阅读说明：本技术 包含三硫代磷酸酯核苷间键的寡核苷酸 (Oligonucleotides comprising phosphorothioate internucleoside linkages ) 是由 K·布雷啻尔 T·科赫 A·朔伊布林 J·J·A·杜施梅尔 M·B·杜什马尔 李美玲 E 于 2019-07-30 设计创作，主要内容包括：本发明涉及一种寡核苷酸,其包含至少一个式(I)的三硫代磷酸酯核苷间键其中(A~1)、(A~2)和R如说明书和权利要求书中所定义。本发明所述的寡核苷酸可用作药物。(The present invention relates to an oligonucleotide comprising at least one phosphorothioate internucleoside linkage of formula (I) Wherein (A) 1 )、(A 2 ) And R is as defined in the description and claims. The oligonucleotides of the invention are useful as medicaments.)

1. An oligonucleotide comprising at least one phosphorothioate internucleoside linkage of formula (I)

Wherein (A)¹) Is a 3' -nucleoside, (A)²) Is a 5' -nucleoside and R is hydrogen or a phosphate protecting group.

2. The oligonucleotide of claim 1, wherein the nucleoside(A¹) Is a DNA nucleoside, an RNA nucleoside or a sugar-modified nucleoside.

3. The oligonucleotide according to claim 1 or 2, wherein the nucleoside (a)¹) Is a DNA nucleoside or a sugar-modified nucleoside.

4. The oligonucleotide according to any one of claims 1 to 3, wherein the nucleoside (A)²) Is a DNA nucleoside, an RNA nucleoside or a sugar-modified nucleoside.

5. The oligonucleotide of any one of claims 1 to 4, wherein the nucleoside (A)²) Is a DNA nucleoside or a sugar-modified nucleoside.

6. The oligonucleotide of any one of claims 2 to 5, wherein the sugar modified nucleoside is a 2' sugar modified nucleoside.

7. The oligonucleotide of any one of claims 1 to 6, wherein the nucleoside (A)²) Comprises the following steps: 2 '-alkoxy-RNA, in particular 2' -methoxy-RNA; 2 '-alkoxyalkoxy-RNA, in particular 2' -methoxyethoxy-RNA; 2' -amino-DNA; 2' -fluoro-RNA; or 2' -fluoro-ANA.

8. The oligonucleotide of any one of claims 1 to 6, wherein the nucleoside (A)²) Is an LNA nucleoside.

9. The oligonucleotide according to claim 8, wherein the LNA nucleoside is independently selected from the group consisting of β -D-oxy LNA, 6' -methyl- β -D-oxy LNA and ENA, in particular β -D-oxy LNA.

10. The oligonucleotide according to claim 1, wherein the nucleoside (A)¹) And nucleosides (A)²) At least one of which is 2' -alkoxyalkoxy-RNA.

11. The oligonucleotide of claim 10, wherein the 2 '-alkoxyalkoxy-RNA is 2' -methoxyethoxy-RNA.

12. The oligonucleotide according to claim 1, wherein the nucleoside (A)¹) And nucleosides (A)²) Both are DNA nucleosides, or both are 2' sugar modified nucleosides, particularly LNA nucleosides.

13. An oligonucleotide according to any one of claims 1 to 12, comprising further internucleoside linkages selected from phosphodiester internucleoside linkages, phosphorothioate internucleoside linkages and trithiophosphate internucleoside linkages of formula (I) as defined in claim 1.

14. An oligonucleotide according to any one of claims 1 to 13, comprising further internucleoside linkages selected from phosphorothioate internucleoside linkages and trithiophosphate internucleoside linkages of formula (I) as defined in claim 1.

15. Oligonucleotide according to any one of claims 1 to 13, comprising between 1 and 15, in particular between 1 and 5, more particularly 1,2, 3, 4 or 5 trithiophosphate internucleoside linkages of formula (I) as defined in claim 1.

16. The oligonucleotide according to any one of claims 1 to 15, wherein the other internucleoside linkages are all of the formula-P (═ S) (OR) O₂A phosphorothioate internucleoside linkage of (a), wherein R is as defined in claim 1.

17. The oligonucleotide of any one of claims 1 to 16, comprising additional nucleosides selected from the group consisting of DNA nucleosides, RNA nucleosides, and sugar-modified nucleosides.

18. The oligonucleotide of any one of claims 1 to 17, wherein one or more nucleosides is a nucleobase modified nucleoside.

19. The oligonucleotide of any one of claims 1 to 18, wherein the oligonucleotide is an antisense oligonucleotide, an siRNA, a microrna mimetic, or a ribozyme.

20. The oligonucleotide of any one of claims 1 to 19, wherein the oligonucleotide is an antisense gapmer oligonucleotide.

21. The oligonucleotide of claim 20, wherein the phosphorothioate internucleoside linkage of formula (I) is in the gap region of the gapmer oligonucleotide.

22. The oligonucleotide of claim 20, wherein the phosphorothioate internucleoside linkage of formula (I) is in a flanking region of the gapmer oligonucleotide.

23. The oligonucleotide according to claims 20 to 22, wherein the gapmer oligonucleotide is a LNA gapmer, a mixed wing gapmer or a2 '-substituted gapmer, in particular a 2' -O-methoxyethyl gapmer.

24. The gapmer oligonucleotide of any one of claims 20 to 23, wherein the gapmer oligonucleotide comprises a contiguous nucleotide sequence of the formula 5' -F-G-F ' -3', wherein G is a region of 5 to 18 nucleosides capable of recruiting ribonuclease H and the region G is flanked 5' and 3' by flanking regions F and F ', respectively, wherein regions F and F ' independently comprise or consist of 1 to 72 ' -sugar modified nucleotides, wherein the nucleotides of region F adjacent to region G are 2' -sugar modified nucleotides and wherein the nucleotides of region F ' adjacent to region G are 2' -sugar modified nucleotides.

25. The gapmer oligonucleotide of claim 24, wherein the at least one phosphorothioate internucleoside linkage of formula (I) as defined in claim 1 is positioned between adjacent nucleosides in region G or between region G and region F'.

26. The oligonucleotide according to any one of claims 1 to 25, wherein the oligonucleotide is an antisense oligonucleotide hybrid or full polymer, in particular a splice-switch oligonucleotide or a microrna inhibitor oligonucleotide.

27. A pharmaceutically acceptable salt, in particular a sodium or potassium salt, of an oligonucleotide according to any one of claims 1 to 26.

28. A conjugate comprising an oligonucleotide or a pharmaceutically acceptable salt according to any one of claims 1 to 27 and at least one conjugate moiety covalently attached to the oligonucleotide or the pharmaceutically acceptable salt, optionally via a linker moiety.

29. A pharmaceutical composition comprising an oligonucleotide, pharmaceutically acceptable salt or conjugate according to any one of claims 1 to 28 and a therapeutically inert carrier.

30. An oligonucleotide, pharmaceutically acceptable salt or conjugate according to any one of claims 1 to 28 for use as therapeutically active substance.

31. An oligonucleotide, pharmaceutically acceptable salt or conjugate according to any one of claims 1 to 28 for use in the treatment or prevention of a cardiac or hematologic disorder.

32. Use of an oligonucleotide, pharmaceutically acceptable salt or conjugate according to any one of claims 1 to 28 for the preparation of a medicament for the treatment or prevention of a cardiac or hematologic disorder.

33. Use of an oligonucleotide, pharmaceutically acceptable salt or conjugate according to any one of claims 1 to 28 in the treatment or prevention of a cardiac or hematologic disorder.

34. A method for treating or preventing a cardiac or hematologic disorder comprising administering to a patient in need thereof an effective amount of the oligonucleotide, pharmaceutically acceptable salt, or conjugate of any one of claims 1 to 28.

35. A method for manufacturing an oligonucleotide according to any one of claims 1 to 28, comprising the steps of:

(a) coupling a nucleoside thiophosphorimide to the terminal 5 'sulfur atom of a 5' S-modified nucleoside or oligonucleotide to produce a dithiophosphite triester intermediate;

(b) sulfur oxidizing the dithiophosphorous triester intermediate obtained in step a); and

(c) optionally further extending the oligonucleotide.

36. An oligonucleotide made according to the method of claim 35.

37. The invention as hereinbefore described.

Examples

Example 1

Oligonucleotide synthesis

Oligonucleotides were synthesized using a MerMade 12 automated DNA synthesizer from Bioautomation. Using controlled-pore glass supports with universal jointsThe synthesis was performed on a 1. mu. mol scale.

In a standard cycling procedure for coupling DNA and LNA phosphoramidites, 3% (w/v) trichloroacetic acid in CH₂Cl₂The solution was applied three times at 200. mu.L for 30 seconds for DMT deprotection. With 100. mu. L0.1M in acetonitrile (or for LNA-^MeC structural unit in acetonitrile/CH₂Cl₂1: 1) and 110 μ L of a 0.1M solution of 5- (3, 5-bis (trifluoromethylphenyl)) -1H-tetrazole in acetonitrile as an activator and coupled for 180 seconds, the corresponding phosphoramidite is coupled three times. For sulfur oxidation, a 0.1M solution of 3-amino-1, 2, 4-dithiazole-5-thione in acetonitrile/pyridine 1:1(3X 190. mu.L, 55 seconds) was used. Using THF/lutidine/Ac₂Capping was performed by treatment for 55 seconds with O8: 1:1(CapA, 75. mu. mol) and THF/N-methylimidazole 8:2(CapB, 75. mu. mol).

The synthesis cycle incorporating 2',5' -deoxy-5 ' -mercaptophosphoramidite involves treatment with 100 μ L of a 0.1M acetonitrile solution and 110 μ L of a 0.1M acetonitrile solution of 5- (3, 5-bis (trifluoromethylphenyl)) -1H-tetrazole for 180 seconds to couple phosphoramidite building blocks. Three couplings were performed. A0.1M solution of 3-amino-1, 2, 4-dithiazole-5-thione in acetonitrile/pyridine 1:1(3X 190. mu. mu.l) was usedL, 55 seconds) was subjected to sulfur oxidation. For capping THF/lutidine/Ac₂O8: 1:1(CapA, 75. mu. mol) and THF/N-methylimidazole 8:2(CapB, 75. mu. mol) solutions were applied for 55 seconds. CH with 3% (w/v) trichloroacetic acid₂Cl₂The solution was applied 15 times at 200 μ L for 30 seconds for DMT deprotection and thiol release. It may also be advantageous to use a catalyst in the presence of 5-30% (v/v) triethylsilane in CH₂Cl₂Solutions and/or 2-10% of p-methoxythiophenol CH₂Cl₂In the case of the solution of (1) to (10) percent (v/v) trifluoroacetic acid or (5) to (10) percent (w/v) trichloroacetic acid, 200. mu.L (45 seconds) was applied 3 to 6 times for DMT deprotection and thiol release.

Thiophosphosphoramide as activator with 100. mu.L of 0.15M 10% (v/v) CH₂Cl₂And 110. mu.L of a 0.1m solution of 5- (3, 5-bis (trifluoromethylphenyl)) -1H-tetrazole in acetonitrile were coupled 3 times for 600 seconds each. Standard sulfur oxidation was performed using a 0.1M solution of 3-amino-1, 2, 4-dithiazole-5-thione in acetonitrile/pyridine 1:1(3X 190. mu.L, 55 sec) and THF/lutidine/Ac₂O8: 1:1(CapA, 75. mu. mol) was capped and a THF/N-methylimidazole 8:2(CapB, 75. mu. mol) solution was applied (55 seconds).

The nucleobase protecting groups were removed and cleaved from the solid support by drying at 55 ℃ for 15-16 hours using a mixture of ammonia (32%) containing 20mM DTT and ethanol (3:1, v: v). The crude DMT-oligonucleotide was purified using a solid phase extraction column and purified by ion exchange chromatography or by RP-HPLC using a C18 column, followed by DMT removal with 80% aqueous acetic acid and ethanol precipitation.

The following molecules were prepared according to the general procedure described above.

Trithioester modification between bold and underlined nucleotides

A、G、^mC. T represents LNA nucleotide

a. g, c, t represent DNA nucleotides

All other bonds being prepared as phosphorothioates

Compounds #1-13 are based on SEQ ID NO 1. As shown in the above table, they differ in the position of the phosphorothioate internucleoside linkage of formula (I).

Example 1

In vitro Activity data

LTK cells were grown in cell culture medium (DMEM [ Sigma, Cat. No. D0819 ]) supplemented with 10% fetal bovine serum [ Sigma, Cat. No. F7524] and 0.025mg/ml gentamicin [ Sigma, Cat. No. G1397 ]. Cells were trypsinized every 5 days, washed with Phosphate Buffered Saline (PBS), [ Sigma Cat. No. 14190-. Cells were kept in culture for up to 15 passages.

For experimental use, 2000 cells per well were seeded in 96-well plates (Nunc catalog No. 167008) in 100 μ L growth medium. Oligonucleotides were prepared from 750 μ M stock solutions. Oligonucleotides dissolved in PBS were added approximately 24 hours after seeding the cells to a final concentration range of 16-50,000 nM. Cells were cultured in the presence of oligonucleotides for 3 days.

The medium was removed and 125. mu.L was addedPro 96 lysis buffer (Invitrogen 12173.001A) and 125. mu.L 70% ethanol, cells were harvested. RNA was purified according to the manufacturer's instructions and eluted in a final volume of 50. mu.L of water to give RNA concentrations of 10-20 ng/. mu.l. Prior to the single-step qPCR reaction, the RNA was diluted 10-fold in water. For the single step qPCR reaction, the qPCR mix (qScriptTMXLE 1-step RT-qPCR from QauntaBio)Low ROX, Cat 95134-500) was mixed with two Taqman probes at a ratio of 10:1:1(qPCR mix: Probe 1: Probe 2) to create a premix. Taqman probes were purchased from Life technologies: MALAT 1: mm 01227912; BCL 2Mm 00477631; GAPDH 4352339E. The master mix (6. mu.L) and RNA (4. mu.L, 1-2 ng/. mu.L) were then incubated at qPCR plate (optical 384 wells, 4309849). After sealing, the plates were spun at 1000g for 1 min at room temperature and then transferred to a Viia 7 system (Applied Biosystems, Thermo) using the following PCR conditions: 50 ℃ for 15 minutes; 3 minutes at 95 ℃; 40 periods: 95 ℃ for 5 seconds, then the temperature is reduced by 1.6 ℃/sec, followed by 60 ℃ for 45 seconds. Data were analyzed using quantstudio Real time PCR software.

The results are shown in figure 1.

The data clearly demonstrates the tolerance of this modification. In addition, such modifications have great potential for optimizing conventional phosphorothioate oligonucleotides. In many cases, a significantly lower IC50 value can be obtained with only a single modification. Introducing this modification to the LNA side wings of the spacer has particular benefits. This clearly demonstrates the value of modification to optimize potency and remove chiral centers from phosphorothioate oligonucleotides.

Example 2

Target mRNA levels in the heart were measured at a dose of 15mg/kg (Malat1)

Mice (C57/BL6) were given a 15mg/kg dose of oligonucleotide subcutaneously as three doses on days 1,2 and 3 (n ═ 5). Mice were sacrificed on day 8 and heart MALAT-1 RNA reduction was measured. The parent compound was administered in two doses of 3x 15mg/kg and 3x 30 mg/kg.

The results are shown in fig. 2.

Example 3

Binding affinity to ApoB RNA

The following ApoB sequences were generated according to the procedure of example 1.

Compound ID number	Sequence of	Tm(℃)
			Reference ApoB	GCattggtatTCA	57
#14	GCattggtatTCA	56
			#15	GCattggtatTCA	56
#16	GCattggtatTCA	53
			#17	GCattggtatTCA	55
#18	GCattggtatTCA	53
			#19	GCattggtatTCA	54
#20	GCattggtatTCA	55
			#21	GCattggtatTCA	53

Trithioester modification between bold and underlined nucleotides

A. G, C, T represents LNA nucleotides

a. g, c, t represent DNA nucleotides

All other bonds being prepared as phosphorothioates

Compounds #14-21 are based on SEQ ID NO 2. As shown in the above table, they differ in the position of the phosphorothioate internucleoside linkage of formula (I).

The melting temperature of the LNA duplexes was measured on a Cary 300(Agilent) equipped with a thermal controller. LNA and reverse strand RNA were annealed in phosphate buffer (20mM Na)₂HPO₄200mM NaCl,0.2mM EDTA, pH 7) at a molar concentration of 1.5 μ M. The absorption at 260nm was measured. The temperature gradient was kept set at1 ℃/min in the range of 25 ℃ to 95 ℃ and the holding time was set at 3 minutes at 25 ℃ and 95 ℃ respectively. Absorbance readings were taken every 30 seconds. The melting curve is fitted to a sigmoid and the Tm is determined by its derivative.