Bispecific cytotoxicity-targeted chimeras

Abstract

This disclosure relates to a heterotrifunctional molecule called a bispecific or bitargeted cytotoxic targeted chimeric molecule (CyTaC) or antibody recruiting molecule (ARM) that can simultaneously bind to one or two target cell surface proteins and exogenous antibody proteins. This disclosure also relates to a drug that can bind to receptors on the surface of pathogenic cells and induce depletion of pathogenic cells in a target for use in the treatment of cancer, inflammatory diseases, autoimmune diseases, viral infections, or bacterial infections.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Patent Application No. 63 / 461,181, filed on 21 April 2023, the entire contents of which are incorporated herein by reference. Reference to electronic sequence listings

[0002] This application includes a sequence listing submitted electronically in ST.26 format, which is incorporated herein by reference in its entirety (a copy of the aforementioned ST.26, created on 17 April 2024, is named "209277_seqlist.xml" and has a size of 15,181 bytes).

[0003] This disclosure relates to a heterotrifunctional molecule called a bispecific or bitargeted cytotoxic targeted chimeric molecule (CyTaC) or antibody recruiting molecule (ARM) that can simultaneously bind to one or two target cell surface proteins and exogenous antibody proteins. This disclosure also relates to a drug that can bind to receptors on the surface of pathogenic cells and induce depletion of pathogenic cells in a target for use in the treatment of cancer, inflammatory diseases, autoimmune diseases, viral infections, or bacterial infections. [Background technology]

[0004] Cell surface proteins and their ligands play crucial roles in various inflammatory, infectious, and autoimmune diseases, as well as in tumor development, growth, and metastasis. Antibody-based therapies possess promising characteristics as drug candidates for these indications due to their selectivity for pathogenic cell surface targets and their ability to induce depletion of pathogenic cells by directing immune surveillance towards target-expressing tissues or cells. Examples of such depletion mechanisms include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and complement-dependent cell-mediated cytotoxicity (CDC). However, antibody-based therapies often suffer from problems such as lack of bioavailability, high cost, thermal instability, and manufacturing difficulties due to their size, complexity, and peptide-based structure. Conversely, small molecule therapies often offer affordability, stability, and the convenience of oral administration, but may suffer from low selectivity and off-target effects, and lack the immunomodulation of therapeutic antibodies.

[0005] Therefore, there is a need for improved therapeutic approaches that target pathogenic cells for use in treating diseases. Such compositions and related methods are provided in this disclosure. [Overview of the Initiative]

[0006] In one embodiment, the disclosure provides a heterotrifunctional molecule called a cytotoxic targeted chimeric molecule (CyTaC) or antibody recruiting molecule (ARM), the ARM comprising a portion that binds to one or two target cell surface proteins on a cell and a portion that binds to an exogenous antibody. In a further embodiment, the ARM comprises a trivalent linker that links the target binding portion to the antibody binding portion. In a further embodiment, the target binding portion is a CC chemokine receptor type 2 (CCR2) binding portion. In a further embodiment, the target binding portion is a CC chemokine receptor type 8 (CCR8) binding portion. In a further embodiment, the target binding portion is a prostate-specific membrane antigen (PSMA) binding portion. In a further embodiment, the exogenous antibody is an anticotinin antibody or its antigen-binding fragment.

[0007] In a further aspect, ARM is a compound of formula (I) as defined below or a pharmaceutically acceptable salt thereof,

Chemical formula

Chemical formula

Chemical formula

[0008] In a further embodiment, ARM is a compound of formula (I) as defined below or a pharmaceutically acceptable salt thereof, [ka] It is represented by or a pharmaceutically acceptable salt thereof. During the ceremony, T 1 and T 2 Each of these is independently a target binding site, R 1 C 1~4 Alkyl or C3~6 It is a cycloalkyl, G is a bond, -CH2CH2NH-, -C(O)CH2CH2OCH2CH2NH-, or -L 3 It is -CH2CH2NH-, G 1 and G 2 These are, independently, -C(O)CH2-, -CH2CH2NHC(O)CH2-, -CH2CH2C(O)NH(CH2CH2O)3CH2CH2-, or -CH2CH2NHC(O)(CH2CH2O)3CH2CH2-, L' and L'' are independent of each other, and are combined. [ka] (wherein each y is an integer from 1 to 9), or [ka] (In the formula, each w is an integer from 0 to 5) Y 1 and Y 2 Each of these is independently a bond or a divalent spacer portion with a length of 1 to 12 atoms. L 1 , L 2 and L 3 Each is independently a bond or divalent linker as described herein, and L 1 , L 2 and L 3 At least one of these is a divalent linker as described herein, Each L' group [ka] Y in equation (I) 1 It represents a covalent bond to the base, or Y 1 If it is a combination, then T in equation (I) 1 This represents a covalent bond to the L' group, and each of the L' groups [ka] L is in equation (I). 1 It represents a covalent bond to the base, or L1 If it is a combination, then G in formula (I) 1 Represents a covalent bond to a base, Each L'' group [ka] Y in equation (I) 2 It represents a covalent bond to the base, or Y 2 If it is a combination, then T in equation (I) 2 This represents a covalent bond to the group, and each of the L'' groups [ka] L is in equation (I). 2 It represents a covalent bond to the base, or L 2 If it is a combination, then G in formula (I) 2 This represents a covalent bond to a base.

[0009] In one embodiment, the present disclosure provides a method for treating and / or preventing a disease or disorder in a patient in need thereof, comprising administering a therapeutically effective amount of a compound of formula (I) disclosed herein and an anticotinin antibody or its antigen-binding fragment to the patient.

[0010] In one embodiment, the present disclosure provides a method for increasing antibody-dependent cell-mediated cytotoxicity (ADCC) of target-expressing cells, comprising contacting the cells with an effective amount of a compound of formula (I) disclosed herein and an anti-cotinin antibody or its antigen-binding fragment.

[0011] In one embodiment, the present disclosure provides a method for increasing cytotoxicity of target expression cells, comprising contacting the cells with an effective amount of a compound of formula (I) disclosed herein and an anti-cotinin antibody or its antigen-binding fragment.

[0012] In one embodiment, the present disclosure provides a method for depleting target expression cells, comprising contacting the cells with an effective amount of a compound of formula (I) disclosed herein and an anti-cotinin antibody or its antigen-binding fragment.

[0013] In one embodiment, the Disclosure provides a compound of formula (I) disclosed herein for therapeutic use. In a further embodiment, the Disclosure provides a combination of a compound of formula (I) disclosed herein and an anti-cotinine antibody or its antigen-binding fragment for therapeutic use.

[0014] In one embodiment, the present disclosure provides a combination comprising a compound of formula (I) disclosed herein and an anticotinin antibody or its antigen-binding fragment for use in the treatment of a disease or disorder.

[0015] In one embodiment, the Disclosure provides the use of a compound of formula (I) disclosed herein in the manufacture of a pharmaceutical product for the treatment of a disease or disorder. In a further embodiment, the Disclosure provides the use of a combination comprising a compound of formula (I) disclosed herein and an anticotinin antibody or its antigen-binding fragment in the manufacture of a pharmaceutical product for the treatment of a disease or disorder.

[0016] In one embodiment, the present disclosure provides a combination comprising a compound of formula (I) disclosed herein and an anticotinin antibody or its antigen-binding fragment. [Brief explanation of the drawing]

[0017] [Figure 1] This is a schematic diagram comparing cytotoxic targeted chimeric (CyTaCs) technology with current antibody technology. [Figure 2A] This is a PK analysis of the compound of formula (I) in mice described in Example 12, and shows the PK analysis of the compound of Example 1 administered in the presence of an anti-cotinine antibody. [Figure 2B]This is a PK analysis of the compound of formula (I) in mice described in Example 12, and shows the PK analysis of the compound of Example 2 administered in the presence of an anti-cotinine antibody. [Figure 2C] This shows the PK analysis of the compound of formula (I) in mice described in Example 12, and the PK analysis of the compound of Example 4 administered in the presence of an anti-cotinine antibody. [Figure 2D] This is a PK analysis of the compound of formula (I) in mice described in Example 12, and shows the PK analysis of the compound of Example 7 administered in the presence of an anti-cotinine antibody. [Figure 2E] This is a PK analysis of the compound of formula (I) in mice described in Example 12, and shows the PK analysis of the compound of Example 8 administered in the presence of an anti-cotinine antibody. [Figure 2F] This is a PK analysis of the compound of formula (I) in mice described in Example 12, and shows the PK analysis of the compound of Example 9 administered in the presence of an anti-cotinine antibody. [Modes for carrying out the invention]

[0018] In one embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof. [ka] It is represented by or a pharmaceutically acceptable salt thereof. During the ceremony, T 1 and T 2 Each of these is independently a target binding site, R 1 C 1~4 Alkyl or C 3~6 It is a cycloalkyl, G is a bond, -CH2CH2NH-, -C(O)CH2CH2OCH2CH2NH-, or -L 3 It is -CH2CH2NH-, G 1 and G 2These are, independently, -C(O)CH2-, -CH2CH2NHC(O)CH2-, -CH2CH2C(O)NH(CH2CH2O)3CH2CH2-, or -CH2CH2NHC(O)(CH2CH2O)3CH2CH2-, L' and L'' are independent of each other, and are combined. [ka] (wherein each y is an integer from 1 to 9), or [ka] (In the formula, each w is an integer from 0 to 5) Y 1 and Y 2 Each of these is independently a bond or a divalent spacer portion with a length of 1 to 12 atoms. L 1 , L 2 and L 3 Each is independently a divalent linker of the combination or formula (La), (Lb), (Lc), (Ld), (Le), (Lf), (Lg), (Lh), (Li), (Lj), (Lk), (Lm), (Lni), (Ln-ii), (Ln-iii), (Ln-iv), (Lp), (Lq), (Lr), or (Ls), however, L 1 , L 2 and L 3 At least one of them is a divalent linker of formula (La), (Lb), (Lc), (Ld), (Le), (Lf), (Lg), (Lh), (Li), (Lj), (Lk), (Lm), (Lni), (Ln-ii), (Ln-iii), (Ln-iv), (Lp), (Lq), (Lr), or (Ls), Each L' group [ka] Y in equation (I) 1 It represents a covalent bond to the base, or Y 1 If it is a combination, then T in equation (I) 1 This represents a covalent bond to the L' group, and each of the L' groups [Chemistry] represents a covalent bond to the L group of formula (I), or when L 1 is a bond, represents a covalent bond to the G group of formula (I), 1 and 1 each of the L'' groups [Chemistry] represents a covalent bond to the Y group of formula (I), or when Y 2 is a bond, represents a covalent bond to the T group of formula (I), and each 2 of the L'' groups 2 [Chemistry] represents a covalent bond to the L group of formula (I), or when L 2 is a bond, represents a covalent bond to the G group of formula (I). 2 and 2 In another embodiment, L

[0019] , L 1 , L 2 or L 3 is a bivalent linker of formula (L-a), [Chemistry] or a stereoisomer thereof, wherein ring A and ring B are each independently C 4~6 cycloalkylene, L 1a is C 3~5 linear alkylene, and one or two methylene units are replaced by -O- or -NR a -, each R a is independently hydrogen or C 1~3 alkyl, L 2a is -O-, -NHC(O)-, or -CH2-O-, L 1 each of the groups [Chemistry] represents a covalent bond to the L' group of formula (I), or when L' is a bond, represents a covalent bond to the Y 1 group of formula (I), or when both L' and Y 1 are bonds, represents a covalent bond to the T 1 group of formula (I), and each 1 of the L [Chemistry] represents a covalent bond to the G 1 group of formula (I), and each 2 of the L [Chemistry] represents a covalent bond to the L'' group of formula (I), or when L'' is a bond, represents a covalent bond to the Y 2 group of formula (I), or when both L'' and Y 2 are bonds, represents a covalent bond to the T 2 group of formula (I), and each 2 of the L [Chemistry] represents a covalent bond to the G 2 group of formula (I), and each 3 of the L [Chemistry] represents a covalent bond to the N atom of formula (I), and each 3 of the L [Chemistry] represents a covalent bond to the methylene group of the G group of formula (I).

[0020] In another embodiment, ring A and ring B of formula (L-a) are each independently [Chemistry] That is the case.

[0021] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lai), [ka] or its stereoisomer, During the ceremony, Ring A is C 4~6 It is a cycloalkylene, L 1a C 3~5 It is a linear alkylene, and one or two methylene units are -O- or -NR a - is replaced, Each R a These are independently hydrogen or C 1~3 It is alkyl, L 2a These are -O-, -NHC(O)-, or -CH2-O-, L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0022] In another embodiment, the ring A of formula (Lai) is [ka] That is the case.

[0023] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (La-ii), [ka] or its stereoisomer, During the ceremony, L 1a C 3~5 It is a linear alkylene, and one or two methylene units are -O- or -NR a - is replaced, Each R a These are independently hydrogen or C 1~3 It is alkyl, L 2a These are -O-, -NHC(O)-, or -CH2-O-, p is either 1 or 2. m is either 1 or 2. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0024] In another embodiment, L of formula (La), (Lai), or (La-ii) 1a teeth, [ka] Selected from, During the ceremony, j is 1, 2, 3, or 4. k is 0, 1, 2, or 3. The sum of j and k is 2, 3, or 4. q is either 1 or 2. r is either 1 or 2. s is either 0 or 1, The sum of q, r, and s is 2 or 3. X 1 and X 2 These are independently -O- or NR a And, Each R a These are independently hydrogen or C 1~3 It is alkyl, [ka] This represents a covalent bond to the C(O) group in formula (La), (Lai), or (La-ii), [ka] This represents a covalent bond to ring B in formula (La) or a covalent bond to the cyclohexylene group in formula (Lai) or (La-ii).

[0025] In another embodiment, L of formula (La), (Lai), or (La-ii) 1a is selected from -(CH2)2O-, -(CH2)3O-, -(CH2)4O-, -(CH2)2OCH2-, -(CH2)3OCH2-, -(CH2)2O(CH2)2-, -CH2OCH2-, -CH2O(CH2)2-, -CH2O(CH2)3-, -CH2OCH2O-, or -CH2OCH2OCH2-. In another embodiment, L of formula (La), (Lai), or (La-ii)1a L is selected from -(CH2)2O-, -(CH2)3O-, -(CH2)2OCH2-, or -(CH2)3OCH2-. In another embodiment, L of formula (La), (Lai), or (La-ii) 1a is -(CH2)2NR a -, -(CH2)3NR a -, -(CH2)4NR a -, -(CH2)2NR a CH2-, -(CH2)3NR a CH2-, -(CH2)2NR a (CH2)2-, -CH2NR a CH2-, -CH2NR a (CH2)2-, -CH2NR a (CH2)3-, -CH2NR a CH2NR a -, or -CH2NR a CH2NR a Selected from CH2-, each R a These are independently hydrogen or C 1~3 It is alkyl. In another embodiment, L of formula (La), (Lai), or (La-ii) 1a is -(CH2)2NR a -, -(CH2)3NR a -, -(CH2)2NR a CH2-, or -(CH2)3NR a Selected from CH2-, R a is hydrogen or C 1~3 It is alkyl. In another embodiment, L of formula (La), (Lai), or (La-ii) 1a is selected from -(CH2)2NH-, -(CH2)3NH-, -(CH2)4NH-, -(CH2)2NHCH2-, -(CH2)3NHCH2-, -(CH2)2NH(CH2)2-, -CH2NHCH2-, -CH2NH(CH2)2-, -CH2NH(CH2)3-, -CH2NHCH2NH-, or -CH2NHCH2NHCH2-. In another embodiment, L of formula (La), (Lai), or (La-ii) 1aL is selected from -(CH2)2NH-, -(CH2)3NH-, -(CH2)2NHCH2-, or -(CH2)3NHCH2-. In another embodiment, L of formula (La), (Lai), or (La-ii) 1a is -CH2OCH2NR a -, -CH2NR a CH2O-, -CH2OCH2NR a CH2-, -CH2NR a Selected from CH2OCH2-, R a These are independently hydrogen or C 1~3 It is alkyl. In another embodiment, L of formula (La), (Lai), or (La-ii) 1a The result is selected from -CH2OCH2NH-, -CH2NHCH2O-, -CH2OCH2NHCH2-, and -CH2NHCH2OCH2-.

[0026] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (La-iii), [ka] or its stereoisomer, During the ceremony, p is either 1 or 2. m is either 1 or 2. n is 1, 2, or 3. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I).1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0027] In another embodiment, L 1 , L 2 or L 3 teeth, [ka] [ka] [ka] It is a divalent linker of formula (La) selected from the group consisting of the following.

[0028] In another embodiment, L 1 , L 2 or L 3This is the divalent linker of formula (Lb), [ka] or its stereoisomer, During the ceremony, Ring A is C 4~6 Cycloalkylene or C 7~9 It is a cross-linked bicyclic cycloalkylene, L 1b These are -CH2-NH-C(O)-, -NHC(O)-, or -C(O)NH-, L 2b is C 6-12 It is a linear alkylene, and one, two, three, or four methylene units are -O-, -NR 1b -, -C(O)NR 1b -, or -NR 1b It is replaced by C(O)-, or L 2b teeth [ka] And n is 1, 2, 3, or 4, [ka] is L 1b This represents a covalent bond to, Each R 1b These are independently hydrogen or C 1~3 It is alkyl, L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I).1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0029] In another embodiment, ring A of formula (Lb) is [ka] That is the case.

[0030] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lbi), [ka] or its stereoisomer, During the ceremony, L 1bThese are -CH2-NH-C(O)-, -NHC(O)-, or -C(O)NH-, L 2b is C 6-12 It is a linear alkylene, and one, two, three, or four methylene units are -O-, -NR 1b -, -C(O)NR 1b -, or -NR 1b It is replaced by C(O)-, or L 2b teeth [ka] And n is 1, 2, 3, or 4, [ka] is L 1b This represents a covalent bond to, Each R 1b These are independently hydrogen or C 1~3 It is alkyl, p is either 1 or 2. m is either 1 or 2. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I).2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0031] In another embodiment, L in formula (Lb) or (Lbi) 2b teeth, [ka] Selected from, During the ceremony, j is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The sum of j and k is 5, 6, 7, 8, 9, 10, or 11. q is 1, 2, 3, 4, 5, 6, 7, 8, or 9. r is 1, 2, 3, 4, 5, 6, 7, 8, or 9. s is 0, 1, 2, 3, 4, 5, 6, 7, or 8. The sum of q, r, and s is 4, 5, 6, 7, 8, 9, or 10. t is 1, 2, 3, 4, 5, 6, or 7. u is 1, 2, 3, 4, 5, 6, or 7. v is 1, 2, 3, 4, 5, 6, or 7. w is 0, 1, 2, 3, 4, 5, or 6. The sum of t, u, v, and w is 3, 4, 5, 6, 7, 8, or 9. a is 1, 2, 3, 4, or 5. b is 1, 2, 3, 4, or 5. c is 1, 2, 3, 4, or 5. d is 1, 2, 3, 4, or 5. e is 0, 1, 2, 3, or 4. The sum of a, b, c, d, and e is 4, 5, 6, 7, or 8. X 1 , X 2 , X 3 , and X 4 These are independent of -O- and -NR 1b -, -C(O)NR 1b -, or -NR 1b C(O)-, Each R 1b These are independently hydrogen or C 1~3 It is alkyl, [ka] L is the L of equation (Lb) or (Lbi). 1b This represents a covalent bond to L 1 Base, L 2 base, or L 3 base [ka] These are G in equation (I), respectively. 1 Base, G of equation (I) 2 This represents a group, or the covalent bond between the G group of formula (I) and the methylene group.

[0032] In another embodiment, L 1 , L 2 or L 3 teeth, [ka] [ka] [ka] It is a divalent linker of formula (Lb) selected from the group consisting of the following.

[0033] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lc). [ka] or its stereoisomer, During the ceremony, L 1c is C 2~10 It is a linear alkylene, and one, two, or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-. Ring A is C 4~6 Cycloalkylene or C 7~9 It is a cross-linked bicyclic cycloalkylene, L 2c is -O- or saturated C 2~10 It is a linear alkylene, and one, two, or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0034] In another embodiment, ring A of formula (Lc) is [ka] That is the case.

[0035] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lci). [ka] or its stereoisomer, During the ceremony, L 1c is C 2~10It is a linear alkylene, and one, two, or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-. L 2c is -O- or saturated C 2~10 It is a linear alkylene, and one, two, or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-. p is either 1 or 2. m is either 1 or 2. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0036] In another embodiment, L in formula (Lc) or (Lci) 1c teeth, [ka] Selected from, During the ceremony, j is 1, 2, 3, 4, 5, 6, 7, 8, or 9. k is 0, 1, 2, 3, 4, 5, 6, 7, or 8. The sum of j and k is 1, 2, 3, 4, 5, 6, 7, 8, or 9. q is 1, 2, 3, 4, 5, 6, or 7. r is 1, 2, 3, 4, 5, 6, or 7. s is 0, 1, 2, 3, 4, 5, or 6. The sum of q, r, and s is 2, 3, 4, 5, 6, 7, or 8. t is 1, 2, 3, 4, or 5. u is 1, 2, 3, 4, or 5. v is 1, 2, 3, 4, or 5. w is 0, 1, 2, 3, or 4. The sum of t, u, v, and w is 3, 4, 5, 6, or 7. X 1 , X 2 and X 3 These are independently -O-, -NH-, -NHC(O)-, or -C(O)NH-, [ka] This represents a covalent bond to the C(O) group in formula (Lc) or (Lci), [ka] represents a covalent bond of equation (Lc) or (Lci) to a ring.

[0037] In another embodiment, L in formula (Lc) or (Lci) 2c teeth, [ka] Selected from, During the ceremony, j is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. k is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. The sum of j and k is 1, 2, 3, 4, 5, 6, 7, 8, or 9. q is 0, 2, 3, 4, 5, 6, or 7. r is 1, 2, 3, 4, 5, 6, 7, or 8. s is 0, 1, 2, 3, 4, 5, 6, or 7. The sum of q, r, and s is 1, 2, 3, 4, 5, 6, 7, or 8. t is 0, 1, 2, 3, 4, or 5. u is 1, 2, 3, 4, 5, or 6. v is 1, 2, 3, 4, 5, or 6. w is 0, 1, 2, 3, 4, or 5. The sum of t, u, v, and w is 2, 3, 4, 5, 6, or 7. X 1 , X 2 and X 3 These are independently -O-, -NH-, -NHC(O)-, or -C(O)NH-, [ka] represents a covalent bond of formula (Lc) or (Lci) to a ring, L 1 Base, L 2 base, or L 3 base [ka] These are G in equation (I), respectively. 1 Base, G of equation (I) 2 This represents a group, or the covalent bond between the G group of formula (I) and the methylene group.

[0038] In another embodiment, L 1 , L 2 or L 3 teeth, [ka] [ka] It is a divalent linker of formula (Lc) selected from the group consisting of the following.

[0039] In another embodiment, L 1 , L 2 or L 3 is a divalent linker of formula (Ld), [ka] During the ceremony, L 1d is C 12-31 In linear alkylenes, one, two, three, four, five, six, seven, eight, nine, ten, or eleven methylene units are replaced by -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I).1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0040] In another embodiment, L 1d C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , C 30 , or C 31In a linear alkylene, one, two, three, four, five, six, seven, eight, nine, ten, or eleven methylene units are replaced by -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-. In another embodiment, L 1d C 12-22 Linear alkylenes, for example, C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , or C 22 These are linear alkylenes, and one, two, three, four, or five methylene units can be replaced by -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-.

[0041] In another embodiment, L of formula (Ld) 1d teeth, [ka] Selected from, During the ceremony, j is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The sum of j and k is 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21. q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. The sum of q, r, and s is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. t is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. u is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. v is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. w is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. The sum of t, u, v, and w is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. b is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. c is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. d is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. The sum of a, b, c, d, and e is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. f is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. g is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. h is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. The sum of f, g, h, i, y, and z is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. X 1 , X 2 , X 3 , X 4 , and X 5 These are independently -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-, [ka] This represents the covalent bond to the C(O) group in formula (Ld), and L 1 Base, L 2 base, or L 3 base [ka] These are G in equation (I), which represents the covalent bond to each other. 1 Base, G of equation (I) 2 It represents a group, or the methylene group of the G group in formula (I).

[0042] In another embodiment, L of formula (Ld) 1d teeth [ka] And n is 4, 5, 6, 7, 8, 9, or 10. [ka] This represents the covalent bond to the C(O) group in formula (Ld), and L 1 Base, L 2 base, or L 3 base [ka] These are G in equation (I), which represents the covalent bond to each other. 1 Base, G of equation (I) 2 It represents a group, or the methylene group of the G group in formula (I).

[0043] In another embodiment, L 1 , L 2 or L 3 teeth, [ka] [ka] It is a divalent linker of formula (Ld) selected from the group consisting of the following.

[0044] In another embodiment, L 1 , L 2 or L 3 is a divalent linker of formula (Le), [ka] During the ceremony, n is an integer between 3 and 50. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I)2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0045] In another embodiment, n in formula (Le) is 3-25, 3-10, 3-8, 3-7, 3-5, or 3-4. In another embodiment, n in formula (Le) is 5-22, 7-15, or 9-13. In another embodiment, n in formula (Le) is 3, 4, 5, 7, 8, 11, 22, or 50.

[0046] In another embodiment, n in formula (Le) is 12-50, 15-30, 17-25, 18-24, 18-20, 20-22, or 22-24. In another embodiment, n in formula (Le) is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and / or 50. In another embodiment, n in formula (Le) is 19 or 23.

[0047] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lf). [ka] or its stereoisomer, During the ceremony, L 1f is a bond, C 1~6 It is a linear alkylene, and the 0, 1, or 2 methylene units are -O-, -NH-, or -C(O)-, or -(C 3~6 Replaced with cycloalkylene)-NHC(O)- L 2f These are bonds, -NHC(O)-, -C(O)NH-, or C 1~6 It is a linear alkylene, and zero, one, or two methylene units are replaced by -O-. Z 1 and Z 2 Each of them is independently N or CH, L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I). In another embodiment, L of equation (Lf) 1f teeth, [ka] Selected from, During the ceremony, j is 1, 2, 3, 4, or 5. k is 0, 1, 2, 3, or 4. The sum of j and k is 1, 2, 3, 4, or 5. q is 1, 2, or 3. r is 1, 2, or 3. s is 0, 1, 2, The sum of q, r, and s is 2, 3, or 4. X 1 and X 2 These are independently -O-, -NH-, or -C(O)-, or -(C 3~6 It is cycloalkylene)-NHC(O)-, [ka] This represents the covalent bond to the C(O) group in formula (Lf), [ka] This represents the covalent bond of equation (Lf) to the ring.

[0048] In another embodiment, L of equation (Lf) 2f teeth, [ka] Selected from, During the ceremony, j is 1, 2, 3, 4, or 5. k is 0, 1, 2, 3, or 4. The sum of j and k is 1, 2, 3, 4, or 5. q is 1, 2, or 3. r is 1, 2, or 3. s is 0, 1, 2, The sum of q, r, and s is 2, 3, or 4. [ka] This represents the covalent bond of equation (Lf) to the ring, L 1 Base, L 2 base, or L 3 base [ka] These are G in equation (I), respectively. 1 Base, G of equation (I) 2 This represents a group, or the covalent bond between the G group of formula (I) and the methylene group.

[0049] In another embodiment, L 1 , L 2 or L 3 teeth, [ka] It is a divalent linker of formula (Lf) selected from the group consisting of the following.

[0050] In another embodiment, L 1 , L 2 or L 3 is a divalent linker of formula (Lg), [ka] During the ceremony, Ring A is a 5-6 membered heteroarylene having one or two nitrogen ring atoms. L 1g These are bonds, -CH2-, -NH-, or -O-, L 2g teeth [ka] And n is 1, 2, 3, 4, or 5. [ka] is L 1g This represents a covalent bond to, L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I). In another embodiment, L 1 , L 2 or L 3 is a divalent linker of formula (Lgi), [ka] During the ceremony, L 1g These are bonds, -CH2-, -NH-, or -O-, L 2g teeth [ka] And n is 1, 2, 3, 4, or 5. [ka] is L 1g This represents a covalent bond to, Z 1 , Z 2 , and Z 3 Each is independently selected from N or CH, except Z 1 , Z 2 , and Z 3 One or two of them is N, L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0051] In another embodiment, L 1 , L 2 or L 3 teeth, [ka] It is a divalent linker of formula (Lg) selected from the group consisting of the following.

[0052] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lh). [ka] or its stereoisomer, During the ceremony, each Z 1 These are independently N or CH, L 1h These are bonds, -C(O)-, -C(O)-NH-, or -NHC(O)-, L 2h is C 2~10 Linear alkylene or [ka] And n is 1, 2, 3, or 4, [ka] is L 1h This represents a covalent bond to, [ka] is L 3h This represents a covalent bond to, L 3h The bond is -C(O)CH2-, -O-(C 3~6 It is cycloalkylene)-O- or -C(O)NH(CH2)3OCH2-, L 4h These are bonds, -C(O)-, -CH2C(O)-, or -C(O)CH2-, m is 1, 2, or 3. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0053] In another embodiment, L 1 , L 2 or L 3 teeth, [ka] [ka] It is a divalent linker of formula (Lh) selected from the group consisting of the following.

[0054] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Li). [ka] During the ceremony, L 1i is a bond, C 1~12 Linear alkylene, or [ka] And n is 1, 2, 3, 4, or 5. [ka] is L 3i This represents a covalent bond to, [ka] This represents a covalent bond to NH, L 2i is a bond, C 1~12 Linear alkylene, or [ka] And n is 1, 2, 3, 4, or 5. [ka] This represents a covalent bond to HN, L 3i is a bond or -C(O)-, L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0055] In another embodiment, L 1 , L 2 or L 3 teeth, [ka] It is a divalent linker of formula (Li) selected from the group consisting of the following.

[0056] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lj). [ka] or its stereoisomer, During the ceremony, Z 1 is C, CH, or N, Z 2 , Z 3 , Z 4 and Z 5 Independently, CH or N, however Z 2 , Z 3 , Z 4 and Z 5 Two or fewer of these are N, L 1j These are -NH-, -C(O)NH-, -NHC(O)-, or -O-. L 2j C 1~6 Linear alkylene or [ka] And n is either 1 or 2, [ka] is L 1j This represents a covalent bond to, [ka] This represents a single bond or a double bond. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0057] In another embodiment, L 1 , L 2 or L 3 teeth, [ka] It is a divalent linker of formula (Lj) selected from the group consisting of the following.

[0058] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lk). [ka] or its stereoisomer, During the ceremony, Ring A is a phenyl or 5- or 6-membered heteroarylene having one or two nitrogen ring atoms. Z 1 and Z 2Each of them is independently either CH or N, L 1k These are bonds, -C(O)-, -C(O)NH-, or -NHC(O)-, L 2k C 3~8 Linear alkylene or [ka] And n is 1, 2, or 3, [ka] is L 1k This represents a covalent bond to, L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0059] In another embodiment, L 1 , L 2 or L 3 teeth, [ka] It is a divalent linker of formula (Lk) selected from the group consisting of the following.

[0060] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lm). [ka] or its stereoisomer, During the ceremony, Z 1 is CH or N, m is either 1 or 2. p is either 1 or 2. [ka] The 0, 1, or 2 hydrogen atoms are replaced by F. L 1m These are bonds, -C(O)-, -C(O)NH-, -NHC(O)-, -S(O)2NH-, or -NHS(O)2-. L 2m C 3~6 Linear alkylene, C 3~6 Cycloalkylene, or [ka] And n is either 1 or 2, [ka] is L 1m This represents a covalent bond to, L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0061] In another embodiment, L 1 , L 2 or L 3 teeth, [ka] It is a divalent linker of formula (Lm) selected from the group consisting of the following.

[0062] In another embodiment, L 1 , L 2 or L 3 is a divalent linker of formula (Lni), [ka] L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I)2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I). In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Ln-ii), [ka] L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I).2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I). In another embodiment, L 1 , L 2 or L 3 is a divalent linker of formula (Ln-iii), [ka] L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0063] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Ln-iv), [ka] L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I).1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0064] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lp), [ka] or its stereoisomer, where y is an integer from 1 to 9. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0065] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lq). [ka] or its stereoisomer, During the ceremony, Rings A, B, C, and D are each independent of each other. C 4~6It is a cycloalkylene, L 1a , L 3a , and L 4a Each of them is independent of C 3~5 It is a linear alkylene, with one or two methylene units, and is -O- or -NR a - is replaced, Each R a These are independently hydrogen or C 1~3 It is alkyl, L 2a These are -O-, -NHC(O)-, or -CH2-O-, L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0066] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lqi). [ka] or its stereoisomer, During the ceremony, L 1a , L 3a , and L 4a Each of them is independent of C 3~5 It is a linear alkylene, with one or two methylene units, and is -O- or -NR a - is replaced, Each R a These are independently hydrogen or C 1~3 It is alkyl, L 2a These are -O-, -NHC(O)-, or -CH2-O-, L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0067] In another embodiment, L 1 , L 2 or L 3 This is the divalent linker of formula (Lq-ii). [ka] or its stereoisomer, During the ceremony, p is 1, 2, or 3. m is 1, 2, or 3. n is 1, 2, or 3. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0068] In another embodiment, L 1 , L 2 or L 3 This is a divalent linker of formula (Lq) having the following structure: [ka] In another embodiment, L 1 , L 2 or L 3 is a divalent linker of formula (Lr), [ka] During the ceremony, n is an integer between 10 and 30. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0069] In another embodiment, n in formula (Lr) is 10-20, 10-18, 12-16, or 13-15. In another embodiment, n in formula (Lr) is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In another embodiment, n in formula (Lr) is 14.

[0070] In another embodiment, L 1 , L 2 or L 3 is a divalent linker of formula (Ls), [ka] During the ceremony, n is an integer between 10 and 30. L 1 Each of the bases [ka] represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). 1 Represents a covalent bond to the base, or L' and Y 1 If both are conjugates, then T in equation (I) 1 Represents a covalent bond to the base, L 1 Each of the bases [ka] This is G in equation (I). 1 Represents a covalent bond to a base, L 2 Each of the bases [ka] represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). 2 Represents a covalent bond to the base, or L'' and Y 2 If both are conjugates, then T in equation (I) 2 Represents a covalent bond to the base, L 2 Each of the bases [ka] This is G in equation (I). 2 Represents a covalent bond to a base, L 3 Each of the bases [ka] This represents the covalent bond to the N atom in formula (I), and L 3 Each of the bases [ka] This represents the covalent bond between the G group and the methylene group in formula (I).

[0071] In another embodiment, n in formula (Ls) is 10-20, 10-18, 12-16, or 13-15. In another embodiment, n in formula (Ls) is 15-30, 17-28, 18-26, 19-25, 20-24, or 21-23. In another embodiment, n in formula (Ls) is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In another embodiment, n in formula (Ls) is 14 or 22.

[0072] In one embodiment of this disclosure, Y 1 and Y 2 Each is independent of the bond, -NH-, and -(C 1~12 Selected from alkylenes, one, two, or three methylene units are -O-, -NH-, -N(CH3)-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C 3~6 Cycloalkylene)-,-(C 3~6Cycloalkenylene)-, 3-10 member heterocycloalkylene, arylene, or heteroarylene, or -(C 2~12 Alkenylene)- is replaced, and one, two, or three methylene units are -O-, -NH-, -N(CH3)-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C 3~6 Cycloalkylene)-,-(C 3~6 It can be replaced with cycloalkenylene, 3- to 10-membered heterocycloalkylene, arylene, or heteroarylene.

[0073] In another embodiment, Y 1 and Y 2 These are, independently, bonded, -NH-, -(C 1~6 Alkylene)-O-, -O-(C 1~6 Alkilen)-,-(C 2~6 Alkenylene)-O-,-(C 1~6 Alkylene)-C(O)-,-(C 2~6 Alkenylene)-C(O)-, Phenylene, Piperidinylene, Hydroxypiperidinylene, Fluoropiperidinylene, Azetidinylene, -C(O)-Piperazinerene-, -(C 1~6 Alkylene)-oxopiperazinylen-, pyrrolidinylene, 7-9 membered bridged bicyclic heterocycloalkylene, -(C 1~6 Alkylene)-O-phenylene-,-(C 2~6 Alkenylene)-O-Piperidylene, -(C 1~5 Selected from alkylene)-NH-, with 0, 1, or 2 methylene units, -O-, -NH-(C 1~5 Alkylene)-NH-,-N(CH3)-(C 1~5 Alkylene)-NH-,-NH-(C 1~5 Alkylene)-N(CH3)-, -N(CH3)-(C 1~5 Alkylene)-N(CH3)-,-(C 3~6 Cycloalkylene)-NH-,-C(O)NH-(C 1~5 Alkylene)-NH-,-C(O)NH-(C 3~6 Cycloalkylene)-NH-,-(C 1~5 Alkylene)-O-(C 3~6-NH-(cycloalkylene); -(C 3~6 -NH-(cycloalkenylene), or

Chem.

[0074] In another embodiment, Y 1 and Y 2 are each independently

Chem.

[0075] In another embodiment, Y 1 or Y 2 is a bond. In another embodiment, Y 1 or Y 2 is NH. In another embodiment, Y 1 or Y 2 is

Chem.

[0076] In another embodiment, R 1 is methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, or t-butyl. In another embodiment, R 1 is methyl. In another embodiment, R 1 is ethyl. In another embodiment, R 1 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0077] In another embodiment, y of L’ or L’’ is independently 2 - 8, 3 - 7, 4 - 7, or 5 - 7. In another embodiment, y of L’ or L’’ is independently 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[0078] In another embodiment, w of L’ or L’’ is independently 0 - 4, 0 - 3, 0 - 2, or 1 - 2. In another embodiment, w of L’ or L’’ is independently 0, 1, 2, 3, 4, or 5.

[0079] In another embodiment, L’ and L’’ are each independently a bond or

Chemical formula

[0080] In another embodiment, L’ and L’’ are each independently a bond or

Chemical formula

[0081] In another embodiment, L’ and L’’ are each independently

Chemical formula

[0082] In another embodiment, L’ and L’’ are each independently a bond.

[0083] In another embodiment, T 1 or T 2 is

Chemical formula

[0084] In another embodiment, T 1 or T 2 teeth, [ka] That is the case.

[0085] In another embodiment, T 1 or T 2 teeth, [ka] That is the case.

[0086] In another embodiment, T 1 or T 2 teeth, [ka] That is the case.

[0087] In another embodiment, T 1 or T 2 teeth, [ka] That is the case.

[0088] In another embodiment, T 1 or T 2 teeth, [ka] That is the case.

[0089] In another embodiment, T 1 or T 2 teeth, [ka] And the R of formulas G1, G2, G3, and G4 2 and R 3 Each of these is independently either F or H.

[0090] In another embodiment, T 1 or T 2 teeth, [ka] And R of formula H 2 is hydrogen or C 1~4 It is alkyl, and R of formula H 3 is hydrogen or C 1~4 It is alkyl. In another embodiment, R of formula H 2 and R 3 Each is independently hydrogen, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, or t-butyl. In another embodiment, R of formula H 2 It is isopropyl, and the R of formula H 3 is methyl. In another embodiment, R of formula H 2 t-butyl is the R of formula H 3 It is hydrogen.

[0091] In another embodiment, T 1 or T 2 teeth, [ka] And Q is C 1~5It is an alkylene, with 0, 1, or 2 methylene units replaced by -O-, and Ar is an optionally substituted 5- to 10-membered aromatic ring or a 9- or 10-membered unsaturated condensed diring.

[0092] In another embodiment, Q is -CH2-, -CH(CH3)-, -CH2CH2CH2-, -CH(CH2CH3)-, or -CH2CH2O-. In another embodiment, Q is -CH2- or -CH(CH3)-. In another embodiment, Q is -CH(CH3)-.

[0093] In another embodiment, Ar is an optionally substituted 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, or 10-membered aromatic ring. In another embodiment, Ar is an optionally substituted 6-membered aromatic ring. In another embodiment, Ar is an optionally substituted 9-membered aromatic ring. In another embodiment, Ar is an optionally substituted 9-membered or 10-membered unsaturated condensed biring. In another embodiment, Ar is an optionally substituted 9-membered unsaturated condensed biring.

[0094] In another embodiment, Ar is phenyl, pyridinyl, indolyl, indolinyl, dihydrobenzofuranyl, or benzofuranyl, and each Ar is substituted with 0, 1, or 2 substituents. In another embodiment, Ar is phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl, 2-dihydrobenzofuranyl, 3-dihydrobenzofuranyl, 4-dihydrobenzofuranyl, 5-dihydrobenzofuranyl, 6-dihydrobenzofuranyl, 7-dihydrobenzofuranyl, 2-benzofuranyl, 3-benzofuranyl, 4-benzofuranyl, 5-benzofuranyl, 6-benzofuranyl, or 7-benzofuranyl, and each Ar is substituted with 0, 1, or 2 substituents.

[0095] In another embodiment, the Ar substituent is independently C 1~3 Alkyl, C 1~3Alkoxy, C 1~3 Haloalkyl, C 1~3 The Ar substituent is selected from haloalkoxy or halo. In another embodiment, the Ar substituent is independently selected from methyl, ethyl, methoxy, ethoxy, bromo, chloro, or trifluoromethyl.

[0096] In another embodiment, Ar [ka] That is the case.

[0097] In another embodiment, compounds of formula (I) are selected from the compounds listed in Table 1: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0098] definition As used herein and in the claims, the singular forms "a" and "the" include the plural forms unless the context clearly indicates otherwise.

[0099] As used herein and in the claims, the term “including” encompasses both “including” and “consisting of,” for example, a composition “including” X may consist of X alone or it may include something additional, such as X + Y.

[0100] The term "essentially consisting of" limits the scope of the feature to the specified material or step, and those that do not substantially affect the fundamental properties of the requested feature.

[0101] The term "consists of" excludes the existence of additional components (or multiple components).

[0102] The term "pathogenic cells" includes a subset of cells that cause or are likely to cause disease. Examples of pathogenic cells include, but are not limited to, pathogenic immune cells, cancer cells or tumor cells, and stromal cells. Pathogenic cells can also be pathogens that can cause infection, such as viruses and bacterial cells.

[0103] The term "pathogenic immune cells" includes specific subsets of immune cells that cause or are likely to cause disease. These cell subsets are either commensal cells or recruited to specific locations to secrete cytokines, chemokines, and other mediators that contribute to the persistence and progression of disease, such as cancer in the tumor microenvironment or chronic inflammation of the lungs in asthma. Examples of pathogenic immune cells include myeloid suppressor cells (MDSCs), regulatory T cells (Tregs), neutrophils, macrophages, regulatory B cells (Bregs), CD8 regulatory cells (CD8regs), and exhausted T cells.

[0104] The term "pharmaceutical composition" refers to a combination of the compound of the present invention with a medium commonly accepted in the art for the delivery of a biologically active compound to a mammal, such as a human. Such a medium may include any pharmaceutically acceptable carrier, diluent, or excipient.

[0105] The terms “effective dose” and “therapeutic dose” refer to the amount of a compound, antibody, or its antigen-binding portion according to the present invention that, when administered to a patient in need, is sufficient to treat a disease condition, symptom, or disorder for which the compound is useful. Such a dose is sufficient to elicit the desired biological or medical response from the tissue system or patient as desired by researchers or clinicians. The amount of the compound of the present invention constituting a therapeutic dose varies depending on factors such as the compound and its biological activity, the composition used for administration, the time of administration, the route of administration, the rate of excretion of the compound, the duration of treatment, the type and severity of the disease condition or disorder being treated, any agents used in combination with or concurrently with the compound of the present invention, and the patient’s age, weight, overall health, sex, and diet. Such a therapeutic dose can be routinely determined by those skilled in the art, taking into account their knowledge, the latest technology, and the present disclosure.

[0106] The term "alkyl" refers to a saturated, straight-chain or branched hydrocarbon moiety having a specified number of carbon atoms. 1-3 The term "alkyl" refers to an unsubstituted alkyl moiety containing one, two, or three carbon atoms. Exemplary alkyls include methyl, ethyl, and propyl.

[0107] The term "alkylene" refers to a saturated straight-chain or branched hydrocarbon moiety having a specified number of carbon atoms and two bonding sites. The two bonding sites may arise from the same carbon atom or from different carbon atoms. 1-3 The term "alkylene" refers to an unsubstituted alkylene moiety containing one, two, or three carbon atoms and having two bonding sites. Exemplary C 1-3 Examples of alkylene groups include methylene, ethylene, and propylene.

[0108] The term "alkenyl" refers to an unsaturated, straight-chain or branched hydrocarbon moiety having a specified number of carbon atoms. 2-6The term "alkenyl" refers to an unsubstituted alkenyl moiety containing two, three, four, five, or six carbon atoms. Exemplary alkenyls include propenyl, butenyl, pentenyl, and hexenyl.

[0109] The term "alkenylene" refers to an unsaturated, straight-chain or branched hydrocarbon moiety having a specified number of carbon atoms and two bonding sites. The two bonding sites may arise from the same carbon atom or from different carbon atoms. 2-6 The term "alkenylene" refers to an unsubstituted alkenylene moiety containing two, three, four, five, or six carbon atoms and having two bonding sites. Exemplary C 2-6 Examples of alkenylene groups include propenylene, butenylene, pentenylene, and hexenylene.

[0110] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon moiety having a specified number of carbon atoms. 3-6 The term "cycloalkyl" refers to an unsubstituted cycloalkyl moiety containing three, four, five, or six carbon atoms, and exemplary cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0111] The term "cycloalkylene" refers to a saturated cyclic hydrocarbon moiety having a specified number of carbon atoms and two bonding sites. The two bonding sites can arise from the same carbon atom or from different carbon atoms. 4-6 The term "cycloalkylene" refers to an unsubstituted cycloalkylene moiety containing four, five, or six carbon atoms and having two bonding sites. Examples of cycloalkylene groups include cyclobutane-1,3-diyl, cyclopentane-1,3-diyl, cyclohexane-1,3-diyl, or cyclohexane-1,4-diyl.

[0112] The term "cycloalkenylene" refers to an unsaturated cyclic hydrocarbon moiety having a specified number of carbon atoms and two bonding sites. The two bonding sites can arise from the same carbon atom or from different carbon atoms. 3-6 The term "cycloalkenylene" refers to an unsubstituted cycloalkenylene moiety containing three, four, five, or six carbon atoms and having two bonding sites.

[0113] The term "heterocycloalkylene" refers to a saturated cyclic hydrocarbon moiety having two bonding sites, comprising one or two heteroatoms independently selected from oxygen, sulfur, or nitrogen atoms. The two bonding sites may arise from the same carbon atom or from different carbon atoms. The term "3-6 membered heterocycloalkylene" refers to a 3-6 membered saturated cyclic moiety having two, three, four, or five carbon atoms in addition to one or two oxygen, sulfur, or nitrogen atoms, and having two bonding sites. Preferably, the 3-6 membered heterocycloalkylene group comprises one oxygen or nitrogen atom. Preferably, such a group comprises three carbon atoms and one oxygen or nitrogen atom, such as azetidinediyl or oxetanediyl. Preferably, such a group comprises four or five carbon atoms and one oxygen or nitrogen atom, such as tetrahydrofranziyl, tetrahydropyrandiyl, pyrrolidinediyl, or piperidinediyl.

[0114] The term "bridged bicyclic cycloalkylene" refers to a saturated bicyclic hydrocarbon moiety having at least one bridge and two bonding sites. A "bridge" is an unbranched chain of multiple atoms, or a single atom, or a valence bond, connecting two bridgeheads, where a "bridgehead" is any skeletal atom in a ring system bonded to three or more skeletal atoms (excluding hydrogen). The two bonding sites may arise from the same carbon atom or from different carbon atoms. 7-9 The term "bridged bicyclic cycloalkylene" refers to an unsubstituted bridged bicyclic cycloalkylene moiety containing seven, eight, or nine carbon atoms and having two bonding sites.

[0115] The term "arylene" refers to a monocyclic or bicyclic ring system in which at least one ring is aromatic and has two bonding sites. Exemplary arylene groups include phenylene, biphenylene, naphthylene, and anthracilene.

[0116] The term "heteroarylene" refers to a monocyclic or bicyclic ring system in which at least one ring is aromatic and has 1 to 5 heteroatoms independently selected from oxygen, sulfur, or nitrogen atoms in addition to carbon atoms, and has 2 bonding sites. The term "5-6 membered heteroarylene" refers to a 5-6 membered cyclic aromatic moiety that has 2, 3, 4, or 5 carbon atoms in addition to 1, 2, or 3 heteroatoms independently selected from oxygen, sulfur, or nitrogen atoms, and has 2 bonding sites.

[0117] A skilled technician will understand that salts of the compound of formula (I), including pharmaceutically acceptable salts, can be prepared. In fact, in certain embodiments of the present invention, salts of the compound of formula (I), including pharmaceutically acceptable salts, may be preferred over the respective free or unsalted compounds. Therefore, the present invention further relates to salts of the compound of formula (I), including pharmaceutically acceptable salts. The present invention further relates to the free or unsalted compounds of the compound of formula (I).

[0118] Salts of the compounds of the present invention (including pharmaceutically acceptable salts) can be readily prepared by those skilled in the art.

[0119] Representative pharmaceutically acceptable acid addition salts include 4-acetamidebenzoate, acetate, adipine, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, tartrate, butyrate, calcium edetate, camphorate, camphor sulfonate (camsylate), caprine (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, and disuccinate. , dodecyl sulfate (Eslate), edetate (ethylenediaminetetraacetate), eslate (lauryl sulfate), ethane-1,2-disulfonate (edisylate), ethanesulfonate (esylate), formate, fumarate, galactarate (mucinate), gentisinate (2,5-dihydroxybenzoate), glucoheptonic acid (gluceptate), gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glycerophosphate, glycolic acid, hexylresorcinic acid, hippuric acid, hydravamin (N,N'-di(dehydro (Bethyl) ethylenediamine, hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoic acid, isobutyric acid, lactate, lactobionic acid, laurate, malic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid (mesylate), methylsulfate, mucinic acid, naphthalene-1,5-disulfonic acid (napadisylate), naphthalene-2-sulfonic acid (napsylate), nicotinic acid, nitrate, oleic acid, palmitic acid, p-aminobenzenesulfonic acid, p-aminosalitic acid, pamoic acid (embonate), pantothenic acid, pectin Examples include, but are not limited to, nitrates, persulfate phenylacetate, phenylethyl barbiturates, phosphates, polygalacturonic acid, propionates, p-toluenesulfonates (tosylates), pyroglutamates, pyruvates, salicylates, sebacinates, stearates, acetates, succinates, sulfamates, sulfates, tannates, tartrates, theocrates (8-chlorotheophylline), thiocyansates, trithiozides, trifluoroacetates, undecanoates, undecylenates, and valersates.

[0120] Typical pharmaceutically acceptable base addition salts include aluminum, 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS, tromethamine), arginine, benetamine (N-benzylphenethylamine), benzathine (N,N'-dibenzylethylenediamine), b / s-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, cremisole (1-p-chlorobenzyl-2-pyrrolidine-1'-ylmethylbenzimidazole), and cyanoacrylate. Examples include, but are not limited to, chlorohexylamine, dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, dopamine, ethanolamine, ethylenediamine, L-histidine, iron, isoquinoline, lepidine, lithium, lysine, magnesium, meglumine (N-methylglucamine), piperazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, t-butylamine, and zinc.

[0121] Compounds of formula (I) may contain one or more chiral centers and therefore may exist as individual enantiomers, diastereomers, other stereoisomers, or mixtures thereof. Chiral centers, such as chiral carbon atoms, may be present in substituents such as alkyl groups. Unless otherwise specified, the stereochemistry of chiral centers present in compounds of formula (I), or in any chemical structure shown herein, is intended to encompass all individual stereoisomers and all mixtures thereof. Thus, compounds of formula (I) containing one or more chiral centers may be used as racemic mixtures, enantiomer-enriched mixtures, or enantiomerically pure individual stereoisomers.

[0122] A mixture of stereoisomers whose relative configurations of all stereocenters are known can be represented using the symbol "&" and an index number (e.g., "&1"). For example, a group of two stereocenters labeled with the symbol "&1" represents a mixture of two possible stereoisomers whose two stereocenters have the relative configurations shown in the diagram.

[0123] A divalent group is a group that has two bonding points. For all divalent groups, unless otherwise specified, the orientation of the group is indicated by the direction in which the formula or structure of the group is written.

[0124] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the invention pertains. Any composition and method similar to or equivalent to those described herein may be used in the practice or testing of the methods of this disclosure, but exemplary compositions and methods are described herein. Any aspects and embodiments of this disclosure described herein may be combined. For example, the subject matter of dependent or independent claims disclosed herein may be combined in multiple ways (for example, one or more descriptions from each dependent claim may be combined into a single claim based on the independent claim to which they depend).

[0125] The scope provided herein includes all values ​​within a specific range described, as well as values ​​for endpoints within that range.

[0126] The concentrations described herein are measured at ambient temperature and pressure. This may be, for example, room temperature or the temperature and pressure at a specific portion of the process stream. Preferably, the concentration is measured under standard conditions of 25°C and 1 bar pressure.

[0127] Target and target binding portion The compounds of formula (I) disclosed herein are heterotrifunctional or bitargeted synthetic agents designed so that two of their terminals interact with one or two cell surface targets and a third terminal binds to a specific antibody. More specifically, the ARM binds to both the cell surface target and the specific antibody simultaneously. This multicomponent complex induces immune surveillance in one or two target-expressing tissues / cells, integrating the mechanism of antibody function and small molecule dosage control. This mechanism may include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cell-mediated cytotoxicity (CDC), preferably ADCC. The same Fc receptor-expressing immune cells that initiate the destruction of ARM / antibody-tagged cells also participate in the presentation of endogenous antigens for the potential of long-term cellular immunity.

[0128] The compounds of formula (I) disclosed herein include a target-binding moiety capable of binding to one or two target proteins (e.g., receptors) present on the cell surface. Those skilled in the art may select molecules known to bind to target proteins for use as target-binding moieties within the ARM.

[0129] In one embodiment, the compound of formula (I) is designed so that one end interacts with a cell surface prostate-specific membrane antigen (PSMA) target, a second end interacts with a cell surface CC chemokine receptor type 2 (CCR2) target, and a third end interacts with a specific antibody. In another embodiment, the compound of formula (I) is designed so that one end interacts with a cell surface prostate-specific membrane antigen (PSMA) target, a second end interacts with a cell surface CC chemokine receptor type 8 (CCR8) target, and a third end interacts with a specific antibody. In yet another embodiment, the compound of formula (I) is designed so that one end interacts with a cell surface CC chemokine receptor type 2 (CCR2) target, a second end interacts with a cell surface CC chemokine receptor type 8 (CCR8) target, and a third end interacts with a specific antibody. In yet another embodiment, the compound of formula (I) is designed so that two ends interact with cell surface PSMA targets and a third end interacts with a specific antibody. In another embodiment, the compound of formula (I) is designed so that two ends interact with a cell surface CCR2 target and a third end interacts with a specific antibody. In yet another embodiment, the compound of formula (I) is designed so that two ends interact with a cell surface CCR8 target and a third end interacts with a specific antibody.

[0130] In one embodiment, the compound of formula (I) includes a PSMA binding site and a CCR2 binding site. In another embodiment, the compound of formula (I) includes a PSMA binding site and a CCR8 binding site. In yet another embodiment, the compound of formula (I) includes a CCR2 binding site and a CCR8 binding site. In yet another embodiment, the compound of formula (I) includes two PSMA binding sites. In yet another embodiment, the compound of formula (I) includes two CCR2 binding sites. In yet another embodiment, the compound of formula (I) includes two CCR8 binding sites.

[0131] In one embodiment, the target of the target-binding region is a cell surface protein. In a further embodiment, the target of the target-binding region is a target protein expressed on pathogenic cells.

[0132] In further embodiments, pathogenic cells are pathogenic immune cells, tumor cells or cancer cells, or stromal cells (including stromal cells present in the tumor microenvironment).

[0133] In further embodiments, the target of the target-binding moiety is located on the surface of a pathogen selected from a viral or bacterial cell. Examples of viruses that express cell surface targets include, but are not limited to, influenza. Examples of cell surface targets of influenza viruses include, but are not limited to, neuraminidase.

[0134] In further embodiments, pathogenic immune cells include monocytes, myeloid-derived suppressor cells (MDSCs), e.g., monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs), regulatory T cells (Tregs), neutrophils (e.g., N2 neutrophils), macrophages (e.g., M2 macrophages), regulatory B cells (Bregs, memory B cells), plasma cells, CD8 cells (e.g., CD8 regulatory cells (CD8regs), memory CD8 cells, effector CD8 cells, naive CD8 T cells, TEMRAs), exhausted T cells, eosinophils, basophils, mast cells, dendritic cells, natural killer (NK) cells, innate lymphoid cells, NK T cells (NKTs), or γδ T cells.

[0135] In further embodiments, pathogenic immune cells include myeloid suppressor cells (MDSCs) such as monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs), regulatory T cells (Tregs), neutrophils (e.g., N2 neutrophils), macrophages (e.g., M2 macrophages), regulatory B cells (Bregs), CD8 regulatory cells (CD8regs), or exhausted T cells.

[0136] In a further embodiment, the tumor cells or cancer cells are solid tumor cells.

[0137] In further embodiments, tumor cells or cancer cells are lung cancer cells (e.g., non-small cell lung cancer (NSCLC) cells), hepatocellular carcinoma (HCC) cells, colorectal cancer (CRC) cells, cervical cancer cells (e.g., cervical squamous cell carcinoma (CESC) cells), head and neck cancer cells (e.g., head and neck squamous cell carcinoma (HNSC) cells), pancreatic cancer cells, prostate cancer cells (e.g., metastatic castration-resistant prostate cancer (mCRPC) cells), ovarian cancer cells, endometrial cancer cells, brain cancer cells, endocrine cancer cells, testicular cancer cells, bladder cancer cells, bone cancer cells, esophageal cancer cells, gastric cancer cells, renal cell carcinoma cells, melanoma cancer cells, thyroid cancer cells, or breast cancer cells, preferably mCRPC cells, breast cancer cells, lung cancer cells, colorectal cancer cells, or renal cell carcinoma cells.

[0138] In further embodiments, the pathogenic cells are endothelial cells associated with tumor neovascularization. In a further embodiment, the stromal cells are cancer-associated fibroblasts (CAFs).

[0139] In one embodiment, the target of the target-binding moiety is selected from G protein-coupled receptors (GPCRs), enzymes (e.g., dehydrogenases, esterases, phosphodiesterases, hydrolases, lipases, phosphatases, kinases, reductases, or transferases), transporters (e.g., ion channels), proteases, or receptors. In further embodiments, the target of the target-binding moiety is selected from GPCRs, enzymes (e.g., dehydrogenases, esterases, phosphodiesterases, hydrolases, lipases, phosphatases, kinases, reductases, or transferases), transporters (e.g., ion channels), proteases, or receptors, and the aforementioned targets are associated with and / or expressed on immune cells (including pathogenic immune cells), tumor cells or cancer cells, or stromal cells (including stromal cells present in the tumor microenvironment).

[0140] In further embodiments, the targets of the target-binding moiety include 15-hydroxyprostaglandin dehydrogenase, 5-hydroxytryptamine receptor, activated leukocyte adhesion molecule, ADAM metallopeptidase, adenosine receptor, adenosine deaminase, adrenergic receptor β, advanced glycation end product specific receptor, membrane alanylaminopeptidase, alkaline phosphatase, calcium voltage-gated channel, cannabinoid receptor, carcinoembryonic antigen-associated cell adhesion molecule, CC motif chemokine receptor, CD14, CD19, CD200 receptor, CD22, and CD27. 4, CD276, CD33, CD37, CD38, CD3e, CD4, CD44, CD48, CD70, CD74, CD80, CD99, muscarinic cholinergic receptor, nicotinic cholinergic receptor, coagulation factor II thrombin receptor, colony-stimulating factor 2 receptor, complement C5a receptor, C-type lectin domain, CXC motif chemokine receptor, cysteinyl leukotriene receptor, cytotoxic T lymphocyte-associated protein, delta-like standard Notch ligand, dipeptidyl peptidase, ectonucleoside triphosphate diphosphohydrolase, e Rislopoietin receptor, F11 receptor, formyl peptide receptor, FXYD domain-containing ion transport regulator, G protein-coupled bile acid receptor, G protein-coupled receptor, γ-aminobutyric acid type A receptor, gastric suppressor polypeptide receptor, glutamate metabolite receptor, platelet glycoprotein, hepatitis A virus cell receptor, histamine receptor, hydroxycarboxylic acid receptor, integrin, intercellular adhesion molecule, interleukin receptor accessory protein, interleukin receptor, killer cell lectin-like receptor, KISS1 receptor, leukotriene receptor, lymphocyte activation gene protein, lymphocyte antigen, mannose receptor, membrane metalloendopeptidase, transmembrane 4 domain, platelet activator receptor, potassium calcium activating channel, potassium voltage-gated channel, programmed cell death protein, prostaglandin receptor, prostaglandin synthase, protein tyrosine phosphatase, purine receptor, pyrimidine receptor, scavenger receptor, selectin, signal transduction lymphocyte activating molecule (SLAM) protein, sodium voltage-gated channel, somatostatin receptor,The receptor is selected from sphingosine-1-phosphate receptors, tumorigenesis inhibitory proteins, T cell immune receptors, thromboxane receptors, TNF receptors, Toll-like receptors, transient receptor potential channels, induced receptors expressed in myeloid cells, or V-set immunomodulatory receptors.

[0141] In further embodiments, the target of the target binding portion is one of the targets listed in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 2-23 Table 2-24 Table 2-25 Table 2-26 Table 2-27 Table 2-28 Table 2-29 Table 2-30 Table 2-31 [Table 2-32] [Table 2-33]

[0142] In further embodiments, the target of the target-binding moiety is a chemokine receptor (CCR). In further embodiments, the target of the target-binding moiety is selected from CCR1, CCR2, CCR3, CCR5, or CCR8.

[0143] In further embodiments, the target of the target binding moiety is CC motif chemokine receptor (CCR) 2 (CCR2), CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXC motif chemokine receptor 1 (CXCR1), CXC motif chemokine receptor 2 (CXCR2), CXC motif chemokine receptor 3 (CXCR3), CXC motif chemokine receptor 4 (CXCR4), CXC motif chemokine receptor 5 (CXCR5), CXC motif chemokine receptor 6 (CXCR6), atypical chemokine receptor Cain receptor 3 (ACKR3), integrin αvβ6, fibroblast-activating protein α (FAPα), prostate-specific membrane antigen (PSMA), folate receptor (folate receptor 1 or folate receptor β), complement C3a receptor 1 (C3AR1), complement C5a receptor 1 (C5AR1), G protein-coupled receptor (GPR) 65 (GPR65), GRP132, GPR84, GPR183, GPR35, GPR42, cholecystokinin A receptor (CCKAR), leukotriene B4 receptor (LTB4R), somatostatin receptor 2 (SSTR2), free fatty acid receptor 1 (FFAR1), purine receptor P2Y2 (P2RY2), prostaglandin D2 receptor (PTGDR), calcitonin receptor (CALCR), CD38, purine receptor P2X7 (P2RX7), integrin subunit αV (ITGAV), integrin subunit α5 (ITGA5), integrin subunit β1 (ITGB1), integrin subunit β6 (ITGB6), integrin subunit β3 (ITGB3), prostaglandin D2 receptor 2 (PTGDR2), gastrin-releasing peptide receptor (GRPR), M ER proto-oncogene tyrosine kinase (MERTK), C-X3-C motif chemokine receptor 1 (CX3CR1), oxidized low-density lipoprotein receptor 1 (OLR1), plasminogen activator urokinase receptor (PLAUR), carbonic anhydrase 9 (CA9), carbonic anhydrase 12 (CA12), mas-related G protein-coupled receptor member X2 (MRGPRX2), heat shock protein 90α family class A member 1 (HSP90AA1), dipeptidyl peptidase 4 (DPP4), formyl peptide receptor 2 (FPR2),And selected from succinate receptor 1 (SUCNR1).

[0144] In further embodiments, the target binding portion T 1 or T 2 These are small molecules that bind to targets listed in Table 2. Those skilled in the art may select small molecules known to bind to target proteins for use as target-binding moieties within the ARM. In one embodiment, the target-binding small molecule is modified to include a functional group such as -NH2 or -COOH to facilitate covalent bonding of the target-binding small molecule to the divalent linker moiety by the formation of an amide bond.

[0145] This disclosure also provides pharmaceutical compositions comprising a compound of formula (I) disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0146] Anti-cotinine antibodies This disclosure provides an antibody or antigen-binding fragment thereof that binds to a cotinine moiety. As used herein, the term “anti-cotinine antibody or antigen-binding fragment” refers to an antibody or antigen-binding fragment that binds to a cotinine moiety. Cotinine has the following structure: [ka]

[0147] As used herein, the term “cotinine moiety” refers to cotinine or a cotinine analog. The compounds of formula (I) described herein include a cotinine moiety that binds via a linker to a target binding site such as a PSMA binding site, a CCR2 binding site, and / or a CCR8 binding site. In one embodiment, the cotinine moiety has the following structure: [ka] In the formula, R 1 C 1-4 Alkyl or C 3-6 It is cycloalkyl. In another embodiment, R 1is methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, or t-butyl. In another embodiment, R 1 is methyl. In another embodiment, R 1 is ethyl. In another embodiment, R 1 These are cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0148] As used herein, the term “antibody” in its broadest sense refers to a molecule having an immunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD, or IgE), including monoclonal antibodies, recombinant antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, multispecific antibodies including bispecific antibodies, and heteroconjugate antibodies; single variable domains (e.g., domain antibodies (DABs)), antigen-binding antibody fragments, Fab, F(ab')2, Fv, disulfide-linked Fv, single-chain Fv, disulfide-linked scFv, diabodies, TANDABS, etc., and any modification of any of the above (for an overview of alternative “antibody” forms, see Holliger and Hudson, Nature Biotechnology, 2005, 23(9):1126–1136).

[0149] The terms “complete antibody,” “whole antibody,” or “intact antibody,” as used interchangeably herein, refer to a heterotetrameric glycoprotein with a molecular weight of approximately 150,000 daltons. The intact antibody consists of two identical heavy chains (HC) and two identical light chains (LC) linked by a covalent disulfide bond. This H2L2 structure folds to form three functional domains, including two antigen-binding fragments known as “Fab” fragments and an “Fc” crystallizable fragment. The Fab fragment consists of a variable domain at the amino terminus, a variable heavy chain (VH) or a variable light chain (VL), and constant domains at the carboxyl terminus, CH1 (heavy chain) and CL (light chain). The Fc fragment consists of two domains formed by the dimerization of paired CH2 and CH3 regions. Fc can induce effector function by binding to receptors on immune cells or by binding to C1q, the first component of the classical complement pathway. The five classes of antibodies (IgM, IgA, IgG, IgE, and IgD) are defined by different heavy chain amino acid sequences called μ, α, γ, ε, and δ, respectively, and each heavy chain can be paired with either a K or λ light chain. The majority of antibodies in serum belong to the IgG class, and human IgG has four isotypes (IgG1, IgG2, IgG3, and IgG4), whose sequences differ mainly in their hinge region.

[0150] "CDR" is defined as the complementarity-determining region amino acid sequence of an antibody or its antigen-binding fragment. These are the hypervariable regions of the immunoglobulin heavy and light chains. The variable region of an immunoglobulin contains three heavy chain CDRs (or CDR regions) and three light chain CDRs. Therefore, as used herein, "CDR" refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

[0151] Throughout this specification, variable domain sequences and amino acid residues within variable domain regions within full-length antigen-binding sequences (e.g., within antibody heavy chain sequences or antibody light chain sequences) are numbered according to Kabat numbering rules. Similarly, the terms “CDR,” “CDRL1,” “CDRL2,” “CDRL3,” “CDRH1,” “CDRH2,” and “CDRH3” used in the examples follow Kabat numbering rules. For further details, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987).

[0152] It will be apparent to those skilled in the art that there are alternative numbering rules for amino acid residues in variable domain sequences and full-length antibody sequences. Some alternative numbering rules for CDR sequences are described, for example, in Chothia et al., Nature, 1989, 342:877-883. The structure and folding of antigen-binding proteins imply that other residues are considered to be part of the CDR sequence, and this will be understood to those skilled in the art.

[0153] Other numbering conventions for CDR sequences available to those skilled in the art include the "AbM" (University of Bath) system and the "contact" (University College London) system.

[0154] Table 3 below shows one definition for each CDR or combined unit using each numbering convention. Note that some CDR definitions may differ depending on the individual publication used. [Table 3]

[0155] In further embodiments, the anti-cotinin antibody is humanized. In further embodiments, the Fc region of the anti-cotinin antibody is modified to enhance ADCC activity, ADCP activity, and / or CDC activity, the appropriate modifications of which are described below. In further embodiments, the Fc region of the anti-cotinin antibody is modified to enhance ADCC activity.

[0156] Fc engineering methods can be applied to modify the functional or pharmacokinetic properties of an antibody. Effector function can be altered by introducing mutations in the Fc region that increase or decrease binding to C1q or Fcγ receptors, thereby altering CDC or ADCC activity, respectively. Effector function can also be altered by changing the glycosylation pattern of the antibody. The in vivo half-life of an antibody can be altered by introducing mutations that affect the binding of Fc to FcRn (neonatal Fc receptor).

[0157] As used herein, the term “effector function” refers to one or more antibody-mediated effects, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-mediated complement activation including complement-dependent cytotoxicity (CDC), complement-mediated cell-mediated phagocytosis (CDCP), antibody-dependent complement-mediated cytolysis (ADCML), and Fc-mediated phagocytosis or antibody-dependent cell phagocytosis (ADCP).

[0158] The interaction between the Fc region of antigen-binding proteins or antibodies and various Fc receptors (FcRs), including FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), FcRn, C1q, and type II Fc receptors, is thought to mediate the effector function of antigen-binding proteins or antibodies. Significant biological effects may be a result of effector function. Typically, the ability to mediate effector function requires the binding of the antigen-binding protein or antibody to an antigen, and not all antigen-binding proteins or antibodies mediate all effector functions.

[0159] Effector function can be evaluated in various ways, including, for example, methods for evaluating the ADCC effector function of antibodies coated on target cells mediated by natural killer (NK) cells via FcγRIII or monocytes / macrophages via FcγRI, or methods for evaluating the CDC effector function of antibodies coated on target cells mediated by the complement cascade via C1q. For example, the antibodies or antigen-binding fragments of the present invention can be evaluated for ADCC effector function in natural killer cell assays. Examples of such assays are described in Shields et al., The Journal of Biological Chemistry, 2001, 276:6591-6604; Chappel et al., The Journal of Biological Chemistry, 1993, 268:25124-25131; and Lazar et al., PNAS, 2006, 103:4005-4010.

[0160] Examples of assays for determining CDC function include those described in J Imm Meth, 1995, 184:29-38.

[0161] The effects of mutations on effector functions (e.g., FcRn binding, FcγR and C1q binding, CDC, ADCML, ADCC, ADCP) can be evaluated as described, for example, in Grevys et al., J Immunol., 2015, 194(11):5497-5508; Tam et al., Antibodies, 2017, 6(3):12; or Monnet et al., mAbs, 2014, 6(2):422-436.

[0162] Throughout this specification, amino acid residues within the Fc region of antibody sequences or full-length antigen-binding protein sequences are numbered according to EU index numbering rules.

[0163] Human IgG1 constant regions containing specific mutations have been shown to enhance binding to the Fc receptor. In some cases, these mutations have also been shown to enhance effector functions such as ADCC and CDC, as described below. The antibodies or antigen-binding fragments of the present invention may contain any of the following mutations.

[0164] Enhanced CDC:Fc engineering can be used to enhance complement-based effector function. For example (see IgG1), K326W / E333S, S267E / H268F / S324T, and IgG1 / IgG3 cross subclasses can increase C1q binding, while E345R (Diebolder et al., Science, 2014, 343:1260-1293) and E345R / E430G / S440Y pre-form IgG hexamers (Wang et al., Protein Cell, 2018, 9(1):63-73).

[0165] Enhanced ADCC: Fc engineering can be used to enhance ADCC. For example (referring to IgG1), F243L / R292P / Y300L / V305I / P396L, S239D / I332E, and S298A / E333A / K334A increase FcγRIIIa binding, S239D / I332E / A330L increases FcγRIIIa binding and decreases FcγRIIb binding, and G236A / S239D / I332E improves binding to FcγRIIa, improves the FcγRIIa / FcγRIIb binding ratio (activation / inhibition ratio), and enhances phagocytosis of antibody-coated target cells by macrophages. In asymmetric Fc, where one heavy chain contains the L234Y / L235Q / G236W / S239M / H268D / D270E / S298A mutation and the other heavy chain contains D270E / K326D / A330M / K334E, the binding affinity to inhibitory FcγRIIb does not increase, but the affinity to FcγRIIIaF158 (low affinity allele) and FcγRIIIaV158 (high affinity allele) increases (Mimoto et al., mAbs, 2013, 5(2):229-236).

[0166] Enhanced ADCP: Fc engineering can be used to enhance ADCP. For example (see IgG1), G236A / S239D / I332E increases FcγRIIa binding and increases FcγRIIIa binding (Richards, J. et al., Mol. Cancer Ther., 2008, 7:2517-2527).

[0167] Improved co-engagement: Fc engineering can be used to improve co-engagement with FcR. For example (see IgG1), S267E / L328F increases FcγRIIb binding, while N325S / L328F increases FcγRIIa binding and decreases FcγRIIIa binding (Wang et al., Protein Cell, 2018, 9(1):63-73).

[0168] In further embodiments, the antibody or antigen-binding fragment of the present invention may include a heavy chain constant region having a modified glycosylation profile, resulting in the antibody or antigen-binding fragment having enhanced effector function, e.g., enhanced ADCC, enhanced CDC, or both enhanced ADCC and CDC. Examples of suitable methods for producing antibodies or antigen-binding fragments having a modified glycosylation profile are described in WO2003 / 011878, WO2006 / 014679 and EP1229125.

[0169] Because the innermost α1,6 fucose residue is absent on the Fc glycan portion at N297 of the IgG1 antibody, its affinity for FcγRIIIA is increased. Therefore, afucosylated or low-fucosylated monoclonal antibodies may have improved therapeutic effects (Shields et al., J Biol Chem., 2002, 277(30):26733-40 and Monnet et al., mAbs, 2014, 6(2):422-436).

[0170] In one embodiment, an antibody or its antigen-binding fragment is provided that includes a chimeric heavy chain constant region. In one embodiment, the antibody or its antigen-binding fragment includes an IgG1 / IgG3 chimeric heavy chain constant region, and as a result, the antibody or its antigen-binding fragment has enhanced effector function, e.g., enhanced ADCC or enhanced CDC, or enhanced ADCC and CDC function. For example, the chimeric antibody or its antigen-binding fragment of the present invention may include at least one IgG3-derived CH2 domain. In such one embodiment, the antibody or its antigen-binding fragment may include one IgG3-derived CH2 domain, or both CH2 domains may be IgG3-derived. In a further embodiment, the chimeric antibody or its antigen-binding fragment includes a CH1 domain of IgG1, a CH2 domain of IgG3, and a CH3 domain of IgG3. In a further embodiment, the chimeric antibody or its antigen-binding fragment includes a CH1 domain of IgG1, a CH2 domain of IgG3, and a CH3 domain of IgG3, excluding position 435 which is histidine.

[0171] In further embodiments, the chimeric antibody or its antigen-binding fragment comprises the CH1 domain of IgG1 and at least one IgG3-derived CH2 domain. In one embodiment, the chimeric antibody or its antigen-binding fragment comprises the CH1 domain of IgG1 and the following residues corresponding to IgG3 residues in the CH2 domain: 274Q, 276K, 296F, 300F, and 339T. In one embodiment, the chimeric antibody or its antigen-binding fragment also comprises 356E corresponding to an IgG3 residue in the CH3 domain. In one embodiment, the antibody or its antigen-binding fragment also comprises one or more of the following residues corresponding to IgG3 residues in the CH3 domain: 358M, 384S, 392N, 397M, 422I, 435R, and 436F.

[0172] Furthermore, according to the present invention, a) A step of culturing recombinant host cells containing an expression vector comprising a nucleic acid sequence encoding a chimeric Fc region having amino acid residues of both the Fc regions of IgG1 and IgG3 (for example, as described above), b) A step of recovering the antibody or its antigen-binding fragment, A method for producing an antibody or its antigen-binding fragment, including the above, is provided.

[0173] Such methods for producing antibodies or antigen-binding fragments having a chimeric heavy chain constant region can be carried out, for example, using the COMPLEGENT technology system available from BioWa, Inc. (Princeton, NJ) and Kyowa Hakko Kirin Co., Ltd. The COMPLEGENT system comprises recombinant host cells containing an expression vector, in which a nucleic acid sequence encoding a chimeric Fc region having amino acid residues from both the Fc regions of IgG1 and IgG3 is expressed to produce an antibody or antigen-binding fragment having enhanced CDC activity, i.e., increased CDC activity compared to other identical antibodies or antigen-binding fragments lacking the chimeric Fc region, as described in WO2007 / 011041 and US2007 / 0148165, respectively, incorporated herein by reference. In another embodiment, CDC activity may be increased by introducing sequence-specific mutations into the Fc region of the IgG chain. Those skilled in the art will also recognize other suitable systems.

[0174] Furthermore, according to the present invention, a) A step of culturing recombinant host cells comprising an expression vector containing a nucleic acid encoding an antibody or an antigen-binding fragment, wherein the FUT8 gene encoding α-1,6-fucosyltransferase is optionally inactivated in the recombinant host cells, b) A step of recovering the antibody or its antigen-binding fragment, A method for producing an antibody or its antigen-binding fragment, including the above, is provided.

[0175] Such methods for the production of antibodies or their antigen-binding fragments can be carried out, for example, using the POTELLIGENT technology system available from BioWa, Inc. (Princeton, NJ), in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies with enhanced ADCC activity compared to the same monoclonal antibodies produced in cells with a functional FUT8 gene, all of which are incorporated herein by reference. Other suitable systems will also be recognized by those skilled in the art.

[0176] In one embodiment, the antibody or its antigen-binding fragment is produced in host cells in which the FUT8 gene has been inactivated. In a further embodiment, the antibody or its antigen-binding fragment is produced in - / -FUT8 host cells. In a further embodiment, the antibody or its antigen-binding fragment is defucosylated at Asn297(IgG1).

[0177] It will be apparent to those skilled in the art that such modifications can be used not only individually to further enhance the effects pedal's functionality, but also in combination with each other.

[0178] In one such embodiment, an antibody or antigen-binding fragment is provided that includes a heavy chain constant region containing both the mutant heavy chain constant region and the chimeric heavy chain constant region described individually above. For example, an antibody or antigen-binding fragment containing at least one IgG3-derived CH2 domain and one IgG1-derived CH2 domain, wherein the CH2 domain of IgG1 has one or more mutations (for example, the mutations may be selected from S239D, I332E, and A330L) at positions selected from 239, 332, and 330, and as a result, the antibody or antigen-binding fragment has enhanced effector function, such as enhanced ADCC or enhanced CDC, or enhanced ADCC and enhanced CDC, compared to an equivalent antibody or antigen-binding fragment having an IgG1 heavy chain constant region lacking the above mutations. In one embodiment, the CH2 domain of IgG1 has mutations S239D and I332E. In another embodiment, the CH2 domain of IgG1 has mutations S239D, A330L, and I332E.

[0179] In another embodiment, an antibody or its antigen-binding fragment is provided that includes both the chimeric heavy chain constant region and the modified glycosylation profile described individually above. In one embodiment, the antibody or its antigen-binding fragment includes a modified glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less. In such an embodiment, the heavy chain constant region includes at least one IgG3-derived CH2 domain and one IgG1-derived CH2 domain, and has a modified glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less, and for example, the antibody or its antigen-binding fragment is defucosylated. The antibody or its antigen-binding fragment has enhanced effector function, such as enhanced ADCC or enhanced CDC, or enhanced ADCC and enhanced CDC, compared to an equivalent antibody or its antigen-binding fragment having an IgG1 heavy chain constant region lacking the glycosylation profile described above.

[0180] In another embodiment, the antibody or its antigen-binding fragment has at least one IgG3 heavy chain CH2 domain and at least one IgG1-derived heavy chain constant domain, and both IgGCH2 domains are mutated according to the limitations described herein.

[0181] In one embodiment, according to the present invention as described herein, a) A step of culturing recombinant host cells containing an expression vector comprising a nucleic acid sequence encoding a chimeric Fc domain having both amino acid residues of the Fc domains of IgG1 and IgG3 (for example, as described above), wherein the FUT8 gene encoding α-1,6-fucosyltransferase is inactivated in the recombinant host cells, and the above step, b) A step of recovering the antibody or its antigen-binding fragment, A method for producing an antibody or its antigen-binding fragment, including the above, is provided.

[0182] Such methods for the production of antibodies or their antigen-binding fragments can be carried out, for example, using the ACCRETAMAB technology system available from BioWa, Inc. (Princeton, NJ), which combines the POTELLIGENT and COMPLEGENT technology systems to produce antibodies or their antigen-binding fragments having both enhanced ADCC activity and enhanced CDC activity compared to other identical monoclonal antibodies lacking a chimeric Fc domain and fucosylated.

[0183] In another embodiment, an antibody or its antigen-binding fragment is provided that includes a mutant chimeric heavy chain constant region, the antibody or its antigen-binding fragment having a modified glycosylation profile, and as a result, the antibody or its antigen-binding fragment having enhanced effector function, such as enhanced ADCC or enhanced CDC, or both enhanced ADCC and CDC. In one embodiment, the mutation is selected from positions 239, 332 and 330, for example, S239D, I332E, and A330L. In a further embodiment, the heavy chain constant region includes at least one IgG3-derived CH2 domain and one IgG1-derived CH1 domain. In one embodiment, the heavy chain constant region has a modified glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less, for example, the antibody or its antigen-binding fragment is defucosylated, and as a result, the antibody or its antigen-binding fragment has enhanced effector function compared to an equivalent non-chimeric antibody or its antigen-binding fragment that lacks the above mutation and the above-described modified glycosylation profile.

[0184] In further embodiments, the anti-cotinin antibody or its antigen-binding fragment comprises a heavy chain CDR1 having SEQ ID NO: 1, a heavy chain CDR2 having SEQ ID NO: 2, a heavy chain CDR3 having SEQ ID NO: 3, a light chain CDR1 having SEQ ID NO: 4, a light chain CDR2 having SEQ ID NO: 5, and a light chain CDR3 having SEQ ID NO: 6. In further embodiments, the anti-cotinin antibody has a heavy chain and a light chain, the heavy chain comprising CDR1 having SEQ ID NO: 1, CDR2 having SEQ ID NO: 2, and CDR3 having SEQ ID NO: 3, and the light chain comprising CDR1 having SEQ ID NO: 4, CDR2 having SEQ ID NO: 5, and CDR3 having SEQ ID NO: 6. In further embodiments, the anti-cotinin antibody is an IgG1 isotype. In further embodiments, the anti-cotinin antibody is an IgG1 isotype that includes substitutions in the Fc region to increase or enhance ADCC activity. In a further embodiment, the anti-cotinin antibody is an IgG1 isotype containing a substitution in the Fc region to increase or enhance ADCC activity, wherein the substitution is S239D / I332E or S239D / I332E / A330L, and the residue numbering follows the EU index.

[0185] In further embodiments, the anti-cotinin antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) having SEQ ID NO: 7 and a light chain variable region (VL) having SEQ ID NO: 8. In further embodiments, the anti-cotinin antibody has a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) having SEQ ID NO: 7 and the light chain comprising a light chain variable region (VL) having SEQ ID NO: 8. In further embodiments, the anti-cotinin antibody is an IgG1 isotype. In further embodiments, the anti-cotinin antibody is an IgG1 isotype comprising a substitution in the Fc region to increase or enhance ADCC activity, the substitution being S239D / I332E or S239D / I332E / A330L, and the residue numbering follows the EU index. In a further embodiment, the anti-cotinin antibody is an IgG1 isotype containing a substitution in the Fc region to increase or enhance ADCC activity, the substitution being S239D / I332E, and the residue numbering following the EU index.

[0186] In a further embodiment, the anti-cotinin antibody has a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO: 10.

[0187] This disclosure also provides pharmaceutical compositions comprising an anti-cotinin antibody or its antigen-binding fragment disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0188] This disclosure also provides combinations comprising a compound of formula (I) disclosed herein and an anti-cotinine antibody or its antigen-binding fragment disclosed herein. The compound of formula (I) and the anti-cotinine antibody or its antigen-binding fragment may be present in the same composition or in separate compositions. In one embodiment, the combination comprises a pharmaceutical composition comprising a compound of formula (I) disclosed herein, an anti-cotinine antibody or its antigen-binding fragment disclosed herein, and a pharmaceutically acceptable carrier, diluent, or excipient. In another embodiment, the combination comprises a first pharmaceutical composition comprising a compound of formula (I) disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient, and a second pharmaceutical composition comprising an anti-cotinine antibody or its antigen-binding fragment disclosed herein and a pharmaceutically acceptable carrier, excipient, or diluent.

[0189] statement of use Compounds of formula (I) and their pharmaceutically acceptable salts can conjugate simultaneously to one or more targets expressed on the cell surface and an anti-cotinin antibody or its antigen-binding fragment to form a multicomponent complex for the treatment and / or prevention of diseases or disorders associated with target-expressing cells.

[0190] In one embodiment, the present disclosure provides a method for treating and / or preventing a disease or disorder in a patient in need thereof, comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anticotinin antibody or an antigen-binding fragment thereof to the patient, wherein the disease or disorder is selected from cancer, inflammatory diseases, autoimmune diseases, viral infections, or bacterial infections.

[0191] In further embodiments, the compound and the antibody or its antigen-binding fragment are administered simultaneously. In further embodiments, the compound and the antibody or its antigen-binding fragment are administered simultaneously from a single composition, for example, as a fixed-dose composition, or by pre-mixing the compound and the antibody or its antigen-binding fragment before administration. For example, the compound and the antibody or its antigen-binding fragment may be pre-mixed before administration for about 2 seconds to about 30 seconds, about 30 seconds to about 2 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 30 minutes, or about 30 minutes to about 2 hours. In further embodiments, the compound and the antibody or its antigen-binding fragment are administered simultaneously from two separate compositions.

[0192] In further embodiments, the compound and the antibody or its antigen-binding fragment are administered sequentially.

[0193] In certain embodiments, the compound and the antibody or its antigen-binding fragment may be administered via the same route or via different routes, whether simultaneously or sequentially. In one embodiment, both the compound and the antibody or its antigen-binding fragment are administered intravenously or subcutaneously in the same composition or in separate compositions. In another embodiment, the compound is administered orally, and the antibody or its antigen-binding fragment is administered intravenously or subcutaneously.

[0194] In further embodiments, the compound and the antibody or its antigen-binding fragment are administered in molar ratios of about 2:1, about 1.8:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1:1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.8, about 1:2, about 2:1 to about 1.5:1, about 1.5:1 to about 1.2:1, about 1.2:1 to about 1:1, about 1:1 to about 1:1.2, about 1:1.2 to about 1:1.5, or about 1:1.5 to about 1:2.

[0195] In further embodiments, the compound and the antibody or its antigen-binding fragment exist as combinations in molar ratios of about 2:1, about 1.8:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1:1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.8, about 1:2, about 2:1 to about 1.5:1, about 1.5:1 to about 1.2:1, about 1.2:1 to about 1:1, about 1:1 to about 1:1.2, about 1:1.2 to about 1:1.5, or about 1:1.5 to about 1:2.

[0196] In further embodiments, the compound and the antibody or its antigen-binding fragment are administered in doses of 0.0001 mg / kg to 1 mg / kg for the compound and 0.01 mg / kg to 100 mg / kg for the antibody or its antigen-binding fragment. For example, in further embodiments, the compound is administered in doses of about 0.0001 mg / kg to about 0.0002 mg / kg, about 0.0002 mg / kg to about 0.0003 mg / kg, about 0.0003 mg / kg to about 0.0004 mg / kg, about 0.0004 mg / kg to about 0.0005 mg / kg, about 0.0005 mg / kg to about 0.001 mg / kg, about 0.001 mg / kg to about 0.002 mg / kg, and about 0 0.002mg / kg to approximately 0.003mg / kg, approximately 0.003mg / kg to approximately 0.004mg / kg, approximately 0.004mg / kg to approximately 0.005mg / kg, approximately 0.005mg / kg to approximately 0.01mg / kg, approximately 0.01mg / kg to approximately 0.02mg / kg, approximately 0.02mg / kg to approximately 0.03mg / kg, approximately 0.03mg / kg to approximately 0.04mg / kg, approximately 0.04mg / kg to approximately 0.05 The antibody or its antigen-binding fragment is administered in doses of approximately 0.01 mg / kg, approximately 0.05 mg / kg to approximately 0.1 mg / kg, approximately 0.1 mg / kg to approximately 0.2 mg / kg, approximately 0.2 mg / kg to approximately 0.3 mg / kg, approximately 0.3 mg / kg to approximately 0.4 mg / kg, approximately 0.4 mg / kg to approximately 0.5 mg / kg, and / or approximately 0.5 mg / kg to approximately 1 mg / kg, and the antibody or its antigen-binding fragment is administered in doses of approximately 0.01 mg / kg to approximately 0.02 mg / kg, about 0.02 mg / kg to about 0.03 mg / kg, about 0.03 mg / kg to about 0.04 mg / kg, about 0.04 mg / kg to about 0.05 mg / kg, about 0.05 mg / kg to about 0.1 m g / kg, about 0.1 mg / kg to about 0.2 mg / kg, about 0.2 mg / kg to about 0.3 mg / kg, about 0.3 mg / kg to about 0.4 mg / kg, about 0.4 mg / kg to about 0.5 mg / kg, about 0.5mg / kg to about 1mg / kg, about 1mg / kg to about 2mg / kg, about 2mg / kg to about 3mg / kg, about 3mg / kg to about 4mg / kg, about 4mg / kg to about 5mg / kg, about 5mg / kg ~10mg / kg, 10mg / kg~15mg / kg, 15mg / kg~20mg / kg, 20mg / kg~25mg / kg, 25mg / kg~30mg / kg, 30mg It is administered in doses of approximately 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, and / or 100 mg / kg.

[0197] In further embodiments, the compound and the antibody or its antigen-binding fragment are administered in doses of 0.007 mg to 70 mg of the compound and 0.7 mg to 7000 mg of the antibody or its antigen-binding fragment. For example, in further embodiments, the compound may be administered in doses of approximately 0.007 mg to approximately 0.01 mg, approximately 0.01 mg to approximately 0.02 mg, approximately 0.02 mg to approximately 0.03 mg, approximately 0.03 mg to approximately 0.04 mg, approximately 0.04 mg to approximately 0.05 mg, approximately 0.05 mg to approximately 0.1 mg, approximately 0.1 mg to approximately 0.2 mg, approximately 0.2 mg to approximately 0.3 mg, approximately 0.3 mg to approximately 0.4 mg, approximately 0.4 mg to approximately 0.5 mg, and approximately 0. The antibody or its antigen-binding fragment is administered in doses of approximately 5 mg to 1 mg, approximately 1 mg to 2 mg, approximately 2 mg to 3 mg, approximately 3 mg to 4 mg, approximately 4 mg to 5 mg, approximately 5 mg to 10 mg, approximately 10 mg to 20 mg, approximately 20 mg to 30 mg, approximately 30 mg to 40 mg, approximately 40 mg to 50 mg, approximately 50 mg to 60 mg, and / or approximately 60 mg to 70 mg, with the antibody or its antigen-binding fragment being approximately 0.7 mg to 1 mg. g, about 1 mg to about 2 mg, about 2 mg to about 3 mg, about 3 mg to about 4 mg, about 4 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 20 mg, about 20 mg to about 30 mg, about 30 mg to about 40 m g, about 40mg to about 50mg, about 50mg to about 100mg, about 100mg to about 500mg, about 500mg to about 1000mg, about 1000mg to about 1500mg, about 1500mg to about 2000mg, It is administered in doses of approximately 2000mg to 2500mg, 2500mg to 3000mg, 3000mg to 3500mg, 3500mg to 4000mg, 4000mg to 4500mg, 4500mg to 5000mg, 5000mg to 5500mg, 5500mg to 6000mg, 6000mg to 6500mg, and / or 6500mg to 7000mg.

[0198] In further embodiments, the compound and the antibody or its antigen-binding fragment are administered at the molar ratios and / or doses described herein, at frequencies of once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks, over a period of one week to one year, for example, one week, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months.

[0199] In further embodiments, the disclosure provides therapeutically effective amounts of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an antibody or an antigen-binding fragment thereof, for therapeutic use. The compound of formula (I) or a pharmaceutically acceptable salt thereof, and the antibody or an antigen-binding fragment thereof may be used to treat or prevent a disease or disorder selected from cancer, inflammatory diseases, autoimmune diseases, viral infections, or bacterial infections.

[0200] In further embodiments, the disclosure provides therapeutically effective amounts of compounds of formula (I) or pharmaceutically acceptable salts thereof, and antibodies or antigen-binding fragments thereof, for the manufacture of pharmaceuticals. These agents can be used to treat or prevent diseases or disorders selected from cancer, inflammatory diseases, autoimmune diseases, viral infections, or bacterial infections.

[0201] In further embodiments, the disease or disorder is mediated by chemokine receptor 2 (CCR2) and / or associated with CCR2-positive pathogenic cells. In further embodiments, the CCR2-positive cell type is identified by testing for CCR2 expression, for example, by immunohistochemistry or flow cytometry.

[0202] In further embodiments, the disease or disorder is mediated by CXC motif chemokine receptor 3 (CXCR3) and / or associated with CXCR3-positive pathogenic cells. In further embodiments, the CXCR3-positive cell type is identified by testing for CXCR3 expression, for example, by immunohistochemistry or flow cytometry.

[0203] In further embodiments, the disease or disorder is mediated by PSMA and / or associated with PSMA-positive pathogenic cells. In further embodiments, the PSMA-positive cell type is identified by testing for PSMA expression, for example, by immunohistochemistry or flow cytometry.

[0204] In further embodiments, the disease or disorder is mediated by integrin αVβ6 and / or associated with integrin αVβ6-positive pathogenic cells. In further embodiments, the integrin αVβ6-positive cell type is identified by testing for integrin αVβ6 expression, for example, by immunohistochemistry or flow cytometry.

[0205] In further embodiments, the disease or disorder is mediated by folate receptor α (FRα) and / or folate receptor β (FRβ) and / or associated with FRα and / or FRβ-positive pathogenic cells. In further embodiments, FRα and / or FRβ-positive cell types are identified by testing for FRα and / or FRβ expression, for example, by immunohistochemistry or flow cytometry.

[0206] In further embodiments, the disease or disorder is mediated by fibroblast-activating protein (FAP) and / or associated with FAP-positive pathogenic cells. In further embodiments, the FAP-positive cell type is identified by testing for FAP expression, for example, by immunohistochemistry or flow cytometry.

[0207] In further embodiments, the disease or disorder is mediated by chemokine receptor 8 (CCR8) and / or associated with CCR8-positive pathogenic cells. In further embodiments, the CCR8-positive cell type is identified by testing for CCR8 expression, for example by immunohistochemistry or flow cytometry.

[0208] In further embodiments, the disease or disorder is a cancer selected from lung cancer (e.g., non-small cell lung cancer (NSCLC)), hepatocellular carcinoma (HCC), colorectal cancer (CRC), cervical cancer (e.g., squamous cell carcinoma of the cervix (CESC)), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (HNSC)), pancreatic cancer, prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), ovarian cancer, endometrial cancer, brain cancer, endocrine cancer, testicular cancer, bladder cancer, bone cancer, esophageal cancer, gastric cancer, renal cell carcinoma, melanoma cancer, thyroid cancer, or breast cancer, preferably a cancer selected from mCRPC, breast cancer, lung cancer, colorectal cancer, or renal cell carcinoma.

[0209] In further embodiments, the disease or disorder is a solid tumor. In further embodiments, the disease or disorder is a solid tumor selected from lung cancer (e.g., NSCLC), HCC, CRC, cervical cancer (e.g., CESC), head and neck cancer (e.g., HNSC), pancreatic cancer, prostate cancer (e.g., mCRPC), ovarian cancer, endometrial cancer, brain cancer, endocrine cancer, testicular cancer, bladder cancer, bone cancer, esophageal cancer, gastric cancer, renal cell carcinoma, melanoma cancer, thyroid cancer, or breast cancer, preferably a solid tumor selected from mCRPC, breast cancer, lung cancer, colorectal cancer, or renal cell carcinoma.

[0210] In further embodiments, the disease or disorder is a PD-1 recurrent or refractory cancer, such as PD-1 recurrent or refractory lung cancer (e.g., NSCLC), HCC, CRC, cervical cancer (e.g., CESC), head and neck cancer (e.g., HNSC), pancreatic cancer, prostate cancer (e.g., mCRPC), ovarian cancer, endometrial cancer, brain cancer, endocrine cancer, testicular cancer, bladder cancer, bone cancer, esophageal cancer, gastric cancer, renal cell carcinoma, melanoma cancer, thyroid cancer, or breast cancer, preferably PD-1 recurrent or refractory breast cancer, lung cancer, head and neck cancer, or cervical cancer.

[0211] In further embodiments, the disease or disorder is a non-solid tumor. In further embodiments, the disease or disorder is leukemia, lymphoma, or myeloma.

[0212] In further embodiments, the disease or disorder is a viral infection. In further embodiments, the viral infection is caused by an influenza virus, a coronavirus (e.g., COVID-19), or a hepatitis B virus.

[0213] In further embodiments, the disease or disorder is a bacterial infection. In further embodiments, the bacterial infection is a chronic bacterial infection.

[0214] In further embodiments, the disease is an autoimmune or inflammatory disease selected from vitiligo and type 1 diabetes.

[0215] In one embodiment, the present disclosure provides a method for increasing antibody-dependent cell-mediated cytotoxicity (ADCC) of target-expressing cells, the method comprising contacting cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinin antibody or an antigen-binding fragment thereof, wherein the target-binding portion of the compound binds to a target expressed on the cell.

[0216] In one embodiment, the present disclosure provides a method for increasing antibody-dependent cell phagocytosis (ADCP) of target-expressing cells, the method comprising contacting cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinin antibody or an antigen-binding fragment thereof, wherein the target-binding portion of the compound binds to a target expressed on the cell.

[0217] In one embodiment, the present disclosure provides a method for increasing complement-dependent cell-mediated cytotoxicity (CDC) of target-expressing cells, the method comprising contacting cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinin antibody or an antigen-binding fragment thereof, wherein the target-binding portion of the compound binds to a target expressed on the cell.

[0218] In one embodiment, the present disclosure provides a method for conditioning a patient for treatment with chimeric antigen receptor (CAR) T-cell therapy or CAR NK-cell therapy, the method comprising administering to the patient an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinine antibody or an antigen-binding fragment thereof. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, and the anti-cotinine antibody or an antigen-binding fragment thereof are administered in combination with CAR-T-cell therapy or CAR-NK-cell therapy. The compound of formula (I) or a pharmaceutically acceptable salt thereof, and the anti-cotinine antibody or an antigen-binding fragment thereof may be administered as conditioning therapy or combination therapy to enhance efficacy in the treatment of solid tumor cancer. In other embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, and the anti-cotinine antibody or an antigen-binding fragment thereof may be administered as neoadjuvant therapy to other therapies, including but not limited to immunotherapy, surgical resection, radiotherapy, and / or chemotherapy.

[0219] In one embodiment, the present disclosure provides a method for increasing cellular killing of target-expressing cells, the method comprising contacting the cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinin antibody or an antigen-binding fragment thereof, wherein the target-binding portion of the compound binds to a target expressed on the cells.

[0220] In one embodiment, the present disclosure provides a method for depleting target-expressing cells, the method comprising contacting the cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinin antibody or an antigen-binding fragment thereof, wherein the target-binding portion of the compound binds to a target expressed on the cells.

[0221] In further embodiments, the target expression cells are CCR2-expressing cells. In further embodiments, the CCR2-expressing cells are myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), neutrophils, macrophages, regulatory B cells (Bregs), CD8 regulatory cells (CD8regs), exhausted T cells, or cancer-associated fibroblasts (CAFs).

[0222] In further embodiments, the target expression cells are CXCR3-expressing cells. In further embodiments, CXCR3-expressing cells are activated T cells, autoreactive T cells, regulatory T cells (Treg), CD4 regulatory T cells (CD4reg), CD8 regulatory T cells (CD8reg), T helper (Th) T cells, Th1 T cells, natural killer T (NKT) cells, natural killer (NK) cells, dendritic cells, B cells, γδ T cells, or tumor cells.

[0223] In a further embodiment, the target expression cells are PSMA-expressing cells. In a further embodiment, the PSMA-expressing cells are tumor cells.

[0224] In a further embodiment, the target expression cells are integrin αVβ6 expressing cells. In a further embodiment, the integrin αVβ6 expressing cells are tumor cells.

[0225] In further embodiments, the target expression cells are FRα and / or FRβ expressing cells. In further embodiments, the FRα and / or FRβ expressing cells are myeloid suppressor cells (MDSCs), macrophages, B cells, or tumor cells.

[0226] In further embodiments, the target expression cells are FAP-expressing cells. In further embodiments, the FAP-expressing cells are cancer-associated fibroblasts (CAFs), macrophages, or tumor cells. In further embodiments, the target expression cells are CCR8-expressing cells. In further embodiments, the CCR8-expressing cells are regulatory T cells (Tregs) or tumor cells.

[0227] In a further embodiment, the target expression cells are pathogenic cells.

[0228] In further embodiments, the pathogenic cells are pathogenic immune cells, tumor cells or cancer cells, or stromal cells.

[0229] In further embodiments, pathogenic immune cells include monocytes, myeloid-derived suppressor cells (MDSCs), e.g., monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs), regulatory T cells (Tregs), neutrophils (e.g., N2 neutrophils), macrophages (e.g., M2 macrophages), regulatory B cells (Bregs, memory B cells), plasma cells, CD8 cells (e.g., CD8 regulatory cells (CD8regs), memory CD8 cells, effector CD8 cells, naive CD8 T cells, TEMRAs), exhausted T cells, eosinophils, basophils, mast cells, dendritic cells, natural killer (NK) cells, innate lymphoid cells, NK T cells (NKTs), or γδ T cells.

[0230] In further embodiments, pathogenic immune cells include myeloid suppressor cells (MDSCs) such as monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs), regulatory T cells (Tregs), neutrophils (e.g., N2 neutrophils), macrophages (e.g., M2 macrophages), regulatory B cells (Bregs), CD8 regulatory cells (CD8regs), and exhausted T cells.

[0231] In a further embodiment, the tumor cells or cancer cells are solid tumor cells.

[0232] In further embodiments, tumor cells or cancer cells are lung cancer cells (e.g., non-small cell lung cancer (NSCLC) cells), hepatocellular carcinoma (HCC) cells, colorectal cancer (CRC) cells, cervical cancer cells (e.g., cervical squamous cell carcinoma (CESC) cells), head and neck cancer cells (e.g., head and neck squamous cell carcinoma (HNSC) cells), pancreatic cancer cells, prostate cancer cells (e.g., metastatic castration-resistant prostate cancer (mCRPC) cells), ovarian cancer cells, endometrial cancer cells, brain cancer cells, endocrine cancer cells, testicular cancer cells, bladder cancer cells, bone cancer cells, esophageal cancer cells, gastric cancer cells, renal cell carcinoma cells, melanoma cancer cells, thyroid cancer cells, or breast cancer cells, preferably selected from mCRPC cells, breast cancer cells, lung cancer cells, colorectal cancer cells, or renal cell carcinoma cells.

[0233] In further embodiments, the pathogenic cells are endothelial cells associated with tumor neovascularization.

[0234] In a further embodiment, the stromal cells are cancer-associated fibroblasts (CAFs).

[0235] Combination therapy The compounds of the present invention may be used alone or in combination with other therapeutic agents. Therefore, combination therapy according to the present invention comprises the administration of at least one compound of formula (I) or a pharmaceutically acceptable salt thereof, and the use of at least one other pharmaceutically active agent. The compounds of the present invention and the other pharmaceutically active agents may be administered together in a single pharmaceutical composition or separately, and if administered separately, they may be administered simultaneously or sequentially in any order. The amounts of the compounds of the present invention and the other pharmaceutically active agents, as well as the relative timing of administration, are selected to achieve the desired combination therapeutic effect.

[0236] When the compound of the present invention is administered in combination with one or more other therapeutic agents that are normally administered by inhalation, intravenous, oral, intranasal, topical or ocular, or other routes, it will be understood that the resulting pharmaceutical composition may be administered by the same route. Alternatively, the individual components of the composition may be administered by different routes.

[0237] In one embodiment, the compounds and pharmaceutical compositions disclosed herein are used in combination with or comprise one or more additional therapeutic agents. In further embodiments, the additional therapeutic agents are checkpoint inhibitors or immunomodulators.

[0238] In further embodiments, the checkpoint inhibitor is selected from PD-1 inhibitors (e.g., anti-PD-1 antibodies including, but not limited to, pembrolizumab, nivolumab, semiprimab, or dostallimab), PD-L1 inhibitors (e.g., anti-PD-L1 antibodies including, but not limited to, atezolizumab, avelumab, or durvalumab), or CTLA-4 inhibitors (e.g., anti-CTLA-4 antibodies including, but not limited to, ipilimumab or toremirumab).

[0239] In further embodiments, the checkpoint inhibitor is selected from CD226 axis inhibitors, including but not limited to TIGIT inhibitors (e.g., anti-TIGIT antibodies), CD96 inhibitors (e.g., anti-CD96 antibodies), and / or PVRIG inhibitors (e.g., anti-PVRIG antibodies).

[0240] In further embodiments, the immunomodulator is an ICOS agonist (e.g., an anti-ICOS antibody including, but not limited to, feradilimab), a PARP inhibitor (e.g., niraparib, olaparib), or a STING agonist.

[0241] Pharmaceutical composition, dosage, and dosage form For administration, in certain embodiments, the ARM described herein is administered as a raw chemical substance or formulated as a pharmaceutical composition. The pharmaceutical compositions disclosed herein comprise the ARM and one or more pharmaceutically acceptable carriers, diluents, or excipients. The ARM is present in the composition in an amount effective to treat a particular disease, disorder, or symptom of interest. The activity of the ARM can be determined by those skilled in the art, for example, as described in the biological assays described below. Appropriate concentrations and dosages can be readily determined by those skilled in the art. In certain embodiments, the ARM is present in the pharmaceutical composition in an amount of about 25 mg to about 500 mg. In certain embodiments, the ARM is present in the pharmaceutical composition in an amount of about 0.01 mg to about 300 mg. In certain embodiments, the ARM is present in the pharmaceutical composition in an amount of about 0.01 mg, 0.1 mg, 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, or about 500 mg.

[0242] The compounds of the present invention or pharmaceutically acceptable salts thereof are administered in pure form or in suitable pharmaceutical compositions by any acceptable method of administering a drug that performs a similar function. Pharmaceutical compositions of the present invention are prepared by combining the compounds of the present invention with suitable pharmaceutically acceptable carriers, diluents or excipients, and in certain embodiments are formulated into solid, semi-solid, liquid or gaseous formulations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral (e.g., intramuscular, subcutaneous, intravenous, or intradermal), sublingual, oral cavity, rectal, vaginal, and nasal cavity. Pharmaceutical compositions of the present invention are formulated so that their active ingredients are bioavailable when administered to a patient.

[0243] The composition administered to the subject or patient may take the form of one or more dosage units; for example, a tablet may be a single dosage unit, and a container of the aerosol form of the compound of the present invention may hold multiple dosage units. Practical methods for preparing such dosage forms are known or obvious to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia, College of Pharmacy and Science, 2000). The composition administered in any case shall contain a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof for treating the disease or condition of interest in accordance with the teachings described herein.

[0244] The pharmaceutical compositions disclosed herein are prepared by methodologies well known in the pharmaceutical industry. For example, in certain embodiments, pharmaceutical compositions intended for injectable administration are prepared by mixing the compounds of the present invention with sterile distilled water to form a solution. In some embodiments, surfactants are added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with the compounds of the present invention to promote the dissolution or homogeneous suspension of the compounds in an aqueous delivery system.

[0245] Conventional antibody therapies have several drawbacks that can be addressed by the ARM approach described herein, including the difficulty in managing adverse events by adjusting dosage and frequency of administration, the difficulty in generating antibodies against specific classes of drug targets (e.g., GPCRs, ion channels, and enzymes), and the potentially time-consuming and costly process of requiring a new cell line for development for each new antibody. Furthermore, the production of different forms of biologics (e.g., bispecific formulations) can be challenging. In contrast, the ARM approach has the advantage of integrating antibody pharmacology with small molecule dosage control, enabling transient cell depletion through dosage-controlled PK / PD, making multimerization easier, and allowing rapid recovery of cell depletion through the administration of antibody-binding components (e.g., cotinine hapten), thereby decoupling therapeutic effects from potential adverse events. [Examples]

[0246] The following examples illustrate the present invention. These examples are not intended to limit the scope of the present invention, but rather to provide guidance to those skilled in the art for the preparation and use of the compounds, compositions, and methods of the present invention. While specific embodiments of the present invention are described, those skilled in the art will see that various changes and modifications are possible. References to preparations performed in a manner similar to other preparations, or in a general manner for other preparations, may include variations in routine parameters such as small changes in time, temperature, work-up conditions, and reagent amounts. All title compounds' chemical names are generated using ChemDraw Plugin version 16.0.1.13c(90) or ChemDraw desktop version 16.0.1.13(90). Those skilled in the art will recognize that compounds of the present invention may have alternative names when different naming software is used.

[0247] compound synthesis Compounds of formula (I) are prepared using conventional organic synthesis methods. Suitable synthetic routes are shown in the general reaction scheme below. All starting materials are commercially available or readily prepared from commercially available starting materials by those skilled in the art. If a substituent described herein is not suitable for the synthetic method described herein, those skilled in the art will understand that such substituent can be protected with a suitable protecting group that is stable under the reaction conditions. The protecting group can be removed at an appropriate time in the reaction sequence to provide the desired intermediate or target compound. Suitable protecting groups and methods for protecting and deprotecting various substituents using such suitable protecting groups are well known to those skilled in the art, and examples are given in T. Greene and P. Wuts, Protecting Groups in Organic Synthesis (4th ed.), John Wiley & Sons, NY (2006). In some cases, substituents may be specifically selected to be reactive under the reaction conditions used. Under these circumstances, the reaction conditions may convert the selected substituent to another substituent useful as an intermediate or a desired substituent in the target compound.

[0248] Scheme 1 Intermediate 1: (2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxylic acid [ka] Commercial racemic trans-4-cotininecarboxylic acid (304 g, 1.38 mol) was purified by chiral preparative HPLC (61 injections) at 500 mL / min using a Chiralpak 1A 20u 101x210 mm substrate with elution in 50% acetonitrile in methanol containing 0.1% formic acid. The desired fraction was collected and concentrated at 45°C. The solid residue was stirred in acetonitrile, filtered, and dried under reduced pressure for 18 hours to obtain the title compound as a solid with a whiteness of 10 (143.6 g, 652 mmol, 94.5% yield). Chiral HPLC for analysis: 95% ee, retention time 2.5 min, Chiralpak AD-H 5u 4.6x150 mm, methanol containing 0.1% formic acid, 1.0 mL / min; αD = +58°C (c = 0.3, CH3OH); VCD analysis was used to assign absolute stereochemistry. LCMS m / z 221.1(M+H)+. 1 H NMR(400MHz,DMSO-d6)δ ppm 2.48-2.49(m,2H)2.53-2.61(m,1H)2.71-2.80(m,1H)3.06-3.15(m,1H)3.34(br s,1H)4.79(d,J=6.3Hz,1H)7.3515-7.57(m,1H)7.74(dt,J=7.9,2.0Hz,1H)8.54(d,J=1.8Hz,1H)8.57(dd,J=4.7,1.7Hz,1H)12.78(br s,1H).

[0249] Intermediate 2: (1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-20 carboxylic acid, hydrochloride. [ka] [ka]

[0250] Step 1: Methyl(E)-4-(((1r,4r)-4-(2-dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoate. [ka]

[0251] At room temperature, a stirred solution of (1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexane-1-ol (250 g, 736 mmol) in toluene (2500 mL) was mixed with methylbuta-2-inoate (140 g, 1423 mmol), triphenylphosphine (19.32 g, 73.6 mmol), and acetic acid (16.86 mL, 295 mmol). The resulting solution was stirred at 115 °C for 16 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain the crude compound. The crude compound was adsorbed onto silica gel (500 g, 60-120 mesh) and purified by manual column chromatography (1.5 kg, 100-200 mesh) with elution using 15% ethyl acetate in petroleum ether to obtain methyl (E&Z)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoate (350 g) as a mixture of E / Z isomers (52.48% and 21.15%). To separate both isomers, the compound was adsorbed onto silica gel (500 g, 100-200 mesh) and purified by manual column chromatography (1.5 kg, 100-200 mesh) with elution using 15% ethyl acetate in petroleum ether to obtain the title compound (240 g, 463 mmol, yield 62.9%, purity 84.45%) as a pale yellow liquid. LC-MS m / z 438.3(M+H)+.

[0252] Step 2: (E)-4-(((1,4-trans)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoic acid [ka] Methyl(E)-4-(((1,4-trans)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoate (9.03 g, 20.64 mmol) was dissolved in tetrahydrofuran (THF) (25 mL), and a 5.089 M sodium hydroxide aqueous solution (4.87 mL, 24.76 mmol) was added. A homogeneous pale yellow reaction mixture was heated under reflux for 1 hour. An additional 5.089 M sodium hydroxide (1.217 mL, 6.19 mmol) was added, and the reaction mixture was refluxed for 50 minutes. The reaction mixture was cooled over 60 minutes and concentrated under vacuum. The residue was azeotropically mixed twice with toluene to facilitate the removal of water. The residue was pumped under high vacuum to obtain the title compound (9.9 g, 22.17 mmol, yield 107%, purity 82%, E / Z mixture) as a yellow solid. LC-MS m / z 424.4(M+H)+.

[0253] Step 3: tert-butyl(1R,4r)-4-((E)-4-(((1r,4R)-4-(2-15(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enamide)cyclohexane-1-carboxylate. [ka]

[0254] (E)-4-(((1,4-trans)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoic acid, sodium salt (9.2 g, 20.60 mmol) was suspended in dry DMF (40 ml) with stirring. HATU (8.62 g, 22.66 mmol) was added as a solid, and a partially dissolved mixture was observed. The mixture was stirred for 30 minutes to obtain a partially dissolved greenish solution. Tert-butyl(1,4-trans)-4-aminocyclohexane-1-carboxylate (4.11 g, 20.60 mmol) was added as a solution in DMF (10 ml), followed by the addition of DIEA (10.80 mL, 61.8 mmol) in DMF (10 ml). An additional 10 ml of DMF was added, and the heterogeneous mixture was stirred at room temperature for 15 hours. An additional HATU (1.724 g, 4.53 mmol) was added, and the nearly homogeneous reaction mixture was stirred for 60 minutes. The turbid reaction mixture was stirred for another 60 minutes. The reaction mixture was diluted with 200 ml of HCl and 200 ml of water, and stirred for 10 minutes. The resulting homogeneous two-phase mixture was transferred to a separatory funnel, and the layers were separated. The aqueous layer was extracted twice more with 150 ml of HCl, and the combined HCl layers were washed four times with water and twice with saturated NaCl to remove DMF. The HCl extract was dried over sodium sulfate, filtered, concentrated under vacuum, and pumped under high vacuum to obtain an orange syrup. The syrup was purified by silica gel chromatography (IscoCombiflash, 330 gram gold column, 0-80% Â:heptane, 45 minutes, flow rate 150 ml / min, loaded as a solution in DCM) to obtain the title compound (4.55 g, 7.52 mmol, yield 36.5%) as a white foamy solid. LC-MS m / z 605.5(M+H)+.

[0255] Step 4: tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate [ka]

[0256] In a 500 ml three-necked RB flask, tert-butyl(1R,4r)-4-((E)-4-(((1r,4R)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enamido)cyclohexane-1-carboxylate (6.40 g, 10.58 mmol) and isopropanol (120 mL) were added, and the suspension was stirred until a homogeneous solution was achieved. 10% wet Pd-C (0.640 g, 6.01 mmol) was added, and the flask was evacuated and placed under two hydrogen balloons attached to the end neck of the flask. The middle neck was covered with a rubber diaphragm. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was degassed and filtered twice through Celite. The filtrate was concentrated under vacuum and pumped under high vacuum to obtain a waxy gray solid with a slight isopropanol odor. The waxy solid was dissolved in DCM and concentrated under vacuum at 54°C for 20 minutes to promote the removal of isopropanol. The residue was pumped under high vacuum to obtain the title compound (4.44 g, 10.41 mmol, 98% yield) as a waxy gray solid. LC-MS m / z 427.4(M+H)+.

[0257] Step 5: tert-butyl(1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate. [ka]

[0258] (2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxylic acid (intermediate 1) (2.287 g, 10.38 mmol) was suspended in 30 ml of DCM in a 500 ml RB flask with stirring at room temperature. HATU (4.34 g, 11.42 mmol) was added, and the suspension was stirred for 15 minutes. A solution of tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (4.43 g, 10.38 mmol) in 20 ml of DCM was added dropwise by pipette over 15 minutes. After the addition was complete, a solution of DIEA (5.44 mL, 31.2 mmol) in DCM (10 ml) was added dropwise over 10 minutes, and the resulting homogeneous dark solution was stirred at room temperature for 16 hours. The reaction mixture was concentrated under vacuum to remove DCM and DIEA. The residue was dissolved in 100 ml of DCM and transferred to a separatory funnel. 20 ml of saturated sodium bicarbonate was added. The layers were separated, the DCM layer was washed with saturated NaCl, dried over sodium sulfate, filtered, concentrated under vacuum, and pumped under high vacuum to obtain an orange syrup. This was purified by silica gel chromatography (IscoCombiflash, 0-10% MeOH:DCM, 60 minutes or more, 330 g gold column, flow rate of 150 ml / min, loaded as 30 ml of DCM solution) to obtain the title compound (4.13 g, 6.57 mmol, 63.2% yield) as a white solid. LC-MS m / z 629.3(M+H)+.

[0259] Step 6: (1R,4r)-4-(4-(((1S,4R)-4-(((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid, hydrochloride. [ka]

[0260] In a 250 ml RB flask, tert-butyl(1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (4.13 g, 6.57 mmol) was dissolved in dry 1,4-dioxane (13 ml) with stirring. 4 M anhydrous HCl (39 mL, 156 mmol) was added to the 1,4-dioxane, and the mixture was stirred at room temperature. Insoluble oil was observed upon addition of HCl. The mixture was stirred at room temperature for 90 minutes. The reaction mixture was concentrated under vacuum (bath temperature 60°C), pumped under high vacuum for 15 hours, and the title compound (4.187 g, 6.87 mmol, yield 105%) was obtained as a white solid. LC-MS m / z 573.4(M+H)+. 1H NMR(400MHz,DMSO-d6)δ1.09-1.37(m,10H),1.58-1.70(m,2H),1.73-1.91(m,7H),2.03-2.16(m,3H),2.43-2.49(m,1H)2.72(dd,J=16.9,9. 5Hz,1H)2.95-3.05(m,1H)3.08-3.23(m,4H),3.27-3.48(m,5H),3.55-3.57(m,3H),4.81(d,J=5.9Hz,1H)7.84-7.92(m,J=4.9Hz,1H)8.05(br t,J=5.9Hz,1H)8.19-8.28(m,1H)8.71-8.76(m,1H)8.76-8.83(m,1H).

[0261] Intermediate 3: Methyl(1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate. [ka] [ka]

[0262] Step 1. Sodium (E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoate (Intermediate 3A).

Chem.

[0263] Methyl (E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoate (5.02 g, 11.47 mmol) was dissolved in THF (14.5 mL), and 5.089 M aqueous sodium hydroxide solution (3.38 ml, 17.21 mmol) was added. The unstirred mixture formed two distinct layers at room temperature. The pale yellow reaction mixture was heated at 80 °C for 18 h, then cooled to room temperature and concentrated under vacuum. The resulting residue was co-evaporated with toluene (2 × 50 mL) and dried under high vacuum to give the title compound (6.16 g, 12.3 mmol, > theoretical yield) as a sticky yellow solid. LCMS m / z 424.2 (M+H)+. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.07 - 1.26 (m, 4H) 1.77 - 1.93 (m, 4H) 2.53 - 2.58 (m, 2H) 3.15 - 3.28 (m, 2H) 3.45 - 3.53 (m, 2H) 3.56 - 3.65 (m, 4H) 3.89 - 4.01 (m, 2H) 5.68 - 5.77 (m, 1H) 6.09 - 6.26 (m, 1H) 7.12 - 7.20 (m, 6H) 7.21 - 7.29 (m, 6H) 7.30 - 7.39 (m, 8H).

[0264] Step 2. Methyl (1R,4r)-4-((E)-4-(((1r,4R)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enamide)cyclohexane-1-carboxylate (Intermediate 3B)

Chem.

[0265] To a solution of (E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoate (2.25 g, 5.05 mmol), methyl(1r,4r)-4-aminocyclohexane-1-carboxylate hydrochloride (1.39 g, 7.18 mmol), and HATU (2.69 g, 7.07 mmol) in DCM (20 mL), DIEA (2.65 ml, 15.2 mmol) was added. The reaction mixture was stirred at room temperature for 3 nights, and then diluted with 10% methanol (100 mL) and saturated sodium bicarbonate aqueous solution (50 mL) in DCM. The layers were separated, and the aqueous layer was back-extracted with 10% methanol (2 × 30 mL) in DCM. The organic fractions were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by silica gel chromatography using elution with 0-100% ethyl acetate in heptane to obtain the title compound as an off-white solid (1.94 g, 3.24 mmol, yield 64.1%). LC-MS m / z 563.34(M+H)+. 1 H NMR(400MHz,chloroform-d)δ ppm1.10-1.23(m,2H)1.30-1.37(m,3H)1.51-1.67(m,2H)1.91-2.15(m,10H)2.21-2.32(m,1H)2.62-2.71(m,2H)3.20 -3.29(m,1H)3.29-3.39(m,1H)3.51-3.60(m,2H)3.66(s,4H)3.69(s,3H)3.78-3.93(m,1H)4.12-4.17(m,1H)5.37(br d,J=7.83Hz,1H)6.01(dt,J=15.28,2.14Hz,1H)6.82-6.90(m,1H)7.20-7.27(m,2H)7.29-7.35(m,4H)7.36-7.42(m,4H).

[0266] Step 3. Methyl(1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (intermediate 3). [ka]

[0267] Under a nitrogen atmosphere, Pd-C (10% wt, 0.366 g, 0.344 mmol) was added to a solution of methyl (1R,4r)-4-(((1r,4R)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enamido)cyclohexane-1-carboxylate (1.94 g, 3.44 mmol). The flask was evacuated, backfilled with a hydrogen gas balloon, and stirred at room temperature overnight for 32 hours. The flask was again evacuated and backfilled with a new hydrogen gas balloon. After 22 hours, the mixture was filtered through a Celite pad and washed with additional methanol (2 x 20 mL). The filtrate was concentrated under vacuum and dried under high vacuum to obtain the title compound as an off-white solid (1.19 g, 3.08 mmol, 89% yield). LCMS m / z 563.34(M+H)+. 1 H NMR(400MHz,DMSO-d6)δ ppm1.06-1.26(m,4H)1.29-1.45(m,2H)1.59-1.71(m,2H)1.74-1.82(m,2H)1.83-1.95(m,4H)2.02-2.12(m,2H)2.19-2.31(m,1 H)2.58-2.64(m,1H)3.13-3.28(m,4H)3.29-3.38(m,8H)3.39-3.54(m,1H)3.59(s,3H)4.04-4.15(m,1H)7.66(d,J=7.82Hz,1H).

[0268] Intermediate 4: Benzyl(1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate [ka] [ka]

[0269] Step 1: Methyl 4-(((1r,4r)-4-(2-aminoethoxy)cyclohexyl)oxy)butanoic acid (intermediate 4A) [ka]

[0270] Under a nitrogen atmosphere, Pd-C (0.608 g, 0.571 mmol) was added to a solution of methyl(E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoate (5.00 g, 11.4 mmol). The flask was evacuated, backfilled with a hydrogen gas balloon, and stirred at room temperature for 3 nights. The flask was then evacuated again and backfilled with nitrogen gas. Additional Pd-C (0.608 g, 0.571 mmol) was added. The flask was again evacuated and backfilled with a new hydrogen gas balloon. The reaction mixture was stirred at room temperature for 24 hours and filtered through a Celite pad. Pd-C (0.608 g, 0.571 mmol) was added to the filtrate. The flask was evacuated again, backfilled with a new hydrogen gas balloon, and stirred at room temperature for 22 hours.

[0271] An additional 0.608 g of Pd-C (0.571 mmol) was added. The flask was evacuated and backfilled with a new hydrogen gas balloon. After another 24 hours, an additional 0.608 g of Pd-C (0.571 mmol) was added. The flask was again evacuated and backfilled with a hydrogen gas balloon. After stirring at room temperature for 5 hours, the mixture was filtered through a Celite pad and washed with additional methanol (2 x 20 mL). The filtrate was concentrated under vacuum and dried under high vacuum to obtain the title compound as a yellow semi-solid (5.22 g, 10.1 mmol, yield 88%). LC-MS m / z 260.28 (M+H)+. 1 H NMR(400MHz,chloroform-d)δ ppm1.17-1.42(m,4H)1.84-1.93(m,2H)1.94-2.05(m,4H)2.35-2.50(m,2H)2.84-2.93(m,2H)3.19-3.37(m,2H)3.41-3.57(m,4H)3.69(s,3H).

[0272] Step 2: Methyl 4-(((1r,4r)-4-(2-((tert-butoxycarbonyl)amino)ethoxy)cyclohexyl)oxy)butanoic acid (intermediate 4B) [ka]

[0273] To a solution of methyl 4-(((1r,4r)-4-(2-aminoethoxy)cyclohexyl)oxy)butanoic acid (1.20 g, 4.63 mmol) in dichloromethane (23 mL), di-tert-butyl dicarbonate (1.52 g, 6.94 mmol) and DIPEA (1.616 ml, 9.25 mmol) were added. The mixture was stirred at room temperature for 19 hours, and then diluted with additional dichloromethane (50 mL). The organic solution was washed with saturated sodium bicarbonate aqueous solution (30 mL) and brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution using 0-10% methanol in dichloromethane to obtain the title compound as a yellow oil (1.22 g, 3.00 mmol, yield 88%). LCMS m / z 360.14(M+H)+. 1 ¹H NMR (400MHz, chloroform-d) δ ppm 1.41-1.51 (m,9H) 1.85-1.93 (m,3H) 1.94-2.05 (m,5H) 2.26-2.33 (m,1H) 2.42 (t,J=7.34Hz,2H) 3.17-3.34 (m,4H) 3.36-3.60 (m,6H) 3.69 (s,3H).

[0274] Step 3: 4-(((1r,4r)-4-(2-((tert-butoxycarbonyl)amino)ethoxy)cyclohexyl)oxy)butanoate sodium (1.23g, 3.35mmol), benzyl(1r,4r)-4-aminocyclohexane-1-carboxylate hydrochloride (intermediate 4C) [ka]

[0275] To a solution of methyl 4-(((1r,4r)-4-(2-((tert-butoxycarbonyl)amino)ethoxy)cyclohexyl)oxy)butanoic acid (1.21 g, 3.36 mmol) in THF (13.4 mL), 5.089 molar aqueous sodium hydroxide solution (0.990 ml, 5.04 mmol) was added. The mixture was stirred at room temperature for 18 hours, then concentrated under vacuum and dried under high vacuum to obtain the title compound as a pale yellow solid (1.234 g, 3.36 mmol, theoretical yield). LCMS m / z 346.18(M+H)+. 1 H NMR (400MHz, DMSO-d6) δ ppm 1.34-1.41 (m, 9H) 1.53-1.68 (m, 4H) 1.77-1.93 (m, 6H) 2.06-2.12 (m, 1H) 2.96-3.06 (m, 2H) 3.29-3.41 (m, 7H).

[0276] Step 4: Benzyl(1R,4r)-4-(4-(((1r,4R)-4-(2-((tert-butoxycarbonyl)amino)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (intermediate 4D) [ka]

[0277] To a solution of 4-(((1r,4r)-4-(2-((tert-butoxycarbonyl)amino)ethoxy)cyclohexyl)oxy)butanoate sodium (1.23 g, 3.35 mmol), benzyl(1r,4r)-4-aminocyclohexane-1-carboxylate hydrochloride (0.903 g, 3.35 mmol), and HATU (1.782 g, 4.69 mmol) in DCM (13.4 mL), DIPEA (1.75 mL, 10.0 mmol) was added. The mixture was stirred at room temperature for 3 nights, and then diluted with an additional DCM (100 mL). The organic solution was washed with saturated sodium bicarbonate aqueous solution (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution in 0-15% methanol in dichloromethane to obtain the title compound as a pale yellow oily substance. LCMS m / z 561.34(M+H)+. 1 H NMR(400MHz,chloroform-d)δ ppm1.42-1.53(m,9H)1.57-1.63(m,2H)1.84-1.92(m,2H)1.96-2.03(m,4H)2.03-2.12(m,4H)2.22-2.31(m,2H)3.16-3.25(m ,4H)3.25-3.35(m,3H)3.44-3.56(m,4H)3.66-3.84(m,5H)4.81-4.95(m,1H)5.13(s,2H)5.58-5.65(m,1H)7.31-7.44(m,5H).

[0278] Step 5: Benzyl(1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (intermediate 4) [ka]

[0279] Benzyl(1R,4R)-4-(4-(((1R,4R)-4-(2-((tert-butoxycarbonyl)amino)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate in DCM (12.6 mL)

[0280] To a solution of (1.77 g, 3.16 mmol), TFA (2.43 mL, 31.6 mmol) was added. The mixture was stirred at room temperature for two nights. Subsequently, the reactants were concentrated, azeotropically mixed with toluene (2x), and dried under high vacuum to obtain the title compound as a pale yellow semi-solid (2.98 g, 1.81 mmol, theoretical yield). LCMS m / z 461.33(M+H)+. 1 H NMR(400MHz,DMSO-d6)δ ppm1.07-1.30(m,28H)1.34-1.48(m,2H)1.59-1.73(m,2H)1.75-1.83(m,2H)1.85-1.98(m,4H)2.03-2.11(m,1H)2.92-2.98(m ,1H)3.10-3.21(m,3H)3.30-3.37(m,2H)3.52-3.68(m,5H)5.09(s,2H)7.29-7.44(m,4H)7.61-7.75(m,2H)8.18-8.32(m,1H).

[0281] Intermediate 5: (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-2-one, 2-hydrochloride salt. [ka] [ka]

[0282] Step 1: Ethyl(1R,2S,5R)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidine-1-yl)-5-(isopropyl(methyl)amino)cyclohexane-1-carboxylate [ka]

[0283] At room temperature, a mixture of ethyl (1R,2S)-2-((S)-3-((benzyloxy)carbonyl)amino)-2-oxopyrrolidine-1-yl)-5-oxocyclohexane-1-carboxylate (28.0 g, 69.6 mmol) and isopropylmethylamine (12.32 mL, 118 mmol) in dichloromethane (DCM) (270 mL) was mixed with titanium(IV) isopropoxide (30.6 mL, 104 mmol). The mixture was stirred under a nitrogen atmosphere at room temperature for 21 hours. 5% Pt / C (4.07 g, 1.044 mmol) was added, and the mixture was stirred under a hydrogen balloon atmosphere at room temperature for 29 hours. The hydrogen balloon was refilled after 7 hours and 16 hours. The mixture was filtered through Celite®, and the catalyst was washed with dichloromethane (DCM). The combined filtrate was concentrated under reduced pressure. The residue was dissolved in dichloromethane (DCM) (80 mL), placed in an ice bath, and ethyl acetate (250 mL) and Celite® (5 g) were added. The mixture was stirred at room temperature for 4 hours and sonicated at room temperature for 20 minutes. The mixture was filtered using Celite® and wet ethyl acetate (4 × 70 mL). The combined filtrate was concentrated under reduced pressure and washed and extracted with dichloromethane (DCM) (3 × 100 mL) to obtain the title compound as a light brown oily foam (24.58 g, 77%). LC-MS m / z 460.5(M+H)+.

[0284] Step 2: (1R,2S,5R)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidine-1-yl)-5-(isopropyl(methyl)amino)cyclohexanecarboxylic acid. [ka]

[0285] Ethyl (1R,2S,5R)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidine-1-yl)-5-(isopropyl(methyl)amino)cyclohexane-1-carboxylate (24.58 g, 53.5 mmol) was dissolved in toluene (140 mL) and dichloromethane (DCM) (5 mL), and the solution was extracted with 2 N HCl (2 x 67 mL). The combined aqueous extract was placed in a metal insert under a nitrogen atmosphere and heated at 63 °C for 22 hours. The mixture was placed in an ice bath and 10 N sodium hydroxide (32 mL) was added. The final temperature of the mixture was 16 °C. The mixture was washed with toluene (150 mL), and the aqueous phase was filtered. The aqueous emulsion (20 mL) was separated and filtered through Celite®. The combined aqueous phase was cooled in an ice bath, and the pH was adjusted to 6-7 with concentrated HCl. The mixture was saturated with NaCl and extracted with dichloromethane (DCM) (200 mL). Concentrated HCl (0.5 mL) was added, and the aqueous phase was extracted with 10% methanol in dichloromethane (DCM) (2 × 100 mL). The combined organic extracts were dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound as a beige foamy solid (14.18 g, 2.74 mmol, 61%). LC-MS m / z 432.4(M+H)+.

[0286] Step 3: tert-butyl((1R,2S,5R)-2-((S)-3-(((benxyloxy)carbonyl)amino)-2-oxopyrrolidine-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)carbamate. [ka]

[0287] (1R,2S,5R)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidine-1-yl)-5-(isopropyl(methyl)amino)cyclohexanecarboxylic acid (14.18 g, 32.9 mmol) was washed and extracted with dichloromethane (DCM) (30 mL) and toluene (3 × 330 mL). Under a nitrogen atmosphere, anhydrous tert-butanol (31.0 mL, 329 mmol) and triethylamine (16.0 mL, 115 mmol) were added to the solution of the residue in anhydrous toluene (121 mL), and the mixture was heated in a metal insert at 85°C for 5 minutes. DPPA (7.79 mL, 36.1 mmol) was added dropwise over 14 minutes, and the mixture was heated under a nitrogen atmosphere at 85°C for 2.75 hours. The mixture was cooled to room temperature, ethyl acetate (280 mL) was added, and the internal temperature was adjusted to 2-3°C using an ice bath. Saturated NaHCO3 (280 mL) was added dropwise over 20 minutes, and the mixture was stirred at room temperature for 2 hours. The organic phase was washed with brine (40 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was left under vacuum at room temperature for 2 days. Tert-butanol (100 mL) was added, and the mixture was sonicated at room temperature for 20 minutes. 1N sodium hydroxide (80 mL) was added, and the mixture was stirred at room temperature for 30 minutes, and concentrated to a volume of 80 mL under reduced pressure. Dichloromethane (DCM) (200 mL) and water (40 mL) were added, the organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was traced and washed with ethyl acetate (2 × 85 mL) for extraction. The residue was stirred with ethyl acetate (30 mL) and heptane (40 mL), filtered, and washed with a 1:10 ethyl acetate / heptane solution to obtain the title compound as a white solid (8.03 g, 15.02 mmol, 48.6%). LC-MS m / z 503.5(M+H)+.

[0288] Step 4: tert-butyl((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidine-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)carbamate [ka]

[0289] To a solution of tert-butyl((1R,2S,5R)-2-((S)-3-(((benxyloxy)carbonyl)amino)-2-oxopyrrolidine-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)carbamate (4.99 g, 9.93 mmol) in ethanol (70 mL), 20 wt% Pd(OH)2 / C (0.7 g, 0.993 mmol) was added, and the mixture was stirred at room temperature under a balloon hydrogen atmosphere for 23 hours. The mixture was filtered through Celite® under a nitrogen atmosphere, and the catalyst was washed with ethanol (3 × 10 mL). The combined filtrate was concentrated under reduced pressure, and the residue was left overnight at room temperature under vacuum to obtain the title compound as a white solid (3.64 g, 9.38 mmol, yield 99%). LC-MS m / z 369.4(M+H)+.

[0290] Step 5: tert-butyl((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazoline-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamate [ka]

[0291] To a solution of tert-butyl((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidine-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl) carbamate (3.64 g, 9.88 mmol) in ethanol (100 mL), commercially available 4-chloro-6-(trifluoromethyl)quinazoline (2.30 g, 9.88 mmol) and DIPEA (2.77 mL, 15.84 mmol) were added, and the mixture was heated at 50°C for 3 hours under a nitrogen atmosphere using a metal insert. The mixture was concentrated under reduced pressure, dichloromethane (DCM) (150 mL) was added, and the organic phase was washed with water (30 mL). The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by eluting DCM in MeOH containing a 0-75% gradient of DCM / 1% NH4OH using ISCO CombiFlash® Rf (220 g RediSep® Rf Gold column, 120 mL / min), followed by washing of the column with MeOH. The desired fractions were combined and dried under reduced pressure to obtain the title compound as a white solid (4.32 g, 7.57 mmol, 77% yield). LC-MS m / z 565.2 (M+H) +

[0292] Step 6: (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-2-one, 2 hydrochloride salt. [ka]

[0293] A mixture of tert-butyl((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamate (3.18 g, 5.63 mmol) in dichloromethane (DCM) (8 mL) was mixed with HCl (7.04 mL, 28.2 mmol). The mixture was stirred at room temperature for 3 hours, concentrated to dryness, and the title compound was obtained as a white solid (3.5 g, 6.51 mmol, yield 116%). LC-MS m / z 465.3(M+H)+.

[0294] Intermediate 6: (S)-1-((1S,2R,4R)-2-amino-4-(tert-butylamino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-2-one [ka] [ka]

[0295] Step 1: Benzyl((S)-1-((7R,8S)-7-acetamido-1,4-dioxaspiro=[4.5]decane-8-yl)-2-oxopyrrolidine-3-yl)carbamate. [ka]

[0296] At room temperature, triethylamine (0.500 L, 3.58 mmol) was added to a solution of (7R,8S)-8-((S)-3-(((benzyloxy)carbonyl)amino-2-oxopyrrolidine-1-yl)-1,4-dioxaspiro[4.5]decane-7-carboxylic acid (1.5 g, 3.58 mmol) in toluene (15 mL). The mixture was cooled to -10°C, isobutyl chloroformate (0.471 mL, 3.58 mmol) was added, and the mixture was stirred at 0°C to -10°C for 30 minutes. Sodium azide (0.419 g, 6.45 mmol) and tetrabutylane bromide were added to water (3.00 mL). A solution of monium (0.058 g, 0.179 mmol) was added, and the mixture was stirred at 0°C to -10°C for 2 hours. Water (50 mL) and toluene (100 mL) were added, and the mixture was stirred for 10 minutes. The organic phase was dried over a molecular sieve (4 Å), and acetic anhydride (0.744 mL, 7.89 mmol) and acetic acid (0.267 mL, 4.66 mmol) were added, and the mixture was stirred at 90°C for 4 hours. The mixture was cooled to room temperature and concentrated under reduced pressure. The residue was pulverized with pentane (20 mL) to obtain the title compound as an off-white solid (0.8 g, 1.714 mmol, yield 47.8%). LC-MS m / z 432.2(M+H)+.

[0297] Step 2: Benzyl((S)-1-((1S,2R)-2-acetamido-4-oxocyclohexyl)-2-oxopyrrolidine-3-yl)carbamate. [ka]

[0298] To a solution of benzyl((S)-1-((7R,8S)-7-acetamido-1,4-dioxaspiro[4.5]decane-8-yl)-2-oxopyrrolidine-3-yl)carbamate (800 mg, 1.854 mmol) in acetone (10 mL), HCl (5 mL, 5.00 mmol) was added, and the mixture was stirred at 50°C for 2 hours. The mixture was cooled to room temperature and concentrated. Water (10 mL) was added, and the mixture was extracted with dichloromethane (DCM) (2 × 50 mL). The combined organic extract was dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated. The residue was pulverized with diethyl ether (10 mL) to obtain the title compound as an off-white solid (600 mg, 1.490 mmol, yield 80%). LC-MS m / z 388.2(M+H)+.

[0299] Step 3: Benzyl((S)-1-((1S,2R,4R)-2-acetamido-4-(tert-butylamino)cyclohexyl)-2-oxopyrrolidine-3-yl)carbamate. [ka]

[0300] TiCl2(i-OiPr)2 was pre-formed at 5-10°C by adding titanium(IV) isopropoxide (0.282 mL, 0.964 mmol) to 1 M TiCl4 in dichloromethane (DCM) (0.964 mL, 0.964 mmol), and the mixture was stirred for 15 minutes. At -20°C, the pre-formed TiCl2(i-OiPr)2 was added to a solution of benzyl((S)-1-((1S,2R)-2-acetamido-4-oxocyclohexyl)-2-oxopyrrolidine-3-yl) carbamate (600 mg, 1.607 mmol) and tert-butylamine (0.851 mL, 8.03 mmol) in dichloromethane (DCM) (10 mL). The mixture was warmed to room temperature and stirred for 2 hours. Add borane-dimethyl sulfide complex (0.153 mL, 1.607 mmol) and stir the mixture at room temperature for 16 hours. Add dichloromethane (DCM) (50 mL) and water (50 mL) and stir the mixture for 10 minutes. Filter the emulsion through Celite® and extract the aqueous phase with dichloromethane (DCM) (50 mL). Add 1N HCl (20 mL) to the combined organic extract and stir the mixture for 10 minutes. Add dichloromethane (DCM) (50 mL) and adjust the pH to 8-9 with ammonium hydroxide solution. Wash the organic phase with ammonium chloride solution (14%) (2 × 25 mL), dry over anhydrous Na₂SO₄, filter, and evaporate the filtrate. The title compound was obtained as an off-white solid by purification using column chromatography (neutral alumina column) with elution in 2% methanol in dichloromethane (DCM) (300 mg, 0.673 mmol, yield 41.9%). LC-MS m / z 445.48(M+H)+.

[0301] Step 4: N-((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidine-1-yl)-5-(tert-butylamino)cyclohexyl)acetamide. [ka]

[0302] A mixture of benzyl((S)-1-((1S,2R,4R)-2-acetamido-4-(tert-butylamino)cyclohexyl)-2-oxopyrrolidine-3-yl)carbamate (1.00 g, 2.249 mmol) and 10% Pd / C (100 mg, 0.094 mmol) in methanol (10 mL) was stirred at room temperature for 2 hours under a balloon hydrogen atmosphere. The mixture was filtered through Celite®, the catalyst was washed with methanol, and the combined filtrate was concentrated under reduced pressure to obtain the title compound as an off-white solid (696.1 mg, 2.242 mmol, 100% yield). LC-MS m / z 311.5(M+H)+.

[0303] Step 5: N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-((2-chloro-6-(trifluoromethyl)quinazolin-4-yl)amino)-2-oxopyrrolidine-1-yl)cyclohexyl)acetamide. [ka]

[0304] A mixture of N-((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidine-1-yl)-5-(tert-butylamino)cyclohexyl)acetamide (683 mg, 2,200 mmol), commercially available 2,4-dichloro-6-(trifluoromethyl)quinazoline (587 mg, 2,200 mmol), and DIPEA (0.615 mL, 3.52 mmol) in ethanol (20 mL) was stirred at room temperature for 2 hours, and the mixture was then concentrated under reduced pressure. Saturated NaHCO3 was added, and the mixture was extracted with ethyl acetate. The combined organic extract was washed with saturated NaCl, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound as a pale yellow solid (1.128 g, 2.085 mmol, 95% yield). LC-MS m / z 541.5(M+H)+.

[0305] Step 6: N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)acetamide. [ka]

[0306] To a solution of N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-((2-chloro-6-(trifluoromethyl)quinazolin-4-yl)amino)-2-oxopyrrolidine-1-yl)cyclohexyl)acetamide (4.61 g, 8.52 mmol) in methanol (150 mL), 10% Pd / C (1.360 g, 1.278 mmol) and Cs2CO3 (4.16 g, 12.78 mmol) were added. The mixture was stirred at room temperature for 3 hours under a balloon hydrogen atmosphere. The mixture was filtered, and the catalyst was washed with methanol and dichloromethane (DCM). The combined filtrate was concentrated to dryness, the residue was washed with dichloromethane (DCM), and the combined filtrate was concentrated to dryness to obtain the title compound as a solid (4.97 g, 9.81 mmol, yield 115%). LC-MS m / z 507.1(M+H)+.

[0307] Step 7: (S)-1-((1S,2R,4R)-2-amino-4-(tert-butylamino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-2-one [ka]

[0308] A mixture of N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)acetamide (814 mg, 1.607 mmol) and 2 M HCl (7.231 mL, 14.46 mmol) was stirred at 50°C for 6 days. The mixture was basicized with saturated NaHCO3 and concentrated to dryness under reduced pressure. The residue was suspended in 10% methanol in dichloromethane (DCM), stirred at room temperature for 1 hour, and filtered. The solid was washed with 10% methanol in dichloromethane (DCM), and the combined filtrate was concentrated. The title compound was obtained as a white solid (300 mg, 0.646 mmol, 40.2% yield) by purification using ISCO CombiFlash® chromatography (80 g RediSep Rf Gold® column, 60 mL / min) with elution using a 0-15% methanol gradient containing ammonium hydroxide (10%) in dichloromethane (DCM). LC-MS m / z 465.4(M+H)+.

[0309] Intermediate 7: (1S,3R)-3-(((benzyloxy)carbonyl)amino)-1-isopropylcyclopentane-1-carboxylic acid [ka] [ka]

[0310] To a solution of methyl(1S,3R)-3-((tert-butoxycarbonyl)amino)-1-isopropylcyclopentane-1-carboxylate (3.64 g, 12.75 mmol, J.Med.Chem.,2013,56(19),7706) in methanol (75 mL), a solution of lithium hydroxide (1.527 g, 63.8 mmol) in water (15 mL) was added, followed by the addition of tetrahydrofuran (THF) (7.5 mL). The mixture was refluxed for 24 hours and cooled to room temperature. 1.0 M HCl (63.8 mL, 63.8 mmol) was slowly added, and the mixture was extracted with dichloromethane (DCM) (3 x 150 mL). The combined organic extract was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in 2,2,2-trifluoroacetic acid (20 mL, 12.75 mmol). After 1 hour, 12M hydrochloric acid (50.0 mL, 600 mmol) was added, and the mixture was heated at 100°C for 16 hours and concentrated under reduced pressure. The residue was dissolved in a mixture of 1,4-dioxane (50 mL) and 1.0M sodium hydroxide (50.0 mL, 50.0 mmol), cooled to 0°C, and Cbz-Cl (2.367 mL, 16.58 mmol) was added dropwise. After stirring at 0°C for 20 hours, the mixture was extracted with ether (100 mL). The aqueous layer was acidified with 1.0M HCl (50.0 mL, 50.0 mmol) and extracted with diethyl ether (150 mL). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound as a brown oily substance (3.32 g, 10.87 mmol, yield 85%). LC-MS m / z 306.3(M+H)+.

[0311] Intermediate 8A: benzyl(R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate, and intermediate 8B: benzyl(S)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate. [ka] [ka]

[0312] Step 1: Benzyl 3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine 6(5H)-carboxylate. [ka]

[0313] A mixture of commercially available 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthiridine dihydrochloride (5.0 g, 18.18 mmol) in 2-methyltetrahydrofuran (2-MeTHF) (100 mL) was stirred at room temperature for 1 minute. 1.0 M sodium hydroxide (72.7 mL, 72.7 mmol) was slowly added, and the mixture was stirred for 5 minutes. Cbz-Cl (2.72 mL, 19.08 mmol) was added dropwise at room temperature, and the mixture was stirred for 1 hour. 2-methyltetrahydrofuran (2-MeTHF) was removed under reduced pressure, and the resulting emulsion was stirred until the oily substance solidified into a yellow solid. The solid was filtered, ground into a fine powder with a spatula, washed with water, and air-dried overnight under vacuum filtration to obtain the title compound as a yellow solid (6.06 g, 18.02 mmol, 99% yield). LC-MS m / z 337.3(M+H)+.

[0314] Step 2: 6-((benzyloxy)carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine 1-oxide [ka]

[0315] At room temperature, a solution of benzyl 3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (6.0 g, 17.84 mmol) in dichloromethane (DCM) (10.0 mL) was mixed with methyltrioxorenium (vii) (0.160 g, 0.642 mmol), followed by the dropwise addition of 30% hydrogen peroxide (3.64 mL, 35.7 mmol). The mixture was stirred at room temperature for 20 hours. Excess hydrogen peroxide was decomposed by the addition of manganese(IV) oxide (1.0 mg, 0.012 mmol), and the mixture was stirred for 30 minutes. Dichloromethane (DCM) (50 mL) and 1.0 M NaOH (50 mL) were added, and the aqueous phase was extracted with dichloromethane (DCM) (50 mL). The combined organic extract was dried over Na2SO4 and filtered. The filtrate was diluted with toluene (50 mL) and concentrated under reduced pressure to obtain the title compound as a white solid (6.20 g, 17.6 mmol, 99% yield). LC-MS m / z 353.3(M+H)+.

[0316] Step 3: Benzyl 8-bromo-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate. [ka]

[0317] At room temperature, over 7 minutes, two solutions were simultaneously added dropwise from two separate syringes to a solution of 6-((benzyloxy)carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthirizine 1-oxide (6.20 g, 17.60 mmol) in chloroform (30 mL): a solution of phosphorus oxybromide (10.09 g, 35.2 mmol) in chloroform (10.0 mL) and a solution of triethylamine (1.385 mL, 9.93 mmol) in chloroform (10.0 mL). The mixture was stirred at room temperature for 45 minutes, poured onto ice, and basicized with 2.0 M Na2CO3. The mixture was extracted with dichloromethane (DCM) (3 x 200 mL). The combined organic extract was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The title compound was obtained as a white crystalline solid by purification using conventional phase chromatography (330 g silica, 200 mL / min) with elution of a 0-40% gradient in heptane with ethyl acetate (2.83 g, 6.82 mmol, yield 38.7%). LC-MS m / z 417.1(M+H)+.

[0318] Step 4: Benzyl(R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthiridine-6(5H)-carboxylate, and benzyl(S)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthiridine-6(5H)-carboxylate. [ka]

[0319] A solution of benzyl 8-bromo-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (2.70 g, 6.50 mmol) in N,N-dimethylformamide (DMF) (25.0 mL) was mixed with sodium azide (1.268 mg, 19.51 mmol) and stirred at room temperature for 18 hours. The flask was purged with nitrogen, and freshly degassed tetrahydrofuran (THF) (30.000 mL) and water (6.000 mL) were added, followed by 1.0 M trimethylphosphine (32.5 mL, 32.5 mmol) in toluene. The mixture was stirred at room temperature for 20 hours and concentrated under reduced pressure. The residue was separated between diethyl ether and water to separate the organic phase, dried over Na2SO4, and concentrated under reduced pressure. The title compound was obtained as a white solid by preparative chiral resolution using a Lux Amylose 2 column (5 μm, 21 x 250 mm, 20 mL / min) with elution in 30% heptane in ethanol containing 0.1% isopropylamine (990.1 mg, 2.82 mmol, yield 43.3%). LC-MS m / z 352.2(M+H)+. Benzyl(S)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate was also isolated as a white solid (888.7 mg, 2.53 mmol, yield 38.9%). LC-MS m / z 352.3(M+H)+. The absolute stereochemistry of both enantiomers was determined by VCD using the following procedure.

[0320] The samples were dissolved in CD3CN (16.8 mM) and placed in a cell with a BaF2 window and a 100 μm optical path length. IR and VCD spectra were recorded using a ChiralIR2X™ VCD spectrometer (BioTools Inc., Jupiter, FL) equipped with a dual PEM accessory, having a resolution of 4 cm⁻¹, with 6 hours of collection for one isomer E1 and 12 hours of collection for the other isomer E2, optimized at 1400 cm⁻¹. Conformational searches for the modeled (R) structure were performed using a Low Mode MOE with an MMFF94x force field and Born solvation, with a dielectric constant of 20 and an external dielectric constant of 47. Shape optimization, frequency, IR, and VCD intensity calculations for 10 conformational isomers obtained from conformational searches were performed using Gaussian 16 (Gaussian Inc., Wallingford, CT) with DFT level b3lyp / 6-31G(d)scrf=(solvent=dimethyl sulfoxide). The Gaussian output files were converted to VCD and IR spectra using BLAIR. The calculated frequencies were scaled to 0.981, and the IR and VCD intensities were converted to Lorentz bands with a full width at half maximum of 8 cm⁻¹ for comparison with experimental spectra. The assignments were evaluated using the CompareVOA program (BioTools Inc., Jupiter, FL), and confidence levels were produced based on the current database containing 88 previous correct assignments for various chiral structures.

[0321] Intermediate 9: ((R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-yl)((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)methanone [ka] [ka]

[0322] Step 1: Benzyl(R)-8-((tert-butoxycarbonyl)amino)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate. [ka]

[0323] Benzyl(R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (steps 3-4) (4.25 g, 12.10 mmol) was mixed with di-tert-butyl dicarbonate (2.90 g, 13.31 mmol) and water (24.00 mL), and the mixture was stirred for 5 minutes. Acetone (24.00 mL) was slowly added. The mixture was stirred for 2 hours, the precipitate was filtered, washed with water, and air-dried to obtain the title compound as a pale yellow solid (4.70 g, 10.41 mmol, yield 86%). LC-MS m / z 452.1(M+H)+.

[0324] Step 2: tert-butyl(R)-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-8-yl)carbamate. [ka]

[0325] A mixture of benzyl(R)-8-((tert-butoxycarbonyl)amino)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (4.60 g, 10.19 mmol) and 20% palladium hydroxide on carbon (2.216 g, 3.06 mmol) in methanol (100.000 mL) was sealed in a flask and purged with nitrogen. Hydrogen was blown into the mixture, and the mixture was stirred under a balloon hydrogen atmosphere for 1.5 hours. The mixture was exposed to air and stirred for 10 minutes. 7.0 M ammonia in methanol (100 mL) was added, the mixture was filtered, and the catalyst was washed with methanol. The combined filtrate was concentrated under reduced pressure. The title compound was obtained as a pale yellow foam (2.66 g, 8.38 mmol, 82% yield) by reverse-phase HPLC chromatography (C18 Aq 275 g gold column, 125 mL / min) by elution with acetonitrile in a 30-60% gradient in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%). LC-MS m / z 318.1(M+H)+.

[0326] Step 3: tert-butyl((R)-6-((1S,3R)-3-((benzyloxy)carbonyl)amino)-1-isopropylcyclopentan-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-8-yl)carbamate. [ka]

[0327] To a solution of (1S,3R)-3-(((benzyloxy)carbonyl)amino)-1-isopropylcyclopentane-1-carboxylic acid (5.12 g, 16.77 mmol) in dichloromethane (DCM), DIPEA (9.15 mL, 52.4 mmol) and HATU (5.90 g, 15.51 mmol) were added, and the mixture was stirred for 24 hours. The mixture was added to tert-butyl(R)-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyrizin-8-yl)carbamate (2.66 g, 8.38 mmol), stirred for 2 minutes, and the resulting solution was divided into two 20 mL vials. The vials were sealed and stirred at 80°C for 22 hours. The mixture was cooled to room temperature, water was added (1,000 mL each), the vials were sealed, and the mixture was stirred at 80°C for 50 minutes. The two mixtures were cooled to room temperature, mixed, and water (100 mL) and dichloromethane (DCM) (100 mL) were added. The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The title compound was obtained as a white foam (2.95 g, 4.88 mmol, yield 58.2%) by ISCO CombiFlash® chromatography (330 g silica, 200 mL / min) with elution of 0-10% gradient in dichloromethane (DCM) with methanol. LC-MS m / z 605.2(M+H)+.

[0328] Step 4: tert-butyl((R)-6-((1S,3R)-3-amino-1-isopropylcyclopentan-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-8-yl)carbamate. [ka]

[0329] A mixture of tert-butyl((R)-6-((1S,3R)-3-(((benzyloxy)carbonyl)amino)-1-isopropylcyclopentan-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-8-yl)carbamate (2.95 g, 4.88 mmol) and 20% palladium hydroxide on carbon (1.028 g, 1.464 mmol) was purged with nitrogen in methanol (50,000 mL). Hydrogen was blown into the mixture, and the mixture was stirred under a balloon hydrogen atmosphere for 1 hour. The mixture was exposed to air and stirred for 15 minutes. 2.0 M ammonia in methanol (100 mL) was added, and the mixture was filtered. The catalyst was washed with methanol, and the combined filtrate was concentrated under reduced pressure to obtain the title compound as a white foam (2.24 g, 4.76 mmol, 98% yield). LC-MS m / z 471.4(M+H)+.

[0330] Step 5: tert-butyl((R)-6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentan-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-8-yl)carbamate, 2 formate. [ka]

[0331] To a solution of tert-butyl((R)-6-((1S,3R)-3-amino-1-isopropylcyclopentan-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-8-yl)carbamate (2.24 g, 4.76 mmol) in isopropyl acetate (50.00 mL), 2-propanol (0.917 mL, 11.90 mmol) and triethylamine (0.664 mL, 4.76 mmol) were added. The mixture was cooled to 0°C, sodium triacetoxyborohydride (2.52 g, 11.90 mmol) was added, followed by the dropwise addition of a solution of (R)-3-methoxytetrahydro-4H-pyran-4-one (0.929 g, 7.14 mmol) in isopropyl acetate (50.00 mL). The mixture was stirred at 0°C for 20 minutes, warmed to room temperature, and stirred at room temperature for 2 hours. 1.0 M NaOH (200 mL) and isopropyl acetate (200 mL) were added, the organic phase was dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure. The title compound was obtained as a white foam (2.73 g, 4.03 mmol, 85% yield) by reverse-phase HPLC chromatography (C18 275 g gold column, 125 mL / min) eluting with 35% acetonitrile in water containing formic acid (0.1%). This compound was used as an intermediate in the next step. LC-MS m / z 585.2(M+H)+. Step 6: ((R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-yl)((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)methanone. [ka]

[0332] A mixture of tert-butyl((R)-6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentan-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-8-yl)carbamate, formate (2.73 g, 4.03 mmol) and trifluoroacetic acid (10 mL, 130 mmol) was stirred at room temperature for 15 minutes and concentrated under reduced pressure. Dichloromethane (DCM) (100 mL) and 1.0 M NaOH (100 mL) were added, and the aqueous phase was extracted with dichloromethane (DCM) (100 mL). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound as a white foam (1.77 g, 3.65 mmol, 91% yield). LC-MS m / z 485.4(M+H)+.

[0333] Intermediate 10: Di-tert-butyl(((S)-6-((S)-2-amino-3-(naphthalene-2-yl)propanamide)-1-(tert-butoxy)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid [ka] [ka]

[0334] Step 1: Di-tert-butyl(((S)-6-((S)-2-amino-3-(naphthalene-2-yl)propanamide)-1-(tert-butoxy)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid To a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(naphthalen-2-yl)propanoic acid (269 mg, 0.615 mmol) and HATU (304 mg, 0.800 mmol) in anhydrous N,N-dimethylformamide (6.2 mL), DIPEA (215 μl, 1.23 mmol) was added. After 10 minutes, di-tert-butyl(((S)-6-amino-1-(tert-butoxy)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid (300 mg, 0.615 mmol) was added, and the reaction mixture was stirred at room temperature for 23 hours. Piperidine (122 μl, 1.23 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with toluene (50 mL), washed with saturated sodium bicarbonate aqueous solution (2 x 30 mL) and brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by ISCO CombiFlash® chromatography, eluting with ethyl acetate in a 0-20% gradient in hexane, to obtain the title compound as a yellow solid (261 mg, 381 mmol, yield 61.9%). LC-MS m / z 685.4(M+H)+.

[0335] Intermediate 11: (S)-5-(6-Methoxy-2-(Piperidine-4-yl)Quinoline-4-yl)-3-(1-((5-Methoxy-6-methylpyridine-2-yl)methyl)Piperidine-4-yl)Oxazolidine-2-one [ka] [ka]

[0336] Step 1: tert-butyl(S)-4-((2-hydroxy-2-(6-methoxyquinoline-4-yl)ethyl)amino)piperidine-1-carboxylate (intermediate 11A) [ka]

[0337] To a solution of (S)-6-methoxy-4-(oxiran-2-yl)quinoline (10.0 g, 49.7 mmol) in ethanol (150 mL), stirred at room temperature under nitrogen, tert-butyl 4-aminopiperidine-1-carboxylate (10.95 g, 54.7 mmol) was added, followed by lithium perchlorate (5.82 g, 54.7 mmol). The reaction mixture was heated and stirred at 80 °C overnight, then concentrated under vacuum and diluted with water (500 mL). The mixture was extracted with ethyl acetate (4 × 500 mL), the combined organic fraction was washed with brine (1000 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title compound as an orange liquid (18.0 g, 34.8 mmol, yield 70.0%). LMCS m / z 402.2(M+H)+. 1 H NMR(400MHz,DMSO-d6)δ 8.70-8.69(m,1H)7.95-7.92(m,1H)7.57-7.56(m,1H)7.41-7.38(m,2H)5.4 0-5.30(m,1H)3.90(s,3H)3.83-3.78(m,4H)2.72-2.63(m,5H)1.90-1.55(m 4H)1.41-1.34(m,4H)1.18-1.01(m 5H).

[0338] Step 2: tert-butyl(S)-4-(5-(6-methoxyquinoline-4-yl)-2-oxoxazolidine-3-yl)piperidine-1-carboxylate (intermediate 11B) [ka]

[0339] To a solution of tert-butyl(S)-4-((2-hydroxy-2-(6-methoxyquinoline-4-yl)ethyl)amino)piperidine-1-carboxylate (350 g, 708 mmol) in dichloromethane (5250 mL) stirred under nitrogen, DMAP (130 g, 1062 mmol) was added, followed by CDI (172 g, 1062 mmol). The reaction mixture was stirred at room temperature for 16 hours, then diluted with water (3000 mL) and extracted with dichloromethane (3 × 2000 mL). The combined organic fraction was washed with brine (3000 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude black liquid was adsorbed onto silica gel (1500 g, 60-120 mesh) and purified by silica gel chromatography with elution using 50-100% ethyl acetate in petroleum ether to obtain a yellow solid. The yellow solid was adsorbed onto silica gel (700 g, 60-120 mesh) and re-purified by silica gel chromatography using elution with 0-5% methanol in dichloromethane to obtain the title compound as a pale yellow solid (130 g, 304 mmol, yield 43.0%). Chiral purity 99.87%. LCMS m / z 482.2(M+H)+. 1 H NMR(400MHz,DMSO-d6)δ 8.78(s,1H)8.02-7.99(m,1H)7.48-7.46(m,2H)7.21-7.20(m,1H)6.38-6.34(m,1 H)4.34-4.29(m,1H)4.05-3.95(m,5H)3.78-3.72(m,1H)3.37-3.34(m,1H)2.78(br s,2H)1.75-1.72(m,1H)1.61-1.54(m,2H)1.45-1.37(m,10H).

[0340] Step 3: (S)-5-(6-methoxyquinoline-4-yl)-3-(piperidine-4-yl)oxazolidine-2-one hydrochloride (intermediate 11C) [ka]

[0341] To a solution of tert-butyl(S)-4-(5-(6-methoxyquinoline-4-yl)-2-oxoxazolidine-3-yl)piperidine-1-carboxylate (6.00 g, 14.0 mmol) in dichloromethane (24 mL) and methanol (12 mL), 3 M HCl in CPME (46.8 mL, 140 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours, followed by the addition of 3 M HCl in CPME (10 mL, 30 mmol). After 1.5 hours, 3 M HCl in CPME (6 mL, 18 mmol) was added. After another 1 hour, 3 M HCl in CPME (5 mL, 15 mmol) was added. The milky reaction mixture was then diluted with methanol and concentrated under a stable flow of nitrogen for 16 hours to obtain the title compound as a fine white powder (6.25 g, 14.0 mmol, theoretical yield). LCMS m / z 328.3(M+H)+. 1 H NMR(400MHz,DMSO-d6)δ9.08-8.98(m,2H),8.94-8.82(m,1H)8.30(d,J=9.3Hz, 1H)7.79(d,J=5.4Hz,1H)7.75(dd,J=9.3,2.4Hz,1H)7.44(d,J=2.4Hz,1H)6.56( dd,J=9.0,6.6Hz,1H)4.40(t,J=9.0Hz,1H)4.01(s,3H),3.97-3.84(m,1H)3.38 (dd,J=8.6,6.6Hz,1H)3.36-3.21(m,2H),3.06-2.91(m,2H),2.07-1.70(m,4H).

[0342] Step 4: (S)-5-(6-methoxyquinoline-4-yl)-3-(1-(2,2,2-trifluoroacetyl)piperidine-4-yl)oxazolidine-2-one (intermediate 11D) [ka]

[0343] To a solution of (S)-5-(6-methoxyquinoline-4-yl)-3-(piperidine-4-yl)oxazolidine-2-one hydrochloride (3.00 g, 3.25 mmol) in dichloromethane (55 mL), TEA (4.60 mL, 33.0 mmol) and trifluoroacetic anhydride (1.51 mL, 10.7 mmol) were added. The reaction mixture was stirred at room temperature for 24 hours, followed by concentration under vacuum. The resulting residue was purified by silica gel chromatography with elution using 0-10% methanol in dichloromethane to obtain the title compound as a yellow solid (3.09 g, 6.42 mmol, yield 78%). LCMS m / z 424.22(M+H)+. 1 H NMR(400MHz,CHLOROFORM-d)δppm1.44-1.57(m,1H)1.70(qd,J=12.72,4.40Hz,1H)1.80-1.93(m,1H) 2.06(ddt,J=8.50,4.22,2.20,2.20Hz,1H)2.84(q,J=13.86Hz,1H)3.13-3.31(m,1H)3.41(td,J=8.56 ,6.36Hz,1H)3.98(s,3H)4.04-4.27(m,3H)4.55-4.78(m,1H)6.07-6.21(m,1H)6.83-6.94(m,1H)7.47 (dd,J=9.29,2.45Hz,1H)7.55(d,J=4.89Hz,1H)8.14(dd,J=9.29,0.98Hz,1H)8.84(d,J=4.40Hz,1H).

[0344] Step 5: tert-butyl(S)-4-(6-methoxy-4-(2-oxo-3-(1-(2,2,2-trifluoroacetyl)piperidine-4-yl)oxazolidine-5-yl)quinoline-2-yl)piperidine-1-carboxylate (intermediate 11E) [ka]

[0345] At room temperature, a solution of (S)-5-(6-methoxyquinoline-4-yl)-3-(1-(2,2,2-trifluoroacetyl)piperidine-4-yl)oxazolidine-2-one (1.50 g, 3.54 mmol), 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (8.12 g, 35.4 mmol), and peroxydisulfate, diammonium salt (4.85 g, 21.3 mmol) in DMSO (35.4 mL) and water (0.059 mL) was bubbling nitrogen gas for 1 hour. The mixture was heated at 50°C for 21 hours, then cooled to room temperature and diluted with water (700 mL). Additional 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (8.12 g, 35.4 mmol) and peroxydisulfate, diammonium salt (4.85 g, 21.3 mmol) were added, and the reaction mixture was heated at 50°C for 25 hours. The mixture was cooled to room temperature and subsequently diluted with dichloromethane (100 mL) and saturated sodium bicarbonate aqueous solution (100 mL). The layers were separated, and the aqueous layer was extracted with dichloromethane (100 mL). The organic fractions were combined and washed with saturated sodium bicarbonate aqueous solution (50 mL) and brine (50 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum. The resulting residue was adsorbed onto silica gel and purified by silica gel chromatography with elution using 0-5% methanol in dichloromethane to obtain the title compound as a yellow solid (0.990 g, 1.13 mmol, yield 31.8%). LCMS m / z 607.27(M+H)+. 1 H NMR(400MHz,chloroform-d)δ ppm1.52(s,9H)1.61-1.74(m,5H)1.83-2.02(m,2H)2.79-2.97(m,5H)3.17-3.44(m,3H)3.97(s,3H)4.01-4 .23(m,3H)4.25-4.40(m,2H)6.06-6.18(m,1H)6.83(d,J=2.45Hz,1H)7.39-7.50(m,2H)8.00-8.12(m,1H).

[0346] Step 6: tert-butyl(S)-4-(6-methoxy-4-(2-oxo-3-(piperidine-4-yl)oxazolidine-5-yl)quinoline-2-yl)piperidine-1-carboxylate (intermediate 11F) [ka]

[0347] In an RB flask, tert-butyl(S)-4-(6-methoxy-4-(2-oxo-3-(1-(2,2,2-trifluoroacetyl)piperidine-4-yl)oxazolidine-5-yl)quinoline-2-yl)piperidine-1-carboxylate (0.989 g, 1.630 mmol) and potassium carbonate (2.25 g, 16.3 mmol) were added, and methanol (16.3 mL) was added. The suspension was stirred at room temperature for 24 hours, then poured into a saturated aqueous solution of ammonium chloride. The aqueous layer was extracted with dichloromethane (5 times), the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the title compound as a yellow solid (569 mg, 0.981 mmol, yield 60.2%). LCMS m / z 511.34(M+H)+. 1 ¹H NMR (400MHz, chloroform-d) δ ppm 1.50-1.53 ​​(m,9H) 1.55-1.67 (m,2H) 1.70-1.80 (m,2H) 1.81-2.03 (m,3H) 2.68-2.97 (m,5H) 2.99-3.11 (m,2H) 3.15-3.23 (m,1H) 3.24-3.31 (m,1H) 3.40-3.4 7(m,1H)3.89-4.00(m,4H)4.18-4.27(m,1H)4.28-4.38(m,1H)6.09(dd,J=8.8 0, 6.85Hz,1H)6.85(d,J=2.93Hz,1H)7.39-7.48(m,2H)8.06(d,J=8.80Hz,1H).

[0348] Step 7: tert-butyl(S)-4-(6-methoxy-4-(3-(1-((5-methoxy-6-methylpyridine-2-yl)methyl)piperidine-4-yl)-2-oxoxazolidine-5-yl)quinoline-2-yl)piperidine-1-carboxylate (intermediate 11G) [ka]

[0349] Under nitrogen, a mixture of tert-butyl(S)-4-(6-methoxy-4-(2-oxo-3-(piperidine-4-yl)oxazolidine-5-yl)quinoline-2-yl)piperidine-1-carboxylate (1.60 g, 3.13 mmol), 5-methoxy-6-methylpicolinaldehyde (568 mg, 3.76 mmol), and sodium triacetoxyborate (1.99 g, 9.40 mmol) was mixed with DCE (31 mL) and titanium(IV) isopropoxide (2.05 g, 2.19 mL, 7.21 mmol). The mixture was then heated at 75°C for 5 hours. The reaction mixture was cooled to room temperature, the reaction was stopped with saturated sodium bicarbonate aqueous solution (30 mL), and the mixture was stirred at room temperature for 30 minutes. The slurry was filtered through a Celite pad to remove solids, and then washed with dichloromethane (30 mL). The filtrate was extracted with dichloromethane (three times), and the combined organic fraction was dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution in 0-10% methanol in dichloromethane to obtain the title compound as a yellow solid (882 mg, 1.34 mmol, yield 42.7%). LCMS m / z 646.21(M+H)+. 1H NMR(400MHz,CHLOROFORM-d)δppm1.51(s,9H)1.58-1.75(m,2H)1.79-1.92(m,4H)1.93-2.00(m,2H)2.11-2.26 (m,2H)2.46(s,3H)2.83-2.97(m,3H)2.99-3.08(m,2H)3.42(dd,J=8.31,6.85Hz,1H)3.59(s,2H)3.83(s,3H)3. 85-3.90(m,1H)3.96(s,3H)4.20(t,J=8.80Hz,1H)4.25-4.41(m,2H)6.07(dd,J=9.05,7.09Hz,1H)6.84(d,J=2. 45Hz,1H)7.01-7.07(m,1H)7.10-7.16(m,1H)7.42(dd,J=9.29,2.93Hz,1H)7.45(s,1H)8.05(d,J=9.29Hz,1H).

[0350] Step 8: (S)-5-(6-Methoxy-2-(Piperidine-4-yl)Quinoline-4-yl)-3-(1-((5-Methoxy-6-methylpyridine-2-yl)methyl)piperidine-4-yl)Oxazolidine-2-one (Intermediate 11) [ka]

[0351] To a solution of tert-butyl(S)-4-(6-methoxy-4-(3-(1-((5-methoxy-6-methylpyridine-2-yl)methyl)piperidine-4-yl)-2-oxoxazolidine-5-yl)quinoline-2-yl)piperidine-1-carboxylate (880 mg, 1 equivalent, 1.36 mmol) in dichloromethane, 4N HCl (497 mg, 3.41 mL, 13.6 mmol) in dioxane HCl was added. The mixture was stirred at room temperature for 20 hours, then concentrated and dried under high vacuum to obtain the title compound as a yellow solid (1.235 g, 2.24 mmol, >theoretical yield). LCMS m / z 546.25(M+H)+. 1H NMR(400MHz,DMSO-d6)δ ppm1.73-1.83(m,1H)1.89-2.01(m,1H)2.02-2.18(m,4H)2.18-2.29(m,1H)2.39-2.46(m,2H)2.82-2.95(m,1H)2.97-3.10(m,2H)3.11-3. 27(m,2H)3.30-3.52(m,5H)3.58-3.78(m,3H)3.87(s,3H)3.93-4.02(m,3H)4.25-4.43(m,3H)6.40-6.49(m,1H)7.24-7.35(m,1H)7.47(br s,1H)7.56-7.65(m,1H)8.02-8.24(m,1H)8.78-8.92(m,1H)9.14-9.31(m,1H)10.55-10.89(m,1H).

[0352] Example 1 (((S)-1-carboxy-5-((S)-2-((1R,4S)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobut (Xy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)-3-(naphthalene-2-yl)propanamide)pentyl)carbamoyl)-L-glutamic acid [ka] [ka]

[0353] Step 1: tert-butyl((benzyloxy)carbonyl)glycinate [ka]

[0354] To a solution of benzyl 2-bromoacetate (20 g, 87 mmol) and tert-butylglycine hydrochloride (16.1 g, 96.0 mmol) in tetrahydrofuran (175 ml), DIPEA (35.1 mL, 201 mmol) was added. The reaction mixture was stirred at room temperature for 44 hours. The mixture was filtered to remove the white solid, which was washed with ethyl acetate. The organic filtrate was diluted with additional ethyl acetate (200 mL), washed with saturated sodium bicarbonate aqueous solution (200 mL) and brine (200 mL), dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution in 0-60% ethyl acetate in hexane to obtain the title compound as a yellow oil (13.97 g, 49.0 mmol, yield 57.3%; 48.0 mmol, purity 98%, yield 56.1%). LC-MS m / z 280.21(M+H)+. 1 ¹H NMR (400MHz, chloroform-d) δ ppm 1.48 (s,9H) 1.91 (br s,1H) 3.38 (s,2H) 3.53 (s,2H) 5.20 (s,2H) 7.32-7.43 (m,5H).

[0355] Step 2: (E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoic acid sodium [ka]

[0356] Methyl(E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoate (5.02 g, 11.5 mmol) dissolved in tetrahydrofuran (14.6 mL) was mixed with aqueous sodium hydroxide solution (5.089 M, 3.38 mL, 17.2 mmol). The unstirred mixture separated into two distinct layers at room temperature.

[0357] A homogeneous pale yellow reaction mixture was heated at 80°C for 17 hours with magnetic stirring. The mixture was concentrated under vacuum, azeotropically mixed with toluene (2 x 50 mL), and dried under high vacuum to obtain the title compound as a viscous pale yellow solid (5.33 g, 11.4 mmol, 95% purity, 99.1% yield). LC-MS m / z 424.23(M+H)+. 1 H NMR(400MHz,DMSO-d6)δ ppm1.11-1.26(m,4H)1.80-1.92(m,4H)2.53-2.58(m,2H)3.16-3.19(m,1H)3.20-3.27(m,1H)3.49(t,J=6.11Hz,2H)3. 57-3.65(m,4H)3.95(dd,J=5.62, 1.71Hz,2H)5.69-5.79(m,1H)6.13-6.30(m,1H)7.20-7.29(m,2H)7.29-7.40(m,8H).

[0358] Step 3: Benzyl N-(2-(tert-butoxy)-2-oxoethyl)-N-((E)-4-(((1r,4r)-4-(2-dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoyl)glycinate [ka]

[0359] To a solution of benzyl (2-(tert-butoxy)-2-oxoethyl)glycinate (500 mg, 1.79 mmol) and (E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoate sodium (797 mg, 1.79 mmol) in dichloromethane (3.58 mL), HATU (885 mg, 2.33 mmol) and N-ethyl-N-isopropylpropan-2-amine (468 μl, 2.68 mmol) were added. The reaction mixture was stirred at room temperature overnight for 19 hours. The mixture was diluted with dichloromethane (100 mL), washed with saturated sodium bicarbonate aqueous solution (50 mL) and brine (50 mL), dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography using elution with 0-50% ethyl acetate in heptane to obtain the title compound as a yellow oily substance (961 mg, 1.19 mmol, purity 85%, yield 66.6%). LC-MS m / z 685.29(M+H)+. 1 H NMR(400MHz,chloroform-d)δ ppm1.25-1.36(m,7H)1.43-1.52(m,11H)1.81-2.04(m,4H)2.69(t,J=6.11Hz,2H)3.13-3.21(m,1H)3.51-3.60(m, 2H)3.65-3.72(m,4H)4.09-4.31(m,4H)5.16-5.24(m,2H)6.82-7.07(m,1H)7.20-7.28(m,2H)7.30-7.47(m,12H).

[0360] Step 4: N-(2-(benzyloxy)-2-oxoethyl)-N-((E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoyl)glycine [ka]

[0361] To a solution of benzyl N-(2-(tert-butoxy)-2-oxoethyl)-N-((E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoyl)glycinate (960 mg, 1.40 mmol) in dichloromethane (5.6 mL), TFA (2.16 mL, 28.0 mmol) was added. The reaction mixture was stirred at room temperature for 20 hours, then concentrated under vacuum and dried under high vacuum to obtain the title compound as an orange oil (1.57 g, 1.39 mmol, 99% yield). LC-MS m / z 629.23(M+H)+. 1 H NMR(400MHz,chloroform-d)δ ppm1.23-1.47(m,3H)1.48-1.58(m,1H)1.61-1.71(m,1H)1.93-2.10(m,3H)2.70(dt,J=9.29,6.36Hz,1H)2.86-3.01(m,1H)3.25-3.43 (m,3H)3.70-3.90(m,2H)4.19-4.43(m,7H)4.51-4.64(m,2H)5.19-5.30(m,2H)6.36-6.52(m,1H)6.92-7.09(m,1H)7.32-7.56(m,15H).

[0362] Step 5: Methyl(1R,4r)-4-(4-(((1r,4R)-4-((2-((E)-N-(2-(benzyloxy)-2-oxoethyl)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)butanamide)acetamide)methoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate [ka]

[0363] To a solution of N-(2-(benzyloxy)-2-oxoethyl)-N-((E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2-enoyl)glycine trifluoroacetate (500 mg, 0.673 mmol) in dichloromethane (6.898 mL), HATU (333 mg, 0.875 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.586 mL, 3.37 mmol) were added. Tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (316 mg, 0.740 mmol) was added, and the reaction mixture was stirred at room temperature for 3 hours. The mixture was diluted with dichloromethane (50 mL), washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL), dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by silica gel chromatography using elution with 0-10% methanol in dichloromethane to obtain the title compound as a yellow oil (581 mg, 0.429 mmol, yield 63.7%). LC-MS m / z 1037.42(M+H)+. 1 H NMR(400MHz,chloroform-d)δppm1.07-1.17(m,4H)1.23-1.37(m,4H)1.39-1.54(m,9 H)1.73(brs,4H)1.83-2.19(m,10H)2.19-2.32(m,2H)3.20(q,J=7.50Hz,4H)3.50 (s,14H)3.74(dt,J=13.45,6.48Hz,2H)4.01-4.34(m,4H)5.32(s,2H)5.59-5.71 (m,1H)6.32-6.41(m,1H)6.97-7.09(m,1H)7.33-7.46(m,15H)8.05-8.13(m,1H).

[0364] Step 6: N-(4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)butanoyl)-N-(2-((((1R,4r)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)methyl)amino)-2-oxoethyl)glycine sodium [ka]

[0365] To a solution of tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-(2-((E)-N-(2-(benzyloxy)-2-oxoethyl)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (580 mg, 0.559 mmol) in tetrahydrofuran (5.6 mL) (5591 μl), 1N sodium hydroxide aqueous solution was added. The reaction mixture was stirred at room temperature for 17 hours. The mixture was neutralized with 1N HCl, concentrated under vacuum, and dried under high vacuum to obtain the title compound as a yellow semi-solid (580 mg, 0.557 mmol, yield 100%). LC-MS 947.44(M+H)+. 1 H NMR(400MHz,chloroform-d)δ ppm1.12-1.38(m,11H)1.42-1.47(m,10H)1.89(br s,13H)2.09-2.18(m,3H)2.21-2.30(m,3H)3.19(q,J=7.66Hz,7H)3.38-3.49(m,4H)3.51-3.58(m,2H)3.58-3.67( m,2H)3.68-3.82(m,8H)5.95-6.03(m,1H)6.28-6.44(m,1H)7.14-7.22(m,2H)7.37-7.37(m,1H)7.37-7.49(m,8H).

[0366] Step 7: tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(4-(((1r,4r)-4-(2-(((dibenzylamino)ethoxy)cyclohexyl)oxy)-N-(2-((((1R,4r)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)methyl)amino)2-oxoethyl)butanamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate [ka]

[0367] Dichloromethane (22.7 mL) contains N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4-(tert-butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-N-((E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)buta-2- DIPEA (0.191 mL, 1.09 mmol) was added to a solution of enoyl)glycine sodium (530 mg, 0.547 mmol), methyl (1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (210 mg, 0.547 mmol), and HATU (270 mg, 0.711 mmol). The reaction mixture was stirred at room temperature for 1 hour, and then diluted with dichloromethane (20 mL). The mixture was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL), dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution using 0-10% methanol in dichloromethane to obtain the title compound as a clear film (174 mg, 0.106 mmol, yield 19.3%). LC-MS m / z1313.64(M+H)+. 1H NMR(400MHz,chloroform-d)δ ppm1.03-1.18(m,5H)1.21-1.33(m,11H)1.39-1.44(m,11H)1.45-1.59(m,4H)1.79-1.87(m, 4H)1.89-2.05(m,20H)2.05-2.14(m,2H)2.17-2.26(m,6H)2.59-2.68(m,2H)3.17-3.18(m,1H )3.17-3.32(m,5H)3.39-3.54(m,14H)3.62-3.66(m,6H)3.67-3.78(m,2H)3.93-3.98(m,1H) 4.05-4.09(m,1H)4.09-4.13(m,1H)5.67-5.86(m,2H)6.30(dt,J=15.04,1.77Hz,1H)6.62(br t,J=5.38Hz,1H)6.85-7.00(m,2H)7.18-7.23(m,1H)7.25-7.32(m,4H)7.33-7.37(m,4H).

[0368] Step 8: tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(4-(((1r,4r)-4-(2-aminoethoxy)cyclohexyl)oxy)-N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)butanamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate [ka]

[0369] Under nitrogen, Pd-C (13.8 mg, 0.013 mmol) was added to a solution of tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-((E)-4-(((1r,4r)-4-(2-(((dibenzylamino)ethoxy)cyclohexyl)oxy)-N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)buta-2-enamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (170 mg, 0.129 mmol) in methanol (22.7 mL). The flask was evacuated, backfilled with a helium gas balloon, and stirred at room temperature for 19 hours. The flask was evacuated and placed under nitrogen. Additional Pd-C (13.8 mg, 0.013 mmol) was added, and the flask was subsequently evacuated and backfilled with a new hydrogen gas balloon. The reaction mixture was stirred at room temperature for 3 nights, then filtered through a Celite plug and washed with additional methanol (20 mL). The filtrate was concentrated and dried under high vacuum to obtain the crude title compound as a yellow film, which was used without further purification or characterization (144 mg, 0.036 mmol, yield 27.4%). LC-MS m / z 1135.98(M+H)+.

[0370] Step 9: tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate [ka]

[0371] To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxylic acid (50 mg, 0.227 mmol) and HATU (112 mg, 0.295 mmol) in dichloromethane (22.7 mL), DIPEA (0.079 mL, 0.454 mmol) was added.

[0372] The reaction mixture was stirred at room temperature for 15 minutes. Tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(((1r,4r)-4-(2-aminoethoxy)cyclohexyl)oxy)-N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)butanamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (196 mg, 0.173 mmol) was added, and the reaction was continued at room temperature for 3 hours. A solution of (2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxylic acid (50 mg, 0.227 mmol), HATU (112 mg, 0.295 mmol), and DIPEA (0.079 ml, 0.454 mmol) in 0.5 mL of DMF was prepared and added to the above mixture. The reaction mixture was stirred at room temperature for 3 nights, and then diluted with an additional 10 mL of dichloromethane. The organic solution was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL), dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution using 0-20% methanol in dichloromethane, and the title compound was obtained as a yellow film (88 mg, 0.057 mmol, yield 28.7%). LC-MS m / z 669.80(M+2H) / 2. 1H NMR(400MHz,chloroform-d)δppm1.03-1.16(m,5H)1.18-1.29(m,10H)1.35-1.38(m,4H)1.41(s,9H)1.4 4-1.59(m,4H)1.79-1.90(m,9H)1.91-2.02(m,15H),2.05-2.14(m,1H)2.18-2.24(m,4H)2.31-2.37( m,2H)2.62-2.67(m,3H)2.75-2.89(m,3H)3.01-3.10(m,1H)3.13-3.31(m,7H)3.34-3.47(m,13H)3.4 8-3.57(m,4H)3.60-3.76(m,6H)3.90(s,1H)4.03(s,1H)4.73-4.84(m,1H)5.70-5.88(m,2H)6.24(br t,J=4.89Hz,1H)6.65(br t,J=5.14Hz,1H)7.35(dd,J=7.83,4.89Hz,1H)7.56(dt,J=7.83,1.96Hz,1H)8.50(d,J=1.96Hz,1H)8.60(dd,J=4.89,1.47Hz,1H)8.93(br t,J=5.14Hz,1H).

[0373] Step 10: (1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4-(Methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid [ka]

[0374] tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(((1S,4r)-4 To a solution of -(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (88 mg, 0.066 mmol), TFA (101 μl, 1.316 mmol) was added. The reaction mixture was stirred at room temperature for 20 hours. Additional TFA (101 μl, 1.316 mmol) was added, and the reaction mixture was stirred at room temperature for 20 hours. The mixture was concentrated and dried under high vacuum to obtain the crude title compound as an orange film (130.1 mg, 0.064 mmol, yield 97%). LC-MS m / z 1281.63(M+H)+. 1 H NMR(400MHz,chloroform-d)δ ppm1.26-1.39(m,14H)1.41-1.49(m,10H)1.57-1.60(m,10H)1.87-2.22(m,24H)2.29-2.51(m,4H)2.53-2.65(m,3H)2.77-2.8 7(m,3H)3.15-3.24(m,2H)3.37-3.58(m,10H)3.62-3.69(m,6H)3.72-3.82(m,6H)4.06-4.10(m,1H)4.30-4.36(m,1H)5.30(br d,J=6.85Hz,1H)7.57-7.73(m,2H)8.64-8.74(m,2H)8.81(dd,J=4.65,1.22Hz,1H)8.90-8.99(m,2H).

[0375] Step 11: Methyl(1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohex Sil)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate [ka]

[0376] Dichloromethane (2.6 mL) contains 2,2,2-trifluoroacetic acid (1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1r,4R)-4-(Methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclo To a solution of xan-1-carboxylic acid compound (180 mg, 0.129 mmol) and (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-2-one hydrochloride (78 mg, 0.155 mmol), 2-(3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (63.8 mg, 0.168 mmol) and N-ethyl-N-isopropylpropan-2-amine (112 μl, 0.645 mmol) were added. The reaction mixture was stirred at room temperature for 1.5 hours and diluted with additional dichloromethane (30 mL). The organic solution was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL), dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography using elution with 0-20% methanol in dichloroethane to obtain the title compound as an orange film (182 mg, 0.066 mmol, yield 51.5%). LC-MS m / z 864.95(M+2H) / 2. 1H NMR(400MHz,chloroform-d)δ ppm1.03-1.30(m,14H)1.34-1.41(m,2H)1.58(br s,5H)1.61-1.72(m,3H)1.75-2.05(m,28H)2.14-2.22(m,6H)2.26(br s,3H)2.38-2.47(m,1H)2.60-2.65(m,4H)2.65-2.71(m,2H)2.73(br s,5H)3.03-3.12(m,1H)3.13-3.26(m,6H)3.33-3.43(m,20H)3.46-3.56(m,6H)3.59-3.70(m,5H) 3.89(s,2H)3.98-4.03(m,2H)4.77(d,J=6.36Hz,1H)4.79-4.86(m,1H)5.01-5.10(m,1H)5.82(br d,J=7.82Hz,1H)5.85-5.92(m,1H)6.32(br t,J=4.89Hz,1H)6.74(dt,J=14.92,5.26Hz,1H)7.33(dd,J=7.82,4.89Hz,1H)7.54(br d,J=7.82Hz,1H)7.85(s,2H)8.45(br d,J=7.34Hz,1H)8.48(d,J=1.96Hz,1H)8.55-8.59(m,1H)8.61(d,J=0.98Hz,1H)8.85(s,1H)8.91-9.01(m,1H)9.12-9.29(m,1H).

[0377] Step 12: 4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazoline-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl) (Amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid [ka]

[0378] Methyl(1R,4r)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino in tetrahydrofuran (2.1 mL) To a solution of cyclohexane-1-carboxylate (180 mg, 0.104 mmol) containing 1N sodium hydroxide aqueous solution (208 μl, 0.208 mmol), cyclohexane-1-carboxylate (180 mg, 0.104 mmol) was added. The reaction mixture was stirred at room temperature for 3 nights. The mixture was neutralized with 1N HCl, concentrated under vacuum, and dried under high vacuum to obtain the crude title compound as a yellow solid (199 mg, 0.083 mmol, yield 79%). LC-MS m / z 857.46(M+2H) / 2. 1H NMR(400MHz,DMSO-d6)δ ppm 0.77-0.92(m,1H)0.99-1.26(m,9H)1.26-1.42(m,3H)1.44-1.56(m,1H)1.58-1.75(m,4H)1.78-1.95(m,7H)1.96-2.15(m,4H)2.18- 2.40(m,3H)2.39-2.58(m,12H)2.58-2.67(m,1H)2.72-3.09(m,3H)3.13-3.47(m,13H)3.49-3.98(m,46H)3.99-4.26(m,6H)4.84(br d,J=5.87Hz,1H)5.38-5.52(m,1H)7.11-7.36(m,2H)7.39-7.50(m,1H)7.64-7.78(m,1H)8.03-8.16(m,2H)8.26- 8.42(m,2H)8.73-8.91(m,2H)9.01(d,J=2.93Hz,1H)9.41-9.63(m,1H)10.15-10.32(m,1H)11.65-12.27(m,3H).

[0379] Step 13: (((S)-1-carboxy-5-((S)-2-((1R,4S)-4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobut Xy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)-3-(naphthalene-2-yl)propanamide)pentyl)carbamoyl)-L-glutamic acid (Example 1) [ka]

[0380] 4-(4-(((1r,4R)-4-(2-(2-(N-(2-((2-(((1R,4r)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropylmethylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl To a solution of )-4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid (175 mg, 0.102 mmol), HATU (58.2 mg, 0.153 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.053 mL, 0.306 mmol) were added. Di-tert-butyl(((S)-6-((S)-2-amino-3-(naphthalene-2-yl)propanamide)-1-(tert-butoxy)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid (69.9 mg, 0.102 mmol) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was diluted with an additional 20 mL of dichloromethane, washed with 15 mL of saturated sodium bicarbonate aqueous solution and 15 mL of brine, dried over sodium sulfate, filtered, concentrated under vacuum, and dried under high vacuum for 1 hour. To the solution of the resulting residue in 6.9 mL of dichloromethane, TFA (0.157 mL, 2.042 mmol) was added. After 19 hours, an additional 0.157 mL of TFA (2.042 mmol) was added. After 2 hours, the mixture was concentrated under vacuum. The resulting residue was purified.

[0381] The resulting residue was purified by MDAP (XSelect® CSH C18 5um column, 40 mL / min) using a 30-85% gradient acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) to obtain the title compound as an off-white solid (47.9 mg, 0.020 mmol, yield 19.3%). LC-MS m / z 1107.30(M+2H) / 2. HPLC: purity 91.8% at 254 nm. 1 H NMR(400MHz,DMSO-d6)δppm0.93(brd,J=6.36Hz,2H)1.01-1.07(m,3H)1.08-1.27(m,11H)1.28-1.53(m ,5H)1.54-1.68(m,7H)1.69-1.75(m,1H)1.77-1.93(m,10H)1.96-2.08(m,4H)2.09-2.18(m,4H)2.22(br t,J=7.09Hz,1H)2.32(dt,J=12.47,6.48Hz,2H)2.38-2.46(m,1H)2.47-2.55(m,26H)2.61(br s,1H)2.64-2.74(m,1H)2.89-3.06(m,3H)3.08-3.25(m,9H)3.27-3.45(m,30H)3.47-3.58(m,2H)3.81-3.91( m,2H)3.93-4.09(m,3H)4.44-4.60(m,2H)4.65(d,J=5.87Hz,1H)4.93-5.04(m,1H)6.13-6.24(m,1H)6.41(br d,J=7.82Hz,1H)7.35-7.51(m,3H)7.60(br d,J=7.34Hz,2H)7.65-7.73(m,2H)7.76-7.82(m,2H)7.85(br d,J=7.34Hz,1H)7.90(d,J=8.80Hz,1H)7.97-8.05(m,2H)8.05-8.12(m,2H)8.25-8.31(m,1H)8.45-8.50(m,1H)8.56(br d,J=4.40Hz,1H)8.59-8.70(m,2H)8.75-8.84(m,1H)8.96(s,2H).

[0382] Example 2 2,2,2-Containing (((S)-1-Carboxy-5-((S)-2-((1R,4S)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobut Xy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)3-(naphthalene-2-yl)propanamide)pentyl)carbamoyl)-L-glutamic acid compound [ka] [ka]

[0383] Step 1: tert-butyl N-(2-methoxy-2-oxoethyl)-N-((1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carbonyl)glycinate [ka]

[0384] To a solution of (1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid (2.00 g, 3.49 mmol), tert-butyl (2-methoxy-2-oxoethyl)glycinate (0.850 g, 4.18 mmol), and HATU (1.72 g, 4.53 mmol), N-ethyl-N-isopropylpropan-2-amine (1.21 mL, 6.97 mmol) was added. The reaction mixture was stirred at room temperature overnight for 22 hours, and then diluted with saturated sodium bicarbonate aqueous solution (50 mL). The layers were separated, the organic fraction was washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography using elution with 0-15% methanol in dichloromethane to obtain the title compound as a pale yellow foam (1.79 g, 1.96 mmol, yield 56.1%). 1 H NMR(400MHz,chloroform-d)δ ppm1.12-1.33(m,7H)1.41-1.54(m,8H)1.71-1.77(m,2H)1.80-2.02(m,8H)2. 03-2.16(m,2H)2.21-2.28(m,2H)2.34(ddd,J=11.62,8.19,3.67Hz,1H)2.69( s,3H)2.74-2.93(m,3H)3.23(td,J=8.80, 4.40Hz,2H)3.41-3.49(m,4H)3.65- 3.86(m,4H)4.07(d,J=1.96Hz,2H)4.17(s,2H)4.81(d,J=6.36Hz,1H)5.51(br d,J=7.82Hz,1H)5.78(br s,1H)7.35-7.42(m,1H)7.54-7.65(m,1H)8.55(d,J=1.47Hz,1H)8.65(dd,J=4.65,1.71Hz,1H).

[0385] Step 2: N-(2-methoxy-2-oxoethyl)-N-((1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carbonyl)glycine [ka]

[0386] To a solution of tert-butyl N-(2-methoxy-2-oxoethyl)-N-((1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carbonyl)glycinate (1.48 g, 1.96 mmol) in anhydrous 1,4-dioxane (9.78 mL), 3 M HCl (9.78 ml, 29.3 mmol) in CPME was added. The reaction mixture was stirred at room temperature for 24 hours. A sticky white solid was scraped off the side of the flask. 3.26 mL, 9.78 mmol of 3 M HCl in additional CPME was added, and the mixture was stirred for a further 27 hours at room temperature. Subsequently, it was concentrated under vacuum and dried under high vacuum to obtain the title compound as a white solid (1.36 g, 1.43 mmol, yield 73.3%). LCMS m / z 702.39(M+H)+. 1 H NMR(400MHz,DMSO-d6)δ ppm1.07-1.25(m,5H)1.31-1.50(m,3H)1.54-1.71(m,5H)1.72-1.91(m,5H)2.07(br t,J=7.58Hz,2H)2.29-2.46(m,2H)2.66-2.77(m,2H)2.93-3.04(m,1H)3.09-3.25(m,4H)3.27-3.51(m,5H)3.60-3.72(m,2H)3.92-4.1 1(m,3H)4.20-4.39(m,3H)4.68-4.77(m,2H)7.60-7.70(m,2H)7.90-7.98(m,1H)8.00-8.06(m,1H)8.57-8.65(m,1H)8.66-8.72(m,1H).

[0387] Step 3: tert-butyl(1R,4R)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-methoxy-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate [ka]

[0388] N-(2-methoxy-2-oxoethyl)-N-((1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carbonyl)glycine (250mg) in dichloromethane (23mL) To a solution of tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (182 mg, 0.427 mmol) and HATU (176 mg, 0.463 mmol), DIPEA (0.124 ml, 0.712 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours, followed by dilution with an additional 20 mL of DCM. The mixture was washed with 20 mL of saturated sodium bicarbonate aqueous solution and brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution in 0-20% methanol in dichloromethane to obtain the title compound as a clear film (197 mg, 0.149 mmol, yield 41.8%). LCMS m / z 1110.39(M+H)+. 1H NMR(400MHz,chloroform-d)δ ppm1.02-1.30(m,12H)1.38-1.41(m,9H)1.42-1.52(m,2H)1.54-1.67(m,2H)1. 70-1.85(m,4H)1.87-2.03(m,8H)2.15-2.26(m,4H)2.61(s,3H)2.71-2.79(m,2 H)2.81-2.90(m,1H)3.11-3.28(m,4H)3.33-3.56(m,20H)3.61-3.72(m,3H)3.7 3-3.80(m,3H)4.02(d,J=13.69Hz,3H)4.79(d,J=6.85Hz,1H)5.85-6.07(m,2H) 6.36-6.47(m,1H)7.29-7.38(m,1H)7.59(dt,J=7.83, 1.96Hz,1H)8.20-8.32(m,1H)8.49(d,J=1.96Hz,1H)8.53-8.63(m,2H).

[0389] Step 4: N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(tert-butoxycarbonyl)cyclohexyl)amino-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-N-((1R,4R)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carbonyl)glycine [ka]

[0390] To a solution of tert-butyl(1R,4R)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-methoxy-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (195 mg, 0.176 mmol), a 1N sodium hydroxide aqueous solution (351 μl, 0.351 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour, followed by neutralization with a 1N HCl aqueous solution. The mixture was concentrated under vacuum and dried under high vacuum to obtain the crude title compound as a white solid (245.4 mg, >theoretical value), which was used without further purification or characterization.

[0391] Step 5: tert-butyl 4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate [ka]

[0392] N-(2-((2-(((1R,4R)-4-(4-(((1r,4R)-4-(tert-butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-N-((1R,4R)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy To a solution of (butanamide)cyclohexane-1-carbonyl)glycine (190 mg, 0.173 mmol) and methyl(1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)(-)butanamide)cyclohexane-1-carboxylate (73.3 mg, 0.191 mmol), HATU (86 mg, 0.225 mmol) and N-ethyl-N-isopropylpropan-2-amine (60.5 μL, 0.347 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours, followed by dilution with additional DCM (20 mL). The mixture was washed with saturated sodium bicarbonate aqueous solution (10 mL) and brine (10 mL), dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution using 0-20% methanol in DCM to obtain the title product as a pale yellow solid. LCMS m / z 732.13(M+2H) / 2. 1 H NMR(400MHz,chloroform-d)δ ppm1.06-1.28(m,12H)1.37-1.64(m,16H)1.76-2.06(m,28H)2.14-2.30(m,8H)2.62(br s,3H)2.72-2.88(m,3H)3.20(br s,6H)3.33-3.55(m,23H)3.59-3.74(m,6H)3.86(br s,2H)4.01(br s,2H)4.77(br d,J=5.87Hz,1H)5.85(br d,J=6.85Hz,3H)6.27(br s,1H)6.56(br s,1H)7.21-7.41(m,1H)7.55(br d,J=7.34Hz,1H)8.37-8.78(m,2H)9.20(br s,1H).

[0393] Step 6: (1R,4r)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid [ka]

[0394] DCM (0.4 mL) contains tert-butyl 4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S, To a solution of 3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (120 mg, 0.082 mmol), TFA (0.3 mL, 4.1 mmol) was added. The reaction mixture was stirred at room temperature for 2.5 hours, concentrated under vacuum, and dried under high vacuum to obtain the crude title compound as a pale yellow oil (225 mg, 0.081 mmol, yield 99%), which was used without further purification or characterization. LCMS m / z 704.17(M+H)+.

[0395] Step 7: Methyl(1R,4r)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazoline-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino) -4-Oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate [ka]

[0396] (1R,4r)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide To a solution of cyclohexane-1-carboxylic acid (125 mg, 0.089 mmol) and N-ethyl-N-isopropylpropan-2-amine (155 μl, 0.889 mmol), 2-(3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (50.7 mg, 0.133 mmol) and (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-2-one hydrochloride (53.4 mg, 0.107 mmol) were added. The reaction mixture was stirred at room temperature for 3 nights, followed by dilution with an additional 30 mL of DCM. The organic mixture was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography using elution with 0-20% methanol in DCM to obtain the title compound as a pale yellow oily substance (158 mg, 0.083 mmol, yield 93.1%). 1¹H NMR (400MHz, chloroform-d) δ ppm 1.04-1.15 (m, 9H) 1.23 (br s,11H)1.35-1.42(m,4H)1.44-1.54(m,3H)1.58-1.69(m,4H)1.76-1.86(m,8H)1.88-2.04(m,10H)2.14-2.25(m,10H)2.26-2.47(m,10H) )2.64(s,3H)2.67-2.72(m,1H)2.73-2.90(m,3H)3.09(q,J=7.34Hz,1H)3.16-3.27(m,4H)3.35-3.53(m,33H)3.58-3.74(m,6H)3.87(br s,2H)3.97-4.10(m,3H)4.79(br d,J=6.36Hz,2H)5.09(br d,J=3.91Hz,1H)5.83-5.99(m,3H)6.34(br d,J=4.40Hz,1H)6.59-6.73(m,1H)7.32-7.42(m,1H)7.56(br d,J=7.82Hz,1H)7.86(s,2H)8.50(br s,2H)8.55-8.67(m,2H)8.83(br s,1H)9.23(br s,1H).

[0397] Step 8: 4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazoline-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-O Xosobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid [ka]

[0398] Methyl(1R,4r)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy To a solution of ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (155 mg, 0.084 mmol), 167 μl, 0.167 mmol, was added. The reaction mixture was stirred at room temperature for 18 hours, concentrated under vacuum, and dried under high vacuum to obtain the crude title compound as a yellow solid (155 mg, 0.076 mmol, yield 91%). LCMS m / z 919.78(M+2H) / 2. 1H NMR(400MHz,METHANOL-d4)δ ppm 0.99-1.05(m,3H)1.12-1.18(m,3H)1.20-1.38(m,16H)1.47-1.63(m,6H)1.66-1.76(m,3H)1.78-1.85(m,6H)1.87-1.91(m,9H)1.98(br d,J=7.34Hz,16H)2.04-2.19(m,2H)2.20-2.25(m,6H)2.26-2.30(m,3H)2.31-2.45(m,2H)2.48-2.60(m,1H)2.64-2.69(m,3H)2.69-2.76(m,2H)2.80-2.90(m,1H)3.04-3.13(m,1H)3.23-3.31(m,4H)3.32-3.38(m,6H)3.39-3.49(m,8H)3.50-3.59(m,6H)3.61-3.69(m,4H)3.71-3.78(m,6H)3.97-4.11(m,3H)4.18-4.28(m,2H)4.65(br d,J=2.93Hz,1H)4.84(d,J=6.36Hz,1H)5.26(t,J=7.83Hz,1H)7.33(dd,J=8.31,4.40Hz,1H)7.54(dd,J=7.83,4.89Hz,1H)7.81(br d,J=7.83Hz,1H)7.90(d,J=8.80Hz,1H)8.05(dd,J=9.05、1.71Hz,1H)8.19(dd,J=8.31,1.47Hz,1H)8.48-8.56(m,2H)8.57-8.61(m,2H)8.81(s,1H)。

[0399] Step 9: Di-tert-butyl(((S)-1-(tert-butoxy)-6-((S)-2-((1R,4S)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy Cyclohexyl oxyethyl amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl oxy)butanamide)cyclohexane-1-carboxamide)-3-(naphthalene-2-yl)propanamide)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid [ka]

[0400] DCM (7mL) contains 4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazoline-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3 To a solution of (130 mg, 71.0 μmol) di-tert-butyl(((S)-6-((S)-2-amino-3-(naphthalene-2-yl)propanamide)-1-(tert-butoxy)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid (48.4 mg, 71.0 μmol) and HATU (40.3 mg, 106 μmol), DIPEA (27.4 mg, 37.0 μL, 212 μmol) and DMF (0.5 mL) were added. The reaction mixture was stirred at room temperature for 1.5 hours, followed by dilution with an additional 20 mL of DCM. The organic mixture was washed with saturated sodium bicarbonate aqueous solution (15 mL) and brine (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography using elution with 0-20% methanol in dichloromethane to obtain the title compound as a white solid (104 mg, 42.0 mmol, yield 58.8%). LC-MS m / z 1253.65(M+2H) / 2. 1H NMR(400MHz,クロロホルム-d)δ ppm 0.80-1.04(m,5H)1.04-1.18(m,9H)1.18-1.33(m,15H)1.35-1.42(m,3H)1.44-1.47(m,16H)1.48-1.63(m,15H)1.64-1.72(m,5H) 1.89(br s,13H)1.91-2.08(m,21H)2.12-2.27(m,11H)2.27-2.36(m,2H)2.37-2.52(m,3H)2.67(s,3H)2.69-2.69(m,1H)2.69-2.73(m,1H)2.86(br s,3H)2.96-3.13(m,2H)3.18-3.32(m,7H)3.48(br s,14H)3.50(br s,6H)3.70(br s,2H)3.93(br s,1H)4.00-4.14(m,3H)4.25(br s,1H)4.53-4.68(m,1H)4.78-4.85(m,1H)4.89(br d,J=4.40Hz,1H)5.08(td,J=7.58,1.96Hz,1H)5.51-5.64(m,1H)5.69(br dd,J=15.65,7.82Hz,2H)6.05(br d,J=2.93Hz,1H)6.31-6.39(m,1H)6.46-6.56(m1H)7.15(br s,1H)7.33-7.51(m,3H)7.58(dt,J=7.83,1.96Hz,2H)7.64(s,1H)7.71(br d,J=8.31Hz,1H)7.89(d,J=1.47Hz,2H)8.39-8.47(m,1H)8.54(d,J=1.96Hz,1H)8.60-8.65(m,1H)8.67(d,J=2.45Hz,1H)8.91(s,1H)9.18-9.34(m,2H)。

[0401] Step 10: Contains 2,2,2-trifluoroacetic acid (((S)-1-carboxy-5-((S)-2-((1R,4S)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobut Xy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)3-(naphthalen-2-yl)propanamide)pentyl)carbamoyl)-L-glutamic acid compound (Example 2) [ka]

[0402] Di-tert-butyl(((S)-1-(tert-butoxy)-6-((S)-2-((1R,4S)-4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazoline-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino) To a solution of -2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)-3-(naphthalene-2-yl)propanamide)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid (104 mg, 0.042 mmol), TFA (0.272 mL, 3.53 mmol) was added. After 24 hours, the mixture was concentrated, and the resulting residue was purified by MDAP (XSelect® CSH C18 5um column, 40 mL / min) using a 15-55% gradient of acetonitrile in water containing trifluoroacetic acid (0.1%) to obtain the title compound as a white solid (61.1 mg, 0.021 mmol, yield 29.4%). LC-MS m / z 1170.25(M+2H) / 2. HPLC: 100% purity at 254 nm. 1H NMR(400MHz, methanol-d4)δ ppm1.40(br s,21H)1.42-1.45(m,2H)1.47-1.64(m,5H)1.66-1.75(m,1H)1.79-1.86(m,8H)1.87-1.91(m,2 H)1.92-2.04(m,14H)2.11-2.28(m,10H)2.35-2.50(m,4H)2.59-2.66(m,1H)2.66-2.76(m,3H) 2.80-2.83(m,2H)2.85-2.93(m,2H)3.00-3.03(m,1H)3.05-3.17(m,3H)3.22 -3.39(m,34H)3.40-3.51(m,8H)3.51-3.60(m,5H)3.61-3.69(m,2H)3.71-3. 79(m,1H)3.80-3.94(m,2H)4.01(d,J=6.85Hz,2H)4.16-4.25(m,3H)4.27-4. 39(m,2H)4.68(dd,J=8.56,6.60Hz,1H)5.03(dd,J=6.60,1.71Hz,1H)5.61(br t,J=9.29Hz,1H)7.35-7.51(m,3H)7.69(s,1H)7.75-7.86(m,3H)7.94-8.06(m,3H)8.33(d,J=8.80Hz,1H)8.40(br d,J=7.34Hz,1H)8.78-8.86(m,2H)8.89(s,1H)9.01(s,1H).

[0403] Example 3 (((S)-1-carboxy-5-((S)-2-(2-((1R,4S)-N-(2-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazoline-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)-3-(naphthalene-2-yl)propanamide)pentyl)carbamoyl)-L-glutamic acid [ka] [ka]

[0404] Step 1: Di-tert-butyl(((S)-1-(tert-butoxy)-6-((S)-2-(2-((1R,4S)-N-(2-methoxy-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)-3-(naphthalene-2-yl)propanamide)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid. [ka]

[0405] N-(2-methoxy-2-oxoethyl)-N-((1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carbonyl)glycine (50 mg, 0.071 mmol) in dichloromethane (22.7 mL) l) Di-tert-butyl(((S)-6-((S)-2-amino-3-(naphthalene-2-yl)propanamide)-1-(tert-butoxy)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid (48.8 mg, 0.071 mmol) and HATU (32.5 mg, 0.085 mmol) were mixed with DIPEA (0.025 ml, 0.142 mmol). The reaction mixture was stirred at room temperature for 19 hours, followed by dilution with an additional 20 mL of DCM. The mixture was washed with saturated sodium bicarbonate (20 mL) and brine (20 mL), dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution in 0-20% methanol in dichloromethane to obtain the title compound as an off-white solid (39.5 mg, 0.026 mmol, yield 40.5%). LCMS m / z 685.15(M+2H) / 2. 1H NMR(400MHz,chloroform-d)δ ppm 0.58-0.73(m,1H)0.81-0.96(m,1H)1.08-1.32(m,11H)1.33-1.48(m,27H)1.55-1.77(m,4H)1.79-2.00(m,12H)2.02- 2.13(m,2H)2.16-2.26(m,2H)2.28-2.43(m,2H)2.67(s,3H)2.76-2.88(m,3H)3.16-3.26(m,3H)3.28-3.35(m,1H)3.38 -3.52(m,7H)3.68-3.82(m,4H)3.89-4.03(m,2H)4.09-4.24(m,2H)4.25-4.34(m,1H)4.37-4.49(m,1H)4.77-4.86(m,1 H)5.43-5.55(m,1H)5.65-5.74(m,1H)5.77-5.94(m,2H)7.26-7.33(m,1H)7.35-7.40(m,1H)7.42-7.50(m,2H)7.58(br d,J=7.82Hz,1H)7.64-7.84(m,3H)8.54(d,J=1.47Hz,1H)8.57-8.69(m,2H).

[0406] Step 2: N-(2-(((7S,11S,18S)-7,11-bis(tert-butoxycarbonyl)-2,2-dimethyl-19-(naphthalene-2-yl)-4,9,17-trioxo-3-oxa-8,10,16-triazanonadecane-18-yl)amino)-2-oxoethyl)-N-((1R,4S)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carbonyl)glycine sodium. [ka]

[0407] To a solution of di-tert-butyl(((S)-1-(tert-butoxy)-6-((S)-2-(2-((1R,4S)-N-(2-methoxy-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)-3-(naphthalene-2-yl)propanamide)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid (39 mg, 0.028 mmol) in THF (2.3 mL), 42.7 μl of 1.0 M aqueous sodium hydroxide solution (0.043 mmol) was added. A homogeneous pale yellow solution was stirred at room temperature for two nights, followed by concentration under vacuum. The resulting residue was azeotropically mixed with toluene (2x), dried under high vacuum, and the title compound was obtained as a clear film (38.6 mg, 0.028 mmol, quantitative yield), which was used without further purification or characterization. LCMS m / z 678.11(M+2H) / 2.

[0408] Step 3: (((S)-1-carboxy-5-((S)-2-(2-((1R,4S)-N-(2-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazoline-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)amino)-2-oxoethyl)-4-(4-( ((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)-3-(naphthalene-2-yl)propanamide)pentyl)carbamoyl)-L-glutamic acid (Example 3). [ka]

[0409] N-(2-(((7S,11S,18S)-7,11-bis(tert-butoxycarbonyl)-2,2-dimethyl-19-(naphthalene-2-yl)-4,9,17-trioxo-3-oxa-8,10,16-triazanonadecane-18-yl)amino)-2-oxoethyl)-N-((1R,4S)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethoxy)cyclo To a solution of hexyl)oxy)butanamide)cyclohexane-1-carbonyl)glycine sodium (19 mg, 0.014 mmol) and (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-2-one hydrochloride (6.91 mg, 0.014 mmol), HATU (6.82 mg, 0.018 mmol) and DIPEA (7.23 μl, 0.041 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours, followed by dilution with an additional 20 mL of DCM. The organic solution was washed with saturated sodium bicarbonate aqueous solution (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was redissolved in 2 mL of DCM. TFA (106 μl, 1.380 mmol) was added, and the reaction mixture was stirred at room temperature for 30 hours. The reaction mixture was concentrated and dried under high vacuum. The resulting residue was purified by MDAP (XSelect® CSH C18 5 μm column, 40 mL / min) using a 15-55% gradient of acetonitrile in water containing trifluoroacetic acid (0.1%) to obtain the title compound as a white solid (11.1 mg, 0.0067 mmol, yield 48.8%). LC-MS m / z 817.19(M+2H) / 2. HPLC: 100% purity at 254 nm. 1H NMR(400MHz, methanol-d4)δ ppm1.22-1.31(m,5H)1.32-1.48(m,12H)1.54-1.54(m,1H)1.54-1.67(m,2H)1.70-1.86(m,6H)1.89-2.05(m,8H)2.08-2.33( m,10H)2.38-2.47(m,3H)2.65-2.74(m,6H)2.76-2.84(m,3H)2.85-2.96(m,2H)3.02-3.13(m,2H)3.14-3.22(m,2H)3.24-3.3 1(m,2H)3.43-3.53(m,6H)3.64-3.78(m,4H)3.80-3.88(m,2H)3.91-4.14(m,4H)4.16-4.36(m,7H)4.71-4.79(m,1H)5.00-5. 07(m,2H)5.59-5.69(m,1H)7.34-7.46(m,3H)7.58-7.69(m,2H)7.72-7.81(m,2H)7.94-8.00(m,2H)8.02-8.11(m,1H)8.31(br d,J=8.80Hz,1H)8.37(br d,J=8.31Hz,1H)8.74-8.84(m,3H)8.87-8.94(m,2H).

[0410] Example 4 (((S)-1-carboxy-5-((S)-2-((1R,4S)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl Carbamoyl cyclohexyl amino-4-oxobutoxy cyclohexyl oxyethyl amino-2-oxoethyl 1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide acetamide ethoxy cyclohexyl oxy butanamide cyclohexane-1-carboxamide-3-(naphthalene-2-yl)propanamide pentyl carbamoyl-L-glutamic acid [ka] [ka]

[0411] Step 1: Benzyl (2-(tert-butoxy)-2-oxoethyl)glycinate [ka]

[0412] To a solution of benzyl 2-bromoacetate (20.0 g, 87.0 mmol) and tert-butylglycine hydrochloride (16.1 g, 96.0 mmol) in THF (175 ml), DIPEA (35.1 ml, 201 mmol) was added. The reaction mixture was stirred at room temperature for two nights. The white solid was removed by filtration and washed with ethyl acetate. The organic filtrate was diluted with ethyl acetate (200 mL), washed with aqueous sodium bicarbonate (200 mL) and brine (200 mL), washed with anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution in 0-60% ethyl acetate in hexane to obtain the title compound as a yellow oily substance. LCMS m / z 280.21(M+H)+. 1 ¹H NMR (400MHz, chloroform-d) δ ppm 1.48 (s,9H) 1.91 (br s,1H) 3.38 (s,2H) 3.53 (s,2H) 5.20 (s,2H) 7.32-7.43 (m,5H).

[0413] Step 2: Benzyl N-(2-(tert-butoxy)-2-oxoethyl)-N-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carbonyl)glycinate [ka]

[0414] To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxylic acid (500 mg, 2.270 mmol), benzyl (2-(tert-butoxy)-2-oxoethyl)glycinate (698 mg, 2.497 mmol), and HATU (1122 mg, 2.95 mmol) in dichloromethane (23 mL), DIPEA (0.793 mL, 4.54 mmol) was added. The reaction mixture was stirred at room temperature for 24 hours, followed by dilution with an additional 50 mL of DCM. The organic solution was washed with saturated sodium bicarbonate aqueous solution (30 mL) and brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography using elution with 0-10% methanol in dichloromethane to obtain the title compound as a yellow oily substance (1.20 g, 2.24 mmol, 99% yield). LCMS m / z 482.17(M+H)+. 1 1H NMR (400 MHz, chloroform-d)δ ppm1.35-1.47(m,9H)2.69-2.71(m,2H)2.74-2.81(m,1H)3.07-3.15(m,1 H)3.17-3.25(m,1H)3.69-3.81(m,2H)3.81-3.96(m,1H)3.98-4.32(m,2H )4.94-5.02(m,1H)5.07(s,1H)5.18(s,1H)7.24-7.30(m,1H)7.31-7.44( m,5H)7.50-7.62(m,1H)8.52-8.58(m,1H)8.64(dd,J=4.65,1.71Hz,1H).

[0415] Step 3: N-(2-(benzyloxy)-2-oxoethyl)-N-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carbonyl)glycine [ka]

[0416] To a solution of benzyl N-(2-(tert-butoxy)-2-oxoethyl)-N-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carbonyl)glycinate (600 mg, 1.246 mmol) in DCM (6 mL), TFA (1.92 mL, 24.9 mmol) was added. The reaction mixture was stirred at room temperature for 24 hours, concentrated, and dried under high vacuum to obtain the title compound as a yellow oil (832 mg, 1.26 mmol, quantitative yield). LCMS m / z 426.13(M+H)+. 1 ¹H NMR (400MHz, chloroform-d) δ ppm 2.78-2.85 (m,3H) 2.93-3.05 (m,1H) 3.11-3.21 (m,1H) 3.29-3.44 (m,1H) 3.67-3.79 (m,1H) 3.88-4.08 (m,2H) 4.10-4.20 (m,1H) 4.28-4.52 (m,1H) 5.07-5.41 (m,3H) 7.25-7.31 (m,1H) 7.32-7.42 (m,4H) 7.95-8.07(m,1H)8.31-8.38(m,1H)8.79-8.79(m,1H)8.79-8.87(m,1H)8.91-8.96(m,1H).

[0417] Step 4: tert-butyl(1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-(benzyloxy)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate [ka]

[0418] To a solution of tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (348 mg, 0.816 mmol) containing 2,2,2-trifluoroacetic acid and N-(2-(benzyloxy)-2-oxoethyl)-N-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carbonyl)glycine compound (400 mg, 0.741 mmol), HATU (367 mg, 0.964 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.387 mL, 2.22 mmol) were added. The reaction mixture was stirred at room temperature for 20 hours, followed by dilution with an additional 50 mL of DCM. The organic solution was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography using elution with 0-10% methanol in dichloromethane to obtain the title compound as a yellow oil (488 mg, 0.556 mmol, yield 75.0%). LCMS m / z 834.39(M+H)+. 1 H NMR(400MHz,chloroform-d)δ ppm1.40-1.49(m,13H) 1.50-1.58(m,3H)1.83-1.92(m,2H)1.93-2.19(m,8H)2.20-2.33(m,2H)2.63-2.75(m,4H)2 .78-2.91(m,1H)3.08-3.19(m,2H)3.22-3.32(m,2H)3.35-3.57(m,6H)3.67-3.84(m,3H)3. 89-4.26(m,3H)4.86-4.99(m,1H)5.05-5.14(m,1H)5.50-5.61(m,1H)7.26-7.32(m,3H)7.3 5-7.46(m,4H)7.49-7.61(m,1H)7.63-7.76(m,1H)8.53(d,J=1.96Hz,1H)8.59-8.72(m,1H).

[0419] Step 5: N-(2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-(tert-butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-N-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carbonyl)glycine [ka]

[0420] Under nitrogen, Pd-C (60.0 mg, 0.056 mmol) was added to a solution of tert-butyl(1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-(benzyloxy)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (470 mg, 0.564 mmol) in methanol (7 mL). The flask was evacuated, backfilled with a hydrogen gas balloon, and stirred at room temperature for 19 hours. The mixture was filtered through a Celite pad and washed with additional methanol. The filtrate was concentrated and dried under high vacuum to obtain the title compound as a clear film (415 mg, 0.463 mmol, yield 82%). LCMS m / z 744.22(M+H)+. 1H NMR(400MHz,chloroform-d)δ ppm1.06-1.21(m,2H)1.21-1.33(m,4H)1.39-1.46(m,9H)1.48-1.59(m,2H)1 .80-1.90(m,2H)1.92-2.06(m,10H)2.08-2.20(m,1H)2.25(td,J=7.09,2.45 Hz,2H)2.62-2.73(m,2H)2.77-2.92(m,4H)3.11(q,J=7.34Hz,2H)3.20-3.42 (m,2H)3.49(s,4H)3.63-3.76(m,2H)4.98(dd,J=13.45,6.11Hz,1H)5.81(br dd,J=15.65,7.83Hz,1H)5.88-6.03(m,1H)7.40(td,J=8.31,4.89Hz,1H)7.58-7.67(m, 1H)8.00-8.09(m,1H)8.50-8.56(m,1H)8.61(td,J=5.62,1.47Hz,1H)8.83-8.93(m,1H).

[0421] Step 6: Benzyl(1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-(tert-butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate [ka]

[0422] Benzyl (1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (180 mg, 0.390 mmol) and N-(2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-(tert-butoxycarbonyl)cyclohexyl)amino)-4-oxobut To a solution of 290 mg, 0.390 mmol of oxy(cyclohexyl)oxy(ethyl)amino)-2-oxoethyl)-N-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carbonyl)glycine (290 mg, 0.390 mmol), HATU (193 mg, 0.507 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.204 mL, 1.17 mmol) were added. The reaction mixture was stirred at room temperature for 28 hours, followed by dilution with an additional 100 mL of DCM. The organic solution was washed with saturated sodium bicarbonate aqueous solution (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution in 0-20% methanol in dichloromethane to obtain the title compound as a clear film (93 mg, 0.078 mmol, yield 20.1%). LCMS m / z 1186.45(M+2H) / 2. 1H NMR(400MHz,chloroform-d)v δ ppm1.05-1.19(m,4H)1.22-1.33(m,4H)1.48-1.64(m,14H)1.79-1.88(m,4H)1.92-2.07( m,16H)2.09-2.15(m,1H)2.18-2.33(m,5H)2.62-2.67(m,3H)2.77-2.88(m,1H)3.05-3.1 9(m,4H)3.21-3.30(m,3H)3.38-3.48(m,8H)3.49-3.57(m,2H)3.62-3.78(m,5H)3.79-3. 85(m,2H)3.86-3.91(m,1H)3.95-4.11(m,1H)4.92(d,J=6.36Hz,1H)5.10(s,2H)5.74(br d,J=3.91Hz,2H)6.72(br d,J=6.85Hz,1H)7.18-7.44(m,6H)7.56(br d,J=6.85Hz,1H)8.49(s,1H)8.61(br s,1H)8.78(br s,1H)11.11(br s,1H).

[0423] Step 7: (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-((benzyloxy)carbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid [ka]

[0424] To a solution of benzyl (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-(tert-butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (93 mg, 0.078 mmol), TFA (302 μl, 3.92 mmol) was added. The reaction mixture was stirred at room temperature for 30 hours, concentrated under vacuum, and dried under high vacuum to obtain the crude title compound as a clear oil (98.0 mg, 0.075 mmol, 95% yield), which was used without further purification or characterization. LCMS m / z 1243.38(M+H)+.

[0425] Step 8: Di-tert-butyl(((S)-6-((S)-2-((1R,4S)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-((benzyloxy)carbonyl)cyclohexyl)amino)-4-oxobutioxy)cyclohexyl)oxy)ethyl)amino)- 2-Oxoethyl)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrroridine-3-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)-3-(naphthalene-2-yl)propanamide)-1-(tert-butoxy)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid [ka]

[0426] Dichloromethane (315 μl) contains 2,2,2-trifluoroacetic acid (1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-((benzyloxy)carbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid compound (98 mg, To a solution of 0.079 mmol) and di-tert-butyl(((S)-6-((S)-2-amino-3-(naphthalene-2-yl)propanamide)-1-(tert-butoxy)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid (13.5 mg, 0.020 mmol), 2-(3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (44.9 mg, 0.118 mmol) and N-ethyl-N-isopropylpropan-2-amine (41.2 μl, 0.236 mmol) were added. The reaction mixture was stirred at room temperature for 3 nights, and additional N-ethyl-N-isopropylpropan-2-amine (41.2 μl, 0.236 mmol) was added until the pH reached approximately 10. After a further 3 hours at room temperature, additional di-tert-butyl(((S)-6-((S)-2-amino-3-(naphthalene-2-yl)propanamide)-1-(tert-butoxy)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid (13.5 mg, 0.020 mmol) was added. The mixture was stirred for a further 20 hours, followed by the addition of additional HATU (44.9 mg, 0.118 mmol). After 24 hours, DMF (1 mL) and additional di-tert-butyl(((S)-6-((S)-2-amino-3-(naphthalene-2-yl)propanamide)-1-(tert-butoxy)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid (13.5 mg, 0.020 mmol) were added.The mixture was stirred at room temperature for a further 24 hours, then diluted with DCM (30 mL), washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution using 0-20% methanol in DCM to obtain the title compound as a clear film (69.4 mg, 0.036 mmol, yield 45.1%). LCMS 899.23(M+H)+. 1 H NMR(400Hz,chloroform-d)δ ppm1.22-1.38(m,2H)1.38-1.49(m,27H)1.51-1.61(m,18H)1.82-1.93(m,8H)1.94-2.10(m,14H)2.17-2.32(m,6H)2.34-2.47(m ,2H)2.60-2.69(m,2H)2.77-2.88(m,1H)2.93-2.97(m,1H)3.04-3.18(m,6H)3.20-3.33(m,5H)3.40-3.55(m,10H)3.63-3.73(m, 4H)3.75-3.91(m,4H)3.98-4.08(m,1H)4.17-4.26(m,1H)4.92-4.98(m,1H)5.10-5.15(m,2H)5.56-5.65(m,1H)5.68-5.75(m,1H) )6.50-6.60(m,1H)7.14-7.25(m,1H)7.31-7.53(m,9H)7.55-7.68(m,3H)7.70-7.78(m,1H)8.48-8.55(m,1H)8.62-8.68(m,1H).

[0427] Step 9: 4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1S,4R)-4-(((7S,11S,18S)-7,11-bis(tert-butoxycarbonyl)-2,2-dimethyl-19-(naphthalene-2-yl)-4,9,17-trioxo-3-oxa-8,10,16 Triazanonadecane-18-yl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid [ka]

[0428] Under nitrogen gas, di-tert-butyl(((S)-6-((S)-2-((1R,4S)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-(((benzyloxy)carbonyl)cyclohexyl)amino)-4-oxobutioxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1-methyl in methanol (154 μl). Pd / C (4.09 mg, 3.84 μmol) was added to a solution of -5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)-3-(naphthalene-2-yl)propanamide)-1-(tert-butoxy)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid (69 mg, 0.038 mmol).

[0429] The flask was evacuated, backfilled with a hydrogen gas balloon, and stirred at room temperature for 5 hours. The flask was then evacuated again and backfilled with nitrogen gas. Additional Pd / C (4.09 mg, 3.84 μmol) was added. The flask was evacuated again, backfilled with a hydrogen gas balloon, and stirred at room temperature for 3 nights. The mixture was filtered through a Celite pad and washed with additional MeOH (10 mL). The filtrate was concentrated and dried under high vacuum to obtain the title compound as a clear oil (65.5 mg, 0.034 mmol, 89% yield). LCMS m / z 854.20(M+2H) / 2. 1 H NMR(400MHz,chloroform-d)δ ppm1.10-1.19(m,4H)1.26-1.35(m,10H)1.38-1.51(m,40H)1.83-1.93(m,5H)1.95-2.11(m,12H)2.19-2.32 (m,5H)2.33-2.45(m,3H)2.62-2.66(m,1H)2.66-2.71(m,3H)2.80-2.89(m,2H)2.93-2.98(m,2H)2.99-3.08( m,4H)3.09-3.18(m,3H)3.23-3.35(m,5H)3.41-3.52(m,9H)3.53-3.65(m,5H)3.73-3.90(m,4H)3.99-4.09( m,1H)4.18-4.39(m,2H)4.45-4.63(m,1H)4.95(d,J=6.85Hz,1H)6.43-6.55(m,1H)7.36-7.49(m,2H)7.59(br d,J=7.82Hz,1H)7.63-7.69(m,1H)7.75(br d,J=7.82Hz,1H)8.52(d,J=1.96Hz,1H)8.64(dd,J=4.89,1.47Hz,1H).

[0430] Step 10: (((S)-1-carboxy-5-((S)-2-((1R,4S)-4-(4-(((1S,4R)-4-(2-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazoline-4-yl)amino)pyrrolidine-1-yl)cyclohexyl (L)Carbamoyl)Cyclohexyl)Amino)-4-Oxobutoxy)Cyclohexyl)Oxy)Ethyl)Amino)-2-Oxoethyl)-1-Methyl-5-Oxo-2-(Pyridine-3-yl)Pyrrolidine-3-Carboxamide)Acetamide)Ethoxy)Cyclohexyl)Oxy)Butanamide)Cyclohexane-1-Carboxamide)-3-(Naphthalene-2-yl)Propanamide)Pentyl)Carbamoyl)-L-Glutamic Acid (Example 4) [ka]

[0431] (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-2-one hydrochloride (11.7 mg, 0.023 mmol) and 4-(4-(((1S,4R)-4-(2-((2S,3S)-N-(2-((2-(((1R,4S)-4-(4-(((1S,4R)-4-(((7S,11S,18S)-7,11-bis(tert-butoxycarbonyl)-2,2-dimethyl-19-(naphthalene-2-yl)-4 in DCM (117 mL) To a solution of 9,17-trioxo-3-oxa-8,10,16-triazanonadecane-18-yl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid (40 mg, 0.023 mmol), HATU (11.6 mg, 0.030 mmol) and DIPEA (12.3 mL, 0.070 mmol) were added. The reaction mixture was stirred at room temperature for 17 hours, followed by dilution with an additional 20 mL of DCM. The organic solution was washed with saturated sodium bicarbonate aqueous solution (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. To the solution of the obtained residue in DCM (2 mL), TFA (90 μl, 1.172 mmol) was added. After 4 hours, an additional TFA (90 μl, 1.172 mmol) was added, and the mixture was stirred at room temperature for 21 hours, followed by concentration under vacuum. The obtained residue was purified by MDAP (XSelect® CSH C18 5 μm column, 40 mL / min) with elution using acetonitrile in a 15-55% gradient in water containing trifluoroacetic acid (0.1%), and the title compound was obtained as a gray solid (13.0 mg, 0.0065 mmol, yield 27.7%). LCMS m / z 993.05 (M+2H) / 2. HPLC: purity >96% at 254 nm. 11H NMR (400 MHz, methanol-d4) δ ppm 1.10 - 1.24 (m, 4H) 1.26 - 1.40 (m, 12H) 1.42 - 1.47 (m, 3H) 1.48 - 1.74 (m, 7H) 1.77 - 1.94 (m, 10H) 1.94 - 2.04 (m, 9H) 2.10 - 2.29 (m, 10H) 2.39 - 2.49 (m, 3H) 2.61 - 2.66 (m, 2H) 2.67 - 2.74 (m, 5H) 2.78 - 2.83 (m, 3H) 2.90 - 3.00 (m, 2H) 3.04 - 3.18 (m, 3H) 3.21 - 3.30 (m, 2H) 3.36 - 3.40 (m, 3H) 3.40 - 3.60 (m, 13H) 3.62 - 3.78 (m, 4H) 3.82 - 3.92 (m, 3H) 3.98 - 4.15 (m, 5H) 4.17 - 4.24 (m, 2H) 4.25 - 4.31 (m, 1H) 4.31 - 4.37 (m, 2H) 4.64 - 4.71 (m, 1H) 5.18 (br d, J = 5.87 Hz, 1H) 5.53 - 5.64 (m, 2H) 7.38 - 7.42 (m, 1H) 7.42 - 7.48 (m, 2H) 7.68 - 7.71 (m, 1H) 7.76 - 7.84 (m, 3H) 7.92 - 7.97 (m, 1H) 7.97 - 8.02 (m, 1H) 8.30 - 8.35 (m, 2H) 8.76 (s, 1H) 8.80 (br d, J = 5.38 Hz, 1H) 8.88 (s, 1H) 8.98 (s, 1H).

[0432] Example 5 (((S)-1-carboxy-5-((S)-2-((1R,4S)-4-(4-(((1S,4R)-4-(2-((2-(((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl) Amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)3-(naphthalene-2-yl)propanamide)pentyl)carbamoyl)-L-glutamic acid [ka] [ka]

[0433] Step 1: (2S,3S)-N-allyl-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide [ka]

[0434] To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxylic acid (800 mg, 3.63 mmol), propa-2-en-1-amine (0.327 ml, 4.36 mmol), and HATU (1.80 g, 4.72 mmol) in dichloromethane (23 mL), DIPEA (1.27 ml, 7.27 mmol) was added. The reaction mixture was stirred at room temperature for 17 hours, followed by dilution with additional dichloromethane (30 mL). The organic solution was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution using 0-20% methanol in dichloromethane to obtain the title compound as a yellow oil (631 mg, 2.36 mmol, yield 65.0%). 1 H NMR(400MHz,chloroform-d)δ ppm2.68(s,3H)2.75-2.94(m,3H)3.82-3.98(m,2H)4.82(d,J=6.85Hz,1H)5.06-5.18(m,2H)5.64(br s,1H)5.79(ddt, J=17.12,10.27,5.87,5.87Hz,1H)7.38(dd,J=7.58,5.14Hz,1H)7.59(dt,J=7.82,1.96Hz,1H)8.54(d,J=1.96Hz,1H)8.64(dd,J=4.65,1.71Hz,1H).

[0435] Step 2: (2S,3S)-1-methyl-5-oxo-N-(2-oxoethyl)-2-(pyridine-3-yl)pyrrolidine-3-carboxamide. [ka]

[0436] To a solution of (2S,3S)-N-allyl-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide (470 mg, 1.81 mmol) in a 3:1 mixture of THF (12 mL) and water (4.00 mL) cooled to 0°C, osmium tetroxide (3.41 mL, 0.272 mmol) was added. After 15 minutes, sodium periodate (853 mg, 3.99 mmol) was added.

[0437] After 1.5 hours, the water bath was removed, and the mixture was warmed to room temperature and stirred for 1 hour. The reaction mixture was then diluted with dichloromethane and stirred at room temperature for 30 minutes. The mixture was filtered to remove solids, washed with an additional 20 mL of dichloromethane, and then washed with 1:1 methanol and dichloromethane (2 × 20 mL). The combined filtrate was concentrated, and the resulting residue was pre-adsorbed onto silica gel. Purification by silica gel chromatography, elution with 0-15% methanol in dichloromethane, yielded the title compound as a clear oil (310 mg, 1.07 mmol, yield 59.0%). LCMS m / z 262.06(M+H)+. 1 ¹H NMR (400MHz, chloroform-d) δ ppm 2.78-2.89 (m,2H) 3.28-3.38 (m,1H) 3.48-3.55 (m,3H) 4.14-4.35 (m,1H) 4.56-4.66 (m,1H) 4.75-4.85 (m,1H) 7.36-7.44 (m,1H) 7.57-7.66 (m,1H) 8.52-8.58 (m,1H) 8.60-8.67 (m,1H) 9.66 (s,1H).

[0438] Step 3: Benzyl N-(2-(tert-butoxy)-2-oxoethyl)-S N-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)glycinate. [ka]

[0439] To a solution of benzyl (2-(tert-butoxy)-2-oxoethyl)glycinate (331 mg, 1.186 mmol) in 1,2-dichloroethane (6 mL), (2S,3S)-1-methyl-5-oxo-N-(2-oxoethyl)-2-(pyridine-3-yl)pyrrolidine-3-carboxamide (310 mg, 1.186 mmol) and acetic acid (3.40 μl, 0.059 mmol) were added. Sodium triacetoxyborate (503 mg, 2.373 mmol) was added to this mixture. The reaction mixture was stirred at room temperature for 19 hours, diluted with dichloromethane (50 mL), washed with saturated sodium bicarbonate aqueous solution (50 mL) and brine (50 mL), dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography using elution with 0-10% methanol in dichloromethane to obtain the title compound as a pale yellow oily substance (343.2 mg, 0.635 mmol, yield 53.5%). LCMS m / z 525.20(M+H)+. 1 H NMR(400MHz,chloroform-d)δ ppm1.43(s,9H)2.69(s,3H)2.73-2.82(m,2H)2.83-2.88(m,2H)3.24(q,J=5.22Hz,2H)3.29 -3.42(m,2H)3.44-3.58(m,3H)4.87(d,J=6.85Hz,1H)5.10(s,2H)7.30-7.43(m,6H)7.55(br t,J=3.91Hz,1H)7.62(dt,J=7.83,1.96Hz,1H)8.56(d,J=1.96Hz,1H)8.59(dd,J=4.89,1.47Hz,1H).

[0440] Step 4: N-(2-(benzyloxy)-2-oxoethyl)-N-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)glycine [ka]

[0441] To a solution of benzyl N-(2-(tert-butoxy)-2-oxoethyl)-S N-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)glycinate (340 mg, 0.648 mmol) in dichloromethane (3.2 mL), TFA (999 μl, 12.96 mmol) was added. The mixture was stirred at room temperature for 16 hours. Additional TFA (999 μl, 12.96 mmol) was added, and the reaction was stirred at room temperature for 5 hours. Additional TFA (999 μl, 12.96 mmol) was added again, and the mixture was stirred at room temperature for 17 hours. The reaction was concentrated and dried under high vacuum to obtain the title compound as a pale yellow oil (699 mg, 0.648 mmol, theoretical yield). LCMS m / z 469.16(M+H + ). 1 H NMR(400MHz,chloroform-d)δ ppm2.78(s,3H)2.89(dd,J=18.10,6.85Hz,1H)3.14(br dd,J=17.85,10.03Hz,1H)3.32-3.51(m,2H)3.62(br s,2H)3.83-3.98(m,1H)4.06-4.17(m,2H)4.22-4.38(m,2H)5.28-5.40(m,3H)7.35-7.53(m,5H)8.09(br dd,J=8.07,5.62Hz,1H)8.44(br d,J=7.83Hz,1H)8.63-8.78(m,1H)8.90(d,J=5.38Hz,1H)9.52(br s,1H).

[0442] Step 5: tert-butyl(1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2-(benzyloxy)-2-oxoethyl)(2-((2S,3S)-1-ethyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate. [ka]

[0443] To a solution of 2,2,2-trifluoroacetic acid-containing N-(2-(benzyloxy)-2-oxoethyl)-N-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)glycine compound (370 mg, 0.635 mmol) in dichloromethane (7 mL), HATU (314 mg, 0.826 mmol), N-ethyl-N-isopropylpropan-2-amine (1.106 mL, 6.35 mmol), and tert-butyl(1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (298 mg, 0.699 mmol) were added. The reaction mixture was stirred at room temperature for 3 hours, followed by dilution with an additional 20 mL of dichloromethane. The organic solution was washed with 20 mL of saturated sodium bicarbonate aqueous solution and 20 mL of brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography with elution using 0-20 percent methanol in dichloromethane to obtain the title compound as a yellow oil (385.7 mg, 0.396 mmol, yield 62.3 percent). LCMS m / z 877.25(M+H)+. 1 H NMR(400MHz,chloroform-d)δ ppm1.42(br s,9H)1.50-1.59(m,3H)1.83-1.92(m,3H)1.92-1.98(m,4H)1.99-2.08(m,3H)2.08-2.19(m,1H)2.26 (t,J=7.34Hz,2H)2.70(s,2H)2.76(t,J=5.62Hz,2H)2.81-2.83(m,2H)3.14(q,J=7.34Hz,8H)3.26(br d,J=1.47Hz,3H)3.46-3.48(m,3H)3.67-3.77(m,8H)4.88(d,J=6.85Hz,1H)5.13(s,2H)5.62-5.73(m,1H)7.01-7.09(m,1H)7.16(br s,1H)7.31-7.46(m,5H)7.61(dt,J=7.83,1.96Hz,1H)8.56(d,J=2.45Hz,1H)8.61(dd,J=4.89,1.47Hz,1H).

[0444] Step 6: N-(2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-(tert-butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-N-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)glycine. [ka]

[0445] Under a nitrogen gas atmosphere, Pd-C (46.7 mg, 0.044 mmol) was added to a solution of tert-butyl(1R,4r)-4-(4-(((1S,4R)-4-(2-(2-((2-(benzyloxy)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (385 mg, 0.439 mmol) in methanol (4.5 mL). The flask was evacuated, backfilled with a hydrogen gas balloon, and stirred at room temperature for 3 nights. The mixture was filtered through a Celite plug and washed with an additional methanol (20 mL). The filtrate was concentrated under vacuum and dried under high vacuum to obtain the title compound as a yellow oil. LCMS m / z 787.31(M+H)+. 1 H NMR(400MHz,chloroform-d)δ ppm1.11-1.28m,4H)1.39-1.48(m,9H)1.85-2.18(m,12H)2.26(br t,J=6.85Hz,2H)2.71(br s,2H)2.83(s,6H)3.15(q,J=7.34Hz,6H)3.26-3.32(m,2H)3.33-3.38(m,2H)3.4 3-3.49(m,2H)3.67-3.80(m,6H)4.90(d,J=6.36Hz,1H)5.65-5.86(m,1H)7.41(br dd,J=7.82,4.89Hz,1H)7.64(br d,J=7.82Hz,1H)8.56-8.63(m,1H)10.28-10.74(m,3H).

[0446] Step 7: tert-butyl(1R,4r)-4-(4-(((1S,4R)-4-(2-((2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate. [ka]

[0447] N-(2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-(tert-butoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-N-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl) To a solution of lysine (345 mg, 0.438 mmol) and methyl(1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (185 mg, 0.482 mmol), HATU (217 mg, 0.570 mmol) and N-ethyl-N-isopropylpropan-2-amine (153 μl, 0.877 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, followed by dilution with additional dichloromethane (20 mL). The organic solution was washed with saturated sodium bicarbonate aqueous solution (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography using elution with 0-20% methanol in dichloromethane to obtain the title compound as a sticky yellow solid (417.9 mg, 0.330 mmol, 75% yield). LCMS m / z 1153.40 (M+H+).1 H NMR(400MHz,chloroform-d)δ ppm 0.93-1.31(m,10H)1.32-1.65(m,14H)1.73-2.14(m,19H)2.15-2.39(m,5H)2.53-3.03(m,8H)3.05-3.35(m,12H)3.42(br s,10H)3.56-3.87(m,6H)3.96-4.42(m,2H)4.86(br d,J=4.89Hz,1H)5.87(br d,J=0.98Hz,2H)7.04(br s,1H)7.19-7.45(m,1H)7.51-7.78(m,2H)8.54(br d, J = 17.61 Hz, 2H).

[0448] Step 8: (1R,4r)-4-(4-(((1S,4R)-4-(2-((2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid. [ka]

[0449] To a solution of tert-butyl(1R,4r)-4-(4-(((1S,4R)-4-(2-((2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (220 mg, 0.191 mmol), TFA (294 μl, 3.81 mmol) was added. The reaction mixture was stirred at room temperature for 1.5 hours, concentrated under vacuum, and dried under high vacuum to obtain the title compound as a yellow oil (475.5 mg, 0.188 mmol, 99% yield). LC-MS m / z 1097.29(M+H)+. 1 H NMR(400MHz,METHANOL-d4)δ ppm1.19-1.36(m,14H)1.37-1.42(m,2H)1.44-1.58(m,4H)1.78-1.89(m,4H)1.92-2.08(m,14H)2.20-2.38(m,6H)2.73(br s,3H)2.77-2.85(m,1H)2.89-2.99(m,1H)3.13-3.27(m,2H)3.28-3.39(m,4H)3.40-3. 52(m,9H)3.53-3.64(m,5H)3.67(s,3H)3.75(s,1H)4.03-4.27(m,4H)5.30(d,J=5.87H z,1H)7.47-7.60(m,1H)8.13(dd,J=8.31,5.87Hz,1H)8.43-8.51(m,1H)8.65(dt,J=8. 31,1.71Hz,1H)8.76(dd,J=4.65,1.22Hz,1H)8.87-8.94(m,1H)9.04(d,J=1.47Hz,1H).

[0450] Step 9: Methyl(1R,4r)-4-(4-(((1S,4R)-4-(2-((2-(((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazoline-4-yl)amino)pyrrolidine-1-yl)cyclohexyl) Rubamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate. [ka]

[0451] (1R,4r)-4-(4-(((1S,4R)-4-(2-((2-((2-(((1R,4S)-4-(4-(((1r,4R)-4-(methoxycarbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid (150 mg, 0.137 mg) in dichloromethane (3 mL) To a solution of (1,1,2,3) and N-ethyl-N-isopropylpropan-2-amine (238 μl, 1.37 mmol), 2-(3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (78.0 mg, 0.205 mmol) and (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-2-one hydrochloride (82.0 mg, 0.164 mmol) were added. The reaction mixture was stirred at room temperature for 1.5 hours, followed by dilution with an additional 30 mL of dichloromethane. The organic solution was washed with saturated sodium bicarbonate aqueous solution (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel chromatography using elution with 0-20% methanol in dichloromethane, and the title compound was obtained as a yellow film (233.6 mg, 0.136 mmol, theoretical yield). LCMS m / z 772.60(M+2H) / 2. 1H NMR(400MHz,クロロホルム-d)δ ppm1.04-1.27(m,10H)1.34-1.43(m,9H)1.44-1.58(m,4H)1.60-1.70(m,2H)1.77-1.85(m,4H)1.86-2.02(m,14H)2.15-2.25(m,6H)2.37-2.49(m,1H)2.60-2.66(m,4H)2.68-2.77(m,3H)2.77-2.89(m,3H)2.95(td,J=9.05,6.85Hz,2H)3.09(q,J=7.66Hz,2H)3.13-3.24(m,9H)3.26-3.35(m,4H)3.38-3.45(m,14H)3.63(s,3H)3.69(dt,J=13.33、6.79Hz,2H)3.97-4.07(m,1H)4.75(br s,1H)4.85(d,J=6.85Hz,1H)5.08(td,J=7.58,4.40Hz,1H)5.90-6.04(m,2H)7.16(dd,J=8.56,4.16Hz,1H)7.23(q,J=5.38Hz,2H)7.32(dd,J=7.83,4.89Hz,1H)7.57(dt,J=7.82,1.96Hz,1H)7.75(t, J=4.89Hz,1H)7.86(d,J=0.98Hz,2H)8.12(dd,J=8.56,1.22Hz,1H)8.46(dd,J=4.16,1.22Hz,1H)8.49(d,J=1.96Hz,1H)8.54(dd,J=4.65,1.71Hz,1H)8.62(s,1H)8.82(s,1H)。

[0452] Step 10: 4-(4-(((1S,4R)-4-(2-((2-(((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazoline-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamate (Iyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid. [ka]

[0453] A solution of methyl(1R,4r)-4-(4-(((1S,4R)-4-(2-((2-(((2-(((1R,4S)-4-(4(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl) in THF (2 mL) contains oxy)ethyl)amino)-2-oxoethyl) To a solution of (2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylate (140 mg, 0.091 mmol), 181 μl, 0.181 mmol, was added. The reaction mixture was stirred at room temperature for 21 hours, concentrated, and dried under high vacuum to obtain the title compound as a white solid (188.2 mg, 0.091 mmol, theoretical yield). LCMS m / z 765.62(M+2H) / 2. 1H NMR(400MHz,chloroform-d)δ ppm 0.84-1.00(m,4H)1.04-1.20(m,14H) 1.61-1.73(m,6H)1.75-2.02(m,22H)2.11-2.25(m,8H)2.39-2.48(m,1H)2.52-2.72(m,7H)2.75-2.90(m,2H)2.99-3.26(m,14H) 3.28-3.45(m,13H)3.49-3.70(m,4H)3.72-3.79(m,1H)3.98-4.09(m,1H)5.05-5.11(m,2H)6.84-7.00(m,1H)7.58(br d,J=6.36Hz,1H)7.81-7.90(m,2H)8.09-8.26(m,1H)8.45(br d,J=15.16Hz,2H)8.59(s,1H)8.86(br s,1H)9.18-9.34(m,1H).

[0454] Step 11: (((S)-1-carboxy-5-((S)-2-((1R,4S)-4-(4-(((1S,4R)-4-(2-((2-(((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazoline-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl) Amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)3-(naphthalene-2-yl)propanamide)pentyl)carbamoyl)-L-glutamic acid (Example 5). [ka]

[0455] Dichloromethane (30 mL) and DMF (3 mL) contain 4-(4-(((1S,4R)-4-(2-((2-(((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrro To a solution of din-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid (100 mg, 0.065 mmol), HATU (37.3 mg, 0.098 mmol), N-ethyl-N-isopropylpropan-2-amine (0.034 mL, 0.196 mmol), and di-tert-butyl[((S)-6-((S)-2-amino-3-(naphthalene-2-yl)propanamide)-1-(tert-butoxy)-1-oxohexane-2-yl)carbamoyl]-L-glutamic acid (44.8 mg, 0.065 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, followed by dilution with an additional 20 mL of DCM. The organic solution was washed with saturated sodium bicarbonate solution (15 mL) and saline solution (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated.The resulting residue was purified by silica gel chromatography using elution with 0-20% methanol in dichloromethane to obtain the intermediate di-tert-butyl(((S)-1-(tert-butoxy)-6-((S)-2-((1R,4S)-4-(4-(((1S,4R)-4-(2-((2-(((2-(((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)cal (Bamoyl)cyclohexyl)(amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyrrolidine-3-carboxamide)ethyl)amino)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)-3-(naphthalene-2-yl)propanamide)-1-oxohexane-2-yl)carbamoyl)-L-glutamic acid was obtained as a white solid (70.5 mg, 0.032 mmol, yield 49.1%). LCMS m / z 1099.25(M+2H) / 2).

[0456] To a solution of the above-mentioned intermediate (70.5 mg, 0.032 mmol) in dichloromethane (30 mL), TFA (0.252 mL, 3.27 mmol) was added. The mixture was stirred at room temperature for 18 hours, and then concentrated under vacuum. The resulting residue was purified by MDAP (XSelect® CSH C18 5 μm column, 40 mL / min) using acetonitrile in a 15-55% gradient in water containing trifluoroacetic acid (0.1%) to obtain the title compound as a white solid (26.0 mg, 0.013 mmol, yield 19.4%). LCMS m / z 1014.75 (M+2H) / 2. HPLC: 100% purity at 254 nm. 1H NMR(400MHz,メタノール-d4)δ ppm1.04-1.40(m,20H)1.41-1.46(m,3H)1.48-1.74(m,6H)1.74-1.94(m,12H )1.95-2.04(m,10H)2.08-2.28(m,11H)2.35-2.56(m,3H)2.59-2.67(m,1H)2 .68-2.80(m,4H)2.80-2.85(m,3H)2.89-2.99(m,1H)3.00-3.23(m,5H)3.23- 3.31(m,3H)3.35-3.38(m,2H)3.39-3.50(m,9H)3.52-3.61(m,6H)3.62-3.82 (m,4H)3.83-3.96(m,2H)4.00-4.15(m,4H)4.16-4.28(m,2H)4.29-4.39(m,2 H)4.68(dd,J=8.56,6.60Hz,1H)5.25(d,J=5.87Hz,1H)5.60-5.70(m,1H)7.3 6-7.50(m,3H)7.69(s,1H)7.76-7.86(m,3H)8.00(d,J=8.80Hz,1H)8.05(dd, J=8.07、5.62Hz,1H)8.32(dd,J=9.05、1.71Hz,1H)8.42-8.49(m,1H)8.54(br d,J=8.31Hz,1H)8.85(d,J=4.40Hz,1H)8.89(s,1H)8.99(s,1H)9.04(s,1H).

[0457] Example 6

change

change

[0458] In DCM (2.5 mL), 4-(4-(((1R,4R)-4-(2-(2-((1R,4R)-N-(2-((2-(((1R,4R)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)amino)-2-oxoethyl)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3-yl)pyro To a solution of lysine-3-carboxamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxamide)acetamide)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid (130.0 mg, 70.68 μmol), (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-2-one hydrochloride (35.4 mg, 70.7 μmol), and HATU (34.9 mg, 91.9 μmol), DIPEA (18.3 mg, 24.6 μL, 141 μmol) was added. The reaction mixture was stirred at room temperature for 30 hours. An additional (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidine-2-one hydrochloride (3.54 mg, 7.07 μmol) was added. The reaction mixture was stirred at room temperature for 19 hours, then diluted with an additional 20 mL of dichloromethane, washed with 10 mL of saturated sodium bicarbonate aqueous solution and 10 mL of brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by MDAP (XSelect® C18 5 μm column, 40 mL / min) elution with acetonitrile in a 50-99% gradient in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) to obtain the title compound as a white solid (48.32 mg...

Claims

1. Compound of formula (I) 【Chemistry 1】 or a pharmaceutically acceptable salt thereof During the ceremony, T １ and T ２ Each of these is independently a target binding site, R １ C １～４ Alkyl or C ３～６ It is a cycloalkyl, G is a bond, -CH ２ CH ２ NH-, -C(O)CH ２ CH ２ OCH ２ CH ２ NH-, or -L ３ -CH ２ CH ２ NH- and is G １ and G ２ Each of these is independently -C(O)CH ２ -ien-CH ２ CH ２ NHC(O)CH ２ -ien-CH ２ CH ２ C(O)NH(CH ２ CH ２ O) ３ CH ２ CH ２ -, or -CH ２ CH ２ NHC(O)(CH ２ CH ２ O) ３ CH ２ CH ２ - and L' and L'' are independent of each other, and can be combined. 【Chemistry 2】 (wherein each y is an integer from 1 to 9), or 【Transformation 3】 (In the formula, each w is an integer from 0 to 5) L １ , L ２ and L ３ Each of these is independently a combination, or a divalent linker of formula (L-a), (L-c), or (L-e), where L １ , L ２ and L ３ At least one of them is a divalent linker of the following formulas (L-a), (L-c), or (L-e): 【Chemistry 4】 or its stereoisomer (In the formula, ring A and ring B are each independently of C ４～６ It is a cycloalkylene, L １ａ C ３～５ It is a linear alkylene, and one or two methylene units are -O- or -NR ａ Replaced with - Each R ａ These are independently hydrogen or C １～３ It is alkyl, L ２ａ These are -O-, -NHC(O)-, or -CH ２ (-O-) 【Transformation 5】 or its stereoisomer (In the formula, L １ｃ is C ２～１０ It is a linear alkylene, and one, two, or three methylene units are replaced with -O-, -NH-, -NHC(O)-, or -C(O)NH-. Ring A is C ４～６ Cycloalkylene or C ７～９ It is a cross-linked bicyclic cycloalkylene, L ２ｃ is -O- or saturated C ２～１０ It is a linear alkylene, and one, two, or three methylene units can be replaced with -O-, -NH-, -NHC(O)-, or -C(O)NH-). 【Transformation 6】 (In the formula, n is an integer between 3 and 50.) Each L' group 【Transformation 7】 Y in equation (I) １ It represents a covalent bond to the base, or Y １ If it is a combination, then T in formula (I) １ This represents a covalent bond to the group, and each of the L' groups 【Transformation 8】 L in equation (I) １ It represents a covalent bond to the base, or L １ If it is a combination, then G in formula (I) １ Represents a covalent bond to a base, Each L'' group 【Chemistry 9】 Y in equation (I) ２ It represents a covalent bond to the base, or Y ２ If it is a combination, then T in formula (I) ２ This represents a covalent bond to the group, and each of the L'' groups 【Chemistry 10】 L in equation (I) ２ It represents a covalent bond to the base, or L ２ If it is a combination, then G in formula (I) ２ Represents a covalent bond to a base, L １ Each of the bases 【Chemistry 11】 represents a covalent bond to the L' group in formula (I), or if L' is a bond, Y in formula (I). １ Represents a covalent bond to the base, or L' and Y １ If both are conjugates, then T in equation (I) １ Represents a covalent bond to the base, L １ Each of the bases 【Chemistry 12】 G in equation (I) １ Represents a covalent bond to a base, L ２ Each of the bases 【Chemistry 13】 represents a covalent bond to the L'' group in formula (I), or if L'' is a bond, then Y in formula (I). ２ Represents a covalent bond to the base, or L'' and Y ２ If both are conjugates, then T in equation (I) ２ Represents a covalent bond to the base, L ２ Each of the bases 【Chemistry 14】 G in equation (I) ２ Represents a covalent bond to a base, L ３ Each of the bases 【Chemistry 15】 This represents the covalent bond to the N atom in formula (I), and L ３ Each of the bases 【Chemistry 16】 This represents the covalent bond between the G group in formula (I) and the methylene group, Y １ and Y ２ Each of these is independently a bond or a divalent spacer portion having a length of 1 to 12 atoms, the compound or a pharmaceutically acceptable salt thereof.

2. R １ ha-CH ３ The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

3. L' and L'' are independent of each other, or are combined or 【Chemistry 17】 The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof.

4. L １ , L ２ or L ３ is a divalent linker of formula (L-a-i) or its stereoisomer, [Chemistry 18] In the formula, ring A and L １ａ L ２ａ , 【Chemistry 19】 The compound described in any one of the prior claims, or a pharmaceutically acceptable salt thereof, is defined for formula (L-a).

5. L １ , L ２ or L ３ is a divalent linker of formula (L- a-ii) or a stereoisomer thereof, 【Chemistry 20】 In the formula, L １ａ L ２ａ , 【Chemistry 21】 The compound according to any one of the prior claims, or a pharmaceutically acceptable salt thereof, is defined as for formula (L-a), where p is 1 or 2 and m is 1 or 2.

6. L １ , L ２ or L ３ is a divalent linker of formula (L-iii) or a stereoisomer thereof, 【Chemistry 22】 In the formula, p is 1 or 2, m is 1 or 2, and n is 1, 2, or 3. 【Chemistry 23】 The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein is defined for formula (L-a).

7. L １ , L ２ or L ３ teeth, 【Chemistry 24-1】 【Chemistry 24-2】 A compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, which is a divalent linker of formula (L-a) selected from the group consisting of the following.

8. L １ , L ２ or L ３ is a divalent linker of formula (L-c-i) or its stereoisomer, 【Chemistry 25】 In the formula, L １ｃ L ２ｃ , 【Chemistry 26】 The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein p is as defined for formula (L-c), and m is 1 or 2.

9. L １ , L ２ or L ３ teeth, 【Chemistry 27】 The compound according to any one of claims 1 to 3, which is a divalent linker of formula (L-c) selected from the group consisting of the following.

10. Y １ and Y ２ These are, independently, combined, -NH-, -(C １～１２ Alkylene) - (where one, two, or three methylene units are -O-, -NH-, N(CH) ３ )-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C ３～６ Cycloalkylene) -, -(C ３～６ (Cycloalkenylene) -, can be replaced with 3- to 10-membered heterocycloalkylene, arylene, or heteroarylene), or - (C ２～１２ Alkenylene) - (where one, two, or three methylene units are -O-, -NH-, N(CH) ３ )-, -C(O)-, NHC(O)-, -C(O)NH-, -(C ３～６ Cycloalkylene) -, -(C ３～６ A compound according to any one of the prior claims, or a pharmaceutically acceptable salt thereof, selected from cycloalkenylene (which can be replaced by a 3- to 10-membered heterocycloalkylene, arylene, or heteroarylene).

11. Y １ and Y ２ These are, independently, combined, -NH-, -(C １～６ Alkylene)-O-,-O-(C １～６ Alkylene) -, -(C ２～６ Alkenylene)-O-,-(C １～６ Alkylene)-C(O)-,-(C ２～６ Alkenylene)-C(O)-, Phenylene, Piperidinylene, Hydroxypiperidinylene, Fluoropiperidinylene, Azetidinylene, -C(O)-Piperadinylene-, -(C １～６ Alkylene)-Oxopiperazinylen-, Pyrrolidinylene, 7-9 membered bridged bicyclic heterocycloalkylene, -(C) １～６ Alkylene)-O-Phenylene-,-(C) ２～６ Alkenylene)-O-Piperidinylene,-(C １～５ Alkylene)-NH- (where 0, 1, or 2 methylene units are replaced by -O-),-NH-(C １～５ Alkylene)-NH-,-N(CH) ３ ) - (C １～５ Alkylene)-NH-,NH-(C １～５ Alkylene)-N(CH) ３ )-,-N(CH ３ ) - (C １～５ Alkylene)-N(CH) ３ ) -, -( C ３～６ Cycloalkylene)-NH-,-C(O)NH-(C １～５ Alkylene)-NH-,-C(O)NH-(C ３～６ Cycloalkylene)-NH-,-(C １～５ Alkylene)-O-(C) ３～６ Cycloalkylene)-NH-,-(C ３～６ Cycloalkenylene)-NH-, or 【Chemistry 28】 (In the formula, Y １ａ is a bond, -O-, -NH-, -NHC(O)-, -C(O)NH-, or C １～３ It is alkylene, Y ２ａ is a bond, -O-, -NH-, -NHC(O)-, -C(O)NH-, or C １～３ A compound according to any one of the prior claims, or a pharmaceutically acceptable salt thereof, selected from (which is alkylene).

12. Y １ and Y ２ Each of them operates independently. 【Chemistry 29】 A compound according to any one of the prior claims, or a pharmaceutically acceptable salt thereof, selected from the group consisting of the above.

13. Y １ and Y ２ Each of these can be performed independently, as a combination, NH, or 【Transformation 30】 The compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof.

14. T １ or T ２ teeth, 【Chemistry 31】 And in the formula, R ２ is hydrogen or C １～４ It is alkyl, R ３ is hydrogen or C １～４ A compound according to any one of the prior claims, which is alkyl, or a pharmaceutically acceptable salt thereof.

15. T １ or T ２ teeth, 【Chemistry 32】 The compound according to claim 14.

16. T １ or T ２ teeth, 【Transformation 33】 A compound according to any one of claims 1 to 13, selected from the group consisting of the above, or a pharmaceutically acceptable salt thereof.

17. T １ or T ２ teeth, 【Transformation 34】 The compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof.

18. T １ or T ２ teeth, 【Chemistry 35】 The compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof.

19. T １ or T ２ teeth, 【Transformation 36】 The compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof.

20. T １ or T ２ teeth, 【Chemistry 37】 The compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof.

21. T １ or T ２ teeth, 【Transformation 38】 Selected from the group consisting of, R ２ and R ３ The compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein each is independently F or H.

22. T １ or T ２ teeth, 【Chemistry 39】 And, R ２ is hydrogen or C １～４ It is alkyl, R ３ is hydrogen or C １～４ A compound according to any one of claims 1 to 13, which is alkyl, or a pharmaceutically acceptable salt thereof.

23. T １ or T ２ teeth, 【Chemistry 40】 And, Q is C １～５ It is an alkylene, and zero, one, or two methylene units are replaced by -O-. The compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein Ar is an optionally substituted 5- to 10-membered aromatic ring or a 9- or 10-membered unsaturated condensed biring.

24. The compound according to claim 1, selected from the compounds listed in Table 1.

25. The target binding portion T １ or T ２ The target is selected from G protein-coupled receptors (GPCRs), enzymes, ion channels, proteases, and receptors, and is a compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof.

26. The target binding portion T １ or T ２ The aforementioned target is a compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, present on the surface of pathogenic immune cells, tumor cells or cancer cells, or stromal cells.

27. The target binding portion T １ or T ２ The aforementioned target is a compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, present on the surface of a pathogen selected from a virus or bacterial cell.

28. The target binding portion T １ or T ２ The aforementioned targets are the compounds according to any one of claims 1 to 13, or pharmaceutically acceptable salts thereof, present on the surface of monocytic myeloid suppressor cells (mMDSCs), regulatory T cells (Treg), neutrophils, macrophages, regulatory B cells (Breg), regulatory CD8 cells (CD8reg), exhausted T cells, polymorphonuclear myeloid suppressor cells (PMN-MDSCs), or cancer-associated fibroblasts (CAFs).

29. The target binding portion T １ or T ２ The aforementioned target is a chemokine receptor (CCR), the compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof.

30. The target binding portion T １ or T ２ The compound according to any one of claims 1 to 13, wherein the target is selected from CCR1, CCR2, CCR3, CCR5, or CCR8.

31. The target binding portion T １ or T ２ The aforementioned targets are C-C motif chemokine receptors (CCR) 2 (CCR2), CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, C-X-C motif chemokine receptor 1 (CXCR1), C-X-C motif chemokine receptor 2 (CXCR2), C-X-C motif chemokine receptor 3 (CXCR3), C-X-C motif chemokine receptor 4 (CXCR4), C-X-C motif chemokine receptor 5 (CXCR5), C-X-C motif chemokine receptor 6 (CXCR6), and atypical chemokine receptors. Integrin receptor 3 (ACKR3), integrin αvβ6, fibroblast-activating protein α (FAPα), prostate-specific membrane antigen (PSMA), folate receptor (folate receptor 1 or folate receptor β), complement C3a receptor 1 (C3AR1), complement C5a receptor 1 (C5AR1), G protein-coupled receptor (GPR) 65 (GPR65), GRP132, GPR84, GPR183, GPR35, GPR42, cholecystokinin A receptor (CCKAR), leukotriene B4 receptor (LTB4R), somatostatin receptor 2 (SSTR2), free fatty acid receptor 1 (FFAR1), purine receptor P2Y2 (P2RY2), prostaglandin D2 receptor (PTGDR), calcitonin receptor (CALCR), CD38, purine receptor P2X7 (P2RX7), integrin subunit αV (ITGAV), integrin subunit α5 (ITGA5), integrin subunit β1 (ITGB1), integrin subunit β6 (ITGB6), integrin subunit β3 (ITGB3), prostaglandin D2 receptor 2 (PTGDR2), gastrin-releasing peptide receptor (GRPR), M ER proto-oncogene tyrosine kinase (MERTK), C-X3-C motif chemokine receptor 1 (CX3CR1), oxidized low-density lipoprotein receptor 1 (OLR1), plasminogen activator urokinase receptor (PLAUR), carbonic anhydrase 9 (CA9), carbonic anhydrase 12 (CA12), mas-related G protein-coupled receptor member X2 (MRGPRX2), heat shock protein 90α family class A member 1 (HSP90AA1), dipeptidyl peptidase 4 (DPP4), formyl peptide receptor 2 (FPR2),A compound selected from and succinate receptor 1 (SUCNR1) according to any one of claims 1 to 13.

32. A method for treating and / or preventing a disease or disorder in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound according to any one of claims 1 to 31 and an anticotinin antibody or its antigen-binding fragment, wherein the disease or disorder is selected from cancer, inflammatory diseases, autoimmune diseases, viral infections, or bacterial infections.

33. The method according to claim 32, wherein the disease or disorder is mediated by chemokine receptor 2 (CCR2) and / or associated with CCR2-positive pathogenic cells.

34. The method according to claim 32, wherein the disease or disorder is mediated by C-X-C motif chemokine receptor 3 (CXCR3) and / or associated with CXCR3-positive pathogenic cells.

35. The method according to claim 32, wherein the disease or disorder is mediated by PSMA and / or associated with PSMA-positive pathogenic cells.

36. The method according to claim 32, wherein the disease or disorder is mediated by integrin αVβ6 and / or associated with integrin αVβ6-positive pathogenic cells.

37. The method according to claim 32, wherein the disease or disorder is mediated by folate receptor α (FRα) and / or folate receptor β (FRβ), and / or is associated with FRα-positive pathogenic cells and / or FRβ-positive pathogenic cells.

38. The method according to claim 32, wherein the disease or disorder is mediated by fibroblast-activating protein (FAP) and / or associated with FAP-positive pathogenic cells.

39. The method according to claim 32, wherein the disease or disorder is mediated by chemokine receptor 8 (CCR8) and / or associated with CCR8-positive pathogenic cells.

40. The method according to any one of claims 32 to 39, wherein the disease is a solid tumor, or cancer.

41. The method according to any one of claims 32 to 40, wherein the cancer is selected from lung cancer (e.g., non-small cell lung cancer (NSCLC)), hepatocellular carcinoma (HCC), colorectal cancer (CRC), cervical cancer (e.g., squamous cell carcinoma of the cervix (CESC)), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (HNSC)), pancreatic cancer, prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), ovarian cancer, endometrial cancer, brain tumor, endocrine cancer, testicular cancer, bladder cancer, bone cancer, esophageal cancer, gastric cancer, renal cell carcinoma, melanoma cancer, thyroid cancer, or breast cancer.

42. The method according to claim 32 or 34, wherein the disease is an autoimmune disease or inflammatory disease selected from vitiligo and type 1 diabetes.

43. The method according to any one of claims 32 to 42, wherein the compound and the antibody or its antigen-binding fragment are administered simultaneously.

44. The method according to any one of claims 32 to 42, wherein the compound and the antibody or its antigen-binding fragment are administered sequentially.

45. A method for enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) of target-expressing cells, the method comprising contacting the cells with an effective amount of a compound according to any one of claims 1 to 31 and an anti-cotinin antibody or its antigen-binding fragment, wherein the target-binding portion of the compound binds to the target expressed on the cells.

46. A method for depleting target-expressing cells, the method comprising contacting the cells with an effective amount of a compound according to any one of claims 1 to 31 and an anti-cotinin antibody or its antigen-binding fragment, wherein the target-binding portion of the compound binds to the target expressed on the cells.

47. The method according to claim 45 or 46, wherein the target expression cells are myeloid-derived suppressor cells (MDSCs), regulatory T cells (Treg), neutrophils, macrophages, regulatory B cells (Breg), regulatory CD8 cells (CD8reg), exhausted T cells, or cancer-associated fibroblasts (CAFs).

48. The method according to any one of claims 45 to 47, wherein the target expression cells are CCR2 expressing cells.

49. The method according to claim 45 or 46, wherein the target expression cells are CXCR3 expressing cells.

50. The method according to claim 45 or 46, wherein the target expression cells are PSMA-expressing cells.

51. The method according to claim 45 or 46, wherein the target expression cells are integrin αVβ6 expressing cells.

52. The method according to claim 45 or 46, wherein the target expression cells are FRα-expressing cells and / or FRβ-expressing cells.

53. The method according to claim 45 or 46, wherein the target expression cells are FAP-expressing cells.

54. The method according to claim 45 or 46, wherein the target expression cells are CCR8 expressing cells.

55. The method according to any one of claims 32 to 54, wherein the anti-cotinin antibody has a heavy chain and a light chain, the heavy chain comprises CDR1 having SEQ ID NO: 1, CDR2 having SEQ ID NO: 2, and CDR3 having SEQ ID NO: 3, and the light chain comprises CDR1 having SEQ ID NO: 4, CDR2 having SEQ ID NO: 5, and CDR3 having SEQ ID NO:

6.

56. The method according to any one of claims 32 to 55, wherein the anti-cotinin antibody has a heavy chain and a light chain, the heavy chain includes a heavy chain variable region (VH) having SEQ ID NO: 7, and the light chain includes a light chain variable region (VL) having SEQ ID NO:

8.

57. The method according to any one of claims 32 to 56, wherein the anti-cotinin antibody is an IgG1 isotype containing a substitution in the Fc region to enhance ADCC activity.

58. The method according to claim 57, wherein the substitution in the Fc region is S239D / I332E, and the residue numbering follows the EU index.

59. The method according to any one of claims 32 to 58, wherein the anti-cotinin antibody comprises a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO:

10.

60. A combination comprising the compound according to any one of claims 1 to 31 and an anti-cotinin antibody or its antigen-binding fragment.

61. The combination according to claim 60, wherein the anti-cotinin antibody has a heavy chain and a light chain, the heavy chain comprises CDR1 having SEQ ID NO: 1, CDR2 having SEQ ID NO: 2, and CDR3 having SEQ ID NO: 3, and the light chain comprises CDR1 having SEQ ID NO: 4, CDR2 having SEQ ID NO: 5, and CDR3 having SEQ ID NO:

6.

62. The combination according to claim 60 or 61, wherein the anti-cotinin antibody has a heavy chain and a light chain, the heavy chain includes a heavy chain variable region (VH) having SEQ ID NO: 7, and the light chain includes a light chain variable region (VL) having SEQ ID NO:

8.

63. The combination according to any one of claims 60 to 62, wherein the anti-cotinin antibody is an IgG1 isotype that includes a substitution in the Fc region to enhance ADCC activity.

64. The combination according to claim 63, wherein the substitution in the Fc region is S239D / I332E, and the residue numbering follows the EU index.

65. The anti-cotinin antibody is the combination according to any one of claims 60 to 64, comprising a heavy chain containing SEQ ID NO: 9 and a light chain containing SEQ ID NO: 10.

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