Activators of effector t cells

Abstract

The present disclosure provides antibody drug conjugates and methods of delivering the conjugates to effector T cells. Also disclosed are novel Cbl-b inhibitors. The compounds and conjugates are useful for treating disease in a subject in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 318,278 filed on March 9, 2022, U.S. Provisional Application No. 63 / 477,053 filed on December 23, 2022, and U.S. Provisional Application No. 63 / 488,211 filed on March 3, 2023, which are incorporated herein by reference in their entirety.

[0003] References to sequence listings submitted electronically

[0004] The contents of the electronically submitted .XML document filed 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. Technical Field

[0005] The present disclosure provides antibody drug conjugates and methods for delivering the conjugates to effector T cells. Also disclosed are novel Cbl-b inhibitors. The compounds and conjugates can be used to treat diseases in subjects in need thereof. Background Art

[0006] Effector T cells are the main players that guide the immune system to perform immune functions. These cells circulate until they encounter their specific antigens. Therefore, the cells play a key role in immunity. The immune system is a powerful weapon against many diseases and conditions (including, for example, cancer), and compounds that have been shown to activate effector T cells, such as inhibitors of E3 ubiquitin ligase Casitas B-lineage lymphoma-b (Casitas B-lineage lymphoma-b, Cbl-b), increase cytokine secretion and proliferation derived from T cells and may have anticancer activity. However, improved Cbl-b inhibitors are needed.

[0007] In addition, compounds that activate effector T cells are likely 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 conjugating immune activation drugs to antibodies that specifically recognize tumor antigens so that immune activation drugs specifically target cancer cells. (See, for example, Ackerman, SE, Pearson, CI, Gregorio, JD et al., Immune-stimulating antibody conjugates elicitrobust 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 indicate direct targeting of the immune system itself. Therefore, although this work is in progress, it is still necessary to activate T cells, especially effector T cells, for example, for the treatment of cancer, while minimizing off-target immune responses. Summary of the invention

[0008] In certain aspects, the present disclosure provides compounds of formula (I):

[0009]

[0010] (I)

[0011] or a pharmaceutically acceptable salt thereof, wherein:

[0012] n is 0, 1, or 2;

[0013] X and Y are each independently CH or N;

[0014] Z is selected from CH(CH 3 ), O and SO 2 ;or

[0015] Z is selected from CH(CH 3 ), NH, N(CH 3 ), O and SO 2 ;

[0016] R 1 Selected from 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 ring; wherein

[0017] R z Selected from

[0018]

[0019] R a and R b are independently selected from hydrogen, C 2 -C 6 Alkenyl, C 1 -C 6 Alkyl, amide (C 1 -C 6 Alkyl), amino (C 1 -C 6 alkyl), azido (C 1 -C 6 Alkyl), C 2 -C 6 Alkynyl, carboxyl (C 1 -C 6 Alkyl), cyano (C 1 -C 6 alkyl), C optionally substituted by cyano 3 -C 6 Cycloalkyl, dimethylamino (C 1 -C 6 alkyl), 3- to 6-membered heterocyclic ring, 3- to 6-membered heterocyclic (C 1 -C 3 Alkyl), hydroxyl (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), where R c and R d are independently selected from hydrogen, C 2 alkenylcarbonyl and methyl; or,

[0020] R aand R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from R a 、-C(O)R a 、-SO 2 R a , azido and cyano group substitution;

[0021] 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 optionally substituted by one, two or three independently selected from C 1 -C 3 Alkyl, C 2 Alkynyl, amide, azido, carboxyl, cyano, dimethylamino, hydroxyl, methoxy, methylamino, HS- and CH 3 and

[0022] R 2 Selected from

[0023] , , , , , , , , and ;in

[0024] m is 0, 1, 2, or 3;

[0025] m'' is 0, 1, 2, 3, or 4;

[0026] B' is a 3-7 membered saturated or unsaturated ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen and sulfur; wherein the ring is optionally substituted by one or two independently selected from -OH, -CH 2 SH, CH 2 SCH 3 、-CH 2 OH, -CH 2 NH 2 and -CH 2 NHCH 3 Substituents are substituted;

[0027] Each R 500 are independently selected from hydrogen, C 1 -C 6 Alkyl, halide, -OH, and -CH2 OH; and

[0028] X 50 Selected from O, NH, NCH 3 and S;

[0029] R 3 Selected from hydrogen, acetyl, amino, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylaminomethyl, C 1 -C 6 Alkylcarbonyl, aminoC 1 -C 6 Alkyl, aminocarbonyl, aminomethyl, carboxyl, cyano, C 3 Cycloalkyl, formyl, hydroxyl, hydroxyl C 1 -C 6 Alkyl, methoxy, Azolyl, -SH, -SCH 3 、-SOCH 3 、-SO 2 CH 3 、-SO(=NH)CH 3 , tetrazolyl, thiazolyl and trifluoromethyl, wherein the C 3 The cycloalkyl group is optionally substituted with hydroxy;

[0030] R 4 Selected from hydrogen, methyl, -CH 2 OH, -CH 2 SH and -CH 2 SCH 3 ;

[0031] R 5 Selected from hydrogen, hydroxyl, -CH 2 SH, -CH 2 SCH 3 and methyl;

[0032] Optionally, the prerequisite is that when R 5 is hydroxy or methyl, and R 4 When it is hydrogen, then R 1 Not C 3 -C 6 Cycloalkyl, 3 to 6 membered heterocyclic ring, hydroxyl, hydroxyl (C 1 -C 6 Alkyl), -OR a , where R a It is C 1 -C 6 alkyl, 3 to 6 membered heterocyclic ring or hydroxyl (C 1 -C6 or -NR a R b , where R a and R b are independently selected from hydrogen, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, hydroxyl (C 1 -C 6 alkyl), a 3- to 6-membered heterocyclic ring, or wherein R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from C 1 -C 6 Alkyl or hydroxyl (C 1 -C 6 alkyl) group; and

[0033] R 6 and R 6' are independently selected from hydrogen, cyclopropyl, -CH 2 OH, -CH 2 SH, -CH 2 SCH 3 and -CH 2 R 200 , where R 200 is a 3-7 membered saturated or unsaturated ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0034] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0035] In certain aspects, the present disclosure provides compounds of formula (IA-1):

[0036]

[0037] (IA-1),

[0038] or a pharmaceutically acceptable salt thereof, wherein:

[0039] n is 0, 1, or 2;

[0040] X and Y are each independently CH or N;

[0041] Z is selected from CH(CH 3 ), O and SO 2 ;

[0042] R 1Selected from 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 ring; wherein

[0043] R z Selected from

[0044]

[0045] R a and R b are independently selected from hydrogen, C 2 -C 6 Alkenyl, C 1 -C 6 Alkyl, amide (C 1 -C 6 Alkyl), amino (C 1 -C 6 alkyl), azido (C 1 -C 6 Alkyl), C 2 -C 6 Alkynyl, carboxyl (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 heterocyclic ring, 3- to 6-membered heterocyclic (C 1 -C 3 Alkyl), hydroxyl (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 CH3 S(C 1 -C 6 alkyl), where R c and R d are independently selected from hydrogen, C 2 alkenylcarbonyl and methyl; or,

[0046] R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from R a 、-C(O)R a 、-SO 2 R a , azido and cyano group substitution;

[0047] 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 optionally substituted by one, two or three independently selected from C 1 -C 3 Alkyl, C 2 Alkynyl, amide, azido, carboxyl, cyano, dimethylamino, hydroxyl, methoxy, methylamino, HS- and CH 3 and

[0048] R 2 yes

[0049] or ;in

[0050] m is 0, 1, 2, or 3;

[0051] R 3 Selected from hydrogen, acetyl, amino, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylaminomethyl, amino C 1 -C 6 Alkyl, aminocarbonyl, aminomethyl, carboxyl, cyano, C 3 Cycloalkyl, formyl, hydroxyl, hydroxyl C 1 -C 6 Alkyl, methoxy, Azolyl, -SH, -SCH 3 , tetrazolyl, thiazolyl and trifluoromethyl, wherein the C 3 The cycloalkyl group is optionally substituted with hydroxy;

[0052] R 4 and R 6 are independently selected from hydrogen, -CH 2 SH and -CH 2 SCH 3 ;and

[0053] R 5 Selected from hydroxyl, -CH 2 SH, -CH 2 SCH 3 and methyl;

[0054] Optionally, the prerequisite is that when R 5 is hydroxy or methyl, and R 4 When it is hydrogen, then R 1 Not C 3 -C 6 Cycloalkyl, 3 to 6 membered heterocyclic ring, hydroxyl, hydroxyl (C 1 -C 6 Alkyl), -OR a , where R a It is C 1 -C 6 alkyl, 3 to 6 membered heterocyclic ring or hydroxyl (C 1 -C 6 or -NR a R b , where R a and R b are independently selected from hydrogen, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, hydroxyl (C 1 -C 6 alkyl), a 3- to 6-membered heterocyclic ring, or wherein R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from C 1 -C 6 Alkyl or hydroxyl (C 1 -C 6 alkyl) groups.

[0055] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0056] In certain aspects, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0057] X is N;

[0058] Y is CH; and

[0059] R 2 yes .

[0060] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0061] In some aspects, Z is O. In some aspects, Z is CH (CH 3 ).

[0062] In some aspects, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein

[0063] R 3 is trifluoromethyl;

[0064] R 4 and R 6 is hydrogen; and

[0065] R 5 It's methyl.

[0066] In some respects, R 1 Selected from -NR a R b 、-NHC(O)R a 、-NHC(S)NHR a and-SR a In some aspects, wherein R 1 Yes-SCH 2 CH 3 In some aspects, R 1 It is -NH(CH 2 ) 2 CN. In some aspects, R 1 It is -NH(CH 2 ) 2 N 3 .

[0067] In some aspects, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, the compound being selected from:

[0068] , and .

[0069] In some aspects, the compounds of Formula (I) are inhibitors of Casitas B-lineage lymphoma proto-oncogene b (Cbl-b).

[0070] In certain aspects, 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 relative to background; increases IFN-γ secretion by about 1.1 to about 2 fold relative to background; and / or increases CD69 levels by about 0.81 to about 1.1 fold relative to background.

[0071] In certain aspects, the present disclosure provides a conjugate or a pharmaceutically acceptable salt thereof, the conjugate or a pharmaceutically acceptable salt thereof comprising a binding portion 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 portion is directly connected to the payload or is connected to the payload via a linker. In some aspects, the binding portion is connected to the payload via a linker. In certain aspects, the binding portion is capable of specifically binding to a protein or glycoprotein on the surface of an effector T cell. In some aspects, the binding portion is capable of specifically binding to programmed cell death protein 1 (PD1).

[0072] In certain aspects, the present disclosure provides 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 linked to the payload or is linked to the payload via a linker.

[0073] In some aspects, the present disclosure provides a conjugate or a pharmaceutically acceptable salt thereof, comprising a binding portion capable of specifically binding to PD1 and a payload that is an inhibitor of Casitas B-lineage lymphoma proto-oncogene b (Cbl-b).

[0074] In some aspects, the conjugates described herein are of Formula (I):

[0075] Bm-[LP] a (I)

[0076] in:

[0077] a is an integer from 1 to 50;

[0078] P is the payload;

[0079] L is a connector; and

[0080] Bm is the binding part.

[0081] In some aspects, the payload is an inhibitor of Casitas B lineage lymphoma proto-oncogene b (Cbl-b). In some aspects, the Cbl-b inhibitor is a compound of formula (I) covalently linked to a binding moiety or linker. In some aspects, the Cbl-b inhibitor is

[0082] , or .

[0083] In some aspects, the Cbl-b inhibitor is Compound 146, Compound 147, Compound 148, or NX-1607.

[0084] In some aspects, the payload is an agonist of toll-like receptor 7 (TLR-7) and / or toll-like receptor 8 (TLR-8). In some aspects, the payload is the TLR-7 / TLR-8 agonist T785. In some aspects, the payload is the TLR-7 / TLR-8 agonist MEDI9197.

[0085] In some aspects, the payload is an inhibitor of hematopoietic progenitor cell kinase 1 (HPK-1).

[0086] In some aspects, 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.

[0087] In some aspects, the payload is an agonist of stimulator of interferon genes (STING).

[0088] In some aspects, the payload is a small molecule. In some aspects, the payload is a peptide.

[0089] In certain aspects, the present disclosure provides a conjugate as described above, wherein L is a non-cleavable linker. In some aspects, L is selected from the group consisting of:

[0090] , , , , , , , , and ;

[0091] in:

[0092] p is an integer from 1 to 10;

[0093] p* is an integer from 1 to 10;

[0094] Y is selected from hydrogen and C 1 -C 6 alkyl;

[0095] is the connection point to the payload; and

[0096] It is the connection point with the binding part.

[0097] In some aspects, L is selected from

[0098] , and ;

[0099] in:

[0100] p is an integer from 1 to 10;

[0101] p* is an integer from 1 to 10;

[0102] is the connection point to the payload; and

[0103] It is the connection point with the binding part.

[0104] In some aspects, L is a cleavable linker. In some aspects, the cleavable linker is cleavable by a protease. In some aspects, L is selected from

[0105] ; ; ; ; ; ; ; ; and ;

[0106] in:

[0107] q is an integer from 2 to 10;

[0108] Z 1 , Z 2 , Z 3 and Z 4 each independently being an amino acid residue that is not present or is naturally present in the L or D configuration, provided that Z 1 , Z 2 , Z 3 and Z 4 At least two of are amino acid residues;

[0109] is the connection point to the payload; and

[0110] It is the connection point with the binding part.

[0111] In some respects, Z 1 , Z 2 , Z 3 and Z 4 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; provided that Z 1 , Z 2 , Z 3 and Z 4 At least two of them are amino acid residues.

[0112] In some ways:

[0113] Z 1 absent or glycine;

[0114] Z 2 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;

[0115] Z 3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine and glycine; and

[0116] Z 4 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.

[0117] In some aspects, L is selected from

[0118] , , , and ,

[0119] in:

[0120] q is an integer from 2 to 10;

[0121] is the connection point to the payload; and

[0122] It is the connection point with the binding part.

[0123] In some aspects, L is a bioreducible linker. In some aspects, L is selected from

[0124]

[0125] in:

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

[0127] R, R', R'' and R''' are each independently selected from 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 may form a cyclobutyl or cyclopropyl ring;

[0128] is the connection point to the payload; and

[0129] It is the connection point with the binding part.

[0130] In some aspects, L is selected from

[0131] and ;

[0132] in:

[0133] q is an integer from 2 to 10;

[0134] R, R', R'' and R''' are each independently selected from 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 may form a cyclobutyl or cyclopropyl ring;

[0135] is the connection point to the payload; and

[0136] It is the connection point with the binding part.

[0137] In certain aspects, L is an acid cleavable linker.

[0138] In some aspects, L is selected from the group consisting of:

[0139]

[0140] in:

[0141] q is an integer from 2 to 10;

[0142] is the connection point to the payload; and

[0143] It is the connection point with the binding part.

[0144] In some aspects, L is a click release linker. In some aspects, L is selected from

[0145]

[0146] in:

[0147] q is an integer from 2 to 10;

[0148] is the connection point to the payload; and

[0149] It is the connection point with the binding part.

[0150] In some aspects, L is a pyrophosphatase cleavable linker. In some aspects, L is

[0151] ;

[0152] in:

[0153] q is an integer from 2 to 10;

[0154] is the connection point to the payload; and

[0155] It is the connection point with the binding part.

[0156] In some aspects, L is a β-glucuronidase cleavable linker. In some aspects, L is selected from

[0157] , , and ;

[0158] in:

[0159] q is an integer from 2 to 10;

[0160] ----Does not exist or is a key;

[0161] is the connection point to the payload; and

[0162] It is the connection point with the binding part.

[0163] In some aspects, L is

[0164] ;

[0165] in:

[0166] q is an integer from 2 to 10;

[0167] ----Does not exist or is a key;

[0168] is the connection point to the payload; and

[0169] It is the connection point with the binding part.

[0170] In some aspects, the present disclosure provides a conjugate selected from:

[0171] ; ; ; ; ; ; ; ; ; ; ; ; ; ; and .

[0172] In some aspects, the linker of the conjugate is connected to a cysteine, lysine, tyrosine or glutamine in Bm. In some aspects, the cysteine ​​or lysine is an engineered cysteine ​​or lysine. In some aspects, the cysteine ​​or lysine is endogenous to Bm.

[0173] In some aspects, the binding moiety is an antibody or an antigen-binding fragment thereof.

[0174] In certain aspects, the disclosure provides conjugates wherein L is linked to an engineered cysteine ​​at heavy chain position S239 and / or K334 according to EU numbering of the antibody, or antigen binding portion thereof.

[0175] In some aspects, L is linked to glutamine at position 295 of the heavy chain of the antibody, or antigen-binding portion thereof, according to EU numbering.

[0176] In certain aspects, the binding moiety 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.

[0177] In some aspects, the binding moiety 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.

[0178] In some aspects, the binding moiety 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.

[0179] In some aspects, the binding moiety is an antibody or antigen-binding fragment thereof comprising the 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.

[0180] In some aspects, the binding moiety 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.

[0181] In some aspects, the binding moiety is capable of specifically binding to CD25.

[0182] In some aspects, the binding moiety 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.

[0183] In some aspects, the binding moiety 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.

[0184] In some aspects, the binding moiety 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.

[0185] In some aspects, the binding moiety 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.

[0186] In some aspects, the binding moiety is an antibody or antigen-binding fragment thereof 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.

[0187] In some aspects, the binding moiety 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.

[0188] In some aspects, the binding moiety is capable of specifically binding to CD7.

[0189] In some aspects, the binding moiety 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.

[0190] In some aspects, the binding moiety 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.

[0191] In some aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region.

[0192] In some aspects, the heavy chain constant region comprises an Fc silent mutation.

[0193] In some aspects, the heavy chain constant region is an IgG heavy chain constant region.

[0194] In some aspects, the IgG heavy chain constant region is an IgG1 heavy chain constant region.

[0195] In some aspects, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:20.

[0196] In some aspects, the IgG heavy chain constant region is an IgG4 heavy chain constant region.

[0197] In some aspects, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:21.

[0198] In some aspects, the present disclosure provides a conjugate as described herein, or a pharmaceutically acceptable salt thereof, wherein (a) the binding moiety is pembrolizumab, or (b) the binding moiety is nivolumab.

[0199] In some aspects, the disclosure provides a conjugate as described herein, or a pharmaceutically acceptable salt thereof, wherein (a) the binding moiety is a686, (b) the binding moiety is MA251, or (c) the binding moiety is humanized MA251.

[0200] In some aspects, the binding moiety is a small molecule.

[0201] In some aspects, the disclosure provides a conjugate as described herein, wherein a is 1 to 40. In some aspects, a is 1 to 10. In some aspects, a is 2 to 8.

[0202] In some aspects, the present disclosure provides a conjugate as described herein, which is capable of increasing effector T cell activity.

[0203] In some aspects, the present disclosure provides a conjugate as described herein, which is capable of increasing effector T cell proliferation.

[0204] In some aspects, the present disclosure provides a conjugate as described herein, which is capable of increasing the migration of effector T cells to tumor cells.

[0205] In some aspects, the present disclosure provides a conjugate as described herein, which is capable of reducing effector T cell exhaustion.

[0206] In some aspects, the disclosure provides a conjugate as described herein, which increases the secretion of IFN-γ from T cells by about 2.5 to about 3 fold relative to background; and / or increases the secretion of IL-2 from T cells by about 6 to about 8 fold relative to background.

[0207] In some aspects, the present disclosure provides a composition comprising a compound as described herein or a pharmaceutically acceptable salt thereof.

[0208] In some aspects, the present disclosure provides a composition comprising a conjugate as described herein or a pharmaceutically acceptable salt thereof.

[0209] In a certain aspect, the present disclosure provides a method for 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 aspects, cancer (i) is resistant or refractory to anti-PD1 therapy, optionally wherein the anti-PD1 therapy is nivolumab, pembrolizumab, and / or cemiplimab; and / or (ii) is resistant or refractory to anti-PDL1 therapy, optionally wherein the anti-PDL1 therapy is durvalumab, atezolizumab, and / or avelumab.

[0210] In some aspects, the present disclosure provides a method of treating a condition that would benefit from an increased immune response 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 aspects, the condition is an infection, optionally wherein the infection is a viral infection, a bacterial infection, or a parasitic infection, an immunosuppressive disease or disorder, or multiple sclerosis.

[0211] In some aspects, the disclosure provides a method of increasing the activity of an immune cell, the method comprising contacting the immune cell with a compound described herein, a conjugate described herein, or a composition described herein.

[0212] In some aspects, the present disclosure provides a method of increasing proliferation of an immune cell, the method comprising contacting the immune cell with a compound described herein, a conjugate described herein, or a composition described herein.

[0213] In some aspects, the present disclosure provides a method of increasing the migration of an immune cell to a tumor cell, the method comprising contacting the immune cell with a compound described herein, a conjugate described herein, or a composition described herein.

[0214] In some aspects, the disclosure provides methods of reducing exhaustion of an immune cell, the method comprising contacting the immune cell with a compound described herein, a conjugate described herein, or a composition described herein.

[0215] In some aspects, the disclosure provides a method for increasing the secretion of IFN-γ or IL-2 from immune cells, the method comprising contacting the immune cells with compounds described herein, conjugates described herein, or compositions described herein. In some aspects, immune cells are T cells, optionally wherein the T cells are effector T cells. In some aspects, immune cells are natural killer (NK) cells.

[0216] In some aspects, the disclosure provides a method of enhancing and / or maintaining an antigen recall response of a T cell, the method comprising contacting the T cell with a compound described herein, a conjugate described herein, or a composition described herein.

[0217] In some aspects, the disclosure provides a method of delivering a payload capable of activating effector T cells to immune cells, the method comprising contacting the effector T cells with a conjugate described herein or a composition comprising a conjugate described herein. In some aspects, the contact is in vitro. In some aspects, the contact is in vivo in a subject, optionally wherein the subject suffers from cancer or a disease that would benefit from an increased immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0218] Figure 1 are histograms showing the protein-ligand interaction scores between Cbl-b and compound 147 and between Cbl-b and compound 118.

[0219] Figure 2 is a graph showing the activity of the conjugate in mixed lymphocyte reaction.

[0220] Figure 3A is a graph showing the activity of the conjugate in a mixed lymphocyte reaction in the presence and absence of TGF-β.

[0221] Figure 3B is a graph showing the activity of the conjugate in mixed lymphocyte reaction in the presence and absence of Treg cells.

[0222] Figure 4 is a graph showing activation of tumor infiltrating lymphocytes (TIL) in vitro.

[0223] Figure 5 and Figure 6 is a graph showing the in vivo activity of the conjugates against B16F10 melanoma cells in mice compared to unconjugated antibody and vehicle controls.

[0224] Figure 7 is a graph showing activation of exhausted T cells by anti-PD-1 - Cbl-B inhibitor conjugate.

[0225] Figure 8 is a graph showing the antigen recall response (as measured by IFN-γ levels) of T cells treated with anti-PD-1 - Cbl-B inhibitor conjugate. DETAILED DESCRIPTION

[0226]

[0227] (I)

[0228] or a pharmaceutically acceptable salt thereof, wherein:

[0229] n is 0, 1, or 2;

[0230] X and Y are each independently CH or N;

[0231] Z is selected from CH(CH 3 ), O and SO 2 ;or

[0232] Z is selected from CH(CH 3 ), NH, N(CH 3 ), O and SO 2 ;

[0233] R 1 Selected from 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 ring; wherein

[0234] R z Selected from

[0235]

[0236] R a and R b are independently selected from hydrogen, C 2 -C 6 Alkenyl, C 1 -C 6 Alkyl, amide (C 1 -C 6 Alkyl), amino (C 1 -C 6 alkyl), azido (C 1 -C 6 Alkyl), C 2 -C 6 Alkynyl, carboxyl (C 1 -C 6 Alkyl), cyano (C 1 -C 6 alkyl), C optionally substituted by cyano 3 -C 6Cycloalkyl, dimethylamino (C 1 -C 6 alkyl), 3- to 6-membered heterocyclic ring, 3- to 6-membered heterocyclic (C 1 -C 3 Alkyl), hydroxyl (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), where R c and R d are independently selected from hydrogen, C 2 alkenylcarbonyl and methyl; or,

[0237] R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from R a 、-C(O)R a 、-SO 2 R a , azido and cyano group substitution;

[0238] 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 optionally substituted by one, two or three independently selected from C 1 -C 3 Alkyl, C 2 Alkynyl, amide, azido, carboxyl, cyano, dimethylamino, hydroxyl, methoxy, methylamino, HS- and CH 3 and

[0239] R 2 Selected from

[0240] , , , , , , , , and ;in

[0241] m is 0, 1, 2, or 3;

[0242] m'' is 0, 1, 2, 3, or 4;

[0243] B' is a 3-7 membered saturated or unsaturated ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen and sulfur; wherein the ring is optionally substituted by one or two independently selected from -OH, -CH 2 SH, CH 2 SCH 3 、-CH 2 OH, -CH 2 NH 2 and -CH 2 NHCH 3 Substituents are substituted;

[0244] Each R 500 are independently selected from hydrogen, C 1 -C 6 Alkyl, halide, -OH, and -CH 2 OH; and

[0245] X 50 Selected from O, NH, NCH 3 and S;

[0246] R 3 Selected from hydrogen, acetyl, amino, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylaminomethyl, C 1 -C 6 Alkylcarbonyl, aminoC 1 -C 6 Alkyl, aminocarbonyl, aminomethyl, carboxyl, cyano, C 3 Cycloalkyl, formyl, hydroxyl, hydroxyl C 1 -C 6 Alkyl, methoxy, Azolyl, -SH, -SCH 3 、-SOCH 3 、-SO 2 CH 3 、-SO(=NH)CH 3 , tetrazolyl, thiazolyl and trifluoromethyl, wherein the C 3 The cycloalkyl group is optionally substituted with hydroxy;

[0247] R 4Selected from hydrogen, methyl, -CH 2 OH, -CH 2 SH and -CH 2 SCH 3 ;

[0248] R 5 Selected from hydrogen, hydroxyl, -CH 2 SH, -CH 2 SCH 3 and methyl;

[0249] Optionally, the prerequisite is that when R 5 is hydroxy or methyl, and R 4 When it is hydrogen, then R 1 Not C 3 -C 6 Cycloalkyl, 3 to 6 membered heterocyclic ring, hydroxyl, hydroxyl (C 1 -C 6 Alkyl), -OR a , where R a It is C 1 -C 6 alkyl, 3 to 6 membered heterocyclic ring or hydroxyl (C 1 -C 6 or -NR a R b , where R a and R b independently selected from hydrogen, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, hydroxyl (C 1 -C 6 alkyl), a 3- to 6-membered heterocyclic ring, or wherein R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from C 1 -C 6 Alkyl or hydroxyl (C 1 -C 6 alkyl) group; and

[0250] R 6 and R 6' are independently selected from hydrogen, cyclopropyl, -CH 2 OH, -CH 2 SH, -CH 2 SCH 3 and -CH 2 R 200 , where R 200is a 3-7 membered saturated or unsaturated ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0251] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0252] In certain aspects, the present disclosure relates to Cbl-b inhibitors of formula (IA-1):

[0253]

[0254] (IA-1),

[0255] or a pharmaceutically acceptable salt thereof, wherein:

[0256] n is 0, 1, or 2;

[0257] X and Y are each independently CH or N;

[0258] Z is selected from CH(CH 3 ), O and SO 2 ;

[0259] R 1 Selected from 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 ring; wherein

[0260] R z Selected from

[0261]

[0262] R a and R b are independently selected from hydrogen, C 2 -C 6 Alkenyl, C 1 -C 6 Alkyl, amide (C 1 -C 6 Alkyl), amino (C 1 -C 6 alkyl), azido (C1 -C 6 Alkyl), C 2 -C 6 Alkynyl, carboxyl (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 heterocyclic ring, 3-6 membered heterocyclic group (C 1 -C 3 ) alkyl, hydroxyl (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), where R c and R d are independently selected from hydrogen, C 2 alkenylcarbonyl and methyl; or,

[0263] R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from R a 、-C(O)R a 、-SO 2 R a , azido and cyano group substitution;

[0264] Each C 3 -C 6 cycloalkyl, each 3-6 membered heterocyclyl ring, and the 3-6 membered heterocyclyl (C 1 -C 3 The heterocyclyl moiety of the alkyl) is optionally substituted by one, two or three independently selected from C 1 -C 3 Alkyl, C 2 Alkynyl, amide, azido, carboxyl, cyano, dimethylamino, hydroxyl, methoxy, methylamino, HS- and CH 3and

[0265] R 2 yes

[0266] or ;in

[0267] m is 0, 1, 2, or 3;

[0268] R 3 Selected from hydrogen, acetyl, amino, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylaminomethyl, amino C 1 -C 6 Alkyl, aminocarbonyl, aminomethyl, carboxyl, cyano, C 3 Cycloalkyl, formyl, hydroxyl, hydroxyl C 1 -C 6 Alkyl, methoxy, Azolyl, -SH, -SCH 3 , tetrazolyl, thiazolyl and trifluoromethyl, wherein the C 3 The cycloalkyl group is optionally substituted with hydroxy;

[0269] R 4 and R 6 are independently selected from hydrogen, -CH 2 SH and -CH 2 SCH 3 ;and

[0270] R 5 Selected from hydroxyl, -CH 2 SH, -CH 2 SCH 3 and methyl.

[0271] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0272] The present disclosure also provides conjugates for activating effector T cells, and further provides methods for treating cancer, methods for increasing T cell activity, methods for increasing T cell proliferation, methods for increasing migration of T cells to tumor cells, methods for reducing T cell exhaustion, and methods for using the conjugates to deliver payloads capable of activating effector T cells.

[0273] I. Definitions

[0274] In order that the description of the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0275] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "nucleotide sequence" should be understood to represent one or more nucleotide sequences. Therefore, the terms "one" (or "a"), "one or more", and "at least one" can be used interchangeably herein. It should be further noted that the claims can be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a precondition for the use of exclusive terms such as "only", "only" and the like associated with the recitation of claim elements or the use of negative limitations.

[0276] In addition, "and / or" as used herein should be considered as specifically disclosing each of two specified features or components with or without the other. Therefore, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following aspects: 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 (alone); B (alone); and C (alone).

[0277] It should be understood that where aspects are described herein with the language “comprising,” other similar aspects described with the terminology “consisting of” and / or “consisting essentially of” are also provided.

[0278] 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 the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press, provide a comprehensive dictionary of many of the terms used in the present disclosure to those skilled in the art.

[0279] Units, prefixes and symbols are expressed in a form recognized by its system International System of Units (Système International de Unites, SI). Numerical ranges include the values ​​defining the ranges. In the case of a range of narrated values, it is understood that each intermediate integer value and each fraction thereof between the upper and lower limits of that range are also specifically disclosed, as well as each subrange between such values. The upper and lower limits of any range may be independently included or excluded in the range, and each range including any limit, excluding two limits or including two limits is also encompassed in the present disclosure. Therefore, the ranges described herein are to be understood as shorthand for all values ​​within the range, with the described endpoints included.

[0280] Where a value is explicitly recited, it is understood that values ​​of approximately the same quantity or amount as the recited value are also within the scope of the present disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and within the scope of the present disclosure. Conversely, where different elements or groups of elements are disclosed individually, their combinations are also disclosed. Where any element of the present disclosure is disclosed as having multiple alternatives, examples of that disclosure excluding each alternative, either individually or in any combination with other alternatives, are also hereby disclosed; more than one element of the disclosure may have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0281] The terms "effector T cell", "T effector cell", "Teff" and "T-eff" are used interchangeably. In some aspects, the "effector T cell" is a CD8+ cytotoxic T cell.

[0282] The "activator" of effector T cells refers to a molecule that can promote one or more functions of effector T cells. The activator of effector T cells can promote effector T cell function, for example, by promoting the proliferation, survival and / or migration of effector T cells, by promoting the production of effector cytokines and / or cytotoxic mediators from effector T cells, and / or by reducing the exhaustion of effector T cells. The "activator" of effector T cells can also activate one or more other cell types, for example, one or more other T cell types, as long as the activator can activate effector T cells. As used herein, the term "interleukin-2 receptor subunit α", "IL2-RA", "IL2RA", "CD-25" or "CD25" refers to mammalian CD25 polypeptides, including but not limited to native CD25 polypeptides and CD25 polypeptide isoforms. These terms cover full-length unprocessed CD25 polypeptides and CD25 polypeptide forms produced by intracellular processing.

[0283] As used herein, the term "human CD25" refers to a polypeptide comprising amino acids 22-272 of SEQ ID NO: 1. SEQ ID NO: 1 provides the sequence of mature human CD25 (amino acids 22-272) and its signal sequence (amino acids 1-21, underlined below).

[0284] MDSYLLMWGLLTFIMVPGCQA ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQ MVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI (SEQ ID NO:1)

[0285] "CD25 polynucleotide," "CD25 nucleotide," or "CD25 nucleic acid" refers to a polynucleotide that encodes a CD25 polypeptide.

[0286] As used herein, the term "programmed cell death protein 1", "PD-1" or "PD1" refers to mammalian PD1 polypeptides, including but not limited to native PD1 polypeptides and PD1 polypeptide isoforms. These terms encompass full-length unprocessed PD1 polypeptides as well as PD1 polypeptide forms produced by intracellular processing.

[0287] As used herein, the term "human PD1" refers to a polypeptide comprising amino acids 24-288 of SEQ ID NO: 2. SEQ ID NO: 2 provides the sequence of mature human PD1 (amino acids 24-288) and its signal sequence (amino acids 1-23, underlined below).

[0288] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAH PSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO:2).

[0289] "PD1 polynucleotide", "PD1 nucleotide" or "PD1 nucleic acid" refers to a polynucleotide encoding a PD1 polypeptide.

[0290] As used herein, the term "T cell antigen CD-7", "CD-7" or "CD7" refers to mammalian CD7 polypeptides, including but not limited to native CD7 polypeptides and CD7 polypeptide isoforms. These terms encompass full-length unprocessed CD7 polypeptides as well as CD7 polypeptide forms produced by intracellular processing.

[0291] 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) as well as its signal sequence (amino acids 1-25, underlined below).

[0292] MAGPPRLLLLPLLLALARGLPGALA AQEVQQSPHCTTVPVGASVNITCSTSGGLRGIYLRQLGPQPQDIIYYEDGVVPTTDRRFRGRIDFSGSQDNLTITMHRLQLSDTGTYTCQAITEVNVYGSGTLVLVT EEQSQGWHRCSDAPPRASALPAPPTGSALPDPQTASALPDPPAASALPAALAVISFLLGLGLGVACVLARTQIKKLCSWRDKNSAACVVYEDMSHSRCNTLSSPNQYQ (SEQ ID NO:3).

[0293] "CD7 polynucleotide," "CD7 nucleotide" or "CD7 nucleic acid" refers to a polynucleotide that encodes a CD7 polypeptide.

[0294] As used herein, the term "Casitas B lineage lymphoma proto-oncogene b", "E3 ubiquitin-protein ligase Cbl-b" or "Cbl-b" refers to mammalian Cbl-b polypeptides, including but not limited to native Cbl-b polypeptides and Cbl-b polypeptide isoforms. These terms encompass full-length unprocessed Cbl-b polypeptides as well as Cbl-b polypeptide forms produced by intracellular processing.

[0295] As used herein, the term "human Cbl-b" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO:25.

[0296] MANSMNGRNPGGRGGNPRKGRILGIIDAIQDAVGPPKQAAADRRTVEKTWKLMDKVVRLCQNPKLQLKNSPPYILDILPDTYQHLRLILSKYDDNQKLAQLSENEYFKIYIDSLMKKSKRAIRLFKEGKERMYEEQSQDRRNLTKLSLIFSHMLAEIKAIFPNGQFQGDNFRITKADAAEFWRKFFGDKTIVPWKVFRQCLHEVHQISSGLEAMALKSTIDLTCNDYISVFEFDIFTRLFQPWGSILRNWNFLAVTHPGYMAFLTYDEVKARLQKYSTKPGSYIFRLSCTRLGQWAIGYVTGDGNILQTIPHNKPLFQALIDGSREGFYLYPDGRSYNPDLTGLCEPTPHDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTAWQESDGQGCPFCRCEIKGTEPIIVDPFDPRDEGSRCCSIIDPFGMPMLDLDDDDDREESLMMNRLANVRKCTDRQNSPVTSPGSSPLAQRRKPQPDPLQIPHLSLPPVPPRLDLIQKGIVRSPCGSPTGSPKSSPCMVRKQDKPLPAPPPPLRDPPPPPPERPPPIPPDNRLSRHIHHVESVPSRDPPMPLEAWCPRDVFGTNQLVGCRLLGEGSPKPGITASSNVNGRHSRVGSDPVLMRKHRRHDLPLEGAKVFSNGHLGSEEYDVPPRLSPPPPVTTLLPSIKCTGPLANSLSEKTRDPVEEDDDEYKIPSSHPVSLNSQPSHCHNVKPPVRSCDNGHCMLNGTHGPSSEKKSNIPDLSIYLKGDVFDSASDPVPLPPARPPTRDNPKHGSSLNRTPSDYDLLIPPLGEDAFDALPPSLPPPPPPARHSLIEHSKPPGSSSRPSSGQDLFLLPSDPFVDLASGQVPLPPARRLPGENVKTNRTSQDYDQLPSCSDGSQAPARPPKPRPRRTAPEIHHRKPHGPEAALENVDAKIAKLMGEGYAFEEVKRALEIAQNNVEVARSILREFAFPPPVSPRLNL (SEQ ID NO:25)。

[0297] "Cbl-b polynucleotide," "Cbl-b nucleotide," or "Cbl-b nucleic acid" refers to a polynucleotide that encodes a Cbl-b polypeptide.

[0298] As used herein, the term "antibody" refers to an immunoglobulin molecule that immunospecifically binds to a target of interest (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing) or a portion thereof. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies, fusion proteins comprising antibodies, and any other modified immunoglobulin molecules, as long as the antibody exhibits the desired biological activity. The antibodies disclosed herein may belong to any class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (isotype) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Immunoglobulins may be derived from any species. In one aspect, immunoglobulins have human, murine, or rabbit origin. Antibodies may be naked antibodies or conjugated to other molecules, such as activators of effector T cells, including small molecule activators of effector T cells.

[0299] "Complete antibodies" are antibodies comprising an antigen-binding variable region and 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.

[0300] "Antibody fragments" include a portion of an intact antibody. "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'), 2 and 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.

[0301] 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, whose name reflects its ability to crystallize readily. Pepsin treatment yields a F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0302] "Fv" is the smallest antibody fragment containing a complete antigen recognition and antigen binding site. This region consists of a dimer of a heavy chain and a light chain variable domain in tight non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. Together, the six hypervariable regions confer antigen binding specificity to antibodies or their antigen-binding fragments. However, even a single variable domain (or only half an Fv comprising three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen, but the affinity is lower than the entire binding site.

[0303] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the hinge region of the antibody. Fab'-SH is herein the nomenclature for Fab' in which the cysteine ​​residues of the constant domains carry at least one free thiol group. F(ab')2 antibody fragments were originally produced as Fab' fragment pairs with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0304] The term "single domain antibody", also known as a 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 a heavy chain variable domain or a light chain. Examples of single domain antibodies include, but are not limited to, V H H fragment and V NAR Fragment.

[0305] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may also comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0306] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which fragments comprise 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 and create two antigen-binding sites.

[0307] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is connected to a heavy chain by a covalent disulfide bond, and the number of disulfide links varies between heavy chains of different immunoglobulin isotypes. Each heavy chain and light chain also have regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its 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. It is believed that specific amino acid residues form the interface between the light chain and the heavy chain variable domain.

[0308] As used herein, the term "monoclonal" antibody or its antigen-binding fragment refers to an antibody or its antigen-binding fragment obtained from a population of substantially homogeneous antibodies or their antigen-binding fragments, that is, the individual antibodies or their antigen-binding fragments constituting the population are identical except for possible naturally occurring mutations that may exist in trace amounts. Monoclonal antibodies or their antigen-binding fragments are highly specific for a single antigenic site. In addition, in contrast to polyclonal antibody preparations comprising different antibodies for different determinants (epitopes), each monoclonal antibody or its antigen-binding fragment is directed to a single determinant on the antigen. In addition to its specificity, the advantage of monoclonal antibodies or their antigen-binding fragments is that they can be synthesized without being contaminated by other antibodies or their antigen-binding fragments. The modifier "monoclonal" indicates the characteristics of antibodies or their antigen-binding fragments obtained from a substantially homogeneous population of antibodies or their antigen-binding fragments, and should not be interpreted as requiring the production of antibodies or their antigen-binding fragments by any particular method. For example, monoclonal antibodies or their antigen-binding fragments to be used according to the present disclosure can be made by hybridoma methods, or can be made by recombinant DNA methods. "Monoclonal" antibodies or their antigen-binding fragments can also be isolated from phage antibody libraries.

[0309] Monoclonal antibodies and antigen-binding fragments thereof herein specifically include "chimeric" antibodies and antigen-binding fragments thereof, wherein a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody or antigen-binding fragment thereof derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the one or more chains is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as the fragment exhibits the desired biological activity. Chimeric antibodies and antigen-binding fragments thereof of interest herein include "primatized" antibodies and antigen-binding fragments thereof comprising variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0310] The "humanized" form of non-human (e.g., rodent) antibodies and antigen-binding fragments thereof is a chimeric antibody and antigen-binding fragment thereof containing minimal sequences derived from non-human immunoglobulins. Humanization is a method of transferring murine antigen binding information to non-immunogenic human antibody recipients, and many therapeutically available drugs have been produced. The humanization method generally begins by transferring all six murine complementary determining regions (CDRs) to a human antibody or antigen-binding fragment framework. These CDR-grafted antibodies or antigen-binding fragments thereof generally do not retain their initial affinity for antigen binding, and in fact, affinity is often severely impaired. In addition to CDRs, selected non-human antibody framework residues must also be incorporated to maintain appropriate CDR configurations. The transfer of key mouse framework residues to human recipients to support the structural configuration of grafted CDRs has been shown to restore antigen binding and affinity. In most cases, humanized antibodies or their antigen-binding fragments are human immunoglobulins or their fragments (acceptor antibodies or their fragments), wherein the residues from the hypervariable region of the acceptor are replaced by residues from the hypervariable region of a non-human species (donor antibody or its fragment) with the desired specificity, affinity and ability, such as mice, rats, rabbits or non-human primates. In some cases, the framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies or their antigen-binding fragments may include residues not found in the acceptor or donor antibodies or their fragments. These modifications are made to further improve the performance of antibodies or their antigen-binding fragments. In general, humanized antibodies or their antigen-binding fragments will include substantially all at least one and usually two variable domains, wherein all or substantially all hypervariable loops correspond to those of non-human immunoglobulins and all or substantially all FRs are those of human immunoglobulin sequences. Humanized antibodies or their antigen-binding fragments will optionally also include at least a portion of immunoglobulin constant region (Fc), typically the constant region of human immunoglobulins.

[0311] Chimeric and humanized antibodies and antigen-binding fragments thereof reduce the possibility of human anti-mouse antibody (HAMA) response by minimizing the non-human portion of the administered antibody. In addition, chimeric and humanized antibodies and antigen-binding fragments thereof may have the additional benefit of activating secondary human immune responses, such as antibody-dependent cellular cytotoxicity (ADCC).

[0312] An antibody or antigen-binding fragment thereof may have one or more "effector functions," which refers 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), etc. 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 does not have ADCC function.

[0313] Depending on the amino acid sequence of the constant domain of its heavy chain, antibodies and their antigen-binding fragments can be designated as different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are referred to as α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0314] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are commonly used in the art. Variable region generally refers to a part of an antibody or its antigen-binding fragment, generally a part of a light chain or a heavy chain, generally about 110 to 120 amino acids or 110 to 125 amino acids at the amino terminal in a mature heavy chain and about 90 to 115 amino acids in a mature light chain, the amino acids are widely different in sequence between antibodies and their antigen-binding fragments and are used in the binding and specificity of a specific antibody or its antigen-binding fragment to its specific antigen. Sequence variability is concentrated in those regions referred to as complementary determining regions (CDRs), while the more highly conserved regions in the variable domains are referred to as framework regions (FRs). It is not desirable to be bound by any particular mechanism or theory, and it is believed that the CDRs of light and heavy chains are primarily responsible for the interaction and specificity of antibodies or their antigen-binding fragments with antigens. In some aspects, the variable region is a human variable region. In some aspects, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some aspects, the variable region is a primate (e.g., non-human primate) variable region. In some aspects, the variable regions comprise rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

[0315] 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.

[0316] 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.

[0317] The term "Kabat numbering" etc. is generally recognized in the art and refers to a system in which the amino acid residues in the heavy chain and light chain variable region of an antibody or its antigen-binding fragment are numbered. In some respects, CDR can be determined according to the Kabat numbering system (see, for example, Kabat EA and Wu TT (1971) Ann NY Acad Sci 190: 382-391, and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, the 5th edition, USDepartment of Health and Human Services, NIH publication number 91-3242). Using the Kabat numbering system, the CDR in the antibody heavy chain molecule is usually present at amino acid positions 31 to 35, and optionally one or two additional amino acids (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1) after 35, amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule typically occur at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3).

[0318] Chothia refers instead to the position of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The ends of the Chothia CDR-H1 loop vary between H32 and H34 when numbered using the Kabat numbering convention, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, 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.

[0319] Table 1: CDR numbers

[0320]

[0321] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their commonly used meanings in the art. The constant region is the portion of an antibody, e.g., the carboxyl terminal portion of a light chain and / or a heavy chain, which is not directly involved in binding the antibody or its antigen-binding fragment to an antigen, but may exhibit various effector functions, such as interactions with Fc receptors. The constant region of an immunoglobulin molecule generally has an amino acid sequence that is more conserved relative to an immunoglobulin variable domain.

[0322] As used herein, the term "specific binding" refers to an interaction between molecules in which (i) binding becomes saturated when the concentration of one of the molecules increases relative to the other molecule, and (ii) binding can be competed for by the presence of excess one of the interacting molecules. A molecule (e.g., a binding domain, such as an antibody or an antigen binding fragment thereof) that can "specifically bind" to a target on the surface of an effector T cell can also bind to a soluble form of the target and / or a target in a different location (e.g., on the surface of another cell). A molecule (e.g., a binding domain, such as an antibody or an antigen binding fragment thereof) that can "specifically bind" to a target from one species (e.g., humans) can also bind to that target from another species (e.g., cynomolgus monkeys, mice, and / or rats), but the extent of binding to an unrelated target is less than about 10% of the binding to the target.

[0323] As used herein, the term "immunospecific binding" indicates that an antibody or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain and that binding requires some complementarity between the antigen-binding domain and the epitope. Thus, an antibody that "immunospecifically binds" to a human protein, such as human PD1, may also bind to that protein from other species (e.g., cynomolgus monkey, mouse and / or rat PD1) or to related proteins, but the extent of binding to unrelated proteins is less than about 10% of the binding of the antibody to PD1 as measured. An antibody that "immunospecifically binds" to an antigen of interest is an antibody that is capable of binding to that antigen with sufficient affinity so that the antibody can be used to target cells expressing the antigen.

[0324] The term "reduction / reduce" or "inhibition / inhibit" refers to a reduction or interruption of any phenotypic characteristic or a reduction or interruption of the incidence, degree or likelihood of that characteristic. "Reduction" or "inhibition" is to reduce, lower or suppress an activity, function and / or amount compared to a reference. In some aspects, "reduction" or "inhibition" means the ability to cause an overall reduction of 20% or more. In some aspects, "reduction" or "inhibition" means the ability to cause an overall reduction of 50% or more. In some aspects, "reduction" or "inhibition" means the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some aspects, the amounts mentioned above are inhibited or reduced over a certain period of time relative to a control over the same period of time.

[0325] A molecule that is an "inhibitor" of a target can reduce the activity, function, and / or amount of the target 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 a "Cbl-b inhibitor" can reduce the activity, function, and / or amount of Cbl-b compared to the activity, function, and / or amount of Cbl-b in the absence of the molecule, compound, or payload.

[0326] A molecule that is an "agonist" of a target can increase the activity, function, and / or amount of the target compared to the activity, function, and / or amount of the target in the absence of the agonist.

[0327] The term "about" is used herein to mean approximately, roughly, about or about. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify the numerical values ​​above and below the value by, for example, a variance of 10% or more (higher or lower). It should be understood that in the case of describing aspects with the language "about" or "approximately" herein, other aspects are also provided that are similar, but refer to numerical values ​​or ranges of specific numerical values ​​or ranges (without "about"). As used herein, the term "administer / administering / administration" and the like refer to methods that can be used to enable the delivery of drugs, such as effector T cell activators (e.g., small molecules or antibody drug conjugates) to desired biological sites of action. The administration techniques that can be used with the agents and methods described herein are found in, for example, Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0328] The terms "antibody-drug conjugate" and "ADC" are used interchangeably and refer to, for example, an antibody or antigen-binding fragment thereof covalently linked to one or more therapeutic agents (sometimes referred to herein as agents, drugs, or active pharmaceutical ingredients). In some aspects of the present disclosure, the biologically active molecule is an antibody-drug conjugate. In some aspects, 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 can also activate one or more other cell types, for example, one or more other T cell types, as long as the ADC can activate effector T cells.

[0329] "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been 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), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution should be considered conservative. In another aspect, a series of amino acids can be structurally similar conservative substitutions that differ in the order and / or composition of side chain family members.

[0330] As used herein, the term "conserved" refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, which are those that have not changed in the same position of two or more sequences being compared. Relatively conservative nucleotides or amino acids are those that are conserved among sequences that are more related than nucleotides or amino acids that occur elsewhere in the sequence.

[0331] In some aspects, if two or more sequences are 100% identical to each other, the sequences are said to be "fully conserved" or "identical". In some aspects, if two or more sequences 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, the sequences are said to be "highly conserved". In some aspects, if two or more sequences 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, the sequences are said to be "conserved". Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or can apply to a portion, region, or feature thereof.

[0332] As used herein, the terms "linked" and "conjugated" are used interchangeably and each refers to the covalent or non-covalent connection of two or more moieties comprising one or more compounds capable of activating effector T cells and a binding moiety. In some aspects, the connection or conjugation may comprise a linker.

[0333] The term "amino acid sequence variant" refers to a polypeptide having an amino acid sequence that is different from a native sequence polypeptide to a certain extent. Typically, an amino acid sequence variant will have at least about 70% sequence identity with at least one receptor binding domain of a native antibody or with at least one ligand binding domain of a native receptor, and typically, it will have at least about 80%, more typically at least about 90% sequence homology with such receptor or ligand binding domain. An amino acid sequence variant has a substitution, a deletion and / or an insertion at some position within the amino acid sequence of the native amino acid sequence. Amino acids are specified by conventional names, single letters and three-letter codes.

[0334] "Sequence identity" is defined as the percentage of identical residues in amino acid sequence variants after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence identity percentage. Methods and computer programs for alignment are well known in the art. One such computer program is "Align 2" written by Genentech, Inc., which was filed with the United States Copyright Office, Washington, DC 20559, on December 10, 1991, along with user files.

[0335] The term "Fc receptor" or "FcR" is used to describe the receptor that is bound to the Fc region of an antibody or its antigen binding fragment. An exemplary FcR is a native sequence human FcR. In addition, FcR can be a FcR (gamma receptor) that binds to an IgG antibody or its antigen binding fragment and includes receptors of FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors") with similar amino acid sequences and mainly differ 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 inhibition motif (ITIM) in its cytoplasmic domain. Other FcRs are covered by the term "FcR" in this article. The term also includes a neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus.

[0336] "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 (Clq) to a molecule (e.g., an antibody or antigen-binding fragment thereof) complexed with a cognate antigen. To assess complement activation, a CDC assay can be performed.

[0337] The "light chains" of antibodies or antigen-binding fragments thereof from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0338] "Isolated" antibodies or AFs thereof are antibodies or AFs thereof that have been identified and separated and / or recovered from components of their natural environment. The pollutant components of their natural environment are materials that will interfere with the diagnostic or therapeutic use of the antibody or AFs thereof, and may include enzymes, hormones, and other proteins or non-protein solutes. In certain aspects, the antibody or AFs thereof will be purified (1) to greater than 95% by weight or greater than 99% by weight of the antibody or AFs thereof as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence measured by using a gas phase protein sequencer, or (3) to homogeneity measured by SDS-PAGE using Coomassie blue or silver stain under reducing or non-reducing conditions. The isolated antibody or AFs thereof include in situ antibodies or AFs thereof within recombinant cells, because at least one component of the natural environment of the antibody or AFs thereof will not be present. Typically, however, the isolated antibody or AFs thereof will be prepared by at least one purification step.

[0339] "Cancer" refers to a large group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors, which invade adjacent tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. As used herein, "cancer" refers to primary, metastatic, and recurrent cancers.

[0340] As used herein, the term "immune response" refers to a biological response in vertebrates to foreign agents that protects the organism from these agents and the diseases caused by them. The 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 any of these cells or the liver, which selectively targets, binds to, damages, destroys, and / or eliminates invading pathogens, cells or tissues infected by pathogens, cancer cells or other abnormal cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation from the vertebrate body. The immune response includes, for example, activation or inhibition of T cells, such as effector T cells or Th cells, such as CD4 + or CD8 +T cells, or suppress Treg cells. As used herein, the terms "T cells" and "T lymphocytes" are interchangeable and refer to any lymphocyte produced or processed by the thymus. In some aspects, the T cells are CD4+ T cells. In some aspects, the T cells are CD8+ T cells. In some aspects, the T cells are NKT cells.

[0341] "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 aspects, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0342] The term "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of an agent (e.g., a conjugate disclosed herein) that provides a desired biological, therapeutic, and / or preventive result. That result can be a reduction, improvement, alleviation, mitigation, delay, and / or alleviation of one or more signs, symptoms, or causes of a disease, or any other desired change in a biological system. With respect to solid tumors, an effective amount comprises an amount sufficient to cause tumor shrinkage and / or reduce tumor growth rate (such as inhibiting tumor growth) or prevent or delay other inappropriate cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. An effective amount of a composition can, for example, (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, hinder, slow down to some extent, and can terminate cancer cell infiltration into peripheral organs; (iv) inhibit, i.e., slow down to some extent, and can terminate tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor occurrence and / or recurrence; and / or (vii) alleviate to some extent one or more symptoms associated with cancer.

[0343] In some aspects, a "therapeutically effective amount" is an amount of the conjugate that is clinically demonstrated to achieve a significant reduction in cancer or a slowing of progression (regression) of a cancer (such as an advanced solid tumor). The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in an in vitro assay.

[0344] As used herein, the term "standard of care" refers to a treatment that is generally recognized by medical experts as an appropriate treatment for a certain type of disease and is widely used by health care professionals. The term may be used interchangeably with any of the following terms: "best practice," "standard of medical care," and "standard therapy."

[0345] For example, an "anti-cancer agent" promotes regression of cancer in a subject or prevents further tumor growth. In certain aspects, a therapeutically effective amount of a drug promotes regression of cancer to the point of eliminating the cancer.

[0346] The terms "effective" and "effectiveness" with respect to treatment include pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote regression of cancer in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ and / or organismal level caused by the administration of the drug.

[0347] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins regulate T cell activation or function. Numerous checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD1 and its ligands PDL1 and PDL2. Pardoll, DM, NatRev Cancer 12(4):252-64 (2012). These proteins are responsible for the co-stimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins regulate and maintain the self-tolerance, duration, and amplitude of physiological immune responses. Immune checkpoint inhibitors include antibodies and antigen-binding fragments thereof.

[0348] The term "treat" or "treatment" refers to both therapeutic treatment and preventive or defensive measures, wherein the purpose is to prevent or slow down (mitigate) undesirable physiological changes or conditions, such as the development or spread of cancer. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, relief of symptoms, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to the expected survival if not receiving treatment. Those in need of treatment include those who already have a disease or condition as well as those who are susceptible to a disease or condition or those for whom a disease or condition is to be prevented.

[0349] In the absence of particular designation, stereocenters within the compounds described herein may be R or S stereochemistry and may be substituted as described elsewhere in the present disclosure. It should be understood that when stereochemistry is not specified, the present disclosure encompasses all stereochemical isomeric forms or mixtures thereof having the ability to inhibit Cbl-b and / or activate T cells. Individual stereoisomers of compounds may be prepared synthetically from commercially available starting materials containing chiral centers, or by preparing a mixture of enantiomeric products, followed by separation, such as conversion into a mixture of diastereoisomers, followed by separation or recrystallization, chromatographic techniques, or direct separation of enantiomers on a chiral chromatographic column. Starting compounds of specific stereochemistry are commercially available or may be prepared and resolved by techniques known in the art.

[0350] II. Cbl-b inhibitors

[0351] In certain aspects, the present disclosure provides Cbl-b inhibitors of Formula (I):

[0352]

[0353] (I)

[0354] or a pharmaceutically acceptable salt thereof, wherein:

[0355] n is 0, 1, or 2;

[0356] X and Y are each independently CH or N;

[0357] Z is selected from CH(CH 3 ), O and SO 2 ;or

[0358] Z is selected from CH(CH 3 ), NH, N(CH 3 ), O and SO 2 ;

[0359] R 1 Selected from 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 ring; wherein

[0360] Rz Selected from

[0361]

[0362] R a and R b are independently selected from hydrogen, C 2 -C 6 Alkenyl, C 1 -C 6 Alkyl, amide (C 1 -C 6 Alkyl), amino (C 1 -C 6 alkyl), azido (C 1 -C 6 Alkyl), C 2 -C 6 Alkynyl, carboxyl (C 1 -C 6 Alkyl), cyano (C 1 -C 6 alkyl), C optionally substituted by cyano 3 -C 6 Cycloalkyl, dimethylamino (C 1 -C 6 alkyl), 3- to 6-membered heterocyclic ring, 3- to 6-membered heterocyclic (C 1 -C 3 Alkyl), hydroxyl (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), where R c and R d are independently selected from hydrogen, C 2 alkenylcarbonyl and methyl; or,

[0363] R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from R a 、-C(O)R a 、-SO 2 Ra , azido and cyano group substitution;

[0364] 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 optionally substituted by one, two or three independently selected from C 1 -C 3 Alkyl, C 2 Alkynyl, amide, azido, carboxyl, cyano, dimethylamino, hydroxyl, methoxy, methylamino, HS- and CH 3 and

[0365] R 2 Selected from

[0366] , , , , , , , , and ;in

[0367] m is 0, 1, 2, or 3;

[0368] m'' is 0, 1, 2, 3, or 4;

[0369] B' is a 3-7 membered saturated or unsaturated ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen and sulfur; wherein the ring is optionally substituted by one or two independently selected from -OH, -CH 2 SH, CH 2 SCH 3 、-CH 2 OH, -CH 2 NH 2 and -CH 2 NHCH 3 Substituents are substituted;

[0370] Each R 500 are independently selected from hydrogen, C 1 -C 6 Alkyl, halide, -OH, and -CH 2 OH; and

[0371] X 50 Selected from O, NH, NCH 3 and S;

[0372] R3 Selected from hydrogen, acetyl, amino, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylaminomethyl, C 1 -C 6 Alkylcarbonyl, aminoC 1 -C 6 Alkyl, aminocarbonyl, aminomethyl, carboxyl, cyano, C 3 Cycloalkyl, formyl, hydroxyl, hydroxyl C 1 -C 6 Alkyl, methoxy, Azolyl, -SH, -SCH 3 、-SOCH 3 、-SO 2 CH 3 、-SO(=NH)CH 3 , tetrazolyl, thiazolyl and trifluoromethyl, wherein the C 3 The cycloalkyl group is optionally substituted with hydroxy;

[0373] R 4 Selected from hydrogen, methyl, -CH 2 OH, -CH 2 SH and -CH 2 SCH 3 ;

[0374] R 5 Selected from hydrogen, hydroxyl, -CH 2 SH, -CH 2 SCH 3 and methyl;

[0375] Optionally, the prerequisite is that when R 5 is hydroxy or methyl, and R 4 When it is hydrogen, then R 1 Not C 3 -C 6 Cycloalkyl, 3 to 6 membered heterocyclic ring, hydroxyl, hydroxyl (C 1 -C 6 Alkyl), -OR a , where R a It is C 1 -C 6 alkyl, 3 to 6 membered heterocyclic ring or hydroxyl (C 1 -C 6 or -NR a R b , where R a and R b independently selected from hydrogen, C 1 -C 6Alkyl, C 3 -C 6 Cycloalkyl, hydroxyl (C 1 -C 6 alkyl), a 3- to 6-membered heterocyclic ring, or wherein R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from C 1 -C 6 Alkyl or hydroxyl (C 1 -C 6 alkyl) group; and

[0376] R 6 and R 6' are independently selected from hydrogen, cyclopropyl, -CH 2 OH, -CH 2 SH, -CH 2 SCH 3 and -CH 2 R 200 , where R 200 is a 3-7 membered saturated or unsaturated ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0377] In some respects, R 4 In some aspects, R 4 In "S" configuration. In certain aspects, the present disclosure provides a Cbl-b inhibitor of formula (IA-1):

[0378]

[0379] (IA-1);

[0380] or a pharmaceutically acceptable salt thereof, wherein:

[0381] n is 0, 1, or 2;

[0382] X and Y are each independently CH or N;

[0383] Z is selected from CH(CH 3 ), O and SO 2 ;

[0384] R 1 Selected from 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 ring; wherein

[0385] R z Selected from

[0386]

[0387] R a and R b are independently selected from hydrogen, C 2 -C 6 Alkenyl, C 1 -C 6 Alkyl, amide (C 1 -C 6 Alkyl), amino (C 1 -C 6 alkyl), azido (C 1 -C 6 Alkyl), C 2 -C 6 Alkynyl, carboxyl (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 heterocyclic ring, 3-6 membered heterocyclic group (C 1 -C 3 ) alkyl, hydroxyl (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), where R c and R d are independently selected from hydrogen, C 2 alkenylcarbonyl and methyl; or,

[0388] R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from R a 、-C(O)R a 、-SO 2 R a , azido and cyano group substitution;

[0389] Each C 3 -C 6 cycloalkyl, each 3-6 membered heterocyclyl ring, and the 3-6 membered heterocyclyl (C 1 -C 3 The heterocyclyl moiety of the alkyl) is optionally substituted by one, two or three independently selected from C 1 -C 3 Alkyl, C 2 Alkynyl, amide, azido, carboxyl, cyano, dimethylamino, hydroxyl, methoxy, methylamino, HS- and CH 3 and

[0390] R 2 yes

[0391] or ;in

[0392] m is 0, 1, 2, or 3;

[0393] R 3 Selected from hydrogen, acetyl, amino, C 1 -C 6 Alkylamino, C 1 -C 6 Alkylaminomethyl, amino C 1 -C 6 Alkyl, aminocarbonyl, aminomethyl, carboxyl, cyano, C 3 Cycloalkyl, formyl, hydroxyl, hydroxyl C 1 -C 6 Alkyl, methoxy, Azolyl, -SH, -SCH 3 , tetrazolyl, thiazolyl and trifluoromethyl, wherein the C 3 The cycloalkyl group is optionally substituted with hydroxy;

[0394] R 4 and R 6 are independently selected from hydrogen, -CH 2 SH and -CH 2 SCH 3 ;and

[0395] R5 Selected from hydroxyl, -CH 2 SH, -CH 2 SCH 3 and methyl;

[0396] Optionally, the prerequisite is that when R 5 is hydroxy or methyl, and R 4 When it is hydrogen, then R 1 Not C 3 -C 6 Cycloalkyl, 3 to 6 membered heterocyclic ring, hydroxyl, hydroxyl (C 1 -C 6 Alkyl), -OR a , where R a It is C 1 -C 6 alkyl, 3 to 6 membered heterocyclic ring or hydroxyl (C 1 -C 6 or -NR a R b , where R a and R b independently selected from hydrogen, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl, hydroxyl (C 1 -C 6 alkyl), a 3- to 6-membered heterocyclic ring, or wherein R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from C 1 -C 6 Alkyl or hydroxyl (C 1 -C 6 alkyl) groups.

[0397] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0398] Compounds of formula (I) do not include those disclosed in WO2019 / 148005, WO2020 / 210508 and WO2021 / 021761.

[0399] In some aspects, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0400] X is N;

[0401] Y is CH; and

[0402] R 2 yes .

[0403] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0404] In some aspects, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein Z is O.

[0405] In some aspects, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein Z is CH(CH 3 ).

[0406] In some aspects, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0407] R 3 is trifluoromethyl;

[0408] R 4 and R 6 is hydrogen; and

[0409] R 5 It's methyl.

[0410] In some aspects, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 Selected from -NR a R b 、-NHC(O)R a 、-NHC(S)NHR a and-SR a .

[0411] In some aspects, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 Yes-SCH 2 CH 3 .

[0412] In some aspects, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is -NH(CH 2 ) 2 CN.

[0413] In some aspects, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 It is -NH(CH 2 ) 2 N 3 .

[0414] As used herein, the term "acetyl" refers to -C(O)CH 3 .

[0415] As used herein, the term “C 2 "Alkenyl" refers to -CH=CH 2 .

[0416] As used herein, the term “C 2 -C 6 "Alkenyl" refers to a group derived from a straight or branched chain hydrocarbon containing from two to six carbon atoms and containing at least one double bond.

[0417] As used herein, the term “C 2 "Alkenylcarbonyl" refers to a C 2 Alkenyl.

[0418] As used herein, the term “C 1 -C 6 "Alkoxy" refers to a C-1-alkyl group attached to the parent molecular moiety through an oxygen atom. 1 -C 6 alkyl.

[0419] As used herein, the term “C 1 -C 6 Alkoxy C 1 -C 6 "Alkyl" refers to a C 1 -C 6 The alkyl group is attached to the C 1 -C 6 Alkoxy.

[0420] As used herein, the term “C 1 -C 6 "Alkyl" refers to a radical derived from a straight or branched chain saturated hydrocarbon containing from one to six carbon atoms.

[0421] As used herein, the term “C 1 -C 6 "Alkylamino" refers to -NHR, where R is C 1 -C 6 alkyl.

[0422] As used herein, the term “C 1 -C 6 "Alkylaminomethyl" refers to a C 1 -C 6 Alkylamino.

[0423] As used herein, the term “C 2 "Alkynyl" means .

[0424] As used herein, the term “C 2 -C 6 "Alkynyl" refers to a radical derived from a straight or branched chain hydrocarbon containing from two to six carbon atoms and containing at least one double bond.

[0425] As used herein, the term "amide" refers to -C(O)NH 2 .

[0426] As used herein, the term "amide group (C 1 -C 6 "alkyl)" refers to C 1 -C 6 The alkyl group is attached to the amide group of the parent molecular moiety.

[0427] As used herein, the term "amino" refers to -NH 2 .

[0428] As used herein, the term "amino (C 1 -C 6 "alkyl)" refers to C 1 -C 6 The alkyl group is attached to the amino group of the parent molecular moiety.

[0429] The term "aminocarbonyl," as used herein, refers to an amino group attached to the parent molecular moiety through a carbonyl group.

[0430] The term "aminomethyl," as used herein, refers to an amino group attached to the parent molecular moiety through a methylene group.

[0431] As used herein, the term "azido" refers to -N 3 .

[0432] As used herein, the term "azido" 1 -C 6 "alkyl)" refers to C 1 -C 6 The alkyl group is attached to the azide group of the parent molecular moiety.

[0433] As used herein, the term "carbonyl" refers to -C(O)-.

[0434] As used herein, the term "carboxyl" refers to -CO 2 H.

[0435] As used herein, the term "carboxyl (C 1 -C 6 "alkyl)" refers to C 1 -C 6 The alkyl group is attached to the carboxyl group of the parent molecular moiety.

[0436] As used herein, the term "cyano" refers to -CN.

[0437] As used herein, the term "cyano (C 1 -C 6 "alkyl)" refers to C 1 -C 6 The alkyl group is attached to the cyano group of the parent molecular moiety.

[0438] As used herein, the term “C 3 "Cycloalkyl" refers to cyclopropyl.

[0439] As used herein, the term “C 3 -C 6 "Cycloalkyl" refers to a saturated monocyclic hydrocarbon ring system having three to six carbon atoms and zero heteroatoms. Representative examples of cycloalkyl include, but are not limited to, cyclobutyl, cyclopentyl, and cyclohexyl.

[0440] As used herein, the term "dimethylamino (C 1 -C 6 "alkyl)" refers to C 1 -C 6 The alkyl group is attached to the dimethylamino group of the parent molecular moiety.

[0441] As used herein, the term "formyl" refers to -CHO.

[0442] As used herein, the term "halo" refers to F, Cl, Br or I.

[0443] As used herein, the term "3 to 6 membered heterocyclyl" refers to a three-, four-, five- or six-membered saturated or unsaturated group containing one, two, three or four heteroatoms independently selected from nitrogen, oxygen and sulfur. Representative examples of 3 to 6 membered heterocyclyls include, but are not limited to, oxetane, pyrrolidine, thiazole, pyridine and piperidine.

[0444] As used herein, the term "3 to 6 membered heterocyclic group (C 1 -C 3 "alkyl)" refers to C 1 -C 3 The alkyl is attached to the parent molecular moiety of a 3- to 6-membered heterocyclyl ring.

[0445] As used herein, the term "hydroxy" refers to -OH.

[0446] As used herein, the term "hydroxy C 1 -C 6 "Alkyl" refers to a C substituted with one, two or three hydroxy groups. 1 -C 6 alkyl.

[0447] As used herein, the term "methoxy (C1 -C 6 "alkyl)" refers to C 1 -C 6 The alkyl group is attached to the methoxy group of the parent molecular moiety.

[0448] As used herein, the term "methylamino (C 1 -C 6 "alkyl)" refers to C 1 -C 6 The alkyl group is attached to the methylamino group of the parent molecular moiety.

[0449] As used herein, the term “NR c R d (C 1 -C 6 "alkyl)" refers to C 1 -C 6 NR with an alkyl group attached to the parent molecular moiety c R d Group. R c and R d As defined herein.

[0450] As used herein, the term "HS(C 1 -C 6 "alkyl)" refers to C 1 -C 6 The alkyl group is attached to the -SH group of the parent molecular moiety.

[0451] In some aspects, the compound of formula (I) is a compound selected from the group consisting of:

[0452] , and .

[0453] In some aspects, the compounds of formula (I) can treat cancer in a subject. In some aspects, the compounds of formula (I) can increase effector T cell activity and / or effector T cell proliferation. In some aspects, the compounds of formula (I) can increase the migration of effector T cells to tumor cells. In some aspects, the compounds of formula (I) can reduce effector T cell exhaustion.

[0454] In some aspects, the compound of formula (I) competitively inhibits the binding of a probe comprising a BODIPY-FL fluorophore conjugated to a Cbl-b inhibitor (see Example 54 in WO20200264398) to Cbl-b, for example, as assessed using the assay described in Example 2 herein. In some aspects, the compound of formula (I) competitively inhibits with an IC50 of about 1 nM to about 5 nM. In some aspects, the compound of formula (I) competitively inhibits with an IC50 of about 5.01 nM to about 20 nM. In some aspects, the compound of formula (I) competitively inhibits with an IC50 of about 20.01 nM to about 100 nM. In some aspects, the compound of formula (I) competitively inhibits with an IC50 of about 100.01 nM to about 1 mM.

[0455] In some aspects, the compounds of formula (I) are capable of increasing the secretion of IL-2 from T cells, e.g., as assessed using the assay described in Example 3 herein. In some aspects, the compounds of formula (I) increase IL-2 secretion by about 0.8 to about 1.4 times relative to background. In some aspects, the compounds of formula (I) increase IL-2 secretion by about 0.31 to about 0.79 times relative to background. In some aspects, the compounds of formula (I) increase IL-2 secretion by about 0.1 to about 0.3 times relative to background.

[0456] In some aspects, the compounds of formula (I) are capable of increasing the secretion of IFN-γ from T cells, e.g., as assessed using the assay described in Example 3 herein. In some aspects, the compounds of formula (I) increase IFN-γ secretion by about 1.1 to about 2 times relative to background. In some aspects, the compounds of formula (I) increase IFN-γ secretion by about 0.31 to about 1 times relative to background. In some aspects, the compounds of formula (I) increase IFN-γ secretion by about 0.1 to about 0.3 times relative to background.

[0457] In some aspects, the compounds of formula (I) are capable of increasing CD69 levels, for example, as assessed using the assay described in Example 3 herein. In some aspects, the compounds of formula (I) increase CD69 levels by about 1.11 to about 1.5 times relative to background. In some aspects, the compounds of formula (I) increase CD69 levels by about 0.81 to about 1.10 times relative to background. In some aspects, the compounds of formula (I) increase CD69 levels by about 0.81 to about 1.1 times relative to background. In some aspects, the compounds of formula (I) increase CD69 levels by about 0.61 to about 0.8 times relative to background. In some aspects, the compounds of formula (I) increase CD69 levels by about 0.3 to about 0.6 times relative to background.

[0458] In some aspects, the compounds of formula (I) interact with His152 of human Cbl-b. In some aspects, the compounds of formula (I) have a terminal nitrogen atom in the R 1 In some aspects, 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 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 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. 1 The 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.

[0459] III. Conjugates

[0460] The present disclosure provides conjugates, the conjugates comprising a binding portion (e.g., an antibody or its antigen binding fragment) 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 portion can be directly connected to the payload or can be connected to the payload via a joint. In some aspects, the payload can be covalently bonded to a joint or a binding portion.

[0461] In certain aspects, where the payload is a compound of formula (I):

[0462]

[0463] (I);

[0464] The compound of formula (I) can be reacted with a group R 1 The heteroatom in is covalently bonded to the linker.

[0465] In some aspects, where the payload is a compound of formula (Ia):

[0466]

[0467] (Ia);

[0468] The compound of formula (Ia) can be reacted with R 1 , R 3 , R 4 , R 5 , R 6 or a covalent bond to the linker through a nitrogen atom of the piperidine ring.

[0469] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0470] In some aspects, where the payload is a compound of formula (Ib):

[0471]

[0472] (Ib);

[0473] The compound of formula (Ib) can be reacted with R 1 , R 3 , R 4 , R 5 , R 6 or a covalent bond to the linker through a nitrogen atom of the piperidine ring.

[0474] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0475] In some aspects, where the payload is a compound of formula (Ic):

[0476]

[0477] (Ic);

[0478] The compound of formula (Ic) can be reacted with R 1 , R 3 , R 4 , R 5 , R 6 or a covalent bond to the linker through a nitrogen atom of the piperidine ring.

[0479] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0480] In some aspects, where the payload is a compound of formula (Id):

[0481]

[0482] (Id),

[0483] The compound of formula (Id) can be reacted with R 1 , R 3 , R 4 , R 5 , R 6 , R 6' or a covalent bond to the linker through a nitrogen atom of the piperidine ring.

[0484] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0485] In some aspects, where the payload is a compound of formula (Ie):

[0486]

[0487] (Ie)

[0488] The compound of formula (Ie) can be reacted with R 1 , R 3 , R 4 , R 500 , B' or a covalent bond to the linker through the nitrogen atom of the piperidine ring.

[0489] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0490] In some aspects, where the payload is a compound of formula (If):

[0491]

[0492] (If),

[0493] The compound of formula (If) can be reacted with R 1 , R 3 , R 4 , B' or a covalent bond to the linker through the nitrogen atom of the piperidine ring.

[0494] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0495] In some aspects, where the payload is a compound of formula (Ig):

[0496]

[0497] (Ig),

[0498] The compound of formula (Ig) can be reacted with R 1 , R 3 , R 4 , R 500 , B' or a covalent bond to the nitrogen atom of B'.

[0499] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0500] In some aspects, where the payload is a compound of formula (Ih):

[0501]

[0502] (Ih),

[0503] The compound of formula (Ig) can be reacted with R 1 , R 3 , R 4 , B' or a covalent bond to the linker through the nitrogen atom of the piperidine ring.

[0504] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0505] In some aspects, where the payload is a compound of formula (Ii):

[0506]

[0507] (ii),

[0508] The compound of formula (Ii) can be reacted with R 1 , R 3 , R 4 , B' or a covalent bond to the linker through the nitrogen atom of the piperidine ring.

[0509] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0510] In some aspects, where the payload is a compound of formula (Ij):

[0511]

[0512] (Ij),

[0513] The compound of formula (Ii) can be reacted with R 1 , R 3 , R 4, B' or any nitrogen atom of the piperazine ring is covalently linked to the linker.

[0514] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0515] In some aspects, where the payload is a compound of formula (Ik):

[0516]

[0517] (Ik),

[0518] The compound of formula (Ik) can be reacted with R 1 , R 3 , R 4 , R 6 , R 6' or a covalent bond to the linker through a nitrogen atom of the piperidine ring.

[0519] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0520] In some aspects, where the payload is a compound of formula (II):

[0521]

[0522] (Il),

[0523] The compound of formula (Ik) can be reacted with R 1 , R 3 , R 4 , R 6 , R 6' or a covalent bond to the linker through a nitrogen atom of the piperidine ring.

[0524] In some respects, R 4 In some aspects, R 4 It is in an "S" shape.

[0525] In some aspects, the conjugates provided herein comprise a binding portion (e.g., an antibody or an antigen binding fragment thereof) that can specifically bind to PD1 and a payload that can activate effector T cells. In some aspects, the conjugates provided herein comprise a binding portion (e.g., an antibody or an antigen binding fragment thereof) that can specifically bind to PD1 and a payload that is an inhibitor of Casitas B lineage lymphoma proto-oncogene b (Cbl-b).

[0526] In some aspects, the conjugates provided herein comprise a binding portion (e.g., an antibody or an antigen binding fragment thereof) that can specifically bind to CD25 (IL2RA) and a payload that can activate effector T cells. In some aspects, the conjugates provided herein comprise a binding portion (e.g., an antibody or an antigen binding fragment thereof) that can specifically bind to PD1 and a payload that is an inhibitor of Casitas B lineage lymphoma proto-oncogene b (Cbl-b).

[0527] In some aspects, 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).

[0528] In some aspects, the conjugates provided herein have formula (II): Bm-[LP] a (II), wherein a is an integer from 1 to 50; P is a payload; L is a linker; and Bm is a binding moiety (e.g., an antibody or antigen-binding fragment thereof). In some aspects, a is from about 1 to about 40. In some aspects, a is from about 1 to about 10. In some aspects, a is from about 2 to about 8.

[0529] In some aspects, a composition (e.g., a pharmaceutical composition) is provided herein, comprising a conjugate (e.g., at least 2 conjugates) provided herein. In some aspects, the composition comprises an average of about 1 to about 50 payloads per binding moiety. In some aspects, the composition comprises an average of about 1 to about 10 payloads per binding moiety. In some aspects, the composition comprises an average of about 2 to about 8 payloads per binding moiety.

[0530] In some aspects, the conjugates or compositions provided herein can treat cancer in a subject. In some aspects, the conjugates or compositions provided herein can increase effector T cell activity and / or effector T cell proliferation. In some aspects, the conjugates or compositions provided herein can increase the migration of effector T cells to tumor cells. In some aspects, the conjugates or compositions provided herein can reduce effector T cell exhaustion. In some aspects, the conjugates or compositions provided herein can deliver a payload capable of activating effector T cells to effector T cells.

[0531] In some aspects, the conjugates or compositions provided herein are capable of blocking PD1 / PDL1, for example, as measured using the assay described in Example 7 herein. In some aspects, the conjugates or compositions block PD1 / PDL1 by about 3.51 to about 5.49 times relative to background. In some aspects, the conjugates or compositions block PD1 / PDL1 by about 2.5 to about 3.5 times relative to background. In some aspects, the conjugates or compositions block PD1 / PDL1 by about 2 to about 2.49 times relative to background. In some aspects, the conjugates or compositions block PD1 / PDL1 by about 1.5 to about 1.99 times relative to background. In some aspects, the conjugates or compositions block PD1 / PDL1 by about 1.1 to about 1.49 times relative to background.

[0532] In some aspects, the conjugates or compositions provided herein can increase the secretion of IFN-γ from T cells, for example, as assessed using the assay described in Example 7 herein. In some aspects, the conjugates or compositions increase IFN-γ secretion by about 1 to about 1.4 times relative to background. In some aspects, the conjugates or compositions increase IFN-γ secretion by about 1 to about 1.49 times relative to background. In some aspects, the conjugates or compositions increase IFN-γ secretion by about 1.5 to about 1.99 times relative to background. In some aspects, the conjugates or compositions increase IFN-γ secretion by about 2 to about 2.49 times relative to background. In some aspects, the conjugates or compositions increase IFN-γ secretion by about 2.5 to about 3 times relative to background.

[0533] In some aspects, the conjugates or compositions provided herein can increase the secretion of IL-2 from T cells, e.g., as assessed using the assay described in Example 7 herein. In some aspects, the conjugates or compositions increase IL-2 secretion by about 2 to about 4 times relative to background. In some aspects, the conjugates or compositions increase IL-2 secretion by about 4 to about 4.99 times relative to background. In some aspects, the conjugates or compositions increase IL-2 secretion by about 5 to about 5.99 times relative to background. In some aspects, the conjugates or compositions increase IL-2 secretion by about 6 to about 8 times relative to background.

[0534] III.A. Payload

[0535] The conjugates of the present disclosure may comprise a payload attached to a binding moiety (i.e., "Bm"). The payload may be a molecule (e.g., a small molecule, a peptide, or a nucleic acid) capable of activating effector T cells. In some aspects, the payload may promote the proliferation, survival, and / or migration of effector T cells, promote the production of effector cytokines and / or cytotoxic mediators from effector T cells, and / or reduce the exhaustion of effector T cells.

[0536] In some aspects, the payload is an inhibitor of Casitas B-lineage lymphoma proto-oncogene b (Cbl-b), hematopoietic progenitor cell kinase 1 (HPK-1), phosphoinositide 3-kinase gamma (PI3Kγ), a mitogen-activated protein kinase (MAPK) pathway protein (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).

[0537] In some aspects, the effective load is a Cbl-b inhibitor. In some aspects, the effective load in the conjugate provided herein is the above-mentioned formula (I) compound, which is covalently bonded to a binding moiety or a joint. In some aspects, the effective load is selected from the group consisting of compound 146, compound 147, and compound 148. In some aspects, the effective load is NX-1607.

[0538] In some aspects, 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 (as discussed in, for example, Ackerman S. et al., Nature Cancer 2: 18-33 (2021), which is incorporated herein by reference in its entirety) and MEDI9197 (as discussed in, for example, Huck B. et al., Angew. Chem. Int. Ed. 57: 4412-4428 (2018), which is incorporated herein by reference in its entirety).

[0539] In some aspects, the payload is an inhibitor of hematopoietic progenitor kinase 1 (HPK-1). HPK-1 inhibitors are known in the art and reviewed in, for example, Linney, ID and Kaila N., Expert Opin. Ther. Pat. 31(10): 893-910 (2021), which is incorporated herein by reference in its entirety.

[0540] In some aspects, the payload is a STING agonist. 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 in, for example, Huck B. et al., Angew. Chem. Int. Ed. 57: 4412-4428 (2018), which is incorporated herein by reference in its entirety.

[0541] In some aspects, the payload is a PI3Kγ inhibitor. PI3Kγ inhibitors, such as IPI-549, are known in the art and are discussed in, for example, Huck B. et al., Angew. Chem. Int. Ed. 57: 4412-4428 (2018), which is incorporated herein by reference in its entirety.

[0542] In some aspects, the payload is an inhibitor of a mitogen-activated protein kinase (MAPK) pathway protein (e.g., MEK and / or B-raf). MAPK pathway protein inhibitors, such as trametinib, cobimetinib, binimetinib, dabrafenib, vemurafenib, and encorafenib, are known in the art and are discussed in, for example, Huck B. et al., Angew. Chem. Int. Ed. 57: 4412-4428 (2018), which are incorporated herein by reference in their entirety.

[0543] In some aspects, 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 in, for example, Huck B. et al., Angew. Chem. Int. Ed. 57: 4412-4428 (2018), which is incorporated herein by reference in its entirety.

[0544] In some aspects, the payload in the conjugate provided herein is a small molecule, a peptide or a nucleotide (e.g., siRNA). In some aspects, the payload in the conjugate provided herein is a small molecule. In some aspects, the payload in the conjugate provided herein is a peptide. Exemplary peptide payloads are disclosed in, for example, EP Publication No. EP3254701, which is incorporated herein by reference in its entirety. In some aspects, the payload in the conjugate provided herein is a nucleotide (e.g., siRNA). Exemplary nucleotide payloads are disclosed in, for example, U.S. Publication No. US20150313931, which is incorporated herein by reference in its entirety.

[0545] III.B. Connectors

[0546] The conjugates of the present disclosure may comprise a linker. As used herein, the term "linker" refers to any chemical moiety capable of connecting a binding moiety (ie, "Bm") to a payload ("P").

[0547] In some aspects, the linker may contain a heterobifunctional group. In the present disclosure, the term "heterobifunctional group" refers to a chemical part of which the linker as a part is connected to a binding part. The heterobifunctional group is characterized by having different reactive groups at either end of the chemical part. The connection with the binding part can be achieved via chemical or enzymatic conjugation or a combination of the two. Chemical conjugation involves a controlled reaction of accessible amino acid residues on the surface of the binding part with a reactive handle on the heterobifunctional group. Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine ​​coupling, and coupling of non-natural amino acids incorporated by genetic engineering, wherein the non-natural amino acid residues with the desired reactive handle are placed on the binding part. In enzymatic conjugation, the enzyme mediates the coupling of the linker to the accessible amino residues on the binding part. Examples of enzymatic conjugation include, but are not limited to, transpeptidation using a sortase, transpeptidation using a microbial transglutaminase, and N-glycan engineering. Chemical conjugation and enzymatic conjugation can also be used in sequence. For example, enzymatic conjugation can also be used to position unique reactive handles on binding moieties for utilization in subsequent chemical conjugation.

[0548] In some aspects, the heterobifunctional group is selected from:

[0549] , , , , , , , , , , , and ;

[0550] in

[0551] is the point of connection to the rest of the joint; and

[0552] It is the connection point with Bm.

[0553] In some aspects, the joint "L" is non-cleavable. As used herein, the term "non-cleavable joint" is any chemical part that can be connected to a payload in a stable covalent manner and does not belong to the species defined as "cleavable joint" herein. Therefore, non-cleavable joints are generally resistant to acid-induced cleavage, light-induced cleavage, bioreductive cleavage, peptidase-induced cleavage, esterase-induced cleavage and disulfide bond cleavage. "Generally resistant to cleavage" means that 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 populations are in the joint or the chemical bond adjacent to the joint remains non-cleavable by acid, light-labile cleavage agents, bioreductants, peptidases, esterases or chemical or physiological compounds, and the chemical bonds (e.g., disulfide bonds) in the cleavable joint are cleaved in a few hours to a few days with any one of the agents described above. In certain aspects, the linker is not susceptible to acid-induced cleavage, light-induced cleavage, bioreductive cleavage, enzymatic cleavage, etc., under conditions where the payload and / or binding moiety can remain active. ADC catabolites generated from non-cleavable linkers contain residual amino acids from the antibody. These catabolites can exert unique and unexpected properties in the target cells to which they are delivered.

[0554] One of ordinary skill in the art will readily distinguish between non-cleavable linkers and cleavable linkers.

[0555] Examples of non-cleavable linkers include, but are not limited to, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) linkers, succinimidyl thioether linkers, and linkers such as:

[0556] , , , , , , , , and ;

[0557] in:

[0558] p is an integer from 1 to 10;

[0559] p* is an integer from 1 to 10;

[0560] Y is selected from hydrogen and C 1 -C 6 alkyl;

[0561] is the connection point to the payload; and

[0562] It is the connection point with the binding part.

[0563] In some aspects, the linker is:

[0564] , and ;

[0565] in:

[0566] p is an integer from 1 to 10;

[0567] p* is an integer from 1 to 10;

[0568] is the connection point to the payload; and

[0569] It is the connection point with the binding part.

[0570] In certain aspects, the linker may be cleavable. In certain aspects, the linker may be susceptible to acid-induced cleavage, light-induced cleavage, bioreductive cleavage, enzymatic cleavage, etc., under conditions where the payload and / or binding moiety can remain active.

[0571] In some aspects, the cleavable linker can be cleaved enzymatically. In some aspects, the cleavable linker can be cleaved by a protease, a peptidase, an esterase, a β-glucuronidase, a glycosidase, a phosphodiesterase, a phosphatase, a pyrophosphatase, or a lipase.

[0572] In some aspects, 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.

[0573] In some aspects, the cleavable linker contains a peptide. In some aspects, the peptide is the cleavage site of the linker, thereby contributing to drug release after exposure to intracellular proteases (such as lysosomal enzymes). The peptide can be designed and optimized to be enzymatically cleaved by a specific enzyme, such as tumor-associated proteases, cathepsins B, C and D or plasmin proteases. The example of a peptide with two amino acids includes but is 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-high lysine (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 may also be present in the reverse order (i.e., cit-val).

[0574] The peptides disclosed herein may comprise L-isomers or D-isomers of amino acid residues. The term "naturally occurring amino acids" 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 amino acids with a "D" (dextrorotatory) configuration, which is opposite to the configuration in naturally occurring ("L-") amino acids. Amino acids described herein may be commercially available (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0575] In certain aspects, the linker ("L") is a protease cleavable linker selected from:

[0576] ; ; ; ; ; ; ; ; and ;

[0577] in:

[0578] q is an integer from 2 to 10;

[0579] Z 1 , Z 2 , Z 3 and Z 4 each independently being an amino acid residue that is not present or is naturally present in the L or D configuration, provided that Z 1 , Z 2 , Z 3 and Z 4 At least two of are amino acid residues;

[0580] is the connection point to the payload; and

[0581] It is the connection point with the binding part.

[0582] In some ways, Z 1 , Z 2 , Z 3 and Z 4 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; provided that Z 1 , Z 2 , Z 3 and Z 4 At least two of them are amino acid residues.

[0583] In some respects, Z 1 Absent or glycine; Z 2 is absent or is 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; and Z 4 Selected from L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine and glycine.

[0584] In some aspects, L is selected from

[0585] , , , and ,

[0586] in:

[0587] q is an integer from 2 to 10;

[0588] is the connection point to the payload; and

[0589] It is the connection point with the binding part.

[0590] In some aspects, the linker is bioreducible. Bioreducible linkers utilize differences in reduction potentials in intracellular compartments versus plasma. Reduced glutathione present in the cytoplasm of tumor cells is up to 1000 times higher than reduced glutathione present in the cytoplasm of normal cells, and tumor cells also contain enzymes that can contribute to reduction in cellular compartments. The linker keeps the conjugate intact during systemic circulation and is selectively cleaved by high intracellular concentrations of glutathione, thereby releasing active drug from non-toxic prodrugs at the tumor site.

[0591] In some aspects, L is a bioreducible linker selected from:

[0592]

[0593] in:

[0594] q is an integer from 2 to 10;

[0595] R, R', R'' and R''' are each independently selected from 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 may form a cyclobutyl or cyclopropyl ring;

[0596] is the connection point to the payload; and

[0597] It is the connection point with the binding part.

[0598] In some aspects, L is selected from

[0599] and ;

[0600] in:

[0601] q is an integer from 2 to 10;

[0602] R, R', R'' and R''' are each independently selected from 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 may form a cyclobutyl or cyclopropyl ring;

[0603] is the connection point to the payload; and

[0604] It is the connection point with the binding part.

[0605] In some ways, L is

[0606] ;

[0607] wherein Q* is a group that can be attached to a binding moiety, and Is the connection point to the payload.

[0608] In some aspects, the present disclosure provides a linker of formula L**

[0609]

[0610] L**

[0611] wherein Q* is a group that can be attached to a binding moiety, and Is the point of attachment to the payload. In some aspects, a linker can be used to attach any payload to any binding moiety.

[0612] In certain aspects, the linker is acid-cleavable. Acid-cleavable linkers are specifically designed to remain stable at the neutral pH of blood circulation, but to undergo hydrolysis and release the cytotoxic drug in the acidic environment of the cellular compartment.

[0613] In some aspects, L is an acid-cleavable linker selected from:

[0614]

[0615] in:

[0616] q is an integer from 2 to 10;

[0617] is the connection point to the payload; and

[0618] It is the connection point with the binding part.

[0619] In certain aspects, L is a click-release linker, where the release of the payload is chemically triggered by a tetrazine or related compound.

[0620] In some aspects, L is a click-release linker selected from:

[0621]

[0622] in:

[0623] q is an integer from 2 to 10;

[0624] is the connection point to the payload; and

[0625] It is the connection point with the binding part.

[0626] In certain aspects, L is a pyrophosphatase cleavable linker.

[0627] In some aspects, L is a pyrophosphatase cleavable linker that is:

[0628] ;

[0629] in:

[0630] q is an integer from 2 to 10;

[0631] is the connection point to the payload; and

[0632] It is the connection point with the binding part.

[0633] In certain aspects, L is a β-glucuronidase cleavable linker.

[0634] In some aspects, L is a β-glucuronidase cleavable linker selected from:

[0635] , , and ;

[0636] in:

[0637] q is an integer from 2 to 10;

[0638] ----Does not exist or is a key;

[0639] is the connection point to the payload; and

[0640] It is the connection point with the binding part.

[0641] In some ways, L is

[0642] ;

[0643] in:

[0644] q is an integer from 2 to 10;

[0645] ----Does not exist or is a key;

[0646] is the connection point to the payload; and

[0647] It is the connection point with the binding part.

[0648] In some aspects, the connection point with the binding portion is cysteine, lysine, tyrosine or glutamine in the binding portion. In some aspects, the connection point with the binding portion is cysteine. In some aspects, the connection point with the binding portion is lysine. In some aspects, the connection point with the binding portion is tyrosine. In some aspects, the connection point with the binding portion is glutamine (e.g., according to EU numbering at the glutamine at position 295 of the heavy chain of the antibody or its antigen-binding portion).

[0649] The cysteine ​​or lysine can be an engineered (i.e., not endogenous to the binding portion) cysteine ​​or lysine, for example, for site-specific conjugation. Site-specific conjugation refers to a unique and specified site connection on a binding portion (e.g., an antibody or its antigen-binding portion). Site-specific conjugation is discussed in, for example, Zhou, Qun. "Site-Specific Antibody Conjugation for ADC and Beyond." Biomedicines Vol. 5 (4), 64. November 9, 2017, doi: 10.3390 / biomedicines5040064, which is incorporated herein by reference in its entirety. In some aspects, the engineered cysteine ​​is at heavy chain position S239 and / or K334 of an antibody or its antigen-binding portion according to EU numbering.

[0650] The cysteine ​​or lysine used as the point of attachment may be a cysteine ​​or lysine endogenous to the binding moiety.

[0651] III.C. Binding part

[0652] As demonstrated herein, a binding moiety (Bm) can deliver a payload, such as an effector T cell activating agent, to an effector T cell.

[0653] As used herein, the term "binding moiety" refers to any molecule that recognizes and binds to a target. A binding moiety capable of specifically binding to a target on the surface of an effector T cell refers to any molecule that recognizes and binds to a target present on an effector T cell. The target may also be present on the surface of other cells (e.g., other T cells).

[0654] In some aspects, the target is expressed on T cells (including effector T cells), but not on other cells that are not T cells. In some aspects, the expression of the target on effector T cells is enhanced compared to the expression of the target on other T cells. For example, in some aspects, the expression of the target on effector T cells is at least 5 times higher on effector T cells than on regulatory T cells. For example, in some aspects, the expression of the target on effector T cells is at least 10 times higher on effector T cells than on regulatory T cells. For example, in some aspects, the expression of the target on effector T cells is at least 20 times higher on effector T cells than on regulatory T cells.

[0655] In some aspects, the target is expressed on regulatory T cells and effector T cells. In some aspects, when the target is expressed on regulatory T cells and effector T cells, the expression of the target on regulatory T cells is higher than the expression on effector T cells. In some aspects, when the target is expressed on regulatory T cells and effector T cells, the expression of the target on regulatory T cells is higher than the expression on helper T cells. In some aspects, when the target is expressed on regulatory T cells and effector T cells, the expression of the target on regulatory T cells is higher than the expression on effector T cells and higher than the expression on helper T cells.

[0656] In some aspects, the target is a protein, glycoprotein, lipid, glycolipid or carbohydrate on the surface of an effector T cell. In some aspects, the target is a protein or glycoprotein.

[0657] In addition to targeting a compound to a specific cell, tissue or location, a binding moiety may also have a therapeutic effect (e.g., a proliferative effect or a migratory effect) on effector T cells. After the binding moiety specifically binds to a target on the surface of an effector T cell, the effector T cell may internalize a conjugate comprising a binding moiety, a payload attached to the binding moiety, and / or a target.

[0658] In certain aspects, a binding moiety may comprise or may be engineered to comprise at least one chemically reactive group, such as a carboxylic acid, an amine, a thiol, or a chemically reactive amino acid moiety or side chain.

[0659] In some aspects, the binding moiety (Bm) can be a peptide or protein that binds to a cell surface receptor or antigen.

[0660] In some aspects, the group "Bm" can be an antibody or its antigen binding fragment. In some aspects, the antibody or its antigen binding fragment is a monoclonal antibody or its antigen binding fragment. The exemplary sequence of the antibody or its antigen binding fragment is provided in Table 2-4 of this article. The antibody or its antigen binding fragment can belong to any immunoglobulin class, including IgG, IgM, IgE, IgA and IgD and any subclass thereof. In some aspects, the antibody or its antigen binding fragment is an IgG antibody. In some aspects, the antibody or its antigen binding fragment is an IgG1 or IgG4 antibody or its antigen binding fragment. In some aspects, the antibody or its antigen binding fragment is an IgG1 antibody or its antigen binding fragment. In some aspects, the antibody or its antigen binding fragment is an IgG4 antibody or its antigen binding fragment.

[0661] Available monoclonal antibodies or their antigen-binding fragments include, but are not limited to, human monoclonal antibodies or their antigen-binding fragments, humanized monoclonal antibodies or their antigen-binding fragments, or chimeric human-mouse (or other species) monoclonal antibodies or their antigen-binding fragments. Human monoclonal antibodies or their antigen-binding fragments can be prepared by any of numerous methods known in the art.

[0662] Useful antigen-binding fragments of antibodies include F(ab')2 fragments, which contain variable regions, light chain constant regions, and heavy chain CH1 domains, and can be produced by pepsin digestion of antibody molecules; and Fab fragments, which can be produced by reducing the disulfide bridges of F(ab')2 fragments. 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 (SCA), or any other molecule having the same specificity as an antibody.

[0663] Additionally, recombinant antibodies and antigen-binding fragments thereof, such as chimeric and humanized monoclonal antibodies and antigen-binding fragments thereof, which contain human and non-human portions and can be made using standard recombinant DNA techniques, are useful antibodies and antigen-binding fragments thereof.

[0664] Transgenic mice that cannot express endogenous immunoglobulin heavy and light chain genes but can express human heavy and light chain genes can be used to produce fully human antibodies or antigen-binding fragments thereof. Transgenic mice are immunized in a normal manner with selected antigens, such as all or part of the polypeptides disclosed herein. Monoclonal antibodies or antigen-binding fragments thereof against antigens can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes carried by transgenic mice are rearranged during B cell differentiation and subsequently undergo class switching and somatic mutation. Therefore, using this 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 purchased from, for example, Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0665] A technique known as "guided selection" can be used to generate fully human antibodies or antigen-binding fragments thereof that recognize a selected epitope. In this method, a selected non-human monoclonal antibody or antigen-binding fragment thereof, such as a mouse antibody or antigen-binding fragment thereof, is used to guide the selection of fully human antibodies or antigen-binding fragments thereof that recognize the same epitope. Human antibodies or antigen-binding fragments thereof can also be generated using various techniques known in the art, including phage display libraries.

[0666] The antibody or its antigen-binding fragment can be a fusion protein of the antibody or its antigen-binding fragment, for example, wherein the antibody or fragment is fused to an amino acid sequence of another protein (or a portion thereof, such as a portion of at least 10, 20 or 50 amino acids of a protein) that is not an antibody or fragment via a covalent bond (e.g., a peptide bond) at the N-terminus or C-terminus. The antibody or its antigen-binding fragment can be covalently linked to other proteins at the N-terminus of the constant domain.

[0667] Antibodies and antigen-binding fragments thereof include analogs and derivatives that are modified (i.e., by covalent attachment of any type of molecule), as long as such covalent attachment allows the antibody or antigen-binding fragment to retain its antigen-binding immunospecificity. By way of example and not limitation, derivatives and analogs of antibodies and antigen-binding fragments thereof include those that have been further modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting groups / blocking groups, proteolytic cleavage, attachment to cell antibody units or other proteins, etc. Any of numerous chemical modifications may be performed by known techniques, including but not limited to specific chemical cleavages, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. In addition, analogs or derivatives may contain one or more unnatural amino acids.

[0668] The antibody or its antigen binding fragment in the conjugate may include an antibody or its antigen binding fragment with a modification (e.g., substitution, deletion or addition) in the amino acid residues that interact with the Fc receptor. In particular, the antibody or its antigen binding fragment includes an antibody or its antigen binding fragment with a modification in the amino acid residues identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor. The antibody or its antigen binding fragment that can specifically bind to the target on the effector T cell surface can be purchased or produced by any method known to those skilled in the art, such as chemical synthesis or recombinant expression technology. The nucleotide sequence encoding the antibody or its antigen binding fragment that can specifically bind to the target on the effector T cell surface can be obtained from, for example, the GenBank database or its similar databases, literature publications, or obtained by conventional cloning and sequencing.

[0669] In some aspects, the antibody or antigen binding fragment thereof of the conjugate can be a monoclonal antibody or antigen binding fragment thereof, for example, 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 aspects, the antibody or antigen binding fragment thereof can be, for example, a Fab fragment.

[0670] As provided herein, a binding moiety (e.g., an antibody or antigen binding fragment thereof) that binds to a target (antigen) on the surface of an effector T cell can be conjugated to a payload. For example, such a binding moiety (Bm), such as an antibody or antigen binding fragment thereof, can bind to PD1, CD25 (IL2RA) or CD7.

[0671] In some aspects, a binding moiety (Bm), such as an antibody or antigen binding fragment thereof, can specifically bind to CD25, also known as interleukin-2 receptor subunit alpha (IL2RA). Such binding moieties are also referred to herein as "anti-IL2RA binding moieties," "IL2RA binding moieties," "anti-CD25 binding moieties," or "CD25 binding moieties," 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 aspects, an anti-IL2RA or anti-CD25 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDR sequences of SEQ ID NOs: 4 and 5, respectively (e.g., Kabat, Chothia, or AbM defined CDRs) (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 aspects, 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 aspects, the anti-IL2RA or anti-CD25 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDR sequences of SEQ ID NOs: 6 and 7, respectively (e.g., Kabat, Chothia, or AbM defined CDRs) (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 aspects, the 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 aspects, 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., Kabat, Chothia, or AbM defined CDRs) (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 aspects, the 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 aspects, the anti-IL2RA or anti-CD25 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDR sequences of SEQ ID NOs: 16 and 17, respectively (e.g., Kabat, Chothia, or AbM defined CDRs) (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 aspects, the 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.

[0672] Table 2: Sequences of Exemplary IL2RA or CD25 Antibodies or Antigen-Binding Fragments thereof

[0673]

[0674] In some aspects, a binding portion (Bm), such as an antibody or an antigen-binding fragment thereof, can specifically bind to PD1. Such binding portions are also referred to herein as "anti-PD1 binding portions" or "PD1 binding portions", for example, anti-PD1 antibodies or antigen-binding fragments thereof or PD1 antibodies or antigen-binding fragments thereof. Exemplary sequences of anti-PD1 antibodies or antigen-binding fragments thereof are provided in Table 3. In some aspects, the anti-PD1 antibody or antigen-binding fragment thereof comprises heavy chain and light chain CDR sequences of SEQ ID NOs: 10 and 11, respectively (e.g., CDRs defined by Kabat, Chothia or AbM) (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 aspects, the 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 aspects, the anti-PD1 antibody or antigen-binding fragment thereof comprises the heavy and light chain CDR sequences of SEQ ID NOs: 12 and 13, respectively (e.g., CDRs defined by Kabat, Chothia, or AbM) (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 aspects, 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 aspects, the anti-PD1 antibody or its antigen-binding fragment comprises the heavy 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 aspects, the anti-PD1 antibody or its antigen-binding fragment 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 aspects, the anti-PD1 antibody or its antigen-binding fragment 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.

[0675] Table 3: Sequences of exemplary anti-PD1 antibodies or antigen-binding fragments thereof

[0676]

[0677] In some aspects, a binding portion (Bm), such as an antibody or an antigen-binding fragment thereof, can specifically bind to CD7. Such binding portions are also referred to herein as "anti-CD7 binding portions" or "CD7 binding portions", for example, anti-CD7 antibodies or antigen-binding fragments thereof or CD7 antibodies or antigen-binding fragments thereof. Exemplary sequences of anti-CD7 antibodies or antigen-binding fragments thereof are provided in Table 4. In some aspects, anti-CD7 antibodies or antigen-binding fragments thereof comprise heavy and light chain CDR sequences of SEQ ID NOs: 14 and 15, respectively (e.g., CDRs defined by Kabat, Chothia or AbM) (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 aspects, anti-CD7 antibodies or antigen-binding fragments thereof comprise: 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.

[0678] Table 4: Sequences of exemplary anti-CD7 antibodies or antigen-binding fragments thereof

[0679]

[0680] In some aspects, the antibody or its antigen binding portion comprises a constant region. The linker can be connected to the amino acids in the constant region. In some aspects, the antibody or its antigen binding portion comprises a CH1 domain. The linker can be connected to the amino acids in the CH1 domain. In some aspects, the antibody or its antigen binding portion comprises a CH2 domain. The linker can be connected to the amino acids in the CH2 domain. In some aspects, the antibody or its antigen binding portion comprises a CH3 domain. The linker can be connected to the amino acids in the CH3 domain. In some aspects, the antibody or its antigen binding portion comprises a CL domain. The linker can be connected to the amino acids in the CL domain.

[0681] In some aspects, 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.

[0682] In some aspects, the antibody or its antigen binding portion comprises a heavy chain constant region, for example, a human heavy chain constant region. The joint can be connected to the heavy chain constant region, for example, the amino acid in the human heavy chain constant region. In some aspects, the antibody or its antigen binding portion comprises an IgG heavy chain constant region, for example, a human IgG heavy chain constant region. The joint can be connected to the IgG heavy chain constant region, for example, the amino acid in the human IgG heavy chain constant region. In some aspects, the antibody or its antigen binding portion comprises an IgG1 heavy chain constant region, for example, a human IgG1 heavy chain constant region. The joint can be connected to the IgG1 heavy chain constant region, for example, the amino acid in the human IgG1 heavy chain constant region. In some aspects, the antibody or its antigen binding portion comprises an IgG4 heavy chain constant region. The joint can be connected to the IgG4 heavy chain constant region, for example, the amino acid in the human IgG4 heavy chain constant region.

[0683] In some aspects, the antibody or its antigen-binding portion comprises a light chain constant region, for example, a human light chain constant region. The linker can be connected to the light chain constant region, for example, an amino acid in a human light chain constant region. In some aspects, the antibody or its antigen-binding portion comprises a κ light chain constant region, for example, a human κ light chain constant region. The linker can be connected to the κ light chain constant region, for example, an amino acid in a human κ light chain constant region. In some aspects, the antibody or its antigen-binding portion comprises a γ light chain constant region, for example, a human γ light chain constant region. The linker can be connected to the γ light chain constant region, for example, an amino acid in a human γ light chain constant region.

[0684] In some aspects, the antibody or its antigen-binding portion thereof comprises an engineered cysteine ​​at heavy chain position S239 according to EU numbering. A linker can be connected to S239C. In some aspects, the antibody or its antigen-binding portion thereof comprises an engineered cysteine ​​at heavy chain position K334 according to EU numbering. A linker can be connected to K334C.

[0685] Thus, the antibody, or antigen binding portion thereof, may comprise the heavy chain constant region of any one of SEQ ID NOs: 20-24.

[0686] IgG1 heavy chain constant region

[0687] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:20).

[0688] IgG1 N297A constant region (CH1 - hinge - CH2 - CH3)

[0689] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:21).

[0690] IgG4 S228P constant region (CH1 - hinge - CH2 - CH3)

[0691] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:22).

[0692] S239C of the IgG1 heavy chain constant region

[0693] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP C VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:23).

[0694] K334C of the IgG1 heavy chain constant region

[0695] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE C TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 24).

[0696] In some aspects, the antibody, or antigen binding portion thereof, may comprise the heavy chain constant region of SEQ ID NO:28.

[0697] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:28).

[0698] In some aspects, the linker can be attached to heavy chain Q295 of the antibody, or antigen-binding portion thereof, according to EU numbering.

[0699] In the present disclosure, the group "Bm" can be conjugated to more than one compound capable of activating effector T cells. In some aspects, "Bm" can be conjugated to 1 to 10 compounds. In some aspects, "Bm" can be conjugated to 1 to 9 compounds. In some aspects, "Bm" can be conjugated to 1 to 8 compounds. In some aspects, "Bm" can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 compounds. In some aspects, "Bm" can be conjugated to 7 or 8 compounds. In some aspects, "Bm" is conjugated to 5 compounds. In some aspects, "Bm" is conjugated to 6 compounds. In some aspects, "Bm" is conjugated to 7 compounds. In some aspects, "Bm" is conjugated to 8 compounds. In some aspects, "Bm" is conjugated to 9 compounds.

[0700] IV. Compositions and Methods of Use

[0701] The conjugates and / or compounds described herein may be in the form of pharmaceutically or pharmaceutically acceptable salts. In some aspects, such salts are derived from inorganic or organic acids or bases.

[0702] Examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, gluconate heptanoate, 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, toluenesulfonate, and undecanoate.

[0703] Examples of suitable base addition salts include ammonium salts; alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine; and salts with amino acids such as arginine, lysine, etc.

[0704] For example, Berge lists the following FDA-approved commercially available salts: anionic 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, glycollylarsanilate / glycollamidophenylarsonate, hexylresorcinate, hydrabamine (N,N'-di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate (2-hydroxyethanesulfonate), lactate, lactobionate, malate, maleate, mandelate, mesylate / methanesulfonate, methyl bromide, methyl nitrate, methyl sulfate, mucinate, naphthylsulfonate (2-naphthalenesulfonate), nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theophylline (8-chlorotheophylline salt), and triethyl iodide; organic cations benzathine (N,N'-diphenylmethylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucosamine) and procaine; and metal cations aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.

[0705] Berge additionally lists the following non-FDA approved commercially available (outside the U.S.) salts: anionic adipates, alginate, aminosalicylate, dehydromethylene citrate, arecoline, aspartate, bisulfate, butyl bromide, camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydroiodide, methylenebis(salicylate), naphthalene disulfonate (1,5-naphthalene disulfonate), oxalate, pectinate, persulfate, phenethylbarbiturate, picrate, propionate, thiocyanate, toluenesulfonate, and undecanoate salts; organic cationic benethamine (N-benzylphenethylamine), clemizole (1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole), diethylamine, piperazine, and tromethamine (tris(hydroxymethyl)aminomethane); and metallic cationic barium and bismuth.

[0706] Pharmaceutical compositions comprising the conjugates described herein may also contain suitable carriers, excipients, and adjuvants, which may vary depending on the mode of administration.

[0707] In some aspects, the pharmaceutical composition can be formulated into 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 modes for parenteral administration include intravenous administration.

[0708] Parenteral compositions are usually aqueous solutions which may contain excipients such as salts, carbohydrates and buffers. However, the compositions may also be formulated as sterile non-aqueous solutions or dry forms for use in combination with suitable vehicles such as sterile pyrogen-free water.

[0709] 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.

[0710] The parenteral formulation may be mixed with other suitable pharmaceutically acceptable excipients used in parenteral dosage forms such as, but not limited to, preservatives.

[0711] Compounds and conjugates described herein can be used to treat various diseases that will benefit from an increase in immune response, including but not limited to cancer. Certain compounds and conjugates disclosed herein can be superior in efficacy expression, pharmacokinetics (e.g., absorption, distribution, metabolism, excretion), solubility (e.g., water-soluble), interactions with other agents (e.g., drug metabolizing enzyme inhibition), safety (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central toxicity) and / or stability (e.g., chemical stability, stability to enzymes), and can be used as medicaments. Therefore, compounds and conjugates disclosed herein, and compositions comprising them, are provided herein for the preparation of medicaments, for example, for the treatment of diseases (including but not limited to cancer) that will benefit from an increase in immune response. Compounds and conjugates disclosed herein, and compositions comprising them, are also provided herein for the treatment of diseases that will benefit from an increase in immune response, including but not limited to cancer.

[0712] The compounds and conjugates of the present disclosure can be used as medicaments, such as agents for preventing or treating diseases, such as cancer. In some aspects, the cancer is a solid tumor cancer. In some aspects, the cancer is a hematological malignancy. In some aspects, the cancer is selected from the group consisting of: unresectable or metastatic microsatellite instability-high (MSI-H) solid tumors, mismatch repair deficient (dMMR) solid tumors, melanoma, non-small cell lung cancer (NSCLC), malignant pleural mesothelioma, renal cell carcinoma (RCC), classical Hodgkin lymphoma (cHL), head and neck squamous cell carcinoma (SCCHN), urothelial carcinoma, colorectal cancer, hepatocellular carcinoma (HCC), esophageal cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, esophageal adenocarcinoma and head and neck squamous cell carcinoma (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 (merkel cell carcinoma (MCC), endometrial cancer, tumor mutation burden-high (TMB-H) cancers, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), and basal cell carcinoma (BCC).

[0713] In some aspects, the cancer is resistant to anti-PD1 therapy. The anti-PD1 therapy can be, for example, nivolumab, pembrolizumab, and / or cemiplimab.

[0714] In some aspects, the cancer is refractory to anti-PD1 therapy. The anti-PD1 therapy can be, for example, nivolumab, pembrolizumab, and / or cemiplimab.

[0715] In some aspects, the cancer is sensitive to anti-PD1 therapy. The anti-PD1 therapy can be, for example, nivolumab, pembrolizumab, and / or cemiplimab.

[0716] In some aspects, the cancer is resistant to anti-PDL1 therapy. The anti-PDL1 therapy can be, for example, durvalumab, atezolizumab, and / or avelumab.

[0717] In some aspects, the cancer is refractory to anti-PDL1 therapy. The anti-PDL1 therapy can be, for example, durvalumab, atezolizumab, and / or avelumab.

[0718] In some aspects, the cancer is sensitive to an anti-PDL1 therapy. The anti-PDL1 therapy can be, for example, durvalumab, atezolizumab, and / or avelumab.

[0719] In some aspects, the compounds or conjugates of the present disclosure can be used in combination with standard care therapy, such as one or more therapeutic agents (e.g., anticancer agents and / or immunomodulators). Therefore, in some aspects, the method for treating tumors disclosed herein includes administering a combination of a compound or conjugate of the present disclosure and one or more additional therapeutic agents. In some aspects, the compounds or conjugates of the present disclosure can be used in combination with one or more anticancer agents so that multiple elements of immune pathways can be targeted. In some aspects, anticancer agents include immune checkpoint inhibitors (i.e., blocking signal transduction via specific immune checkpoint pathways). Non-limiting examples of immune checkpoint inhibitors that can be used in the methods of the present invention 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 inhibitory factor of T-cell activation (VISTA) antagonists, B and T-cell lymphocyte attenuator (BTLA) antagonists, and lymphocyte activation gene-3 (LAG-3) antagonists.

[0720] In some aspects, the compound or conjugate of the present disclosure is administered to the subject before or after the administration of the additional therapeutic agent. In other aspects, the compound or conjugate of the present disclosure is administered to the subject simultaneously with the additional therapeutic agent. In certain aspects, the compound or conjugate of the present disclosure and the additional therapeutic agent can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier. In other aspects, the compound or conjugate of the present disclosure and the additional therapeutic agent are administered simultaneously as separate compositions.

[0721] The compounds and conjugates of the present disclosure can be used as medicaments, such as agents for preventing or treating additional conditions that would benefit from an increased immune response. Conditions that would benefit from an increased immune response include, for example, infections (e.g., viral, bacterial and / or parasitic infections), immunosuppressive diseases or disorders, and multiple sclerosis.

[0722] In some aspects, the subject that can be treated with the compound conjugates of the present disclosure is a non-human animal, such as a rat or mouse. In some aspects, the subject that can be treated is a human.

[0723] In some aspects, 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 contact can be in vitro or in vivo. In terms of in vivo contact, the contact can be in a subject, for example, a human subject with cancer or another disease that would benefit from an increased immune response. Increased immune cell activity can treat cancer or other diseases that would benefit from an increased immune response.

[0724] In some aspects, the compounds or conjugates of the present disclosure can be used to increase immune cell (e.g., NK cells or T cells, including effector T cells) proliferation, for example, by contacting immune cells with compounds or conjugates of the present disclosure. The contact can be in vitro or in vivo. In terms of in vivo contact, the contact can be in a subject, for example, a human subject with a cancer or another disease that will benefit from an increased immune response. Increased immune cell proliferation can treat cancer or other diseases that will benefit from an increased immune response.

[0725] In some aspects, 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 contact can be in vitro or in vivo. In terms of in vivo contact, the contact can be in a subject, for example, a human subject with cancer. Increased immune cell migration can treat cancer.

[0726] In some aspects, the compounds or conjugates of the present disclosure can be used to reduce the exhaustion of immune cells (e.g., NK cells or T cells, including effector T cells), for example, by contacting immune cells with compounds or conjugates of the present disclosure. The contact can be in vitro or in vivo. In terms of in vivo contact, the contact can be in a subject, for example, a human subject with a cancer or another disease that would benefit from an increased immune response. Reduction in immune cell exhaustion can treat cancer or other diseases that would benefit from an increased immune response.

[0727] In some aspects, the compounds or conjugates of the present disclosure can be used to increase the secretion of IFN-γ or IL-2 from immune cells (e.g., NK cells or T cells, including effector T cells), for example, by contacting immune cells with compounds or conjugates of the present disclosure. The contact can be in vitro or in vivo. In terms of in vivo contact, the contact can be in a subject, for example, a human subject with a cancer or another disease that will benefit from an increased immune response. Increased secretion of IFN-γ or IL-2 can treat cancer or other diseases that will benefit from an increased immune response.

[0728] In some aspects of the above, the immune cell is an effector T cell. Thus, in some aspects, the compounds or conjugates of the present disclosure can be used to increase effector T cell activity, for example, by contacting effector T cells with the compounds or conjugates of the present disclosure. The contact can be in vitro or in vivo. In terms of in vivo contact, the contact can be in a subject, for example, a human subject with cancer. Increased effector T cell activity can treat cancer.

[0729] In some aspects, the compounds or conjugates of the present disclosure can be used to increase effector T cell proliferation, for example, by contacting effector T cells with the compounds or conjugates of the present disclosure. The contact can be in vitro or in vivo. In terms of in vivo contact, the contact can be in a subject, for example, a human subject with cancer. Increased effector T cell proliferation can treat cancer.

[0730] In some aspects, the compounds or conjugates of the present disclosure can be used to increase the migration of effector T cells to tumor cells, for example, by contacting the effector T cells with the compounds or conjugates of the present disclosure. The contact can be in vitro or in vivo. In terms of in vivo contact, the contact can be in a subject, for example, a human subject with cancer. Increased effector T cell migration can treat cancer.

[0731] In some aspects, 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 contact can be in vitro or in vivo. In terms of in vivo contact, the contact can be in a subject, for example, a human subject with cancer. Reduction of effector T cell exhaustion can treat cancer.

[0732] In some aspects, the conjugates of the present disclosure can be used to deliver a payload capable of activating effector T cells to effector T cells, for example, by contacting the effector T cells with the conjugates of the present disclosure. The contact can be in vitro or in vivo. In terms of in vivo contact, the contact can be in a subject, for example, a human subject with cancer. The delivery of the payload to the effector T cells can treat cancer.

[0733] V. Methods of Preparing Conjugates

[0734] The present disclosure further provides a method for preparing a conjugate, the process comprising reacting a binding moiety 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 moiety as described herein, connects the binding moiety to the payload.

[0735] In certain aspects, the linker precursor can be selected from:

[0736] , , , , , , and ;in

[0737] p is an integer from 1 to 10;

[0738] p* is an integer from 1 to 10; and

[0739] Is the connection point to the payload.

[0740] In certain aspects, the linker precursor can be selected from:

[0741] ; ; ; ; ; ; ; ; and ;

[0742] in:

[0743] q is an integer from 2 to 10;

[0744] Z 1 , Z 2 , Z 3 and Z 4 each independently being absent or being a naturally occurring amino acid residue in the L or D configuration, provided that Z 1 , Z 2 , Z 3 and Z 4 At least two of are amino acid residues; and

[0745] Is the connection point to the payload.

[0746] In some ways, Z 1 , Z 2 , Z 3and Z 4 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; provided that Z 1 , Z 2 , Z 3 and Z 4 At least two of them are amino acid residues.

[0747] In some respects, Z 1 Absent or glycine; Z 2 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; and Z 4 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.

[0748] In some aspects, the linker precursor can be selected from:

[0749] , , , and ,

[0750] in:

[0751] q is an integer from 2 to 10;

[0752] Is the connection point to the payload.

[0753] In certain aspects, the linker precursor can be selected from

[0754]

[0755] in:

[0756] q is an integer from 2 to 10;

[0757] R, R', R'' and R''' are each independently selected from hydrogen, C 1 -C 6 Alkoxy C1 -C 6 Alkyl, (C 1 -C 6 ) 2 NC 1 -C 6 Alkyl and C 1 -C 6 alkyl, or the two geminal R groups together with the carbon atoms to which they are attached may form a cyclobutyl or cyclopropyl ring; and

[0758] Is the connection point to the payload.

[0759] In certain aspects, the linker precursor can be selected from

[0760] ;

[0761] in:

[0762] q is an integer from 2 to 10; and

[0763] Is the connection point to the payload.

[0764] In certain aspects, the linker precursor can be selected from

[0765]

[0766] in:

[0767] q is an integer from 2 to 10;

[0768] Is the connection point to the payload.

[0769] In some aspects, the linker precursor can be selected from

[0770]

[0771] in:

[0772] q is an integer from 2 to 10; and

[0773] Is the connection point to the payload.

[0774] In some aspects, the linker precursor can be

[0775] ;

[0776] in:

[0777] q is an integer from 2 to 10;

[0778] Is the connection point to the payload.

[0779] In certain aspects, the linker precursor can be selected from

[0780] , , and ;

[0781] in:

[0782] q is an integer from 2 to 10;

[0783] ----Does not exist or is a key;

[0784] Is the connection point to the payload.

[0785] In some aspects, the linker precursor can be

[0786] ;

[0787] in:

[0788] q is an integer from 2 to 10;

[0789] ---- does not exist or is a key; and

[0790] Is the connection point to the payload.

[0791] In some aspects, the binding moiety is pretreated prior to reacting the binding moiety with a payload or payload-linker precursor. In certain aspects, a payload or payload-linker precursor reacts with a binding moiety comprising an antibody or antigen-binding portion thereof. In aspects where the binding moiety is an antibody, the antibody can be pretreated to reduce interchain disulfide bonds prior to reacting with a payload or payload-linker precursor.

[0792] The embodiments described herein are further described in detail by reference to the examples shown below. These examples are provided for illustrative purposes only and the embodiments described herein should in no way be construed as being limited to these examples. In fact, the embodiments should be construed to cover any and all variations that become apparent as a result of the teachings provided herein.

[0793] Example

[0794] General synthetic methods and intermediates

[0795] Compounds of the present disclosure can be prepared by those of ordinary skill in the art in view of the present disclosure and the knowledge in the art and / or by reference to the schemes and synthetic examples shown below. Exemplary synthetic pathways are described in the schemes below and in the examples. It should be understood that the variables (e.g., "R" groups) that appear in the following schemes and examples should be understood independently from those variables that appear elsewhere in the application. Those of ordinary skill in the art will readily understand how the schemes and examples shown below illustrate the preparation of compounds described herein.

[0796] The abbreviations used in the schemes generally follow the conventions used in the art. The chemical abbreviations used in this specification and examples are defined as follows:

[0797] "DMF" stands for N,N-dimethylformamide; "DIBAL-H" or "DIBAl-H" stands for diisobutylaluminum hydride; "DCM" stands for dichloromethane; "Ph" stands for phenyl; "DEAD" stands for diethyl azodicarboxylate; "NMO" stands for N-methylmorpholine N-oxide; "THF" stands for tetrahydrofuran; "t-Bu" stands for tert-butyl; "HATU" stands for 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine hexafluorophosphate 3-oxide or N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethylammonium hexafluorophosphate N-oxide; "DIEA", "DIPEA" and "iPrNEt 2 " represents diisopropylethylamine; "PE" represents petroleum ether; "EA" or "EtOAc" represents ethyl acetate; "Ac" represents acetyl; "BPO" represents benzoyl peroxide; "AIBN" represents azobisisobutyronitrile; "o / n" represents overnight; "rt" or "rt" or "RT" represents room temperature or retention time (as determined by the context); "IBX" represents 2-iodoacetylbenzoic acid; "STAB" represents sodium triacetoxyborohydride; "Me" represents methyl; "NBS" represents N-bromosuccinimide; "dtbbpy" represents 4,4'-di-tert-butyl-2,2'-bipyridine; "TFA" represents trifluoroacetic acid; "DMA" represents N,N-dimethylacetamide; "Et 3"N" and "TEA" represent trimethylamine; "Ts" represents p-toluenesulfonyl; "Trt" represents triphenylmethyl; "TIPS" represents triisopropylsilyl; "Et" represents ethyl; "LiHMDS" represents lithium hexamethyldisilazane; "MeCN" or "ACN" represents acetonitrile; "TMS" represents trimethylsilyl; "NIS" represents N-iodosuccinimide; "BOC" or "Boc" represents tert-butyloxycarbonyl; "DMSO" represents dimethyl sulfoxide; and "HOBt" or "HOBT" represents 1-hydroxybenzotriazole hydrate; "h" represents hours; and "min" represents minutes.

[0798] Example 1: Preparation of Cbl-b inhibitors

[0799] Synthetic INT3

[0800]

[0801] Step 1: Synthesis of compound 2

[0802] To a stirred mixture of 2,4,6-trichloropyridine (Compound 1, 100 g, 548.15 mmol, 1 eq.) and dimethyl malonate (86.90 g, 657.78 mmol, 1.2 eq.) in DMF (1000 mL) was added Cs(II) portionwise at room temperature under air atmosphere. 2 CO 3 (535.80 g, 1644.46 mmol, 3 eq., powder). The resulting mixture was stirred at room temperature under air atmosphere for 2 days. LCMS indicated 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 purified by anhydrous Na 2 SO 4 Dry. After filtration, the filtrate was concentrated under reduced pressure. The resulting mixture was concentrated under vacuum to give 1,3-dimethyl 2-(2,6-dichloropyridin-4-yl)malonate (Compound 2, 95 g, 62%) as a yellow oil. LCMS: (ES, m / s): 278,280 [M+H] + .

[0803] Step 2: Synthesis of compound 3

[0804] 2-(2,6-dichloropyridin-4-yl)malonic acid 1,3-dimethyl ester (Compound 2, 95 g, 341.61 mmol, 1 equivalent) and K were added at 0°C under air atmosphere. 2 CO 3To a stirred mixture of 4-nitro-1-yl pyridine-2 ... 2 SO 4 After 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)malonate (Compound 3, 95 g, 76%) as a yellow oil. LCMS: (ES.m / z): 318,320 [M+1] + .

[0805] Step 3. Synthesis of compound 4

[0806] To a stirred solution of 1,3-dimethyl 2-(2,6-dichloropyridin-4-yl)-2-(prop-2-en-1-yl)malonate (compound 3, 70 g, 220.02 mmol, 1.00 equiv) in DCM (700 mL) was added DIBAl-H (880.09 mL, 1 N in DCM, 880.09 mmol, 4 equiv) dropwise at -40 °C under nitrogen atmosphere. The resulting mixture was stirred at -40 °C under nitrogen atmosphere for 1 hour. LCMS indicated a complete reaction. The reaction was quenched at room temperature by the addition of MeOH (20 mL). The resulting mixture was filtered and the filter cake was washed with EtOAc (3×50 mL). The filtrate was concentrated under reduced pressure to give 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] + .

[0807] Step 4. Synthesis of compound 5

[0808] At 0°C in N 2 2-(2,6-dichloropyridin-4-yl)-2-(prop-2-en-1-yl)propane-1,3-diol (compound 4, 70 g, 267.04 mmol, 1 equivalent) and PPh 3 (140.09 g, 534.08 mmol, 2 eq) was added portionwise to a stirred mixture of toluene (700 mL). 2The resulting mixture was stirred under atmosphere for 10 minutes. Ziram (122.49 g, 400.56 mmol, 1.5 equivalents) and DEAD (93.01 g, 534.08 mmol, 2 equivalents) were added dropwise to the above mixture at 0 ° C for 30 minutes. The resulting mixture was stirred overnight at room temperature. LCMS indicates that the reaction is 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 and eluted with PE / EA (5: 1) to obtain 2,6-dichloro-4-[3-(prop-2-ene-1-yl)oxetane-3-yl]pyridine (compound 5, 37 g, 50%) as a yellow oil. LCMS: (ES.m / z): 244,246[M+1] + .

[0809] Step 5: Synthesis of compound 6

[0810] 2,6-dichloro-4-[3-(prop-2-en-1-yl)oxetan-3-yl]pyridine (Compound 5, 37 g, 151.56 mmol, 1 eq.) in THF (240 mL) and H 2 To a stirred solution of 2% ethanol (100 mL) in 4% CO was added NMO (53.27 g, 454.69 mmol, 3 eq) portionwise and K 2 OSO 4 (2.8 g, 9.00 mmol, 0.05 equiv). The resulting mixture was stirred at room temperature under nitrogen atmosphere overnight. LCMS indicated the reaction was complete. 2 S 2 O 3 The reaction was quenched. 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 gave 3-[3-(2,6-dichloropyridin-4-yl)oxetane-3-yl]propane-1,2-diol (Compound 6, 38 g, 90%) as a black solid. LCMS: (ES.m / z): 278,280 [M+1] + .

[0811] Step 6: Synthesis of compound 7

[0812] NaIO 4 (58.45 g, 273.25 mmol, 2.00 equiv) and silica (76 g) in H 2O (190 mL) in the mixture was stirred for 30 minutes. Then a stirred solution of 3-[3-(2,6-dichloropyridin-4-yl)oxetane-3-yl]propane-1,2-diol (compound 6, 38 g, 136.62 mmol, 1 equivalent) in DCM (380 mL) was added dropwise at room temperature. The resulting mixture was stirred for 2 hours at room temperature. LCMS indicated that 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 gave 2-[3-(2,6-dichloropyridin-4-yl)oxetane-3-yl]acetaldehyde (compound 7, 27 g, 80%) as a brown solid. LCMS: (ES.m / z): 246,248[M+1] + .

[0813] Step 7: Synthesis of Compound 8

[0814] 2-[3-(2,6-dichloropyridin-4-yl)oxetan-3-yl]acetaldehyde (compound 7, 27 g, 109.71 mmol, 1 eq.) in t-BuOH (270 mL) and H 2 O (135 mL) was added portionwise to a stirred solution of NaClO 2 (34.73 g, 384.00 mmol, 3.5 eq.) and NaH 2 PO 4 (26.33 g, 219.43 mmol, 2 eq.) 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 indicated the reaction was complete. The mixture was acidified to pH 3 with 1N HCl. The precipitated solid was collected by filtration and washed with Et 2 O (3 × 100 mL). The filtrate was concentrated under reduced pressure. 2 The purified residue was triturated with 4% paraformaldehyde (50 mL) and 4% paraformaldehyde (50 mL). This gave [3-(2,6-dichloropyridin-4-yl)oxetane-3-yl]acetic acid (Compound 8, 14.4 g, 50%) as a white solid. LCMS: (ES.m / z): 262, 264 [M+1] + .

[0815] Step 9: Synthesis of compound 10

[0816] To a stirred mixture of [3-(2,6-dichloropyridin-4-yl)oxetane-3-yl]acetic acid (compound 8, 14 g, 53.41 mmol, 1 eq) and 1-amino-3-methylthiourea (6.18 g, 58.75 mmol, 1.1 eq) in DMF (120 mL) at 0°C under air atmosphere was added HATU (24.37 g, 64.10 mmol, 1.2 eq) portionwise and DIEA (20.71 g, 160.25 mmol, 3 eq) dropwise. The resulting mixture was stirred at room temperature overnight. NaOH (1N, 100 mL) was added dropwise to the above mixture at 0°C over 10 minutes. The resulting mixture was stirred at 0°C for another hour. LCMS indicated that 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. 2 The purified residue was triturated with 4-[[(3-(2,6-dichloropyridin-4-yl)oxetane-3-yl]methyl]-4-methyl-1,2,4-triazole-3-thiol)-1,4-dichloropyridin-4-yl)oxetane-3-yl]methyl)-1,4-dichloropyridin-4-yl)oxetane-3-yl]methyl)-1,4-dichloropyridin-4-yl)oxetane-3-yl)- ... + .

[0817] Step 9: Synthesize INT3

[0818] 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 eq) and NaNO were added under air atmosphere at -20 °C. 2 To a stirred mixture of 2-nitropropene (7.50 g, 108.69 mmol, 3 eq.) in THF (120 mL) was added HNO dropwise 3 (1N, 119.92 mL, 119.92 mmol, 3.31 equiv.). The resulting mixture was stirred at 0 °C for 2 h. LCMS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The saturated Na 2 CO 3 (aqueous solution) and basified the mixture to pH 8. 2 Cl 2 The resulting mixture was extracted with CH 2 Cl 2 The combined organic layers were washed with anhydrous Na 2 SO 4After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by CH 2 Cl 2 / MeOH (10:1) to obtain 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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).

[0819] Synthetic INT4

[0820]

[0821] Step 1: Synthesis of compound 2

[0822] To a stirred mixture of methyl 2-methyl-3-(trifluoromethyl)benzoate (compound 1, 44 g, 201.67 mmol, 1 eq.) in AcOH (286 mL) at 10 °C under nitrogen atmosphere was added HNO dropwise over 20 min. 3 (127 g, 2015.46 mmol, 9.99 equiv.) To the above mixture was added Br dropwise at 10 °C over 10 min. 2 (35.5 g, 222.14 mmol, 1.10 equiv.). The resulting mixture was stirred at 10°C for another 10 min. To the above mixture was added dropwise a solution of AgNO at 10°C over 25 min. 3 (44.4 g, 261.37 mmol, 1.30 equiv) of water (105 mL) (2.5 mol / L). The resulting mixture was stirred at room temperature overnight. TLC indicated that the reaction was complete. 2 CO 3 The mixture was basified to pH 8 with 4% paraformaldehyde (5% ethanol) (aqueous solution). The resulting mixture was extracted with EtOAc (3 × 800 mL). The combined organic layers were washed with water (3 × 100 mL) and washed with anhydrous Na 2 SO 4 The mixture was dried. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and 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).

[0823] Step 2: Synthesis of compound 3

[0824] 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) at room temperature were added oxalic acid (19.8 g, 219.91 mmol, 1.56 equiv) and Pd(OAc) portionwise. 2 (3.2 g, 14.25 mmol, 0.10 eq) and Xantphos (16.5 g, 28.51 mmol, 0.20 eq). To the mixture was added DIEA (42 mL, 241.12 mmol, 1.71 eq) dropwise at room temperature. The resulting mixture was stirred at 100 °C under nitrogen atmosphere for 4 hours. The mixture was cooled down to room temperature. LCMS indicated the reaction was complete.

[0825] The resulting mixture was quenched with water (1 L) and extracted with EtOAc (3 x 800 mL). The combined organic layers were washed with brine (2 x 100 mL) and purified by anhydrous Na 2 SO 4 After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and 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] - .

[0826] Step 3: Synthesis of compound 4

[0827] To a stirred mixture of 3-(methoxycarbonyl)-4-methyl-5-(trifluoromethyl)benzoic acid (29 g, 110.60 mmol, 1 eq.) in ACN (580 mL) at room temperature was added NBS (29.53 g, 165.91 mmol, 1.50 eq.) and BPO (8.03 g, 33.18 mmol, 0.3 eq.) in portions. The resulting mixture was stirred overnight at 80 °C under a nitrogen atmosphere. The mixture was cooled down to room temperature. LCMS indicated that 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 give 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).

[0828] Step 4: Synthesis of compound 5

[0829] 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) at 0 °C was added BH 3 -THF (235 mL, 1 mol / L, 235 mmol, 2.0 equivalents). The resulting mixture was stirred at room temperature for 6 hours. LCMS indicates that the reaction is complete. The reactant is quenched with MeOH and stirred at room temperature for 1 hour. The resulting mixture is concentrated under reduced pressure. The residue is purified by silica gel column chromatography and eluted with PE / EA (1: 1) to obtain 2- (bromomethyl) -5- (hydroxymethyl) -3- (trifluoromethyl) benzoic acid methyl ester (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 ) δ8.05 (s, 1H), 7.91 (s, 1H), 5.04 (s, 2H), 4.63 (s, 2H), 3.92 (s, 3H).

[0830] Step 5: Synthesis of compound 6

[0831] 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) portionwise at room temperature. The resulting mixture was stirred at 70 °C for 3 h. The mixture was cooled down to room temperature. LCMS indicated the reaction was complete. The resulting mixture was filtered and the filter cake was washed with EtOAc (3 x 50 mL). The residue was washed with saturated Na 2 S 2 The filtrate was washed with O4 (3×30 mL) and then 2 SO 4 After drying, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and 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).

[0832] Step 6: Synthesis of compound 7

[0833] 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) at 0 °C 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) portionwise. The resulting mixture was stirred at room temperature for 2 hours. The reaction was quenched by adding MeOH (5 mL) at 0 °C. 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 purified by anhydrous Na 2 SO 4 After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography using the following conditions: C18 column, water containing ACN (0.1% NH 4 HCO 3), 10% to 80% gradient in 50 minutes; detector, UV 254nm. The collected fractions were concentrated to give 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] + .

[0834] Step 7. Synthesize INT4

[0835] Methyl 2-(bromomethyl)-5-{[(3S)-3-methylpiperidin-1-yl]methyl}-3-(trifluoromethyl)benzoate (Compound 8, 1.7 g, 4.16 mmol, 1 equivalent) was added to a mixture containing NH 3 The mixture was stirred at room temperature for 5 hours. LCMS indicated that the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography using the following conditions: C18 column; mobile phase, water containing ACN (0.05% NH 4 HCO 3 ), 10% to 100% gradient in 40 minutes; detector, UV 254 nm. The resulting mixture was concentrated under reduced pressure. This gave 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindole-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).

[0836] Synthesis of compound 100

[0837]

[0838] Step 1: Synthesis of compound 3

[0839] At room temperature in N 2To a solution of ethylene glycol (compound 2, 124 mg, 2.01 mmol, 3.00 equiv) in THF (10 mL) was added NaH (40 mg, 60%, 1.00 mmol, 1.50 equiv). 2 The resulting mixture was stirred for 0.5 h at room temperature under N 2 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-3-yl}pyridine (INT3, 200 mg, 0.67 mmol, 1.00 equiv) was added under reduced pressure. The resulting mixture was stirred at room temperature overnight. LCMS indicated that 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 purified by anhydrous Na 2 SO 4 Dried. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (DCM: MeOH = 10: 1) to give 2- ((6-chloro-4- (3- ((4-methyl-4H-1,2,4-triazol-3-yl) methyl) oxetanes-3-yl) pyridin-2-yl) oxy) ethane-1-ol (150 mg, 65%) as a white solid. LCMS (ES, m / z): 325,327 [M+H] + .

[0840] Step 2: Synthesis of compound 100

[0841] At 120°C in N 2 2-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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 2 CO 3 (421 mg, 1.29 mmol, 3.00 equiv) and Pd(OAc) 2 (10 mg, 0.04 mmol, 0.10 equiv) in di The solution in alkane (8 mL) was stirred for 1 hour. LCMS indicated that the reaction was complete. After cooling to room temperature, the reactant was filtered. The filtrate was concentrated to dryness under vacuum. The residue was purified by preparative TLC (DCM: MeOH = 10: 1) to give 80 mg of crude product as a white solid. The crude product was re-purified by reverse phase flash chromatography with the following conditions: column: XSelect CSH Prep C18 OBD column, 19 × 150 mm, 5 μm; mobile phase A: water (0.05% FA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 5% B to 35% B, 35% B in 7 minutes; 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).

[0842] Synthesis of compound 101

[0843]

[0844] Step 1: Synthesis of compound 3

[0845] 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 2 CO 3 (1307 mg, 4.01 mmol, 3.00 equiv) and Pd(OAc) 2 (30 mg, 0.13 mmol, 0.10 equiv) in di The solution in alkane (6 mL) was stirred for 1 hour. LCMS indicated that the reaction was complete. After cooling to room temperature. The reactant was filtered. The filtrate was concentrated to dryness under vacuum. The residue was purified by preparative TLC (DCM: MeOH = 10: 1) to obtain N-{[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-3-yl}pyridin-2-yl)carbamoyl]methyl}-N-methylcarbamic acid tert-butyl ester (Compound 3, 390 mg, 63%) as a green solid. LCMS (ES, m / z): 451,453 [M+H] + .

[0846] Step 2: Synthesis of compound 5

[0847] 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 2 CO 3 (607 mg, 1.86 mmol, 3.00 equiv) and Pd(OAc) 2 (14 mg, 0.06 mmol, 0.10 equiv) in di alkane (6mL) solution was stirred for 2 hours.LCMS indicates that the reaction is complete.After cooling to room temperature.The reactant is filtered and concentrated to dryness under vacuum.The residue is purified by preparative TLC (DCM:MeOH=10:1) to obtain N-methyl-N-{[(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetanes-3-yl}-6-(6-{[(3S)-3-methylpiperidin-1-yl]methyl}-1-oxo-4-(trifluoromethyl)-3H-isoindol-2-yl)pyridin-2-yl)carbamoyl]methyl}t-butylcarbamate (Compound 5, 200 mg, 39%).LCMS (ES, m / z): 727 [M+H] + .

[0848] Step 3: Synthesis of compound 101

[0849] 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) were stirred at room temperature for 2 hours. LCMS indicated the reaction was complete. The reaction was concentrated to dryness under vacuum. The residue was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 60% gradient in 30 minutes; 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).

[0850] Synthesis of compound 102

[0851]

[0852] Step 1: Synthesis of compound 1

[0853] 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 eq) and β-mercaptoethanol (53 mg, 0.67 mmol, 1.01 eq) in DMF (2 mL) was added K portionwise at room temperature. 2 CO 3 (186 mg, 1.34 mmol, 2.01 equiv.). The resulting mixture was stirred at 70 ° C under a nitrogen atmosphere for 2 hours. LCMS indicated that the reaction was complete. The reaction mixture was filtered and the filtrate was purified by reverse phase flash chromatography with the following conditions: C18 column; mobile phase, water containing ACN (0.1% FA), 10% to 50% gradient in 40 minutes; detector, UV 254 nm. The collected fractions were concentrated under reduced pressure. This gave 2-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-3-yl}pyridin-2-yl)sulfanyl]ethanol (Compound 1, 130 mg, 57%) as a yellow oil. LCMS: (ES.m / z): 341,343[M+1] + .

[0854] Step 2. Synthesis of compound 102

[0855] To 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 eq.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 124 mg, 0.39 mmol, 1.09 eq.) was added to dihydroisoindole-1-one (INT4, 124 mg, 0.39 mmol, 1.09 eq.) at room temperature. To a stirred mixture in oxane (3 mL) were added Xantphos (42 mg, 0.073 mmol, 0.20 equiv) and Pd(OAc) portionwise. 2 (8 mg, 0.036 mmol, 0.10 equiv) and Cs 2 CO 3 (354 mg, 1.08 mmol, 2.99 equiv.). The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. LCMS indicated that the reaction was complete. The reaction mixture was purified by reverse phase flash chromatography using the following conditions: C18 column; mobile phase, water containing ACN, 10% to 60% gradient in 40 minutes; detector, UV 254 nm. The collected fractions were concentrated under reduced pressure. This gave 2-{6-[(2-hydroxyethyl)sulfanyl]-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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).

[0856] Synthesis of compound 103

[0857]

[0858] Step 1: Synthesis of compound 2

[0859] 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.). The reaction mixture was stirred at 100 ° C under a nitrogen atmosphere for 48 hours. About 20% of the desired product can 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 phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, water containing ACN (0.1% FA), 10% to 60% gradient in 30 minutes; detector, UV 254 nm. The collected fractions were concentrated to dryness to give 6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-3-yl}-N-[2-(methylsulfanyl)ethyl]pyridine-2-amine (compound 2, 44 mg, 35%) as a white solid. LCMS: (ES, m / s): 354,356 [M+H] + .

[0860] Step 2. Synthesis of compound 103

[0861] To 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 eq.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 35 mg, 0.11 mmol, 1.05 eq.) was added to dihydroisoindole-1-one (INT4, 35 mg, 0.11 mmol, 1.05 eq.) at room temperature under nitrogen atmosphere. To the stirred mixture in 2% paraffin (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). The resulting mixture was stirred at 120 °C under nitrogen atmosphere for 1 hour. LCMS indicated 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 preparative HPLC using 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, 48% B in 10 minutes; wavelength: 254 nm); the collected fractions were lyophilized to give 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).

[0862] Synthesis of compound 104

[0863]

[0864] Step 1. Synthesis of compound 2

[0865] 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 eq) and [(tert-butoxycarbonyl)(methyl)amino]acetic acid (190 mg, 1.00 mmol, 1.50 eq) 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 eq.) and BTMG (170 mg, 0.99 mmol, 1.49 eq.) was added dropwise. The resulting mixture was stirred at room temperature and irradiated overnight using a blue LED lamp (365 nm). LCMS indicated that the reaction had 50% 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-3-yl}pyridine and 50% desired product. The reaction mixture was purified by reverse phase flash chromatography using the following conditions: C18 column; mobile phase, water containing ACN (0.1% FA), 10% to 80% gradient in 40 minutes; detector, UV 254 nm. The resulting fractions were concentrated under reduced pressure. This gave 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] + .

[0866] Step 2: Synthesis of compound 3

[0867] To 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 eq.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 50 mg, 0.16 mmol, 1.04 eq.) were added to dihydroisoindole-1-one (INT4, 50 mg, 0.16 mmol, 1.04 eq.) at room temperature. To the stirred mixture in 2-nitropropane (0.5 mL) was added Pd(OAc) portionwise. 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.). The resulting mixture was stirred at 120 ° C under a nitrogen atmosphere overnight. 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 phase flash chromatography using the following conditions: C18 column; mobile phase, water containing ACN (0.1% FA), 10% to 50% gradient in 40 minutes; detector, UV 254 nm. The resulting mixture was concentrated under vacuum. The following conditions were used by preparative HPLC (column: XBridge Shield RP18 OBD column, 30×150 mm, 5 μm; mobile phase A: water (10 mmol / L NH 4 HCO 3 +0.1%NH 3 .H 2 O), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 33% B to 56% B, 56% B in 7 minutes; wavelength: 254 nm) The crude product (37 mg) was repurified; the collected fractions were concentrated to give tert-butyl N-methyl-N-[(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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] + .

[0868] Step 3: Synthesis of compound 104

[0869] 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 eq) in DCM (1200 uL) was added TFA (300 uL) dropwise at room temperature. The resulting mixture was stirred at room temperature under air atmosphere for 1 hour. LCMS indicated 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).

[0870] Synthesis of compound 105

[0871]

[0872] Step 1: Synthesis of compound 2

[0873] To a stirred solution of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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) portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 80 °C under nitrogen atmosphere for 4 days. Approximately 30% of the desired product could be detected by LCMS. The reaction mixture was purified by reverse phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, water with ACN (0.1% FA), 10% to 60% gradient in 30 minutes; detector, UV 254 nm. The resulting mixture was concentrated under vacuum to give 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] + .

[0874] Step 2: Synthesis of compound 105

[0875] To 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 dihydroisoindole-1-one was added 5-(4-(trifluoromethyl)-2-(4-(trifluoromethyl)-1-one) at room temperature under nitrogen atmosphere. To a stirred solution in oxane (1.5 mL) were added portionwise Xantphos (10 mg, 0.016 mmol, 0.4 eq), Pd(OAc) 2 (4 mg, 0.016 mmol, 0.4 eq) and Cs 2 CO 3(43 mg, 0.13 mmol, 3.0 equiv). The resulting mixture was stirred at 120 °C under nitrogen atmosphere for 1 hour. LCMS indicated the reaction was complete. The reaction mixture was purified by preparative HPLC using the following conditions (column: XBridge Shield RP18 OBD column, 30×150 mm, 5 μm; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 23% B to 43% B, 43% B in 7 minutes; wavelength: 254 nm); the 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 as a white solid; formic acid (Compound 105, 2.6 mg, 7%). 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).

[0876] Synthesis of compound 106

[0877]

[0878] Step 1: Synthesis of compound 2

[0879] To a stirred solution of tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate (compound 1, 10 g, 46.44 mmol, 1 eq.) in DCM (250 mL) was added TsCl (17.71 g, 92.89 mmol, 2 eq.) and TEA (14.10 g, 139.34 mmol, 3 eq.) at 0 °C. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. LCMS indicated that the reaction was complete. The reactant was quenched with MeOH at 0 °C. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and eluted with PE / EA (5:1) to give 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] +

[0880] Step 2: Synthesis of compound 3

[0881] To a stirred mixture of tert-butyl 4-{[(4-methylbenzenesulfonyl)oxy]methyl}piperidine-1-carboxylate (Compound 2, 5 g, 13.53 mmol, 1 eq.) and KI (2.25 g, 13.53 mmol, 1 eq.) in EtOH (50 mL) at room temperature was added sodium thiomethoxide (1.90 g, 27.06 mmol, 2 eq.). The resulting mixture was stirred at 80 °C under nitrogen atmosphere for 1 hour. LCMS indicated the reaction was complete. The mixture was cooled down to room temperature. The reaction was quenched with water at room temperature. The reaction was heated to 40 °C for 1 hour. The reaction mixture was heated to 40 °C for 1 hour. The reaction mixture was heated to 40 °C for 2 hours. The reaction mixture was heated to 40 °C for 2 hours. The reaction mixture was heated to 40 °C for 3 ...3 hours. The reaction mixture was heated to 40 °C for 2 hours. The reaction mixture was heated to 40 ° 2 Cl 2 The resulting mixture was extracted with (3× 100 mL). The combined organic layers were washed with brine (100 mL) and washed with 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] + .

[0882] Step 3: Synthesis of compound 4

[0883] A solution of tert-butyl 4-[(methylsulfanyl)methyl]piperidine-1-carboxylate (compound 3, 560 mg, 2.28 mmol, 1 eq.) in HCl (g, 4N in MeOH, 5.6 mL) was stirred at room temperature under nitrogen atmosphere for 1 hour. LCMS indicated that 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 gave 4-[(methylsulfanyl)methyl]piperidine hydrochloride (370 mg, 89%) as a yellow solid. LCMS (ESI, m / z): 146 [M+H-HCl] + .

[0884] Step 4: Synthesis of compound 5

[0885] To a stirred solution of 4-[(methylsulfanyl)methyl]piperidine hydrochloride (compound 4, 350 mg, 1.92 mmol, 1 eq.) in DCM (4 mL) was added 2-(bromomethyl)-5-formyl-3-(trifluoromethyl)benzoic acid methyl ester (compound 6, 626 mg, 1.92 mmol, 1 eq. from the procedure for preparing INT4) at room temperature. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 30 minutes. STAB (1.63 g, 7.70 mmol, 4 eq.) was added portionwise to the above mixture at room temperature. The resulting mixture was stirred at room temperature for another overnight. LCMS indicated that the reaction was complete. The reactant was quenched with MeOH at 0°C and stirred for 30 minutes. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by CH 2 Cl 2 / MeOH (10:1) to give 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] + .

[0886] Step 5: Synthesis of compound 6

[0887] To methyl 2-(bromomethyl)-5-({4-[(methylsulfanyl)methyl]piperidin-1-yl}methyl)-3-(trifluoromethyl)benzoate (Compound 5, 400 mg, 0.88 mmol, 1 eq.) in NH 3 (g, 7N, in MeOH, 4 mL) was added to a stirred solution. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 1 hour. LCMS indicated that the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by CH 2 Cl2 / MeOH (14:1) to give 6-({4-[(methylsulfanyl)methyl]piperidin-1-yl}methyl)-4-(trifluoromethyl)-2,3-dihydroisoindole-1-one (Compound 6, 170 mg, 53%) as a white solid. LCMS (ESI, m / z): 359 [M+H] + .

[0888] Step 6: Synthesis of compound 106

[0889] To 6-({4-[(methylsulfanyl)methyl]piperidin-1-yl}methyl)-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (compound 6, 86 mg, 0.24 mmol, 1 eq) 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 eq) in dihydroisoindole-1-one was added 6-({4-[(methylsulfanyl)methyl]piperidin-1-yl}methyl)-4-(trifluoromethyl)-2,3-dihydroisoindole ... in dihydroisoindole-1-one at room temperature under nitrogen atmosphere. Cs was added to a stirred solution in 2 mL of 2 CO 3(156 mg, 0.48 mmol, 2 eq), di-tert-butyl([2-[2,4,6-tri(propan-2-yl)phenyl]phenyl])phosphane (10 mg, 0.02 mmol, 0.1 eq) and tBuXPhos Pd G3 (19 mg, 0.02 mmol, 0.1 eq). The resulting mixture was stirred at 120 °C under nitrogen atmosphere overnight. LCMS showed 45% product. The reaction mixture was cooled down to room temperature. The reaction mixture was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, water with ACN (0.1% FA), 5% to 60% gradient in 30 minutes; detector, UV 254 nm. The collected fractions were concentrated to give the crude product (40 mg) as a white solid. The crude product (40 mg) was purified by preparative HPLC using the following conditions (column: XBridge Shield RP18 OBD column, 30×150 mm, 5 μm; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 19% B to 39% B, 39% B in 7 minutes; wavelength: 254 nm; RT1 (min): 4.62); the collected fractions were lyophilized to give 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 (10.8 mg, 7%) as a white solid. LCMS (ESI, ms):630[M+H] + ; 1 H 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).

[0890] Synthesis of compound 107

[0891]

[0892] Step 1: Synthesis of compound 2

[0893] To a stirred solution of 2,6-dichloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-3-yl}pyridine (INT3, 2 g, 6.68 mmol, 1 eq) in DMF (20 mL) was added (ethylsulfanyl)sodium (0.62 g, 7.35 mmol, 1.1 eq) portionwise at room temperature. The resulting mixture was stirred at 50 °C under nitrogen atmosphere for 2 hours. LCMS indicated 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 purified by anhydrous Na 2 SO 4 Dry. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, water containing FA, 0% to 50% gradient in 40 minutes; detector, UV 254 nm. This gave 2-chloro-6-(ethylsulfanyl)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-3-yl}pyridine (Compound 2, 1.4 g, 62%) as a yellow solid. LCMS: (ES.m / z): 325,327 [M+1] + .

[0894] Step 2: Synthesis of compound 107

[0895] To 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) was added to dihydroisoindole-1-one (INT4, 96 mg, 0.30 mmol, 1.00 equiv) at room temperature. To the stirred mixture in 2-nitropropane (5 mL) was added Cs(II) portionwise. 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). The resulting mixture was stirred at 120 ° C under a nitrogen atmosphere for 2 hours. The mixture was cooled to room temperature. LCMS indicated that 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 and eluted with DCM / MeOH (10: 1) to give 2-[6-(ethylsulfanyl)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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 ) δ 8.26 (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).

[0896] Synthesis of compound 108

[0897]

[0898] Step 1: Synthesis of compound 1

[0899] 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 eq.) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 328 mg, 1.05 mmol, 1.05 eq.) were added to dihydroisoindole-1-one (INT4, 328 mg, 1.05 mmol, 1.05 eq.) at room temperature under nitrogen atmosphere. To a stirred solution in 2-nitropropane (6 mL) was added Cs 2 CO 3(653 mg, 2.00 mmol, 2 eq), XantPhos (116 mg, 0.20 mmol, 0.2 eq) and Pd(OAc) 2 (22 mg, 0.10 mmol, 0.1 eq.). The resulting mixture was stirred at 120 °C under nitrogen atmosphere for 1 hour. 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 purified by silica gel column chromatography and purified by CH 2 Cl 2 / MeOH (10:1) to give 2-(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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] + .

[0900] Step 2: Synthesis of Compound 2

[0901] To 2-(6-chloro-4-{2-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxirane-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 eq.) and 2-[(triphenylmethyl)sulfanyl]ethanamine hydrochloride (190 mg, 0.53 mmol, 1.2 eq.) was added dichloromethane (2-(6-chloro-4-{2-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxirane-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 eq.) at room temperature under nitrogen atmosphere was added dichloromethane (2-(6-chloro-4-{2-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxirane-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 eq.) and 2-[(triphenylmethyl)sulfanyl]ethanamine hydrochloride (190 mg, 0.53 mmol, 1.2 eq.) in dichloromethane (2-(6-chloro-4-{2-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxirane-2-yl} Cs was added to a stirred solution in 2% paraffin (3 mL). 2 CO 3 (290 mg, 0.89 mmol, 2 eq), XantPhos (51 mg, 0.08 mmol, 0.2 eq) and Pd(OAc) 2 (10 mg, 0.04 mmol, 0.1 eq.). The resulting mixture was stirred at 120 °C under nitrogen atmosphere for 2 hours. LCMS indicated the reaction was complete. The mixture was cooled to room temperature. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and purified by CH 2 Cl 2 / MeOH (10:1) to give 2-(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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] + .

[0902] Step 3: Synthesis of compound 108

[0903] 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 eq) and TFA (2 mL) in DCM (4 mL) was added tri(propyl-2-yl)silane (200 uL) at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 1 hour. LCMS indicated the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography using the following conditions: column, silica gel; mobile phase, MeCN in water (0.1% FA), gradient from 5% to 60% in 30 minutes; detector, UV 254 nm. The collected fractions were lyophilized to give 2-(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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).

[0904] Synthesis of compound 109

[0905]

[0906] Step 1: Synthesis of compound 2

[0907] To 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 eq.) and (S)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one (INT4, 245 mg, 0.786 mmol, 1 eq.) were added to dichloro-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridine (INT3, 235 mg, 0.786 mmol, 1 eq.) at room temperature To the stirred mixture in 2-nitropropane (7 mL) was added Cs(II) portionwise. 2 CO 3 (512 mg, 1.57 mmol, 2 eq.) and Xantphos (90.8 mg, 0.157 mmol, 0.2 eq.) and Pd(OAc) 2 (17.6 mg, 0.0786 mmol, 0.1 eq.). The resulting mixture was stirred at 90 °C under nitrogen atmosphere for 1 hour. The mixture was cooled down to room temperature. LCMS indicated the reaction was complete. The reaction was quenched with saturated sodium bicarbonate at room temperature. The reaction was quenched with CH 2 Cl 2 The resulting mixture was extracted with 4% paraformaldehyde (3 × 50 ml). The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by 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)oxetane-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] + .

[0908] Step 2: Synthesis of compound 109

[0909] To (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 eq.) and propargylamine (5.7 mg, 0.10 mmol, 2 eq.) was added to distilled water at room temperature. To the stirred mixture in 2% ethanol (1 mL) was added Cs 2 CO 3 (113 mg, 0.34 mmol, 2 eq.) and Xantphos (6.0 mg, 0.01 mmol, 0.2 eq.) and Pd(OAc) 2 (1.2 mg, 0.05 mmol, 0.1 eq.). The resulting mixture was stirred at 120 °C under nitrogen atmosphere for 40 min. LCMS indicated the reaction was complete. The resulting mixture was filtered and loaded onto the column as is. The column was dried in vacuo and purified by silica gel column chromatography using 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-(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 = 1.5Hz, 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).

[0910] Synthesis of compound 110

[0911]

[0912] Step 1: Synthesis of compound 2

[0913] 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) at room temperature under nitrogen atmosphere was added K 2 CO 3 (0.92 g, 6.68 mmol, 2.0 equiv.). The resulting mixture was stirred at 120 °C under nitrogen atmosphere for two days. 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. 2 Cl 2 (3 × 100 mL) to extract the aqueous layer. Concentrate the resulting mixture under vacuum. Purify the residue by silica gel column chromatography and use 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] + .

[0914] Step 2: Synthesis of compound 110

[0915] To 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]propionitrile (INT4, 50 mg, 0.15 mmol, 1.0 equiv) in dihydroisoindole was added at room temperature under nitrogen atmosphere. To a stirred solution in 2-nitro-1-oxane (2.0 mL) were added portionwise Xantphos (34 mg, 0.06 mmol, 0.4 eq), Pd(OAc) 2 (13 mg, 0.06 mmol, 0.4 eq) and Cs 2 CO 3 (146 mg, 0.45 mmol, 3.0 equiv). The resulting mixture was stirred at 120 °C under nitrogen atmosphere for 1 hour. LCMS indicated that 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 purified by silica gel column chromatography and purified by CH 2 Cl 2 / MeOH (5: 1) eluted to give a crude product. The crude product (50 mg) was purified by preparative HPLC using the following conditions (column: mobile phase A: water (0.1% FA), mobile phase B: ACN). The collected fractions were freeze-dried to give 3-[(4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-3-yl}-6-(6-{[(3S)-3-methylpiperidin-1-yl]methyl}-1-oxo-4-(trifluoromethyl)-3H-isoindol-2-yl)pyridin-2-yl)amino]propionitrile as a white solid; formic acid (compound 110, 25 mg, 25%). 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).

[0916] Synthesis of compound 111

[0917]

[0918] Step 1: Synthesis of compound 2

[0919] To a stirred mixture of (3S)-3-(hydroxymethyl)piperidine-1-carboxylic acid tert-butyl ester (compound 10 g, 46.44 mmol, 1 eq.) and TEA (14 g, 138.35 mmol, 2.98 eq.) in DCM (100 mL) was added TsCl (17.5 g, 91.79 mmol, 1.98 eq.) dropwise at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature. LCMS indicated that 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 give (3S)-3-{[(4-methylbenzenesulfonyl)oxy]methyl}piperidine-1-carboxylic acid tert-butyl ester (compound 2, 14.2 g, 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).

[0920] Step 2: Synthesis of compound 3

[0921] To a stirred solution of (3S)-3-{[(4-methylbenzenesulfonyl)oxy]methyl}piperidine-1-carboxylic acid tert-butyl ester (compound 2, 5 g, 13.53 mmol, 1 eq) and KI (2.25 g, 13.53 mmol, 1 eq) in EtOH (50 mL) at room temperature under nitrogen atmosphere was added (methylsulfanyl)sodium (1.90 g, 27.06 mmol, 2 eq). The resulting mixture was stirred at 80 °C under nitrogen atmosphere for 1 hour. LCMS indicated the reaction was complete. The resulting mixture was cooled down to room temperature, diluted with water (100 mL), extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (100 mL) and purified by anhydrous Na 2 SO 4 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] + .

[0922] Step 3: Synthesis of compound 4

[0923] Tert-butyl (3S)-3-[(methylsulfanyl)methyl]piperidine-1-carboxylate (Compound 3, 800 mg, 3.26 mmol, 1 eq.) was dissolved in HCl (gas, 4N in 1,4-dihydroquinone) at room temperature under nitrogen atmosphere. The stirred solution in 2% ethyl acetate (30 mL) was stirred for 1 hour. LCMS indicated that the reaction was complete. The resulting mixture was concentrated under reduced pressure. This gave (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] + .

[0924] Step 4: Synthesis of compound 5

[0925] A stirred solution of (3S)-3-[(methylsulfanyl)methyl]piperidine hydrochloride (compound 4, 2.1 g, 11.55 mmol, 1 eq.) and methyl 2-(bromomethyl)-5-formyl-3-(trifluoromethyl)benzoate (3.01 g, 9.24 mmol, 0.8 eq.) in DCM (21 mL) was stirred for 30 minutes at room temperature under a nitrogen atmosphere. STAB (9.80 g, 46.22 mmol, 4 eq.) was added to the above mixture at room temperature. The resulting mixture was stirred for another 12 hours at room temperature. LCMS indicated that the reaction was complete. The reactant was quenched with MeOH and stirred at room temperature for 30 minutes. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by 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] + .

[0926] Step 5: Synthesis of compound 6

[0927] Methyl 2-(bromomethyl)-5-{[(3S)-3-[(methylsulfanyl)methyl]piperidin-1-yl]methyl}-3-(trifluoromethyl)benzoate (Compound 5, 600 mg, 1.32 mmol, 1 equivalent) was dissolved in NH 3 The stirred solution in (g, 7N, in MeOH, 6 mL) was stirred for 2 hours. LCMS indicated that the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography with the following conditions: column, silica gel; mobile phase, water containing MeCN (0.1FA%), 5% to 60% gradient in 30 minutes; detector, UV 254 nm. The collected fractions were concentrated to give 6-{[(3S)-3-[(methylsulfanyl)methyl]piperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindole-1-one (compound 6, 200 mg, 42%) as a yellow oil. LCMS (ESI, ms): 359[M+H] + .

[0928] Step 6: Synthesis of compound 111

[0929] To 6-{[(3S)-3-[(methylsulfanyl)methyl]piperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (Compound 6, 120 mg, 0.33 mmol, 1 eq.) 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 eq.) were added to dihydroisoindole-1-one (Compound 6, 120 mg, 0.33 mmol, 1 eq.) at room temperature under nitrogen atmosphere. Cs was added to a stirred solution in 2,400 mL of 2 CO 3 (218 mg, 0.67 mmol, 2 eq.), RuPhos (31 mg, 0.06 mmol, 0.2 eq.) and RuPhos Palladacycle Gen. 3 (28 mg, 0.03 mmol, 0.1 eq.). The resulting mixture was stirred at 120 °C under a nitrogen atmosphere overnight. LCMS indicated that the reaction was complete. The resulting mixture was cooled down to room temperature. The reaction mixture was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, water containing MeCN (0.1% FA), 5% to 60% gradient in 40 minutes; detector, UV 254 nm. The collected fractions were lyophilized to give 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).

[0930] Synthesis of compound 112

[0931]

[0932] Step 1: Synthesis of compound 2

[0933] 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 minutes at -70 °C under nitrogen atmosphere. Dimethyl carbonate (1.0 g, 11.10 mmol, 1.80 equiv) was added to the above mixture at -70 °C under nitrogen atmosphere. The resulting mixture was stirred for 2 hours at -70 °C under nitrogen atmosphere. About 50% of the desired product could be detected by LCMS. The reaction mixture was heated to 40 °C with saturated NH 4 The reaction was quenched with Cl (aqueous solution, 100 mL). 2 Cl 2 The aqueous layer was extracted with 4-(2-(2,6-dichloropyridin-4-yl)acetate (compound 2, 590 mg, 41%) of 1,2-dichloropyridin-4-yl)acetate (compound 2, 590 mg, 41%). The mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and 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]+.

[0934] Step 2: Synthesis of compound 3

[0935] 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 portionwise NaH (2.73 g, 68.16 mmol, 3.00 equiv, 60%) at 0°C under a nitrogen atmosphere. The resulting mixture was stirred for 30 minutes at 0°C under a nitrogen atmosphere. 1,3-dibromo-2-methylpropane (7.36 g, 34.08 mmol, 1.5 equiv) was added to the above mixture at 0°C. The resulting mixture was stirred for another 2 hours at 10°C. About 30% of the desired product could be detected by LCMS. The reactants were 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 (3×300 mL). The resulting mixture was concentrated under vacuum. The residue was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient 10% to 70% in 30 min; detector, UV 254 nm. 2 Cl 2 (3×300 mL) to extract the isomers. The resulting mixture was concentrated under vacuum to obtain 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] + .

[0936] Step 3: Synthesis of compound 4

[0937] 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 2 (0=9.0 mL). The resulting mixture was stirred at room temperature under nitrogen atmosphere for 1 hour. LCMS indicated the reaction was complete. The reaction mixture was adjusted to pH=5 with 1N HCL. The mixture was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 50% gradient in 30 minutes; detector, UV 254 nm. CH 2 Cl 2The aqueous layer was extracted with 4% paraformaldehyde (3×300 mL). The resulting mixture was concentrated under vacuum to obtain 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] + .

[0938] Step 4: Synthesis of compound 5

[0939] 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) at 0 °C under nitrogen atmosphere 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) portionwise. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 2 hours. LCMS indicated the reaction was complete. The reaction was quenched with water at room temperature. The reaction was quenched with CH 2 Cl 2 (3×200 mL) to extract the aqueous layer. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and eluted with PE / EA (2:3) to give 1-(2,6-dichloropyridin-4-yl)-3-methyl-N-[(methylthiocarbamoyl)amino]cyclobutane-1-carboxamide (2.5 g, 70%) as a white solid. LCMS: (ES, m / s): 347,349 [M+H] + .

[0940] Step 5: Synthesis of compound 6

[0941] To a stirred solution of 1-(2,6-dichloropyridin-4-yl)-3-methyl-N-[(methylthiocarbamoyl)amino]cyclobutane-1-carboxamide (compound 5, 2.3 g, 6.62 mmol, 1.0 equiv) in THF (11 mL) at 0 °C under nitrogen atmosphere was added NaOH (1.0 g, 25.00 mmol, 3.77 equiv) (H 2 (0=11 mL). The resulting mixture was stirred at room temperature under nitrogen atmosphere overnight. Approximately 73% of the desired product could be detected by LCMS. The mixture was acidified to pH 5 with concentrated HCl. 2 Cl 2(3×200 mL) to extract the aqueous layer. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and 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] + .

[0942] Step 6: Synthesis of compound 7

[0943] 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) at 0 °C under nitrogen atmosphere was added NaNO portionwise 2 (0.75 g, 10.93 mmol, 3.0 equiv)(H 2 O=12 mL) and HNO 3 (12 mL, 1.0 mol / L). The resulting mixture was stirred at room temperature under nitrogen atmosphere overnight. LCMS indicated the reaction was complete. The reaction was quenched with water / ice at room temperature. 2 Cl 2 The aqueous layer was extracted with 1% 4% iodine (3 × 200 mL). The resulting mixture was concentrated under vacuum. The residue was purified by reverse phase flash chromatography using the following conditions: column, silica gel; mobile phase, ACN in water (0.05% TFA), 10% to 50% gradient in 30 minutes; detector, UV 254 nm. 2 Cl 2 The aqueous layer was extracted with 4-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyridine (Compound 7, 900 mg, 74%). LCMS: (ES, m / s): 297,299 [M+H] + .

[0944] Step 7: Synthesis of Compound 8

[0945] 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 under nitrogen atmosphere for 5 hours. LCMS indicated the reaction was complete. The resulting mixture was cooled down to room temperature and purified by reverse phase flash chromatography with the following conditions: column, silica gel; mobile phase, ACN in water (0.1% FA), 10% to 60% gradient in 30 minutes; detector, UV 254nm. The resulting mixture was concentrated under vacuum to give 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] + .

[0946] Step 8: Synthesis of compound 112

[0947] To 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 dihydroisoindole-1-one was added 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) at room temperature under nitrogen atmosphere. To a stirred solution in 2-nitropropane (8.0 mL) were added portionwise Xantphos (107 mg, 0.18 mmol, 0.4 eq), Pd(OAc) 2 (41 mg, 0.18 mmol, 0.4 eq) and Cs 2 CO 3 (454 mg, 1.39 mmol, 3.0 equiv). The resulting mixture was stirred at 120 ° C under a nitrogen atmosphere for 1 hour. LCMS indicated that the reaction was complete. The resulting mixture was cooled down to room temperature and purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase TFA, water containing ACN, 10% to 50% gradient in 10 minutes; detector, UV 254 nm. The resulting mixture was concentrated under vacuum to give compound 9. The following conditions were used by chiral HPLC (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 minutes; 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) to separate the product; the first eluting isomer (RT = 10.35 min) was concentrated to dryness and lyophilized to give 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).

[0948] Synthesis of compound 113

[0949]

[0950] Step 1: Synthesis of compound 2

[0951] 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 minutes at -70 °C under nitrogen atmosphere. Dimethyl carbonate (1.0 g, 11.10 mmol, 1.80 equiv) was added to the above mixture at -70 °C under nitrogen atmosphere. The resulting mixture was stirred for 2 hours at -70 °C under nitrogen atmosphere. About 50% of the desired product could be detected by LCMS. The reaction mixture was heated to 40 °C with saturated NH 4 The reaction was quenched with Cl (aqueous solution, 100 mL). 2 Cl 2 The aqueous layer was extracted with 4-(2-(2,6-dichloropyridin-4-yl)acetate (compound 2, 590 mg, 41%) of 1,2-dichloropyridin-4-yl)acetate (compound 2, 590 mg, 41%). The mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and 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] + .

[0952] Step 2: Synthesis of compound 3

[0953] 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%) portionwise at 0°C under nitrogen atmosphere. The resulting mixture was stirred at 0°C under nitrogen atmosphere for 30 minutes. 1,3-dibromo-2-methylpropane (7.36 g, 34.08 mmol, 1.5 equiv) was added to the above mixture at 0°C. The resulting mixture was stirred for another 2 hours at 10°C. About 30% of the desired product could be detected by LCMS. The reactant was quenched with water (200 mL) at room temperature. The mixture was acidified to pH 6 with concentrated HCl. CH 2 Cl 2 The aqueous layer was extracted with 1% 4% iodine (3 × 300 mL). The resulting mixture was concentrated under vacuum. The residue was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 70% gradient in 30 minutes; detector, UV 254 nm. 2 Cl 2(3×300 mL) to extract the isomers. The resulting mixture was concentrated under vacuum to obtain 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] + .

[0954] Step 3. Synthesis of compound 4

[0955] 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) at room temperature under nitrogen atmosphere was added LiOH (0.31 g, 13.13 mmol, 2.0 equiv) (H 2 The resulting mixture was stirred at room temperature under nitrogen atmosphere for 1 hour. LCMS indicated that the reaction was complete. The reaction mixture was adjusted to pH=5 with 1N HCL. The mixture was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 50% gradient in 30 minutes; detector, UV254 nm. CH 2 Cl 2 The aqueous layer was extracted with 4% paraformaldehyde (3×300 mL). The resulting mixture was concentrated under vacuum to obtain 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] + .

[0956] Step 4: Synthesis of compound 5

[0957] 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) at 0 °C under nitrogen atmosphere 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) portionwise. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 2 hours. LCMS indicated the reaction was complete. The reaction was quenched with water at room temperature. The reaction was quenched with CH 2 Cl 2(3×200 mL) to extract the aqueous layer. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and eluted with PE / EA (2:3) to give 1-(2,6-dichloropyridin-4-yl)-3-methyl-N-[(methylthiocarbamoyl)amino]cyclobutane-1-carboxamide (Compound 5, 2.5 g, 70%) as a white solid. LCMS: (ES, m / s): 347,349 [M+H] + .

[0958] Step 5: Synthesis of compound 6

[0959] To a stirred solution of 1-(2,6-dichloropyridin-4-yl)-3-methyl-N-[(methylthiocarbamoyl)amino]cyclobutane-1-carboxamide (compound 5, 2.3 g, 6.62 mmol, 1.0 equiv) in THF (11 mL) at 0 °C under nitrogen atmosphere was added NaOH (1.0 g, 25.00 mmol, 3.77 equiv) (H 2 (0=11 mL). The resulting mixture was stirred at room temperature under nitrogen atmosphere overnight. Approximately 73% of the desired product could be detected by LCMS. The mixture was acidified to pH 5 with concentrated HCl. 2 Cl 2 (3×200 mL) to extract the aqueous layer. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and 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] + .

[0960] Step 6: Synthesis of compound 7

[0961] 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) at 0 °C under nitrogen atmosphere was added NaNO portionwise 2 (0.75 g, 10.93 mmol, 3.0 equiv)(H 2 O=12 mL) and HNO 3 (12 mL, 1.0 mol / L). The resulting mixture was stirred at room temperature under nitrogen atmosphere overnight. LCMS indicated the reaction was complete. The reaction was quenched with water / ice at room temperature. 2 Cl2 The aqueous layer was extracted with 1% 4% iodine (3 × 200 mL). The resulting mixture was concentrated under vacuum. The residue was purified by reverse phase flash chromatography using the following conditions: column, silica gel; mobile phase, ACN in water (0.05% TFA), 10% to 50% gradient in 30 minutes; detector, UV 254 nm. 2 Cl 2 The aqueous layer was extracted with 4-[3-methyl-1-(4-methyl-1,2,4-triazol-3-yl)cyclobutyl]pyridine (Compound 7, 900 mg, 74%). LCMS: (ES, m / s): 297,299 [M+H] + .

[0962] Step 7: Synthesis of Compound 8

[0963] 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 under nitrogen atmosphere for 5 hours. LCMS indicated the reaction was complete. The resulting mixture was cooled down to room temperature and purified by reverse phase flash chromatography with the following conditions: column, silica gel; mobile phase, ACN in water (0.1% FA), 10% to 60% gradient in 30 minutes; detector, UV 254nm. The resulting mixture was concentrated under vacuum to give 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] + .

[0964] Step 8. Synthesis of compound 113

[0965] To 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 dihydroisoindole-1-one was added 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) at room temperature under nitrogen atmosphere. To a stirred solution in 2-nitropropane (8.0 mL) were added portionwise Xantphos (107 mg, 0.18 mmol, 0.4 eq), Pd(OAc) 2 (41 mg, 0.18 mmol, 0.4 eq) and Cs 2 CO 3 (454 mg, 1.39 mmol, 3.0 equiv). The resulting mixture was stirred at 120 °C under a nitrogen atmosphere for 1 hour. LCMS indicated that the reaction was complete. The resulting mixture was cooled down to room temperature and purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase TFA, water containing ACN, 10% to 50% gradient in 10 minutes; detector, UV 254 nm. The resulting mixture was concentrated under vacuum to give compound 9. The following conditions were used by chiral HPLC (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 minutes; 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) to separate the product; the second eluting isomer (RT = 13.03 min) was concentrated to dryness and lyophilized to give 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).

[0966] Synthesis of Compound 114A and Compound 114B

[0967]

[0968] Step 1: Synthesis of compound 2

[0969] To 1-tert-butyl 3-methyl malonate (Compound 11, 35.61 g, 204.44 mmol, 1.25 equiv) and Cs 2 CO 3 2,4,6-trichloropyrimidine (compound 1, 30 g, 163.55 mmol, 1 eq.) (106.58 g, 327.11 mmol, 2 eq.) was added dropwise to a stirred mixture in acetone (550 mL). The resulting mixture was stirred at 25 °C for 4 hours. LCMS indicated 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 gave 1-tert-butyl 3-methyl 2-(2,6-dichloropyrimidin-4-yl)malonate (compound 2, 30 g, 57%) as a yellow oil. LCMS (ES, m / z): 321,323 [M+H] + .

[0970] Step 2. Synthesis of compound 3

[0971] To a stirred mixture of 1-tert-butyl 3-methyl 2-(2,6-dichloropyrimidin-4-yl)malonate (Compound 2, 15 g, 46.70 mmol, 1 eq.) in DCM (150 mL) was added TFA (30 mL) dropwise at 0 °C. The resulting mixture was stirred at 25 °C for 3 hours. LCMS indicated that the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was washed with saturated NaHCO 3The residue was basified to pH 8 using CH 2 Cl 2 The resulting mixture was extracted with (3 × 100 mL). The combined organic layers were washed with brine (100 mL) and washed with anhydrous Na 2 SO 4 After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and 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] + .

[0972] Step 3: Synthesis of compound 4

[0973] To a stirred mixture of methyl 2-(2,6-dichloropyrimidin-4-yl)acetate (compound 3, 5.6 g, 25.33 mmol, 1 eq.) in THF (80 mL) at 0°C under a nitrogen atmosphere was added LiHMDS (12.72 g, 76.00 mmol, 3 eq.). The resulting mixture was stirred at 0°C under a nitrogen atmosphere for 1 hour. Methyl 1,3-dibromo-2-methylpropane (compound 12, 10.94 g, 50.66 mmol, 2 eq.) was added dropwise to the above mixture at 0°C. The resulting mixture was stirred at 25°C for another 16 hours. LCMS indicated that the reaction was complete. The reaction was quenched by adding 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 purified by anhydrous Na 2 SO 4 Dry. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (6:1) to give 1-(2,6-dichloropyrimidin-4-yl)-3-methylcyclobutane-1-carboxylic acid methyl ester (compound 4, 2.8 g, 40%) as a yellow oil. LCMS (ES, m / z): 275,277 [M+H] + .

[0974] Step 4: Synthesis of compound 5

[0975] To a stirred mixture of 1-(2,6-dichloropyrimidin-4-yl)-3-methylcyclobutane-1-carboxylic acid methyl ester (compound 4, 2.5 g, 9.08 mmol, 1 equiv) in THF (20 mL) was added dropwise a solution of 2.0 M ethylamine in THF (20 mL) at 0 ° C. The resulting mixture was stirred at 25 ° C for 4 hours. LCMS indicated that the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (5: 1) to give 1-[2-chloro-6-(ethylamino)pyrimidin-4-yl]-3-methylcyclobutane-1-carboxylic acid methyl ester (compound 5, 2.3 g, 70%) as a yellow oil. LCMS (ES, m / z): 284,286 [M+H] + .

[0976] Step 5: Synthesis of compound 6

[0977] 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 eq) in THF (30 mL) at 0 °C was added LiOH (0.23 g, 9.72 mmol, 1.2 eq) in H 2 O (10 mL). The resulting mixture was stirred at 25 ° C for 16 hours. LCMS indicated that the reaction was complete. The resulting mixture was concentrated under reduced pressure. The mixture was acidified to pH 5 with HCl (aqueous solution). The residue was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, water (0.05% TFA), ACN 5% to 50% gradient in 30 minutes; detector, UV 254 nm. This gave 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] + .

[0978] Step 6: Synthesis of compound 7

[0979] 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) at 0°C 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.). The resulting mixture was stirred at 25°C for 3 hours. LCMS indicated 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] + .

[0980] Step 7: Synthesis of Compound 8

[0981] To the above stirred mixture of 1-[2-chloro-6-(ethylamino)pyrimidin-4-yl]-3-methyl-N-[(methylthiocarbamoyl)amino]cyclobutane-1-carboxamide (Compound 7) was added NaOH (313.82 mg, 7.84 mmol, 2 eq) in H 2 O (50 mL). The resulting mixture was stirred at 50 °C for 1 hour. LCMS indicated the reaction was complete. The mixture was cooled down 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 purified by anhydrous Na 2 SO 4 Dried. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and 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] + .

[0982] Step 8: Synthesis of Compound 9

[0983] 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 eq) in DCM (6 mL) at 0 °C were added AcOH (1.5 g, 24.97 mmol, 14.59 eq), H 2 O 2(0.5 g, 14.70 mmol, 8.59 equiv). The resulting mixture was stirred at room temperature for 1 hour. LCMS indicated the reaction was complete. The reaction was concentrated with saturated NaHCO 3 The reaction mixture was basified to pH 8 with 4% paraformaldehyde (5% ethanol) (aqueous solution). The resulting mixture was extracted with EtOAc (3 × 30 mL). The combined organic layers were washed with brine (30 mL) and purified by anhydrous Na 2 SO 4 After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by 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] + .

[0984] Step 9: Compound 114A and Compound 114B

[0985] 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 eq.), 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 256 mg, 0.82 mmol, 1.05 eq.), Pd(OAc) 2 (17 mg, 0.078 mmol, 0.1 eq.), Xantphos (90 mg, 0.15 mmol, 0.2 eq.) and Cs 2 CO 3 (764 mg, 2.34 mmol, 3 eq.) in di The mixture was stirred in 20% ethanol (5 mL) for 3 h. LCMS indicated 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 × 20 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by CH 2 Cl 2 / MeOH (10:1). The crude product was repurified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, water (0.05% TFA), ACN 5% to 100% gradient in 30 minutes; detector, UV 254 nm. The product was isolated by preparative SFC using the following conditions: column: CHIRALPAK IA, 3 × 25 cm, 5 μm; mobile phase A: CO 2 , mobile phase B: ACN: MeOH = 1:1 (1% 2 M 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 give 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).

[0986] The second eluting isomer (RT1 = 4.88 min) was concentrated and lyophilized to give 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 3 OD, 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).

[0987] Synthesis of compound 115

[0988]

[0989] Step 1: Synthesis of compound 1

[0990] 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) were added to dihydroisoindole-1-one (INT4, 1 g, 3.20 mmol, 1.00 equiv) at room temperature under nitrogen atmosphere. To the stirred mixture in 2-nitropropane (10 mL) was added Cs(II) portionwise. 2 CO 3 (2.09 g, 6.40 mmol, 2 eq.) and Xantphos (0.37 g, 0.64 mmol, 0.2 eq.) and di 2-Hydane (10 mL). The resulting mixture was stirred at 120 °C under nitrogen atmosphere for 2 h. The mixture was cooled down to room temperature. LCMS indicated the reaction was complete. The resulting mixture was filtered and purified by CH 2 Cl 2 (3 × 20 mL) and the filter cake was washed. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by CH 2 Cl 2 / MeOH (10:1) to give 2-(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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] + .

[0991] Step 2: Synthesis of compound 115

[0992] 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 eq) in DMF (2 mL) was added Zn(CN) portionwise at room temperature under nitrogen atmosphere. 2 (73 mg, 0.62 mmol, 1.2 equiv) and Pd(PPh 3 ) 4 (60 mg, 0.05 mmol, 0.1 eq.). The resulting mixture was stirred at 120 °C under a nitrogen atmosphere for 2 hours. The mixture was cooled down to room temperature. LCMS indicated that the reaction was complete. The reaction mixture was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, water containing ACN (0.05% TFA), 0% to 50% gradient in 30 minutes; detector, UV 254 nm. The collected fractions were concentrated under vacuum to give 4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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-d 6) δ 9.72 (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).

[0993] Synthesis of compound 116

[0994]

[0995] Step 1. Synthesis of compound 1

[0996] 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) were added to dihydroisoindole-1-one (INT4, 1 g, 3.20 mmol, 1.00 equiv) at room temperature under nitrogen atmosphere. To the stirred mixture in 2-nitropropane (10 mL) was added Cs(II) portionwise. 2 CO 3 (2.09 g, 6.40 mmol, 2 eq.) and Xantphos (0.37 g, 0.64 mmol, 0.2 eq.) and di 2-Hydane (10 mL). The resulting mixture was stirred at 120 °C under nitrogen atmosphere for 2 h. The mixture was cooled down to room temperature. LCMS indicated the reaction was complete. The resulting mixture was filtered and purified by CH 2 Cl 2 (3 × 20 mL) and the filter cake was washed. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by CH 2 Cl 2 / MeOH (10:1) to give 2-(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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] + .

[0997] Step 2: Synthesis of Compound 2

[0998] 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 eq) in DMF (2 mL) was added Zn(CN) portionwise at room temperature under nitrogen atmosphere. 2 (73 mg, 0.62 mmol, 1.2 equiv) and Pd(PPh 3 ) 4 (60 mg, 0.05 mmol, 0.1 eq.). The resulting mixture was stirred at 120 °C under a nitrogen atmosphere for 2 hours. The mixture was cooled down to room temperature. LCMS indicated that the reaction was complete. The reaction mixture was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, water containing ACN (0.05% TFA), 0% to 50% gradient in 30 minutes; detector, UV 254 nm. The collected fractions were concentrated under vacuum to give 4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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] + .

[0999] Step 3: Synthesis of Compound 3

[1000] 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 eq) in MeOH (4 mL) was added Reney-Ni (20 mg) portionwise at room temperature. The resulting mixture was stirred at room temperature under a hydrogen atmosphere overnight. LCMS indicated the reaction was complete. The resulting mixture was filtered and the filtrate was concentrated under reduced pressure to give 2-[6-(aminomethyl)-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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] + .

[1001] Step 4: Synthesis of compound 116

[1002] 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 eq) in DCM (2 mL) was added acryloyl chloride (28 mg, 0.31 mmol, 1.5 eq) and TEA (64 mg, 0.63 mmol, 3 eq) dropwise at 0°C. The resulting mixture was stirred at 0°C for 2 hours. LCMS indicated the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC using 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 minutes; wavelength: 254 nm; RT1 (min): 6.30); the collected fractions were lyophilized 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)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).

[1003] Synthesis of compound 117

[1004]

[1005] Step 1: Synthesis of compound 3

[1006] To a stirred mixture of 4-(bromomethyl)-3-(methoxycarbonyl)-5-(trifluoromethyl)benzoic acid (Compound 1, 6 g, 17.5 mmol, 1 eq) in THF (300 mL) was added 4-methoxybenzylamine (Compound 2, 6.00 g, 43.80 mmol, 2.49 eq) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. LCMS indicated 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 purified by 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] + .

[1007] Step 2. Synthesis of compound 5

[1008] 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 eq) and HATU (4.01 g, 10.54 mmol, 1.54 eq) in DMF (40 mL) at room temperature was added N,O-dimethylhydroxylamine hydrochloride (compound 4, 1.00 g, 10.26 mmol, 1.50 eq) and DIEA (2.75 g, 21.28 mmol, 3.11 eq). The resulting mixture was stirred at room temperature overnight. LCMS indicated that 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 purified by anhydrous Na 2 SO 4The mixture was dried. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and 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] + .

[1009] Step 3: Synthesis of compound 6

[1010] 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 eq) in THF (20 mL) was added MeMgBr (5 mL, 1 N in THF, 5 mmol) dropwise at -5 °C under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 1 hour. LCMS indicated the reaction was complete. The reaction mixture was purified by addition of saturated NH 4 The reaction was quenched with 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 purified by anhydrous Na 2 SO 4 After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and 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] + .

[1011] Step 4: Synthesis of compound 7

[1012] 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 eq, 50%) in EtOH (10 mL) was added CuBr at room temperature. 2 (1350 mg, 6.04 mmol, 4.99 equiv). The resulting mixture was stirred at 80 °C under nitrogen atmosphere for 1 hour. LCMS indicated 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 purified by anhydrous Na 2 SO4 The mixture was dried. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and 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] + .

[1013] Step 5: Synthesis of compound 8

[1014] To a stirred solution of sodium methanethiolate (compound 7, 114.23 mg, 1.631 mmol, 1.5 eq) 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 eq) dropwise at 0°C under nitrogen atmosphere. The resulting mixture was stirred at 0°C under nitrogen atmosphere for 10 minutes. LCMS indicated the reaction was complete. The reaction was quenched with water / ice at 0°C. The precipitated solid was collected by filtration and washed with Et 2 O (3 × 10 mL). This afforded 2-[(4-methoxyphenyl)methyl]-6-[2-(methylsulfanyl)acetyl]-4-(trifluoromethyl)-3H-isoindol-1-one (Compound 8, 280 mg, 86%) as a yellow solid. LCMS (ESI, ms): 410 [M+H] + .

[1015] Step 6: Synthesis of compound 9

[1016] 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 eq) in DCM (7 mL) was added NaBH portionwise at 0 °C under nitrogen atmosphere. 4 (36.96 mg, 0.97 mmol, 2 eq.). The resulting mixture was stirred at 0 °C under nitrogen atmosphere for 2 h. LCMS indicated the reaction was complete. The reaction was quenched with water at 0 °C. 2 Cl 2 The resulting mixture was extracted with 10 mL (3 × 10 mL). The combined organic layers were washed with brine (50 mL) and purified by anhydrous Na 2 SO 4After 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] + .

[1017] Step 7: Synthesis of compound 10

[1018] 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 eq) and DIEA (188 mg, 1.45 mmol, 3 eq) in DCM (1 mL) at 0° C. was added TsCl (139 mg, 0.72 mmol, 1.5 eq) and DMAP (6 mg, 0.05 mmol, 0.1 eq) dropwise. The resulting mixture was stirred at 25° C. for 16 h. The reaction was quenched by the addition of water (5 mL) at room temperature. The residue was washed with CH 2 Cl 2 The resulting mixture was extracted with 10 mL (3 × 10 mL). The combined organic layers were washed with brine (3 × 10 mL) and purified by anhydrous Na 2 SO 4 The mixture was dried. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (1:1) to give 4-methylbenzenesulfonic acid 1-{2-[(4-methoxyphenyl)methyl]-3-oxo-7-(trifluoromethyl)-1H-isoindol-5-yl}-2-(methylsulfanyl)ethyl ester (Compound 10, 120 mg, 43%) as a yellow solid. LCMS (ES, m / z): 566 [M+H] + .

[1019] Step 8. Synthesis of compound 12

[1020] 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 eq) and (3S)-3-methylpiperidine hydrochloride (43 mg, 0.31 mmol, 1.5 eq) in DCM (3 mL) at 0°C was added TEA (42 mg, 0.42 mmol, 2 eq). The resulting mixture was stirred at 60°C for 16 hours. LCMS indicated the reaction was complete. The reaction was quenched by the addition of water (10 mL) at room temperature. CH 2 Cl 2 The resulting mixture was extracted with 10 mL (3 × 10 mL). The combined organic layers were washed with brine (3 × 15 mL) and washed with anhydrous Na 2 SO 4 After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by 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] + .

[1021] Step 9: Synthesis of compound 13

[1022] 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 eq) in DCM (100 uL) was added TFA (30 uL) at 0°C. The resulting mixture was stirred at 25°C for 2 hours. LCMS indicated that the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, water 0.05% TFA, ACN 5% to 50% gradient in 30 minutes; detector, UV 254 nm. This afforded 6-{1-[(3S)-3-methylpiperidin-1-yl]-2-(methylsulfanyl)ethyl}-4-(trifluoromethyl)-2,3-dihydroisoindole-1-one; trifluoroacetic acid (Compound 13, 50 mg, 67%) as a yellow oil. LCMS (ES, m / z): 373 [M+H] + .

[1023] Step 10: Synthesis of Compound 117

[1024] 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 eq), 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 eq), Ruphos (20 mg, 0.04 mmol, 0.4 eq), RuPhos Palladacycle Gen.3 (18 mg, 0.02 mmol, 0.2 eq) and Cs 2 CO 3 (70 mg, 0.21 mmol, 2 eq.) in di The mixture in 2-(4-(4-nitropropene)-2-nitropropene (1.2 mL) was stirred for 16 hours. LCMS indicated that the reaction was complete. The resulting mixture was cooled to room temperature and 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 phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, water (0.05% TFA), ACN5% to 50% gradient in 30 minutes; detector, UV 254 nm. The crude product was repurified by preparative HPLC using the following conditions: column: XBridgeShield 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 minutes; wavelength: 254 nm; RT1 (min): 4.03. The collected fractions were lyophilized to give 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; trifluoroacetic acid (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).

[1025] Synthesis of compound 118

[1026]

[1027] Step 1: Synthesis of compound 2

[1028] To 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 dihydroisoindole (INT4) was added at room temperature. To the stirred mixture in 2-nitropropane (10 mL) was added Cs(II) portionwise. 2 CO 3 (2.09 g, 6.40 mmol, 2 eq.) and Xantphos (0.37 g, 0.64 mmol, 0.2 eq.) and Pd(OAc) 2 (112 mg, 0.50 mmol, 0.15 equiv). The resulting mixture was stirred at 120 °C under nitrogen atmosphere for 2 h. The mixture was cooled down to room temperature. LCMS indicated the reaction was complete. The resulting mixture was filtered and purified by CH 2 Cl 2 (3 × 20 mL) and the filter cake was washed. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by 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 2, 630 mg, 29%) as a green solid. LCMS: (ES.m / z): 575,577 [M+1] + .

[1029] Step 2: Synthesis of compound 118

[1030] To 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 eq.) and 2-azidoethylamine (Compound 1, 22 mg, 0.26 mmol, 1.5 eq.) in distilled water was added at room temperature. To the stirred mixture in 2% ethanol (1 mL) was added Cs 2 CO 3 (113 mg, 0.34 mmol, 2 eq.) and Xantphos (20 mg, 0.03 mmol, 0.2 eq.) and Pd(OAc) 2 (4 mg, 0.02 mmol, 0.1 eq.). The resulting mixture was stirred at 90 °C under nitrogen atmosphere for 1.5 h. LCMS indicated 50% product on LCMS. The resulting mixture was filtered and the filter cake was washed with DCM (3 x 50 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by CH 2 Cl 2The mixture was eluted with 1% / 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 material). The crude product (30 mg) was purified by preparative HPLC using the following conditions (column: XSelect CSH Prep C18 OBD column, 19×250 mm, 5 μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 20% B to 35% B, 35% B in 10 minutes; wavelength: 254 nm; RT1 (min): 8.77); the collected fractions were lyophilized 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 (Compound 118, 8.5 mg, 8%) as a yellow solid. LCMS: (ES.m / z): 625 [M+1] + ; 1 H NMR (400MHz, 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).

[1031] Synthesis of compound 119

[1032]

[1033] Step 1: Synthesis of compound 4

[1034] To aminoacetonitrile hydrochloride (500 mg, 5.4 mmol, 1 eq.) was added dichloromethane. To a stirred solution in 1,2-dioxane (10 mL) was added 1 M aqueous NaOH (217 mg, 5.4 mmol, 1 eq.). The reaction was cooled to 0 °C and a solution of 5 mL of dioxane was added dropwise. Di-tert-butyl dicarbonate (1.18 g, 5.4 mmol, 1 eq.) in oxane was added. The reaction mixture was stirred vigorously overnight. In the morning, TLC control indicated complete consumption of the starting material. The mixture was heated to 40 ℃ and then dried over medium temperature with saturated NaHCO 3 The reaction was quenched with aqueous solution and extracted with dichloromethane (3 × 100 mL). The combined organic layers were washed with brine (3 × 100 mL) and purified by Na 2 SO 4 Drying and concentration under vacuum gave tert-butyl (cyanomethyl)carbamate (Compound 4, 800 mg, 95%) as a colorless oil. 1 H NMR (400MHz, CDCl 3 ) δ 5.03 (s, 1H), 4.06 (m, 2H), 1.44 (s, 9H).

[1035] Step 2: Synthesis of compound 1

[1036] To 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 eq.) and (S)-6-((3-methylpiperidin-1-yl)methyl)-4-(trifluoromethyl)isoindolin-1-one (INT4, 245 mg, 0.786 mmol, 1 eq.) were added to dichloro-4-(3-((4-methyl-4H-1,2,4-triazol-3-yl)methyl)oxetan-3-yl)pyridine (INT3, 235 mg, 0.786 mmol, 1 eq.) at room temperature To the stirred mixture in 2-nitropropane (7 mL) was added Cs(II) portionwise. 2 CO 3 (512 mg, 1.57 mmol, 2 eq.) and Xantphos (90.8 mg, 0.157 mmol, 0.2 eq.) and Pd(OAc) 2 (17.6 mg, 0.0786 mmol, 0.1 eq.). The resulting mixture was stirred at 90 °C under nitrogen atmosphere for 1 hour. The mixture was cooled down to room temperature. LCMS indicated the reaction was complete. The reaction was quenched with saturated sodium bicarbonate at room temperature. The reaction was quenched with CH 2 Cl 2 The resulting mixture was extracted with 4% paraformaldehyde (3 × 50 ml). The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by 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)oxetane-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] + .

[1037] Step 3: Synthesis of Compound 2

[1038] To (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 eq) and tert-butyl (cyanomethyl)carbamate (Compound 4, 110 mg, 0.7 mmol, 10 eq) was added distilled water at room temperature. To the stirred mixture in 2% ethanol (1.2 mL) was added Cs(II) portionwise. 2 CO 3 (45 mg, 0.14 mmol, 2 eq.) and Xantphos (8.1 mg, 0.014 mmol, 0.2 eq.) and Pd(OAc) 2 (1.6 mg, 0.007 mmol, 0.1 eq.). The resulting mixture was stirred at 120 °C under nitrogen atmosphere for 40 min. LCMS indicated the reaction was complete. The resulting mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and purified by CH 2 Cl 2 / MeOH (10:1) to give (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)carbamic acid tert-butyl ester (Compound 2, 25 mg, 52%) as a white solid. LCMS: (ES.m / z): 695 [M+1] + .

[1039] Step 4: Synthesis of compound 119

[1040] 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 mmmol) was dissolved in distilled water. The product was added to a 1 / 1 mixture of oxane / water (1 ml) and heated in a microwave initiator at 120° C. for 15 minutes. The product was extracted with dichloromethane and purified by silica gel column chromatography using 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 (300MHz, 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).

[1041] Synthesis of compound 120

[1042]

[1043] Step 1: Synthesis of compound 2

[1044] 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 eq) and 3-mercaptopropane-1,2-diol (Compound 1A, 86 mg, 0.80 mmol, 1.2 eq) in DMF (2 mL) was added K 2 CO 3 (184 mg, 1.33 mmol, 2 eq.). The resulting mixture was stirred overnight at 50 °C under a nitrogen atmosphere. LCMS indicated that the reaction was complete. The mixture was cooled down to room temperature. The reaction mixture was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, water containing ACN (0.1% FA), 5% to 50% gradient in 30 minutes; detector, UV 254 nm. The collected fractions were concentrated to give 3-[(6-chloro-4-{3-[(4-methyl-1,2,4-triazol-3-yl)methyl]oxetane-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] + .

[1045] Step 2: Synthesis of compound 120

[1046] 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 eq) and 6-{[(3S)-3-methylpiperidin-1-yl]methyl}-4-(trifluoromethyl)-2,3-dihydroisoindol-1-one (INT4, 92 mg, 0.29 mmol, 1.1 eq) were added to 1,4-dihydroisoindole-1-one at room temperat...

Claims

1. A compound of formula (I): (I)， or a pharmaceutically acceptable salt thereof, wherein: n is 0, 1, or 2; X and Y are each independently CH or N; Z is selected from CH(CH3), O and SO2; or Z is selected from CH(CH3), NH, N(CH3), O and SO2; R 1 Selected from 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 , C3-C6 cycloalkyl and 3 to 6 membered heterocyclyl rings; wherein: R z Selected from R a and R b independently selected from hydrogen, C2-C6 alkenyl, C1-C6 alkyl, amide (C1-C6 alkyl), amino (C1-C6 alkyl), azido (C1-C6 alkyl), C2-C6 alkynyl, carboxyl (C1-C6 alkyl), cyano (C1-C6 alkyl), C3-C6 cycloalkyl optionally substituted with cyano, dimethylamino (C1-C6 alkyl), 3 to 6 membered heterocyclyl ring, 3-6 membered heterocyclyl (C1-C3 alkyl), hydroxy (C1-C6 alkyl), methoxy (C1-C6 alkyl), methylamino (C1-C6 alkyl), NR c R d (C1-C6 alkyl), HS (C1-C6 alkyl) and CH3S (C1-C6 alkyl), where R c and R d are independently selected from hydrogen, C2 alkenylcarbonyl and methyl; or, R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from R a 、-C(O)R a 、-SO2R a , azido and cyano group substitution; wherein each C3-C6 cycloalkyl, each 3 to 6-membered heterocyclyl ring, and the heterocyclyl moiety of the 3 to 6-membered heterocyclyl (C1-C3 alkyl) ring are optionally substituted with one, two or three groups independently selected from C1-C3 alkyl, C2 alkynyl, amide, azido, carboxyl, cyano, dimethylamino, hydroxyl, methoxy, methylamino, HS- and CH3S-; and R 2 Selected from , , , , , , , , and ;in m is 0, 1, 2, or 3; m'' is 0, 1, 2, 3, or 4; B' is a 3-7 membered saturated or unsaturated ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen and sulfur; wherein the ring is optionally substituted with one or two substituents independently selected from -OH, -CH2SH, CH2SCH3, -CH2OH, -CH2NH2 and -CH2NHCH3; Each R 500 are independently selected from hydrogen, C1-C6 alkyl, halo, -OH and -CH2OH; and X 50 is selected from O, NH, NCH3 and S; R 3 is selected from hydrogen, acetyl, amino, C1-C6 alkylamino, C1-C6 alkylaminomethyl, C1-C6 alkylcarbonyl, aminoC1-C6 alkyl, aminocarbonyl, aminomethyl, carboxyl, cyano, C3 cycloalkyl, formyl, hydroxy, hydroxyC1-C6 alkyl, methoxy, Oxazolyl, -SH, -SCH3, -SOCH3, -SO2CH3, -SO(=NH)CH3, tetrazolyl, thiazolyl and trifluoromethyl, wherein the C3 cycloalkyl is optionally substituted with hydroxyl; R 4 is selected from the group consisting of hydrogen, methyl, -CH2OH, -CH2SH and -CH2SCH3; R 5 is selected from hydrogen, hydroxy, -CH2SH, -CH2SCH3 and methyl; Optionally, the prerequisite is that when R 5 is hydroxy or methyl, and R 4 When it is hydrogen, then R 1 Not C3-C6 cycloalkyl, 3- to 6-membered heterocyclyl ring, hydroxy, hydroxy(C1-C6 alkyl), -OR a , where R a is C1-C6 alkyl, 3- to 6-membered heterocyclyl ring or hydroxy(C1-C6 alkyl); or -NR a R b , where R a and R b are independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, hydroxy(C1-C6 alkyl), 3 to 6 membered heterocyclyl ring, or wherein R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted with a group selected from C1-C6 alkyl or hydroxy(C1-C6 alkyl); and R 6 and R 6' independently selected from hydrogen, cyclopropyl, -CH2OH, -CH2SH, -CH2SCH3 and -CH2R 200 , where R 200 is a 3-7 membered saturated or unsaturated ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen and sulfur.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: X is N; Y is CH; and R 2 yes .

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

4. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein Z is CH(CH3).

5. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein: R 3 is trifluoromethyl; R 4 and R 6 is hydrogen; and R 5 It's methyl.

6. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 1 Selected from -NR a R b 、-NHC(O)R a 、-NHC(S)NHR a and-SR a .

7. A compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 6, wherein R 1 It is -SCH2CH3.

8. A compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 6, wherein R 1 It is -NH(CH2)2CN.

9. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein R 1 It is -NH(CH2)2N3.

10. A compound of formula (IA-1): (I)， or a pharmaceutically acceptable salt thereof, wherein: n is 0, 1, or 2; X and Y are each independently CH or N; Z is selected from CH(CH3), O and SO2; R 1 Selected from 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 , C3-C6 cycloalkyl and 3 to 6 membered heterocyclyl rings; wherein: R z Selected from R a and R b independently selected from hydrogen, C2-C6 alkenyl, C1-C6 alkyl, amide (C1-C6 alkyl), amino (C1-C6 alkyl), azido (C1-C6 alkyl), C2-C6 alkynyl, carboxyl (C1-C6 alkyl), cyano (C1-C6 alkyl), C3-C6 cycloalkyl, dimethylamino (C1-C6 alkyl), 3 to 6 membered heterocyclyl ring, 3-6 membered heterocyclyl (C1-C3 alkyl), hydroxy (C1-C6 alkyl), methoxy (C1-C6 alkyl), methylamino (C1-C6 alkyl), NR c R d (C1-C6 alkyl), HS (C1-C6 alkyl) and CH3S (C1-C6 alkyl), where R c and R d are independently selected from hydrogen, C2 alkenylcarbonyl and methyl; or, R a and R b Together with the nitrogen atom to which they are attached, they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a moiety selected from R a 、-C(O)R a 、-SO2R a , azido and cyano group substitution; wherein each C3-C6 cycloalkyl, each 3 to 6-membered heterocyclyl ring, and the heterocyclyl moiety of the 3 to 6-membered heterocyclyl (C1-C3 alkyl) ring are optionally substituted with one, two or three groups independently selected from C1-C3 alkyl, C2 alkynyl, amide, azido, carboxyl, cyano, dimethylamino, hydroxyl, methoxy, methylamino, HS- and CH3S-; and R 2 yes or ;in m is 0, 1, 2, or 3; R 3 is selected from hydrogen, acetyl, amino, C1-C6 alkylamino, C1-C6 alkylaminomethyl, aminoC1-C6 alkyl, aminocarbonyl, aminomethyl, carboxyl, cyano, C3 cycloalkyl, formyl, hydroxy, hydroxyC1-C6 alkyl, methoxy, oxazolyl, -SH, -SCH3, tetrazolyl, thiazolyl and trifluoromethyl, wherein the C3 cycloalkyl is optionally substituted with hydroxy; R 4 and R 6 are independently selected from hydrogen, -CH2SH and -CH2SCH3; and R 5 Selected from hydroxy, -CH2SH, -CH2SCH3 and methyl; Optionally, the prerequisite is that when R 5 is hydroxy or methyl, and R 4 When it is hydrogen, then R 1 Not C3-C6 cycloalkyl, 3- to 6-membered heterocyclyl ring, hydroxy, hydroxy(C1-C6 alkyl), -OR a , where R a is C1-C6 alkyl, 3- to 6-membered heterocyclyl ring or hydroxy(C1-C6 alkyl); or -NR a R b , where R a and R b are independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, hydroxy(C1-C6 alkyl), 3 to 6 membered heterocyclyl ring, or wherein R a and R b Together with the nitrogen atom to which they are attached they form a five- or six-membered ring optionally containing one additional nitrogen atom, wherein the ring is optionally substituted by a group selected from C1-C6 alkyl or hydroxy(C1-C6 alkyl).

11. The compound according to claim 1, selected from and or a pharmaceutically acceptable salt thereof.

12. A compound selected from: ; and .

13. The compound of any one of claims 1 to 12, wherein the compound is an inhibitor of Casitas B-lineage lymphoma proto-oncogene b (Cbl-b).

14. The compound of any one of claims 1 to 13, 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 times relative to background; increases IFN-γ secretion by about 1.1 to about 2 times relative to background; and / or increases CD69 levels by about 0.81 to about 1.1 times relative to background.

15. A conjugate or a pharmaceutically acceptable salt thereof, comprising a binding portion 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 portion is directly linked to the payload or is linked to the payload via a linker.

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

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

18. The conjugate of claim 17 or a pharmaceutically acceptable salt thereof, wherein the binding moiety 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 linked to the payload or is linked to the payload via a linker.

20. 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 according to any one of claims 15 to 20, or a pharmaceutically acceptable salt thereof, wherein the conjugate has formula (I): Bm-[LP] a (I), in: a is an integer from 1 to 50; P is the payload; L is a connector; and Bm is the binding moiety.

22. The conjugate of any one of claims 15 to 19 and 21, or a pharmaceutically acceptable salt thereof, wherein the payload is an inhibitor of Casitas B lineage lymphoma proto-oncogene b (Cbl-b).

23. The conjugate of claim 22 or a pharmaceutically acceptable salt thereof, wherein the Cbl-b inhibitor is a compound according to any one of claims 1 to 10, which is covalently linked to the binding moiety or the linker.

24. The conjugate of claim 22 or a pharmaceutically acceptable salt thereof, wherein the Cbl-b inhibitor is or .

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

26. The conjugate of any one of claims 15 to 19 and 21, 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. The conjugate of claim 26 or a pharmaceutically acceptable salt thereof, wherein the payload is the TLR-7 / TLR-8 agonist T785.

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

29. The conjugate of any one of claims 15 to 19 and 21, or a pharmaceutically acceptable salt thereof, wherein the payload is an inhibitor of hematopoietic progenitor cell kinase 1 (HPK-1).

30. The conjugate of any one of claims 15 to 19 and 21, 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. The conjugate of any one of claims 15 to 20, or a pharmaceutically acceptable salt thereof, wherein the payload is an agonist of stimulator of interferon genes (STING).

32. The conjugate of any one of claims 15 to 22, 26, or 29 to 31, or a pharmaceutically acceptable salt thereof, wherein the payload is a small molecule.

33. The conjugate of any one of claims 15 to 22, 26, or 29 to 31, or a pharmaceutically acceptable salt thereof, wherein the payload is a peptide.

34. The conjugate of any one of claims 21 to 33, or a pharmaceutically acceptable salt thereof, wherein L is a non-cleavable linker.

35. The conjugate of any one of claims 21 to 34, or a pharmaceutically acceptable salt thereof, wherein L is selected from the group consisting of: , , , , , , , , and ; in: p is an integer from 1 to 10; p* is an integer from 1 to 10; Y is selected from hydrogen and C1-C6 alkyl; is the connection point to the payload; and is the point of connection to the binding moiety.

36. The conjugate of any one of claims 21 to 35, or a pharmaceutically acceptable salt thereof, wherein L is selected from , and ; in: p is an integer from 1 to 10; p* is an integer from 1 to 10; is the connection point to the payload; and is the point of connection to the binding moiety.

37. The conjugate of any one of claims 21 to 33, or a pharmaceutically acceptable salt thereof, wherein L is a cleavable linker.

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

39. The conjugate of any one of claims 21 to 33, 37 and 38, or a pharmaceutically acceptable salt thereof, wherein L is selected from ; ; ; ; ; ; ; ; and ; in: q is an integer from 2 to 10; Z 1 , Z 2 , Z 3 and Z 4 each independently being absent or being a naturally occurring amino acid residue in the L or D configuration, provided that Z 1 , Z 2 , Z 3 and Z 4 At least two of are amino acid residues; is the connection point to the payload; and is the point of connection to the binding moiety.

40. The conjugate of claim 39 or a pharmaceutically acceptable salt thereof, wherein Z 1 , Z 2 , Z 3 and Z 4 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; provided that Z 1 , Z 2 , Z 3 and Z 4 At least two of them are amino acid residues.

41. The conjugate of claim 39 or a pharmaceutically acceptable salt thereof, wherein: Z 1 absent or glycine; Z 2 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; and Z 4 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.

42. The conjugate of any one of claims 21 to 33, 37 and 38, or a pharmaceutically acceptable salt thereof, wherein L is selected from , , , and , in: q is an integer from 2 to 10; is the connection point to the payload; and is the point of connection to the binding moiety.

43. The conjugate of any one of claims 21 to 33, or a pharmaceutically acceptable salt thereof, wherein L is a bioreducible linker.

44. The conjugate of any one of claims 21 to 33 and 43, or a pharmaceutically acceptable salt thereof, wherein L is selected from in: q is an integer from 2 to 10; R, R', R'' and R''' are each independently selected from hydrogen, C1-C6 alkoxy, C1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or two geminal R groups together with the carbon atoms to which they are attached are capable of forming a cyclobutyl or cyclopropyl ring; is the connection point to the payload; and is the point of connection to the binding moiety.

45. The conjugate of any one of claims 21 to 33, 43 and 44, or a pharmaceutically acceptable salt thereof, wherein L is selected from and ; in: q is an integer from 2 to 10; R, R', R'' and R''' are each independently selected from hydrogen, C1-C6 alkoxy, C1-C6 alkyl, (C1-C6)2NC1-C6 alkyl and C1-C6 alkyl, or two geminal R groups together with the carbon atoms to which they are attached are capable of forming a cyclobutyl or cyclopropyl ring; is the connection point to the payload; and is the point of connection to the binding moiety.

46. ​​The conjugate of any one of claims 21 to 33, or a pharmaceutically acceptable salt thereof, wherein L is an acid-cleavable linker.

47. The conjugate of any one of claims 21 to 33 and 46, or a pharmaceutically acceptable salt thereof, wherein L is selected from the group consisting of: in: q is an integer from 2 to 10; is the connection point to the payload; and is the point of connection to the binding moiety.

48. The conjugate of any one of claims 21 to 33, or a pharmaceutically acceptable salt thereof, wherein L is a click-release linker.

49. The conjugate of any one of claims 21 to 33 and 48, or a pharmaceutically acceptable salt thereof, wherein L is selected from in: q is an integer from 2 to 10; is the connection point to the payload; and is the point of connection to the binding moiety.

50. The conjugate of any one of claims 21 to 33, or a pharmaceutically acceptable salt thereof, wherein L is a pyrophosphatase cleavable linker.

51. The conjugate of any one of claims 21 to 33 and 50, or a pharmaceutically acceptable salt thereof, wherein L is ； in: q is an integer from 2 to 10; is the connection point to the payload; and is the point of connection to the binding moiety.

52. The conjugate of any one of claims 21 to 33, or a pharmaceutically acceptable salt thereof, wherein L is a β-glucuronidase cleavable linker.

53. The conjugate of any one of claims 21 to 33 and 52, or a pharmaceutically acceptable salt thereof, wherein L is selected from , , and ; in: q is an integer from 2 to 10; ----Does not exist or is a key; is the connection point to the payload; and is the point of connection to the binding moiety.

54. The conjugate of any one of claims 21 to 33, 52 and 53, or a pharmaceutically acceptable salt thereof, wherein L is ； in: q is an integer from 2 to 10; ----Does not exist or is a key; is the connection point to the payload; and is the point of connection to the binding moiety.

55. The conjugate of claim 21, wherein the conjugate is selected from: ; ; ; ; ; ; ; ; ; ; ; ; ; ; and .

56. The conjugate of any one of claims 15 to 55, or a pharmaceutically acceptable salt thereof, wherein the linker is linked to cysteine, lysine, tyrosine or glutamine in the Bm.

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

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

59. The conjugate of any one of claims 15 to 58, or a pharmaceutically acceptable salt thereof, wherein the binding moiety is an antibody or an antigen-binding fragment thereof.

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

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

62. The conjugate of any one of claims 15 to 61, or a pharmaceutically acceptable salt thereof, wherein the binding moiety 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. The conjugate of any one of claims 15 to 61, or a pharmaceutically acceptable salt thereof, wherein the binding moiety 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. The conjugate of any one of claims 15 to 61, or a pharmaceutically acceptable salt thereof, wherein the binding moiety is an antibody or antigen-binding fragment thereof comprising the 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. The conjugate of any one of claims 15 to 61, or a pharmaceutically acceptable salt thereof, wherein the binding moiety 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. The conjugate of any one of claims 15 to 17 and 21 to 61, or a pharmaceutically acceptable salt thereof, wherein the binding moiety is capable of specifically binding to CD25.

67. The conjugate of any one of claims 15 to 17, 21 to 61 and 66, or a pharmaceutically acceptable salt thereof, wherein the binding moiety 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. The conjugate of any one of claims 15 to 17, 21 to 61 and 66, or a pharmaceutically acceptable salt thereof, wherein the binding moiety 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. The conjugate of any one of claims 15 to 17, 21 to 61 and 66, wherein the binding moiety 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. The conjugate of any one of claims 15 to 17, 21 to 61 and 66, or a pharmaceutically acceptable salt thereof, wherein the binding moiety 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. The conjugate of any one of claims 15 to 17, 21 to 61 and 66, or a pharmaceutically acceptable salt thereof, wherein the binding moiety 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: 8 and amino acids 24-33, 49-55 and 88-96 of SEQ ID NO:

9.

72. The conjugate of any one of claims 15 to 17, 21 to 61 and 66, or a pharmaceutically acceptable salt thereof, wherein the binding moiety 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. The conjugate of any one of claims 15 to 17 and 21 to 61, or a pharmaceutically acceptable salt thereof, wherein the binding moiety is capable of specifically binding to CD7.

74. The conjugate of any one of claims 15 to 17, 21 to 61 and 73, or a pharmaceutically acceptable salt thereof, wherein the binding moiety 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. The conjugate of any one of claims 15 to 17, 21 to 61 and 73, or a pharmaceutically acceptable salt thereof, wherein the binding moiety 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. The conjugate of any one of claims 59 to 65, 66 to 72, 74 and 75, or a pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region.

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

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

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. The conjugate of any one of claims 76 to 79, or a pharmaceutically acceptable salt thereof, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

20.

81. 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. The conjugate of any one of claims 76 to 78 and 81, or a pharmaceutically acceptable salt thereof, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

21.

83. The conjugate of any one of claims 15 to 56, 58, 59 and 61, or a pharmaceutically acceptable salt thereof, wherein (a) the binding moiety is pembrolizumab, or (b) the binding moiety is nivolumab.

84. The conjugate of any one of claims 15 to 56, 58, 59 and 61, or a pharmaceutically acceptable salt thereof, wherein (a) the binding moiety is a686, (b) the binding moiety is MA251, or (c) the binding moiety is humanized MA251.

85. The conjugate of any one of claims 15 to 55, or a pharmaceutically acceptable salt thereof, wherein the binding moiety is a small molecule.

86. The conjugate of any one of claims 21 to 85, or a pharmaceutically acceptable salt thereof, wherein a is 1 to 40.

87. The conjugate of any one of claims 21 to 85, or a pharmaceutically acceptable salt thereof, wherein a is 1 to 10.

88. The conjugate of any one of claims 21 to 85, or a pharmaceutically acceptable salt thereof, wherein a is 2 to 8.

89. The conjugate of any one of claims 15 to 88, or a pharmaceutically acceptable salt thereof, wherein the conjugate is capable of increasing effector T cell activity.

90. The conjugate of any one of claims 15 to 89, or a pharmaceutically acceptable salt thereof, wherein the conjugate is capable of increasing effector T cell proliferation.

91. The conjugate of any one of claims 15 to 90, or a pharmaceutically acceptable salt thereof, wherein the conjugate is capable of increasing the migration of effector T cells to tumor cells.

92. The conjugate of any one of claims 15 to 91 or a pharmaceutically acceptable salt thereof, wherein the conjugate is capable of reducing effector T cell exhaustion.

93. The conjugate of any one of claims 15 to 92, or a pharmaceutically acceptable salt thereof, wherein the conjugate increases the secretion of IFN-γ from T cells by about 2.5 to about 3 fold relative to background; and / or increases the secretion of IL-2 from T cells by about 6 to about 8 fold relative to background.

94. A composition comprising a compound of 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 94 or a pharmaceutically acceptable salt thereof.

96. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a compound of any one of claims 1 to 14, a conjugate of any one of claims 15 to 93, or a composition of claim 94 or 95.

97. The method 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. 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 of any one of claims 1 to 14, a conjugate of any one of claims 15 to 93, or a composition of claim 94 or 95.

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

100. A method for increasing the activity of an immune cell, the method comprising contacting the immune cell with a compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 14, a conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 15 to 93, or a composition as described in claim 94 or 95.

101. A method for increasing the proliferation of immune cells, the method comprising contacting the immune cells with a compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 14, a conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 15 to 93, or a composition as described in claim 94 or 95.

102. A method for increasing the migration of immune cells to tumor cells, the method comprising contacting the immune cells with a compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 14, a conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 15 to 93, or a composition as described in claim 94 or 95.

103. A method of reducing exhaustion of immune cells, the method comprising contacting the immune cells with a compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 14, a conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 15 to 93, or a composition as described in claim 94 or 95.

104. A method of increasing the secretion of IFN-γ or IL-2 from an immune cell, the method comprising contacting the immune cell with a compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 14, a conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 15 to 93, or a composition as described in claim 94 or 95.

105. The method of any one of claims 100 to 104, wherein the immune cell is a T cell, optionally wherein the T cell is an effector T cell.

106. The method of any one of claims 100 to 104, wherein the immune cell is a natural killer (NK) cell.

107. A method for enhancing and / or maintaining an antigen recall response of a T cell, the method comprising contacting the T cell with a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, a conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 15 to 93, or a composition according to claim 94 or 95.

108. A method of delivering a payload capable of activating effector T cells to an immune cell, the method comprising contacting the effector T cells with the conjugate or a pharmaceutically acceptable salt thereof of any one of claims 15 to 93 or the composition of claim 95.

109. The method of any one of claims 95 to 108, wherein the contacting is in vitro.

110. The method of any one of claims 100 to 108, wherein the contacting is in a subject, optionally wherein the subject has cancer or a condition that would benefit from an increased immune response.

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