Claims for Patent: 8,932,818

Summary for Patent: 8,932,818

Title:	Screening assays for compounds that modulate programmed ribosomal frameshifting
Abstract:	The present invention relates to compounds that modulate ribosomal frameshifting and nucleic acid constructs for use in methods for identifying or validation of compounds that modulate ribosomal frameshifting. In particular, the present invention relates to the use of nucleic acid constructs to identify or validate compounds capable of modulating the efficiency of programmed ribosomal frameshifting and the use of compounds that modulate the efficiency of programmed ribosomal frameshifting to inhibit the replication or infectivity of viruses that employ programmed ribosomal frameshifting.
Inventor(s):	Paushkin; Sergey V. (Belle Mead, NJ), Naryshkin; Nikolai A. (East Brunswick, NJ), Welch; Ellen (Califon, NJ)
Assignee:	PTC Therapeutics, Inc. (South Plainfield, NJ)
Application Number:	13/058,613
Patent Claims:	1. A method for screening compounds for a compound that modulates the efficiency of viral programmed ribosomal frameshifting comprising: (a) contacting a compound with a host cell containing an mRNA transcript encoded by a nucleic acid construct, wherein said nucleic acid construct comprises, in 5' to 3' order: (i) a start codon; (ii) the nucleic acid residues of exon 7 of SMN, wherein a single guanine residue is inserted after the 48.sup.th nucleotide residue from the 5' end of exon 7 of SMN; (iii) 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; (iv) a fragment of the nucleic acid residues of exon 8 of SMN; and (v) a reporter gene coding sequence lacking a start codon, wherein: (A) 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 start codon are out of frame with each other in the mRNA transcript transcribed from the nucleic acid construct; and (B) 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 (C) the 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; and (b) detecting the activity or amount of a fusion protein translated from the mRNA transcript, wherein an increase of 1.5-fold or more 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 modulates the efficiency of programmed ribosomal frameshifting; and (c) contacting a compound that increases the activity or amount of the fusion protein translated from the mRNA transcript with a cell containing a virus that employs programmed ribosomal frameshifting and assaying the ability of the compound to decrease viral replication, wherein a decrease in viral replication of at least 1.5 log 10 in the presence of the compound indicates that a compound that modulates the efficiency of viral programmed ribosomal frameshifting is identified. 2. The method of claim 1, wherein the compound is selected from compounds of Formula (I) or Formula (II), wherein Formula (I) and Formula (II) have the following structures: ##STR00004## wherein, W is selected from the group consisting of C(O), C(S), and CH.sub.2; B is CH.sub.2 or CH(C.sub.nH.sub.2n+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; R.sub.1 and R.sub.2 are independently selected from the group consisting of H and C.sub.1-C.sub.3 alkyl, or R.sub.1 and R.sub.2 may be taken together with the carbon atom to which they are attached to form a C.sub.3-C.sub.6 cycloalkyl ring or a carbonyl group; R.sub.3 is selected from the group consisting of H, halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, CN, NO.sub.2, heteroaryl, and phenyl optionally substituted with any combination of one to five halogen, NO.sub.2, CN, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, or C.sub.1-C.sub.4 alkoxy substituents; R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are independently selected from the group consisting of H, hydroxyl, halogen, CN, NO.sub.2, sulfonamide, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.6 cycloalkyl, cycloalkyloxy, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 haloalkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.2-C.sub.8 alkenyl, amino, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, C.sub.3-C.sub.6 cycloalkylamino, morpholinyl, heteroaryl, arylamino, arylalkylamino, phenyl, C(O)R', NR'(COR''), NR'SO.sub.2R'' and NR'(CONR''R'''), wherein R', R'' and R''' are independently H, C.sub.1-C.sub.6 alkyl, phenyl, or substituted phenyl, and wherein C.sub.1-C.sub.8 alkyl is optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino, C.sub.1-C.sub.6 alkylamino, cycloalkylamino, and morpholinyl, and the phenyl is optionally substituted with one or more substituents selected from the group consisting of halogen, NO.sub.2, CN, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, and C.sub.1-C.sub.4 alkoxy, or R.sub.4 and R.sub.5, R.sub.5 and R.sub.6, or R.sub.6 and R.sub.7, 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)OR.sub.8, wherein R.sub.8 is H or C.sub.1-C.sub.8 alkyl, and C.sub.1-C.sub.8 alkyl optionally is substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino, C.sub.1-C.sub.6 alkylamino, cycloalkylamino, phenyl, and morpholinyl; C(O)NR.sub.9R.sub.10 or CH.sub.2NR.sub.9R.sub.10, wherein R.sub.9 and R.sub.10 are independently selected from the group consisting of H and C.sub.1-C.sub.6 alkyl, or R.sub.9 and R.sub.10 together with the nitrogen to which they are attached form a heterocyclyl ring; CH.sub.2OR.sub.ii, wherein R.sub.11 is H, C.sub.1-C.sub.8 alkyl, or C.sub.3-C.sub.6 cycloalkyl, wherein C.sub.1-C.sub.8 alkyl is optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino, C.sub.1-C.sub.6 alkylamino, cycloalkylamino, and morpholinyl; CH.sub.2Z, wherein Z is halogen; C(O)NHOH; C(O)NHCN; C(O)N(R.sub.1)SO.sub.2R.sub.13, wherein R.sub.13 is C.sub.1-C.sub.4 alkyl, phenyl, or substituted phenyl; C.sub.1-C.sub.8 alkyl, optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino, and C.sub.1-C.sub.6 alkylamino; and C.sub.2-C.sub.8 alkenyl, optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino, and C.sub.1-C.sub.6 alkylamino. 3. The method of claim 1, wherein the compound is selected from compounds of Formula (Ia) or Formula (IIa), wherein Formula (Ia) and Formula (IIa) have the following structures: ##STR00005## wherein, W.sub.1 is selected from the group consisting of C(O), C(S), and CH.sub.2; B.sub.1 is CH.sub.2 or CH(C.sub.mH.sub.2m+1), wherein m is an integer from 1 to 8; Ring C.sub.1 is selected from the group consisting of a thienyl ring, a pyridinyl ring, a cyclohexyl ring, a benzo[d][1,3]dioxolyl ring and a 2,3-dihydrobenzo[b][1,4]dioxinyl ring, wherein benzo[d][1,3]dioxolyl and 2,3-dihydrobenzo[b][1,4]dioxinyl, each having a benzo ring portion, are fused via said benzo portion, and wherein any of the foregoing rings may optionally be fully or partially saturated; R.sub.20 and R.sub.21 are independently selected from the group consisting of H and C.sub.1-C.sub.3 alkyl, or R.sub.20 and R.sub.21 may be taken together with the carbon atom to which they are attached to form a C.sub.3-C.sub.6 cycloalkyl ring or a carbonyl group; R.sub.22 is selected from the group consisting of H, halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, cyano, nitro, heteroaryl, and phenyl optionally substituted with any combination of one to five halogen, nitro, cyano, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl or C.sub.1-C.sub.4 alkoxy substituents; R.sub.23, R.sub.24, R.sub.25 and R.sub.26 are independently selected from the group consisting of H, hydroxyl, halogen, cyano, nitro, sulfonamide, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkoxyalkoxy, C.sub.1-C.sub.6 alkoxyalkyl, C.sub.1-C.sub.6 haloalkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.4 haloalkenyl, formyl, C.sub.1-C.sub.6 alkylcarbonyl, amino, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylaminoalkyl, C.sub.1-C.sub.4 dialkylaminoalkyl, phenyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.3-C.sub.6 cycloalkylalkyl, C.sub.3-C.sub.6 cycloalkylalkoxy, cycloalkyloxy, heterocyclyl, heterocyclylalkyl, heteroaryl, and phenylcarbonyl, wherein amino is optionally disubstituted with one substituent selected from hydrogen, C.sub.1-C.sub.6 alkyl or phenyl and the other is selected from formyl, phenyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.1-C.sub.6 alkylcarbonyl, aminocarbonyl, C.sub.1-C.sub.6 alkylaminocarbonyl, C.sub.1-C.sub.6 dialkylaminocarbonyl, phenylcarbonyl, phenylaminocarbonyl, N-phenyl-N--C.sub.1-C.sub.6 alkyl-aminocarbonyl, C.sub.1-C.sub.6 alkylsulfonyl, aminosulfonyl, C.sub.1-C.sub.6 alkylaminosulfonyl, C.sub.1-C.sub.6 dialkylaminosulfonyl or phenylsulfonyl, wherein each instance of C.sub.1-C.sub.6 alkylcarbonyl is optionally substituted on the alkyl portion with one or more substituents selected from the group consisting of halogen, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.6 alkylamino, C.sub.1-C.sub.6 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, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl and C.sub.1-C.sub.4 alkoxy, and alternatively, R.sub.23 and R.sub.24, R.sub.24 and R.sub.25 or R.sub.25 and R.sub.26 may be taken together with the carbons to which they are attached to form a C.sub.3-C.sub.6 cycloalkyl ring; X.sub.1 is absent or is selected from the group consisting of H, cyano, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.4 alkoxy, amino, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, carboxy, C.sub.1-C.sub.8 alkoxycarbonyl, aminocarbonyl, C.sub.1-C.sub.8 alkylaminocarbonyl, C.sub.1-C.sub.8 dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl, phenylaminocarbonyl, aminosulfonylaminocarbonyl, C.sub.1-C.sub.8 alkylaminosulfonylaminocarbonyl, C.sub.1-C.sub.8 dialkylaminosulfonylaminocarbonyl, C.sub.1-C.sub.8 alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl and heterocyclylcarbonyl, wherein C.sub.1-C.sub.4 alkoxy and the C.sub.1-C.sub.8 alkoxy portion of C.sub.1-C.sub.8 alkoxycarbonyl is optionally substituted with one or more substituents selected from the group consisting of halogen, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, amino, C.sub.1-C.sub.6 alkylamino, C.sub.1-C.sub.6 dialkylamino, cycloalkylamino, phenyl and heterocyclyl, wherein C.sub.1-C.sub.8 alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, C.sub.1-C.sub.4 haloalkyl, C.sub.2-C.sub.8 alkenyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.4 alkoxyalkoxy, C.sub.3-C.sub.6 cycloalkyloxy, amino, C.sub.1-C.sub.6 alkylamino, C.sub.1-C.sub.6 dialkylamino, cycloalkylamino, aminocarbonyl, C.sub.1-C.sub.6 alkylaminocarbonyl, C.sub.1-C.sub.6 dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl, C.sub.1-C.sub.6 alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl and heterocyclyl, wherein C.sub.1-C.sub.4 alkoxy or C.sub.2-C.sub.8 alkenyl are each further optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, amino, C.sub.1-C.sub.6 alkylamino and C.sub.1-C.sub.6 dialkylamino. 4. The method of claim 3, wherein: (a) m is an integer selected from 1, 2 or 3; and, wherein Ring C.sub.1 is selected from the group consisting of a thienyl ring, a pyridinyl ring, a cyclohexyl ring, a cyclohexenyl ring, a cyclohexa-1,4-dienyl ring, a benzo[d][1,3]dioxolyl ring and a 2,3-dihydrobenzo[b][1,4]dioxinyl ring, wherein benzo[d][1,3]dioxolyl and 2,3-dihydrobenzo[b][1,4]dioxinyl, each having a benzo ring portion, are fused via said benzo portion; (b) R.sub.20 and R.sub.21 are each H; alternatively, R.sub.20 and R.sub.21 are each C.sub.1-C.sub.3 alkyl; (c) R.sub.22 is selected from the group consisting of H, halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, cyano, thienyl, furanyl, pyridinyl, pyrimidinyl and phenyl, wherein phenyl is optionally substituted with one or two halogen, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy substituents; (d) when one, two or three of R.sub.23, R.sub.24, R.sub.25 and R.sub.26 are each H, then three, two or one of R.sub.23, R.sub.24, R.sub.25 and R.sub.26, respectively, are each selected from hydroxyl, halogen, cyano, nitro, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkoxyalkoxy, C.sub.1-C.sub.6 alkoxyalkyl, C.sub.1-C.sub.6 difluoroalkoxy, C.sub.1-C.sub.6 trifluoroalkoxy, C.sub.1-C.sub.4 trifluoroalkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.4 trifluoroalkenyl, amino, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylaminoalkyl or C.sub.1-C.sub.4 dialkylaminoalkyl; (e) when three of R.sub.23, R.sub.24, R.sub.25 and R.sub.26 are each H, then one of R.sub.23, R.sub.24, R.sub.25 and R.sub.26 is selected from phenyl, cyclopentyl, cyclopropyl, benzyloxy, C.sub.1-C.sub.4 cyclopentylalkoxy, C.sub.1-C.sub.4 cyclobutylalkoxy, cyclopentyloxy, pyrrolidinyl, piperidinyl, morpholinyl, C.sub.1-C.sub.4 morpholinylalkyl, thienyl, pyridinyl, pyrimidinyl, or amino, wherein amino is optionally disubstituted with one substituent selected from hydrogen or C.sub.1-C.sub.6 alkyl and the other is selected from phenyl, C.sub.1-C.sub.4 alkylcarbonyl, aminocarbonyl, C.sub.1-C.sub.4 alkylaminocarbonyl, C.sub.1-C.sub.4 dialkylaminocarbonyl, phenylcarbonyl, phenylaminocarbonyl, N-phenyl-N--C.sub.1-C.sub.4 alkyl-aminocarbonyl, C.sub.1-C.sub.6 alkylsulfonyl or phenylsulfonyl, and wherein each instance of phenyl is optionally substituted with one or two substituents selected from halogen, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy; or (f) X.sub.1 is absent or is selected from the group consisting of H, cyano, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 hydroxylalkyl, C.sub.1-C.sub.6 alkoxyalkyl, C.sub.1-C.sub.4 morpholinylalkyl, amino, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylaminoalkyl, C.sub.1-C.sub.4 dialkylaminoalkyl, carboxy, C.sub.1-C.sub.6 alkoxycarbonyl, benzyloxycarbonyl, aminocarbonyl, C.sub.1-C.sub.8 alkylaminocarbonyl, C.sub.1-C.sub.8 dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl, phenylaminocarbonyl, aminosulfonylaminocarbonyl, C.sub.1-C.sub.8 alkylaminosulfonylaminocarbonyl C.sub.1-C.sub.8 dialkylaminosulfonylaminocarbonyl, C.sub.1-C.sub.8 alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl, morpholinylcarbonyl and piperidinylcarbonyl. 5. The method of claim 1, wherein the nucleic acid construct comprises the nucleic acid residues of exon 6 of SMN or a fragment thereof downstream (3') to the start codon and upstream (5') of the nucleic acid residues of exon 7 of SMN, wherein the fragment of the nucleic acid residues of exon 6 of SMN comprises any number of nucleotides of exon 6 of SMN so long as in the mRNA transcript the first start codon and the stop codon upstream of the reporter gene coding sequence are maintained in the same contiguous open reading frame. 6. The method of claim 5, wherein the nucleic acid construct comprises the nucleic acid residues of intron 6 of SMN or a fragment thereof downstream (3') of the nucleic acid residues of exon 6 of SMN or a fragment thereof and upstream (5') of the nucleic acid residues of exon 7 of SMN, wherein the fragment of the nucleic acid residues of intron 6 of SMN comprises any number of nucleotides of intron 6 of SMN required for a functional, minimum intron. 7. The method of claim 1, further comprising contacting the host cell with a positive control. 8. The method of claim 7, wherein said positive control is Compound 1: ##STR00006## 9. A method for screening compounds for a compound that modulates the efficiency of viral programmed ribosomal frameshifting comprising: (a) contacting a compound with a composition comprising a cell-free extract and an mRNA transcript transcribed from a nucleic acid construct, wherein said nucleic acid construct comprises, in 5' to 3' order: (i) a start codon; (ii) the nucleic acid residues of exon 7 of SMN, wherein a single guanine residue is inserted after the 48.sup.th nucleotide residue from the 5' end of exon 7 of SMN; (iii) 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; (iv) a fragment of the nucleic acid residues of exon 8 of SMN; and (v) a reporter gene coding sequence lacking a start codon, wherein: (A) 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 start codon are out of frame with each other in the mRNA transcript transcribed from the nucleic acid construct; and (B) 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 (C) the 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; and (b) detecting the activity or amount of a fusion protein translated from the mRNA transcript, wherein an increase of 1.5-fold or more 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 modulates the efficiency of programmed ribosomal frameshifting; and (c) contacting a compound that increases the activity or amount of the fusion protein translated from the mRNA transcript with a cell containing a virus that employs programmed ribosomal frameshifting and assaying the ability of the compound to decrease viral replication, wherein a decrease in viral replication of at least 1.5 log 10 in the presence of the compound indicates that a compound that modulates the efficiency of viral programmed ribosomal frameshifting is identified. 10. The method of claim 9, wherein the compound is selected from compounds of Formula (I) or Formula (II), wherein Formula (I) and Formula (II) have the following structures: ##STR00007## wherein, W is selected from the group consisting of C(O), C(S), and CH.sub.2; B is CH.sub.2 or CH(C.sub.nH.sub.2n+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; R.sub.1 and R.sub.2 are independently selected from the group consisting of H and C.sub.1-C.sub.3 alkyl, or R.sub.1 and R.sub.2 may be taken together with the carbon atom to which they are attached to form a C.sub.3-C.sub.6 cycloalkyl ring or a carbonyl group; R.sub.3 is selected from the group consisting of H, halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, CN, NO.sub.2, heteroaryl, and phenyl optionally substituted with any combination of one to five halogen, NO.sub.2, CN, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, or C.sub.1-C.sub.4 alkoxy substituents; R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are independently selected from the group consisting of H, hydroxyl, halogen, CN, NO.sub.2, sulfonamide, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.6 cycloalkyl, cycloalkyloxy, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 haloalkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.2-C.sub.8 alkenyl, amino, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, C.sub.3-C.sub.6 cycloalkylamino, morpholinyl, heteroaryl, arylamino, arylalkylamino, phenyl, C(O)R', NR'(COR''), NR'SO.sub.2R'' and NR'(CONR''R'''), wherein R', R'' and R''' are independently H, C.sub.1-C.sub.6 alkyl, phenyl, or substituted phenyl, and wherein C.sub.1-C.sub.8 alkyl is optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino, C.sub.1-C.sub.6 alkylamino, cycloalkylamino, and morpholinyl, and the phenyl is optionally substituted with one or more substituents selected from the group consisting of halogen, NO.sub.2, CN, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, and C.sub.1-C.sub.4 alkoxy, or R.sub.4 and R.sub.5, R.sub.5 and R.sub.6, or R.sub.6 and R.sub.7, 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)OR.sub.8, wherein R.sub.8 is H or C.sub.1-C.sub.8 alkyl, and C.sub.1-C.sub.8 alkyl optionally is substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino, C.sub.1-C.sub.6 alkylamino, cycloalkylamino, phenyl, and morpholinyl; C(O)NR.sub.9R.sub.10 or CH.sub.2NR.sub.9R.sub.10, wherein R.sub.9 and R.sub.10 are independently selected from the group consisting of H and C.sub.1-C.sub.6 alkyl, or R.sub.9 and R.sub.10 together with the nitrogen to which they are attached form a heterocyclyl ring; CH.sub.2OR.sub.ii, wherein R.sub.11 is H, C.sub.1-C.sub.8 alkyl, or C.sub.3-C.sub.6 cycloalkyl, wherein C.sub.1-C.sub.8 alkyl is optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino, C.sub.1-C.sub.6 alkylamino, cycloalkylamino, and morpholinyl; CH.sub.2Z, wherein Z is halogen; C(O)NHOH; C(O)NHCN; C(O)N(R.sub.1)SO.sub.2R.sub.13, wherein R.sub.13 is C.sub.1-C.sub.4 alkyl, phenyl, or substituted phenyl; C.sub.1-C.sub.8 alkyl, optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino, and C.sub.1-C.sub.6 alkylamino; and C.sub.2-C.sub.8 alkenyl, optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 dialkylamino, and C.sub.1-C.sub.6 alkylamino. 11. The method of claim 9, wherein the compound is selected from compounds of Formula (Ia) or Formula (IIa), wherein Formula (Ia) and Formula (IIa) have the following structures: ##STR00008## wherein, W.sub.1 is selected from the group consisting of C(O), C(S), and CH.sub.2; B.sub.1 is CH.sub.2 or CH(C.sub.mH.sub.2m+1), wherein m is an integer from 1 to 8; Ring C.sub.1 is selected from the group consisting of a thienyl ring, a pyridinyl ring, a cyclohexyl ring, a benzo[d][1,3]dioxolyl ring and a 2,3-dihydrobenzo[b][1,4]dioxinyl ring, wherein benzo[d][1,3]dioxolyl and 2,3-dihydrobenzo[b][1,4]dioxinyl, each having a benzo ring portion, are fused via said benzo portion, and wherein any of the foregoing rings may optionally be fully or partially saturated; R.sub.20 and R.sub.21 are independently selected from the group consisting of H and C.sub.1-C.sub.3 alkyl, or R.sub.20 and R.sub.21 may be taken together with the carbon atom to which they are attached to form a C.sub.3-C.sub.6 cycloalkyl ring or a carbonyl group; R.sub.22 is selected from the group consisting of H, halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, cyano, nitro, heteroaryl, and phenyl optionally substituted with any combination of one to five halogen, nitro, cyano, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl or C.sub.1-C.sub.4 alkoxy substituents; R.sub.23, R.sub.24, R.sub.25 and R.sub.26 are independently selected from the group consisting of H, hydroxyl, halogen, cyano, nitro, sulfonamide, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkoxyalkoxy, C.sub.1-C.sub.6 alkoxyalkyl, C.sub.1-C.sub.6 haloalkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.4 haloalkenyl, formyl, C.sub.1-C.sub.6 alkylcarbonyl, amino, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylaminoalkyl, C.sub.1-C.sub.4 dialkylaminoalkyl, phenyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.3-C.sub.6 cycloalkylalkyl, C.sub.3-C.sub.6 cycloalkylalkoxy, cycloalkyloxy, heterocyclyl, heterocyclylalkyl, heteroaryl, and phenylcarbonyl, wherein amino is optionally disubstituted with one substituent selected from hydrogen, C.sub.1-C.sub.6 alkyl or phenyl and the other is selected from formyl, phenyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.1-C.sub.6 alkylcarbonyl, aminocarbonyl, C.sub.1-C.sub.6 alkylaminocarbonyl, C.sub.1-C.sub.6 dialkylaminocarbonyl, phenylcarbonyl, phenylaminocarbonyl, N-phenyl-N--C.sub.1-C.sub.6 alkyl-aminocarbonyl, C.sub.1-C.sub.6 alkylsulfonyl, aminosulfonyl, C.sub.1-C.sub.6 alkylaminosulfonyl, C.sub.1-C.sub.6 dialkylaminosulfonyl or phenylsulfonyl, wherein each instance of C.sub.1-C.sub.6 alkylcarbonyl is optionally substituted on the alkyl portion with one or more substituents selected from the group consisting of halogen, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.6 alkylamino, C.sub.1-C.sub.6 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, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl and C.sub.1-C.sub.4 alkoxy, and alternatively, R.sub.23 and R.sub.24, R.sub.24 and R.sub.25 or R.sub.25 and R.sub.26 may be taken together with the carbons to which they are attached to form a C.sub.3-C.sub.6 cycloalkyl ring; X.sub.1 is absent or is selected from the group consisting of H, cyano, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.4 alkoxy, amino, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, carboxy, C.sub.1-C.sub.8 alkoxycarbonyl, aminocarbonyl, C.sub.1-C.sub.8 alkylaminocarbonyl, C.sub.1-C.sub.8 dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl, phenylaminocarbonyl, aminosulfonylaminocarbonyl, C.sub.1-C.sub.8 alkylaminosulfonylaminocarbonyl, C.sub.1-C.sub.8 dialkylaminosulfonylaminocarbonyl, C.sub.1-C.sub.8 alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl and heterocyclylcarbonyl, wherein C.sub.1-C.sub.4 alkoxy and the C.sub.1-C.sub.8 alkoxy portion of C.sub.1-C.sub.8 alkoxycarbonyl is optionally substituted with one or more substituents selected from the group consisting of halogen, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, amino, C.sub.1-C.sub.6 alkylamino, C.sub.1-C.sub.6 dialkylamino, cycloalkylamino, phenyl and heterocyclyl, wherein C.sub.1-C.sub.8 alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, C.sub.1-C.sub.4 haloalkyl, C.sub.2-C.sub.8 alkenyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.4 alkoxyalkoxy, C.sub.3-C.sub.6 cycloalkyloxy, amino, C.sub.1-C.sub.6 alkylamino, C.sub.1-C.sub.6 dialkylamino, cycloalkylamino, aminocarbonyl, C.sub.1-C.sub.6 alkylaminocarbonyl, C.sub.1-C.sub.6 dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl, C.sub.1-C.sub.6 alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl and heterocyclyl, wherein C.sub.1-C.sub.4 alkoxy or C.sub.2-C.sub.8 alkenyl are each further optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, amino, C.sub.1-C.sub.6 alkylamino and C.sub.1-C.sub.6 dialkylamino. 12. The method of claim 11, wherein: (a) m is an integer selected from 1, 2 or 3; and, wherein Ring C.sub.1 is selected from the group consisting of a thienyl ring, a pyridinyl ring, a cyclohexyl ring, a cyclohexenyl ring, a cyclohexa-1,4-dienyl ring, a benzo[d][1,3]dioxolyl ring and a 2,3-dihydrobenzo[b][1,4]dioxinyl ring, wherein benzo[d][1,3]dioxolyl and 2,3-dihydrobenzo[b][1,4]dioxinyl, each having a benzo ring portion, are fused via said benzo portion; (b) R.sub.20 and R.sub.21 are each H; alternatively, R.sub.20 and R.sub.21 are each C.sub.1-C.sub.3 alkyl; (c) R.sub.22 is selected from the group consisting of H, halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, cyano, thienyl, furanyl, pyridinyl, pyrimidinyl and phenyl, wherein phenyl is optionally substituted with one or two halogen, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy substituents; (d) when one, two or three of R.sub.23, R.sub.24, R.sub.25 and R.sub.26 are each H, then three, two or one of R.sub.23, R.sub.24, R.sub.25 and R.sub.26, respectively, are each selected from hydroxyl, halogen, cyano, nitro, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkoxyalkoxy, C.sub.1-C.sub.6 alkoxyalkyl, C.sub.1-C.sub.6 difluoroalkoxy, C.sub.1-C.sub.6 trifluoroalkoxy, C.sub.1-C.sub.4 trifluoroalkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.4 trifluoroalkenyl, amino, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylaminoalkyl or C.sub.1-C.sub.4 dialkylaminoalkyl; (e) when three of R.sub.23, R.sub.24, R.sub.25 and R.sub.26 are each H, then one of R.sub.23, R.sub.24, R.sub.25 and R.sub.26 is selected from phenyl, cyclopentyl, cyclopropyl, benzyloxy, C.sub.1-C.sub.4 cyclopentylalkoxy, C.sub.1-C.sub.4 cyclobutylalkoxy, cyclopentyloxy, pyrrolidinyl, piperidinyl, morpholinyl, C.sub.1-C.sub.4 morpholinylalkyl, thienyl, pyridinyl, pyrimidinyl, or amino, wherein amino is optionally disubstituted with one substituent selected from hydrogen or C.sub.1-C.sub.6 alkyl and the other is selected from phenyl, C.sub.1-C.sub.4 alkylcarbonyl, aminocarbonyl, C.sub.1-C.sub.4 alkylaminocarbonyl, C.sub.1-C.sub.4 dialkylaminocarbonyl, phenylcarbonyl, phenylaminocarbonyl, N-phenyl-N--C.sub.1-C.sub.4 alkyl-aminocarbonyl, C.sub.1-C.sub.6 alkylsulfonyl or phenylsulfonyl, and wherein each instance of phenyl is optionally substituted with one or two substituents selected from halogen, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy; or (f) X.sub.1 is absent or is selected from the group consisting of H, cyano, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.6 hydroxylalkyl, C.sub.1-C.sub.6 alkoxyalkyl, C.sub.1-C.sub.4 morpholinylalkyl, amino, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylaminoalkyl, C.sub.1-C.sub.4 dialkylaminoalkyl, carboxy, C.sub.1-C.sub.6 alkoxycarbonyl, benzyloxycarbonyl, aminocarbonyl, C.sub.1-C.sub.8 alkylaminocarbonyl, C.sub.1-C.sub.8 dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl, phenylaminocarbonyl, aminosulfonylaminocarbonyl, C.sub.1-C.sub.8 alkylaminosulfonylaminocarbonyl C.sub.1-C.sub.8 dialkylaminosulfonylaminocarbonyl, C.sub.1-C.sub.8 alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl, morpholinylcarbonyl and piperidinylcarbonyl. 13. The method of claim 9, wherein the nucleic acid construct comprises the nucleic acid residues of exon 6 of SMN or a fragment thereof downstream (3') to the start codon and upstream (5') of the nucleic acid residues of exon 7 of SMN, wherein the fragment of the nucleic acid residues of exon 6 of SMN comprises any number of nucleotides of exon 6 of SMN so long as in the mRNA transcript the first start codon and the stop codon upstream of the reporter gene coding sequence are maintained in the same contiguous open reading frame. 14. The method of claim 13, wherein the nucleic acid construct comprises the nucleic acid residues of intron 6 of SMN or a fragment thereof downstream (3') of the nucleic acid residues of exon 6 of SMN or a fragment thereof and upstream (5') of the nucleic acid residues of exon 7 of SMN, wherein the fragment of the nucleic acid residues of intron 6 of SMN comprises any number of nucleotides of intron 6 of SMN required for a functional, minimum intron. 15. The method of claim 9, further comprising contacting the cell-free extract with a positive control. 16. The method of claim 15, wherein said positive control is Compound 1: ##STR00009##

Details for Patent 8,932,818

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2028-08-13
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2028-08-13
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2028-08-13