Activators of effector T cells

Abstract

The present disclosure provides antibody-drug conjugates and methods for delivering the conjugates to effector T cells. Novel Cbl-b inhibitors are also disclosed. The compounds and conjugates are useful for treating diseases in subjects in need thereof. Effector T cells are the main participants in inducing the immune system to exert immune functions. These cells circulate until they encounter their specific antigen. Thus, they play an important role in immunity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 318,278, filed March 9, 2022; U.S. Provisional Application No. 63 / 477,053, filed December 23, 2022; and U.S. Provisional Application No. 63 / 488,211, filed March 3, 2023, which are incorporated by reference in their entireties herein.

[0002] REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING The contents of the electronically submitted sequence listing in the XML file submitted with this application (Name: 4547_023PC03_SeqListing_ST26.xml; Size: 42,283 bytes; and Creation Date: March 8, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure provides antibody-drug conjugates and methods for delivering the conjugates to effector T cells. Novel Cbl-b inhibitors are also disclosed. The compounds and conjugates are useful for treating diseases in subjects in need thereof. [Background technology]

[0004] Effector T cells are the main participants in inducing the immune system to exert immune functions. These cells circulate until they encounter their specific antigen. Thus, they play a key role in immunity. The immune system is a powerful weapon against many diseases and disorders, including cancer, and compounds that activate effector T cells, such as inhibitors of the E3 ubiquitin ligase Casitas B lineage lymphoma-b (Cbl-b), have been shown to increase T cell-derived cytokine secretion and proliferation and may have anti-cancer activity. However, improved inhibitors of Cbl-b are needed. Furthermore, compounds that activate effector T cells have the potential to cause cytokine release syndrome or other off-target immune responses. It has been proposed that such off-target immune responses may be avoided by targeting immune-activating drugs specifically to cancer cells by conjugating them to antibodies that specifically recognize tumor antigens. (See, e.g., Ackerman, SE, Pearson, CI, Gregorio, JDet al. Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity. Nat Cancer 2, 18-33 (2021). doi.org / 10.1038 / s43018-020-00136-x). However, such proposals do not suggest directly targeting the immune system itself. Thus, despite such ongoing research, there exists a need to activate T cells, particularly effector T cells, for example, for the treatment of cancer, while minimizing off-target immune responses. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Ackerman,SE,Pearson,CI,Gregorio,JDet al.Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity.Nat Cancer 2,18-33(2021).doi.org / 10.1038 / s43018-020-00136-x Summary of the Invention [Means for solving the problem]

[0006] In certain embodiments, the present disclosure provides a compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof,

[0007] n is 0, 1, or 2;

[0008] X and Y are each independently CH or N;

[0009] Z is CH(CH 3 ), O, and SO 2 Selected from; or

[0010] Z is CH(CH 3 ), NH, N(CH 3 ), O, and SO 2 Selected from;

[0011] R 1 is hydrogen, -CN, -NHR z , -R a , -NR a R b , -OR a , -NHC(O)R a , -NHC(S)R a , -NHC(O)NHR a , -NHC(S)NHR a , -SR a , C 3 -C 6 cycloalkyl, and 3- to 6-membered heterocyclyl rings;

[0012] R z teeth, [ka] Selected from;

[0013] R a and R b is hydrogen, C 2- C 6 Alkenyl, C 1 -C6 Alkyl, Amide (C 1 -C 6 Alkyl), Amino (C 1 -C 6 Alkyl), Azide (C 1 -C 6 Alkyl), C 2 -C 6 Alkynyl, carboxy (C 1 -C 6 Alkyl), Cyano (C 1 -C 6 alkyl), C optionally substituted with a cyano group 3 -C 6 Cycloalkyl, dimethylamino (C 1 -C 6 alkyl), 3-6 membered heterocyclyl ring, 3-6 membered heterocyclyl (C 1 -C 3 Alkyl), Hydroxy (C 1 -C 6 Alkyl), Methoxy (C 1 -C 6 Alkyl), methylamino (C 1 -C 6 alkyl), NR c R d (C 1 -C 6 alkyl), HS(C 1 -C 6 alkyl), and CH 3 S(C 1 -C 6 alkyl), R c and R d is hydrogen, C 2 alkenylcarbonyl, and methyl; or

[0014] R a and R b together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, the ring being a , -C(O)R a , -SO 2 R a , azido, and cyano;

[0015] Each C 3 -C 6 Cycloalkyl, 3- to 6-membered heterocyclyl ring, and 3- to 6-membered heterocyclyl (C 1 -C 3 The heterocyclyl portion of the alkyl ring is 1 -C 3 Alkyl, C 2 Alkynyl, amide, azido, carboxy, cyano, dimethylamino, hydroxy, methoxy, methylamino, HS-, and CH 3 optionally substituted with 1, 2, or 3 groups independently selected from S-;

[0016] R 2 teeth, [ka] Selected from;

[0017] m is 0, 1, 2, or 3;

[0018] m'' is 0, 1, 2, 3, or 4;

[0019] B' is a 3-7 membered saturated or unsaturated ring optionally containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; the ring is selected from -OH, -CH 2 S.H., C.H. 2 SCH 3 , -CH 2 OH, -CH 2 NH 2 , and -CH 2 NHCH 3 and is optionally substituted with 1 or 2 substituents independently selected from:

[0020] Each R 500 is hydrogen, C 1 -C 6 Alkyl, halo, -OH, and -CH 2 is independently selected from OH;

[0021] X 50are O, NH, and NCH. 3 , and S;

[0022] R 3 is hydrogen, acetyl, amino, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylaminomethyl, C 1 -C 6 Alkylcarbonyl, Amino C 1 -C 6 Alkyl, aminocarbonyl, aminomethyl, carboxy, cyano, C 3 Cycloalkyl, formyl, hydroxy, hydroxyC 1 -C 6 Alkyl, methoxy, oxazolyl, -SH, -SCH 3 , -SOCH 3 , -SO 2 CH 3 , -SO(=NH)CH 3 , tetrazolyl, thiazolyl, and trifluoromethyl; 3 The cycloalkyl is optionally substituted with a hydroxy group;

[0023] R 4 is hydrogen, methyl, -CH 2 OH, -CH 2 SH, and -CH 2 SCH 3 Selected from;

[0024] R 5 is hydrogen, hydroxyl, -CH 2 SH, -CH 2 SCH 3 and methyl;

[0025] Optionally, but R 5 is hydroxy or methyl, and R 4 If is hydrogen, then R 1 is C 3 -C 6 Cycloalkyl, 3-6 membered heterocyclyl ring, hydroxy, hydroxy(C 1 -C6 alkyl), -OR a (In the formula, R a is C 1 -C 6 Alkyl, 3- to 6-membered heterocyclyl ring, or hydroxy (C 1 -C 6 alkyl); or -NR a R b (In the formula, R a and R b is hydrogen, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, hydroxy(C 1 -C 6 alkyl), 3- to 6-membered heterocyclyl ring, or R a and R b together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, the ring being 1 -C 6 Alkyl or hydroxy (C 1 -C 6 alkyl);

[0026] R 6 and R 6’ is hydrogen, cyclopropyl, -CH 2 OH, -CH 2 SH, -CH 2 SCH 3 , and -CH 2 R 200 are independently selected from R 200 is a 3-7 membered saturated or unsaturated ring optionally containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. to provide.

[0027] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0028] In certain embodiments, the present disclosure provides a compound of formula (IA-1): [ka] or a pharma- ceutically acceptable salt thereof,

[0029] n is 0, 1, or 2;

[0030] X and Y are each independently CH or N;

[0031] Z is CH(CH 3 ), O, and SO 2 Selected from;

[0032] R 1 is hydrogen, -CN, -NHR z , -R a , -NR a R b , -OR a , -NHC(O)R a , -NHC(S)R a , -NHC(O)NHR a , -NHC(S)NHR a , -SR a , C 3 -C 6 cycloalkyl, and 3- to 6-membered heterocyclyl rings;

[0033] R z teeth,

[0034] [ka] Selected from;

[0035] R a and R b is hydrogen, C 2- C 6 Alkenyl, C 1 -C 6 Alkyl, Amide (C 1 -C 6 Alkyl), Amino (C 1 -C 6Alkyl), Azide (C 1 -C 6 Alkyl), C 2 -C 6 Alkynyl, carboxy (C 1 -C 6 Alkyl), Cyano (C 1 -C 6 Alkyl), C 3 -C 6 Cycloalkyl, dimethylamino (C 1 -C 6 alkyl), 3-6 membered heterocyclyl ring, 3-6 membered heterocyclyl (C 1 -C 3 Alkyl), Hydroxy (C 1 -C 6 Alkyl), Methoxy (C 1 -C 6 Alkyl), methylamino (C 1 -C 6 alkyl), NR c R d (C 1 -C 6 alkyl), HS(C 1 -C 6 alkyl), and CH 3 S(C 1 -C 6 alkyl), R c and R d is hydrogen, C 2 alkenylcarbonyl, and methyl; or

[0036] R a and R b together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, the ring being a , -C(O)R a , -SO 2 R a , azido, and cyano;

[0037] Each C 3 -C 6 Cycloalkyl, 3- to 6-membered heterocyclyl ring, and 3- to 6-membered heterocyclyl (C1 -C 3 The heterocyclyl portion of the alkyl ring is 1 -C 3 Alkyl, C 2 Alkynyl, amide, azido, carboxy, cyano, dimethylamino, hydroxy, methoxy, methylamino, HS-, and CH 3 optionally substituted with 1, 2, or 3 groups independently selected from S-;

[0038] R 2 teeth,

[0039] [ka] and;

[0040] m is 0, 1, 2, or 3;

[0041] R 3 is hydrogen, acetyl, amino, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylaminomethyl, Amino C 1 -C 6 Alkyl, aminocarbonyl, aminomethyl, carboxy, cyano, C 3 Cycloalkyl, formyl, hydroxy, hydroxyC 1 -C 6 Alkyl, methoxy, oxazolyl, -SH, -SCH 3 , tetrazolyl, thiazolyl, and trifluoromethyl; 3 The cycloalkyl is optionally substituted with a hydroxy group;

[0042] R 4 and R 6 is hydrogen, -CH 2 SH, and -CH 2 SCH 3 are independently selected from;

[0043] R 5 is hydroxy, -CH2 SH, -CH 2 SCH 3 and methyl;

[0044] Optionally, but R 5 is hydroxy or methyl, and R 4 If is hydrogen, then R 1 is C 3 -C 6 Cycloalkyl, 3-6 membered heterocyclyl ring, hydroxy, hydroxy(C 1 -C 6 alkyl), -OR a (In the formula, R a is C 1 -C 6 Alkyl, 3- to 6-membered heterocyclyl ring, or hydroxy (C 1 -C 6 alkyl); or -NR a R b (In the formula, R a and R b is hydrogen, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, hydroxy(C 1 -C 6 alkyl), 3- to 6-membered heterocyclyl ring, or R a and R b together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, the ring being 1 -C 6 Alkyl or hydroxy (C 1 -C 6 alkyl) to provide.

[0045] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0046] In certain aspects, the present disclosure provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof, wherein:

[0047] X is N;

[0048] Y is CH;

[0049] R 2 teeth, [ka] is) to provide.

[0050] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0051] In certain embodiments, Z is O. In some embodiments, Z is CH(CH 3 ).

[0052] In some aspects, the present disclosure provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof, wherein:

[0053] R 3 is trifluoromethyl;

[0054] R 4 and R 6 is hydrogen;

[0055] R 5 is methyl) to provide.

[0056] In certain embodiments, R 1 -NR a R b , -NHC(O)R a , -NHC(S)NHR a , and -SR a In some embodiments, R 1 -SCH 2 CH3 In some embodiments, R 1 is -NH(CH 2 ) 2 CN. In some embodiments, R 1 is -NH(CH 2 ) 2 N 3 It is.

[0057] In some aspects, the present disclosure provides a method for producing a pharmaceutical composition comprising: [ka] The present invention provides a compound of formula (I) selected from:

[0058] In some embodiments, the compounds of formula (I) are inhibitors of Casitas B lineage lymphoma proto-oncogene b (Cbl-b).

[0059] In certain embodiments, the compound interacts with His152 of human Cbl-b; increases IL-2 secretion from T cells by about 0.8 to about 1.4 fold over background; increases IFN-y secretion by about 1.1 to about 2 fold over background; and / or increases CD69 levels by about 0.81 to about 1.1 fold over background.

[0060] In certain embodiments, the present disclosure provides a conjugate, or a pharma- ceutically acceptable salt thereof, comprising a binding moiety capable of specifically binding to a target on the surface of an effector T cell and a payload capable of activating the effector T cell, the binding moiety being directly attached to the payload or attached to the payload via a linker. In some embodiments, the binding moiety is attached to the payload via a linker. In certain embodiments, the binding moiety is capable of specifically binding a protein or glycoprotein on the surface of an effector T cell. In some embodiments, the binding moiety is capable of specifically binding to programmed cell death protein 1 (PD1).

[0061] In certain aspects, the disclosure provides a conjugate, or a pharma- ceutically acceptable salt thereof, comprising a binding moiety capable of specifically binding to PD1 and a payload capable of activating an effector T cell, wherein the binding moiety is directly attached to the payload or is attached to the payload via a linker, or a pharma- ceutically acceptable salt thereof.

[0062] In some aspects, the disclosure provides a conjugate, or a pharma- ceutically acceptable salt thereof, comprising a binding moiety capable of specifically binding to PD1 and a payload that is an inhibitor of Casitas B lineage lymphoma proto-oncogene b (Cbl-b).

[0063] In some embodiments, the conjugates described herein have the formula (I):

[0064] Bm-[LP] a (I) (In the formula,

[0065] a is an integer from 1 to 50;

[0066] P is the payload;

[0067] L is a linker which is

[0068] Bm is the binding site. has.

[0069] In some embodiments, the payload is an inhibitor of Casitas B lineage lymphoma proto-oncogene b (Cbl-b). In some embodiments, the inhibitor of Cbl-b is a compound of formula (I) attached to a binding moiety or linker via a covalent bond. In some embodiments, the inhibitor of Cbl-b is [ka] It is.

[0070] In some embodiments, the inhibitor of Cbl-b is compound 146, compound 147, compound 148, or NX-1607.

[0071] In some embodiments, the payload is an agonist of Toll-like receptor 7 (TLR-7) and / or Toll-like receptor 8 (TLR-8). In some embodiments, the payload is the TLR-7 / TLR-8 agonist T785. In some embodiments, the payload is the TLR-7 / TLR-8 agonist MEDI9197.

[0072] In some embodiments, the payload is an inhibitor of hematopoietic progenitor kinase 1 (HPK-1).

[0073] In some embodiments, the payload is an inhibitor of STING, phosphoinositide-3-kinase gamma (PI3Kγ), CXCR4, CCR5, or a mitogen-activated protein kinase (MAPK) pathway protein, optionally wherein the MAPK pathway protein is MEK or B-raf.

[0074] In some embodiments, the payload is an agonist of stimulator of interferon genes (STING).

[0075] In some embodiments, the payload is a small molecule, hi some embodiments, the payload is a peptide.

[0076] In certain embodiments, the disclosure provides a conjugate as described above, wherein L is a non-cleavable linker. [ka] (In the formula,

[0077] p is an integer from 1 to 10;

[0078] p* is an integer from 1 to 10;

[0079] Y is hydrogen and C 1 -C 6 alkyl;

[0080] [ka] is the attachment point to the payload;

[0081] [ka] is the attachment point to the binding site) is selected from the group consisting of:

[0082] In some embodiments, L is [ka] (In the formula,

[0083] p is an integer from 1 to 10;

[0084] p* is an integer from 1 to 10;

[0085] [ka] is the attachment point to the payload;

[0086] [ka] is the attachment point to the binding site) is selected from the group consisting of:

[0087] In some embodiments, L is a cleavable linker. In some embodiments, the cleavable linker is cleavable by a protease. In certain embodiments, L is [ka] (In the formula,

[0088] q is an integer from 2 to 10;

[0089] Z 1 , Z 2 , Z 3 , and Z 4 are each independently absent or a naturally occurring amino acid residue of L or D configuration, with the proviso that Z 1 , Z 2 , Z 3 , and Z 4 at least two of are amino acid residues;

[0090] [ka] is the attachment point to the payload;

[0091] [ka] is the attachment point to the binding site) is selected from.

[0092] In some embodiments, Z 1 , Z 2 , Z 3 , and Z 4 is independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, and glycine; with the proviso that Z is 1 , Z 2 , Z 3 , and Z 4 At least two of are amino acid residues.

[0093] In some embodiments,

[0094] Z1 is absent or glycine;

[0095] Z 2 is absent or selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine;

[0096] Z 3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine;

[0097] Z 4 is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.

[0098] In certain embodiments, L is [ka] (In the formula,

[0099] q is an integer from 2 to 10;

[0100] [ka] is the attachment point to the payload;

[0101] [ka] is the attachment point to the binding site) is selected from.

[0102] In certain embodiments, L is a bioreducible linker. In some embodiments, L is [ka] (In the formula,

[0103] q is an integer from 2 to 10;

[0104] R, R', R'', and R''' are hydrogen, C 1 -C 6 Alkoxy C 1 -C 6 Alkyl, (C 1 -C 6 ) 2 NC 1 -C 6 Alkyl, and C 1 -C 6 alkyl, or two geminal R groups together with the carbon atom to which they are attached can form a cyclobutyl or cyclopropyl ring;

[0105] [ka] is the attachment point to the payload;

[0106] [ka] is the attachment point to the binding site) is selected from.

[0107] In some embodiments, L is [ka] (In the formula,

[0108] q is an integer from 2 to 10;

[0109] R, R', R'', and R''' are hydrogen, C 1 -C 6 Alkoxy C 1 -C 6 Alkyl, (C 1 -C 6 ) 2 NC 1 -C 6Alkyl, and C 1 -C 6 alkyl, or two geminal R groups together with the carbon atom to which they are attached can form a cyclobutyl or cyclopropyl ring;

[0110] [ka] is the attachment point to the payload;

[0111] [ka] is the attachment point to the binding site) is selected from.

[0112] In certain embodiments, L is an acid-cleavable linker.

[0113] In some embodiments, L is [ka] (In the formula,

[0114] q is an integer from 2 to 10;

[0115] [ka] is the attachment point to the payload;

[0116] [ka] is the attachment point to the binding site) is selected from the group consisting of:

[0117] In some embodiments, L is a click-to-release linker. In some embodiments, L is [ka] (In the formula,

[0118] q is an integer from 2 to 10;

[0119] [ka] is the attachment point to the payload;

[0120] [ka] is the attachment point to the binding site) is selected from.

[0121] In some embodiments, L is a pyrophosphatase-cleavable linker. [ka] (In the formula,

[0122] q is an integer from 2 to 10;

[0123] [ka] is the attachment point to the payload;

[0124] [ka] is the attachment point to the binding site) It is.

[0125] In some embodiments, L is a beta-glucuronidase cleavable linker. [ka] (In the formula,

[0126] q is an integer from 2 to 10;

[0127] ---- is non-existence or union;

[0128] [ka] is the attachment point to the payload;

[0129] [ka] is the attachment point to the binding site) is selected from.

[0130] In some embodiments, L is [ka] (In the formula,

[0131] q is an integer from 2 to 10;

[0132] ---- is non-existence or union;

[0133] [ka] is the attachment point to the payload;

[0134] [ka] is the attachment point to the binding site) It is.

[0135] In some aspects, the present disclosure provides a method for producing a pharmaceutical composition comprising: [ka] [ka] [ka] [ka] [ka] [ka] The present invention provides a conjugate selected from:

[0136] In some embodiments, the linker of the conjugate is attached to a cysteine, lysine, tyrosine, or glutamine in Bm. In some embodiments, the cysteine ​​or lysine is an engineered cysteine ​​or lysine. In some embodiments, the cysteine ​​or lysine is endogenous to Bm.

[0137] In some aspects, the binding site is an antibody or an antigen-binding fragment thereof.

[0138] In certain aspects, the disclosure provides a conjugate, wherein L is attached to an engineered cysteine ​​at heavy chain position S239 and / or K334 of the antibody, or antigen-binding portion thereof, according to EU numbering.

[0139] In some embodiments, L is attached to a glutamine at heavy chain position 295 of the antibody or antigen-binding portion thereof according to EU numbering.

[0140] In certain embodiments, the binding site is an antibody or antigen-binding fragment thereof that comprises the CDR sequences of amino acids 31-35, 50-66, and 99-109 of SEQ ID NO:10 and amino acids 24-38, 54-60, and 93-101 of SEQ ID NO:11.

[0141] In some embodiments, the binding site is an antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:10 and a variable light chain comprising the amino acid sequence of SEQ ID NO:11.

[0142] In some embodiments, the binding site is an antibody or antigen-binding fragment thereof comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:26 and a light chain comprising the amino acid sequence of SEQ ID NO:27.

[0143] In some embodiments, the binding site is an antibody or antigen-binding fragment thereof that comprises heavy and light chain CDR sequences of amino acids 31-35, 50-66, and 99-102 of SEQ ID NO:12 and amino acids 24-34, 50-56, and 89-97 of SEQ ID NO:13.

[0144] In some embodiments, the binding site is an antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:12 and a variable light chain comprising the amino acid sequence of SEQ ID NO:13.

[0145] In some embodiments, the binding site is capable of specifically binding to CD25.

[0146] In some embodiments, the binding site is an antibody or antigen-binding fragment thereof that comprises the CDR sequences of amino acids 31-35, 50-66, and 99-111 of SEQ ID NO:4 and amino acids 24-34, 50-56, and 89-97 of SEQ ID NO:5.

[0147] In some embodiments, the binding site is an antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:4 and a variable light chain comprising the amino acid sequence of SEQ ID NO:5.

[0148] In some embodiments, the binding site is an antibody or antigen-binding fragment thereof that comprises the CDR sequences of amino acids 31-35, 50-65, and 98-108 of SEQ ID NO:6 and amino acids 24-33, 49-55, and 88-96 of SEQ ID NO:7.

[0149] In some embodiments, the binding site is an antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:6 and a variable light chain comprising the amino acid sequence of SEQ ID NO:7.

[0150] In some embodiments, the binding site is an antibody or antigen comprising the CDR sequences of amino acids 31-35, 50-65, or 98-108 of SEQ ID NO:8 and amino acids 24-33, 49-55, and 88-96 of SEQ ID NO:9, or an antigen-binding fragment thereof.

[0151] In some embodiments, the binding site is an antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:8 and a variable light chain comprising the amino acid sequence of SEQ ID NO:9.

[0152] In some embodiments, the binding site is capable of specifically binding to CD7.

[0153] In some embodiments, the binding site is an antibody or antigen-binding fragment thereof that comprises the CDR sequences of amino acids 31-35, 50-66, and 99-112 of SEQ ID NO:14 and amino acids 23-36, 52-58, and 91-99 of SEQ ID NO:15.

[0154] In some embodiments, the binding site is an antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:14 and a variable light chain comprising the amino acid sequence of SEQ ID NO:15.

[0155] In some aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region.

[0156] In some embodiments, the heavy chain constant region comprises Fc silent mutations.

[0157] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region.

[0158] In some embodiments, the IgG heavy chain constant region is an IgG1 heavy chain constant region.

[0159] In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:20.

[0160] In some embodiments, the IgG heavy chain constant region is an IgG4 heavy chain constant region.

[0161] In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:21.

[0162] In some aspects, the disclosure provides a conjugate as described herein, or a pharma- ceutically acceptable salt thereof, wherein (a) the binding moiety is pembrolizumab, or (b) the binding moiety is nivolumab, or a pharma- ceutically acceptable salt thereof.

[0163] In some aspects, the disclosure provides a conjugate as described herein, or a pharma- ceutically acceptable salt thereof, wherein (a) the binding site is a686; (b) the binding site is MA251; or (c) the binding site is humanized MA251.

[0164] In some embodiments, the binding moiety is a small molecule.

[0165] In some embodiments, the disclosure provides a conjugate as described herein, wherein a is 1 to 40. In some embodiments, a is 1 to 10. In some embodiments, a is 2 to 8.

[0166] In some aspects, the present disclosure provides a conjugate as described herein capable of increasing effector T cell activity.

[0167] In some aspects, the present disclosure provides a conjugate as described herein capable of increasing effector T cell proliferation.

[0168] In some aspects, the present disclosure provides a conjugate as described herein capable of increasing the migration of effector T cells into tumor cells.

[0169] In some aspects, the present disclosure provides a conjugate as described herein capable of reducing effector T cell exhaustion.

[0170] In some aspects, the disclosure provides a conjugate as described herein that increases IFN-y secretion from T cells by about 2.5 to about 3 fold over background; and / or increases IL-2 secretion from T cells by about 6 to about 8 fold over background.

[0171] In some aspects, the disclosure provides compositions comprising a compound described herein, or a pharma- ceutically acceptable salt thereof.

[0172] In some aspects, the disclosure provides a composition comprising a conjugate described herein, or a pharma- ceutically acceptable salt thereof.

[0173] In some embodiments, the disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a compound described herein, a conjugate described herein, or a composition described herein. In some embodiments, the cancer is (i) resistant or refractory to anti-PD1 therapy, optionally wherein the anti-PD1 therapy is nivolumab, pembrolizumab, and / or cemiplimab, and / or (ii) resistant or refractory to anti-PDL1 therapy, optionally wherein the anti-PDL1 therapy is durvalumab, atezolizumab, and / or avelumab.

[0174] In some aspects, the disclosure provides a method of treating a condition in a subject in need thereof that would benefit from an increased immune response, the method comprising administering to the subject a compound described herein, a conjugate described herein, or a composition described herein. In some aspects, the condition is an infectious disease, optionally the infectious disease is a viral, bacterial, or parasitic infection, an immunosuppressive disease or disorder, or multiple sclerosis.

[0175] In some aspects, the disclosure provides a method of increasing the activity of an immune cell, comprising contacting the immune cell with a compound described herein, a conjugate described herein, or a composition described herein.

[0176] In some aspects, the disclosure provides a method of increasing proliferation of an immune cell, comprising contacting an immune cell with a compound described herein, a conjugate described herein, or a composition described herein.

[0177] In some aspects, the disclosure provides a method of increasing migration of immune cells into tumor cells, comprising contacting an immune cell with a compound described herein, a conjugate described herein, or a composition described herein.

[0178] In some aspects, the disclosure provides a method of reducing exhaustion of an immune cell, the method comprising contacting an immune cell with a compound described herein, a conjugate described herein, or a composition described herein.

[0179] In some embodiments, the present disclosure provides a method for increasing the secretion of IFN-y or IL-2 from immune cells, comprising contacting immune cells with a compound described herein, a conjugate described herein, or a composition described herein.In some embodiments, the immune cells are T cells, and optionally the T cells are effector T cells.In some embodiments, the immune cells are natural killer (NK) cells.

[0180] In some aspects, the disclosure provides a method of improving and / or prolonging an antigen recall response of T, comprising contacting a T cell with a compound described herein, a conjugate described herein, or a composition described herein.

[0181] In some embodiments, the present disclosure provides a method of delivering a payload capable of activating an effector T cell to an immune cell, the method comprising contacting the effector T cell with a conjugate described herein or a composition comprising a conjugate described herein. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in a subject, optionally the subject has cancer or a condition that would benefit from an increased immune response. [Brief description of the drawings]

[0182] [Figure 1] 13 is a histogram showing the protein-ligand interaction fraction between Cbl-b and compound 147 and between Cbl-b and compound 118.

[0183] [Diagram 2] 1 is a graph showing the activity of the conjugates in a mixed lymphocyte reaction.

[0184] [Figure 3A] 1 is a graph showing the activity of the conjugates in a mixed lymphocyte reaction in the presence and absence of TGF-β.

[0185] [Figure 3B] FIG. 1 is a graph showing the activity of conjugates in a mixed lymphocyte reaction in the presence and absence of Treg cells.

[0186] [Figure 4] 1 is a graph showing in vitro tumor infiltrating lymphocyte (TIL) activation.

[0187] [Diagram 5] FIG. 1 is a graph showing the in vivo activity of the conjugate against B16F10 melanoma cells in mice compared to unconjugated antibody and vehicle control. [Figure 6]1 is a graph showing the in vivo activity of the conjugate against B16F10 melanoma cells in mice compared to unconjugated antibody and vehicle control.

[0188] [Figure 7] 1 is a graph showing activation of exhausted T cells by anti-PD-1-Cbl-B inhibitor conjugates.

[0189] [Figure 8] Graph showing antigen recall responses (as measured by levels of IFN-γ) of T cells treated with anti-PD-1-Cbl-B inhibitor conjugates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0190] [ka] or a pharma- ceutically acceptable salt thereof, n is 0, 1, or 2;

[0191] X and Y are each independently CH or N;

[0192] Z is CH(CH 3 ), O, and SO 2 Selected from; or

[0193] Z is CH(CH 3 ), NH, N(CH 3 ), O, and SO 2 Selected from;

[0194] R 1 is hydrogen, -CN, -NHR z , -R a , -NR a R b , -OR a , -NHC(O)R a , -NHC(S)R a , -NHC(O)NHR a , -NHC(S)NHRa , -SR a , C 3 -C 6 cycloalkyl, and 3- to 6-membered heterocyclyl rings;

[0195] R z teeth, [ka] Selected from;

[0196] R a and R b is hydrogen, C 2- C 6 Alkenyl, C 1 -C 6 Alkyl, Amide (C 1 -C 6 Alkyl), Amino (C 1 -C 6 Alkyl), Azide (C 1 -C 6 Alkyl), C 2 -C 6 Alkynyl, carboxy (C 1 -C 6 Alkyl), Cyano (C 1 -C 6 alkyl), C optionally substituted with a cyano group 3 -C 6 Cycloalkyl, dimethylamino (C 1 -C 6 alkyl), 3-6 membered heterocyclyl ring, 3-6 membered heterocyclyl (C 1 -C 3 Alkyl), Hydroxy (C 1 -C 6 Alkyl), Methoxy (C 1 -C 6 Alkyl), methylamino (C 1 -C 6 alkyl), NR c R d (C 1 -C 6 alkyl), HS(C 1 -C 6 alkyl), and CH 3 S(C 1-C 6 alkyl), R c and R d is hydrogen, C 2 alkenylcarbonyl, and methyl; or

[0197] R a and R b together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, the ring being a , -C(O)R a , -SO 2 R a , azido, and cyano;

[0198] Each C 3 -C 6 Cycloalkyl, 3- to 6-membered heterocyclyl ring, and 3- to 6-membered heterocyclyl (C 1 -C 3 The heterocyclyl portion of the alkyl ring is 1 -C 3 Alkyl, C 2 Alkynyl, amide, azido, carboxy, cyano, dimethylamino, hydroxy, methoxy, methylamino, HS-, and CH 3 optionally substituted with 1, 2, or 3 groups independently selected from S-;

[0199] R 2 teeth, [ka] Selected from;

[0200] m is 0, 1, 2, or 3;

[0201] m'' is 0, 1, 2, 3, or 4;

[0202] B' is a 3-7 membered saturated or unsaturated ring optionally containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; the ring is selected from -OH, -CH2 S.H., C.H. 2 SCH 3 , -CH 2 OH, -CH 2 NH 2 , and -CH 2 NHCH 3 and is optionally substituted with 1 or 2 substituents independently selected from:

[0203] Each R 500 is hydrogen, C 1 -C 6 Alkyl, halo, -OH, and -CH 2 is independently selected from OH;

[0204] X 50 are O, NH, and NCH. 3 , and S;

[0205] R 3 is hydrogen, acetyl, amino, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylaminomethyl, C 1 -C 6 Alkylcarbonyl, Amino C 1 -C 6 Alkyl, aminocarbonyl, aminomethyl, carboxy, cyano, C 3 Cycloalkyl, formyl, hydroxy, hydroxyC 1 -C 6 Alkyl, methoxy, oxazolyl, -SH, -SCH 3 , -SOCH 3 , -SO 2 CH 3 , -SO(=NH)CH 3 , tetrazolyl, thiazolyl, and trifluoromethyl; 3 The cycloalkyl is optionally substituted with a hydroxy group;

[0206] R 4 is hydrogen, methyl, -CH 2 OH, -CH 2 SH, and -CH 2SCH 3 Selected from;

[0207] R 5 is hydrogen, hydroxyl, -CH 2 SH, -CH 2 SCH 3 and methyl;

[0208] Optionally, but R 5 is hydroxy or methyl, and R 4 If is hydrogen, then R 1 is C 3 -C 6 Cycloalkyl, 3-6 membered heterocyclyl ring, hydroxy, hydroxy(C 1 -C 6 alkyl), -OR a (In the formula, R a is C 1 -C 6 Alkyl, 3- to 6-membered heterocyclyl ring, or hydroxy (C 1 -C 6 alkyl); or -NR a R b (In the formula, R a and R b is hydrogen, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, hydroxy(C 1 -C 6 alkyl), 3- to 6-membered heterocyclyl ring, or R a and R b together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, the ring being 1 -C 6 Alkyl or hydroxy (C 1 -C 6 alkyl);

[0209] R 6 and R 6’ is hydrogen, cyclopropyl, -CH 2OH, -CH 2 SH, -CH 2 SCH 3 , and -CH 2 R 200 are independently selected from R 200 is a 3-7 membered saturated or unsaturated ring optionally containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0210] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0211] In certain embodiments, the disclosure provides a Cbl-b inhibitor of formula (IA-1): [ka] or a pharma- ceutically acceptable salt thereof,

[0212] n is 0, 1, or 2;

[0213] X and Y are each independently CH or N;

[0214] Z is CH(CH 3 ), O, and SO 2 Selected from;

[0215] R 1 is hydrogen, -CN, -NHR z , -R a , -NR a R b , -OR a , -NHC(O)R a , -NHC(S)R a , -NHC(O)NHR a , -NHC(S)NHR a , -SR a , C 3 -C 6 cycloalkyl, and 3- to 6-membered heterocyclyl rings;

[0216] Rz teeth, [ka] Selected from;

[0217] R a and R b is hydrogen, C 2- C 6 Alkenyl, C 1 -C 6 Alkyl, Amide (C 1 -C 6 Alkyl), Amino (C 1 -C 6 Alkyl), Azide (C 1 -C 6 Alkyl), C 2 -C 6 Alkynyl, carboxy (C 1 -C 6 Alkyl), Cyano (C 1 -C 6 Alkyl), C 3 -C 6 Cycloalkyl, dimethylamino (C 1 -C 6 alkyl), 3-6 membered heterocyclyl ring, 3-6 membered heterocyclyl (C 1 -C 3 ) Alkyl, Hydroxy (C 1 -C 6 Alkyl), Methoxy (C 1 -C 6 Alkyl), methylamino (C 1 -C 6 alkyl), NR c R d (C 1 -C 6 alkyl), HS(C 1 -C 6 alkyl), and CH 3 S(C 1 -C 6 alkyl), R c and R d is hydrogen, C 2 alkenylcarbonyl, and methyl; or

[0218] R a and R b together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, the ring being a , -C(O)R a , -SO 2 R a , azido, and cyano;

[0219] Each C 3 -C 6 Cycloalkyl, 3- to 6-membered heterocyclyl ring, and 3- to 6-membered heterocyclyl (C 1 -C 3 The heterocyclyl portion of the alkyl group is C 1 -C 3 Alkyl, C 2 Alkynyl, amide, azido, carboxy, cyano, dimethylamino, hydroxy, methoxy, methylamino, HS-, and CH 3 optionally substituted with 1, 2, or 3 groups independently selected from S-;

[0220] R 2 teeth, [ka] and;

[0221] m is 0, 1, 2, or 3;

[0222] R 3 is hydrogen, acetyl, amino, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylaminomethyl, Amino C 1 -C 6 Alkyl, aminocarbonyl, aminomethyl, carboxy, cyano, C 3 Cycloalkyl, formyl, hydroxy, hydroxyC 1 -C 6 Alkyl, methoxy, oxazolyl, -SH, -SCH 3, tetrazolyl, thiazolyl, and trifluoromethyl; 3 The cycloalkyl is optionally substituted with a hydroxy group;

[0223] R 4 and R 6 is hydrogen, -CH 2 SH, and -CH 2 SCH 3 are independently selected from;

[0224] R 5 is hydroxy, -CH 2 SH, -CH 2 SCH 3 and methyl The following is the target.

[0225] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0226] The present disclosure also provides conjugates that activate effector T cells, and further provides methods of treating cancer, increasing T cell activity, increasing T cell proliferation, increasing T cell migration to tumor cells, reducing T cell exhaustion, and using the conjugates to deliver a payload capable of activating effector T cells.

[0227] I. Definition In order that this specification may be more readily understood, certain terms are first defined. Further definitions are set forth throughout the detailed description.

[0228] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a predicate basis for the use of such exclusive terms, such as "solely," "only," and the like, or the use of a negative limitation in connection with the recitation of claim elements.

[0229] Furthermore, "and / or", as used herein, should be construed as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0230] Whenever an embodiment is described herein using the word "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0231] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0232] Units, prefixes, and symbols are indicated in their Systeme International de Unites (SI) accepted form. Numeric ranges include the numbers that define the range. When a range of values ​​is described, it should be understood that each intervening integer value between the stated upper and lower limits of that range, and each fraction thereof, is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can be independently included or excluded from the range, and each range in which either or both of the limits are included or not included is also encompassed by the present disclosure. Thus, it is understood that the ranges described herein are abbreviated to all of the values ​​within the range, including the stated endpoints.

[0233] When a value is explicitly stated, it should be understood that a quantity or amount that is approximately the same as the stated value is also within the scope of this disclosure. When a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of this disclosure. Conversely, when different elements or groups of elements are individually disclosed, their combinations are also disclosed. When any element of the disclosure is disclosed as having multiple alternatives, examples of that disclosure in which each alternative is excluded alone or in any combination with other alternatives are also hereby disclosed; multiple elements of the disclosure may have such exclusions, and all combinations of elements with such exclusions are hereby disclosed.

[0234] The terms "effector T cells," "T effector cells," "Teff," and "T-eff" are used interchangeably. In some embodiments, an "effector T cell" is a CD8+ cytotoxic T cell.

[0235] An "activator" of an effector T cell refers to a molecule capable of promoting one or more functions of an effector T cell. An activator of an effector T cell may promote effector T cell function, for example, by promoting the proliferation, survival, and / or migration of the effector T cell, by promoting the production of effector cytokines and / or cytotoxic mediators from the effector T cell, and / or by reducing exhaustion of the effector T cell. An "activator" of an effector T cell may also be capable of activating one or more other cell types, for example, one or more other T cell types, so long as it is capable of activating an effector T cell. As used herein, the term "interleukin-2 receptor subunit alpha," "IL2-RA," "IL2RA," "CD-25," or "CD25" refers to a mammalian CD25 polypeptide, including, but not limited to, native CD25 polypeptide and isoforms of CD25 polypeptide. These terms encompass full-length unprocessed CD25 polypeptide and forms of CD25 polypeptide that result from processing within a cell.

[0236] As used herein, the term "human CD25" refers to a polypeptide comprising amino acids 22 to 272 of SEQ ID NO: 1. SEQ ID NO: 1 provides the sequence of mature human CD25 (amino acids 22 to 272) and its signal sequence (amino acids 1 to 21, underlined below). [ka]

[0237] A "CD25 polynucleotide," "CD25 nucleotide," or "CD25 nucleic acid" refers to a polynucleotide that encodes a CD25 polypeptide.

[0238] As used herein, the terms "programmed cell death protein 1," "PD-1," or "PD1" refer to mammalian PD1 polypeptides, including, but not limited to, native PD1 polypeptides and isoforms of PD1 polypeptides. These terms encompass full-length, unprocessed PD1 polypeptides and forms of PD1 polypeptides that result from processing within a cell.

[0239] As used herein, the term "human PD1" refers to a polypeptide comprising amino acids 24-288 of SEQ ID NO: 2. SEQ ID NO: 1 provides the sequence of mature human PD1 (amino acids 24-288) and its signal sequence (amino acids 1-23, underlined below). [ka]

[0240] A "PD1 polynucleotide," "PD1 nucleotide," or "PD1 nucleic acid" refers to a polynucleotide that encodes a PD1 polypeptide.

[0241] As used herein, the terms "T cell antigen CD-7," "CD-7," or "CD7" refer to mammalian CD7 polypeptides, including, but not limited to, native CD7 polypeptides and isoforms of CD7 polypeptides. These terms encompass full-length unprocessed CD7 polypeptides and forms of CD7 polypeptides that result from processing within a cell.

[0242] As used herein, the term "human CD7" refers to a polypeptide comprising amino acids 26-240 of SEQ ID NO: 3. SEQ ID NO: 3 provides the sequence of mature human CD7 (amino acids 26-240) and its signal sequence (amino acids 1-25, underlined below). [ka]

[0243] A "CD7 polynucleotide," "CD7 nucleotide," or "CD7 nucleic acid" refers to a polynucleotide that encodes a CD7 polypeptide.

[0244] As used herein, the terms "Casitas B lineage lymphoma proto-oncogene b," "E3 ubiquitin-protein ligase Cbl-b," or "Cbl-b" refer to mammalian Cbl-b polypeptides, including, but not limited to, native Cbl-b polypeptides and isoforms of Cbl-b polypeptides. These terms encompass full-length, unprocessed Cbl-b polypeptides and forms of Cbl-b polypeptides that result from processing within a cell.

[0245] As used herein, the term "human Cbl-b" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO:25. (SEQ ID NO:25)

[0246] A "Cbl-b polynucleotide," "Cbl-b nucleotide," or "Cbl-b nucleic acid" refers to a polynucleotide that encodes a Cbl-b polypeptide.

[0247] The term "antibody" as used herein refers to an immunoglobulin molecule that immunospecifically binds a target antigen (e.g., a protein, a polypeptide, a peptide, a carbohydrate, a polynucleotide, a lipid, or a combination of the foregoing) of interest or a portion thereof. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule, so long as the antibody exhibits the desired biological activity. The antibodies disclosed herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (isotype) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Immunoglobulins can be derived from any species. In one embodiment, the immunoglobulin is of human, murine, or rabbit origin. Antibodies can be naked or conjugated to other molecules, such as activators of effector T cells, including small molecule activators of effector T cells.

[0248] An "intact antibody" comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0249] An "antibody fragment" includes a portion of an intact antibody. An "antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to the portion of an intact antibody that binds to an antigen. Examples of antibody fragments include Fab, Fab', F(ab') 2and Fv fragments; diabodies; linear antibodies; fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), and epitope-binding fragments of any of the above that immunospecifically bind to a target of interest, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0250] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0251] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen binding specificity to the antibody or its antigen-binding fragment. However, even a single variable domain (or even half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0252] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0253] The term "single domain antibody", also known as nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain with a molecular weight of about 12 kDa to about 15 kDa. Single domain antibodies can be based on the heavy chain variable domain or the light chain. Examples of single domain antibodies include the V H H fragment and V NAR These include, but are not limited to, fragments.

[0254] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0255] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which fragment comprises a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, generating two antigen-binding sites.

[0256] A "native antibody" is usually a heterotetrameric glycoprotein of about 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain contains a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0257] The term "monoclonal" antibody or antigen-binding fragment thereof, as used herein, refers to an antibody or antigen-binding fragment thereof obtained from a population of substantially homogeneous antibodies or antigen-binding fragments thereof, i.e., the individual antibodies or antigen-binding fragments thereof that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies or antigen-binding fragments thereof are highly specific and directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody or antigen-binding fragment thereof is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies or antigen-binding fragments thereof are advantageous in that they may be synthesized without contamination by other antibodies or antigen-binding fragments thereof. The modifier "monoclonal" indicates the character of the antibody or antigen-binding fragment thereof as being obtained from a substantially homogeneous population of antibodies or antigen-binding fragments thereof and should not be construed as requiring production of the antibody or antigen-binding fragment thereof by any particular method. For example, the monoclonal antibodies or antigen-binding fragments thereof used in accordance with the present disclosure may be made by hybridoma methods or may be made by recombinant DNA methods. "Monoclonal" antibodies or antigen-binding fragments thereof may also be isolated from phage antibody libraries.

[0258] Monoclonal antibodies and antigen-binding fragments thereof herein specifically include "chimeric" antibodies, and fragments of such antibodies, in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies and antigen-binding fragments thereof from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, so long as the desired biological activity is exhibited. Chimeric antibodies and antigen-binding fragments thereof of interest herein include "primatized" antibodies and antigen-binding fragments thereof that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0259] "Humanized" forms of non-human (e.g., rodent) antibodies and antigen-binding fragments thereof are chimeric antibodies and antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. Humanization is a method for transferring murine antigen-binding information to a non-immunogenic human antibody acceptor, which has resulted in many therapeutically useful drugs. The humanization process usually begins by transferring all six murine complementarity determining regions (CDRs) into a human antibody or antigen-binding fragment framework. These CDR-grafted antibodies or antigen-binding fragments thereof usually do not retain their original affinity for antigen binding, and in fact, affinity is often severely impaired. In addition to the CDRs, select non-human antibody framework residues must also be incorporated to maintain proper CDR conformation. Transfer of key murine framework residues into the human acceptor to support the structural conformation of the grafted CDRs has been shown to restore antigen binding and affinity. In most cases, a humanized antibody or antigen-binding fragment thereof is a human immunoglobulin or fragment thereof (recipient antibody or fragment thereof) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody or fragment thereof) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and performance. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies or antigen-binding fragments thereof may comprise residues that are not found in the recipient or donor antibody or fragment thereof. These modifications are made to further refine antibody or antigen-binding fragment performance. Usually, a humanized antibody or antigen-binding fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. A humanized antibody or antigen-binding fragment thereof will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0260] Chimeric and humanized antibodies and antigen-binding fragments thereof reduce the likelihood of a human anti-mouse antibody (HAMA) response by minimizing the non-human portion of the administered antibody. Furthermore, chimeric and humanized antibodies and antigen-binding fragments thereof may have the added benefit of activating a secondary human immune response, such as antibody-dependent cellular cytotoxicity (ADCC).

[0261] An antibody or antigen-binding fragment thereof may have one or more "effector functions," which refer to those biological activities attributable to the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody or antigen-binding fragment thereof. Examples of effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), and the like. An antibody or antigen-binding fragment thereof may also be "Fc silent," which refers to an antibody or antigen-binding fragment that does not bind to an Fc receptor or has no ADCC function.

[0262] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies and antigen-binding fragments thereof can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0263] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody or antigen-binding fragment thereof, usually a portion of the light or heavy chain, typically about the amino terminal 110-120 or 110-125 amino acids in the mature heavy chain and about 90-115 amino acids in the mature light chain, which differs extensively in sequence among antibodies and their antigen-binding fragments and is used in the binding and specificity of a particular antibody or its antigen-binding fragment to its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs), whereas the more highly conserved regions in the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of an antibody or its antigen-binding fragment with an antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0264] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody or antigen-binding fragment thereof.

[0265] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or antigen-binding fragment thereof.

[0266] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof. In some embodiments, CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically located at amino acid positions 31-35 (which may optionally include one or two additional amino acids after 35 (referred to in the Kabat numbering scheme as 35A and 35B)) (CDR1), amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3).

[0267] Chothia rather refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The ends of the Chothia CDR-H1 loops, when numbered using the Kabat numbering convention, vary from H32 to H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. [Table 1]

[0268] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. The constant region is the carboxyl-terminal portion of the light and / or heavy chain that is not directly involved in binding the antibody or its antigen-binding fragment to an antigen, but may exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule usually has a more conserved amino acid sequence than the immunoglobulin variable domain.

[0269] As used herein, the term "specifically binds" refers to an interaction between molecules where (i) the binding becomes saturated when the concentration of one of the molecules is increased relative to the other molecule, and (ii) the binding can be competed by the presence of excess of one of the interacting molecules. A molecule (e.g., a binding domain, e.g., an antibody or antigen-binding fragment thereof) that is "capable of specifically binding" to a target on the surface of an effector T cell can also bind to a target in soluble form and / or to a target at a different location (e.g., on the surface of another cell). A molecule (e.g., a binding domain, e.g., an antibody or antigen-binding fragment thereof) that is "capable of specifically binding" to a target from one species (e.g., human) can also bind to its target from another species (e.g., cynomolgus monkey, mouse, and / or rat), but the extent of binding to unrelated targets is less than about 10% of the binding to the target.

[0270] As used herein, the term "immunospecifically binds" indicates that an antibody or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. Thus, an antibody that "immunospecifically binds" to a human protein, e.g., human PD1, may also bind to that protein or related proteins from other species (e.g., cynomolgus monkey, mouse, and / or rat PD1), but the extent of binding to unrelated proteins, as measured, is less than about 10% of the antibody's binding to PD1. An antibody that "immunospecifically binds" to an antigen of interest is one that is capable of binding the antigen with sufficient affinity such that the antibody is useful in targeting cells expressing the antigen.

[0271] The term "reduce" or "reducing" or "inhibition" or "inhibiting" refers to the reduction or cessation of any phenotypic characteristic or the reduction or cessation of the frequency, extent, or likelihood of occurrence of that characteristic. "Reduce" or "inhibit" refers to the reduction or cessation of an activity, function, and / or amount when compared to a reference. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 20% or more. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 50% or more. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the amount is inhibited or reduced over a period of time compared to a control over the same period of time.

[0272] A molecule that is an "inhibitor" of a target may decrease the activity, function, and / or amount of the target when compared to the activity, function, and / or amount of the target in the absence of the inhibitor. Thus, a molecule, compound, or payload that is an "inhibitor of Cbl-b" may decrease the activity, function, and / or amount of Cbl-b when compared to the activity, function, and / or amount of Cbl-b in the absence of the molecule, compound, or payload.

[0273] A molecule that is an "agonist" of a target can increase the activity, function, and / or amount of the target when compared to the activity, function, and / or amount of the target in the absence of the agonist.

[0274] The term "about" is used herein to mean approximately, roughly, in the region, or within the region. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. Typically, the term "about" can modify a numerical value by varying a numerical value above and below the stated value, for example, by 10 percent above or below (higher or lower). Whenever an embodiment is described herein using the words "about" or "approximately," it is understood that a numerical value or range that is otherwise similar but refers to a particular numerical value or range (without "about") is also provided. The terms "administer," "administering," "administration," and the like, as used herein, refer to methods that can be used to enable delivery of a drug, e.g., an activator of effector T cells (e.g., a small molecule or an antibody drug conjugate), to a desired site of biological action. Administration techniques that can be used with the agents and methods described herein can be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0275] The terms "antibody-drug conjugate" and "ADC" are used interchangeably and refer to an antibody or antigen-binding fragment thereof linked, e.g., covalently, to a therapeutic agent (sometimes referred to herein as a drug, medication, or active pharmaceutical ingredient) or agent. In some embodiments of the present disclosure, the biologically active molecule is an antibody-drug conjugate. In some embodiments, the ADC comprises an antibody or antigen-binding fragment thereof covalently linked to a payload capable of activating effector T cells. An ADC having a payload capable of activating effector T cells may also be capable of activating one or more other cell types, e.g., one or more other T cell types, so long as it is capable of activating effector T cells.

[0276] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered conservative. In another embodiment, a string of amino acids can be conservatively replaced with a structurally similar string that differs in the order and / or composition of side chain family members.

[0277] As used herein, the term "conserved" refers to nucleotides or amino acid residues of a polynucleotide or polypeptide sequence, respectively, that occur unaltered in the same position in two or more sequences that are being compared. Relatively conserved nucleotides or amino acids are those that are more conserved between related sequences than nucleotides or amino acids that occur elsewhere in the sequences.

[0278] In some embodiments, two or more sequences are referred to as "fully conserved" or "identical" if they are 100% identical to each other. In some embodiments, two or more sequences are referred to as "highly conserved" if they are at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other. In some embodiments, two or more sequences are referred to as "conserved" if they are at least about 30% identical, at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to a portion, region, or feature thereof.

[0279] As used herein, the terms "linking" and "conjugating" are used interchangeably and refer to the covalent or non-covalent attachment of two or more moieties, including one or more compounds and binding moieties capable of activating effector T cells, respectively. In some embodiments, linking or conjugating may include a linker.

[0280] The term "amino acid sequence variant" refers to a polypeptide having an amino acid sequence that differs somewhat from a native sequence polypeptide. Usually, amino acid sequence variants will retain at least about 70% sequence identity with at least one receptor binding domain of a native antibody or at least one ligand binding domain of a native receptor, and typically they will be at least about 80%, more typically at least about 90% homologous in sequence to such receptor or ligand binding domain. Amino acid sequence variants will retain substitutions, deletions, and / or insertions at predetermined positions within the amino acid sequence of the native amino acid sequence. Amino acids are designated by the one-letter and three-letter codes, which are conventional names.

[0281] "Sequence identity" is defined as the percentage of residues in amino acid sequence variants that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignment are well known in the art. One such computer program is "Align2" created by Genentech, Inc., which was submitted with user documentation to the United States Copyright Office, Washington, DC 20559 on December 10, 1991.

[0282] The term "Fc receptor" or "FcR" is used to refer to a receptor that binds to the Fc region of an antibody or antigen-binding fragment thereof. An exemplary FcR is a native sequence human FcR. Additionally, an FcR can be one that binds an IgG antibody or an antigen-binding fragment thereof (a gamma receptor), which includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. Other FcRs are encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus.

[0283] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody or an antigen-binding fragment thereof) complexed with a cognate antigen. To assess complement activation, a CDC assay can be performed.

[0284] The "light chains" of antibodies or antigen-binding fragments from any vertebrate species can be assigned to one of two clearly distinguishable types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0285] An "isolated" antibody or antigen-binding fragment thereof is one that has been identified, separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses of the antibody or antigen-binding fragment thereof, which may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody or antigen-binding fragment thereof will be purified (1) to greater than 95% by weight, or greater than 99% by weight, of the antibody or antigen-binding fragment thereof as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a gas phase protein sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated antibody or antigen-binding fragment thereof includes an antibody or antigen-binding fragment thereof in situ, within a recombinant cell, because at least one component of the antibody's or antigen-binding fragment's natural environment will not be present. Ordinarily, however, isolated antibody or antigen-binding fragment thereof will be prepared by at least one purification step.

[0286] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer," as used herein, refers to primary, metastatic, and recurrent cancers.

[0287] As used herein, the term "immune response" refers to a biological reaction in a vertebrate to foreign substances that protect the organism against these substances and the diseases caused by them. An "immune response" is mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by either these cells or the liver that selectively target, bind to, damage, destroy, and / or eliminate from the vertebrate body an invading pathogen and / or pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues. Immune responses include, for example, T cells, e.g., effector T cells, or Th cells, e.g., CD4 + or CD8 + These include activation or inhibition of T cells, or inhibition of Treg cells. As used herein, the terms "T cell" and "T lymphocyte" are interchangeable and refer to any lymphocyte generated or processed by the thymus. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a NKT cell.

[0288] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates, such as non-human primates, sheep, dogs, and rodents, such as mice, rats, and guinea pigs. In some embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0289] The term "therapeutically effective amount" or "therapeutically effective dosage" refers to an amount of an agent (e.g., a conjugate disclosed herein) that provides a desired biological, therapeutic, and / or prophylactic result. The result may be reduction, amelioration, alleviation, attenuation, delay, and / or amelioration of one or more of the signs, symptoms, or causes of a disease, or any other desired modification of a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to cause tumor shrinkage and / or to reduce the rate of tumor growth (e.g., to inhibit tumor growth) or to prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay tumor progression. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered in one or more administrations. An effective amount of the composition may, for example, (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, delay, slow to some extent, or stop cancer cell invasion into peripheral organs, (iv) inhibit (i.e., slow to some extent or stop) tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay the onset and / or recurrence of tumors, and / or (vii) relieve to some extent one or more of the symptoms associated with cancer.

[0290] In some embodiments, a "therapeutically effective amount" is an amount of a conjugate that has been clinically proven to affect a significant reduction in cancer or slowing the progression (reversal) of cancer, e.g., advanced solid tumors. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those of skill in the art, for example, by assessing the activity of the agent in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0291] As used herein, the term "standard of care" refers to a treatment that is accepted by medical experts as an appropriate treatment for a given type of disease and that is widely used by health care professionals. The term may be used interchangeably with any of the following terms: "best practice," "standard medical care," and "standard therapy."

[0292] By way of example, an "anti-cancer drug" promotes cancer regression or prevents further tumor growth in a subject. In certain embodiments, a therapeutically effective amount of a drug promotes cancer regression to the point of eliminating the cancer.

[0293] The terms "effective" and "effectiveness" in reference to a treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ and / or organism level resulting from administration of the drug.

[0294] As used herein, the term "immune checkpoint inhibitors" refers to molecules that reduce, inhibit, impede or modulate one or more checkpoint proteins, either in whole or in part. Checkpoint proteins control T cell activation or function. A number of checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD1 and its ligands PDL1 and PDL2. Pardoll, DM, Nat Rev Cancer 12(4):252-64(2012). These proteins are responsible for co-stimulatory or inhibitory interactions in T cell responses. Immune checkpoint proteins control and maintain self-tolerance as well as the duration and magnitude of physiological immune responses. Immune checkpoint inhibitors include antibodies and antigen-binding fragments thereof.

[0295] The term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (attenuate) the occurrence or spread of unwanted physiological changes or disorders, such as cancer. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, attenuation of the extent of disease, a stabilized (i.e., not worsening) state of disease, delaying or slowing disease progression, improvement or palliation of the disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment. Those in need of treatment include those already with a condition or disorder as well as those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

[0296] Unless otherwise specified, stereocenters within the compounds described herein may be of R or S stereochemistry and may be substituted as described elsewhere in this disclosure. When stereochemistry is not specified, it should be understood that the disclosure encompasses all stereochemical isomeric forms, or mixtures thereof, that retain the ability to inhibit Cbl-b and / or activate T cells. Individual stereoisomers of the compounds may be prepared synthetically from commercially available starting materials that contain chiral centers, or by preparation of a mixture of enantiomeric products followed by separation, such as conversion to a mixture of diastereomers, followed by separation or recrystallization, chromatographic techniques, or direct separation of the enantiomers on a chiral chromatographic column. Starting compounds of a particular stereochemistry are commercially available or may be made and resolved by techniques known in the art.

[0297] II. Cbl-b inhibitors In certain embodiments, the disclosure provides a Cbl-b inhibitor of formula (I): [ka] or a pharma- ceutically acceptable salt thereof,

[0298] n is 0, 1, or 2;

[0299] X and Y are each independently CH or N;

[0300] Z is CH(CH 3 ), O, and SO 2 Selected from; or

[0301] Z is CH(CH 3 ), NH, N(CH 3 ), O, and SO 2 Selected from;

[0302] R 1 is hydrogen, -CN, -NHR z , -R a , -NR a R b , -OR a , -NHC(O)R a , -NHC(S)R a , -NHC(O)NHR a , -NHC(S)NHR a , -SR a , C 3 -C 6 cycloalkyl, and 3- to 6-membered heterocyclyl rings;

[0303] R z teeth, [ka] Selected from;

[0304] R a and R b is hydrogen, C 2- C 6 Alkenyl, C 1 -C 6 Alkyl, Amide (C 1 -C 6 Alkyl), Amino (C 1 -C 6 Alkyl), Azide (C 1 -C6 Alkyl), C 2 -C 6 Alkynyl, carboxy (C 1 -C 6 Alkyl), Cyano (C 1 -C 6 alkyl), C optionally substituted with a cyano group 3 -C 6 Cycloalkyl, dimethylamino (C 1 -C 6 alkyl), 3-6 membered heterocyclyl ring, 3-6 membered heterocyclyl (C 1 -C 3 Alkyl), Hydroxy (C 1 -C 6 Alkyl), Methoxy (C 1 -C 6 Alkyl), methylamino (C 1 -C 6 alkyl), NR c R d (C 1 -C 6 alkyl), HS(C 1 -C 6 alkyl), and CH 3 S(C 1 -C 6 alkyl), R c and R d is hydrogen, C 2 alkenylcarbonyl, and methyl; or

[0305] R a and R b together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, the ring being a , -C(O)R a , -SO 2 R a , azido, and cyano;

[0306] Each C 3 -C 6 Cycloalkyl, 3- to 6-membered heterocyclyl ring, and 3- to 6-membered heterocyclyl (C 1 -C3 The heterocyclyl portion of the alkyl ring is 1 -C 3 Alkyl, C 2 Alkynyl, amide, azido, carboxy, cyano, dimethylamino, hydroxy, methoxy, methylamino, HS-, and CH 3 optionally substituted with 1, 2, or 3 groups independently selected from S-;

[0307] R 2 teeth, [ka] Selected from;

[0308] m is 0, 1, 2, or 3;

[0309] m'' is 0, 1, 2, 3, or 4;

[0310] B' is a 3-7 membered saturated or unsaturated ring optionally containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; the ring is selected from -OH, -CH 2 S.H., C.H. 2 SCH 3 , -CH 2 OH, -CH 2 NH 2 , and -CH 2 NHCH 3 and is optionally substituted with 1 or 2 substituents independently selected from:

[0311] Each R 500 is hydrogen, C 1 -C 6 Alkyl, halo, -OH, and -CH 2 is independently selected from OH;

[0312] X 50 are O, NH, and NCH. 3 , and S;

[0313] R 3 is hydrogen, acetyl, amino, C1 -C 6 Alkylamino, C 1 -C 6 Alkylaminomethyl, C 1 -C 6 Alkylcarbonyl, Amino C 1 -C 6 Alkyl, aminocarbonyl, aminomethyl, carboxy, cyano, C 3 Cycloalkyl, formyl, hydroxy, hydroxyC 1 -C 6 Alkyl, methoxy, oxazolyl, -SH, -SCH 3 , -SOCH 3 , -SO 2 CH 3 , -SO(=NH)CH 3 , tetrazolyl, thiazolyl, and trifluoromethyl; 3 The cycloalkyl is optionally substituted with a hydroxy group;

[0314] R 4 is hydrogen, methyl, -CH 2 OH, -CH 2 SH, and -CH 2 SCH 3 Selected from;

[0315] R 5 is hydrogen, hydroxyl, -CH 2 SH, -CH 2 SCH 3 and methyl;

[0316] Optionally, but R 5 is hydroxy or methyl, and R 4 If is hydrogen, then R 1 is C 3 -C 6 Cycloalkyl, 3-6 membered heterocyclyl ring, hydroxy, hydroxy(C 1 -C 6 alkyl), -OR a (In the formula, R a is C 1 -C 6Alkyl, 3- to 6-membered heterocyclyl ring, or hydroxy (C 1 -C 6 alkyl); or -NR a R b (In the formula, R a and R b is hydrogen, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, hydroxy(C 1 -C 6 alkyl), 3- to 6-membered heterocyclyl ring, or R a and R b together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, the ring being 1 -C 6 Alkyl or hydroxy (C 1 -C 6 alkyl);

[0317] R 6 and R 6’ is hydrogen, cyclopropyl, -CH 2 OH, -CH 2 SH, -CH 2 SCH 3 , and -CH 2 R 200 are independently selected from R 200 is a 3-7 membered saturated or unsaturated ring optionally containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. to provide.

[0318] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is in the "S" configuration. In certain embodiments, the present disclosure provides a Cbl-b inhibitor of formula (IA-1): [ka] or a pharma- ceutically acceptable salt thereof,

[0319] n is 0, 1, or 2;

[0320] X and Y are each independently CH or N;

[0321] Z is CH(CH 3 ), O, and SO 2 Selected from;

[0322] R 1 is hydrogen, -CN, -NHR z , -R a , -NR a R b , -OR a , -NHC(O)R a , -NHC(S)R a , -NHC(O)NHR a , -NHC(S)NHR a , -SR a , C 3 -C 6 cycloalkyl, and 3- to 6-membered heterocyclyl rings;

[0323] R z teeth, [ka] Selected from;

[0324] R a and R b is hydrogen, C 2- C 6 Alkenyl, C 1 -C 6 Alkyl, Amide (C 1 -C 6 Alkyl), Amino (C 1 -C 6 Alkyl), Azide (C 1 -C 6 Alkyl), C 2 -C 6 Alkynyl, carboxy (C 1 -C 6 Alkyl), Cyano (C 1 -C 6Alkyl), C 3 -C 6 Cycloalkyl, dimethylamino (C 1 -C 6 alkyl), 3-6 membered heterocyclyl ring, 3-6 membered heterocyclyl (C 1 -C 3 ) alkyl, hydroxy (C 1 -C 6 Alkyl), Methoxy (C 1 -C 6 Alkyl), methylamino (C 1 -C 6 alkyl), NR c R d (C 1 -C 6 alkyl), HS(C 1 -C 6 alkyl), and CH 3 S(C 1 -C 6 alkyl), R c and R d is hydrogen, C 2 alkenylcarbonyl, and methyl; or

[0325] R a and R b together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, the ring being a , -C(O)R a , -SO 2 R a , azido, and cyano;

[0326] Each C 3 -C 6 Cycloalkyl, 3- to 6-membered heterocyclyl ring, and 3- to 6-membered heterocyclyl (C 1 -C 3 The heterocyclyl portion of the alkyl group is C 1 -C 3 Alkyl, C 2 Alkynyl, amide, azido, carboxy, cyano, dimethylamino, hydroxy, methoxy, methylamino, HS-, and CH3 optionally substituted with 1, 2, or 3 groups independently selected from S-;

[0327] R 2 teeth,

[0328] [ka] and;

[0329] m is 0, 1, 2, or 3;

[0330] R 3 is hydrogen, acetyl, amino, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylaminomethyl, Amino C 1 -C 6 Alkyl, aminocarbonyl, aminomethyl, carboxy, cyano, C 3 Cycloalkyl, formyl, hydroxy, hydroxyC 1 -C 6 Alkyl, methoxy, oxazolyl, -SH, -SCH 3 , tetrazolyl, thiazolyl, and trifluoromethyl; 3 The cycloalkyl is optionally substituted with a hydroxy group;

[0331] R 4 and R 6 is hydrogen, -CH 2 SH, and -CH 2 SCH 3 are independently selected from;

[0332] R 5 is hydroxy, -CH 2 SH, -CH 2 SCH 3 and methyl; Optionally, but R 5 is hydroxy or methyl, and R 4 If is hydrogen, then R 1 is C3 -C 6 Cycloalkyl, 3-6 membered heterocyclyl ring, hydroxy, hydroxy(C 1 -C 6 alkyl), -OR a (In the formula, R a is C 1 -C 6 Alkyl, 3- to 6-membered heterocyclyl ring, or hydroxy (C 1 -C 6 alkyl); or -NR a R b (In the formula, R a and R b is hydrogen, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, hydroxy(C 1 -C 6 alkyl), 3- to 6-membered heterocyclyl ring, or R a and R b together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, the ring being 1 -C 6 Alkyl or hydroxy (C 1 -C 6 alkyl) to provide.

[0333] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0334] Compounds of formula (I) are excluded from those disclosed in WO2019 / 148005, WO2020 / 210508, and WO2021 / 021761.

[0335] In some aspects, the present disclosure provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof, wherein:

[0336] X is N;

[0337] Y is CH;

[0338] R 2 teeth, [ka] is) to provide.

[0339] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0340] In some aspects, the disclosure provides compounds of formula (I), or a pharma- ceutically acceptable salt thereof, wherein Z is O.

[0341] In some aspects, the present disclosure provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof, wherein Z is CH(CH 3 ) is provided.

[0342] In some aspects, the present disclosure provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof, wherein:

[0343] R 3 is trifluoromethyl;

[0344] R 4 and R 6 is hydrogen;

[0345] R 5 is methyl) to provide.

[0346] In some embodiments, the present disclosure provides a method for the preparation of a compound comprising: 1 But -NR a R b , -NHC(O)R a , -NHC(S)NHR a , and -SR aThe present invention provides a compound of formula (I), selected from:

[0347] In some aspects, the present disclosure provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof, 1 -SCH 2 CH 3 (which is

[0348] In some aspects, the present disclosure provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof, 1 is -NH(CH 2 ) 2 CN).

[0349] In some aspects, the present disclosure provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof, 1 is -NH(CH 2 ) 2 N 3 (which is

[0350] As used herein, the term "acetyl" refers to -C(O)CH 3 Refers to...

[0351] As used herein, the term "C 2 Alkenyl is -CH=CH 2 Refers to...

[0352] As used herein, the term "C 2- C 6 "Alkenyl" refers to a group derived from a straight or branched chain hydrocarbon containing 2 to 6 carbon atoms and containing at least one double bond.

[0353] As used herein, the term "C 2 "Alkenylcarbonyl" is a C bonded to the parent molecular moiety through a carbonyl group. 2 Refers to an alkenyl group.

[0354] As used herein, the term "C 1-C 6 "Alkoxy," as used herein, refers to a C alkyl group attached to the parent molecular moiety through an oxygen atom. 1 -C 6 Refers to an alkyl group.

[0355] As used herein, the term "C 1 -C 6 Alkoxy C 1 -C 6 Alkyl" is C 1 -C 6 C attached to the parent molecular moiety via an alkyl group 1 -C 6 Refers to an alkoxy group.

[0356] As used herein, the term "C 1 -C 6 "Alkyl" refers to a group derived from a straight or branched chain saturated hydrocarbon containing from 1 to 6 carbon atoms.

[0357] As used herein, the term "C 1 -C 6 "Alkylamino" refers to -NHR, where R is C 1 -C 6 It refers to an alkyl group.

[0358] As used herein, the term "C 1 -C 6 "Alkylaminomethyl" is a C alkylamino group attached to the parent molecular moiety through a methylene group. 1 -C 6 Refers to the alkylamino group.

[0359] As used herein, the term "C 2 Alkynyl is [ka] Refers to...

[0360] As used herein, the term "C 2- C 6"Alkynyl" refers to a group derived from a straight or branched chain hydrocarbon containing 2 to 6 carbon atoms and containing at least one double bond.

[0361] As used herein, the term "amide" refers to -C(O)NH 2 Refers to...

[0362] As used herein, the term “amide (C 1 -C 6 "Alkyl)" is C 1 -C 6 Refers to an amide group attached to the parent molecular moiety through an alkyl group.

[0363] As used herein, the term "amino" refers to -NH 2 Refers to...

[0364] As used herein, the term “amino (C 1 -C 6 "Alkyl)" is C 1 -C 6 Refers to an amino group attached to the parent molecular moiety through an alkyl group.

[0365] The term "aminocarbonyl," as used herein, refers to an amino group attached to the parent molecular moiety through a carbonyl group.

[0366] The term "aminomethyl," as used herein, refers to an amino group attached to the parent molecular moiety through a methylene group.

[0367] As used herein, the term "azide" refers to a -N 3 Refers to...

[0368] As used herein, the term "azide (C 1 -C 6 "Alkyl)" is C 1 -C 6 Refers to an azide group attached to the parent molecular moiety through an alkyl group.

[0369] As used herein, the term "carbonyl" refers to -C(O)-.

[0370] As used herein, the term "carboxy" refers to -CO 2 Point to H.

[0371] As used herein, the term “carboxy (C 1 -C 6 "Alkyl)" is C 1 -C 6 It refers to a carboxy group attached to the parent molecular moiety through an alkyl.

[0372] As used herein, the term "cyano" refers to --CN.

[0373] As used herein, the term "cyano (C 1 -C 6 "Alkyl)" is C 1 -C 6 Refers to a cyano group attached to the parent molecular moiety through an alkyl group.

[0374] As used herein, the term "C 3 "Cycloalkyl" refers to a cyclopropyl group.

[0375] As used herein, the term "C 3 -C 6 "Cycloalkyl" refers to a saturated monocyclic hydrocarbon ring system having 3 to 6 carbon atoms and 0 heteroatoms. Representative examples of cycloalkyl groups include, but are not limited to, cyclobutyl, cyclopentyl, and cyclohexyl.

[0376] As used herein, the term “dimethylamino (C 1 -C 6 "Alkyl)" is C 1 -C 6 Refers to a dimethylamino group attached to the parent molecular moiety through an alkyl group.

[0377] As used herein, the term "formyl" refers to --CHO.

[0378] As used herein, the term “halo” refers to F, Cl, Br, or I.

[0379] As used herein, the term "3- to 6-membered heterocyclyl" refers to a 3-, 4-, 5-, or 6-membered saturated or unsaturated group containing 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Representative examples of 3- to 6-membered heterocyclyl groups include, but are not limited to, oxetane, pyrrolidine, thiazole, pyridine, and piperidine.

[0380] As used herein, the term "3- to 6-membered heterocyclyl (C 1 -C 3 "C alkyl" 1 -C 3 Refers to a 3- to 6-membered heterocyclyl ring attached to the parent molecular moiety through an alkyl.

[0381] As used herein, the term "hydroxy" refers to --OH.

[0382] As used herein, the term "hydroxy C 1 -C 6 "Alkyl" refers to a C alkyl group substituted with one, two, or three hydroxy groups. 1 -C 6 Refers to an alkyl group.

[0383] As used herein, the term “methoxy(C 1 -C 6 "C alkyl" 1 -C 6 Refers to a methoxy group attached to the parent molecular moiety through an alkyl group.

[0384] As used herein, the term “methylamino (C 1 -C 6 "C alkyl" 1 -C 6Refers to a methylamino group attached to the parent molecular moiety through an alkyl group.

[0385] As used herein, the term "NR c R d (C 1 -C 6 "Alkyl)" is C 1 -C 6 NR attached to the parent molecular moiety via an alkyl group c R d R c and R d is as defined herein.

[0386] As used herein, the term "HS(C 1 -C 6 "Alkyl)" is C 1 -C 6 Refers to an -SH group attached to the parent molecular moiety through an alkyl group.

[0387] In some embodiments, the compound of formula (I) is [ka] The compound is selected from the group consisting of:

[0388] In some embodiments, the compound of formula (I) can treat cancer in a subject. In some embodiments, the compound of formula (I) can increase effector T cell activity and / or effector T cell proliferation. In some embodiments, the compound of formula (I) can increase effector T cell migration to tumor cells. In some embodiments, the compound of formula (I) can reduce effector T cell exhaustion.

[0389] In some embodiments, the compound of formula (I) competitively inhibits the binding of a probe comprising a BODIPY-FL fluorophore conjugated to a Cbl-b inhibitor to Cbl-b, for example, when evaluated using the assay described herein in Example 2 (see Example 54 in WO20200264398). In some embodiments, the compound of formula (I) competitively inhibits with an IC50 of about 1 nM to about 5 nM. In some embodiments, the compound of formula (I) competitively inhibits with an IC50 of about 5.01 nM to about 20 nM. In some embodiments, the compound of formula (I) competitively inhibits with an IC50 of about 20.01 nM to about 100 nM. In some embodiments, the compound of formula (I) competitively inhibits with an IC50 of about 100.01 nM to about 1 mM.

[0390] In some embodiments, the compounds of formula (I) are capable of increasing IL-2 secretion from T cells, e.g., as assessed using the assay described herein in Example 3. In some embodiments, the compounds of formula (I) increase IL-2 secretion by about 0.8 to about 1.4 fold over background. In some embodiments, the compounds of formula (I) increase IL-2 secretion by about 0.31 to about 0.79 fold over background. In some embodiments, the compounds of formula (I) increase IL-2 secretion by about 0.1 to about 0.3 fold over background.

[0391] In some embodiments, the compounds of formula (I) are capable of increasing IFN-y secretion from T cells, for example, as assessed using the assay described herein in Example 3. In some embodiments, the compounds of formula (I) increase IFN-y secretion by about 1.1 to about 2-fold over background. In some embodiments, the compounds of formula (I) increase IFN-y secretion by about 0.31 to about 1-fold over background. In some embodiments, the compounds of formula (I) increase IFN-y secretion by about 0.1 to about 0.3-fold over background.

[0392] In some embodiments, the compound of formula (I) is capable of increasing CD69 levels, for example, as assessed using the assay described herein in Example 3. In some embodiments, the compound of formula (I) increases CD69 levels by about 1.11 to about 1.5 times over background. In some embodiments, the compound of formula (I) increases CD69 levels by about 0.81 to about 1.10 times over background. In some embodiments, the compound of formula (I) increases CD69 levels by about 0.81 to about 1.1 times over background. In some embodiments, the compound of formula (I) increases CD69 levels by about 0.61 to about 0.8 times over background. In some embodiments, the compound of formula (I) increases CD69 levels by about 0.3 to about 0.6 times over background.

[0393] In some embodiments, the compounds of formula (I) interact with His152 of human Cbl-b. In some embodiments, the compounds of formula (I) include an R 1 In some embodiments, the group R 1 The distance between the terminal nitrogen atom of and His152 of Cbl-b is about 1 to about 5 angstroms in the compound and in the crystal structure of human Cbl-b. 1 The distance between the terminal nitrogen atom of and His152 of Cbl-b is about 1 to about 4 angstroms in the co-crystal structure of the compound and human Cbl-b. 1 The distance between the terminal nitrogen atom of and His152 of Cbl-b is about 2 to about 5 angstroms in the co-crystal structure of the compound and human Cbl-b. 1 The distance between the terminal nitrogen atom of and His152 of Cbl-b is about 2 to about 4 angstroms in the co-crystal structure of the compound and human Cbl-b. 1 The distance between the terminal nitrogen atom of and His152 of Cbl-b is about 2.5 to about 5 angstroms in the co-crystal structure of the compound and human Cbl-b. 1The distance between the terminal nitrogen atom of and His152 of Cbl-b is about 2.5 to about 4 angstroms in the co-crystal structure of the compound and human Cbl-b.

[0394] III. Conjugates The present disclosure provides conjugates that include a binding moiety (e.g., an antibody or antigen-binding fragment thereof) capable of specifically binding to a target on the surface of an effector T cell and a payload capable of activating the effector T cell. The binding moiety can be directly attached to the payload or can be attached to the payload via a linker. In certain embodiments, the payload can be attached to the linker or the binding moiety via a covalent bond.

[0395] The payload is a compound of formula (I): [ka] In certain embodiments, the compound of formula (I) comprises a group R 1 The heteroatom may be attached to the linker via a covalent bond to a heteroatom in

[0396] The payload is a compound of formula (Ia): [ka] In some embodiments, the compound of formula (Ia) is 1 , R 3 , R 4 , R 5 , R 6 or to the linker via a covalent bond to the nitrogen atom of the piperidine ring.

[0397] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0398] The payload is a compound of formula (Ib): [ka] In some embodiments, the compound of formula (Ib) is 1 , R 3 , R 4 , R 5 , R 6 or to the linker via a covalent bond to the nitrogen atom of the piperidine ring.

[0399] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0400] The payload is a compound of formula (Ic): [ka] In some embodiments, the compound of formula (Ic) is 1 , R 3 , R 4 , R 5 , R 6 or to the linker via a covalent bond to the nitrogen atom of the piperidine ring.

[0401] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0402] The payload is a compound of formula (Id): [ka] In some embodiments, the compound of formula (Id) is 1 , R 3 , R 4 , R 5 , R 6 , R 6’ or to the linker via a covalent bond to the nitrogen atom of the piperidine ring.

[0403] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4is an "S" configuration.

[0404] The payload is a compound of formula (Ie): [ka] In some embodiments, the compound of formula (Ie) is 1 , R 3 , R 4 , R 500 , B', or the linker via a covalent bond to the nitrogen atom of the piperidine ring.

[0405] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0406] The payload is a compound of formula (If): [ka] In some embodiments, the compound of formula (If) is R 1 , R 3 , R 4 , B', or the linker via a covalent bond to the nitrogen atom of the piperidine ring.

[0407] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0408] The payload is a compound of formula (Ig): [ka] In some embodiments, the compound of formula (Ig) is 1 , R 3 , R 4 , R 500 , B', or the linker via a covalent bond to a nitrogen atom bonded to B'.

[0409] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0410] The payload is a compound of formula (Ih): [ka] In some embodiments, the compound of formula (Ig) is 1 , R 3 , R 4 , B', or the linker via a covalent bond to the nitrogen atom of the piperidine ring.

[0411] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0412] The payload is a compound of formula (Ii): [ka] In some embodiments, the compound of formula (Ii) is 1 , R 3 , R 4 , B', or the linker via a covalent bond to the nitrogen atom of the piperidine ring.

[0413] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0414] The payload is a compound of formula (Ij): [ka] In some embodiments, the compound of formula (Ii) is 1 , R 3 , R 4 , B', or the piperazine ring can be attached to the linker via a covalent bond to a nitrogen atom of either the piperazine ring.

[0415] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0416] The payload is a compound of formula (Ik): [ka] In some embodiments, the compound of formula (Ik) is 1 , R 3 , R 4 , R 6 , R 6’ or to the linker via a covalent bond to the nitrogen atom of the piperidine ring.

[0417] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0418] The payload is a compound of formula (II): [ka] In some embodiments, the compound of formula (II) is 1 , R 3 , R 4 , R 6 , R 6’ or to the linker via a covalent bond to the nitrogen atom of the piperidine ring.

[0419] In some embodiments, R 4 is in the "R" configuration. In some embodiments, R 4 is an "S" configuration.

[0420] In some embodiments, the conjugates provided herein comprise a binding moiety capable of specifically binding to PD1 (e.g., an antibody or antigen-binding fragment thereof) and a payload capable of activating effector T cells. In some embodiments, the conjugates provided herein comprise a binding moiety capable of specifically binding to PD1 (e.g., an antibody or antigen-binding fragment thereof) and a payload that is an inhibitor of Casitas B lineage lymphoma proto-oncogene b (Cbl-b).

[0421] In some embodiments, the conjugates provided herein comprise a binding moiety (e.g., an antibody or antigen-binding fragment thereof) capable of specifically binding to CD25 (IL2RA) and a payload capable of activating an effector T cell. In some embodiments, the conjugates provided herein comprise a binding moiety (e.g., an antibody or antigen-binding fragment thereof) capable of specifically binding to PD1 and a payload that is an inhibitor of Casitas B lineage lymphoma proto-oncogene b (Cbl-b).

[0422] In some aspects, the conjugates provided herein comprise a binding moiety capable of specifically binding to PD1 (e.g., an antibody or antigen-binding fragment thereof) and a payload that is an inhibitor of Casitas B lineage lymphoma proto-oncogene b (Cbl-b).

[0423] In some embodiments, the conjugates provided herein have the formula (II): Bm-[LP] a (II) (wherein a is an integer of 1 to 50; P is a payload; L is a linker; and Bm is a binding site (e.g., an antibody or an antigen-binding fragment thereof). In some embodiments, a is about 1 to about 40. In some embodiments, a is about 1 to about 10. In some embodiments, a is about 2 to about 8.

[0424] In some embodiments, provided herein are compositions (e.g., pharmaceutical compositions) comprising a conjugate provided herein (e.g., at least two conjugates). In some embodiments, the compositions comprise, on average, about 1 to about 50 payloads per binding site. In some embodiments, the compositions comprise, on average, about 1 to about 10 payloads per binding site. In some embodiments, the compositions comprise, on average, about 2 to about 8 payloads per binding site.

[0425] In some embodiments, the conjugates or compositions provided herein are capable of treating cancer in a subject. In some embodiments, the conjugates or compositions provided herein are capable of increasing effector T cell activity and / or effector T cell proliferation. In some embodiments, the conjugates or compositions provided herein are capable of increasing effector T cell migration to tumor cells. In some embodiments, the conjugates or compositions provided herein are capable of reducing effector T cell exhaustion. In some embodiments, the conjugates or compositions provided herein are capable of delivering a payload to an effector T cell capable of activating the effector T cell.

[0426] In some embodiments, the conjugates or compositions provided herein are capable of blocking PD1 / PDL1, e.g., as measured using the assay described herein in Example 7. In some embodiments, the conjugates or compositions provide about 3.51 to about 5.49 fold blockage of PD1 / PDL1 over background. In some embodiments, the conjugates or compositions provide about 2.5 to about 3.5 fold blockage of PD1 / PDL1 over background. In some embodiments, the conjugates or compositions provide about 2 to about 2.49 fold blockage of PD1 / PDL1 over background. In some embodiments, the conjugates or compositions provide about 1.5 to about 1.99 fold blockage of PD1 / PDL1 over background. In some embodiments, the conjugates or compositions provide about 1.1 to about 1.49 fold blockage of PD1 / PDL1 over background.

[0427] In some embodiments, the conjugates or compositions provided herein are capable of increasing IFN-y secretion from T cells, for example, as assessed using the assay described herein in Example 7. In some embodiments, the conjugates or compositions increase IFN-y secretion about 1 to about 1.4 fold over background. In some embodiments, the conjugates or compositions increase IFN-y secretion about 1 to about 1.49 fold over background. In some embodiments, the conjugates or compositions increase IFN-y secretion about 1.5 to about 1.99 fold over background. In some embodiments, the conjugates or compositions increase IFN-y secretion about 2 to about 2.49 fold over background. In some embodiments, the conjugates or compositions increase IFN-y secretion about 2.5 to about 3 fold over background.

[0428] In some embodiments, the conjugates or compositions provided herein are capable of increasing IL-2 secretion from T cells, e.g., as assessed using the assay described herein in Example 7. In some embodiments, the conjugates or compositions increase IL-2 secretion about 2 to about 4 fold over background. In some embodiments, the conjugates or compositions increase IL-2 secretion about 4 to about 4.99 fold over background. In some embodiments, the conjugates or compositions increase IL-2 secretion about 5 to about 5.99 fold over background. In some embodiments, the conjugates or compositions increase IL-2 secretion about 6 to about 8 fold over background.

[0429] III.A. Payload Conjugates of the present disclosure may include a payload linked to a binding site (i.e., "Bm"). The payload may be a molecule (e.g., a small molecule, a peptide, or a nucleic acid) capable of activating an effector T cell. In some embodiments, the payload is capable of promoting the proliferation, survival, and / or migration of effector T cells, promoting the production of effector cytokines and / or cytotoxic mediators from effector T cells, and / or reducing exhaustion of effector T cells.

[0430] In some embodiments, the payload is an inhibitor of Casitas B lineage lymphoma proto-oncogene b (Cbl-b), hematopoietic progenitor kinase 1 (HPK-1), phosphoinositide-3-kinase gamma (PI3Kγ), mitogen-activated protein kinase (MAPK) pathway proteins (e.g., MEK and / or B-raf), CXCR4, or CCR5, or an agonist of Toll-like receptor 7 (TLR-7), Toll-like receptor 8 (TLR-8), or stimulator of interferon genes (STING).

[0431] In some embodiments, the payload is an inhibitor of Cbl-b. In some embodiments, the payload in the conjugates provided herein is a compound of formula (I) above, attached to a binding moiety or linker via a covalent bond. In some embodiments, the payload is selected from the group consisting of compound 146, compound 147, and compound 148. In some embodiments, the payload is NX-1607.

[0432] In some embodiments, the payload is an agonist of Toll-like receptor 7 (TLR-7) and / or Toll-like receptor 8 (TLR-8). Agonists of TLR-7 and / or TLR-8 are known in the art and include, for example, T785 (discussed in Ackerman S., et al., Nature Cancer 2:18-33 (2021) (incorporated herein by reference in its entirety)) and MEDI9197 (e.g., Huck B., et al., Angew. Chem. Int. Ed. 57:4412-4428 (2018) (incorporated herein by reference in its entirety)).

[0433] In some embodiments, the payload is an inhibitor of hematopoietic progenitor kinase 1 (HPK-1). Inhibitors of HPK-1 are known in the art and are reviewed, for example, in Linney, ID and Kaila N., Expert Opin. Ther. Pat. 31(10):893-910 (2021), which is incorporated herein by reference in its entirety.

[0434] In some embodiments, the payload is an agonist of STING. STING agonists, such as (R,R)-S2-CDA, ADU-S100, MIW815 (Aduro BioTech / Novartis) and the cyclic dinucleotide MK-1454, are known in the art and are discussed, for example, in Huck B., et al., Angew. Chem. Int. Ed. 57:4412-4428 (2018), which is incorporated herein by reference in its entirety.

[0435] In some embodiments, the payload is an inhibitor of PI3K gamma. Inhibitors of PI3K gamma, such as IPI-549, are known in the art and are discussed, for example, in Huck B., et al., Angew. Chem. Int. Ed. 57:4412-4428 (2018) (incorporated herein by reference in its entirety).

[0436] In some embodiments, the payload is an inhibitor of a mitogen-activated protein kinase (MAPK) pathway protein (e.g., MEK and / or B-raf). Inhibitors of MAPK pathway proteins, such as trametinib, cobimetinib, binimetinib, dabrafenib, vemurafenib, and encorafenib, are known in the art and are discussed, for example, in Huck B., et al., Angew. Chem. Int. Ed. 57:4412-4428 (2018), which is incorporated herein by reference in its entirety.

[0437] In some embodiments, the payload is an inhibitor of CCR4 and / or CCR5. Inhibitors of CCR4 and / or CCR5, such as BL-8040, X4P-001, LY2510924, and BMS-813160, are known in the art and are discussed, for example, in Huck B., et al., Angew. Chem. Int. Ed. 57:4412-4428 (2018), which is incorporated herein by reference in its entirety.

[0438] In some embodiments, the payload in the conjugates provided herein is a small molecule, a peptide, or a nucleotide (e.g., siRNA). In some embodiments, the payload in the conjugates provided herein is a small molecule. In some embodiments, the payload in the conjugates provided herein is a peptide. Exemplary peptide payloads are disclosed, for example, in European Publication No. EP3254701, which is incorporated herein by reference in its entirety. In some embodiments, the payload in the conjugates provided herein is a nucleotide (e.g., siRNA). Exemplary nucleotide payloads are disclosed, for example, in US Publication No. US20150313931, which is incorporated herein by reference in its entirety.

[0439] III.B. Linker The conjugates of the present disclosure may include a linker. As used herein, the term "linker" refers to any chemical moiety capable of connecting a binding moiety (i.e., "Bm") to a payload ("P").

[0440] In certain embodiments, the linker may contain a heterobifunctional group. In this disclosure, the term "heterobifunctional group" refers to a chemical moiety that connects the linker of which it is a part to the binding site. Heterobifunctional groups are characterized as having different reactive groups at either end of the chemical moiety. Attachment to the binding site may be accomplished through chemical or enzymatic conjugation, or a combination of both. Chemical conjugation involves the controlled reaction of accessible amino acid residues on the surface of the binding site with reactive handles on the heterobifunctional group. Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine ​​coupling, and coupling via engineered non-natural amino acids, where non-natural amino acid residues with the desired reactive handles are introduced onto the binding site. In enzymatic conjugation, an enzyme mediates the coupling of the linker to an accessible amino residue on the binding site. Examples of enzymatic conjugation include, but are not limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, and N-glycan engineering. Chemical and enzymatic conjugation can also be used sequentially, for example, enzymatic conjugation can also be used to introduce a unique reactive handle on the binding site that is utilized in subsequent chemical conjugation.

[0441] In some embodiments, the heterobifunctional group is [ka] (In the formula, [ka] is the point of attachment to the remainder of the linker; [ka] is the point of attachment to Bm) is selected from.

[0442] In certain embodiments, the linker "L" is non-cleavable. As used herein, the term "non-cleavable linker" refers to any chemical moiety that can link a bond to a payload in a stable covalent manner and does not fall into the category defined herein as a "cleavable linker". Thus, a non-cleavable linker is substantially resistant to acid-induced cleavage, light-induced cleavage, bioreductive cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. "Substantially resistant to cleavage" means that the chemical bond in or adjacent to the linker in at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% of the conjugate population remains non-cleavable within hours to days of treatment with any of the above-mentioned agents by acids, photolabile cleaving agents, bioreductive agents, peptidases, esterases, or chemical or physiological compounds that cleave the chemical bond (e.g., disulfide bond) in the cleavable linker. In certain embodiments, the linker is not susceptible to acid-induced cleavage, photoinduced cleavage, bioreductive cleavage, enzymatic cleavage, etc., under conditions where the payload and / or binding site may still be active. ADC catabolites generated from non-cleavable linkers contain residual amino acids from the antibody. These catabolites may exert unique and unexpected properties in the target cells to which they are delivered.

[0443] One of skill in the art will readily distinguish a non-cleavable linker from a cleavable linker.

[0444] Examples of non-cleavable linkers include SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) linkers, succinimide thioether linkers, and linkers such as [ka] (In the formula,

[0445] p is an integer from 1 to 10;

[0446] p* is an integer from 1 to 10;

[0447] Y is hydrogen and C 1 -C 6 alkyl;

[0448] [ka] is the attachment point to the payload;

[0449] [ka] is the attachment point to the binding site) These include, but are not limited to:

[0450] In some embodiments, the linker is [ka] (In the formula,

[0451] p is an integer from 1 to 10;

[0452] p* is an integer from 1 to 10;

[0453] [ka] is the attachment point to the payload;

[0454] [ka] is the attachment point to the binding site) It is.

[0455] In certain embodiments, the linker may be cleavable, hi some embodiments, the linker may be susceptible to acid-induced cleavage, photoinduced cleavage, bioreductive cleavage, enzymatic cleavage, etc., provided that the payload and / or the binding moiety may still be active.

[0456] In some embodiments, the cleavable linker can be enzymatically cleaved by a protease, peptidase, esterase, beta-glucuronidase, glycosidase, phosphodiesterase, phosphatase, pyrophosphatase, or lipase.

[0457] In some embodiments, the cleavable linker can be cleaved by a protease. Examples of proteases include, but are not limited to, cathepsin B, VAGP tetrapeptide, and the like.

[0458] In certain embodiments, the cleavable linker contains a peptide. In some embodiments, the peptide is the site of cleavage of the linker, thereby facilitating release of the drug upon exposure to intracellular proteases, e.g., lysosomal enzymes. The peptide can be designed and optimized for enzymatic cleavage by a specific enzyme, e.g., tumor-associated proteases, cathepsins B, C and D, or plasmin proteases. Examples of peptides with two amino acids include, but are not limited to, alanine-alanine (ala-ala), valine-alanine (val-ala), valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit). Examples of peptides with three amino acids include, but are not limited to, glycine-valine-citrulline (gly-val-cit), aspartic acid-valine-citrulline (asp-val-cit), alanine-alanine-asparagine (ala-ala-asn), alanine-phenylalanine-lysine (ala-phe-lys), glycine-glycine-phenylalanine (gly-gly-phe), and glycine-glycine-glycine (gly-gly-gly). Examples of peptides with four amino acids include, but are not limited to, glycine-glycine-valine-citrulline (gly-gly-val-cit) and glycine-glycine-phenylalanine-glycine (gly-gly-phe-gly). The above amino acid combinations can also exist in the reverse order (i.e., cit-val).

[0459] The peptides of the present disclosure may include L- or D-isomers of amino acid residues. The term "naturally occurring amino acid" refers to Ala, Asp, Asx, Cit, Cys, Glu, Phe, Glx, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr. "D-" designates an amino acid having a "D" (dextrorotatory) configuration, as opposed to the naturally occurring ("L-") amino acid configuration. The amino acids described herein may be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0460] In certain embodiments, the linker ("L") is [ka] (In the formula,

[0461] q is an integer from 2 to 10;

[0462] Z 1 , Z 2 , Z 3 , and Z 4 are each independently absent or a naturally occurring amino acid residue of L or D configuration, with the proviso that Z 1 , Z 2 , Z 3 , and Z 4 at least two of are amino acid residues;

[0463] [ka] is the attachment point to the payload;

[0464] [ka] is the attachment point to the binding site) is a protease-cleavable linker selected from

[0465] In certain embodiments, Z 1 , Z 2 , Z 3 , and Z 4 is independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, and glycine; with the proviso that Z is 1 , Z 2 , Z 3 , and Z 4 At least two of are amino acid residues.

[0466] In some embodiments, Z 1 is absent or glycine; Z 2 is absent or selected from L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 is selected from L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; Z 4 is selected from L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.

[0467] In some embodiments, L is [ka] (In the formula,

[0468] q is an integer from 2 to 10;

[0469] [ka] is the attachment point to the payload;

[0470] [ka] is the attachment point to the binding site) is selected from.

[0471] In some embodiments, the linker is bioreductive. Bioreductive linkers take advantage of the difference in reduction potential between intracellular compartments and plasma. The reduced glutathione present in the cytoplasm of tumor cells is up to 1000 times higher than that present in the cytoplasm of normal cells, and tumor cells also contain enzymes that can contribute to reduction in the cellular compartment. The linker keeps the conjugate intact during systemic circulation and is selectively cleaved by the high intracellular concentration of glutathione, releasing the active drug from the non-toxic prodrug at the tumor site.

[0472] In some embodiments, L is [ka] (In the formula,

[0473] q is an integer from 2 to 10;

[0474] R, R', R'', and R''' are hydrogen, C 1 -C 6 Alkoxy C 1 -C 6 Alkyl, (C 1 -C 6 ) 2 NC 1 -C 6 Alkyl, and C 1 -C 6 alkyl, or two geminal R groups together with the carbon atom to which they are attached can form a cyclobutyl or cyclopropyl ring;

[0475] [ka] is the attachment point to the payload;

[0476] [ka] is the attachment point to the binding site) is a bioreducible linker selected from

[0477] In some embodiments, L is

[0478] [ka] (In the formula,

[0479] q is an integer from 2 to 10;

[0480] R, R', R'', and R''' are hydrogen, C 1 -C 6 Alkoxy C 1 -C 6 Alkyl, (C 1 -C 6 ) 2 NC 1 -C 6 Alkyl, and C 1 -C 6 alkyl, or two geminal R groups together with the carbon atom to which they are attached can form a cyclobutyl or cyclopropyl ring;

[0481] [ka] is the attachment point to the payload;

[0482] [ka] is the attachment point to the binding site) is selected from.

[0483] In certain embodiments, L is [ka] (wherein Q* is a group that can be attached to the bonding site, [ka] is the attachment point to the payload).

[0484] In some embodiments, the present disclosure provides a linker of formula L** [ka] (wherein Q* is a group that can be attached to the bonding site, [ka] is the point of attachment to the payload). In some embodiments, a linker can be used to connect any payload to any attachment site.

[0485] In certain embodiments, the linker is acid cleavable. Acid cleavable linkers are specifically designed to remain stable at the neutral pH of the blood circulation but undergo hydrolysis in the acidic environment of the cellular compartment, releasing the cytotoxic drug.

[0486] In some embodiments, L is [ka] (In the formula,

[0487] q is an integer from 2 to 10;

[0488] [ka] is the attachment point to the payload;

[0489] [ka] is the attachment point to the binding site) is an acid cleavable linker selected from

[0490] In certain embodiments, L is a click-to-release linker, and release of the payload is chemically triggered by a tetrazine or related compound.

[0491] In some embodiments, L is [ka] (In the formula,

[0492] q is an integer from 2 to 10;

[0493] [ka] is the attachment point to the payload;

[0494] [ka] is the attachment point to the binding site) A click-to-release linker is selected from

[0495] In certain embodiments, L is a pyrophosphatase-cleavable linker.

[0496] In some embodiments, L is [ka] (In the formula,

[0497] q is an integer from 2 to 10;

[0498] [ka] is the attachment point to the payload;

[0499] [ka] is the attachment point to the binding site) is a pyrophosphatase-cleavable linker which is

[0500] In certain embodiments, L is a beta-glucoronidase cleavable linker.

[0501] In some embodiments, L is [ka] (In the formula,

[0502] q is an integer from 2 to 10;

[0503] ---- is non-existence or union;

[0504] [ka] is the attachment point to the payload;

[0505] [ka] is the attachment point to the binding site) is a beta-glucoronidase cleavable linker selected from:

[0506] In certain embodiments, L is

[0507] [ka]

[0508] (In the formula,

[0509] q is an integer from 2 to 10;

[0510] ---- is non-existence or union;

[0511] [ka] is the attachment point to the payload;

[0512] [ka] is the attachment point to the binding site) It is.

[0513] In some embodiments, the attachment point to the binding site is a cysteine, lysine, tyrosine, or glutamine in the binding site. In some embodiments, the attachment point to the binding site is a cysteine. In some embodiments, the attachment point to the binding site is a lysine. In some embodiments, the attachment point to the binding site is a tyrosine. In some embodiments, the attachment point to the binding site is a glutamine (e.g., a glutamine at heavy chain position 295 of an antibody or antigen-binding portion thereof according to EU numbering).

[0514] The cysteine ​​or lysine can be, for example, an engineered (i.e., not endogenous to the binding site) cysteine ​​or lysine for site-specific conjugation. Site-specific conjugation refers to attachment via a unique, defined site on the binding site (e.g., an antibody or antigen-binding portion thereof). Site-specific conjugation is discussed, for example, in Zhou, Qun. “Site-Specific Antibody Conjugation for ADC and Beyond.” Biomedicines vol.5(4), 64.9 Nov.2017, doi:10.3390 / biomedicines5040064, which is incorporated herein by reference in its entirety. In some embodiments, the engineered cysteine ​​is at heavy chain position S239 and / or K334 of an antibody or antigen-binding portion thereof according to EU numbering.

[0515] The attachment point cysteine ​​or lysine can be a cysteine ​​or lysine that is endogenous to the attachment site.

[0516] III.C. Binding site As demonstrated herein, the binding site (Bm) can deliver a payload, for example an effector T cell activator, to an effector T cell.

[0517] The term "binding site" as used herein refers to any molecule that recognizes and binds to a target. The binding site that can specifically bind the target on the surface of effector T cell refers to any molecule that recognizes and binds to the target present on effector T cell. The target can also be present on the surface of other cells (e.g., other T cells).

[0518] In some embodiments, the target is expressed on T cells (including effector T cells) but not on other cells that are not T cells. In some embodiments, the expression of the target on effector T cells is enhanced when compared to the expression of the target on other T cells. For example, in some embodiments, the expression of the target on effector T cells is at least 5 times more abundant on effector T cells than on regulatory T cells. For example, in some embodiments, the expression of the target on effector T cells is at least 10 times more abundant on effector T cells than on regulatory T cells. For example, in some embodiments, the expression of the target on effector T cells is at least 20 times more abundant on effector T cells than on regulatory T cells.

[0519] In some embodiments, the target is expressed on regulatory T cells and effector T cells. In some embodiments where the target is expressed on regulatory T cells and effector T cells, the expression of the target on the regulatory T cells is greater than the expression on the effector T cells. In some embodiments where the target is expressed on regulatory T cells and effector T cells, the expression of the target on the regulatory T cells is greater than the expression on the helper T cells. In some embodiments where the target is expressed on regulatory T cells and effector T cells, the expression of the target on the regulatory T cells is greater than the expression on the effector T cells and greater than the expression on the helper T cells.

[0520] In some embodiments, the target is a protein, glycoprotein, lipid, glycolipid, or carbohydrate on the surface of an effector T cell, hi some embodiments, the target is a protein or glycoprotein.

[0521] The binding moiety may also have a therapeutic effect on the effector T cell (e.g., a proliferative or migratory effect) in addition to targeting the compound(s) to a particular cell, tissue, or location. After specific binding of the binding moiety to a target on the surface of the effector T cell, the effector T cell may internalize a conjugate that includes the binding moiety, a payload bound to the binding moiety, and / or the target.

[0522] In certain embodiments, the binding site may contain, or be engineered to contain, at least one chemically reactive group, such as a carboxylic acid, an amine, a thiol, or a chemically reactive amino acid site or side chain.

[0523] In some embodiments, the binding site (Bm) may be a peptide or protein that binds to a cell surface receptor or antigen.

[0524] In certain embodiments, the group "Bm" can be an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof. Exemplary sequences of antibodies or antigen-binding fragments thereof are provided herein in Tables 2-4. The antibody or antigen-binding fragment thereof can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD and any subclass thereof. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG1 or IgG4 antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG1 antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG4 antibody or antigen-binding fragment thereof.

[0525] Useful monoclonal antibodies or antigen-binding fragments thereof include, but are not limited to, human monoclonal antibodies or antigen-binding fragments thereof, humanized monoclonal antibodies or antigen-binding fragments thereof, or chimeric human-mouse (or other species) monoclonal antibodies or antigen-binding fragments thereof. Human monoclonal antibodies or antigen-binding fragments thereof may be produced by any of a number of techniques known in the art.

[0526] Useful antigen-binding fragments of antibodies include F(ab')2 fragments which contain the variable region, the light chain constant region and the CH1 domain of the heavy chain and can be generated by pepsin digestion of the antibody molecule, and Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragment. Other useful antibodies and antigen-binding fragments thereof are heavy and light chain dimers, or any minimal fragment thereof, such as Fv or single chain antibodies (SCAs), or any other molecule with the same specificity as an antibody.

[0527] Also useful are recombinant antibodies and antigen-binding fragments thereof, such as chimeric and humanized monoclonal antibodies and antigen-binding fragments thereof, containing both human and non-human portions, which can be produced using standard recombinant DNA techniques.

[0528] Fully human antibodies or antigen-binding fragments thereof can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but that can express human heavy and light chain genes. The transgenic mice are immunized in the normal manner with a selected antigen, e.g., all or a portion of a polypeptide of the present disclosure. Monoclonal antibodies or antigen-binding fragments thereof directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B-cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such technology, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies or antigen-binding fragments thereof. For a review of this technology for producing human antibodies or antigen-binding fragments thereof, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). Other human antibodies can be obtained commercially from, for example, Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0529] Fully human antibodies or antigen-binding fragments thereof that recognize a selected epitope can be generated using a technique called "guided selection." In this approach, a selected non-human monoclonal antibody or antigen-binding fragment thereof, e.g., a mouse antibody or antigen-binding fragment thereof, is used to guide the selection of a fully human antibody or antigen-binding fragment thereof that recognizes the same epitope. Human antibodies or antigen-binding fragments thereof can also be generated using a variety of techniques known in the art, including phage display libraries.

[0530] The antibody or antigen-binding fragment thereof may be a fusion protein of the antibody or antigen-binding fragment thereof, e.g., in which the antibody or fragment is fused, either N- or C-terminus, via a covalent bond (e.g., a peptide bond) to the amino acid sequence of another protein (or a portion thereof, e.g., at least a 10, 20 or 50 amino acid portion of the protein) that is not an antibody or fragment. The antibody or antigen-binding fragment thereof may be covalently linked to the other protein at the N-terminus of the constant domain.

[0531] Antibodies and antigen-binding fragments thereof include analogs and derivatives that have been modified, i.e., modified by the covalent attachment of any type of molecule, so long as such covalent attachment enables the antibody or antigen-binding fragment to retain its antigen-binding immunospecificity. For example, and without limitation, analogs and derivatives of antibodies and antigen-binding fragments thereof include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to cellular antibody units or other proteins, etc. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Analogs or derivatives may also contain one or more unnatural amino acids.

[0532] The antibody or antigen-binding fragment thereof in the conjugate may include an antibody or antigen-binding fragment thereof having a modification (e.g., substitution, deletion, or addition) in an amino acid residue that interacts with an Fc receptor. In particular, the antibody or antigen-binding fragment thereof includes an antibody or antigen-binding fragment thereof having a modification in an amino acid residue identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor. An antibody or antigen-binding fragment thereof capable of specifically binding to a target on the surface of an effector T cell may be obtained commercially or produced by any method known to those skilled in the art, such as, for example, chemical synthesis or recombinant expression techniques. A nucleotide sequence encoding an antibody or antigen-binding fragment thereof capable of specifically binding to a target on the surface of an effector T cell may be obtained, for example, from the GenBank database or a database like it, literature publications, or by conventional cloning and sequencing.

[0533] In certain embodiments, the antibody or antigen-binding fragment of the conjugate can be a monoclonal antibody or antigen-binding fragment thereof, such as a murine monoclonal antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof can be, for example, a Fab fragment.

[0534] As provided herein, a binding site (e.g., an antibody or an antigen-binding fragment thereof) that binds to a target (antigen) on the surface of an effector T cell can be conjugated to a payload. By way of example, such a binding site (Bm), e.g., an antibody or an antigen-binding fragment thereof, can bind to PD1, CD25 (IL2RA), or CD7.

[0535] In some embodiments, a binding site (Bm), e.g., an antibody or antigen-binding fragment thereof, may specifically bind CD25 (also known as interleukin-2 receptor subunit alpha (IL2RA)). Such binding sites are also referred to herein as "anti-IL2RA binding sites," "IL2RA binding sites," "anti-CD25 binding sites," or "CD25 binding sites," e.g., anti-IL2RA antibodies or antigen-binding fragments thereof, IL2RA antibodies or antigen-binding fragments thereof, anti-CD25 antibodies or antigen-binding fragments thereof, or CD25 antibodies or antigen-binding fragments thereof. Exemplary sequences of anti-IL2RA or anti-CD25 antibodies or antigen-binding fragments thereof are provided in Table 2. In some embodiments, an anti-IL2RA or anti-CD25 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDR sequences (e.g., CDRs defined by Kabat, Chothia, or AbM) of SEQ ID NOs: 4 and 5, respectively (e.g., amino acids 31-35, 50-66, and 99-111 of SEQ ID NO: 4 and amino acids 24-34, 50-56, and 89-97 of SEQ ID NO: 5). In some embodiments, an anti-IL2RA or anti-CD25 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and / or a variable light chain comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, an anti-IL2RA or anti-CD25 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDR sequences (e.g., CDRs defined by Kabat, Chothia, or AbM) of SEQ ID NOs: 6 and 7, respectively (e.g., amino acids 31-35, 50-65, and 98-108 of SEQ ID NO: 6 and amino acids 24-33, 49-55, and 88-96 of SEQ ID NO: 7). In some embodiments, an anti-IL2RA or anti-CD25 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 6 and / or a variable light chain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-IL2RA or anti-CD25 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDR sequences of SEQ ID NOs: 8 and 9, respectively (e.g., CDRs defined by Kabat, Chothia, or AbM) (e.g., amino acids 31-35, 50-65, and 98-108 of SEQ ID NO: 8 and amino acids 24-33, 49-55, and 88-96 of SEQ ID NO: 9).In some embodiments, an anti-IL2RA or anti-CD25 antibody, or antigen-binding fragment thereof, comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:8 and / or a variable light chain comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, an anti-IL2RA or anti-CD25 antibody, or antigen-binding fragment thereof, comprises heavy and light chain CDR sequences (e.g., CDRs defined by Kabat, Chothia, or AbM) of SEQ ID NOs:16 and 17, respectively (e.g., amino acids 31-35, 50-66, and 99-103 of SEQ ID NO:16 and amino acids 24-39, 55-61, and 94-102 of SEQ ID NO:17). In some embodiments, an anti-IL2RA or anti-CD25 antibody, or antigen-binding fragment thereof, comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:16 and / or a variable light chain comprising the amino acid sequence of SEQ ID NO:17. [Table 2]

[0536] In some embodiments, a binding site (Bm), e.g., an antibody or antigen-binding fragment thereof, can specifically bind PD1. Such a binding site is also referred to herein as an "anti-PD1 binding site" or a "PD1 binding site", e.g., an anti-PD1 antibody or antigen-binding fragment thereof or a PD1 antibody or antigen-binding fragment thereof. Exemplary sequences of anti-PD1 antibodies or antigen-binding fragments thereof are provided in Table 3. In some embodiments, an anti-PD1 antibody or antigen-binding fragment thereof comprises heavy and light chain CDR sequences (e.g., CDRs defined in Kabat, Chothia, or AbM) of SEQ ID NOs: 10 and 11, respectively (e.g., amino acids 31-35, 50-66, and 99-109 of SEQ ID NO: 10 and amino acids 24-38, 54-60, and 93-101 of SEQ ID NO: 11). In some embodiments, an anti-PD1 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and / or a variable light chain comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-PD1 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDR sequences (e.g., CDRs defined by Kabat, Chothia, or AbM) of SEQ ID NOs: 12 and 13, respectively (e.g., amino acids 31-35, 50-66, and 99-102 of SEQ ID NO: 12 and amino acids 24-34, 50-56, and 89-97 of SEQ ID NO: 13). In some embodiments, the anti-PD1 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and / or a variable light chain comprising the amino acid sequence of SEQ ID NO:13. In some embodiments, the anti-PD1 antibody or antigen-binding fragment thereof comprises heavy chain and light chain CDR sequences of SEQ ID NOs: 18 and 19, respectively (e.g., CDRs defined by Kabat, Chothia, or AbM) (e.g., amino acids 31-35, 50-68, and 101-110 of SEQ ID NO: 18 and amino acids 24-33, 49-55, and 88-100 of SEQ ID NO: 19). In some embodiments, the anti-PD1 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and / or a variable light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-PD1 antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 26 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 27. [Table 3]

[0537] In some embodiments, a binding site (Bm), e.g., an antibody or antigen-binding fragment thereof, can specifically bind CD7. Such a binding site is also referred to herein as an "anti-CD7 binding site" or a "CD7 binding site", e.g., an anti-CD7 antibody or antigen-binding fragment thereof or a CD7 antibody or antigen-binding fragment thereof. Exemplary sequences of anti-CD7 antibodies or antigen-binding fragments thereof are provided in Table 4. In some embodiments, an anti-CD7 antibody or antigen-binding fragment thereof comprises heavy and light chain CDR sequences (e.g., CDRs defined in Kabat, Chothia, or AbM) of SEQ ID NOs: 14 and 15, respectively (e.g., amino acids 31-35, 50-66, and 99-112 of SEQ ID NO: 14 and amino acids 23-36, 52-58, and 91-99 of SEQ ID NO: 15). In some embodiments, an anti-CD7 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and / or a variable light chain comprising the amino acid sequence of SEQ ID NO: 15. [Table 4]

[0538] In some embodiments, the antibody or antigen-binding portion thereof comprises a constant region. The linker may be attached to an amino acid in the constant region. In some embodiments, the antibody or antigen-binding portion thereof comprises a CH1 domain. The linker may be attached to an amino acid in the CH1 domain. In some embodiments, the antibody or antigen-binding portion thereof comprises a CH2 domain. The linker may be attached to an amino acid in the CH2 domain. In some embodiments, the antibody or antigen-binding portion thereof comprises a CH3 domain. The linker may be attached to an amino acid in the CH3 domain. In some embodiments, the antibody or antigen-binding portion thereof comprises a CL domain. The linker may be attached to an amino acid in the CL domain.

[0539] In some embodiments, the constant region, CH1 domain, CH2 domain, CH3 domain, or CL domain is an engineered constant region, CH1 domain, CH2 domain, CH3 domain, or CL domain.

[0540] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain constant region, e.g., a human heavy chain constant region. The linker may be attached to an amino acid in the heavy chain constant region, e.g., a human heavy chain constant region. In some embodiments, the antibody or antigen-binding portion thereof comprises an IgG heavy chain constant region, e.g., a human IgG heavy chain constant region. The linker may be attached to an amino acid in the IgG heavy chain constant region, e.g., a human IgG heavy chain constant region. In some embodiments, the antibody or antigen-binding portion thereof comprises an IgG1 heavy chain constant region, e.g., a human IgG1 heavy chain constant region. The linker may be attached to an amino acid in the IgG1 heavy chain constant region, e.g., a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding portion thereof comprises an IgG4 heavy chain constant region. The linker may be attached to an amino acid in the IgG4 heavy chain constant region, e.g., a human IgG4 heavy chain constant region.

[0541] In some aspects, the antibody or antigen-binding portion thereof comprises a light chain constant region, e.g., a human light chain constant region. The linker may be attached to an amino acid in the light chain constant region, e.g., a human light chain constant region. In some aspects, the antibody or antigen-binding portion thereof comprises a kappa light chain constant region, e.g., a human kappa light chain constant region. The linker may be attached to an amino acid in the kappa light chain constant region, e.g., a human kappa light chain constant region. In some aspects, the antibody or antigen-binding portion thereof comprises a gamma light chain constant region, e.g., a human gamma light chain constant region. The linker may be attached to an amino acid in the gamma light chain constant region, e.g., a human gamma light chain constant region.

[0542] In some embodiments, the antibody or antigen-binding portion thereof comprises an engineered cysteine ​​at heavy chain position S239 according to EU numbering. A linker may be attached to S239C. In some embodiments, the antibody or antigen-binding portion thereof comprises an engineered cysteine ​​at heavy chain position K334 according to EU numbering. A linker may be attached to K334C.

[0543] Thus, the antibody or antigen-binding portion thereof can comprise a heavy chain constant region of any of SEQ ID NOs:20-24. IgG1 heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 20) IgG1 N297A constant region (CH1-hinge-CH2-CH3) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21) IgG4 S228P constant region (CH1-hinge-CH2-CH3) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 22) IgG1 heavy chain constant region S239C ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 23) IgG1 heavy chain constant region K334C ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIECTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 24)

[0544] In some embodiments, the antibody or antigen-binding portion thereof may comprise the heavy chain constant region of SEQ ID NO:28. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 28)

[0545] In some embodiments, the linker may be attached to heavy chain Q295 of the antibody or antigen-binding portion thereof according to EU numbering.

[0546] In the present disclosure, the group "Bm" may be conjugated to multiple compounds capable of activating effector T cells. In some embodiments, "Bm" may be conjugated to 1-10 compounds. In some embodiments, "Bm" may be conjugated to 1-9 compounds. In some embodiments, "Bm" may be conjugated to 1-8 compounds. In some embodiments, "Bm" may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compounds. In some embodiments, "Bm" may be conjugated to 7 or 8 compounds. In some embodiments, "Bm" is conjugated to 5 compounds. In some embodiments, "Bm" is conjugated to 6 compounds. In some embodiments, "Bm" is conjugated to 7 compounds. In some embodiments, "Bm" is conjugated to 8 compounds. In some embodiments, "Bm" is conjugated to 9 compounds.

[0547] IV. Compositions and Methods of Use The conjugates and / or compounds described herein may be in the form of a pharma- ceutically or pharma-ceutically acceptable salt. In some embodiments, such salts are derived from inorganic or organic acids or bases.

[0548] Examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate salts.

[0549] Examples of suitable base addition salts include ammonium salts; alkali metal salts, e.g., sodium and potassium salts; alkaline earth metal salts, e.g., calcium and magnesium salts; salts with organic bases, e.g., dicyclohexylamine salts, N-methyl-D-glucamine; and salts with amino acids, e.g., arginine, lysine, and the like.

[0550] For example, Berge lists the following FDA approved commercially available salts of the anions acetate, besylate (benzenesulfonate), benzoate, bicarbonate, bitartrate, bromide, calcium edetate (ethylenediaminetetraacetate), camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate (ethylenediaminetetraacetate), edisylate (1,2 ethanedisulfonate), estolate (lauryl sulfate), esylate (ethanesulfonate), fumarate, gluceptate (glucoheptonate), gluconate, glutamate, glycolyl arsanilate (glycolamidophenylarsonate), hexylresorcinate, hydrabamine (N,N'-di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, ise. The following compounds may be used: thionate (2-hydroxyethanesulfonate), lactate, lactobionate, malate, maleate, mandelate, mesylate (methanesulfonate), methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate (2-naphthalenesulfonate), nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theoclate (8-chlorotheophyllinate), and triethiodide; the organic cations benzathine (N,N'-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine; and the metal cations aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.

[0551] Berge lists the following additional commercially (outside the U.S.) salts that are not approved by the FDA: anionic adipate, alginate, aminosalicylate, anhydrous methylene citrate, arecoline, aspartate, bisulfate, butyl bromide, camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydroiodide, methylene bis(salicylate), napadisilate (1,5-naphthalenedisulfonate dihydrogen phosphate), and 1,2-dihydro-1,2-tetrahydrofuran. sulfonates), oxalates, pectinates, persulfates, phenylethylbarbiturates, picrates, propionates, thiocyanates, tosylates, and undecanoates; the organic cations benethamine (N-benzylphenethylamine), clemizole (1-p-chlorobenzyl-2-pyrrolidine-1'-ylmethylbenzimidazole), diethylamine, piperazine, and tromethamine (tris(hydroxymethyl)aminomethane); and the metal cations barium and bismuth.

[0552] Pharmaceutical compositions containing the conjugates described herein may also contain suitable carriers, excipients, and adjuvants, which may vary depending on the mode of administration.

[0553] In some embodiments, the pharmaceutical composition can be formulated as a suitable parenteral dosage form. The formulation can be prepared by various methods known in the art. The pharmaceutical composition can be administered directly into the bloodstream. Suitable means for parenteral administration include intravenous administration.

[0554] Parenteral compositions are typically aqueous solutions which may contain excipients such as salts, carbohydrates, and buffers. However, the compositions can also be formulated as sterile non-aqueous solutions or dry forms for use in conjunction with a suitable vehicle, such as sterile pyrogen-free water.

[0555] The preparation of parenteral compositions under sterile conditions, for example, by lyophilization, may be readily accomplished using standard techniques well known to those skilled in the art.

[0556] Parenteral preparations may be mixed with other suitable pharma- ceutically acceptable excipients for use in parenteral dosage forms, such as, but not limited to, preservatives.

[0557] The compounds and conjugates described herein may be used to treat a variety of conditions that would benefit from an increased immune response, including but not limited to cancer. Certain compounds and conjugates of the present disclosure may be excellent in terms of efficacy expression, pharmacokinetics (e.g., absorption, distribution, metabolism, excretion), solubility (e.g., aqueous solubility), interactions with other drugs (e.g., drug-metabolizing enzyme inhibitory effects), safety (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central toxicity) and / or stability (e.g., chemical stability, stability against enzymes), and may be useful as pharmaceuticals. Thus, provided herein are compounds and conjugates of the present disclosure for use in the preparation of pharmaceuticals, for example, pharmaceuticals for the treatment of conditions that would benefit from an increased immune response, including but not limited to cancer, as well as compositions comprising the same. Thus, provided herein are compounds and conjugates of the present disclosure for use in treating conditions that would benefit from an increased immune response, including but not limited to cancer, as well as compositions comprising the same.

[0558] The compounds and conjugates of the present disclosure may be used as pharmaceuticals, such as drugs for the prevention or treatment of diseases, e.g., cancer. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is an unresectable or metastatic microsatellite instability-high (MSI-H) solid tumor, mismatch repair deficient (dMMR) solid tumor, melanoma, non-small cell lung cancer (NSCLC), malignant pleural mesothelioma, renal cell carcinoma (RCC), classical Hodgkin's lymphoma (cHL), squamous cell carcinoma of the head and neck (SCCHN), urothelial carcinoma, colorectal cancer, hepatocellular carcinoma (HCC), esophageal cancer, gastric cancer, esophago-gastric junction cancer, esophageal cancer, esophageal adenocarcinoma, and squamous cell carcinoma of the head and neck. (HNSCC), primary mediastinal large B-cell lymphoma (PMBCL), microsatellite instability-high or mismatch repair deficient cancer, microsatellite instability-high or mismatch repair deficient colorectal cancer (CRC), gastric cancer, cervical cancer, Merkel cell carcinoma (MCC), endometrial cancer, tumor mutation burden-high (TMB-H) cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), and basal cell carcinoma (BCC).

[0559] In some embodiments, the cancer is resistant to anti-PD1 therapy, which can be, for example, nivolumab, pembrolizumab, and / or cemiplimab.

[0560] In some embodiments, the cancer is refractory to anti-PD1 therapy, which can be, for example, nivolumab, pembrolizumab, and / or cemiplimab.

[0561] In some embodiments, the cancer is sensitive to anti-PD1 therapy, which can be, for example, nivolumab, pembrolizumab, and / or cemiplimab.

[0562] In some embodiments, the cancer is resistant to an anti-PDL1 therapy, which can be, for example, durvalumab, atezolizumab, and / or avelumab.

[0563] In some embodiments, the cancer is refractory to an anti-PDL1 therapy, which can be, for example, durvalumab, atezolizumab, and / or avelumab.

[0564] In some embodiments, the cancer is susceptible to an anti-PDL1 therapy, which can be, for example, durvalumab, atezolizumab, and / or avelumab.

[0565] In some embodiments, the compounds or conjugates of the present disclosure may be used in combination with standard care therapy, e.g., one or more therapeutic agents (e.g., anti-cancer agents and / or immunomodulatory agents). Thus, in certain embodiments, the methods of treating tumors disclosed herein include administering the compounds or conjugates of the present disclosure in combination with one or more additional therapeutic agents. In some embodiments, the compounds or conjugates of the present disclosure may be used in combination with one or more anti-cancer agents, such that multiple elements of the immune pathway may be targeted. In some embodiments, the anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling through a specific immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that may be used in the present methods include CTLA-4 antagonists (e.g., anti-CTLA-4 antibodies or antigen-binding fragments thereof), PD1 antagonists (e.g., anti-PD1 antibodies or antigen-binding fragments thereof, anti-PD-L1 antibodies or antigen-binding fragments thereof), TIM-3 antagonists (e.g., anti-TIM-3 antibodies or antigen-binding fragments thereof), or combinations thereof. Additional examples of immune checkpoint inhibitors include T cell immunoglobulin and ITIM domain (TIGIT) antagonists, V-domain Ig inhibitor of T cell activation (VISTA) antagonists, B- and T-cell lymphoattenuator (BTLA) antagonists, and lymphocyte-activation gene-3 (LAG-3) antagonists.

[0566] In some embodiments, the compound or conjugate of the present disclosure is administered to a subject before or after administration of an additional therapeutic agent. In other embodiments, the compound or conjugate of the present disclosure is administered to a subject simultaneously with an additional therapeutic agent. In certain embodiments, the compound or conjugate of the present disclosure and the additional therapeutic agent can be administered simultaneously as a single composition in a pharma- ceutically acceptable carrier. In other embodiments, the compound or conjugate of the present disclosure and the additional therapeutic agent are administered simultaneously as separate compositions.

[0567] The compounds and conjugates of the present disclosure may be used as pharmaceuticals, such as drugs for the prevention or treatment of additional conditions that would benefit from an increased immune response, including, for example, infectious diseases (e.g., viral, bacterial, and / or parasitic infections), immunosuppressive diseases or disorders, and multiple sclerosis.

[0568] In some embodiments, the subject that can be treated with the compound conjugates of the present disclosure is a non-human animal, such as a rat or a mouse. In some embodiments, the subject that can be treated is a human.

[0569] In some embodiments, the compounds or conjugates of the present disclosure can be used to increase immune cell activity (e.g., natural killer (NK) cell activity or T cell activity, including effector T cell activity), for example, by contacting immune cells with the compounds or conjugates of the present disclosure. The contacting can be in vitro or in vivo. In embodiments where the contacting is in vivo, the contacting can be in a subject, for example, a human subject with cancer or another condition that would benefit from an increased immune response. The increased immune cell activity can treat cancer or another condition that would benefit from an increased immune response.

[0570] In some embodiments, the compounds or conjugates of the present disclosure can be used to increase immune cell (e.g., NK cell or T cell, including effector T cell) proliferation, for example, by contacting the immune cell with a compound or conjugate of the present disclosure. The contacting can be in vitro or in vivo. In embodiments where the contacting is in vivo, the contacting can be in a subject, for example, a human subject with cancer or another condition that would benefit from an increased immune response. The increased immune cell proliferation can treat cancer or another condition that would benefit from an increased immune response.

[0571] In some embodiments, the compounds or conjugates of the present disclosure can be used to increase the migration of immune cells (e.g., NK cells or T cells, including effector T cells) to tumor cells, for example, by contacting the immune cells with the compounds or conjugates of the present disclosure. The contacting can be in vitro or in vivo. In embodiments where the contacting is in vivo, the contacting can be in a subject, for example, a human subject with cancer. The increased immune cell migration can treat cancer.

[0572] In some embodiments, the compounds or conjugates of the present disclosure can be used to reduce exhaustion of immune cells (e.g., NK cells or T cells, including effector T cells), for example, by contacting the immune cells with the compounds or conjugates of the present disclosure. The contacting can be in vitro or in vivo. In embodiments where the contacting is in vivo, the contacting can be in a subject, for example, a human subject with cancer or another condition that would benefit from an increased immune response. The reduced immune cell exhaustion can treat cancer or another condition that would benefit from an increased immune response.

[0573] In some embodiments, the compounds or conjugates of the present disclosure can be used to increase the secretion of IFN-y or IL-2 from immune cells (e.g., NK cells or T cells, including effector T cells), for example, by contacting the immune cells with the compounds or conjugates of the present disclosure. The contacting can be in vitro or in vivo. In embodiments where the contacting is in vivo, the contacting can be in a subject, for example, a human subject with cancer or another condition that would benefit from an increased immune response. The increased secretion of IFN-y or IL-2 can treat cancer or another condition that would benefit from an increased immune response.

[0574] In some of the above embodiments, the immune cell is an effector T cell.Thus, in some embodiments, the compound or conjugate of the present disclosure can be used to increase effector T cell activity, for example, by contacting effector T cell with the compound or conjugate of the present disclosure.The contacting can be in vitro or in vivo.In the embodiment where the contacting is in vivo, the contacting can be in a subject, for example, a human subject with cancer.The increased effector T cell activity can treat cancer.

[0575] In some embodiments, the disclosed compounds or conjugates can be used to increase effector T cell proliferation, for example, by contacting effector T cells with the disclosed compounds or conjugates. The contacting can be in vitro or in vivo. In embodiments where the contacting is in vivo, the contacting can be in a subject, for example, a human subject with cancer. The increased effector T cell proliferation can treat cancer.

[0576] In some embodiments, the disclosed compounds or conjugates can be used to increase the migration of effector T cells to tumor cells, for example, by contacting effector T cells with the disclosed compounds or conjugates. The contacting can be in vitro or in vivo. In embodiments where the contacting is in vivo, the contacting can be in a subject, for example, a human subject with cancer. The increased effector T cell migration can treat cancer.

[0577] In some embodiments, the compounds or conjugates of the present disclosure can be used to reduce effector T cell exhaustion, for example, by contacting effector T cells with the compounds or conjugates of the present disclosure.The contacting can be in vitro or in vivo.In embodiments where the contacting is in vivo, the contacting can be in a subject, for example, a human subject with cancer.The reduced effector T cell exhaustion can treat cancer.

[0578] In some embodiments, the conjugates of the present disclosure can be used to deliver a payload to an effector T cell that can activate the effector T cell, for example, by contacting the effector T cell with the conjugates of the present disclosure. The contacting can be in vitro or in vivo. In embodiments where the contacting is in vivo, the contacting can be in a subject, for example, a human subject with cancer. The delivery of the payload to the effector T cell can treat cancer.

[0579] V. Methods for Preparing Conjugates The present disclosure further provides a method of preparing a conjugate, the process comprising reacting a binding site with a payload or payload-linker precursor as described herein. As used herein, the term "linker precursor" refers to a compound that, when reacted with a binding site as described herein, connects the binding site to a payload.

[0580] In certain embodiments, the linker precursor comprises:

[0581] [ka] (In the formula,

[0582] p is an integer from 1 to 10;

[0583] p* is an integer from 1 to 10;

[0584] [ka] is the attachment point to the payload) may be selected from:

[0585] In certain embodiments, the linker precursor comprises: [ka] [ka] (In the formula,

[0586] q is an integer from 2 to 10;

[0587] Z 1 , Z 2 , Z 3 , and Z 4 are each independently absent or a naturally occurring amino acid residue of L or D configuration, with the proviso that Z 1 , Z 2 , Z 3 , and Z 4 at least two of are amino acid residues;

[0588] [ka] is the attachment point to the payload) may be selected from:

[0589] In certain embodiments, Z 1 , Z 2 , Z 3 , and Z 4 is independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, and glycine; with the proviso that Z is 1 , Z 2 , Z 3 , and Z 4 At least two of are amino acid residues.

[0590] In some embodiments, Z 1 is absent or glycine; Z 2 is absent or selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; Z 4 is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.

[0591] In some aspects, the linker precursor is [ka] (In the formula,

[0592] q is an integer from 2 to 10;

[0593] [ka] is the attachment point to the payload) may be selected from:

[0594] In certain embodiments, the linker precursor comprises: [ka] (In the formula,

[0595] q is an integer from 2 to 10;

[0596] R, R', R'', and R''' are hydrogen, C 1 -C 6 Alkoxy C 1 -C 6 Alkyl, (C 1 -C 6 ) 2 NC 1 -C 6 Alkyl, and C 1 -C 6 alkyl, or two geminal R groups together with the carbon atom to which they are attached can form a cyclobutyl or cyclopropyl ring;

[0597] [ka] is the attachment point to the payload) may be selected from:

[0598] In certain embodiments, the linker precursor comprises: [ka] (In the formula,

[0599] q is an integer from 2 to 10;

[0600] [ka] is the attachment point to the payload) may be selected from:

[0601] In certain embodiments, the linker precursor comprises: [ka] (In the formula,

[0602] q is an integer from 2 to 10;

[0603] [ka] is the attachment point to the payload) may be selected from:

[0604] In some aspects, the linker precursor is [ka] (In the formula,

[0605] q is an integer from 2 to 10;

[0606] [ka] is the attachment point to the payload) may be selected from:

[0607] In some aspects, the linker precursor is [ka] (In the formula,

[0608] q is an integer from 2 to 10;

[0609] [ka] is the attachment point to the payload) It could be.

[0610] In certain embodiments, the linker precursor comprises: [ka] (In the formula,

[0611] q is an integer from 2 to 10;

[0612] ---- is non-existence or union;

[0613] [ka] is the attachment point to the payload) may be selected from:

[0614] In some aspects, the linker precursor is [ka] (In the formula,

[0615] q is an integer from 2 to 10;

[0616] ---- is non-existence or union;

[0617] [ka] is the attachment point to the payload) It could be.

[0618] In some embodiments, the binding site is pretreated prior to reaction with the payload or payload-linker precursor. In certain embodiments, the payload or payload-linker precursor is reacted with a binding site comprising an antibody or an antigen-binding portion thereof. In embodiments where the binding site is an antibody, the antibody may be pretreated to reduce interchain disulfides prior to reaction with the payload or payload-linker precursor.

[0619] The embodiments described herein will be further described with reference to the examples shown below.These examples are provided for illustrative purposes only, and the embodiments described herein should not be interpreted as being limited to these examples in any way.Rather, the embodiments should be interpreted to include any variations that become apparent as a result of the teachings provided herein. EXAMPLES

[0620] General Synthetic Methods and Intermediates The compounds of the present disclosure can be prepared by those skilled in the art in view of this disclosure and knowledge in the art and / or by reference to the schemes and synthetic examples shown below. Exemplary synthetic routes are shown in the schemes and examples below. It should be understood that variables (e.g., "R" groups) appearing in the schemes and examples below are to be interpreted independently of their appearance elsewhere in this specification. Those skilled in the art will readily understand how the schemes and examples shown below describe the preparation of the compounds described herein.

[0621] Abbreviations used in the schemes generally follow conventions used in the art. Chemical abbreviations used in the specification and examples are defined as follows:

[0622] "DMF" N,N-dimethylformamide; "DIBAL-H" or "DIBAl-H" diisobutylaluminum hydride; "DCM" dichloromethane; "Ph" phenyl; "DEAD" diethyl azodicarboxylate; "NMO" N-methylmorpholine N-oxide; "THF" tetrahydrofuran; "t-Bu" tert-butyl; "HATU" 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate or N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide; "DIEA", "DIPEA", and "iPrNEt 2 "Diisopropylethylamine;"PE"Petroleum ether;"EA" or "EtOAc"Ethyl acetate;"Ac"Acetyl;"BPO"Benzoyl peroxide;"AIBN"Azobisisobutyronitrile;"o / n"Overnight;"Room temperature" or"rt" or"RT"Room temperature or hold time (context indicates);"IBX"2-Iodoxybenzoic acid;"STAB"Sodium triacetoxyborohydride;"Me"Methyl;"NBS"N-Bromosuccinimide;"dtbbpy"4,4'-Di-tert-butyl-2,2'-bipyridyl;"TFA"Trifluoroacetic acid;"DMA"N,N-Dimethylacetamide;"Et 3 "N" and "TEA" trimethylamine; "Ts" p-toluenesulfonyl; "Trt" triphenylmethyl; "TIPS" triisopropylsilyl; "Et" ethyl; "LiHMDS" lithium hexamethyldisilazide; "MeCN" or "ACN" acetonitrile; "TMS" trimethylsilyl; "NIS" N-iodosuccinimide; "BOC" or "Boc" tert-butoxycarbonyl; "DMSO" dimethylsulfoxide; and "HOBt" or "HOBT" 1-hydroxybenzotriazole hydrate, "h" hours; and "min" minutes.

[0623] Example 1: Preparation of Cbl-b inhibitors Synthesis of INT3 [ka] Step 1: Synthesis of Compound 2 To a stirred mixture of 2,4,6-trichloropyridine (compound 1, 100 g, 548.15 mmol, 1 equiv.) and dimethyl malonate (86.90 g, 657.78 mmol, 1.2 equiv.) in DMF (1000 mL) was added Cs 2 CO 3 (535.80 g, 1644.46 mmol, 3 equiv., powder) was added in portions at room temperature under air atmosphere. The resulting mixture was stirred at room temperature under air atmosphere for 2 days. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was washed with EtOAc (3×500 mL). The combined organic layers were washed with water (5×500 mL) and anhydrous Na 2 SO 4 The mixture was dried at 40° C. for 1 hour. After filtration, the filtrate was concentrated under reduced pressure. The resulting mixture was concentrated under vacuum to provide 1,3-dimethyl 2-(2,6-dichloropyridin-4-yl)propanedioate (compound 2, 95 g, 62%) as a yellow oil. LCMS: (ES, m / s): 278,280 [M+H] + .

[0624] Step 2: Synthesis of Compound 3 1,3-Dimethyl 2-(2,6-dichloropyridin-4-yl)propanedioate (compound 2, 95 g, 341.61 mmol, 1 equiv.) in DMF (950 mL) and K 2 CO 3 To a stirred mixture of (141.64 g, 1024.84 mmol, 3 equiv.) was added dropwise allyl bromide (82.66 g, 683.23 mmol, 2 equiv.) at 0° C. under air atmosphere. The resulting mixture was stirred overnight at 0° C. under air atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was washed with EtOAc (3×400 mL). The combined organic layers were washed with water (4×400 mL) and diluted with anhydrous Na 2 SO 4After filtration, the filtrate was concentrated under reduced pressure. This gave 1,3-dimethyl 2-(2,6-dichloropyridin-4-yl)-2-(prop-2-en-1-yl)propanedioate (compound 3, 95 g, 76%) as a yellow oil. LCMS: (ES.m / z): 318,320 [M+1] + .

[0625] Step 3. Synthesis of compound 4 To a stirred solution of 1,3-dimethyl 2-(2,6-dichloropyridin-4-yl)-2-(prop-2-en-1-yl)propanedioate (compound 3, 70 g, 220.02 mmol, 1.00 equiv.) in DCM (700 mL) was added DIBAl-H (880.09 mL, 1N in DCM, 880.09 mmol, 4 equiv.) dropwise at −40° C. under nitrogen atmosphere. The resulting mixture was stirred for 1 h at −40° C. under nitrogen atmosphere. LCMS indicated complete reaction. The reaction was quenched by addition of MeOH (20 mL) at room temperature. The resulting mixture was filtered and the filter cake was washed with EtOAc (3×50 mL). The filtrate was concentrated under reduced pressure to provide 2-(2,6-dichloropyridin-4-yl)-2-(prop-2-en-1-yl)propane-1,3-diol (compound 4, 50 g, 69%) as a yellow oil. LCMS (ESI, ms): 262, 264 [M+H] +

[0626] Step 4. Synthesis of compound 5 2-(2,6-dichloropyridin-4-yl)-2-(prop-2-en-1-yl)propane-1,3-diol (compound 4, 70 g, 267.04 mmol, 1 equiv.) and PPh 3 (140.09 g, 534.08 mmol, 2 equiv.) was added in portions to a stirred mixture at 0 °C with N 2 The resulting mixture was incubated at 0 °C for 10 min under N 2Stirred under atmosphere. To the above mixture was added ziram (122.49 g, 400.56 mmol, 1.5 eq) and DEAD (93.01 g, 534.08 mmol, 2 eq) dropwise over 30 min at 0° C. The resulting mixture was further stirred at room temperature overnight. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was washed with EtOAc (3×100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (5:1) to provide 2,6-dichloro-4-[3-(prop-2-en-1-yl)oxetan-3-yl]pyridine (compound 5, 37 g, 50%) as a yellow oil. LCMS: (ES.m / z): 244, 246 [M+1] + .

[0627] Step 5: Synthesis of Compound 6 THF (240 mL) and H 2 To a stirred solution of 2,6-dichloro-4-[3-(prop-2-en-1-yl)oxetan-3-yl]pyridine (compound 5, 37 g, 151.56 mmol, 1 equiv.) in HO (120 mL) was added NMO (53.27 g, 454.69 mmol, 3 equiv.) in portions and cooled to 50° C. 2 OSO 4 (2.8 g, 9.00 mmol, 0.05 equiv) was added in portions at 0° C. under air atmosphere. The resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction was purified by distillation with Na 2 S 2 O 3 The mixture was quenched with 50 ml of 1,2-dichloropyridin-4-yl at 0° C. The resulting mixture was filtered and the filter cake was washed with DCM (3×100 mL). The filtrate was concentrated under reduced pressure. The crude product was used directly in the next step without further purification. This resulted in 3-[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]propane-1,2-diol (compound 6, 38 g, 90%) as a black solid. LCMS: (ES.m / z): 278,280 [M+1] + .

[0628] Step 6: Synthesis of Compound 7 H2 NaIO in O (190 mL) 4 A mixture of 2-[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]propane-1,2-diol (compound 6, 38 g, 136.62 mmol, 1 equiv.) and silica (76 g) was stirred at room temperature for 30 min under air atmosphere. Then a stirred solution of 3-[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]propane-1,2-diol (compound 6, 38 g, 136.62 mmol, 1 equiv.) in DCM (380 mL) was added dropwise at room temperature. The resulting mixture was stirred at room temperature for 2 h. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was washed with DCM (3×150 mL). The filtrate was concentrated under reduced pressure. The crude product was used directly in the next step without further purification. This resulted in 2-[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]acetaldehyde (compound 7, 27 g, 80%) as a brown solid. LCMS: (ES.m / z): 246, 248 [M+1] + .

[0629] Step 7: Synthesis of Compound 8 t-BuOH (270 mL) and H 2 To a stirred solution of 2-[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]acetaldehyde (compound 7, 27 g, 109.71 mmol, 1 equiv.) in 2HO (135 mL) was added NaClO 2 (34.73 g, 384.00 mmol, 3.5 eq.) and NaH 2 PO 4 (26.33 g, 219.43 mmol, 2 equiv) was added in portions at 0° C. under air atmosphere. To the stirred mixture was added 2-methyl-2-butene (142 mL) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. LCMS showed the reaction was complete. The mixture was acidified to pH 3 with 1N HCl. The precipitated solid was collected by filtration and diluted with Et 2 The mixture was washed with 2×O (3×100 mL). The filtrate was concentrated under reduced pressure. The residue was diluted with Et 2The mixture was purified by trituration with 0 (50 mL). This gave [3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]acetic acid (compound 8, 14.4 g, 50%) as a white solid. LCMS: (ES.m / z): 262,264 [M+1] + .

[0630] Step 9: Synthesis of Compound 10 To a stirred mixture of [3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]acetic acid (compound 8, 14 g, 53.41 mmol, 1 equiv.) and 1-amino-3-methylthiourea (6.18 g, 58.75 mmol, 1.1 equiv.) in DMF (120 mL) was added HATU (24.37 g, 64.10 mmol, 1.2 equiv.) portionwise and DIEA (20.71 g, 160.25 mmol, 3 equiv.) dropwise at 0° C. under air atmosphere. The resulting mixture was stirred overnight at room temperature. To the above mixture was added NaOH (1N, 100 mL) dropwise over 10 min at 0° C. The resulting mixture was stirred for an additional 1 h at 0° C. LCMS showed the reaction was complete. The mixture was acidified to pH 3 with 1N HCl. The aqueous layer was extracted with EtOAc (3×150 mL). The combined organics were concentrated under reduced pressure. The residue was extracted with Et 2 The residue was purified by trituration with 0 (80 mL). This gave 5-{[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]methyl}-4-methyl-1,2,4-triazole-3-thiol (compound 10, 14 g, 77%) as a white solid. LCMS: (ES.m / z): 331,333 [M+1] + .

[0631] Step 9: Synthesis of INT3 5-{[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]methyl}-4-methyl-1,2,4-triazole-3-thiol (compound 10, 12 g, 36.23 mmol, 1 equiv.) and NaNO in THF (120 mL). 2 To a stirred mixture of (7.50 g, 108.69 mmol, 3 equiv.) 3(1N, 119.92 mL, 119.92 mmol, 3.31 equiv) was added dropwise at -20°C under air atmosphere. The resulting mixture was stirred at 0°C for 2 h. LCMS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The mixture was washed with saturated Na 2 CO 3 (aqueous) to pH 8. The resulting mixture was diluted with CH 2 Cl 2 (3×100 mL). The combined organic layers were extracted with CH 2 Cl 2 (2×100 mL), and washed with anhydrous Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 / MeOH (10:1) to give 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 5.6 g, 47%) as a white solid. LCMS: (ES.m / z): 299,301 [M+1] + ; 1 H-NMR (300 MHz, DMSO-d 6 ) 8.37 (s, 1H), 7.58(s, 2H), 4.98-4.70(m, 4H), 3.65(s, 2H), 3.54(s, 2H).

[0632] INT4 Synthesis [ka] Step 1: Synthesis of Compound 2 To a stirred mixture of methyl 2-methyl-3-(trifluoromethyl)benzoate (compound 1, 44 g, 201.67 mmol, 1 equiv.) in AcOH (286 mL) was added HNO 3 (127 g, 2015.46 mmol, 9.99 equiv.) was added dropwise over 20 min at 10 °C under nitrogen atmosphere. 2(35.5 g, 222.14 mmol, 1.10 equiv) was added dropwise over 10 min at 10 °C. The resulting mixture was stirred for an additional 10 min at 10 °C. The above mixture was treated with AgNO in water (105 mL). 3 (44.4 g, 261.37 mmol, 1.30 equiv) (2.5 mol / L) was added dropwise over 25 min at 10° C. The resulting mixture was further stirred overnight at room temperature. TLC showed the reaction was complete. The mixture was diluted with saturated Na 2 CO 3 (aqueous) to pH 8. The resulting mixture was extracted with EtOAc (3×800 mL). The combined organic layers were washed with water (3×100 mL) and diluted with anhydrous Na 2 SO 4 The mixture was dried at 40° C. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (10:1) to give methyl 5-bromo-2-methyl-3-(trifluoromethyl)benzoate (compound 2, 42 g, 70%) as a yellow oil. 1 H NMR (300MHz, DMSO-d 6 ) δ 8.32(s, 1H), 8.08(s, 1H), 3.87(s, 3H), 3.46-3.44(m, 3H).

[0633] Step 2: Synthesis of Compound 3 To a stirred mixture of methyl 5-bromo-2-methyl-3-(trifluoromethyl)benzoate (compound 2, 42 g, 141.38 mmol, 1.00 equiv.) and acetic anhydride (21.7 g, 212.56 mmol, 1.50 equiv.) in DMF (420 mL, 5427.13 mmol, 38.39 equiv.), oxalic acid (19.8 g, 219.91 mmol, 1.56 equiv.) and Pd(OAc) 2 (3.2 g, 14.25 mmol, 0.10 equiv) and Xantphos (16.5 g, 28.51 mmol, 0.20 equiv) were added portionwise at room temperature. To the mixture was added DIEA (42 mL, 241.12 mmol, 1.71 equiv) dropwise at room temperature. The resulting mixture was stirred at 100° C. for 4 h under nitrogen atmosphere. The mixture was cooled to room temperature. LCMS showed the reaction was complete.

[0634] The resulting mixture was quenched with water (1 L) and extracted with EtOAc (3×800 mL). The combined organic layers were washed with brine (2×100 mL) and diluted with anhydrous Na 2 SO 4 The mixture was dried at 70° C. for 1 hour. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (1:1) to give 3-(methoxycarbonyl)-4-methyl-5-(trifluoromethyl)benzoic acid (compound 3, 29 g, 78.24%) as an off-white solid. LCMS: (ES.m / z): 261 [M-1] - .

[0635] Step 3: Synthesis of Compound 4 To a stirred mixture of 3-(methoxycarbonyl)-4-methyl-5-(trifluoromethyl)benzoic acid (29 g, 110.60 mmol, 1 equiv.) in ACN (580 mL) was added NBS (29.53 g, 165.91 mmol, 1.50 equiv.) and BPO (8.03 g, 33.18 mmol, 0.3 equiv.) portionwise at room temperature. The resulting mixture was stirred overnight at 80° C. under nitrogen atmosphere. The mixture was cooled to room temperature. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (1:1) to provide 4-(bromomethyl)-3-(methoxycarbonyl)-5-(trifluoromethyl)benzoic acid (compound 4, 32 g, 84%) as a yellow solid. LCMS: (ES.m / z): 339 [M-1] - . 1 H NMR (300MHz, DMSO-d 6 ) δ 8.57(s, 1H), 8.35(s, 1H), 5.07(s, 2H), 3.99(s, 3H).

[0636] Step 4: Synthesis of Compound 5 To a stirred mixture of 3-(methoxycarbonyl)-4-methyl-5-(trifluoromethyl)benzoic acid (compound 4, 32 g, 122.05 mmol, 1.00 equiv.) in THF (320 mL) was added BH3 -THF (235 mL, 1 mol / L, 235 mmol, 2.0 equiv) was added dropwise at 0 °C. The resulting mixture was stirred at room temperature for 6 h. LCMS showed the reaction was complete. The reaction was quenched with MeOH and stirred at room temperature for 1 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (1:1) to provide methyl 2-(bromomethyl)-5-(hydroxymethyl)-3-(trifluoromethyl)benzoate (compound 5, 23.49 g, 58%) as a yellow oil. LCMS: (ES.m / z): 245 [M-1-Br] - . 1 H NMR (300MHz, DMSO-d 6 ) δ 8.05(s, 1H), 7.91(s, 1H), 5.04(s, 2H), 4.63(s, 2H), 3.92(s, 3H).

[0637] Step 5: Synthesis of Compound 6 To a stirred solution of methyl 2-(bromomethyl)-5-(hydroxymethyl)-3-(trifluoromethyl)benzoate (compound 5, 23.49 g, 71.81 mmol, 1.00 equiv.) in EA (240 mL) was added IBX (30.16 g, 107.72 mmol, 1.5 equiv.) in portions at room temperature. The resulting mixture was stirred for 3 h at 70° C. The mixture was cooled to room temperature. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was washed with EtOAc (3×50 mL). The filtrate was diluted with saturated Na 2 S 2 Wash with 04 (3 × 30 mL) and add anhydrous Na 2 SO 4 The mixture was dried at 70° C. for 1 hour. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (1:1) to give methyl 2-(bromomethyl)-5-formyl-3-(trifluoromethyl)benzoate (compound 6, 20 g, 85%) as a yellow oil. LCMS: (ES.m / z): 323 [M-1] - . 1 H NMR (300MHz, DMSO-d 6) δ 10.13(s, 1H), 8.57(s, 1H), 8.46(s, 1H), 5.08(s, 2H), 3.97(s, 3H).

[0638] Step 6: Synthesis of Compound 7 To a stirred mixture of methyl 2-(bromomethyl)-5-formyl-3-(trifluoromethyl)benzoate (compound 6, 5.17 g, 15.90 mmol, 1.00 equiv.) in DCM (31.7 mL) was added (3S)-3-methylpiperidine hydrochloride (2.37 g, 17.49 mmol, 1.1 equiv.) and STAB (13.48 g, 63.61 mmol, 4 equiv.) in portions at 0° C. The resulting mixture was stirred at room temperature for 2 h. The reaction was quenched at 0° C. by the addition of MeOH (5 mL). The resulting mixture was concentrated under reduced pressure and quenched with water (30 mL). The resulting mixture was extracted with DCM (3×30 mL). The combined organic layers were washed with water (3×10 mL) and diluted with anhydrous Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography using the following conditions: C18 column; water (0.1% NH 4 HCO 3 ), gradient 10% to 80% in 50 min; detector, UV 254 nm. Collected fractions were concentrated to provide methyl 2-(bromomethyl)-5-{[(3S)-3-methylpiperidin-1-yl]methyl}-3-(trifluoromethyl)benzoate (compound 8, 3.0 g, 46%) as an off-white oil. LCMS: (ES.m / z): 408, 410 [M+1] + .

[0639] Step 7. Synthesis of INT4 NH in MeOH at 0 °C 3(g) To a stirred mixture of methyl 2-(bromomethyl)-5-{[(3S)-3-methylpiperidin-1-yl]methyl}-3-(trifluoromethyl)benzoate (compound 8, 1.7 g, 4.16 mmol, 1 equiv.) in (7 M, 17 mL, 119 mmol). The resulting mixture was stirred at room temperature for 5 h. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: C18 column; mobile phase, water (0.05% NH 4 HCO 3 ), gradient 10% to 100% in 40 min; detector, UV 254 nm. The resulting mixture was concentrated under reduced pressure. This afforded 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 507.6 mg, 38%) as a yellow solid. LCMS: (ES.m / z): 313 [M+1] + . 1 H NMR (300MHz, DMSO-d 6 ) δ 8.87(s, 1H), 7.87(d, J=11.4Hz, 2H), 4.53(s, 2H), 3.61(s, 2H), 2.72-2.67(m, 2H), 1.99-1.87(m, 1H), 1.77-1.40(m, 5H), 0.90-0.60(m, 4H).

[0640] Synthesis of Compound 100 [ka] Step 1: Synthesis of Compound 3 A solution of ethylene glycol (compound 2, 124 mg, 2.01 mmol, 3.00 equiv.) in THF (10 mL) was diluted with NaH (40 mg, 60%, 1.00 mmol, 1.50 equiv.) at room temperature with N 2 The resulting mixture was stirred at room temperature for 0.5 h under N 2Then, 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 200 mg, 0.67 mmol, 1.00 equiv.) was added at room temperature and stirred under N 2 The resulting mixture was stirred at room temperature overnight. LCMS showed the reaction was complete. The resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (20 mL) and washed with anhydrous Na 2 SO 4 The mixture was dried at 37° C. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by Prep-TLC (DCM:MeOH=10:1) to give 2-((6-chloro-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-yl)oxy)ethan-1-ol (150 mg, 65%) as a white solid. LCMS (ES, m / z): 325,327 [M+H] +

[0641] Step 2: Synthesis of Compound 100 2-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)oxy]ethanol (compound 3, 140 mg, 0.43 mmol, 1.00 equiv.), 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 135 mg, 0.43 mmol, 1.00 equiv.), XantPhos (50 mg, 0.09 mmol, 0.2 equiv.), Cs in dioxane (8 mL). 2 CO 3 (421 mg, 1.29 mmol, 3.00 equiv.) and Pd(OAc) 2 A solution of (10 mg, 0.04 mmol, 0.10 equiv.) was incubated at 120 °C for 1 h in N 2The mixture was stirred under reduced pressure. LCMS showed the reaction was complete. After cooling to room temperature, the reaction was filtered. The filtrate was concentrated to dryness under vacuum. The residue was purified by Prep-TLC (DCM:MeOH=10:1) to give 80 mg of crude product as a white solid. The crude product was re-purified by reverse flash chromatography with the following conditions: Column: XSelect CSH Prep C18 OBD Column, 19*150mm, 5μm; Mobile phase A: water (0.05% FA), Mobile phase B: ACN; Flow rate: 25mL / min; Gradient: 5%B to 35%B, 35%B in 7min; Wavelength: 254nm; RT1(min): 5.4. The collected fractions were lyophilized to give 2-[6-(2-hydroxyethoxy)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl]-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 100, 52.5 mg, 19%) as a white solid. LCMS (ES, m / z): 601 [M+H] + ; 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.26 (s, 1H), 7.99-7.94 (m, 3H), 6.46 (s, 1H), 5.20 (s, 2H), 4.98-4.86 (s, 5 H), 4.28 (t, J=4.8Hz, 2H), 3.74 (s, 2H), 3.68 (s, 2H), 3.57 (s, 2H), 3.31 (s, 3H), 2.78-2.67 (m, 2H), 1.96-1.91 (m, 1H), 1.68-1.59 (m, 4H), 1.52-1.43 (m, 1H), 0.90-0.81 (m, 4H).

[0642] Synthesis of Compound 101 [ka] Step 1: Synthesis of Compound 3 2,6-Dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 400 mg, 1.34 mmol, 1.00 equiv.), tert-butyl N-(carbamoylmethyl)-N-methylcarbamate (252 mg, 1.34 mmol, 1.00 equiv.), XantPhos (155 mg, 0.27 mmol, 0.20 equiv.), Cs in dioxane (6 mL). 2 CO 3 (1307 mg, 4.01 mmol, 3.00 equiv.) and Pd(OAc) 2 A solution of (30 mg, 0.13 mmol, 0.10 equiv) was stirred at 120° C. for 1 h. LCMS showed the reaction was complete. After cooling to room temperature, the reaction was filtered. The filtrate was concentrated to dryness under vacuum. The residue was purified by Prep-TLC (DCM:MeOH=10:1) to give tert-butyl N-{[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)carbamoyl]methyl}-N-methylcarbamate (compound 3, 390 mg, 63%) as a green solid. LCMS (ES, m / z): 451,453 [M+H] +

[0643] Step 2: Synthesis of Compound 5 tert-Butyl N-{[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)carbamoyl]methyl}-N-methylcarbamate (compound 3, 280 mg, 0.62 mmol, 1.00 equiv.), 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 194 mg, 0.62 mmol, 1.00 equiv.), XantPhos (72 mg, 0.12 mmol, 0.20 equiv.), Cs in dioxane (6 mL). 2 CO 3 (607 mg, 1.86 mmol, 3.00 equiv.) and Pd(OAc) 2A solution of (14 mg, 0.06 mmol, 0.10 equiv) was stirred at 120° C. for 2 h. LCMS showed the reaction was complete. After cooling to room temperature, the reaction was filtered and concentrated to dryness under vacuum. The residue was purified by Prep-TLC (DCM:MeOH=10:1) to give tert-butyl N-methyl-N-{[(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-(6-{[(3S)-3-methylpiperidin-1-yl]methyl}-1-oxo-4-(trifluoromethyl)-3H-isoindol-2-yl)pyridin-2-yl)carbamoyl]methyl}carbamate (compound 5, 200 mg, 39%) as a yellow solid. LCMS (ES, m / z): 727 [M+H] +

[0644] Step 3: Synthesis of Compound 101 tert-Butyl N-methyl-N-{[(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-(6-{[(3S)-3-methylpiperidin-1-yl]methyl}-1-oxo-4-(trifluoromethyl)-3H-isoindol-2-yl)pyridin-2-yl)carbamoyl]methyl}carbamate (compound 5, 250 mg, 0.34 mmol, 1.00 equiv.) in TFA (400 uL) and DCM (4 mL) was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The reaction was concentrated to dryness under vacuum. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 0% to 60% in 30 min; detector, UV 254 nm. The collected fractions were concentrated to give N-(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-(6-{[(3S)-3-methylpiperidin-1-yl]methyl}-1-oxo-4-(trifluoromethyl)-3H-isoindol-2-yl)pyridin-2-yl)-2-(methylamino)acetamide; formic acid (compound 101, 100 mg, 41%) as a white solid. LCMS (ES, m / z): 627 [M+H] + ;1 H NMR (400 MHz, DMSO-d 6 ) δ8.24 (s, 1H), 8.20 (s, 2H), 8.08 (s, 1H), 7.99 (s, 1H), 7.95 (s, 1H), 7.75 (br, 1H), 5.19 (s, 2H), 4.98-4.81 (m, 4H), 3.65 (s, 2H), 3.59 (s, 2H), 3.52-3.50 (m, 2H), 3.33 (s, 3H), 2.74-2.67 (m, 2H), 2.42 (s, 3H), 1.97-1.92 (m, 1H), 1.68-1.59 (m, 4H), 1.52-1.46 (m, 1H), 0.88-0.81 (m, 4H).

[0645] Synthesis of compound 102 [ka] Step 1: Synthesis of Compound 1 To a stirred mixture of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 200 mg, 0.66 mmol, 1 equiv.) and β-mercaptoethanol (53 mg, 0.67 mmol, 1.01 equiv.) in DMF (2 mL) was added K 2 CO 3 (186 mg, 1.34 mmol, 2.01 equiv) was added portionwise at room temperature. The resulting mixture was stirred at 70° C. for 2 h under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction mixture was filtered and the filtrate was purified by reverse flash chromatography with the following conditions: C18 column; mobile phase, ACN in water (0.1% FA), gradient from 10% to 50% in 40 min; detector, UV 254 nm. The collected fractions were concentrated under reduced pressure. This yielded 2-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)sulfanyl]ethanol (compound 1, 130 mg, 57%) as a yellow oil. LCMS: (ES.m / z): 341,343 [M+1] + .

[0646] Step 2. Synthesis of compound 102 To a stirred mixture of 2-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)sulfanyl]ethanol (compound 1, 124 mg, 0.36 mmol, 1 equiv.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 124 mg, 0.39 mmol, 1.09 equiv.) in dioxane (3 mL), was added Xantphos (42 mg, 0.073 mmol, 0.20 equiv.) and Pd(OAc). 2 (8 mg, 0.036 mmol, 0.10 equiv.) and Cs 2 CO 3 (354 mg, 1.08 mmol, 2.99 equiv) was added portionwise at room temperature. The resulting mixture was stirred at room temperature for 1 h under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction mixture was purified by reverse flash chromatography with the following conditions: C18 column; mobile phase, ACN in water, gradient from 10% to 60% in 40 min; detector, UV 254 nm. The collected fractions were concentrated under reduced pressure. This yielded 2-{6-[(2-hydroxyethyl)sulfanyl]-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl}-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 102, 12.2 mg, 5.40%) as an off-white solid. LCMS: (ES.m / z): 617[M+1] + ; 1 H NMR (300MHz, DMSO-d 6) δ 8.26(s, 1H), 8.04(s, 1H), 7.98(s, 1H), 7.94(s, 1H), 6.96(s, 1H), 5.22(s, 2H), 4.94-4.90(m, 3H), 4.83-4.82(m, 2H), 3.66-3.65(m, 4H), 3.57(s, 2H), 3.23-3.22(m, 2H), 2.80-2.65(m, 2H), 2.00-1.90(m, 1H), 1.77-1.55(m, 4H), 1.55-1.40(m, 1H), 0.82-0.81(m,4H).

[0647] Synthesis of compound 103 [ka] Step 1: Synthesis of Compound 2 To a stirred mixture of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 100 mg, 0.33 mmol, 1.00 equiv.) in DMF (2 mL) was added 2-(methylsulfanyl)ethanamine hydrochloride (129 mg, 1.01 mmol, 3.02 equiv.) and K 2 CO 3 (139 mg, 1.01 mmol, 3.0 equiv.) was added. The reaction mixture was stirred at 100° C. for 48 h under nitrogen atmosphere. Approximately 20% of the desired product could be detected by LCMS. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated to dryness under vacuum and the residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 60% in 30 min; detector, UV 254 nm. The collected fractions were concentrated to dryness to provide 6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-N-[2-(methylsulfanyl)ethyl]pyridin-2-amine (compound 2, 44 mg, 35%) as a white solid. LCMS: (ES, m / s): 354,356 [M+H] + .

[0648] Step 2. Synthesis of compound 103 To a stirred mixture of 6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-N-[2-(methylsulfanyl)ethyl]pyridin-2-amine (compound 2, 38 mg, 0.11 mmol, 1 equiv.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 35 mg, 0.11 mmol, 1.05 equiv.) in dioxane (400 uL) was added Cs 2 CO 3 (70 mg, 0.22 mmol, 2.00 equiv.), Xantphos (12 mg, 0.021 mmol, 0.20 equiv.) and Pd(OAc). 2 (24 mg, 0.11 mmol, 1.00 equiv) was added at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 120° C. for 1 h under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated to dryness under vacuum, and the residue was purified by Prep-HPLC under the following conditions (Column: XBridge Shield RP18 OBD Column, 19×250 mm, 10 μm; Mobile phase A: water (0.05% FA), Mobile phase B: MeOH--HPLC; Flow rate: 25 mL / min; Gradient: 38% B to 48% B to 48% B in 10 min; Wavelength: 254 nm; Collected fractions were lyophilized to provide 2-(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-{[2-(methylsulfanyl)ethyl]amino}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 103, 8.1 mg, 11%) as a yellow solid. LCMS: (ES, m / z): 630 [M+1] + ; 1 H-NMR (300 MHz, DMSO-d 6) 8.25 (s, 1H), 7.95-7.91(m, 2H), 7.45(s, 1H), 6.89(t, J=5.7Hz, 1H), 5.95(s, 1H), 5.17(s, 2H), 4.90(d, J=4.5Hz, 2H), 4.78(d, J=4.5Hz,, 2H), 3.64(s, 2H), 3.56-3.46(m, 4H), 3.23(s, 3H), 2.73-2.67(m, 2H), 2.65-2.61(m, 2H), 2.13(s, 3H), 1.96-1.93(m, 1H), 1.67-1.58(m, 4H), 1.49-1.46(m, 1H), 0.87-0.81(m, 4H).

[0649] Synthesis of compound 104 [ka] Step 1. Synthesis of compound 2 To a stirred mixture of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 200 mg, 0.66 mmol, 1 equiv.) and [(tert-butoxycarbonyl)(methyl)amino]acetic acid (190 mg, 1.00 mmol, 1.50 equiv.) in DMSO (12 mL) was added [Ir(dF(CF 3 )ppy) 2 (dtbbpy)]PF 6 (150 mg, 0.13 mmol, 0.20 equiv.) and [Ni(dtbbpy)(H 2 O) 4 ]Cl 2(60 mg, 0.13 mmol, 0.19 equiv) was added portionwise and BTMG (170 mg, 0.99 mmol, 1.49 equiv) was added dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred and irradiated at room temperature overnight using a blue LED lamp (365 nm). LCMS showed the reaction was 50% 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine and 50% desired product. The reaction mixture was purified by reverse flash chromatography using the following conditions: C18 column; mobile phase, ACN in water (0.1% FA), gradient from 10% to 80% in 40 min; detector, UV 254 nm. The resulting fractions were concentrated under reduced pressure. This resulted in tert-butyl N-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)methyl]-N-methylcarbamate (compound 2, 68 mg, 24%) as a yellow oil. LCMS: (ES.m / z): 408,410 [M+1] + .

[0650] Step 2: Synthesis of Compound 3 To a stirred mixture of tert-butyl N-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)methyl]-N-methylcarbamate (compound 2, 63 mg, 0.15 mmol, 1 equiv.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 50 mg, 0.16 mmol, 1.04 equiv.) in dioxane (0.5 mL), Pd(OAc) 2 (4 mg, 0.018 mmol, 0.12 equiv.) and Xantphos (18 mg, 0.03 mmol, 0.20 equiv.) and Cs 2 CO 3(152 mg, 0.47 mmol, 3.02 equiv) was added portionwise at room temperature. The resulting mixture was stirred overnight at 120° C. under nitrogen atmosphere. After cooling to room temperature, the reaction mixture was filtered and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: C18 column; mobile phase, ACN in water (0.1% FA), gradient from 10% to 50% in 40 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum. The crude product (37 mg) was re-purified by Prep-HPLC with the following conditions (Column: XBridge Shield RP18 OBD Column, 30*150 mm, 5 μm; Mobile phase A: Water (NH 4 HCO 3 +0.1%NH 3 .H 2 0), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 33% B to 56% B to 56% B in 7 min; wavelength: 254 nm; collected fractions were concentrated to provide tert-butyl N-methyl-N-[(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-(6-{[(3S)-3-methylpiperidin-1-yl]methyl}-1-oxo-4-(trifluoromethyl)-3H-isoindol-2-yl)pyridin-2-yl)methyl]carbamate (compound 3, 12 mg, 11%) as an off-white solid. LCMS: (ES.m / z): 684[M+1] + .

[0651] Step 3: Synthesis of Compound 104 To a stirred mixture of tert-butyl N-methyl-N-[(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-(6-{[(3S)-3-methylpiperidin-1-yl]methyl}-1-oxo-4-(trifluoromethyl)-3H-isoindol-2-yl)pyridin-2-yl)methyl]carbamate (12 mg, 0.018 mmol, 1 equiv.) in DCM (1200 uL) was added TFA (300 uL) dropwise at room temperature. The resulting mixture was stirred for 1 h at room temperature under air atmosphere. LCMS showed the reaction was complete. The reaction mixture was concentrated to dryness under vacuum. The resulting solid was dried by lyophilization. This gave 2-(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-[(methylamino)methyl]pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one; trifluoroacetic acid (9.0 mg, 69%) as a colorless oil. LCMS: (ES.m / z): 584 [M+1] + ; 1 H NMR (400MHz, CD 3 OD) δ 8.87(s, 1H), 8.48(s, 1H), 8.35(s, 1H), 8.28(s, 1H), 7.27(s, 1H), 5.46(s, 2H), 5.07-5.02(m, 4H), 4.52(s, 2H), 4.44(s, 2H), 3.96(s, 2H), 3.85(s, 3H), 3.77-3.76(m, 1H), 3.63-3.62(m, 1H), 2.97-2.95(m, 1H), 2.89(s, 3H), 2.85-2.71(m, 1H), 2.00-1.61(m, 4H), 1.35-1.29(m, 1H), 1.26-1.10(m, 1H), 1.00(s, 3H).

[0652] Synthesis of compound 105 [ka] Step 1: Synthesis of Compound 2 To a stirred solution of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 100 mg, 0.34 mmol, 1.0 equiv.) and 2-methyl-2-(methylsulfanyl)propan-1-amine (48 mg, 0.40 mmol, 1.2 equiv.) in DMA (2.0 mL) was added DIEA (172 mg, 1.33 mmol, 4.00 equiv.) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 80° C. for 4 days under nitrogen atmosphere. Approximately 30% of the desired product could be detected by LCMS. The reaction mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 60% in 30 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum to provide 6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-N-[2-methyl-2-(methylsulfanyl)propyl]pyridin-2-amine (compound 2, 17 mg, 13%) as a solid. LCMS: (ES, m / s): 382, ​​384 [M+H] + .

[0653] Step 2: Synthesis of Compound 105 To a stirred solution of 6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-N-[2-methyl-2-(methylsulfanyl)propyl]pyridin-2-amine (compound 2, 17 mg, 0.04 mmol, 1.0 equiv.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 14 mg, 0.04 mmol, 1.0 equiv.) in dioxane (1.5 mL), was added Xantphos (10 mg, 0.016 mmol, 0.4 equiv.), Pd(OAc). 2 (4 mg, 0.016 mmol, 0.4 equiv.) and Cs 2 CO 3(43 mg, 0.13 mmol, 3.0 equiv) was added in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 1 h at 120° C. under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction mixture was purified by Prep-HPLC under the following conditions (Column: XBridge Shield RP18 OBD Column, 30*150mm, 5μm; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 23%B to 43%B to 43%B in 7min; Wavelength: 254nm; Collected fractions were lyophilized to give 2-(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-{[2-methyl-2-(methylsulfanyl)propyl]amino}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one; formic acid (compound 105, 2.6mg, 7%) as a white solid. LCMS: (ES, m / s): 658[M+H] + ; 1 H-NMR (300 MHz, DMSO-d 6 ) 8.49 (br s, 1H), 8.24 (s, 1H), 7.95-7.91(m, 2H), 7.39(s, 1H), 6.78(t, J=5.4 Hz, 1H), 6.15(s, 1H), 5.16(s, 2H), 4.96-4.83(m, 2H), 4.81-4.77(m, 2H), 3.62(d, J=6.9 Hz, 2H), 3.56-3.48(m, 4H), 3.20(s, 3H), 2.70-2.68(m, 2H), 2.06(s, 3H), 1.99-1.92(m, 1H), 1.70-1.40(m, 5H), 1.28-1.15(m, 6H), 0.95-0.75(m, 4H).

[0654] Synthesis of compound 106 [ka] Step 1: Synthesis of Compound 2 To a stirred solution of tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate (compound 1, 10 g, 46.44 mmol, 1 equiv.) in DCM (250 mL) was added TsCl (17.71 g, 92.89 mmol, 2 equiv.) and TEA (14.10 g, 139.34 mmol, 3 equiv.) at 0° C. The resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction was quenched with MeOH at 0° C. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography eluted with PE / EA (5:1) to provide tert-butyl 4-{[(4-methylbenzenesulfonyl)oxy]methyl}piperidine-1-carboxylate (compound 2, 12 g, 69%) as an off-white solid. LCMS (ESI, m / z): 370 [M+H] +

[0655] Step 2: Synthesis of Compound 3 To a stirred mixture of tert-butyl 4-{[(4-methylbenzenesulfonyl)oxy]methyl}piperidine-1-carboxylate (compound 2, 5 g, 13.53 mmol, 1 equiv.) and KI (2.25 g, 13.53 mmol, 1 equiv.) in EtOH (50 mL) was added sodium thiomethoxide (1.90 g, 27.06 mmol, 2 equiv.) at room temperature. The resulting mixture was stirred at 80° C. for 1 h under nitrogen atmosphere. LCMS showed the reaction was complete. The mixture was cooled to room temperature. The reaction was quenched with water at room temperature. The resulting mixture was diluted with CH 2 Cl 2 (3×100 mL). The combined organic layers were washed with brine (100 mL) and anhydrous Na 2 SO 4 After filtration, the filtrate was concentrated under reduced pressure. This gave tert-butyl 4-[(methylsulfanyl)methyl]piperidine-1-carboxylate (compound 3, 2.8 g, 84%) as an off-white solid. LCMS (ESI, m / z): 246 [M+H] + .

[0656] Step 3: Synthesis of Compound 4 A solution of tert-butyl 4-[(methylsulfanyl)methyl]piperidine-1-carboxylate (compound 3, 560 mg, 2.28 mmol, 1 equiv.) in HCl (g, 4N in MeOH, 5.6 mL) was stirred for 1 h at room temperature under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The crude product was used directly in the next step without further purification. This resulted in 4-[(methylsulfanyl)methyl]piperidine hydrochloride (370 mg, 89%) as a yellow solid. LCMS (ESI, m / z): 146 [M+H-HCl] +

[0657] Step 4: Synthesis of Compound 5 To a stirred solution of 4-[(methylsulfanyl)methyl]piperidine hydrochloride (compound 4, 350 mg, 1.92 mmol, 1 equiv.) in DCM (4 mL) was added methyl 2-(bromomethyl)-5-formyl-3-(trifluoromethyl)benzoate (compound 6 from the procedure for preparing INT4, 626 mg, 1.92 mmol, 1 equiv.) at room temperature. The resulting mixture was stirred for 30 min at room temperature under nitrogen atmosphere. To the above mixture was added STAB (1.63 g, 7.70 mmol, 4 equiv.) in portions at room temperature. The resulting mixture was further stirred overnight at room temperature. LCMS showed the reaction was complete. The reaction was quenched with MeOH at 0° C. and stirred for 30 min. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 Purification by silica gel column chromatography eluted with 1,2-dichloromethane / MeOH (10:1) afforded methyl 2-(bromomethyl)-5-({4-[(methylsulfanyl)methyl]piperidin-1-yl}methyl)-3-(trifluoromethyl)benzoate (compound 5, 820 mg, 93%) as a yellow oil. LCMS (ESI, m / z): 454, 456 [M+H] + .

[0658] Step 5: Synthesis of Compound 6 NH at room temperature 3To a stirred solution of methyl 2-(bromomethyl)-5-({4-[(methylsulfanyl)methyl]piperidin-1-yl}methyl)-3-(trifluoromethyl)benzoate (compound 5, 400 mg, 0.88 mmol, 1 equiv.) in (g, 7N in MeOH, 4 mL). The resulting mixture was stirred for 1 h at room temperature under a nitrogen atmosphere. LCMS indicated the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by HPLC using HPLC with 5% COOH. 2 Cl 2 Purification by silica gel column chromatography eluted with 1,2-dichloromethane / MeOH (14:1) afforded 6-({4-[(methylsulfanyl)methyl]piperidin-1-yl}methyl)-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (compound 6, 170 mg, 53%) as a white solid. LCMS (ESI, m / z): 359 [M+H] + .

[0659] Step 6: Synthesis of Compound 106 To a stirred solution of 6-({4-[(methylsulfanyl)methyl]piperidin-1-yl}methyl)-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (compound 6, 86 mg, 0.24 mmol, 1 equiv.) and 6-chloro-N-ethyl-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-amine (prepared as described in WO2020210508, 73 mg, 0.24 mmol, 1 equiv.) in dioxane (2 mL), Cs 2 CO 3(156 mg, 0.48 mmol, 2 equiv.), di-tert-butyl([2-[2,4,6-tris(propan-2-yl)phenyl]phenyl])phosphane (10 mg, 0.02 mmol, 0.1 equiv.) and tBuXPhos Pd G3 (19 mg, 0.02 mmol, 0.1 equiv.) were added at room temperature under nitrogen atmosphere. The resulting mixture was stirred overnight at 120° C. under nitrogen atmosphere. LCMS showed 45% product. The reaction mixture was cooled to room temperature. The reaction mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 5% to 60% in 30 min; detector, UV 254 nm. The collected fractions were concentrated to provide the crude product (40 mg) as a white solid. The crude product (40 mg) was purified by Prep-HPLC under the following conditions (column: XBridge Shield RP18 OBD Column, 30*150mm, 5μm; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 19%B to 39%B, 39%B in 7min; Wavelength: 254nm; RT1(min): 4.62; Collected fractions were lyophilized to provide 2-[6-(ethylamino)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl]-6-({4-[(methylsulfanyl)methyl]piperidin-1-yl}methyl)-4-(trifluoromethyl)-3H-isoindol-1-one (10.8mg, 7%) as a white solid. LCMS (ESI, ms): 630[M+H] + ; 1H NMR (400 MHz, DMSO-d6) δ 8.38 (s, 1H), 7.94-7.90 (m, 2H), 7.40 (s, 1H), 6.62 (t, J = 5.2 Hz, 1H), 5.89 (s, 1H), 5.14 (s, 2H), 4.90 (d, J = 6.0 Hz, 2H), 4.78 (d, J = 6.0 Hz, 2H), 3.65 (s, 2H), 3.49 (s, 2H), 3.24-3.17(m, 5H), 2.82-2.79 (m, 2H), 2.41 (d, J = 6.8 Hz, 2H), 2.02-1.96 (m, 5H), 1.75-1.72 (m, 2H), 1.46-1.44 (m, 1H), 1.26 - 1.10 (m, 5H).

[0660] Synthesis of compound 107 [ka] Step 1: Synthesis of Compound 2 To a stirred solution of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 2 g, 6.68 mmol, 1 equiv.) in DMF (20 mL) was added (ethylsulfanyl)sodium (0.62 g, 7.35 mmol, 1.1 equiv.) in portions at room temperature. The resulting mixture was stirred for 2 h at 50° C. under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was diluted with water (100 mL) and extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (3×100 mL) and diluted with anhydrous Na 2 SO 4The mixture was dried at 40° C. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, FA in water, gradient from 0% to 50% in 40 min; detector, UV 254 nm. This resulted in 2-chloro-6-(ethylsulfanyl)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (compound 2, 1.4 g, 62%) as a yellow solid. LCMS: (ES.m / z): 325,327[M+1] + .

[0661] Step 2: Synthesis of Compound 107 To a stirred mixture of 2-chloro-6-(ethylsulfanyl)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (compound 2, 120 mg, 0.36 mmol, 1.20 equiv.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 96 mg, 0.30 mmol, 1.00 equiv.) in dioxane (5 mL), Cs 2 CO 3 (200 mg, 0.61 mmol, 2.00 equiv.) and Xantphos (35 mg, 0.06 mmol, 0.20 equiv.) and Pd(OAc) 2(6.90 mg, 0.03 mmol, 0.10 equiv) was added portionwise at room temperature. The resulting mixture was stirred at 120° C. for 2 h under nitrogen atmosphere. The mixture was cooled to room temperature. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was washed with DCM (3×10 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (10:1) to provide 2-[6-(ethylsulfanyl)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl]-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (17.7 mg, 9%) as a white solid. LCMS: (ES.m / z): 601[M+1] + ; 1 H NMR (400 MHz, DMSO-d 6 ) δ8.26 (s, 1H), 8.05-7.94 (m, 3H), 6.91 (d, J=1.2Hz, 1H), 5.23 (s, 2H), 4.92-4.82 (m, 4H), 3.63-3.57 (m, 4H), 3.34-3.30 (m, 5H), 3.14-3.12 (m, 2H), 2.74-2.68 (m, 2H), 1.98-1.90 (m, 1H), 1.65-1.62 (m, 4H), 1.50-1.45 (m, 1H), 1.35-1.32 (m, 3H), 0.82-0.81 (m, 4H).

[0662] Synthesis of compound 108 [ka] Step 1: Synthesis of Compound 1 To a stirred solution of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 300 mg, 1.00 mmol, 1 equiv.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 328 mg, 1.05 mmol, 1.05 equiv.) in dioxane (6 mL), Cs 2 CO 3 (653 mg, 2.00 mmol, 2 equiv.), XantPhos (116 mg, 0.20 mmol, 0.2 equiv.) and Pd(OAc). 2 (22 mg, 0.10 mmol, 0.1 equiv) was added at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 120° C. for 1 h under nitrogen atmosphere. The desired product could be detected by LCMS. The mixture was cooled to room temperature. The resulting mixture was concentrated under vacuum. The residue was dissolved in CH 2 Cl 2 / MeOH (10:1) to give 2-(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 1, 250 mg, 43%) as a white solid. LCMS (ESI, ms): 575,577 [M+H] + .

[0663] Step 2: Synthesis of Compound 2 To a stirred solution of 2-(6-chloro-4-{2-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxiran-2-yl}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 1, 250 mg, 0.44 mmol, 1 equiv.) and 2-[(triphenylmethyl)sulfanyl]ethanamine hydrochloride (190 mg, 0.53 mmol, 1.2 equiv.) in dioxane (3 mL), Cs 2 CO 3(290 mg, 0.89 mmol, 2 equiv.), XantPhos (51 mg, 0.08 mmol, 0.2 equiv.) and Pd(OAc). 2 (10 mg, 0.04 mmol, 0.1 equiv) was added at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 120° C. for 2 h under nitrogen atmosphere. LCMS showed the reaction was complete. The mixture was cooled to room temperature. The resulting mixture was concentrated in vacuo. The residue was dissolved in CH 2 Cl 2 / MeOH (10:1) to give 2-(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-({2-[(triphenylmethyl)sulfanyl]ethyl}amino)pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 2, 165 mg, 43%) as a yellow solid. LCMS (ESI, m / z): 858 [M+H] + .

[0664] Step 3: Synthesis of Compound 108 To a stirred solution of 2-(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-({2-[(triphenylmethyl)sulfanyl]ethyl}amino)pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (165 mg, 0.19 mmol, 1 equiv.) in DCM (4 mL) and TFA (2 mL) was added tris(propan-2-yl)silane (200 uL) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere. LCMS indicated the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, silica gel; mobile phase, MeCN in water (0.1% FA), gradient from 5% to 60% in 30 min; detector, UV 254 nm. The collected fractions were lyophilized to provide 2-(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-[(2-sulfanylethyl)amino]pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 108, 47.9 mg, 37%) as a white solid. LCMS: (ES.m / z): 616 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 ) δ 8.29-8.23 (m, 1H), 8.01-7.83(m, 2H),7.44(s, 1H), 6.91(t, J=5.7 Hz, 1H), 5.97(s, 1H), 5.24(s, 2H), 4.97-4.84(m, 2H), 4.79-4.74(m, 2H), 3.79-3.77(m, 2H), 3.49(s, 2H), 3.41(s, 2H), 3.23(s, 3H), 2.73-2.63(m, 4H), 2.26(t, J=7.8 Hz, 1H), 1.98-1.95(m, 1H), 1.63-1.50(m, 6H), 0.91-0.81(m, 4H).

[0665] Synthesis of compound 109 [ka] Step 1: Synthesis of Compound 2 To a stirred mixture of 2,6-dichloro-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridine (INT3, 235 mg, 0.786 mmol, 1 equiv.) and (S)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one (INT4, 245 mg, 0.786 mmol, 1 equiv.) in dioxane (7 mL), Cs 2 CO 3 (512 mg, 1.57 mmol, 2 equiv.) and Xantphos (90.8 mg, 0.157 mmol, 0.2 equiv.) and Pd(OAc) 2 (17.6 mg, 0.0786 mmol, 0.1 equiv) was added portionwise at room temperature. The resulting mixture was stirred at 90° C. for 1 h under nitrogen atmosphere. The mixture was cooled to room temperature. LCMS showed the reaction was complete. The reaction was quenched with saturated sodium bicarbonate at room temperature. The resulting mixture was diluted with CH 2 Cl 2 (3×50 ml). The combined organic layers were concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 / MeOH (9:1) to afford (S)-2-(6-chloro-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-yl)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one (compound 2, 300 mg, 66.4%) as a green solid. LCMS: (ES.m / z): 575,577 [M+1] + .

[0666] Step 2: Synthesis of Compound 109 To a stirred mixture of (S)-2-(6-chloro-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-yl)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one (compound 2, 30 mg, 0.052 mmol, 1 equiv.) and propargylamine (5.7 mg, 0.10 mmol, 2 equiv.) in dioxane (1 mL), Cs 2 CO 3 (113 mg, 0.34 mmol, 2 equiv.) and Xantphos (6.0 mg, 0.01 mmol, 0.2 equiv.) and Pd(OAc) 2 (1.2 mg, 0.05 mmol, 0.1 equiv) was added portionwise at room temperature. The resulting mixture was stirred for 40 min at 120° C. under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered and loaded directly onto the column. The column was dried in vacuum and purified by CH 2 Cl 2 / MeOH (3:1) to afford (S)-2-(4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)-6-(prop-2-yn-1-ylamino)pyridin-2-yl)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one (compound 109, 13 mg, 42%) as a yellow solid. LCMS: (ES.m / z): 594 [M+1] + ; 1 H NMR (400 MHz, CDCl 3) δ 8.30 (d, J = 1.5 Hz, 1H), 8.02 (s, 1H), 7.94 (s, 1H), 7.86 (s, 1H), 6.97 (d, J = 1.5 Hz, 1H), 5.25 - 5.20 (m, 2H), 5.11 (d, J = 6.4 Hz, 2H), 5.05 (d, J = 6.4 Hz, 2H), 3.69 (s, 2H), 3.64 (d, J = 7.3 Hz, 1H), 3.60 (s, 4H), 3.26 (s, 3H), 2.75 (dd, J = 17.3, 9.8 Hz, 2H), 1.95 (td, J = 11.1, 3.3 Hz, 1H), 1.47 (t, J = 7.2 Hz, 2H), 1.25 (s, 8H), 1.07 (s, 1H), 0.92 - 0.82 (m, 9H), 0.07 (s, 2H).

[0667] Synthesis of compound 110 [ka] Step 1: Synthesis of Compound 2 To a stirred solution of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 1.0 g, 3.34 mmol, 1.0 equiv.) and β-aminopropionitrile (4.69 g, 66.90 mmol, 20.0 equiv.) in DMA (4.0 mL) was added K 2 CO 3 (0.92 g, 6.68 mmol, 2.0 equiv) was added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 120° C. for 2 days under nitrogen atmosphere. Approximately 70% of the desired product could be detected by LCMS. The reaction was cooled to room temperature and quenched with water at room temperature. The aqueous layer was diluted with CH 2 Cl 2 (3×100 mL). The resulting mixture was concentrated in vacuo. The residue was extracted with CH 2 Cl 2 / MeOH (5:1) to give 3-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)amino]propanenitrile (600 mg, 49%) as a light green solid. LCMS: (ES, m / s): 333,335 [M+H] + .

[0668] Step 2: Synthesis of Compound 110 To a stirred solution of 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (compound 2, 47 mg, 0.15 mmol, 1.0 equiv.) and 3-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)amino]propanenitrile (INT4, 50 mg, 0.15 mmol, 1.0 equiv.) in dioxane (2.0 mL), was added Xantphos (34 mg, 0.06 mmol, 0.4 equiv.), Pd(OAc). 2 (13 mg, 0.06 mmol, 0.4 equiv.) and Cs 2 CO 3 (146 mg, 0.45 mmol, 3.0 equiv) was added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 1 h at 120° C. under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was washed with ACN and DCM. The filtrate was concentrated under reduced pressure. The residue was extracted with CH 2 Cl 2 / MeOH (5:1) to give the crude product. The crude product (50 mg) was purified by Prep-HPLC with the following conditions (Column: Mobile phase A: Water (0.1% FA), Mobile phase B: ACN). The collected fractions were lyophilized to give 3-[(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-(6-{[(3S)-3-methylpiperidin-1-yl]methyl}-1-oxo-4-(trifluoromethyl)-3H-isoindol-2-yl)pyridin-2-yl)amino]propanenitrile; formic acid (Compound 110, 25 mg, 25%) as a white solid. LCMS: (ES, m / s): 609 [M+H] + ,305[M / 2+H] + ; 1 H NMR (300 MHz, DMSO-d 6 ) δ 8.24 (s, 1H), 7.95-7.91(m, 2H), 7.49 (s, 1H), 7.07 (t, J= 5.4 Hz, 1H), 6.05 (s, 1H), 5.20(s, 2H), 4.91 (d, J= 6 Hz, 2H), 4.78(d, J= 6 Hz, 2H), 3.64 (s, 2H), 3.53-3.50 (m, 4H), 3.25(s, 3H), 2.81 (t, J= 6.3 Hz, 2H), 2.72(t, J= 7.4 Hz, 2H), 1.97-1.93(m, 1H), 1.69-1.42 (m, 5H), 0.95-0.78 (m, 4H).

[0669] Synthesis of compound 111 [ka] Step 1: Synthesis of Compound 2 To a stirred mixture of tert-butyl (3S)-3-(hydroxymethyl)piperidine-1-carboxylate (compound 10g, 46.44mmol, 1eq) and TEA (14g, 138.35mmol, 2.98eq) in DCM (100mL) was added TsCl (17.5g, 91.79mmol, 1.98eq) dropwise at 0°C under nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (5:1) to provide tert-butyl (3S)-3-{[(4-methylbenzenesulfonyl)oxy]methyl}piperidine-1-carboxylate (compound 2, 14.2g, 74.47%) as a colorless oil. LCMS: 370[M+H] + ; 1 H NMR (300 MHz, chloroform-d) δ 7.86 - 7.70 (m, 2H), 7.45 - 7.30 (m, 2H), 3.94 - 3.77 (m, 4H), 2.84-2.80 (m, 1H), 2.64 (dd, J = 13.0, 9.7 Hz, 1H), 2.45 (s, 3H), 1.91 - 1.70 (m, 2H), 1.60-1.58 (m, 1H), 1.42 (s,9H).

[0670] Step 2: Synthesis of Compound 3 To a stirred solution of tert-butyl (3S)-3-{[(4-methylbenzenesulfonyl)oxy]methyl}piperidine-1-carboxylate (compound 2, 5 g, 13.53 mmol, 1 equiv.) and KI (2.25 g, 13.53 mmol, 1 equiv.) in EtOH (50 mL) was added (methylsulfanyl)sodium (1.90 g, 27.06 mmol, 2 equiv.) at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 80° C. for 1 h under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was cooled to room temperature, diluted with water (100 mL), and extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (100 mL) and diluted with anhydrous Na 2 SO 4The mixture was dried at 4° C. After filtration, the filtrate was concentrated under reduced pressure. This gave tert-butyl (3S)-3-[(methylsulfanyl)methyl]piperidine-1-carboxylate (3.1 g, 93%) as a yellow oil. LCMS (ESI, ms): 246 [M+H] + .

[0671] Step 3: Synthesis of Compound 4 A stirred solution of tert-butyl (3S)-3-[(methylsulfanyl)methyl]piperidine-1-carboxylate (compound 3, 800 mg, 3.26 mmol, 1 equiv.) in HCl (gas, 4N in 1,4-dioxane, 30 mL) was stirred for 1 h at room temperature under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. This afforded (3S)-3-[(methylsulfanyl)methyl]piperidine hydrochloride (compound 4, 2.1 g, 94%) as a white solid. The crude product was used directly in the next step without further purification. LCMS (ESI, ms): 146 [M+H-HCl] + .

[0672] Step 4: Synthesis of Compound 5 A stirred solution of (3S)-3-[(methylsulfanyl)methyl]piperidine hydrochloride (compound 4, 2.1 g, 11.55 mmol, 1 equiv.) and methyl 2-(bromomethyl)-5-formyl-3-(trifluoromethyl)benzoate (3.01 g, 9.24 mmol, 0.8 equiv.) in DCM (21 mL) was stirred for 30 min at room temperature under nitrogen atmosphere. STAB (9.80 g, 46.22 mmol, 4 equiv.) was added to the above mixture at room temperature. The resulting mixture was stirred for an additional 12 h at room temperature. LCMS showed the reaction was complete. The reaction was quenched with MeOH and stirred for 30 min at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 / MeOH (8:1) to give methyl 2-(bromomethyl)-5-{[(3S)-3-[(methylsulfanyl)methyl]piperidin-1-yl]methyl}-3-(trifluoromethyl)benzoate (compound 5, 1.2 g, 22%) as a yellow oil. LCMS (ESI, ms): 454, 456 [M+H] + .

[0673] Step 5: Synthesis of Compound 6 NH 3 A stirred solution of methyl 2-(bromomethyl)-5-{[(3S)-3-[(methylsulfanyl)methyl]piperidin-1-yl]methyl}-3-(trifluoromethyl)benzoate (compound 5, 600 mg, 1.32 mmol, 1 equiv.) in (g, 7N in MeOH, 6 mL) was stirred for 2 h at room temperature under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, silica gel; mobile phase, MeCN in water (0.1FA%), gradient from 5% to 60% in 30 min; detector, UV 254 nm. The collected fractions were concentrated to provide 6-{[(3S)-3-[(methylsulfanyl)methyl]piperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (compound 6, 200 mg, 42%) as a yellow oil. LCMS(ESI, ms): 359[M+H] +

[0674] Step 6: Synthesis of Compound 111 To a stirred solution of 6-{[(3S)-3-[(methylsulfanyl)methyl]piperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (compound 6, 120 mg, 0.33 mmol, 1 equiv.) and 6-chloro-N-ethyl-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-amine (103 mg, 0.33 mmol, 1 equiv.) in dioxane (2.4 mL), Cs 2 CO 3(218 mg, 0.67 mmol, 2 equiv), RuPhos (31 mg, 0.06 mmol, 0.2 equiv) and RuPhos Palladacycle Gen.3 (28 mg, 0.03 mmol, 0.1 equiv) were added at room temperature under nitrogen atmosphere. The resulting mixture was stirred overnight at 120° C. under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was cooled to room temperature. The reaction mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in water (0.1% FA), gradient from 5% to 60% in 40 min; detector, UV 254 nm. The collected fractions were lyophilized to provide 2-[6-(ethylamino)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl]-6-{[(3S)-3-[(methylsulfanyl)methyl]piperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (70.5 mg, 31%) as an off-white solid. LCMS (ESI, ms): 630 [M+H] + ; 1 H-NMR (300 MHz, DMSO-d 6 ) 8.24 (brs, 1H), 7.95-7.91 (m, 2H), 7.40(s, 1H), 6.63(t, J=4.8Hz, 1H), 5.88(s, 1H), 5.31(s, 2H), 4.91(d, J=6H, 2H), 4.79(d, J=6.6Hz, 2H), 3.72-3.70 (m, 2H), 3.68(s, 2H), 3.22-3.18(m, 5H), 2.85-2.82(m, 1H), 2.72-2.69(m, 1H), 2.40-2.38(m, 2H), 2.02-2.00(m, 4H), 1.82-1.80(m, 3H), 1.78-1.55(m, 2H), 1.12-1.10(m, 3H),1.05-0.98(m, 1H).

[0675] Synthesis of compound 112 [ka] Step 1: Synthesis of Compound 2 To a stirred mixture of 2,6-dichloro-4-methylpyridine (compound 1, 1.0 g, 6.17 mmol, 1.0 equiv.) in THF (10.0 mL) was added LiHMDS (1N in THF, 8.02 mL) at -70° C. under nitrogen atmosphere. The reaction mixture was stirred for 30 min at -70° C. under nitrogen atmosphere. To the above mixture was added dimethyl carbonate (1.0 g, 11.10 mmol, 1.80 equiv.) at -70° C. under nitrogen atmosphere. The resulting mixture was stirred for 2 h at -70° C. under nitrogen atmosphere. Approximately 50% of the desired product could be detected by LCMS. The reaction was diluted with saturated NH 4 The mixture was quenched with Cl (aq, 100 mL) at 0 °C. The aqueous layer was washed with CH 2 Cl 2 (3×200 mL). The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography eluted with PE / EA (3:1) to give methyl 2-(2,6-dichloropyridin-4-yl)acetate (compound 2, 590 mg, 41%) as an oil. LCMS (ES, m / z): 220, 222 [M+1]+.

[0676] Step 2: Synthesis of Compound 3 To a stirred solution of methyl 2-(2,6-dichloropyridin-4-yl)acetate (compound 2, 5.0 g, 22.72 mmol, 1.0 equiv.) in DMF (50 mL) was added NaH (2.73 g, 68.16 mmol, 3.00 equiv., 60%) in portions at 0 °C under nitrogen atmosphere. The resulting mixture was stirred for 30 min at 0 °C under nitrogen atmosphere. To the above mixture was added 1,3-dibromo-2-methylpropane (7.36 g, 34.08 mmol, 1.5 equiv.) at 0 °C. The resulting mixture was stirred for another 2 h at 10 °C. Approximately 30% of the desired product could be detected by LCMS. The reaction was quenched with water (200 mL) at room temperature. The mixture was acidified to pH 6 with concentrated HCl. The aqueous layer was extracted with CH2Cl2 (3 x 300 mL). The resulting mixture was concentrated under vacuum. The residue was purified by reverse flash chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 70% in 30 min; detector, UV 254 nm. Eluting isomers were analyzed by CH 2 Cl 2 (3×300 mL). The resulting mixture was concentrated under vacuum to provide methyl 1-(2,6-dichloropyridin-4-yl)-3-methylcyclobutane-1-carboxylate (compound 3, 1.9 g, 15%) as a green oil. LCMS: (ES, m / s): 274, 276 [M+H] + .

[0677] Step 3: Synthesis of Compound 4 To a stirred solution of methyl 1-(2,6-dichloropyridin-4-yl)-3-methylcyclobutane-1-carboxylate (compound 3, 1.8 g, 6.56 mmol, 1.0 equiv.) in THF (18 mL) was added LiOH (0.31 g, 13.13 mmol, 2.0 equiv.) (H 2HCl (9.0 mL) was added in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction mixture was adjusted to pH=5 with 1N HCL. The mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 50% in 30 min; detector, UV 254 nm. The aqueous layer was purified by CH 2 Cl 2 (3×300 mL). The resulting mixture was concentrated under vacuum to provide 1-(2,6-dichloropyridin-4-yl)-3-methylcyclobutane-1-carboxylic acid (compound 4, 2.0 g, 83%) as a semi-solid. LCMS: (ES, m / s): 260, 262 [M+H] + .

[0678] Step 4: Synthesis of Compound 5 To a stirred solution of 1-(2,6-dichloropyridin-4-yl)-3-methylcyclobutane-1-carboxylic acid (compound 4, 2.4 g, 9.22 mmol, 1.0 equiv.) in DMF (24 mL) was added HATU (5.26 g, 13.84 mmol, 1.5 equiv.), 1-amino-3-methylthiourea (1.16 g, 11.07 mmol, 1.2 equiv.) and DIEA (2.39 g, 18.45 mmol, 2.0 equiv.) in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 2 h under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction was quenched with water at room temperature. The aqueous layer was separated by CH 2 Cl 2 (3×200 mL). The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography eluted with PE / EA (2:3) to give 1-(2,6-dichloropyridin-4-yl)-3-methyl-N-[(methylcarbamothioyl)amino]cyclobutane-1-carboxamide (2.5 g, 70%) as a white solid. LCMS: (ES, m / s): 347, 349 [M+H] + .

[0679] Step 5: Synthesis of Compound 6 To a stirred solution of 1-(2,6-dichloropyridin-4-yl)-3-methyl-N-[(methylcarbamothioyl)amino]cyclobutane-1-carboxamide (compound 5, 2.3 g, 6.62 mmol, 1.0 equiv.) in THF (11 mL) was added NaOH (1.0 g, 25.00 mmol, 3.77 equiv.) (H 2 HCl) was added in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. Approximately 73% of the desired product could be detected by LCMS. The mixture was acidified to pH 5 with concentrated HCl. The aqueous layer was diluted with CH 2 Cl 2 (3×200 mL). The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography eluted with PE / EA (1:1) to give 5-[1-(2,6-dichloropyridin-4-yl)-3-methylcyclobutyl]-4-methyl-1,2,4-triazole-3-thiol (compound 6, 1.2 g, 55%) as a white solid. LCMS: (ES, m / s): 329, 331 [M+H] + .

[0680] Step 6: Synthesis of Compound 7 To a stirred solution of 5-[1-(2,6-dichloropyridin-4-yl)-3-methylcyclobutyl]-4-methyl-1,2,4-triazole-3-thiol (compound 6, 1.2 g, 3.64 mmol, 1.0 equiv) in THF (12 mL) was added NaNO 2 (0.75g, 10.93mmol, 3.0eq)(H 2 O = 12 mL) and HNO 3 (12 mL, 1.0 mol / L) was added in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction was quenched with water / ice at room temperature. The aqueous layer was diluted with CH 2 Cl 2(3×200 mL). The resulting mixture was concentrated in vacuo. The residue was purified by reverse flash chromatography with the following conditions: column, silica gel; mobile phase, ACN in water (0.05% TFA), gradient from 10% to 50% in 30 min; detector, UV 254 nm. The aqueous layer was purified by CH 2 Cl 2 (3×200 mL). The resulting mixture was concentrated under vacuum to provide 2,6-dichloro-4-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyridine (compound 7, 900 mg, 74%) as a green solid. LCMS: (ES, m / s): 297, 299 [M+H] + .

[0681] Step 7: Synthesis of Compound 8 To a stirred solution of 2,6-dichloro-4-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyridine (compound 7, 400 mg, 1.346 mmol, 1.0 equiv.) and (ethylsulfanyl)sodium (147 mg, 1.75 mmol, 1.3 equiv.) in THF (12 mL) at 0° C. under nitrogen atmosphere. The resulting mixture was stirred at 80° C. for 5 h under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was cooled to room temperature and purified by reverse flash chromatography with the following conditions: column, silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 60% in 30 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum to provide 2-chloro-6-(ethylsulfanyl)-4-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyridine (compound 8, 300 mg, 65%) as a light green oil. LCMS: (ES, m / s): 323, 325 [M+H] + .

[0682] Step 8: Synthesis of Compound 112 To a stirred solution of 2-chloro-6-(ethylsulfanyl)-4-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyridine (compound 8, 150 mg, 0.46 mmol, 1.0 equiv.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 145 mg, 0.46 mmol, 1.0 equiv.) in dioxane (8.0 mL), was added Xantphos (107 mg, 0.18 mmol, 0.4 equiv.), Pd(OAc). 2 (41 mg, 0.18 mmol, 0.4 equiv.) and Cs 2 CO 3 (454 mg, 1.39 mmol, 3.0 equiv) was added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 120° C. for 1 h under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was cooled to room temperature and purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase TFA, ACN in water, gradient from 10% to 50% in 10 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum to provide compound 9. The products were separated by chiral-HPLC with the following conditions (column: mobile phase A: Hex(0.1% 2M NH 3 -MeOH)-HPLC, Mobile phase B: EtOH--HPLC; Flow rate: 20 mL / min; Gradient: 70% B to 70% B in 15.5 min; Wavelength: 220 / 254 nm; RT1 (min): 10.35; RT2 (min): 13.03; Sample solvent: Hex(0.1% 2M NH 3 -MeOH)-HPLC; injection volume: 0.5 mL; the first eluting isomer (RT=10.35 min) was concentrated to dryness and lyophilized to provide 2-[6-(ethylsulfanyl)-4-[(1r,3s)-3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyridin-2-yl]-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 112, 16.1 mg, 5.60%) as a white solid. LCMS: (ES, m / s): 658 [M+H] + .1 H-NMR (300 MHz, DMSO-d 6 ) 8.37 (s, 1H), 8.18 (s, 1H), 7.96(d, J=14.8 Hz, 2H), 7.06(s, 1H), 5.22(s, 2H), 4.69-4.62(m, 2H), 4.53(t, J=7.2 Hz, 1H), 3.65(s, 2H), 3.53(s, 3H), 3.15-3.13(m, 2H), 2.98-2.91(m, 1H), 2.85-2.65(m, 3H), 1.93(d, J=9.6 Hz, 1H), 1.80-1.50(m, 8H), 1.36-1.33(m, 3H), 0.95-0.75(m, 4H).

[0683] Synthesis of compound 113 [ka] Step 1: Synthesis of Compound 2 To a stirred mixture of 2,6-dichloro-4-methylpyridine (compound 1, 1.0 g, 6.17 mmol, 1.0 equiv.) in THF (10.0 mL) was added LiHMDS (1N in THF, 8.02 mL) at -70° C. under nitrogen atmosphere. The reaction mixture was stirred for 30 min at -70° C. under nitrogen atmosphere. To the above mixture was added dimethyl carbonate (1.0 g, 11.10 mmol, 1.80 equiv.) at -70° C. under nitrogen atmosphere. The resulting mixture was stirred for 2 h at -70° C. under nitrogen atmosphere. Approximately 50% of the desired product could be detected by LCMS. The reaction was diluted with saturated NH 4 The mixture was quenched with Cl (aq, 100 mL) at 0 °C. The aqueous layer was washed with CH 2 Cl 2 (3×200 mL). The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography eluted with PE / EA (3:1) to give methyl 2-(2,6-dichloropyridin-4-yl)acetate (compound 2, 590 mg, 41%) as an oil. LCMS (ES, m / z): 220, 222 [M+1] + .

[0684] Step 2: Synthesis of Compound 3 To a stirred solution of methyl 2-(2,6-dichloropyridin-4-yl)acetate (compound 2, 5.0 g, 22.72 mmol, 1.0 equiv.) in DMF (50 mL) was added NaH (2.73 g, 68.16 mmol, 3.00 equiv., 60%) in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 30 min at 0° C. under nitrogen atmosphere. To the above mixture was added 1,3-dibromo-2-methylpropane (7.36 g, 34.08 mmol, 1.5 equiv.) at 0° C. The resulting mixture was stirred for another 2 h at 10° C. Approximately 30% of the desired product could be detected by LCMS. The reaction was quenched with water (200 mL) at room temperature. The mixture was acidified to pH 6 with concentrated HCl. The aqueous layer was separated by CH 2 Cl 2 (3×300 mL). The resulting mixture was concentrated under vacuum. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 70% in 30 min; detector, UV 254 nm. Eluting isomers were analyzed by CH 2 Cl 2 (3×300 mL). The resulting mixture was concentrated under vacuum to provide methyl 1-(2,6-dichloropyridin-4-yl)-3-methylcyclobutane-1-carboxylate (compound 3, 1.9 g, 15%) as a green oil. LCMS: (ES, m / s): 274, 276 [M+H] + .

[0685] Step 3. Synthesis of compound 4 To a stirred solution of methyl 1-(2,6-dichloropyridin-4-yl)-3-methylcyclobutane-1-carboxylate (1.8 g, 6.56 mmol, 1.0 equiv.) in THF (18 mL) was added LiOH (0.31 g, 13.13 mmol, 2.0 equiv.) (H 2HCl (9.0 mL) was added in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction mixture was adjusted to pH=5 with 1N HCL. The mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 50% in 30 min; detector, UV 254 nm. The aqueous layer was purified by CH 2 Cl 2 (3×300 mL). The resulting mixture was concentrated under vacuum to provide 1-(2,6-dichloropyridin-4-yl)-3-methylcyclobutane-1-carboxylic acid (compound 4, 2.0 g, 83%) as a semi-solid. LCMS: (ES, m / s): 260, 262 [M+H] + .

[0686] Step 4: Synthesis of Compound 5 To a stirred solution of 1-(2,6-dichloropyridin-4-yl)-3-methylcyclobutane-1-carboxylic acid (compound 4, 2.4 g, 9.22 mmol, 1.0 equiv.) in DMF (24 mL) was added HATU (5.26 g, 13.84 mmol, 1.5 equiv.), 1-amino-3-methylthiourea (1.16 g, 11.07 mmol, 1.2 equiv.) and DIEA (2.39 g, 18.45 mmol, 2.0 equiv.) in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 2 h under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction was quenched with water at room temperature. The aqueous layer was separated by CH 2 Cl 2 (3×200 mL). The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography eluted with PE / EA (2:3) to give 1-(2,6-dichloropyridin-4-yl)-3-methyl-N-[(methylcarbamothioyl)amino]cyclobutane-1-carboxamide (compound 5, 2.5 g, 70%) as a white solid. LCMS: (ES, m / s): 347,349 [M+H] + .

[0687] Step 5: Synthesis of Compound 6 To a stirred solution of 1-(2,6-dichloropyridin-4-yl)-3-methyl-N-[(methylcarbamothioyl)amino]cyclobutane-1-carboxamide (compound 5, 2.3 g, 6.62 mmol, 1.0 equiv.) in THF (11 mL) was added NaOH (1.0 g, 25.00 mmol, 3.77 equiv.) (H 2 HCl) was added in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. Approximately 73% of the desired product could be detected by LCMS. The mixture was acidified to pH 5 with concentrated HCl. The aqueous layer was diluted with CH 2 Cl 2 (3×200 mL). The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography eluted with PE / EA (1:1) to give 5-[1-(2,6-dichloropyridin-4-yl)-3-methylcyclobutyl]-4-methyl-1,2,4-triazole-3-thiol (compound 6, 1.2 g, 55%) as a white solid. LCMS: (ES, m / s): 329, 331 [M+H] + .

[0688] Step 6: Synthesis of Compound 7 To a stirred solution of 5-[1-(2,6-dichloropyridin-4-yl)-3-methylcyclobutyl]-4-methyl-1,2,4-triazole-3-thiol (compound 6, 1.2 g, 3.64 mmol, 1.0 equiv) in THF (12 mL) was added NaNO 2 (0.75g, 10.93mmol, 3.0eq)(H 2 O = 12 mL) and HNO 3 (12 mL, 1.0 mol / L) was added in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction was quenched with water / ice at room temperature. The aqueous layer was diluted with CH 2 Cl 2(3×200 mL). The resulting mixture was concentrated in vacuo. The residue was purified by reverse flash chromatography with the following conditions: column, silica gel; mobile phase, ACN in water (0.05% TFA), gradient from 10% to 50% in 30 min; detector, UV 254 nm. The aqueous layer was purified by CH 2 Cl 2 (3×200 mL). The resulting mixture was concentrated under vacuum to provide 2,6-dichloro-4-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyridine (compound 7, 900 mg, 74%) as a green solid. LCMS: (ES, m / s): 297, 299 [M+H] + .

[0689] Step 7: Synthesis of Compound 8 To a stirred solution of 2,6-dichloro-4-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyridine (compound 7, 400 mg, 1.346 mmol, 1.0 equiv.) and (ethylsulfanyl)sodium (147 mg, 1.75 mmol, 1.3 equiv.) in THF (12 mL) at 0° C. under nitrogen atmosphere. The resulting mixture was stirred at 80° C. for 5 h under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was cooled to room temperature and purified by reverse flash chromatography with the following conditions: column, silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 60% in 30 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum to provide 2-chloro-6-(ethylsulfanyl)-4-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyridine (compound 8, 300 mg, 65%) as a light green oil. LCMS: (ES, m / s): 323, 325 [M+H] + .

[0690] Step 8. Synthesis of compound 113 To a stirred solution of 2-chloro-6-(ethylsulfanyl)-4-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyridine (compound 8, 150 mg, 0.46 mmol, 1.0 equiv.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 145 mg, 0.46 mmol, 1.0 equiv.) in dioxane (8.0 mL), was added Xantphos (107 mg, 0.18 mmol, 0.4 equiv.), Pd(OAc). 2 (41 mg, 0.18 mmol, 0.4 equiv.) and Cs 2 CO 3 (454 mg, 1.39 mmol, 3.0 equiv) was added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 120° C. for 1 h under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was cooled to room temperature and purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase TFA, ACN in water, gradient from 10% to 50% in 10 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum to provide compound 9. The products were separated by chiral-HPLC with the following conditions (column: mobile phase A: Hex(0.1% 2M NH 3 -MeOH)-HPLC, Mobile phase B: EtOH--HPLC; Flow rate: 20 mL / min; Gradient: 70% B to 70% B in 15.5 min; Wavelength: 220 / 254 nm; RT1 (min): 10.35; RT2 (min): 13.03; Sample solvent: Hex(0.1% 2M NH 3 -MeOH)-HPLC; injection volume: 0.5 mL; the second eluting isomer (RT=13.03 min) was concentrated to dryness and lyophilized to provide 2-[6-(ethylsulfanyl)-4-[(1s,3r)-3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyridin-2-yl]-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 113, 39.4 mg, 13.85%) as a white solid. LCMS: (ES, m / s): 658 [M+H] +, 330[M / 2+H] + . ; 1 H-NMR (300 MHz, DMSO-d 6 ) 8.35 (s, 1H), 8.18 (s, 1H), 7.96-7.93(m, 2H), 7.00(s, 1H), 5.23(s, 2H), 3.64(s, 2H), 3.33(s, 3H), 3.24(s, 2H), 3.20-3.13(m, 2H), 2.83-2.82(m, 1H), 2.73-2.70(m, 2H), 2.56-2.54(m, 2H), 1.96-1.89(m, 1H), 1.75-1.42(m, 5H), 1.40-1.32(m, 3H), 1.16-1.02(m, 3H), 0.95-0.70(m, 4H).

[0691] Synthesis of Compound 114A and Compound 114B [ka] Step 1: Synthesis of Compound 2 1-tert-Butyl 3-methylpropanedioate (compound 11, 35.61 g, 204.44 mmol, 1.25 equiv.) and Cs in acetone (550 mL). 2 CO 3 To a stirred mixture of (106.58 g, 327.11 mmol, 2 equiv.) was added 2,4,6-trichloropyrimidine (compound 1, 30 g, 163.55 mmol, 1 equiv.) dropwise at 10° C. The resulting mixture was stirred at 25° C. for 4 h. LCMS showed that the reaction was complete. The resulting mixture was filtered and the filter cake was washed with EtOAc (3×200 mL). The filtrate was concentrated under reduced pressure. This afforded 1-tert-butyl 3-methyl 2-(2,6-dichloropyrimidin-4-yl)propanedioate (compound 2, 30 g, 57%) as a yellow oil. LCMS (ES, m / z): 321,323 [M+H] + .

[0692] Step 2. Synthesis of compound 3 To a stirred mixture of 1-tert-butyl 3-methyl 2-(2,6-dichloropyrimidin-4-yl)propanedioate (compound 2, 15 g, 46.70 mmol, 1 equiv.) in DCM (150 mL) was added TFA (30 mL) dropwise at 0° C. The resulting mixture was stirred for 3 h at 25° C. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was diluted with saturated NaHCO 3 (aqueous) to pH 8. The resulting mixture was diluted with CH 2 Cl 2 (3×100 mL). The combined organic layers were washed with brine (100 mL) and anhydrous Na 2 SO 4 The mixture was dried at 70° C. for 1 hour. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (6:1) to give methyl 2-(2,6-dichloropyrimidin-4-yl)acetate (compound 3, 6 g, 51%) as a yellow oil. LCMS (ES, m / z): 221, 223 [M+H] + .

[0693] Step 3: Synthesis of Compound 4 To a stirred mixture of methyl 2-(2,6-dichloropyrimidin-4-yl)acetate (compound 3, 5.6 g, 25.33 mmol, 1 equiv.) in THF (80 mL) was added LiHMDS (12.72 g, 76.00 mmol, 3 equiv.) at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 1 h at 0° C. under nitrogen atmosphere. To the above mixture was added methyl 1,3-dibromo-2-methylpropane (compound 12, 10.94 g, 50.66 mmol, 2 equiv.) dropwise at 0° C. The resulting mixture was stirred for an additional 16 h at 25° C. LCMS showed the reaction was complete. The reaction was quenched by the addition of water (100 mL) at room temperature. The resulting mixture was extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (100 mL) and diluted with anhydrous Na 2 SO 4The mixture was dried at 37° C. for 1 hour. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (6:1) to give methyl 1-(2,6-dichloropyrimidin-4-yl)-3-methylcyclobutane-1-carboxylate (compound 4, 2.8 g, 40%) as a yellow oil. LCMS (ES, m / z): 275, 277 [M+H] + .

[0694] Step 4: Synthesis of Compound 5 To a stirred mixture of methyl 1-(2,6-dichloropyrimidin-4-yl)-3-methylcyclobutane-1-carboxylate (compound 4, 2.5 g, 9.08 mmol, 1 equiv.) in THF (20 mL) was added dropwise 2.0 M of ethylamine solution in THF (20 mL) at 0° C. The resulting mixture was stirred for 4 h at 25° C. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (5:1) to provide methyl 1-[2-chloro-6-(ethylamino)pyrimidin-4-yl]-3-methylcyclobutane-1-carboxylate (compound 5, 2.3 g, 70%) as a yellow oil. LCMS (ES, m / z): 284, 286 [M+H] + .

[0695] Step 5: Synthesis of Compound 6 To a stirred mixture of methyl 1-[2-chloro-6-(ethylamino)pyrimidin-4-yl]-3-methylcyclobutane-1-carboxylate (compound 5, 2.3 g, 8.10 mmol, 1 equiv.) in THF (30 mL) was added H 2LiOH (0.23 g, 9.72 mmol, 1.2 equiv) in 0 (10 mL) was added dropwise at 0° C. The resulting mixture was stirred at 25° C. for 16 h. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The mixture was acidified to pH 5 with HCl (aq). The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, water (0.05% TFA), ACN, gradient from 5% to 50% in 30 min; detector, UV 254 nm. This resulted in 1-[2-chloro-6-(ethylamino)pyrimidin-4-yl]-3-methylcyclobutane-1-carboxylic acid (compound 6, 1.4 g, 64%) as a yellow solid. LCMS (ES, m / z): 270, 272 [M+H] + .

[0696] Step 6: Synthesis of Compound 7 To a stirred mixture of 1-[2-chloro-6-(ethylamino)pyrimidin-4-yl]-3-methylcyclobutane-1-carboxylic acid (compound 5, 1.2 g, 4.44 mmol, 1 eq.) and HATU (2.54 g, 6.67 mmol, 1.5 eq.) in DMF (24 mL) was added 1-amino-3-methylthiourea (0.56 g, 5.33 mmol, 1.2 eq.) and DIEA (1.73 g, 13.34 mmol, 3 eq.) at 0° C. The resulting mixture was stirred for 3 h at 25° C. LCMS showed the reaction was complete. The reaction mixture was used directly in the next step without further purification. LCMS (ES, m / z): 357, 359 [M+H] + .

[0697] Step 7: Synthesis of Compound 8 To the above stirred mixture of 1-[2-chloro-6-(ethylamino)pyrimidin-4-yl]-3-methyl-N-[(methylcarbamothioyl)amino]cyclobutane-1-carboxamide (compound 7), add H 2NaOH (313.82 mg, 7.84 mmol, 2 equiv.) in 2H2O (50 mL) was added at 0° C. The resulting mixture was stirred at 50° C. for 1 h. LCMS showed the reaction was complete. The mixture was cooled to room temperature. The resulting mixture was extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (3×100 mL) and washed with anhydrous Na 2 SO 4 The mixture was dried at 37° C. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (1:1) to give 5-{1-[2-chloro-6-(ethylamino)pyrimidin-4-yl]-3-methylcyclobutyl}-4-methyl-1,2,4-triazole-3-thiol (compound 8, 0.6 g, 45%) as a yellow solid. LCMS (ES, m / z): 339, 341 [M+H] + .

[0698] Step 8: Synthesis of Compound 9 To a stirred mixture of 5-{1-[2-chloro-6-(ethylamino)pyrimidin-4-yl]-3-methylcyclobutyl}-4-methyl-1,2,4-triazole-3-thiol (compound 8, 0.58 g, 1.71 mmol, 1 equiv.) in DCM (6 mL), AcOH (1.5 g, 24.97 mmol, 14.59 equiv.), H 2 O 2 (0.5 g, 14.70 mmol, 8.59 equiv) was added portionwise at 0° C. The resulting mixture was stirred at room temperature for 1 h. LCMS showed the reaction was complete. The reaction mixture was diluted with saturated NaHCO 3 (aqueous) to pH 8. The resulting mixture was extracted with EtOAc (3×30 mL). The combined organic layers were washed with brine (30 mL) and anhydrous Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 / MeOH (10:1) to give 2-chloro-N-ethyl-6-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyrimidin-4-amine (compound 9, 0.25 g, 47%) as a yellow solid. LCMS (ES, m / z): 307, 309 [M+H] + .

[0699] Step 9: Compound 114A and Compound 114B 2-Chloro-N-ethyl-6-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyrimidin-4-amine (compound 9, 240 mg, 0.78 mmol, 1 equiv.), 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 256 mg, 0.82 mmol, 1.05 equiv.), Pd(OAc) in dioxane (5 mL). 2 (17 mg, 0.078 mmol, 0.1 equiv.), Xantphos (90 mg, 0.15 mmol, 0.2 equiv.) and Cs 2 CO 3 A mixture of (764 mg, 2.34 mmol, 3 equiv) was stirred for 3 h at 120 °C under nitrogen atmosphere. LCMS showed the reaction was complete. The mixture was cooled to room temperature. The resulting mixture was filtered and the filter cake was washed with EtOAc (3 x 20 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 The crude product was purified by silica gel column chromatography eluted with 100% hexane / MeOH (10:1). The crude product was repurified by reverse flash chromatography under the following conditions: column, C18 silica gel; mobile phase, water (0.05% TFA), ACN, gradient from 5% to 100% in 30 min; detector, UV 254 nm. The product was separated by Prep-SFC under the following conditions: column: CHIRALPAK IA, 3×25 cm, 5 μm; mobile phase A: CO 2 , mobile phase B:ACN:MeOH=1:1(1% 2M NH 3-MeOH); flow rate: 100 mL / min; gradient: isocratic 30% B; column temperature: 35° C.; back pressure (bar): 100; wavelength: 220 nm; RT1 (min): 3.58; RT2 (min): 4.88; the first eluting isomer (RT1=3.58 min) was concentrated and lyophilized to provide 2-[4-(ethylamino)-6-[(1s,3R)-3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyrimidin-2-yl]-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 114A, 19.8 mg, 4%) as a white solid. LCMS (ES, m / z): 583 [M+H] + ; 1 H-NMR (CD 3 OD, 400 MHz) δ (ppm):8.40 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 6.20 (brs, 1H), 5.21 (s, 2H), 4.77-4.72 (m, 2H), 4.60-4.56 (m, 1H), 3.86-3.79 (m, 5H), 3.52 (brs, 1H), 3.08-2.84 (m, 4H), 2.12 (brs, 1H), 1.86-1.56 (m, 8H), 1.35-1.20 (m, 4H), 1.05-0.85 (m, 4H).

[0700] The second eluting isomer (RT1=4.88 min) was concentrated and lyophilized to provide 2-[4-(ethylamino)-6-[(1r,3S)-3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyrimidin-2-yl]-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 114B, 31.8 mg, 6%) as a colorless oil. LCMS (ES, m / z): 583 [M+H] + ; 1 H-NMR (CD 3OD, 400 MHz) δ (ppm): 8.56 (s, 1H), 8.35 (s, 1H), 8.26 (s, 1H), 6.42 (s, 1H), 5.32 (s, 2H), 4.70-4.68 (m, 2H), 4.56 (s, 2H), 3.86 (s, 3H), 3.61-3.56 (m, 1H), 3.53-3.48 (m, 1H), 3.46-3.42 (m, 1H), 3.15-2.86 (m, 3H), 2.76-2.65 (m, 1H), 2.00-1.84 (m, 6H), 1.31-1.29 (m, 6H), 1.06-0.94 (m, 3H), 0.96-0.8 (m, 1H).

[0701] Synthesis of compound 115 [ka] Step 1: Synthesis of Compound 1 To a stirred mixture of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 1.05 g, 3.52 mmol, 1.10 equiv.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 1 g, 3.20 mmol, 1.00 equiv.) in dioxane (10 mL), Cs 2 CO 3 (2.09 g, 6.40 mmol, 2 equiv.) and Xantphos (0.37 g, 0.64 mmol, 0.2 equiv.) and dioxane (10 mL) were added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 h at 120° C. under nitrogen atmosphere. The mixture was cooled to room temperature. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was purified by HPLC with CH 2 Cl 2 (3×20 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 / MeOH (10:1) to give 2-(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 1, 630 mg, 29%) as a green solid. LCMS (ES, m / z): 575,577 [M+H] + .

[0702] Step 2: Synthesis of Compound 115 To a stirred solution of 2-(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 1, 300 mg, 0.52 mmol, 1 equiv.) in DMF (2 mL) was added Zn(CN). 2 (73 mg, 0.62 mmol, 1.2 equiv.) and Pd(PPh 3 ) 4 (60 mg, 0.05 mmol, 0.1 equiv) was added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 120° C. for 2 h under nitrogen atmosphere. The mixture was cooled to room temperature. LCMS showed the reaction was complete. The reaction mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), gradient from 0% to 50% in 30 min; detector, UV 254 nm. The collected fractions were concentrated under vacuum to provide 4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-(6-{[(3S)-3-methylpiperidin-1-yl]methyl}-1-oxo-4-(trifluoromethyl)-3H-isoindol-2-yl)pyridine-2-carbonitrile (compound 115, 270 mg, 74%) as an off-white solid. LCMS (ES, m / z): 566 [M+H-TFA] + ; 1 H NMR (400 MHz, DMSO-d6 ) δ9.72 (br s, 1H), 8.84 (d, J=1.2Hz, 1H), 8.48 (s, 1H), 8.32-8.26 (m, 2H), 7.94 (s, 1H), 5.31 (s, 2H), 4.93-4.87 (m, 4H), 4.63-4.54 (m, 2H), 3.71 (s, 2H), 3.49 (s, 3H), 3.40-3.37 (m, 1H), 3.30-3.28 (m, 1H), 2.88-2.85 (m, 1H), 2.62-2.57 (m, 1H), 1.87-1.63 (m, 4H), 1.09-1.05 (m, 1H), 0.94 (d, J=15.6Hz, 3H).

[0703] Synthesis of compound 116 [ka] Step 1. Synthesis of Compound 1 To a stirred mixture of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 1.05 g, 3.52 mmol, 1.10 equiv.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 1 g, 3.20 mmol, 1.00 equiv.) in dioxane (10 mL), Cs 2 CO 3 (2.09 g, 6.40 mmol, 2 equiv.) and Xantphos (0.37 g, 0.64 mmol, 0.2 equiv.) and dioxane (10 mL) were added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 h at 120° C. under nitrogen atmosphere. The mixture was cooled to room temperature. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was purified by HPLC with CH 2 Cl 2 (3×20 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 / MeOH (10:1) to afford 2-(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (630 mg, 29%) as a green solid. LCMS (ES, m / z): 575,577 [M+H] + .

[0704] Step 2: Synthesis of Compound 2 To a stirred solution of 2-(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 1, 300 mg, 0.52 mmol, 1 equiv.) in DMF (2 mL) was added Zn(CN). 2 (73 mg, 0.62 mmol, 1.2 equiv.) and Pd(PPh 3 ) 4 (60 mg, 0.05 mmol, 0.1 equiv) was added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 120° C. for 2 h under nitrogen atmosphere. The mixture was cooled to room temperature. LCMS showed the reaction was complete. The reaction mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), gradient from 0% to 50% in 30 min; detector, UV 254 nm. The collected fractions were concentrated under vacuum to provide 4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-(6-{[(3S)-3-methylpiperidin-1-yl]methyl}-1-oxo-4-(trifluoromethyl)-3H-isoindol-2-yl)pyridine-2-carbonitrile (compound 2, 270 mg, 74%) as an off-white solid. LCMS (ES, m / z): 566 [M+H] + .

[0705] Step 3: Synthesis of Compound 3 To a stirred solution of 4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-(6-{[(3S)-3-methylpiperidin-1-yl]methyl}-1-oxo-4-(trifluoromethyl)-3H-isoindol-2-yl)pyridine-2-carbonitrile (compound 2, 140 mg, 0.24 mmol, 1 equiv.) in MeOH (4 mL) was added Raney·Ni (20 mg) portionwise at room temperature. The resulting mixture was stirred overnight at room temperature under a hydrogen atmosphere. LCMS indicated the reaction was complete. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to provide 2-[6-(aminomethyl)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl]-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 3, 120 mg, 85%) as a yellow oil. The crude product was used directly in the next step without further purification. LCMS (ES, m / z): 570 [M+H] + .

[0706] Step 4: Synthesis of Compound 116 To a stirred solution of 2-[6-(aminomethyl)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl]-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 3, 120 mg, 0.21 mmol, 1 equiv.) in DCM (2 mL) was added acryloyl chloride (28 mg, 0.31 mmol, 1.5 equiv.) and TEA (64 mg, 0.63 mmol, 3 equiv.) dropwise at 0° C. The resulting mixture was stirred for 2 h at 0° C. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by Prep-HPLC with the following conditions (Column: XBridge Prep C18 OBD Column, 19*150 mm, 5 μm; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 25 mL / min; Gradient: 25% B to 53% B, 53% B in 7 min; Wavelength: 254 nm; RT1 (min): 6.30; The collected fractions were lyophilized to provide N-[(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}-6-(6-{[(3S)-3-methylpiperidin-1-yl]methyl}-1-oxo-4-(trifluoromethyl)-3H-isoindol-2-yl)pyridin-2-yl)methyl]prop-2-enamide (compound 116, 7.7 mg, 5%) as a white solid. LCMS (ES, m / z): 624 [M+H] + ; 1 H NMR (400 MHz, DMSO-d 6) δ 8.66 (br s, 1H), 8.20 (s, 2H), 7.99-7.94 (m, 2H), 6.93 (s, 1H), 6.32-6.30 (m, 1H), 6.17-6.17 (s, 1H), 5.67-5.64 (m, 1H), 5.19 (s, 2H), 4.97 (d, J= 6.0Hz, 2H), 4.81 (m, d, J= 6.0Hz, 2H), 4.42 (d, J= 6.0Hz, 2H), 3.65 (s, 2H), 3.56 (s, 2H), 3.22(s, 3H), 2.71-2.70 (m, 2H), 1.98-1.88 (m, 1H), 1.63-1.58(m,4H), 1.50-1.47 (m, 1H), 0.83-0.81 (m, 4H).

[0707] Synthesis of compound 117 [ka] Step 1: Synthesis of Compound 3 To a stirred mixture of 4-(bromomethyl)-3-(methoxycarbonyl)-5-(trifluoromethyl)benzoic acid (compound 1, 6 g, 17.5 mmol, 1 equiv.) in THF (300 mL) was added 4-methoxybenzenemethanamine (compound 2, 6.00 g, 43.80 mmol, 2.49 equiv.) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction was quenched with water. The resulting mixture was extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (100 mL) and diluted with anhydrous Na 2 SO 4 After filtration, the filtrate was concentrated under reduced pressure. This gave 2-[(4-methoxyphenyl)methyl]-3-oxo-7-(trifluoromethyl)-1H-isoindole-5-carboxylic acid (compound 3, 1.6 g, 45%) as a light yellow solid. LCMS (ESI, m / z): 366 [M+H] +

[0708] Step 2. Synthesis of compound 5 To a stirred mixture of 2-[(4-methoxyphenyl)methyl]-3-oxo-7-(trifluoromethyl)-1H-isoindole-5-carboxylic acid (compound 3, 2.5 g, 6.84 mmol, 1 equiv.) and HATU (4.01 g, 10.54 mmol, 1.54 equiv.) in DMF (40 mL) was added N,O-dimethylhydroxylamine hydrochloride (compound 4, 1.00 g, 10.26 mmol, 1.50 equiv.) and DIEA (2.75 g, 21.28 mmol, 3.11 equiv.) at room temperature. The resulting mixture was stirred overnight at room temperature. LCMS showed the reaction was complete. The resulting mixture was diluted with water (5 mL). The resulting mixture was extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (5 mL) and diluted with anhydrous NaCl. 2 SO 4 The mixture was dried at 37° C. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (5:1) to give N-methoxy-2-[(4-methoxyphenyl)methyl]-N-methyl-3-oxo-7-(trifluoromethyl)-1H-isoindole-5-carboxamide (compound 5, 2.5 g, 89%) as a colorless oil. LCMS: (ms, ESI): 409 [M+H]+

[0709] Step 3: Synthesis of Compound 6 To a stirred mixture of N-methoxy-2-[(4-methoxyphenyl)methyl]-N-methyl-3-oxo-7-(trifluoromethyl)-1H-isoindole-5-carboxamide (compound 5, 1 g, 2.44 mmol, 1 equiv.) in THF (20 mL) was added MeMgBr (5 mL, 1N in THF, 5 mmol) dropwise at -5°C under nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere. LCMS indicated the reaction was complete. The reaction was purified with saturated NH 4 The mixture was quenched at room temperature by the addition of Cl(aq) (20 mL). The resulting mixture was extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (20 mL) and anhydrous Na 2 SO 4The mixture was dried at 70° C. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (5:1) to give 6-acetyl-2-[(4-methoxyphenyl)methyl]-4-(trifluoromethyl)-3H-isoindol-1-one (compound 6, 880 mg, 98%) as a yellow oil. LCMS: (ms, ESI): 364 [M+H]+

[0710] Step 4: Synthesis of Compound 7 To a stirred mixture of 6-acetyl-2-[(4-methoxyphenyl)methyl]-4-(trifluoromethyl)-3H-isoindol-1-one (compound 6, 880 mg, 1.21 mmol, 1 equiv, 50%) in EtOH (10 mL) was added CuBr 2 (1350 mg, 6.04 mmol, 4.99 equiv) was added at room temperature. The resulting mixture was stirred at 80° C. for 1 h under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (20 mL) and washed with anhydrous Na 2 SO 4 The mixture was dried at 70° C. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (3:1) to give 6-(2-bromoacetyl)-2-[(4-methoxyphenyl)methyl]-4-(trifluoromethyl)-3H-isoindol-1-one (compound 7, 380 mg, 70%) as a yellow solid. LCMS (ms, ESI): 442, 444 [M+H] +

[0711] Step 5: Synthesis of Compound 8 To a stirred solution of sodium methanethiolate (compound 7, 114.23 mg, 1.631 mmol, 1.5 equiv) in MeOH (7 mL) was added 6-(2-bromoacetyl)-2-[(4-methoxyphenyl)methyl]-4-(trifluoromethyl)-3H-isoindol-1-one (350 mg, 1.08 mmol, 1 equiv) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 10 min at 0° C. under nitrogen atmosphere. LCMS indicated the reaction was complete. The reaction was quenched with water / ice at 0° C. The precipitated solid was collected by filtration and diluted with Et 2 2H-isoindol-1-one (compound 8, 280 mg, 86%) as a yellow solid. LCMS (ESI, ms): 410 [M+H]. +

[0712] Step 6: Synthesis of Compound 9 To a stirred solution of 2-[(4-methoxyphenyl)methyl]-6-[2-(methylsulfanyl)acetyl]-4-(trifluoromethyl)-3H-isoindol-1-one (compound 8, 280 mg, 0.48 mmol, 1 equiv) in DCM (7 mL) was added NaBH 4 (36.96 mg, 0.97 mmol, 2 equiv) was added in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 2 h at 0° C. under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction was quenched with water at 0° C. The resulting mixture was diluted with CH 2 Cl 2 (3×10 mL). The combined organic layers were washed with brine (50 mL) and anhydrous Na 2 SO 4 After filtration, the filtrate was concentrated under reduced pressure. This gave 6-[1-hydroxy-2-(methylsulfanyl)ethyl]-2-[(4-methoxyphenyl)methyl]-4-(trifluoromethyl)-3H-isoindol-1-one (compound 9, 200 mg, 59%) as a white solid. LCMS (ESI, ms): 292 [M+H] +

[0713] Step 7: Synthesis of Compound 10 To a stirred mixture of 6-[1-hydroxy-2-(methylsulfanyl)ethyl]-2-[(4-methoxyphenyl)methyl]-4-(trifluoromethyl)-3H-isoindol-1-one (compound 9, 200 mg, 0.48 mmol, 1 equiv.) and DIEA (188 mg, 1.45 mmol, 3 equiv.) in DCM (1 mL) was added TsCl (139 mg, 0.72 mmol, 1.5 equiv.) and DMAP (6 mg, 0.05 mmol, 0.1 equiv.) dropwise at 0° C. The resulting mixture was stirred for 16 h at 25° C. The reaction was quenched by the addition of water (5 mL) at room temperature. The resulting mixture was diluted with CH 2 Cl 2 (3×10 mL). The combined organic layers were washed with brine (3×10 mL) and extracted with anhydrous Na 2 SO 4 The mixture was dried at 37° C. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EA (1:1) to give 1-{2-[(4-methoxyphenyl)methyl]-3-oxo-7-(trifluoromethyl)-1H-isoindol-5-yl}-2-(methylsulfanyl)ethyl 4-methylbenzenesulfonate (compound 10, 120 mg, 43%) as a yellow solid. LCMS (ES, m / z): 566 [M+H] +

[0714] Step 8. Synthesis of compound 12 To a stirred mixture of 1-{2-[(4-methoxyphenyl)methyl]-3-oxo-7-(trifluoromethyl)-1H-isoindol-5-yl}-2-(methylsulfanyl)ethyl 4-methylbenzenesulfonate (compound 10, 120 mg, 0.21 mmol, 1 equiv.) and (3S)-3-methylpiperidine hydrochloride (43 mg, 0.31 mmol, 1.5 equiv.) in DCM (3 mL) was added TEA (42 mg, 0.42 mmol, 2 equiv.) at 0° C. The resulting mixture was stirred at 60° C. for 16 h. LCMS showed the reaction was complete. The reaction was quenched by the addition of water (10 mL) at room temperature. The resulting mixture was diluted with CH 2 Cl 2 (3×10 mL). The combined organic layers were washed with brine (3×15 mL) and extracted with anhydrous Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 / MeOH (10:1) to give 2-[(4-methoxyphenyl)methyl]-6-{1-[(3S)-3-methylpiperidin-1-yl]-2-(methylsulfanyl)ethyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 12, 80 mg, 76%) as a yellow solid. LCMS (ES, m / z): 493 [M+H] +

[0715] Step 9: Synthesis of Compound 13 To a stirred mixture of 2-[(4-methoxyphenyl)methyl]-6-{1-[(3S)-3-methylpiperidin-1-yl]-2-(methylsulfanyl)ethyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 12, 75 mg, 0.15 mmol, 1 equiv.) in DCM (100 uL) was added TFA (30 uL) at 0° C. The resulting mixture was stirred for 2 h at 25° C. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, water 0.05% TFA, ACN, gradient from 5% to 50% in 30 min; detector, UV 254 nm. This resulted in 6-{1-[(3S)-3-methylpiperidin-1-yl]-2-(methylsulfanyl)ethyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one; trifluoroacetate (compound 13, 50 mg, 67%) as a yellow oil. LCMS (ES, m / z): 373 [M+H] +

[0716] Step 10: Synthesis of Compound 117 6-{1-[(3S)-3-methylpiperidin-1-yl]-2-(methylsulfanyl)ethyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (compound 13, 40 mg, 0.10 mmol, 1 equiv.), 6-chloro-N-ethyl-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-amine (prepared as described in WO2020210508, 36 mg, 0.11 mmol, 1.1 equiv.), Ruphos (20 mg, 0.04 mmol, 0.4 equiv.), RuPhos Palladacycle Gen. 3 (18 mg, 0.02 mmol, 0.2 equiv.) and Cs in dioxane (1.2 mL). 2 CO 3A mixture of (70 mg, 0.21 mmol, 2 equiv) was stirred for 16 h at 120° C. under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was cooled to room temperature, filtered, and the filter cake was washed with EtOAc (3×10 mL). The filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, water (0.05% TFA), ACN, gradient from 5% to 50% in 30 min; detector, UV 254 nm. The crude product was re-purified by Prep-HPLC with the following conditions: Column: XBridge Shield RP18 OBD Column, 19×250 mm, 10 μm; Mobile phase A: water (0.05% TFA), Mobile phase B: ACN; Flow rate: 25 mL / min; Gradient: 32% B to 52% B, 52% B in 7 min; Wavelength: 254 nm; RT1 (min): 4.03. The collected fractions were lyophilized to provide 2-[6-(ethylamino)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl]-6-{1-[(3S)-3-methylpiperidin-1-yl]-2-(methylsulfanyl)ethyl}-4-(trifluoromethyl)-3H-isoindol-1-one; trifluoroacetate (compound 117, 8.8 mg, 10%) as a colorless oil. LCMS (ES, m / z): 644 [M+H] + ; 1 H-NMR (CD 3OD, 400 MHz) δ (ppm):8.98-8.75 (m, 1H), 8.28 (s, 1H), 8.23-8.10 (m, 1H), 7.25-7.11 (m, 1H), 6.05-5.92 (m, 1H), 5.32 (s, 2H), 5.02-4.98 (m, 4H), 4.90 (s, 2H), 4.83-4.80 (m, 1H), 3.83-3.70 (m, 3H), 3.52-3.45 (m, 2H), 3.40-3.38 (m, 4H), 2.86-2.70 (m, 1H), 2.63-2.43 (m, 1H), 2.11 (s, 3H), 2.03-1.72 (m, 4H), 1.27-1.23 (m, 3H), 1.18-1.08 (m, 1H), 1.02-0.96 (m, 3H).

[0717] Synthesis of compound 118 [ka] Step 1: Synthesis of Compound 2 To a stirred mixture of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 1.05 g, 3.52 mmol, 1.10 equiv.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 1 g, 3.20 mmol, 1.00 equiv.) in dioxane (10 mL), Cs 2 CO 3 (2.09 g, 6.40 mmol, 2 equiv.) and Xantphos (0.37 g, 0.64 mmol, 0.2 equiv.) and Pd(OAc) 2 (112 mg, 0.50 mmol, 0.15 equiv) was added portionwise at room temperature. The resulting mixture was stirred at 120° C. for 2 h under nitrogen atmosphere. The mixture was cooled to room temperature. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was purified by HPLC with CH 2 Cl 2 (3×20 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl2 / MeOH (10:1) to give 2-(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 2, 630 mg, 29%) as a green solid. LCMS: (ES.m / z): 575,577 [M+1] + .

[0718] Step 2: Synthesis of Compound 118 To a stirred mixture of 2-(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 2, 100 mg, 0.17 mmol, 1 equiv.) and 2-azidoethanamine (compound 1, 22 mg, 0.26 mmol, 1.5 equiv.) in dioxane (1 mL), Cs 2 CO 3 (113 mg, 0.34 mmol, 2 equiv.) and Xantphos (20 mg, 0.03 mmol, 0.2 equiv.) and Pd(OAc) 2 (4 mg, 0.02 mmol, 0.1 equiv) was added portionwise at room temperature. The resulting mixture was stirred for 1.5 h at 90° C. under nitrogen atmosphere. LCMS showed 50% product by LCMS. The resulting mixture was filtered and the filter cake was washed with DCM (3×50 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 / MeOH (10:1) to give 2-{6-[(2-azidoethyl)amino]-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl}-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (30 mg, crude). The crude product (30 mg) was purified by Prep-HPLC under the following conditions (Column: XSelect CSH Prep C18 OBD Column, 19*250mm, 5μm; Mobile phase A: water (0.05% TFA), Mobile phase B: ACN; Flow rate: 25mL / min; Gradient: 20%B to 35%B to 35%B in 10min; Wavelength: 254nm; RT1(min): 8.77; The collected fractions were lyophilized to provide 2-{6-[(2-azidoethyl)amino]-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl}-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one (compound 118, 8.5mg, 8%) as a yellow solid. LCMS: (ES.m / z): 625[M+1] + ; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.81 (s, 1H), 8.45 (s, 1H), 8.27-8.19 (m, 2H), 7.44 (s, 1H), 6.98 (br s, 1H), 6.07 (s, 1H), 5.24 (s, 2H), 4.91-4.85 (m, 2H), 4.79-4.67 (m, 2H), 4.52 (s, 2H), 3.55 (s, 1H), 3.49-3.48 (m, 4H), 3.39-3.36 (m, 1H), 3.29-3.21 (m, 4H), 2.86-2.84 (m, 1H), 2.63-2.56 (m, 1H), 2.49-2.48 (m, 1H), 1.85-1.62 (m, 4H), 1.10-1.12 (m, 1H), 0.94 (d, J=5.6Hz, 3H)

[0719] Synthesis of compound 119 [ka] Step 1: Synthesis of Compound 4 To a stirred solution of aminoacetonitrile hydrochloride (500 mg, 5.4 mmol, 1 equiv.) in dioxane (10 mL) was added 1M aqueous NaOH (217 mg, 5.4 mmol, 1 equiv.). The reaction was cooled to 0° C. and di-tert-butyl dicarbonate (1.18 gr, 5.4 mmol, 1 equiv.) dissolved in 5 mL of dioxane was added dropwise. The resulting mixture was stirred vigorously overnight. In the morning, a TLC control showed complete consumption of the starting material. The reaction was purified by elution with saturated NaHCO 3 The mixture was quenched with an aqueous solution of 0.1 ml of ethyl acetate and extracted with dichloromethane (3×100 mL). The combined organic layers were washed with brine (3×100 mL) and diluted with Na 2 SO 4 The mixture was dried at rt and concentrated in vacuo to provide tert-butyl (cyanomethyl)carbamate (compound 4, 800 mg, 95%) as a colorless oil. 1 H NMR (400 MHz, CDCl 3 ) δ 5.03 (s, 1H), 4.06 (m, 2H), 1.44 (s, 9H).

[0720] Step 2: Synthesis of Compound 1 To a stirred mixture of 2,6-dichloro-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridine (INT3, 235 mg, 0.786 mmol, 1 equiv.) and (S)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one (INT4, 245 mg, 0.786 mmol, 1 equiv.) in dioxane (7 mL), Cs 2 CO 3 (512 mg, 1.57 mmol, 2 equiv.) and Xantphos (90.8 mg, 0.157 mmol, 0.2 equiv.) and Pd(OAc) 2(17.6 mg, 0.0786 mmol, 0.1 equiv) was added portionwise at room temperature. The resulting mixture was stirred at 90° C. for 1 h under nitrogen atmosphere. The mixture was cooled to room temperature. LCMS showed the reaction was complete. The reaction was quenched with saturated sodium bicarbonate at room temperature. The resulting mixture was diluted with CH 2 Cl 2 (3×50 ml). The combined organic layers were concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 / MeOH (9:1) to give (S)-2-(6-chloro-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-yl)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one (compound 1, 300 mg, 66.4%) as a green solid. LCMS: (ES.m / z): 575,577 [M+1] + .

[0721] Step 3: Synthesis of Compound 2 To a stirred mixture of (S)-2-(6-chloro-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-yl)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one (compound 1, 40 mg, 0.07 mmol, 1 equiv.) and tert-butyl (cyanomethyl)carbamate (compound 4, 110 mg, 0.7 mmol, 10 equiv.) in dioxane (1.2 mL), was added Cs 2 CO 3 (45 mg, 0.14 mmol, 2 equiv.) and Xantphos (8.1 mg, 0.014 mmol, 0.2 equiv.) and Pd(OAc) 2 (1.6 mg, 0.007 mmol, 0.1 equiv) was added portionwise at room temperature. The resulting mixture was stirred for 40 min at 120° C. under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 / MeOH (10:1) to give tert-butyl (S)-(cyanomethyl)(4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)-6-(6-((3-methylpiperidin-1-yl)methyl)-1-oxo-4-(trifluoromethyl)isoindolin-2-yl)pyridin-2-yl)carbamate (compound 2, 25 mg, 52%) as a white solid. LCMS: (ES.m / z): 695[M+1] + .

[0722] Step 4: Synthesis of Compound 119 tert-Butyl (S)-(cyanomethyl)(4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)-6-(6-((3-methylpiperidin-1-yl)methyl)-1-oxo-4-(trifluoromethyl)isoindolin-2-yl)pyridin-2-yl)carbamate (compound 2, 25 mg, 0.042 mmol) was dissolved in a 1 / 1 mixture of dioxane / water (1 ml) and heated at 120 C for 15 min in a microwave initiator. The product was extracted with dichloromethane and eluted in CH 2 Cl 2 / MeOH (3:1) to give (S)-2-((4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)-6-(6-((3-methylpiperidin-1-yl)methyl)-1-oxo-4-(trifluoromethyl)isoindolin-2-yl)pyridin-2-yl)amino)acetonitrile (7 mg, 30%) as a white solid. LCMS: (ES.m / z): 595[M+1] + ; 1 H NMR (300 MHz, DMSO-d 6) δ 8.25 (s, 1H), 7.95 (d, J = 17.2 Hz, 2H), 7.61 (d, J = 1.2 Hz, 1H), 7.29 (t, J = 5.8 Hz, 1H), 6.18 (d, J = 1.2 Hz, 1H), 5.27 - 5.22 (m, 2H), 4.92 (d, J = 6.1 Hz, 2H), 4.80 (d, J = 6.1 Hz, 2H), 4.29 (d, J = 5.7 Hz, 2H), 3.66 (s, 2H), 3.52 (s, 2H), 3.26 (s, 3H), 2.72 (d, J = 8.7 Hz, 2H), 2.01 - 1.88 (m, 1H), 1.66 (d, J = 13.1 Hz, 3H), 1.59 (s, 1H), 1.48 (d, J = 11.8 Hz, 1H), 1.23 (s, 4H), 0.89 (m, 4H).

[0723] Synthesis of compound 120 [ka] Step 1: Synthesis of Compound 2 To a stirred solution of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 200 mg, 0.66 mmol, 1 equiv.) and 3-mercaptopropane-1,2-diol (compound 1A, 86 mg, 0.80 mmol, 1.2 equiv.) in DMF (2 mL) was added K 2 CO 3(184 mg, 1.33 mmol, 2 equiv) was added at room temperature under nitrogen atmosphere. The resulting mixture was stirred overnight at 50° C. under nitrogen atmosphere. LCMS showed the reaction was complete. The mixture was cooled to room temperature. The reaction mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 5% to 50% in 30 min; detector, UV 254 nm. The collected fractions were concentrated to provide 3-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)sulfanyl]propane-1,2-diol (compound 2, 190 mg, 76%) as a white solid. LCMS (ESI, ms): 370,372 [M+H] +

[0724] Step 2: Synthesis of Compound 120 To a stirred solution of 3-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)sulfanyl]propane-1,2-diol (compound 2, 100 mg, 0.27 mmol, 1 equiv.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 92 mg, 0.29 mmol, 1.1 equiv.) in 1,4-dioxane (2 mL), Cs 2 CO 3 (263 mg, 0.810 mmol, 3 equiv.), XantPhos (31 mg, 0.05 mmol, 0.2 equiv.) and Pd(OAc). 2(6 mg, 0.03 mmol, 0.1 equiv) was added at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 120° C. for 1 h under nitrogen atmosphere. LCMS showed the reaction was complete. The mixture was cooled to room temperature. The mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 5% to 50% in 30 min; detector, UV 254 nm. The collected fractions were lyophilized to provide 2-{6-[(2,3-dihydroxypropyl)sulfanyl]-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl}-6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-3H-isoindol-1-one; formic acid (33.8 mg, 17%) as a white solid. LCMS (ESI, ms): 646 [M+H-FA] + ; 1 H NMR (300 MHz, DMSO-d 6 ) δ 8.25 (s, 1H), 8.14 (s, 1H), 8.03 (s, 1H), 7.99 (s, 1H), 7.94 (s, 1H), 6.96 (s, 1H), 5.24 (s, 2H), 4.92 (d, J = 6.3 Hz, 2H), 4.84 (d, J = 6.3 Hz, 2H), 3.67-3.64 (m, 4H), 3.57 (s, 3H), 3.48- 3.41 (m, 4H), 3.07-2.97 (m, 1H), 2.76- 2.70 (m, 2H), 1.96-1.93 (m, 1H), 1.72-1.50 (m, 5H), 0.88-0.75 (m, 4H).

[0725] Synthesis of compound 121 [ka] To a stirred mixture of (S)-2-(4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)-6-(piperazin-1-yl)pyridin-2-yl)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one (compound 140, 35 mg, 0.056 mmol, 1 equiv.) and triethylamine (7.4 mg, 0.073 mmol, 1.3 equiv.) in dichloromethane (1.5 mL) pre-cooled at 0° C. was added acryloyl chloride (6.6 mg, 0.073 mmol, 1.3 equiv.) in dichloromethane (1.5 mL). The resulting mixture was allowed to reach room temperature and stirred for 2 h. LCMS indicated the reaction was complete. The reaction was quenched with saturated sodium bicarbonate at room temperature. The resulting mixture was 2 Cl 2 (3×50 ml). The combined organic layers were concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 / MeOH (3:1) to provide (S)-2-(6-(4-acryloylpiperazin-1-yl)-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridin-2-yl)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one (compound 121, 15 mg, 39%) as a white solid. LCMS: (ES.m / z): 679 [M+1] + ; 1 H NMR (400 MHz, DMSO-d 6) δ 10.72 (s, 1H), 8.34 (s, 1H), 8.30 (s, 1H), 8.23 ​​(s, 1H), 7.56 (d, J = 1.0 Hz, 1H), 6.88 (dd, J = 16.7, 10.4 Hz, 1H), 6.33 (s, 1H), 6.15 (dd, J = 16.7, 2.4 Hz, 1H), 5.71 (dd, J = 10.4, 2.4 Hz, 1H), 5.22 (s, 2H), 4.92 (d, J = 6.2 Hz, 2H), 4.84 (d, J = 6.2 Hz, 2H), 4.49 (s, 1H), 3.69 (s, 2H), 3.64 (s, 2H), 3.56 (s, 2H), 3.53 (s, 4H), 3.18 (d, J = 15.2 Hz, 4H), 3.06 (qd, J = 7.3, 4.8 Hz, 3H), 2.79 (s, 1H), 2.55 (s, 1H), 1.96 (s, 1H), 1.79 (s, 1H), 1.73 (d, J = 13.7 Hz, 2H), 1.25 - 1.14 (m, 6H), 0.85 (t, J = 5.0 Hz, 4H).

[0726] Synthesis of compound 122 [ka] Step 1: Synthesis of Compound 2 To a stirred solution of methyl 3-cyanopropanoate (compound 1, 5.0 g, 44.20 mmol, 1.0 equiv.) in THF (50 mL) at room temperature under a nitrogen atmosphere. The above mixture was added with LiBH 4 (0.58 g, 26 mmol, 0.6 equiv) was added portionwise at room temperature. The resulting mixture was stirred overnight at 80° C. under nitrogen atmosphere. TLC showed the reaction was complete. The reaction was quenched with water / ice at room temperature. The aqueous layer was extracted with EA (3×300 mL). The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography eluted with PE / EA (1:1) to afford 4-hydroxybutanenitrile (700 mg, 18%) as a pale oil. 1H-NMR (300 MHz, DMSO-d 6 ) 4.68(t, J=6 Hz, 1H), 3.49-3.42 (m, 2H), 2.53-2.48 (m, 2H), 1.74-1.65 (m, 2H).

[0727] Step 2: Synthesis of Compound 3 To a stirred solution of 4-hydroxybutanenitrile (compound 2, 63 mg, 0.73 mmol, 1.10 equiv.) in DMF (4.0 mL) was added NaH (54 mg, 1.33 mmol, 2.00 equiv., 60%) at 0° C. under nitrogen atmosphere. To the above mixture was added 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridine (INT3, 200 mg, 0.66 mmol, 1.00 equiv.) in portions over 30 min at 0° C. The resulting mixture was stirred at room temperature for another 2 h. LCMS showed the reaction was complete. The residue was purified by reverse flash chromatography with the following conditions: column, silica gel; mobile phase FA, MeCN in water, gradient from 10% to 50% in 40 min; detector, UV 254 nm. The resulting mixture was concentrated under vacuum to provide 4-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetan-3-yl}pyridin-2-yl)oxy]butanenitrile (compound 3, 180 mg, 69%...

Claims

1. Compounds of formula (I): 【Chemistry 281】 or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, or 2; X and Y are each independently CH or N; Z is CH(CH ３ ), O, and SO ２ or Z is CH(CH ３ ), NH, N(CH ３ ), O, and SO ２ Selected from: R １ represents hydrogen, -CN, -NHR ｚ , -R ａ , -NR ａ R ｂ , -OR ａ , -NHC(O)R ａ , -NHC(S)R ａ , -NHC(O)NHR ａ ,-NHC(S)NHR ａ , -SR ａ , C ３ -C ６ selected from cycloalkyl, and 3- to 6-membered heterocyclyl rings; R ｚ teeth, 【Chemistry 282】 Selected from: R ａ and R ｂ is hydrogen, C ２－ C ６ Alkenyl, C １ -C ６ Alkyl, amide (C １ -C ６ alkyl), amino (C １ -C ６ alkyl), azide (C １ -C ６ alkyl), C ２ -C ６ Alkynyl, carboxy (C １ -C ６ alkyl), cyano (C １ -C ６ alkyl), C optionally substituted with a cyano group ３ -C ６ Cycloalkyl, dimethylamino (C １ -C ６ alkyl), 3- to 6-membered heterocyclyl ring, 3- to 6-membered heterocyclyl (C １ -C ３ alkyl), hydroxy (C １ -C ６ alkyl), methoxy (C １ -C ６ alkyl), methylamino (C １ -C ６ alkyl), NR ｃ R ｄ (C １ -C ６ alkyl), HS(C １ -C ６ alkyl), and CH ３ S (C １ -C ６ alkyl), and R ｃ and R ｄ is hydrogen, C ２ independently selected from alkenylcarbonyl, and methyl; or R ａ and R ｂ together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, said ring being ａ , -C(O)R ａ , -SO ２ R ａ optionally substituted with one group selected from , azido, and cyano; Each C ３ -C ６ cycloalkyl, each 3- to 6-membered heterocyclyl ring, and the 3- to 6-membered heterocyclyl (C １ -C ３ The heterocyclyl portion of the alkyl ring is C １ -C ３ Alkyl, C ２ Alkynyl, amido, azido, carboxy, cyano, dimethylamino, hydroxy, methoxy, methylamino, HS—, and CH ３ optionally substituted with 1, 2, or 3 groups independently selected from S—; R ２ teeth, 【Chemistry 283】 is selected from m is 0, 1, 2, or 3; m″ is 0, 1, 2, 3, or 4; B' is a 3- to 7-membered saturated or unsaturated ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur; said ring is selected from the group consisting of -OH, -CH ２ S.H., C.H. ２ SCH ３ , -CH ２ OH, -CH ２ NH ２ , and -CH ２ NHCH ３ optionally substituted with 1 or 2 substituents independently selected from Each R ５００ is hydrogen, C １ -C ６ Alkyl, halo, —OH, and —CH ２ independently selected from OH; X ５０ is O, NH, NCH ３ and S; R ３ is hydrogen, acetyl, amino, C １ -C ６ Alkylamino, C １ -C ６ Alkylaminomethyl, C １ -C ６ Alkylcarbonyl, Amino C １ -C ６ Alkyl, aminocarbonyl, aminomethyl, carboxy, cyano, C ３ Cycloalkyl, formyl, hydroxy, hydroxy C １ -C ６ Alkyl, methoxy, oxazolyl, -SH, -SCH ３ , -SOCH ３ , -SO ２ CH ３ , -SO(=NH)CH ３ , tetrazolyl, thiazolyl, and trifluoromethyl; ３ The cycloalkyl is optionally substituted with a hydroxy group; R ４ is hydrogen, methyl, -CH ２ OH, -CH ２ SH, and -CH ２ SCH ３ Selected from: R ５ is hydrogen, hydroxy, -CH ２ SH, -CH ２ SCH ３ and methyl; Optionally, provided that R ５ is hydroxy or methyl, and R ４ is hydrogen, then R １ is C ３ -C ６ Cycloalkyl, 3- to 6-membered heterocyclyl ring, hydroxy, hydroxy(C １ -C ６ alkyl), -OR ａ (In the formula, R ａ is C １ -C ６ alkyl, 3- to 6-membered heterocyclyl ring, or hydroxy (C １ -C ６ alkyl); or —NR ａ R ｂ (In the formula, R ａ and R ｂ is hydrogen, C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, hydroxy (C １ -C ６ alkyl), 3- to 6-membered heterocyclyl ring, or R ａ and R ｂ together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, said ring being １ -C ６ Alkyl or hydroxy (C １ -C ６ alkyl); R ６ and R ６’ is hydrogen, cyclopropyl, -CH ２ OH, -CH ２ SH, -CH ２ SCH ３ , and -CH ２ R ２００ and R ２００ is a 3- to 7-membered saturated or unsaturated ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur.

2. X is N; Y is CH; R ２ teeth, 【Chemistry 284】 That is, 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof.

3. 2. The compound of claim 1, wherein Z is O, or a pharmaceutically acceptable salt thereof.

4. Z is CH(CH ３ 2. The compound of claim 1, wherein:

5. R ３ is trifluoromethyl; R ４ and R ６ is hydrogen; R ５ is methyl, 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof.

6. R １ is -NR ａ R ｂ , -NHC(O)R ａ ,-NHC(S)NHR ａ , and -SR ａ 2. The compound of claim 1 selected from:

7. R １ is -SCH ２ CH ３ 2. The compound of claim 1, wherein:

8. R １ is -NH(CH ２ ) ２ CN, or a pharmaceutically acceptable salt thereof.

9. R １ is -NH(CH ２ ) ２ N ３ 2. The compound of claim 1, wherein:

10. Compound of formula (IA-1): 【Chemical 285】 or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, or 2; X and Y are each independently CH or N; Z is CH(CH ３ ), O, and SO ２ Selected from: R １ represents hydrogen, -CN, -NHR ｚ , -R ａ , -NR ａ R ｂ , -OR ａ , -NHC(O)R ａ , -NHC(S)R ａ , -NHC(O)NHR ａ ,-NHC(S)NHR ａ , -SR ａ , C ３ -C ６ selected from cycloalkyl, and 3- to 6-membered heterocyclyl rings; R ｚ teeth, 【Chemistry 286】 Selected from: R ａ and R ｂ is hydrogen, C ２－ C ６ Alkenyl, C １ -C ６ Alkyl, amide (C １ -C ６ alkyl), amino (C １ -C ６ alkyl), azide (C １ -C ６ alkyl), C ２ -C ６ Alkynyl, carboxy (C １ -C ６ alkyl), cyano (C １ -C ６ alkyl), C ３ -C ６ Cycloalkyl, dimethylamino (C １ -C ６ alkyl), 3- to 6-membered heterocyclyl ring, 3- to 6-membered heterocyclyl (C １ -C ３ alkyl), hydroxy (C １ -C ６ alkyl), methoxy (C １ -C ６ alkyl), methylamino (C １ -C ６ alkyl), NR ｃ R ｄ (C １ -C ６ alkyl), HS(C １ -C ６ alkyl), and CH ３ S (C １ -C ６ alkyl), and R ｃ and R ｄ is hydrogen, C ２ independently selected from alkenylcarbonyl, and methyl; or R ａ and R ｂ together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, said ring being ａ , -C(O)R ａ , -SO ２ R ａ optionally substituted with one group selected from , azido, and cyano; Each C ３ -C ６ cycloalkyl, each 3- to 6-membered heterocyclyl ring, and the 3- to 6-membered heterocyclyl (C １ -C ３ The heterocyclyl portion of the alkyl ring is C １ -C ３ Alkyl, C ２ Alkynyl, amido, azido, carboxy, cyano, dimethylamino, hydroxy, methoxy, methylamino, HS—, and CH ３ optionally substituted with 1, 2, or 3 groups independently selected from S—; R ２ teeth, 【Chemistry 287】 and m is 0, 1, 2, or 3; R ３ is hydrogen, acetyl, amino, C １ -C ６ Alkylamino, C １ -C ６ Alkylaminomethyl, Amino C １ -C ６ Alkyl, aminocarbonyl, aminomethyl, carboxy, cyano, C ３ Cycloalkyl, formyl, hydroxy, hydroxy C １ -C ６ Alkyl, methoxy, oxazolyl, -SH, -SCH ３ , tetrazolyl, thiazolyl, and trifluoromethyl; ３ The cycloalkyl is optionally substituted with a hydroxy group; R ４ and R ６ is hydrogen, -CH ２ SH, and -CH ２ SCH ３ are independently selected from R ５ is hydroxy, -CH ２ SH, -CH ２ SCH ３ and methyl; Optionally, provided that R ５ is hydroxy or methyl, and R ４ is hydrogen, then R １ is C ３ -C ６ Cycloalkyl, 3- to 6-membered heterocyclyl ring, hydroxy, hydroxy(C １ -C ６ alkyl), -OR ａ (In the formula, R ａ is C １ -C ６ alkyl, 3- to 6-membered heterocyclyl ring, or hydroxy (C １ -C ６ alkyl); or —NR ａ R ｂ (In the formula, R ａ and R ｂ is hydrogen, C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, hydroxy (C １ -C ６ alkyl), 3- to 6-membered heterocyclyl ring, or R ａ and R ｂ together with the nitrogen atom to which they are attached form a 5- or 6-membered ring optionally containing one additional nitrogen atom, said ring being １ -C ６ Alkyl or hydroxy (C １ -C ６ alkyl). 【Request Item 11】 【Chemistry 288】 2. The compound of claim 1, selected from: or a pharmaceutically acceptable salt thereof. 【Request Item 12】 【Chemistry 289】 A compound selected from:

13. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is an inhibitor of Casitas B-lineage lymphoma proto-oncogene b (Cbl-b).

14. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound interacts with His152 of human Cbl-b; increases IL-2 secretion from T cells by about 0.8 to about 1.4 fold over background; increases IFN-γ secretion by about 1.1 to about 2 fold over background; and / or increases CD69 levels by about 0.81 to about 1.1 fold over background.

15. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a binding moiety capable of specifically binding to a target on the surface of an effector T cell and a payload capable of activating the effector T cell, wherein the binding moiety is directly bound to the payload or is bound to the payload via a linker, or a pharmaceutically acceptable salt thereof.

16. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding moiety is attached to the payload via a linker.

17. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is capable of specifically binding a protein or glycoprotein on the surface of the effector T cell.

18. 18. The conjugate of claim 17, or a pharmaceutically acceptable salt thereof, wherein the binding site is capable of specifically binding to programmed cell death protein 1 (PD1).

19. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a binding moiety capable of specifically binding to PD1 and a payload capable of activating effector T cells, wherein the binding moiety is directly bound to the payload or is bound to the payload via a linker, or a pharmaceutically acceptable salt thereof.

20. 1. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a binding moiety capable of specifically binding to PD1 and a payload that is an inhibitor of Casitas B-lineage lymphoma proto-oncogene b (Cbl-b).

21. The conjugate has the formula (I): B-[L-P] ａ (I) (In the formula, a is an integer from 1 to 50; P is the payload; L is a linker; Bm is the binding site 16. The conjugate of claim 15, having the formula:

22. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the payload is an inhibitor of Casitas B-lineage lymphoma proto-oncogene b (Cbl-b).

23. 23. The conjugate of claim 22, or a pharmaceutically acceptable salt thereof, wherein the inhibitor of Cbl-b is a compound according to any one of claims 1 to 10, attached to the binding site or to the linker via a covalent bond.

24. Inhibitors of Cbl-b include: 【Chemistry 290】 23. The conjugate of claim 22, wherein:

25. 23. The conjugate of claim 22, or a pharmaceutically acceptable salt thereof, wherein the inhibitor of Cbl-b is Compound 146, Compound 147, Compound 148, or NX-1607.

26. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the payload is an agonist of Toll-like receptor 7 (TLR-7) and / or Toll-like receptor 8 (TLR-8).

27. 27. The conjugate of claim 26, or a pharmaceutically acceptable salt thereof, wherein the payload is the TLR-7 / TLR-8 agonist T785.

28. 27. The conjugate of claim 26, or a pharmaceutically acceptable salt thereof, wherein the payload is the TLR-7 / TLR-8 agonist MEDI9197.

29. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the payload is an inhibitor of hematopoietic progenitor kinase 1 (HPK-1).

30. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the payload is an inhibitor of STING, phosphoinositide-3-kinase gamma (PI3Kγ), CXCR4, CCR5, or a mitogen-activated protein kinase (MAPK) pathway protein, optionally wherein the MAPK pathway protein is MEK or B-raf.

31. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the payload is an agonist of stimulator of interferon genes (STING).

32. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the payload is a small molecule.

33. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the payload is a peptide.

34. 22. The conjugate of claim 21, or a pharmaceutically acceptable salt thereof, wherein L is a non-cleavable linker.

35. L is, 【Chemistry 291】 (In the formula, p is an integer from 1 to 10; p* is an integer from 1 to 10; Y is hydrogen and C １ -C ６ alkyl; 【Chemistry 292】 is the attachment point to the payload; 【Chemistry 293】 is the point of attachment to the binding site) 22. The conjugate of claim 21, selected from the group consisting of:

36. L is, 【Chemistry 294】 (In the formula, p is an integer from 1 to 10; p* is an integer from 1 to 10; 【Chemistry 295】 is the attachment point to the payload; 【Chemistry 296】 is the point of attachment to the binding site) 22. The conjugate of claim 21, selected from:

37. 22. The conjugate of claim 21, or a pharmaceutically acceptable salt thereof, wherein L is a cleavable linker.

38. 38. The conjugate of claim 37, or a pharmaceutically acceptable salt thereof, wherein the cleavable linker is cleavable by a protease.

39. L is, 【Chemistry 297】 (In the formula, q is an integer from 2 to 10; Z １ , Z ２ , Z ３ , and Z ４ are each independently absent or a naturally occurring amino acid residue of L or D configuration, with the proviso that Z １ , Z ２ , Z ３ , and Z ４ at least two of are amino acid residues; 【Chemistry 298】 is the attachment point to the payload; 【Chemistry 299】 is the point of attachment to the binding site) 22. The conjugate of claim 21, selected from:

40. Z １ , Z ２ , Z ３ , and Z ４ is independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, and glycine; with the proviso that Z １ , Z ２ , Z ３ , and Z ４ 40. The conjugate of claim 39, or a pharmaceutically acceptable salt thereof, wherein at least two of are amino acid residues.

41. Z １ is absent or glycine; Z ２ is absent or selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z ３ is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; Z ４ is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine; 40. The conjugate of claim 39, or a pharmaceutically acceptable salt thereof.

42. L is, [Chemical 300] (In the formula, q is an integer from 2 to 10; 【Chemical 301】 is the attachment point to the payload; 【Chemical 302】 is the point of attachment to the binding site) 22. The conjugate of claim 21, selected from:

43. 22. The conjugate of claim 21, or a pharmaceutically acceptable salt thereof, wherein L is a bioreducible linker.

44. L is, 【Chemical 303】 (In the formula, q is an integer from 2 to 10; R, R', R'', and R''' are hydrogen, C １ -C ６ Alkoxy C １ -C ６ Alkyl, (C １ -C ６ ) ２ NC １ -C ６ Alkyl, and C １ -C ６ alkyl, or two geminal R groups together with the carbon atoms to which they are attached can form a cyclobutyl or cyclopropyl ring; 【Chemical 304】 is the attachment point to the payload; 【Chemical 305】 is the point of attachment to the binding site) 22. The conjugate of claim 21, selected from:

45. L is, 【Chemical 306】 (In the formula, q is an integer from 2 to 10; R, R', R'', and R''' are hydrogen, C １ -C ６ Alkoxy C １ -C ６ Alkyl, (C １ -C ６ ) ２ NC １ -C ６ Alkyl, and C １ -C ６ alkyl, or two geminal R groups together with the carbon atoms to which they are attached can form a cyclobutyl or cyclopropyl ring; 【Chemical 307】 is the attachment point to the payload; 【Chemical 308】 is the point of attachment to the binding site) 22. The conjugate of claim 21, selected from:

46. 22. The conjugate of claim 21, or a pharmaceutically acceptable salt thereof, wherein L is an acid-cleavable linker.

47. L is, 【Chemical 309】 (In the formula, q is an integer from 2 to 10; 【Chemical 310】 is the attachment point to the payload; 【Chemical 311】 is the point of attachment to the binding site) 22. The conjugate of claim 21, selected from the group consisting of:

48. 22. The conjugate of claim 21, or a pharmaceutically acceptable salt thereof, wherein L is a click-to-release linker.

49. L is, 【Chemical 312】 (In the formula, q is an integer from 2 to 10; 【Chemistry 313】 is the attachment point to the payload; 【Chemical 314】 is the point of attachment to the binding site) 22. The conjugate of claim 21, selected from:

50. 22. The conjugate of claim 21, or a pharmaceutically acceptable salt thereof, wherein L is a pyrophosphatase-cleavable linker.

51. L is, 【Chemical Industry 315】 (In the formula, q is an integer from 2 to 10; 【Chemical 316】 is the attachment point to the payload; 【Chemical 317】 is the point of attachment to the binding site) 22. The conjugate of claim 21, wherein:

52. 22. The conjugate of claim 21, or a pharmaceutically acceptable salt thereof, wherein L is a beta-glucuronidase cleavable linker.

53. L is, 【Chemical 318】 (In the formula, q is an integer from 2 to 10; --- is absent or a bond; 【Chemical 319】 is the attachment point to the payload; 【Chem.320】 is the point of attachment to the binding site) 22. The conjugate of claim 21, selected from:

54. L is, 【Chemistry 321】 (In the formula, q is an integer from 2 to 10; --- is absent or a bond; 【Chemistry 322】 is the attachment point to the payload; 【Chemical 323】 is the point of attachment to the binding site) 22. The conjugate of claim 21, wherein: 【Request Item 55】 【Chemistry 324-1】 【Chemistry 324-2】 【Chemistry 324-3】 【Chemistry 324-4】 【Chemistry 324-5】 【Chemistry 324-6】 22. The conjugate of claim 21 , selected from:

56. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the linker is attached to a cysteine, lysine, tyrosine, or glutamine in the Bm.

57. 57. The conjugate of claim 56, or a pharmaceutically acceptable salt thereof, wherein the cysteine ​​or lysine is an engineered cysteine ​​or lysine.

58. 57. The conjugate of claim 56, or a pharmaceutically acceptable salt thereof, wherein the cysteine ​​or lysine is endogenous to the Bm.

59. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or an antigen-binding fragment thereof.

60. 60. The conjugate of claim 59, or a pharmaceutically acceptable salt thereof, wherein L is attached to an engineered cysteine ​​at heavy chain position S239 and / or K334 of said antibody or antigen-binding portion thereof according to EU numbering.

61. 60. The conjugate of claim 59, or a pharmaceutically acceptable salt thereof, wherein L is attached to glutamine at heavy chain position 295 of said antibody or antigen-binding portion thereof according to EU numbering.

62. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or antigen-binding fragment thereof comprising the CDR sequences of amino acids 31-35, 50-66, and 99-109 of SEQ ID NO: 10 and amino acids 24-38, 54-60, and 93-101 of SEQ ID NO:

11.

63. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or antigen-binding fragment thereof comprising: (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 11; or (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 26 and a light chain comprising the amino acid sequence of SEQ ID NO:

27.

64. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or antigen-binding fragment thereof comprising heavy and light chain CDR sequences of amino acids 31-35, 50-66, and 99-102 of SEQ ID NO: 12 and amino acids 24-34, 50-56, and 89-97 of SEQ ID NO:

13.

65. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a variable light chain comprising the amino acid sequence of SEQ ID NO:

13.

66. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is capable of specifically binding to CD25.

67. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or antigen-binding fragment thereof comprising the CDR sequences of amino acids 31-35, 50-66, and 99-111 of SEQ ID NO:4 and amino acids 24-34, 50-56, and 89-97 of SEQ ID NO:

5.

68. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a variable light chain comprising the amino acid sequence of SEQ ID NO:

5.

69. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or antigen-binding fragment thereof comprising the CDR sequences of amino acids 31-35, 50-65, and 98-108 of SEQ ID NO:6 and amino acids 24-33, 49-55, and 88-96 of SEQ ID NO:

7.

70. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:6 and a variable light chain comprising the amino acid sequence of SEQ ID NO:

7.

71. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or antigen, or an antigen-binding fragment thereof, comprising the CDR sequences of amino acids 31-35, 50-65, and 98-108 of SEQ ID NO:8 and amino acids 24-33, 49-55, and 88-96 of SEQ ID NO:

9.

72. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:8 and a variable light chain comprising the amino acid sequence of SEQ ID NO:

9.

73. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is capable of specifically binding to CD7.

74. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or antigen-binding fragment thereof comprising the CDR sequences of amino acids 31-35, 50-66, and 99-112 of SEQ ID NO: 14 and amino acids 23-36, 52-58, and 91-99 of SEQ ID NO:

15.

75. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding site is an antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a variable light chain comprising the amino acid sequence of SEQ ID NO:

15.

76. 60. The conjugate of claim 59, or a pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region.

77. 77. The conjugate of claim 76, or a pharmaceutically acceptable salt thereof, wherein the heavy chain constant region comprises an Fc silent mutation.

78. 77. The conjugate of claim 76, or a pharmaceutically acceptable salt thereof, wherein the heavy chain constant region is an IgG heavy chain constant region.

79. 79. The conjugate of claim 78, or a pharmaceutically acceptable salt thereof, wherein the IgG heavy chain constant region is an IgG1 heavy chain constant region.

80. 77. The conjugate of claim 76, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof.

81. 79. The conjugate of claim 78, or a pharmaceutically acceptable salt thereof, wherein the IgG heavy chain constant region is an IgG4 heavy chain constant region.

82. 77. The conjugate of claim 76, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 21, or a pharmaceutically acceptable salt thereof.

83. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein (a) the binding moiety is pembrolizumab, or (b) the binding moiety is nivolumab.

84. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein (a) the binding site is a686, (b) the binding site is MA251, or (c) the binding site is humanized MA251.

85. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the binding moiety is a small molecule.

86. 22. The conjugate of claim 21, or a pharmaceutically acceptable salt thereof, wherein a is 1 to 40.

87. 22. The conjugate of claim 21, or a pharmaceutically acceptable salt thereof, wherein a is 1 to 10.

88. 22. The conjugate of claim 21, or a pharmaceutically acceptable salt thereof, wherein a is 2 to 8.

89. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the conjugate is capable of increasing effector T cell activity.

90. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the conjugate is capable of increasing effector T cell proliferation.

91. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the conjugate is capable of increasing effector T cell migration into tumor cells.

92. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the conjugate is capable of reducing effector T cell exhaustion.

93. 16. The conjugate of claim 15, or a pharmaceutically acceptable salt thereof, wherein the conjugate increases IFN-y secretion from T cells by about 2.5 to about 3 fold over background; and / or increases IL-2 secretion from T cells by about 6 to about 8 fold over background.

94. A composition comprising a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof.

95. A composition comprising the conjugate of any one of claims 15 to 22 or 24 to 93, or a pharmaceutically acceptable salt thereof.

96. 94. A composition for treating cancer in a subject in need thereof, said composition comprising a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, or a conjugate according to any one of claims 15 to 22 or 24 to 93, or a pharmaceutically acceptable salt thereof.

97. 97. The composition of claim 96, wherein the cancer is (i) resistant or refractory to anti-PD1 therapy, optionally wherein the anti-PD1 therapy is nivolumab, pembrolizumab, and / or cemiplimab, and / or (ii) resistant or refractory to anti-PDL1 therapy, optionally wherein the anti-PDL1 therapy is durvalumab, atezolizumab, and / or avelumab.

98. 94. A composition for treating a condition in a subject in need thereof that would benefit from an increased immune response, said composition comprising a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, or a conjugate according to any one of claims 15 to 22 or 24 to 93, or a pharmaceutically acceptable salt thereof.

99. 99. The composition of claim 98, wherein the pathological condition is an infectious disease, optionally wherein the infectious disease is a viral, bacterial, or parasitic infection, an immunosuppressive disease or disorder, or multiple sclerosis.

100. 94. An in vitro method of increasing the activity of an immune cell, comprising contacting said immune cell with a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, or a conjugate according to any one of claims 15 to 22 or 24 to 93, or a pharmaceutically acceptable salt thereof.

101. 94. An in vitro method of increasing the proliferation of immune cells, said in vitro method comprising contacting immune cells with a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, or a conjugate according to any one of claims 15 to 22 or 24 to 93, or a pharmaceutically acceptable salt thereof.

102. 94. An in vitro method of increasing the migration of immune cells into tumor cells, comprising contacting immune cells with a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, or a conjugate according to any one of claims 15 to 22 or 24 to 93, or a pharmaceutically acceptable salt thereof.

103. 94. An in vitro method of reducing exhaustion of an immune cell, said in vitro method comprising contacting an immune cell with a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, or a conjugate according to any one of claims 15 to 22 or 24 to 93, or a pharmaceutically acceptable salt thereof.

104. 94. An in vitro method for increasing secretion of IFN-γ or IL-2 from immune cells, said in vitro method comprising contacting said immune cells with a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, or a conjugate according to any one of claims 15 to 22 or 24 to 93, or a pharmaceutically acceptable salt thereof.

105. 101. The in vitro method of claim 100, wherein the immune cell is a T cell, and optionally the T cell is an effector T cell.

106. 101. The in vitro method of claim 100, wherein the immune cells are natural killer (NK) cells.

107. 94. An in vitro method of enhancing and / or prolonging an antigen recall response of T cells, said in vitro method comprising contacting said T cells with a compound of any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, or a conjugate of any one of claims 15 to 22 or 24 to 93, or a pharmaceutically acceptable salt thereof.

108. 94. An in vitro method of delivering a payload capable of activating an effector T cell to an immune cell, said method comprising contacting an effector T cell with the conjugate of any one of claims 15 to 22 or 24 to 93, or a pharmaceutically acceptable salt thereof.

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