Cytotoxic targeting chimeras for CCR2-expressing cells

Abstract

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

Description

[Technical Field]

[0001] The present disclosure relates to heterobifunctional molecules, termed cytotoxic targeting chimeras (CyTaCs) or antibody-recruiting molecules (ARMs), that can simultaneously bind to a target cell surface protein and an exogenous antibody protein. The present disclosure also relates to agents capable of binding to receptors on the surface of pathogenic cells and inducing depletion of the pathogenic cells in a subject for use in treating cancer, inflammatory diseases, autoimmune diseases, viral infections, or bacterial infections. [Background technology]

[0002] Cell surface proteins and their ligands play important roles in inflammatory, infectious, and autoimmune diseases, as well as tumor initiation, growth, and metastasis. Antibody-based therapeutics have promising properties as drug candidates for these indications due to their selectivity for pathogenic cell surface targets and their ability to direct immune surveillance to target-expressing tissues or cells to induce depletion of pathogenic cells. Examples of such depletion mechanisms include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). However, antibody-based therapeutics often suffer from lack of bioavailability, high cost, thermal instability, and difficult manufacturing due to their size, complexity, and peptide-based structure. Conversely, small molecule therapeutics often offer affordability, stability, and the convenience of oral administration, but can suffer from poor selectivity and off-target effects while also lacking the immune control of therapeutic antibodies.

[0003] Thus, there is a need for improved therapeutic approaches that target pathogenic cells for use in treating disease. Such compositions and related methods are provided in the present disclosure. Summary of the Invention

[0004] In one aspect, the present disclosure provides heterobifunctional molecules, termed cytotoxic targeting chimeras (CyTaCs) or antibody-recruiting molecules (ARMs), which comprise a moiety that binds to a target cell surface protein on a cell and a moiety that binds to an exogenous antibody. In a further aspect, the ARM comprises a bivalent linker that connects the target-binding moiety to the antibody-binding moiety. In a further aspect, the target-binding moiety is a CC chemokine receptor type 2 (CCR2)-binding moiety. In a further aspect, the exogenous antibody is an anti-cotinine antibody, or an antigen-binding fragment thereof.

[0005] In a further embodiment, the ARM is a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof; During the ceremony, R 1 is C 1~4 Alkyl or C 3~6 is cycloalkyl, R 2 is hydrogen or C 1~4 is alkyl, R 3 is hydrogen or C 1~4 is alkyl, L is a bivalent linker as described herein.

[0006] In one aspect, provided is 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 compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof.

[0007] In one aspect, the present disclosure provides a method for increasing antibody-dependent cellular cytotoxicity (ADCC) of CCR2-expressing cells, comprising contacting the cells with a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof.

[0008] In one aspect, the present disclosure provides a method for 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.

[0009] In one aspect, the present disclosure provides a compound of formula (I) disclosed herein for use in therapy. In a further aspect, the present disclosure provides a combination comprising a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof for use in therapy.

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

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

[0012] In one aspect, the present disclosure provides a combination comprising a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof. [Brief explanation of the drawings]

[0013] [Figure 1A] 1 is an analysis of MC38 tumor-bearing mice (human CCR2 knock-in C57 mice) administered an ARM compound (compound of Example 36) in the presence of anti-cotinine antibody, as described in Example 204. It shows the tumor volume of tumor-bearing mice treated with an ARM compound in the presence of anti-cotinine antibody at various concentrations of ARM and antibody over time, as described in Example 204. [Figure 1B]1A is an analysis of MC38 tumor-bearing mice (human CCR2 knock-in C57 mice) administered an ARM compound (compound of Example 36) in the presence of anti-cotinine antibodies, as described in Example 204. Blood drug concentrations of the ARM compound in the mice at various time points after administration are shown (see FIG. 1A for legend). [Figure 1C] 1 is an analysis of MC38 tumor-bearing mice (human CCR2 knock-in C57 mice) administered an ARM compound (compound of Example 36) in the presence of anti-cotinine antibody, as described in Example 204. Percent depletion of CCR2-expressing cells as determined by flow cytometry is shown. [Figure 2] Figure 2 shows tumor growth in human CCR2 knock-in mice inoculated with MC38 tumors and administered an anti-cotinine antibody and an ARM compound (the compound of Example 36) with or without depletion of CD4 and CD8 T cells, as described in Example 209. 2A shows tumor growth in the presence of CD4 and CD8 T cells. 2B shows tumor growth with depletion of CD4 and CD8 T cells. The results demonstrate that tumor growth inhibition by the antibody / ARM compound is dependent on the presence of CD4 and CD8 T cells. [Figure 3] Figure 3 shows an alternative administration strategy for the small molecule ARM compound versus the anti-cotinine antibody described in Example 206, in which the anti-cotinine antibody and the small molecule ARM compound (Example 1) were administered intravenously either simultaneously or sequentially. Figure 3A shows the blood concentration of the small molecule ARM compound at various time points after administration (each time point represents three animals tested, except for the 168-hour time point, which represents six animals tested). Figure 3B shows the depletion rate of CCR2+-expressing cells (see Figure 3A for legend). The results demonstrate that simultaneous versus sequential administration of the anti-cotinine antibody and the small molecule ARM compound does not alter PK and PD. [Figure 4]An alternative administration strategy for the small molecule ARM compound versus anti-cotinine antibody described in Example 206, in which the anti-cotinine antibody and small molecule ARM compound (Example 1) were administered intravenously in succession, is shown. Data are presented for sequential administration of the antibody and small molecule ARM, with a 2-hour interval between administration of the small molecule ARM and the antibody. The results demonstrate that administration at a 2:1 molar ratio of ARM to antibody is sufficient to saturate the antibody. [Figure 5] Figure 2 shows an alternative administration strategy for small molecule ARM compound versus anti-cotinine antibody described in Example 206, in which the anti-cotinine antibody was administered intravenously and the ARM compound (Example 1) was administered subcutaneously after a 2-hour delay following antibody administration at various molar ratios of small molecule ARM:antibody. Data shown are blood concentrations of the small molecule ARM compound at various time points after administration. [Figure 6] Figure 6 shows an alternative administration strategy for small molecule ARM compounds versus anti-cotinine antibodies described in Example 206, in which the anti-cotinine antibody was administered intravenously and the ARM compound (Example 36) was administered orally. (A) Blood concentrations of the small molecule ARM compound at various time points after administration. (B) Percent depletion of CCR2-expressing cells determined by flow cytometry (see Figure 6A for legend). [Figure 7] 1 is a cotinine "off switch" study described in Example 208. A shows the blood concentrations of small molecule ARM compounds after administration of (S)-cotinine at the indicated time points. B shows depletion of target-expressing cells. [Figure 8] Figure 2 shows ex vivo T cell proliferation after MDSC depletion as described in Example 205. (A) Flow cytometry data demonstrating depletion of CCR2+ cells upon treatment with an ARM compound (Example 36) in the presence of an anti-cotinine antibody. (B) Percentage of divided CD8+ T cells. CD8+ T cell proliferation was observed in two of the three donor samples in which robust CCR2+ cell depletion was observed. [Figure 9] Cynomolgus monkey study described in Example 207. (A) shows the amount of the compound of Example 1 detected in the blood after administration of the compound and anti-cotinine antibody. (B) shows the depletion of target-expressing cells as determined by flow cytometry. [Figure 10] Analysis of CD8+ T cell and mMDSC depletion and mouse survival upon treatment with an anti-cotinine antibody and an ARM compound (the compound of Example 36), as described in Example 204. (A) shows that the percentage of CD8+ T cells increased and the percentage of mMDSC decreased upon treatment with an ARM compound plus antibody compared to treatment with an ARM compound alone. (B) shows the survival rate of tumor-bearing mice treated with an ARM compound alone or in the presence of an anti-cotinine antibody. [Figure 11A] 2 shows data from CyTOF analysis of depletion of CCR2-expressing cells in PBMCs isolated from healthy donors and cancer patients as described in Example 210. Arrows indicate target cell populations expressing CCR2. Depletion is shown in PBMCs from healthy donors. [Figure 11B] Figure 2 shows data from CyTOF analysis of depletion of CCR2-expressing cells in PBMCs isolated from healthy donors and cancer patients, as described in Example 210. Arrows indicate target cell populations expressing CCR2. Depletion is shown in PBMCs from a bladder cancer patient. The data demonstrate that CCR2 expression is primarily restricted to the target cells of interest for selective depletion (MDSCs), and that CCR2-expressing target cells are selectively depleted in the presence of anti-cotinine antibody plus ARM compound (right panel), but not in the presence of ARM compound alone (left panel). [Figure 12] FIG. 1 is a schematic diagram of cytotoxic targeted chimera (CyTaC) technology compared to current antibody technology. DETAILED DESCRIPTION OF THE INVENTION

[0014] In one aspect, the present disclosure provides a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, R 1 is C 1~4 Alkyl or C 3~6 is cycloalkyl, R 2is hydrogen or C 1~4 is alkyl, R 3 is hydrogen or C 1~4 is alkyl, L is a bivalent linker of formula (La), (Lb), (Lc), (Ld), (Le), (Lf), (Lg), (Lh), (Li), (Lj), (Lk), (Lm), (Lni), (Ln-ii), (Ln-iii), or (Ln-iv).

[0015] In one embodiment of the present disclosure, L is a bivalent linker of formula (La): [ka] or a stereoisomer thereof, During the ceremony, Ring A and ring B each independently represent C 4~6 is cycloalkylene, L 1a is C 3~5 A straight chain alkylene in which one or two methylene units are -O- or -NR a - is replaced by Each R a are independently hydrogen or C 1~3 is alkyl, L 2a is -O-, -NHC(O)-, or -CH2-O-, [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0016] In another embodiment, ring A and ring B of formula (La) are each independently: [ka] is.

[0017] In another embodiment, L is a bivalent linker of formula (Lai): [ka] or a stereoisomer thereof, During the ceremony, Ring A is C 4~6 is cycloalkylene, L 1a is C 3~5 A straight chain alkylene in which one or two methylene units are -O- or -NR a - is replaced by Each R a are independently hydrogen or C 1~3 is alkyl, L 2a is -O-, -NHC(O)-, or -CH2-O-, [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0018] In another embodiment, ring A of formula (Lai) is [ka] is.

[0019] In another embodiment, L is a bivalent linker of formula (La-ii): [ka] or a stereoisomer thereof, During the ceremony, L 1a is C 3~5 A straight chain alkylene in which one or two methylene units are -O- or -NR a - is replaced by Each R a are independently hydrogen or C 1~3 is alkyl, L 2a is -O-, -NHC(O)-, or -CH2-O-, p is 1 or 2; m is 1 or 2; [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0020] In another embodiment, L of formula (La), (Laai), or (La-ii) 1a teeth, [ka] is selected from During the ceremony, j is 1, 2, 3, or 4; k is 0, 1, 2, or 3; the sum of j and k is 2, 3, or 4; q is 1 or 2; r is 1 or 2, s is 0 or 1, the sum of q, r, and s is 2 or 3; X 1 and X 2 are independently -O- or NR a and Each R a are independently hydrogen or C 1~3 is alkyl, [ka] represents a covalent bond to a C(O) group of formula (La), (Laai), or (La-ii), [ka] represents a covalent bond to ring B of formula (La) or to a cyclohexylene group of formula (Lai) or (La-ii).

[0021] In another embodiment, L of formula (La), (Laai), or (La-ii) 1a is selected from -(CH2)2O-, -(CH2)3O-, -(CH2)4O-, -(CH2)2OCH2-, -(CH2)3OCH2-, -(CH2)2O(CH2)2-, -CHOCH2-, -CHO(CH2)2-, -CHO(CH2)3-, -CHOCH2O-, or -CH2OCH2OCH2-. In another embodiment, L of formula (La), (Lai), or (La-ii) is 1a is selected from —(CH2)2O—, —(CH2)3O—, —(CH2)2OCH2—, or —(CH2)3OCH2—. In another embodiment, L of formula (La), (Laai), or (La-ii) 1a is -(CH2)2NR a -, -(CH2)3NR a -, -(CH2)4NR a -, -(CH2)2NR a CH2-, -(CH2)3NR a CH2-, -(CH2)2NR a (CH2)2-, -CH2NR a CH2-, -CH2NR a (CH2)2-, -CH2NR a (CH2)3-, -CH2NR a CH2NR a -, or -CH2NR a CH2NR a CH2-, and each R a are independently hydrogen or C 1~3 In another embodiment, L of formula (La), (La-i), or (La-ii) is alkyl. 1a is -(CH2)2NR a -, -(CH2)3NR a -, -(CH2)2NR a CH2-, or -(CH2)3NRa CH2-, wherein R a is hydrogen or C 1~3 In another embodiment, L of formula (La), (La-i), or (La-ii) is alkyl. 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 is -CH2OCH2NR a -, -CH2NR a CH2O-, -CH2OCH2NR a CH2-, -CH2NR a CH2OCH2-, wherein R a are independently hydrogen or C 1~3 In another embodiment, L of formula (La), (La-i), or (La-ii) is alkyl. 1a is selected from -CH2OCH2NH-, -CH2NHCH2O-, -CH2OCH2NHCH2-, -CH2NHCH2OCH2-.

[0022] In another embodiment, L is a bivalent linker of formula (La-iii): [ka] or a stereoisomer thereof, During the ceremony, p is 1 or 2; m is 1 or 2; n is 1, 2, or 3; [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0023] In another embodiment, L is [ka] [ka] [ka] is a bivalent linker of formula (La) selected from the group consisting of:

[0024] In another embodiment, L is a bivalent linker of formula (Lb): [ka] or a stereoisomer thereof, During the ceremony, Ring A is C 4~6 Cycloalkylene or C 7~9 is a bridged bicyclic cycloalkylene; L 1b is -CH2-NH-C(O)-, -NHC(O)-, or -C(O)NH-, L 2b is C 6~12 A straight chain alkylene, in which 1, 2, 3, or 4 methylene units are -O-, -NR 1b -, -C(O)NR 1b - or -NR 1b replaced by C(O)-, or L 2b teeth, [ka] where n is 1, 2, 3, or 4; [ka] L 1b represents a covalent bond to Each R 1b are independently hydrogen or C 1~3 is alkyl, [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0025] In another embodiment, ring A of formula (Lb) is [ka] is.

[0026] In another embodiment, L is a bivalent linker of formula (Lbi): [ka] or a stereoisomer thereof, During the ceremony, L 1b is -CH2-NH-C(O)-, -NHC(O)-, or -C(O)NH-, L 2b is C 6~12 A straight chain alkylene, in which 1, 2, 3, or 4 methylene units are -O-, -NR 1b -, -C(O)NR 1b - or -NR 1b replaced by C(O)-, or L 2b teeth, [ka] where n is 1, 2, 3, or 4; [ka] L 1b represents a covalent bond to Each R 1b are independently hydrogen or C 1~3 is alkyl, p is 1 or 2; m is 1 or 2; [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0027] In another embodiment, L of formula (Lb) or (Lbi) 2b teeth, [ka] is selected from During the ceremony, j is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the sum of j and k is 5, 6, 7, 8, 9, 10, or 11; q is 1, 2, 3, 4, 5, 6, 7, 8, or 9; r is 1, 2, 3, 4, 5, 6, 7, 8, or 9; s is 0, 1, 2, 3, 4, 5, 6, 7, or 8; the sum of q, r, and s is 4, 5, 6, 7, 8, 9, or 10; t is 1, 2, 3, 4, 5, 6, or 7; u is 1, 2, 3, 4, 5, 6, or 7, v is 1, 2, 3, 4, 5, 6, or 7; w is 0, 1, 2, 3, 4, 5, or 6; the sum of t, u, v, and w is 3, 4, 5, 6, 7, 8, or 9; a is 1, 2, 3, 4, or 5; b is 1, 2, 3, 4, or 5; c is 1, 2, 3, 4, or 5; d is 1, 2, 3, 4, or 5; e is 0, 1, 2, 3, or 4; the sum of a, b, c, d, and e is 4, 5, 6, 7, or 8; X 1 , X 2 , X 3 , and X 4 are independently -O-, -NR 1b -, -C(O)NR 1b -, or -NR 1b C(O)-, Each R 1b are independently hydrogen or C 1~3 is alkyl, [ka] is the L in formula (Lb) or (Lbi) 1b represents a covalent bond to [ka] represents a covalent bond to a methylene group in formula (I).

[0028] In another embodiment, L is [ka] [ka] [ka] is a bivalent linker of formula (Lb) selected from the group consisting of:

[0029] In another embodiment, L is a bivalent linker of formula (Lc): [ka] or a stereoisomer thereof, During the ceremony, L 1c is C 2~10 a straight chain alkylene in which one, two, or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-; Ring A is C 4~6 Cycloalkylene or C 7~9 is a bridged bicyclic cycloalkylene; L 2c is -O- or saturated C 2~10 a straight chain alkylene in which one, two, or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-; [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0030] In another embodiment, ring A of formula (Lc) is [ka] is.

[0031] In another embodiment, L is a bivalent linker of formula (Lci): [ka] or a stereoisomer thereof, During the ceremony, L 1c is C 2~10 a straight chain alkylene in which one, two, or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-; L 2c is -O- or saturated C 2~10a straight chain alkylene in which one, two, or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-; p is 1 or 2; m is 1 or 2; [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0032] In another embodiment, L of formula (Lc) or (Lci) 1c teeth, [ka] is selected from During the ceremony, j is 1, 2, 3, 4, 5, 6, 7, 8, or 9; k is 0, 1, 2, 3, 4, 5, 6, 7, or 8; the sum of j and k is 1, 2, 3, 4, 5, 6, 7, 8, or 9; q is 1, 2, 3, 4, 5, 6, or 7; r is 1, 2, 3, 4, 5, 6, or 7; s is 0, 1, 2, 3, 4, 5, or 6; the sum of q, r, and s is 2, 3, 4, 5, 6, 7, or 8; t is 1, 2, 3, 4, or 5; u is 1, 2, 3, 4, or 5, v is 1, 2, 3, 4, or 5; w is 0, 1, 2, 3, or 4; the sum of t, u, v, and w is 3, 4, 5, 6, or 7; X 1 , X 2 , and X 3are independently —O—, —NH—, —NHC(O)—, or —C(O)NH—; [ka] represents a covalent bond to a C(O) group of formula (Lc) or (Lci), [ka] represents a covalent bond to a ring of formula (Lc) or (Lci).

[0033] In another embodiment, L of formula (Lc) or (Lci) 2c teeth, [ka] is selected from During the ceremony, j is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; k is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; the sum of j and k is 1, 2, 3, 4, 5, 6, 7, 8, or 9; q is 0, 2, 3, 4, 5, 6, or 7; r is 1, 2, 3, 4, 5, 6, 7, or 8; s is 0, 1, 2, 3, 4, 5, 6, or 7; the sum of q, r, and s is 1, 2, 3, 4, 5, 6, 7, or 8; t is 0, 1, 2, 3, 4, or 5; u is 1, 2, 3, 4, 5, or 6, v is 1, 2, 3, 4, 5, or 6; w is 0, 1, 2, 3, 4, or 5; the sum of t, u, v, and w is 2, 3, 4, 5, 6, or 7; X 1 , X 2 , and X 3 are independently —O—, —NH—, —NHC(O)—, or —C(O)NH—; [ka] represents a covalent bond to a ring of formula (Lc) or (Lci), [ka] represents a covalent bond to a methylene group in formula (I).

[0034] In another embodiment, L is [ka] is a bivalent linker of formula (Lc) selected from the group consisting of:

[0035] In another embodiment, L is a bivalent linker of formula (Ld): [ka] During the ceremony, L 1d is C 12~22 a straight chain alkylene in which 1, 2, 3, 4, or 5 methylene units are replaced by -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-; [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0036] In another embodiment, L of formula (Ld) 1d teeth, [ka] is selected from During the ceremony, j is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; the sum of j and k is 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19; r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19; s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; the sum of q, r, and s is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; t is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17; u is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, v is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17; w is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; the sum of t, u, v, and w is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19; a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; b is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; c is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; d is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; the sum of a, b, c, d, and e is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; f is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; g is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; h is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; the sum of f, g, h, i, y, and z is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17; X 1 , X 2 , X 3 , X 4 , and X 5 are independently -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-; [ka] represents a covalent bond to the C(O) group of formula (Ld), [ka] represents a covalent bond to a methylene group in formula (I).

[0037] In another embodiment, L is [ka] [ka] is a bivalent linker of formula (Ld) selected from the group consisting of:

[0038] In another embodiment, L is a bivalent linker of formula (Le): [ka] During the ceremony, n is an integer from 3 to 50, [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0039] In another embodiment, n in formula (Le) is 3 to 25, 3 to 10, 3 to 8, 3 to 7, 3 to 5, or 3 to 4. In another embodiment, n in formula (Le) is 3, 4, 5, 7, 8, 22, or 50.

[0040] In another embodiment, L is a bivalent linker of formula (Lf): [ka] or a stereoisomer thereof, During the ceremony, L 1f is a bond, C 1~6 straight-chain alkylene in which 0, 1, or 2 methylene units are replaced by -O-, -NH-, or -C(O)-; 1~6 Straight chain alkylene, or -(C 3~6 cycloalkylene)-NHC(O)-; L 2f is a bond, -NHC(O)-, -C(O)NH-, or C 1~6 is a straight chain alkylene in which 0, 1, or 2 methylene units are replaced by -O-; Z 1 and Z 2each is independently N or CH; [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0041] In another embodiment, L of formula (Lf) 1f teeth, [ka] is selected from During the ceremony, j is 1, 2, 3, 4, or 5; k is 0, 1, 2, 3, or 4; the sum of j and k is 1, 2, 3, 4, or 5; q is 1, 2, or 3; r is 1, 2, or 3; s is 0, 1, 2, the sum of q, r, and s is 2, 3, or 4; X 1 and X 2 are independently -O-, -NH-, or -C(O)-, or -(C 3~6 cycloalkylene)-NHC(O)-; [ka] represents a covalent bond to the C(O) group of formula (Lf), [ka] represents a covalent bond to the ring of formula (Lf).

[0042] In another embodiment, L of formula (Lf) 2f teeth, [ka] is selected from During the ceremony, j is 1, 2, 3, 4, or 5; k is 0, 1, 2, 3, or 4; the sum of j and k is 1, 2, 3, 4, or 5; q is 1, 2, or 3; r is 1, 2, or 3; s is 0, 1, 2, the sum of q, r, and s is 2, 3, or 4; [ka] represents a covalent bond to the ring of formula (Lf), [ka] represents a covalent bond to a methylene group in formula (I).

[0043] In another embodiment, L is [ka] is a bivalent linker of formula (Lf) selected from the group consisting of:

[0044] In another embodiment, L is a bivalent linker of formula (Lg): [ka] During the ceremony, Ring A is a 5- to 6-membered heteroarylene having 1 or 2 nitrogen ring atoms; L 1g is a bond, -CH2-, -NH-, or -O-, L 2g teeth, [ka] wherein n is 1, 2, 3, 4, or 5; [ka] L 1g represents a covalent bond to [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0045] In another embodiment, L is a bivalent linker of formula (Lgi): [ka] During the ceremony, L 1g is a bond, -CH2-, -NH-, or -O-, L 2g teeth, [ka] wherein n is 1, 2, 3, 4, or 5; [ka] L 1g represents a covalent bond to Z 1 , Z 2 , and Z 3 are each independently selected from N or CH, with the proviso that Z 1 , Z 2 , and Z 3 one or two of the following must be N: [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0046] In another embodiment, L is [ka] is a bivalent linker of formula (Lg) selected from the group consisting of:

[0047] In another embodiment, L is a bivalent linker of formula (Lh): [ka] or a stereoisomer thereof, During the ceremony, each Z 1 are independently N or CH; L 1h is a bond, —C(O)—, —C(O)—NH—, or —NHC(O)—, L 2h is C 2~10 Straight chain alkylene or [ka] wherein n is 1, 2, 3, or 4; [ka] L 1h represents a covalent bond to [ka] L 3h represents a covalent bond to L 3h is a bond, -C(O)CH2-, -O-(C 3~6 -cycloalkylene)-O-, or -C(O)NH(CH2)3OCH2-; L 4h is a bond, —C(O)—, —CHC(O)—, or —C(O)CH—; m is 1, 2, or 3; [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

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

[0049] In another embodiment, L is a bivalent linker of formula (Li): [ka] During the ceremony, L 1i is a bond, C 1~12 straight chain alkylene, or [ka] wherein n is 1, 2, 3, 4, or 5; [ka] L 3i represents a covalent bond to [ka] represents a covalent bond to NH, L 2i is a bond, C 1~12 straight chain alkylene, or [ka] wherein n is 1, 2, 3, 4, or 5; [ka] represents a covalent bond to HN, L 3i is a bond or -C(O)-, [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

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

[0051] In another embodiment, L is a bivalent linker of formula (Lj): [ka] or a stereoisomer thereof, During the ceremony, Z 1 is C, CH, or N, Z 2 , Z 3 , Z 4 , and Z 5 are independently CH or N, with the proviso that Z 2 , Z 3 , Z 4 , and Z 5 provided that no more than two of L 1j is -NH-, -C(O)NH-, -NHC(O)-, or -O-; L 2j is C 1~6 Straight chain alkylene or [ka] wherein n is 1 or 2; [ka] L 1j represents a covalent bond to [ka] represents a single or double bond, [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

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

[0053] In another embodiment, L is a bivalent linker of formula (Lk): [ka] or a stereoisomer thereof, During the ceremony, Ring A is phenyl or a 5- or 6-membered heteroarylene having 1 or 2 nitrogen ring atoms; Z 1 and Z 2 each is independently CH or N; L 1k is a bond, —C(O)—, —C(O)NH—, or —NHC(O)—, L 2k is C 3~8 Straight chain alkylene or [ka] wherein n is 1, 2, or 3; [ka] L 1k represents a covalent bond to [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

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

[0055] In another embodiment, L is a bivalent linker of formula (Lm): [ka] or a stereoisomer thereof, During the ceremony, Z 1 is CH or N, m is 1 or 2; p is 1 or 2; [ka] 0, 1, or 2 hydrogen atoms are replaced by F, L 1m is a bond, —C(O)—, —C(O)NH—, —NHC(O)—, —S(O)2NH—, or —NHS(O)2—, L 2m is C 3~6 Straight chain alkylene, C 3~6 cycloalkylene, or [ka] wherein n is 1 or 2; [ka] L 1m represents a covalent bond to [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

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

[0057] In another embodiment, L is a bivalent linker of formula (Lni): [ka] During the ceremony, [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0058] In another embodiment, L is a bivalent linker of formula (Ln-ii): [ka] During the ceremony, [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0059] In another embodiment, L is a bivalent linker of formula (Ln-iii): [ka] During the ceremony, [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0060] In another embodiment, L is a bivalent linker of formula (Ln-iv): [ka] During the ceremony, [ka] represents a covalent bond to the NH group of formula (I), [ka] represents a covalent bond to a methylene group in formula (I).

[0061] In another embodiment, R 1 is methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, or t-butyl. 1 is methyl. In another embodiment, R 1 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0062] In another embodiment, the compound of formula (I) is selected from the compounds listed in Table 1. [Table 1-1] Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-28

Table 1-33

[0063] definition As used in this specification and claims, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise.

[0064] As used in the specification and claims, the term "comprising" encompasses, for example, "including" or "consisting," e.g., a composition "comprising" X may consist exclusively of X or may include additional, e.g., X+Y.

[0065] The term "consisting essentially of" limits the scope of a feature to particular materials or steps, and those that do not materially affect the basic characteristic(s) of the claimed feature.

[0066] The term "consisting of" excludes the presence of any additional component(s).

[0067] The term "pathogenic cells" includes a cell subset that causes or is capable of causing disease. Examples of pathogenic cells include, but are not limited to, pathogenic immune cells, cancer or tumor cells, and stromal cells. Pathogenic cells can also be pathogens capable of causing infectious diseases, such as viruses or bacterial cells.

[0068] The term "pathogenic immune cells" includes specific immune cell subsets that cause or are capable of causing disease. These cell subsets are resident cells or are recruited to specific locations, secreting cytokines, chemokines, and other mediators that contribute to the persistence and progression of diseases such as cancer in the tumor microenvironment or chronic inflammation in the lungs in asthma. Examples of pathogenic immune cells include, but are not limited to, myeloid-derived suppressor cells (MDSCs), T regulatory cells (Tregs), neutrophils, macrophages, B regulatory cells (Bregs), CD8 regulatory cells (CD8regs), and exhausted T cells.

[0069] The term "pharmaceutical composition" refers to a formulation of a compound of the present invention and a vehicle generally accepted in the art for delivery of the biologically active compound to a mammal, e.g., a human. Such a vehicle includes any pharmaceutically acceptable carrier, diluent, or excipient therefor.

[0070] The terms "effective amount" and "therapeutically effective amount" refer to an amount of a compound according to the present invention, or an antibody, or antigen-binding portion thereof, that, when administered to a patient in need of treatment, is sufficient to provide treatment for the disease state, condition, or disorder for which the compound has utility. Such an amount will be sufficient to elicit the biological or medical response in a tissue system or patient that is desired by the researcher or clinician. The amount of a compound according to the present invention that constitutes a therapeutically effective amount will vary depending on factors such as the compound and its biological activity, the composition used for administration, the time of administration, the route of administration, the rate of excretion of the compound, the duration of treatment, the type and severity of the disease state or disorder being treated, drugs used in combination with or concomitantly with the compound of the present invention, and the patient's age, weight, general health, sex, and diet. Such a therapeutically effective amount can be routinely determined by one of ordinary skill in the art having regard to their own knowledge, the state of the art, and this disclosure.

[0071] The term "alkyl" refers to a saturated, straight-chain, or branched-chain hydrocarbon moiety having the specified number of carbon atoms. 1~3 The term "alkyl" refers to an unsubstituted alkyl moiety containing 1, 2, or 3 carbon atoms; exemplary alkyls include methyl, ethyl, and propyl.

[0072] The term "alkylene" refers to a saturated, straight-chain, or branched-chain 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 The term "alkylene" refers to an unsubstituted alkyl moiety containing 1, 2, or 3 carbon atoms with two points of attachment, exemplary C 1~3 Alkylene groups include methylene, ethylene, and propylene.

[0073] The term "alkenyl" refers to an unsaturated, straight-chain, or branched-chain hydrocarbon moiety having the specified number of carbon atoms. 2~6The term "alkenyl" refers to an unsubstituted alkenyl moiety containing 2, 3, 4, 5, or 6 carbon atoms; exemplary alkenyls include propenyl, butenyl, pentenyl, and hexenyl.

[0074] The term "alkenylene" refers to an unsaturated, straight-chain, or branched-chain 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 The term "alkenylene" refers to an unsubstituted alkenyl moiety containing 2, 3, 4, 5, or 6 carbon atoms with two points of attachment, exemplary C 2~6 Alkenylene groups include propenylene, butenylene, pentenylene, and hexenylene.

[0075] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon moiety having a specified number of carbon atoms. 3~6 The term "cycloalkyl" refers to an unsubstituted cycloalkyl moiety containing 3, 4, 5, or 6 carbon atoms; exemplary cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0076] The term "cycloalkylene" refers to a saturated cyclic hydrocarbon moiety having a specified number of carbon atoms with two points of attachment. The two points of attachment may be from the same or different carbon atoms. 4~6 The term "cycloalkylene" refers to an unsubstituted cycloalkylene moiety containing 4, 5, or 6 carbon atoms with two points of attachment. Exemplary cycloalkylene groups include cyclobutane-1,3-diyl, cyclopentane-1,3-diyl, cyclohexane-1,3-diyl, or cyclohexane-1,4-diyl.

[0077] The term "cycloalkenylene" refers to an unsaturated cyclic hydrocarbon moiety having a specified number of carbon atoms with two points of attachment. The two points of attachment may be from the same or different carbon atoms. 3~6The term "cycloalkenylene" refers to an unsubstituted cycloalkenylene moiety containing 3, 4, 5, or 6 carbon atoms with two points of attachment.

[0078] The term "heterocycloalkylene" refers to a saturated cyclic hydrocarbon moiety containing one or two heteroatoms, independently selected from oxygen, sulfur, or nitrogen atoms, along with two points of attachment. The two points of attachment can 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 containing one or two oxygen, sulfur, or nitrogen atoms, as well as two, three, four, or five carbon atoms, along with two points of attachment. Preferably, the 3- to 6-membered heterocycloalkylene group contains one oxygen or nitrogen atom. Preferably, such groups contain three carbon atoms and one oxygen or nitrogen atom, such as azetidinediyl or oxetanediyl. Preferably, such groups contain four or five carbon atoms and one oxygen or nitrogen atom, such as tetrahydrofurandiyl, tetrahydropyrandiyl, pyrrolidinediyl, or piperidinediyl.

[0079] The term "bridged bicyclic cycloalkylene" refers to a saturated bicyclic hydrocarbon moiety having at least one bridge with two points of attachment. A "bridge" is an unbranched chain of atoms or an atom or valence bond connecting two bridgeheads, and a "bridgehead" is any skeletal atom of the ring system that is bonded to three or more skeletal atoms (excluding hydrogen). The two points of attachment can be from the same or different carbon atoms. "C 7~9 The term "bridged bicyclic cycloalkylene" refers to an unsubstituted bridged bicyclic cycloalkylene moiety containing 7, 8, or 9 carbon atoms with two points of attachment.

[0080] The term "arylene" refers to a monocyclic or bicyclic ring system having two points of attachment in which at least one ring in the system is aromatic. Exemplary arylene groups include phenylene, biphenylene, naphthylene, and anthracylene.

[0081] The term "heteroarylene" refers to a monocyclic or bicyclic ring system in which at least one ring in the system is aromatic and has, along with two points of attachment, carbon atoms and one to five heteroatoms independently selected from oxygen, sulfur, or nitrogen atoms. The term "5- to 6-membered heteroarylene" refers to a 5- to 6-membered cyclic aromatic moiety containing, along with two points of attachment, one, two, or three heteroatoms independently selected from oxygen, sulfur, or nitrogen atoms, plus two, three, four, or five carbon atoms.

[0082] Those skilled in the art will understand that salts, including pharmaceutically acceptable salts, of compounds according to formula (I) can be prepared.In fact, in certain embodiments of the present invention, salts, including pharmaceutically acceptable salts, of compounds according to formula (I) may be preferred over the respective free or unsalted compounds.Therefore, the present invention is further directed to salts, including pharmaceutically acceptable salts, of compounds according to formula (I).The present invention is further directed to free or unsalted compounds of formula (I).

[0083] Salts, including pharmaceutically acceptable salts, of the compounds of the present invention are readily prepared by those of skill in the art.

[0084] Representative pharmaceutically acceptable acid addition salts include 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, tartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, and dodecyl sulfate. Salt (Estolate), Edetate (Ethylenediaminetetraacetic Acid), Estolate (Lauryl Sulfate), Ethan-1,2-Disulfonate (Edisylate), Ethanesulfonate (Esylate), Formate, Fumarate, Galactarate (Mucate), Gentisate (2,5-Dihydroxybenzoate), Glucoheptonate (Gluceptate), Gluconate, Glucuronate, Glutamate, Glutarate, Glycerophosphate, Glycolate, Hexylresorcylate, Hippurate, Hydrabamine (N,N'-Di(dehydroabietyl)-ethyl)- diamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoic acid, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methylsulfate, mucate, naphthalene-1,5-disulfonate (napadisilate), naphthalene-2-sulfonate (napsylate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate, p-aminosalicylate, pamoate (embonate), pantothenate, These include, but are not limited to, pectinate, persulfate, phenylacetate, phenylethylbarbiturate, phosphate, polygalacturonate, propionate, p-toluenesulfonate (tosylate), pyroglutamate, pyruvate, salicylate, sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, teoclate (8-chlorotheophylline), thiocyanate, triethiodide, trifluoroacetate, undecanoate, undecylenate, and valerate salts.

[0085] Representative pharmaceutically acceptable base addition salts include aluminum, 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS, tromethamine), arginine, benethamine (N-benzylphenethylamine), benzathine (N,N'-dibenzylethylenediamine), b / s-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, clemizole (1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole), cyclohexyl benzoate, ... These include, but are not limited to, dibenzohexylamine, dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, dopamine, ethanolamine, ethylenediamine, L-histidine, iron, isoquinoline, lepidine, lithium, lysine, magnesium, meglumine (N-methylglucamine), piperazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, t-butylamine, and zinc.

[0086] Compounds according to formula (I) may contain one or more asymmetric centers (also referred to as chiral centers) and therefore may exist as individual enantiomers, diastereomers, or other stereoisomeric forms, or as mixtures thereof. Chiral centers, such as chiral carbon atoms, may be present in substituents, such as alkyl groups. If the stereochemistry of a chiral center is present in a compound of formula (I) or any chemical structure illustrated herein, and if not specified, the structure is intended to encompass all individual stereoisomers and all mixtures thereof. Thus, 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.

[0087] A divalent group is a group that has two points of attachment. For all divalent groups, unless otherwise specified, orientation of the group is implied by the direction in which the group's formula or structure is written.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any compositions and methods similar or equivalent to those described herein can be used to practice or test the methods of the present disclosure, exemplary compositions and methods are described herein. Any of the aspects and embodiments of the present disclosure described herein may also be combined. For example, the subject matter of any dependent or independent claim disclosed herein may be combined in a composite manner (e.g., one or more recitations from each dependent claim may be combined into a single claim based on the independent claim to which they depend).

[0089] The ranges provided herein include all values ​​within the particular ranges recited, as well as the values ​​relating to the endpoints of the particular ranges.

[0090] The concentrations described herein are determined at ambient temperature and pressure. This may be, for example, at room temperature or at the temperature and pressure of a particular portion of the process stream. Preferably, the concentrations are determined at standard conditions of 25°C and 1 bar pressure.

[0091] CCR2 target site and CCR2 binding site The compounds of formula (I) disclosed herein are heterobifunctional synthetic agents designed so that one end interacts with a cell surface CCR2 target while 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 directs immune surveillance to CCR2-expressing tissues / cells, combining the mechanisms of antibody function with small molecule dose control. This mechanism may include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC), preferably ADCC. The same Fc receptor-expressing immune cells that initiate destruction of ARM / antibody-tagged cells are also involved in presenting endogenous antigens for the potential of long-term cellular immunity.

[0092] The compounds of formula (I) disclosed herein comprise a CCR2 binding moiety capable of binding to CCR2 present on the surface of a cell. In one embodiment, CCR2 is expressed on a pathogenic cell.

[0093] In further embodiments, the pathogenic cell is a pathogenic immune cell, a tumor or cancer cell, or a stromal cell (including stromal cells present in the tumor microenvironment).

[0094] In a further embodiment, the CCR2 target is present on the surface of a pathogen selected from a virus or a bacterial cell. Examples of viruses that express cell surface targets include, but are not limited to, influenza.

[0095] In further embodiments, 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 (Treg), a neutrophil (e.g., an N2 neutrophil), a macrophage (e.g., an M2 macrophage), a B regulatory cell (Breg, a memory B cell), a plasma cell, a CD8 cell (e.g., a CD8 regulatory cell (CD8reg), a memory CD8 cell, an effector CD8 cell, a naive CD8 T cell, a 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.

[0096] In further embodiments, the pathogenic immune cells are myeloid-derived suppressor cells (MDSCs), such as monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs), T regulatory cells (Tregs), neutrophils (e.g., N2 neutrophils), macrophages (e.g., M2 macrophages), B regulatory cells (Bregs), CD8 regulatory cells (CD8regs), exhausted T cells.

[0097] In a further embodiment, the CCR2-expressing pathogenic immune cells are myeloid-derived suppressor cells (MDSCs). In a further embodiment, the CCR2-expressing pathogenic immune cells are selected from monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs).

[0098] In a further embodiment, the tumor or cancer cell is a solid tumor cell.

[0099] In further embodiments, the tumor or cancer cell is a non-small cell lung cancer (NSCLC) cell, a hepatocellular carcinoma (HCC) cell, a colorectal cancer (CRC) cell, a cervical squamous cell carcinoma (CESC) cell, a head and neck squamous cell carcinoma (HNSC) cell, a pancreatic cancer cell, a metastatic castration-resistant prostate cancer (mCRPC) cell, an ovarian cancer cell, a bladder cancer cell, or a breast cancer cell, preferably an NSCLC cell, an HCC cell, or a CRC cell.

[0100] In a further embodiment, the stromal cells are cancer-associated fibroblasts (CAFs).

[0101] The present disclosure also provides pharmaceutical compositions comprising a compound of formula (I) disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0102] Anticotinine antibodies The present disclosure provides an antibody or antigen-binding fragment thereof that binds to a cotinine moiety. As used herein, the term "anti-cotinine antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that binds to a cotinine moiety. Cotinine has the following structure: [ka]

[0103] As used herein, the term "cotinine moiety" refers to cotinine or a cotinine analog. The compounds of formula (I) described herein include a cotinine moiety linked to a CCR2 binding moiety via a linker. In one embodiment, the cotinine moiety has the following structure: [ka] In the formula, R 1 is C 1~4 Alkyl or C 3~6 In another embodiment, R 1 is methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, or t-butyl. 1 is methyl. In another embodiment, R 1 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0104] The term "antibody" is used herein in the broadest sense to refer to a molecule having an immunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD, or IgE), and includes monoclonal antibodies, recombinant antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, multispecific antibodies, single variable domains (e.g., domain antibodies (DABs)), antigen-binding antibody fragments, Fab, F(ab'), Fv, disulfide-linked Fv, single-chain Fv, disulfide-linked scFv, diabodies, TANDABS, etc., including bispecific and heteroconjugate antibodies, as well as modified forms of any of the foregoing (for a review of alternative "antibody" formats, see Holliger and Hudson, Nature Biotechnology, 2005, 23(9):1126-1136).

[0105] The terms "complete," "whole," or "intact" antibody, used interchangeably herein, refer to a heterotetrameric glycoprotein with an approximate molecular weight of 150,000 daltons. An intact antibody is composed of two identical heavy chains (HC) and two identical light chains (LC) linked by covalent disulfide bonds. This H2L2 structure folds to form three functional domains, including two antigen-binding fragments known as "Fab" fragments and an "Fc" crystallizable fragment. The Fab fragment consists of an amino-terminal variable domain, either a variable heavy chain (VH) or a variable light chain (VL), and a carboxyl-terminal constant domain, CH1 (heavy chain) and CL (light chain). The Fc fragment consists of two domains formed by the dimerization of paired CH2 and CH3 regions. The Fc fragment can trigger effector functions by binding to receptors on immune cells or by binding to C1q, the first component of the classical complement pathway. The five classes of antibodies, IgM, IgA, IgG, IgE, and IgD, are defined by distinct heavy chain amino acid sequences, termed μ, α, γ, ε, and δ, respectively, and each heavy chain can pair with either a K or a λ light chain. The majority of antibodies in serum belong to the IgG class, and there are four isotypes of human IgG (IgG1, IgG2, IgG3, and IgG4), which differ in sequence primarily in their hinge region.

[0106] "CDRs" are defined as the complementarity-determining region amino acid sequences of an antibody or antigen-binding fragment thereof. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, "CDRs" as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

[0107] Throughout this specification, amino acid residues within variable domain sequences and variable domain regions within a full-length antigen-binding sequence, e.g., an antibody heavy chain sequence or an antibody light chain sequence, are numbered according to the Kabat numbering convention. Similarly, the terms "CDR," "CDRL1," "CDRL2," "CDRL3," "CDRH1," "CDRH2," and "CDRH3" used in the examples follow the Kabat numbering convention. For further information, see Sequences of Proteins of Immunological Interest, 4th Ed., USDapartment of Health and Human Services, National Institutes of Health (1987).

[0108] It will be apparent to those skilled in the art that alternative numbering conventions exist for amino acid residues within variable domain sequences and full-length antibody sequences. Alternative numbering conventions for CDR sequences also exist, such as those presented in Chothia et al., Nature, 1989, 342:877-883. The structure and protein folding of the antigen-binding protein may mean that other residues are considered part of the CDR sequences and will be understood as such by those skilled in the art.

[0109] Other numbering conventions for CDR sequences available to those skilled in the art include the "AbM" (University of Bath) and "Contact" (University College London) methods.

[0110] Table 2 below presents one definition using each numbering convention for each CDR or binding unit. It should be noted that some of the CDR definitions may vary depending on the particular publication used. [Table 2]

[0111] In a further embodiment, the anti-cotinine antibody is humanized. In a further embodiment, the Fc region of the anti-cotinine antibody is modified to increase ADCC activity, ADCP activity, and / or CDC activity, suitable modifications of which are provided below. In a further embodiment, the Fc region of the anti-cotinine antibody is modified to increase ADCC activity.

[0112] Fc engineering methods can be applied to modify the functional or pharmacokinetic properties of antibodies. Effector function can be altered by making mutations in the Fc region that increase or decrease binding to C1q or Fcγ receptors, modifying CDC or ADCC activity, respectively. Modifications to the glycosylation pattern of antibodies can also be made to alter effector function. The in vivo half-life of antibodies can be altered by making mutations that affect Fc binding to FcRn (neonatal Fc receptor).

[0113] The term "effector function" as used herein refers to one or more of antibody-mediated effects, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-mediated complement activation, including complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated phagocytosis (CDCP), antibody-dependent complement-mediated cytolysis (ADCML), and Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis (ADCP).

[0114] The interaction between the Fc region of an antigen-binding protein or antibody and various Fc receptors (FcRs), including FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), FcRn, C1q, and type II Fc receptors, is thought to mediate the effector functions of the antigen-binding protein or antibody. Significant biological effects can be the result of effector functions. Typically, the ability to mediate effector functions requires binding of the antigen-binding protein or antibody to an antigen, and not all antigen-binding proteins or antibodies mediate all effector functions.

[0115] Effector function can be evaluated in several ways, including, for example, assessing the ADCC effector function of the antibody coated on target cells mediated by natural killer (NK) cells via FcγRIII or monocytes / macrophages via FcγRI, or assessing the CDC effector function of the antibody coated on target cells mediated by the complement cascade via C1q. For example, the antibody of the present invention or its antigen-binding fragment can be evaluated for ADCC effector function in a natural killer cell assay. Examples of such assays can be found in Shields et al., The Journal of Biological Chemistry, 2001, 276:6591-6604; Chappel et al., The Journal of Biological Chemistry, 1993, 268:25124-25131; Lazar et al., PNAS, 2006, 103:4005-4010.

[0116] Examples of assays for determining CDC function include those described in J Imm Meth, 1995, 184:29-38.

[0117] The effect of mutations on effector function (e.g., FcRn binding, FcγR and C1q binding, CDC, ADCML, ADCC, ADCP) can be assessed, for example, as described in Grevys et al., J Immunol., 2015, 194(11):5497-5508; Tam et al., Antibodies, 2017, 6(3):12; or Monnet et al., mAbs, 2014, 6(2):422-436.

[0118] Throughout this specification, amino acid residues within the Fc region of an antibody sequence or full-length antigen binding protein sequence are numbered according to the EU index numbering convention.

[0119] 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 contain any of the following mutations:

[0120] Enhanced CDC: Fc engineering can be used to enhance complement-based effector functions. For example, (for IgG1), K326W / E333S, S267E / H268F / S324T, and IgG1 / IgG3 cross-subclasses can increase C1q binding, and E345R (Diebolder et al., Science, 2014, 343:1260-1293) and E345R / E430G / S440Y result in preformed IgG hexamers (Wang et al., Protein Cell, 2018, 9(1):63-73).

[0121] Enhanced ADCC: Fc engineering can be used to enhance ADCC. For example (for IgG1), F243L / R292P / Y300L / V305I / P396L, S239D / I332E, and S298A / E333A / K334A increase FcγRIIIa binding, S239D / I332E / A330L increase FcγRIIIa binding and decrease FcγRIIb binding, and G236A / S239D / I332E improve FcγRIIa binding, improve the FcγRIIa / FcγRIIb binding ratio (activation / inhibition ratio), and enhance phagocytosis of antibody-coated target cells by macrophages. An asymmetric Fc, in which one heavy chain contains L234Y / L235Q / G236W / S239M / H268D / D270E / S298A mutations and the other heavy chain has D270E / K326D / A330M / K334E, increases affinity for FcγRIIIa F158 (low affinity allele) and FcγRIIIa V158 (high affinity allele) without increasing binding affinity to the inhibitory FcγRIIb (Mimoto et al., mAbs, 2013, 5(2):229-236).

[0122] Enhancement of ADCP: Fc engineering can be used to enhance ADCP. For example (for IgG1), G236A / S239D / I332E increases FcγRIIa binding and increases FcγRIIIa binding (Richards, J. et al., Mol. Cancer Ther., 2008, 7:2517-2527).

[0123] Increased co-engagement: Fc engineering can be used to increase co-engagement with FcRs. For example (for IgG1), S267E / L328F increases FcγRIIb binding, and N325S / L328F increases FcγRIIa binding and decreases FcγRIIIa binding (Wang et al., Protein Cell, 2018, 9(1):63-73).

[0124] In a further 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 WO2003 / 011878, WO2006 / 014679, and EP1229125.

[0125] The absence of the α1,6 innermost fucose residue on the Fc glycan moiety at N297 of IgG1 antibodies enhances affinity for FcγRIIIA. Thus, afucosylated or hypofucosylated monoclonal antibodies may have increased therapeutic efficacy (Shields et al., J Biol Chem., 2002, 277(30):26733-40, and Monnet et al., mAbs, 2014, 6(2):422-436).

[0126] In one embodiment, an antibody or antigen-binding fragment thereof is provided that comprises a chimeric heavy chain constant region. In one embodiment, the antibody or antigen-binding fragment thereof comprises an IgG1 / IgG3 chimeric heavy chain constant region, such that the antibody or antigen-binding fragment thereof has enhanced effector function, such as enhanced ADCC or enhanced CDC, or enhanced ADCC and CDC functions. For example, a chimeric antibody or antigen-binding fragment thereof of the present invention may comprise at least one CH2 domain derived from IgG3. In one such embodiment, the antibody or antigen-binding fragment thereof comprises one CH2 domain derived from IgG3, or both CH2 domains may be derived from IgG3. In a further embodiment, the chimeric antibody or antigen-binding fragment thereof comprises an IgG1 CH1 domain, an IgG3 CH2 domain, and an IgG3 CH3 domain. In a further embodiment, the chimeric antibody or antigen-binding fragment thereof comprises an IgG1 CH1 domain, an IgG3 CH2 domain, and an IgG3 CH3 domain, except for position 435, which is a histidine.

[0127] In further embodiments, the chimeric antibody or antigen-binding fragment thereof comprises an IgG1 CH1 domain and at least one CH2 domain derived from IgG3. In one embodiment, the chimeric antibody or antigen-binding fragment thereof comprises an IgG1 CH1 domain and the following residues within the CH2 domain that correspond to IgG3 residues: 274Q, 276K, 296F, 300F, and 339T. In one embodiment, the chimeric antibody or antigen-binding fragment thereof also comprises 356E within the CH3 domain, which corresponds to IgG3 residues. In one embodiment, the antibody or antigen-binding fragment thereof also comprises one or more of the following residues within the CH3 domain that correspond to IgG3 residues: 358M, 384S, 392N, 397M, 422I, 435R, and 436F.

[0128] Also provided is a method for producing an antibody or antigen-binding fragment thereof according to the invention, comprising the steps of: a) culturing a recombinant host cell comprising an expression vector comprising a nucleic acid sequence encoding a chimeric Fc region having both IgG1 and IgG3 Fc region amino acid residues (e.g., as described above); b) recovering the antibody or antigen-binding fragment thereof.

[0129] Such methods for producing antibodies or antigen-binding fragments thereof having chimeric heavy chain constant regions can be carried out, for example, using the COMPLEGENT technology system available from BioWa, Inc. (Princeton, NJ) and Kyowa Hakko Kirin Co., Ltd. The COMPLEGENT system comprises a recombinant host cell containing an expression vector in which a nucleic acid sequence encoding a chimeric Fc region having both IgG1 and IgG3 Fc region amino acid residues is expressed to produce an antibody or antigen-binding fragment thereof with enhanced CDC activity, i.e., CDC activity is increased relative to an otherwise identical antibody or antigen-binding fragment thereof lacking such a chimeric Fc region, as described in WO 2007 / 011041 and US 2007 / 0148165, each of which is incorporated herein by reference. In alternative embodiments, CDC activity can be increased by introducing sequence-specific mutations into the Fc region of the IgG chain. Those skilled in the art will also recognize other suitable systems.

[0130] The present invention also provides a method for producing an antibody or antigen-binding fragment thereof according to the invention, comprising the steps of: a) culturing a recombinant host cell comprising an expression vector comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof, wherein optionally the FUT8 gene encoding α-1,6-fucosyltransferase is inactivated in the recombinant host cell; b) recovering the antibody or antigen-binding fragment thereof.

[0131] Such methods for producing antibodies or antigen-binding fragments thereof can be carried out, for example, using the POTELLIGENT technology system available from BioWa, Inc. (Princeton, NJ), in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies with enhanced ADCC activity relative to the same monoclonal antibodies produced in cells with a functional FUT8 gene, as described in U.S. Patent No. 7,214,775, U.S. Patent No. 6,946,292, WO 00 / 61739, and WO 02 / 31240, each of which is incorporated herein by reference. Those skilled in the art will also recognize other suitable systems.

[0132] In one embodiment, the antibody or antigen-binding fragment thereof is produced in a host cell in which the FUT8 gene is inactivated. In a further embodiment, the antibody or antigen-binding fragment thereof is produced in a - / -FUT8 host cell. In a further embodiment, the antibody or antigen-binding fragment thereof is afucosylated at Asn297 (IgG1).

[0133] It will be apparent to one skilled in the art that such modifications can be used alone as well as in combination with each other to further enhance effector function.

[0134] In one such embodiment, an antibody or antigen-binding fragment thereof is provided, comprising a heavy chain constant region comprising both the mutated heavy chain constant region and the chimeric heavy chain constant region described individually above. For example, an antibody or antigen-binding fragment thereof comprising at least one CH2 domain derived from IgG3 and one CH2 domain derived from IgG1 is provided, wherein the IgG1 CH2 domain has one or more mutations at positions 239, 332, and 330 (e.g., the mutations can be selected from S239D, I332E, and A330L), such that the antibody or antigen-binding fragment thereof has enhanced effector function, e.g., enhanced ADCC or enhanced CDC, or enhanced ADCC and enhanced CDC, compared to a comparable antibody or antigen-binding fragment thereof having an IgG1 heavy chain constant region lacking the mutations. In one embodiment, the IgG1 CH2 domain has the mutations S239D and I332E. In another embodiment, the IgG1 CH2 domain has the mutations S239D, A330L, and I332E.

[0135] In alternative embodiments, an antibody or antigen-binding fragment thereof is provided that comprises both a chimeric heavy chain constant region and an altered glycosylation profile, as described individually above. In one embodiment, the antibody or antigen-binding fragment thereof comprises an altered glycosylation profile such that the fucose-to-mannose ratio is 0.8:3 or less. In one such embodiment, the heavy chain constant region comprises at least one CH2 domain from IgG3 and one CH2 domain from IgG1, and has an altered glycosylation profile such that the fucose-to-mannose ratio is 0.8:3 or less, e.g., the antibody or antigen-binding fragment thereof is defucosylated. The antibody or antigen-binding fragment thereof has enhanced effector function, e.g., enhanced ADCC or enhanced CDC, or enhanced ADCC and enhanced CDC, compared to a comparable antibody or antigen-binding fragment thereof having an IgG1 heavy chain constant region lacking the glycosylation profile.

[0136] In an alternative embodiment, the antibody or antigen-binding fragment thereof has at least one IgG3 heavy chain CH2 domain and at least one heavy chain constant domain derived from IgG1, and both IgG CH2 domains are mutated according to the restrictions described herein.

[0137] In one aspect, there is provided a method for producing an antibody or antigen-binding fragment thereof according to the invention described herein, comprising the steps of: a) culturing a recombinant host cell comprising an expression vector comprising a nucleic acid sequence encoding a chimeric Fc domain having both IgG1 and IgG3 Fc domain amino acid residues (e.g., as described above), wherein the FUT8 gene encoding α-1,6-fucosyltransferase is inactivated in the recombinant host cell; b) recovering the antibody or antigen-binding fragment thereof.

[0138] Such methods for the production of antibodies or antigen-binding fragments thereof can be performed, for example, using the ACCRETAMAB technology system available from BioWa, Inc. (Princeton, NJ), which combines the POTELLIGENT and COMPLEGENT technology systems to produce antibodies or antigen-binding fragments thereof with both enhanced ADCC and CDC activity relative to an otherwise identical monoclonal antibody that lacks a chimeric Fc domain and is fucosylated.

[0139] In another embodiment, an antibody or antigen-binding fragment thereof is provided comprising a mutation and a chimeric heavy chain constant region, wherein the antibody or antigen-binding fragment thereof has an altered glycosylation profile such that the antibody or antigen-binding fragment thereof has enhanced effector function, e.g., enhanced ADCC or enhanced CDC, or both enhanced ADCC and CDC. In one embodiment, the mutation is selected from positions 239, 332, and 330, e.g., S239D, I332E, and A330L. In a further embodiment, the heavy chain constant region comprises at least one CH2 domain derived from IgG3 and one CH1 domain derived from IgG1. In one embodiment, the heavy chain constant region has an altered glycosylation profile such that the fucose-to-mannose ratio is 0.8:3 or less, e.g., the antibody or antigen-binding fragment thereof is defucosylated such that the antibody or antigen-binding fragment thereof has enhanced effector function compared to a comparable non-chimeric antibody or antigen-binding fragment thereof lacking the mutation and the altered glycosylation profile.

[0140] In a further embodiment, the anti-cotinine antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 having SEQ ID NO: 1, a heavy chain CDR2 having SEQ ID NO: 2, a heavy chain CDR3 having SEQ ID NO: 3, a light chain CDR1 having SEQ ID NO: 4, a light chain CDR2 having SEQ ID NO: 5, and a light chain CDR3 having SEQ ID NO: 6. In a further embodiment, the anti-cotinine antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a CDR1 having SEQ ID NO: 1, a CDR2 having SEQ ID NO: 2, and a CDR3 having SEQ ID NO: 3, and the light chain comprises a CDR1 having SEQ ID NO: 4, a CDR2 having SEQ ID NO: 5, and a CDR3 having SEQ ID NO: 6. In a further embodiment, the anti-cotinine antibody is an IgG1 isotype. In a further embodiment, the anti-cotinine antibody is an IgG1 isotype comprising substitutions in the Fc region to increase or enhance ADCC activity. In a further embodiment, the anti-cotinine antibody is an IgG1 isotype comprising substitutions in the Fc region to increase or enhance ADCC activity, the substitutions being S239D / I332E or S239D / I332E / A330L, where the residue numbering is according to the EU index. In a further embodiment, the anti-cotinine antibody is an IgG1 isotype comprising substitutions in the Fc region to increase or enhance ADCC activity, the substitutions being S239D / I332E, where the residue numbering is according to the EU index.

[0141] In a further embodiment, the anti-cotinine antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) having SEQ ID NO: 7 and a light chain variable region (VL) having SEQ ID NO: 8. In a further embodiment, the anti-cotinine antibody has a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) having SEQ ID NO: 7 and the light chain comprises a light chain variable region (VL) having SEQ ID NO: 8. In a further embodiment, the anti-cotinine antibody is of the IgG1 isotype. In a further embodiment, the anti-cotinine antibody is of the IgG1 isotype comprising substitutions in the Fc region to increase or enhance ADCC activity. In a further embodiment, the anti-cotinine antibody is of the IgG1 isotype comprising substitutions in the Fc region to increase or enhance ADCC activity, wherein the substitutions are S239D / I332E or S239D / I332E / A330L, and the numbering of residues is according to the EU index. In a further embodiment, the anti-cotinine antibody is an IgG1 isotype comprising substitutions in the Fc region to increase or enhance ADCC activity, the substitutions being S239D / I332E, where residue numbering is according to the EU index.

[0142] In a further embodiment, the anti-cotinine antibody has a heavy chain comprising SEQ ID NO:9 and a light chain comprising SEQ ID NO:10.

[0143] The present disclosure also provides a pharmaceutical composition comprising an anti-cotinine antibody or antigen-binding fragment thereof disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0144] The present disclosure also provides a combination comprising a compound of formula (I) disclosed herein and an anti-cotinine antibody or antigen-binding fragment thereof disclosed herein. The compound of formula (I) and the anti-cotinine antibody or antigen-binding fragment thereof can be present in the same composition or in separate compositions. In one embodiment, the combination comprises a pharmaceutical composition comprising 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 comprising a compound of formula (I) disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient, and a second pharmaceutical composition comprising an anti-cotinine antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0145] Description of Use The compounds of formula (I) and their pharmaceutically acceptable salts are capable of simultaneously binding to cell surface-expressed CCR2 and an anti-cotinine antibody or antigen-binding fragment thereof to form a ternary complex for the treatment and / or prevention of diseases or disorders associated with CCR2-expressing cells.

[0146] In one embodiment, the present disclosure provides a method for treating and / or preventing a disease or disorder in a patient in need thereof, comprising administering to the patient therapeutically effective amounts of a compound of formula (I) or a pharmaceutically acceptable salt thereof and an anti-cotinine antibody or antigen-binding fragment thereof, wherein the disease or disorder is selected from cancer, an inflammatory disease, an autoimmune disease, a viral infection, or a bacterial infection.

[0147] In further embodiments, the compound and antibody, or antigen-binding fragment thereof, are administered simultaneously. In further embodiments, the compound and antibody, or antigen-binding fragment thereof, are administered simultaneously from a single composition, including as a fixed-dose composition or by premixing the compound and antibody or antigen-binding fragment thereof prior to administration. For example, the compound and antibody, or antigen-binding fragment thereof, can be premixed about 2 seconds to about 30 seconds, about 30 seconds to about 2 minutes, about 2 minutes to about 10 minutes, about 10 minutes to about 30 minutes, or about 30 minutes to about 2 hours prior to administration. In further embodiments, the compound and antibody, or antigen-binding fragment thereof, are administered simultaneously from two separate compositions.

[0148] In a further embodiment, the compound and the antibody, or antigen-binding fragment thereof, are administered sequentially.

[0149] In certain embodiments, the compound and the antibody, or antigen-binding fragment thereof, can be administered by the same route or by different routes, whether administered simultaneously or sequentially. In one embodiment, the compound and the antibody, or antigen-binding fragment thereof, are both administered intravenously or subcutaneously, either in the same composition or in separate compositions. In another embodiment, the compound is administered orally and the antibody, or antigen-binding fragment thereof, is administered intravenously or subcutaneously.

[0150] In further embodiments, the compound and antibody, or antigen-binding fragment thereof, are administered at a molar ratio of about 2:1, about 1.8:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1:1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.8, about 1:2, about 2:1 to about 1.5:1, about 1.5:1 to about 1.2:1, about 1.2:1 to about 1:1, about 1:1 to about 1:1.2, about 1:1.2 to about 1:1.5, or about 1:1.5 to about 1:2 compound to antibody, or antigen-binding fragment thereof.

[0151] In further embodiments, the compound and antibody, or antigen-binding fragment thereof, are present in combination in a molar ratio of about 2:1, about 1.8:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1:1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.8, about 1:2, about 2:1 to about 1.5:1, about 1.5:1 to about 1.2:1, about 1.2:1 to about 1:1, about 1:1 to about 1:1.2, about 1:1.2 to about 1:1.5, or about 1:1.5 to about 1:2 compound to antibody, or antigen-binding fragment thereof.

[0152] In further embodiments, the compound and antibody, or antigen-binding fragment thereof, are administered at a dosage of 0.0001 mg / kg to 1 mg / kg of compound and 0.01 mg / kg to 100 mg / kg of antibody. For example, in further embodiments, the compound is administered at a dosage of about 0.0001 mg / kg to about 0.0002 mg / kg, about 0.0002 mg / kg to about 0.0003 mg / kg, about 0.0003 mg / kg to about 0.0004 mg / kg, about 0.0004 mg / kg to about 0.0005 mg / kg, about 0.0005 mg / kg to about 0.001 mg / kg, about 0.001 mg / kg to about 0.002 mg / kg, about 0.002mg / kg to about 0.003mg / kg, about 0.003mg / kg to about 0.004mg / kg, about 0.004mg / kg to about 0.005mg / kg, about 0.005mg / kg to about 0.01mg / kg, about 0.01mg / kg to about 0.02mg / kg, about 0.02mg / kg to about 0.03mg / kg, about 0.03mg / kg to about 0.04mg / kg, about 0.04mg / kg to about 0. The antibody or antigen-binding fragment thereof is administered at a dosage of about 0.01 mg / kg to about 0.02 mg / kg, about 0.05 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 0.2 mg / kg, about 0.2 mg / kg to about 0.3 mg / kg, about 0.3 mg / kg to about 0.4 mg / kg, about 0.4 mg / kg to about 0.5 mg / kg, and / or about 0.5 mg / kg to about 1 mg / kg, and the antibody or antigen-binding fragment thereof is administered at a dosage of about 0.01 mg / kg to about 0.02 mg / kg. g / kg, about 0.02mg / kg to about 0.03mg / kg, about 0.03mg / kg to about 0.04mg / kg, about 0.04mg / kg to about 0.05mg / kg, about 0.05mg / kg to about 0.1m g / kg, about 0.1 mg / kg to about 0.2 mg / kg, about 0.2 mg / kg to about 0.3 mg / kg, about 0.3 mg / kg to about 0.4 mg / kg, about 0.4 mg / kg to about 0.5 mg / kg, about 0.5mg / kg to about 1mg / kg, about 1mg / kg to about 2mg / kg, about 2mg / kg to about 3mg / kg, about 3mg / kg to about 4mg / kg, about 4mg / kg to about 5mg / kg, about 5mg / kg ~10mg / kg, 10mg / kg~15mg / kg, 15mg / kg~20mg / kg, 20mg / kg~25mg / kg, 25mg / kg~30mg / kg, 30mg The compound is administered at a dosage of about 100 mg / kg to about 35 mg / kg, about 35 mg / kg to about 40 mg / kg, about 40 mg / kg to about 45 mg / kg, about 45 mg / kg to about 50 mg / kg, about 50 mg / kg to about 60 mg / kg, about 60 mg / kg to about 70 mg / kg, about 70 mg / kg to about 80 mg / kg, about 80 mg / kg to about 90 mg / kg, and / or about 90 mg / kg to about 100 mg / kg.

[0153] In further embodiments, the compound and antibody, or antigen-binding fragment thereof, are administered at a dosage of 0.007 mg to 70 mg of compound and 0.7 mg to 7000 mg of antibody. For example, in further embodiments, the compound is administered at a dosage of about 0.007 mg to about 0.01 mg, about 0.01 mg to about 0.02 mg, about 0.02 mg to about 0.03 mg, about 0.03 mg to about 0.04 mg, about 0.04 mg to about 0.05 mg, about 0.05 mg to about 0.1 mg, about 0.1 mg to about 0.2 mg, about 0.2 mg to about 0.3 mg, about 0.3 mg to about 0.4 mg, about 0.4 mg to about 0.5 mg, about 0. and the antibody or antigen-binding fragment thereof is administered at a dosage of about 0.5 mg to about 1 mg, about 1 mg to about 2 mg, about 2 mg to about 3 mg, about 3 mg to about 4 mg, about 4 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 20 mg, about 20 mg to about 30 mg, about 30 mg to about 40 mg, about 40 mg to about 50 mg, about 50 mg to about 60 mg, and / or about 60 mg to about 70 mg. Approximately 1 mg to approximately 2 mg, approximately 2 mg to approximately 3 mg, approximately 3 mg to approximately 4 mg, approximately 4 mg to approximately 5 mg, approximately 5 mg to approximately 10 mg, approximately 10 mg to approximately 20 mg, approximately 20 mg to approximately 30 mg, approximately 30 mg to approximately 40 mg, Approximately 40mg to approximately 50mg, approximately 50mg to approximately 100mg, approximately 100mg to approximately 500mg, approximately 500mg to approximately 1000mg, approximately 1000mg to approximately 1500mg, approximately 1500mg to approximately 2000mg, approximately It is administered in dosages of 2000 mg to about 2500 mg, about 2500 mg to about 3000 mg, about 3000 mg to about 3500 mg, about 3500 mg to about 4000 mg, about 4000 mg to about 4500 mg, about 4500 mg to about 5000 mg, about 5000 mg to about 5500 mg, about 5500 mg to about 6000 mg, about 6000 mg to about 6500 mg, and / or about 6500 mg to about 7000 mg.

[0154] In further embodiments, the compounds and antibodies, or antigen-binding fragments thereof, are administered at the molar ratios and / or dosages described herein once per week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks for a period of between one week and one year, such as, for example, a period of one week, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months.

[0155] In a further 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 the anti-cotinine antibody or an antigen-binding fragment thereof can be used in treating or preventing a disease or disorder selected from cancer, inflammatory diseases, autoimmune diseases, viral infections, or bacterial infections.

[0156] In a further 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 antigen-binding fragment thereof for the manufacture of a medicament, which can be used in treating or preventing a disease or disorder selected from cancer, inflammatory disease, autoimmune disease, viral infection, or bacterial infection.

[0157] In a further embodiment, the disease or disorder is mediated by chemokine receptor 2 (CCR2) and / or is associated with CCR2-positive pathogenic cells. In a further embodiment, the CCR2-positive cell type is identified by examining CCR expression by immunohistochemistry or flow cytometry.

[0158] In further embodiments, the disease or disorder is a cancer selected from non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), colorectal cancer (CRC), cervical squamous cell carcinoma (CESC), head and neck squamous cell carcinoma (HNSC), pancreatic cancer, metastatic castration-resistant prostate cancer (mCRPC), ovarian cancer, bladder cancer, or breast cancer, preferably a cancer selected from NSCLC, HCC, or CRC.

[0159] In a further embodiment, the disease or disorder is a solid tumor. In a further embodiment, the disease or disorder is a solid tumor selected from NSCLC, HCC, CRC, CESC, HNSC, pancreatic cancer, mCRPC, ovarian cancer, bladder cancer or breast cancer, preferably a solid tumor selected from NSCLC, HCC or CRC.

[0160] In a further embodiment, the disease or disorder is a PD-1 relapsed or refractory cancer, such as PD-1 relapsed or refractory NSCLC, HCC, CRC, CESC, HNSC, pancreatic cancer, mCRPC, ovarian cancer, bladder cancer, or breast cancer, preferably PD-1 relapsed or refractory NSCLC, HCC, or CRC.

[0161] In a further embodiment, the disease or disorder is a non-solid cancer, hi a further embodiment, the disease or disorder is a leukemia, lymphoma, or myeloma.

[0162] In a further embodiment, the disease or disorder is a viral infection. In a further embodiment, the viral infection is caused by an influenza virus, a coronavirus (e.g., COVID-19), or a hepatitis B virus.

[0163] In a further embodiment, the disease or disorder is a bacterial infection. In a further embodiment, the bacterial infection is a chronic bacterial infection.

[0164] In one embodiment, the present disclosure provides a method for increasing antibody-dependent cellular cytotoxicity (ADCC) of CCR2-expressing cells, 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 antigen-binding fragment thereof, wherein the CCR2-binding portion of the compound binds to CCR2 expressed on the cells.

[0165] In one embodiment, the present disclosure provides a method for increasing antibody-dependent cellular phagocytosis (ADCP) of CCR2-expressing cells, 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 antigen-binding fragment thereof, wherein the CCR2-binding portion of the compound binds to CCR2 expressed on the cells.

[0166] In one embodiment, the present disclosure provides a method for increasing complement-dependent cytotoxicity (CDC) of a CCR2-expressing cell, comprising contacting the cell with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and an anti-cotinine antibody or antigen-binding fragment thereof, wherein the CCR2-binding portion of the compound binds to CCR2 expressed on the cell.

[0167] In one embodiment, the present disclosure provides a method for conditioning a patient for treatment with chimeric antigen receptor (CAR) T-cell therapy, comprising administering to the patient an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and an anti-cotinine antibody or antigen-binding fragment thereof. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof and the anti-cotinine antibody or 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 the anti-cotinine antibody or antigen-binding fragment thereof may be administered as conditioning therapy or combination therapy to improve efficacy in the treatment of solid tumors. In other embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof and the anti-cotinine antibody or antigen-binding fragment thereof may be administered as neoadjuvant therapy for other therapies, including, but not limited to, immunotherapy, surgical resection, radiation, and / or chemotherapy.

[0168] In one embodiment, the present disclosure provides a method for depleting CCR2-expressing cells, comprising contacting the cells with a compound of formula (I) or a pharmaceutically acceptable salt thereof and an anti-cotinine antibody or antigen-binding fragment thereof, wherein the CCR2-binding portion of the compound binds to CCR2 expressed on the cells.

[0169] In a further embodiment, the CCR2-expressing cell is a pathogenic cell.

[0170] In further embodiments, the pathogenic cell is a pathogenic immune cell, a tumor or cancer cell, or a stromal cell.

[0171] In further embodiments, 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 (Treg), a neutrophil (e.g., an N2 neutrophil), a macrophage (e.g., an M2 macrophage), a B regulatory cell (Breg, a memory B cell), a plasma cell, a CD8 cell (e.g., a CD8 regulatory cell (CD8reg), a memory CD8 cell, an effector CD8 cell, a naive CD8 T cell, a 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.

[0172] In further embodiments, the pathogenic immune cells are myeloid-derived suppressor cells (MDSCs), such as monocytic MDSCs (mMDSCs) and polymorphonuclear MDSCs (PMN_MDSCs), T regulatory cells (Tregs), neutrophils (e.g., N2 neutrophils), macrophages (e.g., M2 macrophages), B regulatory cells (Bregs), CD8 regulatory cells (CD8regs), exhausted T cells.

[0173] In further embodiments, the tumor or cancer cell is a non-small cell lung cancer (NSCLC) cell, a hepatocellular carcinoma (HCC) cell, a colorectal cancer (CRC) cell, a cervical squamous cell carcinoma (CESC) cell, a head and neck squamous cell carcinoma (HNSC) cell, a pancreatic cancer cell, a metastatic castration-resistant prostate cancer (mCRPC) cell, an ovarian cancer cell, a bladder cancer cell, or a breast cancer cell, preferably an NSCLC cell, an HCC cell, or a CRC cell.

[0174] In a further embodiment, the stromal cells are cancer-associated fibroblasts (CAFs).

[0175] Combination therapy The compounds of the present invention can be used alone or in combination with other therapeutic agents. Thus, the combination therapy according to the present invention comprises the administration of at least one compound of formula (I) or its pharmaceutically acceptable salt and the use of at least one other pharmaceutically active agent. The compounds of the present invention and the other pharmaceutically active agent can be administered together in a single pharmaceutical composition or separately, and when administered separately, this can occur simultaneously or sequentially in any order. The amounts of the compounds of the present invention and the other pharmaceutically active agent, as well as the relative timing of administration, are selected to achieve the desired combined therapeutic effect.

[0176] When the compounds of the present invention are administered in combination with one or more other therapeutically active agents that are normally administered by inhalation, intravenous, oral, intranasal, topical ophthalmic, or other routes, it will be understood that the resulting pharmaceutical composition can be administered by the same route. Alternatively, the individual components of the composition can be administered by different routes.

[0177] In one embodiment, the compounds and pharmaceutical compositions disclosed herein are used in combination with or comprise one or more additional therapeutic agents, hi a further embodiment, the additional therapeutic agent is a checkpoint inhibitor or an immunomodulatory agent.

[0178] In further embodiments, the checkpoint inhibitor is selected from a PD-1 inhibitor (e.g., an anti-PD-1 antibody, including but not limited to, pembrolizumab, nivolumab, cemiplimab, or dostarimab), a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody, including but not limited to, atezolizumab, avelumab, or durvalumab), or a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody, including but not limited to, ipilimumab or tremilumumab).

[0179] In further embodiments, the checkpoint inhibitor is selected from a CD226 axis inhibitor, including but not limited to a TIGIT inhibitor (e.g., an anti-TIGIT antibody), a CD96 inhibitor (e.g., an anti-CD96 antibody), and / or a PVRIG inhibitor (e.g., an anti-PVRIG antibody).

[0180] In further embodiments, the immunomodulatory agent is an ICOS agonist (e.g., an anti-ICOS antibody, including but not limited to, ferazilimab), a PARP inhibitor (e.g., niraparib, olaparib), or a STING agonist.

[0181] Pharmaceutical Compositions, Dosages, and Dosage Forms For purposes of administration, in certain embodiments, the ARMs described herein are administered as raw chemicals or formulated as pharmaceutical compositions. The pharmaceutical compositions disclosed herein comprise an ARM and one or more pharmaceutically acceptable carriers, diluents, or excipients. The ARM is present in the composition in an amount effective to treat the particular disease, disorder, or condition of interest. The activity of the ARM can be determined by one of ordinary skill in the art, for example, as described in the biological assays described below. Appropriate concentrations and dosages can be readily determined by one of ordinary skill in the art. In certain embodiments, the ARM is present in the pharmaceutical composition in an amount of about 25 mg to about 500 mg. In certain embodiments, the ARM is present in the pharmaceutical composition in an amount of about 0.01 mg to about 300 mg. In certain embodiments, the ARM is present in the pharmaceutical composition in an amount of about 0.01 mg, 0.1 mg, 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, or about 500 mg.

[0182] The administration of the compounds of the present invention or their pharmaceutically acceptable salts in pure form or in a suitable pharmaceutical composition can be carried out via any of the accepted modes of administration of drugs to provide similar benefits. Pharmaceutical compositions of the present invention are prepared by combining the compounds of the present invention with suitable pharmaceutically acceptable carriers, diluents, or excipients, and in specific embodiments, are formulated into solid, semi-solid, liquid, or gaseous preparations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Exemplary routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral (e.g., intramuscular, subcutaneous, intravenous, or intradermal), sublingual, buccal, rectal, vaginal, and intranasal. Pharmaceutical compositions of the present invention are formulated to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.

[0183] The composition administered to a subject or patient may take the form of one or more dosage units; for example, a tablet may be a single dosage unit, and a container of the compound of the present invention in aerosol form may hold multiple dosage units.The actual method of preparing such dosage forms will be known or obvious to those skilled in the art.See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia, College of Pharmacy and Science, 2000).The composition to be administered in any case comprises a therapeutically effective amount of the compound of the present invention or its pharmaceutically acceptable salt for treating the target disease or condition according to the teachings described herein.

[0184] The pharmaceutical compositions disclosed herein are prepared by methods well known in the pharmaceutical field.For example, in certain embodiments, the pharmaceutical compositions intended to be administered by injection are prepared by combining the compound of the present invention with sterile distilled water to form a solution.In some embodiments, surfactants are added to promote the formation of a homogeneous solution or suspension.Surfactants are compounds that interact non-covalently with the compound of the present invention to promote the dissolution or homogeneous suspension of the compound in aqueous delivery system.

[0185] Conventional antibody therapeutics have several drawbacks that are addressed by the ARM approach described herein, including difficulties in managing adverse events through adjusting the dose and frequency of administration, challenges in generating antibodies against certain classes of drug targets (e.g., GPCRs, ion channels, and enzymes), and the need for new cell lines for development for each new antibody, which can be slow and expensive. Furthermore, different forms of biologics (e.g., bispecifics) can be difficult to manufacture. In contrast, the ARM approach offers the following advantages: integrating antibody pharmacology with small molecule dose control, dose-controlled PK / PD that allows for timed cell depletion, simpler multimerization, and rapid reversal of cell depletion through administration of the antibody-binding moiety (e.g., cotinine hapten), which can uncouple therapeutic efficacy from potential adverse events. [Example]

[0186] The following examples illustrate the present invention. These examples are not intended to limit the scope of the invention, but rather to provide guidance to those of ordinary skill in the art for preparing and using the compounds, compositions, and methods of the present invention. While specific embodiments of the present invention are described, those skilled in the art will understand that various changes and modifications can be made. References to preparations carried out in a manner similar to, or by common methods for, other preparations may encompass variations in routine parameters such as slight variations in time, temperature, purification conditions, and reagent amounts. Chemical names for all title compounds were generated using ChemDraw Plugin Version 16.0.1.13c(90) or ChemDraw Desktop Version 16.0.1.13(90).

[0187] compound synthesis The compounds of formula (I) are prepared using conventional organic synthesis methods. Suitable synthetic routes are depicted below in the following general reaction schemes. All starting materials are commercially available or can be easily prepared from commercially available starting materials by those skilled in the art.

[0188] Those skilled in the art will understand that if a substituent described herein is not compatible with the synthetic methods described herein, the substituent can be protected with a suitable protecting group that is stable to the reaction conditions. The protecting group can be removed at a suitable point in the reaction sequence to provide the desired intermediate or target compound. Suitable protecting groups and methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art, and examples can be found in T. Greene and P. Wuts, Protecting Groups in Organic Synthesis (4th ed.), John Wiley & Sons, NY (2006). In some cases, a substituent can be specifically selected to be reactive under the reaction conditions used. Under these circumstances, the reaction conditions convert the selected substituent into another substituent that is either useful as an intermediate compound or is a desired substituent in a target compound.

[0189] Scheme 1 [ka] Intermediates Intermediate 1 (1R,2S)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-5-oxocyclohexane-1-carboxylate. [ka] Step 1 Ethyl 8-oxo-1,4-dioxaspiro[4.5]decane-7-carboxylate. [ka] To a solution of 1,4-dioxaspiro[4.5]decan-8-one (125 g, 800 mmol) in tetrahydrofuran (THF) (1300 mL) was added lithium bis(trimethylsilyl)amide (800 mL, 800 mmol, 1 M in THF) at -78 °C for 2 h. Ethyl carbonocyanide (79 g, 800 mmol) was added slowly and stirred at -78 °C for 6 h. Ice-cold water (2 L) was added, and the mixture was extracted with ethyl acetate (2 × 2000 mL). The combined organic extracts were washed with brine (500 mL), dried over anhydrous NaSO, filtered, and the filtrate was evaporated. Purification by normal-phase chromatography (silica gel 60-120 mesh column) eluting with 10% ethyl acetate in hexane provided the title compound as a pale yellow liquid (95 g, 403 mmol, 50.3% yield). LC / MS: m / z 229.09 (M+H) + .

[0190] Step 2 Ethyl (E-8-(((S)-1-phenylethyl)imino)-1,4-dioxaspiro[4.5]decane-7-carboxylate. [ka] To a solution of 8-oxo-1,4-dioxaspiro[4.5]decane-7-carboxylate (100 g, 438 mmol) in toluene (1000 mL) was added (S)-1-phenylethanamine (62.1 mL, 482 mmol) and ytterbium(III) trifluoromethanesulfonate (1.087 g, 1.753 mmol), and the mixture was stirred at reflux for 3 hours using a Dean-Stark trap to remove water. The mixture was concentrated under reduced pressure. Trituration with hexane (500 mL) provided the title compound as a tan solid (110 g, 327 mmol, 74.6% yield). LC-MS m / z 332.28 (M+H). + .

[0191] Step 3 Ethyl (7R,8S)-8-(((S)-1-phenylethyl)amino)-1,4-dioxaspiro[4.5]decane-7-carboxylate. [ka] To a solution of (E)-ethyl 8-(((S)-1-phenylethyl)imino)-1,4-dioxaspiro[4.5]decane-7-carboxylate (110 g, 332 mmol) in acetonitrile (550 mL) and acetic acid (275 mL), sodium triacetoxyborohydride (125 g, 587 mmol) was added portionwise at 0 °C, and the mixture was stirred at room temperature for 16 h. The mixture was evaporated, and the residue was chased with dichloromethane (DCM) (2 × 150 mL). The residue was dissolved in dichloromethane (DCM) (1000 mL) and neutralized with 30% sodium hydroxide solution (150 mL). The organic phase, washed with brine (700 mL), was dried over anhydrous Na2SO4, filtered, and the filtrate was evaporated. Purification by normal phase chromatography (silica gel 100-200 mesh column) eluting with 10% ethyl acetate in petroleum ether provided the title compound as a yellow gummy solid (90 g, 208 mmol, 62.8% yield). LC-MS m / z 334.29 (M+H) + .

[0192] Step 4 (7R,8S)-7-(ethoxycarbonyl)-N-((S)-1-phenylethyl)-1,4-dioxaspiro[4.5]decane-8-aminium, 4-methylbenzenesulfonate. [ka] To a solution of (7R,8S)-8-(((S)-1-phenylethyl)amino)-1,4-dioxaspiro[4.5]decane-7-carboxylate (90 g, 270 mmol) in dichloromethane (DCM) (900 mL) was added p-toluenesulfonic acid monohydrate (51.3 g, 270 mmol) at 0° C., and the mixture was stirred at 26° C. for 16 hours. The mixture was concentrated under reduced pressure, diethyl ether (200 mL) was added, and the mixture was stirred for 30 minutes. The precipitate was collected by filtration and dried to provide the title compound as a pale yellow solid (100 g, 197 mmol, 73.1% yield). LC-MS m / z 334.35 (M-172+H). + .

[0193] Step 5 (7R,8S)-7-(ethoxycarbonyl)-1,4-dioxaspiro-[4.5]-decane-8-aminium, 4-methylbenzenesulfonate. [ka] To a solution of (7R,8S)-7-(ethoxycarbonyl)-N-((S)-1-phenylethyl)-1,4-dioxaspiro[4.5]decane-8-aminium, 4-methylbenzenesulfonate (100 g, 197 mmol) in ethanol (1 L) was added 10% Pd / C (30.5 g, 28.7 mmol) at room temperature, and the mixture was stirred under 40 psi of hydrogen in an autoclave at 40° C. for 16 hours. The mixture was filtered through Celite®, and the filtrate was evaporated to provide the title compound as a pale yellow gummy solid (78 g, 194 mmol, 98% yield). LC-MS m / z 230.1 (M+H) + .

[0194] Step 6 Ethyl (7R,8S)-8-((S)-2-(((benzyloxy)carbonyl)amino)-4-(methylthio)butanamido)-1,4-dioxaspiro[4.5]decane-7-carboxylate. [ka] To a solution of (7R,8S)-7-(ethoxycarbonyl)-1,4-dioxaspiro[4.5]decane-8-aminium, 4-methylbenzenesulfonate (78 g, 194 mmol) in acetonitrile (800 mL) was added EDC·HCl (37.2 g, 194 mmol), HOBt (29.7 g, 194 mmol), and triethylamine (81 mL, 581 mmol) at 26 °C. Commercially available (S)-2-(((benzyloxy)carbonyl)amino)-4-(methylthio)butanoic acid (54.9 g, 194 mmol) was added, and the mixture was stirred at 26 °C for 16 h. Ice-cold water (700 mL) was added, and the mixture was extracted with ethyl acetate (2 × 800 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated to provide the title compound as a brown liquid (95 g, 187 mmol, 96% yield). LC-MS m / z 495.34 (M+H). + .

[0195] Step 7 ((S)-3-(((benzyloxy)carbonyl)amino)-4-(((7R,8S)-7-(ethoxycarbonyl)-1,4-dioxaspiro[4.5]decan-8-yl)amino)-4-oxobutyl)dimethylsulfonium iodide [ka] To ethyl (7R,8S)-8-((S)-2-(((benzyloxy)carbonyl)amino)-4-(methylthio)butanamido)-1,4-dioxaspiro[4.5]decane-7-carboxylate (95 g, 192 mmol) was added iodomethane (120 mL, 1921 mmol) at 26° C., and the mixture was stirred at 26° C. for 24 hours. The mixture was distilled to give the crude product. The crude product was washed with methyl t-butyl ether (MTBE) (300 mL) to provide the title compound as an off-white solid (115 g, 181 mmol, 94% yield). LC-MS m / z 509.2 (M) + .

[0196] Step 8 Ethyl (7R,8S)-8-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-1,4-dioxaspiro[4.5]decane-7-carboxylate. [ka] To a solution of ((S)-3-(((benzyloxy)carbonyl)amino)-4-(((7R,8S)-7-(ethoxycarbonyl)-1,4-dioxaspiro[4.5]decan-8-yl)amino)-4-oxobutyl)dimethylsulfonium iodide (75 g, 118 mmol) in anhydrous DMSO (100 mL) under a nitrogen atmosphere, finely ground cesium carbonate (42.2 g, 130 mmol) was added in small portions every 2–3 minutes over 15 minutes, and the mixture was stirred for 7 hours. The mixture was filtered and washed with ethyl acetate (300 mL). Ethyl acetate (1000 mL, cold) and brine (420 mL) were added to the filtrate, and the organic phase was washed twice with brine (420 mL) and concentrated under reduced pressure to provide the title compound as an orange oil (39.6 g, 88.8 mmol, 75% yield). LC-MS m / z 447.1(M+H) + .

[0197] Step 9 Ethyl (1R,2S)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-5-oxocyclohexane-1-carboxylate. [ka] A mixture of ethyl (7R,8S)-8-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-1,4-dioxaspiro[4.5]decane-7-carboxylate (47.12 g, 106 mmol), ethyl acetate (156 mL), acetone (255 mL), water (287 mL), and concentrated HCl (13 mL) was stirred at 60 °C for 3 h under a nitrogen atmosphere. The mixture was cooled in an ice bath and stored in the refrigerator overnight. The mixture was concentrated under reduced pressure to a suspension and extracted with dichloromethane (DCM) (1 × 400 mL, 1 × 150 mL). The combined organic extracts were washed with brine (80 mL), dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure. The residue was chased with dichloromethane (DCM) (3 x 100 mL), dried under reduced pressure at 36 °C for 1 h, and placed under vacuum at room temperature for 2 days to provide the title compound as a beige waxy solid (40.0 g, 94%). LC-MS m / z 403.3 (M+H) + .

[0198] Intermediate 2 (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one, dihydrochloride. [ka] Step 1 Ethyl (1R,2S,5R)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexane-1-carboxylate. [ka] To a mixture of (1R,2S)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-5-oxocyclohexane-1-carboxylate (Intermediate 1) (28.0 g, 69.6 mmol) and isopropylmethylamine (12.32 mL, 118 mmol) in dichloromethane (DCM) (270 mL) was added titanium(IV) isopropoxide (30.6 mL, 104 mmol) at room temperature. The mixture was stirred at room temperature under a nitrogen atmosphere for 21 hours. 5% Pt / C (4.07 g, 1.044 mmol) was added, and the mixture was stirred at room temperature under a balloon atmosphere of hydrogen for 29 hours. The hydrogen balloon was refilled after 7 and 16 hours. The mixture was filtered through Celite®, and the catalyst was washed with dichloromethane (DCM). The combined filtrates were concentrated under reduced pressure. The residue was dissolved in dichloromethane (DCM) (80 mL), placed in an ice bath, and ethyl acetate (250 mL) and Celite® (5 g) were added. The mixture was stirred at room temperature for 4 hours and sonicated at room temperature for 20 minutes. The mixture was filtered through Celite® with wet ethyl acetate (4 x 70 mL). The combined filtrates were concentrated under reduced pressure and chased with dichloromethane (DCM) (3 x 100 mL) to provide the title compound as a light brown oily foam (24.58 g, 77%). LC-MS m / z 460.5 (M+H) + .

[0199] Step 2 (1R,2S,5R)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexanecarboxylic acid. [ka] Ethyl (1R,2S,5R)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexane-1-carboxylate (24.58 g, 53.5 mmol) was dissolved in toluene (140 mL) and dichloromethane (DCM) (5 mL), and the solution was extracted with 2 N HCl (2 × 67 mL). The combined aqueous extracts were placed in a metal insert under a nitrogen atmosphere and heated at 63 °C for 22 h. The mixture was placed in an ice bath, and 10 N sodium hydroxide (32 mL) was added. The final temperature of the mixture was 16 °C. The mixture was washed with toluene (150 mL), and the aqueous phase was filtered. The aqueous emulsion (20 mL) was separated and filtered through Celite®. The combined aqueous phases were cooled in an ice bath, and the pH was adjusted to 6-7 with concentrated HCl. The mixture was saturated with NaCl and extracted with dichloromethane (DCM) (200 mL). Concentrated HCl (0.5 mL) was added, and the aqueous phase was extracted with 10% methanol in dichloromethane (DCM) (2 x 100 mL). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to provide the title compound as a beige foamy solid (14.18 g, 2.74 mmol, 61%). LC-MS m / z 432.4 (M+H) + .

[0200] Step 3 tert-Butyl ((1R,2S,5R)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)carbamate. [ka] (1R,2S,5R)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexane-1-carboxylic acid (14.18 g, 32.9 mmol) was chased with dichloromethane (DCM) (30 mL) and toluene (330 mL) (3×). To a solution of the residue in anhydrous toluene (121 mL) under a nitrogen atmosphere, anhydrous tert-butanol (31.0 mL, 329 mmol) and triethylamine (16.0 mL, 115 mmol) were added, and the mixture was heated at 85° C. in a metal insert for 5 min. DPPA (7.79 mL, 36.1 mmol) was added dropwise over 14 min, and the mixture was heated at 85° C. under a nitrogen atmosphere for 2.75 h. The mixture was cooled to room temperature, ethyl acetate (280 mL) was added, and the internal temperature was adjusted to 2-3 °C using an ice bath. Saturated NaHCO3 (280 mL) was added dropwise over 20 min, and the mixture was stirred at room temperature for 2 h. The organic phase was washed with brine (40 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was placed under vacuum at room temperature for 2 days. tert-Butanol (100 mL) was added, and the mixture was sonicated at room temperature for 20 min. 1N sodium hydroxide (80 mL) was added, and the mixture was stirred at room temperature for 30 min and concentrated under reduced pressure to a volume of 80 mL. Dichloromethane (DCM) (200 mL) and water (40 mL) were added, and the organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was chased with ethyl acetate (85 mL) (twice). The residue was stirred in ethyl acetate (30 mL) and heptane (40 mL), collected by filtration, and washed with a 1:10 ethyl acetate / heptane solution to provide the title compound as a white solid (8.03 g, 15.02 mmol, 48.6%). LC-MS m / z 503.5 (M+H). + .

[0201] Step 4 tert-Butyl ((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)carbamate. [ka] To a solution of tert-butyl ((1R,2S,5R)-2-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)carbamate (4.99 g, 9.93 mmol) in ethanol (70 mL) was added 20 wt% Pd(OH)2 / C (0.7 g, 0.993 mmol), and the mixture was stirred at room temperature under a balloon atmosphere of hydrogen for 23 h. The mixture was filtered through Celite® under a nitrogen atmosphere, and the catalyst was washed with ethanol (3 x 10 mL). The combined filtrates were concentrated under reduced pressure, and the residue was placed under vacuum at room temperature overnight to provide the title compound as a white solid (3.64 g, 9.38 mmol, 99% yield). LC-MS m / z 369.4 (M+H) + .

[0202] Step 5 tert-Butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate. [ka] To a solution of tert-butyl ((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)carbamate (3.64 g, 9.88 mmol) in ethanol (100 mL) was added commercially available 4-chloro-6-(trifluoromethyl)quinazoline (2.30 g, 9.88 mmol) and DIPEA (2.77 mL, 15.84 mmol), and the mixture was heated at 50 °C under a nitrogen atmosphere in a metal insert for 3 h. The mixture was concentrated under reduced pressure, dichloromethane (DCM) (150 mL) was added, and the organic phase was washed with water (30 mL). The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by ISCO CombiFlash® Rf (220 g RediSep Rf Gold® column, 120 mL / min) eluting with a gradient of 0-75% DCM / DCM in MeOH containing 1% NH4OH, followed by washing the column with MeOH. The desired fractions were combined and dried under reduced pressure to provide the title compound as a white solid (4.32 g, 7.57 mmol, 77% yield). LC-MS m / z 565.2 (M+H). + .

[0203] Step 6 (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one, dihydrochloride. [ka] To a mixture of tert-butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (3.18 g, 5.63 mmol) in dichloromethane (DCM) (8 mL) was added HCl (7.04 mL, 28.2 mmol). The mixture was stirred at room temperature for 3 hours and concentrated to dryness to provide the title compound as a white solid (3.5 g, 6.51 mmol, 116% yield). LC-MS m / z 465.3 (M+H). + .

[0204] Intermediate 3 (S)-1-((1S,2R,4R)-2-amino-4-(tert-butylamino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one. [ka] Step 1 Benzyl ((S)-1-((7R,8S)-7-acetamido-1,4-dioxaspiro[4.5]decan-8-yl)-2-oxopyrrolidin-3-yl)carbamate. [ka] To a solution of (7R,8S)-8-((S)-3-(((benzyloxy)carbonyl)amino)-2-oxopyrrolidin-1-yl)-1,4-dioxaspiro[4.5]decane-7-carboxylic acid (1.5 g, 3.58 mmol) in toluene (15 mL) was added triethylamine (0.500 mL, 3.58 mmol) at room temperature. The mixture was cooled to -10 °C, isobutyl chloroformate (0.471 mL, 3.58 mmol) was added, and the mixture was stirred at 0 °C to -10 °C for 30 min. A solution of sodium azide (0.419 g, 6.45 mmol) and tetrabutylammonium bromide (0.058 g, 0.179 mmol) in water (3.00 mL) was added, and the mixture was stirred at 0 °C to -10 °C for 2 h. Water (50 mL) and toluene (100 mL) were added, and the mixture was stirred for 10 minutes. The organic phase was dried over molecular sieves (4A), and acetic anhydride (0.744 mL, 7.89 mmol) and acetic acid (0.267 mL, 4.66 mmol) were added, and the mixture was stirred at 90° C. for 4 hours. The mixture was cooled to room temperature and concentrated under reduced pressure. The residue was triturated with pentane (20 mL) to provide the title compound as an off-white solid (0.8 g, 1.714 mmol, 47.8% yield). LC-MS m / z 432.2 (M+H) + .

[0205] Step 2 Benzyl ((S)-1-((1S,2R)-2-acetamido-4-oxocyclohexyl)-2-oxopyrrolidin-3-yl)carbamate. [ka] To a solution of benzyl ((S)-1-((7R,8S)-7-acetamido-1,4-dioxaspiro[4.5]decan-8-yl)-2-oxopyrrolidin-3-yl)carbamate (800 mg, 1.854 mmol) in acetone (10 mL), HCl (5 mL, 5.00 mmol) was added, and the mixture was stirred at 50° C. for 2 hours. The mixture was cooled to room temperature and concentrated. Water (10 mL) was added, and the mixture was extracted with dichloromethane (DCM) (2×50 mL). The combined organic extracts were dried over anhydrous NaSO, filtered, and the filtrate was concentrated. The residue was triturated with diethyl ether (10 mL) to provide the title compound as an off-white solid (600 mg, 1.490 mmol, 80% yield). LC-MS m / z 388.2 (M+H) + .

[0206] Step 3 Benzyl ((S)-1-((1S,2R,4R)-2-acetamido-4-(tert-butylamino)cyclohexyl)-2-oxopyrrolidin-3-yl)carbamate. [ka] TiCl(i-OPr) was preformed by adding titanium(IV) isopropoxide (0.282 mL, 0.964 mmol) to 1 M TiCl in dichloromethane (DCM) (0.964 mL, 0.964 mmol) at 5–10 °C, and the mixture was stirred for 15 min. The preformed TiCl(i-OPr) was added to a solution of benzyl ((S)-1-((1S,2R)-2-acetamido-4-oxocyclohexyl)pyrrolidin-3-yl)carbamate (600 mg, 1.607 mmol) and tert-butylamine (0.851 mL, 8.03 mmol) in dichloromethane (DCM) (10 mL) at –20 °C. The mixture was warmed to room temperature and stirred for 2 h. Borane-dimethyl sulfide complex (0.153 mL, 1.607 mmol) was added, and the mixture was stirred at room temperature for 16 h. Dichloromethane (DCM) (50 mL) and water (50 mL) were added, and the mixture was stirred for 10 min. The emulsion was filtered through Celite®, and the aqueous phase was extracted with dichloromethane (DCM) (50 mL). 1 N HCl (20 mL) was added to the combined organic extracts, and the mixture was stirred for 10 min. Dichloromethane (DCM) (50 mL) was added, and the pH was adjusted to 8-9 with ammonium hydroxide solution. The organic phase was washed with ammonium chloride solution (14%) (2 × 25 mL), dried over anhydrous NaSO, filtered, and the filtrate was evaporated. Purification by column chromatography (neutral alumina column) eluting with 2% methanol in dichloromethane (DCM) provided the title compound as an off-white solid (300 mg, 0.673 mmol, 41.9% yield). LC-MS m / z 445.48 (M+H) + .

[0207] Step 4 N-((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidin-1-yl)-5-(tert-butylamino)cyclohexyl)acetamide. [ka] A mixture of benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-(tert-butylamino)cyclohexyl)-2-oxopyrrolidin-3-yl)carbamate (1.00 g, 2.249 mmol) and 10% Pd / C (100 mg, 0.094 mmol) in methanol (10 mL) was stirred at room temperature under a balloon atmosphere of hydrogen for 2 hours. The mixture was filtered through Celite®, the catalyst was washed with methanol, and the combined filtrates were concentrated under reduced pressure to provide the title compound as an off-white solid (696.1 mg, 2.242 mmol, 100% yield). LC-MS m / z 311.5 (M+H) + .

[0208] Step 5 N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-((2-chloro-6-(trifluoromethyl)quinazolin-4-yl)amino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide. [ka] A mixture of N-((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidin-1-yl)-5-(tert-butylamino)cyclohexyl)acetamide (683 mg, 2.200 mmol), commercially available 2,4-dichloro-6-(trifluoromethyl)quinazoline (587 mg, 2.200 mmol), and DIPEA (0.615 mL, 3.52 mmol) in ethanol (20 mL) was stirred at room temperature for 2 hours, and the mixture was concentrated under reduced pressure. Saturated NaHCO was added, and the mixture was extracted with ethyl acetate. The combined organic extracts were washed with saturated NaCl, dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure to provide the title compound as a pale yellow solid (1.128 g, 2.085 mmol, 95% yield). LC-MS m / z 541.5 (M+H) + .

[0209] Step 6 N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)acetamide. [ka] To a solution of N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-3-((2-chloro-6-(trifluoromethyl)quinazolin-4-yl)amino)-2-oxopyrrolidin-1-yl)cyclohexyl)acetamide (4.61 g, 8.52 mmol) in methanol (150 mL) was added 10% Pd / C (1.360 g, 1.278 mmol) and CsCO (4.16 g, 12.78 mmol). The mixture was stirred at room temperature under a balloon atmosphere of hydrogen for 3 hours. The mixture was filtered, and the catalyst was washed with methanol and dichloromethane (DCM). The combined filtrates were concentrated to dryness, the residue was washed with dichloromethane (DCM), and the combined filtrates were concentrated to dryness to provide the title compound as a solid (4.97 g, 9.81 mmol, 115% yield). LC-MS m / z 507.1(M+H) + .

[0210] Step 7 (S)-1-((1S,2R,4R)-2-amino-4-(tert-butylamino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one. [ka] A mixture of N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)acetamide (814 mg, 1.607 mmol) and 2 M HCl (7231 μL, 14.46 mmol) was stirred at 50° C. for 6 days. The mixture was basified with saturated NaHCO and concentrated to dryness under reduced pressure. The residue was suspended in 10% methanol in dichloromethane (DCM), stirred at room temperature for 1 hour, and filtered. The solid was washed with 10% methanol in dichloromethane (DCM), and the combined filtrates were concentrated. Purification by ISCO CombiFlash® chromatography (80 g RediSep Rf Gold® column, 60 mL min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound as a white solid (300 mg, 0.646 mmol, 40.2% yield). LC-MS m / z 465.4 (M+H). + .

[0211] Intermediate 4 (1r,4R)-4-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride. [ka] Step 1 tert-Butyl ((1R,4r)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)carbamate. [ka] To a mixture of (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one, dihydrochloride salt (Intermediate 2) (368 mg, 0.685 mmol) and commercially available (1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexane-1-carboxylic acid (167 mg, 0.685 mmol) in dichloromethane (DCM) (1 mL), triethylamine (0.573 mL, 4.11 mmol) and HATU (312 mg, 0.822 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 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. Purification by ISCO CombiFlash® chromatography (40 g RediSep Rf Gold® column, 40 mL / min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound (356 mg, 0.511 mmol, 74.6% yield). LC-MS m / z 690.4 (M+H) + .

[0212] Step 2 (1r,4R)-4-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexanecarboxamide, dihydrochloride. [ka] To a mixture of tert-butyl ((1R,4r)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)carbamate (355 mg, 0.515 mmol) in dichloromethane (DCM) (0.5 mL) was added HCl (1.029 mL, 4.12 mmol) and the mixture was stirred at room temperature for 2 hours. The mixture was concentrated to dryness to provide the title compound as a white solid (340 mg, 0.513 mmol, 100% yield). LC-MS m / z 590.3 (M+H + ).

[0213] Intermediate 5 (1s,4S)-4-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride. [ka] Step 1 tert-Butyl ((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)carbamate. [ka] A mixture of tert-butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (Intermediate 2, Step 5) (3.022 g, 5.35 mmol), 4 N HCl in 1,4-dioxane (9.37 mL, 37.5 mmol), and dichloromethane (5 mL) was stirred at room temperature for 4 hours and concentrated to dryness. Commercially available (1s,4s)-4-((tert-butoxycarbonyl)amino)cyclohexane-1-carboxylic acid (1.302 g, 5.35 mmol), dichloromethane (DCM) (30.00 mL), triethylamine (3.73 mL, 26.8 mmol), and HATU (2.442 g, 6.42 mmol) were added, and the mixture was stirred at room temperature 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. Purification by ISCO CombiFlash® chromatography (40 g Redisep Rf Gold® column, 40 mL / min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound (3.35 g, 4.86 mmol, 91% yield). LC-MS m / z 690.4(M+H) + .

[0214] Step 2 (1s,4S)-4-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride. [ka] A mixture of tert-butyl ((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)carbamate (3.35 g, 4.86 mmol), 4 N HCl in 1,4-dioxane (7.28 mL, 29.1 mmol), and dichloromethane (DCM) (3 mL) was stirred at room temperature for 4 hours. The mixture was concentrated to dryness to provide the title compound as a white solid (3.5 g, 5.28 mmol, 109% yield). LC-MS m / z 590.2 (M+H) + .

[0215] The following intermediates were or can be prepared using procedures similar to those described for Intermediates 4 and 5. [Table 3]

[0216] Intermediate 9 (1r,4R)-4-amino-N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride. [ka] Step 1 tert-Butyl ((1R,4r)-4-(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)carbamate. [ka] (S)-1-((1S,2R,4R)-2-amino-4-(tert-butylamino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one (105 mg, 0.226 mmol) and commercially available (1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexane-1-carboxylic acid (55.0 mg, 0.226 mmol) in dichloromethane (DCM) (2 mL). To a mixture of 100 mmol of ammonium hydroxide (10%), triethylamine (0.095 mL, 0.678 mmol) and HATU (103 mg, 0.271 mmol) were added, and the mixture was stirred at room temperature 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. Purification by ISCO CombiFlash® chromatography (24 g RediSep Rf Gold® column, 40 mL min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound (120 mg, 0.174 mmol, 77% yield). LC-MS m / z 690.1 (M+H) + .

[0217] Step 2 (1r,4R)-4-amino-N-((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride. [ka] A mixture of tert-butyl ((1R,4r)-4-(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (70 mg, 0.101 mmol), 4 N HCl in 1,4-dioxane (0.2 mL, 0.800 mmol), and dichloromethane (DCM) (0.1 mL) was stirred at room temperature for 3 hours. The mixture was concentrated to provide the title compound (70 mg, 0.106 mmol, 104% yield). LC-MS m / z 590.0 (M+H). + .

[0218] The following intermediates were or can be prepared using procedures similar to those described for intermediate 9. [Table 4]

[0219] Intermediate 11 (1s,3S)-3-(3-(2-(2-aminoethoxy)ethoxy)propanamido)-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclobutanecarboxamide, dihydrochloride. [ka] Step 1 tert-Butyl (2-(2-(3-(((1S,3s)-3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclobutyl)amino)-3-oxopropoxy)ethoxy)ethyl)carbamate. [ka] To a mixture of (1s,3S)-3-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclobutanecarboxamide, dihydrochloride (225 mg, 0.355 mmol) in dichloromethane (DCM) (2 mL) was added commercially available 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecan-14-oic acid (98 mg, 0.355 mmol), triethylamine (0.396 mL, 2.84 mmol) and HATU (162 mg, 0.426 mmol), and the mixture was stirred at room temperature for 1 h. Saturated NaHCO3 was added, and the mixture was extracted with dichloromethane (DCM). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by ISCO CombiFlash® chromatography (24 g RediSep Rf Gold® column, 35 mL / min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound (283 mg, 0.345 mmol, 97% yield). LC-MS m / z 821.1 (M+H) + .

[0220] Step 2 (1s,3S)-3-(3-(2-(2-aminoethoxy)ethoxy)propanamido)-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclobutane-1-carboxamide, dihydrochloride. [ka] A mixture of tert-butyl (2-(2-(3-(((1S,3s)-3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclobutyl)amino)-3-oxopropoxy)ethoxy)ethyl)carbamate (283 mg, 0.345 mmol), 4 N HCl in 1,4-dioxane (0.862 mL, 3.45 mmol), and dichloromethane (DCM) (0.2 mL) was stirred at room temperature for 3 hours. The mixture was concentrated to dryness to provide the title compound (280 mg, 0.353 mmol, 102% yield). LC-MS m / z 721.5 (M+H) + .

[0221] Intermediate 12 (2S,3S)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid. [ka] Racemic (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (200 g) was dissolved in boiling methanol (4000 mL) and acetonitrile (4000 mL) and purified by chiral preparative HPLC (27 injections) (Chiralpak 1A 101 × 210 mm 20 μm column, 500 mL / min) eluting with acetonitrile / methanol / formic acid (50:50:0.1). The desired fractions were collected and concentrated under reduced pressure. Enantiomer E1 was washed with acetonitrile and dried under high vacuum for 18 hours to provide (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid as a white solid (93.1 g). The other enantiomer was also isolated and characterized (76 g). Analytical chiral HPLC of enantiomer-E1: eluting with acetonitrile / methanol / formic acid (50:50:1) with a retention time of 2.3 min and 96% ee (Chiralpak 1A 5 μm 4.6 × 150 mm, 1.0 ml / min). Enantiomer-E2 had a retention time of 7.2 min and 99% ee. Absolute stereochemistry was assigned using VCD analysis.

[0222] (2S,3S)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (Enantiomer-E1), LC-MS m / z 221.0 (M+H) + .

[0223] (2R,3R)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (enantiomer-E2) LC-MS m / z 221.0 (M+H) + .

[0224] Intermediate 13 (2S,3S)-1-Ethyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid [ka] Racemic 1-((2S,3S)-1-ethyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid) (630 mg) was dissolved in methanol (20 mL) and purified by chiral SFC 80 (Chiralpak IG 20 mm x 250 mm 5 μm, 50 g / min, 3 mL injection volume) eluting with 40% ethanol to provide enantiomer-E1 (2.8-3.4 min) and enantiomer-E2 (5.1-7.4 min). These desired fractions were collected and dried under reduced pressure. Samples were transferred to 20 mL vials and dried at 40 °C under a stream of nitrogen. The chiral purity of each enantiomer was determined using analytical chiral SFC using the method described below.

[0225] Chiral SFC Analysis QC Method Equipment: Thar Investigator(1) Column: Chiralpak IG 4.6×150mm, 5μm Co-solvent: 30% EtOH Flow rate: 3g / min Back pressure: 100 bar UV wavelength: 220nm Temperature: 35℃ Injection volume: 5 μL

[0226] Based on the SFC-UV data, the chiral purity of sample Enantiomer-E1 (room temperature, 2.17 min, 0.21 g) was 100%, and that of Enantiomer-E2 (room temperature, 4.47 min, 0.22 g) was 100%.

[0227] (2S,3S)-1-Ethyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (Enantiomer-E1) LC-MS m / z 235.2(M+H) + .RT=0.26 minutes. 1H NMR(400MHz,methanol-d4)δ ppm 1.02(t,J=7.2Hz,3H)2.64-2.84(m,2 H)2.86-2.96(m,1 H)3.15-3.25(m,1 H)3.63(dd,J = 14,7.3 Hz,1 H)5.08(d,J = 6.1 Hz,1 H)7.54(dd,J = 7.6,4.9 Hz,1 H)7.88(dt,J = 8.0,1.9 Hz,1 H)8.58(td,J = 4.1,1.8 Hz,2 H).

[0228] (2R,3R)-1-Ethyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (enantiomer E-2). LC-MS m / z 235.2(M+H) + RT=0.30min. 1 H NMR(400 MHz,METHANOL-d4)δ ppm 1.02(t,J=7.2 Hz,3 H)2.6-2.83(m,2 H)2.86-2.96(m,1 H)3.15-3.25(m,1 H)3.63(dd,J=14,7.2 Hz,1 H)5.08(d,J=6.1 Hz,1 H)7.54(dd,J=7.8,4.9 Hz,1 H)7.88(dt,J=8.1,2.0 Hz,1 H)8.54-8.62(m,2 H).

[0229] The following intermediates were or can be prepared using procedures similar to those described for intermediate 13. [Table 5]

[0230] Intermediate 16 tert-Butyl 4-(4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)piperidine-1-carboxylate. [ka] Step 1 tert-Butyl 4-(4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohex-1-en-1-yl)-3,6-dihydropyridine-1(2H)-carboxylate. [ka] To a solution of tert-butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (70 mg, 0.124 mmol) in dichloromethane (DCM) (1 mL) was added 3 M HCl in cyclopentyl methyl ether (0.6 mL). The mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The residue was suspended in dichloromethane (DCM) (5 mL) and triethylamine (0.060 mL, 0.434 mmol) was added. 4-(1-(tert-butoxycarbonyl)-1,2,3,6-tetrahydropyridin-4-yl)cyclohex-3-ene-1-carboxylic acid (45 mg, 0.146 mmol) and HATU (70.7 mg, 0.186 mmol) were added. The mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. The residue was dissolved in dimethyl sulfoxide (DMSO) (2 mL) and purified by MDAP chromatography (XSelect™ CSH Prep C18 OBD column, 40 mL / min) eluting with a gradient of 15 to 55% acetonitrile in water, both containing formic acid (0.1%), to provide the title compound as an off-white solid (50 mg, 0.065 mmol, 52.4% yield). LC-MS m / z 754.1(M+H) + .

[0231] Step 2 tert-Butyl 4-(4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)piperidine-1-carboxylate. [ka] To a solution of tert-butyl 4-(4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohex-1-en-1-yl)-3,6-dihydropyridine-1(2H)-carboxylate (50 mg, 0.066 mmol) in methanol (5 mL) was added 10% Pd / C (7.06 mg, 6.63 μmol). The flask was recharged with hydrogen several times and the mixture was stirred at room temperature overnight and at 50° C. over the weekend. The mixture was filtered through Celite® and the filtrate was concentrated under reduced pressure to provide a mixture of trans and cis isomers as a white gummy solid (50 mg, 0.063 mmol, 95% yield). LC-MS m / z 758.5(M+H) + .

[0232] Intermediate 17 3-Ethoxy-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)cyclobutyl-3-ene-1,2-dione. [ka] Commercially available 3,4-diethoxycyclobut-3-ene-1,2-dione (73.3 mg, 0.431 mmol) in THF (1 mL) was stirred at −5° C., and (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one (200 mg, 0.431 mmol) in THF (2 mL) was added, followed by the dropwise addition of DIPEA (0.225 mL, 1.292 mmol). The mixture was allowed to warm to room temperature. Purification by MDAP chromatography (XSelect™ CSH Prep C18 5 μm OBD column, 40 mL / min) eluting with a gradient of 30-85% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) provided the title compound as a white solid (220 mg, 0.336 mmol, 78% yield). LC-MS m / z 589.1 (M+H). + .

[0233] Intermediate 18 (2S,3S)-1-Cyclobutyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid [ka] Step 1 (Z)-N-Cyclobutyl-1-(pyridin-3-yl)methanimine. [ka] A mixture of nicotinaldehyde (0.709 mL, 9.34 mmol) and cyclobutanamine (0.833 mL, 9.34 mmol) was stirred at 100 °C under a nitrogen atmosphere for 25 min. The suspension was filtered through a pad of MgSO and washed with deuterated dichloromethane (CD2Cl2). The product was dried over nitrogen to provide the title compound (0.4517 g, 2.82 mmol, 30.2% yield). 1H NMR(400 MHz, dichloromethane-d2)δ ppm 1.84-1.92(m,2 H)2.13-2.25(m,2 H)2.31-2.40(m,2 H)4.17-4.26(m,1 H)7.37(dd,J=7.8,4.9 Hz,1 H)8.12(dt,J=7.8,2.0 Hz,1 H)8.22(d,J=1.5 Hz,1 H)8.63(dd,J=4.9,1.5 Hz,1 H)8.86(d,J=2.0 Hz,1 H).

[0234] Step 2 (2S,3S)-1-Cyclobutyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid [ka] (Z)-N-Cyclobutyl-1-(pyridin-3-yl)methanimine (0.3372 g, 2.105 mmol) and dihydrofuran-2,5-dione (0.2150 g, 2.148 mmol) were stirred at 150 °C for 1 hour. Ethanol was added, and the precipitate was collected by filtration to provide a mixture of diastereomers (0.1649 g, 0.634 mmol, 30.1% yield). The mixture was combined with additional preparations and separated according to the following method.

[0235] Racemic (2S,3S)-1-cyclobutyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid and (2R,3R)-1-cyclobutyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (1.3 g) were dissolved in acetonitrile / methanol / formic acid (70:30:0.1) (120 mL) and purified by chiral preparative HPLC (Chiralpak 1A 30 × 250 mm 5 μm column, 45 mL / min, 25 mL injection volume) eluting with acetonitrile / methanol / formic acid (70:30:0.1). The desired fractions were collected and concentrated under reduced pressure. These fractions were collected and dried under reduced pressure. The chiral purity of each isomer was determined using analytical chiral UPLC using the method described below.

[0236] Chiral UPLC analytical QC method Equipment: Acquity UPLC Column: CHS C18 30 x 2.1 mm, 1.7 μm Elution solvent: 1-99% gradient of acetonitrile (containing 0.1% formic acid) in water (containing 0.1% formic acid) Flow rate: 1.3mL / min Back pressure: 100 bar UV wavelength: 210~350nm Temperature: 55℃ Injection volume: 0.55 μL

[0237] Based on the chiral UPLC-UV data, the chiral purity of Enantiomer-E1 (3.5 min) (room temperature, 3.5 min, 500 mg) was 100%, and that of Enantiomer-E2 (room temperature, 6.5 min, 450 mg) was 100%.

[0238] Enantiomer E1-a D =+22 degrees (c=0.2, CH3OH), LC-MS m / z 261.2(M+H) + . 1 H NMR(400 MHz,DMSO-d6)δ = 12.87(br s,1H),8.63-8.47(m,2H),7.71(td,J = 2.0,7.8 Hz,1H),7.43(dd,J = 4.6,8.1 Hz,1H),5.09(d,J = 4.4 Hz,1H),4.20-3.98(m,1H),2.95(td,J = 4.8,9.0 Hz,1H),2.84-2.72(m,1H),2.52-2.44(m,1H),2.29(quin,J = 10.0 Hz,1H),2.02-1.78(m,2H),1.74-1.58(m,1H),1.56-1.38(m,2H)

[0239] Intermediate 19 tert-Butyl 4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)-[1,4'-bipiperidine]-1'-carboxylate. [ka] A solution of tert-butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (237 mg, 0.420 mmol) and 3 M HCl in cyclopentyl methyl ether (1 mL, 3.00 mmol) in dichloromethane (DCM) (1 mL) was stirred at room temperature for 1 hour and concentrated under reduced pressure. The residue was suspended in dichloromethane (DCM) (7 mL) containing triethylamine (0.205 mL, 1.469 mmol). 1'-(tert-Butoxycarbonyl)-[1,4'-bipiperidine]-4-carboxylic acid (144 mg, 0.462 mmol) and HATU (239 mg, 0.630 mmol) were added, and the mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. Purification by MDAP chromatography (XSelect™ CSH Prep C18 5 μm OBD column, 40 mL / min) eluting with a gradient of 50-99% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to pH 10 with ammonia, provided the title compound as an off-white solid (267 mg, 0.345 mmol, 82% yield). LC-MS m / z 759.4 (M+H). + .

[0240] Intermediate 20 tert-Butyl 2-(2-(((benzyloxy)carbonyl)amino)ethoxy)acetate. [ka] To a solution of benzyl (2-hydroxyethyl)carbamate (12.0 g, 61.5 mmol) and tert-butyl 2-bromoacetate (23.98 g, 123 mmol) in toluene (200 mL) was added tetrabutylammonium hydrogen sulfate (10.44 g, 30.7 mmol). The reaction mixture was stirred vigorously, and a solution of 30% sodium hydroxide (27.2 mL, 61.5 mmol) was slowly added, and the mixture was stirred overnight. Water was added, and the mixture was extracted with ethyl acetate (3 times). The combined organic extracts were washed with brine (2 times), dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by CombiFlash® Rf chromatography (220 g silica column, 150 mL / min) eluting with a gradient of 0 to 40% ethyl acetate in heptane provided the title compound as a colorless oil (16.48 g, 53.3 mmol, 87% yield). LC-MS m / z 332.0(M+Na) + .

[0241] Intermediate 21 2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)acetic acid, hydrochloride. [ka] Step 1 tert-Butyl 2-(2-aminoethoxy)acetate. [ka] To a solution of tert-butyl 2-(2-(((benzyloxy)carbonyl)amino)ethoxy)acetate (7.08 g, 22.89 mmol) in ethanol under a nitrogen atmosphere was added Degussa-type 10% Pd—C (0.244 g, 2.289 mmol). The mixture was evacuated and refilled with hydrogen (three times). The mixture was stirred under a balloon atmosphere of hydrogen over the weekend and filtered through Celite®. The filtrate was concentrated to provide the title compound as a wax (4.8 g, 22.74 mmol, 99% yield). LC-MS m / z 176.2 (M+H) + .

[0242] Step 2 tert-Butyl 2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)acetate. [ka] To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (400 mg, 1.816 mmol) in N,N-dimethylformamide (DMF) (4.0 mL), DIPEA (1.586 mL, 9.08 mmol), HOBt (278 mg, 1.816 mmol), HATU (1036 mg, 2.72 mmol), and tert-butyl 2-(2-aminoethoxy)acetate (460 mg, 2.180 mmol) were added successively, and the mixture was stirred overnight. The mixture was stirred. Water was added, and the mixture was extracted with ethyl acetate (3 times). The combined organic extracts were washed with brine (2 times), dried over Na2SO4, filtered, and the combined filtrates were concentrated. Purification by CombiFlash® Rf chromatography (40 g silica column, 40 mL / min) eluting with a gradient of 0-20% methanol in dichloromethane (DCM) provided the title compound as a pale wax (675 mg, 1.788 mmol, 98% yield) LC-MS m / z 378.3 (M+H). + .

[0243] Step 3 2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)acetic acid, hydrochloride. [ka] To a solution of tert-butyl 2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)acetate (675 mg, 1.788 mmol) in dichloromethane (DCM) (2.0 mL) was added 4 M HCl in dioxane (5.0 mL, 20.00 mmol), the mixture was stirred for 1 h, concentrated and dried to the title compound as an off-white solid (640 mg, 1.789 mmol, 100% yield). LC-MS m / z 322.1 (M+H) + .

[0244] Intermediate 22 tert-Butyl 2-(2-(2-aminoethoxy)ethoxy)acetate. [ka] Step 1 Benzyl (2-(2-hydroxyethoxy)ethyl)carbamate. [ka] To a solution (50 mL) of 2-(2-aminoethoxy)ethan-1-ol (3.0 g, 28.5 mmol) and triethylamine (3.98 mL, 28.5 mmol) in dichloromethane (DCM) at 0 °C, benzyl carbonochloridate (4.87 g, 28.5 mmol) was added dropwise. The mixture was stirred at 0 °C for 2 h and then at room temperature overnight. Saturated NaHCO was added, and the mixture was extracted with dichloromethane (DCM) (twice). The combined organic extracts were washed with brine (three times), dried over NaSO, filtered, and the filtrate was concentrated. Purification by CombiFlash® Rf chromatography (120 g silica column, 80 mL / min) eluting with a gradient of 50–90% ethyl acetate in heptane provided the title compound (6.48 g, 27.1 mmol, 95% yield) as a colorless oil. LC-MS m / z 240.3(M+H) + .

[0245] Step 2 tert-Butyl 3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecane-12-oate. [ka] To a solution of benzyl (2-(2-hydroxyethoxy)ethyl)carbamate (6.48 g, 27.1 mmol) and tert-butyl 2-bromoacetate (10.57 g, 54.2 mmol) in toluene (100 mL) was added tetrabutylammonium hydrogen sulfate (4.60 g, 13.54 mmol). The reaction mixture was stirred vigorously, and a solution of 30% sodium hydroxide (12.0 mL, 27.1 mmol) was slowly added, and the mixture was stirred overnight. Water was added, and the mixture was extracted with ethyl acetate (3 times). The combined organic extracts were washed with brine (2 times), dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by CombiFlash® Rf chromatography (120 g silica column, 80 mL / min) eluting with a gradient of 0 to 40% ethyl acetate in heptane provided the title compound as a colorless oil (7.25 g, 20.51 mmol, 76% yield). LC-MS m / z 354.2 (M+H) + .

[0246] Step 3 tert-Butyl 2-(2-(2-aminoethoxy)ethoxy)acetate. [ka] To a solution of tert-butyl 3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecane-12-oate (4.20 g, 11.88 mmol) in ethyl acetate (40 mL) under a nitrogen atmosphere was added Degussa-type 10% Pd—C (0.126 g, 1.188 mmol). The reaction mixture was evacuated and refilled with hydrogen (three times) and stirred under a balloon atmosphere of hydrogen over the weekend. The mixture was filtered through Celite®, and the filtrate was concentrated to provide the title compound as a colorless oil (2.65 g, 12.08 mmol, 102% yield). LC-MS m / z 220.3 (M+H) + .

[0247] Intermediate 23 tert-Butyl (2-(2-(3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-3-oxopropoxy)ethoxy)ethyl)carbamate. [ka] To a mixture of (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one, 2-trifluoroacetate (124 mg, 0.179 mmol) and 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecane-14-oic acid (49.7 mg, 0.179 mmol) in dichloromethane (DCM) (1 mL), triethylamine (0.200 mL, 1.432 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. HATU (82 mg, 0.215 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Purification by ISCO CombiFlash® chromatography (12 g RediSep Rf Gold® column, 30 mL / min) eluting with a gradient of 0 to 20% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound as a colorless oil (90 mg, 0.124 mmol, 69.4% yield). LC-MS m / z 724.4 (M+H). + .

[0248] Intermediate 24 (1S,4s)-4-(4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)phenyl)cyclohexane-1-carboxylic acid, hydrochloride [ka] Step 1 4'-(tert-butyl) 4-ethyl (S)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4,4'-dicarboxylate. [ka] A mixture of tert-butyl 4-bromobenzoate (0.5 g, 1.945 mmol), ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-ene-1-carboxylate (0.57 g, 2.034 mmol), sodium carbonate (0.824 g, 7.78 mmol), and tetrakis(triphenylphosphine)palladium(0) (0.225 g, 0.194 mmol) in 1,4-dioxane (10 mL) and water (5.0 mL) was degassed by vacuum / nitrogen backfill cycles and stirred at 80–90 °C for 5 h. The mixture was cooled, poured into water (30 mL), and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated. Purification by ISCO CombiFlash® chromatography (80 g Redisep Rf Gold® column, 60 mL / min) eluting with a step gradient of 0 to 50% ethyl acetate in heptane (25 min) and 50% ethyl acetate in heptane (4 min) provided the title compound as a white solid. LC-MS m / z 275.5 (M+-tBu). + .

[0249] Step 2 tert-Butyl 4-(4-(ethoxycarbonyl)cyclohexyl)benzoate. [ka] A mixture of 4'-(tert-butyl) 4-ethyl 2,3,4,5-tetrahydro-[1,1'-biphenyl]-4,4'-dicarboxylate (451 mg, 1.365 mmol) and 10% Pd / C (145 mg, 0.136 mmol) in ethanol (10 mL) was evacuated and refilled with hydrogen several times. The mixture was stirred at room temperature under a balloon atmosphere of hydrogen for 24 hours, filtered through Celite®, and the filtrate was concentrated to provide the title compound as a colorless viscous liquid (461 mg, 1.317 mmol, 97% yield). 1 H NMR (400 MHz, chloroform-d) δ ppm 1.25-1.36 (m, 3 H), 1.42-1.85 (m, 14 H), 2.00 (br dd, J = 13.8, 3.0 Hz, 1 H), 2.11-2.21 (m, 1 H), 2.22-2.31 (m, 1 H), 2.52-2.68 (m, 1 H), 2.73 (br d, J = 3.0 Hz, 1 H), 4.14-4.25 (m, 2 H), 7.24-7.28 (m, 2 H), 7.90-7.96 (m, 2 H). NMR is consistent for a 1:2 mixture of cis and trans isomers.

[0250] Step 3 Ethyl (1S,4s)-4-(4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)phenyl)cyclohexane-1-carboxylate, formate salt. [ka] A mixture of tert-butyl 4-(4-(ethoxycarbonyl)cyclohexyl)benzoate (120 mg, 0.361 mmol) and 3 M HCl in cyclopentyl methyl ether (1 mL, 3 mmol) in dichloromethane (DCM) (3 mL) was stirred at room temperature overnight and concentrated under reduced pressure to provide 4-(4-(ethoxycarbonyl)cyclohexyl)benzoic acid. A mixture of tert-butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (114.3 mg, 0.202 mmol) and 3 M HCl in cyclopentyl methyl ether (0.5 mL, 1.500 mmol) in dichloromethane (DCM) (1 mL) was stirred at room temperature for 1 hour and concentrated under reduced pressure. The residue was dissolved in dichloromethane (DCM) (5 mL) containing triethylamine (0.15 mL, 1.076 mmol), and 4-(4-(ethoxycarbonyl)cyclohexyl)benzoic acid was added, followed by HATU (148 mg, 0.389 mmol). The mixture was stirred at room temperature overnight and concentrated under reduced pressure. Purification by MDAP chromatography (XSelect™ CSH Prep C18 5 μm OBD column, 40 mL / min) eluting with a gradient of 15-55% acetonitrile in water, both containing formic acid (0.1%), provided the title compound as a white solid. LC-MS m / z 723.1 (M+H) + (36.9 mg, 0.047 mmol, 23.23% yield). 1H NMR(400 MHz,methanol-d4)δ ppm 1.28(t,J = 7.0 Hz,3 H)1.33-1.58(m,7 H)1.76(br t,J = 16.1 Hz,2 H)1.99-2.24(m,6 H)2.26-2.46(m,4 H)2.53(br s,3 H)2.80(br s,3 H)3.56-3.69(m,1 H)3.78-3.96(m,2 H)3.99-4.10(m,1 H)4.15(q,J = 7.1 Hz,2 H)4.38(br s,1 H)4.52(br s,1 H)4.74-4.82(m,1 H)5.50(s,1 H)6.91(br d,J = 6.3 Hz,2 H)7.50(br d,J = 7.8 Hz,2 H)7.79(br d,J = 8.5 Hz,1 H)8.07(br d,J = 8.5 Hz,1 H)8.16(br s,1 H)8.36(br s,2 H)8.69(s,1 H).

[0251] Step 4 (1S,4s)-4-(4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)phenyl)cyclohexane-1-carboxylic acid, hydrochloride. [ka] A mixture of ethyl (1S,4s)-4-(4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)phenyl)cyclohexane-1-carboxylate (36.9 mg, 0.051 mmol) and 2 M HCl (1 mL, 2.000 mmol) was stirred at 50-55° C. for 4 hours and concentrated under reduced pressure to provide the title compound as a white solid (39 mg, 0.050 mmol, 98% yield). LC-MS m / z 695.5 (M+H) + .

[0252] Intermediate 25 1-(4-(ethoxycarbonyl)cyclohexyl)piperidine-4-carboxylic acid, trifluoroacetate. [ka] Triethylamine (1.610 ml, 11.55 mmol) was added to a solution of piperidine-4-carboxylic acid, trifluoroacetate (2.96 g, 12.17 mmol) and ethyl 4-oxocyclohexane-1-carboxylate (1.498 ml, 9.40 mmol) in methanol (72.3 ml), and the mixture was stirred at room temperature for 2 minutes. Picoline borane (1.106 g, 10.34 mmol) was added, and the mixture was placed in a preheated aluminum block at 50°C under a nitrogen atmosphere. The reaction was maintained at 50°C for 1.5 hours and stirred at room temperature overnight. The mixture was heated sequentially to 60°C for 2 hours, 70°C for 2 hours, and 75°C for 1 hour. The mixture was concentrated and dissolved in dichloromethane (DCM). The organic phase was washed with NaHCO3, and the aqueous phase was acidified with 1N HCl. The aqueous and organic extracts were concentrated under reduced pressure. Purification by reverse-phase HPLC (100 g TFA C18 column, 75 mL / min) eluting with a gradient of 0 to 50% acetonitrile in water containing 0.01% TFA provided the title compound as a clear oil (2.26 g, 2.218 mmol, 23.59% yield). LC-MS m / z 284.2 (M+H). + .

[0253] The following intermediates were or can be prepared using procedures similar to those described for intermediate 25. [Table 6]

[0254] Intermediate 28 tert-Butyl ((1s,4s)-4-aminocyclohexane-1-carbonyl)glycinate. [ka] Step 1 tert-Butyl ((1s,4s)-4-(((benzyloxy)carbonyl)amino)cyclohexane-1-carbonyl)glycinate. [ka] To a solution of (1s,4s)-4-(((benzyloxy)carbonyl)amino)cyclohexane-1-carboxylic acid (827 mg, 2.98 mmol), HOBt (548 mg, 3.58 mmol), EDC (858 mg, 4.47 mmol) and tert-butyl glycinate, hydrochloride (500 mg, 2.98 mmol) in dichloromethane (DCM) (10.0 mL), DIPEA (2.60 mL, 14.91 mmol) was added and the mixture was stirred overnight. Saturated NaHCO was added and the mixture was extracted with dichloromethane (DCM) (3 times). The combined organic extracts were washed with brine (2 times), dried over NaSO, filtered, and the filtrate was concentrated. Purification by ISCO CombiFlash® chromatography (40 g RediSep Rf Gold® column, 40 mL / min) eluting with a gradient of 0-20% methanol in dichloromethane (DCM) provided the title compound as a colorless oil (1.16 g, 2.97 mmol, 100% yield). LC-MS m / z 391.3 (M+H) + .

[0255] Step 2 tert-Butyl ((1s,4s)-4-aminocyclohexane-1-carbonyl)glycinate. [ka] To a solution of tert-butyl ((1s,4s)-4-(((benzyloxy)carbonyl)amino)cyclohexane-1-carbonyl)glycinate (1.16 g, 2.97 mmol) in ethyl acetate (20 mL) under a nitrogen atmosphere was added Degussa-type 10% Pd—C (0.032 g, 0.297 mmol). The mixture was evacuated and refilled with hydrogen (three times) and stirred under a balloon atmosphere of hydrogen over the weekend. The mixture was filtered through Celite® and the filtrate was concentrated to provide the title compound as a colorless oil (590 mg, 2.302 mmol, 77% yield). LC-MS m / z 257.3 (M+H) + .

[0256] Intermediate 29 3-Azaspiro[5.5]undecan-9-one, trifluoroacetate. [ka] To a mixture of tert-butyl 9-oxo-3-azaspiro[5.5]undecane-3-carboxylate (336 mg, 1.257 mmol) in dichloromethane (DCM) (1.1 mL) was added TFA (1.1 mL, 14.28 mmol). The mixture was stirred at room temperature for 1 hour and concentrated to provide the title compound. LC-MS m / z 168.2 (M+H) + .

[0257] Intermediate 30 3-(2-(2-(2-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)ethoxy)ethoxy)ethoxy)-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)propenamide. [ka] To a mixture of (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one, dihydrochloride (861 mg, 1.5 mmol), 3-(2-(2-(2-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)ethoxy)ethoxy)ethoxy)propanoic acid (518 mg, 1.500 mmol), and DIPEA (2.62 mL, 15.00 mmol) in dichloromethane (DCM) (5 mL), T3P (1.161 mL, 1.950 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Dichloromethane (DCM) (10 mL) and water (10 mL) were added. The organic layer was dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by MDAP chromatography (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 30-85% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) provided the title compound as a white solid (354 mg, 0.443 mmol, 29.5% yield). LC-MS m / z 792.4 (M+H) + .

[0258] Intermediate 31 tert-Butyl ((1R,4r)-4-(2-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-2-oxoethyl)cyclohexyl)carbamate. [ka] To a suspension of (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one, dihydrochloride (85 mg, 0.158 mmol) in dichloromethane (DCM) (3 mL) was added triethylamine (0.110 mL, 0.791 mmol), followed by 2-((trans)-4-((tert-butoxycarbonyl)amino)cyclohexyl)acetic acid (48.8 mg, 0.190 mmol) and HATU (90 mg, 0.237 mmol). The mixture was stirred at room temperature for 2 hours and concentrated under reduced pressure. Purification by MDAP chromatography (XSelect™ CSH Prep C18 5 μm OBD column, 40 mL / min) eluting with a gradient of 50-99% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a white solid (86 mg, 0.120 mmol, 76% yield). LC-MS m / z 704.3 (M+H) + .

[0259] Intermediate 32 1-Azido-4-iodobutane. [ka] Step 1 4-Azidobutan-1-ol. [ka] A mixture of 4-chlorobutan-1-ol (9.19 mL, 92 mmol) and sodium azide (11.98 g, 184 mmol) in dimethyl sulfoxide (DMSO) (65 mL) was heated at 85 °C overnight, cooled, poured into water (75 mL), and extracted with ethyl acetate (3 × 100 mL). The combined organic extracts were washed with brine, dried over MgSO, and concentrated under reduced pressure. Purification by normal-phase column chromatography (40 g RediSep Rf Gold® column, 60 mL / min) eluting with a stepwise gradient of 10–50% ethyl acetate in hexanes (10 column volumes) and 50% ethyl acetate in hexanes (5 column volumes) provided the title compound as a colorless liquid (9.56 g, 74.7 mmol, 81% yield). 1 H NMR (400 MHz, chloroform-d) δ ppm 1.64-1.77 (m, 5 H) 3.36 (t, J = 6.2 Hz, 2 H) 3.72 (t, J = 6.1 Hz, 2 H).

[0260] Step 2 4-Azidobutyl methanesulfonate. [ka] To a mixture of 4-azidobutan-1-ol (5.9 g, 51.2 mmol), triethylamine (7.14 mL, 51.2 mmol), and 4-dimethylaminopyridine (DMAP) (6.26 g, 51.2 mmol) in dichloromethane (DCM) (40 mL) was added methanesulfonyl chloride (3.99 mL, 51.2 mmol) dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 1 h, poured into ice water (75 mL), and extracted with dichloromethane (DCM) (3 × 100 mL). The combined organic extracts were washed with brine, dried over MgSO4, and concentrated under reduced pressure. Purification by normal-phase column chromatography (40 g RediSep Rf Gold® column, 40 mL / min) provided the title product as a pale yellow liquid. 1H NMR (400 MHz, chloroform-d): δ ppm 1.69-1.80 (m, 2 H), 1.85-1.93 (m, 2 H), 3.05 (s, 3 H), 3.39 (t, J = 6.6 Hz, 2 H), 4.30 (t, J = 6.2 Hz, 2 H).

[0261] Step 3 1-Azido-4-iodobutane [ka] To a solution of 4-azidobutyl methanesulfonate (6.545 g, 33.9 mmol) in acetone (40 mL) was added anhydrous sodium iodide (10.15 g, 67.7 mmol). The reaction mixture was stirred at room temperature overnight and diluted with diethyl ether (150 mL). The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was dissolved in diethyl ether (150 mL). The organic phase was washed with saturated NaSO (100 mL), brine, dried over MgSO, and concentrated under reduced pressure to provide the title compound as a light brown oil (7.6 g, 30.4 mmol, 90% yield). 1 H NMR (400 MHz, chloroform-d) δ ppm 1.68-1.81 (m, 2 H), 1.85-2.02 (m, 2 H), 3.24 (t, J = 6.8 Hz, 2 H), 3.35 (t, J = 6.7 Hz, 2 H).

[0262] Intermediate 33 4-(4-(methoxycarbonyl)piperidin-1-yl)benzoic acid. [ka] Step 1 Methyl 1-(4-(tert-butoxycarbonyl)phenyl)piperidine-4-carboxylate. [ka] DIPEA (10.68 mL, 61.2 mmol) was added to a solution of tert-butyl 4-fluorobenzoate (4 g, 20.39 mmol) and methyl piperidine-4-carboxylate (3.30 mL, 24.46 mmol) in dimethyl sulfoxide (DMSO) (20 mL) at room temperature under a nitrogen atmosphere. The mixture was stirred at 145 °C for 16 hours and cooled to room temperature. Water (100 mL) was added, and the mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic extracts were washed with water (2 × 50 mL), brine (1 × 50 mL), dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure. Purification by Biotage® Isolera™ column chromatography (100 g SNAP® column) eluting with a gradient of 5-20% ethyl acetate in hexanes provided the title compound as a white solid (1.4 g, 4.24 mmol, 20.80% yield). LC-MS m / z 320.2 (M+H) + .

[0263] Step 2 4-(4-(methoxycarbonyl)piperidin-1-yl)benzoic acid. [ka] To a solution of methyl 1-(4-(tert-butoxycarbonyl)phenyl)piperidine-4-carboxylate (500 mg, 1.565 mmol) in dichloromethane (DCM) (10 mL) was added HCl in 1,4 dioxane (1.957 mL, 7.83 mmol) at 0° C. The mixture was stirred at room temperature for 16 hours and concentrated under reduced pressure. The residue was triturated with diethyl ether (20 mL) to provide the title compound as a white solid (0.28 g, 0.957 mmol, 61.1% yield). LC-MS m / z 264.0 (M+H) + .

[0264] Intermediate 34 tert-Butyl 4-((1r,4r)-4-(ethoxycarbonyl)cyclohexyl)piperazine-1-carboxylate. [ka] Acetic acid (3.37 ml) was added to a solution of ethyl 4-oxocyclohexane-1-carboxylate (4.68 ml, 29.4 mmol) and tert-butyl piperazine-1-carboxylate (6.57 g, 35.3 mmol) in dichloromethane (DCM) (49.9 ml) under a nitrogen atmosphere, and the mixture was stirred for 5 minutes. Sodium triacetoxyborohydride (9.60 g, 45.3 mmol) was added. The mixture was stirred at 50°C for 1 hour. Water was added, and the mixture was basified to a pH of 8 with 1 M sodium hydroxide and extracted with dichloromethane (DCM) (3 times). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. This was purified by EZ-Prep using a 275 g Hp C18 Aq column in ACN / water to provide the title compound as a yellow oil (1.609 g, 4.58 mmol, 15.61% yield). LC-MS m / z 341.4 (M+H) + .

[0265] Intermediate 35 4-(1-(tert-butoxycarbonyl)-1,2,3,6-tetrahydropyridin-4-yl)cyclohex-3-ene-1-carboxylic acid. [ka] Step 1 tert-Butyl 4-(4-(ethoxycarbonyl)cyclohex-1-en-1-yl)-3,6-dihydropyridine-1(2H)-carboxylate. [ka] A mixture of tert-butyl 4-(((trifluoromethyl)sulfonyl)oxy)-3,6-dihydropyridine-1(2H)-carboxylate (1.11 g, 3.35 mmol), ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-ene-1-carboxylate (1.0 g, 3.57 mmol), sodium carbonate (1.065 g, 10.05 mmol), and tetrakis(triphenylphosphine)palladium(0) (0.387 g, 0.335 mmol) in 1,4-dioxane (12 mL) and water (4.0 mL) was degassed by vacuum / nitrogen backfill cycles and stirred in a microwave reactor at 80° C. for 3 hours. The mixture was cooled, water (30 mL) was added, and the mixture was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over MgSO4, and the filtrate was concentrated. The residue was purified by normal-phase chromatography (40 g ISCO Gold® column, 45 mL / min) eluting with a stepwise gradient of 0 to 30% ethyl acetate in heptane (9 CV) and 30% ethyl acetate in heptane (5 CV) to provide the title compound as a waxy solid (527.2 mg, 1.493 mmol, 44.6% yield).

[0266] Step 2 4-(1-(tert-butoxycarbonyl)-1,2,3,6-tetrahydropyridin-4-yl)cyclohex-3-ene-1-carboxylic acid. [ka] A mixture of tert-butyl 4-(4-(ethoxycarbonyl)cyclohex-1-en-1-yl)-3,6-dihydropyridine-1(2H)-carboxylate (349 mg, 1.040 mmol) and aqueous 2 N sodium hydroxide (1.5 mL, 3.00 mmol) in tetrahydrofuran (THF) (2 mL) and ethanol (2 mL) was stirred at room temperature overnight and concentrated under reduced pressure to remove the organic solvent. The pH was adjusted to 4-5 with 1 N HCl and stirred for 1 h. The precipitate was collected by filtration, washed with water, and dried in a stream of air to provide the title compound as a beige solid (305 mg, 0.992 mmol, 95% yield). 1 H NMR(400 MHz,DMSO-d6)δ ppm 1.41(s,9 H)1.52-1.63(m,1 H)1.92-2.07(m,1 H)2.10-2.46(m,7 H)3.36-3.48(m,2 H)3.91(br s,2 H)5.70(br s,1 H)5.78(br s,1 H)12.19(br s,1 H).

[0267] The following intermediates were or can be prepared using procedures similar to those described for intermediate 35. [Table 7]

[0268] Intermediate 38 tert-Butyl 3-(2-(2-(isopropylamino)ethoxy)ethoxy)propanoate. [ka] To a solution of tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (1.00 g, 4.29 mmol) in acetone (0.373 g, 6.43 mmol) and dichloromethane (DCM) (20 mL) were added two drops of acetic acid followed by sodium triacetoxyborohydride (2.271 g, 10.72 mmol), and the mixture was stirred overnight. Saturated NaHCO was added, and the mixture was extracted with dichloromethane (DCM) (3 times). The combined organic extracts were washed with brine (2 times), dried over NaSO, filtered, and the filtrate was concentrated to provide the title compound as a colorless oil (1.20 g, 4.36 mmol, 102% yield). LC-MS m / z 276.3 (M+H) + . 1 H NMR(400 MHz,DMSO-d6)δ ppm 0.99(d,J=6.4 Hz,6 H)1.40(s,9 H)2.42(t,J=6.4 Hz,2 H)2.68(t,J=5.9 Hz,2 H)2.72-2.81(m,1 H)3.46(t,J=5.6 Hz,2 H)3.49(s,4 H)3.59(t,J=6.4 Hz,2 H)5.77(s,1 H).

[0269] Intermediate 39 2-(2-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-2-oxoethoxy)acetic acid. [ka] To a solution of (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one, dihydrochloride (50 mg, 0.079 mmol) in dichloromethane (DCM) (0.25 mL) was added dropwise 1,4-dioxane-2,6-dione (10.20 mg, 0.079 mmol) and DIPEA (0.028 mL, 0.158 mmol) dissolved in dichloromethane. The mixture was stirred at room temperature for 1 hour and concentrated to provide the title compound (0.0458 mg, 0.079 mmol, 100% yield). LC-MS m / z 581.0 (M+H) + .

[0270] Intermediate 40 tert-Butyl 4-(piperazine-1-carbonyl)piperazine-1-carboxylate. [ka] To a solution of 1-tert-butyl 4-(4-nitrophenyl)piperazine-1,4-dicarboxylate (5.2 g, 14.80 mmol) in tetrahydrofuran (THF) (148 ml) was added piperazine (2.55 g, 29.6 mmol). The mixture was stirred at 70° C. for 16 hours and concentrated under reduced pressure. Purification by normal phase column chromatography eluting with 10% methanol in dichloromethane provided the title compound as a pale yellow solid (3.3 g, 11.06 mmol, 74.7% yield). LC-MS m / z 299.1 (M+H). + .

[0271] Intermediate 41 9-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-9-oxononyl 4-methylbenzenesulfonate. [ka] To a mixture of (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one, 2-trifluoroacetate salt (154 mg, 0.222 mmol) in dichloromethane (DCM) (1 ml), triethylamine (0.248 mL, 1.779 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. 9-(Tosyloxy)nonanoic acid (73.0 mg, 0.222 mmol) and HATU (101 mg, 0.267 mmol) in DCM (1 ml) were added, and the mixture was stirred at room temperature for 15 minutes. The mixture was partitioned between dichloromethane (DCM) and saturated NaHCO3, and the aqueous layer was extracted with dichloromethane (DCM). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by ISCO CombiFlash® chromatography (12 g RediSep Rf Gold® column, 30 mL / min) eluting with a gradient of 0-50% methanol / dichloromethane (80:20) in dichloromethane (DCM) provided the title compound as a pale yellow oil (120 mg, 0.155 mmol, 69.6% yield). LC-MS m / z 775.2 (M+H) + .

[0272] Example 1 (2S,3S)—N-(2-(((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 (1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexanol. [ka] To a solution of commercially available (1r,4r)-cyclohexane-1,4-diol (2.4 g, 20.66 mmol) in N,N-dimethylformamide (DMF) (20 mL) under a nitrogen atmosphere, sodium hydride (60% in mineral oil) (0.826 g, 20.66 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 30 min. N,N-Dibenzyl-2-chloroethan-1-amine (4.29 g, 16.53 mmol) was added at 0 °C, and the mixture was stirred at 80 °C for 6 h. Ice water (50 mL) was added, and the mixture was extracted with ethyl acetate (3 × 60 mL). The combined organic extracts were dried over NaSO and concentrated under reduced pressure. Purification by Biotage® Isolera™ column chromatography (50 g SNAP® column) eluting with a gradient of 0–20% ethyl acetate in hexane provided the title compound as a pale yellow oil. (2.5g, 7.29mmol, 35.3% yield). LC-MS m / z 340.0(M+H) + .

[0273] Step 2 Methyl (E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoate. [ka] To a solution of (1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexanol (2.5 g, 7.36 mmol) in toluene (30 mL) under a nitrogen atmosphere, acetic acid (0.084 mL, 1.473 mmol), triphenylphosphine (0.097 g, 0.368 mmol), and methyl but-2-ynoate (1.084 g, 11.05 mmol) were added at room temperature. The mixture was stirred at 115 °C for 16 h, cooled to room temperature, and ethyl acetate (100 mL) was added. The organic phase was washed with water (2 × 50 mL), dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure. Purification by Biotage® Isolera™ column chromatography (25 g SNAP® column) eluting with a gradient of 0-30% ethyl acetate in hexanes provided the title compound as a yellow oil (1.77 g, 3.56 mmol, 48.3% yield). LC-MS m / z 438.0 (M+H) + .

[0274] Step 3 Methyl 4-(((1r,4r)-4-(2-aminoethoxy)cyclohexyl)oxy)butanoate. [ka] To a stirred solution of methyl (E)-4-(((1r,4r)-4-(2-(dibenzylamino)ethoxy)cyclohexyl)oxy)but-2-enoate (1.7 g, 3.89 mmol) in methanol (20 mL) at room temperature was added acetic acid (0.445 mL, 7.77 mmol) and 10% Pd / C (600 mg, 0.564 mmol). The resulting reaction mixture was stirred at room temperature under a hydrogen atmosphere for 16 hours. Methanol (50 mL) was added, the mixture was filtered through Celite®, the catalyst was washed with methanol (3×20 mL), and the combined filtrates were concentrated under reduced pressure to provide the title compound as a colorless oil (0.9 g, 3.44 mmol, 88% yield). LC-MS m / z 260.0 (M+H) + .

[0275] Step 4 Methyl 4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanoate. [ka] To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (0.75 g, 3.41 mmol) in N,N-dimethylformamide (DMF) (10 mL) under a nitrogen atmosphere, DIPEA (1.190 mL, 6.81 mmol), HATU (1.942 g, 5.11 mmol), and methyl 4-(((1r,4r)-4-(2-aminoethoxy)cyclohexyl)oxy)butanoate (0.883 g, 3.41 mmol) were added, and the mixture was stirred at room temperature for 16 hours. Water (50 mL) was added, and the mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic extracts were dried over NaSO, filtered, and the filtrate was concentrated. Purification by Biotage® Isolera™ column chromatography (25 g SNAP® column) eluting with 5% methanol in dichloromethane (DCM) provided the title compound as a yellow oil (750 mg, 1.397 mmol, 41.0% yield). LC-MS m / z 462.0 (M+H) + .

[0276] Step 5 4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanoic acid. [ka] To a solution of methyl 4-(((1S,4r)-4-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanoate (700 mg, 1.517 mmol) in methanol (5.0 mL) and water (5.00 mL), lithium hydroxide (54.5 mg, 2.275 mmol) was added and the mixture was stirred at room temperature for 2 hours. The methanol was removed under reduced pressure, the pH was adjusted to 6 with 1.5 N HCl solution, and the mixture was concentrated under reduced pressure. Purification by preparative reverse-phase HPLC (YMC-Triart C18 ExRS 5 μm column, 15 mL / min) eluting with a stepwise gradient of 10-50% and 100% acetonitrile in water containing formic acid (0.1%) provided the title compound as a colorless oil (256 mg, 0.566 mmol, 37.3% yield). LC-MS m / z 448.1 (M+H). + .

[0277] Step 6 (2S,3S)—N-(2-(((1R,4S)-4-(4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)oxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] (1r,4R)-4-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride (59.2 mg, 0.089 mmHg) in dichloromethane (DCM) (1 mL). To a mixture of 4-(((1S,4r)-4-(2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)cyclohexyl)oxy)butanoic acid (40 mg, 0.089 mmol) was added triethylamine (0.075 mL, 0.536 mmol) and HATU (40.8 mg, 0.107 mmol). The mixture was stirred at room temperature for 2 hours and concentrated. Purification by reverse-phase HPLC (XSelect™ CSH Prep C18 5 μm OBD column, 40 mL / min) eluting with a gradient of 30 to 85% acetonitrile containing ammonium hydroxide (1%) in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) provided the title compound as a white solid (56.1 mg, 0.055 mmol, 61.6% yield). LC-MS m / z 1019.1 (M) + . 1H NMR(400 MHz, methanol-d4)δ ppm 1.03(br d,J=6.4 Hz,3 H)1.16(br d,J=6.4 Hz,3 H)1.20-1.34(m,7 H)1.58(q,J=12.2 Hz,2 H)1.67-1.89(m,6 H)1.88-2.04(m,10 H)2.05-2.18(m,2 H)2.18-2.26(m,3 H)2.28(s,3 H)2.31-2.39(m,1 H)2.49-2.59(m,1 H)2.66(s,3 H)2.68-2.76(m,2 H)2.77-2.93(m,1 H)3.02-3.10(m,1 H)3.22-3.31(m,2 H)3.34-3.55(m,9 H)3.56-3.69(m,2 H)4.03(dt,J=12.7,3.7 Hz,1 H)4.66(br d,J=2.4 Hz,1 H)4.87-5.06(m,1 H)5.27(t,J=8.1 Hz,1 H)7.53(dd,J=8.8,4.9 Hz,1 H)7.80(dt,J=7.8,2.0 Hz,1 H)7.91(d,J=8.8 Hz,1 H)8.06(dd,J=8.8,2.0 Hz,1 H)8.51(d,J=2.0 Hz,1 H)8.58(dd,J=4.6,1.7 Hz,1H)8.59(s,1H)8.81(s,1H)

[0278] The following compounds were or can be prepared by procedures similar to those described in Example 1. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10] [Table 8-11] [Table 8-12] [Table 8-13] [Table 8-14] [Table 8-15]

[0279] Example 16 (2S,3S)—N-(3-((2-(2-(3-(((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)ethoxy)amino)-3-oxopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 tert-Butyl 3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanoate. [ka] To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (242 mg, 1.101 mmol) in dichloromethane (DCM) (10.0 mL), commercially available tert-butyl 3-aminopropanoate, hydrochloride (200 mg, 1.101 mmol), HOBt (202 mg, 1.321 mmol), EDC (317 mg, 1.651 mmol), and DIPEA (0.961 mL, 5.50 mmol) were added, and the mixture was stirred overnight. Water was added, and the mixture was extracted with dichloromethane (DCM) (3 times). The combined organic extracts were washed with brine (2 times), dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by CombiFlash® Rf chromatography (40 g silica column, 40 mL / min) eluting with a gradient of 0-20% methanol in dichloromethane (DCM) provided the title compound as a wax (370 mg, 1.065 mmol, 97% yield). LC-MS m / z 348.2 (M+H) + .

[0280] Step 2 tert-Butyl 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,5-dioxo-9,12-dioxa-2,6-diazapentadecan-15-oate. [ka] A mixture of tert-butyl 3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanoate (180 mg, 0.518 mmol), 4 M HCl in 1,4-dioxane (4 mL, 16.00 mmol), and dichloromethane (DCM) (2.0 mL) was stirred for 1 hour and concentrated. Dichloromethane (DCM) (2.0 mL), DIPEA (0.452 mL, 2.59 mmol), and HOBt (79 mg, 0.518 mmol), as well as commercially available tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (121 mg, 0.518 mmol), were added. EDC (149 mg, 0.777 mmol) was added, and the mixture was stirred at room temperature overnight. The mixture was diluted with saturated NaHCO3 and extracted with dichloromethane (DCM) (3 times). The combined organic extracts were washed with brine (2 times), dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by CombiFlash® Rf chromatography (40 g silica column, 40 mL / min) eluting with a gradient of 0-20% methanol in dichloromethane (DCM) provided the title compound as an off-white solid (240 mg, 0.474 mmol, 91% yield). LC-MS m / z 507.2 (M+H) + .

[0281] Step 3 (2S,3S)—N-(3-((2-(2-(3-(((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)ethoxy)amino)-3-oxopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] A mixture of tert-butyl 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,5-dioxo-9,12-dioxa-2,6-diazapentadecan-15-oate (49.5 mg, 0.098 mmol), 4 M HCl in 1,4-dioxane (4.0 mL, 16.00 mmol), and dichloromethane (DCM) (2.0 mL) was stirred for 1 hour and concentrated. Dichloromethane (DCM) (2.0 mL), (1s,4S)-4-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride (60 mg, 0.081 mmol), HOBt (12.48 mg, 0.081 mmol), and DIPEA (0.071 mL, 0.407 mmol) were added. EDC (23.43 mg, 0.122 mmol) was added, and the mixture was stirred at room temperature overnight. Saturated NaHCO was added, and the mixture was extracted with dichloromethane (DCM) (3 times). The combined organic extracts were washed with brine (twice), dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by MDAP chromatography (XSelect™ CSH Prep C18 5 μm OBD column, 40 mL / min) eluting with a gradient of 30 to 85% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) provided the title compound as an off-white solid (60.3 mg, 0.056 mmol, 68.8% yield). LC-MS m / z 1022.0 (M) + . 1H NMR(400 MHz,DMSO-d6)δ ppm 0.89(d,J=6.4 Hz,3 H)1.04(d,J=6.9 Hz,3 H)1.44(br d,J=13 Hz,2 H)1.50-1.81(m,9 H)1.87-2.12(m,5 H)2.14(s,3 H)2.22(t,J=6.9 Hz,2 H)2.28(t,J=6.6 Hz,2 H)2.31-2.47(m,2 H)2.49(s,3 H)2.55-2.62(m,1 H)2.63-2.75(m,1 H)2.86-2.95(m,1 H)3.09-3.32(m,5 H)3.35-3.46(m,6 H)3.47-3.60(m,3 H)3.70(br s,1 H)3.89(br d,J=11 Hz,1 H)4.42(br s,1 H)4.63(d,J=5.9 Hz,1 H)5.03(q,J=8.3 Hz,1 H)7.43(dd,J=7.8,4.9 Hz,1 H)7.56-7.75(m,2 H)7.89(d,J=8.8 Hz,1 H)7.93(t,J=5.6 Hz,1 H)8.00-8.15(m,2 H)8.47(d,J=1.5 Hz,1 H)8.55(dd,J=4.7,1.7 Hz,1 H)8.58(s,1 H)8.62-8.77(m,2 H)8.89(s,1 H).

[0282] Example 17 (2S,3S)—N-(4-((3-(3-(((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)propyl)amino)-4-oxobutyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 tert-Butyl 3-(3-(((benzyloxy)carbonyl)amino)propoxy)propanoate. [ka] To a solution of commercially available benzyl (3-hydroxypropyl)carbamate (15.0 g, 71.7 mmol) and tert-butyl acrylate (13.78 g, 108 mmol) in tetrahydrofuran (THF) (200 mL) was added a solution of KOH (4.02 g, 71.7 mmol) in water (2.67 mL), and the mixture was stirred overnight. Water was added, and the mixture was extracted with ethyl acetate (3 times). The combined organic extracts were washed with brine (2 times), dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by CombiFlash® Rf chromatography (300 g silica column, 200 mL / min) eluting with a gradient of 0 to 40% ethyl acetate in heptane provided the title compound as a colorless oil (10.3 g, 30.5 mmol, 42.6% yield). LC-MS m / z 338.1 (M+H) + .

[0283] Step 2 tert-Butyl 3-(3-aminopropoxy)propanoate. [ka] To a solution of tert-butyl 3-(3-(((benzyloxy)carbonyl)amino)propoxy)propanoate (500 mg, 1.482 mmol) in ethyl acetate (10 mL) under a nitrogen atmosphere was added Degussa-type 10% Pd / C (15.77 mg, 0.148 mmol). The mixture was placed under reduced pressure, the reaction flask was refilled with hydrogen (three times), and the mixture was stirred under a balloon atmosphere of hydrogen overnight. The mixture was filtered through Celite®, and the filtrate was concentrated to provide the title compound as a colorless oil (301 mg, 1.481 mmol, 100% yield). LC-MS m / z 204.2 (M+H) + . 1H NMR(400 MHz,DMSO-d6)δ ppm 1.41(s,8 H)1.50-1.61(m,2 H)2.37-2.43(m,2 H)2.56(t,J=6.8 Hz,2 H)3.41(t,J=6.4 Hz,2 H)3.54(t,J=6.4 Hz,2 H).

[0284] Step 3 tert-Butyl 3,8-dioxo-1-phenyl-2,13-dioxa-4,9-diazahexadecan-16-oate. [ka] To a mixture of commercially available 4-((benzyloxy)carbonyl)amino)butanoic acid (420 mg, 1.771 mmol), tert-butyl 3-(3-aminopropoxy)propanoate (300 mg, 1.476 mmol), DIPEA (1.289 mL, 7.38 mmol), and HOBt (271 mg, 1.771 mmol) in dichloromethane (DCM) (4.0 mL), EDC (424 mg, 2.214 mmol) was added and the mixture was stirred overnight. The mixture was diluted with water and extracted with dichloromethane (DCM) (3 times). The combined organic extracts were washed with brine (2 times), dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by CombiFlash® Rf chromatography (40 g silica column, 40 mL / min) eluting with a gradient of 0-20% methanol in dichloromethane (DCM) provided the title compound as a colorless oil (563 mg, 1.332 mmol, 90% yield). LC-MS m / z 423.2 (M+H) + .

[0285] Step 4 tert-Butyl 3-(3-(4-aminobutanamido)propoxy)propanoate. [ka] To a solution of tert-butyl 3,8-dioxo-1-phenyl-2,13-dioxa-4,9-diazahexadecan-16-oate (560 mg, 1.325 mmol) in ethyl acetate (5.0 mL) under a nitrogen atmosphere was added Degussa-type 10% Pd / C (14.10 mg, 0.133 mmol). The mixture was placed under reduced pressure, the reaction flask was refilled with hydrogen (three times), and the mixture was stirred overnight under a balloon atmosphere of hydrogen. The mixture was filtered through Celite®, and the filtrate was concentrated to provide the title compound as a colorless oil (382 mg, 1.325 mmol, 100% yield). LC-MS m / z 289.2 (M+H) + .

[0286] Step 5 tert-Butyl 3-(3-(4-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)butanamido)propoxy)propanoate. [ka] To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (240 mg, 1.090 mmol) in dichloromethane (10.0 mL) was added commercially available tert-butyl 3-(3-(4-aminobutanamido)propoxy)propanoate (377 mg, 1.308 mmol), HOBt (200 mg, 1.308 mmol), and EDC (313 mg, 1.635 mmol). DIPEA (0.952 mL, 5.45 mmol) was added, and the mixture was stirred overnight. Water was added, and the mixture was extracted with dichloromethane (DCM) (3 times). The combined organic extracts were washed with brine (2 times), dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by CombiFlash® Rf chromatography (40 g silica column, 40 mL / min) eluting with a gradient of 0-20% methanol in dichloromethane (DCM) provided the title compound as a wax (480 mg, 0.978 mmol, 90% yield). LC-MS m / z 491.1 (M+H) + .

[0287] Step 6 (2S,3S)—N-(4-((3-(3-(((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)propyl)amino)-4-oxobutyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka] A mixture of tert-butyl 3-(3-(4-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)butanamido)propoxy)propanoate (62.2 mg, 0.127 mmol), 4 M HCl in 1,4-dioxane (4.0 mL, 16.00 mmol), and dichloromethane (2.0 mL) was stirred for 1 hour and concentrated. Dichloromethane (DCM) (2.0 mL), (1s,4S)-4-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride (60 mg, 0.091 mmol), HOBt (16.64 mg, 0.109 mmol), DIPEA (0.079 mL, 0.453 mmol), and EDC (26.0 mg, 0.136 mmol) were added, and the mixture was stirred at room temperature overnight. Saturated NaHCO was added, and the mixture was extracted with dichloromethane (DCM) (3 times). The combined organic extracts were washed with brine (twice), dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by MDAC chromatography (XSelect™ CSH Prep C18 5 μm OBD column, 40 mL / min) eluting with a gradient of 30 to 85% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) provided the title compound as an off-white solid (52.8 mg, 0.050 mmol, 55.1% yield). LC-MS m / z 1006.0 (M) + . 1H NMR(400 MHz,DMSO-d6)δ ppm 0.89(d,J=6.4 Hz,3 H)1.04(d,J=6.8 Hz,3 H)1.36-1.49(m,2 H)1.51-1.76(m,13 H)1.88-2.12(m,7 H)2.14(s,3 H)2.27(t,J=6.4 Hz,2 H)2.31-2.39(m,1 H)2.40-2.49(m,2 H)2.56-2.62(m,1 H)2.67-2.74(m,1 H)2.89(ddd,J=9.5,7.3,6.1 Hz,1 H)3.02(quin,J=6.8 Hz,3 H)3.23-3.29(m,1 H)3.30(s,1 H)3.42(br s,1 H)3.47-3.58(m,3 H)3.71(br s,1 H)3.86-3.93(m,1 H)4.42(br s,1 H)4.65(d,J=6.4 Hz,1 H)5.03(q,J=8.2 Hz,1 H)7.44(dd,J=7.6,5.1 Hz,1 H)7.63(d,J=6.8 Hz,1 H)7.68(dt,J=8.1,1.8 Hz,1 H)7.72(t,J=5.4 Hz,1 H)7.89(d,J=8.8 Hz,1 H)7.99(br t,J=5.6 Hz,1 H)8.07(dd,J=8.8,2.0 Hz,1 H)8.48(d,J=1.5 Hz,1 H)8.56(dd,J=4.9,1.5 Hz,1 H)8.58(s,1 H)8.71(br d,J=8.3 Hz,2 H)8.89(s,1 H).

[0288] Example 18 N 1 -((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)-N 5 -(3-(3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propoxy)propyl)glutaramide. [ka] Step 1 N 1 -(3-(3-aminopropoxy)propyl)-N 5 -((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)glutaramide. [ka] To a suspension of (1s,4S)-4-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride (62 mg, 0.089 mmol) in dichloromethane (DCM) (3 mL) was added triethylamine (0.1 mL, 0.717 mmol) and commercially available dihydro-2H-pyran-2,6(3H)-dione (10.12 mg, 0.089 mmol), and the mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure, and N,N-dimethylformamide (DMF) (1.0 mL), commercially available 3,3'-oxybis(propan-1-amine), and HATU (43 mg, 0.113 mmol) were added, and the mixture was stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure. Purification by MDAP chromatography (XSelect™ CSH C18 5 μm column) eluting with a gradient of 30-85% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a white foamy solid (24 mg, 0.028 mmol, 31.1% yield). LC-MS m / z 818.3 (M+H) + .

[0289] Step 2 N 1-((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)-N 5 -(3-(3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propoxy)propyl)glutaramide. [ka] N in dichloromethane (DCM) (2 mL) 1 -(3-(3-aminopropoxy)propyl)-N 5 A solution of -((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)glutaramide (24 mg, 0.029 mmol) was added to triethylamine (0.012 mL, 0.088 mmol), (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (9.69 mg, 0.044 mmol), and HATU (13.39 mg, 0.035 mmol) at room temperature for 1 hour and concentrated. Purification by MDAP chromatography (XSelect™ CSH C18 5 μm column) eluting with a gradient of 30-85% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a white solid (21 mg, 0.020 mmol, 68.8% yield). LC-MS m / z 1020.3 (M) + . 1H NMR(400 MHz, methanol-d4)δ ppm 1.00(d,J=6.4 Hz,3 H)1.16(d,J=6.4 Hz,3 H)1.52-2.02(m,19 H)2.09-2.40(m,11 H)2.53-2.63(m,1 H)2.66(s,3 H)2.67-2.75(m,2 H)2.79-2.89(m,1 H)3.05(ddd,J=9.3,8.1,6.6 Hz,1 H)3.16-3.37(m,9 H)3.39-3.67(m,5 H)3.89(br s,1 H)4.03(dt,J=12.2,3.7 Hz,1 H)4.61(br d,J=3.4 Hz,1 H)4.82(d,J=6.4 Hz,1 H)5.30(t,J=8.6 Hz,1 H)7.51-7.56(m,1 H)7.80(dt,J=7.8,2.0 Hz,1 H)7.91(d,J=8.8 Hz,1 H)8.06(dd,J=8.8,2.0 Hz,1 H)8.51(d,J=1.5 Hz,1 H)8.57(dd,J=4.9,1.5 Hz,1 H)8.59(s,1 H)8.73(s,1 H).

[0290] The following compounds were or can be prepared by procedures similar to those described in Example 18. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0291] Example 23 (2S,3S)—N-(2-(2-(2-(3-(((1S,4s)-4-(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)ethoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 tert-Butyl 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oate. [ka] To a mixture of commercially available tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate (2 g, 7.21 mmol), (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (1.588 g, 7.21 mmol), and triethylamine (3.02 mL, 21.63 mmol) in dichloromethane (DCM) (20 mL), HATU (3.29 g, 8.65 mmol) was added, and the mixture was stirred for 72 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. Purification by ISCO CombiFlash® chromatography (120 g RediSep Rf Gold® column, 80 mL / min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound (1.91 g, 3.98 mmol, 55.2% yield). LC-MS m / z 480.2 (M+H). + .

[0292] Step 2 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, hydrochloride, [ka] To a mixture of tert-butyl 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oate (1.9 g, 3.96 mmol) in dichloromethane (DCM) (5 mL) at 0° C. was added 4 M HCl (4.95 mL, 19.81 mmol). The mixture was stirred at room temperature for 2 hours and concentrated to dryness to provide the title compound (1.8 g, 3.91 mmol, 99% yield). LC-MS (m / z) 424.2 (M+H). + .

[0293] Step 3 (2S,3S)—N-(2-(2-(2-(3-(((1S,4s)-4-(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)ethoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] tert-Butyl ((1S,4s)-4-(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (56 mg, 0.081 mmol) in dichloromethane (DCM) (0.1 mL) and 4M The mixture was stirred at room temperature for 1 hour, stored in a freezer overnight, and stirred at room temperature for 1 hour. The mixture was concentrated to dryness, and dichloromethane (DCM) (1 mL) was added. 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, hydrochloride salt (37.3 mg, 0.081 mmol), triethylamine (0.091 mL, 0.649 mmol), and HATU (37.0 mg, 0.097 mmol) were added, and the mixture was stirred at room temperature for 1 hour. MDAP chromatography (XSelect™ CSH C18) eluted with a gradient of 30–85% acetonitrile in water containing ammonium bicarbonate (10 mM) was performed. Purification by elution with a 5 μm column (40 mL / min) and adjusting the pH to 10 with ammonia provided the title compound as a white solid (43.2 mg, 0.043 mmol, 53.5% yield). LC-MS m / z 997.0 (M+H) + . 1H NMR(400 MHz, methanol-d4)δ ppm 1.18(s,9 H)1.52-2.09(m,15 H)2.21-2.37(m,2 H)2.41(t,J=6.3 Hz,2 H)2.53-2.63(m,1 H)2.66(s,3 H)2.67-2.75(m,1 H)2.77-2.90(m,1 H)3.05(ddd,J=9.3,8.0,6.5 Hz,1 H)3.23(br s,1 H)3.27-3.32(m,1 H)3.34-3.40(m,1 H)3.41-3.58(m,12 H)3.58-3.65(m,1 H)3.68(t,J=6.4 Hz,2 H)3.87-3.94(m,1 H)4.00(dt,J=12.5,3.6 Hz,1 H)4.59(br d,J=3.0 Hz,1 H)5.32(t,J=8.7 Hz,1 H)7.49-7.54(m,1 H)7.79(dt,J=8.0,1.9 Hz,1 H)7.91(d,J=8.8 Hz,1 H)8.06(dd,J=8.8,1.8 Hz,1 H)8.51(d,J=1.8 Hz,1 H)8.56(dd,J=4.8,1.5 Hz,1 H)8.59(s,1 H)8.73(s,1 H).

[0294] The following compounds may be prepared by procedures similar to those described in Example 23. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8] [Table 10-9] [Table 10-10] [Table 10-11] [Table 10-12]

[0295] Example 36 (2S,3S)-N-(27-(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka] 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-octaoxa-2-azanonacosan-29-oic acid, trifluoroacetate [ka] To a mixture of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (252 mg, 1.144 mmol) and N,N-dimethylformamide (DMF) (1 mL), DIPEA (0.600 mL, 3.43 mmol) and 2-(2,5-dioxopyrrolidin-1-yl)-1,3,3-tetramethylisolonium tetrafluoroborate (344 mg, 1.144 mmol) were added, and the mixture was stirred at room temperature for 2 hours. A slurry of commercially available 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oic acid (505 mg, 1.144 mmol) in N,N-dimethylformamide (DMF) (1 mL) was added, and the mixture was stirred at room temperature for 2 hours. Purification by Gilson® reverse-phase chromatography (acidic Luna® column, 47 mL / min) eluting with a gradient of 5 to 25% acetonitrile in water containing TFA (0.1%) provided the title compound as a colorless oil (865 mg, 1.142 mmol, 100% yield). LC-MS m / z 644.2 (M+H) + .

[0296] Step 2 (2S,3S)-N-(27-(((1R,2S,5R)-5-(tert-butylamino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka] To a mixture 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-octaoxa-2-azanonacosan-29-oic acid, trifluoroacetate (860 mg, 1.135 mmol) and dichloromethane (DCM) (2 mL) was added (S)-1-( (1S,2R,4R)-2-amino-4-(tert-butylamino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one (527 mg, 1.135 mmol), DIPEA (0.793 mL, 4.54 mmol), and HATU (518 mg, 1.362 mmol) were stirred at room temperature for 1 hour. 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. Purification by ISCO CombiFlash® chromatography (80 g RediSep Rf Gold® column) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound (800 mg, 0.734 mmol, 64.7% yield). LC-MS m / z 545.9(M / 2) + . 1H NMR(400 MHz, methanol-d4)δ ppm 1.20(s,9 H)1.30(br d,J=6.4 Hz,4 H)1.69-1.80(m,2 H)1.82-1.93(m,4 H)2.26(qd,J=12.3,5.5 Hz,1 H)2.47-2.53(m,3 H)2.67(s,3 H)2.68-2.76(m,1 H)2.78-2.95(m,1 H)3.02-3.09(m,1 H)3.22-3.27(m,1 H)3.37(s,12 H)3.48-3.59(m,18 H)3.68-3.77(m,2 H)3.85(ddd,J=9.5,7.5,5.0 Hz,1 H)4.02(dt,J=12.4,3.7 Hz,1 H)4.64(br d,J=2.9 Hz,1 H)4.83(d,J=6.6 Hz,2 H)5.31(t,J=8.1 Hz,1 H)7.51-7.56(m,1 H)7.81(dt,J=8.0,1.9 Hz,1 H)7.91(d,J=8.8 Hz,1 H)8.06(dd,J=8.8,2.0 Hz,1 H)8.52(d,J=1.7 Hz,1 H)8.58(dd,J=4.9,1.7 Hz,1 H)8.60(s,1 H) 8.86 (d, J = 0.7 Hz, 1 H).

[0297] The following compounds were or can be prepared by procedures similar to those described in Example 36. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6] [Table 11-7]

[0298] Example 44 (2S,3S)—N-(2-(3-((2-(3-(((1S,3s)-3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclobutyl)amino)-3-oxopropoxy)ethyl)-3-oxopropoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 tert-Butyl 3-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)propanoate. [ka] To a mixture of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (235 mg, 1.067 mmol) and commercially available tert-butyl 3-(2-aminoethoxy)propanoate (202 mg, 1.067 mmol) in dichloromethane (DCM) (4 mL) was added HATU (487 mg, 1.281 mmol) and triethylamine (0.446 mL, 3.20 mmol), and the mixture was stirred at room temperature for 3 h. Purification by ISCO CombiFlash® chromatography (40 g RediSep Rf Gold® column, 40 mL / min) eluting with a gradient of 0 to 15% methanol containing ammonium chloride (10%) in dichloromethane (DCM) provided the title compound (465 mg, 1.188 mmol, 111% yield). LC-MS m / z 392.3(M+H) + .

[0299] Step 2 3-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)propanoic acid, hydrochloride. [ka] A mixture of tert-butyl 3-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)propanoate (465 mg, 1.188 mmol), 4N HCl 1,4-dioxane (1.782 mL, 7.13 mmol), and dichloromethane (0.5 mL) was stirred at room temperature for 4 hours. The mixture was concentrated to dryness to provide the title compound (450 mg, 1.210 mmol, 102% yield). LC-MS m / z 336.1 (M+H) + .

[0300] Step 3 tert-Butyl 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,8-dioxo-5,12-dioxa-2,9-diazapentadecan-15-oate. [ka] To a mixture of commercially available tert-butyl 3-(2-aminoethoxy)propanoate (148 mg, 0.780 mmol) and 3-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)propanoate, hydrochloride (290 mg, 0.780 mmol) in dichloromethane (DCM) (2 mL), EDC (224 mg, 1.170 mmol), HOBt (179 mg, 1.170 mmol), and DIPEA (0.817 mL, 4.68 mmol) were added, and the mixture was stirred at room temperature overnight. 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. Purification by reverse-phase chromatography (50 g C18 Aq Gold column, 40 mL / min) eluting with a gradient of 20 to 55% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) provided the title compound (225 mg, 56.9%). LC-MS m / z 507.1 (M+H) + .

[0301] Step 4 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,8-dioxo-5,12-dioxa-2,9-diazapentadecan-15-oic acid, hydrochloride. [ka] A mixture of tert-butyl 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,8-dioxo-5,12-dioxa-2,9-diazapentadecan-15-oate (225 mg, 0.444 mmol), 4N HCl in 1,4-dioxane (0.666 mL, 2.66 mmol), and dichloromethane (DCM) (0.02 mL) was stirred at room temperature for 3 hours. The mixture was concentrated to provide the title compound (225 mg, 0.462 mmol, 104% yield). LC-MS m / z 451.4 (M+H) + .

[0302] Step 5 (2S,3S)—N-(2-(3-((2-(3-(((1S,3s)-3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclobutyl)amino)-3-oxopropoxy)ethyl)amino)-3-oxopropoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, [ka] (1s,3S)-3-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclobutane-1-carboxamide, dihydrochloride (52.1 mg, 0.082 mmol) and 1-((2S,3S)-1-methyl)cyclobutane-1-carboxamide, dihydrochloride (52.1 mg, 0.082 mmol) in dichloromethane To a mixture of ethyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,8-dioxo-5,12-dioxa-2,9-diazapentadecan-15-oic acid hydrochloride (40 mg, 0.082 mmol), DIPEA (115 μL, 0.657 mmol), EDC (23.62 mg, 0.123 mmol), and HOBt (18.87 mg, 0.123 mmol) were added. The mixture was stirred at room temperature overnight and concentrated. Purification by MDAP (XSelect™ CSH Prep C18 5 μm OBD column, 40 mL / min) eluting with a gradient of 30-85% acetonitrile containing ammonium hydroxide (1%) in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) provided the title compound as a white solid (47.8 mg, 0.048 mmol, 58.5% yield). LC-MS m / z 994.6 (M+H). + . 1H NMR(400 MHz, methanol-d4)δ ppm 1.02(d,J=6.4 Hz,3 H)1.14(br d,J=6.4 Hz,3 H)1.59-1.81(m,3 H)1.88-2.02(m,1 H)2.10-2.24(m,5 H)2.26(s,3 H)2.27-2.45(m,6 H)2.45-2.63(m,4 H)2.66(s,3 H)2.68-2.89(m,5 H)3.05(ddd,J=9.3,8.1,6.6 Hz,1 H)3.26-3.31(m,2 H)3.34-3.57(m,7 H)3.60-3.70(m,5 H)4.02(dt,J=12.2,3.7 Hz,1 H)4.11-4.32(m,1 H)4.59(br d,J=2.9 Hz,1 H)4.82(d,J=6.8 Hz,1 H)5.32(t,J=8.8 Hz,1 H)7.53(dd,J=7.3,4.9 Hz,1 H)7.79(dt,J=7.8,2.0 Hz,1 H)7.91(d,J=8.8 Hz,1 H)8.07(dd,J=8.8,2.0 Hz,1 H)8.51(d,J=2.0 Hz,1 H)8.57(dd,J=4.9,1.5 Hz,1 H)8.59(s,1 H)8.75(s,1 H)

[0303] The following compounds were prepared or can be prepared using procedures similar to those described in Example 44. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4]

[0304] Example 49 (2S,3S)—N-(3-((2-(2-(3-(((1S,3s)-3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclobutyl)amino)-3-oxopropoxy)ethoxy)amino)-3-oxopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka] Step 1 tert-Butyl 3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanoate. [ka] To a mixture of commercially available tert-butyl 3-aminopropanoate hydrochloride (267 mg, 1.471 mmol) and (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (324 mg, 1.471 mmol) in dichloromethane (DCM) (3 mL), triethylamine (1.025 mL, 7.36 mmol) and HATU (671 mg, 1.765 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 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. Purification by ISCO CombiFlash® chromatography (40 g RediSep Rf Gold® column, 40 mL / min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound (525 mg, 1.511 mmol, 103% yield). LC-MS m / z 348.1 (M+H). + .

[0305] Step 2 3-((2S,3S)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanoic acid, hydrochloride. [ka] A mixture of tert-butyl 3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanoate (525 mg, 1.511 mmol), 4N HCl in 1,4-dioxane (3.02 mL, 12.09 mmol), and dichloromethane (0.5 mL) was stirred at room temperature for 3 hours. The mixture was concentrated to provide the title compound (560 mg, 1.537 mmol, 102% yield). LC-MS m / z 292.1 (M+H) + .

[0306] Step 3 (2S,3S)—N-(3-((2-(2-(3-(((1S,3s)-3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclobutyl)amino)-3-oxopropoxy)ethoxy)amino)-3-oxopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka] (1s,3S)-3-(3-(2-(2-aminoethoxy)ethoxy)propanamido)-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclobutane-1-carboxamide in dichloromethane (DCM) (1 mL) To a mixture of boxamide dihydrochloride (67 mg, 0.084 mmol) and 3-(2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanoic acid, hydrochloride (27.7 mg, 0.084 mmol) was added triethylamine (0.094 mL, 0.675 mmol) and HATU (38.5 mg, 0.101 mmol). The mixture was stirred at room temperature for 3 h and concentrated. Purification by MDAP (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 30–85% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a white solid (45.7 mg, 0.046 mmol, 54.5% yield). LC-MS m / z 498.5(M / 2+H) + . 1H NMR(400 MHz,methanol-d4)δ ppm 1.03(br d,J=5.9 Hz,3 H)1.15(br d,J=6.4 Hz,3 H)1.61-1.81(m,3 H)1.89-2.04(m,1 H)2.08-2.25(m,4 H)2.27(s,3 H)2.30-2.46(m,5 H)2.46-2.65(m,3 H)2.64-2.91(m,7 H)2.96-3.07(m,1 H)3.26-3.31(m,1 H)3.35-3.64(m,11 H)3.68(br t,J=6.1 Hz,3 H)4.03(br d,J=12.2 Hz,1 H)4.12-4.35(m,1 H)4.60(br s,1 H)5.30(t,J=8.8 Hz,1 H)7.52(dd,J=7.8,4.9 Hz,1 H)7.78(br d,J=7.8 Hz,1 H)7.91(d,J=8.8 Hz,1 H)8.06(br d,J=9.3 Hz,1 H)8.51(s,1 H)8.57(d,J=4.9 Hz,1 H)8.59(s,1 H)8.75(s,1 H).

[0307] The following compounds were prepared or can be prepared using procedures similar to those described in Example 49. [Table 13-1] [Table 13-2]

[0308] Example 52 (2S,3S)—N-(3-(((1R,4S)-4-(4-(((1S,4S)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)amino)-3-oxopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka] Step 1 tert-Butyl (3-(((1r,4r)-4-hydroxycyclohexyl)amino)-3-oxopropyl)carbamate. [ka] To a mixture of commercially available (1r,4r)-4-aminocyclohexan-1-ol (1.2 g, 10.42 mmol) and 3-((tert-butoxycarbonyl)amino)propanoic acid (1.26 g, 6.66 mmol) in dichloromethane (DCM) (50 mL), triethylamine (3.71 mL, 26.6 mmol), EDC (1.532 g, 7.99 mmol), and HOBt (1.530 g, 9.99 mmol) were added, and the mixture was stirred at room temperature overnight. 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. Purification by ISCO CombiFlash® chromatography (40 g RediSep Rf Gold® column, 40 mL / min) eluting with a gradient of 0-15% methanol in dichloromethane (DCM) provided the title compound (900 mg, 3.14 mmol, 47.2% yield). LC-MS m / z 287.2 (M+H) + .

[0309] Step 2 Ethyl (E)-4-(((1r,4r)-4-(3-((tert-butoxycarbonyl)amino)propanamido)cyclohexyl)oxy)but-2-enoate. [ka] To a mixture of tert-butyl (3-(((1r,4r)-4-hydroxycyclohexyl)amino)-3-oxopropyl)carbamate (317 mg, 1.107 mmol), triphenylphosphine (29.0 mg, 0.111 mmol), and acetic acid (0.056 mL, 0.978 mmol) in toluene (5 mL), commercially available ethyl but-2-ynoate (0.334 mL, 2.87 mmol) was added, and the mixture was stirred at 110 °C overnight. Water was added, and the mixture was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over Na2SO4, filtered, and the filtrate was concentrated. The product was purified via ISCO CombiFlash® chromatography (40 g RediSep Rf Gold® column, 40 mL / min) eluting with a gradient of 0-15% methanol in dichloromethane (DCM) to provide the title compound (163 mg, 0.409 mmol, 37.0% yield). LC-MS m / z 399.2 (M+H) + . 1 H NMR (400 MHz, methanol-d₄) δ ppm 1.22-1.41 (m, 7 H), 1.45 (s, 9 H), 1.95 (br dd, J = 13.4, 2.2 Hz, 2 H), 2.04-2.15 (m, 2 H), 2.34 (t, J = 6.8 Hz, 2 H), 3.27-3.31 (m, 2 H), 3.35-3.39 (m, 1 H), 3.66 (t, J = 10.8, 3.9 Hz, 1 H), 4.15-4.24 (m, 4 H), 6.06 (dt, J = 15.6, 2.2 Hz, 1 H), 6.98 (dt, J = 15.6, 4.2 Hz, 1 H). (The cis isomer was observed by NMR.)

[0310] Step 3 (E)-4-(((1r,4r)-4-(3-((tert-butoxycarbonyl)amino)propanamido)cyclohexyl)oxy)but-2-enoic acid. [ka] A mixture of (E)-4-(((1r,4r)-4-(3-((tert-butoxycarbonyl)amino)propanamido)cyclohexyl)oxy)but-2-enoate (163 mg, 0.409 mmol) and sodium hydroxide (0.409 mL, 2.045 mmol) in methanol (0.4 mL) was stirred at room temperature for 2 hours. The mixture was concentrated, acidified with HCl (0.409 mL, 2.454 mmol), and extracted with methanol (5%) in dichloromethane (DCM). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated to provide the title compound as a white solid (130 mg, 0.351 mmol, 86% yield). LC-MS m / z 371.1 (M+H) + .

[0311] Step 4 tert-Butyl (3-(((1R,4r)-4-(((E)-4-(((1S,4S)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobut-2-en-1-yl)oxy)cyclohexyl)amino)-3-oxopropyl)carbamate. [ka] To a mixture of (1s,4S)-4-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride (125 mg, 0.189 mmol) and (E)-4-(((1r,4r)-4-(3-((tert-butoxycarbonyl)amino)propanamido)cyclohexyl)oxy)but-2-enoic acid (84 mg, 0.226 mmol) in dichloromethane (DCM) (1 mL) was added triethylamine (0.158 mL, 1.132 mmol) and HATU (86 mg, 0.226 mmol), and the mixture was stirred at room temperature for 2 hours. Saturated NaHCO3 was added, and the mixture was extracted with dichloromethane (DCM). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by ISCO CombiFlash® chromatography (12 g RediSep Rf Gold® column, 30 mL / min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound (125 mg, 0.133 mmol, 70.3% yield). LC-MS m / z 942.1 (M) + .

[0312] Step 5 (2S,3S)—N-(3-(((1R,4S)-4-(4-(((1S,4S)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)cyclohexyl)amino)-3-oxopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka] To a mixture of tert-butyl (3-(((1R,4r)-4-(((E)-4-(((1S,4S)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobut-2-en-1-yl)oxy)cyclohexyl)amino)-3-oxopropyl)carbamate (125 mg, 0.133 mmol) in methanol (1 mL), Pd / C (14.12 mg, 0.013 mmol) was added, and the mixture was stirred under a balloon atmosphere of hydrogen at room temperature overnight. The mixture was filtered, and the catalyst was washed with methanol. The combined filtrates were concentrated to dryness. Dichloromethane (DCM) (0.1 mL) and 4N HCl in 1,4-dioxane (0.332 mL, 1.327 mmol) were added, and the mixture was stirred at room temperature for 2 hours and concentrated to dryness. Dichloromethane (DCM) (1.000 mL), (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (29.2 mg, 0.133 mmol), triethylamine (0.111 mL, 0.796 mmol), and HATU (60.5 mg, 0.159 mmol) were added. The mixture was stirred at room temperature for 2 hours and concentrated. Purification by MDAP (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 30-85% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a white solid (64 mg, 0.061 mmol, 46.1% yield). LC-MS m / z 524.3 (M / 2+H). + . 1H NMR(400 MHz, methanol-d4)δ ppm 1.01(d,J=6.4 Hz,3 H)1.16(d,J=6.4 Hz,4 H)1.19-1.33(m,4 H)1.59-1.71(m,3 H)1.71-1.88(m,12 H)1.93-2.03(m,3 H)2.14-2.26(m,4 H)2.27(s,3 H)2.29-2.39(m,4 H)2.54-2.62(m,1 H)2.66(s,3 H)2.68-2.76(m,2 H)2.80-2.89(m,1 H)2.98-3.06(m,1 H)3.17-3.25(m,1 H)3.35-3.37(m,1 H)3.40-3.68(m,9 H)3.86-3.94(m,1 H)4.03(dt,J=12.3,3.6 Hz,1 H)4.62(br d,J=2.4 Hz,1 H)4.84-4.87(m,2 H)5.30(t,J=8.8 Hz,1 H)7.52(dd,J=8.3,4.9 Hz,1 H)7.79(dt,J=8.2,1.8 Hz,1 H)7.91(d,J=8.8 Hz,1 H)8.06(dd,J=8.8,2.0 Hz,1 H)8.51(d,J=2.0 Hz,1 H)8.57(dd,J=4.9,1.5 Hz,1 H)8.59(s,1 H)8.73(s,1 H).

[0313] Example 53 (2S,3S)—N-(2-(((1R,4S)-4-((3-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)methyl)cyclohexyl)methoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka] Step 1 ((1r,4r)-4-((2-(dibenzylamino)ethoxy)methyl)cyclohexyl)methanol. [ka] To a mixture of commercially available ((1r,4r)-cyclohexane-1,4-diyl)dimethanol (308 mg, 2.136 mmol) in N,N-dimethylformamide (DMF) (5.0 mL) at 0 °C, sodium hydride (85 mg, 2.136 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. N,N-Dibenzyl-2-chloroethan-1-amine (555 mg, 2.136 mmol) was added, and the mixture was stirred at room temperature over the weekend. The mixture was cooled to 0 °C, saturated NH4Cl was added, and the mixture was extracted with ethyl acetate. The combined organic extracts were washed with water, dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by ISCO CombiFlash® chromatography (40 g RediSep Rf Gold® column, 40 mL / min) eluting with a gradient of 0-50% ethyl acetate in heptane provided the title compound (197 mg, 0.536 mmol, 25.09% yield). LC-MS m / z 368.5 (M+H) + .

[0314] Step 2 tert-Butyl 3-(((1S,4r)-4-((2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)methyl)cyclohexyl)methoxy)propanoate. [ka] To a mixture of ((1r,4r)-4-((2-(dibenzylamino)ethoxy)methyl)cyclohexyl)methanol (197 mg, 0.536 mmol), tetrabutylammonium hydrogen sulfate (35 mg, 0.103 mmol), and sodium hydroxide (515 mg, 6.43 mmol) in dichloromethane (DCM) (3 mL), tert-butyl acrylate (0.157 mL, 1.072 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Water was added, and the mixture was extracted with dichloromethane (DCM). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated to dryness. To the residue was added ethyl acetate (3.00 mL) and Pd / C (114 mg, 0.107 mmol), and the mixture was stirred under a balloon atmosphere of hydrogen for 16 hours. Additional Pd / C (114 mg, 0.107 mmol) was added, and the mixture was stirred under a balloon atmosphere of hydrogen for 24 hours. The mixture was filtered, the catalyst was washed with methanol, and the combined filtrates were concentrated to dryness. To the residue in dichloromethane (DCM) (3.00 mL) was added (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (118 mg, 0.536 mmol), HATU (245 mg, 0.643 mmol), and triethylamine (0.224 mL, 1.608 mmol), and the mixture was stirred at room temperature for 2 hours. Saturated NaHCO3 was added, and the mixture was extracted with dichloromethane (DCM). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by ISCO CombiFlash® chromatography (12 g RediSep Rf Gold® column, 30 mL / min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound (212 mg, 0.410 mmol, 76% yield). LC-MS m / z 518.1 (M+H). + .

[0315] Step 3 3-(((1S,4r)-4-((2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)methyl)cyclohexyl)methoxy)propanoic acid, hydrochloride [ka] A mixture of tert-butyl 3-(((1S,4r)-4-((2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)methyl)cyclohexyl)methoxy)propanoate (212 mg, 0.410 mmol), 4N HCl in 1,4-dioxane (1.024 mL, 4.10 mmol), and dichloromethane (DCM) (0.1 mL) was stirred at room temperature for 3 hours. The mixture was concentrated to dryness to provide the title compound (210 mg, 0.422 mmol, 103% yield). LC-MS m / z 462.1 (M+H) + .

[0316] Step 4 (2S,3S)—N-(2-(((1R,4S)-4-((3-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)methyl)cyclohexyl)methoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka] A mixture of (1r,4R)-4-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride (47.9 mg, 0.072 mmol) and 3-(((1S,4r)-4 To a mixture of -((2-(2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)methyl)cyclohexyl)methoxyl)propanoic acid hydrochloride (36 mg, 0.072 mmol), DIPEA (0.101 mL, 0.578 mmol), EDC (20.79 mg, 0.108 mmol), and HOBt (16.60 mg, 0.108 mmol) were added. The mixture was stirred at room temperature overnight and concentrated. Purification by MDAP (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 30-85% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a white solid (38.1 mg, 0.037 mmol, 51.0% yield). LC-MS m / z 517.8 (M / 2+H). + . 1H NMR(400 MHz, methanol-d4)δ ppm 0.82-0.98(m,4 H)1.02(br d,J=6.4 Hz,3 H)1.16(br d,J=6.4 Hz,3 H)1.19-1.35(m,2 H)1.44-1.65(m,4 H)1.67-1.81(m,7 H)1.90-2.05(m,5 H)2.06-2.26(m,3 H)2.28(s,3 H)2.33(br s,1 H)2.38(t,J=6.1 Hz,2 H)2.44-2.60(m,1 H)2.66(s,3 H)2.68-2.77(m,2 H)2.78-2.88(m,1 H)3.05(ddd,J=9.3,8.1,6.6 Hz,1 H)3.23(dd,J=16.6,6.4 Hz,4 H)3.34-3.56(m,6 H)3.62-3.69(m,4 H)4.02(dt,J=12.5,3.8 Hz,1 H)4.66(br d,J=2.9 Hz,1 H)4.84-4.88(m,1 H)5.27(t,J=8.1 Hz,1 H)7.52(dd,J=7.8,4.9 Hz,1 H)7.79(dt,J=7.8,2.0 Hz,1 H)7.91(d,J=8.8 Hz,1 H)8.06(dd,J=8.8,2.0 Hz,1 H)8.51(d,J=2.0 Hz,1 H)8.57(dd,J=4.9,1.5 Hz,1 H)8.59(s,1 H)8.81(s,1 H)

[0317] The following compounds were or can be prepared using procedures similar to those described in Example 53. [Table 14-1] [Table 14-2]

[0318] Example 56 N 1-((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)-N 5 -(4-((3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)glutaramide. [ka] Step 1 tert-Butyl (3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)carbamate. [ka] To a mixture of commercially available tert-butyl (3-aminopropyl)carbamate (346 mg, 1.986 mmol) and (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (437 mg, 1.986 mmol) in dichloromethane (DCM) (5 mL), HATU (906 mg, 2.383 mmol) and triethylamine (0.830 mL, 5.96 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Saturated NaHCO was added, and the mixture was extracted with dichloromethane (DCM). The combined organic extracts were washed with brine, dried over NaSO, filtered, and the filtrate was concentrated. Purification by ISCO CombiFlash® chromatography (40 g RediSep Rf Gold® column, 40 mL / min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound (690 mg, 1.833 mmol, 92% yield). LC-MS m / z 377.2 (M+H) + .

[0319] Step 2. (2S,3S)-N-(3-aminopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, dihydrochloride [ka] A mixture of tert-butyl (3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)carbamate (690 mg, 1.833 mmol), 4 M HCl in 1,4-dioxane (2.291 mL, 9.16 mmol), and dichloromethane (DCM) (0.5 mL) was stirred at room temperature for 3 hours and concentrated to dryness to provide the title compound (810 mg, 2.319 mmol, 127% yield). LC-MS m / z 277.2 (M+H) +

[0320] Step 3 tert-Butyl (4-((3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)carbamate. [ka] To a mixture of commercially available 4-((tert-butoxycarbonyl)amino)butanoic acid (152 mg, 0.750 mmol) and (2S,3S)-N-(3-aminopropyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide dihydrochloride (262 mg, 0.750 mmol) in dichloromethane (DCM) (2 mL), triethylamine (0.627 mL, 4.50 mmol) and HATU (342 mg, 0.900 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Water was added, and the mixture was extracted with dichloromethane (DCM). The combined organic extracts were dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by ISCO CombiFlash® chromatography (40 g RediSep Rf Gold® column, 40 mL / min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound (200 mg, 0.433 mmol, 57.8% yield). LC-MS m / z 462.2 (M+H) + .

[0321] Step 4. (2S,3S)-N-(3-(4-aminobutanamido)propyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, dihydrochloride [ka] A mixture of tert-butyl (4-((3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)carbamate (200 mg, 0.433 mmol), 4 M HCl in 1,4-dioxane (0.542 mL, 2.167 mmol), and dichloromethane (DCM) (0.3 mL) was stirred at room temperature for 5 hours and concentrated to dryness to provide the title compound (200 mg, 0.460 mmol, 106% yield). LC-MS m / z 362.2 (M+H) + .

[0322] Step 5 N 1 -((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)-N 5 -(4-((3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propyl)amino)-4-oxobutyl)glutaramide. [ka] To a mixture of (1s,4S)-4-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride (53.4 mg, 0.081 mmol) in dichloromethane (DCM) (0.5 mL) was added commercially available dihydro-2H-pyran-2,6(3H)-dione (9.19 mg, 0.081 mmol) and triethylamine (0.079 mL, 0.564 mmol), and the mixture was stirred at room temperature for 1.5 h. (2S,3S)-N-(3-(4-aminobutanamido)propyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, dihydrochloride (35 mg, 0.081 mmol) and HATU (36.8 mg, 0.097 mmol) were added, and the mixture was stirred at room temperature for 1.5 hours. Triethylamine (50 μL, 0.357 mmol) and HATU (20 mg, 0.053 mmol) were added, and the mixture was stirred for 1 hour. Additional (2S,3S)-N-(3-(4-aminobutanamido)propyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide, dihydrochloride (20 mg, 0.046 mmol) was added, and the mixture was stirred at room temperature overnight and concentrated. Purification by MDAP (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 30-85% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a white solid (37.7 mg, 0.036 mmol, 44.7% yield). LC-MS m / z 1047.6 (M+H). + 1H NMR(400 MHz, methanol-d4)δ ppm 1.01(d,J=6.4 Hz,3 H)1.16(d,J=6.4 Hz,3 H)1.58-1.70(m,6 H)1.71-1.90(m,11 H)1.90-2.02(m,1 H)2.14-2.25(m,8 H)2.27(s,3 H)2.33(br d,J=13.7 Hz,1 H)2.53-2.63(m,1 H)2.67(s,3 H)2.68-2.75(m,2 H)2.80-2.88(m,1 H)2.97-3.06(m,1 H)3.10-3.27(m,7 H)3.40-3.48(m,1 H)3.50-3.59(m,1 H)3.60-3.70(m,1 H)3.89(br s,1 H)4.03(dt,J=12.2,3.7 Hz,1 H)4.61(br d,J=2.9 Hz,1 H)4.83(d,J=6.8 Hz,1 H)5.31(t,J=8.6 Hz,1 H)7.54(dd,J=7.8,4.9 Hz,1 H)7.82(dt,J=7.8,2.0 Hz,1 H)7.91(d,J=8.8 Hz,1 H)8.07(dd,J=8.8,2.0 Hz,1 H)8.53(d,J=1.5 Hz,1 H)8.57(dd,J=4.9,1.5 Hz,1 H)8.59(s,1 H)8.73(s,1 H).

[0323] Example 57 (2S,3S)—N-(2-(((1S,4R)-4-((4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)methyl)cyclohexyl)methoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 ((1s,4s)-4-((2-(dibenzylamino)ethoxy)methyl)cyclohexyl)methanol. [ka] To a solution of ((1s,4s)-cyclohexane-1,4-diyl)dimethanol (4.0 g, 27.7 mmol) in N,N-dimethylformamide (DMF) (20 mL) under a nitrogen atmosphere, sodium hydride (60%) (1.109 g, 27.7 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 30 minutes. N,N-Dibenzyl-2-chloroethan-1-amine (5.76 g, 22.19 mmol) was added at 0 °C, and the mixture was warmed to room temperature and stirred for 16 hours. Water (100 mL) was added, and the mixture was extracted with ethyl acetate (3 × 60 mL). The combined organic extracts were dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure. Purification by Biotage® Isolera™ column chromatography (50 g SNAP® column) eluting with a gradient of 0-20% hexanes in ethyl acetate provided the title compound as a pale yellow oil (1.7 g, 4.47 mmol, 16.11% yield). LC-MS m / z 368 (M+H) + .

[0324] Step 2. Methyl (E)-4-(((1s,4s)-4-((2-(dibenzylamino)ethoxy)methyl)cyclohexyl)methoxy)but-2-enoate. [ka] To a solution of ((1s,4s)-4-((2-(dibenzylamino)ethoxy)methyl)cyclohexyl)methanol (1.7 g, 4.63 mmol) in toluene (20 mL) under a nitrogen atmosphere, acetic acid (0.053 mL, 0.925 mmol), triphenylphosphine (0.061 g, 0.231 mmol), and commercially available methyl but-2-ynoate (0.681 g, 6.94 mmol) were added at room temperature, and the mixture was stirred at 115 °C for 16 h. The mixture was cooled to room temperature, ethyl acetate (50 mL) was added, and the organic phase was washed with water (2 × 50 mL). The organic phase was dried over NaSO and concentrated under reduced pressure. Purification by Biotage® Isolera™ chromatography (25 g SNAP® column) eluting with a gradient of 0-30% ethyl acetate in hexanes provided the title compound as a pale yellow oil (1.2 g, 2.397 mmol, 51.8% yield). LC-MS m / z 466 (M+H) + .

[0325] Step 3 Methyl 4-(((1s,4s)-4-((2-aminoethoxy)methyl)cyclohexyl)methoxy)butanoate. [ka] To a stirred solution of methyl (E)-4-(((1s,4s)-4-((2-(dibenzylamino)ethoxy)methyl)cyclohexyl)methoxy)but-2-enoate (1.2 g, 2.58 mmol) in methanol (20 mL) was added acetic acid (0.295 mL, 5.15 mmol) and 10% Pd / C (0.30 g, 0.282 mmol), and the mixture was stirred at room temperature under a balloon atmosphere of hydrogen for 16 hours. Methanol (50 mL) was added, the mixture was filtered through Celite®, and the combined filtrates were concentrated under reduced pressure to provide the title compound as a colorless liquid (700 mg, 2.433 mmol, 94% yield). LC-MS m / z 288 (M+H) + .

[0326] Step 4 Methyl 4-(((1R,4s)-4-((2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)methyl)cyclohexyl)methoxy)butanoate. [ka] To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (0.650 g, 2.95 mmol) in N,N-dimethylformamide (DMF) (10 mL) under a nitrogen atmosphere, DIPEA (1.031 mL, 5.90 mmol), HATU (1.683 g, 4.43 mmol), and methyl 4-(((1s,4s)-4-((2-aminoethoxy)methyl)cyclohexyl)methoxy)butanoate (0.763 g, 2.66 mmol) were added, and the mixture was stirred at room temperature for 16 h. Water (50 mL) was added, and the mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic extracts were dried over NaSO and concentrated under reduced pressure. Purification by Biotage® Isolera™ chromatography (25 g SNAP® column) eluting with 5% methanol in dichloromethane (DCM) provided the title compound as a pale yellow liquid (800 mg, 1.225 mmol, 41.5% yield). LC-MS m / z 490.1 (M+H) + .

[0327] Step 5 4-(((1R,4s)-4-((2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)methyl)cyclohexyl)methoxy)butanoic acid. [ka] A mixture of methyl 4-(((1R,4s)-4-((2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)methyl)cyclohexyl)methoxy)butanoate (800 mg, 1.634 mmol) and lithium hydroxide (47.0 mg, 1.961 mmol) in tetrahydrofuran (THF) (5.0 mL) and water (5.00 mL) was stirred at room temperature for 2 h, and the mixture was concentrated under reduced pressure. The pH was adjusted to 6 using 1.5 N HCl, and the mixture was concentrated under reduced pressure. The residue was purified by reverse-phase HPLC (YMC-Triart C18 ExRS 5 μm column, 15 mL / min) eluting with a stepwise gradient of 10–50% (12 min) and 100% (4 min) acetonitrile in water containing formic acid (0.1%). A second purification by chiral SFC (YMC Cellulose-C column, 3 mL / min, 15 μL injection volume, 100 bar, 35 °C) eluting with a co-solvent of 40% isopropyl alcohol provided the title compound as a colorless oil (195 mg, 0.408 mmol, 24.99% yield). LC-MS m / z 476.1 (M+H). + .

[0328] Step 6 (2S,3S)—N-(2-(((1S,4R)-4-((4-(((1R,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-4-oxobutoxy)methyl)cyclohexyl)methoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] A mixture of (1r,4R)-4-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, 2-hydrochloride (48.8 mg, 0.074 mmol) and 4-( To a mixture of ((1R,4s)-4-((2-(2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)methyl)cyclohexyl)methoxy)butanoic acid (35 mg, 0.074 mmol), triethylamine (0.062 mL, 0.442 mmol) and HATU (33.6 mg, 0.088 mmol) were added and stirred for 2 h. Purification by MDAP (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 30–85% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a white solid (49.5 mg, 0.047 mmol, 64.2% yield). LC-MS m / z 1047.5(M+H) + . 1H NMR(400 MHz, methanol-d4)δ ppm 1.02(br d,J=6.4 Hz,3 H)1.16(br d,J=6.4 Hz,3 H)1.18-1.31(m,3 H)1.33-1.79(m,16 H)1.79-1.89(m,2 H)1.90-2.02(m,5 H)2.06-2.18(m,2 H)2.19-2.26(m,3 H)2.28(s,3 H)2.34(br d,J=14.2 Hz,1 H)2.44-2.62(m,1 H)2.66(s,3 H)2.68-2.76(m,2 H)2.77-2.89(m,1 H)3.02-3.10(m,1 H)3.28-3.32(m,2 H)3.35-3.54(m,8 H)3.57-3.67(m,2 H)4.02(dt,J=12.5,3.8 Hz,1 H)4.65(br d,J=2.9 Hz,1 H)4.83(d,J=6.4 Hz,1 H)5.28(t,J=8.1 Hz,1 H)7.51-7.56(m,1 H)7.80(dt,J=7.8,2.0 Hz,1 H)7.91(d,J=8.8 Hz,1 H)8.07(dd,J=8.8,2.0 Hz,1 H)8.51(d,J=1.5 Hz,1 H)8.58(dd,J=4.9,1.5 Hz,1 H)8.59(s,1 H)8.81(s,1 H).

[0329] The following compounds were prepared or can be prepared using procedures similar to those described in Example 57. [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5]

[0330] Example 63 (2S,3S)—N-(2-((2-(2-(3-(((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)ethoxy)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide [ka] Step 1 tert-Butyl ((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carbonyl)glycinate. [ka] To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (150 mg, 0.681 mmol) in N,N-dimethylformamide (4.0 mL), DIPEA (440 mg, 3.41 mmol), HOBt (104 mg, 0.681 mmol), HATU (388 mg, 1.022 mmol), followed by tert-butyl glycinate, hydrochloride (114 mg, 0.681 mmol) were added, and the mixture was stirred overnight. Water was added, and the mixture was extracted with ethyl acetate (3 times). The combined organic extracts were washed with brine (2 times), dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by CombiFlash® Rf chromatography (40 g silica column, 40 mL / min) eluting with a gradient of 0-20% methanol in dichloromethane (DCM) provided the title compound as a wax (230 mg, 0.690 mmol, 101% yield). LC-MS m / z 334.0 (M+H) + .

[0331] Step 2 tert-Butyl 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,4-dioxo-8,11-dioxa-2,5-diazatetradecane-14-oate. [ka] A mixture of tert-butyl ((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carbonyl)glycinate (230 mg, 0.690 mmol) and 4 M HCl in 1,4-dioxane (4 mL, 16.00 mmol) was stirred for 1 hour and concentrated to dryness under reduced pressure. The residue was dissolved in N,N-dimethylformamide (4.0 mL), and DIPEA (446 mg, 3.45 mmol), HOBt (106 mg, 0.690 mmol), and HATU (393 mg, 1.035 mmol) were added, followed by commercially available tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (161 mg, 0.690 mmol), and the mixture was stirred overnight. Water was added, and the mixture was extracted with ethyl acetate (three times). The combined organic extracts were washed with brine (twice), dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by CombiFlash® Rf chromatography (40 g silica column, 40 mL / min) eluting with a gradient of 0-20% methanol in dichloromethane (DCM) provided the title compound as an off-white solid (260 mg, 0.528 mmol, 77% yield). LC-MS (m / z) 493.1 (M+H). + .

[0332] Step 3 (2S,3S)—N-(2-((2-(2-(3-(((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)amino)-3-oxopropoxy)ethoxy)ethyl)amino)-2-oxoethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] A mixture of tert-butyl 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,4-dioxo-8,11-dioxa-2,5-diazatetradecane-14-oate (32.1 mg, 0.065 mmol), 4 M HCl in 1,4-dioxane (4 mL, 16.00 mmol), and dichloromethane (DCM) (2.0 mL) was stirred for 1 hour and concentrated to dryness under reduced pressure. Dichloromethane (DCM) (2.0 mL), (1s,4S)-4-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride (40 mg, 0.054 mmol), HOBt (8.32 mg, 0.054 mmol), and DIPEA (0.047 mL, 0.272 mmol) were added. HATU (31.0 mg, 0.081 mmol) was added, and the mixture was stirred at room temperature for 3 hours. Saturated NaHCO was added, and the mixture was extracted with dichloromethane (DCM) (3 times). The combined organic extracts were washed with brine (twice), dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by MDAP (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 30 to 85% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) provided the title compound as an off-white solid (25.6 mg, 0.024 mmol, 44.4% yield). LC-MS m / z 1008.5 (M)+. 1H NMR(400 MHz,DMSO-d6)δ ppm 0.90(d,J=6.4 Hz,3 H)0.98(br s,10 H)1.04(d,J=6.4 Hz,3 H)1.35-1.82(m,11 H)1.90-2.19(m,6 H)2.28(t,J=6.6 Hz,1 H)2.32-2.39(m,1 H)2.40-2.49(m,2 H)2.56-2.63(m,1 H)2.75(dd,J=16.4,10.0 Hz,1 H)3.04(ddd,J=9.5,7.1,5.9 Hz,2 H)3.19(q,J=5.7 Hz,2 H)3.44(s,3 H)3.48-3.60(m,3 H)3.68(d,J=5.9 Hz,2 H)3.85-3.93(m,1 H)4.43(br s,1 H)4.69(d,J=5.9 Hz,1 H)5.03(q,J=8.2 Hz,1 H)7.44(dd,J=7.8,4.9 Hz,1 H)7.65(d,J=6.8 Hz,1 H)7.69(dt,J=7.8,2.0 Hz,1 H)7.82-7.98(m,2 H)8.07(dd,J=8.8,2.0 Hz,1 H)8.23(t,J=5.6 Hz,1 H)8.50(d,J=2.0 Hz,1 H)8.55(dd,J=4.9,1.5 Hz,1 H)8.58(s,1 H)8.63-8.69(m,1 H)8.71(d,J=8.3 Hz,1 H)8.89(s,1 H).

[0333] The following compounds were prepared or can be prepared using procedures similar to those described in Example 63. [Table 16-1] [Table 16-2] [Table 16-3]

[0334] Example 67 ((2S,3S)—N-(1-((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)cyclohexyl)-1-oxo-5,8,11,14-tetraoxa-2-azahexadecan-16-yl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 tert-Butyl (1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14-tetraoxa-2-azahexadecan-16-yl)carbamate. [ka] A mixture of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (122 mg, 0.554 mmol) and commercially available tert-butyl (14-amino-3,6,9,12-tetraoxatetradecyl)carbamate (205 mg, 0.609 mmol) was dried under reduced pressure. HATU (253 mg, 0.665 mmol) was added, and the mixture was dissolved in acetonitrile (4261 μL). DIPEA (387 μL, 2.216 mmol) was slowly added, and the mixture was stirred for 30 minutes and concentrated under reduced pressure. Purification by ISCO CombiFlash® chromatography (24 g Redisep Rf Gold® column, 35 mL / min) eluting with a gradient of 0-20% methanol in dichloromethane (DCM) provided the title compound as an orange oil (323 mg, 0.582 mmol, 105% yield). LC-MS m / z 539.2 (M+H) + .

[0335] Step 2 (2S,3S)-1-Methyl-5-oxo-N-(1-oxo-1-(4-oxocyclohexyl)-5,8,11,14-tetraoxa-2-azahexadecan-16-yl)-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] A mixture of tert-butyl (1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14-tetraoxa-2-azahexadecan-16-yl)carbamate (323 mg, 0.600 mmol), HCl (0.40 mL) and dichloromethane (DCM) (2 mL) was stirred at room temperature for 15 minutes, concentrated under reduced pressure and chased with dichloromethane (DCM) to provide (2S,3S)—N-(14-amino-3,6,9,12-tetraoxatetradecyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide (324 mg, 0.739 mmol). Commercially available 4-oxocyclohexane-1-carboxylic acid (105 mg, 0.739 mmol) was dried under reduced pressure, TSTU (267 mg, 0.886 mmol) was added, and the mixture was dissolved in acetonitrile (5682 μL). Triethylamine (412 μL, 2.95 mmol) was added dropwise. (2S,3S)-N-(14-amino-3,6,9,12-tetraoxatetradecyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide (324 mg, 0.739 mmol) was added, and the mixture was stirred for 30 minutes and concentrated under reduced pressure. Purification by ISCO CombiFlash® chromatography (twice) (24 g Redisep Rf Gold® column, 35 mL / min) eluting with a gradient of 0-20% methanol in dichloromethane (DCM) provided the title compound (217.9 mg, 0.306 mmol, 41.4% yield). LC-MS m / z 563.1 (M+H). + . 1H NMR(400 MHz, methanol-d4)δ ppm 1.63-2.04(m,2 H)2.08-2.21(m,1 H)2.37-2.54(m,1 H)2.68(s,3 H)2.70-2.80(m,1 H)2.84-2.94(m,1 H)3.01-3.21(m,1 H)3.31-3.45(m,13 H)3.50-3.69(m,11 H)7.58(dd,J=7.9,4.8 Hz,1 H)7.85(dt,J=7.9,1.8 Hz,1 H)8.08-8.27(m,1 H)8.54(d,J=1.8 Hz,1 H)8.61(dd,J=4.8,1.3 Hz,1 H).

[0336] Step 3 ((2S,3S)—N-(1-((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)cyclohexyl)-1-oxo-5,8,11,14-tetraoxa-2-azahexadecan-16-yl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one, dihydrochloride (188 mg, 0.349 mmol) and (2S,3S)-1-methyl-5-oxo-N-(1-oxo-1-(4-oxocyclohexyl)-5,8,11,14-tetraoxa-2-azahexadecan-16-yl)-2-(pyridin-3-yl)pyrrolidine-3-carboxamide (217.9 mg, 0.387 mmol) were sonicated (2 min) in N,N-dimethylformamide (DMF) (704 μL). Additional N,N-dimethylformamide (DMF) was added to ensure the solid starting material was dissolved. Sodium triacetoxyborohydride (243 mg, 1.147 mmol) was added, and the mixture was stirred at 50° C. for 3 hours. The mixture was concentrated under reduced pressure, partitioned between dichloromethane (DCM) and water, and basified with 1 M sodium hydroxide. The aqueous layer was extracted with dichloromethane (DCM) (3 times). The aqueous layer contained the desired product, which was concentrated under reduced pressure and dissolved in a 1:1 mixture of methanol and water (6 mL). The isomers were separated by purification by MDAP chromatography (XBridge™ column) eluting with acetonitrile in water containing an ammonium carbonate modifier. The desired isomer was purified by MDAP (Sunfire™ C18 column) eluting with acetonitrile in water containing a TFA modifier. The desired fractions were concentrated under reduced pressure, basified with 1M sodium hydroxide, and extracted with dichloromethane (DCM) to provide the title compound as a white solid (48.5 mg, 0.048 mmol, 12.38% yield). LC-MS m / z 1011.4 (M+H) + . 1H NMR(400 MHz, methanol-d4)δ ppm 1.09-1.15(m,6 H)1.43-1.61(m,2 H)1.66-2.01(m,8 H)2.05-2.21(m,3 H)2.30(s,3 H)2.34-2.44(m,1 H)2.50-2.60(m,1 H)2.63-2.69(m,4 H)2.70-2.76(m,1 H)2.78-2.89(m,2 H)2.99-3.11(m,2 H)3.21(dt,J=12.7,6.3 Hz,1 H)3.33-3.35(m,3 H)3.36-3.41(m,1 H)3.48-3.54(m,4 H)3.54-3.64(m,12 H)3.70-3.81(m,1 H)4.02-4.11(m,1 H)4.30-4.37(m,1 H)4.80-4.86(m,1 H)5.02-5.11(m,1 H)5.52(s,2 H)7.51-7.57(m,1 H)7.81(dt,J=7.9,2.0 Hz,1 H)7.90(d,J=8.6 Hz,1 H)8.06(dd,J=8.9,1.8 Hz,1 H)8.51-8.55(m,1 H)8.57-8.60(m,1 H)8.61-8.63(m,1 H)8.63-8.67(m,1 H).

[0337] The following compounds were prepared or can be prepared using procedures similar to those described in Example 67. [Table 17-1] [Table 17-2]

[0338] Example 70 (2S,3S)—N—((S)-1-(4-((4-((1R,4S)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)piperazin-1-yl)sulfonyl)piperidin-1-yl)-1-oxopropan-2-yl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 tert-Butyl 4-((4-((1r,4r)-4-(ethoxycarbonyl)cyclohexyl)piperazin-1-yl)sulfonyl)piperidine-1-carboxylate. [ka] tert-Butyl 4-((1s,4s)-4-(ethoxycarbonyl)cyclohexyl)piperazine-1-carboxylate (204 mg, 0.599 mmol) was dissolved in dichloromethane (DCM) (2 mL) and TFA (3 mL), and the mixture was stirred at room temperature for 5 minutes. The mixture was concentrated under reduced pressure and chased with dichloromethane (DCM) (3 times). The residue was dissolved in dichloromethane (DCM) (6072 μL), and triethylamine (250 μL, 1.792 mmol) was added. The mixture was cooled to 0° C., and a solution of tert-butyl 4-(chlorosulfonyl)piperidine-1-carboxylate (224 mg, 0.789 mmol) in dichloromethane (DCM) (1 mL) was added dropwise. The mixture was stirred at room temperature for 3 hours and concentrated under reduced pressure. Water was added, and the mixture was extracted with ethyl acetate (4 times). The combined organic extracts were concentrated under reduced pressure. Purification by normal phase column chromatography (24 g silica) eluting with a gradient of 0 to 100% ethyl acetate in heptane provided the title compound as a white crystalline solid (98.4 mg, 0.159 mmol, 20.19% yield). LC-MS m / z 488.2 (M+H) + .

[0339] Step 2 Ethyl (1S,4r)-4-(4-((1-((tert-butoxycarbonyl)-L-alanyl)piperidin-4-yl)sulfonyl)piperazin-1-yl)cyclohexane-1-carboxylate. [ka] tert-Butyl 4-((4-((1r,4r)-4-(ethoxycarbonyl)cyclohexyl)piperazin-1-yl)sulfonyl)piperidine-1-carboxylate (98.4 mg, 0.202 mmol) was dissolved in dichloromethane (DCM) (1 mL) and TFA (2 mL), and the mixture was stirred at room temperature for 10 minutes. The mixture was concentrated under reduced pressure and chased with dichloromethane (DCM) (3 times). A mixture of the residue, (tert-butoxycarbonyl)-L-alanine (40 mg, 0.211 mmol), and HATU (100 mg, 0.263 mmol) was dissolved in acetonitrile (1.626 mL). Triethylamine (0.105 mL, 0.753 mmol) was added dropwise. The mixture was stirred at room temperature for 30 minutes and concentrated under reduced pressure. Purification by normal phase column chromatography (12 g silica) eluting with a gradient of 0-20% methanol in dichloromethane (DCM) provided the title compound (404.8 mg, 0.558 mmol, 264% yield). LC-MS m / z 559.2 (M+H) + .

[0340] Step 3 (1S,4r)-4-(4-((1-(((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carbonyl)-L-alanyl)piperidin-4-yl)sulfonyl)piperazin-1-yl)cyclohexane-1-carboxylic acid. [ka] TFA (2.0 mL) was added to a solution of (1S,4r)-4-(4-((1-((tert-butoxycarbonyl)-L-alanyl)piperidin-4-yl)sulfonyl)piperazin-1-yl)cyclohexane-1-carboxylic acid (404 mg, 0.761 mmol) in dichloromethane (DCM) (1.5 mL), and the mixture was stirred at room temperature for 5 minutes. The mixture was concentrated by reduced pressure and chased three times with dichloromethane (DCM) (3×) to remove excess TFA and provide (1S,4r)-4-(4-((1-(L-alanyl)piperidin-4-yl)sulfonyl)piperazin-1-yl)cyclohexane-1-carboxylic acid, 2-trifluoroacetate salt (389 mg, 0.590 mmol).

[0341] To a mixture of 2-(2,5-dioxopyrrolidin-1-yl)-1,1,3,3-tetramethylisouronium tetrafluoroborate (213 mg, 0.708 mmol) and (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (130 mg, 0.590 mmol) in acetonitrile (4541 μL), triethylamine (329 μL, 2.361 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. (1S,4r)-4-(4-((1-(L-alanyl)piperidin-4-yl)sulfonyl)piperazin-1-yl)cyclohexane-1-carboxylic acid, 2-trifluoroacetate (389 mg, 0.590 mmol) was added. The mixture was concentrated under reduced pressure. Purification by normal phase column chromatography (24 g column, 35 mL / min) eluting with a gradient of 0 to 80% methanol in dichloromethane (DCM) provided the title compound (101 mg, 0.067 mmol, 11.36% yield). LC-MS m / z 633.3 (M+H) + .

[0342] Step 4 (2S,3S)—N—((S)-1-(4-((4-((1R,4S)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)piperazin-1-yl)sulfonyl)piperidin-1-yl)-1-oxopropan-2-yl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] A mixture of tert-butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (108 mg, 0.192 mmol) and 4 M HCl in 1,4-dioxane (0.2 mL, 0.8 mmol) in dichloromethane (DCM) (1.0 mL) was stirred at room temperature for 30 minutes, and the mixture was concentrated by reduced pressure and chased with dichloromethane (DCM). To a mixture of the residue and triethylamine (89 μL, 0.638 mmol) in dichloromethane (DCM) (4314 μL), (1S,4r)-4-(4-((1-(((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carbonyl)-L-alanyl)piperidin-4-yl)sulfonyl)piperazin-1-yl)cyclohexane-1-carboxylic acid (101 mg, 0.160 mmol) and HATU (72.8 mg, 0.192 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. Purification by MDAP (XBridge™ column) eluting with acetonitrile in water containing ammonium carbonate modifier provided the title compound as a white solid (26.1 mg, 0.024 mmol, 14.93% yield). LC-MS m / z 1079.4(M+H) + . 1H NMR(400 MHz, methanol-d4)δ ppm 1.03(br d,J=6.3 Hz,3 H)1.15(br d,J=6.3 Hz,3 H)1.23-1.37(m,5 H)1.50-1.78(m,7 H)1.91-2.12(m,9 H)2.14-2.20(m,1 H)2.27(s,3 H)2.30-2.38(m,2 H)2.50-2.75(m,11 H)2.83-2.91(m,1 H)3.07-3.23(m,2 H)3.33(dt,J=3.3,1.6 Hz,3 H)3.37(br s,2 H)3.40-3.55(m,3 H)3.61-3.68(m,1 H)4.03(dt,J=12.2,3.7 Hz,1 H)4.09-4.20(m,1 H)4.56(br t,J=14.7 Hz,1 H)4.66(br d,J=3.0 Hz,1 H)5.25(t,J=7.8 Hz,1 H)7.52(dd,J=7.8,5.0 Hz,1 H)7.82(dt,J=8.0,1.9 Hz,1 H)7.91(d,J=8.8 Hz,1 H)8.06(dd,J=8.9,1.9 Hz,1 H)8.55-8.58(m,2 H)8.60(s,1 H)8.81(s,1 H)

[0343] The following compounds were or can be prepared using procedures similar to those described in Example 70. [Table 18]

[0344] Example 72 (2S,3S)—N-(2-(2-(3-(4-((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)piperidin-1-yl)-3-oxopropoxy)ethoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 tert-Butyl 3-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)ethoxy)propanoate, trifluoroacetate. [ka] To a solution of tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (1001 mg, 4.29 mmol) in dichloromethane (DCM) (35 mL) was added triethylamine (1.495 mL, 10.73 mmol), followed by (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (945 mg, 4.29 mmol) and HATU (1958 mg, 5.15 mmol). The mixture was stirred overnight at room temperature and concentrated. Purification by MDAP (Method B) eluting with a gradient of 15-55% acetonitrile in water (both containing 0.1% TFA) provided the title compound as a pale yellow liquid (2.22 g, 3.84 mmol, 89% yield). LC-MS m / z 436.3 (M+H). + .

[0345] Step 2 (2S,3S)—N-(2-(2-(3-(4-((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)piperidin-1-yl)-3-oxopropoxy)ethoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] 3M HCl in cyclopentyl methyl ether (0.3 mL, 0.900 mmol) was added to a solution of tert-butyl 4-(4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)piperidine-1-carboxylate (38 mg, 0.050 mmol) in dichloromethane (DCM) (1 mL), and the mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The residue was dissolved in dichloromethane (DCM) (2 mL), and triethylamine (0.035 mL, 0.251 mmol) was added.

[0346] 3M HCl in cyclopentyl methyl ether (0.3 mL, 0.900 mmol) was added to a solution of tert-butyl 3-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)ethoxy)propanoate, trifluoroacetate (35 mg, 0.064 mmol) in dichloromethane (DCM) (0.5 mL), and the mixture was stirred at room temperature overnight and concentrated under reduced pressure. The amine solution obtained above was added, followed by HATU (28.6 mg, 0.075 mmol), and the mixture was stirred at room temperature for 2 hours and concentrated. Purification by MDAP chromatography (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 50-99% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) provided the title compound as a white solid (17.4 mg, 0.017 mmol, 33.4% yield). LC-MS m / z 1019.3 (M+H). + . 1H NMR(400 MHz, methanol-d4)δ ppm 0.96-1.20(m,11 H)1.28-1.38(m,1 H)1.41-1.52(m,2 H)1.67-2.36(m,18 H)2.47-2.57(m,2 H)2.59-2.64(m,2 H)2.65-2.69(m,4 H)2.70-2.78(m,2 H)2.80-2.88(m,1 H)2.94-3.13(m,2 H)3.36-3.42(m,1 H)3.45-3.60(m,8 H)3.68-3.75(m,2 H)3.95-4.08(m,2 H)4.48-4.55(m,1 H) 4.63-4.67 (m, 1 H) 4.80-4.83 (m, 1 H) 5.19-5.30 (m, 1 H) 7.49-7.55 (m, 1 H) 7.76-7.82 (m, 1 H) 7.89-7.94 (m, 1 H) 8.03-8.10 (m, 1 H) 8.50-8.53 (m, 1 H) 8.55-8.61 (m, 2 H) 8.80-8.84 (m, 1 H). (The trans isomer of Example 66 was also isolated and characterized.)

[0347] The following compounds were or can be prepared using procedures similar to those described in Example 72. [Table 19]

[0348] Example 74 (2S,3S)—N-(1-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-1,10,16-trioxo-3,6,12-trioxa-9,15-diazooctadecan-18-yl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 tert-Butyl 3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanoate. [ka] To a solution of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (514 mg, 2.334 mmol) in dichloromethane (DCM) (6 mL), HOBt (429 mg, 2.80 mmol) and EDC (626 mg, 3.27 mmol) were added, and the mixture was stirred for 30 minutes. tert-Butyl 3-aminopropanoate (428 mg, 2.80 mmol) and DIPEA (0.815 mL, 4.67 mmol) were added, and the mixture was stirred overnight. The mixture was washed with saturated NaHCO3, and the aqueous phase was extracted with dichloromethane (DCM) (3 × 20 mL). The combined organic extracts were washed with saturated NaCl, dried over Na2SO4, and concentrated onto silica. Purification by ISCO CombiFlash® chromatography (40 g RediSep Rf Gold® column, 60 mL / min) eluting with a step gradient of 4% (10 min), 7% (20 min), and 10% (30 min) methanol in dichloromethane (DCM) provided the title compound as a clear tan oil (279 mg, 0.763 mmol, 32.7% yield). LC-MS m / z 348.1 (M+H). + .

[0349] Step 2 3-((2S,3S)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanoic acid, hydrochloride. [ka] A mixture of 4M HCl in 1,4-dioxane (4.53 mL, 18.11 mmol) and tert-butyl 3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanoate (276 mg, 0.755 mmol) was stirred at room temperature for 4 hours and concentrated to provide the title compound as a white gum (547 mg, 0.751 mmol, 100% yield). LC-MS m / z 293.0 (M+H). + .

[0350] Step 3 tert-Butyl 2-(2-(3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanamido)ethoxy)acetate. [ka] To a solution of 3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanoic acid, hydrochloride (540 mg, 0.741 mmol) in dichloromethane (DCM) (4 mL) was added HOBt (136 mg, 0.890 mmol) and EDC (199 mg, 1.038 mmol), and the mixture was stirred for 30 min. tert-Butyl 2-(2-aminoethoxy)acetate (188 mg, 0.890 mmol) and DIPEA (0.259 mL, 1.483 mmol) were added, and the mixture was stirred overnight. HOBt (17 mg, 0.12 mmol), 25 mg of EDC (25 mg, 13 mmol), and DIPEA (0.05 mL, 0.29 mmol) were added, and the mixture was stirred for 4 h. The mixture was washed with saturated NaHCO3, and the aqueous phase was extracted with dichloromethane (DCM) (3 x 20 mL). The combined organic phases were washed with saturated brine, dried over Na2SO4, and concentrated onto silica. Purification by ISCO CombiFlash® chromatography (24 g RediSep Rf Gold® column, 35 mL / min) eluting with a stepwise gradient of 0% (5 min), 5% (15 min), and 10% (35 min) methanol in dichloromethane (DCM) provided the title compound as a pale orange wax (268 mg, 0.514 mmol, 69.3% yield). LC-MS m / z 449.1 (M+H) + .

[0351] Step 4 2-(2-(3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanamido)ethoxy)acetic acid, hydrochloride. [ka] tert-Butyl 2-(2-(3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanamido)ethoxy)acetate (260 mg, 0.499 mmol) was suspended in 1,4-dioxane (1 mL) and 4 M HCl in 1,4-dioxane (2.99 mL, 11.96 mmol) was added. The mixture was stirred at room temperature for 4 h and concentrated to provide the title compound as a white gum (769 mg, 0.484 mmol, 97% yield). LC-MS m / z 393.2 (M+H) + .

[0352] Step 5 tert-Butyl 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,5,11-trioxo-9,15,18-trioxa-2,6,12-triazaicosan-20-oate. [ka] To a solution of 2-(2-(3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)propanamido)ethoxy)acetic acid, hydrochloride (380 mg, 0.239 mmol) in dichloromethane (DCM) (2 mL) was added HOBt (44.0 mg, 0.287 mmol) and EDC (64.2 mg, 0.335 mmol), and the mixture was stirred for 30 min. tert-Butyl 2-(2-(2-aminoethoxy)ethoxy)acetate (61.7 mg, 0.239 mmol) and DIPEA (0.084 mL, 0.478 mmol) were added, and the mixture was stirred overnight. The mixture was washed with saturated NaHCO3, and the aqueous phase was extracted with dichloromethane (DCM) (3 × 20 mL). The combined organic extracts were washed with saturated NaCl, dried over Na2SO4, and concentrated onto silica. Purification by ISCO CombiFlash® chromatography (12 g RediSep Rf Gold® column, 30 mL / min) eluting with a stepwise gradient of 0-5% (5 min), 5% (10 min), 5-10% (25 min), and 10% (10 min) methanol in dichloromethane (DCM) provided the title compound as a clear tan oil (135 mg, 0.198 mmol, 83% yield). LC-MS m / z 594.3 (M+H). + .

[0353] Step 6 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,5,11-trioxo-9,15,18-trioxa-2,6,12-triazaicosan-20-oic acid, hydrochloride. [ka] tert-Butyl 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,5,11-trioxo-9,15,18-trioxa-2,6,12-triazaicosan-20-oate (131 mg, 0.192 mmol) was suspended in 1,4-dioxane (1 mL) and 4 M HCl in 1,4-dioxane (1.152 mL, 4.61 mmol) was added. The mixture was stirred at room temperature for 4 hours and concentrated to provide the title compound as a white gum (267 mg, 0.163 mmol, 85% yield). LC-MS m / z 538.2 (M+H) + .

[0354] Step 7 (2S,3S)—N-(1-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-1,10,16-trioxo-3,6,12-trioxa-9,15-diazooctadecan-18-yl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] To a solution of 1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1,5,11-trioxo-9,15,18-trioxa-2,6,12-triazaicosan-20-oic acid, hydrochloride salt (259 mg, 0.158 mmol) in dichloromethane (DCM) (1 mL) was added HOBt (29.1 mg, 0.190 mmol) and EDC (42.4 mg, 0.221 mmol), and the mixture was stirred for 30 min. (S)-1-((1S,2R,4R)-2-amino-4-(isopropyl(methyl)amino)cyclohexyl)-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-2-one, dihydrochloride (50 mg, 0.079 mmol) and DIPEA (0.055 mL, 0.316 mmol) were added and the mixture was stirred overnight. The mixture was washed with saturated NaHCO and the aqueous phase was extracted with dichloromethane (DCM) (3 × 20 mL). The combined organic extracts were washed with saturated NaCl, dried over NaSO, and concentrated onto silica. Purification by MDAP (Xselect™ CSH Prep C18 5 um OBD column, 40 mL / min) eluting with a gradient of 30-85% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonium hydroxide (0.075%) provided the title compound as a white solid (51 mg, 0.049 mmol, 62.3% yield). LC-MS m / z 984.8 (M+H) + . 1H NMR(400 MHz, methanol-d4)δ ppm 1.00(d,J=6.4 Hz,3 H)1.16(br d,J=6.4 Hz,3 H)1.62-1.80(m,2 H)1.89-2.02(m,1 H)2.05-2.16(m,1 H)2.24(s,4 H)2.31-2.43(m,3 H)2.51-2.62(m,1 H)2.65-2.74(m,5 H)2.79-2.88(m,1 H)2.97-3.05(m,1 H)3.35-3.46(m,8 H)3.50-3.64(m,6 H)3.65-3.71(m,2 H)3.72-3.77(m,2 H)3.95-3.98(m,2 H)4.04-4.12(m,3 H)4.71(br d,J=2.9 Hz,1 H)4.83(d,J=6.4 Hz,1 H)5.31(t,J=8.6 Hz,1 H)7.52(dd,J=7.8,4.9 Hz,1 H)7.79(dt,J=7.8,2.0 Hz,1 H)7.90(d,J=8.8 Hz,1 H)8.04-8.09(m,1 H)8.50-8.52(m,1 H)8.57(dd,J=4.9,2.0 Hz,1 H)8.59(s,1 H)8.77-8.81(m,1 H).

[0355] The following compounds were prepared or can be prepared using procedures similar to those described in Example 74. [Table 20-1] [Table 20-2] [Table 20-3] [Table 20-4] [Table 20-5] [Table 20-6] [Table 20-7] [Table 20-8] [Table 20-9] [Table 20-10]

[0356] Example 85 (2S,3S)—N-(17-((2-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3,6,9,12,15-pentaoxaheptadecyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 (2S,3S)-N-(17-amino-3,6,9,12,15-pentaoxaheptadecyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. Dihydrochloride. [ka] To a mixture of (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (370 mg, 1.680 mmol), tert-butyl (14-amino-3,6,9,12-tetraoxatetradecyl)carbamate (840 mg, 2.497 mmol) in dichloromethane (DCM) (10 mL) was added T3P (1.757 mL, 2.95 mmol). The mixture was stirred at room temperature for 2 hours and washed with water (10 mL). The organic phase was dried over Na2SO4 and concentrated under reduced pressure to provide tert-butyl (1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14-tetraoxa-2-azahexadecan-16-yl)carbamate as a white semi-solid (750 mg, 1.39 mmol, 82.7% yield). LC-MS m / z 539.1 (M+H) + .

[0357] Tert-butyl (1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14-tetraoxa-2-azahexadecan-16-yl)carbamate as a white semi-solid was treated with 4N HCl in 1,4-dioxane and added to the above product. The mixture was stirred for 1 hour and concentrated under reduced pressure to provide the title compound (650 mg, 1.482 mmol, 75.6% yield). LC-MS m / z 439.2 (M+H) + .

[0358] Step 2 (2S,3S)—N-(17-((2-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3,6,9,12,15-pentaoxaheptadecyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] A mixture of 3-ethoxy-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)cyclobut-3-ene-1,2-dione (60 mg, 0.102 mmol), (2S,3S)—N-(17-amino-3,6,9,12,15-pentaoxaheptadecyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide (49.2 mg, 0.102 mmol), and DIPEA (0.036 mL, 0.204 mmol) in methanol (2 mL) was stirred at 60° C. for 18 hours in a sealed vial and concentrated. Purification by MDAP (XSelect™ CSH Prep C18 5 um OBD column, 40 mL / min) eluting with a gradient of 15-65% acetonitrile in water containing ammonium bicarbonate (10 mM) and ammonia hydroxide (0.075%) provided the title compound (95.3 mg, 0.088 mmol, 87% yield). LC-MS m / z 1025.4 (M+H). + . 1 H NMR(400 MHz, methanol-d4)δ ppm 1.00-1.20(m,6 H)1.29-1.39(m,1 H)1.68-1.92(m,4 H)2.29(br s,5 H)2.43-2.52(m,1 H)2.55-2.77(m,1 H)2.57-2.75(m,4 H)2.80-2.89(m,2 H)3.02-3.09(m,1 H)3.36-3.77(m,26 H)4.10-4.17(m,1 H)4.72(br d,J=2.9 Hz,1 H)4.83(d,J=6.4 Hz,1 H)5.23-5.34(m,1 H)7.53(dd,J=7.8,4.9 Hz,1 H)7.80(dt,J=7.8,2.0 Hz,1 H)7.91(d,J=8.8 Hz,1 H)8.06(dd,J=8.8,2.0 Hz,1 H)8.50-8.53(m,1 H)8.56-8.59(m,2 H)8.71-8.81(m,1 H).

[0359] Example 86 (2S,3S)—N-(6-(4-(4-(3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-3-oxopropyl)phenyl)piperidin-1-yl)-6-oxohexyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 tert-Butyl (E)-4-hydroxy-4-(4-(3-methoxy-3-oxoprop-1-en-1-yl)phenyl)piperidine-1-carboxylate. [ka] A mixture of tert-butyl 4-(4-bromophenyl)-4-hydroxypiperidine-1-carboxylate (528.3 mg, 1.483 mmol), methyl acrylate (1.344 mL, 14.83 mmol), triethylamine (0.827 mL, 5.93 mmol), palladium(II) acetate (71 mg, 0.316 mmol) and triphenylphosphine (160 mg, 0.610 mmol) in acetonitrile (15 mL) was degassed by vacuum and nitrogen backfill cycles and heated at 100° C. for 24 h using a microwave reactor, cooled, filtered through Celite® and the filtrate was concentrated. Purification by normal phase chromatography (24 g ISCO Gold column, 35 mL / min) eluting with a stepwise gradient of 20–50% (15 column volumes) and 50% (5 column volumes) ethyl acetate provided the title compound as a yellow semi-solid (324.3 mg, 0.897 mmol, 60.5% yield). 1H NMR(400 MHz,chloroform-d)δ ppm 1.44(s,9 H)1.69(br d,J=12.3 Hz,2 H)1.86-1.96(m,2 H)2.99(br s,1 H)3.22(br s,2 H)3.76(s,3 H)3.89-4.03(m,2 H)6.36(d,J=16.1 Hz,1 H)7.41-7.50(m,4 H)7.60(d,J=16.1 Hz,1 H).

[0360] Step 2 tert-Butyl 4-(4-(3-methoxy-3-oxopropyl)phenyl)piperidine-1-carboxylate. [ka] To a solution of tert-butyl (E)-4-hydroxy-4-(4-(3-methoxy-3-oxoprop-1-en-1-yl)phenyl)piperidine-1-carboxylate (337 mg, 0.932 mmol) in methanol (10 mL) was added 10% Pd / C (100 mg, 0.094 mmol). The reaction was purged by vacuum and hydrogen refill cycles. The mixture was stirred at room temperature for 2 h, filtered, and the filtrate was concentrated. Purification by normal-phase chromatography (24 g ISCO Gold column, 35 mL / min) eluting with a stepwise gradient of 20–50% (15 column volumes) and 50% (5 column volumes) ethyl acetate provided the title compound as a colorless film (236.3 mg, 0.666 mmol, 71.5% yield). 1 H NMR(400 MHz,chloroform-d)δ ppm 1.49(s,9 H)1.61(td,J=12.8,4.1 Hz,2 H)1.80(br d,J=13.7 Hz,2 H)2.57-2.65(m,3 H)2.75-2.83(m,2 H)2.92(t,J=7.9 Hz,2 H)3.66(s,3 H)4.24(br d,J=12.9 Hz,2 H)7.13(d,J=1.3 Hz,4 H)

[0361] Step 3 3-(4-(1-(tert-butoxycarbonyl)piperidin-4-yl)phenyl)propanoic acid. [ka] To a solution of tert-butyl 4-(4-(3-methoxy-3-oxopropyl)phenyl)piperidine-1-carboxylate (236 mg, 0.679 mmol) in tetrahydrofuran (THF) (1.5 mL) and ethanol (1.5 mL) was added 2 N sodium hydroxide (1.358 mL, 2.72 mmol). The mixture was stirred at room temperature for 1 h, and the pH was adjusted to 4-5 using 1 N HCl. The mixture was concentrated to remove the organic solvent. The residue was extracted with ethyl acetate (twice). The combined organic extracts were washed with brine, dried over MgSO4, and concentrated to provide the title compound as a white solid (228.2 mg, 0.678 mmol, 100% yield). 1 H NMR(400 MHz,chloroform-d)δ ppm 1.51(s,9 H)1.59-1.69(m,2 H)1.79-1.87(m,2 H)2.59-2.68(m,1 H)2.68-2.74(m,2 H)2.82(td,J=13.1,2.5 Hz,2 H)2.97(t,J=7.9 Hz,2 H)4.26(br d,J=13.0 Hz,2 H)7.17(d,J=4.3 Hz,4 H)

[0362] Step 4 tert-Butyl 4-(4-(3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-3-oxopropyl)phenyl)piperidine-1-carboxylate. [ka] To a solution of tert-butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (80 mg, 0.142 mmol) in dichloromethane (DCM) (1 mL) was added 3 M HCl in cyclopentyl methyl ether (0.5 mL, 1.500 mmol). The mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The residue was suspended in dichloromethane (DCM) (3 mL) containing triethylamine (0.069 mL, 0.496 mmol). 3-(4-(1-(tert-butoxycarbonyl)piperidin-4-yl)phenyl)propanoic acid (55 mg, 0.165 mmol) and HATU (81 mg, 0.213 mmol) were added. The mixture was stirred at room temperature for 2 h and concentrated. Purification by MDAP (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 80-99% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a white solid (92.5 mg, 0.116 mmol, 82% yield). LC-MS m / z 780.2 (M+H) + .

[0363] Step 5 tert-Butyl (6-(4-(4-(3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-3-oxopropyl)phenyl)piperidin-1-yl)-6-oxohexyl)carbamate. [ka] To a solution of tert-butyl 4-(4-(3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-3-oxopropyl)phenyl)piperidine-1-carboxylate (36.5 mg, 0.047 mmol) in dichloromethane (DCM) (1 mL) was added 3 M HCl in cyclopentyl methyl ether (0.5 mL, 1.500 mmol). The mixture was stirred at room temperature for 1 hour and concentrated to dryness. The white solid residue was suspended in dichloromethane (DCM) (3 mL). DIPEA (0.041 mL, 0.234 mmol) was added, followed by 6-((tert-butoxycarbonyl)amino)hexanoic acid (12.99 mg, 0.056 mmol) and HATU (26.7 mg, 0.070 mmol). The resulting mixture was stirred at room temperature for 0.5 h and concentrated. Purification by MDAP (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 50-99% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a white solid (30.6 mg, 0.030 mmol, 79% yield). LC-MS m / z 893.3 (M+H). + .

[0364] Step 6 (2S,3S)—N-(6-(4-(4-(3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-3-oxopropyl)phenyl)piperidin-1-yl)-6-oxohexyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] A solution of tert-butyl (6-(4-(4-(3-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)amino)-3-oxopropyl)phenyl)piperidin-1-yl)-6-oxohexyl)carbamate (34 mg, 0.038 mmol) in dichloromethane (DCM) (1 mL) was treated with 3 M HCl in cyclopentyl methyl ether (0.25 mL, 0.750 mmol). The mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The residue was suspended in dichloromethane (DCM) (3 mL) containing triethylamine (0.027 mL, 0.190 mmol). (2S,3S)-1-Methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (10.06 mg, 0.046 mmol) and HATU (21.71 mg, 0.057 mmol) were added. The mixture was stirred at room temperature for 1 h and concentrated. Purification by MDAP (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 50-99% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a white solid (30.6 mg, 0.030 mmol, 79% yield). LC-MS m / z 995.3 (M+H). + . 1H NMR(400 MHz, methanol-d4)δ ppm 1.00(br d,J=6.1 Hz,3 H)1.13(br d,J=6.3 Hz,3 H)1.28-1.39(m,2 H)1.45-1.72(m,10 H)1.73-1.86(m,2 H)1.95-2.17(m,5 H)2.24-2.55(m,5 H)2.58-2.76(m,8 H)2.79-2.88(m,1 H)2.93(t,J=7.0 Hz,2 H)3.02(ddd,J=9.4,8.4,6.8 Hz,1 H)3.13-3.28(m,4 H)3.36-3.46(m,2 H)3.96-4.10(m,2 H)4.57(br d,J=3.0 Hz,1 H)4.65(br d,J=13.9 Hz,1 H)4.81-4.87(m,1 H)5.18(t,J=8.0 Hz,1 H)7.02-7.13(m,4 H)7.54(dd,J=7.9,4.8 Hz,1 H)7.77-7.85(m,1 H)7.92(d,J=8.9 Hz,1 H)8.08(dd,J=8.9,1.8 Hz,1 H)8.52(d,J=1.8 Hz,1 H)8.57-8.62(m,2 H)8.84-8.88(m,1 H).

[0365] The following compounds were prepared or can be prepared using procedures similar to those described in Example 86. [Table 21]

[0366] Example 88 N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)-4-((1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14-tetraoxa-2-azahexadecan-16-yl)oxy)picolinamide. [ka] Step 1 Ethyl 4-((2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecane-19-yl)oxy)picolinate. [ka] Polymer-bound triphenylphosphine (1.795 mmol) was added to a suspension of tert-butyl (14-hydroxy-3,6,9,12-tetraoxatetradecyl)carbamate (606 mg, 1.795 mmol) and ethyl 4-hydroxypicolinate (150 mg, 0.897 mmol) in tetrahydrofuran (THF) (8973 μL). A 40% solution of diethyl azodicarboxylate (710 μL, 1.795 mmol) in toluene was added dropwise, and the mixture was stirred at reflux for 20 h. Ethyl acetate was added, and the organic phase was washed with saturated NH4Cl, Na2CO3 solution, and brine. The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated. Purification by ISCO CombiFlash® chromatography (80 g RediSep Rf Gold® column, 60 mL / min) eluting with a stepwise gradient of 0% (10 column volumes), 0-7% (25 column volumes), and 7% (4 column volumes) methanol in dichloromethane (DCM) provided the title compound as a colorless oil (220 mg, 0.439 mmol, 48.9% yield). LC-MS m / z 487.2 (M+H). + .

[0367] Step 2 4-((2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecane-19-yl)oxy)picolinic acid. [ka] 10 M sodium hydroxide (265 μL, 2.65 mmol) was added to a solution of ethyl 4-((2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecane-19-yl)oxy)picolinate (215 mg, 0.442 mmol) in a mixture of methanol:tetrahydrofuran (THF):HO (2:1:1) and stirred at 20 °C for 20 h. The pH was adjusted to 7 using 1 N HCl. The mixture was concentrated, dichloromethane (DCM) was added, undissolved solids were removed by filtration, and the filtrate was concentrated under reduced pressure to provide the title compound as a clear, colorless oil (214 mg, 0.443 mmol, 100% yield). LC-MS m / z 459.1 (M+H) + .

[0368] Step 3 tert-Butyl (14-((2-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)pyridin-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)carbamate. [ka] 4 M HCl in 1,4-dioxane (815 μL, 3.26 mmol) was added to a solution of tert-butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (184 mg, 0.326 mmol) in 1,4-dioxane (1086 μL), and the mixture was heated to 45° C. for 30 minutes. The mixture was concentrated under reduced pressure. The residue and 4-((2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecane-19-yl)oxy)picolinic acid (179 mg, 0.391 mmol) were added sequentially to dichloromethane (DCM) (0.5 mL), HOAt (48.8 mg, 0.358 mmol), DIPEA (569 μL, 3.26 mmol), and EDC (63.5 μL, 0.358 mmol), and the mixture was stirred at 37 °C for 20 h. The mixture was diluted with dichloromethane (DCM) and washed with water, saturated NH Cl, saturated Na CO, and brine. The organic phase was passed through a phase separator and concentrated under reduced pressure. Purification by reverse-phase HPLC (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 50-99% acetonitrile in water containing ammonium bicarbonate (10 mM) provided the title compound as an off-white solid (186 mg, 0.203 mmol, 62.4% yield). LC-MS m / z 905.2 (M+H). + .

[0369] Step 4 N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)-4-((1-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidin-3-yl)-1-oxo-5,8,11,14-tetraoxa-2-azahexadecan-16-yl)oxy)picolinamide. [ka] 4 M HCl in 1,4-dioxane (503 μL, 2.011 mmol) was added to a solution of tert-butyl (14-((2-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)pyridin-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)carbamate (182 mg, 0.201 mmol) in 1,4-dioxane (670 μL). A few drops of methanol were added, and the mixture was stirred at 45° C. for 30 minutes. The mixture was concentrated under reduced pressure, and dichloromethane (DCM) was added. The organic phase was washed with saturated Na2CO3 and brine. The organic phase was passed through a phase separator, and the filtrate was concentrated under reduced pressure. The residue was dissolved in 0.4 mL of N,N-dimethylformamide (DMF) (0.4 mL), and (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (53.1 mg, 0.241 mmol) and HOAt (30.1 mg, 0.221 mmol) were added. EDC (39.2 μL, 0.221 mmol) and DIPEA (211 μL, 1.207 mmol) were added, and the mixture was stirred at room temperature for 4 hours. Purification by reverse-phase HPLC (XSelect™ CSH Prep C18 5 μm OBD column, 40 mL / min) eluting with a gradient of 50-99% acetonitrile in water containing ammonium bicarbonate (10 mM) provided the title compound as an off-white solid (126 mg, 0.124 mmol, 61.6% yield). LC-MS m / z 1007.3 (M+H). + . 1H NMR(400 MHz, methanol-d4)δ ppm 0.89-1.42(m,6 H)1.76(br dd,J=8.1,4.6 Hz,4 H)2.26(s,8 H)2.65(s,6 H)2.98-3.12(m,1 H)3.35-3.66(m,18 H)3.80(t,J=4.6 Hz,2 H)4.18(br d,J=7.4 Hz,3 H)4.80(s,2 H)5.32(s,1 H)7.11(dd,J=5.6,2.5 Hz,1 H)7.53(br d,J=5.1 Hz,1 H)7.65(d,J=2.5 Hz,1 H)7.79(d,J=8.1 Hz,1 H)7.93(d,J=8.6 Hz,1 H)8.03-8.21(m,1 H)8.36-8.68(m,4 H)8.84(d,J=0.8 Hz,1 H).

[0370] The following compounds were or can be prepared using procedures similar to those described in Example 88. [Table 22]

[0371] Example 90 N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)-1'-(2-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide)ethoxy)ethoxy)ethyl)-[1,4'-bipiperidine]-4-carboxamide. [ka] Step 1 2-(2-(2-azidoethoxy)ethoxy)ethyl methanesulfonate. [ka] To a solution of 0.5 M 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (10 mL, 5.00 mmol) and triethylamine (2.091 mL, 15.00 mmol) in tert-butyl methyl ether in diethyl ether (10 mL) at 0° C. was added dropwise methanesulfonyl chloride (0.584 mL, 7.50 mmol). The mixture was stirred at 0° C. for 1 h and poured into water (30 mL). The mixture was extracted with diethyl ether (3×30 mL). The combined organic extracts were washed with dilute HCl, dried over MgSO4, and concentrated under reduced pressure to provide the title compound as a colorless liquid. 1 H NMR (400 MHz, chloroform-d) δ ppm 3.03-3.17 (m, 3 H), 3.42 (t, J = 4.9 Hz, 2 H), 3.65-3.75 (m, 6 H), 3.79-3.85 (m, 2 H), 4.35-4.47 (m, 2 H).

[0372] Step 2 1'-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)-[1,4'-bipiperidine]-4-carboxamide. [ka] To a solution of tert-butyl 4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)-[1,4'-bipiperidine]-1'-carboxylate (105 mg, 0.138 mmol) in dichloromethane (DCM) (2 mL) was added 4 M HCl in 1,4-dioxane (1 mL, 4.00 mmol). The mixture was stirred at room temperature for 1 hour and concentrated to dryness. The resulting white solid was suspended in acetonitrile (7.5 mL). 2-(2-(2-azidoethoxy)ethoxy)ethyl methanesulfonate (44.0 mg, 0.174 mmol) was added, followed by potassium carbonate (97 mg, 0.702 mmol). The resulting mixture was stirred at 85 °C for 5 h, filtered, and the filtrate was concentrated. Purification by MDAP (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 50-99% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a pale yellow film (83 mg, 0.100 mmol, 72.0% yield). LC-MS m / z 816.3 (M+H) + .

[0373] Step 3 1'-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)-[1,4'-bipiperidine]-4-carboxamide. [ka] To a solution of 1'-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)-[1,4'-bipiperidine]-4-carboxamide (83 mg, 0.102 mmol) in methanol (7 mL) was added 10% Pd / C (10.82 mg, 10.17 μmol). The mixture was purged by vacuum and refilled with hydrogen. The mixture was stirred at room temperature for 2 h, filtered, and the filtrate was concentrated to provide the title compound as a colorless film (66 mg, 0.082 mmol, 80% yield). LC-MS m / z 790.4 (M+H) + .

[0374] Step 4 N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)-1'-(2-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide)ethoxy)ethoxy)ethyl)-[1,4'-bipiperidine]-4-carboxamide. [ka] To a solution of 1'-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)-[1,4'-bipiperidine]-4-carboxamide (66 mg, 0.084 mmol) in dichloromethane (DCM) (5 mL) was added triethylamine (0.035 mL, 0.251 mmol), followed by (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (22.08 mg, 0.100 mmol) and HATU (47.7 mg, 0.125 mmol). The mixture was stirred at room temperature for 1 h and concentrated. Purification by MDAP (XSelect™ CSH C18 5 μm column, 40 mL / min) eluting with a gradient of 30-85% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a white solid (67.7 mg, 0.067 mmol, 80% yield). LC-MS m / z 992.4 (M+H) + . 1H NMR(400 MHz, methanol-d4)δ ppm 0.91-1.10(m,3 H)1.17(br d,J=6.3 Hz,3 H)1.48-1.64(m,2 H)1.64-1.84(m,7 H)1.89-2.11(m,6 H)2.11-2.35(m,10 H)2.49-2.66(m,3 H)2.66-2.69(m,3 H)2.69-2.76(m,2 H)2.80-2.92(m,1 H)2.92-3.09(m,5 H)3.30-3.45(m,4 H)3.50-3.72(m,10 H)4.04(dt,J=12.5,3.6 Hz,1 H)4.66(br d,J=3.0 Hz,1 H)4.79-4.89(m,1 H)5.30(t,J=8.4 Hz,1 H)7.54(dd,J=8.0,4.9 Hz,1 H)7.81(dt,J=8.0,2.0 Hz,1 H)7.91(d,J=8.6 Hz,1 H)8.07(dd,J=8.9,2.0 Hz,1 H)8.53(d,J=1.5 Hz,1 H)8.58(dd,J=4.9,1.4 Hz,1 H)8.60(s,1 H)8.77(s,1 H).

[0375] The following compounds were or can be prepared using procedures similar to those described in Example 90. [Table 23]

[0376] Example 92 N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)-1-(1-(2-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide)ethoxy)ethoxy)ethyl)piperidin-4-yl)-1H-pyrazole-4-carboxamide. [ka] Step 1 tert-Butyl 4-(4-(ethoxycarbonyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate. [ka] To a stirred solution of ethyl 1H-pyrazole-4-carboxylate (1.0446 g, 7.45 mmol) in N,N-dimethylformamide (DMF) (30 mL) was added 60% sodium hydride mineral oil dispersion (0.328 g, 8.20 mmol) at 0° C. tert-Butyl 4-((methylsulfonyl)oxy)piperidine-1-carboxylate (2.290 g, 8.20 mmol) was added and the reaction mixture was stirred at 50° C. overnight, poured into water (100 mL) and stirred for 2 hours. The precipitate was collected by filtration to provide the title compound as an off-white solid (2.35 g, 6.61 mmol, 89% yield). LC-MS m / z 324.1 (M+H) + .

[0377] Step 2 Ethyl 1-(1-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)piperidin-4-yl)-1H-pyrazole-4-carboxylate, formate. [ka] To a stirred solution of tert-butyl 4-(4-(ethoxycarbonyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (245 mg, 0.682 mmol) in dichloromethane (DCM) (2 mL) was added TFA (2 mL, 26.0 mmol). The mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. N,N-Dimethylformamide (DMF) (3 mL) and potassium carbonate (321 mg, 2.321 mmol) were added, followed by tert-butyl (2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate (207 mg, 0.663 mmol), and the mixture was stirred at 60° C. for 2 hours, cooled, and filtered. Purification by preparative HPLC (Agilent Eclipse Plus C18 5 μm column, 47 mL / min) eluting with a gradient of 5-40% acetonitrile in water containing formic acid (0.1%) provided the title compound as a colorless film (260 mg, 0.493 mmol, 74.4% yield). LC-MS m / z 455.2 (M+H) + .

[0378] Step 3 Ethyl 1-(1-(2-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)ethoxy)ethyl)piperidin-4-yl)-1H-pyrazole-4-carboxylate. [ka] A mixture of 4 M HCl (1 mL, 4.00 mmol) in 1,4-dioxane and ethyl 1-(1-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)piperidin-4-yl)-1H-pyrazole-4-carboxylate, formate (260 mg, 0.519 mmol) in DCM (1.5 mL) was stirred at room temperature for 1 hour and concentrated. The residue was dissolved in dichloromethane (DCM) (3 mL), and DIPEA (0.4 mL, 2.290 mmol), (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (137 mg, 0.623 mmol), and HATU (296 mg, 0.779 mmol) were added sequentially. The mixture was stirred at room temperature for 2 hours and concentrated. Purification by MDAP eluting with a gradient of 15-85% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 with ammonia, provided the title compound as a light brown film (223 mg, 0.381 mmol, 73.3% yield). LC-MS m / z 557.1 (M+H). + .

[0379] Step 4 1-(1-(2-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)ethoxy)ethyl)piperidin-4-yl)-1H-pyrazole-4-carboxylic acid. [ka] A mixture of ethyl 1-(1-(2-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)ethoxy)ethyl)piperidin-4-yl)-1H-pyrazole-4-carboxylate (223 mg, 0.401 mmol) and 2N aqueous sodium hydroxide (0.401 mL, 0.801 mmol) in tetrahydrofuran (THF) (1.500 mL) and ethanol (1.5 mL) was stirred at room temperature for 2 hours and concentrated to remove the organic solvent. The mixture was cooled, the pH was adjusted to 4-5 using 2N HCl, and the mixture was concentrated under reduced pressure. Methanol in dichloromethane (DCM) (2%) (30 mL) was added, and the mixture was stirred, sonicated, and filtered. The filtrate was concentrated under reduced pressure to provide the title compound as a pale yellow film (201 mg, 0.247 mmol, 61.7% yield). LC-MS m / z 529.1 (M+H) + .

[0380] Step 5 N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)-1-(1-(2-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide)ethoxy)ethoxy)ethyl)piperidin-4-yl)-1H-pyrazole-4-carboxamide. [ka] A mixture of 4 M HCl in 1,4-dioxane (1 mL, 4.00 mmol) and tert-butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (100 mg, 0.177 mmol) in dichloromethane (DCM) (1 mL) was stirred at room temperature for 1 hour and concentrated under reduced pressure. The residue was suspended in dichloromethane (DCM) (5 mL). Triethylamine (0.086 mL, 0.620 mmol), 1-(1-(2-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)ethoxy)ethyl)piperidin-4-yl)-1H-pyrazole-4-carboxylic acid (202 mg, 0.248 mmol), and HATU (101 mg, 0.266 mmol) were added. The mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The residue was purified by preparative HPLC (Agilent Eclipse Plus C18 5 μm column, 47 mL / min) eluting with a gradient of 5 to 35% acetonitrile in water containing TFA (0.1%). A second purification by MDAP eluting with a gradient of 30-85% acetonitrile in water containing ammonium bicarbonate (10 mM), and the pH adjusted to 10 using ammonia, provided the title compound as a white powder (59 mg, 0.059 mmol, 33.5% yield). LC-MS m / z 975.3 (M+H). + . 1H NMR(400 MHz,DMSO-d6)δ ppm 0.81-0.90(m,3 H)1.04-1.13(m,3 H)1.55-1.75(m,3 H)1.77-1.97(m,5 H)2.04-2.22(m,8 H)2.26-2.36(m,1 H)2.42-2.47(m,2 H)2.48-2.49(m,3 H)2.60-2.76(m,3 H)2.86-3.00(m,3 H)3.11-3.30(m,3 H)3.36-3.41(m,2 H)3.43-3.55(m,8 H)3.95-4.16(m,2 H)4.45-4.56(m,1 H)4.62-4.72(m,1 H)4.99-5.14(m,1 H)7.40-7.47(m,1 H)7.63-7.71(m,2 H)7.86-7.92(m,1 H)8.01-8.11(m,3 H)8.44-8.49(m,1 H)8.52-8.58(m,2 H)8.74-8.83(m,1 H)8.92-9.03(m,2 H).

[0381] The following compounds were prepared or can be prepared using procedures similar to those described in Example 92. [Table 24-1] [Table 24-2]

[0382] Example 95 N-((1S,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)-3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carbonyl)-3-azaspiro[5.5]undecane-9-carboxamide. [ka] Step 1 tert-Butyl 9-(((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)carbamoyl)-3-azaspiro[5.5]undecane-3-carboxylate. [ka] To a mixture of (1s,4S)-4-amino-N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)cyclohexane-1-carboxamide, dihydrochloride (80 mg, 0.121 mmol) in dichloromethane (DCM) (0.5 mL), triethylamine (0.101 mL, 0.724 mmol), 3-(tert-butoxycarbonyl)-3-azaspiro[5.5]undecane-9-carboxylic acid (35.9 mg, 0.121 mmol), and HATU (55.1 mg, 0.145 mmol) were added successively, and the mixture was stirred at room temperature for 2 hours. Purification by ISCO CombiFlash® chromatography (40 g RediSep Rf Gold® column, 40 mL / min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound as a solid (75 mg, 0.086 mmol, 71.5% yield). LC-MS m / z 869.3 (M+H). + .

[0383] Step 2 N-((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)-3-azaspiro[5.5]undecane-9-carboxamide, dihydrochloride. [ka] A mixture of 4 M HCl (0.170 mL, 0.681 mmol) and tert-butyl 9-(((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)carbamoyl)-3-azaspiro[5.5]undecane-3-carboxylate (74 mg, 0.085 mmol) in dichloromethane (DCM) (0.1 mL) was stirred at room temperature for 3 hours. The mixture was concentrated to provide the title compound as a solid (81 mg, 0.096 mmol, 113% yield). LC-MS m / z 769.4 (M+H) + .

[0384] Step 3 N-((1S,4R)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)-3-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carbonyl)-3-azaspiro[5.5]undecane-9-carboxamide. [ka] N-((1S,4s)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)-3-azaspiro[5.5]undecane-9-carbohydrate in dichloromethane (DCM) (0.5 mL) To a mixture of oxamide dihydrochloride (29 mg, 0.034 mmol), triethylamine (0.029 mL, 0.207 mmol), (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (7.59 mg, 0.034 mmol), and HATU (15.72 mg, 0.041 mmol) were added, and the mixture was stirred at room temperature for 5 h. Purification by ISCO CombiFlash® chromatography (24 g RediSep Rf Gold® column, 35 mL / min) eluting with a gradient of 0 to 15% methanol containing ammonium hydroxide (10%) in dichloromethane (DCM) provided the title compound as a solid (23.6 mg, 0.024 mmol, 70.5% yield). LC-MS m / z 971.3 (M+H). + . 1 H NMR(400 MHz, methanol-d4)δ ppm 0.95-1.09(m,5 H)1.12-1.33(m,6 H)1.42-1.88(m,19 H)1.91-2.02(m,1 H)2.05-2.14(m,1 H)2.16-2.37(m,7 H)2.53-2.60(m,1 H)2.62-2.77(m,5 H)2.86-2.97(m,1 H)3.23-3.29(m,1 H)3.40-3.73(m,6 H)3.81-3.87(m,1 H)3.95-4.05(m,1 H)4.56-4.65(m,1 H)4.97-5.02(m,1 H)5.25-5.34(m,1 H)7.52-7.58(m,1 H)7.83-7.88(m,1 H)7.89-7.94(m,1 H)8.04-8.09(m,1 H)8.54-8.56(m,1 H)8.57-8.61(m,2 H)8.71-8.74(m,1 H).

[0385] Example 96 (2S,3S)—N-(2-(2-(3-(4-((1R,4r)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)piperazin-1-yl)-3-oxopropoxy)ethoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 Ethyl (1r,4r)-4-(4-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecane-14-oyl)piperazin-1-yl)cyclohexane-1-carboxylate. [ka] tert-Butyl 4-((1r,4r)-4-(ethoxycarbonyl)cyclohexyl)piperazine-1-carboxylate (246 mg, 0.721 mmol) was dissolved in dichloromethane (DCM) (1.4 mL) and TFA (1.4 mL) and stirred at room temperature for 10 minutes. The mixture was concentrated under reduced pressure and chased with dichloromethane (DCM) (3 times). The residue, 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecan-14-oic acid (200 mg, 0.721 mmol) and HATU (329 mg, 0.865 mmol) were dissolved in acetonitrile (5548 μL). Triethylamine (402 μL, 2.88 mmol) was added dropwise, and the mixture was stirred at room temperature for 30 minutes. Purification by normal phase chromatography (24 g silica column, 35 mL / min) eluting with a gradient of 0-20% methanol in dichloromethane (DCM) provided the title compound (403 mg, 0.758 mmol, 105% yield). LC-MS m / z 500.5 (M+H) + .

[0386] Step 2 Ethyl (1r,4r)-4-(4-(3-(2-(2-((2S,3S)-1-thyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)ethoxy)propanoyl)piperazin-1-yl)cyclohexane-1-carboxylate. [ka] Ethyl (1r,4r)-4-(1-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecane-14-oyl)piperidin-4-yl)cyclohexane-1-carboxylate (100 mg, 0.201 mmol) was dissolved in dichloromethane (DCM) and TFA (1 mL) and stirred at room temperature for 10 minutes. The mixture was concentrated under reduced pressure and chased with dichloromethane (DCM) (3 times). The residue, (2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (40 mg, 0.182 mmol), and TSTU (65.6 mg, 0.218 mmol) were dissolved in acetonitrile (1400 μL). Triethylamine (201 μL, 1.442 mmol) was added dropwise, and the mixture was stirred at room temperature for 30 min. Purification by normal phase chromatography (12 g silica column, 30 mL / min) eluting with a gradient of 0 to 20% methanol in dichloromethane (DCM) provided the title compound (25.5 mg, 0.024 mmol, 13.30% yield). LC-MS m / z 602.3 (M+H) + .

[0387] Step 3 (1r,4r)-4-(4-(3-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)ethoxy)propanoyl)piperazin-1-yl)cyclohexane-1-carboxylic acid. [ka] Ethyl (1r,4r)-4-(4-(3-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)ethoxy)propanoyl)piperazin-1-yl)cyclohexane-1-carboxylate (25.5 mg, 0.042 mmol) was dissolved in methanol (170 μL), water (170 μL), and tetrahydrofuran (THF) (170 μL), and lithium hydroxide monohydrate (11.17 mg, 0.266 mmol) was added. The mixture was stirred at room temperature overnight and concentrated under reduced pressure. The residue was dissolved in dichloromethane (DCM) using a minimum amount of methanol, ISOLUTE® was added, and concentrated by reduced pressure. Purification by MDAP (XBridge™ column) eluting with acetonitrile in water containing ammonium carbonate modifier provided the title compound (13.2 mg, 0.018 mmol, 27.0% yield). LC-MS m / z 574.2 (M+H) + .

[0388] Step 4 (2S,3S)—N-(2-(2-(3-(4-((1R,4r)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)piperazin-1-yl)-3-oxopropoxy)ethoxy)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] tert-Butyl ((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamate (14 mg, 0.025 mmol) was dissolved in dichloromethane (DCM) (0.5 mL) and 4 M HCl in 1,4-dioxane (0.1 mL, 0.4 mmol), and the mixture was stirred at room temperature for 15 minutes. The reaction was concentrated under reduced pressure and chased with dichloromethane (DCM). The residue was dissolved in dichloromethane (DCM) (670 μL) and triethylamine (13.82 μL, 0.099 mmol), and the mixture was added to (1r,4r)-4-(4-(3-(2-(2-((2S,3S)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamido)ethoxy)ethoxy)propanoyl)piperazin-1-yl)cyclohexanecarboxylic acid (14.22 mg, 0.025 mmol) and stirred for several minutes. HATU (12.26 mg, 0.032 mmol) was added, and the mixture was stirred at room temperature for 4 hours. The mixture was concentrated under reduced pressure. Purification by MDAP (XBridge™ column) eluting with acetonitrile in water containing ammonium carbonate modifier provided the title compound as a white solid (9.8 mg, 9.61 μmol, 38.7% yield). LC-MS m / z 1020.4 (M+H) + . 1H NMR (400 MHz, methanol d4) δ ppm 0.99-1.08(m,3 H)1.12-1.20(m,3 H)1.26-1.39(m,2 H)1.48-1.60(m,2 H)1.64-1.80(m,3 H)1.92-2.36(m,14 H)2.50-2.76(m,13 H)2.79-2.90(m,1 H)3.03-3.14(m,1 H)3.42-3.58(m,12 H)3.62-3.67(m,1 H)3.69-3.77(m,2 H)3.99-4.08(m,1 H)4.64-4.68(m,1 H)4.81-4.85(m,1 H)5.21-5.31(m,1 H)7.51-7.57(m,1 H)7.77-7.85(m,1 H)7.88-7.95(m,1 H)8.04-8.11(m,1 H)8.50-8.54(m,1 H)8.56-8.63(m,2 H)8.79-8.87(m,1 H).

[0389] Example 97 (2S,3S)—N-(2-(3-(4-((1R,4S)-4-(((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-((6-(trifluoromethyl)quinazolin-4-yl)amino)pyrrolidin-1-yl)cyclohexyl)carbamoyl)cyclohexyl)piperazin-1-yl)cyclobutyl)ethyl)-1-methyl-5-oxo-2-(pyridin-3-yl)pyrrolidine-3-carboxamide. [ka] Step 1 Ethyl (1r,4r)-4-(piperazin-1-yl)cyclohexane-1-carboxylate, 2-trifluoroacetate. [ka] tert-Butyl 4-((1r,4r)-4-(ethoxycarbonyl)cyclohexyl)piperazine-1-carboxylate (220 mg, 0.646 mmol) was dissolved in dichloromethane (DCM) (1.40 mL) and T...

Claims

1. Compounds of formula (I): 【Chemical 1】 or a pharmaceutically acceptable salt thereof, During the ceremony, R １ But C １～４ Alkyl or C ３～６ is cycloalkyl, R ２ is hydrogen or C １～４ is alkyl, R ３ is hydrogen or C １～４ is alkyl, L is a bivalent linker of formula (L-a), (L-b), (L-c), (L-d), (L-e), (L-f), (L-g), (L-h), (L-i), (L-j), (L-k), (L-m), (L-ni), (L-n-ii), (L-n-iii), or (L-n-iv): 【Chemistry 2】 or a stereoisomer thereof, During the ceremony, Ring A and ring B are each independently C ４～６ is cycloalkylene, L １ａ But C ３～５ A straight chain alkylene in which one or two methylene units are -O- or -NR ａ is replaced by -, Each R ａ are independently hydrogen or C １～３ is alkyl, L ２ａ is —O—, —NHC(O)—, or —CH ２ the bivalent linker or a stereoisomer thereof, 【Chemistry 3】 or a stereoisomer thereof, During the ceremony, Ring A is C ４～６ Cycloalkylene or C ７～９ is a bridged bicyclic cycloalkylene; L １ｂ But -CH ２ -NH-C(O)-, -NHC(O)-, or -C(O)NH-; L ２ｂ But C ６～１２ A straight chain alkylene in which 1, 2, 3, or 4 methylene units are -O-, -NR １ｂ -, -C(O)NR １ｂ - or -NR １ｂ replaced by C(O)—, or L ２ｂ but, 【Chemistry 4】 and n is 1, 2, 3, or 4; 【Chemistry 5】 But, L １ｂ represents a covalent bond to Each R １ｂ are independently hydrogen or C １～３ the bivalent linker or a stereoisomer thereof, which is alkyl; 【Chemistry 6】 or a stereoisomer thereof, During the ceremony, L １ｃ But C ２～１０ a straight chain alkylene in which one, two, or three methylene units are replaced by —O—, —NH—, —NHC(O)—, or —C(O)NH—; Ring A is C ４～６ Cycloalkylene or C ７～９ is a bridged bicyclic cycloalkylene; L ２ｃ is —O— or saturated C ２～１０ the bivalent linker or a stereoisomer thereof, which is a straight chain alkylene in which one, two, or three methylene units are replaced by -O-, -NH-, -NHC(O)-, or -C(O)NH-; 【Chemistry 7】 And, During the ceremony, L １ｄ But C １２～２２ the bivalent linker which is a straight chain alkylene in which 1, 2, 3, 4, or 5 methylene units are replaced with -NH-, -O-, -C(O)NH-, -NHC(O)-, or -NHC(O)-NH-; 【Chemistry 8】 wherein n is an integer from 3 to 50; 【Chemistry 9】 or a stereoisomer thereof, During the ceremony, L １ｆ is bond, C １～６ straight-chain alkylene in which 0, 1, or 2 methylene units are replaced by —O—, —NH—, or —C(O)—; １～６ Straight chain alkylene, or -(C ３～６ cycloalkylene)-NHC(O)-; L ２ｆ is a bond, —NHC(O)—, —C(O)NH—, or C １～６ is a straight chain alkylene in which 0, 1, or 2 methylene units are replaced by —O—; Z １ and Z ２ each is independently N or CH; 【Chemistry 10】 And, During the ceremony, Ring A is a 5- to 6-membered heteroarylene having 1 or 2 nitrogen ring atoms; L １ｇ is a bond, -CH ２ -, -NH-, or -O-; L ２ｇ but, 【Chemistry 11】 wherein n is 1, 2, 3, 4, or 5; 【Chemistry 12】 But, L １ｇ the bivalent linker representing a covalent bond to 【Chemistry 13】 or a stereoisomer thereof, During the ceremony, Each Z １ is independently N or CH; L １ｈ is a bond, —C(O)—, —C(O)—NH—, or —NHC(O)—; L ２ｈ But C ２～１０ Straight chain alkylene or 【Chemistry 14】 wherein n is 1, 2, 3, or 4; 【Chemistry 15】 But, L １ｈ represents a covalent bond to 【Chemistry 16】 But, L ３ｈ represents a covalent bond to L ３ｈ is a bond, -C(O)CH ２ -, -O-(C ３～６ cycloalkylene)-O-, or -C(O)NH(CH ２ ) ３ OCH ２ - and L ４ｈ is a bond, —C(O)—, —CH ２ C(O)- or -C(O)CH ２ - and the bivalent linker or a stereoisomer thereof, wherein m is 1, 2, or 3; 【Chemistry 17】 And, During the ceremony, L １ｉ is bond, C １～１２ straight chain alkylene, or 【Chemistry 18】 wherein n is 1, 2, 3, 4, or 5; 【Chemistry 19】 But, L ３ｉ represents a covalent bond to 【Chemistry 20】 represents a covalent bond to NH; L ２ｉ is bond, C １～１２ straight chain alkylene, or 【Chemical formula 21】 wherein n is 1, 2, 3, 4, or 5; 【Chemical 22】 represents a covalent bond to HN; L ３ｉ is a bond or —C(O)—; 【Chemical 23】 or a stereoisomer thereof, During the ceremony, Z １ is C, CH, or N; Z ２ , Z ３ , Z ４ , and Z ５ are each independently CH or N, with the proviso that Z ２ , Z ３ , Z ４ , and Z ５ provided that no more than two of L １ｊ is —NH—, —C(O)NH—, —NHC(O)—, or —O—; L ２ｊ But C １～６ Straight chain alkylene or 【Chemistry 24】 wherein n is 1 or 2; 【Chemistry 25】 But, L １ｊ represents a covalent bond to 【Chemical 26】 represents a single bond or a double bond, or a stereoisomer thereof; 【Chemical 27】 or a stereoisomer thereof, During the ceremony, Ring A is phenyl or a 5- or 6-membered heteroarylene having 1 or 2 nitrogen ring atoms; Z １ and Z ２ are each independently CH or N; L １ｋ is a bond, —C(O)—, —C(O)NH—, or —NHC(O)—; L ２ｋ But C ３～８ Straight chain alkylene or 【Chemical Formula 28】 wherein n is 1, 2, or 3; 【Chemical Formula 29】 But, L １ｋ or a stereoisomer thereof, which represents a covalent bond to 【Chemistry 30】 or a stereoisomer thereof, During the ceremony, Z １ is CH or N, m is 1 or 2; p is 1 or 2; 【Chemical 31】 in which 0, 1, or 2 hydrogen atoms are replaced by F, L １ｍ is a bond, -C(O)-, -C(O)NH-, -NHC(O)-, -S(O) ２ NH- or -NHS(O) ２ - and L ２ｍ But C ３～６ Straight chain alkylene, C ３～６ cycloalkylene, or 【Chemical 32】 wherein n is 1 or 2; 【Chemical 33】 But, L １ｍ or a stereoisomer thereof, which represents a covalent bond to 【Chemical 34】 each 【Chemistry 35】 represents a covalent bond to the NH group of formula (I), and each 【Chemical 36】 is said bivalent linker, wherein represents a covalent bond to the methylene group of formula (I), or a pharmaceutically acceptable salt thereof.

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

3. R ２ is isopropyl, and R ３ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein is methyl.

4. R ２ is t-butyl, and R ３ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein is hydrogen.

5. L is a bivalent linker of formula (L-ai): 【Chemical 37】 or a stereoisomer thereof, Wherein, ring A, L １ａ , L ２ａ 、 【Chemical 38】 is as defined for formula (La), or a pharmaceutically acceptable salt thereof.

6. L is a bivalent linker of formula (L-a-ii): 【Chemical 39】 or a stereoisomer thereof, Where, L １ａ , L ２ａ 、 【Chemistry 40】 is as defined for formula (La), p is 1 or 2, and m is 1 or 2, or a pharmaceutically acceptable salt thereof.

7. L is a bivalent linker of formula (L-a-iii): 【Chemistry 41】 or a stereoisomer thereof, wherein p is 1 or 2, m is 1 or 2, and n is 1, 2, or 3; 【Chemistry 42】 is as defined for formula (La), or a pharmaceutically acceptable salt thereof.

8. L, 【Chemistry 43-1】 【Chemistry 43-2】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the bivalent linker has the formula (La) selected from the group consisting of:

9. L, 【Chemical 44】 or a stereoisomer thereof, Where, L １ｂ , L ２ｂ 、 【Chemistry 45】 is as defined for formula (L-b), p is 1 or 2, and m is 1 or 2, or a pharmaceutically acceptable salt thereof.

10. L, 【Chemistry 46-1】 【Chemistry 46-2】 【Chemistry 46-3】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the bivalent linker is of formula (Lb) selected from the group consisting of:

11. L, 【Chemistry 47】 or a stereoisomer thereof, Where, L １ｃ , L ２ｃ 、 【Chemistry 48】 is as defined for formula (Lc), p is 1 or 2, and m is 1 or 2, or a pharmaceutically acceptable salt thereof.

12. L, 【Chemistry 49】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the bivalent linker is of formula (Lc) selected from the group consisting of:

13. (i) L is 【Chemistry 50-1】 【Chemistry 50-2】 a bivalent linker of formula (L-d) selected from the group consisting of: (ii) L is 【Chemistry 51】 a bivalent linker of formula (L-f) selected from the group consisting of: (iii) L is 【Chemistry 52-1】 【Chemistry 52-2】 a bivalent linker of formula (Lh) selected from the group consisting of: (iv) L is 【Chemistry 53】 a bivalent linker of formula (Li) selected from the group consisting of: (v) L is [Chemical 54] a bivalent linker of formula (Lj) selected from the group consisting of: (vi) L is 【Chemistry 55】 a bivalent linker of formula (Lj) selected from the group consisting of: (vii) L is 【Chemical 56】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the bivalent linker has the formula (Lk) selected from the group consisting of:

14. L is a bivalent linker of formula (Lgi), 【Chemical 57】 Where, L １ｇ , L ２ｇ 、 【Chemistry 58】 is as defined for formula (L-g), and Z １ , Z ２ , and Z ３ are each independently selected from N or CH, with the proviso that Z １ , Z ２ , and Z ３ and optionally L is 【Chemical Formula 59】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the bivalent linker is of formula (Lg) selected from the group consisting of:

15. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the compounds listed in Table 1. 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】 【Table 1-7】 【Table 1-8】 【Table 1-9】 【Table 1-10】 【Table 1-11】 【Table 1-12】 【Table 1-13】 【Table 1-14】 【Table 1-15】 【Table 1-16】 【Table 1-17】 【Table 1-18】 【Table 1-19】 【Table 1-20】 【Table 1-21】 【Table 1-22】 【Table 1-23】 【Table 1-24】 【Table 1-25】 【Table 1-26】 【Table 1-27】 【Table 1-28】 【Table 1-29】 【Table 1-30】 【Table 1-31】 【Table 1-32】 【Table 1-33】 【Table 1-34】 【Table 1-35】 【Table 1-36】 【Table 1-37】 【Table 1-38】 【Table 1-39】 【Table 1-40】 【Table 1-41】 【Table 1-42】 【Table 1-43】

16. The compound is 【Chemistry 60】 10. The compound of claim 1, which is:

17. The compound of claim 1 【Hua 61】 10. The compound of claim 1, which is:

18. The compound of claim 17, 【Hua 62】 2. The compound of claim 1, wherein:

19. A pharmaceutical composition comprising: (i) a compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier, diluent, or excipient.

20. A pharmaceutical composition comprising (i) a compound according to claim 17, or a pharmaceutically acceptable salt thereof, or a compound according to claim 18, and (ii) a pharmaceutically acceptable carrier, diluent, or excipient.

21. 20. The pharmaceutical composition of claim 19 for treating and / or preventing a disease or disorder in a patient in need thereof, wherein the patient is also receiving an anti-cotinine antibody, or an antigen-binding fragment thereof, and the disease or disorder is selected from cancer, an inflammatory disease, an autoimmune disease, a viral infection, or a bacterial infection.

22. 22. The pharmaceutical composition of claim 21, wherein the disease or disorder is mediated by chemokine receptor 2 (CCR2) and / or is associated with CCR2-positive pathogenic cells.

23. 22. The pharmaceutical composition of claim 21, wherein the disease or disorder is a cancer that is a solid tumor.

24. 22. The pharmaceutical composition of claim 21, 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.

25. The pharmaceutical composition of claim 21, wherein the disease or disorder is leukemia.

26. The pharmaceutical composition of claim 21, wherein (i) the compound and the antibody, or antigen-binding fragment thereof, are administered to the patient simultaneously, or (ii) the compound and the antibody, or antigen-binding fragment thereof, are administered to the patient sequentially.

27. 20. The pharmaceutical composition of claim 19 for increasing antibody-dependent cellular cytotoxicity (ADCC) of C-C motif chemokine receptor 2 (CCR2)-expressing cells, comprising contacting the cells with an effective amount of the compound and an anti-cotinine antibody, or an antigen-binding fragment thereof, wherein the CCR2-binding portion of the compound binds to the CCR2 expressed on the cells.

28. 20. The pharmaceutical composition of claim 19 for depleting C-C motif chemokine receptor 2 (CCR2)-expressing cells, comprising contacting the cells with an effective amount of the compound and an anti-cotinine antibody, or an antigen-binding fragment thereof, wherein the CCR2-binding portion of the compound binds to the CCR2 expressed on the cells.

29. 29. The pharmaceutical composition of claim 28, 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).

30. The pharmaceutical composition of claim 21, wherein the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising a CDR1 having SEQ ID NO: 1, a CDR2 having SEQ ID NO: 2, and a CDR3 having SEQ ID NO: 3, and the light chain comprising a CDR1 having SEQ ID NO: 4, a CDR2 having SEQ ID NO: 5, and a CDR3 having SEQ ID NO:

6.

31. The pharmaceutical composition of claim 21, wherein the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) having SEQ ID NO: 7, and the light chain comprising a light chain variable region (VL) having SEQ ID NO:

8.

32. The pharmaceutical composition of claim 30, wherein the anti-cotinine antibody is an IgG1 isotype containing a substitution in the Fc region to increase ADCC activity.

33. 33. The pharmaceutical composition of claim 32, wherein the substitutions in the Fc region are S239D / I332E, where residue numbering is according to the EU index.

34. The pharmaceutical composition of claim 21 , wherein the anti-cotinine antibody has a heavy chain comprising SEQ ID NO: 9 and a light chain comprising SEQ ID NO:

10.

35. The pharmaceutical composition described in claim 25, wherein the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising a CDR1 having SEQ ID NO: 1, a CDR3 having SEQ ID NO: 2, and a CDR3 having SEQ ID NO: 3, and the light chain has a CDR1 having SEQ ID NO: 4, a CDR2 having SEQ ID NO: 5, and a CDR3 having SEQ ID NO:

6.

36. The pharmaceutical composition described in claim 28, wherein the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising a CDR1 having SEQ ID NO: 1, a CDR3 having SEQ ID NO: 2, and a CDR3 having SEQ ID NO: 3, and the light chain has a CDR1 having SEQ ID NO: 4, a CDR2 having SEQ ID NO: 5, and a CDR3 having SEQ ID NO:

6.

37. A combination comprising a compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, and an anti-cotinine antibody, or an antigen-binding fragment thereof.

38. The combination of claim 37, wherein the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising CDR1 having SEQ ID NO: 1, CDR2 having SEQ ID NO: 2, and CDR3 having SEQ ID NO: 3, and the light chain comprising CDR1 having SEQ ID NO: 4, CDR2 having SEQ ID NO: 5, and CDR3 having SEQ ID NO:

6.

39. The combination described in claim 37, wherein the anti-cotinine antibody has a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) having SEQ ID NO: 7, and the light chain comprising a light chain variable region (VL) having SEQ ID NO:

8.

40. 39. The combination of claim 38, wherein the anti-cotinine antibody is an IgG1 isotype containing substitutions in the Fc region to increase ADCC activity.

41. 41. The combination of claim 40, wherein the substitutions in the Fc region are S239D / I332E, residue numbering being according to the EU index.

42. The combination of claim 37, 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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