METHODS OF TREATING CANCER USING A CLK INHIBITOR

TECHNICAL FIELD

This present disclosure relates to the fields of cancer biology and molecular biology, and more specifically, to methods of treating cancer using CDC-like kinase (CLK) inhibitor.

BACKGROUND

Carcinogenesis is a multistep transformation of a normal cell into a cancerous cell, which is characterized by unchecked growth. These steps enable a cancer cell's “hallmark capabilities,” including chronic proliferation, resistance to apoptosis, metastatic and angiogenic potential, immune evasion, and replicative immortality (Hanahan and Weinberg, Cell 100:57-70, 2000). Motility, cytostasis and differentiation, proliferation, and viability are the intracellular signaling networks or circuits contributing to the development of these hallmark capabilities of a cancer cell (Hanahan and Weinberg, Cell 144:646-674, 2011). There is robust crosstalk among these pathways which support cancer cell growth. The nexus of these biological processes is changes in gene expression, which can fundamentally inhibit or promote cancer cell hallmark capabilities. One pathway which can directly modulate genes important in multiple cancer signaling networks is the Wnt/β-catenin signaling pathway.

Wnt signaling is an evolutionary conserved pathway which plays an important role in embryonic development, cell viability, and regeneration (Clevers et al., Cell 149:1192-1205, 2012; Clevers, Cell 127:469-480, 2006). Signaling is activated upon Wnt ligand binding to a Frizzled family cell receptor and is transmitted via canonical (β-catenin dependent) or non-canonical (β-catenin-independent) pathways (Clevers, Cell 127(3):469-480, 2006). Activation of canonical Wnt signaling releases β-catenin from the protein complex of GSK3-β, AXIN, and adenomatous polyposis coli (APC), and promotes the proteosomal degradation of the freed β-catenin (Nusse et al., EMBO J. 31:2670-2684, 2012). Upon subsequent translocation into the nucleus, β-catenin interacts with TCF/LEF transcription factors to activate expression of target genes important not only in cell fate, but in cell proliferation and survival (Moon et al., Nat. Rev. Genet. 5:691-701, 2004). Approximately 90% of colorectal cancers (CRC) are characterized by somatic mutations in the WNT/β-catenin signaling pathway; with 80% of those resulting from loss-of-function mutation of the APC gene and to a smaller extent CTNNB1 (Kwong et al., Adv. Exp. Med. Biol. 656:85-106, 2009; Nature 487:330-337, 2012). Loss of APC function causes abnormal activation of the canonical pathway resulting in higher levels of β-catenin which contributes to tumorigenesis. The aberrant activation of Wnt/β-catenin pathway is implicated in other cancer types such as, gastric cancer, breast cancer, liver cancer, pancreatic cancer, and lung cancer (Clevers, Cell 127(3):469-480, 2006; Moon et al., Nat. Rev. Genet. 5:691-701, 2004). There are no approved therapeutic agents targeting Wnt signaling to date (Kahn, Nature Rev. Drug Discov. 13:513-532, 2014).

SUMMARY

The present disclosure is based on the discovery that CLK inhibitors can decrease the level of Wnt/β-catenin signaling activity in a mammalian cell and can modulate mRNA splicing in a mammalian cell. In view of these discoveries, provided herein are methods of treating a cancer in a subject, methods of selecting a treatment for a subject, methods of selecting a subject for treatment, and methods of selecting a subject for participation in a clinical trial, that each include identifying a subject having a cancer cell (e.g., any of the types of cancer cell described herein) that has an elevated level of Wnt pathway activity as compared to a reference level. Also provided herein are methods of determining the efficacy of a CLK inhibitor in a subject that include detecting a level of Wnt/β-catenin signaling activity in a cancer cell obtained from the subject. Also provided are methods of decreasing the activity of one or more of CLK1, CLK2, CLK3, and CLK4 (e.g., in vitro or in a mammalian cell) that include the use of any of the CLK inhibitors or pharmaceutically acceptable salts or solvates thereof described herein. Also provided herein are methods of altering mRNA splicing in a mammalian cell having aberrant mRNA splicing activity that include the use of any of the CLK inhibitors or pharmaceutically acceptable salts or solvates thereof described herein. Also provided herein are methods of treating a cancer using a CLK inhibitor, methods of selecting a treatment including a CLK inhibitor for a subject, methods of selecting a subject for treatment with a CLK inhibitor, and methods of selecting a subject for participation in a clinical trial, that each include the use of a CLK inhibitor, that include a step of identifying a subject having aberrant mRNA splicing activity.

Also provided herein are methods of treating a cancer in a subject that include: identifying a subject having a cancer cell that has an elevated level of Wnt pathway activity as compared to a reference level; and administering to the identified subject a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of treating a cancer in a subject that include administering a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof to a subject identified as having a cancer cell that has an elevated level of Wnt pathway activity as compared to a reference level.

Also provided herein are methods of selecting a treatment for a subject that include: identifying a subject having a cancer cell that has an elevated level of Wnt pathway activity as compared to a reference level; and selecting for the identified subject a treatment including a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of selecting a treatment for a subject that include selecting a treatment including a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof for a subject identified as having a cancer cell that has an elevated level of Wnt pathway activity as compared to a reference level.

Also provided herein are methods of selecting a subject for treatment that include: identifying a subject having a cancer cell that has an elevated level of Wnt pathway activity as compared to a reference level; and selecting an identified subject for treatment with a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of selecting a subject for treatment that include selecting a subject identified as having a cancer cell that has an elevated level of Wnt pathway activity as compared to a reference level, for treatment with a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of selecting a subject for participation in a clinical trial that include: identifying a subject having a cancer cell that has an elevated level of Wnt pathway activity as compared to a reference level; and selecting the identified subject for participation in a clinical trial that includes administration of a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of selecting a subject for participation in a clinical trial that include selecting a subject identified as having a cancer cell that has an elevated level of Wnt pathway activity as compared to a reference level for participation in a clinical trial that includes administration of a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of treating a subject having a cancer that include: (a) administering to the subject a therapeutic agent; (b) after (a), identifying the subject as having a cancer cell that has an elevated level of Wnt pathway activity as compared to a reference level; and (c) administering to the identified subject a treatment including a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of treating a subject having a cancer that include: identifying a subject previously administered a therapeutic agent, as having a cancer cell that has an elevated level of Wnt pathway activity as compared to a reference level; and administering to the identified subject a treatment including a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of treating a subject having a cancer that include administering to a subject previously administered a therapeutic agent and later identified as having an elevated level of Wnt pathway activity as compared to a reference level, a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of determining the efficacy of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof in a subject that include: (a) determining a first level of Wnt pathway activity in a cancer cell obtained from a subject at a first time point; (b) administering to the subject after the first time a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof; (c) determining a second level of Wnt pathway activity in a cancer cell obtained from the subject at a second time point; and (d) determining that the CLK inhibitor is effective in a subject having a second level of Wnt pathway activity that is decreased as compared to the first level of Wnt pathway activity. Some embodiments of any of the methods described herein further include: (e) after (d), administering one or more additional doses of the CLK inhibitor to the subject.

In some embodiments of any of the methods described herein, the level of Wnt pathway activity is the level of CLK1, CLK2, CLK3, CLK4, or β-catenin expression. In some embodiments of any of the methods described herein, the level of CLK1, CLK2, CLK3, CLK4, or β-catenin expression is the level of CLK1, CLK2, CLK3, CLK4, or β-catenin protein. In some embodiments of any of the methods described herein, the level of Wnt pathway activity is the level of β-catenin in the nucleus.

In some embodiments of any of the methods described herein, the Wnt pathway activity is detection of a mutation in a Wnt pathway gene selected from the group of: gain-of-function mutation in a β-catenin gene, a loss-of-function mutation in an AXIN gene, a loss-of-function mutation in an AXIN2 gene, a loss-of-function mutation in a APC gene, a loss-of-function mutation in a CTNNB1 gene, a loss-of-function mutation in a Tsc1 gene, a loss-of-function mutation in a Tsc2 gene, and a loss-of-function mutation GSK3D gene.

In some embodiments of any of the methods described herein, the Wnt pathway activity is detection of an elevated level of expression of one or more Wnt-upregulated genes. In some embodiments of any of the methods described herein, the one or more Wnt-upregulated genes are selected from the group of: CCND1, CSNK2A1, CXCL12, LRP5, MMP7, MMP9, LEF1, AXIN2, MYC, TCF7L2, TCF7, LRP6, DVL2, BIRC, ERRB2, MAPK8, PKN1, AXIN2, ABCB1, ADAM10, ALEX1, ASCL2, BAMBI, BCL2L2, BIRC5, BMI1, BMP4, CCND1, CD44, CDKN2A, CDX1, CEBPD, CLDN1, COX2, DNMT1, EDN1, EFNB1, ENC1, EPHB2, EPHB3, FGF18, FGFBP, FRA1, FSCN1, FZD6, FZD7, FZD8, GAST, HDAC3, HEF1, HES1, ID2, ITF2, JAG1, JUN, LlCAM, LAMC2, LGR5, MENA, MET, MMP14, MYB, MYCBP, NOS2, NOTCH2, NRCAM, PLCB4, PLAU, PLAUR, PPARD, RUVBL1, S100A4, S100A6, SGK1, SMC3, SOX9, SP5, SRSF3, SUZ12, TCF1, TIAM1, TIMP-1. TN-C, VEGF, WNT-5a, WNT-5b, WNT11, and YAP.

In some embodiments of any of the methods described herein, the Wnt pathway activity is detection of a decreased level of expression of one or more of APC, FRZB, CTGF, and GSK3B.

In some embodiments of any of the methods described herein, the cancer is a small cell lung cancer, colorectal cancer, head and neck cancer, ovarian cancer, melanoma, renal cell carcinoma, pancreatic cancer, or non-small cell lung cancer.

Also provided herein are methods of decreasing the activity of one or more of CLK1, CLK2, CLK3, and CLK4, the method includes contacting one or more of CLK1, CLK2, CLK3 and CLK4 with an effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof. In some embodiments of any of the methods described herein, the method includes contacting one or both of CLK2 and CLK3 with an effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of decreasing the activity of one or more of CLK1, CLK2, CLK3 and CLK4 in a mammalian cell that include contacting the mammalian cell with an effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof. In some embodiments of any of the methods described herein, the mammalian cell is a cancer cell. In some embodiments of any of the methods described herein, the cancer cell has been identified as having an elevated level of Wnt pathway activity as compared to a reference level. In some embodiments of any of the methods described herein, the contacting results in a decrease in the activity of one or both of CLK2 and CLK3 in the mammalian cell.

Also provided herein are methods of altering mRNA splicing in a mammalian cell having aberrant mRNA splicing activity that include contacting the mammalian cell with an effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof. In some embodiments of any of the methods described herein, the mammalian cell is a cancer cell. In some embodiments of any of the methods described herein, the cancer cell having aberrant mRNA spicing activity has one or more of: an increased level of phosphorylated SRSF6 as compared to a reference level; an increased level of phosphorylated SRSF5 as compared to a reference level; a mutation in a SF3B1 gene, a SRSF1 gene, a SRSF2 gene, a U2AF1 gene, or a ZRSR2 gene; and an increased level of SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, and SRSF10 as compared to a reference level.

Also provided herein are methods of treating a cancer in a subject that include: identifying a subject having a cancer cell that has aberrant mRNA splicing activity as compared to a reference level; and administering to the identified subject a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of treating a cancer in a subject that include administering a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof to a subject identified as having a cancer cell that has aberrant mRNA splicing activity as compared to a reference level.

Also provided herein are methods of selecting a treatment for a subject that include: identifying a subject having a cancer cell that has aberrant mRNA splicing activity as compared to a reference level; and selecting for the identified subject a treatment including a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of selecting a treatment for a subject that include selecting a treatment including a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof for a subject identified as having a cancer cell that has aberrant mRNA splicing activity as compared to a reference level.

Also provided herein are methods of selecting a subject for treatment that include: identifying a subject having a cancer cell that has aberrant mRNA splicing activity as compared to a reference level; and selecting an identified subject for treatment with a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of selecting a subject for treatment that include selecting a subject identified as having a cancer cell that has aberrant mRNA splicing activity as compared to a reference level, for treatment with a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of selecting a subject for participation in a clinical trial that include: identifying a subject having a cancer cell that has aberrant mRNA splicing activity as compared to a reference level; and selecting the identified subject for participation in a clinical trial that comprises administration of a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of selecting a subject for participation in a clinical trial that include selecting a subject identified as having a cancer cell that has aberrant mRNA splicing activity as compared to a reference level for participation in a clinical trial that includes administration of a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of treating a subject having a cancer that include: (a) administering to the subject a therapeutic agent; (b) after (a), identifying the subject as having a cancer cell that has aberrant mRNA splicing activity as compared to a reference level; and (c) administering to the identified subject a treatment including a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of treating a subject having a cancer that include: identifying a subject previously administered a therapeutic agent, as having a cancer cell that has aberrant mRNA splicing activity as compared to a reference level; and administering to the identified subject a treatment comprising a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of treating a subject having a cancer that include administering to a subject previously administered a therapeutic agent and later identified as having aberrant mRNA splicing activity as compared to a reference level, a therapeutically effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of determining the efficacy of a CLK inhibitor in a subject that include: (a) determining a first level of SRSF6 phosphorylation and/or SRSF5 phosphorylation in a cancer cell obtained from a subject at a first time point; (b) administering to the subject after the first time a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof, (c) determining a second level of SRSF6 phosphorylation and/or SRSF5 phosphorylation in a cancer cell obtained from the subject at a second time point; and (d) determining that the CLK inhibitor is effective in a subject having a second level that is decreased as compared to the first level.

Also provided herein are methods of determining the efficacy of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof in a subject that include: (a) determining a first level of a ˜55 kDa isoform of SRSF6 in a cancer cell obtained from a subject at a first time point; (b) administering to the subject after the first time a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof, (c) determining a second level of the ˜55 kDa isoform of SRSF6 in a cancer cell obtained from the subject at a second time point; and (d) determining that the CLK inhibitor is effective in a subject having a second level of the ˜55 kDa isoform of SRSF6 that is increased as compared to the first level of the ˜55 kDa isoform of SRSF6.

Also provided herein are methods of determining the efficacy of a compound of any one of Formulas III-XI or a pharmaceutically acceptable salt or solvate thereof in a subject that include: (a) determining a first level of a ˜35 kDa isoform of SRSF1 in a cancer cell obtained from a subject at a first time point; (b) administering to the subject after the first time point a compound of any one of Formulas (I)-(XII) or a pharmaceutically acceptable salt or solvate thereof, (c) determining a second level of the ˜35 kDa isoform of SRSF1 in a cancer cell obtained from the subject at a second time point; and (d) determining that the CLK inhibitor is effective in a subject having a second level of the ˜35 kDa isoform of SRSF1 that is increased as compared to the first level of the ˜35 kDa isoform of SRSF1.

Some embodiments of any of the methods described herein further includes: (e) after (d), administering one or more additional doses of the CLK inhibitor to the subject.

In some embodiments of any of the methods described herein, the CLK inhibitor is a multi-isoform CLK inhibitor. In some embodiments of any of the methods described herein, the multi-isoform CLK inhibitor has an IC₅₀of between about 1 nM and about 10 μM for each of CLK2 and CLK3. In some embodiments of any of the methods described herein, the multi-isoform CLK inhibitor has an IC₅₀of between about 1 nM and about 1 μM for each of CLK2 and CLK3. In some embodiments of any of the methods described herein, the multi-isoform CLK inhibitor has an IC₅₀of between about 1 nM and about 100 nM for each of CLK2 and CLK3.

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of any one of Formulas (I)-(XII) or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments of any of the methods described herein, the multi-isoform CLK inhibitor has an IC₅₀of between about 2 nM and about 10 μM for each of CLK1, CLK2, and CLK3. In some embodiments of any of the methods described herein, the multi-isoform CLK inhibitor has an IC₅₀of between about 2 nM and about 1 μM for each of CLK1, CLK2, and CLK3. In some embodiments of any of the methods described herein, the multi-isoform CLK inhibitor has an IC₅₀of between about 2 nM and about 10 μM for each of CLK1, CLK2, CLK3, and CLK4. In some embodiments of any of the methods described herein, the multi-isoform CLK inhibitor has an IC₅₀of between about 2 nM and about 1 μM for each of CLK1, CLK2, CLK3, and CLK4.

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of Formula (I)

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof, wherein:

R¹is selected from the group consisting of H, halide, and unsubstituted —(C_1-3alkyl);

R²is selected from the group consisting of unsubstituted —(C_1-3alkyl), unsubstituted —(C_2-9alkenyl), unsubstituted —(C_1-9haloalkyl), —(C_1-2alkylene)_p(C_3-6carbocyclyl) optionally substituted with 1-12 R⁴, -monocyclic heterocyclyl optionally substituted with 1-10 R⁵, -phenyl substituted with 1-5 R⁶, -heteroaryl optionally substituted with 1-4 R⁷, —CO₂R, —OR⁹, and —(C═O)R¹⁰; wherein heteroaryl selected from the group consisting of pyridinyl, pyrimidinyl, pyrazinyl, oxazolyl, oxadiazolyl, thiazolyl, 2,3-dihydrobenzo[b]dioxinyl, 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazinyl, 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl, isoquinolinyl, and quinolinyl; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein; with the proviso that when L¹is a bond, R²is selected from the group consisting of -phenyl substituted with 1-5 R⁶and -heteroaryl optionally substituted with 1-4 R⁷; wherein heteroaryl selected from the group consisting of pyridinyl, oxazolyl, oxadiazolyl, thiazolyl, 2,3-dihydrobenzo[b]dioxinyl, 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazinyl, 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl, isoquinolinyl, and quinolinyl;

R³is selected from the group consisting of -heterocyclyl substituted with 1-10 R¹, —(C_1-4alkylene)_pphenyl substituted with 1-5 R¹², -heteroaryl optionally substituted with 1-4 R¹³, and —(C_1-4alkylene)OR¹⁴; wherein heteroaryl selected from the group consisting of pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, oxazolyl, oxadiazolyl, thiadiazolyl, indolyl, indazolyl, benzimidazolyl, imidazo[4,5-b]pyridinyl, imidazo[4,5-c]pyridinyl, 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazinyl, 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl, 1,2,3,4-tetrahydroisoquinolinyl, isoquinolinyl, and quinolinyl; wherein

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is only substituted at positions 4 and 7; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

with the proviso that when L²is a bond, R³is selected from -heteroaryl optionally substituted with 1-4 R¹³; wherein heteroaryl selected from the group consisting of pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, oxazolyl, oxadiazolyl, thiadiazolyl, indolyl, indazolyl, benzimidazolyl, imidazo[4,5-b]pyridinyl, imidazo[4,5-c]pyridinyl, 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazinyl, 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl, 1,2,3,4-tetrahydroisoquinolinyl, isoquinolinyl, and quinolinyl; wherein

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is only substituted at positions 4 and 7;

each R⁴is halide;

each R⁵is independently selected from the group consisting of halide, Me, and Et;

each R⁶is independently selected from the group consisting of methyl, —CH₂F, —CHF₂, —CF₃, —OR^15a, and —(C_1-4alkylene)_pN(R^16a)(R^16b); wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R⁷is independently selected from the group consisting of F, methyl, —CH₂F, —CHF₂, —CF₃, —CF₂CH₃, —OR^15a, —CO₂R¹⁷, —NR¹⁸(C═O)R¹⁹, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R^20b, and —(C_1-4alkylene)_pN(R^16a)(R^16b); wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

R⁸is unsubstituted —(C_1-9alkyl);

R⁹is unsubstituted —(C_1-9alkyl);

R¹⁰is -aryl optionally substituted with 1-5 R²¹;

each R¹¹is independently selected from the group consisting of halide, methyl, and ethyl;

each R¹²is independently selected from the group consisting of —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R^20a, -aryl optionally substituted with 1-5 R²², —(C_1-4alkylene)N(R^16a)(R^16b), and —OR^23a; wherein heterocyclyl selected from the group consisting of azetidinyl, pyrrolidinyl, piperidinyl, and piperazinyl; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R¹³is independently selected from the group consisting of F, methyl, —CH₂F, —CHF₂, —CF₃, —(C_1-4alkylene)_pN(R^16a)₂, —OR^23b, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R^20b, -aryl optionally substituted with 1-5 R²², and -heteroaryl substituted with 1-4 R²⁴; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

R¹⁴is selected from the group consisting of unsubstituted —(C_1-4alkyl) and -aryl optionally substituted with 1-5 R²²;

each R^15ais independently selected from the group consisting of unsubstituted —(C_2-3alkyl), and -heterocyclyl optionally substituted with 1-10 R^20b;

each R^15bis independently selected from the group consisting of H, unsubstituted —(C_2-9alkyl), and -heterocyclyl optionally substituted with 1-10 R^20b;

each R^16ais independently selected from the group consisting of H and unsubstituted —(C_1-2alkyl);

each R^16bis unsubstituted —(C_1-2alkyl);

each R¹⁷is unsubstituted —(C_1-9alkyl);

each R¹⁸is independently selected from the group consisting of H and Me;

each R¹⁹is unsubstituted —(C_1-9alkyl);

each R^20ais independently selected from the group consisting of halide and unsubstituted —(C_2-9alkyl);

each R^20bis independently selected from the group consisting of halide and unsubstituted —(C_1-9alkyl);

each R²¹is independently selected from the group consisting of halide and unsubstituted —(C_1-9alkyl);

each R²²is independently selected from the group consisting of halide and unsubstituted —(C_1-9alkyl);

each R^23ais independently selected from the group consisting of unsubstituted —(C_2-9alkyl), —(C_1-4alkylene)OR²⁵, and —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R^20b; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R^23bis independently selected from the group consisting of unsubstituted —(C_1-9alkyl), —(C_1-4alkylene)OR²⁵, and —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R^20b; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R²⁴is independently selected from the group consisting of halide and unsubstituted —(C_1-9alkyl);

each R²⁵is independently selected from the group consisting of H and unsubstituted —(C_1-9alkyl);

L¹is selected from the group consisting of a bond, —CH═CH—, —C≡C—, —(CH₂)_pNR¹⁸(C═O)—, —(C═O)NR¹⁸(CH₂)_p—, —NR¹⁸(C═O)NR¹⁸—, —NH(CH₂)_p—, and —(CH₂)_pNH—;

L²is selected from the group consisting of a bond, —(C═O)NR¹⁸—, —NR¹⁸(C═O)—, —NHCH₂—, and —CH₂NH—; and

each p is independently an integer of 0 or 1.

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of Formula (II)

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof, wherein:

Ring A is a 5-6-membered heteroaryl optionally substituted with 1-4 R¹;

L is -L¹-L²-L³-L⁴-;

L¹is selected from the group consisting of unsubstituted —(C_1-3alkylene)-, —NR²—, —NR³(C═O)—, —(C═O)NR³—, and —O—;

L²is selected from the group consisting of unsubstituted —(C_1-6alkylene)- and —NR²—;

L³is selected from the group consisting of unsubstituted —(C_1-6alkylene)-, —O—, and -carbocyclylene- optionally substituted with one or more halides;

L⁴is selected from the group consisting of unsubstituted —(C_1-6alkylene)-, —O—, —NR²—, —NR³(C═O)—, —(C═O)NR³—, -arylene- optionally substituted with 1-5 R⁴, and -heteroarylene-optionally substituted with 1-4 R⁵;

with the proviso that —NR²— and —O— are not adjacent to each other;

with the proviso that two —NR³(C═O)— and/or —(C═O)NR³—, are not adjacent to each other;

each R¹is selected from the group consisting of halide, unsubstituted —(C_1-3alkyl), unsubstituted —(C_1-3haloalkyl), and —CN;

each R²is selected from the group consisting of H and unsubstituted —(C_1-6alkyl);

each R³is selected from the group consisting of H and unsubstituted —(C_1-6alkyl);

each R⁴is selected from the group consisting of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_1-6haloalkyl), and —CN;

each R⁵is selected from the group consisting of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_1-6haloalkyl), and —CN;

Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶are independently selected from the group consisting of carbon and nitrogen; wherein

if Y¹is nitrogen then Y²and Y³are CH;

if Y²is nitrogen then Y¹and Y³are CH;

if Y³is nitrogen then Y¹and Y²are CH;

if Y⁴is nitrogen then Y⁵and Y⁶are CH;

if Y⁵is nitrogen then Y⁴and Y⁶are CH; and

if Y⁶is nitrogen then Y⁴and Y⁵are CH.

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of Formula (III)

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof, wherein:

R¹is selected from the group of H and halide;

R²is a 6-membered -heteroaryl substituted with 1-4 R³;

each R³is selected from the group of —OR⁴, —NHR⁵, and —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R⁶; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R⁴is independently selected from the group of -heterocyclyl optionally substituted with 1-10 R⁷and —CH₂CH(R)NH₂;

each R is independently selected from the group of —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R⁹and -carbocyclyl optionally substituted with 1-12 R¹⁰; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R⁶is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R⁷is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R⁸is independently selected from the group of —(C_1-4alkylene)aryl optionally substituted with 1-5 R¹and —(C_1-4alkylene)heteroaryl optionally substituted with 1-4 R¹²; wherein

each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁹is independently selected from the group of halide, —OH, —NH₂, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R¹⁰is independently selected from the group of halide, —OH, —NH₂, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R¹¹is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R¹²is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl); and

each p is independently 0 or 1.

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of Formula (IV)

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof, wherein:

R¹is selected from the group of H and halide;

R²is a -heteroaryl optionally substituted with 1-4 R⁴;

R³is selected from the group of -aryl optionally substituted with 1-5 R⁵and -heteroaryl optionally substituted with 1-4 R⁶;

each R⁴is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —(C_1-4alkylene)_pN(R⁷)(R⁸), —NHC(═O)R⁹, —(C_1-4alkylene)_pOR¹⁰, unsubstituted -carbocyclyl, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R¹⁴, —(C_1-4alkylene)_paryl optionally substituted with 1-5 R¹¹, and —(C_1-4alkylene)_pheteroaryl optionally substituted with 1-4 R¹²; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁵is independently selected from the group of halide, —CN, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —(C_1-4alkylene)_paryl optionally substituted with 1-5 R¹³, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R¹⁴, —C(═O)N(R¹⁵)₂, —NHC(═O)R¹⁶, —(C_1-4alkylene)_pN(R¹⁷)(R¹⁸), —SO₂R¹⁹, and —OR²⁰; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁶is independently selected from the group of halide, —CN, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —(C_1-4alkylene)_paryl optionally substituted with 1-5 R¹³, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R¹⁴, —C(═O)N(R¹⁵)₂, —NHC(═O)R¹⁶, —(C_1-4alkylene)_pN(R¹⁷)(R¹⁸), —SO₂R¹⁹, and —OR²⁰; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁷is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), and unsubstituted —(C_2-6alkynyl);

each R⁸is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and -heterocyclyl optionally substituted with 1-10 R²¹;

alternatively, R⁷and R⁸are taken together to form a -heterocyclyl ring optionally substituted with 1-10 R²¹;

each R⁹is independently selected from the group of —N(R²²)₂, -carbocyclyl optionally substituted with 1-12 R²³, -heterocyclyl optionally substituted with 1-10 R²¹, and -aryl optionally substituted with 1-5 R²⁴;

each R¹⁰is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), and -heterocyclyl optionally substituted with 1-10 R²¹;

each R¹¹is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl); each R¹²is independently selected from the group of halide, —(C_1-4alkylene)pOH, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl); wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R¹³is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R¹⁴is independently selected from the group of halide, —(C_1-4alkylene)pOH, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl); wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R⁵is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and -carbocyclyl optionally substituted with 1-12 R²³;

alternatively, two adjacent R¹⁵are taken together to form a -heterocyclyl ring optionally substituted with 1-10 R²¹;

each R¹⁶is independently selected from the group of unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and -carbocyclyl optionally substituted with 1-12 R²³;

each R¹⁷is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), and unsubstituted —(C_2-6alkynyl);

each R¹⁸is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), —(C_1-4alkylene)NMe₂, and -heterocyclyl ring optionally substituted with 1-10 R²¹; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁹is independently selected from the group of unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), and unsubstituted —(C_2-6alkynyl).

each R²⁰is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —CH(CH₂OH)₂, —(C_1-4alkylene)_pheterocyclyl ring optionally substituted with 1-10 R²¹, and -aryl optionally substituted with 1-5 R²⁴; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R²¹is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R²²is independently selected from the group of unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), and unsubstituted —(C_2-6alkynyl);

each R²³is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R²⁴is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl); and

each p is independently 0 or 1.

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of Formula (V)

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or a pharmaceutically acceptable salt or solvate thereof, wherein:

R¹is a -heteroaryl optionally substituted with 1-2 R³;

R²is selected from the group of H, halide, -aryl optionally substituted with 1-5 R⁴-heteroaryl optionally substituted with 1-4 R⁵, and -heterocyclyl ring optionally substituted with 1-10 R⁶;

each R³is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R⁷, —C(═O)N(R⁸)₂, —NHC(═O)R⁹, —(C_1-4alkylene)_pN(R¹⁰)(R¹), —(C_1-4alkylene)_pOR¹², and -carbocyclyl optionally substituted with 1-12 R¹³; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁴is independently selected from the group of halide, —CN, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —(C_1-4alkylene)_pNHSO₂R¹⁴, —NR⁵(C_1-4alkylene)NR¹⁵R¹⁶, —(C_1-4alkylene)_pNR¹⁵R¹⁶, —OR¹⁷, and -heterocyclyl optionally substituted with 1-10 R¹⁹; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁵is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), and —C(═O)R¹⁸;

each R⁶is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R⁷is independently selected from the group of halide, —NH₂, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R⁸is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), -heterocyclyl optionally substituted with 1-10 R¹⁹, —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 R²⁰; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R⁹is independently selected from the group of unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R¹⁹, —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 R²⁰; —(C_1-4alkylene)_paryl optionally substituted with 1-5 R²¹, —(C_1-4alkylene)_pN(R²²)₂; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁰is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), and unsubstituted —(C_2-6alkynyl);

each R¹¹is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 R²⁰; and —(C_1-4alkylene)_paryl optionally substituted with 1-5 R²¹; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R¹²is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R¹⁹, —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 R²⁰; —(C_1-4alkylene)_paryl optionally substituted with 1-5 R²¹, —(C_1-4alkylene)_pN(R²²)₂; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R¹³is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl); each R¹⁴is independently selected from the group of unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), and unsubstituted —(C_2-6alkynyl);

each R⁵is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), and unsubstituted —(C_2-6alkynyl);

each R¹⁶is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), and unsubstituted —(C_2-6alkynyl);

each R¹⁷is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R¹⁹, and, —(C_1-4alkylene)_pN(R²²)₂; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁸is independently selected from the group of unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), and unsubstituted —(C_2-6alkynyl);

each R¹⁹is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R²⁰is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R²¹is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R²²is independently selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), and unsubstituted —(C_2-6alkynyl);

each R²³is independently selected from the group of H and halide;

Y¹, Y², and Y³are independently selected from the group of —CR²³═ and —N═;

Y⁴is selected from the group of —CH═ and —N═;

Z¹, Z², and Z³are independently selected from the group of —CR²³═ and —N═; and

each p is independently 0 or 1.

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of Formula (VI)

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof, wherein:

R¹is selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and -heteroaryl optionally substituted with 1-4 R⁴, -aryl optionally substituted with 1-5 R;

R²is selected from the group of H, —(C_1-4alkylene)_pheteroaryl optionally substituted with 1-4 R⁶, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R⁷, and —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 R; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

R³is selected from the group of -heteroaryl optionally substituted with 1-4 R⁹and -aryl optionally substituted with 1-5 R¹⁰;

each R⁴is independently selected from the group of halide, —CN, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —OR¹¹, —C(═O)N(R¹²)₂, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R¹³, —SO₂R¹⁴, and —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 R¹⁵; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁵is independently selected from the group of halide, —CN, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —OR¹¹, —C(═O)N(R¹²)₂, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R¹³, —SO₂R¹⁴, and —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 R¹⁵; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁷is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R⁸is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl); each R⁹is independently selected from the group of halide, —CN, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —OR¹¹, —C(═O)N(R¹²)₂, and —SO₂R¹⁴;

each R¹⁰is independently selected from the group of halide, —CN, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —OR¹¹, —C(═O)N(R¹²)₂, and —SO₂R¹⁴;

each R¹¹is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

each R¹²is independently selected from the group of H, halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), and unsubstituted —(C_2-6alkynyl);

each R¹³is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl); each R¹⁴is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), and unsubstituted —(C_2-6alkynyl);

each R⁵is independently selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and unsubstituted —(C_1-6haloalkyl);

L is selected from the group of a bond, —O—, and —NH—; and each p is independently 0 or 1.

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of Formula (VII)

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof, wherein:

R¹, R², R⁴, and R⁵are independently absent or selected from the group of H and halide;

R³is selected from the group of -heteroaryl optionally substituted with 1-4 R⁸and -Xheterocyclyl optionally substituted with one or more halides or one or more unsubstituted —(C_1-5alkyl);

R⁶is selected from the group of -aryl substituted with 1-5 R⁹, —(C_2-4alkenylene)aryl substituted with 1-5 R⁹, —(C_1-4alkylene)_pheteroaryl optionally substituted with 1-6 R¹⁰; -heterocyclyl optionally substituted with 1-10 R¹¹, -carbocyclyl optionally substituted with 1-12 R¹², and —(C_2-9alkynyl) optionally substituted with one or more halides; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein; wherein —(C_1-4alkenylene) is, optionally substituted with one or more substituents as defined anywhere herein;

with the proviso that R⁶is heterocyclyl only when R³is a 6-membered heteroaryl;

each R⁸is independently selected from the group of halide, unsubstituted —(C_1-9alkyl), unsubstituted —(C_2-9alkenyl), unsubstituted —(C_2-9alkynyl), unsubstituted —(C_1-9haloalkyl), —CN, —N(R¹⁵)(R¹⁸), —(C_1-4alkylene)_pXR¹⁹, —C(═O)N(R¹⁵)₂, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R²⁰, and -carbocyclyl optionally substituted with 1-12 R²¹; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

alternatively, two adjacent R⁸are taken together to form a ring which is selected from the group of -heterocyclyl optionally substituted with 1-10 R²²and -carbocyclyl optionally substituted with 1-12 R²¹;

each R⁹is independently selected from the group of D, halide, unsubstituted —(C_1-9alkyl), unsubstituted —(C_2-9alkenyl), unsubstituted —(C_2-9alkynyl), unsubstituted —(C_1-9haloalkyl), —XR²³, —(C_1-4alkylene)_pN(R²⁴)₂, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R²²; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁰is independently selected from the group of halide, unsubstituted —(C_1-9alkyl), unsubstituted —(C_2-9alkenyl), unsubstituted —(C_2-9alkynyl), unsubstituted —(C_1-9haloalkyl), —CN, —XR²³, —C(═O)N(R¹⁵)₂, —(C_1-4alkylene)_pN(R²⁴)₂, -heterocyclyl optionally substituted with 1-10 R²², and -carbocyclyl optionally substituted with 1-12 R²¹; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R¹¹is independently selected from the group of halide, unsubstituted —(C_1-9alkyl), unsubstituted —(C_2-9alkenyl), unsubstituted —(C_2-9alkynyl), and unsubstituted —(C_1-9haloalkyl);

each R¹²is independently selected from the group of halide, —(C_1-4alkylene)_pOR¹⁹; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein; each R⁵is selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), and unsubstituted —(C_1-5haloalkyl);

R¹⁸is independently selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), unsubstituted —(C_1-5haloalkyl), and —(C_1-4alkylene)_pheterocyclyl optionally substituted with one or more halides or one or more unsubstituted —(C_1-5alkyl); wherein —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁹is independently selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), unsubstituted —(C_1-5haloalkyl), —(C_1-4alkylene)_pheterocyclyl optionally substituted with one or more halides or one or more unsubstituted —(C_1-5alkyl); wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R²⁰independently is selected from the group of halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), unsubstituted —(C_1-5haloalkyl), and —OH;

each R²¹is independently selected from the group of halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), unsubstituted —(C_1-5haloalkyl), and —CN;

each R²²is independently selected from the group of halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), unsubstituted —(C_1-5haloalkyl), —OH, —N(R⁵)₂, —C(═O)R³⁴, and -carbocyclyl optionally substituted with 1-12 R²¹;

each R²³is independently selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), unsubstituted —(C_1-5haloalkyl), —(C_1-4alkylene)N(R¹⁵)₂, -heterocyclyl optionally substituted with 1-10 R³¹, and -carbocyclyl optionally substituted with 1-12 R²¹; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R²⁴is independently selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), unsubstituted —(C_1-5haloalkyl), —(C_1-4alkylene)_pheterocyclyl optionally substituted with one or more halides or one or more unsubstituted —(C_1-5alkyl), and —(C_1-4alkylene)N(R⁵)₂; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R³¹is independently selected from the group of halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), and unsubstituted —(C_1-5haloalkyl);

each R³⁴is independently selected from the group of —O(C_1-5alkyl) and a heteroaryl optionally substituted with 1-6 R³⁵;

each R³⁵is a -heterocyclyl optionally substituted with one or more halides or one or more unsubstituted —(C_1-5alkyl);

each X is selected from the group of O and S;

Y¹, Y², Y³, and Y⁴are independently selected from the group consisting of carbon and nitrogen; wherein

if Y¹is nitrogen then Y², Y³, and Y⁴are carbon, and R⁴is absent;

if Y²is nitrogen then Y¹, Y³, and Y⁴are carbon, and R⁵is absent;

if Y³is nitrogen then Y¹, Y², and Y⁴are carbon, and R¹is absent;

if Y⁴is nitrogen then Y¹, Y², and Y³are carbon, and R²is absent; and

each p is independently 0 or 1.

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of Formula (VIII)

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof, wherein:

R¹is selected from the group of —(C_1-4alkylene)N(R⁵)₂, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R⁶, and —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 R⁷; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

R²is selected from the group of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), unsubstituted —(C_1-6haloalkyl), —CN, —OR, —C(═O)NHR⁹, —NHC(═O)(R¹⁰), —SO₂R¹⁰, —NHSO₂R¹⁰, and —SO₂NHR⁹;

R³is selected from the group of H, halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), and unsubstituted —(C_L_5 haloalkyl);

R⁴is selected from the group of H, halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), and unsubstituted —(C_1-5haloalkyl);

each R⁵is independently selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C₂_s alkenyl), and unsubstituted —(C₂_s alkynyl);

each R⁶is independently selected from the group of halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), unsubstituted —(C_1-5haloalkyl), —OH, and —CN;

each R⁷is independently selected from the group of halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), unsubstituted —(C_1-5haloalkyl), —OH, and —CN;

R⁸is selected from the group of H, unsubstituted —(C_1-6alkyl), unsubstituted —(C_2-6alkenyl), unsubstituted —(C_2-6alkynyl), and —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R⁶; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R⁹is independently selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), and unsubstituted —(C_2-5alkynyl), and —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R⁶; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁰is independently selected from the group of unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), and unsubstituted —(C_2-5alkynyl), and —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 R⁶; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein; and

each p is independently 0 or 1.

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of Formula (IX)

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof, wherein:

R¹is -heteroaryl optionally substituted with 1-6 R⁴;

each R²is independently selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), and unsubstituted —(C_1-5haloalkyl);

R³is —CH(R⁵)R⁶;

each R⁴is independently selected from the group of halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), unsubstituted —(C_1-5haloalkyl), —CN, —OR⁷, -carbocyclyl optionally substituted with 1-12 R;

R⁵is -aryl optionally substituted with 1-5 R⁹;

R⁶is —(C_1-4alkylene)N(R¹⁰)₂; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R⁷is independently selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), and unsubstituted —(C_1-5haloalkyl);

each R⁸is independently selected from the group of halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), and unsubstituted —(C_1-5haloalkyl);

each R⁹is independently selected from the group of halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), unsubstituted —(C_1-5haloalkyl), —CN, and —OR⁷;

each R¹⁰is independently selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), and unsubstituted —(C_2-5alkynyl); and

X is selected from the group of O, S, and NH.

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of Formula (X)

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof, wherein:

R¹is selected from the group of H, halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_1-5haloalkyl), and —CN;

R²is selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), and unsubstituted —(C_2-5alkynyl);

R³is -aryl optionally substituted with 1-5 R⁴;

R⁵is selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), and unsubstituted —(C_1-5haloalkyl); and

X is selected from the group of N and CR⁵.

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of Formula (XI)

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof, wherein:

R¹is —N(R⁴)₂;

R²is selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), and unsubstituted —(C_1-5haloalkyl);

R³is -heteroaryl optionally substituted with 1-6 R⁵;

each R⁴is independently selected from the group of H, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), and -heterocyclyl optionally substituted with 1-10 R⁶;

alternatively, two adjacent R⁴are taken together to form a ring which is selected from the group of -heterocyclyl optionally substituted with 1-10 R⁶;

each R⁵is independently selected from the group of halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), unsubstituted —(C_1-5haloalkyl), —CN, —OH, and —OMe; and

each R⁶is independently selected from the group of halide, unsubstituted —(C_1-5alkyl), unsubstituted —(C_2-5alkenyl), unsubstituted —(C_2-5alkynyl), and unsubstituted —(C_1-5haloalkyl).

In some embodiments of any of the methods described herein, the CLK inhibitor is a compound of Formula (XII)

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof, wherein:

Ring A is a 5-6-membered heteroaryl optionally substituted with 1-3 R¹;

L is -L¹-L²-L³-L⁴-

L¹is selected from the group consisting of unsubstituted —(C_1-3alkylene)-, —NR²—, —NR³(C═O)—, —(C═O)NR³—, and —O—;

L²is selected from the group consisting of unsubstituted —(C_1-6alkylene)-, —NR²—, —NR³(C═O)—, and —(C═O)NR³—;

L³is selected from the group consisting of unsubstituted —(C_1-6alkylene)-, —O—, and carbocyclylene optionally substituted with one or more halides;

L⁴is selected from the group consisting of unsubstituted —(C_1-6alkylene)-, —O—, —NR²—, —NR³(C═O)—, —(C═O)NR³—, -arylene substituted with 1-5 R⁴, and -heteroarylene optionally substituted with 1-4 R⁵;

with the proviso that —NR²— and —O— are not adjacent to each other;

with the proviso that two —NR³(C═O)— and/or —(C═O)NR³—, are not adjacent to each other;

each R¹is selected from the group consisting of halide, unsubstituted —(C_1-3alkyl), unsubstituted —(C_1-3haloalkyl), and —CN;

each R²is selected from the group consisting of H and unsubstituted —(C_1-6alkyl);

each R³is selected from the group consisting of H and unsubstituted —(C_1-6alkyl);

each R⁴is selected from the group consisting of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_1-6haloalkyl), and —CN;

each R⁵is selected from the group consisting of halide, unsubstituted —(C_1-6alkyl), unsubstituted —(C_1-6haloalkyl), and —CN;

Y¹, Y², and Y³are independently selected from the group consisting of carbon and nitrogen; wherein

if Y¹is nitrogen then Y²and Y³are CH;

if Y²is nitrogen then Y¹and Y³are CH; and

if Y³is nitrogen then Y¹and Y²are CH.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure, as claimed.

As used herein, “Wnt pathway activity” is an art-known term and generally refers to one or more direct Wnt/p-catenin activities in a mammalian cell and/or one or more indirect activities of Wnt/β-catenin (downstream activities resulting from Wnt/p-catenin activity) in a mammalian cell. Non-limiting examples of Wnt pathway activities include the level of expression of one or more Wnt-upregulated genes (e.g., one or more of any of the exemplary Wnt-upregulated genes described herein) in a mammalian cell, the level of β-catenin present in a nucleus of a mammalian cell, the level of expression of one or more of CLK1, CLK2, CLK3, CLK4, and β-catenin in a mammalian cell, detection of a gain-of-function mutation in a β-catenin gene, and detection of one or more of a loss-of-function mutation in one or more of a AXIN gene, a AXIN2 gene, a APC gene, a CTNNB1 gene, a Tsc1 gene, a Tsc2 gene, and a GSK3p gene. Methods for detecting a level of each of these exemplary types of Wnt pathway activity are described herein. Additional examples of Wnt pathway activities are known in the art, as well as methods for detecting a level of the same.

As used herein, “gain-of-function mutation” means one or more nucleotide substitutions, deletions, and/or insertions in a gene that results in: an increase in the level of expression of the encoded protein as compared to the level of the expression by the corresponding wildtype gene, and/or the expression of a protein encoded by the gene that has one or more increased activities in a mammalian cell as compared to the version of the protein encoded by the corresponding wildtype gene.

As used herein, “loss-of-function mutation” means one or more nucleotide substitutions, deletions, and/or insertions in a gene that results in: a decrease in the level of expression of the encoded protein as compared to the level of the expression by the corresponding wildtype gene, and/or the expression of a protein encoded by the gene that has one or more decreased activities in a mammalian cell as compared to the version of the protein encoded by the corresponding wildtype gene.

As used herein, “Wnt-upregulated gene” means a gene that exhibits an increased level of transcription when the Wnt/β-catenin signaling pathway is active in a mammalian cell. Non-limiting examples of Wnt-upregulated genes are described herein. Additional examples of Wnt-upregulated genes are known in the art. Exemplary methods of detecting the level of expression of Wnt-upregulated genes are described herein. Additional methods of detecting the level of expression of Wnt-upregulated genes are known in the art.

As used herein, “CLK inhibitor” refers to an agent (e.g., compound) that decreases the catalytic activity of one or more of CLK1, CLK2, CLK3, and CLK4 with an IC₅₀of about 1 nM to about 10 μM (or any of the subranges of this range described herein) (e.g., determined using the exemplary in vitro assays for determining CLK1, CLK2, CLK3, and CLK4 activities described in the Examples).

As used herein, “a multi-isoform CLK inhibitor” refers to an agent (e.g., a compound that decreases the catalytic activity of two or more of CLK1, CLK2, CLK3, and CLK4 with an IC₅₀of about 1 nM to about 10 μM (or any of the subranges of this range described herein) (e.g., determined using the exemplary in vitro assays for determining CLK1, CLK2, CLK3, and CLK4 activities described in the Examples).

As used herein, “altering mRNA splicing” means (i) changing the relative expression levels of two or more different isoforms of a protein in a mammalian cell that are encoded by the same gene, wherein the different isoforms of the protein result from mRNA splicing in the mammalian cell; and/or (ii) changing the level of activity, phosphorylation, and/or expression of one or more splicing factors in a mammalian cell.

As used herein, “aberrant mRNA splicing” means a mammalian cell that has been identified as having (i) a different relative expression levels of two or more different isoforms of a protein in a mammalian cell that are encoded by the same gene, wherein the different isoforms of the protein result from mRNA splicing in the mammalian cell; and/or (ii) a different level of activity, phosphorylation, and/or expression of one or more splicing factors, e.g., as compared to a reference level (e.g., the level in a healthy, non-cancerous cell or a corresponding non-cancerous cell).

As used herein, “alkyl” means a branched or straight chain chemical group containing only carbon and hydrogen, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, and neo-pentyl. Alkyl groups can either be unsubstituted or substituted with one or more substituents. In some embodiments, alkyl groups include 1 to 9 carbon atoms (for example, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 2 carbon atoms).

As used herein, “alkenyl” means a straight or branched chain chemical group containing only carbon and hydrogen, and containing at least one carbon-carbon double bond, such as ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. In various embodiments, alkenyl groups can either be unsubstituted or substituted with one or more substituents. Typically, alkenyl groups will comprise 2 to 9 carbon atoms (for example, 2 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 carbon atoms).

As used herein, “alkynyl” means a straight or branched chain chemical group containing only carbon and hydrogen, and containing at least one carbon-carbon triple bond, such as ethynyl, 1-propynyl, 1-butynyl, 2-butynyl, and the like. In various embodiments, alkynyl groups can either be unsubstituted or substituted with one or more substituents. Typically, alkynyl groups will comprise 2 to 9 carbon atoms (for example, 2 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 carbon atoms).

As used herein, “alkylene” means a bivalent branched, or straight chain chemical group containing only carbon and hydrogen, such as methylene, ethylene, n-propylene, iso-propylene, n-butylene, iso-butylene, sec-butylene, tert-butylene, n-pentylene, iso-pentylene, sec-pentylene, and neo-pentylene. Alkylene groups can either be unsubstituted or substituted with one or more substituents. In some embodiments, alkylene groups include 1 to 9 carbon atoms (for example, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 2 carbon atoms).

As used herein, “alkenylene” means a bivalent branched, or straight chain chemical group containing only carbon and hydrogen, and containing at least one carbon-carbon double bond, such as ethenylene, 1-propenylene, 2-propenylene, 2-methyl-1-propenylene, 1-butenylene, 2-butenylene, and the like. In various embodiments, alkenylene groups can either be unsubstituted or substituted with one or more substituents. Typically, alkenylene groups will comprise 2 to 9 carbon atoms (for example, 2 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 carbon atoms).

As used herein, “alkynylene” means a bivalent branched, or straight chain chemical group containing only carbon and hydrogen, and containing at least one carbon-carbon triple bond, such as ethynylene, 1-propynylene, 1-butynylene, 2-butynylene, and the like. In various embodiments, alkynylene groups can either be unsubstituted or substituted with one or more substituents. Typically, alkynylene groups will comprise 2 to 9 carbon atoms (for example, 2 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 carbon atoms).

As used herein, “alkoxy” means an alkyl-O— group in which the alkyl group is as described herein. Exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, pentoxy, hexoxy, and heptoxy, and also the linear or branched positional isomers thereof.

As used herein, “haloalkoxy” means a haloalkyl-O— group in which the haloalkyl group is as described herein. Exemplary haloalkoxy groups include fluoromethoxy, difluoromethoxy, and trifluoromethoxy, and also the linear or branched positional isomers thereof.

As used herein, “carbocyclyl” means a cyclic ring system containing only carbon atoms in the ring system backbone, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexenyl. Carbocyclyls may include multiple fused rings. Carbocyclyls may have any degree of saturation provided that none of the rings in the ring system are aromatic. Carbocyclyl groups can either be unsubstituted or substituted with one or more substituents. In some embodiments, carbocyclyl groups include 3 to 10 carbon atoms, for example, 3 to 6 carbon atoms.

As used herein, “aryl” means a mono-, bi-, tri- or polycyclic group with only carbon atoms present in the ring backbone having 5 to 14 ring atoms, alternatively 5, 6, 9, or 10 ring atoms; and having 6, 10, or 14 pi electrons shared in a cyclic array; where at least one ring in the system is aromatic. Aryl groups can either be unsubstituted or substituted with one or more substituents. Examples of aryl include phenyl, naphthyl, tetrahydronaphthyl, 2,3-dihydro-1H-indenyl, and others. In some embodiments, the aryl is phenyl.

As used herein, “arylalkylene” means an aryl-alkylene- group in which the aryl and alkylene moieties are as previously described. In some embodiments, arylalkylene groups contain a C_1-4alkylene moiety. Exemplary arylalkylene groups include benzyl and 2-phenethyl.

As used herein, the term “heteroaryl” means a mono-, bi-, tri- or polycyclic group having 5 to 14 ring atoms, alternatively 5, 6, 9, or 10 ring atoms; and having 6, 10, or 14 pi electrons shared in a cyclic array; wherein at least one ring in the system is aromatic, and at least one ring in the system contains one or more heteroatoms independently selected from the group consisting of N, O, and S. Heteroaryl groups can either be unsubstituted or substituted with one or more substituents. Examples of heteroaryl include thienyl, pyridinyl, furyl, oxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, triazolyl, thiodiazolyl, pyrazolyl, isoxazolyl, thiadiazolyl, pyranyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thiazolyl benzothienyl, benzoxadiazolyl, benzofuranyl, benzimidazolyl, benzotriazolyl, cinnolinyl, indazolyl, indolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, purinyl, thienopyridinyl, pyrido[2,3-d]pyrimidinyl, pyrrolo[2,3-b]pyridinyl, quinazolinyl, quinolinyl, thieno[2,3-c]pyridinyl, pyrazolo[3,4-b]pyridinyl, pyrazolo[3,4-c]pyridinyl, pyrazolo[4,3-c]pyridine, pyrazolo[4,3-b]pyridinyl, tetrazolyl, chromane, 2,3-dihydrobenzo[b][1,4]dioxine, benzo[d][1,3]dioxole, 2,3-dihydrobenzofuran, tetrahydroquinoline, 2,3-dihydrobenzo[b][1,4]oxathiine, isoindoline, and others. In some embodiments, the heteroaryl is selected from thienyl, pyridinyl, furyl, pyrazolyl, imidazolyl, isoindolinyl, pyranyl, pyrazinyl, and pyrimidinyl.

As used herein, “halo”, “halide,” or “halogen” is a chloro, bromo, fluoro, or iodo atom radical. In some embodiments, a halo is a chloro, bromo or fluoro. For example, a halide can be fluoro.

As used herein, “haloalkyl” means a hydrocarbon substituent, which is a linear or branched, alkyl, alkenyl, or alkynyl substituted with one or more chloro, bromo, fluoro, and/or iodo atom(s).

In some embodiments, a haloalkyl is a fluoroalkyls, where one or more of the hydrogen atoms have been substituted by fluoro. In some embodiments, haloalkyls are of 1 to about 3 carbons in length (e.g., 1 to about 2 carbons in length or 1 carbon in length). The term “haloalkylene” means a diradical variant of haloalkyl, and such diradicals may act as spacers between radicals, other atoms, or between a ring and another functional group.

As used herein, “heterocyclyl” means a nonaromatic cyclic ring system comprising at least one heteroatom in the ring system backbone. Heterocyclyls may include multiple fused rings. Heterocyclyls may be substituted or unsubstituted with one or more substituents. In some embodiments, heterocycles have 3-11 members. In six-membered monocyclic heterocycles, the heteroatom(s) are selected from one to three of O, N, or S, and where, when the heterocycle is five-membered, it can have one or two heteroatoms selected from O, N, or S. Examples of heterocyclyl include azirinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, 1,4,2-dithiazolyl, dihydropyridinyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl, morpholinyl, thiomorpholinyl, piperazinyl, pyranyl, pyrrolidinyl, tetrahydrofuryl, tetrahydropyridinyl, oxazinyl, thiazinyl, thiinyl, thiazolidinyl, isothiazolidinyl, oxazolidinyl, isoxazolidinyl, piperidinyl, pyrazolidinyl imidazolidinyl, thiomorpholinyl, and others. In some embodiments, the heterocyclyl is selected from azetidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and tetrahydropyridinyl.

As used herein, “monocyclic heterocyclyl” means a single nonaromatic cyclic ring comprising at least one heteroatom in the ring system backbone. Heterocyclyls may be substituted or unsubstituted with one or more substituents. In some embodiments, heterocycles have 3-7 members. In six-membered monocyclic heterocycles, the heteroatom(s) are selected from one to three of O, N, or S, and where, when the heterocycle is five-membered, it can have one or two heteroatoms selected from O, N, or S. Examples of heterocyclyls include azirinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, 1,4,2-dithiazolyl, dihydropyridinyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl, morpholinyl, thiomorpholinyl, piperazinyl, pyranyl, pyrrolidinyl, tetrahydrofuryl, tetrahydropyridinyl, oxazinyl, thiazinyl, thiinyl, thiazolidinyl, isothiazolidinyl, oxazolidinyl, isoxazolidinyl, piperidinyl, pyrazolidinyl imidazolidinyl, thiomorpholinyl, and others.

As used herein, “bicyclic heterocyclyl” means a nonaromatic bicyclic ring system comprising at least one heteroatom in the ring system backbone. Bicyclic heterocyclyls may be substituted or unsubstituted with one or more substituents. In some embodiments, bicyclic heterocycles have 4-11 members with the heteroatom(s) being selected from one to five of 0, N, or S. Examples of bicyclic heterocyclyls include 2-azabicyclo[1.1.0]butane, 2-azabicyclo[2.1.0]pentane, 2-azabicyclo[1.1.1]pentane, 3-azabicyclo[3.1.0]hexane, 5-azabicyclo[2.1.1]hexane, 3-azabicyclo[3.2.0]heptane, octahydrocyclopenta[c]pyrrole, 3-azabicyclo[4.1.0]heptane, 7-azabicyclo[2.2.1]heptane, 6-azabicyclo[3.1.1]heptane, 7-azabicyclo[4.2.0]octane, 2-azabicyclo[2.2.2]octane, and the like.

As used herein, “spirocyclic heterocyclyl” means a nonaromatic bicyclic ring system comprising at least one heteroatom in the ring system backbone and with the rings connected through just one atom. Spirocyclic heterocyclyls may be substituted or unsubstituted with one or more substituents. In some embodiments, spirocyclic heterocycles have 5-11 members with the heteroatom(s) being selected from one to five of O, N, or S. Examples of spirocyclic heterocyclyls include 2-azaspiro[2.2]pentane, 4-azaspiro[2.5]octane, 1-azaspiro[3.5]nonane, 2-azaspiro[3.5]nonane, 7-azaspiro[3.5]nonane, 2-azaspiro[4.4]nonane, 6-azaspiro[2.6]nonane, 1,7-diazaspiro[4.5]decane, 2,5-diazaspiro[3.6]decane, and the like.

The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more non-hydrogen atoms of the molecule. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. Substituents can include, for example, —(C_1-9alkyl) optionally substituted with one or more of hydroxyl, —NH₂, —NH(C_1-3alkyl), and —N(C_1-3alkyl)₂; —(C_1-9haloalkyl); a halide; a hydroxyl; a carbonyl [such as —C(O)OR, and —C(O)R]; a thiocarbonyl [such as —C(S)OR, —C(O)SR, and —C(S)R]; —(C_1-9alkoxy) optionally substituted with one or more of halide, hydroxyl, —NH₂, —NH(C_1-3alkyl), and —N(C_1-3alkyl)₂; —OPO(OH)₂; a phosphonate [such as —PO(OH)₂and —PO(OR′)₂]; —OPO(OR′)R″; —NRR′; —C(O)NRR′; —C(NR)NR′R″; —C(NR′)R″; a cyano; a nitro; an azido; —SH; —S—R; —OSO₂(OR); a sulfonate [such as —SO₂(OH) and —SO₂(OR)]; —SO₂NR′R″; and —SO₂R; in which each occurrence of R, R′, and R″ are independently selected from H; —(C_1-9alkyl); C_6-10aryl optionally substituted with from 1-3R′″; 5-10 membered heteroaryl having from 1-4 heteroatoms independently selected from N, O, and S and optionally substituted with from 1-3 R′″; C₃_₇carbocyclyl optionally substituted with from 1-3 R′″; and 3-8 membered heterocyclyl having from 1-4 heteroatoms independently selected from N, O, and S, and optionally substituted with from 1-3 R′″; where each R′″ is independently selected from —(C_1-6alkyl), —(C_1-6haloalkyl), a halide (e.g., F), a hydroxyl, —C(O)OR, —C(O)R, —(C_1-6alkoxyl), —NRR′, —C(O)NRR′, and a cyano, in which each occurrence of R and R′ is independently selected from H and —(C_1-6alkyl). In some embodiments, the substituent is selected from —(C_1-6alkyl), —(C_1-6haloalkyl), a halide (e.g., F), a hydroxyl, —C(O)OR, —C(O)R, —(C_1-6alkoxyl), —NRR′, —C(O)NRR′, and a cyano, in which each occurrence of R and R′ is independently selected from H and —(C_1-6alkyl).

As used herein, when two groups are indicated to be “linked” or “bonded” to form a “ring,” it is to be understood that a bond is formed between the two groups and may involve replacement of a hydrogen atom on one or both groups with the bond, thereby forming a carbocyclyl, heterocyclyl, aryl, or heteroaryl ring. The skilled artisan will recognize that such rings can and are readily formed by routine chemical reactions. In some embodiments, such rings have from 3-7 members, for example, 5 or 6 members.

The skilled artisan will recognize that some chemical structures described herein may be represented on paper by one or more other resonance forms; or may exist in one or more other tautomeric forms, even when kinetically, the artisan recognizes that such tautomeric forms represent only a very small portion of a sample of such compound(s). Such compounds are clearly contemplated within the scope of this disclosure, though such resonance forms or tautomers are not explicitly represented herein.

The compounds provided herein may encompass various stereochemical forms. The compounds also encompass diastereomers as well as optical isomers, e.g., mixtures of enantiomers including racemic mixtures, as well as individual enantiomers and diastereomers, which arise as a consequence of structural asymmetry in certain compounds. Separation of the individual isomers or selective synthesis of the individual isomers is accomplished by application of various methods which are well known to practitioners in the art. Unless otherwise indicated, when a disclosed compound is named or depicted by a structure without specifying the stereochemistry and has one or more chiral centers, it is understood to represent all possible stereoisomers of the compound.

The present disclosure includes all pharmaceutically acceptable isotopically labeled compounds of Formulas (I)-(XII) wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature. Examples of isotopes suitable for inclusion in the compounds of the disclosure include, but are not limited to, isotopes of hydrogen, such as ²H (deuterium) and ³H (tritium), carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, such as 18F, iodine, such as ¹²³I and ¹²⁵I, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, and sulfur, such as ³⁵S.

The term “administration” or “administering” refers to a method of providing a dosage of a compound or pharmaceutical composition to a vertebrate or invertebrate, including a mammal, a bird, a fish, or an amphibian, where the method is, e.g., orally, subcutaneously, intravenously, intralymphatic, intranasally, topically, transdermally, intraperitoneally, intramuscularly, intrapulmonarilly, vaginally, rectally, ontologically, neuro-otologically, intraocularly, subconjuctivally, via anterior eye chamber injection, intravitreally, intraperitoneally, intrathecally, intracystically, intrapleurally, via wound irrigation, intrabuccally, intra-abdominally, intra-articularly, intra-aurally, intrabronchially, intracapsularly, intrameningeally, via inhalation, via endotracheal or endobronchial instillation, via direct instillation into pulmonary cavities, intraspinally, intrasynovially, intrathoracically, via thoracostomy irrigation, epidurally, intratympanically, intracisternally, intravascularly, intraventricularly, intraosseously, via irrigation of infected bone, or via application as part of any admixture with a prosthetic device. The method of administration can vary depending on various factors, e.g., the components of the pharmaceutical composition, the site of the disease, the disease involved, and the severity of the disease.

A “diagnostic” as used herein is a compound, method, system, or device that assists in the identification or characterization of a health or disease state. The diagnostic can be used in standard assays as is known in the art.

The term “mammal” is used in its usual biological sense. Thus, it specifically includes humans, cattle, horses, monkeys, dogs, cats, mice, rats, cows, sheep, pigs, goats, and non-human primates, but also includes many other species.

The term “pharmaceutically acceptable carrier”, “pharmaceutically acceptable diluent” or “pharmaceutically acceptable excipient” includes any and all solvents, co-solvents, complexing agents, dispersion media, coatings, isotonic and absorption delaying agents and the like which are not biologically or otherwise undesirable. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

Supplementary active ingredients can also be incorporated into the compositions. In addition, various adjuvants such as are commonly used in the art may be included. These and other such compounds are described in the literature, e.g., in the Merck Index, Merck & Company, Rahway, N.J. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Brunton et al. (Eds.) (2017); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 13th Ed., The McGraw-Hill Companies.

The term “pharmaceutically acceptable salt” refers to salts that retain the biological effectiveness and properties of the compounds provided herein and, which are not biologically or otherwise undesirable. In many cases, the compounds provided herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Many such salts are known in the art, for example, as described in WO 87/05297. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally-occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

The term “subject” is defined herein to include animals such as mammals, including but not limited to, mice, rats, rabbits, dogs, cats, horses, goats, sheep, pigs, goats, cows, primates (e.g., humans), and the like. In preferred embodiments, the subject is a human. In some embodiments of any of the methods described herein, a subject may be referred to as a patient. In some embodiments of any of the methods described herein, the subject is 1 year old or older, 5 years old or older, 10 years old or older, 15 years old or older, 18 years old or older, 20 years old or older, 25 years old or older, 30 years old or older, 35 years old or older, 40 years old or older, 45 years old or older, 50 years old or older, 55 years old or older, 60 years old or older, 65 years old or older, 70 years old or older, 75 years old or older, 80 years old or older, 85 years old or older, 90 years old or older, 95 years old or older, 100 years old or older, or 105 years old or older.

In some embodiments of any of the methods described herein, the subject has been previously diagnosed or identified as having a cancer (e.g., any of the types of cancer described herein or known in the art). In some embodiments of any of the methods described herein, the subject is suspected of having a cancer (e.g., any of the types of cancer described herein or known in the art). In some embodiments of any of the methods described herein, the subject is presenting with one or more (e.g., two, three, four, five, or six) symptoms of a cancer (e.g., any of the types of cancer described herein or known in the art). In some embodiments, the cancer can be selected from the group of: a small cell lung cancer, a colorectal cancer, a head and neck cancer, an ovarian cancer, a melanoma, a renal cell carcinoma, a pancreatic cancer, or a non-small cell lung cancer.

In some embodiments of any of the methods described herein, the subject is a participant in a clinical trial. In some embodiments, the subject has been previously administered a different pharmaceutical composition and the different pharmaceutical composition was determined not to be therapeutically effective.

A “therapeutically effective amount” of a compound as provided herein is one which is sufficient to achieve the desired physiological effect and may vary according to the nature and severity of the disease condition, and the potency of the compound. “Therapeutically effective amount” is also intended to include one or more of the compounds of Formulas (I)-(XII) in combination with one or more other agents that are effective to treat the diseases and/or conditions described herein. The combination of compounds can be a synergistic combination. Synergy, as described, for example, by Chou and Talalay, Advances in Enzyme Regulation (1984), 22, 27-55, occurs when the effect of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a single agent. In general, a synergistic effect is most clearly demonstrated at sub-optimal concentrations of the compounds. It will be appreciated that different concentrations may be employed for prophylaxis than for treatment of an active disease. This amount can further depend upon the patient's height, weight, sex, age and medical history.

A therapeutic effect relieves, to some extent, one or more of the symptoms of the disease.

“Treat,” “treatment,” or “treating,” as used herein refers administering a compound (e.g., any of the compounds described herein) or treatment to a patient already suffering from a disease thus causing a therapeutically beneficial effect, such as ameliorating one or more existing symptoms, ameliorating the underlying metabolic causes of symptoms, postponing the further development of a disorder, and/or reducing the severity of one or more symptoms that will or are expected to develop.

The phrase “an elevated” or “an increased level” as used herein can be an increase of at least 1% (e.g., at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, between 1% and 500%, between 1% and 450%, between 1% and 400%, between 1% and 350%, between 1% and 300%, between 1% and 250%, between 1% and 200%, between 1% and 180%, between 1% and 160%, between 1% and 140%, between 1% and 120%, between 1% and 100%, between 1% and 95%, between 1% and 90%, between 1% and 85%, between 1% and 80%, between 1% and 75%, between 1% and 70%, between 1% and 65%, between 1% and 60%, between 1% and 55%, between 1% and 50%, between 1% and 45%, between 1% and 40%, between 1% and 35%, between 1% and 30%, between 1% and 25%, between 1% and 20%, between 1% and 15%, between 1% and 10%, between 1% and 5%, between 5% and 500%, between 5% and 450%, between 5% and 400%, between 5% and 350%, between 5% and 300%, between 5% and 250%, between 5% and 200%, between 5% and 180%, between 5% and 160%, between 5% and 140%, between 5% and 120%, between 5% and 100%, between 5% and 95%, between 5% and 90%, between 5% and 85%, between 5% and 80%, between 5% and 75%, between 5% and 70%, between 5% and 65%, between 5% and 60%, between 5% and 55%, between 5% and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5% and 15%, between 5% and 10%, between 10% and 500%, between 10% and 450%, between 10% and 400%, between 10% and 350%, between 10% and 300%, between 10% and 250%, between 10% and 200%, between 10% and 180%, between 10% and 160%, between 10% and 140%, between 10% and 120%, between 10% and 100%, between 10% and 95%, between 10% and 90%, between 10% and 85%, between 10% and 80%, between 10% and 75%, between 10% and 70%, between 10% and 65%, between 10% and 60%, between 10% and 55%, between 10% and 50%, between 10% and 45%, between 10% and 40%, between 10% and 35%, between 10% and 30%, between 10% and 25%, between 10% and 20%, between 10% and 15%, between 20% and 500%, between 20% and 450%, between 20% and 400%, between 20% and 350%, between 20% and 300%, between 20% and 250%, between 20% and 200%, between 20% and 180%, between 20% and 160%, between 20% and 140%, between 20% and 120%, between 20% and 100%, between 20% and 95%, between 20% and 90%, between 20% and 85%, between 20% and 80%, between 20% and 75%, between 20% and 70%, between 20% and 65%, between 20% and 60%, between 20% and 55%, between 20% and 50%, between 20% and 45%, between 20% and 40%, between 20% and 35%, between 20% and 30%, between 20% and 25%, between 30% and 500%, between 30% and 450%, between 30% and 400%, between 30% and 350%, between 30% and 300%, between 30% and 250%, between 30% and 200%, between 30% and 180%, between 30% and 160%, between 30% and 140%, between 30% and 120%, between 30% and 100%, between 30% and 95%, between 30% and 90%, between 30% and 85%, between 30% and 80%, between 30% and 75%, between 30% and 70%, between 30% and 65%, between 30% and 60%, between 30% and 55%, between 30% and 50%, between 30% and 45%, between 30% and 40%, between 30% and 35%, between 40% and 500%, between 40% and 450%, between 40% and 400%, between 40% and 350%, between 40% and 300%, between 40% and 250%, between 40% and 200%, between 40% and 180%, between 40% and 160%, between 40% and 140%, between 40% and 120%, between 40% and 100%, between 40% and 95%, between 40% and 90%, between 40% and 85%, between 40% and 80%, between 40% and 75%, between 40% and 70%, between 40% and 65%, between 40% and 60%, between 40% and 55%, between 40% and 50%, between 40% and 45%, between 50% and 500%, between 50% and 450%, between 50% and 400%, between 50% and 350%, between 50% and 300%, between 50% and 250%, between 50% and 200%, between 50% and 180%, between 50% and 160%, between 50% and 140%, between 50% and 120%, between 50% and 100%, between 50% and 95%, between 50% and 90%, between 50% and 85%, between 50% and 80%, between 50% and 75%, between 50% and 70%, between 50% and 65%, between 50% and 60%, between 50% and 55%, between 60% and 500%, between 60% and 450%, between 60% and 400%, between 60% and 350%, between 60% and 300%, between 60% and 250%, between 60% and 200%, between 60% and 180%, between 60% and 160%, between 60% and 140%, between 60% and 120%, between 60% and 100%, between 60% and 95%, between 60% and 90%, between 60% and 85%, between 60% and 80%, between 60% and 75%, between 60% and 70%, between 60% and 65%, between 70% and 500%, between 70% and 450%, between 70% and 400%, between 70% and 350%, between 70% and 300%, between 70% and 250%, between 70% and 200%, between 70% and 180%, between 70% and 160%, between 70% and 140%, between 70% and 120%, between 70% and 100%, between 70% and 95%, between 70% and 90%, between 70% and 85%, between 70% and 80%, between 70% and 75%, between 80% and 500%, between 80% and 450%, between 80% and 400%, between 80% and 350%, between 80% and 300%, between 80% and 250%, between 80% and 200%, between 80% and 180%, between 80% and 160%, between 80% and 140%, between 80% and 120%, between 80% and 100%, between 80% and 95%, between 80% and 90%, between 80% and 85%, between 90% and 500%, between 90% and 450%, between 90% and 400%, between 90% and 350%, between 90% and 300%, between 90% and 250%, between 90% and 200%, between 90% and 180%, between 90% and 160%, between 90% and 140%, between 90% and 120%, between 90% and 100%, between 90% and 95%, between 100% and 500%, between 100% and 450%, between 100% and 400%, between 100% and 350%, between 100% and 300%, between 100% and 250%, between 100% and 200%, between 100% and 180%, between 100% and 160%, between 100% and 140%, between 100% and 120%, between 120% and 500%, between 120% and 450%, between 120% and 400%, between 120% and 350%, between 120% and 300%, between 120% and 250%, between 120% and 200%, between 120% and 180%, between 120% and 160%, between 120% and 140%, between 140% and 500%, between 140% and 450%, between 140% and 400%, between 140% and 350%, between 140% and 300%, between 140% and 250%, between 140% and 200%, between 140% and 180%, between 140% and 160%, between 160% and 500%, between 160% and 450%, between 160% and 400%, between 160% and 350%, between 160% and 300%, between 160% and 250%, between 160% and 200%, between 160% and 180%, between 180% and 500%, between 180% and 450%, between 180% and 400%, between 180% and 350%, between 180% and 300%, between 180% and 250%, between 180% and 200%, between 200% and 500%, between 200% and 450%, between 200% and 400%, between 200% and 350%, between 200% and 300%, between 200% and 250%, between 250% and 500%, between 250% and 450%, between 250% and 400%, between 250% and 350%, between 250% and 300%, between 300% and 500%, between 300% and 450%, between 300% and 400%, between 300% and 350%, between 350% and 500%, between 350% and 450%, between 350% and 400%, between 400% and 500%, between 400% and 450%, or about 450% to about 500%), e.g., as compared to a reference level (e.g., any of the exemplary reference levels described herein).

As used herein, a “first time point” can, e.g., refer to a designated time point, which can, e.g., be used to refer to chronologically later time points (e.g., a second time point). In some examples, a subject may not have yet received a treatment at a first time point (e.g., may not have yet received a dose of a CLK inhibitor (e.g., any of the CLK inhibitors described herein) at a first time point). In some examples, a subject may have already received a treatment that does not include a CLK inhibitor at the first time point. In some examples, the previous treatment that does not include a CLK inhibitor was identified as being ineffective prior to the first time point. In some examples, a subject has previously been identified or diagnosed as having a cancer (e.g., any of the types of cancer described herein or known in the art) at the first time point. In some examples, a subject has previously been suspected of having a cancer (e.g., any of the types of cancer described herein or known in the art) at the first time point. In other examples, a first time point can be a time point when a subject has developed at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) symptom(s) associated with a cancer and has not yet received any treatment for cancer.

As used herein, a “second time point” refers to a time point that occurs chronologically after a first designated time point. In some examples, a subject (e.g., any of the subjects described herein) can receive or has received at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) doses of a treatment (e.g., a CLK inhibitor (e.g., any of the CLK inhibitors described herein)) between the first and the second time points. In some examples, the time difference between a first and a second time point can be, e.g., 1 day to about 12 months, 1 day to about 11 months, 1 day to about 10 months, 1 day to about 9 months, 1 day to about 8 months, 1 day to about 7 months, 1 day to about 6 months, 1 day to about 22 weeks, 1 day to about 20 weeks, 1 day to about 18 weeks, 1 day to about 16 weeks, 1 day to about 14 weeks, 1 day to about 12 weeks, 1 day to about 10 weeks, 1 day to about 8 weeks, 1 day to about 6 weeks, 1 day to about 4 weeks, 1 day to about 3 weeks, 1 day to about 2 weeks, 1 day to about 1 week, about 2 days to about 12 months, about 2 days to about 11 months, about 2 days to about 10 months, about 2 days to about 9 months, about 2 days to about 8 months, about 2 days to about 7 months, about 2 days to about 6 months, about 2 days to about 22 weeks, about 2 days to about 20 weeks, about 2 days to about 18 weeks, about 2 days to about 16 weeks, about 2 days to about 14 weeks, about 2 days to about 12 weeks, about 2 days to about 10 weeks, about 2 days to about 8 weeks, about 2 days to about 6 weeks, about 2 days to about 4 weeks, about 2 days to about 3 weeks, about 2 days to about 2 weeks, about 2 days to about 1 week, about 4 days to about 12 months, about 4 days to about 11 months, about 4 days to about 10 months, about 4 days to about 9 months, about 4 days to about 8 months, about 4 days to about 7 months, about 4 days to about 6 months, about 4 days to about 22 weeks, about 4 days to about 20 weeks, about 4 days to about 18 weeks, about 4 days to about 16 weeks, about 4 days to about 14 weeks, about 4 days to about 12 weeks, about 4 days to about 10 weeks, about 4 days to about 8 weeks, about 4 days to about 6 weeks, about 4 days to about 4 weeks, about 4 days to about 3 weeks, about 4 days to about 2 weeks, about 4 days to about 1 week, about 1 week to about 12 months, about 1 week to about 11 months, about 1 week to about 10 months, about 1 week to about 9 months, about 1 week to about 8 months, about 1 week to about 7 months, about 1 week to about 6 months, about 1 week to about 22 weeks, about 1 week to about 20 weeks, about 1 week to about 18 weeks, about 1 week to about 16 weeks, about 1 week to about 14 weeks, about 1 week to about 12 weeks, about 1 week to about 10 weeks, about 1 week to about 8 weeks, about 1 week to about 6 weeks, about 1 week to about 4 weeks, about 1 week to about 3 weeks, about 1 week to about 2 weeks, about 2 weeks to about 12 months, about 2 weeks to about 11 months, about 2 weeks to about 10 months, about 2 weeks to about 9 months, about 2 weeks to about 8 months, about 2 weeks to about 7 months, about 2 weeks to about 6 months, about 2 weeks to about 22 weeks, about 2 weeks to about 20 weeks, about 2 weeks to about 18 weeks, about 2 weeks to about 16 weeks, about 2 weeks to about 14 weeks, about 2 weeks to about 12 weeks, about 2 weeks to about 10 weeks, about 2 weeks to about 8 weeks, about 2 weeks to about 6 weeks, about 2 weeks to about 4 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 12 months, about 3 weeks to about 11 months, about 3 weeks to about 10 months, about 3 weeks to about 9 months, about 3 weeks to about 8 months, about 3 weeks to about 7 months, about 3 weeks to about 6 months, about 3 weeks to about 22 weeks, about 3 weeks to about 20 weeks, about 3 weeks to about 18 weeks, about 3 weeks to about 16 weeks, about 3 weeks to about 14 weeks, about 3 weeks to about 12 weeks, about 3 weeks to about 10 weeks, about 3 weeks to about 8 weeks, about 3 weeks to about 6 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 12 months, about 4 weeks to about 11 months, about 4 weeks to about 10 months, about 4 weeks to about 9 months, about 4 weeks to about 8 months, about 4 weeks to about 7 months, about 4 weeks to about 6 months, about 4 weeks to about 22 weeks, about 4 weeks to about 20 weeks, about 4 weeks to about 18 weeks, about 4 weeks to about 16 weeks, about 4 weeks to about 14 weeks, about 4 weeks to about 12 weeks, about 4 weeks to about 10 weeks, about 4 weeks to about 8 weeks, about 4 weeks to about 6 weeks, about 6 weeks to about 12 months, about 6 weeks to about 11 months, about 6 weeks to about 10 months, about 6 weeks to about 9 months, about 6 weeks to about 8 months, about 6 weeks to about 7 months, about 6 weeks to about 6 months, about 6 weeks to about 22 weeks, about 6 weeks to about 20 weeks, about 6 weeks to about 18 weeks, about 6 weeks to about 16 weeks, about 6 weeks to about 14 weeks, about 6 weeks to about 12 weeks, about 6 weeks to about 10 weeks, about 6 weeks to about 8 weeks, about 8 weeks to about 12 months, about 8 weeks to about 11 months, about 8 weeks to about 10 months, about 8 weeks to about 9 months, about 8 weeks to about 8 months, about 8 weeks to about 7 months, about 8 weeks to about 6 months, about 8 weeks to about 22 weeks, about 8 weeks to about 20 weeks, about 8 weeks to about 18 weeks, about 8 weeks to about 16 weeks, about 8 weeks to about 14 weeks, about 8 weeks to about 12 weeks, about 8 weeks to about 10 weeks, about 10 weeks to about 12 months, about 10 weeks to about 11 months, about 10 weeks to about 10 months, about 10 weeks to about 9 months, about 10 weeks to about 8 months, about 10 weeks to about 7 months, about 10 weeks to about 6 months, about 10 weeks to about 22 weeks, about 10 weeks to about 20 weeks, about 10 weeks to about 18 weeks, about 10 weeks to about 16 weeks, about 10 weeks to about 14 weeks, about 10 weeks to about 12 weeks, about 12 weeks to about 12 months, about 12 weeks to about 11 months, about 12 weeks to about 10 months, about 12 weeks to about 9 months, about 12 weeks to about 8 months, about 12 weeks to about 7 months, about 12 weeks to about 6 months, about 12 weeks to about 22 weeks, about 12 weeks to about 20 weeks, about 12 weeks to about 18 weeks, about 12 weeks to about 16 weeks, about 12 weeks to about 14 weeks, about 14 weeks to about 12 months, about 14 weeks to about 11 months, about 14 weeks to about 10 months, about 14 weeks to about 9 months, about 14 weeks to about 8 months, about 14 weeks to about 7 months, about 14 weeks to about 6 months, about 14 weeks to about 22 weeks, about 14 weeks to about 20 weeks, about 14 weeks to about 18 weeks, about 14 weeks to about 16 weeks, about 16 weeks to about 12 months, about 16 weeks to about 11 months, about 16 weeks to about 10 months, about 16 weeks to about 9 months, about 16 weeks to about 8 months, about 16 weeks to about 7 months, about 16 weeks to about 6 months, about 16 weeks to about 22 weeks, about 16 weeks to about 20 weeks, about 16 weeks to about 18 weeks, about 18 weeks to about 12 months, about 18 weeks to about 11 months, about 18 weeks to about 10 months, about 18 weeks to about 9 months, about 18 weeks to about 8 months, about 18 weeks to about 7 months, about 18 weeks to about 6 months, about 18 weeks to about 22 weeks, about 18 weeks to about 20 weeks, about 20 weeks to about 12 months, about 20 weeks to about 11 months, about 20 weeks to about 10 months, about 20 weeks to about 9 months, about 20 weeks to about 8 months, about 20 weeks to about 7 months, about 20 weeks to about 6 months, about 20 weeks to about 22 weeks, about 22 weeks to about 12 months, about 22 weeks to about 11 months, about 22 weeks to about 10 months, about 22 weeks to about 9 months, about 22 weeks to about 8 months, about 22 weeks to about 7 months, about 22 weeks to about 6 months, about 24 weeks to about 12 months, about 24 weeks to about 11 months, about 24 weeks to about 10 months, about 24 weeks to about 9 months, about 24 weeks to about 8 months, about 24 weeks to about 7 months, about 7 months to about 12 months, about 7 months to about 11 months, about 7 months to about 10 months, about 7 months to about 9 months, about 7 months to about 8 months, about 8 months to about 12 months, about 8 months to about 11 months, about 8 months to about 10 months, about 8 months to about 9 months, about 9 months to about 12 months, about 9 months to about 11 months, about 9 months to about 10 months, about 10 months to about 12 months, about 10 months to about 11 months, or about 11 months to about 12 months.

“Drug-eluting” and/or controlled release as used herein refers to any and all mechanisms, e.g., diffusion, migration, permeation, and/or desorption by which the drug(s) incorporated in the drug-eluting material pass therefrom over time into the surrounding body tissue.

“Drug-eluting material” and/or controlled release material as used herein refers to any natural, synthetic or semi-synthetic material capable of acquiring and retaining a desired shape or configuration and into which one or more drugs can be incorporated and from which incorporated drug(s) are capable of eluting overtime.

“Elutable drug” as used herein refers to any drug or combination of drugs having the ability to pass over time from the drug-eluting material in which it is incorporated into the surrounding areas of the body.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

Other features and advantages of the disclosure will be apparent from the following detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a graph showing the percent inhibition using CLK2, CLK3, and CDK1 kinase IC₅₀s as determined by the Thermo Fisher Scientific Z-LYTE™ platform. Inhibitory concentration (IC₅₀) values were determined from dose response curves from n=4 experiments.

FIG. 1B is a kinase dendrogram of Compound 12. Kinases with IC₅₀values 0.01-0.05 μM are marked by small circles, whereas larger circles represent more potent IC₅₀s of 0.001-0.01 μM.

FIG. 1C is a graph showing normalized luciferase activity in SW480 colon cancer cells stably expressing the Wnt-responsive TOPflash or the control luciferase reporter under the EFla promoter and treated with Compound 12 following an 8-point dose response. Luciferase activities were measured using Bright-Glo™. Data represent the mean of two or three replicates±standard error of mean (SEM).

FIG. 1D are graphs showing Wnt pathway gene expression (AXIN2 and LEF1) in HEK-293T cells treated with Compound 12 or PRI-724 at the indicated doses for 1 hour before stimulation with Wnt3a (200 ng/mL). Fold-change in gene expression relative to unstimulated DMSO (n=3 biological replicates, Mean±SD, **P<0.01, ***P<0.001, ****P<0.0001, unpaired student's t-test vs. stimulated DMSO).

FIG. 1E are graphs showing Wnt pathway gene expression (AXIN2 and LEF1) in HEK-293T cells treated with Compound 12 or PRI-724 at the indicated doses for 1 hour before stimulation with CHIR99021 (4 μM) for 20 hours. Fold-change in gene expression relative to unstimulated DMSO (n=3 biological replicates, Mean±SD, **P<0.01, ***P<0.001, ****P<0.0001, unpaired student's t-test vs. stimulated DMSO).

FIG. 2A is a graph showing the percent activity in SW480 cells treated with a 3-fold, 10-point titration of Compound 12 or PRI-724 (0.0005-10 μM) for ˜48 hrs. Data is representative from three independent assays performed in quadruplicate.

FIG. 2B is a graph showing LGR5 gene expression in IEC-6 rat small intestinal cells treated with Compound 12 or PRI-724 at various doses and stimulated with Wnt3a for 16 h. The fold-change relative to unstimulated DMSO is shown (n=3, Mean±SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, unpaired student's t-test vs. ligand). Data is representative from two independent assays.

FIG. 2C is a graph showing LGR5 gene expression in IEC-6 cells treated with Compound 12 or PRI-724 at various doses and stimulated with CHIR99021 for 16 h. Fold-change relative to unstimulated DMSO is shown (n=3, Mean±SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, unpaired student's t-test vs. ligand). Data is representative from two independent assays.

FIG. 3A is a set of immunofluorescent images of SW480 cells treated with Compound 12 at test concentration 3, 1, 0.3, 0.1, and 0.03 μM with Compound 12, or with Staurosporine at 0.1 M, and stained with the CellEvent™ Caspase 3/7 Green Detection Reagent to detect activated caspase 3/7 (green) and with Hoechst 33342 to stain nuclei (blue). Images are representative of two independent assays.

FIG. 3B is a bar graph showing the percent of the total number of cells containing active caspase 3/7 following exposure to 3, 1, or 0.3 μM Compound 12 for 48 hours. Data is representative of two independent assays (n=3 biological replicates, Mean±standard deviation (SD), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, unpaired student's t-test).

FIG. 3C is an immunoblot showing survivin, MCL-1, and cleaved PARP protein expression in SW480 cells following treatment with Compound 12 at test concentrations of 10, 3, 1, 0.3, 0.1, or 0.03 μM for 48 hours. R-actin was used as the loading control. Data is representative of two independent assays.

FIG. 4 is an image of SW480 cells treated with Compound 12 at test concentrations of 1, 0.3, 0.1, or 0.03 μM for 72 hours on a 2% agarose gel with a GelRed nucleic acid stain visualized on a UV transilluminator. Cells were also treated with Staurosporine at 1 μM for 24 hours as a positive control. Image shown is from one experiment and is representative of data from two independent assays.

FIG. 5 is an immunoblot showing cytoplasmic and nuclear localization of CLK1 (˜57 kDa), CLK2 (˜60 kDa), CLK3 (˜59 kDa), and CLK4 (˜62 kDa) in SW480 CRC cells. Protein lysates from untreated SW480 cells were separated into nuclear and cytoplasmic fractions. The Western blots were performed with antibodies for CLK1, CLK2, CLK3, and CLK4. β-actin was used as a loading control.

FIG. 6A is an immunoblot showing phosphorylated SRSF6 and SRSF5 in SW480 cells treated as indicated for 1 hour. Total SRSF5 and R-actin blots were used as loading controls. The blots are representative of two experiments.

FIG. 6B is a set of representative immunofluorescence images (×100 magnification) from SW480 cells treated with Compound 12 as indicated for 6 hours. The cells were stained with a phospho-SC35 antibody (green) and a Hoechst 33342 nuclear stain (blue). Scale bar, 10 μm.

FIG. 6C is a set of bar graphs showing qRT-PCR analysis of Wnt pathway genes AXIN2, CTNNB1, LEF1, MYC, TCF7, and TCF7L2 in SW480 cells treated with Compound 12 at indicated concentrations for 24 hours. Fold-change relative to DMSO is shown (n=3 biological replicates per group, Mean±SD, **P<0.01; ***P<0.001, student's two-tailed t-test).

FIG. 6D is an immunoblot showing Wnt pathway-related protein expression in SW480 cells treated as indicated for 24 hours. The proteins were separated into nuclear and cytoplasmic fractions. GAPDH and Lamin B1 represent cytoplasmic and nuclear protein loading controls, respectively. The blots are representative of two experiments.

FIG. 6E is an immunoblot showing Wnt pathway-related protein expression in SW480 cells treated as indicated for 48 hours. The proteins were separated into nuclear and cytoplasmic fractions. GAPDH and Lamin B1 represent cytoplasmic and nuclear protein loading controls, respectively. The blots are representative of two experiments.

FIG. 7A is a graph showing the effects of Compound 12 on Nanostring nCounter® Wnt pathway gene array. Seventeen different CRC cell lines (COLO 320 HSR, C₂BBel, HuTu 80, COLO 205, SQ1417, HT29, RKO, HCT 15, SW620, DLD-1, LoVo, LS123, T84, SW480, LS513, and HCT 116) were treated with 1 μM of Compound 12for 20-24 hrs. Diagonal lines indicating 2-fold changes are shown for both upregulated (blue) and downregulated (red) genes. The genes with absolute fold-changes greater than 2 and significant (FDR adjusted p<0.05) have labels highlighted in green.

FIG. 7B are bar graphs showing qRT-PCR analysis of the top gene hits from FIG. 7A in SW480 cells treated with Compound 12 for 24 hours. The fold-change relative to DMSO is shown (n=3 biological replicates per group, Mean±SD, *P<0.05; **P<0.01; ***P<0.001, student's two-tailed t-test).

FIG. 7C is an immunoblot showing protein expression of hits identified in FIG. 7A. SW480 cells were treated as indicated for 24 hours and proteins were separated into nuclear and cytoplasmic fractions. GAPDH and Lamin B1 represent the cytoplasmic and nuclear protein loading controls, respectively. The blots are representative of two experiments.

FIG. 8A is an immunoblot showing SRSF6 protein expression in SW480 cells treated with Nontarget, SRSF5, or SRSF6 siRNA for 5 days. R-actin is a loading control. Blots are representative of two experiments.

FIG. 8B is an immunoblot showing SRSF5 protein expression in SW480 cells treated with Nontarget, SRSF5 or SRSF6 siRNA for 5 days. R-actin was used as a loading control. The blots are representative of two experiments.

FIG. 8C is an immunoblot showing phospho-SRSF protein expression in SW480 cells treated with Nontarget, SRSF5, or SRSF6 siRNA for 5 days. -actin was used as a loading control. The blots are representative of two experiments.

FIG. 8D is an immunoblot showing phospho-SR protein expression in SW480 cells treated with Nontarget, SRSF6 siRNA for 5 days. -actin was used as a loading control. The blots are representative of two experiments.

FIG. 9A is a set of representative immunofluorescence images (×100 magnification) of SW480 cells treated with indicated concentrations for 6 hours. The cells were stained with a phospho-SC35 antibody (green) and a Hoechst 33342 nuclear stain (blue). Scale bar, 10 μm.

FIG. 9B is two graphs showing percent activity (left) and cell viability (right) of SW480 cells treated with a 3-fold 10-point titration of doses of Compound 12, CC-671, or Harmine (0.0005-10 μM) for 48 hrs (Wnt reporter assay) or 4 days (cell viability assay). The data is representative from three independent assays performed in quadruplicate.

FIG. 9C is a set of bar graphs showing qRT-PCR analysis of Wnt pathway genes in SW480 cells treated with Compound 12 for 24 hours. The data are presented as Mean±SD (n=3 biological replicates per group. *P<0.05; **P<0.01; ***P<0.001, student's two-tailed t test).

FIG. 10A is set of bar graphs showing qRT-PCR analysis of top gene hits from the Nanostring assay in SW480 cells treated with Compound 12 for 24 hours. The data are presented as Mean±SD (n=3 biological replicates per group, ***P<0.001, student's two-tailed t test).

FIG. 10B is an immunoblot of the indicated proteins in cytoplasmic and nuclear fractions from SW480 cells. GAPDH blot is a cytoplasmic loading control and Lamin B1 blot is a nuclear loading control. The blots are representative of two experiments.

FIG. 11A is a bar graph showing CTNNB1 gene expression in SW480 cells treated with Nontarget, or CTN/pBI siRNA for 5 days. The fold-change relative to Nontarget control is shown (n=3 biological replicates per group, Mean±SD, **P<0.01; ***P<0.001, student's two-tailed t-test).

FIG. 11B is a bar graph showing CLK2 gene expression in SW480 cells treated with Nontarget or CLK2 siRNA for 5 days. The fold-change relative to Nontarget control is shown (n=3 biological replicates per group, Mean±SD, **P<0.01; ***P<0.001, student's two-tailed t-test).

FIG. 11C is a bar graph showing CLK3 gene expression in SW480 cells treated with Nontarget or CLK3 siRNA for 5 days. The fold-change relative to Nontarget control is shown (n=3 biological replicates per group, Mean±SD, **P<0.01; ***P<0.001, student's two-tailed t-test).

FIG. 11D is an immunoblot showing CLK2, CLK3, and β-catenin protein expression in siRNA-treated cells. R-actin was used as a loading control.

FIG. 11E is an immunoblot showing protein expression of phosphorylated and total SRSF6 in siRNA-treated cells. R-actin was used as a loading control.

FIG. 11F is a bar graph showing analysis of the TOPflash reporter activity of SW480 cells treated for 5 days as indicated.

FIG. 11G is a bar graph showing cell viability of SW480 cells treated for 5 days as indicated.

FIG. 11H is a set of bar graphs showing qRT-PCR analysis of Wnt pathway-related genes (AXIN2, BTRC, DVL2, LEF1, LRP5, MYC, TCF7, and TCFL2) in siRNA-treated SW480 cells. The fold-change relative to Nontarget control is shown (n=3 biological replicates per group, Mean SD, *P<0.05; **P<0.01; ***P<0.001, student's two-tailed t-test).

FIG. 11I is an immunoblot of nuclear and cytoplasmic-fractionated protein of genes identified in FIG. 11H in siRNA-treated SW480 cells. GAPDH, Lamin B1, and R-actin were used as loading controls. Each panel is representative of three independent experiments.

FIG. 12 is an immunoblot of cytoplasmic and nuclear protein from SW480 cells for CLK1. GAPDH blot was used as a cytoplasmic loading control and Lamin B1 blot was used as a nuclear loading control. The blots are representative of two experiments (n=3 biological replicates per group).

FIG. 13 is a set of bar graphs showing qRT-PCR analysis for LRP6, MAPK8, BTRC, and FRZB in SW480-TOPflash cells treated with Nontarget, CTNNB1, CLK2, or CLK3 siRNA for 5 days. The fold-change relative to DMSO is shown (n=3 biological replicates per group, Mean SD, *P<0.05; **P<0.01; ***P<0.001, student's two-tailed t-test).

FIG. 14A is an immunoblot showing nuclear protein expression of CLK3, CLK2, and CLK1 in CLK3-CRISPR clonal cell lines. Lamin B1 was used as a loading control. The blots are representative of two experiments.

FIG. 14B is an immunoblot showing phosphorylated and total SRSF6 in WT and CLK3 KO SW480 clonal cells. The blots are representative of two experiments.

FIG. 14C is a bar graph showing MYC gene expression levels in CLK3 CRISPR clonal cell lines as determined by qRT-PCR. The fold-change relative to Cas9 WT (n=5 replicates per each group, Mean±SEM, **P<0.01; ****P<0.0001, student's two-tailed t-test).

FIG. 14D is an immunoblot for nuclear protein MYC in CLK3 CRISPR clonal cell lines. Lamin B1 was used as a loading control. The blots are representative of two experiments. The relative band intensity of MYC was determined after normalization with each Lamin B1 band and averaging WT and CLK3 KO clones (Mean±SEM, *P<0.05, student's two-tailed t-test).

FIG. 14E is a graph showing tumor growth curves of SW480 xenografts injected with WT, CLK3 KO clone 3, or CLK3 KO clone 5 cells. Tumor volumes were measured twice per week. Data presented as Mean±SEM (n=8-10 mice per group, ****P<0.0001, student's two-tailed t-test).

FIG. 14F are representative images of tumor pictures of WT and CLK3 KO clonal SW480 tumors at the end of study (day 28).

FIG. 14G is a bar graph showing CLK3 gene expression levels in WT and CLK3 KO clonal SW480 tumors at day 28 as determined by qRT-PCR. The data are presented as Mean±SEM (n=7-10 mice per each group. ****P<0.0001, student's two-tailed t-test).

FIG. 14H is an immunoblot for MYC in WT and CLK3 KO SW480 tumors collected at day 28. β-actin was used as a loading control. The relative band intensity of MYC was determined after normalization with each β-actin band and averaging WT and each CLK3 KO clonal tumors. The data are presented as Mean±SEM (*P<0.05, student's two-tailed t-test).

FIG. 15A is a graph showing cell growth of WT SW480 cells and CLK3 KO cells cultured in 10% FBS. BrdU cell proliferation ELISA was performed at day 4 and day 6 or 7 after plating the cells. The data are presented as Mean±SEM (n=3-10 biological replicates per group, ***P<0.001; ****P<0.0001, student's two-tailed t-test vs. WT).

FIG. 15B is a graph showing cell viability of WT SW480 cells and CLK3 KO cells cultured in 10% FBS. The data are presented as Mean±SEM (n=3-10 biological replicates per group, ***P <0.001; ****P<0.0001, student's two-tailed t-test vs. WT).

FIG. 15C is a graph showing cell growth of WT SW480 cells and CLK3 KO cells cultured in 1% FBS. BrdU cell proliferation ELISA was performed at day 4 and day 6 or 7 after plating the cells. The cells were adjusted to the low serum condition for two weeks before assays. CellTiter-Glo® luminescent cell viability assays were performed at day 4 and day 6 or 7 after plating the cells. The data are presented as Mean±SEM (n=3-10 biological replicates per group, ***P<0.001; ****P<0.0001, student's two-tailed t-test vs. WT).

FIG. 15D is a graph showing cell viability of WT SW480 cells and CLK3 KO cells cultured in 1% FBS. The cells were adjusted to the low-serum condition for two weeks before assays. CellTiter-Glo® luminescent cell viability assays were performed at day 4 and day 6 or 7 after plating the cells. The data are presented as Mean±SEM (n=3-10 biological replicates per group, ***P<0.001; ****P<0.0001, student's two-tailed t-test vs. WT).

FIG. 15E is a set of representative images of WT or CLK3 KO cells cultured for 5 days in 10% FBS media. The images are representative of data from two independent assays.

FIG. 15F are representative images of WT or CLK3 KO cells cultured for 5 days in 1% FBS media. The images are representative of data from two independent assays.

FIG. 16 is a graph showing mean plasma concentration versus time profiles of Compound 12. Following a single Intravenous (IV) Bolus or Oral (PO) Dose to Male Balb/c Mice, approximately 0.1 mL whole blood was collected via the cheek vein (submandibular) according to an alternate bleeding schedule (n=3/time point/route) at 0.083 (IV), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-dose into tubes containing K₂EDTA anticoagulant and plasma was harvested by centrifugation.

FIG. 17A is a graph showing tumor volume in SW480 tumor-bearing mice that were orally administered doses of Compound 12, 6.25-25 mg/kg, at the indicated frequencies (QD=daily; QOD=every other day). Dosing was initiated when tumors were 100-200 mm³in size and measurements performed twice per week. The data are presented as Mean±SEM (n=6-7 mice per each group. *P<0.05, student's t-test).

FIG. 17B is a graph showing tumor volume in HCT-116 tumor-bearing mice that were orally administered doses of Compound 12, 6.25-25 mg/kg, at the indicated frequencies (QD=daily; QOD=every other day). Dosing was initiated when tumors were 100-200 mm3 in size and measurements performed twice per week. The data are presented as Mean±SEM (n=6-7 mice per each group. *P<0.05, student's t-test).

FIG. 17C is a graph showing tumor volume in PDX-CR2545 (Crown Biosciences) tumor-bearing mice that were orally administered doses of Compound 12, 6.25-25 mg/kg, at the indicated frequencies (QD=daily; QOD=every other day). Dosing was initiated when tumors were 100-200 mm3 in size and measurements performed twice per week. The data are presented as Mean±SEM (n=6-7 mice per each group. *P<0.05, student's t-test).

FIG. 17D is an immunoblot showing tumor pharmacodynamics in athymic nude mice bearing SW480 tumors. After a single dose of Compound 12, tumors were harvested at 4, 8, and 24 hours and the effect on SR phosphorylation was evaluated, with total SRSF6, total SRSF5, and R-actin used as loading controls.

FIG. 17E is a set of graphs showing qRT-PCR analysis of Wnt pathways genes on RNA extracted from the SW480 tumors. The fold-change relative to vehicle is shown (n=3 biological replicates per group, Mean±SD, *P<0.05; **P<0.01; ***P<0.001, student's two-tailed t-test).

FIG. 18A is a graph showing the effect of Compound 12 on body weight in CRC-SW480 tumor-bearing athymic nude mice. CRC tumor xenograft-bearing mice were administered Compound 12 or vehicle by oral administration at the indicated doses and frequencies starting on day 0. Body weights in grams (g) were determined every 3-4 days. The data are presented as Mean±SEM (n=6-7 mice per each group.

*P<0.05, student's t-test). The percent body weight change represents the total change in body weight relative to the baseline body weight on day 0 prior to the first dose.

FIG. 18B is a graph showing the effect of Compound 12 on body weight in CRC-HCT116 tumor-bearing athymic nude mice. CRC tumor xenograft-bearing mice were administered Compound 12 or vehicle by oral administration at the indicated doses and frequencies starting on day 0. The body weights in grams (g) were determined every 3-4 days. The data are presented as Mean±SEM (n=6-7 mice per each group. *P<0.05, student's t-test). The percent body weight change represents the total change in body weight relative to the baseline body weight on day 0 prior to the first dose.

FIG. 18C is a graph showing the effect of Compound 12 on body weight in CRC-PDX CR2545 (Crown Biosciences) tumor-bearing Balb/c nude female mice. CRC tumor xenograft-bearing mice were administered Compound 12 or vehicle by oral administration at the indicated doses and frequencies starting on day 0. The body weights in grams (g) were determined every 3-4 days. The data are presented as Mean±SEM (n=6-7 mice per each group. *P<0.05, student's t-test). The percent body weight change represents the total change in body weight relative to the baseline body weight on day 0 prior to the first dose.

FIG. 19 is a set of bar graphs showing qRT-PCR analysis of central Wnt pathway genes on RNA extracted from SW480 tumors isolated 4, 8, and 24 hours after SW480 tumor-bearing athymic nude mice were given a single dose of Compound 12, 25 mg/kg. The data are presented as Mean SD (n=3 biological replicates per group, *P<0.05; ***P<0.001, student's two-tailed t-test).

FIG. 20 is a graph showing tumor volume in NCI-N87 GC tumor xenograft-bearing mice that were administered Compound 12 or vehicle by oral administration at the indicated doses and frequencies starting on day 0 to day 21 (22-day dosing period). QD=daily; QOD=every other day.

The tumor volumes were measured twice a week. Each data point represents Mean±SEM (n=7 mice per group, *P<0.05, student's two-tailed t-test).

FIGS. 21A-O are boxplots representing the distribution of log 2FC values for each compound across multiple cell lines. Compounds on the x-axis are sorted by average viability EC₅₀across 50 cell lines (See Table 18), and each graph represents a single gene biomarker. A significant regression model (p<0.05) suggests gene expression differences are correlated with compound efficacy. Gene biomarkers represented are FIG. 21A, APC; FIG. 21B, TIAM1; FIG. 21C, CSNK2A1; FIG. 21D, CTGF; FIG. 21E, DVL2; FIG. 21F, FRZB; FIG. 21G, FZD6; FIG. 21H, GSK3B; FIG. 21I, HDAC3; FIG. 21J, LRP5; FIG. 21K, MYC; FIG. 21L, PLCB4; FIG. 21M, RUVBL1; FIG. 21N, SRSF5; and FIG. 21O, TCF7.

DETAILED DESCRIPTION

The present disclosure is based on the discovery that Compound 12, a CDC-like kinase (CLK) inhibitor, modulates mRNA splicing in mammalian cells and downregulates Wnt signaling activity in cancer cells. In view of these discoveries, provided herein are methods of treating a cancer in a subject, methods of selecting a treatment for a subject, methods of selecting a subject for treatment, and methods of selecting a subject for participation in a clinical trial, that each include identifying a subject having a cancer cell (e.g., any of the types of cancer cell described herein) that has an elevated level of Wnt pathway activity as compared to a reference level. Also provided herein are methods of determining the efficacy of a CLK inhibitor in a subject that include detecting a level of Wnt/β-catenin signaling activity in a cancer cell obtained from the subject. Also provided are methods of decreasing the activity of one or more of CLK1, CLK2, CLK3, and CLK4 (e.g., in vitro or in a mammalian cell) that include the use of any of the CLK inhibitors or pharmaceutically acceptable salts or solvates thereof described herein. Also provided herein are methods of alternative mRNA splicing in a mammalian cell having aberrant mRNA splicing activity that include the use of any of the CLK inhibitors or pharmaceutically acceptable salts or solvates thereof described herein. Also provided herein are methods of treating a cancer using a CLK inhibitor, methods of selecting a treatment including a CLK inhibitor for a subject, methods of selecting a subject for treatment with a CLK inhibitor, and methods of selecting a subject for participation in a clinical trial, that each include the use of a CLK inhibitor, that include a step of identifying a subject having aberrant mRNA splicing activity.

Non-limiting aspects of these methods are described below and can be used in any combination without limitation. Additional aspects of these methods are known in the art.

Methods of Treating—Type A

Provided herein are methods of treating a cancer (e.g., any of the exemplary cancers described herein or known in the art) in a subject that include: identifying a subject having a cancer cell that has an elevated level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of Wnt pathway activity (e.g., any of the exemplary types of Wnt pathway activity described herein or known in the art) as compared to a reference level (e.g., any of the exemplary reference levels described herein); and administering to the identified subject a therapeutically effective amount of a CLK inhibitor as well as prodrugs and pharmaceutically acceptable salt or solvate thereof (e.g., any of the exemplary CLK inhibitors described herein or known in the art).

Also provided herein are methods of treating a cancer in a subject that include: administering a therapeutically effective amount of a CLK inhibitor as well as prodrugs and pharmaceutically acceptable salt or solvate thereof (e.g., any of the exemplary CLK inhibitors described herein or known in the art) to a subject (e.g., any of the subjects described herein) identified as having a cancer cell that has an elevated level (e.g., an increase of 1% to about 500%, or any of the subranges of this range described herein) of Wnt pathway activity (e.g., any of the exemplary types of Wnt pathway activity described herein or known in the art) as compared to a reference level (e.g., any of the exemplary reference levels described herein).

Also provided herein are methods of treating a subject (e.g., any of the subjects described herein) having a cancer (e.g., any of the exemplary cancers described herein or known in the art) that include: (a) administering to the subject a therapeutic agent (e.g., any therapeutic agent that is not a CLK inhibitor or any therapeutic regimen that does not include a CLK inhibitor as a monotherapy); (b) after (a), identifying the subject as having a cancer cell that has an elevated level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of Wnt pathway activity (e.g., any of the exemplary types of Wnt pathway activity described herein or known in the art) as compared to a reference level (e.g., any of the reference levels described herein); and (c) administering to the identified subject a treatment comprising a therapeutically effective amount of a CLK inhibitor as well as prodrugs and pharmaceutically acceptable salt or solvate thereof (e.g., any of the exemplary CLK inhibitors described herein or known in the art). In some embodiments, the subject is also administered the previously administered therapeutic agent (e.g., any cancer therapeutic agent known in the art, e.g., chemotherapy, radiation therapy, other kinase inhibitors, or a biologic), in addition to the CLK inhibitor or the pharmaceutically acceptable salt of solvate thereof.

Also provided herein are methods of treating a subject (e.g., any of the subjects described herein) having a cancer (e.g., any of the types of cancer described herein or known in the art) that include: identifying a subject previously administered a therapeutic agent (e.g., any therapeutic agent that is not a CLK inhibitor or any therapeutic regimen that does not include a CLK inhibitor as a monotherapy), as having a cancer cell that has an elevated level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of Wnt pathway activity (e.g., any of the exemplary types of Wnt pathway activity described herein or known in the art) as compared to a reference level (e.g., any of the exemplary reference levels described herein); and administering to the identified subject a treatment comprising a therapeutically effective amount of a CLK inhibitor as well as prodrugs and pharmaceutically acceptable salt or solvate thereof (e.g., any of the CLK inhibitors described herein or known in the art). In some embodiments, the subject is also administered the previously administered therapeutic agent (e.g., any cancer therapeutic agent known in the art, e.g., chemotherapy, radiation therapy, other kinase inhibitors, or a biologic), in addition to the CLK inhibitor or the pharmaceutically acceptable salt of solvate thereof.

Also provided herein are methods of treating a subject (e.g., any of the subjects described herein) having a cancer (e.g., any of the exemplary cancers described herein or known in the art) that include: administering to a subject previously administered a therapeutic agent (e.g., any therapeutic agent that is not a CLK inhibitor or any therapeutic regimen that does not include a CLK inhibitor as a monotherapy) and later identified as having an elevated level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of Wnt pathway activity (e.g., any of the exemplary types of Wnt pathway activity described herein or known in the art) as compared to a reference level (e.g., any of the exemplary reference levels described herein), a therapeutically effective amount of a CLK inhibitor as well as prodrugs and pharmaceutically acceptable salt or solvate thereof (e.g., any of the exemplary CLK inhibitors described herein or known in the art). In some embodiments, the subject is also administered the previously administered therapeutic agent (e.g., any cancer therapeutic agent known in the art, e.g., chemotherapy, surgery, radiation therapy, other kinase inhibitors, or a biologic), in addition to the CLK inhibitor or the pharmaceutically acceptable salt of solvate thereof.

Non-limiting types of Wnt pathway activity are described below and can be used in any of the methods of treatment described herein. For example, in some embodiments of any of the methods of treatment described herein, the Wnt pathway activity can be the level of CLK1, CLK2, CLK3, CLK4, or β-catenin expression, where an increased level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of expression of CLK1, CLK2, CLK3, CLK4, or β-catenin protein or mRNA expression, as compared to a reference level (e.g., any of the reference levels described herein) indicates an increased level of Wnt pathway activity.

For example, in some embodiments of any of the methods of treatment described herein, the Wnt pathway activity can be the level of β-catenin in the nucleus of a mammalian cell, where an increased level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of p-catenin in the nucleus of a mammalian cell as compared to a reference level (e.g., any of the reference levels described herein) indicates an increased level of Wnt pathway activity.

For example, in some embodiments of any of the methods of treatment described herein, the Wnt pathway activity can be detection of a mutation in a Wnt pathway gene selected from the group consisting of: gain-of-function mutation in a β-catenin gene (e.g., a gene encoding a β-catenin protein including a 41A, 45F, or 45P amino acid substitution, a mutation in exon 3, or deletion in exon 3) (Le Guellac et al., Modern Pathology 25: 1551, 2012), a loss-of-function mutation in an AXIN gene (e.g., c.178_1597del, c.266_1585del, c.355_1712del, c.1938_2704del, c.2168_3098del, c.2426_3101del, or c.2325_3106del, or a gene encoding an AXIN protein including a P218S, S226C, P263T, A360V, R382C, G433E, V517F, P661L, A740T, F824K, S828G, E842K, K397X, T58M, L101P, R103M, L106R, T122A, K203M, S215L, P263T, N370K, P345L, R349H, R353H, H394N, R395C, E41 iD, M4181, G425S, D495E, G583S, G650S, R841Q, P848L, E852G, W247X, Y305X, or E406X amino acid substitution (Mazzoni and Fearon, Cancer Lett 355(1): 1-8, 2014)), a loss-of-function mutation in an AXIN2 gene (e.g., c.1209insAT (V506X), c.1994delG (L688X), c.2013_2024del, or c.1926insA (E706X), or a gene encoding an AXIN2 protein including a S658C, R659W, Q696R, S738F, S762N, S738F, R656X, or W663X amino acid substitution (Mazzoni and Fearon, Cancer Lett 355(1): 1-8, 2014)), a loss-of-function mutation in a APC gene (e.g., 2-bp deletion in exon 7, 904C-T transition in exon 8, or 1-bp deletion in exon 10, or a gene encoding an APC protein including a R414C, R302X, S280X, Q1338X, Q541X, G1120E, R554X, or Y935X amino acid substitution), a loss-of-function mutation in a CTNNB1 gene (e.g., a gene encoding a CTNN11 protein including a Q558X or R710C amino acid substitution), a loss-of-function mutation in a Tsc1 gene (e.g., 4-bp deletion in exon 15, or a gene encoding a Tsc1 protein including a H732Y, K587R, M224R, L180P, R22W, or R204C amino acid substitution), a loss-of-function mutation in a Tsc2 gene (e.g., del5151OA or del4590C, or a gene encoding a Tsc2 protein including a K12X, R505X, R611Q, L717R, P1675L, Q2503P, R905Q, R905W, R905G, or V1547I amino acid substitution), and a loss-of-function mutation GSK3D gene.

For example, in some embodiments of any of the methods of treatment described herein, the Wnt pathway activity can be detection of an elevated level of expression of one or more Wnt-regulated genes as compared to a reference level (e.g., any of the reference levels described herein). Non-limiting examples of Wnt-upregulated genes include CCND1, CSNK2A1, CXCL12, LRP5, MMP7, MMP9, LEF1, AXIN2, MYC, TCF7L2, TCF7, LRP6, DVL2, BIRC, ERRB2, MAPK8, PKN1, AXIN2, ABCB1, ADAM10, ALEX1, ASCL2, BAMBI, BCL2L2, BIRC5, BMI1, BMP4, CCND1, CD44, CDKN2A, CDX1, CEBPD, CLDN1, COX2, DNMT1, EDN1, EFNB1, ENC1, EPHB2, EPHB3, FGF18, FGFBP, FRA1, FSCN1, FZD6, FZD7, FZD8, GAST, HDAC3, HEF1, HES1, ID2, ITF2, JAG1, JUN, LICAM, LAMC2, LGR5, MENA, MET, MMP14, MYB, MYCBP, NOS2, NOTCH2, NRCAM, PLAU, PLAUR, PLCB4, PPARD, RUVBL1, S100A4, S100A6, SGK1, SMC3, SOX9, SP5, SRSF3, SUZ12, TCF1, TIAM1, TIMP-1. TN-C, VEGF, WNT-5a, WNT-5b, WNT11, and YAP.

For example, in some embodiments of any of the methods of treatment described herein, the Wnt pathway activity can be detection of a decrease level of expression of one or more of APC, FRZB, CTGF, and GSK3B.

Non-limiting examples of Wnt-downregulated genes include secreted frizzled related protein 1 (FRP), disheveled associated activator of morphogenesis 1 (DAAM1) human ortholog of atonal 1 (HATH1), and cadherin 1 (CDH1). See, e.g., Slattery et al., Oncotarget 9(5): 6075-6085, 2018; Herbst et al., BMC Genomics 15:74, 2014. An elevated level of Wnt pathway activity can be detection of a decreased level of expression of one or more of these Wnt-downregulated genes (e.g., any of the Wnt-downregulated genes described herein or known in the art) as compared to a reference level (e.g., any of the reference levels described herein).

In some embodiments of any of the methods of treatment described herein, the cancer is a small cell lung cancer, a colorectal cancer, ahead and neck cancer, an ovarian cancer, a melanoma, a renal cell carcinoma, a pancreatic cancer, or a non-small cell lung cancer.

In some embodiments of any of the methods described herein, the method can result in an increased life span of the subject (e.g., as compared to a similar subject having a similar cancer but receiving a different treatment).

In some embodiments of any of the methods described herein, the cancer can be:

1) Breast cancers, including, for example ER⁺ breast cancer, ER⁻ breast cancer, her2⁻ breast cancer, her2⁺ breast cancer, stromal tumors, such as fibroadenomas, phyllodes tumors, and sarcomas, and epithelial tumors, such as large duct papillomas; carcinomas of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma; and miscellaneous malignant neoplasms. Further examples of breast cancers can include luminal A, luminal B, basal A, basal B, and triple negative breast cancer, which is estrogen receptor negative (ER⁻), progesterone receptor negative, and Her2 negative (Her2⁻). In some embodiments, the breast cancer may have a high risk Oncotype score.

2) Cardiac cancers, including, for example sarcoma, e.g., angiosarcoma, fibrosarcoma, rhabdomyosarcoma, and liposarcoma; myxoma; rhabdomyoma; fibroma; lipoma and teratoma.

3) Lung cancers, including, for example, bronchogenic carcinoma, e.g., squamous cell, undifferentiated small cell, undifferentiated large cell, and adenocarcinoma; alveolar and bronchiolar carcinoma; bronchial adenoma; sarcoma; lymphoma; chondromatous hamartoma; and mesothelioma.

4) Gastrointestinal cancer, including, for example, cancers of the esophagus, e.g., squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, and lymphoma; cancers of the stomach, e.g., carcinoma, lymphoma, and leiomyosarcoma; cancers of the pancreas, e.g., ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, and vipoma; cancers of the small bowel, e.g., adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, and fibroma; cancers of the large bowel, e.g., adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, and leiomyoma.

5) Genitourinary tract cancers, including, for example, cancers of the kidney, e.g., adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, and leukemia; cancers of the bladder and urethra, e.g., squamous cell carcinoma, transitional cell carcinoma, and adenocarcinoma; cancers of the prostate, e.g., adenocarcinoma, and sarcoma; cancer of the testis, e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, and lipoma.

6) Liver cancers, including, for example, hepatoma, e.g., hepatocellular carcinoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hepatocellular adenoma; and hemangioma.

7) Bone cancers, including, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochrondroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors.

8) Nervous system cancers, including, for example, cancers of the skull, e.g., osteoma, hemangioma, granuloma, xanthoma, and osteitis deformans; cancers of the meninges, e.g., meningioma, meningiosarcoma, and gliomatosis; cancers of the brain, e.g., astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, oligodendrocytoma, schwannoma, retinoblastoma, and congenital tumors; and cancers of the spinal cord, e.g., neurofibroma, meningioma, glioma, and sarcoma.

9) Gynecological cancers, including, for example, cancers of the uterus, e.g., endometrial carcinoma; cancers of the cervix, e.g., cervical carcinoma, and pre tumor cervical dysplasia; cancers of the ovaries, e.g., ovarian carcinoma, including serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa theca cell tumors, Sertoli Leydig cell tumors, dysgerminoma, and malignant teratoma; cancers of the vulva, e.g., squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, and melanoma; cancers of the vagina, e.g., clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma, and embryonal rhabdomyosarcoma; and cancers of the fallopian tubes, e.g., carcinoma.

10) Hematologic cancers, including, for example, cancers of the blood, e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, chronic myeloid leukemia, multiple myeloma, and myelodysplastic syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma (malignant lymphoma) and Waldenstrom's macroglobulinemia.

11) Skin cancers and skin disorders, including, for example, malignant melanoma and metastatic melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, and scleroderma.

12) Adrenal gland cancers, including, for example, neuroblastoma.

More particularly, cancer in any of the methods described herein can be:

1) Astrocytic tumors, e.g., diffuse astrocytoma (fibrillary, protoplasmic, gemistocytic, mixed), anaplastic (malignant) astrocytoma, glioblastoma multiforme (giant cell glioblastoma and gliosarcoma), pilocytic astrocytoma (pilomyxoid astrocytoma), pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, and gliomatosis cerebri.

2) Oligodendroglial tumors, e.g., oligodendroglioma and anaplastic oligodendroglioma.

3) Oligoastrocytic tumors, e.g., oligoastrocytoma and anaplastic oligoastrocytoma.

4) Ependymal tumors, e.g., subependymoma, myxopapillary ependymoma, ependymoma, (cellular, papillary, clear cell, tanycytic), and anaplastic (malignant) ependymoma.

5) Choroid plexus tumors, e.g., choroid plexus papilloma, atypical choroid plexus papilloma, and choroid plexus carcinoma.

6) Neuronal and mixed neuronal-glial tumors, e.g., gangliocytoma, ganglioglioma, dysembryoplastic neuroepithelial tumor (DNET), dysplastic gangliocytoma of the cerebellum (Lhermitte-Duclos), desmoplastic infantile astrocytoma/ganglioglioma, central neurocytoma, anaplastic ganglioglioma, extraventricular neurocytoma, cerebellar liponeurocytoma, Papillary glioneuronal tumor, Rosette-forming glioneuronal tumor of the fourth ventricle, and paraganglioma of the filum terminale.

7) Pineal tumors, e.g., pineocytoma, pineoblastoma, papillary tumors of the pineal region, and pineal parenchymal tumor of intermediate differentiation.

8) Embryonal tumors, e.g., medulloblastoma (medulloblastoma with extensive nodularity, anaplastic medulloblastoma, desmoplastic, large cell, melanotic, medullomyoblastoma), medulloepithelioma, supratentorial primitive neuroectodermal tumors, and primitive neuroectodermal tumors (PNETs) such as neuroblastoma, ganglioneuroblastoma, ependymoblastoma, and atypical teratoid/rhabdoid tumor.

9) Neuroblastic tumors, e.g., olfactory (esthesioneuroblastoma), olfactory neuroepithelioma, and neuroblastomas of the adrenal gland and sympathetic nervous system.

10) Glial tumors, e.g., astroblastoma, chordoid glioma of the third ventricle, and angiocentric glioma.

11) Tumors of cranial and paraspinal nerves, e.g., schwannoma, neurofibroma Perineurioma, and malignant peripheral nerve sheath tumor.

12) Tumors of the meninges such as tumors of meningothelial cells, e.g., meningioma (atypical meningioma and anaplastic meningioma); mesenchymal tumors, e.g., lipoma, angiolipoma, hibernoma, liposarcoma, solitary fibrous tumor, fibrosarcoma, malignant fibrous histiocytoma, leiomyoma, leiomyosarcoma, rhabdomyoma, rhabdomyosarcoma, chondroma, chondrosarcoma, osteoma, osteosarcoma, osteochondroma, haemangioma, epithelioid hemangioendothelioma, haemangiopericytoma, anaplastic haemangiopericytoma, angiosarcoma, Kaposi Sarcoma, and Ewing Sarcoma; primary melanocytic lesions, e.g., diffuse melanocytosis, melanocytoma, malignant melanoma, meningeal melanomatosis; and hemangioblastomas.

13) Tumors of the hematopoietic system, e.g., malignant Lymphomas, plasmocytoma, and granulocytic sarcoma.

14) Germ cell tumors, e.g., germinoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma, teratoma, and mixed germ cell tumors.

15) Tumors of the sellar region, e.g., craniopharyngioma, granular cell tumor, pituicytoma, and spindle cell oncocytoma of the adenohypophysis.

Cancers may be solid tumors that may or may not be metastatic. Cancers may also occur, as in leukemia, as a diffuse tissue. Thus, the term “cancer cell,” as provided herein, includes a cell afflicted by any one of the above identified disorders or cancers.

In some embodiments of any of the methods described herein, the cancer is chosen from: hepatocellular carcinoma, colon cancer, breast cancer, pancreatic cancer, chronic myeloid leukemia (CML), chronic myelomonocytic leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia, acute lymphocytic leukemia, Hodgkin lymphoma, lymphoma, sarcoma, and ovarian cancer.

In some embodiments of any of the methods described herein, the cancer is chosen from: lung cancer—non-small cell, lung cancer—small cell, multiple myeloma, nasopharyngeal cancer, neuroblastoma, osteosarcoma, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer—basal and squamous cell, skin cancer -melanoma, small intestine cancer, stomach (gastric) cancers, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, laryngeal or hypopharyngeal cancer, kidney cancer, Kaposi sarcoma, gestational trophoblastic disease, gastrointestinal stromal tumor, gastrointestinal carcinoid tumor, gallbladder cancer, eye cancer (melanoma and lymphoma), Ewing tumor, esophagus cancer, endometrial cancer, colorectal cancer, cervical cancer, brain or spinal cord tumor, bone metastasis, bone cancer, bladder cancer, bile duct cancer, anal cancer and adrenal cortical cancer.

In some embodiments, the cancer is hepatocellular carcinoma.

In some embodiments, the cancer is colon cancer.

In some embodiments, the cancer is colorectal cancer.

In some embodiments, the cancer is breast cancer.

In some embodiments, the cancer is pancreatic cancer.

In some embodiments, the cancer is chronic myeloid leukemia (CML).

In some embodiments, the cancer is chronic myelomonocytic leukemia.

In some embodiments, the cancer is chronic lymphocytic leukemia (CLL).

In some embodiments, the cancer is acute myeloid leukemia.

In some embodiments, the cancer is acute lymphocytic leukemia.

In some embodiments, the cancer is Hodgkin lymphoma.

In some embodiments, the cancer is lymphoma.

In some embodiments, the cancer is tumors of the hematopoietic and lymphoid tissues.

In some embodiments, the cancer is hematological malignancies.

In some embodiments, the cancer is sarcoma.

In some embodiments, the cancer is ovarian cancer.

In some embodiments, the cancer is lung cancer—non-small cell.

In some embodiments, the cancer is lung cancer—small cell.

In some embodiments, the cancer is multiple myeloma.

In some embodiments, the cancer is nasopharyngeal cancer.

In some embodiments, the cancer is neuroblastoma.

In some embodiments, the cancer is osteosarcoma.

In some embodiments, the cancer is penile cancer.

In some embodiments, the cancer is pituitary tumors.

In some embodiments, the cancer is prostate cancer.

In some embodiments, the cancer is retinoblastoma.

In some embodiments, the cancer is rhabdomyosarcoma.

In some embodiments, the cancer is salivary gland cancer.

In some embodiments, the cancer is skin cancer—basal and squamous cell.

In some embodiments, the cancer is skin cancer—melanoma.

In some embodiments, the cancer is small intestine cancer.

In some embodiments, the cancer is stomach (gastric) cancers.

In some embodiments, the cancer is testicular cancer.

In some embodiments, the cancer is thymus cancer.

In some embodiments, the cancer is thyroid cancer.

In some embodiments, the cancer is uterine sarcoma.

In some embodiments, the cancer is vaginal cancer.

In some embodiments, the cancer is vulvar cancer.

In some embodiments, the cancer is Wilms tumor.

In some embodiments, the cancer is laryngeal or hypopharyngeal cancer.

In some embodiments, the cancer is kidney cancer.

In some embodiments, the cancer is Kaposi sarcoma.

In some embodiments, the cancer is gestational trophoblastic disease.

In some embodiments, the cancer is gastrointestinal stromal tumor.

In some embodiments, the cancer is gastrointestinal carcinoid tumor.

In some embodiments, the cancer is gallbladder cancer.

In some embodiments, the cancer is eye cancer (melanoma and lymphoma).

In some embodiments, the cancer is Ewing tumor.

In some embodiments, the cancer is esophagus cancer.

In some embodiments, the cancer is endometrial cancer.

In some embodiments, the cancer is colorectal cancer.

In some embodiments, the cancer is cervical cancer.

In some embodiments, the cancer is brain or spinal cord tumor.

In some embodiments, the cancer is bone metastasis.

In some embodiments, the cancer is bone cancer.

In some embodiments, the cancer is bladder cancer.

In some embodiments, the cancer is bile duct cancer.

In some embodiments, the cancer is anal cancer.

In some embodiments, the cancer is adrenal cortical cancer.

Methods of Selecting a Treatment—Type A

Provided herein are methods of selecting a treatment for a subject (e.g., any of the subjects described herein) that include: identifying a subject having a cancer cell that has an elevated level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of Wnt pathway activity (e.g., any of the exemplary types of Wnt pathway activity described herein or known in the art) as compared to a reference level (e.g., any of the exemplary reference levels described herein); and selecting for the identified subject a treatment comprising a therapeutically effective amount of a CLK inhibitor (e.g., any of the CLK inhibitors described herein) as well as prodrugs and pharmaceutically acceptable salt or solvate thereof. In some embodiments, the selected treatment can further include another treatment or therapeutic agent (e.g., any cancer therapeutic agent known in the art, e.g., chemotherapy, surgery, radiation therapy, other kinase inhibitors, or a biologic), in addition to the CLK inhibitor or the pharmaceutically acceptable salt of solvate thereof.

Provided herein are methods of selecting a treatment for a subject (e.g., any of the subjects described herein) that include selecting a treatment comprising a therapeutically effective amount of a CLK inhibitor (e.g., any of the CLK inhibitors described herein or known in the art)) as well as prodrugs and pharmaceutically acceptable salt or solvate thereof for a subject identified as having a cancer cell that has an elevated level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of Wnt pathway activity (e.g., any of the exemplary types of Wnt pathway activity described herein or known in the art) as compared to a reference level (e.g., any of the exemplary reference levels described herein). In some embodiments, the selected treatment can further include another treatment or therapeutic agent (e.g., any cancer therapeutic agent known in the art, e.g., chemotherapy, surgery, radiation therapy, other kinase inhibitors, or a biologic), in addition to the CLK inhibitor or the pharmaceutically acceptable salt of solvate thereof.

In some embodiments of any of the methods of selecting a treatment described herein, the cancer cell is a small cell lung cancer cell, a colorectal cancer cell, a head and neck cancer cell, an ovarian cancer cell, a melanoma cell, a renal cell carcinoma cell, a pancreatic cancer cell, or a non-small cell lung cancer cell. In some embodiments of any of the methods of selecting a treatment described herein, the cancer can be any of the cancers described herein or known in the art.

Non-limiting types of Wnt pathway activity are described below and can be used in any of the methods of selecting a treatment described herein. For example, in some embodiments of any of the methods of selecting a treatment described herein, the Wnt pathway activity can be the level of CLK1, CLK2, CLK3, CLK4, or β-catenin expression, where an increased level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of expression of CLK1, CLK2, CLK3, CLK4, or β-catenin protein or mRNA expression, as compared to a reference level (e.g., any of the reference levels described herein) indicates an increased level of Wnt pathway activity.

For example, in some embodiments of any of the methods of selecting a treatment described herein, the Wnt pathway activity can be the level of β-catenin in the nucleus of a mammalian cell, where an increased level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of β-catenin in the nucleus of a mammalian cell as compared to a reference level (e.g., any of the reference levels described herein) indicates an increased level of Wnt pathway activity.

For example, in some embodiments of any of the methods of selecting a treatment described herein, the Wnt pathway activity can be detection of a mutation in a Wnt pathway gene selected from the group consisting of: gain-of-function mutation in a β-catenin gene, a loss-of-function mutation in an AXIN gene, a loss-of-function mutation in an AXIN2 gene, a loss-of-function mutation in a APC gene, a loss-of-function mutation in a CTNNB1 gene, a loss-of-function mutation in a Tsc1 gene, a loss-of-function mutation in a Tsc2 gene, and a loss-of-function mutation GSK3D gene.

For example, in some embodiments of any of the methods of selecting a treatment described herein, the Wnt pathway activity can be detection of an elevated level of expression of one or more Wnt-regulated genes as compared to a reference level (e.g., any of the reference levels described herein). Non-limiting examples of Wnt-upregulated genes include CCND1, CSNK2A1, CXCL12, LRP5, MMP7, MMP9, LEF1, AXIN2, MYC, TCF7L2, TCF7, LRP6, DVL2, BIRC, ERRB2, MAPK8, PKN1, AXIN2, ABCB1, ADAM10, ALEX1, ASCL2, BAMBI, BCL2L2, BIRC5, BMI1, BMP4, CCND1, CD44, CDKN2A, CDX1, CEBPD, CLDN1, COX2, DNMT1, EDN1, EFNB1, ENC1, EPHB2, EPHB3, FGF18, FGFBP, FRA1, FSCN1, FZD6, FZD7, FZD8, GAST, HDAC3, HEF1, HES1, ID2, ITF2, JAG1, JUN, LlCAM, LAMC2, LGR5, MENA, MET, MMP14, MYB, MYCBP, NOS2, NOTCH2, NRCAM, PLAU, PLAUR, PLCB4, PPARD, RUVBL1, S100A4, S100A6, SGK1, SMC3, SOX9, SP5, SRSF3, SUZ12, TCF1, TIAM1, TIMP-1. TN-C, VEGF, WNT-5a, WNT-5b, WNT11, and YAP.

For example, in some embodiments of any of the methods of selecting a treatment described herein, the Wnt pathway activity can be detection of a decreased level of expression of one or more of APC, FRZB, CTGF, and GSK3B.

In some embodiments of any of the methods described herein, the cancer is a small cell lung cancer, a colorectal cancer, a head and neck cancer, an ovarian cancer, a melanoma, a renal cell carcinoma, a pancreatic cancer, or a non-small cell lung cancer.

Methods of Selecting a Subject for Treatment—Type A

Provided herein are methods of selecting a subject for participation in a clinical trial that include: identifying a subject (e.g., any of the subjects described herein) having a cancer cell (e.g., any of the types of cancer cells described herein or known in the art) that has an elevated level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of Wnt pathway activity (e.g., any of the exemplary types of Wnt pathway activity described herein or known in the art) as compared to a reference level (e.g., any of the exemplary reference levels described herein); and selecting the identified subject for participation in a clinical trial that comprises administration of a therapeutically effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) as well as prodrugs and pharmaceutically acceptable salt or solvate thereof. In some embodiments, the subject can be selected for a treatment that further includes another treatment or therapeutic agent (e.g., any cancer therapeutic agent known in the art, e.g., chemotherapy, surgery, radiation therapy, other kinase inhibitors, or a biologic), in addition to the CLK inhibitor or the pharmaceutically acceptable salt of solvate thereof.

Provided herein are methods of selecting a subject (e.g., any of the subjects described herein) for participation in a clinical trial that include selecting a subject identified as having a cancer cell (e.g., any of the types of cancer cells described herein or known in the art) that has an elevated level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of Wnt pathway activity (e.g., any of the exemplary types of Wnt pathway activity described herein or known in the art) as compared to a reference level for participation in a clinical trial that comprises administration of a therapeutically effective amount of a CLK inhibitor (e.g., any of the CLK inhibitors described herein or known in the art) as well as prodrugs and pharmaceutically acceptable salt or solvate thereof. In some embodiments, the subject can be selected for a treatment that further includes another treatment or therapeutic agent (e.g., any cancer therapeutic agent known in the art, e.g., chemotherapy, surgery, radiation therapy, other kinase inhibitors, or a biologic), in addition to the CLK inhibitor or the pharmaceutically acceptable salt of solvate thereof.

In some embodiments of any of the methods of selecting a subject for treatment described herein, the cancer cell is a small cell lung cancer cell, a colorectal cancer cell, a head and neck cancer cell, an ovarian cancer cell, a melanoma cell, a renal cell carcinoma cell, a pancreatic cancer cell, or a non-small cell lung cancer cell. In some embodiments of any of the methods of selecting a subject for treatment described herein, the cancer can be any of the cancers described herein or known in the art.

Non-limiting types of Wnt pathway activity are described below and can be used in any of the methods of selecting a subject for treatment described herein. For example, in some embodiments of any of the methods of selecting a subject for treatment described herein, the Wnt pathway activity can be the level of CLK1, CLK2, CLK3, CLK4, or β-catenin expression, where an increased level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of expression of CLK1, CLK2, CLK3, CLK4, or β-catenin protein or mRNA expression, as compared to a reference level (e.g., any of the reference levels described herein) indicates an increased level of Wnt pathway activity.

For example, in some embodiments of any of the methods of selecting a subject for treatment described herein, the Wnt pathway activity can be the level of β-catenin in the nucleus of a mammalian cell, where an increased level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of β-catenin in the nucleus of a mammalian cell as compared to a reference level (e.g., any of the reference levels described herein) indicates an increased level of Wnt pathway activity.

For example, in some embodiments of any of the methods of selecting a subject for treatment described herein, the Wnt pathway activity can be detection of a mutation in a Wnt pathway gene selected from the group consisting of: gain-of-function mutation in a β-catenin gene, a loss-of-function mutation in an AXIN gene, a loss-of-function mutation in an AXIN2 gene, a loss-of-function mutation in a APC gene, a loss-of-function mutation in a CTNNB1 gene, a loss-of-function mutation in a Tsc1 gene, a loss-of-function mutation in a Tsc2 gene, and a loss-of-function mutation GSK3D gene.

For example, in some embodiments of any of the methods of selecting a subject for treatment described herein, the Wnt pathway activity can be detection of an elevated level of expression of one or more Wnt-regulated genes as compared to a reference level (e.g., any of the reference levels described herein). Non-limiting examples of Wnt-upregulated genes include CCND1, CSNK2A1, CXCL12, LRP5, MMP7, MMP9, LEF1, AXIN2, MYC, TCF7L2, TCF7, LRP6, DVL2, BIRC, ERRB2, MAPK8, PKN1, AXIN2, ABCB1, ADAM10, ALEX1, ASCL2, BAMBI, BCL2L2, BIRC5, BMI1, BMP4, CCND1, CD44, CDKN2A, CDX1, CEBPD, CLDN1, COX2, DNMT1, EDN1, EFNB1, ENC1, EPHB2, EPHB3, FGF18, FGFBP, FRA1, FSCN1, FZD6, FZD7, FZD8, GAST, HDAC3, HEF1, HES1, ID2, ITF2, JAG1, JUN, LlCAM, LAMC2, LGR5, MENA, MET, MMP14, MYB, MYCBP, NOS2, NOTCH2, NRCAM, PLAU, PLAUR, PLCB4, PPARD, RUVBL1, S100A4, S100A6, SGK1, SMC3, SOX9, SP5, SRSF3, SUZ12, TCF1, TIAM1, TIMP-1. TN-C, VEGF, WNT-5a, WNT-5b, WNT11, and YAP.

For example, in some embodiments of any of the methods of selecting a subject for treatment described herein, the Wnt pathway activity can be detection of a decreased level of expression of one or more of APC, FRZB, CTGF, and GSK3B.

In some embodiments of any of the methods of selecting a subject for treatment described herein, the cancer is a small cell lung cancer, a colorectal cancer, a head and neck cancer, an ovarian cancer, a melanoma, a renal cell carcinoma, a pancreatic cancer, or a non-small cell lung cancer.

Methods of Selecting a Subject for Participation in a Clinical Study

Also provided herein are methods of selecting a subject (e.g., any of the subjects described herein) for participation in a clinical trial that include: identifying a subject having a cancer cell (e.g., any of the types of cancer cells described herein or known in the art) that has an elevated level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of Wnt pathway activity (e.g., any of the exemplary types of Wnt pathway activity described herein or known in the art) as compared to a reference level (e.g., any of the exemplary reference levels described herein); and selecting the identified subject for participation in a clinical trial that includes administration of a therapeutically effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvate thereof. In some embodiments of any of the methods of selecting a subject for participation in a clinical study, the clinical trial further includes administration of another treatment or therapeutic agent (e.g., any cancer therapeutic agent known in the art, e.g., chemotherapy, surgery, radiation therapy, other kinase inhibitors, or a biologic), in addition to the CLK inhibitor or the pharmaceutically acceptable salt of solvate thereof.

Also provided herein are methods of selecting a subject (e.g., any of the subjects described herein or known in the art) for participation in a clinical trial that include selecting a subject identified as having a cancer cell (e.g., any of the types of cancer cells described herein or known in the art) that has an elevated level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of Wnt pathway activity (e.g., any of the exemplary types of Wnt pathway activity described herein or known in the art) as compared to a reference level (e.g., any of the exemplary reference levels described herein) for participation in a clinical trial that comprises administration of a therapeutically effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvate thereof. In some embodiments of any of the methods of selecting a subject for participation in a clinical study, the clinical trial further includes administration of another treatment or therapeutic agent (e.g., any cancer therapeutic agent known in the art, e.g., chemotherapy, surgery, radiation therapy, other kinase inhibitors, or a biologic), in addition to the CLK inhibitor or the pharmaceutically acceptable salt of solvate thereof.

In some embodiments of any of the methods of selecting a subject for participation in a clinical study described herein, the cancer cell is a small cell lung cancer cell, a colorectal cancer cell, a head and neck cancer cell, an ovarian cancer cell, a melanoma cell, a renal cell carcinoma cell, a pancreatic cancer cell, or a non-small cell lung cancer cell. In some embodiments of any of the methods of selecting a subject for participation in a clinical study described herein, the cancer can be any of the cancers described herein or known in the art.

Non-limiting types of Wnt pathway activity are described below and can be used in any of the methods of selecting a subject for participation in a clinical study described herein. For example, in some embodiments of any of the methods of selecting a subject for participation in a clinical study described herein, the Wnt pathway activity can be the level of CLK1, CLK2, CLK3, CLK4, or β-catenin expression, where an increased level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of expression of CLK1, CLK2, CLK3, CLK4, or β-catenin protein or mRNA expression, as compared to a reference level (e.g., any of the reference levels described herein) indicates an increased level of Wnt pathway activity.

For example, in some embodiments of any of the methods of selecting a subject for participation in a clinical study described herein, the Wnt pathway activity can be the level of β-catenin in the nucleus of a mammalian cell, where an increased level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of β-catenin in the nucleus of a mammalian cell as compared to a reference level (e.g., any of the reference levels described herein) indicates an increased level of Wnt pathway activity.

For example, in some embodiments of any of the methods of selecting a subject for participation in a clinical study described herein, the Wnt pathway activity can be detection of a mutation in a Wnt pathway gene selected from the group consisting of: gain-of-function mutation in a β-catenin gene, a loss-of-function mutation in an AXIN gene, a loss-of-function mutation in an AXIN2 gene, a loss-of-function mutation in a APC gene, a loss-of-function mutation in a CTNNB1 gene, a loss-of-function mutation in a Tsc1 gene, a loss-of-function mutation in a Tsc2 gene, and a loss-of-function mutation GSK3D gene.

For example, in some embodiments of any of the methods of selecting a subject for participation in a clinical study described herein, the Wnt pathway activity can be detection of an elevated level of expression of one or more Wnt-regulated genes as compared to a reference level (e.g., any of the reference levels described herein). Non-limiting examples of Wnt-upregulated genes include CCND1, CSNK2A1, CXCL12, LRP5, MMP7, MMP9, LEF1, AXIN2, MYC, TCF7L2, TCF7, LRP6, DVL2, BIRC, ERRB2, MAPK8, PKN1, AXIN2, ABCB1, ADAM10, ALEX1, ASCL2, BAMBI, BCL2L2, BIRC5, BMI1, BMP4, CCND1, CD44, CDKN2A, CDX1, CEBPD, CLDN1, COX2, DNMT1, EDN1, EFNB1, ENC1, EPHB2, EPHB3, FGF18, FGFBP, FRA1, FSCN1, FZD6, FZD7, FZD8, GAST, HDAC3, HEF1, HES1, ID2, ITF2, JAG1, JUN, L1CAM, LAMC2, LGR5, MENA, MET, MMP14, MYB, MYCBP, NOS2, NOTCH2, NRCAM, PLAU, PLAUR, PLCB4, PPARD, RUVBL1, S100A4, S100A6, SGK1, SMC3, SOX9, SP5, SRSF3, SUZ12, TCF1, TIAM1, TIMP-1. TN-C, VEGF, WNT-5a, WNT-5b, WNT11, and YAP.

In some embodiments of any of the methods of selecting a subject for participation in a clinical study described herein, the cancer is a small cell lung cancer, a colorectal cancer, a head and neck cancer, an ovarian cancer, a melanoma, a renal cell carcinoma, a pancreatic cancer, or a non-small cell lung cancer.

Methods of Determining Efficacy of a CLK Inhibitor—Type A

Also provided herein are methods of determining the efficacy of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvate thereof in a subject (e.g., any of the subjects described herein) that include: (a) determining a first level of Wnt pathway activity (e.g., any of the exemplary types of Wnt pathway activity described herein or known in the art) in a cancer cell (e.g., any of the exemplary cancer cells described herein or known in the art) obtained from a subject at a first time point; (b) administering to the subject after the first time a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof, (c) determining a second level of the Wnt pathway activity in a cancer cell obtained from the subject at a second time point; and (d) determining that the CLK inhibitor is effective in a subject having a second level of Wnt pathway activity that is decreased (e.g., 10% to about 99% decreased, 1% to about 95% decreased, 10% to about 90% decreased, 1% to about 85% decreased, 1% to about 80% decreased, 1% to about 75% decreased, 1% to about 70% decreased, 1% to about 650% decreased, 1% to about 60% decreased, 1% to about 550% decreased, 1% to about 50% decreased, 1% to about 45% decreased, 1% to about 40% decreased, 1% to about 35% decreased, 1% to about 30% decreased, 1% to about 25% decreased, 1% to about 20% decreased, 1% to about 15% decreased, 1% to about 10% decreased, 1% to about 5% decreased, about 5% to about 99% decreased, about 5% to about 95% decreased, about 5% to about 90% decreased, about 5% to about 85% decreased, about 5% to about 80% decreased, about 5% to about 75% decreased, about 5% to about 70% decreased, about 5% to about 65% decreased, about 5% to about 60% decreased, about 5% to about 55% decreased, about 5% to about 50% decreased, about 5% to about 45% decreased, about 5% to about 40% decreased, about 5% to about 35% decreased, about 5% to about 30% decreased, about 5% to about 25% decreased, about 5% to about 20% decreased, about 5% to about 15% decreased, about 5% to about 10% decreased, about 10% to about 99% decreased, about 10% to about 95% decreased, about 10% to about 90% decreased, about 10% to about 85% decreased, about 10% to about 80% decreased, about 10% to about 75% decreased, about 10% to about 70% decreased, about 10% to about 65% decreased, about 10% to about 60% decreased, about 10% to about 55% decreased, about 10% to about 50% decreased, about 10% to about 45% decreased, about 10% to about 40% decreased, about 10% to about 35% decreased, about 10% to about 30% decreased, about 10% to about 25% decreased, about 10% to about 20% decreased, about 10% to about 15% decreased, about 15% to about 99% decreased, about 15% to about 95% decreased, about 15% to about 90% decreased, about 15% to about 85% decreased, about 15% to about 80% decreased, about 15% to about 75% decreased, about 15% to about 70% decreased, about 15% to about 65% decreased, about 15% to about 60% decreased, about 15% to about 55% decreased, about 15% to about 50% decreased, about 15% to about 45% decreased, about 15% to about 40% decreased, about 15% to about 35% decreased, about 15% to about 30% decreased, about 15% to about 25% decreased, about 15% to about 20% decreased, about 20% to about 99% decreased, about 20% to about 95% decreased, about 20% to about 90% decreased, about 20% to about 85% decreased, about 20% to about 80% decreased, about 20% to about 75% decreased, about 20% to about 70% decreased, about 20% to about 65% decreased, about 20% to about 60% decreased, about 20% to about 55% decreased, about 20% to about 50% decreased, about 20% to about 45% decreased, about 20% to about 40% decreased, about 20% to about 35% decreased, about 20% to about 30% decreased, about 20% to about 25% decreased, about 25% to about 99% decreased, about 25% to about 95% decreased, about 25% to about 90% decreased, about 25% to about 85% decreased, about 25% to about 80% decreased, about 25% to about 75% decreased, about 25% to about 70% decreased, about 25% to about 65% decreased, about 25% to about 60% decreased, about 25% to about 55% decreased, about 25% to about 50% decreased, about 25% to about 45% decreased, about 25% to about 40% decreased, about 25% to about 35% decreased, about 25% to about 30% decreased, about 30% to about 99% decreased, about 30% to about 95% decreased, about 30% to about 90% decreased, about 30% to about 85% decreased, about 30% to about 80% decreased, about 30% to about 75% decreased, about 30% to about 70% decreased, about 30% to about 65% decreased, about 30% to about 60% decreased, about 30% to about 55% decreased, about 30% to about 50% decreased, about 30% to about 45% decreased, about 30% to about 40% decreased, about 30% to about 35% decreased, about 35% to about 99% decreased, about 35% to about 95% decreased, about 35% to about 90% decreased, about 35% to about 85% decreased, about 35% to about 80% decreased, about 35% to about 75% decreased, about 35% to about 70% decreased, about 35% to about 65% decreased, about 35% to about 60% decreased, about 35% to about 55% decreased, about 35% to about 50% decreased, about 35% to about 45% decreased, about 35% to about 40% decreased, about 40% to about 99% decreased, about 40% to about 95% decreased, about 40% to about 90% decreased, about 40% to about 85% decreased, about 40% to about 80% decreased, about 40% to about 75% decreased, about 40% to about 70% decreased, about 40% to about 65% decreased, about 40% to about 60% decreased, about 40% to about 55% decreased, about 40% to about 50% decreased, about 40% to about 45% decreased, about 45% to about 99% decreased, about 45% to about 95% decreased, about 45% to about 90% decreased, about 45% to about 85% decreased, about 45% to about 80% decreased, about 45% to about 75% decreased, about 45% to about 70% decreased, about 45% to about 65% decreased, about 45% to about 60% decreased, about 45% to about 55% decreased, about 45% to about 50% decreased, about 50% to about 99% decreased, about 50% to about 95% decreased, about 50% to about 90% decreased, about 50% to about 85% decreased, about 50% to about 80% decreased, about 50% to about 75% decreased, about 50% to about 70% decreased, about 50% to about 65% decreased, about 50% to about 60% decreased, about 50% to about 55% decreased, about 55% to about 99% decreased, about 55% to about 95% decreased, about 55% to about 90% decreased, about 55% to about 85% decreased, about 55% to about 80% decreased, about 55% to about 75% decreased, about 55% to about 70% decreased, about 55% to about 65% decreased, about 55% to about 60% decreased, about 60% to about 99% decreased, about 60% to about 95% decreased, about 60% to about 90% decreased, about 60% to about 85% decreased, about 60% to about 80% decreased, about 60% to about 75% decreased, about 60% to about 70% decreased, about 60% to about 65% decreased, about 65% to about 99% decreased, about 65% to about 95% decreased, about 65% to about 90% decreased, about 65% to about 85% decreased, about 65% to about 80% decreased, about 65% to about 75% decreased, about 65% to about 70% decreased, about 70% to about 99% decreased, about 70% to about 95% decreased, about 70% to about 90% decreased, about 70% to about 85% decreased, about 70% to about 80% decreased, about 70% to about 75% decreased, about 75% to about 99% decreased, about 75% to about 95% decreased, about 75% to about 90% decreased, about 75% to about 85% decreased, about 75% to about 80% decreased, about 80% to about 99% decreased, about 80% to about 95% decreased, about 80% to about 90% decreased, about 80% to about 85% decreased, about 85% to about 99% decreased, about 85% to about 95% decreased, about 85% to about 90% decreased, about 90% to about 99% decreased, about 90% to about 95% decreased, or about 95% to about 99% decreased) as compared to the first level of Wnt pathway activity.

In some embodiments of any of the methods described herein, the method further includes: (e) after (d), administering one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 40, 50, 60, 70, 80, 90, or 100) additional doses of the CLK inhibitor to the subject.

In some embodiments of any of the methods further include a step of selecting a subject having cancer or diagnosing a subject as having cancer. For example, a subject having cancer can have previously been administered a treatment for cancer, and the previous treatment was unsuccessful. Some embodiments of any of the methods described herein can further include obtaining a cancer cell from the subject at the first and second time points.

In some embodiments of any of the methods described herein, the method further includes recording the identified efficacy of the CLK inhibitor in the subject's medical record (e.g., a computer readable medium).

In some embodiments of any of the methods described herein, the method further includes informing the subject, the subject's family, and/or the subject's primary care physician or attending physician of the determined efficacy of the CLK inhibitor.

In some embodiments of any of the methods described herein, the method further includes monitoring the subject. For example, the method can include authorizing a refill of the CLK inhibitor administered to the subject between the first and second time points and determined to be effective.

In some embodiments of any of the methods of determining the efficacy of treatment described herein, the cancer cell is a small cell lung cancer cell, a colorectal cancer cell, a head and neck cancer cell, an ovarian cancer cell, a melanoma cell, a renal cell carcinoma cell, a pancreatic cancer cell, or a non-small cell lung cancer cell. In some embodiments of any of the methods of determining the efficacy of treatment described herein, the cancer can be any of the cancers described herein or known in the art.

Non-limiting types of Wnt pathway activity are described below and can be used in any of the methods of determining the efficacy of treatment described herein. For example, in some embodiments of any of the methods of determining the efficacy of treatment described herein, the Wnt pathway activity can be the level of CLK1, CLK2, CLK3, CLK4, or β-catenin expression, where an increase in the second level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of expression of CLK1, CLK2, CLK3, CLK4, or β-catenin protein or mRNA expression, as compared to the first level of CLK1, CLK2, CLK3, CLK4, or β-catenin protein or mRNA expression indicates that the CLK inhibitor was effective in the subject.

For example, in some embodiments of any of the methods of determining the efficacy of treatment described herein, the Wnt pathway activity can be the level of β-catenin in the nucleus of a mammalian cell, where an increase in the second level (e.g., an increase of 1% to 500%, or any of the subranges of this range described herein) of p-catenin in the nucleus of a mammalian cell as compared to the first level of β-catenin in the nucleus of a mammalian cell indicates that the CLK inhibitor was effective in the subject.

For example, in some embodiments of any of the methods of determining the efficacy of treatment described herein, the Wnt pathway activity can be detection of first and second levels of expression of one or more Wnt-regulated genes, where an decreased second level (e.g., a 1% to a 99% decrease, or any of the subranges of this range described herein) of expression of the one or more Wnt-regulated genes as compared to the first level of expression of the one or more Wnt-regulated genes indicates that the CLK inhibitor was effective in the subject. Non-limiting examples of Wnt-upregulated genes include CCND1, CSNK2A1 CXCL12, LRP5, MMP7, MMP9, LEF1, AXIN2, MYC, TCF7L2, TCF7, LRP6, DVL2, BIRC, ERRB2, MAPK8, PKN1, AXIN2, ABCB1, ADAM10, ALEX1, ASCL2, BAMBI, BCL2L2, BIRC5, BMI1, BMP4, CCND1, CD44, CDKN2A, CDX1, CEBPD, CLDN1, COX2, DNMT1, EDN1, EFNB1, ENC1, EPHB2, EPHB3, FGF18, FGFBP, FRA1, FSCN1, FZD6, FZD7, FZD8, GAST, HDAC3, HEF1, HES1, ID2, ITF2, JAG1, JUN, LICAM, LAMC2, LGR5, MENA, MET, MMP14, MYB, MYCBP, NOS2, NOTCH2, NRCAM, PLAU, PLAUR, PLCB4, PPARD, RUVBL1, S100A4, S100A6, SGK1, SMC3, SOX9, SP5, SRSF3, SUZ12, TCF1, TIAM1, TIMP-1. TN-C, VEGF, WNT-5a, WNT-5b, WNT11, and YAP.

In some embodiments of any of the methods of determining the efficacy of treatment described herein, the Wnt pathway activity can be detection of first and second levels of expression of one or more of APC, FRZB, CTGF, and GSK3B, where an increased (e.g., a 1% to a 500% increase or any of the subranges of this range described herein) second level of expression of the one or more of APC, FRZB, CTGF, and GSK3B, as compared to the first level of expression of one or more of APC, FRZB, CTGF, and GSK3B indicates that the CLK inhibitor was effective in the subject In some embodiments of any of the methods of determining the efficacy of treatment described herein, the cancer is a small cell lung cancer, a colorectal cancer, a head and neck cancer, an ovarian cancer, a melanoma, a renal cell carcinoma, a pancreatic cancer, or a non-small cell lung cancer.

Methods of Determining the Level of Wnt Pathway Activity

In some embodiments of any of the methods described herein, the level of Wnt pathway activity is the level of CLK1, CLK2, CLK3, CLK4, or β-catenin expression. In some embodiments, the level of CLK1, CLK2, CLK3, CLK4, or β-catenin expression is the level of CLK1, CLK2, CLK3, CLK4, or β-catenin protein in any of the cells described herein. In some embodiments, the level of CLK1, CLK2, CLK3, CLK4, or β-catenin expression is the level of CLK1, CLK2, CLK3, CLK4, or β-catenin mRNA in any of the cells described herein.

In some embodiments of any of the methods described herein, the level of Wnt pathway activity is the level of β-catenin in the nucleus of any of the cells described herein.

In some embodiments of any of the methods described herein, the Wnt pathway activity is detection of a mutation in a Wnt pathway gene selected from the group consisting of: gain-of-function mutation in a β-catenin gene (e.g., any of the exemplary gain-of-function mutations in a β-catenin gene described herein), a loss-of-function mutation in an AXIN gene, a loss-of-function mutation in an AXIN2 gene, a loss-of-function mutation in a APC gene, a loss-of-function mutation in a CTNNB1 gene, a loss-of-function mutation in a Tsc1 gene, a loss-of-function mutation in a Tsc2 gene, and a loss-of-function mutation GSK3D gene.

In some embodiments of any of the methods described herein, the Wnt pathway activity is an increased level of expression of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) Wnt-upregulated genes.

In some embodiments, the one or more Wnt-upregulated genes are selected from the group consisting of: cyclin D1 (CCND1), casein kinase 2 alpha 1 (CSNK2A1), C—X—C motif chemokine ligand 12 (CXCL12), low density lipoprotein receptor-related protein 5 (LRP5), matrix metallopeptidase 7 (MMP7), matrix metallopeptidase 9 (MMP9), lymphoid enhancer binding factor 1 (LEF1), axin 2 (AXIN2), MYC proto-oncogene (MYC), transcription factor 7 like 2 (TCF7L2), transcription factor 7 (TCF7), low density lipoprotein receptor-related protein 6 (LRP6), disheveled segment polarity protein 2 (DVL2), NLR family apoptosis inhibitory protein pseudogene (BIRC), estrogen-related receptor beta type 2 (ERRB2), mitogen-activated protein kinase 8 (MAPK8), protein kinase N1 (PKN1), axin 2 (AXIN2), ATP binding cassette subfamily B member 1 (ABCB1), a disintegrin and metallopeptidase domain 10 (ADAM1O), armadillo repeat containing X-linked 1 (ALEX1), achaete-scute family bHLH transcription factor 2 (ASCL2), BMP and activin membrane bound inhibitor (BAMBI), BLCL2-like 2 (BCL2L2), baculoviral IAP repeat containing 5 (BIRC5), BMI1 proto-oncogene (BMI1), bone morphogenetic protein 4 (BMP4), CCND1, CD44 molecule (CD44), cyclin dependent kinase inhibitor 2A (CDKN2A), caudal type homeobox 1 (CDX1), CCAAT enhancer binding protein delta (CEBPD), claudin 1 (CLDN1), cytochrome c oxidase subunit II (COX2), DNA methyltransferase I (DNMT1), endothelin 1 (EDN1), ephrin B1 (EFNB1), ectodermal-neural cortex 1 (ENC1), Eph receptor B2 (EPHB2), Eph receptor B3 (EPHB3), fibroblast growth factor 18 (FGF18), fibroblast growth factor binding protein 1 (FGFBP), FOS-like 1 (FRA1), facin actin-bundling protein 1 (FSCN1), frizzled class receptor 6 (FZD6), frizzled class receptor 7 (FZD7), frizzled class receptor 8 (FZD8), gastrin (GAST), histone deacetylase 3 (HDAC3), neural precursor cell expressed developmentally down-regulated 9 (HEF1), hes family bHLH transcription factor 1 (HES1), inhibitor of DNA binding 2 (ID2), transcription factor 4 (ITF2), jagged 1 (JAG1), Jun proto-oncogene (JUN), L1 cell adhesion molecule (LiCAM), laminin subunit gamma 2 (LAMC2), leucine rich containing G protein coupled receptor 5 (LGR5), ENAH (MENA), MET proto-oncogene (MET), matrix metallopeptidase 14 (MMP14), MYB proto-oncogene (MYB), MYC binding protein (MYCBP), nitric oxide synthase 2 (NOS2), notch 2 (NOTCH2), neuronal cell adhesion molecule (NRCAM), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), phospholipase C beta 4 (PLCB4), peroxisome proliferator activated receptor delta (PPARD), RuvB Like AAA ATPase 1 (RUVBL1), S100 calcium binding protein A4 (S100A4), S100 calcium binding protein A6 (S100A6), serum/glucocorticoid regulated kinase 1 (SGK1), structural maintenance of chromosomes 3 (SMC3), sex determining region Y-box 9 (SOX9), trans-acting transcription factor 5 (SP5), serine and arginine rich splicing factor 3 (SRSF3), SUZ12 polycomb repressive complex 2 subunit (SUZ12), HNF1 homeobox A (TCF1), T cell lymphoma invasion and metastasis 1 (TIAM1), tissue inhibitor of metalloproteinase 1 (TIMP-1), tenascin C (TN-C), vascular endothelial growth factor (VEGF), wingless-type family member 5A (WNT-5a), wingless-type family member 5B (WNT-5b), wingless-type family member 11 (WNT11), and Yes associated protein 1 (YAP).

In some embodiments of any of the methods described herein, the Wnt pathway activity is a decreased level of expression of one or more of APC Regulator of Wnt Signaling Pathway (APC), Frizzled Related Protein (FRZB), Connective Tissue Growth Factor (CTGF), and Glycogen Synthase Kinase 3 Beta (GSK3B).

In some embodiments, the Wnt pathway activity is the activity determined by assessing the expression level (e.g., protein or mRNA) of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) of: AXIN (NCBI Accession NG_012267.1), AXIN2 (NCBI Accession NG_012142.1), APC (NCBI Accession NG_008481.4), CSNK2A1 (NCBI Accession No. BC011668.2), CTGF (NCBI Accession AY395801.1), CTNNB1 (NCBI Accession NG_013302.2), Tsc1 (NCBI Accession NG_012386.1), Tsc2 (NCBI Accession NG_005895.1), GSK3β (NCBI Accession NG_012922.1), CCND1 (NCBI Accession NG_-7375.1), CXCL12 (NCBI Accession NG_016861.1), LRP5 (NCBI Accession NG_015835.1), MMP7 (NCBI Accession NM_002423.4), MMP9 (NCBI Accession NG_004994.2), LEF1 (NCBI Accession NG_015798.1), AXIN2 (NCBI Accession NG_012142.1), MYC (NCBI Accession NG_007161.2), TCF7L2 (NCBI Accession NG_012631.1), TCF7 (NCBI Accession NG_030367.1), LRP6 (NCBI Accession NG_01618.1), DVL2 (NCBI Accession NG_033038.1), BIRC (NCBI Accession NG_008752.1), ERRB2 (NCBI Accession NG_007503.1), MAPK8 (NCBI Accession NG_029053.2), PKN1 (NCBI Accession NG_), AXIN2 (NCBI Accession NG_00019.10), ABCB1 (NCBI Accession NG_011513.1), ADAM10 (NCBI Accession NG_033876.1), ALEX1 (NCBI Accession NG_015846.1), ASCL2 (NCBI Accession NM_005170.2), BAMBI (NCBI Accession NM_012342.2), BCL2L2 (NCBI Accession NM_001199839.1), BIRC5 (NCBI Accession NG_029069.1), BMI1 (NCBI Accession NM_005180.8), BMP4 (NCBI Accession NG_009215.1), CD44 (NCBI Accession NG_008937.1), CDKN2A (NCBI Accession NG_007485.1), CDX1 (NCBI Accession NG_046970.1), CEBPD (NCBI Accession NM_005195.3), CLDN1 (NCBI Accession NG_021418.1), COX2 (NCBI Accession NG_028206.2), DNMT1 (NCBI Accession NG_028016.3), EDN1 (NCBI Accession NG_016196.1), EFNB1 (NCBI Accession NG_008887.1), ENC1 (NCBI Accession NM_001256575.1), EPHB2 (NCBI Accession NG_011804.2), EPHB3 (NCBI Accession NM_004443.3), FGF18 (NCBI Accession NG_029158.1), FGFBP (NCBI Accession NM_005130.4), FRA1 (NCBI Accession NM_005438.4), FRZB (NCBI Accession NM_001463.4), FSCN (NCBI Accession NG_030004.1), FZD6 (NCBI Accession NM_003506.4), FZD7 (NCBI Accession NM_003507.1), FZD8 (NCBI Accession NG_029968.1), GAST (NCBI Accession NM_00805.4), GSK3B (NCBI Accession NM_002093.4), HDAC3 (NCBI Accession NM_001355039.2), HEF1 (NCBI Accession NM_006403.3), HES1 (NCBI Accession NM_005524.3), ID2 (NCBI Accession NM_002166.4), ITF2 (NCBI Accession NG_011716.2), JAG1 (NCBI Accession NG_007496.1), JUN (NCBI Accession NG_047027.1), LICAM (NCBI Accession NG_009645.3), LAMC2 (NCBI Accession NG_007079.2), LGR5 (NCBI Accession NM_003667.3), MENA (NCBI Accession NG_051578.1), MET (NCBI Accession NG_008996.1), MMP14 (NCBI Accession NG_046989.1), MYB (NCBI Accession NG_012330.1), MYCBP (NCBI Accession NM_012333.4), NOS2 (NCBI Accession NG_011470.1), NOTCH2 (NCBI Accession NG_008163.1), NRCAM (NCBI Accession NG_029898.1), PLAU (NCBI Accession NG_011904.1), PLAUR (NCBI Accession NG_032898.1), PLCB4 (NCBI Accession NM_000933.3), PPARD (NCBI Accession NG_012345.1), RUVBL1 (NCBI Accession NM_003707.3), S100A4 (NCBI Accession NG_027993.1), S100A6 (NCBI Accession NM_014624.3), SGK1 (NCBI Accession NM_005627.3), SMC3, (NCBI Accession NG_012217.1), SOX9 (NCBI Accession NG_012490.1), SP5 (NCBI Accession NM_001003845.2), SRSF3 (NCBI Accession NM_003017.4), SUZ12 (NCBI Accession NG_009237.1), TCF1 (NCBI Accession NG_011731.2), TIAM1 (NCBI Accession NM_001353693.1), TIMP-1 (NCBI Accession NG_012533.1), TN-C(NCBI Accession NG_029637.1), VEGF (NCBI Accession NG_008732.1), WNT-5a (NCBI Accession NG_031992.1), WNT-5b (NCBI Accession NM_032642.2), WNT11 (NCBI Accession NG_046931.1), YAP (NCBI Accession NG_029530.1), SRSF1 (NCBI Accession NM_006924.4), SRSF2 (NCBI Accession NG_032905.1), SRSF3 (NCBI Accession NM_003017.4), SF3B1 (NCBI Accession NG_032903.2), SRSF4 (NCBI Accession NM_005626.4), SRSF5 (NCBI Accession NM_001039465.1), SRSF6 (NCBI Accession NM_006275.5), SRSF10 (NCBI Accession NM_006625.5), U2AF1 (NCBI Accession NG_029455.1), and ZRSR2 (NCBI Accession NG_012746.1).

In any of the methods described herein, the level of at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25) Wnt pathway activity can be determined (e.g., in any combination).

The biological activity of the compounds described herein can be tested using any suitable assay known to those of skill in the art, see, e.g., WO 2001/053268 and WO 2005/009997. For example, the activity of a compound may be tested using one or more of the test methods outlined below.

In one example, tumor cells may be screened for Wnt independent growth. In such a method, tumor cells of interest are contacted with a compound (i.e. inhibitor) of interest, and the proliferation of the cells, e.g. by uptake of tritiated thymidine, is monitored. Non-limiting examples of assays that can be used to determine cell proliferation include: BrdU incorporation assay, EdU incorporation assay, MTT assay, XTT cell proliferation assay, proliferating cell nuclear antigen (PCNA) immunohistochemistry assay, Ki67 immunohistochemistry, minichromosome maintenance complex component 2 (MCM2) immunohistochemistry. In some embodiments, cell proliferation is determined by conducting a cell growth curve. In some embodiments, a proliferation assay is carried out using flow cytometry.

In some embodiments, tumor cells may be isolated from a candidate patient who has been screened for the presence of a cancer that is associated with a mutation in the Wnt signaling pathway. Candidate cancers include, without limitation, those described herein.

In another example, one may utilize in vitro assays for Wnt biological activity, e.g., stabilization of β-catenin and promoting growth of stem cells. Assays for biological activity of Wnt include stabilization of p-catenin, which can be measured, for example, by serial dilutions of a candidate inhibitor composition. An exemplary assay for Wnt biological activity contacts a candidate inhibitor with cells containing constitutively active Wnt/β-catenin signaling. The cells are cultured for a period of time sufficient to stabilize p-catenin, usually at least about 1 hour, and lysed. The cell lysate is resolved by SDS PAGE, then transferred to nitrocellulose and probed with antibodies specific for p-catenin.

In a further example, the activity of a candidate compound can be measured in a Xenopus secondary axis bioassay (Leyns, L. et al. Cell (1997), 88(6), 747-756).

In some embodiments, Wnt pathway activity is determined using a Wnt reporter assay. Briefly, cells are transfected with a reporter vector (e.g., a luciferase reporter) in which a reporter gene is operatively-linked to a gene regulatory element (e.g., a promoter, a responsive element) of a Wnt pathway target gene (e.g., TCF/LEF). Untransfected cells can serve as a negative control, while transfected cells cultured in the presence of a known Wnt pathway agonist (e.g., a Wnt pathway ligand) can serve as a positive control.

Determination of expression levels and/or detection of any of the mutations described herein may be performed by any suitable method including, but are not limited to, methods based on analyses of polynucleotide expression, sequencing of polynucleotides, and/or analyses of protein expression. For example, determination of expression levels may be performed by detecting the expression of mRNA expressed from the genes of interest, and/or by detecting the expression of a polypeptide encoded by the genes.

Commonly used methods for the analysis of polynucleotides (e.g., detection of any of the mutations described herein and/or detection of the expression level of any of the mRNAs described herein), include Southern blot analysis, Northern blot analysis, in situ hybridization, RNAse protection assays, and polymerase chain reaction (PCR)-based methods, such as reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), real-time PCR, TaqMan™, TaqMan™ low density array (TLDA), anchored PCR, competitive PCR, rapid amplification of cDNA ends (RACE), and microarray analyses. RT-PCR is a quantitative method that can be used to compare mRNA levels in different samples to examine gene expression profiles. A variation of RT-PCR is real time quantitative PCR, which measures PCR product accumulation through a dual-labeled fluorigenic probe (e.g., TaqMan™ probe). There are many other PCR-based techniques known to one of skill in the art, including but not limited to, differential display, amplified fragment length polymorphism, BeadArray™ technology, high coverage expression profiling (HiCEP) and digital PCR. Representative methods for sequencing-based gene expression analyses include Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS), and NexGen sequencing analysis, including mRNA sequencing.

In certain embodiments, the biomarker expression is determined using a qPCR assay. For example, total RNA is extracted from a fresh frozen (FF) tissue sample or total RNA is extracted from a macro-dissected formalin-fixed paraffin embedded (FFPE) tissue sample. The quantity and quality of the total RNA is assessed by standard spectrophotometry and/or any other appropriate method (e.g., an Agilent Bioanalyzer). Following RNA extraction, the RNA sample is reverse transcribed using standard methods and/or a commercially available cDNA synthesis kit (e.g., Roche Transcriptor First Strand cDNA synthesis kit). The resultant cDNA is pre-amplified using, for example, an ABI pre-amplification kit. Expression of the biomarker(s) are assessed on, for example, a Roche Lightcycler 480 system (Roche Diagnostics) using an ABI TaqMan Gene Expression Mastermix. qPCR reactions are performed in triplicate. For each assay a subset of the samples is run without reverse transcription (the RT-neg control), as well as, control samples run without template. A universal human reference RNA sample is included on each plate to act as a positive control. Suitable reference genes are identified from a standard panel of reference genes. Candidate reference genes are selected with different cellular functions to eliminate risk of co-regulation. The most suitable reference genes are evaluated and selected using specific software and algorithms (e.g., Genex software; GeNorm and Normfinder algorithms). The expression level of each biomarker is normalized using the selected optimum reference genes. In some embodiments, these normalized (or standardized) expression values for each biomarker are used to calculate the decision value of the sample. In some embodiments, these normalized (or standardized) expression values for each biomarker are used to calculate an expression level.

In some embodiments, the detection of any of the mutations described herein and/or detection of the level of any of the mRNAs described herein can be performed using a PCR-based assay comprising specific primers and/or probes. As used herein, the term “probe” refers to any molecule that is capable of selectively binding a specifically intended target biomolecule. Probes can be synthesized by one of skill in the art using known techniques or derived from biological preparations. Probes may include but are not limited to, RNA, DNA, proteins, peptides, aptamers, antibodies, and organic molecules. The term “primer” or “probe” encompasses oligonucleotides that have a sequence of a specific SEQ ID NO or oligonucleotides that have a sequence complementary to a specific SEQ ID NO. In some embodiments, the probe is modified. In some embodiments, the probe is modified with a quencher. In some embodiments, the probe is labeled. Labels can include, but are not limited to, colorimetric, fluorescent, chemiluminescent, or bioluminescent labels.

In some embodiments, the expression level of any of the proteins described herein can be determined by immunohistochemistry (IHC) of formalin fixed paraffin embedded tissue samples or overexpressed gene expression.

In some embodiments, the expression level of any of the mRNAs described herein can be determined by qPCR methods.

In some embodiments, the expression level of any of the proteins described herein or any of the phosphorylated proteins described herein can be determined from tumor biopsy samples by immunohistochemistry (IHC) of formalin fixed paraffin embedded tissue samples.

In some embodiments, the expression level of any of the mRNAs described herein can be determined from tumor biopsy samples by qPCR methods.

Commonly used methods for determining the level of any of the proteins described herein (or the level of any of the phosphorylated proteins described herein), include but are not limited to, immunohistochemistry (IHC)-based, antibody-based, and mass spectrometry-based methods. Antibodies, generally monoclonal antibodies, may be used to detect expression of a gene product (e.g., protein). In some embodiments, the antibodies can be detected by direct labeling of the antibodies themselves. In other embodiments, an unlabeled primary antibody is used in conjunction with a labeled secondary antibody Immunohistochemistry methods and/or kits are well known in the art and are commercially available.

In some embodiments, the level or expression level of any of the proteins described herein (or any of the phosphorylated proteins described herein) can be determined using methods known in the art, including but not limited to, multi-analyte profile test, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescent assay, immunohistochemical assay, dot blot assay, slot blot assay, and SDS-PAGE. In some embodiments, wherein an antibody is used in the assay the antibody is detectably labeled. The antibody labels may include, but are not limited to, immunofluorescent label, chemiluminescent label, phosphorescent label, enzyme label, radiolabel, avidin/biotin, colloidal gold particles, colored particles and magnetic particles.

Other suitable methods for determining the level of any of the proteins described herein (or any of the phosphorylated proteins described herein) include proteomics-based methods. Proteomics includes, among other things, study of the global changes of protein expression in a sample. In some embodiments, a proteomic method comprises the following steps: (1) separation of individual proteins in a sample by 2-D electrophoresis (2-D PAGE), (2) identification of individual proteins recovered from the gel (e.g., by mass spectrometry or N-terminal sequencing), and (3) analysis of the data using bioinformatics. In some embodiments, a proteomic method comprises using a tissue microarray (TMA). Tissue arrays may be constructed according to a variety of techniques known to one of skill in the art. In certain embodiments, a manual tissue arrayer is used to remove a “core” from a paraffin block prepared from a tissue sample. The core is then inserted into a separate paraffin block in a designated location on a grid. Cores from as many as about 400 samples can be inserted into a single recipient block. The resulting tissue array may be processed into thin sections for analysis. In some embodiments, a proteomic method comprises an antibody microarray. In some embodiments, a proteomic method comprises using mass spectrometry, including but not limited to, SELDI, MALDI, electro spray, and surface plasmon resonance methods. In some embodiments, a proteomic method comprises bead-based technology, including but not limited to, antibodies on beads in an array format. In some embodiments, the proteomic method comprises a reverse phase protein microarray (RPPM). In some embodiments, the proteomic method comprises multiplexed protein profiling, including but not limited to, the Global Proteome Survey (GPS) method.

In some embodiments, the level of expression of any of the mRNAs described herein in a mammalian cell (e.g., a cancer cell) obtained from the subject can be compared to a reference level of expression in a control cell (e.g., a non-cancerous cell or a healthy cell from the same subject or from a similar non-cancerous tissue from a similar subject) using gene microarray (e.g., Affimetrix chips). The comparison of the expression level of any of the mRNAs described herein in a cell obtained from a subject as compared to a reference level of expression in a control cell (e.g., a non-cancerous cell) can be determined from gene microarray using statistical methods. The statistical methods may include, but are not limited to, cluster analysis, supported vector machines (SVM) analysis, supported vector machines-recursive feature elimination (SVM-RFE) analysis, Platt scaling, neural networks, and other algorithms, t-test analysis, and paired-sample empirical Baysian analysis.

In some embodiments, the Wnt pathway activity is determined by Western blotting, immunohistochemistry, or immunofluorescence. For example, a readout for increased Wnt pathway activity can be an increase in the level of β-catenin (e.g., an increase in non-phosphorylated β-catenin), an increase in the phosphorylation of Dishevelled, or an increase in the phosphorylation of LRP.

Some embodiments of any of the methods described herein can include a step of performing an assay to determine a level or levels (e.g., a first and a second level) of a Wnt pathway gene in a cancer cell obtained from the subject at a first and a second time point. Non-limiting assays that may be used to detect a level or levels of a Wnt pathway gene are described herein. Additional assays that may be used to detect a level or levels of a Wnt pathway gene are known in the art.

Additional non-limiting assays that can be used to detect a level of a Wnt pathway protein include: immunohistochemistry, immunofluorescence, Western blotting, mass spectrometry, flow cytometry, immunoassays (e.g., sandwich enzyme-linked immunosorbent assays, enzyme-linked immunosorbent assays, and immunoprecipitation).

Additional non-limiting assays that can be used to detect a level of a Wnt pathway gene expression include: reverse transcription polymerase chain reaction (rt-PCR), real time quantitative reverse transcription polymerase chain reaction (qRT-PCR), microarray, next generation sequencing,

Reference Levels

In some embodiments of any of the methods described herein, the reference can be a corresponding level detected in a non-cancerous cell obtained from a subject (e.g., a non-cancerous cell from a similar non-cancerous tissue in a heathy subject who does not have a cancer and does not have a family history of cancer). For example, the reference level can be a corresponding level detected in a non-cancer cell of the same cell type as the cancerous cell. In some embodiments, the reference level can be a corresponding level detected in a non-cancerous skin cell (e.g., a melanocyte), and the cancer cell is a melanoma cell. In some embodiments, a reference level can be a corresponding level detected in a non-cancerous cell obtained from the breast, and the cancer cell is a breast cancer cell. In some embodiments, a reference level can be a corresponding level detected in a non-cancerous cell obtained from the prostate, and the cancer cell is a prostate cancer cell.

In some embodiments, a reference level can be a corresponding level detected in a non-cancerous cell obtained from the subject prior to the subject having been identified and/or diagnosed with a cancer (e.g., any of the cancers described herein). In some embodiments, a reference level can be a corresponding level in an intestinal stem cell (e.g., an intestinal stem cell obtained from the subject).

In some embodiments, a reference level can be a corresponding threshold level.

In some embodiments, a reference level can be a percentile value (e.g., mean value, 99% percentile, 95% percentile, 90% percentile, 85% percentile, 80% percentile, 75% percentile, 70% percentile, 65% percentile, 60% percentile, 55% percentile, or 50% percentile) of the corresponding levels detected in similar samples in a population of healthy subjects (e.g., subjects that are not diagnosed or identified as having a cancer (e.g., any of the cancers described herein), do not present with a symptom of cancer, and are not considered to have an elevated risk of developing cancer). In some embodiments, a reference level can be a threshold numerical value.

In some embodiments, a reference level can be a corresponding level detected in a similar sample obtained from the subject at an earlier time point.

Methods of Decreasing Activity of One or More of CLK1, CLK2, CLK3, and CLK4

Also provided herein are methods of decreasing (e.g., a 1% to 99% decrease, or any of the subranges of this range described herein) the activity of one or more of CLK1, CLK2, CLK3, and CLK4 that include: contacting one or more (e.g., one, two, three, or four) of CLK1, CLK2, CLK3, and CLK4 with an effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the method includes contacting one or both of CLK2 and CLK3 with an effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof.

Provided herein are methods of decreasing (e.g., a 1% to 99% decrease, or any of the subranges of this range described herein) the activity of one or more (e.g., one, two, three, or four) of CLK1, CLK2, CLK3 and CLK4 in a mammalian cell (e.g., any of the types of cells described herein, e.g., any of the types of cancer cells described herein) that include: contacting the mammalian cell with an effective amount of a CLK inhibitor or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the contacting results in a decrease in the activity of one or both of CLK2 and CLK3 in the mammalian cell. In some embodiments of any of the methods described herein, the mammalian cell is a cancer cell (e.g., any of the types of cancer cells described herein or known in the art). For example, the mammalian cell can be a cancer cell (e.g., any of the types of cancer cells described herein or known in the art) that has been identified as having an elevated level of Wnt pathway activity as compared to a reference level.

Various methods are known in the art to determine the activity of one or more (e.g., one, two, three, or four) of CLK1, CLK2, CLK3 and CLK4, including the methods described in the Examples).

CLKs

The CLK family of kinases contains four characterized isoforms (CLK1, CLK2, CLK3 and CLK4). CLKs are proposed to exert their function by directly phosphorylating serine and arginine rich splicing factor (SRSF) proteins. SRSFs are reported to play an important role in spliceosome assembly and regulation of alternative splicing and gene expression.

Exemplary human CLK1, CLK2, CLK3, and CLK4 protein sequences are SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, and 17. Exemplary cDNA sequences that encode CLK1, CLK2, CLK3, and CLK4 are SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

Human CLK1 protein isoform 1

(SEQ ID NO: 1)

MRHSKRTYCPDWDDKDWDYGKWRSSSSHKRRKRSHSSAQENKRCKYNHSKMCDSHYLESRSINEK

DYHSRRYIDEYRNDYTQGCEPGHRQRDHESRYQNHSSKSSGRSGRSSYKSKHRIHHSTSHRRSHG

KSHRRKRTRSVEDDEEGHLICQSGDVLSARYEIVDTLGEGAFGKVVECIDHKAGGRHVAVKIVKN

VDRYCEAARSEIQVLEHLNTTDPNSTFRCVQMLEWFEHHGHICIVFELLGLSTYDFIKENGFLPF

RLDHIRKMAYQICKSVNFLHSNKLTHTDLKPENILFVQSDYTEAYNPKIKRDERTLINPDIKVVD

FGSATYDDEHHSTLVSTRHYRAPEVILALGWSQPCDVWSIGCILIEYYLGFTVFPTHDSKEHLAM

MERILGPLPKHMIQKTRKRKYFHHDRLDWDEHSSAGRYVSRRCKPLKEFMLSQDVEHERL

FDLIQKMLEYDPAKRITLREALKHPFFDLLKKSI

Human CLK1 cDNA isoform 1

(SEQ ID NO: 2)

atgagacac tcaaagagaa cttactgtcc tgattgggat gacaaggatt gggattatgg

aaaatggagg agcagcagca gtcataaaag aaggaagaga tcacatagca gtgcccagga

gaacaagcgc tgcaaataca atcactctaa aatgtgtgat agccattatt tggaaagcag

gtctataaat gagaaagatt atcatagtcg acgctacatt gatgagtaca gaaatgacta

cactcaagga tgtgaacctg gacatcgcca aagagaccat gaaagccggt atcagaacca

tagtagcaag tcttctggta gaagtggaag aagtagttat aaaagcaaac acaggattca

ccacagtact tcacatcgtc gttcacatgg gaagagtcac cgaaggaaaa gaaccaggag

tgtagaggat gatgaggagg gtcacctgat ctgtcagagt ggagacgtac taagtgcaag

atatgaaatt gttgatactt taggtgaagg agcttttgga aaagttgtgg agtgcatcga

tcataaagcg ggaggtagac atgtagcagt aaaaatagtt aaaaatgtgg atagatactg

tgaagctgct cgctcagaaa tacaagttct ggaacatctg aatacaacag accccaacag

tactttccgc tgtgtccaga tgttggaatg gtttgagcat catggtcaca tttgcattgt

ttttgaacta ttgggactta gtacttacga cttcattaaa gaaaatggtt ttctaccatt

tcgactggat catatcagaa agatggcata tcagatatgc aagtctgtga attttttgca

cagtaataag ttgactcaca cagacttaaa gcctgaaaac atcttatttg tgcagtctga

ctacacagag gcgtataatc ccaaaataaa acgtgatgaa cgcaccttaa taaatccaga

tattaaagtt gtagactttg gtagtgcaac atatgatgac gaacatcaca gtacattggt

atctacaaga cattatagag cacctgaagt tattttagcc ctagggtggt cccaaccatg

tgatgtctgg agcataggat gcattcttat tgaatactat cttgggttta ccgtatttcc

aacacacgat agtaaggagc atttagcaat gatggaaagg attcttggac ctctaccaaa

acatatgata cagaaaacca ggaaacgtaa atattttcac cacgatcgat tagactggga

tgaacacagt tctgccggca gatatgtttc aagacgctgt aaacctctga aggaatttat

gctttctcaa gatgttgaac atgagcgtct ctttgacctc attcagaaaa tgttggagta

tgatccagcc aaaagaatta ctctcagaga agccttaaag catcctttct ttgaccttct

gaagaaaagt atatag

Human CLK1 protein isoform 2

(SEQ ID NO: 3)

MAAGRRPASALWPERRGSPLRGDLLGFQNVREPSSCGETLSGMRHSKRTYCPDWDDKDWDYGKWR

SSSSHKRRKRSHSSAQENKRCKYNHSKMCDSHYLESRSINEKDYHSRRYIDEYRNDYTQGCEPGH

RQRDHESRYQNHSSKSSGRSGRSSYKSKHRIHHSTSHRRSHGKSHRRKRIRSVEDDEEGHLICQS

GDVLSARYEIVDTLGEGAFGKVVECIDHKAGGRHVAVKIVKNVDRYCEAARSEIQVLEHLNTTDP

NSTFRCVQMLEWFEHHGHICIVFELLGLSTYDFIKENGFLPFRLDHIRKMAYQICKSVNFLHSNK

LTHTDLKPENILFVQSDYTEAYNPKIKRDERTLINPDIKVVDFGSATYDDEHHSTLVSTRHYRAP

EVILALGWSQPCDVWSIGCILIEYYLGFTVFPTHDSKEHLAMMERILGPLPKHMIQKTRKRKYFH

HDRLDWDEHSSAGRYVSRRCKPLKEFMLSQDVEHERLFDLIQKMLEYDPAKRI

TLREALKHPFFDLLKKSI

Human CLK1 cDNA isoform 2

(SEQ ID NO: 4)

atggc ggctgggcgg aggccggctt cggccctgtg gccggaaagg cgaggctccc

cgttgagggg ggatttgctg gggttccaga atgtgcgtga gccaagcagc tgtggggaaa

cgttgtctgg aatgagacac tcaaagagaa cttactgtcc tgattgggat gacaaggatt

gggattatgg aaaatggagg agcagcagca gtcataaaag aaggaagaga tcacatagca

gtgcccagga gaacaagcgc tgcaaataca atcactctaa aatgtgtgat agccattatt

tggaaagcag gtctataaat gagaaagatt atcatagtcg acgctacatt gatgagtaca

gaaatgacta cactcaagga tgtgaacctg gacatcgcca aagagaccat gaaagccggt

atcagaacca tagtagcaag tcttctggta gaagtggaag aagtagttat aaaagcaaac

acaggattca ccacagtact tcacatcgtc gttcacatgg gaagagtcac cgaaggaaaa

gaaccaggag tgtagaggat gatgaggagg gtcacctgat ctgtcagagt ggagacgtac

taagtgcaag atatgaaatt gttgatactt taggtgaagg agcttttgga aaagttgtgg

agtgcatcga tcataaagcg ggaggtagac atgtagcagt aaaaatagtt aaaaatgtgg

atagatactg tgaagctgct cgctcagaaa tacaagttct ggaacatctg aatacaacag

accccaacag tactttccgc tgtgtccaga tgttggaatg gtttgagcat catggtcaca

tttgcattgt ttttgaacta ttgggactta gtacttacga cttcattaaa gaaaatggtt

ttctaccatt tcgactggat catatcagaa agatggcata tcagatatgc aagtctgtga

attttttgca cagtaataag ttgactcaca cagacttaaa gcctgaaaac atcttatttg

tgcagtctga ctacacagag gcgtataatc ccaaaataaa acgtgatgaa cgcaccttaa

taaatccaga tattaaagtt gtagactttg gtagtgcaac atatgatgac gaacatcaca

gtacattggt atctacaaga cattatagag cacctgaagt tattttagcc ctagggtggt

cccaaccatg tgatgtctgg agcataggat gcattcttat tgaatactat cttgggttta

ccgtatttcc aacacacgat agtaaggagc atttagcaat gatggaaagg attcttggac

ctctaccaaa acatatgata cagaaaacca ggaaacgtaa atattttcac cacgatcgat

tagactggga tgaacacagt tctgccggca gatatgtttc aagacgctgt aaacctctga

aggaatttat gctttctcaa gatgttgaac atgagcgtct ctttgacctc attcagaaaa

tgttggagta tgatccagcc aaaagaatta ctctcagaga agccttaaag catcctttct

ttgaccttct gaagaaaagt atatag

Human CLK2 protein isoform 1

(SEQ ID NO: 5)

MPHPRRYHSSERGSRGSYREHYRSRKHKRRRSRSWSSSSDRTRRRRREDSYHVRSRSSYDDRSSD

RRVYDRRYCGSYRRNDYSRDRGDAYYDTDYRHSYEYQRENSSYRSQRSSRRKHRRRRRRSRTFSR

SSSQHSSRRAKSVEDDAEGHLIYHVGDWLQERYEIVSTLGEGTFGRVVQCVDHRRGGARVALKII

KNVEKYKEAARLEINVLEKINEKDPDNKNLCVQMFDWFDYHGHMCISFELLGLSTFDFLKDNNYL

PYPIHQVRHMAFQLCQAVKFLHDNKLTHTDLKPENILFVNSDYELTYNLEKKRDERSVKSTAVRV

VDFGSATFDHEHHSTIVSTRHYRAPEVILELGWSQPCDVWSIGCIIFEYYVGFTLFQTHDNREHL

AMMERILGPIPSRMIRKTRKQKYFYRGRLDWDENTSAGRYVRENCKPLRRYLTSEAEEHHQLFDL

IESMLEYEPAKRLTLGEALQHPFFARLRAEPPNKLWDSSRDISR

Human CLK2 cDNA isoform 1

(SEQ ID NO: 6)

a tgccgcatcc tcgaaggtac cactcctcag agcgaggcag ccgggggagt

taccgtgaac actatcggag ccgaaagcat aagcgacgaa gaagtcgctc ctggtcaagt

agtagtgacc ggacacgacg gcgtcggcga gaggacagct accatgtccg ttctcgaagc

agttatgatg atcgttcgtc cgaccggagg gtgtatgacc ggcgatactg tggcagctac

agacgcaacg attatagccg ggatcgggga gatgcctact atgacacaga ctatcggcat

tcctatgaat atcagcggga gaacagcagt taccgcagcc agcgcagcag ccggaggaag

cacagacggc ggaggaggcg cagccggaca tttagccgct catcttcgca gcacagcagc

cggagagcca agagtgtaga ggacgacgct gagggccacc tcatctacca cgtcggggac

tggctacaag agcgatatga aatcgttagc accttaggag aggggacctt cggccgagtt

gtacaatgtg ttgaccatcg caggggtggg gctcgagttg ccctgaagat cattaagaat

gtggagaagt acaaggaagc agctcgactt gagatcaacg tgctagagaa aatcaatgag

aaagaccctg acaacaagaa cctctgtgtc cagatgtttg actggtttga ctaccatggc

cacatgtgta tctcctttga gcttctgggc cttagcacct tcgatttcct caaagacaac

aactacctgc cctaccccat ccaccaagtg cgccacatgg ccttccagct gtgccaggct

gtcaagttcc tccatgataa caagctgaca catacagacc tcaagcctga aaatattctg

tttgtgaatt cagactatga gctcacctac aacctagaga agaagcgaga tgagcgcagt

gtgaagagca cagctgtgcg ggtggtagac tttggcagtg ccacctttga ccatgagcac

catagcacca ttgtctccac tcgccattac cgagcaccag aagtcatcct tgagttgggc

tggtcacagc cttgtgatgt gtggagtata ggctgcatca tctttgaata ctatgtggga

ttcaccctct tccagaccca tgacaacaga gagcatctag ccatgatgga aaggatcttg

ggtcctatcc cttcccggat gatccgaaag acaagaaagc agaaatattt ttaccggggt

cgcctggatt gggatgagaa cacatcagct gggcgctatg ttcgtgagaa ctgcaaaccg

ctgcggcggt atctgacctc agaggcagag gaacaccacc agctcttcga tctgattgaa

agcatgctag agtatgaacc agctaagcgg ctgaccttgg gtgaagccct tcagcatcct

ttcttcgccc gccttcgggc tgagccgccc aacaagttgt gggactccag tcgggatatc

agtcggtga

Human CLK2 protein isoform 2

(SEQ ID NO: 7)

MPHPRRYHSSERGSRGSYREHYRSRKHKRRRSRSWSSSSDRTRRRRREDSYHVRSRSSYDDRSSD

RRVYDRRYCGSYRRNDYSRDRGDAYYDTDYRHSYEYQRENSSYRSQRSSRRKHRRRRRRSRTFSR

SSSHSSRRAKSVEDDAEGHLIYHVGDWLQERYEIVSTLGEGTFGRVVQCVDHRRGGARVALKIIK

NVEKYKEAARLEINVLEKINEKDPDNKNLCVQMFDWFDYHGHMCISFELLGLSTFDFLKDNNYLP

YPIHQVRHMAFQLCQAVKFLHDNKLTHTDLKPENILFVNSDYELTYNLEKKRDERSVKSTAVRVV

DFGSATFDHEHHSTIVSTRHYRAPEVILELGWSQPCDVWSIGCIIFEYYVGFTLFQTHDNREHLA

MMERILGPIPSRMIRKTRKQKYFYRGRLDWDENTSAGRYVRENCKPLRRYLTSEAEEHHQLFDLI

ESMLEYEPAKRLTLGEALQHPFFARLRAEPPNKLWDSSRDISR

Human CLK2 cDNA isoform 2

(SEQ ID NO: 8)

a tgccgcatcc tcgaaggtac cactcctcag agcgaggcag ccgggggagt

taccgtgaac actatcggag ccgaaagcat aagcgacgaa gaagtcgctc ctggtcaagt

agtagtgacc ggacacgacg gcgtcggcga gaggacagct accatgtccg ttctcgaagc

agttatgatg atcgttcgtc cgaccggagg gtgtatgacc ggcgatactg tggcagctac

agacgcaacg attatagccg ggatcgggga gatgcctact atgacacaga ctatcggcat

tcctatgaat atcagcggga gaacagcagt taccgcagcc agcgcagcag ccggaggaag

cacagacggc ggaggaggcg cagccggaca tttagccgct catcttcgca cagcagccgg

agagccaaga gtgtagagga cgacgctgag ggccacctca tctaccacgt cggggactgg

ctacaagagc gatatgaaat cgttagcacc ttaggagagg ggaccttcgg ccgagttgta

caatgtgttg accatcgcag gggtggggct cgagttgccc tgaagatcat taagaatgtg

gagaagtaca aggaagcagc tcgacttgag atcaacgtgc tagagaaaat caatgagaaa

gaccctgaca acaagaacct ctgtgtccag atgtttgact ggtttgacta ccatggccac

atgtgtatct cctttgagct tctgggcctt agcaccttcg atttcctcaa agacaacaac

tacctgccct accccatcca ccaagtgcgc cacatggcct tccagctgtg ccaggctgtc

aagttcctcc atgataacaa gctgacacat acagacctca agcctgaaaa tattctgttt

gtgaattcag actatgagct cacctacaac ctagagaaga agcgagatga gcgcagtgtg

aagagcacag ctgtgcgggt ggtagacttt ggcagtgcca cctttgacca tgagcaccat

agcaccattg tctccactcg ccattaccga gcaccagaag tcatccttga gttgggctgg

tcacagcctt gtgatgtgtg gagtataggc tgcatcatct ttgaatacta tgtgggattc

accctcttcc agacccatga caacagagag catctagcca tgatggaaag gatcttgggt

cctatccctt cccggatgat ccgaaagaca agaaagcaga aatattttta ccggggtcgc

ctggattggg atgagaacac atcagctggg cgctatgttc gtgagaactg caaaccgctg

cggcggtatc tgacctcaga ggcagaggaa caccaccagc tcttcgatct gattgaaagc

atgctagagt atgaaccagc taagcggctg accttgggtg aagcccttca gcatcctttc

ttcgcccgcc ttcgggctga gccgcccaac aagttgtggg actccagtcg ggatatcagt

cggtga

Human CLK2 protein isoform 3

(SEQ ID NO: 9)

MFDWFDYHGHMCISFELLGLSTFDFLKDNNYLPYPIHQVRHMAFQLCQAVKFLHDNKLTHTDLKP

ENILFVNSDYELTYNLEKKRDERSVKSTAVRVVDEGSATFDHEHHSTIVSTRHYRAPEVILELGW

SQPCDVWSIGCIIFEYYVGFTLFQTHDNREHLAMMERILGPIPSRMIRKTRKQKYFYRGRLDWDE

NTSAGRYVRENCKPLRRYLTSEAEEHHQLFDLIESMLEYEPAKRLTLGEALQHPFFARLRAEPPN

KLWDSSRDISR

Human CLK2 cDNA isoform 3

(SEQ ID NO: 10)

atgtt tgactggttt gactaccatg gccacatgtg tatctccttt gagcttctgg

gccttagcac cttcgatttc ctcaaagaca acaactacct gccctacccc atccaccaag

tgcgccacat ggccttccag ctgtgccagg ctgtcaagtt cctccatgat aacaagctga

cacatacaga cctcaagcct gaaaatattc tgtttgtgaa ttcagactat gagctcacct

acaacctaga gaagaagcga gatgagcgca gtgtgaagag cacagctgtg cgggtggtag

actttggcag tgccaccttt gaccatgagc accatagcac cattgtctcc actcgccatt

accgagcacc agaagtcatc cttgagttgg gctggtcaca gccttgtgat gtgtggagta

taggctgcat catctttgaa tactatgtgg gattcaccct cttccagacc catgacaaca

gagagcatct agccatgatg gaaaggatct tgggtcctat cccttcccgg atgatccgaa

agacaagaaa gcagaaatat ttttaccggg gtcgcctgga ttgggatgag aacacatcag

ctgggcgcta tgttcgtgag aactgcaaac cgctgcggcg gtatctgacc tcagaggcag

aggaacacca ccagctcttc gatctgattg aaagcatgct agagtatgaa ccagctaagc

ggctgacctt gggtgaagcc cttcagcatc ctttcttcgc ccgccttcgg gctgagccgc

ccaacaagtt gtgggactcc agtcgggata tcagtcggtg a

Human CLK2 protein isoform 4

(SEQ ID NO: 11)

MPHPRRYHSSERGSRGSYREHYRSRKHKRRRSRSWSSSSDRTRRRRREDSYHVRSRSYDDRSSDR

RVYDRRYCGSYRRNDYSRDRGDAYYDTDYRHSYEYQRENSSYRSQRSSRRKHRRRRRRSRTFSRS

SSHSSRRAKSVEDDAEGHLIYHVGDWLQERYEIVSTLGEGTFGRVVQCVDHRRGGARVALKIIKN

VEKYKEAARLEINVLEKINEKDPDNKNLCVQMFDWFDYHGHMCISFELLGLSTFDFLKDNNYLPY

PIHQVRHMAFQLCQAVKFLHDNKLTHTDLKPENILFVNSDYELTYNLEKKRDERSVKSTAVRVVD

FGSATFDHEHHSTIVSTRHYRAPEVILELGWSQPCDVWSIGCIIFEYYVGFTLFQTHDNREHLAM

MERILGPIPSRMIRKTRKQKYFYRGRLDWDENTSAGRYVRENCKPLRRYLTSEAEEHHQLFDLIE

SMLEYEPAKRLTLGEALQHPFFARLRAEPPNKLWDSSRDISR

Human CLK2 cDNA isoform 4

(SEQ ID NO: 12)

a tgccgcatcc tcgaaggtac cactcctcag agcgaggcag ccgggggagt

taccgtgaac actatcggag ccgaaagcat aagcgacgaa gaagtcgctc ctggtcaagt

agtagtgacc ggacacgacg gcgtcggcga gaggacagct accatgtccg ttctcgaagt

tatgatgatc gttcgtccga ccggagggtg tatgaccggc gatactgtgg cagctacaga

cgcaacgatt atagccggga tcggggagat gcctactatg acacagacta tcggcattcc

tatgaatatc agcgggagaa cagcagttac cgcagccagc gcagcagccg gaggaagcac

agacggcgga ggaggcgcag ccggacattt agccgctcat cttcgcacag cagccggaga

gccaagagtg tagaggacga cgctgagggc cacctcatct accacgtcgg ggactggcta

caagagcgat atgaaatcgt tagcacctta ggagagggga ccttcggccg agttgtacaa

tgtgttgacc atcgcagggg tggggctcga gttgccctga agatcattaa gaatgtggag

aagtacaagg aagcagctcg acttgagatc aacgtgctag agaaaatcaa tgagaaagac

cctgacaaca agaacctctg tgtccagatg tttgactggt ttgactacca tggccacatg

tgtatctcct ttgagcttct gggccttagc accttcgatt tcctcaaaga caacaactac

ctgccctacc ccatccacca agtgcgccac atggccttcc agctgtgcca ggctgtcaag

ttcctccatg ataacaagct gacacataca gacctcaagc ctgaaaatat tctgtttgtg

aattcagact atgagctcac ctacaaccta gagaagaagc gagatgagcg cagtgtgaag

agcacagctg tgcgggtggt agactttggc agtgccacct ttgaccatga gcaccatagc

accattgtct ccactcgcca ttaccgagca ccagaagtca tccttgagtt gggctggtca

cagccttgtg atgtgtggag tataggctgc atcatctttg aatactatgt gggattcacc

ctcttccaga cccatgacaa cagagagcat ctagccatga tggaaaggat cttgggtcct

atcccttccc ggatgatccg aaagacaaga aagcagaaat atttttaccg gggtcgcctg

gattgggatg agaacacatc agctgggcgc tatgttcgtg agaactgcaa accgctgcgg

cggtatctga cctcagaggc agaggaacac caccagctct tcgatctgat tgaaagcatg

ctagagtatg aaccagctaa gcggctgacc ttgggtgaag cccttcagca tcctttcttc

gcccgccttc gggctgagcc gcccaacaag ttgtgggact ccagtcggga tatcagtcgg

tga

Human CLK3 protein isoform 1

(SEQ ID NO: 13)

MPVLSARRRELADHAGSGRRSGPSPTARSGPHLSALRAQPARAAHLSGRGTYVRRDTAGGGPGQA

RPLGPPGTSLLGRGARRSGEGWCPGAFESGARAARPPSRVEPRLATAASREGAGLPRAEVAAGSG

RGARSGEWGLAAAGAWETMHHCKRYRSPEPDPYLSYRWKRRRSYSREHEGRLRYPSRREPPPRRS

RSRSHDRLPYQRRYRERRDSDTYRCEERSPSFGEDYYGPSRSRHRRRSRERGPYRTRKHAHHCHK

RRTRSCSSASSRSQQSSKRSSRSVEDDKEGHLVCRIGDWLQERYEIVGNLGEGTFGKVVECLDHA

RGKSQVALKIIRNVGKYREAARLEINVLKKIKEKDKENKFLCVLMSDWFNFHGHMCIAFELLGKN

TFEFLKENNFQPYPLPHVRHMAYQLCHALRFLHENQLTHTDLKPENILFVNSEFETLYNEHKSCE

EKSVKNTSIRVADFGSATFDHEHHTTIVATRHYRPPEVILELGWAQPCDVWSIGCILFEYYRGFT

LFQTHENREHLVMMEKILGPIPSHMIHRTRKQKYFYKGGLVWDENSSDGRYVKENCKPLKSYMLQ

DSLEHVQLFDLMRRMLEFDPAQRITLAEALLHPFFAGLT PEERSFHTSRNPSR

Human CLK3 cDNA isoform 1

(SEQ ID NO: 14)

a atgcccgtc ctctccgcgc gcaggaggga gttggcggac cacgcggggt cggggcgacg

gagcgggccc agccccacgg ccaggtcggg gccccacctc tcggctctga gagcccagcc

ggcccgggcc gcgcacctgt caggtcgggg gacctacgtg cgccgcgaca cggcgggagg

cgggccgggc caggctcgtc ccctcggccc tcccggaact agtctcctag gccgcggcgc

ccgccggagc ggagagggct ggtgccccgg agccttcgag tcgggggcta gagcggccag

gcctccgagc cgggtcgagc cgaggctggc gacggctgcg tcacgcgagg gggcggggct

gccacgggcg gaggtcgcag ccggaagcgg aagaggcgct cggagcgggg agtggggcct

agctgcagcc ggagcctggg agacgatgca tcactgtaag cgataccgct cccctgaacc

agacccgtac ctgagctacc gatggaagag gaggaggtcc tacagtcggg aacatgaagg

gagactgcga tacccgtccc gaagggagcc tcccccacga agatctcggt ccagaagcca

tgaccgcctg ccctaccaga ggaggtaccg ggagcgccgt gacagcgata cataccggtg

tgaagagcgg agcccatcct ttggagagga ctactatgga ccttcacgtt ctcgtcatcg

tcggcgatcg cgggagaggg ggccataccg gacccgcaag catgcccacc actgccacaa

acgccgcacc aggtcttgta gcagcgcctc ctcgagaagc caacagagca gtaagcgcag

cagccggagt gtggaagatg acaaggaggg tcacctggtg tgccggatcg gcgattggct

ccaagagcga tatgagattg tggggaacct gggtgaaggc acctttggca aggtggtgga

gtgcttggac catgccagag ggaagtctca ggttgccctg aagatcatcc gcaacgtggg

caagtaccgg gaggctgccc ggctagaaat caacgtgctc aaaaaaatca aggagaagga

caaagaaaac aagttcctgt gtgtcttgat gtctgactgg ttcaacttcc acggtcacat

gtgcatcgcc tttgagctcc tgggcaagaa cacctttgag ttcctgaagg agaataactt

ccagccttac cccctaccac atgtccggca catggcctac cagctctgcc acgcccttag

atttctgcat gagaatcagc tgacccatac agacttgaaa ccagagaaca tcctgtttgt

gaattctgag tttgaaaccc tctacaatga gcacaagagc tgtgaggaga agtcagtgaa

gaacaccagc atccgagtgg ctgactttgg cagtgccaca tttgaccatg agcaccacac

caccattgtg gccacccgtc actatcgccc gcctgaggtg atccttgagc tgggctgggc

acagccctgt gacgtctgga gcattggctg cattctcttt gagtactacc ggggcttcac

actcttccag acccacgaaa accgagagca cctggtgatg atggagaaga tcctagggcc

catcccatca cacatgatcc accgtaccag gaagcagaaa tatttctaca aagggggcct

agtttgggat gagaacagct ctgacggccg gtatgtgaag gagaactgca aacctctgaa

gagttacatg ctccaagact ccctggagca cgtgcagctg tttgacctga tgaggaggat

gttagaattt gaccctgccc agcgcatcac actggccgag gccctgctgc accccttctt

tgctggcctg acccctgagg agcggtcctt ccacaccagc cgcaacccaa gcagatga

Human CLK3 protein isoform 2

(SEQ ID NO: 15)

MHHCKRYRSPEPDPYLSYRWKRRRSYSREHEGRLRYPSRREPPPRRSRSRSHDRLPYQRRYRERR

DSDTYRCEERSPSFGEDYYGPSRSRHRRRSRERGPYRTRKHAHHCHKRRTRSCSSASSRSQQSSK

RSSRSVEDDKEGHLVCRIGDWLQERYEIVGNLGEGTFGKVVECLDHARGKSQVALKIIRNVGKYR

EAARLEINVLKKIKEKDKENKFLCVLMSDWFNFHGHMCIAFELLGKNTFEFLKENNFQPYPLPHV

RHMAYQLCHALRFLHENQLTHTDLKPENILFVNSEFETLYNEHKSCEEKSVKNTSIRVADFGSAT

FDHEHHTTIVATRHYRPPEVILELGWAQPCDVWSIGCILFEYYRGFTLFQTHENREHLVMMEKIL

GPIPSHMIHRTRKQKYFYKGGLVWDENSSDGRYVKENCKPLKSYMLQDSLEHVQLFDLMR

RMLEFDPAQRITLAEALLHPFFAGLTPEERSFHTSRNPSR

Human CLK3 cDNA isoform 2

(SEQ ID NO: 16)

atgca tcactgtaag cgataccgct cccctgaacc agacccgtac ctgagctacc

gatggaagag gaggaggtcc tacagtcggg aacatgaagg gagactgcga tacccgtccc

gaagggagcc tcccccacga agatctcggt ccagaagcca tgaccgcctg ccctaccaga

ggaggtaccg ggagcgccgt gacagcgata cataccggtg tgaagagcgg agcccatcct

ttggagagga ctactatgga ccttcacgtt ctcgtcatcg tcggcgatcg cgggagaggg

ggccataccg gacccgcaag catgcccacc actgccacaa acgccgcacc aggtcttgta

gcagcgcctc ctcgagaagc caacagagca gtaagcgcag cagccggagt gtggaagatg

acaaggaggg tcacctggtg tgccggatcg gcgattggct ccaagagcga tatgagattg

tggggaacct gggtgaaggc acctttggca aggtggtgga gtgcttggac catgccagag

ggaagtctca ggttgccctg aagatcatcc gcaacgtggg caagtaccgg gaggctgccc

ggctagaaat caacgtgctc aaaaaaatca aggagaagga caaagaaaac aagttcctgt

gtgtcttgat gtctgactgg ttcaacttcc acggtcacat gtgcatcgcc tttgagctcc

tgggcaagaa cacctttgag ttcctgaagg agaataactt ccagccttac cccctaccac

atgtccggca catggcctac cagctctgcc acgcccttag atttctgcat gagaatcagc

tgacccatac agacttgaaa ccagagaaca tcctgtttgt gaattctgag tttgaaaccc

tctacaatga gcacaagagc tgtgaggaga agtcagtgaa gaacaccagc atccgagtgg

ctgactttgg cagtgccaca tttgaccatg agcaccacac caccattgtg gccacccgtc

actatcgccc gcctgaggtg atccttgagc tgggctgggc acagccctgt gacgtctgga

gcattggctg cattctcttt gagtactacc ggggcttcac actcttccag acccacgaaa

accgagagca cctggtgatg atggagaaga tcctagggcc catcccatca cacatgatcc

accgtaccag gaagcagaaa tatttctaca aagggggcct agtttgggat gagaacagct

ctgacggccg gtatgtgaag gagaactgca aacctctgaa gagttacatg ctccaagact

ccctggagca cgtgcagctg tttgacctga tgaggaggat gttagaattt gaccctgccc

agcgcatcac actggccgag gccctgctgc accccttctt tgctggcctg acccctgagg

agcggtcctt ccacaccagc cgcaacccaa gcagatga

Human CLK4 protein

(SEQ ID NO: 17)

MRHSKRTHCPDWDSRESWGHESYRGSHKRKRRSHSSTQENRHCKPHHQFKESDCHYLEARSLNER

DYRDRRYVDEYRNDYCEGYVPRHYHRDIESGYRIHCSKSSVRSRRSSPKRKRNRHCSSHQSRSKS

HRRKRSRSIEDDEEGHLICQSGDVLRARYEIVDTLGEGAFGKVVECIDHGMDGMHVAVKIVKNVG

RYREAARSEIQVLEHLNSTDPNSVERCVQMLEWFDHHGHVCIVFELLGLSTYDFIKENSFLPFQI

DHIRQMAYQICQSINFLHHNKLTHTDLKPENILFVKSDYVVKYNSKMKRDERTLKNIDIKVVDFG

SATYDDEHHSTLVSTRHYRAPEVILALGWSQPCDVWSIGCILIEYYLGFTVFQTHDSKEHLAMME

RILGPIPQHMIQKTRKRKYFHHNQLDWDEHSSAGRYVRRRCKPLKEFMLCHDEEHEKLFD

LVRRMLEYDPTQRITLDEALQHPFFDLLKKK

Human CLK4 cDNA

(SEQ ID NO: 18)

at gcggcattcc aaaagaactc actgtcctga ttgggatagc agagaaagct

ggggacatga aagctatcgt ggaagtcaca agcggaagag gagatctcat agtagcacac

aagagaacag gcattgtaaa ccacatcacc agtttaaaga atctgattgt cattatttag

aagcaaggtc cttgaatgag cgagattatc gggaccggag atacgttgac gaatacagga

atgactactg tgaaggatat gttcctagac attatcacag agacattgaa agcgggtatc

gaatccactg cagtaaatct tcagtccgca gcaggagaag cagtcctaaa aggaagcgca

atagacactg ttcaagtcat cagtcacgtt cgaagagcca ccgaaggaaa agatccagga

gtatagagga tgatgaggag ggtcacctga tctgtcaaag tggagacgtt ctaagagcaa

gatatgaaat cgtggacact ttgggtgaag gagcctttgg caaagttgta gagtgcattg

atcatggcat ggatggcatg catgtagcag tgaaaatcgt aaaaaatgta ggccgttacc

gtgaagcagc tcgttcagaa atccaagtat tagagcactt aaatagtact gatcccaata

gtgtcttccg atgtgtccag atgctagaat ggtttgatca tcatggtcat gtttgtattg

tgtttgaact actgggactt agtacttacg atttcattaa agaaaacagc tttctgccat

ttcaaattga ccacatcagg cagatggcgt atcagatctg ccagtcaata aattttttac

atcataataa attaacccat acagatctga agcctgaaaa tattttgttt gtgaagtctg

actatgtagt caaatataat tctaaaatga aacgtgatga acgcacactg aaaaacacag

atatcaaagt tgttgacttt ggaagtgcaa cgtatgatga tgaacatcac agtactttgg

tgtctacccg gcactacaga gctcccgagg tcattttggc tttaggttgg tctcagcctt

gtgatgtttg gagcataggt tgcattctta ttgaatatta ccttggtttc acagtctttc

agactcatga tagtaaagag cacctggcaa tgatggaacg aatattagga cccataccac

aacacatgat tcagaaaaca agaaaacgca agtattttca ccataaccag ctagattggg

atgaacacag ttctgctggt agatatgtta ggagacgctg caaaccgttg aaggaattta

tgctttgtca tgatgaagaa catgagaaac tgtttgacct ggttcgaaga atgttagaat

atgatccaac tcaaagaatt accttggatg aagcattgca gcatcctttc tttgacttat

taaaaaagaa atga

Methods of Altering mRNA Splicing

Also provided herein are methods of altering mRNA splicing in a mammalian cell (e.g., any of the exemplary mammalian cells described herein, e.g., any of the exemplary types of cancer cells described herein) having aberrant mRNA splicing activity that include: contacting the mammalian cell with an effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvate thereof.

In some aspects, the mammalian cell is a cancer cell (e.g., any of the exemplary types of cancer cells described herein or known in the art). For example, the mammalian cell is a cancer cell having aberrant mRNA spicing activity has one or more (e.g., two, three, four, five, or six) of: an increased level of phosphorylated serine and arginine rich splicing factor 6 (SRSF6) as compared to a reference level (e.g., any of the reference levels described herein); an increased level of phosphorylated serine and arginine rich splicing factor 5 (SRSF5) as compared to a reference level (e.g., any of the reference levels described herein); a mutation in a splicing factor 3b subunit 1 (SF3B1) gene, a serine and arginine rich splicing factor 1 (SRSF1) gene, a serine and arginine rich splicing factor 2 (SRSF2) gene, a small nuclear RNA auxiliary factor 1 (U2AF1) gene, or a zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2 (ZRSR2) gene; and an increased level of SRSF1, SRSF2, serine and arginine rich splicing factor 3 (SRSF3), serine and arginine rich splicing factor 4 (SRSF4), SRSF5, SRSF6, and serine and arginine rich splicing factor 10 (SRSF10) as compared to a reference level (e.g., any of the exemplary reference levels described herein).

In some embodiments of any of the methods described herein, the level of aberrant mRNA splicing is determined by detecting: the level of SRSF6 phosphorylation in the cell; the level of SRSF5 phosphorylation in the cell; the level of a ˜55 kDa isoform of SRSF6 in the cell; or the level of ˜35 kDa isoform of SRSF1 in the cell. Exemplary methods for detecting the level of SRSF6 phosphorylation, the level of SRSF5 phosphorylation, the level of the ˜55 kDa isoform of SRSF6, and the level of the ˜35 kDa isoform of SRSF1 are described in the Examples. Additional methods for determining the level of SRSF6 phosphorylation, the level of SRSF5 phosphorylation, the level of the ˜55 kDa isoform of SRSF6, and the level of the ˜35 kDa isoform of SRSF1 are known in the art. Exemplary sequences for human SRSF1, SRSF2, SRSF3, SF3B1, SRSF4, SRSF5, SRSF6, SRSF10, U2AF1, and ZRSR2 proteins are shown below.

SRSF1 (NCBI Accession NM_006924.4)

(SEQ ID NO: 19)

MSGGGVIRGPAGNNDCRIYVGNLPPDIRTKDIEDVFYKYGAIRDIDLKNRRGGPPFAFVEFEDPR

DAEDAVYGRDGYDYDGYRLRVEFPRSGRGTGRGGGGGGGGGAPRGRYGPPSRRSENRVVVSGLPP

SGSWQDLKDHMREAGDVCYADVYRDGTGVVEFVRKEDMTYAVRKLDNTKFRSHEGETAYIRVKVD

GPRSPSYGRSRSRSRSRSRSRSR SNSRSRSYSPRRSRGSPRYSPRHSRSRSRT

SRSF2 (NCBI Accession NM_001195427.1)

(SEQ ID NO: 20)

MSYGRPPPDVEGMTSLKVDNLTYRTSPDTLRRVFEKYGRVGDVYIPRDRYTKESRGFAFVRFHDK

RDAEDAMDAMDGAVLDGRELRVQMARYGRPPDSHHSRRGPPPRRYGGGGYGRRSRSPRRRRRSRS

RSRSRSRSRSRSRYSRSKSRSRTRSRSRSTSKSRSARRSKSKSSSVSRSRSRSRSRSRSRSPPPV

SKRESKSRSRSKSPPKSPEEEGAVSS

SRSF3 (NCBI Accession NM_003017.4)

(SEQ ID NO: 21)

MHRDSCPLDCKVYVGNLGNNGNKTELERAFGYYGPLRSVWVARNPPGFAFVEFEDPRDAADAVRE

LDGRTLCGCRVRVELSNGEKRSRNRGPPPSWGRRPRDDYRRRSPPPRRRSPRRRSFSRSRSRSLS

RDRRRERSLSRERNHKPSRSFSRSRSRSRSNERK

SF3B1 (NCBI Accession NM_012433.3)

(SEQ ID NO: 22)

MAKIAKTHEDIEAQIREIQGKKAALDEAQGVGLDSTGYYDQEIYGGSDSRFAGYVTSIAATELED

DDDDYSSSTSLLGQKKPGYHAPVALLNDIPQSTEQYDPFAEHRPPKIADREDEYKKHRRTMIISP

ERLDPFADGGKTPDPKMNARTYMDVMREQHLTKEEREIRQQLAEKAKAGELKVVNGAAASQPPSK

RKRRWDQTADQTPGATPKKLSSWDQAETPGHTPSLRWDETPGRAKGSETPGATPGSKIWDPTPSH

TPAGAATPGRGDTPGHATPGHGGATSSARKNRWDETPKTERDTPGHGSGWAETPRTDRGGDSIGE

TPTPGASKRKSRWDETPASQMGGSTPVLTPGKTPIGTPAMNMATPTPGHIMSMTPEQLQAWRWER

EIDERNRPLSDEELDAMFPEGYKVLPPPAGYVPIRTPARKLTATPTPLGGMTGFHMQTEDRTMKS

VNDQPSGNLPFLKPDDIQYFDKLLVDVDESTLSPEEQKERKIMKLLLKIKNGTPPMRKAALRQIT

DKAREFGAGPLFNQILPLLMSPTLEDQERHLLVKVIDRILYKLDDLVRPYVHKILVVIEPLLIDE

DYYARVEGREIISNLAKAAGLATMISTMRPDIDNMDEYVRNTTARAFAVVASALGIPSLLPFLKA

VCKSKKSWQARHTGIKIVQQIAILMGCAILPHLRSLVEIIEHGLVDEQQKVRTISALAIAALAEA

ATPYGIESFDSVLKPLWKGIRQHRGKGLAAFLKAIGYLIPLMDAEYANYYTREVMLILIREFQSP

DEEMKKIVLKVVKQCCGTDGVEANYIKTEILPPFFKHFWQHRMALDRRNYRQLVDTTVELANKVG

AAEIISRIVDDLKDEAEQYRKMVMETIEKIMGNLGAADIDHKLEEQLIDGILYAFQEQTTEDSVM

LNGFGTVVNALGKRVKPYLPQICGTVLWRLNNKSAKVRQQAADLISRTAVVMKTCQEEKLMGHLG

VVLYEYLGEEYPEVLGSILGALKAIVNVIGMHKMTPPIKDLLPRLTPILKNRHEKVQENCIDLVG

RIADRGAEYVSAREWMRICFELLELLKAHKKAIRRATVNTFGYIAKAIGPHDVLATLLNNLKVQE

RQNRVCTTVAIAIVAETCSPFTVLPALMNEYRVPELNVQNGVLKSLSFLFEYIGEMGKDYIYAVT

PLLEDALMDRDLVHRQTASAVVQHMSLGVYGFGCEDSLNHLLNYVWPNVFETSPHVIQAVMGALE

GLRVAIGPCRMLQYCLQGLFHPARKVRDVYWKIYNSIYIGSQDALIAHYPRIYNDDKNTYIRYEL

DYIL

SRSF4 (NCBI Accession NM_005626.4)

(SEQ ID NO: 23)

MPRVYIGRLSYQARERDVERFFKGYGKILEVDLKNGYGFVEFDDLRDADDAVYELNGKDLCGERV

IVEHARGPRRDGSYGSGRSGYGYRRSGRDKYGPPTRTEYRLIVENLSSRCSWQDLKDYMRQAGEV

TYADAHKGRKNEGVIEFVSYSDMKRALEKLDGTEVNGRKIRLVEDKPGSRRRRSYSRSRSHSRSR

SRSRHSRKSRSRSGSSKSSHSKSRSRSRSGSRSRSKSRSRSQSRSRSKKEKSRSPSKEKSRSRSH

SAGKSRSKSKDQAEEKIQNNDNVGKPKSRSPSRHKSKSKSRSRSQERRVEEEKRGSVSRGRSQEK

SLRQSRSRSRSKGGSRSRSRSRSKSKDKRKGRKRSREESRSRSRSRSKSERSRKRGSKRDSKAGS

SKKKKKEDTDRSQSRSPSRSVSKEREHAKSESSQREGRGESENAGTNQETRSRSRSNSKS

KPNLPSESRSRSKSASKTRSRSKSRSRSASRSPSRSRSRSHSRS

SRSF5 (NCBI Accession NM_001039465.1)

(SEQ ID NO: 24)

MSGCRVFIGRLNPAAREKDVERFFKGYGRIRDIDLKRGFGFVEFEDPRDADDAVYELDGKELCSE

RVTIEHARARSRGGRGRGRYSDRFSSRRPRNDRRNAPPVRTENRLIVENLSSRVSWQDLKDFMRQ

AGEVTFADAHRPKLNEGVVEFASYGDLKNAIEKLSGKEINGRKIKLIEGSKRHSRSRSRSRSRTR

SSSRSRSRSRSRSRKSYSRSRSRSRSRSRSKSRSVSRSPVPEKSQKRGSSSRSKSPASVDRQRSR

SRSRSRSVDSGN

SRSF6 (NCBI Accession NM_006275.5)

(SEQ ID NO: 25)

MPRVYIGRLSYNVREKDIQRFFSGYGRLLEVDLKNGYGFVEFEDSRDADDAVYELNGKELCGERV

IVEHARGPRRDRDGYSYGSRSGGGGYSSRRTSGRDKYGPPVRTEYRLIVENLSSRCSWQDLKDFM

RQAGEVTYADAHKERTNEGVIEFRSYSDMKRALDKLDGTEINGRNIRLIEDKPRTSHRRSYSGSR

SRSRSRRRSRSRSRRSSRSRSRSISKSRSRSRSRSKGRSRSRSKGRKSRSKSKSKPKSDRGSHSH

SRSRSKDEYEKSRSRSRSRSPKENGKGDIKSKSRSRSQSRSNSPLPVPPSKARSVSPPPKRATSR

SRSRSRSKS RSRSRSSSRD

SRSF10 (NCBI Accession NM_006625.5)

(SEQ ID NO: 26)

MSRYLRPPNTSLFVRNVADDTRSEDLRREFGRYGPIVDVYVPLDFYTRRPRGFAYVQFEDVRDAE

DALHNLDRKWICGRQIEIQFAQGDRKTPNQMKAKEGRNVYSSSRYDDYDRYRRSRSRSYERRRSR

SRSFDYNYRRSYSPRNSRPTGRPRRSRSHSDNDRPNCSWNTQYSSAYYTSRKI

U2AF1 (NCBI Accession NM_001025203.1)

(SEQ ID NO: 27)

MAEYLASIFGTEKDKVNCSFYFKIGACRHGDRCSRLHNKPTFSQTILIQNIYRNPQNSAQTADGS

HCAVSDVEMQEHYDEFFEEVFTEMEEKYGEVEEMNVCDNLGDHLVGNVYVKFRREEDAEKAVIDL

NNRWFNGQPIHAELSPVTDFREACCRQYEMGECTRGGFCNFMHLKPISRELRRELYGRRRKKHRS

RSRSRERRSRSRDRGRGGGGGGG GGGGGRERDRRRSRDRERSGRF

ZRSR2 (NCBI Accession NM_005089.3)

(SEQ ID NO: 28)

MAAPEKMTFPEKPSHKKYRAALKKEKRKKRRQELARLRDSGLSQKEEEEDTFIEEQQLEEEKLLE

RERQRLHEEWLLREQKAQEEFRIKKEKEEAAKKRQEEQERKLKEQWEEQQRKEREEEEQKRQEKK

EKEEALQKMLDQAENELENGTTNQNPEPPVDFRVMEKDRANCPFYSKTGACRFGDRCSRKHNFPT

SSPTLLIKSMFTTFGMEQCRRDDYDPDASLEYSEEETYQQFLDFYEDVLPEFKNVGKVIQFKVSC

NLEPHLRGNVYVQYQSEEECQAALSLFNGRWYAGRQLQCEFCPVTRWKMAICGLFEIQQCPRGKH

CNFLHVFRNPNNEFWEANRDIYLSPDRTGSSFGKNSERRERMGHHDDYYSRLRGRRNPSPDHSYK

RNGESERKSSRHRGKKSHKRTSKSRERHNSRSRGRNRDRSRDRSRGRGSRSRSRSRSRRS

RRSRSQSSSRSRSRGRRRSGNRDRTVQSPKSK

Exemplary methods for detecting a mutation in a SF3B1 gene, a SRSF1 gene, a SRSF2 gene, a U2AF1 gene, or a ZRSR2 gene are also described herein.

Additional methods of identifying or detecting aberrant mRNA splicing in a mammalian cell are known in the art.

Methods of Treating a Subject—Type B

Also provided herein are methods of treating a cancer (e.g., any of the exemplary types of cancer described herein or known in the art) in a subject (e.g., any of the subjects described herein) that include: identifying a subject having a cancer cell (e.g., any of the exemplary types of cancer cells described herein or known in the art) that has aberrant mRNA splicing activity as compared to a reference level (e.g., any of the exemplary reference levels described herein); and administering to the identified subject a therapeutically effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvate thereof.

Also provided herein are methods of treating a cancer (e.g., any of the exemplary types of cancer described herein or known in the art) in a subject (e.g., any of the subjects described herein) that include administering a therapeutically effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvent thereof to a subject identified as having a cancer cell (e.g., any of the exemplary types of cancer cell described herein or known in the art) that has aberrant mRNA splicing activity as compared to a reference level (e.g., any of the reference levels described herein).

Also provided herein are methods of treating a subject (e.g., any of the subjects described herein) having a cancer (e.g., any of the cancers described herein or known in the art) that include: (a) administering to the subject a therapeutic agent (e.g., any therapeutic agent that is not a CLK inhibitor or any therapeutic regimen that does not include a CLK inhibitor as a monotherapy); (b) after (a), identifying the subject as having a cancer cell (e.g., any of the types of cancer cell described herein or known in the art) that has aberrant mRNA splicing activity as compared to a reference level (e.g., any of the reference levels described herein); and (c) administering to the identified subject a treatment comprising a therapeutically effective amount of a CLK inhibitor (e.g., any of the CLK inhibitors described herein) or a pharmaceutically acceptable salt or solvent thereof.

Also provided herein are methods of treating a subject (e.g., any of the subjects described herein) having cancer (e.g., any of the examples of cancer described herein or known in the art) that include administering to a subject previously administered a therapeutic agent (e.g., any therapeutic agent that is not a CLK inhibitor or any therapeutic regimen that does not include a CLK inhibitor as a monotherapy) and later identified as having aberrant mRNA splicing activity as compared to a reference level (e.g., any of the exemplary reference levels described herein), a therapeutically effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvent thereof.

In some embodiments of any of the methods of treating described herein, the cancer cell having aberrant mRNA spicing activity can have one or more (e.g., two, three, four, five, or six) of: an increased level of phosphorylated serine and arginine rich splicing factor 6 (SRSF6) as compared to a reference level (e.g., any of the reference levels described herein); an increased level of phosphorylated serine and arginine rich splicing factor 5 (SRSF5) as compared to a reference level (e.g., any of the reference levels described herein); a mutation in a splicing factor 3b subunit 1 (SF3B1) gene, a serine and arginine rich splicing factor 1 (SRSF1) gene, a serine and arginine rich splicing factor 2 (SRSF2) gene, a small nuclear RNA auxiliary factor 1 (U2AF1) gene, or a zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2 (ZRSR2) gene; and an increased level of SRSF1, SRSF2, serine and arginine rich splicing factor 3 (SRSF3), serine and arginine rich splicing factor 4 (SRSF4), SRSF5, SRSF6, and serine and arginine rich splicing factor 10 (SRSF10) as compared to a reference level (e.g., any of the exemplary reference levels described herein).

In some embodiments of any of the methods of treating described herein, the level of aberrant mRNA splicing is determined by detecting: the level of SRSF6 phosphorylation in the cancer cell; the level of SRSF5 phosphorylation in the cancer cell; the level of a 55 kDa isoform of SRSF6 in the cancer cell; or the level of ˜35 kDa isoform of SRSF1 in the cancer cell. Exemplary methods for detecting the level of SRSF6 phosphorylation, the level of SRSF5 phosphorylation, the level of the ˜55 kDa isoform of SRSF6, and the level of the ˜35 kDa isoform of SRSF1 are described in the Examples. Additional methods for determining the level of SRSF6 phosphorylation, the level of SRSF5 phosphorylation, the level of the ˜55 kDa isoform of SRSF6, and the level of the ˜35 kDa isoform of SRSF1 are known in the art.

Methods of Selecting a Treatment—Type B

Also provided herein are methods of selecting a treatment for a subject (e.g., any of the subjects described herein) that include: identifying a subject having a cancer cell (e.g., any of the exemplary types of cancer cell described herein or known in the art) that has aberrant mRNA splicing activity as compared to a reference level (e.g., any of the exemplary reference levels described herein); and selecting for the identified subject a treatment including a therapeutically effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvent thereof.

Also provided herein are methods of selecting a treatment for a subject (e.g., any of the subjects described herein) that include selecting a treatment including a therapeutically effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvent thereof for a subject identified as having a cancer cell (e.g., any of the exemplary types of cancer cell described herein or known in the art) that has aberrant mRNA splicing activity as compared to a reference level (e.g., any of the exemplary reference levels described herein).

In some embodiments of any of the methods of selecting a treatment described herein, the cancer cell having aberrant mRNA spicing activity can have one or more (e.g., two, three, four, five, or six) of: an increased level of phosphorylated serine and arginine rich splicing factor 6 (SRSF6) as compared to a reference level (e.g., any of the reference levels described herein); an increased level of phosphorylated serine and arginine rich splicing factor 5 (SRSF5) as compared to a reference level (e.g., any of the reference levels described herein); a mutation in a splicing factor 3b subunit 1 (SF3B1) gene, a serine and arginine rich splicing factor 1 (SRSF1) gene, a serine and arginine rich splicing factor 2 (SRSF2) gene, a small nuclear RNA auxiliary factor 1 (U2AF1) gene, or a zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2 (ZRSR2) gene; and an increased level of SRSF1, SRSF2, serine and arginine rich splicing factor 3 (SRSF3), serine and arginine rich splicing factor 4 (SRSF4), SRSF5, SRSF6, and serine and arginine rich splicing factor 10 (SRSF10) as compared to a reference level (e.g., any of the exemplary reference levels described herein).

In some embodiments of any of the methods of selecting a treatment described herein, the level of aberrant mRNA splicing is determined by detecting: the level of SRSF6 phosphorylation in the cancer cell; the level of SRSF5 phosphorylation in the cancer cell; the level of a 55 kDa isoform of SRSF6 in the cancer cell; or the level of 35 kDa isoform of SRSF1 in the cancer cell. Exemplary methods for detecting the level of SRSF6 phosphorylation, the level of SRSF5 phosphorylation, the level of the ˜55 kDa isoform of SRSF6, and the level of the ˜35 kDa isoform of SRSF1 are described in the Examples. Additional methods for determining the level of SRSF6 phosphorylation, the level of SRSF5 phosphorylation, the level of the ˜55 kDa isoform of SRSF6, and the level of the ˜35 kDa isoform of SRSF1 are known in the art.

Methods of Selecting a Subject for Treatment—Type B

Also provided herein are methods of selecting a subject (e.g., any of the exemplary subjects described herein) for treatment that include: identifying a subject having a cancer cell (e.g., any of the exemplary types of cancer cells described herein or known in the art) that has aberrant mRNA splicing activity as compared to a reference level (e.g., any of the reference levels described herein); and selecting an identified subject for treatment with a therapeutically effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvent thereof.

Also provided herein are methods of selecting a subject (e.g., any of the subjects described herein or known in the art) for treatment that include selecting a subject identified as having a cancer cell (e.g., any of the exemplary types of cancer cell described herein or known in the art) that has aberrant mRNA splicing activity as compared to a reference level (e.g., any of the exemplary reference levels described herein), for treatment with a therapeutically effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvent thereof.

In some embodiments of any of the methods of selecting a subject for treatment described herein, the cancer cell having aberrant mRNA spicing activity can have one or more (e.g., two, three, four, five, or six) of: an increased level of phosphorylated serine and arginine rich splicing factor 6 (SRSF6) as compared to a reference level (e.g., any of the reference levels described herein); an increased level of phosphorylated serine and arginine rich splicing factor 5 (SRSF5) as compared to a reference level (e.g., any of the reference levels described herein); a mutation in a splicing factor 3b subunit 1 (SF3B1) gene, a serine and arginine rich splicing factor 1 (SRSF1) gene, a serine and arginine rich splicing factor 2 (SRSF2) gene, a small nuclear RNA auxiliary factor 1 (U2AF1) gene, or a zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2 (ZRSR2) gene; and an increased level of SRSF1, SRSF2, serine and arginine rich splicing factor 3 (SRSF3), serine and arginine rich splicing factor 4 (SRSF4), SRSF5, SRSF6, and serine and arginine rich splicing factor 10 (SRSF10) as compared to a reference level (e.g., any of the exemplary reference levels described herein).

In some embodiments of any of the methods of selecting a subject for treatment described herein, the level of aberrant mRNA splicing is determined by detecting: the level of SRSF6 phosphorylation in the cancer cell; the level of SRSF5 phosphorylation in the cancer cell; the level of a ˜55 kDa isoform of SRSF6 in the cancer cell; or the level of ˜35 kDa isoform of SRSF1 in the cancer cell. Exemplary methods for detecting the level of SRSF6 phosphorylation, the level of SRSF5 phosphorylation, the level of the ˜55 kDa isoform of SRSF6, and the level of the ˜35 kDa isoform of SRSF1 are described in the Examples. Additional methods for determining the level of SRSF6 phosphorylation, the level of SRSF5 phosphorylation, the level of the ˜55 kDa isoform of SRSF6, and the level of the ˜35 kDa isoform of SRSF1 are known in the art.

Methods of Selecting a Subject for Participation in a Clinical Study—Type B

Also provided herein are methods of selecting a subject (e.g., any of the exemplary subjects described herein) for participation in a clinical trial that include: identifying a subject having a cancer cell (e.g., any of the exemplary types of cancer cells described herein) that has aberrant mRNA splicing activity as compared to a reference level (e.g., any of the exemplary reference levels described herein); and selecting the identified subject for participation in a clinical trial that comprises administration of a therapeutically effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvent thereof.

Also provided herein are methods of selecting a subject (e.g., any of the exemplary subjects described herein) for participation in a clinical trial that include selecting a subject identified as having a cancer cell (e.g., any of the exemplary types of cancer cells described herein or known in the art) that has aberrant mRNA splicing activity as compared to a reference level (e.g., any of the exemplary reference levels described herein) for participation in a clinical trial that comprises administration of a therapeutically effective amount of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvent thereof.

In some embodiments of any of the methods of selecting a subject for participation in a clinical trial described herein, the cancer cell having aberrant mRNA spicing activity can have one or more (e.g., two, three, four, five, or six) of: an increased level of phosphorylated serine and arginine rich splicing factor 6 (SRSF6) as compared to a reference level (e.g., any of the reference levels described herein); an increased level of phosphorylated serine and arginine rich splicing factor 5 (SRSF5) as compared to a reference level (e.g., any of the reference levels described herein); a mutation in a splicing factor 3b subunit 1 (SF3B1) gene, a serine and arginine rich splicing factor 1 (SRSF1) gene, a serine and arginine rich splicing factor 2 (SRSF2) gene, a small nuclear RNA auxiliary factor 1 (U2AF1) gene, or a zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2 (ZRSR2) gene; and an increased level of SRSF1, SRSF2, serine and arginine rich splicing factor 3 (SRSF3), serine and arginine rich splicing factor 4 (SRSF4), SRSF5, SRSF6, and serine and arginine rich splicing factor 10 (SRSF10) as compared to a reference level (e.g., any of the exemplary reference levels described herein).

In some embodiments of any of the methods of selecting a subject for participation in a clinical trial described herein, the level of aberrant mRNA splicing is determined by detecting: the level of SRSF6 phosphorylation in the cancer cell; the level of SRSF5 phosphorylation in the cancer cell; the level of a 55 kDa isoform of SRSF6 in the cancer cell; or the level of 35 kDa isoform of SRSF1 in the cancer cell. Exemplary methods for detecting the level of SRSF6 phosphorylation, the level of SRSF5 phosphorylation, the level of the ˜55 kDa isoform of SRSF6, and the level of the ˜35 kDa isoform of SRSF1 are described in the Examples. Additional methods for determining the level of SRSF6 phosphorylation, the level of SRSF5 phosphorylation, the level of the ˜55 kDa isoform of SRSF6, and the level of the ˜35 kDa isoform of SRSF1 are known in the art.

Methods of Determining the Efficacy of a CLK Inhibitor—Type B

Also provided herein are methods of determining the efficacy of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) or a pharmaceutically acceptable salt or solvate thereof that include: (a) determining a first level of a ˜55 kDa isoform of SRSF6 in a cancer cell (e.g., any of the exemplary types of cancer cell described herein or known in the art) obtained from a subject (e.g., any of the subjects described herein) at a first time point; (b) administering to the subject after the first time a CLK inhibitor or a pharmaceutically acceptable salt or solvent thereof, (c) determining a second level of the ˜55 kDa isoform of SRSF6 in a cancer cell obtained from the subject at a second time point; and (d) determining that the CLK inhibitor is effective in a subject having a second level of the ˜55 kDa isoform of SRSF6 that is increased (e.g., a 1% to 500% increase, or any of the subranges of this range described herein) as compared to the first level of the ˜55 kDa isoform of SRSF6.

Also provided herein are method of determining the efficacy of a CLK inhibitor (e.g., any of the exemplary CLK inhibitors described herein or known in the art) (e.g., a compound of any one of Formulas (I)-(XII) or a pharmaceutically acceptable salt or solvent thereof in a subject (e.g., any of the subjects described herein) that include: (a) determining a first level of a ˜35 kDa isoform of SRSF1 in a cancer cell (e.g., any of the exemplary types of cancer cells described herein or known in the art) obtained from a subject at a first time point; (b) administering to the subject after the first time point a compound of a CLK inhibitor or a pharmaceutically acceptable salt or solvent thereof, (c) determining a second level of the ˜35 kDa isoform of SRSF1 in a cancer cell obtained from the subject at a second time point; and (d) determining that the CLK inhibitor is effective in a subject having a second level of the ˜35 kDa isoform of SRSF1 that is increased (e.g., a 1% to 500% increase, or any of the subranges of this range described herein) as compared to the first level of the ˜35 kDa isoform of SRSF1.

Exemplary methods for detecting the level of SRSF6 phosphorylation, the level of SRSF5 phosphorylation, the level of the ˜55 kDa isoform of SRSF6, and the level of the ˜35 kDa isoform of SRSF1 are described in the Examples. Additional methods for determining the level of SRSF6 phosphorylation, the level of SRSF5 phosphorylation, the level of the ˜55 kDa isoform of SRSF6, and the level of the ˜35 kDa isoform of SRSF1 are known in the art.

Samples

Some embodiments of any of the methods described herein can further including obtaining a cell (e.g., a cancer cell or any of the other types of cells) from the subject. For example, the cell (e.g., cancer cell) can be obtained from the subject in the form of a biological sample, e.g., any clinically relevant tissue sample, such as a tumor biopsy, a core biopsy tissue sample, a fine needle aspirate, a hair follicle, or a sample of bodily fluid, such as blood, plasma, serum, lymph, ascitic fluid, cystic fluid, or urine.

In some embodiments, the biological sample is taken from a patient having a tumor or cancer. In some embodiments, the biological sample is a primary tumor. In some embodiments, the biological sample is a metastasis. The biological sample may be taken from a human, or from non-human mammals such as, mice, rats, non-human primates, canines, felines, ruminants, swine, or sheep. In some embodiments, biological samples are taken from a subject at multiple time points, for example, before treatment, during treatment, and/or after treatment. In some embodiments, biological samples are taken from different locations in the subject, for example, a sample from a primary tumor and a sample from a metastasis in a distant location.

In some embodiments, the biological sample is a paraffin-embedded fixed tissue sample. In some embodiments, the sample is a formalin-fixed paraffin embedded (FFPE) tissue sample. In some embodiments, the sample is a fresh tissue (e.g., tumor) sample. In some embodiments, the sample is a frozen tissue sample. In some embodiments, the sample is a fresh frozen (FF) tissue (e.g., tumor) sample. In some embodiments, the sample is a cell isolated from a fluid. In some embodiments, the sample comprises circulating tumor cells (CTCs). In some embodiments, the sample is an archival tissue sample. In some embodiments, the sample is an archival tissue sample with known diagnosis, treatment, and/or outcome history. In some embodiments, the sample is a block of tissue. In some embodiments, the sample is dispersed cells. In some embodiments, the sample size is from about 1 cell to about 1×10⁶cells or more. In some embodiments, the sample size is about 10 cells to about 1×10⁵cells. In some embodiments, the sample size is about 10 cells to about 10,000 cells. In some embodiments, the sample size is about 10 cells to about 1,000 cells. In some embodiments, the sample size is about 10 cells to about 100 cells. In some embodiments, the sample size is about 1 cell to about 10 cells. In some embodiments, the sample size is a single cell.

In some embodiments, the sample is processed to isolate DNA or RNA.

In some embodiments, RNA is isolated from the sample. In some embodiments, mRNA is isolated from the sample. In some embodiments, RNA is isolated from cells by procedures that involve cell lysis and denaturation of the proteins contained therein. In some embodiments, DNase is added to remove DNA. In some embodiments, RNase inhibitors are added to the lysis buffer. In some embodiments, a protein denaturation/digestion step is added to the protocol.

Methods for preparing total and mRNA are well known in the art and RNA isolation kits are commercially available (e.g., RNeasy mini kit, Qiagen, USA). In some embodiments, the RNA is amplified by PCR-based techniques.

Exemplary CLK Inhibitors

Compound 12 is small molecule CLK inhibitor which acts a Wnt signaling inhibitor by downregulating Wnt pathway gene expression in cancer cells. Compound 12 was phenotypically screened and discovered on its ability to inhibit Wnt reporter activity driven by constitutively active Wnt signaling in SW480 CRC cells. Compound 12's ability to block Wnt signaling was further confirmed by inhibition of Wnt-3a and GSK-3D-inhibitor stimulated Wnt signaling in non-cancerous cell types such as 293T and IEC-6 rat intestinal cells.

In some embodiments, the CLK inhibitor is a multi-isoform CLK inhibitor.

In some embodiments, the CLK inhibitor has an IC₅₀of between about 1 nM and about 10 μM (e.g., between about 1 nM and about 9 μM, between about 1 nM and about 8 μM, between about 1 nM and about 7 μM, between about 1 nM and about 6 μM, between about 1 nM and about 5 μM, between about 1 nM and about 4 μM, between about 1 nM and about 3 μM, between about 1 nM and about 2 μM, between about 1 nM and about 1 μM, between about 1 nM and about 950 nM, between about 1 nM and about 900 nM, between about 1 nM and about 850 nM, between about 1 nM and about 800 nM, between about 1 nM and about 750 nM, between about 1 nM and about 700 nM, between about 1 nM and about 650 nM, between about 1 nM and about 600 nM, between about 1 nM and about 550 nM, between about 1 nM and about 500 nM, between about 1 nM and about 450 nM, between about 1 nM and about 400 nM, between about 1 nM and about 350 nM, between about 1 nM and about 300 nM, between about 1 nM and about 250 nM, between about 1 nM and about 200 nM, between about 1 nM and about 150 nM, between about 1 nM and about 100 nM, between about 1 nM and about 95 nM, between about 1 nM and about 90 nM, between about 1 nM and about 85 nM, between about 1 nM and about 80 nM, between about 1 nM and about 75 nM, between about 1 nM and about 70 nM, between about 1 nM and about 65 nM, between about 1 nM and about 60 nM, between about 1 nM and about 55 nM, between about 1 nM and about 50 nM, between about 1 nM and about 45 nM, between about 1 nM and about 40 nM, between about 1 nM and about 35 nM, between about 1 nM and about 30 nM, between about 1 nM and about 25 nM, between about 1 nM and about 20 nM, between about 1 nM and about 15 nM, between about 1 nM and about 10 nM, between about 1 nM and about 5 nM, between about 1 nM and about 4 nM, between about 1 nM and about 3 nM, between about 1 nM and about 2 nM, between about 2 nM and about 10 μM, between about 2 nM and about 9 μM, between about 2 nM and about 8 μM, between about 2 nM and about 7 μM, between about 2 nM and about 6 μM, between about 2 nM and about 5 μM, between about 2 nM and about 4 μM, between about 2 nM and about 3 μM, between about 2 nM and about 2 μM, between about 2 nM and about 1 μM, between about 2 nM and about 950 nM, between about 2 nM and about 900 nM, between about 2 nM and about 850 nM, between about 2 nM and about 800 nM, between about 2 nM and about 750 nM, between about 2 nM and about 700 nM, between about 2 nM and about 650 nM, between about 2 nM and about 600 nM, between about 2 nM and about 550 nM, between about 2 nM and about 500 nM, between about 2 nM and about 450 nM, between about 2 nM and about 400 nM, between about 2 nM and about 350 nM, between about 2 nM and about 300 nM, between about 2 nM and about 250 nM, between about 2 nM and about 200 nM, between about 2 nM and about 150 nM, between about 2 nM and about 100 nM, between about 2 nM and about 95 nM, between about 2 nM and about 90 nM, between about 2 nM and about 85 nM, between about 2 nM and about 80 nM, between about 2 nM and about 75 nM, between about 2 nM and about 70 nM, between about 2 nM and about 65 nM, between about 2 nM and about 60 nM, between about 2 nM and about 55 nM, between about 2 nM and about 50 nM, between about 2 nM and about 45 nM, between about 2 nM and about 40 nM, between about 2 nM and about 35 nM, between about 2 nM and about 30 nM, between about 2 nM and about 25 nM, between about 2 nM and about 20 nM, between about 2 nM and about 15 nM, between about 2 nM and about 10 nM, between about 2 nM and about 5 nM, between about 2 nM and about 4 nM, between about 2 nM and about 3 nM, between about 5 nM and about M, between about 5 nM and about 9 μM, between about 5 nM and about 8 μM, between about 5 nM and about 7 μM, between about 5 nM and about 6 μM, between about 5 nM and about 5 μM, between about 5 nM and about 4 μM, between about 5 nM and about 3 μM, between about 5 nM and about 2 μM, between about 5 nM and about 1 μM, between about 5 nM and about 950 nM, between about 5 nM and about 900 nM, between about 5 nM and about 850 nM, between about 5 nM and about 800 nM, between about 5 nM and about 750 nM, between about 5 nM and about 700 nM, between about 5 nM and about 650 nM, between about 5 nM and about 600 nM, between about 5 nM and about 550 nM, between about 5 nM and about 500 nM, between about 5 nM and about 450 nM, between about 5 nM and about 400 nM, between about 5 nM and about 350 nM, between about 5 nM and about 300 nM, between about 5 nM and about 250 nM, between about 5 nM and about 200 nM, between about 5 nM and about 150 nM, between about 5 nM and about 100 nM, between about 5 nM and about 95 nM, between about 5 nM and about 90 nM, between about 5 nM and about 85 nM, between about 5 nM and about 80 nM, between about 5 nM and about 75 nM, between about 5 nM and about 70 nM, between about 5 nM and about 65 nM, between about 5 nM and about 60 nM, between about 5 nM and about 55 nM, between about 5 nM and about 50 nM, between about 5 nM and about 45 nM, between about 5 nM and about 40 nM, between about 5 nM and about 35 nM, between about 5 nM and about 30 nM, between about 5 nM and about 25 nM, between about 5 nM and about 20 nM, between about 5 nM and about 15 nM, between about 5 nM and about 10 nM, between about 10 nM and about 10 μM, between about 10 nM and about 9 μM, between about 10 nM and about 8 μM, between about 10 nM and about 7 μM, between about 10 nM and about 6 μM, between about 10 nM and about 5 μM, between about 10 nM and about 4 μM, between about 10 nM and about 3 μM, between about 10 nM and about 2 μM, between about 10 nM and about 1 μM, between about 10 nM and about 950 nM, between about 10 nM and about 900 nM, between about 10 nM and about 850 nM, between about 10 nM and about 800 nM, between about 10 nM and about 750 nM, between about 10 nM and about 700 nM, between about 10 nM and about 650 nM, between about 10 nM and about 600 nM, between about 10 nM and about 550 nM, between about 10 nM and about 500 nM, between about 10 nM and about 450 nM, between about 10 nM and about 400 nM, between about 10 nM and about 350 nM, between about 10 nM and about 300 nM, between about 10 nM and about 250 nM, between about 10 nM and about 200 nM, between about 10 nM and about 150 nM, between about 10 nM and about 100 nM, between about 10 nM and about 95 nM, between about 10 nM and about 90 nM, between about 10 nM and about 85 nM, between about 10 nM and about 80 nM, between about 10 nM and about 75 nM, between about 10 nM and about 70 nM, between about 10 nM and about 65 nM, between about 10 nM and about 60 nM, between about 10 nM and about 55 nM, between about 10 nM and about 50 nM, between about 10 nM and about 45 nM, between about 10 nM and about 40 nM, between about 10 nM and about 35 nM, between about 10 nM and about 30 nM, between about 10 nM and about 25 nM, between about 10 nM and about 20 nM, between about 10 nM and about 15 nM, between about 50 nM and about 10 μM, between about 50 nM and about 9 μM, between about 50 nM and about 8 μM, between about 50 nM and about 7 μM, between about 50 nM and about 6 μM, between about 50 nM and about 5 μM, between about 50 nM and about 4 μM, between about 50 nM and about 3 μM, between about 50 nM and about 2 μM, between about 50 nM and about 6 μM, between about 50 nM and about 950 nM, between about 50 nM and about 900 nM, between about 50 nM and about 850 nM, between about 50 nM and about 800 nM, between about 50 nM and about 750 nM, between about 50 nM and about 700 nM, between about 50 nM and about 650 nM, between about 50 nM and about 600 nM, between about 50 nM and about 550 nM, between about 50 nM and about 500 nM, between about 50 nM and about 450 nM, between about 50 nM and about 400 nM, between about 50 nM and about 350 nM, between about 50 nM and about 300 nM, between about 50 nM and about 250 nM, between about 50 nM and about 200 nM, between about 50 nM and about 150 nM, between about 50 nM and about 100 nM, between about 50 nM and about 95 nM, between about 50 nM and about 90 nM, between about 50 nM and about 85 nM, between about 50 nM and about 80 nM, between about 50 nM and about 75 nM, between about 50 nM and about 70 nM, between about 50 nM and about 65 nM, between about 50 nM and about 60 nM, between about 50 nM and about 55 nM, between about 100 nM and about 10 μM, between about 100 nM and about 9 μM, between about 100 nM and about 8 μM, between about 100 nM and about 7 μM, between about 100 nM and about 6 μM, between about 100 nM and about 5 μM, between about 100 nM and about 4 μM, between about 100 nM and about 3 μM, between about 100 nM and about 2 μM, between about 100 nM and about 1 μM, between about 100 nM and about 950 nM, between about 100 nM and about 900 nM, between about 100 nM and about 850 nM, between about 100 nM and about 800 nM, between about 100 nM and about 750 nM, between about 100 nM and about 700 nM, between about 100 nM and about 650 nM, between about 100 nM and about 600 nM, between about 100 nM and about 550 nM, between about 100 nM and about 500 nM, between about 100 nM and about 450 nM, between about 100 nM and about 400 nM, between about 100 nM and about 350 nM, between about 100 nM and about 300 nM, between about 100 nM and about 250 nM, between about 100 nM and about 200 nM, between about 100 nM and about 150 nM, between about 200 nM and about 10 μM, between about 200 nM and about 9 μM, between about 200 nM and about 8 μM, between about 200 nM and about 7 μM, between about 200 nM and about 6 μM, between about 200 nM and about 5 μM, between about 200 nM and about 4 μM, between about 200 nM and about 3 μM, between about 200 nM and about 2 μM, between about 200 nM and about 1 μM, between about 200 nM and about 950 nM, between about 200 nM and about 900 nM, between about 200 nM and about 850 nM, between about 200 nM and about 800 nM, between about 200 nM and about 750 nM, between about 200 nM and about 700 nM, between about 200 nM and about 650 nM, between about 200 nM and about 600 nM, between about 200 nM and about 550 nM, between about 200 nM and about 500 nM, between about 200 nM and about 450 nM, between about 200 nM and about 400 nM, between about 200 nM and about 350 nM, between about 200 nM and about 300 nM, between about 200 nM and about 250 nM, between about 250 nM and about 10 μM, between about 250 nM and about 9 μM, between about 250 nM and about 8 μM, between about 250 nM and about 7 μM, between about 250 nM and about 6 μM, between about 250 nM and about 5 μM, between about 250 nM and about 4 μM, between about 250 nM and about 3 μM, between about 250 nM and about 2 μM, between about 250 nM and about 1 μM, between about 250 nM and about 950 nM, between about 250 nM and about 900 nM, between about 250 nM and about 850 nM, between about 250 nM and about 800 nM, between about 250 nM and about 750 nM, between about 250 nM and about 700 nM, between about 250 nM and about 650 nM, between about 250 nM and about 600 nM, between about 250 nM and about 550 nM, between about 250 nM and about 500 nM, between about 250 nM and about 450 nM, between about 250 nM and about 400 nM, between about 250 nM and about 350 nM, between about 250 nM and about 300 nM, between about 500 nM and about 10 μM, between about 500 nM and about 9 μM, between about 500 nM and about 8 μM, between about 500 nM and about 7 μM, between about 500 nM and about 6 μM, between about 500 nM and about 5 μM, between about 500 nM and about 4 μM, between about 500 nM and about 3 μM, between about 500 nM and about 2 μM, between about 500 nM and about 1 μM, between about 500 nM and about 950 nM, between about 500 nM and about 900 nM, between about 500 nM and about 850 nM, between about 500 nM and about 800 nM, between about 500 nM and about 750 nM, between about 500 nM and about 700 nM, between about 500 nM and about 650 nM, between about 500 nM and about 600 nM, between about 500 nM and about 550 nM, between about 750 nM and about 10 μM, between about 750 nM and about 9 μM, between about 750 nM and about 8 μM, between about 750 nM and about 7 μM, between about 750 nM and about 6 μM, between about 750 nM and about 5 μM, between about 750 nM and about 4 μM, between about 750 nM and about 3 μM, between about 750 nM and about 2 μM, between about 750 nM and about 1 μM, between about 750 nM and about 950 nM, between about 750 nM and about 900 nM, between about 750 nM and about 850 nM, between about 750 nM and about 800 nM, between about 950 nM and about 10 μM, between about 950 nM and about 9 μM, between about 950 nM and about 8 μM, between about 950 nM and about 7 μM, between about 950 nM and about 6 μM, between about 950 nM and about 5 μM, between about 950 nM and about 4 μM, between about 950 nM and about 3 μM, between about 950 nM and about 2 μM, between about 950 nM and about 1 μM, between about 1 μM and about 10 μM, between about 1 μM and about 9 μM, between about 1 μM and about 8 μM, between about 1 μM and about 7 μM, between about 1 μM and about 6 μM, between about 1 μM and about 5 μM, between about 1 μM and about 4 μM, between about 1 μM and about 3 μM, between about 1 μM and about 2 μM, between about 2 μM and about 10 μM, between about 2 μM and about 9 μM, between bout 2 μM and about 8 μM, between about 2 μM and about 7 μM, between about 2 μM and about 6 μM, between about 2 μM and about 5 μM, between about 2 μM and about 4 μM, between about 2 μM and about 3 μM, between about 4 μM and about 10 μM, between about 4 μM and about 9 μM, between about 4 μM and about 8 μM, between about 4 μM and about 7 μM, between about 4 μM and about 6 μM, between about 4 μM and about 5 μM, between about 5 μM and about 10 μM, between about 5 μM and about 9 μM, between about 5 μM and about 8 μM, between about 5 μM and about 7 μM, between about 5 μM and about 6 μM, between about 6 μM and about 10 μM, between about 6 μM and about 9 μM, between about 6 μM and about 8 μM, between about 6 μM and about 7 μM; between about 7 μM and about 10 μM, between about 7 μM and about 9 μM, between about 7 μM and about 8 μM, between about 8 μM and about 10 μM, between about 8 μM and about 9 μM, or between about 9 μM and about 10 μM) for one or both of CLK2 and CLK3.

In some embodiments, the CLK inhibitor has an IC₅₀of between about 1 nM and about 1 μM (or any of the subranges of this range described herein) for each of CLK3 and CLK4. In some embodiments, the CLK inhibitor has an IC₅₀of between about 1 nM and about 10 μM (or any of the subranges of this range) for each of CLK1 and CLK3. In some embodiments, the CLK inhibitor has an IC₅₀of between about 1 nM and about 10 μM (or any of the subranges of this range described herein) for each of CLK1 and CLK2. In some embodiments, the CLK inhibitor has an IC₅₀of between about 1 nM and about 10 μM (or any of the subranges of this range described herein) for each of CLK1 and CLK4. In some embodiments, the CLK inhibitor has an IC₅₀of between about 1 nM and about 10 μM (or any of the subranges of this range described herein) for each of CLK2 and CLK4. In some embodiments, the CLK inhibitor has an IC₅₀of between about 1 nM and about M (or any of the subranges of this range described herein) for each of CLK1, CLK2, and/or CLK3. In some embodiments, the m CLK inhibitor has an IC₅₀of between about 1 nM and about M (or any of the subranges of this range described herein) for each of CLK1, CLK2 and CLK4. In some embodiments, the CLK inhibitor has an IC₅₀of between about 1 nM and about 10 μM (or any of the subranges of this range described herein) for each of CLK2, CLK3 and CLK4. In some embodiments, the CLK inhibitor has an IC₅₀of between about 1 nM and about 10 μM (or any of the subranges of this range described herein) for each of CLK1, CLK2, CLK3 and CLK4.

In some embodiments, the CLK inhibitor is a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the CLK inhibitor is a compound of Formula II or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the CLK inhibitor is a compound of Formula III or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the CLK inhibitor is a compound of Formula IV or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the CLK inhibitor is a compound of Formula V or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the CLK inhibitor is a compound of Formula VI or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the CLK inhibitor is a compound of Formula VII or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the CLK inhibitor is a compound of Formula VIII or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the CLK inhibitor is a compound of Formula IX or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the CLK inhibitor is a compound of Formula X or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the CLK inhibitor is a compound of Formula XI or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the CLK inhibitor is a compound of Formula XII or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, compounds for use as CLK2 or CLK2/CLK3 inhibitors include the compounds set forth below as described in the following journal articles, U.S. patents and U.S. patent applications.

U.S. provisional applications 62/793,428 and 62/831,478 describe compounds having Formula I and are hereby incorporated by reference in their entirety.

One embodiment disclosed herein includes a compound having the structure of Formula I:

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof.

In some embodiments of Formula (I):

R¹is selected from the group consisting of H, halide (e.g., F, Cl, Br, I), and unsubstituted —(C_1-3alkyl);

with the proviso that when L¹is a bond, R²is selected from the group consisting of -phenyl substituted with 1-5 R⁶and -heteroaryl optionally substituted with 1-4 R⁷; wherein heteroaryl selected from the group consisting of pyridinyl, oxazolyl, oxadiazolyl, thiazolyl, 2,3-dihydrobenzo[b]dioxinyl, 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazinyl, 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl, isoquinolinyl, and quinolinyl;

R³is selected from the group consisting of -heterocyclyl substituted with 1-10 R″, —(C_1-4alkylene)_pphenyl substituted with 1-5 R¹², -heteroaryl optionally substituted with 1-4 R¹³, and —(C_1-4alkylene)OR¹⁴; wherein heteroaryl selected from the group consisting of pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, oxazolyl, oxadiazolyl, thiadiazolyl, indolyl, indazolyl, benzimidazolyl, imidazo[4,5-b]pyridinyl, imidazo[4,5-c]pyridinyl, 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazinyl, 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl, 1,2,3,4-tetrahydroisoquinolinyl, isoquinolinyl, and quinolinyl; wherein

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is only substituted at positions 4 and 7; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

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is only substituted at positions 4 and 7;

each R⁴is halide;

each R⁵is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), Me, and Et;

R⁸is unsubstituted —(C_1-9alkyl);

R⁹is unsubstituted —(C_1-9alkyl);

R¹⁰is -aryl optionally substituted with 1-5 R²¹;

each R¹¹is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), methyl, and ethyl;

R¹⁴is selected from the group consisting of unsubstituted —(C_1-4alkyl) and -aryl optionally substituted with 1-5 R²²;

each R^15ais independently selected from the group consisting of unsubstituted —(C_2-3alkyl), and -heterocyclyl optionally substituted with 1-10 R^20b;

each R^15bis independently selected from the group consisting of H, unsubstituted —(C_2-9alkyl), and -heterocyclyl optionally substituted with 1-10 R^20b;

each R^16ais independently selected from the group consisting of H and unsubstituted —(C_1-2alkyl);

each R^16bis unsubstituted —(C_1-2alkyl);

each R¹⁷is unsubstituted —(C_1-9alkyl);

each R¹⁸is independently selected from the group consisting of H and Me;

each R¹⁹is unsubstituted —(C_1-9alkyl);

each R^20ais independently selected from the group consisting of halide and unsubstituted —(C_2-9alkyl);

each R^20bis independently selected from the group consisting of halide and unsubstituted —(C_1-9alkyl);

each R²¹is independently selected from the group consisting of halide and unsubstituted —(C_1-9alkyl);

each R²²is independently selected from the group consisting of halide and unsubstituted —(C_1-9alkyl);

each R²⁴is independently selected from the group consisting of halide and unsubstituted —(C_1-9alkyl);

each R²⁵is independently selected from the group consisting of H and unsubstituted —(C_1-9alkyl);

L¹is selected from the group consisting of a bond, —CH═CH—,

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(CH₂)_pNR¹⁸(C═O)—, —(C═O)NR¹⁸(CH₂)_p—, —NR¹⁸(C═O)NR¹⁸—, —NH(CH₂)_p—, and —(CH₂)_pNH—;

L²is selected from the group consisting of a bond, —(C═O)NR¹⁸—, —NR¹⁸(C═O)—, —NHCH₂—, and —CH₂NH—; and

each p is independently an integer of 0 or 1.

In some embodiments of Formula I, R¹is selected from the group consisting of H, halide (e.g., F, Cl, Br, I), and unsubstituted —(C_1-3alkyl).

In some embodiments of Formula I, R¹is H.

In some embodiments of Formula I, R¹is F.

In some embodiments of Formula I, R¹is Me.

In some embodiments of Formula I, R²is a -monocyclic heterocyclyl optionally substituted with 1-2 R.

In some embodiments of Formula I, R²is a -monocyclic heterocyclyl optionally substituted with 1 Me.

In some embodiments of Formula I, R³is -heterocyclyl substituted with 1-2 R¹¹.

In some embodiments of Formula I, R³is -heterocyclyl substituted with 1 Me

In some embodiments of Formula I, R is —(C_1-2alkylene)phenyl substituted with 1-2 R¹².

In some embodiments of Formula I, R³is -phenyl substituted with 1-2 R¹².

In some embodiments of Formula I, R³is -heteroaryl optionally substituted with 1-2 R¹³.

In some embodiments of Formula I, R³is -pyridinyl optionally substituted with 1-2 R¹³.

In some embodiments of Formula I, R³is R

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In some embodiments of Formula I, R³is R

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In some embodiments of Formula I, R³is R

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In some embodiments of Formula I, R³is R

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In some embodiments of Formula I, L¹is selected from the group consisting of a bond, —C(═O)NH—, —CH═CH—, and

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In some embodiments of Formula I, L¹is a bond; in some embodiments of Formula I, L¹is —C(═O)NH—; in some embodiments of Formula I, L¹is —CH═CH—; and in some embodiments of Formula I, L¹is

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In some embodiments of Formula I, L²is selected from the group consisting of a bond and —C(═O)NH—.

In some embodiments of Formula I, L²is a bond.

In some embodiments of Formula I, L²is —C(═O)NH—.

U.S. provisional application 62/685,764 describes compounds having Formula II and is hereby incorporated by reference in their entirety.

One embodiment disclosed herein includes a compound having the structure of Formula II:

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof.

In some embodiments of Formula (II):

Ring A is a 5-6-membered heteroaryl optionally substituted with 1-4 R¹;

L is -L¹-L²-L³-L⁴-;

L¹is selected from the group consisting of unsubstituted —(C_1-3alkylene)-, —NR²—, —NR³(C═O)—, —(C═O)NR³—, and —O—;

L²is selected from the group consisting of unsubstituted —(C_1-6alkylene)- and —NR²—;

L³is selected from the group consisting of unsubstituted —(C_1-6alkylene)-, —O—, and -carbocyclylene- optionally substituted with one or more halides;

with the proviso that —NR²— and —O— are not adjacent to each other;

with the proviso that two —NR³(C═O)— and/or —(C═O)NR³—, are not adjacent to each other;

each R¹is selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-3alkyl), unsubstituted —(C_1-3haloalkyl), and —CN;

each R²is selected from the group consisting of H and unsubstituted —(C_1-6alkyl);

each R³is selected from the group consisting of H and unsubstituted —(C_1-6alkyl);

each R⁴is selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl), unsubstituted —(C_1-6haloalkyl), and —CN;

each R is selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl), unsubstituted —(C_1-6haloalkyl), and —CN;

Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶are independently selected from the group consisting of carbon and nitrogen; wherein

if Y¹is nitrogen then Y²and Y³are CH;

if Y²is nitrogen then Yi and Y³are CH;

if Y³is nitrogen then Yi and Y²are CH;

if Y⁴is nitrogen then Y⁵and Y⁶are CH;

if Y⁵is nitrogen then Y⁴and Y⁶are CH; and

if Y⁶is nitrogen then Y⁴and Y⁵are CH.

In some embodiments of Formula II, Ring A is a 5-membered heteroaryl and is selected from the group consisting of

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In some embodiments of Formula II, Ring A is a 6-membered heteroaryl and is selected from the group consisting of

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In some embodiments of Formula II, Ring A is a 5-6-membered heteroaryl and is selected from the group consisting of

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In some embodiments of Formula II, Ring A is a 5-6-membered heteroaryl and is selected from the group consisting of

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In some embodiments of Formula II, L¹is selected from the group consisting of —(CH₂)—, —NH—, —NMe-, —NH(C═O)—, —(C═O)NH—, and —O—; In some embodiments of Formula II, L¹is —(CH₂)—; In some embodiments of Formula II, L¹is —NH—; In some embodiments of Formula II, L¹is —NMe-; In some embodiments of Formula II, L¹is —NH(C═O)—; In some embodiments of Formula II, L¹is —(C═O)NH—; In some embodiments of Formula II, L¹is —O—.

In some embodiments of Formula II, L²is selected from the group consisting of —(CH₂)—, —(CH₂CH₂)—, —(CH₂CH₂CH₂)—, —NH—, and —NMe-; In some embodiments of Formulas II, L²is —(CH₂)—; In some embodiments of Formulas II, L²is —(CH₂CH₂)—; In some embodiments of Formulas II, L²is —(CH₂CH₂CH₂)—; In some embodiments of Formulas II, L²is —NH—; In some embodiments of Formulas II, L²is —NMe-.

In some embodiments of Formula II, L³is selected from the group consisting of —(CH₂)—, —(CH₂CH₂)—, —(CH₂CH₂CH₂)—, —(CH₂CH₂CH₂CH₂)—, —O—, and

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In some embodiments of Formula II, L³is —(CH₂)—; In some embodiments of Formula II, L³is —(CH₂CH₂)—; In some embodiments of Formula II, L³is —(CH₂CH₂CH₂)—; In some embodiments of Formula II, L³is —(CH₂CH₂CH₂CH₂)—; In some embodiments of Formula II, L³is —O—; In some embodiments of Formula II, L³is

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In some embodiments of Formula II, L⁴is selected from the group consisting of —(CH₂)—, —(CH₂CH₂)—, —(CH₂CH₂CH₂)—, —(CH₂CH₂CH₂CH₂)—, —O—, —NH—, —NMe-, —NH(C═O)—, and —(C═O)NH—,

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In some embodiments of Formula II, L⁴is —(CH₂)—; In some embodiments of Formula II, L⁴is —(CH₂CH₂)—; In some embodiments of Formula II, L⁴is —(CH₂CH₂CH₂)—; In some embodiments of Formula II, L⁴is —(CH₂CH₂CH₂CH₂)—; In some embodiments of Formula II, L⁴is —O—; In some embodiments of Formula II, L⁴is —NH—; In some embodiments of Formula II, L⁴is —NMe-; In some embodiments of Formula II, L⁴is —NH(C═O)—; In some embodiments of Formula II, L⁴is —(C═O)NH—; In some embodiments of Formula II, L⁴is

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In some embodiments of Formula II, L⁴is

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In some embodiments of Formula II, L⁴is

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In some embodiments of Formula II, L⁴is

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In some embodiments of Formula II, L⁴is

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Bioorganic & Medicinal Chemistry Letters (2006), 16(14), 3740-3744 and U.S. application Ser. Nos. 10/295,833 and 10/317,914 describe compounds having Formula III and are hereby incorporated by reference in their entirety.

One embodiment disclosed herein includes a compound having the structure of Formula III:

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof.

In some embodiments of Formula (III):

R¹is selected from the group consisting of H and halide (e.g., F, Cl, Br, I);

R²is a 6-membered -heteroaryl substituted with 1-4 (e.g., 1-3, 1-2, 1) R³;

each R³is selected from the group consisting of —OR⁴, —NHR⁵, and —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁶; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R⁴is independently selected from the group consisting of -heterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁷and —CH₂CH(R⁸)NH₂;

each R is independently selected from the group consisting of —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁹and -carbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹⁰; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R⁶is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R⁷is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R⁸is independently selected from the group consisting of —(C_1-4alkylene)aryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R¹¹and —(C^1-4alkylene)heteroaryl optionally substituted with 1-4 (e.g., 1-3, 1-2, 1) R¹²; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁹is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —OH, —NH₂, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R¹⁰is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —OH, —NH₂, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R¹¹is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R¹²is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C₂-s, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁); and

each p is independently 0 or 1.

In some embodiments of Formula III, R¹is halide.

In some embodiments of Formula III, R¹is F.

In some embodiments of Formula III, R¹is H.

In some embodiments of Formula III, R²is pyridinyl substituted with one R³;

In some embodiments of Formula III, R²is pyrazinyl substituted with one R³;

In some embodiments of Formula III, R³is selected from the group consisting of —OR⁴, —NHR⁵, and —(CH₂)heterocyclyl optionally substituted with one R⁶.

In some embodiments of Formula III, R³is —OR⁴; in some embodiments of Formula III, R³is —NHR⁵; and in some embodiments of Formula III, R is —(CH₂)heterocyclyl optionally substituted with one R.

U.S. provisional application 62/634,656 and U.S. Pat. Nos. 9,221,793 and 9,745,271 describe compounds having Formula IV and are hereby incorporated by reference in their entirety.

One embodiment disclosed herein includes a compound having the structure of Formula IV:

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof.

In some embodiments of Formula (IV):

R¹is selected from the group consisting of H and halide (e.g., F, Cl, Br, I);

R²is a -heteroaryl optionally substituted with 1-4 (e.g., 1-3, 1-2, 1) R⁴;

R³is selected from the group consisting of -aryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R⁵and -heteroaryl optionally substituted with 1-4 (e.g., 1-3, 1-2, 1) R⁶;

each R⁴is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), —(C_1-4alkylene)_pN(R⁷)(R⁸), —NHC(═O)R⁹, —(C_1-4alkylene)_pOR¹, unsubstituted -carbocyclyl, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹⁴, —(C_1-4alkylene)_paryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R¹, and —(C_1-4alkylene)_pheteroaryl optionally substituted with 1-4 (e.g., 1-3, 1-2, 1) R¹²; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —CN, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), —(C_1-4alkylene)_paryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R¹³, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹⁴, —C(═O)N(R⁵)₂, —NHC(═O)R¹⁶, —(C_1-4alkylene)_pN(R¹⁷)(R¹⁸), —SO₂R¹⁹, and —OR²⁰; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁶is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —CN, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), —(C_1-4alkylene)_paryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R¹³, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹⁴, —C(═O)N(R⁵)₂, —NHC(═O)R¹⁶, —(C_1-4alkylene)_pN(R¹⁷)(R¹⁸), —SO₂R¹⁹, and —OR²⁰; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁷is independently selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂);

each R⁸is independently selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and -heterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²¹;

alternatively, R⁷and R⁸are taken together to form a -heterocyclyl ring optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²¹;

each R⁹is independently selected from the group consisting of —N(R²²)₂, -carbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²³, -heterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²¹, and -aryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R²⁴;

each R¹⁰is independently selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C₁-s, C_1-4, C_1-3, C_1-2, C₁), and -heterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²¹;

each R¹¹is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C₂-s, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R¹²is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —(C_1-4alkylene)pOH, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_{1_3}, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁); wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R¹³is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R¹⁴is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —(C_1-4alkylene)pOH, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁); wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁵is independently selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and -carbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²³;

alternatively, two adjacent R¹⁵are taken together to form a -heterocyclyl ring optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²¹;

each R¹⁶is independently selected from the group consisting of unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and -carbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²³;

each R¹⁸is independently selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), —(C_1-4alkylene)NMe₂, and -heterocyclyl ring optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²¹; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁹is independently selected from the group consisting of unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂);

each R²⁰is independently selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), —CH(CH₂OH)₂, —(C_1-4alkylene)_pheterocyclyl ring optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²¹, and -aryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R²⁴; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R²¹is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R²²is independently selected from the group consisting of unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂);

each R²³is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R²⁴is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-3, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁); and

each p is independently 0 or 1.

In some embodiments of Formula IV, R¹is halide.

In some embodiments of Formula IV, R¹is F.

In some embodiments of Formula IV, R¹is H.

In some embodiments of Formula IV, R²is a 5-membered -heteroaryl optionally substituted with 1-2 R⁴;

In some embodiments of Formula IV, R²is selected from the group consisting of pyrazolyl, imidazolyl, 1,2,3-triazolyl, isoxazolyl, oxazolyl, isothiazolyl, and thiazolyl; wherein each are optionally substituted with 1-2 R⁴.

In some embodiments of Formula IV, R²is pyrazolyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IV, R²is imidazolyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IV, R²is 1,2,3-triazolyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IV, R²is isoxazolyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IV, R²is oxazolyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IV, R²is isothiazolyl optionally substituted with 1-2 R⁴; and in some embodiments of Formula IV, R²is thiazolyl optionally substituted with 1-2 R⁴.

In some embodiments of Formula IV, R⁴is selected from the group consisting of unsubstituted —(C_1-3alkyl) and -heterocyclyl optionally substituted with one R¹⁴.

In some embodiments of Formula IV, R⁴is unsubstituted —(C_1-3alkyl) and in some embodiments of Formula IV, R⁴is -heterocyclyl optionally substituted with one R¹⁴.

In some embodiments of Formula IV, R²is a 6-membered -heteroaryl optionally substituted with 1-2 R⁴;

In some embodiments of Formula IV, R²is pyridinyl optionally substituted with one R⁴.

In some embodiments of Formula IV, R³is selected from the group consisting of -phenyl optionally substituted with 1-2 R⁵, -pyridinyl optionally substituted with 1-2 R⁶, -pyrimidinyl optionally substituted with 1-2 R⁶, -pyrazinyl optionally substituted with 1-2 R⁶, -pyrazolyl optionally substituted with 1-2 R⁶, -isothiazolyl optionally substituted with 1-2 R⁶, and -thiazolyl optionally substituted with 1-2 R⁶.

In some embodiments of Formula IV, R³is -phenyl optionally substituted with 1-2 R⁵; in some embodiments of Formula IV, R³is -pyridinyl optionally substituted with 1-2 R⁶; in some embodiments of Formula IV, R³is -pyrimidinyl optionally substituted with 1-2 R⁶; in some embodiments of Formula IV, R³is -pyrazinyl optionally substituted with 1-2 R⁶; in some embodiments of Formula IV, R³is -pyrazolyl optionally substituted with 1-2 R⁶; in some embodiments of Formula IV, R³is -isothiazolyl optionally substituted with 1-2 R⁶; and in some embodiments of Formula IV, R³is -thiazolyl optionally substituted with 1-2 R⁶.

In some embodiments of Formula IV, R⁵is selected from the group consisting of F, —(CH₂)N(C_1-3alkyl)(C_1-3alkyl), —(CH₂)_pheterocyclyl optionally substituted with 1-2 R¹⁴, and —O(heterocyclyl optionally substituted with 1-2 R²).

In some embodiments of Formula IV, R⁵is F; in some embodiments of Formula IV, R is —(CH₂)N(C_1-3alkyl)(C_1-3alkyl); in some embodiments of Formula IV, R⁵is —(CH₂)_pheterocyclyl optionally substituted with 1-2 R¹⁴; and in some embodiments of Formula IV, R⁵is —O(heterocyclyl optionally substituted with 1-2 R²¹).

In some embodiments of Formula IV, R⁶is selected from the group consisting of F, Me, —(CH₂)N(C_1-3alkyl)(C_1-3alkyl), —(CH₂)_pheterocyclyl optionally substituted with 1-2 R¹⁴, —OMe, —OCHF₂, —OCF₃, —O(heterocyclyl optionally substituted with 1-2 R²), and —C(═O)N(R⁵)₂.

In some embodiments of Formula IV, R⁶is F; in some embodiments of Formula IV, R⁶is Me; in some embodiments of Formula IV, R⁶is —(CH₂)N(C_1-3alkyl)(C_1-3alkyl); in some embodiments of Formula IV, R⁶is —(CH₂)_pheterocyclyl optionally substituted with 1-2 R¹⁴; in some embodiments of Formula IV, R⁶is —OMe; in some embodiments of Formula IV, R⁶is —OCHF₂; in some embodiments of Formula IV, R⁶is —OCF₃; in some embodiments of Formula IV, R⁶is —O(heterocyclyl optionally substituted with 1-2 R²¹); and in some embodiments of Formula IV, R⁶is —C(═O)N(R⁵)₂.

U.S. application Ser. Nos. 15/749,910, 15/749,922, 15/749,923, and 15/749,929, and U.S. Pat. Nos. 8,252,812, 8,450,340, 8,673,936, 8,883,822, 9,908,867, 9,475,807, 9,475,825, 9,493,487, 9,540,398, 9,546,185, 9,657,016, 9,738,638, and 9,758,531 describe compounds having Formula V and are hereby incorporated by reference in their entirety.

One embodiment disclosed herein includes a compound having the structure of Formula V:

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof.

In some embodiments of Formula (V):

R¹is a -heteroaryl optionally substituted with 1-2 R³;

R²is selected from the group consisting of H, halide (e.g., F, Cl, Br, I), -aryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R⁴-heteroaryl optionally substituted with 1-4 (e.g., 1-3, 1-2, 1) R⁵, and -heterocyclyl ring optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁶;

each R³is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, Cl-3, C_1-2, C₁), —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁷, —C(═O)N(RW)₂, —NHC(═O)R⁹, —(C_1-4alkylene)_pN(R¹⁰)(R¹¹), —(C_1-4alkylene)_pOR¹², and -carbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹³; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁴is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —CN, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), —(C_1-4alkylene)_pNHSO₂R¹⁴, —NR⁵(C_1-4alkylene)NR¹⁵R¹⁶, —(C_1-4alkylene)_pNR¹⁵R¹⁶, —OR¹⁷, and -heterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹⁹; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁵is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), and —C(═O)R¹⁸;

each R⁷is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —NH₂, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R⁹is independently selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), -heterocyclyl optionally substituted with 1-10 (e.g., 1-9 , 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹⁹, —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²⁰; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R⁹is independently selected from the group consisting of unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹⁹, —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²⁰; —(C_1-4alkylene)_paryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R²¹, —(C_1-4alkylene)_pN(R²²)₂; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R¹¹is independently selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²⁰; and —(C_1-4alkylene)_paryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R²¹; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R¹²is independently selected from the group consisting of H unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹⁹, —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²⁰; —(C_1-4alkylene)_paryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R²¹, —(C_1-4alkylene)_pN(R²²)₂; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁴is independently selected from the group consisting of unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), and unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂);

each R¹⁵is independently selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), and unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂);

each R¹⁶is independently selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), and unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂);

each R¹⁷is independently selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹⁹, and, —(C_1-4alkylene)_pN(R²²)₂; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁸is independently selected from the group consisting of unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂);

each R¹⁹is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R²⁰is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R²¹is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C₂-s, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R²²is independently selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂);

each R²³is independently selected from the group consisting of H and halide (e.g., F, Cl, Br, I);

Y¹, Y², and Y³are independently selected from the group consisting of —CR²³═ and —N═;

Y⁴is selected from the group of —CH═ and —N═;

Z¹, Z², and Z³are independently selected from the group consisting of —CR²³═ and —N═; and

each p is independently 0 or 1.

In some embodiments of Formula V, R¹is selected from the group consisting of -pyridinyl optionally substituted with 1-2 R³, -pyrimidinyl optionally substituted with 1-2 R³, -pyrazinyl optionally substituted with 1-2 R³, -pyrazolyl optionally substituted with 1-2 R³, -isothiazolyl optionally substituted with 1-2 R³, and -thiazolyl optionally substituted with 1-2 R³.

In some embodiments of Formula V, R¹is -pyridinyl optionally substituted with 1-2 R³; in some embodiments of Formula V, R¹is -pyrimidinyl optionally substituted with 1-2 R³; in some embodiments of Formula V, R¹is -pyrazinyl optionally substituted with 1-2 R³; in some embodiments of Formula V, R¹is -pyrazolyl optionally substituted with 1-2 R³; in some embodiments of Formula V, R¹is -isothiazolyl optionally substituted with 1-2 R³; and in some embodiments of Formula V, R¹is -thiazolyl optionally substituted with 1-2 R³.

In some embodiments of Formula V, R²is selected from the group consisting of -phenyl optionally substituted with 1-2 R⁴-pyridinyl optionally substituted with one R⁵, -thiophenyl optionally substituted with one R⁵, -furanyl optionally substituted with one R⁵, -piperidinyl ring optionally substituted with one R⁶, and -piperazinyl ring optionally substituted with one R⁶.

In some embodiments of Formula V, R²is -phenyl optionally substituted with 1-2 R⁴; in some embodiments of Formula V, R²is -pyridinyl optionally substituted with one R⁵; in some embodiments of Formula V, R²is -thiophenyl optionally substituted with one R⁵; in some embodiments of Formula V, R²is -furanyl optionally substituted with one R⁵; in some embodiments of Formula V, R²is -piperidinyl ring optionally substituted with one R⁶; and in some embodiments of Formula V, R²is -piperazinyl ring optionally substituted with one R⁶.

In some embodiments of Formula V, R³is selected from the group consisting of unsubstituted —(C_1-3alkyl), —(CH₂)_pheterocyclyl optionally substituted with 1-2 R⁷, —OH, —O((CH₂CH₂)heterocyclyl), —O(heterocyclyl), —O((CH₂)N(C_1-3alkyl)(C_1-3alkyl)), —NH₂, —(CH₂)N(C_1-3alkyl)(C_1-3alkyl), —(CH₂)NH(C_1-3alkyl), —N(C_1-3alkyl)(C_1-3alkyl), —NHC(═O)(C_1-5alkyl), and —NHC(═O)(—(CH₂)_pheterocyclyl).

In some embodiments of Formula V, R³is unsubstituted —(C_1-3alkyl); in some embodiments of Formula V, R³is —(CH₂)_pheterocyclyl optionally substituted with 1-2 R⁷; in some embodiments of Formula V, R³is —OH; in some embodiments of Formula V, R³is —O((CH₂CH₂)heterocyclyl); in some embodiments of Formula V, R³is —O(heterocyclyl); in some embodiments of Formula V, R³is —O((CH₂)N(C_1-3alkyl)(C_1-3alkyl)); in some embodiments of Formula V, R³is —NH₂; in some embodiments of Formula V, R³is —(CH₂)N(C_1-3alkyl)(C_1-3alkyl); in some embodiments of Formula V, R³is —(CH₂)NH(C_1-3alkyl); in some embodiments of Formula V, R³is —N(C_1-3alkyl)(C_1-3alkyl); in some embodiments of Formula V, R³is —NHC(═O)(C_1-5alkyl); and in some embodiments of Formula V, R³is —NHC(═O)(—(CH₂)_pheterocyclyl).

In some embodiments of Formula V, Y¹, Y², Y³, and Y⁴are all —CH═; in some embodiments of Formula V, Y¹is —N═ and Y², Y³, and Y⁴are all —CH═; in some embodiments of Formula V, Y²is —N═ and Y¹, Y³, and Y⁴are all —CH═; in some embodiments of Formula V, Y³is —N═ and Y¹, Y², and Y⁴are all —CH═; in some embodiments of Formula V, Y⁴is —N═ and Y¹, Y², and Y³are all —CH═.

In some embodiments of Formula V, Z¹, Z², and Z³are all —CH═; in some embodiments of Formula V, Z²is —CF═ and Z¹and Z³are both —CH═; in some embodiments of Formula V, Z¹is —N═ and Z²and Z³are both —CH═; in some embodiments of Formula V, Z²is —N═ and Z¹and Z³are both —CH═; in some embodiments of Formula V, Z³is —N═ and Z¹and Z²are both —CH═.

In some embodiments of Formula V, Y¹, Y², Y³, Y⁴, Z¹, Z², and Z³are all —CH═; in some embodiments of Formula V, Z²is —CF═ and Y¹, Y², Y³, Y⁴, Z¹and Z³are all —CH═; in some embodiments of Formula V, Y⁴is —N═ and Y¹, Y², Y³, Z¹, Z²and Z³are all —CH═; in some embodiments of Formula V, Z²is —CF═, Y⁴is —N═ and Y¹, Y², Y³, Z¹and Z³are all —CH═.

In some embodiments of Formula V, Y¹is —N═ and Y², Y³, Y⁴, Z¹, Z², and Z³are all —CH═; in some embodiments of Formula V, Y²is —N═ and Y¹, Y², Y³, Y⁴, Z, Z², and Z³are all —CH═; in some embodiments of Formula V, Y³is —N═ and Y¹, Y², Y⁴, Z¹, Z², and Z³are all —CH═; in some embodiments of Formula V, Y¹is —N═, Z²is —CF═ and Y², Y³, Y⁴, Z¹, and Z³are all —CH═; in some embodiments of Formula V, Y²is —N═, Z²is —CF═ and Y¹, Y³, Y⁴, Z¹, and Z³are all —CH═; and in some embodiments of Formula V, Y³is —N═, Z²is —CF═ and Y¹, Y², Y⁴, Z¹, and Z³are all —CH═; in some embodiments of Formula V, Y¹and Y⁴are —N═ and Y², Y³, Z¹, Z², and Z³are all —CH═; in some embodiments of Formula V, Y²and Y⁴are —N═ and Y¹, Y³, Z¹, Z², and Z³are all —CH═; in some embodiments of Formula V, Y³and Y⁴are —N═ and Y¹, Y², Z¹, Z², and Z³are all —CH═; in some embodiments of Formula V, Y¹and Y⁴are —N═, Z²is —CF═ and Y², Y³, Z¹, and Z³are all —CH═; in some embodiments of Formula V, Y²and Y⁴are —N═, Z²is —CF═ and Y¹, Y³, Z¹, and Z³are all —CH═; and in some embodiments of Formula V, Y³and Y⁴are —N═, Z²is —CF═ and Y¹, Y², Z¹, and Z³are all —CH═.

In some embodiments of Formula V, Z¹is —N═ and Y¹, Y², Y³, Y⁴, Z²and Z³are all —CH═; in some embodiments of Formula V, Z²is —N═ and Y¹, Y², Y³, Y⁴, Z¹and Z³are all —CH═; and in some embodiments of Formula V, Z³is —N═ and Y¹, Y², Y³, Y⁴, Z¹and Z²are all —CH═; in some embodiments of Formula V, Z¹and Y⁴are —N═ and Y¹, Y², Y³, Z²and Z³are all —CH═; in some embodiments of Formula V, Z²and Y⁴are —N═ and Y¹, Y², Y³, Z¹and Z³are all —CH═; and in some embodiments of Formula V, Z³and Y⁴are —N═ and Y¹, Y², Y³, Z¹and Z²are all —CH═.

In some embodiments of Formula V, Y¹and Z¹are —N═ and Y², Y³, Y⁴, Z², and Z³are all —CH═; in some embodiments of Formula V, Y¹and Z²are —N═ and Y², Y³, Y⁴, Z¹, and Z³are all —CH═; Y¹and Z³are —N═ and Y², Y³, Y⁴, Z¹, and Z²are all —CH═; in some embodiments of Formula V, Y²and Z¹are —N═ and Y¹, Y³, Y⁴, Z², and Z³are all —CH═; in some embodiments of Formula V, Y²and Z²are —N═ and Y¹, Y³, Y⁴, Z, and Z³are all —CH═; in some embodiments of Formula V, Y²and Z³are —N═ and Y¹, Y³, Y⁴, Z, and Z²are all —CH═; in some embodiments of Formula V, Y³and Z¹are —N═ and Y¹, Y², Y⁴, Z², and Z³are all —CH═; in some embodiments of Formula V, Y³and Z²are —N═ and Y¹, Y², Y⁴, Z¹, and Z³are all —CH═; and in some embodiments of Formula V, Y³and Z³are —N═ and Y¹, Y², Y⁴, Z¹, and Z²are all —CH═;

in some embodiments of Formula V, Y¹, Z¹, and Y⁴are —N═ and Y², Y³, Z², and Z³are all —CH═; in some embodiments of Formula V, Y¹, Z², and Y⁴are —N═ and Y², Y³, Z¹, and Z³are all —CH═; Y¹, Z³, and Y⁴are —N═ and Y², Y³, Z¹, and Z²are all —CH═; in some embodiments of Formula V, Y², Z¹, and Y⁴are —N═ and Y¹, Y³, Z², and Z³are all —CH═; in some embodiments of Formula V, Y², Z², and Y⁴are —N═ and Y¹, Y³, Z¹, and Z³are all —CH═; in some embodiments of Formula V, Y², Z³, and Y⁴are —N═ and Y¹, Y³, Z¹, and Z²are all —CH═; in some embodiments of Formula V, Y³, Z¹, and Y⁴are —N═ and Y¹, Y², Z², and Z³are all —CH═; in some embodiments of Formula V, Y³, Z², and Y⁴are —N═ and Y¹, Y², Z¹, and Z³are all —CH═; and in some embodiments of Formula V, Y³, Z³, and Y⁴are —N═ and Y¹, Y², Z¹, and Z²are all —CH═.

Kazuho Nishimura, Masahiro Yaguchi, Yukiko Yamamoto, Shunsuke Ebara, Kawakita Yoichi, Ryo Mizojiri, Yusuke Nakayama, Kozo Hayashi, Shuichi Miyakawa, Kenichi Iwai, Toshiyuki Nomura. Takeda Pharmaceutical Company Limited, Cambridge, Mass., Small Molecule Inhibitor of Pre-mRNA Splicing Evokes Antitumor Activity via MDM4-p53. Poster presented at: Molecular Targets and Cancer Therapeutics: Discovery, Biology, and Clinical Applications. AACR-NCI-EORTC International Conference. 2017 Oct. 27-30, Philadelphia, Pa., Journal of Medicinal Chemistry (2017), 60(21), 8989-9002, and U.S. Pat. Nos. 9,346,812 and 9,428,509 describe compounds having Formula VI and are hereby incorporated by reference in their entirety.

One embodiment disclosed herein includes a compound having the structure of Formula VI:

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof.

In some embodiments of Formula (VI):

R¹is selected from the group consisting of H, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and -heteroaryl optionally substituted with 1-4 (e.g., 1-3, 1-2, 1) R⁴, -aryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R⁵;

R²is selected from the group consisting of H, —(C_1-4alkylene)_pheteroaryl optionally substituted with 1-4 (e.g., 1-3, 1-2, 1) R⁶, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁷, and —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

R³is selected from the group consisting of -heteroaryl optionally substituted with 1-4 (e.g., 1-3, 1-2, 1) R⁹and -aryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R¹⁰;

each R⁴is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —CN, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), —OR¹¹, —C(═O)N(R¹²)₂, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹³, —SO₂R¹⁴, and —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁵; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁵is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —CN, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), —OR¹¹, —C(═O)N(R¹²)₂, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹³, —SO₂R¹⁴, and —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁵; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R⁸is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

each R⁹is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —CN, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_{1_3}, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C₂-s, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), —OR¹¹, —C(═O)N(R¹²)₂, and —SO₂R¹⁴;

each R¹⁰is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —CN, unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), —OR¹¹, —C(═O)N(R¹²)₂, and —SO₂R¹⁴;

each R¹²is independently selected from the group consisting of H, halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂);

each R¹³is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-3, C_1-4, C_1-3, C_1-2, C₁₎, unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-3, C_1-4, C_1-3, C_1-2, C₁);

each R¹⁴is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-3, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂);

each R¹⁵is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-6alkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-6alkenyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-6alkynyl) (e.g., C_2-5, C_2-4, C_2-3, C₂), and unsubstituted —(C_1-6haloalkyl) (e.g., C_1-5, C_1-4, C_1-3, C_1-2, C₁);

L is selected from the group consisting of a bond, —O—, and —NH—; and

each p is independently 0 or 1.

In some embodiments of Formula VI, R¹is selected from the group consisting of unsubstituted —(C_1-3alkyl) and -phenyl substituted with 1-2 R.

In some embodiments of Formula VI, R¹is unsubstituted —(C_1-3alkyl); in some embodiments of Formula VI, R¹is Me; and in some embodiments of Formula VI, R¹is -phenyl substituted with 1-2 R¹.

In some embodiments of Formula VI, R²is selected from the group consisting of —(CH₂)_pheteroaryl optionally substituted with 1-2 R⁶and -carbocyclyl optionally substituted with 1-2 R⁸.

In some embodiments of Formula VI, R²is —(CH₂)_pheteroaryl optionally substituted with 1-2 R⁶; in some embodiments of Formula VI, R²is —(CH₂)_ppyridinyl optionally substituted with 1-2 R⁶; in some embodiments of Formula VI, R²is —(CH₂)_ppyrimidinyl optionally substituted with 1-2 R⁶; in some embodiments of Formula VI, R²is —(CH₂)_ppyrazinyl optionally substituted with 1-2 R⁶; in some embodiments of Formula VI, R²is —(CH₂)_ppyrazolyl optionally substituted with 1-2 R⁶; in some embodiments of Formula VI, R²is —(CH₂)_pisothiazolyl optionally substituted with 1-2 R⁶; in some embodiments of Formula VI, R²is —(CH₂)_pthiazolyl optionally substituted with 1-2 R⁶in some embodiments of Formula VI, R²is -carbocyclyl optionally substituted with 1-2 R; in some embodiments of Formula VI, R²is -cyclopropyl optionally substituted with 1-2 R⁸; in some embodiments of Formula VI, R²is -cyclobutyl optionally substituted with 1-2 R⁸; in some embodiments of Formula VI, R²is -cyclopentyl optionally substituted with 1-2 R⁸; and in some embodiments of Formula VI, R²is -cyclohexyl optionally substituted with 1-2 R⁸.

In some embodiments of Formula VI, R³is selected from the group consisting of -heteroaryl optionally substituted with 1-2 R⁹and -phenyl optionally substituted with 1-2 R¹⁰.

In some embodiments of Formula VI, R³is -heteroaryl optionally substituted with 1-2 R⁹; in some embodiments of Formula VI, R³is -pyridinyl optionally substituted with 1-2 R⁹; in some embodiments of Formula VI, R³is -quinolinyl optionally substituted with 1-2 R⁹; in some embodiments of Formula VI, R³is -isoquinolinyl optionally substituted with 1-2 R⁹; in some embodiments of Formula VI, R³is -benzoxazolyl optionally substituted with 1-2 R⁹; in some embodiments of Formula VI, R³is -benzothiazolyl optionally substituted with 1-2 R⁹; in some embodiments of Formula VI, R³is -benzoimidiazolyl optionally substituted with 1-2 R⁹; and in some embodiments of Formula VI, R³is -phenyl optionally substituted with 1-2 R¹⁰.

In some embodiments of Formula VI, L is a bond; in some embodiments of Formula VI, L is —O—, and in some embodiments of Formula VI, L is —NH—.

U.S. provisional applications 62/577,818, 62/578,370, 62/578,691, and 62/579,883, U.S. application Ser. Nos. 15/498,990 and 15/499,013, and U.S. Pat. No. 9,951,048 describe compounds having Formula VII and are hereby incorporated by reference in their entirety.

One embodiment disclosed herein includes a compound having the structure of Formula VII:

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof.

In some embodiments of Formula (VII):

R¹, R², R⁴, and R⁵are independently absent or selected from the group consisting of H and halide (e.g., F, Cl, Br, I);

R³is selected from the group of -heteroaryl optionally substituted with 1-4 (e.g., 1-3, 1-2, 1) R⁸and -Xheterocyclyl optionally substituted with one or more halides (e.g., F, Cl, Br, I) or one or more unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁);

R⁶is selected from the group consisting of -aryl substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R⁹, —(C_2-4alkenylene)aryl substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R⁹, —(C_1-4alkylene)_pheteroaryl optionally substituted with 1-6 (e.g., 1-5, 1-4, 1-3, 1-2, 1) R¹⁰; -heterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹¹, -carbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R¹², and —(C_2-9alkynyl) optionally substituted with one or more halides (e.g., F, Cl, Br, I)s; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein; wherein —(C_1-4alkenylene) is, optionally substituted with one or more substituents as defined anywhere herein;

with the proviso that R⁶is heterocyclyl only when R³is a 6-membered heteroaryl;

each R⁸is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-9alkyl) (e.g., C_1-6, C_1-7, C_1-6, C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-9alkenyl) (e.g., C_2-8, C_2-7, C_2-6, C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-9alkynyl) (e.g., C_{2_8}, C_{2_7}, C_{2_6}, C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-9haloalkyl) (e.g., C_1-8, C_{1_7}, C_1-6, C_1-5, C_1-4, C_1-3, C_1-2, C₁), —CN, —N(R¹⁵)(R¹⁸), —(C_1-4alkylene)_pXR¹⁹, —C(═O)N(R⁵)₂, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²⁰, and -carbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²¹; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

alternatively, two adjacent R⁸are taken together to form a ring which is selected from the group consisting of -heterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²²and -carbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²¹;

each R⁹is independently selected from the group consisting of D, halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-9alkyl) (e.g., C_1-8, C_1-7, C_1-6, C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-9alkenyl) (e.g., C_2-8, C_2-7, C_2-6, C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-9alkynyl) (e.g., C_2-8, C_2-7, C_2-6, C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-9haloalkyl) (e.g., C_1-8, C_1-7, C_1-6, C_1-5, C_1-4, C_1-3, C_1-2, C₁), —XR²³, —(C_1-4alkylene)_pN(R²⁴)₂, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²²; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁰is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-9alkyl) (e.g., C_1-8, C_1-7, C_1-6, C_1-5, C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-9alkenyl) (e.g., C_2-8, C_2-7, C_2-6, C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_2-9alkynyl) (e.g., C_2-8, C_2-7, C_2-6, C_2-5, C_2-4, C_2-3, C₂), unsubstituted —(C_1-9haloalkyl) (e.g., C_1-8, C_1-7, C_1-6, C_1-5, C_1-4, C_1-3, C_1-2, C₁), —CN, —XR²³, —C(═O)N(R⁵)₂, —(C_1-4alkylene)_pN(R²⁴)₂, -heterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²², and -carbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²¹; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R¹¹is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-9alkyl), unsubstituted —(C_2-9alkenyl), unsubstituted —(C_2-9alkynyl), and unsubstituted —(C_1-9haloalkyl);

each R¹²is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), —(C_1-4alkylene)_pOR¹⁹; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁵is selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁);

R¹⁸is independently selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), and —(C_1-4alkylene)_pheterocyclyl optionally substituted with one or more halides (e.g., F, Cl, Br, I) or one or more unsubstituted —(C_1-3alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁); wherein —(C_1-4alkylene) is, independently,

optionally substituted with one or more substituents as defined anywhere herein; each R¹⁹is independently selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-4haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), —(C_1-4alkylene)_pheterocyclyl optionally substituted with one or more halides (e.g., F, Cl, Br, I)s or one or more unsubstituted —(C_1-3alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁); wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R²⁰independently is selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C₂_s alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), and —OH;

each R²¹is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), and —CN;

each R²²is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), —OH, —N(R⁵)₂, —C(═O)R³⁴, and -carbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²¹;

each R²³is independently selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), —(C_1-4alkylene)N(R¹⁵)₂, -heterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R³¹, and -carbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R²¹; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R²⁴is independently selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), —(C_1-4alkylene)_pheterocyclyl optionally substituted with one or more halides (e.g., F, Cl, Br, I) or one or more unsubstituted —(C_1-5alkyl), and —(C_1-4alkylene)N(R⁵)₂; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

each R³¹is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁);

each R³⁴is independently selected from the group consisting of —O(C_1-5alkyl) and a heteroaryl optionally substituted with 1-6 (e.g., 1-5, 1-4, 1-3, 1-2, 1) R³⁵;

each R³⁵is a -heterocyclyl optionally substituted with one or more halides (e.g., F, Cl, Br, I) or one or more unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁);

each X is selected from the group consisting of O and S;

Y¹, Y², Y³, and Y⁴are independently selected from the group consisting of carbon and nitrogen; wherein

if Y¹is nitrogen then Y², Y³, and Y⁴are carbon, and R⁴is absent;

if Y²is nitrogen then Y¹, Y³, and Y⁴are carbon, and R⁵is absent;

if Y³is nitrogen then Y¹, Y², and Y⁴are carbon, and R¹is absent;

if Y⁴is nitrogen then Y¹, Y², and Y³are carbon, and R²is absent; and

each p is independently 0 or 1.

In some embodiments of Formula VII, R¹, R², R⁴, and R⁵are all H or absent; in some embodiments of Formula VII, R¹is F and R², R⁴, and R⁵are all H or absent; in some embodiments of Formula VII, R²is F and R¹, R⁴, and R⁵are all H or absent; in some embodiments of Formula VII, R⁴is F and R¹, R², and R⁵are all H or absent; and in some embodiments of Formula VII, R⁵is F and R¹, R², and R⁴are all H or absent.

In some embodiments of Formula VII, R³is selected from the group consisting of -pyridinyl optionally substituted with 1-2 R, -pyrimidinyl optionally substituted with 1-2 R⁸, -pyrazinyl optionally substituted with 1-2 R, -pyrazolyl optionally substituted with 1-2 R⁸, -isothiazolyl optionally substituted with 1-2 R, -thiazolyl optionally substituted with 1-2 R⁸, -pyrazolyl optionally substituted with 1-2 R⁸, -imidazolyl optionally substituted with 1-2 R⁸, -1,2,3-triazolyl optionally substituted with 1-2 R⁸, -isoxazolyl optionally substituted with 1-2 R⁸, and -oxazolyl optionally substituted with 1-2 R⁸;

In some embodiments of Formula VII, R³is -pyridinyl optionally substituted with 1-2 R⁸; in some embodiments of Formula VII, R³is -pyrimidinyl optionally substituted with 1-2 R⁸; in some embodiments of Formula VII, R³is -pyrazinyl optionally substituted with 1-2 R⁸; in some embodiments of Formula VII, R³is -pyrazolyl optionally substituted with 1-2 R⁸; in some embodiments of Formula VII, R³is -isothiazolyl optionally substituted with 1-2 R⁸; in some embodiments of Formula VII, R is -thiazolyl optionally substituted with 1-2 R⁸; in some embodiments of Formula VII, R³is -pyrazolyl optionally substituted with 1-2 R⁸; in some embodiments of Formula VII, R is -imidazolyl optionally substituted with 1-2 R⁸; in some embodiments of Formula VII, R³is -1,2,3-triazolyl optionally substituted with 1-2 R⁸; in some embodiments of Formula VII, R³is -isoxazolyl optionally substituted with 1-2 R⁸; and in some embodiments of Formula VII, R³is -oxazolyl optionally substituted with 1-2 R⁸.

In some embodiments of Formula VII, R⁶is selected from the group consisting of -phenyl substituted with 1-2 R⁹, -heteroaryl optionally substituted with 1-2 R¹⁰; -heterocyclyl optionally substituted with 1-2 R¹¹, and -carbocyclyl optionally substituted with 1-2 R¹².

In some embodiments of Formula VII, R⁶is -phenyl substituted with 1-2 R⁹; in some embodiments of Formula VII, R⁶is -pyridinyl optionally substituted with 1-2 R¹⁰; in some embodiments of Formula VII, R⁶is -pyrazolyl optionally substituted with 1-2 R¹⁰; in some embodiments of Formula VII, R⁶is -thiazolyl optionally substituted with 1-2 R¹⁰; in some embodiments of Formula VII, R⁶is -imidazolyl optionally substituted with 1-2 R¹⁰; in some embodiments of Formula VII, R⁶is -isoindolinyl optionally substituted with 1-2 R¹⁰; in some embodiments of Formula VII, R⁶is -tetrahydroisoquinolinyl optionally substituted with 1-2 R¹⁰; in some embodiments of Formula VII, R⁶is -1,2,3-triazolyl optionally substituted with 1-2 R¹⁰; in some embodiments of Formula VII, R⁶is -benzimidazolyl optionally substituted with 1-2 R¹⁰; in some embodiments of Formula VII, R⁶is -indazolyl optionally substituted with 1-2 R¹⁰; in some embodiments of Formula VII, R⁶is -cyclopropyl optionally substituted with 1-2 R¹²; in some embodiments of Formula VII, R⁶is -cyclobutyl optionally substituted with 1-2 R¹²; in some embodiments of Formula VII, R⁶is -cyclopentyl optionally substituted with 1-2 R¹²; and in some embodiments of Formula VII, R⁶is -cyclohexyl optionally substituted with 1-2 R¹².

In some embodiments of Formula VII, R⁸is selected from the group consisting of F, unsubstituted —(C_1-3alkyl), —CN, —NH₂, —NH(C_1-4alkyl), —N(C_1-4alkyl)₂, —NH(heterocyclyl), —NH(CH₂heterocyclyl), —OMe, —CH₂OH, and —(CH₂)_pheterocyclyl optionally substituted with 1-2 R²⁰.

In some embodiments of Formula VII, R⁸is F; in some embodiments of Formula VII, R⁸is unsubstituted —(C_1-3alkyl); in some embodiments of Formula VII, R⁸is —NH₂; in some embodiments of Formula VII, R⁸is —NH(C_1-4alkyl); in some embodiments of Formula VII, R⁸is —N(C_1-4alkyl)₂; in some embodiments of Formula VII, R⁸is —NH(heterocyclyl); in some embodiments of Formula VII, R⁸is —NH(CH₂heterocyclyl); in some embodiments of Formula VII, R⁸is —OMe, in some embodiments of Formula VII, R⁸is —CH₂OH; and in some embodiments of Formula VII, R⁸is —(CH₂)_pheterocyclyl optionally substituted with 1-2 R²⁰.

In some embodiments of Formula VII, R¹⁰is selected from the group consisting of F, unsubstituted —(C_1-5alkyl), —CN, —OR²³, —NH(heterocyclyl), —N(C_1-3alkyl)(heterocyclyl), —O(C_1-3alkyl), —O(heterocyclyl), —S(heterocyclyl), —C(═O)NH(C_1-3alkyl), —N(R²⁴)₂, -heterocyclyl optionally substituted with 1-2 R²², and -carbocyclyl optionally substituted with 1-2 R²¹.

In some embodiments of Formula VII, R¹⁰is F; in some embodiments of Formula VII, R¹⁰is unsubstituted —(C_1-5alkyl); in some embodiments of Formula VII, R¹⁰is —CN; in some embodiments of Formula VII, R¹⁰is —OR²³; in some embodiments of Formula VII, R¹⁰is —NH(heterocyclyl); in some embodiments of Formula VII, R¹⁰is —N(C_1-3alkyl)(heterocyclyl); in some embodiments of Formula VII, R¹⁰is —O(C_1-3alkyl); in some embodiments of Formula VII, R¹⁰is —O(heterocyclyl); in some embodiments of Formula VII, R¹⁰is —S(heterocyclyl); in some embodiments of Formula VII, R¹⁰is —C(═O)NH(C_1-3alkyl); in some embodiments of Formula VII, R¹⁰is —N(R²⁴)₂; in some embodiments of Formula VII, R¹⁰is -heterocyclyl optionally substituted with 1-2 R²²; and in some embodiments of Formula VII, R¹⁰is -carbocyclyl optionally substituted with 1-2 R²¹.

In some embodiments of Formula VII, Y¹, Y², Y³, and Y⁴are all carbon; in some embodiments of Formula VII, Y¹is nitrogen and Y², Y³, and Y⁴are all carbon; in some embodiments of Formula VII, Y²is nitrogen and Y¹, Y³, and Y⁴are all carbon; in some embodiments of Formula VII, Y³is nitrogen and Y¹, Y², and Y⁴are all carbon; in some embodiments of Formula VII, Y⁴is nitrogen and Y¹, Y², and Y³are all carbon.

U.S. Pat. No. 8,119,655 describes compounds having Formula VIII and is hereby incorporated by reference in its entirety.

One embodiment disclosed herein includes a compound having the structure of Formula VIII:

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof.

In some embodiments of Formula (VIII):

R¹is selected from the group consisting of —(C_1-4alkylene)N(R⁵)₂, —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁶, and —(C_1-4alkylene)_pcarbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁷; wherein each —(C_1-4alkylene) is, independently, optionally substituted with one or more substituents as defined anywhere herein;

R²is selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), —CN, —OR, —C(═O)NHR⁹, —NHC(═O)(R¹⁰), —SO₂R¹⁰, —NHSO₂R¹⁰, and —SO₂NHR⁹;

R³is selected from the group consisting of H, halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁);

R⁴is selected from the group consisting of H, halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁);

each R⁵is independently selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, Cl-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), and unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂);

each R⁶is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C₂_s alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), —OH, and —CN;

each R⁷is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C₂_s alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), —OH, and —CN;

R⁸is selected from the group consisting of H, unsubstituted —(C_1-3alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁶; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R⁹is independently selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁶; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R¹⁰is independently selected from the group consisting of unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and —(C_1-4alkylene)_pheterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁶; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein; and

each p is independently 0 or 1.

In some embodiments of Formula VIII, R¹is selected from the group consisting of —(C_1-2alkylene)N(C_1-3alkyl)₂, —(CH₂)_pheterocyclyl optionally substituted with 1-2 R⁶, and —(CH₂)_pcarbocyclyl optionally substituted with 1-2 R⁷.

In some embodiments of Formula VIII, R¹is —(C_1-2alkylene)N(C_1-3alkyl)₂; in some embodiments of Formula VIII, R¹is —(CH₂)_pheterocyclyl optionally substituted with 1-2 R⁶; and in some embodiments of Formula VIII, R¹is —(CH₂)_pcarbocyclyl optionally substituted with 1-2 R⁷.

In some embodiments of Formula VIII, R²is selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-4alkyl), unsubstituted —(C_1-4haloalkyl), —CN, —O(C_1-4alkyl), —O(heterocyclyl), —C(═O)NH(C_1-5alkyl), —NHC(═O)(C_1-4alkyl), —SO₂(C_1-4alkyl), —NHSO₂(C_1-4alkyl), and —SO₂NH(C_1-4alkyl).

In some embodiments of Formula VIII, R²is F; in some embodiments of Formula VIII, R²is unsubstituted —(C_1-4alkyl); in some embodiments of Formula VIII, R²is unsubstituted —(C_1-4haloalkyl); in some embodiments of Formula VIII, R²is —CN; in some embodiments of Formula VIII, R²is —O(C_1-4alkyl); in some embodiments of Formula VIII, R²is —O(heterocyclyl); in some embodiments of Formula VIII, R²is —C(═O)NH(C_1-5alkyl); in some embodiments of Formula VIII, R²is —NHC(═O)(C_1-4alkyl); in some embodiments of Formula VIII, R²is —SO₂(C_1-4alkyl); in some embodiments of Formula VIII, R²is —NHSO₂(C_1-4alkyl); and in some embodiments of Formula VIII, R²is —SO₂NH(C_1-4alkyl).

In some embodiments of Formula VIII, R³is selected from the group consisting of H, halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-3alkyl), and unsubstituted —(C_1-3haloalkyl).

In some embodiments of Formula VIII, R³is H; in some embodiments of Formula VIII, R³is F; in some embodiments of Formula VIII, R³is unsubstituted —(C_1-3alkyl); and in some embodiments of Formula VIII, R³is unsubstituted —(C_1-3haloalkyl).

In some embodiments of Formula VIII, R⁴is selected from the group consisting of H, halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-3alkyl), and unsubstituted —(C_1-3haloalkyl).

In some embodiments of Formula VIII, R⁴is H; in some embodiments of Formula VIII, R⁴is F; in some embodiments of Formula VIII, R⁴is unsubstituted —(C_1-3alkyl); and in some embodiments of Formula VIII, R⁴is unsubstituted —(C_1-3haloalkyl).

U.S. Pat. No. 8,067,591 describes compounds having Formula IX and is hereby incorporated by reference in its entirety.

One embodiment disclosed herein includes a compound having the structure of Formula IX:

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof.

In some embodiments of Formula (IX):

R¹is -heteroaryl optionally substituted with 1-6 (e.g., 1-5, 1-4, 1-3, 1-2, 1) R⁴; each R²is independently selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁); R³is —CH(R⁵)R⁶;

each R⁴is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), —CN, —OR⁷, -carbocyclyl optionally substituted with 1-12 (e.g., 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R;

R⁵is -aryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R⁹;

R⁶is —(C_1-4alkylene)N(R¹⁰)₂; wherein —(C_1-4alkylene) is optionally substituted with one or more substituents as defined anywhere herein;

each R⁷is independently selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁);

each R⁸is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁);

each R⁹is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), —CN, and —OR⁷;

each R¹⁰is independently selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), and unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂); and

X is selected from the group consisting of O, S, and NH.

In some embodiments of Formula IX, R¹is -heteroaryl optionally substituted with 1-2 R⁴.

In some embodiments of Formula IX, R¹is a 6-10 membered -heteroaryl optionally substituted with 1-2 R⁴.

In some embodiments of Formula IX, R¹is -pyridinyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IX, R¹is -pyrimidinyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IX, R¹is -pyrazinyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IX, in some embodiments of Formula IX, R¹is -benzimidazolyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IX, R¹is -indazolyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IX, R¹is -thieno[3,2-d]pyrimidinyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IX, R¹is -thiazolo[4,5-d]pyrimidinyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IX, R¹is -benzo[b]thiophenyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IX, R¹is -benzo[d]thiazolyl optionally substituted with 1-2 R⁴; in some embodiments of Formula IX, R¹is -thieno[2,3-c]pyridinyl optionally substituted with 1-2 R⁴; and in some embodiments of Formula IX, R¹is -thieno[3,2-b]pyridinyl optionally substituted with 1-2 R⁴.

In some embodiments of Formula IX, R²is selected from the group consisting of H, unsubstituted —(C_1-3alkyl) and unsubstituted —(C_1-3haloalkyl).

In some embodiments of Formula IX, R²is H; in some embodiments of Formula IX, R²is unsubstituted —(C_1-3alkyl); and in some embodiments of Formula IX, R²is unsubstituted —(C_1-3haloalkyl).

In some embodiments of Formula IX, R³is —CH(phenyl)(C_1-2alkylene)N(C_1-3alkyl)₂.

In some embodiments of Formula IX, X is O; in some embodiments of Formula IX, X is S; and in some embodiments of Formula IX, X is NH.

Bioorganic & Medicinal Chemistry (2007), 15(17), 5837-5844, World Intellectual Property Organization, WO2001083481, and Spanish patent application 2,244,613 describe compounds having Formula X and are hereby incorporated by reference in their entirety.

One embodiment disclosed herein includes a compound having the structure of Formula X:

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof.

In some embodiments of Formula (X):

R¹is selected from the group consisting of H, halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), and —CN;

R²is selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), and unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂);

R³is -aryl optionally substituted with 1-5 (e.g., 1-4, 1-3, 1-2, 1) R⁴;

each R⁴is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C₂_s alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), —NO₂, —CN, and —OMe;

R⁵is selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁); and

X is selected from the group consisting of N and CR⁵.

In some embodiments of Formula X, R¹is selected from the group consisting of H, halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-3alkyl), unsubstituted —(C_1-3haloalkyl), and —CN.

In some embodiments of Formula X, R¹is H; in some embodiments of Formula X, R¹is F; in some embodiments of Formula X, R¹is unsubstituted —(C_1-3alkyl); in some embodiments of Formula X, R¹is unsubstituted —(C_1-3haloalkyl); and in some embodiments of Formula X, R¹is —CN.

In some embodiments of Formula X, R²is selected from the group consisting of H and unsubstituted —(C_1-3alkyl).

In some embodiments of Formula X, R²is H; and in some embodiments of Formula X, R²is unsubstituted —(C_1-3alkyl).

In some embodiments of Formula X, R³is -phenyl optionally substituted with 1-2 R⁴;

In some embodiments of Formula X, X is selected from the group consisting of N and CH.

In some embodiments of Formula X, X is N; and in some embodiments of Formula X, X is CH.

Bioorganic & Medicinal Chemistry (2012), 22(24), 7326-7329, Bioorganic & Medicinal Chemistry Letters (2014), 24(18), 4418-4423 and Nature Communications (2017), 8(7), 1-15 describe compounds having Formula XI and are hereby incorporated by reference in their entirety.

One embodiment disclosed herein includes a compound having the structure of Formula XI:

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof.

In some embodiments of Formula (XI):

R¹is —N(R⁴)₂;

R²is selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁);

R³is -heteroaryl optionally substituted with 1-6 (e.g., 1-5, 1-4, 1-3, 1-2, 1) R⁵;

each R⁴is independently selected from the group consisting of H, unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and -heterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁶;

alternatively, two adjacent R⁴are taken together to form a ring which is selected from the group consisting of -heterocyclyl optionally substituted with 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1) R⁶;

each R⁵is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C₂_s alkynyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), —CN, —OH, and —OMe; and

each R⁶is independently selected from the group consisting of halide (e.g., F, Cl, Br, I), unsubstituted —(C_1-5alkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁), unsubstituted —(C_2-5alkenyl) (e.g., C_2-4, C_2-3, C₂), unsubstituted —(C_2-5alkynyl) (e.g., C_2-4, C_2-3, C₂), and unsubstituted —(C_1-5haloalkyl) (e.g., C_1-4, C_1-3, C_1-2, C₁).

In some embodiments of Formula XI, R¹is selected from the group consisting of —N(C_1-3alkyl)₂, —NH(C_1-3alkyl), —NH(heterocyclyl), and -heterocyclyl optionally substituted with 1-2 R⁶.

In some embodiments of Formula XI, R¹is —N(C_1-3alkyl)₂; in some embodiments of Formula XI, R¹is —NH(C_1-3alkyl); in some embodiments of Formula XI, R¹is —NH(heterocyclyl); and in some embodiments of Formula XI, R¹is -heterocyclyl optionally substituted with 1-2 R⁶.

In some embodiments of Formula XI, R²is selected from the group consisting of H, unsubstituted —(C_1-3alkyl), and unsubstituted —(C_1-3haloalkyl).

In some embodiments of Formula XI, R²is H; in some embodiments of Formula XI, R²is unsubstituted —(C_1-3alkyl); and in some embodiments of Formula XI, R²is unsubstituted —(C_1-3haloalkyl).

In some embodiments of Formula XI, R³is selected from the group consisting of -pyridinyl optionally substituted with 1-2 R⁵, -pyrazolyl optionally substituted with 1-2 R⁵, -thiazolyl optionally substituted with 1-2 R⁵, -imidazolyl optionally substituted with 1-2 R⁵, and -1,2,3-triazolyl optionally substituted with 1-2 R⁵.

In some embodiments of Formula XI, R³is -pyridinyl optionally substituted with 1-2 R⁵; in some embodiments of Formula XI, R³is -pyrazolyl optionally substituted with 1-2 R⁵; in some embodiments of Formula XI, R³is -thiazolyl optionally substituted with 1-2 R⁵; in some embodiments of Formula XI, R³is -imidazolyl optionally substituted with 1-2 R⁵; and in some embodiments of Formula XI, R³is -1,2,3-triazolyl optionally substituted with 1-2 R⁵.

U.S. provisional application 62/685,764 describes compounds having Formula XII and is hereby incorporated by reference in their entirety.

One embodiment disclosed herein includes a compound having the structure of Formula XII:

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as well as prodrugs and pharmaceutically acceptable salt or solvate thereof.

In some embodiments of Formula (XII):