WO2021071982A1 - Composés de modulation d'épissage - Google Patents

Composés de modulation d'épissage Download PDF

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Publication number
WO2021071982A1
WO2021071982A1 PCT/US2020/054630 US2020054630W WO2021071982A1 WO 2021071982 A1 WO2021071982 A1 WO 2021071982A1 US 2020054630 W US2020054630 W US 2020054630W WO 2021071982 A1 WO2021071982 A1 WO 2021071982A1
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WIPO (PCT)
Prior art keywords
substituted
unsubstituted
splicing
compound
mrna
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PCT/US2020/054630
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English (en)
Inventor
Michael Luzzio
Brian Lucas
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Skyhawk Therapeutics, Inc.
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Publication of WO2021071982A1 publication Critical patent/WO2021071982A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D451/00—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof
    • C07D451/02—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof
    • C07D451/04—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof with hetero atoms directly attached in position 3 of the 8-azabicyclo [3.2.1] octane or in position 7 of the 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system

Definitions

  • RNA expression results in a single precursor messenger RNA (pre–mRNA).
  • pre–mRNA messenger RNA
  • splicing a process in which results in the mature messenger RNA (mRNA).
  • mRNA messenger RNA
  • alternative splicing gives rise to multiple mRNAs encoding distinct protein isoforms.
  • the spliceosome an intracellular complex of multiple proteins and ribonucleoproteins, catalyzes splicing.
  • Current therapeutic approaches to direct and control mRNA expression require methods such as gene therapy, genome editing, or a wide range of oligonucleotide technologies (antisense, RNAi, etc.).
  • Oligonucleotides modulate the action of RNA via canonical base/base hybridization.
  • the appeal of this approach is in the design of the basic pharmacophore of an oligonucleotide, which can be defined in a straightforward fashion by known base pairing to the target sequence subject.
  • Each of these therapeutic modalities suffers from substantial technical, clinical, and regulatory challenges.
  • oligonucleotides as therapeutics include unfavorable pharmacokinetics, lack of oral bioavailability, and lack of blood–brain–barrier penetration, with the latter precluding delivery to the brain or spinal cord after parenteral drug administration for the treatment of diseases (e.g., neurological diseases, brain cancers).
  • diseases e.g., neurological diseases, brain cancers.
  • oligonucleotides are not taken up effectively into solid tumors without a complex delivery system such as lipid nanoparticles.
  • oligonucleotide therapies require access to complementary base pairs of the target. This approach assumes that pre–mRNA sequences exist as a linear strand of RNA in the cell. However, pre–mRNA is rarely linear; it has complex secondary and tertiary structure.
  • cis–acting elements e.g., protein binding elements
  • trans–acting factors e.g., splicing complex components
  • SMSMs small molecule splicing modulators
  • RNA transcriptome with small-molecule modulators represents an untapped therapeutic approach to treat a variety of RNA-mediated diseases. Accordingly, there remains a need to develop small-molecule RNA modulators useful as therapeutic agents. There is need in the art for novel modulators of splicing or splicing-dependent processes.
  • each of R A1 and R A2 is independently hydrogen, halogen, -CN, -OR 5 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 haloalkyl, or substituted or unsubstituted C 1 -C 6 heteroalkyl;
  • ring Q is substituted monocyclic aryl or substituted monocyclic heteroaryl; is a single bond or a double bond;
  • X is –NR 21 -, and Z is CR 8 ; or X is and Z is C; or X is , and Z is N o 8 r CR ;
  • R 1 is hydrogen, substituted C 1 -C 6 alkyl, substituted or unsubstituted C 1
  • each of R 15 and R 20 is independently hydrogen or –CH 3
  • W is -CH 2 CH 2 -
  • X is –N(cyclopropyl)-
  • R 15 and R 20 are –CH 3
  • at least one of R, R 8 , R 16 , R 17 , R 18 , and R 19 is not hydrogen or deuterium.
  • each of R 15 and R 20 is independently hydrogen or –CH 3
  • W is -CH 2 CH 2 -
  • X is –N(cyclopropyl)-
  • R 15 and R 20 are –CH 3
  • at least one of R, R 16 , R 17 , R 18 , and R 19 is not hydrogen or deuterium.
  • R 18 and R 19 is F.
  • R 16 and R 17 is F.
  • W is C 1 -C 2 alkylene substituted with one or more F.
  • each of R A1 and R A2 is independently hydrogen, halogen, -CN, -OR 5 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 haloalkyl, or substituted or unsubstituted C 1 -C 6 heteroalkyl;
  • ring Q is substituted monocyclic aryl or substituted monocyclic heteroaryl; is a single bond or a double bond;
  • X is –NR 21 -, and Z is CR 8 ; or X is and Z is C; or X is 8 and Z is N or CR ;
  • R 1 is hydrogen, substituted C 1 -C 6 alky
  • a pharmaceutical composition comprising any of the described compound, or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutically acceptable excipient.
  • the pharmaceutical composition is formulated for intravenous administration, subcutaneous administration, oral administration, inhalation, nasal administration, dermal administration, or ophthalmic administration.
  • the pharmaceutical composition is in a form of a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spray solution, a suppository, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, an ointment, a lotion, an eye drop or an ear drop.
  • the pharmaceutical composition further comprises a second therapeutic agent.
  • a compound, or a pharmaceutically acceptable salt or solvate thereof for use in the treatment, prevention and/or delay of progression of a disease.
  • described herein is a use of a described compound, or a pharmaceutically acceptable salt or solvate thereof, for the preparation of a medicament for the treatment, prevention and/or delay of progression of a disease.
  • described herein is a use of a described compound, or a pharmaceutically acceptable salt or solvate thereof, for the treatment, prevention and/or delay of progression of a disease.
  • described herein is a method for the treatment, prevention and/or delay of progression of a disease comprising administering an effective amount of a described compound, or a pharmaceutically acceptable salt or solvate thereof, to a subject in need thereof.
  • a pharmaceutical composition comprising a described compound, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment, prevention and/or delay of progression of a disease.
  • described herein is a combination comprising a therapeutically effective amount of a described compound, or a pharmaceutically acceptable salt or solvate thereof, and one or more therapeutically active co-agents.
  • described herein is a method of treating a disease or condition comprising administering a described compound, or a pharmaceutically acceptable salt or solvate thereof, to a subject in need thereof.
  • described herein is a method of modulating splicing comprising contacting a described compound, or a pharmaceutically acceptable salt or solvate thereof, to a pre-mRNA.
  • the compound binds to the pre-mRNA and modulates splicing of the pre-mRNA in a cell of a subject.
  • the modulating comprises promoting exon skipping of the pre-mRNA.
  • the modulating alters a ratio of a first splice variant of the pre-mRNA to a second splice variant of the pre- mRNA.
  • the first splice variant is an mRNA encoding a full length protein and the second splice variant is an mRNA encoding a truncated protein.
  • the modulating increases the ratio of the mRNA encoding the truncated protein to the mRNA encoding the full length protein.
  • the modulating decreases the ratio of the mRNA encoding the full length protein to the mRNA encoding the truncated protein.
  • the ratio of the mRNA encoding the truncated protein to the mRNA encoding the full length protein is altered in at least 20%, at least 50%, at least 75%, or at least 90% of the cell.
  • the compound modulates affinity between the pre-mRNA and a splicing complex component.
  • the splicing complex component comprises a snRNA.
  • the snRNA comprises U1 snRNA, U2 snRNA, U4 snRNA, U5 snRNA, U6 snRNA, U11 snRNA, U12 snRNA, U4atac snRNA, U5 snRNA, U6 atac snRNA, or any combination thereof.
  • the snRNA comprises U1 snRNA.
  • the splicing complex component comprises 9G8, A1 hnRNP, A2 hnRNP, ASD-1, ASD-2b, ASF, B1 hnRNP, C1 hnRNP, C2 hnRNP, CBP20, CBP80, CELF, F hnRNP, FBP11, Fox-1, Fox-2, G hnRNP, H hnRNP, hnRNP 1, hnRNP 3, hnRNP C, hnRNP G, hnRNP K, hnRNP M, hnRNP U, Hu, HUR, I hnRNP, K hnRNP, KH-type splicing regulatory protein (KSRP), L hnRNP, M hnRNP, mBBP, muscle-blind like (MBNL), NF45, NFAR, Nova-1, Nova-2, nPTB, P54/SFRS11, polypyrimidine tract binding protein (PTB),
  • the compound binds to a splicing complex. In some embodiments, the compound modulates binding affinity of the splicing complex to the pre-mRNA. In some embodiments, the compound modulates binding affinity of the splicing complex to the pre-mRNA at a splice site sequence. In some embodiments, the compound modulates binding affinity of the splicing complex to the pre-mRNA upstream of a splice site sequence or downstream of a splice site sequence. In some embodiments, the compound interacts with an unpaired bulged nucleobase of an RNA duplex, and the RNA duplex comprises a splice site sequence.
  • the splice site sequence comprises at least one bulged nucleotide or a mutant nucleotide at a -3, - 2, -1, +1, +2, +3, +4, +5 or +6 position of the splice site sequence.
  • the compound modulates a resonance time of the splicing complex with the pre-mRNA. In some embodiments, the compound modulates the resonance time of the splicing complex with the pre- mRNA at the splice site sequence. In some embodiments, the compound modulates the resonance time of the splicing complex with the pre-mRNA upstream of the splice site sequence or downstream of the splice site sequence.
  • the compound modulates steric hindrance between the splicing complex and the pre-mRNA. In some embodiments, the compound modulates steric hindrance between the splicing complex and the pre-mRNA at a splice site sequence. In some embodiments, the compound modulates steric hindrance between the splicing complex and the pre-mRNA upstream of a splice site sequence or downstream of a splice site sequence.
  • the splice site sequence is a 5’ splice site sequence, a 3’ splice site sequence, a branch point splice site sequence, an exonic splicing enhancer (ESE) sequence, an exonic splicing silencer (ESS) sequence, an intronic splicing enhancer (ISE) sequence, an intronic splicing silencer (ISS) sequence, a polypyrimidine tract sequence, a cryptic splice site sequence, or any combination thereof.
  • ESE exonic splicing enhancer
  • ESS exonic splicing silencer
  • ISE intronic splicing enhancer
  • ISS intronic splicing silencer
  • Described herein are compounds for use in modifying splicing of pre-mRNAs Described herein are compounds that modify splicing of pre-mRNAs for use in the treatment, prevention and/or delay of progression of diseases or conditions.
  • the compounds that modify splicing are small molecule splicing modulators (SMSMs).
  • each of R A1 and R A2 is independently hydrogen, halogen, -CN, -OR 5 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 haloalkyl, or substituted or unsubstituted C 1 -C 6 heteroalkyl;
  • ring Q is substituted monocyclic aryl or substituted monocyclic heteroaryl; is a single bond or a double bond;
  • X is –NR 21 -, and Z is CR 8 ; or X is and Z is C; or X is 8 and Z is N or CR ;
  • R 1 is hydrogen, substituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 heteroalkyl, substituted C 3 -C 8 cycloalkyl, or substituted or
  • each of R 15 and R 20 is independently hydrogen or –CH 3
  • W is -CH 2 CH 2 -
  • X is –N(cyclopropyl)-
  • R 15 and R 20 are –CH 3
  • at least one of R, R 8 , R 16 , R 17 , R 18 , and R 19 is not hydrogen or deuterium.
  • each of R 15 and R 20 is independently hydrogen or –CH 3
  • W is -CH 2 CH 2 -
  • X is –N(cyclopropyl)-
  • R 15 and R 20 are –CH 3
  • at least one of R, R 16 , R 17 , R 18 , and R 19 is not hydrogen or deuterium.
  • R 18 and R 19 is F.
  • R 16 and R 17 is F.
  • W is C 1 -C 2 alkylene substituted with one or more F.
  • each of R A1 and R A2 is independently hydrogen, halogen, -CN, -OR 5 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 haloalkyl, or substituted or unsubstituted C 1 -C 6 heteroalkyl;
  • ring Q is substituted monocyclic aryl or substituted monocyclic heteroaryl; is a single bond or a double bond;
  • X is –NR 21 -, and Z is CR 8 ; or X is and Z is C; or X is 8 and Z is N or CR ;
  • R 1 is hydrogen, substituted C 1 -C 6 alky
  • the compound of Formula (I) has the structure of Formula (Ia): [00024] In some embodiments, the compound of Formula (I) has the structure of Formula (Ib): [00025] In some embodiments, compound of Formula (I) has the structure of Formula (Ic): [00026] In some embodiments, R 22 is hydrogen, substituted or unsubstituted C 1 –C 4 alkyl, substituted or unsubstituted C 1 –C 4 haloalkyl, or substituted or unsubstituted C 1 –C 4 heteroalkyl. In some embodiments, R 22 is unsubstituted C 1 –C 4 alkyl.
  • R 22 is substituted C 1 –C 4 alkyl. In some embodiments, R 22 is C 1 –C 4 alkyl substituted with one or more hydroxyl and/or halogen. In some embodiments, R 22 is substituted or unsubstituted C 1 –C 4 haloalkyl. In some embodiments, R 22 is substituted or unsubstituted C 1 –C 4 heteroalkyl.
  • R 22 is hydrogen, -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 OH, -CH 2 CH 2 OH, - CH 2 NHCH 3 , -CH 2 N(CH 3 ) 2 , -CH 2 OCH 3 , -CH 2 F, -CHF 2 , or -CF 3 .
  • R 22 is hydrogen, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OCH 3 , -CH 2 F, -CHF 2 , or -CF 3 .
  • R 22 is hydrogen.
  • R 22 is deuterium. In some embodiments, R 22 is -CH 3 . In some embodiments, R 22 is -CD 3 . In some embodiments, R 22 is -CF 3 . [00027] In some embodiments, the compound of Formula (I) has the structure of Formula (Iaa): [00028] In some embodiments, the compound of Formula (I) has the structure of Formula (Ibb): [00029] In some embodiments, the compound of Formula (I) has the structure of Formula (Icc): [00030] In some embodiments, R 8 is substituted or unsubstituted C 1 -C 6 alkyl.
  • R 8 is substituted or unsubstituted C 1 -C 6 haloalkyl. In some embodiments, R 8 is substituted or unsubstituted C 1 -C 6 heteroalkyl. In some embodiments, R 8 is hydrogen, deuterium, -CH 3 , or -OCH 3 . In some embodiments, R 8 is hydrogen. In some embodiments, R 8 is deuterium. [00031] In some embodiments, X is –NR 21 -.
  • R 21 is hydrogen, substituted or unsubstituted C 1 –C 4 alkyl, substituted or unsubstituted C 1 –C 4 heteroalkyl, substituted or unsubstituted -C 1 -C 4 alkylene-C 3 –C 5 cycloalkyl, substituted or unsubstituted C 3 –C 5 cycloalkyl, or substituted or unsubstituted C 2 –C 3 heterocycloalkyl. In some embodiments, R 21 is substituted or unsubstituted C 1 –C 4 alkyl. In some embodiments, R 21 is substituted or unsubstituted C 1 –C 4 heteroalkyl.
  • R 21 is substituted or unsubstituted C 3 –C 5 cycloalkyl. In some embodiments, R 21 is substituted or unsubstituted C 2 –C 3 heterocycloalkyl. In some embodiments, R 21 is substituted or unsubstituted -C 1 -C 4 alkylene-C 3 –C 5 cycloalkyl. In some embodiments, R 21 is -CH 2 -(C 3 –C 5 cycloalkyl) or - CH 2 -CH 2 -(C 3 –C 5 cycloalkyl), wherein the cycloalkyl is substituted or unsubstituted.
  • R 21 is hydrogen, -CH 3 , -CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 F, -CHF 2 , -CF 3 , cyclopropyl, or oxetanyl. In some embodiments, R 21 is hydrogen, -CH 3 , -CH 2 CH 3 , -CH(CH 3 ) 2 , -CF 3 , cyclopropyl, or oxetanyl. In some embodiments, R 21 is hydrogen, -CH 3 , -CH(CH 3 ) 2 , cyclopropyl, or oxetanyl. In some embodiments, R 21 is hydrogen.
  • R 21 is - CH 3 or cyclopropyl. In some embodiments, R 21 is -CD 3 . In some embodiments, R 21 is -CH 3 . In some embodiments, R 21 is cyclopropyl. [00032] In some embodiments, X is [00033] In some embodiments, X is [00034] In some embodiments, each of R 22a and R 22b is independently hydrogen, substituted or unsubstituted C 1 –C 4 alkyl, substituted or unsubstituted C 1 –C 4 haloalkyl, or substituted or unsubstituted C 1 –C 4 heteroalkyl.
  • R 22a is substituted or unsubstituted C 1 – C 4 alkyl. In some embodiments, R 22a is substituted or unsubstituted C 1 –C 4 haloalkyl. In some embodiments, R 22a is substituted or unsubstituted C 1 –C 4 heteroalkyl. In some embodiments, R 22b is substituted or unsubstituted C 1 –C 4 alkyl. In some embodiments, R 22b is substituted or unsubstituted C 1 –C 4 haloalkyl. In some embodiments, R 22b is substituted or unsubstituted C 1 –C 4 heteroalkyl.
  • each of R 22a and R 22b is independently hydrogen, -CH 3 , - CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 OH, -CH 2 CH 2 OH, -CH 2 NHCH 3 , -CH 2 N(CH 3 ) 2 , - CH 2 OCH 3 , -CH 2 F, -CHF 2 , or -CF 3 .
  • each of R 22a and R 22b is independently hydrogen, -CH 3 , -CH 2 CH 3 , -CH 2 OH, -CH 2 OCH 3 , -CH 2 F, -CHF 2 , or -CF 3 .
  • each of R 22a and R 22b is hydrogen. In some embodiments, each of R 22a and R 22b is -CH 3 . In some embodiments, R 22a is -CH 3. In some embodiments, R 22a is -CD 3. In some embodiments, R 22a is hydrogen. In some embodiments, R 22a is deuterium. In some embodiments, R 22b is -CH 3. In some embodiments, R 22b is -CD 3. In some embodiments, R 22b is hydrogen. In some embodiments, R 22b is deuterium. [00035] In some embodiments, ring Q is substituted monocyclic aryl.
  • Q1 wherein each of R , R Q2 , and R Q3 is independently selected from hydrogen, deuterium, F, Cl, -CN, –OH, -CH 3 , - CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , –CF 3 , –OCH 3 , –OCF 3, -OCH 2 CH 3 , -CH 2 OCH 3 , - OCH 2 CH 2 CH 3 , and -OCH(CH 3 )2.
  • R Q1 is hydrogen, F, Cl, -CN, –OH, – OCH 3 , or -CH 3 .
  • R Q1 is hydrogen.
  • R Q1 is F. In some embodiments, R Q1 is CH 3 . In some embodiments, R Q1 is CD 3 . In some embodiments, R Q1 is deuterium. In some embodiments, R Q2 is hydrogen, F, Cl, -CN, –OH, –OCH 3 , or -CH 3 . In some embodiments, R Q2 is hydrogen. In some embodiments, R Q2 is F. In some embodiments, R Q2 is CH 3 . In some embodiments, R Q2 is CD 3 . In some embodiments, R Q2 is deuterium. In some embodiments, R Q3 is hydrogen, F, Cl, -CN, –OH, –OCH 3 , or -CH 3 .
  • R Q3 is hydrogen. In some embodiments, R Q3 is F. In some embodiments, R Q3 is CH 3 . In some embodiments, R Q3 is CD 3 . In some embodiments, R Q3 is deuterium. [00037] In some embodiments, ring Q is substituted monocyclic heteroaryl. In some embodiments, ring Q is substituted 5- or 6-membered monocyclic heteroaryl. In some embodiments, ring Q is substituted 6-membered monocyclic heteroaryl.
  • each of R Q1 , R Q2 , and R Q3 is independently selected from hydrogen, deuterium, F, Cl, – CN, –OH, -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , –CF 3 , –OCH 3 , –OCF 3, -OCH 2 CH 3 , - CH 2 OCH 3 , -OCH 2 CH 2 CH 3 , and -OCH(CH 3 ) 2 .
  • each of R Q1 , R Q2 , and R Q3 is independently hydrogen, F, Cl, -CN, –OH, -CH 3 ,–CF 3 , or –OCH 3 .
  • each of R Q1 , R Q2 , and R Q3 is independently -CN, –CF 3 , or –OCF 3 . In some embodiments, each of R Q1 , R Q2 , and R Q3 is independently hydrogen, F, –CF 3 , or –OCF 3 . In some embodiments, each of R Q1 , R Q2 , and R Q3 is independently hydrogen, F, or Cl. In some embodiments, R Q1 is hydrogen, F, Cl, -CN, –OH, –OCH 3 , or -CH 3 . In some embodiments, R Q1 is hydrogen. In some embodiments, R Q1 is F. In some embodiments, R Q1 is CH 3 .
  • R Q1 is CD 3 . In some embodiments, R Q1 is deuterium. In some embodiments, R Q2 is hydrogen, F, Cl, -CN, –OH, –OCH 3 , or -CH 3 . In some embodiments, R Q2 is hydrogen. In some embodiments, R Q2 is F. In some embodiments, R Q2 is CH 3 . In some embodiments, R Q2 is CD 3 . In some embodiments, R Q2 is deuterium. In some embodiments, R Q3 is hydrogen, F, Cl, -CN, –OH, –OCH 3 , or -CH 3 . In some embodiments, R Q3 is hydrogen. In some embodiments, R Q3 is F.
  • R Q3 is CH 3 . In some embodiments, R Q3 is CD 3 . In some embodiments, R Q3 is deuterium. [00039] In some embodiments, , , [00040] In some embodiments, each of R 2 and R 3 is independently hydrogen, deuterium, or substituted or unsubstituted C 1 -C 4 alkyl. In some embodiments, each of R 2 and R 3 is independently hydrogen, -CH 3 , -CH 2 CH 3 , -CH(CH 3 ) 2 , or -CF 3 . In some embodiments, each of R 2 and R 3 is independently hydrogen, -CH 3 , -CH 2 CH 3 , or -CH(CH 3 ) 2 .
  • each of R 2 and R 3 is independently hydrogen, -OCH 3 , -OCH 2 CH 3 , or -OCH(CH 3 ) 2 . In some embodiments, each of R 2 and R 3 is independently -CH 3 , -CH 2 CH 3 , or -CH(CH 3 ) 2 , in which one or more hydrogens is substituted with deuterium. In some embodiments, each of R 2 and R 3 is independently hydrogen, -CH 3 , -OCH 3 , or -CF 3 . In some embodiments, each of R 2 and R 3 is hydrogen. In some embodiments, R 2 is substituted or unsubstituted C 1 -C 4 alkyl. In some embodiments, R 2 is hydrogen.
  • R 2 is deuterium.
  • R 3 is substituted or unsubstituted C 1 -C 4 alkyl.
  • R 3 is hydrogen.
  • R 3 is deuterium.
  • R 4 which one or more hydrogens is substituted with deuterium.
  • R 4 is oxazolyl or pyrimidinyl.
  • R 4 is substituted or unsubstituted oxazolyl.
  • R 4 is substituted or unsubstituted pyrimidinyl.
  • R 4 is substituted or unsubstituted triazolyl.
  • R 4 is 5-membered heteroaryl substituted with 1 or 2 substituents each independently selected from halogen and C 1 -C 4 alkyl. In some embodiments, R 4 is 5-membered heteroaryl substituted with 1 or 2 substituents each independently selected from halogen and C 1 -C 4 alkyl. In some embodiments, R 4 is 5-membered heteroaryl substituted with 1 or 2 substituents each independently selected from F, Cl, and -CH 3 . In some embodiments, R 4 is 5-membered heteroaryl substituted with -CH 3 . In some embodiments, the 5- membered heteroaryl comprises 1-4 N ring atoms.
  • the 5-membered heteroaryl comprises 1 O ring atom and 1-2 N ring atoms. In some embodiments, the 5- membered heteroaryl comprises 1 S ring atom and 1-2 N ring atoms. In some embodiments, the 5-membered heteroaryl is selected from pyrazolyl, imidazolyl, triazolyl, and tetrazolyl. In some embodiments, the 5-membered heteroaryl is selected from oxazolyl, thiazolyl, oxadiazolyl, and thiadiazolyl. In some embodiments, R 4 is 6-membered heteroaryl substituted with 1 or 2 substituents each independently selected from F, Cl, and -CH 3 .
  • R 4 is 6- membered heteroaryl substituted with -CH 3 .
  • the 6-membered heteroaryl comprises 1-4 N ring atoms.
  • the 6-membered heteroaryl comprises 1 O ring atom and 1-2 N ring atoms.
  • the 6-membered heteroaryl comprises 1 S ring atom and 1-2 N ring atoms.
  • the R 4 is pyridinyl.
  • the R 4 is pyrimidinyl.
  • the R 4 is pyrazinyl. In some embodiments, the R 4 is triazinyl.
  • R 1 is substituted C 1 -C 3 alkyl. In some embodiments, R 1 is hydrogen. [00044] In some embodiments, W is substituted or unsubstituted C 1 -C 2 alkylene. In some embodiments, W is C 1 -C 2 alkylene substituted with 1, 2, 3, or 4 substituents each independently selected from F, -OH, -OCH 3 , and -CH 3 .
  • W is –CH 2 –, –CHF–, – CH(CH 3 )–, –CH(OH)–, –CH(OCH 3 )–, –CF 2 –, –CH 2 CH 2 –, –CHFCH 2 –, –CH 2 CHF–, – CH(CH 3 )CH 2 –, –CH 2 CH(CH 3 )–, –CH(OH)CH 2 –, –CH 2 CH(OH)–, –CH(OCH 3 )CH 2 –, – CH 2 CH(OCH 3 )–, –CF 2 CH 2 –, or —CH 2 CF 2 –.
  • W is –CH 2 –, –CHF–, – CH(CH 3 )–, –CH(OH)–, –CH(OCH 3 )–, or –CF 2 –.
  • W is –CH 2 CH 2 –, – CHFCH 2 –, –CH 2 CHF–, –CH(CH 3 )CH 2 –, –CH 2 CH(CH 3 )–, –CH(OH)CH 2 –, –CH 2 CH(OH)–, – CH(OCH 3 )CH 2 –, –-CH 2 CH(OCH 3 )–, –CF 2 CH 2 –, or –CH 2 CF 2 –.
  • W is – CH 2 –. In some embodiments, W is –CHF–. In some embodiments, W is –CH 2 CH 2 –. In some embodiments, W is —CHFCH 2 –, –CH 2 CHF–, –CF 2 CH 2 –, or –CH 2 CF 2 –. In some embodiments, W is –CH 2 –. In some embodiments, W is –CHF–. In some embodiments, W is –CH 2 CF 2 –. In some embodiments, W is –CF 2 CH 2 –. In some embodiments, W is –CH 2 CHF–. In some embodiments, W is –CHFCH 2 –.
  • R is hydrogen, substituted or unsubstituted C 1 –C 4 alkyl, substituted or unsubstituted C 1 –C 4 fluoroalkyl, substituted or unsubstituted C 1 –C 4 heteroalkyl, substituted or unsubstituted C 3 –C 5 cycloalkyl, or substituted or unsubstituted C 2 –C 4 heterocycloalkyl.
  • R is substituted or unsubstituted C 1 –C 4 alkyl, substituted or unsubstituted C 1 –C 4 fluoroalkyl, substituted or unsubstituted C 1 –C 4 heteroalkyl, substituted or unsubstituted C 3 –C 5 cycloalkyl, or substituted or unsubstituted C 2 –C 4 heterocycloalkyl.
  • R is substituted or unsubstituted C 1 –C 4 alkyl.
  • R is substituted or unsubstituted C 1 –C 4 fluoroalkyl.
  • R is substituted or unsubstituted C 1 –C 4 heteroalkyl. In some embodiments, R is substituted or unsubstituted C 3 –C 5 cycloalkyl. In some embodiments, R is substituted or unsubstituted C 2 –C 4 heterocycloalkyl.
  • R is hydrogen, -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 OH, - CH 2 F, -CHF 2 , -CF 3 , cyclopropyl, or oxetanyl.
  • R is -CH 3 , -CH 2 CH 3 , - CH 2 F, -CHF 2 , or -CF 3 .
  • R is hydrogen.
  • R is -CH 3 .
  • each of R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , and R 20 is independently selected from the group consisting of hydrogen, deuterium, F, substituted or unsubstituted C 1 -C 3 alkyl, and substituted or unsubstituted C 1 -C 3 fluoroalkyl.
  • each of R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , and R 20 is independently selected from the group consisting of hydrogen, deuterium, F, -OCH 3 , -CH 3 , -CH 2 CH 3 , - CH 2 CH 2 OH, -CH 2 F, -CHF 2 , and -CF 3 .
  • each of R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , and R 20 is independently selected from the group consisting of hydrogen and F.
  • one or more of R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , and R 20 is deuterium. In some embodiments, at least one of R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 and R 20 is F and is present in the compound. In some embodiments, one of R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 and R 20 is F and present in the compound.
  • At least two of R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 and R 20 are F and present in the compound. In some embodiments, at least one of R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 and R 20 is F and present in the compound. In some embodiments, one of R 11 , R 12 , R 13 , R 14 , R 16 , and R 17 is F and present in the compound. In some embodiments, at least two of R 11 , R 12 , R 13 , R 14 , R 16 , and R 17 are F and present in the compound.
  • At least one of R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 and R 20 comprises a fluorine and is present in the compound, e.g., F or C 1 –C 4 fluoroalkyl such as CH 2 F, CF 3 , CHF 2 , and CH 3 CH 2 F.
  • at least one of R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 and R 20 is F or C 1 –C 4 fluoroalkyl and present in the compound.
  • R 15 is -OR 5 , wherein R 5 is hydrogen or substituted or unsubstituted C 1 -C 3 alkyl. In some embodiments, R 15 is substituted or unsubstituted C 1 -C 3 alkyl. In some embodiments, R 15 is substituted or unsubstituted C 1 -C 3 fluoroalkyl. In some embodiments, R 15 is H, F, -OH, -OCH 3 , -OCF 3 , -CH 3 , -CH 2 OH, -CH 2 F, -CHF 2 , and -CF 3 . In some embodiments, R 15 is H, D, or F. In some embodiments, R 15 is D.
  • R 15 is H. In some embodiments, R 15 is F. In some embodiments, R 15 is -OCH 3 . [00048] In some embodiments, R 16 is -OR 5 , wherein R 5 is hydrogen or substituted or unsubstituted C 1 -C 3 alkyl. In some embodiments, R 16 is substituted or unsubstituted C 1 -C 3 alkyl. In some embodiments, R 16 is substituted or unsubstituted C 1 -C 3 fluoroalkyl.
  • R 16 is H, F, -OH, -OCH 3 , -OCF 3 , -CH 3 , -CH 2 OH, -CH 2 F, -CHF 2 , and -CF 3 .
  • R 16 is H, D, or F.
  • R 16 is D.
  • R 16 is H.
  • R 16 is F.
  • R 16 is -OCH 3 .
  • R 17 is -OR 5 , wherein R 5 is hydrogen or substituted or unsubstituted C 1 -C 3 alkyl.
  • R 17 is substituted or unsubstituted C 1 -C 3 alkyl. In some embodiments, R 17 is substituted or unsubstituted C 1 -C 3 fluoroalkyl. In some embodiments, R 17 is H, F, -OH, -OCH 3 , -OCF 3 , -CH 3 , -CH 2 OH, -CH 2 F, -CHF 2 , and -CF 3 . In some embodiments, R 17 is H, D, or F. In some embodiments, R 17 is D. In some embodiments, R 17 is H. In some embodiments, R 17 is F. In some embodiments, R 17 is -OCH 3 .
  • R 18 is -OR 5 , wherein R 5 is hydrogen or substituted or unsubstituted C 1 -C 3 alkyl. In some embodiments, R 18 is substituted or unsubstituted C 1 -C 3 alkyl. In some embodiments, R 18 is substituted or unsubstituted C 1 -C 3 fluoroalkyl. In some embodiments, R 18 is H, F, -OH, -OCH 3 , -OCF 3 , -CH 3 , -CH 2 OH, -CH 2 F, -CHF 2 , and -CF 3 . In some embodiments, R 18 is H, D, or F. In some embodiments, R 18 is D.
  • R 18 is H. In some embodiments, R 18 is F. In some embodiments, R 18 is -OCH 3 .
  • R 19 is -OR 5 , wherein R 5 is hydrogen or substituted or unsubstituted C 1 -C 3 alkyl. In some embodiments, R 19 is substituted or unsubstituted C 1 -C 3 alkyl. In some embodiments, R 19 is substituted or unsubstituted C1-C3 fluoroalkyl.
  • R 19 is H, F, -OH, -OCH 3 , -OCF 3 , -CH 3 , -CH 2 OH, -CH 2 F, -CHF 2 , and -CF 3 .
  • R 19 is H, D, or F.
  • R 19 is D.
  • R 19 is H.
  • R 19 is F.
  • R 19 is -OCH 3 .
  • R 20 is -OR 5 , wherein R 5 is hydrogen or substituted or unsubstituted C 1 -C 3 alkyl.
  • R 20 is substituted or unsubstituted C 1 -C 3 alkyl. In some embodiments, R 20 is substituted or unsubstituted C 1 -C 3 fluoroalkyl. In some embodiments, R 20 is H, F, -OH, -OCH 3 , -OCF 3 , -CH 3 , -CH 2 OH, -CH 2 F, -CHF 2 , and -CF 3 . In some embodiments, R 20 is H, D, or F. In some embodiments, R 20 is D. In some embodiments, R 20 is H. In some embodiments, R 20 is F. In some embodiments, R 20 is -OCH 3 .
  • R 16 is F.
  • R 17 is H or F.
  • both R 17 and R 16 are F.
  • R 18 is F.
  • R 19 is H or F.
  • both R 18 and R 19 are F.
  • each of R A1 and R A2 is independently hydrogen, deuterium, F, Cl, -CN, -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -OH, -OCH 3 , -OCH 2 CH 3 , -OCF 3 , - CH 2 F, -CHF 2 , or -CF 3 .
  • each of R A1 and R A2 is independently hydrogen, F, Cl, -CN, -CH 3 , -OH, -OCH 3 , -OCF 3 , -CH 2 F, -CHF 2 , or -CF 3 .
  • each of R A1 and R A2 is independently hydrogen, F, Cl, -CN, -CH 3 , or -OCH 3 .
  • each of R A1 and R A2 is independently hydrogen, F, Cl, or -CH 3 .
  • each of R A1 and R A2 is hydrogen.
  • R A1 is hydrogen, halogen, -CN, -OR 5 , or substituted or unsubstituted C 1 -C 6 alkyl. In some embodiments, R A1 is halogen. In some embodiments, R A1 is CN. In some embodiments, R A1 is -OR 5 . In some embodiments, R A1 is substituted or unsubstituted C 1 -C 6 alkyl. In some embodiments, R A1 is substituted or unsubstituted C 1 -C 3 alkyl. In some embodiments, R A2 is hydrogen, halogen, -CN, -OR 5 , or substituted or unsubstituted C 1 -C 6 alkyl.
  • R A2 is halogen. In some embodiments, R A2 is CN. In some embodiments, R A2 is -OR 5 . In some embodiments, R A2 is substituted or unsubstituted C 1 -C 6 alkyl. In some embodiments, R A2 is substituted or unsubstituted C 1 -C 3 alkyl. [00055] In some embodiments, each of R 15 and R 20 is independently selected from hydrogen, deuterium, F, –OR 5 , substituted or unsubstituted C 1 –C 3 alkyl, substituted or unsubstituted C 1 –C 3 fluoroalkyl, and substituted or unsubstituted C 1 –C 3 heteroalkyl.
  • each of R 15 and R 20 is independently selected from hydrogen, deuterium, F, -CH 3 , -CH 2 OH, -OCH 2 CN, - OH, -OCH 3 , -OCH 2 CN, -OCF 3 , -CH 2 F, -CHF 2 , and -CF 3 .
  • each of R 15 and R 20 is independently selected from hydrogen, deuterium, -CH 3 , -OCH 3 , -OCF 3 , -CH 2 F, - CHF2, and -CF3.
  • each of R 15 and R 20 is independently selected from hydrogen, deuterium, -CH 3 , and -OCH 3 .
  • each of R 15 and R 20 is independently selected from hydrogen and -CH 3 . In some embodiments, R 15 and R 20 are both - CH 3 . In some embodiments, R 15 is hydrogen and R 20 is -CH 3 . In some embodiments, R 15 is -CH 3 and R 20 is hydrogen. In some embodiments, R 15 and R 20 are both hydrogen. In some embodiments, R 15 and R 20 are both deuterium. In some embodiments, R 15 and R 20 are selected from hydrogen, deuterium, F, -CH 3 , and -OCH 3 . In some embodiments, R 15 is F and R 20 is hydrogen. In some embodiments, R 15 is hydrogen and R 20 is F. In some embodiments, R 15 is hydrogen and R 20 is CH 3 .
  • R 15 is CH 3 and R 20 is hydrogen. In some embodiments, R 15 and R 20 are the same. In some embodiments, R 15 and R 20 are different. [00056] In some embodiments, at least one of W, R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , and R 20 comprises a fluorine, e.g., F or C 1 –C 4 fluoroalkyl such as CH 2 F, CF 3 , CHF 2 , and CH 3 CH 2 F.
  • a fluorine e.g., F or C 1 –C 4 fluoroalkyl
  • one of W, R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , and R 20 comprises a fluorine. In some embodiments, W comprises a fluorine.
  • the abundance of deuterium in each of R A1 , R A2 , R 1 , R 2 , R 3 , R 4 , R 5 , R 5a , R 5b , R 6 , R 8 , R 21 , R 22 , R 22a , R 22b , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , and/or R 20 is independently at least 1%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of a total number of hydrogen and deuterium.
  • one or more of R A1 , R A2 , R 1 , R 2 , R 3 , R 4 , R 5 , R 5a , R 5b , R 6 , R 8 , R 21 , R 22 , R 22a , R 22b , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , and/or R 20 groups comprise deuterium at a percentage higher than the natural abundance of deuterium.
  • each R 15 and R 20 is independently hydrogen or –CH 3
  • W is -CH 2 CH 2 -
  • X is –N(cyclopropyl)-
  • R 15 and R 20 are –CH 3
  • at least one of R, R 8 , R 16 , and R 18 is not hydrogen or deuterium.
  • each of R 15 and R 20 is independently hydrogen or –CH 3
  • W is -CH 2 CH 2 -
  • X is –N(cyclopropyl)-
  • R 15 and R 20 are –CH 3
  • at least one of R, R 16 , and R 18 is not hydrogen or deuterium.
  • at least one of R 16 and R 18 is F.
  • R 16 and R 18 are F.
  • W is C 1 -C 2 alkylene substituted with one or more F.
  • each R A1 and R A2 is independently hydrogen, halogen, or - CH 3 .
  • each R A1 and R A2 is hydrogen.
  • R 21 is hydrogen, C 1 –C 4 alkyl, or C 3 –C 5 cycloalkyl.
  • R 21 is hydrogen.
  • R 21 is -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , or - CH(CH 3 ) 2 .
  • R 21 is -CH 3 . In some embodiments, R 21 is cyclopropyl, cyclobutyl, or cyclopentyl. In some embodiments, R 21 is cyclopropyl. [00062] In some embodiments, wherein each R Q1 , R Q2 , and R Q3 is independently selected from hydrogen, deuterium, F, Cl, and -CH 3 . In some embodiments, each R Q1 , R Q2 , and R Q3 is hydrogen. [00063] In some embodiments, each R 2 and R 3 is independently hydrogen or -CH 3 . In some embodiments, each R 2 and R 3 is hydrogen.
  • R 4 is 5-membered heteroaryl substituted with 1 or 2 substituents each independently selected from halogen and C 1 -C 4 alkyl. In some embodiments, R 4 is 5-membered heteroaryl substituted with 1 or 2 substituents each independently selected from F, Cl, and -CH 3 . In some embodiments, R 4 is 5-membered heteroaryl substituted with -CH 3 . In some embodiments, the 5- membered heteroaryl comprises 1-4 N ring atoms. In some embodiments, the 5-membered heteroaryl comprises 1 O ring atom and 1-2 N ring atoms. In some embodiments, the 5- membered heteroaryl comprises 1 S ring atom and 1-2 N ring atoms.
  • the 5-membered heteroaryl is selected from pyrazolyl, imidazolyl, triazolyl, and tetrazolyl. In some embodiments, the 5-membered heteroaryl is pyrazolyl. In some embodiments, the 5-membered heteroaryl is imidazolyl. In some embodiments, the 5-membered heteroaryl is triazolyl. In some embodiments, the 5-membered heteroaryl is tetrazolyl. In some embodiments, the 5-membered heteroaryl is selected from oxazolyl, thiazolyl, oxadiazolyl, and thiadiazolyl. In some embodiments, the 5-membered heteroaryl is oxazolyl.
  • the 5-membered heteroaryl is thiazolyl. In some embodiments, the 5-membered heteroaryl is oxadiazolyl. In some embodiments, the 5-membered heteroaryl is thiadiazolyl.
  • R 8 is hydrogen or -CH 3 . In some embodiments, R 8 is hydrogen. In some embodiments, R 8 is -CH 3 .
  • R 16 is hydrogen or F. In some embodiments, R 16 is hydrogen. In some embodiments, R 16 is F. In some embodiments, R 18 is hydrogen or F. In some embodiments, R 18 is hydrogen. In some embodiments, R 16 and R 18 are both hydrogen.
  • each R 15 and R 20 is independently selected from hydrogen and -CH 3 . In some embodiments, R 15 and R 20 are both -CH 3 . In some embodiments, R 15 is hydrogen and R 20 is -CH 3 . In some embodiments, R 15 is -CH 3 and R 20 is hydrogen. In some embodiments, R 15 and R 20 are both hydrogen. In some embodiments, R 15 and R 20 are both deuterium. [00068] In some embodiments, W is substituted or unsubstituted C 1 -C 2 alkylene.
  • W is C 1 -C 2 alkylene substituted with 1, 2, 3, or 4 substituents each independently selected from F, -OH, -OCH 3 , and -CH 3 .
  • W is –CH 2 CH 2 –, –CHFCH 2 –, –CH 2 CHF–, –CF 2 CH 2 –, or –CH 2 CF 2 –.
  • W is –CH 2 CH 2 –.
  • W is –CHFCH 2 –.
  • W is –CH 2 CHF–.
  • W is –CF 2 CH 2 –.
  • W is –CH 2 CF 2 –.
  • R is hydrogen or -CH 3 .
  • R is hydrogen.
  • R is -CH 3 .
  • exemplary SMSM compounds are selected from Table 1.
  • described herein is a pharmaceutical composition comprising any of the described compound, or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutically acceptable excipient.
  • the pharmaceutical composition is formulated for intravenous administration, subcutaneous administration, oral administration, inhalation, nasal administration, dermal administration, or ophthalmic administration.
  • the pharmaceutical composition is in a form of a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spray solution, a suppository, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, an ointment, a lotion, an eye drop or an ear drop.
  • the pharmaceutical composition further comprises a second therapeutic agent.
  • described herein is a compound, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment, prevention and/or delay of progression of a disease.
  • described herein is a use of a described compound, or a pharmaceutically acceptable salt or solvate thereof, for the preparation of a medicament for the treatment, prevention and/or delay of progression of a disease.
  • described herein is a use of a described compound, or a pharmaceutically acceptable salt or solvate thereof, for the treatment, prevention and/or delay of progression of a disease.
  • described herein is a method for the treatment, prevention and/or delay of progression of a disease comprising administering an effective amount of a described compound, or a pharmaceutically acceptable salt or solvate thereof, to a subject in need thereof.
  • described herein is a pharmaceutical composition comprising a described compound, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment, prevention and/or delay of progression of a disease.
  • described herein is a combination comprising a therapeutically effective amount of a described compound, or a pharmaceutically acceptable salt or solvate thereof, and one or more therapeutically active co-agents.
  • described herein is a method of treating a disease or condition comprising administering a described compound, or a pharmaceutically acceptable salt or solvate thereof, to a subject in need thereof.
  • described herein is a method of modulating splicing comprising contacting a described compound, or a pharmaceutically acceptable salt or solvate thereof, to a pre-mRNA.
  • the compound binds to the pre-mRNA and modulates splicing of the pre-mRNA in a cell of a subject.
  • the modulating comprises promoting exon skipping of the pre-mRNA.
  • the modulating alters a ratio of a first splice variant of the pre-mRNA to a second splice variant of the pre- mRNA.
  • the first splice variant is an mRNA encoding a full length protein and the second splice variant is an mRNA encoding a truncated protein.
  • the modulating increases the ratio of the mRNA encoding the truncated protein to the mRNA encoding the full length protein.
  • the modulating decreases the ratio of the mRNA encoding the full length protein to the mRNA encoding the truncated protein.
  • the ratio of the mRNA encoding the truncated protein to the mRNA encoding the full length protein is altered in at least 20%, at least 50%, at least 75%, or at least 90% of the cell.
  • the compound modulates affinity between the pre-mRNA and a splicing complex component.
  • the splicing complex component comprises a snRNA.
  • the snRNA comprises U1 snRNA, U2 snRNA, U4 snRNA, U5 snRNA, U6 snRNA, U11 snRNA, U12 snRNA, U4atac snRNA, U5 snRNA, U6 atac snRNA, or any combination thereof.
  • the snRNA comprises U1 snRNA.
  • the splicing complex component comprises 9G8, A1 hnRNP, A2 hnRNP, ASD-1, ASD-2b, ASF, B1 hnRNP, C1 hnRNP, C2 hnRNP, CBP20, CBP80, CELF, F hnRNP, FBP11, Fox-1, Fox-2, G hnRNP, H hnRNP, hnRNP 1, hnRNP 3, hnRNP C, hnRNP G, hnRNP K, hnRNP M, hnRNP U, Hu, HUR, I hnRNP, K hnRNP, KH-type splicing regulatory protein (KSRP), L hnRNP, M hnRNP, mBBP, muscle-blind like (MBNL), NF45, NFAR, Nova-1, Nova-2, nPTB, P54/SFRS11, polypyrimidine tract binding protein (PTB),
  • the compound binds to a splicing complex. In some embodiments, the compound modulates binding affinity of the splicing complex to the pre-mRNA. In some embodiments, the compound modulates binding affinity of the splicing complex to the pre-mRNA at a splice site sequence. In some embodiments, the compound modulates binding affinity of the splicing complex to the pre-mRNA upstream of a splice site sequence or downstream of a splice site sequence. In some embodiments, the compound interacts with an unpaired bulged nucleobase of an RNA duplex, and the RNA duplex comprises a splice site sequence.
  • the splice site sequence comprises at least one bulged nucleotide or a mutant nucleotide at a -3, - 2, -1, +1, +2, +3, +4, +5 or +6 position of the splice site sequence.
  • the compound modulates a resonance time of the splicing complex with the pre-mRNA. In some embodiments, the compound modulates the resonance time of the splicing complex with the pre- mRNA at the splice site sequence. In some embodiments, the compound modulates the resonance time of the splicing complex with the pre-mRNA upstream of the splice site sequence or downstream of the splice site sequence.
  • the compound modulates steric hindrance between the splicing complex and the pre-mRNA. In some embodiments, the compound modulates steric hindrance between the splicing complex and the pre-mRNA at a splice site sequence. In some embodiments, the compound modulates steric hindrance between the splicing complex and the pre-mRNA upstream of a splice site sequence or downstream of a splice site sequence.
  • the splice site sequence is a 5’ splice site sequence, a 3’ splice site sequence, a branch point splice site sequence, an exonic splicing enhancer (ESE) sequence, an exonic splicing silencer (ESS) sequence, an intronic splicing enhancer (ISE) sequence, an intronic splicing silencer (ISS) sequence, a polypyrimidine tract sequence, a cryptic splice site sequence, or any combination thereof.
  • ESE exonic splicing enhancer
  • ESS exonic splicing silencer
  • ISE intronic splicing enhancer
  • ISS intronic splicing silencer
  • the compounds presented herein include suitable diastereomeric, enantiomeric, and epimeric forms as well as the appropriate mixtures thereof.
  • the compounds and methods provided herein include suitable cis, trans, syn, anti, exo, endo,
  • E endo,
  • Z isomers as well as the appropriate mixtures thereof.
  • compounds described herein are prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds/salts, separating the diastereomers and recovering the optically pure enantiomers.
  • resolution of enantiomers is carried out using covalent diastereomeric derivatives of the compounds described herein.
  • diastereomers are separated by separation/resolution techniques based upon differences in solubility.
  • separation of stereoisomers is performed by chromatography or by the forming diastereomeric salts and separation by recrystallization, or chromatography, or any combination thereof.
  • stereoisomers are obtained by stereoselective synthesis.
  • the compounds described herein are labeled isotopically (e.g.
  • isotopes examples include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, such as, for example, 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, 36 Cl.
  • isotopically-labeled compounds described herein for example those into which radioactive isotopes such as 3 H and 14 C are incorporated, are useful in drug and/or substrate tissue distribution assays.
  • substitution with isotopes such as deuterium affords certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements.
  • compositions described herein may be formed as, and/or used as, pharmaceutically acceptable salts.
  • pharmaceutical acceptable salts include, but are not limited to: (1) acid addition salts, formed by reacting the free base form of the compound with a pharmaceutically acceptable: inorganic acid, such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, metaphosphoric acid, and the like; or with an organic acid, such as, for example, acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, trifluoroacetic acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-
  • compounds described herein may coordinate with an organic base, such as, but not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine, dicyclohexylamine, tris(hydroxymethyl)methylamine.
  • compounds described herein may form salts with amino acids such as, but not limited to, arginine, lysine, and the like.
  • Acceptable inorganic bases used to form salts with compounds that include an acidic proton include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.
  • a reference to a pharmaceutically acceptable salt includes the solvent addition forms, particularly solvates.
  • Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol.
  • solvates of compounds described herein are conveniently prepared or formed during the processes described herein.
  • the compounds provided herein can exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the compounds and methods provided herein.
  • heterocycloalkylaryl “haloalkylheteroaryl”, “arylalkylheterocycloalkyl”, or “alkoxyalkyl”.
  • the last member of the combination is the radical which is binding to the rest of the molecule.
  • the other members of the combination are attached to the binding radical in reversed order in respect of the literal sequence, e.g. the combination arylalkylheterocycloalkyl refers to a heterocycloalkyl-radical which is substituted by an alkyl which is substituted by an aryl.
  • the term “one or more” refers to the range from one substituent to the highest possible number of substitution, i.e.
  • C 1 -C x includes C 1 -C 2 , C 1 -C 3 . .
  • C 1 -C x a group designated as “C 1 -C 4 " indicates that there are one to four carbon atoms in the moiety, i.e. groups containing 1 carbon atom, 2 carbon atoms, 3 carbon atoms or 4 carbon atoms.
  • “C 1 -C 4 alkyl” indicates that there are one to four carbon atoms in the alkyl group, i.e., the alkyl group is selected from among methyl, ethyl, propyl, iso-propyl, n-butyl, iso- butyl, sec-butyl, and t-butyl.
  • amino refers to the –NH 2 substituent.
  • hydroxy refers to the –OH substituent.
  • methoxy refers to the –OCH 3 substituent.
  • halo halogen
  • halide are used interchangeably herein and denote fluoro, chloro, bromo, or iodo, most particularly fluoro or chloro.
  • alkyl refers to a straight or branched hydrocarbon chain radical, having from one to twenty carbon atoms, and which is attached to the rest of the molecule by a single bond.
  • An alkyl comprising up to 10 carbon atoms is referred to as a C 1 -C 10 alkyl, likewise, for example, an alkyl comprising up to 6 carbon atoms is a C 1 -C 6 alkyl.
  • Alkyls (and other moieties defined herein) comprising other numbers of carbon atoms are represented similarly.
  • Alkyl groups include, but are not limited to, C 1 -C 10 alkyl, C 1 -C 9 alkyl, C 1 -C 8 alkyl, C 1 -C 7 alkyl, C 1 -C 6 alkyl, C 1 -C 5 alkyl, C 1 -C 4 alkyl, C 1 -C 3 alkyl, C 1 -C 2 alkyl, C 2 -C 8 alkyl, C 3 -C 8 alkyl and C 4 -C 8 alkyl.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (i-propyl), n-butyl, i-butyl, s-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, 1-ethyl-propyl, and the like.
  • the alkyl is methyl or ethyl.
  • the alkyl is –CH(CH 3 )2 or –C(CH 3 )3. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted as described below.
  • alkylene or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group.
  • the alkylene is -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, or -CH 2 CH(CH 3 )CH 2 -.
  • the alkylene is –CH 2 -.
  • the alkylene is –CH 2 CH 2 -.
  • the alkylene is –CH 2 CH 2 CH 2 -.
  • alkoxy refers to a radical of the formula –OR x where R x is an alkyl radical as defined above. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted as described below. Representative alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy. In some embodiments, the alkoxy is methoxy. In some embodiments, the alkoxy is ethoxy. [00109] The term “alkylamino” refers to a radical of the formula -NHR x or -NR x R x where each R x is, independently, an alkyl radical as defined above.
  • alkylamino group may be optionally substituted as described below.
  • alkenyl refers to a type of alkyl group in which at least one carbon- carbon double bond is present.
  • R x is H or an alkyl.
  • an alkenyl is selected from ethenyl (i.e., vinyl), propenyl (i.e., allyl), butenyl, pentenyl, pentadienyl, and the like.
  • alkenylene or alkenylene chain refers to a straight or branched divalent hydrocarbon chain in which at least one carbon-carbon double bond is present linking the rest of the molecule to a radical group.
  • alkynyl refers to a type of alkyl group in which at least one carbon- carbon triple bond is present. In one embodiment, an alkenyl group has the formula -C ⁇ C-R x , wherein R x refers to the remaining portions of the alkynyl group.
  • R x is H or an alkyl.
  • an alkynyl is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
  • Non-limiting examples of an alkynyl group include -C ⁇ CH, - C ⁇ CCH 3 , -C ⁇ CCH 2 CH 3 , and -CH 2 C ⁇ CH.
  • aromatic refers to a planar ring having a delocalized S-electron system containing 4n+2 S electrons, where n is an integer. Aromatics can be optionally substituted.
  • aromatic includes both aryl groups (e.g., phenyl, naphthalenyl) and heteroaryl groups (e.g., pyridinyl, quinolinyl).
  • aryl refers to a radical comprising at least one aromatic ring wherein each of the atoms forming the ring is a carbon atom.
  • Aryl groups can be optionally substituted. Examples of aryl groups include, but are not limited to phenyl, and naphthyl. In some embodiments, the aryl is phenyl.
  • an aryl group can be a monoradical or a diradical (i.e., an arylene group).
  • an aryl group comprises a partially reduced cycloalkyl group defined herein (e.g., 1,2-dihydronaphthalene). In some embodiments, an aryl group comprises a fully reduced cycloalkyl group defined herein (e.g., 1,2,3,4- tetrahydronaphthalene). When aryl comprises a cycloalkyl group, the aryl is bonded to the rest of the molecule through an aromatic ring carbon atom.
  • An aryl radical can be a monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) ring system, which may include fused, spiro or bridged ring systems.
  • haloalkyl denotes an alkyl group wherein at least one of the hydrogen atoms of the alkyl group has been replaced by same or different halogen atoms, particularly fluoro atoms.
  • haloalkyl examples include monofluoro-, difluoro-or trifluoro-methyl, -ethyl or -propyl, for example 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl, or trifluoromethyl.
  • haloalkoxy denotes an alkoxy group wherein at least one of the hydrogen atoms of the alkoxy group has been replaced by same or different halogen atoms, particularly fluoro atoms.
  • haloalkoxyl examples include monofluoro-, difluoro-or trifluoro-methoxy, - ethoxy or -propoxy, for example 3,3,3-trifluoropropoxy, 2-fluoroethoxy, 2,2,2-trifluoroethoxy, fluoromethoxy, or trifluoromethoxy.
  • perhaloalkoxy denotes an alkoxy group where all hydrogen atoms of the alkoxy group have been replaced by the same or different halogen atoms.
  • bicyclic ring system denotes two rings which are fused to each other via a common single or double bond (annelated bicyclic ring system), via a sequence of three or more common atoms (bridged bicyclic ring system) or via a common single atom (spiro bicyclic ring system).
  • Bicyclic ring systems can be saturated, partially unsaturated, unsaturated or aromatic.
  • Bicyclic ring systems can comprise heteroatoms selected from N, O and S.
  • carrier or “carbocycle” refer to a ring or ring system where the atoms forming the backbone of the ring are all carbon atoms.
  • carbocyclic from “heterocyclic” rings or “heterocycles” in which the ring backbone contains at least one atom which is different from carbon.
  • at least one of the two rings of a bicyclic carbocycle is aromatic.
  • both rings of a bicyclic carbocycle are aromatic.
  • Carbocycle includes cycloalkyl and aryl.
  • cycloalkyl refers to a monocyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom.
  • cycloalkyls are saturated or partially unsaturated.
  • cycloalkyls are spirocyclic or bridged compounds. In some embodiments, cycloalkyls are fused with an aromatic ring (in which case the cycloalkyl is bonded through a non-aromatic ring carbon atom). Cycloalkyl groups include groups having from 3 to 10 ring atoms. Representative cycloalkyls include, but are not limited to, cycloalkyls having from three to ten carbon atoms, from three to eight carbon atoms, from three to six carbon atoms, or from three to five carbon atoms.
  • Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • the monocyclic cycloalkyl is cyclopentyl.
  • the monocyclic cycloalkyl is cyclopentenyl or cyclohexenyl.
  • the monocyclic cycloalkyl is cyclopentenyl.
  • Polycyclic radicals include, for example, adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetrainyl, decalinyl, 3,4-dihydronaphthalenyl-1(2H)-one, spiro[2.2]pentyl, norbornyl and bicycle[1.1.1]pentyl. Unless otherwise stated specifically in the specification, a cycloalkyl group may be optionally substituted.
  • the term bridged refers to any ring structure with two or more rings that contains a bridge connecting two bridgehead atoms.
  • the bridgehead atoms are defined as atoms that are the part of the skeletal framework of the molecule and which are bonded to three or more other skeletal atoms.
  • the bridgehead atoms are C, N, or P.
  • the bridge is a single atom or a chain of atoms that connects two bridgehead atoms.
  • the bridge is a valence bond that connects two bridgehead atoms.
  • the bridged ring system is cycloalkyl. In some embodiments, the bridged ring system is heterocycloalkyl. [00121]
  • the term “fused” refers to any ring structure described herein which is fused to an existing ring structure.
  • fused ring is a heterocyclyl ring or a heteroaryl ring
  • any carbon atom on the existing ring structure which becomes part of the fused heterocyclyl ring or the fused heteroaryl ring may be replaced with one or more N, S, and O atoms.
  • fused heterocyclyl or heteroaryl ring structures include 6-5 fused heterocycle, 6-6 fused heterocycle, 5-6 fused heterocycle, 5-5 fused heterocycle, 7-5 fused heterocycle, and 5-7 fused heterocycle.
  • fluoroalkyl refers to an alkyl in which one or more hydrogen atoms are replaced by a fluorine atom.
  • a fluoralkyl is a C 1 -C 6 fluoroalkyl.
  • a fluoroalkyl is selected from trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like.
  • heteroalkyl refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen (e.g. –NH-, - N(alkyl)-, or -N(aryl)-), sulfur (e.g.
  • a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl. In some embodiments, a heteroalkyl is attached to the rest of the molecule at a heteroatom of the heteroalkyl. In some embodiments, a heteroalkyl is a C 1 -C 6 heteroalkyl. Representative heteroalkyl groups include, but are not limited to -OCH 2 OMe, -OCH 2 CH 2 OH, - OCH 2 CH 2 OMe, or –OCH 2 CH 2 OCH 2 CH 2 NH 2 .
  • heteroalkylene or “heteroalkylene chain” refers to a straight or branched divalent heteroalkyl chain linking the rest of the molecule to a radical group. Unless stated otherwise specifically in the specification, the heteroalkyl or heteroalkylene group may be optionally substituted as described below.
  • Representative heteroalkylene groups include, but are not limited to –CH 2 -O-CH 2 -, –CH 2 -N(alkyl)-CH 2 -, –CH 2 -N(aryl)-CH 2 -, -OCH 2 CH 2 O-, – OCH 2 CH 2 OCH 2 CH 2 O-, or –OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 O-.
  • heterocycloalkyl refers to a cycloalkyl group that includes at least one heteroatom selected from nitrogen, oxygen, and sulfur.
  • the heterocycloalkyl radical may be a monocyclic, or bicyclic ring system, which may include fused (when fused with an aryl or a heteroaryl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom) or bridged ring systems.
  • the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized.
  • the nitrogen atom may be optionally quaternized.
  • the heterocycloalkyl radical is partially or fully saturated.
  • heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, t
  • heterocycloalkyl also includes all ring forms of carbohydrates, including but not limited to monosaccharides, disaccharides and oligosaccharides. Unless otherwise noted, heterocycloalkyls have from 2 to 12 carbons in the ring. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring and 1 or 2 N atoms. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring and 3 or 4 N atoms.
  • heterocycloalkyls have from 2 to 12 carbons, 0-2 N atoms, 0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring. In some embodiments, heterocycloalkyls have from 2 to 12 carbons, 1-3 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. It is understood that when referring to the number of carbon atoms in a heterocycloalkyl, the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocycloalkyl (i.e.
  • heterocycle refers to heteroaromatic rings (also known as heteroaryls) and heterocycloalkyl rings (also known as heteroalicyclic groups) that includes at least one heteroatom selected from nitrogen, oxygen and sulfur, wherein each heterocyclic group has from 3 to 12 atoms in its ring system, and with the proviso that any ring does not contain two adjacent O or S atoms.
  • heterocycles are monocyclic, bicyclic, polycyclic, spirocyclic or bridged compounds.
  • Non-aromatic heterocyclic groups include rings having 3 to 12 atoms in its ring system and aromatic heterocyclic groups include rings having 5 to 12 atoms in its ring system.
  • the heterocyclic groups include benzo-fused ring systems.
  • non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, oxazolidinonyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6- tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl
  • aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinox
  • a group derived from pyrrole includes both pyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached).
  • a group derived from imidazole includes imidazol-1-yl or imidazol-3-yl (both N- attached) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-attached).
  • the heterocyclic groups include benzo-fused ring systems.
  • at least one of the two rings of a bicyclic heterocycle is aromatic.
  • both rings of a bicyclic heterocycle are aromatic.
  • heteroaryl refers to an aryl group that includes one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • heteroaryl is monocyclic or bicyclic.
  • monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, furazanyl, indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8- naphthyridine, and pteridine.
  • monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl.
  • bicyclic heteroaryls include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine.
  • heteroaryl is pyridinyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl or furyl.
  • a heteroaryl contains 0-6 N atoms in the ring.
  • a heteroaryl contains 1-4 N atoms in the ring. In some embodiments, a heteroaryl contains 4-6 N atoms in the ring. In some embodiments, a heteroaryl contains 0-4 N atoms, 0-1 O atoms, 0-1 P atoms, and 0- 1 S atoms in the ring. In some embodiments, a heteroaryl contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, heteroaryl is a C1-C9 heteroaryl. In some embodiments, monocyclic heteroaryl is a C 1 -C 5 heteroaryl.
  • monocyclic heteroaryl is a 5-membered or 6-membered heteroaryl.
  • a bicyclic heteroaryl is a C6-C9 heteroaryl.
  • a heteroaryl group comprises a partially reduced cycloalkyl or heterocycloalkyl group defined herein (e.g., 7,8-dihydroquinoline).
  • a heteroaryl group comprises a fully reduced cycloalkyl or heterocycloalkyl group defined herein (e.g., 5,6,7,8-tetrahydroquinoline).
  • heteroaryl comprises a cycloalkyl or heterocycloalkyl group
  • the heteroaryl is bonded to the rest of the molecule through a heteroaromatic ring carbon or hetero atom.
  • a heteroaryl radical can be a monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) ring system, which may include fused, spiro or bridged ring systems.
  • alkyl-aryl refers to a radical of the formula -R y -R x , wherein R x is an alkyl radical as described herein and R y is an aryl radical as described herein.
  • alkyl-heterocycloalkyl refers to a radical of the formula -R y -R x , wherein R x is an alkyl radical as described herein and R y is a heterocycloalkyl radical as described herein.
  • alkyl-heteroaryl refers to a radical of the formula -R y -R x , wherein R x is an alkyl radical as described herein and R y is a heteroaryl radical as described herein.
  • alkoxy-aryl refers to a radical of the formula -R y -R x , wherein R x is an alkoxy radical as described herein and R y is an aryl radical as described herein.
  • alkoxy-heterocycloalkyl refers to a radical of the formula -R y -R x , wherein R x is an alkoxy radical as described herein and R y is a heterocycloalkyl radical as described herein.
  • alkoxy-heteroaryl refers to a radical of the formula -R y -R x , wherein R x is an alkoxy radical as described herein and R y is an heteroaryl radical as described herein.
  • moiety refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.
  • optional substituents are independently selected from D, halogen, -CN, -NH 2 , -OH, -NH(CH 3 ), -N(CH 3 ) 2 , -NH(cyclopropyl), -CH 3 , -CH 2 CH 3 , -CF 3 , - OCH 3 , and -OCF 3 .
  • substituted groups are substituted with one or two of the preceding groups.
  • tautomer refers to a proton shift from one atom of a molecule to another atom of the same molecule.
  • Tautomers are compounds that are interconvertible by migration of a hydrogen atom, accompanied by a switch of a single bond and adjacent double bond. In bonding arrangements where tautomerization is possible, a chemical equilibrium of the tautomers will exist. All tautomeric forms of the compounds disclosed herein are contemplated. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH.
  • tautomeric interconversions include: [00137]
  • modulate means to interact with a target either directly or indirectly so as to alter the activity of the target, including, by way of example only, to enhance the activity of the target, to inhibit the activity of the target, to limit the activity of the target, or to extend the activity of the target.
  • modulator refers to a molecule that interacts with a target either directly or indirectly. The interactions include, but are not limited to, the interactions of an agonist, partial agonist, an inverse agonist, antagonist, degrader, or combinations thereof. In some embodiments, a modulator is an agonist.
  • co-administration or the like, as used herein, are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time.
  • pharmaceutical combination means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients.
  • fixed combination means that the active ingredients, e.g. a compound described herein and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage.
  • non-fixed combination means that the active ingredients, e.g.
  • a compound described herein and a co- agent are administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific intervening time limits, wherein such administration provides effective levels of the two compounds in the body of the patient.
  • cocktail therapy e.g. the administration of three or more active ingredients.
  • mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
  • the mammal is a human.
  • compositions and medicaments containing the compounds of the present disclosure and a therapeutically inert carrier, diluent or excipient, as well as methods of using the compounds of the present disclosure to prepare such compositions and medicaments.
  • the compounds described herein may be formulated by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form.
  • physiologically acceptable carriers i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form.
  • the pH of the formulation depends mainly on the particular use and the concentration of compound, but preferably ranges anywhere from about 3 to about 8.
  • a splice modifying compound described herein is formulated in an acetate buffer, at pH 5.
  • the splice modifying compounds described herein are sterile.
  • the compound may be stored, for example, as a solid or amorphous composition, as a lyophilized composition or as an aqueous solution.
  • Compositions are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the “effective amount” of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to modify pre-mRNA splicing. For example, such amount may be below the amount that is toxic to normal cells, or the mammal as a whole.
  • the compounds of the current disclosure may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the compounds of the present disclosure are formulated for administration to a mammal by intravenous administration, subcutaneous administration, oral administration, inhalation, nasal administration, dermal administration, or ophthalmic administration
  • the compounds of the present disclosure may be administered in any convenient administrative form, e.g., tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc.
  • Such compositions may contain components conventional in pharmaceutical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents.
  • the compounds of the present disclosure are administered in a form of a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spray solution, a suppository, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, an ointment, a lotion, an eye drop or an ear drop.
  • a typical composition is prepared by mixing a compound of the present disclosure and a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C, et al., Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems.
  • compositions may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present disclosure or pharmaceutical composition thereof) or aiding the manufacturing of the pharmaceutical product (i.e., medicament).
  • buffers stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present disclosure or pharmaceutical composition thereof) or aiding the manufacturing of
  • An example of a suitable oral dosage form is a tablet containing about 25mg, 50mg, 100mg, 250mg, or 500mg of the compound of the present disclosure compounded with about 90-30 mg anhydrous lactose, about 5-40 mg sodium croscarmellose, about 5-30mg polyvinylpyrrolidone (PVP) K30, and about 1-10 mg magnesium stearate.
  • the powdered ingredients are first mixed together and then mixed with a solution of the PVP.
  • the resulting composition can be dried, granulated, mixed with the magnesium stearate and compressed to tablet form using conventional equipment.
  • an aerosol composition can be prepared by dissolving the compound, for example 5-400 mg, of the present disclosure in a suitable buffer solution, e.g. a phosphate buffer, adding a tonicifier, e.g. a salt such sodium chloride, if desired.
  • a suitable buffer solution e.g. a phosphate buffer
  • a tonicifier e.g. a salt such sodium chloride
  • the solution may be filtered, e.g., using a 0.2 micron filter, to remove impurities and contaminants.
  • the present disclosure relates to a pharmaceutical composition comprising a pre-mRNA splicing modifier, as described herein or pharmaceutically acceptable salt thereof.
  • the present disclosure relates to a pharmaceutical composition comprising a pre-mRNA splicing modifier, as described herein or pharmaceutically acceptable salt thereof together with one or more pharmaceutically acceptable excipients.
  • the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of a pre-mRNA splicing modifier, as described herein or pharmaceutically acceptable salt thereof together with one or more pharmaceutically acceptable excipients.
  • the present disclosure relates to a combination comprising a therapeutically effective amount of a pre-mRNA splicing modifier, as described herein or pharmaceutically acceptable salt thereof and one or more other therapeutically active pharmaceutical ingredients.
  • the compounds described herein are formulated into pharmaceutical compositions.
  • Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • a summary of pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A.
  • a pharmaceutical composition can be a mixture of an SMSM described herein with one or more other chemical components (i.e.
  • compositions described herein can be administered to the subject in a variety of ways, including parenterally, intravenously, intradermally, intramuscularly, colonically, rectally or intraperitoneally.
  • the small molecule splicing modulator or a pharmaceutically acceptable salt thereof is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject.
  • the pharmaceutical compositions can be administered parenterally, intravenously, intramuscularly or orally.
  • the oral agents comprising a small molecule splicing modulator can be in any suitable form for oral administration, such as liquid, tablets, capsules, or the like.
  • the oral formulations can be further coated or treated to prevent or reduce dissolution in stomach.
  • the compositions of the present invention can be administered to a subject using any suitable methods known in the art. Suitable formulations for use in the present invention and methods of delivery are generally well known in the art.
  • the small molecule splicing modulators described herein can be formulated as pharmaceutical compositions with a pharmaceutically acceptable diluent, carrier or excipient.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions including pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • compositions described herein can be administrable to a subject in a variety of ways by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intralymphatic, intranasal injections), intranasal, buccal, topical or transdermal administration routes.
  • parenteral e.g., intravenous, subcutaneous, intramuscular, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intralymphatic, intranasal injections
  • intranasal buccal
  • topical or transdermal administration routes e.g., topical or transdermal administration routes.
  • the pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.
  • the pharmaceutical formulation is in the form of a tablet.
  • pharmaceutical formulations containing an SMSM described herein are in the form of a capsule.
  • liquid formulation dosage forms for oral administration are in the form of aqueous suspensions or solutions selected from the group including, but not limited to, aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups.
  • an SMSM described herein can be formulated for use as an aerosol, a mist or a powder.
  • the compositions may take the form of tablets, lozenges, or gels formulated in a conventional manner.
  • an SMSM described herein can be prepared as transdermal dosage forms.
  • an SMSM described herein can be formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection.
  • an SMSM described herein can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments.
  • an SMSM described herein can be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas.
  • Extensive posttranscriptional processing occurs before eukaryotic pre-mRNA matures and exits from the nucleus to the cytoplasm, including the addition of a 7-methylguanosine cap at the 5’ end, the cleavage and addition of a poly-A tail at the 3’ end as well as the removal of intervening sequences or introns by the spliceosome.
  • the vast majority of higher eukaryotic genes contain multiple introns that are spliced out with high precision and fidelity in order to maintain the reading frame of the exons.
  • Splicing of pre-mRNA can utilize the recognition of short consensus sequences at the boundaries and within introns and exons by an array of small nuclear ribonucleoprotein (snRNP) complexes (e.g., snRNPs U1, U2, U4, U5, U6, U11, U12m U4atc and U6 atc) and a large number of proteins, including spliceosomal proteins and positively as well as negatively acting splicing modulators.
  • snRNP small nuclear ribonucleoprotein
  • Serine-arginine-rich (SR)-domain-containing proteins generally serve to promote constitutive splicing. They can also modulate alternative splicing by binding to intronic or exonic splicing enhancer (ISE) or ESE, respectively) sequences.
  • ISE intronic or exonic splicing enhancer
  • ESE intronic splicing enhancer
  • SR protein family is a class of at least 10 proteins that have a characteristic serine/arginine rich domain in addition to an RNA-binding. SR proteins are generally thought to enhance splicing by simultaneously binding to U170K, a core component of the U1 snRNP, at the 5’ splice site, and the U2AF35 at the 3’ splice site, thus bridging the two ends of the intron.
  • SR proteins While this particular function of SR proteins seems to be redundant, as any individual SR protein can commit a pre-mRNA for constitutive splicing, the role of the various SR proteins in alternative splicing of specific pre-mRNAs is distinct due in part to their ability to recognize and bind to unique consensus sequences. Phosphorylation of the RS domain of SR proteins can lead to the regulation of their protein interactions, RNA binding, localization, trafficking, and role in alternative splicing.
  • SRPKs SR protein Kinase
  • Clks Cdc2-like kinases
  • PRP4 pre-mRNA processing mutant 4
  • SR proteins may be required for proper functioning as both hypo- and hyperphosphorylation of the RS domains may be detrimental to their role in constitutive and alternative splicing.
  • the vast majority of genes contain one or more introns, which creates a situation in which the exons are spliced together to generate mature mRNA and microRNA (miRNA).
  • miRNA microRNA
  • pre-mRNA splicing is the mechanism by which introns are removed from a pre-mRNA and the exons are ligated together to generate mature mRNAs and pre-miRNA that is then exported to the cytoplasm for translation into the polypeptide gene product.
  • Splicing of pre-mRNA can occur in cis, where two exons derive from two adjacent cotranscribed sequences, or in trans, when the two exons come from different pre-mRNA transcripts.
  • the ratio of the different protein products (isoforms) may be due to the frequency of alternative splicing events within a pre-mRNA that leads to different amounts of distinct splice variants.
  • alternative splicing of a pre-mRNA may lead to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 protein isoforms being expressed. [00161] Aberrations in splicing are thought to be the cause of roughly half of all inherited diseases.
  • splicing due to mutations in consensus sequences involved in exon-intron boundary recognition is responsible for up to 15% of inherited diseases.
  • defects in the splicing machinery itself due to the loss or gain of function of splicing factors and modulators are causes of a wide range of human ailments from cancer to neurodegenerative diseases.
  • Both constitutive and alternative splicing are subject to regulation by upstream signaling pathways. This regulation can be essential during development, in tissue specific expression of certain isoforms, during the cell cycle and in response to extrinsic signaling molecules.
  • Alternative splicing allows for a single gene to express different isoforms of mRNA, thus playing a major role in contributing to the cellular complexity in higher eukaryotes without the need to expand the genome. Splicing can also be subject to regulation by upstream signaling pathways. For example, an upstream signaling pathway may modulate alternative splicing and increase or decrease expression levels of different isoforms of mRNA.
  • Alternative splicing events are highly regulated by numerous splicing factors in a tissue type-, developmental stage-, and signal-dependent manner.
  • non-mutation based causes of splicing defects and defects in the splicing machinery itself e.g., due to the loss/gain of function of splicing factors or their relative stoichiometry, cause of a wide range of human ailments, ranging from cancer to neurodegenerative diseases.
  • the disease state is caused by an alteration of the ratio of different isoforms of two or more proteins expressed from a gene.
  • the alteration in the ratio of the protein products is due to changes in the frequency of alternative splicing events within a pre-mRNA, leading to changes in the ratio of splice variants produced.
  • alternative splicing of a pre-mRNA may lead to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 protein isoforms being expressed.
  • a change in the splice variant ratio is caused by genetic mutation.
  • the vast majority of splicing processes are catalyzed by the spliceosome, an RNA-protein complex that occurs in unique steps and may comprise a subset of several hundred different proteins, in addition to five spliceosomal snRNAs. These factors are responsible for the accurate positioning of the spliceosome on the 5’ and 3’ splice site sequences.
  • mRNA messenger mRNA
  • mRNA messenger mRNA
  • all protein expression derives from mRNAs
  • there is the potential to intervene in protein-mediated diseases by modulating the expression of the relevant protein and by, in turn, modulating the translation of the corresponding upstream mRNA.
  • RNA is only a small portion of the transcriptome: other transcribed RNAs also regulate cellular biology either directly by the structure and function of RNA structures (e.g., ribonucleoproteins) as well as via protein expression and action, including (but not limited to) microRNA (miRNA), long noncoding RNA (lncRNA), long intergenic noncoding RNA (lincRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), small Cajal body- specific RNA (scaRNA), piwi-interacting RNA (piRNA), competing endogenous (ceRNA), and pseudo-genes. Drugs that intervene at this level have the potential of modulating any and all cellular processes.
  • miRNA microRNA
  • lncRNA long noncoding RNA
  • lincRNA long intergenic noncoding RNA
  • small nucleolar RNA small nucleolar RNA
  • snRNA small nuclear RNA
  • scaRNA small Cajal body-specific RNA
  • DNA sequences in the chromosome are transcribed into pre-mRNAs which contain coding regions (exons) and generally also contain intervening non-coding regions (introns).
  • Introns are removed from pre-mRNAs through splicing.
  • Pre-mRNA splicing proceeds by a two- step mechanism. In the first step, the 5’ splice site is cleaved, resulting in a “free” 5’ exon and a lariat intermediate. In the second step, the 5’ exon is ligated to the 3’ exon with release of the intron as the lariat product. These steps are catalyzed in a complex of small nuclear ribonucleoproteins and proteins called the spliceosome. [00167] In most cases, the splicing reaction occurs within the same pre-mRNA molecule, which is termed cis-splicing.
  • Introns are portions of eukaryotic DNA, which intervene between the coding portions, or “exons,” of that DNA. Introns and exons are transcribed into RNA termed “primary transcript, precursor to mRNA” (or “pre-mRNA”). Introns can be removed from the pre-mRNA so that the native protein encoded by the exons can be produced (the term “native protein” as used herein refers to naturally occurring, wild type, or functional protein). The removal of introns from pre- mRNA and subsequent joining of the exons is carried out in the splicing process.
  • RNA RNA that contains both exons and intron(s)
  • mRNA mature mRNA
  • mRNA RNA in which the intron(s) have been removed and the exons joined together sequentially so that the protein may be translated therefrom by the ribosomes.
  • Introns can be defined by a set of “splice elements” that are part of the splicing machinery and may be required for splicing and which are relatively short, conserved RNA segments that bind the various splicing factors, which carry out the splicing reactions.
  • each intron is defined by a 5’ splice site, a 3’ splice site, and a branch point situated there between.
  • Splice elements also comprise exon splicing enhancers and silencers, situated in exons, and intron splicing enhancers and silencers situated in introns at a distance from the splice sites and branch points.
  • RNA transcripts pre-mRNA
  • mRNA messenger RNA
  • the splicing that occurs can vary, so the synthesis of alternative protein products from the same primary transcript can be affected by tissue-specific or developmental signals.
  • spliceosome is a complex comprising ribonucleoprotein (snRNP) particles composed of small nuclear RNAs and proteins.
  • snRNA components of the spliceosome can promote the two transesterification reactions of splicing.
  • Two unique spliceosomes coexist in most eukaryotes: the U2-dependent spliceosome, which catalyzes the removal of U2-type introns, and the less abundant U12-dependent spliceosome, which is present in only a subset of eukaryotes and splices the rare U12-type class of introns.
  • the U2-dependent spliceosome is assembled from the U1, U2, U5, and U4/U6 snRNPs and numerous non-snRNP proteins.
  • RNA splicing typically refers to the editing of the nascent precursor messenger RNA (pre-mRNA) transcript into a mature messenger RNA (mRNA). Splicing is a biochemical process which includes the removal of introns followed by exon ligation.
  • Sequential transesterification reactions are initiated by a nucleophilic attack of the 5’ splice site (5’ss) by the branch adenosine (branch point; BP) in the downstream intron resulting in the formation of an intron lariat intermediate with a 2’, 5’-phosphodiester linkage. This is followed by a 5’ss-mediated attack on the 3’ splice site (3’ss), leading to the removal of the intron lariat and the formation of the spliced RNA product.
  • Splicing can be regulated by various cis-acting elements and trans-acting factors. Cis- acting elements are sequences of the mRNA and can include core consensus sequences and other regulatory elements.
  • Core consensus sequences typically can refer to conserved RNA sequence motifs, including the 5’ss, 3’ss, polypyrimidine tract and BP region, which can function for spliceosome recruitment.
  • BP refers to a partially conserved sequence of pre-mRNA, generally less than 50 nucleotides upstream of the 3’ss. BP reacts with the 5’ss during the first step of the splicing reaction.
  • Other regulatory cis-acting elements can include exonic splicing enhancer (ESE), exonic splicing silencer (ESS), intronic splicing enhancer (ISE), and intronic splicing silencer (ISS).
  • Trans-acting factors can be proteins or ribonucleoproteins which bind to cis-acting elements.
  • Splice site identification and regulated splicing can be accomplished principally by two dynamic macromolecular machines, the major (U2-dependent) and minor (U12-dependent) spliceosomes. Each spliceosome contains five snRNPs: U1, U2, U4, U5 and U6 snRNPs for the major spliceosome (which processes ⁇ 95.5% of all introns); and U11, U12, U4atac, U5 and U 6 atac snRNPs for the minor spliceosome.
  • Spliceosome recognition of consensus sequence elements at the 5’ss, 3’ss and BP sites is one of the steps in the splicing pathway, and can be modulated by ESEs, ISEs, ESSs, and ISSs, which can be recognized by auxiliary splicing factors, including SR proteins and hnRNPs.
  • Polypyrimidine tract-binding protein (PTBP) can bind to the polypyrimidine tract of introns and may promote RNA looping.
  • Alternative splicing is a mechanism by which a single gene may eventually give rise to several different proteins.
  • Alternative splicing can be accomplished by the concerted action of a variety of different proteins, termed “alternative splicing regulatory proteins,” that associate with the pre-mRNA, and cause distinct alternative exons to be included in the mature mRNA. These alternative forms of the gene’s transcript can give rise to distinct isoforms of the specified protein. Sequences in pre-mRNA molecules that can bind to alternative splicing regulatory proteins can be found in introns or exons, including, but not limited to, ISS, ISE, ESS, ESE, and polypyrimidine tract. Many mutations can alter splicing patterns.
  • mutations can be cis-acting elements, and can be located in core consensus sequences (e.g.5’ss, 3’ss and BP) or the regulatory elements that modulate spliceosome recruitment, including ESE, ESS, ISE, and ISS.
  • a cryptic splice site for example, a cryptic 5’ss and a cryptic 3’ss, can refer to a splice site that is not normally recognized by the spliceosome and therefore are in the dormant state.
  • Cryptic splice site can be recognized or activated, for example, by mutations in cis-acting elements or trans-acting factors, or structural configurations, such as bulges.
  • the present invention contemplates use of small molecules with favorable drug properties that modulate the activity of splicing of a target RNA.
  • SMSMs small molecule splicing modulators
  • the SMSMs bind and modulate target RNA.
  • a library of SMSMs that bind and modulate one or more target RNAs.
  • the target RNA is mRNA.
  • the target RNA is mRNA a noncoding RNA.
  • the target RNA is a pre-mRNA.
  • the target RNA is hnRNA.
  • the small molecules modulate splicing of the target RNA. In some embodiments, a small molecule provided herein modulates splicing at a sequence of the target RNA. In some embodiments, a small molecule provided herein modulates splicing at a cryptic splice site sequence of the target RNA. In some embodiments, a small molecule provided herein binds to a target RNA. In some embodiments, a small molecule provided herein binds to a splicing complex component. In some embodiments, a small molecule provided herein binds to a target RNA and a splicing complex component.
  • splicing event in a pre- mRNA molecule
  • methods of preventing or inducing a splicing event in a pre- mRNA molecule comprising contacting the pre-mRNA molecule and/or other elements of the splicing machinery (e.g., within a cell) with a compound provided herein to prevent or induce the splicing event in the pre-mRNA molecule.
  • the splicing event that is prevented or induced can be, e.g., an aberrant splicing event, a constitutive splicing event or an alternate splicing event.
  • a method of identifying a compound capable of preventing or inducing a splicing event in a pre-mRNA molecule comprising contacting the compound with splicing elements and/or factors involved in alternative, aberrant and/or constitutive splicing as described herein (e.g., within cells) under conditions whereby a positive (prevention or induction of splicing) or negative (no prevention or induction of splicing) effect is produced and detected and identifying a compound that produces a positive effect as a compound capable of preventing or inducing a splicing event.
  • a small molecule compound described herein in a pharmaceutically acceptable carrier prevents or induces an alternative or aberrant splicing event in a pre-mRNA molecule.
  • a method of upregulating expression of a native protein in a cell containing a DNA encoding the native protein wherein the DNA contains a mutation or no mutation that causes downregulation of the native protein by aberrant and/or alternate splicing thereof.
  • the DNA can encode a pre-mRNA that has a mutation or an aberrant secondary or tertiary structure that causes downregulation of one or more isoforms of a protein.
  • the method can comprise introducing into the cell a small molecule provided herein that prevents an aberrant splicing event, whereby the native intron is removed by correct splicing and the native protein is produced by the cell.
  • a method comprises introducing into a cell a small molecule provided herein that modulates an alternate splicing event to produce a protein that has a different function than the protein that would be produced without modulation of alternate splicing.
  • provided herein is a method of downregulating expression of a native protein in a cell containing a DNA encoding the native protein, wherein the DNA contains a mutation or no mutation that causes upregulation of the native protein by aberrant and/or alternate splicing thereof.
  • the DNA can encode a pre-mRNA that has a mutation or an aberrant secondary or tertiary structure that causes upregulation of one or more isoforms of a protein.
  • the method can comprise introducing into the cell a small molecule provided herein that prevents an aberrant splicing event, whereby the native intron is removed by correct splicing and the native protein is produced by the cell.
  • a method comprises introducing into a cell a small molecule provided herein that modulates an alternate splicing event to produce a protein that has a different function than the protein that would be produced without modulation of alternate splicing.
  • a method can comprise preventing aberrant splicing in a pre- mRNA molecule containing a mutation or an aberrant secondary or tertiary structure and/or preventing an alternative splicing event.
  • the mutation or aberrant secondary or tertiary structure can cause a pre-mRNA to splice incorrectly and produce an aberrant mRNA or mRNA fragment different from the mRNA ordinarily resulting from a pre-mRNA without the mutation or aberrant secondary or tertiary structure.
  • s pre-mRNA molecule can contain: (i) a first set of splice elements defining a native intron which can be removed by splicing when the mutation or aberrant secondary or tertiary structure is absent to produce a first mRNA molecule encoding a native protein, and (ii) a second set of splice elements induced by the mutation or aberrant secondary or tertiary structure which defines an aberrant intron different from the native intron, which aberrant intron is removed by splicing when the mutation or aberrant secondary or tertiary structure is present to produce an aberrant second mRNA molecule different from the first mRNA molecule.
  • the method can comprise contacting the pre-mRNA molecule and/or other factors and/or elements of the splicing machinery as described herein (e.g., within a cell) with a compound described herein to prevent or promote an aberrant splicing event in a pre-mRNA molecule, whereby the native intron is removed by correct splicing and native protein production is increased in the cell.
  • a method of upregulating expression of a RNA that would otherwise be downregulated by modulating an alternative splicing event in the RNA.
  • the method can comprise contacting a pre-mRNA molecule and/or other elements and/or factors of the splicing machinery with a compound described herein to modulate alternate splicing events, whereby a native splicing event is inhibited and an alternate splicing event is promoted that upregulates expression of a RNA that is otherwise downregulated when under the control of the native splicing event.
  • a method of downregulating expression of a RNA that would otherwise be upregulated by modulating an alternative splicing event in the RNA.
  • the method can comprise contacting a pre-mRNA molecule and/or other elements and/or factors of the splicing machinery with a compound described herein to modulate alternate splicing events, whereby a native splicing event is inhibited and an alternate splicing event is promoted that downregulates expression of a RNA that is otherwise upregulated when under the control of the native splicing event.
  • the methods, compounds and compositions described herein have a variety of uses. For example, they are useful in any process where it is desired to have a means for downregulating expression of a RNA to be expressed until a certain time, after which it is desired to upregulate RNA expression.
  • the RNA to be expressed may be any RNA encoding a protein to be produced so long as the gene contains a native intron.
  • the RNA may be mutated by any suitable means, such as site-specific mutagenesis (see, T. Kunkel, U.S. Pat. No.4,873,192) to deliberately create an aberrant second set of splice elements which define an aberrant intron which substantially downregulates expression of the gene.
  • a sequence encoding the RNA may be inserted into a suitable expression vector and the expression vector inserted into a host cell (e.g., a eukaryotic cell such as a yeast, insect, or mammalian cell (e.g., human, rat)) by standard recombinant techniques.
  • a host cell e.g., a eukaryotic cell such as a yeast, insect, or mammalian cell (e.g., human, rat)
  • the host cell can then be grown in culture by standard techniques.
  • a suitable compound of the present invention in a suitable formulation, can be added to the culture medium so that expression of the gene is upregulated.
  • the method can comprise contacting a pre-mRNA molecule and/or other elements and/or factors of the splicing machinery with a compound or compounds described herein to modulate alternative splicing events.
  • the compound or compounds of this invention can be used to act upon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 alternative splicing events that may occur within a pre-mRNA.
  • a first splice variant may be downregulated or inhibited and/or a second splice variant may be upregulated, resulting in an altered ratio of splice variants of the two or more RNA.
  • a first splice variant may be upregulated while a second splice variant may be unaffected, thereby altering the ratio of the RNA.
  • a first splice variant may be downregulated while a second splicing event may be unaffected thereby altering the ratio of the RNA.
  • the methods, compounds and formulations described herein are also useful as in vitro or in vivo tools to examine and modulate splicing events in human or animal RNAs encoded by genes, e.g., those developmentally and/or tissue regulated (e.g., alternate splicing events).
  • the compounds and formulations described herein are also useful as therapeutic agents in the treatment of disease involving aberrant and/or alternate splicing.
  • a method of treating a subject having a condition or disorder associated with an alternative or aberrant splicing event in a pre-mRNA molecule comprises administering to the subject a therapeutically effective amount of a compound described herein to modulate an alternative splicing event or prevent an aberrant splicing event, thereby treating the subject.
  • the method can, e.g., restore a correct splicing event in a pre-mRNA molecule.
  • the method can, e.g., utilize a small molecule compound described herein in a pharmaceutically acceptable carrier.
  • Formulations containing the small molecules described herein can comprise a physiologically or pharmaceutically acceptable carrier, such as an aqueous carrier.
  • formulations for use in the methods described herein include, but are not limited to, those suitable for oral administration, parenteral administration, including subcutaneous, intradermal, intramuscular, intravenous and intra-arterial administration, as well as topical administration (e.g., administration of an aerosolized formulation of respirable particles to the lungs of a patient afflicted with cystic fibrosis or lung cancer or a cream or lotion formulation for transdermal administration of patients with psoriasis).
  • topical administration e.g., administration of an aerosolized formulation of respirable particles to the lungs of a patient afflicted with cystic fibrosis or lung cancer or a cream or lotion formulation for transdermal administration of patients with psoriasis.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art.
  • the most suitable route of administration in any given case may depend upon the subject, the nature and severity of the condition being treated, and the particular active compound, which is being used, as would be readily determined by one of skill in the art.
  • the medicament upregulates gene expression.
  • the medicament downregulates gene expression.
  • the compound can be admixed with, inter alia, a pharmaceutically acceptable carrier.
  • the carrier may be a solid or a liquid.
  • One or more compounds may be incorporated in any combination in the formulations described herein, which may be prepared by any of the well-known techniques of pharmacy, such as admixing the components, and/or including one or more accessory therapeutic ingredients.
  • the present inventors identify herein low molecular weight compounds (sometimes referred to herein as small molecules, which block mRNA splicing and/or enhance (facilitate, augment) mRNA splicing.
  • the splicing that can be regulated by the methods described herein include alternative splicing, e.g., exon skipping, intron retention, pseudoexons skipping, exon exclusion, partial intron exclusion and others.
  • a method comprises contacting a splice modulating compound (e.g., a SMSM) to a pre-mRNA that modulates splicing of the pre-mRNA to favor expression of a transcript that promotes cell proliferation.
  • a splice modulating compound e.g., a SMSM
  • an SMSM described herein can increase one or more isoforms of a transcript that promotes cell proliferation.
  • an SMSM described herein can decrease expression one or more isoforms of a transcript that prevents or inhibits cell proliferation.
  • a method comprises contacting a splice modulating compound (e.g., a SMSM) to a pre-mRNA that modulates splicing of the pre-mRNA to favor expression of a transcript that prevents or inhibits cell proliferation.
  • a splice modulating compound e.g., a SMSM
  • an SMSM described herein can increase one or more isoforms of a transcript that prevents or inhibits cell proliferation.
  • an SMSM described herein can decrease expression one or more isoforms of a transcript that promotes cell proliferation.
  • a method of modulating splicing of pre-mRNA comprises using an SMSM to decrease expression or functionality of one or more isoforms of a transcript in a subject.
  • the method can comprise administering an SMSM, or a composition comprising an SMSM, to a subject, wherein the SMSM binds to a pre-mRNA or a splicing complex component and modulates splicing of the pre-mRNA to favor expression of one or more isoforms of a transcript.
  • the method can comprise administering an SMSM, or a composition comprising an SMSM, to a subject, wherein the SMSM binds to a pre-mRNA or a splicing complex component and modulates splicing of the pre-mRNA to disfavor expression of one or more isoforms of a transcript.
  • the present invention provides a method of treating a subject afflicted with a disease or condition associated with aberrant splicing of a pre-mRNA.
  • the method can comprise administering an SMSM, or a composition comprising an SMSM, to a subject, wherein the SMSM binds to a pre-mRNA or a splicing complex component and modulates splicing of the pre-mRNA to inhibit expression of one or more isoforms of a transcript.
  • the method can comprise administering an SMSM, or a composition comprising an SMSM, to a subject, wherein the SMSM binds to a pre-mRNA or a splicing complex component and modulates the splicing of the pre-mRNA to increase expression of one or more isoforms of a transcript.
  • a number of diseases are associated with expression of an aberrant gene product (e.g., an RNA transcript or protein) of a gene.
  • an aberrant gene product e.g., an RNA transcript or protein
  • aberrant amounts of a RNA transcript may lead to disease due to corresponding changes in protein expression.
  • Changes in the amount of a particular RNA transcript may be the result of several factors.
  • changes in the amount of RNA transcripts may be due to an aberrant level of transcription of a particular gene, such as by the perturbation of a transcription factor or a portion of the transcription process, resulting in a change in the expression level of a particular RNA transcript.
  • changes in the splicing of particular RNA transcripts can change the levels of a particular RNA transcript.
  • Changes to the stability of a particular RNA transcript or to components that maintain RNA transcript stability, such as the process of poly-A tail incorporation or an effect on certain factors or proteins that bind to and stabilize RNA transcripts may lead to changes in the levels of a particular RNA transcript.
  • the level of translation of particular RNA transcripts can also affect the amount of those transcripts, affecting or upregulating RNA transcript decay processes.
  • RNA transcripts encoded by a pre-mRNA
  • methods for modulating the amount of one, two, three or more RNA transcripts encoded by a pre-mRNA comprising contacting a cell with an SMSM compound or a pharmaceutically acceptable salt thereof.
  • the cell is contacted with an SMSM compound or a pharmaceutically acceptable salt thereof in a cell culture.
  • the cell is contacted with an SMSM compound or a pharmaceutically acceptable salt thereof in a subject (e.g., a non-human animal subject or a human subject).
  • compositions and methods for treatment, prevention and/or delay of progression of a disease or condition comprising administering an effective amount of a small molecule splicing modulator as described herein to a subject, in particular to a mammal.
  • compositions and methods for treating a disease or condition including steric modulator compounds or pharmaceutically acceptable salts thereof that promote prevention or correction of exon skipping of a pre-mRNA.
  • the invention further provides compositions and methods for increasing production of mature mRNA and, in turn, protein, in cells of a subject in need thereof, for example, a subject that can benefit from increased production of protein.
  • the invention further provides compositions and methods for decreasing production of mature mRNA and, in turn, protein, in cells of a subject in need thereof, for example, a subject that can benefit from decreased production of protein.
  • the described methods may be used to treat subjects having a disease or condition caused by a mutation in a gene, including missense, splicing, frameshift and nonsense mutations, as well as whole gene deletions, which result in deficient protein production.
  • the described methods may be used to treat subjects having a disease or condition not caused by gene mutation.
  • the compositions and methods of the present invention are used to treat subjects having a disease or condition, who can benefit from increased production of protein.
  • compositions and methods of the present invention are used to treat subjects having a disease or condition, who can benefit from increased production of protein. In some embodiments, the compositions and methods of the present invention are used to treat subjects having a disease or condition, who can benefit from decreased production of a protein. [00201] In some embodiments, provided herein are methods of treating a disease or condition in a subject in need thereof by increasing the expression of a target protein or functional RNA by cells of the subject, wherein the cells have a mutation that causes, e.g., exon skipping or intron inclusion, or a portion thereof, of pre-mRNA, wherein the pre-mRNA encodes the target protein or functional RNA.
  • the method can comprise contacting cells of a subject with an SMSM compound or a pharmaceutically acceptable salt thereof that targets the pre-mRNA encoding the target protein or functional RNA or splicing complex component, whereby splicing of an exon from a pre-mRNA encoding a target protein or functional RNA is prevented or inhibited, thereby increasing a level of mRNA encoding the target protein or functional RNA, and increasing the expression of the target protein or functional RNA in the cells of the subject.
  • a method of increasing expression of a target protein by cells having a mutation or aberrant secondary or tertiary RNA structure that causes exon skipping of pre-mRNA the pre-mRNA comprising a mutation or aberrant secondary or tertiary RNA structure that causes exon skipping.
  • the method can comprise contacting the cells with an SMSM compound or a pharmaceutically acceptable salt thereof that targets a pre-mRNA encoding a target protein or functional RNA, whereby splicing of an exon from a pre-mRNA encoding a target protein or functional RNA is prevented or inhibited, thereby increasing the level of mRNA encoding functional protein, and increasing the expression of protein in the cells.
  • the target protein is a tumor suppressor. In some embodiments, the target protein is a tumor promoter. In some embodiments, the target protein or the functional RNA is a compensating protein or a compensating functional RNA that functionally augments or replaces a target protein or functional RNA that is deficient in amount or activity in the subject. In some embodiments, the cells are in or from a subject having a condition caused by a deficient amount or activity of the protein.
  • the deficient amount of the target protein is caused by haploinsufficiency of the target protein, wherein the subject has a first allele encoding a functional target protein, and a second allele from which the target protein is not produced, or a second allele encoding a nonfunctional target protein, and wherein an SMSM compound or a pharmaceutically acceptable salt thereof binds to a targeted portion of a pre-mRNA transcribed from the first allele.
  • the target protein is produced in a form that is fully- functional compared to the equivalent protein produced from mRNA in which an exon has been skipped or is missing.
  • the pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a pre-mRNA.
  • an SMSM compound or a pharmaceutically acceptable salt thereof increases the amount of the target protein or the functional RNA by modulating alternative splicing of pre-mRNA transcribed from a gene encoding the functional RNA or target protein.
  • an SMSM compound or a pharmaceutically acceptable salt thereof increases the amount of the target protein or the functional RNA by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or the functional RNA.
  • the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with an SMSM compound or a pharmaceutically acceptable salt thereof is increased at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%,at least about 400%, or at least about 500%, compared to the total amount of the mRNA encoding the target protein or functional RNA produced in a control cell.
  • the total amount of target protein produced by the cell contacted with an SMSMS compound or a pharmaceutically acceptable salt thereof is increased at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%, compared to the total amount of target protein produced by a control cell.
  • a total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with an SMSM compound or a pharmaceutically acceptable salt thereof is increased at least about 1.1-fold, at least about 1.5-fold, at least about 2- fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold compared to the total amount of the mRNA encoding the target protein or functional RNA produced in a control cell.
  • a total amount of target protein produced by a cell contacted with an SMSM compound or a pharmaceutically acceptable salt thereof is increased at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to the total amount of target protein produced by a control cell.
  • the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with an SMSM compound or a pharmaceutically acceptable salt thereof is decreased at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, compared to the total amount of the mRNA encoding the target protein or functional RNA produced in a control cell.
  • binding of an SMSM compound or a pharmaceutically acceptable salt thereof to pre-mRNA prevents splicing out of one or more exons and/or introns and/or proteins thereof, from the population of pre-mRNAs to produce mRNA encoding the target protein or functional RNA.
  • the cell comprises a population of pre-mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of pre-mRNAs comprises a mutation that causes the splicing out of one or more exons, and wherein an SMSM compound or a pharmaceutically acceptable salt thereof binds to the mutation that causes the splicing out of the one or more exons in the population of pre-mRNAs.
  • the binding of an SMSM compound or a pharmaceutically acceptable salt thereof to the mutation that causes the splicing out of the one or more exons prevents splicing out of the one or more exons from the population of pre-mRNAs to produce mRNA encoding the target protein or functional RNA.
  • the condition is a disease or disorder.
  • the method further comprises assessing protein expression.
  • an SMSM compound or a pharmaceutically acceptable salt thereof binds to a targeted portion of a pre-mRNA.
  • the binding of an SMSM compound or a pharmaceutically acceptable salt thereof catalyzes the inclusion of a missing exon or removal of an undesired retained intron or portions thereof, resulting in healthy mRNA and proteins. In some embodiments, the binding of an SMSM compound or a pharmaceutically acceptable salt thereof has minimal to no effect on non-diseased cells.
  • An SMSM can modulate splicing at a splice site of a polynucleotide and does not exhibit significant toxicity. In some embodiments, an SMSM penetrates the blood brain barrier (BBB) when administered to a subject.
  • BBB blood brain barrier
  • an SMSM has a brain/blood AUC of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 40, or higher.
  • SMSM Targets [00211] Aberrant splicing of mRNA, such as pre-mRNA, can result in a defective protein and can cause a disease or a disorder in a subject.
  • the compositions and methods described herein can reduce this aberrant splicing of mRNA, such as pre-mRNA, and treat a disease or a disorder caused by this aberrant splicing.
  • RNA transcripts Diseases associated with changes to RNA transcript amount are often treated with a focus on the aberrant protein expression.
  • the processes responsible for the aberrant changes in RNA levels such as components of the splicing process or associated transcription factors or associated stability factors, could be targeted by treatment with a small molecule, it would be possible to restore protein expression levels such that the unwanted effects of the expression of aberrant levels of RNA transcripts or associated proteins. Therefore, there is a need for methods of modulating the amount of RNA transcripts encoded by certain genes as a way to prevent or treat diseases associated with aberrant expression of the RNA transcripts or associated proteins.
  • Mutations and/or aberrant secondary or tertiary RNA structures in cis-acting elements can induce three-dimensional structural change in pre-mRNA. Mutations and/or aberrant secondary RNA structures in cis-acting elements can induce three-dimensional structural change in pre-mRNA when the pre-mRNA is, for example, bound to at least one snRNA, or at least one snRNP, or at least one other auxiliary splicing factor. For example, non-canonical base pairing of a non-canonical splice site sequence to a snRNA can form a bulge.
  • a bulge can be formed when the 5’ss is bound to U1-U12 snRNA or a portion thereof.
  • a bulge can be induced to form when 5’ss containing at least one mutation is bound to U1-U12 snRNA or a portion thereof.
  • a bulge can be formed when the cryptic 5’ss is bound to U1-U12 snRNA or a portion thereof.
  • a bulge can be induced to form when cryptic 5’ss containing at least one mutation is bound to U1-U12 snRNA or a portion thereof.
  • a bulge can be formed when the 3’ss is bound to U2 snRNA or a portion thereof.
  • a bulge can be induced to form when the 3’ss is bound to U2 snRNA or a portion thereof.
  • a bulge can be formed when the cryptic 3’ss is bound to U2 snRNA or a portion thereof.
  • a bulge can be induced to form when the cryptic 3’ss is bound to U2 snRNA or a portion thereof.
  • the protein components of U1 and U2 may or may not present to form the bulge.
  • a small molecule can bind to a bulge.
  • a bulge is naturally occurring.
  • a bulge is formed by non-canonical base- pairing between the splice site and the small nuclear RNA.
  • a bulge can be formed by non-canonical base-pairing between the 5’ss and U1-U12 snRNA.
  • the bulge can comprise 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides.
  • 3-dimensional structural changes can be induced by a mutation without bulge formation.
  • a bulge may be formed without any mutation in a splice site.
  • a recognition portion can be formed by a mutation in any of the cis-acting elements.
  • a small molecule can bind to a recognition portion that is induced by a mutation.
  • a mutation and/or aberrant secondary or tertiary RNA structure at an authentic 5’ splice site can result in splicing at a cryptic 5’ splice site.
  • a mutation and/or aberrant secondary or tertiary RNA structure can be in one of the regulatory elements including ESEs, ESSs, ISEs, and ISSs.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide in an exon.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide upstream (5’) of the splice site of the splice site sequence.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the -1 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NNN*nnnnn, wherin N* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the -2 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NN*Nnnnnn, wherin N* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the -3 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of N*NNnnnnn, wherin N* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide in an intron. In some embodiments, a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide downstream (3’) of the splice site of the splice site sequence. [00217] In some embodiments, a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +1 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NNNn*nnnn, wherin n* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +2 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NNNnn*nnnn, wherin n* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +3 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NNNnnn*nnn, wherin n* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +4 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NNNnnn*nn, wherin n* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +5 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NNNnnnn*n, wherin n* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +6 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NNNnnnnn*, wherin n* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the +7 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NNNnnnnnn*, wherin n* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with one or more bulged nucleotides at the -1, -2, -3, +1, +2, +3, +4, +5, +6 and/or +7 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NNN*nnnnnn, NN*Nnnnnn, N*NNnnnnn, NNNn*nnnnn, NNNnn*nnnn, NNNnnn*nnn, NNNnnnn*nn, NNNnnnn*n, NNNnnnn*n, NNNnnnnn*n, NNNnnnnnn*, or NNNnnnnnn*, wherin N* or n* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with one or more bulged nucleotides at the -1, -2, and/or -3 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NNN*nnnnn, NN*Nnnnnn, or N*NNnnnnnn, wherin N* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with one or more bulged nucleotides at the +1, +2, +3, +4, +5, +6 and/or +7 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NNNn*nnnn, NNNnn*nnnn, NNNnnn*nn, NNNnnnn*n, NNNnnnn*n, NNNnnnnn*, or NNNnnnnnn*, wherin n* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the -1 position relative to the splice site of the splice site sequence and a bulged nucleotide at the -2 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of NN*N*nnnnn, wherin N* represents a bulged nucleotide.
  • a target of an SMSM is a pre-mRNA comprising a splice site sequence with a bulged nucleotide at the -2 position relative to the splice site of the splice site sequence and a bulged nucleotide at the -3 position relative to the splice site of the splice site sequence.
  • a target of an SMSM can be a pre-mRNA comprising a splice site sequence of N*N*Nnnnnn, wherin N* represents a bulged nucleotide.
  • an SMSM interacts with a bulged nucleotide of an RNA duplex comprising a splice site.
  • the RNA duplex comprises pre-mRNA.
  • an SMSM binds to an RNA duplex and interacts with an unpaired bulged nucleobase of an RNA duplex comprising a splice site.
  • a first portion of the SMSM interacts with the bulged nucleotide on a first RNA strand of the RNA duplex.
  • a second portion of the SMSM interacts with one or more nucleotides of a second RNA strand of the RNA duplex, wherein the first RNA strand is not the second RNA strand.
  • the SMSM forms one or more intermolecular interactions with the duplex RNA, for example, an ionic interaction, a hydrogen bond, a dipole-dipole interaction or a van der Waals interaction.
  • the SMSM forms one or more intermolecular interactions with the bulged nucleotide, for example, an ionic interaction, a hydrogen bond, a dipole-dipole interaction or a van der Waals interaction.
  • the duplex RNA comprises an alpha helix.
  • the bulged nucleotide is located on an external portion of a helix of the duplex RNA.
  • the bulged nucleotide is located within an internal portion of the helix of the duplex RNA. [00224] In some embodiments, a rate of exchange of the bulged nucleotide from within the interior of a helix of the duplex RNA to an exterior portion of the helix is reduced. [00225] In some embodiments, the SMSM modulates a distance of the bulged nucleotide from a second nucleotide of the duplex RNA. In some embodiments, the SMSM reduces the distance of the bulged nucleotide from a second nucleotide of the duplex RNA.
  • the SMSM increases the distance of the bulged nucleotide from a second nucleotide of the duplex RNA.
  • the bulged nucleotide is located within the interior of a helix of the duplex RNA of the complex.
  • the bulged nucleotide has modulated base stacking within an RNA strand of the RNA duplex.
  • the bulged nucleotide has increased base stacking within an RNA strand of the RNA duplex.
  • the bulged nucleotide has decreased base stacking within an RNA strand of the RNA duplex.
  • the SMSM modulates splicing at the splice site of the RNA duplex. In some embodiments, the SMSM increases splicing at the splice site of the RNA duplex. In some embodiments, the SMSM reduces splicing at the splice site of the RNA duplex. In some embodiments, the SMSM reduces a size of a bulge of the RNA duplex. In some embodiments, the SMSM removes a bulge of the RNA duplex. In some embodiments, the SMSM stabilizes a bulge of the RNA duplex.
  • the unpaired bulged nucleotide is free to rotate around a phosphate backbone of an RNA strand of the RNA duplex in the absence of the SMSM. In some embodiments, the SMSM reduces a rate of rotation of the unpaired bulged nucleotide. In some embodiments, the SMSM reduces a rate of rotation of the unpaired bulged nucleotide around a phosphate backbone of an RNA strand of the RNA duplex. [00229] In some embodiments, the SMSM is not an aptamer.
  • a method of modulating splicing comprising contacting a small molecule splicing modulator compound (SMSM) to a cell; wherein the SMSM interacts with an unpaired bulged nucleotide of an RNA duplex in the cell; wherein the duplex RNA comprises a splice site; and wherein the SMSM modulates splicing of the RNA duplex.
  • SMSM small molecule splicing modulator compound
  • a method for modulating the relative position of a first nucleotide relative to a second nucleotide, wherein the first nucleotide and the second nucleotide are within a duplex RNA comprising contacting a small molecule splicing modulator compound (SMSM) to the duplex RNA, or a pharmaceutically acceptable salt thereof, wherein the first nucleotide is a bulged nucleotide of the RNA duplex; wherein the duplex RNA comprises a splice site.
  • SMSM small molecule splicing modulator compound
  • the duplex RNA comprises a helix.
  • the bulged nucleotide is located on an external portion of a helix of the duplex RNA prior to contacting the SMSM.
  • SMSM forms one or more intermolecular interactions with the duplex RNA.
  • the SMSM forms one or more intermolecular interactions with an unpaired bulged nucleotide.
  • the intermolecular interaction is selected from the group comprising an ionic interaction, a hydrogen bond, a dipole-dipole interaction or a van der Waals interaction.
  • a rate of exchange of the unpaired bulged nucleotide from within the interior of a helix of the duplex RNA to an exterior portion of the helix is reduced.
  • a rate of rotation of the unpaired bulged nucleotide is reduced.
  • a rate of rotation of the unpaired bulged nucleotide around a phosphate backbone of an RNA strand of the RNA duplex is reduced.
  • a distance of the unpaired bulged nucleotide from a second nucleotide of the duplex RNA is modulated after contacting the SMSM.
  • the distance of the unpaired bulged nucleotide from a second nucleotide of the duplex RNA is reduced.
  • unpaired bulged nucleotide is located within the interior of the helix of the duplex RNA.
  • a size of a bulge of the RNA duplex is reduced.
  • a bulge of the RNA duplex is removed or maintained.
  • splicing at the splice site of the RNA duplex is promoted.
  • base stacking of the unpaired bulged nucleotide within an RNA strand of the RNA duplex is increased after contacting the SMSM.
  • the distance of the unpaired bulged nucleotide from a second nucleotide of the duplex RNA is increased or maintained. In some embodiments, a bulge of the RNA duplex is stabilized after contacting the SMSM. In some embodiments, the unpaired bulged nucleotide is located on an exterior portion of a helix of the duplex RNA. In some embodiments, a size of a bulge of the RNA duplex is increased. In some embodiments, splicing at the splice site of the RNA duplex is inhibited. In some embodiments, splicing is inhibited at the splice site.
  • base stacking of the unpaired bulged nucleotide within an RNA strand of the RNA duplex is reduced after contacting the SMSM.
  • Exemplary sites targeted by the SMSMs described herein include 5’ splice sites, 3’ splice sites, polypyrimidine tracts, branch sites, splicing enhancers and silencer elements. Mutations or aberrant secondary or tertiary RNA structures at hot spots can create mRNA sites or scaffold sequences that can be targeted. For example, many exons are flanked by the intronic dinucleotides GT and AG at the 5’ and 3’ splice sites, respectively.
  • RNA structures at these sites can cause, e.g., exclusion of an adjacent exon or inclusion of an adjacent intron.
  • Many factors influence the complex pre-mRNA splicing process including several hundred different proteins, at least five spliceosomal snRNAs, sequences on the mRNA, sequence length, enhancer and silencer elements, and strength of splicing signals.
  • Exemplary sites targeted by the SMSMs described herein include secondary and sometimes tertiary structures of RNA.
  • exemplary sites targeted by the SMSMs described herein include a stem loop, hairpin, branch point sequence (BPS), polypyrimidine tract (PPT), 5’ splice site (5’ss) and 3’ splice site (3’ss), duplex snRNA and splice sites and trans acting protein binding to RNA.
  • the target pre-mRNA can comprise a defective sequence, such as a sequence that produces a deficient protein, such as a protein with altered function such as enzyme activity, or expression, such as lack of expression.
  • the defective sequence impacts the structure of the RNA.
  • the defect sequence impacts recognition by snRNP.
  • RNA In addition to consensus splice site sequences, structural constraints, including those resulting from mutations, can affect cis-acting sequences such as exonic/intronic splicing enhancers (ESE/ISE) or silencer elements (ESS/ISS).
  • ESE/ISE exonic/intronic splicing enhancers
  • ESS/ISS silencer elements
  • a mutation in native DNA and/or pre-mRNA, or an aberrant secondary or tertiary structure of RNA creates a new splice site sequence.
  • a mutation or aberrant RNA structure may cause native regions of the RNA that are normally dormant, or play no role as splicing elements, to become activated and serve as splice sites or splice elements.
  • splice sites and elements can be referred to as “cryptic”.
  • a native intron may become divided into two aberrant introns, with a new exon situated there between.
  • a mutation may create a new splice site between a native 5’ splice site and a native branch point.
  • a mutation may activate a cryptic branch point sequence between a native splice site and a native branch point.
  • a mutation may create a new splice site between a native branch point and a native splice site and may further activate a cryptic splice site and a cryptic branch point sequentially upstream from the aberrant mutated splice site.
  • a mutation or misexpression of trans-acting proteins that regulate splicing activity may cause native regions of the RNA that are normally dormant, or play no role as splicing elements, to become activated and serve as splice sites or splice elements.
  • a mutation or misexpression of an SR protein may cause native regions of the RNA that are normally dormant, or play no role as splicing elements, to become activated and serve as splice sites or splice elements.
  • a mutation in native DNA and/or pre-mRNA inhibits splicing at a splice site.
  • a mutation may result in a new splice site upstream from (i.e., 5’ to) a native splice site sequence and downstream from (i.e., 3’ to) a native branch point sequence.
  • the native splice site sequence and the native branch point sequence may serve as members of both the native set of splice site sequences and the aberrant set of splice site sequences.
  • a native splice element e.g., a branch point
  • SMSMs provided herein can block the native element and activate a cryptic element (e.g., a cryptic 5’ss, a cryptic 3’ss or a cryptic branch point), which may recruit remaining members of the native set of splice elements to promote correct splicing over incorrect splicing.
  • a cryptic element e.g., a cryptic 5’ss, a cryptic 3’ss or a cryptic branch point
  • an activated cryptic splice element is in an intron.
  • an activated cryptic splice element is in an exon.
  • the compounds and methods provided herein can be used to block or activate a variety of different splice elements, depending on the type of aberrant splice element (e.g., mutated splice element or non-mutated splice element) and/or depending on regulation of a splice element (e.g., regulation by upstream signaling pathways).
  • the compounds and methods provided herein can block a mutated element, a non-mutated element, a cryptic element, or a native element; it may block a 5’ splice site, a 3’ splice site, or a branch point.
  • an alternate splicing event can be modulated by employing the compounds provided herein.
  • a compound provided herein can be introduced into a cell in which a gene is present that encodes a pre-mRNA that comprises alternate splice sites.
  • a first splicing event occurs to produce a gene product having a particular function.
  • the first splicing event can be inhibited.
  • the first splicing event in the presence of the compound provided herein, can be inhibited and a second or alternate splicing event occurs, resulting in expression of the same gene to produce a gene product having a different function.
  • a first inhibited splicing event e.g., a splicing event inhibited by a mutation, a mutation-induced bulge or a non-mutation induced bulge
  • a first inhibited splicing event is promoted or enhanced in the presence of a compound provided herein.
  • the first inhibited splicing event e.g., a splicing event inhibited by a mutation, a mutation-induced bulge or a non- mutation induced bulge
  • the inhibition of the first splicing event (e.g., a splicing event inhibited by a mutation, a mutation-induced bulge or a non-mutation induced bulge) can be restored to a corresponding first splicing event that is uninhibited, in the presence of a compound provided herein; or the inhibition of the first splicing event can be decreased, in the presence of a compound provided herein.
  • a second or alternate splicing event occurs, resulting in expression of the same gene to produce a gene product having a different function.
  • a method of treating a disease or condition, such as cancer in a mammal in need thereof comprising administering a therapeutically effective amount of a splice modifying compound described herein or a pharmaceutically acceptable salt or solvate thereof, to the mammal in need thereof.
  • a method of treating a disease or condition, such as cancer in a subject in need thereof comprising administering to the subject in need thereof a therapeutically effective amount of a splice modifying compound described herein.
  • the present disclosure relates to the use of a pre-mRNA splicing modifier as described herein for the preparation of a medicament for the treatment, prevention and/or delay of progression of a disease or condition, such as cancer.
  • a pre-mRNA is a human a pre-mRNA.
  • the present disclosure relates to the use of a pre-mRNA splicing modifier as described herein for the treatment, prevention and/or delay of progression of cancer.
  • a pre-mRNA splicing modifier described herein induces a transcriptionally inactive variant.
  • the present disclosure relates to a method for the treatment, prevention and/or delay of progression of a disease or condition, such as central nervous system diseases, comprising administering an effective amount of a pre-mRNA splicing modifier as described herein to a subject, in particular to a mammal.
  • a method for the treatment, prevention and/or delay of progression of a disease or condition, such as cancer comprising administering an effective amount of a pre-mRNA splicing modifier as described herein to a subject, in particular to a mammal.
  • the present disclosure relates to a pharmaceutical composition comprising a pre-mRNA splicing modifier as described herein for use in the treatment, prevention and/or delay of progression of a disease or condition, such as cancer.
  • the present disclosure relates to a pharmaceutical composition comprising a pre-mRNA splicing modifier as described herein for use in the treatment, prevention and/or delay of progression of a disease or condition, such as diseases of the central nervous system.
  • the cancer treated by the compounds of the present disclosure is selected from the group consisting of cancer of the liver, prostate, brain, breast, lung, colon, pancreas, skin, cervix, ovary, mouth, blood and nervous system.
  • the cancer treated by the compounds of the present disclosure is leukemia, acute myeloid leukemia, colon cancer, gastric cancer, macular degeneration, acute monocytic leukemia, breast cancer, hepatocellular carcinoma, cone-rod dystrophy, alveolar soft part sarcoma, myeloma, skin melanoma, prostatitis, pancreatitis, pancreatic cancer, retinitis, adenocarcinoma, adenoiditis, adenoid cystic carcinoma, cataract, retinal degeneration, gastrointestinal stromal tumor, Wegener’s granulomatosis, sarcoma, myopathy, prostate adenocarcinoma, Hodgkin’s lymphoma, ovarian cancer, non-Hodgkin’s lymphoma, multiple myeloma, chronic myeloid leukemia, acute lymphoblastic leukemia, renal cell carcinoma, transitional cell carcinoma, colorectal
  • the cancer prevented and/or treated in accordance with the present disclosure is basal cell carcinoma, goblet cell metaplasia, or a malignant glioma, cancer of the liver, breast, lung, prostate, cervix, uterus, colon, pancreas, kidney, stomach, bladder, ovary, or brain.
  • the cancer prevented and/or treated in accordance with the present disclosure include, but are not limited to, cancer of the head, neck, eye, mouth, throat, esophagus, esophagus, chest, bone, lung, kidney, colon, rectum or other gastrointestinal tract organs, stomach, spleen, skeletal muscle, subcutaneous tissue, prostate, breast, ovaries, testicles or other reproductive organs, skin, thyroid, blood, lymph nodes, kidney, liver, pancreas, and brain or central nervous system.
  • cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas.
  • cancers and conditions associated therewith that are prevented and/or treated in accordance with the present disclosure are breast carcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervical carcinomas, endometrial carcinoma, endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi’s sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma, glioblastoma, Schwannoma, oligodendroglioma, medul
  • the cancer an astrocytoma, an oligodendroglioma, a mixture of oligodendroglioma and an astrocytoma elements, an ependymoma, a meningioma, a pituitary adenoma, a primitive neuroectodermal tumor, a medullblastoma, a primary central nervous system (CNS) lymphoma, or a CNS germ cell tumor.
  • the cancer treated in accordance with the present disclosure is an acoustic neuroma, an anaplastic astrocytoma, a glioblastoma multiforme, or a meningioma.
  • the cancer treated in accordance with the present disclosure is a brain stem glioma, a craniopharyngioma, an ependyoma, a juvenile pilocytic astrocytoma, a medulloblastoma, an optic nerve glioma, primitive neuroectodermal tumor, or a rhabdoid tumor.
  • the disease of the central nervous system treated by the compounds of the present disclosure is selected from the group consisting of Alzheimer's disease, Frontotemporal dementia and parkinsonism linked to chromosome 17, Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), Argyrophilic grain disease, and Pick’s disease.
  • PSP Progressive supranuclear palsy
  • CBD Corticobasal degeneration
  • Argyrophilic grain disease Argyrophilic grain disease
  • Pick Pick’s disease.
  • Combination Treatments [00263] In certain instances, it is appropriate to administer at least one splice modifying compound described herein in combination with another therapeutic agent.
  • a splice modifying compound described herein is co- administered with a second therapeutic agent, wherein the splice modifying compound and the second therapeutic agent modulate different aspects of the disease, disorder or condition being treated, thereby providing a greater overall benefit than administration of either therapeutic agent alone.
  • dosages of the co-administered compounds vary depending on the type of co-drug(s) employed, on the specific drug(s) employed, on the disease or condition being treated and so forth.
  • the compound provided herein is administered either simultaneously with the one or more other therapeutic agents, or sequentially.
  • a pre-mRNA splicing modifier described herein is used in combination with an anti-cancer therapy. In some embodiments, a pre-mRNA splicing modifier is used in combination with conventional chemotherapy, radiotherapy, hormonal therapy, and/or immunotherapy.
  • a pre-mRNA splicing modifier is used in combination with conventional chemotherapeutic agents including alkylating agents (e.g., temozolomide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, etc.), anti-metabolites (e.g., 5-fluorouracil, azathioprine, methotrexate, leucovorin, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, pemetrexed, raltitrexed, etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g., iri
  • a compound in which the absolute stereochemistry of separated enantiomers is undetermined is represented as being either of the single enantiomers, for example (1S,2S,3R,5R) or (1R,2R,3S,5S) or drawn as being either possible single enantiomer. In such cases, the product is pure and a single enantiomer, but absolute stereochemistry is not identified, but relative stereochemistry is known and indicated.
  • Amine Intermediate Syntheses [00274] Synthesis of ( ⁇ ) tert-butyl (1S,2R,3R,5R)-2-fluoro-3-(methylamino)-8- azabicyclo[3.2.1]octane-8-carboxylate (INT 1).
  • Step 1 Synthesis of ( ⁇ ) tert-butyl (1S,2S,5R)-2-fluoro-3-oxo-8- azabicyclo[3.2.1]octane-8-carboxylate.
  • TMSCl (19.2 g, 17.78 mmol) and triethylamine (17.78 g, 17.78 mmol) was added to a stirred solution of tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8- carboxylate (20 g, 8.89 mmol) in 270 mL of DMF. The mixture was stirred at 100 o C for 16 h.
  • Step 3 Synthesis of ( ⁇ ) tert-butyl (1S,2R,3R,5R)-3-(benzylamino)-2-fluoro-8- azabicyclo[3.2.1]octane-8-carboxylate.
  • Step 4 Synthesis of ( ⁇ ) tert-butyl (1S,2R,3R,5R)-3-(benzyl(methyl)amino)-2- fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate.
  • Step 5 Synthesis of ( ⁇ ) tert-butyl (1S,2R,3R,5R)-2-fluoro-3-(methylamino)-8- azabicyclo[3.2.1]octane-8-carboxylate.
  • Step 6 Synthesis of tert-butyl (1S,2R,3R,5R)-3-((6-chloropyridazin-3- yl)(methyl)amino)-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate (INT-1).
  • DIPEA (3.97 g, 30.7 mmol) was added to a solution of 3,6-dichloropyridazine (2.2 g, 14.7 mmol) and ( ⁇ ) (1S,2R,3R,5R)-tert-butyl 3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate (3.0 g, 12.3 mmol) in DMSO (30 mL). The mixture was stirred at 120 o C for 16 h. After cooling to room temperature, the mixture was quenched with H 2 O (50 mL) and extracted with EtOAc (30 mL X 3).
  • Step 1 Synthesis of 4-bromo-2-fluoro-5-methoxybenzaldehyde.
  • 2-bromo-4-fluoro-1- methoxybenzene 130.0 g, 634.1 mmol, 1.0 equiv.
  • DCM 500 mL
  • TiCl 4 140.0 mL, 1.00 mol, 1.59 equiv.
  • dichloromethyl methyl ether 120.0 mL, 1.33 mol, 2.09 equiv.
  • Step 2 Synthesis of 4-bromo-2-fluoro-5-hydroxybenzaldehyde.
  • Step 3 Synthesis of 4-bromo-2-fluoro-5-(methoxymethoxy)benzaldehyde.
  • Step 4 Synthesis of (2E)-3-[4-bromo-2-fluoro-5-(methoxymethoxy)phenyl]prop- 2-enoic acid.
  • Step 7 Synthesis of racemic tert-butyl (1S,3R,5R)-2-fluoro-3-([6-[5-fluoro-2- (methoxymethoxy)-4-[(1E)-2-(methylcarbamoyl)eth-1-en-1-yl]phenyl]pyridazin-3- yl](methyl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate.
  • Step 8 Isolation of tert-butyl (1S,2R,3R,5R)-2-fluoro-3-([6-[5-fluoro-2- (methoxymethoxy)-4-[(1E)-2-(methylcarbamoyl) eth-1-en-1-yl]phenyl]pyridazin-3- yl](methyl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate and tert-butyl (1R,2S,3S,5S)-2- fluoro-3-([6-[5-fluoro-2-(methoxymethoxy)-4-[(1E)-2-(methylcarbamoyl)eth-1-en-1- yl]phenyl]pyridazin-3-yl](methyl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate (INT 8A & 8
  • Racemic tert-Butyl (1S,3R,5R)-2-fluoro-3-([6-[5-fluoro-2-(methoxymethoxy)-4- [(1E)-2-(methylcarbamoyl)eth-1-en-1-yl]phenyl]pyridazin-3-yl](methyl)amino)-8- azabicyclo[3.2.1]octane-8-carboxylate (360 mg, 0.63 mmol) was purified by chiral HPLC using following method: Column: Chiralpak® IF, 2x25cm, 5 Pm; Mobile Phase: MTBE (10 mM NH 3 /MeOH):EtOH 70:30; Flow rate: 20 mL/min; Cycle time: 9 min; Detection wavelength: 340/254 nm.
  • Step 9 Synthesis of (2E)-3-[2-fluoro-4-(6-[[(1R,2R,3S,5S)-2-fluoro-8- azabicyclo[3.2.1]octan-3-yl](methyl)amino]pyridazin-3-yl)-5-hydroxyphenyl]-N- methylprop-2-enamide and (2E)-3-[2-fluoro-4-(6-[[(1S,2S,3R,5R)-2-fluoro-8- azabicyclo[3.2.1]octan-3-yl](methyl)amino]pyridazin-3-yl)-5-hydroxyphenyl]-N- methylprop-2-enamide (Compound 45 & 46).
  • Example A-1 Parenteral Composition
  • 100 mg of a water-soluble salt of a com compopund described herein, or a pharmaceutically acceptable solvate thereof is dissolved in 2% HPMC, 1% Tween 80 in DI water, pH 2.2 with MSA, q.s. to at least 20 mg/mL.
  • the mixture is incorporated into a dosage unit form suitable for administration by injection.
  • Example A-2 Oral Composition
  • 100 mg of a compound described herein, or a pharmaceutically acceptable salt or solvate thereof is mixed with 750 mg of starch.
  • the mixture is incorporated into an oral dosage unit for, such as a hard gelatin capsule, which is suitable for oral administration.
  • BIOLOGY EXAMPLES [00307] KRAS Quantitative Splicing Assay [00308] COR-L-23 lung cancer cells are plated in 96-well plates at 30,000 cells/well. Immediately after plating, cells are dosed with compound for 24 h at concentrations ranging from 10 ⁇ M to 10nM (0.1% DMSO).
  • Treated cells are lysed and cDNA synthesized using the Fast Advanced Cells-to-Ct kit (Thermofisher A35378) according to the manufacturer’s instructions. 2 ⁇ L of each cDNA are used in qPCR reactions to confirm the compound-induced inclusion of a cryptic exon within intron 1 of the KRAS transcripts.
  • the qPCR reactions are prepared in 384-well plates in 10 ⁇ L volume, using TaqManTM Fast Advanced Master Mix [ThermoFisher; 4444965] with primers and probes shown in the table below. Reactions are run in a Quant Studio 7 qPCR instrument with default settings with the primers and probes shown in Table 2 below.
  • Table 2 [00310] A qPCR assay similar to the protocol described above was performed and the results are shown below in Table 3. [00311] Table 3 Fold Change range: 0.01 ⁇ A ⁇ 1; 1.01 ⁇ B ⁇ 2; 2.01 ⁇ C ⁇ 4; 4.01 ⁇ D ⁇ 10; 10.01 ⁇ E ⁇ 20. [00312]

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Abstract

L'invention concerne des agents qui modifient l'épissage de pré-ARNm et des procédés de fabrication et d'utilisation de ceux-ci.
PCT/US2020/054630 2019-10-08 2020-10-07 Composés de modulation d'épissage WO2021071982A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050250713A1 (en) * 2004-04-26 2005-11-10 Bin Zhu 11,12-Cyclic thiocarbamate macrolide antibacterial agents
WO2019028440A1 (fr) * 2017-08-04 2019-02-07 Skyhawk Therapeutics, Inc. Méthodes et compositions permettant de moduler l'épissageé

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050250713A1 (en) * 2004-04-26 2005-11-10 Bin Zhu 11,12-Cyclic thiocarbamate macrolide antibacterial agents
WO2019028440A1 (fr) * 2017-08-04 2019-02-07 Skyhawk Therapeutics, Inc. Méthodes et compositions permettant de moduler l'épissageé

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE PubChem COMPOUND NCBI; 15 January 2019 (2019-01-15), "Branaplam | C22H27N5O2", XP055817250, Database accession no. CID 135565042 *

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