Compounds for treatment of kinase-dependent disorders

Abstract

To provide compounds for treating cancer.SOLUTION: Compounds of Formula I are provided, which inhibit, regulate, and / or modulate Axl and Mer receptor tyrosine kinases. Compositions containing the compounds, methods of using them to treat kinase-dependent diseases and conditions, syntheses of the compounds, and processes for formulating the compounds for pharmaceutical purposes are also provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compounds that modulate cellular activities such as proliferation, differentiation, programmed cell death, migration, and chemical invasion by modulating protein kinase enzymatic activity. Even more specifically, the present invention relates to compounds that inhibit, regulate, and / or modulate Axl and Mer receptor tyrosine kinases, compositions containing these compounds, methods of using them to treat kinase-dependent diseases and conditions, the synthesis of these compounds, and processes for formulating these compounds for pharmaceutical purposes.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 622,702, filed January 26, 2018, and U.S. Provisional Patent Application No. 62 / 758,321, filed November 9, 2018, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Human Axl belongs to the TAM subfamily of Mer-containing receptor tyrosine kinases. TAM kinases are characterized by an extracellular ligand-binding domain consisting of two immunoglobulin-like domains and two fibronectin type III domains. Axl is overexpressed in several tumor cell types and was originally cloned from a patient with chronic myeloid leukemia. When overexpressed, Axl exhibits transforming potential. Axl signaling is thought to initiate tumor growth through activation of proliferative and anti-apoptotic signaling pathways. Axl has been associated with cancers such as lung cancer, myeloid leukemia, uterine cancer, ovarian cancer, glioma, melanoma, thyroid cancer, renal cell carcinoma, osteosarcoma, gastric cancer, prostate cancer, and breast cancer. Overexpression of Axl confers a poor prognosis for patients with the indicated cancers.

[0004] Activation of Mer, like Axl, transduces downstream signaling pathways that initiate tumor growth and activation. Mer binds to ligands such as the soluble protein Gas-6. Gas-6 binding to Mer triggers autophosphorylation of Mer at its intracellular domain, resulting in downstream signal activation. Overexpression of Mer in cancer cells leads to increased metastasis, likely due to the generation of soluble Mer extracellular domain proteins as decoy receptors. Tumor cells secrete a soluble form of the extracellular Mer receptor, which reduces the ability of soluble Gas-6 ligand to activate Mer on endothelial cells, leading to cancer progression.

[0005] Therefore, there is a need for compounds that inhibit TAM receptor tyrosine kinases such as Axl and Mer to treat selected cancers. Summary of the Invention [Means for solving the problem]

[0006] In one aspect, the present invention provides a compound of formula I: [ka] or a pharmaceutically acceptable salt thereof. [In the formula, R1 is selected from the group consisting of -H, -CN, -CO-NR5R6, -CO2R7, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted (C1-C6)alkyl, optionally substituted (C3-C8)cycloalkyl, optionally substituted (C3-C6)heterocycloalkyl, -SON2NR8R9, and -(SO2)-(C1-C6)alkyl; wherein R1 is selected from the group consisting of -CN, -CO-NR5R6, -CO2R7, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted (C3-C8)cycloalkyl, optionally substituted (C3-C6)heterocycloalkyl, -SON2NR8R9, and -(SO2)-(C1-C6)alkyl, then R2 is -H, halo, -NR5R6, or optionally substituted (C1-C6)alkoxy; When R1 is -H, optionally substituted (C1-C6) alkyl, or optionally substituted (C1-C6) alkoxy, R2 is -CO-NR5R6, or -CO2R7; R1 and R2, together with the atoms to which they are attached, form an optionally substituted cycloalkyl or an optionally substituted heterocycloalkyl; R3 is selected from the group consisting of -H, optionally substituted (C1-C6) alkyl, -CN, and halo; R4 is -H or halo; [ka] is optionally substituted with 1, 2, 3, or 4 groups independently selected from the group consisting of halo and (C1-C6)alkyl, wherein [ka] indicates the point of attachment, R5 and R6 are each independently -H, optionally substituted (C1-C6) alkyl, or optionally substituted (C1-C6) alkoxy; R7 is —H or optionally substituted (C1-C6) alkyl; R8 and R9 are each independently —H or optionally substituted (C1-C6) alkyl; R8 and R9 may be linked to form an optionally substituted heterocycle; and Y is selected from the group consisting of O, S, SO, SO2, NH, and N-(C1-C6 alkyl).

[0007] Another embodiment is a compound of formula A: [ka] or a pharmaceutically acceptable salt thereof. [In the formula, (i) R1 is (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, -CN, -NHOH, -C(O)R a , -C(O)NR a R a , -C(O)NHOR a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)NR a R a , C(=NR a )R a , -C(=NOH)R a , -C(=NOH)NR a , -C(=NCN)NR a R a , -C(=NR a )NR a R a , -S(O)NR a R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, and S(O)2NR a R a and R2 is -H, halo, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 ) cycloalkyl-(C1-C4) alkylene-, (5-14 membered heteroaryl)-(C1-C4) alkylene-, (4-14 membered heterocycloalkyl)-(C1-C4) alkylene-, -CN, -NO2, -OR a , -SR a , -NHOR a , -C(O)R a , -C(O)NR a R a , -C(O)NHOR a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)R a , -OC(O)NR a R a , -NHR a , -NR a R a , -NR a C(O)R a , -NR a C(=NR a )R a , -NR a C(O)OR a , -NR a C(O)NR a R a , -C(=NR a )R a , -C(=NOH)R a , -C(=NOH)NR a , -C(=NCN)NR a R a , -NR a C(=NCN)NR a R a , -C(=NR a )NR a R a , -NR a C(=NR a )NR a R a , -NR aS(O)R a , -NR a S(O)2R a , -NR a S(O)NR a R a , -S(O)R a , -S(O)NR a R a , -S(O)2R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, and -S(O)2NR a R a wherein R1 or R2 is selected from the group consisting of (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 )cycloalkyl-(C1-C4)alkylene-, (5- to 14-membered heteroaryl)-(C1-C4)alkylene-, and (4- to 14-membered heterocycloalkyl)-(C1-C4)alkylene- each represent 1, 2, 3, 4, or 5 independently selected R b optionally substituted with substituents, provided that R1 is a 5- to 7-membered heteroaryl or a 5- to 7-membered heterocycloalkyl and R2 is C 1~6 provided that if it is alkoxy, the 5- to 7-membered heteroaryl or 5- to 7-membered heterocycloalkyl is not linked to the fused phenyl ring of the quinoline moiety through a ring nitrogen atom; or (ii) R1 is -H, halo, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, (C6-C 10 ) Aryl, (C3-C 10 ) Cycloalkyl, 5-14 membered heteroaryl alkyl, 4-14 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C10 ) cycloalkyl-(C1-C4) alkylene-, (5-14 membered heteroaryl)-(C1-C4) alkylene-, (4-14 membered heterocycloalkyl)-(C1-C4) alkylene-, -CN, -NO2, -OR a , -SR a , -NHOR a , -C(O)R a , -C(O)NR a R a , -C(O)NHOR a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)R a , -OC(O)NR a R a , -NHR a , -NR a R a , -NR a C(O)R a , -NR a C(=NR a )R a , -NR a C(O)OR a , -NR a C(O)NR a R a , -C(=NR a )R a , -C(=NOH)R a , -C(=NOH)NR a , -C(=NCN)NR a R a , -NR a C(=NCN)NR a R a , -C(=NR a )NR a R a , -NR a C(=NR a )NR a R a , -NR a S(O)R a , -NR a S(O)2R a , -NR a S(O)NR a R a , -S(O)R a, -S(O)NR a R a , -S(O)2R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, and -S(O)2NR a R a wherein R1 or R2 is selected from the group consisting of (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-14 membered heteroaryl, 4-14 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 )cycloalkyl-(C1-C4)alkylene-, (5- to 14-membered heteroaryl)-(C1-C4)alkylene-, and (4- to 14-membered heterocycloalkyl)-(C1-C4)alkylene- each represent 1, 2, 3, 4, or 5 independently selected R b is optionally substituted with a substituent; and R2 is (C2-C6) alkenyl, (C2-C6) alkynyl, -CN, -NHOH, -C(O)R a , -C(O)NR a R a , -C(O)NHOR a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)NR a R a , C(=NR a )R a , -C(=NOH)R a , -C(=NOH)NR a , -C(=NCN)NR a R a , -NR a C(=NCN)NR a R a , -C(=NR a )NRa R a , -S(O)NR a R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, and S(O)2NR a R a and R1 is selected from the group consisting of 5- to 7-membered heteroaryl or 5- to 7-membered heterocycloalkyl, and R2 is C 1~6 provided that, when R1 is alkoxy, the 5- to 7-membered heteroaryl or 5- to 7-membered heterocycloalkyl is not linked to the fused phenyl ring of the quinoline moiety through a ring nitrogen atom; (iii) R1 and R2, together with the atoms to which they are attached, form a fused (C3-C7) cycloalkyl ring or a fused 4-10 membered heterocycloalkyl ring, wherein the fused (C3-C7) cycloalkyl ring and the fused 4-10 membered heterocycloalkyl ring each contain one, two, or three independently selected R b optionally substituted with substituents, with the proviso that the compound is not 1-[2-(4-fluoro-phenyl)-acetyl]-cyclopropanecarboxylic acid [3-fluoro-4-(7,8,10,11,13,14-hexahydro-6,9,12,15-tetraoxa-1-aza-cyclododeca[b]naphthalen-4-yloxy)-phenyl]-amide; R 10 and R 11 are each independently -H, halo, (C1-C6) alkyl, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-14 membered heteroaryl, 4-14 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10) cycloalkyl-(C1-C4) alkylene-, (5-14 membered heteroaryl)-(C1-C4) alkylene-, (4-14 membered heterocycloalkyl)-(C1-C4) alkylene-, -CN, -NO2, -OR a , -SR a , -NHOR a , -C(O)R a , -C(O)NR a R a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)R a , -OC(O)NR a R a , -NHR a , -NR a R a , -NR a C(O)R a , -NR a C(=NR a )R a , -NR a C(O)OR a , -NR a C(O)NR a R a , -C(= NR a )R a , -C(=NOH)R a , -C(=NOH)NR a , -C(=NCN)NR a R a , -NR a C(=NCN)NR a R a , -C(=NR a )NR a R a , -NR a C(=NR a )NR a R a , -NR a S(O)R a , -NR a S(O)2R a , -NR a S(O)NR a R a , -S(O)R a , -S(O)NR aR a , -S(O)2R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, and S(O)2NR a R a wherein R1 or R2 is selected from the group consisting of (C1-C6) alkyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-14 membered heteroaryl, 4-14 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 )cycloalkyl-(C1-C4)alkylene-, (5- to 14-membered heteroaryl)-(C1-C4)alkylene-, and (4- to 14-membered heterocycloalkyl)-(C1-C4)alkylene- each represent 1, 2, 3, 4, or 5 independently selected R b is optionally substituted with a substituent; Each R3 is independently selected from the group consisting of -H, halo, -OH, -CN, optionally substituted (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkoxy, -NH2, -NH(C1-C6)alkyl, -N(C1-C6 alkyl)2, and (C3-C6)cycloalkyl, wherein the (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N(C1-C6 alkyl)2, and (C3-C6)cycloalkyl of R3 each are selected from the group consisting of 1, 2, or 3 independently selected R g is optionally substituted with a substituent; Each R 14are independently halo, -OH, -NH, -CN, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -COOH, -NH(C1-C6)alkyl, -N(C1-C6 alkyl)2, phenyl, phenyl-(C1-C2)alkylene, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl-(C1-C4)alkylene-, 4- to 6-membered heterocycloalkyl, (4- to 6-membered heterocycloalkyl)-(C1-C4)alkylene-, 5- to 6-membered heteroaryl, (5- to 6-membered heteroaryl)-(C1-C4)alkylene-, and -OR e wherein R 14 wherein the (C1-C6)alkyl, phenyl, phenyl-(C1-C2)alkylene, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl-(C1-C4)alkylene-, 4- to 6-membered heterocycloalkyl, (4- to 6-membered heterocycloalkyl)-(C1-C4)alkylene-, 5- to 6-membered heteroaryl, and (5- to 6-membered heteroaryl)-(C1-C4)alkylene- each represent one, two, or three independently selected R g is optionally substituted with a substituent; R 15 is H or C 1~6 is alkyl, Each R4 independently represents -H, halo, -OH, or -COOR e , -CONR e R e , -CN, -NH2, -NH((C1-C6) alkyl), -N((C1-C6) alkyl)2, (C1-C6) alkyl, (C1-C6) alkoxy, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, -CONR a R a , -NR a COR a , -NR a CONR a R a , -SO2R a , -NR a S(O)2R a , -NR a S(O)NR a R a, (C3-C6)cycloalkyl, 4- to 6-membered heterocycloalkyl, phenyl, 5- or 6-membered heteroaryl, (C3-C6)cycloalkyl-(C1-C4)alkylene-, (4- to 6-membered heterocycloalkyl)-(C1-C4)alkylene-, phenyl-(C1-C2)alkylene, and (5- or 6-membered heteroaryl)-(C1-C4)alkylene-, wherein the (C1-C6) alkylene of R4 is alkyl, (C3-C6)cycloalkyl, 4- to 6-membered heterocycloalkyl, phenyl, 5- or 6-membered heteroaryl, (C3-C6)cycloalkyl-(C1-C4)alkylene-, (4- to 6-membered heterocycloalkyl)-(C1-C4)alkylene-, phenyl-(C1-C2)alkylene, and (5- or 6-membered heteroaryl)-(C1-C4)alkylene- each represent one, two, or three independently selected R f is optionally substituted with a substituent; Each R a are independently -H, -CN, (C1-C6) alkyl, (C1-C6) haloalkyl (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-14 membered heteroaryl, 4-14 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 )cycloalkyl-(C1-C4)alkylene-, (5- to 14-membered heteroaryl)-(C1-C4)alkylene-, and (4- to 14-membered heterocycloalkyl)-(C1-C4)alkylene-, wherein R a The (C1-C6) alkyl, (C1-C6) haloalkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-14 membered heteroaryl, 4-14 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10)cycloalkyl-(C1-C4)alkylene-, (5- to 14-membered heteroaryl)-(C1-C4)alkylene-, and (4- to 14-membered heterocycloalkyl)-(C1-C4)alkylene- each represent 1, 2, 3, 4, or 5 independently selected R d is optionally substituted with a substituent; Each R b are independently halo, oxo, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 ) cycloalkyl-(C1-C4) alkylene-, (5-10 membered heteroaryl)-(C1-C4) alkylene-, (4-10 membered heterocycloalkyl)-(C1-C4) alkylene-, -CN, -OH, -NH2, -NO2, -NHOR c , -OR c , -SR c , -C(O)R c , -C(O)NR c R c , -C(O)OR c , -C(O)NR c S(O)2R c , -OC(O)R c , -OC(O)NR c R c , -C(=NOH)R c , -C(=NOH)NR c , -C(=NCN)NR c R c , -NR c C(=NCN)NR c R c , -C(=NR c )NR c R c , -NR c C(=NR c )NR c R c , -NHR c , -NR cR c , -NR c C(O)R c , -NR c C(=NR c )R c , -NR c C(O)OR c , -NR c C(O)NR c R c , -NR c S(O)R c , -NR c S(O)2R c , -NR c S(O)NR c R c , -S(O)R c , -S(O)NR c R c , -S(O)2R c , -S(O)NR c C(O)R c , -Si(R c )3, -P(O)R c R c , -P(O)(OR c )(OR c ), -B(OH)2, -B(OR c )2, and -S(O)2NR c R c wherein R b (C1-C6) alkyl, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 )cycloalkyl-(C1-C4)alkylene-, (5- to 10-membered heteroaryl)-(C1-C4)alkylene-, and (4- to 10-membered heterocycloalkyl)-(C1-C4)alkylene- can each further be selected from one, two, or three independently selected R d is optionally substituted with a substituent; Each R care independently -H, (C1-C6) alkyl, (C1-C6) haloalkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 )cycloalkyl-(C1-C4)alkylene-, (5-10 membered heteroaryl)-(C1-C4)alkylene-, and (4-10 membered heterocycloalkyl)-(C1-C4)alkylene-, wherein R c (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 )cycloalkyl-(C1-C4)alkylene-, (5- to 10-membered heteroaryl)-(C1-C4)alkylene-, and (4- to 10-membered heterocycloalkyl)-(C1-C4)alkylene- are respectively , 1, 2, 3, 4, or 5 independently selected R f is optionally substituted with a substituent; Each R d are independently (C1-C6) alkyl, (C1-C6) haloalkyl, halo, (C6-C 10 ) aryl, 5-10 membered heteroaryl, (C3-C 10 ) cycloalkyl, 4-10 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 ) cycloalkyl-(C1-C4) alkylene-, (5-10 membered heteroaryl)-(C1-C4) alkylene-, (4-10 membered heterocycloalkyl)-(C1-C4) alkylene-, -CN, -NH2, -NHOR e , -OR e , -SR e , -C(O)Re , -C(O)NR e R e , -C(O)OR e , -OC(O)R e , -OC(O)NR e R e , -NHR e , -NR e R e , -NR e C(O)R e , -NR e C(O)NR e R e , -NR e C(O)OR e , -C(=NR e )NR e R e , -NR e C(=NR e )NR e R e , -NR e C(=NOH)NR e R e , -NR e C(=NCN)NR e R e , -S(O)R e , -S(O)NR e R e , -S(O)2R e , -NR e S(O)2R e , -NR e S(O)NR e R e , and -S(O)NR e R e wherein R d (C1-C6) alkyl, (C1-C6) haloalkyl, (C6-C 10 ) aryl, 5-10 membered heteroaryl, (C3-C 10 ) cycloalkyl, 4-10 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10)cycloalkyl-(C1-C4)alkylene-, (5- to 10-membered heteroaryl)-(C1-C4)alkylene-, and (4- to 10-membered heterocycloalkyl)-(C1-C4)alkylene- are each selected from one, two, or three independently selected R f is optionally substituted with a substituent; Each R e are independently -H, (C1-C6) alkyl, (C3-C6) cycloalkyl, (C3-C6) cycloalkyl-(C1-C4) alkylene-, (C6-C 10 ) Aryl, (C6-C 10 )aryl-(C1-C4)alkylene-, 5- or 6-membered heteroaryl, (5- or 6-membered heteroaryl)-(C1-C4)alkylene-, 4- to 7-membered heterocycloalkyl, (4- to 7-membered heterocycloalkyl)-(C1-C4)alkylene-, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, (C2-C4)alkenyl, and (C2-C4)alkynyl, wherein R e (C1-C4) alkyl, (C3-C6) cycloalkyl, (C6-C 10 ) aryl, 5- or 6-membered heteroaryl, 4- to 7-membered heterocycloalkyl, (C6-C 10 )aryl-(C1-C4)alkylene-, (5- or 6-membered heteroaryl)-(C1-C4)alkylene-, (4- to 7-membered heterocycloalkyl)-(C1-C4)alkylene-, (C2-C4)alkenyl, and (C2-C4)alkynyl each have one, two, or three R f optionally substituted with a substituent, or Any two R a The substituents, together with the nitrogen atom to which they are attached, may each be one, two, or three independently selected R f forming a 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered heterocycloalkyl optionally substituted with substituents; Any two R c The substituents, together with the nitrogen atom to which they are attached, may each be one, two, or three independently selected R fforming a 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered heterocycloalkyl optionally substituted with substituents; Any two R e The substituents, together with the nitrogen atom to which they are attached, may each be one, two, or three independently selected R f forming a 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered heterocycloalkyl optionally substituted with substituents; Each R f is independently selected from the group consisting of halo, -OH, -CN, -COOH, -NH, -NH-(C1-C6)alkyl, -N((C1-C6)alkyl), (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)alkylthio, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, phenyl, 5- to 6-membered heteroaryl, 4- to 6-membered heterocycloalkyl, and (C3-C6)cycloalkyl; f (C1-C6) alkyl, phenyl, (C3-C6) cycloalkyl, 4- to 6-membered heterocycloalkyl, and 5- to 6-membered heteroaryl are each halo, —OH, —CN, —COO H, -NH2, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, (C1-C4) haloalkoxy, phenyl, (C3-C 10 ) optionally substituted with 1, 2, or 3 substituents selected from cycloalkyl, 5- to 6-membered heteroaryl, and 4- to 6-membered heterocycloalkyl; Each R g are independently selected from the group consisting of halo, -OH, -CN, -COOH, -COO-(C1-C4)alkyl, -NH2, -NH-(C1-C6)alkyl, -N((C1-C6)alkyl)2, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)alkylthio, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, phenyl, 5- to 6-membered heteroaryl, 4- to 6-membered heterocycloalkyl, and (C3-C6)cycloalkyl; Y is selected from —O—, —S—, —SO—, —SO2—, —NH—, and —N((C1-C6)alkyl)-; The ring nitrogen atom on the quinoline moiety in Formula A is optionally oxidized; the subscript n is an integer of 1, 2, 3, or 4; the subscript m is an integer of 1, 2, 3, 4, or 5; and The subscript p is an integer 0, 1, 2, 3, or 4.

[0008] Another aspect provides a method of using a compound of Formula I, or a pharmaceutically acceptable salt thereof, to treat a disease, disorder, or syndrome mediated at least in part by modulating the in vivo activity of a protein kinase.

[0009] A further aspect provides a process for making compounds of Formula A and Formula I.

[0010] These and other aspects and embodiments are described below. DETAILED DESCRIPTION OF THE INVENTION

[0011] Abbreviations and Definitions The following abbreviations and terms have the meanings indicated below throughout this specification: [Table 5-1] [Table 5-2]

[0012] The symbol "-" means a single bond and "=" means a double bond.

[0013] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0014] Where a variable is generically defined with several possible substituents, each individual radical may be defined with or without a bond. For example, R z can be hydrogen, this means that Rz In the definition of, it may be indicated as "-H" or "H".

[0015] When chemical structures are depicted or described, all carbons are assumed to have hydrogen substitutions in accordance with a valence of four unless expressly stated otherwise. For example, in the structure on the left in the diagram below, nine hydrogens are meant to be present. The nine hydrogens are depicted in the structure on the right. Sometimes, specific atoms in a structure are described in the text formula as having one hydrogen or multiple hydrogens (well-defined hydrogens) as substitutions, e.g., -CHCH-. Those skilled in the art will appreciate that the above-described descriptive techniques are common in the chemical arts to simplify and simplify the description of otherwise complex structures. [ka]

[0016] The group "R" may, for example, be of the formula: [ka] When depicted as "floating" on a ring system, as in, unless otherwise defined, the substituent "R" may be located on any atom of the ring system, and replacement of a shown, implied, or explicitly defined hydrogen from one of the ring atoms is assumed so long as a stable structure is created.

[0017] For example, the expression: [ka] When the group "R" is depicted as floating on a fused ring system, as in, unless otherwise defined, the substituent "R" may be present on any atom of the fused ring system, and replacement of a depicted hydrogen (e.g., -NH- in the formula above), an implied hydrogen (e.g., in the formula above, a hydrogen is not shown but is understood to be present), or an explicitly defined hydrogen (e.g., in the formula above, "Z" equals =CH-) from one of the ring atoms is assumed so long as a stable structure is formed. In the depicted example, the "R" group may be present on either the 5- or 6-membered ring of the fused ring system. When the group "R" is present, for example, in the formula: [ka] When depicted as being present on a ring system containing saturated carbons, such as in: [where in this example, "y" can be more than one], each is assumed to replace a currently depicted, implied, or explicitly defined hydrogen on the ring, and is not otherwise defined. As long as the resulting structure is stable, two "R"s can be on the same carbon. A simple example is when R is a methyl group, and there can be a geminal dimethyl on the carbon of the ring shown (an "annular" carbon). In another example, two Rs on the same carbon, including that carbon, can form a ring, thus, for example, [ka] As in, a spirocyclic ring ("spirocyclyl" group) structure is created that includes the ring shown.

[0018] "Halogen" or "halo" refers to fluorine, chlorine, bromine, or iodine.

[0019] "C n~m " or "C n ~C m The term "" denotes an inclusive range, where n and m are integers and indicate the number of carbons. Examples include C 1~4 , C1~C4, C 1~6, C1 to C6, etc.

[0020] "Alkyl" refers to a branched or straight hydrocarbon chain of 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, hexyl, and heptyl. (C1-C6) alkyl is preferred. n~m alkyl" or (C n ~C m The term alkyl refers to an alkyl group having n to m carbon atoms. When optionally substituted, one or more (e.g., 1 to 4, 1 to 2, or 1) hydrogen atoms of the alkyl group can be replaced with a moiety as described below under "optionally substituted." In some embodiments, the alkyl group is unsubstituted or not optionally substituted.

[0021] "Alkylene" refers to an optionally substituted divalent saturated aliphatic radical having 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 2 carbon atoms. When optionally substituted, one or more (e.g., 1 to 4, 1 to 2, or 1) hydrogen atoms of the alkylene group can be replaced with a moiety as described below under "optionally substituted." In some embodiments, an alkylene group is unsubstituted or not optionally substituted. "C n~m The term "alkylene" refers to an alkylene group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, methylene, ethane-1,2-diyl, propane-1,3-diyl, propane-1,2-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, 2-methyl-propane-1,3-diyl, and the like.

[0022] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more double carbon-carbon bonds. An alkenyl group formally corresponds to an alkene with one C-H bond replaced by the point of attachment of the alkenyl group to the remainder of the compound. n~malkenyl" or (C n ~C m The term alkenyl refers to an alkenyl group having n to m carbons. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.

[0023] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more triple carbon-carbon bonds. An alkynyl group formally corresponds to an alkyne with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. n~m alkynyl" or (C n ~C m The term alkynyl refers to an alkyl group consisting of n to m "Alkynyl" refers to an alkynyl group having 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0024] "Alkoxy" refers to a moiety of the formula -OR', where R' is a (C1-C6) alkyl moiety as defined herein. n~m Alkoxy" or (C n ~C m The term alkoxy refers to an alkoxy group whose alkyl group has n to m carbons. Examples of alkoxy moieties include, but are not limited to, methoxy, ethoxy, isopropoxy, and the like.

[0025] An alkoxy group can be unsubstituted or optionally substituted. When optionally substituted, one or more (e.g., 1 to 4, 1 to 2, or 1) hydrogen atoms of the alkoxy group can be replaced with a moiety as described below under "optionally substituted," provided that the hydrogen atom alpha to the ether oxygen is not replaced with a hydroxy, amino, or thio group. In some embodiments, an alkoxy group is unsubstituted or not optionally substituted.

[0026] "Alkoxycarbonyl" refers to the group -C(O)-R', where R' is (C1-C6)alkoxy as defined herein.

[0027] The term "amino" refers to a group of formula -NH2.

[0028] The term "carbamyl" refers to a group of formula -C(O)NH2.

[0029] The term "carbonyl," used alone or in combination with other terms, refers to a -C(=O)- group, which may also be written as C(O).

[0030] The term "cyano" or "nitrile" refers to a group of formula -C≡N, which may also be written as -CN or CN.

[0031] The term "oxo" refers to an oxygen atom as a divalent substituent, which when attached to carbon forms a carbonyl group, or when attached to a heteroatom forms a sulfoxide or sulfone group, or an N-oxide group. In some embodiments, heterocyclic groups can be optionally substituted with one or two oxo (=O) substituents.

[0032] The term "sulfide" refers to a sulfur atom as a divalent substituent, which when attached to carbon forms a thiocarbonyl group (C=S).

[0033] As used herein, the term "heteroatom" is intended to include boron, phosphorus, sulfur, oxygen, and nitrogen.

[0034] The term "haloalkyl," as used herein, refers to an alkyl group in which one or more of the hydrogen atoms are replaced by a halogen atom. n~m haloalkyl" or (C n ~C m The term haloalkyl refers to a C alkyl group having n to m carbon atoms and at least 1 to {2(n to m)+1} halogen atoms, which may be the same or different. n~m In some embodiments, the halogen atom is a fluoro atom. In some embodiments, the haloalkyl group has 1 to 6 or 1 to 4 carbon atoms. Examples of haloalkyl groups include CF, C2F5, CHF2, CCl3, CHCl2, C2Cl5, and the like. In some embodiments, the haloalkyl group is a fluoroalkyl group.

[0035] The term "haloalkoxy", used alone or in combination with other terms, refers to a radical of the formula -O-haloalkyl, where the haloalkyl radical is as defined above. n~m haloalkoxy" or (C n ~C m The term haloalkoxy refers to a haloalkoxy group where the haloalkyl group has n to m carbon atoms. Examples of haloalkoxy groups include trifluoromethoxy, and the like. In some embodiments, the haloalkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0036] "Aryl" means a monovalent 6- to 14-membered monocyclic or bicyclic carbocyclic ring (e.g., having two fused rings), wherein the monocyclic ring is aromatic and at least one of the rings of the bicyclic ring is aromatic. n~m aryl" or "(C n ~C mThe term "aryl" refers to an aryl group having n to m ring carbon atoms. In some embodiments, an aryl group has 6 to about 10 carbon atoms. In some embodiments, an aryl group has 6 carbon atoms. In some embodiments, an aryl group has 10 carbon atoms. Unless otherwise stated, the valency of the group may be located on any atom of any ring within the radical, valence rules permitting. Representative examples include phenyl, naphthyl, indanyl, and the like.

[0037] An aryl group can be unsubstituted or optionally substituted. When optionally substituted, one or more (e.g., 1 to 5, 1 to 2, or 1) hydrogen atoms of the aryl group can be replaced with a moiety as described below under "optionally substituted." In some embodiments, an alkoxy group is unsubstituted or not optionally substituted.

[0038] "Arylene" means a divalent 6- to 14-membered monocyclic or bicyclic carbocyclic ring, wherein the monocyclic ring is aromatic and at least one of the rings of the bicyclic ring is aromatic. Representative examples include phenylene, naphthylene, and indanylene.

[0039] "Cycloalkyl" refers to a non-aromatic hydrocarbon ring system (monocyclic, bicyclic, or polycyclic) including cyclized alkyl and alkenyl groups. n~m cycloalkyl" or "(C n ~C m The term "cycloalkyl" refers to a cycloalkyl having n to m ring carbon atoms. Cycloalkyl groups can include monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) groups and spirocycles. Cycloalkyl groups can have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ring carbon atoms (C 3~14In some embodiments, the cycloalkyl group has 3 to 14 ring members, 3 to 10 ring members, 3 to 6 ring members, 3 to 5 ring members, or 3 to 4 ring members. In some embodiments, the cycloalkyl group is monocyclic. In some embodiments, the cycloalkyl group is monocyclic or bicyclic. In some embodiments, the cycloalkyl group is C 3~6 Monocyclic cycloalkyl groups. Ring-forming carbon atoms of a cycloalkyl group may be optionally oxidized to form oxo or sulfido groups. Cycloalkyl groups also include cycloalkylidene. In some embodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, bicyclo[1.1.1]pentanyl, bicyclo[2.1.1]hexanyl, and the like. In some embodiments, cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, cycloalkyl includes a single saturated carbocyclic ring of 3 to 8 ring carbons, such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The cycloalkyl may be optionally substituted with one or more substituents, such as 1, 2, or 3 substituents. wherein the cycloalkyl substituent is selected from the group consisting of (C1-C6)alkyl, hydroxy, (C1-C6)alkoxy, halo(C1-C6)alkyl, halo(C1-C6)alkoxy, halo, amino, mono- and di(C1-C6)alkylamino, hetero(C1-C6)alkyl, acyl, aryl, and heteroaryl.

[0040] A cycloalkyl group can be unsubstituted or optionally substituted. When optionally substituted, one or more (e.g., 1 to 4, 1 to 2, or 1) hydrogen atoms of the cycloalkyl group can be replaced with a moiety as described below under "optionally substituted." In some embodiments, a substituted cycloalkyl group can incorporate an exo- or endocyclic alkene (e.g., cyclohex-2-en-1-yl). In some embodiments, a cycloalkyl group is unsubstituted or not optionally substituted.

[0041] "Cycloalkyloxycarbonyl" means the group -C(O)-OR', where R' is (C3-C6)cycloalkyl as defined herein.

[0042] "Phenyloxycarbonyl" refers to the group -C(O)-Ophenyl.

[0043] "Heteroaryl" refers to -O-, -S(O) n"R" means a monovalent monocyclic, fused bicyclic, or fused tricyclic radical of 5 to 14 ring atoms containing one or more, preferably 1, 2, 3, or 4, ring heteroatoms independently selected from -(n is 0, 1, or 2), -N-, and -N(R')-, with the remaining ring atoms being carbon, where the ring containing the monocyclic radical is aromatic and at least one of the fused rings containing the bicyclic or tricyclic radical is aromatic. One or two ring carbon atoms of any non-aromatic ring containing the bicyclic or tricyclic radical may be replaced by a -C(O)-, -C(S)-, or -C(=NH)- group. R' is hydrogen, alkyl, hydroxy, alkoxy, acyl, or alkylsulfonyl. Unless otherwise stated, valence may be located on any atom of any ring of the heteroaryl group, valence rules permitting. In particular, when the point of valence is located on a nitrogen, no additional nitrogen substituents are present.More specifically, the term heteroaryl includes, but is not limited to, 1,2,4-triazolyl, 1,3,5-triazolyl, phthalimidyl, pyridinyl, pyrrolyl, imidazolyl, thienyl, furanyl, indolyl, 2,3-dihydro-1H-indolyl (including, for example, 2,3-dihydro-1H-indol-2-yl or 2,3-dihydro-1H-indol-5-yl), isoindolyl, indolinyl, isoindolinyl, benzimidazolyl, benzodioxol-4-yl, benzofuranyl, cinnolinyl, indolizinyl, naphthyridin-3-yl, phthalazin-3-yl, phthalazin-4-yl, pteridinyl, purinyl, quinazolinyl, quinoxalinyl, tetrazoyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl, oxadiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl (including, for example, tetrahydroisoquinolin-4-yl or tetrahydroisoquinolin-6-yl), pyrrolo[3,2-c]pyridinyl (including, for example, pyrrolo[3,2-c]pyridin-2-yl or pyrrolo[3,2-c]pyridin-7-yl), benzopyranyl, thiazolyl, isothiazolyl, thiadiazolyl, benzothiazolyl, benzothienyl, and derivatives thereof, and N-oxides or protected derivatives thereof.

[0044] A 5-membered heteroaryl ring is a heteroaryl group having 5 ring atoms, where one or more (e.g., 1, 2, 3, or 4) ring atoms are independently selected from N, O, and S. Exemplary 5-membered heteroaryls include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxazolyl, and the like. Includes azolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.

[0045] A 6-membered heteroaryl ring is a heteroaryl group having 6 ring atoms, where one or more (e.g., 1, 2, 3, or 4) ring atoms are independently selected from N, O, and S. Exemplary 6-membered heteroaryl rings are pyridyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl.

[0046] "Heteroarylene" refers to -O-, -S(O) n -(n is 0, 1, or 2), -N-, and -N(R 19 )-, and the remaining ring atoms are carbon, where the ring containing the monocyclic radical is aromatic and at least one of the fused rings containing the bicyclic or tricyclic radical is aromatic. One or two ring carbon atoms of any non-aromatic ring containing the bicyclic or tricyclic radical may be replaced by a -C(O)-, -C(S)-, or -C(=NH)- group. R 19 is hydrogen, alkyl, or alkenyl. Unless otherwise stated, valence may be located on any atom of any ring of the heteroarylene group, if valence rules permit. Specifically, when the point of valence is located on nitrogen, there are no additional nitrogen substituents. More specifically, the term heteroaryl includes, but is not limited to, thien-diyl, benzo[d]isoxazole-diyl, benzo[d]isothiazole-diyl, 1H-indazole-diyl (wherein R is at the N1 position). 19 benzo[d]oxazole-diyl, benzo[d]thiazole-diyl, 1H-benzo[d]imidazole-diyl (optionally substituted at N1 position with R 19 1H-benzo[d][1,2,3]triazole-diyl (optionally substituted at N1 position with R 19optionally substituted with), imidazo[1,2-a]pyridine-diyl, cinnoline-diyl, quinoline-diyl, pyridine-diyl, 1-oxide-pyridine-diyl, [1,2,4]triazolo[4,3-a]pyridine-diyl, and 2,3-dihydroimidazo[1,2-a]pyridine-diyl.

[0047] As used herein, "heterocycloalkyl" or "heterocyclo" refers to a non-aromatic ring or ring system having 4 to 14 ring members, 4 to 10 ring members, 4 to 7 ring members, or 4 to 6 ring members, which may optionally contain one or more alkenylene groups as part of the ring structure and have at least one heteroatom ring member independently selected from boron, nitrogen, sulfur, oxygen, and phosphorus. The term "heterocycloalkyl" includes monocyclic 4-, 5-, 6-, and 7-membered heterocycloalkyl groups. Heterocycloalkyl groups can include monocyclic, bicyclic, or polycyclic (e.g., having 2 or 3 fused or bridged rings) ring systems or spirocycles. In some embodiments, heterocycloalkyl groups are monocyclic groups having 1, 2, or 3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. The ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group may be optionally oxidized to form oxo or sulfido groups or other oxidized bonds (e.g., C(O), S(O), C(S), S(O), N-oxide, etc.), or the nitrogen atom may be quaternized. A heterocycloalkyl group may be bonded through a ring-forming carbon atom or ring-forming heteroatom. In some embodiments, a heterocycloalkyl group contains zero to three double bonds. In some embodiments, a heterocycloalkyl group contains zero to two double bonds. Included within the definition of heterocycloalkyl are moieties having one or more aromatic rings fused to (i.e., having a bond in common with) a heterocycloalkyl ring, e.g., a benzo or thienyl derivative such as piperidine, morpholine, azepine, etc. Heterocycloalkyl groups containing fused aromatic rings may be bonded through any ring-forming atom, including a ring-forming atom of the fused aromatic ring. Heterocycloalkyl Exemplary groups include azetidinyl, azepanyl, dihydrobenzofuranyl, dihydrofuranyl, dihydropyranyl, morpholino, 3-oxa-9-azaspiro[5.5]undecanyl, 1-oxa-8-azaspiro[4.5]decanyl, piperidinyl, piperazinyl, oxopiperazinyl, pyranyl, pyrrolidinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydropyranyl, 1,2,3,4-tetrahydroquinolinyl, tropanyl, 4,5,6,7-tetrahydrothiazolo[5,4-c]pyridinyl, and thiomorpholino.

[0048] A "heterocycloalkyl" or "heterocyclo" can be unsubstituted or optionally substituted. If optionally substituted, one or more (e.g., 1 to 4, 1 to 2, or 1) hydrogen atoms of the group can be replaced with a moiety independently selected from fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some aspects, a substituted heterocyclo group can incorporate an exo- or endocyclic alkene (e.g., cyclohex-2-en-1-yl). In some aspects, a heterocyclo group is unsubstituted or not optionally substituted.

[0049] Optional substitutions Unless otherwise specified, a group is optionally substituted. The term "optionally substituted" refers to substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, carbocycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups are optionally substituted. "Optionally substituted" refers to a group that may be substituted or unsubstituted (e.g., a "substituted" or "unsubstituted" alkyl, a "substituted" or "unsubstituted" alkenyl, a "substituted" or "unsubstituted" alkynyl, a "substituted" or "unsubstituted" "substituted" or "unsubstituted" cycloalkyl, a "substituted" or "unsubstituted" heterocycloalkyl, a "substituted" or "unsubstituted" aryl, or a "substituted" or "unsubstituted" heteroaryl group). In general, the term "substituted" means that at least one hydrogen present on the group is replaced with an acceptable substituent, for example, a substituent that results in a stable compound, for example, a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, or other reaction. Unless otherwise specified, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituents can be the same or different at each position. The term "substituted" is intended to include substitution with all permissible substituents of organic compounds, and includes any of the substituents described herein that result in the formation of stable compounds. The present invention contemplates any and all such combinations in order to yield stable compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituents as described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety. It is not intended that the present invention be limited in any way by the exemplary substituents described herein.

[0050] Exemplary carbon atom substituents include, but are not limited to, halogen (halo), —CN, —NO 2 , —N 3 , —SO 2 H, —SO 3 H, —OH, —OR aa , -ON(Rbb )2、-N(R bb )2、-N(R bb )3 + X -、 -N(OR cc )R bb 、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc )2、-CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3、-C(=S)N(R bb )2、-C(=O)SRaa , -C(=S)SR aa , -SC(=S)SR aa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)2R aa , -OP(=O)2R aa , -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -OP(R cc )2, -OP(R cc )3, -B(OR cc )2, -BR aa (OR cc ), C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, (C3-C 10 ) carbocycloalkyl, 3-14 membered heterocycloalkyl, (C6-C 14 ) aryl, and 5- to 14-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5R dd or substituted with a group Two geminal hydrogens on one carbon atom can be bonded to the groups =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O)2R aa , =NRbb , or =NOR cc has been replaced by R aa Each example is independently (C1~C 10 ) alkyl, (C1-C 10 ) Perhaloalkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, (C3-C 10 ) cycloalkyl, 3-14 membered heterocycloalkyl, (C6-C 14 ) aryl, and 5- to 14-membered heteroaryl, or two R aa The groups taken together form a 3- to 14-membered heterocycloalkyl or 5- to 14-membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R dd is substituted with a group, R bb Each example is independently hydrogen, (C1 to C 10 ) Perhaloalkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, (C3-C 10 ) cycloalkyl, C 6~14 aryl, and 5- to 14-membered heteroaryl, or two R bb The groups taken together form a 3- to 14-membered heterocycloalkyl or 5- to 14-membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R dd is substituted with a group, R cc Each example is independently hydrogen, (C1 to C 10 ) alkyl, (C1-C 10 ) Perhaloalkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, (C3-C 10 ) cycloalkyl, 3-14 membered heterocycloalkyl, (C6-C 14) aryl, and 5- to 14-membered heteroaryl, or two R cc The groups taken together form a 3- to 14-membered heterocycloalkyl or 5- to 14-membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R dd is substituted with a group, R dd Each example is independently a halogen, -CN, -NO2, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3 + X -、 -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO2H, -CO2R ee , -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(R ff )2, -C(=NR ff ) OR ee , -OC(=NR ff )R ee , -OC(=NR ff ) OR ee , -C(=NR ff )N(R ff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R f f )2, -NR ff SO2R ee , -SO2N(Rff )2, -SO2R ee , -SO2OR ee , -OSO2R ee , -S(=O)R ee , -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)2R ee , -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, (C1~C 10 ) alkyl, (C1-C 10 ) Perhaloalkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, (C3-C 10 ) cycloalkyl, 3-10 membered heterocycloalkyl, (C6-C 10 ) aryl, and 5- to 10-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R gg substituted with a group or two geminal R dd the substituents together may form =O or =S; R ee Each example is independently (C1-C6) alkyl, (C1-C6) perhaloalkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C3-C 10 ) cycloalkyl, (C6-C 10 ) aryl, 3- to 10-membered heterocycloalkyl, and 3- to 10-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R gg is substituted with a group, R ffEach example is independently hydrogen, (C1-C6) alkyl, (C1-C6) perhaloalkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C3-C 10 ) cycloalkyl, (C6-C 10 ) aryl, and 5- to 10-membered heteroaryl, or two R ff The groups taken together form a 3- to 10-membered heterocycloalkyl or 5- to 10-membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R gg is substituted with a group, and R gg Each example is independently a halogen, -CN, -NO2, -SO2H, -SO3H, -OH, -OC 1~6 Alkyl, -ON(C 1~6 alkyl)2, -N(C 1~6 alkyl)2, -N(C 1~6 Alkyl)3 + X -、 -NH(C 1~6 alkyl)2 + X -、 -NH2(C 1~6 alkyl) + X -、 -NH3 + X -、 -N(OC 1~6 Alkyl)(C 1~6 alkyl), -N(OH)(C 1~6 alkyl), -NH(OH), -SH, -SC 1~6 Alkyl, -SS(C 1~6 alkyl), -C(=O)(C 1~6 alkyl), -CO2H, -CO2(C 1~6 alkyl), -OC(=O)(C 1~6 alkyl), -OCO2(C 1~6 alkyl), -C(=O)NH2, -C(=O)N(C 1~6 alkyl)2, -OC(=O)NH(C 1~6 alkyl), -NHC(=O)(C 1~6 alkyl), -N(C 1~6 alkyl)C(=O)(C1~6 alkyl), -NHCO2(C 1~6 alkyl), -NHC(=O)N(C 1~6 alkyl)2, -NHC(=O)NH(C 1~6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1~6 alkyl), -OC(=NH)(C 1~6 alkyl), -OC(=NH)OC 1~6 Alkyl, -C(=NH)N(C 1~6 alkyl)2, -C(=NH)NH(C 1~6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1~6 alkyl)2, -OC(NH)NH(C 1~6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1~6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1~6 alkyl), -SO2N(C 1~6 alkyl)2, -SO2NH(C 1~6 alkyl), -SO2NH2, -SO2C 1~6 Alkyl, -SO2OC 1~6 Alkyl, -OSO2C 1~6 Alkyl, -SOC 1~6 Alkyl, -Si(C 1~6 alkyl)3, -OSi(C 1~6 alkyl)3, -C(=S)N(C 1~6 alkyl)2, C(=S)NH(C 1~6 alkyl), C(=S)NH2, -C(=O)S(C 1~6 alkyl), -C(=S)SC 1~6 Alkyl, -SC(=S)SC 1~6 Alkyl, -P(=O)2(C 1~6 alkyl), -P(=O)(C 1~6 alkyl)2, -OP(=O)(C 1~6 alkyl)2, -OP(=O)(OC 1~ 6 alkyl), (C1-C6) alkyl, (C1-C6) perhaloalkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C3-C 10 ) cycloalkyl, (C6-C 10) aryl, 3- to 10-membered heterocycloalkyl, 5- to 10-membered heteroaryl, or two geminal R gg The substituents may be taken together to form =O or =S, where X - is the counter ion.

[0051] As noted above, nitrogen atoms may be substituted or unsubstituted, where valence allows, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, (C1~C 10 ) alkyl, (C1-C 10 ) Perhaloalkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, (C3-C 10 )cycloalkyl, 3-14 membered heterocycloalkyl, (C6-C14)aryl, and 5-14 membered heteroaryl, or two R bonded to the N atom ccand the groups taken together form a 3- to 14-membered heterocycloalkyl or 5- to 14-membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R dd substituted with an R aa , R bb , R cc and R dd is as defined above.

[0052] In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to herein as an "amino protecting group"). Nitrogen protecting groups include, but are not limited to, -OH, -OR aa , -N(R cc )2, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc )R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , (C1~C 10 ) alkyl (e.g., aralkyl, heteroaralkyl), (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, (C3-C 10 ) cycloalkyl, 3-14 membered heterocycloalkyl, (C6-C 14 ) aryl, and 5- to 14-membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R ddsubstituted with an R aa , R bb , R cc , and R dd is as defined herein. Nitrogen protecting groups are well known in the art and include those described in Protecting Groups in Organic Synthesis, T.W. Greene, incorporated herein by reference. and PGMWuts,3 rd edition, John Wiley & Sons, 1999.

[0053] For example, a nitrogen protecting group such as an amide group (e.g., —C(═O)R aa ), including, but not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide , 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0054] Nitrogen protecting groups such as carbamate groups (e.g., -C(=O)OR aa), including, but not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethyl Silylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bu meoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropyl allyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, Alkyl dithiocarbamates, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitribenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthiophenylcarbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzylcarbamate, p-(dihydroxyboryl)benzylcarbamate carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzylthiocarbamate, p -Cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate bamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-, Examples include methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

[0055] Nitrogen protecting groups such as sulfonamide groups (e.g., -S(=O)R aa ), including, but not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0056] Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilyla 1,3-Dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrroline) )-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylamine N-methylthiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylborinic acid derivatives, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidate, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro, These include 4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridine sulfenamide (Npys).

[0057] In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also referred to herein as a "hydroxyl protecting group"). Oxygen protecting groups include, but are not limited to, -R aa , -N(R bb )2, -C(=O)SR aa , -C(=O)R aa , -CO2R aa , -C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb ) OR aa , -C(=NR bb )N(R bb )2, -S(=O)R aa , -SO2R aa , -Si(R aa )3, -P(R cc )2, -P(R cc )3, -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)(OR cc )2, -P(=O)2N(R bb )2, and -P(=O)(NR bb )2, where R aa , Rbb , and R cc is as defined herein. Oxygen protecting groups are well known in the art and include those described in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Buts, 3, incorporated herein by reference. rd edition, John Wiley & Sons, 1999.

[0058] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl. methyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]phenyl] ]-4-Methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloro Ethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-Dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"-tris(benzoyl oxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropyl, Silyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyl-t-hexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxide Acetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyl dithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl Carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl methylthiocarbonate, S-benzylthiocarbonate, 4-ethoxy-1-napthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

[0059] In certain embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also referred to as a "thiol protecting group"). Sulfur protecting groups include, but are not limited to, -R aa , -N(R bb )2, -C(=O)SR aa , -C(=O)R aa , -CO2R aa , -C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb ) OR aa , -C(=NR bb )N(R bb )2, -S(=O)R aa , -SO2R aa , -Si(R aa )3, -P(R cc )2, -P(R cc )3, -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)(OR cc )2, -P(=O)2N(R bb )2, and -P(=O)(NR bb )2, where R aa , R bb , and R ccis as defined herein. Sulfur protecting groups are well known in the art and include those described in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Buts, 3, incorporated herein by reference. rd edition, John Wiley & Sons, 1999.

[0060] As used herein, "leaving group" (LG) is an art-recognized term that refers to a molecular fragment that leaves with a pair of electrons upon heterolytic bond cleavage, where the molecular fragment is an anion or a neutral molecule. As used herein, a leaving group refers to a group that is displaced by a nucleophile. The leaving group may be a displaceable atom or group. See, for example, Smith, March Advanced Organic Chemistry 6th ed. (501-502). Exemplary leaving groups include, but are not limited to, halo (e.g., chloro, bromo, iodo), -OR aa (When the O atom is bonded to a carbonyl group, R aa is as defined herein), —O(C═O)R LG , or -O(SO)2R LG (e.g., tosyl, mesyl, besyl), where R LG is an optionally substituted alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl. In certain embodiments, the leaving group is a halogen.

[0061] The terms defined above are specifically exemplified in the Examples.

[0062] The "yield" for each of the reactions described herein is expressed as a percentage of the theoretical yield.

[0063] For purposes of the present invention, a "patient" includes humans and any other animals, particularly mammals, and other living organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In a preferred embodiment, the patient is a mammal, and in a most preferred embodiment, the patient is human. Examples of preferred mammals include mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, and primates.

[0064] "Kinase-dependent diseases or conditions" refer to pathological conditions that depend on the activity of one or more kinases. Kinases are directly or indirectly involved in the signal transduction pathways of various cellular activities, including proliferation, adhesion, migration, differentiation, and invasion. Diseases associated with kinase activity include tumor growth and pathological angiogenesis that supports solid tumor growth and is associated with other diseases involving excessive local angiogenesis, such as ocular diseases (e.g., diabetic retinopathy, age-related macular degeneration), and inflammation (e.g., psoriasis, rheumatoid arthritis).

[0065] A "therapeutically effective amount" is an amount of a compound of the present invention that, when administered to a patient, ameliorates the symptoms of the disease. The amount of a compound of the present invention that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the age of the patient being treated, etc. A therapeutically effective amount can be routinely determined by one of ordinary skill in the art having regard to their own knowledge and this disclosure.

[0066] "Cancer" includes, but is not limited to: Heart: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; Head and neck: squamous cell carcinoma of the head and neck, laryngeal and hypopharyngeal carcinoma, nasal cavity and paranasal sinus carcinoma, nasopharyngeal carcinoma, salivary gland carcinoma, oral cavity and oropharyngeal carcinoma; Lung: bronchial carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma, non-small cell lung carcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma Tumor, chondromatous hamartoma, mesothelioma, Colon: colorectal carcinoma, adenocarcinoma, gastrointestinal stromal tumor, lymphoma, carcinoid tumor, Turcot's syndrome, Gastrointestinal: gastric cancer, adenocarcinoma of the gastroesophageal junction, Esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), Stomach (carcinoma, lymphoma, leiomyosarcoma), Pancreas (tubular adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), Small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma) , lipoma, neurofibroma, fibroma), colon (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), breast: metastatic breast cancer, ductal carcinoma in situ, invasive ductal carcinoma, tubular carcinoma, medullary carcinoma, mucinous carcinoma, lobular carcinoma in situ, triple-negative breast cancer, genitourinary tract: kidney (adenocarcinoma, Wilms' tumor [nephroblastoma], lymphoma, leukemia, renal cell carcinoma), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, urothelial carcinoma), prostate (adenocarcinoma, sarcoma, castration-resistant prostate adenocarcinoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, chorioepithelioma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenocarcinoma, lipoma), clear cell carcinoma, papillary carcinoma, liver: hepatocellular carcinoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (microglioma), multiple myeloma, malignant giant cell tumor chordoma, Osteochrondroma (osteochondral exostosis), benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid, and giant cell tumor; Thyroid: medullary thyroid carcinoma, differentiated thyroid carcinoma, papillary thyroid carcinoma, follicular thyroid carcinoma, Hürthle cell carcinoma, and anaplastic thyroid carcinoma; Nervous system: skull (osteoma, hemangioma, granuloma, xanthomas, osteitis deformans); Meninges (meningiomas, meningeal sarcomas, gliomatosis); Brain (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor [pinealoma], glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumor), spinal neurofibroma, meningioma, glioma, sarcoma), Gynecology: Uterus (endometrial cancer), Cervix (cervical cancer, preneoplastic cervical dysplasia), Ovarian (ovarian cancer [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified cancer], granulosa-theca cell tumor, "cancerous cell" refers to cell proliferative conditions including: Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonic rhabdomyosarcoma), fallopian tube (carcinoma), blood system: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma], skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis, and adrenal gland: neuroblastoma. Accordingly, the term "cancerous cell" as provided herein includes a cell afflicted with any one of the above-identified conditions.

[0067] The term "pharmaceutically acceptable salts" includes "pharmaceutically acceptable acid addition salts" and "pharmaceutically acceptable base addition salts." A "pharmaceutically acceptable acid addition salt" refers to a salt formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, which retains the biological effectiveness of the free base and is not biologically or otherwise undesirable.

[0068] "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Exemplary salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine (see, e.g., S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977;66:1-19, incorporated herein by reference).

[0069] The term "compound," as used herein, is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures. The term is also intended to refer to compounds of the present invention regardless of the method by which they are prepared, for example, synthetically, by a biological process (e.g., metabolic or enzymatic transformation), or by a combination thereof.

[0070] Compounds of the invention may include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[0071] Any one of the process steps or sequences disclosed and / or claimed herein may be carried out under an inert gas atmosphere, more particularly under argon or nitrogen. Additionally, the method of the present invention may be carried out as a semi-continuous or continuous process, more preferably as a continuous process.

[0072] Additionally, many of the process steps and sequences described herein may be shortened.

[0073] In general, the nomenclature used in this application is based on the naming conventions adopted by the International Union of Pure and Applied Chemistry (IUPAC). The chemical structures shown herein were generated using CHEMDRAW®. Any open valence present on a carbon, oxygen, or nitrogen atom in the structures herein indicates the presence of a hydrogen atom.

[0074] Embodiments of the present invention One embodiment is a compound of formula A: [ka] or a pharmaceutically acceptable salt thereof, wherein the variables and substituents in Formula A are as defined in the Summary of the Invention.

[0075] In one embodiment of this aspect, the compound of formula A is a compound of formula A-1: [ka] is.

[0076] In another embodiment of this aspect, the compound of formula A is a compound of formula A-2: [ka] is.

[0077] In a further embodiment of this aspect, the compound of formula A is a compound of formula A-3: [ka] [Wherein, R a1 is —H or (C1-C6) alkyl].

[0078] In a further embodiment, R 1 in the compound of formula A-3 is —H.

[0079] In a further embodiment of this aspect, the compound of formula A is a compound of formula A-4: [ka] wherein ring A is a 5- to 14-membered heteroaryl and subscript r is 1, 2, 3, or 4.

[0080] In this embodiment, R2 is -H.

[0081] In a further embodiment, r in formula A-4 is 1 or 2.

[0082] In a further embodiment of Formula A: R1 is -H, optionally substituted (C1-C 6) Alkyl, halo, -OR a , -NO2, -NH2, -NHR a , -NR a R a , -SR a , -SOR a , or -S(O)2R a and R2 is (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) Cycloalkyl, -CN, -NHOR a , -C(O)R a , -C(O)NR a R a、 -C(O)NHOR a 、 -C(O)OR a 、 -C(O)NR a S(O)2R a 、 -OC(O)NR a R a 、 NR a C(O)R a 、 -NR a C(=NR a )R a 、 -NR a C(O)OR a 、 -NR a C(O)NR a R a 、 -C(=NR a )R a 、 -C(=NOH)R a 、 -C(=NOR a )R a 、 -C(=NOH)NR a 、 -C(=NCN)NR a R a 、 -NR a C(=NCN)NR a R a 、 -C(=NR a )NR a R a 、 -NR a C(=NR a )NR a R a 、 -NR a S(O)R a 、 -NR a S(O)2R a 、 -NR a S(O)2NR a R a 、 -S(O)R a 、 -S(O)NR a R a 、 -S(O)2R a 、 -S(O)2NR a C(O)R a 、 -P(O)R a R a 、 -P(O)(OR a )(OR a )、 -B(OH)2、 -B(OR a )2、 and -S(O)2NR a R ais selected from the group consisting of:

[0083] In one embodiment of this embodiment, R1 is -H.

[0084] In a further embodiment, R1 is selected from the group consisting of (C2-C6)alkenyl, (C2-C6)alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, -CN, -NHOR a , -C(O)R a , -C(O)NR a R a , -C(O)NHOR a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)NR a R a , -NR a C(O)R a , -NR a C(=NR a )R a , -NR a C(O)OR a , -NR a C(O)NR a R a , -C(=NR a )R a , -C(=NOH)R a , -C(=NOH)NR a , -C(=NOR a )R a , -C(=NCN)NR a R a , -NR a C(=NCN)NR a R a , -C(=NR a )NR a R a , -NR a C(=NR a )NR a R a , -NR a S(O)R a , -NR a S(O)2R a, -NR a S(O)NR a R a , -S(O)R a , -S(O)NR a R a , -S(O)2R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, and -S(O)2NR a R a and R2 is -H, optionally substituted (C1-C6) alkyl, halo, -OR a , -NO2, -NH2, -NHR a , -NR a R a , -SR a , -SOR a , or -S(O)R a is.

[0085] In one embodiment of this embodiment, R2 is -H.

[0086] In another embodiment: R1 is (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, -CN, -NHOR a , -C(O)R a , -C(O)NR a R a , -C(O)NHOR a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)NR a R a , -NR a C(O)R a , -NR a C(=NRa )R a , -NR a C(O)OR a , -NR a C(O)NR a R a , -C(=NR a )R a , -C(=NOH)R a , -C(=NOR a )R a , -C(=NOH)NR a , -C(=NCN)NR a R a , -NR a C(=NCN)NR a R a , -C(=NR a )NR a R a , -NR a C(=NR a )NR a R a , -NR a S(O)R a , -NR a S(O)2R a , -NR a S(O)NR a R a , -S(O)R a , -S(O)NR a R a , -S(O)2R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, or -S(O)2NR a R a and R2 is -H, halo, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, (C 6~C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10) cycloalkyl-(C1-C4) alkylene-, (5-14 membered heteroaryl)-(C1-C4) alkylene-, (4-14 membered heterocycloalkyl)-(C1-C4) alkylene-, -CN, -NO2, -OR a , -SR a , -NHOR a , -C(O)R a , -C(O)NR a R a , -C(O)NHOR a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)R a , -OC(O)NR a R a , -NHR a , -NR a R a , -NR a C(O)R a , -NR a C(=NR a )R a , -NR a C(O)OR a , -NR a C(O)NR a R a , -C(=NR a )R a , -C(=NOH)R a , -C(=NOH)NR a , -C(=NOR a )R a , -C(=NCN)NR a R a , -NR a C(=NCN)NR a R a , -C(=NR a )NR a R a , -NR a C(=NR a )NR a R a , -NR a S(O)R a , -NR a S(O)2R a , -NR a S(O)NR a Ra , -S(O)R a , -S(O)NR a R a , -S(O)2R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, and -S(O)2NR a R a is selected from the group consisting of:

[0087] In one embodiment of this embodiment, R2 is -H.

[0088] In another embodiment: R1 is -H, halo, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-14 membered heteroaryl, 4-14 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 ) cycloalkyl-(C1-C4) alkylene-, (5-14 membered heteroaryl)-(C1-C4) alkylene-, (4-14 membered heterocycloalkyl)-(C1-C4) alkylene-, -CN, -NO2, -OR a , -SR a , -NHOR a , -C(O)R a , -C(O)NR a R a , -C(O)NHOR a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)R a , -OC(O)NR a R a , -NHR a , -NR a Ra , -NR a C(O)R a , -NR a C(=NR a )R a , -NR a C(O)OR a , -NR a C(O)NR a R a , -C(=NR a )R a , -C(=NOH)R a , -C(=NOR a )R a , -C(=NOH)NR a , -C(=NCN)NR a R a , -NR a C(=NCN)NR a R a , -C(=NR a )NR a R a , -NR a C(=NR a )NR a R a , -NR a S(O)R a , -NR a S(O)2R a , -NR a S(O)NR a R a , -S(O)R a , -S(O)NR a R a , -S(O)2R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, and -S(O)2NR a R a is selected from the group consisting of:

[0089] In one embodiment of this embodiment, R1 is -H.

[0090] In another further embodiment: R1 and R2, together with the atoms to which they are attached, form a fused (C3-C7) cycloalkyl ring or a fused 4-10 membered heterocycloalkyl ring, wherein the fused (C3-C7) cycloalkyl ring or the fused 4-10 membered heterocycloalkyl ring each contains one, two, or three independently selected R b Optionally substituted with a substituent, provided that the compound is not 1-[2-(4-fluoro-phenyl)-acetyl]-cyclopropanecarboxylic acid [3-fluoro-4-(7,8,10,11,13,14-hexahydro-6,9,12,15-tetraoxa-1-aza-cyclododeca[b]naphthalen-4-yloxy)-phenyl]-amide.

[0091] In a further embodiment: R1 and R2, together with the atoms to which they are attached, form a fused (C3-C7) cycloalkyl ring or a fused 4-10 membered heterocycloalkyl ring. wherein the fused (C3-C7) cycloalkyl ring or the fused 4- to 10-membered heterocycloalkyl ring each comprises one, two, or three independently selected R b Optionally substituted with a substituent, provided that the compound has the formula: [ka] wherein ring E is a fused 4-10 membered heterocycloalkyl.

[0092] In another embodiment, R in the compound of formula A, A-1, or A-3 is —H, (C-C) alkyl, (C-C) alkenyl, (C-C) alkynyl, —C(═NO—(C-C) alkyl)R a , -CN, -C(O)OR a , -C(O)NR a R a , -C(O)NHOR a , -S(O)NR a R a, phenyl, 5- to 6-membered heteroaryl, (C3-C6)cycloalkyl, and 4- to 6-membered heterocycloalkyl.

[0093] In another embodiment, R1 is -H, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, -C(=NO-(C1-C6) alkyl)R a , -CN, -C(O)OR a , -C(O)NR a R a , -C(O)NHOR a , -S(O)NR a R a , phenyl, 5- to 6-membered heteroaryl, (C-C)cycloalkyl, and 4- to 6-membered heterocycloalkyl; and R2 is H, optionally substituted (C1-C6) alkyl, halo, -OR a , -NO2, -NH2, -NHR a , -NR a R a , -SR a , -SOR a , or -S(O)2R a is.

[0094] In another embodiment, R1 is -H, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, -C(=NO-(C1-C6) alkyl)R a , -CN, -C(O)OR a , -C(O)NR a R a , -C(O)NHOR a , -S(O)NR a R a , phenyl, 5-6 membered heteroaryl, (C-C)cycloalkyl; and 4-6 membered heterocycloalkyl; and R2 is -H, halo, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, (C6-C 10 ) Aryl, (C3-C 10) cycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 ) cycloalkyl-(C1-C4) alkylene-, (5-14 membered heteroaryl)-(C1-C4) alkylene-, (4-14 membered heterocycloalkyl)-(C1-C 4- ) Alkylene -, -CN, -NO2, -OR a , -SR a , -NHOR a , -C(O)R a , -C(O)NR a R a , -C(O)NHOR a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)R a , -OC(O)NR a R a , -NHR a , -NR a R a , -NR a C(O)R a , -NR a C(=NR a )R a , -NR a C(O)OR a , -NR a C(O)NR a R a , -C(=NR a )R a , -C(=NOH)R a , -C(=NOH)NR a , -C(=NOR a )R a , -C(=NCN)NR a R a , -NR a C(=NCN)NR a R a , -C(=NR a )NR a R a , -NR a C(=NR a )NR a R a , -NR a S(O)R a , -NRa S(O)2R a , -NR a S(O)NR a R a , -S(O)R a , -S(O)NR a R a , -S( O)2R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, and -S(O)2NR a R a is selected from the group consisting of:

[0095] In another embodiment, R1 is -H, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, -C(=NO-(C1-C6) alkyl)R a , -CN, -C(O)OR a , -C(O)NR a R a , -C(O)NHOR a , -S(O)NR a R a , phenyl, 5- to 6-membered heteroaryl, -(C3-C6)cycloalkyl, and 4- to 6-membered heterocycloalkyl; and R2 is (C2-C6) alkenyl, (C2-C6) alkynyl, -CN, -NHOR a , -C(O)R a , -C(O)NR a R a , -C(O)NHOR a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)NR a R a , -NR a C(O)R a , -NR a C(=NR a )R a, -NR a C(O)OR a , -NR a C(O)NR a R a , -C(=NR a )R a , -C(=NOH)R a , -C(=NOH)NR a , -C(=NOR a )R a , -C(=NCN)NR a R a , -NR a C(=NCN)NR a R a , -C(=NR a )NR a R a , -NR a C(=NR a )NR a R a , -NR a S(O)R a , -NR a S(O)2R a , -NR a S(O)NR a R a , -S(O)R a , -S(O)NR a R a , -S(O)2R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, or -S(O)2NR a R a is.

[0096] In a further embodiment, R is -H, R a NHC(O)-, R a OC(O)-, (C1-C6) alkyl, (C1-C6) alkoxy, or -C(=NO-CH3)R aand R2 is selected from 2-methoxyethylamino, azetidin-1-yl, methylamino, 3-morpholinopropoxy, 2-methoxyethoxy, 2-hydroxyethoxy, propoxy, 2-hydroxypropoxy, methoxycarbonyl, carboxy, carbamoyl, methylcarbamoyl, (2-hydroxyethoxy)carbamoyl, (2,2-dihydroxyethoxy)carbamoyl, (oxetan-3-yloxy)carbamoyl, methoxycarbamoyl, 2-trimethylsilylethynyl, ethynyl, sulfamoyl, acetyl, and —C(═NOCH3)CH3.

[0097] In a further embodiment of Formula A and A-2, R2 is -H, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, -C(=NO-(C1-C6) alkyl)R a , -CN, -C(O)OR a , -C(O)NR a R a , -C(O)NHOR a , and -S(O)NR a R a is selected from the group consisting of:

[0098] In a further embodiment, R1 is -H, optionally substituted (C1-C6) alkyl, halo, -OR a , -NO2, -NH2, -NHR a , -NR a R a , -SR a , -SOR a , or S(O)2R a and R2 is -H, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, -C(=NO-(C1-C6) alkyl)R a , -CN, -C(O)OR a , -C(O)NR a R a , -C(O)NHOR a , -S(O)NR a R a, phenyl, and (C3-C6)cycloalkyl.

[0099] In a further embodiment, R1 is -H, optionally substituted (C1-C6) alkyl, halo, -OR a , -NO2, -NH2, -NHR a , -NR a R a , -SR a , -SOR a , and -S(O)R a and R2 is -H, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, -C(=NO-(C1-C6) alkyl)R a , -CN, -C(O)OR a , -C(O)NR a R a , -C(O)NHOR a , -S(O)NR a R a A group consisting of are selected.

[0100] In a further embodiment, R1 is selected from the group consisting of (C2-C6)alkenyl, (C2-C6)alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) Cycloalkyl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, -CN, -NHOR a , -C(O)R a , -C(O)NR a R a , -C(O)NHOR a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)NR a R a , -NR a C(O)R a , -NR a C(=NR a )R a , -NR a C(O)OR a , -NR aC(O)NR a R a , -C(=NR a )R a , -C(=NOH)R a , -C(=NOH)NR a , -C(=NOR a )R a , -C(=NCN)NR a R a , -NR a C(=NCN)NR a R a , -C(=NR a )NR a R a , -NR a C(=NR a )NR a R a , -NR a S(O)R a , -NR a S(O)2R a , -NR a S(O)NR a R a , -S(O)R a , -S(O)NR a R a , -S(O)2R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, and -S(O)2NR a R a and R2 is -H, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, -C(=NO-(C1-C6) alkyl)R a , -CN, -C(O)OR a , -C(O)NR a R a , -C(O)NHOR a , -S(O)NR a R a is selected from the group consisting of:

[0101] In a further embodiment, R1 is -H, halo, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-14 membered heteroaryl, 4-14 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 ) cycloalkyl-(C1-C4) alkylene-, (5-14 membered heteroaryl)-(C1-C4) alkylene-, (4-14 membered heterocycloalkyl)-(C1-C4) alkylene-, -CN, -NO2, -OR a , -SR a , -NHOR a , -C(O)R a , -C(O)NR a R a , -C(O)NHOR a , -C(O)OR a , -C(O)NR a S(O)2R a , -OC(O)R a , -OC(O)NR a R a , -NHR a , -NR a R a , -NR a C(O)R a , -NR a C(=NR a )R a , -NR a C(O)OR a , -NR a C(O)NR a R a , -C(=NR a )R a , -C(=NOH)R a , -C(=NOR a )R a , -C(=NOH)NR a , -C(=NCN)NR a R a , -NR a C(=NCN)NR a Ra , -C(=NR a )NR a R a , -NR a C(=NR a )NR a R a , -NR a S(O)R a , -NR a S(O)2R a , -NR a S(O)NR a R a , -S(O)R a , -S(O)NR a R a , -S(O)2R a , -S(O)NR a C(O)R a , -P(O)R a R a , -P(O)(OR a )(OR a ), -B(OH)2, -B(OR a )2, and -S(O)2NR a R a and R2 is -H, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, -C(=NO-(C1-C6) alkyl)R a , -CN, -C(O)OR a , -C(O)NR a R a , -C(O)NHOR a , -S(O)NR a R a is selected from the group consisting of:

[0102] In a further embodiment, R1 is 2-methoxyethylamino, azetidin-1-yl, methylamino, 3-morpholinopropoxy, 2-methoxyethoxy, 2-hydroxyethoxy, propoxy, 2-hydroxypropoxy, methoxycarbonyl, carboxy, carbamoyl, methylcarbamoyl, 2-oxazolyl, pyrazol-3-yl, pyrazol-4-yl, 4-isoxazolyl, 3,5-dimethylisoxazol-4-yl, 1-methyl-pyrazol-4-yl, 2-methyl-pyrazol-3-yl, 2-ethyl-pyrazol-3-yl, 2-(2-hydroxyethyl)-pyrazol-3-yl, 2-( 2,2,2-trifluoroethyl)-pyrazol-3-yl, 2-(2-fluoroethyl)-pyrazol-3-yl, 2-(2,2-difluoroethyl)-pyrazol-3-yl, 2-trifluoromethyl-pyrazol-3-yl, 2-difluoromethyl-pyrazol-3-yl, 1-methyl-imidazol-4-yl, 1-methyl-imidazol-2-yl, 1H-imidazol-2-yl, (2-hydroxyethoxy)carbamoyl, (2,2-dihydroxyethoxy)carbamoyl, (oxetan-3-yloxy)carbamoyl, methoxycarbamoyl, 2-trimethylsilylethynyl, ethynyl, 1,3,4-oxadiazol-3-yl, 1H-1,2,3-triazol-5-yl, sulfamoyl, acetyl, and —C(═NOCH)CH; and R2 is -H, -R a NHC(O)-, -R a OC(O)-, (C1-C6) alkyl, (C1-C6) alkoxy, or -C(=NO-CH3)R a is.

[0103] In a further embodiment of Formula A-4, the subscript r is 1 or 2.

[0104] In a further embodiment of the above aspects and embodiments, R 10 and R 11 are -H, respectively.

[0105] In a further embodiment of the above aspects and embodiments, the subscript n is 1.

[0106] In a further embodiment of the above aspects and embodiments, the subscript m is 1.

[0107] In a further embodiment of the above aspects and embodiments, the subscript p is 1.

[0108] In another embodiment, the compound of formula A is a compound of formula B: [ka] or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are as defined in (i), (ii), or (iii) of Formula A, and R3, R 10 , R 11 , R 14 , R4, n, p, m and Y are as defined below: each R3 is independently selected from the group consisting of -H, halo, -OH, -CN, optionally substituted (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkoxy, -NH2, -NH(C1-C6)alkyl, -N(C1-C6 alkyl)2, and (C3-C6)cycloalkyl, wherein each of (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N(C1-C6 alkyl)2, and (C3-C6)cycloalkyl is optionally substituted; R 10 and R 11 each is independently selected from the group consisting of —H, (C1-C6)alkyl, (C1-C6)alkoxy, and (C1-C6)haloalkoxy; Each R 14 are independently -halo, -OH, -NH2, -CN, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -COOH, -NH(C1-C6)alkyl, -N(C1-C6alkyl)2, phenyl -C1-C2 alkylene, phenyl-(C1-C2) alkylene, (C3-C6) cycloalkyl, (C3-C6) cycloalkyl-(C1-C4) alkylene-, 4- to 6-membered heterocycloalkyl, (4- to 6-membered heterocycloalkyl)-(C1-C4) alkylene-, 5- to 6-membered heteroaryl, (5- to 6-membered heteroaryl)-(C1-C4) alkylene-, and -OR e wherein R 14 wherein the (C1-C6)alkyl, phenyl, phenyl-(C1-C2)alkylene, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl-(C1-C4)alkylene-, 4- to 6-membered heterocycloalkyl, (4- to 6-membered heterocycloalkyl)-(C1-C4)alkylene-, 5- to 6-membered heteroaryl, and (5- to 6-membered heteroaryl)-(C1-C4)alkylene- are each optionally substituted; each R4 is independently selected from -H, halo, -OH, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, and (C1-C6)haloalkoxy, wherein the (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, and (C1-C6)haloalkoxy are each independently optionally substituted; Each R a are independently -H, (C1-C6) alkyl, (C1-C6) haloalkyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-14 membered heteroaryl, 4-14 membered heterocycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 )cycloalkyl-(C1-C4)alkylene-, (5- to 14-membered heteroaryl)-(C1-C4)alkylene-, and (4- to 14-membered heterocycloalkyl)-(C1-C4)alkylene-, wherein (C1-C6)alkyl, (C1-C6)haloalkyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-14 membered heteroaryl, 4-14 membered heterocycloalkyl, (C6-C 10) aryl-(C1-C4) alkylene-, (C3-C 10 ) cycloalkyl-(C1-C4) alkylene-, (5- to 14-membered heteroaryl)-(C1-C4) alkylene-, and (4- to 14-membered heterocycloalkyl)-(C1-C4) alkylene- are each independently optionally substituted; n, p, and m are each independently an integer of 0 to 3, and Y is selected from —O—, —S—, —SO—, —SO 2 —, —NH—, and —N((C 1 -C 6 )alkyl)-.

[0109] In one embodiment of Formulas A and B: (i) R1 is: (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-10 membered heteroaryl, and 4-10 membered heterocycloalkyl (wherein the (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each independently optionally substituted), —CN, —P(O)R a R a , P(O)(OR a )2, B(OH)2, B(OR a )2, X2R a (In this case, X2 is -NHO-, -NH-S(O)-, -N-(C1-C6)alkyl-S(O)-, -NH-S(O)2-, -N-(C1-C6)alkyl-S(O)2R a -, -NH-S(O)-NH-, -N-(C1-C6)alkyl-S(O)NH-, -NH-S(O)2NH-, -N-(C1-C6)alkyl-S(O)2NH-, -S(O)2NHC(O)-, and [ka] (where, [ka] indicates a connection point: Y1 is absent, -NH-, -N-(C1-C6)alkyl-, or -O-; Y2 is absent or is -O-, -NH-, -NHO-, -N-(C1-C6)alkyl-, -N2H2-, -NH-S(O)-, or -NH-S(O)2-; or Y2 is optionally substituted [ka] where: [ka] indicates the point of attachment, ring A is a 3-, 4-, 5-, 6-, or 7-membered ring, and Z 1 is O, NH, N-(C1-C6) alkyl, NOH, NO-(C1-C6) alkyl, or NCN) and R2: -H or: (C1-C6) alkyl, halo, -NO2, and X1R a wherein X1 is a group selected from the group consisting of -O-, -S-, -SO-, -SO2-, -SON2NH-, -SON2NR a -, -NH-, and -N-(C1-C6)alkyl-, wherein (C1-C6)alkyl is optionally substituted.

[0110] In another embodiment of Formulas A and B: (ii) R1 is: (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, 5-10 membered heteroaryl, and 4-10 membered heterocycloalkyl (wherein (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C 10 ) Aryl, (C3-C 10) cycloalkyl, 5- to 14-membered heteroaryl, and 4- to 14-membered heterocycloalkyl are each independently optionally substituted; P(O)R a R a , P(O)(OR a )(OR a ), B(OH)2, B(OR a )2, CN, X2R a (In this case, X2 is -NHO-, -NH-S(O)-, -N-(C1-C6)alkyl-S(O)-, -NH-S(O)2-, -N-(C1-C6)alkyl-S(O)2R a -, -NH-S(O)-NH-, -N-(C1-C6)alkyl-S(O)NH-, -NH-S(O)2NH-, -N-(C1-C6)alkyl-S(O)2NH-, -S(O)-, -S(O)2-, -S(O)2NHC(O), and [ka] (where: Y1 is absent, -NH-, -N-(C1-C6) alkyl-, or -O-. can be, Y2 is absent or is -O-, -NH-, -NHO-, -N-(C1-C6)alkyl-, -N2H2-, -NH-S(O)-, or -NH-S(O)2; or Y2 is optionally substituted [ka] where: [ka] indicates the point of attachment, ring A is a 3-, 4-, 5-, 6-, or 7-membered ring, Z 1 is -O-, -NH-, -N-(C1-C6)alkyl-, -NOH-, -NO-(C1-C6)alkyl-, or -NCN- and R2 is H, halo, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 ) cycloalkyl-(C1-C4) alkylene-, (5-14 membered heteroaryl)-(C1-C4) alkylene-, and (4-14 membered heterocycloalkyl)-(C1-C4) alkylene- (wherein (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, (C6-C 10 ) Aryl, (C3-C 10 ) cycloalkyl, (C6-C 10 ) aryl-(C1-C4) alkylene-, (C3-C 10 ) cycloalkyl-(C1-C4)alkylene-, (5- to 14-membered heteroaryl)-(C1-C4)alkylene-, and (4- to 14-membered heterocycloalkyl)-(C1-C4)alkylene- are each independently optionally substituted; CN, NO2, P(O)R a R a , P(O)(OR a )(OR a ), B(OH)2, B(OR a )2, X1R a (wherein X1 is -O-, -S-, -NH-, or -N-(C1-C6)-), -NHO-, -NH-S(O)-, -N-(C1-C6)alkyl-S(O)-, -NH-S(O)2-, -N-(C1-C6)alkyl-S(O)2-, -NH-S(O)-NH-, -N-(C1-C6)alkyl-S(O)NH-, -NH-S(O)2NH-, -N-(C1-C6)alkyl-S(O)2NH-, -S(O)2NHC(O)-, -NH-S(O)R a -, -N-(C1-C6)alkyl-S(O)R a -, -NH-S(O)R a -, -N-(C1-C6)alkyl-S(O)2R a -, and [ka] (where: Y1 is absent, -NH-, -N-(C1-C6)alkyl-, or -O-; Y2 is absent or is -O-, -NH-, -NHO-, -N-(C1-C6)alkyl-, -N2H2-, -NH-S(O)-, or -NH-S(O)2-; or Y2 is optionally substituted [ka] wherein ring A is a 3-, 4-, 5-, 6-, or 7-membered ring; [ka] indicates the point of attachment, and Z 1 is O, NH, N-(C1-C6) alkyl, NOH, NO-(C1-C6) alkyl, or NCN) is selected from the group consisting of:

[0111] In another embodiment of Formulas A and B: (iii) R1 and R2, together with the atoms to which they are attached, form a 4- to 10-membered heterocycloalkyl ring optionally substituted with one, two, or three groups independently selected from the group consisting of halo, (C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, —CN, —OH, and —NH2, provided that the compound is not 1-[2-(4-fluoro-phenyl)-acetyl]-cyclopropanecarboxylic acid [3-fluoro-4-(7,8,10,11,13,14-hexahydro-6,9,12,15-tetraoxa-1-aza-cyclododeca[b]naphthalen-4-yloxy)-phenyl]-amide.

[0112] In a further embodiment of the compounds of Formula A and B: R1 is -H, -CN, (C1-C6) alkyl, (C3-C 10 ) cycloalkyl, (C6-C10 ) aryl, 4-10 membered heterocycloalkyl, 5-10 membered heteroaryl, -S(O)2NHR a , -P(O)R a R a , -OR a ,or [ka] where: [ka] indicates the attachment point: Y1 is absent, -NH-, -N-(C1-C6)alkyl-, or -O-; Y2 is absent or is -O-, -NH-, -NHO-, -N-(C1-C6)alkyl-, -NH-NH-, -NH-S(O)-, or NH-S(O)2; and Z 1 is —O, —NH, —N—(C1-C6)alkyl, —N—OH, or —NO(C1-C6)alkyl.

[0113] In another embodiment of Formulas A and B: R2 is -H, halo, -X1R a , (C2-C6) alkenyl, (C2-C6) alkynyl, or [ka] where: [ka] indicates the attachment point: Y1 is absent, NH, N—(C1-C6) alkyl, or O; Y2 is absent or is O, NH, NHO, N—(C1-C6) alkyl, N2H2, NH—S(O), or NH—S(O)2, and Z 1 is —O, —NH, —N—(C1-C6)alkyl, —NOH—, or —NO(C1-C6)alkyl.

[0114] In another embodiment of Formulas A and B, R3 is -H or halo.

[0115] In another embodiment of Formulas A and B, R4 is -H or halo.

[0116] In another embodiment of Formulas A and B, R 14 is -H or halo.

[0117] In another embodiment of Formulas A and B, Y is —O—.

[0118] In another embodiment, the compound of B is a compound of either formula B-1 or B-2: [ka] or a pharmaceutically acceptable salt thereof.

[0119] In one embodiment of Formula B-1: R a1 is an optionally substituted (C1-C6) alkyl; R1 is -H, -CN, optionally substituted 3- to 6-membered cycloalkyl, optionally substituted phenyl, optionally substituted 4- to 6-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl, -SO2-(C1-C6)alkyl, -SO2NH2, -SO2-NH(C1-C6)alkyl, P(O)((C1-C6)alkyl)2, or [ka] where: [ka] indicates a connection point: Y1 does not exist, Y2 is absent or is -O-, -NH-, -NHO-, -NH-NH-, -N-(C1-C6)alkyl-, or Y2 is optionally substituted [ka] wherein ring A is a 3-, 4-, 5-, 6-, or 7-membered ring; [ka] indicates the point of attachment, Z 1 is O, NH, N—(C1-C6)alkyl, NHO, or NO—(C1-C6)alkyl, and R a is -H, -(C1-C6)alkyl, 4- to 6-membered heterocycloalkyl, 3- to 6-membered cycloalkyl, -(C2-C6)alkylene-OH, -CH2CHOH-(C2-C6)alkylene-OH, -(C2-C6)alkylene-NH2, -(C2-C6)alkylene-NH(C1-C6), -(C2-C6)alkylene-N(C1-C6)2, or -(C2-C6)alkylene-N-(4- to 6-membered heterocycloalkyl).

[0120] In another embodiment of Formula B-1: R a1 is (C1-C6) alkyl, R1 is -H, -CN, optionally substituted cyclopropyl, optionally substituted phenyl, optionally substituted 4- to 6-membered azetidinyl, optionally substituted pyrrolidinyl, optionally substituted piperidinyl, optionally substituted oxetanyl, optionally substituted oxazolyl, optionally substituted pyridinyl, optionally substituted imidazolyl, optionally substituted pyrrolyl, optionally substituted furanyl, optionally substituted pyrazolyl, optionally substituted oxadiazolyl, -SO2-(C1-C6)alkyl, -SON2NH2, -SO2-NH(C1-C6)alkyl, or P(O)((C1-C6)alkyl)2; R1 is [ka] where: Y1 does not exist, Y2 is O, NH, NHO, NH—NH, or N—(C1-C6) alkyl; or Y2 is optionally substituted azetidinyl; Z 1 is O, NH, or N—(C1-C6) alkyl, and R a is H, (C1-C6)alkyl, —(C2-C6)alkylene-OH, —CH2CHOH-(C2-C6)alkylene-OH, —(C2-C6)alkylene-NH2, —(C2-C6)alkylene-NH(C1-C6)alkyl, —(C2-C6)alkylene-N((C1-C6)alkyl)2, —(C2-C6)alkylene-heterocycloalkyl), or a 4- to 6-membered heterocycloalkyl, wherein the heterocycloalkyl is optionally substituted.

[0121] In one embodiment of Formula B-2: R a2 is an optionally substituted (C1-C6) alkyl; R2 is [ka] where: [ka] indicates a connection point: Y1 does not exist, Y2 is absent or is -O- or -NH-; and Z 1 is O, and R a is —H or —(C1-C6)alkyl.

[0122] In another embodiment of Formula B-2: R a2 is (C1-C6) alkyl, R2 is [ka] where: [ka] indicates a connection point: Y1 does not exist, Y2 is absent or is -O- or -NH-; and Z 1 is O or NO-(C1-C6)alkyl, and R a is —H or —(C1-C6)alkyl.

[0123] In another embodiment of Formula B-1, R a1 is methoxy.

[0124] In another embodiment of Formula B-2, R a2 is methoxy.

[0125] In another embodiment, the compound of formula B is a compound of either formula B-3 or B-4: [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0126] In one embodiment of Formula B-3: R1 is —H or (C1-C6) alkyl, and Y1 does not exist, Y2 is absent or is -O-, -NHO-, or -NH-; and Z 1 is O or NO-(C1-C6)alkyl, and R a is —H or —(C1-C6)alkyl.

[0127] In another embodiment of Formula B-3: R1 is —H or methyl; Y1 does not exist, Y2 is absent or is -O-, -NHO-, or -NH-; and Z 1 is O or NO-Me, and R a is -H or Me.

[0128] In another embodiment of Formula B-3: R1 and R a together with the atoms to which they are attached, Forming a 4-6 membered heterocycloalkyl ring optionally substituted with halo, (C1-C6) alkyl, or (C1-C6) haloalkyl.

[0129] In one embodiment of Formula B-4: Y1 does not exist, Y2 is O, NH, NHO, NH-NH, or N-(C1-C6) alkyl; ,or Y2 is optionally substituted azetidinyl; Z 1 is O, NH, NO—(C1-C6)alkyl, or N—(C1-C6)alkyl, and R a is H, (C1-C6)alkyl, -(C2-C6)alkylene-OH, -CH2CHOH-(C2-C6)alkylene-OH, -(C2-C6)alkylene-NH2, -(C2-C6)alkylene-NH(C1-C6)alkyl, -(C2-C6)alkylene-N((C1-C6)alkyl)2, -(C2-C6)alkylene-optionally substituted 4- to 6-membered heterocycloalkyl), or optionally substituted 4- to 6-membered heterocycloalkyl; R2 is -H, -F, -Cl, -Br, -(C1-C6)alkoxy, -O-(C2-C6)alkylene-OH, -O-(C2-C6)alkylene-O-(C1-C6 alkyl), (C2-C6)alkylene-O-(C1-C6)alkyl, -NH2, -NH-(C1-C6 alkyl), -NH-(C1-C6)alkylene-(optionally substituted 4-6 membered heterocycloalkyl), or -NH-(C2-C6)alkylene-O-(C1-C6 alkyl).

[0130] In another embodiment of Formula B-4: Y1 does not exist, Y2 is O, NH, NHO, NH—NH, or N—(C1-C6) alkyl; or Y2 is optionally substituted azetidinyl; Z 1 is O, NH, NO—(C1-C6) alkyl, N—(C1-C6) alkyl, and R a is -H, methyl, ethyl, -(C2-C6)alkylene-OH, -CH2CHOH-(C2-C6)alkylene-OH, -(C2-C6)alkylene-NH2, -(C2-C6)alkylene-NHMe, -(C2-C6)alkylene-N(Me)2, -(C1-C6)alkylene-morpholinyl), -(C1-C6)alkylene-piperidinyl), (C1-C6)alkylene-(optionally substituted pyrrolidinyl), optionally substituted azetidinyl, or optionally substituted oxetanyl; R2 is -H, -F, -Cl, -Br, methoxy, -O-(C2-C6)alkylene-OH, -O-(C2-C6)alkylene-OMe, -NH2, -NH-(C1-C6 alkyl), -NH-(C2-C6)alkylene-OMe, -NH-(C2-C6)alkylene-(optionally substituted morpholinyl), or -NH-(C2-C6)alkylene-O-(C1-C6 alkyl).

[0131] In another embodiment of Formula B-4: R2 and R atogether with the atoms to which they are attached form a 4- to 6-membered heterocycloalkyl ring optionally substituted with halo, (C1-C6)alkyl, or (C1-C6)haloalkyl.

[0132] In another embodiment, the compound of formula B is a compound of formula B-5: [ka] or a pharmaceutically acceptable salt thereof, wherein ring A in formula B-5 is an optionally substituted 5- to 6-membered heteroaryl or aryl.

[0133] In one embodiment of Formula B-5: Ring A is optionally substituted (C6-C 10 ) aryl, optionally substituted (C3-C 10 ) cycloalkyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 4- to 10-membered heterocycloalkyl; and R2 is H or (C1-C6)alkoxy.

[0134] In another embodiment of Formula B-5: Ring A is optionally substituted phenyl, optionally substituted cyclopropyl, optionally substituted pyridyl, optionally substituted imidazolyl, optionally substituted pyrrolyl, optionally substituted furanyl, optionally substituted pyrazolyl, optionally substituted oxazolyl, optionally substituted azetidinyl, or optionally substituted oxetanyl, and R2 is H or methoxy.

[0135] Another embodiment of the compounds of formula A and B is a compound of formula C: [ka] or a pharmaceutically acceptable salt thereof [In the formula, Y1 does not exist, Y2 is O, NH, NHO, NH—NH, or N—(C1-C6) alkyl; or Y2 is optionally substituted azetidinyl; Z is O, NH, NO—(C1-C6)alkyl, or N—(C1-C6)alkyl; R a is optionally substituted with -H, (C1-C6) alkyl, -(C2-C6) alkylene-OH, -CH2CHOH-(C2-C6) alkylene-OH, -(C2-C6) alkylene-NH2, -(C2-C6) alkylene-NH(C1-C6) alkyl, -(C2-C6) alkylene-N((C1-C6) alkyl)2, -(C2-C6) alkylene- optionally substituted 4- to 6-membered heterocycloalkyl; or optionally substituted 4- to 6-membered heterocycloalkyl; R2 is -H, -F, -Cl, -Br, -(C1-C6)alkoxy, -O-(C2-C6)alkylene-OH, -O-(C2-C6)alkylene-O-(C1-C6 alkyl), (C2-C6)alkylene-O-(C1-C6)alkyl, -NH2, -NH-(C1-C6 alkyl), -NH-(C1-C6)alkylene-(optionally substituted 4-6 membered heterocycloalkyl), -NH-(C2-C6)alkylene-O-(C1-C6 alkyl), and n and m each independently represent an integer of 0 to 3.

[0136] In another embodiment of Formula C: Y1 does not exist, Y2 is O, NH, NHO, NH—NH, or N—(C1-C6) alkyl; or Y2 is optionally substituted azetidinyl; Z 1 is O, NH, NO—(C1-C6)alkyl, or N—(C1-C6)alkyl, R ais -H, methyl, ethyl, -(C2-C6)alkylene-OH, -CH2CHOH-(C2-C6)alkylene-OH, -(C2-C6)alkylene-NH2, -(C2-C6)alkylene-NHMe, -(C2-C6)alkylene-N(Me)2, -(C1-C6)alkylene-morpholinyl), -(C1-C6)alkylene-piperidinyl), (C1-C6)alkylene-(optionally substituted pyrrolidinyl), optionally substituted azetidinyl, or optionally substituted oxetanyl; R2 is -H, -F, -Cl, -Br, methoxy, -O-(C2-C6)alkylene-OH, -O-(C2-C6)alkylene-OMe, -NH2, -NH-(C1-C6 alkyl), -NH-(C2-C6)alkylene-OMe, -NH-(C2-C6)alkylene-(optionally substituted morpholinyl), or -NH-(C2-C6)alkylene-O-(C1-C6 alkyl), and n and m are each 0 or 1.

[0137] In another embodiment of Formula C: R2 and R a together with the atoms to which they are attached form a 4- to 6-membered heterocycloalkyl ring optionally substituted with halo, (C1-C6)alkyl, and (C1-C6)haloalkyl; and n and m each independently represent an integer of 0 to 3.

[0138] Another embodiment of Formula C is a compound of Formula C-1: [ka] or a pharmaceutically acceptable salt thereof: [In the formula, Y2 is O, NH, NHO, NH-NH, or N-(C1-C6) alkyl; ,or Y2 is optionally substituted azetidinyl; Z 1is O, NH, NO—(C1-C6)alkyl, or N—(C1-C6)alkyl, R a is -H, methyl, ethyl, -(C2-C6)alkylene-OH, -CH2CHOH-(C2-C6)alkylene-OH, -(C2-C6)alkylene-NH2, -(C2-C6)alkylene-NHMe, -(C2-C6)alkylene-N(Me)2, -(C1-C6)alkylene-morpholinyl), -(C1-C6)alkylene-piperidinyl)(C1-C6)alkylene-(optionally substituted pyrrolidinyl), optionally substituted azetidinyl, or optionally substituted oxetanyl; R2 is -H, -F, -Cl, -Br, methoxy, -O-(C2-C6)alkylene-OH, -O-(C2-C6)alkylene-OMe, -NH2, -NH-(C1-C6 alkyl), -NH-(C2-C6)alkylene-OMe, -NH-(C2-C6)alkylene-(optionally substituted morpholinyl), or -NH-(C2-C6)alkylene-O-(C1-C6 alkyl), and n and m are each independently 0 or 1.

[0139] Another embodiment of Formulas A and B is a compound of Formula D: [ka] is.

[0140] In one embodiment of Formula D: R1 is —H or (C1-C6) alkyl, and Y1 does not exist, Y2 is absent or is -O-, -NHO-, or -NH-; and Z 1 is O or NO-(C1-C6)alkyl, R a is —H or —(C1-C6)alkyl, and n and m each independently represent an integer of 0 to 3.

[0141] In another embodiment of Formula D: R1 is —H or methyl; Y1 does not exist, Y2 is absent or is -O-, -NHO-, or -NH-; and Z 1 is O or NO-Me, and R a is -H or -Me, n and m each independently represent an integer of 0 to 1.

[0142] In another embodiment of Formula D: R1 and R a together with the atom to which they are attached, halo, (C1-C6) alkyl or a 4-6 membered heterocycloalkyl ring optionally substituted with (C1-C6)haloalkyl; and n and m each independently represent an integer of 0 to 1.

[0143] Another embodiment of formula D is a compound of formula D-1 [ka] is.

[0144] Another embodiment of Formulas A and B is a compound of Formula E: [ka] or a pharmaceutically acceptable salt thereof [In the formula, Ring A is optionally substituted (C6-C 10 ) aryl, optionally substituted (C3-C 10 ) cycloalkyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 4- to 10-membered heterocycloalkyl; and R2 is H or (C1-C6)alkoxy].

[0145] In another embodiment of Formula E: Ring A is optionally substituted phenyl, optionally substituted cyclopropyl, optionally substituted pyridyl, optionally substituted imidazolyl, optionally substituted pyrrolyl, optionally substituted furanyl, optionally substituted pyrazolyl, optionally substituted oxazolyl, optionally substituted azetidinyl, or optionally substituted oxetanyl, and R2 is H or methoxy.

[0146] Another embodiment of formulas A and B is a compound of formula F: [ka] or a pharmaceutically acceptable salt thereof [In the formula, R a1 is an optionally substituted (C1-C6) alkyl; R1 is -H, -CN, optionally substituted 3- to 6-membered cycloalkyl, optionally substituted phenyl, optionally substituted 4- to 6-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl, -SO2-(C1-C6)alkyl, -SO2NH2, -SO2-NH(C1-C6)alkyl, or P(O)((C1-C6)alkyl)2, or [ka] where: [ka] indicates a connection point: Y1 does not exist, Y2 is absent or is -O-, -NH-, -NHO-, -NH-NH-, -N-(C1-C6)alkyl-, or Y2 is optionally substituted [ka] wherein ring A is a 3-, 4-, 5-, 6-, or 7-membered ring; [ka] indicates the point of attachment, Z 1 is O, NH, N—(C1-C6)alkyl, NHO, or NO—(C1-C6)alkyl, and R a is -H, -(C1-C6)alkyl, 4- to 6-membered heterocycloalkyl, 3- to 6-membered cycloalkyl, -(C2-C6)alkylene-OH, -CH2CHOH-(C2-C6)alkylene-OH, -(C2-C6)alkylene-NH2, -(C2-C6)alkylene-NH(C1-C6), -(C2-C6)alkylene-N(C1-C6)2, -(C2-C6)alkylene-N-(4- to 6-membered heterocycloalkyl)].

[0147] In another embodiment of Formula F: R a1 is methyl, R1 is H, -CN, optionally substituted cyclopropyl, optionally substituted phenyl, optionally substituted 4- to 6-membered azetidinyl, optionally substituted pyrrolidinyl, optionally substituted piperidinyl, optionally substituted oxetanyl, optionally substituted oxazolyl, optionally substituted pyridinyl, optionally substituted imidazolyl, optionally substituted pyrrolyl, optionally substituted furnayl, optionally substituted pyrazolyl, optionally substituted oxadiazolyl, -SO2-(C1-C6)alkyl, -SO2NH2, -SO2-NH(C1-C6)alkyl, or P(O)((C1-C6)alkyl)2; R1 is [ka] In this case, Y1 does not exist, Y2 is O, NH, NHO, NH—NH, or N—(C1-C6) alkyl; or Y2 is optionally substituted azetidinyl; Z 1 is O, NH, or N—(C1-C6) alkyl, and R a is H, (C1-C6)alkyl, —(C2-C6)alkylene-OH, —CH2CHOH-(C2-C6)alkylene-OH, —(C2-C6)alkylene-NH2, —(C2-C6)alkylene-NH(C1-C6)alkyl, —(C2-C6)alkylene-N((C1-C6)alkyl)2, —(C2-C6)alkylene-heterocycloalkyl), and 4- to 6-membered heterocycloalkyl, wherein the heterocycloalkyl is optionally substituted.

[0148] Another embodiment of formulas A and B is a compound of formula G: [ka] or a pharmaceutically acceptable salt thereof: [In the formula, R a1 is an optionally substituted (C1-C6) alkyl; R2 is [ka] where: [ka] indicates the point of attachment, Y1 does not exist, Y2 is absent, -O-, or -NH-; and Z 1 is O, and R a is —H or —(C1-C6)alkyl.

[0149] In another embodiment of Formula G: R a2is methyl, R2 is [ka] where: [ka] indicates the point of attachment, Y1 does not exist, Y2 is absent, -O-, or -NH-; and Z 1 is O or NO-(C1-C6)alkyl, and R a is —H, or —(C1-C6) alkyl.

[0150] Another embodiment of Formulas A and B is a compound according to Formula H for modulating kinase activity: [ka] or a pharmaceutically acceptable salt thereof: [In the formula, R1 is selected from the group consisting of -H, -CN, -CO-NR5R6, -CO2R7, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted (C1-C6)alkyl, optionally substituted (C3-C8)cycloalkyl, optionally substituted (C3-C6)heterocycloalkyl, -SON2NR8R9, or -(SO2)(C1-C6)alkyl; when R1 is selected from the group consisting of -CN, -CO-NR5R6, -CO2R7, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted (C3-C8)cycloalkyl, optionally substituted (C3-C6)heterocycloalkyl, -SO2NR8R9, and -(SO2)-(C1-C6)alkyl, R2 is H, halo, NR5R6, or optionally substituted (C1-C6)alkoxy; When R1 is -H, optionally substituted (C1-C6) alkyl, or optionally substituted (C1-C6) alkoxy, R2 is -CO-NR5R6 or -CO2R7; R1 and R2, together with the atoms to which they are attached, form an optionally substituted cycloalkyl or an optionally substituted heterocycloalkyl; , R3 is selected from the group consisting of -H, optionally substituted (C1-C6) alkyl, -CN, and halo; R4 is -H or halo; [ka] is optionally substituted with 1, 2, 3, or 4 groups independently selected from the group consisting of halo and (C1-C6)alkyl, wherein [ka] indicates the point of attachment, R5 and R6 are each independently -H, optionally substituted (C1-C6) alkyl, or optionally substituted C1-C6 alkoxy; R7 is —H or optionally substituted (C1-C6) alkyl; R8 and R9 are each independently —H or optionally substituted (C1-C6) alkyl; R8 and R9 may be linked to form an optionally substituted heterocycle; and Y is selected from the group consisting of O, S, SO, SO2, NH, and N-((C1-C6) alkyl).

[0151] In one embodiment of the compound of formula I, Y is O.

[0152] In another embodiment, R3 is -H.

[0153] In another embodiment, [ka] is not substituted.

[0154] In another embodiment, R4 is halo.

[0155] In another embodiment, R4 is parafluoro.

[0156] In another embodiment, R2 is -H, halo, or optionally substituted (C1-C6)-alkoxy.

[0157] In another embodiment, R1 is -CN.

[0158] In another embodiment, R1 is -CO2H.

[0159] In another embodiment, R1 is -CO2-Me.

[0160] In another embodiment, R1 is -CO-NHR6.

[0161] In another embodiment, R1 is -CO-NH2.

[0162] In another embodiment, R1 is -CO-NMeR6.

[0163] In another embodiment, R3 is -H or halo.

[0164] In another embodiment, R1 is -CN, -(SO2)NH2, -OMe, -(SO2)CH3 [ka] wherein: [ka] is the attachment point.

[0165] In another embodiment, R1 is [ka] wherein: [ka] is the attachment point.

[0166] In another embodiment, R1 is [ka] wherein: [ka] is the attachment point.

[0167] In another embodiment, R2 is -H, -CN, -Br, -F, -Cl, -OMe, -CH3, [ka] wherein: [ka] is the attachment point.

[0168] In another embodiment, R1 is -H, methyl, or methoxy.

[0169] In another embodiment, R2 is -CO2H.

[0170] In another embodiment, R1 is -CO2-Me.

[0171] In another embodiment, R1 is -CO-NHR6.

[0172] In another embodiment, R1 is -CO-NH2.

[0173] In another embodiment, R1 is -CO-NMeR6.

[0174] In another embodiment, R1 is [ka] is selected from the group consisting of:

[0175] In another embodiment, R1 and R2 together represent [ka] is formed.

[0176] In a further embodiment, the compound of formula I is a compound of formula I-1: [ka] wherein R6 is (C1-C6) alkyl, R2 is (C1-C6) alkoxy, R3 is -H or halo, and R4 is halo.

[0177] In another aspect, the present invention provides compounds of formula A or AI as set out in Table 1 below. [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

[0178] Systemic administration 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 or agents to achieve similar utility. Thus, administration can be, for example, oral, nasal, parenteral (intravenous, intramuscular, or subcutaneous), topical, transdermal, intravaginal, intravesical, intracisternal, or rectal, in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, aerosols, and the like, preferably in unit dosage forms suitable for easy administration of precise dosages.

[0179] The compositions include conventional pharmaceutical carriers or excipients and the compounds of the present invention as active agents, and may also include other pharmaceutical agents, pharmaceuticals, carriers, adjuvants, etc. The compositions of the present invention may be used in combination with anticancer drugs or other agents commonly administered to patients undergoing cancer treatment. Adjuvants include preservatives, wetting agents, suspending agents, sweeteners, flavorings, perfuming agents, emulsifying agents, and dispersing agents. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0180] If desired, the pharmaceutical compositions of the present invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants, and the like, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, and the like.

[0181] Compositions suitable for parenteral injection may include physiologically acceptable aqueous or non-aqueous sterile solutions, dispersions, suspensions, or emulsions, as well as sterile powders to be reconstituted into sterile injectable solutions or dispersions. Examples of suitable aqueous or non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (olive oil, etc.), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0182] One of the preferred routes of administration is oral, using a convenient daily dosage regimen that can be adjusted according to the severity of the disease-state being treated.

[0183] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is dispersed in at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as cellulose derivatives, starch, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerol; and (d) disintegrants, such as agar, calcium carbonate, potato, or tapioca. The pharmaceutical composition may be mixed with (e) dissolution retardants such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as cetyl alcohol, glycerol monostearate, magnesium stearate, and the like, (h) adsorbents such as kaolin and bentonite, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0184] The solid dosage forms described above can be prepared with coatings and shells, such as enteric coatings and others known in the art. This may contain a soothing agent and may be a composition that releases the active compound(s) in a delayed manner in a specific part of the intestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. If appropriate, the active compound may be in a microencapsulated form, containing one or more of the above-mentioned additives.

[0185] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving or dispersing the compound(s) of the present invention or a pharmaceutically acceptable salt thereof and optional pharmaceutical adjuvants in a carrier such as water, saline, aqueous dextrose, glycerol, ethanol, solubilizing agents and emulsifying agents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, and dimethylformamide, oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, or mixtures of these substances, thereby forming a solution or suspension. .

[0186] In addition to the active compound, suspensions may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.

[0187] Compositions for rectal administration are, for example, suppositories which can be prepared by mixing a compound of the present invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol or a suppository wax, which is solid at ordinary temperature but liquid at body temperature and therefore melts while in the appropriate body cavity, releasing the active ingredient therein.

[0188] Dosage forms for topical administration of the compounds of the present invention include ointments, powders, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and, if necessary, any preservatives, buffers, or propellants. Ophthalmic formulations, eye ointments, powders, and solutions are also contemplated within the scope of this disclosure.

[0189] Generally, depending on the intended mode of administration, a pharmaceutically acceptable composition will contain from about 1% to about 99% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and 99% to 1% by weight of suitable excipients. In one example, the composition will be between about 5% and about 75% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, with the remainder being suitable excipients.

[0190] Actual methods for preparing such dosage forms will be known or apparent to those skilled in the art; see, for example, Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton, Pa., 1990). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, to treat a disease state in accordance with the teachings of the invention.

[0191] The compounds of the present invention, or pharmaceutically acceptable salts thereof, are administered in therapeutically effective amounts that vary depending on various factors, including the activity of the specific compound used, the metabolic stability and duration of action of the compound, the age, body weight, general health, sex, diet, mode and time of administration, excretion rate, drug combination, the severity of the specific disease state, and the recipient of treatment. The compounds of the present invention can be administered to patients at dosage levels ranging from about 0.1 to about 1,000 mg per day. For a normal human adult weighing approximately 70 kilograms, a dosage range of about 0.01 to about 100 mg per kilogram of body weight per day is exemplary. However, the specific dosage used may vary. For example, the dosage may depend on many factors, including the needs of the patient, the severity of the condition being treated, and the pharmacological activity of the compound used. Determining the optimal dosage for a particular patient is well known to those skilled in the art.

[0192] Combination treatment The compounds as disclosed herein can be administered as a sole therapy or in combination ("co-administration") with one or more additional therapies to treat a disease or disorder, e.g., a disease or disorder associated with hyperproliferation, such as cancer. Treatments that can be used in combination with the compounds disclosed herein include (i) surgery, (ii) radiation therapy (e.g., gamma radiation, neutron radiation therapy, electron beam radiation therapy, proton therapy, brachytherapy, and systemic radioisotopes), (iii) endocrine therapy, (iv) adjuvant therapy, immunotherapy, CAR T-cell therapy, and (v) other chemotherapeutic agents.

[0193] The term "co-administered" ("co-administering") refers to any manner of simultaneous or separate sequential administration of a compound of Formula I' or a salt thereof and additional active pharmaceutical ingredient(s), including cytotoxic agents and radiation treatment. When administration is not simultaneous, the compounds are administered in close time proximity to each other. Furthermore, it is not important whether the compounds are administered in the same dosage form; for example, one compound may be administered topically and the other orally.

[0194] Typically, any agent active against the disease or condition being treated can be co-administered. Examples of such agents for cancer treatment are found, for example, at https: / / www.cancer.gov / about-cancer / treatment / drugs (last accessed January 22, 2019) and in Cancer Principles and Practice of Oncology by V.T.Devita and S. Hellman (editors), 11 th edition (2018), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the disease involved.

[0195] In one embodiment, the method of treatment comprises co-administration of a compound as disclosed herein or a pharmaceutically acceptable salt thereof with at least one immunotherapy. Immunotherapy (also called biological response modifier therapy, biologic therapy, biotherapy, immune therapy, or biological therapy) is a treatment that uses parts of the immune system to fight disease. Immunotherapy can help the immune system recognize cancer cells or enhance the response to cancer cells. Immunotherapy includes active and passive immunotherapy. Active immunotherapy stimulates the body's own immune system, while passive immunotherapy generally uses immune system components generated outside the body.

[0196] Examples of active immunotherapy include, but are not limited to, vaccines including cancer vaccines, tumor cell vaccines (autologous or allogeneic), dendritic cell vaccines, antigen vaccines, anti-idiotype vaccines, DNA vaccines, viral vaccines, or tumor-infiltrating lymphocyte (TIL) vaccines including interleukin-2 (IL-2), or lymphokine-activated killer (LAK) cell therapy.

[0197] Examples of passive immunotherapy include, but are not limited to, monoclonal antibodies and toxin-containing targeted therapeutics. Monoclonal antibodies include naked antibodies and conjugated monoclonal antibodies (also called tagged, labeled, or loaded antibodies). Naked monoclonal antibodies have no drugs or radioactive substances attached, while conjugated monoclonal antibodies are bound to, for example, chemotherapeutic agents (chemical labels), radioactive particles (radiolabels), or toxins (immunotoxins). Examples of these naked monoclonal antibody drugs include, but are not limited to, rituximab (Rituxan), an antibody against the CD20 antigen used, for example, to treat B-cell non-Hodgkin's lymphoma; trastuzumab (Herceptin), an antibody against the HER2 protein used, for example, to treat advanced breast cancer; alemtuzumab (Campath), an antibody against the CD52 antigen used, for example, to treat B-cell chronic lymphocytic leukemia (B-CLL); cetuximab (Erbitux), an antibody against the EGFR protein used, for example, in combination with irinotecan to treat advanced colorectal cancer and head and neck cancer; and bevacizumab (Avastin), an anti-angiogenic therapy that acts against the VEGF protein and is used, for example, in combination with chemotherapy to treat metastatic colorectal cancer. Examples of clonal antibodies include, but are not limited to, the radiolabeled antibody ibritumomab tiuxetan (Zevalin), which delivers radioactivity directly to cancerous B lymphocytes and is used, for example, to treat B-cell non-Hodgkin's lymphoma; the radiolabeled antibody tositumomab (Bexxar), which is used, for example, to treat certain types of non-Hodgkin's lymphoma; and the immunotoxin gemtuzumab ozogamicin (Mylotarg), which contains calicheamicin and is used, for example, to treat acute myeloid leukemia (AML). BL22 is a conjugated monoclonal antibody, for example, to treat hairy cell leukemia; immunotoxins, for example, to treat leukemia, lymphoma, and brain tumors; and radiolabeled antibodies such as OncoScint, for example, for colorectal and ovarian cancer, and ProstaScint, for example, for prostate cancer.

[0198] Further examples of therapeutic antibodies that can be used include, but are not limited to, HERCEPTIN™ (trastuzumab), a humanized anti-HER2 monoclonal antibody (Genentech, Calif.), for treating patients with metastatic breast cancer, and REOPRO™, an anti-glycoprotein IIb / IIIa receptor on platelets to prevent blood clot formation.(abciximab) (Centocor), ZENAPAX™ (daclizumab), an immunosuppressive humanized anti-CD25 monoclonal antibody for preventing acute kidney allograft rejection (Roche Pharmaceuticals, Switzerland), PANOREX™, a murine anti-17-IA cell surface antigen IgG2a antibody (Glaxo Wellcome / Centocor), BEC2, a murine anti-idiotypic (GD3 epitope) IgG antibody (ImClone System), IMC-C225, a chimeric anti-EGFR IgG antibody (ImClone System), VITAXIN™, a humanized anti-alpha V beta 3 integrin antibody (Applied Molecular Evolution / MedImmune), Campath 1H / LDP-03, a humanized anti-CD52 IgG1 antibody (Leukosite), and Smart M195, a humanized anti-CD33 IgG antibody (Protein Design). Lab / Kanebo), RITUXAN™, a chimeric anti-CD20 IgG1 antibody (IDEC Pharm / Genentech, Roche / Zettyaku), LYMPHOCIDE™, a humanized anti-CD22 IgG antibody (Immunomedics), LYMPHOCIDE™ Y-90 (Immunomedics), Lymphoscan (Tc-99m labeled, radioimaging, Immunomedics), Nuvion (directed against CD3, Protein Design Labs), CM3, a humanized anti-ICAM3 antibody (ICOS Pharm), IDEC-114, a primatized anti-CD80 antibody (IDEC Pharm / Mitsubishi), and ZEVALIN™, a radiolabeled murine anti-CD20 antibody (IDEC / Schering). AG), IDEC-131 (IDEC / Eisai), a humanized anti-CD40L antibody, IDEC-151 (IDEC), a primatized anti-CD4 antibody, IDEC-152 (IDEC / Seikagaku), a primatized anti-CD23 antibody, SMART anti-CD3 (Protein Design Lab), a humanized anti-CD3 IgG, and 5G1, a humanized anti-complement factor 5 (C5) antibody.1 (Alexion Pharm), humanized anti-TNF-alpha antibody D2E7 (CAT / BASF), humanized anti-TNF-alpha Fab fragment CDP870 (Celltech), primatized anti-CD4 IgG1 antibody IDEC-151 (IDEC Pharm / SmithKline Beecham), human anti-CD4 IgG antibody MDX-CD4 (Medarex / Eisai / Genmab), CD20 streptavidin (plus biotin-yttrium 90, NeoRx), humanized anti-TNF-alpha IgG4 antibody CDP571 (Celltech), humanized anti-alpha4beta7 antibody LDP-02 (LeukoSite / Genentech), humanized anti-CD4 IgG antibody OrthoClone OKT4A (Ortho Biotech), humanized anti-CD40L IgG antibody ANTOVA™ (Biogen), humanized anti-VLA-4 ANTEGREN™ (Elan), an IgG antibody, and human anti-TGF-β. Examples include the TA2 antibody CAT-152 (Cambridge Ab Tech). Others are listed in the following paragraphs.

[0199] Immunotherapies that can be used in combination with the compounds as disclosed herein include adjuvant immunotherapies. Examples include cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1-alpha, interleukins (including IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27), tumor necrosis factors (including TNF-alpha), and interferons (including IFN-alpha, IFN-beta, and IFN-gamma), aluminum hydroxide (alum), bacillus Calmette-Guerin (BCG), keyhole limpet hemocyanin (KLH), incomplete Freund's adjuvant (IFA), QS-21, DETOX, levamisole, and dinitrophenyl (DNP), as well as combinations thereof, such as combinations of interleukins and other cytokines, such as IL-2 and IFN-alpha.

[0200] In various embodiments, the immunological therapy or immunotherapeutic agent can include one or more of the following: adoptive cell transfer, angiogenesis inhibitors, bacillus Calmette-Guerin therapy, biochemotherapy, cancer vaccines, chimeric antigen receptor (CAR) T-cell therapy, cytokine therapy, gene therapy, immune checkpoint modulators, immunoconjugates, radioconjugates, oncolytic virus therapy, or targeted drug therapy. Collectively, the function or at least one function of an immunological therapy or immunotherapeutic agent is referred to herein as an "immunotherapeutic agent."

[0201] The present disclosure provides methods for preventing, treating, reducing, inhibiting, or controlling neoplasia, tumors, or cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a combination comprising a compound of Formula I' and an immunotherapeutic agent. In one non-limiting embodiment, the method comprises administering a therapeutically effective amount of a combination comprising a compound of Formula I' in combination with an immunotherapeutic agent. In various embodiments, the combination provides a synergistic, additive, or synergistic effect in reducing the number of cancer cells when treated in combination compared to treatment with either alone. In some embodiments, administration of a therapeutically effective amount of a combination comprising a compound of Formula I' and an immunotherapeutic agent results in synergistic anti-tumor activity and / or anti-tumor activity that is more potent than the additive effect of administering a compound of Formula I' or an immunotherapeutic agent alone.

[0202] Human cancers harbor numerous genetic and epigenetic mutations, generating neoantigens potentially recognizable by the immune system (Sjoblom et al. (2006) Science 314:268-74). The adaptive immune system, composed of T and B lymphocytes, possesses potent anticancer potential with a broad ability to respond to diverse tumor antigens and exquisite specificity. Furthermore, the immune system demonstrates considerable plasticity and memory components. Successfully harnessing all of these contributions of the adaptive immune system may make immunotherapy unique among all cancer treatment modalities.

[0203] The present disclosure provides combinations of a compound of Formula I' and an immunotherapeutic agent. These exemplary combinations can be used to treat a subject with cancer. In various embodiments, the immunotherapeutic agent utilized in the compositions, formulations, and methods can include one or more agents or therapies, including adoptive cell transfer, angiogenesis inhibitors, bacillus Calmette-Guerin therapy, biochemotherapy, cancer vaccines, chimeric antigen receptor (CAR) T-cell therapy, cytokine therapy, gene therapy, immune checkpoint modulators, e.g., immune checkpoint inhibitors, immunoconjugates, radioconjugates, oncolytic virus therapy, or targeted drug therapy.

[0204] In certain embodiments of the present disclosure, the therapeutically effective combination comprises a compound of Formula I' and an immunotherapeutic agent. In various related embodiments, the compound of Formula I' enhances the activity of the immunotherapeutic agent.

[0205] In certain embodiments of each of the above aspects, as well as other aspects, and embodiments described elsewhere herein, the immunotherapeutic agent enhances the activity of the compound of formula I'.

[0206] In certain embodiments of each of the above aspects, as well as other aspects, and in embodiments described elsewhere herein, the compound of Formula I' and the immunotherapeutic agent act synergistically. In various embodiments described herein, an exemplary immunotherapeutic agent is an immune cell (e.g., T cell, dendritic cell, natural killer cell, etc.) modulator selected from an agonist or activator of costimulatory molecules, where the modulator is a monoclonal antibody, a bispecific antibody, a trispecific antibody comprising one or more immune checkpoint antigen-binding moieties, or an immune cell-engaging multivalent antibody / fusion protein / construct known in the art. In some embodiments, the immunotherapeutic agent may be an antibody that modulates costimulatory molecules and binds to an antigen on the surface of an immune cell or cancer cell. In each of these various embodiments, the antibody modulator may be a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a trispecific or multispecific format antibody, a fusion protein, or a fragment thereof, such as a diabody, a single chain (sc)-diabody (scFv)2, a miniantibody, a minibody, a barnase-barstar, a scFv-Fc, a sc(Fab)2, a trimeric antibody construct, a triabody antibody construct, a trimericbody antibody construct, a tribody antibody construct, a collabody antibody construct, a (scFv-TNFα)3, or a F(ab)3 / DNL antibody construct.

[0207] In certain embodiments of each of the above aspects, as well as other aspects, and embodiments described elsewhere herein, the immunotherapeutic agent is an agent that modulates the immune response, e.g., a checkpoint inhibitor or checkpoint agonist. In some embodiments, the immunotherapeutic agent is an agent that enhances an anti-tumor immune response. In some embodiments, the immunotherapeutic agent is an agent that increases cell-mediated immunity. In some embodiments, the immunotherapeutic agent is an agent that increases T cell activity. In some embodiments, the immunotherapeutic agent is an agent that increases cytolytic T cell (CTL) activity. In some embodiments, the immunotherapeutic agent is an antibody modulator targeting PD-1, PD-L1, PD-L2, CEACAM (e.g., CEACAM-1, -3, and / or -5), CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFbeta, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, TIM-3, SIRP-alpha, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS, and / or BTNL2, among others known in the art. In some embodiments, the immunotherapeutic agent is an agent that increases natural killer (NK) cell activity. In some embodiments, the immunotherapeutic agent is an agent that inhibits suppression of an immune response. In some embodiments, the immunotherapeutic agent is a suppressor cell or an agent that inhibits suppressor cell activity. In some embodiments, the immunotherapeutic agent is an agent or treatment that inhibits Treg activity. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of an inhibitory immune checkpoint receptor. In some embodiments, the combinations of the present disclosure comprise a compound of Formula I' and an immunotherapeutic agent, wherein the immunotherapeutic agent includes a T cell modulator selected from an agonist or activator of a costimulatory molecule.In one embodiment, the agonist of a costimulatory molecule is an agonist of GITR, OX40, ICOS, SLAM (e.g., SLAMF7), HVEM, LIGHT, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, CD7, NKG2C, NKp80, CD160, B7-H3, or CD83 ligand (e.g., In other embodiments, the effector cell combination includes a bispecific T cell engager (e.g., a bispecific antibody molecule that binds to CD3 and a tumor antigen (e.g., EGFR, PSCA, PSMA, EpCAM, HER2, among others)).

[0208] In some embodiments, the immunotherapeutic is a modulator of PD-1 activity, a modulator of PD-L1 activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4-1BB activity, a modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity , a regulator of IDO1 activity, a regulator of SIRP-alpha activity, a regulator of TIGIT activity, a regulator of VSIG8 activity, a regulator of BTLA activity, a regulator of SIGLEC7 activity, a regulator of SIGLEC9 activity, a regulator of ICOS activity, a regulator of B7H3 activity, a regulator of B7H4 activity, a regulator of FAS activity, a regulator of BTNL2 activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, or an immunostimulatory oligonucleotide. In some embodiments, the immunotherapeutic agent is an immune checkpoint modulator (e.g., an immune checkpoint inhibitor, e.g., an inhibitor of PD-1 activity, a modulator of PD-L1 activity, a modulator of PD-L2 activity, a modulator of CTLA-4, or a CD40 agonist (e.g., an anti-CD40 antibody molecule), (xi) an OX40 agonist (e.g., an anti-OX40 antibody molecule), or (xii) a CD27 agonist (e.g., an anti-CD27 antibody molecule). In one embodiment, the immunomodulator is an inhibitor of PD-1, PD In one embodiment, the inhibitor of an immune checkpoint molecule inhibits PD-1, PD-L1, LAG-3, TIM-3, CEACAM (e.g., CEACAM-1, -3, and / or -5), CTLA-4, or any combination thereof.

[0209] Inhibition of inhibitory molecules can occur at the DNA, RNA, or protein level. In embodiments, inhibitory nucleic acids (e.g., dsRNA, siRNA, or shRNA) can be used to inhibit expression of inhibitory molecules. In other embodiments, the inhibitor of an inhibitory signal is a polypeptide, e.g., a soluble ligand (e.g., PD-1-Ig or CTLA-4 Ig), or an antibody or antigen-binding fragment thereof, e.g., a monoclonal antibody, a bispecific antibody comprising one or more immune checkpoint antigen-binding moieties, a trispecific antibody, or an immune cell-engaging multivalent antibody / fusion protein / construct known in the art that binds to an inhibitory molecule, e.g., an antibody or fragment thereof (also referred to herein as an "antibody molecule") that binds to PD-1, PD-L1, PD-L2, CEACAM (e.g., CEACAM-1, -3, and / or -5), CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGF-beta, or a combination thereof.

[0210] In some embodiments, when the combination comprises a compound of Formula I' and an immunotherapeutic agent, the immunotherapeutic agent is a monoclonal antibody or a bispecific antibody. For example, the monoclonal or bispecific antibody may specifically bind to a member of the c-Met pathway and / or an immune checkpoint regulator (e.g., a bispecific antibody may bind to both hepatocyte growth factor receptor (HGFR) and an immune checkpoint regulator described herein, such as PD-1, PD-L1, PD-L2, or CTLA-4, LAG-3, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, TIM-3, SIRP-alpha, T In certain embodiments, the bispecific antibody specifically binds to human HGFR protein and one of PD-1, PD-L1, and CTLA-4.

[0211] In some embodiments, the immunotherapeutic agent is a cytokine, such as a chemokine, interferon, interleukin, lymphokine, or member of the tumor necrosis factor family, hi some embodiments, the cytokine is IL-2, IL15, or interferon-gamma.

[0212] In some embodiments of any of the above aspects or embodiments described elsewhere herein, the cancer is selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, colon cancer, colorectal cancer, melanoma, gastrointestinal cancer, stomach cancer, renal cancer, ovarian cancer, liver cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, glioma, glioblastoma, glioblastoma multiforme, cervical cancer, gastric cancer, bladder cancer, head and neck cancer, and hepatocellular carcinoma.

[0213] In some embodiments of any of the above aspects or embodiments described elsewhere herein, the subject's cancer or tumor does not respond to immune checkpoint inhibition (e.g., to any immune checkpoint inhibitor described herein, such as a PD-1 antagonist or a PD-L1 antagonist), or the subject's cancer or tumor has progressed after an initial response to immune checkpoint inhibition (e.g., to any immune checkpoint inhibitor described herein, such as a PD-1 antagonist or a PD-L1 antagonist).

[0214] In some embodiments of any of the above aspects or elsewhere herein, the subject is a human.

[0215] A checkpoint inhibitor can be any molecule, agent, treatment, and / or method that inhibits an immune checkpoint and / or promotes the inhibition of an immune checkpoint, for example, by promoting intrinsic immune checkpoint inhibitors, by inhibiting transcription factors involved in the expression of immune checkpoints, and / or by acting in concert with some additional external factors. For example, a checkpoint inhibitor could include a treatment that inhibits transcription factors involved in the expression of immune checkpoint genes or promotes the expression of transcription factors for tumor-suppressor genes, e.g., BACH2 (Luan et al., (2016). Transcription Factors and Checkpoint Inhibitor Expression with Age: Markers of Immunosenescence. Blood, 128(22), 5983). Additionally, checkpoint inhibitors can inhibit the transcription of immune checkpoint genes, the modification and / or processing of immune checkpoint mRNA, the translation of immune checkpoint proteins, and / or the activation of molecules involved in immune or immune checkpoint pathways, e.g., PD-1 transcription factors such as HIF-1, STAT3, NF-κB, and AP-1, or common oncogenic processes such as JAK / STAT, RAS / ERK, or PI3K / AKT / mTOR (Zerdes et al., Genetic, transcriptional and post-translational regulation of the programmed death protein ligand 1 in cancer: biology and clinical correlations, Oncogene volume 37, pages 4639-4661 (2018), the entire disclosure of which is incorporated herein by reference).

[0216] Checkpoint inhibitors can include treatments, molecules, agents, and / or methods that regulate immune checkpoints at the transcriptional level, e.g., using RNA interference pathway co-suppression and / or post-transcriptional gene silencing (PTGS) (e.g., microRNA, miRNA, silencing RNA, small interfering RNA, or short interfering RNA (siRNA)). Transcriptional regulation of checkpoint molecules has been shown to involve mir-16, which has been shown to target the 3'UTR of the checkpoint mRNAs CD80, CD274 (PD-L1), and CD40 (Leibowitz et al., Post-transcriptional regulation of Immune checkpoint genes by mir-16 in melanoma, Annals of Oncology (2017) 28; v428-v448). Mir-33a has also been shown to be involved in regulating PD-1 expression in lung adenocarcinoma (Boldini et al., Role of microRNA-33a in regulating PD-1 expression, the entire disclosure of which is incorporated herein by reference). the expression of PD-1 in lung adenocarcinoma, Cancer Cell Int.2017;17:105).

[0217] T cell-specific aptamer-siRNA chimeras have been suggested as a highly specific method for inhibiting molecules in immune checkpoint pathways (Hossain et al., The aptamer-siRNA conjugates: reprogramming T cells for cancer therapy, Ther. Deliv. 2015 Jan;6(1):1-4, the entire disclosure of which is incorporated herein by reference).

[0218] Alternatively, members of immune checkpoint pathways can be inhibited using treatments that affect related pathways, such as metabolism. For example, excessive supply of the glycolytic intermediate pyruvate in mitochondria from CAD macrophages promoted PD-L1 expression through induction of the bone morphogenetic protein 4 / phosphorylated SMAD1 / 5 / IFN-regulated factor 1 (BMP4 / p-SMAD1 / 5 / IRF1) signaling pathway. Therefore, administering treatments that modulate metabolic pathways can result in subsequent regulation of the immune-inhibitory PD-1 / PD-L1 checkpoint pathway (Watanabe et al., Pyruvate controls the checkpoint inhibitor PD-L1 and suppresses T cell immunity, J Clin Invest. 2017 Jun 30;127(7):2725-2738).

[0219] Checkpoint immunity can be regulated through oncolytic viruses that selectively replicate in tumor cells and induce acute immune responses in the tumor microenvironment, i.e., they act as gene vectors that deliver specific agents (e.g., antibodies, miRNA, siRNA, etc.) to cancer cells, resulting in their tumor lysis and the secretion of cytokines and chemokines, in cooperation with immune checkpoint inhibition (Shi et al., Cancer Immunotherapy: A Focus on the Regulation of Immune Checkpoints, Int J Mol Sci. 2018 May;19(5):1389). Clinical trials are currently underway utilizing the following viruses as checkpoint inhibitors: poliovirus, measles virus, adenovirus, poxvirus, herpes simplex virus (HSV), coxsackievirus, reovirus, Newcastle disease virus (NDV), T-VEC (a herpesvirus co-encoded with GM-CSF (granulocyte-macrophage colony-stimulating factor)), and H101 (Shi et al., supra).

[0220] Checkpoint inhibitors can act at the translational level of immune checkpoint regulation. Translation of mRNA into protein represents a key event in the regulation of gene expression, and therefore, inhibition of immune checkpoint translation can inhibit immune checkpoint pathways. This is a method.

[0221] Inhibition of immune checkpoint pathways can occur at any stage of the immune checkpoint translation process. For example, drugs, molecules, agents, treatments, and / or methods can inhibit the initiation process (whereby the 40S ribosomal subunit is recruited to the 5' end of the mRNA and scans the 5' UTR of the mRNA toward its 3' end). Inhibition can occur by targeting the anticodon of the initiator methionyl-transfer RNA (tRNA) (Met-tRNAi), its base pairing with the start codon, or the recruitment of the 60S subunit to initiate the elongation and sequential addition of amino acids in the translation of immune checkpoint-specific genes. Alternatively, checkpoint inhibitors can inhibit checkpoints at the translational level by preventing the formation of the ternary complex (TC), i.e., eukaryotic translation initiation factor (eIF)2 (or one or more of its α, β, and γ subunits), GTP, and Met-tRNAi.

[0222] Checkpoint inhibition can occur through destabilization of eIF2α by preventing phosphorylation of eIF2α via protein kinase R (PKR), PERK, GCN2, or HRI, or by preventing TCs from associating with the 40S ribosome and / or other initiation factors, thereby preventing the formation of the transcription preinitiation complex (PIC); by inhibiting the eIF4F complex and / or its cap-binding protein eIF4E, the scaffolding protein eIF4G, or the eIF4A helicase. Methods discussing translational control in cancer are discussed in Truitt et al., "New frontiers in translational control of the cancer genome," Nat Rev Cancer. 2016 Apr 26;16(5):288-304, the entire disclosure of which is incorporated herein by reference.

[0223] Checkpoint inhibitors can also include treatments, molecules, agents, and / or methods that modulate immune checkpoints at the cellular and / or protein level, for example, by inhibiting immune checkpoint receptors. Checkpoint inhibition may occur through the use of antibodies, antibody fragments, antigen-binding fragments, small molecules, and / or other drugs, agents, treatments, and / or methods.

[0224] Immune checkpoints refer to inhibitory pathways in the immune system that are responsible for maintaining self-tolerance and regulating the magnitude of immune system responses to minimize damage to peripheral tissues. However, tumor cells can also activate immune system checkpoints to reduce the effectiveness of the immune response against tumor tissues ("block" the immune response). In contrast to the majority of anticancer drugs, checkpoint inhibitors do not directly target tumor cells, but rather target lymphocyte receptors or their ligands to enhance the intrinsic antitumor activity of the immune system (Pardoll, 2012, Nature Reviews Cancer 12:252-264).

[0225] Until recently, cancer immunotherapy has focused considerable efforts on approaches that enhance antitumor immune responses by adoptive transfer of activated effector cells, immunization against relevant antigens, or delivery of nonspecific immunostimulatory agents such as cytokines. However, in the past decade, intensive efforts to develop specific immune checkpoint pathway inhibitors have begun to provide new immunotherapeutic approaches for treating cancer, including the development of ipilimumab (YERVOY®), an antibody (Ab) that binds and inhibits CTLA-4, for the treatment of patients with advanced melanoma (Hodi et al. (2010) N Engl J Med 363:711-23), and nivolumab and pembrolizumab (formerly lambrolizumab, USAN Council S), which specifically bind to the programmed death-1 (PD-1) receptor and block the inhibitory PD-1 / PD-1 ligand pathway. tatement(2013)Pembrolizumab:Statement on a nonproprietary name adopted by the USAN Council(ZZ-165),Nov.27,2013). al. (2014) J Clin Oncol 32(10):1020-30, Hamid et al. (2013) N Engl J Med 369:134-144, Hamid and Carvajal (2013) Expert Opin Biol Ther 13(6):847-61, McDermott and Atkins (2013) Cancer Med 2(5):662-73).

[0226] PD-1 is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression. Nivolumab (formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively blocks the interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of antitumor T cell function (U.S. Patent No. 8,008,449; Wang et al. (2014) Int.). Vitro characterization of the anti-PD-1 The antibody nivolumab, BMS-936558, and in vivo toxicology in non-human primates. Nivolumab is a treatment for patients with unresectable or metastatic melanoma and for patients with ipilimumab and BRAF. It is approved for treating patients with disease progression after BRAF inhibitors if V600 mutation positive, as well as for treating squamous non-small cell lung cancer.

[0227] Recent data suggest a possible secondary mechanism of action of anti-CTLA-4 antibodies within the tumor itself: CTLA-4 has been found to be expressed at higher levels in tumors by regulatory T cells (also referred to herein as "Treg cells") compared with intratumoral effector T cells (also referred to herein as "Teff cells"), leading to the hypothesis that anti-CTLA-4 exerts a preferential and potent effect on Treg cells ("Therapeutic use of anti-CTLA-4 antibodies," Christian U. Blank and Alexander Enk, International Immunology, Vol. 27, No. 1, pp. 3-10). Recent studies of the combination of PD-1 and CTLA-4 have shown that combined blockade of the CTLA-4 and PD-1 pathways also cooperates to increase the ratio of Teff cells to both regulatory T cells and MDSCs, thereby reducing suppression and promoting inflammation in the tumor microenvironment (Curran et al., "Combination of CTLA-4 and PD-1 blockade expands infiltrating T-cells and reduces regulatory T and myeloid cells within B16 melanoma tumors," PNAS | March 2, 2010; vol. 107 (no. 9); pp. 4275-4280, the entire disclosure of which is incorporated herein by reference). The combination of a checkpoint inhibitor and another therapeutic agent(s) can enhance or prolong the antitumor response of the checkpoint inhibitor and / or the effect of the therapeutic agent. In this regard, WO2015 / 069770 discloses a combination treatment based on the activation of the adaptive immune response for treating cancer, specifically the combination of CTLA-4 and PD-1 inhibitors. The disclosure of WO2015 / 069770 is incorporated by reference in its entirety into the disclosure of the present application.

[0228] One mechanism by which checkpoint-blocking anti-CTLA-4 antibodies mediate their antitumor effects is by depleting regulatory T cells. TLA-4 antibodies can be successfully combined with anti-PD1 checkpoint blockade antibodies, which act to abrogate the inhibitory signaling provided to effector T cells. Dual blockade with these antibodies has collectively improved antitumor responses in both preclinical (Proc Natl Acad Sci USA 2010, 107, 4275-4280) and clinical settings (N Engl J Med 2013, 369, 122-133; N Engl J Med 2015, 372, 2006-2017).

[0229] CTLA-4 attenuates the initial activation of naive and memory T cells through interactions with its ligands B7-1 (CD80) and B7-2 (CD86) (Figure 1A). PD-1 is a receptor expressed on the surface of activated mature T cells, activated NK cells, B cells, monocytes, and numerous normal tissues, and plays an important role in maintaining peripheral tolerance [20-21] (Figure 1A). In contrast to CTLA-4, PD-1 acts through interactions with its ligand PD-L1 (also known as B7-H1 or CD274) and is primarily involved in regulating T cell activity in peripheral tissues and also provides a major immune tolerance mechanism within the tumor microenvironment.

[0230] In some embodiments, the immunotherapeutic agent is a modulator of PD-1 activity, a modulator of PD-L1 activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4-1BB activity, a modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a regulator of GITR activity, a regulator of TIGIT activity, a regulator of CD20 activity, a regulator of CD96 activity, a regulator of IDO1 activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, or an immunostimulatory oligonucleotide. In some embodiments, the immune checkpoint regulator is an inhibitor or antagonist, i.e., an activator or agonist, such as a CD28 regulator, a 4-1BB regulator, an OX40 regulator, a CD27 regulator, a CD80 regulator, a CD86 regulator, a CD40 regulator, or a GITR regulator, a Lag-3 regulator, a 41BB regulator, a LIGHT regulator, a CD40 regulator, a GITR regulator, a TGF-beta regulator, a TIM-3 regulator, a SIRP-alpha regulator, a TIGIT regulator, a VSIG8 regulator, a BTLA regulator, a SIGLEC7 regulator, a SIGLEC9 regulator, an ICOS regulator, a B7H3 regulator, a B7H4 regulator, a FAS regulator, and / or a BTNL2 regulator. In some embodiments, the immunotherapeutic is an immune checkpoint modulator as described above (e.g., an immune checkpoint modulator antibody that may be in the form of a monoclonal antibody, a bispecific antibody, a trispecific antibody comprising one or more immune checkpoint antigen-binding moieties, or an immune cell-engaging multivalent antibody / fusion protein / construct known in the art).

[0231] Combination treatment with immune checkpoint inhibitor immunotherapeutics, which may include antibodies that specifically target immune system checkpoints such as CTLA4, PD1, and PD-L1, is one of the most promising new avenues of immunotherapy for cancer and other diseases. Additional checkpoint targets, such as TIM-3, LAG-3, various B-7 ligands, CHK1 and CHK2 kinases, BTLA, and A2aR, are also under investigation. Currently, three checkpoint inhibitors have received accelerated approval from the U.S. Food and Drug Administration for the treatment of cancer, including ipilimumab (Yervoy®), a CTLA-4 inhibitor, and pembrolizumab (Keytruda®) and nivolumab (Opdivo®), both of which are PD-1 inhibitors. In addition, several checkpoint inhibitors are in clinical trials.

[0232] Programmed cell death protein 1, (PD-1 or CD279), a 55-kD type 1 transmembrane protein, binds to the immunoglobulin superfamily members CD28, CTLA- PD-1 is a member of the CD28 family of T cell costimulatory receptors, which includes PD-4, inducible costimulatory molecule (ICOS), and BTLA. PD-1 is highly expressed on activated T cells and B cells. PD-1 expression can also be detected at various expression levels on memory T cell subsets. Two specific ligands for PD-1 have been identified: programmed death-ligand 1 (PD-L1, also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273). PD-L1 and PD-L2 have been shown to downregulate T cell activation upon binding to PD-1 in both mouse and human systems (Okazaki et al., Int Immunol., 2007;19:813-824). The interaction of PD-1 with its ligands, PD-L1 and PD-L2, expressed on antigen-presenting cells (APCs) and dendritic cells (DCs), transmits negative regulatory stimuli to downregulate activated T cell immune responses. Blockade of PD-1 suppresses this negative signal and amplifies T cell responses.

[0233] Numerous studies have shown that the tumor microenvironment manipulates the PD-L1 / PD-1 signaling pathway, and that induction of PD-L1 expression is associated with the inhibition of immune responses against cancer, enabling cancer progression and metastasis. The PD-L1 / PD-1 signaling pathway is a primary mechanism of cancer immune evasion for several reasons. First, and most importantly, this pathway is involved in the negative regulation of immune responses by activated T effector cells found in the periphery. Second, while PD-L1 is upregulated in the tumor microenvironment, PD-1 can also be upregulated on activated tumor-infiltrating T cells, reinforcing a vicious cycle of inhibition. Third, this pathway is intricately involved in both innate and adaptive immune regulation through bidirectional signaling. These factors make the PD-1 / PD-L1 complex a focal point through which cancer can manipulate the immune response and promote its own progression.

[0234] CTLA-4 (also known as cytotoxic T lymphocyte-associated protein 4, CTLA4, CTLA-4, CD152, cluster of differentiation 152, ALPS5, CD, CELIAC3, GRD4, GSE, and IDDM12) is a single-pass type I membrane protein of approximately 24.6 kDa that plays an inhibitory role in T cell function. CTLA-4 was originally identified by differential screening of a mouse cytolytic T cell cDNA library (see Brunet et al., A new member of the immunoglobulin superfamily—CTLA-4, Nature. 1987 Jul 16-22;328(6127):267-70). CTLA-4 has been shown to interact with the b7 family ligands CD80 (also known as cluster of differentiation 80 and B7-1) and CD86 (also known as cluster of differentiation 86 or B7-2). Linsley et al.,CTLA-4 is See, J Exp Med. 1991 Sep 1;174(3):561-9, "A second receptor for the B cell activation antigen B7." Sequence comparison between the human CTLA-4 DNA coding region and the CD28 coding region revealed significant homology between both sequences, with the greatest similarity between the juxtamembrane and cytoplasmic regions; thus, CTLA-4 is involved in the elimination / reduction of T cell activity and antagonizes the activity of CD28. CTLA-4-deficient mice have been shown to exhibit massive lymphoproliferation. Chambers et al., "Lymphoproliferation in CTLA-4-deficient mice is mediated by costimulation-dependent activation of CD4+ T cells," Immunity. 1997 Dec;7(6):885-95. CTLA-4 blockade has been reported to enhance T cell responses both in vitro and in vivo, potentiate induced autoimmune disease, and intensify antitumor immunity (Luhder, J. Exp. Med. 1998;187:427-432; Walunas et al., Immunity. 1994;1:405-413; Kearney, J. Immunol. 1999 5;155:1032-1036; Leach, Science 1996;271:1734-1736). CTLA-4 has also been reported to have alternative and / or additional effects on the initial characteristics of T cell immune responses (Chambers, Curr. Opin. Immunol. 1997;9:396-404; Bluestone, J. Immunol. 1997;158:1989-1993; Thompson, Immunity 1997;7:445-450).

[0235] The first immune checkpoint inhibitor tested in clinical trials was ipilimumab (Yervoy, Bristol-Myers Squibb), a CTLA-4 mAb. CTLA-4 belongs to the immunoglobulin superfamily of receptors that also includes PD-1, BTLA, TIM-3, and V-domain immunoglobulin suppressor of T-cell activation (VISTA). Anti-CTLA-4 mAb is a potent checkpoint inhibitor, releasing the "brake" on both naive and antigen-experienced cells. Treatment enhances the antitumor function of CD8+ T cells, increases the ratio of CD8+ T cells to Foxp3+ T regulatory cells, and inhibits the suppressive function of T regulatory cells. The major drawback of anti-CTLA-4 mAb treatment is the generation of autoimmune toxicity due to the on-target effect of an overproliferating immune system that loses its ability to suppress itself. Up to 25% of patients treated with ipilimumab have been reported to develop severe grade 3-4 adverse events / autoimmune-type side effects, including dermatitis, enterocolitis, hepatitis, endocrinopathy (including hypophysitis, thyroiditis, and adrenal inflammation), arthritis, uveitis, nephritis, and aseptic meningitis. In contrast to the anti-CTLA-4 experience, anti-PD-1 therapy appears to be better tolerated and induces a relatively low rate of autoimmune-type side effects.

[0236] In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of PD-1. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of PD-L1 and / or PD-L2. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of CTLA-4. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of CD80 and / or CD86. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of TIGIT. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of KIR. In some embodiments, the immunotherapeutic agent is an agent that enhances or stimulates the activity of activating immune checkpoint receptors.

[0237] In some embodiments of the methods described herein, the immunotherapeutic agent is a PD-1 antagonist, a PD-L1 antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD80 antagonist, a CD86 antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG3 antagonist, a TIGIT antagonist, a CD20 antagonist, a CD96 antagonist, or an IDO1 antagonist.

[0238] In some embodiments, the PD-1 antagonist is an antibody that specifically binds to PD-1. In some embodiments, the antibody that binds to PD-1 is pembrolizumab (KEYTRUDA®, MK-3475, Merck), pidilizumab (CT-011, Curetech Ltd.), nivolumab (OPDIVO®, BMS-936558, MDX-1106, Bristol Myer Squibb), MEDI0680 (AMP-514, AstraZenenca / MedImmune), REGN2810 (Regeneron Pharmaceuticals), BGB-A317 (BeiGene Ltd.), PDR-001 (Novartis), or STI-A1110 (Sorrento Therapeutics). In some embodiments, the antibody that binds to PD-1 is described in PCT Publication WO2014 / 179664, e.g., the antibodies identified as APE2058, APE1922, APE1923, APE1924, APE1950, or APE1963 (Anaptysbio), or an antibody containing the CDR regions of any of these antibodies. The PD-1 antagonist is a fusion protein containing the extracellular domain of PD-L1 or PD-L2, e.g., AMP-224 (AstraZeneca / MedImmune). In another embodiment, the PD-1 antagonist is a peptide inhibitor, e.g., AUNP-12 (Aurigene).

[0239] In some embodiments, the PD-L1 antagonist is an antibody that specifically binds to PD-L1. In some embodiments, the antibody that binds to PD-L1 is atezolizumab (RG7446, MPDL3280A, Genentech), MEDI4736 (AstraZeneca / MedImmune), BMS-936559 (MDX-1105, Bristol Myers Squibb), avelumab (MSB0010718C, Merck KGaA), KD033 (Kadmon), an antibody portion of KD033, or STI-A1014 (Sorrento Therapeutics). In some embodiments, the antibody that binds to PD-L1 is described in PCT Publication WO2014 / 055897, the entire disclosure of which is incorporated herein by reference, such as Ab-14, Ab-16, Ab-30, Ab-31, Ab-42, Ab-50, Ab-52, or Ab-55, or an antibody containing the CDR regions of any of these antibodies.

[0240] In some embodiments, the CTLA-4 antagonist is an antibody that specifically binds to CTLA-4. In some embodiments, the antibody that binds to CTLA-4 is ipilimumab (YERVOY®, Bristol Myer Squibb) or tremelimumab (CP-675,206, Pfizer). In some embodiments, the CTLA-4 antagonist is a CTLA-4 fusion protein or a soluble CTLA-4 receptor, such as KARR-102 (Kahr Medical Ltd.).

[0241] In some embodiments, the LAG3 antagonist is an antibody that specifically binds to LAG3. In some embodiments, the antibody that binds to LAG3 is IMP701 (Prima BioMed), IMP731 (Prima BioMed / GlaxoSmithKline), BMS-986016 (Bristol Myer Squibb), LAG525 (Novartis), and GSK2831781 (GlaxoSmithKline). In some embodiments, the LAG3 antagonist comprises a soluble LAG3 receptor, e.g., IMP321 (Prima BioMed).

[0242] In some embodiments, the KIR antagonist is an antibody that specifically binds to KIR. In some embodiments, the antibody that binds to KIR is lirilumab (Bristol Myer Squibb / Innate Pharma).

[0243] In some embodiments, the immunotherapeutic agent used in the combinations disclosed herein (e.g., in combination with a compound of Formula I') is an activator or agonist of a costimulatory molecule. In one embodiment, the costimulatory molecule agonist is selected from agonists (e.g., agonistic antibodies or antigen-binding fragments thereof, or soluble fusions) of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligands.

[0244] In some embodiments, the OX40 agonist includes an OX40 ligand, or an OX40-binding portion thereof. For example, the OX40 agonist may be MEDI6383 (AstraZeneca). In some embodiments, the OX40 agonist is an antibody that specifically binds to OX40. In some embodiments, the antibody that binds to OX40 is MEDI6469 (AstraZeneca / MedImmune), MEDI0562 ... In some embodiments, the OX40 agonist is a vector (e.g., an expression vector or a virus, e.g., an adenovirus) capable of expressing an OX40 ligand. In some embodiments, the OX40 expression vector is Delta-24-RGDOX (DNAtrix) or DNX2401 (DNAtrix).

[0245] In some embodiments, the 4-1BB (CD137) agonist is a binding molecule such as anticalin. In some embodiments, the anticalin is PRS-343 (Pieris AG). In some embodiments, the 4-1BB agonist is an antibody that specifically binds to 4-1BB. In some embodiments, the antibody that binds to 4-1BB is PF-2566 (PF-05082566, Pfizer) or urelumab (BMS-663513, Bristol Myer Squibb).

[0246] In some embodiments, the CD27 agonist is an antibody that specifically binds to CD27. In some embodiments, the antibody that binds to CD27 is varlilumab (CDX-1127, Celldex).

[0247] In some embodiments, the GITR agonist comprises a GITR ligand or a GITR-binding portion thereof. In some embodiments, the GITR agonist is an antibody that specifically binds to GITR. In some embodiments, the antibody that binds to GITR is TRX518 (GITR, Inc.), MK-4166 (Merck), or INBRX-110 (Five Prime Therapeutics / Inhibrx).

[0248] TIM-3 has been identified as another important inhibitory receptor expressed by diseased CD8+ T cells. In mouse models of cancer, many dysfunctional tumor-infiltrating CD8+ T cells were indeed found to co-express PD-1 and TIM-3.

[0249] LAG-3 is another recently identified inhibitory receptor that acts to limit effector T cell function and enhance the suppressive activity of T regulatory cells. Recently, it has been shown that PD-1 and LAG-3 are widely co-expressed by tumor-infiltrating T cells in mice, and that combined blockade of PD-1 and LAG-3 elicits potent synergistic antitumor immune responses in mouse models of cancer.

[0250] Blockade of the PD-1 pathway can be combined with vaccines or other compounds of formula I', antibodies, to improve therapeutic efficacy (Hirano, F. et al., Cancer Res., 65(3):1089-1096 (2005); Li, B. et al., Clin. Cancer Res., 15:1507-1509 (2009); and Curran, MA et al., Proc. Natl. Acad. Set, 107(9):4275-4280 (2010)).

[0251] In some embodiments, immunotherapeutics useful in the compositions and methods described herein may include monoclonal antibodies known in the art that specifically target both PD-1 and its ligand PD-L1, bispecific antibodies comprising one or more immune checkpoint antigen-binding moieties, trispecific antibodies, or immune cell-engaging multivalent antibodies / fusion proteins / constructs.

[0252] PD-1 (also known as programmed death 1, CD279, PDCD1) is a cell surface receptor that plays a crucial role in regulating the balance between stimulatory and inhibitory signals in the immune system and maintaining peripheral tolerance (Ishida, Y et al. 1992 EMBO J. 11 3887, Kier, Mary E et al. 20 08 Annu Rev Immunol 26 677-704, Okazaki, Taku et al. 2007 International Immunology 19 813-824). PD-1 is an inhibitory member of the immunoglobulin superfamily with homology to CD28. PD-1 is a monomeric type 1 transmembrane protein consisting of a single immunoglobulin variable-like extracellular domain and a cytoplasmic domain containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based switch motifs (ITSMs). PD-1 expression is inducible on T cells, B cells, natural killer (NK) cells, and monocytes upon lymphocyte activation, e.g., via T cell receptor (TCR) or B cell receptor (BCR) signaling (Kier, Mary E et al. 2008 Annu Rev Immunol 26 677-704; Agata, Y et al. 1996 Int Immunol 8 765-72). PD-1 is the receptor for the ligands CD80, CD86, PD-L1 (B7-H1, CD274), and PD-L2 (B7-DC, CD273), which are cell surface-expressed members of the B7 family (Freeman, Gordon et al. 2000 J Exp Med 192 1027; Latchman, Y et al. 2001 Nat Immunol 2 261). Upon ligand engagement, PD-1 recruits phosphatases, such as SHP-1 and SHP-2, to its intracellular tyrosine motifs, which then dephosphorylate effector molecules activated by TCR or BCR signaling (Chemnitz, J et al. 2004 J Immunol 173 945-954; Riley, James L 2009 Immunological Reviews 229 114-125). Thus, PD-1 transmits inhibitory signals to T and B cells only when simultaneously engaged with TCR or BCR.

[0253] PD-1 has been demonstrated to downregulate effector T cell responses through both cell-intrinsic and cell-extrinsic mechanisms. Inhibitory signaling through PD-1 induces a state of unresponsiveness in T cells, resulting in cells unable to undergo clonal expansion or produce optimal levels of effector cytokines. PD-1 can also induce apoptosis in T cells through its ability to suppress survival signals from costimulation, resulting in reduced expression of key anti-apoptotic molecules such as Bcl-XL (Kier, Mary E et al. 2008 Annu Rev Immunol 26 677-704). In addition to these direct actions, recent publications have implicated PD-1 in suppressing effector cells by promoting the induction and maintenance of regulatory T cells (TREGs). For example, PD-L1 expressed on dendritic cells has been shown to synergize with TGF-β to promote the induction of CD4+ FoxP3+ TREGs with enhanced suppressive function (Francisco, Loise M et al. 2009 J Exp Med 206 3015-3029).

[0254] TIM-3 (also known as T cell immunoglobulin and mucin-domain containing-3, TIM-3, hepatitis A virus cellular receptor 2, HAVCR2, HAVcr-2, KIM-3, TIMD-3, TIMD3, Tim-3, and CD366) is a single-pass type I membrane protein of approximately 33.4 kDa involved in immune responses (Sanchez-Fueyo et al., Tim-3 inhibits T helper type 1-mediated auto- and alloimmune responses and promotes immunological tolerance, Nat. Immunol. 4:1093-1101(2003)).

[0255] TIM-3 is selectively expressed on Th1 cells and phagocytes (e.g., macrophages and dendritic cells). The use of siRNA or blocking antibodies to reduce human expression resulted in increased secretion of interferon-γ (IFN-γ) from CD4+ T cells, implicating an inhibitory role of TIM-3 in human T cells. Analysis of clinical samples from affected patients showed that TIM-3 expression was absent in CD4+ cells. In particular, TIM-3 expression levels in T cell clones obtained from the cerebrospinal fluid of patients with multiple sclerosis were lower than those in clones obtained from normal, healthy individuals, and IFN-γ secretion was higher (Koguchi K et al., J Exp Med. 203:1413-8 (2006)).

[0256] TIM-3 binds to its ligands, galectin-9 (a member of the galectin family that is ubiquitously expressed on various cell types and binds to β-galactoside), phosphatidylserine (PtdSer) (DeKryff et al., T cell / transmembrane, Ig, and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells, J Immunol. 2010 Feb 15;184(4):1918-30), and high-mobility group protein 1 (also known as HMGB1, HMG1, HMG3, SBP-1, HMG-1, and high-mobility group box 1) (Chiba et al., Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1, Nat Immunol. 2012). Sep;13(9):832-42), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, also known as BGP, BGP1, BGP1, carcinoembryonic antigen-related cell adhesion molecule 1) (Huang et al., CEACAM1 regulates TIM-3-mediated tolerance and exhaustion, Nature. 2015 Jan 15;517(7534):386-90).

[0257] BTLA (also known as B and T lymphocyte attenuation factor, BTLA1, CD272, and B and T lymphocyte-associated) is a single-pass type 1 membrane protein of approximately 27.3 kDa involved in lymphocyte inhibition during immune responses. BTLA is constitutively expressed in both B and T cells. BTLA interacts with HVEM (herpesvirus entry mediator), a member of the tumor necrosis factor receptor (TNFR) family (Gonzalez et al., Proc. Natl. Acad. Sci. USA, 2005, 102:1116-21). The interaction of BTLA, which belongs to the CD28 family of the immunoglobulin superfamily, with HVEM, a costimulatory tumor necrosis factor (TNF) receptor (TNFR), is unique in that it defines crosstalk between these two receptor families. BTLA contains a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) and a membrane-distal immunoreceptor tyrosine-based switch motif (ITSM). Disruption of either the ITIM or ITSM eliminates the ability of BTLA to recruit either SHP1 or SHP2, suggesting that BTLA recruits SHP1 and SHP2 in a manner distinct from PD-1 and that both tyrosine motifs are required to block T cell activation. The BTLA cytoplasmic tail also contains a third conserved tyrosine-containing motif within the cytoplasmic domain, whose sequence is similar to the Grb-2 recruitment site (YXN). Furthermore, phosphorylated peptides containing this BTLA N-terminal tyrosine motif can interact with GRB2 and the p85 subunit of PI3K in vitro, although the functional effects of this interaction have not yet been investigated in vivo (Gavrieli et al., Biochem. Biophysi Res Commun. 2003, 312, 1236-43). BTLA is the receptor for the ligands PTPN6 / SHP-1, PTPN11 / SHP-2, TNFRSF14 / HVEM, and B7H4.

[0258] VISTA (V-domain Ig suppressor of T cell activation, VSIR, B7-H5, B 7H5, GI24, PP2135, SISP1, DD1 alpha, VISTA, C10orf54, chromosome 10 open reading frame 54, PD-1H, and V-set immunoregulatory receptor (V-set immunomodulatory receptor) is a single-pass type I membrane protein of approximately 33.9 kDa that is involved in T cell inhibitory responses, embryonic stem cell differentiation via inhibition of BMP4 signaling, and MMP14-mediated MMP2 activation (Yoon et al., Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53, Science. 2015 Jul 31;349(6247):1261669). VISTA interacts with the ligand VSIG-3 (Wang et al., VSIG-3 as a ligand of VISTA inhibits human T-cell function, Immunology.2019 Jan;156(1):74-85).

[0259] LAG-3 (lymphocyte activation gene 3, also known as LAG3, CD223, and lymphocyte activation 3) is a single-pass type I membrane protein of approximately 57.4 kDa that is involved in lymphocyte activation and also binds to HLA class-II antigens. LAG-3 is a member of the immunoglobulin supergene family and is involved in the activation of activated T cells (Huard et al., 1994, Immunogenetics 39:213), NK cells (Triebel et al., 1994, Immunogenetics 39:213), and NK cells (Triebel et al., 1994, Immunogenetics 39:213). LAG-3 is expressed on T cells (Huang et al., 2004, Immunity 21:503-513; Camisaschi et al., 2010, J Immunol. 184:6545-6551; Gagliani et al., 2013, Nat Med 19:739-746), and plasmacytoid dendritic cells (DCs) (Workman et al., 2009, J Immunol 182:1885-1891). LAG-3 is a membrane protein encoded by a gene located on chromosome 12 and is structurally and genetically related to CD4. Like CD4, LAG-3 can interact with MHC class II molecules on the cell surface (Baixeras et al., 1992, J. Exp. Med. 176:327-337; Huard et al., 1996, Eur. J. Immunol. 26:1180-1186). Direct binding of LAG-3 to MHC class II has been suggested to play a role in downregulating antigen-dependent stimulation of CD4+ T lymphocytes (Huard et al., 1994, Eur. J. Immunol. 24:3216-3221), and LAG-3 blockade has been shown to reactivate CD8+ lymphocytes in both tumor or self-antigen (Gross et al., 2007, J. Clin. Invest. 117:3383-3392) and viral models (Blackburn et al., 2009, Nat. Immunol. 10:29-37). Furthermore, the cytoplasmic domain of LAG-3 can interact with LAP (LAG-3-associated protein), a signaling molecule involved in downregulation of the CD3 / TCR activation pathway (Iouzalen et al., 2001, Eur. J. Immunol. 31:2885-2891). Furthermore, CD4+CD25+ regulatory T cells (Tregs) have been shown to express LAG-3 upon activation, which contributes to the suppressor activity of Treg cells (Huang, C. et al., 2004, Immunity 21:503-513).LAG-3 can also negatively regulate T cell homeostasis by Treg cells in both T cell-dependent and -independent mechanisms (Workman, CJ and Vignali, DA, 2005, J. Immunol. 174:688-695).

[0260] LAG-3 has been shown to interact with MHC class II molecules (Huard et al., CD4 / major histocompatibility complex class II interaction analyzed with CD4- and lymphocyte activation gene-3(LA G-3)-Ig fusion proteins, Eur J Immunol.1995 Sep;25(9):2718-21).

[0261] In addition, several kinases are known to be checkpoint inhibitors, for example, CHEK-1, CHEK-2, and A2aR.

[0262] CHEK-1 (also known as CHK1 kinase, CHK1, and checkpoint kinase 1) is an approximately 54.4 kDa serine / threonine-protein kinase involved in checkpoint-mediated cell cycle arrest and activation of DNA repair in response to DNA damage and / or non-replicating DNA.

[0263] CHEK-2 (also known as CHK2 kinase, CDS1, CHK2, HuCds1, LFS2, PP1425, RAD53, hCds1, and checkpoint kinase 2) is an approximately 60.9 kDa serine / threonine-protein kinase involved in checkpoint-mediated cell cycle arrest, DNA repair activation, and double-strand break-mediated apoptosis.

[0264] A2aR (also known as adenosine A2A receptor, ADORA2A, adenosine A2a receptor, A2aR, ADORA2, and RDC8) is an approximately 44.7 kDa multipass membrane receptor for adenosine and other ligands.

[0265] In various embodiments, immunotherapeutics may comprise antibodies or antigen-binding fragments thereof. Within this definition, immune checkpoint inhibitors include bispecific antibodies and immune cell-engaging multivalent antibodies / fusion proteins / constructs known in the art. In some embodiments, immunotherapeutics comprising bispecific antibodies may include bispecific antibodies that are bivalent and bind to either the same epitope of an immune checkpoint molecule, two different epitopes of the same immune checkpoint molecule, or different epitopes of two different immune checkpoints.

[0266] One skilled in the art can realize several bispecific antibody formats known in the art that target one or more of CTLA4, PD1, PD-L1 TIM-3, LAG-3, various B-7 ligands, B7H3, B7H4, CHK1 and CHK2 kinases, BTLA, A2aR, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, SIRP-alpha, TIGIT, VSIG8, SIGLEC7, SIGLEC9, ICOS, FAS, BTNL2, and others for use in the combinations described herein.

[0267] In various embodiments, the immunotherapeutic may comprise an immune cell-engaging multivalent antibody / fusion protein / construct.

[0268] In one embodiment of the disclosure, checkpoint inhibitors are used in combination with compounds of formula I' to reduce or inhibit the metastasis of a primary tumor or cancer to other sites, or the formation or establishment of metastatic tumors or cancers at other sites distal to the primary tumor or cancer, thereby inhibiting or reducing tumor or cancer recurrence or tumor or cancer progression.

[0269] In a further embodiment of the present disclosure, there is provided a combination therapy for treating cancer comprising a compound of Formula I' and blockade of a checkpoint inhibitor that has the ability to induce a potent and durable immune response with enhanced therapeutic efficacy and more manageable toxicity.

[0270] In a further embodiment of the present disclosure, there is provided a combination therapy for treating cancer comprising a compound of formula I' and an immune checkpoint inhibitor. The present invention provides a method for treating cancer and / or preventing the establishment of metastases by using checkpoint inhibitors that act synergistically with the compounds of the present invention.

[0271] In further embodiments, the methods of the present disclosure include one or more of the following: 1) reducing or inhibiting the growth, proliferation, migration or invasiveness of tumor or cancer cells that are likely to give rise to or give rise to metastases; 2) reducing or inhibiting the formation or establishment of metastases that arise from the primary tumor or cancer to one or more other sites, locations or regions distinct from the primary tumor or cancer; 3) reducing or inhibiting the growth or proliferation of metastases at one or more other sites, locations or regions distinct from the primary tumor or cancer after they have formed or established; 4) reducing or inhibiting the formation or establishment of additional metastases after they have formed or established; 5) increasing overall survival; 6) increasing progression-free survival; or 7) disease stabilization.

[0272] In one embodiment of the present disclosure, administration of the immunotherapeutic agent, in combination with a compound of formula I', results in a detectable or measurable improvement in a given subject's condition, for example, a reduction or amelioration of one or more adverse (physical) symptoms or consequences associated with a cell proliferative or hyperproliferative disorder, a neoplasm, a tumor or cancer, or the presence of metastases, i.e., a therapeutic or beneficial effect.

[0273] A therapeutic or beneficial effect is any objective or subjective, transient, temporary, or long-term improvement in a condition or pathology, or a reduction in the onset, severity, duration, or frequency of adverse symptoms associated with or resulting from a cell proliferation or hyperproliferative disorder, such as a neoplasm, tumor, or cancer, or metastasis. These may lead to improved survival. A satisfactory clinical endpoint of a treatment method according to the present disclosure is achieved, for example, when there is a gradual or partial reduction in the severity, duration, or frequency of one or more associated pathologies, adverse symptoms, or complications, or inhibition or reversal of one or more physiological, biochemical, or cellular manifestations or characteristics of a cell proliferation or hyperproliferative disorder, such as a neoplasm, tumor, or cancer, or metastasis. Thus, a therapeutic effect or improvement can be, but is not limited to, the destruction of a target proliferating cell (e.g., a neoplasm, tumor, or cancer, or metastasis), or the elimination of one or more, most or all, pathologies, adverse symptoms, or complications associated with or resulting from a cell proliferation or hyperproliferative disorder, such as a neoplasm, tumor, or cancer, or metastasis. However, a therapeutic effect or improvement need not be a cure or complete destruction of all target proliferating cells (e.g., a neoplasm, tumor or cancer, or metastasis) or the elimination of all pathology, adverse symptoms, or complications associated with or resulting from a cell proliferation or hyperproliferative disorder, such as a neoplasm, tumor or cancer, or metastasis. For example, partial destruction of tumor or cancer cell mass by inhibiting tumor or cancer progression or worsening, or stabilization of tumor or cancer mass, size, or cell number, even if some or most of the tumor or cancer mass, size, or cells remain, may reduce mortality and extend lifespan by even just a few days, weeks, or months.

[0274] Specific non-limiting examples of therapeutic effects include a reduction in the volume (size or cell mass) or cell number of a neoplasm, tumor or cancer, or metastasis; inhibiting or preventing (e.g., stabilizing) an increase in the volume of a neoplasm, tumor or cancer; slowing or inhibiting the progression, worsening, or metastasis of a neoplasm, tumor, or cancer; or inhibiting the proliferation, growth, or metastasis of a neoplasm, tumor, or cancer.

[0275] In one embodiment of the present disclosure, administration of an immunotherapeutic agent in combination treatment with a compound of Formula I' results in: (i) irCR = complete disappearance of all lesions, whether measurable or not, and the absence of new lesions (confirmed by repeated serial assessments no less than 4 weeks from the date of first recording); (ii) irPR = reduction in tumor burden by 50% or more compared to baseline; (confirmed by serial assessments at least 4 weeks after initial documentation) to provide a detectable or measurable improvement or overall response according to the irRC (derived from time point response assessments and based on tumor burden).

[0276] Optionally, any of the methods described herein may not be immediately effective. For example, treatment may be followed by an increase in the number or amount of neoplastic, tumor, or cancer cells, which may be followed over time by an eventual stabilization or reduction in the amount, size, or number of tumor cells in a given subject.

[0277] Additional adverse symptoms and complications associated with neoplasia, tumors, cancer, and metastasis that can be inhibited, reduced, decreased, delayed, or prevented include, for example, nausea, loss of appetite, lethargy, pain, and discomfort. Thus, a partial or complete decrease or reduction in the severity, duration, or frequency of adverse symptoms or complications associated with or resulting from a cell hyperproliferative disorder, improvements in the subject's quality of life and / or health, such as increased energy, appetite, and mental well-being, are all specific, non-limiting examples of therapeutic effects.

[0278] Thus, the therapeutic effect or improvement may also include a subjective improvement in the quality of life of the subject receiving treatment. In a further embodiment, the method extends or increases the lifespan (survival) of the subject. In a further embodiment, the method improves the quality of life of the subject.

[0279] In one embodiment, administration of the immunotherapeutic agent in combination treatment with a compound of Formula I' results in a clinically relevant improvement in one or more markers of disease status and progression selected from one or more of: (i) overall survival; (ii) progression-free survival; (iii) overall response rate; (iv) reduction in metastatic disease; (v) circulating levels of tumor antigens such as carbohydrate antigen 19.9 (CA19.9) and carcinoembryonic antigen (CEA) or others depending on the tumor; (vii) nutritional status (weight, appetite, serum albumin); (viii) pain management or analgesic use; (ix) CRP / albumin ratio.

[0280] Treatment with compounds of Formula I' in combination with immunotherapeutics results in a more complex immune response that includes not only the development of innate and type 1 immunity, but also immunomodulation to more efficiently restore proper immune function.

[0281] In various exemplary methods, a checkpoint inhibitory antibody (monoclonal or polyclonal, bispecific, trispecific, or immune cell-engaging multivalent antibody / fusion protein / construct) directed against a checkpoint molecule of interest (e.g., PD-1) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. A sequence encoding the antibody of interest or its antigen-binding fragment can be maintained in a vector in a host cell, which can then be expanded and frozen for future use. Recombinant monoclonal antibody production in cell culture can be carried out by cloning antibody genes from B cells by means known in the art. See, for example, Tiller et al., 2008, J. Immunol. Methods 329,112; U.S. Patent No. 7,314,622.

[0282] In some embodiments, methods for producing a recombinant antibody may include culturing a host cell containing isolated nucleic acid(s) encoding an antibody of the present disclosure. The method for culturing a host cell containing isolated nucleic acid(s) encoding an antibody of the present disclosure can be carried out in a variety of ways, depending on the nature of the antibody. In some embodiments, when the antibody of the present disclosure is a conventional full-length antibody, for example, the heavy chain variable region and light chain variable region can be isolated under conditions such that the antibody is produced.

[0283] Generally, nucleic acids encoding the antibodies or antigen-binding fragments thereof of the present disclosure are obtained. Such polynucleotides encode both the variable and constant regions of each of the heavy and light chains, although other combinations are contemplated by the present disclosure. The present disclosure also contemplates oligonucleotide fragments derived from the disclosed polynucleotides and nucleic acid sequences complementary to these polynucleotides.

[0284] The polynucleotide may be in the form of RNA, DNA, cDNA, genomic DNA, nucleic acid analogs, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single-stranded, may be the coding (sense) strand or non-coding (antisense) strand. A coding sequence that encodes a polypeptide may be identical to the coding sequence or, as a result of redundancy or degeneracy in the genetic code, may be a different coding sequence that encodes the same polypeptide.

[0285] In some embodiments, the nucleic acid(s) encoding the antibodies of the present disclosure are incorporated into an expression vector, which may be extrachromosomal or may be designed to integrate into the genome of the host cell into which it is introduced. The expression vector may contain any number of appropriate regulatory sequences (including, but not limited to, transcriptional and translational control sequences, promoters, ribosomal binding sites, enhancers, origins of replication, etc.) or other components (such as selection genes), all operably linked, as is well known in the art. In some cases, two nucleic acids are used, each in a different expression vector (e.g., the heavy chain in a first expression vector and the light chain in a second expression vector), or alternatively, they can be in the same expression vector. One of ordinary skill in the art will recognize that the design of the expression vector(s), including the selection of regulatory sequences, may depend on factors such as the choice of host cell, the desired level of protein expression, and the like.

[0286] Generally, nucleic acid and / or expression can be introduced into suitable host cells using any method appropriate for the selected host cell (e.g., transformation, transfection, electroporation, infection) such that the nucleic acid molecule(s) are operably linked to one or more expression control elements (e.g., in a vector, in a construct produced by a cellular process, integrated into the host cell genome) to produce a recombinant host cell. The resulting recombinant host cell can be maintained under conditions appropriate for expression (e.g., in the presence of an inducer, in a suitable non-human animal, in a suitable culture medium supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), thereby producing the encoded polypeptide(s). Optionally, the heavy chain is produced in one cell and the light chain in another cell.

[0287] Mammalian cell lines available as hosts for expression are known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), Manassas, VA USA, including Chinese hamster ovary (CHO) cells, HEK293 cells, NSO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and several other cell lines. Non-mammalian cells, including, but not limited to, bacteria, yeast, insects, and plants, can also be used to express recombinant antibodies. In some embodiments, antibodies can be produced in transgenic animals such as cows or chickens.

[0288] Exemplary and illustrative recombinant methods for antibody molecular biology, expression, purification, and screening are found, for example, in Antibody Engineering, Kontermann & Dubel (eds.), Springer, Heidelberg, 2001 and 2010, Hayhurst & Georgiou, 2001, Curr. Opin. Ch. em. Biol. 5:683-689, Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76, and Morrison, S. (1985) Science 229:1202, the entire disclosures of which are incorporated herein by reference.

[0289] In various embodiments, polynucleotide sequences encoding selected variable heavy and light chains can be used for genetic engineering to humanize the antibody or to improve the affinity or other characteristics of the antibody. Antibodies can also be customized for use in, for example, dogs, cats, primates, horses, and cattle.

[0290] In some embodiments, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response can also be used for humanization or the generation of human antibodies. Examples of such technology are Xenomouse™ from Abgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton, NJ).

[0291] The immune checkpoint modulating antibodies of the present disclosure can be produced recombinantly by first isolating the antibody and antibody-producing cells from a host animal, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in a host cell (e.g., a CHO cell). Another method that can be used is to express the antibody sequence in a plant (e.g., tobacco) or in a yeast cell (e.g., Pichia pastoris or Sacchromyces cerevisiae). Methods for expressing antibodies recombinantly in plants or yeast have been disclosed. See, for example, Peeters, et al., Vaccine 19:2756, 2001; Lonberg, N. and D. Huszar, Int. Rev. Immunol 13:65, 1995; and Horwitz, A. H. et al., Proc. Natl. Acad. Sci. 85:8678-8682, the entire disclosures of which are incorporated herein by reference. Methods for producing antibody derivatives, eg, domains, single chain, etc., are known in the art.

[0292] Immunoassays and flow cytometric sorting techniques such as fluorescence activated cell sorting (FACS) can also be used to isolate antibodies specific for checkpoint molecules.

[0293] In some embodiments, the polynucleotide comprises a sequence encoding the heavy and / or light chain variable region of a checkpoint inhibitor antibody or antigen-binding fragment thereof of the present disclosure. The sequence encoding the antibody of interest or antigen-binding fragment thereof can be maintained in a vector within a host cell, which can then be expanded and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

[0294] The present disclosure includes affinity-matured checkpoint modulating antibodies. For example, affinity-matured antibodies can be generated by procedures known in the art (Marks et al., 1992, Bio / Technology, 10:779-783; Barbas et al., 1994, Proc Nat. Acad. Sci. USA 91:3809-3813). One method for characterizing the CDRs of an antibody and / or altering (e.g., improving) the binding affinity of a polypeptide such as an antibody is referred to as "library scanning mutagenesis." An exemplary method for obtaining affinity-matured antibodies and antigen-binding fragments involves substituting one or more amino acid positions in the CDRs with the corresponding CDRs using art-recognized methods. This may involve substituting two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids. This results in a library of clones, each of which has a complexity of two or more members (when two or more amino acids are substituted at each position). Typically, the library also includes clones containing natural (unsubstituted) amino acids. A small number of clones from each library, for example, about 20-80 clones (depending on the complexity of the library), are screened for binding affinity to the target polypeptide (or other binding target) to identify candidates with increased, identical, decreased, or no binding. Methods for determining binding affinity are well known in the art. Binding affinity can be determined, for example, using Biacore™ surface plasmon resonance analysis, Kinexa® biosensor, scintillation proximity assay, ELISA, ORIGEN® immunoassay, fluorescence quenching, fluorescence transfer, and / or yeast display, which detect differences in binding affinity of about 2-fold or greater. Binding affinity can also be screened using an appropriate bioassay. Biacore™ is particularly useful when the starting antibody already binds with a relatively high affinity, e.g., a KD of about 10 nM or less. The library of clones can then be recombinantly introduced into a selection construct using any method known in the art for selection, including phage display, yeast display, and ribosome display.

[0295] For example, antibodies can be modified in, for example, the heavy and / or light chain variable domains to alter the binding characteristics of the antibody. Changes in the variable regions can alter binding affinity and / or specificity. In some embodiments, no more than one to five conservative amino acid substitutions are made in the CDR domains. In other embodiments, no more than one to three conservative amino acid substitutions are made in the CDR domains. Mutations can be made in one or more of the CDR regions, for example, to increase or decrease the KD of an antibody directed against a checkpoint molecule, to increase or decrease the k, or to alter the binding specificity of the antibody. Site-directed mutagenesis techniques are well known in the art. See, e.g., Sambrook et al. and Ausubel et al.

[0296] Pharmaceutical compositions containing a compound of Formula I' according to the present disclosure typically contain an effective amount of the compound of Formula I', an immunotherapeutic agent, and / or both dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that, where appropriate, do not produce adverse, allergic, or other untoward reactions when administered to an animal, e.g., a human. The preparation of pharmaceutical compositions containing a compound of Formula I' will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990. Furthermore, it will be understood that for administration to animals (e.g., humans), the formulation should meet sterility, pyrogenicity, general safety, and purity standards. Specific examples of pharmacologically acceptable carriers for combination compositions containing a compound of Formula I' mixed with an immunotherapeutic agent as described herein are borate buffer or sterile saline (0.9% NaCl).

[0297] Formulations of immunotherapeutic agents, e.g., immune checkpoint modulating antibodies, used in accordance with the present disclosure can be prepared for storage in the form of a lyophilized formulation or an aqueous solution and / or suspension by mixing the antibody having the desired purity with optional pharmaceutically acceptable carriers, additives, or stabilizers, as described and exemplified in detail in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed.

[1980] . Acceptable carriers, additives, buffers, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include suitable aqueous and / or non-aqueous additives that can be used in pharmaceutical compositions of the present disclosure, e.g., water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, buffers such as phosphate, citric acid, and other organic acids. Antioxidants may include, for example, (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol), low molecular weight (less than about 10 residues). Other exemplary pharmaceutically acceptable excipients may include polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0298] In one exemplary embodiment, the pharmaceutical composition can optionally contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents and toxicity adjusting agents, as needed to approximate physiological conditions, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. In some embodiments, the checkpoint inhibitory antibodies or antigen-binding fragments thereof of the present disclosure can be formulated and lyophilized for storage, and then reconstituted in appropriate additives prior to use according to lyophilization and reconstitution techniques known in the art. In one exemplary pharmaceutical composition containing one or more checkpoint inhibitory antibodies or antigen-binding fragments thereof, the composition is formulated as a preservative-free sterile solution of one or more checkpoint inhibitory antibodies or antigen-binding fragments thereof for intravenous or subcutaneous administration. The formulation can be supplied as a single-use prefilled pen, a single-use prefilled glass syringe containing, for example, about 1 mL, or a single-use institutional vial. Preferably, pharmaceutical compositions containing a checkpoint inhibitor antibody or antigen-binding fragment thereof are clear and colorless and have a pH of about 6.9 to 5.0, preferably 6.5 to 5.0, and even more preferably about 6.0 to about 5.0. In various embodiments, formulations containing the pharmaceutical composition, when reconstituted and administered to a subject, can contain about 500 mg to about 10 mg of the checkpoint inhibitor antibody or antigen-binding fragment thereof per mL of solution, or about 400 mg to about 20 mg, or about 300 mg to about 30 mg, or about 200 mg to about 50 mg. Exemplary injection or infusion additives can include mannitol, citric acid monohydrate, sodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, polysorbate 80, sodium chloride, sodium citrate, and water for parenteral administration, e.g., intravenous, intramuscular, intraperitoneal, or subcutaneous administration.

[0299] In another exemplary embodiment, one or more immunotherapeutic agents, or antigen-binding fragments thereof, are formulated for intravenous or subcutaneous administration as a sterile aqueous solution containing 1-75 mg / mL, or more preferably, about 5-60 mg / mL, or even more preferably, about 10-50 mg / mL, or even more preferably, about 10-40 mg / mL of antibody, along with sodium acetate, polysorbate 80, and sodium chloride, at a pH ranging from about 5-6. Preferably, the intravenous or subcutaneous formulation is a sterile aqueous solution containing 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg / mL of the immunotherapeutic agent, e.g., an immune checkpoint inhibitory antibody or antigen-binding fragment thereof, in 20 mM sodium acetate, 0.2 mg / mL polysorbate 80, and 140 mM sodium chloride at pH 5.5. Additionally, the solution containing the checkpoint inhibitory antibody or antigen-binding fragment thereof can contain, among many other compounds, histidine, mannitol, sucrose, trehalose, glycine, poly(ethylene) glycol, EDTA, methionine, and any combination thereof, as well as many other compounds known in the relevant art.

[0300] In one embodiment, a pharmaceutical composition of the present disclosure comprises the following ingredients: 5-500 mg of an immunotherapeutic or antigen-binding fragment thereof of the present disclosure, 10 mM histidine, 5% sucrose, and 0.01% polysorbate 80 at pH 5.8, with or without a compound of Formula I'. This composition can also be provided as a lyophilized powder. When this powder is reconstituted to the full volume, the composition maintains the same formulation. Alternatively, the powder can be reconstituted to half the volume, in which case the composition comprises 10-500 mg of an immunotherapeutic or antigen-binding fragment thereof of the present disclosure, 20 mM histidine, 10% sucrose, and 0.02% polysorbate 80 at pH 5.8.

[0301] In one embodiment, a portion of the dose of the immunotherapeutic formulation is administered by intravenous bolus, with the remainder administered by infusion. For example, an intravenous injection of about 0.001 to about 200 mg / kg, e.g., about 0.001 mg / kg to about 100 mg / kg, or about 0.001 mg / kg to about 50 mg / kg, or about 0.001 mg / kg to about 10 mg / kg of the immunotherapeutic or antigen-binding fragment thereof can be given as a bolus, and the remainder of the antibody dose can be administered by intravenous injection. A predetermined dose of the immunotherapeutic or antigen-binding fragment thereof can be administered over a period of, for example, 1 hour, 2 hours, or 5 hours.

[0302] In a further embodiment, a portion of the dose of the immunotherapeutic formulation is administered in the form of a bolus via subcutaneous injection and / or infusion, and the remainder via infusion. In some exemplary doses, the immunotherapeutic formulation can be administered subcutaneously at a dose ranging from about 0.001 to about 200 mg / kg, e.g., from about 0.001 mg / kg to about 100 mg / kg, or from about 0.001 mg / kg to about 50 mg / kg, or via intravenous injection of about 0.001 mg / kg to about 10 mg / kg of the immunotherapeutic agent, or antigen-binding fragment thereof. In some embodiments, the dose can be given as a bolus, and the remainder of the immunotherapeutic dose can be administered via subcutaneous or intravenous injection. A given dose of the immunotherapeutic agent, or antigen-binding fragment thereof, can be administered over a period of, for example, 1 hour, 2 hours, or 5 hours.

[0303] The formulations herein may contain more than one active compound, preferably compounds with complementary activities that do not adversely affect each other, as needed to treat a particular indication. For example, it may be desirable to provide one or more immunotherapeutic agents with different specificities. Alternatively, or in addition, the compositions may include anti-inflammatory agents, chemotherapeutic agents, cytotoxic agents, cytokines, growth inhibitors, and / or small molecule antagonists. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

[0304] Formulations to be used for in vivo administration should be sterile or nearly sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0305] In various embodiments, illustrative formulations of the pharmaceutical compositions described herein can be prepared using methods well known in the art of pharmaceutical formulation. In general, such preparation methods include combining the active ingredient with the carrier or one or more other accessory ingredients, and then, if desired, and optionally packaging the product into desired single or multi-dose units.

[0306] In some embodiments, compositions comprising compounds of Formula I' can be delivered in vesicles, and immunotherapeutic agents can be delivered in the same liposomal formulation or in a separate formulation compatible with the liposomal formulation containing the compound of Formula I'. In some instances, liposomes containing one or more liposomal surface moieties, such as polyethylene glycol and antibodies and antibody fragments thereof that target desired tumor surface antigens, receptors, growth factors, glycoproteins, glycolipids, or neoantigens, are selectively transported to specific cells or organs, thus enhancing targeted drug delivery.

[0307] In another embodiment, the compounds of formula I' can be delivered in vesicles, particularly liposomes (Langer, Science 249:1527-1533 (1990); Treat et al., LIPOSOMES IN THE THERAPY OF INFECTIOUS DISEASE AND CANCER, Lopez-Berestein and Fidler (eds.), Liss, NY, pp. 353-365 (1989); see Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0308] In yet another embodiment, the compound of Formula I', or a composition containing a combination, or a composition containing an immunotherapeutic agent, can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, the controlled release of the compound of Formula I' can include polymeric materials to provide sustained, intermediate, pulsatile, or alternating release (MEDICAL APPLICATIONS OF See CONTROLLED RELEASE, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983). See also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989). Other controlled-release systems discussed in the review by Langer (Science 249:1527-1533 (1990)) can also be used.

[0309] The optimum concentration of the active ingredient(s) in the selected medium can be determined empirically according to procedures well known to those skilled in the art and will depend on the final pharmaceutical formulation desired and the intended use.

[0310] The present disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the pharmaceutical composition of the present disclosure, comprising at least a compound of Formula I' as described herein and one or more checkpoint inhibitory antibodies or antigen-binding fragments thereof. In other embodiments, the kit may include one or more additional containers providing pharmaceutically acceptable excipients, e.g., diluents. In one embodiment, the kit may include at least one container that can contain a compound of Formula I' of the present disclosure, a checkpoint inhibitory antibody or antigen-binding fragment thereof. The kit may also include a checkpoint molecular mediator. The composition may also include a set of instructions for preparing and administering to a subject in need thereof a final pharmaceutical composition for treating a medial disease or disorder.

[0311] In some embodiments of the present disclosure, the immunotherapeutic agent is an immune cell population that can be administered in combination with a compound of Formula I' to treat a subject with cancer. In some embodiments, the immunotherapeutic agent is an immune cell population, such as white blood cells (nucleated white blood cells), that contain (e.g., express) a receptor that binds to an antigen of interest. White blood cells of the present disclosure can be, for example, neutrophils, eosinophils, basophils, lymphocytes, or monocytes. In some embodiments, the white blood cells are lymphocytes. Examples of lymphocytes include T cells, B cells, natural killer (NK) cells, or NKT cells. In some embodiments, the T cells are CD4+ Th (T helper) cells, CD8+ cytotoxic T cells, γδ T cells, or regulatory (suppressor) T cells. In some embodiments, the immune cells are dendritic cells.

[0312] The immune cells of the present disclosure are, in some embodiments, genetically engineered to express an antigen-binding receptor. A cell is considered "engineered" if it contains an engineered (exogenous) nucleic acid. The engineered nucleic acid of the present disclosure can be introduced into a cell by any known (e.g., conventional) method. For example, the engineered nucleic acid can be introduced into a cell by electroporation (see, e.g., Heiser WCTranscription Factor Protocols: Methods in Molecular Biology.TM.2000;130:117-134), chemicals (e.g., calcium phosphate or lipids), transfection (see, e.g., Lewis WH, et al., Somatic Cell Genet.1980 May;6(3):333-47; Chen C., et al., Mol Cell Biol.1987 August;7(8):2745-2752), fusion with bacterial protoplasts containing recombinant plasmids (see, e.g., Schaffner W. Proc Natl Acad Sci USA.1980 April;77(4):2163-7), direct microinjection of purified DNA into the nucleus of a cell (see, e.g., Capecchi MR Cell.1980 November;22(2 Pt 2):479-88), or retroviral transduction.

[0313] Some aspects of the present disclosure provide an "adoptive cell" approach, which involves isolating immune cells (e.g., T cells) from a subject with cancer, genetically engineering the immune cells (e.g., to express an antigen-binding receptor such as a chimeric antigen receptor), expanding the cells ex vivo, and then reintroducing the immune cells into the subject. This method results in a larger number of engineered immune cells in the subject than could be achieved by conventional gene delivery and vaccination methods. In some embodiments, the immune cells are isolated from the subject, expanded ex vivo without genetic modification, and then reintroduced into the subject.

[0314] The immune cells of the present disclosure comprise a receptor that binds to an antigen, such as an antigen encoded by an exogenously delivered nucleic acid, as provided herein. In some embodiments, white blood cells are engineered (e.g., genetically modified) to express a receptor that binds to the antigen. The receptor, in some embodiments, may be a naturally occurring antigen receptor (normally expressed on immune cells), a recombinant antigen receptor (not normally expressed on immune cells), or a chimeric antigen receptor (CAR). Naturally occurring and recombinant antigen receptors encompassed by the present disclosure include T cell receptors, B cell receptors, NK cell receptors, NKT cell receptors, and dendritic cell receptors. A "chimeric antigen receptor" refers to an artificial immune cell receptor that has been engineered to recognize and bind to an antigen expressed by a tumor cell. Generally, CARs are designed for T cells and are chimeras of the signaling domain of the T cell receptor (TcR) complex and an antigen recognition domain (e.g., a single-chain fragment (scFv) of an antibody) (Enblad et al., Human Genetics, 1999, 10, 1443-1445, the entire disclosure of which is incorporated herein by reference). Therapy.2015;26(8):498-505).

[0315] In some embodiments, the antigen-binding receptor is a chimeric antigen receptor (CAR). T cells expressing a CAR are referred to as "CAR T cells." In some embodiments, a "CAR T cell receptor" comprises a signaling domain of a T cell receptor (TcR) complex and an antigen recognition domain (e.g., a single-chain fragment (scFv) of an antibody) (Enblad et al., Human Gene Therapy. 2015;26(8):498-505, the entire disclosure of which is incorporated herein by reference).

[0316] There are four generations of CARs, each containing different components. First-generation CARs link an antibody-derived scFv to the CD3 zeta (zeta or z) intracellular signaling domain of the T cell receptor via a hinge and transmembrane domain. Second-generation CARs incorporate additional domains, such as CD28, 4-1BB (41BB), or ICOS, to provide costimulatory signals. Third-generation CARs contain two costimulatory domains fused to the TcR CD3-zeta chain. Third-generation costimulatory domains may include, for example, a combination of CD3z, CD27, CD28, 4-1BB, ICOS, or OX40. In some embodiments, CARs generally contain an endodomain with an ectodomain (e.g., CD3), hinge, transmembrane domain, commonly derived from a single-chain variable fragment (scFv), and one (first generation), two (second generation), or three (third generation) signaling domains derived from CD3Z and / or costimulatory molecules (Maude et al., Blood. 2015;125(26):4017-4023; Kakarla and Gottschalk, Cancer J. 2014;20(2):151-155, the entire disclosures of which are incorporated herein by reference).

[0317] In some embodiments, the chimeric antigen receptor (CAR) is a T-cell redirected for universal cytokine killing (TRUCK), also known as a fourth-generation CAR. TRUCK is a CAR-redirected T cell used as a vehicle to produce and release transgenic cytokines that accumulate in target tissues, such as target tumor tissues. The transgenic cytokines are released when the CAR engages the target. TRUCK cells can deposit various therapeutic cytokines within the target. This can result in the concentration of therapeutic agents at the target site and avoid systemic toxicity.

[0318] CARs typically differ in their functional properties. Upon engagement, the CD3 zeta signaling domain of the T cell receptor activates T cells and induces their proliferation, but can also result in anergy (a lack of response by the body's defense mechanisms, leading to direct induction of peripheral lymphocyte tolerance). Lymphocytes are considered anergic when they are unable to respond to specific antigens. The addition of a costimulatory domain in second-generation CARs improved the replicative capacity and survival of engineered T cells. While similar antitumor effects have been observed in vitro with CD28 CARs or 4-1BB CARs, preclinical in vivo studies suggest that 4-1BB CARs may result in superior proliferation and / or survival. Clinical trials suggest that both of these second-generation CARs can induce substantial T cell proliferation in vivo, but CARs containing the 4-1BB costimulatory domain appear to persist for longer periods. Third-generation CARs combine multiple signaling domains (costimulatory) to enhance efficacy. Fourth generation CARs have additionally been modified with constitutive or inducible expression cassettes for transgenic cytokines released by the CAR T cells to modulate T cell responses. See, e.g., Enblad et al., Human Gene Therapy. 2015;26(8):498-505; Chmielewski and Hi, the entire disclosures of which are incorporated herein by reference. See nrich, Expert Opinion on Biological Therapy. 2015;15(8):1145-1154.

[0319] In some embodiments, an illustrative immunotherapeutic is a first-generation chimeric antigen receptor CAR. In some embodiments, the chimeric antigen receptor is a third-generation CAR. In some embodiments, the chimeric antigen receptor is a second-generation CAR. In some embodiments, the chimeric antigen receptor is a third-generation CAR. In some embodiments, the chimeric antigen receptor is a fourth-generation CAR or universal cytokine-mediated killing redirected T cells (TRUCK).

[0320] In some embodiments, a chimeric antigen receptor (CAR) comprises an extracellular domain comprising an antigen-binding domain, a transmembrane domain, and a cytoplasmic domain. In some embodiments, the CAR is fully human. In some embodiments, the antigen-binding domain of the CAR is specific for one or more antigens. In some embodiments, a "spacer" or "hinge" domain is positioned between the extracellular domain (including the antigen-binding domain) and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. A "spacer domain" refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular domain and / or a cytoplasmic domain within a polypeptide chain. A "hinge domain" refers to any oligopeptide or polypeptide that functions to provide flexibility to a CAR or a domain thereof or to prevent steric hindrance of a CAR or a domain thereof. In some embodiments, a spacer or hinge domain can comprise up to 300 amino acids (e.g., 10-100 amino acids, or 5-20 amino acids). In some embodiments, one or more spacer domain(s) can also be included in other regions of the CAR.

[0321] In some embodiments, a CAR of the present disclosure comprises an antigen-binding domain, such as a single-chain Fv (scFv), specific for a tumor antigen. The choice of binding domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state, such as cancer or an autoimmune disease. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in a CAR of the present disclosure include cell surface markers associated with cancer cells and / or other forms of diseased cells. In some embodiments, a CAR is engineered to target a tumor antigen of interest by engineering a desired antigen-binding domain encoded by an engineered nucleic acid, as provided herein, that specifically binds to the antigen on tumor cells.

[0322] An antigen-binding domain (e.g., an scFv) that "specifically binds" to a target or epitope is a term understood in the art, and methods for determining such specific binding are known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates with a particular target antigen more frequently, rapidly, with a longer duration, and / or with greater affinity than it reacts or associates with alternative targets. An antigen-binding domain (e.g., an scFv) that specifically binds to a first target antigen may or may not specifically bind to a second target antigen. Thus, "specific binding" does not necessarily require (although it can include) exclusive binding.

[0323] In some embodiments, immune cells expressing CARs are genetically engineered to recognize multiple targets or antigens, thereby enabling recognition of unique target or antigen expression patterns on tumor cells. Examples of CARs that can bind to multiple targets include "split-signal CARs" that restrict activation of the complete immune cell to tumors expressing multiple antigens, "tandem CARs" (TanCARs) that contain an ectodomain with two scFvs, and CARs engineered to recognize tumor cells incubated with a tagged monoclonal antibody (Mab). Included are "universal ectodomain CARs" that incorporate vidin- or fluorescein isothiocyanate (FITC)-specific scFvs.

[0324] A CAR is considered "bispecific" if it recognizes two distinct antigens (has two distinct antigen recognition domains). In some embodiments, the bispecific CAR is composed of two distinct antigen recognition domains in tandem on a single transgenic receptor (referred to as a TanCAR; see, e.g., Grada Z et al. Molecular Therapy Nucleic Acids 2013;2:e105, incorporated herein by reference in its entirety). Thus, in some embodiments, a method includes delivering to a tumor a combination comprising a compound of Formula I' and an immunotherapeutic agent that is an engineered nucleic acid encoding an antigen, or delivering to a tumor an engineered nucleic acid that induces expression of an autoantigen, and delivering to the tumor immune cells that express a bispecific CAR that binds to two antigens, one of the antigens being encoded by the engineered nucleic acid.

[0325] In some embodiments, the CAR is an antigen-specific inhibitory CAR (iCAR) (Fedorov, VD et al. Sci. Transl. Med., published online December 11, 2013, which is incorporated herein by reference in its entirety), which can be used to avoid extratumoral toxicity. iCARs contain antigen-specific inhibitory receptors to block nonspecific immune suppression that can result from extratumoral target expression. iCARs can be based on, for example, inhibitory molecules such as CTLA-4 or PD-1. In some embodiments, these iCARs block T cell responses from T cells activated by either their endogenous T cell receptor or activating CAR. In some embodiments, this inhibitory effect is transient.

[0326] In some embodiments, CARs can be used in adoptive cell transfer, in which immune cells are removed from a subject and modified to express a receptor specific for an antigen, e.g., a tumor-specific antigen. The modified immune cells, which can recognize and kill cancer cells, are then reintroduced into the subject (Pule, et al., Cytotherapy. 2003; 5(3):211-226; Maude et al., Blood. 2015; 125(26):4017-4023, each of which is incorporated herein by reference in its entirety).

[0327] In other embodiments of the present disclosure, the tumor antigen component in the vaccine of the present invention is any natural or synthetic tumor-associated protein or peptide, or a combination of tumor-associated proteins and / or peptides, glycoproteins, or glycopeptides. In yet other embodiments, the antigen component may be patient-specific or common to many or most patients with a particular type of cancer. In one embodiment, the antigen component consists of a cell lysate derived from tumor tissue removed from the patient undergoing treatment. In another embodiment, the lysate can be engineered or synthesized from exosomes derived from tumor tissue. In yet another embodiment, the antigen component consists of a cell lysate derived from tumor tissue extracted from one or more unrelated individuals or from a tumor cell line.

[0328] In various embodiments, illustrative immunotherapeutic agents include one or more cancer vaccines for use in combination with the compounds of Formula I'. The tumor-associated antigen component of the vaccine may be produced by any of a variety of well-known techniques. For individual protein components, the antigen protein is isolated from tumor tissue or tumor cell lines by standard chromatographic means, such as high pressure liquid chromatography or affinity chromatography, or alternatively, synthesized by standard recombinant DNA techniques in a suitable expression system, such as E. coli, yeast, or plants. The tumor-associated antigen protein is then purified from the expression system by standard chromatographic means. In the case of peptide antigen components, these are generally prepared by standard automated synthesis. Proteins and peptides are synthesized by amino acid synthesis. Modifications can be made by the addition of acids, lipids, and other agents to improve incorporation into vaccine delivery systems (such as multilamellar liposomes). For tumor-associated antigen components derived from a patient's own tumor, a tumor from another individual, or a cell line, tumor tissue or a single-cell suspension derived from tumor tissue is typically homogenized in an appropriate buffer. The homogenate can also be fractionated, such as by centrifugation, to isolate specific cellular components, such as cell membranes or soluble substances. The tumor material can be used directly, or tumor-associated antigens can be extracted for incorporation into vaccines using a buffer containing a low concentration of a suitable agent, such as detergent. An example of a suitable detergent for extracting antigenic proteins from tumor tissue, tumor cells, and tumor cell membranes is diheptanoylphosphatidylcholine. Exosomes derived from tumor tissue or tumor cells, whether autologous or heterologous to the patient, can be used as starting materials for antigen components for incorporation into vaccines or for extracting tumor-associated antigens.

[0329] In some embodiments of the present disclosure, a cancer vaccine comprises at least one tumor-associated antigen, at least one immunostimulatory agent, and optionally at least one cell-based immunotherapeutic agent. In some embodiments, the immunostimulatory agent component in the cancer vaccine of the present disclosure is any biological response modifier (BRM) capable of enhancing the efficacy of a therapeutic cancer vaccine and inducing humoral and cellular immune responses against cancer cells in patients. According to one aspect, the immunostimulatory agent is a cytokine or a combination of cytokines. Examples of such cytokines include interferons such as IFN-gamma, interleukins such as IL-2, IL-15, and IL-23, colony-stimulating factors such as M-CSF and GM-CSF, and tumor necrosis factors. According to another aspect, the immunostimulatory agent component of the disclosed cancer vaccine comprises one or more adjuvant-type immunostimulatory agents, with or without immunostimulatory cytokines, such as an APC Toll-like receptor agonist or a costimulatory / cell adhesion membrane protein. Examples of Toll-like receptor agonists include lipid A and CpG, as well as costimulatory / adhesion proteins such as CD80, CD86, and ICAM-1.

[0330] In some embodiments, the immunostimulatory agent is selected from the group consisting of IFN-gamma (IFN-γ), IL-2, IL-15, IL-23, M-CSF, GM-CSF, tumor necrosis factor, lipid A, CpG, CD80, CD86, and ICAM-1, or a combination thereof. According to other aspects, the cell-based immunotherapeutic agent is selected from the group consisting of dendritic cells, tumor-infiltrating T lymphocytes, chimeric antigen receptor-modified T effector cells directed against the patient's tumor type, B lymphocytes, natural killer cells, myeloid cells, and any other cells of the patient's immune system, or a combination thereof. In one embodiment, the cancer vaccine immunostimulatory agent comprises one or more cytokines, e.g., interleukin 2 (IL-2), GM-CSF, M-CSF, and interferon-gamma (IFN-γ), one or more Toll-like receptor agonists and / or adjuvants, e.g., monophosphoryl lipid A, lipid A, muramyl dipeptide (MDP) lipid conjugates and double-stranded RNA, or one or more costimulatory membrane proteins and / or cell adhesion proteins, e.g., CD80, CD86, and ICAM-1, or any combination of the above. In one embodiment, the cancer vaccine comprises an immunostimulatory agent that is a cytokine selected from the group consisting of interleukin 2 (IL-2), GM-CSF, M-CSF, and interferon-gamma (IFN-γ). In another embodiment, the cancer vaccine comprises an immunostimulatory agent that is a Toll-like receptor agonist and / or adjuvant selected from the group consisting of monophosphoryl lipid A, lipid A, and muramyl dipeptide (MDP) lipid conjugates and double-stranded RNA. In yet another embodiment, the cancer vaccine comprises an immunostimulatory agent that is a costimulatory membrane protein and / or cell adhesion protein selected from the group consisting of CD80, CD86, and ICAM-1.

[0331] In various embodiments, the immunotherapeutic agent can be directed to any tumor antigen that can potentially be used to construct a fusion protein according to the invention, in particular: (a) NY-ESO-1, SSX2, SCP1, as well as RAGE, BAGE, GAGE ​​and MAGE family polypeptides, e.g., GAGE-1, GAGE-2, MAGE-1, which can be used to address, for example, melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors; (b) testicular cancer antigens including MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12; (b) p53 associated with various solid tumors, e.g., colorectal, lung, and head and neck cancers; p21 / Ras associated with, e.g., melanoma, pancreatic cancer, and colorectal cancer; CDK4 associated with, e.g., melanoma; MUM1 associated with, e.g., melanoma; caspase-8 associated with, e.g., head and neck cancer; CIA0205 associated with, e.g., bladder cancer; HLA-A2-R1701, beta-catenin associated with, e.g., melanoma; TCR associated with, e.g., T-cell non-Hodgkin's lymphoma; BCR-abl associated with, e.g., chronic myeloid leukemia; triosephosphate isomerase; KIA0205; CDC-27, and LDLR-FUT; (c) galectin 4 associated with, e.g., colorectal cancer; overexpressed antigens including galectin 9, associated with, for example, chronic myeloid leukemia; proteinase 3, associated with, for example, chronic myeloid leukemia; WT1, associated with, for example, various leukemias; carbonic anhydrase, associated with, for example, kidney cancer; aldolase A, associated with, for example, lung cancer; PRAME, associated with, for example, melanoma; HER-2 / neu, associated with, for example, breast, colon, lung, and ovarian cancer; mammaglobin, alpha-fetoprotein, associated with, for example, hepatocellular carcinoma; KSA, associated with, for example, colorectal cancer; gastrin, associated with, for example, pancreatic and gastric cancer; telomerase catalytic protein, MUC-1, associated with, for example, breast and ovarian cancer; G-250, associated with, for example, renal cell carcinoma; p53, associated with, for example, breast and colon cancer; and carcinoembryonic antigen, associated with, for example, cancers of the gastrointestinal tract, such as breast, lung, and colorectal cancer. Melanoma-melanocyte differentiation antigens such as A; gp100; MC1R; melanocyte-stimulating hormone receptor; tyrosinase;For example, these may include cancer vaccines incorporating common antigens including tyrosinase-related protein-1 / TRP1 and tyrosinase-related protein-2 / TRP2 associated with melanoma; (e) prostate-associated antigens including PAP, PSA, PSMA, PSH-P1, PSM-P1, and PSM-P2 associated with prostate cancer; and (f) immunoglobulin idiotypes associated with myeloma and B-cell lymphoma. In certain embodiments, the one or more TAAs are selected from the group consisting of pi5, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigens, EBNA, human papillomavirus (HPV) antigens including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, pi6, TAGE, PSCA, CT7, 4 3-9F, 5T4, 791Tgp72, beta-HCG, BCA225, BTAA, CA125, CA15-3 (CA27.29\BCAA), CA195, CA242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, or any combination thereof;

[0332] In some embodiments, the cancer vaccines of the disclosure for use in combination with compounds of Formula I' are directed against the following human proteins: TCTN1 (Gene ID: ENSG00000204852), TCTN2 (Gene ID: ENSG00000168778), TCTN3 (Gene ID: ENSG00000119977), HIGD2A (Gene ID: ENSG00000146066), HIGD2B (Gene ID: ENSG00000175202), C4ORF32 (Gene ID: ENSG00000174749), FAM62A (E -SYT1, gene ID: ENSG00000139641), COLEC11 (gene ID: ENSG00000118004), FSTL5 (gene ID: ENSG00000168843), FAM82A2 (gene ID: ENSG00000137824), SCARA5 (gene ID: ENSG00000168079), VSTM1 (gene ID: ENSG00000189068), RNF5 (gene ID: ENSG00000189068), child ID: ENSG00000183574), UNQ6126 (gene ID: gi|169216088), DPY19L3 (gene ID: ENSG00000178904), SLC39A10 (gene ID: ENSG00000196950), GPR107 (gene ID: ENSG00000148358), COL20A1 (gene ID: ENSG00000101203), GLT25D2 (gene ID :ENSG00000198756), SYTL3 (Gene ID: ENSG00000164674), DENND1B (Gene ID: ENSG00000162701), C6orf98 (Gene ID: EG:387079), FAM69B (Gene ID: ENSG00000165716), EMID1 (Gene ID: OTTHUMG00000030824), KLRG2 (Gene ID: ENSG00000 188883), ERMP1 (Gene ID: ENSG00000099219), VMO1 (Gene ID: ENSG00000182853), C9orf46 (Gene ID: ENSG00000107020), F1137107 (Gene ID: ENSG00000177990), YIPF2 (Gene ID: ENSG00000130733), TRYX3 (PRSS58, ENSG00000258223).2), C14orf135 (Gene ID: ENSG00000126773), ANGPTL7 (Gene ID: ENSG00000171819), TPCN2 (Gene ID: ENSG00000162341), C18orf19 (Gene ID: ENSG00000177150), OLFML1 (Gene ID: ENSG00000183801), LYPD4 (Gene ID: ENSG00000101203), MEGF8 (Gene ID: ENS G00000105429), F1142986 (Gene ID: ENSG00000196460), SLC46A1 (Gene ID: ENSG00000076351), FAM180A (Gene ID: ENSG00000189320), CRISP-3 (Gene ID: ENSG00000096006), or a combination thereof. These tumor antigens are disclosed in WO2010 / 086162, WO2010 / 086163, WO2011 / 051278, WO2011 / 051276, WO2011 / 051277, WO2011 / 051280, WO2011 / 051271, WO2011 / 135068, and WO2014 / 198919, the entire contents of which are incorporated herein by reference.

[0333] In various embodiments, an illustrative immunotherapeutic agent may comprise an mRNA that can be engineered to encode any one or more of the cancer antigens described above that are useful for synthesizing a cancer vaccine. In some illustrative embodiments, the mRNA-based cancer vaccine may have one or more of the following properties: a) the mRNA encoding each cancer antigen is interspersed with cleavage-sensitive sites; b) the mRNA encoding each cancer antigen is directly linked to each other without a linker; c) the mRNA encoding each cancer antigen is linked to each other with a single nucleotide linker; d) each cancer antigen comprises 20-40 amino acids and contains a centrally located SNP mutation; e) at least 40% of the cancer antigens have very high affinity for class I MHC molecules from the subject; f) at least 40% of the cancer antigens have very high affinity for class II MHC molecules from the subject; g) at least 40% of the cancer antigens have predicted binding affinity for HLA-A, HLA-B, and / or DRB1 of IC>500 nM; h) the mRNA encodes 1-15 cancer antigens; i) 10-60% of the cancer antigens have binding affinity for class I MHC and 10-60% of the cancer antigens have binding affinity for class II MHC molecules. have binding affinity for MHC, and / or j) mRNA encoding the cancer antigen is arranged such that the cancer antigen is ordered to minimize pseudo-epitopes.

[0334] In various embodiments, a combination comprising a compound of Formula I' as disclosed herein and a cancer vaccine immunotherapeutic can be used to induce an immune response to a cancer antigen in a subject. This method involves administering to the subject an RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide or immunogenic fragment thereof, in combination with the administration of a compound of Formula I', either in the same composition or in separate compositions administered simultaneously or sequentially, thereby inducing an immune response in the subject specific to the antigenic polypeptide or immunogenic fragment thereof, wherein anti-antigen polypeptide antibody titers in the subject are increased after vaccination compared to anti-antigen polypeptide antibody titers in subjects vaccinated with a prophylactically effective dose of a conventional vaccine against cancer. An "anti-antigen polypeptide antibody" is a serum antibody that specifically binds to an antigenic polypeptide.

[0335] A prophylactically effective dose is a therapeutically effective dose that prevents the progression of cancer at a clinically acceptable level. In some embodiments, a therapeutically effective dose is the dose described in the package insert for the vaccine. As used herein, a conventional vaccine refers to a vaccine other than the mRNA vaccine of the present invention. For example, conventional vaccines include, but are not limited to, live microbial vaccines, killed microbial vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, etc. In exemplary embodiments, a conventional vaccine is one that has regulatory approval and / or is registered with a national drug regulatory agency, e.g., in the United States, the Food and Drug Administration (FDA) or the European Medicines Agency (EMA).

[0336] In some embodiments, the anti-antigen polypeptide antibody titer in the subject increases by 1 log to 10 logs after vaccination compared to the anti-antigen polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a conventional vaccine against cancer. In some embodiments, the anti-antigen polypeptide antibody titer in the subject increases by 1 log after vaccination compared to the anti-antigen polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a conventional vaccine against cancer. In some embodiments, the anti-antigen polypeptide antibody titer in the subject increases by 2 logs after vaccination compared to the anti-antigen polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a conventional vaccine against cancer.

[0337] An aspect of the present invention provides a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigen polypeptide, wherein the RNA polynucleotides are present in a formulation for in vivo administration to a recipient, and the nucleic acid vaccine produces an antibody titer for the first antigen that exceeds a criterion for antibody prevalence in an acceptable percentage of human subjects. In some embodiments, the antibody titer produced by the mRNA vaccine of the present invention is a neutralizing antibody titer. In some embodiments, the neutralizing antibody titer is higher than that produced by a protein vaccine. In other embodiments, the neutralizing antibody titer produced by the mRNA vaccine of the present invention is higher than that produced by an adjuvanted protein vaccine. In still other embodiments, the neutralizing antibody titer generated by the mRNA vaccine of the present invention is 1,000 to 10,000, 1,200 to 10,000, 1,400 to 10,000, 1,500 to 10,000, 1,000 to 5,000, 1,000 to 4,000, 1,800 to 10,000, 2,000 to 10,000, 2,000 to 5,000, 2,000 to 3,000, 2,000 to 4,000, 3,000 to 5,000, 3,000 to 4,000, or 2,000 to 2,500. Neutralizing titers are typically expressed as the highest serum dilution required to achieve a 50% reduction in plaque counts.

[0338] In preferred embodiments, the RNA vaccine immunotherapeutics (e.g., mRNA vaccines) of the present disclosure are prophylactically and / or therapeutically effective in the blood or serum of vaccinated subjects. The antibody titer may result in a level, concentration, and / or titer of antigen-specific antibodies. As defined herein, the term antibody titer refers to the amount of antigen-specific antibodies produced in a subject, e.g., a human subject. In an exemplary embodiment, the antibody titer is expressed as the reciprocal of the highest dilution (in a serial dilution assay) that still gives a positive result. In an exemplary embodiment, the antibody titer is determined or measured by enzyme-linked immunosorbent assay (ELISA). In an exemplary embodiment, the antibody titer is determined or measured by a neutralization assay, e.g., a microneutralization assay. In certain aspects, the antibody titer measurement is expressed as a ratio, such as 1:40, 1:100, etc.

[0339] In exemplary embodiments of the invention, an effective vaccine results in an antibody titer of greater than 1:40, greater than 1:100, greater than 1:400, greater than 1:1000, greater than 1:2000, greater than 1:3000, greater than 1:4000, greater than 1:500, greater than 1:6000, greater than 1:7500, or greater than 1:10000. In exemplary embodiments, the antibody titer occurs or is achieved by 10 days post-vaccination, by 20 days post-vaccination, by 30 days post-vaccination, by 40 days post-vaccination, or 50 days or more post-vaccination. In exemplary embodiments, the titer occurs or is achieved after a single dose of vaccine is administered to the subject. In other embodiments, the titer occurs or is achieved after multiple administrations, for example, after a first and second administration (e.g., booster doses). In exemplary aspects of the invention, antigen-specific antibodies are measured in g / ml, or in IU / L (International Units per Liter) or mIU / ml (milli-International Units per ml). In exemplary embodiments of the invention, an effective vaccine provides >0.5 μg / mL, >0.1 μg / mL, >0.2 μg / mL, >0.35 μg / mL, >0.5 μg / mL, >1 μg / mL, >2 μg / mL, >5 μg / mL, or >10 μg / mL. In exemplary embodiments of the invention, an effective vaccine provides >10 mIU / mL, >20 mIU / mL, >50 mIU / mL, >100 mIU / mL, >200 mIU / mL, >500 mIU / mL, or >1000 mIU / mL. In exemplary embodiments, the antibody level or concentration occurs or is achieved by 10 days post-vaccination, by 20 days post-vaccination, by 30 days post-vaccination, by 40 days post-vaccination, or 50 days or more post-vaccination. In exemplary embodiments, the level or concentration occurs or is achieved after a single dose of vaccine is administered to the subject. In other embodiments, the level or concentration occurs or is achieved after multiple doses, e.g., after a first and second dose (e.g., a booster dose). In exemplary embodiments, the antibody level or concentration is determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, the antibody level or concentration is determined or measured by a neutralization assay, e.g., a microneutralization assay.Also provided are nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide or linear polypeptide, wherein the RNA polynucleotides, when present in a formulation for in vivo administration to a recipient, have a stabilizing element or are formulated with an adjuvant to induce higher and longer-lasting antibody titers than those induced by an mRNA vaccine encoding the first antigenic polypeptide. In some embodiments, the RNA polynucleotides are formulated to generate neutralizing antibodies within one week of a single administration. In some embodiments, the adjuvant is selected from a cationic peptide and an immunostimulatory nucleic acid. In some embodiments, the cationic peptide is protamine.

[0340] An immunotherapeutic comprising a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide or linear polypeptide, with at least one chemical modification, or optionally no nucleotide modification, wherein the RNA polynucleotides are in a formulation for in vivo administration to a recipient such that the level of antigen expression in the recipient significantly exceeds the level of antigen expression produced by an mRNA vaccine encoding the first antigenic polypeptide, the mRNA vaccine having a stabilizing element or formulated with an adjuvant.

[0341] Another aspect provides a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide or linear polypeptide, with at least one chemical modification, or optionally no nucleotide modification, wherein the vaccine has at least 10-fold less RNA polynucleotide than would be required for an unmodified mRNA vaccine to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotide is present in a dosage of 25 to 100 micrograms.

[0342] Aspects of the present invention also provide vaccine dosage units comprising 10 μg to 400 μg of one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide or linear polypeptide, with at least one chemical modification, or optionally no nucleotide modification, and a pharmaceutically acceptable carrier or excipient, formulated for delivery to a human subject. In some embodiments, the vaccine further comprises cationic lipid nanoparticles.

[0343]

[0009] Aspects of the present invention provide methods for creating, maintaining, or restoring antigenic memory against a tumor in an individual or a population of individuals, the method comprising administering to the individual or population of individuals an antigenic memory booster nucleic acid vaccine comprising: (a) at least one RNA polynucleotide, with at least one chemical modification or optionally no nucleotide modification, and comprising two or more codon-optimized open reading frames, the open reading frames encoding a set of reference antigenic polypeptides; and (b) optionally, a pharmaceutically acceptable carrier or excipient. In some embodiments, the vaccine is administered to the individual via a route selected from the group consisting of intramuscular administration, intradermal administration, and subcutaneous administration. In some embodiments, the administering step comprises contacting the subject's muscle tissue with a device suitable for injecting the composition. In some embodiments, the administering step comprises contacting the subject's muscle tissue with a device suitable for injecting the composition in combination with electroporation.

[0344] An embodiment of the present invention provides a method of vaccinating a subject, comprising administering to the subject a single dosage of 25 μg / kg to 400 μg / kg of a nucleic acid vaccine comprising an effective amount of one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide or linear polypeptide, to vaccinate the subject.

[0345] Another aspect provides a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame that includes at least one chemical modification, the open reading frame encoding a first antigenic polypeptide or linear polypeptide, wherein the nucleic acid vaccine comprises at least 10-fold less RNA polynucleotide than would be required for an unmodified mRNA vaccine to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotide is present in a dosage of 25-100 micrograms.

[0346] In some embodiments, illustrative immunotherapeutic agents may include one or more interfering RNAs that can be administered in combination with a compound of Formula I'. As used herein, an "RNA interfering agent" is defined as any agent that interferes with or inhibits the expression of a target biomarker gene by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, nucleic acid molecules comprising RNA molecules cognate to the target biomarker genes of the invention, or fragments thereof, short interfering RNAs (siRNAs), and small molecules that interfere with or inhibit the expression of a target biomarker nucleic acid by RNA interference (RNAi). "Short interfering RNAs" (siRNAs), also referred to herein as "small interfering RNAs," are used in a variety of applications, e.g., siRNA is defined as an agent that functions to inhibit expression of a target biomarker nucleic acid through RNAi. siRNA can be chemically synthesized, generated by in vitro transcription, or generated within a host cell. In one embodiment, siRNA is a double-stranded RNA (dsRNA) molecule about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides, and more preferably about 19, 20, 21, or 22 nucleotides in length, and includes 3' and / or 5' overhangs on each strand having lengths of about 0, 1, 2, 3, 4, or 5 nucleotides. The lengths of the overhangs are independent between the two strands; i.e., the length of the overhang on one strand is independent of the length of the overhang on the second strand. Preferably, siRNA can promote RNA interference by degradation of target messenger RNA (mRNA) or specific post-transcriptional gene silencing (PTGS).

[0347] Antisense oligonucleotides can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. Antisense nucleic acids can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, antisense nucleic acids (e.g., antisense oligonucleotides) can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or increase the physical stability of the duplex formed between antisense and sense nucleic acids, for example, phosphorothioate derivatives and acridine-substituted nucleotides can be used. Examples of modified nucleotides that can be used to generate antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methyl Examples include guanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), wybutoxosine, pseudouracil, cheiosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine.Alternatively, antisense nucleic acids can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., the RNA transcribed from this inserted nucleic acid is in an antisense orientation relative to the target nucleic acid of interest, as further described in the subsection below).

[0348] Antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ so that they hybridize or bind to cellular mRNA and / or genomic DNA encoding a polypeptide corresponding to a selected marker of the invention, thereby inhibiting expression of the marker, e.g., by inhibiting transcription and / or translation. This hybridization may be by conventional nucleotide complementarity to form a stable duplex, or, in the case of antisense nucleic acid molecules that bind to DNA duplexes, through specific interactions in the major groove of the double helix. Exemplary routes of administration of antisense nucleic acid molecules of the invention include direct administration at a tissue site. These methods include intravenous injection or injection of antisense nucleic acids into blood or bone marrow-related fluids. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified to specifically bind to receptors or antigens expressed on the surface of selected cells, for example, by conjugating the antisense nucleic acid molecule to a peptide or antibody that binds to the cell surface receptor or antigen. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is under the control of a strong pol II or pol III promoter are preferred.

[0349] Antigens that can be targeted to synthesize corresponding antisense RNA molecules include any antigen specific to one or more tumors, such as those exemplified above with respect to cancer vaccines.

[0350] In some embodiments, the combination of immunotherapeutic agent and compound of Formula I' may comprise a bispecific antibody immunotherapeutic agent. The bispecific antibody may comprise a protein construct having a first antigen-binding moiety and a second antigen-binding moiety that binds to cytotoxic immune cells. The first antigen-binding moiety may bind to a tumor antigen specifically treated with the combination of the invention. For example, the first antigen-binding moiety may bind to non-limiting examples of tumor antigens selected from EGFR, HGFR, Her2, Ep-CAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin αVβ3, integrin α5β1, MUC1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and tenascin, among others. In some embodiments, the first antigen-binding moiety has specificity for a protein or peptide that is overexpressed on tumor cells compared to corresponding non-tumor cells. In some embodiments, the first antigen-binding moiety has specificity for a protein that is overexpressed on tumor cells compared to corresponding non-tumor cells. As used herein, "corresponding non-tumor cells" refers to non-tumor cells that are the same cell type as the origin of tumor cells. Note that such proteins are not necessarily different from tumor antigens. Non-limiting examples include carcinoembryonic antigen (CEA), which is overexpressed in many colon, rectum, breast, lung, pancreatic, and gastrointestinal cancers; heregulin receptors (HER-2, neu, or c-erbB-2), which are frequently overexpressed in breast, ovarian, colon, lung, prostate, and cervical cancers; epidermal growth factor receptors (EGFR), which are highly expressed in a range of solid tumors, including those of the breast, head and neck, non-small cell lung, and prostate; asialoglycoprotein receptors; transferrin receptors; serpin enzyme complex receptors expressed on hepatocytes; fibroblast growth factor receptors (FGFR), which are overexpressed on pancreatic tubular adenocarcinoma cells; vascular endothelial growth factor receptors (VEGFR) in anti-angiogenic gene therapy; folate receptors, which are selectively overexpressed in 90% of non-mucinous ovarian cancers; cell surface glycocalyx; carbohydrate receptors; and polymeric immunoglobulin receptors.

[0351] The second antigen-binding moiety is any molecule that specifically binds to an antigen or protein or polypeptide expressed on the surface of a cytotoxic immune cell (CIK cell). Exemplary, non-limiting antigens expressed on the surface of cytotoxic immune cells suitable for use in the present disclosure include CD2, CD3, CD4, CD5, CD8, CD11a, CD11b, CD14, CD16a, CD27, CD28, CD45, CD45RA, CD56, CD62L, Fc receptors, LFA, LFA-1, TCRαβ, CCR7, macrophage inflammatory protein 1a, perforin, PD-1, PD-L1, PD-L2, or CTLA-4, LAG-3, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, TIM-3, SIRP-alpha, TIGIT, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS, BTNL2, CD27, and Fas The second antigen-binding moiety may include a ligand. In some embodiments, the second antigen-binding moiety binds to CD3 on a cytotoxic immune cell, e.g., a CIK cell. In some embodiments, the second antigen-binding moiety binds to CD56 on a cytotoxic immune cell. In some embodiments, the second antigen-binding moiety binds to an Fc receptor on a cytotoxic immune cell. In some embodiments, the Fc region of a bispecific antibody binds to an Fc receptor on a cytotoxic immune cell. In some embodiments, the second antigen-binding moiety is any molecule that specifically binds to an antigen expressed on the surface of a cytotoxic immune cell (e.g., a CIK cell). The second antigen-binding moiety is specific for an antigen on a cytotoxic immune cell. Exemplary cytotoxic immune cells include, but are not limited to, CIK cells, T cells, CD8+ T cells, activated T cells, monocytes, natural killer (NK) cells, NK T cells, lymphokine-activated killer (LAK) cells, macrophages, and dendritic cells. The second antigen-binding moiety specifically binds to an antigen expressed on the surface of a cytotoxic immune cell. Exemplary non-limiting antigens expressed on the surface of cytotoxic immune cells suitable for modulation with the present disclosure can include CD2, CD3, CD4, CD5, CD8, CD11a, CD11b, CD14, CD16a, CD27, CD28, CD45, CD45RA, CD56, CD62L, Fc receptors, LFA, LFA-1, TCRαβ, CCR7, macrophage inflammatory protein 1a, perforin, PD-1, PD-L1, PD-L2, or CTLA-4, LAG-3, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, TIM-3, SIRP-alpha, TIGIT, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS, BTNL2, CD27, and Fas ligand. In other embodiments, the bispecific antibody modulator is an activator of a costimulatory molecule (e.g., an OX40 agonist). In one embodiment, the OX40 agonist is a bispecific antibody molecule directed against OX40 and another tumor antigen or costimulatory antigen.The OX40 agonist can be administered alone or in combination with other immunomodulators, for example, in combination with inhibitors (e.g., antibody constructs) of PD-1, PD-L1, CTLA-4, CEACAM (e.g., CEACAM-1, -3, and / or -5), TIM-3, or LAG-3. In some embodiments, the anti-OX40 antibody molecule is a bispecific antibody that binds to GITR and PD-1, PD-L1, CTLA-4, CEACAM (e.g., CEACAM-1, -3, and / or -5), TIM-3, or LAG-3. In one exemplary embodiment, the OX40 antibody molecule is administered in combination with an anti-PD-1 antibody molecule (e.g., an anti-PD-1 molecule as described herein). The OX40 antibody molecule and the anti-PD-1 antibody molecule may be in the form of separate antibody compositions or as a bispecific antibody molecule. In other embodiments, the OX40 agonist can be administered in combination with an agonist of another costimulatory molecule, such as GITR, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligand. In some embodiments, the second antigen-binding moiety binds to an Fc receptor on a cytotoxic immune cell, such as a CIK cell.

[0352] In some embodiments, the bispecific antibody immunotherapeutic agent has specificity for a tumor antigen and CIK cells, thereby bringing tumor antigen-expressing tumor cells into close proximity with CIK cells, resulting in tumor cell elimination via anti-tumor cytotoxicity of the CIK cells. In some embodiments, the bispecific antibody has specificity for a tumor antigen but not for CIK cells, however, the Fc region of the bispecific antibody can bind to the Fc receptor of the CIK cells, thereby bringing tumor cells into close proximity with CIK cells, resulting in tumor cell elimination via anti-tumor cytotoxicity of the CIK cells. In some embodiments, the bispecific antibody has specificity for CIK cells but not for tumor cells, however, the Fc region of the bispecific antibody can bind to the Fc receptor of the tumor cells, thereby bringing tumor cells into close proximity with CIK cells, resulting in tumor cell elimination via anti-tumor cytotoxicity of the CIK cells.

[0353] In some embodiments, the combination of immunotherapeutic and compound of Formula I' may include an immune cell-engaging multivalent antibody / fusion protein / construct immunotherapeutic. In various embodiments, exemplary immunotherapeutic agents may include recombinant constructs, e.g., immune cell-engaging multivalent antibodies / fusion proteins / constructs, which may include all engineered antibodies that do not mimic the original IgG structure. In this case, various strategies for multimerizing antibody fragments are utilized. For example, shortening the peptide linker between the V domains allows scFvs to self-associate into dimers (diabodies, 55 kDa). Bispecific diabodies are formed by the non-covalent association of two VHA-VLB and VHB-VLA fragments expressed in the same cell. This results in the formation of heterodimers with two distinct binding sites. Single-chain diabodies (sc-diabodies) are bispecific molecules in which the VHA-VLB and VHB-VLA fragments are linked by an additional third linker. Tandem diabodies (Tandabs) are tetravalent bispecific antibodies generated by two sc diabodies.

[0354] Also included are di-diabodies, known in the art. This 130 kDa molecule is formed by fusing a diabody to the N-terminus of the CH3 domain of IgG, resulting in an IgG-like structure. Additional diabody derivatives are triabodies and tetrabodies, which fold into trimeric and tetrameric fragments by shortening the linker to <5 or 0-2 residues. Also exemplified is the (scFv)2 construct known as a "bispecific T cell engager" (BITE). BITE is a bispecific single-chain antibody consisting of two scFv antibody fragments linked via a flexible linker, directed against a surface antigen on target cells and CD3 on T cells. Bivalent (Fab)2 and trivalent (Fab)3 antibody formats are also exemplified. Minibodies and trimeric bodies generated from scFvs are also exemplified. Exemplary constructs useful for targeting tumor antigens may include one or more of the following: diabodies, single-chain (sc)-diabodies (scFv)2, miniantibodies, minibodies, barnase-barstar, scFv-Fc, sc(Fab)2, trimeric antibody constructs, triabody antibody constructs, trimeric body antibody constructs, tribody antibody constructs, collabody antibody constructs, (scFv-TNFα)3, and F(ab)3 / DNL. In each of these exemplary constructs, at least one binding moiety can bind to an antigen or protein or polypeptide expressed on the surface of a cytotoxic immune cell, and at least one binding moiety specifically binds to an antigen on the cytotoxic immune cell. Exemplary cytotoxic immune cells include, but are not limited to, CIK cells, T cells, CD8+ T cells, activated T cells, monocytes, natural killer (NK) cells, NK T cells, lymphokine-activated killer (LAK) cells, macrophages, and dendritic cells.

[0355] In some embodiments, the combination of an immunotherapeutic agent and a compound of Formula I' can include a radioconjugated immunotherapeutic agent.

[0356] In various embodiments, the radioconjugate is a small or large molecule (also referred to herein as a "cell-targeting agent"), such as a polypeptide, antibody, or antibody fragment thereof, that is coupled to or otherwise immobilized with one or more radionuclides, such that binding of the radioconjugate to its target (a protein or molecule on or within a cancer cell) results in the death or disease of the cancer cell. In various embodiments, the radioconjugate may be a cell-targeting agent labeled with a radionuclide, or the cell-targeting agent may be coupled to or otherwise immobilized with a particle, microparticle, or nanoparticle containing multiple radionuclides, where the radionuclides are the same or different. Methods for synthesizing radioconjugates are known in the art and may include groups of immunoglobulins, or antigen-binding portions thereof, that are conjugated to toxic radionuclides.

[0357] In some embodiments, molecules that bind to cancer cells may also be known as "cell-targeting drugs." As used herein, exemplary cell-targeting drugs may enable drug-containing nanoparticles or radionuclides to target specific cell types of interest. Examples of cell-targeting drugs include, but are not limited to, small molecules (e.g., folic acid, adenosine, purines) and large molecules (e.g., peptides or antibodies) that bind to or target tumor-associated antigens. Examples of tumor-associated antigens include, but are not limited to, adenosine receptor, alpha v beta 3, aminopeptidase P, alpha fetoprotein, cancer antigen 125, carcinoembryonic antigen, c-caveolin-1, chemokine receptor, clusterin, carcinoembryonic antigen, CD20, epithelial tumor antigen, melanoma-associated antigen, Ras, p53, Her2 / Neu, ErbB2, ErbB3, ErbB4, folate receptor, prostate-specific membrane antigen, prostate-specific antigen, purinergic receptor, radiation-inducible cell surface receptor, serpin B3, serpin B4, squamous cell carcinoma antigen, thrombospondin, tumor antigen 4, tumor-associated glycoprotein 72, thiocinase, and tyrosine kinase. In some embodiments, the cell-targeting agent is folic acid or a folic acid derivative that specifically binds to the folate receptor (FR). In some embodiments, the cell-targeting agent is an antibody, bispecific antibody, trispecific antibody or antigen-binding construct thereof that specifically binds to a cancer antigen selected from EGFR, HGFR, Her2, Ep-CAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin αVβ3, integrin α5β1, MUC1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and tenascin, among others.

[0358] The use of folic acid as a targeting agent in radioconjugates also allows both tumor cells and regulatory T (Treg) cells to be targeted for destruction. It is well-known that large numbers of Treg cells suppress tumor immunity. Specifically, Treg cells suppress (foreign and auto) reactive T cells without killing them through contact-dependent or cytokine (e.g., IL-10, TGF-beta, etc.) secretion. FR4 is selectively upregulated in Treg cells. Antibody blockade of FR4 has been shown to deplete Treg cells and induce tumor immunity in tumor-bearing mice. Therefore, folic acid-coated PBM nanoparticles carrying cytotoxic drugs will target and destroy FR-expressing cells, and their destruction will inhibit tumor progression both directly (i.e., BrCa cells) and indirectly (i.e., breast tumor-associated and peripheral Treg cells).

[0359] In another further embodiment, the targeting agent is an antibody or peptide or an immune cell engaging multivalent antibody / fusion protein / construct capable of binding to a tumor associated antigen such as, but not limited to: adenosine receptor, alpha v beta 3, aminopeptidase P, alpha fetoprotein, cancer antigen 125, carcinoembryonic antigen, caveolin-1, chemokine receptor, clusterin, carcinoembryonic antigen, CD20, human growth factor receptor (HGFR), epithelial tumor antigen, melanoma associated antigen, MUC1, Ras, p53, Her2 / Neu, ErbB2, ErbB3, ErbB4, folate receptor, prostate specific membrane antigen, prostate specific antigen, purinergic receptor, radiation inducible cell surface receptor, serpin B3, serpin B4, squamous cell carcinoma antigen, thrombospondin, tumor antigen 4, tumor associated glycoprotein 72, tyrosinase, and tyrosine kinase.

[0360] In one embodiment, the method of treatment comprises co-administration of a compound as disclosed herein or a pharmaceutically acceptable salt thereof with at least one cytotoxic agent. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and Lu radioisotopes); chemotherapy drugs; proliferation These include inhibitors; enzymes such as nucleases and fragments thereof; and toxins (including fragments and / or variants thereof) such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin.

[0361] Exemplary cytotoxic agents may be selected from anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotics, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutics, pro-apoptotic agents, inhibitors of LDH-A; inhibitors of fatty acid biosynthesis; cell cycle signaling inhibitors; HDAC inhibitors, proteasome inhibitors; and inhibitors of cancer metabolism.

[0362] "Chemotherapeutic agents" include chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrokinase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer / Sugen), letrozole (FEMARA®, Novartis), imatinib mesylate, and fluticasone. Other examples include nib (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafamib (SCH66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), AG1478; alkylating agents such as thiotepa and CYTOXAN®; cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metuledopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine ethylenimines and methylamelamines, including trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); corticosteroids (including prednisone and prednisolone);Cyproterone acetate; 5-alpha-reductase inhibitors, including finasteride and dutasteride; vorinostat, romidespine, panobinostat, valproic acid, mocetinostat; dolastatins; aldesleukin, talc; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatins; chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethrin nitrogen mustards such as melphalan, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; enediyne antibiotics such as calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega 1I (Angew Chem. Intl. Ed. Engl. 1994 33:183-186); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomysin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin leucine, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, Mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; fludarabine, 6-mercaptopurine, and thiamiprine Purine analogues such as thiamiprine and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid;Eniluracil; Amsacrine; Bestravcil; Bisantrene; Edatrexate; Defofamine; Demecolcine; Diaziquone; Elfomithine; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids such as maytansine and ansamitocins; Mitoguazone; Mitoxantrone; Mopidamnol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Losoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK® Polysaccharide Complex (JHS Natural; Products, Eugene, Ore.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as taxol (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® (docetaxel, Sanofi-Aventis); chlorambucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVE LBINE® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0363] Chemotherapeutic agents include (i) antiestrogens and select anti-estrogens, including, for example, tamoxifen (including NOLVADEX®, tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate). (ii) antihormonal drugs that act to modulate or inhibit hormone action on tumors, such as selective estrogen receptor modulators (SERMs), for example, 4(5)-imidazole, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane, Pfizer), formestani, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole, Novartis), and ARIMIDEX® (anastrozole). (iii) aromatase inhibitors, such as benzodiazepine (Zol, AstraZeneca), which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands; (iv) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans retinoic acid, fenretinide, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog), (v) benzodiazepines, such as benzodiazepines (Zol, AstraZeneca), which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands; (iv) protein kinase inhibitors, (v) lipid kinase inhibitors, (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-alpha, Ralf, and H-Ras, (vii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors, (viii) gene therapy vaccines, such as, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®;Also included are topoisomerase 1 inhibitors such as PROLEUKIN®, rIL-2; LURTOTECAN®; ABARELIX®, and (ix) pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0364] Chemotherapy agents also include antibodies as described above, including alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Additional humanized monoclonal antibodies having therapeutic potential as agents in combination with the compounds of the invention include apolizumab, acelizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, levoflu ... mab), fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nivolumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab tuzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan These include tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and anti-interleukin-12 (ABT-8744695, Wyeth Research and Abbott Laboratories), a recombinant, entirely human sequence, full-length IgG1 lambda antibody genetically engineered to recognize the interleukin-12p40 protein.

[0365] Chemotherapeutic agents include EGFR inhibitors; small molecule HER2 tyrosine kinase inhibitors such as mubritonib (TAK165, Takeda); CP-724.714 (Axon Medchem BV, an oral selective inhibitor of ErbB2 receptor tyrosine kinase); dual HER inhibitors such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR overexpressing cells; lapatinib (GSK572016, available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033, Pharmacia); Raf-1 inhibitors such as ISIS-5132, an antisense drug available from ISIS Pharmaceuticals that inhibits Raf-1 signaling; imatinib mesylate (GLEEVEC®, Glaxo) non-HER-targeted TK inhibitors such as sunitinib (SUTENT®, available from Pfizer); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, available from Novartis / Schering AG); MAPK extracellular regulated kinase 1 inhibitor CI-1040 (available from Pharmacia); quinazolines such as PD153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP59326, CGP60261 and CGP62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2, 3-d]pyrimidines; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines containing a nitrothiophene moiety; antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxalines (U.S. Patent No. 5,804,396); tryphostins (U.S. Patent No. 5,804,396); Affinitac (ISIS 3521, Isis / Lilly); PKI166 (Novartis); semaxinib (Pfizer);INC-1C11 (Imclone), rapamycin (sirolimus, RAPAMUNE®); or the following patent publications: U.S. Pat. No. 5,804,396, WO1999 / 09016 (American Cyanamid), WO1998 / 43960 (American Cyanamid), WO1997 / 38983(Warner Lambert), Also included are "tyrosine kinase inhibitors," including those described in any of WO1999 / 06378 (Warner Lambert), WO1999 / 06396 (Warner Lambert), WO1996 / 30347 (Pfizer, Inc.), WO1996 / 33978 (Zeneca), WO1996 / 3397 (Zeneca), and WO1996 / 33980 (Zeneca). Tyrosine kinase inhibitors include erlotinib (Tarceva®), gefitinib (Iressa®), dasatinib (Sprycel®), nilotinib (Tasigna®), crizotinib (Xalkori®), ruxolitinib (Jakafi®), vemurafenib (Zelboraf®), vandetanib (Caprelsa®), pazopanib (Votrient®), afatinib, alisertib, amuvatinib, axitinib, bosutinib, brivanib, canertinib, cabozantinib, cediranib, clevostatinib ... Also included are nolanib, dabrafenib, dacomitinib, danusertib, dovitinib, foretinib, ganetespib, ibrutinib, iniparib, lenvatinib, linifanib, linsitinib, masitinib, momelotinib, motesanib, neratinib, niraparib, oprozomib, olaparib, pictilisib, ponatinib, quizartinib, regorafenib, rigosertib, rucaparib, saracatinib, salidegib, tandutinib, tasocitinib, telatinib, tivantinib, tivozanib, tofacitinib, trametinib, veliparib, vismodegib, volasertib, and cobimetinib (Cotellic®).

[0366] Chemotherapy drugs include dexamethasone, interferon, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, live BCG, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, erotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, and ibuprofen. Also included are interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.

[0367] Chemotherapy drugs include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-butyrate, and hydrocortisone-17-butyrate. Lutisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone capronate, fluocortolone pivalate, and fluprednidene acetate; phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN) Immunoselective anti-inflammatory peptides (ImSAIDs) such as BioTherapeutics, LLC; antirheumatic drugs such as azathioprine, cyclosporin (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, minocycline, sulfasalazine, etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), and golimumab (Simponi), interleukin 1 (IL-1) blockers such as anakinra (Kineret), T-cell costimulation blockers such as abatacept (Orencia), interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®); interleukin 13 (IL-13) blockers such as lebrikizumab; interferon alpha (IFN) blockers such as lontalizumab; beta 7 integrin blockers such as rhuMAb beta 7 IgE pathway blockers such as anti-M1 prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / 132 blockers such as anti-lymphotoxin alpha (LTa); various investigational drugs such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavin, flavanols, procyanidins, and betulin polyphenols such as carboxylic acid and its derivatives; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin; podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®); clodronate (e.g., BONE bisphosphonates such as FOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®) or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines such as the THERATOPE® vaccine;Also included are perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); CCI-779; Bcl-2 inhibitors such as tipifarnib (R11577); orafenib, ABT510; oblimersen sodium (GENASENSE®); pixantrone; farnesyltransferase inhibitors such as lonafarnib (SCH6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; as well as many combinations of two or more of the above, such as CHOP, an abbreviation for combination therapy with cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen including oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin.

[0368] Chemotherapeutic agents also include poly ADP-ribose polymerase (PARP) inhibitors: olaparib (Lynparza®), rucaprib (Rubraca®), niraparib (Zejula®), and tarzoparib (Talzenna®).

[0369] Effective combinations of compounds of Formula I' or any formula as described herein with other agents can be identified through preclinical and clinical trials of the combination, and will depend on many factors, including the type and stage of development of the disease, the patient's general health, the toxicity and side effects of the agents, etc.

[0370] In some embodiments, the compounds as disclosed herein can be used in combination therapy with any of the kinase inhibitors disclosed herein to treat diseases such as cancer. Exemplary kinase inhibitors include imatinib, baricitinib, gefitinib, erlotinib, sorafenib, dasatinib, sunitinib, lapatinib, nilotinib, pirfenidone, pazopanib, crizotinib, vemurafenib, vandetanib, ruxolitinib, and azithromycin. These include xanthomonas globulin, bosutinib, regorafenib, tofacitinib, cabozantinib, ponatinib, trametinib, dabrafenib, afatinib, ibrutinib, ceritinib, idelalisib, nintedanib, palbociclib, lenvatinib, cobimetinib, XL-147, XL-765, XL-499, and XL-880. In some embodiments, compounds as described herein can be used in combination with an HSP90 inhibitor (e.g., XL888), a liver X receptor (LXR) modulator, a retinoid-related orphan receptor gamma (RORy) modulator, a CK1 inhibitor, a CK1-α inhibitor, a Wnt pathway inhibitor (e.g., SST-215), or a mineralocorticoid receptor inhibitor, (e.g., esaxerenone or XL-550) to treat a disease disclosed herein, such as cancer.

[0371] In some embodiments, a compound as disclosed herein is administered in combination with an inhibitor of PD-1 or an inhibitor of PD-L1, such as an anti-PD-1 monoclonal antibody or an anti-PD-L1 monoclonal antibody, e.g., nivolumab (Opdivo), pembrolizumab (Keytruda, MK-3475), atezolizumab, avelumab, AMP-224, AMP-514, PDR001, durvalumab, pidilizumab, or the like, to treat cancer. It can be used in combination with other anti-cancer drugs such as CTLA-4 inhibitors, such as anti-CTLA-4 antibodies, e.g., ipilimumab (Yervoy) and tremelimumab; and phosphatidylserine inhibitors, e.g., bavituximab (PGN401); antibodies against cytokines (e.g., IL-10, TGF-β); and cemiplimab.

[0372] In some embodiments, the compounds as described herein can be used in combination with vaccination protocols to treat cancer. In some embodiments, the compounds as described herein can be used in combination with vaccines to stimulate immune responses against pathogens, toxins, and self-antigens. Examples of pathogens for which this therapeutic approach may be particularly useful include those for which no effective vaccine currently exists or for which conventional vaccines are not fully effective. These include, but are not limited to, HIV, hepatitis (types A, B, and C), influenza, Herpes, Giardia, malaria, leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0373] In some embodiments, the compounds as disclosed herein can be used in combination with inhibitors of PARP, such as olaparib (Lynparza®), rucaprib (Rubraca®), niraparib (Zejula®), talzoparib (Talzenna®).

[0374] The amounts of both a compound disclosed herein or a salt thereof and one or more additional therapeutic agents (in compositions containing additional therapeutic agents, as described above) that can be combined with the carrier materials to produce a single dosage form will vary depending on the host treated and the particular mode of administration. In certain embodiments, the compositions of the present invention are formulated so that a dosage of 0.01 to 100 mg / kg body weight / day of the present invention can be administered.

[0375] The additional therapeutic agent and the compound disclosed herein may act synergistically. Thus, the amount of the additional therapeutic agent in such compositions may be less than that required in a monotherapy utilizing only that therapeutic agent, or a lower dose may be used, so that fewer side effects may occur in the patient. In certain embodiments, the additional therapeutic agent in such compositions may be administered at a dosage of between 0.01 and 10,000 μg / kg body weight / day.

[0376] Labeled Compounds and Assay Methods Another aspect of the present invention relates to labeled compounds (radiolabeled, fluore...

Claims

[Claim 1] Kinase-dependent disorder.

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