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WO2007145593A1 - Thymidine à pont 2'-n,4'-c-éthylène (aza-ena-t) à contrainte conformationnelle - Google Patents

Thymidine à pont 2'-n,4'-c-éthylène (aza-ena-t) à contrainte conformationnelle Download PDF

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WO2007145593A1
WO2007145593A1 PCT/SE2007/050522 SE2007050522W WO2007145593A1 WO 2007145593 A1 WO2007145593 A1 WO 2007145593A1 SE 2007050522 W SE2007050522 W SE 2007050522W WO 2007145593 A1 WO2007145593 A1 WO 2007145593A1
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ena
aza
aon
rna
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Jyoti Chattopadhyaya
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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
    • 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
    • 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
    • C07H19/06—Pyrimidine radicals
    • C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
    • 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
    • C07H19/16—Purine radicals
    • C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • New 2',4'-piperdino fused aza-ENA-thymine, -guanine, -adenine, -cytosine or -uarcil (Aza-ENA) and fluoro-nucleobase analogs (Aza-ENA, for example, 13, IS or 18 in Scheme 2) nucleosides are conformationally-constrained nucleosides (North- type) .
  • the Aza-ENA block(s) has/have been incorporated into antisense oligonucleotides (AON, See Table 1 for example) and their antisense properties as gene-directed agent has been evaluated in order to selectively arrest translation of mRNA to protein product.
  • Aza-ENA modified AONs have shown high target affinity to complementary RNA strand (T m increase of +2.5 to +9 0 C per modification, some examples are given in Table 1), depending upon the substitution site in the AON sequence, compared to the native counterpart.
  • All the aza-ENA modified AONs offered greater protection towards 3' exonucleases compared to the native sequence. In fact, all the modified AONs cleaved at one nucleotide before the modification towards 3'-end and did not degrade any further. These residual AONs have been found to be stable for over 48 h in human serum and for over 24 h with the snake venom phosphodiesterase. This result clearly suggests that a single modification at the second position from the 3 '-end can give even more substantial stability towards 3' exonucleases.
  • New 2',4'-piperdino fused aza-ENA-thymine, -guanine, -adenine, -cytosine or -uarcil (Aza-ENA) and its derivatives such as mono- di or tri-phosphates and fluoro analogs can be specifically used to inhibit virus- or -tumor specific proteins, and thereby inactivate the pathogen/tumor growth.
  • These analogs have also found to be useful in various diagnostic applications and assays including polymerase chain reaction.
  • Drawing Figure 1 shows the structure of one compound according to the invention, namely aza-ENA-thymine.
  • aza-ENA having 2-aza-6-oxabicyclo[3.2.1]octane skeleton
  • aza-ENA has been synthesized using a key cyclization step involving 2'- ⁇ ra-trifluoromemylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-ZjZ 1 S-OBn protected diastereomerically pure aza-ENAs (12a and 12b) with the chair conformation of the piperidino skeleton, whereas the pentofuranosyl moiety is locked in the North-type conformation (7° ⁇ P ⁇ 27°, 44° ⁇ ⁇ m ⁇ 52°).
  • AON mer antisense oligonucleotides
  • the relative rates of the RNase Hl cleavage of the aza-ENA-modified AON/RNA heteroduplexes were very comparable to that of the native counterpart, but the RNA cleavage sites of the modified AON/RNA were found to be very different.
  • the aza-ENA modifications also made the AONs very resistant to 3 '-degradation (stable over 48 h) in the blood serum compared to the unmodified AON (fully degraded in 4 h).
  • the aza-ENA modification in the AON fulfilled three important antisense criteria, compared to the native: (i) improved RNA target affinity, (ii) comparable RNase H cleavage rate, and (iii) higher blood serum stability.
  • Modified Oligonucleotides have successfully been used as valuable tools to inhibit the gene expression by utilizing various mechanisms of actions. 1"7
  • the most matured method is the antisense technology 6 ' 8 which exploits ability of a single stranded DNA-oligonucleotide to bind to the target messenger RNA (mRNA) via Watson-Crick base pairing in a sequence specific manner. Once bound to the target RNA, antisense agent either sterically blocks the synthesis of ribosomal proteins or induces RNase H mediated degradation of the target mRNA.
  • RNA interference RNA interference
  • Figure 1 Structures of North-type conformationally-constrained ⁇ / ⁇ -D/L-pentofuranosyl nucleoside derivatives
  • LNA incorporated oligonucleotides showed unprecedented level of affinity towards complementary RNA (AT m ⁇ +4° to +8 0 C per modification) and a moderate increase of +3 to +5 0 C per modification towards complementary DNA.
  • ENA modified AONs have approximately 55 times higher stability towards 3'-exonuclease compared to the LNA analog. 27
  • This ability of ENA for efficient target binding and high nuclease resistance have been well exploited to evaluate their antisense, antigene and RNAi 43"47 properties: High target affinity has also been reported 46 for the triplex formation by ENA monosubstituted oligopyrimidine 2'-deoxynucleotides efficiently binding to dsDNA at physiological pH. 46 ENA/DNA chimeric AONs have also been used to study the RNase H mediated antisense activity.
  • VEGF vascular endothelial growth factor
  • RNA/ENA chimera duplexes were also found to induce skipping of the exon-41 containing the nonsense mutation which suggested that such design of ENA incorporated AONs could be used to promote dystrophin expression in mycocytes of Duchenne muscular dystrophy (DMD) patients.
  • DMD Duchenne muscular dystrophy
  • JDP2 Jun dimerization protein-2
  • ENA modified AONs namely high target affinity, sequence selectivity towards ssRNA/dsDNA targets and high nuclease resistance (in vivo and in vitro), prompted us to design the 2'-amino modified ENA analogs (aza-ENA, compound E in Scheme 1), as a new class of conformationally-constrained AON which could potentially be an important analog for effective gene- directed therapeutics and diagnostics.
  • aza-ENA based AONs may have three clear advantages over the corresponding ENA counterpart: First, the endocyclic amino functionality of the aza-ENA analog could be utilized as a well defined conjugation site 50 and thereby we can control the hydrophilic, hydrophobic and steric requirements of a minor groove of the duplex. Second, the amine-derivatized
  • AONs have displayed increased thermal affinities 51 ' 52 towards complementary RNA possibly because of the presence of positively charged moieties at physiological pH, and thus could influence partial neutralization of the negatively charged phosphates in the duplexes. 36 ' 42 Third, introduction of a fluorescence probe connected to this nitrogen moiety will enable us for real-time in vivo imaging of RNA and can therefore be used for specific detection of nucleic acids while maintaining their hybridization properties. 53 ' 54
  • the aza-ENA nucleotides have been incorporated in 15-mer AON as single modification at four different sites to give four mono aza-ENA substituted AONs #2-5 (sequences shown in Table 1). These AONs have shown an increase in the thermal stability from 2.5 to 4 0 C per modification towards complementary RNA depending upon the substitution site. We also show that the relative rates of the RNase Hl promoted cleavage of the aza-ENA-modified AON/RNA heteroduplexes are comparable to that of the native counterpart, and, quite interestingly, the aza-ENA modifications also results in significant increase of AON/RNA resistance to 3'-exonucleases degradation in the blood serum compared to the native counterpart.
  • aza-ENA derivative was started with known sugar precursor 32 1, which was converted to 3,5-di- ⁇ 9-benzyl-4-C- hydroxymethyl-l,2-0-isopropylidene- ⁇ -D-ribofuranose 2 32 .
  • Compound 2 was then converted to the 4- triflyloxymethylene derivative 3 using triflic anhydride in dichloromethane-pyridine mixture (3:1, v/v) at 0 0 C.
  • the crude product obtained after aqueous workup was subsequently treated with 3 equivalents of LiCN in DMF, and stirred at r.t. for 3 days, which afforded cyano-sugar 4 in an overall yield of 56% from 2, along with some unidentified minor compounds.
  • NC(CH 2 ) 2 OP(Cl)NCPr) 2 OP(Cl)NCPr) 2 , DIPEA, THF, r.t., 3 h, (overnight for 20);
  • Ac acetyl
  • Tf trifluoromethylsulfonyl
  • PAC phenoxyacetyl
  • THF tetrahydrofuran
  • DBU l,8-diazabicyclo[5.4.0]undec-7-en
  • DMTr 4,4'-dimethoxytrityl
  • DIPEA diisopropylethylamine
  • TFA trifluoroacetyl.
  • Insets (A) and (B) are the 1 H- 13 C HMBC spectra showing the long range through-bond connectivities between C77H2' and C2'/H7 e ' for the two diastereomers 12a and 12b.
  • the crude product 5 (4 ⁇ 5 was almost quantitative) was subjected to modified Vorbruggen reaction 42 ' 56 using in situ silylation of thymine and subsequent trimethylsilyl triflate mediated coupling to give the /3-conf ⁇ gured thymine nucleoside 6 in 80% yield.
  • the ⁇ configuration of 6 was confirmed by ID differential nOe experiment which showed 8% nOe enhancement of H-6 upon
  • the upfield H6 e proton ( ⁇ l .31) of 12a was distinguished from the FI6 a proton (52.02) by the fact that the former has only a smaller JH 1 H coupling of 4.8 Hz beside a geminal coupling of 13 Hz, whereas H6 a proton has a large trans Jn 1H coupling of 11.6 Hz and a cisoid J H1 H coupling of 6.7 Hz.
  • This assignment of H6 a /H6 e protons allowed us to identify immediately the H7 a /H7 e protons (at 53.13 and 53.02) by a
  • H6 e proton 51.53 of 12b was distinguished from the H6 a proton (52.04) by the fact that the former has only a smaller 3 J H , H coupling of 5.3 Hz beside a geminal coupling of 13.4 Hz, whereas H6 a proton has a large trans 3 J H1H coupling of 11.9 Hz and cisoid 3 J H>H coupling of 6.7 Hz.
  • a set of double and single decoupling experiments at the centre of multiplets of H6 a and/or H6 e protons gave the assignment of both the H7 a and H7 e protons. From the geometrical point of view the H7 a is expected to have different
  • H7 e and H6 a are expected to be medium ( ⁇ [H7 e -C7-C6-H6 a ] is about 40°), while that
  • H7 a has been
  • C2'j is in the region of «90°.
  • the H6 e proton (51.17) of 13 was distinguished from the H-6 a (51.78) by the fact that the former
  • H6 a proton has only a smaller 3 J H1H coupling of 4.6 Hz besides a geminal coupling of 12.9 Hz, whereas H6 a proton has a large trans 3 J H1H coupling of 13.0 Hz and cisoid 3 J H1H coupling of 6.8 Hz.
  • a set of double and single decouplings at the centre of multiplets of H6 a and/or H6 e protons gave both the assignment of H7 a (A) : Major isomer (12a) with N-H 6 (B) : Minor isomer (12b) with N-H 3 (C): nOe contacts (D): nOe contacts
  • Figure 4 ID selective NOESY spectra of 12a and 12b.
  • No couplings were observed between H7 e and H6 e confirmed by 2D COSY experiments. This shows that the dihedral angle between H6 e and H7 e , ⁇ [H7 e -
  • the ⁇ G* was found to be 25.4 kcal mol "1 at 298 K ( Figure S38 in SI). The complete conversion in pyridine-t/ 5 with 2 kcal mol "1 lower ⁇ G* suggests that the conversion of 12b to 12a is base catalyzed.
  • the NIR barriers increase with a decrease of the ring size in azabicylces.
  • the presence of a 5-membered ring as a component of a rigid nitrogen-bridged bicyclic skeleton increase the NER. barrier by ca 3 kcal/mol per ring, thereby showing a relationship between azacycle geometry and the NIR barrier.
  • the phosphoramidite 17 was successfully incorporated into the mixed 15mer sequence (Table 1) but to our surprise the PAC protecting group was very stable and could not be deprotected even in 33% aqueous ammonia and AMA (33% aqueous ammonia/methylamine 1:1 v/v) at 65°C for 2 days, which was clear from the mass measurement using MALDI-TOF mass spectroscopy [expected mass with PAC protection m/z 4624.7 and observed 4624.9].
  • the PAC protected nucleoside 15, on the other hand, could be deprotected with aqueous ammonia at 55 0 C overnight.
  • Figure 7 The CD spectra of aza-ENA modified 15mer AONs (AON 2-5) as duplex with (A) complementary DNA, and (B) with complementary RNA in comparison with the native counterpart (AON 1).
  • Escherichia coli RNase Hl has been used in this work because of two reasons: first, it is commercially available in a pure form, and second its cleavage properties are not very different from those of the mammalian enzyme. 70 Hence, the antisense properties of aza-ENA modified oligonucleotides duplex with the complementary RNA were compared with the native as well as with the identical oxetane- modified counterparts 71 with Escherichia coli RNase Hl as a model system.
  • RNA cleavage patterns of all aza-ENA modified AONs were found to be uniquely different from those of the isosequential oxetane modified AONs (AON 6 - 9; Figure 9).
  • AON 2 showed only one prominent cleavage site at A8 position of the complementary RNA ( Figure 8) unlike the oxetane-modified AON 6 which showed cleavages at A7, A8, AlO and UI l with no clear preferences (Figure 9).
  • Comparison of AON 4 versus AON 8 and AON 5 versus AON 9 clearly shows the absence of single RNA cleavage site in aza-ENA modified AONs/RNA duplexes compared to those of the oxetane-modified counterparts (A7 of AON 8 and A9 of AON 9).
  • AON 3 and its isosequential oxetane analog AON 7 showed identical cleavage footprint pattern of 5 nucleotide gap. This shows that the local structures of all aza-ENA modified AONs/RNA duplexes are not the same.
  • RNase H enzyme indeed can finely discriminate these local variations of the stereochemical properties of the microstructure brought about by various type and incorporation site of the North-type modification (aza-ENA versus oxetane modifications) in the AON.
  • FIG. 8 Autoradiograms of 20% denaturing PAGE, showing the cleavage kinetics of 5'- 32 P- labeled target RNA by E.coli RNase Hl in native AON 1/RNA (lane 5) and the aza-ENA modified AONs (2-4)/RNA hybrid duplexes (lane 1 to 4) after 2, 5, 15, 35 and 60 min of incubation.
  • Conditions of cleavage reactions RNA (0.8 ⁇ M) and AONs (4 ⁇ M) in buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM KCl, 10 mM MgC12 and 0.1 mM DTT at 21 0 C; 0.08 U of RNase H. Total reaction volume 30 ⁇ L.
  • Figure 9 Insets (A and B) are autoradiograms of 20% denaturing PAGE, showing the cleavage kinetics of 5'- 32 P-labeled target RNA by E. coli RNase Hl in native AON 1/RNA and the oxetane modified AONs (6-9)/RNA hybrid duplexes after 30 min, 1 and 2 h of incubation.
  • Conditions of cleavage reactions RNA (0.8 ⁇ M) and AONs (4 ⁇ M) in buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM KCl, 10 mM MgC12 and 0.1 mM DTT at 21 0 C; 0.08 U of RNase H. Total reaction volume 30 ⁇ L.
  • Figure 10 Autoradiograms of 20% denaturing PAGE, showing the degradation pattern of 5'- 32 P-labeled AON 1 to 5 in human serum. Time points are taken after 0, 15, 30 min, 1, 2 and 9 h of incubation. The % of AON remaining after 9 h of incubation: 0% of AON 1, 0% of AON 2, 8% of AON 3, 15% of AON 4, 20% of AON 5.
  • aza-ENA modified AONs 1 to 5 towards 3' exonuclease was investigated using snake venom phosphodiesterase (SVPDE) over a period of 24 h at 21 0 C. Time points were taken at O, 1, 2 and 24 h to examine the cleavage pattern ( Figure 11).
  • the enzyme digestion pattern was similar to that obtained from the digestion with human serum except the fact that these aza-ENA modified AONs offered resistance to degradation even after 24 h. Note that native AON 1 was completely degraded in 1 h, whereas the full length AONs 2 to 5 were 18%, 20%, 14% and 10% left undegraded respectively after 24 h.
  • Figure 11 Denaturing PAGE analysis of snake venom phosphodiesterase (SVPDE) degradation pattern of 5'- 32 P-labeled AON 1 to 5. Time points are taken after O, 1, 2 and 24 h of incubation with enzyme. The % of AON remaining after 24 h of incubation: 0% of AON 1, 18% of AON 2, 20% of AON 3, 14% of AON 4, 10% of AON 5
  • SVPDE snake venom phosphodiesterase
  • Step (ii) Perform NMR constrained molecular dynamics (MD) simulation ( 0.5 ns,10 steps) simulated annealing (SA) followed by 0.5 ns NMR constrained simulations at 298 K using the NMR derived torsional constraints from Step (i) to yield NMR defined molecular structures of 3',5'-bis-OBn protected (12a, 12b) and de-protected aza- ENA (13).
  • MD NMR constrained molecular dynamics
  • SA simulated annealing
  • NMR constrained simulations at 298 K using the NMR derived torsional constraints from Step (i) to yield NMR defined molecular structures of 3',5'-bis-OBn protected (12a, 12b) and de-protected aza- ENA (13).
  • the MD simulations were performed using Amber force field (AMBER 7 75 ) and explicit TIP3P 76 aqueous medium (see details in Experimental section), (iii) Acquire 6-31G** Hartree-Fock optimized ab inilio gas phase geometries (by Gaussian 98 77 ) in order to compare the NMR derived torsions with the ab initio geometry, (iv) Refine the Karplus parameters with the help of the NMR and ab initio derived torsions, (v) Analyze the full conformational hyperspace using 2 ns NMR/ ⁇ initio constrained MD simulations of compounds 12a, 12b and 13 followed by full relaxation of the constraints. The results of these studies are summarized in Table 3.
  • V 0 C4'-O4'-Cr-C2' 3.38 (4.2 ⁇ 6.0) 3.21 (4.2 ⁇ 5.6) -0.91 (-0.5 ⁇ 6.8)
  • V 1 O4'-C1'-C2'-C3' -31.88 (-32.9 ⁇ 4.9) -31.28 (-32.9 ⁇ 4.6) -28.21 (-29.5 ⁇ 5.5)
  • V 2 C1'-C2'-C3'-C4 T 46.00 (45.2 ⁇ 3.2) 45.07 (45.1 ⁇ 3.2) 43.87 (44.8 ⁇ 3.4)
  • V 3 C2'-C3'-C4'-O4' -45.21 (-45.5 ⁇ 3.8) .43.87 (-45.4 ⁇ 3.8) .45.14 (47.6 ⁇ 4.0)
  • V 4 C3'-C4'-O4'-C1' 26.83 (26.6 ⁇ 5.5) 26.19 (26.5 ⁇ 5.3) 29.65
  • Figure 12 Experimental 3 JHi ' ,H 2' and 3 JH 2' ,H3 ' vicinal coupling constants of the ENA (compound (I) in Figure 1), aza-ENA (12a,12b,13), LNA and 2'-amino LNA analogs (compounds (A) and (B) in Figure 1) shown together with contours of theoretical 3 J H r,H2 ' vs. 3 JH 2' ,H 3' dependencies at fixed sugar puckering amplitudes (from 35° to 65°) calculated using algorithm and Haasnoot-de Leeuw-Altona generalized Karplus equation reported in Ref. 73 ' 74 and using the same parameters as described in the text and in the caption of Table 1.
  • Aza-ENA modified AONs have shown high target affinity to complementary RNA strand (T m increase of +2.5 to +4 0 C per modification), depending upon the substitution site, compared to the native counterpart, while hybridization with the complementary DNA sequence lead to substantial destabilization of the duplexes (T m drop of -0.5 to -3°C per modification).
  • All the aza-ENA modified AONs offered greater protection towards 3' exonucleases compared to the native sequence. In fact, all the modified AONs cleaved at one nucleotide before the modification towards 3 '-end and did not degrade any further. These residual AONs have been found to be stable for over 48 h in human serum and for over 24 h with the snake venom phosphodiesterase. This result clearly suggests that a single modification at the second position from the 3 '-end can give even more substantial stability towards 3' exonucleases.
  • the organic phase was dried over anhydrous MgS O 4 , evaporated under reduced pressure followed by co evaporation with toluene 3 times and dichloromethane 3 times.
  • the crude reaction product was dissolved in 150 mL dry DMF and 80 mL of IM LiCN in DMF was added and stirred for 3 days at r.t. Solvent was carefully evaporated and the residue was dissolved in dichloromethane, saturated aqueous NaHCO 3 was added and extracted with dichloromethane (3 times).
  • the organic phase was dried over anhydrous MgSO 4 and evaporated under reduced pressure.
  • the crude product was co-evaporated several times with dry toluene till the product solidifies to give 5 (more than 90% pure by NMR).
  • the crude product was dissolved in 150 mL of anhydrous CH 3 CN, thymine (2.24 g, 17.7 mmol) and N,O-bis(trimethylsilyl)acetamide (10.2 mL, 41.4 mmol) was added and stirred at 80 0 C for 1 h under nitrogen atmosphere.
  • the reaction mixture was cooled to r.t, TMSOTf (3.48 mL, 19.24 mmol) was added and again warmed to 80 0 C and stirred overnight under nitrogen atmosphere.
  • the reaction was quenched with saturated aqueous NaHCO 3 and extracted with dichloroniethane.
  • Nucleoside 6 (6.14 g, 11.8 mmol) was dissolved in methanol 60 mL, 18 mL of 1 M sodium methoxide was added and stirred at r.t. for 3 h. The solvent was partially evaporated under reduced pressure and extracted with dichloromethane. The combined organic phase was dried over anhydrous MgS ⁇ 4 , evaporated to give 7 (more than 90% pure by NMR) as a white solid. The crude product was co- evaporated with dry pyridine 3 times to remove traces of moisture and dissolved in 60 mL of the same solvent.
  • Nucleoside 8 (6.2 g, 11.2 mmol) was dissolved in 70 mL of anhydrous CH 3 CN; DBU (1.75 mL, 11.76 mmol) was added drop wise and stirred at r.t. for 1 h. The reaction was quenched with water and extracted with dichloromethane. Combined organic phase was dried over anhydrous MgSO 4 and evaporated under reduced pressure.
  • oligonucleotides were synthesized using an automated DNA/RNA synthesizer by Applied Biosystems, model 392.
  • fast deprotecting phosphoramidites nucleobases were protected using the following groups: Ac for C, 1 Pr-PAC for G, and PAC for A
  • the AONs were deprotected at room temperature by aqueous NH 3 treatment for 24 h.
  • All AONs and the target RNA were purified by 20% polyacrylamide/7M urea) PAGE, extracted with 0.3 M NaOAc, desalted with C18-reverse phase catridges and their purity (greater than 95%) was confirmed by PAGE.
  • T m of the AON/RNA hybrids was carried out in the following buffer: 57 mM Tris-HCl (pH 7.5), 57 mM KCl, 1 mM MgCl 2 . Absorbance was monitored at 260 run in the temperature range from 20 0 C to 70 0 C using UV spectrophotometer equipped with Peltier temperature programmer with the heating rate of 1 0 C per minute. Prior to measurements, samples (1 ⁇ M of AON and 1 ⁇ M RNA mixture) were preannealed by heating to 80 0 C for 5 min followed by slow cooling to 4 0 C and 30 min equilibration at this temperature.
  • CD spectra were recorded from 300 to 200 run in 0.2 cm path length cuvettes. Spectra were obtained with a AON/RNA duplex concentration of 5 ⁇ M in 57 mM Tris-HCl (pH 7.5), 57 mM KCl, 1 mM MgCl 2 . All the spectra were measured at 25 0 C and each spectrum is an average of 5 experiments from which CD spectrum of the buffer was subtracted.
  • oligonucleotides 32 P Labeling of Oligonucleotides.
  • the oligoribonucleotide, oligodeoxyribonucleotides were 5'-end labeled with P using T4 polynucleotide kinase and [ ⁇ - 32 P] ATP by standard procedure.
  • Labeled AONs and RNA were purified by 20% denaturing PAGE and specific activities were measured using Beckman LS 3801 counter.
  • the source of RNase Hl was Escherichia coli containing clone of RNase H gene.
  • the percentage of RNA cleavage was monitored by gel electrophoreses as a function of time (0-60 min), using 0.08 U and 0.12 U of RNase H.
  • AONs (6 ⁇ L) at 2 ⁇ M concentration (5'-end 32 P labeled with specific activity 90 000 cpm) were incubated in 26 ⁇ L of human serum (male AB) at 21 0 C (total reaction volume was 36 ⁇ L). Aliquots (3 ⁇ L) were taken at 0, 15 and 30 min, 1, 2 and 9 h, and quenched with 7 ⁇ L quenching solution containing 50 mM EDTA in 80% formamide, resolved in 20% polyacrylamide denaturing (7 M urea) gel electrophoresis and visualized by autoradiography. Theoretical calculations.
  • 2',4' conformationally constrained aza-ENA (3',5'-bis-OBn protected compounds 12a and 12b and de- protected 13) nucleosides have theoretically simulated to build up their molecular structures using the following protocol: (i) Derive Initial dihedral angles from the observed 3 J H1H using Haasnoot-de Leeuw- Altona generalized Karplus equation 73 ' 74 (ii) Perform NMR constrained molecular dynamics (MD) simulation ( 0.5 ns,10 steps) simulated annealing (SA) followed by 0.5 ns NMR constrained simulations at 298 K using the NMR derived torsional constraints from Step (i) to yield NMR defined molecular structures of 3',5'-bis-OBn protected (12a, 12b) and de-protected aza-ENA (13).
  • MD constrained molecular dynamics
  • SA simulated annealing
  • NMR constrained simulations at 298 K using the NMR derived
  • the atomic charges and optimized geometries of compounds 12a, 12b, and 13 were then used as AMBER 75 force field parameters employed in the MD simulations.
  • the protocol of the MD simulations is based on Cheathan-Kollman's procedure employing modified version of Amber 1994 force field as it is implemented in AMBER 7 program package.
  • T m increase by 2.5° to 4°C per T modification vis-a-vis Native.

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  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)

Abstract

La présente invention concerne un composé selon la formule : la thymidine à pont 2'-désoxy-2'-N,4'-C-éthylène (aza-ENA-T) a été synthétisée en utilisant une étape de cyclisation clé pour obtenir un squelette pipéridino condensé en conformation de chaise, tandis que le fragment pentofuranosyle est bloqué dans la conformation de type nord. Le composé est utilisé pour préparer des oligonucléotides antisens qui sont utilisés pour le traitement du cancer et d'infections virales.
PCT/SE2007/050522 2006-06-15 2007-07-12 Thymidine à pont 2'-n,4'-c-éthylène (aza-ena-t) à contrainte conformationnelle Ceased WO2007145593A1 (fr)

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SE0601316 2006-06-15
SE0601316-3 2006-06-15

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WO2017047816A1 (fr) * 2015-09-18 2017-03-23 田辺三菱製薬株式会社 Acide nucléique réticulé guna, procédé de production de ce dernier, et composé intermédiaire
US10358458B2 (en) 2014-09-26 2019-07-23 Riboscience Llc 4′-vinyl substituted nucleoside derivatives as inhibitors of respiratory syncytial virus RNA replication
CN112996522A (zh) * 2018-11-12 2021-06-18 田边三菱制药株式会社 桥连型人工核酸alna
US11098077B2 (en) 2016-07-05 2021-08-24 Chinook Therapeutics, Inc. Locked nucleic acid cyclic dinucleotide compounds and uses thereof

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MORITA K. ET AL.: "Synthesis and Properties of 2'-O-4'-C-Ethylene-Bridged Nucleic Acids (ENA) as Effective Antisense Oligonucleotides", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 11, 2003, pages 2211 - 2226, XP002432951 *
SINGH S.K. ET AL.: "Synthesis of 2'-Amino-LNA: A Novel Conformationally Restricted High-Affinity Oligonucleotide Analogue with a Handle", J. ORG. CHEM., vol. 63, 1998, pages 10035 - 10039, XP002252481 *
VARGHESE O.P. ET AL.: "Conformationally Constrained 2'-N,4'-C-Ethylene-Bridged Thymidine (Aza-ENA-T): Synthesis, Structure, Physical, and Biochemical Studies of Aza-ENA-T-Modified Oligonucleotides", J. AM. CHEM. SOC., vol. 128, 2006, pages 15173 - 15187, XP003018390 *

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Publication number Priority date Publication date Assignee Title
US10358458B2 (en) 2014-09-26 2019-07-23 Riboscience Llc 4′-vinyl substituted nucleoside derivatives as inhibitors of respiratory syncytial virus RNA replication
JP2022033869A (ja) * 2015-09-18 2022-03-02 田辺三菱製薬株式会社 架橋型核酸ＧｕＮＡ、その製造方法および中間体化合物
JP2023075302A (ja) * 2015-09-18 2023-05-30 田辺三菱製薬株式会社 架橋型核酸ＧｕＮＡ、その製造方法および中間体化合物
CN108137638A (zh) * 2015-09-18 2018-06-08 田边三菱制药株式会社 桥连型核酸GuNA、其制造方法及中间体化合物
US10961269B2 (en) 2015-09-18 2021-03-30 Mitsubishi Tanabe Pharma Corporation Bridged nucleic acid GuNA, method for producing same, and intermediate compound
EP3974436A1 (fr) * 2015-09-18 2022-03-30 Mitsubishi Tanabe Pharma Corporation Acide nucléique réticulé guna, son procédé de production et composé intermédiaire
JPWO2017047816A1 (ja) * 2015-09-18 2018-07-05 田辺三菱製薬株式会社 架橋型核酸ＧｕＮＡ、その製造方法および中間体化合物
JP2024153835A (ja) * 2015-09-18 2024-10-29 田辺三菱製薬株式会社 架橋型核酸ＧｕＮＡ、その製造方法および中間体化合物
JP6994197B2 (ja) 2015-09-18 2022-01-14 田辺三菱製薬株式会社 架橋型核酸ＧｕＮＡ、その製造方法および中間体化合物
CN108137638B (zh) * 2015-09-18 2022-05-03 田边三菱制药株式会社 桥连型核酸GuNA、其制造方法及中间体化合物
WO2017047816A1 (fr) * 2015-09-18 2017-03-23 田辺三菱製薬株式会社 Acide nucléique réticulé guna, procédé de production de ce dernier, et composé intermédiaire
US11098077B2 (en) 2016-07-05 2021-08-24 Chinook Therapeutics, Inc. Locked nucleic acid cyclic dinucleotide compounds and uses thereof
CN112996522A (zh) * 2018-11-12 2021-06-18 田边三菱制药株式会社 桥连型人工核酸alna
CN112996522B (zh) * 2018-11-12 2024-09-20 田边三菱制药株式会社 桥连型人工核酸alna
EP3881851A4 (fr) * 2018-11-12 2023-02-15 Mitsubishi Tanabe Pharma Corporation Acide nucléique artificiel réticulé alna
US12338265B2 (en) 2018-11-12 2025-06-24 Mitsubishi Tanabe Pharma Corporation Crosslinked artificial nucleic acid ALNA

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