Methods for treating spinal muscular atrophy

A method for the identification or validation of a compound that produces a stabilized SMNΔEx7 protein comprising:

• (A) contacting a compound with either a host cell containing an mRNA transcript transcribed from a nucleic acid construct, or a composition comprising a cell-free extract and an mRNA transcript transcribed from a nucleic acid construct, wherein the nucleic acid construct comprises, in 5′ to 3′ order:
• (a) a start codon;
• (b) the nucleic acid residues of exon 7 of wherein any number of nucleotides are inserted after the 48th nucleotide residue from the 5′ end of exon 7 of SMN as long as the native stop codon of exon 7 of SMN is inactivated and any additional stop codon is not generated;
• (c) a fragment of the nucleic acid residues of exon 8 of SMN:
• and
• (d) a reporter gene coding sequence lacking a start codon, wherein:
• (i) the reporter gene coding sequence is fused to the fragment of the nucleic acid residues of exon 8 of SMN such that the first codon of the reporter gene coding sequence and the first codon of the fragment are out of frame with each other in the mRNA transcript transcribed from the nucleic acid construct and there is a stop codon upstream of the reporter gene coding sequence in the region of the mRNA transcript that corresponds to the fragment of the nucleic acid residues of exon 8 of SMN;
• and
• (ii) the first start codon and the stop codon upstream from the reporter gene coding sequence in the mRNA transcript are in the same contiguous open reading frame without any interruption;
• and
• (B) detecting the activity or amount of a fusion protein translated from the mRNA transcript, wherein an increase in the activity or amount of the fusion protein translated from the mRNA transcript in the presence of a compound when compared to:
• (i) a previously determined reference range for a negative control,
• (ii) the activity or amount of the fusion protein translated from the mRNA transcript in the absence of the compound, or
• (iii) the activity or amount of the fusion protein translated from the mRNA transcript in the presence of a negative control indicates that the compound produces a stabilized SMNΔEx7 protein.

A method for the identification or validation of a compound that produces a stabilized SMNΔEx7 protein comprising:

• (A) contacting a compound with either a host cell containing an mRNA transcript transcribed from a nucleic acid construct, or a composition comprising a cell-free extract and an mRNA transcript transcribed from a nucleic acid construct, in 5′ to 3′ order:
• (a) a start codon;
• (b) the nucleic acid residues of exon 7 of SMN, wherein a single guanine residue is inserted after the 48111 nucleotide residue from the 5′ end of exon 7 of SAN;
• (c) the nucleic acid residues of intron 7 of SMN or a fragment thereof, wherein the fragment of the nucleic acid residues of intron 7 comprises any number of nucleotides of intron 7 of SMN required for a functional, minimum intron;
• (d) a fragment of the nucleic acid residues of exon 8 of SMN;
• and
• (e) a reporter gene coding sequence lacking a start codon, wherein:
• (i) the reporter gene coding sequence is fused to the fragment of the nucleic acid residues of exon 8 of SMN such that the first codon of the reporter gene coding sequence and the first codon of the fragment are out of frame with each other in the mRNA transcript transcribed from the nucleic acid construct;
• and
• (ii) the production of the mRNA transcript generates a stop codon upstream from the reporter gene coding sequence in the region of the mRNA transcript that corresponds to the fragment of the nucleic acid residues of exon 8 of SMN;
• and
• (iii) the first start codon and the stop codon upstream from the reporter gene coding sequence in the mRNA transcript are in the same contiguous open reading frame without any interruption;
• and
• (B) detecting the activity or amount of a fusion protein translated from the mRNA transcript, wherein an increase in the activity or amount of the fusion protein translated from the mRNA transcript in the presence of a compound when compared to:
• (i) a previously determined reference range for a negative control,
• (ii) the activity or amount of the fusion protein translated from the mRNA transcript in the absence of the compound, or
• (iii) the activity or amount of the fusion protein translated from the mRNA transcript in the presence of a negative control indicates that the compound produces a stabilized SMNΔEx7 protein.

A method for the identification or validation of a compound that produces a stabilized SMNΔEx7 protein comprising:

• (A) contacting a compound with either a host cell containing an mRNA transcript transcribed from a nucleic acid construct, or a composition comprising a cell-free extract and an mRNA transcript transcribed from a nucleic acid construct, in 5′ to 3′ order:
• (a) a start codon;
• (b) a minimum of one nucleotide;
• (c) a fragment of the nucleic acid residues of exon 7 of SMN, wherein (i) the fragment of the nucleic acid residues of exon 7 of SMN comprises a minimum of the first six nucleotides from the 3′ end of exon 7 of SMN and wherein a single guanine residue is inserted into the fragment of the nucleic acid residues of exon 7 of SMN at the location that corresponds to the location in exon 7 of SMN that is after the 48th nucleotide from the 5′ end of exon 7 of SMN or (ii) the fragment of the nucleic acid residues of exon 7 of SMN consists of the first six nucleotides from the 3′ end of exon 7 of SMN and wherein a single guanine residue is inserted upstream (5′) of the fragment of the nucleic acid residues of exon 7 of SMN;
• (d) the nucleic acid residues of intron 7 of SMN or a fragment thereof, wherein the fragment of the nucleic acid residues of intron 7 of SMN comprises any number of nucleotides of intron 7 required for a functional, minimum intron;
• (e) a fragment of the nucleic acid residues of exon 8 of SMN;
• and
• (f) a reporter gene coding sequence lacking a start codon, wherein
• (i) the reporter gene coding sequence is fused to the fragment of the nucleic acid residues of exon 8 of SMN such that the first codon of the reporter gene coding sequence and the first codon of the fragment are out of frame with each other in the mRNA transcript transcribed from the nucleic acid construct;
• and
• (ii) the production of the mRNA transcript generates a stop codon upstream from the reporter gene coding sequence in the region of the mRNA transcript that corresponds to the fragment of the nucleic acid residues of exon 8 of SMN;
• and
• (iii) the first start codon and the stop codon upstream from the reporter gene coding sequence in the mRNA transcript are in the same contiguous open reading frame without any interruption;
• and
• (B) detecting the activity or amount of a fusion protein translated from the mRNA transcript, wherein an increase in the activity or amount of the fusion protein translated from the mRNA transcript in the presence of a compound when compared to:
• (i) a previously determined reference range for a negative control,
• (ii) the activity or amount of the fusion protein translated from the mRNA transcript in the absence of the compound, or
• (iii) the activity or amount of the fusion protein translated from the mRNA transcript in the presence of a negative control indicates that the compound produces a stabilized SMNΔEx7 protein.

A method for producing a stabilized SMNΔEx7 protein in a human subject in need thereof, comprising administering to the human subject an effective amount of a compound or a pharmaceutical composition thereof. wherein the compound is selected from compounds of Formula (I) or a form thereof or Formula (II) or a form thereof, wherein Formula (I) and Formula (II) have the following structures:

• wherein,
• W is selected from the group consisting of C(O), C(S), and CH2;
• B is CH2 or CH(CnH2n+1), wherein n is an integer from 1 to 8;
• Ring C is selected from the group consisting of a fused thienyl ring, a fused pyridinyl ring, and a fused cyclohexyl ring, any of which can be saturated or contain, one or two non-conjugated double bonds;
• R1 and R2 are independently selected from the group consisting of H and C1-C3 alkyl, or R1 and R2 may be taken together with the carbon atom to which they are attached to form a C3-C6 cycloalkyl ring or a carbonyl group;
• R3 is selected from the group consisting of H, halogen, C1-C4;
• alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, CN, NO2, heteroaryl, and phenyl optionally substituted with any combination of one to five halogen, NO2, CN, C1-C4 alkyl, C1-C4 haloalkyl, or C1-C4 alkoxy substituents;
• R4, R5, R6 and R7 are independently selected from the group consisting of H, hydroxyl, halogen, CN, NO2, sulfonamide, C1-C8 alkyl, C3-C6 cycloalkyl, cycloalkyloxy, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C4 haloalkyl, C2-C8 alkenyl, amino, C1-C4 alkylamino, C1-C4 dialkylamino, C3-C6 cycloalkylamino, morpholinyl, heteroaryl, arylamino, arylalkylamino, phenyl, C(O)R′, NR′(COR″), NR′SO2R″ and NR′(CONR″R′″), wherein R′, R″ and R″′ are independently H, C1-C6 alkyl, phenyl, or substituted phenyl, and wherein C1-C8 alkyl is optionally substituted with one or more substituents selected from the group consisting of C1-C4 alkoxy, C1-C4 haloalkyl, C1-C6 dialkylamino, C1-C6 alkylamino, cycloalkylamino, and morpholinyl, and the phenyl is optionally substituted with one or more substituents selected from the group consisting of halogen, NO2, CN, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy, or R4 and R5, R5 and R6, or R6 and R7, taken together with the carbon to which they are attached, form a ring;
• X is selected from the group consisting of H;
• CN;
• C(O)OR8, wherein R8 is H or C1-C8 alkyl, and C1-C8 alkyl optionally is substituted with one or more substituents selected from the group consisting of C1-C4 alkoxy, haloalkyl, C1-C6 dialkylamino, C1-C6 alkylamino, cycloalkylamino, phenyl, and morpholinyl;
• C(O)NR9R10 or CH2NR9R10, wherein R9 and R10 are independently selected from the group consisting of H and C1-C6 alkyl, or R9 and R10 together with the nitrogen to which they are attached form a heterocyclyl ring;
• CH2OR11, wherein R11 is H, C1-C8 alkyl, or C3-C6 cycloalkyl, wherein C1-C8 alkyl is optionally substituted with one or more substituents selected from the group consisting of C1-C4 alkoxy, C1-C4 haloalkyl, C1-C6 dialkylamino, C1-C6 alkylamino, cycloalkylamino, and morpholinyl;
• CH2Z, wherein Z is halogen;
• C(O)NHOH;
• C(O)NHCN;
• C(O)N(R1)SO2R13, wherein R13 is C1-C4 alkyl, phenyl, or substituted phenyl;
• C1-C8 alkyl, optionally substituted with one or more substituents selected from the group consisting of C1-C4 alkoxy, C1-C4 haloalkyl, C1-C6 dialkylamino, and C1-C6 alkylamino;
• and C2-C8 alkenyl, optionally substituted with one or more substituents selected from the group consisting of C1-C4 alkoxy, C1-C4 haloalkyl, C1-C6 dialkylamino, and C1-C6 alkylamino.

A method for producing a stabilized SMNΔEx7 protein in a human subject in need thereof, comprising administering to the human subject an effective amount of a compound or a pharmaceutical composition thereof, wherein the compound is selected from compounds of Formula (Ia) or a form thereof or Formula (IIa) or a form thereof, wherein Formula (Ia) and Formula (IIa) have the following structures:

• wherein,
• W1 is selected from the group consisting of C(O), C(S), and CH2;
• B1 is CH, or CH(CmH2m+1), wherein m is an integer from 1 to 8;
• Ring C1 is selected from the group consisting of a thienyl ring, a pyridinyl ring, a cyclohexyl ring, a benzo[a][1,3]dioxolyl ring and a 2,3-dihydrobenzo[b][1,4]dioxinyl ring, each of said rings fused to the moiety of Formula (IIa), wherein benzo[d][1,3]dioxolyl and 2,3-dihydrobenzo[b][1,4]dioxinyl, each having a benzo ring portion, are fused via said benzoportion, and wherein any of the foregoing rings may optionally be fully or partially saturated;
• R20 and R21 are independently selected from the group consisting of H and C1-C3 alkyl, or R20 and R21 may be taken together with the carbon atom to which they are attached to form a C3-C6 cycloalkyl ring or a carbonyl group;
• R22 is selected from the group consisting of H, halogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, cyano, nitro, heteroaryl, and phenyl optionally substituted with any combination of one to five halogen, nitro, cyano, C1-C4 alkyl, C1-C4 haloalkyl or C1-C4 alkoxy substituents;
• R23, R24, R25 and R26 are independently selected from the group consisting of H, hydroxyl, halogen, cyano, nitro, sulfonamide, C1-C8 alkyl, C1-C6 alkoxy, C1-C6 alkoxyalkoxy, C1-C6 alkoxyalkyl, C1-C6 haloalkoxy, C1-C4 haloalkyl, C2-C6 alkenyl, C2-C4 haloalkenyl, formyl, C1-C6 alkylcarbonyl, amino, C1-C4 alkylamino, C1-C4 dialkylamino, C1-C4 aminoalkyl, C1-C4 alkylaminoalkyl, C1-C4 dialkylaminoalkyl phenyl, C3-C6 cycloalkyl, C3-C6 cycloalkylalkyl, C3-C6 cycloalkylalkoxy, cycloalkyloxy, heterocyclyl, heterocyclylalkyl, heteroaryl, and phenylcarbonyl,
• wherein amino is optionally disubstituted with one substituent selected from hydrogen, C1-C6 alkyl or phenyl and the other is selected from formyl, phenyl, C3-C6 cycloalkyl, C1-C6 alkylcarbonyl, aminocarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 dialkylaminocarbonyl, phenylcarbonyl, phenylaminocarbonyl, N-phenyl-N—C1-C6 alkyl-aminocarbonyl, C1-C6 alkylsulfonyl, aminosulfonyl, C1-C6 alkylaminosulfonyl, C1-C6 dialkylaminosulfonyl or phenylsulfonyl,
• wherein each instance of C1-C6 alkylcarbonyl is optionally substituted on the alkyl portion with one or more substituents selected from the group consisting of halogen, C1-C4 alkoxy, C1-C6 alkylamino, C1-C6 dialkylamino, cycloalkylamino and heterocyclyl,
• wherein each instance of phenyl is optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, cyano, C1-C4 alkyl, C1-C4 haloalkyl and C1-C4 alkoxy, and
• alternatively, R23 and R24, R24 and R25 or R25 and R26 may be taken together with the carbon to which they are attached to form a C3-C6 cycloalkyl ring fused to the moiety of Formula (Ia);
• X1 is absent or is selected from the group consisting of H, cyano, C1-C8 alkyl, C1-C4 alkoxy, amino, C1-C4 alkylamino, C1-C4 dialkylamino, carboxy, C1-C8 alkoxycarbonyl, aminocarbonyl, C1-C8 alkylaminocarbonyl, C1-C8 dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl, phenylaminocarbonyl, aminosulfonylaminocarbonyl, C1-C8 alkylaminosulfonylaminocarbonyl, C1-C8 dialkylaminosulfonylaminocarbonyl, C1-C8 alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl and heterocyclylcarbonyl,
• wherein C1-C4 alkoxy and the C1-C8 alkoxy portion of C1-C8 alkoxycarbonyl is optionally substituted with one or more substituents selected from the group consisting of halogen, C1-C4 alkoxy. C1-C4 haloalkyl amino, C1-C6 alkylamino, C1-C6 dialkylamino, cycloalkylamino, phenyl and heterocyclyl,
• wherein C1-C8 alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, C1-C4 haloalkyl, C2-C8 alkenyl, C1-C6 alkoxy, C1-C4 alkoxyalkoxy, C3-C6 cycloalkyloxy, amino, C1-C6 alkylamino, C1-C6 dialkylamino, cycloalkylamino, aminocarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl, C1-C6 alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl and heterocyclyl, wherein C1-C4 alkoxy or C2-C8 alkenyl are each further optionally substituted with one or more substituents selected from the group consisting of C1-C4 alkoxy, C1-C4 haloalkyl, amino, C1-C6 alkylamino and C1-C6 dialkylamino.