Cytotoxic targeting chimeras for C-C chemokine receptor 2 expressing cells

Abstract

The present disclosure relates to heterobifunctional molecules, referred to as cytotoxic targeting chimeras (CyTaCs) or antibody recruitment molecules (ARM), capable of binding both target cell surface proteins and exogenous antibody proteins. The disclosure also relates to agents capable of binding to receptors on the surface of pathogenic cells and inducing depletion of pathogenic cells in a subject for use in the treatment of cancer, inflammatory diseases, autoimmune diseases, viral infections or bacterial infections.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Patent Application No. 63 / 314,034, filed on February 25, 2022, which is incorporated herein by reference in its entirety.

[0003] FIELD OF THE DISCLOSURE

[0004] The present disclosure relates to heterobifunctional molecules capable of simultaneously binding to target cell surface proteins and exogenous antibody proteins, which are referred to as cytotoxic targeting chimeras (CyTaCs) or antibody recruiting molecules (ARMs). The present disclosure also relates to agents capable of binding to receptors on the surface of pathogenic cells and inducing depletion of pathogenic cells in a subject, which are used to treat cancer, inflammatory diseases, autoimmune diseases, viral infections or bacterial infections.

[0005] background

[0006] Cell surface proteins and their ligands play a key role in a series of inflammatory, infectious and autoimmune diseases as well as tumor initiation, growth and metastasis. Antibody-based therapeutic agents have promising performance as drug candidates for these indications because they are selective for pathogenic cell surface targets, and they can guide immune surveillance to tissues or cells expressing targets to induce the exhaustion of pathogenic cells. Examples of such exhaustion mechanisms include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). However, antibody-based therapeutic agents often lack bioavailability, high cost, thermal instability and manufacturing difficulties due to their size, complexity and peptide-based structures. On the contrary, small molecule therapeutic agents often provide affordability, stability and convenience of oral administration, but may have poor selectivity and off-target effects, while also lacking the immune control of therapeutic antibodies.

[0007] Therefore, there is a need for improved therapeutic regimens that target pathogenic cells for treating disease. Such compositions and related methods are provided in the present disclosure. SUMMARY OF THE INVENTION

[0009] In one aspect, the present disclosure provides heterobifunctional molecules referred to as cytotoxic targeting chimeras (CyTaCs) or antibody recruiting molecules (ARMs), wherein the ARM comprises a portion that binds a target cell surface protein on a cell and a portion that binds an exogenous antibody. In another aspect, the ARM comprises a bivalent linker that connects the target binding portion to the antibody binding portion. In another aspect, the target binding portion is a CC chemokine receptor type 2 (CCR2)-binding portion. In another aspect, the exogenous antibody is an anti-cotinine antibody or an antigen-binding fragment thereof.

[0010] In another aspect, the ARM is a compound of formula (I):

[0011]

[0012] or a pharmaceutically acceptable salt thereof,

[0013] in:

[0014] T is

[0015] R 1 It is C 1-4 Alkyl or C 3-6 Cycloalkyl;

[0016] Y is a bond or a divalent spacer moiety of 1 to 12 atoms in length; and

[0017] L is a divalent linking group as described herein.

[0018] In one aspect, the present disclosure provides a method of treating and / or preventing a disease or disorder in a patient in need thereof, the method comprising administering to the patient a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof.

[0019] In one aspect, the present disclosure provides a method of increasing antibody-dependent cell cytotoxicity (ADCC) of CCR2-expressing cells, the method comprising contacting the cells with a compound of formula (I) disclosed herein and an anti-cotinine antibody or an antigen-binding fragment thereof.

[0020] In one aspect, the disclosure provides a method of depleting CCR2 expressing cells, the method comprising contacting the cells with a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof.

[0021] In one aspect, the disclosure provides a compound of formula (I) disclosed herein for use in therapy. In another aspect, the disclosure provides a combination for use in therapy comprising a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof.

[0022] In one aspect, the present disclosure provides a combination for use in treating a disease or disorder comprising a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof.

[0023] In one aspect, the present disclosure provides the use of a compound of formula (I) disclosed herein in the preparation of a medicament for treating a disease or disorder. In another aspect, the present disclosure provides the use of a combination comprising a compound of formula (I) disclosed herein and an anti-cotinine antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating a disease or disorder.

[0024] 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.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A , Figure 1B and Figure 1C shows the analysis of various syngeneic mouse tumors administered CyTaCs compounds (compounds of Example 22) in the presence of anti-cotinine antibodies; Figure 1A and Figure 1B Tumor volumes of tumor-bearing mice treated with the CyTaC compound of Example 22 and anti-cotinine antibody or PBS as a control are shown; the tumor volume of each tumor model on the last day of the study is shown; Figure 1C Shown are tumor volumes of mice bearing EMT-6 tumors when administered PBS or the CyTaC compound of Example 22 in combination with an anti-cotinine antibody; each line represents a single mouse in the same experiment.

[0027] Figure 2A and Figure 2B shows depletion of monocytes in the blood of tumor-bearing animals after treatment with CyTaC compound (compound of Example 22) and anti-cotinine antibody; Figure 2A Depletion of monocytic (Ly6Chi / CD11b+) cells in various tumor models was demonstrated; Figure 2B Shown are examples of flow gating of CD11b and Ly6C in the blood of MC38 tumor-bearing animals from mice dosed with PBS (left) and mice dosed with CyTaC compound and antibody (right).

[0028] Figure 3 Shown is the effect of treatment with CyTaC compound (compound of Example 22) in combination with an anti-cotinine antibody on intratumoral CD8+ T-cell levels of lymphocytes from tumors excised from syngeneic models as determined by flow cytometry.

[0029] Figure 4A , Figure 4B , Figure 4C and Figure 4D Characterization of various cell populations in the blood and tumors of HEPA1-6 bearing mice by flow cytometry is shown; Figure 4A and Figure 4C Analysis of F480+ positive cells from blood and intratumoral lymphocytes is shown; Figure 4B and Figure 4D Analysis of Ly6G-monocytes from blood and intratumoral lymphocytes is shown.

[0030] Figure 5 Shown is flow cytometric analysis of macrophages in tumor samples excised from subcutaneously grown KPC tumors.

[0031] Fig. 6A and Figure 6B Flow cytometry analysis of tumor samples from subcutaneously grown Hepa1-6 or KPC tumors is shown; Fig. 6A shows the effect of treatment with the CyTaC compound of Example 22 in combination with an anti-cotinine antibody on the percentage of the T cell population present in KPC tumors; Figure 6B The effect of treatment with the CyTaC compound of Example 22 in combination with an anti-cotinine antibody on the percentage of T cell subsets present in Hepal-6 tumors is shown.

[0032] Figure 7 The therapeutic effect of the CyTaC compound of Example 22 in combination with an anti-cotinine antibody, either alone or further in combination with a STING agonist treatment, is shown.

[0033] Fig. 8A and Figure 8B The therapeutic effect of the CyTaC compound of Example 22 in combination with an anti-cotinine antibody, alone or further in combination with anti-PD1 treatment, is shown; Fig. 8A Tumor volumes for each individual animal treated over time are shown; Figure 8B Overall survival of animals across treatment groups is shown.

[0034] Fig. 9The therapeutic effect of the CyTaC compound of Example 22 in combination with an anti-cotinine antibody in a collagen-induced arthritis model is shown; animals were dosed as described in Example 32, and inflammation was assessed to generate a plotted composite clinical score.

[0035] Fig. 10A and Fig. 10B The antagonism of monocyte chemotaxis by CyTaC compounds when administered with an anti-cotinine antibody without ADCC activity was shown; chemotaxis of immune cells was induced using thioglycolate; cells were isolated using intraperitoneal lavage and subjected to flow cytometry to identify monocytes (Ly6Chi / CD11b+); Fig. 10A A comparison between the activities of the compounds of Examples 2 and 14 is shown; Fig. 10B The activity of the compound of Example 14 is shown.

[0036] Fig.11 : Schematic representation of cytotoxic targeting chimeras (CyTaCs) technology compared to current antibody technology.

[0037] Detailed Description

[0038] In one aspect, the present disclosure provides compounds of formula (I):

[0039]

[0040] or a pharmaceutically acceptable salt thereof,

[0041] in:

[0042] T is

[0043] R 1 It is C 1-4 Alkyl or C 3-6 Cycloalkyl;

[0044] Y is a bond or a divalent spacer moiety of 1 to 12 atoms in length; and

[0045] L is a divalent linking group of formula (La), (Lb), (Lc), (Ld), (Le), (Lf), (Lg), (Lh), (Li), (Lj), (Lk), (Lm), (Lni), (Ln-ii), (Ln-iii) or (Ln-iv).

[0046] In one embodiment of the present disclosure, L is a divalent linking group of formula (La):

[0047] or a stereoisomer thereof,

[0048] in:

[0049] Ring A and Ring B are each independently C 4-6 Cycloalkylene;

[0050] L 1a It is C 3-5 A straight chain alkylene group in which one or two methylene units are replaced by -O- or -NR a -replace;

[0051] Each R a are independently hydrogen or C 1-3 alkyl; and

[0052] L 2a is -O-, -NHC(O)- or -CH2-O-;

[0053] in 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), and represents a covalent bond to the methylene group of formula (I).

[0054] In another embodiment, Ring A and Ring B of Formula (La) are each independently

[0055] In another embodiment, L is a divalent linking group of formula (Lai):

[0056] or a stereoisomer thereof,

[0057] in:

[0058] Ring A is C 4-6 Cycloalkylene;

[0059] L 1a It is C 3-5 A straight chain alkylene group in which one or two methylene units are replaced by -O- or -NR a -replace;

[0060] Each R a are independently hydrogen or C 1-3 alkyl; and

[0061] L 2a is -O-, -NHC(O)- or -CH2-O-;

[0062] in 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), and represents a covalent bond to the methylene group of formula (I).

[0063] In another embodiment, Ring A of formula (Lai) is

[0064] In another embodiment, L is a divalent linking group of formula (La-ii):

[0065] or a stereoisomer thereof,

[0066] in:

[0067] L 1a It is C 3-5 A straight chain alkylene group in which one or two methylene units are replaced by -O- or -NR a -replace;

[0068] Each R a are independently hydrogen or C 1-3 alkyl;

[0069] L 2a is -O-, -NHC(O)- or -CH2-O-;

[0070] p is 1 or 2; and

[0071] m is 1 or 2;

[0072] in 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), and represents a covalent bond to the methylene group of formula (I).

[0073] In another embodiment, L of formula (La), (Lai) or (La-ii) 1a Selected from

[0074] in:

[0075] j is 1, 2, 3, or 4;

[0076] k is 0, 1, 2, or 3;

[0077] The sum of j and k is 2, 3 or 4;

[0078] q is 1 or 2;

[0079] r is 1 or 2;

[0080] s is 0 or 1;

[0081] The sum of q, r and s is 2 or 3;

[0082] X 1 and X 2 independently -O- or NR a ;and

[0083] Each R a are independently hydrogen or C 1-3 alkyl;

[0084] in represents a covalent bond to the C(O) group of formula (La), (Lai) or (La-ii), and represents a covalent bond to Ring B of Formula (La) or to a cyclohexylene group of Formula (Lai) or (La-ii).

[0085] In another embodiment, L of formula (La), (Lai) or (La-ii) 1a is selected from -(CH2)2O-, -(CH2)3O-, -(CH2)4O-, -(CH2)2OCH2-, -(CH2)3OCH2-, -(CH2)2O(CH2)2-, -CH2OCH2-, -CH2O(CH2)2-, -CH2O(CH2)3-, -CH2OCH2O- or -CH2OCH2OCH2-. In another embodiment, L of formula (La), (Lai) or (La-ii) 1a is selected from -(CH2)2O-, -(CH2)3O-, -(CH2)2OCH2- or -(CH2)3OCH2-. In another embodiment, L of formula (La), (Lai) or (La-ii) 1a Selected from -(CH2)2NR a -、-(CH2)3NR a -、-(CH2)4NR a -、-(CH2)2NR a CH2-、-(CH2)3NR a CH2-、-(CH2)2NR a (CH2)2-、-CH2NR a CH2-、-CH2NR a (CH2)2-、-CH2NR a (CH2)3-、-CH2NR a CH2N a -or-CH2NR a CH2N a CH2-, where each R a are independently hydrogen or C 1-3In another embodiment, L of formula (La), (Lai) or (La-ii) 1a Selected from -(CH2)2NR a -、-(CH2)3NR a -、-(CH2)2NR a CH2- or -(CH2)3NR a CH2-, where R a is hydrogen or C 1-3 In another embodiment, L of formula (La), (Lai) or (La-ii) 1a is selected from -(CH2)2NH-, -(CH2)3NH-, -(CH2)4NH-, -(CH2)2NHCH2-, -(CH2)3NHCH2-, -(CH2)2NH(CH2)2-, -CH2NHCH2-, -CH2NH(CH2)2-, -CH2NH(CH2)3-, -CH2NHCH2NH- or -CH2NHCH2NHCH2-. In another embodiment, L of formula (La), (Lai) or (La-ii) 1a is selected from -(CH2)2NH-, -(CH2)3NH-, -(CH2)2NHCH2- or -(CH2)3NHCH2-. In another embodiment, L of formula (La), (Lai) or (La-ii) 1a Selected from -CH2OCH2NR a -、-CH2NR a CH2O-、-CH2OCH2NR a CH2-、-CH2NR a CH2OCH2-, where R a are independently hydrogen or C 1-3 In another embodiment, L of formula (La), (Lai) or (La-ii) 1a Selected from -CH2OCH2NH-, -CH2NHCH2O-, -CH2OCH2NHCH2-, -CH2NHCH2OCH2-.

[0086] In another embodiment, L is a divalent linking group of formula (La-iii):

[0087] or a stereoisomer thereof,

[0088] in:

[0089] p is 1 or 2;

[0090] m is 1 or 2; and

[0091] n is 1, 2, or 3;

[0092] in 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), and represents a covalent bond to the methylene group of formula (I).

[0093] In another embodiment, L is a divalent linking group of formula (La) selected from:

[0094]

[0095]

[0096] In another embodiment, L is a divalent linking group of formula (Lb): or a stereoisomer thereof, wherein:

[0097] Ring A is C 4-6 Cycloalkylene or C 7-9 bridged bicyclic cycloalkylene;

[0098] L 1b is -CH2-NH-C(O)-, -NHC(O)- or -C(O)NH-;

[0099] L 2b It is C 6-12 A straight chain alkylene group in which 1, 2, 3 or 4 methylene units are replaced by -O-, -NR 1b -、-C(O)NR 1b -or-NR 1b C(O)-substitution; or

[0100] L 2b yes where n is 1, 2, 3, or 4, and Represents and L 1b covalent bonds; and

[0101] Each R 1b are independently hydrogen or C 1-3 alkyl;

[0102] in 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), and represents a covalent bond to the methylene group of formula (I).

[0103] In another embodiment, Ring A of formula (Lb) is

[0104] In another embodiment, L is a divalent linking group of formula (Lbi):

[0105] or a stereoisomer thereof,

[0106] in:

[0107] L 1b is -CH2-NH-C(O)-, -NHC(O)- or -C(O)NH-;

[0108] L 2b It is C 6-12 A straight chain alkylene group in which 1, 2, 3 or 4 methylene units are replaced by -O-, -NR 1b -、-C(O)NR 1b -or-NR 1b C(O)-substitution; or

[0109] L 2b yes where n is 1, 2, 3, or 4, and Represents and L 1b covalent bond

[0110] Each R 1b are independently hydrogen or C 1-3 alkyl;

[0111] p is 1 or 2; and

[0112] m is 1 or 2;

[0113] in 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), and represents a covalent bond to the methylene group of formula (I).

[0114] In another embodiment, L of formula (Lb) or (Lbi) 2b Selected from

[0115]

[0116] in:

[0117] j is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0118] k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0119] The sum of j and k is 5, 6, 7, 8, 9, 10 or 11;

[0120] q is 1, 2, 3, 4, 5, 6, 7, 8, or 9;

[0121] r is 1, 2, 3, 4, 5, 6, 7, 8, or 9;

[0122] s is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0123] The sum of q, r and s is 4, 5, 6, 7, 8, 9 or 10;

[0124] t is 1, 2, 3, 4, 5, 6, or 7;

[0125] u is 1, 2, 3, 4, 5, 6, or 7;

[0126] v is 1, 2, 3, 4, 5, 6, or 7;

[0127] w is 0, 1, 2, 3, 4, 5, or 6;

[0128] The sum of t, u, v and w is 3, 4, 5, 6, 7, 8 or 9;

[0129] a is 1, 2, 3, 4, or 5;

[0130] b is 1, 2, 3, 4, or 5;

[0131] c is 1, 2, 3, 4, or 5;

[0132] d is 1, 2, 3, 4, or 5;

[0133] e is 0, 1, 2, 3, or 4;

[0134] The sum of a, b, c, d and e is 4, 5, 6, 7 or 8;

[0135] X 1 , X 2 , X 3 and X 4 are independently -O-, -NR 1b -、-C(O)NR 1b -or-NR 1b C(O)-; and

[0136] Each R 1b are independently hydrogen or C 1-3 alkyl;

[0137] in represents L in formula (Lb) or (Lbi) 1b covalent bonds, and represents a covalent bond to the methylene group of formula (I).

[0138] In another embodiment, L is a divalent linking group of formula (Lb) selected from the following:

[0139]

[0140]

[0141] In another embodiment, L is a divalent linking group of formula (Lc):

[0142] or a stereoisomer thereof,

[0143] in:

[0144] L 1c It is C 2-10 Straight chain alkylene in which 1, 2 or 3 methylene units are replaced by -O-, -NH-, -NHC(O)- or -C(O)NH-;

[0145] Ring A is C 4-6 Cycloalkylene or C 7-9 a bridged bicyclic cycloalkylene; and

[0146] L 2c Is -O- or saturated C 2-10 Straight chain alkylene in which 1, 2 or 3 methylene units are replaced by -O-, -NH-, -NHC(O)- or -C(O)NH-;

[0147] in 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), and represents a covalent bond to the methylene group of formula (I).

[0148] In another embodiment, Ring A of formula (Lc) is

[0149] In another embodiment, L is a divalent linking group of formula (Lci):

[0150] or a stereoisomer thereof,

[0151] in:

[0152] L 1c It is C 2-10 Straight chain alkylene in which 1, 2 or 3 methylene units are replaced by -O-, -NH-, -NHC(O)- or -C(O)NH-;

[0153] L 2c Is -O- or saturated C 2-10Straight chain alkylene in which 1, 2 or 3 methylene units are replaced by -O-, -NH-, -NHC(O)- or -C(O)NH-;

[0154] p is 1 or 2; and

[0155] m is 1 or 2;

[0156] in 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), and represents a covalent bond to the methylene group of formula (I).

[0157] In another embodiment, L of formula (Lc) or (Lci) 1c Selected from

[0158]

[0159] in:

[0160] j is 1, 2, 3, 4, 5, 6, 7, 8, or 9;

[0161] k is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0162] The sum of j and k is 1, 2, 3, 4, 5, 6, 7, 8 or 9;

[0163] q is 1, 2, 3, 4, 5, 6, or 7;

[0164] r is 1, 2, 3, 4, 5, 6, or 7;

[0165] s is 0, 1, 2, 3, 4, 5, or 6;

[0166] The sum of q, r and s is 2, 3, 4, 5, 6, 7 or 8;

[0167] t is 1, 2, 3, 4, or 5;

[0168] u is 1, 2, 3, 4, or 5;

[0169] v is 1, 2, 3, 4, or 5;

[0170] w is 0, 1, 2, 3, or 4;

[0171] the sum of t, u, v and w is 3, 4, 5, 6 or 7; and

[0172] X 1 , X 2 and X 3 are independently -O-, -NH-, -NHC(O)- or -C(O)NH-;

[0173] in represents a covalent bond to the C(O) group of formula (Lc) or (Lci), and represents a covalent bond to the ring of formula (Lc) or (Lci).

[0174] In another embodiment, L of formula (Lc) or (Lci) 2c Selected from

[0175]

[0176] in:

[0177] j is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9;

[0178] k is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9;

[0179] The sum of j and k is 1, 2, 3, 4, 5, 6, 7, 8 or 9;

[0180] q is 0, 2, 3, 4, 5, 6, or 7;

[0181] r is 1, 2, 3, 4, 5, 6, 7, or 8;

[0182] s is 0, 1, 2, 3, 4, 5, 6, or 7;

[0183] The sum of q, r and s is 1, 2, 3, 4, 5, 6, 7 or 8;

[0184] t is 0, 1, 2, 3, 4, or 5;

[0185] u is 1, 2, 3, 4, 5, or 6;

[0186] v is 1, 2, 3, 4, 5, or 6;

[0187] w is 0, 1, 2, 3, 4, or 5;

[0188] the sum of t, u, v and w is 2, 3, 4, 5, 6 or 7; and

[0189] X 1 , X 2 and X 3 are independently -O-, -NH-, -NHC(O)- or -C(O)NH-;

[0190] in represents a covalent bond to the ring of formula (Lc) or (Lci), and represents a covalent bond to the methylene group of formula (I).

[0191] In another embodiment, L is a divalent linking group of formula (Lc) selected from the following:

[0192]

[0193]

[0194] In another embodiment, L is a divalent linking group of formula (Ld):

[0195]

[0196] in:

[0197] L 1d It is C 12-31 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-;

[0198] in 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), and represents a covalent bond to the methylene group of formula (I). 1d It 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 linear alkylene group 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-. 1d It is C 12-22 Straight chain alkylene, for example, C 12 , C 13 , C 14 , C 15 , C 16, C 17 , C 18 , C 19 , C 20 , C 21 or C 22 , wherein 1, 2, 3, 4 or 5 methylene units are replaced by -NH-, -O-, -C(O)NH-, -NHC(O)- or -NHC(O)-NH-.

[0199] In another embodiment, L of formula (Ld) 1d Selected from

[0200]

[0201] in:

[0202] j is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;

[0203] k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;

[0204] The sum of j and k is 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21;

[0205] q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19;

[0206] r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19;

[0207] s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18;

[0208] the sum of q, r, and s is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;

[0209] t is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17;

[0210] u is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17;

[0211] v is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17;

[0212] w is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16;

[0213] the sum of t, u, v and w is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19;

[0214] a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;

[0215] b is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;

[0216] c is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;

[0217] d is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;

[0218] e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14;

[0219] The sum of a, b, c, d and e is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18;

[0220] f is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13;

[0221] g is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13;

[0222] h is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13;

[0223] i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13;

[0224] y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13;

[0225] z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;

[0226] the sum of f, g, h, i, y and z is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17; and

[0227] X 1 , X 2 , X 3 , X 4 and X 5 are independently -NH-, -O-, -C(O)NH-, -NHC(O)- or -NHC(O)-NH-;

[0228] in represents a covalent bond to the C(O) group of formula (Ld), and represents a covalent bond to the methylene group of formula (I).

[0229] In another embodiment, L of formula (Ld) 1d yes wherein n is 4, 5, 6, 7, 8, 9 or 10;

[0230] in represents a covalent bond to the C(O) group of formula (Ld), and represents a covalent bond to the methylene group of formula (I).

[0231] In another embodiment, L is a divalent linking group of formula (Ld) selected from the following:

[0232]

[0233]

[0234] In another embodiment, L is a divalent linking group of formula (Le):

[0235]

[0236] in:

[0237] n is an integer from 3 to 50;

[0238] in 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), and represents a covalent bond to the methylene group of formula (I).

[0239] In another embodiment, n of 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 of Formula (Le) is 5 to 22, 7 to 15, or 9 to 13. In another embodiment, n of Formula (Le) is 3, 4, 5, 7, 8, 11, 22, or 50.

[0240] In another embodiment, L is a divalent linking group of formula (Lf):

[0241] or a stereoisomer thereof,

[0242] in:

[0243] L 1f is the key; C 1-6 A straight chain alkylene group in which 0, 1 or 2 methylene units are replaced by -O-, -NH- or -C(O)-; or -(C 3-6 Cycloalkylene)-NHC(O)-;

[0244] L 2f is a bond, -NHC(O)-, -C(O)NH- or C 1-6 Straight chain alkylene wherein 0, 1 or 2 methylene units are replaced by -O-; and

[0245] Z 1 and Z 2 Each of is independently N or CH;

[0246] in 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), and represents a covalent bond to the methylene group of formula (I).

[0247] In another embodiment, L of formula (Lf) 1f Selected from

[0248]

[0249] in:

[0250] j is 1, 2, 3, 4, or 5;

[0251] k is 0, 1, 2, 3, or 4;

[0252] The sum of j and k is 1, 2, 3, 4 or 5;

[0253] q is 1, 2, or 3;

[0254] r is 1, 2, or 3;

[0255] s is 0, 1, 2;

[0256] the sum of q, r and s is 2, 3 or 4; and

[0257] X 1 and X 2 are independently -O-, -NH- or -C(O)-; or -(C 3-6Cycloalkylene)-NHC(O)-;

[0258] in represents a covalent bond to the C(O) group of formula (Lf), and represents a covalent bond to the ring of formula (Lf).

[0259] In another embodiment, L of formula (Lf) 2f Selected from

[0260]

[0261] in:

[0262] j is 1, 2, 3, 4, or 5;

[0263] k is 0, 1, 2, 3, or 4;

[0264] The sum of j and k is 1, 2, 3, 4 or 5;

[0265] q is 1, 2, or 3;

[0266] r is 1, 2, or 3;

[0267] s is 0, 1, 2; and

[0268] The sum of q, r and s is 2, 3 or 4;

[0269] in represents a covalent bond to the ring of formula (Lf), and represents a covalent bond to the methylene group of formula (I).

[0270] In another embodiment, L is a divalent linking group of formula (Lf) selected from the following:

[0271]

[0272] In another embodiment, L is a divalent linking group of formula (Lg):

[0273]

[0274] in:

[0275] Ring A is a 5- to 6-membered heteroarylene group having 1 or 2 nitrogen ring atoms;

[0276] L 1g is a bond, -CH2-, -NH-, or -O-; and

[0277] L 2g yes where n is 1, 2, 3, 4, or 5, and Represents and L1g covalent bonds;

[0278] in 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), and represents a covalent bond to the methylene group of formula (I).

[0279] In another embodiment, L is a divalent linking group of formula (Lgi):

[0280]

[0281] in:

[0282] L 1g is a bond, -CH2-, -NH-, or -O-;

[0283] L 2g yes where n is 1, 2, 3, 4, or 5, and Represents and L 1g covalent bonds;

[0284] Z 1 , Z 2 and Z 3 are each independently selected from N or CH, provided that Z 1 , Z 2 and Z 3 One or two of them are N;

[0285] in 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), and represents a covalent bond to the methylene group of formula (I).

[0286] In another embodiment, L is a divalent linking group of formula (Lg) selected from the following:

[0287]

[0288]

[0289] In another embodiment, L is a divalent linking group of formula (Lh):

[0290] or a stereoisomer thereof,

[0291] in:

[0292] Each Z 1 are independently N or CH;

[0293] L 1h is a bond, -C(O)-, -C(O)-NH-, or -NHC(O)-;

[0294] L 2h It is C 2-10 Straight chain alkylene or where n is 1, 2, 3, or 4, and Represents and L 1h covalent bonds and Represents and L 3h covalent bonds;

[0295] L 3h is a bond, -C(O)CH2-, -O-(C 3-6 Cycloalkylene)-O- or -C(O)NH(CH2)3OCH2-;

[0296] L 4h is a bond, -C(O)-, -CH2C(O)-, or -C(O)CH2-; and

[0297] m is 1, 2, or 3;

[0298] in 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), and represents a covalent bond to the methylene group of formula (I).

[0299] In another embodiment, L is a divalent linking group of formula (Lh) selected from the following:

[0300]

[0301]

[0302] In another embodiment, L is a divalent linking group of formula (Li):

[0303]

[0304] in:

[0305] L 1i Yes key, C 1-12 Straight chain alkylene or where n is 1, 2, 3, 4, or 5, and Represents and L 3i covalent bonds and represents a covalent bond with NH;

[0306] L 2i Yes key, C 1-12 Straight chain alkylene or where n is 1, 2, 3, 4, or 5, and represents a covalent bond to HN; and

[0307] L 3i is a bond or -C(O)-;

[0308] in 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), and represents a covalent bond to the methylene group of formula (I).

[0309] In another embodiment, L is a divalent linking group of formula (Li) selected from:

[0310]

[0311] In another embodiment, L is a divalent linking group of formula (Lj):

[0312] or a stereoisomer thereof,

[0313] in:

[0314] Z 1 is C, CH or N;

[0315] Z 2 , Z 3 , Z 4 and Z 5 Each of Z is independently CH or N, provided that 2 , Z 3 , Z 4 and Z 5 No more than two of them are N;

[0316] L 1j is -NH-, -C(O)NH-, -NHC(O)- or -O-;

[0317] L 2j It is C 1-6 Straight chain alkylene or where n is 1 or 2, and Represents and L 1j covalent bonds; and

[0318] represents a single bond or a double bond;

[0319] in 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), and represents a covalent bond to the methylene group of formula (I).

[0320] In another embodiment, L is a divalent linking group of formula (Lj) selected from the following:

[0321]

[0322] In another embodiment, L is a divalent linking group of formula (Lk):

[0323] or a stereoisomer thereof,

[0324] in:

[0325] Ring A is phenyl or a 5- or 6-membered heteroarylene group having 1 or 2 nitrogen ring atoms;

[0326] Z 1 and Z 2 Each of is independently CH or N;

[0327] L 1k is a bond, -C(O)-, -C(O)NH-, or -NHC(O)-; and

[0328] L 2k It is C 3-8 Straight chain alkylene or where n is 1, 2, or 3, and Represents and L 1k covalent bonds;

[0329] in 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), and represents a covalent bond to the methylene group of formula (I).

[0330] In another embodiment, L is a divalent linking group of formula (Lk) selected from the following:

[0331]

[0332] In another embodiment, L is a divalent linking group of formula (Lm):

[0333] or a stereoisomer thereof,

[0334] in:

[0335] Z 1 is CH or N;

[0336] m is 1 or 2;

[0337] p is 1 or 2;

[0338] 0, 1 or 2 hydrogen atoms of are replaced by F;

[0339] L 1m is a bond, -C(O)-, -C(O)NH-, -NHC(O)-, -S(O)2NH-, or -NHS(O)2-; and

[0340] L 2m It is C 3-6 Straight chain alkylene, C 3-6 Cycloalkylene or where n is 1 or 2, and Represents and L 1m covalent bonds;

[0341] in 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), and represents a covalent bond to the methylene group of formula (I).

[0342] In another embodiment, L is a divalent linking group of formula (Lm) selected from the following:

[0343]

[0344]

[0345] In another embodiment, L is a divalent linking group of formula (Lni):

[0346]

[0347] in 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), and represents a covalent bond to the methylene group of formula (I).

[0348] In another embodiment, L is a divalent linking group of formula (Ln-ii):

[0349]

[0350] in 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), and represents a covalent bond to the methylene group of formula (I).

[0351] In another embodiment, L is a divalent linking group of formula (Ln-iii):

[0352]

[0353] in 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), and represents a covalent bond to the methylene group of formula (I).

[0354] In another embodiment, L is a divalent linking group of formula (Ln-iv):

[0355]

[0356] in 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), and represents a covalent bond to the methylene group of formula (I).

[0357] In one embodiment of the present disclosure, Y is selected from a bond; -NH-; -(C 1-12 alkylene)-, wherein 1, 2 or 3 methylene units are replaced by -O-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C 3-6 Cycloalkylene)-, -(C 3-6 -(C 2-12 alkenyl)-, wherein 1, 2 or 3 methylene units are replaced by -O-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C 3-6 Cycloalkylene)-, -(C 3-6 The invention is replaced by a 3- to 6-membered heterocycloalkylene group, an arylene group or a heteroarylene group.

[0358] In another embodiment, Y is selected from 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 -(C 1-5 -NH-(C 1-5 Alkylene)-NH-; -(C 3-6 Cycloalkylene)-NH-; -(C3-6 Cycloalkenylene)-NH-; or where Y 1a is a bond, -O-, -NH-, -NHC(O)-, -C(O)NH- or C 1-3 Alkylene; and Y 2a is a bond, -O-, -NH-, -NHC(O)-, -C(O)NH- or C 1-3 In another embodiment, Y is -NH-.

[0359] In another embodiment, Y is selected from:

[0360] In another embodiment, R 1 is methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl or tert-butyl. 1 In another embodiment, R 1 is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0361] In another embodiment, the compound of formula (I) is selected from the compounds listed in Table 1:

[0362] Table 1

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370] definition

[0371] As used herein and in the claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise.

[0372] As used herein and in the claims, the term "comprising" encompasses "including" or "consisting of," for example, a composition "comprising" X may consist exclusively of X, or may include additional things, such as X+Y.

[0373] The term "consisting essentially of limits the scope of a feature to the specified materials or steps and those that do not materially affect the basic characteristics of the claimed feature.

[0374] The term "consisting of excludes the presence of any additional components.

[0375] The term "pathogenic cell" includes a subset of cells 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 pathogenic bodies that are capable of causing infection, such as viral or bacterial cells.

[0376] The term "pathogenic immune cells" includes specific immune cell subsets that cause or can cause disease. These cell subsets are resident cells, or are recruited to a specific location and secrete cytokines, chemokines and other mediators, and contribute to the continuation and progression of the disease, such as cancer in the case of a tumor microenvironment or chronic inflammation of the lungs in the case of asthma. The example of pathogenic immune cells includes, but is not limited to, suppressor cells (MDSC) of bone marrow origin, T regulatory cells (Tregs), neutrophils, macrophages, B regulatory cells (Bregs), CD8 regulatory cells, (CD8regs) and exhausted T cells.

[0377] The term "pharmaceutical composition" refers to a preparation of a compound of the present invention and a medium generally accepted in the art for delivering the biologically active compound to a mammal (e.g., a human). Such a medium includes all pharmaceutically acceptable carriers, diluents, or excipients thereof.

[0378] The terms "effective amount" and "therapeutically effective amount" represent the amount of the compound or antibody or its antigen-binding portion thereof according to the present invention: when administered to a patient in need thereof, the amount is sufficient to achieve the treatment of the disease state, illness or disorder for which the compound is useful. Such an amount will be sufficient to induce the biological or medical response of the tissue system or patient sought by the researcher or clinician. The amount of the compound according to the present invention constituting the therapeutically effective amount will vary according to factors such as the following: the compound and its biological activity, the composition for administration, the time of administration, the route of administration, the excretion rate of the compound, the duration of treatment, the type of disease state or disorder treated and its severity, the drug used in combination with the compound of the present invention or simultaneously, and the age, weight, general health, sex and diet of the patient. Such a therapeutically effective amount can be routinely determined by those of ordinary skill in the art taking into account their own knowledge, technical level and the disclosure.

[0379] The term "alkyl" represents a saturated straight or branched chain hydrocarbon moiety having the specified number of carbon atoms. 1-3"Alkyl" means an unsubstituted alkyl moiety containing 1, 2 or 3 carbon atoms; exemplary alkyl groups include methyl, ethyl and propyl.

[0380] The term "alkylene" represents a saturated straight or branched hydrocarbon moiety having a specified number of carbon atoms with two points of attachment. The two points of attachment may be from the same or different carbon atoms. 1-3 "Alkylene" means an unsubstituted alkyl moiety having two points of attachment and containing 1, 2 or 3 carbon atoms; exemplary C 1-3 Alkylene groups include methylene, ethylene and propylene.

[0381] The term "alkenyl" represents an unsaturated straight or branched chain hydrocarbon moiety having the specified number of carbon atoms. 2-6 "Alkenyl" means an unsubstituted alkenyl moiety containing 2, 3, 4, 5 or 6 carbon atoms; exemplary alkenyl groups include propenyl, butenyl, pentenyl and hexenyl.

[0382] The term "alkenylene" represents an unsaturated straight or branched hydrocarbon moiety having a specified number of carbon atoms with two points of attachment. The two points of attachment may be from the same or different carbon atoms. 2-6 "Alkenylene" means 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.

[0383] The term "cycloalkyl" represents a saturated cyclic hydrocarbon moiety having the specified number of carbon atoms. 3-6 "Cycloalkyl" means an unsubstituted cycloalkyl moiety containing 3, 4, 5 or 6 carbon atoms; exemplary cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0384] The term "cycloalkylene" represents a saturated cyclic hydrocarbon moiety having a specified number of carbon atoms with two points of attachment. The two points of attachment may be from the same or different carbon atoms. 4-6 "Cycloalkylene" means an unsubstituted cycloalkylene moiety containing 4, 5 or 6 carbon atoms having 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.

[0385] The term "cycloalkenylene" represents an unsaturated cyclic hydrocarbon moiety having a specified number of carbon atoms with two points of attachment. The two points of attachment may be from the same or different carbon atoms. 3-6"Cycloalkenylene" refers to an unsubstituted cycloalkenylene moiety containing 3, 4, 5 or 6 carbon atoms having two points of attachment.

[0386] The term "heterocycloalkylene" refers to a saturated cyclic hydrocarbon moiety having two attachment points, containing 1 or 2 heteroatoms independently selected from oxygen, sulfur or nitrogen atoms. The two attachment points may be from the same or different carbon atoms. The term "3- to 6-membered heterocycloalkylene" refers to a 3- to 6-membered saturated cyclic moiety having two attachment points, containing 2, 3, 4 or 5 carbon atoms in addition to 1 or 2 oxygen, sulfur or nitrogen atoms. Suitably, 3- to 6-membered heterocycloalkylene groups contain 1 oxygen or nitrogen atom. Suitably, such groups contain 3 carbon atoms and 1 oxygen or nitrogen atom, such as azetidine diyl or oxetane diyl. Suitably, such groups contain 4 or 5 carbon atoms and 1 oxygen or nitrogen atom, such as tetrahydrofuran diyl, tetrahydropyran diyl, pyrrolidine diyl or piperidine diyl.

[0387] The term "bridged bicyclic cycloalkylene" refers to a saturated bicyclic hydrocarbon moiety with at least one bridge having two points of attachment. A "bridge" is an unbranched chain of atoms or an atom or valence bond connecting two bridgeheads, wherein a "bridgehead" is any backbone atom of a ring system that is bonded to three or more backbone atoms (excluding hydrogen). The two points of attachment may be from the same or different carbon atoms. The term "C 7-9 "Bridged bicyclocycloalkylene" means an unsubstituted bridged bicyclocycloalkylene moiety having two points of attachment and containing 7, 8 or 9 carbon atoms.

[0388] The term "arylene" means a monocyclic or bicyclic ring system having two points of attachment, wherein at least one ring in the system is aromatic. Exemplary arylene groups include phenylene, biphenylene, naphthylene, and anthracene.

[0389] The term "heteroarylene" means a monocyclic or bicyclic ring system having two points of attachment, wherein at least one ring in the system is aromatic and has, in addition to carbon atoms, from 1 to 5 heteroatoms independently selected from oxygen, sulfur or nitrogen atoms. The term "5- to 6-membered heteroarylene" means a 5- to 6-membered cyclic aromatic moiety having two points of attachment and containing, in addition to 1, 2 or 3 heteroatoms independently selected from oxygen, sulfur or nitrogen atoms, 2, 3, 4 or 5 carbon atoms.

[0390] It will be appreciated by those skilled in the art that salts according to compounds of formula (I) may be prepared, including pharmaceutically acceptable salts. In fact, in certain embodiments of the present invention, salts (including pharmaceutically acceptable salts according to compounds of formula (I)) may be superior to respective free or unsalified compounds. Therefore, the present invention further relates to salts according to compounds of formula (I), including pharmaceutically acceptable salts. The present invention further relates to compounds of free or unsalified formula (I).

[0391] Salts of the compounds of the present invention, including pharmaceutically acceptable salts, are readily prepared by those skilled in the art.

[0392] Representative pharmaceutically acceptable acid addition salts include, but are not limited to, 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (benzenesulfonate), benzoate, bisulfate, bitartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (camphorsulfonate), decanoate (decanoate), hexanoate (caproate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, hydrogen succinate disuccinate, dodecyl sulfate (etopoate), edetate (ethylenediaminetetraacetate), etopoate (lauryl sulfate), ethane-1,2-disulfonate (ethanedisulfonate), ethanesulfonate (ethanesulfonate), formate, fumarate, galactarate (mucate), gentisate (2,5-dihydroxybenzoate), glucoheptonate (glucose), gluconate, glucuronate, glutamate, glutarate, glycerophosphate, glycolate, hexylresorcinate, hippurate, hydrabamine (N,N′-di(dehydroabietyl)-ethylenediamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methylsulfate, mucate, naphthalene-1,5-disulfonate (naphthalene disulfonate), naphthalene-2-sulfonate (naphthalene sulfonate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate, p-aminobenzenesulfonate Aminosalicylate, pamoate (pamoate), pantothenate, pectinate, persulfate, phenylacetate, phenylethylbarbiturate, phosphate, polygalacturonate, propionate, p-toluenesulfonate (toluenesulfonate), pyroglutamate, pyruvate, salicylate, sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, theophylline (8-chlorotheophylline salt), thiocyanate, triethyl iodide, trifluoroacetate, undecanoate, undecenoate, and valerate.

[0393] Representative pharmaceutically acceptable base addition salts include, but are not limited to, aluminum, 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS, tromethamine), arginine, phenethamine (N-benzylphenethylamine), dibenzylethylenediamine (N,N′-dibenzylethylenediamine), b / s-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, clemizole (1-p-chlorobenzyl-2-pyrrolidin-1′-ylmethylbenzimidazole), cyclohexylamine, dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, dopamine, ethanolamine, ethylenediamine, L-histidine, iron, isoquinoline, lepidol, lithium, lysine, magnesium, meglumine (N-methylglucosamine), piperazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, tert-butylamine, and zinc.

[0394] Compounds according to formula (I) may contain one or more asymmetric centers (also referred to as chiral centers), and therefore may exist as each enantiomer, diastereomer 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 there is the stereochemistry of a chiral center in a compound of formula (I) or any chemical structure shown herein, if not specifically stated, the structure is intended to encompass all individual stereoisomers and all mixtures thereof. Therefore, compounds according to formula (I) containing one or more chiral centers may be used as racemic mixtures, enantiomerically enriched mixtures or enantiomerically pure individual stereoisomers.

[0395] The symbol "&" can be used with an index number (e.g., "&1") to depict a mixture of stereoisomers where the relative configurations of all stereocenters are known. For example, a set of two stereocenters labeled with the symbol "&1" represents a mixture of two possible stereoisomers where the two stereocenters have the depicted relative configurations.

[0396] A divalent group is a group with two points of attachment.For all divalent groups, unless otherwise indicated, the orientation of the group is implied by the direction in which the formula or structure of the group is written.

[0397] Unless otherwise defined, all technical and scientific terms used in this article have the same meanings as those of ordinary skill in the art to which the present invention belongs. Although any compositions and methods similar or equivalent to those described herein can be used to practice or test the method of the present disclosure, 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 claim or independent claim disclosed herein can be combined in multiple combinations (for example, one or more narrations of each dependent claim can be combined into a single claim based on the independent claim on which they rely).

[0398] The ranges provided herein include all values ​​within the particular range described as well as values ​​about the endpoints of the particular range.

[0399] The concentrations described herein are determined at ambient temperature and pressure. For example, this may be at room temperature or at the temperature and pressure in a particular part of a process stream. Preferably, the concentrations are determined at standard conditions of 25°C and 1 bar pressure.

[0400] CCR2 Target and CCR2 Binding Moieties

[0401] The compound of formula (I) disclosed herein is a heterobifunctional synthetic agent, which is designed so that one end interacts with the cell surface CCR2 target, and the other end binds to a specific antibody. More specifically, ARM simultaneously binds to the cell surface CCR2 target and the specific antibody. This ternary complex guides immune surveillance to tissues / cells expressing CCR2, and combines the antibody functional mechanism with the dosage control of small molecules. The mechanism may include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC), and preferably includes ADCC. Immune cells expressing the same Fc receptor (which initiates the destruction of ARM / antibody-labeled cells) also participate in the presentation of endogenous antigens to achieve the potential for long-term cellular immunity.

[0402] The compounds of formula (I) disclosed herein include a CCR2 binding portion capable of binding to CCR2 present on the surface of a cell. In one embodiment, the CCR2 is expressed on a pathogenic cell.

[0403] In another embodiment, the pathogenic cell is a pathogenic immune cell, a tumor cell or a cancer cell, or a stromal cell (including a stromal cell present in a tumor microenvironment).

[0404] In another embodiment, the CCR2 target is present on the surface of a pathogen selected from a virus or a bacterial cell. Examples of viruses expressing cell surface targets include, but are not limited to, influenza.

[0405] In another embodiment, the pathogenic immune cell is a monocyte, a myeloid-derived suppressor cell (MDSC) such as a monocytic MDSC (mMDSC) and a polymorphonuclear MDSC (PMN-MDSC), a T regulatory cell (Tregs), a neutrophil (e.g., N2 neutrophil), a macrophage (e.g., M2 macrophage), a B regulatory cell (Bregs, memory B cells), a plasma cell, a CD8 cell (e.g., CD8 regulatory cells (CD8regs), memory CD8 cells, effector CD8 cells, naive CD8 T cells, 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.

[0406] In another embodiment, 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.

[0407] In another embodiment, the pathogenic immune cells expressing CCR2 are myeloid-derived suppressor cells (MDSC). In another embodiment, the pathogenic immune cells expressing CCR2 are selected from mononuclear MDSC (mMDSC) and polymorphonuclear MDSC (PMN_MDSC).

[0408] In another embodiment, the tumor cell or cancer cell is a solid tumor cell.

[0409] In another embodiment, the tumor cell 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 a NSCLC cell, a HCC cell, or a CRC cell.

[0410] In another embodiment, the stromal cells are cancer associated fibroblasts (CAFs).

[0411] The present disclosure also provides a pharmaceutical composition comprising a compound of formula (I) as disclosed herein and a pharmaceutically acceptable excipient, carrier or diluent.

[0412] Anti-cotinine antibodies

[0413] The present disclosure provides antibodies or antigen binding fragments thereof that bind to a cotinine portion. 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 portion. Cotinine has the following structure:

[0414]

[0415] The term "cotinine moiety" as used herein refers to cotinine or an analog of cotinine. The compounds of formula (I) described herein comprise a cotinine moiety connected to a CCR2 binding moiety via a linker. In one embodiment, the cotinine moiety has the following structure:

[0416]

[0417] Where R 1 It 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 tert-butyl. In another embodiment, R 1 In another embodiment, R 1 is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0418] The term "antibody" is used herein in the broadest sense to refer to molecules having immunoglobulin-like domains (e.g., IgG, IgM, IgA, IgD or IgE), and includes monoclonal, recombinant, polyclonal, chimeric, human, humanized, multispecific antibodies, including bispecific antibodies and heteroconjugate antibodies; single variable domains (e.g., domain antibodies (DAB)), antigen-binding antibody fragments, Fab, F(ab')2, Fv, disulfide-linked Fv, single-chain Fv, disulfide-linked scFv, bispecific antibodies, TANDABS, etc., and modified versions of any of the above (for a summary of alternative "antibody" formats, see Holliger and Hudson, Nature Biotechnology, 2005, 23(9): 1126-1136).

[0419] The terms "complete antibody", "whole antibody" or "intact antibody" used interchangeably herein refer to heterotetrameric glycoproteins with an approximate molecular weight of 150,000 Daltons. A complete antibody consists 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 (called "Fab" fragments) and an "Fc" crystallizable fragment. The Fab fragment consists of the variable domains at the amino terminus, the heavy chain variable region (VH) or the light chain variable region (VL), and the constant domains CH1 (heavy chain) and CL (light chain) at the carboxyl terminus. The Fc fragment consists of two domains formed by dimerization of paired CH2 and CH3 regions. The Fc can elicit 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 different heavy chain amino acid sequences, which are called μ, α, γ, ε and δ, respectively, and each heavy chain can be paired with a kappa or lambda light chain. Most antibodies in serum belong to the IgG class, and human IgG has four isotypes (IgG1, IgG2, IgG3 and IgG4), whose sequences differ mainly in their hinge regions.

[0420] "CDR" is defined as the complementarity determining region amino acid sequence of an antibody or its antigen binding fragment. These are the hypervariable regions of the heavy and light chains of immunoglobulins. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Therefore, "CDR" as used herein means all three heavy chain CDRs, all three light chain CDRs, all heavy chain and light chain CDRs, or at least two CDRs.

[0421] Throughout this specification, the amino acid residues in the variable domain sequence and variable domain region within the full-length antigen binding sequence (e.g., within the antibody heavy chain sequence or the 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 more information, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987).

[0422] It will be apparent to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable domain sequences and full-length antibody sequences. There are also alternative numbering conventions for CDR sequences, such as those set forth 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 sequence and the skilled artisan will understand this.

[0423] Other numbering conventions for CDR sequences available to the skilled artisan include the "AbM" (University of Bath) and "contact" (University College London) methods.

[0424] Table 2 below represents one definition for each CDR or binding unit using each numbering convention. It should be noted that certain CDR definitions may vary depending on the individual publications used.

[0425] Table 2

[0426] Kabat CDR Chothia CDR AbM CDR Contact CDR H1 31-35 / 35A / 35B 26-32 / 33 / 34 26-35 / 35A / 35B 30-35 / 35A / 35B H2 50-65 52-56 50-58 47-58 H3 95-102 95-102 95-102 93-101 L1 24-34 24-34 24-34 30-36 L2 50-56 50-56 50-56 46-55 L3 89-97 89-97 89-97 89-96

[0427] In another embodiment, the anti-cotinine antibody is humanized. In another embodiment, the Fc region of the anti-cotinine antibody is modified to increase ADCC activity, ADCP activity and / or CDC activity, suitable modifications of which are provided below. In another embodiment, the Fc region of the anti-cotinine antibody is modified to increase ADCC activity.

[0428] Fc engineering methods can be applied to modify the functional or pharmacokinetic properties of antibodies. Effector functions can be altered by generating mutations in the Fc region that increase or decrease binding to C1q or Fcγ receptors and alter CDC or ADCC activity, respectively. The glycosylation pattern of the antibody can also be modified to alter effector functions. The in vivo half-life of an antibody can be altered by generating mutations that affect the binding of the Fc to FcRn (neonatal Fc receptor).

[0429] As used herein, the term "effector function" refers to one or more of the following: 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 cell lysis (ADCML) and Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis (ADCP).

[0430] It is believed that the interaction between the Fc region of an antigen binding protein or antibody and various Fc receptors (FcR), including FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), FcRn, C1q, and type II Fc receptors, mediates the effector functions of the antigen binding protein or antibody. Significant biological effects can be the result of effector functionality. In general, the ability to mediate effector functions requires binding of the antigen binding protein or antibody to the antigen, and not all antigen binding proteins or antibodies mediate every effector function.

[0431] Effector function can be assessed in a variety of ways, including, for example, assessing the ADCC effector function of an antibody coated to a target cell mediated by natural killer (NK) cells via FcγRIII, or coated to monocytes / macrophages via FcγRI, or assessing the CDC effector function of an antibody coated to a target cell mediated by the complement cascade via C1q. For example, the antibodies or antigen-binding fragments thereof of the invention can be assessed 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.

[0432] Examples of assays to determine CDC function include those described in J Imm Meth, 1995, 184:29-38.

[0433] The effect of mutations on effector function (e.g., FcRn binding, FcγRs 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.

[0434] Throughout the present specification, amino acid residues in the Fc region in the antibody sequence or full-length antigen binding protein sequence are numbered according to the EU index numbering convention.

[0435] 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.

[0436] Enhanced CDC: Fc engineering can be used to enhance complement-based effector functions. For example (refer to IgG1), K326W / E333S; S267E / H268F / S324T; and IgG1 / IgG3 cross-subclass can increase C1q binding; E345R (Diebolder et al., Science, 2014, 343: 1260-1293) and E345R / E430G / S440Y produce preformed IgG hexamer (Wang et al., Protein Cell, 2018, 9 (1): 63-73).

[0437] Enhanced ADCC :Fc engineering can be used to enhance ADCC. For example (refer to IgG1), F243L / R292P / Y300L / V305I / P396L; S239D / I332E; and S298A / E333A / K334A increase FcγRIIIa binding; S239D / I332E / A330L increases FcγRIIIa binding and reduces FcγRIIb binding; G236A / S239D / I332E increases binding to FcγRIIa, increases FcγRIIa / FcγRIIb binding ratio (activation / inhibition ratio), and enhances macrophage phagocytosis of antibody-coated target cells. An asymmetric Fc in which one heavy chain contains L234Y / L235Q / G236W / S239M / H268D / D270E / S298A mutations and D270E / K326D / A330M / K334E in the opposing heavy chain increases affinity for FcγRIIIa F158 (a lower affinity allele) and has no increased binding affinity for FcγRIIIa V158 (a higher affinity allele) to inhibitory FcγRIIb (Mimoto et al., mAbs, 2013, 5(2):229-236).

[0438] Enhanced ADCP : Fc engineering can be used to enhance ADCP. For example (refer to IgG1), G236A / S239D / I332E increases FcγRIIa binding and increases FcγRIIIa binding (Richards, J. et al., Mol. Cancer Ther., 2008, 7:2517-2527).

[0439] Increased common connections :Fc engineering can be used to increase co-engagement with FcR. For example (refer to IgG1), S267E / L328F increases FcγRIIb binding; N325S / L328F increases FcγRIIa binding and reduces FcγRIIIa binding (Wang et al., Protein Cell, 2018, 9(1):63-73).

[0440] In another embodiment, the antibody or antigen-binding fragment thereof of the present invention may comprise a heavy chain constant region with an altered glycosylation profile, such that the antibody or antigen-binding fragment thereof has 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 WO 2003 / 011878, WO 2006 / 014679, and EP1229125.

[0441] The lack of the innermost α1,6 fucose residue on the Fc glycan portion at N297 of IgG1 antibodies enhances affinity for FcγRIIIA. Therefore, defucosylated 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).

[0442] In one embodiment, an antibody or antigen binding fragment thereof comprising a chimeric heavy chain constant region is provided. In one embodiment, the antibody or antigen binding fragment thereof comprises an IgG1 / IgG3 chimeric heavy chain constant region, so that the antibody or antigen binding fragment thereof has an enhanced effector function, such as enhanced ADCC or enhanced CDC, or enhanced ADCC and CDC functions. For example, the chimeric antibody or antigen binding fragment thereof of the present invention may comprise at least one CH2 domain from IgG3. In such an embodiment, the antibody or antigen binding fragment thereof comprises a CH2 domain from IgG3, or two CH2 domains may be from IgG3. In another embodiment, the chimeric antibody or antigen binding fragment thereof comprises an IgG1 CH1 domain, an IgG3 CH2 domain, and an IgG3 CH3 domain. In another embodiment, the chimeric antibody or antigen binding fragment thereof comprises an IgG1 CH1 domain, an IgG3 CH2 domain, and an IgG3 CH3 domain, but position 435 is histidine.

[0443] In another embodiment, the chimeric antibody or antigen-binding fragment thereof comprises an IgG1 CH1 domain and at least one CH2 domain from IgG3. In one embodiment, the chimeric antibody or antigen-binding fragment thereof comprises an IgG1 CH1 domain and the following residues in the CH2 domain, which correspond to IgG3 residues: 274Q, 276K, 296F, 300F, and 339T. In one embodiment, the chimeric antibody or antigen-binding fragment thereof also comprises 356E in the CH3 domain, which corresponds to an IgG3 residue. In one embodiment, the antibody or antigen-binding fragment thereof also comprises one or more of the following residues, which correspond to IgG3 residues in the CH3 domain: 358M, 384S, 392N, 397M, 422I, 435R, and 436F.

[0444] Also provided is a method for producing an antibody or antigen-binding fragment thereof according to the present invention, the method comprising the following steps:

[0445] a) culturing a recombinant host cell comprising an expression vector comprising a nucleic acid sequence encoding a chimeric Fc region having amino acid residues of IgG1 and IgG3 Fc regions (e.g., as described above); and

[0446] b) recovering the antibody or antigen-binding fragment thereof.

[0447] For example, using the COMPLEGENT technology system available from BioWa, Inc. (Princeton, NJ) and Kyowa Hakko Kirin Co., Ltd, such a method for producing an antibody or its antigen-binding fragment with a chimeric heavy chain constant region can be performed. The COMPLEGENT system comprises a recombinant host cell comprising an expression vector, wherein a nucleic acid sequence encoding a chimeric Fc region with amino acid residues of IgG1 and IgG3 Fc regions is expressed to produce an antibody or its antigen-binding fragment with enhanced CDC activity, i.e., an increase in CDC activity relative to an otherwise identical antibody or its antigen-binding fragment lacking such a chimeric Fc region, as described in WO 2007 / 011041 and US 2007 / 0148165, each of which is incorporated herein by reference. In an alternative embodiment, CDC activity can be increased by introducing sequence-specific mutations into the Fc region of an IgG chain. One of ordinary skill in the art will also recognize other suitable systems.

[0448] The present invention also provides a method for producing the antibody or antigen-binding fragment thereof according to the present invention, the method comprising the following steps:

[0449] a) culturing a recombinant host cell comprising an expression vector comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof, optionally wherein a FUT8 gene encoding α-1,6-fucosyltransferase has been inactivated in the recombinant host cell; and

[0450] b) recovering the antibody or antigen-binding fragment thereof.

[0451] For example, using the POTELLIGENT technology system available from BioWa, Inc. (Princeton, NJ), a method for producing antibodies or antigen-binding fragments thereof can be performed in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies with enhanced ADCC activity, which is increased relative to the same monoclonal antibody produced in cells with a functional FUT8 gene, as described in U.S. Pat. No. 7,214,775, U.S. Pat. No. 6,946,292, WO 00 / 61739, and WO 02 / 31240, all of which are incorporated herein by reference. Those of ordinary skill in the art will also recognize other suitable systems.

[0452] In one embodiment, the antibody or antigen-binding fragment thereof is produced in a host cell in which the FUT8 gene has been inactivated. In another embodiment, the antibody or antigen-binding fragment thereof is produced in a - / - FUT8 host cell. In another embodiment, the antibody or antigen-binding fragment thereof is defucosylated at Asn297 (IgG1).

[0453] It is obvious to those skilled in the art that such modifications can be used not only alone but also in combination with each other to further enhance effector function.

[0454] In one such embodiment, an antibody or antigen-binding fragment thereof is provided, which comprises a heavy chain constant region, which comprises a mutated and chimeric heavy chain constant region described above, respectively. For example, an antibody or antigen-binding fragment thereof comprising at least one CH2 domain from IgG3 and one CH2 domain from IgG1, and wherein the IgG1 CH2 domain has one or more mutations at positions selected from 239, 332 and 330 (e.g., the mutation may be selected from S239D, I332E and A330L), such that compared to an equivalent antibody or antigen-binding fragment thereof having an IgG1 heavy chain constant region lacking the mutation, the antibody or antigen-binding fragment thereof has an enhanced effector function, such as enhanced ADCC or enhanced CDC, or enhanced ADCC and enhanced CDC. In one embodiment, the IgG1 CH2 domain has mutations S239D and I332E. In another embodiment, the IgG1 CH2 domain has mutations S239D, A330L and I332E.

[0455] In an alternative embodiment, an antibody or antigen binding fragment thereof is provided, which comprises a chimeric heavy chain constant region and an altered glycosylation profile, each described 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 such an 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 such that the ratio of fucose to mannose is 0.8:3 or less, for example, wherein the antibody or antigen binding fragment thereof is defucosylated. Compared to an equivalent antibody or antigen binding fragment thereof having an IgG1 heavy chain constant region lacking the glycosylation profile, the antibody or antigen binding fragment thereof has an enhanced effector function, such as enhanced ADCC or enhanced CDC, or enhanced ADCC and enhanced CDC.

[0456] 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 from IgG1, wherein both IgG CH2 domains are mutated according to the restrictions described herein.

[0457] In one aspect, there is provided a method of producing an antibody or antigen-binding fragment thereof according to the invention described herein, the method comprising the steps of:

[0458] a) culturing a recombinant host cell containing an expression vector comprising a nucleic acid sequence encoding a chimeric Fc domain having IgG1 and IgG3 Fc domain amino acid residues (e.g., as described above); and wherein a FUT8 gene encoding α-1,6-fucosyltransferase has been inactivated in said recombinant host cell; and

[0459] b) recovering the antibody or antigen-binding fragment thereof.

[0460] For example, such a method for producing an antibody or antigen-binding fragment thereof can be performed using the ACCRETAMAB technology system available from BioWa, Inc. (Princeton, NJ), which combines the POTELLIGENT and COMPLEGENT technology systems to produce an antibody or antigen-binding fragment thereof having enhanced ADCC and CDC activity compared to an otherwise identical monoclonal antibody that lacks a chimeric Fc domain and is fucosylated.

[0461] In another embodiment, an antibody or antigen-binding fragment thereof comprising a mutated and chimeric heavy chain constant region is provided, wherein the antibody or antigen-binding fragment thereof has an altered glycosylation profile such that the antibody or antigen-binding fragment thereof has an enhanced effector function, such as enhanced ADCC or enhanced CDC or enhanced ADCC and CDC. In one embodiment, the mutation is selected from positions 239, 332, and 330, such as S239D, I332E, and A330L. In another 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, such as the antibody or antigen-binding fragment thereof is defucosylated such that the antibody or antigen-binding fragment thereof has an enhanced effector function compared to an equivalent non-chimeric antibody or antigen-binding fragment thereof lacking the mutation and lacking the altered glycosylation profile.

[0462] In another 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 another embodiment, 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. In another embodiment, the anti-cotinine antibody is of the IgG1 isotype. In another embodiment, the anti-cotinine antibody is of the IgG1 isotype and comprises a substitution in the Fc region to increase or enhance ADCC activity. In another embodiment, the anti-cotinine antibody is of the IgG1 isotype comprising a substitution in the Fc region to increase or enhance ADCC activity, wherein the substitution is S239D / I332E or S239D / I332E / A330L, wherein the residue numbering is according to the EU index. In another embodiment, the anti-cotinine antibody is of the IgG1 isotype comprising a substitution in the Fc region to increase or enhance ADCC activity, wherein the substitution is S239D / I332E, wherein the residue numbering is according to the EU index.

[0463] In another embodiment, the anti-cotinine antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) having SEQ ID NO: 7, a light chain variable region (VL) having SEQ ID NO: 8. In another embodiment, the anti-cotinine antibody has a heavy chain comprising a heavy chain variable region (VH) having SEQ ID NO: 7, and a light chain comprising a light chain variable region (VL) having SEQ ID NO: 8. In another embodiment, the anti-cotinine antibody is of the IgG1 isotype. In another embodiment, the anti-cotinine antibody is of the IgG1 isotype, comprising a substitution in the Fc region to increase or enhance ADCC activity. In another embodiment, the anti-cotinine antibody is of the IgG1 isotype, comprising a substitution in the Fc region to increase or enhance ADCC activity, wherein the substitution is S239D / I332E or S239D / I332E / A330L, wherein the residue numbering is according to the EU index. In another embodiment, the anti-cotinine antibody is of the IgG1 isotype comprising a substitution in the Fc region to increase or enhance ADCC activity, wherein the substitution is S239D / I332E, wherein residue numbering is according to the EU index.

[0464] In another embodiment, the anti-cotinine antibody has a heavy chain comprising SEQ ID NO:9 and a light chain comprising SEQ ID NO:10.

[0465] The present disclosure also provides a pharmaceutical composition comprising an anti-cotinine antibody or antigen-binding fragment thereof as disclosed herein and a pharmaceutically acceptable excipient, carrier or diluent.

[0466] The present disclosure also provides a group of combinations comprising a compound of formula (I) as disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof as disclosed herein. The compound of formula (I) and the anti-cotinine antibody or antigen-binding fragment thereof may be present in the same composition or in separate compositions. In one embodiment, the combination comprises a pharmaceutical composition comprising a compound of formula (I) disclosed herein and an 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 and a second pharmaceutical composition, wherein the first pharmaceutical composition comprises a compound of formula (I) disclosed herein and a pharmaceutically acceptable carrier, diluent or excipient, and the second pharmaceutical composition comprises an anti-cotinine antibody or antigen-binding fragment thereof as disclosed herein and a pharmaceutically acceptable carrier, excipient or diluent.

[0467] Instructions

[0468] The compound of formula (I) and its pharmaceutically acceptable salt can simultaneously bind to CCR2 expressed on the cell surface and anti-cotinine antibody or its antigen-binding fragment to form a ternary complex for treating and / or preventing diseases or disorders associated with CCR2-expressing cells.

[0469] In one embodiment, the present disclosure provides a method of treating and / or preventing a disease or disorder in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinine antibody or an antigen-binding fragment thereof, wherein the disease or disorder is selected from cancer, inflammatory disease, autoimmune disease, viral infection or bacterial infection.

[0470] In another embodiment, the compound and the antibody or its Fab are administered simultaneously. In another embodiment, the compound and the antibody or its Fab are administered simultaneously from a single composition, including as a fixed dose composition or by premixing the compound and the antibody or its Fab before administration. For example, the compound and the antibody or its Fab can be premixed for about 2 seconds to about 30 seconds, about 30 seconds to about 2 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 30 minutes, or about 30 minutes to about 2 hours before administration. In another embodiment, the compound and the antibody or its Fab are administered simultaneously from two separate compositions.

[0471] In another embodiment, the compound and the antibody or antigen-binding fragment thereof are administered sequentially.

[0472] 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 may be administered by different routes. In one embodiment, the compound and the antibody or antigen-binding fragment thereof are administered intravenously or subcutaneously in the same composition or in separate compositions. In another embodiment, the compound is administered orally and the antibody or antigen-binding fragment thereof is administered intravenously or subcutaneously.

[0473] In another embodiment, the compound and the antibody or antigen-binding fragment thereof are administered at a molar ratio of compound to antibody or antigen-binding fragment thereof 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.

[0474] In another embodiment, the compound and the antibody or antigen-binding fragment thereof are present as a combination at a molar ratio of compound to antibody or antigen-binding fragment thereof 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.

[0475] In another embodiment, the compound and the antibody or antigen-binding fragment thereof are administered at a dose of 0.0001 mg / kg to 1 mg / kg of the compound and 0.01 mg / kg to 100 mg / kg of the antibody. For example, in another embodiment, the compound and the antibody or antigen-binding fragment thereof are administered at a dose 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 .002 mg / kg to about 0.003 mg / kg, about 0.003 mg / kg to about 0.004 mg / kg, about 0.004 mg / kg to about 0.005 mg / kg, about 0.005 mg / kg to about 0.01 mg / kg, about 0.01 mg / kg to about 0.02 mg / kg, about 0.02 mg / kg to about 0.03 mg / kg, about 0.03 mg / kg to about 0.04 mg / kg, about 0.04 mg / kg The compound is administered at a dose of about 0.05 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 at a dose of about 0.01 mg / kg to about 0.02 mg / kg, About 0.02 mg / kg to about 0.03 mg / kg, about 0.03 mg / kg to about 0.04 mg / kg, about 0.04 mg / kg to about 0.05 mg / kg, about 0.05 mg / kg to about 0.1 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, about 0.5 mg / kg to about 1 mg / kg, about 1 mg / kg to about 2 mg / kg, about 2 mg / kg to about 3 mg / kg, about 3 mg / kg to about 4 mg / kg, about 4 mg / kg to about 5 mg / kg, about 5 mg / kg to about 10 mg / kg, about 10 mg / kg to about 15 mg / kg, about 15 mg / kg to about 20 mg / kg, about 20 mg / kg to about 25 mg / kg, about 25 mg / kg to about 30 mg / kg, about 30 mg / kg The antibody or antigen-binding fragment thereof is administered at a dose of about 35 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.

[0476] In another embodiment, the compound and the antibody or antigen-binding fragment thereof are administered at a dose of 0.007 mg to 70 mg of the compound and 0.7 mg to 7000 mg of the antibody. For example, in another embodiment, 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 0.5 The compound is administered at a dose of about 0.7 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, and the compound is administered at a dose of about 0.7 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 100 mg, about 100 mg to about 500 mg, about 500 mg to about 1000 mg, about 1000 mg to about 1500 mg, about 1500 mg to about 2000 mg, about 2000 mg to about The antibody or antigen-binding fragment thereof is administered at a dose of 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.

[0477] In another embodiment, the compound and the antibody or antigen-binding fragment thereof are administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks at a molar ratio and / or dosage as described herein for a period of one week to one year, such as 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.

[0478] In another embodiment, the present disclosure provides a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinine antibody or an antigen-binding fragment thereof for use in therapy. The compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinine antibody or an antigen-binding fragment thereof can be used to treat or prevent a disease or disorder selected from cancer, inflammatory disease, autoimmune disease, viral infection, or bacterial infection.

[0479] In another embodiment, the present disclosure provides a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinine antibody or an antigen-binding fragment thereof for preparing a medicament. The medicament can be used to treat or prevent a disease or disorder selected from cancer, inflammatory diseases, autoimmune diseases, viral infections, or bacterial infections.

[0480] In another embodiment, the disease or disorder is mediated by chemokine receptor 2 (CCR2) and / or is associated with CCR2-positive pathogenic cells. In another embodiment, the expression of CCR is tested by immunohistochemistry or flow cytometry to identify CCR-positive cell types.

[0481] In another embodiment, 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.

[0482] In another embodiment, the disease or disorder is a solid tumor. In another 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.

[0483] In another 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.

[0484] In another embodiment, the disease or disorder is a non-solid cancer. In another embodiment, the disease or disorder is a leukemia, lymphoma or myeloma.

[0485] In another embodiment, the disease or disorder is a viral infection. In another embodiment, the viral infection is caused by influenza virus, coronavirus (e.g., COVID-19) or hepatitis B virus.

[0486] In another embodiment, the disease or disorder is a bacterial infection. In another embodiment, the bacterial infection is a chronic bacterial infection.

[0487] In one embodiment, the present disclosure provides a method for increasing antibody-dependent cell-mediated cytotoxicity (ADCC) of a CCR2-expressing cell, the method comprising contacting the cell with an effective amount of a compound of formula (I) or a pharmaceutically 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 cell.

[0488] In one embodiment, the present disclosure provides a method for increasing antibody-dependent cellular phagocytosis (ADCP) of CCR2-expressing cells, the method comprising contacting the cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinine antibody or an antigen-binding fragment thereof, wherein the CCR2-binding portion of the compound binds to CCR2 expressed on the cells.

[0489] In one embodiment, the present disclosure provides a method for increasing complement-dependent cytotoxicity (CDC) of CCR2-expressing cells, the method comprising contacting the cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinine antibody or an antigen-binding fragment thereof, wherein the CCR2-binding portion of the compound binds to CCR2 expressed on the cells.

[0490] In one embodiment, the present disclosure provides a method of adapting a patient to chimeric antigen receptor (CAR) T cell therapy treatment, the method comprising administering to the patient an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinine antibody or an antigen binding fragment thereof. In certain embodiments, the compound of formula (I) or a pharmaceutically 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 pharmaceutically acceptable salt thereof, and an anti-cotinine antibody or an antigen binding fragment thereof can be administered as a conditioning therapy or a combination therapy to improve the efficacy in the treatment of solid tumor cancers. In other embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, and an anti-cotinine antibody or an antigen binding fragment thereof can be administered as a neoadjuvant therapy for other therapies, including, but not limited to, immunotherapy, surgical resection, radiation and / or chemotherapy.

[0491] In one embodiment, the present disclosure provides a method for depleting CCR2-expressing cells, the method comprising contacting the cells with a compound of formula (I) or a pharmaceutically 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.

[0492] In another embodiment, the CCR2 expressing cell is a pathogenic cell.

[0493] In another embodiment, the pathogenic cell is a pathogenic immune cell, a tumor cell or a cancer cell or a stromal cell.

[0494] In another embodiment, the pathogenic immune cell is a monocyte, a myeloid-derived suppressor cell (MDSC) such as a monocytic MDSC (mMDSC) and a polymorphonuclear MDSC (PMN-MDSC), a T regulatory cell (Tregs), a neutrophil (e.g., N2 neutrophil), a macrophage (e.g., M2 macrophage), a B regulatory cell (Bregs, memory B cells), a plasma cell, a CD8 cell (e.g., CD8 regulatory cells (CD8regs), memory CD8 cells, effector CD8 cells, naive CD8 T cells, 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.

[0495] In another embodiment, 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.

[0496] In another embodiment, the tumor cell 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 a NSCLC cell, a HCC cell, or a CRC cell.

[0497] In another embodiment, the stromal cells are cancer associated fibroblasts (CAFs).

[0498] Combination therapy

[0499] The compounds of the present invention can be used alone or in combination with other therapeutic agents. Therefore, the combination therapy according to the present invention comprises administering at least one compound of formula (I) or a pharmaceutically acceptable salt thereof, and using at least one other pharmaceutically active agent. The compounds of the present invention and other pharmaceutically active agents can be administered together or administered alone in a single pharmaceutical composition, and when administered alone, this can occur simultaneously or sequentially in any order. In order to achieve the desired combined therapeutic effect, the amount of the compounds of the present invention and other pharmaceutically active agents and the relative timing of administration will be selected.

[0500] It should be understood that when the compounds of the present invention are administered in combination with one or more other therapeutically active agents that are typically administered by inhalation, intravenous, oral, intranasal, ocular topical or other routes, the resulting pharmaceutical composition can be administered by the same route. Alternatively, the individual components of the composition can be administered by different routes.

[0501] In one embodiment, the compounds and pharmaceutical compositions disclosed herein are used in combination with or include one or more additional therapeutic agents. In another embodiment, the additional therapeutic agent is a checkpoint inhibitor or an immunomodulator.

[0502] In another embodiment, 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 dostarlimab), 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).

[0503] In another embodiment, 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).

[0504] In another embodiment, the immunomodulator is an ICOS agonist (eg, an anti-ICOS antibody, including but not limited to feladilimab), a PARP inhibitor (eg, niraparib, olaparib), or a STING agonist.

[0505] Pharmaceutical compositions, dosages and dosage forms

[0506] For the purpose of administration, in certain embodiments, the ARM described herein is administered as a raw chemical or formulated as a pharmaceutical composition. Pharmaceutical compositions disclosed herein include ARM and one or more of the following: a pharmaceutically acceptable carrier, diluent or excipient. ARM is present in the composition in an amount effective to treat a particular disease, disorder or condition of interest. The activity of ARM can be determined by those skilled in the art, for example, as described in the following bioassays. Those skilled in the art can easily determine appropriate concentrations and dosages. In certain embodiments, ARM is present in a pharmaceutical composition in an amount of about 25 mg to about 500 mg. In certain embodiments, ARM is present in a pharmaceutical composition in an amount of about 0.01 mg to about 300 mg. In certain embodiments, ARM is present in a 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.

[0507] The compound of the present invention or its pharmaceutically acceptable salt is administered in pure form or in an appropriate pharmaceutical composition by any accepted mode of administration of a pharmaceutical agent for serving similar purposes. The pharmaceutical composition of the present invention is prepared by combining the compound of the present invention with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and in a specific embodiment, is formulated into a product in the form of a solid, semisolid, liquid or gas, such as a tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant, gel, microsphere and aerosol. Exemplary routes of administering such a pharmaceutical composition 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 so that the active ingredient contained therein is bioavailable after the composition is administered to the patient.

[0508] The composition to be administered to the subject or patient is in the form of one or more dosage units, where, for example, a tablet can be a single dosage unit, and a container of the compound of the invention in aerosol form can accommodate multiple dosage units. The actual method of preparing such dosage forms is known or obvious to those skilled in the art; for example, see Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia. College of Pharmacy and Science, 2000). In any case, the composition to be administered will contain a therapeutically effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof for treating a disease or condition of interest according to the teachings described herein.

[0509] Pharmaceutical compositions disclosed herein are prepared by methods well known in the pharmaceutical field. For example, in certain embodiments, a pharmaceutical composition intended to be administered by injection is prepared by combining the compound of the present invention with sterile distilled water to form a solution. In certain embodiments, a surfactant is added to promote the formation of a uniform solution or suspension. A surfactant is a compound that interacts non-covalently with the compound of the present invention, thereby promoting the dissolution or uniform suspension of the compound in an aqueous delivery system. Traditional antibody therapeutics have several disadvantages, which are addressed by the ARM scheme described herein, including the difficulty of managing adverse events by adjusting the dosage and dosage frequency of administration, the challenge of producing antibodies for certain categories of drug targets (e.g., GPCRs, ion channels, and enzymes), and each new antibody requires a new cell line to develop, which can be slow and expensive. In addition, the manufacture of different forms of biologics (e.g., bispecific agents) may be challenging. In contrast, the ARM approach offers the following advantages: combining the pharmacology of antibodies with the dose control of small molecules, dose-controlled PK / PD allowing temporary cell depletion, simpler multimerization, and rapid reversal of cell depletion by administration of an antibody-binding component (e.g., cotinine hapten), which can uncouple therapeutic effects from potential adverse events. Example

[0510] The following examples illustrate the present invention. These examples are not intended to limit the scope of the invention, but rather provide guidance for the skilled artisan to prepare and use the compounds, compositions and methods of the present invention. Although specific embodiments of the present invention have been described, it will be appreciated by the skilled artisan that various changes and modifications may be made. References to preparations made in a manner similar to other preparations or by the general methods of other preparations may encompass changes in conventional parameters, such as time, temperature, post-processing conditions, minor changes in reagent amounts, etc. The chemical names of all title compounds were generated using ChemDraw plug-in version 16.0.1.13c (90) or ChemDraw desktop version 16.0.1.13 (90). One of ordinary skill in the art will recognize that the compounds of the present invention may have alternative names when using different naming software.

[0511] Compound synthesis

[0512] 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 readily prepared from commercially available starting materials by those skilled in the art.

[0513] The skilled artisan will appreciate that if the substituents described herein are incompatible with the synthetic methods described herein, the substituents may be protected with suitable protecting groups that are stable to the reaction conditions. The protecting groups may be removed at appropriate points in the reaction sequence to provide the desired intermediates or target compounds. Suitable protecting groups and methods of protecting and deprotecting various substituents using such suitable protecting groups are well known to those skilled in the art; examples of which may be found in T. Greene and P. Wuts, Protecting Groups in Organic Synthesis (4th edition), John Wiley & Sons, NY (2006). In some cases, the substituent can be specifically selected to be reactive under the reaction conditions used. In these cases, the reaction conditions convert the selected substituent into another substituent, which can be used as an intermediate compound or a desired substituent in the target compound.

[0514] Solution 1

[0515] Intermediates

[0516] Intermediate 1: (1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid methyl ester.

[0517]

[0518] Step 1: (1R,4S)-4-aminocyclopent-2-ene-1-carboxylic acid methyl ester, hydrochloride.

[0519]

[0520] SOCl2 (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 using a steady stream of nitrogen to afford the title compound (25 g, 99 mmol, 99% yield) as a white solid. LC / MS m / z 142.2 (M+H) + .

[0521] Step 2: Methyl (1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate.

[0522]

[0523] (1R,4S)-4-aminocyclopent-2-ene-1-carboxylic acid methyl ester, 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 aqueous NH4Cl solution (2 x 50 mL), brine (3 x 50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated to provide the title compound (21.835 g, 90 mmol, 90% yield) as a red oil. LC / MS m / z 220.2 (M+H) + .

[0524] Intermediate 2: (R)-3-methoxytetrahydro-4H-pyran-4-one

[0525]

[0526] Step 1: 4,4-Dimethoxytetrahydro-2H-pyran-3-ol.

[0527]

[0528] Potassium hydroxide (80g, 1428mmol) was dissolved in methanol (1L) at room temperature under stirring. The homogeneous mixture was cooled to 0°C. Commercially available tetrahydro-4H-pyran-4-one (50g, 499mmol) was dissolved in methanol (250mL), and potassium hydroxide solution was added dropwise while maintaining the temperature <5°C. After stirring for another 1.5h, iodine (139g, 549mmol) was added portionwise over 1.5h while maintaining the temperature <5°C. The reaction mixture was stirred at room temperature for 18h. The reactant was concentrated, toluene (500mL) was added, and the mixture was stirred for 30min. A small amount of precipitate was removed by filtration, and the filtrate was evaporated to provide the title compound (60.8g, 375mmol, 76% yield) as a chestnut oil. 1 H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.74-1.82 (m, 1H) 1.96 (ddd, J=14, 12, 4.9 Hz, 1H) 2.37 (s, 1H) 3.26 (s, 3H) 3.28 (s, 3H) 3.50 (td, J=12, 2.7 Hz, 1H) 3.66-3.72 (m, 2H) 3.77-3.88 (m, 2H).

[0529] Step 2: 4,4-Dimethoxydihydro-2H-pyran-3(4H)-one.

[0530]

[0531] 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. Molecular sieves (60.8 g, 375 mmol) were added, followed by 4-methylmorpholine N-oxide (110 g, 937 mmol) and tetra-n-propylammonium perruthenate (vii) (5.47 g, 15.57 mmol). The mixture was stirred for 13 h. The reactants were passed through Filter and concentrate. Purification by normal phase chromatography eluting with 10% ethyl acetate in hexanes provided 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, 2H) 3.16 (s, 6H) 3.84-3.88 (m, 2H) 3.91-3.98 (m, 2H).

[0532] Step 3: (R)-4,4-Dimethoxytetrahydro-2H-pyran-3-ol.

[0533]

[0534] To a solution of 4,4-dimethoxydihydro-2H-pyran-3(4H)-one (45 g, 281 mmol) in potassium phosphate buffer (7.0 pH) (1.125 L) was added 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). The mixture was stirred at 25 ° C for 6 h. At 2 h intervals, the pH was adjusted to 7 using saturated KHCO3 solution. Potassium chloride (50 g) was added and the mixture was stirred for 10 min. The mixture was extracted with acetonitrile (3x500 mL) and the organic extract was concentrated to near dryness. The residue was dissolved in dichloromethane (DCM) (250 mL) and the residual water was separated. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. Purification by normal phase chromatography with a gradient elution of 10 to 50% ethyl acetate in hexane provided the title compound (35 g, 209 mmol, 74.5% yield) as a colorless liquid. Specific rotation [α] 25 589 :-16.2°(1% in methanol). GC-MS m / z 162.1(M) + . 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.74-1.82 (m, 1H) 1.90-1.99 (m, 1H) 2.15 (d, 6 Hz, 1H) 3.26 (s, 3H) 3.28 (s, 3H) 3.50 (td, J=12, 2.7 Hz, 1H) 3.66-3.72 (m, 2H) 3.77-3.88 (m, 2H).

[0535] Step 4: (R)-3,4,4-Trimethoxytetrahydro-2H-pyran.

[0536]

[0537] A 60% mineral oil dispersion of sodium hydride (17.26 g, 432 mmol) in tetrahydrofuran (THF) (220 mL) was stirred under a nitrogen atmosphere 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, iodomethane (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 slowly poured into ice-cold water (500 mL) and extracted with ethyl acetate (500 mL). The aqueous phase was extracted with dichloromethane (DCM) (2 x 500 mL), the combined organic extracts were dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated. Purification by normal phase chromatography eluting with 30% ethyl acetate in hexanes provided the title compound (34 g, 191 mmol, 89% yield) as a light brown liquid. Specific rotation [α] 25 589 :-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, 1H) 1.90-2.05 (m, 1H) 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).

[0538] Step 5: (R)-3-Methoxytetrahydro-4H-pyran-4-one.

[0539]

[0540] To a mixture solution of (R)-3,4,4-trimethoxytetrahydro-2H-pyran (35 g, 199 mmol) in tetrahydrofuran (THF) (105 mL) and water (35.0 mL) was added dropwise concentrated HCl aqueous solution (280 mL, 3248 mmol) at 0 ° C for 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 x 1 L). The combined organic extracts were washed with saturated NaHCO3 solution (100 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated to provide the title compound (25 g, 184 mmol, 93% yield) as an off-white solid. Specific rotation [α] 25 589 :+85.000°(1% in methanol). GC-MS m / z 130.1(M) + . 1 H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.58-2.62 (m, 1H) 3.50 (s, 3H) 3.55-3.58 (m, 1H) 3.70-3.82 (m, 2H) 4.08-4.17 (m, 1H) 4.19-4.23 (m, 1H).

[0541] Intermediate 3: (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid

[0542]

[0543] Commercially available racemic trans-4-cotinine carboxylic acid (304 g, 1.38 mol) was purified by chiral preparative HPLC (61 injections) on Chiralpak 1A 20u101 x 210 mm at 500 mL / min, eluting with 50% acetonitrile in methanol containing 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 reduced pressure for 18 h to provide the title compound (143.6 g, 652 mmol, 94.5% yield) as a white solid. Analytical chiral HPLC: 95% ee at retention time 2.5 min, Chiralpak AD-H 5u 4.6 x 150 mm, methanol containing 0.1% formic acid at 1.0 mL / min; Alpha D = +58 degrees (c = 0.3, CH3OH); VCD analysis was used to assign absolute stereochemistry. LC-MS m / z 221.1(M+H) + .

[0544] 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, 3 hydrochloride.

[0545]

[0546] Step 1: tert-Butyl 4-(2-iodoethyl)piperidine-1-carboxylate.

[0547]

[0548] 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 pressure. The residue was partitioned between ethyl acetate and water, and the organic phase was washed with 50% (v / v) aqueous Na2S2O5, brine, dried over MgSO4, filtered, and the filtrate was concentrated under reduced pressure. By ISCO Chromatography (220 g silica Gold column) eluting with a gradient of 0 to 50% ethyl acetate in heptane provided the title compound as a colorless oil (23.49 g, 65.8 mmol, 93% yield). LCMS (m / z) 284.0 (M-(t-Bu)+H) + .

[0549] 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.

[0550]

[0551] To a solution of 1M lithium bis(trimethylsilyl)amide (LiHMDS) in tetrahydrofuran (THF) (70.2 mL, 70.2 mmol) in tetrahydrofuran (THF) (70 mL) was added dropwise a solution of (1R, 4S)-methyl 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) at -78°C under a nitrogen atmosphere, and the mixture was stirred for 40 min. 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 min. The mixture was warmed to 0°C over 1.5 h and stirred at 0°C for 40 min. Saturated NH4Cl aqueous solution (250 mL) and water (150 mL) were added and the aqueous layer was extracted with ethyl acetate (2x). The combined organic phases were washed with saturated NaHCO3 aqueous solution, brine, dried over MgSO4, filtered, and the filtrate was concentrated. The residue was divided into two parts and purified by ISCO Chromatography (330 g silica Gold cartridge) eluting with a step gradient of 0 to 15% (15 min), 15% (6 min) and 15 to 50% (10 min) ethyl acetate in heptane to provide the title compound (19.75 g, 45.0 mmol, 70.4% yield) as a light brown oil. LCMS (m / z) 431.4 (M+H) + .

[0552] Step 3: (1R,4S)-1-(2-(1-(tert-Butyloxycarbonyl)piperidin-4-yl)ethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid.

[0553]

[0554] 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 (20 mL) was added 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 (x 5), and dried in a vacuum oven at 45 °C for 2 days to provide the title compound (19.26 g, 43.9 mmol, 90% yield) as a light brown solid. LCMS (m / z) 417.5 (M+H) + .

[0555] 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.

[0556]

[0557] 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) were added HATU (3.78 g, 9.93 mmol) and DIPEA (6.31 mL, 36.1 mmol) dropwise. The mixture was stirred at room temperature for 15 min, commercially available 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine, 2 hydrochloride (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), saturated NHCl4 (30 mL) was added, and the precipitate was collected by filtration. The precipitate was washed with water and dried under reduced pressure to afford the title compound (7.23 g, 9.03 mmol, 100% yield). LCMS (m / z) 601.2 (M+H) + .

[0558] 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.

[0559]

[0560] 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) was added hydroxylamine hydrochloride (3.76 g, 54.2 mmol), hydroxylamine (50 wt % in aqueous solution) (3.60 mL, 58.7 mmol) and water (30.0 mL) under nitrogen atmosphere and the suspension was stirred at 95°C for 30 min. Methanol (30 mL) was added and the mixture was stirred at 95°C for 18 h. The mixture was cooled to room temperature and concentrated to remove methanol. Water (50 mL) was added and the pH was adjusted to 11 with 1.0 N sodium hydroxide. The aqueous phase was extracted with ethyl acetate (3x), the combined extracts were washed with brine, dried over MgSO4, filtered, and the filtrate was concentrated to provide the title compound (6.29 g, 9.03 mmol, 100% yield). LCMS (m / z) 523.4 (M+H) + .

[0561] 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.

[0562]

[0563] 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) at 0° C. under nitrogen atmosphere, and the mixture was stirred at 0° C. for 40 min. (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 h. Saturated NaHCO3 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 (x 2). The combined organic extracts were washed with saturated NaHCO3 (100 mL), water (100 mL) and brine (100 mL), dried over MgSO4, filtered, and the filtrate was concentrated. The product was purified by ISCO Purification by Rf chromatography (220 g silica Gold column) eluting with a step gradient of 0 to 5%, 5% and 5 to 20% methanol in dichloromethane (DCM) provided the title compound (4.75 g, 6.94 mmol, 62.6% yield) as a light beige solid. LCMS (m / z) 637.2 (M+H) + .

[0564] 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.

[0565]

[0566] 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 at room temperature under a hydrogen balloon atmosphere for 90 min, and 18 h. The mixture was passed through Filter, wash the catalyst with methanol, and concentrate the combined filtrates under reduced pressure to afford the title compound as a light beige solid (1.653 g, 2.381 mmol, 92% yield). LCMS (m / z) 639.3 (M+H) + .

[0567] 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, 3 hydrochloride.

[0568]

[0569] 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 slowly added 4M HCl (5.00 mL, 20.00 mmol). The mixture was stirred at room temperature for 1 h and concentrated under reduced pressure to afford the title compound (1.73 g, 2.483 mmol, 100% yield) as a light brown solid. LCMS (m / z) 539.4 (M+H) + .

[0570] The following intermediates were prepared using procedures analogous to those described for Intermediate 4:

[0571]

[0572] 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, 3 hydrochloride.

[0573]

[0574] Step 1: Methyl (1S,4S)-1-(2-bromoethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate.

[0575]

[0576] 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 (1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid methyl ester (Intermediate 1) (4.39 g, 20 mmol) in tetrahydrofuran (THF) (20.00 mL). The mixture was stirred at -78°C for 1 h and a solution of 1,2-dibromoethane (5.17 mL, 60.0 mmol) in tetrahydrofuran (THF) (20.00 mL) was added dropwise at -78°C. The mixture was stirred at -78°C for 30 minutes, warmed to 0°C over 3.5 h, and stirred at 0°C for 60 min. Saturated aqueous NH4Cl solution (100 mL) and ethyl acetate were added, and the aqueous phase was extracted with ethyl acetate (3x). The combined organic phases were washed with saturated aqueous NaHCO3 (1x), with brine (1x), dried over MgSO4, filtered, and the filtrate was concentrated to afford the title compound as a brown oil (8.09 g, 11.66 mmol, 58.3% yield). LCMS (m / z) 326.1 (M+H) + .

[0577] 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.

[0578]

[0579] To a solution of (1S,4S)-1-(2-bromoethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid methyl ester (8.09 g, 11.66 mmol) in N,N-dimethylformamide (DMF) (100 mL) was added di-tert-butyl iminodicarboxylate (10.86 g, 50 mmol) and Cs2CO3 (20.36 g, 62.5 mmol). 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 ether (5x). The combined organic extracts were concentrated. The residue was purified by ISCO Chromatography (120 g silica Gold column, 150 mL / min) eluting with a gradient of 0 to 100% ethyl acetate in heptane provided the title compound (2.65 g, 1.375 mmol, 11.80% yield). LCMS (m / z) 463.4 (M+H) + .

[0580] Step 3: (1R,4S)-1-(2-((tert-Butyloxycarbonyl)amino)ethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid

[0581]

[0582] To a solution of (1R,4S)-1-(4-((di-tert-butoxycarbonyl)amino)ethyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid methyl ester (2.65 g, 1.375 mmol) in methanol (10.31 mL) and water (3.44 mL) was added 5 M aqueous sodium hydroxide solution (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 1 N HCl. The white solid precipitate was collected by filtration, washed with cold water (100 mL), with hexanes (100 mL), and chased with toluene (3x) to provide the title compound (797 mg, 0.549 mmol, 39.9% yield) as an orange solid. LCMS (m / z) 349.3 (M+H) + .

[0583] 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

[0584]

[0585] 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) was dissolved in N,N-dimethylacetamide (DMA) (13.75 mL) under nitrogen atmosphere. 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, 2 hydrochloride (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 NH4Cl aqueous solution (30 mL) were added. The precipitate was collected by filtration, washed with water, saturated aqueous NaHCO 3 solution and water to provide the title compound as a brown solid (3.85 g, 1.375 mmol, 100% yield). LCMS (m / z) 455.4 (M+H) + .

[0586] 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

[0587]

[0588] 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) were added hydroxylamine hydrochloride (562 mg, 8.09 mmol), hydroxylamine (50 wt% in aqueous solution) (0.550 mL, 9.14 mmol) and water (5.00 mL), and the mixture was stirred at 75 ° C for 18.5 h. The mixture was cooled to room temperature and concentrated. Purification by EZ Prep chromatography (C18 100 g Gold column, 60 mL / min) eluting with a gradient of 10 to 50% acetonitrile in water containing formic acid (0.1%) provided the title compound (266.5 mg, 0.516 mmol, 37.5% yield) as a yellow oil. LCMS (m / z) 455.4 (M+H) + .

[0589] 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

[0590]

[0591] 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) and 2-propanol (0.113 mL, 1.463 mmol) was added sodium triacetoxyborohydride (0.310 g, 1.463 mmol) at 5°C, and the mixture was stirred for 1 h. The reaction was cooled to 1°C, (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 aqueous NaHCO3 (5 mL), water (10 mL) and ethyl acetate (10 mL) were added. The aqueous phase was extracted with ethyl acetate (5x). The combined organic phases were washed with 1M sodium hydroxide (1x), with brine (1x), dried over Na2SO4, filtered, and the filtrate was concentrated. The residue was placed under high vacuum overnight to provide the title compound (257.6 mg, 0.367 mmol, 75% yield) as a tan solid. LCMS (m / z) 569.5 (M+H) + .

[0592] 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

[0593]

[0594] 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 at room temperature under hydrogen atmosphere for 1 h. The reactant was passed through Filter, wash with methanol, and concentrate the filtrate to provide the title compound as a white solid (0.1493 g, 0.235 mmol, 52.0% yield). LCMS (m / z) 571.5 (M+H) + .

[0595] 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, 3-hydrochloride

[0596]

[0597] 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 afford the title compound (0.147 g, 0.2 mmol, 100% yield) as a white solid. LCMS (m / z) 471.4 (M+H) + .

[0598] 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, 3-acetate

[0599]

[0600]

[0601] Step 1: Methyl (1R,4S)-1-((Benzyloxy)methyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate

[0602]

[0603] To a solution of 1M LHMDS in tetrahydrofuran (THF) (25 mL, 25.00 mmol) was added dropwise a solution of (1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid methyl ester (4.39 g, 20 mmol) in 2-methyltetrahydrofuran (2-MeTHF) (20.00 mL) at -78 °C, 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 aqueous NH4Cl solution (100 mL) and ethyl acetate were added, and the aqueous phase was extracted with ethyl acetate (3x). The combined organic extracts were washed with saturated aqueous NaHCO3 (1x), with brine (1x), dried over MgSO4, filtered, and the filtrate was concentrated. Chromatography (120 g silica Gold column, 85 mL / min) eluting with a gradient of 0 to 20% ethyl acetate in heptane provided the title compound (4.64 g, 12.44 mmol, 62.2% yield) as a colorless oil. LCMS (m / z) 340.3 (M+H) + .

[0604] Step 2: (1R,4S)-1-((Benzyloxy)methyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid

[0605]

[0606] To a mixture of (1R,4S)-1-((benzyloxy)methyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid methyl ester (4.64 g, 13.67 mmol) in methanol (34.2 mL) and water (11.39 mL) was added 5 M aqueous sodium hydroxide solution (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 reduced pressure to provide the title compound (3.48 g, 9.84 mmol, 72.0% yield) as an orange solid. LCMS (m / z) 326.3 (M+H) + .

[0607] 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

[0608]

[0609] 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 in N,N-dimethylacetamide (DMA) (26.7 mL) (4.47 g, 11.76 mmol) was added DIPEA (7.47 mL, 42.8 mmol) dropwise under nitrogen atmosphere, and the mixture was stirred at room temperature for 15 min. Commercially available 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine, 2 hydrochloride (2.94 g, 10.69 mmol) was added and the mixture was stirred at room temperature for 30 minutes. The mixture was poured into a solution of water and saturated aqueous NH4Cl (1:1) at 0°C. The precipitate was collected by filtration and washed with water to provide the title compound as a red-tan wet solid (6.05 g, 10.69 mmol, 100% yield). LCMS (m / z) 510.4 (M+H) + .

[0610] 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

[0611]

[0612] 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 aqueous solution) (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 (5x). The combined organic extracts were washed with brine and concentrated under reduced pressure. The residue was chased with dichloromethane (DCM) to provide the title compound (6.15 g, 7.84 mmol, 73.3% yield) as a tan solid. LCMS (m / z) 432.4 (M+H) + .

[0613] 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

[0614]

[0615] 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) was added sodium triacetoxyborohydride (3.07 g, 14.46 mmol) at 0°C and the mixture was stirred for 1 hour. (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 aqueous NaHCO3 solution (20 mL), water (30 mL) and ethyl acetate (20 mL) were added and the aqueous phase was extracted with ethyl acetate (5x). The combined organic extracts were washed with 1M sodium hydroxide (1x), with brine (1x), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to afford the title compound as a tan solid (3.46 g, 4.82 mmol, 100% yield). LCMS (m / z) 546.3 (M+H) + .

[0616] 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.

[0617]

[0618] A mixture 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 at room temperature for 2.5 days under a hydrogen atmosphere. The mixture was passed through Filter, wash the catalyst with methanol, and concentrate the combined filtrates to provide the title compound as a yellow solid (1.674 g, 2.109 mmol, 43.8% yield). LCMS (m / z) 548.3 (M+H) + .

[0619] 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.

[0620]

[0621] 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) was added Pd(OH)2 (2.085 g, 2.97 mmol), hydrogen 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 hours. The mixture was opened to the atmosphere for 15 min and passed through with methanol. Filter. Combine this solution with similar reactants 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). Chromatography (24 g silica Gold column, 35 mL / min) eluting with a gradient of 0 to 100% methanol in dichloromethane (DCM) containing ammonium hydroxide (10%) provided the title compound (265 mg, 0.441 mmol, 16.32% yield) as a white solid. LCMS (m / z) 458.4 (M+H) + .

[0622] 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

[0623]

[0624] 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 aqueous NH4Cl (10 mL) and dichloromethane (DCM) were added and the organic phase was concentrated under a steady stream of nitrogen. The product was purified by ISCO. Purification by chromatography (12 g silica Gold column, 30 mL / min) eluting with a gradient of 0 to 100% methanol in dichloromethane (DCM) provided the title compound (51 mg, 0.075 mmol, 32.1% yield) as a brown oil. LCMS (m / z) 497.7 (M+H) + .

[0625] 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, 3-acetate.

[0626]

[0627] 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 hydrogen atmosphere for 1 h. The mixture was stirred open to air for 10 minutes. This mixture was combined with similar reactants 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 passed through Filtration and concentration provided the title compound as an off-white solid (314 mg, 0.302 mmol, 100% yield). LCMS (m / z) 501.4 (M+H) + .

[0628] Intermediate 10: (1r, 4r)-4-(2-(dibenzylamino)ethoxy)cyclohexane-1-ol

[0629]

[0630] 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-chloroethane-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. NH4Cl (10 g) was added and the mixture was extracted with ethyl acetate (3 x 150 mL). The combined organic extracts were washed with brine (100 mL), dried over MgSO4, 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 Chromatography (120 g Gold column, 85 mL / min) eluting with a gradient of 30 to 70% ethyl acetate in heptane provided the title compound (5.8227 g, 17.15 mmol, 50.8% yield). LCMS (m / z) 340.4 (M+H) + .

[0631] 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.

[0632]

[0633] Step 1: 3-(Trifluoromethyl)-1,6-naphthyridin-2-ol.

[0634]

[0635] 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 to a solution of 3,3,3-trifluoropropionyl chloride (9.18 g, 62.6 mmol) in dichloromethane (DCM) (200 mL) at 20 °C for 20 min. The mixture was stirred for 1.5 h, saturated aqueous NaHCO3 solution (200 mL) was added, and the mixture was vigorously stirred at room temperature for 5 min. The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in N,N-dimethylformamide (DMF) (100 mL), K2CO3 (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 the 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 dried in air to provide the title compound (7.24 g, 33.8 mmol, 81% yield) as a dark yellow solid. LC-MS m / z 215.1 (M+H) + .

[0636] Step 2: 6-Benzyl-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ol.

[0637]

[0638] Benzyl bromide (4.07 mL, 34.2 mmol) was added to a suspension of 3-(trifluoromethyl)-1,6-naphthyridine-2-ol (6.66 g, 31.1 mmol) in acetonitrile (50 mL). 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, sodium borohydride (5.88 g, 156 mmol) was added portionwise over 30 min, and the mixture was stirred at room temperature under a nitrogen atmosphere for 1 h. Methanol (200 mL) was added, followed by sodium borohydride (5.88 g, 156 mmol) over 30 minutes. The reaction mixture was stirred under a nitrogen atmosphere for 30 minutes, and additional sodium borohydride (5.88 g, 156 mmol) was added portionwise over 30 minutes. 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. (120g RediSep Column, 85 mL / min) with a gradient elution of 0 to 75% dichloromethane (DCM): methanol (4:1) in dichloromethane (DCM) to provide the title compound as a yellow-orange solid (3.75 g, 12.16 mmol, 39% yield). LC-MS m / z 309.3 (M+H) + .

[0639] Step 3: tert-Butyl (4-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butyl)carbamate.

[0640]

[0641] To a solution of 6-benzyl-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-2-ol (3.75 g, 12.16 mmol) in dichloromethane (DCM) (200 mL) was added dropwise a solution of trifluoromethanesulfonic anhydride (2.260 mL, 13.38 mmol) in dichloromethane (DCM) (20 mL) at 0 ° C for 5 min. The mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. Bis(triphenylphosphine)palladium(II) dichloride (0.171 g, 0.243 mmol) and cuprous(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 but-3-yn-1-ylcarbamate (3.09 g, 18.24 mmol) and DIPEA (6.37 mL, 36.5 mmol) were added in sequence. The mixture was stirred at 80 °C for 15 h and concentrated under reduced pressure. 20% palladium hydroxide / 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 air for 15 min and filtered. The catalyst was washed with a solution of 2M NH3 in methanol (100 mL) and with methanol (100 mL). 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 dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. (220g RediSep Column purification (150 mL / min) eluting with 5% methanol (containing ammonium hydroxide) in dichloromethane (DCM) provided the title compound (3.0 g, 8.03 mmol, 66% yield) as a light yellow oil. LC-MS m / z 374.1 (M+H)+ .

[0642] 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.

[0643]

[0644] To (1S,3R)-3-(((benzyloxy)carbonyl)amino)-1-isopropylcyclopentane-1-carboxylic acid (2.62 g, 8.57 mmol) (dried by azeotroping with toluene before use) was added dichloromethane (DCM) (15 mL), HATU (1.751 g, 4.61 mmol) and DIPEA (5.99 mL, 34.3 mmol) in sequence, 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 evenly divided into two 20 mL sealed test tubes and heated at 80 °C for 25 h. The mixture was combined, dichloromethane (DCM) (100 mL), water (50 mL) and 1.0 M NaOH (50 mL) were added, and the mixture was vigorously stirred for 5 min. The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. (80g RediSep Column, 22 mL / min) with a gradient elution of 0 to 22% ethyl acetate:ethanol (3:1) in hexanes provided the title compound as a light yellow foam (2.61 g, 3.95 mmol, 92% yield). LC-MS m / z 661.4 (M+H) + .

[0645] 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.

[0646]

[0647] 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 with hydrogen. Hydrogen was bubbled through the mixture for 2 min, and the reaction mixture was stirred under a hydrogen balloon atmosphere for 30 min. The mixture was opened to the air, a 7.0 M solution of ammonia in methanol (50 mL) was added, and stirred for 15 min. The mixture was filtered, the catalyst was washed with methanol, and the combined filtrate was 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 and 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 cooling 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 dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by ISCO (120g RediSep Column purification (column, 85 mL / min) eluting with a gradient of 0 to 2% methanol in dichloromethane (DCM) provided the title compound as a foam. LC-MS m / z 641.2 (M+H) + .

[0648] 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, 2-hydrochloride, 1,4-dioxane

[0649]

[0650] 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 under vacuum to give the title product (223.2 mg, 0.318 mmol, 97% yield) LC / MS (M+H) + 541.4m / z.

[0651] 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.

[0652]

[0653] Step 1: Benzyl 3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate.

[0654]

[0655] A mixture of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine, 2 hydrochloride (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 for 5 minutes under a nitrogen atmosphere 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 x 30 mL). The aqueous phase was extracted with ethyl acetate (2 x 30 mL). The combined organic extracts were dried over MgSO4, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by normal phase silica gel chromatography (40 g RediSep Column) purification with a step gradient of 30% and 50% ethyl acetate in hexanes provided the title compound as a yellow waxy solid (1100 mg, 3.27 mmol, 90% yield). LC-MS m / z 337.1 (M+H) + .

[0656] Step 2: Benzyl 2-(4-hydroxybutyl)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate.

[0657]

[0658] To a mixture of 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′-bipyridyl-N1,N1′]bis[3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridyl-N]phenyl-C]iridium(III) hexafluorophosphate, [Ir{dF(CF3)ppy}2(dtbpy)]PF6 (0.125 g, 0.112 mmol), 6M HCl (1.363 mL, 8.18 mmol) and trifluoroacetic acid (4.01 mL, 52.0 mmol) in methanol (9 mL) were added benzoyl peroxide, A75 (5.40 g, 16.73 mmol) and tetrahydrofuran (THF) (45.0 mL).

[0659] The mixture was irradiated with a H150-Blue Kessil lamp at 1 inch while wrapped in aluminum foil. Room air was blown onto the reaction vessel and the mixture was stirred at room temperature for 48h. The lamp was removed and the mixture was stirred overnight. The mixture was cooled in an ice bath and saturated NaHCO3 aqueous solution (100mL) was added. The aqueous phase was extracted with ethyl acetate. The organic extract was washed with brine (50mL). The aqueous phase was extracted with ethyl acetate again, and the combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. By ISCO (120g RediSep Column) purification with a step gradient of 30 to 50% and 75 to 100% ethyl acetate in hexanes provided the title compound (1780 mg, 4.361 mmol, 58.7% yield) as a partially solidified light orange oil. LC-MS m / z 409.2 (M+H) + .

[0660] Step 3: Benzyl 2-(4-(3-(methoxycarbonyl)phenoxy)butyl)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate.

[0661]

[0662] 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 (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 with brine. The organic phase was dried over MgSO4 and concentrated. The reaction mixture was purified by ISCO. (80g RediSep Column) purification with a gradient of 10 to 50% ethyl acetate in heptane provided the title compound (1580 mg, 2.91 mmol, 66.8% yield). LC-MS m / z 543.2 (M+H) + .

[0663] Step 4: Methyl 3-(4-(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)butoxy)benzoate.

[0664]

[0665] 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. (40g RediSep Column) purification with a gradient of 30 to 70% methanol in dichloromethane (DCM) containing ammonium hydroxide (1%) provided the title compound (1150 mg, 2.017 mmol, 69.3% yield). LC-MS m / z 409.2 (M+H) + .

[0666] 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.

[0667]

[0668] 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 minutes, 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 aqueous NaHCO3 solution (15 mL) was added, and the aqueous phase was extracted with dichloromethane (DCM) (2 x 150 mL). The combined organic extracts were dried over MgSO4, filtered, and the filtrate was concentrated under reduced pressure. (40g RediSep Column) purification with a gradient of 15 to 70% ethyl acetate in heptane provided the title compound (1100 mg, 1.496 mmol, 53.1% yield). LC-MS m / z 662.5 (M+H) + .

[0669] 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.

[0670]

[0671] 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 x 3 mL) and dried under high vacuum to afford the title compound (1.35 g, 1.710 mmol, 99% yield) as an orange oil. LC-MS m / z 562.3 (M+H) + .

[0672] 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.

[0673]

[0674] 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 (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. Activated molecular sieve powder (~325 mesh, Aldrich) (1 g, 1.701 mmol), (R)-3-methoxytetrahydro-4H-pyran-4-one (0.266 g, 2.041 mmol), followed by sodium triacetoxyborohydride (0.541 g, 2.55 mmol), and the mixture was stirred at room temperature for 22 h under a nitrogen atmosphere. Dichloromethane (DCM) (20 mL) was added. The organic phase was washed with saturated aqueous NaHCO3 solution (25 mL) and with brine (25 mL). The combined aqueous phases were extracted with dichloromethane (DCM) (20 mL). The combined organic extracts were dried over MgSO4, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by ISCO Lumen (80g RediSep Column, 60 mL / min) with a gradient elution of 10 to 90% dichloromethane (DCM): methanol (4:1) in dichloromethane (DCM) to provide the title compound (505 mg, 0.747 mmol, 43.9% yield) as a colorless oil. LC-MS m / z 676.4 (M+H) + .

[0675] 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.

[0676]

[0677] 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 dried over MgSO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was placed under high vacuum to afford the title compound as a foam (380 mg, 0.574 mmol, 78% yield). LC-MS m / z 662.4 (M+H) + .

[0678] 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

[0679]

[0680] Step 1: (1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-ene-1-carboxylic acid methyl ester

[0681]

[0682] To a mixture of (1R, 4S)-4-(2,5-dimethyl-1H-pyrrole-1-yl)cyclopent-2-ene-1-formic acid methyl ester (1000 mg, 4.56 mmol) in THF (10 mL) was added dropwise LHMDS (7.30 mL, 7.30 mmol) in THF at -20 ° C. The resulting mixture was stirred at -20 ° C for 30 minutes, followed by the addition of 2-iodopropane (0.912 mL, 9.12 mmol) in THF (10 mL). The mixture was stirred at room temperature for 1 hour, then warmed to room temperature until LC-MS showed that the starting material was exhausted. The reactant was quenched with 6% NH4Cl aqueous solution (50 mL) and diluted with ethyl acetate. The organic layer was separated, then washed with water (1X 50 mL), dried over sodium sulfate and concentrated in vacuo. The crude material was then purified by column chromatography (RediSepRf High Efficiency GOLD 24g HP Silica with CH2Cl2 / 20% MeOH in CH2Cl2 as mobile phase) to yield (1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-ene-1-carboxylic acid methyl ester (1.06 g, 80% yield) with 90% purity as a light brown oil. LCMS MS (m / z) 262.2 (M+H) +

[0683] Step 2: (1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-ene-1-carboxylic acid

[0684]

[0685] In a 20 mL microwave vial was added (1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-ene-1-carboxylic acid methyl ester (2000 mg, 7.65 mmol), methanol (3 mL) and potassium hydroxide (3.06 mL, 30.6 mmol). The resulting mixture was heated to 120 ° C for 3 hours. LC-MS showed that the product was completely formed. The MeOH was removed under vacuum. The reaction mixture was then placed on an ice bath. The pH of the solution was adjusted to ~2 with 12N HCl. The light brown precipitate was difficult to filter. The reaction solution was then extracted with ethyl acetate (2X 30 mL). It was then purified on a column chromatograph using RediSepRf High Efficiency GOLD 24g HP Silica with a 0-100% gradient of DCM / 20% MeOH in DCM as the mobile phase to yield the desired product (1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-ene-1-carboxylic acid (1600 mg, 5.82 mmol, 76% yield) as a light brown solid. LCMS (m / z) 248.2 (M+H) + .

[0686] 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

[0687]

[0688] To a 100 mL round bottom flask, (1S,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-isopropylcyclopent-2-ene-1-carboxylic acid (1 g, 4.04 mmol) was added as a red amber slurry. The slurry was dissolved in tetrahydrofuran (THF) (10 mL). DIEA (1.412 mL, 8.09 mmol) was added to the dark solution. The reaction mixture was cooled to 0 ° C. Ms-Cl (0.347 mL, 4.45 mmol) was added dropwise over 2 minutes. The reactants were warmed to room temperature. It was stirred at room temperature for 4 hours. The reaction mixture was cooled in ice for 3 minutes, followed by the addition of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine, 2 hydrochloride (1.008 g, 3.66 mmol). The reactants were gradually warmed to ambient temperature. Add additional volume of DIEA (0.706mL, 4.04mmol). The reactant was stirred at room temperature for 45 minutes. The reaction mixture was added to a separatory funnel with 50mL of 5% NaHCO3. The two layers were distributed, and the aqueous layer obtained was extracted with ethyl acetate twice. The organic layers were merged and washed with water. The organic solutions obtained were merged and concentrated under vacuum to provide a dark brown oil. The oil was pumped overnight and purified using the gold 40g silica gel cartridge on ISCO using 10% MeOH / DCM mobile phase. LCMS analysis indicated that it was contaminated by raw carboxylic acid. In order to remove the carboxylic acid starting material, each fraction was merged and washed 5 times with saturated bicarbonate. The resulting organic layer was washed with brine and dried over sodium sulfate, then filtered, concentrated, and purified using an ISCO, 40 g silica gel column with 5% MeOH / DCM mobile phase to provide ((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) + .

[0689] Step 4: ((1S,4S)-4-amino-1-isopropylcyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, 2-hydrochloride

[0690]

[0691] Hydroxylamine hydrochloride (1.025 g, 14.75 mmol) was added to a 50 mL round bottom flask and dissolved in water. Hydroxylamine 50% w / w (aqueous solution) (6.56 mL, 2.508 mmol) was added to the solution. ((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 to the hydroxylamine solution via pipette. The reactants were 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. Thereafter, the solution was diluted with water and transferred to a 250 mL separatory funnel. The aqueous layer was thoroughly extracted 4 times with 10 mL of EtOAc each. The organic layers were combined and concentrated. The resulting orange oil was purified on HPH reverse phase ISCO to provide ((1S,4S)-4-amino-1-isopropylcyclopent-2-en-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone, 2 hydrochloride (.717 g, 1.682 mmol, 67.1% yield) as an orange oil. LCMS (m / z) 354.28 (M+H) + .

[0692] 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

[0693]

[0694] 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, 2 hydrochloride (0.350 g, 0.821 mmol) dissolved in isopropyl acetate (3.40 ml). The solution was cooled to 0 °C. 2-Propanol (0.278 ml, 3.61 mmol) was then added followed by sodium triacetoxyborohydride (0.766 g, 3.61 mmol). The reaction was stirred at 0 °C for 1 h 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 h. 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 on ISCO using a 0-5% DCM / MeOH gradient elution. ((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) + .

[0695] 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

[0696]

[0697] To a small 2-5 mL 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. α-Methylpyridine 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 1 N HCl. The suspension was stirred for 30 min. It was then basified with 2.5 g of sodium carbonate dissolved in 10 mL of water. The solution was extracted with DCM (3x15 mL), washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude oil was combined with another batch and purified on a 40g ISCO cartridge, eluting with a gradient elution method (0-20% MeOH / DCM). The desired fractions were combined and concentrated to provide ((1S, 4S) -4- ((3- (dibenzylamino) propyl) ((4S) -3-methoxytetrahydro -2H- pyrans -4- base) amino) -1- isopropylcyclopent-2-ene-1-yl) (3- (trifluoromethyl) -7,8- dihydro -1,6- naphthyridine -6 (5H) -yl) ketone (30 mg, 0.043 mmol, 4.42% yield) as an orange solid. LCMS (m / z) 705.57 (M+H) + .

[0698] 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

[0699]

[0700] Pd-C (85 mg, 0.040 mmol) was added to a 50 mL round bottom flask. 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-ene-1-yl)(3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-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 through a balloon. The reactants were stirred at room temperature for 16 h. After completion, the reaction mixture was passed through a pad of celite and concentrated to provide 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 (13 mg, 0.025 mmol, 61.7% yield) as a clear oil. LCMS (m / z) 527.44 (M+H) + .

[0701] Intermediate 15: 3-(Dibenzylamino)propan-1-ol

[0702]

[0703] Step 1: 3-(Dibenzylamino)propan-1-ol

[0704]

[0705] To a 250 mL round bottom flask was added 3-aminopropane-1-ol (5.06 ml, 66.6 mmol) dissolved in acetone (119 ml). To the solution was added (bromomethyl)benzene (16.23 ml, 136 mmol) followed by K2CO3 (19.32 g, 140 mmol). The reactants were stirred at room temperature for 16 hours. After completion, the reaction mixture was filtered. The solids were washed with acetone. The filtrate was concentrated to provide a dark brown oil and purified on a 330 g silica gel column on ISCO with a gradient of 10-50% EtOAc / heptane mobile phase to provide 3-(dibenzylamino)propane-1-ol (15.016 g, 58.8 mmol, 88% yield) as a yellow oil. LCMS (m / z) 256.6 (M+H) + .

[0706] Step 2: 3-(Dibenzylamino)propanal

[0707]

[0708] Oxalyl chloride DCM solution (6.31 mL, 3.16 mmol, 0.5 M) was added to a dried 100 mL round bottom flask at -78 °C. Dimethyl sulfoxide (0.466 mL, 6.57 mmol) was then added dropwise over 1 minute. The reactants were stirred for 15 minutes. After activation, 3-(dibenzylamino)propane-1-ol (4.90 mL, 1.958 mmol) dissolved in 4.9 mL of DCM was added dropwise over 5 minutes. The reactants were stirred for 15 minutes. TLC (30% EtOAc: heptanes) monitored the complete formation of the sulfur oxygen bond indicated by the loss of alcohol spots. Triethylamine (0.718 mL, 5.15 mmol) was added dropwise to the reactants over 1 minute. The reactants were stirred for 15 minutes. After completion, the reactants were 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 provide 3-(dibenzylamino)propanal (412 mg, 1.626 mmol, 83% yield) as a crude product. 1 H NMR(400MHz, DMSO-d6)δppm 2.55-2.65(m,2H)2.69-2.79(m,2H)3.52(s,4H)7.21-7.39(m,10H)9.50(t,J=2.20Hz,1H)

[0709] Intermediate 16: (1S,3R)-3-(((Benzyloxy)carbonyl)amino)-1-isopropylcyclopentane-1-carboxylic acid

[0710]

[0711] To a solution of (1S,3R)-3-((tert-butoxycarbonyl)amino)-1-isopropylcyclopentane-1-carboxylic acid methyl ester (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 slowly added and the mixture was extracted with dichloromethane (DCM) (3 x 150 mL). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in 2,2,2-trifluoroacetic acid (20 mL, 12.75 mmol). After 1 h, 12 M hydrochloric acid (50.0 mL, 600 mmol) was added, 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 dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to provide the title compound (3.32 g, 10.87 mmol, 85% yield) as a brown oil. LC-MS m / z 306.3 (M+H) + .

[0712] Intermediate 17A: (R)-benzyl 8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate; and Intermediate 17B: (S)-benzyl 8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate.

[0713]

[0714]

[0715] Step 1: Benzyl 3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate.

[0716]

[0717] 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 slowly added 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 to a fine powder with a spatula, washed with water, and air-dried overnight under vacuum filtration to provide the title compound (6.06 g, 18.02 mmol, 99% yield) as a yellow solid. LC-MS m / z 337.3 (M+H) + .

[0718] Step 2: 6-((Benzyloxy)carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine 1-oxide.

[0719]

[0720] 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) was added methyltrioxorhenium (vii) (0.160 g, 0.642 mmol) at room temperature, followed by dropwise addition of 30% hydrogen peroxide (3.64 mL, 35.7 mmol) and the mixture was stirred at room temperature for 20 h. Excess hydrogen peroxide was destroyed by addition of manganese (IV) oxide (1.0 mg, 0.012 mmol) and the mixture was stirred for 30 min. 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 dried over Na2SO4 and filtered. The filtrate was diluted with toluene (50 mL) and concentrated under reduced pressure to provide the title compound (6.20 g, 17.6 mmol, 99% yield) as a white solid. LC-MS m / z 353.3 (M+H) + .

[0721] Step 3: Benzyl 8-bromo-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate.

[0722]

[0723] The following two solutions were added dropwise simultaneously from two separate syringes 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) over 7 min at room temperature: a solution of phosphorus oxybromide (10.09 g, 35.2 mmol) in chloroform (10.0 mL) and a solution of triethylamine (1.385 mL, 9.93 mmol) in chloroform (10.0 mL). The mixture was stirred at room temperature for 45 min, poured on ice, and basified with 2.0 M Na2CO3. The mixture was extracted with dichloromethane (DCM) (3 x 200 mL). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. Purification by normal phase chromatography (330 g silica gel, 200 mL / min) eluting with a gradient of 0 to 40% ethyl acetate in heptane provided the title compound (2.83 g, 6.82 mmol, 38.7% yield) as a white crystalline solid. LC-MS m / z 417.1 (M+H) + .

[0724] Step 4: (R)-benzyl 8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate; and (S)-benzyl 8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate.

[0725]

[0726] 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 a 1.0 M solution of 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, dried over Na2SO4, and concentrated under reduced pressure. Preparative chiral separation using a Lux Amylose 2 column (5 μm, 21 x 250 mm, 20 mL / min) and eluting with 30% heptane in ethanol containing 0.1% isopropylamine provided the title compound as a white solid (990.1 mg, 2.82 mmol, 43.3% yield). LC-MS m / z 352.2 (M+H) +(S)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylic acid benzyl ester (888.7 mg, 2.53 mmol, 38.9% yield) was also isolated as a white solid. LC-MS m / z 352.3 (M+H) + The absolute stereochemistry of the two enantiomers was determined by VCD using the following procedure:

[0727] The samples were dissolved in CD3CN (16.8 mM) and placed in a 100 μm path length cell with BaF2 windows. TM IR and VCD spectra were recorded on a VCD spectrometer (BioTools Inc., Jupiter, FL) with a resolution of 4 cm -1 One isomer, E1, was collected for 6 hours, and the other isomer, E2, was collected for 12 hours and was concentrated at 1400 cm -1 Optimize the instrument. Use MMFF94x force field in low mode (LowMode) to perform MOE, adopt Born solvation, dielectric constant set to 20 and external dielectric constant set to 47, and perform conformational search on the modeled (R) structure. Use Gaussian 16 (Gaussian Inc., Wallingford, CT) at DFT level b3lyp / 6-31G(d)scrf=(solvent=dimethyl sulfoxide) to perform geometry optimization, frequency and IR and VCD intensity calculation on 10 conformers obtained from conformational search. Use BLAIR to convert Gaussian output files to VCD and IR spectra. Scale the calculated frequencies by 0.981, and convert IR and VCD intensities 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, FL) to generate a confidence level based on the current database, which includes 88 previous correct assignments to different chiral structures.

[0728] 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

[0729]

[0730]

[0731] Step 1: (R)-benzyl 8-((tert-butoxycarbonyl)amino)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate.

[0732]

[0733] To (R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylic acid benzyl ester (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, the precipitate was filtered, washed with water, and dried in air to provide the title compound (4.70 g, 10.41 mmol, 86% yield) as a light yellow solid. LC-MS m / z 452.1 (M+H) + .

[0734] Step 2: (R)-tert-Butyl(3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-8-yl)carbamate.

[0735]

[0736] A mixture of (R)-8-((tert-butoxycarbonyl)amino)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylic acid benzyl ester (4.60 g, 10.19 mmol) and 20% palladium hydroxide / 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 hydrogen balloon atmosphere for 1.5 h. The mixture was opened to the air and stirred for 10 min. A 7.0 M solution of ammonia in methanol (100 mL) was added, the mixture was filtered, and the catalyst was washed with methanol. The combined filtrate was concentrated under reduced pressure. Purification by reverse phase HPLC chromatography (C18 Aq 275g Gold column, 125 mL / min) eluting with a gradient of 30 to 60% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) provided the title compound (2.66 g, 8.38 mmol, 82% yield) as a light yellow foam. LC-MS m / z 318.1 (M+H) + .

[0737] 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.

[0738]

[0739] To a solution of (1S,3R)-3-(((benzyloxy)carbonyl)amino)-1-isopropylcyclopentane-1-carboxylic acid (5.12 g, 16.77 mmol) in dichloromethane (DCM) was added DIPEA (9.15 mL, 52.4 mmol) and HATU (5.90 g, 15.51 mmol) 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 vial was 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 dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. Purification by chromatography (330 g silica gel, 200 mL / min) eluting with a gradient of 0 to 10% methanol in dichloromethane (DCM) provided the title compound (2.95 g, 4.88 mmol, 58.2% yield) as a white foam. LC-MS m / z 605.2 (M+H) + .

[0740] 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.

[0741]

[0742] 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 hydrogen balloon atmosphere for 1 h. The mixture was opened to the air and the mixture was stirred for 15 min. A 2.0 M solution of 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 provide the title compound (2.24 g, 4.76 mmol, 98% yield) as a white foam. LC-MS m / z 471.4 (M+H) + .

[0743] 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, 2-carboxylate.

[0744]

[0745] 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, sodium triacetoxyborohydride (2.52 g, 11.90 mmol) was added, followed by a solution of (R)-3-methoxytetrahydro-4H-pyran-4-one (0.929 g, 7.14 mmol) in isopropyl acetate (50.00 mL) dropwise. 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, the organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. Purification by reverse phase HPLC chromatography (C18 275 g Gold column; 125 mL / min) eluting with 35% acetonitrile in water containing formic acid (0.1%) provided the title compound (2.73 g, 4.03 mmol, 85% yield) as a white foam. The compound was used as an intermediate in the next step. LC-MS m / z 585.2 (M+H) + .

[0746] 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.

[0747]

[0748] 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, 2-formate (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 dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to provide the title compound (1.77 g, 3.65 mmol, 91% yield) as a white foam. LC-MS m / z 485.4 (M+H) + .

[0749] The following intermediates were prepared using procedures analogous to those described for Intermediate 18:

[0750]

[0751] Example 1

[0752] (2S,3S)-N-(4-((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-dodeca-40-azatricarboxylic acid)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide

[0753]

[0754] 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-dodecano-2-azahexadecane-41-oic acid, ammonium salt

[0755]

[0756] In a round-bottom flask, (2S, 3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (0.357g, 1.619mmol) was dissolved in acetonitrile (10ml). TSTU (0.487g, 1.619mmol) and DIPEA (1.131ml, 6.48mmol) were then added and the reactants were stirred at room temperature. After acid activation, 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-12oxatrinonadecan-39-acid (1g, 1.619mmol) was added as a solid, and N,N-dimethylformamide (DMF) (3ml) was added to enhance the solubility of the acid. The reactants were stirred at room temperature and monitored by LCMS. After completion, the reactants were concentrated to provide a viscous oil. The crude material was purified by reverse phase chromatography (loaded as a solution in approximately 6 mL of 10 mM aqueous ammonium bicarbonate solution (adjusted to pH 10 with ammonia), C18 100 g Gold column, 60 mL / min, gradient 5-50% 10 mM aqueous ammonium bicarbonate solution (adjusted to pH 10 with ammonia) to MeCN over 23 min) to provide the title compound (1.3 g, 1.351 mmol, 83% yield) as a colorless oil (87% purity by NMR, the material contained CH2Cl2). LC-MS m / z 820.1 (M+H) + .

[0757] Step 2: ((2S,3S)-N-(4-((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-dodeca-40-azatricarboxylic acid)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide

[0758]

[0759] 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, 3 hydrochloride (89 mg, 0.15 mmol) in dichloromethane (DCM) (1500 μL) was added 1-((2S,3S)-1-methyl-5-oxo-2- A solution of (pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35,38-dodeca-2-azahexadecane-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). The mixture was stirred at room temperature for 2 h, methanol was added, and the mixture was concentrated. The residue was purified by Purification was performed by EZ Prep chromatography (30 g C18 column, 35 mL / min) using a gradient of 10 to 70% acetonitrile in water containing ammonium bicarbonate (10 mM) and adjusted to pH 10 with ammonia. TM Further purification (CSH C18 column, 40 mL / min) with a gradient elution of 15 to 55% acetonitrile in water containing ammonium bicarbonate (10 mM) and adjusted to pH 10 with ammonia provided the title compound (79.2 mg, 0.061 mmol, 40.6% yield) as a clear viscous oil. LCMS m / z (M / 2+H) + 644.1. 1H NMR (400MHz, DMSO-d6) δppm 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=12.7,6.8Hz,1H),2.03-2.14(m,1H),2.22(br t,J=6.4Hz,2H),2.44(dd,J=16.9,7.6Hz,2H),2.50(br s,4H),2.70(br dd,J=16.6,9.3Hz,2H),2.86-3.04(m,5H),3.12-3.28(m,8H),3.38(td,J=5.7,1.7Hz,2H),3.4 2-3.57(m,44H),3.66-3.76(m,2H),3.86(dt,J=7.7,3.7Hz,3H),4.65(d,J=5.9Hz,1H),4.79(br s,2H),7.45(dd,J=7.8,4.9Hz,1H),7.68(dt,J=7.8,2.0Hz,1H),7.74(br s,1H),8.07(t,J=5.4Hz,1H),8.18(br s,1H),8.44-8.52(m,1H),8.56(dd,J=4.6,1.7Hz,1H),8.77(s,1H)

[0760] 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

[0761]

[0762]

[0763] Step 1

[0764] (E)-methyl 4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoate.

[0765]

[0766] To a stirred solution of (1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexane-1-ol (250 g, 736 mmol) in toluene (2500 mL) was added but-2-ynoic acid methyl ester (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 a crude compound. The crude compound was adsorbed on silica gel (500 g, 60-120 mesh) and purified by manual column chromatography (1.5 kg, 100-200 mesh) eluted with 15% EtOAc in petroleum ether to provide (E&Z)-methyl 4-(((lr,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 the two isomers, the compound was adsorbed on silica gel (500 g, 100-200 mesh) and purified by manual column chromatography (1.5 kg, 100-200 mesh) eluted with 15% EtOAc in petroleum ether to provide the title compound (240 g, 463 mmol, 62.9% yield, 84.45% purity) as a light yellow liquid. LC-MS m / z 438.3 (M+H) + .

[0767] Step 2

[0768] (E)-4-(((1,4-trans)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoic acid

[0769]

[0770] (E)-methyl 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 molar aqueous sodium hydroxide solution (4.87 mL, 24.76 mmol) was added. The homogeneous light yellow reaction was heated at reflux for 1 hour. Additional 5.089 M sodium hydroxide (1.217 mL, 6.19 mmol) was added and the reaction was refluxed for 50 minutes. The reaction was cooled for 60 minutes and concentrated in vacuo. The residue was azeotroped twice with toluene to assist in the removal of water. The residue was extracted under high vacuum to provide the title compound (9.9 g, 22.17 mmol, 107% yield, 82% purity, E / Z mixture) as a yellow solid. LC-MS m / z 424.4 (M+H) + .

[0771] Step 3

[0772] Tert-butyl (1R,4r)-4-((E)-4-(((1r,4R)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoylamino)cyclohexane-1-carboxylate.

[0773]

[0774] (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 a partially dissolved mixture was observed. The mixture was stirred for 30 minutes to produce a partially dissolved light green 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 hours. Add additional HATU (1.724 g, 4.53 mmol) and stir the almost homogeneous reactant for 60 minutes. Stir the turbid reactant for another 60 minutes. Dilute the reactant with 200 ml of EtOAc and 200 ml of water and stir for 10 minutes. The resulting homogeneous two-phase mixture is transferred to a separating funnel and the layers are separated. The water layer is extracted another 2 times with 150 ml of EtOAc, and the combined EtOAc layers are washed 4 times with water and 2 times with saturated NaCl to remove DMF. The EtOAc extracts were dried over sodium sulfate, filtered, and concentrated in vacuo and drawn under high vacuum to produce an orange syrup which was purified by silica gel chromatography (Isco Combiflash, 330 gram gold column, 0-80% EtOAc:heptane over 45 minutes, 150 ml / min flow rate, loaded as a solution in DCM) to produce the title compound (4.55 g, 7.52 mmol, 36.5% yield) as a white foamy solid. LC-MS m / z 605.5 (M+H) + .

[0775] Step 4

[0776] tert-Butyl (1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butyrylamino)cyclohexane-1-carboxylate

[0777]

[0778] A 500 ml 3-neck RB flask was charged with tert-butyl (1R, 4r)-4-((E)-4-(((1r, 4R)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoylamino)cyclohexane-1-carboxylate (6.40 g, 10.58 mmol) and isopropanol (120 mL), and the suspension was stirred until a homogeneous solution was obtained. 10% wet Pd-C (0.640 g, 6.01 mmol) was added and the flask was evacuated and placed under 2 hydrogen balloons connected to the end of the neck of the flask. The middle neck was covered with a rubber septum. The reactants were stirred at room temperature for 16 hours. The reactants were degassed and filtered through diatomaceous earth twice. The filtrate was concentrated in vacuo and extracted under high vacuum to produce a waxy gray solid with a slight odor of isopropanol. The waxy solid was dissolved in DCM and concentrated in vacuo at 54 °C for 20 minutes to aid in the removal of isopropanol. The residue was extracted under high vacuum to provide the title compound (4.44 g, 10.41 mmol, 98% yield) as a waxy grey solid. LC-MS m / z 427.4 (M+H) + .

[0779] Step 5

[0780] 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)butyrylamino)cyclohexane-1-carboxylate.

[0781]

[0782] (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 DCM in a 500 ml RB flask at room temperature with stirring. HATU (4.34 g, 11.42 mmol) was added and the suspension was stirred for 15 minutes. A solution of tert-butyl (1R,4r)-4-(4-(((1r,4R)-4-(2-aminoethoxy)cyclohexyl)oxy)butyrylamino)cyclohexane-1-carboxylate (4.43 g, 10.38 mmol) in DCM (20 ml) was added dropwise over 15 minutes via a pipette. After the addition was complete, a solution of DIEA (5.44 mL, 31.2 mmol) in DCM (10 ml) was added dropwise over 10 minutes and the resulting homogeneous dark solution was stirred at room temperature for 16 hours. The reaction 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 drawn under high vacuum to produce an orange slurry, which was purified by silica gel chromatography (Isco Combiflash, 0-10% MeOH:DCM over 60 minutes, 330 grams of gold column, 150 ml / min flow rate, loaded as a solution in 30 ml DCM) to provide the title compound (4.13 g, 6.57 mmol, 63.2% yield) as a white solid. LC-MS m / z 629.3 (M+H) + .

[0783] Step 6

[0784] (1R,4r)-4-(4-(((1S,4R)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butyrylamino)cyclohexane-1-carboxylic acid, hydrochloride.

[0785]

[0786] 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)butyrylamino)cyclohexane-1-carboxylate (4.13 g, 6.57 mmol) was dissolved in dry 1,4-dioxane (13 ml) in a 250 ml RB flask with stirring. A 4M solution of anhydrous HCl (39 mL, 156 mmol) in 1,4-dioxane was added and the mixture was stirred at room temperature. After the addition of HCl, an insoluble oil was observed. The mixture was stirred at room temperature for 90 minutes. The reaction was concentrated in vacuo (at 60 degree bath temperature) and pulled under high vacuum for 15 hours to provide the title compound as a white solid (4.187 g, 6.87 mmol, 105% yield). LC-MS m / z 573.4 (M+H) + .

[0787] 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

[0788]

[0789] 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)butyrylamino)cyclohexane-1-carboxylic acid, hydrochloride (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 minutes. ((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, 3 hydrochloride (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 h. Dichloromethane (DCM) (6 mL) and saturated NaHCO3 (6 mL) were added and the mixture was stirred for 10 min. The organic phase was washed with saturated NaCl, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by ISCO Purification by chromatography (12 g silica Gold column, 30 mL / min) eluting with a gradient of 0 to 30% methanol in dichloromethane (DCM) provided the title compound (63.5 mg, 0.0508 mmol, 64.7% yield) as a white solid. LCMS (m / z) 1093.7 (M+H) + . 1H NMR (400MHz, methanol-d4) δppm 1.22-1.43(m,8H)1.50-1.77(m,8H)1.83(dt,J=14,6.7Hz,4H)1.90-2.05(m,8H)2.11-2.20(m,1H)2.25(t,J=7.3Hz,2H)2.60(br d,J=11Hz,2H)2.66(s,3H)2.68-2.76(m,2H)2.80-2.91(m,2H)2.83-2.83(m,1H)2.96(br s,1H)3.03-3.16(m,2H)3.24-3.31(m,2H)3.35-3.53(m,17H)3.56-3.78(m,2H)3.83-4.10(m,6H)4.15(br d,J=13Hz,2H)4.43(br s,2H)4.80-4.85(m,1H)7.54(dd,J=8.8,4.9Hz,1H)7.81(dt,J=8.3,2.0Hz,1H)8.09(br s,1H)8.52(d,J=2.0Hz,1H)8.59(dd,J=4.9,1.5Hz,1H)8.72(s,1H).

[0790] The following compounds were prepared using a procedure similar to that described in Example 2:

[0791]

[0792]

[0793]

[0794]

[0795]

[0796]

[0797]

[0798]

[0799]

[0800]

[0801]

[0802]

[0803]

[0804]

[0805]

[0806] 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

[0807]

[0808] Step 1: tert-Butyl (3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)carbamate.

[0809]

[0810] 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% solution in ethyl acetate) (5.95 mL, 9.99 mmol). The mixture was stirred at room temperature for 2 h, washed with water (10 mL), dried over Na2SO4, and concentrated under reduced pressure. Purification by normal phase chromatography eluting with a gradient of 0 to 10% methanol in dichloromethane (DCM) provided the title compound (3430 mg, 8.20 mmol, 90% yield) as a light yellow liquid. LCMS (m / z) 377.1 (M+H) + .

[0811] Step 2: (2S,3S)-N-(3-aminopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, 2-hydrochloride.

[0812]

[0813] 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 afford the title compound (3143 mg, 8.10 mmol, 89% yield) as a white solid. LCMS (m / z) 277.1 (M+H) + .

[0814] Step 3: tert-Butyl (4-((3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)carbamate.

[0815]

[0816] To a mixture of (2S,3S)-N-(3-aminopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, 2 hydrochloride (3143 mg, 9 mmol), 4-((tert-butoxycarbonyl)amino)butanoic acid (1829 mg, 9.00 mmol) in dichloromethane (DCM) (30 mL) was added DIPEA (4.70 mL, 27.0 mmol) followed by a 50% solution of T3P 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 dried over Na2SO4 and concentrated under reduced pressure. Purification by normal phase chromatography eluting with a gradient of 0 to 10% methanol in dichloromethane (DCM) provided the title compound (900 mg, 1.852 mmol, 20.58% yield) as a light yellow liquid to provide the title compound (900 mg, 1.852 mmol, 20.58%) as a light yellow liquid. LCMS m / z 462.5 (M+H) + .

[0817] Step 4: (2S,3S)-N-(3-(4-aminobutyrylamino)propyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, 2 hydrochloride.

[0818]

[0819] 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 afford the title compound (847 mg, 1.852 mmol, 20.58% yield) as a white solid. LCMS (m / z) 362.1 (M+H) + .

[0820] Step 5: (1s,4s)-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)cyclohexane-1-carboxylate.

[0821]

[0822] To a mixture of (2S,3S)-N-(3-(4-aminobutyrylamino)propyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, 2 hydrochloride (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)-4-aminocyclohexanecarboxylic acid tert-butyl ester, hydrochloride (437 mg, 1.852 mmol). The mixture was stirred at room temperature for 2 h, washed with water (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The product was purified by ISCO. Purification by chromatography (24 g Gold column) eluting with a gradient of 0 to 20% dichloromethane (DCM) in methanol provided the title compound (1200 mg, 1.644 mmol, 89% yield) as an off-white solid. LCMS (m / z) 657.2 (M+H) + .

[0823] 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.

[0824]

[0825] 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) for 1 h. It was then concentrated under vacuum to give the title compound (1155 mg, 1.631 mmol, 88% yield) as a white solid. LCMS (m / z) 601.1 (M+H) +

[0826] 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

[0827]

[0828] To ((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, 3 hydrochloride (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. The residue was purified by MDAP (XSelect TMCSH C18 column, 40 mL / min) and eluted with a gradient of 15 to 55% acetonitrile in water containing ammonium bicarbonate (10 mM) and adjusted to pH 10 with ammonia to provide 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 (400MHz, methanol-d4) δ1.38 (br d,J=8.3Hz,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=12.2,5.9Hz,2H),4.17(br d,J=11.2Hz,1H),4.84(d,J=6.4Hz,1H),7.54(dd,J=7.6,5.1Hz,1H),7.83(dt,J=7.9,1.9Hz,1H),8.06(br s,1H),8.54(d,J=1.5Hz,1H),8.58(dd,J=4.9,1.5Hz,1H),8.72(s,1H)

[0829] Embodiment 17

[0830] (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

[0831]

[0832]

[0833] 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.

[0834]

[0835] 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) and triethylamine (25.8 mL, 185 mmol) and the mixture was stirred at room temperature under a nitrogen balloon atmosphere for 2 h. Saturated NaHCO was added and the mixture was extracted with dichloromethane (DCM). The combined organic extracts were dried over NaSO, filtered, and the filtrate was concentrated. The product was purified by ISCO Chromatography (330 g silica Gold column, 200 mL / min) eluting with a gradient of 0 to 15% methanol (containing 10% NH4OH) in dichloromethane (DCM) provided the title compound (31.5 g, 65.7 mmol, 92% yield) as a yellow oil. LCMS (m / z) 480.2 (M+H) + .

[0836] Step 2: 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

[0837]

[0838] 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-ate (31 g, 64.6 mmol) in dichloromethane (DCM) (40 mL) was added 4N HCl in 1,4-dioxane (81 mL, 323 mmol) at 0°C. The mixture was stirred at room temperature for 2 h and concentrated to provide the title compound (29 g, 63.1 mmol, 98% yield). LCMS (m / z) 424.2 (M+H) + .

[0839] Step 3: (1s,4s)-tert-butyl 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

[0840]

[0841] To a mixture of commercially available (1s,4s)-4-aminocyclohexane-1-carboxylic acid tert-butyl ester, 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, dichloromethane (DCM) and saturated NaHCO3 were added, and the aqueous phase was extracted with dichloromethane (DCM). The combined organic phases were dried over Na2SO4, filtered, and the filtrate was concentrated. The product was purified by ISCO Chromatography (120 g silica Gold column, 60 mL / min) eluting with a gradient of 0 to 15% methanol (containing 10% NH4OH) in dichloromethane (DCM) provided the title compound (5.46 g, 9.03 mmol, 83% yield). LCMS (m / z) 605.3 (M+H) + .

[0842] 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

[0843]

[0844] 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) was added 4N HCl / 1,4-dioxane (15.41 mL, 61.6 mmol) at 0°C. The mixture was stirred at room temperature for 5 h and concentrated to afford the title compound (10 g, 17.09 mmol, 111% yield). LCMS (m / z) 549.3 (M+H) + .

[0845] 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

[0846]

[0847] To ((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, 3 hydrochloride (59.4 mg, 0.100 mmol) and (1s,4s)-4-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridine-3- To a solution of 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. The HPLC was performed by MDAP (XSelect TMCSH C18 column, 40 mL / min) and gradient elution with 5 to 35% acetonitrile in water containing formic acid (0.1%) provided the title compound (44.5 mg, 0.039 mmol, 39.4% yield) as a white solid. LCMS (m / z) 1015.7 (M+H) + . 1 H NMR (400 MHz, methanol-d4) δ ppm 1.29-1.50 (m, 2H), 1.52-2.05 (m, 13H), 2.22 (br d,J=5.9Hz,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=9.8Hz,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=13.2Hz,1H),4.84(d,J=6.4Hz,1H),7.54(dd,J=7.8,4.9Hz,1H),7.78-7.92(m,1H),8.07(br s,1H),8.51-8.61(m,2H),8.74(s,1H)

[0848] The following compounds were prepared using a procedure similar to that described in Example 17:

[0849]

[0850]

[0851] Embodiment 19

[0852] (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

[0853]

[0854]

[0855] Step 1: tert-Butyl 3,7-dioxo-1-phenyl-2,11,14-trioxa-4,8-diazaheptadecan-17-oate

[0856]

[0857] 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)propionic acid (2.51 g, 11.25 mmol) in dichloromethane (DCM) (35.7 ml) was added DIPEA (5.61 ml, 32.1 mmol) and HATU (4.28 g, 11.25 mmol) at room temperature and the mixture was stirred under nitrogen atmosphere for 20 h. Dichloromethane (DCM) was added and the organic phase was washed with saturated NH4Cl, with saturated NaHCO3 and with brine. The organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. The product was purified by ISCO Purification by Rf chromatography (80 g silica gel column, 60 mL / min) eluting with a gradient of 50% (5 column volumes), 50 to 90% (25 column volumes) and 90% (5 column volumes) ethyl acetate in heptane provided the title compound (4.69 g, 10.70 mmol, 100% yield) as a colorless oil. LCMS (m / z) 383.4 (M+H) + .

[0858] Step 2: 3,7-dioxo-1-phenyl-2,11,14-trioxa-4,8-diazaheptadecan-17-oic acid

[0859]

[0860] 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 a 4M solution of HCl in 1,4-dioxane (12.60 mL, 50.4 mmol) and the mixture was stirred at room temperature for 20 h. A 4M solution of HCl in 1,4-dioxane (1 mL) was added twice at 2 h intervals, and the mixture was concentrated under reduced pressure to afford the title compound (5.69 g, 13.39 mmol, 126% yield) as an off-white solid. LCMS (m / z) 383.4 (M+H) + .

[0861] Step 3: tert-Butyl (1s,4s)-4-(3,7-dioxo-1-phenyl-2,11,14-trioxa-4,8-diazaheptadecan-17-amido)cyclohexane-1-carboxylate

[0862]

[0863] 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 (1s,4s)-4-aminocyclohexane-1-carboxylic acid tert-butyl ester, hydrochloride (2.499 g, 10.60 mmol) in dichloromethane (DCM) (35.3 mL) was added DIPEA (7.59 mL, 43.5 mmol) and HATU (4.23 g, 11.13 mmol) and the mixture was stirred at room temperature under nitrogen for 20 h. Dichloromethane (DCM) was added and the organic phase was washed with saturated NH4Cl, with saturated NaHCO3 and with brine. The organic phase was dried over NaSO4, filtered, and concentrated under reduced pressure. The product was purified by ISCO Combi Rf (80 g silica gel, 60 mL / min) was purified with a step gradient elution of 0% (5 column volumes), 0 to 25% (25 column volumes) and 25% (5 column volumes) methanol in dichloromethane (DCM) to provide the desired product (5.06 g, 8.08 mmol, 76% yield) as a clear colorless oil. LCMS (m / z) 564.5 (M+H) + .

[0864] Step 4: tert-Butyl (1s,4s)-4-(3-(2-(2-(3-aminopropanecarboxamido)ethoxy)ethoxy)propanecarboxamido)cyclohexane-1-carboxylate

[0865]

[0866] A suspension of tert-butyl (1s,4s)-4-(2,6-dioxo-1-phenoxy-10,13-dioxa-3,7-diazahexadecane-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 at room temperature for 20 h under a hydrogen balloon atmosphere. 10% Pd—C (300 mg) was added and the mixture was stirred under a hydrogen balloon atmosphere for 1 h. The mixture was passed through Filter and concentrate the filtrate under reduced pressure to afford the title compound as a brown oil (3.43 g, 7.19 mmol, 81% yield). LCMS (m / z) 430.5 (M+H) + .

[0867] Step 5: (1s,4s)-tert-butyl 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

[0868]

[0869] 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 (1s,4s)-tert-butyl 4-(3-(2-(2-(3-aminopropaneamido)ethoxy)ethoxy)propaneamido)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 NH4Cl, with saturated NaHCO3, and with brine. The organic phase was dried over NaSO4, filtered, and concentrated under reduced pressure. The product was purified by ISCO Combi Rf (80 g silica gel, 60 mL / min) was purified with a step gradient elution of 0% (5 column volumes), 0 to 25% (25 column volumes) and 25% (5 column volumes) methanol in dichloromethane (DCM) to provide the title compound (1.74 g, 2.479 mmol, 62.6% yield) as a clear colorless oil. LCMS (m / z) 632.5 (M+H) + .

[0870] 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

[0871]

[0872] 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 atmosphere for 20 h. 4M HCl in 1,4-dioxane (3.03 mL, 12.1 mmol) was added and the mixture was stirred for 4 h. A 4M solution of 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 provide the title compound (1.88 g, 3.07 mmol, 114% yield) as a sticky off-white solid. LCMS (m / z) 576.5 (M+H) + .

[0873] 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

[0874]

[0875] To ((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, 3 hydrochloride (59.4 mg, 0.100 mmol) and (1s,4s)-4-(1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl) To a solution of (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. The HPLC was performed by MDAP (XSelect TM CSH C18 column, 40 mL / min) and eluted with a gradient of 15 to 55% acetonitrile in water containing ammonium bicarbonate (10 mM) and adjusted to pH 10 with ammonia to provide 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-d4) 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=6.7,1.7Hz,2H),2.48(t,J=6.1Hz,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=5.1Hz,3H),3.61(s,4H),3.75(t,J=6.1Hz,3H),3.88-4.10(m,5H),4.16(br d,J=13.7Hz,2H),4.84(d,J=6.4Hz,1H),7.54(dd,J=7.6,5.1Hz,1H),7.80(dt,J=7.8,2.0Hz,2H),8.06(br s,1H),8.52(d,J=2.0Hz,1H),8.58(dd,J=4.9,2.0Hz,1H),8.72(s,1H)

[0876] Embodiment 20

[0877] ((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

[0878]

[0879]

[0880] Step 1: N,N-dibenzyl-2-(((1r,4r)-4-(vinyloxy)cyclohexyl)oxy)ethan-1-amine.

[0881]

[0882] 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)cyclohexane-1-ol (3.15 g, 9.28 mmol) and the mixture was distributed into two vials. The vials were sealed and stirred at 75 °C for 3.5 h. The reaction mixture was purified by ISCO. Chromatography (80 g silicaGold column, 60 mL / min) eluting with a gradient of 0 to 20% ethyl acetate in heptane provided the title compound (2.3956 g, 6.55 mmol, 70.6% yield). LCMS (m / z) 366.4 (M+H) + .

[0883] Step 2: 2-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)ethan-1-ol.

[0884]

[0885] 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 a 1 M solution of borane in tetrahydrofuran (THF) (9.64 mL, 9.64 mmol) and the mixture was stirred at room temperature for 40 min. 5N sodium hydroxide (12.86 mL, 64.3 mmol) and H2O2 (30%) (13.13 mL, 129 mmol) were added and the mixture was stirred at room temperature for 23 h. The mixture was extracted with ethyl acetate (3 x 30 mL) and the combined organic extracts were washed with brine (30 mL), dried over MgSO4, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by ISCO Chromatography (40 g silica Gold column, 40 mL / min) eluting with a gradient of 0 to 70% ethyl acetate in heptane provided the title compound (2.1723 g, 4.86 mmol, 76% yield). LCMS (m / z) 384.4 (M+H) + .

[0886] Step 3: N,N-dibenzyl-2-(((1r,4r)-4-(2-(vinyloxy)ethoxy)cyclohexyl)oxy)ethan-1-amine.

[0887]

[0888] 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. The product was purified by ISCO Chromatography (40 g silica Gold column, 40 mL / min) eluting with a gradient of 0 to 30% ethyl acetate in heptane provided the title compound (1.9085 g, 3.79 mmol, 78% yield). LCMS (m / z) 410.1 (M+H) + .

[0889] Step 4: 2-(2-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)ethoxy)ethan-1-ol.

[0890]

[0891] To a solution of N,N-dibenzyl-2-(((1r,4r)-4-(2-(vinyloxy)ethoxy)cyclohexyl)oxy)ethan-1-amine (1.9 g, 3.78 mmol) in tetrahydrofuran (THF) (40 mL) was added borane (1 M solution in THF) (5.66 mL, 5.66 mmol) and the mixture was stirred at room temperature for 30 min. 5N sodium hydroxide (7.55 mL, 37.8 mmol) and H2O2 (30%) (7.71 mL, 76 mmol) were added and the mixture was stirred at room temperature for 19 h. The mixture was extracted with ethyl acetate (3 x 30 mL) and the combined organic extracts were washed with brine (30 mL), dried over MgSO4, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by ISCO Chromatography (40 g silica Gold column, 40 mL / min) eluting with a gradient of 0 to 80% ethyl acetate in heptane provided the title compound (0.9820 g, 2.297 mmol, 60.8% yield). LCMS (m / z) 428.4 (M+H) + .

[0892] Step 5: tert-Butyl 3-(2-(2-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)ethoxy)ethoxy)propanoate.

[0893]

[0894] To 2-(2-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)ethoxy)ethane-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 h and concentrated under reduced pressure. Dichloromethane (DCM) (3 mL) and water (0.181 mL) were added, the mixture was stirred at room temperature for 23 h, and concentrated under reduced pressure. The residue was purified by ISCO Chromatography (24 g silica Gold column, 35 mL / min) eluting with a step gradient of 0 to 35% (15 min) and 35 to 80% (10 min) ethyl acetate in heptane provided the title compound (0.8950 g, 1.610 mmol, 71.0% yield). LCMS (m / z) 556.6 (M+H) + .

[0895] Step 6: tert-Butyl 3-(2-(2-(((1r,4r)-4-(2-aminoethoxy)cyclohexyl)oxy)ethoxy)ethoxy)propanoate.

[0896]

[0897] 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 at room temperature for 4 h under a hydrogen atmosphere. The mixture was passed through Filter and concentrate under reduced pressure to provide the title compound (0.5599 g, 1.491 mmol, 97% yield). LCMS (m / z) 376.4 (M+H) + .

[0898] 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

[0899]

[0900] A mixture of 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) was stirred at room temperature for 93 h. Saturated aqueous NaHCO3 (5 drops) was added and the mixture was concentrated under reduced pressure. The residue was purified by ISCO. Chromatography (24 g silica Gold column, 35 mL / min) eluting with a gradient of 0 to 55% ethyl acetate in heptane provided the title compound (0.9152 g, 1.381 mmol, 94% yield). LCMS (m / z) 578.5 (M+H) + .

[0901] 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

[0902]

[0903] 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 afford the title compound (0.8810 g, 1.085 mmol, 79% yield). LCMS (m / z) 522.4 (M+H) + .

[0904] 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

[0905]

[0906] 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 on a biotage V10 evaporator under vacuum, dissolved in dichloromethane (DCM) (0.200 mL) and then HCl (4M in p-dioxane) (0.235 mL, 0.941 mmol) was added. The resulting reaction mixture was stirred at ambient temperature for 2 hours. The reaction mixture was evaporated on a biotage V10 evaporator under vacuum, 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 hours. The reaction was quenched with water (~3 drops), evaporated on a biotage V10 evaporator under vacuum, 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, 7H), 1.46-1.84 (m, 5H), 1.84-2.00 (m, 6H), 2.16-2.24 (m, 2H), 2.37 (br t,J=6.11Hz,2H),2.47-2.60(m,1H),2.65(s,3H),2.78-2.85(m,1H),3.01-3.15(m,5H),3.21-3.30(m,5H ),3.32-3.41(m,5H),3.41-3.56(m,8H),3.58(s,6H),3.69(t,J=6.11Hz,2H),3.93-4.03(m,3H),4.24(br d, J = 12.72 Hz, 1H), 4.82 (d, J = 6.85 Hz, 1H), 7.52 (dd, J = 7.83, 4.89 Hz, 1H), 7.79 (dt, J = 8.31, 1.96 Hz, 1H), 8.06 (br s, 1H), 8.47-8.59 (m, 3H), 8.72 (s, 1H); 19F NMR (400 MHz, methanol-d4) δ ppm -63.69; LC / MS (TFA, M+H) +989.5 m / z, retention time 0.56 min.

[0907] The following compounds were prepared using a procedure similar to that described in Example 20:

[0908]

[0909]

[0910] Embodiment 22:

[0911] (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-azatriacontane-31-yl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide.

[0912]

[0913] Step 1: 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-azatriacontane-31-yl)carbamate, trifluoroacetate.

[0914]

[0915] 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) was added DIPEA (1.404 mL, 8.04 mmol) dropwise under nitrogen atmosphere and the mixture was stirred for 15 min. Tert-butyl (29-amino-3,6,9,12,15,18,21,24,27-nonaoxaconicosyl)carbamate (1969 mg, 3.54 mmol) in N,N-dimethylformamide (DMF) was added and the mixture was stirred at room temperature for 1.5 h. The reaction mixture was purified by ISCO. Purification by Rf chromatography (C18 100 g column, 60 mL / min) with a gradient elution of 10 to 100% acetonitrile in water containing trifluoroacetic acid (0.1%) provided the title compound (2.8 g, 3.21 mmol, 100% yield). LCMS (m / z) 759.6 (M+H) + .

[0916] Step 2: (2S,3S)-N-(29-amino-3,6,9,12,15,18,21,24,27-nonaoxaconicosyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, 2-trifluoroacetate.

[0917]

[0918] 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-azatriacontane-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 Rf chromatography (C18 100 g column, 60 mL / min) with a gradient elution of 10 to 100% acetonitrile in water containing trifluoroacetic acid (0.1%) provided the title compound (1.32 g, 1.49 mmol, 46.4% yield). LCMS (m / z) 659.5 (M+H) + .

[0919] 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-azatriacontane-31-yl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide.

[0920]

[0921] 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 (1.1 g, 1.236 mmol) was dissolved in DMSO (20 mL) and triethylamine (0.7 ml, 5.02 mmol). After 5 min, a solution of TSTU (0.447 g, 1.483 mmol) in DMSO (2 mL) was added and the mixture was stirred for 30 min. (2S,3S)-N-(29-amino-3,6,9,12,15,18,21,24,27-nonaoxaconicosyl)-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 h and acidified using 0.5 N HCl. The mixture was purified by ISCO Purify by Rf chromatography (C18 100 g column, 60 mL / min) eluting with a gradient of 0 to 100% acetonitrile in water containing trifluoroacetic acid (0.1%). The desired fractions were concentrated under reduced pressure to remove acetonitrile and the pH was adjusted to ~9 using ammonium hydroxide. The residue was purified by Rf chromatography (C18 100 g column, 60 mL / min) eluting with a gradient of 0 to 100% acetonitrile in water containing ammonium hydroxide (0.1%) to provide the title compound (817 mg, 0.627 mmol, 50.7% yield) as clear toffee. LCMS (m / z) 652.3 (M / 2+H) + . 1 H NMR (400MHz, methanol-d4) δppm 8.58 (dd, J=4.8, 1.5Hz, 1H) 8.53 (d, J=1.8Hz, 1H) 7.97 (br s,1H)7.82(dt,J=8.0,1.9Hz,1H)7.51-7.59(m,1H)7.30-7.46(m,3H)7.09(ddd,J=7.7,2.6,1.4Hz ,1H)4.79-4.86(m,3H)4.03-4.17(m,4H)3.84-3.95(m,2H)3.49-3.72(m,37H)3.35-3.45(m,6H)3. 12-3.20(m,1H)2.95-3.10(m,5H)2.79-2.90(m,2H)2.68-2.76(m,1H)2.67(s,3H)2.48-2.58(m,1H )2.27-2.41(m,1H)2.12-2.21(m,1H)1.85-1.97(m,5H)1.57-1.77(m,4H)1.18-1.32(m,1H)0.97(br d,J=6.3Hz,3H)0.77-0.86(m,3H).

[0922] Embodiment 23:

[0923] (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-dodecaoxatriacontanoyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, 3-carboxylate.

[0924]

[0925]

[0926] 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-dodeca-2-azahexadecane-41-oic acid

[0927]

[0928] 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 min. A portion of commercially available 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodeca-triaconta-39-oic acid (1 g, 1.619 mmol) in acetonitrile (10.00 mL) and dimethyl sulfoxide (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 EZprep chromatography (C18 50 g Gold column, 40 mL / min) eluting with a gradient of 10 to 70% acetonitrile in water containing ammonium hydroxide (0.1%) provided the title compound (0.9445 g, 1.21 mmol, 67.6% yield) as a clear light yellow oil. LC-MS m / z 820.6 (M+H) + .

[0929] Step 2: (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-dodeca-2-azahexadecane-41-amido)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylic acid benzyl ester, 2-carboxylate.

[0930]

[0931] 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-dodeca-2-azahetradecane-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. (R)-8-amino-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylic acid benzyl ester (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) eluting with a gradient of 30 to 60% acetonitrile in water containing formic acid (0.1%) provided the title compound (104.3 mg, 0.084 mmol, 65.4% yield) as a pale yellow oil. LC-MS m / z 577.3 (M / 2+H) + .

[0932] 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-dodecaoxatriacontadecyl)-2-(pyridin-3-yl)pyrrolidine-3-carboxamide.

[0933]

[0934] A mixture of (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-dodeca-2-azatetriacontane-41-amido)-3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylic acid benzyl ester, 2-formate (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, and then hydrogen from a balloon was bubbled through the mixture for 5 min. The mixture was stirred at room temperature under a hydrogen balloon for 50 min. The mixture was filtered, the catalyst was washed with methanol, and the combined filtrates were concentrated under reduced pressure to afford the title compound as a colorless oil (84 mg, 0.082 mmol, 98% yield). LC-MS m / z 1019.1 (M) + .

[0935] 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-dodeca-2-azahexadecane-41-amido)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-6-carbonyl)cyclopentyl)carbamate.

[0936]

[0937] To (1S,3R)-3-(((benzyloxy)carbonyl)amino)-1-isopropylcyclopentane-1-carboxylic acid (50.3 mg, 0.165 mmol) (freshly dried over 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, then 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-dodecaoxatriacontadecyl)-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 and the residue was stirred at 80°C for 2 h. Another portion of the preactivated acid (prepared as above) was added and heating at 80°C was continued for 20 h. The vial was cooled, water (0.50 mL) was added and heating at 100°C was continued for 2 h. The mixture was partitioned between 1.0 M NaOH and dichloromethane (DCM). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by reverse phase HPLC chromatography (XSelect TM CSH Prep C18 5 μm OBD column, 40 mL / min) and gradient elution with 30 to 85% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) provided the title compound (37.1 mg, 0.028 mmol, 34.5% yield) as a yellow oil. LC-MS m / z 653.8 (M / 2) + .

[0938] 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-dodecaoxatriacontanoyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, 3-carboxylate.

[0939]

[0940] 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-dodeca-2-azatahedrine-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) followed by methanol (1.00 mL) was sealed in a flask and purged with nitrogen. Hydrogen was bubbled through the mixture for 2 min. The mixture was stirred at room temperature for 30 min under a hydrogen balloon atmosphere. The mixture was opened to the air, a 7.0 M solution of ammonia in methanol (50 mL) was added, the mixture was stirred for 15 min and filtered. The catalyst was washed with methanol and the combined filtrate was 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 min, and 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 min, warmed to room temperature, and stirred for 1 h. The mixture was diluted with dichloromethane (DCM) and washed with 1.0 M NaOH. The aqueous phase was extracted with dichloromethane (DCM), the combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by reverse phase HPLC chromatography (XSelect TM CSH Prep C18 5 μm OBD column, 40 mL / min) and gradient elution with 15 to 55% acetonitrile in water containing formic acid (0.1%) provided the title compound (4.3 mg, 3.02 μmol, 10.63% yield) as a colorless foam. LC-MS m / z 644.3 (M / 2+H) + . 1HNMR (400MHz, methanol-d4) δppm 0.84-1.06 (range, 6H) 1.66 (br s, 1H) 1.81 (br d, J = 9.3Hz, 1H) 2.06 (s, 4H) 2.14-2.29 (m, 2H) 2.35 (br s, 2H) 2.58 (br s,3H)2.67(s,3H)2.68-2.75(m,2H)2.78-2.92(m,1H)3.00-3.11(m,1H)3.45(s,3H)3.50-3.55(m,3H)3.55-3.70(m,48H)3.82(br t,J=6.1Hz,3H)4.01(br dd,J=12,4.7Hz,1H)4.29(br d,J=14Hz,1H)4.47(brd,J=14Hz,2H)5.12(br s,2H)7.55(dd,J=7.9,4.9Hz,1H)7.82(dt,J=7.9,1.9Hz,1H)8.15(s,1H)8.30(br s,3H)8.53(br s,1H)8.59(br d,J=4.4Hz,1H)8.84(s,1H).

[0941] The following compounds were prepared using a procedure similar to that described in Example 23:

[0942]

[0943]

[0944] Biological assays

[0945] Example Compounds 1-24, which are compounds of Formula (I) having a CCR2 binding moiety, were tested in a variety of biological assays as described in more detail below.

[0946] Example 25: Antibody-dependent cellular cytotoxicity (ADCC) reporter assay

[0947] The antibody-dependent cytotoxicity reporter assay was performed using the following four assay components: (i) an ARM compound of formula (I) targeting CCR2 (at a concentration range of 1 pM to 10 μM), (ii) an anti-cotinine antibody (rabbit variable region with a human IgG1 Fc domain containing a DE mutation (S239D / I332E)) having a heavy chain sequence of SEQ ID NO: 11 and a light chain sequence of SEQ ID NO: 12 (at a concentration range of 0.01 μg / mL to 200 μg / mL); (iii) target cells: CHOK1 cells engineered to overexpress human CCR2 (typically 1000-20,000 cells / well), and (iv) reporter cells: Jurkat cells engineered to express FcγRIIIa with a reporter gene luciferase under the control of the NFAT promoter (typically 3000-75,000 cells / well). The reagents were combined in a 384-well tissue culture treated plate with a final volume of 20 μL. All four assay components were incubated together for approximately 12-18 hours. Thereafter, BioGlo detection reagent (Promega) was added to the wells to lyse the cells and provide a substrate for the luciferase reporter protein. The luminescent signal was measured on a microplate reader, and the signal was calculated by dividing the signal of the test well by the signal obtained when the heterodivalent compound of formula (I) was not added: background. GraphpadPrism software, particularly nonlinear regression curve fitting (Y=bottom+(top-bottom) / (1+10^((Log EC50-X)*slope))), was used to complete EC50 calculations.

[0948] The ADCC activity of the ARM compound of formula (I) of the present invention was tested in one or more experimental runs in the above assay, and the results are shown in Table 3 below. The potency of the compound of formula (I) of the present invention is reported with pEC50 values. The pEC50 value is the negative logarithm of the EC50 value, where the EC50 value is the half-maximum effective concentration measured in moles (M). For compounds tested in more than one experimental run, the pEC50 value is reported with an average value.

[0949] Example 26: PBMC in vitro depletion assay

[0950] 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 incorporates endogenous natural killer cells to deplete endogenous target monocytes.

[0951] To screen compounds in human PBMC monocyte depletion flow cytometry assays, 11-point dose-response curves were prepared from 1 mM stock solutions in pure DMSO by serially diluting the ARM compounds of formula (I) four-fold in a row into 384-well plates. These serially diluted compounds were then stamped into flat-bottom polypropylene 384-well plates (Greiner Bio-One) at 100 nL / well. Fifty microliters of 30 μg / mL of an anti-cotinine antibody (rabbit variable region with human IgG1 Fc domain containing DE mutation (S239D / I332E)) having a heavy chain sequence of SEQ ID NO: 11 and a light chain sequence of SEQ ID NO: 12, which had been diluted in assay medium (RPMI 1640 (Gibco)) containing 10% fetal bovine serum (Gibco), 1% Glutamax (Gibco) and 1% sodium pyruvate (Corning), were added to the wells of the plate. The plate was centrifuged at 500 rpm (revolutions per minute) for 1 minute and then shaken at room temperature for 5 minutes. The plate was incubated at 37°C / 5% CO2 for 15 minutes.

[0952] Frozen PBMC cells (HemaCare BioResearch) were thawed and centrifuged at 1,200 rpm for 4 minutes. Cells were diluted to 2 x 10 in assay medium. 6 The cells were diluted to a cell density of 10 cells / mL. Fifty 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% CO2 for 18-24 hours.

[0953] After incubation for 18-24 hours, 6.25 μ g people FC blockers (BD Pharmingen) of 2 μ L diluted in assay culture medium are added to the holes of flat plates. Flat plates are centrifuged at 500 rpm for 1 minute, and then incubated at room temperature for 15 minutes. 5 microliters of 750nM spiral NP NIR (Biolegend) diluted in assay culture medium are added to the holes of flat plates. Then 9 microliters of antibody mixtures containing the anti-CD45 antibody (Beckman Coulter) of fluorescein isothiocyanate (FITC) mark of 1 / 5 dilution, the anti-CD14 antibody (Beckman Coulter) of PC7 mark and the anti-CD4 antibody (Beckman Coulter) of phycoerythrin (PE) are added to the holes of flat plates. Flat plates are centrifuged at 500 rpm for 1 minute, and incubated at 4-8 ℃ for 30 minutes. Samples from flat plates are run on BD FACS Canto II flow cytometers, and FlowJo software analysis data is used.

[0954] The ability of the ARM compounds of formula (I) of the present invention to deplete monocytes was tested in one or more experimental runs in the above assays, 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-maximum effective concentration measured in moles (M). For compounds tested in more than one experimental run, the pEC50 value is reported as an average or a single value measured.

[0955] Table 3: Results of ADCC reporter assay (Example 25) and PBMC ex vivo depletion assay (Example 26)

[0956]

[0957]

[0958] Example 27: In vivo depletion assay

[0959] PBS solutions containing compounds of formula (I) were intravenously administered to human CCR2 (hCCR2) knock-in mice (C57 background) to measure the depletion of CCR2 expressing cells in peripheral blood. Peripheral blood from IV dosed mice was also analyzed to determine the PK properties of the ARM compounds of formula (I).

[0960] Stock solution preparation: Stock solutions of the compound of formula (I) at 20 mg / mL and 100 mg / mL were prepared in DMSO for PK / PD and dose tolerance studies, respectively. The stock solutions were stored at -20°C until further use.

[0961] Preparation of formulations: On the day of the experiment, the stock solution of the compound of formula (I) was taken out from -20°C storage and thawed at room temperature. If necessary, the anti-cotinine antibody (rabbit variable region sequence with mouse IgG2a Fc domain) having the heavy chain sequence of SEQ ID NO: 13 and the light chain sequence of SEQ ID NO: 14 was taken out from -80°C storage 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 experimental requirements.

[0962] Preparation composition : The formulation consisted of saline:DMSO:PBS. Saline was added as needed, and 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 mice. 1 to 2% (v / v) DMSO was used in the final formulation.

[0963] Administration to animals: Solution formulations of the antibody and the compound of formula (I) were injected (bolus injection) into the right / left tail vein of restrained mice. The animals were dosed with 0.1 mg / kg (mpk) of the compound of formula (I) and 10 mpk of anti-cotinine antibody.

[0964] Collect blood for PK: Under light isoflurane anesthesia, blood was collected by retro-orbital bleeding at time points of 0.25 hours, 2 hours, and 4 hours after administration (50 μL / time point).

[0965] Terminal bleeding at the end of the experiment (48 hours): Under deep isoflurane anesthesia, approximately 250 μL of blood was collected from each mouse in a K2EDTA tube and approximately 250 μL of blood in a SST (serum separator tube) by retro-orbital bleeding. After bleeding, each mouse was sacrificed by cervical dislocation. The blood distribution at termination was determined as follows:

[0966] ●For PK: Transfer 50 μL of K2EDTA blood to another tube for PK

[0967] • Flow cytometry: The remaining blood (approximately 200 μL) was used for flow cytometry analysis.

[0968] • For serum collection: Centrifuge serum separator tubes (BD) at 5000 rpm for 15 minutes at 4°C. Separate approximately 100 μL of serum and store at -80°C for further use.

[0969] • Blood drug concentration: Samples collected at different time points (0.25 hours, 2 hours, 24 and 48 hours) were analyzed to determine blood drug concentration.

[0970] Flow cytometry analysis:

[0971] CCR2 depletion in peripheral blood was determined by flow cytometry. In short, peripheral blood was collected from all animals at the end of the study and processed. Blood samples were lysed using 1X RBC lysis buffer, and the resulting cell pellet was washed twice in FACS buffer (HBSS containing 5% FBS). Fc blocking was performed on cells using mouse FC blocker (Trustain, anti-mouse CD16 / 32, catalog number: 101319) from Biolegend to block nonspecific binding, and then surface staining was performed on immune markers (CD45, CD3, CD11b, Ly6C, human CCR2). Surface staining was performed in ice for 30 minutes, and cells were washed twice to remove unbound antibodies. Cells were stained with 7AAD to determine viability and collected on a BDFACSVerse flow cytometer. Appropriate FMO controls and single-color controls were included in the experiment for gating and compensation, respectively.

[0972] CCR2+ monocytes were gated as CD11b+Ly6C+Hi, medium and low populations, and the reduction of treatment-induced CD11b+Ly6c+Hi cells (i.e., cells with high CCR2 expression) was evaluated. The percentage depletion of CD11b+Ly6c+Hi cells and CCR2 expression was determined for the different treatment groups. The results are shown in Table 4 below and are reported as the percentage depletion of CCR2 expressing cells (%).

[0973] Blood drug concentration analysis:

[0974] The drug concentration in blood samples was determined by a bioanalytical method based on LC-MS / MS developed at Syngene. The samples were analyzed using a Q-Trap, API-5500LC-MS / MS system coupled to an Exion UHPLC system from SCIEX, USA (operated in multiple reaction monitoring mode using positive electrospray ionization technology). Using 10mM ammonium acetate in Milli-Q water as the A phase and 0.1% formic acid in acetonitrile as the B phase, the analyte and internal standard peaks were resolved on Synergi Polar, 75×2.0mm, 4μ columns. Gradient elution was performed as follows: 95% A phase was used at 0.0min, maintained for 0.2 minutes, raised to 5% before 1.0 minutes, maintained the same in the next 0.5 minutes, and returned to 95% before 1.6 minutes. The total running time was 2 minutes.

[0975] Working dilutions of calibration curves and quality control standards were prepared by serial dilution of 20 mg / mL stock solution with DMSO. The spiked concentration of the whole blood calibration curve ranged from 1 ng / mL to 1000 ng / mL. A working solution of internal standard (verapamil, 25 ng / mL) was prepared in acetonitrile. 10 μL of research samples and calibration curves, quality control and blank whole blood samples were aliquoted into 96 deep well plates for processing. 10 μL of Milli-Q water was added to all samples and vortexed briefly to induce complete hemolysis. 10 μL of 20 mM dithiothreitol (DTT) was added to all samples and incubated at 37°C for 30 minutes. The addition of DTT enhances the recovery of ARM compounds of formula (I) from biological matrices. 300 μL of working internal standard solution was added to all samples except the total blank and wash samples (wherein 300 μL of acetonitrile was added). All samples were vortexed for 5 minutes and then centrifuged at 4°C for 10 minutes at 4000 rpm. 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 post-dose.

[0976] Table 4: Results of in vivo depletion assay - % depletion of CCR2 expressing cells and PK analysis of ARM compounds of formula (I) after 48 hours exposure

[0977]

[0978]

[0979] 1 nd = not determined

[0980] Example 28: Binding of anti-cotinine antibodies to ARM compounds of formula (I) measured by surface plasmon resonance (SPR)

[0981] Anti-cotinine antibodies (rabbit variable regions with human IgG1 Fc domains containing DE mutations (S239D / I332E)) having a heavy chain sequence of SEQ ID NO: 11 and a light chain sequence of SEQ ID NO: 12 were captured on one or more flow cells of a protein A sensor chip (Cytiva) using a Biacore T200, while flow cell 1 was retained as a reference. After capture, a 3000 second wait step was included to reduce drift during compound analysis. The ARM compound of formula (I) was then injected at 100 μL / min, with association and dissociation times of 200 and 2000 seconds, respectively. The entire experiment was run at 37°C, with a running buffer containing 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% P20, and 1% DMSO. The ARM compound of formula (I) was titrated at a maximum concentration of 200 nM using a 3-fold 5-point dilution series, and the corresponding 5 injection buffer cycles were run for blank subtraction. Data were double referenced by subtracting the response of the reference flow cell from the response of the flow cell containing the antibody and then subtracting the reference blank sensorgram. After compound analysis, pH 1.5 glycine was injected for 30 seconds at a flow rate of 30 μL / min to regenerate the surface and then recapture the antibody. Experiments were run using Biacore T200 control software and evaluated using Biacore T200 evaluation software. Curves were fitted using a 1:1 kinetic binding model to obtain k on (1 / Ms), k off (1 / s), K d (M)(determined as k off / k on ) and residence time (determined as 1 / k off (s)).

[0982] In the above assay, anti-cotinine antibodies were tested for binding to the ARM compounds of formula (I) of the invention in one or more experimental runs and the results are shown in Table 5 below. Affinity is reported as pKd Value. d The value is K d The negative logarithm of the value, where K d Values ​​are measured dissociation constants in molar (M) units, as described above. For compounds tested in more than one experimental run, pK d Values ​​are reported as averages.

[0983] Table 5: Binding of anti-cotinine antibodies to ARM compounds of formula (I) measured by SPR

[0984]

[0985]

[0986] Example 29: Nicotinic Acetylcholine Receptor Antagonism Assay

[0987] Use the buffer solution containing trypsin to wash and dissociate the cells expressing human α1 nicotinic acetylcholine (nACh) receptor (nAChR).Then wash the cells and inoculate with a density of 3-5 million cells / mL and allow to grow for 3 days.Then, 60-80 μL cells are added to the plate and incubated with the dose response of the ARM compound of the formula (I) of the highest concentration of 10 μM or 100 μM.Then stimulate the receptor by adding acetylcholine (ACh), and monitor the activation of the receptor by the diaphragm clamp system automated by IONFlux.IonFlux Data Analyzer software is utilized to perform leak deduction and measure (minimum) peak current amplitude.Then these data are exported to Excel.

[0988] Data processing: (1) The current values ​​recorded by applying only the vehicle in a single trap / pattern were deducted from the corresponding current amplitude values ​​for the control, test article, and control+test article. (2) The control response was calculated as the percentage of the average response of the third of a total of 3 single ACh (10mM) applications. In rare cases, if the third ACh response was obscured by technical error, the second and / or first ACh (10mM) applications were considered as controls. The peak current amplitude produced in the presence of each test article concentration was then used to calculate the % inhibition compared to the control response (10mMACh alone). These estimates were further corrected for the vehicle response and normalized by the time-matched control response (10mMACh alone). The corrected percentages were then fitted to a nonlinear function in GraphPad Prism 6 (or subsequent versions) to determine the IC50.

[0989] Exclusion criteria: (i) any n with poor recording quality, (ii) control current amplitude <500 pA or (iii) significant outliers as determined by Grubbs' test.

[0990] In the above assay, the ARM compounds of formula (I) of the present invention were tested for antagonism of nAChR in one or more experimental runs, and the results are shown in Table 6 below. 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 percentage observed at the highest concentration of the compound of formula (I) tested, as shown in Table 6. For compounds tested in more than one experimental run, the IC50 values ​​are reported as average values.

[0991] Table 6: Results of nAChR antagonism assay

[0992]

[0993] Example 30: In vivo mouse studies (muScreen)

[0994] The effect of treatment with a CyTaC compound of Formula (I) in combination with an 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.

[0995] Dosing and analysis of tumor-bearing animals: Tumor cells were injected subcutaneously into the flank of mice (syngeneic to the injected tumor cells). Tumor volume was measured using a digital vernier caliper, measuring the length (l) and width (w) of the tumor. Tumor volume was calculated using the following formula: Tumor volume (mm 3)=L×W2 / 2, where L=length (mm); W=width (mm). 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) and CyTaC compound of Example 22 were diluted in saline and administered intravenously to produce a final concentration of 0.1 or 0.2 mpk CyTAC compound and 10 mpk anti-cotinine antibody. Flow cytometry of peripheral blood was performed as described in Example 27 above. For flow cytometry analysis of tumors: Tumor isolation procedures were performed in a laminar flow clean bench following aseptic technique. All dissection tools used during the procedure were sterilized. Tools were stored in 70% ethanol between uses. Mice bearing subcutaneous tumors 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 with dissecting scissors to expose the subcutaneous tumor. The exposed tumor was cut open and excised using small dissecting scissors. The tumor sample was placed in a 50 mL falcon filled with RPMI 1640 serum-free medium and further processed for flow analysis. Antibody staining was performed on blood samples.

[0996] Survival analysis--when the tumor reaches >1500mm 3 Or when tumor ulceration was visible, the animals were humanely euthanized.

[0997] The results are shown in Figures 1-6.

[0998] Example 31: In vivo studies in mice - thioglycolate-induced peritonitis model

[0999] The ability of CyTaC compounds of formula (I) to block CCR2-mediated chemotaxis in combination with anti-cotinine antibodies was evaluated in a thioglycollate-induced peritonitis mouse model to demonstrate the ability of the combination to antagonize CCR2 activity in vivo.

[1000] Thioglycolate medium (Sigma,B2551-500G) Preparation: Dissolve Brewer Thioglycolate Medium Powder (4-6 g*) in 100 mL of MilliQ water and place in a water bath to allow the medium to dissolve completely. The medium is then autoclaved at 121°C for 15 minutes. The autoclaved medium is wrapped in aluminum foil and stored at 4-8°C for two weeks before administration. The aging process of the medium turning brown is critical for effective induction of peritonitis. (*Note: In some studies, 4% w / v Thioglycolate Medium was used, while in others 6% w / v was used).

[1001] diseaseInduction: Mice were injected intraperitoneally (ip) with 1 ml of 4% w / v or 0.7 mL of 6% w / v thioglycolate medium aged for two weeks.

[1002] An anti-cotinine antibody with rabbit variable region sequences and mouse IgG2a Fc domain whose ADCC activity was abolished by p-mutation (L234A / L235A) was used in the study. Mice were administered 10 mpk or 50 mpk of anti-cotinine antibody and 0.001 mpk to 0.5 mpk of CyTaC compound.

[1003] Collection of peritoneal lavage fluid :At different time points (6 hours, 24 hours, 48 ​​hours and 72 hours) after the administration of thioglycolate, mice were subjected to peritoneal lavage. For peritoneal lavage, 10 ml of ice-cold PBS (containing 3% v / v FBS) was injected intraperitoneally into mice using a 27 g needle. After injection, the abdominal cavity was gently massaged for 2 minutes to allow adherent cells to fall off into the lavage fluid and to make the cell suspension in the peritoneal cavity uniform. After the abdominal massage was completed, the lavage fluid was collected by suction. A 25G needle connected to a 10 mL syringe was placed into the abdominal cavity, and the peritoneal fluid was drawn into the syringe as much as possible. The collected peritoneal lavage fluid was kept on ice until TLC and DLC analysis.

[1004] Flow cytometry and cytokine analysis : 2 ml peritoneal lavage fluid was taken for flow cytometry. 1 ml peritoneal lavage fluid was centrifuged at 10000 rpm at 4°C and the supernatant was stored at -80°C for cytokine analysis.

[1005] The results are shown in Figures 10A-10B middle.

[1006] Example 32: In vivo studies in mice - collagen-induced arthritis (CIA) mouse model

[1007] The efficacy of depleting CCR2 monocytes by combination therapy of CyTaC compounds of formula (I) and anti-cotinine antibodies was evaluated in a collagen-induced arthritis model. The results suggest that combination therapy of CyTaC compounds of formula (I) and anti-cotinine antibodies may have therapeutic benefits in autoimmune disorders.

[1008] Preparation of 0.05M glacial acetic acid : Sterile water for injection (10 mL) was measured and added to a 15 mL falcon tube, and 28.6 μl was removed and replaced with 28.6 μl of glacial acetic acid.

[1009] Preparation of Immunization Grade Type II Collagen (CII): One day before immunization, 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. The vials were stored at 4°C overnight. 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.

[1010] Immunization schedule on day 0 :

[1011] ● Emulsion preparation: 2mg / mL of collagen (CII) was emulsified with 2mg / mL of complete Freund's adjuvant (CFA) in a ratio of 1:1, with a final concentration of 1mg / mL of CII and 1mg / mL of CFA. One volume of CFA was added to a sealed 5mL glass syringe. Then, an equal volume of collagen (CII) was gradually added dropwise to the CFA suspension while mixing at a low speed using a homogenizer. After complete mixing, homogenization was performed at maximum rpm for 2 minutes. The emulsion was then cooled by keeping the syringe in ice for 5 minutes. The procedure was repeated three times to obtain a uniform and stable emulsion (which would not disperse when a drop of emulsion was placed on water). The emulsion was transferred from the glass syringe to a 1mL syringe (BD).

[1012] Administration of the emulsion: The animals were lightly anesthetized with isoflurane and 100 μL of the emulsion was injected subcutaneously approximately 2 cm from the base of the tail.

[1013] Immunization schedule on day 21:

[1014] ● Emulsion preparation: 2mg / mL of collagen (CII) was emulsified with incomplete Freund's adjuvant (IFA) in a ratio of 1: 1, with a final concentration of 1mg / mL of CII. One volume of IFA was added to a sealed 5mL glass syringe. Then, an equal volume of collagen (CII) was gradually added dropwise to the CFA suspension while mixing at a low speed using a homogenizer. After complete mixing, homogenization was performed at maximum rpm for 2 minutes. The emulsion was then cooled by keeping the syringe in ice for 5 minutes. The procedure was repeated three times to obtain a uniform and stable emulsion (a drop of emulsion will not disperse when placed on water). The emulsion was transferred from the glass syringe to a 1mL syringe (BD).

[1015] Administration of the emulsion: The animals were lightly anesthetized with isoflurane and 100 μL of the emulsion was injected subcutaneously approximately 1 cm from the base of the tail.

[1016] • Scoring: Paw inflammation was scored qualitatively for disease severity using the criteria listed in Table 7 below.

[1017] Table 7: Scoring criteria

[1018]

[1019] Treatment start: On the day of illness onset (day 26 to day 54)

[1020] o Dexamethasone (control drug) - 0.1 mg / kg orally daily.

[1021] o Enbrel (control drug) - 10 mg / kg intraperitoneal

[1022] o Compound of formula (I) (Compound of Example 22) + anti-cotinine antibody. - Once a week intraperitoneally.

[1023] ●Clinical signs: Day 22 (the day after the booster dose) to the end of the experiment (Day 54).

[1024] - Paw thickness: measured at baseline, Day 36, and Day 54 on Day 21 (day of booster dose) using a Mitutoyo micrometer ruler.

[1025] Paw weight: The weight of all four paws (i.e., hind and forelimbs) removed at the hairline at the end time (day 54)

[1026] - Cytokine analysis: 14-plex mouse inflammation panel, 14 plexes were performed in serum and paw homogenates of forelimbs.

[1027] • Cartilage degradation markers in serum: CTXII and COMP were detected in serum obtained on day 54 using commercially available ELISA kits.

[1028] ● Autoantibody levels Serum: Mouse anti-bovine type II collagen IgG antibody assay using ELISA

[1029] ●Flow cytometry: Peripheral blood - CD45, CD3, CD11b, Ly6c, mCCR2, CD4, PD1, CXCR5

[1030] ●Flow cytometry: Synovial lavage fluid - CD45, CD3, CD11b, Ly6c, mCCR2, CD4, PD1, CXCR5

[1031] - Histopathology - Hindlimb ankle joints were collected in 10% NBF. Joints were decalcified and then stained to detect cartilage degradation and infiltration.

[1032] The results are shown in Fig. 9 In. Fig. 9 In the figure, "sham control" refers to mice that were not induced with collagen and therefore did not develop an "arthritic" phenotype; and "pathological control" refers to mice that were injected with collagen but did not receive any therapeutic intervention.

[1033] Example 33: In vivo studies in mice - efficacy of combination therapy with anti-PD-1 or STING agonists

[1034] The efficacy of the CyTaC compound of Example 22 in combination with an anti-cotinine antibody, further co-administered with an anti-PD-1 or STING agonist, was evaluated in vivo.

[1035] Combination with anti-PD-1

[1036] Administration and analysis of tumor-bearing animals: EMT-6 tumor cells were injected subcutaneously into the flank of mice (homologous to the injected tumor cells). Tumor volume was measured using a digital vernier caliper, measuring the length (l) and width (w) of the tumor. Tumor volume was calculated using the following formula: Tumor volume (mm 3 )=L×W2 / 2, where L=length (mm); W=width (mm). When the tumor volume is 50-100mm 3 Animals were randomized between 2 and 3 months and treatment was initiated after randomization. 3 The animals were sacrificed at 1:1. The anti-cotinine antibody and the CyTAC compound of formula (I) (Example 22) were diluted in saline and administered intravenously to produce a final concentration of 0.5 mpk CyTAC BIW and 10 mpk anti-cotinine antibody, which was administered once a week. The dose of anti-PD1 antibody was 10 mpk once a week.

[1037] The results are shown in Figures 8A-8B middle.

[1038] Combination with STING agonists

[1039] The efficacy of the CyTaC compound of Example 22 in combination with an anti-cotinine antibody, further administered in combination with a STING agonist, was evaluated in vivo in the CT26 mouse model, which is a syngeneic mouse model of murine colon cancer.

[1040] Six to eight week old female BALB / c mice were used in this study. Mice were inoculated subcutaneously with 5e4 CT-26 cells in the right hind flank and tumors were grown until they reached approximately 100 mm 3 (measured with a digital caliper) and applying the formula tumor volume TV = 0.52*L*W 2 (Day 0). On day 0, mice were randomized into study groups based on tumor volume, n=10 mice per group, and the first dose was administered. Mice were dosed as shown in the table below. Aspartate was administered as a vehicle control for STING agonist treatment. Tumor volume and body weight were measured 3 times per week throughout the study, and when tumor volume >2000mm 3 Mice reached the endpoint when open ulcers appeared on the tumor or the mice lost >20% of their body weight.

[1041]

[1042] The results are shown in Figure 7 middle.

[1043] Sequence Listing

[1044] Heavy chain CDR1 amino acid sequence

[1045] SEQ ID NO:1

[1046] NYWMS

[1047] Heavy chain CDR2 amino acid sequence

[1048] SEQ ID NO:2

[1049] DIHGNRGFNYHASWAKG

[1050] Heavy chain CDR3 amino acid sequence

[1051] SEQ ID NO:3

[1052] ADDSGSHDI

[1053] Light chain CDR1 amino acid sequence

[1054] SEQ ID NO:4

[1055] QSSQSVYSAKLS

[1056] Light chain CDR2 amino acid sequence

[1057] SEQ ID NO:5

[1058] YGSTLAS

[1059] Light chain CDR3 amino acid sequence

[1060] SEQ ID NO:6

[1061] QGTFYGPDWYFA

[1062] Variable heavy chain amino acid sequence

[1063] SEQ ID NO:7

[1064] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVGDIHGNRGFNYHASWAKGRFTVSRSKNTLYLQMNSLRAEDTAVYYCAKADDSGSHDIW GQGTLVTVSS

[1065] Variable light chain amino acid sequence

[1066] SEQ ID NO:8

[1067] DIQMTQSPSSLSASVGDRVTITCQSSQSVYSAKLSWYQQKPGKAPKLLIYYGSTLASGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCQGTFYGPDWYFAFGGGTKVEIK

[1068] Heavy chain amino acid sequence

[1069] SEQ ID NO:9

[1070] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVGDIHGNRGFNYHASWAKGRFTVSRSKNTLYLQMNSLRAEDTAVYYCAKADDSGSHDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1071] Light chain amino acid sequence

[1072] SEQ ID NO:10

[1073] DIQMTQSPSSLSASVGDRVTITCQSSQSVYSAKLSWYQQKPGKAPKLLIYYGSTLASGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCQGTFYGPDWYFAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG

[1074] EC

[1075] Heavy chain amino acid sequence

[1076] SEQ ID NO:11

[1077] QQQLVESGGR LVTPGGSLTL TCTASGFSLN NYWMSWVRQAPGKGLEWIGD IHGNRGFNYH ASWAKGRFTV SRTSTTVDLRMTSLTTEDTA IYFCARADDS GSHDIWGPGT LVTVSS ASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFP EPVTVSWNSGALTSGVHTFP AVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPDVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKCKVSNKALPAPEEKTISKAKG QPREPQVYTL PPSRDELTKNQVSLTCLVKG FYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSLSLSPGK

[1078] Light chain amino acid sequence

[1079] SEQ ID NO:12

[1080] ELDLTQTPSPVSAAVGDTVTINCQSSQSVYSAKLSWYQQKPGQPPKLLIYYGSTLASGVPSRFKGSGSG TQFSLTISDVQCADAATYYCQGTYYGPDWYFAFGGGTEVVVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1081] Heavy chain amino acid sequence

[1082] SEQ ID NO:13

[1083] QQQLVESGGRLVTPGGSLTLTCTASGFSLNNYWMSWVRQAPGKGLEWIGDIHGNRGFNYHASWAKGRFT VSRTSTTVDLRMTSLTTEDTAIYFCARADDSGSHDIWGPGTLVTVSS AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVLTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKALPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[1084] Light chain amino acid sequence

[1085] SEQ ID NO:14

[1086] ELDLTQTPSPVSAAVGDTVTINCQSSQSVYSAKLSWYQQKPGQPPKLLIYYGSTLASGVPSRFKGSGSG TQFSLTISDVQCADAATYYCQGTYYGPDWYFAFGGGTEVVVK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

Claims

1. Compounds of formula (I): or a pharmaceutically acceptable salt thereof, in: T is R 1 It is C 1-4 Alkyl or C 3-6 Cycloalkyl; L is a divalent linking group of formula (La), (Lb), (Lc), (Ld) or (Le): or a stereoisomer thereof, in: Ring A and Ring B are each independently C 4-6 Cycloalkylene; L 1a It is C 3-5 A straight chain alkylene group in which one or two methylene units are replaced by -O- or -NR a -replace; Each R a are independently hydrogen or C 1-3 alkyl; and L 2a is -O-, -NHC(O)- or -CH2-O-; or a stereoisomer thereof, in: Ring A is C 4-6 Cycloalkylene or C 7-9 bridged bicyclic cycloalkylene; L 1b is -CH2-NH-C(O)-, -NHC(O)- or -C(O)NH-; L 2b It is C 6-12 A straight chain alkylene group in which 1, 2, 3 or 4 methylene units are replaced by -O-, -NR 1b -、-C(O)NR 1b -or-NR 1b C(O)-substitution; or L 2b yes where n is 1, 2, 3, or 4, and Represents and L 1b covalent bonds; and Each R 1b are independently hydrogen or C 1-3 alkyl; or a stereoisomer thereof, in: L 1c It is C 2-10 Straight chain alkylene in which 1, 2 or 3 methylene units are replaced by -O-, -NH-, -NHC(O)- or -C(O)NH-; Ring A is C 4-6 Cycloalkylene or C 7-9 a bridged bicyclic cycloalkylene; and L 2c Is -O- or saturated C 2-10 Straight chain alkylene in which 1, 2 or 3 methylene units are replaced by -O-, -NH-, -NHC(O)- or -C(O)NH-; in: L 1d It is C 12-30 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-; or wherein n is an integer from 3 to 50; Each of these 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), and each represents a covalent bond to a methylene group of formula (I); and Y is a bond or a divalent spacer moiety of 1 to 12 atoms in length.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 It is -CH3.

3. The compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein L is a divalent linking group of formula (Lai): or a stereoisomer thereof, Among them, ring A, L 1a , L 2a , As defined for formula (La).

4. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein L is a divalent linking group of formula (La-ii): or a stereoisomer thereof, Where L 1a , L 2a , As defined for formula (La); p is 1 or 2; and m is 1 or 2.

5. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein L is a divalent linking group of formula (La-iii): or a stereoisomer thereof, wherein p is 1 or 2; m is 1 or 2; n is 1, 2 or 3; and As defined for formula (La).

6. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein L is a divalent linking group of formula (La) selected from the following:

7. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein L is a divalent linking group of formula (Lbi): or a stereoisomer thereof, Where L 1b , L 2b , As defined for Formula (Lb); p is 1 or 2; and m is 1 or 2.

8. The compound of any one of claims 1 to 2 or 7, wherein L is a divalent linking group of formula (Lb) selected from the following:

9. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein L is a divalent linking group of formula (Lci): or a stereoisomer thereof, Where L 1c , L 2c , As defined for Formula (Lc); p is 1 or 2; and m is 1 or 2.

10. The compound of claim 1 or 2, wherein L is a divalent linking group selected from the following formula (Lc):

11. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein L is a divalent linking group selected from the following formula (Ld):

12. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein Y is selected from a bond; -NH-; -(C 1-12 alkylene)-, wherein 1, 2 or 3 methylene units are replaced by -O-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C 3-6 Cycloalkylene)-, -(C 3-6 -(C 2-12 alkenyl)-, wherein 1, 2 or 3 methylene units are replaced by -O-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-, -(C 3-6 Cycloalkylene)-, -(C 3-6 The invention is replaced by a 3- to 6-membered heterocycloalkylene group, an arylene group or a heteroarylene group.

13. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein Y is selected from 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 -(C 1-5 -NH-(C 1-5 Alkylene)-NH-; -(C 3-6 Cycloalkylene)-NH-; -(C 3-6 Cycloalkenylene)-NH-; or where Y 1a is a bond, -O-, -NH-, -NHC(O)-, -C(O)NH- or C 1-3 Alkylene; and Y 2a is a bond, -O-, -NH-, -NHC(O)-, -C(O)NH- or C 1-3 Alkylene.

14. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein Y is selected from:

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

16. The compound of claim 1, wherein the compound is selected from: (Example No.: 1), (Example No.: 2) and (Example No.: 22).

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

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

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 cell-mediated 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 of 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 CCR2 expressed on the cell.

24. A method for depleting CC motif chemokine receptor 2 (CCR2) expressing cells, the method comprising: contacting the cells with an effective amount of a compound of 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 CCR2 expressed on the cells.

25. The method of claim 23 or 24, wherein the CCR2-expressing cells are myeloid-derived suppressor cells (MDSCs), T regulatory cells (Tregs), neutrophils, macrophages, B regulatory cells (Bregs), CD8 regulatory cells, (CD8regs), exhausted T cells, or cancer-associated fibroblasts (CAFs).

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

27. The method of any one of claims 17 to 26, wherein the anti-cotinine antibody has a heavy chain comprising a heavy chain variable region (VH) having SEQ ID NO:7 and a light chain comprising a light chain variable region (VL) having SEQ ID NO:

8.

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

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

30. The method of 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 as claimed in any one of claims 1 to 16 and an anti-cotinine antibody or antigen-binding fragment thereof.

32. The combination of claim 31, wherein the anti-cotinine antibody has a 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 a 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 of claim 31 or 32, wherein the anti-cotinine antibody has a heavy chain comprising a heavy chain variable region (VH) having SEQ ID NO:7 and a light chain comprising a light chain variable region (VL) having SEQ ID NO:

8.

34. The combination of any one of claims 31 to 33, wherein the anti-cotinine antibody is of the IgGl isotype comprising a substitution in the Fc region to increase ADCC activity.

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

36. The combination of 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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