Cytotoxic targeting chimeras for CC chemokine receptor 2-expressing cells

Abstract

The present disclosure relates to heterobifunctional molecules, termed cytotoxic targeting chimeras (CyTaC) or antibody recruiting molecules (ARM), that can simultaneously bind to a target cell surface protein and an exogenous antibody protein. The present disclosure also relates to agents capable of binding to receptors on the surface of pathogenic cells and inducing depletion of the pathogenic cells in a subject for use in the treatment of cancer, inflammatory diseases, autoimmune diseases, viral infections, or bacterial infections.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 63 / 314,034, filed February 25, 2022, which is incorporated by reference in its entirety.

[0002] The present disclosure relates to heterobifunctional molecules, termed cytotoxic targeting chimeras (CyTaC) or antibody recruiting molecules (ARM), that can simultaneously bind to a target cell surface protein and an exogenous antibody protein. The present disclosure also relates to agents capable of binding to receptors on the surface of pathogenic cells and inducing depletion of the pathogenic cells in a subject for use in the treatment of cancer, inflammatory diseases, autoimmune diseases, viral infections, or bacterial infections. [Background technology]

[0003] Cell surface proteins and their ligands play important roles in inflammatory, infectious and autoimmune diseases, as well as tumor initiation, growth and metastasis. Antibody-based therapeutics have promising properties as drug candidates for these indications due to their selectivity for pathogenic cell surface targets and their ability to direct immune surveillance to target-expressing tissues or cells to induce depletion of pathogenic cells. Examples of such depletion mechanisms include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). However, antibody-based therapeutics often suffer from lack of bioavailability, high cost, thermolability, and difficult manufacture due to their size, complexity, and peptide-based structure. Conversely, small molecule therapeutics often offer affordability, stability, and convenience of oral administration, but suffer from poor selectivity and off-target effects while also lacking the immune control of therapeutic antibodies.

[0004] Thus, there is a need for improved therapeutic approaches that target pathogenic cells for use in the treatment of disease. Such compositions and associated methods are provided in the present disclosure. Summary of the Invention

[0005] In one aspect, the present disclosure provides a heterobifunctional molecule, referred to as a cytotoxic targeting chimera (CyTaC) or antibody recruiting molecule (ARM), in which the ARM comprises a portion that binds to a target cell surface protein on a cell and a portion that binds to an exogenous antibody. In a further aspect, the ARM comprises a bivalent linker that links the target binding portion to the antibody binding portion. In a further aspect, the target binding portion is a CC chemokine receptor type 2 (CCR2) binding portion. In a further aspect, the exogenous antibody is an anti-cotinine antibody, or an antigen-binding fragment thereof.

[0006] In a further aspect, the ARM is a compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony, T is [ka] and R 1 is C 1~4 Alkyl or C 3~6 is cycloalkyl, Y is a bond or a divalent spacer moiety 1 to 12 atoms in length; L is a bivalent linker as described herein.

[0007] In one aspect, a method of treating and / or preventing a disease or disorder in a patient in need of such treatment and / or prevention is provided, comprising administering to the patient a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof.

[0008] In one aspect, the disclosure provides a method for increasing antibody-dependent cellular cytotoxicity (ADCC) of a CCR2-expressing cell, comprising contacting the cell with a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof.

[0009] In one aspect, the disclosure provides a method of depleting CCR2-expressing cells, comprising contacting the cells with a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof.

[0010] In one aspect, the disclosure provides a compound of formula (I) as disclosed herein for use in therapy. In a further aspect, the disclosure provides a combination comprising a compound of formula (I) as disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof for use in therapy.

[0011] In one aspect, the present disclosure provides a combination comprising a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof, for use in treating a disease or disorder.

[0012] In one aspect, the present disclosure provides the use of a compound of formula (I) disclosed herein in the manufacture of a medicament for the treatment of a disease or disorder. In a further aspect, the present disclosure provides the use of a combination comprising a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment of a disease or disorder.

[0013] In one aspect, the disclosure provides a combination comprising a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof. [Brief description of the drawings]

[0014] [Figure 1A]1 shows an analysis of various syngeneic mouse tumors administered with a CyTaC compound (compound of Example 22) in the presence of an anti-cotinine antibody. Tumor volumes of tumor-bearing mice treated with the CyTaC compound of Example 22 and an anti-cotinine antibody or PBS (control) are shown, with tumor volumes shown on the final day of the study for each tumor model. [Figure 1B] 1 shows an analysis of various syngeneic mouse tumors administered with a CyTaC compound (compound of Example 22) in the presence of an anti-cotinine antibody. Tumor volumes of tumor-bearing mice treated with the CyTaC compound of Example 22 and an anti-cotinine antibody or PBS (control) are shown, with tumor volumes shown on the final day of the study for each tumor model. [Figure 1C] Figure 2 shows the analysis of various syngeneic mouse tumors administered with CyTaC compound (compound of Example 22) in the presence of anti-cotinine antibody. Tumor volumes of EMT-6 tumor-bearing mice administered with PBS or the combination of CyTaC compound of Example 22 and anti-cotinine antibody. Each line represents an individual mouse in the same experiment. [Figure 2A] Figure 2 shows depletion of monocytes from the blood of tumor-bearing animals after treatment with CyTaC compound (compound of Example 22) and anti-cotinine antibody. Figure 2 shows depletion of monocytic (Ly6Chi / CD11b+) cells in multiple tumor models. [Figure 2B] Figure 2 shows depletion of monocytes from blood of tumor-bearing animals after treatment with CyTaC compound (compound of Example 22) and anti-cotinine antibody. An example of flow gating of CD11b and Ly6C in blood from MC38 tumor-bearing animals in mice treated with PBS (left) and mice treated with CyTaC compound and antibody (right). [Diagram 3] 1 shows the effect of treatment with a combination of a CyTaC compound (compound of Example 22) and an anti-cotinine antibody on intratumoral levels of CD8+ T cells as quantified by flow cytometry of lymphocytes from tumors excised from a syngeneic model. [Figure 4A]1 shows the flow cytometric characterization of different cell populations in the blood and tumors of mice bearing HEPA1-6. Analysis of F480+ positive cells from blood and intratumoral lymphocytes is shown. [Figure 4B] 1 shows the flow cytometric characterization of different cell populations in the blood and tumors of HEPA1-6 bearing mice. 2 shows the analysis of Ly6G- monocytes derived from blood and intratumoral lymphocytes. [Figure 4C] 1 shows the flow cytometric characterization of different cell populations in the blood and tumors of mice bearing HEPA1-6. Analysis of F480+ positive cells from blood and intratumoral lymphocytes is shown. [Figure 4D] 1 shows the flow cytometric characterization of different cell populations in the blood and tumors of HEPA1-6 bearing mice. 2 shows the analysis of Ly6G- monocytes derived from blood and intratumoral lymphocytes. [Diagram 5] 1 shows flow cytometry analysis of macrophages in tumor samples excised from subcutaneously grown KPC tumors. [Figure 6A] 1 shows flow cytometry analysis of tumor samples from subcutaneously grown Hepa1-6 or KPC tumors, and shows the effect of treatment with the combination of CyTaC compound of Example 22 and anti-cotinine antibody on the percentage of T cell population present in KPC tumors. [Figure 6B] Figure 6B shows the flow cytometry analysis of tumor samples from subcutaneously grown Hepa1-6 tumors or KPC tumors. Figure 6B shows the effect of treatment with the combination of the CyTaC compound of Example 22 and anti-cotinine antibody on the percentage of T cell subpopulations present in Hepa1-6 tumors. [Figure 7] The CyTaC compound of Example 22 shows efficacy of treatment in combination with anti-cotinine antibodies, alone or in further combination with STING agonist therapy. [Figure 8]Figure 2 shows the efficacy of treatment with the CyTaC compound of Example 22 when combined with anti-cotinine antibody, alone, or further combined with anti-PD1 treatment. A shows the tumor volume of individual treated animals over time. B shows the overall survival of animals across all treatment groups. [Figure 9] 1 shows the efficacy of a combination treatment with the CyTaC compound of Example 22 and an anti-cotinine antibody in a collagen-induced arthritis model. Animals were dosed and assessed for inflammation as described in Example 32 to generate a conglomerate clinical score, which is plotted. [Figure 10A] Figure 1 shows the antagonism of monocyte chemotaxis by CyTaC compounds when administered with non-ADCC active anti-cotinine antibody, thioglycollate is used to induce immune cell chemotaxis, cells are isolated using peritoneal lavage, and cells are subjected to flow cytometry to identify monocytes (Ly6Chi / CD11b+).The activity of the compounds of Example 2 and Example 14 is compared. [Figure 10B] This shows the antagonism of monocyte chemotaxis by CyTaC compounds when administered with non-ADCC active anti-cotinine antibody, using thioglycollate to induce immune cell chemotaxis, isolating cells using peritoneal lavage, and subjecting cells to flow cytometry to identify monocytes (Ly6Chi / CD11b+).The activity of the compound of Example 14 is shown. [Figure 11] FIG. 1 is a schematic diagram of the cytotoxic targeted chimera (CyTaC) technology compared to current antibody technology. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In one aspect, the present disclosure provides a compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, During the ceremony, T is [ka] and R 1 is C 1~4 Alkyl or C 3~6 is cycloalkyl, Y is a bond or a divalent spacer moiety 1 to 12 atoms in length; L is a bivalent linker of formula (La), (Lb), (Lc), (Ld), (Le), (Lf), (Lg), (Lh), (Li), (Lj), (Lk), (Lm), (Lni), (Ln-ii), (Ln-iii), or (Ln-iv).

[0016] In one embodiment of the disclosure, L is a bivalent linker of formula (La): [ka] or a stereoisomer thereof, During the ceremony, Ring A and ring B each independently represent C 4~6 is cycloalkylene, L 1a is C 3~5 A straight chain alkylene, in which one or two methylene units are -O- or -NR a - is replaced by Each R a are independently hydrogen or C 1~3 is alkyl, L 2a is -O-, -NHC(O)-, or -CH 2 -O-, [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0017] In another embodiment, ring A and ring B of formula (La) are each independently [ka] It is.

[0018] In another embodiment, L is a bivalent linker of formula (Lai): [ka] or a stereoisomer thereof, During the ceremony, Ring A is C 4~6 is cycloalkylene, L 1a is C 3~5 A straight chain alkylene, in which one or two methylene units are -O- or -NR a - is replaced by Each R a are independently hydrogen or C 1~3 is alkyl, L 2a is -O-, -NHC(O)-, or -CH 2 -O-, [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0019] In another embodiment, ring A of formula (Lai) is [ka] It is.

[0020] In another embodiment, L is a bivalent linker of formula (La-ii): [ka] or a stereoisomer thereof, During the ceremony, L 1a is C 3~5 A straight chain alkylene, in which one or two methylene units are -O- or -NR a - is replaced by Each R a are independently hydrogen or C 1~3 is alkyl, L 2a is -O-, -NHC(O)-, or -CH 2 -O-, p is 1 or 2; m is 1 or 2; [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0021] In another embodiment, L of formula (La), (Lai), or (La-ii) 1a teeth, [ka] is 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 1 or 2; r is 1 or 2; s is 0 or 1; the sum of q, r, and s is 2 or 3; X 1 and X 2 are independently -O- or NR a and Each R a are independently hydrogen or C 1~3 is alkyl, [ka] represents a covalent bond to a C(O) group of formula (La), (Lai), or (La-ii), [ka] represents a covalent bond to ring B of formula (La) or to a cyclohexylene group of formula (Lai) or (La-ii).

[0022] In another embodiment, L of formula (La), (Lai), or (La-ii) 1a is -(CH 2 ) 2 O-, -(CH 2 ) 3 O-, -(CH 2 ) 4 O-, -(CH 2 ) 2 OCH 2 -, -(CH 2 ) 3 OCH 2 -, -(CH 2 ) 2 O(CH 2 ) 2 -, -CH 2 OCH 2 -, -CH 2 O(CH 2 ) 2 -, -CH 2 O(CH 2 ) 3 -, -CH 2 OCH 2 O-, or -CH 2 OCH 2 OCH 2 In another embodiment, L of formula (La), (Lai), or (La-ii) is selected from 1a is -(CH 2 ) 2 O-, -(CH 2 ) 3O-, -(CH 2 ) 2 OCH 2 - or -(CH 2 ) 3 OCH 2 In another embodiment, L of formula (La), (Lai), or (La-ii) is selected from 1a is -(CH 2 ) 2 NR a -, -(CH 2 ) 3 NR a -, -(CH 2 ) 4 NR a -, -(CH 2 ) 2 NR a CH 2 -, -(CH 2 ) 3 NR a CH 2 -, -(CH 2 ) 2 NR a (CH 2 ) 2 -, -CH 2 NR a CH 2 -, -CH 2 NR a (CH 2 ) 2 -, -CH 2 NR a (CH 2 ) 3 -, -CH 2 NR a CH 2 NR a -, or -CH 2 NR a CH 2 NR a CH 2 - selected from, each R a are independently hydrogen or C 1~3 In another embodiment, L of formula (La), (Lai), or (La-ii) is alkyl. 1a is -(CH 2 ) 2 NR a -, -(CH 2) 3 NR a -, -(CH 2 ) 2 NR a CH 2 - or -(CH 2 ) 3 NR a CH 2 -, wherein R a is hydrogen or C 1~3 In another embodiment, L of formula (La), (Lai), or (La-ii) is alkyl. 1a is -(CH 2 ) 2 NH-, -(CH 2 ) 3 NH-, -(CH 2 ) 4 NH-, -(CH 2 ) 2 NHCH 2 -, -(CH 2 ) 3 NHCH 2 -, -(CH 2 ) 2 NH(CH 2 ) 2 -, -CH 2 NHCH 2 -, -CH 2 NH(CH 2 ) 2 -, -CH 2 NH(CH 2 ) 3 -, -CH 2 NHCH 2 NH- or -CH 2 NHCH 2 NHCH 2 In another embodiment, L of formula (La), (Lai), or (La-ii) is selected from 1a is -(CH 2 ) 2 NH-, -(CH 2 ) 3 NH-, -(CH 2 ) 2 NHCH 2 - or -(CH 2 ) 3 NHCH 2In another embodiment, L of formula (La), (Lai), or (La-ii) is selected from 1a is -CH 2 OCH 2 NR a -, -CH 2 NR a CH 2 O-, -CH 2 OCH 2 NR a CH 2 -, -CH 2 NR a CH 2 OCH 2 -, wherein R a are independently hydrogen or C 1~3 In another embodiment, L of formula (La), (Lai), or (La-ii) is alkyl. 1a is -CH 2 OCH 2 NH-, -CH 2 NHCH 2 O-, -CH 2 OCH 2 NHCH 2 -, -CH 2 NHCH 2 OCH 2 - is selected.

[0023] In another embodiment, L is a bivalent linker of formula (La-iii): [ka] or a stereoisomer thereof, During the ceremony, p is 1 or 2; m is 1 or 2; n is 1, 2, or 3; [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0024] In another embodiment, L is [ka] [ka] [ka] is a bivalent linker of formula (La) selected from the group consisting of:

[0025] In another embodiment, L is a bivalent linker of formula (Lb): [ka] or a stereoisomer thereof, During the ceremony, Ring A is C 4~6 Cycloalkylene or C 7~9 is a bridged bicyclic cycloalkylene; L 1b is -CH 2 -NH-C(O)-, -NHC(O)-, or -C(O)NH-; L 2b is C 6~12 A straight chain alkylene having 1, 2, 3, or 4 methylene units, such as -O-, -NR 1b -, -C(O)NR 1b - or -NR 1b replaced by C(O)-, or L 2b teeth, [ka] where n is 1, 2, 3, or 4; [ka] L 1b represents a covalent bond to Each R1b are independently hydrogen or C 1~3 is alkyl, [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0026] In another embodiment, ring A of formula (Lb) is [ka] It is.

[0027] In another embodiment, L is a bivalent linker of formula (Lbi): [ka] or a stereoisomer thereof, During the ceremony, L 1b is -CH 2 -NH-C(O)-, -NHC(O)-, or -C(O)NH-; L 2b is C 6~12 A straight chain alkylene having 1, 2, 3, or 4 methylene units, such as -O-, -NR 1b -, -C(O)NR 1b - or -NR 1b replaced by C(O)-, or L 2b teeth, [ka] where n is 1, 2, 3, or 4; [ka] L 1b represents a covalent bond to Each R 1b are independently hydrogen or C 1~3 is alkyl, p is 1 or 2; m is 1 or 2; [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0028] In another embodiment, L of formula (Lb) or (Lbi) 2b teeth, [ka] is 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 are independently -O-, -NR 1b -, -C(O)NR 1b -, or -NR 1b C(O)-, Each R 1b are independently hydrogen or C 1~3 is alkyl, [ka] is the L of formula (Lb) or (Lbi) 1b represents a covalent bond to [ka] represents a covalent bond to the methylene group of formula (I).

[0029] In another embodiment, L is [ka] [ka] [ka] is a bivalent linker of formula (Lb) selected from the group consisting of:

[0030] In another embodiment, L is a bivalent linker of formula (Lc): [ka] or a stereoisomer thereof, During the ceremony, L 1c is C 2~10 a straight chain alkylene in which 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 is a bridged bicyclic cycloalkylene; L 2c is -O- or saturated C 2~10 a straight chain alkylene in which one, two or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-; [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0031] In another embodiment, ring A of formula (Lc) is [ka] It is.

[0032] In another embodiment, L is a bivalent linker of formula (Lci): [ka] or a stereoisomer thereof, During the ceremony, L 1c is C 2~10 a straight chain alkylene in which one, two or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-; L 2c is -O- or saturated C 2~10a straight chain alkylene in which one, two or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-; p is 1 or 2; m is 1 or 2; [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0033] In another embodiment, L of formula (Lc) or (Lci) 1c teeth, [ka] is 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 X3 are independently -O-, -NH-, -NHC(O)-, or -C(O)NH-; [ka] represents a covalent bond to the C(O) group of formula (Lc) or (Lci), [ka] represents a covalent bond to a ring of formula (Lc) or (Lci).

[0034] In another embodiment, L of formula (Lc) or (Lci) 2c teeth, [ka] is 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 are independently -O-, -NH-, -NHC(O)-, or -C(O)NH-; [ka] represents a covalent bond to a ring of formula (Lc) or (Lci), [ka] represents a covalent bond to the methylene group of formula (I).

[0035] In another embodiment, L is [ka] [ka] is a bivalent linker of formula (Lc) selected from the group consisting of:

[0036] In another embodiment, L is a bivalent linker of formula (Ld): [ka] During the ceremony, L 1d is C 12~31 a straight chain alkylene in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 methylene units are replaced by -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-; [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I). 1d is 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 31 In another embodiment, L is a straight chain alkylene in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 methylene units are replaced with -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-. 1d is C 12~22 Straight chain alkylene, e.g., C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 wherein 1, 2, 3, 4, or 5 methylene units are replaced by -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-.

[0037] In another embodiment, L of formula (Ld) 1d teeth, [ka] is 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 are independently -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-; [ka] represents a covalent bond to the C(O) group of formula (Ld), [ka] represents a covalent bond to the methylene group of formula (I).

[0038] In another embodiment, L of formula (Ld) 1d teeth, [ka] and n is 4, 5, 6, 7, 8, 9, or 10; [ka] represents a covalent bond to the C(O) group of formula (Ld), [ka] represents a covalent bond to the methylene group of formula (I).

[0039] In another embodiment, L is [ka] [ka] is a bivalent linker of formula (Ld) selected from the group consisting of:

[0040] In another embodiment, L is a bivalent linker of formula (Le): [ka] During the ceremony, n is an integer from 3 to 50, [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0041] In another embodiment, n in formula (Le) is 3 to 25, 3 to 10, 3 to 8, 3 to 7, 3 to 5, or 3 to 4. In another embodiment, n in formula (Le) is 5 to 22, 7 to 15, or 9 to 13. In another embodiment, n in formula (Le) is 3, 4, 5, 7, 8, 11, 22, or 50.

[0042] In another embodiment, L is a bivalent linker of formula (Lf): [ka] or a stereoisomer thereof, During the ceremony, L 1f is a bond, C 1~6Straight-chain alkylene in which 0, 1, or 2 methylene units are replaced by -O-, -NH-, or -C(O)-; 1~6 Linear alkylene, or -(C 3~6 cycloalkylene)-NHC(O)-; L 2f is a bond, -NHC(O)-, -C(O)NH-, or C 1~6 is a straight chain alkylene in which 0, 1, or 2 methylene units are replaced by -O-; Z 1 and Z 2 each is independently N or CH; [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0043] In another embodiment, L of formula (Lf) 1f teeth, [ka] is 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 are independently -O-, -NH-, or -C(O)-, or -(C 3~6 cycloalkylene)-NHC(O)-; [ka] represents a covalent bond to the C(O) group of formula (Lf), [ka] represents a covalent bond to the ring of formula (Lf).

[0044] In another embodiment, L of formula (Lf) 2f teeth, [ka] is 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] represents a covalent bond to the ring of formula (Lf), [ka] represents a covalent bond to the methylene group of formula (I).

[0045] In another embodiment, L is [ka] is a bivalent linker of formula (Lf) selected from the group consisting of:

[0046] In another embodiment, L is a bivalent linker of formula (Lg): [ka] During the ceremony, Ring A is a 5- to 6-membered heteroarylene having 1 or 2 nitrogen ring atoms; L 1g is a bond, -CH 2 -, -NH-, or -O-; L 2g teeth, [ka] where n is 1, 2, 3, 4, or 5; [ka] L 1g represents a covalent bond to [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0047] In another embodiment, L is a bivalent linker of formula (Lgi): [ka] During the ceremony, L 1g is a bond, -CH 2 -, -NH-, or -O-; L 2g teeth, [ka] where n is 1, 2, 3, 4, or 5; [ka] L 1g represents a covalent bond to Z 1, Z 2 , and Z 3 are each independently selected from N or CH, with the proviso that Z 1 , Z 2 , and Z 3 is N, [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0048] In another embodiment, L is [ka] is a bivalent linker of formula (Lg) selected from the group consisting of:

[0049] In another embodiment, L is a bivalent linker of formula (Lh): [ka] or a stereoisomer thereof, During the ceremony, each Z 1 are independently N or CH; L 1h is a bond, -C(O)-, -C(O)-NH-, or -NHC(O)-, L 2h is C 2~10 Linear alkylene or [ka] where n is 1, 2, 3, or 4; [ka] L 1h represents a covalent bond to [ka] L 3h represents a covalent bond to L 3h is a bond, -C(O)CH 2 -, -O-(C 3~6 Cycloalkylene)-O-, or -C(O)NH(CH 2 ) 3 OCH 2 - and L 4h is a bond, -C(O)-, -CH 2 C(O)- or -C(O)CH 2 - and m is 1, 2, or 3; [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0050] In another embodiment, L is [ka] [ka] is a bivalent linker of formula (Lh) selected from the group consisting of:

[0051] In another embodiment, L is a bivalent linker of formula (Li): [ka] During the ceremony, L 1i is a bond, C 1~12 Linear alkylene, or [ka] where n is 1, 2, 3, 4, or 5; [ka] L 3i represents a covalent bond to [ka] represents a covalent bond to NH, L 2i is a bond, C 1~12 Linear alkylene, or [ka] where n is 1, 2, 3, 4, or 5; [ka] represents a covalent bond to HN, L 3i is a bond or -C(O)-, [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0052] In another embodiment, L is [ka] is a bivalent linker of formula (Li) selected from the group consisting of:

[0053] In another embodiment, L is a bivalent linker of formula (Lj): [ka] or a stereoisomer thereof, During the ceremony, Z 1 is C, CH, or N, Z 2 , Z 3 , Z 4 , and Z 5 Each of is independently CH or N, with the proviso that Z 2 , Z 3 , Z 4 , and Z 5 provided that no more than two of are N, L 1j is -NH-, -C(O)NH-, -NHC(O)-, or -O-, L 2j is C 1~6 Linear alkylene or [ka] where n is 1 or 2; [ka] L 1j represents a covalent bond to [ka] represents a single or double bond, [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0054] In another embodiment, L is [ka] is a bivalent linker of formula (Lj) selected from the group consisting of:

[0055] In another embodiment, L is a bivalent linker of formula (Lk): [ka] or a stereoisomer thereof, During the ceremony, Ring A is phenyl or a 5- or 6-membered heteroarylene having 1 or 2 nitrogen ring atoms; Z 1 and Z 2 each is independently CH or N; L 1k is a bond, -C(O)-, -C(O)NH-, or -NHC(O)-, L 2k is C 3~8 Linear alkylene or [ka] where n is 1, 2, or 3; [ka] L 1k represents a covalent bond to [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0056] In another embodiment, L is [ka] is a bivalent linker of formula (Lk) selected from the group consisting of:

[0057] In another embodiment, L is a bivalent linker of formula (Lm): [ka] or a stereoisomer thereof, During the ceremony, Z 1 is CH or N, m is 1 or 2; p is 1 or 2; [ka] 0, 1, or 2 hydrogen atoms are replaced by F, L 1m is a bond, -C(O)-, -C(O)NH-, -NHC(O)-, -S(O) 2 NH-, or -NHS(O) 2 - and L 2m is C 3~6 Straight chain alkylene, C 3~6 cycloalkylene, or [ka] where n is 1 or 2; [ka] L 1m represents a covalent bond to [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0058] In another embodiment, L is [ka] is a bivalent linker of formula (Lm) selected from the group consisting of:

[0059] In another embodiment, L is a bivalent linker of formula (Lni): [ka] During the ceremony, [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0060] In another embodiment, L is a bivalent linker of formula (Ln-ii): [ka] During the ceremony, [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0061] In another embodiment, L is a bivalent linker of formula (Ln-iii): [ka] During the ceremony, [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0062] In another embodiment, L is a bivalent linker of formula (Ln-iv): [ka] During the ceremony, [ka] represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); [ka] represents a covalent bond to the methylene group of formula (I).

[0063] In one embodiment of the present disclosure, Y is a bond, —NH—, —(C 1~12 alkylene)-, in which one, two or three methylene units are selected from the group consisting of -O-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C 3~6 Cycloalkylene)-, -(C 3~6 cycloalkenylene)-, 3- to 6-membered heterocycloalkylene, arylene, or heteroarylene; -(C 1~12 alkylene)-, or -(C 2~12 alkenylene)-, in which one, two or three methylene units are selected from the group consisting of -O-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C 3~6 Cycloalkylene)-, -(C 3~6 cycloalkenylene)-, 3- to 6-membered heterocycloalkylene, arylene, or heteroarylene; -(C 2~12 alkenylene).

[0064] In another embodiment, Y is a bond, —NH—, —(C 1~6 alkylene)-O-, -(C 2~6 alkenylene)-O-, -(C1~6 alkylene)-C(O)-, -(C 2~6 alkenylene)-C(O)-, phenylene, piperidinylene, -(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 -O-, -NH-(C 1~5 alkylene)-NH-, -(C 3~6 Cycloalkylene)-NH-, -(C 3~6 cycloalkenylene)-NH-, or [ka] Replaced by -(C 1~5 (alkylene)-NH-, 1a is a bond, -O-, -NH-, -NHC(O)-, -C(O)NH-, or C 1~3 is alkylene, and Y 2a is a bond, -O-, -NH-, -NHC(O)-, -C(O)NH-, or C 1~3 In another embodiment, Y is -NH-.

[0065] In another embodiment, Y is [ka] is selected from the group consisting of:

[0066] In another embodiment, R 1 is methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, or t-butyl. 1 is methyl. In another embodiment, R 1 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0067] In another embodiment, the compound of formula (I) is selected from the compounds listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0068] definition As used in this specification and claims, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise.

[0069] As used in the specification and claims, the term "comprising" includes, for example, "including" or "consisting", e.g., a composition "comprising" X may consist exclusively of X or may include additional, e.g., X+Y.

[0070] The term "consisting essentially of" limits the scope of a feature to particular materials or steps and those that do not materially affect the basic characteristic(s) of the claimed feature.

[0071] The term "consisting of" excludes the presence of any additional component or components.

[0072] The term "pathogenic cells" includes cell subsets that cause or are capable of causing disease. Examples of pathogenic cells include, but are not limited to, pathogenic immune cells, cancer or tumor cells, and stromal cells. Pathogenic cells can also be pathogens capable of causing infectious diseases, such as viruses or bacterial cells.

[0073] The term "pathogenic immune cells" includes certain immune cell subsets that cause or can cause disease. These cell subsets are resident cells or are recruited to specific locations and secrete cytokines, chemokines, and other mediators that contribute to the persistence and progression of diseases, such as cancer in the case of tumor microenvironment or chronic inflammation of the lung in the case of asthma. Examples of pathogenic immune cells include, but are not limited to, myeloid-derived suppressor cells (MDSCs), T regulatory cells (Tregs), neutrophils, macrophages, B regulatory cells (Bregs), CD8 regulatory cells, (CD8regs), and exhausted T cells.

[0074] The term "pharmaceutical composition" refers to a formulation of a compound of the invention and a vehicle generally accepted in the art for delivery of the biologically active compound to a mammal, e.g., a human. Such a vehicle includes any pharma- ceutically acceptable carrier, diluent, or excipient therefor.

[0075] The terms "effective amount" and "therapeutically effective amount" refer to an amount of a compound according to the invention, or an antibody, or an antigen-binding portion thereof, that, when administered to a patient in need of treatment, is sufficient to provide treatment for a disease state, condition, or disorder for which the compound has utility. Such an amount will be sufficient to elicit a biological or medical response in a tissue system or patient that is desired by a researcher or clinician. The amount of a compound according to the invention that constitutes a therapeutically effective amount will vary 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 state or disorder being treated, the drugs used in combination with or concurrently with the compound of the invention, and the age, weight, general health, sex, and diet of the patient. Such a therapeutically effective amount can be routinely determined by one skilled in the art having regard to his or her own knowledge, the state of the art, and this disclosure.

[0076] The term "alkyl" refers to a saturated, straight-chain, or branched-chain hydrocarbon moiety having a specified number of carbon atoms. 1~3 The term "alkyl" refers to an unsubstituted alkyl moiety containing 1, 2, or 3 carbon atoms; exemplary alkyls include methyl, ethyl, and propyl.

[0077] The term "alkylene" refers to a saturated, straight or branched chain hydrocarbon moiety having a specified number of carbon atoms with two points of attachment. The two points of attachment may come from the same or different carbon atoms. 1~3 The term "alkylene" refers to an unsubstituted alkyl moiety containing 1, 2, or 3 carbon atoms with two points of attachment, exemplary C 1~3 Alkylene groups include methylene, ethylene, and propylene.

[0078] The term "alkenyl" refers to an unsaturated, straight-chain, or branched-chain hydrocarbon moiety having the specified number of carbon atoms. 2~6The term "alkenyl" refers to an unsubstituted alkenyl moiety containing 2, 3, 4, 5, or 6 carbon atoms, exemplary alkenyls include propenyl, butenyl, pentenyl, and hexenyl.

[0079] The term "alkenylene" refers to an unsaturated, straight or branched chain hydrocarbon moiety having a specified number of carbon atoms with two points of attachment. The two points of attachment may come from the same or different carbon atoms. 2~6 The term "alkenylene" refers to an unsubstituted alkenyl moiety containing 2, 3, 4, 5, or 6 carbon atoms with two points of attachment, exemplary C 2~6 Alkenylene groups include propenylene, butenylene, pentenylene, and hexenylene.

[0080] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon moiety having the specified number of carbon atoms. 3~6 The term "cycloalkyl" refers to an unsubstituted cycloalkyl moiety containing 3, 4, 5, or 6 carbon atoms, exemplary cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0081] The term "cycloalkylene" refers to a saturated cyclic hydrocarbon moiety having a specified number of carbon atoms with two points of attachment. The two points of attachment may come from the same or different carbon atoms. 4~6 The term "cycloalkylene" refers to an unsubstituted cycloalkylene moiety containing 4, 5, or 6 carbon atoms with two points of attachment. Exemplary cycloalkylene groups include cyclobutane-1,3-diyl, cyclopentane-1,3-diyl, cyclohexane-1,3-diyl, or cyclohexane-1,4-diyl.

[0082] The term "cycloalkenylene" refers to an unsaturated cyclic hydrocarbon moiety having a specified number of carbon atoms with two points of attachment. The two points of attachment may come from the same or different carbon atoms. 3~6The term "cycloalkenylene" refers to an unsubstituted cycloalkenylene moiety containing 3, 4, 5, or 6 carbon atoms with two points of attachment.

[0083] The term "heterocycloalkylene" refers to a saturated cyclic hydrocarbon moiety containing one or two heteroatoms, independently selected from oxygen, sulfur, or nitrogen atoms, together with two points of attachment. The two points of attachment may be from the same or different carbon atoms. The term "3-6 membered heterocycloalkylene" refers to a 3-6 membered saturated cyclic moiety containing one or two oxygen, sulfur, or nitrogen atoms, as well as two, three, four, or five carbon atoms, together with two points of attachment. Preferably, the 3-6 membered heterocycloalkylene group contains one oxygen or nitrogen atom. Preferably, such groups contain three carbon atoms and one oxygen or nitrogen atom, such as azetidinediyl or oxetanediyl. Preferably, such groups contain four or five carbon atoms and one oxygen or nitrogen atom, such as tetrahydrofurandiyl, tetrahydropyrandiyl, pyrrolidinediyl, or piperidinediyl.

[0084] The term "bridged bicyclic cycloalkylene" refers to a saturated bicyclic hydrocarbon moiety having at least one bridge with two points of attachment. A "bridge" is an unbranched chain of atoms or an atom or valence bond connecting two bridgeheads, and a "bridgehead" is any skeletal atom of the ring system that is attached to three or more skeletal atoms (except hydrogen). The two points of attachment may come from the same or different carbon atoms. "C 7~9 The term "bridged bicyclic cycloalkylene" refers to an unsubstituted bridged bicyclic cycloalkylene moiety containing 7, 8, or 9 carbon atoms with two points of attachment.

[0085] The term "arylene" refers to a monocyclic or bicyclic ring system having two points of attachment in which at least one ring in the system is aromatic. Exemplary arylene groups include phenylene, biphenylene, naphthylene, and anthracylene.

[0086] The term "heteroarylene" refers to a monocyclic or bicyclic ring system in which at least one ring in the system is aromatic and has, along with two points of attachment, carbon atoms and 1-5 heteroatoms independently selected from oxygen, sulfur, or nitrogen atoms. The term "5- to 6-membered heteroarylene" refers to a 5- to 6-membered cyclic aromatic moiety that contains, along with two points of attachment, 1, 2, or 3 heteroatoms independently selected from oxygen, sulfur, or nitrogen atoms, plus 2, 3, 4, or 5 carbon atoms.

[0087] Those skilled in the art will understand that salts, including pharma- ceutically acceptable salts, of compounds according to formula (I) can be prepared.Indeed, in certain embodiments of the present invention, salts, including pharma- ceutically acceptable salts, of compounds according to formula (I) may be preferred over the respective free or unsalted compounds.Therefore, the present invention is further directed to salts, including pharma- ceutically acceptable salts, of compounds according to formula (I).The present invention is further directed to free or unsalted compounds of formula (I).

[0088] Salts, including pharma- ceutically acceptable salts, of the compounds of the present invention are readily prepared by those of skill in the art.

[0089] Representative pharma- ceutically acceptable acid addition salts include 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, acid tartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, and dodecyl sulfate. Salt (Estolate), Edetate (Ethylenediaminetetraacetate), Estolate (Lauryl Sulfate), Ethane-1,2-disulfonate (Edisylate), Ethanesulfonate (Esylate), Formate, Fumarate, Galactarate (Mucate), Gentisate (2,5-Dihydroxybenzoate), Glucoheptonate (Gluceptate), Gluconate, Glucuronate, Glutamate, Glutarate, Glycerophosphate, Glycolate, Hexylresorcylate, Hippurate, Hydrabamine (N,N'-Di(dehydroabietyl)-ethyl)- diamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoic acid, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methylsulfate, mucate, naphthalene-1,5-disulfonate (napadisilate), naphthalene-2-sulfonate (napsilate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate, p-aminosalicylate, pamoate (embonate), pantothenate, These include, but are not limited to, pectinate, persulfate, phenylacetate, phenylethylbarbiturate, phosphate, polygalacturonate, propionate, p-toluenesulfonate (tosylate), pyroglutamate, pyruvate, salicylate, sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, theoclate (8-chlorotheophylline), thiocyanate, triethiodide, trifluoroacetate, undecanoate, undecylenate, and valerate.

[0090] Representative pharma- ceutically acceptable base addition salts include aluminum, 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS, tromethamine), arginine, benethamine (N-benzylphenethylamine), benzathine (N,N'-dibenzylethylenediamine), b / s-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, clemizole (1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole), cyclohexyl ether, ... These include, but are not limited to, diethylamine, 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.

[0091] Compounds according to formula (I) may contain one or more asymmetric centers (also referred to as chiral centers) and therefore may exist as individual enantiomers, diastereomers, or other stereoisomeric forms, or as mixtures thereof. Chiral centers, such as chiral carbon atoms, may be present in substituents, such as alkyl groups. When the stereochemistry of a chiral center is present in a compound of formula (I) or any chemical structure illustrated herein, if not specified, the structure is intended to encompass all individual stereoisomers and all mixtures thereof. Thus, compounds according to formula (I) that contain one or more chiral centers may be used as racemic mixtures, enantiomerically enriched mixtures, or enantiomerically pure individual stereoisomers.

[0092] Mixtures of stereoisomers in which the relative configurations of all stereocenters are known can be designated using the symbol "&" along with an index number (e.g., "&1"). For example, a group of two asymmetric centers designated with the symbol "&1" represents a mixture of the two possible stereoisomers with the relative configurations of the two asymmetric centers as shown.

[0093] A divalent group is a group that has two points of attachment. For all divalent groups, unless otherwise specified, orientation of the group is implied by the direction in which the formula or structure of the group is written.

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Any compositions and methods similar or equivalent to those described herein can be used to practice or test the method of the present disclosure, but exemplary compositions and methods are described herein. Any of the aspects and embodiments of the present disclosure described herein can also be combined. For example, the subject matter of any dependent or independent claims disclosed herein can be combined in a composite manner (e.g., one or more recitations from each dependent claim can be combined into a single claim based on the independent claim to which they depend).

[0095] The ranges provided herein include all values ​​within the particular ranges recited, as well as the values ​​relating to the end points of the particular ranges.

[0096] The concentrations described herein are determined at ambient temperature and pressure. This may be, for example, at room temperature or at the temperature and pressure of a particular portion of the process stream. Preferably, the concentrations are determined at standard conditions of 25° C. and 1 bar pressure.

[0097] CCR2 Targets and CCR2 Binding Moieties The compounds of formula (I) disclosed herein are heterobifunctional synthetic agents designed to interact with cell surface CCR2 targets at one end while binding to specific antibodies at the other end. More specifically, ARM binds to cell surface CCR2 targets and specific antibodies simultaneously. This triple complex directs immune surveillance to CCR2-expressing tissues / cells and combines the mechanism of antibody function with small molecule dose control. This mechanism may include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC), and preferably includes ADCC. The same Fc receptor-expressing immune cells that initiate the destruction of ARM / antibody-tagged cells are also involved in the presentation of endogenous antigens for the potential of long-term cellular immunity.

[0098] The compounds of formula (I) disclosed herein comprise a CCR2 binding moiety capable of binding to CCR2 present on the surface of a cell. In one embodiment, CCR2 is expressed on a pathogenic cell.

[0099] In further embodiments, the pathogenic cell is a pathogenic immune cell, a tumor or cancer cell, or a stromal cell (including stromal cells present in the tumor microenvironment).

[0100] In a further embodiment, the CCR2 target is present on the surface of a pathogen selected from a virus or a bacterial cell. An example of a virus that expresses a cell surface target includes, but is not limited to, influenza.

[0101] In further embodiments, the pathogenic immune cell is a monocyte, a myeloid-derived suppressor cell (MDSC), such as monocytic MDSC (mMDSC) and polymorphonuclear MDSC (PMN_MDSC), a T regulatory cell (Treg), a neutrophil (e.g., N2 neutrophil), a macrophage (e.g., M2 macrophage), a B regulatory cell (Breg, memory B cell), a plasma cell, a CD8 cell (e.g., CD8 regulatory cell (CD8reg), memory CD8 cell, effector CD8 cell, naive CD8 T cell, TEMRA), an exhausted T cell, an eosinophil, a basophil, a mast cell, a dendritic cell, a natural killer (NK cell), an innate lymphoid cell, a NK T cell (NKT), or a γδ T cell.

[0102] In further embodiments, the pathogenic immune cells are myeloid-derived suppressor cells (MDSCs), such as monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs), T regulatory cells (Tregs), neutrophils (e.g., N2 neutrophils), macrophages (e.g., M2 macrophages), B regulatory cells (Bregs), CD8 regulatory cells (CD8regs), exhausted T cells.

[0103] In a further embodiment, the pathogenic immune cells expressing CCR2 are myeloid-derived suppressor cells (MDSCs). In a further embodiment, the pathogenic immune cells expressing CCR2 are selected from monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs).

[0104] In further embodiments, the tumor or cancer cells are solid tumor cells.

[0105] In further embodiments, the tumor or cancer cell is a non-small cell lung cancer (NSCLC) cell, a hepatocellular carcinoma (HCC) cell, a colorectal cancer (CRC) cell, a cervical squamous cell carcinoma (CESC) cell, a head and neck squamous cell carcinoma (HNSC) cell, a pancreatic cancer cell, a metastatic castration-resistant prostate cancer (mCRPC) cell, an ovarian cancer cell, a bladder cancer cell, or a breast cancer cell, preferably an NSCLC cell, an HCC cell, or a CRC cell.

[0106] In a further embodiment, the stromal cells are cancer-associated fibroblasts (CAFs).

[0107] The present disclosure also provides a pharmaceutical composition comprising a compound of formula (I) disclosed herein and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0108] Anticotinine antibodies The present 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 thereof" refers to an antibody or antigen-binding fragment thereof that binds to a cotinine moiety. Cotinine has the following structure: [ka]

[0109] As used herein, the term "cotinine moiety" refers to cotinine or an analog of cotinine. The compounds of formula (I) described herein include a cotinine moiety linked to a CCR2 binding moiety via a linker. In one embodiment, the cotinine moiety has the following structure: [ka] In the formula, R 1 is C 1~4 Alkyl or C 3~6 In another embodiment, R 1 is methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, or t-butyl. 1 is methyl. In another embodiment, R 1 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0110] The term "antibody" is used in the broadest sense herein to refer to a molecule having an immunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD, or IgE), and includes monoclonal antibodies, recombinant antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, multispecific 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., including bispecific and heteroconjugate antibodies, as well as modified forms of any of the foregoing (for a review of alternative "antibody" formats, see Holliger and Hudson, Nature Biotechnology, 2005, 23(9):1126-1136).

[0111] The terms complete, whole, or intact antibody, used interchangeably herein, refer to a heterotetrameric glycoprotein with an approximate molecular weight of 150,000 daltons. An intact antibody is composed of two identical heavy chains (HC) and two identical light chains (LC) linked by covalent disulfide bonds. 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 is composed of an amino-terminal variable domain, a variable heavy chain (VH) or a variable light chain (VL), and a carboxyl-terminal constant domain, CH1 (heavy chain) and CL (light chain). The Fc fragment is composed of two domains formed by dimerization of paired CH2 and CH3 regions. Fc can trigger effector functions 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 distinct heavy chain amino acid sequences, called μ, α, γ, ε and δ, respectively, and each heavy chain can pair with either a K or a λ light chain. The majority of antibodies in serum belong to the IgG class, and there are four isotypes of human IgG (IgG1, IgG2, IgG3 and IgG4), which differ primarily in sequences in their hinge region.

[0112] "CDR" is defined as the complementarity determining region amino acid sequences of an antibody or antigen-binding fragment thereof. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, "CDR" as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

[0113] Throughout this specification, amino acid residues within variable domain sequences and variable domain regions within a full-length antigen-binding sequence, e.g., within an antibody heavy chain sequence or an antibody light chain sequence, are numbered according to the Kabat numbering convention. Similarly, the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2", "CDRH3" used in the examples follow the Kabat numbering convention. For further information, see Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987).

[0114] It will be apparent to the skilled artisan that there are alternative numbering conventions for amino acid residues within variable domain sequences and full-length antibody sequences. There are also alternative numbering conventions for CDR sequences, such as those provided in Chothia et al., Nature, 1989, 342:877-883. The structure and protein folding of the antigen-binding protein may mean that other residues are considered part of the CDR sequences and will be understood as such by the skilled artisan.

[0115] Other numbering conventions for CDR sequences available to those skilled in the art include the "AbM" (University of Bath) and "Contact" (University College London) methods.

[0116] Table 2 below presents one definition using each numbering convention for each CDR or binding unit. It should be noted that some of the CDR definitions may vary depending on the particular publication used. [Table 2]

[0117] In a further embodiment, the anti-cotinine antibody is humanized. In a further embodiment, the Fc region of the anti-cotinine antibody is modified to increase ADCC activity, ADCP activity, and / or CDC activity, and suitable modifications are provided below. In a further embodiment, the Fc region of the anti-cotinine antibody is modified to increase ADCC activity.

[0118] Fc engineering methods can be applied to modify the functional or pharmacokinetic properties of antibodies. Effector functions can be altered by making mutations in the Fc region that increase or decrease binding to C1q or Fcγ receptors, modifying CDC or ADCC activity, respectively. Modifications to the glycosylation pattern of the antibody can also be made to alter effector functions. The in vivo half-life of the antibody can be altered by making mutations that affect the binding of Fc to FcRn (neonatal Fc receptor).

[0119] The term "effector function" as used herein refers to one or more of antibody-mediated effects, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-mediated complement activation, including complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated phagocytosis (CDCP), antibody-dependent complement-mediated cytolysis (ADCML), and Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis (ADCP).

[0120] The interaction between the Fc region of an antigen-binding protein or antibody and various Fc receptors (FcRs), including FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), FcRn, C1q, and type II Fc receptors, is believed to mediate the effector functions of the antigen-binding protein or antibody. A significant biological effect can be the result of the effector function. Usually, the ability to mediate an 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.

[0121] Effector function can be evaluated in several ways, including, for example, evaluating the ADCC effector function of the antibody coated on the target cell mediated by natural killer (NK) cells via FcγRIII or monocytes / macrophages via FcγRI, or evaluating the CDC effector function of the antibody coated on the target cell mediated by the complement cascade via C1q. For example, the antibody or antigen-binding fragment of the present invention can be evaluated for ADCC effector function in a natural killer cell assay. Examples of such assays can be found 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; Lazar et al., PNAS, 2006, 103:4005-4010.

[0122] Examples of assays for determining CDC function include those described in J Imm Meth, 1995, 184:29-38.

[0123] The effect of mutations on effector function (e.g., FcRn binding, FcγR and C1q binding, CDC, ADCML, ADCC, ADCP) can be assessed, for example, as described 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.

[0124] Throughout this specification, amino acid residues within the Fc region of an antibody sequence or full length antigen binding protein sequence are numbered according to the EU index numbering convention.

[0125] Human IgG1 constant regions containing specific mutations have been shown to enhance binding to Fc receptors. 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 thereof of the present invention may include any of the following mutations:

[0126] Enhanced CDC: Fc engineering can be used to enhance complement-based effector functions. For example (for 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 result in preformed IgG hexamers (Wang et al., Protein Cell, 2018, 9(1):63-73).

[0127] Enhancement of ADCC: Fc engineering can be used to enhance ADCC. For example (for IgG1), F243L / R292P / Y300L / V305I / P396L, S239D / I332E, and S298A / E333A / K334A increase FcγRIIIa binding, S239D / I332E / A330L increase FcγRIIIa binding and decrease FcγRIIb binding, 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. An asymmetric Fc, in which one heavy chain contains L234Y / L235Q / G236W / S239M / H268D / D270E / S298A mutations and the opposite heavy chain has D270E / K326D / A330M / K334E, has increased affinity for FcγRIIIa F158 (low affinity allele) and FcγRIIIa V158 (high affinity allele) without increased binding affinity to the inhibitory FcγRIIb (Mimoto et al., mAbs, 2013, 5(2):229-236).

[0128] Enhancement of ADCP: Fc engineering can be used to enhance ADCP. For example (for IgG1), G236A / S239D / I332E increases FcγRIIa binding and increases FcγRIIIa binding (Richards, J. et al., Mol. Cancer Ther., 2008, 7:2517-2527).

[0129] Increased co-engagement: Fc engineering can be used to increase co-engagement with FcRs. For example (for IgG1), S267E / L328F increases FcγRIIb binding, and N325S / L328F increases FcγRIIa binding and decreases FcγRIIIa binding (Wang et al., Protein Cell, 2018, 9(1):63-73).

[0130] In further embodiments, the antibodies or antigen-binding fragments thereof of the invention may comprise a heavy chain constant region with an altered glycosylation profile, such that the antibodies or antigen-binding fragments thereof have 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 thereof with altered glycosylation profiles are described in WO2003 / 011878, WO2006 / 014679 and EP1229125.

[0131] The absence of the α1,6 innermost fucose residue on the Fc glycan moiety on N297 of IgG1 antibodies enhances affinity for FcγRIIIA. Thus, afucosylated or hypofucosylated monoclonal antibodies may have increased therapeutic efficacy (Shields et al., J Biol Chem., 2002, 277(30):26733-40, and Monnet et al., mAbs, 2014, 6(2):422-436).

[0132] In one embodiment, an antibody or antigen-binding fragment thereof is provided that comprises a chimeric heavy chain constant region. In one embodiment, the antibody or antigen-binding fragment thereof comprises an IgG1 / IgG3 chimeric heavy chain constant region, such that the antibody or antigen-binding fragment thereof has enhanced effector function, e.g., enhanced ADCC or enhanced CDC, or enhanced ADCC and CDC function. For example, the chimeric antibody or antigen-binding fragment thereof of the present invention may comprise at least one CH2 domain derived from IgG3. In one such embodiment, the antibody or antigen-binding fragment thereof may comprise one CH2 domain derived from IgG3, or both CH2 domains may be derived from IgG3. In a further embodiment, the chimeric antibody or antigen-binding fragment thereof comprises an IgG1 CH1 domain, an IgG3 CH2 domain, and an IgG3 CH3 domain. In a further embodiment, the chimeric antibody or antigen-binding fragment thereof comprises an IgG1 CH1 domain, an IgG3 CH2 domain, and an IgG3 CH3 domain, except for position 435, which is a histidine.

[0133] In a further embodiment, the chimeric antibody or antigen-binding fragment thereof comprises an IgG1 CH1 domain and at least one CH2 domain derived from IgG3. In one embodiment, the chimeric antibody or antigen-binding fragment thereof comprises an IgG1 CH1 domain 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 antigen-binding fragment thereof also comprises 356E, which corresponds to IgG3 residues in the CH3 domain. In one embodiment, the antibody or antigen-binding fragment thereof 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.

[0134] Also provided is a method for producing an antibody or antigen-binding fragment thereof according to the invention, comprising the steps of: a) culturing a recombinant host cell comprising an expression vector comprising a nucleic acid sequence encoding a chimeric Fc region having both IgG1 and IgG3 Fc region amino acid residues (e.g., as described above); b) recovering the antibody or antigen-binding fragment thereof.

[0135] Such methods of producing antibodies or antigen-binding fragments thereof having chimeric heavy chain constant regions 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 a recombinant host cell comprising an expression vector in which a nucleic acid sequence encoding a chimeric Fc region having both IgG1 and IgG3 Fc region amino acid residues is expressed to produce an antibody or antigen-binding fragment thereof with enhanced CDC activity, i.e., CDC activity is increased relative to an otherwise identical antibody or antigen-binding fragment thereof lacking such a chimeric Fc region, as described in WO2007 / 011041 and US2007 / 0148165, each of which is incorporated herein by reference. In alternative embodiments, CDC activity can 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.

[0136] The present invention also provides a method for producing an antibody or antigen-binding fragment thereof according to the invention, comprising the steps of: a) culturing a recombinant host cell comprising an expression vector comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof, optionally wherein the FUT8 gene encoding α-1,6-fucosyltransferase is inactivated in the recombinant host cell; b) recovering the antibody or antigen-binding fragment thereof A method is also provided, including:

[0137] Such methods for the production of antibodies or antigen-binding fragments thereof 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 that is increased relative to the same monoclonal antibodies produced in cells with a functional FUT8 gene, as described in U.S. Patent No. 7,214,775, U.S. Patent No. 6,946,292, WO00 / 61739, and WO02 / 31240, each of which is incorporated herein by reference. Those of skill in the art will also recognize other suitable systems.

[0138] In one embodiment, the antibody or antigen-binding fragment thereof is produced in a host cell in which the FUT8 gene is inactivated. In a further embodiment, the antibody or antigen-binding fragment thereof is produced in a - / -FUT8 host cell. In a further embodiment, the antibody or antigen-binding fragment thereof is afucosylated at Asn297 (IgG1).

[0139] It will be apparent to one of skill in the art that such modifications can be used not only alone but also in combination with each other to further enhance effector function.

[0140] In one such embodiment, an antibody or antigen-binding fragment thereof is provided, comprising a heavy chain constant region comprising both the mutated heavy chain constant region and the chimeric heavy chain constant region described individually above. For example, an antibody or antigen-binding fragment thereof comprising at least one CH2 domain from IgG3 and one CH2 domain from IgG1, wherein the IgG1 CH2 domain has one or more mutations at positions selected from 239, 332, and 330 (e.g., the mutations can be selected from S239D, I332E, and A330L), such that the antibody or antigen-binding fragment thereof has enhanced effector function, e.g., enhanced ADCC or enhanced CDC, or enhanced ADCC and enhanced CDC, compared to a comparable antibody or antigen-binding fragment thereof having an IgG1 heavy chain constant region lacking said mutations. In one embodiment, the IgG1 CH2 domain has the mutations S239D and I332E. In another embodiment, the IgG1 CH2 domain has the mutations S239D, A330L, and I332E.

[0141] In alternative embodiments, an antibody or antigen-binding fragment thereof is provided that comprises both a chimeric heavy chain constant region and an altered glycosylation profile, as described individually above. In one embodiment, the antibody or antigen-binding fragment thereof comprises an altered glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less. In one such embodiment, the heavy chain constant region comprises at least one CH2 domain from IgG3 and one CH2 domain from IgG1, and has an altered glycosylation profile, e.g., the antibody or antigen-binding fragment thereof is defucosylated such that the ratio of fucose to mannose is 0.8:3 or less. The antibody or antigen-binding fragment thereof has enhanced effector function, e.g., enhanced ADCC or enhanced CDC, or enhanced ADCC and enhanced CDC, compared to a comparable antibody or antigen-binding fragment thereof having an IgG1 heavy chain constant region lacking the glycosylation profile.

[0142] In an alternative embodiment, the antibody or antigen-binding fragment thereof has at least one IgG3 heavy chain CH2 domain and at least one heavy chain constant domain derived from IgG1, and both IgG CH2 domains are mutated according to the restrictions described herein.

[0143] In one aspect, there is provided a method for producing an antibody or antigen-binding fragment thereof according to the invention described herein, comprising the steps of: a) culturing a recombinant host cell comprising an expression vector comprising a nucleic acid sequence encoding a chimeric Fc domain having both IgG1 and IgG3 Fc domain amino acid residues (e.g., as described above), wherein the FUT8 gene encoding α-1,6-fucosyltransferase is inactivated in the recombinant host cell; b) recovering the antibody or antigen-binding fragment thereof.

[0144] Such methods for the production of antibodies or antigen-binding fragments thereof can be performed, 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 antigen-binding fragments thereof that have both enhanced ADCC and CDC activity relative to an otherwise identical monoclonal antibody that lacks a chimeric Fc domain and is fucosylated.

[0145] In another embodiment, an antibody or antigen-binding fragment thereof is provided comprising a mutation and a chimeric heavy chain constant region, the antibody or antigen-binding fragment thereof having an altered glycosylation profile such that the antibody or antigen-binding fragment thereof has enhanced effector function, e.g., enhanced ADCC or enhanced CDC, or both enhanced ADCC and CDC. In one embodiment, the mutation is selected from positions 239, 332, and 330, e.g., S239D, I332E, and A330L. In a further embodiment, the heavy chain constant region comprises at least one CH2 domain from IgG3 and one CH1 domain from IgG1. In one embodiment, the heavy chain constant region has an altered glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less, e.g., the antibody or antigen-binding fragment thereof is defucosylated such that the antibody or antigen-binding fragment thereof has enhanced effector function compared to a comparable non-chimeric antibody or antigen-binding fragment thereof lacking the mutation and lacking the altered glycosylation profile.

[0146] In a further embodiment, the anti-cotinine antibody or antigen-binding fragment thereof 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 a further embodiment, the anti-cotinine antibody comprises a heavy chain and a light chain, the heavy chain comprises a CDR1 having SEQ ID NO: 1, a CDR2 having SEQ ID NO: 2, and a CDR3 having SEQ ID NO: 3, and the light chain comprises a CDR1 having SEQ ID NO: 4, a CDR2 having SEQ ID NO: 5, and a CDR3 having SEQ ID NO: 6. In a further embodiment, the anti-cotinine antibody is an IgG1 isotype. In a further embodiment, the anti-cotinine antibody is an IgG1 isotype comprising a substitution in the Fc region to increase or enhance ADCC activity. In a further embodiment, the anti-cotinine 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, the numbering of the residues being according to the EU index. In a further embodiment, the anti-cotinine antibody is an IgG1 isotype comprising a substitution in the Fc region to increase or enhance ADCC activity, the substitution being S239D / I332E, the numbering of the residues being according to the EU index.

[0147] In a further embodiment, the anti-cotinine antibody or antigen-binding fragment thereof 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 a further embodiment, the anti-cotinine antibody comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region (VH) having SEQ ID NO: 7 and the light chain comprises a light chain variable region (VL) having SEQ ID NO: 8. In a further embodiment, the anti-cotinine antibody is an IgG1 isotype. In a further embodiment, the anti-cotinine antibody is an IgG1 isotype comprising a substitution in the Fc region for increasing or enhancing ADCC activity. In a further embodiment, the anti-cotinine antibody is an IgG1 isotype comprising a substitution in the Fc region for increasing or enhancing ADCC activity, the substitution being S239D / I332E or S239D / I332E / A330L, the numbering of the residues being according to the EU index. In a further embodiment, the anti-cotinine antibody is an IgG1 isotype comprising substitutions in the Fc region to increase or enhance ADCC activity, the substitutions being S239D / I332E, where the numbering of residues is according to the EU index.

[0148] In a further embodiment, the anti-cotinine antibody has a heavy chain comprising SEQ ID NO:9 and a light chain comprising SEQ ID NO:10.

[0149] The present disclosure also provides a pharmaceutical composition comprising an anti-cotinine antibody or antigen-binding fragment thereof disclosed herein and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0150] The present disclosure also provides a combination comprising the compound of formula (I) disclosed herein and the anti-cotinine antibody or antigen-binding fragment thereof disclosed herein. The compound of formula (I) and the anti-cotinine antibody or antigen-binding fragment thereof can be present in the same composition or in separate compositions. In one embodiment, the combination comprises a pharmaceutical composition comprising the compound of formula (I) disclosed herein and the anti-cotinine antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier, diluent, or excipient. In another embodiment, the combination comprises a first pharmaceutical composition comprising the compound of formula (I) disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient, and a second pharmaceutical composition comprising the anti-cotinine antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0151] Description of Use The compounds of formula (I) and their pharma- ceutically acceptable salts are capable of simultaneously binding to cell surface-expressed CCR2 and to an anti-cotinine antibody or an antigen-binding fragment thereof to form a ternary complex for the treatment and / or prevention of diseases or disorders associated with CCR2-expressing cells.

[0152] In one embodiment, the disclosure provides a method for treating and / or preventing a disease or disorder in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt thereof and an anti-cotinine antibody or antigen-binding fragment thereof, wherein the disease or disorder is selected from cancer, an inflammatory disease, an autoimmune disease, a viral infection, or a bacterial infection.

[0153] In further embodiments, the compound and the antibody, or antigen-binding fragment thereof, are administered simultaneously. In further embodiments, the compound and the antibody, or antigen-binding fragment thereof, are administered simultaneously from a single composition, including as a fixed dose composition or by premixing the compound and the antibody or antigen-binding fragment thereof prior to administration. For example, the compound and the antibody, or antigen-binding fragment thereof, can be premixed 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 prior to administration. In further embodiments, the compound and the antibody, or antigen-binding fragment thereof, are administered simultaneously from two separate compositions.

[0154] In a further embodiment, the compound and the antibody, or antigen-binding fragment thereof, are administered sequentially.

[0155] In certain embodiments, the compound and the antibody, or antigen-binding fragment thereof, whether administered simultaneously or sequentially, may be administered by the same route or by different routes. In one embodiment, the compound and the antibody, or antigen-binding fragment thereof, are both administered intravenously or subcutaneously, either in the same composition or in separate compositions. In another embodiment, the compound is administered orally and the antibody, or antigen-binding fragment thereof, is administered intravenously or subcutaneously.

[0156] In further embodiments, the compound and antibody, or antigen-binding fragment thereof, are administered in a molar ratio 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 of compound to antibody, or antigen-binding fragment thereof.

[0157] In further embodiments, the compound and antibody, or antigen-binding fragment thereof, are present in combination in a molar ratio 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 of compound to antibody, or antigen-binding fragment thereof.

[0158] In further embodiments, the compound and antibody, or antigen-binding fragment thereof, are administered at a dosage of 0.0001 mg / kg to 1 mg / kg of compound and 0.01 mg / kg to 100 mg / kg of antibody. For example, in further embodiments, the compound is administered at a dosage 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, about 0.002mg / kg to about 0.003mg / kg, about 0.003mg / kg to about 0.004mg / kg, about 0.004mg / kg to about 0.005mg / kg, about 0.005mg / kg to about 0.01mg / kg, about 0.01mg / kg to about 0.02mg / kg, about 0.02mg / kg to about 0.03mg / kg, about 0.03mg / kg to about 0.04mg / kg, about 0.04mg / kg to about 0. The antibody or antigen-binding fragment thereof is administered at a dosage of about 0.01 mg / kg to about 0.02 mg / kg, about 0.05 mg / kg to about 0.1 mg / 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, and / or about 0.5 mg / kg to about 1 mg / kg, and the antibody or antigen-binding fragment thereof is administered at a dosage of about 0.01 mg / kg to about 0.02 mg / kg, about 0.05 mg / kg to about 0.1 mg / 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, and / or about 0.5 mg / kg to about 1 mg / kg. g / kg, about 0.02mg / kg to about 0.03mg / kg, about 0.03mg / kg to about 0.04mg / kg, about 0.04mg / kg to about 0.05mg / kg, about 0.05mg / kg to about 0.1m 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 The compound is administered at a dosage of about 100 mg / kg to about 35 mg / kg, about 35 mg / kg to about 40 mg / kg, about 40 mg / kg to about 45 mg / kg, about 45 mg / kg to about 50 mg / kg, about 50 mg / kg to about 60 mg / kg, about 60 mg / kg to about 70 mg / kg, about 70 mg / kg to about 80 mg / kg, about 80 mg / kg to about 90 mg / kg, and / or about 90 mg / kg to about 100 mg / kg.

[0159] In further embodiments, the compound and antibody, or antigen-binding fragment thereof, are administered in a dosage of 0.007 mg to 70 mg of compound and 0.7 mg to 7000 mg of antibody. For example, in further embodiments, the compound is administered in a dosage of about 0.007 mg to about 0.01 mg, about 0.01 mg to about 0.02 mg, about 0.02 mg to about 0.03 mg, about 0.03 mg to about 0.04 mg, about 0.04 mg to about 0.05 mg, about 0.05 mg to about 0.1 mg, about 0.1 mg to about 0.2 mg, about 0.2 mg to about 0.3 mg, about 0.3 mg to about 0.4 mg, about 0.4 mg to about 0.5 mg, about 0. and the antibody or antigen-binding fragment thereof is administered at a dosage of about 0.5 mg to about 1 mg, 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 mg, about 40 mg to about 50 mg, about 50 mg to about 60 mg, and / or about 60 mg to about 70 mg. Approximately 1 mg to approximately 2 mg, approximately 2 mg to approximately 3 mg, approximately 3 mg to approximately 4 mg, approximately 4 mg to approximately 5 mg, approximately 5 mg to approximately 10 mg, approximately 10 mg to approximately 20 mg, approximately 20 mg to approximately 30 mg, approximately 30 mg to approximately 40 mg, Approximately 40mg to approximately 50mg, approximately 50mg to approximately 100mg, approximately 100mg to approximately 500mg, approximately 500mg to approximately 1000mg, approximately 1000mg to approximately 1500mg, approximately 1500mg to approximately 2000mg, approximately The compound is administered in a dosage of 2000 mg to about 2500 mg, about 2500 mg to about 3000 mg, about 3000 mg to about 3500 mg, about 3500 mg to about 4000 mg, about 4000 mg to about 4500 mg, about 4500 mg to about 5000 mg, about 5000 mg to about 5500 mg, about 5500 mg to about 6000 mg, about 6000 mg to about 6500 mg, and / or about 6500 mg to about 7000 mg.

[0160] In further embodiments, the compounds and antibodies, or antigen-binding fragments thereof, are administered in the molar ratios and / or dosages described herein once per week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks for a period of one week to one year, such as, for example, a period of 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.

[0161] In a further embodiment, the present disclosure provides a therapeutically effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt thereof and an anti-cotinine antibody or an antigen-binding fragment thereof for use in treatment. The compound of formula (I) or a pharma- ceutically acceptable salt thereof and the anti-cotinine antibody or an antigen-binding fragment thereof can be used in treating or preventing a disease or disorder selected from cancer, inflammatory disease, autoimmune disease, viral infection, or bacterial infection.

[0162] In a further embodiment, the present disclosure provides a therapeutically effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt thereof and an anti-cotinine antibody or an antigen-binding fragment thereof for the manufacture of a medicament, which can be used in treating or preventing a disease or disorder selected from cancer, an inflammatory disease, an autoimmune disease, a viral infection, or a bacterial infection.

[0163] In a further embodiment, the disease or disorder is mediated by chemokine receptor 2 (CCR2) and / or is associated with CCR2-positive pathogenic cells. In a further embodiment, the CCR-positive cell type is identified by testing for expression of CCR by immunohistochemistry or flow cytometry.

[0164] In further embodiments, the disease or disorder is a cancer selected from non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), colorectal cancer (CRC), cervical squamous cell carcinoma (CESC), head and neck squamous cell carcinoma (HNSC), pancreatic cancer, metastatic castration-resistant prostate cancer (mCRPC), ovarian cancer, bladder cancer, or breast cancer, preferably a cancer selected from NSCLC, HCC, or CRC.

[0165] In a further embodiment, the disease or disorder is a solid tumor.In a further embodiment, the disease or disorder is a solid tumor selected from NSCLC, HCC, CRC, CESC, HNSC, pancreatic cancer, mCRPC, ovarian cancer, bladder cancer or breast cancer, preferably a solid tumor selected from NSCLC, HCC or CRC.

[0166] In a further embodiment, the disease or disorder is a PD-1 relapsed or refractory cancer, such as PD-1 relapsed or refractory NSCLC, HCC, CRC, CESC, HNSC, pancreatic cancer, mCRPC, ovarian cancer, bladder cancer, or breast cancer, preferably PD-1 relapsed or refractory NSCLC, HCC, or CRC.

[0167] In a further embodiment, the disease or disorder is a non-solid cancer, hi a further embodiment, the disease or disorder is a leukemia, lymphoma, or myeloma.

[0168] In a further embodiment, the disease or disorder is a viral infection. In a further embodiment, the viral infection is caused by an influenza virus, a coronavirus (e.g., COVID-19), or a hepatitis B virus.

[0169] In a further embodiment, the disease or disorder is a bacterial infection. In a further embodiment, the bacterial infection is a chronic bacterial infection.

[0170] In one embodiment, the disclosure provides a method for increasing antibody-dependent cellular cytotoxicity (ADCC) of a CCR2-expressing cell, comprising contacting the cell with an effective amount of a compound of formula (I) or a pharma- ceutical acceptable salt thereof and an anti-cotinine antibody or antigen-binding fragment thereof, wherein the target-binding portion of the compound binds to CCR2 expressed on the cell.

[0171] In one embodiment, the disclosure provides a method for increasing antibody-dependent cellular phagocytosis (ADCP) of a CCR2-expressing cell, comprising contacting the cell with an effective amount of a compound of formula (I) or a phagocytosis or phagocytosis or antigen-binding fragment thereof, wherein the target binding portion of the compound binds to CCR2 expressed on the cell.

[0172] In one embodiment, the disclosure provides a method for increasing complement dependent cytotoxicity (CDC) of a CCR2-expressing cell, comprising contacting the cell with an effective amount of a compound of formula (I) or a pharma- ceutical acceptable salt thereof and an anti-cotinine antibody or antigen-binding fragment thereof, wherein the CCR2-binding portion of the compound binds to CCR2 expressed on the cell.

[0173] In one embodiment, the present disclosure provides a method of conditioning a patient for treatment with chimeric antigen receptor (CAR) T-cell therapy, comprising administering to the patient an effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt thereof and an anti-cotinine antibody or an antigen-binding fragment thereof. In some embodiments, the compound of formula (I) or a pharma-ceutically acceptable salt thereof and an anti-cotinine antibody or an antigen-binding fragment thereof are administered in combination with CAR-T-cell therapy. The compound of formula (I) or a pharma-ceutically acceptable salt thereof and an anti-cotinine antibody or an antigen-binding fragment thereof may be administered as a conditioning therapy or a combination therapy to improve efficacy in the treatment of solid tumors. In other embodiments, the compound of formula (I) or a pharma-ceutically acceptable salt thereof and an anti-cotinine antibody or an antigen-binding fragment thereof may be administered as a neoadjuvant therapy for other therapies, including but not limited to immunotherapy, surgical resection, radiation, and / or chemotherapy.

[0174] In one embodiment, the disclosure provides a method for depleting CCR2-expressing cells, comprising contacting the cells with a compound of formula (I) or a pharma- ceutical acceptable salt thereof and an anti-cotinine antibody or an antigen-binding fragment thereof, wherein the CCR2-binding portion of the compound binds to CCR2 expressed on the cells.

[0175] In a further embodiment, the CCR2 expressing cell is a pathogenic cell.

[0176] In further embodiments, the pathogenic cell is a pathogenic immune cell, a tumor or cancer cell, or a stromal cell.

[0177] In further embodiments, the pathogenic immune cell is a monocyte, a myeloid-derived suppressor cell (MDSC), such as monocytic MDSC (mMDSC) and polymorphonuclear MDSC (PMN_MDSC), a T regulatory cell (Treg), a neutrophil (e.g., N2 neutrophil), a macrophage (e.g., M2 macrophage), a B regulatory cell (Breg, memory B cell), a plasma cell, a CD8 cell (e.g., CD8 regulatory cell (CD8reg), memory CD8 cell, effector CD8 cell, naive CD8 T cell, TEMRA), an exhausted T cell, an eosinophil, a basophil, a mast cell, a dendritic cell, a natural killer (NK cell), an innate lymphoid cell, a NK T cell (NKT), or a γδ T cell.

[0178] In further embodiments, the pathogenic immune cells are myeloid-derived suppressor cells (MDSCs), such as monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs), T regulatory cells (Tregs), neutrophils (e.g., N2 neutrophils), macrophages (e.g., M2 macrophages), B regulatory cells (Bregs), CD8 regulatory cells (CD8regs), exhausted T cells.

[0179] In further embodiments, the tumor or cancer cell is a non-small cell lung cancer (NSCLC) cell, a hepatocellular carcinoma (HCC) cell, a colorectal cancer (CRC) cell, a cervical squamous cell carcinoma (CESC) cell, a head and neck squamous cell carcinoma (HNSC) cell, a pancreatic cancer cell, a metastatic castration-resistant prostate cancer (mCRPC) cell, an ovarian cancer cell, a bladder cancer cell, or a breast cancer cell, preferably an NSCLC cell, an HCC cell, or a CRC cell.

[0180] In a further embodiment, the stromal cells are cancer-associated fibroblasts (CAFs).

[0181] Combination therapy The compounds of the present invention can be employed alone or in combination with other therapeutic agents. Thus, the combination therapy according to the present invention includes the administration of at least one compound of formula (I) or its pharma- ceutically acceptable salt, and the use of at least one other pharma- ceutical active agent. The compounds of the present invention and the other pharma- ceutical active agent can be administered together in a single pharmaceutical composition or separately, and when administered separately, this can occur simultaneously or sequentially in any order. The amounts of the compounds of the present invention and the other pharma- ceutical active agent, as well as the relative timing of administration, are selected to achieve the desired combined therapeutic effect.

[0182] When the compounds of the present invention are administered in combination with one or more other therapeutically active agents that are normally administered by inhalation, intravenous, oral, intranasal, topical ophthalmic, or other routes, it will be understood that the resulting pharmaceutical composition can be administered by the same route. Alternatively, the individual components of the composition can be administered by different routes.

[0183] In one embodiment, the compounds and pharmaceutical compositions disclosed herein are used in combination with or include one or more additional therapeutic agents. In a further embodiment, the additional therapeutic agent is a checkpoint inhibitor or an immunomodulatory agent.

[0184] In further embodiments, the checkpoint inhibitor is selected from a PD-1 inhibitor (e.g., an anti-PD-1 antibody, including but not limited to, pembrolizumab, nivolumab, cemiplimab, or dostarimab), a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody, including but not limited to, atezolizumab, avelumab, or durvalumab), or a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody, including but not limited to, ipilimumab or tremilumumab).

[0185] In further embodiments, the checkpoint inhibitor is selected from a CD226 axis inhibitor, including but not limited to a TIGIT inhibitor (e.g., an anti-TIGIT antibody), a CD96 inhibitor (e.g., an anti-CD96 antibody), and / or a PVRIG inhibitor (e.g., an anti-PVRIG antibody).

[0186] In further embodiments, the immunomodulatory agent is an ICOS agonist (e.g., an anti-ICOS antibody, including but not limited to, ferazilimab), a PARP inhibitor (e.g., niraparib, olaparib), or a STING agonist.

[0187] Pharmaceutical Compositions, Dosages, and Formulations For purposes of administration, in certain embodiments, the ARM described herein is administered as a raw chemical or formulated as a pharmaceutical composition. The pharmaceutical compositions disclosed herein include an ARM and one or more of a pharma- ceutically acceptable carrier, diluent, or excipient. The ARM is present in the composition in an amount effective to treat a particular disease, disorder, or condition of interest. The activity of the ARM can be determined by one of skill in the art, for example, as described in the biological assays described below. Appropriate concentrations and dosages can be readily determined by one of skill 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.

[0188] The administration of the compound of the present invention or its pharmaceutically acceptable salt in pure form or in a suitable pharmaceutical composition is carried out through any of the accepted administration modes of drugs to provide similar utility. The pharmaceutical composition of the present invention is prepared by combining the compound of the present invention with a suitable pharmaceutically acceptable carrier, diluent, or excipient, and in a specific embodiment, is formulated into a solid, semi-solid, liquid, or gaseous form of preparation, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Exemplary 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, buccal, rectal, vaginal, and intranasal. The pharmaceutical composition of the present invention is formulated to allow the active ingredient contained therein to be bioavailable upon administration of the composition to a patient.

[0189] 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 compound of the present invention in aerosol form may hold multiple dosage units.The actual method of preparing such dosage forms will be 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 comprises a therapeutically effective amount of the compound of the present invention or its pharma-ceutically acceptable salt for treating the disease or condition of interest, according to the teachings described herein.

[0190] The pharmaceutical compositions disclosed herein are prepared by methods well known in the pharmaceutical field.For example, in certain embodiments, pharmaceutical compositions intended to be administered by injection are prepared by combining the compound of the present invention with sterile distilled water to form a solution.In some embodiments, surfactants are added to promote the formation of a homogeneous solution or suspension.Surfactants are compounds that non-covalently interact with the compound of the present invention to promote the dissolution or homogeneous suspension of the compound in aqueous delivery system.

[0191] Traditional antibody therapeutics have several drawbacks that are addressed by the ARM approach described herein, including difficulties in managing adverse events through adjusting the dose and frequency of administration, challenges in generating antibodies against certain classes of drug targets (e.g., GPCRs, ion channels, and enzymes), and the need for a new cell line for development, which can be slow and expensive, for each new antibody. Furthermore, different forms of biologics (e.g., bispecifics) can be difficult to manufacture. In contrast, the ARM approach offers the following advantages: integrating antibody pharmacology with small molecule dose control, dose-controlled PK / PD that allows for timed cell depletion, simpler multimerization, and rapid reversal of cell depletion through administration of an antibody-binding moiety (e.g., cotinine hapten) that can uncouple therapeutic effects from potential adverse events. EXAMPLES

[0192] 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 of skill in the art for preparing and using the compounds, compositions, and methods of the present invention. Although specific embodiments of the present invention are described, those of skill in the art will understand that various changes and modifications can be made. References to preparations carried out in a similar manner to other preparations, or by general methods of other preparations, may encompass variations in routine parameters such as slight changes in time, temperature, purification conditions, reagent amounts, etc. All title compound chemical names were generated using ChemDraw Plugin Version 16.0.1.13c(90) or ChemDraw Desktop Version 16.0.1.13(90). Those of skill in the art will recognize that compounds of the present invention may have alternative names when different naming software is used.

[0193] compound synthesis The compounds according to formula (I) are prepared using conventional organic synthesis methods.Suitable synthetic routes are depicted below in the following general reaction schemes.All starting materials are commercially available or can be easily prepared from commercially available starting materials by those skilled in the art.

[0194] Those skilled in the art will understand that if a substituent described herein is not compatible with the synthetic methods described herein, the substituent can be protected with a suitable protecting group that is stable to the reaction conditions. The protecting group can be removed at a suitable point in the reaction sequence to provide the desired intermediate or target compound. Suitable protecting groups and methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art, examples of which can be found in T. Greene and P. Wuts, Protecting Groups in Organic Synthesis (4th ed.), John Wiley & Sons, NY (2006). In some cases, a substituent can be specifically selected to be reactive under the reaction conditions used. Under these circumstances, the reaction conditions convert the selected substituent into another substituent that is either useful as an intermediate compound or is a desired substituent in a target compound.

[0195] Scheme 1 Intermediates Intermediate 1: Methyl (1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate. [ka]

[0196] Step 1: Methyl (1R,4S)-4-aminocyclopent-2-ene-1-carboxylate, hydrochloride salt. [ka]

[0197] SOCl 2(14.60 mL, 200 mmol) was added dropwise to methanol (100 mL) at 0° C., commercially available (1S,4R)-2-azabicyclo[2.2.1]hept-5-en-3-one (10.91 g, 100 mmol) was added, and the mixture was stirred at 0° C. for 2 h. The mixture was concentrated at 50° C. with a steady stream of nitrogen to give the title compound as a white solid (25 g, 99 mmol, 99% yield). LC / MS m / z 142.2 (M+H) + .

[0198] Step 2: Methyl (1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate. [ka]

[0199] Methyl (1R,4S)-4-aminocyclopent-2-ene-1-carboxylate, hydrochloride (17.76 g, 100 mmol) was dissolved in methanol (50.0 mL) at room temperature under nitrogen atmosphere. DIPEA (20 mL, 115 mmol) was added followed by hexane-2,5-dione (12.90 mL, 110 mmol). The reaction was stirred at room temperature overnight and diluted with ethyl acetate. The organic layer was washed with saturated NH 4 Wash with aqueous Cl (2×50 mL), brine (3×50 mL), and Na 2 SO 4 The mixture was dried at 4° C., filtered, and the filtrate was concentrated to give the title compound as a red oil (21.835 g, 90 mmol, 90% yield). LC / MS m / z 220.2 (M+H). + .

[0200] Intermediate 2: (R)-3-Methoxytetrahydro-4H-pyran-4-one [ka]

[0201] Step 1: 4,4-Dimethoxytetrahydro-2H-pyran-3-ol. [ka]

[0202] Potassium hydroxide (80 g, 1428 mmol) was dissolved in methanol (1 L) with stirring at room temperature. The homogeneous mixture was cooled to 0° C. Commercially available tetrahydro-4H-pyran-4-one (50 g, 499 mmol) was dissolved in methanol (250 mL) and added dropwise to the potassium hydroxide solution while maintaining the temperature below 5° C. After stirring for an additional 1.5 h, iodine (139 g, 549 mmol) was added dropwise over 1.5 h while maintaining the temperature below 5° C. The reaction mixture was stirred at room temperature for 18 h. The reaction was concentrated, toluene (500 mL) was added, and the mixture was stirred for 30 min. A small amount of precipitate was removed by filtration, and the filtrate was evaporated to give the title compound as a maroon oil (60.8 g, 375 mmol, 76% yield). 1 H NMR (400 MHz, chloroform-d) δ ppm 1.74 - 1.82 (m, 1 H) 1.96 (ddd,J=14, 12, 4.9 Hz, 1 H) 2.37 (s, 1H) 3.26 (s, 3 H) 3.28 (s, 3 H) 3.50 (td,J=12, 2.7 Hz, 1 H) 3.66 - 3.72 (m, 2 H) 3.77 - 3.88 (m, 2 H).

[0203] Step 2: 4,4-Dimethoxydihydro-2H-pyran-3(4H)-one. [ka]

[0204] A solution of 4,4-dimethoxytetrahydro-2H-pyran-3-ol (60.8 g, 375 mmol) in dichloromethane (DCM) (500 mL) was stirred at room temperature under nitrogen atmosphere. 4 Å molecular sieves (60.8 g, 375 mmol) was added, followed by 4-methylmorpholine N-oxide (110 g, 937 mmol) and tetra-n-propylammonium perthenate (vii) (5.47 g, 15.57 mmol). The mixture was stirred for 13 hours. The reaction was filtered through Celite® and concentrated. Purification by normal phase chromatography (eluting with 10% ethyl acetate in hexanes) afforded the title compound as a yellow oil (57.73 g, 360 mmol, 96% yield). 1 H NMR (400 MHz, chloroform-d) δ ppm 2.11 - 2.15 (m, 2 H) 3.16 (s, 6 H) 3.84 - 3.88 (m, 2 H) 3.91 - 3.98 (m, 2 H).

[0205] Step 3: (R)-4,4-Dimethoxytetrahydro-2H-pyran-3-ol. [ka]

[0206] A solution of 4,4-dimethoxydihydro-2H-pyran-3(4H)-one (45 g, 281 mmol) in potassium phosphate buffer (pH 7.0) (1.125 L) at room temperature was added with NADPH + (7.92 g, 281 mmol), D-glucose (67.5 g, 375 mmol), KRED-101 (2.25 g, 281 mmol), and GDH (2.25 g, 281 mmol) were added. The mixture was stirred at 25 °C for 6 h. At 2-h intervals, saturated KHCO 3 The pH was adjusted to 7 using a solution of potassium chloride (50 g) and the mixture was stirred for 10 min. The mixture was extracted with acetonitrile (3×500 mL) and the organic extract was concentrated to near dryness. The residue was dissolved in dichloromethane (DCM) (250 mL) and the remaining water was separated. The organic layer was washed with anhydrous Na 2 SO 4The mixture was dried at 40° C., filtered, and concentrated. Purification by normal phase chromatography (eluting with a gradient of 10-50% ethyl acetate in hexanes) afforded the title compound as a colorless liquid (35 g, 209 mmol, 74.5% yield). Specific rotation [α] 25589 : -16.2° (1% in methanol). GC-MS m / z 162.1 (M) + . 1 H NMR (400 MHz, chloroform-d) δ ppm 1.74 - 1.82 (m, 1 H) 1.90-1.99(m, 1 H)2.15 (d,6Hz, 1H)3.26 (s, 3 H) 3.28 (s, 3 H) 3.50 (td,J=12, 2.7 Hz, 1 H) 3.66 - 3.72 (m, 2 H) 3.77 - 3.88 (m, 2 H).

[0207] Step 4: (R)-3,4,4-Trimethoxytetrahydro-2H-pyran. [ka]

[0208] A 60% mineral oil dispersion of sodium hydride (17.26 g, 432 mmol) in tetrahydrofuran (THF) (220 mL) was stirred under nitrogen and cooled to 0° C. (R)-4,4-Dimethoxytetrahydro-2H-pyran-3-ol (35 g, 216 mmol) was added dropwise over 15 min. The mixture was stirred at 25° C. for 1 h, methyl iodide (27.0 mL, 432 mmol) was added dropwise over 15 min, and the mixture was stirred at 25° C. for 4 h. The mixture was poured slowly into ice-cold water (500 mL) and extracted with ethyl acetate (500 mL). The aqueous phase was extracted with dichloromethane (DCM) (2×500 mL) and the combined organic extracts were washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered, and the filtrate was concentrated. Purification by normal phase chromatography (eluted with 30% ethyl acetate in hexane) afforded the title compound as a light brown liquid (34 g, 191 mmol, 89% yield). Specific rotation [α] 25589 :-29.400° (1% in methanol). GC-MS m / z 176.2 (M+ ). 1 H NMR (400 MHz, chloroform-d) δ ppm 1.74 - 1.79(m, 1 H) 1.90-2.05(m, 1 H) 3.19-3.28(m,9H) 3.45-3.51(m,2H) 3.55- 3.60(m,1H) 3.75- 3.83(m,1H)3.95 - 4.05 (m, 1H).

[0209] Step 5: (R)-3-Methoxytetrahydro-4H-pyran-4-one. [ka]

[0210] To a solution of (R)-3,4,4-trimethoxytetrahydro-2H-pyran (35 g, 199 mmol) in tetrahydrofuran (THF) (105 mL) and water (35.0 mL) at 0° C., concentrated aqueous HCl (280 mL, 3248 mmol) was added dropwise over 30 min. The mixture was stirred at 0° C. for 1 h and concentrated to remove tetrahydrofuran (THF). Water (100 mL) was added and the mixture was extracted with dichloromethane (DCM) (2×1 L). The combined organic extracts were washed with saturated NaHCO 3 Wash with aqueous solution (100 mL) of anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered, and the filtrate was concentrated to give the title compound as an off-white solid (25 g, 184 mmol, 93% yield). 25589 :+85.000°(1% in methanol). GC-MSm / z130.1 (M) + . 1 H NMR (400 MHz, chloroform-d) δ ppm2.58- 2.62(m, 1 H)3.50(s,3H) 3.55-3.58(m,1H) 3.70- 3.82(m,2H)4.08-4.17(m, 1 H)4.19- 4.23(m, 1H).

[0211] Intermediate 3: (2S,3S)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid [ka]

[0212] Commercially available racemic trans-4-cotinine carboxylic acid (304 g, 1.38 mol) was purified by chiral preparative HPLC (61 injections) (Chiralpak 1A 20u 101x210mm, 500mL / min, eluted with 50% acetonitrile in methanol with 0.1% formic acid). The desired fractions were collected and concentrated at 45°C. The solid residue was stirred in acetonitrile, filtered and dried under vacuum for 18 hours to give the title compound as a white solid (143.6 g, 652 mmol, 94.5% yield). Analytical chiral HPLC: 95% ee, retention time 2.5 min, Chiralpak AD-H 5u 4.6x150mm, 0.1% formic acid in methanol, 1.0mL / min, Alpha D=+58deg(c=0.3, CH 3 OH); VCD analysis was used to assign absolute stereochemistry. LC-MS m / z 221.1 (M+H) + .

[0213] Intermediate 4: ((1S,3R)-3-(((3S,4S)-3-Methoxytetrahydro-2H-pyran-4-yl)amino)-1-(2-(piperidin-4-yl)ethyl)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, trihydrochloride salt. [ka]

[0214] Step 1: tert-Butyl 4-(2-iodoethyl)piperidine-1-carboxylate. [ka]

[0215] A mixture of commercially available tert-butyl 4-(2-bromoethyl)piperidine-1-carboxylate (20.65 g, 70.7 mmol), sodium iodide (37.1 g, 247 mmol), and sodium bicarbonate (2.97 g, 35.3 mmol) in acetone (236 mL) was heated at 60° C. for 20 h. The mixture was cooled to room temperature and concentrated under reduced vacuum. The residue was partitioned between ethyl acetate and water, and the organic phase was extracted with 50% (v / v) Na in water. 2 S 2 O 5 , washed with brine and MgSO 4 The mixture was dried at 75° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by ISCO CombiFlash® chromatography (220 g silica Gold column) (eluting with a gradient of 0-50% ethyl acetate in heptane) afforded the title compound as a colorless oil (23.49 g, 65.8 mmol, 93% yield). LCMS (m / z) 284.0 (M-(t-Bu)+H). + .

[0216] Step 2: tert-Butyl 4-(2-((1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-(methoxycarbonyl)cyclopent-2-en-1-yl)ethyl)piperidine-1-carboxylate. [ka]

[0217] To a solution of 1M lithium bis(trimethylsilyl)amide (LiHMDS) (70.2 mL, 70.2 mmol) in tetrahydrofuran (THF) (70 mL) at −78° C. under nitrogen atmosphere, methyl (1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate (Intermediate 1) (14.0 g, 63.8 mmol) in tetrahydrofuran (THF) (30.0 mL) was added dropwise and the mixture was stirred for 40 minutes. tert-Butyl 4-(2-iodoethyl)piperidine-1-carboxylate (22.74 g, 67.0 mmol) in tetrahydrofuran (THF) (30 mL) was added dropwise and the mixture was stirred at −78° C. for 30 minutes. The mixture was warmed to 0° C. over 1.5 hours and stirred at 0° C. for 40 minutes. Saturated NH 4 Aqueous Cl (250 mL) and water (150 mL) were added and the aqueous layer was extracted with ethyl acetate (twice). The combined organic phases were washed with saturated NaHCO 3 aqueous solution, washed with brine, and MgSO 4 The mixture was dried at 70° C., filtered, and the filtrate was concentrated. The residue was purified in two portions by ISCO Combiflash® chromatography (330 g silica Gold cartridge) (eluting with a stepwise gradient of 0-15% (15 min), 15% (6 min), and 15-50% (10 min) ethyl acetate in heptane) to give the title compound as a light brown oil (19.75 g, 45.0 mmol, 70.4% yield). LCMS (m / z) 431.4 (M+H). + .

[0218] Step 3: (1R,4S)-1-(2-(1-(tert-butoxycarbonyl)piperidin-4-yl)ethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid. [ka]

[0219] To a suspension of tert-butyl 4-(2-((1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-(methoxycarbonyl)cyclopent-2-en-1-yl)ethyl)piperidine-1-carboxylate (21.05 g, 48.9 mmol) in methanol (120 mL) and water (100 mL) was added 5 M sodium hydroxide (98 mL, 489 mmol) and the mixture was stirred at 60° C. for 1.5 h. Methanol was added (20 mL) and the mixture was stirred at 80° C. for 1 h. The mixture was cooled to room temperature, poured into water (500 mL) and the pH was adjusted to 3 with 6N HCl. The precipitate was collected by filtration, washed with water (5 times) and dried in a vacuum oven at 45° C. for 2 days to give the title compound as a light brown solid (19.26 g, 43.9 mmol, 90% yield). LCMS (m / z) 417.5 (M+H) + .

[0220] Step 4: tert-Butyl 4-(2-((1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)piperidine-1-carboxylate. [ka]

[0221] To a solution of (1R,4S)-1-(2-(1-(tert-butoxycarbonyl)piperidin-4-yl)ethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid (4.0 g, 9.03 mmol) in N,N-dimethylacetamide (DMA) (19 mL), HATU (3.78 g, 9.93 mmol) and DIPEA (6.31 mL, 36.1 mmol) were added dropwise. The mixture was stirred at room temperature for 15 min, and commercially available 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine, dihydrochloride (2.61 g, 9.48 mmol) was added, and the mixture was stirred at room temperature for 30 min. The mixture was slowly added to ice water (150 mL) and saturated NHCl 4(30 mL) was added and the precipitate was collected by filtration. The precipitate was washed with water and dried under vacuum to give the title compound (7.23 g, 9.03 mmol, 100% yield). LCMS (m / z) 601.2 (M+H). + .

[0222] Step 5: tert-Butyl 4-(2-((1R,4S)-4-amino-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)piperidine-1-carboxylate. [ka]

[0223] To a solution of tert-butyl 4-(2-((1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)piperidine-1-carboxylate (7.23 g, 9.03 mmol) in methanol (60 mL) under nitrogen atmosphere, hydroxylamine hydrochloride (3.76 g, 54.2 mmol), hydroxylamine (50 wt % in water) (3.60 mL, 58.7 mmol), and water (30.0 mL) were added and the suspension was stirred at 95° C. for 30 minutes. Methanol (30 mL) was added and the mixture was stirred at 95° C. for 18 hours. The mixture was cooled to room temperature and concentrated to remove methanol. Water was added (50 mL) and the pH was adjusted to 11 with 1.0 N sodium hydroxide. The aqueous phase was extracted with ethyl acetate (3 times) and the combined extracts were washed with brine and MgSO 4 The mixture was dried at 4° C., filtered, and the filtrate was concentrated to give the title compound (6.29 g, 9.03 mmol, 100% yield). LCMS (m / z) 523.4 (M+H). + .

[0224] Step 6: tert-Butyl 4-(2-((1R,4S)-4-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)piperidine-1-carboxylate. [ka]

[0225] To a solution of tert-butyl 4-(2-((1R,4S)-4-amino-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)piperidine-1-carboxylate (5.80 g, 11.09 mmol) in isopropyl acetate (55.5 mL) was added 2-propanol (2.56 mL, 33.3 mmol) and sodium triacetoxyborohydride (7.05 g, 33.3 mmol) under nitrogen atmosphere at 0° C., and the mixture was stirred at 0° C. for 40 minutes. (R)-3-Methoxytetrahydro-4H-pyran-4-one (Intermediate 2) (1.804 g, 13.86 mmol) in isopropyl acetate (5 mL) was added, and the mixture was stirred at 0° C. for 1.5 hours. Saturated NaHCO 3 Aqueous solution (30 mL) and water (50 mL) were added, the pH was adjusted to 10 with 1N sodium hydroxide (75 mL) and the aqueous phase was extracted with ethyl acetate (2 times). The combined organic extracts were washed with saturated NaHCO 3 (100 mL), water (100 mL), brine (100 mL), and MgSO 4 The mixture was dried at 75° C., filtered, and the filtrate was concentrated. Purification by ISCO Combiflash® Rf chromatography (220 g silica Gold column) (eluting with a stepwise gradient of 0-5%, 5%, and 5-20% methanol in dichloromethane (DCM)) afforded the title compound as a light beige solid (4.75 g, 6.94 mmol, 62.6% yield). LCMS (m / z) 637.2 (M+H). + .

[0226] Step 7: tert-Butyl 4-(2-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)ethyl)piperidine-1-carboxylate. [ka]

[0227] A mixture of tert-butyl 4-(2-((1R,4S)-4-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)piperidine-1-carboxylate (1.64 g, 2.58 mmol) and Pd / C (0.548 g, 0.258 mmol) (wet) in methanol (13.0 mL) was stirred under a balloon atmosphere of hydrogen at room temperature for 90 minutes and 18 hours. The mixture was filtered through Celite®, the catalyst was washed with methanol, and the combined filtrate was concentrated under reduced pressure to give the title compound as a pale beige solid (1.653 g, 2.381 mmol, 92% yield). LCMS (m / z) 639.3 (M+H) + .

[0228] Step 8: ((1S,3R)-3-(((3S,4S)-3-Methoxytetrahydro-2H-pyran-4-yl)amino)-1-(2-(piperidin-4-yl)ethyl)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, trihydrochloride salt. [ka]

[0229] To a solution of tert-butyl 4-(2-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)ethyl)piperidine-1-carboxylate (1.58 g, 2.473 mmol) in dichloromethane (DCM) (12.37 mL) was added 4M HCl (5.00 mL, 20.00 mmol) slowly. The mixture was stirred at room temperature for 1 h and concentrated under reduced pressure to give the title compound as a light brown solid (1.73 g, 2.483 mmol, 100% yield). LCMS (m / z) 539.4 (M+H) + .

[0230] The following intermediates were or can be prepared using procedures similar to those described for Intermediate 4. [Table 3-1] [Table 3-2]

[0231] Intermediate 8: ((1R,3R)-1-(2-aminoethyl)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, trihydrochloride salt. [ka] [ka]

[0232] Step 1: Methyl (1S,4S)-1-(2-bromoethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate. [ka]

[0233] To a 1M solution of lithium bis(trimethylsilyl)amide (LiHMDS) in (tetrahydrofuran)THF (25 mL, 25.00 mmol) at -78°C was added dropwise a solution of methyl (1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate (Intermediate 1) (4.39 g, 20 mmol) in tetrahydrofuran (THF) (20.00 mL). The mixture was stirred at -78°C for 1 h and added dropwise to a solution of 1,2-dibromoethane (5.17 mL, 60.0 mmol) in tetrahydrofuran (THF) (20.00 mL) at -78°C. The mixture was stirred at -78°C for 30 min, warmed to 0°C over 3.5 h and stirred at 0°C for 60 min. Saturated NH 4 Aqueous Cl (100 mL) and ethyl acetate were added and the aqueous phase was extracted with ethyl acetate (3 times). The combined organic phase was washed with saturated NaHCO 3 Washed once with aqueous solution, once with brine, and then with MgSO 4 The mixture was dried at 4° C., filtered, and the filtrate was concentrated to give the title compound as a brown oil (8.09 g, 11.66 mmol, 58.3% yield). LCMS (m / z) 326.1 (M+H). + .

[0234] Step 2: Methyl (1R,4S)-1-(2-(bis(tert-butoxycarbonyl)amino)ethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate. [ka]

[0235] A solution of methyl (1S,4S)-1-(2-bromoethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate (8.09 g, 11.66 mmol) in N,N-dimethylformamide (DMF) (100 mL) was added with di-tert-butyliminodicarboxylate (10.86 g, 50 mmol) and Cs 2 CO3 (20.36 g, 62.5 mmol) was added. The mixture was heated to 80° C. for 2 h. The mixture was cooled to room temperature, water was added and the aqueous phase was extracted with diethyl ether (5 times). The combined organic extracts were concentrated. Purification by ISCO CombiFlash® chromatography (120 g silica Gold column, 150 mL / min) (eluting with a gradient of 0-100% ethyl acetate in heptane) gave the title compound (2.65 g, 1.375 mmol, 11.80% yield). LCMS (m / z) 463.4 (M+H). + .

[0236] Step 3: (1R,4S)-1-(2-((tert-butoxycarbonyl)amino)ethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid [ka]

[0237] To a solution of methyl (1R,4S)-1-(4-((bis-tert-butoxycarbonyl)amino)ethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate (2.65 g, 1.375 mmol) in methanol (10.31 mL) and water (3.44 mL) was added 5 M aqueous sodium hydroxide (5.50 mL, 27.5 mmol). The mixture was stirred at 90° C. for 2 h and concentrated under a steady stream of nitrogen. Water (150 mL) was added. The mixture was cooled in an ice bath and adjusted to pH 4 with 1N HCl. The white solid precipitate was collected by filtration, washed with cold water (100 mL), hexane (100 mL), and chased with toluene (3 times) to give the title compound as an orange solid (797 mg, 0.549 mmol, 39.9% yield). LCMS (m / z) 349.3 (M+H) + .

[0238] Step 4: tert-Butyl (2-((1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)carbamate [ka]

[0239] A mixture of (1R,4S)-1-(2-((tert-butoxycarbonyl)amino)ethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid (1.996 g, 1.375 mmol) and HATU (0.784 g, 2.063 mmol) under nitrogen atmosphere was dissolved in N,N-dimethylacetamide (DMA) (13.75 mL). DIPEA (0.961 mL, 5.50 mmol) was added dropwise and the mixture was stirred at room temperature for 15 min. Commercially available 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine, dihydrochloride (0.416 g, 1.513 mmol) was added and the mixture was stirred at room temperature for 30 min. Ice water (30 mL) and saturated NH 4 Aqueous Cl (30 mL) was added. The precipitate was collected by filtration and washed with water, saturated NaHCO 3 Washing with aqueous solution and water gave the title compound as a brown solid (3.85 g, 1.375 mmol, 100% yield). LCMS (m / z) 455.4 (M+H) + .

[0240] Step 5: tert-Butyl (2-((1R,4S)-4-amino-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)carbamate [ka]

[0241] To a solution of tert-butyl (2-((1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)carbamate (732 mg, 1.375 mmol) in methanol (10 mL) was added hydroxylamine hydrochloride (562 mg, 8.09 mmol), hydroxylamine (50 wt % in water) (0.550 mL, 9.14 mmol), and water (5.00 mL) and the mixture was stirred at 75° C. for 18.5 hours. The mixture was cooled to room temperature and concentrated. Purification by CombiFlash® EZ Prep chromatography (C18 100 g Gold column, 60 mL / min) (eluting with a gradient of 10-50% acetonitrile in water containing formic acid (0.1%)) afforded the title compound as a yellow oil (266.5 mg, 0.516 mmol, 37.5% yield). LCMS (m / z) 455.4 (M+H). + .

[0242] Step 6: tert-Butyl (2-((1R,4S)-4-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)carbamate [ka]

[0243] To a mixture of tert-butyl (2-((1R,4S)-4-amino-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)carbamate (0.2665 g, 0.488 mmol), isopropyl acetate (10 mL), triethylamine (0.204 mL, 1.463 mmol), 2-propanol (0.113 mL, 1.463 mmol) at 5° C. was added sodium triacetoxyborohydride (0.310 g, 1.463 mmol) and the mixture was stirred for 1 h. The reaction was cooled to 1° C. and (R)-3-methoxytetrahydro-4H-pyran-4-one (0.105 g, 0.807 mmol) in isopropyl acetate (1 mL) was added and the mixture was stirred for 1 h. Saturated NaHCO 3 Aqueous solution (5 mL), water (10 mL), and ethyl acetate (10 mL) were added. The aqueous phase was extracted with ethyl acetate (5 times). The combined organic phase was washed with 1 M sodium hydroxide (1 time), brine (1 time) and diluted with Na 2 SO 4 The mixture was dried at 40° C., filtered, and the filtrate was concentrated. The residue was placed under high vacuum overnight to give the title compound as a tan solid (257.6 mg, 0.367 mmol, 75% yield). LCMS (m / z) 569.5 (M+H). + .

[0244] Step 7: tert-Butyl (2-((1R,4S)-4-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)carbamate [ka]

[0245] To a solution of tert-butyl (2-((1R,4S)-4-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopent-2-en-1-yl)ethyl)carbamate (0.2576 g, 0.453 mmol) in methanol (4.53 mL) was added 10% Pd / C (0.121 g, 0.113 mmol) and the mixture was stirred under hydrogen atmosphere at room temperature for 1 h. The reaction was filtered through Celite®, washed with methanol and the filtrate was concentrated to give the title compound as a white solid (0.1493 g, 0.235 mmol, 52.0% yield). LCMS (m / z) 571.5 (M+H) + .

[0246] Step 8: ((1R,3R)-1-(2-aminoethyl)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, trihydrochloride [ka]

[0247] To a mixture of tert-butyl (2-((1R,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)ethyl)carbamate (0.114 g, 0.200 mmol) in dichloromethane (DCM) (2.000 mL) was added 4M HCl in 1,4-dioxane (1 mL, 4.00 mmol). The mixture was stirred at room temperature for 15 minutes. Methanol (1 mL) was added and the mixture was concentrated under a steady stream of nitrogen to give the title compound as a white solid (0.147 g, 0.2 mmol, 100% yield). LCMS (m / z) 471.4 (M+H) + .

[0248] Intermediate 9: ((1S,3R)-1-((2-aminoethoxy)methyl)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, triacetate [ka] [ka]

[0249] Step 1: Methyl (1R,4S)-1-((benzyloxy)methyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate [ka]

[0250] To a 1M solution of LHMDS in tetrahydrofuran (THF) (25 mL, 25.00 mmol) at -78°C, a solution of methyl (1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate (4.39 g, 20 mmol) in 2-methyltetrahydrofuran (2-MeTHF) (20.00 mL) was added dropwise and the mixture was stirred at -78°C for 1 h. A solution of commercially available ((chloromethoxy)methyl)benzene (5.56 mL, 40.0 mmol) in 2-methyltetrahydrofuran (2-MeTHF) (20.00 mL) was added dropwise and the mixture was stirred at -78°C for 30 min. The mixture was warmed to 0°C over 3.5 h and stirred at 0°C for 60 min. Saturated NH 4 Aqueous Cl (100 mL) and ethyl acetate were added and the aqueous phase was extracted with ethyl acetate (3 times). The combined organic extracts were washed with saturated NaHCO 3 Washed once with aqueous solution, once with brine, and then with MgSO 4The mixture was dried at 70° C., filtered, and the filtrate was concentrated. Purification by ISCO CombiFlash® chromatography (120 g silica Gold column, 85 mL / min) (eluting with a gradient of 0-20% ethyl acetate in heptane) afforded the title compound as a colorless oil (4.64 g, 12.44 mmol, 62.2% yield). LCMS (m / z) 340.3 (M+H). + .

[0251] Step 2: (1R,4S)-1-((benzyloxy)methyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid [ka]

[0252] To a mixture of methyl (1R,4S)-1-((benzyloxy)methyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate (4.64 g, 13.67 mmol) in methanol (34.2 mL) and water (11.39 mL) was added 5 M sodium hydroxide (27.3 mL, 137 mmol) and the mixture was heated at 90° C. for 1.5 h. The mixture was concentrated under a steady stream of nitrogen. Water (200 mL) was added and the mixture was placed in an ice bath. The pH was adjusted to 4 with 1N HCl. The precipitate was collected by filtration and dried under vacuum to give the title compound as an orange solid (3.48 g, 9.84 mmol, 72.0% yield). LCMS (m / z) 326.3 (M+H). + .

[0253] Step 3: ((1R,4S)-1-((benzyloxy)methyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone [ka]

[0254] To a solution of (1R,4S)-1-((benzyloxy)methyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid (3.48 g, 10.69 mmol) and HATU (4.47 g, 11.76 mmol) in N,N-dimethylacetamide (DMA) (26.7 mL) under nitrogen atmosphere, DIPEA (7.47 g, 42.8 mmol) was added dropwise and the mixture was stirred at room temperature for 15 minutes. Commercially available 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine, dihydrochloride (2.94 g, 10.69 mmol) was added and the mixture was stirred at room temperature for 30 minutes. The mixture was diluted with water and saturated NH 4 Poured into a solution of aqueous Cl (1:1) at 0° C. The precipitate was collected by filtration and washed with water to give the title compound as a red-tan wet solid (6.05 g, 10.69 mmol, 100% yield). LCMS (m / z) 510.4 (M+H). + .

[0255] Step 4: ((1R,4S)-1-((benzyloxy)methyl)-4-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone [ka]

[0256] A mixture of ((1R,4S)-1-((benzyloxy)methyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone (5.45 g, 10.69 mmol), methanol (71.3 mL), hydroxylamine hydrochloride (4.37 g, 62.9 mmol), hydroxylamine (50 wt % in water) (4.28 mL, 71.1 mmol), and water (35.6 mL) was stirred at 75° C. for 22.5 h. The mixture was cooled to room temperature, diluted with water, the pH was adjusted to 11 with 1 M sodium hydroxide, and the aqueous phase was extracted with ethyl acetate (5 times). The combined organic extracts were washed with brine and concentrated under reduced pressure. The residue was chased with dichloromethane (DCM) to give the title compound as a tan solid (6.15 g, 7.84 mmol, 73.3% yield). LCMS (m / z) 432.4 (M+H). + .

[0257] Step 5: ((1R,4S)-1-((benzyloxy)methyl)-4-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone [ka]

[0258] To a mixture of ((1R,4S)-4-amino-1-((benzyloxy)methyl)cyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone (2.08 g, 4.82 mmol), isopropyl acetate (48.2 mL), and 2-propanol (1.114 mL, 14.46 mmol) at 0° C., sodium triacetoxyborohydride (3.07 g, 14.46 mmol) was added and the mixture was stirred for 1 h. (R)-3-Methoxytetrahydro-4H-pyran-4-one (0.941 g, 7.23 mmol) in isopropyl acetate (6 mL) was added and the mixture was stirred at 0° C. for 1 h. Saturated NaHCO 3 Aqueous solution (20 mL), water (30 mL), and ethyl acetate (20 mL) were added and the aqueous phase was extracted with ethyl acetate (5 times). The combined organic extracts were washed with 1 M sodium hydroxide (1 time), brine (1 time) and diluted with Na 2 SO 4 The mixture was dried at 40° C., filtered, and the filtrate was concentrated under reduced pressure to give the title compound as a brown solid (3.46 g, 4.82 mmol, 100% yield). LCMS (m / z) 546.3 (M+H). + .

[0259] Step 6: ((1S,3R)-1-((benzyloxy)methyl)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone. [ka]

[0260] A solution of ((1R,4S)-1-((benzyloxy)methyl)-4-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone (2.63 g, 4.82 mmol) and Pd / C (0.513 g, 0.482 mmol) in methanol (48.2 mL) was stirred under hydrogen atmosphere at room temperature for 2.5 days. The mixture was filtered through Celite®, the catalyst was washed with methanol, and the combined filtrate was concentrated to give the title compound as a yellow solid (1.674 g, 2.109 mmol, 43.8% yield). LCMS (m / z) 548.3 (M+H) + .

[0261] Step 7: ((1S,3R)-1-(hydroxymethyl)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone. [ka]

[0262] To a solution of ((1S,3R)-1-((benzyloxy)methyl)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone (1.479 g, 2.7 mmol) in methanol (26 mL), Pd(OH) 2(2.085 g, 2.97 mmol) was added, hydrogen gas was bubbled through the mixture for 5 min, and the mixture was stirred at room temperature for 1 h. Acetic acid (3.25 mL) was added, and the mixture was stirred under a hydrogen atmosphere for 5.5 h. The mixture was opened to the atmosphere for 15 min and filtered through Celite® with methanol. This solution was combined with a similar reaction starting with ((1S,3R)-1-((benzyloxy)methyl)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone (0.290 g, 0.530 mmol). Purification by ISCO CombiFlash® chromatography (24 g silica Gold column, 35 mL / min) (eluting with a gradient of 0-100% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM)) afforded the title compound as a white solid (265 mg, 0.441 mmol, 16.32% yield). LCMS (m / z) 458.4 (M+H). + .

[0263] Step 8: 2-(((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)methoxy)acetonitrile [ka]

[0264] A mixture of sodium hydride (60% in mineral oil) (0.060 g, 1.500 mmol) and ((1S,3R)-1-(hydroxymethyl)-3-(((4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone (0.107 g, 0.234 mmol) in tetrahydrofuran (THF) (2.339 mL) was stirred at 0° C. for 10 min. Commercially available 2-bromoacetonitrile (0.050 mL, 0.718 mmol) was added dropwise and the mixture was stirred at 0° C. for 30 min. Saturated NH 4 Aqueous Cl (10 mL) and dichloromethane (DCM) were added and the organic phase was concentrated under a steady stream of nitrogen. Purification by ISCO CombiFlash® chromatography (12 g silica Gold column, 30 mL / min) (eluting with a gradient of 0-100% methanol in dichloromethane (DCM)) afforded the title compound as a brown oil (51 mg, 0.075 mmol, 32.1% yield). LCMS (m / z) 497.7 (M+H). + .

[0265] Step 9: ((1S,3R)-1-((2-aminoethoxy)methyl)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, triacetate. [ka]

[0266] To a solution of 2-(((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)methoxy)acetonitrile (0.250 g, 0.503 mmol) in isopropanol (4 mL) and acetic acid (2.000 mL) was added Pd / C (0.268 g, 0.252 mmol) and the reaction was stirred under a hydrogen atmosphere for 1 h. The mixture was stirred open to air for 10 min. This mixture was combined with a similar reaction starting with 2-(((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)methoxy)acetonitrile (0.050 g, 0.100 mmol). The combined mixture was filtered through Celite® and concentrated to give the title compound as an off-white solid (314 mg, 0.302 mmol, 100% yield). LCMS (m / z) 501.4 (M+H). + .

[0267] Intermediate 10: (1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexan-1-ol [ka]

[0268] A mixture of 5N sodium hydroxide (67.5 mL, 338 mmol), solid sodium hydroxide (13.50 g, 338 mmol), 1,4-dioxane (100 mL), toluene (50 mL), tetrabutylammonium iodide (1.247 g, 3.38 mmol), commercially available (1r,4r)-cyclohexane-1,4-diol (5.88 g, 50.6 mmol), and commercially available N,N-dibenzyl-2-chloroethan-1-amine hydrochloride (10 g, 33.8 mmol) was stirred at 55° C. for 19.5 h, at 60° C. for 24.5 h, and at 65° C. for 24 h. NH 4Cl (10 g) was added and the mixture was extracted with ethyl acetate (3×150 mL). The combined organic extracts were washed with brine (100 mL) and MgSO 4 The mixture was dried at 75° C., filtered, and the filtrate was concentrated under reduced pressure. Dichloromethane (DCM) (100 mL) was added, the mixture was filtered, and the filtrate was purified by ISCO CombiFlash® chromatography (120 g Gold column, 85 mL / min) (eluting with a gradient of 30-70% ethyl acetate in heptane) to give the title compound (5.8227 g, 17.15 mmol, 50.8% yield). LCMS (m / z) 340.4 (M+H). + .

[0269] Intermediate 11: tert-butyl (4-(6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butyl)carbamate. [ka]

[0270] Step 1: 3-(trifluoromethyl)-1,6-naphthyridin-2-ol. [ka]

[0271] A solution of 4-aminonicotinaldehyde (5.1 g, 41.8 mmol) and DIPEA (10.94 mL, 62.6 mmol) in dichloromethane (DCM) (100 mL) was added dropwise over 20 min to a solution of 3,3,3-trifluoropropanoyl chloride (9.18 g, 62.6 mmol) in dichloromethane (DCM) (200 mL) at 20° C. The mixture was stirred for 1.5 h and saturated NaHCO 3 Aqueous solution (200 mL) was added and the mixture was stirred vigorously at room temperature for 5 min. The organic phase was diluted with Na 2 SO 4The residue was dissolved in N,N-dimethylformamide (DMF) (100 mL) and diluted with K 2 CO 3 (5.77 g, 41.8 mmol) was added and the mixture was heated to 70° C. for 15 min and cooled to room temperature. Water (100 mL) was added followed by slow addition of 1.0 M hydrochloric acid (84 mL, 84 mmol). The mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. Water (200 mL) was added to the residue. The resulting precipitate was collected by filtration, rinsed with water and air-dried to give the title compound as a deep yellow solid (7.24 g, 33.8 mmol, 81% yield). LC-MS m / z 215.1 (M+H) + .

[0272] Step 2: 6-Benzyl-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ol. [ka]

[0273] To a suspension of 3-(trifluoromethyl)-1,6-naphthyridin-2-ol (6.66 g, 31.1 mmol) in acetonitrile (50 mL) was added benzyl bromide (4.07 mL, 34.2 mmol). The mixture was refluxed for 2 h and concentrated under reduced pressure. The residue was dissolved in methanol (200 mL) and water (60 mL) was added. The flask was purged with nitrogen and sodium borohydride (5.88 g, 156 mmol) was added portionwise over 30 min and the mixture was stirred at room temperature under nitrogen for 1 h. Methanol (200 mL) was added followed by sodium borohydride (5.88 g, 156 mmol) over 30 min. The reaction mixture was stirred under nitrogen for 30 min and additional sodium borohydride (5.88 g, 156 mmol) was added portionwise over 30 min. The reaction mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. The residue was suspended in water (300 mL) and the resulting precipitate was collected by filtration. Purification by ISCO CombiFlash® (120 g RediSep Gold® column, 85 mL / min) (eluting with a gradient of 0-75% dichloromethane (DCM):methanol (4:1) in dichloromethane (DCM)) afforded the title compound as a yellow-orange solid (3.75 g, 12.16 mmol, 39% yield). LC-MS m / z 309.3 (M+H) + .

[0274] Step 3: tert-Butyl (4-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butyl)carbamate. [ka]

[0275] To a solution of 6-benzyl-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ol (3.75 g, 12.16 mmol) in dichloromethane (DCM) (200 mL) at 0° C., a solution of trifluoromethanesulfonic anhydride (2.260 mL, 13.38 mmol) in dichloromethane (DCM) (20 mL) was added dropwise over 5 min. The mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. Bis(triphenylphosphine)palladium(II) chloride (0.171 g, 0.243 mmol) and copper(I) iodide (0.069 g, 0.365 mmol) were added to the residue. The mixture was purged with nitrogen and degassed N,N-dimethylformamide (DMF) (100 mL), tert-butyl bu-3-yn-1-ylcarbamate (3.09 g, 18.24 mmol), and DIPEA (6.37 mL, 36.5 mmol) were added sequentially. The mixture was stirred at 80° C. for 15 h and concentrated under reduced pressure. 20% palladium hydroxide on carbon (8.54 g, 12.16 mmol) and methanol (100 mL) were added to the residue, hydrogen was bubbled through the mixture for 10 min, and the mixture was stirred for 5.5 h. The mixture was stirred open to the atmosphere for 15 min and filtered. The catalyst was removed using 2M NH 3 (100 mL) and methanol (100 mL), and the combined filtrate was concentrated under reduced pressure. The residue was partitioned between dichloromethane (DCM) (200 mL) and 1.0 M NaOH (100 mL), and the aqueous layer was extracted with dichloromethane (DCM) (200 mL). The combined organic extracts were washed with Na 2 SO 4 The mixture was dried at 40° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by ISCO CombiFlash® (220 g RediSep Gold® column, 150 mL / min) eluted with 5% methanol (containing ammonium hydroxide) in dichloromethane (DCM) afforded the title compound as a pale yellow oil (3.0 g, 8.03 mmol, 66% yield). LC-MS m / z 374.1 (M+H) + .

[0276] Step 4: Benzyl ((1R,3S)-3-(2-(4-((tert-butoxycarbonyl)amino)butyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)-3-isopropylcyclopentyl)carbamate. [ka]

[0277] To (1S,3R)-3-(((benzyloxy)carbonyl)amino)-1-isopropylcyclopentane-1-carboxylic acid (2.62 g, 8.57 mmol) (dried by azeotroping with toluene prior to use), dichloromethane (DCM) (15 mL), HATU (1.751 g, 4.61 mmol), and DIPEA (5.99 mL, 34.3 mmol) were added sequentially and the mixture was stirred at room temperature for 3 h. A solution of tert-butyl (4-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butyl)carbamate (1.60 g, 4.28 mmol) in dichloromethane (DCM) (7.50 mL) was added dropwise and the mixture was stirred at room temperature for 67 h. The reaction mixture was divided equally between two 20 mL sealed tubes and heated at 80° C. for 25 h. The mixtures were combined, dichloromethane (DCM) (100 mL), water (50 mL), and 1.0 M NaOH (50 mL) were added, and the mixture was stirred vigorously for 5 min. The organic phase was washed with Na 2 SO 4 The mixture was dried at 75° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by ISCO CombiFlash® (80 g RediSep Gold® column, 22 mL / min) (eluting with a gradient of 0-22% ethyl acetate:ethanol (3:1) in hexanes) afforded the title compound as a pale yellow foam (2.61 g, 3.95 mmol, 92% yield). LC-MS m / z 661.4 (M+H). + .

[0278] Step 5: tert-Butyl (4-(6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butyl)carbamate. [ka]

[0279] A mixture of benzyl ((1R,3S)-3-(2-(4-((tert-butoxycarbonyl)amino)butyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)-3-isopropylcyclopentyl)carbamate (2.61 g, 3.95 mmol) and 20% palladium hydroxide on carbon (0.832 g, 1.185 mmol) in methanol (100 mL) was purged with nitrogen and hydrogen. Hydrogen was bubbled through the mixture for 2 minutes and the reaction mixture was stirred under a hydrogen balloon atmosphere for 30 minutes. The mixture was opened to the atmosphere and 7.0 M ammonia in methanol (50 mL) was added and stirred for 15 minutes. The mixture was filtered, the catalyst washed with methanol and the combined filtrates were concentrated under reduced pressure. Isopropyl acetate (50 mL), 2-propanol (0.761 mL, 9.87 mmol), and triethylamine (0.551 mL, 3.95 mmol) were added to the residue, the mixture was cooled to 0° C., sodium triacetoxyborohydride (1.842 g, 8.69 mmol) was added, followed by dropwise addition of a solution of (R)-3-methoxytetrahydro-4H-pyran-4-one (0.900 g, 6.91 mmol) in isopropyl acetate (20 mL). The mixture was stirred at 0° C. for 10 min, the cold bath was removed, and the mixture was stirred at room temperature for 1 h. Ethyl acetate was added and the organic phase was washed with 1.0 M NaOH. The aqueous layer was extracted with ethyl acetate and the combined organic extracts were washed with Na 2 SO 4The mixture was dried at 40° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by ISCO CombiFlash® (120 g RediSep Gold® column, 85 mL / min) (eluting with a gradient of 0-2% methanol in dichloromethane (DCM)) afforded the title compound as a foam. LC-MS m / z 641.2 (M+H) + .

[0280] Intermediate 12: (2-(4-aminobutyl)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)methanone, dihydrochloride, 1,4-dioxane [ka]

[0281] To a mixture of tert-butyl (4-(6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butyl)carbamate (210 mg, 0.328 mmol) in 1,4-dioxane (3 mL) was added HCl (4M in p-dioxane) (0.819 mL, 3.28 mmol). The resulting reaction mixture was stirred at ambient temperature for 3 h. The reaction mixture was evaporated to dryness under vacuum to give the title product (223.2 mg, 0.318 mmol, 97% yield). LC / MS (M+H) + 541.4 m / z. Intermediate 13: 3-(4-(6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)benzoic acid. [ka]

[0282] Step 1: Benzyl 3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate. [ka]

[0283] A mixture of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine, dihydrochloride (1000 mg, 3.64 mmol) and DIPEA (1.905 mL, 10.91 mmol) in N,N-dimethylformamide (DMF) (20 mL) was stirred at room temperature under nitrogen for 5 min and benzyl chloroformate (0.519 mL, 3.64 mmol) was added dropwise over 5 min. The mixture was stirred at room temperature for 3 h and water was added. The organic phase was washed with brine (2×30 mL). The aqueous phase was extracted with ethyl acetate (2×30 mL). The combined organic extracts were diluted with MgSO 4 The mixture was dried at 70° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by normal phase silica gel chromatography (40 g RediSep Gold® column) (eluted with a stepwise gradient of 30% and 50% ethyl acetate in hexanes) afforded the title compound as a yellow waxy solid (1100 mg, 3.27 mmol, 90% yield). LC-MS m / z 337.1 (M+H). + .

[0284] Step 2: Benzyl 2-(4-hydroxybutyl)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate. [ka]

[0285] Benzyl 3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (2.5 g, 7.43 mmol), [4,4'-bis(1,1-dimethylethyl)-2,2'-bipyridine-N1,N1']bis[3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridinyl-N]phenyl-C]iridium(III) hexafluorophosphate, [Ir{ To a mixture of dF(CF3)ppy}2(dtbpy)]PF6 (0.125 g, 0.112 mmol), 6 M HCl (1.363 mL, 8.18 mmol), and trifluoroacetic acid (4.01 mL, 52.0 mmol) in methanol (9 mL) was added benzoyl peroxide, Luperox® A75 (5.40 g, 16.73 mmol), and tetrahydrofuran (THF) (45.0 mL).

[0286] The mixture was wrapped in aluminum foil and irradiated with a 1 inch H150-Blue Kessil lamp. House air was blown into the reaction vessel and the mixture was stirred at room temperature for 48 hours. The light source was removed and the mixture was stirred overnight. The mixture was cooled in an ice bath and saturated NaHCO 3 Aqueous solution (100 mL) was added. The aqueous phase was extracted with ethyl acetate. The organic extracts were washed with brine (50 mL). The aqueous phase was extracted again with ethyl acetate and the combined organic extracts were washed with Na 2 SO 4 The mixture was dried at 4° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by ISCO CombiFlash® (120 g RediSep Gold® column) (eluted with a stepwise gradient of 30-50% and 75-100% ethyl acetate in hexanes) afforded the title compound as a partially solidified light orange oil (1780 mg, 4.361 mmol, 58.7% yield). LC-MS m / z 409.2 (M+H). + .

[0287] Step 3: Benzyl 2-(4-(3-(methoxycarbonyl)phenoxy)butyl)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate. [ka]

[0288] To a solution of benzyl 2-(4-hydroxybutyl)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (1780 mg, 4.36 mmol), methyl 3-hydroxybenzoate (995 mg, 6.54 mmol), and triphenylphosphine (1715 mg, 6.54 mmol) in tetrahydrofuran (THF) (40 mL) was added diisopropyl (E)-diazene-1,2-dicarboxylate (1.271 mL, 6.54 mmol). The mixture was stirred overnight and concentrated. Ethyl acetate was added and the organic phase was washed with water and brine. The organic phase was diluted with MgSO 4 The mixture was dried at 40° C. and concentrated. Purification by ISCO CombiFlash® (80 g RediSep Gold® column) (eluted with a gradient of 10-50% ethyl acetate in heptane) afforded the title compound (1580 mg, 2.91 mmol, 66.8% yield). LC-MS m / z 543.2 (M+H) + .

[0289] Step 4: Methyl 3-(4-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)benzoate. [ka]

[0290] 33% HBr in acetic acid (2.64 mL, 14.56 mmol) was added to a solution of benzyl 2-(4-(3-(methoxycarbonyl)phenoxy)butyl)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (1580 mg, 2.91 mmol) in dichloromethane (DCM) (20 mL) and the mixture was stirred at room temperature for 2 h and concentrated. Purification by ISCO CombiFlash® (40 g RediSep Gold® column) (eluted with a gradient of 30-70% methanol in dichloromethane (DCM) containing ammonium hydroxide (1%)) gave the title compound (1150 mg, 2.017 mmol, 69.3% yield). LC-MS m / z 409.2 (M+H) + .

[0291] Step 5: Methyl 3-(4-(6-((1S,3R)-3-((tert-butoxycarbonyl)amino)-1-isopropylcyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)benzoate. [ka]

[0292] To a mixture of methyl 3-(4-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)benzoate (1150 mg, 2.82 mmol) in dichloromethane (DCM) (2 mL) was added DIPEA (2.459 mL, 14.08 mmol). The solution was stirred for 5 min, then DMAP (275 mg, 2.253 mmol), (1S,3R)-3-((tert-butoxycarbonyl)amino)-1-isopropylcyclopentane-1-carboxylic acid (1375 mg, 5.07 mmol) were added, followed by PyBroP (3282 mg, 7.04 mmol) and the mixture was stirred at room temperature for 4 days. Saturated NaHCO 3 Aqueous solution (15 mL) was added and the aqueous phase was extracted with dichloromethane (DCM) (2×150 mL). The combined organic extracts were washed with MgSO4 The mixture was dried at 40° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by ISCO CombiFlash® (40 g RediSep Gold® column) (eluted with a gradient of 15-70% ethyl acetate in heptane) afforded the title compound (1100 mg, 1.496 mmol, 53.1% yield). LC-MS m / z 662.5 (M+H). + .

[0293] Step 6: Methyl 3-(4-(6-((1S,3R)-3-amino-1-isopropylcyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)benzoate, 2-trifluoroacetate salt. [ka]

[0294] To a solution of methyl 3-(4-(6-((1S,3R)-3-((tert-butoxycarbonyl)amino)-1-isopropylcyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)benzoate (1.14 g, 1.723 mmol) in dichloromethane (DCM) (10 mL) was added trifluoroacetic acid (2.65 mL, 34.5 mmol). The reaction mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. The residue was chased with dichloromethane (DCM) (3×3 mL) and dried under high vacuum to give the title compound as an orange oil (1.35 g, 1.710 mmol, 99% yield). LC-MS m / z 562.3 (M+H) + .

[0295] Step 7: Methyl 3-(4-(6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)benzoate. [ka]

[0296] To a solution of methyl 3-(4-(6-((1S,3R)-3-amino-1-isopropylcyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)benzoate, 2 trifluoroacetate salt (1.34 g, 1.701 mmol) in dichloromethane (DCM) (17 mL) was added DIPEA (0.891 mL, 5.10 mmol) and the mixture was stirred for 5 min. 4 Å powdered activated molecular sieves (ca. 325 mesh, Aldrich) (1 g, 1.701 mmol), (R)-3-methoxytetrahydro-4H-pyran-4-one (0.266 g, 2.041 mmol) were added, followed by sodium triacetoxyhydride (0.541 g, 2.55 mmol) and the mixture was stirred under nitrogen atmosphere at room temperature for 22 h. Dichloromethane (DCM) (20 mL) was added. The organic phase was washed with saturated NaHCO 3 The combined aqueous phase was extracted with dichloromethane (DCM) (20 mL). The combined organic extracts were washed with MgSO 4 The mixture was dried at 75° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by ISCO Lumen CombiFlash® (80 g RediSep Gold® column, 60 mL / min) (eluting with a gradient of 10-90% dichloromethane (DCM):methanol (4:1) in dichloromethane (DCM)) afforded the title compound as a colorless oil (505 mg, 0.747 mmol, 43.9% yield). LC-MS m / z 676.4 (M+H) + .

[0297] Step 8: 3-(4-(6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)benzoic acid. [ka]

[0298] To a solution of methyl 3-(4-(6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)benzoate (496 mg, 0.734 mmol) in ethanol (5 mL) was added a solution of lithium hydroxide (92 mg, 2.202 mmol) in water (5.00 mL). The reaction mixture was stirred at room temperature for 68 h and most of the ethanol was removed under reduced pressure. The resulting aqueous mixture was adjusted to pH 7.2 with 1N HCl (~2.1 mL) and extracted with dichloromethane (DCM) (3 x 20 mL). The combined organic extracts were washed with MgSO 4 The mixture was dried at 40° C., filtered, and the filtrate was concentrated under reduced pressure. The residue was placed under high vacuum to give the title compound as a foam (380 mg, 0.574 mmol, 78% yield). LC-MS m / z 662.4 (M+H). + .

[0299] Intermediate 14: ((1S,3S)-3-((3-aminopropyl)((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-isopropylcyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone [ka]

[0300] Step 1: Methyl (1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-ene-1-carboxylate [ka]

[0301] To a mixture of methyl (1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate (1000 mg, 4.56 mmol) in THF (10 mL) at -20°C was added LHMDS (7.30 mL, 7.30 mmol) in THF dropwise. The resulting mixture was stirred at -20°C for 30 min, then 2-iodopropane (0.912 mL, 9.12 mmol) in THF (10 mL) was added. The mixture was stirred at room temperature for 1 h and then warmed to room temperature until LC-MS indicated consumption of starting material. The reaction was purified by NH 4 The mixture was quenched with a 6% aqueous solution of Cl (50 mL) and diluted with ethyl acetate. The organic layer was separated and then washed with water (1×50 mL), dried over sodium sulfate, and concentrated in vacuo. The crude material was then purified by column chromatography (RediSepRf high performance GOLD 24 g HP Silica, CH as mobile phase). 2 Cl 2 / CH 2 Cl 2 in 20% MeOH) to give methyl (1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-ene-1-carboxylate as a light brown oil in 90% purity (1.06 g, 80% yield). LCMS MS (m / z) 262.2 (M+H). +

[0302] Step 2: (1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-ene-1-carboxylic acid [ka]

[0303] To a 20 mL microwave vial was added methyl (1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-ene-1-carboxylate (2000 mg, 7.65 mmol), methanol (3 mL), and potassium hydroxide (3.06 mL, 30.6 mmol). The resulting mixture was heated at 120° C. for 3 h. LC-MS showed complete formation of the product. MeOH was removed under vacuum. The reaction mixture was then placed in an ice bath. The pH of the solution was adjusted to about 2 with 12 N HCl. A light brown precipitate was difficult to filter. The reaction solution was then extracted with ethyl acetate (2×30 mL). This was then purified by column chromatography (RediSepRf high performance GOLD 24g HP silica, using DCM / 20% MeOH in DCM as mobile phase, gradient 0-100%) to give the desired product (1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-ene-1-carboxylic acid as a light brown solid (1600mg, 5.82mmol, 76% yield). LCMS (m / z) 248.2 (M+H). + .

[0304] Step 3: ((1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone [ka]

[0305] To a 100 mL round bottom flask was added (1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-ene-1-carboxylic acid (1 g, 4.04 mmol) as a red amber syrup. The syrup was dissolved in tetrahydrofuran (THF) (10 mL). To the dark solution was added DIEA (1.412 mL, 8.09 mmol). The reaction mixture was cooled to 0° C. Ms-Cl (0.347 mL, 4.45 mmol) was added dropwise over 2 min. The reaction was allowed to warm to room temperature. It was stirred at room temperature for 4 h. The reaction mixture was cooled in ice for 3 min, then 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine, dihydrochloride (1.008 g, 3.66 mmol) was added. The reaction was allowed to warm gradually to ambient temperature. An additional amount of DIEA (0.706 mL, 4.04 mmol) was added. The reaction was stirred at room temperature for 45 min. The reaction mixture was diluted with 50 mL of 5% NaHCO 3 The two layers were partitioned and the resulting aqueous layer was extracted twice with ethyl acetate. The organic layers were combined and washed with water. The resulting organic solutions were combined and concentrated under vacuum to give a dark brown oil. The oil was pumped overnight and purified by ISCO (10% MeOH / DCM mobile phase) using a gold 40 g silica gel column cartridge. LCMS analysis showed contamination with starting carboxylic acid. The fractions were combined and washed five times with saturated bicarbonate to remove the carboxylic acid starting material. The resulting organic layer was washed with brine, dried over sodium sulfate, then filtered, concentrated and purified using an ISCO, 40 g silica gel column gold with 5% MeOH / DCM mobile phase to give ((1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone. LCMS (m / z) 432.35 (M+H). + .

[0306] Step 4: ((1S,4S)-4-Amino-1-isopropylcyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, dihydrochloride [ka]

[0307] A 50 mL round bottom flask was charged with hydroxylamine hydrochloride (1.025 g, 14.75 mmol) and dissolved in water. To the solution was added hydroxylamine 50% w / w (aqueous) (6.56 mL, 2.508 mmol). ((1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone (1.0823 g, 2.508 mmol) was dissolved in methanol (10.35 mL) and added via pipette to the solution of hydroxylamine. The reaction was refluxed at 71.5° C. for 16 hours. The reaction mixture was cooled to room temperature. The pH was adjusted to 11 using 10 N NaOH. The solution was then diluted with water and transferred to a 250 mL separatory funnel. The aqueous layer was exhaustively extracted 4 times with 10 mL EtOAc. The organic layers were combined and concentrated. The resulting orange oil was purified by HPH reverse phase ISCO to give ((1S,4S)-4-amino-1-isopropylcyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, dihydrochloride salt as an orange oil (.717 g, 1.682 mmol, 67.1% yield). LCMS (m / z) 354.28 (M+H). + .

[0308] Step 5: ((1S,4S)-1-isopropyl-4-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone [ka]

[0309] To a 100 mL round bottom flask was added ((1S,4S)-4-amino-1-isopropylcyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, dihydrochloride (0.350 g, 0.821 mmol) dissolved in isopropyl acetate (3.40 mL). The solution was cooled to 0° C. Then 2-propanol (0.278 ml, 3.61 mmol) was added followed by sodium triacetoxyborohydride (0.766 g, 3.61 mmol). The reaction was stirred at 0° C. for 1 hour and (R)-3-methoxytetrahydro-4H-pyran-4-one (0.46 g, 3.53 mmol) was added as an oil. The reaction was stirred at 0° C. for 2 hours. Upon completion, the reaction was quenched with ammonium chloride, diluted with 5 mL of water and extracted with DCM (2x20 mL). The resulting organic solutions were combined, concentrated and purified by ISCO (using a gradient elution of 0-5% DCM / MeOH). ((1S,4S)-1-isopropyl-4-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone (312 mg, 0.667 mmol, 81% yield) was obtained as an orange solid. LCMS (m / z) 468.44 (M+H). + .

[0310] Step 6: ((1S,4S)-4-((3-(dibenzylamino)propyl)((4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-isopropylcyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone [ka]

[0311] To a 2-5 mL small microwave vial was added ((1S,4S)-1-isopropyl-4-(((4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone (450 mg, 0.962 mmol) dissolved in a solution of methanol (9021 μl) and acetic acid (902 μl). 2-(Dibenzylamino)acetaldehyde (2073 mg, 8.66 mmol) was added as an oil. Alpha-picoline borane (759 mg, 6.74 mmol) was added. The reaction was capped and stirred at room temperature for 16 hours. Upon completion, the reaction was concentrated and suspended in 1N HCl. The suspension was stirred for 30 minutes. This was then basified with 2.5 g of sodium carbonate dissolved in 10 mL of water. The solution was extracted with DCM (3×15 mL), washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude oil was combined with another batch and purified on a 40 g ISCO cartridge eluted with gradient elution (0-20% MeOH / DCM). The desired fractions were combined and concentrated to give ((1S,4S)-4-((3-(dibenzylamino)propyl)((4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-isopropylcyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone as an orange solid (30 mg, 0.043 mmol, 4.42% yield). LCMS (m / z) 705.57 (M+H) + .

[0312] Step 7: ((1S,3S)-3-((3-aminopropyl)((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-isopropylcyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone [ka]

[0313] To a 50 mL round bottom flask was added Pd-C (85 mg, 0.040 mmol). The catalyst was wetted with 0.1 mL of methanol. To the round bottom flask containing the catalyst was added ((1S,4R)-4-((3-(dibenzylamino)propyl)((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-isopropylcyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone (28.2 mg, 0.040 mmol) dissolved in 0.4 mL of methanol. The reaction flask was degassed and exposed to hydrogen gas via a balloon. The reaction was stirred at room temperature for 16 hours. Upon completion, the reaction mixture was passed through a pad of Celite and concentrated to give the product ((1S,3S)-3-((3-aminopropyl)((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-isopropylcyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone as a clear oil (13 mg, 0.025 mmol, 61.7% yield). LCMS (m / z) 527.44 (M+H). + .

[0314] Intermediate 15: 3-(dibenzylamino)propan-1-ol [ka]

[0315] Step 1: 3-(dibenzylamino)propan-1-ol [ka]

[0316] To a 250 mL round bottom flask was added 3-aminopropan-1-ol (5.06 mL, 66.6 mmol) dissolved in acetone (119 mL). To this solution was added (bromomethyl)benzene (16.23 mL, 136 mmol) followed by K2CO3 (19.32 g, 140 mmol). The reaction was stirred at room temperature for 16 h. Upon completion, the reaction mixture was filtered. The solids were washed with acetone. The filtrate was concentrated to give a dark brown oil which was purified by ISCO on a 330 g silica gel cartridge using a 10-50% EtOAc / Heptane mobile phase gradient to give 3-(dibenzylamino)propan-1-ol as a yellow oil (15.016 g, 58.8 mmol, 88% yield). LCMS (m / z) 256.6 (M+H) + .

[0317] Step 2: 3-(dibenzylamino)propanal [ka]

[0318] To an oven dried 100 mL round bottom flask was added oxalyl chloride in DCM (6.31 mL, 3.16 mmol, 0.5 M) at -78 °C and dimethyl sulfoxide (0.466 mL, 6.57 mmol) was added dropwise over 1 min. The reaction was stirred for 15 min. After activation, 3-(dibenzylamino)propan-1-ol (4.90 mL, 1.958 mmol) dissolved in 4.9 mL of DCM was added dropwise over 5 min. The reaction was stirred for 15 min. Monitoring by TLC (30% EtOAc:heptane) indicates complete formation of the sulfur oxygen bond via disappearance of the alcohol spot. To the reaction was added triethylamine (0.718 mL, 5.15 mmol) dropwise over 1 min. The reaction was stirred for 15 min. Upon completion, the reaction was quenched with saturated sodium bicarbonate. The bicarbonate layer was extracted 3 times with DCM. The DCM layers were combined, dried over sodium sulfate, filtered and concentrated to give 3-(dibenzylamino)propanal (412 mg, 1.626 mmol, 83% yield) as crude product. 1 H NMR (400 MHz, DMSO-d6 ) δ ppm 2.55 - 2.65 (m, 2 H) 2.69 - 2.79 (m, 2 H) 3.52 (s, 4 H) 7.21 - 7.39 (m, 10 H) 9.50 (t, J=2.20 Hz, 1 H)

[0319] Intermediate 16: (1S,3R)-3-(((benzyloxy)carbonyl)amino)-1-isopropylcyclopentane-1-carboxylic acid [ka]

[0320] 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) was added a solution of lithium hydroxide (1.527 g, 63.8 mmol) in water (15 mL) followed by tetrahydrofuran (THF) (7.5 mL). The mixture was refluxed for 24 h and cooled to room temperature. 1.0 M HCl (63.8 mL, 63.8 mmol) was added slowly and the mixture was extracted with dichloromethane (DCM) (3 x 150 mL). The combined organic extracts were washed with Na 2 SO 4 The mixture was dried over 100° C., 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 h, 12 M hydrochloric acid (50.0 mL, 600 mmol) was added, and the mixture was heated at 100° C. for 16 h and concentrated under reduced pressure. The residue was dissolved in a mixture of 1,4-dioxane (50 mL) and 1.0 M 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 h, the mixture was extracted with ether (100 mL). The aqueous layer was acidified with 1.0 M HCl (50.0 mL, 50.0 mmol) and extracted with diethyl ether (150 mL). The combined organic extracts were diluted with Na 2 SO 4The mixture was dried at 40° C., filtered, and the filtrate was concentrated under reduced pressure to give the title compound as a brown oil (3.32 g, 10.87 mmol, 85% yield). LC-MS m / z 306.3 (M+H). + .

[0321] Intermediate 17A: benzyl (R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate; and Intermediate 17B: benzyl (S)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate. [ka]

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

[0323] A mixture of commercially available 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine, dihydrochloride (5.0 g, 18.18 mmol) in 2-methyltetrahydrofuran (2-MeTHF) (100 mL) was stirred at room temperature for 1 min. 1.0 M sodium hydroxide (72.7 mL, 72.7 mmol) was added slowly and the mixture was stirred for 5 min. Cbz-Cl (2.72 mL, 19.08 mmol) was added dropwise at room temperature and the mixture was stirred for 1 h. 2-Methyltetrahydrofuran (2-MeTHF) was removed under reduced pressure and the resulting emulsion was stirred until the oil solidified to a yellow solid. The solid was filtered off, crushed with a spatula to a fine powder, washed with water, and air-dried overnight under vacuum filtration to give the title compound as a yellow solid (6.06 g, 18.02 mmol, 99% yield). LC-MS m / z 337.3 (M+H) + .

[0324] Step 2: 6-((benzyloxy)carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine 1-oxide. [ka]

[0325] To 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) at room temperature, methyltrioxorhenium(vii) (0.160 g, 0.642 mmol) was added, followed by dropwise addition of 30% hydrogen peroxide (3.64 mL, 35.7 mmol) and the mixture was stirred at room temperature for 20 hours. Excess hydrogen peroxide was destroyed 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 extracts were washed with Na 2 SO 4 The mixture was dried at 40° C. and filtered. The filtrate was diluted with toluene (50 mL) and concentrated under reduced pressure to give the title compound as a white solid (6.20 g, 17.6 mmol, 99% yield). LC-MS m / z 353.3 (M+H). + .

[0326] Step 3: Benzyl 8-bromo-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate. [ka]

[0327] To a solution of 6-((benzyloxy)carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-1-oxide (6.20 g, 17.60 mmol) in chloroform (30 mL) were added the following two solutions: phosphorus oxybromide (10.09 g, 35.2 mmol) in chloroform (10.0 mL) and triethylamine (1.385 mL, 9.93 mmol) in chloroform (10.0 mL) dropwise simultaneously from two separate syringes over 7 min at room temperature. The mixture was stirred at room temperature for 45 min, poured onto ice, and diluted with 2.0 M Na 2 CO 3 The mixture was extracted with dichloromethane (DCM) (3×200 mL). The combined organic extracts were washed with Na 2 SO 4 The mixture was dried at 75° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by normal phase chromatography (330 g silica, 200 mL / min) (eluting with a gradient of 0-40% ethyl acetate in heptane) afforded the title compound as a white crystalline solid (2.83 g, 6.82 mmol, 38.7% yield). LC-MS m / z 417.1 (M+H). + .

[0328] Step 4: benzyl (R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate; and benzyl (S)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate. [ka]

[0329] To 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 added sodium azide (1.268 mg, 19.51 mmol) and the mixture was stirred at room temperature for 18 h. 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 in toluene (32.5 mL, 32.5 mmol). The mixture was stirred at room temperature for 20 h and concentrated under reduced pressure. The residue was partitioned between diethyl ether and water, the organic phase was separated and sodium 2 SO 4 The mixture was dried at 40° C. and concentrated under reduced pressure. Preparative chiral separation on a Lux Amylose 2 column (5 μm, 21×250 mm, 20 mL / min) eluted with 30% heptane in ethanol containing 0.1% isopropylamine afforded the title compound as a white solid (990.1 mg, 2.82 mmol, 43.3% yield). 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, 38.9% yield). LC-MS m / z 352.3 (M+H) + The absolute stereochemistry of both enantiomers was determined by VCD using the following procedure.

[0330] The sample is 3 Dissolve BaF in CN (16.8 mM) 2 The samples were placed in a 100 μm path length cell with a window plate. IR and VCD spectra were recorded on a ChiralIR2X™ VCD spectrometer (BioTools Inc., Jupiter, FL) equipped with a dual PEM accessory (resolution 4 cm). -1 One isomer, E1, was collected for 6 hours, and the other isomer, E2, was collected for 12 hours. The instrument was set at 1400 cm -1(optimized with 100% NMR). A conformational search against the modeled (R)-structure was performed with MOE in LowMode (using MMFF94x force field and Born solvation, with a dielectric constant of 20 and an external dielectric constant of 47). Geometry optimization, vibrational frequency, and IR and VCD intensity calculations of the 10 conformers resulting from the conformational search were performed with Gaussian 16 (Gaussian Inc., Wallingford, CT) at the DFT level b3lyp / 6-31G(d)scrf= (solvent=dimethylsulfoxide). Gaussian output files were converted to VCD and IR spectra using BLAIR. Calculated frequencies were scaled by 0.981, and IR and VCD intensities were converted to Lorentzian bands with a half-width of 8-cm-1 for comparison with experimental spectra. The assignments were evaluated by the CompareVOA program (BioTools Inc., Jupiter, Fla.) to generate a confidence level based on the current database, which contains 88 previous correct assignments in different chiral structures.

[0331] Intermediate 18: ((R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)methanone [ka]

[0332] Step 1: Benzyl (R)-8-((tert-butoxycarbonyl)amino)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate. [ka]

[0333] To benzyl (R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (step 3-4) (4.25 g, 12.10 mmol) was added di-tert-butyl dicarbonate (2.90 g, 13.31 mmol) and water (24.00 mL), and the mixture was stirred for 5 min. Acetone (24.00 mL) was added slowly. The mixture was stirred for 2 h, and the precipitate was filtered, washed with water, and air-dried to give the title compound as a pale yellow solid (4.70 g, 10.41 mmol, 86% yield). LC-MS m / z 452.1 (M+H) + .

[0334] Step 2: tert-Butyl (R)-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)carbamate. [ka]

[0335] 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 bubbled through the mixture and the mixture was stirred under a balloon atmosphere of hydrogen for 1.5 hours. The mixture was opened to the atmosphere 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 filtrates were concentrated under reduced pressure. Purification by reverse-phase HPLC chromatography (C18 Aq 275 g Gold column, 125 mL / min) (eluting with a gradient of 30-60% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonia hydroxide (0.075%)) afforded the title compound as a pale yellow foam (2.66 g, 8.38 mmol, 82% yield). LC-MS m / z 318.1 (M+H). + .

[0336] Step 3: tert-Butyl ((R)-6-((1S,3R)-3-(((benzyloxy)carbonyl)amino)-1-isopropylcyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)carbamate. [ka]

[0337] 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 h. The mixture was added to tert-butyl (R)-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)carbamate (2.66 g, 8.38 mmol), stirred for 2 min, and the resulting solution was divided into two 20 mL vials. The vials were sealed and stirred at 80° C. for 22 h. The mixture was cooled to room temperature, water (1.000 mL each) was added, the vials were sealed, and the mixture was stirred at 80° C. for 50 min. The two mixtures were cooled to room temperature, combined and water (100 mL) and dichloromethane (DCM) (100 mL) were added. The organic phase was washed with Na 2 SO 4 The mixture was dried at 75° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by ISCO CombiFlash® chromatography (330 g silica, 200 mL / min) (eluting with a gradient of 0-10% methanol in dichloromethane (DCM)) afforded the title compound as a white foam (2.95 g, 4.88 mmol, 58.2% yield). LC-MS m / z 605.2 (M+H). + .

[0338] Step 4: tert-Butyl ((R)-6-((1S,3R)-3-amino-1-isopropylcyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)carbamate. [ka]

[0339] A mixture of tert-butyl ((R)-6-((1S,3R)-3-(((benzyloxy)carbonyl)amino)-1-isopropylcyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)carbamate (2.95 g, 4.88 mmol) and 20% palladium hydroxide on carbon (1.028 g, 1.464 mmol) in methanol (50.000 mL) was purged with nitrogen. Hydrogen was bubbled through the mixture and the mixture was stirred under a balloon atmosphere of hydrogen for 1 h. The mixture was opened to the atmosphere and the mixture was stirred for 15 min. 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 give the title compound as a white foam (2.24 g, 4.76 mmol, 98% yield). LC-MS m / z 471.4 (M+H) + .

[0340] Step 5: tert-Butyl ((R)-6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)carbamate, diformate salt. [ka]

[0341] To a solution of tert-butyl ((R)-6-((1S,3R)-3-amino-1-isopropylcyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)carbamate (2.24 g, 4.76 mmol) in isopropyl acetate (50.00 mL) was added 2-propanol (0.917 mL, 11.90 mmol) and triethylamine (0.664 mL, 4.76 mmol). The mixture was cooled to 0° C. and sodium triacetoxyborohydride (2.52 g, 11.90 mmol) was added, followed by 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 min, warmed to room temperature, and stirred at room temperature for 2 h. 1.0 M NaOH (200 mL) and isopropyl acetate (200 mL) were added and the organic phase was washed with Na 2 SO 4 The mixture was dried at 75° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by reverse phase HPLC chromatography (C18 275 g Gold column, 125 mL / min) (eluted with 35% acetonitrile in water containing formic acid (0.1%)) afforded the title compound as a white foam (2.73 g, 4.03 mmol, 85% yield). This compound was used as an intermediate in the next step. LC-MS m / z 585.2 (M+H) + .

[0342] Step 6: ((R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)methanone. [ka]

[0343] A mixture of tert-butyl ((R)-6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)carbamate, diformate (2.73 g, 4.03 mmol) and trifluoroacetic acid (10 mL, 130 mmol) was stirred at room temperature for 15 min 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 washed with Na 2 SO 4 The mixture was dried at 40° C., filtered, and the filtrate was concentrated under reduced pressure to give the title compound as a white foam (1.77 g, 3.65 mmol, 91% yield). LC-MS m / z 485.4 (M+H). + .

[0344] The following intermediates were or can be prepared using procedures similar to those described for intermediate 18. [Table 4]

[0345] Example 1 (2S,3S)-N-(43-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)-39-oxo-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxa-40-azatritetracontyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka]

[0346] Step 1: 1-((2S,3S)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2-azahenetetracontan-41-oic acid, ammonia salt [ka]

[0347] In a round bottom flask, (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (0.357 g, 1.619 mmol) was dissolved in acetonitrile (10 mL). TSTU (0.487 g, 1.619 mmol) and DIPEA (1.131 ml, 6.48 mmol) were then added and the reaction was stirred at room temperature. Once the acid was activated, 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanotriacontan-39-oic acid (1 g, 1.619 mmol) was added as a solid and N,N-dimethylformamide (DMF) (3 mL) was added to aid in dissolving the acid. The reaction was stirred at room temperature and monitored by LCMS. Upon completion, the reaction was concentrated to give a viscous oil. The crude material was purified by reverse phase chromatography (loaded as a solution into approximately 6 mL of 10 mM aqueous ammonium bicarbonate in H20 (adjusted to pH 10 with ammonia), C18 100 g Gold column, 60 mL / min, gradient: 5-50% 10 mM aqueous ammonium bicarbonate in H20 (adjusted to pH 10 with ammonia):MeCN, 23 min) to give the title compound as a colorless oil (1.3 g, 1.351 mmol, 83% yield) (87% purity by NMR, material was CH 2 Cl 2 LC-MS m / z 820.1 (M+H) + .

[0348] Step 2: ((2S,3S)-N-(43-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)-39-oxo-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxa-40-azatritetracontyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka]

[0349] To a solution of ((1S,3R)-1-(3-aminopropyl)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, trihydrochloride (89 mg, 0.15 mmol) in dichloromethane (DCM) (1500 μL) was added 1-((2S,3S)-1-methyl-5-oxo- A solution of 2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2-azahentetracontan-41-oic acid, ammonium salt (123 mg, 0.150 mmol) in dichloromethane (DCM) (1500 μL), HATU (86 mg, 0.225 mmol), and DIPEA (262 μL, 1.500 mmol) were added. The mixture was stirred at room temperature for 2 h, methanol was added, and the mixture was concentrated. The residue was purified by CombiFlash® EZ Prep chromatography (30 g C18 column, 35 mL / min) (eluted with 10-70% acetonitrile in water containing ammonium bicarbonate (10 mM)) and adjusted to pH 10 with ammonia. Further purification by MDAP (XSelect™ CSH C18 column, 40 mL / min) (eluting with a gradient of 15-55% acetonitrile in water containing ammonium bicarbonate (10 mM) adjusted to pH 10 with ammonia) afforded the title compound as a clear viscous oil (79.2 mg, 0.061 mmol, 40.6% yield). LCMS m / z (M / 2+H) + 644.1. 1 H NMR (400 MHz, DMSO-d 6) δ ppm 1.11 - 1.28 (m, 4H), 1.29 - 1.39 (m, 1H), 1.41 - 1.53 (m, 3H), 1.53 - 1.68 (m, 2H), 1.69 - 1.86 (m, 2H), 1.99 (br dd, J = 12.7, 6.8 Hz, 1H), 2.03 - 2.14 (m, 1H), 2.22 (br t, J = 6.4 Hz, 2H), 2.44 (dd, J = 16.9, 7.6 Hz, 2H), 2.50 (br s, 4H), 2.70 (br dd, J = 16.6, 9.3 Hz, 2H), 2.86 - 3.04 (m, 5H), 3.12 - 3.28 (m, 8H), 3.38 (td, J = 5.7, 1.7 Hz, 2H), 3.42 - 3.57 (m, 44H), 3.66 - 3.76 (m, 2H), 3.86 (dt, J = 7.7, 3.7 Hz, 3H), 4.65 (d, J = 5.9 Hz, 1H), 4.79 (br s, 2H), 7.45 (dd, J = 7.8, 4.9 Hz, 1H), 7.68 (dt, J = 7.8, 2.0 Hz, 1H), 7.74 (br s, 1H), 8.07 (t, J = 5.4 Hz, 1H), 8.18 (br s, 1H), 8.44 - 8.52 (m, 1H), 8.56 (dd, J = 4.6, 1.7 Hz, 1H), 8.77 (s, 1H)

[0350] Example 2: (2S,3S)-N-(2-(((1R,4S)-4-(4-(((1S,4R)-4-(4-(2-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)ethyl)piperidine-1-carbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka]

[0351] Step 1 Methyl (E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoate. [ka]

[0352] To a stirred solution of (1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexan-1-ol (250 g, 736 mmol) in toluene (2500 mL) was added methyl but-2-ynoate (140 g, 1423 mmol), triphenylphosphine (19.32 g, 73.6 mmol), and acetic acid (16.86 mL, 295 mmol) at room temperature, and the resulting solution was stirred at 115° C. for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to give 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) (eluting with 15% EtOAc in petroleum ether) to give methyl (E&Z)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-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) (eluting with 15% EtOAc in petroleum ether) to give the title compound as a pale yellow liquid (240 g, 463 mmol, 62.9% yield, 84.45% purity). LC-MS m / z 438.3 (M+H) + .

[0353] Step 2 (E)-4-(((1,4-trans)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoic acid [ka]

[0354] Methyl (E)-4-(((1,4-trans)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoate (9.03 g, 20.64 mmol) was dissolved in tetrahydrofuran (THF) (25 mL) and 5.089 M aqueous sodium hydroxide (4.87 mL, 24.76 mmol) was added. The homogeneous pale yellow reaction was heated to reflux for 1 h. Additional 5.089 M sodium hydroxide (1.217 mL, 6.19 mmol) was added and the reaction was refluxed for 50 min. The reaction was cooled over 60 min and concentrated in vacuo. The residue was azeotroped twice with toluene to aid in the removal of water. The residue was pumped under high vacuum to give the title compound as a yellow solid (9.9 g, 22.17 mmol, 107% yield, 82% purity, E / Z mixture). LC-MS m / z 424.4 (M+H) + .

[0355] Step 3 tert-Butyl (1R,4r)-4-((E)-4-(((1r,4R)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enamido)cyclohexane-1-carboxylate. [ka]

[0356] (E)-4-(((1,4-trans)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-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 the mixture was observed to partially dissolve. The mixture was stirred for 30 min to give 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 a solution 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 h. Additional HATU (1.724 g, 4.53 mmol) was added and the nearly homogeneous reaction was stirred for 60 min. The cloudy reaction was stirred for an additional 60 min. The reaction was diluted with 200 mL EtOAc and 200 mL water and stirred for 10 min. The resulting homogeneous biphasic mixture was transferred to a separatory funnel and the layers separated. The aqueous layer was extracted two more times with 150 mL EtOAc and the combined EtOAc layers were washed four times with water and two times with saturated NaCl to remove DMF. The EtOAc extracts were dried over sodium sulfate, filtered, concentrated in vacuo, and pumped under high vacuum to give an orange syrup which was purified by silica gel chromatography (Isco Combiflash, 330 gram gold column, 0-80% EtOAc:heptane, 45 min, flow rate 150 mL / min, loaded as a solution in DCM) to give the title compound as a white foamy solid (4.55 g, 7.52 mmol, 36.5% yield). LC-MS m / z 605.5 (M+H) + .

[0357] Step 4 tert-Butyl (1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylate [ka]

[0358] A 500 mL 3-neck round bottom flask was charged with tert-butyl (1R,4r)-4-((E)-4-(((1r,4R)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enamido)cyclohexane-1-carboxylate (6.40 g, 10.58 mmol) and isopropanol (120 mL) and the suspension was stirred until a homogenous solution was obtained. 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 center neck was covered with a rubber septum. The reaction was stirred at room temperature for 16 h. The reaction was degassed and filtered through Celite twice. The filtrate was concentrated in vacuo and pumped under high vacuum to give a grey waxy solid with a slight odor of isopropanol. The waxy solid was dissolved in DCM and concentrated in vacuo at 54° C. for 20 min to aid in the removal of isopropanol. The residue was pumped under high vacuum to give the title compound as a grey waxy solid (4.44 g, 10.41 mmol, 98% yield). LC-MS m / z 427.4 (M+H). + .

[0359] Step 5 tert-Butyl (1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylate. [ka]

[0360] (2S,3S)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (2.287 g, 10.38 mmol) was suspended in 30 mL of DCM in a 500 mL round bottom flask with stirring at room temperature. HATU (4.34 g, 11.42 mmol) was added and the suspension was stirred for 15 min. A solution of tert-butyl (1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylate (4.43 g, 10.38 mmol) in DCM (20 ml) was added dropwise by pipette over 15 min. After the addition was complete, a solution of DIEA (5.44 mL, 31.2 mmol) in DCM (10 mL) was added dropwise over 10 min and the resulting homogeneous dark solution was stirred at room temperature for 16 h. The reaction was concentrated in vacuo to remove DCM and DIEA. The residue was dissolved in 100 ml DCM and transferred to a separatory funnel. 20 ml of saturated sodium bicarbonate was added. The layers were separated and the DCM layer was washed with saturated NaCl, dried over sodium sulfate, filtered, concentrated in vacuo and pumped under high vacuum to give an orange syrup which was purified by silica gel chromatography (Isco Combiflash, 0-10% MeOH:DCM, 60 min, 330 gram gold column, flow rate 150 ml / min, loaded as a solution in 30 ml DCM) to give the title compound as a white solid (4.13 g, 6.57 mmol, 63.2% yield). LC-MS m / z 629.3 (M+H) + .

[0361] Step 6 (1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid, hydrochloride. [ka]

[0362] tert-Butyl (1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamido)cyclohexane-1-carboxylate (4.13 g, 6.57 mmol) was dissolved in dry 1,4-dioxane (13 ml) with stirring in a 250 ml round bottom flask. Anhydrous HCl 4M in 1,4-dioxane (39 mL, 156 mmol) was added and the mixture was stirred at room temperature. Upon addition of HCl an insoluble oil was observed. The mixture was stirred at room temperature for 90 min. The reaction was concentrated in vacuo (bath temperature 60° C.) and pumped under high vacuum for 15 h to give the title compound as a white solid (4.187 g, 6.87 mmol, 105% yield). LC-MS m / z 573.4 (M+H). + .

[0363] Step 7: (2S,3S)-N-(2-(((1R,4S)-4-(4-(((1S,4R)-4-(4-(2-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)ethyl)piperidine-1-carbonyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka]

[0364] A mixture of (1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanamide)cyclohexane-1-carboxylic acid, hydrochloride salt (54.7 mg, 0.090 mmol) and HATU (37.6 mg, 0.099 mmol) in dichloromethane (DCM) (1.0 mL) was stirred at room temperature for 15 min. ((1S,3R)-3-(((3S,4S)-3-Methoxytetrahydro-2H-pyran-4-yl)amino)-1-(2-(piperidin-4-yl)ethyl)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, trihydrochloride (58.2 mg, 0.090 mmol) was added as a solid. DIPEA (0.125 mL, 0.718 mmol) and dichloromethane (DCM) (1.5 mL) were added and the mixture was stirred at room temperature for 16 hours. Dichloromethane (DCM) (6 mL) and saturated NaHCO 3 (6 mL) was added and the mixture was stirred for 10 min. The organic phase was washed with saturated NaCl and Na 2 SO 4 The mixture was dried at 75° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by ISCO CombiFlash® chromatography (12 g silica Gold column, 30 mL / min) (eluting with a gradient of 0-30% methanol in dichloromethane (DCM)) afforded the title compound as a white solid (63.5 mg, 0.0508 mmol, 64.7% yield). LCMS (m / z) 1093.7 (M+H). + . 1 H NMR (400 MHz, methanol-d 4) δ ppm 1.22 - 1.43 (m, 8 H) 1.50 - 1.77 (m, 8 H) 1.83 (dt, J=14, 6.7 Hz, 4 H) 1.90 - 2.05 (m, 8 H) 2.11 - 2.20 (m, 1 H) 2.25 (t, J=7.3 Hz, 2 H) 2.60 (br d, J=11 Hz, 2 H) 2.66 (s, 3 H) 2.68 - 2.76 (m, 2 H) 2.80 - 2.91 (m, 2 H) 2.83 - 2.83 (m, 1 H) 2.96 (br s, 1 H) 3.03 - 3.16 (m, 2H) 3.24 - 3.31 (m, 2 H) 3.35 - 3.53 (m, 17 H) 3.56 - 3.78 (m, 2 H) 3.83 - 4.10 (m, 6 H) 4.15 (br d, J=13 Hz, 2 H) 4.43 (br s, 2 H) 4.80 - 4.85 (m, 1 H) 7.54 (dd, J=8.8, 4.9 Hz, 1 H) 7.81 (dt, J=8.3, 2.0 Hz, 1 H) 8.09 (br s, 1 H) 8.52 (d, J=2.0 Hz, 1 H) 8.59 (dd, J=4.9, 1.5 Hz, 1 H) 8.72 (s, 1 H).

[0365] The following compounds were prepared or can be prepared by procedures similar to those described in Example 2. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11] [Table 5-12] [Table 5-13] [Table 5-14] [Table 5-15] [Table 5-16] [Table 5-17] [Table 5-18]

[0366] Example 16: N 1 -((1R,4s)-4-((3-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)propyl)carbamoyl)cyclohexyl)-N 5-(4-((3-((2S,3S)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)glutaramide [ka]

[0367] Step 1: tert-Butyl (3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)carbamate. [ka]

[0368] To a mixture of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (2000 mg, 9.08 mmol) and commercially available tert-butyl (3-aminopropyl)carbamate (1741 mg, 9.99 mmol) in dichloromethane (DCM) (30 mL) was added DIPEA (1.740 mL, 9.99 mmol) followed by T3P (50% in ethyl acetate) (5.95 mL, 9.99 mmol). The mixture was stirred at room temperature for 2 h, washed with water (10 mL) and diluted with Na 2 SO 4 The mixture was dried at 4° C. and concentrated under reduced pressure. Purification by normal phase chromatography (eluting with a gradient of 0-10% methanol in dichloromethane (DCM)) afforded the title compound as a pale yellow liquid (3430 mg, 8.20 mmol, 90% yield). LCMS (m / z) 377.1 (M+H). + .

[0369] Step 2: (2S,3S)—N-(3-aminopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, dihydrochloride. [ka]

[0370] To tert-butyl (3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)carbamate (3430 mg, 8.20 mmol) was added 4N HCl in 1,4-dioxane (6.81 mL, 27.2 mmol). The mixture was stirred for 1 h and concentrated under reduced pressure to give the title compound as a white solid (3143 mg, 8.10 mmol, 89% yield). LCMS (m / z) 277.1 (M+H). + .

[0371] Step 3: tert-Butyl (4-((3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)carbamate. [ka]

[0372] To a mixture of (2S,3S)-N-(3-aminopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, dihydrochloride (3143 mg, 9 mmol), 4-((tertbutoxycarbonyl)amino)butanoic acid (1829 mg, 9.00 mmol) in dichloromethane (DCM) (30 mL) was added DIPEA (4.70 mL, 27.0 mmol) followed by T3P 50% in ethyl acetate (6.43 mL, 10.80 mmol). The mixture was stirred at room temperature for 2 h and washed with water (30 mL). The organic phase was washed with Na 2 SO 4 The mixture was dried at 4°C for 20 min and concentrated under reduced pressure. Purification by normal phase chromatography (eluting with a gradient of 0-10% methanol in dichloromethane (DCM)) afforded the title compound as a pale yellow liquid (900 mg, 1.852 mmol, 20.58% yield). LCMS m / z 462.5 (M+H) + .

[0373] Step 4: (2S,3S)—N-(3-(4-aminobutanamido)propyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, dihydrochloride. [ka]

[0374] To tert-butyl (4-((3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)carbamate (900 mg, 1.852 mmol, 20.58% yield) was added 4N HCl in 1,4-dioxane, the mixture was stirred for 1 h and concentrated under reduced pressure to give the title compound as a white solid (847 mg, 1.852 mmol, 20.58% yield). LCMS (m / z) 362.1 (M+H). + .

[0375] Step 5: tert-Butyl (1s,4s)-4-(5-((4-((3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)amino)-5-oxopentanamido)cyclohexane-1-carboxylate. [ka]

[0376] To a mixture of (2S,3S)-N-(3-(4-aminobutanamido)propyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, dihydrochloride (804 mg, 1.852 mmol) and commercially available dihydro-2H-pyran-2,6(3H)-dione (211 mg, 1.852 mmol) in dichloromethane (DCM) (3 mL) was added DIPEA (1.613 mL, 9.26 mmol) and the mixture was stirred at room temperature for 10 min. HATU (845 mg, 2.222 mmol) was added followed by commercially available (1s,4s)-tert-butyl-4-aminocyclohexanecarboxylate, hydrochloride (437 mg, 1.852 mmol). The mixture was stirred at room temperature for 2 h, washed with water (10 mL) and diluted with Na 2 SO 4 The mixture was dried at 40° C. and concentrated under reduced pressure. Purification by ISCO CombiFlash® chromatography (24 g Gold column) (gradient elution with 0-20% dichloromethane (DCM) in methanol) afforded the title compound as an off-white solid (1200 mg, 1.644 mmol, 89% yield). LCMS (m / z) 657.2 (M+H). + .

[0377] Step 6: (1s,4s)-4-(5-((4-((3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)amino)-5-oxopentanamido)cyclohexane-1-carboxylic acid, hydrochloride. [ka]

[0378] To a solution of (1s,4s,cis)-tert-butyl 4-(5-((4-((3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)amino)-5-oxopentanamido)cyclohexanecarboxylate (1200 mg, 1.644 mmol) in dichloromethane (DCM) (3 mL) was added 4M HCl in dioxane (3 mL) over 1 h. It was then concentrated under vacuum to give the title compound as a white solid (1155 mg, 1.631 mmol, 88% yield). LCMS (m / z) 601.1 (M+H). +

[0379] Step 7: N 1 -((1R,4s)-4-((3-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)propyl)carbamoyl)cyclohexyl)-N 5 -(4-((3-((2S,3S)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)glutaramide [ka]

[0380] ((1S,3R)-1-(3-aminopropyl)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, trihydrochloride (59.4 mg, 0.100 mmol) and (1s,4s)-4-(5-((4-((3-((2S,3S)-1-methyl-5-oxo-2- To a solution of (pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)amino)-5-oxopentanamido)cyclohexane-1-carboxylic acid (60.1 mg, 0.100 mmol) in dichloromethane (DCM) (2000 μL) was added HATU (57.0 mg, 0.150 mmol) and DIPEA (175 μL, 1.000 mmol) and the mixture was stirred at room temperature for 1.5 h. Methanol was added and the mixture was concentrated under a steady stream of nitrogen. Purification by MDAP (XSelect™ CSH C18 column, 40 mL / min) (eluting with a gradient of 15-55% acetonitrile in water containing ammonium bicarbonate (10 mM) adjusted to pH 10 with ammonia) afforded the title compound as a white solid (11.1 mg, 9.88 μmol, 9.88% yield). LCMS (m / z) 1067.7 (M+H) + . 1 H NMR (400 MHz, methanol-d 4) δ 1.38 (br d, J = 8.3 Hz, 3H), 1.52 - 1.69 (m, 6H), 1.70 - 1.84 (m, 7H), 1.85 - 1.96 (m, 2H), 1.96 - 2.10 (m, 2H), 2.13 - 2.29 (m, 6H), 2.51 - 2.63 (m, 1H), 2.67 (s, 2H), 2.68 - 2.77 (m, 1H), 2.80 - 2.91 (m, 1H), 2.98 - 3.06 (m, 1H), 3.07 - 3.29 (m, 8H), 3.31 - 3.35 (m, 14H), 3.36 - 3.52 (m, 5H), 3.88 - 3.96 (m, 2H), 4.01 (br dd, J = 12.2, 5.9 Hz, 2H), 4.17 (br d, J = 11.2 Hz, 1H), 4.84 (d, J = 6.4 Hz, 1H), 7.54 (dd, J = 7.6, 5.1 Hz, 1H), 7.83 (dt, J = 7.9, 1.9 Hz, 1H), 8.06 (br s, 1H), 8.54 (d, J = 1.5 Hz, 1H), 8.58 (dd, J = 4.9, 1.5 Hz, 1H), 8.72 (s, 1H)

[0381] Example 17 (2S,3S)-N-(2-(2-(2-(3-(((1R,4s)-4-((3-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)propyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)ethoxy)ethoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, formate [ka]

[0382] Step 1: tert-Butyl 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oate. [ka]

[0383] To a mixture of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (15.69 g, 71.3 mmol) and commercially available tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate (19.7638 g, 71.3 mmol) in dichloromethane (DCM) (200 mL) was added HATU (32.5 g, 86 mmol), triethylamine (25.8 mL, 185 mmol) and the mixture was stirred at room temperature under a balloon atmosphere of nitrogen for 2 h. Saturated NaHCO 3 was added and the mixture was extracted with dichloromethane (DCM). The combined organic extracts were diluted with Na 2 SO 4 The extract was dried over 1000 ml of water, filtered, and the filtrate was concentrated. ISCO CombiFlash® chromatography (330 g silica Gold column, 200 mL / min) (0-15% methanol (10% NH 4 The mixture was purified by elution with a gradient of 0.1% hexanes (containing OH) to give the title compound as a yellow oil (31.5 g, 65.7 mmol, 92% yield). LCMS (m / z) 480.2 (M+H). + .

[0384] Step 2: 1-((2S,3S)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, hydrochloride [ka]

[0385] To a mixture of tert-butyl 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oate (31 g, 64.6 mmol) in dichloromethane (DCM) (40 mL) at 0° C. was added 4N HCl in 1,4-dioxane (81 mL, 323 mmol). The mixture was stirred at room temperature for 2 hours and concentrated to give the title compound (29 g, 63.1 mmol, 98% yield). LCMS (m / z) 424.2 (M+H) + .

[0386] Step 3: tert-Butyl (1s,4s)-4-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-amido)cyclohexane-1-carboxylate [ka]

[0387] To a mixture of commercially available tert-butyl (1S,4S)-4-aminocyclohexane-1-carboxylate, hydrochloride (2.56 g, 10.87 mmol) and 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid, hydrochloride (5 g, 10.87 mmol) in dichloromethane (DCM) (100 mL) was added triethylamine (7.58 mL, 54.4 mmol) and HATU (4.96 g, 13.05 mmol). The mixture was stirred at room temperature for 4 h and then diluted with dichloromethane (DCM) and saturated NaHCO 3 The aqueous phase was extracted with dichloromethane (DCM). 2 SO 4 The extract was dried over 1000 ml of ethyl acetate, filtered, and the filtrate was concentrated. ISCO CombiFlash® chromatography (120 g silica Gold column, 60 mL / min) (0-15% methanol (10% NH 4The mixture was purified by elution with a gradient of 0.1% hexanes (containing OH) to give the title compound (5.46 g, 9.03 mmol, 83% yield). LCMS (m / z) 605.3 (M+H). + .

[0388] Step 4: (1s,4s)-4-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-amido)cyclohexane-1-carboxylic acid, hydrochloride [ka]

[0389] To a mixture of tert-butyl (1s,4s)-4-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-amido)cyclohexane-1-carboxylate (9.32 g, 15.41 mmol) in dichloromethane (DCM) (10 mL) at 0° C. was added 4N HCl in 1,4-dioxane (15.41 mL, 61.6 mmol). The mixture was stirred at room temperature for 5 hours and concentrated to give the title compound (10 g, 17.09 mmol, 111% yield). LCMS (m / z) 549.3 (M+H) + .

[0390] Step 5: (2S,3S)-N-(2-(2-(2-(3-(((1R,4s)-4-((3-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)propyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)ethoxy)ethoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, formate [ka]

[0391] ((1S,3R)-1-(3-aminopropyl)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, trihydrochloride (59.4 mg, 0.100 mmol) and (1s,4s)-4-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridinyl) To a solution of (58.5 mg, 0.100 mmol)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-amido)cyclohexane-1-carboxylic acid, hydrochloride (58.5 mg, 0.100 mmol) in dichloromethane (DCM) (2000 μL) was added HATU (57.0 mg, 0.150 mmol) and DIPEA (175 μL, 1.000 mmol) and the mixture was stirred at room temperature for 1.5 h. Methanol was added and the mixture was concentrated under a steady stream of nitrogen. Purification by MDAP (XSelect™ CSH C18 column, 40 mL / min) (eluting with a gradient of 5-35% acetonitrile in water containing formic acid (0.1%)) afforded the title compound as a white solid (44.5 mg, 0.039 mmol, 39.4% yield). LCMS (m / z) 1015.7 (M+H) + . 1 H NMR (400 MHz, methanol-d 4) δ ppm 1.29 - 1.50 (m, 2H), 1.52 - 2.05 (m, 13H), 2.22 (br d, J = 5.9 Hz, 3H), 2.31 - 2.41 (m, 1H), 2.42 - 2.50 (m, 2H), 2.51 - 2.62 (m, 1H), 2.65 - 2.68 (m, 3H), 2.68 - 2.77 (m, 1H), 2.83 (d, J = 9.8 Hz, 1H), 2.99 - 3.24 (m, 5H), 3.34 - 3.44 (m, 3H), 3.44 - 3.48 (m, 3H), 3.51 (ddd, J = 5.9, 4.6, 2.7 Hz, 3H), 3.54 - 3.63 (m, 9H), 3.65 - 3.76 (m, 3H), 3.88 - 4.08 (m, 4H), 4.27 (br d, J = 13.2 Hz, 1H), 4.84 (d, J = 6.4 Hz, 1H), 7.54 (dd, J = 7.8, 4.9 Hz, 1H), 7.78 - 7.92 (m, 1H), 8.07 (br s, 1H), 8.51 - 8.61 (m, 2H), 8.74 (s, 1H)

[0392] The following compounds were prepared by procedures similar to those described in Example 17: [Table 6-1] [Table 6-2]

[0393] Example 19 (2S,3S)-N-(3-((2-(2-(3-(((1s,4s)-4-((3-((1R,3S)-3-(((3R,4R)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)propyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)ethoxy)ethyl)amino)-3-oxopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka]

[0394] Step 1: tert-Butyl 3,7-dioxo-1-phenyl-2,11,14-trioxa-4,8-diazaheptadecan-17-oate [ka]

[0395] To a mixture of commercially available tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (2.5 g, 10.72 mmol) and commercially available 3-(((benzyloxy)carbonyl)amino)propanoic acid (2.51 g, 11.25 mmol) in dichloromethane (DCM) (35.7 mL) at room temperature, DIPEA (5.61 ml, 32.1 mmol) and HATU (4.28 g, 11.25 mmol) were added and the mixture was stirred under nitrogen atmosphere for 20 h. Dichloromethane (DCM) was added and the organic phase was washed with saturated NH 4 Cl, saturated NaHCO 3 The organic phase was washed with Na 2 SO 4The mixture was dried at 70° C., filtered, and concentrated under reduced pressure. Purification by ISCO CombiFlash® Rf chromatography (80 g silica column, 60 mL / min) (eluting with a gradient of 50% (5 column volumes), 50-90% (25 column volumes), and 90% (5 column volumes) ethyl acetate in heptane) afforded the title compound as a colorless oil (4.69 g, 10.70 mmol, 100% yield). LCMS (m / z) 383.4 (M+H). + .

[0396] Step 2: 3,7-dioxo-1-phenyl-2,11,14-trioxa-4,8-diazaheptadecan-17-oic acid [ka]

[0397] To a suspension of tert-butyl 3,7-dioxo-1-phenyl-2,11,14-trioxa-4,8-diazaheptadecan-17-oate (4.65 g, 10.60 mmol) in 1,4-dioxane (35.3 mL) was added 4M HCl in 1,4-dioxane (12.60 mL, 50.4 mmol) and the mixture was stirred at room temperature for 20 h. 4M HCl in 1,4-dioxane (1 mL) was added twice at 2 h intervals and the mixture was concentrated under reduced pressure to give the title compound as an off-white solid (5.69 g, 13.39 mmol, 126% yield). LCMS (m / z) 383.4 (M+H) + .

[0398] Step 3: tert-Butyl (1s,4s)-4-(3,7-dioxo-1-phenyl-2,11,14-trioxa-4,8-diazaheptadecan-17-amido)cyclohexane-1-carboxylate [ka]

[0399] To a suspension of 3,7-dioxo-1-phenyl-2,11,14-trioxa-4,8-diazaheptadecan-17-oic acid (4.05 g, 10.60 mmol) and commercially available tert-butyl (1s,4s)-4-aminocyclohexane-1-carboxylate, hydrochloride (2.499 g, 10.60 mmol) in dichloromethane (DCM) (35.3 mL) were added DIPEA (7.59 mL, 43.5 mmol) and HATU (4.23 g, 11.13 mmol) and the mixture was stirred under nitrogen at room temperature for 20 h. Dichloromethane (DCM) was added and the organic phase was washed with saturated NH 4 Cl, saturated NaHCO 3 The organic phase was washed with NaSO 4 The residue was dried at 4° C., filtered and concentrated under reduced pressure. Purification by ISCO Combi Flash® Rf (80 g silica, 60 mL / min) (eluting with a stepwise gradient of 0% (5 column volumes), 0 to 25% (25 column volumes), and 25% (5 column volumes) methanol in dichloromethane (DCM)) afforded the desired product as a clear, colorless oil (5.06 g, 8.08 mmol, 76% yield). LCMS (m / z) 564.5 (M+H). + .

[0400] Step 4: tert-Butyl (1s,4s)-4-(3-(2-(2-(3-aminopropanamido)ethoxy)ethoxy)propanamido)cyclohexane-1-carboxylate [ka]

[0401] A suspension of tert-butyl (1s,4s)-4-(2,6-dioxo-1-phenoxy-10,13-dioxa-3,7-diazahexadecan-16-amido)cyclohexane-1-carboxylate (5.0 g, 8.87 mmol) and 10% Pd-C (0.944 g, 0.887 mmol) in ethyl acetate (29.6 mL) was stirred under a balloon atmosphere of hydrogen at room temperature for 20 h. 10% Pd-C (300 mg) was added and the mixture was stirred under a balloon atmosphere of hydrogen for 1 h. The mixture was filtered through Celite® and the filtrate was concentrated under reduced pressure to give the title compound as a brown oil (3.43 g, 7.19 mmol, 81% yield). LCMS (m / z) 430.5 (M+H) + .

[0402] Step 5: tert-Butyl (1s,4s)-4-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,5-dioxo-9,12-dioxa-2,6-diazapentadecan-15-amido)cyclohexane-1-carboxylate [ka]

[0403] To a suspension of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (0.872 g, 3.96 mmol) and tert-butyl (1s,4s)-4-(3-(2-(2-(3-aminopropanamido)ethoxy)ethoxy)propanamido)cyclohexane-1-carboxylate (1.7 g, 3.96 mmol) in dichloromethane (DCM) (13.19 mL) was added DIPEA (2.76 mL, 15.83 mmol) and HATU (1.580 g, 4.16 mmol) and the mixture was stirred at room temperature for 20 h. Dichloromethane (DCM) was added and the organic phase was washed with saturated NH 4 Cl, saturated NaHCO 3 The organic phase was washed with NaSO 4The mixture was dried at 70° C., filtered and concentrated under reduced pressure. Purification by ISCO Combi Flash® Rf (80 g silica, 60 mL / min) (eluting with a stepwise gradient of 0% (5 column volumes), 0 to 25% (25 column volumes), and 25% (5 column volumes) methanol in dichloromethane (DCM)) afforded the title compound as a clear, colorless oil (1.74 g, 2.479 mmol, 62.6% yield). LCMS (m / z) 632.5 (M+H). + .

[0404] Step 6: (1s,4s)-4-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,5-dioxo-9,12-dioxa-2,6-diazapentadecan-15-amido)cyclohexane-1-carboxylic acid, hydrochloride [ka]

[0405] To a suspension of tert-butyl (1s,4s)-4-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,5-dioxo-9,12-dioxa-2,6-diazapentadecan-15-amido)cyclohexane-1-carboxylate (1.7 g, 2.69 mmol) in 1,4-dioxane (8.97 mL) was added 4M HCl in 1,4-dioxane (2.018 mL, 8.07 mmol). Acetonitrile (3 mL) was added and the mixture was stirred at room temperature under nitrogen for 20 hours. 4M HCl in 1,4-dioxane (3.03 mL, 12.1 mmol) was added and the mixture was stirred for 4 hours. 4M HCl in 1,4-dioxane (6.05 mL, 24.2 mmol) was added and the mixture was stirred at room temperature for 20 h. The mixture was concentrated under reduced pressure to give the title compound as a sticky off-white solid (1.88 g, 3.07 mmol, 114% yield). LCMS (m / z) 576.5 (M+H). + .

[0406] Step 7: (2S,3S)-N-(3-((2-(2-(3-(((1S,4s)-4-((3-((1R,3S)-3-(((3R,4R)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)propyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)ethoxy)ethyl)amino)-3-oxopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka]

[0407] ((1S,3R)-1-(3-aminopropyl)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentyl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, trihydrochloride (59.4 mg, 0.100 mmol) and (1s,4s)-4-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridine- To a solution of (3-yl)pyrrolidin-3-yl)-1,5-dioxo-9,12-dioxa-2,6-diazapentadecan-15-amido)cyclohexane-1-carboxylic acid, hydrochloride (61.2 mg, 0.100 mmol) in dichloromethane (DCM) (2000 μL) was added HATU (57.0 mg, 0.150 mmol) and DIPEA (175 μL, 1.000 mmol) and the mixture was stirred at room temperature for 1.5 h. Methanol was added and the mixture was concentrated under a steady stream of nitrogen. Purification by MDAP (XSelect™ CSH C18 column, 40 mL / min) (eluting with a gradient of 15-55% acetonitrile in water containing ammonium bicarbonate (10 mM) adjusted to pH 10 with ammonia) afforded the title compound as a white solid (42.3 mg, 0.039 mmol, 38.6% yield). LCMS (m / z) 1042.7 (M+H) + . 1 H NMR (400 MHz, methanol-d 4) δ ppm 1.29 - 1.48 (m, 3H), 1.53 - 1.84 (m, 12H), 1.93 - 2.09 (m, 2H), 2.14 - 2.29 (m, 2H), 2.40 (td, J = 6.7, 1.7 Hz, 2H), 2.48 (t, J = 6.1 Hz, 2H), 2.53 - 2.64 (m, 1H), 2.64 - 2.74 (m, 4H), 2.79 - 2.91 (m, 2H), 2.97 - 3.19 (m, 5H), 3.34 - 3.48 (m, 11H), 3.52 (q, J = 5.1 Hz, 3H), 3.61 (s, 4H), 3.75 (t, J = 6.1 Hz, 3H), 3.88 - 4.10 (m, 5H), 4.16 (br d, J = 13.7 Hz, 2H), 4.84 (d, J = 6.4 Hz, 1H), 7.54 (dd, J = 7.6, 5.1 Hz, 1H), 7.80 (dt, J = 7.8, 2.0 Hz, 2H), 8.06 (br s, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.58 (dd, J = 4.9, 2.0 Hz, 1H), 8.72 (s, 1H)

[0408] Example 20 ((2S,3S)-N-(2-(((1r,4S)-4-(2-(2-(3-((3-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)propyl)amino)-3-oxopropoxy)ethoxy)ethoxy)cyclohexyl)oxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, formate [ka]

[0409] Step 1: N,N-Dibenzyl-2-(((1r,4r)-4-(vinyloxy)cyclohexyl)oxy)ethan-1-amine. [ka]

[0410] To a mixture of palladium(II) trifluoroacetate (0.154 g, 0.464 mmol) and bathophenanthroline (0.154 g, 0.464 mmol) in ethyl vinyl ether (27.5 mL, 278 mmol) was added DIPEA (1.621 mL, 9.28 mmol) and (1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexan-1-ol (3.15 g, 9.28 mmol) and the mixture was separated into two vials. The vials were sealed and stirred at 75 °C for 3.5 h. Purification by ISCO CombiFlash® chromatography (80 g silica Gold column, 60 mL / min) (eluting with a gradient of 0-20% ethyl acetate in heptane) gave the title compound (2.3956 g, 6.55 mmol, 70.6% yield). LCMS (m / z) 366.4 (M+H) + .

[0411] Step 2: 2-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)ethan-1-ol. [ka]

[0412] To a solution of N,N-dibenzyl-2-(((1r,4r)-4-(vinyloxy)cyclohexyl)oxy)ethan-1-amine (2.35 g, 6.43 mmol) in tetrahydrofuran (THF) (50 mL) was added 1 M borane in tetrahydrofuran (THF) (9.64 mL, 9.64 mmol) and the mixture was stirred at room temperature for 40 minutes. 5N sodium hydroxide (12.86 mL, 64.3 mmol) and H 2 O 2(30%) (13.13 mL, 129 mmol) was added and the mixture was stirred at room temperature for 23 h. The mixture was extracted with ethyl acetate (3×30 mL) and the combined organic extracts were washed with brine (30 mL) and MgSO 4 The mixture was dried at 75° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by ISCO CombiFlash® chromatography (40 g silica Gold column, 40 mL / min) (eluting with a gradient of 0-70% ethyl acetate in heptane) afforded the title compound (2.1723 g, 4.86 mmol, 76% yield). LCMS (m / z) 384.4 (M+H). + .

[0413] Step 3: N,N-Dibenzyl-2-(((1r,4r)-4-(2-(vinyloxy)ethoxy)cyclohexyl)oxy)ethan-1-amine. [ka]

[0414] To a mixture of palladium(II) trifluoroacetate (0.081 g, 0.243 mmol) and bathophenanthroline (0.081 g, 0.243 mmol) in ethyl vinyl ether (14.39 mL, 146 mmol) was added DIPEA (0.848 mL, 4.85 mmol) and N,N-dibenzyl-2-(((1r,4r)-4-(2-(vinyloxy)ethoxy)cyclohexyl)oxy)ethan-1-amine (2.17 g, 4.85 mmol) and the mixture was stirred in a sealed vial at 75° C. for 4 h. The mixture was cooled to room temperature, filtered, and concentrated under reduced pressure. Purification by ISCO CombiFlash® chromatography (40 g silica Gold column, 40 mL / min) (eluting with a gradient of 0-30% ethyl acetate in heptane) afforded the title compound (1.9085 g, 3.79 mmol, 78% yield). LCMS (m / z) 410.1 (M+H). + .

[0415] Step 4: 2-(2-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)ethoxy)ethan-1-ol. [ka]

[0416] To a solution of N,N-dibenzyl-2-(((1r,4r)-4-(2-(vinyloxy)ethoxy)cyclohexyl)oxy)ethane-1-amineamine (1.9 g, 3.78 mmol) in tetrahydrofuran (THF) (40 mL) was added borane (1 M in THF) (5.66 mL, 5.66 mmol) and the mixture was stirred at room temperature for 30 minutes. 5N sodium hydroxide (7.55 mL, 37.8 mmol) and H 2 O 2 (30%) (7.71 mL, 76 mmol) was added and the mixture was stirred at room temperature for 19 h. The mixture was extracted with ethyl acetate (3×30 mL) and the combined organic extracts were washed with brine (30 mL) and MgSO 4 The mixture was dried at 75° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by ISCO CombiFlash® chromatography (40 g silica Gold column, 40 mL / min) (eluting with a gradient of 0-80% ethyl acetate in heptane) afforded the title compound (0.9820 g, 2.297 mmol, 60.8% yield). LCMS (m / z) 428.4 (M+H). + .

[0417] Step 5: tert-Butyl 3-(2-(2-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)ethoxy)ethoxy)propanoate. [ka]

[0418] To 2-(2-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)ethoxy)ethan-1-ol (970 mg, 2.269 mmol) in dichloromethane (DCM) (16 mL) was added tert-butyl acrylate (0.665 mL, 4.54 mmol), tetrabutylammonium hydrogen sulfate (77 mg, 0.227 mmol), and 5N sodium hydroxide (0.181 mL, 0.907 mmol). The mixture was stirred at room temperature for 21 hours and concentrated under reduced pressure. Dichloromethane (DCM) (3 mL) and water (0.181 mL) were added and the mixture was stirred at room temperature for 23 hours and concentrated under reduced pressure. Purification by ISCO CombiFlash® chromatography (24 g silica Gold column, 35 mL / min) (eluting with a stepwise gradient of 0-35% (15 min) and 35-80% (10 min) ethyl acetate in heptane) afforded the title compound (0.8950 g, 1.610 mmol, 71.0% yield). LCMS (m / z) 556.6 (M+H). + .

[0419] Step 6: tert-Butyl 3-(2-(2-(((1r,4r)-4-(2-aminoethoxy)cyclohexyl)oxy)ethoxy)ethoxy)propanoate. [ka]

[0420] A mixture of tert-butyl 3-(2-(2-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)ethoxy)ethoxy)propanoate (850 mg, 1.529 mmol) and 10% Pd / C (163 mg, 0.153 mmol) in methanol (30 mL) was stirred under hydrogen atmosphere at room temperature for 4 h. The mixture was filtered through Celite® and concentrated under reduced pressure to give the title compound (0.5599 g, 1.491 mmol, 97% yield). LCMS (m / z) 376.4 (M+H) + .

[0421] Step 7: tert-Butyl 3-(2-(2-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)ethoxy)ethoxy)propanoate [ka]

[0422] tert-Butyl 3-(2-(2-(((1r,4r)-4-(2-aminoethoxy)cyclohexyl)oxy)ethoxy)ethoxy)propanoate (550 mg, 1.465 mmol), (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (323 mg, 1.465 mmol), DIPEA (0.767 mL, 4.39 mmol), and T3P (50% wt in ethyl acetate) (1.308 mL, 2.197 mmol) were stirred at room temperature for 93 h. 3 Aqueous solution (5 drops) was added and the mixture was concentrated under reduced pressure. Purification by ISCO CombiFlash® chromatography (24 g silica Gold column, 35 mL / min) (eluting with a gradient of 0-55% ethyl acetate in heptane) afforded the title compound (0.9152 g, 1.381 mmol, 94% yield). LCMS (m / z) 578.5 (M+H). + .

[0423] Step 8: 3-(2-(2-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)ethoxy)ethoxy)propanoic acid, hydrochloride [ka]

[0424] To a solution of tert-butyl 3-(2-(2-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)ethoxy)ethoxy)propanoate (0.910 g, 1.374 mmol) in 1,4-dioxane (2 mL) was added 4M HCl in 1,4-dioxane (1.717 mL, 6.87 mmol) and the mixture was stirred at room temperature for 18 h. The mixture was concentrated under reduced pressure to give the title compound (0.8810 g, 1.085 mmol, 79% yield). LCMS (m / z) 522.4 (M+H) + .

[0425] Step 9: ((2S,3S)-N-(2-(((1r,4S)-4-(2-(2-(3-((3-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)propyl)amino)-3-oxopropoxy)ethoxy)ethoxy)cyclohexyl)oxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, formate [ka]

[0426] To a solution of tert-butyl (3-((1S,3R)-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)-1-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)propyl)carbamate (55 mg, 0.094 mmol) in 1,4-dioxane (0.2 mL) was added HCl (4M in p-dioxane) (0.235 mL, 0.941 mmol). The resulting reaction mixture was heated at ambient temperature for 20 hours. The reaction mixture was evaporated under vacuum using a biotage V10 evaporator and dissolved in dichloromethane (DCM) (0.200 mL) followed by addition of HCl (4M in p-dioxane) (0.235 mL, 0.941 mmol). The resulting reaction mixture was stirred at ambient temperature for 2 hours. The reaction mixture was evaporated under vacuum on a biotage V10 evaporator, then dichloromethane (DCM) (2 mL), 3-(2-(2-((trans-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)ethoxy)ethoxy)propanoic acid (76 mg, 0.094 mmol), DIEA (0.099 mL, 0.564 mmol), and HATU (53.7 mg, 0.141 mmol) were added. The resulting reaction mixture was stirred at ambient temperature for 91 h. The reaction was quenched with water (approximately 3 drops), evaporated under vacuum on a biotage V10 evaporator, and purified by reverse phase HPLC (HpH) to give the title compound (50.5 mg, 0.049 mmol, 51.9% yield).1H NMR (400 MHz, methanol-d4) δ ppm 1.13 - 1.44 (m, 7 H), 1.46 - 1.84 (m, 5 H), 1.84 - 2.00 (m, 6 H), 2.16 - 2.24 (m, 2 H), 2.37 (br t, J=6.11 Hz, 2 H), 2.47 - 2.60 (m, 1 H), 2.65 (s, 3 H), 2.78 - 2.85 (m, 1 H), 3.01 - 3.15 (m, 5 H), 3.21 - 3.30 (m, 5 H), 3.32 - 3.41 (m, 5 H), 3.41 - 3.56 (m, 8 H), 3.58 (s, 6 H), 3.69 (t, J=6.11 Hz, 2 H), 3.93 - 4.03 (m, 3 H), 4.24 (br d, J=12.72 Hz, 1 H), 4.82 (d, J=6.85 Hz, 1 H), 7.52 (dd, J=7.83, 4.89 Hz, 1 19F NMR (400 MHz, methanol-d4) δ ppm -63.69;LC / MS (TFA, M+H)+ 989.5 m / z, retention time 0.56 min.

[0427] The following compounds were prepared by procedures similar to those described in Example 20: [Table 7]

[0428] Example 22: (2S,3S)-N-(1-(3-(4-(6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)phenyl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azahentriacontan-31-yl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka]

[0429] Step 1: t-Butyl (1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azahentriacontan-31-yl)carbamate, trifluoroacetate. [ka]

[0430] To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (708 mg, 3.21 mmol) and HATU (1345 mg, 3.54 mmol) in N,N-dimethylformamide (DMF) under nitrogen atmosphere, DIPEA (1.404 mL, 8.04 mmol) was added dropwise and the mixture was stirred for 15 minutes. t-Butyl (29-amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosyl)carbamate (1969 mg, 3.54 mmol) in N,N-dimethylformamide (DMF) was added and the mixture was stirred at room temperature for 1.5 hours. Purification by ISCO Combiflash® Rf chromatography (C18 100 g column, 60 mL / min) (eluting with a gradient of 10-100% acetonitrile in water, both containing 0.1% trifluoroacetic acid) gave the title compound (2.8 g, 3.21 mmol, 100% yield). LCMS (m / z) 759.6 (M+H). + .

[0431] Step 2: (2S,3S)—N-(29-amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, 2-trifluoroacetate salt. [ka]

[0432] A solution of tert-butyl (1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azahentriacontan-31-yl)carbamate (2.8 g, 3.21 mmol) in dichloromethane (8 mL) and trifluoroacetic acid (8.53 mL, 111 mmol) was stirred for 1.5 h and concentrated under reduced pressure. Purification by ISCO Combiflash® Rf chromatography (C18 100 g column, 60 mL / min) (eluting with a gradient of 10-100% acetonitrile in water, both containing 0.1% trifluoroacetic acid) afforded the title compound (1.32 g, 1.49 mmol, 46.4% yield). LCMS (m / z) 659.5 (M+H) + .

[0433] Step 3: (2S,3S)-N-(1-(3-(4-(6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)phenyl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azahentriacontan-31-yl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka]

[0434] 3-(4-(6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)benzoic acid, 2-trifluoroacetate salt (1.1 g, 1.236 mmol) was dissolved in DMSO (20 mL) and triethylamine (0.7 ml, 5.02 mmol). A solution of TSTU (0.447 g, 1.483 mmol) in DMSO (2 mL) was added after 5 min and the mixture was stirred for 30 min. (2S,3S)-N-(29-amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, 2-trifluoroacetate (1.316 g, 1.483 mmol) dissolved in DMSO (4 mL) was added. The mixture was stirred at room temperature for 1.5 min and acidified with 0.5 N HCl. The mixture was purified by ISCO Combiflash® Rf chromatography (C18 100 g column, 60 mL / min) (eluted with a gradient of 10-100% acetonitrile in water, both containing trifluoroacetic acid (0.1%)). The desired fractions were concentrated under reduced pressure to remove acetonitrile and the pH was adjusted to approximately 9 with ammonium hydroxide. The residue was purified by ISCO Combiflash® Rf chromatography (C18 100 g column, 60 mL / min) (eluting with a gradient of 0-100% acetonitrile in water containing ammonium hydroxide (0.1%)) to give the title compound as a clear taffy (817 mg, 0.627 mmol, 50.7% yield). LCMS (m / z) 652.3 (M / 2+H). + . 1 H NMR (400 MHz, methanol-d 4) δ ppm 8.58 (dd, J=4.8, 1.5 Hz, 1 H) 8.53 (d, J=1.8 Hz, 1 H) 7.97 (br s, 1 H) 7.82 (dt, J=8.0, 1.9 Hz, 1 H) 7.51 - 7.59 (m, 1 H) 7.30 - 7.46 (m, 3 H) 7.09 (ddd, J=7.7, 2.6, 1.4 Hz, 1 H) 4.79 - 4.86 (m, 3 H) 4.03 - 4.17 (m, 4 H) 3.84 - 3.95 (m, 2 H) 3.49 - 3.72 (m, 37 H) 3.35 - 3.45 (m, 6 H) 3.12 - 3.20 (m, 1 H) 2.95 - 3.10 (m, 5 H) 2.79 - 2.90 (m, 2 H) 2.68 - 2.76 (m, 1 H) 2.67 (s, 3 H) 2.48 - 2.58 (m, 1 H) 2.27 - 2.41 (m, 1 H) 2.12 - 2.21 (m, 1 H) 1.85 - 1.97 (m, 5 H) 1.57 - 1.77 (m, 4 H) 1.18 - 1.32 (m, 1 H) 0.97 (br d, J=6.3 Hz, 3 H) 0.77 - 0.86 (m, 3 H).

[0435] Example 23: (2S,3S)-N-(39-(((R)-6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)amino)-39-oxo-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, triformate salt. [ka]

[0436] Step 1: 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2-azahenetetracontan-41-oic acid [ka]

[0437] N-Methylmorpholine (0.71 mL, 6.46 mmol) and 2-(2,5-dioxopyrrolidin-1-yl)-1,1,3,3-tetramethylisouronium tetrafluoroborate (0.487 g, 1.619 mmol) were added to a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (0.357 g, 1.619 mmol) in acetonitrile (10 mL). The vial was capped and the reaction was stirred at room temperature for 25 minutes. A partial slurry of commercially available 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanotriacontan-39-oic acid (1 g, 1.619 mmol) in acetonitrile (10.00 mL) and dimethylsulfoxide (DMSO) (6 mL) was added. The reaction was stirred at room temperature for 1.35 h. It was concentrated to remove acetonitrile and water (5 mL) was added. Purification by Combiflash® EZ prep chromatography (C18 50 g Gold column, 40 mL / min) (eluting with a gradient of 10-70% acetonitrile in water containing ammonium hydroxide (0.1%)) afforded the title compound as a clear pale yellow oil (0.9445 g, 1.21 mmol, 67.6% yield). LC-MS m / z 820.6 (M+H) + .

[0438] Step 2: Benzyl (R)-8-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2-azahentetracontan-41-amido)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate, diformate salt. [ka]

[0439] To a stirred solution of 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2-azahentetracontan-41-oic acid (116 mg, 0.141 mmol) in N,N-dimethylformamide (DMF) (1.0 mL) was added HATU (53.6 mg, 0.141 mmol) followed by DIPEA (0.067 mL, 0.384 mmol) and the mixture was stirred at room temperature for 10 min. Benzyl (R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (45 mg, 0.128 mmol) was added and the mixture was stirred for 45 min. Purification by reverse-phase HPLC chromatography (C18 Aq 50 g Gold column, 40 mL / min) (eluted with a gradient of 30-60% acetonitrile in water containing formic acid (0.1%)) afforded the title compound as a pale yellow oil (104.3 mg, 0.084 mmol, 65.4% yield). LC-MS m / z 577.3 (M / 2+H) + .

[0440] Step 3: (2S,3S)-1-Methyl-5-oxo-N-(39-oxo-39-(((R)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)amino)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontyl)-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka]

[0441] Benzyl (R)-8-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2- A mixture of (azahentetracontan-41-amido)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylic acid, diformate (104.3 mg, 0.084 mmol) and 20% palladium hydroxide on carbon (58.8 mg, 0.084 mmol) in methanol (2.0 mL) was sealed in a vial and purged with nitrogen, then hydrogen from a balloon was bubbled through the mixture for 5 minutes. The mixture was stirred under a hydrogen balloon at room temperature for 50 minutes. The mixture was filtered, the catalyst was washed with methanol, and the combined filtrate was concentrated under reduced pressure to give the title compound as a colorless oil (84 mg, 0.082 mmol, 98% yield). LC-MS m / z 1019.1 (M) + .

[0442] Step 4: Benzyl ((1R,3S)-3-isopropyl-3-((R)-8-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2-azahenetetracontan-41-amido)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)carbamate. [ka]

[0443] To (1S,3R)-3-(((benzyloxy)carbonyl)amino)-1-isopropylcyclopentane-1-carboxylic acid (50.3 mg, 0.165 mmol) (freshly dried by toluene azeotrope) was added dichloromethane (DCM) (1.00 mL) at room temperature. HATU (37.6 mg, 0.099 mmol) was added and the mixture was stirred for 1 min, after which DIPEA (0.115 mL, 0.659 mmol) was added. The mixture was stirred at room temperature for 18 h. A solution of (2S,3S)-1-methyl-5-oxo-N-(39-oxo-39-(((R)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)amino)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontyl)-2-(pyridin-3-yl)pyrrolidine-3-carboxamide (84 mg, 0.082 mmol) in dichloromethane (DCM) (0.50 mL) was added dropwise at room temperature and the mixture was stirred at 80° C. for 3 days. The organic solvent was boiled off and the residue was stirred at 80° C. for 2 hours. Another portion of preactivated acid (prepared as above) was added and heating was continued at 80° C. for 20 hours. The vial was cooled, water (0.50 mL) was added and heating was continued at 100° C. for 2 h. The mixture was partitioned between 1.0 M NaOH and dichloromethane (DCM). The organic phase was diluted with Na 2 SO 4 The residue was dried at 40° C., filtered and concentrated under reduced pressure. Purification by reverse-phase HPLC chromatography (XSelect™ CSH Prep C18 5 μm OBD column, 40 mL / min) (eluting with a gradient of 30-85% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonia hydroxide (0.075%)) afforded the title compound as a yellow oil (37.1 mg, 0.028 mmol, 34.5% yield). LC-MS m / z 653.8 (M / 2) + .

[0444] Step 5: (2S,3S)-N-(39-(((R)-6-((1S,3R)-1-isopropyl-3-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)amino)cyclopentane-1-carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)amino)-39-oxo-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, triformate salt. [ka]

[0445] A mixture of benzyl ((1R,3S)-3-isopropyl-3-((R)-8-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2-azahentetracontan-41-amido)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)carbamate (37.1 mg, 0.028 mmol) and 20% palladium hydroxide on carbon (19.94 mg, 0.028 mmol) was then added to a sealed flask and purged with nitrogen. Hydrogen was bubbled through the mixture for 2 minutes. The mixture was stirred at room temperature under a balloon atmosphere of hydrogen for 30 minutes. The mixture was opened to the atmosphere, 7.0 M ammonia in methanol (50 mL) was added, the mixture was stirred for 15 minutes, and filtered. The catalyst was washed with methanol, and the combined filtrates were concentrated under reduced pressure. Isopropyl acetate (1.00 mL), dichloromethane (DCM) (0.50 mL), 2-propanol (0.016 mL, 0.213 mmol), and triethylamine (0.012 mL, 0.085 mmol) were added, the mixture was placed in an ice bath for 5 minutes, sodium triacetoxyborohydride (39.7 mg, 0.187 mmol) was added, followed by dropwise addition of a solution of (R)-3-methoxytetrahydro-4H-pyran-4-one (22.17 mg, 0.170 mmol) in isopropyl acetate (1.00 mL). The mixture was stirred at 0° C. for 10 minutes, warmed to room temperature, and stirred for 1 hour. The mixture was diluted with dichloromethane (DCM) and washed with 1.0 M NaOH. The aqueous phase was extracted with dichloromethane (DCM) and the combined organic extracts were washed with Na 2 SO 4 The residue was dried at 40° C., filtered, and the filtrate was concentrated under reduced pressure. Purification by reverse-phase HPLC chromatography (XSelect™ CSH Prep C18 5 μm OBD column, 40 mL / min) (eluted with a gradient of 15-55% acetonitrile in water containing formic acid (0.1%)) afforded the title compound as a colorless foam (4.3 mg, 3.02 μmol, 10.63% yield). LC-MS m / z 644.3 (M / 2+H). + . 1H NMR (400 MHz, methanol-d 4 ) δ ppm 0.84 - 1.06 (range, 6 H) 1.66 (br s, 1 H) 1.81 (br d, J=9.3 Hz, 1 H) 2.06 (s, 4 H) 2.14 - 2.29 (m, 2 H) 2.35 (br s, 2 H) 2.58 (br s, 3 H) 2.67 (s, 3 H) 2.68 - 2.75 (m, 2 H) 2.78 - 2.92 (m, 1 H) 3.00 - 3.11 (m, 1 H) 3.45 (s, 3 H) 3.50 - 3.55 (m, 3 H) 3.55 - 3.70 (m, 48 H) 3.82 (br t, J=6.1 Hz, 3 H) 4.01 (br dd, J=12, 4.7 Hz, 1 H) 4.29 (br d, J=14 Hz, 1 H) 4.47 (br d, J=14 Hz, 2 H) 5.12 (br s, 2 H) 7.55 (dd, J=7.9, 4.9 Hz, 1 H) 7.82 (dt, J=7.9, 1.9 Hz, 1 H) 8.15 (s, 1 H) 8.30 (br s, 3 H) 8.53 (br s, 1 H) 8.59 (br d, J=4.4 Hz, 1 H) 8.84 (s, 1 H).

[0446] The following compounds were prepared by procedures similar to those described in Example 23: [Table 8-1] [Table 8-2]

[0447] Biological assays Example compounds 1-24, which are compounds of formula (I) having a CCR2 binding moiety, were tested in a variety of biological assays, which are described in more detail below.

[0448] Example 25: Antibody-dependent cellular cytotoxicity (ADCC) reporter assay An antibody-dependent cellular cytotoxicity reporter assay was performed using the following four assay components: (i) an ARM compound of formula (I) targeting CCR2 (at concentrations ranging from 1 pM to 10 μM), (ii) an anti-cotinine antibody having a heavy chain sequence of SEQ ID NO: 11 and a light chain sequence of SEQ ID NO: 12 (rabbit variable region with human IgG1 Fc domain containing DE mutations (S239D / I332E)) (at concentrations ranging from 0.01 μg / mL to 200 μg / mL), (iii) target cells: CHOK1 cells engineered to overexpress either human CCR2 (typically 1000-20,000 cells per well), and (iv) reporter cells: Jurkat cells engineered to express FcγRIIIa together with the reporter gene luciferase under the control of the NFAT promoter (typically 3000-75,000 cells per well). The reagents were combined in a final volume of 20 μL in a 384-well tissue culture treated plate. All four assay components were incubated together for approximately 12-18 hours. BioGlo detection reagent (Promega) was then added to the wells to lyse the cells and provide a substrate for the luciferase reporter protein. Luminescence signals were measured on a microplate reader and signal:background was calculated by dividing the signal in the test well by the signal obtained when no heterobivalent compound of formula (I) was added. EC50 calculations were performed using Graphpad Prism Software, specifically, nonlinear regression curve fitting (Y=Bottom+(Top-Bottom) / (1+10^((Log EC50-X)*HillSlope))).

[0449] The ARM compounds of formula (I) of the present invention were tested for ADCC activity in the above assay in one or more experimental runs, and the results are shown in Table 3 below. The potency of the compounds of formula (I) of the present invention is reported as pEC50 values. The pEC50 value is the negative logarithm of the EC50 value, where the EC50 value is the half-maximal effective concentration measured in molar (M). For compounds tested in more than one experimental run, the pEC50 value is reported as the average.

[0450] Example 26: PBMC ex vivo depletion assay A human PBMC (peripheral blood mononuclear cell) monocyte depletion flow cytometry assay was developed to screen compounds in a 384-well plate format. The assay incorporated endogenous natural killer cells to deplete endogenous target monocytes.

[0451] To screen compounds in the human PBMC monocyte depletion flow cytometry assay, 11-point dose-response curves were generated from 1 mM stocks in pure DMSO by serially diluting ARM compounds of formula (I) 4-fold per row in a 384-well plate. These serially diluted compounds were then loaded at 100 nL / well into flat-bottom polypropylene 384-well plates (Greiner Bio-One). Fifty microliters of 30 μg / mL anti-cotinine antibody with heavy chain sequence of SEQ ID NO: 11 and light chain sequence of SEQ ID NO: 12 (rabbit variable region with human IgG1 Fc domain containing DE mutations (S239D / I332E)) diluted in assay medium (RPMI 1640 (Gibco) containing 10% fetal bovine serum (Gibco), 1% Glutamax (Gibco), and 1% sodium pyruvate (Corning) was added to the wells of the plate. The plate was centrifuged at 500 rpm (revolutions per minute) for 1 minute and then shaken for 5 minutes at room temperature. The plate was incubated at 37° C. / 5% CO 2 The mixture was incubated at RT for 15 min.

[0452] Frozen PBMC cells (HemaCare BioResearch) were thawed and centrifuged at 1,200 rpm for 4 min. Cells were diluted to 2 × 10 in assay medium. 6 The cells were diluted to a cell density of 1000 cells / mL. 50 microliters of the diluted cells were added to the wells of the plate. The plate was centrifuged at 500 rpm for 1 minute and incubated at 37°C / 5% CO 2 The mixture was incubated at 4°C for 18 to 24 hours.

[0453] After 18-24 hours of incubation, 2 μL of 6.25 μg human FC block (BD Pharmingen) diluted in assay medium was added to the wells of the plate. The plate was centrifuged at 500 rpm for 1 minute and then incubated at room temperature for 15 minutes. Five microliters of 750 nM Helix NP NIR (Biolegend) diluted in assay medium was added to the wells of the plate. Then, 9 microliters of an antibody cocktail containing 1 / 5 diluted fluorescein isothiocyanate (FITC)-labeled anti-CD45 antibody (Beckman Coulter), PC7-labeled anti-CD14 antibody (Beckman Coulter), and phycoerythrin (PE) anti-CD4 antibody (Beckman Coulter) was added to the wells of the plate. The plate was centrifuged at 500 rpm for 1 minute and incubated at 4-8 °C for 30 minutes. Samples from the plate were run on a BD FACS Canto II flow cytometer and data was analyzed using FlowJo software.

[0454] The ARM compounds of formula (I) of the present invention were tested for their ability to deplete monocytes in the above assay in one or more experimental runs, and the results are shown in Table 3 below. The potency of the compounds of the present invention is reported as pEC50 values. The pEC50 value is the negative logarithm of the EC50 value, where the EC50 value is the half-maximal effective concentration measured in molar (M). For compounds tested in more than one experimental run, the pEC50 value is reported as the average or as the individual values ​​measured. [Table 9]

[0455] Example 27: In vivo depletion assay Human CCR2 (hCCR2) knock-in mice (C57 background) were intravenously administered a PBS solution containing the compound of formula (I) to measure depletion of CCR2 expressing cells in the peripheral blood. Peripheral blood from IV-administered mice was also analyzed to determine the PK properties of the ARM compound of formula (I).

[0456] Stock solution preparation: Stock solutions of the compound of formula (I) were prepared in DMSO at 20 mg / mL and 100 mg / mL for PK / PD and dose tolerability studies, respectively. The stock solutions were stored at -20°C until further use.

[0457] Preparation of formulations: On the day of the experiment, the stock solution of the compound of formula (I) was removed from storage at -20°C and thawed at room temperature. If necessary, the anti-cotinine antibody having the heavy chain sequence of SEQ ID NO: 13 and the light chain sequence of SEQ ID NO: 14 (rabbit variable region sequence with mouse IgG2a Fc domain) was removed from storage at -80°C and thawed at room temperature. The antibody vial was immediately transferred to wet ice after thawing. The compound of formula (I) was further diluted in DMSO according to the experiment requirements.

[0458] Formulation composition: The formulation composition was saline:DMSO:PBS. Saline was added based on the amount required, then a stock solution of the compound of formula (I) prepared in DMSO was added, followed by the antibody in PBS. The formulation was incubated at room temperature for 30 minutes before administration to the mice. DMSO was used at 1-2% (v / v) in the final formulation.

[0459] Animal dosing: Solution formulations of antibody and compound of formula (I) were injected (bolus injection) into restrained mice in the right / left lateral tail vein. Animals were dosed with 0.1 milligram per kilogram (mpk) of compound of formula (I) and 10 mpk of anti-cotinine antibody.

[0460] Blood collection for PK: Blood was collected (50 μL / time point) at 0.25, 2, and 4 hours post-dose via retro-orbital bleed under light isoflurane anesthesia.

[0461] Terminal bleeding at the end of the experiment (48 hours): K 2Approximately 250 μL of blood in EDTA tubes and 250 μL of blood in SST (serum separator tubes) were collected from each mouse through retro-orbital bleeding under deep isoflurane anesthesia. After bleeding, each mouse was sacrificed by cervical dislocation. Blood distribution at termination was determined as follows: For PK: 50μL of K 2 The EDTA blood was transferred to a separate tube for PK. ● Flow cytometry: The remaining blood (approximately 200 μL) was used for flow cytometry analysis. For serum collection: Serum separator tubes (BD) were centrifuged at 5000 rpm for 15 minutes at 4° C. Approximately 100 μL of serum was separated and stored at -80° C. for further use. - Blood drug concentrations: Samples collected at various time points (0.25 h, 2 h, 24 h, and 48 h) were analyzed to determine blood drug concentrations.

[0462] Flow cytometry analysis: CCR2 depletion in peripheral blood was determined by flow cytometry. Briefly, peripheral blood was collected and processed from all animals at the end of the study. Blood samples were lysed using 1X RBC lysis buffer and the resulting cell pellet was washed twice in FACS buffer (HBSS containing 5% FBS). Cells were subjected to Fc blocking using mouse Fc block from Biolegend (Trustain, anti-mouse CD16 / 32, Cat:101319) to block non-specific binding, followed by surface staining for immune markers (CD45, CD3, CD11b, Ly6C, human CCR2). Surface staining was performed for 30 minutes in ice and cells were washed twice to remove unbound antibodies. Cells were stained with 7AAD to determine viability and acquired on a BD FACSVerse flow cytometer. Appropriate FMO and single-color controls were included in the experiments for gating and compensation, respectively.

[0463] CCR2+ monocytes were gated as CD11b+Ly6C+ high, medium, and low populations, and treatment induced a reduction in CD11b+Ly6c+ high cells (i.e., cells with high CCR2 expression). The depletion rate of CD11b+Ly6c+ high cells and CCR2 expression was determined for the different treatment groups. The results are shown in Table 4 below and are reported as the depletion rate (%) of CCR2 expressing cells.

[0464] Blood Drug Concentration Analysis: Drug concentrations in blood samples were determined by an LC-MS / MS based bioanalytical method developed at Syngene. Samples were analyzed on a Q-Trap, API-5500 LC-MS / MS system coupled with an Exion UHPLC system from SCIEX (USA) operated in multiple reaction monitoring mode employing electrospray ionization technique in positive polarity. Analyte and internal standard peaks were resolved on a Synergi Polar, 75×2.0 mm, 4μ column using mobile phases, i.e., 10 mM ammonium acetate in Milli-Q water as A phase and 0.1% formic acid in acetonitrile as B phase. Gradient elution was performed with an initial composition of 95% A phase at 0.0 min, held for 0.2 min, ramped to 5% by 1.0 min, maintained the same for the next 0.5 min, and returned to 95% by 1.6 min. The total run time was 2 min.

[0465] Working dilutions for the calibration curve and quality control standards were prepared by serially diluting a 20 mg / mL stock solution with DMSO. Spiking concentrations for the calibration curve in whole blood ranged from 1 ng / mL to 1000 ng / mL. A working solution of the internal standard (Verapamil, 25 ng / mL) was prepared in acetonitrile. 10 μL of the study samples, as well as the calibration curve, quality control, and blank whole blood samples, were aliquoted in a 96 deep-well plate for processing. 10 μL of Milli-Q water was added to all samples and vortexed briefly to initiate complete hemolysis. 10 μL of 20 mM dithiothreitol (DTT) was added to all samples and incubated at 37 °C for 30 min. The addition of DTT enhanced the recovery of the ARM compound of formula (I) from biological matrices. 300 μL of the working internal standard solution was added to all samples except for all blanks and wash samples, which were added with 300 μL of acetonitrile. All samples were vortex mixed for 5 minutes, followed by centrifugation at 4000 rpm for 10 minutes at 4° C. The supernatant was transferred to a loading plate and 3 μL was injected into the LC-MS / MS system for analysis. The results are shown in Table 4 below and are reported as compound exposure 48 hours after dosing. [Table 10]

[0466] Example 28: Binding of anti-cotinine antibodies to ARM compounds of formula (I) as measured by surface plasmon resonance (SPR) Anti-cotinine antibody with heavy chain sequence of SEQ ID NO: 11 and light chain sequence of SEQ ID NO: 12 (rabbit variable region with human IgG1 Fc domain containing DE mutations (S239D / I332E)) was captured on one or more flow cells of a Protein A sensor chip (Cytiva) using a Biacore T200 while retaining flow cell 1 as a reference. After capture, a waiting step of 3000 seconds was included to reduce drift during compound analysis. ARM compounds of formula (I) were then injected at 100 μL / min with association and dissociation times of 200 and 2000 seconds. The entire experiment was carried out at 37° C. using a running buffer containing 10 mM HEPES, 150 mM NaCl, 0.005% P20, and 1% DMSO at pH 7.4. ARM compounds of formula (I) were titrated at a top concentration of 200 nM using a 3-fold 5-point dilution series, and the corresponding 5 injection buffer cycles were performed for blank subtraction. Data were double referenced by subtracting the response of the reference flow cell from the response of the antibody-containing flow cell, followed by subtraction of the reference blank sensorgram. Following compound analysis, the surface was regenerated using a 30 second injection of glycine, pH 1.5, at a flow rate of 30 μL / min, after which the antibody was recaptured. Experiments were run using the Biacore T200 Control Software and evaluated using the Biacore T200 Evaluation Software. Curves were fitted to a 1:1 kinetic binding model to determine k on (1 / Ms), k off (1 / s), k off / k on k is determined as d (M), and 1 / k off The residence time, determined as (s), was obtained.

[0467] Anti-cotinine antibodies were tested for binding to the ARM compounds of formula (I) of the invention in the above assay in one or more experimental runs, and the results are shown in Table 5 below. Affinity is expressed as pK d It is reported as a pK value. d The value is K d is the negative logarithm of the value of K dValues ​​are dissociation constants measured in molar (M) as above. For compounds tested in more than one experimental run, the pK d Values ​​are reported as averages. [Table 11]

[0468] Example 29: Nicotinic acetylcholine receptor antagonism assay Cells expressing human alpha1 nicotinic acetylcholine (nACh) receptors (nAChRs) were washed and dissociated using a trypsin-containing buffer. Cells were then washed and plated at a density of 3-5 million cells / mL and allowed to grow for 3 days. 60-80 μL of cells were then added to the plate and incubated with a dose response of ARM compound of formula (I) with a highest concentration of either 10 μM or 100 μM. Receptors were then stimulated via addition of acetylcholine (ACh) and receptor activation was monitored via an IONFlux automated patch clamp system. Leak subtraction was performed and (minimum) peak current amplitudes were measured utilizing IonFlux Data Analyzer software. These data were then exported to Excel.

[0469] Data processing: (1) Current values ​​recorded for vehicle-only applications in each trap / pattern were subtracted from the corresponding current amplitude values ​​for control, test article, and control + test article. (2) Control responses were calculated as % of the average response of the third of the total three ACh (10 mM) applications alone. In rare circumstances, when the third ACh response was obscured due to technical artifacts, the second and / or first ACh (10 mM) applications were considered as controls. The peak current amplitudes generated in the presence of each test article concentration were then used to calculate % inhibition relative to the control response (10 mM ACh alone). These estimates were further corrected for vehicle responses and rundown corrected by normalizing with the time-matched control response (10 mM ACh alone). The corrected percentages were then fitted with a nonlinear function in GraphPad Prism 6 (or later versions) to determine IC50.

[0470] Exclusion criteria: (i) any n with poor recording quality, (ii) control current amplitude <500 pA, or (iii) significant outliers as determined by Grubbs' test.

[0471] The ARM compounds of formula (I) of the present invention were tested for nAChR antagonism in the above assay in one or more experimental runs, and the results are shown in Table 6 below. As shown in Table 6, the antagonistic activity of the compounds of formula (I) of the present invention is reported as IC50 values ​​(half-maximal inhibitory concentration), and the maximum antagonism is observed at the highest concentration of the compounds of formula (I) tested. For compounds tested in more than one experimental run, the IC50 values ​​are reported as averages. [Table 12]

[0472] Example 30: In vivo mouse study (muScreen) The effect of treatment with a combination of CyTaC compound of formula (I) and anti-cotinine antibody on tumor growth was evaluated in multiple in vivo mouse models, including KPC, pan02, b16BL6, H22, RENCA, RM1, B16F10, EMT-6, LLC, CT26, MC38, A20, and Hepa1-6 models.

[0473] Treatment and analysis of tumor-bearing mice: Tumor cells were injected subcutaneously into the flank of mice (syngeneic to the tumor cells being injected). Tumor volume was measured using a digital Vernier caliper to measure the length (l) and width (w) of the tumor. Tumor volume was calculated using the following formula: tumor volume (mm 3 )=L×W2 / 2 (L=length (mm), W=width (mm)). Anti-cotinine antibody having a heavy chain sequence of SEQ ID NO:13 and a light chain sequence of SEQ ID NO:14 (rabbit variable region sequence with mouse IgG2a Fc domain) and CyTaC compound of Example 22 were diluted in saline and administered intravenously to a final concentration of 0.1 or 0.2 mpk of CyTAC compound and 10 mpk of anti-cotinine antibody. Flow cytometry of peripheral blood was performed as described above in Example 27. Flow cytometric analysis of tumors: The tumor isolation procedure was performed in a laminar flow hood according to aseptic technique. All dissection instruments used during the procedure were sterilized. After use, the instruments were stored in 70% ethanol until further use. Subcutaneous tumor-bearing mice were humanely euthanized using carbon dioxide asphyxiation, sprayed with 70% ethanol, and then wiped with betadine to remove loose hair. The skin at the tumor site was lifted using forceps and cut off with a pair of dissecting scissors to expose the subcutaneous tumor. The exposed tumor was cut and removed using a small pair of dissecting scissors. The tumor samples were placed in a 50 mL Falcon filled with RPMI 1640 serum-free medium and further processed for flow analysis. For blood-derived samples, antibody staining was performed.

[0474] Survival analysis: Tumors 1500 mm 3 Once tumor ulceration was reached or tumor ulceration was visible, animals were humanely euthanized.

[0475] The results are shown in Figures 1 to 6.

[0476] Example 31: In vivo mouse study - Thioglycollate-induced peritonitis model The ability of the CyTaC compound of formula (I) in combination with an anti-cotinine antibody to inhibit CCR2-mediated chemotaxis was evaluated in a thioglycollate-induced peritonitis mouse model to demonstrate the ability of this combination to antagonize CCR2 activity in vivo.

[0477] Preparation of Thioglycollate Medium (Sigma, B2551-500G): Thioglycollate medium powder (4-6 grams * ) was dissolved in 100 mL of MilliQ water and kept in a water bath to completely dissolve the medium. The medium was then autoclaved at 121°C for 15 minutes. The autoclaved medium was wrapped in aluminum foil and stored at 4-8°C for 2 weeks before administration. The aging process, during which the medium turns brown, is essential for efficient induction of peritonitis. ( * NOTE: Some studies have used 4% w / v thioglycollate medium, others 6% w / v).

[0478] Induction of disease: Mice were injected intraperitoneally (ip) with thioglycollate medium aged for 2 weeks at a dose of 1 ml of 4% w / v thioglycollate medium or 0.7 mL of 6% w / v thioglycollate medium.

[0479] The anti-cotinine antibody used in this study had a rabbit variable region sequence and a mouse IgG2a Fc domain, and had its ADCC activity abolished by p-mutation (L234A / L235A). Mice were administered 10 mpk or 50 mpk of anti-cotinine antibody and CyTaC compounds ranging from 0.001 mpk to 0.5 mpk.

[0480] Collection of peritoneal lavage fluid: Peritoneal lavage of mice was performed at different time points (6 h, 24 h, 48 h, and 72 h) after thioglycollate administration. For peritoneal lavage, mice were injected with 10 ml of ice-cold PBS (containing 3% v / v FBS) using a 27 g needle. After injection, the peritoneal cavity was gently massaged for 2 min to remove attached cells and place them in the lavage fluid, ensuring a homogenous cell suspension in the peritoneal cavity. After completion of abdominal massage, the lavage fluid was collected by aspiration. A 25 G needle attached to a 10 mL syringe was inserted into the peritoneal cavity, and as much peritoneal fluid as possible was pumped into the syringe by aspiration. The collected peritoneal lavage fluid was stored on ice until TLC and DLC analysis was performed.

[0481] Flow cytometry and cytokine analysis: 2 ml of peritoneal lavage fluid was collected for flow cytometry. 1 ml of peritoneal lavage fluid was centrifuged at 10,000 rpm at 4°C, and the supernatant was stored at -80°C for cytokine analysis.

[0482] The results are shown in Figures 10A-10B.

[0483] Example 32: In vivo mouse studies - Collagen-induced arthritis (CIA) mouse model The effectiveness of depleting CCR2 monocytes by combined treatment of CyTaC compound of formula (I) and anti-cotinine antibody was evaluated in collagen-induced arthritis model.The results suggest that combined treatment of CyTaC compound of formula (I) and anti-cotinine antibody may provide therapeutic benefit to autoimmune disorders.

[0484] Preparation of 0.05M Glacial Acetic Acid: Sterile water for injection (10 mL) was measured and added to a 15 mL Falcon tube, 28.6 μl was removed and replaced with 28.6 μl of Glacial Acetic Acid.

[0485] Preparation of immune grade type II collagen (CII): Type II collagen was prepared by adding an appropriate amount of 0.05 M acetic acid solution to make a 4 mg / mL type II collagen solution one day before immunization. The vial was stored overnight at 4°C. On the day of the experiment (day 0), the solution was diluted (1:1) with 0.05 M acetic acid to obtain a final concentration of 2 mg / mL collagen.

[0486] Day 0 Immunization Procedure: ● Preparation of emulsion: 2 mg / mL collagen (CII) was emulsified with 2 mg / mL Freund's complete adjuvant (CFA) in a 1:1 ratio to give a final concentration of 1 mg / mL CII and 1 mg / mL CFA. One portion of the CFA was added to a sealed 5 mL glass syringe. An equal volume of collagen (CII) was then slowly added dropwise to the CFA suspension while mixing at low speed with a homogenizer. Once thoroughly mixed, homogenization was carried out at maximum speed for 2 minutes. The emulsion was then cooled by keeping the syringe in ice for 5 minutes. This procedure was repeated three times to obtain a stable (droplet of emulsion does not disperse when placed in water) homogenous emulsion. The emulsion was transferred from the glass syringe to a 1 mL syringe (BD). Administration of emulsion: Animals were lightly anesthetized with isoflurane. 100 μL of emulsion was injected subcutaneously approximately 2 cm from the base of the tail.

[0487] Immunization Procedure on Day 21: Preparation of emulsion: 2 mg / mL collagen (CII) was emulsified with Freund's incomplete adjuvant (IFA) in a 1:1 ratio to a final concentration of 1 mg / mL CII. One portion of the IFA was added to a sealed 5 mL glass syringe. An equal volume of collagen (CII) was then slowly added dropwise to the CFA suspension while mixing at low speed with a homogenizer. Once thoroughly mixed, homogenization was carried out at maximum speed for 2 minutes. The emulsion was then cooled by keeping the syringe in ice for 5 minutes. This procedure was repeated three times to obtain a stable (droplet of emulsion does not disperse when placed in water) homogenous emulsion. The emulsion was transferred from the glass syringe to a 1 mL syringe (BD). Administration of emulsion: Animals were lightly anesthetized with isoflurane. 100 μL of emulsion was injected subcutaneously approximately 1 cm from the base of the tail. Scoring: A qualitative scoring of the severity of paw inflammation was performed using the criteria shown in Table 7 below. [Table 13] ● Start of treatment: On the day of disease onset (day 26-54) Dexamethasone (comparator) - 0.1 mg / kg, orally, daily Enbrel (comparison) - 10mg / kg, intraperitoneal administration A compound of formula (I) (compound of Example 22) + anti-cotinine antibody - administered intraperitoneally once a week. Clinical signs: from day 22 (the day after booster administration) to the end of the study (day 54) Foot thickness: Using a Mitutoyo micrometer at baseline, day 21 (booster day), day 36, and day 54 Paw weight: weight of all four paws (i.e. both hind and forelimbs), excised at the hairline at termination (day 54) Cytokine analysis: 14-plex mouse 14-plex inflammatory panel in forelimb serum and paw homogenates Serum cartilage degradation markers: CTXII and COMP in serum obtained on day 54 (using a commercially available ELISA kit) ●Autoantibody level serum: Mouse anti-bovine type II collagen IgG antibody assay (using ELISA) ●Flow: Peripheral blood - CD45, CD3, CD11b, Ly6c, mCCR2, CD4, PD1, CXCR5 ●Flow: Synovial lavage fluid - CD45, CD3, CD11b, Ly6c, mCCR2, CD4, PD1, CXCR5 Histopathological examination: The ankle joints of the hind limbs were harvested and decalcified in 10% NBF, then stained for cartilage degradation and infiltration.

[0488] The results are shown in Figure 9. In Figure 9, "Sham control" refers to mice that did not undergo collagen induction so as not to develop the "arthritic" phenotype, and "pathological control" refers to mice that were injected with collagen but did not receive any therapeutic intervention.

[0489] Example 33: In vivo mouse studies - Efficacy of combination treatment with anti-PD-1 or STING agonists The CyTaC compound of Example 22 was evaluated for efficacy in vivo when combined with an anti-cotinine antibody and further with anti-PD-1 or STING agonists.

[0490] Combination with anti-PD-1 Dosing and analysis of tumor-bearing mice: EMT-6 tumor cells were injected subcutaneously into the flank of mice (syngeneic to the tumor cells being injected). Tumor volume was measured using a digital Vernier caliper to measure the length (l) and width (w) of the tumor. Tumor volume was calculated using the following formula: tumor volume (mm 3 ) = L × W2 / 2 (L = length (mm), W = width (mm)). Tumor volume is 50-100 mm 3 Animals were randomized when tumor volumes reached 3000 mm and treatment was initiated after randomization. 3Animals exceeding this limit were sacrificed. Anti-cotinine antibody and CyTAC compound of formula (I) (Example 22) were diluted in saline and administered intravenously to a final concentration of 0.5 mpk CyTAC (administered twice a week) and 10 mpk anti-cotinine antibody (administered once a week). Anti-PD1 antibody was administered at 10 mpk once a week.

[0491] The results are shown in Figures 8A-8B.

[0492] Combination with STING agonists The efficacy of the CyTaC compound of Example 22 in combination with an anti-cotinine antibody and further in combination with a STING agonist was evaluated in vivo in the CT26 mouse model (a syngeneic mouse model of murine colon cancer).

[0493] Female BALB / c mice aged 6-8 weeks were used in this study. Mice were subcutaneously inoculated with 5e4 CT-26 cells in the right hind flank, and tumors were measured by digital calipers to obtain tumor volume TV=0.52*L*W. 2 Applying this formula, it is about 100 mm. 3 Tumors were allowed to grow until the tumors reached 2000 mm3 (day 0). On day 0, mice were randomized into test groups, n=10 per group, by tumor volume and administered the first dose. Mice were dosed as shown in the table below. Aspartic acid was administered as a vehicle control for STING agonist treatment. Tumor volumes and body weights were measured three times a week during the study period, and mice were maintained at 2000 mm3 or higher when tumor volumes reached 2000 mm3. 3 The endpoint was reached when the tumor volume exceeded 100 μg / kg, when tumors developed open ulcers, or when mice lost more than 20% body weight. [Table 14]

[0494] The results are shown in FIG.

[0495] Sequence Listing Heavy chain CDR1 amino acid sequence SEQ ID NO:1 NYWMS

[0496] Heavy chain CDR2 amino acid sequence SEQ ID NO:2 DIHGNRGFNYHASWAKG

[0497] Heavy chain CDR3 amino acid sequence SEQ ID NO:3 ADDSGSHDI

[0498] Light chain CDR1 amino acid sequence SEQ ID NO:4 QSSQSVYSAKLS

[0499] Light chain CDR2 amino acid sequence SEQ ID NO:5 YGSTLAS

[0500] Light chain CDR3 amino acid sequence SEQ ID NO:6 QGTFYGPDWYFA

[0501] Variable heavy chain amino acid sequence SEQ ID NO:7 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVGDIHG NRGFNYHASWAKGRFTVSRSKNTLYLQMNSLRAEDTAVYYCAKADDSGSHDIW GQGTLVTVSS

[0502] Variable light chain amino acid sequence SEQ ID NO:8 DIQMTQSPSSLSASVGDRVTITCQSSQSVYSAKLSWYQQKPGKAPKLLIYYGSTLASGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCQGTFYGPDWYFAFGGGTKVEIK

[0503] Heavy chain amino acid sequence SEQ ID NO:9 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVGDIHGNRGFNYHASWAKGRFTVSRSKNTLYLQMNSLRAEDTAVYYCAKADDSGSHDIWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0504] Light chain amino acid sequence SEQ ID NO:10 DIQMTQSPSSLSASVGDRVTITCQSSQSVYSAKLSWYQQKPGKAPKLLIYYGST LASGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCQGTFYGPDWYFAFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

[0505] Heavy chain amino acid sequence SEQ ID NO:11 [ka]

[0506] Light chain amino acid sequence SEQ ID NO:12 [ka]

[0507] Heavy chain amino acid sequence SEQ ID NO:13 [ka]

[0508] Light chain amino acid sequence SEQ ID NO:14 [ka]

Claims

1. Compounds of formula (I): 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof, During the ceremony, T, 【Chemistry 2】 and R １ But, C １～４ Alkyl or C ３～６ is cycloalkyl, L is a bivalent linker of formula (La), (Lb), (Lc), (Ld), or (Le): 【Chemistry 3】 or a stereoisomer thereof, During the ceremony, Ring A and ring B are each independently represented by C ４～６ is cycloalkylene, L １ａ But, C ３～５ A straight chain alkylene having one or two methylene units, -O- or -NR ａ is replaced by - Each R ａ are independently hydrogen or C １～３ is alkyl, L ２ａ is -O-, -NHC(O)-, or -CH ２ or a stereoisomer thereof; 【Chemistry 4】 or a stereoisomer thereof, During the ceremony, Ring A is C ４～６ Cycloalkylene or C ７～９ is a bridged bicyclic cycloalkylene; L １ｂ But -CH ２ -NH-C(O)-, -NHC(O)-, or -C(O)NH-; L ２ｂ But, C ６～１２ A straight chain alkylene in which 1, 2, 3, or 4 methylene units are -O-, -NR １ｂ --, --C(O)NR １ｂ - or -NR １ｂ is replaced by C(O)-, or L ２ｂ but 【Chemistry 5】 where n is 1, 2, 3, or 4; 【Chemistry 6】 But, L １ｂ represents a covalent bond to Each R １ｂ are independently hydrogen or C １～３ the bivalent linker or a stereoisomer thereof which is alkyl; 【Chemistry 7】 or a stereoisomer thereof, During the ceremony, L １ｃ But, C ２～１０ a straight chain alkylene in which 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 is a bridged bicyclic cycloalkylene; L ２ｃ is -O- or saturated C ２～１０ a bivalent linker which is a straight chain alkylene in which one, two or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-, or a stereoisomer thereof; 【Chemistry 8】 And, During the ceremony, L １ｄ But, C １２～３０ the bivalent linker which is a straight chain alkylene in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 methylene units are replaced with -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-; or 【Chemistry 9】 wherein n is an integer from 3 to 50; each 【Chemistry 10】 represents a covalent bond to the Y group of formula (I) or, when Y is a bond, represents a covalent bond to the T group of formula (I); 【Chemistry 11】 represents a covalent bond to the methylene group of formula (I), The compound, or a pharma- ceutically acceptable salt thereof, wherein Y is a bond or a divalent spacer moiety 1 to 12 atoms in length.

2. R １ But -CH ３ 2. The compound of claim 1, wherein:

3. L is a bivalent linker of formula (L-ai): 【Chemistry 12】 or a stereoisomer thereof, Wherein, ring A, L １ａ , L ２ａ , 【Chemistry 13】 is as defined for formula (La), or a pharma- ceutically acceptable salt thereof.

4. L is a bivalent linker of formula (L-a-ii): 【Chemistry 14】 or a stereoisomer thereof, Where, L １ａ , L ２ａ , 【Chemistry 15】 is as defined for formula (La), p is 1 or 2, and m is 1 or 2, or a pharma- ceutically acceptable salt thereof.

5. L is a bivalent linker of formula (L-a-iii): 【Chemistry 16】 or a stereoisomer thereof, In the formula, p is 1 or 2, m is 1 or 2, and n is 1, 2, or 3; 【Chemistry 17】 is as defined for formula (La), or a pharma- ceutically acceptable salt thereof.

6. L, 【Chemistry 18-1】 【Chemistry 18-2】 The compound according to any one of claims 1 to 4, wherein the bivalent linker has the formula (La) selected from the group consisting of:

7. L is a bivalent linker of formula (L-bi): 【Chemistry 19】 or a stereoisomer thereof, Where, L １ｂ , L ２ｂ , 【Chemistry 20】 is as defined for formula (L-b), p is 1 or 2, and m is 1 or 2, or a pharma- ceutically acceptable salt thereof.

8. L, 【Chemistry 21-1】 【Chemistry 21-2】 【Chemistry 21-3】 The compound of any one of claims 1, 2, or 7, wherein the bivalent linker has the formula (Lb) selected from the group consisting of:

9. L is a bivalent linker of formula (L-ci): 【Chemical 22】 or a stereoisomer thereof, Where, L １ｃ , L ２ｃ , 【Chemistry 23】 is as defined in formula (Lc), p is 1 or 2, and m is 1 or 2, or a pharma- ceutically acceptable salt thereof.

10. L, 【Chemistry 24-1】 【Chemistry 24-2】 The compound according to claim 1 or 2, wherein the bivalent linker is of formula (Lc) selected from the group consisting of:

11. L, 【Chemistry 25-1】 【Chemistry 25-2】 3. The compound according to claim 1 or 2, wherein the bivalent linker is of the formula (L-d) selected from the group consisting of:

12. Y is a bond, -NH-, -(C １～１２ alkylene)-, in which one, two or three methylene units are -O-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C ３～６ Cycloalkylene)-, -(C ３～６ cycloalkenylene)-, 3- to 6-membered heterocycloalkylene, arylene, or heteroarylene; １～１２ alkylene)- or -(C ２～１２ alkenylene)-, in which one, two or three methylene units are -O-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C ３～６ Cycloalkylene)-, -(C ３～６ cycloalkenylene)-, 3- to 6-membered heterocycloalkylene, arylene, or heteroarylene; ２～１２ 2. A compound according to any one of the preceding claims, selected from: -, -(alkylene)-, or a pharma- ceutically acceptable salt thereof.

13. Y is a bond, -NH-, -(C １～６ alkylene)-O-, -(C ２～６ alkenylene)-O-, -(C １～６ alkylene)-C(O)-, -(C ２～６ alkenylene)-C(O)-, phenylene, piperidinylene, -(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 -O-, -NH-(C １～５ alkylene)-NH-, -(C ３～６ Cycloalkylene)-NH-, -(C ３～６ cycloalkenylene)-NH-, or 【Chemistry 26】 The -(C １～５ (alkylene)-NH-, １ａ is a bond, —O—, —NH—, —NHC(O)—, —C(O)NH—, or C １～３ is alkylene, and Y ２ａ is a bond, —O—, —NH—, —NHC(O)—, —C(O)NH—, or C １～３ 2. A compound according to any one of the preceding claims, or a pharma- ceutically acceptable salt thereof, which is alkylene.

14. Y, 【Chemistry 27】 13. A compound according to any one of the preceding claims, selected from the group consisting of:

15. 2. The compound of claim 1, wherein the compound is selected from the compounds set forth in Table 1.

16. The compound is 【Chemistry 28】 2. The compound of claim 1 selected from the group consisting of:

17. A method for treating and / or preventing a disease or disorder in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound described in any one of the preceding claims and an anti-cotinine antibody, or an antigen-binding fragment thereof, wherein the disease or disorder is selected from cancer, an inflammatory disease, an autoimmune disease, a viral infection, or a bacterial infection.

18. 18. The method of claim 17, wherein the disease or disorder is mediated by chemokine receptor 2 (CCR2) and / or is associated with CCR2-positive pathogenic cells.

19. 19. The method of claim 17 or 18, wherein the disease is a cancer that is a solid tumor.

20. 20. The method of any one of claims 17 to 19, wherein the cancer is selected from non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), colorectal cancer (CRC), cervical squamous cell carcinoma (CESC), head and neck squamous cell carcinoma (HNSC), pancreatic cancer, metastatic castration-resistant prostate cancer (mCRPC), ovarian cancer, bladder cancer, or breast cancer.

21. The method of any one of claims 17 to 20, wherein the compound and the antibody, or antigen-binding fragment thereof, are administered simultaneously.

22. The method of any one of claims 17 to 20, wherein the compound and the antibody, or antigen-binding fragment thereof, are administered sequentially.

23. A method for increasing antibody-dependent cellular cytotoxicity (ADCC) of a C-C motif chemokine receptor 2 (CCR2) expressing cell, the method comprising contacting the cell with an effective amount of a compound according to any one of claims 1 to 16 and an anti-cotinine antibody, or an antigen-binding fragment thereof, wherein the CCR2 binding portion of the compound binds to the CCR2 expressed on the cell.

24. 19. A method for depleting C-C motif chemokine receptor 2 (CCR2) expressing cells, the method comprising contacting the cells with an effective amount of a compound according to any one of claims 1 to 16 and an anti-cotinine antibody, or an antigen-binding fragment thereof, wherein the CCR2 binding portion of the compound binds to the CCR2 expressed on the cells.

25. 25. The method of claim 23 or 24, wherein the CCR2 expressing cell is a myeloid derived suppressor cell (MDSC), a T regulatory cell (Treg), a neutrophil, a macrophage, a B regulatory cell (Breg), a CD8 regulatory cell, (CD8reg), an exhausted T cell, or a cancer associated fibroblast (CAF).

26. The method according to any one of claims 17 to 25, wherein the anti-cotinine 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.

27. The method according to any one of claims 17 to 26, wherein the anti-cotinine 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.

28. The method according to any one of claims 17 to 27, wherein the anti-cotinine antibody is of the IgG1 isotype, comprising substitutions in the Fc region to increase ADCC activity.

29. 29. The method of claim 28, wherein the substitutions in the Fc region are S239D / I332E, where residue numbering is according to the EU index.

30. The method according to any one of claims 17 to 29, wherein the anti-cotinine antibody has a heavy chain comprising SEQ ID NO:9 and a light chain comprising SEQ ID NO:

10.

31. A combination comprising a compound according to any one of claims 1 to 16 and an anti-cotinine antibody, or an antigen-binding fragment thereof.

32. The combination described in claim 31, wherein the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising a CDR1 having SEQ ID NO: 1, a CDR2 having SEQ ID NO: 2, and a CDR3 having SEQ ID NO: 3, and the light chain comprising a CDR1 having SEQ ID NO: 4, a CDR2 having SEQ ID NO: 5, and a CDR3 having SEQ ID NO:

6.

33. The combination described in claim 31 or 32, wherein the anti-cotinine 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.

34. A combination according to any one of claims 31 to 33, wherein the anti-cotinine antibody is of the IgG1 isotype, comprising substitutions in the Fc region to increase ADCC activity.

35. 35. The combination of claim 34, wherein the substitutions in the Fc region are S239D / I332E, where residue numbering is according to the EU index.

36. The combination according to any one of claims 31 to 35, wherein the anti-cotinine antibody has a heavy chain comprising SEQ ID NO:9 and a light chain comprising SEQ ID NO:10.

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