WO2018156056A1 - Oligonucléotides modifiés activant l'arnase n - Google Patents

Oligonucléotides modifiés activant l'arnase n Download PDF

Info

Publication number
WO2018156056A1
WO2018156056A1 PCT/RU2018/050022 RU2018050022W WO2018156056A1 WO 2018156056 A1 WO2018156056 A1 WO 2018156056A1 RU 2018050022 W RU2018050022 W RU 2018050022W WO 2018156056 A1 WO2018156056 A1 WO 2018156056A1
Authority
WO
WIPO (PCT)
Prior art keywords
modified
oligonucleotide
group
oligonucleotides
carrier
Prior art date
Application number
PCT/RU2018/050022
Other languages
English (en)
Russian (ru)
Inventor
Дмитрий Александрович СТЕЦЕНКО
Борис Павлович ЧЕЛОБАНОВ
Алеся Анатольевна ФОКИНА
Екатерина Анатольевна БУРАКОВА
Original Assignee
Дмитрий Александрович СТЕЦЕНКО
Борис Павлович ЧЕЛОБАНОВ
Алеся Анатольевна ФОКИНА
Екатерина Анатольевна БУРАКОВА
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Дмитрий Александрович СТЕЦЕНКО, Борис Павлович ЧЕЛОБАНОВ, Алеся Анатольевна ФОКИНА, Екатерина Анатольевна БУРАКОВА filed Critical Дмитрий Александрович СТЕЦЕНКО
Publication of WO2018156056A1 publication Critical patent/WO2018156056A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • 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
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • the invention relates to the field of molecular medicine and bioorganic chemistry.
  • the subject of the invention is new biologically stable derivatives of oligonucleotides with modified phosphate groups, capable of forming a strong complementary duplex with RNA, penetrating into cells in the presence of a transfection agent or in its absence, and activating the enzyme RNase N.
  • Modified oligonucleotides are promising agents for targeting the genomes of living organisms and viruses [Therapeutic Oligonucleotides, J. Goodchild (ed.), Methods Mol. Biol., 764, Humana Press, 2011]. Derivatives of oligonucleotides are also widely used as molecular diagnostic tools [Bell, N.M .; Micklefield, J. ChemBioChem, 2009, 10, 2691]. A number of therapeutic drugs based on modified oligonucleotides have entered clinical practice. This is, for example, the antiviral drug Vitravene (Fomivirsen, ISIS 2922) [Mulamba, G.B .; Hu, A .; Azad, R.F.
  • the requirements for drugs based on modified oligonucleotides include:
  • derivatives of oligonucleotides are able to inhibit both stages of the transfer of genetic information: both from genomic DNA to mRNA (transcription) and from mRNA to protein (translation). Inhibition of transcription is carried out by binding of DNA to triplex-forming oligonucleotides [Duca, M .; Vekhoff, P .; Oussedik, K .; Halby, L .; Arimondo, R.V. Nucl. Acids Res. 2008, 36, 5123], in particular, peptide nucleic acids (PNAs) [Nielsen, P.E. Curr. Opin. Mol. Ther. 2010, 12, 184].
  • PNAs peptide nucleic acids
  • oligonucleotide derivatives are capable of modulating gene expression by binding to proteins [Lee, J.F .; Stovall, G.M .; Ellington, A.D. Curr. Opin. Chem. Biol. 2006, 10, 282; Davydova, A.C .; Vorobyova, M.A .; Venyaminova, A. G. Acta Naturae 2011, 3, 13].
  • antisense agents based on oligonucleotide analogues act on the principle of spatial translation blocking, binding to mRNA with high affinity [Du, L .; Gatti, RA Curr. Opin. Mol. Ther. 2009, 11, 116; Perez, B .; Laura Rodriguez-Pascau, L .; Vilageliu, L .; Grinberg, D .; Ugarte, M .; Desviat, LRJ Inherit. Metabol. Disease 2010, 33, 397].
  • oligo-2'-ribofluoro-2'-deoxynucleotides include derivatives with a modified ribose residue, namely, oligo-2'-ribofluoro-2'-deoxynucleotides [Ruckman, J .; Green, LS; Beeson, J .; Waugh, S .; Gillette, WL; Henninger, DD; Claesson- Welsh, L .; Janjic, NJ Biol. Chem. 1998, 273, 20556], oligo-2'-O-methylribonucleotides [Lamond, AI; Sproat, BS FEBS Lett.
  • oligo-2'-O-P-methoxyethylribonucleotides (2'-MOE) [Chi, KN; Eisenhauer, E .; Jones, EC; Goldenberg, SL, Powers, J .; Tu, D .; Gleave, MEJ Natl. Cancer Inst. 2005, 97, 1287] and "locked nucleic acids” (eng. Locked nucleic acids, LNA) [Kaur, H .; Babu, BR; Maiti, S. Chem. Rev. 2007, 107, 4672].
  • 2'-MOE oligo-2'-O-P-methoxyethylribonucleotides
  • oligonucleotides with a rearranged sugar phosphate backbone for example, peptide nucleic acids (PNA) [Egholm. M .; Buchardt, O .; Nielsen, R.E .; Berg, RHJ Am. Chem. Soc. 1992, 114, 1895] and phosphorus diamide morpholine oligonucleotides (FMO) [Summerton, J .; Weller, D. Antisense Nucl. Acid Drug Dev. 1997, 7, 187].
  • PNA peptide nucleic acids
  • FMO phosphorus diamide morpholine oligonucleotides
  • modified oligonucleotide analogues are compounds with a modified internucleotide phosphate group.
  • modified internucleotide phosphate group include, for example, oligodeoxynucleotides with thiophosphate [Stec, W.J .; Zon, G .; Egan, W .; Stec, B. J. Am. Chem. Soc. 1984, 106, 6077], dithiophosphate [Seeberger, P.H .; Caruthers, M.H. J. Am. Chem. Soc. 1995, 117, 1472] and boranophosphate groups [Rait, V.K .; Shaw, B. R., Antisense Nucleic Acid Drug Dev., 1999, 9, 53].
  • oligodeoxynucleotide derivatives are capable of causing irreversible inactivation of mRNA by activating the cellular enzyme RNase H, whose biological role is to catalyze the selective hydrolytic cleavage of RNA as part of a DNA heteroduplex: RNA [Yang, W .; Hendricson, W.A .; Crouch, R.J .; Satow, Y., Science, 1990, 249, 1398].
  • siRNAs cause catalytic hydrolysis of mRNA by recruiting an RNA-protein RISC complex with nuclease activity, and ribozymes and deoxyribozymes do not need cell proteins, being themselves catalysts for RNA hydrolytic cleavage.
  • Providing a catalytic effect can reduce the therapeutic dose of the antisense drug due to the inactivation of several mRNA molecules with one antisense oligonucleotide molecule.
  • oligonucleotides capable of causing RNase H activation are oligodeoxynucleotide (DNA) derivatives (Fig. 1, 1) with a modified internucleotide phosphate group.
  • oligodeoxynucleotides in which the natural phosphate groups are replaced by thiophosphate groups (Fig. 1, la) [Stein, C.A .; Subasinghe, C; Shinozuka, K .; Cohen, J.S. Nucl. Acids Res. 1988, 16, 3209].
  • Thiophosphate oligonucleotide analogues PS oligonucleotides are most commonly used as antisense oligonucleotides [Crooke, S.T., Annu Rev. Med., 2004, 55, 61;
  • thiophosphate groups into oligonucleotides is primarily necessary to increase the resistance to the action of nucleases: in this case, the half-life
  • oligonucleotides in human serum are increased to 9-10 hours [Campbell, J.M .; Bacon, T.A .; Wickstrom, E., J. Biochem. Biophys. Methods, 1990, 20, 259; Phillips, M.I .; Zhang, Y. C., Methods Enzymol, 2000, 313, 46].
  • PS-oligonucleotides form stable duplexes with RNA due to Watson-Crick base pairs, have a total negative charge, which facilitates
  • transfection agents based on cationic lipids, for example, lipofectamine 3000, and have attractive pharmacokinetic properties.
  • Their disadvantage is the increased ability to bind to certain proteins, such as heparin-binding proteins [Guvakova, M.A .; Yakubov, L.A .; Vlodavsky, I .; Tonkinson, J.L .;
  • PS oligonucleotides also have a relatively high toxicity and somewhat reduced RNA affinity compared to other analogues. These flaws hold back
  • Dithiophosphate analogues of oligodeoxynucleotides are capable of activating RNase H, similarly to thiophosphate analogues, although with less efficiency, and provide even greater resistance to the action of nucleases.
  • DNA dithiophosphates bind to proteins more strongly than PS oligonucleotides, and at the same time are less specific in the inhibition of translation.
  • their chemical synthesis in comparison with thiophosphates is more complicated.
  • dithiophosphates do not have great advantages over PS oligonucleotides [Verma, S .;
  • Boranophosphate oligodeoxynucleotides are relatively stable compounds that protect them from nuclease degradation, as is the case with the P-S group of thiophosphates [Rait, V.K .; Shaw, B. R., Antisense Nucleic Acid Drug Dev., 1999, 9, 53]. Boranophosphates are resistant to
  • nucleases upon the action of nucleases, they form stable duplexes with RNA targets and activate RNase H, but less efficiently than phosphodiester and thiophosphate oligodeoxynucleotides, which reduces their attractiveness for further use as antisense agents [Rait, V .; Sergueev, D .; Summers, J .; He, K .; Huang, F .; Krzyzanowska, B .; Shaw, B.R., Nucleosides
  • ANA arabinonucleic acid
  • Fig. 1, 2a Damha et al. derivatives of arabinonucleic acid (ANA) (Fig. 1, 2a) have been shown to form a duplex with RNA, which is substrate 155 for RNase H [Damha, M.J .; Wilds, C.J .; Noronha, A. et al, J. Am. Chem. Soc, 1998, 120, 12976].
  • ANA PHK duplexes have less stability than DNA: RNA and P8-DNA: RNA duplexes, which significantly reduces their interest.
  • oligo-2'-ara-fluoro-2'-deoxynucleotides form duplexes with RNA, the thermal stability of which, as a rule, is 160 greater than that of DNA: RNA duplexes [ Patureau, V.M .; Hudson, RHE; Damha, MJ, Bioconjugate Chem., 2007, 18, 421].
  • Duplex FANA PHK has a similar helical conformation as the DNA: RNA hybrid. In this case, the fluorine atom is located in a large groove, i.e. where it should not interfere with the activation of RNase H [Kalota, A., Karabon, L., Swider, CR, Viazovkina, E. et al., Nucleic Acids
  • RNA 170 increase in affinity for RNA can be used in the form of so-called “Hapmers” including both a separate RNase H activating domain and strongly RNA binding domains, for example, based on 2'-O-alkylribonucleosides [Crooke, S.T., Methods Enzymol, 2000, 313, 3].
  • 175 phosphate groups are their relatively moderate cost due to the use of natural 2'-deoxyribonucleosides and highly effective solid-phase phosphitamide chemistry.
  • analogues of oligodeoxynucleotides with an uncharged internucleotide phosphate group for example, methylphosphonate [Jager, A .;
  • phosphate group modified oligonucleotide analogues 190 affinity for RNA and differ in resistance to enzymatic cleavage, which must be taken into account when assessing the prospects of using certain oligonucleotide derivatives with modified phosphate group as therapeutic agents.
  • phosphate group modified oligonucleotide analogues 190 affinity for RNA and differ in resistance to enzymatic cleavage, which must be taken into account when assessing the prospects of using certain oligonucleotide derivatives with modified phosphate group as therapeutic agents.
  • phosphate group modified oligonucleotide analogues phosphate group modified oligonucleotide analogues
  • Cyclohexene nucleic acids (CeNA) (Fig. 1, 3) are a separate class of nucleic acids in which the sugar residue is replaced by a cyclohexene ring [Herdewijn, P., De Clercq, E., Bioorg. Med. Chem. Lett., 2001, 11, 1591]. Cyclohexene Oligonucleotides were Synthesized by Herdewijn
  • CeNAs form stable duplexes with complementary DNA or RNA and protect oligonucleotides from degradation by nucleases.
  • CeNA can be considered as a new class of oligonucleotides, combining the advantage of stabilizing the duplex with RNA and resistance in serum with the potential for activating RNase N.
  • these oligonucleotide analogues are candidates for use as
  • antisense agents in cellular systems reduced ability to activate RNase H and their complex and laborious synthesis, and, as a result, high cost, reduce interest in the use of cyclohexene oligonucleotides as antisense agents in therapy.
  • 215 oligonucleotides belonging to the class of C-phosphonates and containing instead of a phosphate group an anionic phosphonate group.
  • derivatives of phosphonoacetates 4a and thiophosphonoacetates 46 [Dellinger et al., J. Am. Chem. Soc. 2003, 125, 940] (Fig. 1). According to the literature, the above derivatives have a set of properties that make them suitable for
  • the approach used in the present invention is to use for chemical modification of oligonucleotides bearing when
  • oligonucleotides with aromatic K- (sulfonyl) -phosphoramide group (Fig. 2, 5a), disclosed in the article [Heindl, D .; Kessler, D .; Schube, A .; Thuer, W .; Giraut, A. Nucleic Acids Symp. Ser. 2008, 52, 405] and the patent [Heindl, D., F. Hoffman-La Roche AG WO 2008/128686 Al dated 04/18/2007].
  • Staudinger reaction [Staudinger, N .; Meyer, J., Helv. Chim. Acta,
  • the present invention is based on the approach developed by the authors to design promising candidates for the role of therapeutic oligonucleotides with enhanced enzymatic stability, capable of forming a strong complementary complex (duplex) with the target RNA,
  • oligonucleotides as potential therapeutic agents:
  • the present invention discloses new analogues of oligonucleotides that meet the above requirements for therapeutic oligonucleotides, in particular, have high chemical and biological resistance, the ability to form a strong duplex with RNA and cause it
  • these analogues belong to the class of imides of phosphoric acid and sulfonic acids. Accordingly, in
  • a preferred application of the present invention discloses oligonucleotides containing a K- (sulfonyl) phosphoramide group, preferably a K- (alkanesulfonyl) phosphoramide group.
  • the present invention includes derivatives of oligonucleotides with N-th (sulfonyl) -phosphoramide groups that contain other 320 modified phosphate groups, for example, thiophosphate or phosphorylguanidine groups, as well as other chemical modifications, for example, in carbohydrate residues, in particular, leading to the formation of “hapmers”.
  • the technical result of the invention is to obtain new 325 promising for use in medicine derivatives of oligonucleotides capable of activating RNase H, in which the natural phosphate group is replaced by - (sulfonyl) -phosphoramide while maintaining, modifying or replacing the natural carbohydrate residue with its analogue.
  • oligonucleotides resulting from this 330 invention contain one or more (two or more, up to exhaustive substitution by them of all natural phosphate groups) of N- (sulfonyl) phosphoramide groups corresponding to general formula (I) (taking into account the possibilities of ionization and salt formation due to proton separation in the NH-group):
  • modified phosphate groups are very interesting because of their physicochemical properties, since they can lead to the formation of 340 compounds that retain a negative charge under physiological conditions (pH about 7). This causes interest in these compounds in order to obtain close analogues of natural oligonucleotides suitable for use in medicine as therapeutic agents and molecular diagnostics, as well as tools in scientific research. 345
  • the present inventors have found that these modified phosphate groups can be introduced into oligonucleotides using methods compatible with many known and biologically active oligonucleotide sequences and derivatives.
  • a modified phosphate group may correspond to 350 formula (II):
  • each of the substituents R 3 and R 4 is independently selected from the group consisting of —H, —C-20 alkyl, —C2-20 alkenyl, —C2-20 alkynyl, —Cb-u aryl, or —C5-10 heteroaryl;
  • alkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocycle may be further substituted.
  • the present invention may consist in 365 obtaining derivatives of oligonucleotides corresponding to the formula (III):
  • 370 Deputy R is independently selected from the group consisting of a nucleotide, modified nucleotide, nucleotide analog, oligonucleotide, modified oligonucleotide, or oligonucleotide analog; a hydrogen atom —H, —PG, where PG is a protecting group; linker, monophosphate group, diphosphate group, triphosphate group,
  • R 1 is independently selected from the group consisting of a nucleotide, modified nucleotide, nucleotide analog, oligonucleotide, modified oligonucleotide or oligonucleotide analog; a hydrogen atom —H, —PG, where PG is a protecting group;
  • Deputy R 2 independently selected from the group including alkyl, -C2-20 alkenyl, -C2-20 alkynyl, -Cb-aryl, -C5-10 heteroaryl, -F, -N3, -CN or -NR 3 R 4 ;
  • each of the substituents R 3 and R 4 is independently selected from the group consisting of —H, —C-20 alkyl, —C2-20 alkenyl, —C2-20 alkynyl, —Cb-aryl, or —C5-10 heteroaryl;
  • R 2 is selected from —NR 3 R 4 , —OR 3, and —SR 3 . In some applications, R 2 is —NR 3 R 4 .
  • the oligonucleotide disclosed in this invention may be a modified oligonucleotide.
  • modified in this context refers to a modified phosphate group, for example, the corresponding formula (II).
  • each subsequent nucleoside may independently be a nucleoside analog and, additionally or vice versa, each subsequent phosphate group, if any, may be modified in accordance with the present invention.
  • R 3 and R 4 may independently be selected from the group consisting of —H, —C-20 alkyl, —C2-20 alkenyl, —C2-20 alkynyl, —Cb-aryl, or —C5-10 heteroaryl, where each alkyl, aryl, heteroaryl, alkylene or heteroalkylene may be further substituted.
  • R 3 and R 4 may be a hydrogen atom or a methyl group.
  • R 4 is independently selected from the group consisting of —H and —C — b alkyl optionally substituted with one or more substituents from the series —F, —CI, —Br, —I, —CN, —N3, —OH, —C-6 alkoxy, -NH 2 , -NH (Ci-6 alkyl) and -N (Ci-6 alkyl) g; preferably from —H and —Cl — balkyl; even more preferably from a hydrogen atom —H and a methyl group.
  • R 4 is independently selected from the group consisting of —H and —C — b alkyl optionally substituted with one or more substituents from the series —F, —CI, —Br, —I, —CN, —N3, —OH, —C-6 alkoxy, -NH 2 , -NH (Ci-6 alkyl) and -N (Ci-6 alkyl) g
  • each substituent R 3 and R 4 is independently
  • 425 is selected from the group consisting of —H and —O-b alkyl optionally substituted
  • R 2 is -NR 3 R 4, preferably -NMe2.
  • R 2 is —NH 2 or —NMe2.
  • R 3 and R 4 together with the atom to which they are attached, form a 5-8 membered heterocycle.
  • the heterocycle is piperazine.
  • the present invention encompasses oligonucleotides containing at least one modified phosphate group corresponding to formula (III), where R, R 1 and R 2 are defined, 445 as described above.
  • the present invention encompasses oligonucleotides in which the modified phosphate group connecting adjacent nucleosides or nucleoside analogs is a K- (sulfonyl) phosphoramide group.
  • Modified oligonucleotides that are the subject of the present invention can be used to obtain various results.
  • oligonucleotide preferably single-stranded
  • hybridization i.e. the formation of a complementary complex, preferably double-stranded (duplex).
  • the aforementioned method may include detection
  • the method can be used to detect a specific oligonucleotide, for example, mutated, with a changed sequence or containing a single nucleotide polymorphism (SNP). This method can be used to diagnose a disease in a patient, for example,
  • oligonucleotides that are the subject of the present invention can be used to obtain primers for the amplification of nucleic acids by the method of polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • oligonucleotides that are the subject of the present invention can be used to obtain oligonucleotide chips (DNA chips), followed by their use in methods requiring the use of DNA chips.
  • DNA chips oligonucleotide chips
  • oligonucleotides that are the subject of the present invention can be used to obtain therapeutic agents based on
  • oligonucleotides such as siRNA [Angell & Baulcombe, EMBO J., 1997, 16, 3675; Voinnet & Baulcombe, Nature, 1997, 389, 553; Fire, A. et al., Nature, 1998, 391; Fire, A., Trends Genet., 1999, 15, 358, Sharp, Genes Dev., 2001, 15, 485; Hammond et al, Nature Rev. Genet., 2001, 2, 1110; Tuschl, ChemBioChem, 2001, 2, 239], ribozymes, DNAzymes, aptamers [Gould, L. et al., Science, 1990, 249, 505; WO patent
  • antisense antisense oligonucleotides (including “hapmers”).
  • Oligonucleotide therapeutic agents are used to treat a number of diseases, including viral infections, cancer, eye diseases, including age-related diseases, to prevent unwanted neovascularization, diseases caused by splicing disorder, such as Duchenne muscular dystrophy, and
  • a therapeutic oligonucleotide may include a covalently attached peptide, including to improve penetration into cells, for example, as described in patent WO 2009/147368.
  • the oligonucleotide 495 of the subject invention is intended for use in medicine as a medicine or therapeutic agent.
  • the oligonucleotide of the invention is intended to provide a medicament or dosage form for use in the treatment of a disease.
  • a method of treating a disease comprising administering to the patient an oligonucleotide of the invention to treat a disease.
  • the oligonucleotide which is the subject of the present invention, may be part of a medicament or dosage form. Medication or
  • 505 dosage form may include the oligonucleotide, which is the subject of the present invention, in isolated or purified form, as well as pharmaceutically acceptable additives.
  • Medicines or dosage forms comprising oligonucleotides that are the subject of the present invention may be
  • Medicines and dosage forms may be in liquid or solid form. Liquid forms may be administered by injection into an appropriate part of the human or animal body.
  • the administration is in a therapeutically effective amount (dose), i.e. in an amount sufficient to produce a therapeutically beneficial effect.
  • dose a therapeutically effective amount administered and the time schedule for administration will depend on the nature and severity of the disease. Appropriate therapeutic solutions, as well as dosages, are found in
  • Methods of using the oligonucleotides of the present invention may include both their in vitro and in vivo use.
  • the term "in vitro” in this case means experiments with materials, biological samples, cells and / or tissues in the laboratory or in cultures of cells and / or tissues.
  • the objects of use of the oligonucleotides that are the subject of the present invention can be plants, animals, preferably mammals, and more preferably humans, including male 535 or female patients.
  • nucleotide is used to denote a chemical compound containing a nucleoside or a modified nucleoside, and at least 545 one phosphate group or a modified phosphate group attached to it by a covalent bond.
  • a covalent bond independently and without limitation, is an ether bond between a 2'-, 3'- or 5'-hydroxyl group of a nucleoside and a phosphate group or a modified phosphate group.
  • oligonucleotide is used to refer to a chemical compound consisting of two or more nucleotides linked together in a polymer chain.
  • the oligonucleotide may be a DNA or RNA fragment.
  • Oligonucleotides can be single-stranded or double-stranded, i.e. contain two chains with a high degree of complementarity, and also include
  • any of the chains, as well as two or more included in the double-stranded complex, triplex or four-chain complex can be modified, including, according to the present invention.
  • An oligonucleotide as a polymer of two or more nucleotides may have
  • an oligonucleotide may have a minimum length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides. Additionally, the oligonucleotide may have a maximum length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
  • oligonucleotide consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 can be obtained , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 ,
  • oligonucleotide ah which is the subject of the present invention, one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides, or all nucleotides may contain a modified phosphate group in
  • modified nucleotide and “modified oligonucleotide” are used to denote a nucleotide or oligonucleotide, respectively, which contain a chemical modification, for example, substituents in the sugar residue, in the phosphate group and / or in the heterocyclic
  • An example of a chemical modification is the introduction of a modified nucleotide, an additional chemical grouping at the 3 ′ and / or 5 ′ end of the oligonucleotide (for example, the 3 ′ “inverted” nucleoside residue), conjugation with the remainder of a high molecular weight compound of low immunogenicity, for example, polyethylene glycol (PEG ), conjugation with
  • 590 low molecular weight compounds for example, cholesterol, mannose or folic acid, conjugation with peptides, for example, peptides that facilitate penetration into cells ("vector" peptides), substitution in the phosphate group, for example, its transformation into a thiophosphate group.
  • Chemical modification of heterocyclic bases may include, in
  • Modification sugar residue may include the introduction of 2'-aminonucleotide, 2'-fluoronucleotide, 2'-O-methylribonucleotide, 2'-O-allylribonucleotide, 2'- ⁇ - ⁇ -
  • nucleoside can end, including with a hydroxyl group, as in the natural nucleoside, with a 3 'amino group ( ⁇ 3'- ⁇ 5' phosphoramide) or a 3'-mercapto group (3'-thiophosphate).
  • Nucleoside analogs may also be part of modified nucleotides or modified oligonucleotides.
  • Nucleotides or oligonucleotides that are the subject of the present invention can be isolated or obtained in purified form.
  • nucleoside is used to denote a chemical compound containing a sugar residue and a heterocyclic base residue.
  • examples of nucleosides may include, but are not limited to, ribose, 2-deoxyribose, 2-O-
  • heterocyclic bases may include, but not limited to, thymine, uracil, cytosine, adenine, guanine, purine, hypoxanthine, xanthine, 2-aminopurine, 2,6-diaminopurin, 5-methylcytosine.
  • 620 thiouracil, 2-thiothymine, 4-thiothymine, 5-propynyluracil, 5-propinylcytosine, 7-deazaadenin, 7-deazaguanine, 7-deaza-8-azaadenine, 7-deaza-8-azaguanine, isocytosine, isoguanine, etc. .
  • nucleoside analogue is used to mean a modified nucleoside in which the sugar residue is replaced by another 625 cyclic or acyclic structure.
  • nucleoside analogues in which the sugar residue is replaced by a different cyclic structure may include, but not limited to, monomers of phosphordiamide morpholine oligonucleotides (FMO, phosphordiamidate morpholino oligonucleotides, PMO) and tricyclo-DNA (tricyclo-DNA, tcDNA).
  • nucleoside analogues in which the sugar residue 630 is replaced by a different acyclic structure may include, but are not limited to, peptide nucleic acid monomers (PNAs) and glycerol nucleic acids (GNAs).
  • PNAs peptide nucleic acid monomers
  • GAAs glycerol nucleic acids
  • nucleoside analogue is used to mean a nucleoside containing a chemical modification, for example, a substituent in
  • nucleoside analogs may include, but are not limited to, 2'-substituted 2'-deoxynucleosides, such as 2'-amino and 2'-fluoro, and ribonucleosides, such as 2'-O-methyl, 2'-O allyl, 2'-O-P-methoxyethylribonucleosides, "closed" nucleosides (LNA), and the like.
  • 2'-substituted 2'-deoxynucleosides such as 2'-amino and 2'-fluoro
  • ribonucleosides such as 2'-O-methyl, 2'-O allyl, 2'-O-P-methoxyethylribonucleosides, "closed" nucleosides (LNA), and the like.
  • Including nucleoside analogs may include analogues in which the sugar residue is replaced by a morpholine ring, as shown in the formula below:
  • Base is a heterocyclic base.
  • the hydroxymethyl substituent in the morpholine ring corresponds to the 5 ′ end
  • the third valency of the nitrogen atom corresponds to the 3 ′ end.
  • nucleoside analogues that may be encompassed by the present invention are also tricyclo-DNAs containing a residue of 650 tricyclic sugars corresponding to the structure:
  • oligonucleotide analogs is used to refer to modified oligonucleotides containing, including chemical modification of the phosphate group and / or those in which nucleosides are replaced
  • oligonucleotide analogs may include, but not limited to, thiophosphates (PS), selenophosphates, dithiophosphates, phosphoramides, boranophosphates, phosphordiamide morpholine oligonucleotides (FMO, PMO), tricyclic DNA and peptide nucleic acids (PNA).
  • PS thiophosphates
  • selenophosphates selenophosphates
  • dithiophosphates dithiophosphates
  • phosphoramides boranophosphates
  • FMO phosphordiamide morpholine oligonucleotides
  • PNA tricyclic DNA and peptide nucleic acids
  • peptide nucleic acids usually refers to analogues
  • oligonucleotides in which, including phosphate groups are replaced by peptide bonds.
  • peptide nucleic acids may also include compounds that contain modified phosphate groups that are the subject of the present invention. Therefore, it should be considered that such compounds may also be encompassed.
  • phosphate group is used herein to mean the phosphoric acid residue NzRO 4 , in which one or more hydrogen atoms are replaced by an organic radical to produce, respectively, a phosphomonoester, phosphodiester or phosphotriether.
  • modified phosphate group is used herein to mean a phosphate group in which any of the oxygen atoms is replaced by any chemical group.
  • substituents include, but are not limited to, sulfur or selenium atoms, an amino group, or a borane residue (BH3).
  • Preferred examples of the modified phosphate group are
  • the phosphate group and the modified phosphate group may be chiral. If the stereochemical configuration is not indicated, the structure includes both Rp and Sp configuration, both separately and in the form of a mixture, for example, a racemic mixture
  • These compounds may also include more than one chiral center. In this case, it should be considered that the structure covers all possible stereoisomers: enantiomers and diastereomers.
  • polymeric carrier is used herein to mean a polymeric carrier used in solid-phase oligonucleotide synthesis.
  • polymeric carriers can include, but not limited to, pore size glass (CPG), polystyrene resins, TentaGel®, TSK Gel® Toyopearl®, polyvinyl alcohol, cellulose acetate, and the like.
  • CPG pore size glass
  • polystyrene resins TentaGel®, TSK Gel® Toyopearl®
  • polyvinyl alcohol cellulose acetate
  • polymer carrier is also used with respect to varieties of substrates for parallel oligonucleotide synthesis, independently including, without limitation, filter paper disks, multipin systems, multi-well plates, and the like.
  • protected oligonucleotide is used herein to mean an oligonucleotide or a modified oligonucleotide containing one or more protecting groups.
  • unprotected oligonucleotide is used herein to mean an oligonucleotide or a modified oligonucleotide from which one or more protecting groups have been removed.
  • protecting group means a chemical group that is used to temporarily block a reaction site in an organic compound and can be removed under certain conditions.
  • protecting groups may include, but are not limited to, acetyl (Ac), benzoyl (Bz), isobutyryl (Ibu), tert-butylphenoxyacetyl (Tac), levulinyl (Lev), methyl (Me), ⁇ -cyanoethyl (CE), allyl (AN), o-chlorophenyl (o-ClPh), 4,4'-dimethoxytrityl (DMTr), 4-methoxytrityl (MMTg), tert-butyldimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM) and other groups.
  • protecting group is also used here to the designation of a chemical group linker for attaching an organic compound to a polymer carrier, cleavable under special conditions with the removal of the corresponding organic compounds from the corresponding polymer carrier.
  • linkers may include, but not limited to, 715 succinyl, diglycolyl, oxalyl, hydroquinone-O, O'-diacetyl (Q-linker), phthaloyl, 4,5-dichlorophthaloyl, malonyl, glutaryl, diisopropylsilyl, 1, 1,3, 3-tetraisopropyl disiloxane-1,3-diyl and other linkers.
  • linker may also refer to non-nucleotide chemical
  • internucleotide linkers groups introduced into the modified oligonucleotide (internucleotide linkers), or non-nucleotide chemical groups connecting the nucleotide with a different chemical modification, for example, a fluorescent label or fluorescence quencher.
  • linkers include, but are not limited to, 1,2-dodecanediol phosphate residue.
  • organic radical is used to mean a chemical group containing one or more carbon atoms attached to any other free valence atoms on a carbon atom.
  • examples of other atoms may include, but are not limited to, hydrogen, nitrogen, oxygen, fluorine, silicon, phosphorus, sulfur, chlorine, bromine, iodine, and others.
  • alkyl and alkyl are used to mean branched or unbranched, cyclic or acyclic substituents based on saturated hydrocarbons with a free valency on a carbon atom.
  • C 1-4 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl and tert-butyl radicals.
  • alkenyl and alkenyl are used to mean branched or unbranched acyclic substituents with a free valence on a carbon atom based on unsaturated hydrocarbons containing at least one carbon-carbon double bond, and, in some applications, not containing carbon -carbon triple bonds.
  • alkenyl may mean C2-10 alkenyl, in some applications C2-6 alkenyl, in other C2-4 alkenyl.
  • alkynyl and alkynyl are used to mean branched or unbranched acyclic substituents with a free valence on a carbon atom based on unsaturated hydrocarbons,
  • alkynyl may mean C2-10 alkynyl, in some applications C2-6 alkynyl, in other C2-4 alkynyl.
  • aromatic radicals with a free valency on a carbon atom or, in some applications, on a heteroatom.
  • aromatic radicals may include, but are not limited to, phenyl and naphthyl (1-naphthyl or 2-naphthyl).
  • Aryl radicals can be monocyclic, for example, as phenyl, or polycyclic, for example, as 1-
  • Heteroaromatic radicals contain one or more (two or more) heteroatoms, including, for example, nitrogen, oxygen, sulfur, selenium, etc.
  • heteroaromatic radicals may include, but are not limited to, five-membered heteroaromatic radicals based on furan, thiophene, or pyrrole, six-membered heteroaromatic radicals, for example, pyridyl (2-
  • heteroaromatic radicals contain 5-10 membered rings with 1, 2, 3, or 4 heteroatoms that are independently selected from the group consisting of nitrogen, oxygen, sulfur, and selenium.
  • substituted is used to refer to organic radicals, which, in turn, can be substituted by one or more substituents up to the maximum number of free valencies in a given radical.
  • substituents may be selected, including from the group consisting of:
  • the substituted radicals are selected from the group consisting of, inter alia, —Cl-4 alkyl, —F, —CI, —Br, —I, —CN, —N3, —CF3, —OH, —OLL alkyl, - NH 2 , -NHCi-4 alkyl and -N (Ci-4 alkyl) g.
  • room temperature is used to mean a temperature range 790 from 14 to 29 ° C, preferably a temperature range from 20 to 25 ° C inclusive.
  • a particular advantage of the present invention is the convenience of 795 introducing modified phosphate groups in the framework of the present invention.
  • the - (sulfonyl) -phosphoramide group corresponding to formula (I) may be introduced into the oligonucleotide during amidophosphite or H-phosphonate oligonucleotide synthesis.
  • the present invention includes a method for producing oligonucleotides that are 800 subject of the present invention.
  • this production method can be carried out, inter alia, using reagents immobilized on a polymer carrier.
  • this method can be carried out using an automatic DNA / RNA synthesizer.
  • the N-phosphonate oligonucleotide synthesis method is a convenient method for producing oligonucleotides [Froehler, B.C .; Ng, P.G .; Matteucci, M. D., Nucleic Acid Res., 1986, 14, 5399].
  • H-phosphonate method is a convenient method for producing oligonucleotides [Froehler, B.C .; Ng, P.G .; Matteucci, M. D., Nucleic Acid Res., 1986, 14, 5399].
  • H-phosphonate method is a convenient method for producing oligonucleotides [Froehler, B.C .; Ng, P.G .; Matteucci, M. D., Nucleic Acid Res., 1986, 14, 5399].
  • H-phosphonate method is a convenient method for producing oligonucleotides [Froehler, B.C .; N
  • RNA 810 is used in the solid phase embodiment, even more preferably using an automated DNA / RNA synthesizer.
  • a DMTr-nucleoside ester immobilized using an appropriate linker on a polymer carrier is subjected to sequential detritilation and condensation with the monoester of the N-phosphonate derivative of nucleoside in
  • nucleoside 815 in the presence of an appropriate condensing reagent to form a dinucleoside-N-phosphonate diester.
  • nucleosides can be introduced similarly by repeating the indicated operations of detritylation and H-phosphonate condensation until the synthesis of the oligonucleotide sequence is completed, followed by oxidation of the internucleotide diester H-
  • 830 phosphate groups can be introduced at any desired position within the target oligonucleotide sequence when it is gradually expanded.
  • amidophosphite method is the most effective and widely used method for the synthesis of oligonucleotides [Sinha, ND; Biernat, J .; McManus, J .; Koster, H., Nucleic Acid Res., 1984, 12, 4539].
  • the protocols of the amidophosphite method are known to those skilled in the art of producing oligonucleotides and, in a preferred application, are carried out in
  • the oxidation step of the phosphitriethether with iodine is replaced, for example, by the Staudinger reaction of dinucleoside phosphite with the corresponding sulfonylazide.
  • an appropriate silylating reagent may be added.
  • the modified phosphate groups can be introduced at any desired position within the target oligonucleotide sequence when it
  • a process for preparing the compounds encompassed by the present invention, 865 includes a variant of the Staudinger reaction between dialkylsilylphosphite, for example, dinucleosidesilylphosphite obtained in situ from the corresponding H-phosphonate and the corresponding silylating agent, and a sulfonylazide corresponding to the formula (V): ABOUT
  • R 2 is determined in accordance with the description for structure (II).
  • a base can be used, for example triethylamine or pyridine.
  • reaction 875 is suitable for the preparation of oligonucleotides with various N- (sulfonyl) -phosphoramide groups.
  • sulfonyl azides may have structure (VI):
  • each of the substituents R 3 and R 4 is a hydrogen atom; that is, the resulting modified phosphate group is - (sulfamoyl) -phosphoramide group.
  • each of the substituents R 3 and R 4 is methyl; that is, the resulting the modified phosphate group is the 1CH- (dimethylsulfamoyl) - phosphoramide group.
  • R 3 and R 4 together with the atom to which they are attached, form a 5-8 membered heterocycle.
  • the 900 atom to which they are attached form a 5-6 membered heterocycle
  • the heterocycle is piperazine; that is, the resulting modified phosphate group is an N- (l-piperazinosulfonyl) phosphoramide group.
  • Oligonucleotides encompassed by the present invention may be any method of producing modified oligonucleotides according to the formula (III) Oligonucleotides encompassed by the present invention.
  • the authors of the present invention have found that oligonucleotides with a modified phosphate group according to formula (III) can be obtained by
  • silylphosphite obtained by treating N-phosphonate with a silylating agent.
  • both process variants can be carried out using polymer-immobilized reagents, more preferably, using an automatic DNA synthesizer, as illustrated
  • a variant of the method for producing oligonucleotides according to the formula (III) includes:
  • the obtained modified oligonucleotide can be isolated and purified using 945 known methods for the isolation and purification of oligonucleotides.
  • This production method is applied at a temperature in the range of -10 to 120 ° C., preferably at a temperature in the range of 0 to 100 ° C., even more preferably at a temperature in the range of 15 to 80 ° C.
  • the concentration of sulfonylazide is in the range of 0.05-2 M, 950 is preferably in the range of 0.1-1 M, even more preferably in the range of 0.15 - 0.5 M, inclusive.
  • the list of solvents includes acetonitrile, ⁇ , ⁇ -dimethylformamide (DMF), ⁇ , ⁇ -dimethylacetamide (DMA),] H [-methyl-2-pyrrolidone (NMP), 1,3-dimethyl-2-imidazolidinone, tetramethylurea, 955 hexamethylphosphoric triamide (HMFTA), sulfolane, acetone, ethyl acetate, tetrahydrofuran (THF), 1,4-dioxane, 1,2-dimethoxyethane (DME), pyridine and the like.
  • acetonitrile is used as a solvent.
  • ⁇ , ⁇ -dimethylformamide is used as solvent 960.
  • NMP 1CH-methyl-2-pyrrolidone
  • a mixture of two or more solvents may be used.
  • the silylating agent is independently selected from the group consisting of O-bis (trimethylsilyl) acetamide (BSA), ⁇ , ⁇ -bis (trimethylsilyl) trifluoroacetamide (BSTFA), trimethylchlorosilane, trimethyl bromosilane, trimethyl iodosilane trifluoromethyl, trimethylsilyl trisulfonyltrimethanesulfonyltrimethanes
  • O-bis (trimethylsilyl) acetamide (BSA) is used as the silylating agent.
  • the concentration of the silylating agent is in the range of 0.1% to 30% by volume.
  • a mixture of two or more silylating agents may be used.
  • a base may be used, for example,
  • a mixture of two or more bases may be used.
  • the concentration of said base is in the range of 0.1% to 30% by volume.
  • triethylamine is used as the base.
  • Oligonucleotides were synthesized on an ASM-800 automatic DNA synthesizer (Biosset LLC, Russia) according to the ⁇ -cyanethyl phosphitamide protocol [Sinha, ND; Biernat, J .; McManus, J .; 1045 Koster, H. Nucleic Acids Res. 1984, 12, 4539] from the corresponding -cyanethyl- - diisopropylamidophosphites of deoxynucleosides using other amidophosphite monomers for oligonucleotides of mixed composition, and polymer carriers with immobilized deoxynucleosides or other functionalities on a scale of 0.2-0.4 ⁇ mol using standard
  • the sulfonyl azides obtained can preferably be isolated in pure form (Example 1) or used in situ as a solution in an organic solvent, preferably acetonitrile (Examples 2-4).
  • the reaction of introducing a modified phosphate group can be
  • phosphate group modification reactions were carried out in continuous solid-phase synthesis using an automatic DNA synthesizer with a solution placement
  • the polymer carrier was transferred from the reactor into a 1.5 ml plastic tube with a screw cap and a rubber O-ring, 200 ⁇ l conc. (approx. 25%) aq. ammonia solution based on 5 mg of the carrier and the oligonucleotide was cleaved from
  • 1155 test tube with a volume of 1.5 ml.
  • the suspension was vigorously shaken for 5 min in an argon atmosphere, then left on a shaker at 1400 rpm for 3 hours at 25 ° C.
  • a suspension of sodium chloride was precipitated by centrifugation for 2 min at 14500 rpm, the mesylazide solution was stored in the dark under argon and used for no more than 2 weeks.
  • Example 3 The use of buzilazide (1-butanesulfonyl azide) in the form of a ⁇ 0.5 M solution in acetonitrile obtained in situ.
  • Example 4 The use of hesylazide (1-hexanesulfonyl azide) in the form of a ⁇ 0.5 M solution in acetonitrile obtained in situ.
  • Hezyl chloride (1-hexanesulfonyl chloride) (923.4 mg, 5 mmol, 1 equiv) was added 1175 with vigorous shaking in an argon atmosphere to a suspension of sodium azide NaN3 (1.2 equiv, 390 mg, 6 mmol) in 10 ml of abs. acetonitrile in a 15 ml plastic tube.
  • the suspension was vigorously shaken for 5 min in an argon atmosphere, then left on a shaker at 1400 rpm for 24 hours at 25 ° C.
  • a suspension of sodium chloride was precipitated by centrifugation for 15 min at 1180–3500 rpm, a solution of 1-hexanesulfonylazide was stored in the dark under argon and used for no more than 72 hours.
  • Example 5 Obtaining oligodeoxynucleotide 5'- ⁇ ( ⁇ ⁇ ⁇ ), modified mesylphosphoramide (- (methanesulfonyl) -phosphoramide)
  • Example 6 Obtaining oligodeoxynucleotide 5 '- (1 ( ⁇ ⁇ ), modified by the mesylphosphoramide group (formula VII); the symbol ( ⁇ ) indicates the position of the modification.
  • Example 7 Obtaining oligodeoxynucleotide 5'-d (GCGCCA tl AACA), 1235 modified mesylphosphoramide group (formula VII); the symbol ( ⁇ ) indicates the modification position.
  • ⁇ 10 mg of a polymer carrier based on porous glass CPG with a pore size of 500-1000 A s was loaded into a solid-phase synthesis reactor immobilized 5'-DMTr-dA Bz (nucleoside loading of 30-40 ⁇ mol / g) and 1240 launched the automated solid-phase DNA synthesis protocol according to the ⁇ -cyanoethyl phosphitamide scheme at a scale of 0.4 ⁇ mol.
  • a modified synthesis schedule was used, which combined oxidation with iodine for ordinary phosphate groups and treatment with a 0.5 M solution of mesylazide in acetonitrile for 15 min to introduce the mesylphosphoramide group.
  • the carrier reactor was dried in a SpeedVac vacuum concentrator for 15 minutes, and the carrier was transferred into a 1.5 ml plastic tube for subsequent release.
  • Example 8 Obtaining oligodeoxynucleotide 5 '- (1 ( ⁇ ⁇ ), modified by two mesylphosphoramide groups (formula VII); the symbol ( ⁇ ) indicates the position of the modification.
  • the reactor with the carrier was dried in a SpeedVac vacuum concentrator for 15 min, and the carrier was transferred into a 1.5 ml plastic tube for subsequent release.
  • Example 11 Obtaining oligodeoxynucleotide 5'- modified mesylphosphoramide group (formula VII); the symbol ( ⁇ ) indicates the modification position.
  • 1310 carriers based on porous glass CPG with a pore size of 500-1000 A with immobilized 5'-DMTr-dA Bz (loading of a nucleoside of 30-40 ⁇ mol / g) and a protocol of automated solid-phase DNA synthesis using the ⁇ -cyanethyl phosphitamide scheme at a scale of 0.4 ⁇ mol was launched .
  • a modified synthesis schedule was used in which iodine oxidation was replaced
  • ⁇ 10 mg of a polymer carrier based on porous glass CPG with a pore size of 500-1000 A with 1330 immobilized 5'-DMTr-dA Bz (loading of nucleoside 30-40 ⁇ mol / g) was loaded into the solid-phase synthesis reactor and the automated solid-phase DNA synthesis protocol was launched according to the ⁇ -cyanethyl phosphitamide scheme on a scale of 0.4 ⁇ mol.
  • a modified synthesis schedule was used in which iodine oxidation was replaced treatment with a 0.5 M solution of mesylazide in acetonitrile for 15 min, with 1335 followed by capping.
  • the reactor with the carrier was dried in a SpeedVac vacuum concentrator for 15 min, and the carrier was transferred into a 1.5 ml plastic tube for subsequent release.
  • Example 13 Obtaining an oligo-2'-O-methylribonucleotide 2'-OM- ⁇ ( ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) modified with a mesylphosphoramide group (formula VII);
  • Example 14 Obtaining oligodeoxynucleotide 5'-d (T
  • T 3 TTTTT
  • a buzylphosphoramide [- (1-butanesulfonyl) phosphoramide) group (formula VIII); the symbol (P) indicates the position of the modified phosphate group.
  • Example 15 Obtaining oligodeoxynucleotide 5'-d (GCGCCAAAC
  • the HPLC elution profile is shown in FIG. 6.
  • the ESI LC-MS / MS mass spectrum is shown in FIG. 22.
  • Example 16 Obtaining oligodeoxynucleotide 5'-d (GCGCCA
  • the HPLC elution profile is shown in FIG. 6.
  • the ESI LC-MS / MS mass spectrum is shown in FIG. 23.
  • the HPLC elution profile is shown in FIG. 6.
  • the ESI LC-MS / MS mass spectrum is shown in FIG. 24.
  • Example 18 Obtaining oligodeoxynucleotide 5'-d (T
  • the reactor with the carrier was dried in a SpeedVac vacuum concentrator for 15 min, and the carrier was transferred into a 1.5 ml plastic tube for subsequent release.
  • ⁇ 10 mg of a polymer carrier based on porous glass CPG with a pore size of 500-1000 A with immobilized 5'-DMTr-dA Bz (loading of a nucleoside of 30-40 ⁇ mol / g) was loaded into a solid-phase synthesis reactor and 1470 the automated solid-phase DNA synthesis protocol was launched according to the ⁇ -cyanethyl phosphitamide scheme on a scale of 0.4 ⁇ mol.
  • a modified synthesis schedule was used, which combined oxidation with iodine for ordinary phosphate groups and treatment with a 0.5 M solution of buzylazide in acetonitrile for 20 min to introduce the buzyl phosphoramide group.
  • the carrier reactor was dried in a SpeedVac vacuum concentrator for 15 min, and the carrier was transferred into a 1.5 ml plastic tube for subsequent release.
  • Example 20 Obtaining oligodeoxynucleotide 5'-d (T h TTTTT), 1485 modified hesylphosphoramide (] H [- (1-hexanesulfonyl) phosphoramide) group (formula IX); the symbol ( h ) indicates the position of the modified phosphate group.
  • Example 21 Obtaining oligodeoxynucleotide 5'-d (T h T h T h T h T h T T) modified with hesylphosphoramide (] H [- (1-hexanesulfonyl) phosphoramide) group (formula IX); symbol ( h ) indicates position
  • Example 22 Obtaining oligodeoxynucleotide 5'-d (GCGCCAAAC h A), modified hesylphosphoramide group (formula IX); the symbol ( h ) indicates the modification position.
  • Example 23 Obtaining oligodeoxynucleotide 5'-d (GCGCCA h AACA), 1545 modified hesylphosphoramide group (formula IX); the symbol ( h ) indicates the modification position.
  • ⁇ 10 mg of a polymer carrier based on porous glass CPG with a pore size of 500-1000 A with immobilized 5'-DMTr-dA Bz (loading of a nucleoside of 30-40 ⁇ mol / g) was loaded into a reactor for solid-phase synthesis and 1550 the automated solid-phase DNA synthesis protocol was launched according to the ⁇ -cyanethyl phosphitamide scheme on a scale of 0.4 ⁇ mol.
  • a modified synthesis schedule was used, which combined oxidation with iodine for ordinary phosphate groups and treatment with a 0.5 M solution of hesylazide in acetonitrile for 20 minutes to introduce the hesylphosphoramide group.
  • the reactor with the support was dried in a vacuum concentrator.
  • Example 24 Obtaining oligodeoxynucleotide 5'-d (GCGCCA h AAC h A), modified with two hesylphosphoramide groups (formula IX); the symbol ( h ) indicates the modification position.
  • Example 25 Preparation of oligodeoxynucleotide 5'-d (T T TTTTT) , modified tozilfosforamidnoy (- (p-toluenesulfonyl) - phosphoramidite) group (Formula X); the symbol ( ⁇ ) indicates the position of the modified phosphate group.
  • Example 26 Obtaining oligodeoxynucleotide 5 '-d (T ⁇ TTTTT), modified nosylphosphoramide (- (o-nitrobenzenesulfonyl) - phosphoramide) group (formula XI); symbol ( v ) indicates position 1615 of the modified phosphate group.
  • Example 27 Obtaining oligodeoxynucleotide 5'-d (T v T v T v T v T v T v v T) modified with a nosylphosphoramide group (formula XI); symbol ( v ) indicates the position of the modified phosphate group.
  • Example 28 Obtaining oligodeoxynucleotide 5'-d (GCGCCAAAC v A), modified nosylphosphoramide group (formula XI); character ( v )
  • Example 29 Obtaining oligodeoxynucleotide 5'-d (GCGCCA v AAC v A), modified by two nosylphosphoramide groups (formula XI); the symbol ( v ) indicates the modification position.
  • Example 30 Obtaining oligodeoxynucleotide 5'-d (T v GT v T v T v G v G v C v GT), 1690 modified nosylphosphoramide group (formula XI); symbol ( v ) indicates the position of the modified phosphate group.
  • a solid-phase synthesis reactor 5 mg of a CPG porous glass polymer carrier with a pore size of 500-1000 A with immobilized 5'-DMTr-dT (nucleoside loading of 30-40 ⁇ mol / g) was loaded and 1695 automated ⁇ solid-phase DNA synthesis protocol was launched -cyanethyl phosphitamide scheme on a scale of 0.2 ⁇ mol.
  • a modified synthesis schedule was used in which iodine oxidation for ordinary phosphate groups was replaced by treating with a 0.15 M solution of nosylazide in acetonitrile for 15 min to introduce the nosyl phosphoramide group.
  • the carrier reactor 1700 was dried in a SpeedVac vacuum concentrator for 15 min, and the carrier was transferred into a 1.5 ml plastic tube for subsequent release.
  • RNA modified oligonucleotides containing - (sulfonyl) - phosphoramide groups Duplexes formed by modified oligonucleotides with RNA and DNA were melted in a temperature-controlled chamber with a UV-1740 detector using a UV-1800 UV spectrophotometer (Shimadzu, Japan). Duplexes were formed by stoichiometric mixing of oligonucleotides with concentrations of 10 "5 M. Thermal denaturation studies were carried out in a buffer containing 10 mM Na-cacodylate, 5 mM magnesium chloride and 100 mM sodium chloride.
  • Example 33 Determination of the stability of mesylphosphoramide 1755 oligodeoxynucleotide compared with unmodified oligodeoxynucleotide in the presence of fetal calf serum.
  • oligonucleotides modified by the mesylphosphoramide group 700 ⁇ l of IMDM cell medium containing 10% fetal calf serum was heated in 1760 for 10 min at 37 ° C. The warmed medium was added to 1 about. e.
  • Example 34 Determination of the stability of an oligodeoxynucleotide containing one 3'-terminal hesylphosphoramide group compared to unmodified oligodeoxynucleotide in the presence of fetal calf serum.
  • Example 35 Determination of the cytotoxicity of modified oligonucleotides in relation to the cell culture of human breast adenocarcinoma MDA-MB-231.
  • MTT test To determine the cytotoxicity of oligonucleotides in relation to the cell culture of human mammary adenocarcinoma MDA-MB-231 1800 was used MTT test. For this, cells in 96-well plates were incubated for 24 hours with oligonucleotides in various concentrations (from 0.39 to 50 ⁇ M), then washed, incubated for 4 h with 1.2 mM MTT in a medium that does not contain phenol red. Then, SDS-HC1 solution was added, and after 6 hours of incubation, the optical density at a wavelength of 570 nm was measured on a 1805 Multiscan FC flat-bed spectrophotometer (Thermo Fisher Scientific, USA).
  • Example 36 Determination of the ability of a mesylphosphoramide oligodeoxynucleotide to cause RNA cleavage in a complementary duplex due to activation of RNase N.
  • RNAse H was used to duplicate RNA in a modified oligonucleotide duplex.
  • RNA in duplexes with oligodeoxyribonucleotide modified by mesylphosphoramide groups is cleaved by RNase H almost to the same extent as in the case of duplexes with unmodified
  • RNA in duplexes with oligodeoxynucleotide modified by mesylphosphoramide groups and hexylphosphoramide groups is cleaved by RNase H in the same way as the control RNA duplex with unmodified oligodeoxynucleotide.
  • the hesylphosphoramide oligodeoxynucleotide activated the cleavage of complementary RNA i860 21 nt long by the RNase H enzyme by slightly less effective than the corresponding mesylphosphoramide oligonucleotide.
  • Buzylphosphoramide oligodeoxynucleotide activated RNase H much less efficiently than the control unmodified oligodeoxynucleotide and a mesylphosphoramide oligodeoxynucleotide, less effective, a hesylphosphoramide oligonucleotide (Fig. 38).
  • Example 38 The study of the penetration of mesylphosphoramide and hesylphosphoramide oligonucleotides into HEK293 cells in the absence of a transfection agent (hymnosis) using laser confocal 1870 microscopy.
  • the HEK 293 cell line Human Embryonic Kidney, human kidney embryonic epithelium
  • DMEM fetal calf serum
  • FBS Fetal Bovine Serum
  • penicillin and streptomycin 100 units / ml
  • Example 39 The study of the penetration of mesylphosphoramide, 1910 buzylphosphoramide and hesylphosphoramide oligonucleotides into MDA MB 231 cells in the absence of a transfection agent (hymnosis) using laser confocal microscopy.
  • 1925 cells were washed three times with IMDM medium with 10% FBS and incubated in 200 ⁇ l IMDM medium with 10% FBS containing one of four different oligonucleotides at a concentration of 5 ⁇ M for 24 hours at 37 ° C in a CO2 incubator. After incubation, the cells were washed three times from excess reagent with serum-free DMEM, fixed with 3.7% formalin in DMEM for
  • the mesylphosphoramide oligonucleotide was localized in the form of granules around the nucleus and diffusely in the nucleus itself.
  • the penetration level of the buzylphosphoramide oligonucleotide was significantly higher than that of the mesylphosphoramide oligonucleotide.
  • Buzylphosphoramide oligonucleotide was localized in
  • hesylphosphoramide oligonucleotide penetrated significantly better than the rest and accumulated in large quantities in the cytoplasm of cells in the form of granular and vesicle-like structures (Fig. 40).
  • Example 40 Study of the penetration of mesylphosphoramide and hesylphosphoramide oligonucleotides using a transfection agent lipofectamine 3000 into MDA MB 231 cells using laser confocal microscopy.
  • the MDA MB 231 breast cancer cell line was used.
  • Cells were cultured in IMDM medium containing 10% FBS and antibiotics penicillin and streptomycin (100 u / ml) in an atmosphere of 5% CO2 at 37 ° C.
  • Cell viability was determined by trypan blue stain.
  • cells were scattered on 8-well Nunc TM Lab-Tek TM Chamber Slide System
  • control oligodeoxynucleotide in complex with lipofectamine 3000 accumulated in the form of granules in the cytoplasm.
  • Lipofectamine for the delivery of hesylphosphoramide oligonucleotide observed accumulation of oligonucleotides in significant quantities in the cytoplasm and, in part, in the nuclei of cells.
  • lipofectamine 3000 to deliver the mesylphosphoramide oligonucleotide, the penetration rate was only slightly lower than in the case of the hesylphosphoramide oligonucleotide complex
  • the permeated mesylphosphoramide oligonucleotide was localized mainly in the nuclei of cells with a small content in the cytoplasm in the form of granules or vesicle-like structures (Fig. 41).
  • FIG. 2 Analogues of oligonucleotides with ionized - (sulfonyl) -phosphoramide group.
  • FIG. 3 Elution profile of HPLC of crude 5'-d oligodeoxynucleotides (TPTTTTT) (black line) and 5 '- (1 ( ⁇ ⁇ ⁇ ) (gray line) modified with the 2010 buzylphosphoramide (K- (1-butanesulfonyl) phosphoramide) group ( ⁇ ) or mesylphosphoramide (- (methanesulfonyl) -phosphoramide) group ( ⁇ ), respectively.
  • TPTTTTT crude 5'-d oligodeoxynucleotides
  • FIG. 4 Elution profile of HPLC of crude oligonucleotides 5'- 2015 ( ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) (black line) and 5 '- ⁇ - ⁇ ( ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) ( gray line) modified mesylphosphoramide (- (methanesulfonyl) -phosphoramide) group ( ⁇ ).
  • FIG. 5 Elution profile of the HPLC of the crude 5'-DMTr- oligonucleotide (1 ( ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) (gray line) and purified 2020 of HPLC and detritylated oligonucleotide 5'-
  • FIG. 6 Elution profile of HPLC of crude 5'-DMTr-d oligonucleotides (GCGCCAAAC p A) (gray line), 5'-DMTr-d (GCGCC ⁇ ⁇ ⁇ AC A) (dashed line) and 5'-2025 DMTr-d (GCGCCA p AAC
  • FIG. 7 Elution profile of HPLC of the crude oligonucleotides 5'-DMTr- ⁇ ( ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) (black line) and 5'-DMTr-d (G ' 3 Cl 3 Gl 3 Cl 3 Cl 3 Al 3 Al 3 Al 3 Cl 3 A) (gray line), modified with the buzylphosphoramide group (P).
  • FIG. 8 Elution profile of HPLC of the crude oligonucleotides 5'-d (T h TTTTT) (black line) and 5'_d (T h T h T h T h T h T) (gray line) modified with hesylphosphoramide (- (1-hexanesulfonyl ) -phosphoramide) group ( h ).
  • FIG. 9 Elution profile of HPLC of the crude oligonucleotides 5'-DMTr-d (GCGCCAAAC h A) (gray line), 5 '-DMTr-d (GCGCC A h AAC A) (dashed line) and 5'-2035 DMTr-d (GCGCCA h AAC h A) (black line) modified by the hesylphosphoramide group.
  • FIG. 10 Elution profile of HPLC of the crude oligonucleotides 5′-d (TTTTTT) (dashed line), 5′-d (T T TTTTT) (black line) and 5′-DMTr-d (T T TTTTT) (gray line), modified tosylphosphoramide (N- (w-2040 toluenesulfonyl) -phosphoramide) group ( t ).
  • FIG. 11 Elution profile of HPLC of crude oligonucleotides 5'-d (T v TTTTT) (thin black line), 5 '-DMTr-d (T v TTTTT) (gray line), 5'-d (T v T v T v T T v T) (bold black line) and 5 '-DMTr-d (T v T v T v T v T v T) (dotted line), modified nosylphosphoramide (- (o-nitrobenzenesulfonyl) - phosphoramide) group ( v ).
  • FIG. 12 Elution profile of the HPLC of the crude oligonucleotides 5'-DMTr-d (GCGCCAAAC v A) (gray line), 5'-DMTr-d (GCGCCA v AAC v A) (dashed line) and 5'-d (T v G v T v T v T v G G v G v C v G v T) (black line) modified by the nosylphosphoramide group ( v ).
  • FIG. 14 Mass spectrum of the ESI LC-MS / MS oligodeoxynucleotide 5'-d (GCGCCAAAC tl A) modified with the mesylphosphoramide group ( ⁇ ).
  • FIG. 15 Mass spectrum of the ESI LC-MS / MS oligodeoxynucleotide 5′-2055 d (GCGCCA tl AACA) modified with the mesylphosphoramide group ( ⁇ ).
  • FIG. 16 Mass spectrum of ESI LC-MS / MS oligodeoxynucleotide 5'-d (GCGCCA tl AAC tl A) modified with a mesylphosphoramide group ( ⁇ ).
  • FIG. 17 Mass spectrum of ESI LC-MS / MS oligodeoxynucleotide 5'- ⁇ ( ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) modified by the mesylphosphoramide group ( ⁇ ).
  • FIG. 18 Mass spectrum of ESI LC-MS / MS oligodeoxynucleotide 5'-d (G ⁇ T ⁇ C ⁇ C ⁇ A ⁇ G ⁇ C ⁇ C ⁇ C ⁇ C ⁇ A ⁇ T ⁇ G ⁇ G ⁇ A) modified mesylphosphoramide group ( ⁇ ).
  • FIG. 19 Mass spectrum ESI LC-MS / MS oligodeoxynucleotide 5'-d (A ⁇ l A ⁇ l C ⁇ l G ⁇ l T ⁇ l A ⁇ l A ⁇ l G ⁇ l C ⁇ l C ⁇ l C ⁇ l A ⁇ l T ⁇ l G ⁇ l G ⁇ l A ⁇ l T ⁇ l G ⁇ l A ⁇ l T ⁇ l G ⁇ l A ⁇ l T ⁇ l G l A l A l T ⁇ l G ⁇ l A ⁇ l T ⁇ l G ⁇ l A ⁇ l T ⁇ l G ⁇ l A ⁇ l T ⁇ l G ⁇ l A ⁇ l T ⁇ l T), modified with the 2065 mesylphosphoramide group ( ⁇ ).
  • FIG. 20 Mass spectrum of ESI LC-MS / MS oligo-2'-O-methylribonucleotide 5'- ⁇ ( ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) modified by the mesylphosphoramide group ( ⁇ ).
  • FIG. 21 Mass spectrum of an ESI LC-MS / MS 5'-d oligonucleotide (T p TTTTT) modified with a buzyl phosphoramide group (P). 2070 Fig. 22. Mass Spectrum of ESI LC-MS / MS 5'-d Oligonucleotide (GCGCCAAAC p A) Modified with the Buzyl Phosphoramide Group (P).
  • FIG. 23 Mass Spectrum of ESI LC-MS / MS 5'-d Oligonucleotide (GCGCCAPAACA) Modified with the Buzyl Phosphoramide Group (P).
  • FIG. 24 Mass spectrum of ESI LC-MS / MS oligonucleotide 5'-d (GCGCCAPAAC p A), 2075 modified with a buzyl phosphoramide group (P).
  • FIG. 25 Mass spectrum of an ESI LC-MS / MS oligonucleotide (Z ⁇ T ⁇ T ⁇ T) modified with a buzyl phosphoramide group (P).
  • FIG. 26 Mass spectrum of an ESI LC-MS / MS oligonucleotide 5'-d (G p C , 3 G , 3 C , 3 C , 3 A , 3 A , 3 A , 3 C , 3 A) modified with a buzyl phosphoramide group ( ⁇ ).
  • FIG. 27 Mass ESI LC-MS / MS of the oligonucleotide 5'-d (T T TTTTT) , modified tozilfosforamidnoy group ( ⁇ ).
  • FIG. 28 Mass spectrum of the ESI LC-MS / MS oligodeoxynucleotide 5'-d (T ⁇ TTTTT) modified with the nosylphosphoramide group ( v ).
  • FIG. 29 Mass spectrum of ESI LC-MS / MS oligodeoxynucleotide 5′-2085 d (GCGCCAAAC v A) modified with a nosylphosphoramide group ( v ).
  • FIG. 30 Mass spectrum of the ESI LC-MS / MS oligodeoxynucleotide 5'-d (GCGCCA v AAC v A) modified with the nosyl phosphoramide group ( v ).
  • FIG. 31 Mass spectrum of ESI LC-MS / MS oligodeoxynucleotide 5'- ⁇ (G TTT) modified with the nosylphosphoramide group ( v ).
  • FIG. 33 Electrophoretic analysis of the mobility of oligonucleotides in 20% SDS page. Tracks: 1) S'-dCGT ⁇ CG ⁇ TC TC ⁇ A); 2) 5'-d (GCGCCA T AAC T A); 3) 5'-d (GCGCCA T AACA); 4) 5'-d (GCGCCAAAC T A); 5) 5'- 2095 d (G ⁇ G ⁇ C ⁇ A ⁇ A ⁇ A); 6) 5'-d (GCGCCA ⁇ AAC ⁇ A); 7) 5'-d (GCGCCA ⁇ AACA); 8) 5'-d (GCGCCAAAC ⁇ ); 9) 5'-d (GCGCCAAACA) (unmodified control); 10) 5'-d (T T T T T T T T T T T T T T T); 11) 5'-d (T T TTTTT); 12) 5 ' ⁇ ( ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ); ( ⁇ ) 5
  • FIG. 34 Electrophoretic analysis in 20% PAG of oligonucleotide stability in the presence of 10% calf fetal serum: (a) Control unmodified oligonucleotide 5'-d (A ACGT A AGCCCC ATGG ATG AT); (b) Oligonucleotide 5'- dCA ⁇ A ⁇ G ⁇ A ⁇ A ⁇ G ⁇ T ⁇ G j ⁇ T ⁇ G ⁇ T), modified
  • FIG. 35 Stability of an oligodeoxynucleotide containing a hexylphosphoramide group at the 3 'end position in DMEM culture medium for HEK293 cells containing 10% fetal calf serum 2110 at 37 ° C (15% PAAG, stained with AN-Stain). Lanes 0, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h - oligonucleotides after incubation for a specified period of time.
  • FIG. 36 Cytotoxicity of the modified oligonucleotides for cell culture of human breast adenocarpins MDA-MB-231 (MTT test). 2115 Designations: thio - oligodeoxynucleoside thiophosphate, dFGO - phosphorylguanidine oligodeoxynucleotide, mesyl - mesylphosphoramide oligodeoxynucleotide, nosyl - nosylphosphoramide oligodeoxyphosphonide oligodeoxyphosphonide - 2-oligodeoxyphosphonide
  • RNA + modified oligonucleotide 60 min; buffer: 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCh, 0.1 mM EDTA, 0.1 mM DTT, 0.8 units RNase H, 100 nM RNA, 1 ⁇ M oligonucleotide.
  • FIG. 38 Radio-autograph for activation of RNase H by mesylphosphoramide oligo deoxynucleotide 5 '-
  • FIG. 39 The influence of the nature of the side chain of the K- (sulfonyl) -phosphoramide group on the penetration of modified oligonucleotides into HEK293 cells in the absence of a transfection agent (hymnosis). Concentrations of oligonucleotides 1 ⁇ M, incubation time 8 hours. Panel a) control unmodified
  • FIG. 40 The influence of the nature of the side chain of the - (sulfonyl) -phosphoramide group on the penetration of modified oligonucleotides into MDA MB 231 cells in
  • FIG. 41 The influence of the nature of the side chain of the - (sulfonyl) -phosphoramide group on the penetration of modified oligonucleotides into MDA MB 231 cells in the presence of the transfection agent lipofectamine 3000. Concentrations of oligonucleotides 1 ⁇ M, incubation time 24 hours. Panel a) mesylphosphoramide
  • R 3 and R 4 together with the atom to which they are bonded form a 5-8 membered heterocycle; where each alkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocycle may be further substituted.
  • R 2 , R 3 and R 4 are independently selected from the group consisting of —H and —Cyo alkyl, optionally substituted with one or more substituents from the series —F, —CI, —Br, —I, CN, N 3 , —OH, —OCi-b alkyl, —OC 2 -b alkenyl, —OC 2-6 alkynyl, —OCb aryl, —OCs-io heteroaryl, —SCi- 6 alkyl, —SC 2- 6 alkenyl, -SC 2- 6 alkynyl, aryl -BSb th, -SCs-io heteroaryl, -NH 2, -NHCi-b alkyl and -N (Ci -6 alkyl) 2.
  • R 2 is selected from the group consisting of —Ci -6 alkyl, —C 2-6 alkenyl, —C 2-6 alkynyl.
  • R 2 is preferably a methyl, n-butyl or n-hexyl group.
  • R 2 is preferably an n-dodecyl, n-hexadecyl, 4- (1-dodecyl) phenyl or perfluoro-n-octyl group.
  • R 2 is NR 3 R 4
  • R 3 and R 4 are each independently selected from the group consisting of —H and —Ci- 4 alkyl.
  • oligonucleotide according to claim 1 in which the phosphate group that binds residues of adjacent nucleosides, modified nucleosides or nucleoside analogs is a modified phosphate group corresponding to formula (III).
  • the residues of nucleosides, modified nucleosides or nucleoside analogs linked by a modified phosphate group corresponding to formula (III) comprise a continuous sequence of 6 to 30 or more nucleotides, modified nucleotides or nucleotide analogs.
  • oligonucleotide according to claim 1 which contains a sequence of 4 to 10 or more nucleotides corresponding to formula (III), which is flanked by modified nucleotides, preferably 2'-modified ribonucleotides, which improve binding to RNA (hapmer).
  • oligonucleotide according to claim 1 in the form of a salt, which is obtained by ionizing the ⁇ -group in the formula (III), with a counter, which preferably, but not exclusively, is NH 4 + , Na + , K + or Li + .
  • modified phosphate group corresponding to formula (III) and linking the residues of adjacent nucleosides, modified nucleosides or nucleoside analogs is an individual stereoisomer or a mixture of stereoisomers.
  • oligonucleotide according to claim 1 in medicine as a medicine, based on penetration into cells, binding to RNA and activation of RNase N.
  • oligonucleotide according to claim 1 in a pharmaceutical composition comprising the oligonucleotide according to claim 1 and a pharmaceutically acceptable component or components for the treatment of any disease, where the treatment is associated with exposure to oligonucleotide according to claim 1 for a biological RNA molecule and activation of RNase N.
  • oligonucleotide according to claim 16 comprising administering to the patient in need of treatment the oligonucleotide according to claim 1 for the treatment of any disease, where the treatment is associated with the action of the oligonucleotide according to claim 1 on the biological RNA molecule and RNase activation N.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne le domaine de la médecine moléculaire et de chimie bioorganique. L'objet de l'invention est constitué par de nouveaux dérivés biologiquement stables d'oligonucléotides avec groupes phosphatiques modifiés capables de former un duplex complémentaire avec les ARN, pénétrer dans les cellules par elles-mêmes (c'està dire, dans des conditions d'hypnose) ou au moyen d'un agent de transfection, et activer le ferment ARNase N.
PCT/RU2018/050022 2017-02-21 2018-02-21 Oligonucléotides modifiés activant l'arnase n WO2018156056A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
RU2017105743 2017-02-21
RU2017105743A RU2740501C2 (ru) 2017-02-21 2017-02-21 МОДИФИЦИРОВАННЫЕ ОЛИГОНУКЛЕОТИДЫ, АКТИВИРУЮЩИЕ РНКазу Н

Publications (1)

Publication Number Publication Date
WO2018156056A1 true WO2018156056A1 (fr) 2018-08-30

Family

ID=63254434

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/RU2018/050022 WO2018156056A1 (fr) 2017-02-21 2018-02-21 Oligonucléotides modifiés activant l'arnase n

Country Status (2)

Country Link
RU (1) RU2740501C2 (fr)
WO (1) WO2018156056A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10450568B2 (en) 2015-10-09 2019-10-22 Wave Life Sciences Ltd. Oligonucleotide compositions and methods thereof
US11208430B2 (en) 2014-08-22 2021-12-28 Noogen Llc Modified oligonucleotides and methods for their synthesis
CN114555621A (zh) * 2019-08-15 2022-05-27 Ionis制药公司 键修饰的寡聚化合物及其用途
US11603532B2 (en) 2017-06-02 2023-03-14 Wave Life Sciences Ltd. Oligonucleotide compositions and methods of use thereof
WO2023076710A1 (fr) * 2021-11-01 2023-05-04 A2Tbio Llc Agents d'arn stabilisés

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2115658C1 (ru) * 1992-07-23 1998-07-20 Ворсестер фаундейшн фор байомедикал рисеч, Инк. Гибридный олигонуклеотид, содержащий фосфоротиоатную и/или фосфородитиоатную связи, терапевтическая фармацевтическая композиция, способ ингибирования генной экспрессии
WO2007059816A1 (fr) * 2005-11-23 2007-05-31 Roche Diagnostics Gmbh Polynucleotide contenant un mimetique de phosphate
WO2008128686A1 (fr) * 2007-04-18 2008-10-30 Roche Diagnostics Gmbh Nucléotide à mimétique alpha-phosphate
WO2008141799A1 (fr) * 2007-05-24 2008-11-27 Roche Diagnostics Gmbh Oligophosphoramidates

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2115658C1 (ru) * 1992-07-23 1998-07-20 Ворсестер фаундейшн фор байомедикал рисеч, Инк. Гибридный олигонуклеотид, содержащий фосфоротиоатную и/или фосфородитиоатную связи, терапевтическая фармацевтическая композиция, способ ингибирования генной экспрессии
WO2007059816A1 (fr) * 2005-11-23 2007-05-31 Roche Diagnostics Gmbh Polynucleotide contenant un mimetique de phosphate
WO2008128686A1 (fr) * 2007-04-18 2008-10-30 Roche Diagnostics Gmbh Nucléotide à mimétique alpha-phosphate
WO2008141799A1 (fr) * 2007-05-24 2008-11-27 Roche Diagnostics Gmbh Oligophosphoramidates

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE CA [O] 19 June 2007 (2007-06-19), retrieved from STN Database accession no. 937803-19-5 *
DATABASE CA 19 June 2007 (2007-06-19), retrieved from STN Database accession no. 937803-20-8 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11208430B2 (en) 2014-08-22 2021-12-28 Noogen Llc Modified oligonucleotides and methods for their synthesis
US10450568B2 (en) 2015-10-09 2019-10-22 Wave Life Sciences Ltd. Oligonucleotide compositions and methods thereof
US11603532B2 (en) 2017-06-02 2023-03-14 Wave Life Sciences Ltd. Oligonucleotide compositions and methods of use thereof
CN114555621A (zh) * 2019-08-15 2022-05-27 Ionis制药公司 键修饰的寡聚化合物及其用途
US11629348B2 (en) 2019-08-15 2023-04-18 Ionis Pharmaceuticals, Inc. Linkage modified oligomeric compounds and uses thereof
EP4013767A4 (fr) * 2019-08-15 2023-10-25 Ionis Pharmaceuticals, Inc. Composés oligomères modifiés par liaison et leurs utilisations
WO2023076710A1 (fr) * 2021-11-01 2023-05-04 A2Tbio Llc Agents d'arn stabilisés

Also Published As

Publication number Publication date
RU2017105743A3 (fr) 2018-08-21
RU2017105743A (ru) 2018-08-21
RU2740501C2 (ru) 2021-01-14

Similar Documents

Publication Publication Date Title
RU2708237C2 (ru) Модифицированные олигонуклеотиды и способ их получения
JP5572090B2 (ja) テトラヒドロピラン核酸類似体
CN101490074B (zh) 5’-修饰的双环核酸类似物
AU2005328382C1 (en) Oligonucleotides comprising a modified or non-natural nucleobase
CN103154014B (zh) 修饰核苷、其类似物以及由它们制备的寡聚化合物
WO2018156056A1 (fr) Oligonucléotides modifiés activant l'arnase n
RU2567664C2 (ru) Антисмысловые нуклеиновые кислоты
CN102766630B (zh) 6-修饰的双环核酸类似物
KR100782896B1 (ko) Ｌ-리보-ｌｎａ 유사체
JP2021046396A (ja) ホスホルアミダイト化学を使用する骨格修飾モルホリノオリゴヌクレオチド及びキメラの合成
AU2005327517A1 (en) Oligonucleotides comprising a non-phosphate backbone linkage
CN110023322B (zh) 新的双环核苷和由其制备的寡聚体
JP2013520438A (ja) 逆方向合成ｒｎａのためのホスホルアミダイト
JP2018109015A (ja) 三環ヌクレオシドおよびこれらから調製したオリゴマー化合物
JP2023546199A (ja) アシアロ糖タンパク質受容体のための新規リガンド
US8247540B2 (en) Caged nucleotides and oligonucleotides and their application
WO2022081046A1 (fr) Composé chimique avec un groupe triazine et procédé de production
JP2024508780A (ja) オリゴヌクレオチドおよび炭水化物をコンジュゲートするための組成物
RU2799452C2 (ru) Химическое соединение с триазиновой группой и способ его получения
CN101410406B (zh) 6-修饰的双环核酸类似物
Malek-Adamian Synthesis of Novel 2′, 4′-Modified Nucleoside Analogues and Their Properties in Oligonucleotides and Nanoparticle Development for Targeted Delivery to Glioblastoma Multiformes Drr-Expressing Cells
WO2006043521A1 (fr) Composé d'acide oligonucléique optiquement actif contenant une liaison phosphorothioate
Piperakis Synthesis and Properties of Nucleic Acid Duplexes and Quadruplexes containing 3'-S-Phosphorothiolate Linkages
AU2012201664A1 (en) 6-modified bicyclic nucleic acid analogs

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18756620

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18756620

Country of ref document: EP

Kind code of ref document: A1