WO1993009127A1 - Formation amelioree d'une triple helice, grace a des oligonucleotides contenant la 2'-desoxy-7-desazaxanthosine, la 2'-desoxy-7-desazaguanosine et des composes analogues - Google Patents

Formation amelioree d'une triple helice, grace a des oligonucleotides contenant la 2'-desoxy-7-desazaxanthosine, la 2'-desoxy-7-desazaguanosine et des composes analogues Download PDF

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WO1993009127A1
WO1993009127A1 PCT/US1992/009195 US9209195W WO9309127A1 WO 1993009127 A1 WO1993009127 A1 WO 1993009127A1 US 9209195 W US9209195 W US 9209195W WO 9309127 A1 WO9309127 A1 WO 9309127A1
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oligomer
oligomers
triplex
allyl
alkyl
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PCT/US1992/009195
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Mark Matteucci
Steve Krawczyk
John Milligan
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Gilead Sciences, Inc.
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Publication of WO1993009127A1 publication Critical patent/WO1993009127A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/14—Pyrrolo-pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays

Definitions

  • the invention relates generally to novel bases, oligonucleotide-based therapy and diagnosis through triplex binding. More specifically, the invention concerns nucleoside analogs such as 2'-deoxy-7- deazaxanthosine and 2' -deoxy-7-deazaguanosine or related nucleoside analogs and oligomers containing same.
  • Duplex D ⁇ A can be specifically bound by oligomers based on a recognizable nucleotide sequence.
  • Two major recognition motifs have been recognized. In an earlier description of a "CT” motif, cytosine residues recognize G-C basepairs while thymine residues recognize A-T basepairs in the duplex. These recognition rules are outlined by Maher III, L.J., et al. , Science (1989) 245:725-730; Moser, H.E., et al. , Science (1987) 238:645- 650. More recently, an additional motif, called herein "GT” recognition, was described by Cooney, M. , et al.
  • the recognition sequence In both of these binding motifs, the recognition sequence must align with a sequence as played out on one of the chains of the duplex; thus, recognition, for example, of an A-T pair by a thy ine depends on " he location of repeated adenyl residues along one chain of the duplex and thymine series on the other. The recognition does not extend to alternating A-T-A-T sequences; only the adenyl residues on one chain or the other would be recognized.
  • the two motifs exhibit opposite binding orientations with regard to homopurine target chains in the duplex.
  • the targeting oligonucleotide is oriented parallel to the target sequence; in the GT motif, it is oriented antiparallel (Beal, P. ., et al., Science (1990) 251:1360-1363).
  • recognition sequences in the CT motif are read with respect to target 5' ⁇ 3' sequences so that in the 5'-*3' direction, synthetic oligonucleotides contain the required sequence of C or T residues with respect to the guanyl or adenyl residues in the target.
  • the targeted sequence is read 5'-3' in order to design the 3' ⁇ 5' sequence of the targeting oligonucleotide.
  • GT motif oligomers for "binding to physiological targets such as the human c-myc gene promoter (Postel, E.H. et al., Proc Natl Acad Sci (1991) :8227-8231) or the epidermal growth factor receptor gene promoter (Durland, R.H. et al., Biochemistry (1991) 3_8:9246-9255) have been described. Binding of oligomers to the promoter region was reported to affect the expression of the target gene. This effect was ascribed to interference with promoter activity mediated by the oligomers. In vitro analyses demonstrated that, under conditions that favored triplex formation, relatively stable triplex structures were formed.
  • triplex structures were formed under nonphysiological ion conditions (no K + ion present) .
  • the present inventors have shown that GT mode oligomers containing only thymine and guanine do not form triplex structures under physiological ion conditions (i.e., 140 mM KC1) .
  • the presence of high levels of K + ion is postulated by the present inventors to interfere with the formation of triplex structures mediated by guanine-rich oligomers due to the formation of "G-quartets" or similar complex structures (Williamson, J.R. et al. , Cell (1989) 59:871- 880) .
  • the oligomers of the present invention that diminish this postulated ion-induced aggregation phenomenon for G-rich oligomers, due to the absence of the nitrogen atom at position 7 of either guanine or xanthine (as a thymine substitute) . While not intending to be bound by any theory, the inventors believe this atcm mediates chelation of K + ion which leads to the postulated aggregation phenomenon.
  • the present inventors endeavored to substitute ⁇ the nucleoside analog deoxyxanthosine (shown below as ⁇ structure A) for thymidine in the G-T motif in order to enhance the association of such oligomers with their complementary duplex DNA.
  • ⁇ structure A nucleoside analog deoxyxanthosine
  • the desired triplex association effect was only achieved under conditions which are more acidic than physiological pH. This shortcoming was rationalized by invoicing the ionization of the N-3 proton of the compound of structural formula A at a physiological pH and above.
  • the present inventors sought to overcome the above-referred to limitation with respect to obtaining association of a given oligomer with DNA duplexes in order to form triplexes at physiological pH. This was done by substituting an analog of the compound A which did not ionize to the same degree under equivalent conditions as did the compound of structure A. (Seela, F., Driller, H., and Liman, U. , Liebicrs Ann Chemie 1985, 312-320.)
  • An additional problem relates to the stability of the triplex. Covalent crosslinking to the duplex provides one answer to this problem.
  • Vlassov, V.V. et al., Gene (1988) 313-322 and Fedorova, O.S. et al., FEBS (1988) 228.:273- 276, describe targeting duplex DNA with a 5'-phospho- linked oligonucleotide.
  • oligonucleotides of inverted polarity are provided. Provision of such oligomers is described in U.S. application serial no. 07/559,958, filed 30 July 1990, and incorporated herein by reference. Briefly, by "inverted polarity" is meant that the oligonucleotide contains tandem sequences which have opposite polarity, i.e., one having polarity 5' ⁇ 3' followed by another with polarity 3' ⁇ 5', or vice versa.
  • the present invention is directed to deazapurine bases and oligomers containing those bases which oligomers are capable of binding to a variety of desired DNA duplexes and thereby forming triplexes.
  • the oligomers include a nucleoside or nucleotide analog having structural formula I or II.
  • the analog I is defined by the following general structural formula I
  • R 1 is H, OH, F, Cl, O-allyl, S-allyl, OR or SR, wherein R is alkyl (1-4C) ;
  • R 2 is H, alkyl(1- 4C) , CN, Br, Cl, F, CONR 2 , lower alkenyl(1-4C) or lower alkynyl(l-4C) ;
  • R 3 is H or a lower alkyl(1-4C) ; with the proviso that if R 2 and R 3 are both H, R 1 cannot be H or OH, and wherein each X is independently H, -P0 3 ⁇ 2 or a group useful in oligomer synthesis.
  • R 2 is CH 3
  • R 1 is H or OH
  • R 3 is H.
  • the compounds of formula I are nucleotides, i.e., deoxyribonucleotides or ribonucleotides.
  • the nucleotides are, of course, formed by the attachment of the phosphate group to the 5' and/or 3' position of the sugar ring--the phosphate group being normally present to link nucleotides to each other and form an oligomer.
  • each X is independently a group useful in oligomer synthesis such as DMT, MMT, H-phosphonate, methyl phosphonate, ⁇ - cyanoethylphosphoramidite, or methylphosphoramidite.
  • the analog II is defined by the following general structural formula II wherein R 4 is H, OH, F, Cl, O-allyl, S-allyl, OR or SR, wherein R is lower alkyl (1-4C) ; R 5 is H, lower alkyl (1-4C), CN, Br, Cl, F, CONR 2 , lower alkenyl (1-4C) or lower alkynyl (1-4C) ; with the proviso that R 5 is not H when R 4 is H or OH; wherein each X is independently H, -P0 3 ⁇ 2 or a group useful in oligomer synthesis.
  • each X is independently a group useful in synthesis such as DMT, MMT, H-phosphonate, methyl phosphonate, ⁇ - cyanoethylphosphoramidite, or methylphosphoramidite.
  • oligomers designed for triple-helix formation with a complementary duplex DNA strand are incorporated into oligomers designed for triple-helix formation with a complementary duplex DNA strand.
  • the oligomers have enhanced ability to form triplexes as compared with oligomers containing only conventional bases.
  • All of the nucleotides in the oligomer may contain the deazapurine bases of the invention.
  • oligomers of the invention may be comprised of residues of both analogs I and II and their analogs.
  • the oligomers of the invention are capable of forming triplexes with various target sequences such as HER-2 and HIV sequences by coupling into the major groove of a target DNA duplex at physiological pH. When the triplexes are formed they can prevent or inhibit transcription.
  • the oligomers of the invention may be incorporated into pharmaceutically acceptable carriers and may be constructed to have any desired sequence, provided the sequence includes the deazapurine base residue which enhances the ability of the oligomer to form a triplex with the target sequence.
  • compositions of the invention can be used as pharmaceuticals to treat various diseases such as cancers (associated with HER-2) and viruses (HIV) and can be used for diagnostic purposes in order to detect the presence of sequences generally known to be associated with neoplastic growth, viruses and a variety of disease conditions.
  • diseases such as cancers (associated with HER-2) and viruses (HIV)
  • HIV viruses
  • the invention is also directed to oligomers which are capable of triple-helix formation containing residues of analogs I and/or II.
  • the invention is directed to oligomers capable of forming triplexes with various target sequences such as HER-2 and HIV sequences by coupling into the major groove of a target DNA duplex under physiological pH and ion conditions.
  • oligomers are preferably included in a pharmaceutically acceptable carrier and can have any desired sequence provided the sequence can be modified to include one or more residues of the analogs of structural formula I or II.
  • the invention is directed to a method to form a triplex using the oligomers of the invention to target DNA duplexes and to the resulting DNA triplexes.
  • Other aspects of the invention include pharmaceutical and diagnostic compositions which contain the oligomers of the invention, to methods to diagnose and treat diseases characterized by various target sequences such as HER-2 and HIV duplexes using these compositions.
  • a primary object of the invention is to provide novel nucleosides and nucleotides and oligomers which include deazapurine bases.
  • a feature of the invention is that the oligomers of the invention can be comprised of a variety of different sequences and thereby used to target a variety of different target sequences.
  • Another feature of the invention is that the oligomers may be used by themselves or bound to other compounds such as labels or active pharmaceuticals such as anti-viral or anti-neoplastic agents.
  • An advantage of the present invention is that the oligomers of the invention are capable of forming triplexes under physiological pH and ion conditions.
  • novel nucleosides described herein are more hydrophobic than the unmodified parent compounds containing thymine, xanthine or guanine, which hydrophobicity is generally associated with enhanced - cellular permeation or uptake when the compounds are administered as therapeutic agents.
  • oligonucleotide or “oligomer” is generic to polydeoxyribonucleotides (containing 2'- deoxy-D-ribose or modified forms thereof), i.e., DNA, to polyribonucleotides (containing D-ribose or modified forms thereof), i.e., RNA, and to any other type of polynucleotide which is an N-glycoside or C-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base.
  • the oligomers of the invention may be formed using conventional phosphodiester-linked nucleotides and synthesized using standard solid phase (or solution phase) oligonucleotide synthesis techniques, which are now commercially available.
  • the oligomers " of the invention may also contain one or more substitute linkages as is generally understood in the art. These conventional alternative linkages are synthesized as described in the generally available literature.
  • linking groups include, but are not limited to embodiments wherein a moiety of the formula P(0)S, P(0)NR' 2 , P(0)R', P(0)0R 2 , CO, or C0NR' 2 , wherein R' is H (or a salt) or alkyl (1-20C) and R 2 is alkyl (1-20C) is joined to adjacent nucleotides through - 0- or -S- . Not all such linkages in the same oligomer need to be identical.
  • Nonphosphorous-based linkages may also be used, such as the formacetal (-0-CH 2 -0-), 3' -thioformacetal (- S-CH 2 -0 -), and 3' -amino (-NH-CH 2 -0 2 -) type linkages described and claimed in copending applications U.S. Serial Nos. 690,786 and 763,130, both assigned to the same assignee as the present application and both incorporated herein by reference.
  • "Nucleoside” and “nucleotide” include those moieties which contain not only the known purine and pyrimidine bases, but also heterocyclic bases which have been modified. Such modifications include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Such "analogous purines” and
  • analogous pyrimidines are those generally known in the art, many of which are used as chemotherapeutic agents.
  • An exemplary but not exhaustive list includes 5-methyl cytosine, pseudoisocytosine, 8-hydroxy-N-methyladenine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylamino- methyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, dihydrouracil, inosine, N6-isopentenyl-adenine, l-methyl- adenine, l-methylpseudouracil, 1-methylguanine, l-methyl- inosine, 2,2-dimethylguanine, 2-methyladenine, 2-methyl- guanine, 3-methylcytosine, 5-methylcytosine, N6-methyl- adenine, 7-methylguanine, 5-methylamino
  • Nucleosides or “nucleotides” also include those which contain modifications in the sugar moiety, for example, wherein one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or functionalized as ethers, amines, and the like.
  • the oligomers of the present invention may contain analogous forms of ribose or deoxyribose that are generally known in the art.
  • An exemplary, but not exhaustive, list includes 2'-substituted sugars such as 2'-0-methyl-, 2'-fluoro- or 2 '-azido-ribose, alpha- ano eric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • one or more nucleotides may contain this linkage or a domain thereof. (Praseuth, D. r et al., Proc Natl Acad Sci (USA) (1988) 85:1349-1353) .
  • nucleoside or “nucleotide” will similarly be generic to ribonucleosides or ribonucleotides, deoxyribonucleosides or deoxyribonucleotides, or to any other nucleoside which is an N-glycoside or C-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base.
  • the stereochemistry of the sugar carbons may be other than that of D-ribose in one or more residues.
  • analogs where the ribose or deoxyribose moiety is replaced by an alternate structure such as the 6-member morpholino ring described in U.S.
  • the oligomers of the present invention also may be of any length, but lengths of 2 to 30 nucleotides are preferred, and those containing about 10-30 nucleotides are more preferred. However, the longer oligonucleotides may also be made, particularly those of greater than 50 nucleotides or greater than 100 nucleotides.
  • oligo ⁇ nucleotides include those conventionally recognized in the art.
  • the oligonucleotides may be covalently linked to various moieties such as intercalators, substances which interact specifically with the minor groove of the DNA double helix and other arbitrarily chosen conjugates, such as labels (radioactive, fluorescent, enzyme, etc.). These additional moieties may be derivatized through any convenient linkage.
  • intercalators such as acridine can be linked through any available -OH or -SH, e.g., at the terminal 5' position of RNA or DNA, the 2' positions of RNA, or an OH, NH 2 , COOH or SH engineered into the 5 position of pyrimidines, e.g., instead of the 5 methyl of cytosine, a derivatized form which contains, for example, -CH 2 CH 2 NH 2 , -CH 2 CH 2 CH 2 0H or -CH 2 CH 2 CH 2 SH in the 5 position.
  • substituents can be attached, including those bound through conventional linkages.
  • the indicated -OH moieties in the oligomers may be replaced by phosphonate groups, protected by standard protecting groups, or activated to prepare additional linkages to other nucleotides, or may be bound to the conjugated substituent.
  • the 5' terminal OH may be phosphorylated; the 2' -OH or OH substituents at the 3' terminus may also be phosphorylated.
  • the hydroxyls may also be derivatized to standard protecting groups.
  • Oligonucleotides or the segments thereof of are conventionally synthesized. Methods for such synthesis are found, for example, in Froehler, B., et al., Nucleic Acids Res (1986) 14:5399-5467; Nucleic Acids Res (1988) 16:4831-4839; Nucleosides and Nucleotides (1987) 6.:287- 291; Froehler, B., Tet Lett (1986) 27:5575-5578.
  • oligonucleotides may also be synthesized using solution phase methods such as triester synthesis. These methods are workable, but in general, less efficient for oligonucleotides of any substantial length.
  • the invention in one aspect, is directed to the inclusion within an oligomer of a moiety of formula I(a) or II(a) , including one or both of these moieties.
  • an oligomer of a moiety of formula I(a) or II(a) including one or both of these moieties.
  • the invention involves the synthesis, use and incorporation into an oligomer of one or more moieties of formula 1(a) or 11(a) .
  • moieties of formula 1(a) are residues of the compounds of formula I as follows: wherein R 1 is H, OH, F, Cl, O-allyl, S-allyl, OR or SR, wherein R is alkyl (1-4C) ; R 2 is H, alkyl (1-4C), CN, Br, Cl, F, CONR 2 , lower alkenyl (1-4C) or lower alkynyl(1-4C) ; and R 3 is H or a lower alkyl (1-4C).
  • R 2 is CH 3
  • R 1 is H or OH
  • R 3 is H.
  • R 4 is H, OH, F, Cl, O-allyl, S-allyl, OR or SR, wherein R is lower alkyl (1-4C) ; and R 5 is H or lower alkyl(l-4C), CN, Br, Cl, F, CONR 2 , lower alkenyl (1-4C) or lower alkynyl(l-4C) .
  • R 4 is H or OH and R 5 is CH 3 .
  • Oligomers containing the moiety of formula I(a) can be synthesized by following the general reaction scheme exemplified by 2' -deoxy-7-deazaxanthosine:
  • the intermediate is incorporated into an oligomer designed to bind a target duplex in the GT antiparallel motif. (It has been found that 7-deazaxanthine and 7- deazaguanine containing moieties do not participate in CT mode binding in either direction.)
  • the 2' -deoxy-7-deazaguanosine analog is also protected and phosphorylated and incorporated into oligomers.
  • the analogous nucleosides of the invention wherein, for example, at least one of R 2 and R 3 or R 5 is other than H, are incorporated in a similar manner.
  • sequences designed to bind the target in the GT mode are provided substituting the monomers of formula I in place of the thymine- containing monomers and monomers of formula II in place of guanine-containing monomers.
  • nucleotide monomers comprising a base which is a substituted or unsubstituted 7-deazaxanthine will replace nucleotides containing a thymine residue; nucleotides containing a substituted or unsubstituted 7-deazaguanine base will replace nucleotides containing guanine residues.
  • GX oligomers The oligomers wherein only thymine bases are replaced by substituted or unsubstituted 7-deazaxanthine bases may be referred to as "GX oligomers" which correspond in sequence, except for the indicated base substitution, to the conventionally designed GT oligomer.
  • G*T oligomers oligomers which correspond in sequence to GT oligomers designed for triplex binding but wherein the G residues are replaced by nucleotides containing the 7- deazaguanine bases, designated G .
  • the oligomer in the conventional system used to effect GT binding would be 5' GGTGGTGGTTGTGGT 3'.
  • the corresponding GX oligomer of the invention would then be
  • oligomers of the invention which would be suitable for binding to this target sequence would include:
  • Oligomers were synthesized to target the HER2 sequence above; these were of the formulas:
  • GX 5' GGTGGTGGTTGTGGTY 3'
  • GX 5' GGXGGXGGXXGXGGXY 3', where Y is anthraquinone as further described below.
  • the GX oligomer showed enhanced triplex formation as compared to the native sequences.
  • the inverted polarity oligonucleotides contain at least one segment along their length of the formula:
  • 5' 3' indicates a stretch of oligomer in the opposite orientation wherein the linkages are formed between the 3' hydroxyl of the ribosyl residue of the left nucleotide and the 5' hydroxyl of the ribosyl residue of the nucleotide on the right, thus leaving the 3' hydroxyl of the rightmost nucleotide ribosyl residue free for additional conjugation.
  • the linkage symbolized by -C-, may be formed so as to link the 5' hydroxyls of the adjacent ribosyl residues in formula (1) or the 3' hydroxyls of the adjacent ribosyl residues in formula (2), or the "-C-" linkage may conjugate other portions of the adjacent nucleotides so as to link the inverted polarity strands.
  • "-C-" may represent a linker moiety, or simply a covalent bond.
  • either the 3' or 2' position can be involved in the linkage, and either of these positions may be in either R or S configuration.
  • the choice of configuration will in part determine the geometry of the oligomer in the vicinity of the linkage. Thus, for example, if adjacent 3' positions are used to effect a covalent linkage, less severe deformation of the oligonucleotide chain will generally occur if both 3' hydroxyls involved in the linkage are in the conventional R configuration. If they are both in the S configuration, this will result in a formable "kink" in the chain.
  • the characterizing aspect of the switchback oligonucleotides of the invention is that they comprise tandem regions of inverted polarity, so that a region of 3'-»5' polarity is followed by one of 5' ⁇ 3' polarity, or vice versa, or both.
  • this coupling may be effected by insertion of a dimeric nucleotide wherein the appropriate 3' positions of each member of the dimer or the 5' positions of each member of the dimer are activated for inclusion of the dimer in the growing chain, or the conventional synthesis can be continued but using for the condensing nucleotide a nucleotide which is protected/activated in the inverse manner to that which would be employed if the polarity of the chain were to remain the same.
  • This additional nucleotide may also contain a linker moiety which may be included before or after condensation to extend the chain.
  • oligonucleotides having modified residues and/or inverted polarity may be accomplished utilizing standard solid phase synthesis methods.
  • solid phase-based approaches to the synthesis of oligonucleotides containing conventional 3' ⁇ 5' or 5' ⁇ 3' linkages one involving intermediate phosphoramidites and the other involving intermediate phosphonate linkages.
  • a suitably protected nucleotide having a cyanoethylphosphoramidite at the position to be coupled is reacted with the free hydroxyl of a growing nucleotide chain derivatized to a solid support.
  • the reaction yields a cyanoethyl- phosphite, which linkage must be oxidized to the cyanoethylphosphate at each intermediate step, since the reduced form is unstable to acid.
  • the H-phosphonate- based synthesis is conducted by the reaction of a suitably protected nucleoside containing an H-phosphonate moiety at a position to be coupled with a solid phase- derivatized nucleotide chain having a free hydroxyl group, in the presence of a suitable activator to obtain an H-phosphonate diester linkage, which is stable to acid.
  • the oxidation to the phosphate or thiophosphate can be conducted at any point during the synthesis of the oligonucleotide or after synthesis of the oligonucleotide is complete.
  • the H-phosphonates can also be converted to phosphoramidate derivatives by reaction with a primary or secondary amine in the presence of carbon tetrachloride.
  • the incoming nucleoside is regarded as having an "activated phosphite/phosphate" group.
  • Nonphos- phorous based linkages may also be used, such as the formacetal, 3' -thioformacetal, and 5' -amino type linkages described and claimed in copending application U.S. Serial No. 690,786 referred to above.
  • oligonucleotide segment which has a 3' ⁇ 5' polarity
  • a nucleotide protected at the 5' position and containing an activated phosphite/phosphate group at the 3 " ' position is reacted with the hydroxyl at the 5' position of a nucleoside coupled to a solid support through its 3' -hydroxyl.
  • the resulting condensed oligomer is deprotected and the reaction repeated with an additional 5' -protected, 3- 'phosphite/phosphate activated nucleotide.
  • a nucleotide protected in the 3' position and containing an activated phosphite/phosphate in the 5' position is reacted with a nucleotide oligomer or nucleoside attached to a solid support through the 5' position, leaving the 3' -hydroxyl available to react.
  • the 3' group is deprotected and reacted with an additional 3' -protected, 5' -activated nucleotide. The sequence is continued until the desired number of nucleotides have been added.
  • oligonucleotides may also be synthesized using solution phase methods such as triester synthesis. These methods are workable, but in general, less efficient for oligonucleotides of any substantial length. This oligonucleotide chain elongation will proceed in conformance with a predetermined sequence in a series of condensations, each one of which results in the addition of another nucleotide. Prior to the addition of a nucleoside having an activated phosphite/ phosphate, the protecting group on the solid support-bound nucleotide is removed.
  • DMT dimethoxytrityl
  • the carrier bound nucleotide is preferably washed with anhydrous pyridine/ acetonitrile (1:1, v/v) , again deprotected, and the condensation reaction is completed in as many cycles as are required to form the desired number of congruent polarity internucleotide bonds which will be converted to phosphoramidates, phosphoro- dithioates, phosphorothioates or phosphodiesters as desired.
  • the incoming activated, protected nucleoside is provided in the opposite polarity to the support-bound oligomers.
  • the support-bound oligomer is 3'-»5 * "
  • the deprotected 5' hydroxyl is reacted with a 3 ' -protected, 5' -activated monomer, and the synthesis continued with monomers activated at the 5 ' position and protected at the 3' position.
  • a dinucleoside synthon containing the linker element having one end which is activated for condensation (such as a hydrogen phosphonate) to the support-bound oligonucleotide and another end which is a protected hydroxyl group (or protected thio group) is condensed onto the support-bound oligonucleotide.
  • the linked dinucleoside is condensed and deprotected using the same conditions as those used to condense and deprotect the protected nucleoside hydrogen phosphonate.
  • Subsequent extension of the oligonucleotide chain then uses oligonucleotide residues which are activated and protected in the opposite manner from those used to synthesize the previous portion of the chain.
  • FIG. 2 One approach to this synthesis, using a linker already derivatized to two nucleotide/nucleoside residues which will be included in each portion of the strand is illustrated in Figure 2.
  • the 5' ⁇ 3' nucleotide portion of the strand is coupled using the 3' DMT-5' -activated phosphate nucleosides, as conventionally, to solid support.
  • the linker is derivatized to two nucleotide residues through their 3' positions; the remaining 5' positions are derivatized by the protecting group DMT in one nucleotide residue and a phosphonate residue in the other.
  • the derivatized linker is coupled to the solid supported strand under standard reagent conditions and then deprotected conventionally. Further standard nucleotide coupling results in extension of the chain in the 3' ⁇ 5' orientation.
  • a particularly preferred dimer synthon used to mediate the switchback in an oligomer is the o-xyloso linker.
  • This o-xyloso linker consists of two xylose- nucleosides linked to each other by o-xylene at the 3' position of each xylose sugar.
  • the switchback linker synthon was synthesized using a,a' -dibromoxylene and 5'- DMT nucleoside to give the dimer.
  • the dimer was converted to the H-phosphonate and was used in solid phase synthesis to generate oligomers.
  • switchback linker dimers may also be synthesized as mixed heterodimers that are separated chromatographically or electrophoret ⁇ cally.
  • oligomers of the present invention is mixed binding motif oligomers.
  • the target sequence for such oligomers is characterized by a G-rich ( ⁇ ⁇ 65% guanosine) polypurine region that is adjacent to an A-rich ( ⁇ - 65% adenosine) region.
  • the A- rich and G-rich target regions may lie on either the same or on opposite strands, and where opposite strands are to be targeted, a switchback linker may optimally be used in the oligomer.
  • Targets such as these would be efficiently bound by oligomers containing, in one region that binds to the G-rich region of the target, thymidine, guanosine, 2 '-deoxy-7-deazaxanthosine, 2' -deoxy-7-deazaguanidine or related nucleotide analogs of the present invention. Such binding would be mediated in the GT binding motif.
  • the other region would consist of nucleotides or nucleotide analogs such as 2'-deoxythymidine, 2'-deoxy-5- methylcytosine, 2 '-deoxycytosine, 2'-deoxy- pseudoisocytosine, 2 '-deoxy-8-oxo-N -methyladenosine or 2 ' -deoxy-N 4 -N 4 -ethanocytosine, that bind to A-rich target sequences in the CT binding motif.
  • 2' -deoxy-8-oxo-N - methyladenosine is disclosed and claimed in commonly assigned U.S. application serial no. 643,382, which is incorporated herein by reference.
  • oligomers containing one region that binds to DNA via CT motif binding may be incorporated adjacent to a region that contains the novel compounds of the present invention.
  • Such oligomers would have continuous polarity but would bind to an A-rich target on one strand that is adjacent to a G-rich target on the other strand.
  • Covalent bonding moieties would preferentially be incorporated into the CT region of the mixed binding motif oligomer.
  • Oligomers of the invention are capable of significant binding activity to form triplexes or other forms of stable associations. Accordingly, these oligomers are useful in diagnosis and therapy of diseases characterized by the presence of a given genetic sequence such as neoplastic growth (HER-2) and viral infections (HIV) .
  • HER-2 neoplastic growth
  • HIV viral infections
  • the oligomers utilized in a manner appropriate for treatment of the particular disease can be formulated for a variety of modes of administration, including systemic, topical or localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA, latest edition.
  • the oligomer itself may be the only active ingredient and is generally combined with a carrier such as a diluent or excipient which may include fillers, extenders, binders, wetting agents, disintegrants, surface-active agents, or ⁇ lubricants, depending on the nature of the mode of administration and dosage forms.
  • Typical dosage forms include tablets, powders, liquid preparations including suspensions, emulsions and solutions, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.
  • injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous.
  • the oligomers of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • Dosages that may be used for systemic administration preferably range from about 0.01 mg/kg to 50 mg/Kg administered once or twice per day. However, different dosing schedules may be utilized depending on (i) the potency of an individual oligomer at inhibiting the activity of its target gene, (ii) the severity or extent of a pathological disease state associated with a given target gene, or (iii) the pharmacokinetic behavior or a given oligomer.
  • Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucosal administration.
  • detergents may be used to facilitate permeation.
  • Transmucosal adminis ⁇ tration may be through use of nasal sprays, for example, or suppositories.
  • the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
  • the oligomers of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art.
  • the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of a given target such as HER-2 or HIV sequences to which they specifically bind. Such diagnostic tests are conducted by hybridization through triple helix formation which is then detected by conventional means.
  • the oligomers may be labeled using any detectable label such as radioactive, fluorescent, or chromogenic labels and the presence of label bound to solid support detected.
  • the presence of a triple helix may be detected by antibodies which specifically recognize these forms.
  • Means for conducting assays using such oligomers as probes are generally known.
  • oligomers as diagnostic agents by triple helix formation is advantageous since triple helices form under mild conditions and the assays may thus be carried out without subjecting test specimens to harsh conditions.
  • Diagnostic assays based on detection of RNA for identification of bacteria, fungi or protozoa sequences often require isolation of RNA from samples or organisms grown in the laboratory, which is laborious and time consuming; as RNA is extremely sensitive to ubiquitous nucleases.
  • the oligomer probes may also incorporate additional modifications such as altered internucleotide linkages that render the oligomer especially nuclease stable, and would thus be useful for assays conducted in the presence of cell or tissue extracts which normally contain nuclease activity. Oligonucleotides containing terminal modifications often retain their capacity to bind to complementary sequences without loss of specificity (Uhlmann et al., Chemical Reviews (1990) 9_0:543-584) .
  • the invention probes may also contain - linkers that permit specific binding to alternate DNA strands by incorporating a linker that permits such binding (Home et al. , J Am Chem Soc (1990) 112:2435- 2437) .
  • incorporation of base analogs of the present invention into probes that also contain covalent crosslinking agents has the potential to increase sensitivity and reduce background in diagnostic or detection assays.
  • the use of crosslinking agents will permit novel assay modifications such as (1) the use of the crosslink to .increase probe discrimination, (2) incorporation of a denaturing wash step to reduce background and (3) carrying out hybridization and crosslinking at or near the melting temperature of the hybrid to reduce secondary structure in the target and to increase probe specificity. Modifications of hybridization conditions have been previously described (Gamper et al., Nucleic Acids Res (1986) 14:9943) .
  • the ability of the oligomers to inhibit gene expression can be verified in in vitro systems by measuring the levels of expression in recombinant systems.
  • the cold solution was treated with 1.5 mL of a 1 M solution of 2-chloro-4H-1,3,2- benzodioxaphosphorin-4-one in methylene chloride. After 15 min, the solution was quenched with 40 mL of ice-cold triethyl ammonium bicarbonate buffer pH 7.5, and the mixture was extracted with methylene chloride. The organic extracts were evaporated and the residue was chromatographed on a silica gel column using acetonitrile/water 9:1 v:v (2% triethylamine) to afford 360 mg of the phosphonate. The protected H-phosphonate was then used in solid phase synthesis to generate oligomers.
  • Example 3 Preparation of Oligomers Containing 7-deaza-2' -deoxyguanosine In a manner similar to that set forth in Example 2, 2'-deoxy-7-deazaguanosine was converted to the 5'-DMT-blocked 3' -H-phosphonate monomer incorporated into oligomers.
  • the GT oligomer sequence used was:
  • the GX oligomer sequence used was: 5' GGXGGXGGXXGXGGXY 3' .
  • Y is anthraquinone and was incorporated into oligomers as described (Lin, K. et al., Nucleic Acids Res (1991) 19 . :3111-3114) .
  • the anthraquinone moiety did not affect binding of the oligomer to duplex target DNA.
  • Both oligomers were footprinted, the GX oligomer footprinted at 1 ⁇ m; no binding was observed with the GT control oligomer.
  • Triplex formation conditions were 20 mM MOPS, pH 7.2, 140 mM KC1, 1 mM spermine, and 1 mM MgCl 2 . Thus, triplex formation was observed under physiological ion conditions due to the presence of 2' -deoxy-7-deazaxanthosine residues.
  • a G*T oligomer synthesized was 20 mM MOPS, pH 7.2, 140 mM KC1, 1 mM spermine, and 1 mM MgCl 2

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Abstract

Les oligomères contenant les bases désazapurine ont une plus grande aptitude à former des hélices triples que les oligomères ne contenant que les bases conventionnelles. Les bases désazapurine sont des formes substituées et non substituées de la 7-désazaxanthine et de la 7-désazaguanine. Les oligomères selon l'invention peuvent former au pH physiologique des hélices triples avec différentes séquences cibles telles que des séquences de HER-2 et de HIV par couplage dans le sillon principal de la double hélice cible d'ADN. Les oligomères de l'invention peuvent être combinés à des vecteurs acceptables sur le plan pharmaceutique et ils peuvent être réalisés avec une quelconque séquence souhaitée, à condition que celle-ci comprenne au moins une désazapurine augmentant l'aptitude de l'oligomère à former une triple hélice avec la séquence cible. Les compositions de l'invention peuvent être utilisées comme produits pharmaceutiques pour traiter différentes maladies associées au matériel génétique et elles peuvent être utilisées pour diagnostiquer différentes maladies.
PCT/US1992/009195 1991-11-07 1992-10-21 Formation amelioree d'une triple helice, grace a des oligonucleotides contenant la 2'-desoxy-7-desazaxanthosine, la 2'-desoxy-7-desazaguanosine et des composes analogues WO1993009127A1 (fr)

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WO1993012135A1 (fr) 1991-12-12 1993-06-24 Gilead Sciences, Inc. Oligomeres stables a la nuclease et aptes aux liaisons et methodes d'utilisation
WO1994024144A2 (fr) * 1993-04-19 1994-10-27 Gilead Sciences, Inc. Formation a helice triple et double a l'aide d'oligomeres contenant des purines modifiees
EP0710667A2 (fr) 1994-11-04 1996-05-08 Hoechst Aktiengesellschaft Oligonucléotides modifiés, leur préparation et leur utilisation
US5594121A (en) * 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
GB2323357A (en) * 1997-03-20 1998-09-23 Amersham Pharm Biotech Inc Derivatives of 7-deaza-2'-deoxy-guanosine-5'-triphosphate, preparation and use thereof
US6093807A (en) * 1999-03-19 2000-07-25 Isis Pharmaceuticals, Inc. Sugar-modified 7-deaza-7-substituted oligonucleotides
US6150510A (en) * 1995-11-06 2000-11-21 Aventis Pharma Deutschland Gmbh Modified oligonucleotides, their preparation and their use
US6670393B2 (en) 1995-06-07 2003-12-30 Promega Biosciences, Inc. Carbamate-based cationic lipids
US6906185B1 (en) 1997-03-20 2005-06-14 Amersham Biosciences Corp. Derivatives of 7-deaza -2′-deoxyguanosine-5'-triphosphate, preparation and use thereof
US7070933B2 (en) 2001-09-28 2006-07-04 Gen-Probe Incorporated Inversion probes
US8367628B2 (en) 2005-12-01 2013-02-05 Pronai Therapeutics, Inc. Amphoteric liposome formulation
WO2013179289A1 (fr) * 2012-05-31 2013-12-05 Bio-Lab Ltd. Analogues de pyrazolotriazolyl-nucléosides et oligonucléotides les comprenant
US8815599B2 (en) 2004-06-01 2014-08-26 Pronai Therapeutics, Inc. Methods and compositions for the inhibition of gene expression

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Volume 89, issued 01 May 1992, S.T. KRAWCZYK et al., "Oligonucleotide-Mediated Triple Helix Formation Using an N3-Protonated Deoxycytidine Analog Exhibitin pH-Independent Binding Within the Physiological Range", pages 3761-3763. *
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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5594121A (en) * 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
WO1993012135A1 (fr) 1991-12-12 1993-06-24 Gilead Sciences, Inc. Oligomeres stables a la nuclease et aptes aux liaisons et methodes d'utilisation
WO1994024144A2 (fr) * 1993-04-19 1994-10-27 Gilead Sciences, Inc. Formation a helice triple et double a l'aide d'oligomeres contenant des purines modifiees
WO1994024144A3 (fr) * 1993-04-19 1995-03-16 Gilead Sciences Inc Formation a helice triple et double a l'aide d'oligomeres contenant des purines modifiees
EP0710667A2 (fr) 1994-11-04 1996-05-08 Hoechst Aktiengesellschaft Oligonucléotides modifiés, leur préparation et leur utilisation
EP0710667A3 (fr) * 1994-11-04 1997-09-10 Hoechst Ag Oligonucléotides modifiés, leur préparation et leur utilisation
US6987177B2 (en) 1994-11-04 2006-01-17 Aventis Pharma Deutschland Gmbh Modified oligonucleotides, their preparation and their use
US5844106A (en) * 1994-11-04 1998-12-01 Hoechst Aktiengesellschaft Modified oligonucleotides, their preparation and their use
US6479651B1 (en) 1994-11-04 2002-11-12 Aventis Pharma Deutschland Gmbh Modified oligonucleotides, their preparation and their use
US6670393B2 (en) 1995-06-07 2003-12-30 Promega Biosciences, Inc. Carbamate-based cationic lipids
US6150510A (en) * 1995-11-06 2000-11-21 Aventis Pharma Deutschland Gmbh Modified oligonucleotides, their preparation and their use
GB2323357B (en) * 1997-03-20 1999-09-29 Amersham Pharm Biotech Inc Derivatives of 7-deaza-2-deoxy-guanosine-5'-triphosphate, preparation and use thereof
US6906185B1 (en) 1997-03-20 2005-06-14 Amersham Biosciences Corp. Derivatives of 7-deaza -2′-deoxyguanosine-5'-triphosphate, preparation and use thereof
GB2323357A (en) * 1997-03-20 1998-09-23 Amersham Pharm Biotech Inc Derivatives of 7-deaza-2'-deoxy-guanosine-5'-triphosphate, preparation and use thereof
US6093807A (en) * 1999-03-19 2000-07-25 Isis Pharmaceuticals, Inc. Sugar-modified 7-deaza-7-substituted oligonucleotides
US7070933B2 (en) 2001-09-28 2006-07-04 Gen-Probe Incorporated Inversion probes
US8815599B2 (en) 2004-06-01 2014-08-26 Pronai Therapeutics, Inc. Methods and compositions for the inhibition of gene expression
US9393258B2 (en) 2004-06-01 2016-07-19 Pronai Therapeutics, Inc. Methods and compositions for the inhibition of gene expression
US8367628B2 (en) 2005-12-01 2013-02-05 Pronai Therapeutics, Inc. Amphoteric liposome formulation
WO2013179289A1 (fr) * 2012-05-31 2013-12-05 Bio-Lab Ltd. Analogues de pyrazolotriazolyl-nucléosides et oligonucléotides les comprenant
US9994604B2 (en) 2012-05-31 2018-06-12 Bio-Lab Ltd. Pyrazolotriazolyl nucleoside analogues and oligonucleotides comprising them

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