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WO2025151487A2 - Small-molecule inhibitors of adar1 - Google Patents

Small-molecule inhibitors of adar1

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Publication number
WO2025151487A2
WO2025151487A2 PCT/US2025/010693 US2025010693W WO2025151487A2 WO 2025151487 A2 WO2025151487 A2 WO 2025151487A2 US 2025010693 W US2025010693 W US 2025010693W WO 2025151487 A2 WO2025151487 A2 WO 2025151487A2
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WO
WIPO (PCT)
Prior art keywords
compound
alkyl
group
optionally substituted
pharmaceutically acceptable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/010693
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French (fr)
Other versions
WO2025151487A3 (en
Inventor
Shaomeng Wang
Weiping Zou
Pulla Reddy BOGGU
Gaopeng LI
Peng LIAO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Michigan System
University of Michigan Ann Arbor
Original Assignee
University of Michigan System
University of Michigan Ann Arbor
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Publication of WO2025151487A2 publication Critical patent/WO2025151487A2/en
Publication of WO2025151487A3 publication Critical patent/WO2025151487A3/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • C07D215/42—Nitrogen atoms attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • C07D215/42—Nitrogen atoms attached in position 4
    • C07D215/44—Nitrogen atoms attached in position 4 with aryl radicals attached to said nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • ADAR1 inhibitors are useful for treating cancer when administered to a subject as a single agent or in combination with one or more immune checkpoint inhibitors.
  • AD ARI Adenosine deaminase acting on RNA 1
  • AD ARI is an RNA editing enzyme that catalyzes the deamination of adenosine to inosine (A-to-I) in double-stranded RNAs (dsRNAs).
  • AD ARI pl50 the full-length AD ARI pl50 (150 kDa)
  • AD ARI pl 10 the shorter AD ARI pl 10 (110 kDa).
  • AD ARI pl 10 is constitutively expressed in the nucleus, while ADAR1 pl50 is localized in the cytoplasm. Nishikura, Nat Rev Mol Cell Biol 17, 83-96 (2016).
  • ADAR 1 -mediated A-to-I editing disrupts dsRNA structures and protects dsRNAs from dsRNA-sensing molecules, such as MDA5, thereby inhibiting the type-I and type-III-IFN-mediated response and innate immunity. Liddicoat et al., Science 349, 1115-1120 (2015). ADAR1 plays pivotal roles in viral infection (Pfaller et al., Annu Rev Virol 8, 239-264 (2021)), autoimmune disease (Chung, H. et al., Cell 172, 811-824 e814 (2016)), and cancer immunity.
  • IFNy type-II IFN transcriptionally stimulates ADAR1 expression. Patterson et al., Mol Cell Biol 15, 5376-5388 (1995). However, the posttranslational modification (PTM), endogenous regulation, and pharmacological manipulation of AD ARI remain largely unknown.
  • PTM posttranslational modification
  • TME tumor microenvironment
  • the present disclosure provides compounds represented by any one of Formulae (I)-(VII) or (IX)-(XVII), and the pharmaceutically acceptable salts and solvates thereof, and compounds of Table 1A and Table 2A, and the pharmaceutically acceptable salts and solvates thereof.
  • These compounds, and the pharmaceutically acceptable salts and solvates thereof are collectively referred to herein as "Compounds of the Disclosure” or individually as a “Compound of the Disclosure.”
  • Compounds of the Disclosure are adenosine deaminase acting on RNA 1 (ADAR1) inhibitors and are thus useful in treating diseases or conditions, e.g., cancer, wherein inhibition of ADAR1 provides a therapeutic benefit to a subject.
  • ADAR1 adenosine deaminase acting on RNA 1
  • compositions comprising a Compound of the Disclosure and a pharmaceutically acceptable excipient.
  • the present disclosure provides methods of treating cancer in a subject by administering a therapeutically effective amount of a Compound of the Disclosure to a subject in need thereof.
  • the present disclosure provides a Compound of the Disclosure for use in treating cancer in a subject in need thereof.
  • the present disclosure provides the use of a Compound of the Disclosure in the manufacture of a medicament for treating cancer in a subject in need thereof.
  • the present disclosure provides methods of treating cancer in a subject by administering a therapeutically effective amount of a Compound of the Disclosure in combination with one or more immune checkpoint inhibitors to a subject in need thereof.
  • Immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, anti-LAG3 antibodies, anti- TIM3 antibodies, anti-VISTA antibodies, anti-TIGIT antibodies, and anti-cd47 antibodies.
  • the present disclosure provides methods of inhibiting ADAR1 in a subject in need thereof by administering a therapeutically effective amount of a Compound of the Disclosure to the subject.
  • the present disclosure provides methods of sensitizing tumor response to immune checkpoint blockade (ICB) by administering a therapeutically effective amount of a Compound of the Disclosure to a subject in need thereof.
  • IRB immune checkpoint blockade
  • present disclosure provides kits for carrying out the methods of the present disclosure.
  • the present disclosure provides compounds represented by any one of Formulae (XVIII)-(XXV), and the pharmaceutically acceptable salts and solvates thereof, and the compounds of Table 8, and the pharmaceutically acceptable salts and solvates thereof.
  • These compounds are synthetic intermediates that can be used, for example, to prepare Compounds of the Disclosure.
  • FIG. 1c is six bar graphs showing human cancer cells (A2058, SW480, and CAOV3) or mouse cancer cells (B16, CT26, and ID8) treated with BPTES in the presence or absence of IFNy for 48 hours.
  • Surface staining of MHC-I was determined by FACS. Data are mean ⁇ SD.
  • n 3 biological independent samples. P values by two-sided t test.
  • FIG. le is four bar graphs showing CHE1 cells treated with BPTES and/or IFNy for 36 hours.
  • FIG. li is four bar graphs showing wild type (WT), type-I IFN receptor knock out (Ifnarl KO), or type-III IFN receptor knockout (Ifnlrl KO) B16 cells were treated with the indicated conditions for 36 hours.
  • TPM Transcripts per million
  • ED FIGS. Ik-1 is volcano map or scatter plot, respectively, showing the differentially expressed genes between BPTES plus IFNy group and IFNy group.
  • FIG. 2c is a graph showing Ifihl KO B16 cells carrying indicated shRNAs treated with IFNy for 48 hours. IFN
  • 3 in the culture medium was determined by EEISA, n 3 biological independent samples.
  • FIG. 2d is a schematic diagram showing the purification of cytosolic RNA samples and transfection into untreated cells.
  • FIGS. 2e-f is a dot plot and bar graph, respectively, showing B16 cells treated with BPTES and/or IFNy for 24 hours.
  • FIGS. 2g-h is a line graph and Kaplan-Meier curve, respectively, showing tumor growth curves and mouse overall survival.
  • WT B16 cells carrying shFluc or shGdh inoculated into C57BE/6 mice. The mice were treated with IgG or anti-PD-El antibody, n 5 mice per group. P values by two-sided t test.
  • FIG. 2a is a Western blot showing WT or Ifihl KO B16 cells treated with
  • FIG. 2b is a Western blot showing WT or Ifihl KO B16 cells were treated with the indicated concentrations of BPTES in the presence or absence of IFNy for 24 hours. Protein levels of the indicated genes were determined by Western blot. 1 of 2 blots is shown.
  • FIG. 3a is a schematic diagram showing the RNA editing of endogenous retrovirus (ERV) regulates MDA5 activation and IFN production.
  • ERP endogenous retrovirus
  • FIG. 3b is a schematic diagram showing the construction of the RNA editing reporter (RE-Euc).
  • FIGS. 3p-q is a Western blot and line graph, respectively, showing WT or C2A B16 cells treated with increasing concentrations of BPTES (0, 1, 2, 5, 10 pM) for 24 hours.
  • the Zinc binding proteins were enriched by Zn-NTA beads via the pulldown assay.
  • Adarl protein was detected in the pulldown products and input. 1 of 2 blots is shown (FIG. 3p).
  • Relative Zinc affinity was calculated by the enrichment of Adarl protein upon Zn-NTA pulldown assay (FIG. 3q).
  • FIG. 3b is a Western blot showing B16 cells treated with IFNy for indicated hours. Adarl protein was determined by Western blot. 1 of 3 blots is shown.
  • FIG. 3d is a Western blot showing B16 cells were treated with the indicated concentrations of BPTES in the presence or absence of IFNy for 24 hours. Adarl protein was determined by Western blot. 1 of 2 blots is shown.
  • FIG. 3e is an image of B16 cells carrying Adarl -OE treated with BPTES for 24 hours. Adarl protein was enriched by Co-IP with Flag antibody. Post-translational modifications were detected in the IP products.
  • ED FIG. 3f is two bar graphs showing B16 cells treated with BPTES and/ or IFNy in the presence or absence of 2-BP. Ifnbl transcripts were determined at 24 hours. Surface expression of H2-K b was determined at 48 hours.
  • FIG. 4e is five bar graphs showing Zdhhc2-overexpressed B16 cells treated with IFNy for 36 hours.
  • the indicated IFN signaling gene transcripts were determined by qPCR. Data are mean ⁇ SD.
  • n 3 biological independent samples. P values by two-sided t test.
  • R la , R 2 , R 3 , R 4 , and R 6a are as defined in connection with Formula (III).
  • the present disclosure provides a compound having any one of Formulae (I)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein R 3 is hydrogen or C 1 -C 6 alkyl. In another embodiment, R 3 is hydrogen. In another embodiment, R 3 is methyl. In another embodiment, R 3 is ethyl.
  • the present disclosure provides a compound having any one of Formulae (I)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein R 3 is C 3 -C 6 cycloalkyl.
  • R 4 is cyclopropyl.
  • the present disclosure provides a compound having any one of Formulae (I)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein R 4 is hydrogen or cyano. In another embodiment, R 4 is hydrogen. In another embodiment, R 4 is cyano.
  • Compounds of the Disclosure are compounds having Formula (IX): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • X is selected from the group consisting of -O- and -NR a -;
  • R a is selected from the group consisting to hydrogen and C1-C4 alkyl
  • R la is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR 8 R 9 ;
  • R lb is selected from the group consisting of hydrogen and C 1 -C 6 alkyl
  • R 9 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, and C 3 -C 6 cycloalkyl; or
  • R 2 is selected from the group consisting of hydrogen, halo, C 1 -C 6 alkyl, and -OR 7 ;
  • R 7 is selected from the group consisting of C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, (optionally substituted C 3 -C 6 cycloalkyl)C 1 -C 6 -alkyl, (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4-to 7-membered heterocyclo)C 1 -C 6 alkyl;
  • R 3 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl;
  • R 4 is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, and C 3 -C 6 cycloalkyl; and [0165] R 5 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, optionally substituted aryl, (heterocyclo)alkyl, and optionally substituted 5- to 10- membered heteroaryl.
  • Compounds of the Disclosure are compounds having Formula (IX), or a pharmaceutically acceptable salt or solvate thereof, wherein R 5 is selected from the group consisting of:
  • Compounds of the Disclosure are compounds having Formula (X): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • R 6a is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and C 3 -C 6 cycloalkyl;
  • R 6b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and C 3 -C 6 cycloalkyl;
  • R la , R 2 , R 3 , R 4 , and X are as defined in connection with Formula (IX);
  • the present disclosure provides a compound having Formula (VIII), or pharmaceutically acceptable salts or solvates thereof, wherein R 4 is hydrogen or cyano. In another embodiment, R 4 is hydrogen. In another embodiment, R 4 is cyano.
  • X is selected from the group consisting of -O- and -NR a -;
  • R a is selected from the group consisting to hydrogen and C1-C4 alkyl
  • LG is a leaving group
  • R 7 is selected from the group consisting of C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, (optionally substituted C 3 -C 6 cycloalkyl)C 1 -C 6 -alkyl, (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4-to 7-membered heterocyclo)C 1 -C 6 alkyl;
  • R 3 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl;
  • R 4 is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, and C 3 -C 6 cycloalkyl;
  • R 5 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, optionally substituted aryl, (heterocyclo)alkyl, and optionally substituted 5- to 10- membered heteroaryl.
  • the present disclosure provides compounds having Formula (XVIII), or a pharmaceutically acceptable salt or solvate thereof, wherein R 5 is selected from the group consisting of:
  • the present disclosure provides compounds having Formula (XIX): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • R 6a is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and C 3 -C 6 cycloalkyl;
  • R 6b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and C 3 -C 6 cycloalkyl;
  • R 6C is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl; C1-C4 haloalkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, (optionally substituted 4- to 7-membered heterocyclo)-O-, (aryl)alkyl-O-, and (heteroaryl)alkyl-O-; and
  • LG, R 2 , R 3 , R 4 , and X are as defined in connection with Formula (VXIII).
  • the present disclosure provides compounds having Formula (XIX), or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • R 6b is hydrogen
  • R 6C is selected from the group consisting of:
  • the present disclosure provides compounds having Formula (XX): (XX), or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • R 5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl
  • LG, R 2 , R 3 , R 4 , and X are as defined in connection with Formula (XVIII);
  • R 6a and R 6b are as defined in connection with Formula (XIX).
  • Compounds of the Disclosure are compounds having Formula (XX), or a pharmaceutically acceptable salt or solvate thereof, wherein R 5a is optionally substituted 5-membered heteroaryl, e.g., imidazole.
  • the present disclosure provides compounds having Formula (XXI): (XXI), or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • R 5b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C2-C6 alkenyl, C 3 -C 6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo;
  • LG, R 2 , R 3 , R 4 , and X are as defined in connection with Formula (XVIII); and
  • R 6a is as defined in connection with Formula (XIX).
  • Compounds of the Disclosure are compounds having Formula (XXII): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • R 5b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C2-C6 alkenyl, C 3 -C 6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo;
  • LG, R 2 , R 3 , R 4 , and X are as defined in connection with Formula (XVIII);
  • R 6a is as defined in connection with Formula (XIX).
  • the present disclosure provides compounds having Formula (XXIII): (XXIII), or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • R 5b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C2-C6 alkenyl, C 3 -C 6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo;
  • LG, R 2 , R 3 , R 4 , and X are as defined in connection with Formula (XVIII);
  • R 6a is as defined in connection with Formula (XIX).
  • R 5b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C2-C6 alkenyl, C 3 -C 6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo;
  • LG, R 2 , R 3 , R 4 , and X are as defined in connection with Formula (XVIII); and [0295] R 6a is as defined in connection with Formula (XIX).
  • the present disclosure provides compounds having Formula (XXV): (XXV), or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • the cancer is a leukemia, for example, a leukemia selected from acute monocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia and mixed lineage leukemia (MLL).
  • the cancer is NUT-midline carcinoma.
  • the cancer is multiple myeloma.
  • the cancer is a lung cancer such as small cell lung cancer (SCLC).
  • SCLC small cell lung cancer
  • the cancer is a neuroblastoma.
  • the cancer is Burkitt's lymphoma.
  • the cancer is cervical cancer.
  • the cancer is esophageal cancer.
  • the cancer is ovarian cancer.
  • the cancer is colorectal cancer.
  • the cancer is prostate cancer.
  • the cancer is breast cancer.
  • the cancer is selected from the group consisting of acute monocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia mixed lineage leukemia, NUT-midline carcinoma, multiple myeloma, small cell lung cancer, non- small cell lung cancer, neuroblastoma, Burkitt's lymphoma, cervical cancer, esophageal cancer, ovarian cancer, colorectal cancer, prostate cancer, breast cancer, bladder cancer, ovary cancer, glioma, sarcoma, esophageal squamous cell carcinoma, and papillary thyroid carcinoma.
  • a Compound of the Disclosure are administered to a subject in need thereof to treat breast cancer, ovarian cancer, or prostate cancer.
  • the cancer is breast cancer.
  • the cancer is ovarian cancer.
  • the cancer is prostate cancer.
  • the cancer is metastatic castration-resistant prostate cancer.
  • the methods of the present disclosure can be accomplished by administering a Compound of the Disclosure as the neat compound or as a pharmaceutical composition.
  • Administration of a pharmaceutical composition, or neat Compound of the Disclosure can be performed during or after the onset of the disease or condition of interest.
  • the pharmaceutical compositions are sterile, and contain no toxic, carcinogenic, or mutagenic compounds that would cause an adverse reaction when administered.
  • the second therapeutic agent is one or more ICIs, e.g., one or more anti-PD-1 antibodies, one or more anti-PD-Ll antibodies, one or more anti-CTLA-4 antibodies, one or more anti-LAG3 antibodies, one or more anti-TIM3 antibodies, one or more anti- VISTA antibodies, one or more anti-TIGIT antibodies, or one or more anti- cd47 antibodies, or a combination thereof, e.g., nivolumab, pembrolizumab, dostarlimab, retifanlimab, cemiplimab, avelumab, atezolizumab, or durvalumab, or a combination thereof.
  • ICIs e.g., one or more anti-PD-1 antibodies, one or more anti-PD-Ll antibodies, one or more anti-CTLA-4 antibodies, one or more anti-LAG3 antibodies, one or more anti-TIM3 antibodies, one or more anti- VISTA antibodies, one or more anti-TIGIT antibodies,
  • the second therapeutic agent is administered in an amount to provide its desired therapeutic effect.
  • the effective dosage range for each second therapeutic agent is known in the art, and the second therapeutic agent is, for example, administered to an individual in need thereof within such established ranges.
  • administration of a Compound of the Disclosure sensitizs the cancer cells to ICB and thus the effective dosage of one or more ICIs co-administered with the Compound of the Disclosure is less, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% less, than the established ranges for the one or more ICIs known in the art.
  • a Compound of the Disclosure and the second therapeutic agent can be administered together as a single-unit dose or separately as multi-unit doses, wherein the Compound of the Disclosure is administered before the second therapeutic agent or vice versa.
  • One or more doses of the Compound of the Disclosure and/or one or more doses of the second therapeutic agent can be administered.
  • the Compound of the Disclosure therefore can be used in conjunction with one or more second therapeutic agents, for example, but not limited to, anticancer agents.
  • a therapeutically effective amount of a Compound of the Disclosure is administered to a subject, e.g., a human cancer patient, in need thereof.
  • a therapeutically effective amount of a Compound of the Disclosure is administered to a subject in need thereof in combination with a therapeutically effective amount of an ICI. Whether such a treatment is indicated depends on the individual case and is subject to medical assessment (diagnosis) that takes into consideration signs, symptoms, and/or malfunctions that are present, the risks of developing particular signs, symptoms and/or malfunctions, and other factors.
  • a Compound of the Disclosure can be administered by any suitable route, for example by oral, buccal, inhalation, sublingual, rectal, vaginal, intracistemal or intrathecal through lumbar puncture, transurethral, nasal, percutaneous, i.e., transdermal, or parenteral (including intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a particular site) administration.
  • Parenteral administration can be accomplished using a needle and syringe or using a high pressure technique.
  • compositions include those wherein a Compound of the Disclosure is administered in an effective amount to achieve its intended purpose.
  • the exact formulation, route of administration, and dosage is determined by an individual physician in view of the diagnosed condition or disease. Dosage amount and interval can be adjusted individually to provide levels of a Compound of the Disclosure that is sufficient to maintain therapeutic effects.
  • Toxicity and therapeutic efficacy of the Compounds of the Disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the maximum tolerated dose (MTD) of a compound, which defines as the highest dose that causes no toxicity in animals.
  • MTD maximum tolerated dose
  • the dose ratio between the maximum tolerated dose and therapeutic effects (e.g. inhibiting of tumor growth) is the therapeutic index.
  • the dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • a therapeutically effective amount of a Compound of the Disclosure required for use in therapy varies with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the patient, and ultimately is determined by the attendant physician. Dosage amounts and intervals can be adjusted individually to provide plasma levels of the ADAR1 inhibitor that are sufficient to maintain the desired therapeutic effects.
  • the desired dose conveniently can be administered in a single dose, or as multiple doses administered at appropriate intervals, for example as one, two, three, four or more subdoses per day. Multiple doses often are desired, or required.
  • a Compound of the Disclosure can be administered at a frequency of: four doses delivered as one dose per day at four-day intervals (q4d x 4); four doses delivered as one dose per day at three-day intervals (q3d x 4); one dose delivered per day at five-day intervals (qd x 5); one dose per week for three weeks (qwk3); five daily doses, with two days rest, and another five daily doses (5/2/5); or, any dose regimen determined to be appropriate for the circumstance.
  • a Compound of the Disclosure used in a method of the present disclosure can be administered in an amount of, for example, about 0.005 to about 5000 milligrams per dose, about 10 to about 1000 milligrams per dose, about 10 to about 500 milligrams per dose, or about 10 to about 100 milligrams per dose.
  • a Compound of the Disclosure can be administered, per dose, in an amount of about 0.005, about 0.05, about 0.5, about 5, about 10, about 20, about 30, about 40, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, or about 1000 milligrams, including all doses between 0.005 and 5000 milligrams.
  • the dosage of a composition containing a Compound of the Disclosure, or a composition containing the same can be from about 1 ng/kg to about 200 mg/kg, about 1 pg/kg to about 100 mg/kg, or about 1 mg/kg to about 50 mg/kg.
  • the dosage of a composition can be at any dosage including, but not limited to, about 1 pg/kg.
  • the dosage of a composition may be at any dosage including, but not limited to, about 1 pg/kg, about 10 pg/kg, about 25 pg/kg, about 50 pg/kg, about 75 pg/kg, about 100 pg/kg, about 125 pg/kg, about 150 pg/kg, about 175 pg/kg, about 200 pg/kg, about
  • Compounds of the Disclosure typically are administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • Pharmaceutical compositions for use in accordance with the present disclosure are formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and/or auxiliaries that facilitate processing of Compound of the Disclosure.
  • Compounds of the Disclosure also can be formulated in rectal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases.
  • the Compound of the Disclosure also can be formulated as a depot preparation.
  • Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • the Compound of the Disclosure can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins.
  • the Compounds of the Disclosure can be administered orally, buccally, or sublingually in the form of tablets containing excipients, such as starch or lactose, or in capsules or ovules, either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents.
  • excipients such as starch or lactose
  • capsules or ovules either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents.
  • Such liquid preparations can be prepared with pharmaceutically acceptable additives, such as suspending agents.
  • Compound of the Disclosure also can be injected parenterally, for example, intravenously, intramuscularly, subcutaneously, or intracoronarily.
  • the Compound of the Disclosure are typically used in the form of a sterile aqueous solution which can contain other substances, for example, salts or monosaccharides, such as mannitol or glucose, to make the solution isotonic with blood.
  • a sterile aqueous solution which can contain other substances, for example, salts or monosaccharides, such as mannitol or glucose, to make the solution isotonic with blood.
  • Immune checkpoint inhibitors are therapies that blockade immune system inhibitor checkpoints.
  • Immune checkpoints can be stimulatory or inhibitory. Blockade of inhibitory immune checkpoint activates immune system function and can be used for cancer immunotherapy. Pardoll, Nature Reviews. Cancer 12:252-64 (2012). Tumor cells turn off activated T cells when they attach to specific T-cell receptors. ICIs prevent tumor cells from attaching to T cells, which results in T cells remaining activated. In effect, the coordinated action by cellular and soluble components combats pathogens and injuries by cancers.
  • the modulation of immune system pathways may involve changing the expression or the functional activity of at least one component of the pathway to then modulate the response by the immune system.
  • ICIs include, but are not limited to, TIGIT inhibitors, PD-1 inhibitors, PD-L1 (B7-H1) inhibitors, CTLA-4 inhibitors, LAG3 inhibitors, TIM3 inhibitors, and cd47 inhibitors.
  • TIGIT inhibitors PD-1 inhibitors
  • PD-L1 (B7-H1) inhibitors CTLA-4 inhibitors
  • LAG3 inhibitors LAG3 inhibitors
  • TIM3 inhibitors cd47 inhibitors.
  • the effective dosage range for ICIs is generally known in the art, and the immune checkpoint inhibitor(s) is(are) administered to a subject in need thereof within, for example, such established ranges.
  • methods and uses of the present disclosure comprise administering a therapeutically effective amount of a Compound of the Disclosure to a subject in combination with a therapeutically effective amount of one or more ICIs to treat cancer.
  • methods of the present disclosure comprise administering a therapeutically effective amount of a Compound of the Disclosure to a subject in combination with one or more PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG3 inhibitors, TIM3 inhibitors, TIGIT inhibitors, or cd47 inhibitors, or a combination thereof.
  • the one or more ICIs comprise a PD-L1 (also known as B7-H1 or CD274) inhibitor.
  • PD-L1 inhibitors include antibodies that specifically bind to PD-L1.
  • Particular anti-PD-Ll antibodies include, but are not limited to, avelumab, atezolizumab, durvalumab, KN035, and BMS-936559.
  • the one or more ICIs comprise a CTLA-4 inhibitor.
  • CTLA-4 also known as cytotoxic T-lymphocyte antigen 4
  • CTLA-4 is a protein receptor that downregulates the immune system.
  • CTLA-4 is characterized as a "brake” that binds costimulatory molecules on antigen-presenting cells, which prevents interaction with CD28 on T cells and also generates an overtly inhibitory signal that constrains T cell activation.
  • CTLA-4 inhibitors include antibodies that specifically bind to CTLA-4.
  • Particular anti-CTLA-4 antibodies include, but are not limited to, ipilimumab and tremelimumab. Lor a general discussion of the availability, methods of production, mechanism of action, and clinical studies, see U.S. 6,984,720, U.S. 6,207,156, and Naido et al., British Journal of Cancer 111:2214-19 (2014).
  • the one or more ICIs comprise a LAG3 inhibitor.
  • LAG3, Lymphocyte Activation Gene 3 is a negative co-simulatory receptor that modulates T cell homeostatis, proliferation, and activation.
  • LAG3 has been reported to participate in regulatory T cells (Tregs) suppressive function. A large proportion of LAG3 molecules are retained in the cell close to the microtubule-organizing center, and only induced following antigen specific T cell activation.
  • Regs regulatory T cells
  • Examples of LAG3 inhibitors include antibodies that specifically bind to LAG3. Particular anti- LAG3 antibodies include, but are not limited to, GSK2831781.
  • the one or more ICIs comprise a TIM3 inhibitor.
  • TIM3, T-cell immunoglobulin and mucin domain 3 is an immune checkpoint receptor that functions to limit the duration and magnitude of TH1 and TCI T-cell responses.
  • the TIM3 pathway is considered a target for anticancer immunotherapy due to its expression on dysfunctional CD8+ T cells and Tregs, which are two reported immune cell populations that constitute immunosuppression in tumor tissue.
  • Examples of TIM3 inhibitors include antibodies that specifically bind to TIM3. Lor a general discussion of the availability, methods of production, mechanism of action, and studies of TIM3 inhibitors, see U.S. 20150225457, U.S.
  • the one or more ICIs comprise a cd47 inhibitor. See Unanue, E.R., PNAS 110:10886-87 (2013).
  • the one or more ICIs comprise a TIGIT inhibitor.
  • T-cell immunoreceptor with immunoglobulin and ITIM domains is an inhibitory receptor expressed on several immune cell types, including CD8+ T cells, natural killer, or NK, cells, T regulatory cells, or Tregs, and follicular T helper cells.
  • TIGIT interacts with CD155 expressed on antigen-presenting cells or tumor cells to down-regulate T cell and natural killer (NK) cell functions.
  • NK natural killer
  • TIGIT has been shown to be a mediator of resistance to existing checkpoint inhibitors, including anti-PD-1. TIGIT also directly suppresses the antitumor effector function on CD8 T cells.
  • 840 mg may be administered by intravenous infusion every two weeks, or 1200 mg may be administered by intravenous infusion every three weeks, or 1680 mg may be administered by intravenous infusion every four weeks.
  • the present disclosure provides a kit for carrying out the methods and uses described herein, the kit comprising: (i) a Compound of the Disclosure; (ii) one or more immune checkpoint inhibitors; and (iii) a label with instructions for how to use the kit.
  • Embodiment 1 A compound having Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • R la is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR 8 R 9 ;
  • R 8 and R 9 are each independently selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, and (C 1 -C 6 alkoxy)C 1 -C 6 alkyl; or
  • R 8 and R 9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo;
  • R 2 is selected from the group consisting of hydrogen, halo, C 1 -C 6 alkyl, and -OR 7 ;
  • R 7 is selected from the group consisting of C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, (optionally substituted C 3 -C 6 cycloalkyl)C 1 -C 6 -alkyl, (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4-to 7-membered heterocyclo)C 1 -C 6 alkyl;
  • R 3 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl;
  • R 4 is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, and C 3 -C 6 cycloalkyl;
  • Embodiment 2 The compound any Embodiment 1, or a pharmaceutically acceptable salt or solvate thereof, with the following provisos:
  • R 8 and R 9 are each independently selected from the group consisting of hydrogen, C2-C6 alkyl, C 3 -C 6 cycloalkyl, and (Ci- C ⁇ > alkoxy)C 1 -C 6 alkyl; or R 8 and R 9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo; or
  • R 4 is selected from the group consisting of hydrogen, halogen, C 1 -C 6 alkyl, and C 3 -C 6 cycloalkyl; or
  • Embodiment 3 The compound of Embodiments 1 or 2 having Formula (II): (II), or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • R 6a is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and C 3 -C 6 cycloalkyl;
  • R 6b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and C 3 -C 6 cycloalkyl;
  • R 6C is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl; C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, (optionally substituted heterocyclo)-O-,
  • Embodiment 4 The compound of Embodiment 3 having Formula (III): or a pharmaceutically acceptable salt or solvate thereof, wherein R 5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl.
  • Embodiment 11 The compound of any one of Embodiments 1-9, or a pharmaceutically acceptable salt or solvate thereof, wherein R la is C1-C4 alkyl.
  • Embodiment 12 The compound of any one of Embodiments 1-9, or a pharmaceutically acceptable salt or solvate thereof, wherein R la is -CH2NR 8 R 9 .
  • Embodiment 14 The compound of Embodiment 13, or a pharmaceutically acceptable salt or solvate thereof, wherein R 8 and R 9 are methyl.
  • Embodiment 15 The compound of Embodiment 12, or a pharmaceutically acceptable salt or solvate thereof, wherein R 8 and R 9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo.
  • Embodiment 17 The compound of any one of Embodiments 1-16, or a pharmaceutically acceptable salt or solvate thereof, wherein R 2 is -OR 7 .
  • Embodiment 18 The compound of Embodiment 17, or a pharmaceutically acceptable salt or solvate thereof, wherein R 7 is methyl, ethyl, or propyl.
  • Embodiment 19 The compound of Embodiment 18, or a pharmaceutically acceptable salt or solvate thereof, wherein R 7 is ethyl.
  • Embodiment 20 The compound of any one of Embodiments 1-19, or a pharmaceutically acceptable salt or solvate thereof, wherein R 3 is hydrogen or C 1 -C 6 alkyl.
  • Embodiment 21 The compound of Embodiment 20, or a pharmaceutically acceptable salt or solvate thereof, wherein R 3 is hydrogen.
  • Embodiment 22 The compound of Embodiment 20, or a pharmaceutically acceptable salt or solvate thereof, wherein R 3 is methyl.
  • Embodiment 23 The compound of Embodiment 20, or a pharmaceutically acceptable salt or solvate thereof, wherein R 3 is ethyl.
  • Embodiment 24 The compound of any one of Embodiments 1-23, or a pharmaceutically acceptable salt or solvate thereof, wherein R 4 is hydrogen or cyano.
  • Embodiment 25 The compound of Embodiment 24, or a pharmaceutically acceptable salt or solvate thereof, wherein R 4 is hydrogen.
  • Embodiment 26 The compound of Embodiment 24, or a pharmaceutically acceptable salt or solvate thereof, wherein R 4 is cyano.
  • Embodiment 27 The compound of any one of Embodiments 3-26, or a pharmaceutically acceptable salt or solvate thereof, wherein R 6a is hydrogen or halogen.
  • Embodiment 28 The compound of Embodiment 27, or a pharmaceutically acceptable salt or solvate thereof, wherein R 6a is hydrogen.
  • Embodiment 29 The compound of Embodiment 27, or a pharmaceutically acceptable salt or solvate thereof, wherein R 6a is chloro.
  • Embodiment 30 The compound of Embodiment 1, or a pharmaceutically acceptable salt or solvate thereof, selected from any one or more of the compounds of Table 1A.
  • Embodiment 31 The compound of Embodiment 30, or a pharmaceutically acceptable salt or solvate thereof, selected from any one or more of:
  • Embodiment 32 A pharmaceutical composition comprising the compound of any one of Embodiments 1-31 and a pharmaceutically acceptable excipient.
  • Embodiment 34 The method of Embodiment 34 further comprising administering a therapeutically effective amount one or more immune checkpoint inhibitors to the subject.
  • Embodiment 35 The method of Embodiment 34, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies, one or more anti-PD-Ll antibodies, one or more anti-CTLA-4 antibodies, one or more anti-LAG3 antibodies, one or more anti-TIM3 antibodies, one or more anti- VISTA antibodies, one or more anti-TIGIT antibodies, or one or more anti-cd47 antibodies, or a combination thereof.
  • Embodiment 36 The method of Embodiment 35 wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies.
  • Embodiment 37 The method of Embodiment 36, wherein the one or more anti-PD-1 antibodies comprise nivolumab, pembrolizumab, dostarlimab, retifanlimab, cemiplimab, vopratelimab (JTX-4014), spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), INCMGA00012 (MGA012), AMP-224, AMP-514 (MEDI0680) and/or Acrixolimab (YBL-006).
  • the one or more anti-PD-1 antibodies comprise nivolumab, pembrolizumab, dostarlimab, retifanlimab, cemiplimab, vopratelimab (JTX-4014), spartalizumab (PDR001), camrelizumab (SHR1210)
  • Embodiment 38 The method any one of Embodiments 35-37, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-Ll antibodies.
  • Embodiment 39 The method of Embodiment 38, wherein the one or more anti-PD-Ll antibodies comprise avelumab, atezolizumab, durvalumab, KN035, and/or cosibelimab (CK-301).
  • Embodiment 43 The method of Embodiment 42, wherein the one or more anti-LAG3 antibodies comprise relatlimab.
  • Embodiment 50 The method of any one of Embodiments 33-49, wherein the cancer is any one or more of the cancers of Table 2 and/or Table 3.
  • Embodiment 52 A method of providing immuno-oncology therapy to a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Embodiments 1-31, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of Embodiment 32.
  • Embodiment 53 A method of sensitizing tumor response to immunotherapy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Embodiments 1-31, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of Embodiment 42.
  • Embodiment 54 The method of Embodiment 53, wherein the tumor is resistant to immune checkpoint blockade.
  • Embodiment 58 A kit for carrying out the method of any one of Embodiments 33-55, the kit comprising: (i) a compound having Formula (I), or a pharmaceutically acceptable salt or solvate thereof; (ii) one or more immune checkpoint inhibitors; and (iii) a label with instructions for how to use the kit.
  • Embodiment 59 The kit of any one of Embodiments 56-58, wherein the label is approved by the United States Food and Drug Administration (FDA), the European Medicines Agency (EMA), the China Food and Drug Administration (CFDA), or the Japanese Ministry of Health Labor and Welfare (MHLW).
  • FDA United States Food and Drug Administration
  • EMA European Medicines Agency
  • CFDA China Food and Drug Administration
  • MHLW Japanese Ministry of Health Labor and Welfare
  • the present disclosure also provides the following particular embodiments relating to compounds having Formula (VIII) and Compounds of the Disclosure having any one of Formula (IX)-(XVI).
  • Embodiment 1 A compound having Formula (IX): (IX), or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • X is selected from the group consisting of -O- and -NR a -;
  • R a is selected from the group consisting to hydrogen and C1-C4 alkyl
  • R la is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR 8 R 9 ;
  • R lb is selected from the group consisting of hydrogen and C 1 -C 6 alkyl
  • R 8 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted 4-to 7-membered heterocyclo, C2-C12 alkynyl, and (C 1 -C 6 alkoxy)C 1 -C 6 alkyl;
  • R 9 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, and C 3 -C 6 cycloalkyl; or
  • R 8 and R 9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo;
  • R 7 is selected from the group consisting of C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, (optionally substituted C 3 -C 6 cycloalkyl)C 1 -C 6 -alkyl, (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4-to 7-membered heterocyclo)C 1 -C 6 alkyl;
  • R 3 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl;
  • R 5 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, optionally substituted aryl, (heterocyclo)alkyl, and optionally substituted 5- to 10- membered heteroaryl.
  • Embodiment 2 The compound Embodiment 1, or a pharmaceutically acceptable salt or solvate thereof, with the following provisos:
  • R 4 is cyano and R 1 is -CH2NR 8 R 9 , then R 8 is hydrogen, C2-C6 alkyl, C 3 -C 6 cycloalkyl, optionally substituted 4-to 7-membered heterocyclo, C2-C12 alkynyl, and (Ci- Ce alkoxy)C 1 -C 6 alkyl; R 9 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, and C 3 -C 6 cycloalkyl; or R 8 and R 9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo; or
  • R 1 is -CH2NR 8 R 9 , R 8 and R 9 are each methyl, and R 4 is cyano, then R 3 is C 1 -C 6 alkyl or C 3 -C 6 cycloalkyl.
  • Embodiment 3 The compound of Embodiments 1 or 2 having Formula
  • R 6C is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl; C1-C4 haloalkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, (optionally substituted 4- to 7-membered heterocyclo)-O-, (aryl)alkyl-O-, and (heteroaryl)alkyl-O-.
  • Embodiment 8 The compound of Embodiment 7, or a pharmaceutically acceptable salt or solvate thereof, having Formula (XIII): (XIII).
  • Embodiment 10 The compound of Embodiment 7, or a pharmaceutically acceptable salt or solvate thereof, having Formula (XV): (XV).
  • Embodiment 14 The compound of Embodiment 11, or a pharmaceutically acceptable salt or solvate thereof, wherein R 5 is optionally substituted 5- to 10-membered heteroaryl.
  • Embodiment 19 The compound of Embodiment 18, or a pharmaceutically acceptable salt or solvate thereof, wherein R 8 and R 9 are C 1 -C 6 alkyl.
  • Embodiment 24 The compound of Embodiment 18, or a pharmaceutically acceptable salt or solvate thereof, wherein R 8 and R 9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo.
  • Embodiment 25 The compound of Embodiment 24, or a pharmaceutically acceptable salt or solvate thereof, wherein R 8 and R 9 taken together with the nitrogen atom to which they are attached form:
  • Embodiment 26 The compound of any one of Embodiments 1-25, or a pharmaceutically acceptable salt or solvate thereof, wherein R 2 is -OR 7 .
  • Embodiment 27 The compound of Embodiment 26, or a pharmaceutically acceptable salt or solvate thereof, wherein R 7 is selected from the group consisting of methyl, ethyl, and propyl.
  • Embodiment 28 The compound of Embodiment 27, or a pharmaceutically acceptable salt or solvate thereof, wherein R 7 is ethyl.
  • Embodiment 29 The compound of any one of Embodiments 1-28, or a pharmaceutically acceptable salt or solvate thereof, wherein R 3 is selected from the group consisting of hydrogen and C 1 -C 6 alkyl.
  • Embodiment 30 The compound of Embodiment 29, or a pharmaceutically acceptable salt or solvate thereof, wherein R 3 is hydrogen.
  • Embodiment 31 The compound of Embodiment 29, or a pharmaceutically acceptable salt or solvate thereof, wherein R 3 is methyl.
  • Embodiment 32 The compound of Embodiment 29, or a pharmaceutically acceptable salt or solvate thereof, wherein R 3 is ethyl.
  • Embodiment 33 The compound of any one of Embodiments 1-32, or a pharmaceutically acceptable salt or solvate thereof, wherein R 4 is selected from the group consisting of hydrogen and cyano.
  • Embodiment 36 The compound of any one of Embodiments 3-10 or 15-35, or a pharmaceutically acceptable salt or solvate thereof, wherein R 6a is selected from the group consisting of hydrogen and halogen.
  • Embodiment 37 The compound of Embodiment 36, or a pharmaceutically acceptable salt or solvate thereof, wherein R 6a is hydrogen.
  • R 6b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and C 3 -C 6 cycloalkyl
  • R 6C is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl; C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, (optionally substituted heterocyclo)-O-, (aryl)alkyl-O-, and (heteroaryl)alkyl-O-.
  • Embodiment 3 The method of Embodiment 2, wherein the compound having Formula (I) is a compound having Formula (III): or a pharmaceutically acceptable salt or solvate thereof, wherein R 5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl.
  • Embodiment 5 The method of Embodiment 3, wherein the compound having Formula (III) is a compound having Formula (IV): wherein R 5b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C2-C6 alkenyl, C 3 -C 6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo.
  • R 5b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C2-C6 alkenyl, C 3 -C 6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo.
  • Embodiment 6 The method of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (V): [0559] Embodiment 7. The method of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (VI):
  • Embodiment 8 The method of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (VII): (VII).
  • Embodiment 9 The method of any one of Embodiments 1-8, wherein R la is hydrogen.
  • Embodiment 17 The method of Embodiment 16, wherein R 7 is methyl, ethyl, or propyl.
  • Embodiment 19 The method of any one of Embodiments 1-18, wherein R 3 is hydrogen or C 1 -C 6 alkyl.
  • Embodiment 23 The method of any one of Embodiments 1-22, wherein R 4 is hydrogen or cyano.
  • Embodiment 24 The method of Embodiment 23, wherein R 4 is hydrogen.
  • Embodiment 27 The method of Embodiment 26, wherein R 6a is hydrogen.
  • Embodiment 28 The method of Embodiment 26, wherein R 6a is chloro.
  • Embodiment 29 The method of Embodiment 1, selected from any one or more of the compounds of Table 1 A.
  • Embodiment 30 The method of Embodiment 29, or a pharmaceutically acceptable salt or solvate thereof, selected from any one or more of:
  • Embodiment 31 The method of any one of Embodiments 1-30, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies, one or more anti-PD-Ll antibodies, one or more anti-CTLA-4 antibodies, one or more anti-LAG3 antibodies, one or more anti-TIM3 antibodies, one or more anti- VISTA antibodies, one or more anti-TIGIT antibodies, or one or more anti-cd47 antibodies, or a combination thereof.
  • Embodiment 38 The method of any one of Embodiments 31-37, wherein the one or more immune checkpoint inhibitors comprise one or more anti-LAG3 antibodies.
  • Embodiment 40 The method of any one of Embodiments 31-39 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIM3 antibodies.
  • Embodiment 42 The method of any one of Embodiments 31-41 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIGIT antibodies.
  • Embodiment 43 The method of any one of Embodiments 31-42 wherein the one or more immune checkpoint inhibitors comprise one or more anti-cd47 antibodies.
  • Embodiment 44 The method of Embodiment 31 comprising administering a therapeutically effective amount of nivolumab, pembrolizumab, dostarlimab, retifanlimab, or cemiplimab to the subject.
  • Embodiment 45 The method of Embodiments 31 or 44 comprising administering a therapeutically effective amount of avelumab, atezolizumab, or durvalumab to the subject.
  • Embodiment 46 The method of any one of Embodiments 1-45, wherein the cancer is any one or more of the cancers of Table 2 and/or Table 3.
  • Embodiments 1-46 the kit comprising: (i) the compound pharmaceutically acceptable salt or solvate thereof; and (ii) a label with instructions for how to use the kit.
  • Embodiment 48 A kit for carrying out the method of any one of Embodiments 1-46, the kit comprising: (i) one or more immune checkpoint inhibitors; and (ii) a label with instructions for how to use the kit.
  • R la is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR 8 R 9 ,;
  • R 8 and R 9 are each independently selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, and (C 1 -C 6 alkoxy)C 1 -C 6 alkyl; or
  • R 8 and R 9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo;
  • R 2 is selected from the group consisting of hydrogen, halo, C 1 -C 6 alkyl, and -OR 7 ;
  • R 7 is selected from the group consisting of C 1 -C 6 alkyl, optionally substituted C3-
  • R 3 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, and C 3 -C 6 cycloalkyl;
  • R 5 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted heterocyclo, optionally substituted aryl, and optionally substituted heteroaryl,
  • R 6b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and C 3 -C 6 cycloalkyl;
  • Embodiment 3 The compound for use of Embodiment 2, wherein the compound having Formula (I) is a compound having Formula (III): (III), or a pharmaceutically acceptable salt or solvate thereof, wherein R 5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl.
  • Embodiment 4 The compound for use of Embodiment 3, wherein R 5a is optionally substituted 5-membered heteroaryl.
  • Embodiment 5 The compound for use of Embodiment 3, wherein the compound having Formula (III) is a compound having Formula (IV): wherein R 5b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C2-C6 alkenyl, C 3 -C 6 cycloalkyl, and optionally substituted 5- or 6-membered heterocyclo.
  • R 5b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C2-C6 alkenyl, C 3 -C 6 cycloalkyl, and optionally substituted 5- or 6-membered heterocyclo.
  • Embodiment 6 The compound for use of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (V):
  • Embodiment 8 The compound for use of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (VII): (VII).
  • Embodiment 9 The compound for use of any one of Embodiments 1-8, wherein R la is hydrogen.
  • Embodiment 10 The compound for use of any one of Embodiments 1-8, wherein R la is C1-C4 alkyl.
  • Embodiment 11 The compound for use of any one of Embodiments 1-8, wherein R la is -CH2NR 8 R 9 .
  • R 9 are C 1 -C 6 alkyl.
  • Embodiment 13 The compound for use of Embodiment 12, wherein R 8 and
  • R 9 are methyl.
  • R 9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo.
  • Embodiment 15 The compound for use of Embodiment 14, wherein R 8 and R 9 taken together with the nitrogen atom to which they are attached form:
  • Embodiment 16 The compound for use of any one of Embodiments 1-15, wherein R 2 is -OR 7 .
  • Embodiment 17 The compound for use of Embodiment 16, wherein R 7 is methyl, ethyl, or propyl.
  • Embodiment 18 The compound for use of Embodiment 17, wherein R 7 is ethyl.
  • Embodiment 19 The compound for use of any one of Embodiments 1-18, wherein R 3 is hydrogen or C 1 -C 6 alkyl.
  • Embodiment 20 The compound for use of Embodiment 19, wherein R 3 is hydrogen.
  • Embodiment 21 The compound for use of Embodiment 19, wherein R 3 is methyl.
  • Embodiment 22 The compound for use of Embodiment 19, wherein R 3 is ethyl.
  • Embodiment 23 The compound for use of any one of Embodiments 1-22, wherein R 4 is hydrogen or cyano.
  • Embodiment 24 The compound for use of Embodiment 23, wherein R 4 is hydrogen.
  • Embodiment 25 The compound for use of Embodiment 23, wherein R 4 is cyano.
  • Embodiment 26 The compound for use of any one of Embodiments 2-25, wherein R 6a is hydrogen or halogen.
  • Embodiment 28 The compound for use of Embodiment 26, wherein R 6a is chloro.
  • Embodiment 30 The compound for use of Embodiment 29, or a pharmaceutically acceptable salt or solvate thereof, selected from any one or more of:
  • Embodiment 36 The compound for use of any one of Embodiments 31-35, wherein the one or more immune checkpoint inhibitors comprise one or more anti- CTLA-4 antibodies.
  • Embodiment 37 The compound for use of Embodiment 36, wherein the one or more anti-CTLA-4 antibodies comprise ipilimumab and/or tremelimumab.
  • Embodiment 38 The compound for use of any one of Embodiments 31-37, wherein the one or more immune checkpoint inhibitors comprise one or more anti-LAG3 antibodies.
  • Embodiment 39 The compound for use of Embodiment 38, wherein the one or more anti-LAG3 antibodies comprise relatlimab.
  • Embodiment 41 The compound for use of any one of Embodiments 31-40 wherein the one or more immune checkpoint inhibitors comprise one or more anti- VISTA antibodies.
  • Embodiment 43 The compound for use of any one of Embodiments 31-42 wherein the one or more immune checkpoint inhibitors comprise one or more anti-cd47 antibodies.
  • Embodiment 44 The compound for use of Embodiment 31 comprising administering a therapeutically effective amount of nivolumab, pembrolizumab, dostarlimab, retifanlimab, or cemiplimab to the subject.
  • Embodiment 45 The compound for use of Embodiments 31 or 44 comprising administering a therapeutically effective amount of avelumab, atezolizumab, or durvalumab to the subject.
  • Embodiment 46 The compound for use of any one of Embodiments 1-45, wherein the cancer is any one or more of the cancers of Table 2 and/or Table 3.
  • Embodiment 1 Use of a compound having Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • R la is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR 8 R 9 ,;
  • R lb is selected from the group consisting of hydrogen and C 1 -C 6 alkyl
  • R 8 and R 9 are each independently selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, and (C 1 -C 6 alkoxy)C 1 -C 6 alkyl; or
  • R 8 and R 9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo;
  • R 2 is selected from the group consisting of hydrogen, halo, C 1 -C 6 alkyl, and -OR 7 ;
  • R 7 is selected from the group consisting of C 1 -C 6 alkyl, optionally substituted C3-
  • R 3 is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, and C 3 -C 6 cycloalkyl;
  • Embodiment ! The use of Embodiment 1, wherein the compound having Formula (I) is a compound having Formula (II): (ID, or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • R 6a is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and C 3 -C 6 cycloalkyl;
  • R 6b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and C 3 -C 6 cycloalkyl;
  • R 6C is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl; C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl, (optionally substituted heterocyclo)-O-, (aryl)alkyl-O-, and (heteroaryl)alkyl-O-.
  • Embodiment 3 The use of Embodiment 2, wherein the compound having Formula (I) is a compound having Formula (III): or a pharmaceutically acceptable salt or solvate thereof, wherein R 5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl.
  • Embodiment 4 The use of Embodiment 3, wherein R 5a is optionally substituted 5-membered heteroaryl.
  • Embodiment 5 The use of Embodiment 3, wherein the compound having
  • Formula (III) is a compound having Formula (IV): wherein R 5b is selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C2-C6 alkenyl, C 3 -C 6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo.
  • Embodiment 6 The use of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (V):
  • Embodiment 7 The use of Embodiment 5, wherein the compound having
  • Formula (IV) is a compound having Formula (VI): (VI).
  • Embodiment 8 The use of Embodiment 5, wherein the compound having
  • Formula (IV) is a compound having Formula (VII): (VII).
  • Embodiment 9 The use of any one of Embodiments 1-8, wherein R la is hydrogen.
  • Embodiment 10 The use of any one of Embodiments 1-8, wherein R la is Ci-
  • Embodiment 11 The use of any one of Embodiments 1-8, wherein R la is -CH 2 NR 8 R 9 .
  • Embodiment 12 The use of Embodiment 11, wherein R 8 and R 9 are C 1 -C 6 alkyl.
  • Embodiment 13 The use of Embodiment 12, wherein R 8 and R 9 are methyl.
  • Embodiment 14 The use of Embodiment 11, wherein R 8 and R 9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo.
  • Embodiment 15 The use of Embodiment 14, wherein R 8 and R 9 taken together with the nitrogen atom to which they are attached form:
  • Embodiment 16 The use of any one of Embodiments 1-15, wherein R 2 is -
  • Embodiment 17 The use of Embodiment 16, wherein R 7 is methyl, ethyl, or propyl.
  • Embodiment 18 The use of Embodiment 17, wherein R 7 is ethyl.
  • Embodiment 19 The use of any one of Embodiments 1-18, wherein R 3 is hydrogen or C 1 -C 6 alkyl.
  • Embodiment 20 The use of Embodiment 19, wherein R 3 is hydrogen.
  • Embodiment 21 The use of Embodiment 19, wherein R 3 is methyl.
  • Embodiment 22 The use of Embodiment 19, wherein R 3 is ethyl.
  • Embodiment 23 The use of any one of Embodiments 1-22, wherein R 4 is hydrogen or cyano.
  • Embodiment 24 The use of Embodiment 23, wherein R 4 is hydrogen.
  • Embodiment 25 The use of Embodiment 23, wherein R 4 is cyano.
  • Embodiment 26 The use of any one of Embodiments 2-25, wherein R 6a is hydrogen or halogen.
  • Embodiment 27 The use of Embodiment 26, wherein R 6a is hydrogen.
  • Embodiment 28 The use of Embodiment 26, wherein R 6a is chloro.
  • Embodiment 29 The use of Embodiment 1, selected from any one or more of the compounds of Table 1A.
  • Embodiment 32 The use of Embodiment 31 wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies.
  • Embodiment 36 The use of any one of Embodiments 31-35, wherein the one or more immune checkpoint inhibitors comprise one or more anti-CTLA-4 antibodies.
  • CTLA-4 antibodies comprise ipilimumab and/or tremelimumab.
  • Embodiment 38 The use of any one of Embodiments 31-37, wherein the one or more immune checkpoint inhibitors comprise one or more anti-LAG3 antibodies.
  • Embodiment 39 The use of Embodiment 38, wherein the one or more anti-
  • LAG3 antibodies comprise relatlimab.
  • Embodiment 40 The use of any one of Embodiments 31-39 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIM3 antibodies.
  • Embodiment 43 The use of any one of Embodiments 31-42 wherein the one or more immune checkpoint inhibitors comprise one or more anti-cd47 antibodies.
  • Embodiment 44 The use of Embodiment 31 comprising administering a therapeutically effective amount of nivolumab, pembrolizumab, dostarlimab, retifanlimab, or cemiplimab to the subject.
  • Embodiment 45 The use of Embodiments 31 or 44 comprising administering a therapeutically effective amount of avelumab, atezolizumab, or durvalumab to the subject.
  • halo or halogen as used herein by itself or as part of another group refers to -Cl, -F, -Br, or -I.
  • alkyl refers to a straight- or branched-chain aliphatic hydrocarbon containing one to twelve carbon atoms, i.e., a C1-C12 alkyl, or the number of carbon atoms designated, e.g., a Ci alkyl such as methyl, a C2 alkyl such as ethyl, etc.
  • the alkyl is a C1-C10 alkyl.
  • the alkyl is a C 1 -C 6 alkyl.
  • the alkyl is a C1-C4 alkyl.
  • alkynyl refers to an alkyl group containing one or two carbon-to-carbon triple bonds.
  • the alkynyl is a C2-C6 alkynyl.
  • the alkynyl is a C2-C4 alkynyl.
  • the alkynyl has one carbon-to-carbon triple bond.
  • Non-limiting exemplary alkynyl groups include ethynyl, propynyl, butynyl, 2-butynyl, pentynyl, and hexynyl groups.
  • Non-limiting exemplary haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3,3-trifluoropropyl, 4,4,4-trifluorobutyl, and trichloromethyl groups.
  • Non-limiting exemplary (hydroxyl) alkyl groups include hydroxymethyl, hydroxyethyl, hydroxypropyl and hydroxybutyl groups, such as 1 -hydroxy ethyl, 2-hydroxyethyl, 1,2-dihydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 3-hydroxybutyl, 4-hydroxybutyl, 2-hydroxy-l- methylpropyl, and l,3-dihydroxyprop-2-yl.
  • haloalkoxy refers to a haloalkyl group attached to a terminal oxygen atom.
  • the haloalkyl group is a C 1 -C 6 haloalkyl.
  • the haloalkyl group is a C1-C4 haloalkyl group.
  • Non-limiting exemplary haloalkoxy groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.
  • the "(alkoxy)alkyl” is a (C 1 -C 6 alkoxy)C 1 -C 6 alkyl;
  • Non-limiting exemplary alkoxyalkyl groups include methoxymethyl, methoxyethyl, methoxypropyl, methoxybutyl, ethoxymethyl, ethoxyethyl, ethoxypropyl, ethoxybutyl, propoxymethyl, iso-propoxymethyl, propoxyethyl, propoxypropyl, butoxymethyl, tert-butoxymethyl, isobutoxymethyl, secbutoxymethyl, and pentyloxy methyl.
  • cycloalkyl refers to saturated and partially unsaturated, e.g., containing one or two double bonds, monocyclic, bicyclic, or tricyclic aliphatic hydrocarbons containing three to twelve carbon atoms, i.e., a C3-12 cycloalkyl, or the number of carbons designated, e.g., a C3 cycloalkyl such a cyclopropyl, a C4 cycloalkyl such as cyclobutyl, etc.
  • the cycloalkyl is bicyclic, i.e., it has two rings.
  • the cycloalkyl is monocyclic, i.e., it has one ring.
  • the cycloalkyl is a C3-8 cycloalkyl.
  • the cycloalkyl is a C3-6 cycloalkyl, i.e., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
  • the cycloalkyl is a C5 cycloalkyl, i.e., cyclopentyl or cyclopentenyl.
  • the cycloalkyl is a Ce cycloalkyl, i.e., cyclohexyl or cyclohexenyl.
  • Non-limiting exemplary C3-12 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbomyl, decalin, adamantyl, cyclohexenyl, and spiro[3.3]heptane.
  • optionally substituted cycloalkyl refers to a cycloalkyl group that is either unsubstituted or substituted with one, two, or three substituents, wherein each substituent is independently halo, nitro, cyano, hydroxy, amino haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, or alkoxyalkyl.
  • optionally substituted cycloalkyl also includes cycloalkyl groups having fused optionally substituted aryl or optionally substituted heteroaryl groups such as:
  • (cycloalkyl)alkyl refers to an alkyl substituted with one optionally substituted cycloalkyl group.
  • the (cycloalkyl)alkyl group is a C 1 -C 6 alkyl substituted with one optionally substituted C 3 -C 6 cycloalkyl group, i.e., an "(optionally substituted C 3 -C 6 cycloalkyl)Ci- C ⁇ > alkyl.”
  • the (cycloalkyl)alkyl group is a C1-C4 alkyl substituted with one optionally substituted optionally substituted C 3 -C 6 cycloalkyl, i.e., an "(optionally substituted C 3 -C 6 cycloalkyl)Ci-C4 alkyl.”
  • the (cycloalkyl)alkyl group is a C1-C2 alkyl substituted
  • heterocyclo refers to saturated and partially unsaturated, e.g., containing one or two double bonds, monocyclic, bicyclic, e.g., spirocyclic, or tricyclic groups containing three to eighteen ring members, i.e., a 3- to 18-membered heterocyclo, comprising one, two, three, or four heteroatoms, or the number of ring atoms designated.
  • Each heteroatom is independently oxygen, sulfur, or nitrogen.
  • heterocyclo also includes groups having fused optionally substituted aryl or optionally substituted heteroaryl groups such as indoline, indolin-2-one, 2,3-dihydro- lH-pyrrolo[2,3-c]pyridine, 2,3,4,5-tetrahydro-lH-benzo[d]azepine, or 1,3,4,5-tetrahydro- 2H-benzo [d] azepin-2-one .
  • the heterocyclo group is a 8- to 12-membered cyclic group containing two rings and one or two nitrogen atoms.
  • the heterocyclo can be linked to the rest of the molecule through any available carbon or nitrogen atom.
  • Non-limiting exemplary heterocyclo groups include:
  • optionally substituted heterocyclo refers to a heterocyclo group that is either unsubstituted or substituted with one to four substituents, wherein each substituent is independently halo, nitro, cyano, hydroxy, amino haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, or alkoxyalkyl. Substitution may occur on any available carbon or nitrogen atom of the heterocyclo group.
  • (heterocyclo)alkyl refers to an alkyl substituted with one optionally substituted heterocyclo group.
  • the (heterocyclo)alkyl group is a C 1 -C 6 alkyl substituted with one optionally substituted 4- to 7-membered heterocyclo group, i.e., an "(optionally substituted 4- to 7-membered heterocyclo)C 1 -C 6 alkyl.”
  • the (heterocyclo)alkyl group is a C1-C4 alkyl substituted with one optionally substituted optionally substituted heterocyclo group, i.e., an "(optionally substituted heterocyclo group)Ci-C4 alkyl.”
  • the (heterocyclo group)alkyl group is a C1-C2 alkyl substituted with one optionally substituted heterocyclo group group, i.e., an "(optionally substituted
  • aryl refers to an aromatic ring system having six to fourteen carbon atoms, i.e., Ce-Cu aryl.
  • Non-limiting exemplary aryl groups include phenyl (abbreviated as "Ph"), naphthyl, phenanthryl, anthracyl, indenyl, azulenyl, biphenyl, biphenylenyl, and fluorenyl groups.
  • the aryl group is phenyl or naphthyl.
  • the aryl group is phenyl.
  • aryl that is either unsubstituted or substituted with one to five substituents, wherein the substituents are each independently halo, nitro, cyano, hydroxy, amino haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxyalkyl, aryloxy, aralkyloxy, hetero aryloxy, (heterocyclo)alkyloxy, heterocyclooxy, (aryl)alkyl, (heteroaryl)alkyl, or (hetero aryl) alkyloxy .
  • the optionally substituted aryl is an optionally substituted phenyl. In another embodiment, the optionally substituted phenyl has four substituents. In another embodiment, the optionally substituted phenyl has three substituents. In another embodiment, the optionally substituted phenyl has two substituents. In another embodiment, the optionally substituted phenyl has one substituent.
  • Non-limiting exemplary optionally substituted aryl groups include 2-methylphenyl, 2-methoxyphenyl,
  • optionally substituted aryl includes aryl groups having fused optionally substituted cycloalkyl groups and fused optionally substituted heterocyclo groups.
  • Non-limiting examples include: 2,3-dihydro- IH-inden-l-yl, 1,2,3,4-tetrahydronaphthalen-l-yl, l,3,4,5-tetrahydro-2H-benzo[c]azepin- 2-yl, 1,2,3,4-tetrahydroisoquinolin-l-yl, and 2-oxo-2,3,4,5-tetrahydro-lH- benzo [d] azepin- 1 -y 1.
  • heteroaryl refers to monocyclic and bicyclic aromatic ring systems having five to 14 fourteen ring members, i.e., a 5- to 14-membered heteroaryl, comprising one, two, three, or four heteroatoms.
  • Each heteroatom is independently oxygen, sulfur, or nitrogen.
  • the heteroaryl has three heteroatoms.
  • the heteroaryl has two heteroatoms.
  • the heteroaryl has one heteroatom.
  • the heteroaryl is a 5- to 10-membered heteroaryl.
  • the heteroaryl has 5 ring atoms, e.g., thienyl, a 5-membered heteroaryl having four carbon atoms and one sulfur atom.
  • the heteroaryl has 6 ring atoms, e.g., pyridyl, a 6-membered heteroaryl having five carbon atoms and one nitrogen atom.
  • Non-limiting exemplary heteroaryl groups include thienyl, benzo [b] thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, benzofuryl, pyranyl, isobenzofuranyl, benzooxazonyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, isoindolyl, 377-indolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, cinnolinyl, quinazolinyl, pteridinyl, 4a77-carbazolyl, carbazolyl, P-carboliny
  • the heteroaryl is chosen from thienyl (e.g., thien-2-yl and thien-3-yl), furyl (e.g., 2-furyl and 3-furyl), pyrrolyl (e.g., lH-pyrrol-2-yl and lH-pyrrol-3-yl), imidazolyl (e.g., 2H-imidazol-2-yl and 2H- imidazol-4-yl), pyrazolyl (e.g., lH-pyrazol-3-yl, lH-pyrazol-4-yl, and lH-pyrazol-5-yl), pyridyl (e.g., pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl), pyrimidinyl (e.g., pyrimidin-2- yl, pyrimidin-4-yl, and pyrimidin-5-yl), thienyl
  • optionally substituted heteroaryl refers to a heteroaryl that is either unsubstituted or substituted with one to four substituents, wherein the substituents are independently halo, nitro, cyano, hydroxy, amino haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxyalkyl, aryloxy, aralkyloxy, heteroaryloxy, (heterocyclo)alkyloxy, heterocyclooxy, (aryl)alkyl, (heteroaryl)alkyl, or (hetero aryl) alkyloxy .
  • the optionally substituted heteroaryl has two substituents. In another embodiment, the optionally substituted heteroaryl has one substituent. Any available carbon or nitrogen atom can be substituted.
  • (heteroaryl)alkyl refers to an alkyl substituted with one optionally substituted heteroaryl group.
  • the alkyl group is a C 1 -C 6 alkyl substituted with one optionally substituted 5- to 14-membered heteroaryl group, i.e., an "(optionally substituted 5- to 14-membered heteroaryl)C 1 -C 6 alkyl.”
  • the alkyl group is a C 1 -C 6 alkyl substituted with one optionally substituted 5- to 9-membered heteroaryl group, i.e., an "(optionally substituted 5- to 9-membered heteroaryl)C 1 -C 6 alkyl.”
  • the alkyl group is a C 1 -C 6 alkyl substituted with one optionally substituted 5- or 6-membered heteroaryl group, i.e., an "(optionally substituted 5- or
  • aralkyl or "(aryl)alkyl” as used herein by themselves or as part of another group refers to an alkyl substituted with one optionally substituted aryl group.
  • the aryl is an optionally substituted phenyl.
  • the alkyl is a C 1 -C 6 alkyl, i.e., an "(optionally substituted aryl)C 1 -C 6 alkyl.”
  • the alkyl is a C1-C4 alkyl, i.e., an "(optionally substituted aryl)Ci-C4 alkyl.”
  • the alkyl is a Ci or C2 alkyl, i.e., an "(optionally substituted aryl)Ci-C2 alkyl.”
  • the alkyl is a Ci or C2 alkyl and the optionally substituted aryl is an optionally substituted phenyl i.e., an "(optionally substituted phenyl)Ci-C2 alkyl.”
  • Non-limiting exemplary (aryl)alkyl groups include benzyl and phenethyl.
  • aryloxy refers to an optionally substituted aryl group attached to a terminal oxygen atom.
  • the optionally substituted aryl is an optionally substituted phenyl.
  • a non-limiting exemplary aryloxy group is PhO-.
  • heteroaryloxy as used herein by itself or as part of another group refers to an optionally substituted heteroaryl group attached to a terminal oxygen atom.
  • a non-limiting exemplary hetereo aryloxy group is:
  • heterocyclooxy refers to an optionally substituted heterocyclo group attached to a terminal oxygen atom.
  • the optionally substituted heterocyclo group is an optionally substituted 4- to 7-membered heterocyclo group and resulting heterocyclooxy is thus referred to as a "4- to 7-membered heterocyclooxy.”
  • Non-limiting exemplary heterocyclooxy groups include:
  • aralkyloxy refers to an aralkyl group attached to a terminal oxygen atom.
  • a non-limiting exemplary aralkyloxy group is PhCfTO-.
  • (heteroaryl)alkyloxy refers to an (heteroaryl)alkyl group attached to a terminal oxygen atom.
  • Non-limiting exemplary (hetero aryl) alkyloxy groups include:
  • (heterocyclo)alkyloxy refers to an (heterocyclo)alkyl group attached to a terminal oxygen atom.
  • Non-limiting exemplary (hetero aryl) alkyloxy groups include:
  • amino refers to a radical of the formula -NR aa R bb , wherein R aa and R bb are independently hydrogen, optionally substituted alkyl, haloalkyl, (hydroxy)alkyl, (alkoxy)alkyl, (amino)alkyl, heteroalkyl, optionally substituted cycloalkyl, optionally substituted heterocyclo, optionally substituted aryl, optionally substituted heteroaryl, (aryl)alkyl, (cycloalkyl)alkyl, (heterocyclo)alkyl, or (heteroaryl)alkyl.
  • the amino is -NH2.
  • the amino is an "alkylamino," i.e., an amino group wherein R aa is C1-6 alkyl and R bb is hydrogen. In one embodiment, R aa is C1-C4 alkyl.
  • Non-limiting exemplary alkylamino groups include -N(H)CH3 and -N(H)CH2CH3.
  • the amino is a "dialkylamino," i.e., an amino group wherein R aa and R bb are each independently C1-6 alkyl.
  • R 55a and R 55b are each independently C1-C4 alkyl.
  • Non-limiting exemplary dialkylamino groups include -N(CH 3 ) 2 and -N(CH3)CH 2 CH(CH 3 )2.
  • a disease or condition wherein ADAR1 inhibition provides a benefit pertains to a disease or condition in which AD ARI is important or necessary, e.g., for the onset, progress, expression of that disease or condition, or a disease or a condition which is known to be treated by an ADAR1 inhibitor.
  • AD ARI is important or necessary, e.g., for the onset, progress, expression of that disease or condition, or a disease or a condition which is known to be treated by an ADAR1 inhibitor.
  • Examples of such conditions include, but are not limited to, a cancer.
  • One of ordinary skill in the art is readily able to determine whether a compound treats a disease or condition mediated by an ADAR1 inhibitor for any particular cell type, for example, by assays which conveniently can be used to assess the activity of particular compounds.
  • ADAR1 inhibitors and can be used in treating or preventing diseases and conditions wherein ADAR1 inhibition provides a benefit.
  • the terms “treat,” “treating,” “treatment,” and the like refer to eliminating, reducing, or ameliorating a disease or condition, e.g., cancer, and/or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated.
  • the term “treat” and synonyms contemplate administering a therapeutically effective amount of a Compound of the Disclosure and optionally an immune checkpoint inhibitor to a subject need of such treatment.
  • the treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, be oriented over a medium term, or can be a long-term treatment, for example within the context of a maintenance therapy.
  • prevent refers to a method of preventing the onset of a disease or condition and/or its attendant symptoms or barring a subject from acquiring a disease.
  • prevent also include delaying the onset of a disease and/or its attendant symptoms and reducing a subject's risk of acquiring a disease.
  • prevent may include “prophylactic treatment,” which refers to reducing the probability of redeveloping a disease or condition, or of a recurrence of a previously- controlled disease or condition, in a subject who does not have, but is at risk of or is susceptible to, redeveloping a disease or condition or a recurrence of the disease or condition.
  • terapéuticaally effective amount refers to an amount of the active ingredient(s) that is(are) sufficient, when administered by a method of the present disclosure, to efficaciously deliver the active ingredient(s) for the treatment of condition or disease of interest to a subject in need thereof.
  • the therapeutically effective amount of the agent may reduce (i.e., retard to some extent or stop) unwanted cellular proliferation; reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., retard to some extent or stop) cancer cell infiltration into peripheral organs; inhibit (i.e., retard to some extent or stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve, to some extent, one or more of the symptoms associated with the cancer.
  • the administered compound or composition prevents growth and/or kills existing cancer cells, it may be cytostatic and/or cytotoxic.
  • the term "container” means any receptacle and closure therefore suitable for storing, shipping, dispensing, and/or handling a pharmaceutical product.
  • the term "insert” means information accompanying a pharmaceutical product that provides a description of how to administer the product, along with the safety and efficacy data required to allow the physician, pharmacist, and patient to make an informed decision regarding use of the product.
  • the package insert generally is regarded as the "label" for a pharmaceutical product.
  • a Compound of the Disclosure and the second therapeutic agent are not administered concurrently, it is understood that they can be administered in any order to a subject in need thereof.
  • a Compound of the Disclosure can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent treatment modality, e.g., an ICI, to a subject in need thereof.
  • a second therapeutic agent treatment modality e.g., an ICI
  • a Compound of the Disclosure and the second therapeutic agent are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart.
  • the components of the combination therapies are administered at about 1 minute to about 24 hours apart.
  • the present disclosure encompasses any Compound of the Disclosure being isotopically-labelled (i.e., radiolabeled) by having one or more atoms replaced by an atom having a different atomic mass or mass number.
  • isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2 H (or deuterium (D)), 3 H, nC, 13 C, 14 C, 15 N, 18 O, 17 0, 31 P, 32 P, 35 S, 18 F, and 36 C1, respectively, e.g., 3 H, n C, and 14 C.
  • compositions wherein substantially all of the atoms at a position within the Compound of the Disclosure are replaced by an atom having a different atomic mass or mass number.
  • Isotopically-labelled Compounds of the Disclosure can be prepared by methods known in the art.
  • Compounds of the Disclosure may contain one or more asymmetric carbon atoms and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms.
  • the present disclosure encompasses the use of all such possible forms, as well as their racemic and resolved forms and mixtures thereof.
  • the individual enantiomers can be separated according to methods known in the art in view of the present disclosure.
  • the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that they include both E and Z geometric isomers. All tautomers are also encompassed by the present disclosure. All conformational isomers, i.e., stereoisomers produced by rotation about a c bond, are also encompassed by the present disclosure.
  • stereoisomers is a general term for all isomers of individual molecules that differ only in the orientation of their atoms in space. It includes enantiomers and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereomers).
  • chiral center or "asymmetric carbon atom” refers to a carbon atom to which four different groups are attached.
  • enantiomer and “enantiomeric” refer to a molecule that cannot be superimposed on its mirror image and hence is optically active wherein the enantiomer rotates the plane of polarized light in one direction and its mirror image compound rotates the plane of polarized light in the opposite direction.
  • racemic refers to a mixture of equal parts of enantiomers and which mixture is optically inactive.
  • Compounds of the Disclosure are racemic.
  • absolute configuration refers to the spatial arrangement of the atoms of a chiral molecular entity (or group) and its stereochemical description, e.g., R or S.
  • enantiomeric excess or "ee” refers to a measure for how much of one enantiomer is present compared to the other.
  • percent enantiomeric excess is defined as
  • *100, where R and 5 are the respective mole or weight fractions of enantiomers in a mixture such that R + 5 1.
  • the term “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other.
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
  • the term “and/or” as used in a phrase such as "A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).
  • the present disclosure encompasses the preparation and use of salts of Compounds of the Disclosure.
  • Salts of Compounds of the Disclosure can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation.
  • Salts of Compounds of the Disclosure can be acid addition salts formed with acceptable acids. Examples of acids which can be employed to form salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric.
  • available amino groups present in the compounds of the disclosure can be quatemized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides.
  • any reference to Compounds of the Disclosure appearing herein is intended to include Compounds of the Disclosure as well as salts, hydrates, or solvates thereof.
  • solvate as used herein is a combination, physical association and/or solvation of a compound of the present disclosure with a solvent molecule such as, e.g., a disolvate, monosolvate or hemisolvate, where the ratio of solvent molecule to compound of the present disclosure is about 2:1, about 1:1 or about 1:2, respectively.
  • a solvent molecule such as, e.g., a disolvate, monosolvate or hemisolvate
  • This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding.
  • the solvate can be isolated, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid.
  • a typical, non-limiting, process of preparing a solvate would involve dissolving a Compound of the Disclosure in a desired solvent (organic, water, or a mixture thereof) at temperatures above 20°C to about 25°C, then cooling the solution at a rate sufficient to form crystals, and isolating the crystals by known methods, e.g., filtration.
  • Analytical techniques such as infrared spectroscopy can be used to confirm the presence of the solvent in a crystal of the solvate.
  • Neratinib or Neratinib maleate is administered to a subject in a daily dose of 240 mg or less, e.g., 200 mg or less, 160 mg or less, or 120 mg or less. In some embodiments, Neratinib or Neratinib maleate is administered to a subject in a daily dose of 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, or 110 mg.
  • the mobile phase was a gradient of solvent A (H2O with 0.1% TFA) and solvent B (CH3CN with 0.1% of TFA) at a flow rate of 60 mL/min and 1%/min increase of solvent B. All final compounds have purity >95% as determined by Waters ACQUITY ultraperformance liquid chromatograph (UPLC) using reversed- phase column (SunFire, C18, 5 pm, 4.6 x 150 mm) and a solvent gradient of A (H2O with 0.1% of TFA) and solvent B (CH3CN with 0.1% of TFA). Electrospray ionization (ESI) mass spectral (MS) analysis was performed on a Thermo Scientific LCQ Fleet mass spectrometer.
  • ESI Electrospray ionization
  • Condition 1A of Step A.l Sodium hydride (60%, 5.69 mmol) in dry DMF (3 mL) under a nitrogen atmosphere and the resulting mixture is cooled in ice water. To above suspension is added a compound having Formula I (2.85 mmol) in anhydrous DMF (2 mL). 2-Chloro-l-fluoro-4-nitrobenzene (compound II) (2.85 mmol) was added to the above solution and the resulting solution was stirred at rt for 6 h. Cold water was added slowly to the reaction mixture, and a yellow precipitate resulted. The yellow solid is isolated by vacuum filtration, washed with cold water, and air dried to give the title compound as yellow solids.
  • Condition IB of Step A.2. A mixture of Formula III (2.51 mmol), iron (12.56 mmol) and NH4CI (25.11 mmol) in 70% ethanol in water was heated at 70 °C for 1 h. The reaction mixture was allowed to cool to rt, and then filtered over celite®. The filtrate was evaporated under vacuum giving an orange solid which was stirred with EtOAc, and then the resulting mixture was filtered. The filtrate was dried over anhydrous Na2SO4 and evaporated under vacuum to afford the desired compound IV as brown solid. The crude compound was used for the next step without further purification.
  • Condition 2B of Step A.2. A mixture of Formula III (2.51 mmol), and iron (12.56 mmol) in acetic acid (10 mF) was stirred at rt for 3 h. The reaction mixture was then filtered over celite® and washed with ethyl acetate. The filtrate was evaporated under vacuum giving an orange solid which was stirred with EtOAc and saturated NaHCOa. The ethyl acetate was separated, and the aqueous layer was extracted with ethyl acetate. The combined extracts were washed with brine and dried over anhydrous Na2SO4 and evaporated under vacuum to afford the desired compound having Formula IV
  • intermediate Compound VII (-Br and -Cl mixture) was isolated and treated with appropriate amine in the presence of CS2CO3 in acetonitrile at 60 °C for 2 h to afford desired compounds such as Cpd. No. 5 (Step C.2), Cpd. No. 7 (Step C.3), and Cpd. No. 8 (Step C.4) in higher yields.
  • NMP 4 vol.
  • tertbutyl (4-(methylamino)butyl)carbamate 1.1 eq.
  • Step D.3 A solution of Compound IX (1 eq.) in DMF (1 mL) was added at room temperature to a solution of acid Biotin (1 eq.) DIPEA (2 eq.) and HATU (1.1 eq.) in DMF (1 mL). The mixture was then stirred at room temperature for 12 h. The compound was purified by preparative HPLC to afford Cpd. No.
  • Step D.4 A solution of Compound IX (1 eq.) in DMF (1 mL) was added at room temperature to a solution of Biotin-PEG4-acid (1 eq.), DIPEA (2 eq.) and HATU (1.1 eq) in DMF (1 mL). The mixture was then stirred at room temperature for 12 h. The compound was purified by preparative HPLC to afford Cpd. No.
  • Step D.5 A solution of Compound IX (53.4 mg, 0.087 mmol) in DMF (0.5 mL) was added at room temperature to a solution of acid X (30 mg, 0.087 mmol) DIPEA (30.3
  • Step E.l and Step E.2 5-(5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4- d]imidazol-4-yl)pentanamido)pentanoic acid (Compound X).
  • Benzyl 5-aminopentanoate hydrogen chloride XI 49.9 mg, 0.205 mmol was added at room temperature to a solution of Biotin (50 mg, 0.205 mmol), DIPEA (71 pL, 0.409 mmol) and HATU (85.7 mg, 0.226 mmol) in DMF (1 mL).
  • Step 1 A mixture of N-(4-amino-2-ethoxyphenyl)acetamide (20.59 mmol), and ethyl (Z)-2-cyano-3-ethoxybut-2-enoate (26.77 mmol) in toluene was refluxed for 16 h. The reaction mixture was allowed to cool to rt, and then the reaction mixture was evaporated under vacuum giving a light brown solid (Yield - 95%) of ethyl (Z)-3-((4- acetamido-3-ethoxyphenyl)amino)-2-cyanobut-2-enoate which was used for the next step without further purification.
  • Step 2 To a solution of ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2- cyanobut-2-enoate (19.62 mmol) in Dowtherm was irradiated with micro wave (10 - 15V) at 270 °C for 30 min. The reaction mixture was allowed to cool to rt, and then the precipitated compound was triturated with n-hexane giving a light yellow solid (Yield - 90%) of N-(3-cyano-7-ethoxy-2-methyl-4-oxo-l,4-dihydroquinolin-6-yl)acetamide.
  • Step 3 To a solution of N-(3-cyano-7-ethoxy-2-methyl-4-oxo-l,4- dihydroquinolin-6-yl)acetamide (17.54 mmol) in 1, 4-dioxane (50 mL) was added POCI 3 (87.72 mmol) and then the resulting mixture was refluxed for 5 ⁇ 6 h.
  • Step 1 A mixture of A-(4-amino-2-ethoxyphenyl)acetamide (20.59 mmol), and ethyl (Z)-2-cyano-3-ethoxypent-2-enoate (26.77 mmol) in toluene was refluxed for 16 h. The reaction mixture was allowed to cool to rt, and then the reaction mixture was evaporated under vacuum giving a light brown solid (Yield - 97%) of ethyl (Z)-3-((4- acetamido-3-ethoxyphenyl)amino)-2-cyanopent-2-enoate which was used for the next step without further purification.
  • Step 2 To a solution of ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2- cyanopent-2-enoate (19.98 mmol) in Dowtherm was irradiated with microwave (10 - 15V) at 270 °C for 30 min. The reaction mixture was allowed to cool to rt, and then the precipitated compound was triturated with n-hexane giving a light yellow solid (Yield 84%) of N-(3-cyano-7-ethoxy-2-ethyl-4-oxo-l,4-dihydroquinolin-6-yl)acetamide.
  • Step 3 To a solution of N-(3-cyano-7-ethoxy-2-ethyl-4-oxo-l,4-dihydroquinolin- 6-yl)acetamide (16.70 mmol) in 1, 4-dioxane (50 mL) was added POCI3 (83.52 mmol) and then the resulting mixture was refluxed for 5 - 6 h.
  • Step 1 Methansulfonic acid (0.197 mmol) was added at room temperature to a suspension of N-(4-chloro-3-cyano-7-ethoxy-2-methylquinolin-6-yl)acetamide (0.066 mmol) and 3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3-yl)methoxy)aniline (0.079 mmol) in anhydrous ethyl alcohol. The mixture was then refluxed for 4 ⁇ 6 h.
  • Step 2 Synthesis of Cpd. No. 24: (E)-4-(dimethylainino)but-2-enoyl chloride hydrochloride (0.067 mmol) was added at 0 °C to a suspension of 6-amino-4-((3-chloro- 4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3-yl)methoxy)phenyl)amino)-7 -ethoxy-2- methylquinoline-3-carbonitrile (0.045 mmol) in N- methyl pyrrolidine (0.3 mL). The mixture was then allowed to ambient temperature and stirred for 2 h. The compound was purified by preparative HPLC to yield Cpd. No.
  • Step 1 Methansulfonic acid (0.189 mmol) was added at room temperature to a suspension of A-(4-chloro-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)acetamide (0.063 mmol) and 3-chloro-4-(pyridin-2-ylmethoxy)aniline (0.076 mmol) in anhydrous ethyl alcohol. The mixture was then refluxed for 4 - 6 h.
  • Step 2 Synthesis of Cpd. No. 27: (E)-A-(4-((3-chloro-4-((6-(tetrahydro-2H- pyran-4-yl)pyridin-3-yl)methoxy)phenyl)amino)-3-cyano-7-ethoxy-2-methylquinolin-6- yl)-4-(dimethylamino)but-2-enamide.
  • Step 1 Methansulfonic acid (0.197 mmol) was added at room temperature to a suspension of A-(4-chloro-3-cyano-7-ethoxy-2-methylquinolin-6-yl)acetamide (0.066 mmol) and 3-chloro-4-(pyridin-2-ylmethoxy)aniline (0.079 mmol) in anhydrous ethyl alcohol. The mixture was then refluxed for 4 - 6 h. The solution was cooled to ambient temperature, then concentrated in vacuo, and purified by preparative HPLC to afford 6- amino-4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7-ethoxy-2-methylquinoline-
  • Step 1 Sodium hydride (60%, 5.69 mmol) in dry DMF (3 mL) under a nitrogen atmosphere and the resulting mixture is cooled in ice water. To above suspension is added appropriate alcohol (2.85 mmol) in anhydrous DMF (2 mL). 2-Chloro-l-fluoro-4- nitrobenzene (2.85 mmol) was added to the above solution and the resulting solution was stirred at rt. Cold water was added slowly to the reaction mixture, and a yellow precipitate resulted. The yellow solid is isolated by vacuum filtration, washed with cold water, and air dried to give the title compound as yellow solid.
  • Step 2 A mixture of nitro compound (2.51 mmol), iron (12.56 mmol) and NH4CI (25.11 mmol) in 70% ethanol in water was heated at 70 °C for 1 h. The reaction mixture was allowed to cool to rt, and then filtered over celite®. The filtrate was evaporated under vacuum giving an orange solid which was stirred with EtOAc, and then the resulting mixture was filtered. The filtrate was dried over anhydrous Na2SO4 and evaporated under vacuum to afford the desired amine compound. The crude compound was used for the next step without further purification.
  • Step 3 Methansulfonic acid (1.035 mmol) was added at room temperature to a suspension of the corresponding acetamide (0.345 mmol) and the appropriate amine (0.345 mmol) in anhydrous ethyl alcohol. The mixture was then refluxed. The solution was cooled to ambient temperature, then concentrated in vacuo, and purified by preparative HPLC to yield the desired compounds.
  • Step 4 (E)-4-(Dimethylamino)but-2-enoyl chloride hydrochloride (0.067 mmol) was added at 0 °C to a suspension of the appropriate amine (0.045 mmol) in N-methylpyrrolidine. The mixture was then allowed to ambient temperature and stirred for ⁇ 2 h. The compound was purified by preparative HPLC to yield the desired compounds.

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Abstract

The present disclosure provides compounds represented by Formula (IX): and the pharmaceutically acceptable salts and/or solvates thereof, wherein R1, R2, R3, R4, R5, and X are as defined in the specification. Compounds having Formula (I) are adenosine deaminase acting on RNA 1 (ADAR1) inhibitors useful for the treatment of cancer and other diseases. Compounds having Formula (I) can be administered to a subject as a single agent or in combination with immune checkpoint inhibitors.

Description

SMALL-MOLECULE INHIBITORS OF AD ARI
STATEMENT REGARDING SEQUENCE LISTING
[0001] This application contains, as a separate part of the disclosure, a Sequence Listing in computer readable form (Filename: 2024194_SeqListing; Size: 127,902 bytes; Created January 8, 2025), which is incorporated by reference in its entirety.
BACKGROUND
Field
[0002] The present disclosure provides adenosine deaminase acting on RNA 1 (AD ARI) inhibitors. ADAR1 inhibitors are useful for treating cancer when administered to a subject as a single agent or in combination with one or more immune checkpoint inhibitors.
Background
[0003] Adenosine deaminase acting on RNA 1 (AD ARI) is an RNA editing enzyme that catalyzes the deamination of adenosine to inosine (A-to-I) in double-stranded RNAs (dsRNAs). There are two protein isoforms of AD ARI, the full-length AD ARI pl50 (150 kDa) and the shorter AD ARI pl 10 (110 kDa). AD ARI pl 10 is constitutively expressed in the nucleus, while ADAR1 pl50 is localized in the cytoplasm. Nishikura, Nat Rev Mol Cell Biol 17, 83-96 (2016). ADAR 1 -mediated A-to-I editing disrupts dsRNA structures and protects dsRNAs from dsRNA-sensing molecules, such as MDA5, thereby inhibiting the type-I and type-III-IFN-mediated response and innate immunity. Liddicoat et al., Science 349, 1115-1120 (2015). ADAR1 plays pivotal roles in viral infection (Pfaller et al., Annu Rev Virol 8, 239-264 (2021)), autoimmune disease (Chung, H. et al., Cell 172, 811-824 e814 (2018)), and cancer immunity. Ishizuka et al., Nature 565, 43-48 (2019); Gannon et al., Nat Commun 9, 5450 (2018); Liu et al., Nat Med 25, 95-102 (2019); Quin et al., Biochem Sci 46, 758-771 (2021). IFNy (type-II IFN) transcriptionally stimulates ADAR1 expression. Patterson et al., Mol Cell Biol 15, 5376-5388 (1995). However, the posttranslational modification (PTM), endogenous regulation, and pharmacological manipulation of AD ARI remain largely unknown. [0004] Active cancer metabolic reprogramming supplies energy and nutrient materials for tumor cell growth and fosters immunosuppression. Zou et al., Cell Metab 35, 1101-1113 (2023); O'Sullivan et al., Nat Rev Immunol 19, 324-335 (2019); Lim et al., Elife 9 (2020); Tsai et al., Cell Metab 35, 118-133 el 17 (2023); Arner et al., Cancer Cell 41, 421-433 (2023). Targeting cancer metabolism can potentiate immunotherapy response in tumor bearing animal models. Bader et al., Mol Cell 78, 1019-1033 (2020); Stine et al., Nat Rev Drug Discov 21, 141-162 (2022). It is posited and tested that metabolic restriction induces tumor regression and improves cancer outcome. Meynet et al., Trends Mol Med 20, 419-427 (2014); Vidoni et al., J Cancer Prev 26, 224-236 (2021). However, the underlying cellular and molecular mechanisms and the potential connection between tumor metabolic reprogramming and RNA-editing activity in the tumor microenvironment (TME) are largely unexplored. There exists a need in the art for safe and efficacious small molecule therapeutics that target cancer metabolism.
BRIEF SUMMARY
[0005] In one aspect, the present disclosure provides compounds represented by any one of Formulae (I)-(VII) or (IX)-(XVII), and the pharmaceutically acceptable salts and solvates thereof, and compounds of Table 1A and Table 2A, and the pharmaceutically acceptable salts and solvates thereof. These compounds, and the pharmaceutically acceptable salts and solvates thereof, are collectively referred to herein as "Compounds of the Disclosure" or individually as a "Compound of the Disclosure." Compounds of the Disclosure are adenosine deaminase acting on RNA 1 (ADAR1) inhibitors and are thus useful in treating diseases or conditions, e.g., cancer, wherein inhibition of ADAR1 provides a therapeutic benefit to a subject.
[0006] In another aspect, the present disclosure provides pharmaceutical compositions comprising a Compound of the Disclosure and a pharmaceutically acceptable excipient.
[0007] In another aspect, the present disclosure provides methods of treating cancer in a subject by administering a therapeutically effective amount of a Compound of the Disclosure to a subject in need thereof.
[0008] In another aspect, the present disclosure provides a Compound of the Disclosure for use in treating cancer in a subject in need thereof. [0009] In another aspect, the present disclosure provides the use of a Compound of the Disclosure in the manufacture of a medicament for treating cancer in a subject in need thereof.
[0010] In another aspect, the present disclosure provides methods of treating cancer in a subject by administering a therapeutically effective amount of a Compound of the Disclosure in combination with one or more immune checkpoint inhibitors to a subject in need thereof. Immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, anti-LAG3 antibodies, anti- TIM3 antibodies, anti-VISTA antibodies, anti-TIGIT antibodies, and anti-cd47 antibodies.
[0011] In another aspect, the present disclosure provides a Compound of the Disclosure for use in treating cancer in combination with one or more immune checkpoint inhibitors.
[0012] In another aspect, the present disclosure provides the use of a Compound of the Disclosure in the manufacture of a medicament for treating cancer in combination with one or more immune checkpoint inhibitors.
[0013] In another aspect, the present disclosure provides methods of inhibiting ADAR1 in a subject in need thereof by administering a therapeutically effective amount of a Compound of the Disclosure to the subject.
[0014] In another aspect, the present disclosure provides methods of sensitizing tumor response to immune checkpoint blockade (ICB) by administering a therapeutically effective amount of a Compound of the Disclosure to a subject in need thereof.
[0015] In another aspect, present disclosure provides kits for carrying out the methods of the present disclosure.
[0016] In another aspect, the present disclosure provides compounds represented by Formula (VIII), and the pharmaceutically acceptable salts and solvates thereof, and compounds of Table 1C, and the pharmaceutically acceptable salts and solvates thereof. These compounds are synthetic intermediates that can be used, for example, to prepare Compounds of the Disclosure.
[0017] In another aspect, the present disclosure provides compounds represented by any one of Formulae (XVIII)-(XXV), and the pharmaceutically acceptable salts and solvates thereof, and the compounds of Table 8, and the pharmaceutically acceptable salts and solvates thereof. These compounds are synthetic intermediates that can be used, for example, to prepare Compounds of the Disclosure. BRIEF DESCRIPTION OF DRAWINGS
[0018] FIGS, la-e are box plots showing the calculated by Z score in the indicated metabolic pathways (FIGS, la-d) and IFN signaling pathway (FIG. le) in each group (based on the immunotherapy responses, cancer patients receiving immune checkpoint blockade (ICB) were divided into CR (complete response), PR/SD (partial response/stable disease) and PD (progressive disease) groups). Nutrient metabolism (FIG. Id) was calculated by the sum of glucose (FIG. la), glutamine (FIG. lb), and fatty acids (FIG. 1c) metabolism, n = 50 patients, P values were determined by one-way ANOVA.
[0019] FIG. If is a diagram showing the IFN cycling and magnified IFN response during metabolic restriction.
[0020] FIGS. Ig-h are heat maps showing CHL1 (FIG. 1g) or B16 (FIG. Ih) cells treated with BPTES in the presence or absence of IFNy for 24 hours. Transcripts of the indicated IFN signaling genes were determined by qPCR. n = 3 biological independent samples.
[0021] FIG. li are four bar graphs showing B16 cells treated with the indicated conditions for 36 hours. Transcripts of the indicated IFN signaling genes were determined by qPCR. Data are mean ± SD. n = 3 biological independent samples. P values by 2-sided t-test.
[0022] FIG. Ij is a bar graph showing B16 cells carrying the indicated shRNAs treated with IFNy for 48 hours. IFN|3 levels in the culture medium were determined by ELISA. Data are mean ± SD. n = 3 biological independent samples.
[0023] FIG. Ik is a Venn diagram showing B16 cells treated with BPTES in the presence or absence of IFNy for 36 hours. RNA-seq was performed in these B16 cells. The overlapping of differentially expressed genes (DEGs) between groups is shown. The indicated IFN signaling genes were enriched upon BPTES plus IFNy treatment.
[0024] Fig 11 is a graph showing an ontology analysis performed with the differentially expressed genes between BPTES plus IFNy and IFNy treatments. Arrows indicate immune-related biological processes enriched in BPTES plus IFNy group compared with IFNy alone group.
[0025] Extended Data ("ED") FIG. la is a bar graph showing CHL1 cells treated with the indicated inhibitors in the presence or absence of IFNy for 48 hours. Cell surface expression of HLA-ABC was determined by FACS. Data are mean ± SD. n = 3 biological independent samples. [0026] ED FIG. lb is a bar graph showing CHL1 cells treated with the indicated concentrations of BPTES in the presence or absence of IFNy for 48 hours. Cell surface expression of HLA-ABC was determined by FACS. Data are mean ± SD. n = 3 biological independent samples. P values by one-way ANOVA.
[0027] ED FIG. 1c is six bar graphs showing human cancer cells (A2058, SW480, and CAOV3) or mouse cancer cells (B16, CT26, and ID8) treated with BPTES in the presence or absence of IFNy for 48 hours. Surface staining of MHC-I was determined by FACS. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0028] ED FIG. Id is a bar graph showing B16 cells carrying the indicated shRNAs were treated with IFNy for 48 hours. Surface expression of MHC-I (H2-Kb) was determined by FACS. Data are mean ± SD. n = 3 biological independent samples.
[0029] ED FIG. le is four bar graphs showing CHE1 cells treated with BPTES and/or IFNy for 36 hours. The MHC-I-related gene transcripts were determined by qPCR. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0030] ED FIGS. If-g is a Western blot image and graph, respectively, showing B16 control cells (shFluc) or metabolically restricted cells (shGdh, shPkm) treated with IFNy for the indicated hours. The indicated proteins were determined by Western blot (only 1 of 2 blots is shown). Surface expression of H2-Kb was determined by FACS, n = 3 biological independent samples.
[0031] ED FIG. Ih is two bar graphs showing B16 cells or mouse embryonic fibroblasts (MEF) treated with the indicated conditions for 24 hours. Ifiibl transcripts were determined by qPCR. Data are mean ± SD. n = 3 biological independent samples.
[0032] ED FIG. li is four bar graphs showing wild type (WT), type-I IFN receptor knock out (Ifnarl KO), or type-III IFN receptor knockout (Ifnlrl KO) B16 cells were treated with the indicated conditions for 36 hours. The indicated IFN signaling gene transcripts were determined by qPCR. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0033] ED FIG. Ij is seven bar graphs showing RNA-seq performed in B16 cells treated with BPTES in the presence or absence of IFNy for 40 hours. Transcripts per million (TPM) of the indicated IFN signaling genes are shown, n = 2 biological independent samples. [0034] ED FIGS. Ik-1 is volcano map or scatter plot, respectively, showing the differentially expressed genes between BPTES plus IFNy group and IFNy group.
[0035] ED FIG. Im is a graph showing the gene ontology analysis of the differentially expressed genes between BPTES plus IFNy group and BPTES group. Arrows indicate the enriched immune-related processes.
[0036] FIG. 2a is a schematic diagram showing the common signaling pathways that trigger IFN production.
[0037] FIG. 2b is a graph showing WT or the indicated KO B16 cells treated with BPTES and/ or IFNy for 24 hours. Ifnbl transcripts were determined by qPCR. n = 3 biological independent samples.
[0038] FIG. 2c is a graph showing Ifihl KO B16 cells carrying indicated shRNAs treated with IFNy for 48 hours. IFN|3 in the culture medium was determined by EEISA, n = 3 biological independent samples.
[0039] FIG. 2d is a schematic diagram showing the purification of cytosolic RNA samples and transfection into untreated cells.
[0040] FIGS. 2e-f is a dot plot and bar graph, respectively, showing B16 cells treated with BPTES and/or IFNy for 24 hours. Cytosolic RNA samples were incubated with/without RNase III and dot-plotted on membrane. DsRNA was determined by anti- dsRNA antibody (FIG. 2e). Cytosolic RNA samples were transfected into B16 cells for 12 hours. Ifnbl transcripts were determined by qPCR (FIG. 2f). Data are mean ± SD. n = 3 biological independent samples.
[0041] FIGS. 2g-h is a line graph and Kaplan-Meier curve, respectively, showing tumor growth curves and mouse overall survival. WT B16 cells carrying shFluc or shGdh inoculated into C57BE/6 mice. The mice were treated with IgG or anti-PD-El antibody, n = 5 mice per group. P values by two-sided t test.
[0042] FIGS. 2i-j is a line graph and Kaplan-Meier curve, respectively, showing tumor growth curves and mouse overall survival. Ifihl KO B16 cells carrying shFluc or shGdh were inoculated into C57BE/6 mice. The mice were treated with IgG or anti-PD-El antibody, n = 5 mice per group. P values by two-sided t test.
[0043] ED FIG. 2a is a Western blot showing WT or Ifihl KO B16 cells treated with
IFNy for 24 hours. Protein levels of the indicated genes were determined by Western blot.
1 of 2 blots is shown. [0044] ED FIG. 2b is a Western blot showing WT or Ifihl KO B16 cells were treated with the indicated concentrations of BPTES in the presence or absence of IFNy for 24 hours. Protein levels of the indicated genes were determined by Western blot. 1 of 2 blots is shown.
[0045] FIG. 3a is a schematic diagram showing the RNA editing of endogenous retrovirus (ERV) regulates MDA5 activation and IFN production.
[0046] FIG. 3b is a schematic diagram showing the construction of the RNA editing reporter (RE-Euc).
[0047] FIG. 3c is a bar graph showing B16 cells transfected with RE-Euc and treated with BPTES and/or IFNy for 36 hours. Relative luminescence was determined by dual luciferase reporter assay. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0048] FIG. 3d is a Western blot showing WT or Adar 1 KO B16 cells treated with IFNy for 24 hours. Protein levels of the indicated genes were determined by Western blot. Adarl pl50 and pl 10 were detected. 1 of 3 blots is shown.
[0049] FIGS. 3e-g are bar graphs showing WT or Adarl KO B16 cells carrying RE-Euc were treated with BPTES and/or IFNy. Relative luminescence (FIG. 3e) or Ifnbl transcripts (FIG. 3f) were determined at 24 hours. Surface expression of H2-Kb (FIG. 3g) was determined at 48 hours. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0050] FIG. 3h is a Western blot showing Adarl -overexpressing B16 cells (Adarl-OE) incubated with BPTES for 24 hours. Palmitoylated Adarl was determined by pulldown assay following Click-IT reaction. 1 of 2 blots is shown.
[0051] FIG. 3i is a Western blot showing B16 cell forced expression of Adarl incubated with BPTES and/or 2-BP for 24 hours. Palmitoylated Adarl was determined by pulldown assay following Click-IT reaction. 1 of 2 blots is shown.
[0052] FIG. 3j is a schematic diagram showing ADAR1 domains and putative palmitoylation sites. The amino acid sequence around Cysio8i/io82 on ADAR1 is conserved across multiple species.
[0053] FIG. 3k Adarl KO B16 cells stably transfected with wild type Adarl (WT) or Cysio8i/io82 mutant Adarl (C2A) and treated with BPTES for 24 hours. Palmitoylation of Adarl were detected by Click-IT assay. 1 of 2 blots is shown. [0054] FIG. 31 is a Western blot showing WT or C2A B16 cells transfected with RE-Luc and treated with increasing concentrations of BPTES for 24 hours. Relative luminescence was determined by dual luciferase reporter assay.
[0055] FIGS. 3m-n are bar graphs showing WT or C2A B16 cells were treated with BPTES in the presence or absence of IFNy. Ifnbl transcripts were determined at 24 hours (FIG. 3m). Surface expression of H2-Kb was determined at 48 hours (FIG. 3n). Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0056] FIG. 3o is a schematic diagram showing the purification of the Zinc binding proteins with Zn-NTA agarose.
[0057] FIGS. 3p-q is a Western blot and line graph, respectively, showing WT or C2A B16 cells treated with increasing concentrations of BPTES (0, 1, 2, 5, 10 pM) for 24 hours. The Zinc binding proteins were enriched by Zn-NTA beads via the pulldown assay. Adarl protein was detected in the pulldown products and input. 1 of 2 blots is shown (FIG. 3p). Relative Zinc affinity was calculated by the enrichment of Adarl protein upon Zn-NTA pulldown assay (FIG. 3q).
[0058] FIG. 3r is a schematic diagram showing S-palmitoylation regulates ADAR1 RNA editing activity.
[0059] ED FIG. 3a is a schematic diagram showing domains of the two ADAR1 isoforms.
[0060] ED FIG. 3b is a Western blot showing B16 cells treated with IFNy for indicated hours. Adarl protein was determined by Western blot. 1 of 3 blots is shown.
[0061] ED FIG. 3c is two bar graphs showing B16 cells transfected with RE-Luc and/ or Adarl -OE for 48 hours (left). WT or Adarl KO B16 cells were transfected with RE-Luc for 48 hours. Relative luminescence was determined by dual luciferase reporter assay. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0062] ED FIG. 3d is a Western blot showing B16 cells were treated with the indicated concentrations of BPTES in the presence or absence of IFNy for 24 hours. Adarl protein was determined by Western blot. 1 of 2 blots is shown.
[0063] ED FIG. 3e is an image of B16 cells carrying Adarl -OE treated with BPTES for 24 hours. Adarl protein was enriched by Co-IP with Flag antibody. Post-translational modifications were detected in the IP products. [0064] ED FIG. 3f is two bar graphs showing B16 cells treated with BPTES and/ or IFNy in the presence or absence of 2-BP. Ifnbl transcripts were determined at 24 hours. Surface expression of H2-Kb was determined at 48 hours.
[0065] ED FIG. 3g is a table showing predicted palmitoylation sites on AD ARI by CSS- Palm.
[0066] ED FIG. 3h is a Western blot showing B16 cells carrying Adarl treated with the increasing concentrations of BPTES (0, 1, 2, 5, 10 pM) in the presence or absence of 25pM 2-BP for 24 hours. Adarl protein was determined by pulldown assay with Zn- NTA.
[0067] FIG. 4a is a graph showing B16 cells treated with BPTES for 12 hours. The indicated gene transcripts were determined by qPCR. n = 3 biological independent samples.
[0068] FIG. 4b is bar graph showing B16 cells transfected with RE-Luc, PRL-SV40, and the indicated Zdhhc plasmids overnight and treated with IFNy for 24 hours. Relative luminescence was determined. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0069] FIG. 4c-d are bar graphs showing B16 cells ectopically expressed with the indicated Zdhhc genes overnight and treated with IFNy. Ifnbl transcripts were determined at 24 hours (FIG. 4c). Surface expression of H2-Kb was determined at 48 hours (FIG. 4d).
[0070] FIG. 4e is five bar graphs showing Zdhhc2-overexpressed B16 cells treated with IFNy for 36 hours. The indicated IFN signaling gene transcripts were determined by qPCR. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0071] FIG. 4f is a Western blot showing B16 cells treated with BPTES for the indicated hours. Zdhhc2 protein was determined by Western blot. 1 of 2 blots is shown.
[0072] FIG. 4g is a Western blot showing WT or Zdhhc2 KO B16 cells treated with BPTES at the indicated concentrations for 24 hours. Zdhhc2 protein was determined by Western blot. 1 of 2 blots is shown.
[0073] FIG. 4h is three bar graphs showing Zdhhc2 KO B16 cells treated with BPTES and/or IFNy for 36 hours. The indicated gene transcripts were determined by qPCR. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test. [0074] FIG. 4i is a Western blot showing Adar 1 -overexpressing B16 cells forced to express Zdhhc2 at different levels for 48 hours. Palmitoylation of Adarl was determined by Click-IT assay.
[0075] FIG. 4j-k are Western blots showing B16 cells forced to express Adarl (Flag- tagged) and/or Zdhhc2 (HA-tagged) for 48 hours. Co-Immunoprecipitation (Co-IP) was performed with antibody against HA (Fig, 4j) or Flag (FIG. 4k). The indicated proteins were determined in the IP products or input samples. 1 of 2 blots is shown.
[0076] FIG. 41 is an image showing B16 cells forced to express Adarl (Flag-tagged) and/or Zdhhc2 (HA-tagged) for 48 hours. Immunofluorescence staining was performed with antibody against HA (Alexa Fluor 488) or Flag (Alexa Fluor 647). 1 of 4 captures is shown.
[0077] FIG. 4m is a Western blot showing the indicated proteins determined in WT or Srebpl KO B16 cells.
[0078] FIG. 4n is three bar graphs showing WT or Srepbl KO B16 cells were treated with IFNy for 36 hours. The indicated gene transcripts were determined by qPCR. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0079] FIG. 4o is a schematic diagram showing metabolic restriction enables IFN signaling amplification through the SREBP1-ZDHHC2-ADAR1 axis.
[0080] ED FIG. 4a is a bar graph showing CHL1 cells transfected with the indicated shRNAs for 24 hours. ZDHHC2 transcripts were determined by qPCR. Data are mean ± SD. n = 3 biological independent samples.
[0081] ED FIG. 4b is an image showing ChlP-seq in GM78 cells with antibody against SREPB1 or SREPB2 shows the enrichment of SREBP1 but not SREBP2 in the ZDHHC2 promoter. The binding motif of SREBP1 in the human and mouse ZDHHC2 promoter is shown.
[0082] ED FIG. 4c is two bar graphs showing CHL1 cells treated with SREBP1 inhibitor (Fatostatin) or SREPB2 inhibitor (Betulin) at the indicated concentrations for 24 hours. ZDHHC2 transcripts were determined by qPCR. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0083] ED FIG. 4d is a Western blot showing Adarl expressing B16 cells treated with Fatostatin at the indicated concentrations for 24 hours. Palmitoylation of Adarl was determined by Click-IT assay. 1 of 2 blots is shown. [0084] ED FIG. 4e-g are bar graphs showing CHL1 cells were treated with the indicated concentrations of Fatostatin in the presence or absence of IFNy. RNA editing efficacy (ED FIG. 4e) and IFNB1 transcripts (ED FIG. 4f) were determined at 24 hours. Surface expression of HEA-ABC (ED FIG. 4g) was determined at 48 hours. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0085] ED FIG. 4h is a bar graph showing ZDHHC2 transcripts were determined by qPCR in WT or Srebpl KO B16 cells. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0086] ED FIG. 4i and 4j are bar graphs showing WT or Srebpl KO B16 cells treated with BPTES and/or IFNy. Ifnbl transcripts (ED FIG. 4i) were determined at 24 hours. Surface expression of H2-Db (ED FIG. 4j) were determined at 48 hours.
[0087] ED FIG. 4k is a schematic diagram showing the working model of SREBP1 and small activating RNAs (saRNAs) targeting the ZDHHC2 promoter.
[0088] ED FIG. 41 and 4m are three bar graphs and a Western blot, respectively, showing B16 cells transfected with Zdhhc2 saRNAs for 24 hours. Expression of the indicated genes was determined by qPCR (ED FIG. 41) or Western blot (ED FIG. 4m). Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test. 1 of 2 blots is shown.
[0089] ED FIG. 4n-o are bar graphs showing B16 cells transfected with Zdhhc2 saRNAs and treated with IFNy. Expression of the indicated genes was determined at 24 hours (ED FIG. 4n) or 48 hours (ED FIG. 4o). Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test. 1 of 2 blots is shown.
[0090] ED FIG. 4p-q are bar graphs showing Zdhhc2 KO B16 cells transfected with Zdhhc2 saRNAs and treated with IFNy. Expression of the indicated genes was determined at 24 hours (ED FIG. 4p) or 48 hours (ED FIG. 4q). Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test. 1 of 2 blots is shown.
[0091] FIG. 5a is a schematic diagram showing the screening of the cysteine-targeting covalent inhibitors for their potential modification in ADAR 1 -RNA editing activity.
[0092] FIG. 5b-d are bar graphs showing B16 cells pretreated with IFNy overnight and subsequently treated with the indicated inhibitors at I pM. RNA editing efficacy (FIG. 5b) and Ifnbl transcripts (FIG. 5c) were determined at 24 hours. Surface expression of H2-Kb (FIG. 5d) was determined at 48 hours. Data are mean ± SD. n = 3 biological independent samples.
[0093] FIG. 5e is the structure of Neratinib. Rectangle indicates the active cysteine- targeting group.
[0094] FIG. 5f-g are line graphs showing IFNy-pretreated B16 cells treated with 1 pM Neratinib at the indicated times (FIG. 5f) or with different concentrations of Neratinib at 8 hours (FIG. 5g). Ifnbl transcripts were determined by qPCR.
[0095] FIG. 5h-i are bar graphs showing WT, Adarl KO (h), or Adar 1 C2A mutant B16 cells treated with IFNy overnight and subsequently incubated with Neratinib at IpM for 8 hours. Ifnbl transcripts were determined by qPCR. Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0096] FIG. 5j is the structure of Neratinib-inactive. Rectangle indicates the inactive cysteine-targeting group.
[0097] FIG. 5k-n are bar graphs showing IFNy-pretreated B16 cells treated with Neratinib or Neratinib-inactive at I pM. Expression of the indicated genes were determined at 24 (FIG. 5k), 36 (FIG. 51, FIG. 5m), and 48 hours (FIG. 5n). Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0098] FIG. 5o is a Western blot showing B16-Adarl KO cells forced to express Adarl for 48 hours. Protein samples were incubated with biotin-Neratinib or Neratinib overnight, followed by pulldown assay with Streptavidin. Adarl and Gapdh proteins were detected in the Pulldown products or input samples. 1 of 2 blots is shown.
[0099] FIG. 5p is a Western blot showing B16-Adarl KO cells transfected with WT Adarl or C2A mutant plasmids for 48 hours. Protein samples were incubated with Biotin-Neratinib overnight, followed by pulldown assay with Streptavidin. Adarl and Gapdh proteins were detected in the Pulldown products or input samples. 1 of 2 blots is shown.
[00100] FIG. 5q is a schematic working model showing Neratinib regulates ADAR1 activity at Cysiosi/1082.
[0100] FIG. 5r-s is four line graphs and a Kaplan-Meier curve, respectively, showing C57BL/6 mice carrying B16 tumors treated with Neratinib and/or anti-PD-Ll antibody. Tumor growth curves (FIG. 5r) or mouse overall survival (FIG. 5s) were plotted. Data are mean ± SD. n = 12-14 mice. P values by two-sided t test. [0101] ED FIG. 5a is a table showing information on the FDA-approved Cysteine- targeting covalent inhibitors.
[0102] ED FIG. 5b-c are each three line graphs showing human cancer cells (CHE1, SW480, and CAOV3) treated with Neratinib and/or IFNy. Expression of the indicated gene transcripts was determined at 24 (ED FIG. 5b) or 48 hours (ED FIG. 5c). Data are mean ± SD. n = 3 biological independent samples. P values by two-sided t test.
[0103] ED FIG. 5d is a Western blot showing Adar 1 -overexpressing B16 cells (IFNy pretreated) treated with different concentrations of Neratinib (0, 0.1, 0.2, 0.5, 1, 2, 5, and 10 pM) for 24 hours. Adarl protein was determined in the Zn-NTA pulldown products or input samples. 1 of 2 blots is shown.
[0104] ED FIG. 5e is the structure of biotin-Neratinib.
[0105] ED FIG. 5f is a Western blot showing expression levels of the indicated proteins in SKBR3 or B16F0 cells determined by Western blot. 1 of 2 blots is shown.
[0106] ED FIG. 5g is two Western blots showing SKBR3 or B16F0 cells treated with the indicated concentrations of Neratinib for 4 hours. Phosphorylated AKT levels were determined by Western blot. 1 of 2 blots is shown.
[0107] ED FIG. 5h is a line graph showing B16 tumor-bearing mice treated with anti- PD-E1 antibody and/or Neratinib. Tumor growth curves were plotted. Data are shown as mean ± SD. n =12 -14 mice per group.
[0108] ED FIG. 5i-j are representative dot plots (ED FIG. 5i) and the percentages (ED FIG. 5j) of intra-tumoral IFNy+, TNFD+, and Granzyme B+ CD8+ T cells in mice bearing B16 tumors treated with the indicated drugs, n = 6, P value by t-test.
[0109] ED FIG. 5k is a schematic working model suggesting that during metabolic restriction or therapeutic targeting, the Zinc cofactor binding site of ADAR1 can be occupied by the ZDHHC2-mediated palmitoylation or the Neratinib-mediated covalent modification, respectively, thereby diminishing ADAR1-RNA editing activity and eliciting type-I/III IFN production.
DETAILED DESCRIPTION
I. Compounds of the Disclosure
[0110] In one embodiment, the present disclosure provides a compound having
Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0111] R1 is selected from the group consisting of -CH=CHRla and -C=CRlb;
[0112] Rla is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR8R9,;
[0113] Rlb is selected from the group consisting of hydrogen and C1-C6 alkyl;
[0114] R8 and R9 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, and (C1-C6 alkoxy)C1-C6 alkyl; or
[0115] R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo;
[0116] R2 is selected from the group consisting of hydrogen, halo, C1-C6 alkyl, and -OR7;
[0117] R7 is selected from the group consisting of C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, (optionally substituted C3-C6 cycloalkyl)C1-C6-alkyl, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4- to 7-membered heterocyclo)C1-C6 alkyl;
[0118] R3 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl;
[0119] R4 is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, and C3-C6 cycloalkyl; and
[0120] R5 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted heterocyclo, optionally substituted aryl, and optionally substituted heteroaryl.
[0121] In another embodiment, the present disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is -CH=CHRla.
[0122] In another embodiment, the present disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is -C=CRlb. [0123] In another embodiment, the present disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, with the following provisos:
[0124] (i) if R4 is cyano and R1 is -CH2NR8R9, then R8 and R9 are each independently selected from the group consisting of hydrogen, C2-C6 alkyl, C3-C6 cycloalkyl, and (C1-C6 alkoxy )C1-C6 alkyl; or R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo; or
[0125] (ii) if R1 is -CH2NR8R9 and R8 and R9 are each methyl, then R4 is selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, and C3-C6 cycloalkyl; or
[0126] (iii) if R1 is -CH2NR8R9, R8 and R9 are each methyl, and R4 is cyano, then R3 is C1-C6 alkyl or C3-C6 cycloalkyl.
[0127] In another embodiment, the present disclosure provides a compound having Formula (II): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0128] R6a is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl;
[0129] R6b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl; and
[0130] R6C is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl; C1-C6 alkoxy, C3-C6 cycloalkyl, (optionally substituted heterocyclo)-O-, (aryl)alkyl-O-, and (heteroaryl)alkyl-O-; and
[0131] Rla, R2, R3, and R4 are as defined in connection with Formula (I).
[0132] In another embodiment, the present disclosure provides a compound having Formula (III): (III), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0133] R5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl; and
[0134] Rla, R2, R3, R4, R6a, and R6b are as defined in connection with Formula (II).
[0135] In another embodiment, the present disclosure provides a compound having Formula (III), or a pharmaceutically acceptable salt or solvate thereof, wherein R5a is optionally substituted 5-membered heteroaryl.
[0136] In another embodiment, the present disclosure provides a compound havin Formula (IV): (IV), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0137] R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo; and
[0138] Rla, R2, R3, R4, and R6a are as defined in connection with Formula (III).
[0139] In another embodiment, the present disclosure provides a compound having Formula (V): (V), or a pharmaceutically acceptable salt or solvate thereof, wherein Rla, R2, R3, R4, R5b, and R6a are as defined in connection with Formula (IV).
[0140] In another embodiment, the present disclosure provides a compound having Formula (VI): or a pharmaceutically acceptable salt or solvate thereof, wherein Rla, R2, R3, R4, R5b, and R6a are as defined in connection with Formula (IV).
[0141] In another embodiment, the present disclosure provides a compound having Formula (VII): or a pharmaceutically acceptable salt or solvate thereof, wherein Rla, R2, R3, R4, R5b, and R6a are as defined in connection with Formula (IV).
[0142] In another embodiment, the present disclosure provides a compound having any one of Formulae (I)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein Rla is hydrogen.
[0143] In another embodiment, the present disclosure provides a compound having any one of Formulae (I)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein Rla is C1-C4 alkyl.
[0144] In another embodiment, the present disclosure provides a compound having any one of Formulae (I)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein Rla is optionally substituted 4- to 7-membered heterocyclo.
[0145] In another embodiment, the present disclosure provides a compound having any one of Formulae (I)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein Rla is -CH2NR8R9. In another embodiment, R8 and R9 are C1-C6 alkyl. In another embodiment, R8 and R9 are methyl. In another embodiment, R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo. In another embodiment, R8 and R9 taken together with the nitrogen atom to which they are attached form:
[0146] In another embodiment, the present disclosure provides a compound having any one of Formulae (I)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein R2 is -OR7. In another embodiment, R7 is methyl, ethyl, or propyl. In another embodiment, R7 is ethyl. In another embodiment, R7 is C3-C6 cycloalkyl. In another embodiment, R7 is (C3-C6 cycloalkyl)C1-C6-alkyl. In another embodiment, R7 is (C1-C6- alkoxy)C1-C6 alkyl. In another embodiment, R7 is optionally substituted 4- to 7-membered heterocyclo. In another embodiment, R7 is(optionally substituted 4- to 7-membered heterocyclo)C1-C6 alkyl.
[0147] In another embodiment, the present disclosure provides a compound having any one of Formulae (I)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein R3 is hydrogen or C1-C6 alkyl. In another embodiment, R3 is hydrogen. In another embodiment, R3 is methyl. In another embodiment, R3 is ethyl.
[0148] In another embodiment, the present disclosure provides a compound having any one of Formulae (I)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein R3 is C3-C6 cycloalkyl. In another embodiment, R4 is cyclopropyl.
[0149] In another embodiment, the present disclosure provides a compound having any one of Formulae (I)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein R4 is hydrogen or cyano. In another embodiment, R4 is hydrogen. In another embodiment, R4 is cyano.
[0150] In another embodiment, the present disclosure provides a compound having any one of Formulae (I)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein R6a is hydrogen or halogen. In another embodiment, R6a is hydrogen. In another embodiment, R6a is chloro. [0151] In another embodiment, the present disclosure provides a compound having any one of Formulae (IV)-(VII), or pharmaceutically acceptable salts or solvates thereof, wherein R5b is hydrogen or C1-C6 alkyl.
[0152] In another embodiment, Compounds of the Disclosure are compounds having Formula (IX): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0153] X is selected from the group consisting of -O- and -NRa-;
[0154] Ra is selected from the group consisting to hydrogen and C1-C4 alkyl;
[0155] R1 is selected from the group consisting of -CH=CHRla and -C=CRlb;
[0156] Rla is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR8R9;
[0157] Rlb is selected from the group consisting of hydrogen and C1-C6 alkyl;
[0158] R8 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted 4-to 7-membered heterocyclo, C2-C12 alkynyl, and (C1-C6 alkoxy)C1-C6 alkyl;
[0159] R9 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; or
[0160] R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo;
[0161] R2 is selected from the group consisting of hydrogen, halo, C1-C6 alkyl, and -OR7;
[0162] R7 is selected from the group consisting of C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, (optionally substituted C3-C6 cycloalkyl)C1-C6-alkyl, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4-to 7-membered heterocyclo)C1-C6 alkyl;
[0163] R3 is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl;
[0164] R4 is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, and C3-C6 cycloalkyl; and [0165] R5 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, optionally substituted aryl, (heterocyclo)alkyl, and optionally substituted 5- to 10- membered heteroaryl.
[0166] In another embodiment, Compounds of the Disclosure are compounds having Formula (IX), or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is selected from the group consisting of:
[0167] In another embodiment, Compounds of the Disclosure are compounds having Formula (X): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0168] R6a is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl;
[0169] R6b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl; and
[0170] R6C is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl; C1-C4 haloalkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, (optionally substituted 4- to 7-membered heterocyclo)-O-, (aryl)alkyl-O-, and (heteroaryl)alkyl-O-; and
[0171] Rla, R2, R3, R4, and X are as defined in connection with Formula (IX).
[0172] In another embodiment, Compounds of the Disclosure are compounds having
Formula (X), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0173] R6b is hydrogen; and
[0174] R6C is selected from the group consisting of:
[0175] In another embodiment, Compounds of the Disclosure are compounds having Formula (XI): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0176] R5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl;
[0177] Rla, R2, R3, R4, and X are as defined in connection with Formula (IX); and
[0178] R6a and R6b are as defined in connection with Formula (X).
[0179] In another embodiment, Compounds of the Disclosure are compounds having
Formula (XI), or a pharmaceutically acceptable salt or solvate thereof, wherein R5a is optionally substituted 5-membered heteroaryl, e.g., imidazole.
[0180] In another embodiment, Compounds of the Disclosure are compounds having Formula (XII): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0181] R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo; [0182] Rla, R2, R3, R4, and X are as defined in connection with Formula (IX); and
[0183] R6a is as defined in connection with Formula (X).
[0184] In another embodiment, Compounds of the Disclosure are compounds having Formula (XIII): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0185] R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo;
[0186] Rla, R2, R3, R4, and X are as defined in connection with Formula (IX); and
[0187] R6a is as defined in connection with Formula (X).
[0188] In another embodiment, Compounds of the Disclosure are compounds having Formula (XIV): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0189] R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo;
[0190] Rla, R2, R3, R4, and X are as defined in connection with Formula (IX); and
[0191] R6a is as defined in connection with Formula (X).
[0192] In another embodiment, Compounds of the Disclosure are compounds having Formula (XV):
(XV), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0193] R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo;
[0194] Rla, R2, R3, R4, and X are as defined in connection with Formula (IX); and
[0195] R6a is as defined in connection with Formula (X).
[0196] In another embodiment, Compounds of the Disclosure are compounds having Formula (XVI): (XVI), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0197] R5 is selected from the group consisting of optionally substituted 5- to 7-membered heterocylo and optionally substituted 5- to 10-membered heteroaryl; and
[0198] Rla, R2, R3, R4, and X are as defined in connection with Formula (IX).
[0199] In another embodiment, Compounds of the Disclosure are compounds having
Formula (XVI), or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is optionally substituted 5- to 7-membered heterocylo. In another embodiment, R5 is selected from the group consisting of:
[0200] In another embodiment, Compounds of the Disclosure are compounds having
Formula (XVI), or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is optionally substituted 5- to 10-membered heteroaryl. In another embodiment, R5 is selected from the group consisting of:
[0201] In another embodiment, Compounds of the Disclosure are compounds having any one of Formulae (IX)-(XVI), or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is hydrogen.
[0202] In another embodiment, Compounds of the Disclosure are compounds having any one of Formulae (IX)-(XVI), or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is C1-C4 alkyl.
[0203] In another embodiment, Compounds of the Disclosure are compounds having any one of Formulae (IX)-(XVI), or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is -CFhNR8R9; and R8 and R9 are C1-C6 alkyl. In another embodiment, R8 and R9 are methyl. In another embodiment, R8 is optionally substituted 4-to 7-membered heterocyclo. In another embodiment, R8 is selected from the group consisting of:
In another embodiment, R8 is C2-C10 alkynyl. In another embodiment, R8 is selected from the group consisting of:
[0204] In another embodiment, Compounds of the Disclosure are compounds having any one of Formulae (IX)-(XVI), or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is -CFhNR8R9; and R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo. In another embodiment, R8 and R9 taken together with the nitrogen atom to which they are attached form:
[0205] In another embodiment, Compounds of the Disclosure are compounds having any one of Formulae (IX)-(XVI), or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is selected from the group consisting of:
[0206] In another embodiment, Compounds of the Disclosure are compounds having any one of Formulae (IX)-(XVI), or a pharmaceutically acceptable salt or solvate thereof, wherein R2is -OR7. In another embodiment, R7 is selected from the group consisting of methyl, ethyl, and propyl. In another embodiment, R7 is ethyl.
[0207] In another embodiment, Compounds of the Disclosure are compounds having any one of Formulae (IX)-(XVI), or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is selected from the group consisting of hydrogen and C1-C6 alkyl. In another embodiment, R3 is hydrogen. In another embodiment, R3 is methyl. In another embodiment, R3 is ethyl.
[0208] In another embodiment, Compounds of the Disclosure are compounds having any one of Formulae (IX)-(XVI), or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is selected from the group consisting of hydrogen and cyano. In another embodiment, R4 is hydrogen. In another embodiment, R4 is cyano.
[0209] In another embodiment, Compounds of the Disclosure are compounds having any one of Formulae (X)-(XV), or a pharmaceutically acceptable salt or solvate thereof, wherein R6a is selected from the group consisting of hydrogen and halogen. In another embodiment, R6a is hydrogen. In another embodiment, R6a is chloro.
[0210] In another embodiment, Compounds of the Disclosure are compounds having any one of Formulae (IX)-(XVI), wherein X is -O-.
[0211] In another embodiment, Compounds of the Disclosure are compounds having any one of Formulae (IX)-(XVI), wherein X is -NH-.
[0212] In another embodiment, Compounds of the Disclosure are compounds having Formula (XVII): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0213] R3 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, and cyclopropyl;
[0214] R5 is selected from the group consisting of optionally substituted 4- to 7-membered heterocyclo, optionally substituted aryl, (heterocyclo)alkyl, and optionally substituted 5- to 10-membered heteroaryl;
[0215] R8 is selected from the group consisting of C1-C6 alkyl, optionally substituted 4-to 7-membered heterocyclo, and C2-C12 alkynyl; and
[0216] R9 is C1-C6 alkyl; or
[0217] R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo. [0218] In another embodiment, Compounds of the Disclosure are compounds having any one of Formulae (IX)-(XVII), or a pharmaceutically acceptable salt or solvate thereof, with the following provisos:
[0219] (i) if R4 is cyano and R1 is -CH2NR8R9, then R8 is selected from the group consisting of hydrogen, C2-C6 alkyl, C3-C6 cycloalkyl, optionally substituted 4-to 7-membered heterocyclo, C2-C12 alkynyl, and (C1-C6 alkoxy )C1-C6 alkyl; R9 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; or R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo; or
[0220] (ii) if R1 is -CH2NR8R9 and R8 and R9 are each methyl, then R4 is selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, and C3-C6 cycloalkyl; or
[0221] (iii) if R1 is -CH2NR8R9, R8 and R9 are each methyl, and R4 is cyano, then R3 is C1-C6 alkyl or C3-C6 cycloalkyl.
[0222] In another embodiment, the present disclosure provides any one or more of the compounds of Table 1A, or pharmaceutically acceptable salts or solvates thereof.
Table 1A
[0223] In another embodiment, the present disclosure provides any one or more of the compounds of Table 2A, or pharmaceutically acceptable salts or solvates thereof.
Table 2 A [0224] In another embodiment, the present disclosure provides any one or more of the compounds of Table 1 A and 2A, or pharmaceutically acceptable salts or solvates thereof.
[0225] In another embodiment, the present disclosure provides any one or more of the following compounds:
[0226] In another embodiment, the present disclosure provides any one or more of the following compounds:
[0227] In another embodiment, the present disclosure provides a pharmaceutical composition comprising a Compound of the Disclosure and a pharmaceutically acceptable excipient.
[0228] In another embodiment, the present disclosure provides any one or more of the compounds of Table IB, or pharmaceutically acceptable salts or solvates thereof.
Table IB
II. Synthetic Intermediates [0229] In another embodiment, the present disclosure provides compounds having Formula (VIII): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0230] R10 is selected from the group consisting of chloro and -N(H)R5;
[0231] R11 is selected from the group consisting of hydrogen and -C(=O)CH3; and
[0232] R2, R3, R4, and R5 are as defined in connection with Formula (I) or Formula (IX).
[0233] In another embodiment, the present disclosure provides a compound having Formula (VIII), or a pharmaceutically acceptable salt or solvate thereof, wherein R10 is chloro.
[0234] In another embodiment, the present disclosure provides a compound having Formula (VIII), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0235] R10 is -N(H)R5;
[0236]
[0237] R6a is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl;
[0238] R6b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl; and
[0239] R6C is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl; C1-C6 alkoxy, C3-C6 cycloalkyl, (optionally substituted heterocyclo)-O-, (aryl)alkyl-O-, and (heteroaryl)alkyl-O-.
[0240] In another embodiment, the present disclosure provides a compound having Formula (VIII), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0241] R10 is -N(H)R5;
[0242] [0243] R5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl;
[0244] R6a is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl; and
[0245] R6b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl.
[0246] In another embodiment, the present disclosure provides a compound having Formula (VIII), or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is selected from the group consisting of optionally substituted 5- to 7-membered heterocylo and optionally substituted 5- to 10-membered heteroaryl.
[0247] In another embodiment, Compounds of the Disclosure are compounds having Formula (VIII), or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is optionally substituted 5- to 7-membered heterocylo. In another embodiment, R5 is selected from the group consisting of:
[0248] In another embodiment, Compounds of the Disclosure are compounds having Formula (VIII), or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is optionally substituted 5- to 10-membered heteroaryl. In another embodiment, R5 is selected from the group consisting of:
[0249] In another embodiment, the present disclosure provides a compound having Formula (VIII), or pharmaceutically acceptable salts or solvates thereof, wherein R2is -OR7. In another embodiment, R7 is methyl, ethyl, or propyl. In another embodiment, R7 is ethyl. In another embodiment, R7 is C3-C6 cycloalkyl. In another embodiment, R7 is (C3-C6 cycloalkyl)C1-C6-alkyl. In another embodiment, R7 is (C1-C6- alkoxy)C1-C6 alkyl. In another embodiment, R7 is optionally substituted 4- to 7-membered heterocyclo. In another embodiment, R7 is(optionally substituted 4- to 7-membered heterocyclo)C1-C6 alkyl.
[0250] In another embodiment, the present disclosure provides a compound having Formula (VIII), or pharmaceutically acceptable salts or solvates thereof, wherein R3 is hydrogen or C1-C6 alkyl. In another embodiment, R3 is hydrogen. In another embodiment, R3 is methyl. In another embodiment, R3 is ethyl.
[0251] In another embodiment, the present disclosure provides a compound having Formula (VIII), or pharmaceutically acceptable salts or solvates thereof, wherein R3 is C3-C6 cycloalkyl. In another embodiment, R4 is cyclopropyl.
[0252] In another embodiment, the present disclosure provides a compound having Formula (VIII), or pharmaceutically acceptable salts or solvates thereof, wherein R4 is hydrogen or cyano. In another embodiment, R4 is hydrogen. In another embodiment, R4 is cyano.
[0253] In another embodiment, the present disclosure provides a compound having Formula (VIII), or pharmaceutically acceptable salts or solvates thereof, wherein R6a is hydrogen or halogen. In another embodiment, R6a is hydrogen. In another embodiment, R6a is chloro.
[0254] In another embodiment, the present disclosure provides a compound having Formula (VIII), or pharmaceutically acceptable salts or solvates thereof, wherein R11 is hydrogen.
[0255] In another embodiment, the present disclosure provides a compound having Formula (VIII), or pharmaceutically acceptable salts or solvates thereof, wherein R11 is -C(=O)CH3.
[0256] In another embodiment, the present disclosure provides any one or more of the compounds of Table 1C, or pharmaceutically acceptable salts or solvates thereof. Compounds having Formula (VIII) and the compounds of Table 1C are synthetic intermediates that can be used, for example, to prepare certain Compounds of the Disclosure.
Table 1C
[0257] In another embodiment, the present disclosure provides compounds having
Formula (XVIII): (XVIII), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0258] X is selected from the group consisting of -O- and -NRa-;
[0259] Ra is selected from the group consisting to hydrogen and C1-C4 alkyl;
[0260] LG is a leaving group;
[0261] R2 is selected from the group consisting of hydrogen, halo, C1-C6 alkyl, and -OR7;
[0262] R7 is selected from the group consisting of C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, (optionally substituted C3-C6 cycloalkyl)C1-C6-alkyl, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4-to 7-membered heterocyclo)C1-C6 alkyl;
[0263] R3 is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl;
[0264] R4 is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, and C3-C6 cycloalkyl; and
[0265] R5 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, optionally substituted aryl, (heterocyclo)alkyl, and optionally substituted 5- to 10- membered heteroaryl.
[0266] In another embodiment, the present disclosure provides compounds having Formula (XVIII), or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is selected from the group consisting of:
[0267] In another embodiment, the present disclosure provides compounds having Formula (XIX): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0268] R6a is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl;
[0269] R6b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl; and
[0270] R6C is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl; C1-C4 haloalkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, (optionally substituted 4- to 7-membered heterocyclo)-O-, (aryl)alkyl-O-, and (heteroaryl)alkyl-O-; and
[0271] LG, R2, R3, R4, and X are as defined in connection with Formula (VXIII).
[0272] In another embodiment, the present disclosure provides compounds having Formula (XIX), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0273] R6b is hydrogen; and
[0274] R6C is selected from the group consisting of:
[0275] In another embodiment, the present disclosure provides compounds having Formula (XX): (XX), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0276] R5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl;
[0277] LG, R2, R3, R4, and X are as defined in connection with Formula (XVIII); and
[0278] R6a and R6b are as defined in connection with Formula (XIX).
[0279] In another embodiment, Compounds of the Disclosure are compounds having Formula (XX), or a pharmaceutically acceptable salt or solvate thereof, wherein R5a is optionally substituted 5-membered heteroaryl, e.g., imidazole.
[0280] In another embodiment, the present disclosure provides compounds having Formula (XXI): (XXI), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0281] R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo; [0282] LG, R2, R3, R4, and X are as defined in connection with Formula (XVIII); and
[0283] R6a is as defined in connection with Formula (XIX).
[0284] In another embodiment, Compounds of the Disclosure are compounds having Formula (XXII): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0285] R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo;
[0286] LG, R2, R3, R4, and X are as defined in connection with Formula (XVIII); and
[0287] R6a is as defined in connection with Formula (XIX).
[0288] In another embodiment, the present disclosure provides compounds having Formula (XXIII): (XXIII), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0289] R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo;
[0290] LG, R2, R3, R4, and X are as defined in connection with Formula (XVIII); and
[0291] R6a is as defined in connection with Formula (XIX).
[0292] In another embodiment, the present disclosure provides compounds having Formula (XXIV):
(XXIV), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0293] R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo;
[0294] LG, R2, R3, R4, and X are as defined in connection with Formula (XVIII); and [0295] R6a is as defined in connection with Formula (XIX).
[0296] In another embodiment, the present disclosure provides compounds having Formula (XXV): (XXV), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0297] R5 is selected from the group consisting of optionally substituted 5- to 7-membered heterocylo and optionally substituted 5- to 10-membered heteroaryl; and
[0298] LG, R2, R3, R4, and X are as defined in connection with Formula (XVIII).
[0299] In another embodiment, the present disclosure provides compounds having
Formula (XXV), or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is optionally substituted 5- to 7-membered heterocylo. In another embodiment, R5 is selected from the group consisting of:
[0300] In another embodiment, the present disclosure provides compounds having
Formula (XXV), or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is optionally substituted 5- to 10-membered heteroaryl. In another embodiment, R5 is selected from the group consisting of:
[0301] In another embodiment, the present disclosure provides compounds having any one of Formulae (XVIII)-(XXV), or a pharmaceutically acceptable salt or solvate thereof, wherein LG is chloro.
[0302] In another embodiment, the present disclosure provides any one or more of the compounds of Table 8, see EXAMPLE 59, or pharmaceutically acceptable salts or solvates thereof. Compounds having Formulae (XVIII)-(XXV), and the compounds of Table 8 are synthetic intermediates that can be used, for example, to prepare certain Compounds of the Disclosure.
III. Therapeutic Methods and Uses
[0303] Compounds of the Disclosure inhibit adenosine deaminase acting on RNA 1 enzymes (ADAR1) and are thus useful in the treatment of a variety of diseases and conditions. In particular, Compounds of the Disclosure are useful in methods of treating a disease or condition wherein ADAR1 inhibition provides a benefit, for example, cancers and proliferative diseases, viral infections, and autoimmune diseases. The therapeutic methods of the present disclosure comprise administering a therapeutically effective amount of a Compound of the Disclosure to a subject, e.g., a cancer patient, in need thereof. The present methods also encompass administering a second therapeutic agent, e.g., an immune checkpoint inhibitor (ICI), to the subject in combination with the Compound of the Disclosure.
[0304] The present disclosure provides Compounds of the Disclosure as ADAR1 inhibitors for the treatment of a variety of diseases and conditions wherein inhibition of ADAR1 has a beneficial effect. Compounds of the Disclosure typically have IC50 (the drug concentration that results in 50% ADAR1 inhibition) values of less than 100 pM, e.g., less than 50 pM, less than 25 pM, and less than 5 pM, less than about 1 pM, less than about 0.5 pM, or less than about 0.1 pM. In some embodiments, Compounds of the Disclosure typically have IC50 values of less than about 0.01 pM. In some embodiments, Compounds of the Disclosure typically have IC50 values of less than about 0.001 pM. In one embodiment, the present disclosure relates to a method of treating an individual suffering from a disease or condition wherein ADAR1 provides a benefit comprising administering a therapeutically effective amount of a Compound of the Disclosure and optionally one or more ICIs to an individual in need thereof
[0305] Since Compounds of the Disclosure are AD ARI inhibitors, a number of diseases and conditions mediated by ADAR1 can be treated by employing these compounds. The present disclosure is thus directed generally to a method for treating a condition or disorder responsive to ADAR1 in an animal, e.g., a human, suffering from, or at risk of suffering from, the condition or disorder, the method comprising administering to the animal an effective amount of one or more Compounds of the Disclosure.
[0306] The present disclosure is further directed to a method of inhibiting ADAR1 in a subject in need thereof, said method comprising administering to the subject an effective amount of a Compound of the Disclosure.
[0307] The present disclosure is further directed to a method of sensitizing tumor response to immunotherapy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a Compound or the Disclosure and one or more ICIs. Is some aspects, the tumor is resistant to immune checkpoint blockade.
[0308] In another aspect, the present disclosure provides a method of treating cancer in a subject comprising administering a therapeutically effective amount of a Compound of the Disclosure and optionally one or more ICIs. In another aspect, the present disclosure provides a method of treating cancer in a subject comprising administering a therapeutically effective amount of a Compound of the Disclosure. In another aspect, the present disclosure provides a method of treating cancer in a subject comprising administering a therapeutically effective amount of a Compound of the Disclosure in combination with a therapeutically effective amount of one or more ICIs. While not being limited to a specific mechanism, in some embodiments, Compounds of the Disclosure treat cancer by inhibiting ADAR1 and/or sensitizing cancer cells administration of ICIs. Examples of treatable cancers include, but are not limited to, any one or more of the cancers of Table 2. Table 2
[0309] In another embodiment, the cancer is a solid tumor. In another embodiment, the cancer a hematological cancer. Exemplary hematological cancers include, but are not limited to, the cancers listed in Table 3. In another embodiment, the hematological cancer is acute lymphocytic leukemia, chronic lymphocytic leukemia (including B-cell chronic lymphocytic leukemia), or acute myeloid leukemia.
Table 3
[0310] In another embodiment, the cancer is a leukemia, for example, a leukemia selected from acute monocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia and mixed lineage leukemia (MLL). In another embodiment, the cancer is NUT-midline carcinoma. In another embodiment, the cancer is multiple myeloma. In another embodiment, the cancer is a lung cancer such as small cell lung cancer (SCLC). In another embodiment, the cancer is a neuroblastoma. In another embodiment, the cancer is Burkitt's lymphoma. In another embodiment, the cancer is cervical cancer. In another embodiment, the cancer is esophageal cancer. In another embodiment, the cancer is ovarian cancer. In another embodiment, the cancer is colorectal cancer. In another embodiment, the cancer is prostate cancer. In another embodiment, the cancer is breast cancer.
[0311] In another embodiment, the cancer is selected from the group consisting of acute monocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia mixed lineage leukemia, NUT-midline carcinoma, multiple myeloma, small cell lung cancer, non- small cell lung cancer, neuroblastoma, Burkitt's lymphoma, cervical cancer, esophageal cancer, ovarian cancer, colorectal cancer, prostate cancer, breast cancer, bladder cancer, ovary cancer, glioma, sarcoma, esophageal squamous cell carcinoma, and papillary thyroid carcinoma.
[0312] In another embodiment, a Compound of the Disclosure are administered to a subject in need thereof to treat breast cancer, ovarian cancer, or prostate cancer. In another embodiment, the cancer is breast cancer. In another embodiment, the cancer is ovarian cancer. In another embodiment, the cancer is prostate cancer. In another embodiment, the cancer is metastatic castration-resistant prostate cancer.
[0313] The methods of the present disclosure can be accomplished by administering a Compound of the Disclosure as the neat compound or as a pharmaceutical composition. Administration of a pharmaceutical composition, or neat Compound of the Disclosure, can be performed during or after the onset of the disease or condition of interest. Typically, the pharmaceutical compositions are sterile, and contain no toxic, carcinogenic, or mutagenic compounds that would cause an adverse reaction when administered.
[0314] In one embodiment, a Compound of the Disclosure is administered as a single agent to treat a disease or condition wherein inhibition of ADAR1 provides a benefit, e.g., cancer. In another embodiment, a Compound of the Disclosure is administered in combination with a second therapeutic agent, e.g., one or more ICIs, useful in the treatment of a disease or condition wherein inhibition of ADAR1 provides a benefit. A Compound of the Disclosure and the second therapeutic agent can be administered simultaneously or sequentially to achieve the desired effect. In addition, the Compound of the Disclosure and second therapeutic agent can be administered as a single pharmaceutical composition or two separate pharmaceutical compositions. In some embodiments, the second therapeutic agent is one or more ICIs, e.g., one or more anti-PD-1 antibodies, one or more anti-PD-Ll antibodies, one or more anti-CTLA-4 antibodies, one or more anti-LAG3 antibodies, one or more anti-TIM3 antibodies, one or more anti- VISTA antibodies, one or more anti-TIGIT antibodies, or one or more anti- cd47 antibodies, or a combination thereof, e.g., nivolumab, pembrolizumab, dostarlimab, retifanlimab, cemiplimab, avelumab, atezolizumab, or durvalumab, or a combination thereof.
[0315] The second therapeutic agent is administered in an amount to provide its desired therapeutic effect. The effective dosage range for each second therapeutic agent is known in the art, and the second therapeutic agent is, for example, administered to an individual in need thereof within such established ranges. In some embodiments, administration of a Compound of the Disclosure sensitizs the cancer cells to ICB and thus the effective dosage of one or more ICIs co-administered with the Compound of the Disclosure is less, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% less, than the established ranges for the one or more ICIs known in the art.
[0316] A Compound of the Disclosure and the second therapeutic agent can be administered together as a single-unit dose or separately as multi-unit doses, wherein the Compound of the Disclosure is administered before the second therapeutic agent or vice versa. One or more doses of the Compound of the Disclosure and/or one or more doses of the second therapeutic agent can be administered. The Compound of the Disclosure therefore can be used in conjunction with one or more second therapeutic agents, for example, but not limited to, anticancer agents.
[0317] In some methods of the present disclosure, a therapeutically effective amount of a Compound of the Disclosure, typically formulated in accordance with pharmaceutical practice, is administered to a subject, e.g., a human cancer patient, in need thereof. In some methods of the present disclosure, a therapeutically effective amount of a Compound of the Disclosure, typically formulated in accordance with pharmaceutical practice, is administered to a subject in need thereof in combination with a therapeutically effective amount of an ICI. Whether such a treatment is indicated depends on the individual case and is subject to medical assessment (diagnosis) that takes into consideration signs, symptoms, and/or malfunctions that are present, the risks of developing particular signs, symptoms and/or malfunctions, and other factors. [0318] A Compound of the Disclosure can be administered by any suitable route, for example by oral, buccal, inhalation, sublingual, rectal, vaginal, intracistemal or intrathecal through lumbar puncture, transurethral, nasal, percutaneous, i.e., transdermal, or parenteral (including intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a particular site) administration. Parenteral administration can be accomplished using a needle and syringe or using a high pressure technique.
[0319] Pharmaceutical compositions include those wherein a Compound of the Disclosure is administered in an effective amount to achieve its intended purpose. The exact formulation, route of administration, and dosage is determined by an individual physician in view of the diagnosed condition or disease. Dosage amount and interval can be adjusted individually to provide levels of a Compound of the Disclosure that is sufficient to maintain therapeutic effects.
[0320] Toxicity and therapeutic efficacy of the Compounds of the Disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the maximum tolerated dose (MTD) of a compound, which defines as the highest dose that causes no toxicity in animals. The dose ratio between the maximum tolerated dose and therapeutic effects (e.g. inhibiting of tumor growth) is the therapeutic index. The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
[0321] A therapeutically effective amount of a Compound of the Disclosure required for use in therapy varies with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the patient, and ultimately is determined by the attendant physician. Dosage amounts and intervals can be adjusted individually to provide plasma levels of the ADAR1 inhibitor that are sufficient to maintain the desired therapeutic effects. The desired dose conveniently can be administered in a single dose, or as multiple doses administered at appropriate intervals, for example as one, two, three, four or more subdoses per day. Multiple doses often are desired, or required. For example, a Compound of the Disclosure can be administered at a frequency of: four doses delivered as one dose per day at four-day intervals (q4d x 4); four doses delivered as one dose per day at three-day intervals (q3d x 4); one dose delivered per day at five-day intervals (qd x 5); one dose per week for three weeks (qwk3); five daily doses, with two days rest, and another five daily doses (5/2/5); or, any dose regimen determined to be appropriate for the circumstance.
[0322] A Compound of the Disclosure used in a method of the present disclosure can be administered in an amount of, for example, about 0.005 to about 5000 milligrams per dose, about 10 to about 1000 milligrams per dose, about 10 to about 500 milligrams per dose, or about 10 to about 100 milligrams per dose. For example, a Compound of the Disclosure can be administered, per dose, in an amount of about 0.005, about 0.05, about 0.5, about 5, about 10, about 20, about 30, about 40, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, or about 1000 milligrams, including all doses between 0.005 and 5000 milligrams.
[0323] The dosage of a composition containing a Compound of the Disclosure, or a composition containing the same, can be from about 1 ng/kg to about 200 mg/kg, about 1 pg/kg to about 100 mg/kg, or about 1 mg/kg to about 50 mg/kg. The dosage of a composition can be at any dosage including, but not limited to, about 1 pg/kg. The dosage of a composition may be at any dosage including, but not limited to, about 1 pg/kg, about 10 pg/kg, about 25 pg/kg, about 50 pg/kg, about 75 pg/kg, about 100 pg/kg, about 125 pg/kg, about 150 pg/kg, about 175 pg/kg, about 200 pg/kg, about
225 pg/kg, about 250 pg/kg, about 275 pg/kg, about 300 pg/kg, about 325 pg/kg, about
350 pg/kg, about 375 pg/kg, about 400 pg/kg, about 425 pg/kg, about 450 pg/kg, about
475 pg/kg, about 500 pg/kg, about 525 pg/kg, about 550 pg/kg, about 575 pg/kg, about
600 pg/kg, about 625 pg/kg, about 650 pg/kg, about 675 pg/kg, about 700 pg/kg, about
725 pg/kg, about 750 pg/kg, about 775 pg/kg, about 800 pg/kg, about 825 pg/kg, about
850 pg/kg, about 875 pg/kg, about 900 pg/kg, about 925 pg/kg, about 950 pg/kg, about
975 pg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, or more. The above dosages are exemplary of the average case, but there can be individual instances in which higher or lower dosages are merited, and such are within the scope of this disclosure. In practice, the physician determines the actual dosing regimen that is most suitable for an individual subject, which can vary with the age, weight, and response of the particular subject.
[0324] Compounds of the Disclosure typically are administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions for use in accordance with the present disclosure are formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and/or auxiliaries that facilitate processing of Compound of the Disclosure.
[0325] These pharmaceutical compositions can be manufactured, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of the Compound of the Disclosure is administered orally, the composition typically is in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the composition additionally can contain a solid carrier, such as a gelatin or an adjuvant. The tablet, capsule, and powder contain about 0.01% to about 95%, and preferably from about 1% to about 50%, of a Compound of the Disclosure. When administered in liquid form, a liquid carrier, such as water, petroleum, or oils of animal or plant origin, can be added. The liquid form of the composition can further contain physiological saline solution, dextrose or other saccharide solutions, or glycols. When administered in liquid form, the composition contains about 0.1% to about 90%, and preferably about 1% to about 50%, by weight, of a Compound of the Disclosure.
[0326] When a therapeutically effective amount of a Compound of the Disclosure is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred composition for intravenous, cutaneous, or subcutaneous injection typically contains, an isotonic vehicle.
[0327] Compounds of the Disclosure can be readily combined with pharmaceutically acceptable carriers well-known in the art. Standard pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 19th ed. 1995. Such carriers enable the active agents to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding the Compound of the Disclosure to a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
[0328] Suitable excipients include fillers such as saccharides (for example, lactose, sucrose, mannitol or sorbitol), cellulose preparations, calcium phosphates (for example, tricalcium phosphate or calcium hydrogen phosphate), as well as binders such as starch paste (using, for example, maize starch, wheat starch, rice starch, or potato starch), gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, one or more disintegrating agents can be added, such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Buffers and pH modifiers can also be added to stabilize the pharmaceutical composition.
[0329] Auxiliaries are typically flow-regulating agents and lubricants such as, for example, silica, talc, stearic acid or salts thereof (e.g., magnesium stearate or calcium stearate), and polyethylene glycol. Dragee cores are provided with suitable coatings that are resistant to gastric juices. For this purpose, concentrated saccharide solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate can be used. Dye stuffs or pigments can be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
[0330] Compound of the Disclosure can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
[0331] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agent in water-soluble form. Additionally, suspensions of a
- I l l - Compound of the Disclosure can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds and allow for the preparation of highly concentrated solutions. Alternatively, a present composition can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0332] Compounds of the Disclosure also can be formulated in rectal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases. In addition to the formulations described previously, the Compound of the Disclosure also can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the Compound of the Disclosure can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins.
[0333] In particular, the Compounds of the Disclosure can be administered orally, buccally, or sublingually in the form of tablets containing excipients, such as starch or lactose, or in capsules or ovules, either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents. Such liquid preparations can be prepared with pharmaceutically acceptable additives, such as suspending agents. Compound of the Disclosure also can be injected parenterally, for example, intravenously, intramuscularly, subcutaneously, or intracoronarily. For parenteral administration, the Compound of the Disclosure are typically used in the form of a sterile aqueous solution which can contain other substances, for example, salts or monosaccharides, such as mannitol or glucose, to make the solution isotonic with blood.
IV. Immune Checkpoint Inhibitors
[0334] Immune checkpoint inhibitors (ICIs) are therapies that blockade immune system inhibitor checkpoints. Immune checkpoints can be stimulatory or inhibitory. Blockade of inhibitory immune checkpoint activates immune system function and can be used for cancer immunotherapy. Pardoll, Nature Reviews. Cancer 12:252-64 (2012). Tumor cells turn off activated T cells when they attach to specific T-cell receptors. ICIs prevent tumor cells from attaching to T cells, which results in T cells remaining activated. In effect, the coordinated action by cellular and soluble components combats pathogens and injuries by cancers. The modulation of immune system pathways may involve changing the expression or the functional activity of at least one component of the pathway to then modulate the response by the immune system. U.S. 2015/0250853. Examples of ICIs include, but are not limited to, TIGIT inhibitors, PD-1 inhibitors, PD-L1 (B7-H1) inhibitors, CTLA-4 inhibitors, LAG3 inhibitors, TIM3 inhibitors, and cd47 inhibitors. The effective dosage range for ICIs is generally known in the art, and the immune checkpoint inhibitor(s) is(are) administered to a subject in need thereof within, for example, such established ranges.
[0335] In one embodiment, methods and uses of the present disclosure comprise administering a therapeutically effective amount of a Compound of the Disclosure to a subject in combination with a therapeutically effective amount of one or more ICIs to treat cancer.
[0336] In another embodiment, methods of the present disclosure comprise administering a therapeutically effective amount of a Compound of the Disclosure to a subject in combination with one or more PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG3 inhibitors, TIM3 inhibitors, TIGIT inhibitors, or cd47 inhibitors, or a combination thereof.
[0337] In another embodiment, the one or more ICIs comprise a programmed cell death (PD-1) inhibitor. PD-1 is a T-cell coinhibitory receptor that plays a pivotal role in the ability of tumor cells to evade the host's immune system. Blockage of interactions between PD-1 and PD-L1, a ligand of PD-1, enhances immune function and mediates antitumor activity. Examples of PD-1 inhibitors include antibodies that specifically bind to PD-1. Particular anti-PD-1 antibodies include, but are not limited to nivolumab, pembrolizumab, STI-A1014, and pidilzumab. For a general discussion of the availability, methods of production, mechanism of action, and clinical studies of anti-PD- 1 antibodies, see U.S. 2013/0309250, U.S. 6,808,710, U.S. 7,595,048, U.S. 8,008,449, U.S. 8,728,474, U.S. 8,779,105, U.S. 8,952,136, U.S. 8,900,587, U.S. 9,073,994, U.S. 9,084,776, and Naido et al., British Journal of Cancer 111:2214-19 (2014).
[0338] In another embodiment, the one or more ICIs comprise a PD-L1 (also known as B7-H1 or CD274) inhibitor. Examples of PD-L1 inhibitors include antibodies that specifically bind to PD-L1. Particular anti-PD-Ll antibodies include, but are not limited to, avelumab, atezolizumab, durvalumab, KN035, and BMS-936559. For a general discussion of the availability, methods of production, mechanism of action, and clinical studies, see U.S. 8,217,149, U.S. 2014/0341917, U.S. 2013/0071403, WO 2015036499, and Naido et al., British Journal of Cancer 111:2214-19 (2014).
[0339] In another embodiment, the one or more ICIs comprise a CTLA-4 inhibitor. CTLA-4, also known as cytotoxic T-lymphocyte antigen 4, is a protein receptor that downregulates the immune system. CTLA-4 is characterized as a "brake" that binds costimulatory molecules on antigen-presenting cells, which prevents interaction with CD28 on T cells and also generates an overtly inhibitory signal that constrains T cell activation. Examples of CTLA-4 inhibitors include antibodies that specifically bind to CTLA-4. Particular anti-CTLA-4 antibodies include, but are not limited to, ipilimumab and tremelimumab. Lor a general discussion of the availability, methods of production, mechanism of action, and clinical studies, see U.S. 6,984,720, U.S. 6,207,156, and Naido et al., British Journal of Cancer 111:2214-19 (2014).
[0340] In another embodiment, the one or more ICIs comprise a LAG3 inhibitor. LAG3, Lymphocyte Activation Gene 3, is a negative co-simulatory receptor that modulates T cell homeostatis, proliferation, and activation. In addition, LAG3 has been reported to participate in regulatory T cells (Tregs) suppressive function. A large proportion of LAG3 molecules are retained in the cell close to the microtubule-organizing center, and only induced following antigen specific T cell activation. U.S. 2014/0286935. Examples of LAG3 inhibitors include antibodies that specifically bind to LAG3. Particular anti- LAG3 antibodies include, but are not limited to, GSK2831781. Lor a general discussion of the availability, methods of production, mechanism of action, and studies, see, U.S. 2011/0150892, U.S. 2014/0093511, U.S. 2015/0259420, and Huang et al., Immunity 21:503-13 (2004).
[0341] In another embodiment, the one or more ICIs comprise a TIM3 inhibitor. TIM3, T-cell immunoglobulin and mucin domain 3, is an immune checkpoint receptor that functions to limit the duration and magnitude of TH1 and TCI T-cell responses. The TIM3 pathway is considered a target for anticancer immunotherapy due to its expression on dysfunctional CD8+ T cells and Tregs, which are two reported immune cell populations that constitute immunosuppression in tumor tissue. Anderson, Cancer Immunology Research 2:393-98 (2014). Examples of TIM3 inhibitors include antibodies that specifically bind to TIM3. Lor a general discussion of the availability, methods of production, mechanism of action, and studies of TIM3 inhibitors, see U.S. 20150225457, U.S. 20130022623, U.S. 8,522,156, Ngiow et al., Cancer Res 71: 6567-71 (2011), Ngiow, et al., Cancer Res 71:3540-51 (2011), and Anderson, Cancer Immunology Res 2:393-98 (2014).
[0342] In another embodiment, the one or more ICIs comprise a cd47 inhibitor. See Unanue, E.R., PNAS 110:10886-87 (2013).
[0343] In another embodiment, the one or more ICIs comprise a TIGIT inhibitor.
[0344] T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) is an inhibitory receptor expressed on several immune cell types, including CD8+ T cells, natural killer, or NK, cells, T regulatory cells, or Tregs, and follicular T helper cells. TIGIT interacts with CD155 expressed on antigen-presenting cells or tumor cells to down-regulate T cell and natural killer (NK) cell functions. See, e.g., Harjunpaa, Clinical Experimental Immunology 200(2): 108- 19 (2020). TIGIT has been shown to be a mediator of resistance to existing checkpoint inhibitors, including anti-PD-1. TIGIT also directly suppresses the antitumor effector function on CD8 T cells. TIGIT inhibitors may include antibodies and small molecules. Non-limiting exemplary TIGIT inhibitor antibodies include vibostolimab (MK-7684), tiragolumab (RG6058), EOS_448, BMS- 986207, BGB-A1217, MTIG7192A, AB 154, ASP8374, and MK-7684.
[0345] The term "antibody" is meant to include intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. In another embodiment, "antibody" is meant to include soluble receptors that do not possess the Fc portion of the antibody. In one embodiment, the antibodies are humanized monoclonal antibodies and fragments thereof made by means of recombinant genetic engineering.
[0346] Another class of ICIs include polypeptides that bind to and block PD-1 receptors on T-cells without triggering inhibitor signal transduction. Such peptides include B7-DC polypeptides, B7-H1 polypeptides, B7-1 polypeptides and B7-2 polypeptides, and soluble fragments thereof, as disclosed in U.S. Pat. 8,114,845.
[0347] Another class of ICIs include compounds with peptide moieties that inhibit PD-1 signaling. Examples of such compounds are disclosed in U.S. Pat. 8,907,053 and have the structure: or a pharmaceutically acceptable salt thereof, wherein the compound comprises at least 5 amino acids useful as therapeutic agents capable of inhibiting the PD-1 signaling pathway.
[0348] Another class of ICIs include inhibitors of certain metabolic enzymes, such as indoleamine 2,3 dioxygenase (IDO), which is expressed by infiltrating myeloid cells and tumor cells. The IDO enzyme inhibits immune responses by depleting amino acids that are necessary for anabolic functions in T cells or through the synthesis of particular natural ligands for cytosolic receptors that are able to alter lymphocyte functions. Pardoll, Nature Reviews. Cancer 12:252-64 (2012); Lob, Cancer Immunol Immunother 58:153-57 (2009). Particular IDO blocking agents include, but are not limited to, levo-1- methyl typtophan (L-1MT) and 1-methyl-tryptophan (1MT). Qian et al., Cancer Res 69:5498-504 (2009); and Lob et al., Cancer Immunol Immunother 58:153-7 (2009).
[0349] In one embodiment, the one or more ICIs comprise nivolumab, pembrolizumab, pidilizumab, STLA1110, avelumab, atezolizumab, durvalumab, STI A1014, ipilimumab, tremelimumab, cemiplimab, tislelizumab, toripalimab, camrelizumab, GSK2831781, BMS-936559 or MED 14736, or a combination thereof.
[0350] Therapeutically effective amounts of a Compound of the Disclosure and, optionally, the one or more ICIs formulated in accordance with standard pharmaceutical practices, are administered to a human patient in need thereof. Whether such a treatment is indicated depends on the individual case and is subject to medical assessment (diagnosis) that takes into consideration signs, symptoms, and/or malfunctions that are present, the risks of developing particular signs, symptoms and/or malfunctions, and other factors.
[0351] A Compound of the Disclosure and, optionally, the one or more ICIs can be administered by any suitable route, for example by oral, buccal, inhalation, sublingual, rectal, vaginal, intracisternal or intrathecal through lumbar puncture, transurethral, nasal, percutaneous, i.e., transdermal, or parenteral (including intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a particular site) administration. Parenteral administration can be accomplished using a needle and syringe or using a high pressure technique. In one embodiment, a Compound of the Disclosure is administered orally to the subject. In one embodiment, a Compound of the Disclosure is administered intraveneously to the subject.
[0352] Pharmaceutical compositions include those wherein a Compound of the Disclosure and, optionally, the one or more ICIs are administered in an effective amount to achieve their intended purpose. The exact formulation, route of administration, and dosage of each combination partner is determined by an individual physician in view of the diagnosed condition or disease. Dosage amount and interval can be adjusted individually to provide levels of a Compound of the Disclosure, and, optionally, the one or more ICIs that is sufficient to maintain therapeutic effects.
[0353] Toxicity and therapeutic efficacy of a Compound of the Disclosure and/or the one or more ICIs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the maximum tolerated dose (MTD) of a compound, which defines as the highest dose that causes no unacceptable toxicity in a patient. The dose ratio between the maximum tolerated dose and therapeutic effects (e.g. inhibiting of tumor growth) is the therapeutic index. The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
[0354] A therapeutically effective amount of a Compound of the Disclosure, and, optionally, the one or more ICIs required for use in therapy varies with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the subject, and ultimately is determined by the attendant physician. For example, dosage amounts and intervals can be adjusted individually to provide plasma levels of a Compound of the Disclosure and/or the one or more ICIs that are sufficient to maintain the desired therapeutic effects. The desired dose conveniently can be administered in a single dose, or as multiple doses administered at appropriate intervals, for example as one, two, three, four or more subdoses per day. Multiple doses of the therapeutic agent(s) may be desired or required. For example, a Compound of the Disclosure and/or the one or more ICIs can be administered at a frequency of: one dose per day; four doses delivered as one dose per day at four-day intervals (q4d x 4); four doses delivered as one dose per day at three-day intervals (q3d x 4); one dose delivered per day at five-day intervals (qd x 5); one dose per week for three weeks (qwk3); five daily doses, with two days rest, and another five daily doses (5/2/5); or, any dose regimen determined to be appropriate for the circumstance.
[0355] The one or more ICIs are administered to the subject in therapeutically effective amounts. These amounts are guided by standard clinical practice, e.g., according to the prescribing information associated with the ICIs. For example, when the immune checkpoint inhibitor is a monoclonal antibody, about 1 to about 2000 mg can be administered to the subject as an intravenous infusion every 2-4 weeks. For example, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg of the antibody can be administered.
[0356] For example, when the one or more ICIs is nivolumab, 240 mg may be administered to the subject by intravenous infusion every two weeks, or 480 mg may be administered by intravenous infusion every for weeks.
[0357] For example, when the one or more ICIs is pembrolizumab, 200 mg may be administered to the subject by intravenous infusion every three weeks, or 400 mg may be administered to the subject by intravenous infusion every six weeks.
[0358] For example, when the one or more ICIs is avelumab, 800 mg may be administered to the subject by intravenous infusion every two weeks.
[0359] For example, when the one or more ICIs is atezolizumab, 840 mg may be administered by intravenous infusion every two weeks, or 1200 mg may be administered by intravenous infusion every three weeks, or 1680 mg may be administered by intravenous infusion every four weeks.
[0360] For example, when the one or more ICIs is ipilumumab, 3 mg/kg may be administered to the subject by intravenous infusion every three weeks.
[0361] For example, when the one or more ICIs is tremelimumab, 3 to 20 mg/kg may be administered to the subject by intravenous infusion every four weeks.
[0362] For example, when the one or more ICIs is GSK2831781, 1.5 to 5 mg/kg may be administered to the subject by intravenous infusion every 2-4 weeks. [0363] Representative dosing regimens for certain ICIs to treat certain cancers are provided in Table 4.
Table 4 [0364] In one embodiment, the one or more ICIs is an antibody, and 1-20 mg/kg is administered to the subject by intravenous infusion every 2-4 weeks. In another embodiment, 20-2000 mg of the antibody is administered to the subject by intravenous infusion every 2-4 weeks. In another embodiment, a Compound of the Disclosure is administered prior to administration of the antibody. In another embodiment, a Compound of the Disclosure is administered to the subject 1, 2, 3, 4, 5, 6, or 7 days prior to the day of administration of the antibody. In another embodiment, a Compound of the Disclosure is administered to the subject the day the antibody is administered. In another embodiment, a Compound of the Disclosure is administered to the subject 1, 2, 3, 4, 5, 6, or 7 days after the day of administration of the antibody.
[0365] For example, the subject receives pembrolizumab administered by intravenous infusion every three weeks and a Compound of the Disclosure is administered orally, wherein the first dose of a Compound of the Disclosure is administered prior to the first dose of pembrolizumab, the first dose of a Compound of the Disclosure is administered on the same day as the first dose of pembrolizumab, or the first dose of a Compound of the Disclosure is administered after to the first dose of pembrolizumab, e.g., until disease progression or until there is no therapeutic benefit.
[0366] For example, the subject receives nivolumab administered by intravenous infusion every two weeks and a Compound of the Disclosure orally administered 3 to 7 times a week, wherein the first dose of a Compound of the Disclosure is administered prior to the first dose of nivolumab, the first dose of a Compound of the Disclosure is administered on the same day as the first dose of nivolumab, or the first dose of a Compound of the Disclosure is administered after to the first dose of nivolumab, e.g., until disease progression or until there is no therapeutic benefit.
[0367] In another embodiment, the treatment of the cancer patient with a Compound of the Disclosure and one or more ICIs induces anti-proliferative response faster than when the immune checkpoint inhibitor is administered alone, e.g., the Compound of the Disclosure sensitizes the cancer cells to treatment is the immune checkpoint inhibitor.
IV. Kits
[0368] In another embodiment, the present disclosure provides kits for carrying out the methods and uses described herein, the kit comprising: (i) a Compound of the Disclosure; and (ii) a label with instructions for how to use the kit. [0369] In another embodiment, the present disclosure provides a kit for carrying out the methods and uses described herein, the kit comprising: (i) one or more immune checkpoint inhibitors; and (ii) a label with instructions for how to use the kit.
[0370] In another embodiment, the present disclosure provides a kit for carrying out the methods and uses described herein, the kit comprising: (i) a Compound of the Disclosure; (ii) one or more immune checkpoint inhibitors; and (iii) a label with instructions for how to use the kit.
[0371] In another embodiment, the label is approved by the United States Food and Drug Administration (FDA), the European Medicines Agency (EMA), the China Food and Drug Administration (CFDA), or the Japanese Ministry of Health Labor and Welfare (MHLW).
V. Particular Embodiments
[0372] The disclosure provides the following particular embodiments relating to Compounds of the Disclosure having any one of Formula (I)-(VII).
[0373] Embodiment 1. A compound having Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0374] R1 is selected from the group consisting of -CH=CHRla and -C=CRlb;
[0375] Rla is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR8R9;
[0376] Rlb is selected from the group consisting of hydrogen and C1-C6 alkyl;
[0377] R8 and R9 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, and (C1-C6 alkoxy)C1-C6 alkyl; or
[0378] R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo;
[0379] R2 is selected from the group consisting of hydrogen, halo, C1-C6 alkyl, and -OR7;
[0380] R7 is selected from the group consisting of C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, (optionally substituted C3-C6 cycloalkyl)C1-C6-alkyl, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4-to 7-membered heterocyclo)C1-C6 alkyl;
[0381] R3 is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl;
[0382] R4 is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, and C3-C6 cycloalkyl; and
[0383] R5 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted heterocyclo, optionally substituted aryl, and optionally substituted heteroaryl.
[0384] Embodiment 2. The compound any Embodiment 1, or a pharmaceutically acceptable salt or solvate thereof, with the following provisos:
[0385] (i) if R4 is cyano and R1 is -CH2NR8R9, then R8 and R9 are each independently selected from the group consisting of hydrogen, C2-C6 alkyl, C3-C6 cycloalkyl, and (Ci- C<> alkoxy)C1-C6 alkyl; or R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo; or
[0386] (ii) if R1 is -CH2NR8R9 and R8 and R9 are each methyl, then R4 is selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, and C3-C6 cycloalkyl; or
[0387] (iii) if R1 is -CH2NR8R9, R8 and R9 are each methyl, and R4 is cyano, then R3 is C1-C6 alkyl or C3-C6 cycloalkyl.
[0388] Embodiment 3. The compound of Embodiments 1 or 2 having Formula (II): (II), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0389] R6a is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl;
[0390] R6b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl; and
[0391] R6C is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl; C1-C6 alkoxy, C3-C6 cycloalkyl, (optionally substituted heterocyclo)-O-,
(aryl)alkyl-O-, and (heteroaryl)alkyl-O-. [0392] Embodiment 4. The compound of Embodiment 3 having Formula (III): or a pharmaceutically acceptable salt or solvate thereof, wherein R5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl.
[0393] Embodiment 5. The compound of Embodiment 4, or a pharmaceutically acceptable salt or solvate thereof, wherein R5a is optionally substituted 5-membered heteroaryl.
[0394] Embodiment 6. The compound of Embodiment 4, or a pharmaceutically acceptable salt or solvate thereof, having Formula (IV): wherein R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo.
[0395] Embodiment 7. The compound of Embodiment 6, or a pharmaceutically acceptable salt or solvate thereof, having Formula (V):
[0396] Embodiment 8. The compound of Embodiment 6, or a pharmaceutically acceptable salt or solvate thereof, having Formula (VI):
[0397] Embodiment 9. The compound of Embodiment 6, or a pharmaceutically acceptable salt or solvate thereof, having Formula (VII): (VII).
[0398] Embodiment 10. The compound of any one of Embodiments 1-9, or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is hydrogen.
[0399] Embodiment 11. The compound of any one of Embodiments 1-9, or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is C1-C4 alkyl.
[0400] Embodiment 12. The compound of any one of Embodiments 1-9, or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is -CH2NR8R9.
[0401] Embodiment 13. The compound of Embodiment 12, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 and R9 are C1-C6 alkyl.
[0402] Embodiment 14. The compound of Embodiment 13, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 and R9 are methyl.
[0403] Embodiment 15. The compound of Embodiment 12, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo.
[0404] Embodiment 16. The compound of Embodiment 15, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 and R9 taken together with the nitrogen atom to which they are attached form:
[0405] Embodiment 17. The compound of any one of Embodiments 1-16, or a pharmaceutically acceptable salt or solvate thereof, wherein R2 is -OR7.
[0406] Embodiment 18. The compound of Embodiment 17, or a pharmaceutically acceptable salt or solvate thereof, wherein R7 is methyl, ethyl, or propyl.
[0407] Embodiment 19. The compound of Embodiment 18, or a pharmaceutically acceptable salt or solvate thereof, wherein R7 is ethyl.
[0408] Embodiment 20. The compound of any one of Embodiments 1-19, or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is hydrogen or C1-C6 alkyl.
[0409] Embodiment 21. The compound of Embodiment 20, or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is hydrogen.
[0410] Embodiment 22. The compound of Embodiment 20, or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is methyl.
[0411] Embodiment 23. The compound of Embodiment 20, or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is ethyl.
[0412] Embodiment 24. The compound of any one of Embodiments 1-23, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is hydrogen or cyano.
[0413] Embodiment 25. The compound of Embodiment 24, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is hydrogen.
[0414] Embodiment 26. The compound of Embodiment 24, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is cyano.
[0415] Embodiment 27. The compound of any one of Embodiments 3-26, or a pharmaceutically acceptable salt or solvate thereof, wherein R6a is hydrogen or halogen.
[0416] Embodiment 28. The compound of Embodiment 27, or a pharmaceutically acceptable salt or solvate thereof, wherein R6a is hydrogen.
[0417] Embodiment 29. The compound of Embodiment 27, or a pharmaceutically acceptable salt or solvate thereof, wherein R6a is chloro.
[0418] Embodiment 30. The compound of Embodiment 1, or a pharmaceutically acceptable salt or solvate thereof, selected from any one or more of the compounds of Table 1A. [0419] Embodiment 31. The compound of Embodiment 30, or a pharmaceutically acceptable salt or solvate thereof, selected from any one or more of:
[0420] Embodiment 32. A pharmaceutical composition comprising the compound of any one of Embodiments 1-31 and a pharmaceutically acceptable excipient.
[0421] Embodiment 33. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Embodiments 1-31 or the pharmaceutical composition of Embodiment 32.
[0422] Embodiment 34. The method of Embodiment 34 further comprising administering a therapeutically effective amount one or more immune checkpoint inhibitors to the subject.
[0423] Embodiment 35. The method of Embodiment 34, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies, one or more anti-PD-Ll antibodies, one or more anti-CTLA-4 antibodies, one or more anti-LAG3 antibodies, one or more anti-TIM3 antibodies, one or more anti- VISTA antibodies, one or more anti-TIGIT antibodies, or one or more anti-cd47 antibodies, or a combination thereof.
[0424] Embodiment 36. The method of Embodiment 35 wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies.
[0425] Embodiment 37. The method of Embodiment 36, wherein the one or more anti-PD-1 antibodies comprise nivolumab, pembrolizumab, dostarlimab, retifanlimab, cemiplimab, vopratelimab (JTX-4014), spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), INCMGA00012 (MGA012), AMP-224, AMP-514 (MEDI0680) and/or Acrixolimab (YBL-006).
[0426] Embodiment 38. The method any one of Embodiments 35-37, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-Ll antibodies.
[0427] Embodiment 39. The method of Embodiment 38, wherein the one or more anti-PD-Ll antibodies comprise avelumab, atezolizumab, durvalumab, KN035, and/or cosibelimab (CK-301).
[0428] Embodiment 40. The method of any one of Embodiments 35-39, wherein the one or more immune checkpoint inhibitors comprise one or more anti-CTLA-4 antibodies.
[0429] Embodiment 41. The method of Embodiment 40, wherein the one or more anti-CTLA-4 antibodies comprise ipilimumab and/or tremelimumab.
[0430] Embodiment 42. The method of any one of Embodiments 35-41, wherein the one or more immune checkpoint inhibitors comprise one or more anti-LAG3 antibodies.
[0431] Embodiment 43. The method of Embodiment 42, wherein the one or more anti-LAG3 antibodies comprise relatlimab.
[0432] Embodiment 44. The method of any one of Embodiments 35-43 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIM3 antibodies.
[0433] Embodiment 45. The method of any one of Embodiments 35-44 wherein the one or more immune checkpoint inhibitors comprise one or more anti- VISTA antibodies.
[0434] Embodiment 46. The method of any one of Embodiments 35-45 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIGIT antibodies.
[0435] Embodiment 47. The method of any one of Embodiments 35-46 wherein the one or more immune checkpoint inhibitors comprise one or more anti-cd47 antibodies. [0436] Embodiment 48. The method of Embodiment 34 comprising administering a therapeutically effective amount of nivolumab, pembrolizumab, dostarlimab, retifanlimab, or cemiplimab to the subject.
[0437] Embodiment 49. The method of Embodiments 34 or 48 comprising administering a therapeutically effective amount of avelumab, atezolizumab, or durvalumab to the subject.
[0438] Embodiment 50. The method of any one of Embodiments 33-49, wherein the cancer is any one or more of the cancers of Table 2 and/or Table 3.
[0439] Embodiment 51. A method of inhibiting adenosine deaminase acting on RNA 1 (ADAR1) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Embodiments 1-31, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of Embodiment 32.
[0440] Embodiment 52. A method of providing immuno-oncology therapy to a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Embodiments 1-31, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of Embodiment 32.
[0441] Embodiment 53. A method of sensitizing tumor response to immunotherapy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Embodiments 1-31, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of Embodiment 42.
[0442] Embodiment 54. The method of Embodiment 53, wherein the tumor is resistant to immune checkpoint blockade.
[0443] Embodiment 55. The method of any one of Embodiments 51-54 comprising administering to the subject a therapeutically effective amount of Neratinib: [0444] Embodiment 56. A kit for carrying out the method of any one of Embodiments 33-55, the kit comprising: (i) a compound having Formula (I), or a pharmaceutically acceptable salt or solvate thereof; and (ii) a label with instructions for how to use the kit.
[0445] Embodiment 57. A kit for carrying out the method of any one of Embodiments 33-55, the kit comprising: (i) one or more immune checkpoint inhibitors; and (ii) a label with instructions for how to use the kit.
[0446] Embodiment 58. A kit for carrying out the method of any one of Embodiments 33-55, the kit comprising: (i) a compound having Formula (I), or a pharmaceutically acceptable salt or solvate thereof; (ii) one or more immune checkpoint inhibitors; and (iii) a label with instructions for how to use the kit.
[0447] Embodiment 59. The kit of any one of Embodiments 56-58, wherein the label is approved by the United States Food and Drug Administration (FDA), the European Medicines Agency (EMA), the China Food and Drug Administration (CFDA), or the Japanese Ministry of Health Labor and Welfare (MHLW).
[0448] The present disclosure also provides the following particular embodiments relating to compounds having Formula (VIII) and Compounds of the Disclosure having any one of Formula (IX)-(XVI).
[0449] Embodiment 1. A compound having Formula (IX): (IX), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0450] X is selected from the group consisting of -O- and -NRa-;
[0451] Ra is selected from the group consisting to hydrogen and C1-C4 alkyl;
[0452] R1 is selected from the group consisting of -CH=CHRla and -C=CRlb;
[0453] Rla is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR8R9;
[0454] Rlb is selected from the group consisting of hydrogen and C1-C6 alkyl;
[0455] R8 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted 4-to 7-membered heterocyclo, C2-C12 alkynyl, and (C1-C6 alkoxy)C1-C6 alkyl; [0456] R9 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; or
[0457] R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo;
[0458] R2 is selected from the group consisting of hydrogen, halo, C1-C6 alkyl, and -OR7;
[0459] R7 is selected from the group consisting of C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, (optionally substituted C3-C6 cycloalkyl)C1-C6-alkyl, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4-to 7-membered heterocyclo)C1-C6 alkyl;
[0460] R3 is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl;
[0461] R4 is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, and C3-C6 cycloalkyl; and
[0462] R5 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, optionally substituted aryl, (heterocyclo)alkyl, and optionally substituted 5- to 10- membered heteroaryl.
[0463] Embodiment 2. The compound Embodiment 1, or a pharmaceutically acceptable salt or solvate thereof, with the following provisos:
[0464] (i) if R4 is cyano and R1 is -CH2NR8R9, then R8 is hydrogen, C2-C6 alkyl, C3-C6 cycloalkyl, optionally substituted 4-to 7-membered heterocyclo, C2-C12 alkynyl, and (Ci- Ce alkoxy)C1-C6 alkyl; R9 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; or R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo; or
[0465] (ii) if R1 is -CH2NR8R9 and R8 and R9 are each methyl, then R4 is selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, and C3-C6 cycloalkyl; or
[0466] (iii) if R1 is -CH2NR8R9, R8 and R9 are each methyl, and R4 is cyano, then R3 is C1-C6 alkyl or C3-C6 cycloalkyl.
[0467] Embodiment 3. The compound of Embodiments 1 or 2 having Formula
(X): (X), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0468] R6a is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl;
[0469] R6b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl; and
[0470] R6C is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl; C1-C4 haloalkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, (optionally substituted 4- to 7-membered heterocyclo)-O-, (aryl)alkyl-O-, and (heteroaryl)alkyl-O-.
[0471] Embodiment 4. The compound of Embodiment 3, or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0472] R6b is hydrogen; and
[0473] R6C is selected from the group consisting of: or a pharmaceutically acceptable salt or solvate thereof, wherein R5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl. [0474] Embodiment 6. The compound of Embodiment 5, or a pharmaceutically acceptable salt or solvate thereof, wherein R5a is optionally substituted 5-membered heteroaryl.
[0475] Embodiment 7. The compound of Embodiment 5, or a pharmaceutically acceptable salt or solvate thereof, having Formula (XII): (XII), wherein R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo.
[0476] Embodiment 8. The compound of Embodiment 7, or a pharmaceutically acceptable salt or solvate thereof, having Formula (XIII): (XIII).
[0477] Embodiment 9. The compound of Embodiment 7, or a pharmaceutically acceptable salt or solvate thereof, having Formula (XIV): (XIV).
[0478] Embodiment 10. The compound of Embodiment 7, or a pharmaceutically acceptable salt or solvate thereof, having Formula (XV): (XV).
[0479] Embodiment 11. The compound of Embodiments 1 or 2 having Formula (XVI): or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is selected from the group consisting of optionally substituted 5- to 7-membered heterocylo and optionally substituted 5- to 10-membered heteroaryl.
[0480] Embodiment 12. The compound of Embodiment 11, or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is optionally substituted 5- to 7-membered heterocylo.
[0481] Embodiment 13. The compound of Embodiment 12, or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is selected from the group consisting of:
[0482] Embodiment 14. The compound of Embodiment 11, or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is optionally substituted 5- to 10-membered heteroaryl.
[0483] Embodiment 15. The compound of Embodiment 14, or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is selected from the group consisting of:
[0484] Embodiment 16. The compound of any one of Embodiments 1-15, or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is hydrogen.
[0485] Embodiment 17. The compound of any one of Embodiments 1-15, or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is C1-C4 alkyl.
[0486] Embodiment 18. The compound of any one of Embodiments 1-15, or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is -CH2NR8R9.
[0487] Embodiment 19. The compound of Embodiment 18, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 and R9 are C1-C6 alkyl.
[0488] Embodiment 20. The compound of Embodiment 19, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 and R9 are methyl.
[0489] Embodiment 21. The compound of Embodiment 18, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 is optionally substituted 4-to 7-membered heterocyclo.
[0490] Embodiment 22. The compound of Embodiment 21, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 is selected from the group consisting of:
[0491] Embodiment 23. The compound of Embodiment 18, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 is C2-C10 alkynyl.
[0492] Embodiment 24. The compound of Embodiment 18, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo. [0493] Embodiment 25. The compound of Embodiment 24, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 and R9 taken together with the nitrogen atom to which they are attached form:
[0494] Embodiment 26. The compound of any one of Embodiments 1-25, or a pharmaceutically acceptable salt or solvate thereof, wherein R2 is -OR7.
[0495] Embodiment 27. The compound of Embodiment 26, or a pharmaceutically acceptable salt or solvate thereof, wherein R7 is selected from the group consisting of methyl, ethyl, and propyl.
[0496] Embodiment 28. The compound of Embodiment 27, or a pharmaceutically acceptable salt or solvate thereof, wherein R7 is ethyl.
[0497] Embodiment 29. The compound of any one of Embodiments 1-28, or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is selected from the group consisting of hydrogen and C1-C6 alkyl.
[0498] Embodiment 30. The compound of Embodiment 29, or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is hydrogen.
[0499] Embodiment 31. The compound of Embodiment 29, or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is methyl.
[0500] Embodiment 32. The compound of Embodiment 29, or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is ethyl.
[0501] Embodiment 33. The compound of any one of Embodiments 1-32, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is selected from the group consisting of hydrogen and cyano.
[0502] Embodiment 34. The compound of Embodiment 33, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is hydrogen. [0503] Embodiment 35. The compound of Embodiment 33, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is cyano.
[0504] Embodiment 36. The compound of any one of Embodiments 3-10 or 15-35, or a pharmaceutically acceptable salt or solvate thereof, wherein R6a is selected from the group consisting of hydrogen and halogen.
[0505] Embodiment 37. The compound of Embodiment 36, or a pharmaceutically acceptable salt or solvate thereof, wherein R6a is hydrogen.
[0506] Embodiment 38. The compound of Embodiment 36, or a pharmaceutically acceptable salt or solvate thereof, wherein R6a is chloro.
[0507] Embodiment 39. The compound of any one of Embodiments 1-38, or a pharmaceutically acceptable salt or solvate thereof, wherein X is -O-.
[0508] Embodiment 40. The compound of any one of Embodiments 1-38, or a pharmaceutically acceptable salt or solvate thereof, wherein X is -NH-.
[0509] Embodiment 41 The compound of Embodiment 1, or a pharmaceutically acceptable salt or solvate thereof, selected from any one or more of the compounds of Table 1A and/or Table 2 A.
[0510] Embodiment 42. The compound of Embodiment 41, or a pharmaceutically acceptable salt or solvate thereof, selected from the group consisting of:
[0511] Embodiment 43. A pharmaceutical composition comprising the compound of any one of Embodiments 1-42 and a pharmaceutically acceptable excipient.
[0512] Embodiment 44. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Embodiments 1-42 or the pharmaceutical composition of Embodiment 32.
[0513] Embodiment 45. The method of Embodiment 44 further comprising administering a therapeutically effective amount one or more immune checkpoint inhibitors to the subject.
[0514] Embodiment 46. The method of Embodiment 44, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies, one or more anti-PD-Ll antibodies, one or more anti-CTLA-4 antibodies, one or more anti-LAG3 antibodies, one or more anti-TIM3 antibodies, one or more anti- VISTA antibodies, one or more anti-TIGIT antibodies, or one or more anti-cd47 antibodies, or a combination thereof.
[0515] Embodiment 47. The method of Embodiment 46 wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies.
[0516] Embodiment 48. The method of Embodiment 47, wherein the one or more anti-PD-1 antibodies comprise nivolumab, pembrolizumab, dostarlimab, retifanlimab, cemiplimab, vopratelimab (JTX-4014), spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), INCMGA00012 (MGA012), AMP-224, AMP-514 (MEDI0680) and/or Acrixolimab (YBL-006).
[0517] Embodiment 49. The method any one of Embodiments 46-48, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-Ll antibodies.
[0518] Embodiment 50. The method of Embodiment 49, wherein the one or more anti-PD-Ll antibodies comprise avelumab, atezolizumab, durvalumab, KN035, and/or cosibelimab (CK-301). [0519] Embodiment 51. The method of any one of Embodiments 46-50, wherein the one or more immune checkpoint inhibitors comprise one or more anti-CTLA-4 antibodies.
[0520] Embodiment 52. The method of Embodiment 51, wherein the one or more anti-CTLA-4 antibodies comprise ipilimumab and/or tremelimumab.
[0521] Embodiment 53. The method of any one of Embodiments 46-52, wherein the one or more immune checkpoint inhibitors comprise one or more anti-LAG3 antibodies.
[0522] Embodiment 54. The method of Embodiment 53, wherein the one or more anti-LAG3 antibodies comprise relatlimab.
[0523] Embodiment 55. The method of any one of Embodiments 46-54 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIM3 antibodies.
[0524] Embodiment 56. The method of any one of Embodiments 46-55 wherein the one or more immune checkpoint inhibitors comprise one or more anti- VISTA antibodies.
[0525] Embodiment 57. The method of any one of Embodiments 46-56 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIGIT antibodies.
[0526] Embodiment 58. The method of any one of Embodiments 46-57 wherein the one or more immune checkpoint inhibitors comprise one or more anti-cd47 antibodies.
[0527] Embodiment 59. The method of Embodiment 45 comprising administering a therapeutically effective amount of nivolumab, pembrolizumab, dostarlimab, retifanlimab, or cemiplimab to the subject.
[0528] Embodiment 60. The method of Embodiments 45 or 59 comprising administering a therapeutically effective amount of avelumab, atezolizumab, or durvalumab to the subject.
[0529] Embodiment 61. The method of any one of Embodiments 44-60, wherein the cancer is any one or more of the cancers of Table 2 and/or Table 3.
[0530] Embodiment 62. A method of inhibiting adenosine deaminase acting on
RNA 1 (ADAR1) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Embodiments 1-42, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of Embodiment 43.
[0531] Embodiment 63. A method of providing immuno-oncology therapy to a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Embodiments 1-42, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of Embodiment 43.
[0532] Embodiment 64. A method of sensitizing tumor response to immunotherapy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Embodiments 1-42, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of Embodiment 43.
[0533] Embodiment 65. The method of Embodiment 64, wherein the tumor is resistant to immune checkpoint blockade.
[0534] Embodiment 66. The method of any one of Embodiments 62-65 comprising administering to the subject a therapeutically effective amount of Neratinib:
Embodiment 67. A kit for carrying out the method of any one of Embodiments 44-66, the kit comprising: (i) a compound having Formula (IX), or a pharmaceutically acceptable salt or solvate thereof; and (ii) a label with instructions for how to use the kit.
Embodiment 68. A kit for carrying out the method of any one of Embodiments 44-66, the kit comprising: (i) one or more immune checkpoint inhibitors; and (ii) a label with instructions for how to use the kit.
Embodiment 69. A kit for carrying out the method of any one of Embodiments 44-66, the kit comprising: (i) a compound having Formula (IX), or a pharmaceutically acceptable salt or solvate thereof; (ii) one or more immune checkpoint inhibitors; and (iii) a label with instructions for how to use the kit.
Embodiment 70. The kit of any one of Embodiments 67-69, wherein the label is approved by the United States Food and Drug Administration (FDA), the European Medicines Agency (EMA), the China Food and Drug Administration (CFDA), or the Japanese Ministry of Health Labor and Welfare (MHLW).
Embodiment 71. A compound having Formula (VIII): or a pharmaceutically acceptable salt or solvate thereof, wherein:
R10 is selected from the group consisting of chloro and -N(H)R5;
R11 is selected from the group consisting of hydrogen and -C(=0)CH3;
R2 is selected from the group consisting of hydrogen, halo, C1-C6 alkyl, and -OR7;
R7 is selected from the group consisting of C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, (optionally substituted C3-C6 cycloalkyl)C1-C6-alkyl, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4-to 7-membered heterocyclo)C1-C6 alkyl;
R3 is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl;
R4 is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, and C3-C6 cycloalkyl; and
[0535] R5 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, optionally substituted aryl, (heterocyclo)alkyl, and optionally substituted 5- to 10- membered heteroaryl.
[0536] The present disclosure also provides the following particular embodiments relating to therapeutic methods of treatment, purpose-limited products, and purposelimited processes.
A. Therapeutic Method of Treatment Embodiments
[0537] Embodiment 1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject:
[0538] (a) compound having Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0539] R1 is selected from the group consisting of -CH=CHRla and -C=CRlb; [0540] Rla is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR8R9,;
[0541] Rlb is selected from the group consisting of hydrogen and C1-C6 alkyl;
[0542] R8 and R9 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, and (C1-C6 alkoxy) C1-C6 alkyl; or
[0543] R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo;
[0544] R2 is selected from the group consisting of hydrogen, halo, C1-C6 alkyl, and -OR7;
[0545] R7 is selected from the group consisting of C1-C6 alkyl, optionally substituted C3-
C<> cycloalkyl, (optionally substituted C3-C6 cycloalkyl)C1-C6-alkyl, (C1-C6-alkoxy)C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4- to 7-membered heterocyclo)C1-C6 alkyl;
[0546] R3 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl;
[0547] R4 is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, and C3-C6 cycloalkyl; and
[0548] R5 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted heterocyclo, optionally substituted aryl, and optionally substituted heteroaryl; and
[0549] (b) a therapeutically effective amount one or more immune checkpoint inhibitors.
[0550] Embodiment !. The method of Embodiment 1, wherein the compound having Formula (I) is a compound having Formula (II):
[0551] or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0552] R6a is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl;
[0553] R6b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl; and [0554] R6C is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl; C1-C6 alkoxy, C3-C6 cycloalkyl, (optionally substituted heterocyclo)-O-, (aryl)alkyl-O-, and (heteroaryl)alkyl-O-.
[0555] Embodiment 3. The method of Embodiment 2, wherein the compound having Formula (I) is a compound having Formula (III): or a pharmaceutically acceptable salt or solvate thereof, wherein R5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl.
[0556] Embodiment 4. The method of Embodiment 3, wherein R5a is optionally substituted 5-membered heteroaryl.
[0557] Embodiment 5. The method of Embodiment 3, wherein the compound having Formula (III) is a compound having Formula (IV): wherein R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo.
[0558] Embodiment 6. The method of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (V): [0559] Embodiment 7. The method of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (VI):
[0560] Embodiment 8. The method of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (VII): (VII).
[0561] Embodiment 9. The method of any one of Embodiments 1-8, wherein Rla is hydrogen.
[0562] Embodiment 10. The method of any one of Embodiments 1-8, wherein Rla is
Ci-C4 alkyl.
[0563] Embodiment 11. The method of any one of Embodiments 1-8, wherein Rla is -CH2NR8R9.
[0564] Embodiment 12. The method of Embodiment 11, wherein R8 and R9 are C1-C6 alkyl.
[0565] Embodiment 13. The method of Embodiment 12, wherein R8 and R9 are methyl.
[0566] Embodiment 14. The method of Embodiment 11, wherein R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo.
[0567] Embodiment 15. The method of Embodiment 14, wherein R8 and R9 taken together with the nitrogen atom to which they are attached form:
[0568] Embodiment 16. The method of any one of Embodiments 1-15, wherein R2 is -OR7.
[0569] Embodiment 17. The method of Embodiment 16, wherein R7 is methyl, ethyl, or propyl.
[0570] Embodiment 18. The method of Embodiment 17, wherein R7 is ethyl.
[0571] Embodiment 19. The method of any one of Embodiments 1-18, wherein R3 is hydrogen or C1-C6 alkyl.
[0572] Embodiment 20. The method of Embodiment 19, wherein R3 is hydrogen.
[0573] Embodiment 21. The method of Embodiment 19, wherein R3 is methyl.
[0574] Embodiment 22. The method of Embodiment 19, wherein R3 is ethyl.
[0575] Embodiment 23. The method of any one of Embodiments 1-22, wherein R4 is hydrogen or cyano.
[0576] Embodiment 24. The method of Embodiment 23, wherein R4 is hydrogen.
[0577] Embodiment 25. The method of Embodiment 23, wherein R4 is cyano.
[0578] Embodiment 26. The method of any one of Embodiments 2-25, wherein R6a is hydrogen or halogen.
[0579] Embodiment 27. The method of Embodiment 26, wherein R6a is hydrogen.
[0580] Embodiment 28. The method of Embodiment 26, wherein R6a is chloro.
[0581] Embodiment 29. The method of Embodiment 1, selected from any one or more of the compounds of Table 1 A.
[0582] Embodiment 30. The method of Embodiment 29, or a pharmaceutically acceptable salt or solvate thereof, selected from any one or more of:
[0583] Embodiment 31. The method of any one of Embodiments 1-30, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies, one or more anti-PD-Ll antibodies, one or more anti-CTLA-4 antibodies, one or more anti-LAG3 antibodies, one or more anti-TIM3 antibodies, one or more anti- VISTA antibodies, one or more anti-TIGIT antibodies, or one or more anti-cd47 antibodies, or a combination thereof.
[0584] Embodiment 32. The method of Embodiment 31 wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies.
[0585] Embodiment 33. The method of Embodiment 32, wherein the one or more anti-PD-1 antibodies comprise nivolumab, pembrolizumab, dostarlimab, retifanlimab, cemiplimab, vopratelimab (JTX-4014), spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), INCMGA00012 (MGA012), AMP-224, AMP-514 (MEDI0680) and/or Acrixolimab (YBL-006).
[0586] Embodiment 34. The method any one of Embodiments 31-33, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-Ll antibodies. [0587] Embodiment 35. The method of Embodiment 34, wherein the one or more anti-PD-Ll antibodies comprise avelumab, atezolizumab, durvalumab, KN035, and/or cosibelimab (CK-301).
[0588] Embodiment 36. The method of any one of Embodiments 31-35, wherein the one or more immune checkpoint inhibitors comprise one or more anti-CTLA-4 antibodies.
[0589] Embodiment 37. The method of Embodiment 36, wherein the one or more anti-CTLA-4 antibodies comprise ipilimumab and/or tremelimumab.
[0590] Embodiment 38. The method of any one of Embodiments 31-37, wherein the one or more immune checkpoint inhibitors comprise one or more anti-LAG3 antibodies.
[0591] Embodiment 39. The method of Embodiment 38, wherein the one or more anti-LAG3 antibodies comprise relatlimab.
[0592] Embodiment 40. The method of any one of Embodiments 31-39 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIM3 antibodies.
[0593] Embodiment 41. The method of any one of Embodiments 31-40 wherein the one or more immune checkpoint inhibitors comprise one or more anti- VISTA antibodies.
[0594] Embodiment 42. The method of any one of Embodiments 31-41 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIGIT antibodies.
[0595] Embodiment 43. The method of any one of Embodiments 31-42 wherein the one or more immune checkpoint inhibitors comprise one or more anti-cd47 antibodies.
[0596] Embodiment 44. The method of Embodiment 31 comprising administering a therapeutically effective amount of nivolumab, pembrolizumab, dostarlimab, retifanlimab, or cemiplimab to the subject.
[0597] Embodiment 45. The method of Embodiments 31 or 44 comprising administering a therapeutically effective amount of avelumab, atezolizumab, or durvalumab to the subject.
[0598] Embodiment 46. The method of any one of Embodiments 1-45, wherein the cancer is any one or more of the cancers of Table 2 and/or Table 3.
[0599] Embodiment 47. A kit for carrying out the method of any one of
Embodiments 1-46, the kit comprising: (i) the compound pharmaceutically acceptable salt or solvate thereof; and (ii) a label with instructions for how to use the kit. [0600] Embodiment 48. A kit for carrying out the method of any one of Embodiments 1-46, the kit comprising: (i) one or more immune checkpoint inhibitors; and (ii) a label with instructions for how to use the kit.
[0601] Embodiment 49. A kit for carrying out the method of any one of Embodiments 1-46, the kit comprising: (i) the compound, or a pharmaceutically acceptable salt or solvate thereof; (ii) one or more immune checkpoint inhibitors; and (iii) a label with instructions for how to use the kit.
[0602] Embodiment 50. The kit of any one of Embodiments 47-9, wherein the label is approved by the United States Food and Drug Administration (FDA), the European Medicines Agency (EMA), the China Food and Drug Administration (CFDA), or the Japanese Ministry of Health Labor and Welfare (MHLW).
B. Purpose-Limited Product Embodiments
[0603] Embodiment 1. A compound having Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0604] R1 is selected from the group consisting of -CH=CHRla and -C=CRlb;
[0605] Rla is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR8R9,;
[0606] Rlb is selected from the group consisting of hydrogen and C1-C6 alkyl;
[0607] R8 and R9 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, and (C1-C6 alkoxy)C1-C6 alkyl; or
[0608] R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo;
[0609] R2 is selected from the group consisting of hydrogen, halo, C1-C6 alkyl, and -OR7;
[0610] R7 is selected from the group consisting of C1-C6 alkyl, optionally substituted C3-
C<> cycloalkyl, (optionally substituted C3-C6 cycloalkyl)C1-C6-alkyl, (C1-C6-alkoxy)C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4- to 7-membered heterocyclo)C1-C6 alkyl; [0611] R3 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl;
[0612] R4 is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, and C3-C6 cycloalkyl; and
[0613] R5 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted heterocyclo, optionally substituted aryl, and optionally substituted heteroaryl,
[0614] for use in treating cancer in a subject in need thereof, wherein the compound having Formula (I) is to be administered in combination with one or more immune checkpoint inhibitors.
Embodiment 2. The compound for use of Embodiment 1, wherein the compound having Formula (I) is a compound having Formula (II): (II), or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0615] R6a is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl;
[0616] R6b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl; and
[0617] R6C is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl; C1-C6 alkoxy, C3-C6 cycloalkyl, (optionally substituted heterocyclo)-O-, (aryl)alkyl-O-, and (heteroaryl)alkyl-O-.
Embodiment 3. The compound for use of Embodiment 2, wherein the compound having Formula (I) is a compound having Formula (III): (III), or a pharmaceutically acceptable salt or solvate thereof, wherein R5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl.
[0618] Embodiment 4. The compound for use of Embodiment 3, wherein R5a is optionally substituted 5-membered heteroaryl.
[0619] Embodiment 5. The compound for use of Embodiment 3, wherein the compound having Formula (III) is a compound having Formula (IV): wherein R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6-membered heterocyclo.
[0620] Embodiment 6. The compound for use of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (V):
[0621] Embodiment 7. The compound for use of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (VI):
[0622] Embodiment 8. The compound for use of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (VII): (VII).
[0623] Embodiment 9. The compound for use of any one of Embodiments 1-8, wherein Rla is hydrogen.
[0624] Embodiment 10. The compound for use of any one of Embodiments 1-8, wherein Rla is C1-C4 alkyl.
[0625] Embodiment 11. The compound for use of any one of Embodiments 1-8, wherein Rla is -CH2NR8R9.
[0626] Embodiment 12. The compound for use of Embodiment 11, wherein R8 and
R9 are C1-C6 alkyl.
[0627] Embodiment 13. The compound for use of Embodiment 12, wherein R8 and
R9 are methyl.
[0628] Embodiment 14. The compound for use of Embodiment 11, wherein R8 and
R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo.
[0629] Embodiment 15. The compound for use of Embodiment 14, wherein R8 and R9 taken together with the nitrogen atom to which they are attached form:
[0630] Embodiment 16. The compound for use of any one of Embodiments 1-15, wherein R2 is -OR7.
[0631] Embodiment 17. The compound for use of Embodiment 16, wherein R7 is methyl, ethyl, or propyl.
[0632] Embodiment 18. The compound for use of Embodiment 17, wherein R7 is ethyl.
[0633] Embodiment 19. The compound for use of any one of Embodiments 1-18, wherein R3 is hydrogen or C1-C6 alkyl.
[0634] Embodiment 20. The compound for use of Embodiment 19, wherein R3 is hydrogen. [0635] Embodiment 21. The compound for use of Embodiment 19, wherein R3 is methyl.
[0636] Embodiment 22. The compound for use of Embodiment 19, wherein R3 is ethyl.
[0637] Embodiment 23. The compound for use of any one of Embodiments 1-22, wherein R4 is hydrogen or cyano.
[0638] Embodiment 24. The compound for use of Embodiment 23, wherein R4 is hydrogen.
[0639] Embodiment 25. The compound for use of Embodiment 23, wherein R4 is cyano.
[0640] Embodiment 26. The compound for use of any one of Embodiments 2-25, wherein R6a is hydrogen or halogen.
[0641] Embodiment 27. The compound for use of Embodiment 26, wherein R6a is hydrogen.
[0642] Embodiment 28. The compound for use of Embodiment 26, wherein R6a is chloro.
[0643] Embodiment 29. The compound for use of Embodiment 1, selected from any one or more of the compounds of Table 1 A.
[0644] Embodiment 30. The compound for use of Embodiment 29, or a pharmaceutically acceptable salt or solvate thereof, selected from any one or more of:
[0645] Embodiment 31. The compound for use of any one of Embodiments 1-30, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies, one or more anti-PD-Ll antibodies, one or more anti-CTLA-4 antibodies, one or more anti-LAG3 antibodies, one or more anti-TIM3 antibodies, one or more anti- VISTA antibodies, one or more anti-TIGIT antibodies, or one or more anti-cd47 antibodies, or a combination thereof.
[0646] Embodiment 32. The compound for use of Embodiment 31 wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies.
[0647] Embodiment 33. The compound for use of Embodiment 32, wherein the one or more anti-PD-1 antibodies comprise nivolumab, pembrolizumab, dostarlimab, retifanlimab, cemiplimab, vopratelimab (JTX-4014), spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IB 1308), tislelizumab (BGB-A317), INCMGA00012 (MGA012), AMP-224, AMP-514 (MEDI0680) and/or Acrixolimab (YBL-006).
[0648] Embodiment 34. The compound for use any one of Embodiments 31-33, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-Ll antibodies. [0649] Embodiment 35. The compound for use of Embodiment 34, wherein the one or more anti-PD-Ll antibodies comprise avelumab, atezolizumab, durvalumab, KN035, and/or cosibelimab (CK-301).
[0650] Embodiment 36. The compound for use of any one of Embodiments 31-35, wherein the one or more immune checkpoint inhibitors comprise one or more anti- CTLA-4 antibodies.
[0651] Embodiment 37. The compound for use of Embodiment 36, wherein the one or more anti-CTLA-4 antibodies comprise ipilimumab and/or tremelimumab.
[0652] Embodiment 38. The compound for use of any one of Embodiments 31-37, wherein the one or more immune checkpoint inhibitors comprise one or more anti-LAG3 antibodies.
[0653] Embodiment 39. The compound for use of Embodiment 38, wherein the one or more anti-LAG3 antibodies comprise relatlimab.
[0654] Embodiment 40. The compound for use of any one of Embodiments 31-39 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIM3 antibodies.
[0655] Embodiment 41. The compound for use of any one of Embodiments 31-40 wherein the one or more immune checkpoint inhibitors comprise one or more anti- VISTA antibodies.
[0656] Embodiment 42. The compound for use of any one of Embodiments 31-41 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIGIT antibodies.
[0657] Embodiment 43. The compound for use of any one of Embodiments 31-42 wherein the one or more immune checkpoint inhibitors comprise one or more anti-cd47 antibodies.
[0658] Embodiment 44. The compound for use of Embodiment 31 comprising administering a therapeutically effective amount of nivolumab, pembrolizumab, dostarlimab, retifanlimab, or cemiplimab to the subject.
[0659] Embodiment 45. The compound for use of Embodiments 31 or 44 comprising administering a therapeutically effective amount of avelumab, atezolizumab, or durvalumab to the subject.
[0660] Embodiment 46. The compound for use of any one of Embodiments 1-45, wherein the cancer is any one or more of the cancers of Table 2 and/or Table 3. C. Purpose-limited Process Embodiments
[0661] Embodiment 1. Use of a compound having Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0662] R1 is selected from the group consisting of -CH=CHRla and -C=CRlb;
[0663] Rla is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR8R9,;
[0664] Rlb is selected from the group consisting of hydrogen and C1-C6 alkyl;
[0665] R8 and R9 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, and (C1-C6 alkoxy)C1-C6 alkyl; or
[0666] R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo;
[0667] R2 is selected from the group consisting of hydrogen, halo, C1-C6 alkyl, and -OR7;
[0668] R7 is selected from the group consisting of C1-C6 alkyl, optionally substituted C3-
C<> cycloalkyl, (optionally substituted C3-C6 cycloalkyl)C1-C6-alkyl, (C1-C6-alkoxyjC1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4- to 7-membered heterocyclo)C1-C6 alkyl;
[0669] R3 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl;
[0670] R4 is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, and C3-C6 cycloalkyl; and
[0671] R5 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted heterocyclo, optionally substituted aryl, and optionally substituted heteroaryl,
[0672] in the manufacture of a medicament for treating cancer in a subject in need thereof, wherein the compound having Formula (I) is to be administered in combination with one or more immune checkpoint inhibitors.
[0673] Embodiment !. The use of Embodiment 1, wherein the compound having Formula (I) is a compound having Formula (II): (ID, or a pharmaceutically acceptable salt or solvate thereof, wherein:
[0674] R6a is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl;
[0675] R6b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl; and
[0676] R6C is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl; C1-C6 alkoxy, C3-C6 cycloalkyl, (optionally substituted heterocyclo)-O-, (aryl)alkyl-O-, and (heteroaryl)alkyl-O-.
[0677] Embodiment 3. The use of Embodiment 2, wherein the compound having Formula (I) is a compound having Formula (III): or a pharmaceutically acceptable salt or solvate thereof, wherein R5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl.
[0678] Embodiment 4. The use of Embodiment 3, wherein R5a is optionally substituted 5-membered heteroaryl.
[0679] Embodiment 5. The use of Embodiment 3, wherein the compound having
Formula (III) is a compound having Formula (IV): wherein R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo.
[0680] Embodiment 6. The use of Embodiment 5, wherein the compound having Formula (IV) is a compound having Formula (V):
[0681] Embodiment 7. The use of Embodiment 5, wherein the compound having
Formula (IV) is a compound having Formula (VI): (VI).
[0682] Embodiment 8. The use of Embodiment 5, wherein the compound having
Formula (IV) is a compound having Formula (VII): (VII).
[0683] Embodiment 9. The use of any one of Embodiments 1-8, wherein Rla is hydrogen.
[0684] Embodiment 10. The use of any one of Embodiments 1-8, wherein Rla is Ci-
C4 alkyl.
[0685] Embodiment 11. The use of any one of Embodiments 1-8, wherein Rla is -CH2NR8R9. [0686] Embodiment 12. The use of Embodiment 11, wherein R8 and R9 are C1-C6 alkyl.
[0687] Embodiment 13. The use of Embodiment 12, wherein R8 and R9 are methyl.
[0688] Embodiment 14. The use of Embodiment 11, wherein R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo.
[0689] Embodiment 15. The use of Embodiment 14, wherein R8 and R9 taken together with the nitrogen atom to which they are attached form:
[0690] Embodiment 16. The use of any one of Embodiments 1-15, wherein R2 is -
OR7.
[0691] Embodiment 17. The use of Embodiment 16, wherein R7 is methyl, ethyl, or propyl.
[0692] Embodiment 18. The use of Embodiment 17, wherein R7 is ethyl.
[0693] Embodiment 19. The use of any one of Embodiments 1-18, wherein R3 is hydrogen or C1-C6 alkyl.
[0694] Embodiment 20. The use of Embodiment 19, wherein R3 is hydrogen.
[0695] Embodiment 21. The use of Embodiment 19, wherein R3 is methyl.
[0696] Embodiment 22. The use of Embodiment 19, wherein R3 is ethyl.
[0697] Embodiment 23. The use of any one of Embodiments 1-22, wherein R4 is hydrogen or cyano.
[0698] Embodiment 24. The use of Embodiment 23, wherein R4 is hydrogen.
[0699] Embodiment 25. The use of Embodiment 23, wherein R4 is cyano.
[0700] Embodiment 26. The use of any one of Embodiments 2-25, wherein R6a is hydrogen or halogen.
[0701] Embodiment 27. The use of Embodiment 26, wherein R6a is hydrogen.
[0702] Embodiment 28. The use of Embodiment 26, wherein R6a is chloro.
[0703] Embodiment 29. The use of Embodiment 1, selected from any one or more of the compounds of Table 1A.
[0704] Embodiment 30. The use of Embodiment 29, or a pharmaceutically acceptable salt or solvate thereof, selected from any one or more of:
[0705] Embodiment 31. The use of any one of Embodiments 1-30, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies, one or more anti-PD-Ll antibodies, one or more anti-CTLA-4 antibodies, one or more anti- LAG3 antibodies, one or more anti-TIM3 antibodies, one or more anti-VISTA antibodies, one or more anti-TIGIT antibodies, or one or more anti-cd47 antibodies, or a combination thereof.
[0706] Embodiment 32. The use of Embodiment 31 wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies.
[0707] Embodiment 33. The use of Embodiment 32, wherein the one or more anti-PD-1 antibodies comprise nivolumab, pembrolizumab, dostarlimab, retifanlimab, cemiplimab, vopratelimab (JTX-4014), spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), INCMGA00012 (MGA012), AMP-224, AMP-514 (MEDI0680) and/or Acrixolimab (YBL-006).
[0708] Embodiment 34. The use any one of Embodiments 31-33, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-Ll antibodies. [0709] Embodiment 35. The use of Embodiment 34, wherein the one or more anti- PD-L1 antibodies comprise avelumab, atezolizumab, durvalumab, KN035, and/or cosibelimab (CK-301).
[0710] Embodiment 36. The use of any one of Embodiments 31-35, wherein the one or more immune checkpoint inhibitors comprise one or more anti-CTLA-4 antibodies.
[0711] Embodiment 37. The use of Embodiment 36, wherein the one or more anti-
CTLA-4 antibodies comprise ipilimumab and/or tremelimumab.
[0712] Embodiment 38. The use of any one of Embodiments 31-37, wherein the one or more immune checkpoint inhibitors comprise one or more anti-LAG3 antibodies.
[0713] Embodiment 39. The use of Embodiment 38, wherein the one or more anti-
LAG3 antibodies comprise relatlimab.
[0714] Embodiment 40. The use of any one of Embodiments 31-39 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIM3 antibodies.
[0715] Embodiment 41. The use of any one of Embodiments 31-40 wherein the one or more immune checkpoint inhibitors comprise one or more anti- VISTA antibodies.
[0716] Embodiment 42. The use of any one of Embodiments 31-41 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIGIT antibodies.
[0717] Embodiment 43. The use of any one of Embodiments 31-42 wherein the one or more immune checkpoint inhibitors comprise one or more anti-cd47 antibodies.
[0718] Embodiment 44. The use of Embodiment 31 comprising administering a therapeutically effective amount of nivolumab, pembrolizumab, dostarlimab, retifanlimab, or cemiplimab to the subject.
[0719] Embodiment 45. The use of Embodiments 31 or 44 comprising administering a therapeutically effective amount of avelumab, atezolizumab, or durvalumab to the subject.
[0720] Embodiment 46. The use of any one of Embodiments 1-45, wherein the cancer is any one or more of the cancers of Table 2 and/or Table 3.
VII. Definitions
[0721] The term "halo" or "halogen" as used herein by itself or as part of another group refers to -Cl, -F, -Br, or -I.
[0722] The term "nitro" as used herein by itself or as part of another group refers to -NO2. [0723] The term "cyano" as used herein by itself or as part of another group refers to -CN.
[0724] The term "hydroxy" as herein used by itself or as part of another group refers to -OH.
[0725] The term "alkyl" as used herein by itself or as part of another group refers to a straight- or branched-chain aliphatic hydrocarbon containing one to twelve carbon atoms, i.e., a C1-C12 alkyl, or the number of carbon atoms designated, e.g., a Ci alkyl such as methyl, a C2 alkyl such as ethyl, etc. In one embodiment, the alkyl is a C1-C10 alkyl. In another embodiment, the alkyl is a C1-C6 alkyl. In another embodiment, the alkyl is a C1-C4 alkyl. In another embodiment, the alkyl is a C1-C3 alkyl, i.e., methyl, ethyl, propyl, or isopropyl. Non-limiting exemplary C1-C12 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, zso-butyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, and decyl.
[0726] The term "alkenyl" as used herein by itself or as part of another group refers to an alkyl group containing one, two, or three carbon-to-carbon double bonds. In one embodiment, the alkenyl group is a C2-C6 alkenyl group. In another embodiment, the alkenyl group is a C2-C4 alkenyl group. In another embodiment, the alkenyl group has one carbon-to-carbon double bond. Non-limiting exemplary alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, pentenyl, and hexenyl.
[0727] The term "alkynyl" as used herein by itself or as part of another group refers to an alkyl group containing one or two carbon-to-carbon triple bonds. In one embodiment, the alkynyl is a C2-C6 alkynyl. In another embodiment, the alkynyl is a C2-C4 alkynyl. In another embodiment, the alkynyl has one carbon-to-carbon triple bond. Non-limiting exemplary alkynyl groups include ethynyl, propynyl, butynyl, 2-butynyl, pentynyl, and hexynyl groups.
[0728] The term "haloalkyl" as used herein by itself or as part of another group refers to an alkyl group substituted by one or more fluorine, chlorine, bromine, and/or iodine atoms. In one embodiment, the alkyl is substituted by one, two, or three fluorine and/or chlorine atoms. In another embodiment, the alkyl is substituted by one, two, or three fluorine atoms. In another embodiment, the alkyl is a C1-C6 alkyl. In another embodiment, the alkyl is a C1-C4 alkyl. In another embodiment, the alkyl group is a Ci or C2 alkyl. Non-limiting exemplary haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3,3-trifluoropropyl, 4,4,4-trifluorobutyl, and trichloromethyl groups.
[0729] The terms "hydroxy alkyl" or "(hydroxy)alkyl" as used herein by themselves or as part of another group refer to an alkyl group substituted with one, two, or three hydroxy groups. In one embodiment, the alkyl is a C1-C6 alkyl. In another embodiment, the alkyl is a C1-C4 alkyl. In another embodiment, the alkyl is a Ci or C2 alkyl. In another embodiment, the hydroxyalkyl is a monohydroxyalkyl group, i.e., substituted with one hydroxy group. In another embodiment, the hydroxy alkyl group is a dihydroxy alkyl group, i.e., substituted with two hydroxy groups. Non-limiting exemplary (hydroxyl) alkyl groups include hydroxymethyl, hydroxyethyl, hydroxypropyl and hydroxybutyl groups, such as 1 -hydroxy ethyl, 2-hydroxyethyl, 1,2-dihydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 3-hydroxybutyl, 4-hydroxybutyl, 2-hydroxy-l- methylpropyl, and l,3-dihydroxyprop-2-yl.
[0730] The term "alkoxy" as used herein by itself or as part of another group refers to an alkyl group attached to a terminal oxygen atom. In one embodiment, the alkyl is a C1-C6 alkyl and resulting alkoxy is thus referred to as a "C1-C6 alkoxy." In another embodiment, the alkyl is a C1-C4 alkyl group and resulting alkoxy is thus referred to as a "C1-C4 alkoxy." Non-limiting exemplary alkoxy groups include methoxy, ethoxy, and tert-butoxy. The term "haloalkoxy" as used herein by itself or as part of another group refers to a haloalkyl group attached to a terminal oxygen atom. In one embodiment, the haloalkyl group is a C1-C6 haloalkyl. In another embodiment, the haloalkyl group is a C1-C4 haloalkyl group. Non-limiting exemplary haloalkoxy groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.
[0731] The terms "alkoxyalkyl" or "(alkoxy)alkyl" as used herein by themselves or as part of another group refers to an alkyl group substituted with one alkoxy group. In one embodiment, the alkoxy is a C1-C6 alkoxy. In another embodiment, the alkoxy is a C1-C4 alkoxy. In another embodiment, the alkyl is a C1-C6 alkyl. In another embodiment, the alkyl is a C1-C4 alkyl. Thus, in some embodiments, the "(alkoxy)alkyl" is a (C1-C6 alkoxy)C1-C6 alkyl; Non-limiting exemplary alkoxyalkyl groups include methoxymethyl, methoxyethyl, methoxypropyl, methoxybutyl, ethoxymethyl, ethoxyethyl, ethoxypropyl, ethoxybutyl, propoxymethyl, iso-propoxymethyl, propoxyethyl, propoxypropyl, butoxymethyl, tert-butoxymethyl, isobutoxymethyl, secbutoxymethyl, and pentyloxy methyl. [0732] The term "cycloalkyl" as used herein by itself or as part of another group refers to saturated and partially unsaturated, e.g., containing one or two double bonds, monocyclic, bicyclic, or tricyclic aliphatic hydrocarbons containing three to twelve carbon atoms, i.e., a C3-12 cycloalkyl, or the number of carbons designated, e.g., a C3 cycloalkyl such a cyclopropyl, a C4 cycloalkyl such as cyclobutyl, etc. In one embodiment, the cycloalkyl is bicyclic, i.e., it has two rings. In another embodiment, the cycloalkyl is monocyclic, i.e., it has one ring. In another embodiment, the cycloalkyl is a C3-8 cycloalkyl. In another embodiment, the cycloalkyl is a C3-6 cycloalkyl, i.e., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In another embodiment, the cycloalkyl is a C5 cycloalkyl, i.e., cyclopentyl or cyclopentenyl. In another embodiment, the cycloalkyl is a Ce cycloalkyl, i.e., cyclohexyl or cyclohexenyl. Non-limiting exemplary C3-12 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbomyl, decalin, adamantyl, cyclohexenyl, and spiro[3.3]heptane.
[0733] The term "optionally substituted cycloalkyl" as used herein by itself or as part of another group refers to a cycloalkyl group that is either unsubstituted or substituted with one, two, or three substituents, wherein each substituent is independently halo, nitro, cyano, hydroxy, amino haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, or alkoxyalkyl. The term optionally substituted cycloalkyl also includes cycloalkyl groups having fused optionally substituted aryl or optionally substituted heteroaryl groups such as:
[0734] The term "(cycloalkyl)alkyl" as used herein by itself or as part of another group refers to an alkyl substituted with one optionally substituted cycloalkyl group. In one embodiment, the (cycloalkyl)alkyl group is a C1-C6 alkyl substituted with one optionally substituted C3-C6 cycloalkyl group, i.e., an "(optionally substituted C3-C6 cycloalkyl)Ci- C<> alkyl." In another embodiment, the (cycloalkyl)alkyl group is a C1-C4 alkyl substituted with one optionally substituted optionally substituted C3-C6 cycloalkyl, i.e., an "(optionally substituted C3-C6 cycloalkyl)Ci-C4 alkyl." In another embodiment, the (cycloalkyl)alkyl group is a C1-C2 alkyl substituted with one optionally substituted C3-C6 cycloalkyl group, i.e., an "(optionally substituted C3-C6 cycloalkyl heteroaryl)Ci-C2 alkyl." In another embodiment, the (cycloalkyl)alkyl group is a C1-C2 alkyl substituted with one cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl group.
[0735] The term "heterocyclo" as used herein by itself or as part of another group refers to saturated and partially unsaturated, e.g., containing one or two double bonds, monocyclic, bicyclic, e.g., spirocyclic, or tricyclic groups containing three to eighteen ring members, i.e., a 3- to 18-membered heterocyclo, comprising one, two, three, or four heteroatoms, or the number of ring atoms designated. Each heteroatom is independently oxygen, sulfur, or nitrogen. Each sulfur atom is independently oxidized to give a sulfoxide, i.e., S(=O), or sulfone, i.e., S(=O)2. The term heterocyclo includes groups wherein one or more -CH2- groups is replaced with one or more -C(=O)- groups, including cyclic ureido groups such as imidazolidinyl-2-one, cyclic amide groups such as pyrrolidin-2-one or piperidin-2-one, and cyclic carbamate groups such as oxazolidinyl-2- one. The term heterocyclo also includes groups having fused optionally substituted aryl or optionally substituted heteroaryl groups such as indoline, indolin-2-one, 2,3-dihydro- lH-pyrrolo[2,3-c]pyridine, 2,3,4,5-tetrahydro-lH-benzo[d]azepine, or 1,3,4,5-tetrahydro- 2H-benzo [d] azepin-2-one .
[0736] In one embodiment, the heterocyclo group is a 4- to 8-membered cyclic group containing one ring and one or two oxygen atoms, e.g., tetrahydrofuran or tetrahydropyran, or one or two nitrogen atoms, e.g., pyrrolidine, piperidine, or piperazine, or one oxygen and one nitrogen atom, e.g., morpholine, and, optionally, one -CH2- group is replaced with one -C(=O)- group, e.g., pyrrolidin-2-one or piperazin-2-one. In another embodiment, the heterocyclo group is a 5- to 8-membered cyclic group containing one ring and one or two nitrogen atoms and, optionally, one -CH2- group is replaced with one -C(=O)- group. In another embodiment, the heterocyclo group is a 4- to 7-membered cyclic group containing one ring and one or two nitrogen atoms and, optionally, one -CH2- group is replaced with one -C(=O)- group. In another embodiment, the heterocyclo group is a 5- or 6-membered cyclic group containing one ring and one or two nitrogen atoms and, optionally, one -CH2- group is replaced with one -C(=O)- group. In another embodiment, the heterocyclo group is a 8- to 12-membered cyclic group containing two rings and one or two nitrogen atoms. The heterocyclo can be linked to the rest of the molecule through any available carbon or nitrogen atom. Non-limiting exemplary heterocyclo groups include:
[0737] The term "optionally substituted heterocyclo" as used herein by itself or part of another group refers to a heterocyclo group that is either unsubstituted or substituted with one to four substituents, wherein each substituent is independently halo, nitro, cyano, hydroxy, amino haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, or alkoxyalkyl. Substitution may occur on any available carbon or nitrogen atom of the heterocyclo group.
[0738] The term "(heterocyclo)alkyl" as used herein by itself or as part of another group refers to an alkyl substituted with one optionally substituted heterocyclo group. In one embodiment, the (heterocyclo)alkyl group is a C1-C6 alkyl substituted with one optionally substituted 4- to 7-membered heterocyclo group, i.e., an "(optionally substituted 4- to 7-membered heterocyclo)C1-C6 alkyl." In another embodiment, the (heterocyclo)alkyl group is a C1-C4 alkyl substituted with one optionally substituted optionally substituted heterocyclo group, i.e., an "(optionally substituted heterocyclo group)Ci-C4 alkyl." In another embodiment, the (heterocyclo group)alkyl group is a C1-C2 alkyl substituted with one optionally substituted heterocyclo group group, i.e., an "(optionally substituted heterocyclo group heteroaryl)Ci-C2 alkyl."
[0739] The term "aryl" as used herein by itself or as part of another group refers to an aromatic ring system having six to fourteen carbon atoms, i.e., Ce-Cu aryl. Non-limiting exemplary aryl groups include phenyl (abbreviated as "Ph"), naphthyl, phenanthryl, anthracyl, indenyl, azulenyl, biphenyl, biphenylenyl, and fluorenyl groups. In one embodiment, the aryl group is phenyl or naphthyl. In another embodiment, the aryl group is phenyl.
[0740] The term "optionally substituted aryl" as used herein by itself or as part of another group refers to aryl that is either unsubstituted or substituted with one to five substituents, wherein the substituents are each independently halo, nitro, cyano, hydroxy, amino haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxyalkyl, aryloxy, aralkyloxy, hetero aryloxy, (heterocyclo)alkyloxy, heterocyclooxy, (aryl)alkyl, (heteroaryl)alkyl, or (hetero aryl) alkyloxy .
[0741] In one embodiment, the optionally substituted aryl is an optionally substituted phenyl. In another embodiment, the optionally substituted phenyl has four substituents. In another embodiment, the optionally substituted phenyl has three substituents. In another embodiment, the optionally substituted phenyl has two substituents. In another embodiment, the optionally substituted phenyl has one substituent. Non-limiting exemplary optionally substituted aryl groups include 2-methylphenyl, 2-methoxyphenyl,
2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3- fluorophenyl, 3-chlorophenyl, 4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 2,6-di-fluorophenyl, 2,6-di-chlorophenyl, 2-methyl,
3-methoxyphenyl, 2-ethyl, 3-methoxyphenyl, 3,4-di-methoxyphenyl, 3,5-di-fluorophenyl 3,5-di-methylphenyl, 3,5-dimethoxy, 4-methylphenyl, 2-fluoro-3-chlorophenyl, 3-chloro-
4-fluorophenyl, and 2-phenylpropan-2-amine. The term optionally substituted aryl includes aryl groups having fused optionally substituted cycloalkyl groups and fused optionally substituted heterocyclo groups. Non-limiting examples include: 2,3-dihydro- IH-inden-l-yl, 1,2,3,4-tetrahydronaphthalen-l-yl, l,3,4,5-tetrahydro-2H-benzo[c]azepin- 2-yl, 1,2,3,4-tetrahydroisoquinolin-l-yl, and 2-oxo-2,3,4,5-tetrahydro-lH- benzo [d] azepin- 1 -y 1.
[0742] The term "heteroaryl" as used herein by itself or as part of another group refers to monocyclic and bicyclic aromatic ring systems having five to 14 fourteen ring members, i.e., a 5- to 14-membered heteroaryl, comprising one, two, three, or four heteroatoms. Each heteroatom is independently oxygen, sulfur, or nitrogen. In one embodiment, the heteroaryl has three heteroatoms. In another embodiment, the heteroaryl has two heteroatoms. In another embodiment, the heteroaryl has one heteroatom. In another embodiment, the heteroaryl is a 5- to 10-membered heteroaryl. In another embodiment, the heteroaryl has 5 ring atoms, e.g., thienyl, a 5-membered heteroaryl having four carbon atoms and one sulfur atom. In another embodiment, the heteroaryl has 6 ring atoms, e.g., pyridyl, a 6-membered heteroaryl having five carbon atoms and one nitrogen atom. Non-limiting exemplary heteroaryl groups include thienyl, benzo [b] thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, benzofuryl, pyranyl, isobenzofuranyl, benzooxazonyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, isoindolyl, 377-indolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, cinnolinyl, quinazolinyl, pteridinyl, 4a77-carbazolyl, carbazolyl, P-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, thiazolyl, isothiazolyl, phenothiazolyl, isoxazolyl, furazanyl, and phenoxazinyl. In one embodiment, the heteroaryl is chosen from thienyl (e.g., thien-2-yl and thien-3-yl), furyl (e.g., 2-furyl and 3-furyl), pyrrolyl (e.g., lH-pyrrol-2-yl and lH-pyrrol-3-yl), imidazolyl (e.g., 2H-imidazol-2-yl and 2H- imidazol-4-yl), pyrazolyl (e.g., lH-pyrazol-3-yl, lH-pyrazol-4-yl, and lH-pyrazol-5-yl), pyridyl (e.g., pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl), pyrimidinyl (e.g., pyrimidin-2- yl, pyrimidin-4-yl, and pyrimidin-5-yl), thiazolyl (e.g., thiazol-2-yl, thiazol-4-yl, and thiazol-5-yl), isothiazolyl (e.g., isothiazol-3-yl, isothiazol-4-yl, and isothiazol-5-yl), oxazolyl (e.g., oxazol-2-yl, oxazol-4-yl, and oxazol-5-yl) and isoxazolyl (e.g., isoxazol-3- yl, isoxazol-4-yl, and isoxazol-5-yl). The term heteroaryl also includes N-oxides. A nonlimiting exemplary N-oxide is pyridyl N-oxide.
[0743] The term "optionally substituted heteroaryl" as used herein by itself or as part of another group refers to a heteroaryl that is either unsubstituted or substituted with one to four substituents, wherein the substituents are independently halo, nitro, cyano, hydroxy, amino haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxyalkyl, aryloxy, aralkyloxy, heteroaryloxy, (heterocyclo)alkyloxy, heterocyclooxy, (aryl)alkyl, (heteroaryl)alkyl, or (hetero aryl) alkyloxy .
[0744] In one embodiment, the optionally substituted heteroaryl has two substituents. In another embodiment, the optionally substituted heteroaryl has one substituent. Any available carbon or nitrogen atom can be substituted.
[0745] The term "(heteroaryl)alkyl" as used herein by itself or as part of another group refers to an alkyl substituted with one optionally substituted heteroaryl group. In one embodiment, the alkyl group is a C1-C6 alkyl substituted with one optionally substituted 5- to 14-membered heteroaryl group, i.e., an "(optionally substituted 5- to 14-membered heteroaryl)C1-C6 alkyl." In another embodiment, the alkyl group is a C1-C6 alkyl substituted with one optionally substituted 5- to 9-membered heteroaryl group, i.e., an "(optionally substituted 5- to 9-membered heteroaryl)C1-C6 alkyl." In another embodiment, the alkyl group is a C1-C6 alkyl substituted with one optionally substituted 5- or 6-membered heteroaryl group, i.e., an "(optionally substituted 5- or 6-membered heteroaryl)C1-C6 alkyl." In another embodiment, the alkyl group is a C1-C4 alkyl substituted with one optionally substituted 5- to 9-membered heteroaryl group, i.e., an "(optionally substituted 5- to 9-membered heteroaryl)Ci-C4 alkyl." In another embodiment, the alkyl group is a C1-C4 alkyl substituted with one optionally substituted 5- or 6-membered heteroaryl group, i.e., an "(optionally substituted 5- or 6-membered heteroaryl)Ci-C4 alkyl." In another embodiment, the alkyl group is a C1-C2 alkyl substituted with one optionally substituted 5- or 6-membered heteroaryl group, i.e., an "(optionally substituted 5- or 6-membered heteroaryl)Ci-C2 alkyl." In another embodiment, the alkyl group is a C1-C2 alkyl substituted with one optionally substituted pyridyl group, i.e., an "(optionally substituted pyridyl)Ci-C2 alkyl." Non-limiting examples include:
[0746] The terms "aralkyl" or "(aryl)alkyl" as used herein by themselves or as part of another group refers to an alkyl substituted with one optionally substituted aryl group. In another embodiment, the aryl is an optionally substituted phenyl. In one embodiment, the alkyl is a C1-C6 alkyl, i.e., an "(optionally substituted aryl)C1-C6 alkyl." In another embodiment, the alkyl is a C1-C4 alkyl, i.e., an "(optionally substituted aryl)Ci-C4 alkyl." In another embodiment, the alkyl is a Ci or C2 alkyl, i.e., an "(optionally substituted aryl)Ci-C2 alkyl." In another embodiment, the alkyl is a Ci or C2 alkyl and the optionally substituted aryl is an optionally substituted phenyl i.e., an "(optionally substituted phenyl)Ci-C2 alkyl." Non-limiting exemplary (aryl)alkyl groups include benzyl and phenethyl.
[0747] The term "aryloxy" as used herein by itself or as part of another group refers to an optionally substituted aryl group attached to a terminal oxygen atom. In one embodiment, the optionally substituted aryl is an optionally substituted phenyl. A non-limiting exemplary aryloxy group is PhO-.
[0748] The term "heteroaryloxy" as used herein by itself or as part of another group refers to an optionally substituted heteroaryl group attached to a terminal oxygen atom. A non-limiting exemplary hetereo aryloxy group is:
[0749] The term "heterocyclooxy" as used herein by itself or as part of another group refers to an optionally substituted heterocyclo group attached to a terminal oxygen atom. In one embodiment, the optionally substituted heterocyclo group is an optionally substituted 4- to 7-membered heterocyclo group and resulting heterocyclooxy is thus referred to as a "4- to 7-membered heterocyclooxy." Non-limiting exemplary heterocyclooxy groups include:
[0750] The term "aralkyloxy" as used herein by itself or as part of another group refers to an aralkyl group attached to a terminal oxygen atom. A non-limiting exemplary aralkyloxy group is PhCfTO-.
[0751] The term "(heteroaryl)alkyloxy" as used herein by itself or as part of another group refers to an (heteroaryl)alkyl group attached to a terminal oxygen atom. Non-limiting exemplary (hetero aryl) alkyloxy groups include:
[0752] The term "(heterocyclo)alkyloxy" as used herein by itself or as part of another group refers to an (heterocyclo)alkyl group attached to a terminal oxygen atom. Non-limiting exemplary (hetero aryl) alkyloxy groups include:
[0753] The term "amino" as used by itself or as part of another group refers to a radical of the formula -NRaaRbb, wherein Raa and Rbb are independently hydrogen, optionally substituted alkyl, haloalkyl, (hydroxy)alkyl, (alkoxy)alkyl, (amino)alkyl, heteroalkyl, optionally substituted cycloalkyl, optionally substituted heterocyclo, optionally substituted aryl, optionally substituted heteroaryl, (aryl)alkyl, (cycloalkyl)alkyl, (heterocyclo)alkyl, or (heteroaryl)alkyl. [0754] In one embodiment, the amino is -NH2.
[0755] In another embodiment, the amino is an "alkylamino," i.e., an amino group wherein Raa is C1-6 alkyl and Rbb is hydrogen. In one embodiment, Raa is C1-C4 alkyl. Non-limiting exemplary alkylamino groups include -N(H)CH3 and -N(H)CH2CH3.
[0756] In another embodiment, the amino is a "dialkylamino," i.e., an amino group wherein Raa and Rbb are each independently C1-6 alkyl. In one embodiment, R55a and R55b are each independently C1-C4 alkyl. Non-limiting exemplary dialkylamino groups include -N(CH3)2 and -N(CH3)CH2CH(CH3)2.
[0757] The term "a disease or condition wherein ADAR1 inhibition provides a benefit" and the like pertains to a disease or condition in which AD ARI is important or necessary, e.g., for the onset, progress, expression of that disease or condition, or a disease or a condition which is known to be treated by an ADAR1 inhibitor. Examples of such conditions include, but are not limited to, a cancer. One of ordinary skill in the art is readily able to determine whether a compound treats a disease or condition mediated by an ADAR1 inhibitor for any particular cell type, for example, by assays which conveniently can be used to assess the activity of particular compounds.
[0758] The term "disease" or "condition" denotes disturbances and/or anomalies that as a rule are regarded as being pathological conditions or functions, and that can manifest themselves in the form of particular signs, symptoms, and/or malfunctions. Compounds of the Disclosure are ADAR1 inhibitors and can be used in treating or preventing diseases and conditions wherein ADAR1 inhibition provides a benefit.
[0759] As used herein, the terms "treat," "treating," "treatment," and the like refer to eliminating, reducing, or ameliorating a disease or condition, e.g., cancer, and/or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated. The term "treat" and synonyms contemplate administering a therapeutically effective amount of a Compound of the Disclosure and optionally an immune checkpoint inhibitor to a subject need of such treatment. The treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, be oriented over a medium term, or can be a long-term treatment, for example within the context of a maintenance therapy.
[0760] As used herein, the terms "prevent," "preventing," and "prevention" refer to a method of preventing the onset of a disease or condition and/or its attendant symptoms or barring a subject from acquiring a disease. As used herein, "prevent," "preventing," and "prevention" also include delaying the onset of a disease and/or its attendant symptoms and reducing a subject's risk of acquiring a disease. The terms "prevent," "preventing" and "prevention" may include "prophylactic treatment," which refers to reducing the probability of redeveloping a disease or condition, or of a recurrence of a previously- controlled disease or condition, in a subject who does not have, but is at risk of or is susceptible to, redeveloping a disease or condition or a recurrence of the disease or condition.
[0761] The term "therapeutically effective amount" or "effective dose" as used herein refers to an amount of the active ingredient(s) that is(are) sufficient, when administered by a method of the present disclosure, to efficaciously deliver the active ingredient(s) for the treatment of condition or disease of interest to a subject in need thereof. In the case of a cancer or other proliferation disorder, the therapeutically effective amount of the agent may reduce (i.e., retard to some extent or stop) unwanted cellular proliferation; reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., retard to some extent or stop) cancer cell infiltration into peripheral organs; inhibit (i.e., retard to some extent or stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve, to some extent, one or more of the symptoms associated with the cancer. To the extent the administered compound or composition prevents growth and/or kills existing cancer cells, it may be cytostatic and/or cytotoxic.
[0762] The term "container" means any receptacle and closure therefore suitable for storing, shipping, dispensing, and/or handling a pharmaceutical product.
[0763] The term "insert" means information accompanying a pharmaceutical product that provides a description of how to administer the product, along with the safety and efficacy data required to allow the physician, pharmacist, and patient to make an informed decision regarding use of the product. The package insert generally is regarded as the "label" for a pharmaceutical product.
[0764] Concurrent administration," "administered in combination," "simultaneous administration," and similar phrases mean that two or more agents are administered concurrently to the subject being treated. By "concurrently," it is meant that each agent is administered either simultaneously or sequentially in any order at different points in time. However, if not administered simultaneously, it is meant that they are administered to a subject in a sequence and sufficiently close in time so as to provide the desired therapeutic effect and can act in concert. For example, a Compound of the Disclosure can be administered at the same time or sequentially in any order at different points in time as a second therapeutic agent. A Compound of the Disclosure and the second therapeutic agent can be administered separately, in any appropriate form and by any suitable route. When a Compound of the Disclosure and the second therapeutic agent are not administered concurrently, it is understood that they can be administered in any order to a subject in need thereof. For example, a Compound of the Disclosure can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent treatment modality, e.g., an ICI, to a subject in need thereof. In various embodiments, a Compound of the Disclosure and the second therapeutic agent are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart. In one embodiment, the components of the combination therapies are administered at about 1 minute to about 24 hours apart.
[0765] The present disclosure encompasses any Compound of the Disclosure being isotopically-labelled (i.e., radiolabeled) by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2H (or deuterium (D)), 3H, nC, 13C, 14C, 15N, 18O, 170, 31P, 32P, 35S, 18F, and 36C1, respectively, e.g., 3H, nC, and 14C. In one embodiment, provided is a composition wherein substantially all of the atoms at a position within the Compound of the Disclosure are replaced by an atom having a different atomic mass or mass number. In another embodiment, provided is a composition wherein a portion of the atoms at a position within the Compound of the disclosure are replaced, i.e., the Compound of the Disclosure is enriched at a position with an atom having a different atomic mass or mass number." Isotopically-labelled Compounds of the Disclosure can be prepared by methods known in the art.
[0766] Compounds of the Disclosure may contain one or more asymmetric carbon atoms and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. The present disclosure encompasses the use of all such possible forms, as well as their racemic and resolved forms and mixtures thereof. The individual enantiomers can be separated according to methods known in the art in view of the present disclosure. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that they include both E and Z geometric isomers. All tautomers are also encompassed by the present disclosure. All conformational isomers, i.e., stereoisomers produced by rotation about a c bond, are also encompassed by the present disclosure.
[0767] As used herein, the term "stereoisomers" is a general term for all isomers of individual molecules that differ only in the orientation of their atoms in space. It includes enantiomers and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereomers).
[0768] The term "chiral center" or "asymmetric carbon atom" refers to a carbon atom to which four different groups are attached.
[0769] The terms "enantiomer" and "enantiomeric" refer to a molecule that cannot be superimposed on its mirror image and hence is optically active wherein the enantiomer rotates the plane of polarized light in one direction and its mirror image compound rotates the plane of polarized light in the opposite direction.
[0770] The term "racemic" refers to a mixture of equal parts of enantiomers and which mixture is optically inactive. In one embodiment, Compounds of the Disclosure are racemic.
[0771] The term "absolute configuration" refers to the spatial arrangement of the atoms of a chiral molecular entity (or group) and its stereochemical description, e.g., R or S.
[0772] The stereochemical terms and conventions used in the specification are meant to be consistent with those described in Pure & Appl. Chem 65:2193 (1996), unless otherwise indicated.
[0773] The term "enantiomeric excess" or "ee" refers to a measure for how much of one enantiomer is present compared to the other. For a mixture of R and 5 enantiomers, the percent enantiomeric excess is defined as | R - 5 | *100, where R and 5 are the respective mole or weight fractions of enantiomers in a mixture such that R + 5 = 1. With knowledge of the optical rotation of a chiral substance, the percent enantiomeric excess is defined as ([cx]obs/[cx]max)*100, where [cc]obs is the optical rotation of the mixture of enantiomers and [cx]max is the optical rotation of the pure enantiomer. Determination of enantiomeric excess is possible using a variety of analytical techniques, including NMR spectroscopy, chiral column chromatography or optical polarimetry.
[0774] The term "about," as used herein, includes the recited number ± 10%. Thus, "about 10" means 9 to 11.
[0775] As used herein, the term "and/or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).
[0776] The term "leaving group" as used herein refers to an atom or group of atoms that becomes detached from an atom or group of atoms in what is considered to be the residual or main part of the molecule in a specified reaction. Non-limiting exemplary leaving groups include -Cl, -I, -Br, -OTf, -OMs, and -OTs.
[0777] The word "comprising" is used in a manner consistent with its open-ended meaning, that is, to mean that a given product, composition, or process can optionally also have additional features or elements beyond those expressly described. It is understood that wherever embodiments are described with the language "comprising," otherwise analogous embodiments described in terms of "consisting of" and/or "consisting essentially of" are also contemplated and within the scope of this disclosure.
[0778] Pharmaceutically acceptable salts and solvates, e.g., hydrates, of the Compounds of the Disclosure can also be used in the methods disclosed herein.
[0779] The present disclosure encompasses the preparation and use of salts of Compounds of the Disclosure. Salts of Compounds of the Disclosure can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation. Salts of Compounds of the Disclosure can be acid addition salts formed with acceptable acids. Examples of acids which can be employed to form salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric. Non-limiting examples of salts of compounds of the disclosure include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzene sulfonate, and p-toluenesulfonate salts. In addition, available amino groups present in the compounds of the disclosure can be quatemized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference to Compounds of the Disclosure appearing herein is intended to include Compounds of the Disclosure as well as salts, hydrates, or solvates thereof.
[0780] The present disclosure encompasses the preparation and use of solvates of Compounds of the Disclosure. The term "solvate" as used herein is a combination, physical association and/or solvation of a compound of the present disclosure with a solvent molecule such as, e.g., a disolvate, monosolvate or hemisolvate, where the ratio of solvent molecule to compound of the present disclosure is about 2:1, about 1:1 or about 1:2, respectively. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances, the solvate can be isolated, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. Thus, "solvate" encompasses both solution-phase and isolatable solvates. Compounds of the Disclosure can be present as solvated forms with a solvent, such as water, methanol, and ethanol, and it is intended that the disclosure includes both solvated and unsolvated forms of Compounds of the Disclosure.
[0781] One type of solvate is a hydrate. A "hydrate" relates to a particular subgroup of solvates where the solvent molecule is water. Preparation of solvates is known in the art. See, for example, M. Caira et al, J. Pharmaceut. Sci., 93(3):6Q1-611 (2004), which describes the preparation of solvates of fluconazole with ethyl acetate and with water. Similar preparation of solvates, hemisolvates, hydrates, and the like are described by van Tender el al., AAPS Pharm. Sci. Tech., 5( 7): Article 12 (2004), and A.L. Bingham el al., Chem. Commun. 603-604 (2001). A typical, non-limiting, process of preparing a solvate would involve dissolving a Compound of the Disclosure in a desired solvent (organic, water, or a mixture thereof) at temperatures above 20°C to about 25°C, then cooling the solution at a rate sufficient to form crystals, and isolating the crystals by known methods, e.g., filtration. Analytical techniques such as infrared spectroscopy can be used to confirm the presence of the solvent in a crystal of the solvate.
[0782] The term "Neratinib" refers to (E)-N-{4-[3-chloro-4-(pyridin-2-yl methoxy)anilino]-3-cyano-7-ethoxyquinolin-6-yl}-4-(dimethylamino)but-2-enamide.
In some embodiments in connection with the methods and/or uses of the present disclosure, Neratinib or Neratinib maleate is administered to a subject in a daily dose of 240 mg or less, e.g., 200 mg or less, 160 mg or less, or 120 mg or less. In some embodiments, Neratinib or Neratinib maleate is administered to a subject in a daily dose of 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, or 110 mg.
EXAMPLES
General Procedures:
[0783] Unless otherwise specified, all commercial reagents were used as supplied without further purification, and all reactions were performed under a nitrogen atmosphere in a dry solvent under anhydrous conditions. NMR spectra were obtained on a Bruker 400 Ascend spectrometer at a frequency of 400 MHz. Chemical shifts (5) are reported in parts per million (ppm) relative to an internal standard. The final products were purified on a preparative high-performance liquid chromatography (HPLC) column (Waters 2545, Quaternary Gradient Module) with a SunFire Prep C18 OBD 5 pm 50 x 100 mm reversed-phase column. The mobile phase was a gradient of solvent A (H2O with 0.1% TFA) and solvent B (CH3CN with 0.1% of TFA) at a flow rate of 60 mL/min and 1%/min increase of solvent B. All final compounds have purity >95% as determined by Waters ACQUITY ultraperformance liquid chromatograph (UPLC) using reversed- phase column (SunFire, C18, 5 pm, 4.6 x 150 mm) and a solvent gradient of A (H2O with 0.1% of TFA) and solvent B (CH3CN with 0.1% of TFA). Electrospray ionization (ESI) mass spectral (MS) analysis was performed on a Thermo Scientific LCQ Fleet mass spectrometer.
EXAMPLE 1
General Procedure A - Preparation of compounds having Formula III
Scheme 1
NaH, DMF, rt (n = 1)
(or) ep . IV
[0784] Condition 1A of Step A.l. Sodium hydride (60%, 5.69 mmol) in dry DMF (3 mL) under a nitrogen atmosphere and the resulting mixture is cooled in ice water. To above suspension is added a compound having Formula I (2.85 mmol) in anhydrous DMF (2 mL). 2-Chloro-l-fluoro-4-nitrobenzene (compound II) (2.85 mmol) was added to the above solution and the resulting solution was stirred at rt for 6 h. Cold water was added slowly to the reaction mixture, and a yellow precipitate resulted. The yellow solid is isolated by vacuum filtration, washed with cold water, and air dried to give the title compound as yellow solids.
[0785] Condition IB of Step A.l. To a solution of compound having Formula I (2.85 mmol) and CS2CO3 (5.7 mmol) in dry acetonitrile (10 mL) under a nitrogen atmosphere was added 2-chloro-l-fluoro-4-nitrobenzene (compound II) (2.85 mmol) and the resulting solution was stirred at 60 °C for 12 h. The reaction solution was cool to rt, filtered and washed with ethyl acetate. The filtrate was evaporated to give the compounds as yellow solids.
[0786] The following compounds having Formula III were prepared using this general procedure:
EXAMPLE 2
General Procedure B
Preparation of compounds having Formula IV
[0787] Condition IB of Step A.2. A mixture of Formula III (2.51 mmol), iron (12.56 mmol) and NH4CI (25.11 mmol) in 70% ethanol in water was heated at 70 °C for 1 h. The reaction mixture was allowed to cool to rt, and then filtered over celite®. The filtrate was evaporated under vacuum giving an orange solid which was stirred with EtOAc, and then the resulting mixture was filtered. The filtrate was dried over anhydrous Na2SO4 and evaporated under vacuum to afford the desired compound IV as brown solid. The crude compound was used for the next step without further purification.
[0788] Condition 2B of Step A.2. A mixture of Formula III (2.51 mmol), and iron (12.56 mmol) in acetic acid (10 mF) was stirred at rt for 3 h. The reaction mixture was then filtered over celite® and washed with ethyl acetate. The filtrate was evaporated under vacuum giving an orange solid which was stirred with EtOAc and saturated NaHCOa. The ethyl acetate was separated, and the aqueous layer was extracted with ethyl acetate. The combined extracts were washed with brine and dried over anhydrous Na2SO4 and evaporated under vacuum to afford the desired compound having Formula IV
[0789] The following compounds were prepared using this general procedure:
EXAMPLE 3
General Procedure C - Preparation of Compounds having Formula VI
Scheme 2
Cpd Nos 2 and 29
[0790] Methansulfonic acid (3 eq.) was added at room temperature to a suspension of amide compound having a Formula V (1 eq.) and the appropriate amine having Formula IV (1.2 eq.) in anhydrous ethyl alcohol. The mixture was then refluxed for 3 ~ 6 h. The solution was cooled to ambient temperature, then concentrated in vacuo, and purified by preparative HPLC to yield the desired compounds having Formula VI as a light-yellow solid.
[0791] The following compounds of Formula VI were prepared using this general procedure: EXAMPLE 4
General Procedure D - Preparation of Cpd. Nos. 1, 3, 4, 6, 12-23, 25, 26, 28, and 31-32
[0792] ('E)-4-(Dimethylamino)but-2-enoyl chloride hydrochloride (1.1 eq.) was added at 0 °C to a suspension of a compound having Formula VI (1 eq.) in N-methylpyrrolidine (4 vol.). See Scheme 2. The mixture was then allowed to ambient temperature and stirred for 2 h. The compound was purified by preparative HPLC to yield Cpd. Nos. 1, 3, 4, 6, 12-23, 25-26, 28, 31 and 32.
[0793] Cpd. No. 1: (E)-N-(4-((3-chloro-4-methoxyphenyl)amino)-3-cyano-7- ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4- methoxyphenyl)amino)-7 -ethoxy quinoline-3-carbonitrile and (E)-4-(dimethylamino)but- 2-enoyl chloride hydrochloride. Yield 58%; LC-MS calculated for C25H26CIN5O3 [M + H]+ = 480.18, found 480.12; 1H NMR (400 MHz, CDCI3) 8 9.20 (s, 1H), 8.46 (s, 1H), 8.07 (s, 1H), 7.74 (br. s„ 1H), 7.23 (s, 1H), 7.16 (d, J = 2.57 Hz, 1H), 6.94 - 7.09 (m, 2H), 6.81 (d, J = 8.68 Hz, 1H), 6.21 (d, J = 15.28 Hz, 1H), 4.28 (q, J = 6.97 Hz, 2H), 3.92 (s, 3H), 3.10 - 3.18 (m, 2H), 2.30 (s, 6H), 1.59 (t, J= 6.97 Hz, 3H).
[0794] Cpd. No. 3: (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano- 7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4-(pyridin- 2-ylmethoxy)phenyl)amino)-7-ethoxyquinoline-3-carbonitrile and (E)-4- (dimethylamino)but-2-enoyl chloride hydrochloride. Yield 68%; LC-MS calculated for C30H29CIN6O3 [M + H]+ = 557.21, found 557.06; 1H NMR (400 MHz, DMSO-d6) 8 9.62 (s, 1H), 9.49 (s, 1H), 8.98 (s, 1H), 8.57 - 8.63 (m, 1H), 8.48 (s, 1H), 7.88 (dt, J = 1.71, 7.70 Hz, 1H), 7.59 (d, J = 7.82 Hz, 1H), 7.33 - 7.45 (m, 3H), 7.26 (d, J = 8.93 Hz, 1H), 7.21 (dd, J = 2.45, 8.80 Hz, 1H), 6.79 (td, J = 5.96, 15.34 Hz, 1H), 6.60 (d, J = 15.41 Hz, 1H), 5.29 (s, 2H), 4.31 (q, J = 6.97 Hz, 2H), 3.08 (dd, J = 1.04, 5.93 Hz, 2H), 1.48 (t, J = 6.97 Hz, 3H).
[0795] Cpd. No. 4: (E)-N-(4-((3-chlorophenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-4- (dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7- ethoxyquinoline-3-carbonitrile and (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 62%; LC-MS calculated for C30H29CIN6O3 [M + H]+ = 557.21, found 557.06; 1H NMR (400 MHz, DMSO-d6) 8 9.62 (s, 1H), 9.49 (s, 1H), 8.98 (s, 1H), 8.57 - 8.63 (m, 1H), 8.48 (s, 1H), 7.88 (dt, J = 1.71, 7.70 Hz, 1H), 7.59 (d, J = 7.82 Hz, 1H), 7.33 - 7.45 (m, 3H), 7.26 (d, J = 8.93 Hz, 1H), 7.21 (dd, J = 2.45, 8.80 Hz, 1H), 6.79 (td, J = 5.96, 15.34 Hz, 1H), 6.60 (d, J = 15.41 Hz, 1H), 5.29 (s, 2H), 4.31 (q, J = 6.97 Hz, 2H), 3.08 (dd, J= 1.04, 5.93 Hz, 2H), 1.48 (t, J = 6.97 Hz, 3H).
[0796] Cpd. No. 6: (E)-N-(3-cyano-7-ethoxy-4-((4-methoxyphenyl)amino)quinolin-6-yl)- 4-(dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between 6-amino-7-ethoxy-4-((4- methoxyphenyl)amino)quinoline-3-carbonitrile and (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 56%; LC-MS calculated for C25H27N5O3 [M + H]+ = 446.22, found 446.27; 1 H NMR (400 MHz, CDCI3) 8 9.08 (s, 1H), 8.57 (s, 1H), 7.79 (br. s„ 1H), 7.27 - 7.29 (m, 2H), 7.01 (m, 3H), 6.72 (d, J = 15.16 Hz, 1H), 4.39 (q, J = 6.52 Hz, 2H), 3.87 (s, 3H), 2.89 (s, 6H), 1.57 (t, J = 6.79 Hz, 3H).
[0797] Cpd. No. 12: (E)-N-(4-((3-chloro-4-((6-methylpyridin-2- yl)methoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2- enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4-((6-methylpyridin-2-yl)methoxy)phenyl)amino)-7- ethoxyquinoline-3-carbonitrile and (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 55%; LC-MS calculated for C31H31CIN6O3 [M + H]+ = 571.22, found 571.12; 1H NMR (400 MHz, CD3OD) 8 9.24 (s, 1H), 8.81 (s, 1H), 8.20 (t, J = 7.89 Hz, 1H), 7.83 (d, J = 7.82 Hz, 1H), 7.62 - 7.67 (m, 2H), 7.43 - 7.49 (m, 2H), 7.35 (d, J = 8.80 Hz, 1H), 6.96 - 7.06 (m, 1H), 6.82 - 6.89 (m, 1H), 5.47 (s, 2H), 4.45 (q, J = 6.97 Hz, 2H), 4.04 (d, J = 6.60 Hz, 2H), 2.96 (s, 6H), 2.74 (s, 3H), 1.61 (t, J = 6.97 Hz, 3H).
[0798] Cpd. No. 13: (E)-N-(4-((3-chloro-4-(pyridin-4-yloxy)phenyl)amino)-3-cyano-7- ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4-(pyridin- 4-yloxy)phenyl)amino)-7-ethoxyquinoline-3-carbonitrile and (E)-4-(dimethylamino)but- 2-enoyl chloride hydrochloride. Yield 65%; LC-MS calculated for C29H27CIN6O3 [M + H]+ = 543.19, found 543.15; 1H NMR (400 MHz, CD3OD) 8 9.22 (s, 1H), 8.94 (s, 1H), 8.36 - 8.44 (m, 2H), 7.80 - 7.88 (m, 2H), 7.65 (dd, J = 2.32, 8.56 Hz, 1H), 7.54 (s, 1H), 7.11 - 7.18 (m, 2H), 6.98 (td, J = 7.05, 15.25 Hz, 1H), 6.87 (d, J = 15.16 Hz, 1H), 4.46 (q, J = 6.97 Hz, 2H), 4.05 (dd, J = 0.73, 6.97 Hz, 2H), 2.97 (s, 6H), 1.61 (t, J = 6.97 Hz, 3H). [0799] Cpd. No. 14: (E)-N-(4-((3-chloro-4-((5-isopropylpyridin-2- yl)methoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2- enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4-((5-isopropylpyridin-2-yl)methoxy)phenyl)amino)-7- ethoxyquinoline-3-carbonitrile and (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 72%; LC-MS calculated for C33H35CIN5O3 [M + H]+ = 599.26, found 599.27; 1H NMR (400 MHz, CD3OD) 8 9.24 (s, 1H), 8.81 (s, 1H), 8.72 (d, J = 2.08 Hz, 1H), 8.38 (dd, J = 2.08, 8.31 Hz, 1H), 8.03 (d, J = 8.19 Hz, 1H), 7.65 (d, J = 2.57 Hz, 1H), 7.44 - 7.52 (m, 2H), 7.36 (d, J = 8.93 Hz, 1H), 6.96 - 7.08 (m, 1H), 6.82 - 6.90 (m, 1H), 5.55 (s, 2H), 4.44 (q, J = 6.97 Hz, 2H), 4.05 (dd, J = 0.92, 7.15 Hz, 2H), 3.20 (td, J= 6.92, 13.91 Hz, 1H), 2.96 (s, 6H), 1.57 - 1.63 (m, 3H), 1.39 (d, J= 6.97 Hz, 6H).
[0800] Cpd. No.15: (E)-N-(4-((3-chloro-4-((6-ethylpyridin-2-yl)methoxy)phenyl)amino)-
3-cyano-7 -ethoxy quinolin-6-yl)-4-(dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4- ((6-ethylpyridin-2-yl)methoxy)phenyl)amino)-7 -ethoxy quinoline-3-carbonitrile and (E)-
4-(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 74%; LC-MS calculated for C32H33CIN6O3 [M + H]+ = 585.24, found 585.21; 1H NMR (400 MHz, CD3OD) 8 9.25 (s, 1H), 8.82 (s, 1H), 8.36 (t, J = 7.95 Hz, 1H), 7.93 (d, J = 7.70 Hz, 1H), 7.79 (d, J = 7.82 Hz, 1H), 7.65 (d, J = 2.45 Hz, 1H), 7.43 - 7.52 (m, 2H), 7.37 (d, J = 8.80 Hz, 1H), 6.94 - 7.06 (m, 1H), 6.82 - 6.91 (m, 1H), 5.54 (s, 2H), 4.44 (q, J = 6.85 Hz, 2H), 4.04 (d, J = 6.97 Hz, 2H), 3.08 (q, J = 7.50 Hz, 2H), 2.96 (s, 6H), 1.60 (t, J = 6.91 Hz, 3H), 1.43 (t, J = 7.58 Hz, 3H).
[0801] Cpd. No.16: (E)-N-(4-((3-chloro-4-((2-methylpyridin-4-yl)oxy)phenyl)amino)-3- cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4- ((2-methylpyridin-4-yl)oxy)phenyl)amino)-7 -ethoxyquinoline-3-carbonitrile and (E)-4- (dimethylamino)but-2-enoyl chloride hydrochloride. Yield 62%; LC-MS calculated for C30H29CIN6O3 [M + H]+ = 557.21, found 557.03; 1H NMR (400 MHz, CD3OD) 8 9.31 (s, 1H), 8.88 (s, 1H), 8.65 (d, J = 6.85 Hz, 1H), 7.91 (d, J = 2.32 Hz, 1H), 7.70 (dd, J = 2.38, 8.62 Hz, 1H), 7.62 (d, J = 8.68 Hz, 1H), 7.55 (s, 1H), 7.48 (dd, J = 2.57, 6.85 Hz, 1H), 7.39 (d, J = 2.45 Hz, 1H), 6.98 - 7.07 (m, 1H), 6.84 - 6.93 (m, 1H), 4.45 (q, J = 6.97 Hz, 2H), 4.06 (d, J = 6.97 Hz, 2H), 2.97 (s, 6H), 2.71 (s, 3H), 1.61 (t, J = 6.97 Hz, 3H). [0802] Cpd. No. 17: (E)-N-(4-((3-chloro-4-((6-isopropylpyridin-3- yl)methoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2- enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4-((6-isopropylpyridin-3-yl)methoxy)phenyl)amino)-7- ethoxyquinoline-3-carbonitrile and (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 44%; LC-MS calculated for C33H35CIN6O3 [M + H]+ = 599.26, found 598.99; 1H NMR (400 MHz, CD3OD) 8 9.25 (s, 1H), 8.79 - 8.85 (m, 2H), 8.65 (dd, J = 2.02, 8.38 Hz, 1H), 8.04 (d, J = 8.44 Hz, 1H), 7.64 (d, J = 2.45 Hz, 1H), 7.44 - 7.50 (m, 2H), 7.36 - 7.41 (m, 1H), 6.96 - 7.05 (m, 1H), 6.83 - 6.90 (m, 1H), 5.51 (s, 2H), 4.45 (q, J = 6.97 Hz, 2H), 4.05 (dd, J = 0.86, 7.09 Hz, 2H), 3.41 (td, J = 6.92, 13.91 Hz, 1H), 2.96 (s, 6H), 1.61 (t, J = 6.97 Hz, 3H), 1.48 (d, J = 6.97 Hz, 6H).
[0803] Cpd. No. 18: (E)-N-(4-((3-chloro-4-((2-isopropylpyridin-4-yl)oxy)phenyl)amino)-
3-cyano-7 -ethoxy quinolin-6-yl)-4-(dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4- ((2-isopropylpyridin-4-yl)oxy)phenyl)amino)-7-ethoxyquinoline-3-carbonitrile and (E)-
4-(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 61%; LC-MS calculated for C32H33CIN6O3 [M + H]+ = 585.24, found 585.02; 1 H NMR (400 MHz, CD3OD) 8 9.30 (s, 1H), 8.86 (s, 1H), 8.66 (d, J = 6.85 Hz, 1H), 7.90 (d, J = 2.32 Hz, 1H), 7.69 (dd, J = 2.38, 8.62 Hz, 1H), 7.63 (d, J = 8.56 Hz, 1H), 7.47 - 7.54 (m, 2H), 7.41 (d, J = 2.69 Hz, 1H), 7.02 (td, J = 7.08, 15.19 Hz, 1H), 6.88 (d, J = 15.28 Hz, 1H), 4.46 (q, J = 7.05 Hz, 2H), 4.05 (dd, J = 0.98, 7.09 Hz, 2H), 3.23 - 3.31 (m, 1H), 2.97 (s, 6H), 1.62 (t, J = 6.97 Hz, 3H), 1.40 (d, J = 6.97 Hz, 6H).
[0804] Cpd. No. 19: (E)-N-(4-((3-chloro-4-((6-(prop-l-en-2-yl)pyridin-2- yl)methoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2- enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4-((6-(prop- l-en-2-yl)pyridin-2- yl)methoxy)phenyl)amino)-7-ethoxyquinoline-3-carbonitrile and (E)-4-
(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 53%; LC-MS calculated for C33H33CIN6O3 [M + H]+ = 597.24, found 597.26; 1 H NMR (400 MHz, CD3OD) 8 9.23 (s, 1H), 8.81 (s, 1H), 7.88 (t, J = 7.89 Hz, 1H), 7.55 - 7.65 (m, 3H), 7.45 (s, 1H), 7.41 (dd, J = 2.57, 8.80 Hz, 1H), 7.31 (d, J = 8.93 Hz, 1H), 6.95 - 7.05 (m, 1H), 6.82 - 6.89 (m, 1H), 5.87 - 5.93 (m, 1H), 5.34 - 5.42 (m, 3H), 4.44 (q, J = 6.97 Hz, 2H), 4.04 (dd, J = 1.04, 7.15 Hz, 2H), 2.96 (s, 6H), 2.20 - 2.28 (m, 3H), 1.61 (t, J = 6.97 Hz, 3H). [0805] Cpd. No. 20: (E)-N-(4-((3-chloro-4-((6-isopropylpyridin-2- yl)methoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2- enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4-((6-isopropylpyridin-2-yl)methoxy)phenyl)amino)-7- ethoxyquinoline-3-carbonitrile and (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 67%; LC-MS calculated for C33H35CIN6O3 [M + H]+ = 599.26, found 599.23; 1H NMR (400 MHz, CD3OD) 8 9.25 (s, 1H), 8.82 (s, 1H), 8.32 (t, J = 7.95 Hz, 1H), 7.89 (d, J = 7.09 Hz, 1H), 7.74 - 7.79 (m, 1H), 7.65 (d, J = 2.57 Hz, 1H), 7.45 - 7.50 (m, 2H), 7.37 (d, J = 8.93 Hz, 1H), 7.01 (td, J = 7.12, 15.22 Hz, 1H), 6.83 - 6.89 (m, 1H), 5.53 (s, 2H), 4.45 (q, J = 6.97 Hz, 2H), 4.04 (dd, J = 0.98, 7.09 Hz, 2H), 3.29-3.36 (m, 1H), 2.96 (s, 6H), 1.61 (t, J = 6.97 Hz, 3H), 1.45 (d, J= 6.97 Hz, 6H).
[0806] Cpd. No. 21: (E)-N-(4-((4-((6-(tert-butyl)pyridin-3-yl)methoxy)-3- chlorophenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between 6- amino-4-((4-((6-(tert-butyl)pyridin-3-yl)methoxy)-3-chlorophenyl)amino)-7- ethoxyquinoline-3-carbonitrile and (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 42%; LC-MS calculated for C34H37CIN6O3 [M + H]+ = 613.27, found 613.25; 1H NMR (400 MHz, CD3OD) 8 9.24 (s, 1H), 8.73 - 8.88 (m, 2H), 8.56 (dd, J = 1.71, 8.44 Hz, 1H), 8.07 (d, J = 8.44 Hz, 1H), 7.63 (d, J = 2.45 Hz, 1H), 7.43 - 7.52 (m, 2H), 7.34 - 7.43 (m, 1H), 6.95 - 7.07 (m, 1H), 6.86 (d, J = 15.16 Hz, 1H), 5.50 (s, 2H), 4.44 (q, J = 6.89 Hz, 2H), 4.04 (d, J = 6.97 Hz, 2H), 2.96 (s, 6H), 1.60 (t, J = 6.85 Hz, 3H), 1.54 (s, 9H).
[0807] Cpd. No. 22: (E)-N-(4-((3-chloro-4-((6-isobutylpyridin-3- yl)methoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2- enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4-((6-isobutylpyridin-3-yl)methoxy)phenyl)amino)-7- ethoxyquinoline-3-carbonitrile and (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 61%; LC-MS calculated for C34H37CIN6O3 [M + H]+ = 613.27, found 613.16; 1H NMR (400 MHz, CD3OD) 8 9.25 (s, 1H), 8.82 - 8.88 (m, 1H), 8.80 (s, 1H), 8.61 (dd, J = 1.90, 8.25 Hz, 1H), 7.97 (d, J = 8.31 Hz, 1H), 7.64 (d, J = 2.57 Hz, 1H), 7.48 (td, J = 2.69, 5.87 Hz, 2H), 7.36 - 7.41 (m, 1H), 6.96 - 7.06 (m, 1H), 6.81 - 6.91 (m, 1H), 5.51 (s, 2H), 4.45 (q, J = 7.01 Hz, 2H), 4.04 (d, J = 6.85 Hz, 2H), 2.89 - 3.01 (m, 8H), 2.17 (quind, J= 6.86, 13.63 Hz, 1H), 1.61 (t, J= 6.97 Hz, 3H), 1.03 (d, J = 6.72 Hz, 6H).
[0808] Cpd. No. 23: (E)-N-(4-((3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3- yl)methoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2- enamide was prepared in a manner according to general procedure D by coupling between 6-amino-4-((3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3- yl)methoxy)phenyl)amino)-7-ethoxyquinoline-3-carbonitrile and (E)-4-
(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 66%; LC-MS calculated for C35H37CIN6O4 [M + H]+ = 641.27, found 641.15; 1H NMR (400 MHz, CD3OD) 8 9.25 (s, 1H), 8.79 - 8.86 (m, 2H), 8.56 (dd, J = 1.90, 8.38 Hz, 1H), 7.96 (d, J= 8.44 Hz, 1H), 7.64 (d, J = 2.57 Hz, 1H), 7.43 - 7.50 (m, 2H), 7.36 - 7.41 (m, 1H), 6.96 - 7.05 (m, 1H), 6.86 (d, J = 15.16 Hz, 1H), 5.49 (s, 2H), 4.45 (q, J = 6.97 Hz, 2H), 4.12 (td, J = 3.01, 11.34 Hz, 2H), 4.04 (d, J = 6.60 Hz, 2H), 3.27-3.35 (m, 1H), 3.56 - 3.69 (m, 2H), 3.23 - 3.31 (m, 1H), 1.91 - 2.02 (m, 4H), 1.61 (t, J = 6.97 Hz, 3H)
[0809] Cpd. No. 25: (E)-N-(4-((3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3- yl)methoxy)phenyl)amino)-7-ethoxy-2-methylquinolin-6-yl)-4-(dimethylamino)but-2- enamide was prepared in a manner according to general procedure D by coupling between N4-(3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3-yl)methoxy)phenyl)-7- ethoxy-2-methylquinoline-4,6-diamine and (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 60%; LC-MS calculated for C35H40CIN5O4 [M + H]+ = 630.29, found 630.56; 1H NMR (400 MHz, CD3OD) 8 9.10 (s, 1H), 8.74 (d, J = 1.56, 1H), 8.26 (dd, J = 1.83, 8.19 Hz, 1H), 7.71 (d, J = 8.19 Hz, 1H), 7.56 (s, 1H), 7.41 (d, J = 1.22 Hz, 2H), 7.30 (s, 1H), 6.94 - 7.05 (m, 1H), 6.79 - 6.88 (m, 1H), 6.58 (s, 1H), 5.39 (s, 2H), 4.41 (q, J = 6.97 Hz, 2H), 4.11 (dd, J = 2.75, 10.33 Hz, 2H), 4.05 (d, J = 7.09 Hz, 2H), 3.62 (dt, J = 3.30, 11.25 Hz, 2H), 3.08 - 3.26 (m, 1H), 2.97 (s, 6H), 2.60 (s, 3H), 1.91 - 2.00 (m, 4H), 1.61 (t, J = 6.97 Hz, 3H)
[0810] Cpd. No. 26: (E)-N-(4-((3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3- yl)methoxy)phenyl)amino)-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between N4-(3- chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3-yl)methoxy)phenyl)-7- ethoxyquinoline-4,6-diamine and (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 55%; LC-MS calculated for C34H38CIN5O4 [M + H]+ = 616.27, found 616.52; 1H NMR (400 MHz, CD3OD) 8 9.17 (s, 1H), 8.79 (s, 1H), 8.33 (m, 1H), 8.26 (d, J= 8.19 Hz, 1H), 7.76 (d, J = 8.19 Hz, 1H), 7.56 (s, 1H), 7.43 (m, 2H), 7.41 (s, 1H), 6.94 - 7.05 (m, 1H), 6.79 - 6.88 (m, 1H), 6.75 (d, J = 4.0 Hz, 1H), 5.41 (s, 2H), 4.41 (q, J = 6.97 Hz, 2H), 4.11 (dd, J = 2.75, 10.33 Hz, 2H), 4.05 (d, J = 7.09 Hz, 2H), 3.62 (dt, J= 3.30, 11.25 Hz, 2H), 3.18 - 3.26 (m, 1H), 2.97 (s, 6H), 1.87 - 2.03 (m, 4H), 1.61 (t, 7= 6.97 Hz, 3H).
[0811] Cpd. No. 28: (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7- ethoxy-2-ethylquinolin-6-yl)-4-(dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between N4-(3-chloro-4-(pyridin-2- ylmethoxy)phenyl)-7-ethoxy-2-ethylquinoline-4,6-diamine and (E)-4-
(dimethylamino)but-2-enoyl chloride hydrochloride. Yield 72%; LC-MS calculated for C31H34CIN5O3 [M + H]+ = 560.25, found 560.45; 1H NMR (400 MHz, CD3OD) 8 9.10 (s, 1H), 8.59 - 8.67 (m, 1H), 7.99 - 8.06 (m, 1H), 7.76 - 7.83 (m, 1H), 7.57 (d, J = 2.32 Hz, 1H), 7.46 - 7.53 (m, 1H), 7.28 - 7.45 (m, 3H), 6.95 - 7.04 (m, 1H), 6.81 - 6.89 (m, 1H), 6.60 (s, 1H), 5.38 (s, 2H), 4.41 (q, J = 6.89 Hz, 2H), 4.05 (dd, J = 0.92, 7.15 Hz, 2H), 2.97 (s, 6H), 2.84 - 2.90 (m, 2H), 1.61 (t, J= 6.97 Hz, 3H), 1.34 (t, 7= 7.58 Hz, 3H).
[0812] Cpd. No. 31: (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7- ethoxy-2-methylquinolin-6-yl)-4-(dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between N4-(3-chloro-4-(pyridin- 2-ylmethoxy )phenyl)-7 -ethoxy-2-methylquinoline-4,6-diamine and 4-
(dimethylamino)butanoyl chloride hydrochloride. Yield 73%; LC-MS calculated for C30H32CIN5O3 [M + H]+ = 546.23, found 546.44; 1 H NMR (400 MHz, CD3OD) 8 9.09 (s, 1H), 8.59 - 8.67 (m, 1H), 8.01 (dt, 7 = 1.77, 7.73 Hz, 1H), 7.56 (d, 7 = 2.20 Hz, 1H), 7.49 (dd, 7 = 5.14, 6.48 Hz, 1H), 7.33 - 7.41 (m, 2H), 7.29 (s, 1H), 6.95 - 7.04 (m, 1H), 6.85 (d, 7 = 15.16 Hz, 1H), 6.58 (s, 1H), 5.38 (s, 2H), 4.41 (q, 7 = 6.97 Hz, 2H), 4.05 (dd, 7 = 0.98, 7.09 Hz, 2H), 2.97 (s, 6H), 2.60 (s, 3H), 1.61 (t, 7 = 6.97 Hz, 3H).
[0813] Cpd. No. 32: (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7- ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide was prepared in a manner according to general procedure D by coupling between N4-(3-chloro-4-(pyridin-2- ylmethoxy)phenyl)-7 -ethoxy quinoline-4,6-diamine and 4-(dimethylamino)butanoyl chloride hydrochloride. Yield 56%; LC-MS calculated for C29H30CIN5O3 [M + H]+ = 532.21, found 532.43; 1H NMR (400 MHz, CD3OD) 8 9.17 (s, 1H), 8.73 - 8.77 (m, 1H),
8.27 - 8.28 (m, 2H), 7.99 (d, 7 = 8.0 Hz, 1H), 7.71- 7.74 (m, 1H), 7.58 - 7.62 (m, 1H), 7.33 - 7.41 (m, 3H), 6.95 - 7.04 (m, 1H), 6.85 (d, J= 15.16 Hz, 1H), 6.77 (d, J= 4.0 Hz, 1H), 5.49 (s, 2H), 4.42 (q, J = 6.97 Hz, 2H), 4.05 (dd, J = 0.98, 7.09 Hz, 2H), 2.97 (s, 6H), 1.61 (t, 7 = 6.97 Hz, 3H).
EXAMPLE 5
General procedure F - Preparation of Cpds Nos. 5, 7, and 8
Scheme 3
[0814] To a solution of (E)-4-bromobut-2-cnoic acid (0.204 mmol) in CH2Q2 (1 mL) was added 2.0 M solution of oxalyl chloride in CH2Q2 (0.204 mmol) followed by 1 drop of DMF. The solution was stirred for 2 h during which time gas evolution ceased. The solvent was evaporated giving the carboxylic acid chloride. A solution of 6-amino-4-((3- chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7-ethoxyquinoline-3-carbonitrile (0.157 mmol) in NMP was stirred under nitrogen in an ice bath as a solution of the acid chloride prepared above in NMP (0.2 mL) was added. The mixture was then allowed to ambient temperature and stirred for 2 h. Then, appropriate amine (0.204 mmol) was added, and the resulting mixture was stirred at rt for 12 h. The compounds were purified by preparative HPLC to yield the desired compound as a light-yellow solid. In addition to this one pot method, intermediate Compound VII (-Br and -Cl mixture) was isolated and treated with appropriate amine in the presence of CS2CO3 in acetonitrile at 60 °C for 2 h to afford desired compounds such as Cpd. No. 5 (Step C.2), Cpd. No. 7 (Step C.3), and Cpd. No. 8 (Step C.4) in higher yields.
[0815] (E)-4-bromo-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7- ethoxyquinolin-6-yl)but-2-enamide VII. LCMS calculated for C28H23BrClNsO3 [M + H]+ = 592.08, found 592.25 and 593.72.
[0816] (E)-4-chloro-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7- ethoxyquinolin-6-yl)but-2-enamide VII. LCMS calculated for C28H23CI2N5O3 [M + H]+ = 548.13, found 547.81
[0817] Cpd No. 5: (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano- 7-ethoxyquinolin-6-yl)-4-morpholinobut-2-enamide was prepared in a manner according to general procedure F by SN2 reaction of Compound VII with morpholine in NMP (or) CS2CO3 in acetonitrile. Yield 78%; LC-MS calculated for C32H31CIN6O4 [M + H]+ = 599.22, found 599.46; 1H NMR (400 MHz, CDCI3) 8 9.06 (s, 1H), 8.62 (br. s„ 2H), 7.86 - 7.93 (m, 1H), 7.83 (br. s„ 1H), 7.75 (d, J = 7.70 Hz, 1H), 7.45 (d, J = 2.20 Hz, 1H), 7.34 - 7.41 (m, 1H), 7.21 - 7.27 (m, 1H), 7.12 (d, J = 8.56 Hz, 1H), 6.94 - 7.04 (m, 1H), 6.68 (d, J = 15.28 Hz, 1H), 5.38 (s, 2H), 4.40 (d, J = 6.60 Hz, 2H), 3.99 (br. s„ 4H), 3.87 (d, J= 5.99 Hz, 2H), 3.25 (br. s„ 4H), 1.57 (t, J= 6.24 Hz, 3H).
[0818] Cpd. No. 7: (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano- 7-ethoxyquinolin-6-yl)-4-(piperidin-l-yl)but-2-enamide was prepared in a manner according to general procedure F by SN2 reaction of Compound VII with piperidine in NMP (or) CS2CO3 in acetonitrile. Yield 72%; LC-MS calculated for C33H33CIN6O3 [M + H]+ = 597.24, found 597.40; 1H NMR (400 MHz, CD3OD) 8 8.94 (s, 1H), 8.53 - 8.61 (m, 1H), 8.37 (s, 1H), 7.92 (dt, J = 1.77, 7.73 Hz, 1H), 7.72 (d, J = 7.83 Hz, 1H), 7.40 (ddd, J = 1.10, 4.98, 7.49 Hz, 1H), 7.37 (d, J = 2.32 Hz, 1H), 7.24 (s, 1H), 7.11 - 7.20 (m, 2H), 7.01 (td, J = 6.92, 15.25 Hz, 1H), 6.63 (d, J = 15.28 Hz, 1H), 5.28 (s, 2H), 4.31 (q, J = 6.97 Hz, 2H), 3.55 (d, J = 6.85 Hz, 2H), 2.84 (br. s„ 4H), 1.74 (quin, J = 5.65 Hz, 4H), 1.56 (t, 7= 6.97 Hz, 5H).
[0819] Cpd. No. 8: (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano- 7-ethoxyquinolin-6-yl)-4-(4-methylpiperazin-l-yl)but-2-enamide was prepared in a manner according to general procedure F by SN2 reaction of VII with 1 -methylpiperazine in NMP (or) CS2CO3 in acetonitrile. Yield 70%; LC-MS calculated for C33H34CIN7O3 [M + H]+ = 612.25, found 612.34; 1H NMR (400 MHz, CD3OD) 8 8.97 (s, 1H), 8.55 - 8.64 (m, 1H), 8.37 (s, 1H), 7.93 (dt, J = 1.77, 7.73 Hz, 1H), 7.73 (d, J = 7.82 Hz, 1H), 7.42 (ddd, J = 1.10, 5.01, 7.58 Hz, 1H), 7.35 (d, J = 2.08 Hz, 1H), 7.20 (s, 1H), 7.10 - 7.18 (m, 2H), 7.00 (td, J = 6.02, 15.34 Hz, 1H), 6.55 (d, J = 15.28 Hz, 1H), 5.28 (s, 2H), 4.30 (q, J = 7.05 Hz, 2H), 3.17 (br. s„ 4H), 2.79 (s, 4H), 2.73 (br. s„ 3H), 1.57 (t, J = 7.03 Hz, 3H).
EXAMPLE 6
General Procedure E - Preparation of Cpd. Nos. 2 and 29
[0820] 4-(Dimethylamino)butanoyl chloride hydrochloride (0.067 mmol) was added at 0 °C to a suspension of VI (0.045 mmol) in N-methylpyrrolidine. The mixture was then allowed to ambient temperature and stirred for 2 h. The compound was purified by preparative HPLC to yield the Cpd. Nos. 2 and 29.
[0821] Cpd. No. 2: N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7- ethoxyquinolin-6-yl)-4-(dimethylamino)butanamide was prepared in a manner according to general procedure E by coupling between 6-amino-4-((3-chloro-4-(pyridin-2- ylmethoxy)phenyl)amino)-7-ethoxyquinoline-3-carbonitrile and
4-(dimethylamino)butanoyl chloride hydrochloride. Yield 62%; LC-MS calculated for C30H31CIN6O3 [M + H]+ = 559.22, found 559.15; 1H NMR (400 MHz, CD3OD) 8 8.84 (s, 1H), 8.54 - 8.61 (m, 1H), 8.42 (s, 1H), 7.92 (dt, J = 1.71, 7.76 Hz, 1H), 7.73 (d, J = 7.95 Hz, 1H), 7.38 - 7.44 (m, 2H), 7.33 (s, 1H), 7.16 - 7.25 (m, 2H), 5.30 (s, 2H), 4.34 (q, J = 6.97 Hz, 2H), 2.87 - 2.93 (m, 2H), 2.61 - 2.70 (m, 8H), 2.05 (quin, J = 7.43 Hz, 2H), 1.57 (t, 7= 7.03 Hz, 3H).
[0822] Cpd No. 29: N-(4-((3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3- yl)methoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-4- (dimethylamino)butanamide was prepared in a manner according to general procedure E by coupling between 6-amino-4-((3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3- yl)methoxy)phenyl)amino)-7-ethoxyquinoline-3-carbonitrile and 4-
(dimethylamino)butanoyl chloride hydrochloride. Yield 59%; LC-MS calculated for C35H39CIN6O4 [M + H]+ = 643.28, found 643.18; 1H NMR (400 MHz, CD3OD) 8 9.16 (s, 1H), 8.77 - 8.83 (m, 2H), 8.49 (dd, J = 2.08, 8.31 Hz, 1H), 7.90 (d, J= 8.31 Hz, 1H), 7.62 (d, J = 2.45 Hz, 1H), 7.41 - 7.48 (m, 2H), 7.34 - 7.40 (m, 1H), 5.47 (s, 2H), 4.43 (q, J = 6.97 Hz, 2H), 4.12 (td, J = 3.00, 11.25 Hz, 2H), 3.57 - 3.67 (m, 2H), 3.22 - 3.30 (m, 3H), 2.76 (t, J = 6.97 Hz, 2H), 2.08 - 2.20 (m, 2H), 1.87 - 2.03 (m, 4H), 1.60 (t, J = 6.97 Hz, 3H).
EXAMPLE 7 Synthesis of Cpd Nos. 9-11
Scheme 4
[0823] Step D.l and Step D.2.- (E)-4-((4-aminobutyl)(methyl)amino)-N-(4-((3-chloro-4- (pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)but-2-enamide (Compound IX). To a solution of Compound VII (1 eq.) in NMP (4 vol.) was added tertbutyl (4-(methylamino)butyl)carbamate (1.1 eq.) and the resulting mixture was stirred at rt for 12 h. The reaction mixture was quenched with trifluoracetic acid and stirred an additional 30 min at room temperature. The compound was purified by preparative HPLC to yield the amine product Compound IX as a light brown oil in 93% yield. LC-MS calculated for C33H36CIN7O3 [M + H]+ = 614.27, found 614.25.
[0824] Step D.3: A solution of Compound IX (1 eq.) in DMF (1 mL) was added at room temperature to a solution of acid Biotin (1 eq.) DIPEA (2 eq.) and HATU (1.1 eq.) in DMF (1 mL). The mixture was then stirred at room temperature for 12 h. The compound was purified by preparative HPLC to afford Cpd. No. 9: N-(4-(((E)-4-((4-((3-chloro-4- (pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)amino)-4-oxobut-2- en-l-yl)(methyl)amino)butyl)-5-((3aS,4S,6aR)-2-oxohexahydro-177-thieno[3,4- d]imidazol-4-yl)pentanamide, as a light-yellow solid in 56% yield. LC-MS calculated for C43H50CIN9O5S [M + H]+ = 840.34, found 840.29. 1 H NMR (400 MHz, CD3OD) 8 9.24 (s, 1H), 8.82 (s, 1H), 8.68 (d, J = 4.89 Hz, 1H), 8.13 (dt, J = 1.65, 7.79 Hz, 1H), 7.88 (d, J = 7.82 Hz, 1H), 7.64 (d, J = 2.57 Hz, 1H), 7.60 (dd, J = 5.69, 7.03 Hz, 1H), 7.41 - 7.50 (m, 2H), 7.33 (d, J= 8.93 Hz, 1H), 6.96 - 7.09 (m, 1H), 6.82 - 6.92 (m, 1H), 5.44 (s, 2H), 4.38 - 4.57 (m, 3H), 4.30 (dd, J = 4.52, 7.83 Hz, 1H), 4.07 (br. s„ 2H), 3.13 - 3.31 (m, 5H), 2.84 - 3.01 (m, 4H), 2.71 (d, J = 12.72 Hz, 1H), 2.23 (t, J = 7.27 Hz, 2H), 1.54 - 1.84 (m, 11H), 1.40 - 1.50 (m, 2H).
[0825] Step D.4: A solution of Compound IX (1 eq.) in DMF (1 mL) was added at room temperature to a solution of Biotin-PEG4-acid (1 eq.), DIPEA (2 eq.) and HATU (1.1 eq) in DMF (1 mL). The mixture was then stirred at room temperature for 12 h. The compound was purified by preparative HPLC to afford Cpd. No. 10: A-(4-(((E)-4-((4-((3- chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)amino)-4- oxobut-2-en-l-yl)(methyl)amino)butyl)-l-(5-((3aS,4S,6aR)-2-oxohexahydro-177- thieno[3,4-d]imidazol-4-yl)pentanamido)-3,6,9, 12-tetraoxapentadecan- 15-amide, as a light-yellow solid in 36% yield. LC-MS calculated for C54H71CIN10O10S [M + H]+ = 1087.49, found 1087.40. 1H NMR (400 MHz, CD3OD) 8 9.27 (s, 1H), 8.83 (s, 1H), 8.75 (d, J = 4.65 Hz, 1H), 8.29 (dt, J = 1.47, 7.83 Hz, 1H), 7.99 (d, J = 7.95 Hz, 1H), 7.72 - 7.86 (m, 1H), 7.66 (d, J = 2.45 Hz, 1H), 7.48 (dd, J = 2.51, 8.74 Hz, 1H), 7.42 (s, 1H), 7.36 (d, J = 8.80 Hz, 1H), 7.02 (td, J = 7.26, 14.95 Hz, 1H), 6.87 (d, J = 15.28 Hz, 1H), 5.51 (s, 2H), 4.42 - 4.55 (m, 3H), 4.32 (td, J = 5.07, 7.70 Hz, 1H), 3.72 - 3.78 (m, 2H), 3.59 - 3.71 (m, 16H), 3.55 (q, J = 5.62 Hz, 2H), 3.36 - 3.40 (m, 2H), 3.28 (t, J = 6.60 Hz, 2H), 3.22 (dd, J = 5.20, 8.01 Hz, 1H), 2.87 - 3.00 (m, 4H), 2.68 - 2.76 (m, 1H), 2.57 (t, J = 6.24 Hz, 1H), 2.47 (t, J = 6.05 Hz, 2H), 2.23 (q, J = 7.46 Hz, 2H), 1.54 - 1.84 (m, 11H), 1.40 - 1.50 (m, 2H)
[0826] Step D.5: A solution of Compound IX (53.4 mg, 0.087 mmol) in DMF (0.5 mL) was added at room temperature to a solution of acid X (30 mg, 0.087 mmol) DIPEA (30.3 |lL, 0.174 mmol) and HATU (39.9 mg, 0.105 mmol) in DMF (1 mL). The mixture was then stirred at room temperature for 16 h. The compound was purified by preparative HPLC to afford Cpd. No. 11: A-(4-(((E)-4-((4-((3-chloro-4-(pyridin-2- ylmethoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)amino)-4-oxobut-2-en-l- yl)(methyl)amino)butyl)-5-(5-((3aS,4S,6aR)-2-oxohexahydro-177-thieno[3,4-d]imidazol- 4-yl)pentanamido)pentanamide, (21.3 mg, 26% yield) as a light-yellow solid. LC-MS calculated for C48H59CIN10O6S [M + H]+ = 939.41, found 939.21. 1H NMR (400 MHz, CD3OD) > 9.26 (s, 1H), 8.83 (s, 1H), 8.73 (d, J = 4.89 Hz, 1H), 8.26 (dt, J = 1.59, 7.76 Hz, 1H), 7.96 (d, J = 7.82 Hz, 1H), 7.69 - 7.75 (m, 1H), 7.66 (d, J = 2.69 Hz, 1H), 7.47 (dd, J = 2.69, 8.80 Hz, 1H), 7.40 (s, 1H), 7.35 (d, J = 9.05 Hz, 1H), 6.97 - 7.08 (m, 1H), 6.81 - 6.93 (m, 1H), 5.45 - 5.56 (m, 2H), 4.41 - 4.54 (m, 3H), 4.31 (dd, J= 4.40, 7.82 Hz, 1H), 4.10 (br. s„ 2H), 3.13 - 3.29 (m, 6H), 2.90 - 3.02 (m, 4H), 2.70 (d, J = 12.72 Hz, 1H), 2.15 - 2.29 (m, 4H), 1.38 - 1.83 (m, 18H)
Scheme 5 x
[0827] Step E.l and Step E.2: 5-(5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4- d]imidazol-4-yl)pentanamido)pentanoic acid (Compound X). Benzyl 5-aminopentanoate hydrogen chloride XI (49.9 mg, 0.205 mmol) was added at room temperature to a solution of Biotin (50 mg, 0.205 mmol), DIPEA (71 pL, 0.409 mmol) and HATU (85.7 mg, 0.226 mmol) in DMF (1 mL). The reaction mixture was stirred at room temperature for 5 h until completion by LCMS analysis. Water and ethyl acetate were added to quench the reaction. The aqueous layer was extracted with ethyl acetate (3x20 mL), and combined organic extracts were washed with water and brine and dried over sodium sulfate and then concentrated in vacuo to afford Compound XII (LC-MS calculated for C22H31N3O4S [M + H]+ = 434.21, found 434.01). The crude residue Compound XII was dissolved in methanol under an atmosphere of nitrogen. Palladium on carbon was added, and the reaction vessel was sparged with hydrogen for 15 min and then stirred for a further 12 h at room temperature until the starting material was consumed by LCMS analysis. The reaction mixture was filtered through Celite® and washed with methanol, then concentrated in cacuo, and purified by preparative HPLC to afford the desired Compound X (36.6 mg, 52% yield) as an off white solid. LC-MS calculated for C15H25N3O4S [M + H]+ = 344.17, found 344.32
EXAMPLE 8
Synthesis of A-(4-chloro-3-cyano-7-ethoxy-2-methylquinolin-6-yl)acetamide
Scheme 6
[0828] Step 1. A mixture of N-(4-amino-2-ethoxyphenyl)acetamide (20.59 mmol), and ethyl (Z)-2-cyano-3-ethoxybut-2-enoate (26.77 mmol) in toluene was refluxed for 16 h. The reaction mixture was allowed to cool to rt, and then the reaction mixture was evaporated under vacuum giving a light brown solid (Yield - 95%) of ethyl (Z)-3-((4- acetamido-3-ethoxyphenyl)amino)-2-cyanobut-2-enoate which was used for the next step without further purification.
[0829] Step 2. To a solution of ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2- cyanobut-2-enoate (19.62 mmol) in Dowtherm was irradiated with micro wave (10 - 15V) at 270 °C for 30 min. The reaction mixture was allowed to cool to rt, and then the precipitated compound was triturated with n-hexane giving a light yellow solid (Yield - 90%) of N-(3-cyano-7-ethoxy-2-methyl-4-oxo-l,4-dihydroquinolin-6-yl)acetamide.
[0830] Step 3. To a solution of N-(3-cyano-7-ethoxy-2-methyl-4-oxo-l,4- dihydroquinolin-6-yl)acetamide (17.54 mmol) in 1, 4-dioxane (50 mL) was added POCI3 (87.72 mmol) and then the resulting mixture was refluxed for 5 ~ 6 h. The solvent was concentrated under vacuum and then purified by flash column chromatography giving an off white solid of N-(4-chloro-3-cyano-7-ethoxy-2-methylquinolin-6-yl)acetamide in 80% yield; LC-MS calculated for C15H14CIN3O2 [M + H]+ = 304.09, found 304.01; 1H NMR (400 MHz, DMSO) 5 9.44 (s, 1H), 9.00 (s, 1H), 7.49 (s, 1H), 4.37 (q, J = 7.0 Hz, 2H), 2.77 (s, 3H), 2.24 (s, 3H), 1.50 (t, J= 7.0 Hz, 3H).
EXAMPLE 9
Synthesis of N-(4-chloro-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)acetamide Scheme 7
[0831] Step 1. A mixture of A-(4-amino-2-ethoxyphenyl)acetamide (20.59 mmol), and ethyl (Z)-2-cyano-3-ethoxypent-2-enoate (26.77 mmol) in toluene was refluxed for 16 h. The reaction mixture was allowed to cool to rt, and then the reaction mixture was evaporated under vacuum giving a light brown solid (Yield - 97%) of ethyl (Z)-3-((4- acetamido-3-ethoxyphenyl)amino)-2-cyanopent-2-enoate which was used for the next step without further purification.
[0832] Step 2. To a solution of ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2- cyanopent-2-enoate (19.98 mmol) in Dowtherm was irradiated with microwave (10 - 15V) at 270 °C for 30 min. The reaction mixture was allowed to cool to rt, and then the precipitated compound was triturated with n-hexane giving a light yellow solid (Yield 84%) of N-(3-cyano-7-ethoxy-2-ethyl-4-oxo-l,4-dihydroquinolin-6-yl)acetamide.
[0833] Step 3. To a solution of N-(3-cyano-7-ethoxy-2-ethyl-4-oxo-l,4-dihydroquinolin- 6-yl)acetamide (16.70 mmol) in 1, 4-dioxane (50 mL) was added POCI3 (83.52 mmol) and then the resulting mixture was refluxed for 5 - 6 h. The solvent was concentrated under vacuum and then purified by flash column chromatography giving an off white solid of N-(4-chloro-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)acetamide in 65% yield; LC- MS calculated for C16H16CIN3O2 [M + H]+ = 318,08 found 318.08; 1H NMR (400 MHz, DMSO) 5 9.45 (s, 1H), 9.03 (s, 1H), 7.51 (s, 1H), 4.39 (q, J = 7.0 Hz, 2H), 3.08 (q, J = 7.5 Hz, 2H), 2.24 (s, 3H), 1.50 (t, J = 7.0 Hz, 3H), 1.36 (t, J = 7.5 Hz, 3H).
EXAMPLE 10
Synthesis of (E)-N-(4-((3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3- yl)methoxy)phenyl)amino)-3-cyano-7-ethoxy-2-methylquinolin-6-yl)-4- (dimethylamino)but-2-enamide (Cpd. No. 24)
Scheme 8
[0834] Step 1. Methansulfonic acid (0.197 mmol) was added at room temperature to a suspension of N-(4-chloro-3-cyano-7-ethoxy-2-methylquinolin-6-yl)acetamide (0.066 mmol) and 3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3-yl)methoxy)aniline (0.079 mmol) in anhydrous ethyl alcohol. The mixture was then refluxed for 4 ~ 6 h. The solution was cooled to ambient temperature, then concentrated in vacuo, and purified by preparative HPLC to afford 6-amino-4-((3-chloro-4-((6-(tetrahydro-2H-pyran-4- yl)pyridin-3-yl)methoxy)phenyl)amino)-7-ethoxy-2-methylquinoline-3-carbonitrile as a yellow solid in 76% yield. LC-MS calculated for C30H30CIN5O3 [M + H]+ = 544.21, found 544.08.
[0835] Step 2. Synthesis of Cpd. No. 24: (E)-4-(dimethylainino)but-2-enoyl chloride hydrochloride (0.067 mmol) was added at 0 °C to a suspension of 6-amino-4-((3-chloro- 4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3-yl)methoxy)phenyl)amino)-7 -ethoxy-2- methylquinoline-3-carbonitrile (0.045 mmol) in N- methyl pyrrolidine (0.3 mL). The mixture was then allowed to ambient temperature and stirred for 2 h. The compound was purified by preparative HPLC to yield Cpd. No. 24 as a light yellow solid in 50% yield. LC-MS calculated for C36H39CIN6O4 [M + H]+ = 655.28, found 655.50; 1H NMR (400 MHz, CD3OD) 8 9.19 (s, 1H), 8.75 - 8.81 (m, 1H), 8.39 (dd, J = 1.83, 8.19 Hz, 1H), 7.81 (d, J = 8.19 Hz, 1H), 7.61 (d, J = 2.45 Hz, 1H), 7.42 - 7.47 (m, 1H), 7.33 - 7.40 (m, 2H), 6.95 - 7.04 (m, 1H), 6.80 - 6.89 (m, 1H), 5.44 (s, 2H), 4.45 (q, J = 6.97 Hz, 2H), 4.08 - 4.16 (m, 2H), 4.04 (d, J= 7.09 Hz, 2H), 3.58 - 3.67 (m, 2H), 3.21 - 3.28 (m, 1H), 2.96 (s, 6H), 2.80 (s, 3H), 1.91 - 2.00 (m, 4H), 1.61 (t, J = 6.97 Hz, 3H). EXAMPLE 11
Synthesis of (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7- ethoxy-2-ethylquinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 27) Scheme 9
[0836] Step 1. Methansulfonic acid (0.189 mmol) was added at room temperature to a suspension of A-(4-chloro-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)acetamide (0.063 mmol) and 3-chloro-4-(pyridin-2-ylmethoxy)aniline (0.076 mmol) in anhydrous ethyl alcohol. The mixture was then refluxed for 4 - 6 h. The solution was cooled to ambient temperature, then concentrated in vacuo, and purified by preparative HPLC to afford 6- amino-4-((3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3- yl)methoxy)phenyl)amino)-7-ethoxy-2-methylquinoline-3-carbonitrile as a yellow solid in 79% yield. LC-MS calculated for C26H24CIN5O2 [M + H]+ = 474.17, found 474.16.
[0837] Step 2. Synthesis of Cpd. No. 27: (E)-A-(4-((3-chloro-4-((6-(tetrahydro-2H- pyran-4-yl)pyridin-3-yl)methoxy)phenyl)amino)-3-cyano-7-ethoxy-2-methylquinolin-6- yl)-4-(dimethylamino)but-2-enamide. ( E) -4-(dimethylamino)but-2-enoy I chloride hydrochloride (0.046 mmol) was added at 0 °C to a suspension of 6-amino-4-((3-chloro- 4-(pyridin-2-ylmethoxy)phenyl)amino)-7-ethoxy-2-ethylquinoline-3-carbonitrile (0.042 mmol) in A-methylpyrrolidine (0.3 mL). The mixture was then allowed to ambient temperature and stirred for 2 h. The compound was purified by preparative HPLC to yield Cpd. No. 27 as a light yellow solid in 62% yield. LC-MS calculated for C32H33CIN6O3 [M + H]+ = 585.24, found 585.29; 1H NMR (400 MHz, CD3OD) 8 9.18 (s, 1H), 8.64 (dd, J = 0.73, 5.01 Hz, 1H), 8.05 (dt, J = 1.65, 7.79 Hz, 1H), 7.82 (d, J= 7.83 Hz, 1H), 7.62 (d, J = 2.57 Hz, 1H), 7.53 (dd, J = 5.20, 6.66 Hz, 1H), 7.38 - 7.45 (m, 2H), 7.31 (d, J= 8.80 Hz, 1H), 6.94 - 7.04 (m, 1H), 6.79 - 6.88 (m, 1H), 5.41 (s, 2H), 4.45 (q, J = 6.93 Hz, 2H), 4.04 (dd, J = 0.86, 7.09 Hz, 2H), 3.10 (q, J = 7.58 Hz, 2H), 2.96 (s, 6H), 1.61 (t, 7 = 6.97 Hz, 3H), 1.44 (t, 7 = 7.64 Hz, 3H).
EXAMPLE 12
Synthesis of (E)-A-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7- ethoxy-2-methylquinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 30)
Scheme 10
[0838] Step 1. Methansulfonic acid (0.197 mmol) was added at room temperature to a suspension of A-(4-chloro-3-cyano-7-ethoxy-2-methylquinolin-6-yl)acetamide (0.066 mmol) and 3-chloro-4-(pyridin-2-ylmethoxy)aniline (0.079 mmol) in anhydrous ethyl alcohol. The mixture was then refluxed for 4 - 6 h. The solution was cooled to ambient temperature, then concentrated in vacuo, and purified by preparative HPLC to afford 6- amino-4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7-ethoxy-2-methylquinoline-
3-carbonitrile as a yellow solid in 73% yield. LC-MS calculated for C25H22CIN5O2 [M + H]+ = 460.16, found 460.18.
[0839] Step 2. Synthesis of Cpd. No. 30: ('E')-4-(dimethylamino)but-2-enoyl chloride hydrochloride (0.048 mmol) was added at 0 °C to a suspension of 6-amino-4-((3-chloro-
4-(pyridin-2-ylmethoxy)phenyl)amino)-7-ethoxy-2-methylquinoline-3-carbonitrile (0.044 mmol) in A-methylpyrrolidine (0.3 mL). The mixture was then allowed to ambient temperature and stirred for 2 h. The compound was purified by preparative HPLC to yield Cpd. No. 30 as a light yellow solid in 68% yield. LC-MS calculated for C31H31CIN6O3 [M + H]+ = 571.22, found 571.46; 1H NMR (400 MHz, CD3OD) 8 9.18 (s, 1H), 8.63 - 8.69 (m, 1H), 8.07 (dt, J = 1.65, 7.79 Hz, 1H), 7.83 (d, J = 7.82 Hz, 1H), 7.61 (d, J = 2.57 Hz, 1H), 7.53 - 7.58 (m, 1H), 7.42 (dd, J = 2.57, 8.80 Hz, 1H), 7.37 (s, 1H), 7.28 - 7.34 (m, 1H), 6.95 - 7.04 (m, 1H), 6.80 - 6.89 (m, 1H), 5.42 (s, 2H), 4.44 (q, J = 6.97 Hz, 2H), 4.04 (dd, J = 0.86, 7.09 Hz, 2H), 2.96 (s, 6H), 2.80 (s, 3H), 1.61 (t, J= 6.97 Hz, 3H).
EXAMPLE 13
General Syntheses of Compounds of the Disclosure
[0840] The synthesis of synthetic intermediates and/or Compounds of the Disclosure can be achieved according to Schemes 11-23 using the methods described above and/or methods known in the art.
Scheme 11
[0841] Step 1: Sodium hydride (60%, 5.69 mmol) in dry DMF (3 mL) under a nitrogen atmosphere and the resulting mixture is cooled in ice water. To above suspension is added appropriate alcohol (2.85 mmol) in anhydrous DMF (2 mL). 2-Chloro-l-fluoro-4- nitrobenzene (2.85 mmol) was added to the above solution and the resulting solution was stirred at rt. Cold water was added slowly to the reaction mixture, and a yellow precipitate resulted. The yellow solid is isolated by vacuum filtration, washed with cold water, and air dried to give the title compound as yellow solid.
[0842] Step 2: A mixture of nitro compound (2.51 mmol), iron (12.56 mmol) and NH4CI (25.11 mmol) in 70% ethanol in water was heated at 70 °C for 1 h. The reaction mixture was allowed to cool to rt, and then filtered over celite®. The filtrate was evaporated under vacuum giving an orange solid which was stirred with EtOAc, and then the resulting mixture was filtered. The filtrate was dried over anhydrous Na2SO4 and evaporated under vacuum to afford the desired amine compound. The crude compound was used for the next step without further purification.
[0843] Step 3: Methansulfonic acid (1.035 mmol) was added at room temperature to a suspension of the corresponding acetamide (0.345 mmol) and the appropriate amine (0.345 mmol) in anhydrous ethyl alcohol. The mixture was then refluxed. The solution was cooled to ambient temperature, then concentrated in vacuo, and purified by preparative HPLC to yield the desired compounds.
[0844] Step 4: (E)-4-(Dimethylamino)but-2-enoyl chloride hydrochloride (0.067 mmol) was added at 0 °C to a suspension of the appropriate amine (0.045 mmol) in N-methylpyrrolidine. The mixture was then allowed to ambient temperature and stirred for ~2 h. The compound was purified by preparative HPLC to yield the desired compounds.
Scheme 12
[0845] Methansulfonic acid (1.035 mmol) was added at room temperature to a suspension of the corresponding acetamide (0.345 mmol) and the appropriate amine (0.345 mmol) in anhydrous ethyl alcohol. The mixture was then refluxed. The solution was cooled to ambient temperature, then concentrated in vacuo, and purified by preparative HPLC to yield the desired compounds.
[0846] (E)-4-(Dimethylamino)but"2-enoyl chloride hydrochloride (0.067 mmol) was added at 0 °C to a suspension of the appropriate amine (0.045 mmol) in N- methylpyrrolidine. The mixture was then allowed to ambient temperature and stirred for ~2 h. The compound was purified by preparative HPLC to yield the desired compounds.
Scheme 13
Scheme 15
1, 4-dioxane, H2O,
90 °C, 12 h
Scheme 19
Cpd. No. 33, R2 = -H
Cpd. No. 34, R2 = -CH3
Cpd. No. 35, R2 = -Cl
Scheme 22
EXAMPLE 14
Synthesis of N-(4-chloro-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)acetamide
[0847] Step 1. Synthesis of ethyl (Z)-2-cyano-3-ethoxypent-2-enoate: Acetic acid (1.3 g, 0.5 eq) was added at room temperature to a solution of ethyl 2-cyanoacetate (5 g, 44.2 mmol, 1.0 eq) in 1,1,1 -triethoxypropane (15.6 g, 88.4 mmol, 2.0 eq). The mixture was then stirred at 125 °C for 16 h. The solution was cooled to ambient temperature, then concentrated in vacuo to afford 9.0 g of ethyl (Z)-2-cyano-3-ethoxypent-2-enoate in quantitative yield as a yellow oil. LC-MS calculated for C10H15NO3 [M + H]+ = 198.12, found 198.23. The crude compound was used for the next step without further purification.
[0848] Step 2. Synthesis of ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2- cyanopent-2-enoate: To a solution of N-(4-amino-2-ethoxyphenyl)acetamide (2.0 g, 10.3 mmol, 1.0 eq) in toluene (20 mL) was added ethyl (Z)-2-cyano-3-ethoxypent-2-enoate (2.03 g, 10.3 mmol, 1.0 eq) at room temperature. The mixture was then stirred at 90 °C for 16 h. The solution was cooled to ambient temperature, then concentrated in vacuo and purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 3.02 g of ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2-cyanopent-2- enoate in 85% yield as off white solid. LC-MS calculated for C18H23N3O4 [M + H]+ = 346.18, found 346.37. [0849] Step 3. Synthesis of N-(3-cyano-7-ethoxy-2-ethyl-4-oxo-l,4-dihydroquinolin-6- yl)acetamide: Ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2-cyanopent-2-enoate (3.0 g, 8.69 mmol, 1 eq) was dissolved in Dowtherm (15 mL) and then the solution was irradiated under microwave at 260 °C for 2 h. The reaction was cooled to room temperature and triturated with n-hexane to afford 2.2 g of the N-(3-cyano-7-ethoxy-2- ethyl-4-oxo-l,4-dihydroquinolin-6-yl)acetamide in 85% yield as light brown solid. LC- MS calculated for C16H17N3O3 [M + H]+ = 300.14, found 300.32. The crude compound was used for the next step without further purification.
[0850] Step 4. Synthesis of N-(4-chloro-3-cyano-7-ethoxy-2-ethylquinolin-6- yl)acetamide: To a solution of N-(3-cyano-7-ethoxy-2-ethyl-4-oxo-l,4-dihydroquinolin- 6-yl)acetamide (2.0 g, 6.69 mmol, 1 eq) in diglyme or 1, 4-dioxane (20 mL) was added phosphorus oxychloride (3.59 g, 23.4 mmol, 3.5 eq). The mixture was then stirred at 100 °C for 1 h. The solution was cooled to ambient temperature, then filtered through celite® and the celite® was washed with diglyme and the filtrate was concentrated in vacuo. The crude compound was quenched with saturated sodium bicarbonate (50 mL) and the compound was extracted with ethyl acetate (3x 100 mL). The combined extracts were washed with brine solutions (50 mL) and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 1.95 g of N-(4-chloro-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)acetamide in 92% yield as a white solid. LC-MS calculated for C16H16CIN3O2 [M + H]+ = 318.10, found 318.35; 1H NMR (400 MHz, CDCI3) 8 9.21 (s, 1H), 8.06 (br. s„ 1H), 7.38 (s, 1H), 4.35 (q, J = 7.05 Hz, 2H), 3.16 (q, J = 7.54 Hz, 2H), 2.32 (s, 3H), 1.61 (t, J = 7.03 Hz, 3H), 1.39 - 1.49 (m, 3H).
EXAMPLE 15
Synthesis of N-(4-chloro-3-cyano-7-ethoxy-2-methylquinolin-6-yl)acetamide
[0851] Step 1. Synthesis of ethyl (Z)-2-cyano-3-ethoxybut-2-enoate: Acetic acid (1.3 g, 0.5 eq) was added at room temperature to a solution of ethyl 2-cyanoacetate (5 g, 44.2 mmol, 1.0 eq) in 1,1,1 -triethoxy ethane (14.3 g, 88.4 mmol, 2.0 eq). The mixture was then stirred at 125 °C for 16 h. The solution was cooled to ambient temperature, then concentrated in vacuo to afford 8.5 g of ethyl (Z)-2-cyano-3-ethoxybut-2-enoate in quantitative yield as a yellow oil. LC-MS calculated for C9H13NO3 [M + H]+ = 184.1, found 184.35. The crude compound was used for the next step without further purification.
[0852] Step 2. Synthesis of ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2- cyanobut-2-enoate: To a solution of N-(4-amino-2-ethoxyphenyl)acetamide (2.0 g, 10.3 mmol, 1.0 eq) in toluene (20 mL) was added ethyl (Z)-2-cyano-3-ethoxybut-2-enoate (1.89 g, 10.3 mmol, 1.0 eq) at room temperature. The mixture was then stirred at 90 °C for 16 h. The solution was cooled to ambient temperature, then concentrated in vacuo and purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 3.2 g of (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2-cyanobut-2-enoate in 94% yield as off white solid. LC-MS calculated for C17H21N3O4 [M + H]+ = 332.16, found 332.36. [0853] Step 3. Synthesis of N-(3-cyano-7-ethoxy-2-methyl-4-oxo-l,4-dihydroquinolin-6- yl)acetamide: Ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2-cyanobut-2-enoate (3.0 g, 9.06 mmol, 1 eq) was dissolved in Dowtherm (18 mL) and then the solution was irradiated under microwave at 260 °C for 2 h. The reaction was cooled to room temperature and triturated with n-hexane to afford 2.1 g of the N-(3-cyano-7-ethoxy-2- methyl-4-oxo-l,4-dihydroquinolin-6-yl)acetamide in 82% yield as light green solid. LC- MS calculated for C15H15N3O3 [M + H]+ = 286.12, found 286.37. The crude compound was used for the next step without further purification.
[0854] Step 4. Synthesis of N-(4-chloro-3-cyano-7-ethoxy-2-methylquinolin-6- yl)acetamide: To a solution of N-(3-cyano-7-ethoxy-2-methyl-4-oxo-l,4- dihydroquinolin-6-yl)acetamide (2.0 g, 7.0 mmol, 1 eq) in diglyme or 1, 4-dioxane (20 mL) was added phosphorus oxychloride (3.76 g, 24.55 mmol, 3.5 eq). The mixture was then stirred at 100 °C for 1 h. The solution was cooled to ambient temperature, then filtered through celite® and the celite® was washed with diglyme and the filtrate was concentrated in vacuo. The crude compound was quenched with saturated sodium bicarbonate (50 mL) and the compound was extracted with ethyl acetate (3x 100 mL). The combined extracts were washed with brine solutions (50 mL) and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 1.98 g of N-(4-chloro-3-cyano-7-ethoxy-2- methylquinolin-6-yl)acetamide in 93% yield as a white solid. LC-MS calculated for C15H14CIN3O2 [M + H]+ = 304.09, found 304.27; 1H NMR (400 MHz, DMSO-d6) 8 9.44 (s, 1H), 9.00 (s, 1H), 7.49 (s, 1H), 4.37 (q, J = 6.97 Hz, 2H), 2.77 (s, 3H), 2.24 (s, 3H), 1.50 (t, 7= 6.97 Hz, 3H).
EXAMPLE 16
Synthesis of N-(4-chloro-3-cyano-7-ethoxy-2-isopropylquinolin-6-yl)acetamide
Step 4
[0855] Step 1. Synthesis of ethyl (Z)-2-cyano-3-methoxy-4-methylpent-2-enoate: Acetic acid (1.3 g, 0.5 eq) was added at room temperature to a solution of ethyl 2-cyanoacetate (5 g, 44.2 mmol, 1.0 eq) in l,l,l-trimethoxy-2-methylpropane (14.5 g, 88.4 mmol, 2.0 eq). The mixture was then stirred at 125 °C for 16 h. The solution was cooled to ambient temperature, then concentrated in vacuo to afford 8.9 g of ethyl (Z)-2-cyano-3-methoxy- 4-methylpent-2-enoate in quantitative yield as a yellow oil. LC-MS calculated for C10H15NO3 [M + H]+ = 198.12, found 198.22. The crude compound was used for the next step without further purification.
[0856] Step 2. Synthesis of ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2-cyano- 4-methylpent-2-enoate: To a solution of N-(4-amino-2-ethoxyphenyl)acetamide (2.0 g, 10.3 mmol, 1.0 eq) in toluene (20 mL) was added ethyl (Z)-2-cyano-3-methoxy-4- methylpent-2-enoate (2.03 g, 10.3 mmol, 1.0 eq) at room temperature. The mixture was then stirred at 90 °C for 16 h. The solution was cooled to ambient temperature, then concentrated in vacuo and purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 3.29 g of ethyl (Z)-3-((4-acetamido-3- ethoxyphenyl)amino)-2-cyano-4-methylpent-2-enoate in 89% yield as off white solid. LC-MS calculated for C19H25N3O4 [M + H]+ = 360.19, found 359.99. [0857] Step 3. Synthesis of N-(3-cyano-7-ethoxy-2-isopropyl-4-oxo-l,4- dihydroquinolin-6-yl)acetamide: ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2- cyano-4-methylpent-2-enoate (3.0 g, 8.35 mmol, 1 eq) was dissolved in Dowtherm (18 mL) and then the solution was irradiated under microwave at 260 °C for 2 h. The reaction was cooled to room temperature and triturated with n-hexane to afford 2.25 g of the N-(3- cyano-7-ethoxy-2-isopropyl-4-oxo-l,4-dihydroquinolin-6-yl)acetamide in 86% yield as light green solid. LC-MS calculated for C17H19N3O3 [M + H]+ = 314.15, found 314.28. The crude compound was used for the next step without further purification.
[0858] Step 4. Synthesis of N-(4-chloro-3-cyano-7-ethoxy-2-isopropylquinolin-6- yl)acetamide: To a solution of N-(3-cyano-7-ethoxy-2-isopropyl-4-oxo-l,4- dihydroquinolin-6-yl)acetamide (2.0 g, 6.4 mmol, 1 eq) in diglyme or 1, 4-dioxane (20 mL) was added phosphorus oxychloride (3.43 g, 22.35 mmol, 3.5 eq). The mixture was then stirred at 100 °C for 1 h. The solution was cooled to ambient temperature, then filtered through celite® and the celite® was washed with diglyme and the filtrate was concentrated in vacuo. The crude compound was quenched with saturated sodium bicarbonate (50 mL) and the compound was extracted with ethyl acetate (3x 100 mL). The combined extracts were washed with brine solutions (50 mL) and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 1.73 g of N-(4-chloro-3-cyano-7-ethoxy-2- isopropylquinolin-6-yl)acetamide in 82% yield as a white solid. LC-MS calculated for C17H18CIN3O2 [M + H]+ = 332.12, found 332.27; 1H NMR (400 MHz, DMSO-d6) 8 9.43 (br. s„ 1H), 9.02 (br. s„ 1H), 7.46 (br. s„ 1H), 4.39 (q, J = 6.52 Hz, 2H), 3.47 - 3.53 (m, 1H), 2.24 (s, 3H), 1.50 (t, J = 6.91 Hz, 3H), 1.36 (d, J = 6.60 Hz, 6H).
EXAMPLE 17
Synthesis of N-(4-chloro-3-cyano-2-cyclopropyl-7-ethoxyquinolin-6-yl)acetamide
- 2Z1 -
[0859] Step 1. Synthesis of ethyl (Z)-2-cyano-3-cyclopropyl-3-ethoxyacrylate: Acetic acid (1.3 g, 0.5 eq) was added at room temperature to a solution of ethyl 2-cyanoacetate (5 g, 44.2 mmol, 1.0 eq) in (trimethoxymethyl)cyclopropane (12.92 g, 88.4 mmol, 2.0 eq). The mixture was then stirred at 125 °C for 16 h. The solution was cooled to ambient temperature, then concentrated in vacuo to afford 9.3 g of ethyl (Z)-2-cyano-3- cyclopropyl-3-ethoxyacrylate in quantitative yield as a yellow oil. LC-MS calculated for C11H15NO3 [M + H]+ = 210.12, found 210.27. The crude compound was used for the next step without further purification.
[0860] Step 2. Synthesis of ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2-cyano- 3-cyclopropylacrylate: To a solution of N-(4-amino-2-ethoxyphenyl)acetamide (2.0 g, 10.3 mmol, 1.0 eq) in toluene (20 mL) was added ethyl (Z)-2-cyano-3-cyclopropyl-3- ethoxyacrylate (2.16 g, 10.3 mmol, 1.0 eq) at room temperature. The mixture was then stirred at 90 °C for 16 h. The solution was cooled to ambient temperature, then concentrated in vacuo and purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 2.13 g of ethyl (Z)-3-((4-acetamido-3- ethoxyphenyl)amino)-2-cyano-3-cyclopropylacrylate in 58% yield as light brown solid. LC-MS calculated for C19H23N3O4 [M + H]+ = 358.18, found 358.19. [0861] Step 3. Synthesis of N-(3-cyano-2-cyclopropyl-7-ethoxy-4-oxo-l,4- dihydroquinolin-6-yl)acetamide: Ethyl (Z)-3-((4-acetamido-3-ethoxyphenyl)amino)-2- cyano-3-cyclopropylacrylate (2.0 g, 5.60 mmol, 1 eq) was dissolved in Dowtherm (10 mL) and then the solution was irradiated under microwave at 260 °C for 2 h. The reaction was cooled to room temperature and triturated with n-hexane to afford 1.4 g of the N-(3- cyano-2-cyclopropyl-7-ethoxy-4-oxo-l,4-dihydroquinolin-6-yl)acetamide in 81% yield as light brown solid. LC-MS calculated for C17H17N3O3 [M + H]+ = 312.14, found 314.27. The crude compound was used for the next step without further purification.
[0862] Step 4. Synthesis of N-(4-chloro-3-cyano-2-cyclopropyl-7 -ethoxy quinolin-6- yl)acetamide: To a solution of N-(3-cyano-2-cyclopropyl-7-ethoxy-4-oxo-l,4- dihydroquinolin-6-yl)acetamide (1.2 g, 3.86 mmol, 1 eq) in diglyme or 1, 4-dioxane (10 mL) was added phosphorus oxychloride (2.07 g, 13.5 mmol, 3.5 eq). The mixture was then stirred at 100 °C for 1 h. The solution was cooled to ambient temperature, then filtered through celite® and the celite® was washed with diglyme and the filtrate was concentrated in vacuo. The crude compound was quenched with saturated sodium bicarbonate (40 mL) and the compound was extracted with ethyl acetate (3x 70 mL). The combined extracts were washed with brine solutions (30 mL) and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 1.09 g of N-(4-chloro-3-cyano-2-cyclopropyl-7- ethoxyquinolin-6-yl)acetamide in 86% yield as a white solid. LC-MS calculated for C17H16CIN3O2 [M + H]+ = 330.10, found 330.15.
EXAMPLE 18
Synthesis of (R)-N-(4-chloro-3-cyano-2-ethyl-7-((tetrahydrofuran-3-yl)oxy)quinolin-6- yl)acetamide
[0863] Step 1. Synthesis of ethyl (R,Z)-3-((4-acetamido-3-((tetrahydrofuran-3- yl)oxy)phenyl)amino)-2-cyanopent-2-enoate: To a solution of (R)-N-(4-amino-2- ((tetrahydrofuran-3-yl)oxy)phenyl)acetamide (2.0 g, 8.5 mmol, 1.0 eq) in toluene (20 mL) was added ethyl (Z)-2-cyano-3-ethoxypent-2-enoate (1.7 g, 8.5 mmol, 1.0 eq) at room temperature. The mixture was then stirred at 90 °C for 16 h. The solution was cooled to ambient temperature, then concentrated in vacuo and purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 2.36 g of ethyl (R,Z)-3-((4-acetamido-3-((tetrahydrofuran-3-yl)oxy)phenyl)amino)-2-cyanopent-2- enoate in 72% yield as off white solid. LC-MS calculated for C20H25N3O5 [M + H]+ = 388.19, found 388.32.
[0864] Step 2. Synthesis of (R)-N-(3-cyano-2-ethyl-4-oxo-7-((tetrahydrofuran-3-yl)oxy)- l,4-dihydroquinolin-6-yl)acetamide: (R,Z)-3-((4-acetamido-3-((tetrahydrofuran-3- yl)oxy)phenyl)amino)-2-cyanopent-2-enoate (2.0 g, 5.17 mmol, 1 eq) was dissolved in Dowtherm (12 mL) and then the solution was irradiated under micro wave at 260 °C for 2 h. The reaction was cooled to room temperature and triturated with n-hexane to afford 1.27 g of the N-(3-cyano-7-ethoxy-2-ethyl-4-oxo-l,4-dihydroquinolin-6-yl)acetamide in 72% yield as light brown solid. LC-MS calculated for C18H19N3O4 [M + H]+ = 342.15, found 341.91. The crude compound was used for the next step without further purification.
[0865] Step 3. Synthesis of (R)-N-(4-chloro-3-cyano-2-ethyl-7-((tetrahydrofuran-3- yl)oxy)quinolin-6-yl)acetamide: To a solution of (R)-N-(3-cyano-2-ethyl-4-oxo-7- ((tetrahydrofuran-3-yl)oxy)-l,4-dihydroquinolin-6-yl)acetamide (1.0 g, 2.93 mmol, 1 eq) in diglyme or 1, 4-dioxane (10 mL) was added phosphorus oxychloride (1.57 g, 10.26 mmol, 3.5 eq). The mixture was then stirred at 100 °C for 1 h. The solution was cooled to ambient temperature, then filtered through celite® and the celite® was washed with diglyme and the filtrate was concentrated in vacuo. The crude compound was quenched with saturated sodium bicarbonate (50 mL) and the compound was extracted with ethyl acetate (3x 100 mL). The combined extracts were washed with brine solutions (50 mL) and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 0.88 g of (R)-N-(4- chloro-3-cyano-2-ethyl-7 -((tetrahydrofuran-3-yl)oxy)quinolin-6-yl)acetamide in 84% yield as a pale yellow solid. LC-MS calculated for C18H18CIN3O3 [M + H]+ = 360.11, found 359.93; 1H NMR (400 MHz, DMSO-d6) 8 9.34 (s, 1H), 9.02 (s, 1H), 7.45 (s, 1H), 5.38 (t, J = 5.32 Hz, 1H), 3.93 - 4.13 (m, 3H), 3.80 (dt, J = 5.01, 8.13 Hz, 1H), 3.39 - 3.47 (m, 1H), 3.06 (q, J= 7.46 Hz, 2H), 2.33 - 2.44 (m, 1H), 2.13 - 2.30 (m, 4H), 1.35 (t, 7 = 7.52 Hz, 3H).
EXAMPLE 19
Synthesis of (S)-N-(4-chloro-3-cyano-2-ethyl-7-((tetrahydrofuran-3-yl)oxy)quinolin-6- yl) acetamide
[0866] Step 1. Synthesis of ethyl (S,Z)-3-((4-acetamido-3-((tetrahydrofuran-3- yl)oxy)phenyl)amino)-2-cyanopent-2-enoate: To a solution of (S)-N-(4-amino-2- ((tetrahydrofuran-3-yl)oxy)phenyl)acetamide (2.0 g, 8.5 mmol, 1.0 eq) in toluene (20 mL) was added ethyl (Z)-2-cyano-3-ethoxypent-2-enoate (1.7 g, 8.5 mmol, 1.0 eq) at room temperature. The mixture was then stirred at 90 °C for 16 h. The solution was cooled to ambient temperature, then concentrated in vacuo and purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 2.79 g of ethyl (S,Z)-3-((4-acetamido-3-((tetrahydrofuran-3-yl)oxy)phenyl)amino)-2-cyanopent-2-enoate in 85% yield as off white solid. LC-MS calculated for C20H25N3O5 [M + H]+ = 388.19, found 388.34.
[0867] Step 2. Synthesis of (S)-N-(3-cyano-2-ethyl-4-oxo-7-((tetrahydrofuran-3-yl)oxy)- l,4-dihydroquinolin-6-yl)acetamide: (S,Z)-3-((4-acetamido-3-((tetrahydrofuran-3- yl)oxy)phenyl)amino)-2-cyanopent-2-enoate (2.0 g, 5.17 mmol, 1 eq) was dissolved in Dowtherm (12 mL) and then the solution was irradiated under micro wave at 260 °C for 2 h. The reaction was cooled to room temperature and triturated with n-hexane to afford 1.39 g of the N-(3-cyano-7-ethoxy-2-ethyl-4-oxo-l,4-dihydroquinolin-6-yl)acetamide in 79% yield as light brown solid. LC-MS calculated for C18H19N3O4 [M + H]+ = 342.15, found 342.34. The crude compound was used for the next step without further purification.
[0868] Step 3. Synthesis of (S)-N-(4-chloro-3-cyano-2-ethyl-7-((tetrahydrofuran-3- yl)oxy)quinolin-6-yl)acetamide: To a solution of (S)-N-(3-cyano-2-ethyl-4-oxo-7- ((tetrahydrofuran-3-yl)oxy)-l,4-dihydroquinolin-6-yl)acetamide (1.0 g, 2.93 mmol, 1 eq) in diglyme or 1, 4-dioxane (10 mL) was added phosphorus oxychloride (1.57 g, 10.26 mmol, 3.5 eq). The mixture was then stirred at 100 °C for 1 h. The solution was cooled to ambient temperature, then filtered through celite® and the celite® was washed with diglyme and the filtrate was concentrated in vacuo. The crude compound was quenched with saturated sodium bicarbonate (50 mL) and the compound was extracted with ethyl acetate (3x 100 mL). The combined extracts were washed with brine solutions (50 mL) and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 0.91 g of (S)-N-(4- chloro-3-cyano-2-ethyl-7 -((tetrahydrofuran-3-yl)oxy)quinolin-6-yl)acetamide in 86% yield as a pale yellow solid. LC-MS calculated for C18H18CIN3O3 [M + H]+ = 360.11, found 359.90; 1H NMR (400 MHz, DMSO-d6) 8 9.33 (s, 1H), 9.01 (s, 1H), 7.44 (s, 1H), 5.38 (t, J = 5.44 Hz, 1H), 3.91 - 4.16 (m, 3H), 3.80 (dt, J = 5.07, 8.16 Hz, 1H), 3.06 (q, J = 7.50 Hz, 2H), 2.33 - 2.44 (m, 1H), 2.10 - 2.30 (m, 4H), 1.35 (t, J = 7.46 Hz, 3H).
EXAMPLE 20
Synthesis of Intermediates cr Py
[0869] Step 1. To a solution of 5-nitro-lH-indazole (3.0 g, 18.4 mmol, 1.0 eq) and K2CO3 (5.09 g, 36.8 mmol, 2.0 eq) in DMF (30 mL) was added py-CFhCl (2.6 g, 20.24 mmol, 1.1 eq) at room temperature. The mixture was then stirred at 80 °C for 12 h. The solution was cooled to ambient temperature, then filtered through celite® and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford Isomer A and Isomer B.
(Or)
[0870] To a solution of 5-nitro-lH-indazole (3.0 g, 18.4 mmol, 1.0 eq) in DMF (30 mL) was added 60% NaH (1.47 g, 36.8 mmol, 2.0 eq) at 0 °C. The solution was stirred for 10 min at 0 °C. And then py-CFLCl (2.6 g, 20.24 mmol, 1.1 eq) was added to the above reaction solution and stirred for 2 h at rt. The solution was cooled to ambient temperature, then quenched with ice water and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford Isomer A and Isomer B.
[0871] 5-Nitro-l-(pyridin-2-ylmethyl)-lH-indazole. Yield 55%; brown solid; LC-MS calculated for C13H10N4O2 [M + H]+ = 255.09, found 255.22; 1H NMR (400 MHz, DMSO) 5 8.84 (d, J = 2.2 Hz, 1H), 8.57 - 8.33 (m, 2H), 8.23 (dd, J = 9.3, 2.2 Hz, 1H), 7.91 (d, J = 9.2 Hz, 1H), 7.76 (td, J = 7.7, 1.9 Hz, 1H), 7.38 - 7.22 (m, 1H), 7.16 (d, J = 7.8 Hz, 1H), 5.86 (s, 2H).
[0872] 5-Nitro-2-(pyridin-2-ylmethyl)-2H-indazole. Yield 31%; brown solid; LC-MS calculated for C13H10N4O2 [M + H]+ = 255.09, found 255.22; 1H NMR (400 MHz, DMSO) 5 8.97 (d, J = 0.9 Hz, 1H), 8.93 (dd, J = 2.3, 0.7 Hz, 1H), 8.53 (ddd, J = 4.9, 1.9,
0.9 Hz, 1H), 8.01 (dd, J = 9.5, 2.3 Hz, 1H), 7.96 (s, 1H), 7.82 (td, J = 7.7, 1.8 Hz, 1H),
7.77 (dt, J = 9.4, 0.9 Hz, 1H), 7.39 - 7.27 (m, 2H), 5.86 (s, 2H).
[0873] 5 -Nitro- l-(pyridin-3-ylmethyl)-lH-indazole.Yield 32%; brown solid; LC-MS calculated for C13H10N4O2 [M + H]+ = 255.09, found 255.13.
[0874] 5-Nitro-2-(pyridin-3-ylmethyl)-2H-indazole. Yield 25%; brown solid; LC-MS calculated for C13H10N4O2 [M + H]+ = 255.09, found 255.13.
[0875] 5 -Nitro-l-(pyridin-4-ylmethyl)-lH-indazole. Yield 29%; brown solid; LC-MS calculated for C13H10N4O2 [M + H]+ = 255.09, found 255.13.
[0876] 5 -Nitro-2-(pyridin-4-ylmethyl)-2H-indazole. Yield 22%; brown solid; LC-MS calculated for C13H10N4O2 [M + H]+ = 255.09, found 255.13.
[0877] l-((6-Methylpyridin-2-yl)methyl)-5-nitro-lH-indazole.Yield 38%; yellow solid; LC-MS calculated for C14H12N4O2 [M + H]+ = 269.11, found 269.01; 1H NMR (400 MHz, DMSO) 5 8.84 (dd, J = 2.2, 0.6 Hz, 1H), 8.45 (d, J = 0.9 Hz, 1H), 8.23 (dd, J = 9.3,
2.2 Hz, 1H), 7.91 (dt, J = 9.2, 0.8 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.15 (d, J = 7.7 Hz, 1H), 6.79 (d, J = 7.7 Hz, 1H), 5.80 (s, 2H), 2.41 (s, 3H).
[0878] 2-((6-Methylpyridin-2-yl)methyl)-5-nitro-2H-indazole. Yield 23%; yellow solid; LC-MS calculated for C14H12N4O2 [M + H]+ = 269.11, found 269.01;^ NMR (400 MHz, DMSO) 5 8.96 (d, J = 0.9 Hz, 1H), 8.92 (dd, J = 2.3, 0.7 Hz, 1H), 8.01 (dd, J = 9.4,
2.3 Hz, 1H), 7.96 (s, 1H), 7.77 (dt, J = 9.5, 0.8 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.05 (dt, J = 7.7, 0.8 Hz, 1H), 5.80 (s, 2H), 2.44 (s, 3H).
[0879] l-((6-Methylpyridin-3-yl)methyl)-5-nitro-lH-indazole. Yield 32%; yellow solid; LC-MS calculated for C14H12N4O2 [M + H]+ = 269.11, found 269.01; 1H NMR (400 MHz, DMSO) 5 8.83 (dd, J = 2.2, 0.6 Hz, 1H), 8.47 (dd, J = 2.4, 0.8 Hz, 1H), 8.44 (d, J = 0.9 Hz, 1H), 8.24 (dd, J = 9.3, 2.2 Hz, 1H), 8.01 (dt, J = 9.3, 0.8 Hz, 1H), 7.54 (dd, J = 8.0, 2.4 Hz, 1H), 7.23 - 7.13 (m, 1H), 5.76 (s, 2H), 2.41 (s, 3H).
[0880] 2-((6-Methylpyridin-3-yl)methyl)-5-nitro-2H-indazole. Yield 27%; yellow solid; LC-MS calculated for C14H12N4O2 [M + H]+ = 269.11, found 269.01; 1H NMR (400 MHz, DMSO) 5 8.95 (d, J = 0.9 Hz, 1H), 8.90 (dd, J = 2.2, 0.7 Hz, 1H), 8.55 (dd, J = 2.4, 0.8 Hz, 1H), 8.00 (dd, J = 9.5, 2.3 Hz, 1H), 7.77 (dt, J = 9.4, 0.8 Hz, 1H), 7.68 (dd, J = 8.0, 2.4 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 5.75 (s, 2H), 2.44 (s, 3H).
[0881] Step 2. To a solution of appropriate nitro compound (0.79 mmol, 1.0 eq) and NH4CI (0.79 mmol, 1.0 eq) in ethanol (5 mL) was added Fe (3.95 mmol, 5.0 eq) at room temperature. The mixture was then stirred at 80 °C for 2 h. The solution was cooled to ambient temperature, then filtered through celite® and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford desired compound.
[0882] l-(Pyridin-2-ylmethyl)-lH-indazol-5-amine. Yield 85%; brown solid; LC-MS calculated for C13H12N4 [M + H]+ = 225.12, found 255.01; 1H NMR (400 MHz, MeOD) 5
8.49 (d, J = 5.0 Hz, 1H), 7.85 (s, 1H), 7.63 (td, J = 7.7, 1.8 Hz, 1H), 7.41 - 7.17 (m, 2H), 7.03 (d, J = 2.0 Hz, 1H), 6.96 (dd, J = 8.9, 2.1 Hz, 1H), 6.83 (d, J = 7.9 Hz, 1H), 5.64 (s, 2H).
[0883] 2-(Pyridin-2-ylmethyl)-2H-indazol-5-amine. Yield 79%; brown solid; LC-MS calculated for C13H12N4 [M + H]+ = 225.12, found 255.01; 1H NMR (400 MHz, MeOD) 5
8.50 (ddd, J = 4.9, 1.8, 0.9 Hz, 1H), 7.98 (d, J = 1.0 Hz, 1H), 7.71 (td, J = 7.7, 1.8 Hz, 1H), 7.43 (dt, J = 9.1, 0.9 Hz, 1H), 7.28 (ddd, J = 7.6, 4.9, 1.1 Hz, 1H), 7.04 (dt, J = 7.9, 1.0 Hz, 1H), 6.94 (dd, J = 9.1, 2.1 Hz, 1H), 6.85 (dd, J = 2.1, 0.8 Hz, 1H), 5.63 (s, 2H).
[0884] l-(Pyridin-3-ylmethyl)-lH-indazol-5-amine. Yield 72%; brown solid; LC-MS calculated for C13H12N4 [M + H]+ = 225.12, found 255.01.
[0885] 2-(Pyridin-3-ylmethyl)-2H-indazol-5-amine. Yield 65%; brown solid; LC-MS calculated for C13H12N4 [M + H]+ = 225.12, found 255.01.
[0886] l-(Pyridin-4-ylmethyl)-lH-indazol-5-amine. Yield 68%; brown solid; LC-MS calculated for C13H12N4 [M + H]+ = 225.12, found 255.01.
[0887] 2-(Pyridin-4-ylmethyl)-2H-indazol-5-amine. Yield 63%; brown solid; LC-MS calculated for C13H12N4 [M + H]+ = 225.12, found 255.01.
EXAMPLE 21
Synthesis of Intermediates
[0888] Step 1. To a solution of 5-nitro-lH-indole (1.0 g, 6.17 mmol, 1.0 eq) in DMF (10 mL) was added 60% NaH (0.49 g, 12.34 mmol, 2.0 eq) at 0 °C. The solution was stirred for 10 min at 0 °C. Py-CH2C1 (0.86 g, 6.79 mmol, 1.1 eq) was added to the above reaction solution and stirred for 2 h at rt. The solution was cooled to ambient temperature, then quenched with ice water and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate/hexanes (0 - 100%) to afford desired compound.
[0889] 5 -Nitro- l-(pyridin-2-ylmethyl)-lH-indole: Yield 78%; brown solid; LC-MS calculated for C14H11N3O2 [M + H]+ = 254.10, found 254.07.
[0890] 5-Nitro-l-(pyridin-3-ylmethyl)-lH-indole. Yield 85%; brown solid; LC-MS calculated for C14H11N3O2 [M + H]+ = 254.10, found 254.06.
[0891] 5 -nitro-l-(pyridin-4-ylmethyl)-lH-indole. Yield 82%; brown solid; LC-MS calculated for C14H11N3O2 [M + H]+ = 254.10, found 254.06.
[0892] Step 2. To a solution of appropriate nitro compound (0.79 mmol, 1.0 eq) and
NH4CI (0.79 mmol, 1.0 eq) in ethanol (5 mL) was added Fe (3.95 mmol, 5.0 eq) at room temperature. The mixture was then stirred at 80 °C for 2 h. The solution was cooled to ambient temperature, then filtered through celite® and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford desired compound.
[0893] l-(Pyridin-2-ylmethyl)-lH-indol-5-amine. Yield 72%; brown solid; LC-MS calculated for C14H13N3 [M + H]+ = 224.12, found 223.94; 1H NMR (400 MHz, MeOD) 5 8.59 - 8.26 (m, 1H), 7.61 (td, J = 7.7, 1.8 Hz, 1H), 7.31 (dd, J = 11.9, 2.7 Hz, 2H), 7.27 - 7.17 (m, 2H), 6.93 - 6.74 (m, 2H), 6.57 - 6.36 (m, 1H), 5.41 (s, 2H).
[0894] l-(Pyridin-3-ylmethyl)-lH-indol-5-amine. Yield 74%; brown solid; LC-MS calculated for C14H13N3 [M + H]+ = 224.12, found 223.97; 1H NMR (400 MHz, MeOD) 5 8.54 (d, J = 37.4 Hz, 2H), 7.88 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 2.1 Hz, 2H), 7.58 - 7.50 (m, 2H), 7.17 (dd, J = 8.7, 2.2 Hz, 1H), 6.68 (dd, J = 3.3, 0.9 Hz, 1H), 5.63 (s, 2H).
[0895] l-(pyridin-4-ylmethyl)-lH-indol-5-amine. Yield 69%; brown solid; LC-MS calculated for C14H13N3 [M + H]+ = 224.12, found 223.98.
EXAMPLE 22
Synthesis of (E)-N-(4-((3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3- yl)methoxy)phenyl)amino)-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)-4- (dimethylamino)but-2-enamide (Cpd. No. 36)
[0896] Step 1. Methanesulfonic acid (3.0 eq) was added at room temperature to a suspension of N-(4-chloro-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)acetamide (1.0 eq) and 3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3-yl)methoxy)aniline (1.5 eq) in anhydrous ethyl alcohol. The mixture was then stirred at 70 °C for 6 h. The solution was cooled to ambient temperature, then concentrated in vacuo, and purified by preparative HPLC to afford 6-amino-4-((3-chloro-4-((6-(tetrahydro-2H-pyran-4-yl)pyridin-3- yl)methoxy)phenyl)amino)-7-ethoxy-2-ethylquinoline-3-carbonitrile as a yellow solid in 68% yield. LC-MS calculated for C31H32CIN5O3 [M + H]+ = 558.23, found 558.16.
[0897] Step 2. Synthesis of Cpd. No. 36: (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride (1.5 eq) was added at 0 °C to a suspension of 6-amino-4-((3-chloro-4-((6- (tetrahydro-2H-pyran-4-yl)pyridin-3-yl)methoxy)phenyl)amino)-7-ethoxy-2- ethylquinoline-3-carbonitrile (1.0 eq) in N- methyl pyrrolidine (6 vol.). The mixture was then allowed to ambient temperature and stirred for 1 h. The compound was purified by preparative HPLC to yield Cpd. No. 36 as a light yellow solid in 54% yield. LC-MS calculated for C37H41CIN6O4 [M + H]+ = 669.30, found 669.30; 1H NMR (400 MHz, MeOD) 5 9.20 (s, 1H), 8.89 - 8.76 (m, 1H), 8.55 (dd, J = 8.3, 2.1 Hz, 1H), 7.96 (d, J = 8.3 Hz, 1H), 7.61 (d, J = 2.5 Hz, 1H), 7.52 - 7.41 (m, 2H), 7.37 (d, J = 8.9 Hz, 1H), 7.01 (dt, J = 15.3, 7.1 Hz, 1H), 6.86 (dt, J = 15.1, 1.2 Hz, 1H), 5.49 (s, 2H), 4.44 (q, J = 7.0 Hz, 2H), 4.12 (dt, J = 11.4, 3.2 Hz, 2H), 4.04 (dd, J = 7.1, 1.1 Hz, 2H), 3.68 - 3.55 (m, 2H), 3.09 (q, J = 7.6 Hz, 2H), 2.96 (s, 6H), 2.04 - 1.90 (m, 4H), 1.61 (t, J = 7.0 Hz, 3H), 1.43 (t, 7= 7.6 Hz, 3H). EXAMPLE 23
Synthesis of (E)-N-(4-((3-chloro-4-((6-isopropylpyridin-3-yl)methoxy)phenyl)amino)-3- cyano-7-ethoxy-2-ethylquinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 39)
[0898] Step 1. Methanesulfonic acid (3.0 eq) was added at room temperature to a suspension of A-(4-chloro-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)acetamide (1.0 eq) and 3-chloro-4-((6-isopropylpyridin-3-yl)methoxy)aniline (1.5 eq) in anhydrous ethyl alcohol. The mixture was then stirred at 70 °C for 6 h. The solution was cooled to ambient temperature, then concentrated in vacuo, and purified by preparative HPLC to afford 6-amino-4-((3-chloro-4-((6-isopropylpyridin-3-yl)methoxy)phenyl)amino)-7- ethoxy-2-ethylquinoline-3-carbonitrile as a yellow solid in 83% yield. LC-MS calculated for C29H30CIN5O2 [M + H]+ = 516.22, found 516.19.
[0899] Step 2. Synthesis of Cpd. No. 39: €-4-(dimethylamino)but-2-enoyl chloride hydrochloride (1.5 eq) was added at 0 °C to a suspension of 6-amino-4-((3-chloro-4-((6- isopropylpyridin-3-yl)methoxy)phenyl)amino)-7-ethoxy-2-ethylquinoline-3-carbonitrile (1.0 eq) in /V- methyl pyrrolidine (6 vol.). The mixture was then allowed to ambient temperature and stirred for 1 h. The compound was purified by preparative HPLC to yield Cpd. No. 39 as a light yellow solid in 62% yield. LC-MS calculated for C35H39CIN6O3 [M + H]+ = 627.29, found 627.40; 1H NMR (400 MHz, MeOD) 5 9.20 (s, 1H), 8.86 - 8.78 (m, 1H), 8.59 (dd, J = 8.4, 2.1 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 2.5 Hz, 1H), 7.49 - 7.42 (m, 2H), 7.38 (d, J= 8.8 Hz, 1H), 7.00 (dt, J = 15.3, 7.1 Hz, 1H), 6.85 (dt, J = 15.2, 1.2 Hz, 1H), 5.50 (s, 2H), 4.45 (q, J = 7.0 Hz, 2H), 4.04 (dd, J = 7.1, 1.2 Hz, 2H), 3.43 - 3.35 (m, 1H), 3.08 (q, J = 7.6 Hz, 2H), 2.96 (s, 6H), 1.61 (t, J = 7.0 Hz, 3H), 1.51 - 1.40 (m, 9H).
EXAMPLE 24
Synthesis of (E)-N-(4-((3-chloro-4-((6-isopropylpyridin-3-yl)methoxy)phenyl)amino)-3- cyano-7-ethoxy-2-methylquinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 47)
[0900] Step 1. Methanesulfonic acid (3.0 eq) was added at room temperature to a suspension of N-(4-chloro-3-cyano-7-ethoxy-2-methylquinolin-6-yl)acetamide (1.0 eq) and 3-chloro-4-((6-isopropylpyridin-3-yl)methoxy)aniline (1.5 eq) in anhydrous ethyl alcohol. The mixture was then stirred at 70 °C for 6 h. The solution was cooled to ambient temperature, then concentrated in vacuo, and purified by preparative HPLC to afford 6-amino-4-((3-chloro-4-((6-isopropylpyridin-3-yl)methoxy)phenyl)amino)-7- ethoxy-2-methylquinoline-3-carbonitrile as a yellow solid in 78% yield. LC-MS calculated for C28H28CIN5O2 [M + H]+ = 502.20, found 502.16.
[0901] Step 2. Synthesis of Cpd. No. 47: (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride (1.5 eq) was added at 0 °C to a suspension of 6-amino-4-((3-chloro-4-((6- isopropylpyridin-3-yl)methoxy)phenyl)amino)-7-ethoxy-2-methylquinoline-3- carbonitrile (1.0 eq) in A-methy 1 pyrrolidine (6 vol.). The mixture was then allowed to ambient temperature and stirred for 1 h. The compound was purified by preparative HPLC to yield Cpd. No. 47 as a light yellow solid in 73% yield. LC-MS calculated for C34H37CIN6O3 [M + H]+ = 613.27, found 613.27; 1H NMR (400 MHz, MeOD) 5 9.20 (s, 1H), 8.81 (d, J = 2.0 Hz, 1H), 8.57 (dd, J = 8.4, 2.1 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 2.5 Hz, 1H), 7.45 (dd, J = 8.7, 2.5 Hz, 1H), 7.43 - 7.32 (m, 2H), 7.00 (dt, J = 15.2, 7.1 Hz, 1H), 6.85 (dt, 7 = 15.1, 1.2 Hz, 1H), 5.49 (s, 2H), 4.44 (q, 7 = 7.0 Hz, 2H), 4.04 (dd, 7 = 7.2, 1.2 Hz, 2H), 3.43 - 3.35 (m, 1H), 2.96 (s, 6H), 2.79 (s, 3H), 1.61 (t, 7 = 7.0 Hz, 3H), 1.46 (d, 7= 7.0 Hz, 6H).
EXAMPLE 25
Synthesis of (E)-N-(4-((3-chloro-4-methoxyphenyl)amino)-3-cyano-7-ethoxy-2- methylquinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 51)
[0902] Synthesis of Cpd. No. 51: ('E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride (21.6 mg, 0.12 mmol, 1.5 eq) was added at 0 °C to a suspension of 6- amino-4-((3-chloro-4-methoxyphenyl)amino)-7-ethoxy-2-methylquinoline-3-carbonitrile (30 mg, 0.08 mmol, 1.0 eq) in A^- methyl pyrrolidine (1 mL). The mixture was then allowed to ambient temperature and stirred for 1 h. The compound was purified by preparative HPLC to yield Cpd. No. 51 (22 mg, 57% yield) as a light yellow solid. LC- MS calculated for C26H28CIN5O3 [M + H]+ = 494.20, found 494.18; 1H NMR (400 MHz, MeOD) 5 9.16 (s, 1H), 7.53 (d, J = 2.6 Hz, 1H), 7.43 - 7.35 (m, 2H), 7.21 (d, J = 8.8 Hz, 1H), 6.99 (dt, J = 15.3, 7.1 Hz, 1H), 6.84 (dt, J = 15.1, 1.2 Hz, 1H), 4.44 (q, 7 = 7.0 Hz, 2H), 4.04 (dd, J = 7.2, 1.2 Hz, 2H), 3.99 (s, 3H), 2.96 (s, 6H), 2.80 (s, 3H), 1.61 (t, J = 7.0 Hz, 3H).
EXAMPLE 26
Synthesis of (E)-N-(4-((3-chloro-4-methoxyphenyl)amino)-3-cyano-7-ethoxy-2- ethylquinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 59)
[0903] Synthesis of Cpd. No. 59: (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride (17.4 mg, 0.1 mmol, 1.5 eq) was added at 0 °C to a suspension of 6-amino- 4-((3-chloro-4-methoxyphenyl)amino)-7-ethoxy-2-ethylquinoline-3-carbonitrile (25 mg, 0.06 mmol, 1.0 eq) in methyl pyrrolidine (1 mL). The mixture was then allowed to ambient temperature and stirred for 1 h. The compound was purified by preparative HPLC to yield Cpd. No. 59 (25 mg, 78% yield) as a yellow solid. LC-MS calculated for C27H30CIN5O3 [M + H]+ = 508.21, found 508.18; 1H NMR (400 MHz, MeOD) 5 9.17 (s, 1H), 7.52 (d, J = 2.6 Hz, 1H), 7.45 (s, 1H), 7.40 (dd, J = 8.8, 2.6 Hz, 1H), 7.21 (d, J = 8.9 Hz, 1H), 7.00 (dt, J = 15.3, 7.1 Hz, 1H), 6.85 (dt, J = 15.1, 1.2 Hz, 1H), 4.43 (q, J = 7.0 Hz, 2H), 4.04 (dd, J = 7.1, 1.2 Hz, 2H), 3.99 (s, 3H), 3.09 (q, J = 7.6 Hz, 2H), 2.96 (s, 6H), 1.60 (t, 7 = 7.0 Hz, 3H), 1.43 (t, J = 7.6 Hz, 3H).
EXAMPLE 27
Synthesis of (E)-N-(4-((3-chloro-4-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)amino)-3- cyano-7-ethoxy-2-ethylquinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 62)
[0904] Synthesis of Cpd. No. 62: (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride (17.8 mg, 0.1 mmol, 1.5 eq) was added at 0 °C to a suspension of 6-amino- 4-((3-chloro-4-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)amino)-7-ethoxy-2- ethylquinoline-3-carbonitrile (30 mg, 0.064 mmol, 1.0 eq) in A-methylpyrrolidine (1 mL). The mixture was then allowed to ambient temperature and stirred for 1 h. The compound was purified by preparative HPLC to yield Cpd. No. 62 (23 mg, 62% yield) as a yellow solid. LC-MS calculated for C31H36CIN5O4 [M + H]+ = 578.26, found 578.25; 1H NMR (400 MHz, MeOD) 5 9.18 (s, 1H), 7.56 (d, J = 2.6 Hz, 1H), 7.49 - 7.34 (m, 2H), 7.28 (d, J = 8.9 Hz, 1H), 6.99 (dt, J = 15.3, 7.1 Hz, 1H), 6.84 (dt, J = 15.2, 1.2 Hz, 1H), 4.79 (dq, J = 7.4, 3.7 Hz, 1H), 4.44 (q, J = 7.0 Hz, 2H), 4.13 - 3.95 (m, 4H), 3.65 (ddd, J = 11.3, 7.5, 3.5 Hz, 2H), 3.09 (q, J = 7.7 Hz, 2H), 2.96 (s, 6H), 2.15 - 2.04 (m, 2H), 1.85 (ddq, J = 13.2, 7.5, 3.6 Hz, 2H), 1.61 (t, J = 7.0 Hz, 3H), 1.43 (t, J = 7.7 Hz, 3H).
EXAMPLE 28
Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-(methylamino)quinolin-6-yl)-4-
(dimethylamino)but-2-enamide (Cpd. No. 63)
[0905] Step 1. Synthesis of N-(3-cyano-7-ethoxy-2-ethyl-4-(methylamino)quinolin-6- yl)acetamide: To a solution of N-(4-chloro-3-cyano-7-ethoxy-2-ethylquinolin-6- yl)acetamide (35 mg, 0.13 mmol, 1 eq) in DMF was added methyl amine (7.9 mg, 0.25, 2 eq). The reaction mixture was stirred at 40 °C for 5 h. The solution was quenched with water and the aqueous layer was extracted with ethyl acetate. The combined organic layer was evaporated under vacuo. The crude compound was purified by preparative HPLC to afford 41.7 mg of the N-(3-cyano-7-ethoxy-2-ethyl-4-(methylamino)quinolin-6- yl)acetamide as yellow solid in 98% yield. LC-MS calculated for C17H20N4O2 [M + H]+ = 313.17, found 313.08.
[0906] Step 2. Synthesis of 6-amino-7-ethoxy-2-ethyl-4-(methylamino)quinoline-3- carbonitrile: A solution of N-(3-cyano-7-ethoxy-2-ethyl-4-(methylamino)quinolin-6- yl)acetamide (40 mg, 0.13 mmol, 1 eq) in Cone. HCI was heated at 50 °C for 12 h. The reaction was quenched with water and purified directly by preparative HPLC to afford 30 mg of the 6-amino-7-ethoxy-2-ethyl-4-(methylamino)quinoline-3-carbonitrile as yellow solid in 86.7% yield. LC-MS calculated for C15H18N4O [M + H]+ = 271.16, found 271.09. Step 3. Synthesis of Cpd. No. 63: (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride (28.6 mg, 0.16 mmol, 1.5 eq) was added at 0 °C to a suspension of 6- amino-7-ethoxy-2-ethyl-4-(methylamino)quinoline-3-carbonitrile (28 mg, 0.104 mmol, 1.0 eq) in Y- methyl pyrrolidine (1 mL). The mixture was then allowed to ambient temperature and stirred for 1 h. The compound was purified by preparative HPLC to yield Cpd. No. 63 (32 mg, 81% yield) as a yellow solid. LC-MS calculated for C21H27N5O2 [M + H]+ = 382.23, found 382.32; 1H NMR (400 MHz, MeOD) 5 9.06 (s, 1H), 7.34 (s, 1H), 7.00 (dt, J = 15.3, 7.1 Hz, 1H), 6.84 (dt, J = 15.2, 1.2 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 4.04 (dd, J = 7.1, 1.2 Hz, 2H), 3.66 (s, 3H), 3.14 (q, J = 7.7 Hz, 2H), 2.97 (s, 6H), 1.59 (t, J = 7.0 Hz, 3H), 1.50 (t, J= 7.7 Hz, 3H).
EXAMPLE 29
Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-(isopropylamino)quinolin-6-yl)-4- (dimethylamino)but-2-enamide (Cpd. No. 64)
68%
[0907] Step 1. Synthesis of N-(3-cyano-7-ethoxy-2-ethyl-4-(isopropylamino)quinolin-6- yl)acetamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 1. Yield 78%; yellow solid; LC-MS calculated for C19H24N4O2 [M + H]+ = 341.20, found 341.19.
Step 2. Synthesis of 6-amino-7-ethoxy-2-ethyl-4-(isopropylamino)quinoline-3- carbonitrile: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 2. Yield 82%; yellow solid; LC-MS calculated for C17H22N4O [M + H]+ = 299.19, found 299.12.
Step 3. Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-(isopropylamino)quinolin-6-yl)- 4-(dimethylamino)but-2-enamide: The synthesis of the Step 3 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3. Yield 68%; yellow solid; LC-MS calculated for C23H31N5O2 [M + H]+ = 410.26, found 410.31; !H NMR (400 MHz, MeOD) 5 9.05 (s, 1H), 7.33 (s, 1H), 7.01 (dt, J = 15.3, 7.1 Hz, 1H), 6.84 (dt, J = 15.2, 1.2 Hz, 1H), 5.11 (hept, J = 6.3 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 4.05 (dd, J= 7.1, 1.3 Hz, 2H), 3.15 (q, J = 7.7 Hz, 2H), 2.97 (s, 6H), 1.62 - 1.46 (m, 12H).
EXAMPLE 30
Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-(((tetrahydro-2H-pyran-4- yl)methyl)amino)quinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 118)
[0908] Step 1. Synthesis of N-(3-cyano-7-ethoxy-2-ethyl-4-(((tetrahydro-2H-pyran-4- yl)methyl)amino)quinolin-6-yl)acetamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 1. Yield 61%; yellow solid; LC-MS calculated for C22H28N4O3 [M + H]+ = 397.23, found 397.12.
[0909] Step 2. Synthesis of 6-amino-7-ethoxy-2-ethyl-4-(((tetrahydro-2H-pyran-4- yl)methyl)amino)quinoline-3-carbonitrile: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 2. Yield 61%; yellow solid; LC-MS calculated for C20H26N4O2 [M + H]+ = 355.22, found 355.14.
[0910] Step 3. Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-(((tetrahydro-2H-pyran- 4-yl)methyl)amino)quinolin-6-yl)-4-(dimethylamino)but-2-enamide: The synthesis of the Step 3 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3. Yield 78%; yellow solid; LC-MS calculated for C26H35N5O3 [M + H]+ = 466.28, found 466.29; 1H NMR (400 MHz, MeOD) 5 9.07 (s, 1H), 7.35 (s, 1H), 7.00 (dt, J = 15.3, 7.1 Hz, 1H), 6.83 (dt, J = 15.2, 1.2 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 4.10 - 3.91 (m, 6H), 3.46 (td, J = 11.8, 2.1 Hz, 2H), 3.15 (q, J = 7.6 Hz, 2H), 2.97 (s, 6H), 2.24 (dqd, J= 10.6, 7.2, 3.3 Hz, 1H), 1.85 (ddd, J= 12.9, 4.0, 1.9 Hz, 2H), 1.59 (t, J = 7.0 Hz, 3H), 1.50 (t, J = 7.7 Hz, 5H).
EXAMPLE 31
Synthesis of (E)-N-(3-cyano-4-(cyclopropylamino)-7-ethoxy-2-ethylquinolin-6-yl)-4-
(dimethylamino)but-2-enamide (Cpd. No. 119)
[0911] Step 1. Synthesis of N-(3-cyano-4-(cyclopropylamino)-7-ethoxy-2-ethylquinolin- 6-yl)acetamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 1. Yield 71%; yellow solid; LC-MS calculated for C19H22N4O2 [M + H]+ = 339.18, found 339.17.
[0912] Step 2. Synthesis of 6-amino-4-(cyclopropylamino)-7-ethoxy-2-ethylquinoline-3- carbonitrile: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 2. Yield 84%; yellow solid; LC-MS calculated for C17H20N4O [M + H]+ = 297.17, found 297.09.
[0913] Step 3. Synthesis of (E)-N-(3-cyano-4-(cyclopropylamino)-7-ethoxy-2- ethylquinolin-6-yl)-4-(dimethylamino)but-2-enamide: The synthesis of the Step 3 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3. Yield 58%; yellow solid; LC-MS calculated for C23H29N5O2 [M + H]+ = 408.24, found 408.33; XH NMR (400 MHz, MeOD) 5 9.10 (s, 1H), 7.37 (s, 1H), 7.00 (dt, J= 15.3, 7.2 Hz, 1H), 6.84 (dt, J = 15.2, 1.2 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 4.04 (dd, J = 7.1, 1.2 Hz, 2H), 3.42 (tt, J = 7.0, 3.7 Hz, 1H), 3.18 (q, J = 7.8 Hz, 2H), 2.96 (s, 6H), 1.58 (t, J = 7.0 Hz, 3H), 1.51 (t, J = 7.7 Hz, 3H), 1.20 (qd, J = 6.6, 4.3 Hz, 2H), 1.11 (dq, 7 = 7.8, 4.2 Hz, 2H).
EXAMPLE 32 Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-((tetrahydro-2H-pyran-4- yl)amino)quinolin-6-yl)-4-(4-methylpiperazin-l-yl)but-2-enamide (Cpd. No. 125)
[0914] Step 1. Synthesis of N-(3-cyano-7-ethoxy-2-ethyl-4-((tetrahydro-2H-pyran-4- yl)amino)quinolin-6-yl)acetamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 1. Yield 78%; yellow solid; LC-MS calculated for C21H26N4O3 [M + H]+ = 383.21, found 383.28.
[0915] Step 2. Synthesis of 6-amino-7-ethoxy-2-ethyl-4-((tetrahydro-2H-pyran-4- yl)amino)quinoline-3-carbonitrile: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 2. Yield 72%; light brown oil; LC-MS calculated for C19H24N4O2 [M + H]+ = 341.20, found 341.22.
[0916] Step 3. Synthesis of (E)-4-chloro-N-(3-cyano-7-ethoxy-2-ethyl-4-((tetrahydro- 2H-pyran-4-yl)amino)quinolin-6-yl)but-2-enamide: (E)-4-chlorobut-2-enoyl chloride (24.3 mg, 0.18 mmol, 1.5 eq) was added at 0 °C to a suspension of 6-amino-7-ethoxy-2- ethyl-4-((tetrahydro-2H-pyran-4-yl)amino)quinoline-3-carbonitrile (40 mg, 0.12 mmol, 1.0 eq) in Y- methyl pyrrolidine (1 mL). The mixture was then allowed to ambient temperature and stirred for 1 h. The compound was purified by preparative HPLC to yield (E)-4-chloro-N-(3-cyano-7-ethoxy-2-ethyl-4-((tetrahydro-2H-pyran-4-yl)amino) quinolin-6-yl)but-2-enamide (29 mg, 56% yield) as a yellow solid. LC-MS calculated for C23H27CIN4O3 [M + H]+ = 443.19, found 443.27.
[0917] Step 4. Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-((tetrahydro-2H-pyran-4- yl)amino)quinolin-6-yl)-4-(4-methylpiperazin-l-yl)but-2-enamide: 1 -Methylpiperazine (11.3 mg, 0.11 mmol, 2.0 eq) was added at room temperature to a suspension of (E)-4- chloro-N-(3-cyano-7-ethoxy-2-ethyl-4-((tetrahydro-2H-pyran-4-yl)amino)quinolin-6- yl)but-2-enamide (25 mg, 0.06 mmol, 1.0 eq) and CS2CO3 (36.8 mg, 0.11 mmol, 2.0 eq) in acetonitrile (2 mL). The mixture was then stirred at 60 °C for 2 h. The compound was purified by preparative HPLC to yield (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-((tetrahydro- 2H-pyran-4-yl)amino)quinolin-6-yl)-4-(4-methylpiperazin-l-yl)but-2-enamide (19 mg, 67% yield) as a yellow solid. LC-MS calculated for C28H38N6O3 [M + H]+ = 507.31, found 507.26; 1H NMR (400 MHz, MeOD) 5 9.05 (s, 1H), 7.35 (s, 1H), 7.02 (dt, J = 15.4, 6.2 Hz, 1H), 6.65 (dt, J= 15.3, 1.5 Hz, 1H), 4.98 (dd, J= 7.6, 3.4 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 4.14 - 4.05 (m, 2H), 3.61 (td, J = 12.0, 2.0 Hz, 2H), 3.48 (dd, J = 6.3, 1.6 Hz, 2H), 3.40 (s, 4H), 3.16 (q, J = 7.7 Hz, 2H), 2.94 (s, 7H), 2.21 - 2.11 (m, 2H), 2.01 (qd, J= 12.0, 4.4 Hz, 2H), 1.59 (t, J = 7.0 Hz, 3H), 1.50 (t, J = 7.6 Hz, 3H).
EXAMPLE 33
[0918] Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-(((tetrahydro-2H-pyran-4- yl)methyl)amino)quinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 93)
The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3 starting from 6-amino-4-((3-chloro-4-(pyridin-2- ylmethoxy)phenyl)amino)-7-ethoxy-2-ethylquinoline-3-carbonitrile. Yield 45%; yellow solid; LC-MS calculated for C29H26CIN5O3 [M + H]+ = 528.18, found 528.17; 1H NMR (400 MHz, MeOD) 5 9.16 (s, 1H), 8.67 (ddd, J = 5.2, 1.7, 0.9 Hz, 1H), 8.11 (td, J = 7.8, 1.7 Hz, 1H), 7.86 (dt, J = 7.9, 1.0 Hz, 1H), 7.62 (d, J = 2.5 Hz, 1H), 7.59 (ddd, J = 7.7, 5.1, 1.2 Hz, 1H), 7.43 (dd, J = 8.8, 2.6 Hz, 1H), 7.39 (s, 1H), 7.32 (d, J = 8.8 Hz, 1H), 6.69 (dd, J = 16.9, 10.2 Hz, 1H), 6.48 (dd, J = 17.0, 1.7 Hz, 1H), 5.89 (dd, J = 10.2, 1.7 Hz, 1H), 5.44 (s, 2H), 4.44 (q, J = 7.0 Hz, 2H), 3.09 (q, J = 7.6 Hz, 2H), 1.62 (t, J = 7.0 Hz, 3H), 1.44 (t, J= 7.6 Hz, 3H).
EXAMPLE 34
Synthesis of (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7- ethoxy-2-ethylquinolin-6-yl)but-2-enamide (Cpd. No. 94)
[0919] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3 starting from 6-amino-4-((3-chloro-4-(pyridin-2- ylmethoxy)phenyl)amino)-7-ethoxy-2-ethylquinoline-3-carbonitrile. Yield 78%; yellow solid; LC-MS calculated for C30H28CIN5O3 [M + H]+ = 542.20, found 541.98; 1H NMR (400 MHz, MeOD) 5 9.13 (s, 1H), 8.67 (ddd, J = 5.1, 1.7, 0.9 Hz, 1H), 8.11 (td, J = 7.8, 1.7 Hz, 1H), 7.86 (dt, 7 = 7.9, 1.1 Hz, 1H), 7.65 - 7.54 (m, 2H), 7.42 (dd, J = 8.7, 2.6 Hz, 1H), 7.37 (s, 1H), 7.31 (d, J = 8.8 Hz, 1H), 7.08 (dq, J = 15.1, 6.9 Hz, 1H), 6.39 (dt, J = 15.2, 1.7 Hz, 1H), 5.43 (s, 2H), 4.43 (q, J = 7.0 Hz, 2H), 3.09 (q, J = 7.6 Hz, 2H), 1.97 (dd, 7= 6.9, 1.7 Hz, 3H), 1.62 (t, 7 = 7.0 Hz, 3H), 1.44 (t, 7 = 7.7 Hz, 3H).
EXAMPLE 35
Synthesis of (E)-N-(4-((3-chloro-4-((tetrahydro-2H-pyran-4-yl)methoxy)phenyl)amino)- 3-cyano-7-ethoxy-2-ethylquinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 95)
[0920] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3 starting from 6-amino-4-((3-chloro-4-((tetrahydro-2H-pyran-4- yl)methoxy)phenyl)amino)-7-ethoxy-2-ethylquinoline-3-carbonitrile. Yield 61%; light yellow solid; LC-MS calculated for C32H38CIN5O4 [M + H]+ = 592.27, found 592.25; 1H NMR (400 MHz, MeOD) 5 9.17 (s, 1H), 7.53 (d, 7 = 2.6 Hz, 1H), 7.46 - 7.34 (m, 2H), 7.20 (d, 7 = 8.9 Hz, 1H), 6.99 (dt, 7 = 14.6, 7.1 Hz, 1H), 6.89 - 6.80 (m, 1H), 4.44 (q, 7 = 7.0 Hz, 2H), 4.11 - 3.97 (m, 6H), 3.52 (td, 7 = 11.8, 2.1 Hz, 2H), 3.08 (q, 7 = 7.6 Hz, 2H), 2.96 (s, 6H), 2.25 - 2.14 (m, 1H), 1.84 (ddd, 7 = 12.7, 4.0, 1.9 Hz, 2H), 1.63 - 1.51 (m, 5H), 1.43 (t, 7 = 7.6 Hz, 3H).
EXAMPLE 36 Synthesis of (E)-N-(4-((3-chloro-4-methoxyphenyl)amino)-3-cyano-7-ethoxy-2- ethylquinolin-6-yl)-4-(piperidin-l-yl)but-2-enamide (Cpd. No. 96)
[0921] Step 1. Synthesis of (E)-4-chloro-N-(4-((3-chloro-4-methoxyphenyl)amino)-3- cyano-7-ethoxy-2-ethylquinolin-6-yl)but-2-enamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 3 from 6-amino-4-((3-chloro-4-methoxyphenyl)amino)-7-ethoxy-2- ethylquinoline-3-carbonitrile. Yield 48%; yellow solid; LC-MS calculated for C25H24CI2N4O3 [M + H]+ = 499.13, found 499.12.
[0922] Step 2. Synthesis of (E)-N-(4-((3-chloro-4-methoxyphenyl)amino)-3-cyano-7- ethoxy-2-ethylquinolin-6-yl)-4-(piperidin-l-yl)but-2-enamide: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 4 from Step 1 compound. Yield 52%; light yellow solid; LC-MS calculated for C30H34CIN5O3 [M + H]+ = 548.25, found 548.24; 1H NMR (400 MHz, MeOD) 5 9.15 (s, 1H), 7.54 (d, J = 2.6 Hz, 1H), 7.43 - 7.37 (m, 2H), 7.21 (d, J = 8.8 Hz, 1H), 7.00 (dt, J = 15.3, 7.2 Hz, 1H), 6.83 (dt, J = 15.2, 1.2 Hz, 1H), 4.44 (q, J = 7.0 Hz, 2H), 4.08 - 3.88 (m, 5H), 3.58 (d, J= 12.1 Hz, 2H), 3.15 - 2.91 (m, 4H), 2.09 - 1.72 (m, 5H), 1.61 (t, J = 7.0 Hz, 4H), 1.44 (t, J= 7.6 Hz, 3H).
EXAMPLE 37
Synthesis of (E)-N-(4-((3-chloro-4-methoxyphenyl)amino)-3-cyano-7-ethoxy-2- ethylquinolin-6-yl)-4-morpholinobut-2-enamide (Cpd. No. 97)
[0923] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 4 from (E)-4-chloro-N-(4-((3-chloro-4-methoxyphenyl)amino)-3- cyano-7-ethoxy-2-ethylquinolin-6-yl)but-2-enamide. Yield 71%; yellow solid; LC-MS calculated for C29H32CIN5O4 [M + H]+ = 550.22, found 550.16; 1H NMR (400 MHz, MeOD) 5 9.16 (s, 1H), 7.53 (d, J = 2.6 Hz, 1H), 7.43 - 7.37 (m, 2H), 7.21 (d, J = 8.9 Hz, 1H), 7.01 (dt, J = 15.2, 7.1 Hz, 1H), 6.88 - 6.82 (m, 1H), 4.44 (q, J = 7.0 Hz, 2H), 4.05 (dd, J = 7.2, 1.2 Hz, 2H), 3.99 (s, 6H), 3.48 - 3.33 (m, 4H), 3.09 (q, J = 7.7 Hz, 2H), 1.61 (t, J= 6.9 Hz, 3H), 1.43 (t, J= 7.6 Hz, 3H).
EXAMPLE 38
Synthesis of (E)-N-(4-((3-chloro-4-methoxyphenyl)amino)-3-cyano-7-ethoxy-2- ethylquinolin-6-yl)-4-(4-methylpiperazin-l-yl)but-2-enamide (Cpd. No. 98)
[0924] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 4 from (E)-4-chloro-N-(4-((3-chloro-4-methoxyphenyl)amino)-3- cyano-7-ethoxy-2-ethylquinolin-6-yl)but-2-enamide. Yield 82%; yellow solid; LC-MS calculated for C30H35CIN6O3 [M + H]+ = 563.26, found 563.25; 1H NMR (400 MHz, MeOD) 5 9.15 (s, 1H), 7.54 (d, J = 2.6 Hz, 1H), 7.45 - 7.36 (m, 2H), 7.22 (d, J = 8.8 Hz, 1H), 7.00 (dt, J = 15.3, 6.1 Hz, 1H), 6.63 (dt, J = 15.3, 1.6 Hz, 1H), 4.44 (q, J = 7.0 Hz, 2H), 3.99 (s, 3H), 3.54 - 3.34 (m, 6H), 3.09 (q, J = 7.6 Hz, 2H), 3.05 - 2.42 (m, 7H), 1.62 (t, J= 7.0 Hz, 3H), 1.44 (t, J= 7.6 Hz, 3H).
EXAMPLE 39
Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-(phenylamino)quinolin-6-yl)-4-
(dimethylamino)but-2-enamide (Cpd. No. 107)
[0925] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3 starting from 6-amino-7-ethoxy-2-ethyl-4-
(phenylamino)quinoline-3-carbonitrile. Yield 72%; light yellow solid; LC-MS calculated for C26H29N5O2 [M + H]+ = 444.24, found 443.68; 1H NMR (400 MHz, MeOD) 5 9.12 (s, 1H), 7.52 (dd, J = 8.3, 6.7 Hz, 2H), 7.48 - 7.38 (m, 4H), 6.98 (dt, J = 15.2, 7.1 Hz, 1H), 6.88 - 6.81 (m, 1H), 4.44 (q, J = 7.0 Hz, 2H), 4.04 (dd, J = 7.2, 1.2 Hz, 2H), 3.07 (q, J = 7.7 Hz, 2H), 2.96 (s, 6H), 1.62 (t, J= 7.0 Hz, 3H), 1.43 (t, J= 7.6 Hz, 3H).
EXAMPLE 40
Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-(phenylamino)quinolin-6-yl)-4- morpholinobut-2-enamide (Cpd. No. 108)
Step 2
[0926] Step 1. Synthesis of (E)-4-chloro-N-(3-cyano-7-ethoxy-2-ethyl-4-
(phenylamino)quinolin-6-yl)but-2-enamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 3 from 6-amino-7-ethoxy-2-ethyl-4-(phenylamino)quinoline-3-carbonitrile. Yield 52%; yellow solid; LC-MS calculated for C24H23CIN4O2 [M + H]+ = 435.16, found 435.21.
[0927] Step 2. Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-(phenylamino)quinolin- 6-yl)-4-morpholinobut-2-enamide: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 4 from Step 1 compound. Yield 65%; light yellow solid; LC-MS calculated for C28H31N5O3 [M + H]+ = 486.25, found 485.71; 1H NMR (400 MHz, MeOD) 5 9.15 (s, 1H), 7.56 - 7.51 (m, 2H), 7.50 - 7.42 (m, 3H), 7.40 (s, 1H), 7.01 (dt, J = 14.6, 7.1 Hz, 1H), 6.84 (d, J = 15.3 Hz, 1H), 4.45 (q, J = 7.0 Hz, 2H), 4.10 - 3.82 (m, 6H), 3.32 (s, 4H), 3.08 (q, J = 7.7 Hz, 2H), 1.62 (t, J = 7.0 Hz, 3H), 1.43 (t, J = 7.6 Hz, 3H).
EXAMPLE 41
Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-(phenylamino)quinolin-6-yl)-4- (piperidin-l-yl)but-2-enamide (Cpd. No. 109) Acetonitrile CS CO
[0928] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 4 from (E)-4-chloro-N-(3-cyano-7-ethoxy-2-ethyl-4- (phenylamino)quinolin-6-yl)but-2-enamide. Yield 68%; yellow solid; LC-MS calculated for C29H33N5O2 [M + H]+ = 484.27, found 483.85; 1H NMR (400 MHz, MeOD) 5 9.15 (s, 1H), 7.57 - 7.50 (m, 2H), 7.50 - 7.43 (m, 3H), 7.41 (s, 1H), 6.99 (dt, J = 14.7, 7.2 Hz, 1H), 6.82 (dt, J = 15.1, 1.2 Hz, 1H), 4.44 (q, J = 7.0 Hz, 2H), 4.00 (dd, J = 7.1, 1.2 Hz, 2H), 3.56 (s, 2H), 3.18 - 2.88 (m, 4H), 1.91 (dt, J = 67.5, 15.0 Hz, 5H), 1.61 (t, J = 7.0 Hz, 4H), 1.43 (t, J= 7.6 Hz, 3H).
EXAMPLE 42
Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-(phenylamino)quinolin-6-yl)-4-(4- methylpiperazin-l-yl)but-2-enamide (Cpd. No. 110)
Acetonitrile CS CO
[0929] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 4 from (E)-4-chloro-N-(3-cyano-7-ethoxy-2-ethyl-4- (phenylamino)quinolin-6-yl)but-2-enamide. Yield 51%; yellow solid; LC-MS calculated for C29H34N6O2 [M + H]+ = 499.28, found 499.25; 1H NMR (400 MHz, MeOD) 5 9.17 (s, 1H), 7.58 - 7.45 (m, 5H), 7.44 (s, 1H), 7.02 (dt, J = 15.3, 6.5 Hz, 1H), 6.75 (dt, J = 15.3, 1.4 Hz, 1H), 4.46 (q, J = 7.0 Hz, 2H), 3.72 (dd, J = 6.6, 1.4 Hz, 2H), 3.52 (s, 4H), 3.32 - 3.01 (m, 6H), 2.98 (s, 3H), 1.63 (t, J= 7.0 Hz, 3H), 1.44 (t, J= 7.6 Hz, 3H).
EXAMPLE 43
Synthesis of (E)-N-(4-((3-chlorophenyl)amino)-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)- 4-(dimethylamino)but-2-enamide (Cpd. No. I l l)
[0930] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3 starting from 6-amino-4-((3-chlorophenyl)amino)-7-ethoxy-2- ethylquinoline-3-carbonitrile. Yield 85%; yellow solid; LC-MS calculated for C26H28CIN5O2 [M + H]+ = 478.20, found 478.19; 1H NMR (400 MHz, MeOD) 5 9.19 (s, 1H), 7.61 - 7.35 (m, 5H), 6.99 (dt, J = 15.2, 7.1 Hz, 1H), 6.84 (dt, J = 15.3, 1.2 Hz, 1H), 4.45 (q, J = 7.0 Hz, 2H), 4.04 (dd, J = 7.1, 1.2 Hz, 2H), 3.12 (q, J = 7.6 Hz, 2H), 2.96 (s,
6H), 1.61 (t, 7 = 7.0 Hz, 3H), 1.45 (t, 7 = 7.7 Hz, 3H).
EXAMPLE 44
Synthesis of (E)-N-(4-((3-chlorophenyl)amino)-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)-
4-morpholinobut-2-enamide (Cpd. No. 112)
[0931] Step 1. Synthesis of (E)-4-chloro-N-(4-((3-chlorophenyl)amino)-3-cyano-7- ethoxy-2-ethylquinolin-6-yl)but-2-enamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 3 from 6-amino-4-((3-chlorophenyl)amino)-7 -ethoxy-2-ethylquinoline-3-carbonitrile. Yield 49%; yellow solid; LC-MS calculated for C24H22CI2N4O2 [M + H]+ = 469.12, found 469.14.
[0932] Step 2. Synthesis of (E)-N-(4-((3-chlorophenyl)amino)-3-cyano-7-ethoxy-2- ethylquinolin-6-yl)-4-morpholinobut-2-enamide: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 4 from Step 1 compound. Yield 72%; yellow solid; LC-MS calculated for C28H30CIN5O3 [M + H]+ = 520.21, found 520.37; 1H NMR (400 MHz, MeOD) 5 9.15 (s, 1H), 7.62 - 7.29 (m, 5H), 7.00 (dt, J = 14.6, 7.1 Hz, 1H), 6.84 (d, J = 15.2 Hz, 1H), 4.45 (q, J= 7.0 Hz, 2H), 4.23 - 3.71 (m, 6H), 3.38 (s, 4H), 3.11 (q, J= 7.6 Hz, 2H), 1.61 (t, J = 7.0 Hz, 3H), 1.45 (t, J = 7.6 Hz, 3H).
EXAMPLE 45
Synthesis of (E)-N-(4-((3-chlorophenyl)amino)-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)-
4-(4-methylpiperazin-l-yl)but-2-enamide (Cpd. No. 113)
[0933] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 4 from Step 1 compound. Yield 63%; yellow solid; LC-MS calculated for C29H33CIN6O2 [M + H]+ = 533.25, found 532.85; 1H NMR (400 MHz, MeOD) 5 9.18 (s, 1H), 7.63 - 7.46 (m, 3H), 7.46 - 7.37 (m, 2H), 7.00 (dt, J = 15.3, 6.2 Hz, 1H), 6.64 (dt, J = 15.3, 1.5 Hz, 1H), 4.45 (q, J = 7.0 Hz, 2H), 3.50 - 3.34 (m, 6H), 3.12 (q, J= 7.6 Hz, 2H), 2.93 (s, 7H), 1.62 (t, J = 7.0 Hz, 3H), 1.45 (t, J= 7.6 Hz, 3H).
EXAMPLE 46
Synthesis of (E)-N-(4-((3-chloro-4-((tetrahydro-2H-pyran-4-yl)methoxy)phenyl)amino)-
3-cyano-7-ethoxy-2-ethylquinolin-6-yl)-4-(4-methylpiperazin-l-yl)but-2-enamide
Step 2 [0934] Step 1. Synthesis of (E)-4-chloro-N-(4-((3-chloro-4-((tetrahydro-2H-pyran-4- yl)methoxy)phenyl)amino)-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)but-2-enamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 3. Yield 56%; yellow solid; LC-MS calculated for C30H32CI2N4O4 [M + H]+ = 583.19, found 583.19.
[0935] Step 2. Synthesis of (E)-N-(4-((3-chloro-4-((tetrahydro-2H-pyran-4- yl)methoxy)phenyl)amino)-3-cyano-7-ethoxy-2-ethylquinolin-6-yl)-4-(4- methylpiperazin-l-yl)but-2-enamide: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 4 from Step 1 compound. Yield 68%; yellow solid; LC-MS calculated for C35H43CIN6O4 [M + H]+ = 647.31, found 647.37.
EXAMPLE 47
Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-((4-methoxyphenyl)amino)quinolin-6- yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 114)
[0936] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3 starting from 6-amino-7-ethoxy-2-ethyl-4-((4- methoxyphenyl)amino)quinoline-3-carbonitrile. Yield 72%; yellow solid; LC-MS calculated for C27H31N5O3 [M + H]+ = 474.25, found 474.27; 1H NMR (400 MHz, MeOD) 5 9.16 (s, 1H), 7.47 - 7.34 (m, 3H), 7.13 - 7.04 (m, 2H), 6.99 (dt, J = 15.3, 7.1 Hz, 1H), 6.84 (dt, J = 15.2, 1.2 Hz, 1H), 4.44 (q, J = 7.0 Hz, 2H), 4.04 (dd, J = 7.2, 1.2 Hz, 2H), 3.89 (s, 3H), 3.08 (t, J= 7.6 Hz, 2H), 2.96 (s, 6H), 1.61 (t, J= 7.0 Hz, 3H), 1.43 (t, 7= 7.6 Hz, 3H).
EXAMPLE 48
Synthesis of (E)-N-(3-cyano-7-ethoxy-4-((4-methoxyphenyl)amino)-2-methylquinolin-6- yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 115)
[0937] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3 starting from 6-amino-7-ethoxy-4-((4-methoxyphenyl)amino)-2- methylquinoline-3-carbonitrile. Yield 69%; yellow solid; LC-MS calculated for C26H29N5O3 [M + H]+ = 460.24, found 459.59; 1H NMR (400 MHz, MeOD) 5 9.15 (s, 1H), 7.42 - 7.35 (m, 3H), 7.09 - 7.04 (m, 2H), 6.99 (dt, J = 15.2, 7.1 Hz, 1H), 6.84 (dt, J = 15.1, 1.2 Hz, 1H), 4.43 (q, J = 7.0 Hz, 2H), 4.05 (d, J = 1.2 Hz, 2H), 3.89 (s, 3H), 2.96 (s, 6H), 2.78 (s, 3H), 1.60 (t, J= 7.0 Hz, 3H).
EXAMPLE 49
Synthesis of (E)-N-(4-((3-chlorophenyl)amino)-3-cyano-7-ethoxy-2-methylquinolin-6- yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 116)
[0938] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3 starting from 6-amino-4-((3-chlorophenyl)amino)-7-ethoxy-2- methylquinoline-3-carbonitrile. Yield 52%; yellow solid; LC-MS calculated for C25H26CIN5O2 [M + H]+ = 464.19, found 464.18; 1H NMR (400 MHz, MeOD) 5 9.18 (s, 1H), 7.56 - 7.46 (m, 3H), 7.44 - 7.37 (m, 2H), 6.99 (dt, J = 15.2, 7.1 Hz, 1H), 6.84 (dt, J = 15.3, 1.2 Hz, 1H), 4.45 (q, J = 7.0 Hz, 2H), 4.04 (dd, J = 7.1, 1.2 Hz, 2H), 2.96 (s, 6H), 2.82 (s, 3H), 1.61 (t, J = 7.0 Hz, 3H).
EXAMPLE 50
Synthesis of (E)-N-(3-cyano-7-ethoxy-2-methyl-4-(phenylamino)quinolin-6-yl)-4- (dimethylamino)but-2-enamide (Cpd. No. 117)
[0939] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3 starting from 6-amino-7-ethoxy-2-methyl-4- (phenylamino)quinoline-3-carbonitrile. Yield 59%; yellow solid; LC-MS calculated for C25H27N5O2 [M + H]+ = 430.23, found 430.35; 1H NMR (400 MHz, MeOD) 5 9.18 (s, 1H), 7.62 - 7.42 (m, 5H), 7.39 (s, 1H), 6.99 (dt, J= 15.2, 7.1 Hz, 1H), 6.84 (dt, J= 15.2, 1.2 Hz, 1H), 4.44 (q, J = 7.0 Hz, 2H), 4.04 (dd, J= 7.2, 1.2 Hz, 2H), 2.96 (s, 6H), 2.79 (s, 3H), 1.61 (t, 7 = 7.0 Hz, 3H).
EXAMPLE 51
Synthesis of (E)-N-(4-(3-chloro-4-methoxyphenoxy)-3-cyano-7-ethoxy-2- methylquinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 120)
[0940] Step 1. Synthesis of N-(4-(3-chloro-4-methoxyphenoxy)-3-cyano-7-ethoxy-2- methylquinolin-6-yl)acetamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 1. Yield 38%; pale yellow solid; LC-MS calculated for C22H20CIN3O4 [M + H]+ = 426.12, found 426.16.
[0941] Step 2. Synthesis of 6-amino-4-(3-chloro-4-methoxyphenoxy)-7-ethoxy-2- methylquinoline-3-carbonitrile: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 2. Yield 58%; yellow solid; LC-MS calculated for C20H18CIN3O3 [M + H]+ = 384.11, found 384.20.
[0942] Step 3. Synthesis of (E)-N-(4-(3-chloro-4-methoxyphenoxy)-3-cyano-7-ethoxy-2- methylquinolin-6-yl)-4-(dimethylamino)but-2-enamide: The synthesis of the Step 3 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3. Yield 42%; LC-MS calculated for C26H27CIN4O4 [M + H]+ = 495.18, found 495.14.
EXAMPLE 52
Synthesis of (E)-N-(4-(3-chloro-4-methoxyphenoxy)-3-cyano-7-ethoxy-2-ethylquinolin- 6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 123)
[0943] Step 1. Synthesis of N-(4-(3-chloro-4-methoxyphenoxy)-3-cyano-7-ethoxy-2- ethylquinolin-6-yl)acetamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 1. Yield 38%; pale yellow solid; LC-MS calculated for C23H22CIN3O4 [M + H]+ = 440.14, found 440.27.
[0944] Step 2. Synthesis of 6-amino-4-(3-chloro-4-methoxyphenoxy)-7-ethoxy-2- ethylquinoline-3-carbonitrile: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 2. Yield 58%; yellow solid; LC-MS calculated for C21H20CIN3O3 [M + H]+ = 398.13, found 398.21.
[0945] Step 3. Synthesis of (E)-N-(4-(3-chloro-4-methoxyphenoxy)-3-cyano-7-ethoxy-2- ethylquinolin-6-yl)-4-(dimethylamino)but-2-enamide: The synthesis of the Step 3 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3. Yield 45%; LC-MS calculated for C27H29CIN4O4 [M + H]+ = 509.20, found 509.20; 1H NMR (400 MHz, MeOD) 5 8.92 (s, 1H), 7.07 (s, 1H), 6.97 - 6.88 (m, 2H), 6.85 - 6.74 (m, 2H), 6.69 (dd, J = 8.8, 2.9 Hz, 1H), 4.30 (q, J = 7.0 Hz, 2H), 4.03 (dd, J= 7.1, 1.2 Hz, 2H), 3.80 (s, 3H), 3.00 - 2.91 (m, 8H), 1.55 (t, J= 7.0 Hz, 3H), 1.44
(t, 7= 7.6 Hz, 3H).
EXAMPLE 53
Synthesis of (E)-N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenoxy)-3-cyano-7-ethoxy-2- ethylquinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 265)
[0946] Step 1. Synthesis of N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenoxy)-3-cyano-7- ethoxy-2-ethylquinolin-6-yl)acetamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 1. Yield 69%; off white solid; LC-MS calculated for C28H25CIN4O4 [M + H]+ = 517.17, found 517.18.
[0947] Step 2. Synthesis of 6-amino-4-(3-chloro-4-(pyridin-2-ylmethoxy)phenoxy)-7- ethoxy-2-ethylquinoline-3-carbonitrile: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 2. Yield 66%; yellow solid; LC-MS calculated for C26H23CIN4O3 [M + H]+ = 475.16, found 475.17.
[0948] Step 3. Synthesis of (E)-N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenoxy)-3- cyano-7-ethoxy-2-ethylquinolin-6-yl)-4-(dimethylamino)but-2-enamide: The synthesis of the Step 3 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3. Yield 75%; LC-MS calculated for C32H32CIN5O4 [M + H]+ = 586.22, found 586.17; 1H NMR (400 MHz, MeOD) 5 8.91 (s, 1H), 8.80 - 8.72 (m, 1H), 8.39 (td, J = 7.8, 1.6 Hz, 1H), 8.06 - 7.98 (m, 1H), 7.82 (ddd, J = 7.2, 5.5, 1.2 Hz, 1H), 7.04 (d, J = 8.5 Hz, 2H), 6.98 - 6.85 (m, 2H), 6.78 (dt, J = 15.3, 1.3 Hz, 1H), 6.71 (dd, J = 8.9, 2.9 Hz, 1H), 5.34 (s, 2H), 4.29 (q, J= 7.0 Hz, 2H), 4.03 (dd, J= 7.1, 1.2 Hz, 2H), 3.03 - 2.87 (m, 8H), 1.54 (t, J= 7.0 Hz, 3H), 1.43 (t, J= 7.6 Hz, 3H).
EXAMPLE 54
Synthesis of (E)-N-(4-(3-chloro-4-methoxyphenoxy)-3-cyano-7-ethoxy-2-ethylquinolin-
6-yl)-4-(4-methylpiperazin-l-yl)but-2-enamide (Cpd. No. 124).
[0949] Step 1. Synthesis of (E)-4-chloro-N-(4-(3-chloro-4-methoxyphenoxy)-3-cyano-7- ethoxy-2-ethylquinolin-6-yl)but-2-enamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 3. Yield 35%; yellow solid; LC-MS calculated for C25H23CI2N3O4 [M + H]+ = 500.12, found 500.12.
[0950] Step 2. Synthesis of (E)-N-(4-(3-chloro-4-methoxyphenoxy)-3-cyano-7-ethoxy-2- ethylquinolin-6-yl)-4-(4-methylpiperazin-l-yl)but-2-enamide: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 4 from Step 1 compound. Yield 42%; yellow solid; LC-MS calculated for C30H34CIN5O4 [M + H]+ = 564.24, found 564.24.
EXAMPLE 55
Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-((l-methyl-lH-indazol-5- yl)amino)quinolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 121).
[0951] The synthesis was carried out in a manner similar to the synthetic procedure used for Cpd. No. 63, Step 3. Yield 63%; yellow solid; LC-MS calculated for C28H31N7O2 [M + H]+ = 498.26, found 498.29; 1H NMR (400 MHz, MeOD) 5 9.20 (s, 1H), 8.12 (d, J = 0.9 Hz, 1H), 7.90 (d, J = 1.9 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.51 (dd, J = 8.9, 2.0 Hz, 1H), 7.40 (s, 1H), 6.96 (dt, J = 15.3, 7.1 Hz, 1H), 6.88 - 6.78 (m, 1H), 4.45 (q, J = 7.0 Hz, 2H), 4.16 (s, 3H), 4.03 (dd, J = 7.0, 1.2 Hz, 2H), 3.05 (q, J = 7.6 Hz, 2H), 2.96 (s, 6H), 1.62 (t, 7 = 7.0 Hz, 3H), 1.41 (t, J = 7.6 Hz, 3H).
EXAMPLE 56
Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-((l-methyl-lH-indazol-5- yl)amino)quinolin-6-yl)-4-(4-methylpiperazin-l-yl)but-2-enamide (Cpd. No. 122).
Step 2
[0952] Step 1. Synthesis of (E)-4-chloro-N-(3-cyano-7-ethoxy-2-ethyl-4-((l-methyl-lH- indazol-5-yl)amino)quinolin-6-yl)but-2-enamide: The synthesis of the Step 1 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 3. Yield 68%; yellow solid; LC-MS calculated for C26H25CIN6O2 [M + H]+ = 489.18, found 489.18.
[0953] Step 2. Synthesis of (E)-N-(3-cyano-7-ethoxy-2-ethyl-4-((l-methyl-lH-indazol-5- yl)amino)quinolin-6-yl)-4-(4-methylpiperazin-l-yl)but-2-enamide: The synthesis of the Step 2 compound was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 4 from Step 1 compound. Yield 68%; yellow solid; LC-MS calculated for C31H36N8O2 [M + H]+ = 553.31, found 553.35; 1H NMR (400 MHz, MeOD) 5 9.18 (s, 1H), 8.12 (d, J = 0.9 Hz, 1H), 7.90 (dd, J = 2.0, 0.7 Hz, 1H), 7.79 - 7.71 (m, 1H), 7.52 (dd, 7 = 8.9, 2.0 Hz, 1H), 7.37 (s, 1H), 6.98 (dt, 7 = 15.4, 6.1 Hz, 1H), 6.61 (dt, 7 = 15.4, 1.6 Hz, 1H), 4.45 (q, 7 = 7.0 Hz, 2H), 4.16 (s, 3H), 3.38 (dd, 7 = 6.1, 1.6 Hz, 6H), 3.05 (q, J = 7.6 Hz, 2H), 2.92 (s, 3H), 2.62 (s, 4H), 1.62 (t, J = 7.0 Hz, 3H), 1.41 (t, 7= 7.6 Hz, 3H).
EXAMPLE 57
[0954] The synthesis of the following examples was carried out in a manner similar to the synthetic procedure used for Cpd. No. 125, Step 4 from (E)-4-chloro-N-(3-cyano-7- ethoxy-2-ethyl-4-((l-methyl-lH-indazol-5-yl)amino)quinolin-6-yl)but-2-enamide
(wherein "amine" is R9R8NH and "R" is R9R8N-). EXAMPLE 58
[0955] Synthesis of the following examples: (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride (0.15 mmol, 1.5 eq) was added at 0 °C to a suspension of appropriate aniline (0.1 mmol, 1.0 eq) in A-methylpyrrolidine (1 mL). The mixture was then allowed to ambient temperature and stirred for 1 h. The compound was purified by preparative
HPLC to yield desired compound.
EXAMPLE 59
[0956] (E)-4-chlorobut-2-enoyl chloride (0.18 mmol, 1.5 eq) was added at 0 °C to a suspension of appropriate aniline (0.12 mmol, 1.0 eq) in Y- methyl pyrrolidine (1 mL). The mixture was then allowed to ambient temperature and stirred for 1 h. The compound was purified by preparative HPLC to yield the chloro compounds of Table 8.
Table 8
-2T2-
EXAMPLE 60
[0957] Amine (0.11 mmol, 2.0 eq) was added at room temperature to a suspension of appropriate chloro compound (0.06 mmol, 1.0 eq) and CS2CO3 (0.11 mmol, 2.0 eq) in acetonitrile (2 mL). The mixture was then stirred at 60 °C for 2 h. The compound was purified by preparative HPLC to yield the desired compounds in good yields.
EXAMPLE 61
Synthesis of (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7-ethoxy-2- methylquinazolin-6-yl)-4-(dimethylamino)but-2-enamide (Cpd. No. 157)
Cpd. No. 157
Step 4
[0958] Step 1. Synthesis of 4-chloro-7-ethoxy-2-methyl-6-nitroquinazoline: To a solution of 7-ethoxy-2-methyl-6-nitroquinazolin-4(lH)-one (1.0 g, 4.02 mmol, 1 eq) in diglyme or 1, 4-dioxane (10 mL) was added phosphorus oxychloride (2.15 g, 14.05 mmol, 3.5 eq). The mixture was then stirred at 100 °C for 1 h. The solution was cooled to ambient temperature, then filtered through celite® and the celite® was washed with diglyme and the filtrate was concentrated in vacuo. The crude compound was quenched with saturated sodium bicarbonate (60 mL) and the compound was extracted with ethyl acetate (3x 80 mL). The combined extracts were washed with brine solutions (40 mL) and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 0.91 g of 4-chloro-7-ethoxy-2-methyl-6- nitroquinazoline in 85% yield as a white solid. LC-MS calculated for C11H10CIN3O3 [M + H]+ = 268.05, found 268.15.
[0959] Step 2. Synthesis of N-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)-7-ethoxy-2- methyl-6-nitroquinazolin-4-amine: To a solution of 4-chloro-7-ethoxy-2-methyl-6- nitroquinazoline (100 mg, 0.37 mmol, 1 eq) and pyridinium chloride (43 mg, 0.37 mmol, 1 eq) in isopropyl alcohol was added 3-chloro-4-(pyridin-2-ylmethoxy)aniline (105 mg, 0.45 mmol, 1.2 eq). The mixture was then stirred at 80 °C for 2 h. The solution was cooled to ambient temperature, then concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 215 mg of N-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)-7-ethoxy-2-methyl-6- nitroquinazolin-4- amine in 81% yield as a light yellow solid. LC-MS calculated for C23H20CIN5O4 [M + H]+ = 466.13, found 466.27.
[0960] Step 3. Synthesis of N4-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)-7-ethoxy-2- methylquinazoline-4,6-diamine. To a solution of N-(3-chloro-4-(pyridin-2- ylmethoxy)phenyl)-7-ethoxy-2-methyl-6-nitroquinazolin-4-amine (50 mg, 0.11 mmol, 1.0 eq) and NH4CI (5.7 ng, 0.11 mmol, 1.0 eq) in ethanol (5 mL) was added Fe (30 mg, 0.54 mmol, 5.0 eq) at room temperature. The mixture was then stirred at 80 °C for 2 h. The solution was cooled to ambient temperature, then filtered through celite® and concentrated in vacuo. The crude compound was purified by flash silica gel column chromatography in ethyl acetate in hexanes (0 - 100%) to afford 30 mg of N4-(3-chloro- 4-(pyridin-2-ylmethoxy)phenyl)-7-ethoxy-2-methylquinazoline-4,6-diamine in 65% yield as a brown solid. LC-MS calculated for C23H22CIN5O2 [M + H]+ = 436.16, found 436.27.
[0961] Step 4. Synthesis of Cpd. No. 157: (E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride (12.7 mg, 0.07 mmol, 1.5 eq) was added at 0 °C to a suspension of (20 mg, 0.05 mmol, 1.0 eq) in A-methylpyrrolidine (0.5 mL). The mixture was then allowed to ambient temperature and stirred for 1 h. The compound was purified by preparative HPLC to yield Cpd. No. 157 as a light yellow solid in (13 mg) 52% yield. LC-MS calculated for C29H31CIN6O3 [M + H]+ = 547.22, found 547.23; 1H NMR (400 MHz, DMSO) 5 9.60 (d, J = 9.9 Hz, 2H), 8.82 (s, 1H), 8.60 (dt, J = 4.8, 1.4 Hz, 1H), 8.05 (d, J = 2.6 Hz, 1H), 7.89 (td, J = 7.7, 1.8 Hz, 1H), 7.81 (dd, J = 9.0, 2.6 Hz, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.38 (ddd, J = 7.7, 4.9, 1.2 Hz, 1H), 7.25 (d, J = 9.1 Hz, 1H), 7.19 (s, 1H), 6.88 - 6.74 (m, 1H), 6.66 (d, J = 15.1 Hz, 1H), 5.29 (s, 2H), 4.27 (q, J = 7.0 Hz, 2H), 1.45 (t, J= 6.9 Hz, 3H), 1.24 (s, 4H).
EXAMPLE 62
Metabolic restriction primes tumor for type-I/III IFN signaling amplification
[0962] To explore a potential impact of tumor metabolic programming on tumor immunity and therapeutic response to immune checkpoint blockade (ICB) (Zou et al., Cell Metab 35, 1101-1113 (2023)), glucose, glutamine, and fatty acid-related metabolic pathways were analzyed in a cohort of melanoma patients who received ICB at the University of Michigan. Li et al., Cancer Cell 41, 304-322 e307 (2023). Based on clinical responses, patients were divided into 3 groups: complete response (CR), partial response and stable disease (PR/SD), and progressive disease (PD). Individual metabolic pathways or combined metabolic pathways uniformly manifested negative correlations with clinical responses to ICB (FIG.la-d). In line with previous reports (Grasso et al., Cancer Cell 39, 122 (2021)), the interferon (IFN) signaling pathway positively correlated with ICB response (FIG.le). The data assert a potential relationship between cancer metabolism, IFN signaling, and ICB response.
[0963] To test this possibility, CHL1, a human melanoma cell line, was treated with IFNy in the presence of different types of metabolic inhibitors, including BPTES and R162 (glutaminolysis inhibitors), PFK15 and Shikonin (glycolysis inhibitors), and Etomoxir (a fatty acid oxidation inhibitor) (ED FIG. la). These inhibitors had no effect on basial MHC-I expression, but increased MHC-I expression in response to IFNy in CHL1 cells. As a control, Palbociclib (a CDK4/6 inhibitor) did not affect MHC-I expression (ED FIG. la). Notably, metabolic inhibitors enhanced MHC-I induction in a dose-dependent manner (ED FIG. lb). Given that MHC-I is a typical IFN responsive gene, the data suggest that metabolic restriction may enhance IFN signaling in tumor cells.
[0964] Studies were extended to additional human cancer cell lines (including A2058, SW480, and CAOV3) and mouse cancer cell lines (including B16, CT26, and ID8 cells). Again, BPTES enhanced MHC-I expression in these tumor cells in response to IFNy (ED FIG. 1c). Thus, metabolic restriction can enhance MHC-I expression in many types of tumor cells. To genetically validate this observation, stable B16 cells carrying short hairpin RNAs (shRNAs) targeting key enzymes in the metabolic pathways were established, including glutaminolysis enzymes (Gls and Gdh\ glycolysis enzymes (Pfkp and Pkm), and fatty acid oxidation enzyme (Cpt). In line with biochemical metabolic inhibition (ED FIG. la-c), knocking down metabolic enzymes boosted MHC-I expression in B16 cells in response to IFNy (ED FIG. Id). The transcripts of MHC-I-related genes were elevated in metabolic -restricted CHL-1 cells in response to IFNy, indicating a transcriptional regulation (ED FIG. le). It was assumed that metabolic restriction promoted early STAT1 activation, thereby enhancing MHC-I expression. Surprising, shGdh and shPkm cancer cells showed similar levels of STAT1 phosphorylation compared with control cancer cells in response to IFNy within 4 hours (ED FIG. If). Next, MHC-I expression at different time points was kinetically monitored. It was found that MHC-I expression in shGdh and shPkm tumor cells peaked at 48 hours (ED FIG. 1g). Hence, metabolic restriction affects the IFN-signaling pathway at a relatively late stage.
[0965] Cancer cells can produce typc-I/III IFNs. Chiappinelli et al., Cell 162, 974-986 (2015); Sheng et al., Cell 174, 549-563 e519 (2018). Given that metabolic restriction can promote MHC-I gene transcription at late stage and type-I IFN signaling contributes to ICB response (Zitvogel et al., Nat Rev Immunol 15, 405-414 (2015)), it was hypothesized that metabolic restriction may prime tumor cells to produce type Fill IFN, in return stimulating MHC-I expression (FIG. If). To test this possibility, type Fill IFN transcripts were quantified in CHE1 and B16 cells upon treatment with BPTES and IFNy. As expected, CHE1 and B16 cells expressed high levels of type Fill IFN gene transcripts in response to BPTES and IFNy, as compared to BPTES or IFNy treatment alone (FIG. 1g, h). Furthermore, higher levels of multiple IFN signaling genes (including Ccl5, CxcllO, H2-dl, and H2-k!) were detected in B16 cells in response to BPTES and IFNy, as compared with vehicle control or single treatment (FIG. li). IFN|3 protein was detected in metabolically restricted B16 cells in response to IFNy (FIG. Ij). BPTES failed to enhance the IFN signaling gene expression in non-tumor cells, such as mouse embryonic fibroblasts (ED FIG. Ih). The data suggests that metabolic restriction enabled- IFN signaling amplification is relatively specific to tumor cells.
[0966] To define the biological relevance of type Fill IFN in metabolically restricted tumor cells, Ifnarl (type-I IFN receptor) or Ifnlrl (type-III IFN receptor) knock out (KO) B16 cells were generated. BTPES and IFNy combined treatment induced a large amount of Ccl5, CxcllO, H2-kl, and H2-dl transcripts in wild type cells, but not in KO cells (ED FIG. li). The data indicate that metabolically restricted tumor cells produce biologically active type Fill IFN.
[0967] To examine the global impact of metabolic restriction at the transcriptomic level, an RNA-sequencing study on B16 cells treated with BPTES and IFNy was conducted. As expected, multiple IFN signaling genes (such as Ccl5, Cxcl9, CxcllO, H2-dl, H2-K1, B2m, and Cilla) were enriched in the BPTES plus IFNy group, compared with vehicle control, BPTES alone, or IFNy alone groups (FIG. Ik; ED FIG. Ij). Other differentially expressed genes (DEGs) were observed between tumor cells treated with BPTES plus IFNy versus IFNy (ED FIG. Ik, 1). Gene Ontology analysis revealed that multiple immune-related processes were enriched in tumor cells treated with BPTES plus IFNy, as compared with tumor cells treated with IFNy (FIG. 11) or BPTES alone (ED FIG. Im). Altogether, these results indicate that metabolic restriction primes tumor cell IFN responsiveness by inducing the type Pill IFNs and prepares and bridges the interplay between type-II and typc-I/III IFNs, thereby resulting in IFN signaling amplification. Thus, abundant metabolic activity in tumor cells disables this IFN signaling amplification, contributing to tumor immune evasion.
EXAMPLE 63
Metabolic restriction triggers cytosolic dsRNA sensing
[0968] Typc-I/III IFN production is governed by the innate immune sensing systems, including the pathogen-associated molecular patterns (PAMPs) recognition by the TLR pathway, cytosolic DNA sensing by the cGAS/STING pathway, and cytosolic RNA sensing by the RIGP MDA5 pathway (FIG. 2a). Hur et al., Annu Rev Immunol 37, 349- 375 (2019); Hertzog et al., Trends Immunol 24, 534-539 (2003); Barber et al., Trends Immunol 35, 88-93 (2014). To explore which pathway is involved in the regulation of type Pill IFN production in response to metabolic restriction, the key signaling genes in the innate immune sensing systems - including Myd88, Ticaml , Cgas, Stingl , Ddx58, and Ifihl was genetically knocked out in B16 cells. These genes encode the proteins of Myd88, Trif, Cgas, Sting, Rigi, and Mda5, respectively (FIG. 2a). Interestingly, in response to IFNy, BPTES treatment induced Ifiibl transcripts in WT, Myd88 KO, Ticaml KO, Cgas KO, Stingl KO, and Ddx58 KO B16 cells, but not in Ifihl KO B16 cells (FIG. 2b). To validate this, similar with WT cells (FIG. Ik), Ifihl KO B16 cells were transfected with metabolic gene targeting shRNAs and established metabolically restricted Ifihl KO B16 cells. Metabolically restricted Ifihl KO cells were no longer able to produce IFN|3 in response to IFNy (FIG. 2c). In line with this, treatment of BPTES plus IFNy resulted in IRF7 phosphorylation, a downstream effect of MDA5 activation, in B16 WT cells, but not in B16 Ifihl KO cells (ED FIG. 2a, b). Thus, MDA5-mediated dsRNA sensing and signaling may be involved in the IFN-signaling amplification during metabolic restriction. [0969] MDA5 senses cytosolic dsRNAs and triggers type-I/III IFN production. Dias Junior et al., Trends Microbiol 27, 75-85 (2019). To directly demonstrate the involvement of dsRNAs in the IFN-I/III production triggered by metabolic restriction, the cytosolic RNAs from cells treated with vehicle, BPTES, IFNy, or BPTES plus IFNy were purified. These cytosolic RNAs were transfected into B16 cells. Elevated levels of cytosolic dsRNA were detected in B16 cells treated with BPTES plus IFNy, compared to B16 cells treated with vehicle, BPTES alone, or IFNy alone (FIG. 2e). Accordingly, high levels of Ifiibl expression in WT B16 cells transfected with dsRNAs from B16 cells previously treated with BPTES plus IFNy, compared to B16 cells treated with vehicle, BPTES alone, or IFNy alone (FIG. 2f). This effect was abolished when the RNA samples were treated with dsRNA-specific RNase (FIG. 2f). Thus, metabolic restriction triggers cytosolic dsRNA sensing, resulting in type-I/III IFN production.
[0970] The role of metabolic restriction triggered cytosolic RNA sensing in antitumor immunity and ICB response was examined. Control or shGdh B16 WT and Ifihl KO cells were inoculated into wild type (immunocompetent) mice and treated them with ICB. In line with previous findings (Lin et al., J Clin Invest 128, 805-815 (2018)), B16 WT cells were relatively resistant to anti-PD-Ll antibody treatment, while shGdh B16 tumors were dramatically shrunken in response to ICB (FIG. 2g, h). The data suggest that metabolic restriction potentiates cancer immunotherapy. However, when the experiment in Ifihl KO B16 tumors was repeated, metabolic restriction by knocking down Gdh failed to enhance immunotherapy efficacy (FIG. 2i, j). Hence, metabolic restriction triggers cytosolic dsRNA sensing, sensitizing ICB response.
EXAMPLE 64
Metabolic restriction induces ADAR1 palmitoylation
[0971] Metabolic restriction triggers MDA5 dependent cytosolic RNA sensing (Fig 2-3, FIG. 3a). ADAR1 is the predominant RNA-editing enzyme in. Nishikura, Annu Rev Biochem 79, 321-349 (2010). ADAR1 has two isoforms, a constitutively expressed isoform with 110 KD molecular weight (pl 10) and an IFN-inducible isoform with 150 KD molecular weight (pl 50) (ED FIG. 3a). It was hypothesized that metabolic restriction affected ADARl-medaited RNA-editing, thereby triggering type-I/III IFN production. To evaluate ADAR 1 -mediated RNA-editing activity, an RNA editing luciferase reporter (RE-Luc) was constructed. To this end, a stop codon as UAG at the RNA-editing site of an Adarl-substrate was designed. Park et al. Nat Commun 11, 5130 (2020). After RNA- editing, UAG was converted to UGG and the downstream luciferase coding sequences can be translated (FIG. 3b). Then, the luciferase activity was recorded as the RNA- editing efficacy. This ADAR1 reporter in RE- Luc expressing cells was validated with ADAR1 overexpression (Adarl OE) and Adarl knockout (ADAR KO) cells. Adarl OE and ADAR KO cells showed potent and minimal luciferase expression, respectively (ED FIG. 3c). B16 cells were transfected with this AD ARI reporter and treated them with BPTES and IFNy. As expected, BPTES did not affect RNA-editing efficacy in basal condition, but strongly reduced RNA-editing efficacy in response to IFNy (FIG. 3c). The data suggests that metabolic restriction may target ADARL To test the dependence of ADAR1, Adarl KO B16 cells (FIG. 3d) were established and treated with BPTES and/ or IFNy. BPTES reduced ADAR1 RE-Luc activity and augmented Ifnbl and MHC-I expression in WT cells, but not in Adarl KO cells (FIG. 3e-g). Therefore, metabolic restriction abolishes ADAR 1 -mediated RNA editing efficacy.
[0972] How metabolic restriction regulated ADAR 1 -mediated RNA editing was explored. BPTES treatment did not affect ADAR1 protein expression with or without IFNy (ED FIG. 3d). It was determined if BPTES treatment resulted in a post-translational modification (PTM) in ADARL BPTES treatment did not affect ADAR1 acetylation, methylation, phosphorylation, or ubiquitination (ED FIG. 3e), but strongly induced palmitoylation, as determined by Click-IT assay (FIG. 3h). In further support of this, BPTES-induced ADAR1 palmitoylation was blocked by 2-BP, an inhibitor for protein palmitoylation (FIG. 3i). Furthermore, 2-BP treatment abolished the effect of BPTES on the induction of Ifnbl and MHC-I in B16 cells (ED FIG. 3f). Collectively, metabolic restriction induces ADAR1 palmitoylation, thereby disabling ADAR 1 -RNA editing capacity and triggering the typc-I/III IFN response.
[0973] GPS-Palm predicts that the cysteineiosi/1082 residues of ADAR1 were the palmitoylation sites (ED FIG. 3g). The potential palmitoylation sites were conserved across multiple species (FIG. 3j). To functionally validate the palmitoylation sites, an AD ARI mutant by replacing cysteines at 1081/1082 with alanine (C2A) was constructed. BPTES failed to induce ADAR1 palmitoylation (FIG. 3k), reduce RNA-editing activity (FIG. 31), or enhance the expression of IFN|3 (FIG. 3m) and MHC-I (FIG. 3n) in cells carrying AD ARI C2A mutant. Thus, cysteines at 1081/1082 are the AD ARI palmitoylation sites, being responsive to metabolic restriction. [0974] Cysteines 1081/1082 are a zinc binding site of AD ARI. Zinc is a cofactor for the enzymatic activity of ADAR. Park et al. Nat Commun 11, 5130 (2020). The Zn-NTA (Zinc ion coupled with nitrilotriacetic acid beads) pulldown assay was used to detect the zinc binding affinity in WT AD ARI or C2A AD ARI mutant (FIG. 3o). BPTES treatment repressed the zinc binding affinity to ADAR1 in a dose dependent manner but failed to do so in ADAR1 C2A mutant (FIG. 3p, q). In addition, palmitoylation inhibitor 2-BP treatment abolished the effect of BPTES on the zinc affinity to ADAR1 (ED FIG. 3h). Collectively, the data suggests that metabolic restriction induces ADARlpalmitoylation at cysteines 1081/1082, thereby blocking cofactor Zinc binding and reducing the RNA- editing activity of ADAR1 (FIG. 3s).
EXAMPLE 65
Metabolic restriction stimulates ZDHHC2 expression
[0975] To explore the enzyme that catalyzes ADAR1 palmitoylation, the transcripts of ZDHHC family members in B16 cells were analyzed upon BPTES treatment. BPTES treatment induced the expression of Zdhhc2, Zdhhc6, Zdhhcl3, Zdhhcl7, and Zdhhc20 in B16 cells (FIG. 4a). These five Zdhhc family members were ectopically expressed in B16 cells and treated them with IFNy. Upon IFNy treatment, Zdhhc2, but not the others, reduced ADAR1 reporter activity (FIG. 4b), elevated Ifiibl expression (FIG. 4c), and enhanced MHC-I induction (FIG. 4d). Furthermore, overexpression of Zdhhc2 magnified the induced IFN signaling gene expression in B16 cells (FIG. 4e). Zdhhc2 was induced by BPTES in a time and dose dependent manner in B16 cells (FIG. 4f, g). In Zdhhc2 KO B16 cells, BPTES failed to induce Zdhhc2 expression and did not change Ifiibl and IFN signaling gene expression (FIG. 4g, h). The data suggests that Zdhhc2 is the metabolically regulated enzyme catalyzing ADAR1 palmitoylation.
[0976] To further validate this, Zdhhc2 was overexpressed in B16 cells and performed Click-IT assay. ADAR1 palmitoylation was induced by Zdhhc2-OE in a dose dependent manner (FIG.4i). Reciprocal co-immunoprecipitation (Co-IP) experiments indicated the physical interaction between Zdhhc2 and Adarl (FIG. 4j, k). Immunofluorescence staining revealed a co-localization of Zdhhc2 and Adarl in the cytoplasm (FIG. 41). Thus, Zdhhc2 catalyzes ADAR1 palmitoylation.
[0977] Next, the link between metabolic restriction and Zdhhc2 expression was explored. Using the ENCODE Project ChlP-seq database at UCSC, it was found that multiple potential metabolism-related transcription factors may target the promoter of ZDHHC2 (ED FIG. 4a). These transcription factors were genetically knocked down. It was found that knocking down of SREBP1 (Sterol regulatory element-binding protein 1), but not the other transcription factors, resulted in an increase in ZDHHC2 expression (ED FIG. 4a). In the ChlP-seq dataset (Consortium, Nature 489, 57-74 (2012)), there was an enrichment of SREBP1 binding, but not SREBP2 binding, in the ZDHHC2 promoter (ED FIG. 4b). CHL cells were treated with Fatostatin, an inhibitor for SREBP1, and Betulin, an inhibitor for SREBP2. Inhibition of SREBP1, but not SREBP2, stimulated ZDHHC2 expression in a dose dependent manner (ED FIG. 4c). In line with this, Fatostatin dose- dependently enhanced ADAR1 palmitoylation (ED FIG. 4d), reduced its RNA editing efficacy (ED FIG. 4e), and augmented IFNB1 and MHC-I expression (ED FIG. 4f, g). Genetically knocking out Srebpl (Srebpl KO) enhanced Zdhhc2 expression at both RNA (ED FIG. 4h) and protein levels (FIG. 4m), and triggered Ifiibl and IFN signaling gene expression upon IFNy treatment (FIG. 4n). Moreover, BPTES failed to enhance Ifiibl production and signaling in Srebpl KO B16 cells upon IFNy treatment (ED FIG. 4i, j). Thus, metabolic restriction targets SREBP1 to induce the type Fill IFN production.
[0978] To further solidify the notion that SREBP1 suppressed ZDHHC2 transcription, multiple small RNAs targeting the region of Zdhhc2 promoter (saZdhhc2) were designed and synthesized (ED FIG. 4k). It was found the small RNA saZdhhc2-b blocked the SREBP1 binding site of Zdhhc2 promoter, strongly enhanced Zdhhc2 expression, but did not affect SREBP1 signaling gene expression such as Edlr and Hmgcs (ED FIG. 41, m). Shimano et al., Nat Rev Endocrinol 13, 710-730 (2017). The data suggest saZdhhc2 blocks SREBP1 -mediated ZDHHC2 repression but does not affect SREBP1 global signaling. When B16 cells were transfected with saZdhhc2-b and treated with IFNy, saZdhhc2-b dramatically enhanced the expression of Ifiibl and H2-Kb (ED FIG. 4n-o) in WT cells, but not in Zd.hh.c2 KO B16 cells (ED FIG. 4p-q). The data suggests that disruption of the SREBP1 binding site on ZDHHC2 promoter may stimulate ZDHHC2 transcription and IFN signaling amplification. Thus, metabolic restriction stimulates ZDHHC2 expression by targeting SREBP1, promoting ADAR palmitoylation, and enabling IFN amplification (FIG. 4o).
EXAMPEE 66
A cysteine-targeting covalent inhibitor modulates AD ARI activity [0979] ADAR1 is considered a therapeutic target in cancers and autoimmune diseases. Chung, H. et al., Cell 172, 811-824 e814 (2018); Ishizukaet al., Nature 565, 43-48 (2019). However, there is no reliable ADAR1 inhibitor for research or clinical translation. Given that the cysteine 1081/1082 site in AD ARI is palmitoylated and functionally critical for ADAR1-RNA editing, a cysteine-targeting compound to modulate the ADAR1-RNA editing activity was needed.
[0980] There are seven FDA-approved covalent cysteine-targeting inhibitors, to date (ED FIG. 5a). Boike et al., Nat Rev Drug Discov 21, 881-898 (2022). IFNy-pretreated B16 cells were cultured with these 7 inhibitors (FIG. 5a). Neratinib, but not the others, reduced ADAR1 RE-Luc activity (FIG. 5b), triggered Ifiibl production (FIG. 5c), and increased MHC-I expression (FIG. 5d). Neratinib could enhance Ifiibl production in a time- and dose-dependent manner (FIG. 5e-g). In ADAR1 KO or ADAR1 C2A mutant cells, Neratinib failed to stimulate Ifiibl (FIG. 5h, i). Similarly, Neratinib induced 1FNB1 and MHC-I induction in multiple cancer cells upon IFNy treatment (ED FIG. 5b, c). Furthermore, Neratinib dramatically reduced the Zinc affinity of ADAR1 (ED FIG. 5d). The data suggests that Neratinib may target the cysteine 1081/1082 site of AD ARI.
[0981] To explore how Neratinib regulates ADAR1, the structure of Neratinib was modified by reducing the Michael acceptor double bond that covalently targets the thiol group (-SH) of the cysteine residue and generates Neratinib-Inactive (FIG. 5j). Interestingly, unlike Neratinib, Neratinib-Inactive failed to alter Ifiibl and IFN signaling gene expression in IFNy-pretreated B 16 cells (FIG. 5k-n). The data suggest that Neratinib covalently targets the cysteine residues of AD ARI. To solidify this conclusion, biotin labeled Neratinib (ED FIG. 5e) was synthesized and astreptavidin pulldown assay in B16 ADAR1 OE cells was performed. ADAR1, but not GAPDH, was enriched in the pulldown products with biotin-Neratinib. As negative controls, unlabeled Neratinib failed to enrich with Adarl or Gapdh (FIG. 5o). Furthermore, biotin-Neratinib interacted with wild type ADAR1, but not ADAR1 C2A mutant (FIG. 5p). The data suggest that Neratinib interacts with AD ARI and targets the cysteinel081/1082 site on AD ARI protein in a covalent manner (FIG. 5q).
[0982] Neratinib is used to treat HER2 overexpressed/amplified breast cancer. As a pan- HER inhibitor, Neratinib binds to and inhibits tyrosine kinases (such as EGFR, HER2, HER4) and SRC, resulting in the reduced phosphorylation of downstream signaling pathways. Unlike the HER2-positive SKBR3 cells, B16 cells expressed negligible levels of HER2 (ED FIG. 5f). Neratinib inhibited AKT phosphorylation in SKBR3 cells (ED FIG. 5f), but not in B16 cells (ED FIG. 5g). Thus, it is likely that the effect of Neratinib on B16 cells is kinase independent.
[0983] Given that Neratinib may serve as an inhibitor for ADAR1, it was hypothesized that Neratinib elicits tumor autocrine IFNs and promotes anti-tumor immunity in vivo. To test this hypothesis, B16 tumor-bearing mice were treated with low doses of Neratinib, anti-PD-Ll, and their combination. As expected, anti-PD-Ll or Neratinib alone had minimal effect on B16 tumor growth and mouse survival, while their combination dramatically sensitized tumor response to anti-PD-Ll therapy (FIG. 5r, s, ED FIG. 5h). Furthermore, the levels of tumor infiltrating IFNy+, TNFoc+, and granzyme B+ CD8+ T cells were higher in mice treated with the combination compared to single therapy (ED FIG. 5i, j). The data suggest that Neratinib targets ADAR1 and sensitizes therapeutic efficacy to ICB.
[0984] Collectively, cancer metabolic restriction targets the SREBP1-ZDHHC2 pathway to mediate AD ARI palmitoylation at the cysteinel081/1082 site. AD ARI palmitoylation blocks the interaction between AD ARI and its cofactor Zinc, thereby reducing the RNA- editing activity of ADAR1, eliciting tumor autocrine IFN Fill amplification, and potentiating immunotherapy response. Interestingly, Neratinib physically interacts with AD ARI at the cysteinel081/1082 site and blocks its interaction with zinc cofactor, thereby diminishing ADAR1 enzymatic activity and sensitizing immunotherapy response. Thus, Neratinib surprisingly acts as a cysteine-targeting covalent inhibitor of ADAR1 (ED FIG. 5k).
[0985] Active cancer metabolic reprogramming results in nutrition deprivation and accumulation of onco-immune metabolites in the TME, contributing to tumor progression and therapeutic resistance. Zou et al., Cell Metab 35, 1101-1113 (2023); O'Sullivan et al., Nat Rev Immunol 19, 324-335 (2019); Lim et al., Elife 9 (2020); Tsai et al., Cell Metab 35, 118-133 el 17 (2023); Arner et al., Cancer Cell 41, 421-433 (2023); DePeaux et al., Nat Rev Immunol 21, 785-797 (2021); Chapman et al., Immunity 55, 14-30 (2022). In the studies described herein, it was surprisinly discovered that high levels of tumor metabolism prevent IFN signaling amplification via sustaining ADAR1 RNA-editing enzymatic activity, thereby diminishing innate immunity and causing resistance to ICB.
[0986] ADAR1 mediates dsRNA editing, affecting immune responses in infectious diseases, autoimmune diseases, and cancers. However, if and how ADAR1 is regulated, and the immunological significance of its regulation remain to be defined. This work demonstrated that cancer metabolic restriction controls ADAR1 enzymatic activity via the SREBP1-ZDHHC2-ADAR1 axis. A negative correlation between active tumor metabolism and therapeutic efficacy in patients receiving ICB was initially found. Given the significance of the type-I and type-II IFN signaling pathways in ICB (Du et al., Cell Mol Immunol 19, 23-32 (2022); Gajewski et al., Nat Immunol 14, 1014-1022 (2013)), this clinical observation prompted the exploratrion of a potential relationship between tumor metabolic reprogram and IFN-signaling pathways. It was found that metabolic restriction amplifies the type-I and -III IFN signaling pathways in tumors. This signaling amplification is attributed to an increase in MDA5-mediated dsRNA sensing due to diminished ADAR1 enzymatic activity. It turns out that metabolic restriction inhibits SREBP1, causing ZDHHC2-mediated palmitoylation of ADAR1 at the Cysteine 1081/1082 site. This palmitoylation blocks the cofactor zinc binding to the Cysteine 1081/1082 site within AD ARI catalytic domain and thereby reduces ADAR 1 -mediated dsRNA editing capacity. Thus, active tumor metabolic reprogramming targets ADAR1, resulting in innate immune signaling sensing failure and ICB resistance. While defining this previously undocumented tumor immune evasion mechanism, the studies of the present disclosure have generated several unexpect and surprising insights into ADAR1 biology and tumor immunotherapy.
[0987] How immunotherapy clears cancer cells in vivo remains unclear. It is generally thought that the interplay between innate (such as type-I and III IFNs) and adaptive (such as type-II IFN, IFNy) immune responses play a decisive role in a successful protective immunity. This goal can be reached in infectious immunity due to pathogen-mediated direct activation of innate immune cells and/or adaptive immune cells. Along this vein, meaningful interaction between innate (type-I/III IFN) and adaptive (type-II IFNs) immune signaling networks in the TME is poorly understood. Zou et al., Nat Rev Cancer 5, 263-274 (2005). It was found that during metabolic restriction, type II IFN can magnify the IFN-I and -III signaling pathways via diminishing ADAR 1 -mediated RNA editing. Thus, RNA editing serves as an immune metabolic checkpoint that connects type-II and type-I/III IFNs in the TME. In support of this concept, it has been shown that reduced tumor metabolic pathways correlate with clinical responses to ICB in patients with cancer. Furthermore, tumor metabolic restriction can sensitize ICB -resistant tumors to immunotherapy in the animal models in the MDA5-sensing dependent manner. Thus, in the absence of infectious pathogens, enabling IFN signaling amplification via metabolic reprogramming may be crucial for inducing complete clinical response in patients receiving ICB, while regulating ADAR 1 -mediated RNA editing plays a critical role in this process.
[0988] Prior to the studies described in the present disclosure, it was unknown if and how ADAR1 was regulated at the levels of post-translational modification PTM. It has been found that metabolic restriction results in ADAR1 palmitoylation via a defined molecular pathway. Surprisingly, the palmitoylation of the cysteine residues alters AD ARl's ability to interact with its Zinc cofactor, consequently abolishing its enzymatic activity. Hence, these findings provide the initial evidence showing a PTM for ADAR1. Furthermore, a role of palmitoylation was uncovered beyond the current understanding that palmitoylation usually affects protein stability, trafficking, and interaction. Linder et al., Nat Rev Mol Cell Biol 8, 74-84 (2007); Smotrys et al., Annu Rev Biochem 73, 559-587 (2004). Moreover, as Zinc can be a cofactor for many enzymes, this work raises the question of whether similar mechanism may be shared by other enzymes. Therefore, the discovery of ADAR1 palmitoylation, its underlying molecular mechanism, and its impact on ADAR1 enzymatic activity not only fills a knowledge gap of ADAR1 PTM but also generates important scientific insight into the therapeutic relevance of ADAR1 PTM.
[0989] ADRA1 mediates dsRNA-editing, disabling innate immune signaling sensing and diminishing tumor immunogenicity. In addition to this work, recent genetic studies show that AD ARI is a cancer immunotherapy target and loss of tumor AD ARI can overcome resistance to ICBs. Given that ADAR1 catalyzes the deamination of adenosine, analogs of adenosine (such as 8-Azaadenosine, 8-chloroadenosine, and ZYS-1) may serve as inhibitors for ADAR1. Ding et al., Cell Transplant 29 (2020); Zipeto et al., Cell Stem Cell 19, 177-191 (2016); Ramirez-Moya et al., Oncogene 39, 3738-3753 (2020). However, the specificity and potency of these analogs are uncertain. It has been reported that these molecules have no effect on A-to-I editing of multiple ADAR1 substrates. Cottrell et al., Cancer Res Commun 1, 56-64 (2021). This questions whether using analogs of adenosine is an ideal strategy to reduce ADAR1 enzymatic activity. For example, AD ARI targets adenosines within the dsRNAs. Free nucleotides may not block the deaminase activity of ADAR1. Moreover, adenosine is an intermediate in the methionine cycle and may affect the synthesis of S-adenosyl methionine (SAM), thereby altering RNA/DNA methylation in T cells. Bian et al., Nature 585, 277-282 (2020). Interestingly, rebecsinib, a spliceosome inhibitor (Villa et al., J Med Chem 56, 6576-6582 (2013)), is proposed to be an ADAR1 inhibitor by suppressing ADAR1 splicing. Crews et al., Cell Stem Cell 30, 250-263 e256 (2023). However, E7107, a spliceosome inhibitor was discontinued in Phase I clinical trial due to unacceptable toxicity. Hong et al., Invest New Drugs 32, 436-444 (2014); Eskens et al., Clin Cancer Res 19, 6296-6304 (2013). It was found that an FDA-approved drug, Neratinib, can bind to the cysteine 1081/1082 site of ADAR1 by covalent modification and blocks its interaction with Zinc, thus reducing the RNA-editing efficacy of AD ARI. Interestingly, administration of low dose Neratinib sensitizes ICB -resistant tumors to immunotherapy in preclinical models. The anti-tumor effect of Neratinib is accompanied with potent T cell responses, manifested no signs of toxicity, and is independent of tumor kinase activity. Thus, these studies identify a previously unknown mechanism and an alternative strategy to target ADAR1.
EXAMPLE 67
General Methods used in EXAMPLES 62-66
Animal experiments
[0990] Six- to eight-week-old female C57BL/6 (C57BL/6J, Stock# 000664) mice were obtained from the Jackson Laboratory. All mice were maintained under pathogen-free conditions. The animal room temperature (18-23°C) and humidity (40-60%) controlled with a 12:12 light-dark cycle. B16 cells (1 x 105) were subcutaneously injected into the right flank of the mice. For anti-PD-Ll treatment in B16 model, 5 mg/kg anti-PD-Ll (ZzzVzvoMAb, 10F.9G2) and control antibody (In VzvoM Ab, LTF-2) were intraperitoneally administered on day 0, 3, 6, 9, 12, and 15 post tumor inoculation. For Neratinib treatment in B16 model, 5 mg/kg of Neratinib were intraperitoneally administered on day 3, 6, 9, 12, and 15 post tumor inoculation. Tumor diameters were measured using calipers. Tumor volume was calculated by Length x Width x Width/2. Animal studies were conducted under the approval of the Institutional Animal Care and Use Committee at the University of Michigan (PRG00010169). The study is compliant with all relevant ethical regulations regarding animal research. In none of the experiments did xenograft tumor size surpass 2.5 cm in any dimensions, and no animal had severe abdominal distension (> 10% original body weight increase). Sample size was chosen based on preliminary data. After tumor inoculation, mice were randomized and assigned to different groups for treatment. Reagents
[0991] BPTES, R162, PFK15, Shikonin, Etomoxir, Fatostatin, Betulin, Palbociclib, and Neratinib were purchased from Cayman Chemical. Recombinant human IFNy (285-IF) and mouse IFNy (485-MI) were from R&D Systems. The synthesis of Neratinib-Inactive and Biotin-Neratinib compounds, e.g., compounds of Table IB, is outlined above. Plasmids
[0992] To generate the ADAR1 reporter (RE-Euc), DNA sequences corresponding to ADAR1 substrates and luciferase were synthesized, annealed, PCR amplified, and ligated into pCI-neo (Promega) plasmid. PRE-SV40 was purchased from Promega. To construct Flag-tagged Adarl plasmid, the coding sequences of Adarl were PCR amplified from the cDNA generated from IFNy pretreated B16 cells and subsequently inserted into PCI- Flag plasmid. Site directed mutagenesis was conducted to generate Adarl C2A mutant plasmid. PCI-Flag plasmid was prepared by inserting the following Kozak sequence plus Flag tag plus 5 x Glycine sequence into the PCI-neo (Promega) plasmid between Nhel and Xhol. shRNAs were designed and inserted into PEKO.l plasmid (Addgene #10879) to knock down mouse Gls, Gd, Pfkp, Pkm, and Cpt. To disrupt Srebpl binding to the Zdhhc2 promoter, saRNAs were designed and inserted into PEKO.l plasmid (Addgene #10879). The shRNA targeting firefly luciferase (shEuc) served as a negative control. sgRNAs were designed and inserted into PX459 plasmid (Addgene #48139) to knock out mouse Ifnarl, Ifnlrl, Myd.88, Trif eGas, Sting, Rigi, Mda5, Adarl, Zdhhc2, and Srebpl. The target sequences are listed in Extended Data Table 1.
Extended Data Table 1
[0993] The primer sequences are listed in Extended Data Table 2.
Extended Data Table 2
Cell Culture
[0994] Human melanoma cell line CHL1 (CRL-1619), A2058 (CRL-3601), human colon cancer cell line SW480 (CCL-228), human ovarian cancer cell line CAOV3 (HTB-75), mouse melanoma cell line B16-F0 (CRL-6322), mouse colon cancer cell line CT26 (CRL-2638), and 293T (CRL-3216) were purchased from the American Type Culture Collection (ATCC). Mouse ovarian cancer cell line ID8 was used previously in the Zou laboratory. Wang et al., Nature 569, 270-274 (2019). B16 Ifriarl KO, Ifrilrl KO, Myd88 KO, Trif KO, Cgas KO, Sting KO, Rigi KO, Ifihl KO, Adarl KO, Zdhhc2 KO, Srebpl KO, shLuc, shGls, shGdh, shPfkp, shPkm, and shCpt cell lines were generated in this study. All cell lines were tested for mycoplasma contamination routinely and confirmed negative for mycoplasma. Cells were cultured in RPMI medium (Gibco #11875) supplemented with 10% FBS, except for CHL1 and 293T cells; the latter 2 lines were cultured in DMEM (Gibco #11965) supplemented with 10% FBS. All cells were maintained in 37°C and 5% CO2. To generate knock down cell lines, lentiviral particles were produced by transfection of PLKO.l shRNA plasmid with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) (4:3: 1) into 293T cells, and subsequently transduced into tumor cells with polybrene (Sigma- Aldrich, 8 pg/ml) overnight. 48 hours after transfection, cells were selected with puromycin (1-2 pg/ml) for additional 2 weeks. To establish knock out cell lines, PX459-sgRNA plasmids were transfected into tumor cells for 2 days and selected by puromycin (1-2 pg/ml) for additional 2 days. The cells were then serially diluted and seeded into 96 well plates. After 2-3 weeks, single cell colonies were dissociated and re-plated into 6 well plates. Upon cell confluency, half of the cells were harvested and validated for knock out (KO) efficiency via Western blotting. All transfections were conducted with lipofectamine 2000 (ThermoFisher Scientific) at a ratio of 1 pg plasmid:2 pl transfection regent. The transfection dosage was determined by titration.
Luciferase activity assay
[0995] Cells were transfected with RE-Luc and PRL-SV40 for 24 hours and then treated with multiple conditions. Luciferase activity for firefly luciferase (RE-Luc) and renilla luciferase (PRL-SV40) were measured with Dual-Luciferase Reporter Assay System (Promega). Relative firefly luciferase activity was normalized with renilla luciferase activity.
Surface staining and flow cytometry analysis (FACS)
[0996] Cells were trypsinized and washed with MACS buffer (PBS, 2%FBS, 1 mM EDTA). Surface staining was performed by adding the following antibodies to the cell suspension in 50 pl MACS buffer: anti-HLA-ABC (G46-2.6, BD Biosciences), anti-H2- Db (KH95, BD Biosciences), anti-H2-Dd (34-2-12, BD Biosciences), and anti-H2-Kb (AF6-88.5, BD Biosciences). After 30 minutes incubation, cells were washed with MACS buffer and analyzed on BD Fortessa flow cytometer.
Quantitative PCR (qPCR)
[0997] Total RNA was isolated from cells by column purification (Direct-zol RNA Miniprep Kit, Zymo Research) with DNase treatment. cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific) with random hexamer primers. Quantitative PCR (qPCR) was performed on cDNA using Fast SYBR Green Master Mix (ThermoFisher Scientific) on a StepOnePlus Real-Time PCR System (ThermoFisher Scientific). Fold changes in mRNA expression were calculated by the AACt method using ACTB as an endogenous control. Results are expressed as fold change by normalizing to the controls. Gene expression was quantified using the primers listed in Extended Data Table 3.
Extended Data Table 3 dsRNA dot blotting
[0998] To measure the dsRNA amount in the cytoplasm, cytosolic fraction was prepared with cell fractionation kit (CST, 9038) with ribonuclease inhibitor (ThermoFisher Scientific, 15518012). The cytosolic RNA was isolated by column purification (Direct- zol RNA Miniprep Kit, Zymo Research) with DNase treatment. The Cyto-RNA samples were diluted in nuclease-free water to final concentrations of 200 ng/ p.L, and half of the Cyto-RNA samples were digested with RNase III (Ambion, AM2290) for 1 hour at 37°C. Then, 6pl aliquots were spotted to a positively charged nylon membrane (Roche, 11209299001) and leave the membrane air-dried at room temperature. The membrane was blocked in 5% (w/v) non-fat dried milk in Tris-buffered saline (TBS)-T buffer (20 mM Tris [pH 7.4], 150 mM NaCl, and 0.1% (v/v) Tween-20). For the detection of dsRNAs, the membrane was incubated with J2 anti-dsRNA murine antibody (SCICONS, 10010200, 1:5000) at 4°C overnight and HRP-conjugated secondary antibodies (CST) for 1 hour at room temperature. Signal was detected using Clarity and Clarity Max Western ECL Blotting Substrates (Bio-Rad) and captured using ChemiDoc Imaging System (Bio-Rad).
Western blotting
[0999] Cells were washed in cold PBS and lysed in 1 x RIPA lysis buffer (ThermoFisher Scientific) with 1 x protease inhibitor (ThermoFisher Scientific). Lysates were incubated on ice for 10 minutes and cleared by centrifugation at 15,000g for 15 minutes. Protein concentration was quantified using a BCA protein assay kit (ThermoFisher Scientific). Thirty microgram protein was mixed with sample buffer (ThermoFisher Scientific) with P-ME and denatured at 95 °C for 5 minutes. For detection of Zdhhc2, protein samples were denatured at 50°C for 10 minutes. Sample was separated by SDS-PAGE and transferred to a Nitrocellulose Membrane (Bio-Rad). Membranes were blocked with 5% w/v non-fat dry milk and incubated with primary antibodies overnight at 4 °C and HRP- conjugated secondary antibodies (CST) for 1 hour at room temperature. Signal was detected using Clarity and Clarity Max Western ECL Blotting Substrates (Bio-Rad) and captured using ChemiDoc Imaging System (Bio-Rad). Antibodies were as follows: anti- phospho-STATl(CST, 9167, 1: 1000), anti-STATl (CST, 9172, 1: 1,000), anti-Gdh, anti-Pkm, anti-GAPDH (Proteintech, 60004, 1: 5,000), anti-MDA5 (CST, 5321, 1:1000), anti-phospho-IRF7 (CST, 24129, 1:1000), anti-IRF7 (CST, 35659, 1:1000), anti- AD ARI (SCBT, 73408, 1:100), anti-ZDHHC2 (SCBT, 515204, 1:100), anti-HMGCS (CST, 42201, 1:1000), anti-SREBPl (SCBT, 13551, 1:100), anti-Tubulin (CST, 2148, 1:1000), anti-Acetyl-Lys (CST, 9441, 1:1000), anti-Mono-Methyl-Arg (CST, 14679, 1:1000), anti-phospho-Ser/Thr (CST, 9631, 1:1000), anti-phospho-Tyr (CST, 9411, 1:1000), and anti-ubiquitin (CST, 3936, 1:1000). Co-Immunoprecipitation (Co-IP)
[01000] Cells were collected with IP lysis buffer (ThermoFisher Scientific, 87787) plus protease inhibitor. Protein concentration was determined with BCA protein assay kit. 200-500 pg protein samples were added to 1-3 pg primary antibodies anti-Flag (CST, 14739) or anti-HA (CST, 12972), and incubated with gentle rocking at 4°C overnight. Then, samples were further incubated with 20 pl Protein A/G PLUS-Agarose (Santa Cruz, sc-2003) for 2 hours at 4°C; and centrifuged at 7500 xg for 30 seconds at 4°C. Cell pellets were washed 4 times with IP lysis buffer, resuspended with 40 pl 2 x sample buffer with P-ME, and heated for 5 minutes at 95°C. The denatured protein samples were analyzed by Western blot. For Flag IP, cells lysates were incubated with 20 pl EZview Red ANTI-FLAG M2 Affinity Gel (Sigma Aldrich) and washed, denatured, and analyzed as described above.
Zinc -binding protein purified by Zn-NTA
[01001] To purify the Zinc -binding proteins, cells were lysed with IP lysis buffer (ThermoFisher Scientific, 87787) plus protease inhibitor. Protein concentration was determined with BCA protein assay kit (ThermoFisher Scientific, 23225). 500 pg protein samples were added to 1 ml of His Binding/Wash Buffer (G-biosciences, 786-542) containing 50 pl of Zinc Chelating Resin (G-biosciences, 786-287). The samples were rotated for 30 minutes at room temperature. The resin was pelleted by centrifugation at 500 xg for 5 minutes, followed by washing 3 times with 1 ml of His Binding/Wash Buffer and 5-minute rotation. Then, the resin was pelleted by centrifugation at 500 xg for 5 minutes. To elute the Zinc -binding protein, the resin was resuspended in 200 pl of His Elution Buffer (G-biosciences, 786-543) with 20-minute rotation. After pelleting the resin by centrifugation at 500 xg for 5 minutes, the supernatant was carefully collected and mixed with sample buffer (ThermoFisher Scientific) with P-ME and denatured at 95°C for 5 minutes. Adarl protein was detected by Western blotting.
Biotin pulldown assay with streptavidin beads
[01002] To purify the biotin-labeled proteins, pulldown assay was performed with Pierce™ Biotinylated Protein Interaction Pull-Down Kit (ThermoFisher Scientific, 21115) as per manufacturer protocols. Briefly, 200 pg of protein samples was loaded with 150 pL of Streptavidin Agarose Resin in binding buffer. After washing 3 times with wash buffer, resin-bound proteins were eluted with elution buffer and perform western blotting as above.
Protein palmitoylation detected by Click- IT assay
[01003] Cells were treated with different conditions, and 100 pM palmitic acid- azide (ThermoFisher Scientific, C 10265) was added to the cell medium with gentle mixing, then incubated at 37 °C and under 5% CO2 for 6 hours. Then, the medium was removed and lysis buffer (1% SDS in 50 mM Tris-HCl, pH 8.0) containing protease and phosphatase inhibitors at appropriate concentrations was added. Cell lysates were put on ice for 20 minutes, then pipetted into a 1.5 ml microcentrifuge tube. After centrifuging the cell lysate at 12,000xg at 4°C for 5 minutes, the supernatant was transferred to a clean tube and determined the protein concentration. Then, Biotin Alkyne solution (ThermoFisher Scientific, B 10185) was added directly to the Click-iT® reaction buffer (ThermoFisher Scientific, C10276) (Component A) for a final concentration of 40 pM. 200 pg protein samples were transferred in a volume of 50 pL of azide-labeled protein in 50 mM Tris-HCl, pH 8.0, containing up to 1% SDS, 100 pL of Click-IT reaction buffer, and 10 pL of molecular water in 1.5 mL Eppendorf tubes; and were vortexed for 5 seconds. 10 pL of CuSCE (Component B) was added and vortexed for 5 seconds. 10 pL of Click-IT® reaction buffer additive 1 solution was added and vortexed for 5 seconds. After 2-3 minutes of incubation, 20 pL of Click-IT® reaction buffer additive 2 solution was added and vortexed for 5 seconds. This solution turned bright orange. After Click-IT reaction, pulldown assay was performed as aforementioned to capture the palmitoylated proteins.
Immunofluorescence staining
[01004] B16 cells were mounted on cover slips and transfected with HA-tagged Zdhhc2 or
Flag-tagged Adarl for 48 hours. After washing twice with PBS, cells were fixed with 4% PFA for 15 minutes and washed 2 times with PBS for 5 minutes each. Then, the cells were permeabilized with 0.5% triton x-100 in PBS for 10 minutes and rinsed twice with PBS for 5 minutes each. Antigens were blocked with 10% normal goat serum in PBS for 30 minutes. Then, primary antibodies were added at 1:50 dilutions of anti-HA (Alexa Fluor 488, CST) and anti-Flag (Alexa Fluor 647, CST), and incubated at 4°C overnight. Then, the cells were washed and mounted on glass slides with ProLong Gold reagent containing DAPI. Confocal fluorescence images were taken using a 63X oil-immersion objective (Leica SP5 Inverted 2-Photon FLIM confocal).
Tumor infiltrating immune cell profiling
[01005] To quantify tumor infiltrating T cells and T cell effector gene expression, singlecell suspensions were prepared from fresh tumor tissues by physically passing through 100 pm cell strainers. Immune cells were enriched by density gradient centrifugation. For membrane and intracellular staining, tumor infiltrating immune cells were incubated in RPMI culture medium containing PMA (5 ng/ml), ionomycin (500 ng/ml), brefeldin A (1:1,000), and Monessen (1:1,000) at 37°C for 4 hours. 2-3 pl of anti-CD45 (30-F11, BD Biosciences), anti-CD90 (53-2.1, BD Biosciences), anti-CD3 (145-2C11, BD Biosciences), anti-CD4 (RM4-5, BD Biosciences), and anti-CD8 (53-6.7, BD Biosciences) antibodies were added for 20 minutes for surface staining. The cells were then washed and resuspended in 1 ml of freshly prepared Fix/Perm solution (BD Biosciences) at 4 °C overnight. After being washed with Perm/Wash buffer (BD Biosciences), the cells were stained with 2-3 pl anti-IFNy (XMG1.2, BD Biosciences), anti-TNFoc (MP6-XT22, BD Biosciences), and anti-granzyme B (GZMB) (GB11, BD Biosciences) for 30 minutes, washed, and fixed in 4% formaldehyde (Sigma Aldrich). All samples were read on an LSR Fortessa cytometer and analyzed with FACS DIVA software v. 8.0 (BD Biosciences).
Gene signature score computation
[01006] Normalized expression of genes was used to define the following signatures: CD8+ T cell infiltration (CD8A, CD8B, PRF1, and GZMB), MHC-I expression (HLA-A, HLA-B, and HLA-C), IFN signaling (STAT1, IRF1, GBP1, GBP2, GBP3, GBP4, GBP5, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, TAPI, B2M, CXCL9, CXCL10, and CXCL11), glucose metabolism (SLC2A1, PFKP, PKM, ENO1, GAPDH, PFKL, PFKM, HK1, HK2, and HK3), glutamine metabolism (GLS, GOT2, GLS2, GLUL, GLUD1, GLUD2, GPT2, and SLC1A5), and fatty acid metabolism (ACADM, ACADS, ACADVL, CPT1A, CPT1B, CPT2, ECHI, ECHS1, HMGCS1, and HMGCS2). Statistical analysis
[01007] Biological triplicates were performed for each cell-based experiment, unless otherwise stated. For animal studies, no less than 5 replicates per group were employed. Animals were randomized into different groups after tumor cell inoculation. The investigators were not blinded to allocation during experiments and outcome assessment. Data are shown as mean values with standard derivation. Statistical analysis was performed using GraphPad Prism8 software (GraphPad Software, Inc.). Two-tailed 2-sided t-test was used to compare treatment groups with control groups. Survival function was estimated by Kaplan-Meier methods and log-rank test was used to calculate statistical differences.
Data availability
[01008] The RNA-seq data (GSE) were deposited in Gene Expression Omnibus (GEO). The TCGA cancer datasets were obtained from UCSC Xena (http://xena.UCSC.edu/). The ChlP-seq datasets were obtained from UCSC ENCODE (https://genome.ucsc.edu/ENCODE/). The RNA-seq data and clinical information on patients receiving ICB were previously reported. Li et al., Cancer Cell 41, 304-322 e307 (2023).
EXAMPLE 68
IFNB1 qPCR Assay
[01009] B16 cells were seeded in 6 well plate at 150,000 cells/ well for 24 hours. 10 ng/ml of IFNg were added and incubate for 18 hours (Overnight). Without changing medium, Multiple doses of Neratinib or representative Compounds of the Disclosure were added and incubated for another 24 hours. Total RNA were prepared with Direct-zol RNA miniprep (ZYMO, R2051) and the RNA levels of Ifnbl were determined by qPCR. Gene expression was quantified using the primers listed as follows:
[01010] MS Actb QF - CACTGTCGAGTCGCGTCCA (SEQ ID NO. 1)
[01011] MS Actb QR - GACCCATTCCCACCATCACA (SEQ ID NO. 2)
[01012] MS Ifnbl QF - GCCTTTGCCATCCAAGAGATGC (SEQ ID NO. 3)
[01013] MS Ifnbl QR - ACACTGTCTGCTGGTGGAGTT (SEQ ID NO. 4)
[01014] The results are shown in Table 5. "N.T." means the compound was not tested. Table 5
EXAMPLE 69
Cell Viability Assay [01015] 104 of SKBR3 cells or 5000 of B16 cells/well were seeded in 96 well plate overnight. Then, multiple doses of Neratinib or representative Compounds of the Disclosure were added and incubated for 48 hours. To determine the effect of treatment on cell growth and viability, 10% volume of alamarBlue (Bio-Rad) was added directly into the medium and incubated for 4 hours. Absorbance at wavelengths of 570 and 600 nm was measured. Results were normalized to untreated controls and shown as relative cell viability (%). The results are shown in Table 6. "N.T." means the compound was not tested.
Table 6
EXAMPLE 70
Phospho-AKT Determination by Western Blotting
[01016] SKBR3 or B16 cells were seeded in 24 well plate at 5xl04 cells/ well for 24 hours. Neratinib and representative Compounds of the Disclosure were incubated for 1 hour. Cells were washed in cold PBS and lysed in 1 x RIPA lysis buffer (Pierce) with 1 x protease inhibitor (Pierce). Lysates were incubated on ice for 10 min and cleared by centrifugation at 15,000g for 15 minutes. Protein concentration was quantified using a BCA protein assay kit (Thermo Fisher). Thirty micrograms of protein was mixed with sample buffer (Thermo Fisher) with P-ME and denatured at 95°C for 5 minutes. Sample was separated by SDS-PAGE and transferred to a Nitrocellulose Membrane (Bio-Rad). Membranes were blocked with 5% w/v non-fat dry milk and incubated with primary antibodies overnight at 4 °C and HRP-conjugated secondary antibodies (CST) for 1 hour at room temperature. Signal was detected using Clarity and Clarity Max Western ECL Blotting Substrates (Bio-Rad) and captured using ChemiDoc Imaging System (Bio-Rad). Antibodies were as follows: anti-pAKT (CST, 4060, 1:1000) and anti-GAPDH (SCBT, sc-47724, 1:1000). The results are shown in Table 7. "N.T." means the compound was not tested.
Table 7
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[01067] Having now fully described the compounds, compositions, the methods herein, it will be understood by those of skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the methods, compounds, and compositions provided herein or any embodiment thereof. All patents, patent applications, and publications cited herein are fully incorporated by reference herein in their entirety.

Claims

What is claimed is:
1. A compound having Formula (IX): or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is selected from the group consisting of -O- and -NRa-;
Ra is selected from the group consisting to hydrogen and C1-C4 alkyl;
R1 is selected from the group consisting of -CH=CHRla and -C=CRlb;
Rla is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and -CH2NR8R9;
Rlb is selected from the group consisting of hydrogen and C1-C6 alkyl;
R8 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted 4-to 7-membered heterocyclo, C2-C12 alkynyl, and (C1-C6 alkoxy)C1-C6 alkyl;
R9 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; or
R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo;
R2 is selected from the group consisting of hydrogen, halo, C1-C6 alkyl, and -OR7;
R7 is selected from the group consisting of C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, (optionally substituted C3-C6 cycloalkyl)C1-C6-alkyl, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4-to 7-membered heterocyclo)C1-C6 alkyl;
R3 is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl;
R4 is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, and C3-C6 cycloalkyl; and
R5 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, optionally substituted aryl, (heterocyclo)alkyl, and optionally substituted 5- to 10- membered heteroaryl.
2. The compound claim 1, or a pharmaceutically acceptable salt or solvate thereof, with the following provisos:
(i) if R4 is cyano and R1 is -CH2NR8R9, then R8 is hydrogen, C2-C6 alkyl, C3-C6 cycloalkyl, optionally substituted 4-to 7-membered heterocyclo, C2-C12 alkynyl, and (Ci- Ce alkoxy )C1-C6 alkyl; R9 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; or R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo; or
(ii) if R1 is -CH2NR8R9 and R8 and R9 are each methyl, then R4 is selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, and C3-C6 cycloalkyl; or
(iii) if R1 is -CH2NR8R9, R8 and R9 are each methyl, and R4 is cyano, then R3 is C1-C6 alkyl or C3-C6 cycloalkyl.
3. The compound of claims 1 or 2 having Formula (X): (X), or a pharmaceutically acceptable salt or solvate thereof, wherein:
R6a is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl;
R6b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl; and
R6C is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl; C1-C4 haloalkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, (optionally substituted 4- to 7-membered heterocyclo)-O-,
(aryl)alkyl-O-, and (heteroaryl)alkyl-O-.
4. The compound of claim 3, or a pharmaceutically acceptable salt or solvate thereof, wherein:
R6b is hydrogen; and
R6C is selected from the group consisting of:
, or a pharmaceutically acceptable salt or solvate thereof, wherein R5a is optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl.
6. The compound of claim 5, or a pharmaceutically acceptable salt or solvate thereof, wherein R5a is optionally substituted 5-membered heteroaryl.
7. The compound of claim 5, or a pharmaceutically acceptable salt or solvate thereof, having Formula (XII): (XII), wherein R5b is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, and optionally substituted 5- or 6- membered heterocyclo.
8. The compound of claim 7, or a pharmaceutically acceptable salt or solvate thereof, having Formula (XIII): (XIII).
9. The compound of claim 7, or a pharmaceutically acceptable salt or solvate thereof, having Formula (XIV): (XIV).
10. The compound of claim 7, or a pharmaceutically acceptable salt or solvate thereof, having Formula (XV): (XV).
11. The compound of claims 1 or 2 having Formula (XVI): (XVI), or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is selected from the group consisting of optionally substituted 5- to 7-membered heterocylo and optionally substituted 5- to 10-membered heteroaryl.
12. The compound of claim 11, or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is optionally substituted 5- to 7-membered heterocylo.
13. The compound of claim 12, or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is selected from the group consisting of:
14. The compound of claim 11, or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is optionally substituted 5- to 10-membered heteroaryl.
15. The compound of claim 14, or a pharmaceutically acceptable salt or solvate thereof, wherein R5 is selected from the group consisting of:
16. The compound of any one of claims 1-15, or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is hydrogen.
17. The compound of any one of claims 1-15, or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is C1-C4 alkyl.
18. The compound of any one of claims 1-15, or a pharmaceutically acceptable salt or solvate thereof, wherein Rla is -CH2NR8R9.
19. The compound of claim 18, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 and R9 are C1-C6 alkyl.
20. The compound of claim 19, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 and R9 are methyl.
21. The compound of claim 18, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 is optionally substituted 4-to 7-membered heterocyclo.
22. The compound of claim 21, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 is selected from the group consisting of:
23. The compound of claim 18, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 is C2-C10 alkynyl.
24. The compound of claim 18, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 and R9 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclo.
25. The compound of claim 24, or a pharmaceutically acceptable salt or solvate thereof, wherein R8 and R9 taken together with the nitrogen atom to which they are attached form:
26. The compound of any one of claims 1-25, or a pharmaceutically acceptable salt or solvate thereof, wherein R2 is -OR7.
27. The compound of claim 26, or a pharmaceutically acceptable salt or solvate thereof, wherein R7 is selected from the group consisting of methyl, ethyl, and propyl.
28. The compound of claim 27, or a pharmaceutically acceptable salt or solvate thereof, wherein R7 is ethyl.
29. The compound of any one of claims 1-28, or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is selected from the group consisting of hydrogen and C1-C6 alkyl.
30. The compound of claim 29, or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is hydrogen.
31. The compound of claim 29, or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is methyl.
32. The compound of claim 29, or a pharmaceutically acceptable salt or solvate thereof, wherein R3 is ethyl.
33. The compound of any one of claims 1-32, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is selected from the group consisting of hydrogen and cyano.
34. The compound of claim 33, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is hydrogen.
35. The compound of claim 33, or a pharmaceutically acceptable salt or solvate thereof, wherein R4 is cyano.
36. The compound of any one of claims 3-10 or 15-35, or a pharmaceutically acceptable salt or solvate thereof, wherein R6a is selected from the group consisting of hydrogen and halogen.
37. The compound of claim 36, or a pharmaceutically acceptable salt or solvate thereof, wherein R6a is hydrogen.
38. The compound of claim 36, or a pharmaceutically acceptable salt or solvate thereof, wherein R6a is chloro.
39. The compound of any one of claims 1-38, or a pharmaceutically acceptable salt or solvate thereof, wherein X is -O-.
40. The compound of any one of claims 1-38, or a pharmaceutically acceptable salt or solvate thereof, wherein X is -NH-.
41 The compound of claim 1, or a pharmaceutically acceptable salt or solvate thereof, selected from any one or more of the compounds of Table 1 A and/or Table 2A.
42. The compound of claim 41, or a pharmaceutically acceptable salt or solvate thereof, selected from the group consisting of:
43. A pharmaceutical composition comprising the compound of any one of claims 1-42 and a pharmaceutically acceptable excipient.
44. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1-42 or the pharmaceutical composition of claim 32.
45. The method of claim 44 further comprising administering a therapeutically effective amount one or more immune checkpoint inhibitors to the subject.
46. The method of claim 44, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies, one or more anti-PD-Ll antibodies, one or more anti-CTLA-4 antibodies, one or more anti-LAG3 antibodies, one or more anti-TIM3 antibodies, one or more anti- VISTA antibodies, one or more anti- TIGIT antibodies, or one or more anti-cd47 antibodies, or a combination thereof.
47. The method of claim 46 wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-1 antibodies.
48. The method of claim 47, wherein the one or more anti-PD-1 antibodies comprise nivolumab, pembrolizumab, dostarlimab, retifanlimab, cemiplimab, vopratelimab (JTX-4014), spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), INCMGA00012 (MGA012), AMP-224, AMP-514 (MED 10680) and/or Acrixolimab (YBL-006).
49. The method any one of claims 46-48, wherein the one or more immune checkpoint inhibitors comprise one or more anti-PD-Ll antibodies.
50. The method of claim 49, wherein the one or more anti-PD-Ll antibodies comprise avelumab, atezolizumab, durvalumab, KN035, and/or cosibelimab (CK-301).
51. The method of any one of claims 46-50, wherein the one or more immune checkpoint inhibitors comprise one or more anti-CTLA-4 antibodies.
52. The method of claim 51, wherein the one or more anti-CTLA-4 antibodies comprise ipilimumab and/or tremelimumab.
53. The method of any one of claims 46-52, wherein the one or more immune checkpoint inhibitors comprise one or more anti-LAG3 antibodies.
54. The method of claim 53, wherein the one or more anti-LAG3 antibodies comprise relatlimab.
55. The method of any one of claims 46-54 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIM3 antibodies.
56. The method of any one of claims 46-55 wherein the one or more immune checkpoint inhibitors comprise one or more anti- VISTA antibodies.
57. The method of any one of claims 46-56 wherein the one or more immune checkpoint inhibitors comprise one or more anti-TIGIT antibodies.
58. The method of any one of claims 46-57 wherein the one or more immune checkpoint inhibitors comprise one or more anti-cd47 antibodies.
59. The method of claim 45 comprising administering a therapeutically effective amount of nivolumab, pembrolizumab, dostarlimab, retifanlimab, or cemiplimab to the subject.
60. The method of claims 45 or 59 comprising administering a therapeutically effective amount of avelumab, atezolizumab, or durvalumab to the subject.
61. The method of any one of claims 44-60, wherein the cancer is any one or more of the cancers of Table 2 and/or Table 3.
62. A method of inhibiting adenosine deaminase acting on RNA 1 (ADAR1) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1-42, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of claim 43.
63. A method of providing immuno-oncology therapy to a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1-42, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of claim 43.
64. A method of sensitizing tumor response to immunotherapy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1-42, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of claim 43.
65. The method of claim 64, wherein the tumor is resistant to immune checkpoint blockade.
66. The method of any one of claims 62-65 comprising administering to the subject a therapeutically effective amount of Neratinib:
67. A kit for carrying out the method of any one of claims 44-66, the kit comprising: (i) a compound having Formula (IX), or a pharmaceutically acceptable salt or solvate thereof; and (ii) a label with instructions for how to use the kit.
68. A kit for carrying out the method of any one of claims 44-66, the kit comprising: (i) one or more immune checkpoint inhibitors; and (ii) a label with instructions for how to use the kit.
69. A kit for carrying out the method of any one of claims 44-66, the kit comprising: (i) a compound having Formula (IX), or a pharmaceutically acceptable salt or solvate thereof; (ii) one or more immune checkpoint inhibitors; and (iii) a label with instructions for how to use the kit.
70. The kit of any one of claims 67-69, wherein the label is approved by the United States Food and Drug Administration (FDA), the European Medicines Agency (EMA), the China Food and Drug Administration (CFDA), or the Japanese Ministry of Health Labor and Welfare (MHLW).
71. A compound having Formula (VIII): or a pharmaceutically acceptable salt or solvate thereof, wherein:
R10 is selected from the group consisting of chloro and -N(H)R5;
R11 is selected from the group consisting of hydrogen and -C(=O)CH3; R2 is selected from the group consisting of hydrogen, halo, C1-C6 alkyl, and -OR7;
R7 is selected from the group consisting of C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, (optionally substituted C3-C6 cycloalkyl)C1-C6-alkyl, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4-to 7-membered heterocyclo)C1-C6 alkyl;
R3 is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl;
R4 is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, and C3-C6 cycloalkyl; and
R5 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, optionally substituted aryl, (heterocyclo)alkyl, and optionally substituted 5- to 10- membered heteroaryl.
72. A compound having Formula (XVIII): (XVIII), or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is selected from the group consisting of -O- and -NRa-;
Ra is selected from the group consisting to hydrogen and C1-C4 alkyl;
LG is a leaving group;
R2 is selected from the group consisting of hydrogen, halo, C1-C6 alkyl, and -OR7;
R7 is selected from the group consisting of C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, (optionally substituted C3-C6 cycloalkyl)C1-C6-alkyl, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted 4- to 7-membered heterocyclo, and (optionally substituted 4-to 7-membered heterocyclo)C1-C6 alkyl;
R3 is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl;
R4 is selected from the group consisting of hydrogen, halogen, cyano, C1-C6 alkyl, and C3-C6 cycloalkyl; and R5 is selected from the group consisting of hydrogen, C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted 4- to 7-membered heterocyclo, optionally substituted aryl, (heterocyclo)alkyl, and optionally substituted 5- to 10- membered heteroaryl.
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