Inhibitors of LIN28 and methods of using the same

Abstract

The present disclosure relates to compounds of formula (I) and compositions comprising same. The present disclosure further relates to methods of treating cancer. [Formula 1] TIFF2023506951000172.tif27165

Description

Technical Field

[0001] Related Applications This application claims priority and the benefit thereof to U.S. Provisional Patent Application No. 62 / 949,873, filed on December 18, 2019, the entire disclosure of which is incorporated herein by reference.

[0002] Government Support This invention was made with government support under grant number TR001881 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background Art

[0003] Acute myeloid leukemia (AML) is a hematological malignancy characterized by clonal proliferation of myeloblasts that results in a lethal outcome for the majority of affected adults (1). Even under very aggressive multi-drug chemotherapy regimens, newer targeted therapies, and myeloablative allogeneic hematopoietic cell transplantation, the majority of patients die from AML within 5 years. Treatment-resistant leukemia stem cells (LSCs) are thought to be the root cause of the high relapse rate and treatment failure (2-4). Therefore, the development of novel treatment strategies capable of eradicating LSCs occupies a major area of unmet medical need.

[0004] Small molecules have been found to be therapeutic agents that are effective in clinical applications by targeting proteins that are thought to be involved in pathogenesis. However, current FDA-approved drugs directed at G protein-coupled receptors, kinases, peptidases, nuclear receptors, proteases, ion channels, enzymes, etc. modulate less than 700 human genome-derived proteins (63). This implies that less than 0.5% of the proteome and less than 0.05% of the genome have been explored as targets for therapeutic approaches. In addition, most small molecule drugs in clinical use utilize structured binding pockets on the protein surface. Allosteric and / or conformational changes in remote catalytic or drug-binding regions lead to drug resistance and ultimately drug treatment failure (64). Therefore, the development of new drugs that can target hitherto unexplored signaling pathways and overcome resistant mutations occupies a major area of unmet medical need.

Summary of the Invention

[0005] The present disclosure provides a compound of formula (I),

Chem.

Chem.

Chem.

Chem.

[0006] In certain embodiments, the disclosure provides compounds of formula (II) and pharmaceutically acceptable salts thereof

Chemical formula

[0007] In certain embodiments, the disclosure relates to a pharmaceutical composition comprising a compound disclosed herein and a pharmaceutically acceptable excipient.

[0008] In certain embodiments, the disclosure relates to a method of inhibiting Lin28 in a cell, the method comprising contacting a cell comprising Lin28 with a compound or composition disclosed herein.

[0009] In certain embodiments, the disclosure relates to a method of treating cancer, the method comprising administering to a subject in need thereof a compound or composition disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0010]

Figure 1

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Mode for Carrying Out the Invention

[0011] Compound The present disclosure provides a compound of formula (I),

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0012] In some embodiments of formula (I),

Chemical formula

Chemical formula

Chemical formula

[0013] In some embodiments of formula (I),

Chemical formula

Chemical formula

Chemical formula

[0014] As generally defined above, X 1 is selected from N and C-R x In some embodiments of any of formula (I), (I-a), and (I-b), X 1 is N. Thus, in some embodiments, the present disclosure provides a compound of formula (I-a-i) or (I-b-i),

Chemical formula

[0015] In some embodiments of any of Formula (I), (I-a), and (I-b), X 1 is C-R x Accordingly, in some embodiments, the present disclosure provides a compound of Formula (I-a-ii) or (I-b-ii),

Chemical Formula

[0016] As generally defined above with respect to Formula (I), Ring B is selected from phenyl and 5- to 6-membered heteroaryl rings having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-b), (I-b-i), and (I-b-ii), Ring B is phenyl. Accordingly, in some embodiments, the present disclosure provides a compound of Formula (I-a-iii), (I-a-iv), (I-a-v), (I-b-iii), (I-b-iv), and (I-b-v),

Chemical Formula

[0017] In some embodiments of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-b), (I-b-i), and (I-b-ii), Ring B is [Chemistry] is as follows.

[0018] In some embodiments of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-b), (I-b-i), and (I-b-ii), Ring B is a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments of Formula (I), (I-a), I-a-i), I-a-ii), (I-b), (I-b-i), and (I-b-ii), Ring B is a 5-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-b), (I-b-i), and (I-b-ii), Ring B is a 6-membered heteroaryl ring having 1 to 2 nitrogen atoms, for example, pyridyl.

[0019] As generally defined above with respect to Formula (I), X 3 is selected from N and C-R x . In some embodiments of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), and (I-a-v), X 3 is N. In other embodiments of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), and (I-a-v), X 3 is C-R x .

[0020] As generally defined above with respect to Formula (I), X 4 is selected from N and C-R x . In some embodiments of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), X 4is N. In any other embodiment of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), X 4 is C-R x wherein.

[0021] As generally defined above with respect to formula (I), R 1 is hydrogen or is an optionally substituted group selected from C 1-6 aliphatic, phenyl, and 5- to 6-membered heteroaryl rings having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R 1 is hydrogen. In any other embodiment of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R 1 is an optionally substituted group selected from C 1-6 aliphatic, phenyl, and 5- to 6-membered heteroaryl rings having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In any other embodiment of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R 1 is an optionally substituted C 1-6It is aliphatic. In any other embodiment of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R 1 is optionally substituted C 1-3 aliphatic, such as CH3, CH2CH3, CH2CH2CH3, or CH(CH3)2. In any other embodiment of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R 1 is optionally substituted phenyl. In any other embodiment of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R 1 is an optionally substituted 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In any other embodiment of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R 1 is an optionally substituted 5-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In any other embodiment of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R 1 is an optionally substituted 6-membered heteroaryl ring having 1 to 2 nitrogen atoms, such as pyridyl or pyrimidinyl.

[0022] As generally defined above for formula (I), R 2 is selected from halogen, NO2, N(R)2, OR, N(R)C(O)R, CO2R, C(O)N(R)2, and optionally substituted C 1-6 selected from aliphatic. In some embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), at least one R 2 is halogen. In other embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), at least one R 2 is NO2. In other embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), at least one R 2 is OR, for example, OMe.

[0023] In other embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), at least one R 2 is N(R)2. In other embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), at least one R 2 is NHR, for example, NH2.

[0024] In another embodiment of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), at least one R 2 is N(R)C(O)R, for example, N(CH3)C(O)CH3. In another embodiment of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), at least one R 2 is NHC(O)R, for example, NHC(O)CH3.

[0025] In another embodiment of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), at least one R 2 is CO2R, for example, CO2H.

[0026] In another embodiment of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), at least one R 2 is C(O)N(R)2. In another embodiment of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), at least one R 2 is C(O)N(H)R, for example, C(O)NHCH3.

[0027] In any other embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), at least one R 2 is optionally substituted C 1-6 is aliphatic. In any other embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), at least one R 2 is optionally substituted C 1-3 is aliphatic.

[0028] As generally defined above for formula (I), each R x is independently selected from hydrogen, halogen, and optionally substituted C 1-6 aliphatic. In some embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R x is hydrogen. In any other embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R x is independently selected from halogen and optionally substituted C 1-6 aliphatic. In any other embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R xis a halogen, for example, fluoro or chloro.

[0029] In another embodiment of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R x is optionally substituted C 1-6 aliphatic. In another embodiment, R x is optionally substituted C 1-3 aliphatic, for example, CH3, CH2CH3, CH2CH2CH3, or CH(CH3)2.

[0030] As generally defined above for formula (I), each R is independently hydrogen, or C 1-6 aliphatic, a 3- to 7-membered monocyclic carbocycle, a 3- to 7-membered monocyclic heterocycle having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, phenyl, or a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, selected from optionally substituted groups. In some embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R is hydrogen. In some embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R is independently C 1-6A group which is optionally substituted and is selected from an aliphatic group, a 3- to 7-membered monocyclic carbocyclic ring, a 3- to 7-membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a phenyl group, and a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R is optionally substituted C 1-6 is aliphatic. In some embodiments of any of Formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), R is optionally substituted C 1-3 is aliphatic. In some such embodiments, R is CH3 or CH2CH3.

[0031] As generally defined above with respect to formula (I), n is from 0 to 3. In some embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), n is from 1 to 2. In other embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), n is 0. In other embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), n is 1. In other embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), n is 2. In other embodiments of any of formula (I), (I-a), (I-a-i), (I-a-ii), (I-a-iii), (I-a-iv), (I-a-v), (I-b), (I-b-i), (I-b-ii), (I-b-iii), (I-b-iv), and (I-b-v), n is 3.

[0032] In some embodiments of any of the disclosed compounds, R 1 is 1-6 aliphatic, for example, methyl or propyl. In other embodiments, R 1 is phenyl. In other embodiments, R 1 is a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In other embodiments, wherein R 1is a 5-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, in the formula, R 1 is a 6-membered heteroaryl ring having 1 to 3 nitrogen atoms. In other embodiments, R 1 is a 6-membered heteroaryl ring having 1 to 2 nitrogen atoms, for example,

Chemical formula

[0033] In some embodiments of any of the disclosed compounds, R x is hydrogen. In other embodiments, R x is halogen or optionally substituted C 1-6 aliphatic. In other embodiments, R x is optionally substituted C 1-6 aliphatic. In other embodiments, R x is unsubstituted C 1-6 aliphatic, for example, methyl.

[0034] In some embodiments of any of the disclosed compounds, R 2 is selected from halogen, NO2, N(R)2, OR, N(R)C(O)R, CO2R, C(O)N(R)2, and optionally substituted C 1-6 aliphatic. In other embodiments, R 2 is halogen, for example, fluoro. In other embodiments, R 2 is NO2. In other embodiments, R 2 is OR, for example, OCH3. In other embodiments, in the formula, R 2 is N(R)2, for example, NH2. In other embodiments, R 2 is N(R)C(O)R, for example, NHC(O)CH3 or N(CH3)C(O)CH3. In other embodiments, R 2 is CO2R, for example, CO2H. In other embodiments, R 2is C(O)N(R)2, for example, C(O)NHCH3. In other embodiments, R 2 is optionally substituted C 1-6 aliphatic, for example, CF3.

[0035] In some embodiments of any of the disclosed compounds, R is hydrogen. In other embodiments, R 1-6 is an optionally substituted group selected from C 1-6 aliphatic, a 3- to 7-membered monocyclic carbocyclic ring, a 3- to 7-membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, phenyl, and a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R 1-6 is optionally substituted C

[0036] In some embodiments of any of the disclosed compounds, R 3 is hydrogen. In other embodiments, R 3 is C 1-6 aliphatic, a 3- to 7-membered monocyclic carbocyclic ring, a 3- to 7-membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, phenyl, and a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R 3 is optionally substituted C 1-6 aliphatic. In other embodiments, R 3 is unsubstituted C 1-6 aliphatic, for example, methyl.

[0037] In some embodiments of any of the disclosed compounds, R 3 is hydrogen. In other embodiments, R 3 is C 1-6A group which is optionally substituted and is selected from an aliphatic group, a 3- to 7-membered monocyclic carbocyclic ring, a 3- to 7-membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a phenyl group, and a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R 3 is optionally substituted C 1-6 aliphatic. In some embodiments, R 3 is unsubstituted C 1-6 aliphatic, for example, methyl.

[0038] In some embodiments of any of the disclosed compounds, n is 0. In other embodiments, n is 1. In other embodiments, n is 2. In some embodiments, the present disclosure provides a compound selected from the following,

Chemical formula

[0039] In some embodiments, the present disclosure provides a compound selected from the following,

Chemical formula

Chemical formula

[0040] In some embodiments, the present disclosure provides a compound of formula (II),

Chemical formula

[0041] In some embodiments, at least one of X 1 、X 3 、and X 4 is N. In other embodiments, at least two of X 1 、X 3 、and X 4 are N. In other embodiments, each of X 1 、X 3 、and X 4 is N. In other embodiments, X 1 and X 3 are each N, and X 4 is CH.

[0042] In some embodiments, R 1 is unsubstituted C 1-6 alkyl, for example, methyl. In other embodiments, R 1 is methyl optionally substituted by halogen. In other embodiments, R 1 is C 2-6 alkyl or C 3-6 cycloalkyl.

[0043] In some embodiments, R 2 is H, amino, nitro, or -N(R 5 )C(O)R 6 , R 5 is H or C 1-5 alkyl, R 6 is C 1-6 alkyl. In other embodiments, R 2 is -N(R 5 )C(O)R 6 , R 5 is H, and R 6 is C 1-6 alkyl. In other embodiments, R 2 is -N(R 5)C(O)R 6 wherein R 5 is H, and R 6 is CH3. In other embodiments, R 2 is H, amino, or nitro. In other embodiments, R 2 is NO2 or -N(R 5 )C(O)R 6 .

[0044] In some embodiments, the compound is

Chem.

[0045] In some embodiments, the compound is JGJ002, JGJ003, JGJ004, JGJ005, JGJ007, or JGJ008, or a pharmaceutically acceptable salt thereof.

[0046] In some embodiments, the compound is JGJ007 or JGJ088, or a pharmaceutically acceptable salt thereof.

[0047] In some embodiments of formula (II), X 1 is N. Thus, in some embodiments, the present disclosure provides a compound of formula (II-a),

Chem.

[0048] In some embodiments of formula (II), X 3 is N. Thus, in some embodiments, the present disclosure provides a compound of formula (I-b),

Chem.

[0049] In some embodiments of formula (II-a), X 3 is N. Thus, in some embodiments, the disclosure provides a compound of formula (I-a-i),

Chemical formula

[0050] As generally defined above with respect to formula (II), R 1 is C 1-6 alkyl or C 3-6 cycloalkyl. In other embodiments of any of formulae (II), (II-a), (II-b), and (II-a-i), R 1 is C 1-6 alkyl. In other embodiments of any of formulae (II), (II-a), (II-b), and (II-a-i), R 1 is C 1-3 alkyl, for example, R 1 is CH3, CH2CH3, CH2CH2CH3, or CH(CH3)2.

[0051] In other embodiments of any of formulae (II), (II-a), (II-b), and (II-a-i), R 1 is C 3-6It is a cycloalkyl, for example, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In another embodiment of any of Formulas (II), (II-a), (II-b), and (II-a-i), R 1 is cyclopropyl or cyclobutyl. In another embodiment of any of Formulas (II), (II-a), (II-b), and (II-a-i), R 1 is cyclopentyl or cyclohexyl.

[0052] As generally defined above with respect to Formula (II), R 2 is H, amino, nitro, or acylamino. In some embodiments of any of Formulas (II), (II-a), (II-b), and (II-a-i), R 2 is H. In another embodiment of any of Formulas (II), (II-a), (II-b), and (II-a-i), R 2 is amino, nitro, or acylamino. In another embodiment of any of Formulas (II), (II-a), (II-b), and (II-a-i), R 2 is amino. In another embodiment of any of Formulas (II), (II-a), (II-b), and (II-a-i), R 2 is nitro. In another embodiment of any of Formulas (II), (II-a), (II-b), and (II-a-i), R 2 is acylamino

Chemical formula

[0053] In some embodiments, the amino is N(R)2.

[0054] In some embodiments, the acylamino is N(R)C(O)R.

[0055] In some embodiments of any of the disclosed compounds, the compound is [Chem.] is not.

[0056] Pharmaceutical Compositions and Their Use In some embodiments, the present disclosure provides the recognition that methods targeting ribonucleic acid (RNA)-RNA binding protein (RBP) interactions constitute a new alternative approach for significantly expanding the druggable proteome and genome and overcoming intrinsic and acquired resistance.

[0057] In certain aspects, the present disclosure further provides the recognition that RBPs play an essential role in cell physiology by controlling RNA processing, translation, and turnover rates. In neoplasms, dysregulated expression of RBPs supports the expression of alternatively spliced, modified, and stabilized RNA transcripts that are relevant to cancer self-renewal, proliferation, and adaptation to stress. In some embodiments, the present disclosure provides compounds that represent a novel therapeutic approach for modulating specific RBP-protein interactions and thus treating cancer and other diseases associated with dysfunctional RNA control.

[0058] MicroRNAs (miRNAs) are short non-coding RNAs of 19–22 nucleotides (nt) that hybridize to complementary mRNA targets, resulting in either their degradation, cleavage, or transcriptional inhibition (5–7). Aberrant miRNA expression has been shown to play an active role in malignant transformation, including leukemia (8–10). Specifically, in acute myeloid leukemia (AML), let-7b and let-7c miRNAs have been found to be significantly downregulated in core-binding factor (CBF) leukemias associated with inv(16), t(8;21), and MLL / t(11q23) (11)(12). In a systematic evaluation of the prognostic value of miRNA expression levels in many human cancers, including several AML subtypes, a decrease in let-7 miRNA expression has often been found to be associated with poor prognosis (10, 13, 14). The let-7 tumor suppressor miRNA family is differentially transcribed from eight chromosomal loci and contains 12 members that suppress several cancer stem cell oncogenes, including KRAS, MYC, IL6, and HMGA1 / 2, as well as cell cycle regulators such as CCND1 / 2 and E2F (Figure 1) (15, 16). In 2008, papers describing LIN28A and its homolog LIN28B (hereinafter referred to as LIN28) as major regulators of let-7 biogenesis by directly binding to either pre-let-7 and / or pri-let-7, thereby impairing their processing into functional mature miRNAs, began to appear (17–21). Indeed, LIN28 is upregulated in more than 15% of human cancers (22) and cancer stem cells (CSCs) (23–27).

[0059] Structural studies have revealed that the C-terminal zinc-knuckle domain (ZKD) of Lin28 binds to the highly conserved GGAG motif within the 3′-terminal loop of pri- / pre-let-7 (28-30). This binding recruits TUTases to polyuridylate pre / pri-let-7, thereby interfering with the maturation of let-7 miRNA (19, 31). Thus, reduced let-7 miRNA leads to overexpression of oncogenic target genes that are directly regulated by them.

[0060] RNA-binding proteins LIN28A and LIN28B are overexpressed in many cancers, and high LIN28 protein correlates with reduced patient survival (54). LIN28A / B (hereinafter referred to as Lin28) impairs the processing of functional mature let-7 microRNA (miRNA) by binding to the highly conserved GGAG motif within the 3′-terminal loop of pri- / pre-let-7 via its C-terminal zinc-knuckle domain (ZKD) (17-21, 28-30). As a result, in some embodiments, reduced let-7 miRNA leads to overexpression of their direct oncogenic target genes such as MYC, KRAS, and CCND1. In addition to its ability to suppress let-7 miRNA biogenesis, Lin28 has been shown to bind to the mRNA transcripts of insulin-like growth factor 2 protein (Igf2), thereby affecting their abundance and / or translation (69, 70).

[0061] In a variety of cancers, there is increasing evidence that LIN28 overexpression (32 - 34) and let-7 loss (35 - 37) are associated with radiation- and chemotherapy-resistant CSCs, ultimately leading to reduced overall survival. Specifically, in AML, deregulated LIN28 / let-7 has been shown to promote leukemogenesis via an LSC-like transcriptional program and is associated with poor clinical outcomes (38). In bone marrow aspirates from patients with refractory AML, let-7a has been found to confer Ara-C chemotherapy resistance via BCL-XL, a member of the BCL-2 family (39). Importantly, several studies have highlighted that overexpression of BCL-2 and BCL-XL, particularly in AML and LSCs, is associated with chemotherapy resistance and poor overall / disease-free survival (40 - 43). Additionally, the let-7 miRNA targets IL6 and RAS, two well-known genetic drivers of the NF-κB pathway, another important regulator of LSC homeostasis (44) (Figure 1).

[0062] NF-κB and BCL-2 have emerged as evidence indicating that they are activated in LSCs but not in hematopoietic stem cells (HSCs) as central components of the inflammation-promoting cellular stress response (45, 46). Thus, therapeutic inhibition of LIN28 and the resultant upregulation of let-7 may selectively kill LSCs. Considering the fundamental role of Lin28 / let-7 in leukemia and other CSCs and its relevance to treatment resistance, targeted inhibition of LIN28 could be a novel approach towards precision AML therapy. Notably, studies in conditional Lin28a and Lin28b knockout mice have revealed that Lin28 deficiency in the fetus (but not neonates or adults) results in growth defects (47), suggesting that Lin28 has a heterochronic effect. Moreover, in mice, the expression of Lin28b was found to decrease in hematopoietic stem cells (48, 49), consistent with the accumulation of mature let-7 in myeloid common progenitor cells during hematopoietic cell maturation (50). Therefore, therapeutic inhibition of LIN28 and the resultant upregulation of the let-7 miRNA may selectively kill LSCs but are likely to have high tolerance for healthy tissues.

[0063] To date, five high-throughput screens (HTS) have been reported with the aim of identifying pharmacologically active compounds that interfere with the binding of LIN28 to pre-let-7 miRNA. The inventors screened 16,000 drug-like organic compounds using FRET-HTS and identified the first hit compound 501632 (hereinafter referred to as LN1632) that binds to LIN28B, selectively upregulates let-7 miRNA levels, and induces differentiation in mouse embryonic stem cells (51). Lim et al. (52) screened an in-house library and found benzopyranyl pyrazole-based compounds as primary hit molecules, while Lightfoot et al. used a biophysical assay to identify 6-hydroxy-DL-DOPA and benzo[a]phenoxazine that inhibit the Lin28 / let-7 interaction in vitro. The Sliz group developed a fluorescence polarization HTS to identify LI71 and TPEN, the latter of which is a potent ZKD domain inhibitor (53). Despite the increasing reports on small molecule inhibitors of the Lin28 / let-7 interaction, the pharmacological inhibition of LIN28 in vivo for targeted AML and LSC therapies is not established. In addition, small molecule inhibitors with high specificity for LIN28 and that inhibit its activity remain unexplored.

[0064] This disclosure reports on the in vitro and in vivo inhibition of Lin28 and Lin28 / let-7 by compounds of formula (I) or (II).

[0065] As described herein, the compounds of formula (I) and (II) exhibit Lin28 / let-7 inhibitory activity in an in vitro FRET assay and in LSC and LSC-like Kasumi-1 cells. The FRET assay was performed as previously described (51).

[0066] Similarly, the compounds of formula (I) and (II) demonstrate in vitro and in vivo inhibition of protein-RNA interactions, particularly in Lin28 / let-7 and PRPF31 / U4.

[0067] The present disclosure provides a method for treating cancer, the method comprising administering to a subject having cancer or presenting symptoms of cancer a compound or composition described herein. In some embodiments, the method comprises treating or ameliorating one or more symptoms of cancer. In some embodiments, the cancer is a blood cancer, such as acute myeloid leukemia. In some embodiments, the method comprises administering the compound or composition in an amount determined to achieve inhibition of cancer cells and / or reduction of their proliferation or according to such a dosing regimen. In some embodiments, the cancer cells include cancer stem cells. In some embodiments, the cancer stem cells include leukemia stem cells (LSCs). In some embodiments, the method comprises administering the compound or composition in an amount determined to achieve inhibition of cancer cells and / or reduction of their proliferation or according to such a dosing regimen, wherein the inhibition of cancer cells and / or reduction of their proliferation is evaluated using the assays shown in Example 3 or 5 or similar assays.

[0068] In some embodiments, the present disclosure provides a method for modulating splicing, the method comprising contacting a splicing-competent system with a compound as described herein.

[0069] In some embodiments, the present disclosure comprises contacting a splicing-competent system with a compound as described herein and, in the system, (i) the presence or level of splicing products (e.g., transcripts that have undergone splicing), (ii) the expression or localization of RNA, and / or (iii) the expression or folding of polypeptides, and evaluating, and provides a method comprising.

[0070] In some embodiments, the present disclosure provides a method of modulating splicing in a system having splicing ability by contacting the system with a compound as described herein, whereby, hereinafter, (i) reduced splicing of RNA, (ii) altered expression or localization of RNA, and / or (iii) altered expression or folding of a polypeptide, is observed for one or more of.

[0071] In some embodiments, the present disclosure provides a method comprising contacting a splicing-capable system with a compound as described herein, the compound being characterized by reducing the proliferation of cancer cells when contacted therewith as compared to that observed in its absence. In some embodiments, splicing is reduced when the compound is present as compared to when it is absent. In some embodiments, the method further comprises evaluating splicing in the system as compared to a reference condition. In some embodiments, the reference condition is the absence of the compound. In some embodiments, the reference condition is the presence of a control compound. In some embodiments, the reference condition is a historical condition. In some embodiments, the compound inhibits one or more attributes of a component of the splicing mechanism and / or the compound inhibits an interaction between or within components of the splicing mechanism. In some embodiments, the compound binds directly to one or more components of the splicing mechanism, or a complex thereof. In some embodiments, the components of the splicing mechanism are RNA components. In some embodiments, the components of the splicing mechanism are polypeptide components. In some embodiments, the components of the splicing mechanism are selected from RNA components, polypeptide components, and complexes thereof or complexes between them. In some embodiments, the RNA component is or includes small nuclear RNA (snRNA). In some embodiments, the snRNA is selected from U1, U2, U4, U5, and U6. In some embodiments, the polypeptide component is or includes an Sm polypeptide or an Lsm polypeptide. In some embodiments, the polypeptide component is selected from Prp3, Prp31, Prp4, CypH, 15.5K, Prp8, Brr2, Snu114, Prp6, Prp28, 40K, Dib1, Snu66, Sad1, and 27K. In some embodiments, the components of the splicing mechanism include the Prp31 polypeptide. In some embodiments, the components of the splicing mechanism include U4 snRNA, U6 snRNA, and the Prp31 polypeptide.In some embodiments, the compound inhibits the interaction between U6 snRNA and Prp31 polypeptide, or between U4 snRNA and Prp31 polypeptide. In some embodiments, the compound inhibits the activity of Prp31 polypeptide.

[0072] In some embodiments, the contact occurs in vitro, ex vivo, or in vivo. In some embodiments, the splicing-capable system is a cancer cell. In some embodiments, the cancer cells with splicing ability include cancer stem cells. In some embodiments, the cancer stem cells with splicing ability include leukemia stem cells (LSCs).

[0073] The compositions and methods of the present invention can be used to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human or a non-human mammal. When administered to an animal such as a human, the composition or compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiological buffered saline, or oils such as glycols, glycerol, olive oil, or other solvents or vehicles such as injectable organic esters. In a preferred embodiment, when such a pharmaceutical composition is for administration to humans, particularly for invasive routes of administration (i.e., routes such as injection or implantation that bypass transport or diffusion through the epithelial barrier), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to provide for delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition can be in unit dosage forms such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophiles for reconstitution, powders, solutions, syrups, suppositories, injections, etc. The composition can also be present in a transdermal delivery system, for example, in a skin patch. The composition can also be present in a solution suitable for topical administration such as a lotion, cream, or ointment.

[0074] A pharmaceutically acceptable carrier may contain a physiologically acceptable agent that acts to stabilize a compound such as a compound of the present invention, increase its solubility, or increase its absorption. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or excipients. The selection of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, is influenced, for example, by the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) can also be, for example, a liposome or other polymeric matrix that can incorporate the compound of the present invention therein. For example, liposomes containing phospholipids or other lipids are non-toxic and physiologically acceptable biodegradable carriers that are relatively simple to prepare and administer.

[0075] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0076] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0077] The pharmaceutical composition (preparation) can be administered to a subject by any of several routes of administration, including, for example, orally (such as in a drinkable medicine, tablet, capsule (including sprinkle capsules and gelatin capsules), bolus, powder, granule, paste for application to the tongue); absorption through the oral mucosa (such as sublingually); subcutaneously; transdermally (such as as a patch applied to the skin); and topically (such as as a cream, ointment, or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, the compound may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions that are suitable for such are found, for example, in U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and the patents cited therein.

[0078] The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound capable of producing a therapeutic effect. Generally, out of 100 percent, this amount will range from about 1 percent to about 99 percent, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent of the active ingredient.

[0079] The methods of preparing these formulations or compositions include the step of associating an active compound, such as a compound of the invention, with a carrier and optionally one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately associating the compound of the invention with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0080] The formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (flavored bases, usually using sucrose and acacia or tragacanth), lyophilized agents, powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, or as water-in-oil or oil-in-water liquid emulsions, or as elixirs or syrups, or as troches (using inert bases such as gelatin and glycerin, or sucrose and acacia), and / or as mouthwashes, etc., each containing a predetermined amount of the compound of the present invention as an active ingredient. The composition or compound may also be administered as a bolus, a pastille, or a paste.

[0081] To prepare solid dosage forms for oral administration (including capsule forms (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid, (2) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia, (3) humectants such as glycerol, (4) disintegrants such as agar - agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (5) solution retarding agents such as paraffin, (6) absorption promoters such as quaternary ammonium compounds, (7) wetting agents such as cetyl alcohol and glycerol monostearate, (8) absorbents such as kaolin and bentonite clay, (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof, (10) complexing agents such as modified and unmodified cyclodextrins, and (11) coloring agents. In the case of capsule forms (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also contain a buffering agent. Similar types of solid compositions may also be used as excipients such as lactose or milk sugar, and as fillers in soft - filled and hard - filled gelatin capsules using high - molecular - weight polyethylene glycol, etc.

[0082] Tablets can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross - linked sodium carboxymethylcellulose), a surfactant, or a dispersing agent. Molded tablets can be prepared by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.

[0083] Tablets of the pharmaceutical composition, as well as other solid dosage forms such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, may optionally be scored or may be prepared with coatings and shells such as enteric coatings and other coatings well - known in the pharmaceutical formulation art. They may also be formulated to provide for slow or controlled release of the active ingredient therein, using, for example, various ratios of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release profile. They can be sterilized, for example, by filtration through a bacteria - retaining filter or by incorporating a sterilizing agent in the form of a sterilized solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain an opacifying agent and may belong to compositions that release the active ingredient(s) only, or preferentially, in a particular part of the digestive tract, optionally in a delayed manner. Examples of implantable compositions that can be used include polymeric substances and waxes. The active ingredient can also, if appropriate, be in microencapsulated form together with one or more of the excipients described above.

[0084] Useful liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, lyophilized agents for reconstitution, microparticle emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain solubilizing and emulsifying agents such as, for example, inert diluents commonly used in the art such as water or other solvents, cyclodextrins and their derivatives, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0085] In addition to the inert diluent, oral compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents, and preservatives.

[0086] Suspending agents may contain, in addition to the active compound, suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, agar-agar and tragacanth, and mixtures thereof.

[0087] Dosage forms for topical or transdermal administration include powders, aerosols, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed, under sterile conditions, with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0088] Ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0089] In addition to the active compound, powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0090] Transdermal patches have the additional advantage of providing controlled delivery of the compounds of the invention to the body. Such dosage forms can be prepared by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the influx of the compound through the skin. The rate of such influx can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymeric matrix or gel.

[0091] As used herein, the terms "parenteral administration" and "administered parenterally" mean a mode of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, and intrasternal injections and infusions. Pharmaceutical compositions suitable for parenteral administration may contain one or more pharmaceutically acceptable, isotonic aqueous or non-aqueous sterile solutions, dispersions, suspensions, or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions immediately prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0092] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersants, and by the use of surfactants.

[0093] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be accomplished by the incorporation of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include in the composition isotonic agents such as sugars, sodium chloride, etc. In addition, long-term absorption of injectable dosage forms can be achieved by the incorporation of agents that delay absorption such as aluminum monostearate and gelatin.

[0094] In some cases, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection in order to extend the effect of the drug. This can be accomplished by the use of a liquid suspension of a poorly water-soluble crystalline or amorphous material. Then, the absorption rate of the drug is dependent on its dissolution rate, which in turn can be dependent on the crystal size and crystal form. Alternatively, delayed absorption of parenterally administered drugs is accomplished by dissolving or suspending the drug in an oily vehicle.

[0095] Injectable depot forms are prepared by forming a microencapsulation matrix of the subject compound in a biodegradable polymer such as polylactide-polyglycolide. The drug release rate can be controlled depending on the drug-to-polymer ratio and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues.

[0096] When used in the method of the present invention, the active compound can be administered, by itself or in combination with a pharmaceutically acceptable carrier, as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably about 0.5 to about 90%) of the active ingredient.

[0097] The method of introducing the compounds of the present invention can also be enabled by a rechargeable device or a biodegradable device. In order to obtain controlled delivery of drugs, including proteinaceous biological agents, various sustained release polymeric devices have been developed and tested in vivo in recent years. Using various biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, an implant can be formed to achieve sustained release of the compound at a specific target site.

[0098] The actual dosage level of the active ingredient in the pharmaceutical composition may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0099] The selected dosage level will be influenced by a variety of factors including the activity of the specific compound or combination of compounds used, or its ester, salt, or amide, the route of administration, the time of administration, the excretion rate of the specific compound(s) used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific compound(s) used, the age, sex, weight, medical condition, general health, and prior medical history of the patient being treated, as well as similar factors well known in the medical arts.

[0100] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe a therapeutically effective amount of the required pharmaceutical composition. For example, a physician or veterinarian can initiate dosing of the pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. "Therapeutically effective amount" means a concentration of the compound sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of a compound will vary depending on the weight, sex, age, and medical history of the subject. Other factors that can affect the effective amount can include, but are not limited to, the severity of the patient's medical condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered in combination with the compounds of the present invention. Higher total doses can be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those of ordinary skill in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0101] In general, a suitable daily dosage of the active compounds used in the compositions and methods of the present invention will be that amount of the compound that is the lowest dosage effective to produce a therapeutic effect. Such effective dosages will generally be determined by the factors described above.

[0102] If desired, the effective daily dosage of the active compound can be administered as one, two, three, four, five, six, or more sub-doses, optionally in unit dosage forms, at appropriate intervals throughout the day. In certain embodiments of the invention, the active compound can be administered two or three times a day. In a preferred embodiment, the active compound will be administered once a day.

[0103] The patient being treated is any animal in need thereof, including primates, particularly humans, as well as other mammals such as horses, cows, pigs, sheep, cats, and dogs, poultry, and pets generally.

[0104] In certain embodiments, the compounds of the invention may be used alone or co-administered with another type of therapeutic agent.

[0105] The present disclosure includes the use of pharmaceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. In certain embodiments, the salts contemplated by the present invention include, but are not limited to, alkyl salts, dialkyl salts, trialkyl salts, or tetra-alkylammonium salts. In certain embodiments, the salts contemplated by the present invention include, but are not limited to, L-arginine salts, benenthamine salts, benzathine salts, betaine salts, calcium hydroxide salts, choline salts, deanol salts, diethanolamine salts, diethylamine salts, 2-(diethylamino)ethanol salts, ethanolamine salts, ethylenediamine salts, N-methylglucamine salts, hydrabamine salts, 1H-imidazole salts, lithium salts, L-lysine salts, magnesium salts, 4-(2-hydroxyethyl)morpholine salts, piperazine salts, potassium salts, 1-(2-hydroxyethyl)pyrrolidine salts, sodium salts, triethanolamine salts, tromethamine salts, and zinc salts. In certain embodiments, the salts contemplated by the present invention include, but are not limited to, Na salts, Ca salts, K salts, Mg salts, Zn salts, or other metal salts.In certain embodiments, the contemplated salts of the invention include, but are not limited to, the acid salts of 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0106] Pharmaceutically acceptable acid addition salts may also exist as various solvates with, for example, water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be inherent in the preparation or crystallization solvent or may be derived from an adventitious crystallization solvent in such solvents.

[0107] Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition.

[0108] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium dithionite, and sodium sulfite; (2) fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0109] Definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those skilled in the art. In general, the nomenclature and techniques described herein in connection with chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry are well known and commonly used in the art.

[0110] The methods and techniques of the present disclosure are generally carried out in accordance with conventional methods well known in the art, unless otherwise indicated, and as described in various general and more specific references cited and discussed throughout this specification. See, for example, “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000), Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995), Lodish et al., “Molecular Cell Biology, 4th ed.”, W.H. Freeman & Co., New York (2000), Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W.H. Freeman & Co., N.Y. (1999), and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0111] Chemical terms used herein are used according to customary usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985), unless otherwise defined herein.

[0112] All of the above and any other publications, patents, and published patent applications referred to in this application are hereby expressly incorporated by reference into this specification. In case of conflict, this specification, including its specific definitions, will control.

[0113] As used herein, the term "agent" refers to a chemical compound (organic or inorganic compound, mixture of chemical compounds, etc.), a biopolymer (nucleic acid, antibody (including portions thereof, as well as humanized, chimeric, and human antibodies, and monoclonal antibodies), protein or a portion thereof, such as a peptide, lipid, carbohydrate, etc.), or an extract made from a biological material such as bacteria, plant, fungal, or animal (especially mammalian) cells or tissues. Agents include, for example, agents with known structures and agents with unknown structures.

[0114] The terms "patient", "subject", or "individual" are used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, domestic animals (including cows, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0115] "Treating" a condition or patient refers to taking steps to obtain a beneficial or desired result, including a clinical result. As used herein and as commonly understood in the art, "treatment" is an approach to obtain a beneficial or desired result, including a clinical result. Beneficial or desired clinical results can include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, whether detectable or undetectable, attenuation of the degree of a disease, a stabilized (i.e., not worsening) disease state, prevention of the spread of a disease, delay or deceleration of disease progression, improvement or temporary alleviation of a disease state, and remission (whether partial or complete). "Treatment" can also mean extending the survival period as compared to the expected survival period if the treatment had not been received.

[0116] The term "preventing" is recognized in the art and, when used in connection with a condition such as local recurrence (e.g., pain), a disease such as cancer, a complex syndrome such as heart failure, or any other medical condition, is well understood in the art to include administration of a composition that reduces the frequency of symptoms of the medical condition, or delays its onset, in a subject as compared to a subject not receiving the composition. Thus, prevention of cancer, for example, includes reducing the number of detectable cancer tumors, statistically and / or clinically significantly, e.g., in a population of patients receiving a prophylactic treatment as compared to an untreated control population, and / or delaying the appearance of detectable cancer tumors in a treated population as compared to an untreated control population.

[0117] "Administering" or "the administration" of a substance, compound, or agent to a subject can be effected using one of a variety of methods known to those of skill in the art. For example, a compound or agent can be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ophthalmically, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracranially, and transdermally (e.g., by absorption through a skin patch). A compound or agent can also be appropriately introduced by a refillable or biodegradable polymeric device or other device, such as a patch and a pump, or a formulation, that provides for sustained release, slow release, or controlled release of the compound or agent. Administration can also be effected, for example, once, a plurality of times, and / or over one or more extended periods of time.

[0118] The appropriate method of administering a substance, compound, or agent to a subject will also depend, for example, on the age and / or physical condition of the subject, as well as the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, a compound or agent is administered to a subject orally, e.g., by ingestion. In some embodiments, an orally administered compound or agent is in a sustained release or slow release formulation or is administered using a device for such slow or sustained release.

[0119] As used herein, the phrase "co - administration" refers to the administration of two or more different therapeutic agents in any form such that a second agent is administered while a previously administered therapeutic agent is still effective in the body (e.g., such that the two agents are simultaneously effective in a patient, which may include a synergistic effect of the two agents). For example, different therapeutic compounds may be administered in the same formulation, in separate formulations, simultaneously or sequentially. Thus, an individual undergoing such treatment can benefit from the combined effects of the different therapeutic agents.

[0120] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is an amount of the drug or agent that will have the intended therapeutic effect when administered to a subject. A complete therapeutic effect is not necessarily produced by the administration of a single dose and may occur only after a series of doses. Thus, a therapeutically effective amount may be administered in one or multiple administrations. The exact effective amount required for a subject will depend, for example, on the size, health, and age of the subject, as well as the nature and extent of the condition being treated, such as cancer or MDS. One of ordinary skill in the art can readily determine the effective amount for a given situation by routine experimentation.

[0121] Related: As used herein, two events or entities are "related" to each other if the presence, level, degree, type, and / or form of one is correlated with those of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered related to a particular disease, disorder, or pathological condition if its presence, level, and / or form is correlated (e.g., across a relevant population) with the incidence and / or susceptibility of that disease, disorder, or pathological condition. In some embodiments, two or more entities are physically "related" to each other if they interact directly or indirectly such that they are physically proximate to each other and / or remain physically proximate. In some embodiments, two or more entities that are physically related to each other are covalently linked to each other, and in some embodiments, two or more entities that are physically related to each other are not covalently linked to each other but are non-covalently related using, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetic interactions, and combinations thereof. Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, states, sets of conditions, etc., which may not be identical to each other, but are similar enough to allow for comparison such that a person of ordinary skill in the art would be able to reasonably draw conclusions based on the observed differences or similarities. In some embodiments, a set of equivalent conditions, states, individuals, or populations is characterized by a plurality of substantially identical features and one or a few varying features. A person of ordinary skill in the art will understand, in context, what degree of identity is required for two or more such agents, entities, situations, sets of conditions, etc. to be considered equivalent in any given situation. For example, a person of ordinary skill in the art will understand that sets of situations, individuals, or populations are equivalent to each other if, under different sets of circumstances, or differences in results or observed phenomena obtained by different sets of circumstances, individuals, or populations, are characterized by a sufficient number and variety of substantially identical features to warrant the reasonable conclusion that the variation is caused by, or indicative of, variation in the varying features.

[0122] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence is accompanied by one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation), (3) translation of the RNA into a polypeptide or protein, and / or (4) post-translational modification of the polypeptide or protein.

[0123] Inhibitor: As used herein, the term "inhibitor" (or "inhibitory agent") refers to an entity, condition, or event whose presence, level, or degree correlates with a decrease in the level or activity of a target. In some embodiments, the inhibitor may act directly (in which case it exerts its effect directly on the target, for example, by binding to the target), and in some embodiments, the inhibitor may act indirectly (in which case it exerts its effect by interacting with a regulator of the target and / or altering it in other ways such that the level and / or activity of the target is reduced). In some embodiments, the inhibitor is one whose presence or level correlates with a target level or activity that is reduced compared to a particular reference level or activity (e.g., that observed under appropriate reference conditions such as the presence of a known inhibitor or the absence of the inhibitor).

[0124] Reference: As used herein, describes a standard or control against which a comparison is made. For example, in some embodiments, a drug, animal, individual, population, sample, sequence, or value of interest is compared to a drug, animal, individual, population, sample, sequence, or value that serves as a reference or control. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control optionally embodied in a tangible medium. Typically, as will be understood by one of ordinary skill in the art, the reference or control is determined or characterized under conditions or circumstances equivalent to those being evaluated. One of ordinary skill in the art will understand when there is sufficient similarity to demonstrate the reliability and / or validity of the comparison to a particular possible reference or control.

[0125] Small molecule: As used herein, the term "small molecule" means a low molecular weight organic and / or inorganic compound. Generally, a "small molecule" is a molecule with a size of less than about 5 kilodaltons (kD). In some embodiments, the small molecule is less than about 4 kD, 3 kD, about 2 kD, or less than about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), less than about 600 D, less than about 500 D, less than about 400 D, less than about 300 D, less than about 200 D, or less than about 100 D. In some embodiments, the small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, the small molecule is not a polymer. In some embodiments, the small molecule does not contain a polymer moiety. In some embodiments, the small molecule is not and / or does not contain a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, the small molecule is not and / or does not contain a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, the small molecule is not and / or does not contain a polysaccharide, e.g., in some embodiments, the small molecule is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, the small molecule is not a lipid. In some embodiments, the small molecule is a modulator (e.g., an inhibiting / inhibitory agent or activator). In some embodiments, the small molecule is biologically active. In some embodiments, the small molecule is detectable (e.g., contains at least one detectable moiety). In some embodiments, the small molecule is a therapeutic agent. One of ordinary skill in the art, upon reading the present disclosure, will understand that a particular small molecule compound described herein may be provided and / or utilized in any of a variety of forms, such as crystalline form, salt form, protected form, prodrug form, ester form, isomeric form (e.g., optical isomer and / or structural isomer), isotopic form, etc. One of ordinary skill in the art will understand that a particular small molecule compound may have a structure that can exist in one or more stereoisomeric forms.In some embodiments, such small molecules may be utilized in the form of individual enantiomers, diastereomers, or geometric isomers, or in the form of a mixture of stereoisomers, according to the present disclosure. In some embodiments, such small molecules may be utilized in the form of a racemic mixture, according to the present disclosure. One of ordinary skill in the art will understand that certain small molecule compounds may have structures that can exist in one or more tautomeric forms. In some embodiments, such small molecules may be utilized in the form of individual tautomers or in a form that interconverts between tautomeric forms, according to the present disclosure. One of ordinary skill in the art will understand that certain small molecule compounds may be isotopically substituted (e.g., instead of H, 2 H or 3 H, instead of 12C 11 C, 13 C, or 14 C, instead of 14N 13 N or 15 N, instead of 16O 17 O or 18 O, instead of XXC 36 Cl, instead of XXF 18Understand that it has a structure that allows for 131I (such as instead of F, XXXI). In some embodiments, such small molecules may be utilized in one or more isotopically modified forms, or mixtures thereof, in accordance with the present disclosure. In some embodiments, reference to a particular small molecule compound may relate to a specific form of that compound. In some embodiments, a particular small molecule compound may be provided and / or utilized in salt form (e.g., in acid addition or base addition salt form depending on the compound), and in some such embodiments, the salt form may be a pharmaceutically acceptable salt form. In some embodiments, if a small molecule compound is one that exists in nature or is found in nature, the compound may be provided and / or utilized in a form different from the form in which it exists in nature or is found in nature. One of ordinary skill in the art will understand that in some embodiments, a preparation of a particular small molecule compound containing an absolute or relative (e.g., relative to another component of the preparation, including a different form of the compound) amount of the compound or form thereof that is different from the absolute or relative amount of the compound or form thereof present in a target reference preparation (e.g., in a primary sample from a target source such as a biological or environmental source) is clearly different from the compound as it exists in the reference preparation or source. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered a different form of the compound from a racemic mixture of the compound, a particular salt of a small molecule compound may be considered a different form from another salt form of the compound, a preparation containing only a form of the compound containing one conformational isomer ((Z) or (E)) of a double bond may be considered a different form of the compound from one containing the other conformational isomer ((E) or (Z)) of the double bond, a preparation in which one or more atoms are isotopes different from those present in the reference preparation may be considered a different form, and so on.

[0126] Splicing components: Those skilled in the art will understand, upon reading the present disclosure, that a "splicing component" is an agent or entity involved in the splicing reaction. In some embodiments, the splicing component is or includes a component of the spliceosome. In some embodiments, the splicing component is or includes a splicing regulator. In some embodiments, the splicing component is or includes an RNA, a polypeptide, and / or a complex thereof or a complex between them. In some embodiments, one or more of U1 snRNA, U2 snRNA, U4 snRNA, U5 snRNA, U6 snRNA, Sm polypeptide, Lsm polypeptide, Prp3 polypeptide, Prp31 polypeptide, Prp4 polypeptide, CypH polypeptide, 15.5K polypeptide, Prp8 polypeptide, Brr2 polypeptide, Snu114 polypeptide, Prp6 polypeptide, Prp28 polypeptide, 40K polypeptide, Dib1 polypeptide, Snu66 polypeptide, Sad1 polypeptide, or 27K polypeptide can be or be part of a splicing component.

[0127] Splicing-capable system: Those skilled in the art will understand, upon reading the present disclosure, that a "splicing-capable system" is a system that includes all the components necessary to perform one or more splicing events (e.g., of one or more specific RNAs). In some embodiments, the splicing-capable system can be an in vitro system or an ex vivo system. In some embodiments, the splicing-capable system can be or include one or more cells (e.g., in a culture, in a tissue, or in an organism).

[0128] The term "acyl" is recognized in the art and refers to a group represented by the general formula hydrocarbyl C(O)-, preferably alkyl C(O)-.

[0129] The term "acylamino" is recognized in the art and refers to an amino group substituted with an acyl group, which can be represented, for example, by the formula hydrocarbyl C(O)NH-.

[0130] The term "acyloxy" is recognized in the art and refers to a group represented by the general formula hydrocarbyl C(O)O-, preferably alkyl C(O)O-.

[0131] The term "alkoxy" refers to an alkyl group bonded to oxygen. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0132] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and can be represented by the general formula alkyl-O-alkyl.

[0133] The term "alkyl" refers to a saturated aliphatic group including a straight-chain alkyl group, a branched-chain alkyl group, a cycloalkyl (alicyclic) group, an alkyl-substituted cycloalkyl group, and a cycloalkyl-substituted alkyl group. In a preferred embodiment, the straight-chain or branched-chain alkyl has 30 or fewer carbon atoms (e.g., C in the case of a straight chain 1-30 , C in the case of a branched chain 3-30 ), more preferably 20 or fewer carbon atoms.

[0134] Furthermore, the term "alkyl" as used throughout this specification, the examples, and the claims is intended to include both unsubstituted alkyl groups and substituted alkyl groups, where the latter refers to an alkyl moiety in which a substituent has replaced one or more hydrogens on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0135] As used herein with respect to the compounds of formula (I), the term "aliphatic" refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or contains one or more unsaturated units, or a monocyclic or bicyclic hydrocarbon that is completely saturated or contains one or more unsaturated units, but is not aromatic (also referred to herein as a "carbocyclic ring" or "cycloaliphatic compound").

[0136] Unless otherwise specified, aliphatic groups contain from 1 to 6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain from 1 to 5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain from 1 to 4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain from 1 to 3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain from 1 to 2 aliphatic carbon atoms. In some embodiments, a "cycloaliphatic compound" (or "carbocyclic ring") is a monocyclic C3-C8 hydrocarbon or a bicyclic C7-C hydrocarbon that is completely saturated or contains one or more unsaturated units, but is not aromatic. 10 Refers to a hydrocarbon that is not aromatic. Suitable aliphatic groups include, but are not limited to, straight-chain or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene groups, and hybrids thereof.

[0137] As described herein, the compounds of formula (I) may contain a "optionally substituted" moiety. In general, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens of the designated moiety are replaced by a suitable substituent. "Substituted" applies to one or more hydrogens that are either explicit or implicit in the structure (e.g.,

Chemical Structure

Chemical Structure

Chem.

Chem.

[0138] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are, independently, halogen; -(CH2) 0-4 R°; -(CH2) 0-4 OR°; -O(CH2) 0-4 R°, -O-(CH2) 0-4 C(O)OR°; -(CH2) 0-4 CH(OR°)2; -(CH2) 0-4 SR°; -(CH2) 0-4 Ph (which may be substituted with R°); -(CH2) 0-4 O(CH2) 0-1 Ph (which may be substituted with R°); -CH=CHPh (which may be substituted with R°); -(CH2) 0-4 O(CH2) 0-1 -pyridyl (which may be substituted with R°); -NO2; -CN; -N3; -(CH2) 0-4 N(R°)2; -(CH2) 0-4N(R°)C(O)R°; -N(R°)C(S)R°; -(CH2) 0-4 N(R°)C(O)NR°2; -N(R°)C(S)NR°2; -(CH2) 0-4 N(R°)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2) 0-4 C(O)R°; -C(S)R°; -(CH2) 0-4 C(O)OR°; -(CH2) 0-4 C(O)SR°; -(CH2) 0-4 C(O)OSiR°3; -(CH2) 0-4 OC(O)R°; -OC(O)(CH2) 0-4 SR°; -(CH2) 0-4 SC(O)R°; -(CH2) 0-4 C(O)NR°2; -C(S)NR°2; -C(S)SR°; -SC(S)SR°; -(CH2) 0-4 OC(O)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)R°; -C(NOR°)R°; -(CH2) 0-4 SSR°; -(CH2) 0-4 S(O)2R°; -(CH2) 0-4 S(O)(NH)R°; -(CH2) 0-4 S(O)2OR°; -(CH2) 0-4 OS(O)2R°; -S(O)2NR°2; -(CH2) 0-4 S(O)R°; -N(R°)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NR°2; -P(O)2R°; -P(O)R°2; -OP(O)R°2; -OP(O)(OR°)2; SiR°3; -(C 1-4 linear or branched alkylene)O-N(R°)2; or -(C 1-4 linear or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined below, independently, hydrogen, C 1-6 aliphatic, -CH2Ph, -O(CH2) 0-1Ph, -CH2-(a 5- to 6-membered heteroaryl ring), a 5- to 6-membered saturated ring, a partially unsaturated ring, or an aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8- to 10-membered bicyclic aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, regardless of the above definition, two independent occurrences of R° together with their intervening atom(s) form a 3- to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0139] Suitable monovalent substituents on R° (or the ring formed by two independent occurrences of R° together with their intervening atom(s)) are, independently, halogen, -(CH2) 0-2 R ● , -(haloR ● ), -(CH2) 0-2 OH, -(CH2) 0-2 OR ● , -(CH2) 0-2 CH(OR ● ), -O(haloR ● ), -CN, -N3, -(CH2) 0-2 C(O)R ● , -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● , -(CH2) 0-2 SR ● , -(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR ● , -(CH2) 0-2 NR ● 2, -NO2, -SiR ● 3, -OSiR ● 3, -C(O)SR ● 、 -(C 1-4 linear or branched alkylene)C(O)OR ● , or -SSR ●and in the formula, each R ● is unsubstituted or, when preceded by "halo", is substituted only with one or more halogens and is independently selected from C 1-4 aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 3- to 6-membered saturated ring, partially unsaturated ring, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on the saturated carbon atoms of R° include =O and =S.

[0140] Suitable divalent substituents on the saturated carbon atoms of a "optionally substituted" group include the following: =O ("oxo"), =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O-, or -S(C(R * 2)) 2-3 S- (wherein each independent occurrence of R * is selected from hydrogen, C 1-6 aliphatic (which may be substituted as defined below), or an unsubstituted 5- to 6-membered saturated ring, partially unsaturated ring, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur). Suitable divalent substituents bonded to the substitutable carbon in the vicinal position of a "optionally substituted" group include -O(CR * 2) 2-3 O- is included, and in the formula, each independent occurrence of R * is selected from hydrogen, C 1-6 aliphatic (which may be substituted as defined below), or an unsubstituted 5- to 6-membered saturated ring, partially unsaturated ring, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur).

[0141] R *Suitable substituents on the aliphatic group include halogen, -R ● , -(haloR ● ), -OH, -OR ● , -O(haloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, wherein each R ● is unsubstituted or, when preceded by "halo", is substituted with one or more halogens only and is independently C 1-4 aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0142] Suitable substituents on the nitrogen of a "optionally substituted" group include -R † , -NR † 2, -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CH2C(O)R † , -S(O)2R † , -S(O)2NR † 2, -C(S)NR † 2, -C(NH)NR † 2, or -N(R † )S(O)2R † , wherein each R † is independently hydrogen, C 1-6 aliphatic (which may be substituted as defined below), unsubstituted -OPh, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, regardless of the above definition, R †Two independent occurrences thereof, together with their intervening atom(s), form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0143] R † Suitable substituents on the aliphatic group of are, independently, halogen, -R ● , -(haloR ● ), -OH, -OR ● , -O(haloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, wherein each R ● is unsubstituted or, when preceded by "halo", is substituted only with one or more halogens and is independently C 1-4 aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0144] The term "C x-y " or "C x ~C y ", when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is intended to include a group containing x to y carbons in the chain. C0 alkyl represents hydrogen when the group is in a terminal position and a bond when internal. For example, a C 1-6 alkyl group contains 1 to 6 carbon atoms in the chain.

[0145] As used herein, the term "alkylamino" refers to an amino group substituted with at least one alkyl group.

[0146] As used herein, the term "alkylthio" refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0147] As used herein, the term "amide" refers to the following group:

Chemical formula

[0148] The terms "amine" and "amino" are recognized in the art and refer to both unsubstituted and substituted amines, as well as their salts, for example, moieties that may be represented by the following:

Chemical formula

[0149] As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

[0150] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0151] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings, wherein two or more carbons are common to two adjacent rings, and at least one of these rings is aromatic. For example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0152] The term "carbamate" is recognized in the art and refers to the following group:

Chemical formula

[0153] As used herein, the term "carbocyclic alkyl" refers to an alkyl group substituted with a carbocyclic group.

[0154] As used herein, the terms "carbocyclic", "carbocyclyl", and "carbocyclic" refer to non-aromatic saturated or unsaturated rings in which each atom of the ring is carbon. Preferably, the carbocyclic ring contains 3 to 10 atoms, more preferably 5 to 7 atoms.

[0155] As used herein, the term "carbocyclic alkyl" refers to an alkyl group substituted with a carbocyclic group.

[0156] The term "carbonate" is recognized in the art and refers to the group -OCO2-.

[0157] As used herein, the term "carboxy" refers to the group represented by the formula -CO2H.

[0158] As used herein, the term "ester" refers to a -C(O)OR 9 group, where R 9 represents a hydrocarbyl group.

[0159] As used herein, the term "ether" refers to a hydrocarbyl group linked to another hydrocarbyl group through oxygen. Thus, an ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. The ether may be either symmetric or asymmetric. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include the "alkoxyalkyl" group which can be represented by the general formula: alkyl-O-alkyl.

[0160] As used herein, the terms "halo" and "halogen" mean halogen and include chloro, fluoro, bromo, and iodo.

[0161] As used herein, the terms "hetaralkyl" and "heteroalkyl" refer to an alkyl group substituted with a hetaryl group.

[0162] The terms "heteroaryl" and "heteroaryl" refer to substituted or unsubstituted aromatic monocyclic structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "heteroaryl" also include polycyclic ring systems having two or more cyclic rings, wherein two or more carbons are common to two adjacent rings, and at least one of these rings is heteroaromatic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0163] As used herein, the term "heteroatom" means an atom that is any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0164] As used herein, the term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclic group.

[0165] The terms "heterocyclyl", "heterocyclic", and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings, wherein two or more carbons are common to two adjacent rings, and at least one of these rings is heterocyclic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, etc.

[0166] As used herein, the term "hydrocarbyl" refers to a group that is bonded through a carbon atom having no =O or =S substituents, typically has at least one carbon-hydrogen bond and a predominantly carbon backbone, and may optionally contain heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has an =O substituent on the linking carbon) and ethoxy (which is linked through oxygen rather than carbon) are not considered hydrocarbyl. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, alkynyl, and combinations thereof.

[0167] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0168] The term "lower", when used in combination with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is intended to include groups having 10 or fewer, preferably 6 or fewer, atoms in the substituent. For example, "lower alkyl" refers to an alkyl group containing 10 or fewer, preferably 6 or fewer, carbon atoms. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents as defined herein, such as in the recitation of hydroxyalkyl and aralkyl, are each, whether they appear alone or in combination with other substituents, lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy (in this case, for example, when counting carbon atoms in an alkyl substituent, atoms within an aryl group are not counted).

[0169] The terms "polycyclyl", "polycyclic", and "polycyclic ring" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more carbons are common to two adjacent rings, for example, where the rings are "fused rings". Each of the rings of the polycyclic ring may be substituted or unsubstituted. In certain embodiments, each ring of the polycyclic ring contains from 3 to 10, preferably 5 to 7, atoms in the ring.

[0170] The term "sulfate" is recognized in the art and refers to the group -OSO3H or a pharmaceutically acceptable salt thereof.

[0171] The term "sulfonamide" is recognized in the art and refers to a group represented by the following general formula:

Chemical formula

[0172] The term "sulfoxide" is recognized in the art and refers to the group -S(O)-.

[0173] The term "sulfonate" is recognized in the art and refers to the group SO3H or a pharmaceutically acceptable salt thereof.

[0174] The term "sulfone" is recognized in the art and refers to the group -S(O)2-.

[0175] The term "substituted" refers to the portion where a substituent replaces hydrogen on one or more carbons of the backbone. The terms "substitution" or "substituted with" are understood to imply that such substitution conforms to the valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not undergo spontaneous conversion, such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all permissible substituents of an organic compound. In one broad aspect, possible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. Permissible substituents can be one or more for a suitable organic compound, and can be the same or different. For the purposes of the present invention, a heteroatom such as nitrogen may have a hydrogen substituent and / or any permissible substituent of the organic compounds described herein that satisfies the valence of the heteroatom. Substituents include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that, where appropriate, the moiety substituted on the hydrocarbon chain itself may be substituted.

[0176] As used herein, the term "thioalkyl" refers to an alkyl group substituted with a thiol group.

[0177] As used herein, the term "thioester" refers to a -C(O)SR 9 or -SC(O)R 9 group, wherein R 9 represents a hydrocarbyl.

[0178] As used herein, the term "thioether" is equivalent to an ether in which oxygen is replaced by sulfur.

[0179] The term "urea" is recognized in the art and may be represented by the following general formula: [Chemical formula] In the formula, R 9 and R 10 each independently represents hydrogen or hydrocarbyl.

[0180] As used herein, the term "modulate" includes inhibition or suppression of a function or activity (such as cell proliferation), as well as enhancement of a function or activity.

[0181] The phrase "pharmaceutically acceptable" is recognized in the art. In certain embodiments, this term refers to compositions, excipients, adjuvants, polymers, and other materials and / or dosage forms that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio.

[0182] "Pharmaceutically acceptable salt" or "salt" is used herein to refer to acid addition salts or basic addition salts that are suitable or appropriate for the treatment of a patient.

[0183] As used herein, the term "pharmaceutically acceptable acid addition salt" means any non-toxic organic or inorganic salt of any basic compound represented by Formula I. Exemplary inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as metal salts such as sodium monohydrogen orthophosphate and potassium bisulfate. Exemplary organic acids that form suitable salts include monocarboxylic acids, dicarboxylic acids, and tricarboxylic acids such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, and salicylic acid, as well as sulfonic acids such as p-toluenesulfonic acid and methanesulfonic acid. Either monoacid salts or diacid salts may be formed, and such salts may exist in any of their hydrated, solvated, or substantially anhydrous forms. Generally, the acid addition salts of the compounds of Formula I have higher solubility in water and various hydrophilic organic solvents and generally exhibit higher melting points compared to their free base forms. The selection of suitable salts will be known to those skilled in the art. Other salts that are not pharmaceutically acceptable, such as oxalates, may be used in the isolation of the compounds of Formula I, for example, for use in the laboratory or for subsequent conversion to pharmaceutically acceptable acid addition salts.

[0184] As used herein, the term "pharmaceutically acceptable basic addition salt" means any non-toxic organic or inorganic base addition salt of any acid compound represented by Formula I or any of their intermediates. Exemplary inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide. Exemplary organic bases that form suitable salts include aliphatic, cycloaliphatic, or aromatic organic amines such as methylamine, trimethylamine, and picoline or ammonia. The selection of suitable salts is known to those skilled in the art.

[0185] Many of the compounds useful in the methods and compositions of the present disclosure have at least one stereocenter in their structure. This stereocenter may be present in the R configuration or the S configuration, and the R and S notations are used in accordance with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The present disclosure contemplates all stereoisomeric forms, such as the enantiomeric and diastereoisomeric forms of the compounds, their salts, prodrugs, or mixtures (including all possible mixtures of stereoisomers).

[0186] Furthermore, certain compounds containing an alkenyl group may exist as the Z (zusammen) or E (entgegen) isomers. In each instance, the present disclosure includes both mixtures and the separate individual isomers.

[0187] Some of the compounds may also exist in tautomeric forms. Such forms are not explicitly shown in the formulas described herein but are intended to be included within the scope of the present disclosure.

[0188] "Prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized, for example, hydrolyzed or oxidized, in a host after administration to form a compound of the present disclosure (e.g., a compound of formula I). Typical examples of prodrugs include compounds having a biologically labile or cleavable (protecting) group on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolized, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to yield the active compound. Examples of prodrugs using esters or phosphoramidates as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patent Nos. 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of the present disclosure are metabolized to produce the compounds of formula I. The present disclosure includes within its scope prodrugs of the compounds described herein. Conventional procedures for selecting and preparing suitable prodrugs are described, for example, in "Design of Prodrugs" Ed. H. Bundgaard, Elsevier, 1985.

[0189] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, useful in formulating a drug for pharmaceutical or therapeutic use.

[0190] As used herein, the terms "logarithm of solubility", "LogS", or "logS" are used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound has a significant effect on its absorption and distribution characteristics. Poor solubility is often associated with poor absorption. The LogS value is the logarithm (base 10) with the units of solubility measured in moles / liter removed.

Examples

[0191] The present invention has been generally described heretofore, but it will be more readily understood with reference to the following examples. These examples are included for the purpose of illustrating and explaining certain aspects and embodiments of the present invention and are not intended to limit the present invention.

[0192] Example 1: Synthesis General experimental method. All reactions were carried out under an argon atmosphere unless otherwise specified. Tetrahydrofuran (THF) was distilled from benzoquinone ketyl radical under an argon atmosphere. Dichloromethane and triethylamine were distilled from calcium hydride under an argon atmosphere. All other solvents and reagents were purified according to the procedures in the literature or purchased from Sigma-Aldrich, Acros, Oakwood and Fisher Scientific Co.. 1 1H NMR spectra were recorded at 400 or 500 MHz and reported relative to the signals of the deuterated solvents. 1 Data for 1H NMR spectra are reported as follows: chemical shift (δ ppm), multiplicity, coupling constant (Hz), and integration. Splitting patterns are denoted as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad. 13 13C NMR spectra were recorded at 100 or 125 MHz. 13 Data for 13C NMR spectra are reported in terms of chemical shift. Chemical shifts are reported in parts per million (ppm, δ) units. Thin layer chromatography (TLC) was carried out using pre-coated silica gel sheets. Visual detection was carried out using potassium permanganate or ammonium cerium nitrate dyes. Flash chromatography was carried out using SilicaFlash P60 (60A, 40 - 63 μm) silica gel with compressed air.

[0193] [Chemical formula] 3-Chloro-6-hydrazinylpyridazine. To a solution of 3,6-dichloropyridazine (400 mg, 2.686 mmol) in EtOH (8 mL) was added hydrazine monohydrate (148 mg, 2.954 mmol), and the mixture was stirred at 100 °C for 3 h. After the mixture was cooled to 23 °C, the resulting solid was collected and washed with Et2O. The mother liquor was concentrated, and the precipitate was washed with Et2O. The combined solids were washed with dichloromethane to give the desired product (pale yellow, 320.2 mg, 2.216 mmol, 82%) and used in the next step without further purification. 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (br s, 1H), 7.41 (d, J = 9.6 Hz, 1H), 7.09 (d, J = 9.2 Hz, 1H), 4.37 (br s, 2H); 13 C NMR (100 MHz, DMSO-d6) δ 161.8, 145.4, 128.7, 116.1. The spectroscopic data is consistent with the literature data. [Reference: Heterocycles, 2009, 78(4) 961-975]

[0194]

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[0195]

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[0196]

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[0197]

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[0198]

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[0199]

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[0200]

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[0201]

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[0202]

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[0203]

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[0204]

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[0205]

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[0206]

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[0207]

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[0208] [Chemical formula] N-(2-(3-Methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ011. Using the same procedure as described for JGJ004, the reaction of 2-(3-methylimidazo[1,2-b]pyridazin-6-yl)aniline (JGJ010, 39.4 mg, 0.176 mmol), triethylamine (21.3 mg, 0.211 mmol), and acetyl chloride (16.5 mg, 0.211 mmol) in dichloromethane (0.8 mL) gave the desired product JGJ011 (35 mg, 0.131 mmol, 75%) as an off-white solid. 1 1H NMR (400 MHz, CDCl3) δ 10.57 (br s, NH), 8.47 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 9.6 Hz, 1H), 7.61 (s, 1H), 7.60 (dd, J = 8.0, 0.8 Hz, 1H), 7.44 (ddd, J = 8.8, 7.2, 0.8 Hz, 1H), 7.34 (d, J = 9.2 Hz, 1H), 7.20 (ddd, J = 8.0, 7.2, 0.8 Hz, 1H), 2.60 (s, 3H), 2.17 (s, 3H); 1313C NMR (100 MHz, CDCl3) δ 168.1, 152.0, 137.3, 136.4, 132.8, 130.6, 129.5, 126.3, 124.6, 124.0, 123.5, 122.4, 116.7, 25.1, 8.9.

[0209]

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[0210]

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[0211]

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[0212]

Chem.

[0213] [Chemical formula] N-Methyl-3-(3-methylimidazo[1,2-b]pyridazin-6-yl)benzamide, JGJ016. To a solution of JGJ013 (20.1 mg, 0.079 mmol) and methylamine hydrochloride (10.7 mg, 0.159 mmol) in dichloromethane (0.3 mL) and DMF (0.5 mL) were added hydroxybenzotriazole (HOBT, 16.1 mg, 0.159 mmol), (3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl, 30.4 mg, 0.159 mmol), and N,N-diisopropylethylamine (DIPEA, 102.6 mg, 0.794 mmol). The mixture was stirred at 23 °C for 12 h. After adding water to the reaction, it was extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (dichloromethane:MeOH = 6:1) to give the desired product JGJ016 (8.6 mg, 0.032 mmol, 41%) as a pale yellow solid. 11H NMR (400 MHz, CDCl3) δ 8.39 (t, J = 1.6 Hz, 1H), 8.10 (dddd, J = 8.0, 1.6, 1.2, 0.8 Hz, 1H), 7.91 (d, J = 9.6 Hz, 1H), 7.86 (ddd, J = 7.6, 1.6, 1.2 Hz, 1H), 7.58 (s, 1H), 7.55 (dd, J = 8.0, 7.6 Hz, 1H), 7.41 (d, J = 9.6 Hz, 1H), 6.75 (m, NH), 3.06 (d, J = 4.8 Hz, 3H), 2.59 (d, J = 0.4 Hz, 3H); 13 13C NMR (100 MHz, CDCl3) δ 167.7, 153.3, 138.0, 136.3, 135.5, 132.3, 129.7, 129.2, 128.0, 125.7, 125.5, 125.4, 114.4, 26.9, 8.7.

[0214] [Chemical formula] 3-Methyl-6-(pyridin-3-yl)imidazo[1,2-b]pyridazine, JGJ017. Using the same procedure as described for JGJ002, the reaction of 6-chloro-3-methylimidazolo[1,2-b]pyridazine (58.8 mg, 0.351 mmol), 3-pyridineboronic acid (47.4 mg, 0.386 mmol), K2CO3 (72.7 mg, 0.526 mmol), and Pd(PPh3)4 (40.6 mg, 0.035 mmol) in 1,4-dioxane / water (5:1 v / v, 0.6 mL) gave the desired product JGJ017 (37.2 mg, 0.177 mmol, 50%) as a pale yellow solid. 11H NMR (400 MHz, CDCl3) 9.20 (d, J = 1.6 Hz, 1H), 8.69 (dd, J = 4.8, 1.6 Hz, 1H), 8.29 (ddd, J = 8.0, 2.0, 1.6 Hz, 1H), 7.98 (d, J = 9.2 Hz, 1H), 7.60 (d, J = 0.4 Hz, 1H), 7.42 (ddd, J = 8.0, 4.8, 0.8 Hz, 1H), 7.41 (d, J = 9.6 Hz, 1H), 2.60 (d, J = 0.8 Hz, 3H); 13 13C NMR (100 MHz, CDCl3) δ 150.6, 148.6, 148.2, 137.9, 134.2, 132.7, 131.6, 125.7, 125.5, 123.6, 113.7, 8.6.

[0215]

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[0216]

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[0217]

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[0218]

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[0219]

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[0220] [Chemical formula] N-(3-(2-Methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ021. Using the same procedure as described for JGJ002, the reaction of 6-chloro-2-methylimidazo[1,2-b]pyridazine (35.3 mg, 0.211 mmol), 3-aminophenylboronic acid (35.9 mg, 0.232 mmol), K2CO3 (43.7 mg, 0.316 mmol), and Pd(PPh3)4 (24.4 mg, 0.021 mmol) in 1,4-dioxane / water (5:1 v / v, 0.5 mL) gave 3-(2-methylimidazo[1,2-b]pyridazin-6-yl)aniline (49.6 mg, quantitative) as a pale yellow solid. Then, using the same procedure as described for JGJ004, acetylation gave the desired product JGJ021 (27.2 mg, 0.102 mmol, 46%) as an ivory solid. 1 H NMR (400 MHz, CDCl3) δ 8.92 (s, 1H), 8.16 (s, 1H), 7.73 (d, J = 9.6 Hz, 1H), 7.63 (m, 2H), 7.54 (d, J = 7.6 Hz, 1H), 7.33 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 10.0 Hz, 1H), 2.44 (s, 3H), 2.19 (s, 3H); 13 C NMR (100 MHz, CDCl3) δ 169.2, 150.7, 143.8, 139.0, 137.7, 136.1, 129.4, 123.9, 122.3, 121.1, 118.2, 115.7, 114.3, 24.4, 14.5.

[0221]

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[0222]

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[0224]

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[0225]

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[0227]

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[0228]

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[0229] [Chemical formula] N-(3-(3-(Pyridin-3-yl)imidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ028. Using the same procedure as described for JGJ002, the reaction of 6-chloro-3-(pyridin-3-yl)imidazo[1,2-b]pyridazine (41.5 mg, 0.180 mmol), 3-aminophenyl-boronic acid (30.7 mg, 0.198 mmol), K2CO3 (37.3 mg, 0.270 mmol), and Pd(PPh3)4 (10.4 mg, 0.009 mmol) in 1,4-dioxane / water (5:1 v / v, 0.4 mL) gave 3-(3-(pyridin-3-yl)imidazo[1,2-b]pyridazin-6-yl)aniline (50.0 mg, 0.174 mmol, 96%) as an off-white solid. Then, using the same procedure as described for JGJ004, acetylation gave the desired product JGJ028 (18.2 mg, 0.055 mmol, 32%) as a pale yellow solid. 11H NMR (400 MHz, CD3OD) δ 9.29 (d, J = 1.2 Hz, 1H), 8.60 (ddd, J = 8.0, 2.0, 1.6 Hz, 1H), 8.49 (d, J = 4.0 Hz, 1H), 8.25 (dd, J = 2.0, 1.6 Hz, 1H), 8.18 (s, 1H), 8.02 (d, J = 9.6 Hz, 1H), 7.68 (d, J = 9.6 Hz, 1H), 7.60 - 7.65 (m, 2H), 7.55 (dd, J = 8.0, 4.8 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 2.16 (s, 3H); 13 13C NMR (100 MHz, CD3OD) δ 172.1, 153.6, 149.2, 148.0, 141.5, 141.2, 137.1, 135.9, 134.1, 130.8, 127.1, 127.0, 126.9, 125.7, 123.8, 123.0, 119.5, 118.6, 24.3.

[0230] [Chemical formula] 6-Chloro-3-(pyrimidin-5-yl)imidazo[1,2-b]pyridazine. Using the same procedure as described for 6-chloro-3-phenylimidazo[1,2-b]pyridazine, the reaction of 6-chloro-3-iodoimidazo[1,2-b]pyridazine (83.6 mg, 0.299 mmol), pyrimidine-5-boronic acid (40.8 mg, 0.329 mmol), K2CO3 (62 mg, 0.449 mmol), and Pd(PPh3)4 (17.3 mg, 0.015 mmol) in 1,4-dioxane / water (5:1 v / v, 1 mL) at 100 °C gave the desired product (9.8 mg, 0.042 mmol, 14%) as a pale yellow solid. 1 1H NMR (400 MHz, CDCl3) δ 9.42 (s, 2H), 9.23 (s, 1H), 8.18 (s, 1H), 8.04 (d, J = 9.6 Hz, 1H), 7.20 (d, J = 9.6 Hz, 1H); 1313C NMR (100 MHz, CDCl3) δ 157.7, 154.0, 147.7, 133.7, 132.1, 128.5, 127.7, 123.0, 119.8.

[0231]

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[0232]

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[0234]

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[0235] [Chemical formula] N-(3-(3-Methylimidazo[1,2-a]pyridin-6-yl)phenyl)acetamide, JGJ031. Using the same procedure as described for JGJ002, 6-Bromo-3-methylimidazo[1,2-a]pyridine (35 mg, 0.166 mmol), 3-aminophenylboronic acid (28.3 mg, 0.182 mmol), K2CO3 (34.4 mg, 0.249 mmol), and Pd(PPh3)4 (9.6 mg, 0.008 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL) were reacted to give 3-(3-Methylimidazo[1,2-a]pyridin-6-yl)aniline (28.1 mg, 0.106 mmol, 64%) as an off-white solid. Then, using the same procedure as described for JGJ004, acetylation gave the desired product JGJ031 (15.8 mg, 0.060 mmol, 56%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 8.30 (br s, 1H), 8.12 (s, 1H), 7.87 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.36-7.43 (m, 3H), 7.27 (m, 1H), 2.49 (s, 3H), 2.23 (s, 3H);

[0236]

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[0237]

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[0238]

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[0239]

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[0240]

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[0241]

Chem.

[0242] [Chemical formula] N-(3-(3-Propyl-1H-pyrrolo[3,2-b]pyridin-5-yl)phenyl)acetamide, JGJ036. Using the same procedure as described for JGJ004, the reaction of 3-(3-propyl-1H-pyrrolo[3,2-b]pyridin-5-yl)aniline (JGJ035, 34.5 mg, 0.137 mmol), triethylamine (20.8 mg, 0.206 mmol), and acetyl chloride (16.2 mg, 0.206 mmol) in dichloromethane (3 mL) gave the desired product JGJ036 (28.8 mg, 0.098 mmol, 72%) as an off-white solid. 11H NMR (400 MHz, CD3OD) δ 8.11 (dd, J = 2.0, 1.6 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.64 - 7.67 (m, 2H), 7.49 (d, J = 8.8 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.32 (s, 1H), 2.85 (t, J = 7.2 Hz, 2H), 2.15 (s, 3H), 1.80 (m, 2H), 1.01 (t, J = 7.2 Hz, 3H); 13 13C NMR (100 MHz, CD3OD) δ 171.8, 151.4, 146.4, 143.1, 140.1, 130.3, 129.9, 127.9, 124.2, 120.6, 120.4, 120.2, 117.4, 115.7, 27.1, 24.5, 23.9, 14.5.

[0243] [Chemical formula] N-(3-Fluoro-5-(3-phenyl-1H-pyrrolo[3,2-b]pyridin-5-yl)phenyl)acetamide, JGJ037. Using the same procedure as described for JGJ002, 3-Fluoro-5-(3-phenyl-1H-pyrrolo[3,2-b]pyridin-5-yl)aniline (18.1 mg, 0.060 mmol, 70%) was obtained as an off-white solid by the reaction of 5-chloro-3-phenyl-1H-pyrrolo[3,2-b]pyridine (19.4 mg, 0.085 mmol), 3-fluoro-5-aminophenylboronic acid (14.5 mg, 0.093 mmol), K2CO3 (17.6 mg, 0.127 mmol), and Pd(PPh3)4 (9.8 mg, 0.009 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL). Then, using the same procedure as described for JGJ004, the desired product JGJ037 (13.8 mg, 0.040 mmol, 67%) was obtained as an off-white solid by acetylation. 11H NMR (400 MHz, CD3OD) δ 8.25 (m, 2H), 7.98 (t, J = 1.6 Hz, 1H), 7.88 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.58 (m, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.21 (td, J = 7.6, 1.2 Hz, 1H), 2.15 (s, 3H); 13 13C NMR (100 MHz, CD3OD) δ 171.9, 164.6 (d, J = 239.6 Hz), 150.2 (d, J = 2.9 Hz), 145.1, 144.7 (d, J = 8.9 Hz), 141.7 (d, J = 11.5 Hz), 136.0, 130.9, 129.4, 127.8, 127.6, 126.6, 120.5, 117.3, 115.3, 114.7 (d, J = 3.2 Hz), 109.8 (d, J = 23.1 Hz), 107.3 (d, J = 27.0 Hz), 24.0.

[0244]

Chem.

[0245]

Chemical Structure

[0246]

Chem.

[0247] Example 2: LIN28 is significantly overexpressed in human and mouse AML and drives MLL leukemia induction. Analysis of databases of AML and healthy hematopoietic cells (HSC, Blood Spot (55)) showed that Lin28b expression was significantly enriched in various AML karyotypes when compared to healthy HSCs (Figure 2A). Additionally, Lin28 was independently found to be a major driver in MLL-related leukemias (56). To further characterize the role of Lin28 / let-7 in the control of AML, LSC proliferation, and treatment resistance in vivo, a doxocycline (DOX)-inducible transgenic mouse model of MLL-AF9-driven AML was used (iMLL-AF9 (57)). In this model, long-term HSCs (LT-HSCs, Lin - CD34 - Sca-1 - c-Kit + CD150 + CD48 - )-derived AML blasts closely reflect an LSC-like phenotype that gives rise to particularly aggressive cytarabine (Ara-C)-resistant AML (57). The inventors transplanted whole bone marrow (WBM) cells or fluorescence-activated cell sorting (FACS) LT-HSCs from non-inducible iMLL-AF9 mice into congenic mice (B6.SJL, CD45.1) and maintained the recipients with DOX. mRNA analysis of AML cells at day +35 (d+35) showed that Lin28b expression was significantly increased in WBM-derived and LT-HSC-derived AML cells (LSCs) when compared to healthy non-DOX-induced LT-HSCs (Figure 2B). Moreover, relapsed AML cells arising from rLSCs at d+60 after treatment with Ara-C (100 mg / kg) had even more enriched Lin28b expression (Figure 2B). Additionally, miRNA levels of both let-7a and let-7b were negatively correlated with Lin28b in rLSCs (Figure 2C) (10, 39). The inventors' findings are consistent with papers reporting that increased Lin28 correlates with disease relapse after chemotherapy in colon and liver cancer stem cells (22, 58).

[0248] Example 3: Lin28 inhibition overcomes treatment resistance in relapsed AML. Since Lin28 is overexpressed in human AML, LSC, and rLSC, the inventors sought to determine whether genetic Lin28b or pharmacological Lin28 / let-7 inhibition by LN1632 could suppress LSC proliferation and thus overcome their treatment resistance. The inventors isolated LT-HSCs by FACS, incubated 500 cells with DOX, and simultaneously transduced them with shLin28b or its corresponding control shScramble, or treated the cells with 200 nM Ara-C, 30 μM 1632, or a control for 48 hours. Ara-C did not change the number of colony-forming cells (CFC), but genetic silencing of Lin28 (shLin28b) or its pharmacological inhibition by LN1632 significantly suppressed the CFC of LSCs (Figure 2D).

[0249] Example 4: Targeted LIN28 / let-7 inhibition reduces tumor burden in AML in vivo. Considering that genetic Lin28b inhibition and treatment with LN1632 suppress the CFC of LSCs, the inventors sought to explore the effect of LN1632 in human AML. By Western blotting, the inventors confirmed that the Lin28 inhibitor LN1632 dose-dependently decreased the LIN28B protein level in AML with t(8;21) (Kasumi-1) and MLL rearrangement (THP-1) (Figure 3A). Notably, the proteasome inhibitor bortezomib was able to inhibit the decrease in LIN28B protein level in TF1-alpha cells after treatment with 1632 (Figure 3B), suggesting that 1632 can directly target LIN28B and lead to its proteasomal degradation. Therefore, the inventors investigated the effect of targeted Lin28 / let-7 inhibition in in vivo AML. The inventors demonstrated that intermittent dosing of 100 mg / kg every other day for 21 days was non-toxic and well-tolerated in healthy C57BL / 6 mice as they showed normal weight gain, complete blood count (CBC), and behavior. Thus, the inventors implanted THP-1 (high LIN28B) cells or MOLM-13 (LIN28B-negative) subcutaneously (subQ) into NSG mice, and 12 days later (tumor size = 40 mm 2 ), initiated intraperitoneal administration of 100 mg / kg of 1632 every other day. These results showed that tumor growth was significantly reduced in THP-1 xenografts but not in MOLM-13 xenografts (Figures 4A - B). The inventors further evaluated the effect of 1632 in systemic Kasumi-1 cell line xenografts (LSC-like CD34 + CD38 - , high LIN28B, AML t(8;21)). Intraperitoneal injection with 100 mg / kg of 1632 every other day for 21 days significantly prolonged the survival of the animals (Figure 4B). Bioluminescence imaging (BLI) confirmed a decrease in tumor burden in 1632-treated mice compared to the vehicle (Figure 4C, photograph).

[0250] Example 5: Targeted inhibition of LIN28 downregulates NF-κB and BCL-2 in primary AML. To determine the full extent to which LN1632 regulates gene expression, we performed RNA sequencing (RNAseq) in LSC-like Kasumi-1 cells. As illustrated in the heatmap in Figure 5A, we observed significant downregulation of a panel of direct let-7 target genes (44), including CCND1 / 2, E2F2, HMGA1, LIN28B, MYC, NFKB1, MRAS, IL6, and STAT5 (green, Figure 5A). Importantly, we confirmed this gene expression pattern in primary AML cells from three relapsed patients (validated for LIN28B overexpression compared to healthy WBM). At 72 hours post-treatment, we observed a dose-dependent significant upregulation of mature let-7a / b and downregulation of multiple let-7 target genes, including NFκB1 (Figure 5B). This is important because NFκB1 (23), which is regulated by let-7 via IL6, along with other BCL-2 family members (BAX, BCL2L15, and BMF, Fig. 5A), are well-characterized genes associated with unique properties of LSC survival and AML relapse rates (45, 59, 60). Consistent with this, gene set enrichment analysis revealed widespread changes in gene expression signatures previously shown to distinguish LSCs from non-self-renewing leukemia cell populations (61) and poor prognosis in pediatric AML relapse (62) (Fig. 5C). We next explored the effect of LN1632 on primary AML cells. CFC assays in Fig. 6A show that treatment with LN1632 significantly increased the expression of CD34 + Colony formation of cells from AML patient #13 versus healthy CD34 + These results indicate that LN1632 exerts a significantly greater effect on LSCs than on BM cells. Moreover, ex vivo treatment of cells from AML patient #13 with 1632 or control inhibited their AML repopulation ability in vivo (Figure 6B-C). Thus, these results suggest that the effect of LN1632 is greater on LSCs than on HSCs.

[0251] Example 6: Lin28 / let-7 inhibitory activity of exemplary compounds. To improve the binding and inhibitory ability of compounds against LIN28b, the inventors predicted the binding mode of LN1632 to LIN28B. By a close-up of the crystal structure of the LIN28 protein, the possible binding mode of LN1632 to the GGAG-RNA sequence binding pocket of the CCHC domain of LIN28 was revealed (not shown). Having obtained this model, the inventors synthesized new compounds (JGJ002 - JGJ008, Figure 8) with improved binding ability to Lin28b. The compounds were screened using a previously described FRET assay that used EGFP-tagged LIN28B as the donor and BHQ-1 quencher-labeled pre-let-7a-2 (pre-let-7a-2-BHQ1) as the acceptor (51). Briefly, recombinant LIN28B-EGFP was harvested from stably transfected HEK cells and diluted in binding buffer (300 mM NaCl, 25 mM HEPES (pH 7.2), 10 μM ZnCl2, 1% Odyssey blocking buffer, 0.05% Tween 20, 0.5 mM TCEP) to adjust the ideal FRET quenching signal intensity. Protein lysates and compounds (JGJ001 - JGJ008) were pre-incubated for 20 minutes at doses in the range of 1.25 μM to 20 μM in 100 μL of diluted protein lysate. Then, pre-let-7a-2-BHQ1 was added to the mixture (at 6.25 nM), and EGFP-LIN28B donor emission was measured using a Tecan Spark plate reader (bandwidth 20 nM, excitation 488 nM, emission reading 545 nM, 30 flashes / second). The results show that, in particular, compounds JGJ005, JGJ007, and JGJ008 inhibit the FRET signal intensity to a greater extent than the original hit compound LN1632. From these results, the inventors conclude that JGJ005, JGJ007, and JGJ008 show greater inhibition of LIN28B / pre-let-7a2 binding than the original compound LN1632 (Figure 7), and thus it is expected that LIN28 is inhibited from binding to the pre-let-7 microRNA, thereby preventing their degradation.Increased endogenous let-7 miRNA levels can thus inhibit tumor growth by targeting a set of LCSs and cancer stem cell hallmark genes.

[0252] Example 7: Evaluation of LN1632 Activity In Vitro and In Vivo Using a target high-throughput fluorescence resonance energy transfer (FRET) screen, triazolopyridazines were identified as a class of small molecules that interfere with the interaction between the RBP LIN28 and pre-let-7 miRNA (51). To study how LN1632 interacts with the LIN28 protein, in silico molecular docking studies were performed using the crystal structure of the LIN28B pre-let-7a complex (PDB ID: 5UDZ) (28). Based on the ability of LN1632 to compete with the LIN28B-pre-let-7 complex in the FRET assay, a hypothesis was put forward that this binding site is likely to be shared with the ZKD RNA-binding motif of LIN28. The results from docking showed that LN1632 binds to the pocket originally occupied by the GGAG motif of pre-let-7a. These results also demonstrated that the amide group of the phenyl ring of LN1632 is positioned in a pocket near the binding site of the zinc ion by H-bonding interactions with LIN28B (Figure 8A).

[0253] To test the structure-activity relationship, 39 LN1632-related analogs (JGJ001-39) were synthesized and their potency and specificity to inhibit LIN28B-RNA binding activity and upregulate mature let-7 miRNA levels were measured. By performing a previously published FRET assay, it was observed that JGJ023, JGJ026, JGJ032, and JGJ034 inhibited the RNA binding ability of LIN28 significantly more than compound LN1632 (Figure 8B). Additionally, in HepG2 cells, JGJ023, JGJ026, and JGJ034 upregulated mature let-7 miRNA at significantly lower doses than LN1632 as measured by a dual luciferase reporter assay (Figure 8C). The dual luciferase reporter assay was performed as previously described (89).

[0254] To determine the extent to which LN1632 controls gene expression, RNA sequencing was performed in human Kasumi-1 AML cells. The data in Figures 9A-9B showed that treatment of cells with LN1632 significantly downregulated genes of the HALLMARK_MYC-TARGET_V1 gene signature (70), leukemia stem cells, and relapse prognostic signature (61, 62). In addition, Ingenuity pathway analysis predicted suppression of upstream signaling molecules IL6 and MYC (Figure 9C).

[0255] Next, the tumor-suppressive effect of LN1632 in vivo was investigated. The maximum tolerated dose (MTD) was evaluated in healthy female C57Bl / 6 mice. A daily dosing schedule of 100 mg / kg for 12 days followed by alternate-day dosing for 9 days was well tolerated and the mice showed a normal complete blood count (CBC) profile without any leukopenia or thrombocytopenia, mild anemia, and normal weight gain (Figures 10A-B).

[0256] Subsequently, the tumor-suppressive effect of LN1632 in cancer in vivo was evaluated. High LIN28B-expressing THP-1 AML cells (2×10 6Cells) were implanted into NSGS mice (cell suspension in Matrigel, 3:1), and on d+12 or d+8 (tumor size = 50 mm 2 ), daily intraperitoneal injection of 100 mg / kg of LN1632 was initiated. The results showed significantly reduced tumor growth 19 days after injection (Figure 11A). These results are consistent with recent reports showing that LN1632 selectively inhibits LIN28B-expressing Ewing sarcoma (EwS), but not LIN28B-depleted EwS (72) and LIN28B-expressing TNBC cells (73). The effect of LN1632 in systemic Kasumi-1 xenografts was also evaluated. Alternate-day intraperitoneal injection with 100 mg / kg of LN1632 over 21 days significantly extended the survival of the animals (Figure 11B). Bioluminescence imaging (BLI) confirmed a decrease in tumor burden in LN1632-treated mice compared to the vehicle (Figure 11B, photograph). The effect of LN1632 on cytarabine chemotherapy (Ara-C) was also compared as previously described (74). THP-1 AML cells were implanted subcutaneously into NSGS mice (1.5×10 6 cells, high LIN28B). Daily intraperitoneal injection with 100 mg / kg of LN1632, 60 mg / kg of cytarabine chemotherapy (Ara-C), or vehicle was initiated on d+3 after implantation of the AML cells, and the vehicle group was continued until the maximum allowable tumor size (250 mm 2 ) was reached. LN1632-treated mice showed increased inhibition of AML tumor growth compared to Ara-C- or vehicle-treated mice (Figure 11C).

[0257] Since LN1632 showed a significant anti-proliferative effect in a cancer in vivo model, additional functional interaction partners of LN1632 were evaluated. A cellular thermal shift assay by mass spectrometry (MS-CETSA, Figure 12A) as described in (75), and immunoprecipitation using biotinylated LN1632 (Figure 12B) were performed. These experiments demonstrated that LN1632 interacts with additional RNA-binding proteins, particularly pre-mRNA processing factor 31 (Figure 12C, PRPF31). PRPF31 is a component of the spliceosome complex, is significantly overexpressed in embryonic stem cells (76), and is downregulated during differentiation (77). PRPF31 is recruited to introns where its highly conserved Nop domain modulates U4 snRNA-15.5K protein interactions. Subsequently, PRPF31 stabilizes the U4 / U6.U5 tri-snRNP by interacting with PRPF6 simultaneously, inducing the transition of the spliceosome complex to an activated state (78).

[0258] As shown in Figure 13, PRPF31 overexpression is correlated with poor prognosis in various tumors including lung adenocarcinoma, gastric adenocarcinoma, and triple-negative breast cancer (TNBC) (Figure 13). Dysregulation of components of the U4 / U6.U5 tri-snRNP complex has been shown to drive tumorigenesis in colorectal cancer (79), TNBC (80 - 82), hepatocellular carcinoma (83), and lung cancer. Dysfunctional RNA splicing and overexpression of splicing factors are essential mechanisms for tumor cell survival and are seen across many hallmarks of cancer (84 - 86). In some embodiments, studies have emerged showing that components of the spliceosome are essential for driving cancer progression by the oncogenic protein MYC. Without wishing to be bound by any particular theory, since MYC is the most frequently amplified oncogene in human cancers and plays an essential role in malignant transformation, in some embodiments, therapies that utilize the spliceosome and particularly target PRPF31 and the U4 / U6 spliceosome complex would be very attractive.

[0259] Using MDA-MB-231 TNBC cells, it was evaluated whether LN1632 targets PRPF31. Overexpression of PRPF31 increased cell proliferation, while genetic silencing of PRPF31 significantly reduced the cell number when evaluated over 7 days (Figure 14A). Importantly, overexpression of PRPF31 (pLenti-C-mGFP-P2A-Puro-PRPF31, Origene) rescued the anti-proliferative effect of LN1632, indicating that LN1632 targets PRPF31. https: / / www.origene.com / catalog / vectors / lentiviral-gene-expression-vectors / ps100093 / plenti-c-mgfp-p2a-puro-lentiviral-gene-expression-vector Additionally, genetic silencing of PRPF31 via small hairpin-mediated RNA (shRNA, ThermoFisher Scientific, TRCN0000001180) inhibited the apoptosis-promoting effect of LN1632. In summary, these results indicate that LN1632 targets PRPF31 (Figure 14A).

[0260] To test whether LN1632 and its novel analogs affect cancer cell growth, cell viability assays and cell counting assays were performed in TNBC (Figure 14B - D), castration-resistant prostate cancer cells (CRPC, Figure 15A - C), and colorectal cancer cells (CRC, Figure 15A - C). The data showed that LN1632 and the novel analogs JGJ034 and JGJ037 preferentially reduced proliferation and induced apoptosis in MYC-driven cancers, including TNBC, CRPC, lung adenocarcinoma, and colorectal adenocarcinoma cells (Table 1).

[0261] To measure cell viability, the CellTiter-Glo (CTG, Promega CellTiter-Glo 2.0 assay) and MTT assay (Sigma Aldrich, Cell Proliferation Kit I) were performed. Briefly, cells were serum-starved overnight and then seeded into 96-well plates. After 24 hours of incubation, the cells were treated with increasing concentrations of the JGJ compound for 96 hours. At the time of assay reading, CellTiter-Glo reagent was added and the luminescence was measured after a 10-minute incubation at room temperature. For the MTT assay, the MTT labeling reagent was added and incubated for 4 hours. Then, the medium was removed, 50 μL of DMSO was added to solubilize the crystals, and the absorbance was measured at 570 nm. Cell viability was calculated as (sample - background) / (control - background). Enzalutamide, palbociclib, and cetuximab, which are drugs for current standard treatment, were used as comparison controls.

Table 1-1

Table 1-2

Table 1-3

[0262] The in vitro ADME properties of the selected analogs are summarized in Tables 2 and 3.

Table 2

Table 3

[0263] Example 8: Synthesis of LN1632 Analog (JGJ Compound) General Experimental Methods All reactions were carried out under an argon atmosphere unless otherwise specified. Tetrahydrofuran (THF) was distilled from benzoquinone ketyl radical under an argon atmosphere. Dichloromethane and triethylamine were distilled from calcium hydride under an argon atmosphere. All other solvents and reagents were purified according to literature procedures or purchased from Sigma-Aldrich, Acros, Oakwood and Fisher Scientific Co.. 1 1H NMR spectra were recorded at 400 or 500 MHz and reported relative to the signals of the deuterated solvents. 1 Data for 1H NMR spectra are reported as follows: chemical shift (δ ppm), multiplicity, coupling constant (Hz), and integration. Splitting patterns are denoted as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad. 13 13C NMR spectra were recorded at 100 or 125 MHz. 13 Data for 13C NMR spectra are reported in terms of chemical shift. Chemical shifts are reported in parts per million (ppm, δ) units. Thin-layer chromatography (TLC) was performed using pre-coated silica gel sheets. Visual detection was performed using potassium permanganate or cerium ammonium nitrate dyes. Flash chromatography was performed using SilicaFlash P60 (60A, 40 - 63 μm) silica gel with compressed air.

[0264]

Chemical Structure

[0265]

Chemical formula

[0266]

Chemical formula

[0267]

Chemical Structure

[0268]

Chemical Structure

[0269]

Chem.

[0270]

Chem.

[0271]

Chem.

[0272]

Chem.

[0273]

Chemical Structure

[0274]

Chem.

[0275]

Chem.

[0276]

Chem.

[0277]

Chemical Structure

[0278]

Chemical formula

[0279]

Chem.

[0280]

Chem.

[0281]

Chem.

[0282]

Chemical Structure

[0283] [Chemical formula] 6-(3-Fluorophenyl)-3-methylimidazo[1,2-b]pyridazine, JGJ015. Using the same procedure as described for JGJ002, the reaction of 6-chloro-3-methylimidazolo[1,2-b]pyridazine (51.5 mg, 0.307 mmol), 3-fluorophenylboronic acid (47.3 mg, 0.338 mmol), K2CO3 (63.7 mg, 0.461 mmol), and Pd(PPh3)4 (35.5 mg, 0.031 mmol) in 1,4-dioxane (0.5 mL) and water (100 μL) gave the desired product JGJ015 (38.2 mg, 0.168 mmol, 55%) as an off-white solid. 1 1H NMR (400 MHz, CDCl3) 7.98 (d, J = 9.2 Hz, 1H), 7.75 (m, 2H), 7.61 (s, 1H), 7.48 (m, 1H), 7.41 (d, J = 9.2 Hz, 1H), 7.18 (m, 1H), 2.63 (s, 3H); 1313C NMR (100 MHz, CDCl3) δ 163.2 (d, J = 244.9 Hz), 149.8 (d, J = 2.6 Hz), 138.2, 138.1, 132.6, 130.5 (d, J = 8.1 Hz), 125.5, 122.6 (d, J = 2.9 Hz), 116.7 (d, J = 21.2 Hz), 114.2, 113.9 (d, J = 23.1 Hz), 8.7. (One low-field carbon was not observed).

[0284] [Chemical formula] N-Methyl-3-(3-methylimidazo[1,2-b]pyridazin-6-yl)benzamide, JGJ016. To a solution of JGJ013 (20.1 mg, 0.079 mmol) and methylamine hydrochloride (10.7 mg, 0.159 mmol) in dichloromethane (0.3 mL) and DMF (0.5 mL) were added hydroxybenzotriazole (HOBT, 16.1 mg, 0.159 mmol), (3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl, 30.4 mg, 0.159 mmol), and N,N-diisopropylethylamine (DIPEA, 102.6 mg, 0.794 mmol). The mixture was stirred at 23 °C for 12 h. After adding water to the reaction, it was extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (dichloromethane:MeOH = 6:1) to give the desired product JGJ016 (8.6 mg, 0.032 mmol, 41%) as a pale yellow solid. 11H NMR (400 MHz, CDCl3) δ 8.39 (t, J = 1.6 Hz, 1H), 8.10 (dddd, J = 8.0, 1.6, 1.2, 0.8 Hz, 1H), 7.91 (d, J = 9.6 Hz, 1H), 7.86 (ddd, J = 7.6, 1.6, 1.2 Hz, 1H), 7.58 (s, 1H), 7.55 (dd, J = 8.0, 7.6 Hz, 1H), 7.41 (d, J = 9.6 Hz, 1H), 6.75 (m, NH), 3.06 (d, J = 4.8 Hz, 3H), 2.59 (d, J = 0.4 Hz, 3H); 13 13C NMR (100 MHz, CDCl3) δ 167.7, 153.3, 138.0, 136.3, 135.5, 132.3, 129.7, 129.2, 128.0, 125.7, 125.5, 125.4, 114.4, 26.9, 8.7.

[0285]

Chem.

[0286] [Chemical formula] 6-(2-Fluorophenyl)-3-methylimidazo[1,2-b]pyridazine, JGJ018. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazolo[1,2-b]pyridazine (27.5 mg, 0.164 mmol), 2-fluorophenylboronic acid (25.3 mg, 0.181 mmol), K2CO3 (34.0 mg, 0.246 mmol), and Pd(PPh3)4 (19.0 mg, 0.016 mmol) in 1,4-dioxane / water (5:1 v / v, 0.5 mL) were reacted to obtain the desired product JGJ018 (18.1 mg, 0.080 mmol, 49%) as an off-white solid. 1 1H NMR (400 MHz, CDCl3) 7.96 (d, J = 9.6 Hz, 1H), 7.91 (ddd, J = 8.0, 7.6, 2.0 Hz, 1H), 7.60 (s, 1H), 7.43-7.49 (m, 2H), 7.30 (ddd, J = 8.0, 7.6, 1.2 Hz, 1H), 7.21 (ddd, J = 11.2, 8.4, 0.8 Hz, 1H), 2.61 (d, J = 0.8 Hz, 3H);13 13C NMR (100 MHz, CDCl3) δ 160.4 (d, J = 249.3 Hz), 148.2, 137.9, 132.2, 131.4 (d, J = 8.5 Hz), 130.7 (d, J = 2.6 Hz), 125.3, 124.7, 124.6 (d, J = 3.6 Hz), 124.3 (d, J = 11.7 Hz), 117.5 (d, J = 7.9 Hz), 116.4 (d, J = 22.2 Hz), 8.7.

[0287]

Chem.

[0288]

Chem.

[0289]

Chemical Structure

[0290]

Chem.

[0291]

Chemical Structure

[0292]

Chem.

[0293]

Chemical Structure

[0294]

Chemical Structure

[0295]

Chemical Structure

[0296]

Chemical Structure

[0297]

Chemical Structure

[0298]

Chemical Structure

[0299]

Chemical Structure

[0300]

Chem.

[0301]

Chem.

[0302]

Chem.

[0303]

Chem.

[0304]

Chem.

[0305]

Chemical Structure

[0306]

Chemical formula

[0307]

Chemical Structure

[0308]

Chemical Structure

[0309]

Chemical Structure

[0310]

Chem.

[0311]

Chemical Structure

[0312]

Chem.

[0313]

Chemical formula

[0314] [Chemical formula] N-(3-Fluoro-5-(3-phenyl-1H-pyrrolo[3,2-b]pyridin-5-yl)phenyl)acetamide, JGJ037. Using the same procedure as described for JGJ002, 3-fluoro-5-(3-phenyl-1H-pyrrolo[3,2-b]pyridin-5-yl)aniline (18.1 mg, 0.060 mmol, 70%) was obtained as an off-white solid by the reaction of 5-chloro-3-phenyl-1H-pyrrolo[3,2-b]pyridine (19.4 mg, 0.085 mmol), 3-fluoro-5-aminophenylboronic acid (14.5 mg, 0.093 mmol), K2CO3 (17.6 mg, 0.127 mmol), and Pd(PPh3)4 (9.8 mg, 0.009 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL). Then, using the same procedure as described for JGJ004, the desired product JGJ037 (13.8 mg, 0.040 mmol, 67%) was obtained as an off-white solid by acetylation. 11H NMR (400 MHz, CD3OD) δ 8.25 (m, 2H), 7.98 (t, J = 1.6 Hz, 1H), 7.88 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.58 (m, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.21 (td, J = 7.6, 1.2 Hz, 1H), 2.15 (s, 3H); 13 13C NMR (100 MHz, CD3OD) δ 171.9, 164.6 (d, J = 239.6 Hz), 150.2 (d, J = 2.9 Hz), 145.1, 144.7 (d, J = 8.9 Hz), 141.7 (d, J = 11.5 Hz), 136.0, 130.9, 129.4, 127.8, 127.6, 126.6, 120.5, 117.3, 115.3, 114.7 (d, J = 3.2 Hz), 109.8 (d, J = 23.1 Hz), 107.3 (d, J = 27.0 Hz), 24.0.

[0315]

Chem.

[0316]

Chemical Structure

[0317]

Chem.

[0318] References 1. Dohner H, Weisdorf DJ, Bloomfield CD. Acute Myeloid Leukemia. The New England journal of medicine. 2015;373:1136-52. 2. Jordan CT. Unique molecular and cellular features of acute myelogenous leukemia stem cells. Leukemia. 2002;16:559-62. 3. Dick JE. Acute myeloid leukemia stem cells. Annals of the New York Academy of Sciences. 2005;1044:1-5. 4. Rosen JM, Jordan CT. The increasing complexity of the cancer stem cell paradigm. Science (New York, NY). 2009;324:1670-3. 5. Ambros V. microRNAs: tiny regulators with great potential. Cell. 2001;107:823-6. 6. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281-97. 7. Ambros V. The functions of animal microRNAs. Nature. 2004;431:350-5. 8. Calin GA, Croce CM. MicroRNA signatures in human cancers. Nature reviews Cancer. 2006;6:857-66. 9. Calin GA, Croce CM. MicroRNA-cancer connection: the beginning of a new tale. Cancer research. 2006;66:7390-4. 10. Jongen-Lavrencic M, Sun SM, Dijkstra MK, Valk PJM, Lowenberg B. MicroRNA expression profiling in relation to the genetic heterogeneity of acute myeloid leukemia. Blood. 2008;111:5078-85. 11. Garzon R, Volinia S, Liu CG, Fernandez-Cymering C, Palumbo T, Pichiorri F, Fabbri M, Coombes K, Alder H, Nakamura T, Flomenberg N, Marcucci G, Calin GA, Kornblau SM, Kantarjian H, Bloomfield CD, Andreeff M, Croce CM. MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia. Blood. 2008;111(6):3183-9. Epub 2008 / 01 / 12. doi: 10.1182 / blood-2007-07-098749. PubMed PMID: 18187662;PMCID: PMC2265455. 12. Chen L, Sun Y, Wang J, Jiang H, Muntean AG. Differential regulation of the c-Myc / Lin28 axis discriminates subclasses of rearranged MLL leukemia. Oncotarget. 2016;7(18):25208-23. Epub 2016 / 03 / 24. doi: 10.18632 / oncotarget.8199. PubMed PMID: 27007052;PMCID: PMC5041898. 13. Nair VS, Maeda LS, Ioannidis JPA. Clinical outcome prediction by microRNAs in human cancer: a systematic review. Journal of the National Cancer Institute. 2012;104:528-40. 14. Dixon-McIver A, East P, Mein CA, Cazier J-B, Molloy G, Chaplin T, Andrew Lister T, Young BD, Debernardi S. Distinctive patterns of microRNA expression associated with karyotype in acute myeloid leukaemia. PloS one. 2008;3:e2141. 15. Johnson CD, Esquela-Kerscher A, Stefani G, Byrom M, Kelnar K, Ovcharenko D, Wilson M, Wang X, Shelton J, Shingara J, Chin L, Brown D, Slack FJ. The let-7 microRNA represses cell proliferation pathways in human cells. Cancer Res. 2007;67(16):7713-22. Epub 2007 / 08 / 19. doi: 10.1158 / 0008-5472.CAN-07-1083. PubMed PMID: 17699775. 16. Roush S, Slack FJ. The let-7 family of microRNAs. Trends in cell biology. 2008;18:505-16. 17. Viswanathan SR, Daley GQ, Gregory RI. Selective blockade of microRNA processing by Lin28. Science (New York, NY). 2008;320:97-100. 18. Newman MA, Thomson JM, Hammond SM. Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing. RNA (New York, NY). 2008;14:1539-49. 19. Heo I, Joo C, Cho J, Ha M, Han J, Kim VN. Lin28 mediates the terminal uridylation of let-7 precursor MicroRNA. Molecular cell. 2008;32:276-84. 20. Piskounova E, Viswanathan SR, Janas M, LaPierre RJ, Daley GQ, Sliz P, Gregory RI. Determinants of microRNA processing inhibition by the developmentally regulated RNA-binding protein Lin28. The Journal of biological chemistry. 2008;283:21310-4. 21. Rybak A, Fuchs H, Smirnova L, Brandt C, Pohl EE, Nitsch R, Wulczyn FG. A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment. Nature cell biology. 2008;10:987-93. 22. Viswanathan SR, Powers JT, Einhorn W, Hoshida Y, Ng TL, Toffanin S, O'Sullivan M, Lu J, Phillips LA, Lockhart VL, Shah SP, Tanwar PS, Mermel CH, Beroukhim R, Azam M, Teixeira J, Meyerson M, Hughes TP, Llovet JM, Radich J, Mullighan CG, Golub TR, Sorensen PH, Daley GQ. Lin28 promotes transformation and is associated with advanced human malignancies. Nature genetics. 2009;41:843-8. 23. Iliopoulos D, Hirsch HA, Struhl K. An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation. Cell. 2009;139:693-706. 24. Albino D, Civenni G, Dallavalle C, Roos M, Jahns H, Curti L, Rossi S, Pinton S, D'Ambrosio G, Sessa F, Hall J, Catapano CV, Carbone GM. Activation of the Lin28 / let-7 Axis by Loss of ESE3 / EHF Promotes a Tumorigenic and Stem-like Phenotype in Prostate Cancer. Cancer research. 2016;76:3629-43. 25. King CE, Cuatrecasas M, Castells A, Sepulveda AR, Lee J-S, Rustgi AK. LIN28B promotes colon cancer progression and metastasis. Cancer research. 2011;71:4260-8. 26. Zhang WC, Shyh-Chang N, Yang H, Rai A, Umashankar S, Ma S, Soh BS, Sun LL, Tai BC, Nga ME, Bhakoo KK, Jayapal SR, Nichane M, Yu Q, Ahmed DA, Tan C, Sing WP, Tam J, Thirugananam A, Noghabi MS, Pang YH, Ang HS, Mitchell W, Robson P, Kaldis P, Soo RA, Swarup S, Lim EH, Lim B. Glycine decarboxylase activity drives non-small cell lung cancer tumor-initiating cells and tumorigenesis. Cell. 2012;148:259-72. 27. Kong D, Banerjee S, Ahmad A, Li Y, Wang Z, Sethi S, Sarkar FH. Epithelial to mesenchymal transition is mechanistically linked with stem cell signatures in prostate cancer cells. PloS one. 2010;5:e12445. 28. Nam Y, Chen C, Gregory RI, Chou JJ, Sliz P. Molecular basis for interaction of let-7 microRNAs with Lin28. Cell. 2011;147(5):1080-91. Epub 2011 / 11 / 15. doi: 10.1016 / j.cell.2011.10.020. PubMed PMID: 22078496;PMCID: PMC3277843. 29. Loughlin FE, Gebert LF, Towbin H, Brunschweiger A, Hall J, Allain FH. Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28. Nat Structmol Biol. 2011;19(1):84-9. Epub 2011 / 12 / 14. doi: 10.1038 / nsmb.2202. PubMed PMID: 22157959. 30. Wang L, Nam Y, Lee AK, Yu C, Roth K, Chen C, Ransey EM, Sliz P. LIN28 Zinc Knuckle Domain Is Required and Sufficient to Induce let-7 Oligouridylation. Cell Rep. 2017;18(11):2664-75. Epub 2017 / 03 / 16. doi: 10.1016 / j.celrep.2017.02.044. PubMed PMID: 28297670. 31. Hagan JP, Piskounova E, Gregory RI. Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells. Nat Structmol Biol. 2009;16(10):1021-5. Epub 2009 / 08 / 29. doi: 10.1038 / nsmb.1676. PubMed PMID: 19713958;PMCID: PMC2758923. 32. Yan BX, Ma JX, Zhang J, Guo Y, Riedel H, Mueller MD, Remick SC, Yu JJ. PSP94 contributes to chemoresistance and its peptide derivative PCK3145 represses tumor growth in ovarian cancer. Oncogene. 2014;33(45):5288-94. Epub 2013 / 11 / 05. doi: 10.1038 / onc.2013.466. PubMed PMID: 24186202. 33. Wang T, Han P, He Y, Zhao C, Wang G, Yang W, Shan M, Zhu Y, Yang C, Weng M, Wu D, Gao L, Jin X, Wei Y, Cui B, Shen G, Li X. Lin28A enhances chemosensitivity of colon cancer cells to 5-FU by promoting apoptosis in a let-7 independent manner. Tumour Biol. 2016;37(6):7657-65. Epub 2015 / 12 / 22. doi: 10.1007 / s13277-015-4559-8. PubMed PMID: 26687759. 34. Teng R, Hu Y, Zhou J, Seifer B, Chen Y, Shen J, Wang L. Overexpression of Lin28 Decreases the Chemosensitivity of Gastric Cancer Cells to Oxaliplatin, Paclitaxel, Doxorubicin, and Fluorouracil in Part via microRNA-107. PLoS One. 2015;10(12):e0143716. Epub 2015 / 12 / 05. doi: 10.1371 / journal.pone.0143716. PubMed PMID: 26636340;PMCID: PMC4670127. 35. Chaudhry MA, Sachdeva H, Omaruddin RA. Radiation-induced micro-RNA modulation in glioblastoma cells differing in DNA-repair pathways. DNA Cell Biol. 2010;29(9):553-61. Epub 2010 / 04 / 13. doi: 10.1089 / dna.2009.0978. PubMed PMID: 20380575. 36. Yang X, Cai H, Liang Y, Chen L, Wang X, Si R, Qu K, Jiang Z, Ma B, Miao C, Li J, Wang B, Gao P. Inhibition of c-Myc by let-7b mimic reverses mutidrug resistance in gastric cancer cells. Oncol Rep. 2015;33(4):1723-30. Epub 2015 / 01 / 31. doi: 10.3892 / or.2015.3757. PubMed PMID: 25633261. 37. Boyerinas B, Park SM, Murmann AE, Gwin K, Montag AG, Zillhardt M, Hua YJ, Lengyel E, Peter ME. Let-7 modulates acquired resistance of ovarian cancer to Taxanes via IMP-1-mediated stabilization of multidrug resistance 1. Int J Cancer. 2012;130(8):1787-97. Epub 2011 / 05 / 28. doi: 10.1002 / ijc.26190. PubMed PMID: 21618519;PMCID: PMC3230767. 38. Zhou J, Chan Z-L, Bi C, Lu X, Chong PSY, Chooi J-Y, Cheong L-L, Liu S-C, Ching YQ, Zhou Y, Osato M, Tan TZ, Ng CH, Ng S-B, Wang S, Zeng Q, Chng W-J. LIN28B Activation by PRL-3 Promotes Leukemogenesis and a Stem Cell-like Transcriptional Program in AML. Molecular cancer research : MCR. 2017;15:294-303. 39. Chen Y, Jacamo R, Konopleva M, Garzon R, Croce C, Andreeff M. CXCR4 downregulation of let-7a drives chemoresistance in acute myeloid leukemia. J Clin Invest. 2013;123(6):2395-407. Epub 2013 / 05 / 17. doi: 10.1172 / JCI66553. PubMed PMID: 23676502;PMCID: PMC3668829. 40. Campos M, Llorens C, Sempere JM, Futami R, Rodriguez I, Carrasco P, Capilla R, Latorre A, Coque TM, Moya A, Baquero F. A membrane computing simulator of trans-hierarchical antibiotic resistance evolution dynamics in nested ecological compartments (ARES). Biol Direct. 2015;10:41. Epub 2015 / 08 / 06. doi: 10.1186 / s13062-015-0070-9. PubMed PMID: 26243297;PMCID: PMC4526193. 41. Mehta SV, Shukla SN, Vora HH. Overexpression of Bcl2 protein predicts chemoresistance in acute myeloid leukemia: its correlation with FLT3. Neoplasma. 2013;60(6):666-75. Epub 2013 / 08 / 03. doi: 10.4149 / neo_2013_085. PubMed PMID: 23906301. 42. Fennell DA, Corbo MV, Dean NM, Monia BP, Cotter FE. In vivo suppression of Bcl-XL expression facilitates chemotherapy-induced leukaemia cell death in a SCID / NOD-Hu model. Br J Haematol. 2001;112(3):706-13. Epub 2001 / 03 / 22. PubMed PMID: 11260076. 43. Lagadinou ED, Sach A, Callahan K, Rossi RM, Neering SJ, Minhajuddin M, Ashton JM, Pei S, Grose V, O'Dwyer KM, Liesveld JL, Brookes PS, Becker MW, Jordan CT. BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells. Cell Stem Cell. 2013;12(3):329-41. Epub 2013 / 01 / 22. doi: 10.1016 / j.stem.2012.12.013. PubMed PMID: 23333149;PMCID: PMC3595363. 44. Agarwal V, Bell GW, Nam JW, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. Elife. 2015;4. Epub 2015 / 08 / 13. doi: 10.7554 / eLife.05005. PubMed PMID: 26267216;PMCID: PMC4532895. 45. Guzman ML, Neering SJ, Upchurch D, Grimes B, Howard DS, Rizzieri DA, Luger SM, Jordan CT. Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells. Blood. 2001;98(8):2301-7. Epub 2001 / 10 / 06. PubMed PMID: 11588023. 46. Kagoya Y, Yoshimi A, Kataoka K, Nakagawa M, Kumano K, Arai S, Kobayashi H, Saito T, Iwakura Y, Kurokawa M. Positive feedback between NF-kappaB and TNF-alpha promotes leukemia-initiating cell capacity. J Clin Invest. 2014;124(2):528-42. Epub 2014 / 01 / 03. doi: 10.1172 / JCI68101. PubMed PMID: 24382349;PMCID: PMC3904603. 47. Shinoda G, Shyh-Chang N, Soysa TYd, Zhu H, Seligson MT, Shah SP, Abo-Sido N, Yabuuchi A, Hagan JP, Gregory RI, Asara JM, Cantley LC, Moss EG, Daley GQ. Fetal deficiency of lin28 programs life-long aberrations in growth and glucose metabolism. Stem cells (Dayton, Ohio). 2013;31:1563-73. 48. Yuan J, Nguyen CK, Liu X, Kanellopoulou C, Muljo SA. Lin28b reprograms adult bone marrow hematopoietic progenitors to mediate fetal-like lymphopoiesis. Science. 2012;335(6073):1195-200. Epub 2012 / 02 / 22. doi: 10.1126 / science.1216557. PubMed PMID: 22345399;PMCID: PMC3471381. 49. Copley MR, Babovic S, Benz C, Knapp DJHF, Beer PA, Kent DG, Wohrer S, Treloar DQ, Day C, Rowe K, Mader H, Kuchenbauer F, Humphries RK, Eaves CJ. The Lin28b-let-7-Hmga2 axis determines the higher self-renewal potential of fetal haematopoietic stem cells. Nature cell biology. 2013;15:916-25. 50. Rowe RG, Wang LD, Coma S, Han A, Mathieu R, Pearson DS, Ross S, Sousa P, Nguyen PT, Rodriguez A, Wagers AJ, Daley GQ. Developmental regulation of myeloerythroid progenitor function by the Lin28b-let-7-Hmga2 axis. J Exp Med. 2016;213(8):1497-512. Epub 2016 / 07 / 13. doi: 10.1084 / jem.20151912. PubMed PMID: 27401346;PMCID: PMC4986532. 51. Roos M, Pradere U, Ngondo RP, Behera A, Allegrini S, Civenni G, Zagalak JA, Marchand J-R, Menzi M, Towbin H, Scheuermann J, Neri D, Caflisch A, Catapano CV, Ciaudo C, Hall J. A Small-Molecule Inhibitor of Lin28. ACS chemical biology. 2016;11:2773-81. 52. Lim D, Byun WG, Koo JY, Park H, Park SB. Discovery of a Small-Molecule Inhibitor of Protein-MicroRNA Interaction Using Binding Assay with a Site-Specifically Labeled Lin28. J Am Chem Soc. 2016. Epub 2016 / 09 / 27. doi: 10.1021 / jacs.6b06965. PubMed PMID: 27668966. 53. Wang L, Rowe RG, Jaimes A, Yu C, Nam Y, Pearson DS, Zhang J, Xie X, Marion W, Heffron GJ, Daley GQ, Sliz P. Small-Molecule Inhibitors Disrupt let-7 Oligouridylation and Release the Selective Blockade of let-7 Processing by LIN28. Cell Rep. 2018;23(10):3091-101. Epub 2018 / 06 / 07. doi: 10.1016 / j.celrep.2018.04.116. PubMed PMID: 29874593. 54. Balzeau J, Menezes MR, Cao S, Hagan JP. The LIN28 / let-7 Pathway in Cancer. Front Genet. 2017;8:31. Epub 2017 / 04 / 13. doi: 10.3389 / fgene.2017.00031. PubMed PMID: 28400788;PMCID: PMC5368188. 55. Bagger FO, Kinalis S, Rapin N. BloodSpot: a database of healthy and malignant haematopoiesis updated with purified and single cell mRNA sequencing profiles. Nucleic Acids Res. 2019;47(D1):D881-D5. Epub 2018 / 11 / 06. doi: 10.1093 / nar / gky1076. PubMed PMID: 30395307;PMCID: PMC6323996. 56. Jiang X, Huang H, Li Z, Li Y, Wang X, Gurbuxani S, Chen P, He C, You D, Zhang S, Wang J, Arnovitz S, Elkahloun A, Price C, Hong G-M, Ren H, Kunjamma RB, Neilly MB, Matthews JM, Xu M, Larson RA, Le Beau MM, Slany RK, Liu PP, Lu J, Zhang J, He C, Chen J. Blockade of miR-150 maturation by MLL-fusion / MYC / LIN-28 is required for MLL-associated leukemia. Cancer cell. 2012;22:524-35. 57. Stavropoulou V, Kaspar S, Brault L, Sanders MA, Juge S, Morettini S, Tzankov A, Iacovino M, Lau I-J, Milne TA, Royo H, Kyba M, Valk PJM, Peters AHFM, Schwaller J. MLL-AF9 Expression in Hematopoietic Stem Cells Drives a Highly Invasive AML Expressing EMT-Related Genes Linked to Poor Outcome. Cancer cell. 2016;30:43-58. 58. Pang M, Wu G, Hou X, Hou N, Liang L, Jia G, Shuai P, Luo B, Wang K, Li G. LIN28B promotes colon cancer migration and recurrence. PLoS One. 2014;9(10):e109169. Epub 2014 / 11 / 02. doi: 10.1371 / journal.pone.0109169. PubMed PMID: 25360631;PMCID: PMC4215835. 59. Dai Y, Guzman ML, Chen S, Wang L, Yeung SK, Pei XY, Dent P, Jordan CT, Grant S. The NF (Nuclear factor)-kappaB inhibitor parthenolide interacts with histone deacetylase inhibitors to induce MKK7 / JNK1-dependent apoptosis in human acute myeloid leukaemia cells. Br J Haematol. 2010;151(1):70-83. Epub 2010 / 08 / 13. doi: 10.1111 / j.1365-2141.2010.08319.x. PubMed PMID: 20701602;PMCID: PMC2950247. 60. Guzman ML, Rossi RM, Karnischky L, Li X, Peterson DR, Howard DS, Jordan CT. The sesquiterpene lactone parthenolide induces apoptosis of human acute myelogenous leukemia stem and progenitor cells. Blood. 2005;105(11):4163-9. Epub 2005 / 02 / 03. doi: 10.1182 / blood-2004-10-4135. PubMed PMID: 15687234;PMCID: PMC1895029. 61. Gal H, Amariglio N, Trakhtenbrot L, Jacob-Hirsh J, Margalit O, Avigdor A, Nagler A, Tavor S, Ein-Dor L, Lapidot T, Domany E, Rechavi G, Givol D. Gene expression profiles of AML derived stem cells;similarity to hematopoietic stem cells. Leukemia. 2006;20(12):2147-54. Epub 2006 / 10 / 14. doi: 10.1038 / sj.leu.2404401. PubMed PMID: 17039238. 62. Yagi T, Morimoto A, Eguchi M, Hibi S, Sako M, Ishii E, Mizutani S, Imashuku S, Ohki M, Ichikawa H. Identification of a gene expression signature associated with pediatric AML prognosis. Blood. 2003;102(5):1849-56. Epub 2003 / 05 / 10. doi: 10.1182 / blood-2003-02-0578. PubMed PMID: 12738660. 63. Santos, R. et al. A comprehensive map of molecular drug targets. Nat Rev Drug Discov 16, 19-34, doi:10.1038 / nrd.2016.230 (2017). 64. Barouch-Bentov, R. & Sauer, K. Mechanisms of drug resistance in kinases. Expert Opin Investig Drugs 20, 153-208, doi:10.1517 / 13543784.2011.546344 (2011). 65. Warner, K. D., Hajdin, C. E. & Weeks, K. M. Principles for targeting RNA with drug-like small molecules. Nat Rev Drug Discov 17, 547-558, doi:10.1038 / nrd.2018.93 (2018). 66. Connelly, C. M., Moon, M. H. & Schneekloth, J. S., Jr. The Emerging Role of RNA as a Therapeutic Target for Small Molecules. Cell Chem Biol 23, 1077-1090, doi:10.1016 / j.chembiol.2016.05.021 (2016). 67. Hentze, M. W., Castello, A., Schwarzl, T. & Preiss, T. A brave new world of RNA-binding proteins. Nat Rev Mol Cell Biol 19, 327-341, doi:10.1038 / nrm.2017.130 (2018). 68. Elcheva, I. A. & Spiegelman, V. S. Targeting RNA-binding proteins in acute and chronic leukemia. Leukemia, doi:10.1038 / s41375-020-01066-4 (2020). 69. Polesskaya, A. et al. Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency. Genes Dev 21, 1125-1138, doi:10.1101 / gad.415007 (2007). 70. Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129-141, doi:10.1016 / j.cell.2010.03.009 (2010). 71. Liberzon, A. et al. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst 1, 417-425, doi:10.1016 / j.cels.2015.12.004 (2015). 72. Keskin, T. et al. LIN28B Underlies the Pathogenesis of a Subclass of Ewing Sarcoma. Cell Rep 31, 107539, doi:10.1016 / j.celrep.2020.107539 (2020). 73. Chen, C. et al. Targeting LIN28B reprograms tumor glucose metabolism and acidic microenvironment to suppress cancer stemness and metastasis. Oncogene 38, 4527-4539, doi:10.1038 / s41388-019-0735-4 (2019). 74. Zuber, J. et al. Mouse models of human AML accurately predict chemotherapy response. Genes Dev 23, 877-889, doi:10.1101 / gad.1771409 (2009). 75. Huber, K. V. M. et al. Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling. Nature methods 12, 1055-1057 (2015). 76. Kwon, S. C. et al. The RNA-binding protein repertoire of embryonic stem cells. Nat Struct Mol Biol 20, 1122-1130, doi:10.1038 / nsmb.2638 (2013). 77. Liu, Z., Scannell, D. R., Eisen, M. B. & Tjian, R. Control of embryonic stem cell lineage commitment by core promoter factor, TAF3. Cell 146, 720-731, doi:10.1016 / j.cell.2011.08.005 (2011). 78. Liu, S. et al. Binding of the human Prp31 Nop domain to a composite RNA-protein platform in U4 snRNP. Science 316, 115-120, doi:10.1126 / science.1137924 (2007). 79. Adler, A. S. et al. An integrative analysis of colon cancer identifies an essential function for PRPF6 in tumor growth. Genes Dev 28, 1068-1084, doi:10.1101 / gad.237206.113 (2014). 80. Chan, S. et al. Basal-A Triple-Negative Breast Cancer Cells Selectively Rely on RNA Splicing for Survival. Mol Cancer Ther 16, 2849-2861, doi:10.1158 / 1535-7163.MCT-17-0461 (2017). 81. Dvinge, H., Guenthoer, J., Porter, P. L. & Bradley, R. K. RNA components of the spliceosome regulate tissue- and cancer-specific alternative splicing. Genome Res 29, 1591-1604, doi:10.1101 / gr.246678.118 (2019). 82. Park, S. et al. PRPF4 is a novel therapeutic target for the treatment of breast cancer by influencing growth, migration, invasion, and apoptosis of breast cancer cells via p38 MAPK signaling pathway. Mol Cell Probes 47, 101440, doi:10.1016 / j.mcp.2019.101440 (2019). 83. Song, H. et al. Splicing factor PRPF6 upregulates oncogenic androgen receptor signaling pathway in hepatocellular carcinoma. Cancer Sci, doi:10.1111 / cas.14595 (2020). 84. Wang, E. T. et al. Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470-476, doi:10.1038 / nature07509 (2008). 85. Islam, S. U., Shehzad, A., Sonn, J. K. & Lee, Y. S. PRPF overexpression induces drug resistance through actin cytoskeleton rearrangement and epithelial-mesenchymal transition. Oncotarget 8, 56659-56671, doi:10.18632 / oncotarget.17855 (2017). 86. Wang, E. & Aifantis, I. RNA Splicing and Cancer. Trends Cancer 6, 631-644, doi:10.1016 / j.trecan.2020.04.011 (2020). 87. Berg, K. C. G. et al. Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies. Mol Cancer 16, 116, doi:10.1186 / s12943-017-0691-y (2017). 88. Augenlicht, L. H. et al. Low-level c-myc amplification in human colonic carcinoma cell lines and tumors: a frequent, p53-independent mutation associated with improved outcome in a randomized multi-institutional trial. Cancer Res 57, 1769-1775 (1997). 89. Cinkornpumin, J. et al. A small molecule screen to identify regulators of let-7 targets.Sci Rep 7, 15973, doi:10.1038 / s41598-017-16258-9 (2017)

[0319] Incorporation by reference All publications and patents mentioned in this specification are hereby incorporated by reference in their entirety as if each individual publication and patent were specifically and individually indicated to be incorporated by reference. In case of conflict, this specification, including any definitions herein, shall prevail.

[0320] Equivalents Specific embodiments of the present invention have been considered, but the above specification is illustrative and not restrictive. Upon reviewing this specification and the appended claims, many variations of the present invention will become apparent to those skilled in the art. The full scope of the present invention should be determined by reference to the claims, along with the full scope of their equivalents, as well as this specification in light of such variations. The following is one of the embodiments of the present invention. (1) A compound of formula (I),

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Claims

【Claim 1】 【Fig. 1】 A compound selected from, or a pharmaceutically acceptable salt thereof.

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