COMPOUNDS AND METHODS FOR MODULATING SPLICING

The present disclosure features compounds and related compositions that, inter alia, modulate nucleic acid splicing, e.g., splicing of a pre-mRNA, as well as methods of use thereof.

BACKGROUND

Alternative splicing is a major source of protein diversity in higher eukaryotes and is frequently regulated in a tissue-specific or development stage-specific manner. Disease associated alternative splicing patterns in pre-mRNAs are often mapped to changes in splice site signals or sequence motifs and regulatory splicing factors (Faustino and Cooper (2003), Genes Dev 17(4):419-37). Current therapies to modulate RNA expression involve oligonucleotide targeting and gene therapy; however, each of these modalities exhibit unique challenges as currently presented. As such, there is a need for new technologies to modulate RNA expression, including the development of small molecule compounds that target splicing.

SUMMARY

The present disclosure features compounds and related compositions that, inter alia, modulate nucleic acid splicing, e.g., splicing of a pre-mRNA, as well as methods of use thereof. In an embodiment, the compounds described herein are compounds of Formula (I) or (II) (e.g., a compound of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m)) and pharmaceutically acceptable salts, solvates, hydrates, tautomers, or stereoisomers thereof. The present disclosure additionally provides methods of using the compounds of the invention (e.g., compounds of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m), and pharmaceutically acceptable salts, solvates, hydrates, tautomers, stereoisomers thereof), and compositions thereof, e.g., to target, and in embodiments bind or form a complex with, a nucleic acid (e.g., a pre-mRNA or nucleic acid component of a small nuclear ribonucleoprotein (snRNP) or spliceosome), a protein (e.g., a protein component of an snRNP or spliceosome, e.g., a member of the splicing machinery, e.g., one or more of the U1, U2, U4, U5, U6, U11, U12, U4atac, U6atac snRNPs), or a combination thereof. In another aspect, the compounds described herein may be used to alter the composition or structure of a nucleic acid (e.g., a pre-mRNA or mRNA (e.g., a pre-mRNA and the mRNA which arises from the pre-mRNA), e.g., by increasing or decreasing splicing at a splice site. In some embodiments, increasing or decreasing splicing results in modulating the level of a gene product (e.g., an RNA or protein) produced.

In another aspect, the compounds described herein may be used for the prevention and/or treatment of a disease, disorder, or condition, e.g., a disease, disorder or condition associated with splicing, e.g., alternative splicing. In some embodiments, the compounds described herein (e.g., compounds of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m), and pharmaceutically acceptable salts, solvates, hydrates, tautomers, stereoisomers thereof) and compositions thereof are used for the prevention and/or treatment of a proliferative disease, disorder, or condition (e.g., a disease, disorder, or condition characterized by unwanted cell proliferation, e.g., a cancer or a benign neoplasm) in a subject. In some embodiments, the compounds described herein (e.g., compounds of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m), and pharmaceutically acceptable salts, solvates, hydrates, tautomers, stereoisomers thereof) and compositions thereof are used for the prevention and/or treatment of a non-proliferative disease, disorder, or condition. In some embodiments, the compounds described herein (e.g., compounds of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m), and pharmaceutically acceptable salts, solvates, hydrates, tautomers, stereoisomers thereof) and compositions thereof are used for the prevention and/or treatment of a neurological disease or disorder, an autoimmune disease or disorder, immunodeficiency disease or disorder, a lysosomal storage disease or disorder, a cardiovascular disease or disorder, a metabolic disease or disorder, a respiratory disease or disorder, a renal disease or disorder, or an infectious disease in a subject.

In another aspect, the present disclosure features a compound of Formula (I):

In another aspect, the present disclosure features a compound of Formula (II):

In another aspect, the present disclosure provides methods for modulating splicing, e.g., splicing of a nucleic acid (e.g., a DNA or RNA, e.g., a pre-mRNA) with a compound of Formula (I) or (II) (e.g., a compound of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m)) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof. In another aspect, the present disclosure provides compositions for use in modulating splicing, e.g., splicing of a nucleic acid (e.g., a DNA or RNA, e.g., a pre-mRNA) with a compound of Formula (I) or (II) (e.g., a compound of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m)) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof. Modulation of splicing may comprise impacting any step involved in splicing and may include an event upstream or downstream of a splicing event. For example, in some embodiments, the compound of Formula (I) or (II) binds to a target, e.g., a target nucleic acid (e.g., DNA or RNA, e.g., a precursor RNA, e.g., a pre-mRNA), a target protein, or combination thereof (e.g., an snRNP and a pre-mRNA). A target may include a splice site in a pre-mRNA or a component of the splicing machinery, such as the U1 snRNP. In some embodiments, the compound of Formula (I) or (II) alters a target nucleic acid (e.g., DNA or RNA, e.g., a precursor RNA, e.g., a pre-mRNA), target protein, or combination thereof. In some embodiments, the compound of Formula (I) or (II) increases or decreases splicing at a splice site on a target nucleic acid (e.g., an RNA, e.g., a precursor RNA, e.g., a pre-mRNA) by about 0.5% or more (e.g., about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more), relative to a reference (e.g., the absence of a compound of Formula (I) or (II), e.g., in a healthy or diseased cell or tissue). In some embodiments, the presence of a compound of Formula (I) or (II) results an increase or decrease of transcription of a target nucleic acid (e.g., an RNA) by about 0.5% or more (e.g., about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more), relative to a reference (e.g., the absence of a compound of Formula (I) or (II), e.g., in a healthy or diseased cell or tissue).

In another aspect, the present disclosure provides methods for preventing and/or treating a disease, disorder, or condition in a subject by administering a compound of Formula (I) or (II) (e.g., a compound of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m)) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or related compositions. In some embodiments, the disease or disorder entails unwanted or aberrant splicing. In some embodiments, the disease or disorder is a proliferative disease, disorder, or condition. Exemplary proliferative diseases include cancer, a benign neoplasm, or angiogenesis. In other embodiments, the present disclosure provides methods for treating and/or preventing a non-proliferative disease, disorder, or condition. In still other embodiments, the present disclosure provides methods for treating and/or preventing a neurological disease or disorder, autoimmune disease or disorder, immunodeficiency disease or disorder, lysosomal storage disease or disorder, cardiovascular disease or disorder, metabolic disease or disorder, respiratory disease or disorder, renal disease or disorder, or infectious disease.

In another aspect, the present disclosure provides methods of down-regulating the expression of (e.g., the level of or the rate of production of) a target protein with a compound of Formula (I) or (II) (e.g., a compound of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m)) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof in a biological sample or subject. In another aspect, the present disclosure provides methods of up-regulating the expression of (e.g., the level of or the rate of production of) a target protein with a compound of Formula (I) or (II) (e.g., a compound of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m)) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof in a biological sample or subject. In another aspect, the present disclosure provides methods of altering the isoform of a target protein with a compound of Formula (I) or (II) (e.g., a compound of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m))) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof in a biological sample or subject. Another aspect of the disclosure relates to methods of inhibiting the activity of a target protein in a biological sample or subject. In some embodiments, administration of a compound of Formula (I) or (II) to a biological sample, a cell, or a subject comprises inhibition of cell growth or induction of cell death.

In another aspect, the present disclosure provides compositions for use in preventing and/or treating a disease, disorder, or condition in a subject by administering a compound of Formula (I) or (II) (e.g., a compound of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m)) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or related compositions. In some embodiments, the disease or disorder entails unwanted or aberrant splicing. In some embodiments, the disease or disorder is a proliferative disease, disorder, or condition. Exemplary proliferative diseases include cancer, a benign neoplasm, or angiogenesis. In other embodiments, the present disclosure provides methods for treating and/or preventing a non-proliferative disease, disorder, or condition. In still other embodiments, the present disclosure provides methods for treating and/or preventing a neurological disease or disorder, autoimmune disease or disorder, immunodeficiency disease or disorder, lysosomal storage disease or disorder, cardiovascular disease or disorder, metabolic disease or disorder, respiratory disease or disorder, renal disease or disorder, or infectious disease.

In another aspect, the present disclosure provides compositions for use in down-regulating the expression of (e.g., the level of or the rate of production of) a target protein with a compound of Formula (I) or (II) (e.g., a compound of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m)) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof in a biological sample or subject. In another aspect, the present disclosure provides compositions for use in up-regulating the expression of (e.g., the level of or the rate of production of) a target protein with a compound of Formula (I) or (II) (e.g., a compound of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m)) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof in a biological sample or subject. In another aspect, the present disclosure provides compositions for use in altering the isoform of a target protein with a compound of Formula (I) or (II) (e.g., a compound of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m))) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof in a biological sample or subject. Another aspect of the disclosure relates to compositions for use in inhibiting the activity of a target protein in a biological sample or subject. In some embodiments, administration of a compound of Formula (I) or (II) to a biological sample, a cell, or a subject comprises inhibition of cell growth or induction of cell death.

In another aspect, the present disclosure features kits comprising a container with a compound of Formula (I) or (II) (e.g., a compound of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), or (II-m)), or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer thereof, or a pharmaceutical composition thereof. In certain embodiments, the kits described herein further include instructions for administering the compound of Formula (I) or (II) or the pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer thereof, or the pharmaceutical composition thereof.

In any and all aspects of the present disclosure, in some embodiments, the compound, target nucleic acid (e.g., DNA, RNA, e.g., pre-mRNA), or target protein described herein is a compound, target nucleic acid (e.g., DNA, RNA, e.g., pre-mRNA), or target protein other than a compound, target nucleic acid (e.g., DNA, RNA, e.g., pre-mRNA), or target protein described one of U.S. Pat. No. 8,729,263, U.S. Publication No. 2015/0005289, WO 2014/028459, WO 2016/128343, WO 2016/196386, WO 2017/100726, WO 2018/232039, WO 2018/098446, WO 2019/028440, WO 2019/060917, WO 2019/199972, and WO 2021/174165. In some embodiments, the compound, target nucleic acid (e.g., DNA, RNA, e.g., pre-mRNA), or target protein described herein is a compound, target nucleic acid (e.g., DNA, RNA, e.g., pre-mRNA), or target protein described one of U.S. Pat. No. 8,729,263, U.S. Publication No. 2015/0005289, WO 2014/028459, WO 2016/128343, WO 2016/196386, WO 2017/100726, WO 2018/232039, WO 2018/098446, WO 2019/028440, WO 2019/060917, WO 2019/199972, and and WO 2021/174165, each of which is incorporated herein by reference in its entirety. The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Examples, and the Claims.

DETAILED DESCRIPTION

Selected Chemical Definitions

The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the present invention. As used herein, “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 24 carbon atoms (“C1-C24 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1-C12 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1-C8 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1-C6 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-C6 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1 alkyl”). Examples of C1-C6alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. Each instance of an alkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents; e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In certain embodiments, the alkyl group is unsubstituted C1-C10 alkyl (e.g., —CH3). In certain embodiments, the alkyl group is substituted C1-C6 alkyl.

As used herein, “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (“C2-C24 alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2-C10 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2-C8 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2-C6 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-C4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Each instance of an alkenyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In certain embodiments, the alkenyl group is unsubstituted C1-C10 alkenyl. In certain embodiments, the alkenyl group is substituted C2-C6 alkenyl.

As used herein, the term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon triple bonds (“C2-C24 alkenyl”). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2-C10 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-C8 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-C6 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-C4 alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Each instance of an alkynyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In certain embodiments, the alkynyl group is unsubstituted C2-10 alkynyl. In certain embodiments, the alkynyl group is substituted C2-6 alkynyl.

As used herein, the term “haloalkyl,” refers to a non-cyclic stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one halogen selected from the group consisting of F, Cl, Br, and I. The halogen(s) F, Cl, Br, and I may be placed at any position of the haloalkyl group. Exemplary haloalkyl groups include, but are not limited to: —CF3, —CCl3, —CH2—CF3, —CH2—CCl3, —CH2—CBr3, —CH2—CI3, —CH2—CH2—CH(CF3)—CH3, —CH2—CH2—CH(Br)—CH3, and —CH2—CH═CH—CH2—CF3. Each instance of a haloalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted haloalkyl”) or substituted (a “substituted haloalkyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent

As used herein, the term “heteroalkyl,” refers to a non-cyclic stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any position of the heteroalkyl group. Exemplary heteroalkyl groups include, but are not limited to: —CH2—CH2—O—CH3, —CH2—CH2—NH—CH3, —CH2—CH2—N(CH3)—CH3, —CH2—S—CH2—CH3, —CH2—CH2, —S(O)—CH3, —CH2—CH2—S(O)2—CH3, —CH═CHO—CH3, —Si(CH3)3, —CH2—CH═N—OCH3, —CH═CH—N(CH3)—CH3, —O—CH3, and —O—CH2—CH3. Up to two or three heteroatoms may be consecutive, such as, for example, —CH2—NH—OCH3 and —CH2—O—Si(CH3)3. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as —CH2O, —NRCRD, or the like, it will be understood that the terms heteroalkyl and —CH2O or —NRCRD are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as —CH2O, —NRCRD, or the like. Each instance of a heteroalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent

As used herein, “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracyl). An aryl group may be described as, e.g., a C6-C10-membered aryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of an aryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is unsubstituted C6-C14 aryl. In certain embodiments, the aryl group is substituted C6-C14 aryl.

As used herein, “heteroaryl” refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). A heteroaryl group may be described as, e.g., a 6-10-membered heteroaryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Each instance of a heteroaryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent

As used herein, “cycloalkyl” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3-C10 cycloalkyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-C8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-C6 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-C6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-C10 cycloalkyl”). A cycloalkyl group may be described as, e.g., a C4-C7-membered cycloalkyl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Exemplary C3-C6 cycloalkyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-C8 cycloalkyl groups include, without limitation, the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C8), bicyclo[1.1.1]pentanyl (C5), bicyclo[2.2.2]octanyl (C8), bicyclo[2.1.1]hexanyl (C6), bicyclo[3.1.1]heptanyl (C7), and the like. Exemplary C3-C10 cycloalkyl groups include, without limitation, the aforementioned C3-C8 cycloalkyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the cycloalkyl group is either monocyclic (“monocyclic cycloalkyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic cycloalkyl”) and can be saturated or can be partially unsaturated. “Cycloalkyl” also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, the cycloalkyl group is unsubstituted C3-C10 cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C3-C10 cycloalkyl.

“Heterocyclyl” as used herein refers to a radical of a 3- to 16-membered non-aromatic ring system having ring carbon atoms and 1 to 8 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-16 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described as, e.g., a 3-7-membered heterocyclyl, wherein the term “membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, within the moiety. Each instance of heterocyclyl may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is unsubstituted 3-16 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-16 membered heterocyclyl.

The terms “alkylene,” “alkenylene,” “alkynylene,” “haloalkylene,” “heteroalkylene,” “cycloalkylene,” or “heterocyclylene,” alone or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl, haloalkylene, heteroalkylene, cycloalkyl, or heterocyclyl respectively. For example, the term “alkenylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene. An alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, or heterocyclylene group may be described as, e.g., a C1-C6-membered alkylene, C2-C6-membered alkenylene, C2-C6-membered alkynylene, C1-C6-membered haloalkylene, C1-C6-membered heteroalkylene, C3-C8-membered cycloalkylene, or C3-C8-membered heterocyclylene, wherein the term “membered” refers to the non-hydrogen atoms within the moiety. In the case of heteroalkylene and heterocyclylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula —C(O)2R′— may represent both —C(O)2R′— and —R′C(O)2—.

As used herein, the terms “cyano” or “—CN” refer to a substituent having a carbon atom joined to a nitrogen atom by a triple bond, e.g., C≡N.

As used herein, the terms “halogen” or “halo” refer to fluorine, chlorine, bromine or iodine.

As used herein, the term “hydroxy” refers to —OH.

As used herein, the term “nitro” refers to a substitutent having two oxygen atoms bound to a nitrogen atom, e.g., —NO2.

As used herein, the term “nucleobase” as used herein, is a nitrogen-containing biological compounds found linked to a sugar within a nucleoside—the basic building blocks of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The primary, or naturally occurring, nucleobases are cytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA), thymine (DNA) and uracil (RNA), abbreviated as C, G, A, T, and U, respectively. Because A, G, C, and T appear in the DNA, these molecules are called DNA-bases; A, G, C, and U are called RNA-bases. Adenine and guanine belong to the double-ringed class of molecules called purines (abbreviated as R). Cytosine, thymine, and uracil are all pyrimidines. Other nucleobases that do not function as normal parts of the genetic code, are termed non-naturally occurring. In an embodiment, a nucleobase may be chemically modified, for example, with an alkyl (e.g., methyl), halo, —O-alkyl, or other modification.

As used herein, the term “nucleic acid” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. The term “nucleic acid” includes a gene, cDNA, pre-mRNA, or an mRNA. In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized) or recombinant. Unless specifically limited, the term encompasses nucleic acids containing analogues or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementarity sequences as well as the sequence explicitly indicated.

As used herein “oxo” refers to a carbonyl, i.e., —C(O)—.

The symbol “” as used herein in relation to a compound of Formula (I) or (II) refers to an attachment point to another moiety or functional group within the compound. Alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, such as any of the substituents described herein that result in the formation of a stable compound. The present disclosure contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.

As used herein, a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, an “S” form of the compound is substantially free from the “R” form of the compound and is, thus, in enantiomeric excess of the “R” form. The term “enantiomerically pure” or “pure enantiomer” denotes that the compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight, or more than 99.9% by weight, of the enantiomer. In certain embodiments, the weights are based upon total weight of all enantiomers or stereoisomers of the compound.

In the compositions provided herein, an enantiomerically pure compound can be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising an enantiomerically pure R-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure R-compound. In certain embodiments, the enantiomerically pure R-compound in such compositions can, for example, comprise, at least about 95% by weight R-compound and at most about 5% by weight S-compound, by total weight of the compound. For example, a pharmaceutical composition comprising an enantiomerically pure S-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure S-compound. In certain embodiments, the enantiomerically pure S-compound in such compositions can, for example, comprise, at least about 95% by weight S-compound and at most about 5% by weight R-compound, by total weight of the compound.

In some embodiments, a diastereomerically pure compound can be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising a diastereometerically pure exo compound can comprise, for example, about 90% excipient and about 10% diastereometerically pure exo compound. In certain embodiments, the diastereometerically pure exo compound in such compositions can, for example, comprise, at least about 95% by weight exo compound and at most about 5% by weight endo compound, by total weight of the compound. For example, a pharmaceutical composition comprising a diastereometerically pure endo compound can comprise, for example, about 90% excipient and about 10% diastereometerically pure endo compound. In certain embodiments, the diastereometerically pure endo compound in such compositions can, for example, comprise, at least about 95% by weight endo compound and at most about 5% by weight exo compound, by total weight of the compound.

In some embodiments, an isomerically pure compound can be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising a isomerically pure exo compound can comprise, for example, about 90% excipient and about 10% isomerically pure exo compound. In certain embodiments, the isomerically pure exo compound in such compositions can, for example, comprise, at least about 95% by weight exo compound and at most about 5% by weight endo compound, by total weight of the compound. For example, a pharmaceutical composition comprising an isomerically pure endo compound can comprise, for example, about 90% excipient and about 10% isomerically pure endo compound. In certain embodiments, the isomerically pure endo compound in such compositions can, for example, comprise, at least about 95% by weight endo compound and at most about 5% by weight exo compound, by total weight of the compound.

In certain embodiments, the active ingredient can be formulated with little or no excipient or carrier.

Compound described herein may also comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D or deuterium), and 3H (T or tritium); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; N may be in any isotopic form, including 14N and 15N; F may be in any isotopic form, including 18F, 19F, and the like.

Other Definitions

The following definitions are more general terms used throughout the present disclosure. The articles “a” and “an” refer to one or more than one (e.g., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. The term “and/or” means either “and” or “or” unless indicated otherwise.

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

“Acquire” or “acquiring” as used herein, refer to obtaining possession of a value, e.g., a numerical value, or image, or a physical entity (e.g., a sample), by “directly acquiring” or “indirectly acquiring” the value or physical entity. “Directly acquiring” means performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity. “Indirectly acquiring” refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value). Directly acquiring a value or physical entity includes performing a process that includes a physical change in a physical substance or the use of a machine or device. Examples of directly acquiring a value include obtaining a sample from a human subject. Directly acquiring a value includes performing a process that uses a machine or device, e.g., mass spectrometer to acquire mass spectrometry data.

The terms “administer,” “administering,” or “administration,” as used herein refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing an inventive compound, or a pharmaceutical composition thereof.

As used herein, the terms “condition,” “disease,” and “disorder” are used interchangeably.

An “effective amount” of a compound of Formula (I) or (II) refers to an amount sufficient to elicit the desired biological response, i.e., treating the condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound of Formula (I) or (II) may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment. For example, in treating cancer, an effective amount of an inventive compound may reduce the tumor burden or stop the growth or spread of a tumor.

A “therapeutically effective amount” of a compound of Formula (I) or (II) is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. In some embodiments, a therapeutically effective amount is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition, or enhances the therapeutic efficacy of another therapeutic agent.

The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprised therein. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.

“Prevention,” “prevent,” and “preventing” as used herein refers to a treatment that comprises administering a therapy, e.g., administering a compound described herein (e.g., a compound of Formula (I) or (II)) prior to the onset of a disease, disorder, or condition in order to preclude the physical manifestation of said disease, disorder, or condition. In some embodiments, “prevention,” “prevent,” and “preventing” require that signs or symptoms of the disease, disorder, or condition have not yet developed or have not yet been observed. In some embodiments, treatment comprises prevention and in other embodiments it does not.

A “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and/or other non-human animals, for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys). In certain embodiments, the animal is a mammal. The animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal.

As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of one or more of a symptom, manifestation, or underlying cause of a disease, disorder, or condition (e.g., as described herein), e.g., by administering a therapy, e.g., administering a compound described herein (e.g., a compound of Formula (I) or (II)). In an embodiment, treating comprises reducing, reversing, alleviating, delaying the onset of, or inhibiting the progress of a symptom of a disease, disorder, or condition. In an embodiment, treating comprises reducing, reversing, alleviating, delaying the onset of, or inhibiting the progress of a manifestation of a disease, disorder, or condition. In an embodiment, treating comprises reducing, reversing, alleviating, reducing, or delaying the onset of, an underlying cause of a disease, disorder, or condition. In some embodiments, “treatment,” “treat,” and “treating” require that signs or symptoms of the disease, disorder, or condition have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition, e.g., in preventive treatment. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. In some embodiments, treatment comprises prevention and in other embodiments it does not.

A “proliferative disease” refers to a disease that occurs due to abnormal extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990). A proliferative disease may be associated with: 1) the pathological proliferation of normally quiescent cells; 2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); 4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis; or 5) evasion of host immune surveillance and elimination of neoplastic cells. Exemplary proliferative diseases include cancers (i.e., “malignant neoplasms”), benign neoplasms, and angiogenesis.

A “non-proliferative disease” refers to a disease that does not primarily extend through the abnormal multiplication of cells. A non-proliferative disease may be associated with any cell type or tissue type in a subject. Exemplary non-proliferative diseases include neurological diseases or disorders (e.g., a repeat expansion disease); autoimmune disease or disorders; immunodeficiency diseases or disorders; lysosomal storage diseases or disorders; inflammatory diseases or disorders; cardiovascular conditions, diseases, or disorders; metabolic diseases or disorders; respiratory conditions, diseases, or disorders; renal diseases or disorders; and infectious diseases.

Compounds

The present disclosure features a compound of Formula (I):

In another aspect, the present disclosure features a compound of Formula (II):

In some embodiments, each of A and B are independently a monocyclic ring, e.g., monocyclic cycloalkyl, monocyclic heterocyclyl, monocyclic aryl, or monocyclic heteroaryl. The monocyclic ring may be saturated, partially unsaturated, or fully unsaturated (e.g., aromatic). In some embodiments, A or B are independently a monocyclic ring comprising between 3 and 10 ring atoms (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 ring atoms). In some embodiments, A is a 4-membered monocyclic ring. In some embodiments, B is a 4-membered monocyclic ring. In some embodiments, A is a 5-membered monocyclic ring. In some embodiments, B is a 5-membered monocyclic ring. In some embodiments, A is a 6-membered monocyclic ring. In some embodiments, B is a 6-membered monocyclic ring. In some embodiments, A is a 7-membered monocyclic ring. In some embodiments, B is a 7-membered monocyclic ring. In some embodiments, A is an 8-membered monocyclic ring. In some embodiments, B is an 8-membered monocyclic ring. In some embodiments, A or B are independently a monocyclic ring optionally substituted with one or more R1.

In some embodiments, A or B are independently a bicyclic ring, e.g., bicyclic cycloalkyl, bicyclic heterocyclyl, bicyclic aryl, or bicyclic heteroaryl. The bicyclic ring may be saturated, partially unsaturated, or fully unsaturated (e.g., aromatic). In some embodiments, A or B are independently a bicyclic ring comprising a fused, bridged, or spiro ring system. In some embodiments, A or B are independently a bicyclic ring comprising between 4 and 18 ring atoms (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 ring atoms). In some embodiments, A is a 6-membered bicyclic ring. In some embodiments, B is a 6-membered bicyclic ring. In some embodiments, A is a 7-membered bicyclic ring. In some embodiments, B is a 7-membered bicyclic ring. In some embodiments, A is an 8-membered bicyclic ring. In some embodiments, B is an 8-membered bicyclic ring. In some embodiments, A is a 9-membered bicyclic ring. In some embodiments, B is a 9-membered bicyclic ring. In some embodiments, A is a 10-membered bicyclic ring. In some embodiments, B is a 10-membered bicyclic ring. In some embodiments, A is an 11-membered bicyclic ring. In some embodiments, B is an 11-membered bicyclic ring. In some embodiments, A is a 12-membered bicyclic ring. In some embodiments, B is a 12-membered bicyclic ring. In some embodiments, A or B are independently a bicyclic ring optionally substituted with one or more R1.

In some embodiments, A or B are independently a tricyclic ring, e.g., tricyclic cycloalkyl, tricyclic heterocyclyl, tricyclic aryl, or tricyclic heteroaryl. The tricyclic ring may be saturated, partially unsaturated, or fully unsaturated (e.g., aromatic). In some embodiments, A or B are independently a tricyclic ring that comprises a fused, bridged, or spiro ring system, or a combination thereof. In some embodiments, A or B are independently a tricyclic ring comprising between 6 and 24 ring atoms (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 ring atoms). In some embodiments, A is an 8-membered tricyclic ring. In some embodiments, B is an 8-membered tricyclic ring. In some embodiments, A is a 9-membered tricyclic ring. In some embodiments, B is a 9-membered tricyclic ring. In some embodiments, A is a 10-membered tricyclic ring. In some embodiments, B is a 10-membered tricyclic ring. In some embodiments, A or B are independently a tricyclic ring optionally substituted with one or more R1.

In some embodiments, A or B are independently monocyclic cycloalkyl, monocyclic heterocyclyl, monocyclic aryl, or monocyclic heteroaryl. In some embodiments, A or B are independently bicyclic cycloalkyl, bicyclic heterocyclyl, bicyclic aryl, or bicyclic heteroaryl. In some embodiments, A or B are independently tricyclic cycloalkyl, tricyclic heterocyclyl, tricyclic aryl, or tricyclic heteroaryl. In some embodiments, A is monocyclic heterocyclyl. In some embodiments, B is monocyclic heterocyclyl. In some embodiments, A is bicyclic heterocyclyl. In some embodiments, B is bicyclic heterocyclyl. In some embodiments, A is monocyclic heteroaryl. In some embodiments, B is monocyclic heteroaryl. In some embodiments, A is bicyclic heteroaryl. In some embodiments, B is bicyclic heteroaryl.

In some embodiments, A or B are independently a nitrogen-containing heterocyclyl, e.g., heterocyclyl comprising one or more nitrogen atom. The one or more nitrogen atom of the nitrogen-containing heterocyclyl may be at any position of the ring. In some embodiments, the nitrogen-containing heterocyclyl is monocyclic, bicyclic, or tricyclic. In some embodiments, A or B are independently heterocyclyl comprising at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 nitrogen atoms. In some embodiments, A is heterocyclyl comprising 1 nitrogen atom. In some embodiments, B is heterocyclyl comprising 1 nitrogen atom. In some embodiments, A is heterocyclyl comprising 2 nitrogen atoms. In some embodiments, B is heterocyclyl comprising 2 nitrogen atoms. In some embodiments, A is heterocyclyl comprising 3 nitrogen atoms. In some embodiments, B is heterocyclyl comprising 3 nitrogen atoms. In some embodiments, A is heterocyclyl comprising 4 nitrogen atoms. In some embodiments, B is heterocyclyl comprising 4 nitrogen atoms. In some embodiments, A or B are independently a nitrogen-containing heterocyclyl comprising one or more additional heteroatoms, e.g., one or more of oxygen, sulfur, boron, silicon, or phosphorus. In some embodiments, the one or more nitrogen of the nitrogen-containing heterocyclyl is substituted, e.g., with R1.

In some embodiments, A or B are independently a nitrogen-containing heteroaryl, e.g., heteroaryl comprising one or more nitrogen atom. The one or more nitrogen atom of the nitrogen-containing heteroaryl may be at any position of the ring. In some embodiments, the nitrogen-containing heteroaryl is monocyclic, bicyclic, or tricyclic. In some embodiments, A or B are independently heteroaryl comprising at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 nitrogen atoms. In some embodiments, A is heteroaryl comprising 1 nitrogen atom. In some embodiments, B is heteroaryl comprising 1 nitrogen atom. In some embodiments, A is heteroaryl comprising 2 nitrogen atoms. In some embodiments, B is heteroaryl comprising 2 nitrogen atoms. In some embodiments, A is heteroaryl comprising 3 nitrogen atoms. In some embodiments, B is heteroaryl comprising 3 nitrogen atoms. In some embodiments, A is heteroaryl comprising 4 nitrogen atoms. In some embodiments, B is heteroaryl comprising 4 nitrogen atoms. In some embodiments, A or B are independently a nitrogen-containing heteroaryl comprising one or more additional heteroatoms, e.g., one or more of oxygen, sulfur, boron, silicon, or phosphorus. In some embodiments, the one or more nitrogen of the nitrogen-containing heteroaryl is substituted, e.g., with R1.

In some embodiments, A is a 6-membered nitrogen-containing heterocyclyl, e.g., a 6-membered heterocyclyl comprising one or more nitrogen. In some embodiments, A is a 6-membered heterocyclyl comprising 1 nitrogen atom. In some embodiments, A is a 6-membered heterocyclyl comprising 2 nitrogen atoms. In some embodiments, A is a 6-membered heterocyclyl comprising 3 nitrogen atoms. In some embodiments, A is a 6-membered heterocyclyl comprising 4 nitrogen atoms. The one or more nitrogen atom of the 6-membered nitrogen-containing heterocyclyl may be at any position of the ring. In some embodiments, A is a 6-membered nitrogen-containing heterocyclyl optionally substituted with one or more R1. In some embodiments, the one or more nitrogen of the 6-membered nitrogen-containing heterocyclyl is substituted, e.g., with R1. In some embodiments, A is a 6-membered nitrogen-containing heterocyclyl comprising one or more additional heteroatoms, e.g., one or more of oxygen, sulfur, boron, silicon, or phosphorus.

In some embodiments, B is a 5-membered nitrogen-containing heterocyclyl or heteroaryl, e.g., a 5-membered heterocyclyl or heteroaryl comprising one or more nitrogen. In some embodiments, B is a 5-membered heterocyclyl comprising 1 nitrogen atom. In some embodiments, B is a 5-membered heteroaryl comprising 1 nitrogen atom. In some embodiments, B is a 5-membered heterocyclyl comprising 2 nitrogen atoms. In some embodiments, B is a 5-membered heteroaryl comprising 2 nitrogen atoms. In some embodiments, B is a 5-membered heterocyclyl comprising 3 nitrogen atoms. In some embodiments, B is a 5-membered heteroaryl comprising 3 nitrogen atoms. The one or more nitrogen atom of the 5-membered nitrogen-containing heterocyclyl or heteroaryl may be at any position of the ring. In some embodiments, B is a 5-membered nitrogen-containing heterocyclyl optionally substituted with one or more R1. In some embodiments, B is a 5-membered nitrogen-containing heteroaryl optionally substituted with one or more R1. In some embodiments, the one or more nitrogen of the 5-membered nitrogen-containing heterocyclyl or heteroaryl is substituted, e.g., with R1. In some embodiments, B is a 5-membered nitrogen-containing heterocyclyl or heteroaryl comprising one or more additional heteroatoms, e.g., one or more of oxygen, sulfur, boron, silicon, or phosphorus.

In some embodiments, B is a nitrogen-containing bicyclic heteroaryl (e.g., a 9-membered nitrogen-containing bicyclic heteroaryl), that is optionally substituted with one or more R1. In some embodiments, B is a 9-membered bicyclic heteroaryl comprising 1 nitrogen atom. In some embodiments, B is a 9-membered bicyclic heteroaryl comprising 2 nitrogen atoms. In some embodiments, B is a 9-membered bicyclic heteroaryl comprising 3 nitrogen atoms. In some embodiments, B is a 9-membered bicyclic heteroaryl comprising 4 nitrogen atoms. The one or more nitrogen atom of the 9-membered bicyclic heteroaryl may be at any position of the ring. In some embodiments, B is a 9-membered bicyclic heteroaryl substituted with one or more R1.

In some embodiments, each of A and B are independently selected from:

wherein each R1 is as defined herein. In an embodiment, A and B are each independently a saturated, partially saturated, or unsaturated (e.g., aromatic) derivative of one of the rings described above. In an embodiment, A and B are each independently a stereoisomer of one of the rings described above.

In some embodiments, each of A and B are independently selected from:

wherein each R1 is as defined herein. In an embodiment, A and B are each independently a saturated, partially saturated, or unsaturated (e.g., aromatic) derivative of one of the rings described above. In an embodiment, A and B are each independently a stereoisomer of one of the rings described above.

In some embodiments, one of A and B is independently selected from

wherein R1 is as described herein. In some embodiments, one of A and B is independently selected from

wherein each R1a is independently C1-C6-alkyl, C1-C6-heteroalkyl, C1-C6-haloalkyl, halo, cyano, or —ORA, and each alkyl, heteroalkyl, and haloalkyl is optionally substituted with one or more R7. In some embodiments, one of A and B is independently

wherein each R1a is independently C1-C6-alkyl, C1-C6-heteroalkyl, C1-C6-haloalkyl, halo, cyano, or —ORA, and each alkyl, heteroalkyl, and haloalkyl is optionally substituted with one or more R7.

In some embodiments, one of A and B is independently selected from N

In some embodiments, one of A and B is independently selected from

In some embodiments, one of A and B is independently

In some embodiments, one of A and B is independently a monocyclic heterocyclyl or bicyclic heterocyclyl, each of which is optionally substituted with one or more R1. In some embodiments, one of A and B is independently a nitrogen-containing heterocyclyl optionally substituted with one or more R1. In some embodiments, one of A and B is independently a 4-8 membered heterocyclyl optionally substituted with one or more R1. In some embodiments, one of A and B is independently selected from

wherein R1 is as described herein. In some embodiments, one of A and B is independently selected from

wherein R1 is as described herein. In some embodiments, one of A and B is

wherein R1 is as described herein. In some embodiments, A is selected from

wherein R1 is as described herein. In some embodiments, B is selected from

wherein Rt is as described herein. In some embodiments, B is selected from

wherein Rt is as described herein. In some embodiments, A is selected

In some embodiments, A is selected one of A and B is independently selected from,

In some embodiments, one of A and Bis

As generally described herein, for Formulas (I) and (II), L1 and L2 each independently may be absent or refer to a C1-C6-alkylene, C1-C6-heteroalkylene, —O—, —C(O)—, —N(R4)—, —N(R4)C(O)—, or —C(O)N(R4)— group, wherein each alkylene and heteroalkylene is optionally substituted with one or more R5.

In some embodiments, L1 is absent, C1-C6-alkylene, C1-C6-heteroalkylene, —N(R4)C(O)—, or —C(O)N(R4)—, wherein each alkylene and heteroalkylene is optionally substituted with one or more R5.

In some embodiments, L1 is C1-C6 heteroalkylene (e.g., C1-heteroalkylene, C2-heteroalkylene, C3-heteroalkylene, C4-heteroalkylene, C5-heteroalkylene, or C6-heteroalkylene). In some embodiments, L1 is unsubstituted C1-C6 heteroalkylene. In some embodiments, L1 is substituted heteroalkylene, e.g., C1-C6 heteroalkylene substituted with one or more R5. In some embodiments, the heteroalkylene comprises 1 or more heteroatoms. In some embodiments, the heteroalkylene comprises one or more of oxygen, sulfur, nitrogen, boron, silicon, or phosphorus. In some embodiments, L1 is —N(R4)C(O)—. In some embodiments, L1 is —C(O)N(R4)—. In some embodiments, L1 is —C(O)N(H)—.

In some embodiments, L1 is oxygen. In some embodiments, L1 is nitrogen which is optionally substituted with R4. In some embodiments, L1 is nitrogen substituted with R4. In some embodiments, L1 is —N(R4)—, e.g., —N(CH3)—. In some embodiments, L1 is —NH—. In some embodiments, L1 is —O—.

In some embodiments, L2 is absent, C1-C6-alkylene, C1-C6-heteroalkylene, —N(R4)C(O)—, or —C(O)N(R4)—, wherein each alkylene and heteroalkylene is optionally substituted with one or more R5. In some embodiments, L2 is unsubstituted C1-C6 heteroalkylene. In some embodiments, L2 is substituted heteroalkylene, e.g., C1-C6 heteroalkylene substituted with one or more R5. In some embodiments, the heteroalkylene comprises 1 or more heteroatoms. In some embodiments, the heteroalkylene comprises one or more of oxygen, sulfur, nitrogen, boron, silicon, or phosphorus. In some embodiments, L2 is —N(R4)C(O)—. In some embodiments, L2 is —C(O)N(R4)—. In some embodiments, L2 is —C(O)N(H)—.

In some embodiments, L2 is nitrogen which is optionally substituted with R4. In some embodiments, L2 is nitrogen substituted with R4. In some embodiments, L2 is —N(R4)—, e.g., —N(CH3)—. In some embodiments, L2 is —NH—.

As generally described herein, for Formula (I), X, Y, and Z each independently refer to C(R3a), C(R3a)(R3b), N, or N(R3c), or O. In some embodiments, at least one of X, Y, and Z is either N or N(R3c). In some embodiments, at least one of X, Y, and Z is O. In some embodiments, at least two of X, Y, and Z is N or N(R3c). In some embodiments, X is N. In some embodiments, X is N(R3c). In some embodiments, X is O. In some embodiments, X is C(R3a) (e.g., CH). In some embodiments, X is C(R3a)(R3b). In some embodiments, Y is N. In some embodiments, Y is N(R3c). In some embodiments, Y is C(R3a) (e.g., CH). In some embodiments, Y is C(R3a)C(R3b). In some embodiments, Z is N. In some embodiments, Z is N(R3c). In some embodiments, Z is C(R3a) (e.g., CH). In some embodiments, Z is C(R3a)C(R3b) In some embodiments, two of X, Y, and Z are N, and the other of X, Y, and Z is C(R3a) (e.g., CH). In some embodiments, one of X, Y, and Z is C(R3a) (e.g., CH), and the others of X, Y, and Z are each independently N. In some embodiments, X and Y are each independently N, and Z is C(R3a) (e.g., CH). In some embodiments, X is C(R3a) (e.g., CH), and Y and Z are each independently N.

In some embodiments, X, Y, and Z are each independently N or C(R3a), wherein at least one of X, Y, and Z is N and the bonds in the ring comprising X, Y, and Z may be single or double bonds as valency permits.

In some embodiments, X is C(R3a), Y is C(R3a), and Z is O. In some embodiments, X is C(R3a), Y is C(R3a), Z is O, and y is 0. In some embodiments, X is C(R3a), Y is C(R3a), Z is O, and the bond between X and Y is a double bond. In some embodiments, X is C(R3a), Y is C(R3a), Z is O, and the bond between Y and Z is a single bond.

In some embodiments,

is selected from

In some embodiments,

In some embodiments,

In some embodiments,

In some embodiments for Formulas (I) and (II), R1 is hydrogen. In some embodiments, R1 is C1-C6-alkyl. In some embodiments, R1 is C2-C6-alkenyl. In some embodiments, R1 is C2-C6-alkynyl. In some embodiments, R1 is C1-C6-heteroalkyl. In some embodiments, R1 is C1-C6-haloalkyl (e.g., —CF3). In some embodiments, R1 is C1-alkyl (e.g., methyl). In some embodiments, R1 is unsubstituted C1-C6-alkyl, unsubstituted C2-C6-alkenyl, unsubstituted C2-C6-alkynyl, unsubstituted C1-C6-heteroalkyl, or unsubstituted C1-C6-haloalkyl. In some embodiments, R1 is C1-C6-alkyl substituted with one or more R6. In some embodiments, R1 is C2-C6-alkenyl substituted with one or more R6. In some embodiments, R1 is C2-C6-alkynyl substituted with one or more R6. In some embodiments, R1 is C1-C6-heteroalkyl substituted with one or more R6. In some embodiments, R1 is C1-C6-haloalkyl substituted with one or more R6. In some embodiments, R1 is methyl.

In some embodiments, R1 is cycloalkyl (e.g., 3-7 membered cycloalkyl). In some embodiments, R1 is heterocyclyl (e.g., 3-7 membered heterocyclyl). In some embodiments, R1 is aryl. In some embodiments, R1 is C1-C6 alkylene-aryl (e.g., benzyl). In some embodiments, R1 is C1-C6 alkenylene-aryl. In some embodiments, R1 is C1-C6 alkylene-heteroaryl. In some embodiments, R1 is heteroaryl. In some embodiments, R1 is unsubstituted cycloalkyl, unsubstituted heterocyclyl, unsubstituted aryl, unsubstituted C1-C6 alkylene-aryl, unsubstituted C1-C6 alkenylene-aryl, unsubstituted C1-C6 alkylene-heteroaryl, or unsubstituted heteroaryl. In some embodiments, R1 is cycloalkyl substituted with one or more R6. In some embodiments, R1 is heterocyclyl substituted with one or more R6. In some embodiments, R1 is aryl substituted with one or more R6. In some embodiments, R1 is C1-C6 alkylene-aryl substituted with one or more R6. In some embodiments, R1 is C1-C6 alkenylene-aryl substituted with one or more R6. In some embodiments, R1 is C1-C6 alkylene-heteroaryl substituted with one or more R6. In some embodiments, R1 is heteroaryl substituted with one or more R6.

In some embodiments, R1 is —ORA. In some embodiments, R1 is —NRBRC (e.g., NH2 or NMe2). In some embodiments, R1 is —NRBC(O)RD. In some embodiments, R1 is-C(O)NRBRC. In some embodiments, R1 is —C(O)RD. In some embodiments, R1 is —C(O)ORD. In some embodiments, R1 is —SRE. In some embodiments, R1 is —S(O)xRD. In some embodiments, R1 is halo, e.g., fluoro, chloro, bromo, or iodo. In some embodiments, R1 is cyano. In some embodiments, R1 is nitro (—NO2). In some embodiments, R1 is oxo.

In some embodiments, two R1 groups, together with the atoms to which they are attached, form a 3-7-membered cycloalkyl. In some embodiments, two R1 groups, together with the atoms to which they are attached, form a 3-7-membered heterocyclyl. In some embodiments, two R1 groups, together with the atoms to which they are attached, form a 5- or 6-membered aryl. In some embodiments, two R1 groups, together with the atoms to which they are attached, form a 5- or 6-membered heteroaryl. The cycloalkyl, heterocyclyl, aryl, or heteroaryl may be substituted with one or more R6.

In some embodiments for Formulas (I) and (II), R2 is hydrogen. In some embodiments, R2 is halo (e.g., fluoro, chloro, bromo, or iodo). In some embodiments, R2 is cyano. In some embodiments, R2 is C1-C6-alkyl. In some embodiments, R2 is C2-C6-alkenyl. In some embodiments, R2 is C2-C6-alkynyl. In some embodiments, R2 is —ORA (e.g., —OH). In some embodiments, R3a, R3b, or both are independently hydrogen, C1-C6-alkyl, C1-C6-heteroalkyl, C1-C6-haloalkyl, halo, cyano, —ORA, —NRBRC, —C(O)RD, or —C(O)ORD. In some embodiments, R3a and R3b are each independently hydrogen or C1-C6-alkyl. In some embodiments, R3a is hydrogen. In some embodiments, R3b is hydrogen. In some embodiments, R3a is C1-C6-alkyl (e.g., methyl). In some embodiments, R3b is C1-C6-alkyl (e.g., methyl). In some embodiments, R3a is halo (e.g., fluoro, chloro, bromo, or iodo). In some embodiments, R3b is halo (e.g., fluoro, chloro, bromo, or iodo). In some embodiments, R3a is cyano. In some embodiments, R3b is cyano. In some embodiments, R3a is —ORA (e.g., —OH). In some embodiments, R3b is —ORA (e.g., —OH). In some embodiments, R3a is —NRBRC. In some embodiments, R3b is —NRBRC. In some embodiments, R3a is —C(O)RD. In some embodiments, R3b is —C(O)RD. In some embodiments, R3a is —C(O)ORD. In some embodiments, R3b is —C(O)ORD In some embodiments, each of R3a and R3b, together with the carbon atom to which they are attached, form an oxo group.

In some embodiments, R3c is hydrogen. In some embodiments, R3c is C1-C6-alkyl. In some embodiments, R3c is methyl. In some embodiments, R3c is not hydrogen. In some embodiments, R3c is not methyl. In some embodiments, R3c is C1-C6 alkyl. In some embodiments, R3c is C1-C6 substituted with one or more R′.

In some embodiments, R5 is hydrogen. In some embodiments, R5 is C1-C6-alkyl. In some embodiments, R5 is C1-C6-heteroalkyl. In some embodiments, R5 is C1-C6-haloalkyl. In some embodiments, R5 is cycloalkyl. In some embodiments, R5 is halo (e.g., fluoro, chloro, bromo, or iodo). In some embodiments, R5 is cyano. In some embodiments, R5 is oxo. In some embodiments, R5 is —ORA. In some embodiments, R5 is —NRBRC. In some embodiments, R5 is —C(O)RD or —C(O)ORD.

In some embodiments, R6 is C1-C6-alkyl. In some embodiments, R6 is C2-C6-alkenyl. In some embodiments, R6 is C2-C6-alkynyl. In some embodiments, R6 is C1-C6-heteroalkyl. In some embodiments, R6 is C1-C6-haloalkyl. In some embodiments, R6 is unsubstituted C1-C6-alkyl, unsubstituted C2-C6-alkenyl, unsubstituted C2-C6-alkynyl, unsubstituted C1-C6-haloalkyl, or unsubstituted C1-C6-heteroalkyl. In some embodiments, R6 is C1-C6-alkyl substituted with one or more R11. In some embodiments, R6 is C2-C6-alkenyl substituted with one or more R11. In some embodiments, R6 is C2-C6-alkynyl substituted with one or more R11. In some embodiments, R6 is C1-C6-haloalkyl substituted with one or more R11. In some embodiments, R6 is C1-C6-heteroalkyl substituted with one or more R11.

In some embodiments, R6 is cycloalkyl. In some embodiments, R6 is heterocyclyl. In some embodiments, R6 is aryl. In some embodiments, R6 is heteroaryl. In some embodiments, R6 is unsubstituted cycloalkyl, unsubstituted heterocyclyl, unsubstituted aryl, or unsubstituted heteroaryl. In some embodiments, R6 is cycloalkyl substituted with one or more R11. In some embodiments, R6 is heterocyclyl substituted with one or more R11. In some embodiments, R6 is aryl substituted with one or more R11. In some embodiments, R6 is heteroaryl substituted with one or more R11.

In some embodiments, R6 is halo (e.g., fluoro, chloro, bromo, or iodo). In some embodiments, R6 is cyano. In some embodiments, R6 is oxo. In some embodiments, R6 is —ORA. In some embodiments, R6 is —NRBRC. In some embodiments, R6 is —NRBC(O)RD. In some embodiments, R6 is —NO2. In some embodiments, R6 is —C(O)NRBRC. In some embodiments, R6 is —C(O)RD. In some embodiments, R6 is —C(O)ORD. In some embodiments, R6 is —SRE. In some embodiments, R6 is —S(O)xRD.

In some embodiments, R11 is C1-C6-alkyl. In some embodiments, R11 is C1-C6-heteroalkyl. In some embodiments, R11 is C1-C6-haloalkyl (e.g., —CF3). In some embodiments, R11 is cycloalkyl. In some embodiments, R11 is heterocyclyl. In some embodiments, R11 is aryl. In some embodiments, R11 is heteroaryl. In some embodiments, R11 is halo. In some embodiments, R11 is cyano. In some embodiments, R11 is oxo. In some embodiments, R11 is —ORA.

In some embodiments for Formulas (I) and (II), RA is hydrogen. In some embodiments, RA is C1-C6 alkyl (e.g., methyl). In some embodiments, RA is C1-C6 haloalkyl. In some embodiments, RA is aryl. In some embodiments, RA is heteroaryl. In some embodiments, RA is C1-C6 alkylene-aryl (e.g., benzyl). In some embodiments, RA is C1-C6 alkylene-heteroaryl. In some embodiments, RA is C(O)RD. In some embodiments, RA is —S(O)xRD.

In some embodiments, RB, RC, or both are independently hydrogen, C1-C6-alkyl, C1-C6-heteroalkyl, cycloalkyl, heterocyclyl, or —ORA. In some embodiments, each of RB and RC is independently hydrogen. In some embodiments, each of RB and RC is independently C1-C6 alkyl. In some embodiments, one of RB and RC is hydrogen, and the other of RB and RC is C1-C6 alkyl. In some embodiments, RB and RC together with the atom to which they are attached form a 3-7-membered heterocyclyl ring optionally substituted with one or more of R7.

In some embodiments, RD, RE, or both are independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, C1-C6 alkylene-aryl (e.g., benzyl), or C1-C6 alkylene-heteroaryl. In some embodiments, each of RD and RE is independently hydrogen. In some embodiments, each of RD and RE is independently C1-C6 alkyl. In some embodiments, RD is hydrogen. In some embodiments, RE is hydrogen. In some embodiments, RD is C1-C6 alkyl (e.g., methyl). In some embodiments, RE is C1-C6 alkyl (e.g., methyl). In some embodiments, RD is C1-C6 heteroalkyl. In some embodiments, RE is C1-C6 heteroalkyl. In some embodiments, RD is C1-C6 haloalkyl. In some embodiments, RE is C1-C6 haloalkyl. In some embodiments, RD is cycloalkyl. In some embodiments, RE is cycloalkyl. In some embodiments, RD is heterocyclyl. In some embodiments, RE is heterocyclyl. In some embodiments, RD is aryl. In some embodiments, RE is aryl. In some embodiments, RD is heteroaryl. In some embodiments, RE is heteroaryl. In some embodiments, RD is C1-C6 alkylene-aryl (e.g., benzyl). In some embodiments, RE is C1-C6 alkylene-aryl (e.g., benzyl). In some embodiments, RD is C1-C6 alkylene-heteroaryl. In some embodiments, RE is C1-C6 alkylene-heteroaryl.

In some embodiments, m is 0, 1, or 2. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, x is 0, 1, or 2. In some embodiments, x is 0. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments y is 0 or 1. In some embodiments, y is 0.

In some embodiments, the compound of Formula (I) is a compound of Formula (I-d):

In some embodiments, R2 is halo. In some embodiments, R2 is fluoro. In some embodiments, R2 is —ORA. In some embodiments, R2 is —N(RB)(RC).

In some embodiments, the compound of Formula (I) is a compound of Formula (I-g):

In some embodiments, the compound of Formula (I) is selected from a compound in Table 1, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

Exemplary compounds of Formula (I)

Compound

In some embodiments, for Formula (I), A is monocyclic heterocyclyl (e.g., piperidinyl); B is bicyclic heteroaryl (e.g., imidazo[1,2-b]pyridazinyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X and Y are each independently C(R3a) (e.g., CH); Z is O; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-c), (I-e), (I-f), (I-h) and (I-i) is Compound 119, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is monocyclic heterocyclyl (e.g., piperidinyl); B is bicyclic heteroaryl (e.g., imidazo[1,2-b]pyridazinyl); L1 is absent or —N(R4)—; and L2 is absent or —C(O)N(R4)— (e.g., —C(O)N(H)—). In some embodiments, for Formula (I), the compound is selected from Compound 118, 141, 228, 229, 242, 243, 269, and 277.

In some embodiments, for Formula (I), A is monocyclic heterocyclyl (e.g., piperazinyl); B is bicyclic heteroaryl (e.g., 8-fluoro-2-methylimidazo[1,2-a]pyridinyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is N; Y is C(R3a) (e.g., C(CH3)); Z is O; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-e), (I-f), and (I-i) is Compound 140, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is monocyclic heterocyclyl (e.g., piperazinyl); B is bicyclic heteroaryl (e.g., 8-fluoro-2-methylimidazo[1,2-a]pyridinyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is 0; Y is C(R3a) (e.g., C(CH3)); Z is N; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-e), (I-f), and (I-i) is Compound 141, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is monocyclic heterocyclyl (e.g., piperazinyl); B is bicyclic heteroaryl (e.g., 8-fluoro-2-methylimidazo[1,2-a]pyridinyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is N(R3c) (e.g., NH); Y is C(R3a) (e.g., C(CH3)); Z is N; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-e), (I-f), and (I-i) is Compound 146, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is monocyclic heterocyclyl (e.g., piperazinyl); B is bicyclic heteroaryl (e.g., 8-fluoro-2-methylimidazo[1,2-a]pyridinyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is N(R3c) (e.g., NH); Y is N; Z is N; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-e), (I-f), and (I-i) is Compound 147, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is monocyclic heterocyclyl (e.g., piperazinyl); B is bicyclic heteroaryl (e.g., 8-fluoro-2-methylimidazo[1,2-a]pyridinyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is N; Y is C(R3a) (e.g., CH); Z is O; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-e), (I-f), and (I-i) is Compound 150, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is monocyclic heterocyclyl (e.g., piperidinyl); B is bicyclic heteroaryl (e.g., 8-fluoro-2-methylimidazo[1,2-a]pyridinyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is 0; Y is C(R3a) (e.g., C(CH3)); Z is N; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-e), (I-f), and (I-i) is Compound 187, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is monocyclic heterocyclyl (e.g., piperidinyl); B is bicyclic heteroaryl (e.g., 8-fluoro-2-methylimidazo[1,2-a]pyridinyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is C(R3a)(R3b) (e.g., CH2); Y is C(R3a)(R3b) (e.g., CH2); Z is O; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-e), (I-f), and (I-i) is Compound 188, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is monocyclic heterocyclyl (e.g., 1-methylpiperidinyl); B is bicyclic heteroaryl (e.g., 8-fluoro-2-methylimidazo[1,2-a]pyridinyl); L1 is —N(R4)— (e.g., —N(CH3)—); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is C(R3a) (e.g., CH); Y is C(R3a) (e.g., C(CH3)); Z is O; y is 0; and m is 0. In some embodiments, the compound of Formulas (I) and (I-a) is Compound 200, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is bicyclic heterocyclyl (e.g., 2,2,6,6-tetramethylpiperidinyl); B is bicyclic heteroaryl (e.g., 8-fluoro-2-methylimidazo[1,2-a]pyridinyl); L1 is —N(R4)— (e.g., —N(CH3)—); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is C(R3a) (e.g., CH); Y is C(R3a) (e.g., C(CH3)); Z is O; y is 0; and m is 0. In some embodiments, the compound of Formulas (I) and (I-a) is Compound 201 or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is bicyclic heterocyclyl (e.g., piperazinyl); B is monocyclic heteroaryl (e.g., pyrazolyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is C(R3a) (e.g., CH); Y is N(R3c) (e.g., N(CH3)); Z is N; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-d), and (I-e) is Compound 270, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is monocyclic heterocyclyl (e.g., piperidinyl); B is bicyclic heteroaryl (e.g., 8-fluoro-2-methylimidazo[1,2-a]pyridinyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is C(R3a) (e.g., CH); Y is N(R3c) (e.g., N(CH3)); Z is N; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-d), and (I-e) is Compound 280, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is bicyclic heteroaryl (e.g., 2,8-dimethylimidazo[1,2-b]pyridazinyl); B is monocyclic heterocyclyl (e.g., piperidinyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is C(R3a) (e.g., CH); Y is N(R3c) (e.g., N(CH3)); Z is N; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-d), and (I-e is Compound 281, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is bicyclic heteroaryl (e.g., 2,8-dimethylimidazo[1,2-b]pyridazinyl); B is monocyclic heterocyclyl (e.g., piperidinyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is C(R3a) (e.g., CH); Y is C(R3a) (e.g., C(CH3)); Z is O; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-c), and (I-e) is Compound 282, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

In some embodiments, for Formula (I), A is bicyclic heteroaryl (e.g., 8-fluoro-2-methylimidazo[1,2-a]pyridinyl); B is monocyclic heterocyclyl (e.g., piperidinyl); L2 is —C(O)N(R4)— (e.g., —C(O)N(H)—); X is C(R3a) (e.g., CH); Y is C(R3a) (e.g., C(CH3)); Z is O; y is 0; and m is 0. In some embodiments, the compound of Formulas (I), (I-a), (I-b), (I-c), and (I-e) is Compound 283, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

The present disclosure further features compounds of Formula (II). In some embodiments, the compound of Formula (II) is a compound of Formula (II-a):

In some embodiments of Formula (II),

is selected from

In some embodiments,

In some embodiments,

In some embodiments,

In some embodiments,

In some embodiments,

In some embodiments,

In some embodiments,

In some embodiments,

In some embodiments, the compound of Formula (II) is selected from a compound in Table 2, or a pharmaceutically acceptable salt thereof.

Pharmaceutical Compositions, Kits, and Administration

The present invention provides pharmaceutical compositions comprising a compound of Formula (I) or (II), e.g., a compound of Formula (I) or (II) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer, as described herein, and optionally a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition described herein comprises a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient. In certain embodiments, the compound of Formula (I) or (II) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount.

Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing the compound of Formula (I) or (II) (the “active ingredient”) into association with a carrier and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

The term “pharmaceutically acceptable excipient” refers to a non-toxic carrier, adjuvant, diluent, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable excipients useful in the manufacture of the pharmaceutical compositions of the invention are any of those that are well known in the art of pharmaceutical formulation and include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Pharmaceutically acceptable excipients useful in the manufacture of the pharmaceutical compositions of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

Compositions of the present invention may be administered orally, parenterally (including subcutaneous, intramuscular, intravenous and intradermal), by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. In some embodiments, provided compounds or compositions are administrable intravenously and/or orally.

The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intraocular, intravitreal, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intraperitoneal intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, subcutaneously, intraperitoneally, or intravenously. Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Pharmaceutically acceptable compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. In some embodiments, a provided oral formulation is formulated for immediate release or sustained/delayed release. In some embodiments, the composition is suitable for buccal or sublingual administration, including tablets, lozenges and pastilles. A provided compound can also be in micro-encapsulated form.

Alternatively, pharmaceutically acceptable compositions of this invention may be administered in the form of suppositories for rectal administration. Pharmaceutically acceptable compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

For ophthalmic use, provided pharmaceutically acceptable compositions may be formulated as micronized suspensions or in an ointment such as petrolatum.

The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like. The desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).

In certain embodiments, an effective amount of a compound for administration one or more times a day to a 70 kg adult human may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosage form.

In certain embodiments, the compounds of Formula (I) or (II) may be at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

It will be also appreciated that a compound or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents. The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects.

The compound or composition can be administered concurrently with, prior to, or subsequent to, one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the inventive compound with the additional pharmaceutical agents and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

Also encompassed by the invention are kits (e.g., pharmaceutical packs). The inventive kits may be useful for preventing and/or treating a proliferative disease or a non-proliferative disease, e.g., as described herein. The kits provided may comprise an inventive pharmaceutical composition or compound and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of an inventive pharmaceutical composition or compound. In some embodiments, the inventive pharmaceutical composition or compound provided in the container and the second container are combined to form one-unit dosage form.

Thus, in one aspect, provided are kits including a first container comprising a compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a pharmaceutical composition thereof. In certain embodiments, the kit of the disclosure includes a first container comprising a compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the kits are useful in preventing and/or treating a disease, disorder, or condition described herein in a subject (e.g., a proliferative disease or a non-proliferative disease). In certain embodiments, the kits further include instructions for administering the compound, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, or a pharmaceutical composition thereof, to a subject to prevent and/or treat a proliferative disease or a non-proliferative disease.

METHODS OF USE

Described herein are compounds useful for modulating splicing. In some embodiments, a compound of Formula (I) or (II) may be used to alter the amount, structure, or composition of a nucleic acid (e.g., a precursor RNA, e.g., a pre-mRNA, or the resulting mRNA) by increasing or decreasing splicing at a splice site. In some embodiments, increasing or decreasing splicing results in modulating the level or structure of a gene product (e.g., an RNA or protein) produced. In some embodiments, a compound of Formula (I) or (II) may modulate a component of the splicing machinery, e.g., by modulating the interaction with a component of the splicing machinery with another entity (e.g., nucleic acid, protein, or a combination thereof). The splicing machinery as referred to herein comprises one or more spliceosome components. Spliceosome components may comprise, for example, one or more of major spliceosome members (U1, U2, U4, U5, U6 snRNPs), or minor spliceosome members (U11, U12, U4atac, U6atac snRNPs) and their accessory splicing factors.

In another aspect, the present disclosure features a method of modifying of a target (e.g., a precursor RNA, e.g., a pre-mRNA) through inclusion of a splice site in the target, wherein the method comprises providing a compound of Formula (I) or (II). In some embodiments, inclusion of a splice site in a target (e.g., a precursor RNA, e.g., a pre-mRNA, or the resulting mRNA) results in addition or deletion of one or more nucleic acids to the target (e.g., a new exon, e.g. a skipped exon). Addition or deletion of one or more nucleic acids to the target may result in an increase in the levels of a gene product (e.g., RNA, e.g., mRNA, or protein).

In another aspect, the present disclosure features a method of modifying a target (e.g., a precursor RNA, e.g., a pre-mRNA, or the resulting mRNA) through exclusion of a splice site in the target, wherein the method comprises providing a compound of Formula (I) or (II). In some embodiments, exclusion of a splice site in a target (e.g., a precursor RNA, e.g., a pre-mRNA) results in deletion or addition of one or more nucleic acids from the target (e.g., a skipped exon, e.g. a new exon). Deletion or addition of one or more nucleic acids from the target may result in a decrease in the levels of a gene product (e.g., RNA, e.g., mRNA, or protein). In other embodiments, the methods of modifying a target (e.g., a precursor RNA, e.g., a pre-mRNA, or the resulting mRNA) comprise suppression of splicing at a splice site or enhancement of splicing at a splice site (e.g., by more than about 0.5%, e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more), e.g., as compared to a reference (e.g., the absence of a compound of Formula (I) or (II), or in a healthy or diseased cell or tissue).

In some embodiments, the gene encoding a target sequence comprises the HTT gene. In some embodiments, the gene encoding a target sequence comprises the MYB gene. In some embodiments, the gene encoding a target sequence comprises the SMN2 gene. In some embodiments, the gene encoding a target sequence comprises the FOAM1 gene.

In an embodiment, a gene sequence or splice site sequence provided herein is related to a proliferative disease, disorder, or condition (e.g., cancer, benign neoplasm, or inflammatory disease). In an embodiment, a gene sequence or splice site sequence provided herein is related to a non-proliferative disease, disorder, or condition. In an embodiment, a gene sequence or splice site sequence provided herein is related to a neurological disease or disorder; autoimmune disease or disorder; immunodeficiency disease or disorder; lysosomal storage disease or disorder; cardiovascular condition, disease or disorder; metabolic disease or disorder; respiratory condition, disease, or disorder; renal disease or disorder; or infectious disease in a subject. In an embodiment, a gene sequence or splice site sequence provided herein is related to a neurological disease or disorder (e.g., Huntington's disease). In an embodiment, a gene sequence or splice site sequence provided herein is related to an immunodeficiency disease or disorder. In an embodiment, a gene sequence or splice site sequence provided herein is related to a lysosomal storage disease or disorder. In an embodiment, a gene sequence or splice site sequence provided herein is related to a cardiovascular condition, disease or disorder. In an embodiment, a gene sequence or splice site sequence provided herein is related to a metabolic disease or disorder. In an embodiment, a gene sequence or splice site sequence provided herein is related to a respiratory condition, disease, or disorder. In an embodiment, a gene sequence or splice site sequence provided herein is related to a renal disease or disorder. In an embodiment, a gene sequence or splice site sequence provided herein is related to an infectious disease.

In an embodiment, a gene sequence or splice site sequence provided herein is related to a mental retardation disorder. In an embodiment, a gene sequence or splice site sequence provided herein is related to a mutation in the SETD5 gene. In an embodiment, a gene sequence or splice site sequence provided herein is related to an immunodeficiency disorder. In an embodiment, a gene sequence and splice site sequence provided herein is related to a mutation in the GATA2 gene. In an embodiment, a gene sequence or splice site sequence provided herein is related to a lysosomal storage disease.

In one aspect, the compounds of Formula (I) or (II) and pharmaceutically acceptable salts, solvates, hydrates, tautomers, stereoisomers, and compositions thereof, may modulate (e.g., increase or decrease) a splicing event of a target nucleic acid sequence (e.g., DNA, RNA, or a pre-mRNA), for example, a nucleic acid encoding a gene described herein, or a nucleic acid encoding a protein described herein, or a nucleic acid comprising a splice site described herein. In an embodiment, the splicing event is an alternative splicing event.

In another aspect, the present disclosure features a method of forming a complex comprising a component of a spliceosome (e.g., a major spliceosome component or a minor spliceosome component), a nucleic acid (e.g., a DNA, RNA, e.g., a pre-mRNA), and a compound of Formula (I) or (II) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, or composition thereof, comprising contacting the nucleic acid (e.g., a DNA, RNA, e.g., a pre-mRNA) with said compound of Formula (I) or (II). In an embodiment, the component of a spliceosome is selected from the U1, U2, U4, U5, U6, U11, U12, U4atac, U6atac small nuclear ribonucleoproteins (snRNPs), or a related accessory factor. In an embodiment, the component of a spliceosome is recruited to the nucleic acid in the presence of the compound of Formula (I) or (II) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, or composition thereof.

In another aspect, the present disclosure features a method of altering the conformation of a nucleic acid (e.g., a DNA, RNA, e.g., a pre-mRNA) comprising contacting the nucleic acid with a compound of Formula (I) or (II) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, or composition thereof. In an embodiment, the altering comprises forming a bulge or kink in the nucleic acid. In an embodiment, the altering comprises stabilizing a bulge or a kink in the nucleic acid. In an embodiment, the altering comprises reducing a bulge or a kink in the nucleic acid. In an embodiment, the nucleic acid comprises a splice site. In an embodiment, the compound of Formula (I) or (II) interacts with a nucleobase, ribose, or phosphate moiety of a nucleic acid (e.g., a DNA, RNA, e.g., pre-mRNA).

The present disclosure also provides methods for the treatment or prevention of a disease, disorder, or condition. In an embodiment, the disease, disorder or condition is related to (e.g., caused by) a splicing event, such as an unwanted, aberrant, or alternative splicing event. In an embodiment, the disease, disorder or condition comprises a proliferative disease (e.g., cancer, benign neoplasm, or inflammatory disease) or non-proliferative disease. In an embodiment, the disease, disorder, or condition comprises a neurological disease, autoimmune disorder, immunodeficiency disorder, cardiovascular condition, metabolic disorder, lysosomal storage disease, respiratory condition, renal disease, or infectious disease in a subject. In another embodiment, the disease, disorder, or condition comprises a haploinsufficiency disease, an autosomal recessive disease (e.g., with residual function), or a paralogue activation disorder. In another embodiment, the disease, disorder, or condition comprises an autosomal dominant disorder (e.g., with residual function). Such methods comprise the step of administering to the subject in need thereof an effective amount of a compound of Formula (I) or (II), or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer thereof, or a pharmaceutical composition thereof. In certain embodiments, the methods described herein include administering to a subject an effective amount of a compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

In certain embodiments, the subject being treated is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal such as a dog or cat. In certain embodiments, the subject is a livestock animal such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal such as a rodent, dog, or non-human primate. In certain embodiments, the subject is a non-human transgenic animal such as a transgenic mouse or transgenic pig.

A proliferative disease may also be associated with inhibition of apoptosis of a cell in a biological sample or subject. All types of biological samples described herein or known in the art are contemplated as being within the scope of the disclosure. The compounds of Formula (I) or (II) and pharmaceutically acceptable salts, solvates, hydrates, tautomers, stereoisomers, and compositions thereof, may induce apoptosis, and therefore, be useful in treating and/or preventing proliferative diseases.

In some embodiments, the proliferative disease is associated with a benign neoplasm. For example, a benign neoplasm may include adenoma, fibroma, hemangioma, tuberous sclerosis, and lipoma. All types of benign neoplasms disclosed herein or known in the art are contemplated as being within the scope of the disclosure.

In some embodiments, the proliferative disease is associated with angiogenesis. All types of angiogenesis disclosed herein or known in the art are contemplated as being within the scope of the disclosure.

In some embodiments, the compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, or compositions comprising such compound or pharmaceutically acceptable salt thereof, is used to prevent or treat a non-proliferative disease. Exemplary non-proliferative diseases include a neurological disease, autoimmune disorder, immunodeficiency disorder, lysosomal storage disease, cardiovascular condition, metabolic disorder, respiratory condition, inflammatory disease, renal disease, or infectious disease.

In certain embodiments, the non-proliferative disease is a neurological disease. In certain embodiments, the compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, or compositions comprising such compound or pharmaceutically acceptable salt thereof, is used to prevent or treat a neurological disease, disorder, or condition. A neurological disease, disorder, or condition may include a neurodegenerative disease, a psychiatric condition, or a musculoskeletal disease. A neurological disease may further include a repeat expansion disease, e.g., which may be characterized by the expansion of a nucleic acid sequence in the genome. For example, a repeat expansion disease includes myotonic dystrophy, amyotrophic lateral sclerosis, Huntington's disease, a trinucleotide repeat disease, or a polyglutamine disorder (e.g., ataxia, fragile X syndrome). In some embodiments, the neurological disease comprises a repeat expansion disease, e.g., Huntington's disease. Additional neurological diseases, disorders, and conditions include Alzheimer's disease, Huntington's chorea, a prion disease (e.g., Creutzfeld-Jacob disease, bovine spongiform encephalopathy, Kuru, or scrapie), a mental retardation disorder (e.g., a disorder caused by a SETD5 gene mutation, e.g., intellectual disability-facial dysmorphism syndrome, autism spectrum disorder), Lewy Body disease, diffuse Lewy body disease (DLBD), dementia, progressive supranuclear palsy (PSP), progressive bulbar palsy (PBP), psuedobulbar palsy, spinal and bulbar muscular atrophy (SBMA), primary lateral sclerosis, Pick's disease, primary progressive aphasia, corticobasal dementia, Parkinson's disease, Down's syndrome, multiple system atrophy, spinal muscular atrophy (SMA), progressive spinobulbar muscular atrophy (e.g., Kennedy disease), post-polio syndrome (PPS), spinocerebellar ataxia, pantothenate kinase-associated neurodegeneration (PANK), spinal degenerative disease/motor neuron degenerative diseases, upper motor neuron disorder, lower motor neuron disorder, Hallervorden-Spatz syndrome, cerebral infarction, cerebral trauma, chronic traumatic encephalopathy, transient ischemic attack, Lytigo-bodig (amyotrophic lateral sclerosis-parkinsonism dementia), Guam-Parkinsonism dementia, hippocampal sclerosis, corticobasal degeneration, Alexander disease, Apler's disease, Krabbe's disease, neuroborreliosis, neurosyphilis, Sandhoff disease, Tay-Sachs disease, Schilder's disease, Batten disease, Cockayne syndrome, Kearns-Sayre syndrome, Gerstmann-Straussler-Scheinker syndrome and other transmissible spongiform encephalopathies, hereditary spastic paraparesis, Leigh's syndrome, a demyelinating diseases, neuronal ceroid lipofuscinoses, epilepsy, tremors, depression, mania, anxiety and anxiety disorders, sleep disorders (e.g., narcolepsy, fatal familial insomnia), acute brain injuries (e.g., stroke, head injury), autism, Machado-Joseph disease, or a combination thereof. In some embodiments, the neurological disease comprises Friedrich's ataxia or Sturge Weber syndrome. In some embodiments, the neurological disease comprises Huntington's disease. In some embodiments, the neurological disease comprises spinal muscular atrophy. All types of neurological diseases disclosed herein or known in the art are contemplated as being within the scope of the disclosure.

In certain embodiments, the non-proliferative disease is a cardiovascular condition. In certain embodiments, the compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, or compositions comprising such compound or pharmaceutically acceptable salt thereof, is used to prevent or treat a cardiovascular disease, disorder, or condition. A cardiovascular disease, disorder, or condition may include a condition relating to the heart or vascular system, such as the arteries, veins, or blood. Exemplary cardiovascular diseases, disorders, or conditions include angina, arrhythmias (atrial or ventricular or both), heart failure, arteriosclerosis, atheroma, atherosclerosis, cardiac hypertrophy, cardiac or vascular aneurysm, cardiac myocyte dysfunction, carotid obstructive disease, endothelial damage after PTCA (percutaneous transluminal coronary angioplasty), hypertension including essential hypertension, pulmonary hypertension and secondary hypertension (renovascular hypertension, chronic glomerulonephritis), myocardial infarction, myocardial ischemia, peripheral obstructive arteriopathy of a limb, an organ, or a tissue; peripheral artery occlusive disease (PAOD), reperfusion injury following ischemia of the brain, heart or other organ or tissue, restenosis, stroke, thrombosis, transient ischemic attack (TIA), vascular occlusion, vasculitis, and vasoconstriction. All types of cardiovascular diseases, disorders, or conditions disclosed herein or known in the art are contemplated as being within the scope of the disclosure.

In certain embodiments, the non-proliferative disease is a metabolic disorder. In certain embodiments, the compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, or compositions comprising such compound or pharmaceutically acceptable salt thereof, is used to prevent or treat a metabolic disease, disorder, or condition. A metabolic disease, disorder, or condition may include a disorder or condition that is characterized by abnormal metabolism, such as those disorders relating to the consumption of food and water, digestion, nutrient processing, and waste removal. A metabolic disease, disorder, or condition may include an acid-base imbalance, a mitochondrial disease, a wasting syndrome, a malabsorption disorder, an iron metabolism disorder, a calcium metabolism disorder, a DNA repair deficiency disorder, a glucose metabolism disorder, hyperlactatemia, a disorder of the gut microbiota. Exemplary metabolic conditions include obesity, diabetes (Type I or Type II), insulin resistance, glucose intolerance, lactose intolerance, eczema, hypertension, Hunter syndrome, Krabbe disease, sickle cell anemia, maple syrup urine disease, Pompe disease, and metachromatic leukodystrophy. All types of metabolic diseases, disorders, or conditions disclosed herein or known in the art are contemplated as being within the scope of the disclosure.

In certain embodiments, the non-proliferative disease is a respiratory condition. In certain embodiments, the compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, or compositions comprising such compound or pharmaceutically acceptable salt thereof, is used to prevent or treat a respiratory disease, disorder, or condition. A respiratory disease, disorder, or condition can include a disorder or condition relating to any part of the respiratory system, such as the lungs, alveoli, trachea, bronchi, nasal passages, or nose. Exemplary respiratory diseases, disorders, or conditions include asthma, allergies, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease (COPD), lung cancer, oxygen toxicity, emphysema, chronic bronchitis, and acute respiratory distress syndrome. All types of respiratory diseases, disorders, or conditions disclosed herein or known in the art are contemplated as being within the scope of the disclosure.

In certain embodiments, the non-proliferative disease is a renal disease. In certain embodiments, the compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, or compositions comprising such compound or pharmaceutically acceptable salt thereof, is used to prevent or treat a renal disease, disorder, or condition. A renal disease, disorder, or condition can include a disease, disorder, or condition relating to any part of the waste production, storage, and removal system, including the kidneys, ureter, bladder, urethra, adrenal gland, and pelvis. Exemplary renal diseases include acute kidney failure, amyloidosis, Alport syndrome, adenovirus nephritis, acute lobar nephronia, tubular necrosis, glomerulonephritis, kidney stones, urinary tract infections, chronic kidney disease, polycystic kidney disease, and focal segmental glomerulosclerosis (FSGS). In some embodiments, the renal disease, disorder, or condition comprises HIV-associated nephropathy or hypertensive nephropathy. All types of renal diseases, disorders, or conditions disclosed herein or known in the art are contemplated as being within the scope of the disclosure.

In certain embodiments, the non-proliferative disease is an infectious disease. In certain embodiments, the compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, or compositions comprising such compound or pharmaceutically acceptable salt thereof, is used to prevent or treat an infectious disease, disorder, or condition. An infectious disease may be caused by a pathogen such as a virus or bacteria. Exemplary infectious diseases include human immunodeficiency syndrome (HIV), acquired immunodeficiency syndrome (AIDS), meningitis, African sleeping sickness, actinomycosis, pneumonia, botulism, chlamydia, Chagas disease, Colorado tick fever, cholera, typhus, giardiasis, food poisoning, ebola hemorrhagic fever, diphtheria, Dengue fever, gonorrhea, streptococcal infection (e.g., Group A or Group B), hepatitis A, hepatitis B, hepatitis C, herpes simplex, hookworm infection, influenza, Epstein-Barr infection, Kawasaki disease, kuru, leprosy, leishmaniasis, measles, mumps, norovirus, meningococcal disease, malaria, Lyme disease, listeriosis, rabies, rhinovirus, rubella, tetanus, shingles, scarlet fever, scabies, Zika fever, yellow fever, tuberculosis, toxoplasmosis, or tularemia. In some embodiments, the infectious disease comprises cytomegalovirus. All types of infectious diseases, disorders, or conditions disclosed herein or known in the art are contemplated as being within the scope of the disclosure.

In certain embodiments, the disease, disorder, or condition is an autosomal recessive disease, e.g., with residual function. In certain embodiments, the compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, or compositions comprising such compound or pharmaceutically acceptable salt thereof, is used to prevent or treat an autosomal recessive disease, disorder, or condition. An autosomal recessive disease with residual function may refer to a monogenic disease with either homozygous recessive or compound heterozygous heritability. These diseases may also be characterized by insufficient gene product activity (e.g., a level of gene product greater than 0%). In an embodiment, a compound of Formula (I) or (II) may increase the expression of a target (e.g., a gene) related to an autosomal recessive disease with residual function. Exemplary autosomal recessive diseases with residual function include Friedreich's ataxia, Stargardt disease, Usher syndrome, chlorioderma, fragile X syndrome, achromatopsia 3, Hurler syndrome, hemophilia B, alpha-1-antitrypsin deficiency, Gaucher disease, X-linked retinoschisis, Wiskott-Aldrich syndrome, mucopolysaccharidosis (Sanfilippo B), DDC deficiency, epidermolysis bullosa dystrophica, Fabry disease, metachromatic leukodystrophy, and odontochondrodysplasia.

In certain embodiments, the disease, disorder, or condition is an autosomal dominant disease. In certain embodiments, the compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, or compositions comprising such compound or pharmaceutically acceptable salt thereof, is used to prevent or treat an autosomal dominant disease, disorder, or condition. An autosomal dominant disease may refer to a monogenic disease in which the mutated gene is a dominant gene. These diseases may also be characterized by insufficient gene product activity (e.g., a level of gene product greater than 0%). In an embodiment, a compound of Formula (I) or (II) may increase the expression of a target (e.g., a gene) related to an autosomal dominant disease. Exemplary autosomal dominant diseases include Huntington's disease, achondroplasia, antithrombin III deficiency, Gilbert's disease, Ehlers-Danlos syndrome, hereditary hemorrhagic telangiectasia, intestinal polyposis, hereditary elliptosis, hereditary spherocytosis, marble bone disease, Marfan's syndrome, protein C deficiency, Treacher Collins syndrome, Von Willebrand's disease, tuberous sclerosis, osteogenesis imperfecta, polycystic kidney disease, neurofibromatosis, and idiopathic hypoparathyroidism.

In certain embodiments, the disease, disorder, or condition is a paralogue activation disorder. In certain embodiments, the compound of Formula (I) or (II), or a pharmaceutically acceptable salt thereof, or compositions comprising such compound or pharmaceutically acceptable salt thereof, is used to prevent or treat a paralogue activation disease, disorder, or condition. A paralogue activation disorder may comprise a homozygous mutation of genetic locus leading to loss-of-function for the gene product. In these disorders, there may exist a separate genetic locus encoding a protein with overlapping function (e.g. developmental paralogue), which is otherwise not expressed sufficiently to compensate for the mutated gene. In an embodiment, a compound of Formula (I) or (II) activates a gene connected with a paralogue activation disorder (e.g., a paralogue gene).

The cell described herein may be an abnormal cell. The cell may be in vitro or in vivo. In certain embodiments, the cell is a proliferative cell. In certain embodiments, the cell is a cancer cell. In certain embodiments, the cell is a non-proliferative cell. In certain embodiments, the cell is a blood cell. In certain embodiments, the cell is a lymphocyte. In certain embodiments, the cell is a benign neoplastic cell. In certain embodiments, the cell is an endothelial cell. In certain embodiments, the cell is an immune cell. In certain embodiments, the cell is a neuronal cell. In certain embodiments, the cell is a glial cell. In certain embodiments, the cell is a brain cell. In certain embodiments, the cell is a fibroblast. In certain embodiment, the cell is a primary cell, e.g., a cell isolated from a subject (e.g., a human subject).

In some embodiments, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has improved cell permeability over a reference compound, e.g., in a standard assay for measuring cell permeability. Cell permeability may be investigated, for example, using a standard assay run in either Madin-Darby Canine Kidney (MDCK) cells expressing Breast Cancer Resistance Protein (BCRP) or subclone MDCKII cells expressing Multidrug Resistance Protein 1 (MDR1); see, e.g., Drug Metabolism and Disposition 36, 268-275 (2008) and Journal of Pharmaceutical Sciences 107 2225-2235 (2018). In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a cell permeability measurement (Papp) of <2×10−6 cm s−1. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a cell permeability measurement (Papp) of between 2-6×10−6 cm s−1 In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a cell permeability measurement (Papp) of Papp greater than 6×10−6 cm s−1. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a cell permeability greater than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, e.g., compared with a reference compound.

In some embodiments, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, exhibits decreased cell efflux, e.g., over a reference compound, e.g., in a standard assay for measuring cell efflux. Cell efflux may be investigated, for example, using a standard assay run in either Madin-Darby Canine Kidney (MDCK) cells expressing Breast Cancer Resistance Protein (BCRP) or subclone MDCKII cells expressing Multidrug Resistance Protein 1 (MDR1); see, e.g., Drug Metabolism and Disposition 36, 268-275 (2008) and Journal of Pharmaceutical Sciences 107 2225-2235 (2018). In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a cell efflux ratio of less than 1.5. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a cell efflux ratio of between 1.5 and 5. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a cell efflux ratio greater than 5. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a cell efflux ratio less than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, e.g., compared with a reference compound.

In some embodiments, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, modulates the expression of a target protein (e.g., HTT or MYB) in a reference cell or sample. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, increases the expression of a target protein (e.g., HTT or MYB) in a reference cell or sample. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, decreases the expression of a target protein (e.g., HTT or MYB) in a reference cell or sample. The effect of an exemplary compound of Formula (I) or (II) on protein abundance may be measured using a standard assay for measuring protein abundance, such as the HiBit-assay system (Promega). In this assay, percent response for each respective cell line may be as calculated at each compound concentration as follows: % response=100*(S−PC)/(NC−PC). For the normalized response at each concentration, a four-parameter logistical regression may be fit to the data and the response may be interpolated at the 50% value to determine a concentration for protein abundance at 50% (IC50) an untreated control. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a protein abundance response less than 100 nM. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a protein abundance response between 100-1000 nM. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a protein abundance response greater than 1000 nM. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a protein abundance response greater than 10 uM. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, modulates the protein abundance of a target protein by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, e.g., compared with a reference compound.

In some embodiments, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, modulates the viability of a target cell in a subject or sample. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, increases the viability of a target cell in a subject or sample. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, decreases the viability of a target cell in a subject or sample. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, does not impact the viability of a cell (e.g., is non-toxic) in a subject or sample. The effect an exemplary compound of Formula (I) or (II) on cell viability may be measured using a standard assay for measuring cell toxicity, such as the Cell Titer Glo 2.0 assay in either K562 (human chronic myelogenous leukemia) or SH-SY5Y (human neuroblastoma) cells. The concentration at which cell viability is measured may be based on the particular assay used. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, is tolerated by a target cell at a concentration of less than 100 nM. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, is tolerated by a target cell at a concentration of between 100-1000 nM. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, is tolerated by a target cell at a concentration of greater than 1000 nM. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, is tolerated by a target cell at a concentration of greater than 10 uM. In some embodiments, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has improved brain permeability over a reference compound, e.g., in a standard assay for measuring brain permeability. Brain permeability may be measured, for example, by determining the unbound partition coefficient (Kpuu), brain. In such an assay, the unbound brain partition coefficient (Kp,uu,bmin) may be defined as the ratio of unbound brain-free compound concentration to unbound plasma concentration. It is calculated using the following equation

K
   
    p
    ,
    uu
    ,
    brain
   
  
  =
  
   
    
     f
     
      u
      ,
      brain
     
    
    ×
    
     C
     brain
    
   
   
    
     f
     
      u
      ,
      plasma
     
    
    ×
    
     C
     plasma

Cbrain and Cplasma represent the total concentrations in brain and plasma, respectively. In this assay, the fu,brain and fu,plasma may be the unbound fraction of the compound in brain and plasma, respectively. Both fu,brain and fu,plasma may be determined in vitro via equilibrium dialysis. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a Kp value of greater than 5. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a Kp value between 1 and 5. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a Kp value between 0.2-1.

In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a Kp value of less than 0.2. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a Kpuu value of greater than 2.5. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a Kpuu value between 0.5-2.5. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a Kpuu value between 0.1-0.5. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a Kpuu value of less than 0.1. In an embodiment, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof, e.g., as described herein, has a brain permeability greater than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, e.g., compared with a reference compound.

In certain embodiments, the methods described herein comprise the additional step of administering one or more additional pharmaceutical agents in combination with the compound of Formula (I) or (II), a pharmaceutically acceptable salt thereof, or compositions comprising such compound or pharmaceutically acceptable salt thereof. Such additional pharmaceutical agents include, but are not limited to, anti-proliferative agents, anti-cancer agents, anti-diabetic agents, anti-inflammatory agents, immunosuppressant agents, and a pain-relieving agent. The additional pharmaceutical agent(s) may synergistically augment the modulation of splicing induced by the inventive compounds or compositions of this disclosure in the biological sample or subject. Thus, the combination of the inventive compounds or compositions and the additional pharmaceutical agent(s) may be useful in treating, for example, a cancer or other disease, disorder, or condition resistant to a treatment using the additional pharmaceutical agent(s) without the inventive compounds or compositions.

EXAMPLES

The compounds provided herein can be prepared from readily available starting materials using modifications to the specific synthesis protocols set forth below that would be well known to those of skill in the art. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in Greene et al., Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.

Reactions can be purified or analyzed according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance (NMR) spectroscopy (e.g., 1H or 13C), infrared (IR) spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry (MS), or by chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

Proton NMR: 1H NMR spectra were recorded in CDCl3 solution in 5-mm o.d. tubes (Wildmad) at 24° C. and were collected on a BRUKER AVANCE NEO 400 at 400 MHz for 1H. The chemical shifts (6) are reported relative to tetramethylsilane (TMS=0.00 ppm) and expressed in ppm.

Preparative HPLC purification: prep-HPLC purification was performed using one of the following HPLC conditions:

Reverse flash chromatography: purification by reverse flash chromatography was performed using one of the following conditions:

Thin Layer chromatography: purification by thin layer chromatography was performed using one of the following conditions:

General Synthetic Schemes

Compounds of the present disclosure may be prepared using a synthetic protocol illustrated in one of Schemes A, B, or C.

General Synthetic Schemes

Compounds of the present disclosure may be prepared using a synthetic protocol illustrated in one of Schemes A, B, or C.

Scheme A. An exemplary method of preparing a compound of Formula (I); wherein A, B, W, X, Y, Z, R2, and m are as defined herein; and LG1, LG2, and LG3 are each independently a leaving group (e.g., halo, —B(OR12)2). In some embodiments of the application, y is 0.

An exemplary method of preparing a compound described herein, e.g., a compound of Formula (II-I) is provided in Scheme A. In Step 1, B-2 is prepared by treating B-1 with a mixture of 2,2,6,6-tetramethylpiperidine, isopropylmagnesium chloride (iPrMgCl), lithium chloride (LiCl), iodine (I2), and zinc chloride (ZnCl2) in tetrahydrofuran (THF), or with a similar combination of reagents or solvent. In Step 2, B-3 is prepared by incubating B2 with 1,1′-bis(diphenylphosphino)ferrocene)palladium(II) dichloride (Pd(dppf)Cl2), carbon monoxide (CO), and triethylamine (TEA), in a mixture of methanol (MeOH) and dichloromethane (CH2Cl2) or a similar mixture of solvents. Alternative catalysts to Pd(dppf)Cl2 may also be used, such as a suitable palladium catalyst, and/or using alternative reagents sufficient to provide B-3.

In Step 3, B-5 is prepared by incubating B-3 with B-4 in the presence of RuPhos-Pd(II) (e.g., RuPhos-Pd(II)-G2 or RuPhos-Pd(II)-G3), and cesium carbonate (Cs2CO3) or a similar reagent. Step 3 may also be carried out using an alternative catalyst to RuPhos-Pd(II), such as another ruthenium catalyst. The reaction may be conducted in dioxane or a similar solvent, at 100° C. or a temperature sufficient to provide B-5. B-5 is then converted to B-6 by treatment with a mixture of ammonia and methanol, at 100° C. or a temperature sufficient to provide B-6.

B-6 and B-7 are coupled to provide a compound of Formula (II-I) in Step 5. This coupling reaction may be conducted in the presence of tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3, XantPhos, and cesium carbonate or a suitable alternative. Step 5 may also be carried out using an alternative catalyst to Pd2(dba)3, such as another palladium catalyst, and/or an alternative ligand to XantPhos (e.g., a different phosphine ligand). The reaction may be conducted in dioxane or a similar solvent, at 100° C. or a temperature sufficient to provide the compound of Formula (II-I). Each starting material and/or intermediate in Scheme B may be protected and deprotected using standard protecting group methods. In addition, purification and characterization of each intermediate as well as the final compound of Formula (II) may be afforded by any accepted procedure.

Scheme B. An exemplary method of preparing a compound of Formula (I); wherein A is as defined herein.

Scheme C. An exemplary method of preparing a compound of Formula (I); wherein B is as defined herein.

Exemplary protocols for the synthesis of compounds in Tables 1 and 2, e.g., Compounds 1-287, can be found in Examples 1-41 in WO 2021/174165, which is incorporated herein by reference in its entirety.

Example 42: Synthesis of Compound 303

Synthesis of Intermediate C1

Synthesis of Intermediate C2

Synthesis of Intermediate C3

Methyl 2-amino-4-bromo-5-fluoro-3-methylbenzoate (1.2 g, 4.579 mmol, 1.0 equiv) and Ac2O (0.6 g, 5.953 mmol, 1.3 equiv) were combined at 25° C. The resulting mixture was stirred for 1 h at 25° C. To the reaction mixture was added potassium acetate (0.13 g, 1.374 mmol, 0.3 equiv) and isoamyl nitrite (1.1 g, 10.074 mmol, 2.2 equiv). The resulting mixture was stirred for an additional 2 h at 80° C., then quenched with water (100 mL) at 25° C. and extracted with CH2C12 (2×100 mL). The organic layers were combined, dried by Na2SO4, filtered, and the filtrate concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 4-bromo-5-fluoro-1H-indazole-7-carboxylate (0.18 g, 12%) as a solid. LCMS (ES, m/z): 273 [M+H]+.

Synthesis of Intermediate C4

Methyl 4-bromo-5-fluoro-1H-indazole-7-carboxylate (50.0 mg, 0.183 mmol, 1.0 equiv), ethyl acetate (2 mL), and boron trifluoride trimethyloxidanium fluoride (108.3 mg, 0.732 mmol, 4.0 equiv) were combined at room temperature. The resulting mixture was stirred for 16 h at room temperature, then quenched with water and extracted with ethyl acetate (2×5 mL). The organic layers were combined, dried by Na2SO4, filtered, and the filtrate concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 4-bromo-5-fluoro-2-methylindazole-7-carboxylate (51 mg, 87%) as a solid. LCMS (ES, m/z): 287 [M+H]+.

Synthesis of Intermediate C5

Synthesis of Intermediate C6

Synthesis of Intermediate C7

Synthesis of Compound 303

Example 43: Synthesis of Compound 290

Synthesis of Intermediate C8

Synthesis of Intermediate C9

Synthesis of Intermediate C10

Synthesis of Intermediate C11

Synthesis of Compound 290

Example 44: Synthesis of Compound 295

Synthesis of Intermediate C12

To a stirred mixture of methyl 4-bromo-2H-indazole-7-carboxylate (650 mg, 2.54 mmol, 1.0 equiv) and 3-iodooxetane (937.6 mg, 5.09 mmol, 2 equiv) in DMF (6.5 mL) was added K2CO3 (704.3 mg, 5.09 mmol, 2 equiv) at room temperature. The resulting mixture was stirred overnight at 100° C. under nitrogen atmosphere, then cooled to room temperature. The resulting mixture was diluted with water (6 mL) and extracted with ethyl acetate (3×6 mL). The organic layers were combined, washed with brine (lx 10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 4-bromo-2-(oxetan-3-yl)indazole-7-carboxylate (200 mg, 25%) as a solid. LCMS (ES, m/z): 311 [M+H]+.

Synthesis of Intermediate C13

Synthesis of Intermediate C14

A solution of methyl 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-(oxetan-3-yl)indazole-7-carboxylate (200 mg, 0.4 mmol, 1.0 equiv) in tetrahydrofuran (2 mL) was treated with lithiumol hydrate (80.6 mg, 1.9 mmol, 4.0 equiv) in water (2 mL) at room temperature. The resulting mixture was stirred for 2 h at 50° C. under nitrogen atmosphere, then cooled to 0° C. The resulting mixture was acidified to pH 4 with HCl (1 M) and extracted with ethyl acetate (2×10 mL). The organic layers were combined, washed with brine (1×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA to afford 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-(oxetan-3-yl)indazole-7-carboxylic acid (160 mg, 83%) as a solid. LCMS (ES, m/z): 403 [M+H]+.

Synthesis of Intermediate C15

To a stirred mixture of 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-(oxetan-3-yl)indazole-7-carboxylic acid (160 mg, 0.39 mmol, 1.0 equiv) and 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (72.2 mg, 0.43 mmol, 1.1 equiv) in DMF (1 mL) was added DIEA (154.1 mg, 1.19 mmol, 3.0 equiv) and HATU (226.7 mg, 0.59 mmol, 1.5 equiv) at room temperature. The resulting mixture was stirred for 2 h at room temperature under nitrogen atmosphere, then quenched with water and extracted with ethyl acetate (2×10 mL). The organic layers were combined, washed with brine (1×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:2) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(oxetan-3-yl)indazol-4-yl]piperazine-1-carboxylate (100 mg, 46%) as a solid. LCMS (ES, m/z): 550 [M+H]+.

Synthesis of Compound 295

Example 45: Synthesis of Compound 299

Synthesis of Intermediate C16

Synthesis of Intermediate C17

A mixture of methyl 4-[1-(tert-butoxycarbonyl)-1,6-diazaspiro[3.4]octan-6-yl]-2-methylindazole-7-carboxylate (170.0 mg, 0.424 mmol, 1.0 equiv), NaOH (169.7 mg, 4.240 mmol, 10.0 equiv), methanol (3 mL), and water (3 mL) was stirred for 5 h at 50° C. The resulting mixture was dilute d with water (50 mL), acidified to pH 5 with 1 N of HCl, and extracted with ethyl acetate (2×50 mL). The resulting mixture was concentrated under reduced pressure to afford 4-I[1-(tert-butoxycarbonyl)-1,6-diazaspiro[3.4]octan-6-yl]-2-methylindazole-7-carboxylic acid (160 mg, 88%) as a solid. LCMS (ES, m/z): 387 [M+H]+.

Synthesis of Intermediate C18

Synthesis of Compound 299

Example 46: Synthesis of Compound 316

Synthesis of Intermediate C19

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100.0 mg, 0.20 mmol, 1.0 equiv) and 2,2,2-trifluoroethyl trifluoromethanesulfonate (70.5 mg, 0.30 mmol, 1.5 equiv) in DMF (1 mL) was added Cs2CO3 (198.0 mg, 0.60 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 5 h at room temperature, then quenched with water (2 mL) and extracted with ethyl acetate (3×5 mL). The organic layers were combined, washed with brine (1×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2,2,2-trifluoroethyl)indazol-4-yl]piperazine-1-carboxylate (52 mg, 44.59%) as a solid. LCMS (ES, m/z): 576 [M+H]+.

Synthesis of Compound 316

Example 47: Synthesis of Compound 304

Synthesis of Intermediate C20

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100 mg, 0.20 mmol, 1.0 equiv) and 4-iodooxane (64.4 mg, 0.30 mmol, 1.5 equiv) in DMF (1 mL) was added Cs2CO3 (198.0 mg, 0.61 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature, then quenched with water at room temperature and extracted with ethyl acetate (3×5 mL). The organic layers were combined, washed with brine (1×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(oxan-4-yl)indazol-4-yl]piperazine-1-carboxylate (41 mg, 35%) as a solid. LCMS (ES, m/z): 578 [M+H]+.

Synthesis of Compound 394

Example 48: Synthesis of Compound 357

Synthesis of Intermediate C22

A solution of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2-oxopropyl)indazol-4-yl]piperazine-1-carboxylate (90.0 mg, 0.16 mmol, 1.0 equiv) in methanol (1 mL) was treated with NaBH4 (7.4 mg, 0.19 mmol, 1.2 equiv) at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature, then quenched with sat. NH4Cl (aq.) at 0° C. and extracted with ethyl acetate (2×5 mL). The organic layers were combined, washed with brine (1×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:4) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2-oxopropyl)indazol-4-yl]piperazine-1-carboxylate (57 mg, 63%) as a solid. LCMS (ES, m/z): 552 [M+H]+.

Synthesis of Compound 357

Example 49: Synthesis of Compound 354

Synthesis of Intermediate C23

A mixture of 1-chloro-2-methyl-2-propanol (1.5 g, 13.816 mmol, 1.0 equiv), DCM (15 mL), imidazole (1.8 g, 27.615 mmol, 2.0 equiv), and t-butyldimethylchlorosilane (3.1 g, 20.701 mmol, 1.5 equiv) was stirred for 3 h at room temperature. The reaction mixture was quenched with water (100 mL) at room temperature and extracted with CH2Cl2 (2×100 mL). The organic layers were combined, dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (5:1) to afford tert-butyl[(1-chloro-2-methylpropan-2-yl)oxy]dimethylsilane (1.2 g, 35%) as an oil.

Synthesis of Intermediate C24

Synthesis of Compound 354

Example 50: Synthesis of Compound 358

Synthesis of Intermediate C25

Synthesis of Intermediate C26

Methyl 1-(oxan-2-yloxy)cyclopropane-1-carboxylate (400.0 mg, 1.998 mmol, 1.0 equiv), tetrahydrofuran (10 mL), and LiAlH4 (113.7 mg, 2.997 mmol, 1.5 equiv) were combined at 0° C. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere, quenched with water (100 mL) at 0° C. and extracted with ethyl acetate (2×100 mL). The organic layers were combined, dried over Na2SO4, and filtered. The resulting mixture was concentrated under reduced pressure to give a solid.

Synthesis of Intermediate C27

Synthesis of Intermediate C28

Synthesis of Compound 358

Example 51: Synthesis of Compound 305

Synthesis of Intermediate C29

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100.0 mg, 0.20 mmol, 1 equiv) and 1-fluoro-2-iodo-ethane, (52.8 mg, 0.30 mmol, 1.5 equiv) in DMF (1 mL) was added Cs2CO3 (198.0 mg, 0.60 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature, then diluted with water and extracted with ethyl acetate (3×5 mL). The organic layers were combined, washed with brine (1×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2-fluoroethyl)indazol-4-yl]piperazine-1-carboxylate (41 mg, 38%) as a solid. LCMS (ES, m/z): 540 [M+H]+.

Synthesis of Compound 305

Example 52: Synthesis of Compound 302

Synthesis of Intermediate C30

Synthesis of Intermediate C31

A solution of methyl 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-211-indazole-7-carboxylate (10.4 g, 28.8 mmol, 1 equiv) in THE (100 mL) was treated with LiOH·H2O (2.7 g, 115.4 mmol, 4 equiv) in water (100 mL) at room temperature. The resulting mixture was stirred for 2 h at 50° C. under nitrogen atmosphere, then cooled to 0° C. The resulting mixture was acidified to pH 4 with HCl (1 M) and extracted with ethyl acetate (3×150 mL). The organic layers were combined, washed with brine (1×100 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (15:1) to afford 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2H-indazole-7-carboxylic acid (9.6 g, 96%) as a solid. LCMS (ES, m/z): 347 [M+H]f.

Synthesis of Intermediate C32

To a stirred mixture of 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2H-indazole-7-carboxylic acid (9.6 g, 27.7 mmol, 1.0 equiv), 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (5.0 g, 30.4 mmol, 1.1 equiv), and NMI (9.1 g, 110.8 mmol, 4 equiv) in acetonitrile (100 mL) was added TCFH (9.3 g, 33.2 mmol, 1.2 equiv) at room temperature. The resulting mixture was stirred for 2 h at room temperature under nitrogen atmosphere, then diluted with water and extracted with ethyl acetate (3×100 mL). The organic layers were combined, washed with brine (1×100 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:4) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (10.6 g, 77%) as a solid. LCMS (ES, m/z): 494 [M+H]f.

Synthesis of Intermediate C33

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (60 mg, 0.12 mmol, 1 equiv) and 1-bromo-3-methoxypropane (27.9 mg, 0.18 mmol, 1.5 equiv) in DMF (0.6 mL) was added Cs2CO3 (118.8 mg, 0.37 mmol, 3 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature, then diluted with water and extracted with ethyl acetate (3×5 mL). The organic layers were combined, washed with brine (1×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(3-methoxypropyl)indazol-4-yl]piperazine-1-carboxylate (34 mg, 49%) as a solid. LCMS (ES, m/z): 566 [M+H]+.

Synthesis of Compound 302

Example 53: Synthesis of Compound 338

Synthesis of Intermediate C34

To a stirred solution of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (110 mg, 0.223 mmol, 1.0 equiv) and 3-(iodomethyl)oxetane (66.20 mg, 0.335 mmol, 1.5 equiv) in DMF (2.2 mL) was added Cs2CO3 (217.8 mg, 0.669 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 1 h at room temperature, then diluted with water (10 mL) and extracted with ethyl acetate (3×10 mL). The organic layer were combined, washed with water (3×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA (100%) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(oxetan-3-ylmethyl)indazol-4-yl]piperazine-1-carboxylate (65 mg, 52%) as a solid. LCMS (ES, m/z): 423.2 [M+H]+.

Synthesis of Compound 338

Example 54: Synthesis of Compound 317

Synthesis of Intermediate C35

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100.0 mg, 0.20 mmol, 1.0 equiv) and epibromohydrin (41.6 mg, 0.30 mmol, 1.5 equiv) in DMF (1 mL) was added Cs2CO3 (198.0 mg, 0.60 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature, then diluted with water (2 mL) and extracted with ethyl acetate (3×5 mL). The organic layers were combined, washed with brine (1×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(oxiran-2-ylmethyl)indazol-4-yl]piperazine-1-carboxylate (55 mg, 49%) as a solid. LCMS (ES, m/z): 550 [M+H]+.

Synthesis of Compound 317

A solution of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(oxiran-2-ylmethyl)indazol-4-yl]piperazine-1-carboxylate (55 mg, 0.10 mmol, 1.0 equiv) in DCM (0.5 mL) was treated with TFA (0.1 mL) at 0° C. The resulting mixture was stirred for 30 min at 0° C., then concentrated under reduced pressure to give a residue. The residue was purified by Prep-HPLC (Condition 2, Gradient 3) to afford N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}-2-(oxiran-2-ylmethyl)-4-(piperazin-1-yl)indazole-7-carboxamide (2 mg, 4%) as a solid. LCMS (ES, m/z): 450 [M+H]+.

Example 55: Synthesis of Compound 339

Synthesis of Intermediate C21

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (300.0 mg, 0.61 mmol, 1.0 equiv) and 1-chloropropan-2-one (67.4 mg, 0.73 mmol, 1.2 equiv) in DMF (3 mL) was added Cs2CO3 (594.1 mg, 1.82 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 1 h at room temperature, then diluted with water and extracted with ethyl acetate (3×6 mL). The organic layers were combined, washed with brine (1×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:3) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2-oxopropyl)indazol-4-yl]piperazine-1-carboxylate (135 mg, 40%) as a solid. LCMS (ES, m/z): 550 [M+H]+.

Synthesis of Compound 339

Example 56: Synthesis of Compound 308

Synthesis of Intermediate C36

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100 mg, 0.20 mmol, 1 equiv) and 3-(bromomethyl)-3-methyloxetane (50.1 mg, 0.30 mmol, 1.5 equiv) in DMF (1 mL) was added Cs2CO3 (198.0 mg, 0.60 mmol, 3 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature, then diluted with water (2 mL) and extracted with ethyl acetate (3×2 mL). The organic layers were combined, washed with brine (1×2 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-[(3-methyloxetan-3-yl)methyl] indazol-4-yl]piperazine-1-carboxylate (42 mg, 36%) as a solid. LCMS (ES, m/z): 578 [M+H]+.

Synthesis of Compound 308

Example 57: Synthesis of Compound 331

Synthesis of Intermediate C37

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (120.0 mg, 0.24 mmol, 1.0 equiv) and methyl chloroacetate (39.5 mg, 0.36 mmol, 1.5 equiv) in DMF (1.2 mL) was added Cs2CO3 (237.6 mg, 0.73 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature, then diluted with water (3 mL) and extracted with ethyl acetate (3×2 mL). The organic layers were combined, washed with brine (1×2 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2-methoxy-2-oxoethyl)indazol-4-yl]piperazine-1-carboxylate (48 mg, 35%) as a solid. LCMS (ES, m/z): 566 [M+H]+.

Synthesis of Intermediate C38

A solution of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2-methoxy-2-oxoethyl)indazol-4-yl]piperazine-1-carboxylate (48.0 mg, 0.08 mmol, 1.0 equiv) in tetrahydrofuran (0.5 mL) was treated with LiOH·H2O (10.1 mg, 0.42 mmol, 5.0 equiv) in water (0.5 mL) at room temperature. The resulting mixture was stirred for 2 h at 50° C. under nitrogen atmosphere, then cooled to 0° C., acidified to pH 4 with HCl (1 M), and extracted with ethyl acetate (2×5 mL). The organic layers were combined, washed with brine (1×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford {4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)indazol-2-yl}acetic acid (34 mg, 73%) as a solid. LCMS (ES, m/z): 552 [M+H]+.

Synthesis of Compound 331

Example 58: Synthesis of Compound 309

Synthesis of Intermediate C39

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100.0 mg, 0.20 mmol, 1.0 equiv) and 2-chloro-N,N-dimethylacetamide (36.9 mg, 0.30 mmol, 1.5 equiv) in DMF (1 mL) was added Cs2CO3 (198.0 mg, 0.60 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature, then diluted with water (3 mL) and extracted with ethyl acetate (3×5 mL). The organic layers were combined, washed with brine (1×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-{2-[(dimethylcarbamoyl)methyl]-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)indazol-4-yl}piperazine-1-carboxylate (45 mg, 38%) as a solid. LCMS (ES, m/z): 579 [M+H]+.

Synthesis of Compound 309

Example 59: Synthesis of Compound 355

Synthesis of Intermediate C40

Synthesis of Intermediate C41

A mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(3-oxocyclobutyl)indazol-4-yl]piperazine-1-carboxylate (50.0 mg, 0.089 mmol, 1.0 equiv), methanol (1 mL), and NaBH4 (6.7 mg, 0.178 mmol, 2.0 equiv) was stirred for 1 h at 0° C. under nitrogen atmosphere. The reaction was quenched with water (5 mL) and extracted with ethyl acetate (2×5 mL). The organic layers were combined, dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (30:1) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(3-hydroxycyclobutyl)indazol-4-yl]piperazine-1-carboxylate (45 mg, 81%) as a solid. LCMS (ES, m/z): 564 [M+H]+.

Synthesis of Compound 355

Example 60: Synthesis of Compound 311

Synthesis of Intermediate C42

A mixture of methyl 2-amino-4-bromo-5-fluoro-3-methylbenzoate (1.2 g, 4.579 mmol, 1.0 equiv) and Ac2O (0.6 g, 5.953 mmol, 1.3 equiv) was stirred for 1 h at 25° C. To the reaction mixture was added potassium acetate (0.13 g, 1.374 mmol, 0.3 equiv) and isoamyl nitrite (1.1 g, 10.074 mmol, 2.2 equiv). The resulting mixture was stirred for an additional 2 h at 80° C., then quenched with water (100 mL) and extracted with CH2Cl2 (2×100 mL). The organic layers were combined, dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl methyl 5-(acetyloxy)-4-bromo-1H-indazole-7-carboxylate (800 g, 50%) as a solid. LCMS (ES, m/z): 313 [M+H]+.

Synthesis of Intermediate C43

A mixture of methyl 5-(acetyloxy)-4-bromo-2H-indazole-7-carboxylate (0.8 g, 2.555 mmol, 1.0 equiv), ethyl acetate (15 mL), and (CH3)3O+BF4− (1.5 g, 10.220 mmol, 4.0 equiv) was stirred for 16 h at room temperature. The reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (2×50 mL). The organic layer were combined, dried over Na2SO4, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 5-(acetyloxy)-4-bromo-2-methylindazole-7-carboxylate (780 mg, 84%) as a solid. LCMS (ES, m/z): 327 [M+H]+.

Synthesis of Intermediate C44

A mixture of methyl 5-(acetyloxy)-4-bromo-2-methylindazole-7-carboxylate (0.8 g, 2.445 mmol, 1.0 equiv), methanol (10 mL), water (5 mL), and K2CO3 (1.0 g, 7.335 mmol, 3.0 equiv) was stirred for 1 h at room temperature. The reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (2×100 mL). The organic layers were combined, dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 4-bromo-5-hydroxy-2-methylindazole-7-carboxylate (410 mg, 53%) as a solid. LCMS (ES, m/z): 285 [M+H]+.

Synthesis of Intermediate C45

A mixture of methyl 4-bromo-5-hydroxy-2-methylindazole-7-carboxylate (0.8 g, 2.806 mmol, 1.0 equiv), K2CO3 (0.8 g, 5.612 mmol, 2.0 equiv), DMF (15 mL), and methyl iodide (0.8 g, 5.612 mmol, 2.0 equiv) was stirred for 1 h at room temperature. The reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (2×100 mL). The organic layers were combined, dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 4-bromo-5-methoxy-2-methylindazole-7-carboxylate (750 mg, 79%) as a solid. LCMS (ES, m/z): 299 [M+H]+.

Synthesis of Intermediate C46

Synthesis of Intermediate C47

A mixture of methyl 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-5-methoxy-2-methylindazole-7-carboxylate (0.9 g, 2.225 mmol, 1.0 equiv), THE (10 mL), water (5 mL), and LiOH (0.5 g, 22.250 mmol, 10.0 equiv) was stirred for 3 h at 50° C. The resulting mixture was diluted with water (100 mL), acidified to pH 5 with HCl (aq.), and extracted with ethyl acetate (2×100 mL). The organic layers were combined, dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to afford 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-5-methoxy-2-methylindazole-7-carboxylic acid (0.63 g, 73%) as a solid. LCMS (ES, m/z): 391 [M+H]+.

Synthesis of Intermediate C48

Synthesis of Compound 311

Example 61: Synthesis of Compound 312

Synthesis of Intermediate C49

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100 mg, 0.20 mmol, 1 equiv) and butyl iodide (55.9 mg, 0.30 mmol, 1.5 equiv) in DMF (1 mL) was added Cs2CO3 (198.0 mg, 0.60 mmol, 3 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature, then diluted with water (3 mL) and extracted with ethyl acetate (3×5 mL). The organic layers were combined, washed with brine (1×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-[2-butyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)indazol-4-yl]piperazine-1-carboxylate (42 mg, 38%) as a solid. LCMS (ES, m/z): 550 [M+H]+.

Synthesis of Compound 312

Example 62: Synthesis of Compound 313

Synthesis of Intermediate C50

Synthesis of Compound 313

Example 63: Synthesis of Compound 318

Synthesis of Intermediate C51

Synthesis of Compound 318

Example 64: Synthesis of Compound 341

Synthesis of Intermediate C52

Synthesis of Intermediate C53

Synthesis of Compound 341

Example 65: Synthesis of Compound 326

Synthesis of Intermediate C54

To a stirred mixture of methyl 4-bromo-2-methylindazole-7-carboxylate (1.6 g, 5.946 mmol, 1 equiv) in THE (1.5 mL) and water (0.5 mL) was added lithiumol hydrate (0.50 g, 11.892 mmol, 2 equiv) at room temperature. The resulting mixture was stirred overnight at room temperature. The resulting solution was concentrated under reduced pressure and acidified to pH 6 with citric acid to afford a precipitate. The precipitated solid was collected by filtration and dried under infrared light to afford 4-bromo-2-methylindazole-7-carboxylic acid (1.3 g, 86%) as a solid. LCMS (ES, m/z): 255 [M+H]+.

Synthesis of Intermediate C55

Synthesis of Intermediate C56

Synthesis of Compound 326

Example 66: Synthesis of Compound 345

Synthesis of Intermediate C57

Synthesis of Compound 345

Example 67: Synthesis of Compound 335

Synthesis of Intermediate C58

Synthesis of Compound 335

Example 68: Synthesis of Compound 327

Synthesis of Intermediate C59

Synthesis of Compound 327

Example 69: Synthesis of Compound 328

Synthesis of Compound 328

Example 70: Synthesis of Compound 336

Synthesis of Intermediate C60

Synthesis of Compound 336

Example 71: Synthesis of Compound 346

Synthesis of Intermediate C61

Synthesis of Compound 346

Example 72: Synthesis of Compound 329

Synthesis of Compound 329

Example 73: Synthesis of Compound 356

Synthesis of Intermediate C62

A mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100.0 mg, 0.2 mmol, 1.0 equiv), 3-bromooxolane (45.8 mg, 0.3 mmol, 1.5 equiv), and Cs2CO3 (198.0 mg, 0.6 mmol, 3.0 equiv) in DMF (1 mL) was stirred for 1 h at room temperature under nitrogen atmosphere. The reaction mixture was diluted with water (3 mL) and extracted with ethyl acetate (2×5 mL). The organic layers were combined, washed with brine (1×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:4) to afford tert-butyl-4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(oxolan-3-yl)indazol-4-yl]piperazine-1-carboxylate (52.0 mg, 46%) as a solid. LCMS (ES, m/z): 564 [M+H]+.

Synthesis of Compound 356

Example 74: Synthesis of Compounds 350 and 351 Synthesis of Compounds 350 and 351

Example 75: Synthesis of Compound 288

Synthesis of Intermediate C63

To a stirred mixture of 2-amino-4-bromo-3-methylbenzoic acid (10 g, 43.467 mmol, 1.0 equiv) and Cs2CO3 (21.2 g, 65.201 mmol, 1.5 equiv) in DMF (100 mL) was added CH3I (7.4 g, 52.160 mmol, 1.2 equiv) in portions at 0° C. under N2 atmosphere. The resulting mixture was stirred for 2 h at room temperature under N2 atmosphere. The resulting mixture was diluted with water (300 mL) and extracted with ethyl acetate (2×300 mL). The organic layers were combined, washed with water (3×400 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to afford methyl 2-amino-4-bromo-3-methylbenzoate (10.4 g, 98%) as a solid. LCMS (ES, m/z): 244 [M+H]+.

Synthesis of Intermediate C64

Synthesis of Intermediate C65

To a stirred solution of methyl 4-bromo-2H-indazole-7-carboxylate (1 g, 3.920 mmol, 1.00 equiv) in ethyl acetate (7.5 mL) was added Et3O+BF4− (3724.20 mg, 19.600 mmol, 5.0 equiv) in portions at room temperature. The resulting mixture was stirred for 3 h at room temperature. The resulting mixture was diluted with water (10 mL) and extracted with ethyl acetate (3×20 mL). The organic layers were combined, washed with water (2×20 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 4-bromo-2-ethylindazole-7-carboxylate (610 mg, 55%) as a solid. LCMS (ES, m/z): 283 [M+H]+.

Synthesis of Intermediate C66

Synthesis of Intermediate C67

To a stirred mixture of methyl 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-ethylindazole-7-carboxylate (210 mg, 0.541 mmol, 1.00 equiv) in THE (1.1 mL) and water (0.55 mL) was added LiOH (51.78 mg, 2.164 mmol, 4.0 equiv) at room temperature. The resulting mixture was stirred for 1 h at 50° C. The resulting mixture was concentrated under reduced pressure, diluted with water (2 mL), acidified to pH 7 with HCl (1 N), and extracted with ethyl acetate (3×3 mL). The organic layers were combined, dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to afford 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-ethylindazole-7-carboxylic acid (200 mg, 99%) as a solid. LCMS (ES, m/z): 375 [M+H]+.

Synthesis of Intermediate C68

To a stirred mixture of 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-ethylindazole-7-carboxylic acid (50 mg, 0.134 mmol, 1.00 equiv) and 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (22.06 mg, 0.134 mmol, 1.0 equiv) in DMF (1 mL) was added DIEA (34.5 mg, 0.268 mmol, 2.0 equiv) and HATU (60.9 mg, 0.161 mmol, 1.2 equiv) dropwise at room temperature. The resulting mixture was stirred for 3 h at room temperature. The resulting mixture was diluted with water (3 mL) and extracted with ethyl acetate (3×3 mL). The organic layers were combined, washed with water (3×3 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 4-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)indazol-4-yl]piperazine-1-carboxylate (58 mg, 83%) as a solid. LCMS (ES, m/z): 522 [M+H]+.

Synthesis of Compound 288

Example 76: Synthesis of Compound 289

Synthesis of Intermediate C69

Synthesis of Intermediate C70

To a stirred solution of methyl 4-[(1R,5S)-8-(tert-butoxycarbonyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]-2-ethylindazole-7-carboxylate (280 mg, 0.676 mmol, 1.0 equiv) in THE (1.4 mL) and water (1.4 mL) was added LiOH·H2O (113.4 mg, 2.702 mmol, 4.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at 50° C., then concentrated under reduced pressure to give a residue. The resulting mixture was diluted with water (2 mL) and extracted with ethyl acetate (3×3 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to afford 4-[(1R,5S)-8-(tert-butoxycarbonyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]-2-ethylindazole-7-carboxylic acid (250 mg, 92%) as a solid. LCMS (ES, m/z): 401 [M+H]+.

Synthesis of Intermediate C71

To a stirred solution of 4-[(1R,5S)-8-(tert-butoxycarbonyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]-2-ethylindazole-7-carboxylic acid (50.0 mg, 0.125 mmol, 1.0 equiv) and 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (20.6 mg, 0.125 mmol, 1.0 equiv) in DMF (1 mL) was added DIEA (48.4 mg, 0.375 mmol, 3.0 equiv) and HATU (85.5 mg, 0.225 mmol, 1.8 equiv) at room temperature. The resulting mixture was stirred for 4 h at room temperature, then diluted with water (3 mL) and extracted with ethyl acetate (3×3 mL). The organic layers were combined, washed with water (3×3 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl (1R,5S)-3-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)indazol-4-yl]-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (60 mg, 88%) as a solid. LCMS (ES, m/z): 548 [M+H]+.

Synthesis of Compound 289

Example 77: Synthesis of Compound 293

Synthesis of Intermediate C72

A mixture of 3-methylpyrazin-2-amine (2.00 g, 18.326 mmol, 1.0 equiv) and NBS (3.59 g, 20.159 mmol, 1.1 equiv) in DMF (40 mL) was stirred for 1.5 h at room temperature. The resulting mixture was diluted with water (100 mL) and extracted with ethyl acetate (3×80 mL). The organic layers were combined, washed with brine (3×80 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford 5-bromo-3-methylpyrazin-2-amine (2.13 g, 62%) as a solid. LCMS (ES, m/z): 188.1 [M+H]+.

Synthesis of Intermediate C73

To a stirred mixture of 5-bromo-3-methylpyrazin-2-amine (2.1 g, 11.169 mmol, 1.0 equiv) and 1-bromo-2,2-dimethoxypropane (2.45 g, 13.403 mmol, 1.2 equiv) in isopropanol (41 mL) was added PPTS (196.5 mg, 0.782 mmol, 0.07 equiv) in portions at room temperature. The resulting mixture was stirred for 16 h at 80° C., then diluted with water (40 mL) and extracted with ethyl acetate (4×40 mL). The organic layers were combined, dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA to afford 6-bromo-2,8-dimethylimidazo[1,2-a]pyrazine (890 mg, 35%) as a solid. LCMS (ES, m/z): 226 [M+H]+. Synthesis of Intermediate C74

Synthesis of Intermediate C75

To a stirred solution of N-{2,8-dimethylimidazo[1,2-a]pyrazin-6-yl}-1,1-diphenylmethanimine (620 mg, 1.899 mmol, 1.0 equiv) in THE (12 mL) was added HCl (6 mL, con) dropwise at room temperature. The resulting mixture was stirred for 2 h at room temperature. The resulting mixture was diluted with water (15 mL), basified to pH 8 with saturated Na2CO3 (aq.), and extracted with ethyl acetate (3×20 mL). The organic layers were combined, dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (10:1) to afford 2,8-dimethylimidazo[1,2-a]pyrazin-6-amine (160 mg, 52%) as a solid. LCMS (ES, m/z): 163 [M+H]+.

Synthesis of Intermediate C76

To a stirred solution of methyl 4-bromo-2H-indazole-7-carboxylate (500 mg, 1.960 mmol, 1.00 equiv) and in ethyl acetate (7.5 mL) was added Me3OBF4 (1449.67 mg, 9.800 mmol, 5.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature, then diluted with water (10 mL) and extracted with ethyl acetate (3×10 mL). The organic layers were combined, washed with water (2×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA to afford methyl 4-bromo-2-methylindazole-7-carboxylate (410 mg, 78%) as a solid. LCMS (ES, m/z): 269 [M+H]+.

Synthesis of Intermediate C77

Synthesis of Intermediate C78

To a stirred mixture of methyl 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-methylindazole-7-carboxylate (300 mg, 0.801 mmol, 1.00 equiv) in THE (1.5 mL) and water (1.5 mL) was added LiOH·H2O (95.9 mg, 4.005 mmol, 5 equiv) at room temperature. The resulting mixture was stirred for 3 h at 50° C., then concentrated under vacuum. The resulting mixture was diluted with water (6 mL), acidified to pH 7 with concentrated HCl, and extracted with ethyl acetate (4×10 mL). The organic layers were combined, dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to afford 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-methylindazole-7-carboxylic acid (200 mg, 69%) as a solid. LCMS (ES, m/z): 361 [M+H]+.

Synthesis of Intermediate C79

To a stirred mixture of 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-methylindazole-7-carboxylic acid (80.0 mg, 0.222 mmol, 1.0 equiv) and 2,8-dimethylimidazo[1,2-a]pyrazin-6-amine (36.0 mg, 0.222 mmol, 1.0 equiv) in DMF (1.6 mL) was added DIEA (86.1 mg, 0.666 mmol, 3.0 equiv) and HATU (151.9 mg, 0.400 mmol, 1.8 equiv) at room temperature. The resulting mixture was stirred for 7 h at room temperature, then diluted with water (3 mL) and extracted with ethyl acetate (3×5 mL). The organic layers were combined, washed with water (3×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 4-[7-({2,8-dimethylimidazo[1,2-a]pyrazin-6-yl}carbamoyl)-2-methylindazol-4-yl]piperazine-1-carboxylate (60 mg, 54%) as a solid. LCMS (ES, m/z): 505 [M+H]+. Synthesis of Compound 293

Example 78: Synthesis of Compound 291

Synthesis of Compound 291

Example 79: Synthesis of Compound 292

Synthesis of Intermediate C80

To a stirred mixture of methyl 4-bromo-2H-indazole-7-carboxylate (600.0 mg, 2.352 mmol, 1.0 equiv) and K2CO3 (650.0 mg, 4.704 mmol, 2.0 equiv) in DMF (6 mL) was added 2-bromoethyl methyl ether (490.4 mg, 3.528 mmol, 1.5 equiv) at room temperature. The resulting mixture was stirred for 16 h at 80° C., then diluted with water (15 mL) and extracted with ethyl acetate (3×20 mL). The organic layers were combined, washed with water (3×15 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 4-bromo-2-(2-methoxyethyl)indazole-7-carboxylate (200 mg, 27%) as a solid. LCMS (ES, m/z): 312 [M+H]+.

Synthesis of Intermediate C81

Synthesis of Intermediate C82

To a stirred mixture of methyl 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-(2-methoxyethyl)indazole-7-carboxylate (320.0 mg, 0.765 mmol, 1.0 equiv) in THE (1.6 mL) and water (1.6 mL) was added LiOH·H2O (91.6 mg, 3.825 mmol, 5.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature, then concentrated under vacuum. The resulting mixture was diluted with water (5 mL), acidified to pH 6 with HCl (1 N), and extracted with ethyl acetate (4×30 mL). The organic layers were combined, dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure. to afford 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-(2-methoxyethyl)indazole-7-carboxylic acid (220 mg, 71%) as a solid. LCMS (ES, m/z): 405 [M+H]+.

Synthesis of Intermediate C83

To a stirred mixture of 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-(2-methoxyethyl)indazole-7-carboxylic acid (220.0 mg, 0.544 mmol, 1.0 equiv) and 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (89.8 mg, 0.544 mmol, 1.0 equiv) in DMF (4.4 mL) was added DIEA (210.9 mg, 1.632 mmol, 3.0 equiv) and HATU (372.3 mg, 0.979 mmol, 1.8 equiv) at room temperature. The resulting mixture was stirred for 4 h at room temperature, then diluted with water (12 mL) and extracted with ethyl acetate (3×10 mL). The organic layers were combined, washed with brine (3×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2-methoxyethyl)indazol-4-yl]piperazine-1-carboxylate (170 mg, 34%) as a solid. LCMS (ES, m/z): 552 [M+H]+.

Synthesis of Compound 292

Example 80: Synthesis of Compound 296

Synthesis of Intermediate C84

Synthesis of Intermediate C85

To a stirred solution of methyl 4-{3-[(tert-butoxycarbonyl)(cyclopropyl)amino]pyrrolidin-1-yl}-2-methylindazole-7-carboxylate (100.0 mg, 0.241 mmol, 1.0 equiv) in THE (1.25 mL) and water (1.25 mL) was added LiOH·H2O (81.0 mg, 1.928 mmol, 8.0 equiv) at room temperature. The resulting mixture was stirred for 2 h at 50° C., then concentrated under vacuum. The resulting mixture was diluted with water (2 mL), acidified to pH 7 with HCl (1 N), and extracted with ethyl acetate (3×2 mL). The organic layers were combined, dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to afford 4-{3-[(tert-butoxycarbonyl)(cyclopropyl)amino]pyrrolidin-1-yl}-2-methylindazole-7-carboxylic acid (80 mg, 83%) as a solid. LCMS (ES, m/z): 401 [M+H]+.

Synthesis of Intermediate C86

To a stirred solution of 4-{3-[(tert-butoxycarbonyl)(cyclopropyl)amino]pyrrolidin-1-yl}-2-methylindazole-7-carboxylic acid (80.0 mg, 0.200 mmol, 1.0 equiv) and 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (33.0 mg, 0.200 mmol, 1.0 equiv) in DMF (1.4 mL) was added DIEA (77.5 mg, 0.600 mmol, 3.0 equiv) and HATU (113.9 mg, 0.300 mmol, 1.5 equiv) at room temperature. The resulting mixture was stirred for 3 h at 50° C., then diluted with water (1 mL) and extracted with ethyl acetate (3×2 mL). The organic layers were combined, washed with water (3×2 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA (100%) to afford tert-butyl N-cyclopropyl-N-{1-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-methylindazol-4-yl]pyrrolidin-3-yl}carbamate (80 mg, 73%) as an oil. LCMS (ES, m/z): 548 [M+H]+.

Synthesis of Compound 296

Example 81: Synthesis of Compound 298

Synthesis of Intermediate C87

To a stirred solution of methyl 4-bromo-2H-indazole-7-carboxylate (0.5 g, 1.960 mmol, 1.0 equiv) in ethyl acetate (7.5 mL) was added tetrafluoroboranuide; trimethyloxidanium (1.45 g, 9.800 mmol, 5.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature, then diluted with water (10 mL) and extracted with ethyl acetate (3×10 mL). The organic layers were combined, washed with water (2×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA to afford methyl 4-bromo-2-methylindazole-7-carboxylate (560 mg, 99%) as a solid. LCMS (ES, m/z): 269 [M+H]+.

Synthesis of Intermediate C88

Synthesis of Intermediate C89

To a stirred solution of methyl 4-{3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl}-2-methylindazole-7-carboxylate (290.0 mg, 0.747 mmol, 1.0 equiv) in THF (3.7 mL) was added water (3.7 mL) and lithiumol hydrate (156.6 mg, 3.735 mmol, 5.0 equiv) at room temperature. The resulting mixture was stirred for 16 h at 50° C. The resulting mixture was concentrated under vacuum, diluted with water (10 mL), acidified to pH 7 with concentrated HCl, and extracted with ethyl acetate (3×10 mL). The organic layers were combined, washed with brine (3×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to afford 4-{3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl}-2-methylindazole-7-carboxylic acid (270 mg, 97%) as a solid. LCMS (ES, m/z): 375 [M+H]+.

Synthesis of Intermediate C90

To a stirred solution of 4-{3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl}-2-methylindazole-7-carboxylic acid (70.0 mg, 0.187 mmol, 1.0 equiv) in DMF (1.4 mL) was added HATU (106.6 mg, 0.280 mmol, 1.5 equiv), DIEA (72.5 mg, 0.561 mmol, 3.0 equiv), and 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (30.9 mg, 0.187 mmol, 1 equiv) in portions at room temperature. The resulting mixture was stirred for 7 h at room temperature, then diluted with water (5 mL) and extracted with ethyl acetate (3×5 mL). The organic layers were combined, washed with water (3×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA (100%) to afford tert-butyl N-{1-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-methylindazol-4-yl]pyrrolidin-3-yl}-N-methylcarbamate (77 mg, 79%) as a solid. LCMS (ES, m/z): 522 [M+H]+.

Synthesis of Compound 298

Example 82: Synthesis of Compound 301

Synthesis of Intermediate C91

Synthesis of Intermediate C92

To a stirred mixture of tert-butyl 7-[7-(methoxycarbonyl)-2-methylindazol-4-yl]-1,7-diazaspiro[3.5]nonane-1-carboxylate (180.0 mg, 0.434 mmol, 1.0 equiv) in THE (2.5 mL) and water (2.5 mL) was added LiOH·H2O (52.0 mg, 2.170 mmol, 5.0 equiv) at room temperature. The resulting mixture was stirred for 4 h at 50° C., then concentrated under vacuum, diluted with water (5 mL), acidified to pH 6 with HCl (1 N), and extracted with ethyl acetate (3×5 mL). The organic layers were combined, dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to afford 4-[1-(tert-butoxycarbonyl)-1,7-diazaspiro[3.5]nonan-7-yl]-2-methylindazole-7-carboxylic acid (160 mg, 92%) as a solid. LCMS (ES, m/z): 401 [M+H]+.

Synthesis of Intermediate C93

To a stirred solution of 4-[1-(tert-butoxycarbonyl)-1,7-diazaspiro[3.5]nonan-7-yl]-2-methylindazole-7-carboxylic acid (160.0 mg, 0.400 mmol, 1.0 equiv) in DMF (3.2 mL) was added DIEA (206.5 mg, 1.600 mmol, 4.0 equiv), HATU (227.8 mg, 0.600 mmol, 1.5 equiv), and 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (66.0 mg, 0.400 mmol, 1.0 equiv) in portions at room temperature. The resulting mixture was stirred for 3 h at 50° C., then diluted with water (10 mL) and extracted with EtOAc (3×10 mL). The organic layers were combined, washed with water (3×5 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with ethyl acetate to afford tert-butyl 7-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-methylindazol-4-yl]-1,7-diazaspiro[3.5]nonane-1-carboxylate (140 mg, 64%) as a solid. LCMS (ES, m/z): 548 [M+H]+.

Synthesis of Compound 301

Example 83: Synthesis of Compound 337

Synthesis of Intermediate C94

Synthesis of Compound 337

Example 84: Synthesis of Compound 338

Synthesis of Intermediate C95

Synthesis of Compound 338

Example 85: Synthesis of Compound 306

Synthesis of Intermediate C96

To a stirred mixture of methyl 4-bromo-2H-indazole-7-carboxylate (100 mg, 0.392 mmol, 1 equiv) and 2-bromoethyl methyl ether (81.74 mg, 0.588 mmol, 1.5 equiv) in DMF (2.5 mL) was added K2CO3 (108.37 mg, 0.784 mmol, 2 equiv) at room temperature. The resulting mixture was stirred for 5 h at 80° C., then cooled to room temperature. The resulting mixture was quenched with water (5 mL) and extracted with ethyl acetate (2×3 mL). The organic layers were combined, washed with brine (2×2 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 4-bromo-2-(2-methoxyethyl)indazole-7-carboxylate (93.5 mg, 76%) as a solid. LCMS (ES, m/z): 313 [M+H]+.

Synthesis of Intermediate C97

A mixture of methyl 4-bromo-2-(2-methoxyethyl) indazole-7-carboxylate (20 mg, 0.064 mmol, 1 equiv) and LiOH (5 mg, 0.192 mmol, 3 equiv) in water (0.25 mL), THF (0.5 mL) and methanol (0.5 mL) was stirred for 3 h at room temperature. The resulting mixture was concentrated under vacuum to give a residue. The residue was acidified to pH 3 with 1 N HCl. A solid precipitated that was collected by filtration, then washed with water (0.25 mL) to afford 4-bromo-2-(2-methoxyethyl) indazole-7-carboxylic acid (10 mg, 52%) as a solid. LCMS (ES, m/z): 299 [M+H]+.

Synthesis of Intermediate C98

Synthesis of Intermediate C99

Synthesis of Compound 306

Example 86: Synthesis of Compound 294

Synthesis of Intermediate C100

Synthesis of Intermediate C101

Synthesis of Intermediate C102

Synthesis of Intermediate C103

Synthesis of Intermediate C104

Synthesis of Intermediate C105

To a stirred solution of 4-bromo-2-cyclopropylindazole-7-carboxylic acid (150 mg, 0.53 mmol, 1.00 equiv) and Cs2CO3 (260.8 mg, 0.8 mmol, 1.50 equiv) in DMF (2 mL) was added CH3I (90.89 mg, 0.64 mmol, 1.20 equiv) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 8 h at room temperature under nitrogen atmosphere. The resulting mixture was extracted with EtOAc (3×40 mL). The combined organic layers were washed with half saturated aqueous NaCl (3×20 mL) and 1×50 ml of saturated aqueous NaCl, dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in methyl 4-bromo-2-cyclopropylindazole-7-carboxylate (150 mg, 95%) as a solid. LCMS (ES, m/z): 295 [M+H]+.

Synthesis of Intermediate C106

Synthesis of Intermediate C107

Synthesis of Intermediate C108

Synthesis of Compound 294

Example 87: Synthesis of Compound 275

Synthesis of Intermediate C109

Synthesis of Compound 275

Example 88: Synthesis of Compound 307

Synthesis of Intermediate C110

Synthesis of Compound 307

Example 89: Synthesis of Compounds 310 and 330

Synthesis of Intermediate C111

Synthesis of Intermediate C112

A mixture of tert-butyl N-{1-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2-methoxyethyl) indazol-4-yl]pyrrolidin-3-yl}carbamate (100 mg, 0.181 mmol, 1 equiv) and TFA (0.8 mL) in DCM (2 mL) was stirred for 1 h at room temperate. The resulting mixture was concentrated under vacuum to give a residue. The residue was basified to pH 8 with 7 M NH3(g) in methanol, then purified by reverse flash chromatography (Condition 1, Gradient 4) to afford 4-(3-aminopyrrolidin-1-yl)-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}-2-(2-methoxyethyl) indazole-7-carboxamide (36 mg, 44%) as a solid. LCMS (ES, m/z): 452 [M+H]+.

Synthesis of Compounds 310 and 330

Example 90: Synthesis of Compound 340

Synthesis of Intermediate C113

To a stirred solution of 5-methyl-1H-indazole (500 mg, 3.783 mmol, 1 equiv) in DCM (10 mL) was added Et3N (1.15 g, 11.349 mmol, 3 equiv), Boc2O (908.2 mg, 4.161 mmol, 1.1 equiv), and DMAP (46.2 mg, 0.378 mmol, 0.1 equiv) in portions at room temperature. The resulting mixture was stirred for 16 h at room temperature, then washed with water (2×10 mL). The organic layer was dried over anhydrous Na2SO4 and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (2:1) to afford tert-butyl 5-methylindazole-1-carboxylate (500 mg, 57%) as a solid. LCMS (ES, m/z): 233 [M+H]+.

Synthesis of Intermediate C114

Synthesis of Intermediate C115

To a stirred solution of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (70.0 mg, 0.142 mmol, 1.0 equiv) and tert-butyl 5-(bromomethyl) indazole-1-carboxylate (66.2 mg, 0.213 mmol, 1.5 equiv) in DMF (1.7 mL) was added Cs2CO3 (138.6 mg, 0.426 mmol, 3 equiv) at room temperature. The resulting mixture was stirred for 1 h at room temperature, then diluted with water (6 mL) and extracted with ethyl acetate (3×6 mL). The organic layers were combined, washed with water (3×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with ethyl acetate to afford tert-butyl 5-({4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-2-yl}methyl) indazole-1-carboxylate (40 mg, 39%) as a solid. LCMS (ES, m/z): 724 [M+H]+.

Synthesis of Compound 340

Example 91: Synthesis of Compound 314

Synthesis of Intermediate C116

Synthesis of Compound 314

Example 92: Synthesis of Compound 359

Synthesis of Intermediate C117

Synthesis of Intermediate C118

Synthesis of Intermediate C119

A mixture of methyl 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-methylindazole-7-carboxylate (2.5 g, 6.677 mmol, 1 equiv) and NH3(g) in methanol (70 mL) was stirred for 2 days at 100° C. The resulting mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-(7-carbamoyl-2-methylindazol-4-yl)piperazine-1-carboxylate (1.35 g, 56%) as a solid. LCMS (ES, m/z): 360 [M+H]+.

Synthesis of Intermediate C120

Synthesis of Compound 359

Example 93: Synthesis of Compound 332

Synthesis of Intermediate C121

Synthesis of Compound 332

Example 94: Synthesis of Compound 343

Synthesis of Intermediate C122

Synthesis of Compound 343

Example 95: Synthesis of Compound 319

Synthesis of Intermediate C123

Synthesis of Compound 319

Example 96: Synthesis of Compound 320

Synthesis of Intermediate C124

Synthesis of Compound 320

Example 97: Synthesis of Compound 321

Synthesis of Intermediate C125

Synthesis of Compound 321

Example 98: Synthesis of Compound 322

Synthesis of Intermediate C126

Synthesis of Compound 322

Example 99: Synthesis of Compound 323

Synthesis of Intermediate C127

Synthesis of Compound 323

Example 100: Synthesis of Compound 324

Synthesis of Intermediate C128

Synthesis of Compound 324

Example 101: Synthesis of Compound 325

Synthesis of Intermediate C129

Synthesis of Compound 325

Example 102: Synthesis of Compound 315

Synthesis of Intermediate C130

Example 103: Synthesis of Compound 344

Synthesis of Intermediate C131

Synthesis of Compound 344

Example 104: Synthesis of Compound 333

Synthesis of Intermediate C132

Synthesis of Compound 333

Example 105: Synthesis of Compound 334

Synthesis of Intermediate C133

Synthesis of Intermediate C134

Synthesis of Compound 334

Example 106: Synthesis of Compound 347

Synthesis of Intermediate C135

Synthesis of Compound 347

Synthesis of Intermediate C136

A mixture of sodium sulfate (111.38 g, 784.152 mmol, 8 equiv), hydroxylamine hydrochloride (23.84 g, 343.067 mmol, 3.5 equiv), and chloral (21.67 g, 147.029 mmol, 1.5 equiv) was dissolved in water (500 mL). To the reaction mixture was added a solution of 3-bromo-5-fluoro-2-methylaniline (20 g, 98.019 mmol, 1 equiv) in a mixture of water (540 mL), ethanol (70 mL), and concentrated HCl (17 mL). The reaction mixture was stirred overnight at 60° C., then cooled to room temperature. A precipitate formed that was collected by filtration and washed with water (2×50 mL). The resulting solid was dried in an oven under reduced pressure to afford (2E)-N-(3-bromo-5-fluoro-2-methylphenyl)-2-(N-hydroxyimino)acetamide (22 g, 82%) as a solid. LCMS (ES, m/z): 275[M+H]+.

Synthesis of Intermediate C137

(2E)-N-(3-bromo-5-fluoro-2-methylphenyl)-2-(N-hydroxyimino)acetamide (22 g, 79.978 mmol, 1 equiv) was added to sulfuric acid (170 mL) in portions at 60° C. The resulting mixture was stirred for 1 h at 60° C., then cooled to room temperature and slowly added to ice water. A precipitate formed that was collected by filtration and washed with water (2×20 mL). The resulting solid was dried under vacuum to afford 6-bromo-4-fluoro-7-methyl-1H-indole-2,3-dione (18.5 g, 90%) as a solid. LCMS (ES, m/z): 258[M+H]+.

Synthesis of Intermediate C138

To a mixture of 6-bromo-4-fluoro-7-methyl-1H-indole-2,3-dione (18.5 g, 71.693 mmol, 1 equiv) and NaOH (2 M) (91 mL, 645.237 mmol, 9 equiv) was added H2O2 (15.4 mL, 358.465 mmol, 5 equiv) dropwise over 15 min at room temperature. The resulting mixture was stirred for an additional 3 h at room temperature, then quenched with saturated sodium sulfite (aq.) at room temperature and neutralized to pH 7 with HCl (2 M). The resulting mixture was filtered, and the filter cake washed with water (2×20 mL). The filtrate was concentrated under reduced pressure to give a residue. The residue was acidified to pH 4 with HCl (2 M). The precipitated solids were collected by filtration and washed with water (2×20 mL). The resulting solid was dried under infrared light to afford 2-amino-4-bromo-6-fluoro-3-methylbenzoic acid (16 g, 90%) as a solid. LCMS (ES, m/z): 249[M+H]+.

Synthesis of Intermediate C139

To a stirred solution of 2-amino-4-bromo-6-fluoro-3-methylbenzoic acid (6 g, 24.189 mmol, 1 equiv) in methanol (60 mL) was added sulfuric acid (23.72 g, 241.890 mmol, 10 equiv) dropwise at room temperature. The resulting mixture was stirred for 16 h at 80° C., then concentrated under reduced pressure to give a residue. The residue was quenched with a mixture of water and ice (50 mL) at room temperature. The resulting mixture was extracted with DCM (3×50 mL). The organic layers were combined, washed with brine (lx 50 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 2-amino-4-bromo-6-fluoro-3-methylbenzoate (2.48 g, 39%) as an oil. LCMS (ES, m/z): 262[M+H]+.

Synthesis of Intermediate C140

Synthesis of Intermediate C141

To a stirred solution of methyl 4-bromo-6-fluoro-2H-indazole-7-carboxylate (1.7 g, 6.226 mmol, 1 equiv) in ethyl acetate (50 mL) was added trimethyloxonium tetrafluoroborate (1.38 g, 9.339 mmol, 1.5 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature, then diluted with ethyl acetate (50 mL) and washed with brine (3×50 mL). The organic phase was concentrated under reduced pressure to afford methyl 4-bromo-6-fluoro-2-methylindazole-7-carboxylate (1.7 g, 95%) as a solid. LCMS (ES, m/z): 287[M+H]+.

Synthesis of Intermediate C142

To a stirred mixture of methyl 4-bromo-6-fluoro-2-methylindazole-7-carboxylate (1.6 g, 5.573 mmol, 1 equiv) in THF (12 mL) and water (4 mL) was added lithiumol hydrate (0.47 g, 11.146 mmol, 2 equiv) at room temperature. The resulting mixture was stirred overnight at room temperature, then concentrated under reduced pressure, diluted with water (20 mL), and acidified to pH 3 with citric acid. A precipitate formed that was collected by filtration and washed with water (2×10 mL) to afford 4-bromo-6-fluoro-2-methylindazole-7-carboxylic acid (1.5 g, 99%) as a solid. LCMS (ES, m/z): 273[M+H]+.

Synthesis of Intermediate C143

Synthesis of Intermediate C144

Synthesis of Compound 360

Example 108: Synthesis of Compound 348

Synthesis of Intermediate C145

Synthesis of Compound 348

Example 109: Synthesis of Compound 349

Synthesis of Intermediate C146

Synthesis of Compound 349

Example 110: Synthesis of Compound 361

Synthesis of Compound 361

Example 111: Synthesis of Compound 362

Synthesis of Intermediate C147

Synthesis of Compound 362

Example 112: Synthesis of Compound 363

Synthesis of Intermediate C148

Synthesis of Compound 363

Example 113: Synthesis of Compounds 352 and 353

Synthesis of Compounds 352 and 353

Example 114: Synthesis of Compound 364

Synthesis of Intermediate C149

A mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2-oxopropyl) indazol-4-yl]piperazine-1-carboxylate (60.0 mg, 0.10 mmol, 1.0 equiv) in DCM (2 mL) was treated with DAST (35.1 mg, 0.21 mmol, 2.0 equiv) at 0° C. The resulting mixture was stirred for 3 h at room temperature, then quenched with ice-water at 0° C. The resulting mixture was extracted with ethyl acetate (3×10 mL). The organic layers were combined, washed with brine (1×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 4-[2-(2,2-difluoropropyl)-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]piperazine-1-carboxylate (29 mg, 46%) as a solid. LCMS (ES, m/z): 572 [M+H]+.

Synthesis of Compound 364

Example 115: Synthesis of Compound 365

Synthesis of Intermediate C150

Synthesis of Compound 365

Example 116: Synthesis of Compound 366

Synthesis of Intermediate C151

Synthesis of Intermediate C152

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (600.0 mg, 1.216 mmol, 1.0 equiv) in DMF (12 mL) was added Cs2CO3 (1.18 g, 3.648 mmol, 3.0 equiv) and 1,3-oxazol-5-ylmethyl methanesulfonate (215.4 mg, 1.216 mmol, 1.0 equiv) at room temperature. The resulting mixture was stirred for 16 h at 50° C., then cooled to room temperature. The resulting mixture was diluted with water (20 mL) and extracted with ethyl acetate (3×20 mL). The organic layers were combined, washed with water (3×20 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with ethyl acetate to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(1,3-oxazol-5-ylmethyl) indazol-4-yl]piperazine-1-carboxylate (80 mg, 11%) as a solid. LCMS (ES, m/z): 575 [M+H]+.

Synthesis of Compound 366

Example 117: Synthesis of Compound 367

Synthesis of Intermediate C153

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (120.0 mg, 0.24 mmol, 1.0 equiv) and 1-bromo-2-methoxypropane (55.8 mg, 0.36 mmol, 1.5 equiv) in DMF (1.5 mL) was added Cs2CO3 (237.6 mg, 0.73 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 4 h at 40° C., then diluted with water and extracted with ethyl acetate (3×10 mL). The organic layers were combined, washed with brine (1×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2-methoxypropyl) indazol-4-yl]piperazine-1-carboxylate (70 mg, 51%) as a solid. LCMS (ES, m/z): 566 [M+H]+.

Synthesis of Compound 367

Example 118: Synthesis of Compound 368

Synthesis of Intermediate C154

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100.0 mg, 0.20 mmol, 1.0 equiv) and 4-(chloromethyl)-1-methyl-1,2,3-triazole (31.9 mg, 0.24 mmol, 1.2 equiv) in DMF (1 mL) was added Cs2CO3 (198.0 mg, 0.60 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 1 h at room temperature, then diluted with water and extracted with ethyl acetate (2×15 mL). The organic layers were combined, washed with brine (1×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with ethyl acetate to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-[(1-methyl-1,2,3-triazol-4-yl)methyl]indazol-4-yl]piperazine-1-carboxylate (65 mg, 55%) as a solid. LCMS (ES, m/z): 589 [M+H]+.

Synthesis of Compound 368

Example 119: Synthesis of Compound 369

Synthesis of Intermediate C155

Synthesis of Compound 369

Example 120: Synthesis of Compound 370

Synthesis of Intermediate C156

Synthesis of Intermediate C157

Synthesis of Intermediate C158

A mixture of tert-butyl 4-(benzylamino)-4-(trifluoromethyl)piperidine-1-carboxylate (200 mg, 0.558 mmol, 1 equiv) and HCl (gas) in 1,4-dioxane (2 mL) was stirred for 2 h at room temperature under nitrogen atmosphere. The resulting mixture was concentrated under vacuum to afford N-benzyl-4-(trifluoromethyl)piperidin-4-amine dihydrochloride (180 mg, 97%) as a solid. LCMS (ES, m/z): 259[M+H]+.

Synthesis of Intermediate C159

Synthesis of Compound 370

Example 121: Synthesis of Compound 371

Synthesis of Intermediate C160

Synthesis of Compound 371

Example 122: Synthesis of Compound 372

Synthesis of Intermediate C161

Synthesis of Compound 372

Example 123: Synthesis of Compound 373

Synthesis of Compound 373

Example 124: Synthesis of Compound 374

Synthesis of Intermediate C162

A mixture of methyl 4-bromo-2H-indazole-7-carboxylate (8 g, 31.36 mmol, 1 equiv) and tetrafluoroboranuide; triethyloxidanium (17.9 g, 94.09 mmol, 3 equiv) in ethyl acetate (100 mL) was stirred overnight at room temperature. The reaction mixture was quenched with water (200 mL) at room temperature, then extracted with ethyl acetate (3×50 mL). The organic layers were combined, washed with brine (1×100 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to afford methyl 4-bromo-2-ethylindazole-7-carboxylate (6.36 g, 72%) as a solid. LCMS (ES, m/z): 283 [M+H]+.

Synthesis of Intermediate C163

A mixture of methyl 4-bromo-2-ethylindazole-7-carboxylate (5.4 g, 18.93 mmol, 1 equiv) and LiOH·H2O (7.9 g, 189.3 mmol, 10 equiv) in THF (50 mL), methanol (50 mL) and water (25 mL) was stirred for 2 h at room temperature. The resulting mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in ethyl acetate (100 mL) and diluted with water (200 mL). The resulting mixture was acidified to pH 2 with 1M HCl (aq.) and extracted with ethyl acetate (3×50 mL). The organic layers were combined, washed with brine (1×100 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to afford 4-bromo-2-ethylindazole-7-carboxylic acid (5 g, 98%) as a solid. LCMS (ES, m/z): 269 [M+H]+.

Synthesis of Intermediate C164

Synthesis of Intermediate C165

Synthesis of Compound 374

Example 125: Synthesis of Compound 375

Synthesis of Compound 375

Example 126: Synthesis of Compound 376

Synthesis of Intermediate C166

To a stirred solution of 4-bromo-2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}indazole-7-carboxamide (190 mg, 0.46 mmol, 1 equiv) and tert-butyl N-cyclopropyl-N-(piperidin-4-yl)carbamate (110 mg, 0.46 mmol, 1 equiv) in dioxane (10 mL) was added Cs2CO3 (446 mg, 1.38 mmol, 3 equiv), RuPhos (43 mg, 0.092 mmol, 0.2 equiv), and RuPhos Palladacycle Gen.3 (38 mg, 0.046 mmol, 0.1 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred overnight at 90° C. under nitrogen atmosphere, then cooled to room temperature. The resulting mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in water (20 mL) and extracted with ethyl acetate (3×5 mL). The organic layers were combined, washed with brine (1×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by reverse flash chromatography (Condition 2, Gradient 2) to afford tert-butyl N-cyclopropyl-N-{1-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]piperidin-4-yl}carbamate (150 mg, 57%) as a solid. LCMS (ES, m/z): 576 [M+H]+.

Synthesis of Compound 376

Example 127: Synthesis of Compound 377

Synthesis of Intermediate C167

To a stirred mixture of 4-bromo-2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}indazole-7-carboxamide (90 mg, 0.22 mmol, 1 equiv) and tert-butyl (2R,6S)-2,6-dimethylpiperazine-1-carboxylate (50 mg, 0.24 mmol, 1.0 equiv) in dioxane (5 mL) was added Cs2CO3 (211 mg, 0.648 mmol, 3 equiv), RuPhos (20 mg, 0.044 mmol, 0.2 equiv), and RuPhos Palladacycle Gen.3 (18 mg, 0.022 mmol, 0.1 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred overnight at 90° C. under nitrogen atmosphere, then cooled to room temperature. The resulting mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in water (20 mL) and extracted with ethyl acetate (3×5 mL). The organic layers were combined, washed with brine (1×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by reverse flash chromatography (Condition 2, Gradient 2) to afford tert-butyl (2R,6S)-4-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]-2,6-dimethylpiperazine-1-carboxylate (65 mg, 55%) as a solid. LCMS (ES, m/z): 550 [M+H]+.

Synthesis of Compound 377

Example 128: Synthesis of Compound 378

Synthesis of Compound 378

Example 129: Synthesis of Compound 379

Synthesis of Intermediate C168

Synthesis of Compound 379

Example 130: Synthesis of Compound 380

Synthesis of Intermediate C169

To a stirred solution of 4-bromo-2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}indazole-7-carboxamide (90 mg, 0.216 mmol, 1 equiv) and tert-butyl N-ethyl-N-(piperidin-4-yl)carbamate (49 mg, 0.216 mmol, 1 equiv) in dioxane (5 mL) was added Cs2CO3 (211 mg, 0.648 mmol, 3 equiv), RuPhos (11 mg, 0.044 mmol, 0.2 equiv), and RuPhos Palladacycle Gen.3 (10 mg, 0.022 mmol, 0.1 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred overnight at 90° C. under nitrogen atmosphere, then cooled to room temperature, and concentrated under reduced pressure to give a residue. The residue was dissolved in water (20 mL) and extracted with ethyl acetate (3×5 mL). The organic layers were combined, washed with brine (1×10 mL), dried over anhydrous Na2SO4, and filtered. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by reverse flash chromatography (Condition 2, Gradient 2) to afford tert-butyl N-ethyl-N-{1-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]piperidin-4-yl}carbamate (45 mg, 37%) as a solid. LCMS (ES, m/z): 564 [M+H]+.

Synthesis of Compound 380

Example 131: Synthesis of Compound 381

Synthesis of Compound 381

Example 132: Synthesis of Compound 382

Synthesis of Compound 382

Example 133: Synthesis of Compound 383

Synthesis of Intermediate C162

Synthesis of Compound 383

Example 134: Synthesis of Compound 384

Synthesis of Intermediate C163

Synthesis of Compound 384

Example 135: Synthesis of Compound 385

Synthesis of Intermediate C164

Synthesis of Intermediate C165

Into a 40 mL round-bottom flask were added methyl 4-bromo-3-hydroxy-2-nitrobenzoate (300 mg, 1.087 mmol, 1 equiv), acetic acid (6 mL) and zinc (213.16 mg, 3.260 mmol, 3.00 equiv) at 25 degrees C. The resulting mixture was stirred for 12 h at 25 degrees C. The reaction was quenched by the addition of sodium bicarbonate aqueous solution (50 mL) at 25 degrees C. The aqueous layer was extracted with ethyl acetate (3×20 mL). The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 2-amino-4-bromo-3-hydroxybenzoate (C165, 200 mg, 74%) as a solid. LCMS (ES, m/z): 246 [M+H]+

Synthesis of Intermediate C166

Synthesis of Intermediate C167

Into a 40 mL vial were added methyl 7-bromo-2-oxo-3H-1,3-benzoxazole-4-carboxylate (200 mg, 0.735 mmol, 1 equiv), triphenylphosphine (289.24 mg, 1.103 mmol, 1.5 equiv), DCM (4 mL) and 2-methoxyethanol (67.13 mg, 0.882 mmol, 1.2 equiv) at room temperature. The resulting mixture was stirred for 1 hr at 0° C. under nitrogen atmosphere. To the above mixture was added DEAD (192.05 mg, 1.103 mmol, 1.5 equiv). The resulting mixture was stirred for additional 2 h at 25 degrees C. The reaction was quenched by the addition of water (10 mL) at room temperature. The aqueous layer was extracted with ethyl acetate (2×10 mL). The resulting liquid was dried Na2SO4. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (3:1) to afford methyl 7-bromo-2-(2-methoxyethoxy)-1,3-benzoxazole-4-carboxylate (C167, 160 mg, 65%) as a solid.

Synthesis of Intermediate C168

Synthesis of Intermediate C169

Into a 40 mL vial were added methyl 7-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-(2-methoxyethoxy)-1,3-benzoxazole-4-carboxylate (120 mg, 0.276 mmol, 1 equiv), tetrahydrofuran (2 mL), water (2 mL) and LiOH (9.90 mg, 0.414 mmol, 1.5 equiv) at room temperature. The resulting mixture was stirred for 3 h at 40° C. The resulting mixture was diluted with water (30 mL). The mixture was acidified to pH 5 with HCl (aq.). The aqueous layer was extracted with ethyl acetate (2×50 mL). The resulting mixture was concentrated under reduced pressure. This resulted in 7-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-(2-methoxy ethoxy)-1,3-benzoxazole-4-carboxylic acid (C169, 100 mg, 86%) as a solid. LCMS (ES, m/z): 422 [M+H]+

Synthesis of Intermediate C170

Synthesis of Compound 367

Example 136: Synthesis of Compound 386

Synthesis of Intermediate C171

To a solution of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-methylindazol-4-yl]piperazine-1-carboxylate (50 mg, 0.099 mmol, 1 equiv) in DMF (1 mL) was added sodium hydride (60% in oil, 7.88 mg, 2 equiv) at 0° C. The mixture was stirred for 15 min. CH3I (13.28 mg, 0.094 mmol, 0.95 equiv) was added and the mixture was allowed to warm to room temperature and stirred for 2 hr. The reaction mixture was quenched by water and extracted with DCM (3×25 mL). The organic phase was concentrated under reduced pressure to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}(methyl)carbamoyl)-2-methylindazol-4-yl]piperazine-1-carboxylate (C171, 40 mg, 77%) as a solid. LCMS (ES, m/z): 522 [M+H]+

Synthesis of Compound 386

Example 137: Synthesis of Compound 387

Synthesis of Intermediate C172

Synthesis of Intermediate C173

To a stirred solution of methyl 4-[(3R)-3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl]-2-methylindazole-7-carboxylate (398.0 mg, 1.02 mmol, 1.0 equiv) in THF (4 mL) were added H2O (4 mL) and LiOH (214.9 mg, 5.12 mmol, 5.0 equiv) at room temperature. The resulting mixture was stirred for 16 hr at 50° C. The resulting mixture was concentrated under vacuum and diluted with water (10 mL). The mixture was acidified to pH 7 with HCl (2 M) and was extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with brine (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-[(3R)-3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl]-2-methylindazole-7-carboxylic acid (C173, 312 mg, 81%) as a solid. LCMS (ES, m/z): 375 [M+H]+

Synthesis of Intermediate C174

Synthesis of Compound 387

Example 138: Synthesis of Compound 388

Synthesis of Intermediate C175

Synthesis of Intermediate C176

To a stirred mixture of methyl (S)-4-(3-((tert-butoxycarbonyl)(methyl)amino)pyrrolidin-1-yl)-2-methyl-2H-indazole-7-carboxylate (650.0 mg, 1.673 mmol, 1 equiv) in THF (8 mL) were added H2O (8 mL) and lithium hydroxide hydrate (561.7 mg, 13.384 mmol, 8.0 equiv) at room temperature. The resulting mixture was stirred for 16 hr at 50° C. The resulting mixture was concentrated under vacuum. The resulting mixture was diluted with water (10 mL). The mixture was acidified to pH 6 with HCl (2 M). The resulting mixture was extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with water (2×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford (S)-4-(3-((tert-butoxycarbonyl)(methyl)amino)pyrrolidin-1-yl)-2-methyl-2H-indazole-7-carboxylic acid (C176, 570 mg, 90%) as a solid. LCMS (ES, m/z): 375 [M+H]+

Synthesis of Intermediate C177

To a stirred mixture of (S)-4-(3-((tert-butoxycarbonyl)(methyl)amino)pyrrolidin-1-yl)-2-methyl-2H-indazole-7-carboxylic acid (200.0 mg, 0.534 mmol, 1.0 equiv) and 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (132.3 mg, 0.801 mmol, 1.5 equiv) in DMF (2.5 mL) were added DIEA (276.1 mg, 2.136 mmol, 4.0 equiv) and HATU (243.7 mg, 0.641 mmol, 1.2 equiv) at room temperature. The resulting mixture was stirred for 16 hr at 50° C. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (10 mL). The resulting mixture was extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl (S)-(1-(7-((8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl)carbamoyl)-2-methyl-2H-indazol-4-yl)pyrrolidin-3-yl)(methyl)carbamate (C177, 130 mg, 46%) as a solid. LCMS (ES, m/z): 522 [M+H]+

Synthesis of Compound 388

Example 139: Synthesis of Compound 389

Synthesis of Intermediate C178

Synthesis of Compound 389

Example 140: Synthesis of Compound 387

Synthesis of Compound 387

Example 141: Synthesis of Compound 388

Synthesis of Compound 388

Example 142: Synthesis of Compound 391

Synthesis of Intermediate C179

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100.0 mg, 0.20 mmol, 1.0 equiv) and iodocyclobutane (55.3 mg, 0.30 mmol, 1.5 equiv) in DMF (2 mL) was added Cs2CO3 (198.0 mg, 0.60 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature. The resulting mixture was diluted with water. The resulting mixture was extracted with ethyl acetate (3×15 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:4) to afford tert-butyl 4-[2-cyclobutyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl] piperazine-1-carboxylate (C179, 59 mg, 53%) as a solid. LCMS (ES, m/z): 548 [M+H]+

Synthesis of Compound 391

Example 143: Synthesis of Compound 392

Synthesis of Intermediate C180

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100.0 mg, 0.20 mmol, 1.0 equiv) and allyl bromide (36.7 mg, 0.30 mmol, 1.5 equiv) in DMF (2 mL) was added Cs2CO3 (198.0 mg, 0.61 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature. The resulting mixture was diluted with water. The resulting mixture was extracted with ethyl acetate (2×10 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:4) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(prop-2-en-1-yl) indazol-4-yl] piperazine-1-carboxylate (C180, 52 mg, 48%) as a solid. LCMS (ES, m/z): 534 [M+H]+

Synthesis of Compound 392

Example 144: Synthesis of Compound 394

Synthesis of Intermediate C181

To a stirred solution of 4-bromo-2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}indazole-7-carboxamide (90 mg, 0.22 mmol, 1 equiv) and tert-butyl 1,7-diazaspiro[3.5]nonane-1-carboxylate (49 mg, 0.22 mmol, 1 equiv) in dioxane (10 mL) were added Cs2CO3 (211 mg, 0.66 mmol, 3 equiv), RuPhos (20 mg, 0.044 mmol, 0.2 equiv) and RuPhos Palladacycle Gen.3 (18 mg, 0.022 mmol, 0.1 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred overnight at 90° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in water (20 mL). The resulting mixture was extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography (Condition 5, Gradient 1) to afford tert-butyl 7-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]-1,7-diazaspiro[3.5]nonane-1-carboxylate (75 mg, 61%) as a solid. LCMS (ES, m/z): 562 [M+H]+

Synthesis of Compound 394

Example 145: Synthesis of Compound 395

Example 146: Synthesis of Compound 396

Synthesis of Intermediate C182

To a stirred solution of 4-bromo-2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}indazole-7-carboxamide (180 mg, 0.43 mmol, 1 equiv) and tert-butyl N-ethyl-N-(pyrrolidin-3-yl)carbamate (93 mg, 0.43 mmol, 1 equiv) in dioxane (10 mL) were added Cs2CO3 (423 mg, 1.29 mmol, 3 equiv), RuPhos (40 mg, 0.086 mmol, 0.2 equiv) and RuPhos Palladacycle Gen.3 (36 mg, 0.043 mmol, 0.1 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for overnight at 90° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in water (20 mL). The resulting mixture was extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography (Condition 5, Gradient 2) to afford tert-butyl N-ethyl-N-{1-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]pyrrolidin-3-yl}carbamate (C182, 100 mg, 42%) as a solid. LCMS (ES, m/z): 550 [M+H]+

Synthesis of Compound 396

Example 147: Synthesis of Compound 397

Synthesis of Intermediate C183

To a stirred solution of 4-[(tert-butoxycarbonyl)amino]piperidine-4-carboxylic acid (1.5 g, 6.140 mmol, 1.0 equiv) and sodium methaneperoxoate sodium (1.3 g, 12.280 mmol, 2.0 equiv) in tetrahydrofuran (15 mL), water (15 mL) was added benzyl chloroformate (1.2 g, 7.368 mmol, 1.2 equiv) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 2 hr at room temperature. The resulting mixture was diluted with water (30 mL). The mixture was acidified to PH 5 with HCl (aq.). The resulting mixture was extracted with ethyl acetate (2×50 mL). The combined organic layers were washed with brine (1×30 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (10:1) to afford 1-[(benzyloxy)carbonyl]-4-[(tert-butoxycarbonyl)amino]piperidine-4-carboxylic acid (C183, 1.4 g, 55%) as an oil. LCMS (ES, m/z): 379 [M+H]+

Synthesis of Intermediate C184

To a stirred solution of 1-[(benzyloxy)carbonyl]-4-[(tert-butoxycarbonyl)amino]piperidine-4-carboxylic acid (1.4 g, 3.700 mmol, 1.0 equiv) in tetrahydrofuran (15 mL) was added borane-tetrahydrofuran complex, 1M (40 mL) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 16 hr at 0° C. under nitrogen atmosphere. The reaction was quenched with methanol at 0° C. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (10:1) to afford benzyl 4-amino-4-(hydroxymethyl)piperidine-1-carboxylate (C184, 0.78 g, 71%) as an oil. LCMS (ES, m/z): 265 [M+H]+

Synthesis of Intermediate C185

Synthesis of Intermediate C186

To a stirred solution of benzyl 4-[(tert-butoxycarbonyl)amino]-4-(hydroxymethyl)piperidine-1-carboxylate (700.0 mg, 1.921 mmol, 1.0 equiv) in DCM (8 mL) was added Diethylaminosulfur trifluoride (464.4 mg, 2.881 mmol, 1.5 equiv) dropwise at 0° C. under nitrogen atmosphere. The reaction was quenched with sat. NaHCO3 (aq.) at 0° C. The resulting mixture was extracted with ethyl acetate (2×20 mL). The combined organic layers were washed with brine (1×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.

Synthesis of Intermediate C187

To a solution of benzyl 4-[(tert-butoxycarbonyl)amino]-4-(fluoromethyl)piperidine-1-carboxylate (290.0 mg, 0.791 mmol, 1.0 equiv) in 10 mL methanol was added Pd/C (10%, 250 mg) under nitrogen atmosphere in a mL round-bottom flask. The mixture was hydrogenated at room temperature for overnight under hydrogen atmosphere using a hydrogen balloon. The mixture was filtered through a Celite pad and concentrated under reduced pressure to yield tert-butyl (4-(fluoromethyl)piperidin-4-yl)carbamate (C187, 130 mg, 65%) as a solid. LCMS (ES, m/z): 233 [M+H]+

Synthesis of Intermediate C188

Synthesis of Intermediate Compound 397

Example 148: Synthesis of Compound 398

Synthesis of Intermediate C189

Synthesis of Compound 398

Example 149: Synthesis of Compounds 399 and 400

Compound No.
Analysis Data

Example 150: Synthesis of Compound 401

Example 151: Synthesis of Compound 402

Synthesis of Intermediate C190

Synthesis of Compound 402

Example 152: Synthesis of Compound 403

Synthesis of Intermediate C191

Synthesis of Compound 403

A solution of tert-butyl 4-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]-2-(hydroxymethyl)piperazine-1-carboxylate (50 mg, 0.091 mmol, 1 equiv) and TFA (0.2 mL) in DCM (2 mL) was stirred for 1 hr at room temperature. The mixture was neutralized to pH 8 with ammonia in methanol. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography (Condition 5, Gradient 3) to afford 2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}-4-[3-(hydroxymethyl)piperazin-1-yl]indazole-7-carboxamide (Compound 403, 10 mg, 24%) as a solid.

Example 153: Synthesis of Compound 404

Synthesis of Intermediate 192

Synthesis of Compound 404

Example 154: Synthesis of Compound 405

Synthesis of Intermediate C193

Synthesis of Compound 405

Example 155: Synthesis of Compound 406

Synthesis of Intermediate C194

Synthesis of Intermediate C195

A solution of tert-butyl 4-[7-({2-[(acetyloxy)methyl]-4-fluoro-1,3-benzoxazol-6-yl}carbamoyl)-2-methylindazol-4-yl]piperazine-1-carboxylate (63 mg, 0.111 mmol, 1 equiv) in methanol (2 mL) was treated with potassium methaneperoxoate potassium (46.44 mg, 0.333 mmol, 3 equiv) for 2 hr at room temperature. The resulting mixture was filtered, the filter cake was washed with DCM. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE:EA=1:1 to afford tert-butyl 4-(7-{[4-fluoro-2-(hydroxymethyl)-1,3-benzoxazol-6-yl]carbamoyl}-2-methylindazol-4-yl)piperazine-1-carboxylate (C195, 42 mg, 72%) as a solid. LCMS (ES, m/z): 525 [M+H]+

Synthesis of Compound 406

Example 156: Synthesis of Compounds 407 and 408

Synthesis of Intermediate C196

Synthesis of Compounds 407 and 408

Compound

No.
Analysis Data

Example 157: Synthesis of Compounds 398, 409, and 410

Synthesis of Intermediate C197

Synthesis of Compound 398

Synthesis of Compounds 409 and 410

Compound No.
Analysis Data

Example 158: Synthesis of Compounds 411 and 412

Synthesis of Intermediate C198

To a stirred solution of ethanamine hydrochloride (22.32 g, 273.672 mmol, 3 equiv) in DCM (300 mL) was added triethyl amine (27.69 g, 273.672 mmol, 3.0 equiv) at room temperature. The mixture was stirred for 10 min at room temperature. To the above mixture was added benzyl 3-oxopyrrolidine-1-carboxylate (20 g, 91.224 mmol, 1.0 equiv), NaBH(OAc)3 (29.00 g, 136.836 mmol, 1.5 equiv) in portions over 10 min at 0° C. The resulting mixture was stirred for additional 16 hr at room temperature. The reaction was quenched with water at 0° C. and diluted with water (200 mL). The resulting mixture was extracted with CH2Cl2 (3×200 mL). The combined organic layers were washed with water (3×200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford benzyl 3-(ethylamino)pyrrolidine-1-carboxylate (C198, 47 g, 99%) as an oil. LCMS (ES, m/z): 249 [M+H]

Synthesis of Intermediate C199

To a stirred mixture of benzyl 3-(ethylamino)pyrrolidine-1-carboxylate (47.00 g, 189.267 mmol, 1 equiv) in DCM (940 mL) were added Et3N (57.46 g, 567.801 mmol, 3 equiv) and Boc2O (61.96 g, 283.900 mmol, 1.5 equiv) in portions at room temperature. The resulting mixture was stirred for 4 h at room temperature. The resulting mixture was diluted with water (900 mL). The resulting mixture was extracted with CH2Cl2 (3×500 mL). The combined organic layers were washed with water (2×400 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford benzyl 3-[(tert-butoxycarbonyl)(ethyl)amino]pyrrolidine-1-carboxylate (C199, 27 mg, 40%) as an oil. LCMS (ES, m/z): 349 [M+H]+

Synthesis of Intermediate C200

To a solution of benzyl 3-[(tert-butoxycarbonyl)(ethyl)amino]pyrrolidine-1-carboxylate (10 g, 28.699 mmol, 1 equiv) in methanol (100 mL) was added Pd/C (2 g 20% W) in a pressure tank. The mixture was hydrogenated at room temperature under 30 psi of hydrogen pressure for 16 hr. The resulting mixture was filtered and the precipitated solids was washed with MeOH (3×50 mL). The combined filtrate was concentrated under vacuum to afford tert-butyl N-ethyl-N-(pyrrolidin-3-yl)carbamate (C200, 5.1 g, 82%) as an oil. LCMS (ES, m/z): 214 [M+H]+ Synthesis of Intermediate C201

Synthesis of Intermediate C202

To a stirred mixture of methyl 4-{3-[(tert-butoxycarbonyl)(ethyl)amino]pyrrolidin-1-yl}-2H-indazole-7-carboxylate (3.2 g, 8.237 mmol, 1 equiv) in THF (32 mL) were added H2O (32 mL) and LiOH·H2O (1.58 g, 65.896 mmol, 8 equiv) at room temperature. The resulting mixture was stirred for 3 hr at 50° C. The mixture was acidified to pH 6 with 1 N of HCl. The resulting mixture was extracted with ethyl acetate (4×40 mL). The combined organic layers were washed with water (3×30 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-{3-[(tert-butoxycarbonyl) (ethyl)amino]pyrrolidin-1-yl}-2H-indazole-7-carboxylic acid (C202, 2.48 g, 80%) as a solid. LCMS (ES, m/z): 375 [M+H]+

Synthesis of Intermediate C203

To a stirred mixture of 4-{3-[(tert-butoxycarbonyl)(ethyl)amino]pyrrolidin-1-yl}-2H-indazole-7-carboxylic acid (1.8 g, 4.807 mmol, 1.00 equiv) and 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (0.95 g, 5.768 mmol, 1.2 equiv) in pyridine (36 mL) was added EDCI (1.38 g, 7.211 mmol, 1.5 equiv) in portions at room temperature. The resulting mixture was stirred for 16 hr at room temperature. The resulting mixture was diluted with water (60 mL). The resulting mixture was extracted with ethyl acetate (3×60 mL). The combined organic layers were washed with water (3×50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl N-ethyl-N-{1-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]pyrrolidin-3-yl}carbamate (C203, 420 mg, 16%) as a solid. LCMS (ES, m/z): 522 [M+H]+

Synthesis of Intermediate C203

To a stirred mixture of tert-butyl N-ethyl-N-{1-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]pyrrolidin-3-yl}carbamate (400 mg, 0.767 mmol, 1 equiv) and 2-bromoethyl methyl ether (159.88 mg, 1.151 mmol, 1.5 equiv) in DMF (8 mL, 103.372 mmol, 134.80 equiv) was added Cs2CO3 (749.59 mg, 2.301 mmol, 3 equiv) at room temperature. The resulting mixture was stirred for 1 hr at room temperature. The resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with water (3×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl N-ethyl-N-{1-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2-methoxyethyl) indazol-4-yl]pyrrolidin-3-yl}carbamate (210 mg, 47.24%) as a solid. LCMS (ES, m/z): 580[M+H]+

Synthesis of Intermediates C204 and C205

Intermediate C203 was separated by Chiral-Prep HPLC (Condition 4, Gradient 1) to yield intermediates C204 and C205.

Intermediate No.
Analysis Data

Synthesis of Compound 412

Synthesis of Compound 411

Example 159: Synthesis of Compound 413

Synthesis of Intermediate C206

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100.0 mg, 0.20 mmol, 1.0 equiv) and 2-bromoacetonitrile (36.4 mg, 0.30 mmol, 1.5 equiv) in DMF (1 mL) was added Cs2CO3 (198.0 mg, 0.61 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature. The resulting mixture was diluted with water. The resulting mixture was extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with brine (1×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 4-[2-(cyanomethyl)-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]piperazine-1-carboxylate (C206, 56 mg, 51%) as a solid. LCMS (ES, m/z): 533 [M+H]+ Synthesis of Compound 413

Example 160: Synthesis of Compound 414

Synthesis of Intermediate C207

To a stirred mixture of methyl 3-hydroxybicyclo[1.1.1]pentane-1-carboxylate (500.0 mg, 3.51 mmol, 1.0 equiv) and imidazole (478.9 mg, 7.03 mmol, 2.0 equiv) in DMF (5 mL) was added TBSCl (636.1 mg, 4.22 mmol, 1.2 equiv) at room temperature. The resulting mixture was stirred for overnight at room temperature. The reaction was monitored by TLC. The resulting mixture was diluted with water. The resulting mixture was extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The crude product was used in the next step directly without further purification to afford methyl 3-[(tert-butyldimethylsilyl)oxy]bicyclo[1.1.1]pentane-1-carboxylate (C207, 900 mg, 99%) as a solid. 1H NMR (400 MHz, Chloroform-d) δ 3.69 (s, 3H), 2.22 (s, 6H), 0.90 (s, 9H), 0.12 (s, 6H).

Synthesis of Intermediate C208

Synthesis of Intermediate C209

To a stirred mixture of {3-[(tert-butyldimethylsilyl)oxy]bicyclo[1.1.1]pentan-1-yl}methanol (300 mg, 1.31 mmol, 1.0 equiv) and Et3N (199.3 mg, 1.97 mmol, 1.5 equiv) in DCM (3 mL) was added MsCl (165.4 mg, 1.44 mmol, 1.1 equiv) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 1 hr at room temperature under nitrogen atmosphere. The reaction was quenched with water. The resulting mixture was extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford {3-[(tert-butyldimethylsilyl)oxy]bicyclo[1.1.1]pentan-1-yl}methyl methanesulfonate (C209, 235 mg, 58%) as a colorless oil without further purification.

Synthesis of Intermediate C210

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (235.0 mg, 0.476 mmol, 1.0 equiv) and {3-[(tert-butyldimethylsilyl)oxy]bicyclo[1.1.1]pentan-1-yl}methyl methanesulfonate (175.1 mg, 0.57 mmol, 1.2 equiv) in DMF (1.5 mL) was added Cs2CO3 (465.4 mg, 1.42 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 3 hr at room temperature. The resulting mixture was diluted with water. The resulting mixture was extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 4-[2-({3-[(tert-butyldimethylsilyl)oxy]bicyclo[1.1.1]pentan-1-yl}methyl)-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]piperazine-1-carboxylate hydrofluoride (C210, 130 mg, 37%) as a solid. LCMS (ES, m/z): 704 [M+H]+

Synthesis of Compound 414

Example 161: Synthesis of Compound 415

Example 162: Synthesis of Compound 416

Synthesis of Intermediate 211

Synthesis of Compound 416

Example 163: Synthesis of Compound 417

Synthesis of Intermediate C212

Synthesis of Intermediate C213

To a stirred mixture of methyl 4-{3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl}-2-methylindazole-7-carboxylate (2.4 g, 6.178 mmol, 1 equiv) in NH3(g) (7 M in MeOH) (200 mL) at room temperature. The resulting mixture was stirred for 72 hr at sealed 100° C. under NH3 atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl N-[1-(7-carbamoyl-2-methylindazol-4-yl)pyrrolidin-3-yl]-N-methylcarbamate (C214, 1.87 g, 62%) as a solid.

Synthesis of Intermediate C215

To a stirred mixture of tert-butyl N-[1-(7-carbamoyl-2-methylindazol-4-yl)pyrrolidin-3-yl]-N-methylcarbamate (280.0 mg, 0.577 mmol, 1 equiv) and 6-bromo-8-methoxy-2-methylimidazo[1,2-a]pyrazine (209.6 mg, 0.865 mmol, 1.5 equiv) in Dioxane (5.39 mL) were added Cs2CO3 (564.3 mg, 1.731 mmol, 3.0 equiv) and XantPhos (66.8 mg, 0.115 mmol, 0.2 equiv) and Pd2(dba)3 (52.9 mg, 0.058 mmol, 0.1 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 hr at 100° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (5 mL). The resulting mixture was extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with brine (lx 5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl N-{1-[7-({8-methoxy-2-methylimidazo[1,2-a]pyrazin-6-yl}carbamoyl)-2-methylindazol-4-yl]pyrrolidin-3-yl}-N-methylcarbamate (C215, 170 mg, 55%) as a solid. LCMS (ES, m/z): 535 [M+H]+

Synthesis of Compound 417

Example 164: Synthesis of Compounds 418, 431, and 432

Synthesis of Intermediate C216

To a stirred solution/mixture of methyl 4-bromo-2H-indazole-7-carboxylate (3.0 g, 11.761 mmol, 1.0 equiv) and Cs2CO3 (7.6 g, 23.522 mmol, 2.0 equiv) in dimethylformamide (50 mL) were added 2-bromoethyl methyl ether (2.45 g, 17.642 mmol, 1.5 equiv) dropwise at room temperature. The resulting mixture was stirred for 2 hr at room temperature. The resulting mixture was diluted with water (50 mL). The resulting mixture was extracted with ethyl acetate (2×50 mL). The combined organic layers were washed with brine (1×50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 4-bromo-2-(2-methoxyethyl) indazole-7-carboxylate (1.2 g, 29%) as a solid. LCMS (ES, m/z): 313 [M+H]+

Synthesis of Intermediate C217

Synthesis of Intermediate C218

To a solution of methyl 4-{3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl}-2-(2-methoxyethyl) indazole-7-carboxylate (500.0 mg, 1.156 mmol, 1.0 equiv) was added NH3(g) in methanol (50 mL) in a pressure tank. The resulting mixture was stirred for 2 days at 100° C. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (30:1) to afford tert-butyl N-{1-[7-carbamoyl-2-(2-methoxyethyl) indazol-4-yl]pyrrolidin-3-yl}-N-methylcarbamate (C218, 450 mg, 74%) as a solid. LCMS (ES, m/z): 418 [M+H]+

Synthesis of Intermediate C219

Synthesis of Compound 418

Synthesis of Compound 431

Synthesis of Compound 432

Example 165: Synthesis of Compound 419

Synthesis of Intermediate C220

To a stirred mixture of 3,5-dibromopyrazin-2-amine (10.00 g, 39.542 mmol, 1 equiv) and Dimethylamine hydrochloride (3.55 g, 43.496 mmol, 1.1 equiv) in DMSO (100 mL) was added DIEA (15.33 g, 118.626 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at 110° C. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (200 mL). The resulting mixture was extracted with ethyl acetate (3×200 mL). The combined organic layers were washed with water (2×200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (3:1) to afford 6-bromo-N2, N2-dimethylpyrazine-2,3-diamine (C220, 6.5 g, 75%) as a solid. LCMS (ES, m/z): 217 [M+H]+

Synthesis of Intermediate C221

To a stirred mixture of 6-bromo-N2,N2-dimethylpyrazine-2,3-diamine (6.50 g, 29.944 mmol, 1 equiv) and 1-bromo-1,1-dimethoxyethane (6.07 g, 35.933 mmol, 1.2 equiv) in i-PrOH (130 mL) was added PPTS (0.75 g, 2.994 mmol, 0.1 equiv) in portions at room temperature. The resulting mixture was stirred for 16 hr at 80° C. The mixture was allowed to cool down to room temperature. The resulting mixture was filtered and the filter cake was washed with i-PrOH (3×100 mL). The filter cake was dried to afford 6-bromo-N,N,2-trimethylimidazo[1,2-a]pyrazin-8-amine (C221, 5.1 g, 66%) as a solid. LCMS (ES, m/z): 255 [M+H]+

Synthesis of Intermediate C222

To a stirred mixture of tert-butyl N-[1-(7-carbamoyl-2-methylindazol-4-yl)pyrrolidin-3-yl]-N-methylcarbamate (280.0 mg, 0.577 mmol, 1.0 equiv) and 6-bromo-N,N,2-trimethylimidazo[1,2-a]pyrazin-8-amine (220.9 mg, 0.865 mmol, 1.5 equiv) in dioxane (6 mL) were added Cs2CO3 (564.3 mg, 1.731 mmol, 3 equiv) and XantPhos (66.8 mg, 0.115 mmol, 0.2 equiv) and Pd2(dba)3 (52.9 mg, 0.058 mmol, 0.1 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 hr at 100° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (5 mL). The resulting mixture was extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with brine (1×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl N-[1-(7-{[8-(dimethylamino)-2-methylimidazo[1,2-a]pyrazin-6-yl]carbamoyl}-2-methylindazol-4-yl)pyrrolidin-3-yl]-N-methylcarbamate (C222, 170 mg, 53%) as a solid. LCMS (ES, m/z): 548 [M+H]+

Synthesis of Compound 419

Example 166: Synthesis of Compound 421

Synthesis of Intermediate C223

Synthesis of Compound 421

Example 167: Synthesis of Compound 422

Synthesis of Intermediate C224

Synthesis of Intermediate C225

To a stirred solution of tert-butyl 1-methyl-1,7-diazaspiro[3.5]nonane-7-carboxylate (35 mg, 0.146 mmol, 1 equiv) in DCM (0.5 mL) was added TFA (0.5 mL, 6.732 mmol, 46.23 equiv) at room temperature. The resulting mixture was stirred for 2 hr at room temperature. The resulting mixture was concentrated under reduced pressure. The crude product was used in the next step directly without further purification. LCMS (ES, m/z): 141 [M+H]+

Synthesis of Compound 422

Example 168: Synthesis of Compound 427

Synthesis of Intermediate C226

To a stirred solution of 3-bromobenzene-1,2-diamine (4.0 g, 21.386 mmol, 1.0 equiv) in acetic acid (20 mL) and water (20 mL) were added sodium nitrite (1.6 g, 23.525 mmol, 1.1 equiv) in portions at room temperature. The resulting mixture was stirred for 1 hr at room temperature. The precipitated solids were collected by filtration and washed with water (2×20 mL). The solid was dried and this resulted in 4-bromo-2H-1,2,3-benzotriazole (C226, 3.2 g, 71%) as a solid. LCMS (ES, m/z): 198 [M+H]+

Synthesis of Intermediate C227

To a stirred mixture of 4-bromo-2H-1,2,3-benzotriazole (2.7 g, 13.635 mmol, 1.0 equiv) and K2CO3 (3.76 g, 27.270 mmol, 2.0 equiv) in dimethylformamide (60 mL) were added methyl iodide (2.9 g, 20.453 mmol, 1.5 equiv) dropwise at 0° C. The resulting mixture was stirred for 1 hr at room temperature. The resulting mixture was diluted with water (200 mL). The resulting mixture was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with water (2×200 mL), brine (1×200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/PE (5:1) to afford 4-bromo-2-methyl-1,2,3-benzotriazole (C227, 0.9 g, 28%) as a solid. LCMS (ES, m/z): 212 [M+H]+

Synthesis of Intermediate C228

Synthesis of Intermediate C229

To a stirred solution of tert-butyl 4-(2-methyl-1,2,3-benzotriazol-4-yl) piperazine-1-carboxylate (850.0 mg, 2.678 mmol, 1.0 equiv) in ACN (15 mL) were added NBS (524.3 mg, 2.946 mmol, 1.1 equiv) in portions at room temperature. The resulting mixture was stirred for 1 hr at room temperature. The resulting mixture was diluted with water (30 mL). The resulting mixture was extracted with ethyl acetate (2×40 mL). The combined organic layers were washed with water (2×30 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-(7-bromo-2-methyl-1,2,3-benzotriazol-4-yl) piperazine-1-carboxylate (C229, 820.0 mg, 73%) as a solid. LCMS (ES, m/z): 396 [M+H]+

Synthesis of Intermediate C230

To a solution of tert-butyl 4-(7-bromo-2-methyl-1,2,3-benzotriazol-4-yl) piperazine-1-carboxylate (250.0 mg, 0.631 mmol, 1.0 equiv) in MeOH (20 mL) was added Pd(dppf)Cl2 (46.1 mg, 0.063 mmol, 0.1 equiv), TEA (191.5 mg, 1.893 mmol, 3.0 equiv) in a pressure tank. The mixture was purged with nitrogen for 2 min and then was pressurized to 2 Mpa with carbon monoxide at 80° C. for 16 h. The reaction mixture was cooled to room temperature and filtered to remove insoluble solids. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (30:1) to afford methyl 7-[4-(tert-butoxycarbonyl) piperazin-1-yl]-2-methyl-1,2,3-benzotriazole-4-carboxylate as a solid. LCMS (ES, m/z): 376 [M+H]+

Synthesis of Intermediate C231

To a stirred mixture of methyl 7-[4-(tert-butoxycarbonyl) piperazin-1-yl]-2-methyl-1,2,3-benzotriazole-4-carboxylate (170.0 mg, 0.453 mmol, 1.0 equiv) in tetrahydrofuran (3 mL) and water (3 mL) was added LiOH·H2O (108.4 mg, 4.530 mmol, 10.0 equiv) in portions at room temperature. The resulting mixture was stirred for 3 hr at 50° C. The resulting mixture was diluted with deionized water (20 mL). The mixture was acidified to pH 6 with HCl (1 N). The resulting mixture was extracted with ethyl acetate (2×30 mL). The combined organic layers were washed with brine (1×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The resulting mixture was concentrated under reduced pressure. This resulted in 7-[4-(tert-butoxycarbonyl) piperazin-1-yl]-2-methyl-1,2,3-benzotriazole-4-carboxylic acid (C231, 130 mg, 73%) as a solid. LCMS (ES, m/z): 362 [M+H]+

Synthesis of Intermediate C232

Synthesis of Compound 427

Example 169: Synthesis of Compound 428

Synthesis of Intermediate C233

To a solution of methyl 3-hydroxy-2-nitrobenzoate (2 g, 10.145 mmol, 1 equiv) in HOAc (40 mL) was added with Br2 (2.4 g, 15.018 mmol, 1.48 equiv) dropwise at 0° C. The resulting was stirred for 12 h at room temperature. The reaction was quenched with aq. Na2S2O3 (50 mL) at room temperature. The resulting mixture was extracted with EA (3×50 mL). The combined organic layers were washed with brine (1×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE:EA (4:1) to afford methyl 4-bromo-3-hydroxy-2-nitrobenzoate (C233, 1 g, 35%) as a solid. LCMS (ES, m/z): 276 [M+H]+

Synthesis of Intermediate C234

To a stirred solution of methyl 4-bromo-3-hydroxy-2-nitrobenzoate (1 g, 3.623 mmol, 1 equiv) in THF (15 mL) was added a solution of Na2S2O4 (3.15 g, 18.115 mmol, 5.0 equiv) in H2O (15 mL) dropwise at room temperature. The resulting mixture was stirred at room temperature for 12 h. The resulting mixture was extracted with EA (3×20 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE:EA (4:1) to afford methyl 2-amino-4-bromo-3-hydroxybenzoate (C234, 500 mg, 56%) as a solid. LCMS (ES, m/z): 246 [M+H]+

Synthesis of Intermediate C235

Synthesis of Intermediate C236

Synthesis of Intermediate C237

A solution of methyl 7-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-methyl-1,3-benzoxazole-4-carboxylate (250 mg, 0.666 mmol, 1 equiv) in MeOH (1 mL) and THF (1 mL) was treated with a solution of LiOH·H2O (167 mg, 3.996 mmol, 6 equiv) in H2O (1 mL) for 1 hr at room temperature. The mixture was acidified to pH 3 with 1 M HCl. The resulting mixture was extracted with EA (2×5 mL). The combined organic layers were washed with brine (1×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography (Condition 3, Gradient 1) to afford 7-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-methyl-1,3-benzoxazole-4-carboxylic acid (C237, 200 mg, 83%) as a solid. LCMS (ES, m/z): 362 [M+H]+

Synthesis of Intermediate C238

Synthesis of Intermediate C239

Synthesis of Compound 428

Example 170: Synthesis of Compound 429

Synthesis of Intermediate C240

Synthesis of Intermediate C241

Example 171: Synthesis of Compound 430

Synthesis of Intermediate C241

Synthesis of Compound 430

Example 172: Synthesis of Compound 469

Synthesis of Intermediate C242

Synthesis of Compound 469

Example 173: Synthesis of Compound 437

Synthesis of Intermediate C243

Synthesis of Compound 437

Example 174: Synthesis of Compounds 438 and 439

Synthesis of Compound 439

Synthesis of Compound 438

Example 175: Synthesis of Compound 443

Synthesis of Intermediate C244

Synthesis of Intermediate C245

To a solution of methyl 4-{4-[(tert-butoxycarbonyl) (ethyl)amino] piperidin-1-yl}-2-methylindazole-7-carboxylate (300.0 mg, 0.720 mmol, 1.0 equiv) was added NH3(g) in MeOH (50 mL) in a pressure tank. The resulting mixture was stirred for 2 days at 100° C. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (30:1) to afford tert-butyl N-[1-(7-carbamoyl-2-methylindazol-4-yl) piperidin-4-yl]-N-ethylcarbamate (C245, 280 mg, 87%) as a solid. LCMS (ES, m/z): 402 [M+H]+

Synthesis of Intermediate C246

Synthesis of Compound 443

Example 176: Synthesis of Compound 444

Synthesis of Intermediate C247

Synthesis of Compound 444

Example 177: Synthesis of Compound 445

Synthesis of Intermediate C248

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (300.0 mg, 0.608 mmol, 1.0 equiv) and Cs2CO3 (396.1 mg, 1.216 mmol, 2.0 equiv) in DMF (6 mL) was added propylene oxide (52.9 mg, 0.912 mmol, 1.5 equiv) dropwise at room temperature. The resulting mixture was stirred for 16 h at 80° C. The resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with ethyl acetate (2×20 mL). The combined organic layers were washed with water (2×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:9) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(2-hydroxypropyl) indazol-4-yl]piperazine-1-carboxylate (C248, 200 mg, 59%) as a solid. LCMS (ES, m/z): 552 [M+H]+

Synthesis of Intermediate C249

Synthesis of Intermediate C250

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-[2-(methanesulfonyloxy)propyl]indazol-4-yl]piperazine-1-carboxylate (210.0 mg, 0.333 mmol, 1.0 equiv) in THF (2 mL) was added t-BuOK (74.8 mg, 0.666 mmol, 2.0 equiv) at room temperature. The resulting mixture was stirred for 16 hr at room temperature. The resulting mixture was diluted with water (4 mL). The resulting mixture was extracted with ethyl acetate (2×4 mL). The combined organic layers were washed with water (1×4 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:9) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-[(1E)-prop-1-en-1-yl]indazol-4-yl]piperazine-1-carboxylate (C250, 130 mg, 73%) as a solid. LCMS (ES, m/z): 534 [M+H]+

Synthesis of Compound 445

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-[(1E)-prop-1-en-1-yl]indazol-4-yl]piperazine-1-carboxylate (80.0 mg, 0.150 mmol, 1.0 equiv) in DCM (2 mL) was added ZnBr2 (337.6 mg, 1.500 mmol, 10.0 equiv) at room temperature. The resulting mixture was stirred for 16 hr at room temperature. The resulting mixture was concentrated under vacuum. The crude product was purified by Prep-HPLC (Condition 10, Gradient 9) to afford N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}-4-(piperazin-1-yl)-2-[(1E)-prop-1-en-1-yl]indazole-7-carboxamide (Compound 445, 18 mg, 27%) as a solid.

Example 178: Synthesis of Compound 446 and 447

Synthesis of Intermediate C251

Synthesis of Compound 446

Synthesis of Intermediate C252

Synthesis of Compound 447

Example 179: Synthesis of Compound 455

Synthesis of Intermediate C253

Synthesis of Compound 455

Example 180: Synthesis of Compound 460

Synthesis of Intermediate C254

Synthesis of Compound 460

Example 181: Synthesis of Compound 440

Synthesis of Intermediate C255

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (800.0 mg, 1.621 mmol, 1.0 equiv) and methyl 2-bromopropanoate (406.0 mg, 2.431 mmol, 1.5 equiv) in DMF (20 mL) was added Cs2CO3 (1056.2 mg, 3.242 mmol, 2.0 equiv) at room temperature. The resulting mixture was stirred for 1 hr at room temperature. The resulting mixture was diluted with water (60 mL). The resulting mixture was extracted with ethyl acetate (3×50 mL). The combined organic layers were washed with water (3×50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(1-methoxy-1-oxopropan-2-yl) indazol-4-yl]piperazine-1-carboxylate (C255, 520 mg, 55%) as a solid. LCMS (ES, m/z): 580 [M+H]+

Synthesis of Intermediate C256

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(1-methoxy-1-oxopropan-2-yl) indazol-4-yl]piperazine-1-carboxylate (520.0 mg, 0.897 mmol, 1.0 equiv) in THF (10 mL) was added LiBH4 (58.6 mg, 2.691 mmol, 3.0 equiv) at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 1 hr at 0° C. under nitrogen atmosphere. The reaction was quenched with water/ice at 0° C. The resulting mixture was extracted with ethyl acetate (2×20 mL). The combined organic layers were washed with water (1×30 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:9) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(1-hydroxypropan-2-yl) indazol-4-yl]piperazine-1-carboxylate (C256, 420 mg, 84%) as a solid. LCMS (ES, m/z): 552 [M+H]+

Synthesis of Intermediate C257

Synthesis of Intermediate C258

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-[1-(methanesulfonyloxy)propan-2-yl]indazol-4-yl]piperazine-1-carboxylate (450.0 mg, 0.715 mmol, 1.0 equiv) in THF (9 mL) was added tert-butoxypotassium (160.4 mg, 1.430 mmol, 2.0 equiv) at room temperature. The resulting mixture was stirred for 16 h at room temperature. The resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with ethyl acetate (2×20 mL). The combined organic layers were washed with water (1×40 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:9) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(prop-1-en-2-yl) indazol-4-yl]piperazine-1-carboxylate (C258, 210 mg, 55%) as a solid. LCMS (ES, m/z): 534 [M+H]+

Synthesis of Compound 440

Example 182: Synthesis of Compound 450

Synthesis of Intermediate C259

Synthesis of Intermediate C260

A solution of methyl 2-ethyl-4-(4-methylpiperazin-1-yl) indazole-7-carboxylate (200 mg, 0.661 mmol, 1 equiv) in 7 N NH3(g) in MeOH (40 mL) was stirred for 3 days at 100° C. The resulting mixture was concentrated under reduced pressure. The crude product was used in the next step directly without further purification. LCMS (ES, m/z): 288 [M+H]+

Synthesis of Intermediate C261

Synthesis of Compound 450

Example 183: Synthesis of Compound 451

Synthesis of Intermediate C262

Synthesis of Intermediate C263

Synthesis of Intermediate C264

Synthesis of Compound 451

Example 184: Synthesis of Compound 453

Example 185: Synthesis of Compound 454

Synthesis of Intermediate C265

Synthesis of Intermediate C266

Synthesis of Intermediate C267

To a stirred solution of 2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}-4-formylindazole-7-carboxamide (40 mg, 0.109 mmol, 1 equiv) and tert-butyl N-(1,3-dihydroxy-2-methylpropan-2-yl)carbamate (80 mg, 0.390 mmol, 3.56 equiv) in DCM (2 mL) and THF (0.2 mL) were addedp-TsOH (56 mg, 0.325 mmol, 2.97 equiv) and Na2SO4 (80 mg, 0.563 mmol, 5.14 equiv) in portions at room temperature. The resulting mixture was stirred for 12 h at room temperature. The resulting mixture was diluted with H2O (5 mL). The resulting mixture was extracted with EA (3×5 mL). The combined organic layers were washed with brine (1×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The crude product was used in the next step directly without further purification. LCMS (ES, m/z): 553 [M+H]+

Synthesis of Compound 454

Example 186: Synthesis of Compounds 456 and 484

Synthesis of Intermediate C268

Synthesis of Intermediate C269

Synthesis of Intermediate C270

Synthesis of Intermediate C271

A mixture of 3-bromo-6-fluoro-2-methoxy-5-methylbenzaldehyde O-methyl (6 g, 21.82 mmol, 1 equiv) in DMSO (70 mL) and N2H4·H2O (70 mL) was stirred for 4 h at 140° C. The reaction was quenched with water (100 mL) at room temperature. The resulting mixture was extracted with ethyl acetate (3×100 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford 5-bromo-4-methoxy-7-methyl-2H-indazole (C271, 1.0 g, 19%) as a solid. LCMS (ES, m/z): 241 [M+H]+

Synthesis of Intermediate C272

Synthesis of Intermediate C273

Synthesis of Compound 456

Synthesis of Compound 484

Example 187: Synthesis of Compound 457

Synthesis of Intermediate C274

A solution of 2-methyl-6-nitroimidazo[1,2-a]pyridine (150 mg, 0.847 mmol, 1 equiv) in MeOH (20 mL) was treated with PtO2 (75 mg, 0.330 mmol, 0.39 equiv) for 8 hr at 80° C. under 20 atm hydrogen atmosphere. The resulting mixture was filtered and the filter cake was washed with methanol. The filtrate was concentrated under reduced pressure. The crude product 2-methyl-5H,6H,7H,8H-imidazo[1,2-a]pyridin-6-amine (C274, 146 mg, 66%) was used in the next step directly without further purification. LCMS (ES, m/z): 152[M+H]+

1. Synthesis of Intermediate c275

Synthesis of Compound 457

Example 188: Synthesis of Compound 458

Synthesis of Intermediate C276

Synthesis of Intermediate C277

Synthesis of Compound 458

Example 189: Synthesis of Compound 459

Synthesis of Intermediate C278

Synthesis of Intermediate Compound 459

Example 190: Synthesis of Compound 460

Synthesis of Intermediate C279

To a stirred solution of methyl 4-{4-[(tert-butoxycarbonyl)(ethyl)amino]piperidin-1-yl}-6-fluoro-2-methylindazole-7-carboxylate (145 mg, 0.334 mmol, 1 equiv) in THF (1.2 mL) and H2O (0.4 mL) was added lithiumol hydrate (42.01 mg, 1.002 mmol, 3 equiv) at room temperature. The resulting mixture was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum. The resulting mixture was diluted with water. The mixture was acidified to pH 4 with citric acid. The resulting mixture was extracted with DCM (3×10 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-{4-[(tert-butoxycarbonyl)(ethyl)amino]piperidin-1-yl}-6-fluoro-2-methylindazole-7-carboxylic acid (C280, 130 mg, 92%) as a solid. LCMS (ES, m/z): 421 [M−H]−

Synthesis of Intermediate C281

Synthesis of Intermediate C282

Synthesis of Compound 460

Example 191: Synthesis of Compound 462

Synthesis of Intermediate C283

Synthesis of Intermediate C284

Synthesis of Compound 462

Example 192: Synthesis of Compound 463

Example 193: Synthesis of Compound 464

Example 194: Synthesis of Compound 470

Synthesis of Intermediate C285

Synthesis of Intermediate C286

Synthesis of Intermediate C287

Synthesis of Intermediate C288

Synthesis of Compound 470

Example 195: Synthesis of Compound 472

Example 196: Synthesis of Compound 473

Synthesis of Intermediate C289

Synthesis of Intermediate C290

To a stirred solution of methyl 4-[(3R)-3-(dimethylamino)pyrrolidin-1-yl]-2-ethyl-6-fluoroindazole-7-carboxylate (135 mg, 0.404 mmol, 1 equiv) in THF (1.2 mL) and H2O (0.4 mL) was added lithiumol hydrate (33.88 mg, 0.808 mmol, 2 equiv) at room temperature. The resulting mixture was stirred overnight at 30° C. The resulting mixture was concentrated under vacuum. The residue was dissolved in methanol (5 mL). The solution was added HCl(g) in MeOH (1 mL) dropwise at 0° C. The resulting mixture was stirred for 10 min at room temperature. The resulting mixture was concentrated under vacuum to afford 4-[(3R)-3-(dimethylamino)pyrrolidin-1-yl]-2-ethyl-6-fluoroindazole-7-carboxylic acid (C290, 150 mg) as a solid. LCMS (ES, m/z): 319 [M−H]−

Synthesis of Compound 473

Example 197: Synthesis of Compound 474

Synthesis of Intermediate C290

Synthesis of Intermediate C291

To a stirred solution of methyl 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-ethyl-6-fluoroindazole-7-carboxylate (90 mg, 0.221 mmol, 1 equiv) in THF (1.2 mL) and H2O (0.4 mL) was added lithiumol hydrate (18.58 mg, 0.442 mmol, 2 equiv) at room temperature. The resulting mixture was stirred overnight at 30° C. The resulting mixture was concentrated under vacuum. The resulting mixture was diluted with H2O (5 mL). The mixture was acidified to pH 4 with citric acid. The resulting mixture was extracted with DCM (3×10 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-ethyl-6-fluoroindazole-7-carboxylic acid (C291, 65 mg, 74%) as a solid. LCMS (ES, m/z): 391 [M−H]−

Synthesis of Intermediate C292

Synthesis of Compound 474

Example 198: Synthesis of Compound 476

Synthesis of Intermediate C293

Synthesis of Compound 476

Example 199: Synthesis of Compound 477

Synthesis of Intermediate C294

Synthesis of Compound 477

A solution of tert-butyl 6-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]-2,6-diazaspiro[3.3]heptane-2-carboxylate (60 mg, 0.112 mmol, 1 equiv) and TFA (0.2 mL) in DCM (2 mL) was stirred for 1 h at room temperature. The mixture was basified to pH 8 with NH3(g) in MeOH. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, CH3CN in water (0.05% NH3·H2O), 20% to 60% gradient in 10 min; detector, UV 254 nm. This resulted in 4-{2,6-diazaspiro[3.3]heptan-2-yl}-2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}indazole-7-carboxamide (27.4 mg, 56.21%) as a light yellow solid.

Example 200: Synthesis of Compound 478

Synthesis of Intermediate C295

Synthesis of Intermediate C296

To a stirred solution of methyl 4-[(3S)-3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl]-2-ethyl-6-fluoroindazole-7-carboxylate (85 mg, 0.202 mmol, 1 equiv) in THF (1.2 mL) and H2O (0.4 mL) was added lithium hydroxide hydrate (18.35 mg, 0.438 mmol, 2 equiv) at room temperature. The resulting mixture was stirred overnight at 30° C. The resulting mixture was concentrated under vacuum. The resulting mixture was diluted with H2O (5 mL). The mixture was acidified to pH 4 with citric acid. The resulting mixture was extracted with DCM (3×10 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-[(3S)-3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl]-2-ethyl-6-fluoroindazole-7-carboxylic acid (C296, 60 mg, 73%) as solid. LCMS (ES, m/z): 405 [M−H]−

Synthesis of Intermediate C297

Synthesis of Compound 478

Example 201: Synthesis of Compound 479

Synthesis of Intermediate C298

Synthesis of Intermediate C299

To a stirred solution of methyl 4-[(3R)-3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl]-2-ethyl-6-fluoroindazole-7-carboxylate (80 mg, 0.190 mmol, 1 equiv) in THF (1.2 mL) and H2O (0.4 mL) was added lithiumol hydrate (15.97 mg, 0.380 mmol, 2 equiv) at room temperature. The resulting mixture was stirred overnight at 30° C. The resulting mixture was concentrated under vacuum. The resulting mixture was diluted with H2O (5 mL). The mixture was acidified to pH 4 with citric acid. The resulting mixture was extracted with DCM (3×10 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-[(3R)-3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl]-2-ethyl-6-fluoroindazole-7-carboxylic acid (C299, 55 mg, 71%) as a solid. LCMS (ES, m/z): 405 [M−H]−

Synthesis of Intermediate C300

Synthesis of Compound 479

Example 202: Synthesis of Compound 480

Synthesis of Intermediate C301

Synthesis of Intermediate C302

Synthesis of Intermediate C303

Synthesis of Intermediate C304

Synthesis of Intermediate C305

Synthesis of Compound 480

Example 203: Synthesis of Compound 481

Synthesis of Intermediate C306

Synthesis of Intermediate C307

To a stirred solution of methyl 2-ethyl-6-fluoro-4-(4-methylpiperazin-1-yl) indazole-7-carboxylate (90 mg, 0.281 mmol, 1 equiv) in THF (1.2 mL) and H2O (0.4 mL) was added lithium hydroxide hydrate (23.58 mg, 0.562 mmol, 2 equiv) at room temperature. The resulting mixture was stirred overnight at 30° C. The resulting mixture was concentrated under vacuum. The residue was dissolved in methanol (5 mL). The solution was added HCl(gas) in MeOH (1 mL) dropwise at 0° C. The resulting mixture was stirred for 10 min at room temperature. The resulting mixture was concentrated under vacuum to afford 2-ethyl-6-fluoro-4-(4-methylpiperazin-1-yl) indazole-7-carboxylic acid (C307, 145 mg, 84%) as a solid. LCMS (ES, m/z): 307 [M+H]+

Synthesis of Compound 481

Example 204: Synthesis of Compound 482

Synthesis of Intermediate C308

Synthesis of Compound 482

Example 205: Synthesis of Compound 390

Example 206: Synthesis of Compound 143

Synthesis of Intermediate C309

Synthesis of Compound 143

Example 207: Synthesis of Compound 468

Synthesis of Intermediate C310

Synthesis of Compound 460

A solution of tert-butyl N-{1-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]-4-methylpiperidin-4-yl}-N-methylcarbamate (200 mg, 0.355 mmol, 1 equiv) in TFA (2 mL) and DCM (2 mL) was stirred for 2 hr at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was basified to pH 8 with 7 M NH3(g) in MeOH. The resulting mixture was concentrated under vacuum. The residue was purified by reverse flash chromatography (Condition 3, Gradient 8) to afford 2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}-4-[4-methyl-4-(methylamino)piperidin-1-yl]indazole-7-carboxamide (Compound 460, 50 mg, 30%) as a solid.

Example 208: Synthesis of Compound 496

Synthesis of Intermediate C311

To a stirred mixture of Cis-tert-butyl 4-(7-((8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl)carbamoyl)-2-((1s,3s)-3-hydroxycyclobutyl)-2H-indazol-4-yl)piperazine-1-carboxylate (270.0 mg, 0.479 mmol, 1.0 equiv) and K2CO3 (133.3 mg, 0.958 mmol, 2.0 equiv), DMF (5 mL) were added methyl iodide (101.9 mg, 0.718 mmol, 1.5 equiv) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 6 hr at 50° C. under nitrogen atmosphere. The reaction was quenched by the addition of water (20 mL) at room temperature. The resulting mixture was extracted with ethyl acetate (2×20 mL). The combined organic layers were washed with brine (1×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (20:1) to afford Cis-tert-butyl 4-(7-((8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl)carbamoyl)-2-((1s,3s)-3-methoxycyclobutyl)-2H-indazol-4-yl)piperazine-1-carboxylate (C311, 150 mg, 49%) as a solid. LCMS (ES, m/z): 578 [M+H]+

Synthesis of Compound 496

Example 209: Synthesis of Compound 420

Synthesis of Intermediate C312

To a stirred solution of NaH (1.42 g, 59.313 mmol, 1.5 equiv) in THF (100 mL) was added ethanol (2.19 g, 47.450 mmol, 1.2 equiv) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 0.5 h at room temperature under nitrogen atmosphere. To the above mixture was added 3,5-dibromopyrazin-2-amine (10.0 g, 39.542 mmol, 1.0 equiv) at room temperature. The resulting mixture was stirred for additional 16 hr at 50° C. The reaction was quenched with water at 0° C. The resulting mixture was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with water (1×200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (2:1) to afford 5-bromo-3-ethoxypyrazin-2-amine (C312, 6 g, 69%) as a solid. LCMS (ES, m/z): 218 [M+H]+

Synthesis of Intermediate C313

To a stirred mixture of 5-bromo-3-ethoxypyrazin-2-amine (3.40 g, 15.592 mmol, 1 equiv) and 1-bromo-2,2-dimethoxypropane (3.42 g, 18.710 mmol, 1.2 equiv) in i-PrOH (60 mL) was added PPTS (0.39 g, 1.559 mmol, 0.1 equiv) at room temperature. The resulting mixture was stirred for 16 hr at 80° C. under nitrogen atmosphere. The mixture was neutralized to pH 7 with saturated NaHCO3 (aq.). The organic solvent was concentrated under vacuum. The residue was extracted with ethyl acetate (3×100 mL). The combined organic layers were washed with water (1×200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (3:1) to afford 6-bromo-8-ethoxy-2-methylimidazo[1,2-a]pyrazine (C313, 1.9 g, 47%) as a solid. LCMS (ES, m/z): 256 [M+H]+

Synthesis of Intermediate C314

To a stirred mixture of tert-butyl N-[1-(7-carbamoyl-2-methylindazol-4-yl)pyrrolidin-3-yl]-N-methylcarbamate (280.0 mg, 0.577 mmol, 1 equiv, 77%) and 6-bromo-8-ethoxy-2-methylimidazo[1,2-a]pyrazine (221.8 mg, 0.865 mmol, 1.5 equiv) in dioxane (6 mL) were added Cs2CO3 (564.3 mg, 1.731 mmol, 3 equiv) and XantPhos (66.8 mg, 0.115 mmol, 0.2 equiv) and Pd2(dba)3 (52.9 mg, 0.058 mmol, 0.1 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 hr at 100° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (5 mL). The resulting mixture was extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with brine (1×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl N-{1-[7-({8-ethoxy-2-methylimidazo[1,2-a]pyrazin-6-yl}carbamoyl)-2-methylindazol-4-yl]pyrrolidin-3-yl}-N-methylcarbamate (C314, 180 mg, 56%) as a solid. LCMS (ES, m/z): 549 [M+H]+

Synthesis of Compound 420

Example 210: Synthesis of Compound 483

Synthesis of Intermediate C315

Synthesis of Intermediate C316

Synthesis of Intermediate C317

Synthesis of Intermediate C318

Synthesis of Intermediate C319

Synthesis of Intermediate C320

Synthesis of Intermediate C321

A solution of 1-(4-bromo-3-fluoro-1H-pyrrol-2-yl)ethanone (700 mg, 3.398 mmol, 1 equiv) and bromoacetone (698.13 mg, 5.097 mmol, 1.5 equiv) in ACN (7 mL) was stirred for 12 hr at 50° C. under N2 atmosphere. The resulting mixture was extracted with EA (10 mL×2). The combined organic layers were washed with brine (5 mL×2), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure The residue was purified by silica gel column chromatography, eluted with PE:EA (0%˜50%) to afford 1-(2-acetyl-4-bromo-3-fluoropyrrol-1-yl)propan-2-one (C321, 430 mg, 48%) as a solid. LCMS (ES, m/z): 262 [M+H]+

Synthesis of Intermediate C322

Synthesis of Intermediate C323

Synthesis of Compound 483

Example 211: Synthesis of Compound 485

Synthesis of Intermediate C234

Synthesis of Compound 485

Example 212: Synthesis of Compound 486

Example 213: Synthesis of Compound 487

Example 214: Synthesis of Compound 488

Synthesis of Intermediate C235

Synthesis of Compound 488

Example 215: Synthesis of Compound 489

Synthesis of Intermediate C236

Synthesis of Compound 489

Example 216: Synthesis of Compound 490

Example 217: Synthesis of Compound 491

Synthesis of Intermediate C237

Synthesis of Compound 491

Example 218: Synthesis of Compound 492

Synthesis of Intermediate C238

Synthesis of Compound 492

Example 219: Synthesis of Compound 493

Synthesis of Intermediate C339

Synthesis of Intermediate C340

To a stirred solution of methyl 4-[(3R,5S)-4-(tert-butoxycarbonyl)-3,5-dimethylpiperazin-1-yl]-2-ethyl-6-fluoroindazole-7-carboxylate (95 mg, 0.219 mmol, 1 equiv) in THF (1.2 mL) and H2O (0.4 mL) was added lithiumol hydrate (18.35 mg, 0.438 mmol, 2 equiv) at room temperature. The resulting mixture was stirred overnight at 30° C. The resulting mixture was concentrated under vacuum. The resulting mixture was diluted with H2O (5 mL). The mixture was acidified to pH 4 with citric acid and extracted with DCM (3×10 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-[(3R,5S)-4-(tert-butoxycarbonyl)-3,5-dimethylpiperazin-1-yl]-2-ethyl-6-fluoroindazole-7-carboxylic acid (C340, 85 mg, 92%) as a solid. LCMS (ES, m/z): 419 [M−H]−

Synthesis of Intermediate C341

Synthesis of Compound 493

Example 220: Synthesis of Compound 494

Synthesis of Intermediate C342

Synthesis of Intermediate C343

Synthesis of Intermediate C344

Synthesis of Compound 494

Example 221: Synthesis of Compound 495

Synthesis of Intermediate C345

Synthesis of Intermediate C346

Synthesis of Intermediate C347

Synthesis of Intermediate C348

Synthesis of Compound 495

A solution of tert-butyl-4-[2-methyl-7-({2-methyl-5H,6H,7H,8H-[1,2,4]triazolo[1,5-a]pyridin-7-yl}carbamoyl) indazol-4-yl]piperazine-1-carboxylate (100 mg, 0.202 mmol, 1 equiv) in DCM (5 mL) was treated with HCl (gas) in 1,4-dioxane (10 mL, 329.128 mmol, 1627.87 equiv) for 30 mins at 20° C. Desired product could be detected by LCMS. The mixture was neutralized to pH 7 with NaHCO3. The resulting solution was dried N2 gas. The residue was purified by prep-(Condition 16, Gradient 1) to afford 2-methyl-N-{2-methyl-5H,6H,7H,8H-[1,2,4]triazolo[1,5-a]pyridin-7-yl}-4-(piperazin-1-yl) indazole-7-carboxamide (Compound 495, 6 mg, 7%) as a solid.

Example 222: Synthesis of Compound 435

Synthesis of Intermediate C349

To a stirred solution of 4-bromo-2H-1,2,3-benzotriazole (2.7 g, 13.635 mmol, 1.0 equiv) and K2CO3 (3.8 g, 27.270 mmol, 2.0 equiv) in dimethylformamide (60 mL) were added methyl iodide (2.9 g, 20.453 mmol, 1.5 equiv) dropwise at 0° C. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was diluted with water (200 mL). The resulting mixture was extracted with ethyl acetate (2×200 mL). The combined organic layers were washed with water (2×200 mL), brine (2×200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/PE (5:1) to afford 4-bromo-1-methyl-1,2,3-benzotriazole (C349, 0.8 g, 25%) as a solid. LCMS (ES, m/z): 212 [M+H]+

Synthesis of Intermediate C350

Synthesis of Intermediate C351

To a stirred solution of tert-butyl 4-(1-methyl-1,2,3-benzotriazol-4-yl) piperazine-1-carboxylate (850.0 mg, 2.678 mmol, 1.0 equiv) in ACN (15 mL) were added NBS (524.3 mg, 2.946 mmol, 1.1 equiv) in portions at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was diluted with deionized water (30 mL). The resulting mixture was extracted with ethyl acetate (2×40 mL). The combined organic layers were washed with water (2×30 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-(7-bromo-1-methyl-1,2,3-benzotriazol-4-yl) piperazine-1-carboxylate (C351 800 mg, 69%) as a solid. LCMS (ES, m/z): 396 [M+H]+

Synthesis of Intermediate C352

To a solution of tert-butyl 4-(7-bromo-1-methyl-1,2,3-benzotriazol-4-yl)piperazine-1-carboxylate (250.0 mg, 0.631 mmol, 1.0 equiv) in MeOH (20 mL) was added Pd(dppf)Cl2 (46.1 mg, 0.063 mmol, 0.1 equiv), TEA (191.5 mg, 1.893 mmol, 3.0 equiv) in a pressure tank. The mixture was purged with nitrogen for 2 min and then was pressurized to 2 Mpa with carbon monoxide at 80° C. for 16 hr. The reaction mixture was cooled to room temperature and filtered to remove insoluble solids. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (30:1) to afford methyl 7-[4-(tert-butoxycarbonyl) piperazin-1-yl]-3-methyl-1,2,3-benzotriazole-4-carboxylate (240.0 mg, 94.24%) as a solid. LCMS (ES, m/z): 376 [M+H]+

Synthesis of Intermediate C353

To a stirred mixture of methyl 7-[4-(tert-butoxycarbonyl) piperazin-1-yl]-3-methyl-1,2,3-benzotriazole-4-carboxylate (170.0 mg, 0.453 mmol, 1.0 equiv) in tetrahydrofuran (3 mL) and water (3 mL) was added LiOH·H2O (108.4 mg, 4.530 mmol, 10.0 equiv) in portions at room temperature. The resulting mixture was stirred for 3 hr at 50° C. The resulting mixture was diluted with water (20 mL). The mixture was acidified to pH 6 with HCl (1 N). The resulting mixture was extracted with ethyl acetate (2×30 mL). The combined organic layers were washed with brine (1×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The resulting mixture was concentrated under reduced pressure. This resulted in 7-[4-(tert-butoxycarbonyl) piperazin-1-yl]-3-methyl-1,2,3-benzotriazole-4-carboxylic acid (C353, 150 mg, 85%) as a solid. LCMS (ES, m/z): 362 [M+H]+

Synthesis of Intermediate C354

Synthesis of Compound 435

Example 223: Synthesis of Compound 436

Synthesis of Intermediate C355

To a stirred solution of 4-bromo-2H-1,2,3-benzotriazole (2.7 g, 13.635 mmol, 1.0 equiv) and K2CO3 (3.8 g, 27.270 mmol, 2.0 equiv) in dimethylformamide (60 mL) were added methyl iodide (2.9 g, 20.453 mmol, 1.5 equiv) dropwise at 0° C. The resulting mixture was stirred for 2 h at room temperature. The resulting mixture was diluted with deionized water (100 mL). The resulting mixture was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with brine (2×100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/PE (5:1) to afford 7-bromo-1-methyl-1,2,3-benzotriazole (C355, 1 g, 31%) as a solid. LCMS (ES, m/z): 212 [M+H]+

Synthesis of Intermediate C356

Synthesis of Intermediate C357

To a stirred solution of tert-butyl 4-(3-methyl-1,2,3-benzotriazol-4-yl) piperazine-1-carboxylate (850.0 mg, 2.678 mmol, 1.0 equiv) in ACN (15 mL) were added NBS (524.3 mg, 2.946 mmol, 1.1 equiv) in portions at room temperature. The resulting mixture was diluted with water (30 mL). The resulting mixture was extracted with ethyl acetate (2×40 mL). The combined organic layers were washed with brine (1×30 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-(7-bromo-3-methyl-1,2,3-benzotriazol-4-yl)piperazine-1-carboxylate (C357, 830 mg, 72%) as a solid. LCMS (ES, m/z): 396 [M+H]+

Synthesis of Intermediate C358

To a solution of tert-butyl 4-(7-bromo-3-methyl-1,2,3-benzotriazol-4-yl) piperazine-1-carboxylate (250 mg, 0.631 mmol, 1 equiv) in 20 mL MeOH was added Pd(dppf)Cl2CH2Cl2 (46.1 mg, 0.063 mmol, 0.1 equiv), TEA (191.5 mg, 1.893 mmol, 3.0 equiv) in a pressure tank. The mixture was purged with nitrogen for 2 min and then was pressurized to 2 Mpa with carbon monoxide at 80° C. for 16 hr. The reaction mixture was cooled to room temperature and filtered to remove insoluble solids. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (30:1) to afford methyl 7-[4-(tert-butoxycarbonyl)piperazin-1-yl]-1-methyl-1,2,3-benzotriazole-4-carboxylate (C358, 230 mg, 87%) as a solid. LCMS (ES, m/z): 376 [M+H]+

Synthesis of Intermediate C359

To a stirred mixture of methyl 7-[4-(tert-butoxycarbonyl) piperazin-1-yl]-1-methyl-1,2,3-benzotriazole-4-carboxylate (170.0 mg, 0.453 mmol, 1.0 equiv) in tetrahydrofuran (3 mL) and water (3 mL) was added LiOH·H2O (108.4 mg, 4.530 mmol, 10.0 equiv) in portions at room temperature. The resulting mixture was stirred for 3 hr at 50° C. The resulting mixture was diluted with deionized water (20 mL). The mixture was acidified to pH 6 with HCl (1 N). The resulting mixture was extracted with ethyl acetate (2×30 mL). The combined organic layers were washed with brine (1×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The resulting mixture was concentrated under reduced pressure. This resulted in 7-[4-(tert-butoxycarbonyl) piperazin-1-yl]-1-methyl-1,2,3-benzotriazole-4-carboxylic acid (C359, 145 mg, 83%) as a solid. LCMS (ES, m/z): 362 [M+H]+

Synthesis of Intermediate C360

Synthesis of Compound 436

Example 224: Synthesis of Compound 448

Synthesis of Intermediate C361

Synthesis of Compound 448

Example 225: Synthesis of Compound 449

Example 226: Synthesis of Compound 461

Example 227: Synthesis of Compounds

Example 228: Synthesis of Compound 304

Synthesis of Intermediate C363

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (100 mg, 0.20 mmol, 1.0 equiv) and 4-iodooxane (64.4 mg, 0.30 mmol, 1.5 equiv) in DMF (1 mL) were added Cs2CO3 (198.0 mg, 0.61 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 3 h at room temperature. The reaction was quenched by the addition of water at room temperature. The resulting mixture was extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (1×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(oxan-4-yl) indazol-4-yl]piperazine-1-carboxylate (41 mg, 35%) as a solid. LCMS (ES, m/z): 578 [M+H]+

Synthesis of Compound 304

Example 229: Synthesis of Compound 342

Synthesis of Intermediate C365

Synthesis of Intermediate C366

Synthesis of Intermediate C367

A solution of methyl 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-methylindazole-7-carboxylate (2.5 g, 6.677 mmol, 1 equiv) in NH3(g) in MeOH (70 mL) was stirred for 2 days at 100° C. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-(7-carbamoyl-2-methylindazol-4-yl)piperazine-1-carboxylate (1.35 g, 56%) as an solid. LCMS (ES, m/z): 360[M+H]+

Synthesis of Intermediate C368

Synthesis of Compound 342

Example 230: Synthesis of Compound 393

Synthesis of Intermediate C370

Synthesis of Compound 393

Example 231: Synthesis of Compound 423

Synthesis of Intermediate C371

Synthesis of Compound 423

Example 232: Synthesis of Compound 452

Synthesis of Intermediate C373

Synthesis of Compound 452

Example 233: Synthesis of Compound 475

Synthesis of Intermediate C375

Synthesis of Intermediate C376

To a stirred solution of methyl 4-[(3R,5S)-4-(tert-butoxycarbonyl)-3,5-dimethylpiperazin-1-yl]-2-ethyl-6-fluoroindazole-7-carboxylate (95 mg, 0.219 mmol, 1 equiv) in THF (1.2 mL) and H2O (0.4 mL) was added lithiumol hydrate (18.35 mg, 0.438 mmol, 2 equiv) at room temperature. The resulting mixture was stirred overnight at 30° C. The resulting mixture was concentrated under vacuum. The resulting mixture was diluted with H2O (5 mL). The mixture was acidified to pH 4 with citric acid and extracted with DCM (3×10 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-[(3R,5S)-4-(tert-butoxycarbonyl)-3,5-dimethylpiperazin-1-yl]-2-ethyl-6-fluoroindazole-7-carboxylic acid (85 mg, 92%) as a solid. LCMS (ES, m/z): 419 [M−H]−

Synthesis of Intermediate C377

Synthesis of Compound 475

Example 234: Synthesis of Compound 497

Synthesis of Intermediate C378

To a stirred solution of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperazine-1-carboxylate (110 mg, 0.223 mmol, 1.0 equiv) and 3-(iodomethyl)oxetane (66.20 mg, 0.335 mmol, 1.5 equiv) in DMF (2.2 mL) was added Cs2CO3 (217.8 mg, 0.669 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was diluted with water (10 mL) and extracted with EtOAc (3×10 mL). The combined organic layer was washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA (100%) to afford tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-(oxetan-3-ylmethyl) indazol-4-yl]piperazine-1-carboxylate (65 mg, 52%) as a solid. LCMS (ES, m/z): 423.2 [M+H]+

Synthesis of Compound 497

Example 235: Synthesis of Compound 498

Synthesis of Intermediate C379

Synthesis of Compound 498

Example 236: Synthesis of Compound 499

Synthesis of Intermediate C380

Synthesis of Compound 499

Example 237: Synthesis of Compound 500

Synthesis of Intermediate C381

A solution of methyl 4-bromo-2-methylindazole-7-carboxylate (550 mg, 2.044 mmol, 1.0 equiv) in THF (6 mL) was treated with DIBAL-H (6.13 mL, 6.132 mmol, 3.0 equiv) at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 2 h at 0° C. under nitrogen atmosphere. The reaction was quenched with water at 0° C. The resulting mixture was extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford (4-bromo-2-methylindazol-7-yl)methanol (505 mg, 100%) as a solid. LCMS (ES, m/z): 241 [M+H]+

Synthesis of Intermediate C382

A solution of (4-bromo-2-methylindazol-7-yl)methanol (505 mg, 2.095 mmol, 1 equiv) in DCM (5 mL) was treated with manganese dioxide (1821.0 mg, 20.950 mmol, 10 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 4 h at room temperature under nitrogen atmosphere. The resulting mixture was filtered and the filter cake was washed with DCM (2×5 mL). The filtrate was concentrated under reduced pressure to afford 4-bromo-2-methylindazole-7-carbaldehyde (440 mg, 88%) as a solid. LCMS (ES, m/z): 239 [M+H]+

Synthesis of Intermediate C383

To a stirred mixture of 4-bromo-2-methylindazole-7-carbaldehyde (440 mg, 1.840 mmol, 1.0 equiv) and 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (364.8 mg, 2.208 mmol, 1.2 equiv) in DCM (5 mL) was added NaBH(OAc)3 (780.1 mg, 3.680 mmol, 2.0 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 h at room temperature under nitrogen atmosphere. The reaction was quenched with Water at room temperature. The resulting mixture was extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford N-[(4-bromo-2-methylindazol-7-yl)methyl]-8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (610 mg, 85%) as a solid. LCMS (ES, m/z): 388 [M+H]+

Synthesis of Intermediate C385

Synthesis of Compound 500

Example 238: Synthesis of Compound 501

Synthesis of Intermediate C387

Synthesis of Intermediate C388

A mixture of methyl 4-bromo-2-(prop-2-yn-1-yloxy)benzoate (5.0 g, 18.581 mmol, 1 equiv) and CsF (2.82 g, 18.581 mmol, 1.0 equiv) in DMA (50 mL) was irradiated with microwave for 4 h at 190° C. The reaction was quenched with water (200 mL). The resulting mixture was extracted with EA (3×100 mL). The combined organic layers were washed with brine (2×100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (3:1) to afford methyl 4-bromo-2-methyl-1-benzofuran-7-carboxylate (2.0 g, 40%) as a solid. LCMS (ES, m/z): 269 [M+H]+

Synthesis of Intermediate C389

Synthesis of Intermediate C390

To a stirred solution of methyl 4-{4-[(tert-butoxycarbonyl)(ethyl)amino]piperidin-1-yl}-2-methyl-1-benzofuran-7-carboxylate (720 mg, 1.729 mmol, 1 equiv) in H2O (5 mL), MeOH (10 mL) and THF (10 mL) were added LiOH (248.40 mg, 10.374 mmol, 6.0 equiv) in portions at room temperature. The resulting mixture was stirred for 2 h at 50° C. The resulting mixture was concentrated under reduced pressure. The residue was acidified to pH 6 with 1 N HCl. The precipitated solids were collected by filtration and washed with water (2×20 mL). The resulting solids were dried to afford 4-{4-[(tert-butoxycarbonyl)(ethyl)amino]piperidin-1-yl}-2-methyl-1-benzofuran-7-carboxylic acid (650 mg, 93%) as a solid. LCMS (ES, m/z): 403 [M+H]+

Synthesis of Intermediate C391

Synthesis of Intermediate C392

Synthesis of Compound 501

Example 239: Synthesis of Compound 502

Synthesis of Intermediate C393

Synthesis of Intermediate C394

Synthesis of Intermediate C395

Synthesis of Intermediate C396

Synthesis of Compound 502

Example 240: Synthesis of Compound 503

Synthesis of Intermediate C397

Synthesis of Intermediate C398

To a stirred solution of methyl 4-{4-[(tert-butoxycarbonyl)(ethyl)amino]piperidin-1-yl}-2H-indazole-7-carboxylate (1 g, 2.485 mmol, 1 equiv) in THF (9 mg) and MeOH (9 mL) was added a solution of LiOH·H2O (0.6 g, 24.850 mmol, 10 equiv) in H2O (9 mL) dropwise at room temperature. The resulting mixture was stirred for 1 h at room temperature. The mixture was acidified to pH 2 with 2 M HCl. The precipitated solids were collected by filtration and washed with H2O (1×10 mL). This resulted in 4-{4-[(tert-butoxycarbonyl)(ethyl)amino]piperidin-1-yl}-2H-indazole-7-carboxylic acid (0.8 g, 83%) as a solid. LCMS (ES, m/z): 389[M+H]+

Synthesis of Intermediate C399

Synthesis of Intermediate C400

To a stirred solution of tert-butyl N-ethyl-N-{1-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2H-indazol-4-yl]piperidin-4-yl}carbamate (180 mg, 0.336 mmol, 1 equiv) and 1-chloro-2-iodoethane (95.9 mg, 0.504 mmol, 1.5 equiv) in DMF (3 mL) was added KOH (113.1 mg, 2.016 mmol, 6.0 equiv) in portions at room temperature. The resulting mixture was stirred for 12 h at 80° C. The mixture was allowed to cool down to room temperature. The resulting mixture was purified by reverse flash chromatography (Condition 5, Gradient 2) to afford tert-butyl N-{1-[2-ethenyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]piperidin-4-yl}-N-ethylcarbamate (80 mg, 42%) as a solid. LCMS (ES, m/z): 562[M+H]+

Synthesis of Compound 503

Example 241: Synthesis of Compound 504

Synthesis of Intermediate C402

A solution of 2-fluoro-6-methoxyaniline (15 g, 106.274 mmol, 1 equiv) in CH3CN (200 mL) was treated with NBS (28.37 g, 159.411 mmol, 1.5 equiv) for 8 hours at 20° C. The mixture was neutralized to pH 7 with NaHCO3. The aqueous layer was extracted with DCM 500 mL×3 times. The combined organic layers were washed with water (1×500 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (3:1) to afford 4-bromo-2-fluoro-6-methoxyaniline (5 g, 21%) is given with solid. LCMS (ES, m/z): 220 [M+H]+

Synthesis of Intermediate C403

To a solution of 4-bromo-2-fluoro-6-methoxyaniline (5 g, 22.723 mmol, 1 equiv) in DCM (50 mL) was added BBr3 (8.54 g, 34.084 mmol, 1.5 equiv) dropwise at 0° C. The resulting mixture was stirred for 16 h at room temperature. The resulting mixture was diluted with water (100 mL). The mixture was neutralized to pH 7 with NaHCO3. The resulting mixture was extracted with EtOAc (3×200 mL). The combined organic layers were washed with water (1×500 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with MeOH/DCM (1/20) to afford 2-amino-5-bromo-3-fluorophenol (3.7 g, 79%) as an oil. LCMS (ES, m/z): 206 [M+H]+

Synthesis of Intermediate C404

Synthesis of Intermediate C405

A solution of 6-bromo-2-(chloromethyl)-4-fluoro-1,3-benzoxazole (2.26 g, 8.545 mmol, 1 equiv) in MeOH (20 mL) was treated with sodium methoxide (4.62 g, 85.450 mmol, 10 equiv) for 8 hours at 26° C. The resulting mixture was diluted with water (100 mL). The aqueous layer was extracted with DCM (3×100 mL). The resulting mixture was washed with 2×200 mL of brine. The resulting organic layer was dried by Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (3/1) to afford 6-bromo-4-fluoro-2-(methoxymethyl)-1,3-benzoxazole (2 g, 90%) as a solid. LCMS (ES, m/z): 260 [M+H]+

Synthesis of Intermediate C406

Synthesis of Compound 504

Example 242: Synthesis of Compound 505

Synthesis of Intermediate C407

Synthesis of Compound 505

Example 243: Synthesis of Compound 506

Synthesis of Intermediate C408

Synthesis of Compound 506

Example 244: Synthesis of Compound 507

Synthesis of Intermediate C410

Synthesis of Intermediate C411

Synthesis of Intermediate C412

Synthesis of Compound 507

Example 245: Synthesis of Compound 508

Example 246: Synthesis of Compound 510

Synthesis of Intermediate C413

Synthesis of Intermediate C414

Synthesis of Compound 510

Example 247: Synthesis of Compound 511

Synthesis of Intermediate C415

Into a 100 mL 3-necked round-bottom flask were added 2,6-dichloro-4-methylpyridin-3-amine (5.0 g, 28.244 mmol, 1.0 equiv) and acetic anhydride (30 mL) at room temperature. The resulting mixture was stirred for 1 h at 90° C. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford N-(2,6-dichloro-4-methylpyridin-3-yl) acetamide (2.5 g, 37%) as a solid. LCMS (ES, m/z): 219 [M+H]+

Synthesis of Intermediate C416

Synthesis of Intermediate C417

Synthesis of Compound 511

Example 248: Synthesis of Compound 512

Synthesis of Intermediate C418

To a stirred mixture of 4-bromo-2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}indazole-7-carboxamide (150 mg, 0.360 mmol, 1 equiv) and tert-butyl 6-(trifluoro-lambda4-boranyl)-3-azabicyclo[4.1.0]heptane-3-carboxylate potassium (120.2 mg, 0.396 mmol, 1.1 equiv) in Toluene (7.5 mL) and H2O (0.75 mL) were added Cs2CO3 (176.1 mg, 0.540 mmol, 1.5 equiv) and Cata Pd G3 (26.2 mg, 0.036 mmol, 0.1 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 10 h at 90° C. under nitrogen atmosphere. The resulting mixture was diluted with water (10 mL). The resulting mixture was extracted with EtOAc (3×10 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 6-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]-3-azabicyclo[4.1.0]heptane-3-carboxylate (90 mg, 47%) as a solid. LCMS (ES, m/z): 533 [M+H]+

Synthesis of Compound 512

Example 249: Synthesis of Compound 514

Synthesis of Intermediate C419

A solution of ethyl 2-fluoroethanimidate hydrochloride (140 mg, 0.989 mmol, 1 equiv) and 2-amino-5-bromo-3-fluorophenol (142.6 mg, 0.692 mmol, 0.7 equiv) in EtOH (4 mL) was stirred for 1 h at 60° C. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under reduced pressure. The crude product was used in the next step directly without further purification. LCMS (ES, m/z): 248 [M+H]+

Synthesis of Intermediate C420

Synthesis of Compound 514

Example 250: Synthesis of Compound 515

Synthesis of Intermediate C421

Synthesis of Intermediate C422

A solution of methyl 4-{4-[(tert-butoxycarbonyl)amino]-4-ethylpiperidin-1-yl}-2-ethylindazole-7-carboxylate (300 mg, 0.697 mmol, 1 equiv) in DMF (4 mL) was treated with NaH (55.7 mg, 1.394 mmol, 2.0 equiv, 60%) for 30 min at 0° C. followed by the addition of CH3I (148.3 mg, 1.045 mmol, 1.5 equiv) dropwise at 0° C. The resulting mixture was stirred for 2 h at room temperature. The resulting mixture was diluted with H2O (5 mL). The resulting mixture was extracted with EA (3×10 mL). The combined organic layers were washed with brine (1×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography (Condition 5, Gradient 2) to afford methyl 4-{4-[(tert-butoxycarbonyl)(methyl)amino]-4-ethylpiperidin-1-yl}-2-ethylindazole-7-carboxylate (170 mg, 55%) as a solid. LCMS (ES, m/z): 445 [M+H]+

Synthesis of Intermediate C423

To a solution of methyl 4-{4-[(tert-butoxycarbonyl)(methyl)amino]-4-ethylpiperidin-1-yl}-2-ethylindazole-7-carboxylate (170 mg, 0.382 mmol, 1 equiv) in MeOH (2 mL) and THF (2 mL) was added a solution of LiOH·H2O (96.2 mg, 2.292 mmol, 6.0 equiv) in H2O (2 mL) at room temperature. The resulting mixture was stirred for 1 h at room temperature. The mixture was acidified to pH 2 with 2 M HCl. The resulting mixture was extracted with EA (3×5 mL). The combined organic layers were washed with brine (1×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography (Condition 5, Gradient 6) to afford 4-{4-[(tert-butoxycarbonyl)(methyl)amino]-4-ethylpiperidin-1-yl}-2-ethylindazole-7-carboxylic acid (141 mg, 85%) as a solid. LCMS (ES, m/z): 431 [M+H]+

Synthesis of Intermediate C424

To a stirred solution of 4-{4-[(tert-butoxycarbonyl)(methyl)amino]-4-ethylpiperidin-1-yl}-2-ethylindazole-7-carboxylic acid (140 mg, 0.325 mmol, 1 equiv) and TCFH (118.6 mg, 0.423 mmol, 1.3 equiv) in CH3CN (3 mL) were added NMI (93.4 mg, 1.137 mmol, 3.5 equiv) and 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (64.4 mg, 0.390 mmol, 1.2 equiv) in portions at room temperature. The resulting mixture was stirred for 2 h at room temperature. The precipitated solids were collected by filtration and washed with H2O (1×5 mL). The crude product was used in the next step directly without further purification. LCMS (ES, m/z): 578 [M+H]+

Synthesis of Compound 515

Example 251: Synthesis of Compounds 516 and 517

Synthesis of Intermediate C425

Synthesis of Intermediate C426

A solution of tert-butyl N-{1-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]-4-methylpiperidin-4-yl}carbamate (250 mg, 0.455 mmol, 1 equiv) and trifluoroacetic acid (3 mL) in DCM (6 mL) was stirred for 2 h at room temperature under nitrogen atmosphere. The resulting mixture was concentrated under vacuum. The residue was purified by reverse flash chromatography (Condition 3, Gradient 9) to afford 4-(4-amino-4-methylpiperidin-1-yl)-2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}indazole-7-carboxamide (130 mg, 64%) as a solid. LCMS (ES, m/z): 450 [M+H]+

Synthesis of Compounds 516 and 517

Example 252: Synthesis of Compound 518

Synthesis of Intermediate C427

Synthesis of Compound 518

Example 253: Synthesis of Compound 520

Synthesis of Intermediate C428

Synthesis of Compound 520

Example 254: Synthesis of Compound 522

Example 255: Synthesis of Compound 524

Synthesis of Intermediate C429

Synthesis of Compound 524

Example 256: Synthesis of Compound 525

Synthesis of Intermediate C430

Synthesis of Intermediate C431

Into a 40 mL vial were added tert-butyl 4-(cyclopropylamino)-4-methylpiperidine-1-carboxylate (260 mg, 1.022 mmol, 1 equiv) and HCl(gas) in 1,4-dioxane (5 mL) at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under vacuum. The crude product was used in the next step directly without further purification. LCMS (ES, m/z): 155 [M+H]+

Synthesis of Compound 525

Example 256: Synthesis of Compound 526

Synthesis of Intermediate C432

Synthesis of Intermediate C433

A solution of methyl 4-[1-(tert-butoxycarbonyl)-4,5-dihydropyrrol-3-yl]-2-ethylindazole-7-carboxylate (500 mg, 1.346 mmol, 1 equiv) in MeOH (15 mL) was added Pd/C (200 mg, 10% w/w). The mixture was stirred for 3 h at room temperature under hydrogen atmosphere. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluted with PE/EA (1/1) to afford methyl 4-[1-(tert-butoxycarbonyl) pyrrolidin-3-yl]-2-ethylindazole-7-carboxylate (270 mg, 54%) as an oil. LCMS (ES, m/z): 374 [M+H]+

Synthesis of Intermediate C434

A solution of methyl 4-[1-(tert-butoxycarbonyl) pyrrolidin-3-yl]-2-ethylindazole-7-carboxylate (270 mg, 0.723 mmol, 1 equiv) in THF/MeOH/H2O (1:1:1) (6 mL) was added LiOH·H2O (303.36 mg, 7.230 mmol, 10.0 equiv). The mixture was stirred for 1 h at 50° C. to give a yellow mixture. The reaction was monitored by LCMS. The resulting mixture was concentrated under vacuum and acidified to pH 5-6 with 1M HCl. The resulting mixture was extracted with EtOAc (3×30 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-[1-(tert-butoxycarbonyl) pyrrolidin-3-yl]-2-ethylindazole-7-carboxylic acid (170 mg, 65%) as a oil. LCMS (ES, m/z): 360 [M+H]+

Synthesis of Intermediate C435

Synthesis of Intermediate C436

Synthesis of Compound 526

Example 257: Synthesis of Compound 527

Synthesis of Intermediate C437

Synthesis of Intermediate C438

To a solution of methyl 4-{[1-(tert-butoxycarbonyl)piperidin-4-yl]amino}-2-methylindazole-7-carboxylate (110 mg, 0.283 mmol, 1 equiv) in THF (2 mL) was added NaH (17.0 mg, 0.424 mmol, 1.5 equiv, 60%) at 0° C. The mixture was stirred for 30 min and ethyl iodide (44.2 mg, 0.283 mmol, 1.0 equiv) was added and the mixture was allowed to warm to rt and stirred for 2 h. The reaction was quenched with MeOH. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford methyl 4-{[1-(tert-butoxycarbonyl)piperidin-4-yl](ethyl)amino}-2-methylindazole-7-carboxylate (120 mg, 92%) as a solid. LCMS (ES, m/z): 417 [M+H]+

Synthesis of Intermediate C439

To a stirred mixture of methyl 4-{[1-(tert-butoxycarbonyl)piperidin-4-yl](ethyl)amino}-2-methylindazole-7-carboxylate (120 mg, 0.288 mmol, 1 equiv) in THF (1.5 mL) and H2O (1.5 mL) was added lithiumol hydrate (96.71 mg, 2.304 mmol, 8 equiv) at room temperature. The resulting mixture was stirred for 2 h at 50° C. The mixture was acidified to pH 5 with HCl (2M). The resulting mixture was extracted with EtOAc (3×5 mL). The combined organic layers were washed with water (3×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-{[1-(tert-butoxycarbonyl)piperidin-4-yl](ethyl)amino}-2-methylindazole-7-carboxylic acid (120 mg, 99%) as a solid. LCMS (ES, m/z): 403 [M+H]+

Synthesis of Intermediate C440

To a stirred mixture of 4-{[1-(tert-butoxycarbonyl)piperidin-4-yl](ethyl)amino}-2-methylindazole-7-carboxylic acid (100 mg, 0.248 mmol, 1 equiv) and 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-amine (49.24 mg, 0.298 mmol, 1.2 equiv) in DMF (1 mL) were added NMI (81.60 mg, 0.992 mmol, 4 equiv) and TCFH (104.57 mg, 0.372 mmol, 1.5 equiv) at room temperature. The resulting mixture was stirred for 4 h at room temperature. The resulting mixture was diluted with water (6 mL). The resulting mixture was extracted with EtOAc (3×6 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (10:1) to afford tert-butyl 4-{ethyl[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-methylindazol-4-yl]amino}piperidine-1-carboxylate (50 mg, 37%) as a solid. LCMS (ES, m/z): 550 [M+H]+

Synthesis of Compound 527

Example 258: Synthesis of Compound 528

Synthesis of Intermediate C441

To a stirred mixture of tert-butyl 4-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-methylindazol-4-yl]piperazine-1-carboxylate (85.0 mg, 0.167 mmol, 1.0 equiv) in DCE (2 mL) was added PCl5 (45.3 mg, 0.217 mmol, 1.3 equiv) in portions at room temperature. The resulting mixture was stirred for 5 h at 60° C. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The crude product mixture was used in the next step directly without further purification. LCMS (ES, m/z): 541 [M+H]+

Synthesis of Compound 528

Example 259: Synthesis of Compound 529

Synthesis of Intermediate C442

To a stirred mixture of 6-bromo-2-methoxypyridin-3-amine (9 g, 44.326 mmol, 1 equiv) in DCM (90 mL) was added NCS (7.1 g, 53.191 mmol, 1.2 equiv) in portions at 0° C. The resulting mixture was stirred for overnight at room temperature. The resulting mixture was diluted with water (100 mL). The resulting mixture was extracted with CH2Cl2 (2×100 mL). The combined organic layers were washed with brine (2×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (10:1) to afford 6-bromo-4-chloro-2-methoxypyridin-3-amine (6 g, 57%) as a solid. LCMS (ES, m/z): 237 [M+H]+

Synthesis of Intermediate C443

Synthesis of Intermediate C444

Synthesis of Intermediate C445

Synthesis of Compound 529

Example 260: Synthesis of Compound 531

Example 261: Synthesis of Compound 533

Example 262: Synthesis of Compound 535

Synthesis of Intermediate C446

Synthesis of Compound 535

Example 263: Synthesis of Compounds 537 and 538

Synthesis of Compound 537

Synthesis of Compound 538

Example 264: Synthesis of Compound 539

Synthesis of Intermediate C447

Synthesis of Compound 539

Example 265: Synthesis of Compounds 541 and 542

Synthesis of Intermediate C448

Synthesis of Compound 541

Synthesis of Compound 542

Example 266: Synthesis of Compound 543

Synthesis of Intermediate C449

Synthesis of Compound 543

Example 267: Synthesis of Compounds 544, 547, and 548

Synthesis of Intermediate C450

To a stirred solution of tert-butyl N-(3-methylpyrrolidin-3-yl)carbamate (2.0 g, 9.986 mmol, 1.0 equiv) and DIEA (2.5 g, 19.972 mmol, 2.0 equiv) in DCM (30 mL) was added benzyl chloroformate (2.0 g, 11.983 mmol, 1.2 equiv) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 5 h at room temperature. The reaction was monitored by LCMS. The resulting mixture was diluted with deionized water (50 mL). The resulting mixture was extracted with CH2Cl2 (2×50 mL). The combined organic layers were washed with brine (1×50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (5:1) to afford benzyl 3-[(tert-butoxycarbonyl)amino]-3-methylpyrrolidine-1-carboxylate (3.4 g, 92%) as a solid. LCMS (ES, m/z): 335 [M+H]+

Synthesis of Intermediate C451

To a stirred solution of benzyl 3-[(tert-butoxycarbonyl)amino]-3-methylpyrrolidine-1-carboxylate (750.0 mg, 2.243 mmol, 1.0 equiv) and NaH (107.6 mg, 4.486 mmol, 2.0 equiv) in dimethylformamide (15 mL) was added methyl iodide (636.6 mg, 4.486 mmol, 2.0 equiv) dropwise at 0° C. The resulting mixture was stirred for 1 h at room temperature. The reaction was quenched with Water at 0° C. The resulting mixture was extracted with EtOAc (2×50 mL). The combined organic layers were washed with brine (1×50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. LCMS (ES, m/z): 349 [M+H]+

Synthesis of Intermediate C452

To a solution of benzyl 3-[(tert-butoxycarbonyl)(methyl)amino]-3-methylpyrrolidine-1-carboxylate (650.0 mg, 1.865 mmol, 1.0 equiv) in 15 mL MeOH was added Pd/C (10%, 59.5 mg) under nitrogen atmosphere in a 100 mL round-bottom flask. The mixture was hydrogenated at room temperature for overnight under hydrogen atmosphere using a hydrogen balloon, filtered through a Celite pad and concentrated under reduced pressure. This resulted in tert-butyl N-methyl-N-(3-methylpyrrolidin-3-yl) carbamate (350 mg, 79%) as an oil. LCMS (ES, m/z): 215 [M+H]+

Synthesis of Intermediate C453

Synthesis of Compound 544

Synthesis of Compound 547

Synthesis of Compound 548

Example 268: Synthesis of Compound 545

Synthesis of Intermediate C454

Synthesis of Compound 545

Example 269: Synthesis of Compound 550

Synthesis of Intermediate C455

Synthesis of Intermediate C456

To a stirred solution of methyl 4-{[(1R,5S)-8-(tert-butoxycarbonyl)-8-azabicyclo[3.2.1]octan-3-yl]amino}-2-ethylindazole-7-carboxylate (500 mg, 1.167 mmol, 1 equiv) in DMF (6 mL) was added NaH (93.33 mg, 2.334 mmol, 2.0 equiv, 60%) in portions at 0° C. The resulting mixture was stirred for 0.5 h at 0° C. To the above mixture was added CH3I (165.6 mg, 1.167 mmol, 1.0 equiv) dropwise at 0° C. The resulting mixture was stirred for additional 2 h at room temperature. The reaction mixture was quenched by the addition of MeOH (5 mL) at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography (Condition 5, Gradient 2) to afford methyl 4-{[(1R,5S)-8-(tert-butoxycarbonyl)-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino}-2-ethylindazole-7-carboxylate (500 mg, 97%) as a solid. LCMS (ES, m/z): 443 [M+H]+

Synthesis of Intermediate C457

Synthesis of Intermediate C458

Synthesis of Compound 550

Example 270: Synthesis of Compound 552

Synthesis of Intermediate C459

A solution of benzyl (3S)-3-[(4-methylbenzenesulfonyl)oxy]pyrrolidine-1-carboxylate (500 mg, 1.332 mmol, 1.0 equiv) in DMSO (5 mL) was treated with morpholine (580.1 mg, 6.66 mmol, 5.0 equiv) at room temperature. The resulting mixture was stirred for overnight at 70° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was extracted with EtOAc (3×30 mL). The combined organic layers were washed with brine (1×30 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford benzyl (3R)-3-(morpholin-4-yl)pyrrolidine-1-carboxylate (395 mg, 98%) as a solid. LCMS (ES, m/z): 291 [M+H]+

Synthesis of Intermediate C460

Synthesis of Compound 552

Example 271: Synthesis of Compound 554

Synthesis of Intermediate C461

To a stirred solution of allylamine (2.0 g, 35.029 mmol, 1.0 equiv) and DIEA (9.0 g, 70.058 mmol, 2.0 equiv) in DCM (30 mL) was added 4-nitrobenzenesulfonyl chloride (7.7 g, 35.029 mmol, 1.0 equiv) dropwise at room temperature. The resulting mixture was stirred for 5 h at room temperature. The resulting mixture was extracted with CH2Cl2 (2×50 mL). The combined organic layers were washed with brine (1×50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (5:1) to afford 4-nitro-N-(prop-2-en-1-yl)benzenesulfonamide (5 g, 53%) as a solid. LCMS (ES, m/z): 243 [M+H]+

Synthesis of Intermediate C462

To a stirred solution of 4-nitro-N-(prop-2-en-1-yl)benzenesulfonamide (1.5 g, 6.192 mmol, 1.0 equiv), triphenylphosphine (2.7 g, 10.526 mmol, 1.7 equiv) and tert-butyl (3S)-3-hydroxypyrrolidine-1-carboxylate (1.7 g, 9.288 mmol, 1.5 equiv) in tetrahydrofuran (20 mL) was added DEAD (2.1 g, 12.384 mmol, 2.0 equiv) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 3 h at room temperature under nitrogen atmosphere. The reaction was monitored by LCMS. The resulting mixture was diluted with deionized water (50 mL). The resulting mixture was extracted with EtOAc (2×50 mL). The combined organic layers were washed with brine (1×50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (5:1) to afford tert-butyl (3R)-3-[N-(prop-2-en-1-yl)4-nitrobenzenesulfonamido]pyrrolidine-1-carboxylate (1.6 g, 56%) as an oil. LCMS (ES, m/z): 412 [M+H]+

Synthesis of Intermediate C463

To a stirred solution of tert-butyl (3R)-3-[N-(prop-2-en-1-yl)4-nitrobenzenesulfonamido]pyrrolidine-1-carboxylate (800.0 mg, 1.944 mmol, 1.0 equiv) in DCM (3 mL) was added HCl (gas) in 1,4-dioxane (1 mL, 4 M) dropwise at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was neutralized to PH=7 with NaHCO3 aqueous and extracted with DCM (3×5 mL). The combined organic layers were dried by anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo. This resulted in 4-nitro-N-(prop-2-en-1-yl)-N-[(3R)-pyrrolidin-3-yl] benzenesulfonamide (450 mg, 68%) as a solid. LCMS (ES, m/z): 312 [M+H]+

Synthesis of Intermediate C464

Synthesis of Compound 554

Example 272: Synthesis of Compound 556

Synthesis of Intermediate C465

Synthesis of Intermediate C466

To a stirred mixture of methyl 4-[(3R)-3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl]-2-ethylindazole-7-carboxylate (1.9 g, 4.721 mmol, 1 equiv) in THF (20 mL) and H2O (20 mL) was added LiOH·H2O (0.99 g, 23.605 mmol, 5 equiv) at room temperature. The resulting mixture was stirred for 2 h at 50° C. The mixture was acidified to pH 4 with HCl (2M). The resulting mixture was extracted with EtOAc (3×40 mL). The combined organic layers were washed with water (3×40 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-[(3R)-3-[(tert-butoxycarbonyl)(methyl)amino]pyrrolidin-1-yl]-2-ethylindazole-7-carboxylic acid (1.4 g, 76%) as a solid. LCMS (ES, m/z): 389 [M+H]+

Synthesis of Intermediate C467

Synthesis of Intermediate C468

Synthesis of Compound 556

Example 273: Synthesis of Compound 557

Synthesis of Intermediate C469

To a stirred solution of methyl 4-bromo-6-fluoro-2H-indazole-7-carboxylate (2 g, 7.324 mmol, 1 equiv) in EA (20 mL) was added tetrafluoroboranuide; triethyloxidanium (2.8 g, 14.648 mmol, 2 equiv) in portions at room temperature. The resulting mixture was stirred for 3 h at room temperature. The reaction was quenched by the addition of saturated aqueous NaHCO3 (100 mL) at room temperature. The resulting mixture was extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (1×50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford methyl 4-bromo-2-ethyl-6-fluoroindazole-7-carboxylate (2.1 g, 95%) as a solid. LCMS (ES, m/z): 301 [M+H]+

Synthesis of Intermediate C470

Synthesis of Intermediate C471

A mixture of methyl 4-{4-[(tert-butoxycarbonyl)amino]-4-ethylpiperidin-1-yl}-2-ethyl-6-fluoroindazole-7-carboxylate (376 mg, 0.838 mmol, 1 equiv) and LiOH (201 mg, 8.38 mmol, 10 equiv) in H2O (4 mL), THF (4 mL) and MeOH (4 mL) was stirred for overnight at room temperature. The resulting mixture was concentrated under vacuum. The resulting mixture was diluted with water (20 mL). The mixture was acidified to pH 2 with 1 M HCl (aq.). The resulting mixture was extracted with CH2Cl2 (3×5 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-{4-[(tert-butoxycarbonyl)amino]-4-ethylpiperidin-1-yl}-2-ethyl-6-fluoroindazole-7-carboxylic acid (136 mg, 37%) as a solid. LCMS (ES, m/z): 435 [M+H]+

Synthesis of Intermediate C472

Synthesis of Compound 557

Example 274: Synthesis of Compounds 559 and 560

Synthesis of Intermediate C473

Synthesis of Intermediate C474

A solution of 4-{1,4-dioxa-8-azaspiro[4.5]decan-8-yl}-2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}indazole-7-carboxamide (370 mg, 0.773 mmol, 1 equiv) in Acetone (4 mL) and H2O (2 mL) was treated with PPTS (3.89 g, 15.460 mmol, 20 equiv) at room temperature. The resulting mixture was stirred for 48 h at 70° C. The mixture was allowed to cool down to room temperature. The mixture was neutralized to pH 7 with saturated NaHCO3 (aq.). The resulting mixture was extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}-4-(4-oxopiperidin-1-yl) indazole-7-carboxamide (230 mg, 68.47%) as a yellow solid. LCMS (ES, m/z): 435 [M+H]+

Synthesis of Compound 559

Synthesis of Compound 560

Example 275: Synthesis of Compound 562

Synthesis of Intermediate C475

A solution of benzyl (3S)-3-[(4-methylbenzenesulfonyl)oxy]pyrrolidine-1-carboxylate (500 mg, 1.332 mmol, 1.0 equiv) in DMSO (5 mL) was treated with tert-butyl piperazine-1-carboxylate (1.24 g, 6.660 mmol, 5.0 equiv) at room temperature. The resulting mixture was stirred for overnight at 70° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was extracted with EtOAc (3×30 mL). The combined organic layers were washed with brine (1×30 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (2:1) to afford tert-butyl 4-[(3R)-1-[(benzyloxy)carbonyl]pyrrolidin-3-yl]piperazine-1-carboxylate (510 mg, 98%) as an oil. LCMS (ES, m/z): 390 [M+H]+

Synthesis of Intermediate C476

To a solution of tert-butyl 4-[(3R)-1-[(benzyloxy)carbonyl]pyrrolidin-3-yl]piperazine-1-carboxylate (510 mg, 1.309 mmol, 1.0 equiv) in 10 mL MeOH was added Pd/C (10%, 50 mg) in a pressure tank. The mixture was hydrogenated at room temperature under 5 psi of hydrogen pressure for overnight, filtered through a Celite pad and concentrated under reduced pressure to afford tert-butyl 4-[(3R)-pyrrolidin-3-yl]piperazine-1-carboxylate (350 mg, 105%) as an oil.

Synthesis of Intermediate C477

Synthesis of Compound 562

Example 276: Synthesis of Compound 563

Synthesis of Intermediate C448

Synthesis of Compound 563

Example 277: Synthesis of Compound 564

Synthesis of Intermediate C449

Synthesis of Compound 564

Example 278: Synthesis of Compound 565

Synthesis of Intermediate C450

Synthesis of Intermediate C451

To a stirred solution of methyl 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-ethylindazole-7-carboxylate (1.1 g, 2.832 mmol, 1 equiv) in THF/H2O (9 mL/3 mL) was added lithiumol hydrate (0.36 g, 8.496 mmol, 3 equiv) at room temperature. The resulting mixture was stirred overnight at 40° C. The resulting mixture was concentrated under vacuum. The resulting mixture was diluted with H2O (10 mL). The mixture was acidified to pH 4 with citric acid. The precipitated solids were collected by filtration and dried under infrared light to afford 4-[4-(tert-butoxycarbonyl)piperazin-1-yl]-2-ethylindazole-7-carboxylic acid (850 mg, 80%) as a solid. LCMS (ES, m/z): 375 [M+H]

Synthesis of Intermediate C452

Synthesis of Intermediate C453

To a stirred solution of 6-bromo-8-fluoro-2-methylimidazo[1,2-a]pyridine (1.0 g, 4.366 mmol, 1 equiv) in THF (20 mL) was added NaH (0.16 g, 6.549 mmol, 1.5 equiv) at 0° C. The resulting mixture was stirred for 0.5 h at 0° C. under nitrogen atmosphere. To the above mixture was added F-TEDA-BF4 (2.32 g, 6.549 mmol, 1.5 equiv) in portions at 0° C. The resulting mixture was stirred for additional 16 h at 60° C. The reaction was quenched with MeOH at room temperature. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (10:1) to afford 6-bromo-3,8-difluoro-2-methylimidazo[1,2-a]pyridine (400 mg, 37.09%) as a solid. LCMS (ES, m/z): 247 [M+H]+

Synthesis of Intermediate C454

Synthesis of Compound 565

Example 279: Synthesis of Compound 566

Synthesis of Intermediate C455

To a solution of phenol (4.30 g, 45.671 mmol, 1.1 equiv) in THF (55 mL) was added sodium hydride (60% in oil, 1.83 g, 1.1 equiv) at 0° C. The mixture was stirred for 30 min. The solution of 3,5-dibromopyrazin-2-amine (10.5 g, 41.519 mmol, 1 equiv) in THF (50 mL) was added and the mixture was allowed to warm to 70° C. and stirred overnight. The mixture was allowed to cool down to room temperature. The reaction was quenched with water/ice. The resulting mixture was extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (1×100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford 5-bromo-3-phenoxypyrazin-2-amine (2.3 g, 21%) as a solid. LCMS (ES, m/z): 266 [M+H]+

Synthesis of Intermediate C456

To a stirred solution of 5-bromo-3-phenoxypyrazin-2-amine (2.3 g, 8.643 mmol, 1 equiv) in isopropanol (23 mL) were added 1-bromo-2,2-dimethoxypropane (1.90 g, 10.372 mmol, 1.2 equiv) and PPTS (0.22 g, 0.864 mmol, 0.1 equiv) at room temperature. The resulting mixture was stirred overnight at 80° C. The mixture was allowed to cool down to room temperature. The precipitated solids were collected by filtration and the solids was dried under vacuum to afford 6-bromo-2-methyl-8-phenoxyimidazo[1,2-a]pyrazine (800 mg, 30%) as a solid. LCMS (ES, m/z): 304 [M+H]+

Synthesis of Intermediate C457

Synthesis of Compound 566

Example 280: Synthesis of Compound 568

Synthesis of Intermediate C458

To a stirred mixture of bicyclo[1.1.1]pentan-1-amine hydrochloride (550 mg, 4.599 mmol, 1 equiv) in DCM (10 mL) were added Et3N (1.40 g, 13.797 mmol, 3 equiv) and 4-nitrobenzene-1-sulfonyl chloride (1.12 g, 5.059 mmol, 1.1 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 5 h at room temperature under nitrogen atmosphere. The resulting mixture was washed with 3×10 mL of water. dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford N-{bicyclo[1.1.1]pentan-1-yl}-4-nitrobenzenesulfonamide (780 mg, 63%) as a solid. LCMS (ES, m/z): 267 [M−H]−

Synthesis of Intermediate C459

Synthesis of Intermediate C460

To a stirred mixture of tert-butyl (3R)-3-(N-{bicyclo[1.1.1]pentan-1-yl}4-nitrobenzenesulfonamido)pyrrolidine-1-carboxylate (320 mg, 0.731 mmol, 1 equiv) in DCM (2 mL) was added HCl(gas) in 1,4-dioxane (0.5 mL, 4M) dropwise at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under vacuum. The mixture basified to pH 8 with saturated NaHCO3 (aq.). The resulting mixture was extracted with EtOAc (3×2 mL). The combined organic layers were washed with water (3×2 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford N-{bicyclo[1.1.1]pentan-1-yl}-4-nitro-N-[(3R)-pyrrolidin-3-yl]benzenesulfonamide (230 mg, 93%) as a solid. LCMS (ES, m/z): 338 [M+H]+

Synthesis of Intermediate C461

To a stirred mixture of 4-bromo-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}-2-methylindazole-7-carboxamide (110 mg, 0.273 mmol, 1.0 equiv) and N-{bicyclo[1.1.1]pentan-1-yl}-4-nitro-N-[(3R)-pyrrolidin-3-yl]benzenesulfonamide (119.95 mg, 0.355 mmol, 1.3 equiv) in DMF (2 mL) were added Cs2CO3 (222.7 mg, 0.683 mmol, 2.5 equiv) and RuPhos (25.5 mg, 0.055 mmol, 0.2 equiv) and RuPhos Palladacycle Gen.3 (22.9 mg, 0.027 mmol, 0.1 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 h at 90° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (5 mL). The resulting mixture was extracted with EtOAc (3×5 mL). The combined organic layers were washed with water (3×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford 4-[(3R)-3-(N-{bicyclo[1.1.1]pentan-1-yl}4-nitrobenzenesulfonamido)pyrrolidin-1-yl]-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}-2-methylindazole-7-carboxamide (60 mg, 33%) as a solid. LCMS (ES, m/z): 658 [M+H]+

Synthesis of Compound 568

Example 281: Synthesis of Compound 569

Synthesis of Intermediate C462

To a stirred solution of methyl 4-bromo-2-ethyl-6-fluoroindazole-7-carboxylate (500 mg, 1.66 mmol, 1 equiv), Cs2CO3 (1082 mg, 3.32 mmol, 2 equiv) and tert-butyl N-cyclopropyl-N-(pyrrolidin-3-yl)carbamate (752 mg, 3.32 mmol, 2 equiv) in dioxane (10 mL) were added RuPhos (775 mg, 1.66 mmol, 1 equiv) and RuPhos Palladacycle Gen.3 (278 mg, 0.332 mmol, 0.2 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for overnight at 100° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The reaction was quenched by the addition of water (30 mL) at room temperature. The resulting mixture was extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography (Condition 3, Gradient 1) to afford methyl 4-{3-[(tert-butoxycarbonyl)(cyclopropyl)amino]pyrrolidin-1-yl}-2-ethyl-6-fluoroindazole-7-carboxylate (150 mg, 20%) as a solid. LCMS (ES, m/z): 447 [M+H]+

Synthesis of Intermediate C463

A mixture of methyl 4-{3-[(tert-butoxycarbonyl)(cyclopropyl)amino]pyrrolidin-1-yl}-2-ethyl-6-fluoroindazole-7-carboxylate (150 mg, 0.336 mmol, 1 equiv) and LiOH·H2O (282 mg, 6.720 mmol, 20 equiv) in H2O (2 mL) and THF (4 mL) was stirred for 24 h at room temperature. The resulting mixture was concentrated under vacuum. The residue was diluted with water (5.0 mL), then adjusted to pH 5 with 1 mol/L aq. HCl. The resulting mixture was extracted with CH2Cl2 (3×5 mL). The combined organic layers were washed with brine (1×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-{3-[(tert-butoxycarbonyl)(cyclopropyl)amino]pyrrolidin-1-yl}-2-ethyl-6-fluoroindazole-7-carboxylic acid (130 mg, 89%) as an oil. LCMS (ES, m/z): 433 [M+H]+

Synthesis of Intermediate C464

Synthesis of Compound 569

Example 282: Synthesis of Compound 571

Synthesis of Intermediate C465

Synthesis of Intermediate C466

Synthesis of Intermediate C467

Synthesis of Compound 571

Example 283: Synthesis of Compound 573

Synthesis of Intermediate C468

To a stirred mixture of 4-bromo-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}-2-methylindazole-7-carboxamide (220 mg, 0.547 mmol, 1 equiv) and tert-butyl N-[(3R)-pyrrolidin-3-yl]carbamate (122.3 mg, 0.656 mmol, 1.2 equiv) in DMF (5.5 mL) were added Cs2CO3 (534.6 mg, 1.641 mmol, 3 equiv) and Ruphos (51.1 mg, 0.109 mmol, 0.2 equiv) and RuPhos Palladacycle Gen.3 (45.8 mg, 0.055 mmol, 0.1 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 16 h at 90° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (15 mL). The resulting mixture was extracted with EtOAc (3×15 mL). The combined organic layers were washed with water (3×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2/MeOH (10:1) to afford tert-butyl N-[(3R)-1-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-methylindazol-4-yl]pyrrolidin-3-yl]carbamate (200 mg, 72%) as a solid. LCMS (ES, m/z): 508 [M+H]+

Synthesis of Intermediate C469

To a stirred mixture of tert-butyl N-[(3R)-1-[7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl)-2-methylindazol-4-yl]pyrrolidin-3-yl]carbamate (170 mg, 0.335 mmol, 1 equiv) in DCM (2.5 mL) was added HCl(gas) in 1,4-dioxane (1 mL) dropwise at room temperature. The resulting mixture was stirred for 2 h at room temperature. The resulting mixture was concentrated under reduced pressure to afford 4-[(3R)-3-aminopyrrolidin-1-yl]-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}-2-methylindazole-7-carboxamide hydrochloride (140 mg, 103%) as a solid. LCMS (ES, m/z): 408 [M+H]+

Synthesis of Compound 573

Example 284: Synthesis of Compound 575

Synthesis of Intermediate C470

To a stirred mixture of tert-butyl 4-(7-carbamoyl-2-ethylindazol-4-yl)piperazine-1-carboxylate (100 mg, 0.268 mmol, 1 equiv) and 6-bromo-8-fluoroquinoline (78.7 mg, 0.348 mmol, 1.3 equiv) in dioxane (2 mL) were added Cs2CO3 (261.7 mg, 0.804 mmol, 3 equiv) and XantPhos (31 mg, 0.054 mmol, 0.2 equiv) and Pd2(dba)3 (24.5 mg, 0.027 mmol, 0.1 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 h at 100° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (5 mL). The resulting mixture was extracted with EtOAc (3×5 mL). The combined organic layers were washed with water (3×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl 4-{2-ethyl-7-[(8-fluoroquinolin-6-yl)carbamoyl]indazol-4-yl}piperazine-1-carboxylate (100 mg, 72%) as a solid. LCMS (ES, m/z): 519 [M+H]+

Synthesis of Compound 575

Example 285: Synthesis of Compound 576

Example 286: Synthesis of Compound 577

Example 287: Synthesis of Compound 579

Synthesis of Intermediate C471

Synthesis of Intermediate C472

A solution of benzyl (3R)-3-(phenylamino) pyrrolidine-1-carboxylate (120 mg, 0.405 mmol, 1 equiv) in MeOH (10 mL) was added Pd/C (20 mg, 10%). The mixture was stirred for overnight at 50° C. under hydrogen atmosphere. The resulting mixture was filtered and the filtrate was concentrated under reduced pressure to afford (3R)—N-phenylpyrrolidin-3-amine (60 mg, 91.34%) as an oil. LCMS (ES, m/z): 163 [M+H]+

Synthesis of Compound 579

Example 288: Synthesis of Compound 580

Synthesis of Intermediate C473

To a stirred mixture of 2-ethyl-N-{8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}-4-(4-oxopiperidin-1-yl) indazole-7-carboxamide (100.0 mg, 0.230 mmol, 1.0 equiv) and tert-butyl 6-amino-3-azabicyclo[3.1.0]hexane-3-carboxylate (68.4 mg, 0.345 mmol, 1.5 equiv) in DCM (1 mL) was added NaBH(AcO)3 (97.6 mg, 0.460 mmol, 2.0 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature. The reaction was quenched with water at room temperature. The resulting mixture was extracted with CH2Cl2 (3×10 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford tert-butyl 6-({1-[2-ethyl-7-({8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl}carbamoyl) indazol-4-yl]piperidin-4-yl}amino)-3-azabicyclo[3.1.0]hexane-3-carboxylate (113.8 mg, 80%) as a solid. LCMS (ES, m/z): 617 [M+H]+

Synthesis of Compound 580

Example 289: Synthesis of Compound 581

Example 290: Synthesis of Compound 582

Synthesis of Intermediate C474

Synthesis of Intermediate C475

Synthesis of Compound 582

Example 291: Synthesis of Compound 584

Synthesis of Intermediate C476

Synthesis of Compound 584

Example 292: Synthesis of Compound 585

Synthesis of Intermediate C477

Synthesis of Compound 585

Example 293: Synthesis of Compound 587

Synthesis of Intermediate C478

Synthesis of Compound 587

Example 294: Synthesis of Compound 589

Synthesis of Intermediate C488

Synthesis of Compound 589

Example 295: Synthesis of Compound 594

Example 296: Synthesis of Compound 596

Synthesis of Intermediate C489

To a stirred solution of 1-aminocyclopropane-1-carbonitrile hydrochloride (2.0 g, 16.869 mmol, 1.0 equiv) and DIEA (4.3 g, 33.738 mmol, 2.0 equiv) in DCM (30 mL) was added 4-nitrobenzene-1-sulfonyl chloride (3.4 g, 15.182 mmol, 0.9 equiv) at room temperature. The resulting mixture was stirred for 5 h at room temperature. The resulting mixture was diluted with deionized water (50 mL). The resulting mixture was extracted with CH2Cl2 (2×50 mL). The combined organic layers were washed with brine (1×50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (5:1) to afford N-(1-cyanocyclopropyl)-4-nitrobenzenesulfonamide (2.5 g, 50%) as a solid. LCMS (ES, m/z): 268 [M+H]+

Synthesis of Intermediate C490

To a stirred solution of N-(1-cyanocyclopropyl)-4-nitrobenzenesulfonamide (1.5 g, 5.613 mmol, 1.0 equiv) and tert-butyl (3S)-3-hydroxypyrrolidine-1-carboxylate (1.4 g, 7.297 mmol, 1.3 equiv) in THF (30 mL) were added DEAD (1.7 g, 10.103 mmol, 1.8 equiv) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 h at room temperature under nitrogen atmosphere. The resulting mixture was diluted with water (50 mL). The resulting mixture was extracted with EtOAc (2×50 mL). The combined organic layers were washed with brine (1×50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford tert-butyl (3R)-3-[N-(1-cyanocyclopropyl)4-nitrobenzenesulfonamido]pyrrolidine-1-carboxylate (1 g, 38%) as a solid. LCMS (ES, m/z): 437 [M+H]+

Synthesis of Intermediate C491

To a stirred solution of tert-butyl (3R)-3-[N-(1-cyanocyclopropyl)4-nitrobenzenesulfonamido] pyrrolidine-1-carboxylate (1.0 g, 2.291 mmol, 1.0 equiv) in DCM (10 mL) was added TFA (5 mL) dropwise at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under reduced pressure. The mixture was basified to pH 8 with saturated NaHCO3 (aq.). The resulting mixture was extracted with EtOAc (2×30 mL). The combined organic layers were washed with brine (1×50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in N-(1-cyanocyclopropyl)-4-nitro-N-[(3R)-pyrrolidin-3-yl]benzenesulfonamide (0.7 g, 84%) as a solid. LCMS (ES, m/z): 337 [M+H]+

Synthesis of Intermediate C492

Synthesis of Compound 596

Example 297: Synthesis of Compound 600

Example 298: Synthesis of Compound 604

Synthesis of Intermediate C493

To a stirred solution of tert-butyl 4-(7-carbamoyl-2-ethylindazol-4-yl)piperazine-1-carboxylate (120 mg, 0.321 mmol, 1 equiv) and 6-bromo-8-chloro-2-methylimidazo[1,2-a]pyrazine (95 mg, 0.385 mmol, 1.2 equiv) in dioxane (2.4 mL) were added Cs2CO3 (314.1 mg, 0.963 mmol, 3 equiv) and XantPhos (37.2 mg, 0.064 mmol, 0.2 equiv) and Pd2(dba)3 (29.4 mg, 0.032 mmol, 0.1 equiv) at room temperature. The resulting mixture was stirred for 2 h at 90° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (3 mL). The resulting mixture was extracted with EtOAc (3×5 mL). The combined organic layers were washed with water (3×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 4-[7-({8-chloro-2-methylimidazo[1,2-a]pyrazin-6-yl}carbamoyl)-2-ethylindazol-4-yl]piperazine-1-carboxylate (150 mg, 87%) as a solid. LCMS (ES, m/z): 539 [M+H]+

Synthesis of Compound 604

Example 299: Synthesis of Compounds 605, 606, and 607

Synthesis of Intermediate C494

Synthesis of Compound 605

Synthesis of Compound 606

Synthesis of Compound 607

Example 300: Synthesis of Compound 610

Synthesis of Intermediate C495

Synthesis of Compound 610

Example 301: Synthesis of Compound 612

Synthesis of Intermediate C496

Synthesis of Compound 612

Example 302: Synthesis of Compound 614

Synthesis of Intermediate C497

Synthesis of Compound 614

Example 303: Synthesis of Compound 615, 616, and 617

Synthesis of Compound 615

Synthesis of Compound 616

Synthesis of Compound 617

Example 304: Synthesis of Compound 619

Synthesis of Intermediate C498

To a solution of 4-bromo-6-chloro-2H-indazole (2.0 g, 8.651 mmol, 1 equiv) in EA (30 mL) was added triethyloxonium tetrafluoroborate (1.98 g, 10.389 mmol, 1.2 equiv) in portions at 0 degrees C. The resulting mixture was stirred for 5 h at room temperature. The reaction mixture was quenched with saturated aqueous NaHCO3 (50 mL). The resulting mixture was extracted with EA (3×20 mL). The combined organics were washed with brine (2×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 4-bromo-6-chloro-2-ethyl-2H-indazole (1.8 g, 80%) as a solid. LCMS (ES, m/z): 259 [M+H]+

Synthesis of Intermediate C499

Synthesis of Intermediate C500

Synthesis of Intermediate C501

To a solution of (3R)-1-(7-bromo-6-chloro-2-ethylindazol-4-yl)-N,N-dimethylpyrrolidin-3-amine (350 mg, 0.942 mmol, 1.0 equiv) in MeOH (4 mL) were added Pd(dppf)Cl2 (68 mg, 0.094 mmol, 0.1 equiv) and TEA (476 mg, 4.710 mmol, 5.0 equiv) in a pressure tank. The mixture was purged with nitrogen for 5 min and then was pressurized to 20 atm with carbon monoxide at 100° C. for overnight. The reaction mixture was cooled to room temperature and filtered to remove insoluble solids. The filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography (Condition 3, Gradient 1) to afford methyl 6-chloro-4-[(3R)-3-(dimethylamino)pyrrolidin-1-yl]-2-ethylindazole-7-carboxylate (310 mg, 94%) as a solid. LCMS (ES, m/z): 351 [M+H]+

Synthesis of Intermediate C502

Synthesis of Compound 619

Example 305: Synthesis of Compound 621

Synthesis of Intermediate C503

A solution of benzyl (3S)-3-[(4-methylbenzenesulfonyl)oxy]pyrrolidine-1-carboxylate (2 g, 5.328 mmol, 1.0 equiv) in DMSO (5 mL) was treated with tert-butyl (2R,6S)-2,6-dimethylpiperazine-1-carboxylate (3.42 g, 15.67 mmol, 3.0 equiv) at room temperature. The resulting mixture was stirred for 16 h at 70° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water. The resulting mixture was extracted with EtOAc (3×50 mL). The combined organic layers were washed with brine (1×100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:2) to afford tert-butyl (2R,6S)-4-[(3R)-1-[(benzyloxy)carbonyl]pyrrolidin-3-yl]-2,6-dimethylpiperazine-1-carboxylate (710 mg, 52%) as an oil. LCMS (ES, m/z): 418 [M+H]+

Synthesis of Intermediate C504

A solution of tert-butyl (2R,6S)-4-[(3R)-1-[(benzyloxy)carbonyl]pyrrolidin-3-yl]-2,6-dimethylpiperazine-1-carboxylate (710 mg, 0.431 mmol, 1 equiv) in MeOH (10 mL) was treated with Pd/C (71 mg) at room temperature. The resulting mixture was stirred for 16 h at room temperature under H2 atmosphere. The resulting mixture was filtered and the filter cake was washed with MeOH (20 mL). The filtrate was concentrated under reduced pressure to afford tert-butyl (2R,6S)-2,6-dimethyl-4-[(3R)-pyrrolidin-3-yl]piperazine-1-carboxylate (322 mg, 83%) as an oil. LCMS (ES, m/z): 284 [M+H]+

Synthesis of Intermediate C505

Synthesis of Compound 621

Example 306: Synthesis of Compound 622

Synthesis of Intermediate C506

To a stirred mixture of benzyl 3-oxopyrrolidine-1-carboxylate (550 mg, 2.509 mmol, 1.0 equiv) and tert-butyl (3R,4R)-3-amino-4-hydroxypyrrolidine-1-carboxylate (608.8 mg, 3.011 mmol, 1.2 equiv) in DCE (5.5 mL) was added NaBH(AcO)3 (797.5 mg, 3.763 mmol, 1.5 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature. The reaction was quenched with water at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (80%) to afford tert-butyl 3-({1-[(benzyloxy)carbonyl]pyrrolidin-3-yl}amino)-4-hydroxypyrrolidine-1-carboxylate (802 mg, 79%) as a solid. LCMS (ES, m/z): 406 [M+H]+

Synthesis of Intermediate C507

To a stirred mixture of tert-butyl 3-({1-[(benzyloxy)carbonyl]pyrrolidin-3-yl}amino)-4-hydroxypyrrolidine-1-carboxylate (800 mg, 1.973 mmol, 1.0 equiv) and TEA (399.2 mg, 3.946 mmol, 2.0 equiv) in DCM (8 mL) was added MsCl (271.1 mg, 2.368 mmol, 1.2 equiv) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 3 h at room temperature under nitrogen atmosphere. The reaction was quenched with Water at room temperature. The resulting mixture was extracted with CH2Cl2 (3×10 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford tert-butyl 6-{1-[(benzyloxy)carbonyl]pyrrolidin-3-yl}-3,6-diazabicyclo[3.1.0]hexane-3-carboxylate (623 mg, 82%) as a solid. LCMS (ES, m/z): 388 [M+H]+

Synthesis of Intermediate C508

A solution of tert-butyl 6-{1-[(benzyloxy)carbonyl]pyrrolidin-3-yl}-3,6-diazabicyclo[3.1.0]hexane-3-carboxylate (623 mg, 1.608 mmol, 1 equiv) in MeOH (20 mL) was treated with Pd/C (62 mg) at room temperature. The resulting mixture was stirred for 16 h at room temperature under H2 atmosphere. The resulting mixture was filtered and the filter cake was washed with MeOH (10 mL). The filtrate was concentrated under reduced pressure to afford tert-butyl 6-(pyrrolidin-3-yl)-3,6-diazabicyclo[3.1.0]hexane-3-carboxylate (280 mg, 69%) as an oil. LCMS (ES, m/z): 254 [M+H]+

Synthesis of Intermediate C509

Synthesis of Compound 622

Example 307: Synthesis of Compound 624

Synthesis of Intermediate C510

A solution of methyl 1-methyl-4-nitropyrazole-3-carboxylate (2 g, 10.803 mmol, 1 equiv) in 7M NH3(g) in MeOH (32 mL) was stirred for overnight at 50° C. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under reduced pressure to afford 1-methyl-4-nitropyrazole-3-carboxamide (1.8 g, 98%) as a solid. LCMS (ES, m/z): 171 [M+H]+

Synthesis of Intermediate C511

To a solution of 1-methyl-4-nitropyrazole-3-carboxamide (1.9 g, 11.168 mmol, 1 equiv) in MeOH (20 mL) was added Pd/C (10%, 1.19 g) under nitrogen atmosphere in a 50 mL round-bottom flask. The mixture was hydrogenated at room temperature for overnight under hydrogen atmosphere using a hydrogen balloon, filtered through a Celite pad and concentrated under reduced pressure to afford 4-amino-1-methylpyrazole-3-carboxamide (1.5 g, 96%) as a solid. LCMS (ES, m/z): 141 [M+H]+

Synthesis of Intermediate C512

To a stirred solution of 4-amino-1-methylpyrazole-3-carboxamide (1.5 g, 10.703 mmol, 1 equiv) in dimethylformamide (20 mL) was added NaH (1.54 g, 64.218 mmol, 6 equiv) in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for 0.5 h at 0° C. under nitrogen atmosphere. To the above mixture was added CDI (5.21 g, 32.109 mmol, 3 equiv) at 0° C. The resulting mixture was stirred for additional 3 h at 75° C. The mixture was allowed to cool down to room temperature. The reaction was quenched by the addition of water (50 mL) at room temperature. The residue was purified by reverse flash chromatography (Condition 5, Gradient 7) to afford 2-methyl-4H,6H-pyrazolo[4,3-d]pyrimidine-5,7-dione (300 mg, 17%) as a solid. LCMS (ES, m/z): 167 [M+H]+

Synthesis of Intermediate C513

A solution of 2-methyl-4H,6H-pyrazolo[4,3-d]pyrimidine-5,7-dione (300 mg, 1.806 mmol, 1 equiv) in POCl3 (2.8 g, 18.060 mmol, 10 equiv) was stirred for 2 h at 50° C. To the above mixture was added DBU (1.65 g, 10.836 mmol, 6 equiv) at 50° C. The resulting mixture was stirred for additional 8 h at 80° C. The mixture was allowed to cool down to room temperature. The reaction was quenched by the addition of water (20 mL) at room temperature. The mixture was basified to pH 8 with saturated aq NaHCO3. The resulting mixture was extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:10) to afford 5,7-dichloro-2-methylpyrazolo[4,3-d]pyrimidine (250 mg, 68%) as a solid. LCMS (ES, m/z): 203 [M+H]+

Synthesis of Intermediate C514

To a stirred solution of 5,7-dichloro-2-methylpyrazolo[4,3-d]pyrimidine (250 mg, 1.231 mmol, 1 equiv) in tetrahydrofuran (5 mL) was added sodium methoxide (63 mg, 1.169 mmol, 0.95 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere. The reaction was quenched by the addition of water (20 mL) at room temperature. The resulting mixture was extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (1×10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1:1) to afford 5-chloro-7-methoxy-2-methylpyrazolo[4,3-d]pyrimidine (180 mg, 74%) as a solid. LCMS (ES, m/z): 199 [M+H]+

Synthesis of Intermediate C515

To a stirred solution of tert-butyl N-[(3R)-1-(7-carbamoyl-2-methylindazol-4-yl)pyrrolidin-3-yl]-N-methylcarbamate (100 mg, 0.268 mmol, 1 equiv) and 5-chloro-7-methoxy-2-methylpyrazolo[4,3-d]pyrimidine (53 mg, 0.268 mmol, 1 equiv) in dioxane (3 mL) were added Cs2CO3 (175 mg, 0.536 mmol, 2 equiv), RuPhos (25 mg, 0.054 mmol, 0.2 equiv) and Pd2(dba)3 (25 mg, 0.027 mmol, 0.1 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 3 h at 100° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The reaction was quenched by the addition of water (20 mL) at room temperature. The resulting mixture was extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (1×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography (Condition 10, Gradient 3) to afford tert-butyl N-[(3R)-1-[7-({7-methoxy-2-methylpyrazolo[4,3-d]pyrimidin-5-yl}carbamoyl)-2-methylindazol-4-yl]pyrrolidin-3-yl]-N-methylcarbamate (90 mg, 63%) as a solid. LCMS (ES, m/z): 536 [M+H]+

Synthesis of Compound 624

Example 308: Synthesis of Compound 626

Synthesis of Intermediate C516

Synthesis of Compound 626

Example 309: Synthesis of Compound 631

Synthesis of Intermediate C517

A solution of tert-butyl N-[(3R)-1-[7-({8-chloro-2-methylimidazo[1,2-a]pyrazin-6-yl}carbamoyl)-2-methylindazol-4-yl]pyrrolidin-3-yl]-N-methylcarbamate (200 mg, 0.371 mmol, 1 equiv) in 2 M methylamine in THF (2 mL) was stirred for overnight at 80° C. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl N-methyl-N-[(3R)-1-(2-methyl-7-{[2-methyl-8-(methylamino)imidazo[1,2-a]pyrazin-6-yl]carbamoyl}indazol-4-yl)pyrrolidin-3-yl]carbamate (150 mg, 76%) as a solid. LCMS (ES, m/z): 534 [M+H]+

Synthesis of Compound 631

Example 310: Synthesis of Compound 656

Synthesis of Intermediate C518

To a stirred solution of tert-butyl 4-(7-carbamoyl-2-ethylindazol-4-yl)piperazine-1-carboxylate (120 mg, 0.321 mmol, 1 equiv) and 6-bromo-8-chloro-2-methylimidazo[1,2-a]pyrazine (95 mg, 0.385 mmol, 1.2 equiv) in dioxane (2.4 mL) were added Cs2CO3 (314.1 mg, 0.963 mmol, 3 equiv) and XantPhos (37.2 mg, 0.064 mmol, 0.2 equiv) and Pd2(dba)3 (29.4 mg, 0.032 mmol, 0.1 equiv) at room temperature. The resulting mixture was stirred for 2 h at 90° C. under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (3 mL). The resulting mixture was extracted with EtOAc (3×5 mL). The combined organic layers were washed with water (3×5 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EA to afford tert-butyl 4-[7-({8-chloro-2-methylimidazo[1,2-a]pyrazin-6-yl}carbamoyl)-2-ethylindazol-4-yl]piperazine-1-carboxylate (150 mg, 87%) as a solid. LCMS (ES, m/z): 539 [M+H]+

Synthesis of Compound 656

Example 311: Synthesis of Compound 658

Synthesis of Intermediate C519

Synthesis of Compound 658

Example 312: Exemplary Splicing Assay for Monitoring Expression Levels of Splice Variants

Compounds described herein were used to modulate RNA transcript abundance in cells. The expression of a target mRNA was measured by detecting the formation of an exon-exon junction in the canonical transcript (CJ). A compound mediated exon-inclusion event was detected by observing an increase in formation of a new junction with an alternative exon (AJ). Real-time qPCR assays were used to detect these splicing switches and interrogate the potency of various compounds towards different target genes. A high-throughput real time quantitative PCR (RT-qPCR) assay was developed to measure these two isoforms of the mRNA (CJ and AJ) for exemplary genes, such as HTT, SMN2, and MYB, together with a control housekeeping gene, GAPDH or GUSB or PPIA, used for normalization. Briefly, the A673 or K562 cell line was treated with various compounds described herein (e.g., compounds of Formula (I)). After treatment, the levels of the HTT, MYB, or SMN2 mRNA targets were determined from each sample of cell lysate by cDNA synthesis followed by qPCR.

Alternative junction (AJ)

Description

The A673 cell line was cultured in DMEM with 10% FBS. Cells were diluted with full growth media and plated in a 96-well plate (15,000 cells in 100 ul media per well). The plate was incubated at 37° C. with 5% CO2 for 24 hours to allow cells to adhere. An 11-point 3-fold serial dilution of the compounds was made in DMSO then diluted in media in an intermediate plate. Compounds were transferred from the intermediate plate to the cell plate with the top dose at a final concentration of 10 uM in the well. Final DMSO concentration was kept at or below 0.25%. The cell plate was returned to the incubator at 37° C. with 5% CO2 for an additional 24 hours.

The K562 cell line was cultured in IMDM with 10% FBS. For K562, cells were diluted with full growth media and plated in either a 96-well plate (50,000 cells in 50 uL media per well) or a 384-well plate (8,000-40,000 cells in 45 uL media per well). An 11-point 3-fold serial dilution of the compounds were made in DMSO then diluted in media in an intermediate plate. Compound was transferred from the intermediate plate to the cell plate with the top dose at a final concentration of 10 uM in the well. Final DMSO concentration was kept at or below 0.25%. Final volume was 100 uL for 96-well plate and 50 uL for 384-well plate. The cell plate was then placed in an incubator at 37° C. with 5% CO2 for 24 hours.

The cells were then gently washed with 50 uL-100 uL cold PBS before proceeding to addition of lysis buffer. 30 uL-50 uL of room temperature lysis buffer with DNAse I (and optionally RNAsin) was added to each well. Cells were shaken/mixed thoroughly at room temperature for 5-10 minutes for lysis to take place and then 3 uL-5 uL of room temperature stop solution was added and wells were shaken/mixed again. After 2-5 minutes, the cell lysate plate was transferred to ice for RT-qPCR reaction setup. The lysates could also be frozen at −80° C. for later use.

In some cases, a direct lysis buffer was used. An appropriate volume of 3× lysis buffer (10 mM Tris, 150 mM NaCl, 1.5%-2.5% Igepal and 0.1-1 U/uL RNAsin, pH 7.4) was directly added to either K562 or A673 cells in media and mixed by pipetting 3 times. The plates were then incubated at room temperature with shaking/rocking for 20-50 minutes to allow for lysis to take place. After this time, the cell lysate plate was transferred to ice to set up for the RT-qPCR reactions. The lysates could also be frozen at −80° C. for later use.

To set up 10 uL RT-qPCR reactions, cell lysates were transferred to 384-well qPCR plates containing the master mix according to the table below. The plates were sealed, gently vortexed, and spun down before the run. The volumes were adjusted accordingly in some instances where the reaction was carried in 20 μL. The table below summarizes the components of the RT-qPCR reactions:

Total volume
10

The RT-qPCR reaction was performed using a QuantStudio (ThermoFisher) under the following fast cycling conditions. All samples and standards were analyzed at least in duplicate. In some instances, bulk room temperature (RT) step of 5-10 minutes was completed for all plates before proceeding with qPCR. The table below summarizes the PCR cycle:

Step
# cycles
Temp.
Time

The data analysis was performed by first determining the ΔCt vs the housekeeper gene. This ΔCt was then normalized against the DMSO control (ΔΔCt) and converted to RQ (relative quantification) using the 2{circumflex over ( )}(−ΔΔCt) equation. The RQ were then converted to a percentage response by arbitrarily setting an assay window of 3.5 and 4.0 ΔCt for HTT-CJ and MYB-CJ respectively and an assay window of 9 and 3 ΔCt for HTT-AJ and MYB-AJ in 96 well format (50,000 K562 cells/well and 15,000 A673 cells per well) and an assay window of 3 and 4 ΔCt for HTT-CJ and MYB-CJ respectively and an assay window of 5 and 3 ΔCt for HTT-AJ and MYB-AJ respectively in 384 well format (8,000 K562 cells/well example). These assay windows correspond to the maximal modulation observed at high concentration of the most active compounds. The percentage response was then fitted to the 4 parametric logistic equation to evaluate the concentration dependence of compound treatment. The increase in AJ mRNA is reported as AC50 (compound concentration having 500 response in AJ increase) while the decrease in CJ mRNA levels is reported as IC50 (compound concentration having 50% response in CJ decrease).

A summary of these results is illustrated in Tables 3A and 3B, wherein “A” represents an AC50/IC50 of less than 100 Nm; “B” represents an AC50/IC50 of between 100 Nm and 1 μM; and “C” represents an AC50/IC50 of between 1 μM and 10 μM; and “D” represents an AC50/IC50 of greater than 10 μM.

Modulation of RNA Splicing by Exemplary Compounds

No.
CJ
AJ
CJ
AJ

119
C
D
C
C

141
A
A
A
A

142
C
C
B
B

145
C
C
B
B

148
C
C
C
C

149
C
C
B
B

188
D
D
C
C

189
C
C
B
B

190
B
B
A
A

191
B
C
B
B

192
C
C
B
B

193
C
C
B
B

195
D
D
B
C

197
D
D
C
C

199
B
B
B
B

200
C
C
C
C

201
D
D
D
D

202
D
C
B
A

204
C
C
B
B

205
B
C
B
B

206
C
C
B
B

209
B
B
A
A

210
B
B
A
A

212
C
D
B
C

217
D
D
D
D

219
D
D
B
C

229
B
B
A
A

230
D
D
D
D

231
D
D
D
D

235
C
C
B
B

237
C
C
B
A

238
C
C
B
B

239
B
B
A
B

240
B
B
A
A

242
B
C
A
B

243
B
B
A
A

244
D
D
D
D

245
B
B
B
B

246
C
C
A
A

247
D
D
D
D

249
D
D
B
B

253
D
D
D
D

255
C
C
B
A

256
C
C
B
B

257
C
C
B
B

258
C
B
A
A

259
C
C
B
C

260
B
B
A
A

261
D
D
D
D

262
C
C
B
B

263
C
C
B
B

264
D
D
D
D

265
D
D
D
D

266
C
C
B
A

267
D
C
C
B

268
D
D
D
D

269
B
B
A
A

270
D
D
D
D

271
D
D
C
C

272
B
B
B
A

273
D
D
B
B

274
C
D
B
B

275
C
C
B
A

278
C
C
B
A

280
C
C
D
C

281
C
C
C
C

282
C
C
D
D

283
C
D
C
D

286
D
D
B
C

289
C
D
B
B

290
D
D
B
B

291
C
C
B
B

292
C
C
A
A

293
C
B
A
A

294
B
C
A
A

295
D
C
B
B

296
D
D
A
A

297
C
C
C
C

298
C
C
A
B

299
D
D
B
B

300
D
D
C
B

301
B
B
A
A

302
C
C
A
A

303
C
C
B
B

304
C
D
B
B

305
C
C
B
B

306
C
C
B
A

307
C
C
B
B

308
D
D
D
D

309
D
D
C
D

310
C
C
A
A

311
D
D
B
C

312
C
C
B
A

313
D
D
C
C

314
C
C
A
A

315
B
B
A
A

316
D
D
C
B

318
D
D
B
B

319
C
C
A
A

320
C
C
B
B

321
D
D
D
D

322
D
D
C
D

323
A
B
A
A

324
D
C
B
B

325
D
D
C
C

326
B
B
A
A

327
D
D
B
B

328
D
D
C
D

329
D
D
D
D

330
D
D
B
B

332
D
D
D
D

333
C
C
B
B

334
D
D
B
B

335
D
D
C
C

336
B
B
A
A

337
C
C
B
B

338
C
C
B
B

339
D
D
C
C

340
D
D
C
C

341
D
D
C
C

342
D
D
C
D

343
A
A
A
A

344
D
D
C
C

347
C
C
A
A

348
C
C
A
A

349
B
B
A
A

350
C
C
A
A

351
C
C
A
A

352
B
B
A
A

353
B
B
A
A

354
D
D
C
C

355
D
C
A
A

356
D
D
B
B

358
C
D
B
B

362
D
D
C
C

364
C
D
C
B

366
C
C
A
A

367
C
C
B
B

368
D
D
B
B

369
B
B
A
A

370
D
D
B
C

371
B
B
A
A

372
C
C
A
A

373
C
C
A
A

374
D
D
A
A

375
B
B
B
B

376
B
C
B
B

377
C
C
B
B

378
C
C
B
B

379
C
D
B
C

380
C
C
A
A

381
D
D
A
A

382
C
C
A
A

383
D
D
A
B

384
D
D
D
C

385
D
D
D
D

387
C
C
A
A

388
C
C

389
D
D
B
C

390
D
D
D
D

391
D
C
A
B

392
C
C
A
A

393
D
D
B
B

394
C
C
B
B

395
C
C
A
A

396
D
D
B
A

397
C
C
B
B

398
D
D
B
B

399
D
D
B
B

400
D
D
A
A

401
B
B
A
A

402
B
B
A
A

403
C
C
B
B

405
C
C
A
B

406
B
B
A
A

407
D
D
B
B

408
D
D
B
B

409
D
D
B
A

410
D
D
B
C

411
C
C
A
A

412
C
D
A
A

413
C
C
A
A

414
D
D
C
C

415
B
B
B
B

416
D
D
D
D

417
C
D
A
A

418
D
D
B
C

419
D
D
D
D

420
D
D
B
B

421
D
D
D
D

423
B
B
A
A

424
D
D
D
D

425
D
D
D
D

426
D
D
D
D

427
B
B
B
B

428
A
A
A
A

429
C
C
B
B

430
C
C
A
A

431
D
D
B
B

432
D
D
A
A

434
C
C
B
C

435
D
D
D
D

436
D
D
B
B

437
C
C
A
A

441
C
C
A
A

442
C
D
B
B

443
C
C
A
A

444
C
D
B
B

446
C
C
B
B

447
B
B
A
A

448
B
C
A
A

450
D
D
D
D

451
D
D
C
C

452
D
D
C
C

453
D
D
C
C

454
C
C
B
C

455
D
D
D
D

456
D
D
D
C

457
D
D
D
D

458
A
B
A
A

459
B
B
A
A

460
A
B
A
A

461
C
D
A
B

462
B
B
A
A

463
B
B
A
A

464
B
B
A
A

469
D
D
D
D

470
D
D
D
D

472
D
C
A
A

473
C
C
A
A

474
A
B
A
A

475
B
B
A
A

476
B
C
C
C

477
C
D
B
B

478
C
D
A
B

479
C
C
A
A

481
B
C
A
B

482
D
D
C
C

484
D
D
D
D

486
B
B
B
B

487
C
C
C
C

488
D
D
D
D

489
D
D
D
D

490
D
D
C
C

491
C
D
B
C

492
C
D
B
C

494
D
D
D
D

496
D
D
D
D

497
B
B
A
A

498
B
C
B
B

499
D
D
D
D

500
D
D
D
D

501
A
A
A
A

502
A
B
A
A

503
B
A
A
A

504
C
C
C
C

505
C
C
B
B

506
B
C
B
B

507
C
C
B
B

508
A
B
A
A

509
D
D
C
C

510
C
C
B
B

511
B
B
B
B

513
C
D
B
B

514
B
B
A
A

515
B
B
A
A

517
C
D
B
B

518
C
C
B
A

520
C
C
A
A

522
C
C
A
B

524
D
D
A
A

525
B
B
A
A

526
D
D
C
C

527
D
D
B
B

528
D
D
B
B

529
A
B
A
A

531
C
C
B
C

533
C
C
A
A

535
D
D
B
B

537
D
D
C
C

538
C
C
C
C

539
D
D
C
C

541
D
D
B
B

542
C
C
B
B

543
C
C
A
A

544
D
D
C
C

545
C
D
B
B

547
D
D
D
D

548
D
D
D
D

550
D
D
C
C

552
B
B
B
B

554
D
D
B
A

556
D
D
C
D

557
B
B
A
A

559
C
C
A
A

560
C
C
B
B

562
D
D
C
C

563
B
B
A
A

564
B
B
A
A

565
D
D
D
D

566
C
C
B
B

568
C
C
C
C

569
D
C
A
A

571
B
B
A
A

573
D
D
B
B

574
D
D
B
B

575
D
D
D
D

576
C
D
B
B

577
B
B
B
B

579
D
D
D
D

580
D
D
B
C

581
C
C
B
B

582
B
B
B
B

584
C
C
B
B

585
D
D
B
B

587
B
B
B
B

589
D
D
B
C

590
C
C
B
B

592
C
D
B
B

594
D
D
B
C

596
D
D
C
C

598
C
C
A
B

600
D
D
C
C

602
C
C
A
B

603
D
D
C
D

604
D
D
C
C

606
B
C
B
B

607
C
C
B
B

610
D
C
B
B

612
D
D
D
D

614
D
D
B
B

616
B
C
B
B

617
C
D
B
C

619
D
D
B
B

621
D
D
C
B

622
D
D
C
D

624
C
C
C
C

626
C
C
B
B

627
C
C
B
B

629
C
C
C
C

631
D
D
C
C

633
C
D
B
B

635
D
D
B
C

637
C
C
B
C

639
D
D
B
B

641
C
C
A
A

643
D
D
B
B

645
C
D
B
B

649
D
D
B
B

651
D
D
D
D

653
D
D
D
D

No.
CJ
AJ
CJ
AJ

908
B
B
A
A

916
B
B
A
A

932
D
D
C
C

940
A
B
A
A

941
B
B
A
A

942
B
C
B
B

943
B
C
A
A

944
C
C
B
B

945
C
C
A
A

946
A
B
A
A

949
A
A
A
A

950
B
B
A
B

952
B
B
B
B

953
C
C
B
B

954
C
C
B
B

955
B
C
A
A

956
A
B
A
A

958
B
C
A
A

959
B
B
A
A

960
B
B
B
B

961
B
B
A
A

963
C
C
B
B

965
A
A
A
A

966
A
A
A
A

968
B
C
A
B

969
C
D
A
A

971
B
B
A
A

973
C
C
A
A

974
B
B
A
A

976
B
C
A
A

978
B
B
A
A

979
B
B
A
A

Additional studies were carried out for a larger panel of genes using the protocol provided above. The junction between flanking upstream and downstream exons was used to design canonical junction qPCR assays. At least one of the forward primer, reverse primer or the CY5-labeled 5′ nuclease probe (with 3′ quencher such as ZEN/Iowa Black FQ) was designed to overlap with the exon junction to capture the CJ mRNA transcript. BLAST was used to confirm the specificity of the probeset and parameters such as melting temperature, GC content, amplicon size, and primer dimer formation are considered during their design. Data for the decrease in CJ mRNA levels for three exemplary genes (HTT, SMN2, and Target C) analyzed in this panel are reported as IC50 (compound concentration having 5000 response in CJ decrease).

A summary of the results from the panel is illustrated in Tables 4A and 4B3, wherein “A” represents an IC50 of less than 100 nM; “B” represents an IC50 of between 100 nM and 1 μM; and “C” represents an IC50 of between 1 μM and 10 μM; and “D” represents an IC50 of greater than 10 μM.

Modulation of RNA Splicing by Exemplary Compounds

Compound

Target

118
C
B
D
B

119
C
B
D
C

140
C
B
C
C

141
A
A
B
A

142
C
A
B
B

143
A
A
B
A

145
C
A
B
B

148
C
B
C
C

149
C
A
D
B

187
B
A
C
B

188
D
B
C
C

189
C
A
C
B

190
B
B
C
A

191
B
A
C
B

192
C
A
C
B

193
C
A
D
B

194
C
A
D
B

195
D
A
D
B

196
C
A
C
B

197
D
B
D
C

198
C
A
C
A

199
B
A
C
B

200
C
A
C
C

201
D
C
D
D

202
D
A
D
B

203
D
A
D
B

204
C
A
C
B

205
B
A
C
B

206
C
A
D
B

207
B
A
C
A

208
B
A
B
A

209
B
C
B
A

210
B
A
C
A

211
C
A
C
B

212
C
B
D
B

217
D
C
D
D

218
C
A
C
B

219
D
B
D
B

228
B
A
B
A

229
B
A
C
A

230
D
D
D
D

231
D
D
D
D

234
C
A
C
A

235
C
A
D
B

237
C
A
D
B

238
C
A
D
B

239
B
A
C
A

240
B
A
C
A

241
B
A
C
B

242
B
A
C
A

243
B
A
C
A

244
D
B
D
D

245
B
A
B
B

246
C
A
D
A

247
D
D
D
D

249
D
A
D
B

250
A
A
B
A

251
B
A
C
A

252
B
A
B
A

253
D
C
D
D

255
C
A
D
B

256
C
A
D
B

257
C
A
D
B

258
C
A
C
A

259
C
A
C
B

260
B
C
C
A

261
D
C
D
D

262
C
A
D
B

263
C
A
C
B

264
D
D
D
D

265
D
C
D
D

266
C
A
D
B

267
D
A
D
C

268
D
D
D
D

269
B
A
C
A

270
D
D
D
D

271
D
B
D
C

272
B
A
B
B

273
D
A
D
B

274
C
A
D
B

275
C
A
D
B

277
B
A
C
A

278
C
A
C
B

279
C
A
C
A

280
C
C
D
D

281
C
B
C
C

282
C
B
D
D

283
C
B
D
C

284
C
A
C
A

285
B
A
B
A

286
D
B
D
B

288
C
A
C
A

289
C
A
D
B

290
D
A
D
B

291
C
A
C
B

292
C
A
C
A

293
C
A
D
A

294
B
A
D
A

295
D
A
D
B

296
D
A
D
A

297
C
B
C
C

298
C
A
C
A

299
D
A
D
B

300
D
B
D
C

301
B
A
C
A

302
C
A
D
A

303
C
A
D
B

304
C
A
D
B

305
C
A
D
B

306
C
A
D
B

307
C
A
D
B

308
D
D
D
D

309
D
C
D
C

310
C
A
D
A

311
D
A
D
B

312
C
A
C
B

313
D
B
C
C

314
C
A
D
A

315
B
A
B
A

316
D
B
D
C

318
D
A
D
B

319
C
A
C
A

320
C
A
D
B

321
D
D
D
D

322
D
C
D
C

323
A
A
C
A

324
D
A
D
B

325
D
A
D
C

326
B
A
C
A

327
D
B
D
B

328
D
B
D
C

329
D
C
D
D

330
D
A
D
B

332
D
D
D
D

333
C
A
D
B

334
D
A
D
B

335
D
B
D
C

336
B
A
B
A

337
C
A
D
B

338
C
A
D
B

339
D
B
D
C

340
D
B
D
C

341
D
B
D
C

342
D
D
D
C

343
A
A
B
A

344
D
B
D
C

347
C
A
C
A

348
C
A
C
A

349
B
A
B
A

350
C
A
D
A

351
C
A
D
A

352
B
A
C
A

353
B
A
C
A

354
D
B
D
C

355
D
A
D
A

356
D
A
D
B

358
C
A
D
B

361
A
A
A
A

362
D
B
D
C

363
A
A
A
A

364
C
A
D
C

366
C
A
D
A

367
C
A
D
B

368
D
A
D
B

369
B
A
C
A

370
D
B
D
B

371
D
A
D
A

372
D
A
D
A

373
C
A
D
A

374
D
A
D
A

375
B
A
B
B

376
C
A
D
A

377
C
A
C
A

378
C
A
D
A

379
C
A
C
B

380
C
A
C
A

381
D
A
D
A

382
C
A
D
A

383
D
A
D
A

384
D
B
D
D

385
D
D
D
D

386
D
D
D
D

387
C
A
D
A

388
D
A
D
A

389
D
A
D
B

407
D
A
D
B

408
D
A
D
B

409
D
A
D
B

410
D
A
D
B

411
D
A
D
A

412
C
A
D
A

413
C
A
D
A

414
D
C
D
C

415
B
B
B
B

416
D
C
D
D

417
D
A
D
A

418
D
A
D
B

419
D
D
D
D

420
D
A
D
B

421
D
D
D
D

422
A
A
B
A

423
B
A
C
A

424
D
C
D
D

425
D
D
D
D

426
D
D
D
D

427
B
A
C
B

428
A
A
B
A

429
C
A
D
B

430
C
A
D
A

431
C
A
C
A

432
D
A
D
B

433
C
A
C
B

434
D
A
D
A

435
C
A
C
B

436
D
D
D
D

437
D
A
D
B

440
A
A
C
A

441
C
A
D
A

442
C
A
D
A

443
C
A
D
B

444
C
A
C
A

445
A
A
B
A

446
C
A
C
B

447
C
A
C
B

448
B
A
C
A

449
C
A
D
A

450
B
A
C
A

451
D
D
D
D

452
D
B
D
C

453
D
B
D
C

454
D
A
D
C

455
C
B
C
B

456
D
D
D
D

457
D
D
D
D

458
D
D
D
D

459
A
A
C
A

460
B
A
B
A

461
C
A
D
A

462
B
A
D
A

463
B
A
D
A

464
B
A
C
A

469
D
D
D
D

470
D
D
D
D

471
A
A
A
A

472
D
A
D
A

473
C
A
D
A

474
B
A
C
A

475
B
A
C
A

476
B
B
C
C

477
C
B
D
B

Compound

Target

908
B
A
C
A

916
B
A
B
A

932
D
B
D
C

940
A
A
B
A

941
B
A
C
A

942
B
A
C
B

943
B
A
C
A

944
C
A
C
B

945
C
A
C
A

946
A
A
B
A

949
A
A
A
A

950
B
A
D
A

952
B
A
B
B

953
C
A
C
B

954
C
A
C
B

955
B
A
D
A

956
A
A
B
A

958
B
A
D
A

959
B
A
B
A

960
B
A
B
B

961
B
A
B
A

963
C
A
D
B

965
A
A
B
A

966
A
A
A
A

968
B
A
C
A

969
C
A
C
A

971
B
A
B
A

973
C
A
C
A

974
B
A
B
A

976
B
A
C
A

978
B
A
C
A

979
B
A
C
A

Example 313: Evaluating Effect of Exemplary Compounds on Protein Abundance

Compounds described herein were used to screen for effects on quantitative protein abundance using a HiBit assay system (Promega). Quantitative protein abundance was determined by measuring the protein levels of HiBit-tagged protein targets expressed in cell culture via luminescence using the Nano-Glo HiBiT Lytic Detection System, which uses a split complementation assay format to reconstitute NanoBiT enzyme to generate a luminescent signal. A protein abundance assay was developed such that endogenous protein targets could be modified with the HiBiT peptide tag and their abundance could be assessed after compound treatment. Briefly, K562 cell lines containing a HiBiT-modification were treated with various compounds described herein (e.g., compounds of Formulas (I) or (II)). After treatment for 24 hours, the protein abundance of a specific target was determined by measuring luminescence.

Design of genetically modified HiBiT cell lines

Guide
Guide

RNA
RNA cut

Line
Gene
Modification
Sequence
location
Donor Sequence

Cells were maintained in EIDM with 10% FBS. Before the assay, cells were diluted with phenolphthalein-free growth media (EIDM+1% FBS media) and were seeded in a 384-well plate at a density of 10000 cells/well (for each cell line listed in Table 5). Each compound was prepared as a 10-point 3-fold serial dilution in DMSO with the top dose at a final concentration of 10 μM in the well. Unmodified K562 cells were added at the previously specified density with DMSO to serve as an assay baseline and positive control (PC) and DMSO only with the respective modified cell lines was added to the negative control (NC) columns. Final DMSO concentration was kept at or below 0.25%. Treated cell plates were placed in an incubator at 37° C. with 5% CO2 for 24 hours. After 24 hours, 25 μL of Complete HiBit Lytic reagent was added to each well at room temperature (e.g. one plate requiring 10 mL Lytic Buffer, 100 μL LgBiT Protein, 200 μL Lytic Substrate), shaken for 5 minutes at 600 RPM, then left to sit for 10 minutes for signal to stabilize before reading on a Spark Cyto plate reader (Tecan) with a 500 ms measurement time.

To determine compound effects on protein abundance of each target in Table 6, the percent response for each respective cell line was calculated at each compound concentration as follows:

For the normalized response at each concentration, a four-parameter logistical regression was fit to the data and the response was interpolated at the 50% value to determine a concentration for protein abundance at 5000 (IC50) the untreated control.

A summary of the results for protein abundance is illustrated in Table 6, wherein A represents <100 nM; B represents 100-1000 nM; C represents 1000-9999 nM; and D represents greater than 10 μM.

Compound No.
HTT
MYB
Target C

118
B
A
C

119
C
C
D

140
C
C
C

141
B
B
C

142
B
A
C

143
A
A
A

145
C
A
C

148
C
C
C

149
C
A
C

187
A
B
B

189
D
B
C

190
B
A
C

191
B
A
C

192
C
B
C

193
C
B
D

194
C
B
D

195
D
C
C

196
B
A
C

197
D
C
D

198
C
A
D

199
C
A
D

200
C
C
C

187
C
A
C

190
B
A
C

191
C
B
C

192
C
B
C

193
C
B
D

194
C
B
D

197
D
C
D

198
C
A
D

199
C
A
D

200
A
A
B

208
B
A
B

217
D
D
D

556
C
C
C

557
B
A
B

559
B
A
B

560
C
A
C

562
D
D
D

563
B
A
B

564
B
B
B

565
C
C
D

566
C
B
C

568
C
C
C

569
C
A
C

571
B
A
C

573
D
C
D

574
D
B
D

575
C
C
D

Example 314: Investigating Effect of Exemplary Compounds on Cell Viability

Compounds described herein were screened for toxicity in K562 (human chronic myelogenous leukemia) and SH-SY5Y (human neuroblastoma) cells using a Cell Titer Glo 2.0 assay.

Cells were plated at 500 cells/well (K562 cells) in 45 μL of IMDM supplemented with 10% FBS in a 384-well opaque plate. Wells containing only medium were used as a blank control. Test compounds (e.g., compounds of Formula (I) or (II)) were first serially diluted in DMSO then diluted 1:100 with EIDM+10% FBS. The final concentration of DMSO was 0.1% in each well. The cells were incubated for 72 hours at 37° C. and 5% CO2 before assaying with Cell Titer Glo 2.0 reagent.

The compounds tested exhibited the following range as shown in Table 7, wherein A represents <100 nM; B represents 100-1000 nM; C represents 1000-9999 nM; and D represents greater than 10 μM in K562 cells.

119
C

140
C

148
C

196
A

198
A

200
C

557
A

559
A

560
A

562
C

565
C

573
C

EQUIVALENTS AND SCOPE