Methods for treating viral infections

Abstract

The present invention relates to compounds that modulate ribosomal frameshifting and nucleic acid constructs for use in methods for identifying or validation such compounds. 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 said compounds to inhibit the replication or infectivity of viruses that employ programmed ribosomal frameshifting.

1 .- 14 . (canceled) 
     
     
         15 . A nucleic acid construct comprising, 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 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 48 th  nucleotide from the 5′ end 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. 
     
     
         16 . (canceled) 
     
     
         17 . The nucleic acid construct of  claim 15 , 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 without any interruption. 
     
     
         18 . The nucleic acid construct of  claim 17 , 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. 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . An mRNA transcript encoded by the nucleic acid construct of  claim 15 . 
     
     
         22 . A method for the identification or validation of a compound that increases ribosomal frameshifting comprising: (a) contacting a compound with either a host cell containing an mRNA transcript encoded by the nucleic acid construct of  claim 15 , or a composition comprising a cell-free extract and an mRNA transcript transcribed from a nucleic acid construct of  claim 15 ; 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 modulates ribosomal frameshifting. 
     
     
         23 . A method for the identification or validation of a compound that modulates the efficiency of programmed ribosomal frameshifting comprising: (a) contacting a compound with either a host cell containing an mRNA transcript encoded by the nucleic acid construct of  claim 15 , or a composition comprising a cell-free extract and an mRNA transcript transcribed from a nucleic acid construct of  claim 15 ; 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 modulates the efficiency of programmed ribosomal frameshifting. 
     
     
         24 . A method for reducing or inhibiting a viral infection, comprising contacting a cell containing a virus or provirus that employs programmed ribosomal frameshifting with a compound, wherein the compound in vitro or in cells increases the amount or activity of a fusion protein encoded by the nucleic acid construct of  claim 15  or translated from a RNA transcript transcribed from the nucleic acid construct of  claim 15 . 
     
     
         25 . A method for reducing or inhibiting a viral infection, comprising contacting a compound with a mixture of a cell or a population of cells and a virus that employs programmed ribosomal frameshifting, wherein the compound in vitro or in cells increases the amount or activity of a fusion protein encoded by the nucleic acid construct of  claim 15  or translated from a RNA transcript transcribed from the nucleic acid construct of  claim 15 . 
     
     
         26 . A method for inhibiting or reducing viral replication or infectivity in a subject, comprising administering to a subject in need thereof an effective amount of a compound or pharmaceutical composition thereof, wherein the compound modulates the efficiency of programmed ribosomal frameshifting as measured in vitro or in cells by an increase in the amount or activity of a fusion protein encoded by the nucleic acid construct of  claim 15  or translated from a RNA transcript transcribed from the nucleic acid construct of  claim 15 . 
     
     
         27 . A method for reducing viral titers in a subject, comprising administering to a subject in need thereof an effective amount of a compound or pharmaceutical composition thereof, wherein the compound modulates the efficiency of programmed ribosomal frameshifting as measured in vitro or in cells by an increase in the amount or activity of a fusion protein encoded by the nucleic acid construct of  claim 15  or translated from a RNA transcript transcribed from the nucleic acid construct of  claim 15 . 
     
     
         28 . A method for treating a viral infection in a subject, comprising administering to a subject in need thereof an effective amount of a compound or pharmaceutical composition thereof, wherein the compound modulates the efficiency of programmed ribosomal frameshifting as measured in vitro or in cells by an increase in the amount or activity of a fusion protein encoded by the nucleic acid construct of  claim 15  or translated from a RNA transcript transcribed from the nucleic acid construct of  claim 15 . 
     
     
         29 . A method for preventing or treating a viral disease in a subject, comprising administering to a subject in need thereof an effective amount of a compound or pharmaceutical composition thereof, wherein the compound modulates the efficiency of programmed ribosomal frameshifting as measured in vitro or in cells by an increase in the amount or activity of a fusion protein encoded by the nucleic acid construct of  claim 15  or translated from a RNA transcript transcribed from the nucleic acid construct of  claim 15 . 
     
     
         30 . The method of  claim 23 , 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 CH 2 ; 
         B is CH 2  or CH(C n H 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 1  and R 2  are independently selected from the group consisting of H and C 1 -C 3  alkyl, or R 1  and R 2  may be taken together with the carbon atom to which they are attached to form a C 3 -C 6  cycloalkyl ring or a carbonyl group; 
         R 3  is selected from the group consisting of H, halogen, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  haloalkyl, CN, NO 2 , heteroaryl, and phenyl optionally substituted with any combination of one to five halogen, NO 2 , CN, C 1 -C 4  alkyl, C 1 -C 4  haloalkyl, or C 1 -C 4  alkoxy substituents; 
         R 4 , R 5 , R 6  and R 7  are independently selected from the group consisting of H, hydroxyl, halogen, CN, NO 2 , sulfonamide, C 1 -C 8  alkyl, C 3 -C 6  cycloalkyl, cycloalkyloxy, C 1 -C 6  alkoxy, C 1 -C 6  haloalkoxy, C 1 -C 4  haloalkyl, C 2 -C 8  alkenyl, amino, C 1 -C 4  alkylamino, C 1 -C 4  dialkylamino, C 3 -C 6  cycloalkylamino, morpholinyl, heteroaryl, arylamino, arylalkylamino, phenyl, C(O)R′, NR′(COR″), NR′SO 2 R″ and NR′(CONR″R′″), wherein R′, R″ and R′″ are independently H, C 1 -C 6  alkyl, phenyl, or substituted phenyl, and wherein C 1 -C 8  alkyl is optionally substituted with one or more substituents selected from the group consisting of C 1 -C 4  alkoxy, C 1 -C 4  haloalkyl, C 1 -C 6  dialkylamino, C 1 -C 6  alkylamino, cycloalkylamino, and morpholinyl, and the phenyl is optionally substituted with one or more substituents selected from the group consisting of halogen, NO 2 , CN, C 1 -C 4  alkyl, C 1 -C 4  haloalkyl, and C 1 -C 4  alkoxy, or R 4  and R 5 , R 5  and R 6 , or R 6  and R 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 8 , wherein R 8  is H or C 1 -C 8  alkyl, and C 1 -C 8  alkyl optionally is substituted with one or more substituents selected from the group consisting of C 1 -C 4  alkoxy, C 1 -C 4  haloalkyl, C 1 -C 6  dialkylamino, C 1 -C 6  alkylamino, cycloalkylamino, phenyl, and morpholinyl; C(O)NR 9 R 10  or CH 2 NR 9 R 10 , wherein R 9  and R 10  are independently selected from the group consisting of H and C 1 -C 6  alkyl, or R 9  and R 10  together with the nitrogen to which they are attached form a heterocyclyl ring; CH 2 OR 11 , wherein R 11  is H, C 1 -C 8  alkyl, or C 3 -C 6  cycloalkyl, wherein C 1 -C 8  alkyl is optionally substituted with one or more substituents selected from the group consisting of C 1 -C 4  alkoxy, C 1 -C 4  haloalkyl, C 1 -C 6  dialkylamino, C 1 -C 6  alkylamino, cycloalkylamino, and morpholinyl; CH 2 Z, wherein Z is halogen; C(O)NHOH; C(O)NHCN; C(O)N(R 1 )SO 2 R 13 , wherein R 13  is C 1 -C 4  alkyl, phenyl, or substituted phenyl; C 1 -C 8  alkyl, optionally substituted with one or more substituents selected from the group consisting of C 1 -C 4  alkoxy, C 1 -C 4  haloalkyl, C 1 -C 6  dialkylamino, and C 1 -C 6  alkylamino; and C 2 -C 8  alkenyl, optionally substituted with one or more substituents selected from the group consisting of C 1 -C 4  alkoxy, C 1 -C 4  haloalkyl, C 1 -C 6  dialkylamino, and C 1 -C 6  alkylamino. 
       
     
     
         31 . The method of  claim 23 , 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, 
         W 1  is selected from the group consisting of C(O), C(S), and CH 2 ; 
         B 1  is CH 2  or CH(C m H 2m+1 ), wherein m is an integer from 1 to 8; 
         Ring C 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, 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; 
 
         R 20  and R 21  are independently selected from the group consisting of H and C 1 -C 3  alkyl, or R 20  and R 21  may be taken together with the carbon atom to which they are attached to form a C 3 -C 6  cycloalkyl ring or a carbonyl group; 
         R 22  is selected from the group consisting of H, halogen, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  haloalkyl, cyano, nitro, heteroaryl, and phenyl optionally substituted with any combination of one to five halogen, nitro, cyano, C 1 -C 4  alkyl, C 1 -C 4  haloalkyl or C 1 -C 4  alkoxy substituents; 
         R 23 , R 24 , R 25  and R 26  are independently selected from the group consisting of H, hydroxyl, halogen, cyano, nitro, sulfonamide, C 1 -C 8  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkoxyalkoxy, C 1 -C 6  alkoxyalkyl, C 1 -C 6  haloalkoxy, C 1 -C 4  haloalkyl, C 2 -C 6  alkenyl, C 2 -C 4  haloalkenyl, formyl, C 1 -C 6  alkylcarbonyl, amino, C 1 -C 4  alkylamino, C 1 -C 4  dialkylamino, C 1 -C 4  aminoalkyl, C 1 -C 4  alkylaminoalkyl, C 1 -C 4  dialkylaminoalkyl, phenyl, C 3 -C 6  cycloalkyl, C 3 -C 6  cycloalkylalkyl, C 3 -C 6  cycloalkylalkoxy, cycloalkyloxy, heterocyclyl, heterocyclylalkyl, heteroaryl, and phenylcarbonyl, 
         wherein amino is optionally disubstituted with one substituent selected from hydrogen, C 1 -C 6  alkyl or phenyl and the other is selected from formyl, phenyl, C 3 -C 6  cycloalkyl, C 1 -C 6  alkylcarbonyl, aminocarbonyl, C 1 -C 6  alkylaminocarbonyl, C 1 -C 6  dialkylaminocarbonyl, phenylcarbonyl, phenylaminocarbonyl, N-phenyl-N—C 1 -C 6  alkyl-aminocarbonyl, C 1 -C 6  alkylsulfonyl, aminosulfonyl, C 1 -C 6  alkylaminosulfonyl, C 1 -C 6  dialkylaminosulfonyl or phenylsulfonyl, 
         wherein each instance of C 1 -C 6  alkylcarbonyl is optionally substituted on the alkyl portion with one or more substituents selected from the group consisting of halogen, C 1 -C 4  alkoxy, C 1 -C 6  alkylamino, C 1 -C 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 1 -C 4  alkyl, C 1 -C 4  haloalkyl and C 1 -C 4  alkoxy, and 
         alternatively, R 23  and R 24 , R 24  and R 25  or R 25  and R 26  may be taken together with the carbon to which they are attached to form a C 3 -C 6  cycloalkyl ring fused to the moiety of Formula (Ia); 
         X 1  is absent or is selected from the group consisting of H, cyano, C 1 -C 8  alkyl, C 1 -C 4  alkoxy, amino, C 1 -C 4  alkylamino, C 1 -C 4  dialkylamino, carboxy, C 1 -C 8  alkoxycarbonyl, aminocarbonyl, C 1 -C 8  alkylaminocarbonyl, C 1 -C 8  dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl, phenylaminocarbonyl, aminosulfonylaminocarbonyl, C 1 -C 8  alkylaminosulfonylaminocarbonyl, C 1 -C 8  dialkylaminosulfonylaminocarbonyl, C 1 -C 8  alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl and heterocyclylcarbonyl, 
         wherein C 1 -C 4  alkoxy and the C 1 -C 8  alkoxy portion of C 1 -C 8  alkoxycarbonyl is optionally substituted with one or more substituents selected from the group consisting of halogen, C 1 -C 4  alkoxy, C 1 -C 4  haloalkyl, amino, C 1 -C 6  alkylamino, C 1 -C 6  dialkylamino, cycloalkylamino, phenyl and heterocyclyl, 
         wherein C 1 -C 8  alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, C 1 -C 4  haloalkyl, C 2 -C 8  alkenyl, C 1 -C 6  alkoxy, C 1 -C 4  alkoxyalkoxy, C 3 -C 6  cycloalkyloxy, amino, C 1 -C 6  alkylamino, C 1 -C 6  dialkylamino, cycloalkylamino, aminocarbonyl, C 1 -C 6  alkylaminocarbonyl, C 1 -C 6  dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl, C 1 -C 6  alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl and heterocyclyl, wherein C 1 -C 4  alkoxy or C 2 -C 8  alkenyl are each further optionally substituted with one or more substituents selected from the group consisting of C 1 -C 4  alkoxy, C 1 -C 4  haloalkyl, amino, C 1 -C 6  alkylamino and C 1 -C 6  dialkylamino. 
       
     
     
         32 . The method of  claim 31 , wherein m is an integer selected from 1, 2 or 3; and, wherein Ring C 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, 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. 
     
     
         33 . The method of  claim 31 , wherein R 20  and R 21  are each H; alternatively, R 20  and R 21  are each C 1 -C 3  alkyl; or wherein R 22  is selected from the group consisting of H, halogen, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  haloalkyl, cyano, thienyl, furanyl, pyridinyl, pyrimidinyl and phenyl, wherein phenyl is optionally substituted with one or two halogen, C 1 -C 4  alkyl or C 1 -C 4  alkoxy substituents. 
     
     
         34 . (canceled) 
     
     
         35 . The method of  claim 31 , wherein, when one, two or three of R 23 , R 24 , R 25  and R 26  are each H, then three, two or one of R 23 , R 24 , R 25  and R 26 , respectively, are each selected from hydroxyl, halogen, cyano, nitro, C 1 -C 8  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkoxyalkoxy, C 1 -C 6  alkoxyalkyl, C 1 -C 6  difluoroalkoxy, C 1 -C 6  trifluoroalkoxy, C 1 -C 4  trifluoroalkyl, C 2 -C 6  alkenyl, C 2 -C 4  trifluoroalkenyl, amino, C 1 -C 4  alkylamino, C 1 -C 4  dialkylamino, C 1 -C 4  aminoalkyl, C 1 -C 4  alkylaminoalkyl or C 1 -C 4  dialkylaminoalkyl; or wherein when three of R 23 , R 24 , R 25  and R 26  are each H, then one of R 23 , R 24 , R 25  and R 26  is selected from phenyl, cyclopentyl, cyclopropyl, benzyloxy, C 1 -C 4  cyclopentylalkoxy, C 1 -C 4  cyclobutylalkoxy, cyclopentyloxy, pyrrolidinyl, piperidinyl, morpholinyl, C 1 -C 4  morpholinylalkyl, thienyl, pyridinyl, pyrimidinyl, or amino, wherein amino is optionally disubstituted with one substituent selected from hydrogen or C 1 -C 6  alkyl and the other is selected from phenyl, C 1 -C 4  alkylcarbonyl, aminocarbonyl, C 1 -C 4  alkylaminocarbonyl, C 1 -C 4  dialkylaminocarbonyl, phenylcarbonyl, phenylaminocarbonyl, N-phenyl-N—C 1 -C 4  alkyl-aminocarbonyl, C 1 -C 6  alkylsulfonyl or phenylsulfonyl, and wherein each instance of phenyl is optionally substituted with one or two substituents selected from halogen, C 1 -C 4  alkyl or C 1 -C 4  alkoxy. 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 31 , wherein X 1  is absent or is selected from the group consisting of H, cyano, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  haloalkyl, C 1 -C 6  hydroxylalkyl, C 1 -C 6  alkoxyalkyl, C 1 -C 4  morpholinylalkyl, amino, C 1 -C 4  alkylamino, C 1 -C 4  dialkylamino, C 1 -C 4  aminoalkyl, C 1 -C 4  alkylaminoalkyl, C 1 -C 4  dialkylaminoalkyl, carboxy, C 1 -C 6  alkoxycarbonyl, benzyloxycarbonyl, aminocarbonyl, C 1 -C 8  alkylaminocarbonyl, C 1 -C 8  dialkylaminocarbonyl, hydroxylaminocarbonyl, cyanoaminocarbonyl, phenylaminocarbonyl, aminosulfonylaminocarbonyl, C 1 -C 8  alkylaminosulfonylaminocarbonyl C 1 -C 8  dialkylamino sulfonylaminocarbonyl, C 1 -C 8  alkylsulfonylaminocarbonyl, phenylsulfonylaminocarbonyl, morpholinylcarbonyl and piperidinylcarbonyl. 
     
     
         38 . The method of  claim 30 , wherein the compound is 2-(4-isopropylphenyl)-6-methoxyisoindolin-1-one. 
     
     
         39 . The method of  claim 23 , wherein the virus is human immunodeficiency virus type I, human immunodeficiency virus type II, feline immunodeficiency virus, rous sarcoma virus, mouse mammary tumor virus, simian retrovirus type 1, human T cell leukemia virus type I, human T cell leukemia virus type II, infectious bronchitis virus, human coronavirus, transmissible gastroenteritis virus, berne virus, equine arteritis virus, human astrovirus serotype-1,  Giardia lamblia  virus,  Saccharomyces cerevisiae  dsRNA virus L-A,  S. cerevisiae  dsRNA virus L1, bacteriophage T7, bacteriophage lambda, barley yellow dwarf virus, beet western yellows virus, potato leaf roll virus, severe acute respiratory syndrome coronavirus, herpes simplex virus, or red clover necrotic mosaic virus. 
     
     
         40 . (canceled)