water (10 mL) and extracted with EtOAc (2×50 mL). The combined EtOAc layers were dried over sodium sulfate and concentrated. The crude compound was purified by flash column chromatography (eluent: 2% MeOH+DCM) to give (R)-(5-(2-(difluoromethoxy)benzamido)-5-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)tert-butylcarbamate (45 mg, 61.88%) as an off-white solid. LC-MS m / z(M+H): 545.1.

[0128] Step 6: Synthesis of (R)-N-(5-amino-1-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)-2-(difluoromethoxy)benzamide. To a stirred solution of (R)-(5-(2-(difluoromethoxy)benzamido)-5-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)pentyl)tert-butylcarbamate (40 mg, 0.0735 mmol) in MeOH (2 mL) was added dropwise at 0° C. Acetyl chloride (2 mL) was then added dropwise at room temperature for 5 h and concentrated. The crude compound obtained was washed with diethyl ether (2×10 mL) and dried on a rotary evaporator. The crude compound obtained was further purified by preparative HPLC to give (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. 1H 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.

[0129] Amide coupling between the target molecule and the E2 binder can be used to prepare the next decomposition molecule. [ka] [ka] [ka]

[0130] Biological assays UBE2K dissociation constant (Kd) of E2 binder compound 1 Binding of mono-Ub UBE2K and UBE2K ligands to UBE2K was measured by surface plasma resonance using a Biacore SPR S200 instrument. Proteins and ligands were prepared for experimental measurements as described below.

[0131] Avi-tagged proteins were biotinylated and immobilized on Series S / Sensor Chip SA. Proteins were immobilized based on their molar weights, such as 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 1XHBS-N buffer + 0.05% Tween 20 (250 mL). Compound 1 stocks were prepared in affinity buffer consisting of 1% DMSO, 1XPBS-P+ buffer. Further dilutions of the stock were prepared in 0% DMSO, 1XPBS-P+ buffer or 3% DMSO, 1XPBS-P+ buffer and used as running buffer. Compound 1 was prepared as 2 mM or 750 μM stocks in the above buffers. Titrations were performed using a liquid handler / TECAN EVO (WALL-E) as follows: To perform the titrations, one or two 96-deep well plates (1.2 mL) were used (aliquot 3% DMSO, 1XPBS-P+buffer). For the remaining titrations, a 1:2 dilution was performed with WALL-E (250 μL+250 μL) to 3.12 μM. Compound 1 titrations were then transferred (125 μL) side-by-side in duplicate to a 384-deep well plate. The plate was then covered with a 384-well S200 compatible foil and used for the affinity method. For each experimental run, a solvent correction buffer containing DMSO concentrations + and - 0.5% DMSO running buffer was run.

[0132] Data was collected and processed according to the method described in the Biacore S200 evaluation software. The software was used to perform steady-state fitting on the data, and the data was represented as a sensorgram to deconvolute the binding and dissociation constants as described in Dahl, G et al., SLAS Discovery, 2017, Vol. 22 (2) 203-209. The resulting Kd (μM) of compound 1 was 2.11. This data demonstrated that compound 1 is a useful UBE2K binder for subsequent PROTAC generation.

[0133] Dose-dependent degradation of BRD4 in HeLa cells The 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 their ability to bind to the E2 enzyme UBE2K.

[0134] All compounds bind to UBE2K-Ub except compound 2. See Figure 1.

[0135] Compounds 6, 7, and 9 were then examined for their ability to degrade BRD4. As shown in Figure 2, compounds 7 and 9 exhibited a concentration-dependent loss of BRD4. This data indicates that targeting E2, particularly UBE2K, is a viable strategy for generating novel PROTACs with therapeutic relevance.

[0136] While a number of embodiments have been described, it will be apparent that the basic examples of the invention can be modified to provide other embodiments which utilize the compounds and methods of the invention. It will therefore be appreciated that the scope of the invention is to be defined by the appended claims rather than by the specific embodiments which have been represented by way of example.

[0137] The contents of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein are given the meaning 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) that covalently bonds the E2 binding portion to the target protein binding portion.

2. The compound according to claim 1, wherein the E2 binding portion is selected from those that bind to an E2 enzyme selected from 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 according to claim 1 or 2, wherein the E2 bond portion is bonded to UBE2K.

4. The E2 bond portion is a compound of formula I, 【Chemistry 1】 During the ceremony, Z １ and Z ２ Each is independently either N or CH, R １ is (C １ ~C ６ ) alkyl, halo(C １ ~C ６ ) alkyl, (C １ ~C ６ ) alkoxy, halo(C １ ~C ６ ) alkoxy, or -NR ｃ R ｄ wherein, the two available hydrogen atoms on the halo(C １ ~C ６ ) alkyl and the halo(C １ ~C ６ ) alkoxy together with the carbon atom to which they are attached can optionally form a 3- to 6-membered cycloalkyl substituted with 1 to 3 groups selected from halo, (C １ ~C ６ ) alkyl, halo(C １ ~C ６ ) alkyl, (C １ ~C ６ ) alkoxy, and halo(C １ ~C ６ ) alkoxy, R ２ , CN, Halo, OH, (C １ ~C ６ ) alkyl, halo(C １ ~C ６ ) alkyl, (C １ ~C ６ ) Alkoxy, or halo(C) １ ~C ６ ) is an alkoxy, or R １ and R ２ When they are on adjacent carbon atoms, they form a halo together with the carbon atoms to which they are bonded, (C １ ~C ６ ) alkyl and halo(C １ ~C ６ ) Forming a 5-membered or 6-membered oxygen-containing heterocycline which is optionally substituted with 1 to 3 groups selected from alkyl groups, R ３ is hydrogen, (C １ ~C ６ ) alkyl, or halo(C １ ~C ６ ) is alkyl, Y is CH ２ ----CHR ａ , -CR ａ R ｂ , S, or SO, p is either 0 or 1, R ａ and R ｂ These are, independently, Halo, (C １ ~C ６ ) alkyl, or halo(C １ ~C ６ ) alkyl, or R ａ and R ｂ These, together with the carbon atoms to which they are bonded, form halos, (C １ ~C ６ ) alkyl, halo(C １ ~C ６ ) alkyl, (C １ ~C ６ ) Alkoxy, Halo (C １ ~C ６ ) Alkoxy, (C １ ~C ６ ) alkylOH, (C １ ~C ６ ) Alkyl O (C １ ~C ６ ) Forming a 3-6 membered cycloalkyl or 3-6 membered heterocycline which is optionally substituted with 1-3 groups selected from alkyl and OH, R ｃ and R ｄ are each independently hydrogen (C １ to C ６ )alkyl, halo(C １ to C ６ )alkyl, (C １ to C ６ )alkyloxy(C １ to C ６ )alkyl, halo(C １ to C ６ )alkyloxy(C １ to C ６ )alkyl, (C １ to C ６ )alkyl-O-halo(C １ to C ６ )alkyl, halo(C １ to C ６ )alkyl-O-halo(C １ to C ６ )alkyl, or (C １ to C ６ )alkanol, and R ｃ and R ｄ together with the nitrogen atom to which they are attached form a 4- to 7-membered heterocyclyl optionally substituted with 1 to 3 groups selected from halo, (C １ to C ６ )alkyl, halo(C １ to C ６ )alkyl, (C １ to C ６ )alkoxy, halo(C １ to C ６ )alkoxy, and oxo, Z is a tricyclic fused ring having the following equation: 【Chemistry 2】 Ring A is aromatic, The wavy bond on ring A represents the bond point to Y, The wavy link next to W represents the link point to the linker (L). X, X １ , and X ２ Each of them, as long as the valence allows, is -CR ７ , independently selected from N, O, and S, The dotted lines within ring B represent single or double bonds. W is NH, -N(C １ ~C ６ ) Alkyl, O, or S, R ５ is hydrogen, (C １ ~C ６ ) alkyl, halo(C １ ~C ６ ) alkyl, (C １ ~C ６ ) Alkoxy, Halo (C １ ~C ６ ) Alkoxy, -S(C １ ~C ６ ) Alkyl, -SH, OH, (C ３ ~C ６ ) Cycloalkyl, (C ４ ~C ７ ) Heterocyclyl, and -NR ｅ R ｆ Selected from, where (C ３ ~C ６ ) Cycloalkyl and the above (C ４ ~C ７ ) Heterocyclines are, respectively, Halo, (C １ ~C ６ ) alkyl, halo(C １ ~C ６ ) alkyl, (C １ ~C ６ ) Alkoxy, Halo (C １ ~C ６ ) optionally substituted with 1 to 3 groups selected from alkoxy and oxo groups, If the dotted line within ring B is a single bond, then R ６ is hydrogen or (C １ ~C ６ ) is alkyl, or the dotted line in ring B is a double bond, R ６ It does not exist. d, d １ d ２ , and d ３ Each of them is independently CR ８ Selected from N, R ｅ and R ｆ Each is independently of hydrogen, (C １ ~C ４ ) alkyl, (C １ ~C ４ ) Alkyl NH(C １ ~C ４ ) alkyl, (C １ ~C ４ ) Alkyl N[(C １ ~C ４ ) Alkyl] ２ , (C １ ~C ４ ) Alkyl O (C １ ~C ４ ) is alkyl, R ７ is hydrogen or (C １ ~C ６ ) is alkyl, R ８ is halogen, hydrogen, (C １ ~C ６ ) alkyl, halo(C １ ~C ６ The compound according to claim 1 or 2, wherein it is alkyl, CN, or OH.

5. The compound according to claim 4, wherein the E2 bond portion is a compound of formula II. 【Transformation 3】

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

7. R ３ The compound according to claim 4, wherein is hydrogen.

8. Z １ and Z ２ The compound according to claim 4, wherein each of them is CH.

9. Y is CH ２ The compound according to claim 4.

10. The compound according to claim 4, wherein p is 0.

11. R １ is, (C １ ~C ６ ) alkyl, halo(C １ ~C ６ ) alkyl, halo(C １ ~C ６ ) Alkoxy, (C １ ~C ６ ) Alkyl, or -NR ｃ R ｄ And R ｃ and R ｄ The compound according to claim 4, wherein these atoms, together with the nitrogen atoms to which they are bonded, form a 5-6 membered heterocycline optionally substituted with 1-3 halos.

12. R １ is, (C １ ~C ３ ) alkyl, halo(C １ ~C ３ ) alkyl, halo(C １ ~C ６ ) Alkoxy, (C １ ~C ３ ) Alkyl, or -NR ｃ R ｄ And R ｃ and R ｄ The compound according to claim 4, wherein these atoms, together with the nitrogen atoms to which they are bonded, form a 5-6 member nitrogen-containing heterocycline optionally substituted with 1-3 halos.

13. R １ OCF ３ , OCHF ２ , OCH ３ ,CH ３ The compound according to claim 4, wherein the compound is pyrrolidinyl or piperidinyl, and the pyrrolidinyl or piperidinyl is optionally substituted with 1 to 3 halos.

14. The compound according to claim 4, wherein X is N.

15. X １ The compound according to claim 4, wherein is CH or N.

16. X ２ The compound according to claim 4, wherein is CH, N, S, or O.

17. X is N, and X １ N is X ２ If is CH, or if X is N, then X １ N is X ２ If is N, or if X is N, then X １ CH is, X ２ Either X is S, or X is N, and X １ CH is, X ２ The compound according to claim 4, wherein is O.

18. d is N or -CR ８ The compound according to claim 4.

19. d ２ is N or -CR ８ The compound according to claim 4.

20. d １ ga-CR ８ The compound according to claim 4.

21. d ３ ga-CR ８ The compound according to claim 4.

22. Z is 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 A compound according to claim 4, selected from the above.

23. Z is 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 A compound according to claim 4, selected from the above.

24. R ８ The compound according to claim 4, wherein is hydrogen or a halo.

25. R ８ The compound according to claim 4, wherein is hydrogen.

26. R ５ The compound according to claim 4, wherein is hydrogen.

27. R ６ is hydrogen and (C １ ~C ４ The compound according to claim 4, selected from alkyl groups.

28. R ６ The compound according to claim 4, wherein is selected from hydrogen and methyl.

29. The E2 bond portion is a compound of formula III, 【Chemistry 13】 In the formula, d, d １ d ２ , and d ３ Each of them is independently CR ８ The compound according to claim 1 or 2.

30. The compound according to claim 29, wherein p is 0.

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

32. The compound according to claim 1 or 2, wherein the E2 bond portion has the following structure. 【Chemistry 14】

33. The compound according to claim 1 or 2, wherein the target protein binding site binds to a protein selected from STAT3, BCL2, WRN, and BRD4.

34. The compound according to claim 1 or 2, wherein the target protein binding site binds to BRD4.

35. The target protein binding portion has the following structure: 【Chemistry 15】 The compound according to claim 1 or 2, wherein the wavy bond represents a bond site to the linker (L).

36. The compound according to claim 1 or 2, wherein the linker (L) comprises an optionally substituted linear or branched alkyl group, which is optionally interrupted by one or more heteroatoms selected from O, N, and S.

37. The compound according to claim 1 or 2, wherein the linker (L) is a linear or branched alkyl group substituted with one or more oxo groups (=O), and is interrupted by one or more heteroatoms selected from O and N.

38. The linker (L) has the following structure: 【Chemistry 16】 The compound according to claim 1 or 2, wherein an asterisk (*) represents a bond point to the E2 bond portion, 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 according to claim 38, wherein j is 1.

41. The compound according to claim 38, wherein the linker (L) is selected from one of the following structures. 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】

42. The aforementioned compound is a compound with the following structural formula, 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】 The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof.

43. A pharmaceutical composition comprising a compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

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